Probiotics as Functional Foods in Enhancing Gut Immunity
Probiotics as microbes when administered in sufficient amounts as functional food impose beneficial effects on gut microbiota and thus enhance health of host. The indigenous microflora of gastrointestinal tract acts as an anatomic barrier against antigens present in food, invading microorganisms which regulates the immunophysiologic mechanism. Many factors may lower the resistance of the body which may lead to inflammatory, infectious, neoplastic, and degenerative conditions. There are other means of treatment like using antibiotics, irradiation, and immunosuppressive therapy which may change normal composition of gut flora. A variety of functional properties of probiotics bound their consideration as conventional, medicinal foods, and dietary supplements.
The most commonly used probiotics are of two genera, Lactobacillus and Bifidobacterium. Healthy microflora is the chief basis of probiotic therapy in literature. Probiotic bacteria demonstrate various immunomodulatory effects and therefore may be treated as novel tool to reduce inflammation in the intestine and dysfunction of gut mucosa, including acute gastroenteritis, inflammatory bowel disease, and food allergy, and downregulate hypersensitivity reactions. A large number of probiotic effects are explained by regulating immunity, especially the balance between anti-inflammatory and proinflammatory cytokines. Probiotics stabilize microbial environment of the gut and the intestinal permeability barrier. This leads to enhanced mucosal IgA responses which promote further the immunological barrier and responses of gut mucosa. In addition, providing immunomodulatory effect on gut mucosa, probiotic therapy is now also being used to cure infections in other organs such as respiratory tract, urogenital tract, and others. This chapter focuses on roles of probiotics as functional foods.
KeywordsProbiotics Functional food Immunomodulation Gastrointestinal tract Respiratory tract Urogenital tract Oral infection
In recent years, the beneficiary health reasons of functional food have been felt worldwide. Probiotic is preferred as a new and healthy component in functional food market because of its functional properties and consumer’s preference. Today, probiotics as functional food ingredients are being consumed by humans as fermented milks, yogurts, baby foods, energy drinks, confectionery, and chewing gum [1, 2]. Several efforts have been attempted to affect the intestinal microbiota by functional food which is beneficiary for host’s health.
The human body is a macrocosm of microorganisms that reside at different body sites. These sites provide an environment where specific microorganisms are more favored than others. These resident microorganisms participate in commensal, mutualistic, and parasitic relationships with the host . The diverse group of commensal (nonpathogenic) bacteria may be differentiated into normal flora (native inhabitants) and transient flora. Native microorganisms colonize specific sites in the human body. Transient flora colonizes the body from the external environment and can persist until some sites are filled with native flora . The normal flora may prevent the colonization of pathogenic microorganisms and thus gives health benefits to host. This phenomenon is called microbial antagonism (Fig. 4.1). When the balance between the normal microbiota and pathogenic microbes is disturbed, it may lead to diseases .These microorganisms colonize mainly in the mucosal surfaces of gastrointestinal (GI) tract, the upper respiratory tract, and the urogenital tract, on the skin and oral cavity [9, 10, 11, 12, 13]. The present chapter enlightens the relationship between probiotics and immunity at the gastrointestinal tract because probiotics primarily affect the gut and improve its morphology and functions. However, probiotics also aid in improving immune components at other mentioned mucosal surfaces. The next section of the chapter focuses the same.
4.2 Microflora of Gastrointestinal Tract and Other Organs
4.2.1 The Gastrointestinal Tract
220.127.116.11 Role of Gut Mucosa and Gut-Associated Lymphoid Tissue in Host Immune System
The main role of the GI tract is to digest and absorb the nutrients to fulfill the metabolic and physiological needs for normal growth of human beings. GI tract is constantly exposed to large number of microorganisms. GI tract provides anatomical and physiological barriers against pathogens such as mucus, saliva, stomach acid, digestive enzymes, and intestinal flora . In the tract (GI), many antigens from enteric route are present in the small intestine. Moreover, antigen load composition in the small intestine constantly and rapidly changes. Mucus secreted by goblet cell of the GI tract acts as anatomical or physical barrier and is the first line of defense. It excludes most of the antigens in nonspecific manner . Besides the gut defense, the villous epithelium has a special antigen transport mechanisms. Antigens are moved by transcytosis across the epithelial layer. Lysosomal processing of the antigen occurs in the main degradative pathway. As second line of defense, immune system removes antigens which penetrate the intestinal mucosa and secrete immune products to combat these antigens from microorganisms in the intestinal lumen .
In a newborn baby, there is a rapid change in function of gut barrier which occurs at time of birth. This happens when the gut received digesting milk in place of amniotic fluid during early days. Milk consumption starts the releasing of trophic hormones and its motility and absorption. During this postnatal period, the mucosal proteins are formed, digestive enzymes are released, and the presence of intestinal flora  strengthened the gut defense. Gastric acidity as first line of defense also starts secreting during the first month after birth .
In healthy, adult, and normal human body, the presence of good bacterial populations is able to control overgrowth of opportunistic bacteria in GI tract . Maturation of small intestinal brush border further influences the epithelial cell membranes which is considered as major interface between the intracellular environment and the luminal contents. The ability of antigens to attach with the epithelial cells is dependent on rate and route of antigen transfer. This ability indicates the potency of mucosal immune responses. The intestinal mucosal layer is treated as major immune part of the body that contains various immunocompetent cells .
Local specific or adaptive immune system, known as gut-associated lymphoid tissue (GALT), protects the surface of mucosal membrane. GALT is the largest secondary or peripheral lymphoid tissue of the human body. It is divided into the lamina propria, which lies just below the epithelial layer and the organized lymphoid tissues, including Peyer’s patches and mesenteric lymph nodes (MLNs) where lymphocytes are scattered throughout the epithelium. The outer mucosal epithelial layer contains intraepithelial lymphocytes (IELs). Many of these lymphocytes are T cells bearing gamma-delta T-cell receptors (γ∂ TCRs) which interact with epithelial cells and protect the mucosa by killing infected cells and invite other immune cells to combat pathogens . Further, the IELs mainly exhibit a suppressor and cytotoxic phenotype (CD8+ T cells), whereas the lamina propria cells exhibit a helper and inducer phenotype (CD4+ T cells). The lamina propria is enriched with lymphocytes belonging to the B-cell lineage, TH cells, and macrophages present in loose clusters or lymphoid follicles [14, 17, 18]. Moreover, lymphocyte activation involves intestinal antigen transport to dendritic cells (DCs) and macrophages in Peyer’s patches and present antigens to adjacent T helper and inducer lymphocytes. These cells distinguished into various effector cells which mediate immune suppression and also promote the differentiation of immunoglobulin A (IgA)-secreting B cells . Dietary antigens, such as food proteins and probiotics, are changed into a tolerogenic form when absorb across the intestinal mucosa. This immunologic unresponsiveness toward such antigens is called oral tolerance . The mechanisms of oral tolerance are not fully known. However, the major mechanisms are supposed to carry out through clonal anergy of antigen-specific T cells and B cells. MHC class I-restricted CD4+ T cells and cytokines (interleukin; IL-10 and transforming growth factor; TGF-β) may intervene this mechanism with hyporesponsive functions . In contrast, stimulation of antigen-specific CD8+ T cells that produce inhibitory cytokines helps in gaining the tolerance. In addition of immune tolerance, protective immune responses are also the part of GI tract. The production of secretory IgA (sIgA) is the local immunoglobulin response of the mucosa of the intestine which is induced by multiple cytokines including IL-4, IL-5, IL-6, IL-10, and TGF-β . These cytokines are also essential for maintaining tolerance against food antigen. Probiotics boost IgA antibody responses followed by intestinal immune exclusion and triggering subsequent elimination. Furthermore, probiotic bacteria downregulate hypersensitivity reactions to harmless antigens by modulating the immune responses. The modulation of immune response mechanisms for sIgA activation or tolerance by probiotics is highly dependent on the strains [23, 24]. T helper (TH) lymphocytes of lamina propria have potential of cytokine production from both subsets, TH1 (IL-12, IFN-γ) and TH2 (IL-10, IL-4, IL-5), that express different immune responses .
The certain B lymphocytes appear in the blood 2–4 days after antigenic exposure. It reaches the highest concentration after 6–8 days and persists in the blood for 2–3 weeks. Mucosal surfaces have IgA antibody production in abundance [14, 23]. However, upper part of the human gut-associated immune system (small intestine) secretes IgA1-producing cells predominantly, while IgA2-producing cells are present in lower-part gut-associated immune system (colon). IgA2-producing cells are more resistant to bacterial proteases .
18.104.22.168 Intestinal Microflora and Development of the Host Immune System
To maintain good health, it is desired to have a normal and functional GI tract. The GI tract has the second largest surface area after surface area of respiratory tract, which is rich in flora containing more than 1500 various species of bacteria [4, 26]. Microflora of the GI tract is critical in the development of the host’s anatomy, immunology, and physiology. It aggravates the immune system to react rapidly to infection caused by pathogens and through bacterial antagonism. It competitively inhibits the colonization of the gut by pathogenic bacteria and helping digestion [27, 28].
The gut flora develops after birth and remains almost stable for rest of the life which is for human homeostasis. The major challenge with the mucosal immune system is to differentiate between pathogens and non-pathogenic microorganisms to develop protective immunity and to maintain the integrity of the GI mucosa [5, 29].
The GI tract of a newborn is almost sterile. As infant exposes with environment, indigenous microflora colonizes the mucosal surfaces. This differs from the adult microflora . The microflora expands quickly after birth and is markedly dependent on mode of delivery, hygiene level, mode of feeding, the mother’s flora, genetic factors, and medication use. Infants born through vaginal delivery develop microbial colony in gut from the birth canal of the mother and the environment. These microorganisms include streptococci, coliforms, and gram-positive, nonspore-forming anaerobic bacilli . An intestinal tract of infant is quickly populated with enterobacteria during the first 2 days after birth. In breastfed infants, 60%–90% of the total flora is of bifidobacteria counts. The most common species in healthy breastfed infants are bifidobacterial species which includes Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium breve, and Bifidobacterium longum which predominate Lactobacillus and Bacteroides increase to a smaller degree. Enterobacteria reduce in these infants [29, 32].
However, large numbers of Lactobacillus, Clostridium, and Bacteroides and relatively less Bifidobacterium species are found in formula-fed infants. In both breastfed and formula-fed infants, the microflora becomes similar when food supplements are started. Bacteroides and anaerobic gram-positive cocci predominate in this state . Populations of Bacteroides and anaerobic cocci increase and sometimes exceed to Bifidobacterium population after the infant reaches 2 years of age, which is same as normal adult’s flora. The gram-negative anaerobic counts increase, while coliforms, clostridia, and streptococci reduced to the levels as found in healthy adults. Permanent colonizing of bacteria is attained in children by 4 years of age. Infection, illness, stress, climate, changes in diet, and medication may lead to changes in these levels and physiological progression process [26, 34].
Though the composition of the microflora varies among individuals, microfloral composition in individual remains steady over a long period. The antigenic stimuli provided by colonization of commensal bacteria in gut are crucial for the development of functionally matured and balanced immune system . This includes homing of B and T lymphocytes to the lamina propria, development of IgA plasmocytes, IgA secretion, and induction of tolerance toward food and microfloral antigens . The microflora constitutes a large number of diverse species; many of them are recognized as pathogens. Approximately 80% of all immunologically active cells in body are present in the GALT. This is because of the development of interaction between microbe and gut immune system. Thus, microbial flora is vital for the development of immunocompetent GALT in infants [8, 27]. Intestinal bacteria are the main cause of epithelium cell functions, signaling through toll-like receptors (TLRs), which aid in T-cell activation and differentiation and B-cell responses to T-cell-dependent antigens and thus regulate the gut immune response . sIgA is one of the most important components of B-cell response to gut lumen protein and pathogen antigens. Colonization also reduces the ratio of TH2 (proallergic) to TH1(suppressive) responses that could decrease the possibilities of immune hyper-reactivity, such as in allergic diseases [28, 35].
Resident bacteria, like lactobacilli and bifidobacteria, can activate antimicrobial activities by inhibiting colonization of potentially pathogenic microbes and thereby influencing both local and systemic immunities . They have also been associated with the release of substances which have antimicrobial properties and the release of mucins (considered as the intestinal physiological barrier). Mucins inhibit the attachment of pathogens to mucosal lining . Some bifidobacteria and lactobacilli are given orally to develop immunologic tolerance to antigens and can decrease allergic-type immune responses by enhancing the production of a balanced TH cell response and stimulating the production of IL-10 and TGF-β [32, 37]. Another major effect of gut bacteria is the boosting of secretion of sIgA at mucosal surface and thus helps in providing protection against antigens, virulence factors, and toxins . The development of IgA plasmocytes in the mucosa is most affected by the microflora. Breast milk contains sufficient amount of sIgA which is fed to the infant. Additionally, bifidobacteria stimulate the biosynthesis and secretion of sIgA .
Macrophage infections are effective inducers of IL-12 and IFN-γ production, whereas extracellular infections, for example, by intestinal parasites, are strong inducers of IL-4 and IL-10 production which effectively enhance cellular immune responses. The mucosal flora has the capacity to restrict or kill certain transient microbial pathogens in their habitat by competing for nutrition and releasing of suppressive factors like bacteriocins in a process called microbial interference whose mechanism needs more research . Bacteriocins are proteins produced by endogenous flora which is a suitable example of intraspecies antagonistic effects .
22.214.171.124 Colorectal Cancer
Colorectal cancer (CRC) is one of the most common causes of cancer mortality worldwide. The development of colorectal cancer is a multifactorial process influenced by physiological, environmental, and genetic factors and to a large extent by lifestyle including diet . Due to genetic mutations, thousands of abnormal cells are generated daily in our body. Our immune system clears these mutated cells to suppress the carcinogenesis. Gut flora may influence the defense against CRC by modulating immune components of the host [40, 41, 42]. Studies on fecal bacterial composition showed increased population of Bacteroides and Prevotella in CRC patients, while Dorea spp. and Faecalibacterium spp. in colonic microbiota in patients with colorectal adenoma. These species may generate carcinogens and tumor-promoting substances including heterocyclic amines and secondary bile acids [43, 44]. Other studies suggested the less diverse or altered bacterial community or high colonization of oral anaerobic bacteria Fusobacterium nucleatum in CRC . Other gut microbiota produces beneficial metabolites such as short-chain fatty acids and is equal to prevent cancer [46, 47]. Studies investigate that the supplementation with symbiotic composition of Lactobacillus rhamnosus GG, Bifidobacterium lactis Bb12, and oligofructose-enriched inulin for 12 weeks resulted in favorable changes in the gut microbiome with high levels of lactobacilli and bifidobacteria and low levels of Clostridium perfringens in colorectal cancer patients [48, 49]. Intake of synbiotics also helps in reducing proliferation and DNA damage in colonic mucosal cells and fecal water-induced necrosis in colonic cells of the patients . Several studies have demonstrated that Lactobacilli casei enhances the immunoresponsive activities of T cells, macrophages, natural killer cells, and T cells against cancer [51, 52, 53]. Probiotics have also been able to reduce side effects of radiotherapy and chemotherapy to treat CRC. In animal studies, L. rhamnosus GG is found to ameliorate intestinal damage from radiation in a TLR2- and COX2-dependent and MyD88-independent manner [54, 55].
126.96.36.199 Adhesion of Probiotics to Target Sites
A major selection criterion for probiotics is considered to be exclusion of pathogens competitively. Probiotics compete directly or obstruct the adhesion sites of pathogens present on gastrointestinal surface. They also affect the development of intestinal microbiota in infants. Adhesion on GI surface increases the retention time of a probiotic which is important because of the short residence time of intestinal material in it . Probiotics may have a high turnover rate on the mucosal surface because of continuous displacement . On the other hand, a probiotic also penetrates the mucosal layer to adhere with the epithelial cells. Once consumed, probiotics travel along the whole length of GI tract. An effective probiotic is that which must stay on desired target sites for sufficient time with sufficient concentration to obtain probiotic effects . The adhesion and temporary multiplication of probiotics at the target sites result in high counts of probiotics at site of action. This achieves the desirable response even at a lower dosage .
As probiotic supplements, lactobacilli and bifidobacteria primarily populated at the small and the large intestine . Both are capable to change immunologic responses related to allergic inflammation. Lactobacilli are investigated as ineffective against allergy generated by cow’s milk . Binding of probiotics preferentially on the specific antigen-processing cells including dendritic cells, macrophages, and epithelial cells may be proved as more vital than the site of adhesion . L. rhamnosus GG has been reported effective in treating diarrhea in infants caused by rotavirus which adhere and colonize to the small intestine [57, 60]. Likewise, Bifidobacterium lactis Bb12 is effective in preventing and treating acute diarrhea in infants as it has strong adherent property, and Lactobacillus bulgaricus adhere to and colonize the intestine and help in treating antibiotic-associated diarrhea [58, 61].
4.2.2 Respiratory Tract
The most common illness among humans is viral respiratory tract infections. Probiotics may have therapeutic efficacy for treating viral respiratory tract infections as they are known to improve the immune system [62, 63]. It has been shown that Bacillus coagulans significantly induced TNF-α production by T cells when healthy adult is exposed to adenovirus exposure and influenza A (H3N2 Texas strain); however, B. coagulans did not exert much effect on other influenza strains . Probiotic strains of Lactobacillus plantarum and Lactobacillus paracasei reduce the risk of acquiring common cold infections when orally administered .
Patients who received external ventilation are at risk to have ventilator-associated pneumonia (VAP). VAP has complex pathogenesis. This develops colonies of pathogenic bacteria in the aerodigestive tract which lead to formation of biofilms and microaspiration of contaminated secretions. Since VAP is associated with high risk of mortality and morbidity , therefore, new efficient VAP prevention strategies work on combating colonization and effective aspiration. It includes elevation of the head of patient’s bed to drain subglottic secretion. To reduce the risk of VAP, intensive oral care and duration of mechanical ventilation should be minimized . Probiotics help in reducing the occurrence of VAP through local and systemic immune defenses [68, 69]. These effects are reduction in colonization of potential pathogens, improvement in gut mucosal barrier function, reduction in bacterial translocation, and TLR-mediated enhancement of immune response. However, evidences for such prophylactic treatment through probiotics are limited but promising. Lactobacillus rhamnosus GG administration appears safe and effective in a patient who is at high risk for VAP . The therapy may be used to avoid ICU complications like Clostridium difficile and ICU-associated diarrhea .
4.2.3 Urogenital Tract
Urinary tract infection (UTI) is the most common health problem especially in women. Members of Enterobacteriaceae family including E. coli, normal inhabitants of human intestines, are found to be the most common causative agent of UTI in several countries . There is a close relation between reduction of the normal genital microflora, mainly Lactobacillus species and higher incidence of genital and bladder infections . Various bacterial species responsible for UTI are E. coli, Staphylococcus saprophyticus, Proteus, and Klebsiella. Viruses, fungi, and parasites can also cause UTI but rarely. Although antimicrobial drugs are generally effective in eradicating the infections, chances of recurrence is high . Modern concept of treating UTI is based on the use of Lactobacillus species which produces hydrogen peroxide, lactic acid, and bacteriocins and thereby maintains low pH of the genital area and retards growth of E. coli. Lactobacilli also activate TLR-2 which produces IL-10 and myeloid differentiation factor 88 .
The vaginal microbiota protects the area from invading pathogens that cause urinary tract infections, sexually transmitted diseases, and other diseases. Lactobacillus acidophilus group and L. fermentum are dominant in this habitat in healthy premenopausal women. L. plantarum, L. jensenii, L. brevis, L. casei, L. salivarius, and L. delbrueckii are also present . Lactobacilli are supposed to interfere with pathogens by different mechanisms. The primary mechanism is competitive elimination of genitourinary pathogens from the epithelial surface receptors, whereas coaggregation of lactobacilli with some uropathogenic bacteria would result in the growth retardation of the pathogen which is the second mechanism [13, 75]. The process of coaggregation is linked to the production of antimicrobial compounds, such as lactic acid, hydrogen peroxide, and bacteriocin-like substances. In this respect, previous studies showed that Candida albicans and Gardnerella vaginalis adhere to vaginal epithelial cell surfaces and produce pathology primarily at the vaginal level. L. acidophilus and these pathogens compete for the same binding sites on receptors present on vaginal epithelial cells and therefore prevent pathogenesis by these microorganisms [76, 77]. The affinity of L. acidophilus for these receptors is stronger than the pathogens. On the other hand, E. coli and Streptococcus agalactiae do not adhere to the vaginal epithelial cells and thus are just opportunistic pathogens . Except S. agalactiae, vaginal lactobacilli coaggregate with all of the pathogens. This suggests that coaggregation is specific because this process also hinders the access of pathogens to surface receptors present on vaginal epithelia which is another reason for the reduced adherence of C. albicans and G. vaginalis to vaginal epithelia in the presence of lactobacilli .
4.2.4 Oral Infection
In oral cavity, many resident bacteria include lactobacilli, streptococci, staphylococci, corynebacteria, and various anaerobes particularly bacteroides and spirochetes. The oral cavity of newborn infant rapidly becomes colonized with bacteria such as Streptococcus salivarius . After eruption of teeth during the first year, Streptococcus mutans, Streptococcus sanguinis, and Streptococcus gordonii are major species which colonize the tooth surfaces, coronal plaque, in gingival crevices (supporting structures of the teeth), and on buccal and pharyngeal mucosal surfaces since the childhood . They may also be found in abscesses and are etiological agents in bacterial endocarditis. During puberty bacteroides and spirochetes such as Treponema denticola colonize the oral cavity . At all sites of colonization or infection, adherence is a prerequisite for streptococcal proliferation. In vitro adherence assays have demonstrated that S. gordonii has diverse adherence abilities since these bacteria adhere to surfaces coated with salivary components, such as mucins, proline-rich proteins, agglutinin, and fibronectin and bind to amylase, immunoglobulin A, and serum agglutinin .
Many studies suggest that probiotic Lactobacillus reuteri Prodentis could be effective in the treatment of both gingivitis and plaque because of its anti-inflammatory and antimicrobial effects [81, 82]. Oral administration of L. reuteri Prodentis improves initial-to-moderate chronic periodontitis . L. reuteri also found high salivary Streptococcus mutans counts in young women .
4.3 Immunomodulation by Probiotics
Probiotic bacteria are shown to elevate the endogenous host defense mechanisms. In addition to stabilization of the gut microflora (nonimmunologic gut defense), probiotic bacteria enhance humoral immune responses and thus promote the immunologic barrier of the intestine .
Probiotics modulate non-specific or innate immunity in various ways including decreasing gut permeability, promoting production of mucin, competing with and inhibiting growth of opportunistic pathogens and increasing cytotoxic activity of natural killer cell, and activating macrophage and its phagocytic activity [36, 59]. Probiotics enhance specific or adaptive immune response by modulating inflammatory gut immune responses and raise IgA-, IgG-, and IgM-secreting plasma cells. Probiotics also increase total and specific sIgA in serum and gut lumen .
Oral supplements of lactobacilli can increase innate immunity of host against microbial pathogens and thereby facilitate the elimination of pathogens in the gut. A number of strains of live lactic acid bacteria (LAB) are found to stimulate the release of the proinflammatory cytokines such as TNF-α, IL-6, and IL-10 which reflect stimulation of` nonspecific immunity . Oral intake of Lactobacillus casei and L. bulgaricus activates the biosynthesis of macrophages, while phagocytic activity is stimulated by L. helveticus and L. acidophilus. Phagocytosis triggers the inflammatory response before antibody production by releasing lethal agents including reactive oxygen and nitrogen intermediates and degradative enzymes [37, 52, 86]. Probiotics are capable of modulating phagocytosis differently in healthy and allergic persons: in healthy people immunostimulatory effects have been detected, whereas in allergic persons, downregulation of inflammatory response was shown . In rotavirus diarrhea, proinflammatory mediator fecal urease activity gets increased that leads to ammonia-induced destruction of gut mucosa and thus promotes overgrowth of urease-producing bacteria . In patients with rheumatoid arthritis, bacterial composition is altered in gut as compared to healthy persons. This suggests that the intestinal microflora is responsible for inflammation beyond the gut . Therapeutic use of probiotics thus may help to stabilize the gut microflora and thereby prevent the generation of inflammatory mediators by GALT, which are triggered in response to exposure of intestinal lumen by potentially harmful antigens [26, 87]. Proinflammatory cytokines such as IL-6, TNF-α, and IFN- γ may play a key role in inflammation and also stimulate production of adaptive immune cells. Intake of lactobacilli in fermented milk products or as live-attenuated bacteria potentiates production of IFN- γ by peripheral blood mononuclear cells. IFN promotes the uptake of antigens in Peyer’s patches, where IgA-committed cells are generated in follicles [90, 91]. Oral administration of Lactobacillus rhamnosus GG (LGG) was shown to reduce high fecal concentrations of TNF-α in patients suffering from atopic dermatitis and cow milk allergy [35, 92]. In this way, probiotic bacteria may stabilize the immunologic barrier of the gut mucosa by reducing the generation of local proinflammatory molecule TNF-α and by reinforcing the systemic production of IFN. However, abnormal production of IFN interferes with the oral tolerance and disturbs gut epithelial cell integrity. Thus, immunomodulating effects shown by probiotics may depend on the immunological status of the host [20, 21].
It has been postulated that partial lactose digestion and stimulation of the intestinal lactase activity act as a mechanism against some types of diarrhea. In case of acute gastroenteritis or recurrent abdominal distress, disaccharidase activity in the small intestine produces osmotic diarrhea as transport of monosaccharides is affected . Lactobacilli have active β-galactosidase which is used to decrease the lactose concentration in dairy products that may improve osmotic diarrhea due to pathogenic organisms [88, 94]. Many metabolites produced by LAB act as antimicrobial substances, such as organic acids, hydrogen peroxide, ammonia, free fatty acids, and bacteriocins. These substances are used in dairy to extend the shelf life of food and to suppress food spoilage . L. casei strain GG (LGG) which has unique colonial morphology that makes it easy to identify in a mixed culture of other lactobacilli and Streptococci has the ability to produce a low-molecular-weight antibacterial substance that inhibits both gram-positive and gram-negative enteric bacteria in mice . Similarly, it has been observed that IgA-specific antibody-secreting cell counts are increased in most patients suffering with rotavirus diarrhea when they received viable LGG at the convalescent stage .
Another mechanism used by probiotics is to block toxin-mediated diseases by modifying toxin receptors and. For example, S. boulardii disintegrates Clostridium difficile toxin receptors in the human colon and blocks cholera-induced secretion in rat jejunum by the production of polyamines [97, 98]. It has been shown that LGG and L. plantarum competitively inhibit the adhesion site of enteropathogenic E. coli 0157H7 to human colonic cancer cell HT-29 . Similarly, S. boulardii also decreases attachment of Entamoeba histolytica trophozoites to erythrocytes in vitro . Different strains of LAB stimulate production of IFN-γ, IL-12, and IL-18 by human blood lymphocytes . Mucosal-associated lactobacilli (mainly L. paracasei) can translocate over the gut barrier and enable to influence gut mucosal immune by strongly stimulating IL-12 secretion which in turn stimulates cytotoxic activity of T cells and natural killer cells. However, IL-12 may also downregulate the TH-2 response, decreasing IL-4 and IgE production .
Whole bacterial cells are able to induce proinflammatory cytokines, such as TNF-α and IL-6 as well as accelerate proliferation of immune cells . Contrary to this, probiotic bacteria mediate suppress cytokine production and lymphocyte proliferation which indicates that probiotic bacteria possess considerable anti-inflammatory properties as good as therapeutic efficacy. For example, Feacalibacterium prausnitzii suppresses release of proinflammatory cytokines IL-12 and IL-17 and TH-17 cells which play role in inflammatory bowel disease [102, 103]. Oral administration of LGG has been shown to elevate serum IL-10 levels in atopic children. This implies that specific probiotics may exhibit anti-inflammatory effects which may be mediated through modification in intestinal microfloral niche [32, 57].
Bifidobacterium longum was shown to increase antibody response when orally incorporated in patients of allergen ovalbumin-induced lung inflammation and also stimulates IgA response to cholera toxin [104, 105]. Likewise, humoral immune response (rotavirus-specific IgA) was increased by LGG administration in children who were suffering with acute rotavirus diarrhea [106, 107]. Cow milk proteins cause type 1 hypersensitivity and lead to defective generation of local IgA responses in human infants . In addition to certain gastrointestinal microfloral species which release low-molecular-weight peptides, incorporation of lactobacilli and bifidobacteria in diet stimulates immune responses through several mechanisms. However, in healthy individuals, milk protein degradative enzymes are released from intestinal bacteria into tolerogenic peptides and thereby exert suppressive effects on the lymphocyte proliferation and downregulate cow’s milk allergy .
4.4 Sources of Probiotics as Functional Food
Various probiotic-based dairy and nondairy products
Cultured dairy/nondairy product
L. bulgaricus, L. acidophilus, S. thermophilus
L. casei, L. brevis, L. lactis, L. acidophilus, L. plantarum, S. cremoris, S. faecium
Fermented probiotic milk
L. casei, L. acidophilus, L. rhamnosus, L. johnsonii, B. lactis, B. bifidum, B. breve
P. acidilactici, P. pentosaceus, L. sakei, L. curvatus
Leuconostoc mesenteroides, Pediococcus cerevisiae, L. brevis, L. plantarum
Leuconostoc mesenteroids, L. plantarum, L. pentosus
P. acidilactici, L. plantarum
L. casei, L. acidophilus
L. acidophilus, L. plantarum, L. salivarius, B. lactis
L. delbrueckii, L. acidophilus
Selection of nontoxic and nonpathogenic organism 
Isolation from the same species as its targeted host 
Not carrying transferable antibiotic resistance genes and susceptible to antibiotics.
Ability to stabilize the normal intestinal microbiota 
Ability to produce antimicrobial substances to antagonize pathogens 
Potential to show beneficial effect on the host 
Durability to endure the complexity of commercial handling 
Abovementioned criterion is useful for selecting probiotic strains, especially at the time for preparation of function food processing and commercial packaging. It has been identified in researches that viability of probiotics during food processing on commercial scale is an important thought for health benefits which is mentioned in the next section of the chapter.
4.5 Viability of Probiotic in Probiotic Foods
Several reports show that many manufactured health products available in the market worldwide have poor viability of probiotic than mentioned and claimed on label of packaging. As the shelf life is less, it is important to make the product sustainable in market. Therefore, it becomes important to ensure higher viability of probiotic and their ability to show probiotic effect for their long-term existence as functional foods.
Study and market survey reveal the functional foods, and other health-care products had either undetectable level or very low microbial contents. Sometimes, the identified strains in the packages do not match in quantity as declared on the label. Many surveys reveal that a number of products for which companies claim to be “probiotic” often do not fulfill the standard criteria for viable count at end of shelf life. Seeing the situation, International Scientific Association for Probiotics and Prebiotics decided that the term “probiotic” should be referred only to the products which have live and well-defined strains of microorganisms with sufficient counts .
Probiotic-based milk products must maintain suitable population of viable microorganisms during whole shelf life to exhibit prophylactic properties . The biotherapeutic effect of probiotic is dose-dependent with a daily recommended dosage (106–109 cfu/ml)  to balance viability loss due to heat, pressure, and high acidity  and to sustain dosage up to 21 days/5 °C . Fecal lactobacilli and bifidobacteria are elevated by ingesting various cultured milk products which contain 106–109 cfu/g that declines coliforms and consecutively alleviates certain human diseases. Viability of LAB is influenced by many physiological factors including gastric acidity, bile salts, and digestive enzymes . It is observed that only 20–40% of the cultures survive in gastric transit in gut environment. High tolerance to low pH and bile salts is exhibited by L. plantarum G1, and L. casei G3 strains in the GI tract of rats suggest the use of these strains as functional food . Thus, it is significant to retain viability of probiotic strains in all phases, i.e., during processing, storage, and transit, through GIT to manifest clinical effects, but researches indicate that even in nonviable or heat-killed probiotic strains, components of dead probiotic cells from culture can confer anti-inflammatory responses [124, 125, 126, 127]. It is also noted that resting cells and dead cells of lactobacilli and Bifidobacterium strains can be able to remove cholesterol from a medium .
Amount of acids and other substances like hydrogen peroxide produced by cultures in yogurt.
Amount of dissolved oxygen contents in the product.
Amount of acids such as lactic acid and acetic acid in the product.
Quantity of fat in milk.
Heat treatment of milk during process.
Temperature of incubation.
Concentration of buffers, for instance, whey protein concentrate.
Physiological condition of probiotic cultures.
Oxygen permeability through the packaging of the final probiotic product.
Physical status of product storage.
4.6 Existing Regulations for Probiotics
Summary of standards adopted by major countries worldwide
Mode of intake
Definition key points
Intended for the prevention, alleviation, cure, diagnosis of disease 
Containing a virus, serum, toxin for prevention and treatment 
Dietary management of a disease, medical evaluation, supervision under physician 
Live organisms, such as bacteria; for prevention and treatment of a disease and not vaccine 
Functional food and drugs
FSSA, PFA, FDA
Health beneficial and able to regulate health body functions 
The live microorganisms, administered in sufficient amounts for health benefit 
Functional food and nutraceuticals
Beneficially affects one or more functions and consumed as improved state of health and reduction of risk of disease not as medicine 
New Zealand and Australia
Physiological roles beyond simple nutrient requirement 
Healthy food, physiological function, enhanced with added ingredients than normal food, beyond nutritional value 
The USA regulates probiotics as intended usage and dealing bodies such as the Dietary Supplement Health and Education Act (DSHEA) and Food and Drug Administration (FDA). In the USA, FDA enlists microorganisms and their secreting substances, used as probiotics and in foods. DSHEA regulates a food in which probiotic is used in the form of dietary supplement. The probiotics as drugs for therapeutic purposes are regulated by FDA for ensuring safe and effective usage . In case of biological products, an approval from Biologic License Application (BLA) is applied in the USA. In the USA, National Yogurt Association (NYA) forces to use “live and active culture seal.” It is mandatory to declare the bacterial genera and species on the labels of probiotic-containing food products including amount of live and active cultures; however differentiation between LAB and other probiotic bacteria or assurance of viability of cultures at end of shelf life is not provided.
In the European Union, microbial cultures present in food need qualified presumption of safety (QPS) assessment in foods and food supplements . European regulatory framework is still not considered as probiotics as food supplement or as are regulated by the Food Products Directive and Regulation (Regulation 178/2002/EC; Directive 2000/13/EU) and under as traditional herbal products by Herbal Medicinal Products Directive (2004/24/EC). Finally, probiotics as herbal medicinal product and registered drugs are covered under the Drug Law (65/65/EC, amended) [134, 135].
As per Japanese regulations, these probiotic products are listed in separate category of foods by the Foods for Specified Health Uses (FOSHU). The Ministry of Health and Welfare (MHLW) allows mentioning the efficacy claims of probiotic food product labels only through special permission . For adopting the FOSHU label, the product must contain dietary ingredients with beneficial effects on the physiological functions and enhance and improve health issue. However, FOSHU does not allow claims of disease-risk reduction on the labels.
In China, the State Food and Drug Administration (SFDA) is the body for regulating functional foods and nutraceuticals in China. The functional food is considered as food which has special health functions or is able to supply mineral and vitamin health benefits .
Brazil becomes the first country to issue legislation for functional food. Probiotics are considered as functional foods. It considered being different from food and legislation address for safety and efficacy labels on food products by registering and approving health authority called National Health Surveillance Agency Brazil (ANVISA) .
It is concluded that probiotic bacteria have various immunomodulatory effects and therefore may be treated as effective means to examine intestinal inflammation and gut mucosal dysfunction including acute gastroenteritis, inflammatory bowel disease and food allergy and downregulate hypersensitivity reactions. Several probiotic effects are exhibited through keeping a balance between secretion of proinflammatory and anti-inflammatory cytokines which play indispensable role in immune regulation. Probiotics may help to balance the gut microbial milieu and mucosal permeability barrier and increase systemic and mucosal IgA immune reactions, therefore mediating the immunological barrier of the gut mucosa. In addition, providing immunomodulating effect on the gut mucosa, probiotic therapy is now also being used to cure infections in other organs such as respiratory tract, urogenital tract, and others.
It is further noted that probiotics as functional food do not exist not only as human health beneficial agents as there are evidences in literature that exist for their side effects. Although the present products in the market are excellent for human health, still there is scope for researchers to study the safety aspects involving the use of probiotics as functional food. Many epidemiological and clinical studies are done to ensure and monitor on consumer safety and their nutritional aspects too. Most of the available probiotics are “generally recognized as safe” (GRAS), but their selection and scrutiny are crucial, especially in the case of patients of immune disorders, GI disorders, or any other critical illnesses. Hence, the focus is needed to be given on identification of responders and non-responders, determining effective size, identifying strain-specific effects, and determining mechanisms to recommend future dietary usage.
The whole process of probiotic manufacturing and its trading also plays an important role in health beneficiary efficacy. Therefore, almost all countries have adopted standards for probiotic products in global markets. The viability and success of probiotic in the future as functional foods for consumers depend on many factors. Consumer’s acceptance and choice of such products are the main issues. It is important to establish the effectiveness of probiotic products and the claims of manufacturers through clear, truthful, and unambiguous information on the labels of the products.
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