Keywords

2.1 Introduction

Living microorganisms that can be incorporated into foods, dietary supplements, functional foods, and drugs in adequate amount for conferring health benefits to the host are called as probiotics. These live microorganisms provide health benefits to the host animal beyond their inherent nutrition. Earliest idea of the beneficial effects of bacteria to human health was provided by Nobel Laureate Metchnikoff, who tried to find out the possible effects of microbes on human health. He reported that the auto-intoxification caused by the intestinal microbes can be prevented by the intake of lactic acid producing bacteria present in yogurt. In his book, “The prolongation of life; optimistic studies,” he suggested that the growth of proteolytic microbes can be prevented by intake of dairy products because they lower the colon pH by breaking lactose. He supported his hypothesis by providing the fact that villagers in Bulgaria who consumed fermented sour milk had longer life expectancy compared to general population. A pediatrician, Henry Tissier, from the Pasteur Institute reported the discovery of Bifidobacterium spp. in the gut of breast-fed infant. He reported that the gastroenteritis could be prevented by Bifidobacterium spp. Stamen Grigorov in 1905 identified Lactobacilli spp. in the starter culture of fermented Bulgarian diary product. Years later, in 1917, a German professor, Alfred Nissle, isolated non-pathogenic E. coli from the feces of two soldiers not affected by the epidemic of shigellosis. He reported that presence of non-pathogenic E. coli in these soldiers prevented the adherence of pathogenic bacteria by the secretion of bacteriocins (Sonnenborn and Schulze 2009).

Earlier the term probiotics was limited to the Lactobacillus and Bifidobacterium spp. With identification of new bacteria and increase in research in probiotics, bacterial spp. belonging to genera Streptococcus, Leuconostoc, Pediococcus, Propionibacterium, and Enterococcus are being used as probiotics. Yeast and molds like Aspergillus niger, A. oryzae, Candida pintolopesii, Saccharomyces boulardii, and S. cerevisiae are also regarded as probiotics.

Human gut is inhibited by both good (probiotics) and bad bacteria. Good bacteria confirm various health benefits to humans. They improve food digestion by fermenting the carbohydrates into simpler structures, producing vitamin-K, and compensating various deficiencies in the body. Probiotics play an important role in proliferation of epithelial cells, development of immune system (Cammarota et al. 2009). Bad bacteria, usually present, but are apparently non-existing, because they are under the pressure of good bacteria. Good bacteria are able to compete with these bad bacteria and prevent their colonization. In the conditions of stress, high alcohol consumption, high fats, genetic disorders, high chlorine and fluoride concentration in diet can cause decrease in the number of good bacteria and bad bacteria will take their position and produce toxins deteriorating human health. In that case, probiotics need to be administrated in higher dosage (Amara 2012).

2.2 Sources of Probiotics

Since ages humans are taking probiotics in diet through fermented food products such as beverages, yogurt, and cheese (Amara 2012). Intake of fermented foods or the dietary supplements are the two ways in which we can take probiotics. Fermented foods are regarded well over dietary supplements because their use is advocated to treat certain diseases and are not recommended for daily use. Fermented dairy products are one of the best sources of probiotics (Liong 2011). Kefir, sauerkraut, pickles, tempeh, miso, kimchi, sourdough bread, and some cheese are important fermented products which are rich sources of probiotics.

  1. 1.

    Yogurt—The “Yogurt” is the Turkish word which means to get thick. Turks are believed to be the earliest users of yogurt to treat gastrointestinal diseases (Atalay 2006). Yogurt, “the milk of eternal life” prepared from milk of goat was used to treat severe gastrointestinal diseases in France (Ozen and Dinleyici 2015). Yogurt is the fermented milk by the fermentation of milk by bacteria called as “Yogurt cultures.” Yogurt culture is the combination of Lactobacillus bulgaricus and Streptococcus thermophilus. S. thermophile is responsible for the lactic acid fermentation and consequent cuddling of the milk to give it a gel like consistency. L. bulgaricus, on the other hand imparts flavor, hydrolyses the casein proteins into smaller peptides. Yogurt is considered as the best carrier food that can be used to deliver L. acidophilus and Bifidobacterium bifidum to human gut, the most common probiotics present in the dairy products. Many other probiotic bacteria are also present in the yogurt. These include B. animalis, B. lactis, B. longum, Enterococcus faecium, Lactobacillus casei, L. delbrueckii, L. johnsonii, L. gasseri, L. plantarum, L. rhamnosus, and S. boulardii. Probiotics bacteria present in yogurt are associated with the prevention of acute diarrhea, allergies, inflammation, genitourinary infections, cardiovascular diseases, vaginitis. The yogurt has attracted the interest of scientific communities due to its antimicrobial, antihypersensitivity, immunomodulatory, lipid-lowering properties, anti-inflammatory properties (Ebringer et al. 2008; Zemel et al. 2005; Ricci-Cabello et al. 2012). Clinical trials have shown the lower risk of antibiotic induced diarrhea with the consumption of yogurt (Beniwal et al. 2003). Lactoferrin, the peptide formed by the digestion of proteins by the probiotic bacteria, present in the yogurt is associated with eradication of Helicobacter pylori infections (Sachdeva et al. 2014).

  2. 2.

    Kefir—It is a fermented milk product similar to yogurt. There has been an increased interest in the commercial use of kefir as a health promoting beverage due to the probiotics present in it. Kefir has a long history of being recommended for health benefits in Soviet nations. The health benefits of kefir are linked both to the consortia of probiotics in it and also due to the presence of organic acids. In addition to being a rich resource of probiotics, it can also be effectively used as a matrix for the delivery of beneficiary microorganisms to the human body (Oliveira et al. 2013; Medrano et al. 2008). Lactose fermenting yeast, non-lactose fermenting yeast, heterofermentative and homofermentative lactic acid bacteria, lactic acid bacteria such as L. kefiri, L. kefiranofaciens, L. paracasei, L. acidophilus, L. parabuchneri, L. delbrueckii, L. bulgaricus, L. plantarum, Acetobacter aceti, A. lovaniensis, A. rasens, A. syzygii, Leuconostoc mesenteroides, Lactococcus lactis, Enterococcus durans, the fungus such as Geotrichum candidum, S. cerevisiae, S. unisporus, Candida kefyr, and Kluyveromyces marxianus (Zanirati et al. 2015; Witthuhn et al. 2004) are present in the kefir. The probiotic population in Kefir varies according to the kefir origin (Gao et al. 2012). Benefits of kefir like hypocholesterolemic effects, antiallergenic properties, food preservative properties, antitumor, and reduction in blood pressure are related to the biological properties of the probiotics present in Kefir (Liu et al. 2002; Chen et al. 2008; Yanping et al. 2009). Antibacterial compounds and antibiotics produced by probiotics present in kefir, inhibit the growth of pathogenic bacteria such as Helicobacter, Shigella sonnei, Staphylococcus, Salmonella, Helicobacter, Escherichia coli, Enterobacter aerogenes, Micrococcus luteus, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus faecalis and fungus like Candida albicans, Fusarium graminearum, Clostridium difficile (Lopitz et al. 2006; Ismaiel et al. 2011).

  3. 3.

    Sauerkraut—It is one of the oldest fermented foods prepared from the cabbage and has a long history of human nutrition. It is regarded as a wonder food due to the presence of probiotics in it. Sauerkraut does not need any culture to ferment as the cabbage growing in a healthy soil has all the bacteria needed to start fermentation. The fermented end product is rich is Lactic acid bacteria (LAB). Predominant species of LAB found in it are identified as L. brevis, L. plantarum, Leuconostoc mesenteroides, Leuconostoc fallax, Pediococcus pentosaceus, L. curvatus, L. sakei, Lactococcus lactis (Björkroth et al. 2002; Murcia-Martinez and Collins 1990; Vogel et al. 1991).These bacteria have established therapeutic role in the treatment of constipation, diarrhea, irritable bowel syndrome, urinary tract infections, and certain cancers. Presence of LAB in sauerkraut has an established role to enhance immunity, prevent certain diseases, and promote lactose tolerance in human body (Orgeron II et al. 2016).

  4. 4.

    Fermented Vegetables—Fermentation is the oldest and the widest used biotechnological method to preserve the perishable vegetable products (Fleming et al. 1995). It is regarded as the natural method of enhancing the nutritional quality and organoleptic properties of the vegetables. Species of LAB such as Lactobacillus, Lactococcus, Pediococcus, and Leuconostoc are naturally present in the vegetables that carry out natural fermentation. Different fermented vegetables have different types of probiotic bacteria (Table 2.1). Probiotic LAB spp. isolated from vegetables are resistant to natural bile and gastric juices tolerating gut stress, therefore vegetables are considered as another important source of probiotics. These probiotic bacteria have found to have antimicrobial activity against Salmonella spp., against gram-positive and gram-negative pathogens (Wang et al. 2010).

  5. 5.

    Kombucha—Kombucha is a fermented black tea which is slightly carbonated and sweet in taste. The fermented tea was very popular in China, Germany, and Russia for preventing metabolic diseases, arthritis, hemorrhoids, and rheumatism, reducing blood pressure, increasing immune response, and curing cancer (Greenwalt et al. 2000). The health benefits of Kombucha are attributed to the presence of vitamins, polyphenols, organic acids produced during the process of fermentation and presence of various lactic acid, gluconic acid, and acetic acid producing bacteria with probiotic attributes (Greenwalt et al. 2000; Malbasa et al. 2008). The Acetobacter xylinum, A. aceti, A. xylinoides, A. pasteurianus, and Gluconobacter are the predominant bacteria present in Kombucha, while yeasts species includes Brettanomyces, Saccharomyces, Pichia, Zygosaccharomyces, and Candida species (Malbasa et al. 2008; Malbasa et al. 2011). However, the yeast population is found to be variable in the Kombucha from different regions (Table 2.2).

  6. 6.

    Tempeh—Tempeh is a fermented soy product, popularly found in Indonesia and is rich in fibers. Tempeh consumption enhances the human gut health by enhancing the number of A. muciniphila and Bifidobacterium significantly (Stephanie et al. 2019). Indonesian tempeh is rich in probiotic microbial consortia consisting of Acetobacter indonesiensis, Flavobacterium sp., Bacillus subtilis, L. agilis, L. fermentum, and Enterobacter cecorum, Brevundimonas sp., Pseudomonas putida, and Acinetobacter spp. (Barus et al. 2008; Radita et al. 2017).

  7. 7.

    Probiotic supplements—WHO documented that most of the today’s diseases are life style related diseases and advocated to switching over to the use of probiotics for treating these diseases. With enhancing health concerns and inclination towards the benefits of probiotics, the demand for “probiotic functional foods” and the probiotic drug supplements is increasing, especially among the younger generation. Probiotic-infused juices, yogurt-based drinks, liquid probiotics are the biggest probiotic supplements present in the market. Mother Diary, Amul, Nestle, Unique Biotech, Tablets India, Danone Yakult, Dr. Reddy Laboratories, Polchem Hygiene Laboratories are the major players in probiotic industry in India. Probiotic preparations are also sold in the form of capsules, syrups and pills. Some of the probiotics available in Indian markets is summarized in the Table 2.3.

  8. 8.

    Human Breast milk—Human breast milk is rich in prebiotics and probiotics, and is considered very important for the development of microbiota in the infant gut. Traditional breast milk was regarded to be sterile, but development of molecular and culture techniques confirms that breast milk continuously supply commensals and rarely, can also transfer infectious microbes to the infant gut from the mother’s skin and gut.

    Approximately 600 different species belonging to Bifidobacteria, Lactobacilli, Enterococcus, Peptostreptococcus, Staphylococcus, Streptococcus, Corynebacterium, and/or an occasional Escherichia species are acknowledged to be present in breast milk that helps in the establishment of infant gut microbiota. Medically important probiotic strains present in human breast milk and their consequent effect on the neonatal growth are summarized in the Fig. 2.1.

Table 2.1 Different probiotics present in fermented vegetable products
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Table 2.2 Variable yeast species present in Kombucha
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Table 2.3 Probiotic functional food and Probiotic drug supplements available in market
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Fig. 2.1
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Various probiotic bacteria present in Breast milk and their beneficial effects (Matsuzaki and Chin 2000; Donnet-Hughes et al. 2010; Gilliland and Speck 1977; Olivares et al. 2006; Vendt et al. 2006)

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2.3 Selection Criteria for the Probiotics

The selection of a probiotic strain for human use should follow a systematic approach. This should be done with the objective that a safe and desired probiotic product is reached to the end users. General aspects like origin of the probiotic, genus and strain identification, biosafety, functional aspects like ability to sustain in gastrointestinal tract, health aspects and production aspects like acid production, proteolysis, and viability should be kept in mind before choosing a probiotic strain. Guidelines provided by ICMR-DBT, for the evaluation of probiotics in food, make it clear that it needs a “step-by-step” approach to test the functional ability of the microbe to be called as a “probiotic.” It is recommended that the strain should be isolated from the targeted animal only, i.e., probiotics for human use should be ideally isolated from human intestine or breast milk. This is because they are better able to adhere to intestinal walls and proved to be safer for humans. Also, they can be isolated from various fermented food products discussed in the above section. The isolated strain should be tested for its probiotic potential and identified up to genus and strain level. The primary identification techniques should be followed by molecular and genetic techniques such as fatty acid methyl ester (FAME), 16SrRNA DNA Sequencing, Polymerase chain reaction (PCR) amplification and DNA and RNA hybridization, and DNA-DNA hybridization technique. For selecting a microbe to be used as probiotic, it should meet certain characteristics that are summarized in Fig. 2.2 and enlisted below:

  1. 1.

    Host associated stress tolerance ability—Upon ingestion the probiotic should be able to sustain through the different parts of digestive tract and tolerate various stress conditions in human body that includes the action of various digestive enzymes like lysozymes, amylase, pepsin, and chymotrypsin. The bacterial strain should have acid and bile tolerance, mild heat shock tolerance caused by internal body temperature. Different probiotic bacteria have found to have varying degree of resistance to the stress conditions. Ogunremi et al. 2015, Psani and Kotzekidou (2006) in their studies summarized that probiotic bacteria should be able to tolerate bile concentrations of 0.3–2.0% and pH range of 2–5.

  2. 2.

    Adhesive properties—The adhesion of the probiotic strain to the intestinal wall is very important in defining its probiotic potential because it ensures that the bacteria is not washed away, auto-aggregates to increase its cell density and biomass in the digestive tract. Adhesion is also important to ensure that the microbial cell forms a better interaction with human epithelial cells in order to produce various host associated functional effects on the host (defined below).

  3. 3.

    Antimicrobial activity—The probiotic strain should be able to fight and survive against the potential pathogenic microbes that may be present in the intestine. The prevention of pathogen survival in the gut is related to the competitive behavior of probiotic strain, where it prevent the adhesion of pathogens to the epithelial cells in the human body and due to the secretion of lacticins, alyteserin-1 a, bacteriocins Abp118, bacteriocin sakacin A, lactic acid, antibodies. Probiotic bacteria also employ increasing leukocyte phagocytic activity, increasing transepithelial resistance, enhancement of cytoskeletal, and tight junctional protein phosphorylation activity to kill various pathogenic bacteria (Mathipa and Thantsha 2017).

  4. 4.

    Immune modulating response—Probiotic strain should be involved in production of metabolites that stimulate the maturation and functioning of the immune cells. These bacteria enhance the secretion of immunoglobulins, cytokine production (Rocha-Ramírez et al. 2017), IgA and IgM-secreting cells (Lammers et al. 2003). The probiotic strain should be selected depending upon the target host to stimulate the systemic immune response.

  5. 5.

    Host Associated functional criteria—One of the most important characteristics of the probiotic is the health benefits that it can provide to the host. The viability and colonization of the probiotic in the human gut should offer anticarcinogenic, anti-cholesterol activity, anti-depression, anti-anxiety, anti-obesity, anti-diabetic, and antioxidant activities to host besides protecting it against irritable bowel syndrome, gastroenteritis, and inflammatory bowel disease, diarrhea, infant allergies, cancer, lactase digestion, diabetes, and hyperlipidemia. Probiotic strains should be selected to inactivate enzymes (nitrate reductase, β-glucosidase, β-glucuronidase) involved in the activation of precarcinogens (Kumar et al. 2013). Additionally, the probiotic strain should be screened for secretion of conjugated linoleic acid production (dos Reis et al. 2017) and apoptosis induction activity (Ewaschuk et al. 2006) for possible anticarcinogenic activity.

    The efficiency of the probiotic strains for the reversal of depression and anxiety can also be studied in vivo on animal models. The anti-diabetic and anti-obesity activities of the potential strain can be analyzed by testing its ability to inhibit mast cell activation (Niers et al. 2005) and ability to break lipopolysaccharides (Alokail et al. 2013).

    Another major characteristic feature of the probiotic is the secretion of antioxidant enzymes (superoxide dismutase, glutathione dismutase), functional molecules (glutathione, melatonin, ascorbic acid), Vitamin-K, biotin, riboflavin, nicotinic acid, thiamine, pantothenic acid, and bioactive enzymes (lipases and amylases). The in vitro production of these components should be tested depending upon the objective of the product.

  6. 6.

    Good technological properties—The extent to which a probiotic can effectively deliver its benefits to the host will depend upon the capability of the strain to survive the storage period and maintain the same efficiency and viability. The probiotic bacteria should be able to grow quickly in different nutrient supplements (preferably cheap fermentation media), food matrices, and microaerophilic conditions. The stress adaptation of bacteria strain to withstand different physical handling techniques during food processing without losing the viability and efficiency is also important for its selection as the probiotic.

Fig. 2.2
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Characteristics of probiotics

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2.4 Conclusion

Fermented and non-fermented dairy products, fermented perishable vegetables and fruits, fermented cereals and soya products should be incorporated into the diet due to the presence of probiotic bacteria in it. The rise in the market potential of probiotics in India is driven by the rising lifestyle disorders and subsequently increasing the awareness of the consumers. The availability of the large number of commercial platforms providing probiotic functional foods and probiotic drug supplements has also contributed to the exponential increase in the use of probiotics in India. In the absence of any regulatory guidelines governing the probiotic industry, it is quite possible that the fake products with low or no health benefits are being marketed to the end users. To avoid such spurious products, the product must fulfill the above prerequisite conditions to be called or labeled as the “probiotic.” Apart from this, efficacy studies should also be carried to prove its health benefits in humans. Manufacturing and handling practices should follow quality assurance so that the end users should get the product with claimed benefits.