health benefits of probiotics research paper

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Advances in probiotics research: mechanisms of action, health benefits, and limitations in applications

• Published: 19 October 2023
• Volume 4 , pages 386–406, ( 2024 )

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• Karan Prajapati   ORCID: orcid.org/0000-0002-5799-7732 1 ,
• Krithika Bisani   ORCID: orcid.org/0000-0001-8335-1102 1 ,
• Harsh Prajapati   ORCID: orcid.org/0000-0002-7821-8240 1 ,
• Siddharth Prajapati   ORCID: orcid.org/0000-0003-2669-6950 1 ,
• Dharmista Agrawal   ORCID: orcid.org/0000-0003-4682-1798 1 ,
• Sweta Singh   ORCID: orcid.org/0000-0002-3740-6907 1 ,
• Meenu Saraf   ORCID: orcid.org/0000-0003-4964-9452 1 &
• Dweipayan Goswami   ORCID: orcid.org/0000-0003-0165-0294 1  

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The human gastrointestinal tract harbors a complex microbiota, pivotal in maintaining health equilibrium. Disruption of this microbial balance has implications for myriad health conditions. Probiotics, beneficial microbial entities, have demonstrated potential in rectifying gut microbiota imbalances, offering health benefits and disease prevention. This review elucidates the nuanced roles of probiotics, emphasizing their interactions with both pathogenic and commensal gut microorganisms. Recent breakthroughs in the identification of potent probiotic strains and their prospective applications in biomedical research are delineated. Comprehensive analyses of clinical studies underscore the safety, efficacy, and applicability of probiotics in diverse food and therapeutic avenues. As probiotic research burgeons, this review amalgamates current insights with future directions, accentuating the transformative prospects of probiotics in contemporary biomedical paradigms. The gut's microflora plays a cardinal role in nutrient metabolism, immune modulation, and protection against pathogens. Disturbances in this microflora can lead to dysbiosis, with potential repercussions for digestive and systemic health. Probiotics exert their beneficial effects through multiple mechanisms, including competitive exclusion of pathogens, production of antimicrobial substances, and modulation of the host's immune response. Their health benefits encompass not only gastrointestinal health, such as in the management of diarrhea, irritable bowel syndrome, and inflammatory bowel diseases but also systemic effects in areas like immune modulation and mental health. The growing recognition of these benefits has led to a surge in the market demand for probiotic supplements and fortified foods, with research continually unveiling novel strains and applications.

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Prajapati, K., Bisani, K., Prajapati, H. et al. Advances in probiotics research: mechanisms of action, health benefits, and limitations in applications. Syst Microbiol and Biomanuf 4 , 386–406 (2024). https://doi.org/10.1007/s43393-023-00208-w

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Article Contents

Introduction, properties essential for effective and successful probiotics, potential attributes and benefits of probiotics, molecular characterization of probiotics marker genes and surface layer protein (slpa), mechanism of action of probiotics, prospective applications of probiotics in developing healthful foods, carriers for probiotics, legislation and safety regarding probiotics, validation of health claims, future prospects: toward genetically modified designer probiotics, acknowledgements, probiotics, their health benefits and applications for developing healthier foods: a review.

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Ravinder Nagpal, Ashwani Kumar, Manoj Kumar, Pradip V. Behare, Shalini Jain, Hariom Yadav, Probiotics, their health benefits and applications for developing healthier foods: a review, FEMS Microbiology Letters , Volume 334, Issue 1, September 2012, Pages 1–15, https://doi.org/10.1111/j.1574-6968.2012.02593.x

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In the industrialized world, functional foods have become a part of an everyday diet and are demonstrated to offer potential health benefits beyond the widely accepted nutritional effects. Currently, the most important and frequently used functional food compounds are probiotics and prebiotics, or they are collectively known as ‘synbiotics’. Moreover, with an already healthy image, dairy products appear to be an excellent mean for inventing nutritious foods. Such probiotic dairy foods beneficially affect the host by improving survival and implantation of live microbial dietary supplements in the gastrointestinal flora, by selectively stimulating the growth or activating the catabolism of one or a limited number of health-promoting bacteria in the intestinal tract, and by improving the gastrointestinal tract's microbial balance. Hence, the paper reviews the current scenario of probiotics and their prospective potential applications for functional foods for better health and nutrition of the society.

Probiotics are defined as ‘live microorganisms which when administered in adequate amount confer health benefits to the host’ ( FAO/WHO, 2002 ). Alternatively, probiotics have been defined as live microbial feed supplements that beneficially affect the host animal by improving its intestinal microbial balance ( Fuller, 1989 ). Probiotics were originally used to improve the health of both animals and humans through the modulation of the intestinal microbiota. At present, several well-characterized strains of Lactobacilli and Bifidobacteria are available for human use to reduce the risk of gastrointestinal (GI) infections or treat such infections ( Salminen et al. , 2005 ). Some of the beneficial effects of probiotic consumption include improvement of intestinal health by the regulation of microbiota, and stimulation and development of the immune system, synthesizing and enhancing the bioavailability of nutrients, reducing symptoms of lactose intolerance, and reducing the risk of certain other diseases ( Fig. 1 ; Kumar et al. , 2009a , b , 2010 , 2011a , b ; Nagpal et al. , 2007 , 2010 , 2011 ; Yadav et al. , 2007a , b , 2008 ). The primary clinical interest in the application of probiotics has been in the prevention of and treatment for GI infections and diseases ( Parvez et al. , 2006 ). Gut microbiota deviations have been associated with enhanced risk of specific diseases; therefore, modulation of an unbalanced indigenous microbiota forms the rationale of probiotic therapy ( Turnbaugh et al. , 2006 ). Also, the development of adjuvant or alternative therapies based on bacterial replacement is becoming important owing to the rapid emergence of antibiotic-resistant pathogenic strains and the adverse consequences of antibiotic therapies on the protective flora, which enhances the risk of infection ( Forestier et al. , 2001 ). However, the use of probiotics should be further investigated for their benefits and possible side effects, if any. As the knowledge about intestinal microbiota, nutrition, immunity, and genetics in health and disease has increased in the past years, such information could certainly help to develop new probiotic strains with disease-specific functions and could also facilitate the understanding of when to use probiotics and how they affect specific pathological states. However, it is important that the probiotic strains for human use should undergo animal studies followed by human clinical trials in order to authenticate the suitability, safety, and benefits of probiotics for human consumption and development of functional foods.

Projected prospective health attributes of probiotics.

It is of utmost importance that the probiotic strain survives the site where it is presumed to be active. For maximum activity, the strain should be able to proliferate and colonize at this specific location. Besides, it should also be tolerated by the immune system. It should not be pathogenic, allergic, or mutagenic/carcinogenic ( Toma & Pokrotnieks, 2006 ; Ohashi & Ushida, 2009 ). Probiotics for human should have ‘generally regarded as safe’ status, with a proven low risk of inducing or being associated with the etiology of disease. The probiotic organisms should preferably be of human origin ( Collins et al. , 1998 ), must be able to survive and grow in the in vivo conditions of the desired site of administration, and thus must be able to tolerate low pH and high concentration of both conjugated and deconjugated bile acids. For successful application in foods, the probiotic used should also be technologically compatible with the food-manufacturing process. In addition to that, the foods containing the probiotic bacteria must maintain the characteristic sensory attributes of the traditional food.

It is now an established fact that the indigenous microbial communities is host specific, location specific, very complex in composition and has beneficial properties to the host. However, it is not precisely known which species of microorganisms play the principal part in these beneficial properties. Some major health benefits of probiotics and their proposed mechanisms are illustrated in Table 1 . Several probiotic bacteria have been introduced in the market, and the range of products in which probiotic bacteria are added is increasing ( Table 2 ). Some of the major health attributes of probiotics are discussed in the following sections.

Health benefits of probiotic bacteria to the host, and speculated mechanisms involved

Some commercial probiotic strains used by various industries

Antimicrobial properties

The intestinal microbial community is a complex ecosystem, and introducing new organisms into this highly competitive environment is difficult. Thus, organisms that can produce a product that inhibits the growth of existing organisms have a characteristic advantage. The ability of probiotics to establish in the GI tract is enhanced by their ability to eliminate competitors. Some antimicrobials with producer organisms are enlisted in Table 3 . In different studies on humans and animals, beneficial microorganisms are used to improve the colonization resistance on body surfaces, such as GI, the urogenital, and the respiratory tract. Bifidobacteria produce acetic and lactic acids in a molar ratio of 3 : 2 ( Desjardins & Roy, 1990 ). Lactobacillus acidophilus and Lactobacillus casei produce lactic acid as the main end product of fermentation. In addition to lactic and acetic acids, probiotic organisms produce other acids, such as hippuric and citric acid. Lactic acid bacteria also produce hydrogen peroxide, diacetyl, and bacteriocin as antimicrobial substances. These inhibitory substances create antagonistic environments for foodborne pathogens and spoilage organisms. Yoghurt bacteria are reported to produce bacteriocin against probiotic bacteria and vice versa ( Dave & Shah, 1997 ).

Antimicrobial substances produced by probiotic bacteria ( Fuller, 1992 )

Anticarcinogenic properties

Goldin & Gorbach (1980 ) reported that the introduction of L. acidophilus into the diet lowers the incidence of chemically induced colon tumors in rats. Later, the same authors also suggested that diet and antibiotics can lower the generation of carcinogens in the colon and reduce chemically induced tumors ( Goldin & Gorbach, 1984 ). These effects appear to be mediated through the intestinal microbial communities. A possible mechanism for these anticancer effects relies on inhibiting intestinal bacterial enzymes that convert procarcinogens to more proximal carcinogens ( Kumar et al. , 2011a , b ). This approach can be expanded in the future by testing probiotics for their ability to inhibit the growth of organisms normally found in the flora that have high activities of enzymes such as β-glucuronidase ( Reddy, 1999 ), nitroreductase, azoreductase, and β-glycosidase or the capability for nitrosation.

The sixth most commonly diagnosed cancer in the world is hepatitis B virus. Consumption of foods, contaminated with aflatoxins, is also established causes of liver cancer. Aflatoxin B1 (AFB1) causes characteristic genetic changes in the p53 tumor suppressor gene and ras protooncogenes. Some probiotic bacterial strains have been successfully shown to bind and neutralize AFB1 in vivo and thus reduce the bioabsorption of the toxin from the gut ( Haskard et al. , 2000 ; Kumar et al. , 2011a , b ). Addition of probiotic Bifidobacterium longum to the diet of rats has been shown to exert a strong antitumor activity on colonic mucosa by reducing the expression level of ras-p21 expression and cell proliferation ( Reddy, 1998 ). Lactobacillus GG administration determined the up- and downregulation of 334 and 92 genes, respectively, by affecting the expression of genes involved in immune response and inflammation [transforming growth factor-beta (TGF-β) and tumor necrosis factor (TNF) family members, cytokines, nitric oxide synthase 1, defensin alpha-1], apoptosis, cell growth and cell differentiation (cyclins and caspases, oncogenes), cell—cell signaling (intracellular adhesion molecules and integrins), cell adhesion (cadherins), signal transcription and transduction ( Caro et al. , 2005 ).

Probiotics have also been found by several researchers to decrease fecal concentrations of enzymes (glycosidase, B-glucuronidase, azoreductase, and nitroreductase) and secondary bile salts and reduce the absorption of harmful mutagens that may contribute to colon carcinogenesis ( Rafter, 1995 ). Normal intestinal flora can influence carcinogenesis by producing enzymes (glycosidase, B-glucuronidase, azoreductase, and nitroreductase) that transform precarcinogens into active carcinogens ( Goldin, 1990 ; Pedrosa et al. , 1995 ). Lactobacillus acidophilus and L. casei supplementation in humans helped to decrease the levels of these enzymes ( Lidbeck et al. , 1991 ). In mice, these bacterial enzymes were suppressed with the administration of Lactobacillus GG ( Drisko et al. , 2003 ). Several mechanisms have been proposed as to how lactic acid bacteria may inhibit colon cancer, which includes enhancing the host's immune response, altering the metabolic activity of the intestinal microbial communities, binding and degrading carcinogens, producing antimutagenic compounds, and altering the physiochemical conditions in the colon ( Hirayama & Rafter, 2000 ; Kumar et al. , 2011a , b ). Oral administration of LAB has been shown to effectively reduce DNA damage, induced by chemical carcinogens, in gastric and colonic mucosa in rats ( Li & Li, 2003 ). By comet assay, L. acidophilus , Lactobacillus gasseri , Lactobacillus confusus , Streptococcus thermophilus , Bifidobacterium breve , and B. longum were antigenotoxic toward N ′-nitro- N -nitrosoguanidine (MNNG; Pool-Zobel et al. , 1996 ). These bacteria were also protective toward 1, 2-dimethylhydrazine (DMH)-induced genotoxicity. Metabolically active L. acidophilus cells, as well as an acetone extract of the culture, prevented MNNG-induced DNA damage, while heat-treated L. acidophilus was not antigenotoxic. Azomethane-induced colon tumor development was also suppressed with a decrease in colonic mucosal cell proliferation and tumor ornithine decarboxylase and ras-p21 activities ( Hirayama & Rafter, 2000 ). There was a report on the antitumorigenic activity of the prebiotic inulin, enriched with oligofructose, in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis in the azoxymethane (AOM)-induced colon carcinogenesis rat model ( Femia et al. , 2002 ). Other lactic acid bacteria have also shown the ability to lower the risk of colon cancer; however, the relationship between enzyme activity and cancer risk needs further investigation.

Immunologic enhancement

There have been several reports indicating that lactobacilli used in dairy products can enhance the immune response of the host. Organisms that have been identified as having this property are B. longum , L. acidophilus , L. casei subsp. rhamnosum , and Lactobacillus helveticus ( Isolauri, 2001 ). However, prospective probiotics should be tested in the future for the enhancement of the immunologic response. The measurements that should be considered are lymphocyte proliferation, interleukins 1, 2, and 6, TNF, prostaglandin E production, and serum total protein, albumin, globulin, and gamma interferon. The intrinsic properties of lactobacilli to modulate the immune system make them attractive for health applications. Enhanced phagocytic activity of granulocytes, cytokine excretion in lymphocytes, and increased immunoglobulin-secreting cells in blood are typical responses to probiotics, all of which are indicative of changes in the immune system. An inflammatory immune response produced cytokine-activated monocytes and macrophages, causing the release of cytotoxic molecules capable of lysing tumor cells in vitro ( Philip & Epstein, 1986 ). The inflammatory cytokines IL-1 and TNF-α exerted cytotoxic and cytostatic effects on neoplastic cells in in vitro models ( Raitano & Kore, 1993 ). Aatourri et al. (2002 ) observed increased lymphocyte proliferation in the spleen, peripheral blood, and Peyer's patches and also increased IFN-γ production in Peyer's patches and spleen of rats fed yogurt containing L. bulgaricus 100158 and S. thermophilus 001158. Because immune function declines with age, enhancing immunity in the elderly with probiotics would be of particular use ( Gill & Rutherfurd, 2001 ). Regardless of the mechanisms involved, probiotics cultures have been shown to stimulate both nonspecific immunity and specific immunity. Possible stimulation of an immune response by probiotic bacteria may explain potential therapeutic and prophylactic applications of such cultures in the treatment for infections and carcinogenesis.

Enhancement of short-chain fatty acid production

Because the improved intestinal microbial communities with probiotics primarily involve the stimulation of intestinal fermentation, the stimulation of short-chain fatty acid (SCFA) production is one of the essential factors for the beneficial effects exerted by probiotics. A significant increase in indigenous lactobacilli in the large intestine as a result of probiotic Lactobacillus has been reported ( Tsukahara & Ushida, 2001 ). Although increases in lactobacilli stimulate lactate production, lactate does not accumulate in the large intestine, except in those patients with short bowel syndrome and dyspeptic diarrhea ( Tsukahara & Ushida, 2001 ). Rather, lactate is normally metabolized to acetate, propionate, or butyrate by lactate-utilizing bacteria ( Bourriaud et al. , 2005 ; Belenguer et al. , 2006 ). Lactate-utilizing bacteria from the human flora have been previously identified as belonging to the Clostridia cluster XIVa, based on their 16S rRNA gene sequences ( Duncan et al. , 2004 ). The increase in fecal SCFA by probiotic Lactobacillus would be due to this mechanism ( Tsukahara et al. , 2006 ). In fact, the oral administration of the lactate-utilizing and butyrate-producing bacterium, Megasphaera elsdenii , with Lactobacillus plantarum has been shown to increase the butyrate production in the large intestine ( Tsukuhara et al. , 2002 ). Thus, the administration of probiotics with other lactate-utilizing bacteria, butyrate-producing bacteria, in particular, could be a more effective way to achieve maximum health benefits.

Antiatherogenic and cholesterol-lowering attributes of probiotics

Coronary heart diseases and cardiovascular diseases (CVD), major causes of most death in adults, are conditions in which the main coronary arteries supplying the heart are no longer able to supply sufficient blood and oxygen to the heart muscle (myocardium). Although low-fat diets offer an effective means of reducing blood cholesterol concentrations, these appear to be less effective, largely due to poor compliance, attributed to low palatability and acceptability of these diets by the consumers. Therefore, attempts have been made to identify other dietary components that can reduce blood cholesterol levels. Individuals with CVD and those with a higher risk of developing the condition are treated in a number of ways to help lower their LDL cholesterol and triacylglycerol (TAG) concentrations while elevating their high-density lipoprotein cholesterol. The role of fermented milk products as hypocholesterolemic agents in human nutrition is still equivocal, as the studies performed have been of varying quality, and statistically analysis with incomplete documentation being the major limitation of most studies. However, since 1974 when Mann & Spoerry (1974 ) showed an 18% fall in plasma cholesterol levels after feeding 4–5 liters of fermented milk per day for 3 weeks to Maasai warriors, there has been a considerable interest in the effect of probiotics on human lipid metabolism. Supplementation of diet with dairy products fermented with LAB has the potential to reduce serum cholesterol levels in humans and animals ( Pulusoni & Rao, 1983 ). A significant decrease in serum cholesterol level in rats fed milk fermented with L. acidophilus has been reported ( Grunewald, 1982 ). Mann (1977 ) showed that large dietary intake of yogurt lowered the cholesterolemia in humans.

Experiments by Gilliland et al. (1985 ) have shown that dietary elevation of plasma cholesterol levels can be prevented by the introduction of a L. acidophilus strain that is bile resistant and assimilates cholesterol. These findings were supported by Pereira & Gibson (2002 ) who demonstrated that probiotic strains were able to assimilate cholesterol in the presence of bile into their cellular membranes. Results, however, were influenced greatly by the bacterial growth stage, and inoculum using resting cells did not interact with cholesterol as also shown by studies conducted by Dambekodi & Gilliland (1998 ). St-Onge et al. (2000 ) extensively reviewed the existing studies from animal and human studies which detected that moderate cholesterol lowering was attributable to the consumption of fermented products containing probiotic bacteria. Studies by Gopal et al. (1996 ) also showed cholesterol removal by Bifidobacterium spp. and L. acidophilus . The possible mechanisms of action of probiotics are cholesterol assimilation by bacteria, deconjugation of bile salts, cholesterol binding to bacterial cell walls, and reduction in cholesterol biosynthesis ( Pulusoni & Rao, 1983 ; Pereira & Gibson, 2002 ).

Probiotics in diabetes and obesity

The role of gut flora in the pathology of insulin resistance (type 2 diabetes) and obesity has been well documented by Ley et al. (2005 ). Animal and human studies have suggested that gut flora enhances the body weight gain and increases the insulin resistance, and these phenotypes are transmittable with gut flora during the implantation studies of microbiota from obese to normal and germ-free mice ( Ley et al. , 2006 ; Turnbaugh et al. , 2006 ). The mechanisms associated with gut flora—mediated pathology of obesity and diabetes are through (1) increased energy harvest, (2) increased blood LPS levels (endotoxemia), and (3) low-grade inflammation ( Delzenne et al. , 2011 ). Therefore, modulation of gut flora has been considered as a potential target to treat against obesity and diabetes. Probiotics are novel gut flora modulators, and their role in the prevention of and treatment for diabetes and obesity has been implicated in recent past by Yadav et al. (2007a , b , 2008 ). Yadav et al. (2007b , 2008 ) suggested that probiotic-supplemented fermented milk product called dahi (yogurt) dramatically suppressed diet-induced insulin resistance and protected from streptozotocin-induced diabetes in animal models. It was also observed that probiotic dahi suppressed the diabetes progression and its complication through enhancing antioxidant system ( Yadav et al. , 2008 ). Though, the actual link between probiotic-mediated pathology of obesity and diabetes has been debated on the basis of farm animal's data ( Raoult, 2008 ; Delzenne & Reid, 2009 ; Ehrlich, 2009 ). In relation to these controversies, Bifidobacteria , one of the important classes of probiotic organisms, have been found to be decreased in overweight women in comparison with normal weight women ( Santacruz et al. , 2009 ). Recent studies have suggested that probiotic-based selective strains of Lactobacilli and Bifidobacteria show beneficial effects on obesity and type-2 diabetes ( Aronsson et al. , 2010 ). Andreasen et al. (2010 ) reported that L. acidophilus decreased the insulin resistance and inflammatory markers in human subjects. More recently, Vajro et al. (2011 ) and others ( Kang et al. , 2010 ; An et al. , 2011 ; Chen et al. , 2011 ; Naito et al. , 2011 ) showed that feeding of specific strains of Lactobacilli and Bifidobacteria ameliorate the progression of obesity and diabetes, suggesting that probiotic-mediated modulation of gut flora can be a potential therapy against obesity and diabetes. Although animal studies have shown promising results in probiotic-mediated suppression of obesity and diabetes, very few studies in humans showed the significant effects. Hence, it is required to conduct well-designed studies for examining the efficacy of probiotic-based formulation in the treatment for obesity and diabetes. Also, the mechanism(s) of action for probiotic-based formulation is not completely understood; therefore, future studies should also be focused on describing the probiotic action—targeted molecules and organs in physiologic models.

Other potential benefits

Certain functional foods containing probiotic provide preformed lactase to gut and allow better digestion of lactose. The regulatory role of probiotics in allergic disease was demonstrated by a suppressive effect on lymphocytes' proliferation and interleukin-4 generation in vitro ( Sutas et al. , 1996 ). Subsequently, the immune inflammatory responses to dietary antigens in allergic individuals were shown to be alleviated by probiotics, this being partly attributable to enhance the production of anti-inflammatory cytokines ( Pessi et al. , 2000 ) and transferring growth factor-β ( Haller et al. , 2000 ). Probiotic bacteria also possess prophylactic and therapeutic properties. Other potential benefits include protection against vaginal or urinary tract infections, reduction in ulcers and intestinal tract infections, increased nutritional value, maintenance of mucosal integrity, reduction in catabolic products eliminated by kidney and liver, stimulation of repair mechanism of cells, breaking down and rebuilding hormones, relieving anxiety and depression, formation, maintenance, or reconstruction of a well-balanced indigenous intestinal and/or respiratory microbial communities, inhibiting decalcification of the bones in elderly people, and synthesis of vitamins and predigestion of proteins.

In view of high stakes involved in the exploration of their commercial value, particularly in the booming functional/health food market, the correct identification of probiotic cultures has become extremely important to rule out the possibility of false claims and to resolve disputes concerning their identity in probiotic preparations ( Mohania et al. , 2008 ). The phylogenetic information encoded by 16S rRNA gene has enabled the development of molecular biology techniques, which allow the characterization of the whole human gut microbiota ( Lawson, 1999 ). These techniques have been used in monitoring the specific strains as they have high discriminating power. Numerous molecular techniques have been exploited for the identification of various putative probiotic marker genes such as bile salt hydrolase (BSH), mucus-binding protein (mub), fibronectin-binding protein (fbp) for the screening of probiotic strains.

Bile salt hydrolase (BSH) gene

BSH, an intracellular enzyme found commonly in certain intestinal bacteria, plays a vital role. BSH catalyzes the hydrolysis of glycine- or taurine-conjugated bile acids into the amino acid residue and deconjugated bile acid. The ability of probiotic strains to hydrolyze bile salts has often been included among the criteria for the selection of probiotic strain, and a number of BSHs have been identified and characterized. It has been investigated that Lactobacillus isolates of human origin along with Bifidobacterium also possess bsh homologs in their genome. Sequence analysis of these bsh homologs establishes intraspecies heterogeneity and interspecies homogeneity, which might be due to the horizontal transfer of bsh gene from one species to other. With the completion of some probiotic genome projects, analyses of sequenced probiotic ( Lactobacilli and Bifidobacteria ) strains reveal that many possess more than one bsh homolog and each BSH may respond to different types of bile or perhaps different length of exposure to bile. Therefore, BSH activity by a probiotic bacterium may be a desirable property because it could maximize its prospects of survival in hostile environment of GI tract and hence can be used as one of the potential markers for the screening of probiotic strains. Because large amounts of deconjugated bile salts may have undesirable effects for the human host, concerns may arise over the safety of administering a BSH-positive probiotic strain. However, the bacterial genera that would most likely to be used as probiotics ( Lactobacilli and Bifidobacteria ) are not capable of dehydroxylating deconjugated bile salts, and so the majority of the breakdown products of BSH activity by a probiotic strain may be precipitated and excreted in feces. Hence, the ability of probiotic strains to hydrolyze conjugated bile salts has often been included among the criteria for probiotic strain selection ( FAO/WHO, 2002 ).

Mucus-binding protein (Mub), Fibronectin-binding protein (FbpA), and surface layer protein (SlpA)

Roos & Jonsson (2002 ) identify the mub gene encoding mucus-binding protein in Lactobacillus reuteri ATCC 53608 (strain 1023). Using the immunoglobulin G (IgG) fraction of an antiserum against cell surface proteins of L. reuteri ATCC 53608 (strain 1023), they screened a phage library and identified a number of clones that were reactive with the antiserum and adhered to mucus. Subcloning resulted in the identification of the mub gene, encoding a very large sortase-dependent protein (SDP) with a highly repetitive structure (3000 residues). Domains with the two main types of repeats, that is, Mub1 and Mub2, were shown to adhere to mucus after recombinant expression in Escherchia coli . In another L. reuteri strain, 100-23, a similar approach using an antiserum against the surface proteins was used to identify the lsp (large cell surface protein) gene, which encodes a high molecular mass cell wall protein, Lsp ( Walter et al. , 2005 ). Mutational analysis showed a reduced ecological performance of the lsp mutant in the murine gastro intestinal tract (GIT). Boekhorst et al. (2005 ) performed an in silico search for potential mucus-binding proteins present in several publicly available databases. They reported that a total of 48 proteins containing at least one MUB domain were identified in 10 lactic acid bacterial species. Callanan et al. (2008 ) reported that these mucus-binding proteins are involved mainly in GIT colonization as observed from the genome sequence of the dairy isolate L. helveticus DPC4571. A striking difference between the various mucus-binding proteins is the number of repeats of the MUB domain, and it might be interesting to investigate whether the number of repeats correlates with the capacity of binding to mucus ( Boekhorst et al. , 2006 ).

Buck et al. (2005 ) reported the genes encoding FbpA, Mub, and SlpA all contribute to the ability of L. acidophilus NCFM to adhere to Caco-2 cells in vitro , confirming that adhesion is determined by multiple factors. mub and fbpA mutations resulted in 65% and 76% decreases in adherence, respectively. In a similar study, VanPijkeren et al. (2006 ) mined the genome of L. salivarius UCC118 for the presence of sortase gene homologs and genes encoding SDPs. The sortase gene srtA was deleted, three genes encoding SDPs (large surface protein lspA , lspB , and lspD ) were disrupted, and the capacity of adherence of these mutants to HT-29 and Caco-2 cells was investigated. Both the srtA and the lspA mutant showed a significant decrease in adherence. While the adherence of the srtA mutant was on average 50% of wild-type levels, the lspA mutant adhered at around 65%, only slightly better than the Sortase srtA mutant, indicating that LspA plays a key role in adherence to these intestinal cells.

Probiotic bacteria have multiple and diverse influences on the host. Different organisms can influence the intestinal luminal environment, epithelial and mucosal barrier function, and the mucosal immune system. The numerous cell types affected by probiotics involve epithelial cells, dendritic cells, monocytes/macrophages, B cells, T cells. There are significant differences between probiotic bacterial genera and species. These differences may be due to various mechanism of action of probiotics. It is crucial that each strain be tested on its own or in products designed for a specific function. Molecular research on these probiotics pays attention to these strain-specific properties. Different probiotic strains have been associated with different effects related to their specific capacities to express particular surface molecules or to secrete proteins and metabolites directly interacting with host cells.

The effectiveness of probiotics is related to their ability to survive in the acidic and alkaline environment of gut as well as their ability to adhere and colonize the colon. The mechanisms for the improved mucosal barrier are achieved by providing a means of limiting access, with respect to pH, redox potential, hydrogen sulfide production, and antimicrobial compounds/molecules, to enteric pathogens or by several interrelated system such as mucous secretion, chloride and water secretion, and binding together of epithelial cells. Hydrogen peroxide in combination with lactoperoxidase—thiocyanate milk system exerts a bactericidal effect on most pathogens ( Kailasapathy & Chin, 2000 ). Bacillus clausii constitute < 1% of gut microbial communities, stimulate CD4 proliferation, and produce bacteriocins to limit the growth of potential pathogens. Microbial communities also enhance nutritive value by producing several enzymes for the fermentation of nondigestible dietary residue and endogenously secreted mucus ( Roberfroid et al. , 1995 ) and help in recovering lost energy in form of short-chain fatty acids. They also have a role in the synthesis of vitamins ( Conly et al. , 1994 ) and in the absorption of calcium, magnesium, and iron ( Younes et al. , 2001 ). Some examples of host benefit and suspected mechanism have been summarized in Table 1 .

A growing public awareness of diet-related health issues and mounting evidence regarding health benefits of probiotics have increased consumers demand for probiotic foods. A number of food products including yoghurt, frozen fermented dairy deserts, spray-dried milk powder, cheeses, ice cream, freeze-dried yoghurt ( Nagpal et al. , 2007 ; Kumar et al. , 2009a ; Nagpal & Kaur, 2011 ), and fruit juices ( Nagpal et al. , 2012 ) have been suggested as delivery vehicles for probiotic to consumer. It has been suggested that approximately 10 9 CFU per day of probiotic microorganisms is necessary to elicit health effects. Based on the daily consumption of 100 g or mL of probiotic food, it has been suggested that a product should contain at least 10 7 cells per g or mL of a food, a level that was also recommended in Japan ( Ross et al. , 2002 ). The most popular food delivery systems for probiotic have been fermented milk and yoghurt. A few studies have shown that many commercial yoghurt products have failed to successfully deliver the required level of viable cells of probiotic bacteria ( Dave & Shah, 1997 ). Cheeses have a number of advantages over fresh fermented products (such as yoghurt) as a delivery system for viable probiotic to GI tract. Cheeses tend to have a higher pH and more solid consistency where the matrix of the cheese and its relatively high fat content may offer protection to probiotic bacteria during passage through the GI tract. Cheese also has high buffering capacity than yoghurt ( Gardiner et al. , 1998 ). Overall, the major points to be addressed while incorporating probiotics into foods are the selection of a compatible probiotic strain/food type combination; using food processing conditions that are compatible with probiotic survival; ensuring that the food matrix supports probiotic growth (if fermentation is required); selecting a product matrix, packaging, and environmental conditions to ensure adequate probiotic survival over the product's supply chain and during shelf storage; and finally ensuring that addition of the probiotic does not adversely impact on the taste and texture of the product.

Probiotics are normally added to foods as a part of the fermentation process. The emphasis for prolonged survival of probiotics in the food matrix has resulted in the alteration in the functionality and efficacy of the food product. In order to exert health benefits, probiotic bacteria must remain viable in the food carriers and survive the harsh condition of GI tract, with a minimum count of 10 6 CFU g −1 . The nature of food carrier can affect the stability of the probiotic microorganisms during GI transit. Although dairy-based products are suggested to be the main carriers for the delivery of probiotics, other nondairy-based products such as soy and fruits can be exploited as a potential carrier of probiotic microorganisms because of the increasing demand for new flavor and taste among consumers. A brief idea about the variety of products that serve as carriers for probiotics is given in Table 4 .

Details of the products that serve as carriers for probiotics

The regulatory status of probiotics as a component in food has to be established on an international level. A regulatory framework should be established to better address probiotic issues, including efficacy, safety, labeling, fraud, and claims. Probiotic products shown to confer defined health benefits on the host should be permitted to describe these specific health benefits. Surveillance systems (trace-back, postmarketing) should be put in place to record and analyze adverse events associated with probiotics in food and monitor long-term health benefits. Probiotic products should be made more widely available, especially for relief work and to populations at high risk of morbidity and mortality. Foods that could be regarded as functional foods are subject to regulations drawn up for other food groups. The US Food and Drug Administration (FDA) defined four food categories: conventional foods, constituting the largest category and including articles of food and drink that do not fall into the other three categories such as foods for special dietary use; medical foods; and dietary supplements. According to Berner & O'Donnell (1998 ), it is possible to envision ‘functional foods’ in any of the categories of foods and supplements mentioned above. From a legislative standpoint, probiotic-containing foods could fit into several of the four categories of foods described by the FDA; however, there is no explicit recognition of any health benefits of probiotic-, prebiotic-, or culture-added dairy foods in the United States.

Government regulations regarding safety assessment differ among countries, and the status of probiotics as a component in food is currently not established on an international basis. For the most part, probiotics come under food and dietary supplements because most are delivered by mouth as foods and, as such, are allowed to make only general health claims. The factors that must be addressed in the evaluation of safety of probiotics include pathogenicity, infectivity, and virulence factors comprising toxicity, metabolic activity, and the intrinsic properties of the microorganisms. Donohue & Salminen (1996 ) provided some methods for assessing the safety of lactic acid bacteria through the use of in vitro studies, animal studies, and human clinical studies and indicated that some current probiotic strains are reported to fulfill the required safety standards. Salminen & Marteau (1997 ) also proposed studies on intrinsic properties, pharmacokinetics, and interactions between the host and probiotics as means to assess the safety of probiotics. It was recognized that there is a need to accurately enumerate the probiotic bacteria in food products to include them on a label and that proper manufacture and handling procedures be employed to ensure the maintenance of viability and probiotic activity through processing, handling, and storage of probiotic foods, including powdered milk products. Good evidence exists that specific strains of probiotics are safe for human use and able to confer some health benefits on the host, but such benefits cannot be extrapolated to other strains without experimentation. As there has been an increased influx of probiotic products in the Indian market during the last decade, therefore an initiative was taken by the Indian Council of Medical Research and Department of Biotechnology, Government of India, to formulate guidelines for the regulation of probiotic products in the country ( Ganguly et al. , 2011 ), defining a set of parameters required for a product/strain to be termed as ‘probiotic’. These include the identification of the strain, in vitro screening for probiotic characteristics, and in vivo animal and human studies to establish efficacy, requirements for labeling of the probiotic products with strain specification, viable numbers at the end of shelf-life, storage conditions, etc., so as to help the consumers to safeguard their awareness.

To validate or substantiate a health-related claim, the proposed relationship between the product and the health-related end point should be identified, and appropriate measurements of both should be indicated. The interests of patients and consumer involvement are becoming integral part of clinical development and should be taken into consideration. For regulatory purposes, health-related claims require sound evidence from all available sources. Positive evidence should not be outweighed by negative evidence, and sufficient evidence based on human experience should be available to support the safety and efficacy, including pre- and postmarketing experience. The greater the consistency of evidence from different sources, the stronger the evidence will be.

The Nutrition Labeling and Education Act of 1990 gives the US Food and Drug Administration (FDA) the authority to regulate health claims on food labels. These claims describe the link between specific nutrients or substances in food, and a particular disease or health-related condition. The process of reviewing the scientific evidence of health claims involves the following steps: define the substance—disease relationship that is the subject of the claim, identify relevant studies, classify the studies, rate the studies on the basis of quality, rate the studies on the basis of the strength of their body of evidence, and report the studies' rank order.

Genetic manipulation offers the potential to enhance the existing probiotic properties of an organism or to load an organism with probiotic properties ( Steidler, 2003 ). Elucidation of mechanisms of activity of a probiotic could enable the manipulation of organisms to create specific and targeted probiotics. Although consumer resistance to genetically modified organisms is such that GMO probiotic foods are unlikely in the near future, potential clinical applications to ameliorate or prevent chronic intractable diseases may be more readily accepted. For instance, Steidler (2003 ) treated mice with genetically modified Lactococcus lactis to deliver mouse cytokine IL-10 at the intestinal mucosa to prevent colitis, demonstrating that probiotics can be designed to produce potent bioactive chemicals. Braat et al. (2006 ) also constructed a biologically contained L. lactis to produce human IL-10 and treated Crohn's disease patients with this GM L. lactis in a phase-1 placebo-uncontrolled trial. A decrease in disease activity was observed with minor adverse effects, and containment of the organism was achieved through its dependency on thymidine for growth and IL-10 production.

Another possibility of gut microbial community management is the use of synbiotics, where probiotics and prebiotics are used in combination. A prebiotic is a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, thus improving the host health ( Gibson & Roberfroid, 1995 ). The combination of suitable probiotics and prebiotics enhances the survival and activity of the organism. The combination of prebiotic and probiotic has synergistic effects because in addition to promoting the growth of existing strains of beneficial bacteria in the colon, synbiotics also act to improve the survival, implantation, and growth of newly added probiotic strains. The synbiotic concept has been widely used by European dairy drink and yoghurt manufacturers such as Aktifit (Emmi, Switzerland), Proghurt (Ja Naturlich Naturprodukte, Austria), Vifit (Belgium, UK), and Fysiq (the Netherlands; Niness, 1999 ). The combination of Bifidobacterium and oligofructose was reported to synergistically improve colon carcinogenesis in rats compared to when both were given individually ( Gallaher & Khil, 1999 ). Another study reported that a synbiotic containing Pediococcus pentoseceus , Leuconostoc mesenteroides , Lactobacillus paracasei , and L. plantarum with four fermentable fibers namely β-glucan, inulin, pectin, and resistant starch reduced the occurrence of postoperation infections from 48% to 13% in 66 liver transplant patients ( Rayes et al. , 2005 ). Most of the claims on benefits of different synbiotics are on general health ( Gibson & Roberfroid, 1995 ). There have yet been any clinical trials on suitable combinations of synbiotics that specifically target reduction in serum cholesterol level in animals and humans. Bifidobacteria and Lactobacilli are the most frequent target organisms for prebiotics. Although there is growing interesting development of new functional foods with synbiotics, the concept of synbiotics has been studied to a limited extent and needs further investigations. Only a few human studies have been carried out on the effectiveness of synbiotics ( Morelli et al. , 2003 ).

There are evidences from well-conducted clinical trials of beneficial health effects from probiotics in a range of clinical conditions. The concept of ‘synbiotics’ has recently been proposed to characterize health-enhancing food and supplements used as functional food ingredients in humans, and with the advent of the functional food concept, it is clear that there is an important niche for these probiotic-based approaches. Although from the ongoing research, more of promising potential health effects of probiotics are being observed, more standardized and verifiable clinical studies are needed to demonstrate the safety, efficacy, and limitations of a putative probiotic, to determine effects on the immune system in healthy and diseased individuals and effects of long-term consumption, and to resolve whether it is superior to existing therapies. Also, the prospect of GM probiotics targeted for clinical conditions demands a rigorous safety strategy to prevent spread into the environment and dissemination of the genetic modification.

The authors report no conflicts of interest.

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Health benefits of probiotics: a review

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• 1 Microbiology Department, Sismanoglion General Hospital of Athens, 15126 Athens, Greece.
• 2 Internal Medicine Department, APPK, Laiko Hospital of Athens, 11527 Athens, Greece.
• PMID: 24959545
• PMCID: PMC4045285
• DOI: 10.5402/2013/481651

Probiotic bacteria have become increasingly popular during the last two decades as a result of the continuously expanding scientific evidence pointing to their beneficial effects on human health. As a result they have been applied as various products with the food industry having been very active in studying and promoting them. Within this market the probiotics have been incorporated in various products, mainly fermented dairy foods. In light of this ongoing trend and despite the strong scientific evidence associating these microorganisms to various health benefits, further research is needed in order to establish them and evaluate their safety as well as their nutritional aspects. The purpose of this paper is to review the current documentation on the concept and the possible beneficial properties of probiotic bacteria in the literature, focusing on those available in food.

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‘Gut-Healing’ Probiotic Supplements Are Everywhere Right Now. Here’s What the Science Says

If you haven’t noticed, we’re living in the “heal your gut” era. Within the past few years, there’s been a wild influx of influencers, documentaries, cookbooks, and news articles breaking down how you can eat this or take that to “fix” your gut, and subsequently, a range of health problems. As someone who deals with horrible stomach issues, I have lurked in Reddit “ microbiome ” threads for tips and clicked on catchy ads for probiotic supplements that claim to get rid of uncomfortable bloating or cure chronic indigestion. And I’ll admit it: They intrigue me. I would love nothing more than to throw money at a pill or powder that makes it easier (and less painful) for me to digest food. But sadly, my gastroenterologist has told me multiple times (because I’ve asked multiple times) that probiotic supplements likely won’t fix my messed up tummy.

Linda Lee, MD, the chief of the division of gastroenterology at Northwell Health’s North Shore University Hospital in Manhasset, New York, tells SELF that, over the years, lots of her patients have asked about probiotics too. So much research is being published on the importance of the gut microbiome—the trillions of microorganisms, like bacteria, viruses, and fungi, that naturally reside in your GI tract—and how it both contributes to and protects against the development of chronic diseases like diabetes and cancer, disorders like irritable bowel syndrome ( IBS ), and mental health conditions like depression and anxiety. As scientists have learned more, there’s been a rapid push to figure out how these microbes can be strategically used to improve our well-being.

So naturally, “gut health” and “microbiome” swiftly became buzzwords in wellness spaces on and offline. “Companies have tapped into that interest—and maybe even fear—and convinced people that they need to take a probiotic supplement to correct what’s ‘wrong’ in there,” Dr. Lee says. There are gummies and powders and capsules that can, allegedly, do it all—and we gobble them up (spending billions on probiotic products every year in the process). But here’s the catch: Scientists don’t have a solid grasp on whether probiotic supplements have legit benefits or even how they might work—at least not yet. As Aparna Church, PhD , the codirector of the Goodman-Luskin Microbiome Center with UCLA Health, tells SELF: “It’s a hot mess.”

If you, like me, find yourself enchanted by the potential benefits of probiotic supplements, keep reading. We take a deep dive into what we do (and don’t) know about these encapsulated little critters—and when, if ever, it’s a good idea to take them.

What even are probiotics?

Probiotics are bacteria and yeasts that, simply put, are presumed to have some kind of health benefit. These good bugs are naturally found in fermented foods, like yogurt, kombucha, kefir, sauerkraut, and kimchi, among others. And when they make their way into your GI tract, they can alter your microbiome and support things like digestion, brain health, and immune function . Different bugs are being studied for different effects: Bifidobacterium are thought to inhibit the growth of harmful pathogens and potentially fight cancer cells, for example, while Lactobacillus might reduce gut inflammation (a common precursor to chronic diseases) and ease digestive woes like diarrhea .

Dr. Church, who’s dedicated her career to studying the gut microbiome, believes probiotics—as in, the actual bugs in fermented foods—are (generally) great. They have so much potential that it’s no surprise companies decided to cash in and pack them into capsules and sell them as catch-all wonder-workers. But probiotic-rich foods and probiotic supplements are not one and the same. Fermented foods typically contain a wide variety of good bacteria that are “alive and active in natural environments,” says Dr. Church, whereas supplements usually contain a high concentration of specific strains that were freeze-dried and stuffed into a pill. “One’s more controlled, one’s more natural,” she says (more on why that matters below).

Probiotic supplements do show some promise—but researchers haven’t totally proven their worth yet.

We are in the golden age of probiotics research. Growing evidence suggests certain types of good bacteria can ease GI symptoms like bloating, shorten the duration of some infections, support mental health, ease inflammatory skin issues like eczema, and improve metabolic conditions like diabetes. “We know that probiotics are good. We know that,” Dr. Church says.

However, there are only a few specific scenarios that probiotic supplementation is currently recommended for, like pouchitis, which is inflammation of an artificial “pouch” that’s surgically placed in the colon of certain folks with inflammatory bowel disease (IBD), like Crohn’s or ulcerative colitis . Probiotics are also often recommended for preterm infants to prevent certain complications that are caused, in part, by microbiome disturbances. And they’re occasionally used to restore gut bacteria in kids and adults taking antibiotics, as these meds can wipe out both good and bad germs, often leading to a bad case of diarrhea. But this route is hotly debated, even among experts and accredited organizations. So far, the potential benefit of taking probiotic supplements while on antibiotics has largely been seen in people with a higher risk of C. difficile infection, a harmful bacteria that can colonize the gut once the good guys are knocked out. (Even in this situation, there are varying criteria for “high-risk” among doctors.)

Experts can’t say for sure if these good bugs alleviate symptoms of or prevent digestive conditions like IBD or pancreatitis, and while some data show they might help improve immune-related issues like allergies and asthma, it’s too soon to start shelling out supplements to folks diagnosed with them. Even with IBS, probiotics are only recommended to those involved in a clinical trial (so things like safety can be controlled while side effects and efficacy are carefully monitored). It’s not that probiotic supplements are total BS—Dr. Church is hopeful that one day we’ll have the rigorous science needed to create expansive guidance, but we’re just not there yet. Case in point: If you look at the American Gastroenterology Association’s guidance on probiotics , you’ll see its recommendations (or lack thereof) are often chalked up to a “knowledge gap” or a varying “quality of evidence.”

As for the average, generally healthy person who grabs a bottle of probiotics tablets at Whole Foods? “There’s no evidence that taking a probiotic [supplement] is actually beneficial to you if it’s not for a specific condition,” Dr. Lee says.

Despite the strides in science, it’s worth noting that a lot of probiotics research is sloppy and unreliable.

Here’s where we’ve gotten ahead of ourselves: There isn’t one perfect gut microbiome everyone should strive for, says Dr. Lee. In fact, there are likely different kinds of bacterial communities that are good for human health. You probably have a mix of bacteria that’s different from the germs camping out in my intestines. Remember: This idea that you can take any ol’ probiotic to “fix” your gut health is an extremely appealing but faulty one. “We don’t even know if a probiotic you take is really ideal for you versus somebody else,” says Dr. Lee.

The major shortfall of probiotic supplements is they operate off the claims that these bugs can help treat, say, a vaginal infection or persistent bloating for basically anyone. Again, don’t get us wrong—some supportive evidence suggests they might ease certain symptoms. But what people often don’t realize, according to Dr. Lee, is that the strains examined in scientific trials are very specific; there are trillions of organisms that come in various strains, each of which can have vastly different health effects in people.

For example, when Dr. Lee’s IBS patients ask if they should be taking a probiotic, she’ll walk them through the existing research and explain how, for example, some studies discovered that Bifidobacterium helped people with IBS feel less bloated—but she’ll stress this doesn’t mean it’ll help them specifically, even if they have IBS, even if they take a pill loaded with Bifidobacterium . “Just because one strain [used in a study] was helpful for IBS does not mean another Bifidobacterium strain is going to confer the same benefits,” Dr. Lee says. To make matters more complicated, there are different types of IBS —certain folks largely deal with constipation, others diarrhea, and some get a combo of the two—that might influence how their bodies react to specific bugs. Your genetics, biological sex, diet, general health history, and even where you live might affect your response to certain probiotics too. As a result, “what might work for one person might not work for another,” says Dr. Church.

Supplements, shampoos, and serums that offer impressive results are everywhere right now. Experts have thoughts.

The body of growing research, at large, is also all over the place. “Different studies often use different strains of probiotics at varying dosages, making it really hard to compare the results and draw definitive conclusions,” says Dr. Church. There’s no standardization across the board. Plus, a lot of studies often use a small, mostly white group of people, lack a placebo (which ensures any reported effects are legitimate), and only look at the short-term risks and benefits. For context, studies should include a lot of people (not just rats!) who come from different backgrounds and are studied over a lengthy period of time—at least a few months, ideally longer. Without these factors in place, the findings aren’t reliable, says Dr. Church.

Companies want to get their supplements out on the market fast because probiotics are hot right now—but they can’t do that if they take the time to conduct a high-quality yearslong trial. Dr. Church says that we desperately need long-term safety and efficacy data before we can come to a consensus, before we can say any particular probiotic works for a certain health issue and why. The only consensus, it seems, is that the jury is still out.

Not to mention, the probiotics market is an unregulated free-for-all.

To make matters stickier, supplements aren’t regulated by the FDA. Unlike pharmaceutical medications, probiotic pills and capsules aren’t required to undergo rigorous safety and efficacy testing (hence the whole research dilemma detailed above). They can, pretty much, be created and thrown onto shelves—the FDA will only go after a brand if, after the fact, the supplement seems to be causing health issues for people who’ve taken it. It’s illegal for a manufacturer to claim its supplement can treat or cure diseases, but they can say something nebulous like “This pill supports your gut health.” So they do.

The lack of federal regulation also means there’s no telling what’s actually in your probiotic pill. Studies have found that the ingredients included in these capsules don’t always match what’s listed on their packaging. Some contain significantly lower levels of the bacteria they claim to have, completely different types of microbes than the label specifies, and, in some cases, pathogens that can potentially make you sick. (This is particularly concerning for people who are immunocompromised. Probiotic supplements, in general, aren’t recommended for folks with weakened immune systems because they can increase the risk of an infection.)

There’s also the looming question of whether the bugs can even survive the journey from the manufacturing plant, into the capsule, onto the (likely hot) truck, into the store, from your car to your house, into your acidic stomach, and finally, through the rest of your gut. These factors can absolutely reduce the potential benefits of probiotics, says Dr. Church, so supplements need to be formulated and stored in a way that’ll keep them alive and viable before the bugs even brave the journey through your body. (This might look like freeze-drying and encapsulating them, which may wind up killing off bugs, or creating time-release formulas that are more resilient to heat—not like there’s any credible oversight on that!).

Before you add that “miracle” pill or powder to your cart, read this.

Even if you create and store them perfectly, the clock is working against you. “The number of live bacteria [in a supplement] decreases over time,” says Dr. Church. Right now, there’s no easy way for scientists to look inside a person’s intestines or measure fecal samples and determine if the probiotics survived all of the roadblocks, but in vitro experiments have shown viability plummets as they weather the harsh environment in your gut. Some manufacturers pack their pills with extra bacteria to try to account for this decline, but it’s not clear whether that’s an effective workaround.

Yet the biggest dilemma, at least in Dr. Church’s eyes, is misinformation. A lot of influencers share captivating stories about how probiotic supplements cleared their acne, eased their allergies, or kept their vagina clear of persistent infections—sometimes, they have megabucks deals with brands and reach millions of eyeballs. Much of what you see about any type of supplement on social media is inaccurate—but that doesn’t stop it from having an enormous influence over what people put into their bodies.

Probiotic supplements aren’t too risky, but they’re also not the cure-alls some people swear them to be.

According to Dr. Lee, eating a diet rich in diverse and nutritious foods is the best way to support your gut microbiome. She gives this same advice to her patients, friends, and family: “If you really care about your gut health, pay attention to what you eat.” Every time you swallow a snack or meal, it tinkers with your gut bacteria. So start with probiotic-rich foods and toss some cheese into your salad, or reach for some Greek yogurt when you’re hankering for a snack.

Research has also shown that people’s gut bacteria expanded dramatically when they shifted from an omnivorous diet to a vegetarian one, and that a high-fiber diet (roughly 25 to 35 grams a day) can feed beneficial gut bacteria. (Good bugs feed off prebiotics , or special plant fibers, to grow, thrive, and survive.) Dr. Lee likens noshing on fiber to growing a garden: “You’re trying to cultivate good plants so you give it good soil and nutrients.”

If you want to try a probiotic supplement , clue in your doctor or a registered dietitian. They can walk you through the possible risks and benefits based on your individual health. Probiotic supplements are pretty low-risk if you’re generally healthy, so they likely won’t lead to any major side effects or issues (aside from less money in your bank account), says Dr. Lee. If anything, you might get gassy or bloated, in which case you can just stop taking them. The most important thing, according to Dr. Lee, is to temper your expectations since the most likely scenario is that they’ll do a whole lot of nothing.

When Dr. Lee sees people who really want to give probiotic supplements a shot, she suggests only taking them for a month. “If you’re feeling no better after a month, it’s probably not doing anything for you,” she tells them. And hey, maybe they seem like they do something for you (or you end up riding the high of a powerful placebo effect). If you feel like it’s making you burp less or reducing your acid reflux, it might be worth sticking with them if your doctor agrees it’s the best option for you. The reason: Studies have shown that once you stop taking a probiotic supplement, that bacteria disappears from your microbiome. “It’s not like you’re recolonizing yourself permanently by taking a probiotic for a shorter period of time,” Dr. Lee says.

This is not a shot at probiotics as a whole. Even the scientists who question the whole gut health movement think probiotics—and the promise they might hold—are pretty wonderful. It’s just easy to get swayed by flashy advertising and the buzz around these good bugs when they’re neatly packaged into a pill. Until we have the science to back supplements specifically, it’s crucial to recognize that no one knows if that pack of probiotic chews is doing anything for your gut or if it’ll only give you a quick case of the toots.

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• 1 Department of Human Nutrition and Dietetics, School of Food and Agricultural Sciences, University of Management and Technology, Lahore, Pakistan
• 2 UniLaSalle, Univ. Artois, ULR7519 - Transformations & Agro-resources, Normandie Université, Mont-Saint-Aignan, France
• 3 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China
• 4 National Institute of Food Science and Technology, University of Agriculture, Faisalabad, Pakistan
• 5 School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China
• 6 School of Food and Biological Engineering, Jiangsu University, Zhenjiang, Jiangsu, China
• 7 College of Food and Biological Engineering, Jimei University, Xiamen, China
• 8 Department of Seafood Processing Technology, Faculty of Fisheries, Cukurova University, Adana, Türkiye
• 9 Biotechnology Research and Application Center, Cukurova University, Adana, Türkiye
• 10 CBQF - Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal
• 11 LEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Porto, Portugal
• 12 ALiCE—Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal
• 13 Department of Food Development and Food Quality, Institute of Food Science and Human Nutrition, Gottfried Wilhelm Leibniz University Hannover, Hannover, Germany
• 14 Department of Food Science, Faculty of Agriculture, Zagazig University, Zagazig, Egypt
• 15 School of Food Science and Engineering, South China University of Technology, Guangzhou, China

Probiotics, like lactic acid bacteria, are non-pathogenic microbes that exert health benefits to the host when administered in adequate quantity. Currently, research is being conducted on the molecular events and applications of probiotics. The suggested mechanisms by which probiotics exert their action include; competitive exclusion of pathogens for adhesion sites, improvement of the intestinal mucosal barrier, gut immunomodulation, and neurotransmitter synthesis. This review emphasizes the recent advances in the health benefits of probiotics and the emerging applications of probiotics in the food industry. Due to their capability to modulate gut microbiota and attenuate the immune system, probiotics could be used as an adjuvant in hypertension, hypercholesterolemia, cancer, and gastrointestinal diseases. Considering the functional properties, probiotics are being used in the dairy, beverage, and baking industries. After developing the latest techniques by researchers, probiotics can now survive within harsh processing conditions and withstand GI stresses quite effectively. Thus, the potential of probiotics can efficiently be utilized on a commercial scale in food processing industries.

1. Introduction

Probiotics, in the form of supplements or food products, have emerged as the most prominent ingredient in the era of functional foods. Probiotics have always been a vital component and commercial target for providing potential health benefits ( Sanz et al., 2016 ; Hamad et al., 2022 ). The term “probiotic” was first presented by Werner Kollath in 1953, which is known to be a derivative of the Latin word pro and the Greek word βιο meaning “for life.” Kollath defined probiotics as active bodies with essential functions for promoting various health aspects ( Gasbarrini et al., 2016 ). Food and Agriculture Organization (FAO) and World Health Organization (WHO) described them as “live microbes when administered in adequate quantities, confer health benefits on host organisms” ( Munir et al., 2022 ). Several bacteria belonging to the genera Pediococcus, Lactococcus, Enterococcus, Streptococcus, Propionibacterium , and Bacillus are considered potential microbes for probiotic status ( de Brito Alves et al., 2016 ; Hamad et al., 2022 ).

The frequently used strains belong to the divergent group of Bifidobacterium and Lactobacillus that significantly affect health with various actions. They detoxify xenobiotics and environmental pollutants ( Reid, 2015 ), bio-transform mycotoxins in foods ( Hamad et al., 2022 ), synthesize vitamin K, riboflavin, and folate ( Reid, 2015 ; Hamad et al., 2022 ), and ferment undigested fiber in the colon ( Warman et al., 2022 ). Probiotics prevent pathogenic bacteria by restricting binding sites on mucosal epithelial cells and modulating the host immune response, thus improving intestinal barrier integrity ( Fusco et al., 2023 ). The advantages of probiotics are related to the modulation of gut microbiota, mitigation of nutritional intolerances (lactose intolerance), increase in bioavailability of macro and micronutrients, and alleviation of allergic incidences in susceptible individuals ( Roobab et al., 2020 ).

Probiotics can be consumed either by incorporating them into foods or drinks in the form of dairy or non-dairy foodstuffs or as supplements ( Fenster et al., 2019 ). Various fermented foods have active microbes genetically similar to the strains utilized as probiotics. It has been observed that fermented foods enhance the functional and nutritional aspects by transforming substrates and producing bioactive and bioavailable end-products ( Marco et al., 2017 ). The approximate consumption of 10 9 colony-forming unit (CFU)/day have been revealed as an effective dose ( Hill et al., 2014 ). By keeping in view, the effective dosage, probiotics are being incorporated into many foods like beverages, ice cream, yogurt, bread, and many others by the food industry. The most significant barrier associated with probiotics in the food industry is their susceptibility to processing conditions and sensitivity to gastrointestinal (GI) stresses. However, regarding their health benefits, the consumer always showed an inclined interest in probiotic products ( Konuray and Erginkaya, 2018 ). Now scientists have developed new and innovative methods like nanoencapsulation and genetic modification, which enable probiotics to withstand harsh conditions of both processing and GI stresses in the body ( Putta et al., 2018 ). This review paper provides a profound insight into the mechanistic approach and current perspective on the beneficial aspects of probiotics in preventing and treating various diseases. The application and safe utilization of probiotics in major food industries have also been described.

2. Mechanisms of action

Outstanding advances have been made in the field of probiotics, but there has yet to be a key breakthrough in the documentation of their mechanism of action. Probiotics possibly exert a positive potential on the human body through these main mechanisms; competitive exclusion of pathogens, improvement in intestinal barrier functions, immunomodulation in the host’s body, and production of neurotransmitters ( Figure 1 ; Plaza-Diaz et al., 2019 ). Probiotics compete with pathogens for nutrients and receptor-binding sites, making their survival difficult in the gut ( Plaza-Diaz et al., 2019 ). Probiotics also act as anti-microbial agents by producing substances; short chain fatty acids (SCFA), organic acids, hydrogen peroxide ( Ahire et al., 2021 ), and bacteriocins ( Fantinato et al., 2019 ) thus decreasing pathogenic bacteria in the gut. Moreover, probiotics improve the intestinal barrier function by stimulating the production of mucin proteins ( Chang et al., 2021 ), regulating the expression of tight junction proteins, including occluding and claudin 1, and regulating the immune response in the gut ( Bu et al., 2022 ; Ma et al., 2022 ).

Figure 1 . Mechanism of action of probiotics. 1. Probiotics perform their function by competing with pathogens for nutrients and receptors for binding thereby making their survival and adherence to gut mucosa difficult. 2. Probiotics produce anti-microbial substances which inhibit pathogens growth. 3. Probiotics promote epithelial barrier function by enhancing mucus production and increasing the expression of tight junction proteins which prevents the translocation of pathogens from intestine into the blood. 4. Probiotics regulate immunity of the host by modulating maturation and function of dendritic cells subsequently increasing the activity of T cells which play important role in immune homeostasis. 5. Probiotics also regulate the production of neurotransmitters including serotonin, dopamine and gamma aminobutyric acid (GABA).

Probiotics also regulate the innate and adaptive immune response modulating dendritic cells (DC), macrophages B and T lymphocytes. Probiotics also increase the production of anti-inflammatory cytokines while interacting with intestinal epithelial cells and attracting macrophages and mononuclear cells ( Petruzziello et al., 2023 ). Furthermore, probiotics can produce neurotransmitters in the gut through the gut-brain axis. Specific probiotic stains can modulate the serotonin, gamma-aminobutyric acid (GABA), and dopamine levels, affecting mood, behavior, gut motility, and stress-related pathways ( Srivastav et al., 2019 ; Sajedi et al., 2021 ; Gangaraju et al., 2022 ).

3. Health attributes of probiotics

The health benefits of probiotics are associated with preventing and reducing many diseases, i.e., allergic diseases, cancer, hypercholesterolemia, lactose intolerance, inflammatory bowel disease, diarrhea, and irritable bowel syndrome ( Grom et al., 2020 ), as shown in Figure 2 . Table 1 shows different studies regarding the application of probiotics in different diseases.

Figure 2 . Health attributes of probiotics. Probiotics help in the prevention and management of allergic diseases, cancer, hypercholesterolemia, irritable bowel syndrome, diarrhea, lactose intolerance, inflammatory bowel disease.

Table 1 . Therapeutic effect of probiotics in gastrointestinal disorders.

3.1. Antiallergic effect of probiotics

Allergy is a hypersensitive disorder of the immune system, termed as type I hypersensitivity and defined as a “disease following a response by the immune system to an antigen.” With escalating incidence rate, allergies affect nearly half of the population of Europe and North America. These allergic reactions occur due to one or more common environmental substances or antigens ( Prakash et al., 2014 ). The most common allergic reactions include asthma, rhinitis, atopic eczema, dermatitis, urticaria, angioedema, hay fever, and food, drug, and insect hypersensitivity ( Lopez-Santamarina et al., 2021 ). The gut microbiome is a viable therapeutic target for managing allergic diseases ( Harata et al., 2016 ), as they modulate the immunological and inflammatory response that consequently affects the development of sensitization and allergy ( Fiocchi et al., 2015 ).

Allergic diseases are characterized by an imbalance in lymphocyte-governed immunity in which the immune response becomes overly biased toward T helper 2 lymphocytes dominated response (Th2 cells) ( Di Costanzo et al., 2016 ). Allergen-sensitized Th2 cells produce various interleukins such as IL-1, IL-4, and IL-5, thus recruiting granular effector cells, i.e., mast cells, eosinophils, and basophils toward the site of allergic inflammation. In addition, the interleukins switch B lymphocyte immunoglobulin isotype, which upsurges the circulating level of total and allergen-specific IgE ( Galli et al., 2020 ). Although the precise mechanism is not entirely known, it is expected that the probiotics improve mucosal barrier functions, stimulate the immune system, reduce leakage of antigen through the mucosa, produce anti-inflammatory cytokines, increase the production of secretory IgA (exclude antigens from intestinal mucosa), degrade dietary antigen and up-regulate anti-inflammatory cytokines as IL-10 ( Liang et al., 2022 ).

The proposed mechanism for the antiallergic effect of probiotics is the augmentation of T helper cells (Th)1/Th2 immune balance by suppressing Th2 skewed immune response and favoring Th1 cell response ( Di Costanzo et al., 2016 ). Ma et al. (2019) explain that probiotics modulate the function of dendritic cells, which in turn have the ability peripheral Tregs. Tregs control the excess immune response and maintain a balance between Th1 and Th2 cells ( Figure 3 ). Besides, lactobacilli stimulate regulatory T cells which play a paramount role in balancing immune response through the production of immunosuppressive cytokines and modulation of IgE, IgA, and IgG production ( Owaga et al., 2014 ).

Figure 3 . Anti-allergic effect of probiotics. Tregs, T regulatory cells; Th 1, T helper cells type 1; Th 2, T helper cell type 2; IL, interleukin; IFN α, interferon α. Probiotics help in the migration and maturation of dendritic cells via modulating the composition of gut microbiota. Dendritic cells in the gut-associated lymphoid tissues have the ability to induce the development of peripheral Tregs and to play a central role in the development of immune homeostasis. Tregs maintain the proper level of Th 1, Th 2 cells as well as anti-allergy and pro-allergy cytokines.

The antiallergic effect of Lactiplantibacillus plantarum SY12 and L. plantarum SY11 was studied using RAW 264.7 (murine macrophage) cell line. Both species showed a reduction in the production of nitric oxide, T helper 2 linked cytokines, tumor necrosis factor-α, and cyclooxygenase-2 as well as inducible nitric oxide synthase compared to the control group ( Lee et al., 2014 ). In this regard, the Limosilactobacillus reuteri effect was also investigated against the food allergy in ovalbumin (OVA)-sensitized BALB/c mice. Oral intake of L. reuteri helped restore the deteriorated profile of colonic microflora and attenuated allergic diarrhea. It also increased the activation of mast cells, enhanced the production of serum immunoglobulin E (IgE), suppressed the T helper 1 and 2 cytokines production, down-regulated the GATA3 expression, and increased the expression of TGF-b, IL-10, and Foxp3. The findings confirmed the anti-allergic activities of L. reuteri promoted by the modulation of enteric flora and enhancement of tolerogenic immune responses ( Huang et al., 2017 ).

3.2. Cancer suppressor activity of probiotics

Probiotics could be used as an adjuvant for various types of cancers based on their potential to modulate enteric flora and enhance local and systematic immunity. They prevent the initiation, progression, and metastasis of transplantable or chemically induced tumors ( Samanta, 2022 ). The effect of probiotics can be observed in suppressing both intestinal and extraintestinal cancers ( So et al., 2017 ). The interaction of probiotics and their metabolites (bacteriocin, peptides, and organic acids) with critical metabolic pathways such as cellular proliferation, inflammation, apoptosis, angiogenesis, and metastasis has been revealed by many researchers ( Harikumar et al., 2013 ). Moreover, the probiotics inhibit carcinogenesis by inhibiting pathogens through competitive exclusion, increasing short-chain fatty acid production ( Chong, 2014 ), reducing carcinogenic bile salts production, binding carcinogens and mutagens, down-regulating NF-kappa B dependent genes products for cell proliferation (Cox-2, cyclin D1) and cell survivability (Bcl-3, Bcl-xL) and enhancing apoptosis ( Konishi et al., 2016 ). Probiotics also upregulate TNF-related apoptosis-inducing ligand (TRAIL) ( Klłonowska-Olejnik, 2004 ), modulate cell cycle by rapamycin (mTOR)/4EBP1 ( Islam et al., 2014 ) and inhibit the formation of aberrant crypt foci ( Yu and Li, 2016 ). Figure 4 describes the anti-cancer effect of probiotics.

Figure 4 . Cancer suppressor activity of probiotics. Probiotics use different pathways to fight against cancer. Probiotics inhibit β glucuronidase activity, produce folate which ultimately modulate DNA methylation patterns protecting the integrity of genome, produce short chain fatty acids (SCFA) enhancing cell differentiation and apoptosis of cancerous cells, exclude pathogens involved in chronic inflammation which may lead to cancer development.

Previous studies have scrutinized that the ERK1/2 pathway modulates cell survival, proliferation, differentiation, and cell motility by regulating the BCL-2 protein family in mitochondria ( Passaniti et al., 2022 ). Saccharomyces boulardii, both in vitro and in vivo , inhibited the activation of ERK1/2 mitogen-associated protein kinase. In the same way, probiotic L. reuteri induced apoptosis in human myeloid leukemia-derived cells by modulating NF-kappa B and MAPK signaling pathways ( Saber et al., 2017 ). The colonic microflora has also been related to the development of liver disorders such as liver fibrosis ( De Minicis et al., 2014 ), nonalcoholic fatty liver diseases ( Zhuge et al., 2022 ), and more recently, liver cancer ( So et al., 2017 ). Probiotics have been demonstrated to inhibit hepatocellular carcinoma (HCC) progression by reducing liver tumor size and down-regulating angiogenic factors. The mechanistic approach to this is the level of T helper (Th) 17 cells in the gut and its recruitment to tumor sites was lower in probiotic-treated mice ( Li et al., 2016 ). In breast cancer apart from immunomodulation, the hypoxia-inducible factor (HIF) pathway was also reported to be significantly suppressed by Lactobacillus cultures supernatant ( Esfandiary et al., 2016 ).

In addition to this, experimental studies were carried out to reduce the mutagenic potential of a powerful carcinogen; N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG) by Lacticaseibacillus rhamnosus Vc. Oral feeding of L. rhamnosus Vc (10 9  CFU) to Gallus gallus (chicks) for 30 days significantly detoxified the parent compound reducing its mutagenicity (61%) and genotoxicity (69%) ( Pithva et al., 2015 ). In another study, the role of Saccharomyces cerevisiae on the activation of apoptotic pathway Akt/NF-kB was explored in cancer. Heat-killed S. cerevisiae induced apoptosis in cancer cells, the SW480 cell line, by up-regulating Bax, cleaved caspase 3 and cleaved caspase 9, and down-regulating p-Akt1, Bcl-XL, Rel A, procaspase 3 and procaspase 9 expressions. Hence, it was concluded that probiotics modulate Akt/NF-kB pathway following the apoptotic cascade and play an essential role in cancer prevention ( Shamekhi et al., 2020 ).

3.3. Hypocholesterolemic effect of probiotics

Probiotics can be used as an effective tool for lowering blood cholesterol levels. They can act directly or indirectly to decrease cholesterol levels in the body. The direct mechanism includes the inhibition of de novo synthesis of cholesterol by hypocholesterolemia factors like uric acid, lactose, orotic acid, and whey protein as well as the reduction in intestinal absorption of dietary cholesterol in three ways- assimilation, binding, and degradation ( Thakkar et al., 2016 ). The indirect mechanism for curtailing cholesterol by probiotics is deconjugating bile salts (conjugated glycodeoxycholic acid and taurodeoxycholic acid) via bile salt hydrolase (BSH) production. Deconjugated bile salts are less reabsorbed through the intestine, thus inhibiting enterohepatic circulation of the bile and higher excretion in the feces ( Figure 5 ; Rezaei et al., 2017 ).

Figure 5 . Mechanism of lowering cholesterol level by probiotics. Probiotics breakdown or deconjugate bile salts into free choline, glycine and amino group by synthesizing bile salt hydrolase. Free choline excreted via choline, amino acid group is absorbed in the intestine, and free taurine and glycine return back to the liver. This increases the elimination of bile from body and more cholesterol is used to synthesize bile thereby, reducing the cholesterol level in the blood.

Human and animal studies have provided evidence for the hypocholesterolemic properties of probiotics. In a study, the hypocholesterolemic properties of Levilactobacillus brevis MT950194 and L. brevis MW365351 were observed both in vitro and in vivo. The strains reduced cholesterol content, increased fecal cholesterol excretion, and converted bile into free cholic acid ( Munir et al., 2022 ). The potential of a probiotic complex comprising Pediococcus , Lactobacillus , and Bifidobacteria was also investigated in lipid metabolism. After 10 weeks of the experimental period, the results showed significantly reduced cholesterol levels in medium and high-dose groups ( Galli et al., 2020 ). The cholesterol reduction potential of a new strain, L. plantarum DMDL 9010, was investigated by using in vivo model. The intake of strain resulted in the reduction of serum cholesterol, hepatic cholesterol, triglycerides, and an increase in fecal excretion of bile acids. A significant decrease in total cholesterol, low-density lipoprotein, and atherosclerosis index by 23.03, 28.00, and 34.03%, respectively was observed with the use of L. plantarum DMDL 9010 (10 9 cells per day) ( Liu et al., 2017 ).

Recently, research regarding gene expression by probiotics in hypercholesterolemia was conducted by Dehkohneh and his colleagues. The role of Lacticaseibacillus paracasei TD3 was examined in modulating two significant genes involved in cholesterol metabolism; 3-hydroxy-3-methyl glutaryl coenzyme (HMGCR) and cytochrome P450 7A1 (CYP7A1). A dose of 1 × 10 10  CFU was given to male Wistar rats for 21 days. The cholesterol level was significantly decreased along with the reduction of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. The dramatic decline of HMGCR and CYP7A1 genes in adipose tissues was also observed using real-time polymerase chain reaction ( Dehkohneh et al., 2019 ).

3.4. Impact of probiotics on intestinal diseases

The gut plays a pivotal role in the digestion and absorption of nutrients and maintains mucosal barrier integrity. Numerous commensal bacteria reside in the human GI tract constituting an active community, which strongly affects human physiology ( Shehata et al., 2022 ). The modification in intestinal microflora can be achieved by administering antibiotics, probiotics, prebiotics, and fecal transplant ( Shahverdi, 2016 ).

The metabolic activity of the intestinal microbiome affects the host’s health, both favorably and unfavorably ( Saber et al., 2017 ). The exact balance in the microflora (eubiosis), when disturbed, results in acute and chronic clinical disorders like antibiotic-associated diarrhea (AAD), ulcers, inflammatory bowel disease (IBD), and irritable bowel syndrome (IBS) ( Saber et al., 2017 ). In addition, several researchers have supported the theory that microbial dysbiosis participates in the etiology of some human cancers ( Su et al., 2021 ), especially GI cancers ( Pereira-Marques et al., 2019 ). Restoring healthy gut microbiota can be used as a practical approach to managing intestinal diseases. Probiotics can increase microbial richness and diversity, increase enzyme (Lactase) production, improve immune micro-environment ( Jang et al., 2019 ), and improves intestinal permeability ( Stratiki et al., 2007 ). In this way, probiotics can alleviate intestinal diseases. Studies regarding the use of probiotics in intestinal diseases are given in Table 1 .

4. Application of probiotics in the food industry

The public awareness of diet-related issues and ever-increasing evidence about probiotic health benefits have increased consumer interest in probiotic foods. A large number of food items, including yogurt, powdered milk, frozen fermented dairy desserts, cheese and cheese products, ice creams, baby foods, cereals, and fruit juices, are among numerous probiotic foods ( Papademas and Kotsaki, 2019 ). The most prominent barrier to using probiotics in the food industry is their sensitivity toward heat treatments during processing and GI stresses in the human body. However, researchers and food industries are trying to find new and innovative methods and techniques to overcome the issues ( Zhang et al., 2022 ). The global increase in sales of probiotics-based products is estimated to reach 75 billion dollars by 2025. This exponential growth in sales of probiotic products has already gained much interest from food producers to develop new products with probiotics. Probiotics are commonly used in dairy, beverage, baking, and edible film industries ( Reque and Brandelli, 2021 ).

4.1. Probiotics in the dairy industry

Food producers have been showing great interest in developing new probiotics products due to their large acceptability among consumers. Dairy-based products are prepared as natural products to promote health and prevent diseases ( Nami et al., 2019 ). Lactic acid bacteria (LAB) in dairy products help increase the shelf life of fermented products. LAB act as antimicrobial agents against many pathogens living inside the human body, thus improving human health ( de Souza da Motta et al., 2022 ). Table 2 refers to the application of probiotics in the dairy industry. Considering the demand for functional dairy products in markets, it has been estimated and forecasted that the industry will jump up to a market value of 64.3 billion USD globally by the end of 2023, apart from traditional dairy products ( Iqbal et al., 2017 ; FAO, 2022 ).

Table 2 . Application of probiotics in food industries.

Many products, such as pasteurized milk, infant formula, fermented milk, and ice creams are being produced and consumed worldwide as probiotic-based dairy products. Some products like cheese and fermented milk are preferred as probiotics carriers because their pH buffering capacity and fat contents give additional protection to probiotics while passing through the GI tract ( Meybodi and Mortazavian, 2017 ). Yogurt, including reduced lactose or lactose-free, functional ingredient-supplemented yogurts such as vitamins, minerals, sterols, stanols, conjugated linoleic acids, prebiotics, and probiotics have also gained good market success for quite a long period ( Fernandez and Marette, 2017 ).

Nowadays, probiotics-based dairy products have been recommended as safe and healthy due to their beneficial effects on health, such as aiding mineral absorptions in the body, being efficient against Helicobacter pylori infection, and preventing diarrhea and constipation ( Gao et al., 2021 ). Nami and his team ( Nami et al., 2019 ) found the hypocholesterolemic effects of L. plantarum from homemade yogurt. They found the most substantial cholesterol-removing potential in growing cells (84%), moderate removal of cholesterol in the resting cell (41.1%), and the lowest in dead cells (32.7%). L. plantarum showed a positive potential for controlling serum cholesterol. At the same time, it was found that L. plantarum was resistant to BSH activity, antibiotics, and hemolytic activity ( Nami et al., 2019 ). Lee et al. (2020) prepared L. plantarum B710 containing fermented milk, which showed bone-protective effects. Moreover, Prezzi et al. (2020) examined that the addition of L. rhamnosus inhibited the growth of Listeria monocytogenes in Minas Frescal cheese . L. rhamnosus showed no negative effect on the textural and physiochemical properties of cheese and survived during storage and after simulated gastrointestinal conditions.

Arbex et al. (2018) investigated six Leuconostoc mesenteroides strains from three different sources of dairy and non-dairy products provided each sample showing probiotic properties. One strain of L. mesenteroids from camel milk coded as CM9 showed high dextran production and the best resistance to intestinal stresses. CM9 had a strong antimicrobial potential against Staphylococcus aureus and Escherichia coli ( Arbex et al., 2018 ; Azam et al., 2021 ). In another research, the effect of Lactobacillus acidophilus and L. rhamnosus were investigated on soft cheese. It was found that L. acidophilus had good overall quality with a better immune-modulation response in mice. At the same time, they also controlled pro-inflammatory cytokines and interleukin regulation and enhanced the secretion of secretory immunoglobulin A ( Cuffia et al., 2019 ). In a study, Nguyen et al. (2019) and Riaz et al. (2019) investigated the survival of Bifidobacterium bifidum encapsulated in zein. The results suggested that probiotic bacteria survived well after 32 days of storage ( Nguyen et al., 2019 ).

4.2. Probiotics in the beverage industry

The demand for non-dairy probiotic foods has been increasing steadily, especially when the consumer has become aware of the side effects associated with medicine. Consuming probiotic food is more readily acceptable to consumers as it is a more natural way of receiving their daily dose of probiotics ( Reque and Brandelli, 2021 ). Fruit juices supplemented with probiotics have been reported as a more unique and appropriate method in the probiotic beverage industry. Fruit juices have been accepted widely among all consumers regardless of age, gender, and geographic region around the globe due to the presence of essential nutrients ( Mantzourani et al., 2018a , b ). The viability of probiotics is shorter in non-dairy foods when compared to dietary supplements due to the harsh environments faced by probiotics in beverages. Processors must consider many factors in the production of probiotic juices, such as pH, temperature, anthocyanins, and most importantly a vegetative form of probiotics ( Min et al., 2019 ; Azam et al., 2022 ).

To overcome these complexities, microencapsulation techniques have been introduced. Using these techniques, probiotics can be employed as an essential ingredient in the functional food industry. The micro or nanoencapsulation of probiotics allows them to withstand harsh processing and storage environments due to the protective coating around them ( Afzaal et al., 2022 ). It was reported that the acid sensitivity of Bifidobacterium and Lactobacillus was improved after their microencapsulation with gelatin or plant gums ( Ozturk et al., 2021 ). Besides this, low-temperature processing is also an effective strategy to control metabolic activity and protect probiotic cell viability throughout the shelf life of juices so that an adequate and safe dose of microbes is delivered to the consumer ( Tyutkov et al., 2022 ). Some studies regarding probiotics in the beverage industry are shown in Table 2 .

Miranda et al. (2019) have investigated the direct addition of an activated and microencapsulated form of probiotics in orange juice to check their effect on physical, chemical, rheological, microbial, and sensory parameters. They found that in the inactivated state, the level of organic acids was increased, but the essential volatile compounds were decreased. On the other hand, the encapsulated probiotics showed improved consistency and rheological parameters but their sensory attributes were not up to the mark due to changes in taste. The most optimum treatment was found to be the direct addition of probiotics to juice based on good physicochemical and sensory acceptance that was more similar to the natural pure product having many essential volatile compounds (octanol, o-cymene, α-cubebene, and 1-hexanol, etc.) ( Miranda et al., 2019 ). Secondary packaging is another important technique used to produce shelf-stable beverage products. In this technique, the probiotics are in a separate compartment from food, i.e., bottle cap or straw, and are released only into juices immediately before consumption ( Fenster et al., 2019 ).

In another research, water kefir grains were used to ferment soy whey (a byproduct of tofu) to prepare a functional beverage. After 2 days of fermentation, the polyphenol contents and antioxidant properties increased significantly, supported by good sensory scores and overall acceptability ( Fenster et al., 2019 ). Laali et al. (2018) used L. plantarum to make a beverage from coconut water after fermentation. This process not only enhanced the vitamin and mineral (potassium, calcium, and sodium) contents but also improved anti-hypertensive, antioxidant, and antimicrobial properties making it suitable for use ( Laali et al., 2018 ). The beverage prepared from whey, germinated millet flour, and barley extract was treated with L. acidophilus in another study, and it was found to be effective in controlling the pathogenicity induced by Shigella in mice models. The beverage stimulated the immune response and enhanced the IgA level, thus controlling pathogenicity ( Ganguly et al., 2019 ).

4.3. Probiotics in bakery

Bakery products (bread, biscuits, doughnuts, cookies, etc.) contribute to several major food components such as carbohydrates, proteins, fats, dietary fiber, vitamins, and minerals in varying amounts ( Niesche and Haase, 2012 ; El-Sohaimy et al., 2019 ). Researchers have been trying to incorporate probiotics in baked products by developing new techniques to deliver thermo-durable bioactive materials so that probiotics can survive high temperatures during baking ( Mirzamani et al., 2021 ).

The microencapsulation technique and the sourdough method have been studied as an alternative to increasing the nutritional value and cell viability of probiotics in bread during baking ( Ganguly et al., 2019 ) and in GI conditions ( Champagne et al., 2018 ; Ashraf et al., 2022 ). In a study, L. rhamnosus was encapsulated in sodium alginate, and higher cell viability was observed during the baking of pan bread and in simulated gastrointestinal conditions ( Hauser and Matthes, 2017 ). Zhang et al. (2018) analyzed the encapsulation of L. plantarum into bread-making using different matrices (reconstituted skim milk, gum arabic, maltodextrin, and inulin). The results suggested that bacterial survival was better in gum arabic and reconstituted skim milk than in the other two heating methods ( Zhang et al., 2018 ). Another research studied the incorporation of L. plantarum under different baking temperatures (175, 205, and 235°C) and its survival during storage. The bacterial cell viability was counted every 2 min during baking and a decline from 10 9  CFU/g to 10 4–5 CFU/g was observed after baking. The storage results were remarkable as the probiotic viability was increased by 2–3 logarithmic cycles to 10 8 , which was attributed to the decline in the pH of bread during storage ( Zhang et al., 2018 ). Table 2 illustrates the use of probiotics using different strains in the baking industry.

4.4. Probiotics in edible food coatings

Bioactive food packaging is the latest approach promoting the concept of functional foods due to its extraordinary health-promoting benefits. This technique is quite helpful in overcoming the stability and GIT stresses faced by probiotics ( Khodaei and Hamidi-Esfahani, 2019 ). Studies on the use of probiotics with some biopolymers for edible coating are illustrated in Table 3 .

Table 3 . Use of probiotics in edible film.

The encapsulation of probiotics into edible films protects them from premature degradation and increases their viability in the human body ( Singh et al., 2019 ). The technique of edible films is being used nowadays as a tool for the effective delivery of probiotics to consumers. Still, at the same time, it also enhances the stability and safety of food by inhibiting the growth of spoilage microorganisms ( Pavli et al., 2018 ). The prime difference between active packaging and edible coating or bioactive packaging is that active packaging is usually done to enhance the safety and quality of packaged food, while on the other hand, bioactive packaging affects the health of consumers directly generating healthier packaged foods through edible coated bioactive material which upon consumption promote health ( Gagliarini et al., 2019 ).

Many researchers have shown keen interest in film-forming materials, for instance, biopolymers including cellulose, zein, seaweed extracts, pectins, alginates, and chitosan for entrapping probiotics to enhance the nutritional values of foods ( Pop et al., 2019 ). Therefore, bacterial microorganisms are being incorporated into films and coatings to confer probiotics’ ability to the food products or act as antimicrobial agents ( Afsah-Hejri et al., 2013 ). As an example, the fabricated cellulose-based edible films in combination with L. rhamnosus using sodium carboxymethyl cellulose (CMC) and hydroxymethyl cellulose (HEC) with citric acid as a crosslinker to control the consistency of film loaded with L. rhamnosus ( Singh et al., 2019 ). Moreover, cellulose-based edible films showed the therapeutic effects of probiotics ( Singh et al., 2019 ). The film effect provides a suitable environment to encapsulate bacteria from transport to delivery in the GIT system effectively.

Four probiotic strains ( L. acidophilus , L. casei , L. rhamnosus , and B. bifidum ) were investigated using CMC-based edible coatings in this regard and their effects on storage under refrigerated conditions were also checked. The results suggested that L. acidophilus showed the highest viable count during storage with more water vapor permeability and opacity and decreased tensile strength and elongation at break values of film structure. The physical and mechanical properties of edible films remained the same ( Ebrahimi et al., 2018 ). Another research found that after incorporating L. plantarum into CMC-based edible coating, the physicochemical properties and microbial characteristics of fresh strawberries were significantly improved. The probiotics population remained constant throughout the storage period, which controlled mold and yeast growth and helped to improve the shelf life of strawberries ( Khodaei and Hamidi-Esfahani, 2019 ).

Bambace et al. (2019) incorporated L. rhamnosus into an alginate prebiotic fiber solution to enhance the shelf life of minimally processed and ready-to-eat blueberries by fourteen days. L. rhamnosus showed good antimicrobial properties with alginate and sensory acceptability for coated food ( Bambace et al., 2019 ). In another work, kefiran polysaccharides-based films were used to deliver probiotics ( L. paracasei and Kluyveromyces marxianus ) to the gut. These films exhibited good antimicrobial properties and protected the probiotics from GIT stresses. L. paracasei showed better mechanical properties and good viable count than K. marxianus ( Gagliarini et al., 2019 ).

5. Delivery systems and the strategies to extend viability

The association between probiotics and human health has been well-known for an extended period. When consumed orally, probiotics can regulate the composition of intestinal microbiota ( Sharma et al., 2023 ). However, the severe physicochemical stresses (high temperatures and acidity during processing, storage, and passage to the large intestine) can drastically reduce the viability of probiotics. Researchers have used different encapsulating techniques to overcome these stresses and enhance the viability of probiotics within the human body ( Luo et al., 2022 ). The traditional and most widely used technique is microencapsulation. Microencapsulation is classified into four methods, namely; spray drying, freeze drying, emulsification, and extrusion. One can improve the ability of probiotics to withstand the harsh environment of processing and the human body. Still, these methods have certain limitations, such as extreme temperatures and acidity can ultimately affect the size, stability, and ultimately viability of microstructures of microcapsules ( Razavi et al., 2021 ).

These hindrances paved the way to find new encapsulation strategies to enhance the durability and viability of probiotics. In recent years, the nanoencapsulation technique has been used widely to enhance probiotics-loaded nanoparticles’ ability to face severe processing and in-vivo stresses. These techniques also facilitate the targeted delivery and control release of probiotics in the intestine ( Xu et al., 2022 ). The unique biological and physicochemical characteristics of nanocapsules, such as smaller particle sizes, higher surface areas, and increased reactivities, improve the efficiency of encapsulated probiotics, thus, providing a logical solution to human health and safety ( Singh et al., 2022 ). The ability of nanoencapsulation to entrap probiotics is analyzed by the potential of electrospun nanofibers, hydrogels, nanocoating, nanoliposomes, and other nanomaterials ( Garcia-Brand et al., 2022 ).

Mojaveri and his colleagues, in their recent work, attempted to improve the viability of Bifidobacterium animalis Bb12 by using a nanofiber technique made from chitosan and poly (vinyl alcohol) and inulin as prebiotics. The simulated results of the GI tract showed that the encapsulation of probiotics in electrospun nanofibers significantly enhanced the physicochemical behavior with increased stability of nanoparticles within the human body ( Mojaveri et al., 2020 ). In another study, Li et al. (2019) studied the cellulose-based gels for control release of encapsulated L. plantarum with better storage and concluded that cellulose-based gels provide better storage stability and much-enhanced control release pattern in simulated intestinal fluids ( Li et al., 2019 ).

Encapsulation of probiotics with the help of biomaterial-based nanocoating can also protect these beneficial microbes from antibiotics and GI conditions, facilitating the retention of probiotics within the GI tract. It was found that metal-phenolic network-based nano-coating made from iron (III) and tannic acid can help protect probiotic microbes from the detrimental effect of antibiotics ( Ashraf et al., 2023 ; Guo and Wu, 2023 ). Due to their physicochemical parameters, smaller structures, and thermodynamic properties, nanoliposomes enjoy vast applications for a wide range of products. The stability of L. rhamnosus was analyzed by loading them into chitosan-gelatin coated nanoliposomes. The characterization study suggested the successful coating of bifidobacteria with coated nanoliposomes. Further supported by the results of simulated GI fluids with a significant amount of viable cells present in the fluid guiding toward the suitability of nanoliposomes as a potential carrier of probiotics in developing nutraceutical foods ( Hosseini et al., 2022 ).

6. Conclusion

Probiotics have well-documented physiological effects with a definitive mechanism. However, the exact mechanism of how they work to enhance health and prevent different diseases must be explored. Evidence from well-documented clinical trials has revealed that probiotics can potentially alleviate different GI and other disorders. Despite our understanding of some molecular mechanisms underlying beneficial aspects of probiotics, we are still far from clinically proven efficacy in many autoimmune and inflammatory diseases. Moreover, many studies have been done on the animal model, so there is an emergent need to translate these results into humans. Currently, genetically modified commensal lactic acid bacteria are being used to deliver special health-interest compounds. But most of the work regarding recombinant bacteria is related to vaccines. However, genetically modified bacteria can be used for exploring innovative strategies to deliver bioactive molecules to mucosal tissues. More consistent and reproducible clinical trials are required to reveal probiotics efficacy, limitations, and safety, determining their effects on the immune system. Considering all the methodologies discussed in this review, probiotics can be applied easily by food producers to make novel functional foods to promote human health.

Author contributions

All authors wrote the manuscript, read and agreed to the published version of the manuscript.

This work was based upon the work from COST Action 18101 SOURDOMICS—Sourdough biotechnology network toward novel, healthier, and sustainable food and bioprocesses ( https://sourdomics.com/; https://www.cost.eu/actions/CA18101/ , accessed on 12 April 2023), where TE was member of the working groups 4, 6, 7, and 8, FÖ was the leader of the working group 8, “Food safety, health-promoting, sensorial perception and consumers’ behavior” and JR was the Chair and Grant Holder Scientific Representative and is supported by COST (European Cooperation in Science and Technology) ( https://www.cost.eu/ , accessed on 12 April 2023). COST was a funding agency for research and innovation networks. JR also acknowledged the Universidade Católica Portuguesa, CBQF—Centro de Biotecnologia e Química Fina—Laboratório Associado, Escola Superior de Biotecnologia, Porto, Portugal, as well as the support made by LA/P/0045/2020 (Alice) and UIDB/00511/2020-UIDP/00511/2020 (LEPABE) funded by national funds through FCT/MCTES (PIDDAC).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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Keywords: probiotics, lactic acid bacteria, immunomodulation, anti-allergic and gastrointestinal diseases, functional foods

Citation: Latif A, Shehzad A, Niazi S, Zahid A, Ashraf W, Iqbal MW, Rehman A, Riaz T, Aadil RM, Khan IM, Özogul F, Rocha JM, Esatbeyoglu T and Korma SA (2023) Probiotics: mechanism of action, health benefits and their application in food industries. Front. Microbiol . 14:1216674. doi: 10.3389/fmicb.2023.1216674

Received: 04 May 2023; Accepted: 04 August 2023; Published: 17 August 2023.

Reviewed by:

Copyright © 2023 Latif, Shehzad, Niazi, Zahid, Ashraf, Iqbal, Rehman, Riaz, Aadil, Khan, Özogul, Rocha, Esatbeyoglu and Korma. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: João Miguel Rocha, [email protected] ; Tuba Esatbeyoglu, [email protected] ; Imran Mahmood Khan, [email protected]

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• Published: 25 August 2024

Impact of probiotics on gut microbiome of extremely preterm or extremely low birthweight infants

• Lauren C. Beck 1 ,
• Janet E. Berrington   ORCID: orcid.org/0000-0002-6185-2843 1 , 2 &
• Christopher J. Stewart 1  

Pediatric Research ( 2024 ) Cite this article

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Meta-analysis of probiotic administration to very preterm or very low birthweight (VP/VLBW) infants shows reduced risk of necrotising enterocolitis (NEC).

Separately reported outcomes for extremely preterm infants (<28 weeks) or extremely low birth weight infants (<1000 g) (EP/ELBW) are lacking meaning some clinicians do not administer probiotics to EP/ELBW infants despite their high risk of NEC.

We present data showing the gut microbiome is impacted in EP/ELBW infants in a similar manner to VP/VLBW infants, suggesting that risk reduction for necrotising enterocolitis that is microbiome driven will also be seen in EP/ELBW infants, making probiotic administration beneficial.

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The use of probiotics in preterm infants has been extensively studied with at least 60 randomised controlled trials (RCTs) and 30 non-randomised studies, overall showing clinical benefit in necrotising enterocolitis (NEC) reduction by up to 50% 1 , 2 Methodological issues and feeding regimes may explain variations seen with clinical use 3 and concerns remain around practical aspects of production and use. In response to these various organisations have produced guidance/recommendations for their use including the European Society for Paediatric Gastroenterology Hepatology and Nutrition (ESPGHAN), 4 the World Health Organisation (WHO), 5 the American Academy of Paediatrics 6 and the Canadian Paediatric Society. 7 Recent intervention by the US Food and Drug Administration (FDA) 8 after a preterm infant with birthweight <1000 g died in association with proven probiotic sepsis, and associated responses by ESPGHAN 9 and UK physicians 10 have once again made this a controversial area. This issue is particularly significant for the most preterm or low birthweight infants ( < 28 weeks or <1000 g), where risks of both NEC and bacterial translocation from the gut are higher compared to infants between 28 and 31 weeks, or weighing more than 1000 g.

Despite probiotics being extensively studied, much data is presented for the whole cohort <32 weeks or <1500 g, and less specifically for EP/ELBW. Although any study with inclusion criteria <32 weeks will also include a proportion of infants also <28 / < 1000 g (for instance, in SIFT (the Speed of Increasing milk Feeds Trial) this was 39% 11 ) the exact proportion of EP/ELBW infants contributing to the overall meta-analysis of probiotic outcomes remains unknown. The most recent Cochrane analysis (2023) identified ten trials where some outcome measures were explicitly presented separately for infants <28 weeks gestation or <1000 g showing little or no impact on NEC (Risk Ratio (RR) 0.92, 95% Confidence Interval (CI) 0.69 to 1.22, 10 trials, 1836 infants; low certainty) in contrast to infants <32 weeks or <1500 g with NEC RR 0.54 (95% CI 0.46 to 0.65; 57 trials, 10,918 infants; low certainty). 1

Multi-omic research may help clarify whether biological markers of probiotic efficacy are seen in the most preterm infants. We recently showed the significant and strain-dependant impact of probiotics on the gut microbiome of healthy preterm infants (all <32 weeks gestation), demonstrating that probiotic receipt was the most important driver of all co-variates. 12 Here, we analyse samples from these infants, divided into <28 weeks and/or <1000 g (EP/ELBW) and compare them to those from infants born 29–31 weeks gestation and ≥1000 g (referred to as VP/VLBW) to help address whether probiotics differentially impact preterm infants depending on gestational age and birthweight.

For detail see Beck et al. 12 Of the 123 < 32 weeks/<1500 g preterm infants included in the original study we identified 91 born at <28 weeks gestation and/or <1000 g leaving 32 of ≥28 weeks or ≥1000 g. Briefly the original samples and data were collected as part of a Research Ethics Committee approved study and all infants cared for in the Royal Victoria Infirmary, Newcastle, with standardised feeding, antibiotic and antifungal guidelines (prophylactic fluconazole). Between 2013 and 2016, infants received the probiotic Infloran ( B. bifidum NCDO 2203 1 × 109 c.f.u. and L. acidophilus NCDO 1784 1 × 109 c.f.u.); then after mid-2016 Labinic ( B. bifidum Bb-06 0.67 × 109 c.f.u., B. longum subsp. infantis Bi-26 0.67 × 109 c.f.u. and L. acidophilus NCFM 0.67 × 109 c.f.u.) was used. Stool samples were collected longitudinally (day 0 to 120), alongside extensive demographic and treatment/feed exposures. Variables fixed through time are described on a per-infant basis; other variables were categorised to reflect exposure in relation to time and therefore on a per-sample basis. DNA was extracted from ~0.1 g of stool using the DNeasy PowerSoil Kit (QIAGEN) and sequencing was performed on the HiSeq X Ten (Illumina) with a read length of 150-bp paired-end reads. Taxonomic profiling of metagenomic samples was performed using MetaPhlAn v.2.0. The five previously identified Preterm Gut Community Types (PGCTs) were used for this analysis. PERMANOVA was performed using the ‘adonis’ function and performed in cross-sectional timepoints, each with 1 sample per patient to account for repeated measures. Timepoints were selected for relevance to exposures and to give similar numbers of samples. A generalised linear mixed effects model was fit to assess whether low birthweight/gestational age (i.e. EP/ELBW vs. VP/VLBW) was associated with Shannon diversity, whilst controlling for the variables included in the beta diversity analysis and patient. Ordinations were performed using non-metric multidimensional scaling based on Bray-Curtis dissimilarity on samples collected during probiotic use. An Area Under the Curve (AUC) analysis based on the relative abundance of probiotic species during probiotic use was used to classify infants as responders or non-responders. Infants falling below 1 standard deviation from the mean, were classified as non-responders. Z scores were calculated from the same AUCs, normalised by the sampling time period for each infant. The thresholds were similar for labinic and infloran (0.1 relative abundance for Labinic infants, 0.12 for Infloran infants) and 0.1 overall and are presented combined for labinic or infloran at the combined threshold of 0.1 in Fig.  1d type.

a Significance and explained variance of clinical co-variates modelled by ‘adonis’ for EP/ELBW infants only. Bubbles show the amount of variance (%) explained by each co-variate at a given timepoint and significant results (FDR < 0.05) are surrounded by a red box. MOM = Mothers own milk, BMF = breast milk fortifier, Season = Spring, Summer, Autumn, Winter and antibiotics 7 days = whether the infant had received antibiotics within 7 days b NMDS plot of taxonomic profiles during the use of probiotics, showing the mean centroid for each group. c Number of samples per PGCT during probiotic use for each group. d Number of infants classified as responders and non-responders for each group and z scores, both based on an AUC analysis of probiotic species relative abundance during probiotic use.

Table  1 shows important demographics of the included 123 infants, by EP/ELBW and VP/VLBW cohorts, and relevant sample information. In total 1431 samples were analysed across 9 time points (days 0–9, 10–14, 15–19, 20–24, 25–29, 30–34, 35–39, 40–49 and 50–69) selected for relevance to exposures and to give similar numbers of samples. As in the original analysis of all <32 weeks or <1500 g 12 probiotic receipt remained the most important identified driver of the microbiome of exclusively EP/ELBW infants (Fig.  1a ).

No significant differences were seen in Shannon diversity ( P  = 0.175) or overall beta diversity between EP/ELBW and VP/VLBW groups at any time point during the use of probiotics (all P  > 0.05) (Fig.  1b ). Furthermore, a similar proportion of samples from each group were classified as probiotic-associated PGCTs 4 and 5 (28% vs. 27%) (Fig.  1c ). Using AUC analysis to define responders and non-responders based on the relative abundance of probiotic species, there was again no significant difference between groups (86% vs. 87% responders; P  = 1) (Fig.  1d ), reflected in the z score medians per group ( P  = 0.48) (Fig.  1d ).

Microbiome differences can act as indicators of whether probiotic administration results in changes that may be mechanistically important in disease prevention. 13 The PiPS trial 14 that administered Bifidobacterium breve strain BBG-001 did not identify clinical benefit and also found no differences in gut microbiome between probiotic vs. placebo. 15 Conversely the ProPrems trial 16 which reported a 54% reduction in NEC in infants receiving Bifidobacterium longum subsp. infantis BB-02, Streptococcus thermophilus TH-4 and Bifidobacterium animalis subsp. lactis BB-12 did identify gut microbiome differences between probiotic vs. placebo. 17 Having previously shown that in healthy preterm infants <32 weeks gestation probiotics are the dominant driver of the microbiome, 12 we confirm here that this remains the case when considering only EP/ELBW infants. Any specific differences in the impact on the gut microbiome between the two probiotic products used are not presented here. Mechanisms by which probiotics exert their effect are variable, and some are species and strain-specific. Common or widespread microbiome-mediated effects are through competitive exclusion of other organisms or through production of beneficial metabolites (e.g. short chain fatty acids) or vitamins. Our data suggest that where probiotics do exert their effect through the microbiome it is likely that an effect seen in a cohort of VP/VLBW infants will also be seen in EP/ELBW infants.

In conclusion, although trials reporting EP/ELBW infants separately are relatively limited, we show that impacts on the infant gut microbiome seen in EP/ELBW infants are similar to those seen in VP/VLBW infants, and this should be included in decision making about probiotic administration to these infants who are at the highest risk of NEC.

Data availability

Suitably anonymised data may be available on reasonable request. All metagenomic sequencing data generated and analysed in the present study have been deposited in the European Nucleotide Archive under study accession no. PRJEB49383 .

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Acknowledgements

We thank the staff involved in the sample collection, particularly J. Groombridge. We thank the families for their willingness to help and support research. We also thank D. Smith, K. Hoffman, M. Wong and J. Petrosino (Baylor College of Medicine) for support with bioinformatic processing of raw data.

This work was supported by the Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and The Royal Society (grant no. 221745/Z/20/Z), a Newcastle University Academic career Track Fellowship and the 2021 Lister Institute Prise Fellow Award. Astarte Medical provided funding for stool sample retrieval and shipment for metagenomic sequencing. Tiny Lives supported set-up of the initial study and the biobanking fees. The funders played no part in the study design, analysis, interpretation or reporting.

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Lauren C. Beck, Janet E. Berrington & Christopher J. Stewart

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L.C.B., J.E.B., C.J.S. orchestrated the study, J.E.B. undertook the original clinical study, L.C.B. and C.J.S. acted as laboratory scientific leads and conducted the analysis. L.C.B. and J.E.B. draughted the initial manuscript and all authors approved the final manuscript as submitted and agree to be accountable for the data presented in this manuscript.

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Dr Berrington report grants to their institutions from Prolacta Biosciences US (Duarte, CA), and grants from Danone Early Life Nutrition (Paris, France). Dr Stewart declares lecture honoraria from Nestlé Nutrition Institute (La Tour-de-Peilz, Switzerland).

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Beck, L.C., Berrington, J.E. & Stewart, C.J. Impact of probiotics on gut microbiome of extremely preterm or extremely low birthweight infants. Pediatr Res (2024). https://doi.org/10.1038/s41390-024-03520-w

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Ohio State study shows benefits of taking probiotics during pregnancy

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A mouse study led by The Ohio State University Wexner Medical Center and The Ohio State University College of Medicine recently revealed that when probiotics were given to pregnant mice, both the mother mouse and its offspring benefited.

The results suggest that, for pregnant humans and their children, certain probiotics can improve the metabolism of common amino acids in our diets when the probiotics are given during pregnancy, says Tamar Gur, MD, PhD, the senior study author.

“Probiotics may also help counteract the negative effects of prenatal stress ,” Dr. Gur says.

How probiotics work during fetal development

Probiotics, live microorganisms that are found in naturally fermented foods, can help support healthy digestive systems and immune systems. They’re considered safe to take in supplement form during pregnancy.

One particular probiotic, Bifidobacterium dentium, might change the way our bodies process certain amino acids like tryptophan, says first study author Jeffrey Galley, PhD.

Tryptophan is perhaps most well-known for being in turkey and making you feel tired. Turkey doesn’t actually have large amounts of tryptophan compared to other foods, and your Thanksgiving-dinner sluggishness may really be due to eating a large meal. But tryptophan does affect newborn sleep rhythm development, as well as the development of their brains and food intake regulation. It’s one of nine essential proteins the body needs to stay healthy, and it’s a major building block for newborn development.

During pregnancy, tryptophan can help control inflammation, too.

What Ohio State has learned about probiotics and pregnancy

Previous research from the Gur Laboratory at Ohio State found that microbes that help metabolize tryptophan decrease with stress . They’ve also examined how stress in pregnancy can lead to abnormal brain development and behavioral changes in offspring.

“We have strong evidence this specific probiotic [Bifidobacterium dentium] helped reduce stress-related problems in both mothers and their offspring, including helping the babies gain weight and improving their social behavior,” Dr. Gur says.

What’s next in research

The team’s next goal, Dr. Gur says, is to understand the mechanisms behind the changes they observed in mice, and look at ways to prevent those changes or treat them.

“Since prenatal stress is common in many pregnancies, we want to develop methods to reduce its negative effects.”

The study is published online in the journal Brain, Behavior, and Immunity .

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• Volume 14, Issue 8
• Effect of probiotic administration to breastfeeding mothers with very low birthweight neonates on some neonatal and maternal outcomes: study protocol for a randomised, double-blind, placebo-controlled trial
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• Maryam Alikamali 1 ,
• http://orcid.org/0000-0003-4785-9333 Sakineh Mohammad-Alizadeh-Charandabi 2 ,
• Mojgan Mirghafourvand 3 ,
• Manizheh Mostafa Gharehbaghi 4 ,
• Aziz Homayouni-Rad 5 ,
• Zahra Fardiazar 6 ,
• http://orcid.org/0000-0001-9676-8492 Mahnaz Shahnazi 7
• 1 Student Research Committee, Faculty of Nursing and Midwifery , Tabriz University of Medical Sciences , Tabriz , Iran (the Islamic Republic of)
• 2 Faculty of Nursing and Midwifery, Social Determinants of Health Research Center , Tabriz University of Medical Sciences , Tabriz , Iran (the Islamic Republic of)
• 3 Faculty of Nursing and Midwifery, Social Determinants of Health Research Center, Department of Midwifery , Tabriz University of Medical Sciences , Tabriz , Iran (the Islamic Republic of)
• 4 Pediatric Health Research Center , Tabriz University of Medical Sciences , Tabriz , East Azerbaijan , Iran (the Islamic Republic of)
• 5 Faculty of Nutrition and Food Science, Department of Food Science and Technology , Tabriz University of Medical Sciences , Tabriz , Iran (the Islamic Republic of)
• 6 Women’s Reproductive Health Research Center , Tabriz University of Medical Sciences , Tabriz , East Azerbaijan , Iran (the Islamic Republic of)
• 7 Faculty of Nursing and Midwifery, Department of Midwifery , Tabriz University of Medical Sciences , Tabriz , Iran (the Islamic Republic of)
• Correspondence to Dr Mahnaz Shahnazi; shahnazimahnaz{at}gmail.com

Introduction Premature birth and very low birth weight (VLBW) are leading causes of neonatal mortality. Almost all premature infants experience hyperbilirubinaemia. Administering probiotics to breastfeeding mothers may positively affect infant outcomes. This trial aims to investigate whether probiotic supplementation for mothers with VLBW infants affects total serum bilirubin levels and postpartum depression scores (primary outcomes), as well as some other neonatal and maternal outcomes (secondary outcomes).

Methods and analysis This is a randomised, double-blind, placebo-controlled superiority trial with two parallel arms. Participants, caregivers and outcome assessors will be blinded. A total of 122 breastfeeding mothers of neonates with a birth weight of 1000–1500 g, along with their infants within 48 hours of birth, will be assigned to either the probiotic or placebo group in a 1:1 ratio through block randomisation, stratified by singleton and twin births. The intervention will involve oral administration of probiotics containing Lactobacillus paracasei 431 and Bifidobacterium lactis BB-12, or an indistinguishable placebo, for 42–45 days. Outcomes will be assessed through daily observations, laboratory assessments and the Edinburgh Postpartum Depression Scale. Adverse events will also be documented. Modified intention-to-treat analyses will be employed for the primary and secondary outcomes, excluding participants lost to follow-up from all postintervention assessments.

Ethics and dissemination This study protocol has been approved by the Medical University of Tabriz Ethics Committee (IR.TBZMED.REC.1401.735). Findings will be disseminated through publication in a peer-reviewed journal and presentations at relevant conferences.

Trial registration number IRCT20100414003706N42.

• Clinical Trial
• Neonatal intensive & critical care
• Depression & mood disorders

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See:  http://creativecommons.org/licenses/by-nc/4.0/ .

https://doi.org/10.1136/bmjopen-2023-079526

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STRENGTHS AND LIMITATIONS OF THIS STUDY

A low risk of selection bias due to the appropriate random assignment of participants to study groups and allocation concealment.

A low risk of performance and detection biases due to the implementation of blinding for participants, care providers and outcome assessors.

Conducting the trial in a tertiary hospital that covers very low birthweight infants from diverse geographical areas enhances the generalisability of the study results.

A lack of long-term assessment of intervention effects, primarily due to financial, time and logistical constraints.

The omission of biological collections from the protocol due to financial constraints limits the understanding of the probiotics’ effects on the maternal and neonatal microbiome.

Introduction

Premature birth and very low birth weight (VLBW, defined as weighing less than 1500 g) are the leading causes of infant mortality. 1 About 60% of infant deaths are attributed to VLBW. In Iran, infants weighing between 1000 and 1500 g have shown survival rates of 45% on discharge and 58% at 28 days after birth. 2 Over the past three decades, advancements in perinatal and neonatal intensive care, such as the utilisation of antenatal steroids, surfactants and innovative mechanical ventilation therapies, have markedly enhanced the survival rates of VLBW infants. 3–5 Despite these improvements, complications like sepsis, necrotising enterocolitis, bronchopulmonary dysplasia, intraventricular haemorrhage and retinopathy of prematurity are still common among these vulnerable infants. 6 7

Jaundice affects nearly 80% of premature neonates within the first week of life. 8 Despite the progress made in the treatment of hyperbilirubinaemia, it remains a major cause of neonatal morbidity and mortality. 9 The current approach to managing hyperbilirubinaemia in premature neonates focuses on determining the age-specific bilirubin threshold necessitating the initiation of phototherapy. However, concerns exist regarding the potential adverse effects of invasive phototherapy in premature neonates. 10 In addition to causing stress for parents, phototherapy impedes breastfeeding and contributes to other problems, such as fluctuating neonate body temperature and dehydration. Therefore, complementary approaches may offer valuable support in this context. 11

Pregnancy and childbirth are transitional periods in women’s lives. However, they can lead to permanent and long-term depression, with an estimated 9–16% of women experiencing postpartum depression (PPD). 12 The prevalence of PPD in Iran is reported to be approximately 25% (95% CI 23% to 28%). 13 The results of a recent systematic review have shown that preterm birth increases the risk of PPD in women by about 29%. 14 However, most women with PPD lack access to psychotherapeutic interventions or are unwilling to take antidepressants. Thus, there is a pressing need for safe and effective preventive and therapeutic interventions to address this critical issue. 15

Probiotics are live microorganisms that resemble those found in the human gut and are known to confer health benefits when administered in appropriate doses. Studies examining the safety of probiotics have not revealed any significant adverse effects on breastfeeding mothers and their infants. 16 17

When administered to breastfeeding mothers of VLBW infants, probiotics may affect bacterial colonisation of the infants. 18 According to the entero-mammary pathway theory, beneficial intestinal bacteria are transferred to the mammary glands via dendritic cells during late pregnancy and breastfeeding. 19 20 Moreover, probiotics can affect the concentration of bilirubin in the liver-intestinal circulation by inhibiting the degradation of conjugated bilirubin, as well as by enhancing intestinal peristalsis and defecation, thereby increasing bilirubin excretion. 16 21 In addition, probiotics may influence mothers’ mental health by reducing stress and depression. 22

The Cochrane systematic review, 18 published in 2018, found only one trial on 49 mothers of 58 infants concerning the effect of postnatal probiotic administration to breastfeeding women on the morbidity of VLBW neonates, in which the effect on hyperbilirubinaemia was not studied. 23 The review authors emphasised the need for further trials in the field. Although our recent trial in this field 24 showed some promising results, its sample size was limited (25 in each group) and the administered probiotics contained only one strain ( Lacticaseibacillus paracasei subsp paracasei , 1.5×10 9 colony-forming units (CFU)/day), and it did not assess maternal mental health outcomes.

A recent systematic review (2022) identified only two studies examining the effect of probiotic administration from pregnancy through the postpartum period on PPD. The meta-analysis from these studies revealed no statistically significant difference in women’s depression between the probiotic and control groups. This indicates a lack of evidence supporting the effect of probiotics on PPD, emphasising the need for further investigation into their effect on maternal mental health. 25

To the best of our knowledge, the present study represents the first trial with a relatively large sample size, aiming to investigate the effects of orally administering a combination of two probiotic strains to breastfeeding mothers of VLBW neonates on neonatal outcomes. Additionally, it is one of the few trials assessing the intervention’s effect on PPD without considering the administering period, and it is the first trial assessing the effect of postpartum administration on PPD.

Methods and analysis

Study objectives.

This trial aims to assess the effects of orally administering probiotics containing L. paracasei 431 and Bifidobacterium lactis BB-12 at a dose of 1×10 9 CFU/day each to breastfeeding mothers of neonates with VLBW on total serum bilirubin (TSB) levels and PPD scores, which are the primary outcomes. Additionally, we will assess the effects of the supplementation on rate of phototherapy, any serious complications, and feeding intolerance on the 7th day of intervention, as well as weight gain, length of and duration of total parenteral nutrition (TPN) as infant secondary outcomes. Also, mastitis and postpartum anxiety were assessed as maternal secondary outcomes.

Supplementing breastfeeding mothers with L. casei 431 and B. lactis BB-12 probiotics reduces the TSB levels in VLBW infants by a minimum of 25%.

Supplementing breastfeeding mothers with L. casei 431 and B. lactis BB-12 probiotics reduces PPD symptom scores by at least 30%.

Study design and setting

This is a randomised, double-blind, placebo-controlled trial with two parallel arms aiming for superiority. The study will take place at Al-Zahra Educational Hospital in Tabriz, Iran, which serves as the primary referral centre in northwest Iran for VLBW infant deliveries and hospitalisation of VLBW infants born in the other hospitals. Participant recruitment commenced on 30 December 2022, and the anticipated completion date is October 2023.

Participants

Participants include breastfeeding women and their VLBW infants, whether singletons or twins. In the case of twin infants, both will be included if eligible.

Inclusion criteria

Infants with a birth weight between 1000 and 1500 g who can receive breast milk.

Delivery of a singleton or twins within the past 48 hours.

Women who desire and are capable of breastfeeding their babies and can be present at the hospital where the baby is admitted at least once a week.

Infants hospitalised for at least 7 days after the intervention commences.

Exclusion criteria

Contraindication to breastfeeding.

Obvious anomalies and/or poor conditions in infants diagnosed by a neonatologist.

Known serious illnesses in women.

Regular use of probiotics (in any form) by the woman.

History of an allergy to probiotics.

Immunodeficiency in the mother and/or infant(s).

The principal investigator (PI; first author, MA) will explain the study aims and procedures to the mothers (and, if possible, to their husbands or another legal guardian), assess the eligibility criteria and ask them to sign written informed consent forms (see online supplemental material ). Women will also be informed that they can leave the study at any time without providing a reason. After obtaining informed consent and collecting baseline data, the participants will be randomised into one of two groups: maternal probiotic supplementation or control ( figure 1 presents a flow chart of the trial process).

Supplemental material

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Flow chart of the trial process. TSB, total serum bilirubin.

Randomisation and blinding

The allocation sequence will be generated using block randomisation, with randomly varying block sizes of 4 and 6 and an allocation ratio of 1:1 in each stratum referring to the random.org computer program. Stratification will be based on whether the infants are singletons or twins. Identical, opaque and sequentially numbered (within each stratum) bottles containing identical capsules (probiotic/placebo) will be used to conceal the sequence and to maintain blinding. Participants within each stratum will receive the bottles in the sequence corresponding to their enrolment in the study. Trial participants, care providers, outcome assessors and data analysts will be blinded. The allocation sequence will be generated and the bottles prepared by a person not involved in participant recruitment, allocation or data collection. Unblinding is permissible in the unlikely event of an adverse occurrence. In such a case, the study coordinator who generated the allocation sequence should inform the neonatologist colleague (MMG, a highly experienced faculty member) of the participant’s intervention assignment.

Intervention

Participants will receive either probiotics or a placebo for 42–45 days. One package of L. paracasei 431 and one package of B. lactis BB-12 at a concentration of 10 12 CFU/g each were sourced from ‘Hansen Co., Denmark’. Corn starch was acquired from ‘Glucose Co, Iran’. Afterwards, 2.56 g of each strain will be mixed with 1281 g of corn starch in the laboratory of the pharmacy department at Tabriz University of Medical Sciences and filled into 2562 capsules (each containing 500 mg) as a probiotic supplement. Additionally, 2562 capsules will be filled with corn starch to serve as the placebo. This probiotic is lyophilised, meaning that it is slowly ground in a sterile mortar to produce very fine and uniform particles, which are then mixed with a filler to ensure that 500 mg contains 1×10 9 probiotic bacteria per strain. Before filling the capsules, the powder will be sampled and cultured for microorganisms. Microbial culture and counting will also be performed on the sample of the filled capsules to ensure quality.

To ensure consumption, capsules will be provided to breastfeeding mothers daily while their infants are in the hospital. If daily access to the mothers is not feasible, the packages will be delivered, with no more than one package per week. If an infant is discharged before the 42–45 days of intervention period, all remaining capsules will be provided to the women and they will be instructed on home supplementation and contacted daily for follow-up. All capsules are taken and any adverse events will be recorded in a diary. Participants will be instructed to take one capsule daily, preferably before or during a meal, store the capsules in a cool, dry place (below 25ᵒC) and take the supplement at least 1 hour before or after other supplements.

The newborns’ TSB levels will be measured at baseline, after obtaining informed consent, as well as on days 4 and 7 after intervention at the laboratory of Al-Zahra Educational Hospital. To ensure result validity, 10 blood samples will be sent for testing both to the comprehensive reference laboratory in Tabriz and to the hospital laboratory before the study commences, to establish the intraclass correlation coefficient (ICC).

A checklist has been developed to meticulously document the commencement and cessation of each phototherapy type and any blood exchange transfusions administered during the 42–45 days following the intervention. In initiating and discontinuing phototherapy, the neonatologists at the hospital follow the Queensland Maternity and Neonatal Clinical Guidelines while consulting the Plot TSB levels on the nomogram, considering gestational age, weight and age appropriateness. Phototherapy is discontinued once the bilirubin level lowers to a safe threshold based on the guidelines. 26 In severe cases with bilirubin levels exceeding 25–30 mg/dL, a blood exchange transfusion is performed to prevent kernicterus. 27 The PI will complete the checklist, also document the occurrence of any significant neonatal issues such as sepsis and necrotising enterocolitis daily based on the infant’s medical record and confirmation of the neonatologist colleague. The infant’s anthropometric measurements (weight, height and head circumference) at birth, on the seventh day after the intervention and at discharge will be recorded based on the infant’s medical record. The infant’s weight will be measured using a calibrated scale in the neonatal intensive care unit (NICU) department. At the 42–45 days of in-person meeting, the PI will record the infant’s anthropometric characteristics, rehospitalisation and phototherapy needs after discharge, infant mortality, other neonatal problems, and maternal health (assessed using the Edinburgh Postnatal Depression, Anxiety and Mastitis Scales), and any side effects. Both study groups will receive standard care provided by health centres and the hospital. Table 1 shows the schedule of enrolment, interventions and assessments.

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Schedule of enrolment, interventions and assessments

Follow-up assessments will occur daily over the period of 42–45 days, either in person or via phone calls, depending on the infant’s hospitalisation status and the woman’s condition. The women will be encouraged to attend the hospital daily during their infant hospitalisation for capsule consumption and maternal mastitis symptom assessment. Any adverse events during the study period will be documented. Serious adverse events will be promptly addressed by a specialist. Mothers observing any adverse events in themselves or their infants will call the PI, who will arrange for a specialist or neonatologist visit at the hospital, free of charge.

Primary outcomes are the TSB levels on the fourth and seventh days following the intervention, as well as the depression score at 6 weeks post partum.

Secondary infant outcomes include the duration of phototherapy, the infant’s weight on the seventh day and the 42–45 days of postintervention, the time taken to achieve full oral feeding, a composite variable of occurrence of serious neonatal problems (such as sepsis, necrotising enterocolitis, bronchopulmonary dysplasia, retinopathy of prematurity and intraventricular haemorrhage) up to 42–45 days of infancy and duration of infant hospitalisation.

Secondary maternal outcomes include the occurrence of mastitis during the 42–45 days of intervention period and the anxiety score at 6 weeks post partum.

Adverse events

Reported adverse events of probiotics include sepsis, vomiting, loose stools, abdominal distension and bloating. These events will be monitored daily throughout the 42–45 days of intervention period using a diary. Any additional adverse events occurring within this period will also be recorded in the diary. In the event of severe complications during the study leading to significant discomfort, treatment allocation will be promptly unblinded to the specialist and/or the person in charge in the ward if requested, to facilitate the immediate provision of necessary support and treatment, provided free of charge under the direct supervision of a designated perinatologist or neonatologist from the research team. To maintain transparency and accountability, any unblinding due to severe complications will be promptly reported to the research ethics committee. This communication serves to keep the committee informed of significant events and enables them to offer guidance or take necessary actions to protect the interests of all participants.

Sample size

The sample size was calculated using G*Power software based on the TSB level and PPD score as the primary outcomes. Referring to Matin et al ’s study 24 regarding the TSB level on the seventh day after intervention, with parameters m1=6.0 mg/dL, m2=4.5 mg/dL (assuming a 25% reduction due to the intervention), SD1=SD2=2.3, two-sided alpha=0.05 and 90% power, the sample size was calculated to be 51 individuals per group. Additionally, based on Vaziri et al ’s study 28 regarding depression, with parameters m1=7.18, m2=5.03 (assuming a 30% reduction due to the intervention), SD1=SD2=3.99, two-sided alpha=0.05 and 80% power, the sample size was calculated as 55 per group. Therefore, due to the larger sample size calculated based on the PPD variable and accounting for a 10% attrition rate, this study will include 61 women in each group, totalling 122 breastfeeding mothers with VLBW infants and their eligible infants.

Interim analysis

There will be no interim analysis conducted in this study.

Data collection

All data will be collected by the PI, a doctoral candidate in midwifery who has received thorough training from supervisors, including the neonatologist on the study team. The neonatologist, a highly experienced faculty member who is present in the study setting every working day, will directly oversee the data collection process. The following tools will be used for data collection:

Maternal baseline assessment questionnaire: It includes information on the mother’s education level, date of birth, gestational age at delivery, number of pregnancies, number of abortions, number of live children, number of stillbirths, number of child deaths after birth, history of caesarean section, number of prenatal care visits, pre-pregnancy complications (such as thyroid disorder, chronic hypertension, pre-eclampsia), smoking and drug use during pregnancy, spouse’s smoking, complications during pregnancy (such as vaginal bleeding, placental abruption, amniotic sac rupture, oligohydramnios, polyhydramnios) and predelivery magnesium sulfate usage and corticosteroid intake. The PI will complete the questionnaire through interviews with the participants and by reviewing her medical records after enrolment.

Characteristics of hospitalised infant questionnaire: TSB levels will be measured using a venous blood sample at baseline and on the fourth and seventh days after intervention. Other data which will be recorded include infant’s hospitalisation date, blood group, gender, first and fifth-minute Apgar score, birth weight, height and head circumference at birth, nutrition status assessment during the first day of enrolment, surfactant usage, continuous positive airway pressure use, need for phototherapy, duration and type of phototherapy, infant death, age at death, the time that the infant can tolerate more than half of the oral feeding with breast milk, the time that the infant can tolerate 100% oral feeding with breast milk, cessation of intravenous feeding (TPN), duration of antibiotic treatment, neonatal complications (such as necrotising enterocolitis, intraventricular haemorrhage, sepsis, positive blood culture for sepsis, pulmonary dysplasia), examination on the seventh day of the intervention (including weight, height, head circumference, nutritional status), assessments on the seventh day and at discharge (infant’s age, weight, height, head circumference and nutritional status) and assessments on the 40th–45th days (including infant’s death, age at death, weight, height, head circumference, retinopathy of prematurity, blood transfusion, rehospitalisation, type of feeding in the last 24 hours). The PI will collect these data via neonate medical records, interviewing the corresponding neonatologist, consulting the neonatologist colleague and interviewing the mothers.

The Edinburgh Postnatal Depression Scale (EPDS): The enrolled mothers will complete the scale at baseline (before the intervention) and 42–45 days after delivery. The EPDS was developed by Cox et al in 1987 and is used to measure depression during pregnancy and after delivery. This scale consists of 10 items with four options. The options for each item are assigned a score from 0 to 3 based on severity of the symptom, and the total score is obtained from the sum of the scores, which can vary from 0 to 30. The concurrent correlation coefficient of the original version of the scale with the Beck Depression Scale was 0.78. The reliability of this scale was estimated at 0.75 using Cronbach’s alpha and the split-half method. 29 Its Persian version has been validated in Iran, and its internal consistency using Cronbach’ s alpha ranges from 0.77 to 0.86 . 30

The Spielberger State Anxiety Scale (SSAS) will be administered at baseline, and the Postpartum Specific Anxiety Scale Research Short-Form (PSAS-RSF) will be administered on the 40th–45th days after delivery. The SSAS assesses state and trait anxiety; however, only state anxiety will be assessed in this study using 20 questions. The questions are arranged with four options (not at all, somewhat, moderate, very much), and the total scale ranges from 20 to 80 (20=no anxiety, 21–39=mild anxiety, 40–59=moderate anxiety, 60–79=severe anxiety, 80=very severe anxiety). This questionnaire demonstrates a high level of validity and reliability. The validity of its Persian version has been confirmed in Iran by Mahram, with an internal consistency of 0.91 using Cronbach’s alpha. 31 The initial version of the PSAS includes 51 items. 32 However, the 16-item version of the PSAS-RSF is considered the strongest version in terms of theory and psychometrics. The PSAS-RSF is the first validated short form specifically designed to measure postpartum anxiety. This scale can be used for up to 12 months after delivery. Each item is scored on a ‘Four-State Ordinal Continuum’ between 1 (never) and 4 (always), and the total scale score ranges from 16 to 64. The scale has demonstrated good reliability (McDonald’s ω=0.88) for the entire instrument. 33 The Persian version of this scale has been validated in Tabriz, Iran, with high internal consistency (Cronbach’s alpha 0.72) and test–retest reliability (ICC 0.97 (95% CI 0.98 to 0.93)). 34

A mastitis checklist will be used to assess signs and symptoms in the breasts (redness, pain, tenderness and existence of a hard mass), as well as influenza (fatigue, fever, shivering or chills). If the woman reports at least two symptoms of breast and one symptom of influenza, a diagnosis of mastitis will be made.

The use of probiotic/placebo supplements and any adverse events will be recorded daily by the mothers in a diary.

Content validity of the tools used in this study (except the validated ones) will be determined using the opinions of experts, including obstetric specialists, neonatologists, midwives and nurses.

All collected data will be transparently reported to an independent auditor assigned by the Ethics Committee of Tabriz University of Medical Science whenever it is requested or necessary.

Data management

Data entry will occur in the software immediately after data collection. To ensure accuracy, range checks will be implemented for the data values. Additionally, the data from the first five participants and approximately 10% of the remaining participants (randomly selected) will undergo a thorough review by another individual.

Regular reminders will be sent to enhance both protocol adherence and participant retention. All required data will be collected from all participants, including those who are non-adherents. Reasons for non-adherence and non-retention will be inquired directly from participants or, if a participant is inaccessible, from a designated support person. The reasons will be reported by the study group. In the informed consent form, participants’ consent will be obtained for the use of their and their babies’ electronic and paper medical records, which may facilitate the collection of some important data from those lost to follow-up.

Confidentiality

To maintain complete confidentiality, we will not include the identifiable data from potential and enrolled participants in their questionnaires or in the computer software where data are entered. Participants will be identified by unique codes. Identifiable participant characteristics, along with their corresponding codes, will be stored separately in a secure location accessible only to the data collector, corresponding author and the auditor assigned by the ethics committee. Under specific circumstances, other members of research team and ethics committee representatives can access these details with a valid rationale.

Statistical analysis

Modified intention-to-treat analyses will be conducted for the primary and secondary outcomes, excluding those lost to follow-up from all postintervention assessments of the outcomes.

The normal distribution of quantitative outcomes across groups will be examined using the Kolmogorov-Smirnov test. If the data are normally distributed, repeated measures analysis of variance will be employed to compare the groups concerning TSB levels assessed on the fourth and seventh postintervention days, adjusting for the baseline values. The interaction effect of group and time will also be evaluated. Analysis of covariance will be used to compare the other quantitative outcomes, such as PPD and anxiety scores assessed once after intervention, adjusting for baseline values.

Assumptions, such as sphericity and the absence of spurious outliers, will be verified before interpreting the results. Non-parametric tests, such as the Mann-Whitney U test, will be used if parametric assumptions are not met. Binary logistic regression will be used to compare the groups regarding qualitative outcomes, such as the need for phototherapy, infant food tolerance, the occurrence of important neonatal problems and maternal mastitis. A p value level of <0.05 will be considered statistically significant, and all analyses will be conducted using IBM SPSS (V.24).

Trial results will be disseminated through publication in peer-reviewed journals and presentations at professional society meetings.

Patient and public involvement

Patients and the public were not involved in the design of the study or the recruitment of participants. At the end of the study, the main results will be disseminated to participants through peer-reviewed journals and presented at international conferences.

Ethics and dissemination

This study protocol has been approved by the Medical University of Tabriz Ethics Committee (IR.TBZMED.REC.1401.735). Participants will give informed consent to participate in the study before taking part. Any protocol amendments will be notified to the ethics committee and the Iranian Registry of Clinical Trials. Findings will be disseminated through publication in a peer-reviewed journal and presentations at relevant conferences.

Low birth weight is one of the most common contributors to increased physiological bilirubin levels in infants. Elevated bilirubin production in premature infants increases the risk of mortality and long-term neurodevelopmental impairment due to bilirubin neurotoxicity. 35 36 VLBW infants are particularly vulnerable to brain damage from hyperbilirubinaemia and generally receive treatment at lower thresholds than normal birthweight infants. 37 Two common treatments for neonatal hyperbilirubinaemia (ie, phototherapy and blood transfusion) are associated with diverse side effects. 38 Therefore, considering the high prevalence of hyperbilirubinaemia and the importance of its prevention and rapid treatment in premature infants, it is essential to use alternative or supportive methods that have minimal or no side effects. The results of a systematic review and meta-analysis revealed evidence of the effect of probiotic supplements on reducing the duration of phototherapy, TSB at 96 hours and on the seventh day after birth, as well as the duration of hospitalisation in infants with hyperbilirubinaemia. However, the authors stated that the certainty of this evidence is low, and that further robust studies are necessary to confirm their efficacy. 39 This trial seeks to provide evidence for such an alternative therapy, which may have other beneficial effects on infants and their mothers.

In our study, we will use two strains, L. paracasei 431 and B. lactis BB-12, well-studied strains with documented safety and effectiveness in various health aspects. 40 41 Numerous clinical trials involving these strains have demonstrated their positive impact on gut microbiota, immune function and gastrointestinal health. 42 43 L. paracasei 431 has been noted for its ability to modulate immune responses and enhance barrier function in the gut, 44 while B. lactis BB-12 is known for its positive effects on microbial balance and its potential to reduce pathogenic bacteria. 45 Our research team and others have successfully used these strains in previous studies, which provided us with a foundation of experience and data to build on. 24 46–48 In this study, these strains were sourced from Chr Hansen and meet the necessary international standards. The formulations containing these strains are currently available in the Iranian market and have been approved by the Iranian Food and Drug Administration of the Ministry of Health and Medical Education. This approval ensures that the strains are safe, effective and manufactured according to good manufacturing practices. Products containing these strains, which can be found in pharmacies and health food stores in Iran, are often marketed for their benefits to digestive health and immune function.

There are no strict recommended guidelines for the dosage. However, the dosage of 1×10 9 CFU/day from each strain falls within the range commonly used in clinical trials and aligns with guidelines for probiotic administration. This dosage has been effective in various studies without causing adverse effects. 49 Additionally, the promising results in our previous study, where only L. paracasei subsp paracasei at 1.5×10 9 CFU/day was used, 24 provided the rationale for selecting the dosage in the current investigation.

Strengths and limitations

Recruiting participants within the first 48 hours after delivery in this study can make it possible to thoroughly investigate the preventive effect of probiotic supplements on hyperbilirubinaemia in VLBW infants. The primary outcome of infants’ TSB levels will be measured at baseline, as well as on the fourth and seventh days after intervention. This timeline aligns with the peak and resolution of neonatal hyperbilirubinaemia in the VLBW infant population. 38 50

Adhering rigorously to clinical trial standards, including proper randomisation and allocation concealment, blinding of participants, care providers, and outcome assessors, comprehensive participant follow-up, strict adherence to the study protocol in its implementation and transparent reporting of all results, we aim to minimise the risk of bias such as selection, performance, detection, attrition and reporting biases. Also, conducting the trial in a tertiary hospital covering VLBW infants from diverse geographical areas enhances the generalisability of the study results.

Due to budgetary constraints, this trial does not incorporate the collection and analysis of biological samples such as blood, maternal stool, maternal milk or fetal stool, which could offer valuable insights into the probiotic effects on the maternal and neonatal microbiome. This omission limits our ability to fully understand the mechanisms behind any observed effects on neonatal and maternal outcomes. Future research should prioritise including microbiome analysis to enhance our comprehension of probiotic roles in maternal and neonatal health, particularly in the context of VLBW infants.

In this trial, we are going to assess the short-term intervention’s effects on a few maternal short-term outcomes. Thus, it may not fully capture the intervention’s impact on the overall well-being of women. Future studies should assess broader maternal outcomes, such as sleep quality, to provide a more holistic view of the probiotic intervention’s influence on maternal health.

The primary outcome of PPD scores will be assessed once after intervention, that is, at the end of the 42–45 days of intervention period, aligning with the conventional time frame for assessing PPD. This time frame allows for examination of the immediate impact of probiotic supplementation on maternal mental health. 51 Assessing PPD at two time points (baseline and 42–45 days after delivery) may not capture fluctuations adequately, and more frequent evaluations, coupled with clinical assessments, could yield comprehensive results. However, due to the vulnerability of women with VLBW infants, frequent assessments could impose a burden on participants. Therefore, a more frequent assessment of PPD is recommended in future studies on less vulnerable women.

Since most VLBW infants admitted to the hospital from which we will be recruiting participants come from other cities, including other provinces, it may be challenging to retain participants in the long term after discharge due to limited physical access. However, previous studies have demonstrated the positive effects of short-term direct probiotic administration on newborns, including a reduction in the duration of phototherapy and TSB levels. 39 In a trial, administering B. breve and L. rhamnosus orally at a concentration of 2×10 6 CFU/day, starting from the first hour of life, resulted in rapid and substantial colonisation by days 5 and 6. 52 To our knowledge, no studies have investigated the effects of postpartum probiotic administration on PPD. Nevertheless, a trial has shown a positive effect from 4 weeks of probiotic supplementation on certain cognitive functions in patients with major depressive disorder. 53 Therefore, based on the evidence supporting the effectiveness of short-term interventions and the practical challenges involved, our research team chose a period of 42–45 days of intervention. This duration strikes a balance between providing sufficient treatment time and addressing the logistical challenges of the study. Long-term interventions, coupled with extended assessments of maternal and infant outcomes in future studies, could provide deeper insights into the impacts of intervention.

Ethics statements

Patient consent for publication.

Consent obtained directly from patient(s)

Acknowledgments

We thank the Vice-Chancellor for Research at Tabriz University of Medical Sciences for their financial support.

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Contributors MA, SM-A-C, MM, MMG, AH-R, ZF and MS contributed to the design of the protocol, critically read the paper, provided inputs and revisions and approved the final manuscript. SM-A-C and MA contributed to the implementation and analysis plan and have written the first draft of this paper. MS is the corresponding author of this article. SM-A-C is the guarantor.

Funding This study is funded by Tabriz University of Medical Sciences.

Disclaimer The funding source had no role in the design and conduct of the study or the decision on writing and submitting this manuscript.

Competing interests None declared.

Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

Provenance and peer review Not commissioned; externally peer reviewed.

Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

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Functional properties of foods and beverages.

1. Introduction

2. overview of the contributions, acknowledgments, conflicts of interest, list of contributions.

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Share and Cite

Abbasi, A.M.; Guo, X.; Chen, Y. Functional Properties of Foods and Beverages. Foods 2024 , 13 , 2763. https://doi.org/10.3390/foods13172763

Abbasi AM, Guo X, Chen Y. Functional Properties of Foods and Beverages. Foods . 2024; 13(17):2763. https://doi.org/10.3390/foods13172763

Abbasi, Arshad Mehmood, Xinbo Guo, and Yongsheng Chen. 2024. "Functional Properties of Foods and Beverages" Foods 13, no. 17: 2763. https://doi.org/10.3390/foods13172763

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Effects of Probiotics, Prebiotics, and Synbiotics on Human Health

The human gastrointestinal tract is colonised by a complex ecosystem of microorganisms. Intestinal bacteria are not only commensal, but they also undergo a synbiotic co-evolution along with their host. Beneficial intestinal bacteria have numerous and important functions, e.g., they produce various nutrients for their host, prevent infections caused by intestinal pathogens, and modulate a normal immunological response. Therefore, modification of the intestinal microbiota in order to achieve, restore, and maintain favourable balance in the ecosystem, and the activity of microorganisms present in the gastrointestinal tract is necessary for the improved health condition of the host. The introduction of probiotics, prebiotics, or synbiotics into human diet is favourable for the intestinal microbiota. They may be consumed in the form of raw vegetables and fruit, fermented pickles, or dairy products. Another source may be pharmaceutical formulas and functional food. This paper provides a review of available information and summarises the current knowledge on the effects of probiotics, prebiotics, and synbiotics on human health. The mechanism of beneficial action of those substances is discussed, and verified study results proving their efficacy in human nutrition are presented.

1. Introduction

Nowadays, besides the basic role of nutrition consisting in the supply of necessary nutrients for growth and development of the organism, some additional aspects are becoming increasingly important, including the maintenance of health and counteracting diseases. In the world of highly processed food, particular attention is drawn to the composition and safety of consumed products. The quality of food is very important because of, i.e., the problem of food poisoning, obesity, allergy, cardiovascular diseases, and cancer—the plague of the 21st century. Scientific reports point to the health benefits of using probiotics and prebiotics in human nutrition. The word “probiotic” comes from Greek, and it means “for life”. Most probably, it was Ferdinand Vergin who invented the term “probiotic” in 1954, in his article entitled “Anti-und Probiotika” comparing the harmful effects of antibiotics and other antibacterial agents on the intestinal microbiota with the beneficial effects (“probiotika”) of some useful bacteria [ 1 ]. Some time after that, in 1965, Lilly and Stillwell described probiotics as microorganisms stimulating the growth of other microorganisms [ 2 ]. The definition of probiotics has been modified and changed many times. To emphasise their microbial origin, Fuller (1989) stated that probiotics must be viable microorganisms and must exert a beneficial effect on their host [ 3 ]. On the other hand, Guarner and Schaafsma (1998) indicated the necessary use of an appropriate dose of probiotic organisms required to achieve the expected effect [ 4 ]. The current definition, formulated in 2002 by FAO (Food and Agriculture Organization of the United Nations) and WHO (World Health Organization) working group experts, states that probiotics are “live strains of strictly selected microorganisms which, when administered in adequate amounts, confer a health benefit on the host” [ 5 ]. The definition was maintained by the International Scientific Association for Probiotics and Prebiotics (ISAPP) in 2013 [ 6 ].

Results of clinical studies confirm the positive effect of probiotics on gastrointestinal diseases (e.g., irritable bowel syndrome, gastrointestinal disorders, elimination of Helicobacter , inflammatory bowel disease, diarrhoeas) and allergic diseases (e.g., atopic dermatitis). Many clinical studies have proven the effectiveness of probiotics for treatment of diseases such as obesity, insulin resistance syndrome, type 2 diabetes, and non-alcoholic fatty liver disease. Furthermore, the positive effects of probiotics on human health have been demonstrated by increasing the body’s immunity (immunomodulation). Scientific reports also show the benefits of the prophylactic use of probiotics in different types of cancer and side effects associated with cancer. Many clinical studies have proven the effectiveness of probiotics, and recommended doses of probiotics are those that have been used in a particular case. Keep in mind that how probiotics work may depend on the strain, dose, and components used to produce a given probiotic product.

In 1995, prebiotics were defined by Gibson and Roberfroid as non-digested food components that, through the stimulation of growth and/or activity of a single type or a limited amount of microorganisms residing in the gastrointestinal tract, improve the health condition of a host [ 7 ]. In 2004, the definition was updated and prebiotics were defined as selectively fermented components allowing specific changes in the composition and/or activity of microorganisms in the gastrointestinal tract, beneficial for host’s health and wellbeing [ 8 ]. Finally, in 2007, FAO/WHO experts described prebiotics as a nonviable food component that confers a health benefit on the host associated with modulation of the microbiota [ 9 ].

Prebiotics may be used as an alternative to probiotics or as an additional support for them. However different prebiotics will stimulate the growth of different indigenous gut bacteria. Prebiotics have enormous potential for modifying the gut microbiota, but these modifications occur at the level of individual strains and species and are not easily predicted a priori. There are many reports on the beneficial effects of prebiotics on human health.

High potential is attributed to the simultaneous use of probiotics and prebiotics. In 1995, Gibson and Roberfroid introduced the term “synbiotic” to describe a combination of synergistically acting probiotics and prebiotics [ 7 ]. A selected component introduced to the gastrointestinal tract should selectively stimulate growth and/or activate the metabolism of a physiological intestinal microbiota, thus conferring beneficial effect to the host’s health [ 10 ]. As the word “synbiotic” implies synergy, the term should be reserved for those products in which a prebiotic component selectively favours a probiotic microorganism [ 11 ]. The principal purpose of that type of combination is the improvement of survival of probiotic microorganisms in the gastrointestinal tract.

Synbiotics have both probiotic and prebiotic properties and were created in order to overcome some possible difficulties in the survival of probiotics in the gastrointestinal tract [ 12 ]. Therefore, an appropriate combination of both components in a single product should ensure a superior effect, compared to the activity of the probiotic or prebiotic alone [ 13 , 14 ].

The aim of the review was to discuss the mechanisms of action of probiotics, prebiotics, and synbiotics, as well as the current insight into their effect on human health. The selection of probiotic strains, prebiotics, and their respective dosages is crucial in obtaining a therapeutic effect, so separate sections are dedicated to this topic. Further research into the acquisition of new probiotic strains, the selection of probiotics and prebiotics for synbiotics, dose setting, safety of use, and clinical trials documenting the desired health effects is necessary. Effects should be confirmed in properly scheduled clinical trials conducted by independent research centres.

2. Probiotics

The knowledge of the beneficial effects of lactic acid fermentation on human health dates back to ancient times. The Bible mentions sour milk several times. Ancient Romans and Greeks knew various recipes for fermented milk. A specific type of sour milk, called “leben raib”, prepared from buffalo, cow, or goat milk, was consumed in ancient Egypt. A similar “jahurt” was also commonly consumed by people inhabiting the Balkans. In India, fermented milk drinks were known already 800–300 years B.C., and in Turkey in the 8th century. A milk drink called “ajran” was consumed in Central Russia in the 12th century, and “tarho” was consumed in Hungary in the 14th century [ 15 ].

A particular interest in lactic acid fermentation was expressed in the beginning of the 20th century by the Russian scientist and immunologist working for the Pasteur Institute in Paris, awarded with the Nobel Prize in medicine for his work on immunology (in 1907), Ilia Miecznikow. Here is a quote from his book “Studies on Optimism”: “with various foods undergoing lactic acid fermentation and consumed raw (sour milk, kefir, sauerkraut, pickles) humans introduced huge amounts of proliferating lactic acid bacteria to their alimentary tracts” [ 16 ].

2.1. Selection Criteria and Requirements for Probiotic Strains

According to the suggestions of the WHO, FAO, and EFSA (the European Food Safety Authority), in their selection process, probiotic strains must meet both safety and functionality criteria, as well as those related to their technological usefulness ( Table 1 ). Probiotic characteristics are not associated with the genus or species of a microorganism, but with few and specially selected strains of a particular species [ 6 ]. The safety of a strain is defined by its origin, the absence of association with pathogenic cultures, and the antibiotic resistance profile. Functional aspects define their survival in the gastrointestinal tract and their immunomodulatory effect. Probiotic strains have to meet the requirements associated with the technology of their production, which means they have to be able to survive and maintain their properties throughout the storage and distribution processes [ 17 ]. Probiotics should also have documented pro-health effects consistent with the characteristics of the strain present in a marketed product. Review papers and scientific studies on one strain may not be used for the promotion of other strains as probiotics. It has to be considered, as well, that the studies documenting probiotic properties of a particular strain at a tested dose do not constitute evidence of similar properties of a different dose of the same strain. Also, the type of carrier/matrix is important, as it may reduce the viability of a particular strain, thus changing the properties of a product [ 18 , 19 ].

Selection criteria of probiotic strains [ 5 , 20 ].

2.2. Probiotic Microorganisms

Probiotic products may contain one or more selected microbial strains. Human probiotic microorganisms belong mostly to the following geni: Lactobacillus , Bifidobacterium , and Lactococus , Streptococcus , Enterococcus . Moreover, strains of Gram-positive bacteria belonging to the genus Bacillus and some yeast strains belonging to the genus Saccharomyces are commonly used in probiotic products [ 21 ].

Probiotics are subject to regulations contained in the general food law, according to which they should be safe for human and animal health. In the USA, microorganisms used for consumption purposes should have the GRAS (Generally Regarded As Safe) status, regulated by the FDA (Food and Drug Administration). In Europe, EFSA introduced the term of QPS (Qualified Presumption of Safety). The QPS concept involves some additional criteria of the safety assessment of bacterial supplements, including the history of safe usage and absence of the risk of acquired resistance to antibiotics [ 22 , 23 ]. Table 2 presents probiotic microorganisms contained in pharmaceutical products and as food additives.

Probiotic microorganisms used in human nutrition [ 24 , 25 , 26 ].

(a) Mostly as pharmaceutical products; (b) mostly as food additives; * QPS (Qualified Presumption of Safety) microorganisms.

2.3. Mechanism of Action of Probiotics

A significant progress has been observed lately in the field of studies on probiotics, mostly in terms of the selection and characteristics of individual probiotic cultures, their possible use, and their effect on health.

Probiotics have numerous advantageous functions in human organisms. Their main advantage is the effect on the development of the microbiota inhabiting the organism in the way ensuring proper balance between pathogens and the bacteria that are necessary for a normal function of the organism [ 27 , 28 ]. Live microorganisms meeting the applicable criteria are used in the production of functional food and in the preservation of food products. Their positive effect is used for the restoration of natural microbiota after antibiotic therapy [ 29 , 30 ]. Another function is counteracting the activity of pathogenic intestinal microbiota, introduced from contaminated food and environment. Therefore, probiotics may effectively inhibit the development of pathogenic bacteria, such as Clostridium perfringens [ 31 ], Campylobacter jejuni [ 32 ], Salmonella Enteritidis [ 33 ], Escherichia coli [ 34 ], various species of Shigella [ 35 ], Staphylococcus [ 36 ], and Yersinia [ 37 ], thus preventing food poisoning. A positive effect of probiotics on digestion processes, treatment of food allergies [ 38 , 39 ], candidoses [ 40 ], and dental caries [ 41 ] has been confirmed. Probiotic microorganisms such as Lactobacillus plantarum [ 42 ], Lactobacillus reuteri [ 43 ], Bifidobacterium adolescentis , and Bifidobacterium pseudocatenulatum [ 44 ] are natural producers of B group vitamins (B1, B2, B3, B6, B8, B9, B12). They also increase the efficiency of the immunological system, enhance the absorption of vitamins and mineral compounds, and stimulate the generation of organic acids and amino acids [ 18 , 45 , 46 , 47 ]. Probiotic microorganisms may also be able to produce enzymes, such as esterase, lipase, and co-enzymes A, Q, NAD, and NADP. Some products of probiotics’ metabolism may also show antibiotic (acidophiline, bacitracin, lactacin), anti-cancerogenic, and immunosuppressive properties [ 45 , 48 , 49 , 50 ].

Molecular and genetic studies allowed the determination of the basics of the beneficial effect of probiotics, involving four mechanisms:

• (1) Antagonism through the production of antimicrobial substances [ 51 ];
• (2) Competition with pathogens for adhesion to the epithelium and for nutrients [ 52 ];
• (3) Immunomodulation of the host [ 53 ];
• (4) Inhibition of bacterial toxin production [ 54 ].

The first two mechanisms are directly associated with their effect on other microorganisms. Those mechanisms are important in prophylaxis and treatment of infections, and in the maintenance of balance of the host’s intestinal microbiota. The ability of probiotic strains to co-aggregate, as one of their mechanisms of action, may lead to the formation of a protective barrier preventing pathogenic bacteria from the colonisation of the epithelium [ 27 ]. Probiotic bacteria may be able to adhere to epithelial cells, thus blocking pathogens. That mechanism exerts an important effect on the host’s health condition. Moreover, the adhesion of probiotic microorganisms to epithelial cells may trigger a signalling cascade, leading to immunological modulation. Alternatively, the release of some soluble components may cause a direct or indirect (through epithelial cells) activation of immunological cells. This effect plays an important role in the prevention and treatment of contagious diseases, as well as in chronic inflammation of the alimentary tract or of a part thereof [ 28 ]. There are also suggestions of a possible role of probiotics in the elimination of cancer cells [ 55 ].

Results of in vitro studies indicate the role of low-molecular-weight substances produced by probiotic microorganisms (e.g., hydroperoxide and short-chain fatty acids) in inhibiting the replication of pathogens [ 28 ]. For example, Lactobacillus genus bacteria may be able to produce bacteriocins, including low-molecular-weight substances (LMWB—antibacterial peptides), as well as high-molecular-weight ones (class III bacteriocins), and some antibiotics. Probiotic bacteria (e.g., Lactobacillus and Bifidobacterium ) may produce the so-called de-conjugated bile acids (derivatives of bile acids), demonstrating stronger antibacterial effect than the bile salts produced by their host [ 28 , 56 ]. Further studies are necessary to explain the mechanism of acquiring resistance to their own metabolites by Lactobacillus genus bacteria. The nutrient essential for nearly all bacteria, except for lactic acid bacteria, is iron. It turns out that Lactobacillus bacteria do not need iron in their natural environment, which may be their crucial advantage over other microorganisms [ 57 ]. Lactobacillus delbrueckii affects the function of other microbes by binding iron hydroxide to its cellular surface, thus making it unavailable to other microbes [ 58 ].

The immunomodulatory effect of the intestinal microbiota, including probiotic bacteria, is based on three, seemingly contradictory phenomena [ 53 , 59 ]:

• (1) Induction and maintenance of the state of immunological tolerance to environmental antigens (nutritional and inhalatory);
• (2) Induction and control of immunological reactions against pathogens of bacterial and viral origin;
• (3) Inhibition of auto-aggressive and allergic reactions.

Probiotic-induced immunological stimulation is also manifested by the increased production of immunoglobulins, enhanced activity of macrophages and lymphocytes, and stimulation of γ-interferon production. Probiotics may influence the congenital and acquired immunological system through metabolites, components of the cellular wall, and DNA, recognised by specialised cells of the host (e.g., those equipped with receptors) [ 28 ]. The principal host cells that are important in the context of the immune response are intestinal epithelial cells and intestinal immune cells. Components of the cellular wall of lactic acid bacteria stimulate the activity of macrophages. Those, in turn, are able to destroy microbes rapidly by the increased production of free oxygen radicals and lysosomal enzymes. Probiotic bacteria are also able to stimulate the production of cytokines by immunocompetent cells of the gastrointestinal tract [ 60 ]. On the other hand, the immunological activity of yeast is associated with the presence of glucans in their cellular wall. Those compounds stimulate the response of the reticuloendothelial system [ 61 ].

The last of the abovementioned probiotic effects—inhibition of the production of bacterial toxins—is based on actions leading to toxin inactivation and help with the removal of toxins from the body. Help in detoxification from the body can take place by adsorption (some strains can bind toxins to their cell wall and reduce the intestinal absorption of toxins), but can also result from the metabolism of mycotoxins (e.g., aflatoxin) by microorganisms [ 62 , 63 , 64 ]. However, not all probiotics exhibit detoxifying properties, as it is a strain-related characteristic. Studies should therefore be conducted to select strains with such characteristics. The effectiveness of some probiotics in combating diarrhoea is probably associated with their ability to protect the host from toxins. The reduction of metabolic reactions leading to the production of toxins is also associated with the stimulation of pathways leading to the production of native enzymes, vitamins, and antimicrobial substances [ 28 ].

Gut microbiota play a significant role in host metabolic processes (e.g., the regulation of cholesterol absorption, blood pressure (BP), and glucose metabolism), and recent metagenomic surveys have revealed that they are involved in host immune modulation and that they influence host development and physiology (organ development) [ 65 , 66 , 67 ]. Nutritional programming to manipulate the composition of the intestinal microbiota through the administration of probiotics continues to receive much attention for the prevention or attenuation of the symptoms of metabolic-related diseases. Currently, studies are exploring the potential for expanded uses of probiotics for improving health conditions in metabolic disorders that increase the risk of developing cardiovascular diseases such as hypertension. Further investigations are required to evaluate the targeted and effective use of the wide variety of probiotic strains in various metabolic disorders to improve the overall health status of the host [ 65 ].

In order to confirm the beneficial role of probiotics in improving cardiovascular health and in the reduction of BP, more extensive studies are needed to understand the mechanisms underlying probiotic action. Most probably, all of the abovementioned mechanisms of probiotic action have an effect on the protection against infections, cancer, and the stabilization of balance of the host’s intestinal microbiota. However, it seems unlikely that each of the probiotic microorganisms has properties of all four aspects simultaneously and constitutes a universal remedy to multiple diseases. An important role in the action of probiotics is played by species- and strain-specific traits, such as: cellular structure, cell surface, size, metabolic properties, and substances secreted by microorganisms. The use of a combination of probiotics demonstrating various mechanisms of action may provide enhanced protection offered by a bio-therapeutic product [ 68 ]. Figure 1 summarises the mechanisms and effects of action of probiotics.

Mechanisms of action of synbiotics and their effects.

2.4. Probiotics for Humans

In the face of widespread diseases and ageing societies, the use of knowledge on microbiocenosis of the gastrointestinal tract and on the beneficial effect of probiotic bacteria is becoming increasingly important. The consumption of pre-processed food (fast food), often containing excessive amounts of fat and insufficient amounts of vegetables, is another factor of harmful modification of human intestinal microbiota. There is currently no doubt about the fact that the system of intestinal microorganisms and its desirable modification with probiotic formulas and products may protect people against enteral problems, and influence the overall improvement of health.

Probiotics may be helpful in the treatment of inflammatory enteral conditions, including ulcerative colitis, Crohn’s disease, and non-specific ileitis. The aetiology of those diseases is not completely understood, but it is evident that they are associated with chronic and recurrent infections or inflammations of the intestine. Clinical studies have demonstrated that probiotics lead to the remission of ulcerative colitis, but no positive effect on Crohn’s disease has been observed [ 69 , 70 ]. Numerous studies assessed the use of probiotics in the treatment of lactose intolerance [ 71 , 72 ], irritable bowel syndrome, and the prevention of colorectal cancer [ 73 ] and peptic ulcers [ 74 ].

Considering their role in the inhibition of some bacterial enzymes, probiotics may reduce the risk of colorectal carcinoma in animals. However, the same effect in humans has not been confirmed in clinical trials [ 75 ]. On the other hand, a positive effect on the urogenital system (prevention and treatment of Urinary Tract Infections (UTIs) and bacterial vaginitis) constitutes an excellent example of the benefits associated with the use of probiotics [ 76 , 77 , 78 ]. There were attempts to apply probiotics to pregnant women and neonates in order to prevent allergic diseases such as atopic dermatitis. However, the scope of action is controversial in this kind of case [ 79 ]. There is evidence that the consumption of probiotics-containing dairy products results in the reduction of blood cholesterol, which may be helpful in the prevention of obesity, diabetes, cardiovascular diseases, and cerebral stroke [ 80 ]. The reduction of cholesterol level achieved due to probiotics is less pronounced compared to the effect of pharmaceutical agents, but leads to a significant minimisation of side effects [ 80 ]. Other studies confirmed the effect of the probiotic formula VSL#3 and of the Oxalobacter formigenes bacterial strain on the elimination of oxalates with urine, which may potentially reduce the risk of urolithiasis [ 81 ]. Studies on animals demonstrated that orally administered Lactobacillus acidophilus induces expression of μ -opioid and cannabinoid receptors in intestinal cells and mediate analgesic functions in the intestine, and that the observed effect is comparable to the effect of morphine [ 82 ]. However, the effect has not been demonstrated in humans.

There are many reports on the application of probiotics in the treatment of diarrhoea. The application of Saccharomyces boulardii yeast to patients with acute, watery diarrhoea resulted in the cure and reduced frequency of that type of complaints in two subsequent months [ 83 ]. The efficacy of probiotic strains in the therapy of nosocomial, non-nosocomial, and viral diarrhoeas has also been documented. It turns out that probiotics may increase the amount of IgA antibodies, which leads to the arrest of a viral infection [ 84 ].

Antibiotic-associated diarrhoea (AAD) is a common complication of most antibiotics and Clostridium difficile disease (CDD), which also is incited by antibiotics, and is a leading cause of nosocomial outbreaks of diarrhoea and colitis. The use of probiotics for these two related diseases remains controversial. A variety of different types of probiotics show promise as effective therapies for these two diseases. Using meta-analyses, three types of probiotics ( Saccharomyces boulardii , Lactobacillus rhamnosus GG, and probiotic mixtures) significantly reduced the development of antibiotic-associated diarrhoea. Only S. boulardii was effective for CDD [ 85 ].

Studies performed in a foster home in Helsinki (Finland) demonstrated that the regular use of Lactobacillus rhamnosus GG in the form of a probiotic resulted in a reduced number of respiratory tract infections [ 86 ]. Other studies demonstrated that the application of a diet depleted of fermented foods caused a reduction of congenital immunological response, as well as a significant reduction of stool Lactobacillus count and of the stool amount of short-chain fatty acids. Moreover, the reduction of phagocytic activity of leukocytes was observed after two weeks of the diet, which could have a negative impact on the organism’s ability to protect against infections [ 87 ]. The effect of a fermented product containing Lactobacillus gasseri CECT5714 and Lactobacillus coryniformis CECT5711 strains on blood and stool parameters was studied in a randomised, double-blind trial on 30 healthy volunteers. No negative effects were observed in the group of subjects receiving the probiotic strains. Some positive effects were observed, including: the production of short-chain fatty acids, humidity, frequency and volume of stools, and subjective improvement of intestinal function [ 88 ]. Studies by Alvaro et al. (2007) demonstrated a significant reduction of Enterobacteriaceae count and increased galactosidase activity in the alimentary tract of yoghurt consumers, compared to those who did not eat yoghurt [ 89 ]. Table 3 lists the results of studies focusing on the effect of probiotics on human health. There are examples of clinical trials during which the probiotics group received the probiotic prophylactically or in addition to the standard therapy.

Examples of clinical trials regarding the effect of probiotics on human health.

Abbreviations: AD—atopic dermatitis; ALT—alanine amino transferase; ASP—aspartate amino transferase; BMI—body mass index; BP—blood pressure; CRP—C-reactive protein; γ-GTP—γ-glutamyltranspeptidase; GLP1—glucagon-like peptide 1; HDL-C—high-density lipoprotein cholesterol; HOMA-IR—homeostasis model assessment of insulin resistance; IL-10—interleukin 10; LDL-C—low-density lipoprotein cholesterol; NAFLD—non-alcoholic fatty liver disease; PBMC—peripheral blood mononuclear cell; PM—postmenopausal; SCORAD—SCORing Atopic Dermatitis; SOD—superoxide dismutase, sVCAM-1—soluble vascular cell adhesion molecule-1; TC—total cholesterol; T2D—type 2 diabetes; VFA—visceral fat area; 8-OHDG—8-hydroxy-2′-deoxyguanosine.

3. Prebiotics

Different prebiotics will stimulate the growth of different indigenous gut bacteria. Prebiotics have enormous potential for modifying the gut microbiota, but these modifications occur at the level of individual strains and species and are not easily predicted a priori. Furthermore, the gut environment, especially pH, plays a key role in determining the outcome of interspecies competition. Both for reasons of efficacy and of safety, the development of prebiotics intended to benefit human health has to take account of the highly individual species profiles that may result [ 129 ].

Fruit, vegetables, cereals, and other edible plants are sources of carbohydrates constituting potential prebiotics. The following may be mentioned as such potential souces: tomatoes, artichokes, bananas, asparagus, berries, garlic, onions, chicory, green vegetables, legumes, as well as oats, linseed, barley, and wheat [ 130 ]. Some artificially produced prebiotics are, among others: lactulose, galactooligosaccharides, fructooligosaccharides, maltooligosaccharides, cyclodextrins, and lactosaccharose. Lactulose constitutes a significant part of produced oligosaccharides (as much as 40%). Fructans, such as inulin and oligofructose, are believed to be the most used and effective in relation to many species of probiotics [ 131 ].

3.1. Prebiotic Selection Criteria

According to Wang (2009), there are five basic criteria for the classification of food components such as prebiotics ( Figure 2 ) [ 132 ]. The first criterion assumes that prebiotics are not digested (or just partially digested) in the upper segments of the alimentary tract. As a consequence, they reach the colon, where they are selectively fermented by potentially beneficial bacteria (a requirement of the second criterion) [ 133 ]. The fermentation may lead to the increased production or a change in the relative abundance of different short-chain fatty acids (SCFAs), increased stool mass, a moderate reduction of colonic pH, reduction of nitrous end products and faecal enzymes, and an improvement of the immunological system [ 134 ], which is beneficial for the host (the requirement of the third criterion). Selective stimulation of growth and/or activity of the intestinal bacteria potentially associated with health protection and wellbeing is considered another criterion [ 8 ]. The last criterion of the classification assumes that a prebiotic must be able to withstand food processing conditions and remained unchanged, non-degraded, or chemically unaltered and available for bacterial metabolism in the intestine [ 132 ]. Huebner et al. (2008) tested several commercially available prebiotics using various processing conditions. They found no significant changes of the prebiotic activity of the tested substances in various processing conditions [ 135 ]. Meanwhile, Ze et al. (2012) showed that it was possible to alter the ability of gut bacteria by utilising starch in vitro [ 136 ]. The structure of prebiotics should be appropriately documented, and components used as pharmaceutical formulas, food, or feed additives should be relatively easy to obtain at an industrial scale [ 137 ].

Requirements for potential prebiotics [ 132 , 138 ].

Prebiotics may be used as an alternative to probiotics or as an additional support for them. Long-term stability during the shelf-life of food, drinks, and feed, resistance to processing, and physical and chemical properties that exhibit a positive effect on the flavour and consistence of products may promote prebiotics as a competition to probiotics. Additionally, resistance to acids, proteases, and bile salts present in the gastrointestinal tract may be considered as other favourable properties of prebiotics. Prebiotic substances selectively stimulate microorganisms present in the host’s intestinal ecosystem, thus eliminating the need for competition with bacteria. Stimulation of the intestinal microbiota by prebiotics determines their fermentation activity, simultaneously influencing the SCFA level, which confers a health benefit on the host [ 139 , 140 ]. Moreover, prebiotics cause a reduction of intestinal pH and maintain the osmotic retention of water in the bowel [ 134 ]. However, it should be considered that an overdose of prebiotics may lead to flatulence and diarrhoea—these effects are absent in the case of excessive consumption of probiotics. Prebiotics may be consumed on a long-term basis and for prophylactic purposes. Moreover, when used at correct doses, they do not stimulate any adverse effects, such as diarrhoea, susceptibility to UV light, or hepatic injuries caused by antibiotics. Prebiotic substances are not allergenic and do not proliferate the abundance of antibiotic-resistance genes. Of course, the effect of the elimination of selected pathogens achieved by the use of prebiotics may be inferior to antibiotics, but the properties mentioned above make them a natural substitute for antibiotics [ 134 ].

3.2. Prebiotic Substances

The majority of identified prebiotics are carbohydrates of various molecular structures, naturally occurring in human and animal diets. The physiological properties of potential prebiotics determine their beneficial effect on the host’s health. Prebiotics may be classified according to those properties as [ 134 ]:

• not digested (or only partially digested);
• not absorbed in the small intestine;
• poorly fermented by bacteria in the oral cavity;
• well fermented by seemingly beneficial intestinal bacteria;
• poorly fermented by potential pathogens in the bowel.

Carbohydrates, such as dietary fibre, are potential prebiotics. Prebiotic and dietary fibre are terms used alternatively for food components that are not digested in the gastrointestinal tract. A significant difference between those two terms is that prebiotics are fermented by strictly defined groups of microorganisms, and dietary fibre is used by the majority of colonic microorganisms [ 141 ]. Therefore, considering one of the basic classification criteria, it turns out that using those terms alternatively is not always correct. Prebiotics may be a dietary fibre, but dietary fibre is not always a prebiotic [ 138 ]. The following non-starch polysaccharides are considered to be dietary fibre: cellulose, hemicellulose, pectins, gums, substances obtained from marine algae, as well as lactulose, soy oligosaccharides, inulins, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and isomaltooligosaccharides. Based on the number of monomers bound together, prebiotics may be classified as: disaccharides, oligosaccharides (3–10 monomers), and polysaccharides. The most promising and fulfilling criteria for the classification of prebiotic substances, as evidenced by in vitro and in vivo studies, are oligosaccharides, including [ 142 , 143 ]: fructooligosaccharides (FOS), galactooligosaccharides (GOS), isomaltooligosaccharides (IMO), xylooligosaccharides (XOS), transgalactooligosaccharides (TOS), and soybean oligosaccharides (SBOS).

Also, polysaccharides such as inulin, reflux starch, cellulose, hemicellulose, or pectin may potentially be prebiotics. Examples of prebiotics that are most commonly used in human nutrition are presented in Table 4 . The use of glucooligosaccharides, glicooligosaccharides, lactitol, izomaltooligosaccharides, stachyose, raffinose, and saccharose as prebiotics requires further studies [ 144 ].

Examples of prebiotics and synbiotics used in human nutrition [ 134 , 145 , 146 ].

Abbreviations: FOS—fructooligosaccharides; GOS—galactooligosaccharides; TOS—transgalactooligosaccharides; XOS—xylooligosaccharides.

3.3. Mechanism of Action of Prebiotics

Prebiotics are present in natural products, but they may also be added to food. The purpose of these additions is to improve their nutritional and health value. Some examples are: inulin, fructooligosaccharides, lactulose, and derivatives of galactose and β -glucans. Those substances may serve as a medium for probiotics. They stimulate their growth, and contain no microorganisms.

Figure 2 presents the principal mechanisms of prebiotic action and some of their effects on the host’s health. Prebiotics are not digested by host enzymes and reach the colon in a practically unaltered form, where they are fermented by saccharolytic bacteria (e.g., Bifidobacterium genus). The consumption of prebiotics largely affects the composition of the intestinal microbiota and its metabolic activity [ 147 ]. This is due to the modulation of lipid metabolism, enhanced absorbability of calcium, effect on the immunological system, and modification of the bowel function [ 147 ]. It is highly probable that providing an energy source that only specific species in the microbiota can utilize has a greater impact on microbiota composition and metabolism than these other factors. The molecular structure of prebiotics determines their physiological effects and the types of microorganisms that are able to use them as a source of carbon and energy in the bowel [ 134 ]. It was demonstrated that, despite the variety of carbohydrates that exhibit the prebiotic activity, the effect of their administration is an increased count of beneficial bacteria, mostly of the Bifidobacterium genus [ 148 , 149 ].

The mechanism of a beneficial effect of prebiotics on immunological functions remains unclear. Several possible models have been proposed [ 150 ]:

• (1) Prebiotics are able to regulate the action of hepatic lipogenic enzymes by influencing the increased production of short-chain fatty acids (SCFAs), such as propionic acid.
• (2) The production of SCFAs (especially of butyric acid) as a result of fermentation was identified as a modulator of histone acetylation, thus increasing the availability of numerous genes for transcription factors.
• (3) The modulation of mucin production.
• (4) It was demonstrated that FOS and several other prebiotics cause an increased count of lymphocytes and/or leukocytes in gut-associated lymphoid tissues (GALTs) and in peripheral blood.
• (5) The increased secretion of IgA by GALTs may stimulate the phagocytic function of intra-inflammatory macrophages.

The main aim of prebiotics is to stimulate the growth and activity of beneficial bacteria in the gastrointestinal tract, which confers a health benefit on the host. Through mechanisms including antagonism (the production of antimicrobial substances) and competition for epithelial adhesion and for nutrients, the intestinal microbiota acts as a barrier for pathogens. Final products of carbohydrate metabolism are mostly SCFAs, namely: acetic acid, butyric acid, and propionic acid, which are subsequently used by the host as a source of energy [ 151 ]. As a result of the fermentation of carbohydrates, Bifidobacterium or Lactobacillus may produce some compounds inhibiting the development of gastrointestinal pathogens, as well as cause a reduction in the intestinal pH [ 152 ]. Moreover, Bifidobacterium genus bacteria demonstrate tolerance to the produced SCFAs and reduced pH. Therefore, due to their favourable effect on the development of beneficial intestinal bacteria, the administration of prebiotics may participate in the inhibition of the development of pathogens. There are very few documented study results regarding the inhibition of the development of pathogens by prebiotics. In 1997 and 2003, Bovee-Oudenhoven et al. studied the use of lactulose in the prevention of Salmonella Enteritidis infections on a rat model. Their results indicated that the acidification of the intestine occurring as a result of lactulose fermentation caused the reduced development of pathogens and increased translocation of pathogens from the bowel [ 153 ]. It was also demonstrated that the administration of prebiotics increases the absorption of minerals, mostly of magnesium and calcium [ 154 , 155 ].

3.4. Prebiotics for Humans

The presence of prebiotics in the diet may lead to numerous health benefits. Studies on colorectal carcinoma demonstrated that the disease occurs less commonly in people who often eat vegetables and fruit. This effect is attributed mostly to inulin and oligofructose [ 156 ]. Among the advantages of those prebiotics, one may also mention the reduction of the blood LDL (low-density lipoprotein) level, stimulation of the immunological system, increased absorbability of calcium, maintenance of correct intestinal pH value, low caloric value, and alleviation of symptoms of peptic ulcers and vaginal mycosis [ 157 ]. Other effects of inulin and oligofructose on human health are: the prevention of carcinogenesis, as well as the support of lactose intolerance or dental caries treatment [ 131 ]. Rat studies demonstrated that administration of inulin for five weeks caused a significant reduction of blood triacylglycerol levels [ 156 ]. Human studies demonstrated that the daily use of 12 g of inulin for one month led to the reduction of blood VLDL (very low-density lipoprotein) levels (the reduction of triacylglycerols by 27%, and of cholesterol by 5%). This effect is associated with the effect of the prebiotic on hepatic metabolism and the inhibition of acetyl-CoA carboxylase and of glukose-6-phosphate dehydrogenase. It is also supposed that oligofructose accelerates lipid catabolism [ 157 ].

Asahara et al. (2001) demonstrated a protective effect of galactooligosaccharides (GOS) in the prevention of Salmonella Typhimurium infections in a murine model [ 158 ]. Buddington et al. (2002) confirmed a positive effect of fructooligosaccharides (FOS) on protection against Salmonella Typhimurium and Listeria monocytogenes infections [ 159 ]. Moreover, prebiotics are helpful in combating pathogenic microorganisms, such as Salmonella Enteritidis and Escherichia coli , and reduce odour compounds [ 160 ]. There are many reports regarding the positive effect of prebiotics on the carcinogenesis process. Results of rat studies proved that a prebiotic-enriched diet leads to significantly reduced indexes of carcinogenesis. Scientific research demonstrated that butyric acid may be a chemopreventive factor in carcinogenesis [ 161 ], or an agent protecting against the development of colorectal carcinoma through the promotion of cell differentiation [ 162 ]. Besides butyric acid, propionic acid also may possess anti-inflammatory properties in relation to colorectal carcinoma cells. In vitro studies on human L97 and HT29 cell lines (representing early and late stages of colorectal carcinoma) demonstrated that inulin fractions in plasma supernatant caused a significant inhibition of growth and induction of apoptosis in human colorectal carcinoma [ 163 ]. According to scientific reports, the administration of inulin and oligofructose to rats caused the inhibition of azoxymethane-induced colorectal carcinoma at the growth stage [ 164 ]. The supplementation of inulin and oligofructose at the dose of 5%–15% had also an effect on reduced occurrence of breast cancer in rats and of metastases to lungs [ 165 ]. However, those results have to be confirmed in humans. Table 5 lists the results of studies focusing on the effect of prebiotics on human health. There are examples of clinical trials during which the prebiotics group received the prebiotic prophylactically or in addition to the standard therapy.

Examples of clinical trials regarding the effect of prebiotics on human health.

Abbreviations: AD—atopic dermatitis; AX—arabinoxylan; FOS—fructooligosaccharides; GOS—galactooligosaccharides; IgA—immunoglobulin A; OFS—oligofructose.

4. Synbiotics

Synbiotics are used not only for the improved survival of beneficial microorganisms added to food or feed, but also for the stimulation of the proliferation of specific native bacterial strains present in the gastrointestinal tract [ 179 ]. The effect of synbiotics on metabolic health remains unclear. It should be mentioned that the health effect of synbiotics is probably associated with the individual combination of a probiotic and prebiotic [ 180 ]. Considering a huge number of possible combinations, the application of synbiotics for the modulation of intestinal microbiota in humans seems promising [ 181 ].

4.1. Synbiotic Selection Criteria

The first aspect to be taken into account when composing a synbiotic formula should be a selection of an appropriate probiotic and prebiotic, exerting a positive effect on the host’s health when used separately. The determination of specific properties to be possessed by a prebiotic to have a favourable effect on the probiotic seems to be the most appropriate approach. A prebiotic should selectively stimulate the growth of microorganisms, having a beneficial effect on health, with simultaneous absent (or limited) stimulation of other microorganisms.

4.2. Synbiotics in Use

Previous sections discussed probiotic microorganisms and prebiotic substances most commonly used in human nutrition. A combination of Bifidobacterium or Lactobacillus genus bacteria with fructooligosaccharides in synbiotic products seems to be the most popular. Table 4 presents the most commonly used combinations of probiotics and prebiotics.

4.3. Mechanism of Action of Synbiotics

Considering the fact that a probiotic is essentially active in the small and large intestine, and the effect of a prebiotic is observed mainly in the large intestine, the combination of the two may have a synergistic effect [ 182 ]. Prebiotics are used mostly as a selective medium for the growth of a probiotic strain, fermentation, and intestinal passage. There are indications in the literature that, due to the use of prebiotics, probiotic microorganisms acquire higher tolerance to environmental conditions, including: oxygenation, pH, and temperature in the intestine of a particular organism [ 183 ]. However, the mechanism of action of an extra energy source that provides higher tolerance to these factors is not sufficiently explained. That combination of components leads to the creation of viable microbiological dietary supplements, and ensuring an appropriate environment allows a positive impact on the host’s health. Two modes of synbiotic action are known [ 184 ]:

• (1) Action through the improved viability of probiotic microorganisms;
• (2) Action through the provision of specific health effects.

The stimulation of probiotics with prebiotics results in the modulation of the metabolic activity in the intestine with the maintenance of the intestinal biostructure, development of beneficial microbiota, and inhibition of potential pathogens present in the gastrointestinal tract [ 180 ]. Synbiotics result in reduced concentrations of undesirable metabolites, as well as the inactivation of nitrosamines and cancerogenic substances. Their use leads to a significant increase of levels of short-chain fatty acids, ketones, carbon disulphides, and methyl acetates, which potentially results in a positive effect on the host’s health [ 184 ]. As for their therapeutic efficacy, the desirable properties of synbiotics include antibacterial, anticancerogenic, and anti-allergic effects. They also counteract decay processes in the intestine and prevent constipation and diarrhoea. It turns out that synbiotics may be highly efficient in the prevention of osteoporosis, reduction of blood fat and sugar levels, regulation of the immunological system, and treatment of brain disorders associated with abnormal hepatic function [ 185 ]. The concept of mechanisms of synbiotic action, based on the modification of intestinal microbiota with probiotic microorganisms and appropriately selected prebiotics as their substrates, is presented in Figure 1 .

4.4. Synbiotics for Humans

Synbiotics have the following beneficial effects on humans [ 186 ]:

• (1) Increased Lactobacillus and Bifidobacterium genus count and maintenance of balance of the intestinal microbiota;
• (2) Improved hepatic function in patients suffering from cirrhosis;
• (3) Improved immunomodulative abilities;
• (4) Prevention of bacterial translocation and reduced incidence of nosocomial infections in patients’ post-surgical procedures and similar interventions.

The translocation of bacterial metabolism products, such as lipopolysaccharides (LPSs), ethanol, and short-chain fatty acids (SFCAs), leads to their penetration of the liver. SCFAs also stimulate the synthesis and storage of hepatic triacylglycerols. Those processes may intensify the mechanisms of hepatic detoxication, which may result in hepatic storage of triacylglycerol (IHTG), and intensify steatosis of the organ. A randomised trial on the use of a synbiotic containing five probiotics ( Lactobacillus plantarum , Lactobacillus delbrueckii spp. bulgaricus , Lactobacillus acidophilus , Lactobacillus rhamnosus , Bifidobacterium bifidum ) and inulin as a prebiotic in adult subjects with NASH (non-alcoholic steatohepatisis) demonstrated a significant reduction of IHTG (intrahepatic triacylglycerol) within six months [ 187 ]. It is also known that LPSs induce proinflammatory cytokines, such as the tumour necrosis factor alpha (TNF- α ), playing a crucial role in insulin resistance and inflammatory cell uptake in NAFLD (non-alcoholic fatty liver disease). In the study on the effect of the synbiotic product containing a blend of probiotics ( Lactobacillus casei , Lactobacillus rhamnosus , Streptococcus thermophilus , Bifidobacterium breve , Lactobacillus acidophilu s, Bifidobacterium longum , Lactobacillus bulgaricus ) and fructooligosccharides, 52 adults participated for 28 weeks. It was found that supplementation with the synbiotic resulted in the inhibition of NF-κB (nuclear factor κB) and reduced production of TNF- α (tumour necrosis factor α) [ 188 ].

In rat studies, an increased level of intestinal IgA was found, following the introduction of the synbiotic product containing Lactobacillus rhamnosus and Bifidobacterium lactis , and inulin and oligofructose as prebiotics to the diet. Synbiotics lead to reduced blood cholesterol levels and lower blood pressure [ 157 ]. Moreover, synbiotics are used in the treatment of hepatic conditions [ 189 ] and improve the absorption of calcium, magnesium, and phosphorus [ 190 ].

Danq et al. (2013), in a meta-analysis, evaluated published studies on pro/prebiotics for eczema prevention, investigating bacterial strain efficacy and changes to the allergy status of the children involved. This meta-analysis found that probiotics or synbiotics may reduce the incidence of eczema in infants aged <2 years. Systemic sensitization did not change following probiotic administration [ 191 ].

Studies carried out within the framework of the SYNCAN project funded by the European Union verified the anti-carcinogenic properties of synbiotics. The effect of fructooligosaccharides (SYN1) combined with two probiotic strains ( Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis Bb12) on the health of patients at risk of colorectal cancer was studied. As a result, a change of biomarkers (genotoxicity, labelling index, labelled cells/crypt, transepithelial resistance, necrosis, interleukin 2, interferon γ) indicating the development of the disease in cancer patients, and in patients post polyp excision, was observed [ 192 ]. It was concluded that the application of the studied synbiotic may reduce the risk of colorectal carcinoma. A lower level of DNA damage was also observed, as well as a lower colonocyte proliferation ratio [ 147 ]. Table 6 lists the results of studies focusing on the effect of synbiotics on human health. There are examples of clinical trials during which the synbiotics group received the synbiotic prophylactically or in addition to the standard therapy.

Examples of clinical trials regarding the effect of synbiotics on human health.

Abbreviations: BMI—body mass index; CFU—colony-forming-unit; CRP—C-reactive protein; FOS—fructo-oligossacharides; IBS-QoL—quality of life with IBS; HDL-C—high-density lipoprotein cholesterol; HOMA-IR—homeostasis model assessment of insulin resistance; IHTG—intrahepatic triacylglycerol; IRS—insulin resistance syndrome; LDL-C—low-density lipoprotein cholesterol; LI—lactose intolerance; NAFLD—non-alcoholic fatty liver disease; NF-κB—nuclear factor κB; T2D—type 2 diabetes; TAG—triacylglycerols; TC—total cholesterol; TGL—total glutathione levels; TNF- α —tumour necrosis factor α; UC—ulcerative colitis.

Probiotic organisms are crucial for the maintenance of balance of human intestinal microbiota. Numerous scientific reports confirm their positive effect in the host’s health. Probiotic microorganisms are attributed a high therapeutic potential in, e.g., obesity, insulin resistance syndrome, type 2 diabetes, and non-alcohol hepatic steatosis [ 207 ]. It seems also that probiotics may be helpful in the treatment of irritable bowel syndrome, enteritis, bacterial infections, and various gastrointestinal disorders and diarrhoeas. Probiotic microorganisms are also effective in the alleviation of lactose intolerance and the treatment of atopic dermatitis. A positive effect of probiotics in the course of various neoplastic diseases and side effects associated with anti-cancer therapies is also worth noting. Prebiotics may be used as an alternative to probiotics, or as an additional support for them. It turns out that the development of bio-therapeutic formulas containing both appropriate microbial strains and synergistic prebiotics may lead to the enhancement of the probiotic effect in the small intestine and the colon. Those “enhanced” probiotic products may be even more effective, and their protective and stimulatory effect superior to their components administered separately [ 208 ]. It seems that we will see further studies on combinations of probiotics and prebiotics, and further development of synbiotics. Future studies may explain the mechanisms of actions of those components, which may confer a beneficial effect on human health.

Acknowledgments

We would like to thank the National Centre for Research and Development for the financial support of publication of this paper within the project PBS3/A8/32/2015 realized within the framework of the Program of Applied Studies.

Conflicts of Interest

The authors declare no conflict of interest.