Probiotics are live microbes which serve as excellent functional food ingredients, to improve human health. They provide essential metabolites with dietary and therapeutic characteristics which confers numerous health benefits. They ensure a proper maintenance of the gut health through complex interactions across the gut-brain axis. The probiotics enter into the body through food. Probiotics can exist naturally or be infused in food. Food products containing probiotics are viable modes for a healthy gut. Understanding the mechanism of action of the probiotic strains and their interaction with the gut microflora is absolutely necessary. This review elaborates on the applications of probiotics in food .It also describes the possible mechanism of action and its clinical significance.
Keywords: probiotics food formulations gastroenteritis clinical implicationsProbiotics are a group of health promoting functional foods with an emerging commercial interest 1 . They are incorporated as living microorganisms in food to enhance its nutritional content and protect the gut. Clinically, probiotics improve the intestinal microbiome contributing towards the immune potency of the host. They also counteract the pathogenic activity in the gut. Multiple mechanisms of actions are reported, including production of antimicrobial agents, competition for space or nutrients and immunomodulation 2 . FAO/WHO define probiotics as “live microbial food supplements or components of bacteria which when taken up in adequate amounts, confers a health benefit to the host” 3 . Probiotics have emerged as medical therapies for gastrointestinal and non-gastrointestinal diseases such as diarrhea, constipation inflammatory bowel disease, irritable bowel syndrome, asthma, atopic dermatitis, peptic ulcer, colon cancer, coronary heart disease and urinary tract infections 1 .Evidences have supported the consumption of fermented milk especially yogurt, due to its nutritional properties in maintenance of gut health, due to its probiotic content.
This article summarizes the applications of probiotics as a functional ingredient in food, mechanism of action and its clinical significance in gut health.
The global market of functional foods including probiotics is growing exponentially. The emerging demand of consumers is incentivizing world markets to manufacture dairy and non-dairy products containing probiotic bacteria. Figure 1 and Table 1 show some dairy and non-dairy probiotic products available in world markets. Therefore, putting probiotic bacteria into products is an important issue with industrial and commercial consequences. This led to a shift in industries, focusing on the probiotics based food products, to fulfill consumer demand. Probiotic cells via food products are available in three different types for direct or indirect human consumption such as fermented or non-fermented form, dried or deep-frozen form for industrial or home uses and drugs in powder, capsule or tablet forms 4 . Some of the commercially available probiotic strains are listed in Table 2 5, 6 .
2.1. Dairy Based Products
Dairy products serve as a good vehicle for probiotics. Some examples of dairy based products are; butter milk, normal milk, flavored liquid milk, fermented milks, dairy fermented beverage, milk powder, Whey-protein-based drinks, yogurt drink, ice cream, sour cream, yogurt, cheese, frozen dairy desserts and baby foods are the most popular vehicles for probiotics 7, 8 . Dairy foods are ideal for performing the delivery function because of their inherent environment stabilizing properties that promote the growth of probiotic bacteria, which can then be stored at refrigerated temperatures. In addition to probiotic strains, several useful compounds such as tryptone, yeast extracts, certain amino acids, nucleotide precursors, whey protein concentrate (WPC), fructooligosaccahrides (FOS) and caseinomacro peptides (CMP) are used in dairy products to enhance the growth and viability of the strains. Citrus fiber in these products enhances bacterial growth and survival of probiotic bacteria, whereas soy germ powder releases bioactive compound that protect probiotic strains from bile salt toxicity 9, 10 . Recently, probiotic cheese, marketed in various forms (fresh, semi-hard and hard cheese), which are highly nutritious and have high energy and fat content, has shown a high rate of survival of probiotics at the end of its shelf life 11, 12 . The utilization of probiotics in the cheese faces some challenges, such as low moisture content, the presence of salt, the development of acid and the influence on flavor during maturation; starter cultures can compete for nutrients and redox potential. Nevertheless, it acts as a potential carrier of probiotics 11 . Cheddar cheese, Feta cheese, Canestrato pugliese hard Cheese, Cottage cheese, white-brined cheese, Minas Fresco cheese, Minas fresh cheese, Argentine Fresco cheese, Iranian white-brined Cheese, Turkish Beyaz cheese are some of probiotic cheese products available on the world market 12, 13 . That having been said, yoghurts with high fat content are the most common vehicles for probiotics among dairy products. Probiotic yoghurt contains cultures of L. acidophilus, L. delbrueckii subsp. Bulgaricus and Streptococcus salivarius subsp. thermophilus bacteria 14 . Some examples of Probiotic yoghurt widely marketed for human consumption are Corn milk yogurt 15 ; Stirred fruit yogurts 13 , Mango soy fortified probiotic yogurt, Iranian yogurt drink (Doogh) and Traditional Greek yogurt 12, 16 . Other dairy-based products, used to carry probiotics are ice cream and frozen dairy desserts composed of milk proteins, fat and lactose as well as other compounds that are required for bacterial growth. These products are infused with certain commercialized probiotic strains such as L. acidophilus La-5 and B. animaliss sp. lactis Bb-12, L. acidophilus and B. lactis. Probiotic chocolate mousse is a good medium for L. paracasei subsp. paracasei LBC 82 which can exist by itself or together with inulin.
Non-diary based products are classified as fermented and non- fermented products. A limitation in the use of dairy products to deliver probiotics is due to the presence of allergens. This led to the development of other food matrices especially non-dairy probiotic products. Cereals are good nutrient substrates for the growth of probiotic strains. The fermentation of cereals in presence of probiotic microorganisms could be beneficial for health due to their availability of the vitamin B, the decrease of non-digestible carbohydrates and the improvement of the quality and level of lysine 17 . Boza is a fermented product based on maize, wheat and other cereals containing several lactic acid bacteria with probiotic characteristics. Malt medium supports the growth of all examined strains (L. plantarum, L. fermentum, L. acidophilus and L. reuteri) better than barley and wheat media due to its chemical composition. Malt, wheat and barley extracts exhibit a significant protective effect on the viability of above probiotic strains under acidic conditions 12 . Oat, an important cereal contains high levels of β-glucans and provides the right nutrient for the growth of L. reuteri, L. acidophilus and B. bifidum 18 . Probiotic cassava-flour product, Starch-saccharified probiotic drink, Meat based products and Dry-fermented sausages are also the name of some other non-dairy probiotic foods 19, 20 . Fruits are rich in several nutrients such as minerals, vitamins, dietary fibers, antioxidants and do not contain any allergens. This advantage of fruits has encouraged several food industries to manufacture healthy probiotic fruit juices like pineapple, apple, orange, grape, watermelon juice, cranberry, cashewapple juice, blackcurrant juice and many more. L. plantarum, L. delbruekii, L. casei, L. paracasei and L. acidophilus are main optimal probiotic bacteria widely used for the production of probiotic juices 21 . Probiotic beverage production from cashew apple juice fermented with Lactobacillus casei NRRL B-442 whereas watermelon juice is produced using four strains of Lactobacilli namely Lactobacillus casei, L. acidophillus, L. fermentum and L. plantarum. Probiotic tomato juice is produced by inoculating lactic acid bacteria namely Latobacillus acidophilus LA39, Lactobacillus plantarum C3, Lactobacillus casei A4 and Lactobacillus delbrueckii D7. Probiotic strains are able to grow without nutrient supplementation and pH adjustment in the tomato juice 22, 23 . Similarly, probiotic bacteria such as L. plantarum, L. casei and L. delbrueckii grow in both beet and cabbage juices too 23, 24 . Some examples of dairy and non dairy-based probiotic products along with their probiotic strains are listed in Table 3 and Table 4 7, 8, 25, 26, 27 .
Micro-encapsulation serves as an important means for the survival of the probiotic cells. The encapsulated cells are introduced into different food matrices e.g. Yoghurt, cheddar cheese, ice-cream, yogurt-covered raisins, nutrient bars, chocolate bars, cocoa butter, biscuits, vegetable and frozen cranberry juice 28, 29 . The encapsulated probiotic cells available in a tablet/capsule form are Forever Active Probiotic, Probiotic 7, Multi-probiotic or in the form of a powder e.g. Pure Baby Probiotic, Cernivet LBC ME10, Geneflora™ and ThreeLac TM . Additionally, Probio-Tec capsules, encapsulated probiotic orange juice termed “Dawn”, chocolate bar named “Attune”, Innovance Probiotiques and an encapsulated yoghurt is available in the market under the brand name Doctor-Capsule. Syntol AMD and Ganeden BC 30 deliver probiotic spores rather than living bacteria is known to be spore-germinated probiotics prepared by using spore germination technology. Though the use of these genetically engineered products has been quite limited but certain genetically modified probiotics strains have been used to increase physiological or immunological properties within the organism which can be useful in mucosal delivery system or in development of vaccine vector 30 . The use of any engineered strains has to be rigorously assessed for its safety before human use.
2.2. Mechanisms of Action of Probiotic Strains
About 10 trillion microbes of 500-1000 different microbial species colonize in the GI tract and remain in a complex equilibrium. These include Bacteroides, Lactobacillus, Clostridium, Fusobacterium, Bifidobacterium, Eubacterium, Peptococcus, Peptostreptococcus, Escherichia and Veillonella. Colonization of these microbes in the human gut start at birth and eventually get exposed to foreign microbial population and antigens derived from digested foods. Therefore, the intestine acts as an interface between the host and exogenous agents such as, pathogenic bacteria, viruses, allergens. The intestinal mucosa may play a central role in host microbiota-pathogen interactions 31 .
Gut microbiota influences human health through an impact on the gut defense barrier, immune function, and nutrient utilization and potentially by direct signaling with the gastrointestinal epithelium 32 . The interaction between the host microbata and exogenous agents may disturb or alter the normal microbial balance or their activity in GIT. Alteration of such microflora is implicated in the pathogenesis of various diseases. Enteric diseases are caused by several pathogens like Escherichia coli, Salmonella spp., Shigella along with various other food borne pathogenic strains such as Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes and Vibrio cholera 33 . They may cause infections in two steps. During the first step of the infection process, the pathogens may attach themselves to the surfaces of intestinal epithelial cell through certain adhesive receptors like glycoproteins and glycolipids and later on, in second step they cause direct cytotoxic injury, intracellular migration, and finally disrupt the epithelial tight junctions that leads to mucosal infection 34 .
Probiotics promote the GIT homeostasis and stimulate the growth of indigenous beneficial gut microbata by inhibiting the growth of pathogenic or opportunistic pathogenic microbes. Therefore, probiotics are recommended as alternative bio therapeutic agents for intestinal pathogenic infections. These may act via several mechanisms such as, production of antimicrobial compounds, competition for nutrient substrates, competitive exclusion, enhancement of intestinal barrier function and immunomodulation 1, 35, 36, 37 .
2.2.1. Production of Antimicrobial Substances
LAB produces antimicrobial substances, such as organic acids, fatty free acids, ammonia, hydrogen peroxide and bio surfactant. It also produces a low-molecular-weight antibacterial peptide- bacteriocins that inhibits both gram positive and gram-negative enteric pathogens 38, 39 . For example, probiotic L. rhamnosus GG inhibits the growth of pathogenic Salmonella enterica by producing lactic acid and other secreted antimicrobial molecules 40 . In order to exert a strong effect on a pathogen in vivo, the probiotic-derived antimicrobial compounds are produced in the right location in the intestinal tract at higher levels. Organic acids produced by LAB or any probiotic strains account for the alteration of bacterial flora due to the acidification of the colon by nutrient fermentation.
Homo-fermentative LAB strains produce lactic acid whereas hetero-fermentative strains produce the short chain fatty acids viz. acetic and propionic acids in addition to lactic acid by their respective metabolic pathways. Organic acids produced by certain probiotic strains lower the external pH that causes acidification of the cell cytoplasm. Nevertheless, these acids are partially in their undissociated at lower pH values. The undissociated organic acids are lipophilic and diffuse passively across the membrane that may cause intracellular acidification. On the contrary, at high intracellular pH values, they dissociate to produce hydrogen ions that interfere with essential bacterial metabolic functions. It also denatures protein and collapses the electrochemical proton gradient resulting in the disruption of substrate transport systems of infectious pathogens. Thus, it also alters the cell membrane permeability of pathogenic bacteria 41 .
Production of free radicals along with hydrogen peroxide (H 2 O 2 ) acts as a precursor that damages the DNA. In certain cases, the antimicrobial activity of H 2 O 2 may result from the oxidation of sulphydryl groups thereby causing denaturation of a number of enzymes. The peroxidation of membrane lipids by H 2 O 2 also causes the increased membrane permeability 41 . Carbon dioxide (CO 2 ) as end products of fermentative metabolism creates an anaerobic environment that may inhibit enzymatic decarboxylation reaction. In certain cases, it causes a malfunction in permeability due to the heavy accumulation of CO 2 in the membrane lipid bilayer. LAB also produces significant amounts of fatty acid, named as Reuterin, that show their antimicrobial potential under specific conditions 41 . Lactobacillus paracasei produces certain bactericidal bio surfactant that inhibits the growth of several pathogens 39 .
2.2.2. Bacteriocins (Antimicrobial peptide)
Bacteriocins are ribosomally synthesized low-molecular-weight antimicrobial peptides with a good functional therapeutic activity against gastrointestinal pathogenic infections 42, 43 . Bacteriocin activity adsorbs specific or non-specific receptors on the target cell surface and alters the membrane permeability, thus disturbing membrane transport, and finally inhibiting energy production. Numerous bacteriocins, such as nisin, lactobrevin, acidophilin, acidolin, lactobacillin, lactocidin and lactolin have been reported to be produced by Lactobacilli 42, 43, 44 . Table 5 presents the class of bacteriocins and its characteristics, based on structural, physicochemical and molecular properties.
Antimicrobial peptides (AMP) get attached to the anionic components of bacterial cell wall because of their cationic nature and cause disruption of cell envelope to reach into cytoplasmic membrane in a process known to be self-promoted uptake. A numbers of models including barrel stave model, carpet, toroidal or aggregate channel model represent different mechanisms of action. AMP become stave in a barrel like cluster around the outer membrane of pathogenic flora and the hydrophilic surfaces of peptides point facilitate pores. Leakage of cellular components through these transmembrane pores depletes proton motive force that ultimately interferes with cellular biosynthesis causing cell death 44, 45 .
2.2.3. Colonization Resistance and Competitive Exclusion
Adherence to human intestinal cells and intestinal mucus glycoproteins (mucin) as well as competitive exclusion of pathogens are most important characterization of probiotic strains to deactivate the pathogen in the intestine. Such competition may occur either for adhesion or for nutritional substrates and biogenic growth metabolites. Infection begins with the binding of the pathogen to intestinal epithelial cells via carbohydrate moieties of glycoconjugate receptor molecules. Probiotic strains compete with invading pathogens for the glycoconjugate receptors at the infection site of intestinal epithelium cells, which may block adhesion and penetration of infectious pathogens 46 . Gastrointestinal cell surface constituents, such as several glygoconjucates, can serve as receptors for bacterial adherence 47 . Adhesion may be specific or non-specific based on physico-chemical factors and involves adhesin molecules on the surfaces of adherent bacteria and receptor molecules on epithelial cell. Probiotic agents also compete for receptors or adhesion to intestinal epithelium that could prevent the colonization of pathogenic microorganisms from occupying this living space (colonization resistance or competitive inhibition) 36 . Escherichia coli binds to epithelial cells via mannose receptors in human intestinal epithelial cells; Probiotic L. acidophilus ATCC 4356 has shown similar adherence capabilities that could inhibit pathogen colonization at the same sites, thereby protecting the host against the infection 48, 49 .
Probiotics may also compete for an ecological niche creating an unfavorable condition for the invading pathogens to take hold in the intestinal tract and impair their colonization ability. A competition for nutritional substrates and biogenic growth metabolites (amino acid, methylamines, vitamins, short-chain fatty acids (SCFA) and bioactive peptides) amongst intestinal microbiota, probiotics and pathogens may occur. Bifidobacterium adolescentis S2-1 can better utilize vitamin K and inhibit the growth of Porphyromonas gingivalis by competing for the growth factor 50 . Lactobacillus plantarum 423 is able to colonize intestinal epithelial cells, thus preventing the adhesion of pathogenic Clostridium sporogenes and Enterococcus faecalis 46 .
2.2.4. Intestinal Barrier Function
The mucus layer is the first line barrier where pathogens penetrate it to reach the epithelial cells during infection, thus it provides protection by shielding the epithelium from potentially harmful antigens 51 . Disruption of epithelial barrier function or a loss of tight junction formation in the intestinal epithelium cells are another major reasons of intestinal pathogenic infections. Probiotics maintain a tight junction protein expression and enhancement of host mucin production, which improves the ability of the mucus layer to act as an antibacterial shield by preventing the increase of apoptotic ratio 36, 52, 53 . Probiotics can reduce the epithelial injury that follows exposure to E. coliO157:H7 and E. coli O127:H6 54 .
2.2.5. Immunomodulation
Several studies have shown an interesting physiological action of probiotics that modulate the immune system. Probiotics might be able to modulate the host's defenses including the innate as well as the acquired immune system 55 . One of the more precise mechanisms of action of probiotics is modulation of GI immunity by altering inflammatory cytokine profiles and down regulating proinflammatory cascades 56 . They enhance the production of serum IgA secretory IgA, as well as natural killer cells and phagocytosis, which play a crucial role in intestinal immunity. They prevent apoptosis and suppress T cell proliferation, thus preventing various inflammatory conditions 56, 57, 58 . Lactobacillus reuteri strains from human breast milk, either stimulate the key pro inflammatory cytokine, human tumor necrosis factor (TNF), or suppress its production by human myeloid cells 56, 58 . Both in vitro production of g-IFN, IL-12, and IL-18 by lymphocytes and enhanced capacity to produce g-IFN in response to different lactic bacteria strains. L. paracasei is a potent stimulator of IL-12. However, IL-12 may down regulate the Th-2 response, decreasing IL-4 and IgE production. It may prevent an allergic tendency in humans.
Probiotics can also modulate toxin receptors and block toxin-mediated pathology by using enzymatic mechanisms. For example; S. boulardii degrades Clostridium difficile toxin receptors and blocks cholera-induced secretion in rat jejunum by the production of polyamines 59 . The presence of several probiotic bacteria regulate human beta defensin 2 (hBD-2) via the transcription factor NF-κB that would strengthen intestinal defenses 60 .
