Whitepaper-on-Prebiotics-and-Immunity

1. White Paper | July 2023 Prebiotics and Immunity Special focus on Fructo-Oligosaccharides

2. Contents: 2 3 Understanding Immunity • Types of Immunity 6 Immune System Dysfunction: Role of Nutrition 7 Immune System and Supporting Nutrients 8 Immune Function & Gut Microbiota 9 9 Gut Microbiome Modulators • Role of Gut Microbiota • Immuno-modulation by Increase in Beneficial Gut Microbiota • Poor Gut Health Linked to Infections 10 11 13 Available Evidences 15 Conclusions

3. Understanding Immunity Living a healthy life throughout ones lifespan is everybody’s desire. And in today’s evolving world, with exponential improvements in safe drinking water facilities, sanitization, vaccinations, affordability to food, etc. people are living longer. On the other hand, with growing populations, there are also increased disease risk conditions across life stages. And protecting oneself becomes important. Mature and Functional immune system Adult (12-50 yrs) Decline in immune system; increased risk of infections Immune system evolves and matures Immature immune system; Develops rapidly in response to external stimuli Kids (0-12 yrs) Adult (>50 yrs) Geriatric (>65 yrs) The function of all components of innate immunity is weak at birth compared to later in life. The immune system is relatively immature at birth and evolves during a lifetime of exposure to multiple foreign challenges through childhood, via young and mature adulthood (including pregnancy), to the decline of old age. The development and decline of immunity in an individual is dependent on many factors such as age, dietary habits, environmental exposures and physiological conditions. Optimal Nutrition with key nutrients is important to enhance the ability of the human body to play a defensive role in combating infections. In this document we are going to understand the role of nutrition to build immunity. The cells of the immune system produced by the human body are antibodies, B-lymphocytes, T-lymphocytes, and Antigen- Presenting Cells (APCs). Their brief functions1 are mentioned below White Blood Cells Functions Neutrophils Lymphocytes Monocytes Early responders, phagocytosis and local killing Adaptive immunity, subdivided in T-cells and B-cells Early responder, phagocytosis and antigen presentation, Mature as Macrophages in the tissues Bind IgE, defence against parasites, allergy Basophils and Eosinophils Table 1: Cells of the immune system Page 2

4. Types of Immunity InnateImmunity AdaptiveImmunity Takes longer to take effect and has a ‘memory’ which allows mounting a much stronger response when encountering the pathogen the second time First line of defense, infectious agents are ‘eaten up’ by scavenger cells Innate Immunity (fast and general effectiveness) It is the first-line defense from pathogens entering our bodies, achieved through protective barriers. We have immunity that is innate, i.e. from within the system, a response that our body provides to infections, if any, naturally. Various cells are involved in the immune system. These cells develop and mature during foetal life, but at different times. When infection enters the body, the phagocytes (also known as scavenger cells; special kind of white blood cells, leukocytes) enclose them, ‘digest’ them, thus neutralizing them. Proteins (enzymes) are also involved in the innate immune response. These enzymes help in marking germs as targets for scavenger cells, attracting other immune system cells from the bloodstream, destroying bacteria cell walls to kill them, and fighting viruses by destroying the microorganism envelope (the outermost layer of a virus) or cells that have been infected with microorganism. And lastly, the Natural Killer cells (NK cells) identify infected cells and then destroy their cell surface using cell toxins.2 Page 3

5. Adaptive Immunity It is a system that learns to recognize a pathogen. This system takes over if the innate immune system is not able to destroy the germs. It specifically targets the type of microorganism that is causing the infection. But to do that it first needs to identify the germ. This means that it is slower to respond than the innate immune system, but when it does it is more accurate. It also has the advantage of being able to ‘remember’ germs, so the next time a known germ is encountered, the adaptive immune system can respond faster. It may take a few days for the adaptive immune system to respond the first time it comes into contact with the microorganism, but the next time the body can react immediately. The second infection is then usually not even noticed, or is at least milder. The adaptive immune system is made up of T-lymphocytes (T-cells), B-lymphocytes (B-cells), and the antibodies. T-cells activate other immune system cells in order to start the adaptive immune system (T helper cells). They detect the infected cells and destroy them Infection Labels infections for killing Blocks new infections Kills infection Antigen Presenting Cells Helps B-Cells make antibodies T-Cells Figure 1: T-Cells and B-Cells attenuating infection Page 4

6. The doctor of the future will no longer treat the human frame with drugs, but rather will cure and prevent disease with NUTRITION. - Thomas Edison

7. Immune System Dysfunction: Role of Nutrition Adequate and appropriate nutrition is important for the optimal function of all the cells in the human body. Optimal nutrition, comprising of a balanced diet inclusive of all nutrients (also dietary fiber) further supports appropriate functioning of the immune cells. Although there is no single immune marker that accurately reflects/predicts an individual’s resistance to infection yet some immune markers to measure immuno-modulation by dietary intervention in human subjects were identified by International Life Sciences Institute (ILSI) Europe activity).3 Optimal Nutrition is important as it initiates effective response against pathogens, it resolves the response rapidly and avoids underlying chronic inflammation, if any. Poor nutrition (food shortages or malnutrition) has been shown to result in increased infections, slow healing from injury and infections, and complications due to immune system dysfunction.4 Thus optimal nutrition becomes critical. Page 6

8. Immune System and Supporting Nutrients Some micronutrients (Minerals and Vitamins) have established and specific roles in the development and maintenance of an effective immune system. For example, minerals like Copper, Selenium Zinc, and Iron contribute to the normal function of the immune system. Similarly, vitamins such as Folate, Vitamin A, Vitamin B6, Vitamin B12, Vitamin C, and Vitamin D are necessary for the normal functioning of the immune system. The table below shares an overview of nutrients that play a key role in the immune system. Some phytonutrients such as garlic, ginger, black pepper, cinnamon, fennel, turmeric, clove, cardamom, holy basil, asafoetida etc. have also been shown to positively support the immune system. Nutrient Support in Immune Function Scientific Evidence Folate Necessary for nucleotide synthesis, immune cell proliferation, and responsiveness EFSA, 20095 B9 Vitamin Contributes to a normal function of the immune system Stimulates the proliferation of T-lymphoid cells that fight pathogens EFSA, 20096 A EFSA, 20097 Contributes to normal function of the immune system Contributes to normal red blood cell formation Vitamin B6 Involved in nucleic acid and protein biosynthesis Important for NK cell activity Important for synthesis of specific immunoglobulins EFSA, 20098 Vitamin B12 Important for innate and adaptive immune responses Contributes to a normal function of the immune system Contributes to the protection of cell constituents from oxidative damage EFSA, 20099 Vitamin C EFSA, 201010, EFSA, 201511 Supports overall health and wellbeing and bone health Maintains serum calcium and phosphorus concentration Vitamin D contributes to the normal function of the immune system by suppressing inflammatory response Vitamin D Contributes to normal function of the immune system Important for copper related enzymes that needed for energy production of immune cells; protection of immune cells against reactive oxygen species EFSA, 200912 Copper Cu EFSA, 201013 Contributes to normal function of the immune system Regulates immune response Selenium Se EFSA, 200914 Contributes to a normal function of the immune system Supports B-cell and T-cell development Necessary for NK cells’ function Zinc Zn Contributes to normal functioning of the immune system Required for the regeneration of new CD4+ T lymphocytes and maintenance of T cell cytolytic processes EFSA, 200915 Iron Fe Are these nutrients enough to maintain or build the immune system, or do we need something more, particularly when we are aware that our physiological state also plays a crucial role in building/maintaining a good immune system Page 7

9. Immune Function & Gut Microbiota The intestinal microflora plays a significant role in the overall functioning of the immune system.We know that almost 70 - 80% of immune cells are found within the gut-associated lymphoid tissue (GALT).16 This emphasises the importance of gut tissues. Within the gut lumen itself, the human gut microbiome (the 100 trillion gut bacteria17) will provide antigens and signals with the potential to interact with resident and systemic immune cells. The gut is a major entrance for pathogens, toxins, and allergens and one of the major roles of the immune system in the gut is to distinguish between harmless antigens, such as food, and health hazards. The development of a healthy immune system, therefore, depends on a healthy gut microbiota, which is directly linked to nutrition. The composition of the gut microbiome changes over the course of life, in response to dietary components and environmental factors such as antibiotic exposure. Dietary interventions targeted at the gut microbiome include probiotics and prebiotics. Prebiotics can either directly affect the number and composition of the intestinal microflora, or their metabolites, generated after fermentation, may induce additional influence on the gut-associated lymphoid tissue. Page 8

10. Gut Microbiome Modulators The major gut microbiome modulators are probiotics and prebiotics. Live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.18 Major probiotics include Lactobacillus and Bifidobacteria strains, among many others. Prebiotics are the substrate selectively utilised by host microorganisms conferring a health benefit.19These are resistant to digestion in the human body and reach the colon intact where they are metabolised completely by the colonic microflora, through fermentation, producing metabolites i.e. Short chain fatty acids (SCFA; butyrate, acetate, propionate), and gases (CO2, H2, methane). Thus enhancing the abundance of the bacteria that are correlated to various health benefits such as digestive health, metabolic health, bone health, among others. SCFA plays an important role in regulating the integrity of the epithelial barrier through coordinated regulation of tight junction proteins. These regulate the intracellular molecular highway between the lumen and hepatic portal system, and thus help in managing the permeability. This helps in limiting the entrance of the pathogens in our systems, supporting a healthy immune cell system. Role of Gut Microbiota Intake of prebiotic fibers has been shown to increase beneficial bacteria in the gut, mostly Bifidobacteria and Lactobacillus while a decrease in pathogenic bacteria, a selective stimulation of growth of probiotics in the gut, more called as the prebiotic effect. Production of short chain fatty acids (SCFAs) such as acetic acid, propionic acid, butyric acid and the immediate effect of accumulation of these acids in the gut is reduction in luminal pH, which inhibits the growth of many pathogenic bacteria.20 Additionally, the pathogens’ growth is inhibited through the crowding out effect i.e. the dominant growth of the beneficial bacteria preventing the binding of pathogens to the intestinal wall.21 Page 9

11. Immuno-modulation by increase in beneficial Gut Microbiota Bifidobacteria Lactobacillus Increased numbers of a particular microbial genus/species (such as Bifidobacteria and Lactobacillus), may change the collective immuno- interactive profile of the microbiota. Through pattern-recognition receptors, such as the toll-like receptors (TLR), both immune cells and enterocytes interact with the so-called pathogen associated such as lipopolysaccharides (LPS, a membrane component of Gram-negative bacteria), lipoteichoic acids and un-methylated C-phosphate-G (CpG) DNA that are present on all the microorganisms surface regardless of pathogenicity. These interactions result in a variety of downstream events eventually leading to cytokine production steering towards an appropriate immune response for the microbial event.22,23,24 molecular patterns, The short chain fatty acids serve as ligands (a molecule that coordinate bonding) for G-protein coupled receptors (GPCRs) that activate anti-inflammatory signalling cascades that control Immune functions. Such binding results in cytokines and chemokines production that helps in combating with pathogens. As an example, butyrate supports the immune response in the following ways.25 Inhibiting histone acetylation, affecting pro-inflammatory response (e.g. TNF-a, IL-6, IL-12, iNOS expression); Increases anti-inflammatory mediators e.g IL-10 and contributes to intestinal homeostasis; thus decrease inflammation Oxidised to Acetyle CoA main energy source entering the TCA cycle Promotes the epithelial barrier function Antimicrobial peptide production Increasing cell proliferation Page 10

12. Poor Gut Health linked to Infections Poor gut health has been widely linked with various infection and disease conditions. Studies have shown that the gut microbiota of diseased individuals had a significant reduction in bacterial diversity in gut microbiota, as compared to the controls. Among the patients, there was an enrichment of opportunistic pathogens and depletion in the abundance of beneficial bacteria (Ruminococcaceae and Lachnospiraceae families) was observed, indicating as a typical sign of dysbiosis and an unhealthy gut.26 Another study27 reported a severity of COVID-19 disease was inversely associated with the abundance of beneficial bacteria as Faecalibacterium P. Strong correlation between COVID-19 risk and an altered gut microbiota have been reported.28 Thus it becomes important to support a healthy gut which may be achieved by a healthy lifestyle, including nutrition. Intake of prebiotic fibers is a way of improved gut health. Page 11

13. A healthy gut keeps many opportunistic infections away

14. Available Evidences Other investigators have also reported concepts of fructo-oligosaccharides and immune markers. Many clinical studies have assessed the impact of Fructo-oligosaccharides supplementation on immunity. FOS consumption increases immune response: A trial wherein full term formula-fed infants (0-7 days old; n=61), were recruited for a study infant formula with or without fructo-oligosaccharides was provided. Secretory IgA, a marker for diet-induced changes in immune function, was assessed in the fecal samples. Anti-poliomyelitis vaccine was chosen to assess the effects of formula on anti-polio virus-specific immune response. Results showed that fecal levels of anti polio-virus specific IgA were higher in fructo-oligosaccharides fortified formula-fed infants as compared to the controls (fructo-oligosaccharides group: 7,852±12,107 vs. control: 2,334±3,896 ng in humid feces) at 4 months of infant age. Additionally, there was a significant increase in the Bifidobacteria count in the fructo-oligosaccharides formula fed infants.29 Possibly, changes in the colonic microflora during the first months of life influence the development and expression of the gut mucosal immune system, both in the short and long term.30 FOS intake increases secretory IgA: Animal studies have shown that fructo-oligosaccharides increase the expression of polymeric Ig receptors at the ileum and colonic level. An increase at the colonic level plays a role in the transport of intestinal IgA onto the mucosal surface and thus contributes to increased levels of sIgA in digestive contents during fructo-oligosaccharides intake.31 FOS supplementation reduces the inflammatory process in the elderly: In a study, 19 elderly nursing home patients were given 8 g fructo-oligosaccharides for 3 weeks (4 g*2/d) after a washout period of 3 weeks. As expected, there was an increase in the fecal Bifidobacteria composition after fructo-oligosaccharides supplementation. Further, there was a decreased phagocytic activity of granulocytes and monocytes, as well as a decreased expression of interleukin-6 (IL-6) mRNA in peripheral blood monocytes. These results suggested a possible decrease in inflammatory fructo-oligosaccharides supplementation.32 process in elderly subjects after Page 13

15. FOS consumption by pregnant and lactating women increases the expression of the immune-regulatory cytokine IL-27 in breast milk: Healthy pregnant women were supplemented with either fructo-oligosaccharides (4 g; n=41) or the placebo (sucrose) (4g; n=43) twice daily from 26 weeks of gestation till 1 month after delivery. Samples of colostrum after childbirth and breast milk (at 1 month after delivery) were collected from the enrolled women along with maternal blood samples after childbirth. Results revealed that concentrations of IL-27 in colostrum samples were significantly higher in the fructo-oligosaccharides group (n=35; range: <0.156–46.6 ng/ml; median: 2.4 ng/ml) than in the placebo group (n=29; range:<0.156–95.8 ng/ml; median:0.2 ng/ml) (p=0.027). Breast milk samples in the fructo-oligosaccharides group also had significantly higher concentrations of IL-27 (p=0.04). Serum concentrations of IL-27 tended to be higher in the fructo-oligosaccharides group than in the placebo group, however not significantly different (p=0.084). The study demonstrated that the consumption of fructo-oligosaccharides by pregnant and lactating women increases the expression of various molecules in cells present in breast milk, with enhanced expression of the immune-regulatory cytokine IL-27.33 In the immune system of the digestive tract, IL-27 is responsible for maintaining epithelial barrier function and is involved in anti-inflammatory and antibacterial activities.34 Page 14

16. Conclusions Good nutrition is fundamental to improving the immune system which is the body’s defence against disease and infection caused by bacteria, viruses and toxins.35 Adequate and appropriate nutrition is required for all cells to function optimally, including cells in the immune system. A diet high in nutritious foods rich in vitamins and minerals support optimal immune function by providing antioxidants to slow damage of cells caused by free radicals36 or assisting in T-cell production37 which are responsible for eliminating infected cells and activating other immune cells, producing cytokines and regulating immune response. Almost 70% of our immune cells live in the gastrointestinal tract (GIT) along with the 100 trillion gut bacteria that make up the gut microbiota.38 The development of a healthy immune system therefore depends on a healthy gut microbiota, which is directly linked to nutrition. Dietary interventions targeted at the gut microbiome such as prebiotic fibers support a healthy gut microbiome. The metabolites produced due to the metabolism of prebiotic fibers have shown to stimulate immune cell activity. Prebiotic / probiotic / synbiotic interventions have shown to improve immune markers in infants39, reduction in risk of infections among young children40, and decrease in inflammatory processes among elderly.41 Page 15

17. References 1.Nicholson LB. The immune system. Essays Biochem. 2016 Oct 31;60(3):275-301. doi: 10.1042/EBC20160017. PMID: 27784777; PMCID: PMC5091071. 2.InformedHealth.org [Internet]. Cologne, Germany: Institute for Quality and Efficiency in Health Care (IQWiG); 2006-. The innate and adaptive immune systems. [Updated 2020 Jul 30]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK279396/ 3.Roberfroid M, et al. Prebiotic effects: metabolic and health benefits. Br J Nutr. 2010 Aug;104 Suppl 2:S1-63. doi: 10.1017/S0007114510003363. PMID: 20920376. 4.Childs CE, et al. Diet and Immune Function. Nutrients. 2019; 11(8):1933. https://doi.org/10.3390/nu11081933 5.EFSA Journal 2009; 7(9):1213. [22 pp.]. 6.EFSA Journal 2009; 7(9):1221. [25 pp.]. doi:10.2903/j.efsa.2009.1221. 7.EFSA Journal 2009; 7(9):1225 [20 pp.]. doi:10.2903/j.efsa.2009.1225. 8.EFSA Journal 2009; 7(9): 1223. [16 pp.]. doi:10.2903/j.efsa.2009.1223. 9.EFSA Journal 2009; 7(9):1226. [28 pp.]. doi:10.2903/j.efsa.2009.1226. 10.EFSA Journal 2010; 8(2):1468. [17 pp.]. doi:10.2903/j.efsa.2010.1468. 11.EFSA Journal 2015;13(5):4096, 7 pp. doi:10.2903/j.efsa.2015.4096 12.EFSA Journal 2009; 7(9):1211. [21 pp.]. doi:10.2903/j.efsa.2009.1211. 13.EFSA Journal 2010;8(10):1727. [18 pp.]. doi:10.2903/j.efsa.2010.1727. 14.EFSA Journal 2009; 7(9):1229. [34 pp.]. doi:10.2903/j.efsa.2009.1229 15.EFSA Journal 2009; 7(9):1215. [20 pp.]. doi:10.2903/j.efsa.2009.1215. 16.Childs CE., et al. Diet and Immune Function. Nutrients. 2019; 11(8):1933. https://doi.org/10.3390/nu11081933 17.West CE, et al. The gut microbiota and inflammatory noncommunicable diseases: Associations and potentials for gut microbiota therapies. J Allergy Clin Immunol, 2015. 135:3-13. doi: 10.1016/j.jaci.2014.11.012 18.Hill C., et al. Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol. 2014 Aug;11(8):506-14. doi: 10.1038/nrgastro.2014.66. Epub 2014 Jun 10. PMID: 24912386. 19.Gibson GR, et al. Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics. Nat Rev Gastroenterol Hepatol. 2017 Aug;14(8):491-502. doi: 10.1038/nrgastro.2017.75. Epub 2017 Jun 14. PMID: 28611480. 20.Ríos-Covian, D et al. Intestinal short chain fatty acids and their link with diet and human health. Front. Microbiol, 2016; 7, 185 21.Roberfroid, MB. Prebiotics and probiotics: are they functional foods? Am. J. Clin. Nutr. 2000; 71, 1682S–1687S, 65 22.Medzhitov R. Recognition of microorganisms and activation of the immune response. Nature. 2007 Oct 18;449(7164):819-26. doi: 10.1038/nature06246. PMID: 17943118. 23.Rakoff-Nahoum S, et al. Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell. 2004 Jul 23;118(2):229-41. doi: 10.1016/j.cell.2004.07.002. PMID: 15260992. 24.Vance RE, et al. Isberg RR, Portnoy DA. Patterns of pathogenesis: discrimination of pathogenic and nonpathogenic microbes by the innate immune system. Cell Host Microbe. 2009 Jul 23;6(1):10-21. doi: 10.1016/j.chom.2009.06.007. PMID: 19616762; PMCID: PMC2777727. 25.Parada Venegas D, et al. Short Chain Fatty Acids (SCFAs)-Mediated Gut Epithelial and Immune Regulation and Its Relevance for Inflammatory Bowel Diseases. Front Immunol. 2019 Mar 11;10:277. doi: 10.3389/fimmu.2019.00277.

18. 26.Gu S, et al. Alterations of the Gut Microbiota in Patients With Coronavirus Disease 2019 or H1N1 Influenza. Clin Infect Dis. 2020 Dec 17;71(10):2669-2678. doi: 10.1093/cid/ciaa709. PMID: 32497191; PMCID: PMC7314193. 27.Zuo T et al. Alterations in Gut Microbiota of Patients With COVID-19 During Time of Hospitalization. Gastroenterology. 2020 Sep;159(3):944-955.e8. doi: 10.1053/j.gastro.2020.05.048. Epub 2020 May 20. PMID: 32442562; PMCID: PMC7237927. 28.Gou, W. et al. (2020). Gut microbiota may underlie the predisposition of healthy individuals to COVID-19. medRxiv.doi: https://doi.org/10.1101/2020.04.22.20076091. https://www.medrxiv.org/content/10.1101/2020. 04.22.20076091v1. 29.Paineau D et al. Effects of short-chain fructooligosaccharides on faecal bifidobacteria and specific immune response in formula-fed term infants: a randomized, double-blind, placebo-controlled trial. J Nutr Sci Vitaminol, 2014; 60:167-75. doi: 10.3177/jnsv.60.167 30.Cebra JJ. Influences of microbiota on intestinal immune system development. Am J Clin Nutr, 1999; 69:1046s-51s. doi: 10.1093/ajcn/69.5.1046s 31.Nakamura Y, et al. Dietary fructooligosaccharides up-regulate immunoglobulin A response and polymeric immunoglobulin receptor expression in intestines of infant mice. Clin Exp Immunol, 2004; 137(1):52-8. doi: 10.1111/j.1365-2249.2004.02487.x 32.Guigoz Y, et al. Effects of oligosaccharide on the faecal flora and non-specific immune system in elderly people. Nutr Res, 2002; 22:13-25. doi: 10.1016/S0271-5317(01)00354-2 33.Kubota T, et.al. Prebiotic consumption in pregnant and lactating women increases IL-27 expression in human milk. Br J Nutr, 2014. 111:625-32. doi: 10.1017/S0007114513003036 34.Diegelmann J, et al. A novel role for interleukin-27 (IL-27) as mediator of intestinal epithelial barrier protection mediated via differential signal transducer and activator of transcription (STAT) protein signaling and induction of antibacterial and anti-inflammatory proteins. J Biol Chem, 2012; 287:286-98. doi: 10.1074/jbc.M111.294355 35.Gombart AF, Pierre A, Maggini S. A Review of Micronutrients and the Immune System-Working in Harmony to Reduce the Risk of Infection. Nutrients. 2020;12(1):236. Published 2020 Jan 16. doi:10.3390/nu12010236. 36.Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: Impact on human health. Pharmacogn Rev. 2010;4(8):118–126 37.Cohen S, et al. Danzaki K, MacIver NJ. Nutritional effects on T-cell immunometabolism. Eur J Immunol. 2017;47(2):225–235. 38.West CE, et al. The gut microbiota and inflammatory noncommunicable diseases: Associations and potentials for gut microbiota therapies. J Allergy Clin Immunol, 2015 Jan;135(1):3-13. 39.Nakamura et al. Dietary fructooligosaccharides up-regulate immunoglobulin A response and polymeric immunoglobulin receptor expression in intestines of infant mice. Clinical & Experimental Immunology. 2004; 137(1):52-8. 40.Chatchatee et al. Effects of growing-up milk supplemented with prebiotics and LCPUFAs on infections in young children. Journal of pediatric gastroenterology and nutrition. 2014; 58(4):428. 41.Guigoz et al. Effects of oligosaccharide on the faecal flora and non-specific immune system in elderly people. Nutrition Research. 2002 Feb 28;22(1):13-25.