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PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF LACTIC ACID
BACTERIA ISOLATED FROM TRADITIONAL FOODS
A
Synopsis
Submitted in
Partial fulfillment for the degree
Of
Doctor of Philosophy
(Microbiology)
Supervised by Submitted by
Dr. Richa Sharma Aditya Chaudhary
Department of Agriculture, Food & Biotechnology
Faculty of Engineering & Technology
Jayoti Vidyapeeth Women’s University
Jaipur (Rajasthan), India
June, 2017
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INTRODUCTION
Probiotics
Probiotics are defined as “live microorganisms which when administered in appropriate amounts, give benefit to the health of host”. It helps in preserving the immune-function of human and improving the properties of commensal GI flora (FAO/WHO, 2002). The term “Probiotic” is derived from the Greek word “Probios” which means “for life”. It is different from the term “Antibiotics” which means “against life” (Longdet et al., 2011).
Lactic acid bacteria
Lactic acid bacteria belong to the phylum Firmicutes (Fooks et al., 1999) and to the family Lactobacteriaceae (Orla-Jensen, 1919). The genus Lactobacillus comprises major groups of lactic acid bacteria which are used in food fermentation and has great economical importance (Schillinger et al., 1999). LAB is a group of gram-positive, acid tolerant, non-sporulant, non-respiring but aero-tolerant, bacillus (rod shaped) or coccus (spherical) bacteria that are devoid of cytochromes but strictly fermentative (Axelsson, 1993).Both viable and non-viable forms of probiotics exert health promoting effects. The probiotics should have following properties and functions (Ouwehand et al., 2002):
Adherence to epithelial cells of intestine of host
Resistance to acid and bile
Reduction in adherence of pathogens to host epithelial cells
Production of acids and H2O2
Production of bacteriocins
Non-pathogenic
Non-carcinogenic
Improve the intestinal microflora of host
Lactic acid bacteria (LAB) helps in preserving the food and are of significant importance to food industries due to their generally regarded as safe status (GRAS) (Tassell and Miller, 2011). The probiotic activity of LAB makes it a good probiotic organism (Mishra and Sharma, 2014). Either the probiotic has been given as a supplement or as an active component of medication; it should be capable of surviving through the passage of digestive tract along with proliferation in the gut. Benefits of the probiotics must be exerted on the host through growth and metabolic activities in the human body.
Classification of lactic acid bacteria
Lactic acid bacteria are classified into four genera on the basis of sugar fermentation and growth at particular temperature which includes Lactobacillus, Betacoccus, Streptococcus and Tetracoccus (Schleifer et al., 1991). Stiles and Holzapfel (1997) and Ercolini et al.,
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(2001) have classified lactic acid bacteria into 13 genera: Lactosphaera, Melissococcus, Oenococcus, Carnobacterium, Leuconostoc, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus, Weissella, Lactobacillus, Lactococcus and Enterococcus. Other classification of lactic acid bacteria are on the basis of metabolic pathway which includes two cultures, i.e. homofermentative (Streptococcus, Pediococcus and Lactobacilli) and heterofermantative (Leuconostoc and Weissella) (Caplice and Fitzgerald, 1999; Kuipers et al., 2000; Jay, 1992). Physiologically lactic acid bacteria are grouped into four genera: Streptococcus, Pediococcus, Leuconostoc, Lactobacillus (Carr et al., 2002; Frank et al., 2002). DNA-DNA hybridization (Yashima et al., 1996), 16s and 23s intergeneric spacer region sequencing (Bourget et al., 1996) and genus specific and species-specific probes (Hensiek et al., 1992; Timisjarvi and Alatossava, 1997) are regarded as techniques used for identification and characterization of LAB strain. PCR (Polymerase Chain Reaction) based techniques such as RFLP (Restriction Fragment Length Polymorphism) as well as PFGE (Pulse Field Gel Electrophoresis) are used for specific characterization of LAB strains (Gevers et al., 2001).
Importance of lactic acid bacteria
Protection against various diseases - The consumption of probiotic bacteria helps in modifying the residential intestinal microflora and balances it in beneficial “rebalancing” manner and thus, improves the digestive health and helps in curing irritable bowel syndrome, heart disease, gut disorders, Crohn’s disease, lactose intolerance, autism, ulcerative colitis and allergies (Borruel et al., 2002).
Therapeutic potential – Lactic acid bacteria are widely used probiotic bacteria in the production of beverages and fermented food and contribute to both sensory qualities of the food and the prevention of spoilage (Sgouras et al., 2004). The human gastrointestinal tract constitute a complex ecosystem of bacterial population (Simon and Gorbach, 1986) and the microflora of the intestine can be altered by introducing LAB for the nutritional and therapeutic benefits (Gorbach, 1990). ). Probiotics that were part of food in the past now have begun to be used by clinicians in regular diets for therapeutic benefits (Floch and Kim, 2014). Bacterial supplement feed has benefits such as increased nutrient utilization, alleviation of lactose intolerance, treatment of intestinal infections and inhibition of carcinogen in the GI tract (Finegold et al., 1977).
Medical property - Enzymes, Vitamins, antioxidants and bacteriocins are synthesized by LAB (Knorr, 1998) and these properties help the body in detoxifying the foreign substances (Salminen, 1990). LAB helps in increasing muscle mass and reducing body fat content (Chin et al., 1992; Akahoshi et al., 2002). It also helps in altering low density lipoprotein/ high density lipoprotein cholesterol ratio (Lee et al., 1994).
Nutraceutical property - LAB helps in improving the digestibility of protein and fat food supplements (Friend and Shahani, 1984). Lactobacillus plantarum produces excess of folate in human host that maintains cognitive function by maintaining normal plasma homocysteine level (Sybesma et al., 2003; Jagerstad et al., 2004).
Anticarcinogenic property – Lactic acid bacteria in fermented dairy foods, dairy proteins and dairy fats have anticarcinogenic and Antimutagenic properties (Lidbeck et al., 1992;
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McIntosh et al., 1995). Fermented dairy fats have organoleptic properties and consumption of conjugated fatty acids such as linolenic acid inhibits the initiation of carcinogenesis (Ip et al., 1991; Devery et al., 2001; Pariza et al., 2001).
Table 1. The probiotic effect of lactic acid bacteria on the human host
LAB STRAIN TARGETL. rhamnosus GG Food allergy prevention (Sütas et al., 1996), reduce
intestinal disorders and pouchitis (Kuisma et al., 2003), treatment of atopic disease (Kalliomaki et al., 2001), prevention of caries formation (Näse et al., 2001)
L. lactis ESI 561, E. faecalis INIA 4-07, E. faecalis EFS 2
Blocking the formation of biogenic amines (Joosten et al., 1996)
L. acidophilus, L. rhamnosus GG, L. acidophilus LB
Alleviate lactose intolerance, prevention of traveller’s diarrhea and Clostridium difficile colitis (Gilliland and Kim, 1984; Fooks et al., 1999; Sanders, 2003)
L. acidophilus Treatment of infection by Helicobacter pylori (Conducci et al., 2000), stimulation of anticarcinogenic activity (Hirayma and Rafter, 2000), treatment of coronary heart disease and anticholesterolaemic effects (Schaafsma et al., 1998; Gilliland et al., 1985)
L. plantarum, L. acidophilus, L. brevis, S. thermophilus, B. infantis
Prevention of kidney stones (Campieri et al., 2001)
L. rhamnosus GG, B. infantis UCC35624
Treatment of Crohn’s disease, ulcerative colitis and inflammatory bowel disease (Gupta et al., 2000; Wright et al., 2002)
L. plantarum Protect against tetanus toxin (Grangette et al., 2001)
Market potential
Nowadays, more attention is given in obtaining new probiotic bacterial strains from traditional foods and pharmaceutical industries (Zielinska et al., 2015). Globally, interest has been developed in the consumption of functional foods or nutraceuticals with potential probiotic microorganisms and the estimation of the global market is above US$28.8 billion (Quinto et al., 2014). Functional food market was started in Japan in late 1980s. Probiotic bacterial products are gaining popularity as it has added ingredient to increase health. Japan and Europe has been the most active countries in the production of probiotic dairy products (Lee et al., 1999; Sanders and Huis In’t Veld, 1999; Stanton et al., 2001). Functional foods, valued at US$889 million are accounting for 65% of the European market. It is followed by spreads which accounts for 23% of the market and valued at US$320 million (Hilliam, 1998). In 1997, a market for probiotic yogurt in United Kingdom, France, Finland, Sweden, Netherlands, Denmark, Belgium, Germany and Spain was reviewed by Leatherhead Food RA and accounted for more than 250 million Kg (Hilliam, 1998). France represents the largest market, valued at US$219 million. A probiotic drink “Bikkle” contains Bifidobacteria species achieved a sale of 11 billion Yen in its first year of launching in Osaka, Japan, 1993 (Young, 1996). During 1996-1997, German market and UK market for probiotic yogurts is increased by 150% and 26% respectively. In a study of 9 countries, Denmark accounted for 20% of the
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probiotic yogurts followed by Germany and UK, accounted for 13%, France at 11%, Netherlands and Belgium at 6% and at last Finland and Sweden at 5% (Hilliam, 1998). Functional food market in Europe accounts for ~5% of total expenditure in Europe which is equal to ~US$30 million based on current prices (Young, 1996). Functional food market in US will gain the fastest growth rates than other countries (Young, 1996). The advancement in technology in food industry improves the composition and nutritional characteristics of food products which provide them attributes beyond nourishing properties (Behrens et al., 2001). Chance of re-buying the product can be enhanced only if it offers a measurable benefit to consumers in long run (Childs, 1997). The probiotic strain of commercialized product should be studied and confirmed for its viability and effectivity at the target site (Vandenplasa et al., 2015).
Prebiotics are defined as non digestible food ingredients that help in improving the activity of the residents of the colon of the host by selectively stimulating the growth of one or more bacterial species (Gibson and Roberfroid, 1995). Utilization of synbiotics gains popularity among consumers globally and demand for such products are increasing as they provide health benefits beyond nutrition.
Preservation of probiotics
The quality of the food product is rated by the viability of the cultures and for maintaining the viability, different techniques are employed.
1. Freeze drying – Lactic acid bacteria starter cultures which are used in dairy and food fermentation are preserved satisfactorily by the technique of freeze drying under vacuum (Kearney et al., 1990). Freeze trying is a technique where a solution is being frozen and then the quantity of the solvent is reduced by sublimation and desorption. This process helps in maintenance of technological properties along with preserving the life of lactic acid bacteria (Corthier et al., 1998). Gram-negative bacteria show lower degrees of freeze drying survival than gram-positive bacteria (Peter and Reichart, 2001). A minimum concentration of survival of microorganisms after freeze drying is 107 CFU/ml (Pocard et al., 1994). Addition of Tween 80 in the fermentation medium enhances the survival of lactic acid bacteria after freezing by modifying the membrane permeability (Gomez-Zavaglia et al., 2000; Beal et al., 2001). Freeze drying technique results in loss of viability during processing and storage (Potts, 1994; Castro et al., 1995; Miyamoto-Shinohara et al., 2000). Studies have shown that freezing and thawing damages the membrane and cause death of lactic acid bacteria. Therefore, to maintain the cell viability, it is necessary to use protective solutes such as non-reducing disaccharides, sugar alcohols, polysaccharides, amino acids, proteins and skimmed milk in the freeze drying media (Costa et al., 2000). Recovery of freeze dried microorganisms is difficult by rehydration. If the cells are rehydrated under inappropriate conditions, it is difficult to repair the damage caused by sub-lethal injury (Champagne et al., 1991). Moderate stress conditions before freezing helps the bacteria to resist better to unfavourable conditions. Lactic acid bacteria react better to such conditions by production of stress proteins (Whitaker and Batt, 1991; Panoff et al., 1998; Broadbent and Lin, 1999 and Roberfroid, 2000) or by modification of composition of membrane (Broadbent and Lin, 1999; Beal, 2001).
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2. Spray drying – In a closed chamber, slurry solution has been converted to granulated powders by atomizing at high velocity (Corcoran et al., 2004). It is the widely accepted method of preservation in dairy industry. Cost of spray drying is six times lower (per Kg of water extracted) than the cost of freeze drying (Knorr, 1998). Spray dried powders are in a stable form for prolonged periods.
3. Foam formation – Biological suspensions are transformed into mechanically stable dry foams by protective sugar matrics. Stability of dry foam has been increases by further heating at high and increased temperatures (Bronshtein, 2004).
4. Fluidized bed drying – Fluidized effect is created in a solid product by mechanical shaking with flow of heated air moving upwards (Larena et al., 2003).
After freeze drying and thawing, death of cell occurs by rupture and leakage of cell membrane (Pringle and Chapman, 1981). Cell recovery after freezing and thawing can be done by stabilization of cell membrane (Hoekstra et al., 1997) by disaccharides (Crowe, 1998). A naturally occurring disaccharide of glucose ‘α, α-Trehalose’ helps in stabilizing the membrane during freezing and drying (Miller et al., 1997).
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OBJECTIVES
The present study will be carried out with the following objectives:
1. To collect the traditional food samples viz. Yogurt, Jalebi batter, seera, dough, kefir,
pickles, soy milk, olive, saukerkraut.
2. To isolate the probiotic strains from the collected food samples.
3. Maintenance and pure culture of isolated probiotic strains.
4. To identify the purified strains on the basis of colonial morphology and biochemical tests.
5. To study the effect of temperature on the growth of isolated lactic acid bacteria.
6. To study the probiotic bacteria against food borne pathogens.
7. To study genotypic characterization of probiotic strains.
8. To study the toxigenicity and antagonistic property of screened probiotic bacteria.
9. To prepare the functional foods using probiotic strains.
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REVIEW OF LITERATURE
Bacterial species of the human gut has important metabolic and immune functions; all accelerate the nutritional and health status of the host (Laparra and Sanz, 2010). Probiotics were originated to favourably affect human health by altering the intestinal microbiota. The effects of probiotics on the human health have been explained by including them in functional foods as single or mixed microbial culture preparation (Nissen et al., 2009). Probiotics and their effects gives positive response on human health in prevention and treatment of various diseases such as diarrhea, lactose intolerance, IBD (Inflammatory Bowel Disease) (Minelli and Benini, 2008), AIDS (Trois et al., 2008), inflammatory diseases i.e. allergies (Yao et al., 2009; Kalliomaki et al., 2010) and cancer (Kumar et al., 2010). Diet which includes food supplements developed with probiotics, prebiotics and synbiotics provides nutritional and therapeutic benefits, helps in attaining the proper human physiology. Awareness of the consumers towards the positive role of foods has been increased over the past decades; make a transformation in food industry for the production of functional foods (Papadimitriou et al., 2015). Benefit of bacterial strains results either from growth and action during manufacturing of foods or from growth and action in the intestinal tract after ingestion of foods (Ali, 2010). For proper maturation and function of the immune system and GIT, the host requires diverse and high number of microorganisms. Homeostasis in the human GIT is maintained by the commensal microflora and changes in this microflora effects the health of the host. Modulating and maintaining commensal flora influences the human health positively. Probiotics which are used as dietary supplements are viable microorganisms with potential for improving the health of human after consumption.
Probiotic microorganisms should be capable of surviving the passage of digestive tract. They must be capable for fast multiplication along with the property of exhibiting acid and bile tolerance. Lactic acid bacteria has inhibitory activity which occurs due to decrease in pH, competition among microbes for substrates and production of antimicrobial substances such as lactic acid, acetic acid, hydrogen peroxide, carbon dioxide and bacteriocins (Indira et al., 2011).
Probiotic bacteria in dairy fermentation help in:
(i) Generation of lactic acid and antimicrobial compounds helps in preservation of the milk
(ii) The production of flavour compounds (acetaldehyde) and other metabolites (extracellular polysaccharides) gives organoleptic property to the product
(iii) The release of free amino acids or the synthesis of vitamins improves the nutritional value of food
(iv) Control of serum cholesterol levels (Parvez et al., 2006).
History of probiotics
Father of medicine and the Greek philosopher Hippocrates wrote: ‘Let food be thy medicine, and medicine be thy food’ (Chow, 2002). The concept of food which has therapeutic benefits has been renamed as “functional foods”. The ingestion of live bacteria by human was
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recorded over 2000 years ago. Tissier (1906), a French paediatrician observed that stool sample of healthy children has more number of bacteria in comparison with children affected with diarrhea. The concept of probiotics was first observed by noble prize winning Elie Metchnikoff in 1907 who hypothesised that the consumption of “soured milk” provide healthy lives of Bulgarian peasants as it exerts beneficial effects on normal gut flora. In 1930s, first clinical trials were performed on patients with constipation. In 1950s, United States Department of Agriculture licensed a probiotic product for the treatment of scour (E.coli infection) among pigs (Orrhage et al., 1994). Kollath (1953) used probiotics on malnourished patients for restoration of the health. Later Ferdinand Vergen (1954) had proposed that the probiotic rich diet could be used to treat the microbial imbalance caused by the antibiotic treatment. The term probiotic was proposed by Lilley and Stillwell (1965) as “microorganisms favoring the growth of other microorganisms” and as an “antonym to antibiotics”. Probiotics are those tissue extracts with the property to promote microbial growth (Sperti, 1971). The Roman historian Plinius explained the treatment of gastroenteritis by administration of fermented milk products (Bottazzi, 1983). Later Fuller (1991) described probiotics as a “live microbial feed supplement which improves the intestinal microbial balance, affecting the host in beneficial terms”. Probiotics are defined as “a live mono or mixed culture of microorganisms which on ingestion gives a beneficial effect in improving the properties of indigenous microflora” (Havenaar and Huis In’t Veld et al., 1992). Guarner and Schaafsma (1998) redefined probiotics as “living microorganisms exerts health benefits beyond inherent basic nutrition when ingest in appropriate numbers”. The definition of probiotics was further reframed as “a preparation containing viable and defined microorganisms in sufficient numbers that are capable to alter the microflora of GI Tract which exerts health benefits in the host”. Probiotic microorganisms which may not be constant inhabitants of the gut but they should provide beneficial effects on the health of man and animal (Holzapfel et al., 2001; Belhadj et al., 2010). Probiotics are “non-pathogenic microorganisms which when ingested in adequate numbers gives a positive influence on the health of the host beyond inherent nutrition (Ouwehand et al., 2002). The joint FAO/WHO (2002) have proposed probiotics as “live microorganisms which when administered in appropriate amounts, give benefit to the health of host”. It helps in preserving the immune-function of human and improving the properties of commensal GI flora. Dr Minoru Shirota marketed Yakult; the fermented milk drink containing a specific strain of Lactobacillus casei Shirota (LeS) for improving the intestinal health (Levin, 2011).
Essential characteristics of probiotics
Requirements for probiotics (Salminen and von Wright, 1998):
1. Survive the location where it is presumed to be active (Resistance to gastric acidity and bile toxicity)
2. Adhesion and proliferation at its active site (for successful colonization)
3. Non-pathogenic and non-toxic
4. Possess the anti-mutagenic or anti-carcinogenic activity
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5. Have genetic stability
6. No plasmid transfer
7. Easy production and reproducible
8. Maintain the viability during processing and storage
Probiotics possess properties like good survival capability during freeze-drying or spray drying, proper growth and viability in fermented food products, phage resistivity and high stability during long-term storage. Ability to reach, survive and persist in the gut environment is the essential determinants for the probiotic organisms (Jankovic et al., 2010).
According to Shobharani and Agarwal (2011), the probiotic strain should be:
1. Capable of exerting a beneficial effect on the host animal
2. Non-pathogenic and non-toxic
3. Able to survive and metabolize in the gut environment
4. Stable
5. Capable of maintaining the viability during storage
6. Able to modulate the immune response
7. Viable cells in large numbers
Mechanism of action
Probiotics exert their effect on host by a hypothetical mechanism (Walker, 2008). According to Kumar and Vaghese (2006), probiotics have direct antagonistic effect against pathogens by acting through production of antimicrobial substances such as cytokines or by reducing gut pH (by stimulation of production of lactic acid bacteria). They compete with pathogenic microorganisms for receptor sites and stimulate immunomodulatory cells. The support mechanisms of the probiotic microorganisms on the host health have not been explained properly (Holzapfel et al., 2001). However, some mechanisms have been hypothesized (Hatcher and Lambrecht, 1993; Ouwehand, 1998; Jacobsen et al., 1999; Boirivant and Strober, 2007; Allan, 2008; Cakir, 2003; Salminen et al., 1998; Fooks et al., 1999)
1. Biochemical effects
a) Production of inhibitory substances include organic acids, hydrogen peroxide, diacetyl and bacteriocins that helps in inhibiting the growth of bacteria (both gram-positive and gram-negative)
b) Production of short chain fatty acids having antagonistic property against pathogenic microorganisms
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c) Reduction in gut pH which creates the unsuitable environmental conditions for survival of pathogens
2. Competition for nutrients
Probiotic microorganisms consume the nutrients which pathogen require for their survival and growth.
3. Stimulating immune system
a) Adherence to pathogens has been reduced by attachment of surface IgA with mucosal membrane
b) Cell mediated response has been stimulated by increasing macrophage phagocytic activity
c) Some studies showed that humoral immune response has been increased as some specific components of cell wall acts as adjuvants
4. Blockage of adhesion sites
Probiotics and pathogenic bacteria are in a competition with each other. Probiotics block the adhesion sites for pathogens to adhere to the intestinal epithelium.
5. Suppression of toxin production
Probiotics degrade the toxin receptors on the intestinal mucosa
Table 2. Established health benefits with their mode of action
Health effects Proposed mechanismRelieve lactose intolerance Break down of lactose into glucose and galactose by
bacterial β-galactosidase that slowly passes via GI transit that shortens the colonic transit time of lactose (Leroy et al., 2008; Heyman, 2000; Hove et al., 1999)
Prevention of bacterial, viral and antibiotic associated diarrhea
Enhancement of local immune defence through specific IgA response, exert a controlling influence on gut microbiota, produce antimicrobial substances, competition for binding sites (Ehrmann et al., 2002; Leroy et al., 2008; Heyman, 2008; Majamaa and Isolauri, 1997)
Prevention of synthesis of colonic carcinogens
Production of antimutagenic compounds, inhibit the transformation of pro-carcinogens to active carcinogens by suppressing the growth of pro-carcinogenic bacteria, altering the metabolic and physiochemical conditions of intestinal bacteria, modulation of host’s immune function, reduce absorption of carcinogens (Hirayama and Rafter, 2000; Rolfe, 2000; Sanders, 1998, Leroy et al., 2008)
Prevention of allergies and atopic eczema
Enhancing immune response due to secretion of serum antibodies IgG, IgA and IgM (Cross, 2002; Perdigon et al., 1999; Majamaa and Isolauri, 1997)
Prevention of pathogenic Production of antimicrobic substances, mucus secretion is
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bacterial infections especially Helicobacter pylori infections
stimulated, modulation of specific and non-specific immune responses, competition for binding sites (Leroy et al., 2008; Elliason and Tatini, 1999; Nurmi and Rantala, 1973; O’Sullivan et al., 2002)
Treatment of irritable bowel syndrome
Reduced production of intestinal gas, stimulation of gut microbiota (Leroy et al., 2008)
Management of heart diseases/ control on cholesterol levels
Digestion and absorption of cholesterol by bacterial cells, bacterial acid hydrolases deconjugates the bile acids, production of hepatic cholesterol synthesis is reduced, binding of cholesterol to bacterial cell walls (Leroy et al., 2008)
Criteria for selection of probiotic strains
There is increasing demand for functional foods or nutraceutical products infused with probiotic bacteria after the safe use of probiotic lactic acid bacteria in fermented food products and positive impacts on human health. Guidelines have been formulated by FAO/WHO (2002), recommending that probiotic strains should be tested for different parameters like antibiotic resistance assays, toxin production, screening for virulence factors and induction of hemolysis. Number of aspects should be considered for the selection of a probiotic strain which include safety, functional and technological aspects (Salminen et al., 1998; Adams, 1999; Saarela et al., 2000). Below are several aspects for selection of a preferable probiotic strain:-
1. It should be non-pathogenic and preferably be of human origin for human use
2. Survival within the GIT: Strains must be able to survive through GI transit and must be active at its site of action. Strains must be able to survive in acidic rich environment of the stomach and bile rich environment of the intestine (Tuomola et al., 2001). Acid tolerance id mediated by membrane ATPases (Lorca and Font de Valez, 2001) and bile resistance is mediated by conjugation of bile salts (Ahn et al., 2003; Ashar and Prajapathi, 1998) or by bile salt hydrolase activity (De Boever et al., 2000).
3. Adhesion property: Strains must adhere to the cell lining of intestinal epithelium, proliferate and colonize its mucosal surface. Prerequisite for adhesion is colonization and for stimulation of immune system (Alander et al., 1997; Tuomola et al., 2001). Mechanism of adherence involves:-
- Receptor specific binding, hydrophobic interaction and adhesion to hydrocarbons (Strus et al., 2001; Wojnicz and Jankowski, 2007)
- Binding to extracellular matrix (Aleljung et al., 1994; Howard et al., 2000; Lorca et al., 2002)
- Adhesion to mucus (Roos and Jonsoon, 2002)
4. Strains must be genetically stable
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5. Strains should remain viable throughout the shelf-life of the fermented products
6. Strain should be non-pathogenic and non-toxic
Table 3 . Desirable criteria for the selection of probiotics in commercial applications (Vasiljevic and Shah, 2008)
Criteria PropertySafety 1. Origin (Human origin for human use)
2. Non-pathogenic3. Non-infectious4. Virulence factors (toxicity, metabolic activity and antibiotic resistance)
Technological 1. Genetically stable strains2. Desirable viability during processing and storage3. Good sensory property4. Phage resistance5. Easy large production
Functional 1. Tolerable to gastric acid and juices2. Bile resistant3. Ability to adhere to mucosal surfaces4. Validated and documented beneficiary health effects
Physiological 1. Immunomodulation2. Antagonism towards gastrointestinal pathogens3. Lactose intolerant4. Reduce levels of cholesterol5. Anticarcinogenic and antimutagenic
Efficacy and technological attributes are assessed after the selection of probiotic strains. Efficacy of the final product is not only indicated by the quality control parameter of colony-forming units per gram but factors such as probiotic growth during manufacturing process, preservation techniques, metabolic state of the probiotic strain and the presence of other functional ingredients at the end of the product plays important role in the effectiveness of the product. More research has to be done to find out in vivo efficacy (Sanders, 2008). For the commercial approval of the probiotic strain, its internal properties, interactions with the host and pharmacokinetics should be accounted (Marteau et al., 1993; Pelletier et al., 1996; Saxelin, 1996).
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Figure 1. Guidelines for the evaluation of probiotics for food use (Collado et al., 2009)
Probiotics and gut health
The gut microbiome- The human gastrointestinal tract is populated by around 500-1000 microbial species. They remain in a complex equilibrium. The human colon is inhabited by 35-50% of the bacterial content. These bacteria include Bacteroides, Bifidobacterium, Clostridium, Eubacterium, Escherichia, Fusobacterium, Lactobacillus, Peptobacillus, Peptococcus and Veillonella. The microbial content is stable during adulthood. Although, the microbial pattern are unique to each individual. The microbial component of the gut has been influenced by many external factors such as diet, age, host genetics, antibiotic treatments and other organisms such as probiotics (Collado et al., 2009). The gut microbiota is
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STRAINIDENTIFICATION
Genus, Species and strainInternational Culture Collection
FUNCTIONALCHARACTERIZATION
In vitro testand/or animal
SAFETYASSESMENT
In vitro test and/or animalPhase 1 Human study
EFFICACY ASSESMENTPhase 2 Human study
Double blind randomized,placebo
EFFECTIVENESSASSESMENT
Phase 3 Human StudyCompare probiotic with standardtreatments of a specific condition
heterogeneous. It shows a continuation, that starts from stomach and duodenum (101 to 103 bacteria per gram), progressing to jejunum and ileum (104 to 107 bacteria per gram) and ends at colon (1011 to 1012 bacteria per gram). This is known is longitudinal heterogeneity. The microbiota of the GI Transit also shows latitudinal variations the microflora of the intestinal lumen differs from the microflora embedded in the mucus layer and the epithelial surface. (Sekirov et al., 2010).
Figure 2a. Composition across the length of the gastrointestinal tract
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ProximalGI tract
LactobacillusVeillonella
Helicobacter
BacilliStreptococcaceae
ActinobacteriaActinomycinaceaeCoryoebacteriacea
e
LachnospiraceaeBacteroides
Stomach
Duodenum
Jejunum
Ileum
Colon
Incr
easi
ng n
umbe
rsIn
crea
sing
div
ersi
ty
DistalGI tract
101
103
107
1012
cells/gm
104
Figure 2b. Longitudinal variations in microbial compositions in the intestine
The mucosal surface of the intestinal epithelium is the largest of the human body. It is the main surface of contact with the exogenous agents such as foreign microbes or antigens obtained from digested food. The gut epithelium acts as a natural resistance factor. The intestinal mucosal cells helps in attachment of probiotic bacterial cells with the host cells via some receptors which leads to competitive exclusion of pathogenic microorganisms and inhibit their colonization at the binding site, thereby protecting the host against infection (Marco et al., 2006). For instance, probiotic strains inhibit the binding of Escherichia coli by binding to intestinal epithelium through mannose receptors.
The mucosal surfaces of GIT are coated by a hydrated gel formed mucin. They are secreted by special epithelial cells (gastric foveolar mucous cells) and intestinal goblet cells, either localized to the cell membrane or secreted into the lumen to form mucous membrane (Turner, 2009). This gel layer acts as the first barrier and gives protection to the epithelium against harmful microorganisms as the pathogens are not able to penetrate the mucous membrane and does not get access to the epithelial cells (Ohland and MacNaughton, 2010). Probiotics increases the mucous secretion to improve the barrier function (Mack et al., 2003; Mattar et al., 2002).
The gut of the foetus is sterile until birth (Mack et al., 2013). A colonization of the gut with microbes starts at birth especially Bifidobacteria (Wolin et al., 1998) from the maternal vagina and external environmental factors (Mutai and Tanaka, 1987). The microbial content of human intestine is very simple during the first year of life. After that, the gut resembles to that of the young adult (Sekirov et al., 2010). The composition of human faecal flora changes with the changes in age (Naidu et al., 1998). Detection of Clostridium paraputrificum and Bacteroides fragilis had been isolated from the fecal sample on the 1st and 2nd day of life respectively. Bifidobacterium species were detected from the faecal sample of 3rd to 7th day of age, accounting from 1010 to 1011 organisms per gram of faeces (Benno and Mitsuoka, 1986).
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Epithelial Surface Mucus Layer Intestinal Lumen
ClostridiumLactobacillusEnterococcus
BacteroidsBifidobacteriumStreptococcus
EnterobacteriaceaeEnterococcusClostridium
LactobacillusRuminococcus
Medical importance: Protection against diseases
1. Gastrointestinal tract problem
A large number of reviews suggest that probiotic therapy decreases the duration and severity of gastric and intestinal illnesses (Pedone et al., 1999; Huang et al., 2002; Weizman et al., 2005). Lactobacillus GG, Lactobacillus reuteri, Saccharomyces boulardii, Bifidobacteria species shows significant benefit in the treatment of diarrhoea (Isolauri et al., 1991; Gorbach, 2000; Hilton et al., 1977), traveller’s diarrhoea (Hilton et al., 1977), diarrhoea caused by rotavirus (Saavedra et al., 1994; Vanderhoof, 2000) and diarrhoea in children (Hilton et al., 1977; Saavedra et al., 1994; Salminen et al., 1996; Gorbach, 2000; Isolauri et al., 1991).
Probiotics helps in treating viral diarrhoea by increasing the concentration of antibodies like secretory IgA, IgM and IgG, decreasing viral shedding and enhancing immune response (Kimura et al., 1997; Grangette et al., 2001). Probiotics can prevent diarrhoeal infection by competing with pathogenic microorganisms for binding sites present on intestinal epithelium (DeSimone, 1986; O’Sullivan et al., 1992). Production of bacteriocins (nisin) by probiotic bacteria inhibits the growth of pathogenic bacteria (Dodd and Gasson, 1994).
Inflammatory Bowel Disease (IBD) is chronic inflammation of terminal ileum. It includes Crohn’s disease and Ulcerative colitis. Causal factors are environmental, genetic susceptibility and immune dysfunction (Shanahan, 2004). Probiotic therapy helps in restoration of epithelium barrier by reducing the secretion of TNF-α and INF-X (Madsen et al., 2001). DNA of probiotic bacteria produces anti-inflammatory effect by giving signals through TLR9 (Rachmilewitz et al., 2004).
2. Lactose intolerance
This disorder occurs due to decline in the activity of lactase which causes lactose malabsorption. The enzyme lactase is present within the brush border mucosa of intestinal epithelium. Symptoms of the lactose malabsorption include flatus, bloating, nausea, abdominal cramps and diarrhoea. Yogurt containing Lactobacillus bulgaricus and Streptococcus thermophilus helps in the relieving the symptoms and increase lactase in lactase-activity deficient individuals (Kim and Gilliland, 1983; Kolars et al., 1984; Alm, 1982; Kilara and Shahani, 1975). ). β-galactosidase catalyzes the hydrolysis of lactose into glucose and galactose. β-galactosidase activity is important for lactose digestion (Lin et al., 1991). This enzyme is used in sweetened, condensed and frozen dairy products for avoiding lactose crystallization (Artolozaga et al., 1998). Microbial activity of β-galactosidase partly helps in the digestion of lactose from yogurt containing Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus salivarius ssp. thermophilus (Martini et al., 1991). Syal and Vohra (2013) qualitatively determined β-galactosidase activity of twenty probiotic yeast strains isolated from traditional Indian fermented foods. All of the selected strains were considered positive by showing blue colour production on agar plates containing IPTG (Isopropyl β-D-1-thiogalatopyranoside, Sigma) and X-gal (5-bromo-4-chloroindolyl-β-D-galactopyranoside, Sigma) solutions. Only one strain J 18 was tested negative for β-galactosidase activity.
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3. Cancer
Probiotics suppresses the growth of bacteria which ultimately reduces the production of enzymes that convert pro-carcinogens into carcinogens (Hosoda et al., 1996; Aso and Akazan, 1992). Probiotic bacteria might bind to mutagenic compounds in the intestine (Motta et al., 1991; Lidbeck et al., 1992; Murch, 2001; Isolauri, 2004) and helps in prevention or recurrence of tumour (Aso and Akazan, 1992). Cause of colorectal cancer (CRC) is associated with the environmental factors mainly the diet. Fermented milks containing probiotic cultures have a potential protective role in preventing CRC (Rowland, 2004; Saikali et al., 2004). Lactobacillus acidophilus superstrain DDS1 exhibited the strongest anti-tumour activity (Shahani, 1983). Fermented milk containing Lactobacillus delbrueckii ssp. bulgaricus produced antimutangenic activity against an mutagen 4NQO in vitro assay (Hosono, 1986). Lactobacillus casei and Lactobacillus lactis showed antimutangenic activity towards aberrant crypts of putative pre-neoplastic lesions (Marotta et al., 2003; Pool-Zobel et al., 1993).
4. Allergies/ Eczema
In patients with atopic eczema, food allergy, allergic rhinitis and bronchial asthma; probiotic bacteria helps in reducing the inflammatory cytokines characteristic of local and systemic inflammation related to hypersensitivity reactions (Majamaa and Isolauri, 1997; Isolauri et al., 1992; Isolauri et al., 2000; Isolauri, 2004). Occurrence of eczema decreases by one-half in at-risk infants who were administered perinatally by Lactobacillus rhamnosus GG (Isolauri, 2000). Probiotics helps in treating food allergy and reducing intestinal inflammation by enhancing the endogenous barrier mechanism of the gastrointestinal tract (Kalliomaki and Isolauri, 2004). Anti-inflammatory cytokines such as interleukin-10 has been produced by probiotic bacteria (Pessi et al., 2000) which enhances immunostimulatory effect in healthy people and reduces immunoinflammatory effect in hypersensitive patients.
5. Hepatic disease
Hepatic encephalopathy is a life threatening liver disease. The probiotics Streptococcus thermophilus, Bifidobacteria, Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus delbrueckii shows the disruption of pathogenesis of hepatic encephalopathy and reduces the portal pressure that ultimately helps in reducing the bleeding (Nanji et al., 1994; Gorbach, 2000; Shanahan, 2001; Solga, 2003).
6. Cholesterol lowering and control of hyperlipidaemia
Cholesterol is essential for human body as it acts as a precursor for some vitamins and hormones. It is an important component of cell membrane and nerve cell. Cholesterol helps in replacing lost bile acid molecule during excretion by converting itself to bile acids, leading in reducing the serum cholesterol level. This conversion is operated by colonic microbes and lactic acid bacteria with active bile salt hydrolase (BSH) (De Smet et al., 1998). Probiotic bacteria are able to produce short chain fatty acids in the intestine by the carbohydrate fermentation. These short chain fatty acids inhibit the synthesis of cholesterol in liver and redistribute it from plasma to the liver. Some strains of probiotic bacteria inhibit the
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absorption of cholesterol from the gut by deconjugation of bile salts (Gilliland et al., 1985; De Boever et al., 2000; Ahn et al., 2003). Although, high plasma level of cholesterol causes coronary heart disease and myocardial infarction. Some studies showed that probiotic bacteria could reduce the serum cholesterol and LDL (low density lipoprotein) cholesterol.
Consumption of Lactobacillus sporogenes by hyperlipidaemia patients for 3 month period showed 32% reduction in total cholesterol and 35% reduction in LDL cholesterol (Mohan, 1990). Some studies showed that Serum cholesterol level can be lowered by dairy fermented products containing several strains of probiotic bacteria (Larsen et al., 2000; Sindhu and Khetarpaul, 2003; Parvez et al., 2006). Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, Micrococcus luteus and Staphylococcus aureus had been isolated from cow milk and investigated for cholesterol lowering capacity. Lactobacillus plantarum was found to be an effective isolate for cholesterol reduction (Amutha and Kokila, 2015). Lactobacillus acidophilus (P106, P110), Lactobacillus plantarum (P164) and Lactobacillus pentosus (P191) isolated from Egyptian infants. They were tested for the capability of lowering the serum cholesterol. Strains which were grown in broth supplemented with 0.2% bile salts show good capability in comparison with those strains grown in simple broth (Mahrous, 2011). Lactobacillus brevis BCG 07-28 isolated from Croatian Fresh Soft Cheese and Serbian White Pickled Cheese showed the highest percentage of cholesterol assimilation in the presence of bile salts (Uroic et al., 2014).
7. Infection of genitourinary tract
Four different strains of lactobacilli showed inhibitory activity against bacterial species isolated from women having recurrent infection of bacterial vaginosis. Recurrent urinary tract infection can be controlled by both oral probiotics and vaginal suppositories of probiotics (McLean and Rosenstein, 2000). Vaginitis caused by Candida can be reduced three-fold by yogurt containing Lactobacillus acidophilus (Hilton et al., 1992).
8. Infection of Helicobacter pylori
The infection is associated with gastritis, peptic ulcers and gastric cancer (Aiba et al., 1998; McFarland, 2000). Helicobacter pylori growth can be inhibited by Lactobacillus salivarius. Urease production by Helicobacter pylori has been reduced by high levels of lactic acid produced by Lactobacillus resulting in poor colonization (Aiba et al., 1998). Lactobacillus johnsonii also showed inhibitory activity towards Helicobacter pylori (Michetti et al., 1999; Marteau et al., 2001).
Probiotic attributes of lactic acid bacteria
1. Acid tolerance
Human stomach secretes approximately 2.5 litres of gastric juice each day which makes the pH as 1.5 (Lankaputhra and Shah, 1995). The pH of stomach secretions can be as high as 6.0 or above after ingestion of food (Johnson, 1977), but ranges from pH 2.5 to 3.5 (Holzapfel et al., 1998). The transit of food through the human stomach depends upon the nature of food and average time of food transit is about 90-180 minute (Begley et al., 2005). Liquids leave
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the stomach faster than solids and takes about 20 minutes to pass through the stomach (Gastro Net Australia, 2001). Acid tolerance of Lactobacillus, Bifidobacterium and propionibacterial strains were used for screening in the range of pH 1.0 to 5.0 (Conway et al., 1987; Lankaputhra and Shah, 1995; Chou and Weimer, 1999; Chung et al., 1999; Zarate et al., 2000). Lactobacillus acidophilus has more acid and bile tolerance than other lactic acid bacteria (Mirlohi et al., 2008). Intracellular pH (pHi) of most LAB decreases as the extracellular pH (pHo) decreases due to build up of acidic end products. Acid tolerance response (ATR) is inducible mechanism where the bacteria acquire the ability to survive lethal acid conditions on exposure to mild acidic conditions (Goodson and Rowbury, 1989). It was observed in Lactococci (Kashket and Kashket, 1985), Lactobacilli and Leuconostoc mesenteroides (McDonald et al., 1990), Clostridia (Baronofsky et al., 1985), Listeria monocytogenes (Kroll and Patchett, 1992).
Lactobacillus acidophilus had been studied for acid tolerance. It was incubated in artificial gastric juice (prepared by supplementing MRS broth at pH 2.5 with pepsin) for 1, 2 and 3 hour. It was observed that Lactobacillus acidophilus survived under high acidic conditions (Oh et al., 2000). Lactobacilli species were isolated from yogurt and observed their capability to survive at extreme conditions (acidic pH 2.5-3.5 and basic pH 7.5-8.5) (Hoque et al., 2010). Lactic acid bacteria isolated from khadi was tested for acid tolerance. 1 ml of bacterial isolate was inoculated with 9 ml of sterile MRS broth at pH 3.5 with 0.5 N HCl. After incubation period of 4 hours, the survival rate with optical density of 0.280 at 600 nm observed to be 87.74% at pH 3.5 (Sukumar and Ghosh, 2010). Lactic acid bacteria was grown in MRS broth at cell density adjusting to A620 of 2.0 and at pH 2.5 with 2 M HCl. After 90 minutes of incubation, it was found that isolates Di7, BRMV1 and HML1 presented viability ˃90% after 30, 60 and 90 minutes of exposure to pH 2.5 respectively (Sadrani et al., 2014). Varsha et al., (2014) isolated the sample from rotten jackfruit and guava, tiger puke, Indian gaur and deer faeces and studied their ability to survive and tolerate the environment at 2.0.
2. Bile tolerance
Bile salts are surface active agents and amphipathic molecules. Bile acids are products of cholesterol metabolism and synthesized in liver. It is secreted in conjugated form (either with glycine or taurine) from gall bladder to duodenum (500-700 ml/day). Bile acids play an important role in digestive process (emulsification of fat). Bile concentration of intestine is 0.3% w/v (Prasad et al., 1998). The average time of food transit through the small intestine varies generally from 1-4 hour (Smith, 1995). pH of small intestine is about 8.0 (Keele and Neil, 1965). Presence of bile salts and pancreatin makes adverse conditions for survival in small intestine (Floch et al., 1972; Levay, 1988). The probiotic bacteria must exhibit bile salt hydrolase activity for the tolerance of bile salts (De Smet et al., 1994; Moser and Savage, 2001). Some studies showed that bile salt resistance cannot be associated with this enzyme in lactobacilli (Gilliland and Speck, 1977; Moser and Savage, 2001; Schmidt et al., 2001). Bile salt resembles that of detergents such as SDS and disrupts the phospholipids and proteins of cell membranes and affects the cellular homeostasis. Therefore, for the survival and colonization; the pathogenic and commensal bacteria have to tolerate bile in gastrointestinal
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tract (Begley et al., 2005; Begley et al., 2001). For selecting probiotic bacteria for human use, a concentration of 0.15-0.3% of bile salt has been suggested (Goldin and Gorbach, 1992).
Different concentrations of bile salts are inoculated in selective growth medium for testing the survivability of lactic acid bacteria (Gilliland et al., 1984; Chung et al., 1999; Clark and Martin, 1994). Five strains of Lactobacillus acidophilus were grown in selective medium with 0.3% bile acids. After incubation of 24 hours, all five strains exhibit bile tolerance (Oh et al., 2000). Lactobacillus species isolated from yogurts tolerated bile acids at 0.3% (Houque et al., 2010). Lactic acid bacteria grown in MRS broth containing 0.3, 0.5 and 1% sodium deoxycholate. It was subjected to incubation for 24 hours at 37oC. Lactic acid bacteria showed growth in 0.3 and 0.5% bile salt solutions but no reduction in growth was seen at 1% (Bhattacharya and Das, 2010). Conjugated bile acids show less inhibition than free bile acids towards intestinal bacteria (Floch, 1972). 2% bile salt concentration had been adopted by Leuconostoc paramesenteroides after sub-culturing for 3-4 generations. After 15 days of incubation and storage, the strain was observed and showed no variations in cell viability (Shobharani and Agarwal, 2011). Lactic acid bacteria strains were treated with 0.1, 0.2 and 0.3% bile salt for 1.5 or 3.0 hours respectively. Highest viable counts and survival rates were estimated for Lactobacillus casei BCRC 14023 (5.94 - 7.11 log CFU/ml; 0.54 – 7.99%). Lactobacillus acidophilus BCRC 10695 had higher survival rates (2.79 – 4.23 log CFU/ml; 0.00035 – 0.0093%) than that of Bifidobacterium bifidum BCRC 14615 (0 – 2.57 log CFU/ml; 0 – 0.0002%) at 0.2 and 0.3% bile salt concentration. No difference in survival was observed at 0.1% bile salt concentration (Huang et al., 2014).
3. Adherence property
Bacterial adhesion to the host cell is the important stage. Cell membrane of probiotic strain must have the ability to adhere to the intestinal surface. The physio-chemical property of the cell surface depends upon hydrophobicity (due to the surface components of bacterial cell) and electrical mobility (due to bacterial surface charges). Initially the interactions appear between two surfaces are non-specific and then it change into a specific interaction between adhesion protein and complementary receptors (Beachey, 1981). Probiotic strains which are well adhered to the hydrocarbons are reviewed as hydrophobic and strains which are poorly adhered are reviewed as hydrophilic (Duary et al., 2011). Gastrointestinal cell surface constituents (glycoconjugates) serves as receptors for bacterial adherence (Servin and Coconnier, 2003; Pretzer et al., 2005). Epithelial cells of gastrointestinal tract constitute a family of transmembrane receptors that is host pattern recognition receptors (PRRS) such as toll-like receptors (TLR). These receptors help in recognizing repetitive patterns i.e. pathogen associated molecular patterns present in various microbes (Takeda et al., 2013; Backhed and Hornef, 2003).
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Table 4. Some receptors along with their functions
Recognition receptors Function
TLR 2 Responds to microbial components of gram positive bacteria such as peptidoglycan and lipoteichoic acid (Takeda et al., 2003; Matsuguchi et al., 2003)
TLR 4 Activated by lipopolysaccharide and gram negative bacteria (Takeda et al., 2003; Matsuguchi et al., 2003)
TLR 5 Recognize bacterial flagella (Rhee et al., 2005)
TLR 9 Responds to short DNA fragments of bacteria which contain CpG sequences (Pedersen et al., 2005)
Nucleotide binding oligomerization domain proteins (located in cell cytoplasm)
Recognizes both gram negative and gram positive bacteria and helps in production of defensins (Lu and Walker, 2001; Berkes, 2003)
The main site for bacterial adhesion and colonization is the epithelial cells which are covered by a layer of mucus and it helps in ingestion of microorganisms passing through the mammalian gut (Mikelsaar et al., 1998). The major component of mucus is mucin glycoproteins and is degraded continuously. New mucin are constantly secreted. There is poorly known mechanism which involves attachment of probiotic strains with the mucus glycoproteins. Some studies reviewed that bacterial proteins are involved in adhesion as the adhesion ability was decreased by protease treatment (Chauviere et al., 1992; Coconnier et al., 1992; Bernet et al., 1993; Greene and Klaenhammer, 1994; Adlerberth et al., 1996). The bacteria that adhere to the mucus but unable to reach the epithelial cells are removed from the mucosal surface with the degraded mucin. Adherence of bacterial strains with intestinal mucus can be evaluated by immobilization of commercially available mucin on micro-well plate surface (Tuomola et al., 1999; Izquierdo et al., 2008). Lactobacillus amylolyticus, Lactobacillus panis and Lactobacillus pontis were isolated from wet wheat distillers’ grain and observed under inverted microscope about the capability to bind to gastric mucin of pig and proved their ability for colonization of the gastrointestinal tract of pigs (Pedersen et al., 2004). Balakrishna (2013) used crystal violet method for determining the adhesion ability of four strains isolated from the skin, gill, gut and intestine of common guppy, Poecilia reticulata and five indicator strains. The results revealed that the selected strains adhered well to the intestinal mucus than the indicator strains.
Antimicrobials from lactic acid bacteria
Lactic acid bacteria exerts antagonistic activity towards intestinal and food borne pathogens by producing antimicrobial substances such as organic acids, free fatty acids, diacetyl, ammonia, hydrogen peroxide and bacteriocin (Gibson et al., 1987; Vandenbergh, 1993; Jack et al., 1995).
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According to Havenaar et al., (1992) and Sanders (1993), the antagonistic properties shown by lactic acid bacteria may be exhibited by
1. Producing some inhibitory substances such as bacteriocins
2. By decreasing the redox potentials of intestinal lumen
3. Producing hydrogen peroxide
4. Decreasing the pH of intestinal lumen by producing short chain fatty acids (SCFA) like acetic acid, lactic acid or propionic acid
5. Providing specific nutrients to pathogens
Lactic acid bacteria produce antimicrobial substances which are of two types:
a) Low molecular mass substances – These substances have low molecular mass of < 1000 Daltons. For example- Non-bacteriocin antimicrobial substances
b) High molecular mass substances – These substances have molecular mass of ˃ 1000 Daltons. For example- Bacteriocins
Low molecular mass antimicrobial substances
1. Organic acids
The most important antimicrobial substances produced by lactic acid bacteria are lactic acid and acetic acid. Inhibitory effects of organic acids are caused by
a) Undissociated form of the molecule (Hydrogen ions) which diffuses from cell membrane to cytosol interferes with the metabolic functions of the cell such as substrate translocation and oxidative phosphorylation
b) Acidic pH – Accumulation of organic acids reduces the intracellular pH (Ingram et al., 1956; Kashket, 1987; Baird-Parker, 1980; Lorca and Valdez, 2009)
On the basis of dissociation constant, acetic acid (pK 4.75) has more bactericidal activity than lactic acid (pK 3.86) (Rasic and Kurmann, 1983). Enterococcus faecalis strains isolated from meconium inhibited the growth of gram-positive as well as gram-negative bacteria. The inhibitory activity was shown through the production of lactic acid bacteria (7.06g/l after 24 hours of incubation) (Atya et al., 2015).
2. Hydrogen peroxide (H2O2)
Through electron transport chain (flavoprotein oxidase or nicotinamide adenine dinucleotide reduced peroxidase), lactic acid bacteria produce hydrogen peroxide in the presence of oxygen (Emitan et al., 2011). Afterwards, destructive hydroxyl radicals form superoxide (O-2) and hydroxyl (OH-) anions in the presence of H2O2. These superoxide and hydroxyl anions lead to the process of per-oxidation of membrane lipids (Morris, 1979) and increased membrane permeability (Kong and Davidson, 1980). The bactericidal activity of these
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oxygen metabolites has strong oxidizing effects on the bacterial cell such as nucleic acids and cell proteins (Dahl et al., 1989; Lindgren and Dobrogosz, 1990; Piard and Desmazeaud, 1992). These oxidizing properties attributed to the anti-microbial activity of H2O2.
MRS agar plates enriched with 0.25 mg/ml of tetramethylbenzidine (TMB) and 0.01 mg/ml of horseradish peroxidase and tested for H2O2 production. Lactobacillus gasseri strains produced less H2O2 under aerobic conditions and Lactobacillus vaginalis and Lactobacillus reuteri strains produced H2O2 under both aerobic and anaerobic conditions (Delgado et al., 2015). Pediococcus species isolated from Traditional Fermented Cereal Gruel and Milk in Nigeria and studied for H2O2 production. Pediococcus pentosaceus CM 1 showed the highest value of H2O2 (5.12 ml/L), Pediococcus acidilactici OB 4 and Pediococcus pentosaceus SM 3 showed same H2O2 value (4.84 mg/L) while Pediococcus pentosaceus WO 3 showed least H2O2 value (4.64 mg/L) (Banwo et al., 2013). Hydrogen peroxide production was tested at 24 h, 48 h, 72 h and 96 h by titrated with 0.1 M potassium permanganate (KMnO4). Highest H2O2 production was observed by Leuconostoc mesenteroides FL 15 at 48 h (0.034 g/L) and the lowest quantity was observed by Lactobacillus brevis FL 18 and Pediococcus acidilactici FL 6 (0.013 g/L) (Wakil and Osamwonyi, 2012).
3. Diacetyl, acetoin and acetaldehyde
Decarboxylation of pyruvate produces active acetaldehyde by heterofermantative lactic acid bacteria. This acetaldehyde condenses with pyruvate, form α-acetolactate and then it is converted to diacetyl by an enzyme α-acetolactate synthases. Acetoin is a reducing form of diacetyl (Collins et al., 2009; Jyoti et al., 2003). Chemically diacetyl is 2,3-butanedione and it imparts buttery aroma to fermented dairy products. It is used in high concentrations for preservation of food. Acetaldehyde used in fermented dairy products helps in controlling the growth of spoilage organisms by inhibiting the growth of undesirable microbes (Venderbergh, 1993).
4. Carbon dioxide (CO2)
CO2 has antifungal activity. It replaces existent molecular oxygen by creating an anaerobic environmental condition. Dysfunction in lipid bilayer membrane permeability occurs by the accumulation of CO2 (Lindgren and Dobrogosz, 1990).
5. Reuterin and reutericyclin
Lactobacillus reuteri produces two substances i.e. reuterin and reutericyclin. Reutericyclin is active towards gram-positive bacteria and is a tetrameric acid derivative of β-hydroxypropionaldehyde. Reuterin is active towards gram-negative, fungi and protozoa and is a mixture of monomeric, hydrated monomeric and cyclic dimeric forms of β-hydroxypropionaldehyde (Kuleasan and Çakmakçi, 2002; Ganzle and Vogel, 2003; Leroy et al., 2006).
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Table 5. Antimicrobial metabolites of lactic acid bacteria (Vanderbergh, 1993; Lindgren and Dobrogosz, 1990; Kashket, 1987; Daechel, 1989; Helander et al., 1997; Knorr, 1998; Lorca and Valdez, 2009)
Antimicrobial substances Microorganisms producers
Lactic acid All lactic acid bacteria
Acetic acid Heterofermentative lactic acid bacteria
Diacetyl, Acetaldehyde and Acetoin
Genera of lactic acid bacteria includes: Lactococcus, Leuconostoc, Lactobacillus and Pediococcus
Hydrogen peroxide All lactic acid bacteria
Carbon dioxide Heterofermentative lactic acid bacteria
Reuterin Lactobacillus reuteri
Reutericyclin Lactobacillus reuteri
Cyclic dipeptides Lactobacillus plantarum, Lactobacillus pentosus
3-phenyllactic acid4-hydroxyphenyllactic acid
Lactobacillus plantarum, Lactobacillus alimentarius,Lactobacillus rhamnosus, Lactobacillus sanfranciscensis,Lactobacillus hilgardii, Leuconostoc citreum, Lactobacillus brevis, Lactobacillus acidophilus, Leuconostoc mesenteroides
Lactobacillus plantarum
Benzoic acid methylhydantoinmevalonolactone
Lactobacillus plantarum
High molecular mass antimicrobial substances
Bacteriocins
Lactic acid bacteria produce antimicrobial proteinaceous substances having bactericidal activity against related species (showing narrow spectrum of activity) or across genera (showing broad spectrum of activity) (Rogelj and Bogovič-Matijašič, 1994; Cotter et al., 2005). Bacteriocins are ribosomally synthesized single polypeptides with antimicrobial activity produced by many gram-positive and gram-negative bacteria, whereas producer bacterial cells are immune to their own bacteriocins (Klaenhammer, 1998; De Vuyst and Vandamme, 1994; Chen and Hoover, 2003). Production of bacteriocins can either be spontaneous or induced. The genetic determinants of bacteriocins are mostly located on plasmids but can also be chromosomally encoded. Lactic acid bacteria must have detergent resistant phospholipase A in its outer membrane for the release of bacteriocins.
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Mode of action of bacteriocins
Lethal activity of antimicrobial agents are exerted through
1. Adsorption to specific receptors present on the external surface of microbes. This adsorption leads to morphological, biological and metabolic changes which results in killing of the bacteria (Toomula et al., 2011; Alakomi et al., 2000; Elliason and Tatini, 1999)
2. Bacteriocin electrostatically binds to the anionic surface and leads to increase in local concentration which disturbs lipid dynamics of the membrane and causes localized membrane strain. This forces the entry of the nisin molecule into the membrane (Driessen et al., 1995). The entry of nisin molecule produces the voltage-dependent pores, leading to the dissipation of PMF (Proton motive force). PMF helps in the synthesis of ATP and transport of ions and with the loss of this force, energy dependent reactions got depleted and cell death occurs (Breukink and De Kruijff, 1999; Klaenhammer, 1993).
3. Bacteriocin (nisin) interacts with cell wall precursors (Lipid I and lipid II) and inhibits synthesis of peptidoglycan (Wiedemann et al., 2004).
4. Bacteriocin prevents post-germination swelling and spore outgrowth by inactivating endospore formation (Hitchins et al., 1963; Thomas et al., 2000)
5. Inactivation of the gram-negative bacterial cell membrane in concurrence with antimicrobial environmental factors such as organic acid, low temperature and detergents
6. Modulation of enzyme activity (Chen and Hoover, 2003; Moll et al., 1999)
7. Inhibition of outgrowth of spores (Chen and Hoover, 2003; Moll et al., 1999)
Classification of bacteriocins
There are many proposed classification of bacteriocins divided into 3 or 4 classes. According to Klaenhammer (1993), bacteriocins are classified into four groups. This classification is based on numerous factors such as molecular mass, thermo-stability, enzymatic activity, mechanism of action and presence of modified amino acids.
Class I bacteriocins: Lantibiotics: These are small (<5kDa), heat-stable, membrane active single and two peptides bacteriocins, produced by many gram-positive and gram-negative bacteria. They contain unusual amino acids such as lanthionine, dehydroalanine, α-dehydroalanine and dehydrobutyrine. Post-translational modification occurs in biologically inactive pre-peptides of these bacteriocins (Klaenhammer, 1988; De Vuyst and Vandamme, 1994; Chen and Hoover, 2003).
On the basis of their mode of action and chemical structure, Lantibiotics are divided into two subgroups:
Type A Lantibiotics – They consists of elongated, amphiphilic peptides having cationic properties. They have screw shaped structure. They have unspecific interaction with the
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target cell membrane and produce voltage-dependent pores, leading to dissipation of membrane potential (Klaenhammer, 1993).
Example- nisin, lactocin S, lacticin 481 (Cotter et al., 2005; De Vuyst and Leroy, 2007; Klaenhammer, 1993; Nes et al., 1996)
Type B Lantibiotics – They consists of smaller peptides having anionic or neutral properties. They have globular shaped structure. They are active through the inhibition of specific enzymes (Cotter et al., 2005; Chen and Hoover, 2003; Jung, 1991).
Example- mersacidin (Cotter et al., 2005; De Vuyst and Leroy, 2007; Klaenhammer, 1993; Nes et al., 1996)
Class II bacteriocins: It encompasses non-lanthionine bacteriocins. It is a heterogeneous group of heat-stable, small (<10kDa) and non-modified peptides. It is further divided into 3 subgroups:
Class II a – It consists of pediocin-like peptides showing the consensus sequence –Tyr-Gly-Asn-Gly-Val-Xaa-Cys-. These peptides are anti-listerial, having a specific activity towards a food pathogen Listeria monocytogenes (Ennahar et al., 2000; Klaenhammer, 1993). Their antimicrobial activity depends on mannose permease of the phospho-transferase system (PTS) as a target (Cotter et al., 2005). This group has a large potential in food industry.
Example- pediocin PA1, sakacin A, sakacin P, leuocin A, curvacin A (Cotter et al., 2005; De Vuyst and Leroy, 2007; Klaenhammer, 1993; Nes et al., 1996)
Class II b – These consists of two different peptides for this activity and thus are known as two-peptide bacteriocins. They act by forming cation or anion specific pores which leads to dissipation of the proton motive force (PMF) (Cotter et al., 2005).
Example - lactococcin G, lactococcin M, lactacin F, plantaricin A (Cotter et al., 2005; De Vuyst and Leroy, 2007; Klaenhammer, 1993; Nes et al., 1996)
Class III c – It contains remaining peptides of this class. They are circular bacteriocins. It includes sec-dependent bacteriocins (Chen and Hoover, 2003). These have various mode of action such as pheromone activity of target cells, membrane permeabilisation and cell wall formation by inhibition of septum formation (Cotter et al., 2005).
Example- acidocin B, enterocin P, enterocin B, reuterin 6 (Cotter et al., 2005; De Vuyst and Leroy, 2007; Klaenhammer, 1993; Nes et al., 1996)
Class III bacteriocins: Bacteriolysins: These are large bacteriocins having molecular mass of ˃30 kDa. They are heat-labile peptide antibiotics with domain-type structure. They catalyze the hydrolysis of cell wall and leads to the lysis of sensitive cells (Cotter et al., 2005).
Example- lysostaphin, ebterolysin A, helveticin J, helveticin V-1829 (Cotter et al., 2005; De Vuyst and Leroy, 2007; Klaenhammer, 1993; Nes et al., 1996)
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Class IV bacteriocins: They are complex bacteriocins consists of either glycoproteins or lipoproteins (Zhao et al., 2010; Kozak et al., 1977). They require non-proteinaceous moieties like carbohydrates or lipid for their activity (Klaenhammer, 1993).
Example- plantaricin S, leuconocin S, lactocin 27, pediocin SJ1 (Cotter et al., 2005; De Vuyst and Leroy, 2007; Klaenhammer, 1993; Nes et al., 1996)
Bacteriocin production by Lactobacillus species suppresses the growth of Escherichia coli ATCC 25922 and Bacillus subtilis NCIB3610. Optimum conditions were pH 6.0, temperature 34oC with 4% phenyl acetamide showing the maximum growth inhibition zones . MRS medium supplemented with 1% K2HPO4, 1% Tween 80, 1% beef extract, glucose, cysteine and 1% peptone extract helps in the synthesis of large amount of bacteriocin from Lactobacillus acidophilus isolate CH1 (Mahrous et al., 2013).
Table 6. Bacteriocins produced by lactic acid bacteria (Dave and Prajapathi, 1994)
Bacteriocins Microorganisms producer
Fermenticin Lactobacillus fermentiPlantacin B Lactobacillus plantarumLactocidin Lactobacillus acidophilusAcidphilin Lactobacillus acidophilusAcidolin Lactobacillus acidophilusNisin Lactococcus lactis Lactococcin I Lactococcus lactis ssp. cremoris Lactoccin Lactococcus lactis ssp. lactisMesenterocin Leuconostoc mesenteroides Leucocin S Leuconostoc paramesenteroidesCarnocin Leuconostoc cornosumPediocin AcH Pediococcus acidolacticiPediocin A Pediococcus pentosaceus
Bacteriocin-producing lactic acid bacteria in food industry
Bacteriocins are gaining the immediate attention in food industry as bio-preservatives and they can be used in food products without any concentration or purification (Cotter et al., 2005). Bacteriocin-producing starter cultures are used in industries as they inhibit the growth of autochthonous lactic acid bacteria that produces off-flavour and contribute to food safety. This property leads to a more controlled and standardized fermentation process by improving the competitiveness of the starter cultures (Leroy et al., 2006; Ross et al., 2000; De Vuyst et al., 2004; Beganović, 2005). Bacteriocin-producing adjunct cultures have bacteriocinogenic property along with the property of giving a characteristic flavour of traditional fermented products. Example- Lactococcus lactis strain isolated from raw ewe’s milk, produces both nisin and lacticin 481 and used as adjunct culture in the manufacturing of dairy products to destroy the growth of undesirable microorganisms (Bravo et al., 2009). Adjunct cultures do not contribute to the flavour. Bacteriocin-producing protective cultures are used during the
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shelf life of non-fermented foods, to inhibit pathogenic and spoilage bacteria. According to Quinane et al., (2005), two such preparations are present in the market:
ALTATM 2341 – It is obtained from Pediococcus acidilactici and contains pediocin PA1
MicrogardTM - It is commercially available fermented milk product and contains antimicrobial substances.
Activity of bacteriocins can be enhanced through the combination with other antimicrobial factors such as chelating agents (EDTA), inorganic salts (Sodium chloride), organic acids, essential oils, phenolic compounds , natural antimicrobials, physio-chemical treatments, pulsed electric field or pulsed magnetic field, gamma irradiation and high hydrostatic pressure (Gálvez et al., 2007; Chen and Hoover, 2003; Ross et al., 2003; Deegan et al., 2006). Activity of bacteriocin destabilizes the outer membrane of bacteria especially gram-negative bacteria when exposed to hurdles like chelating agents (Gálvez et al., 2007; Fang and Tsai, 2003; Omar et al., 2006; Cotter et al., 2004).
Application of nisin in food industry
Nisin began to be commercially isolated from Lactococcus lactis ssp. lactis in England in 1953. It is the best known bacteriocin isolated from lactic acid bacteria (Hurst, 1981). It was approved by food additive legislating bodies in the US (FDA) and in the EU for use in foodstuffs (Cotter et al., 2004; Thomas et al., 2000). Nisin was approved by US FDA (Food and Drug Administration) in 1988 for use in processed, pasteurized cheese spreads and then as a food additive across 50 countries (Cotter et al., 2005). Nisin is naturally produced by Lactococcus lactis ssp. lactis and is the only bacteriocin licensed as food preservative (Anonymous, 1994). Nisin are active against gram-positive bacteria (Listeria species and Micrococcus species) and spore-forming bacteria (Bacillus species and Clostridium species) (McAuliffe et al., 2001; Thomas et al., 2000; Zendo et al., 2003). Nisin extends the shelf life of the product when used as a food preservative by inhibiting the growth of gram-positive spoilage bacteria such as Listeria, Staphylococcus and Mycobacterium and spore-forming bacteria such as Bacillus and Clostridium (O’Sullivan et al., 2002; Zottola et al., 1994; Kalyanchand et al., 2004; Olasupo et al., 2004; Black et al., 2005; Mauriello et al., 2005; Stergiou et al., 2006). Most available form of nisin is Nisaplin TM. A new nisin has been isolated from Lactococcus lactis strain and present in river water in Japan and named it as nisin Q. It differs by four amino acids as a mature peptide and by two amino acids of the leader sequence (Zendo et al., 2003).
Sources, isolation and identification of probiotic bacteria
Hypothetically, soil, plants and gut of herbivorous animals are considered as the first niche of the lactic acid bacteria (Morelli et al., 2012). Probiotic lactic acid bacteria are prevalent microbes found in environment rich mainly in carbohydrates such as mucosal surfaces of humans and animals. Lactic acid bacteria are normal microbiota in the human and animal bodies that naturally inhabits the gastrointestinal and gastrourinary tract which comprises different bacterial species (Aureli et al., 2011; Barinov et al., 2011). The probiotic lactic acid
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bacteria colonize the intestine of the mammalian system to exert the beneficial effects to the target area (Hooper and Macpherson, 2010; Kamada et al., 2013).
Most common sources of probiotics are yogurt, cheese, cultured buttermilk and kefir. Other foods which are produced by fermentation are beer, bread, chocolate, Japanese miso, kimchi, olives, pickles, sauerkraut, sour dough and tempeh (Roy and Kalicki, 2009). Non-dairy fermented substrates also have probiotic strains (Schrezenmeir and De Vrese, 2001). Some non-dairy probiotic products are cabbage, cereal, legume, maize, pearl millet, sorghum and vegetable (Rivera-Espinoza and Gallardo-Navarro, 2010).
The probiotic microorganisms are isolated from different habitats and cultivated on solid or liquid media. Techniques of microbiology like streaking or successive dilutions are used for obtaining pure cultures and then cultivated on solidified agar medium. Strains are characterized on morphological, biochemical, immunological and toxicological studies. The strain is then preserved at low temperature (by lyophilisation or storage in freezer below -80oC or in liquid nitrogen) (Stefana et al., 2004).
Isolation from yogurt and other dairy products
Lactobacillus delbrueckii ssp. bulgaricus is isolated from commercially available yogurt and dairy products using MRS agar and modified MRS agar (MRS agar + L-cysteine + LiCl + Na propionate) anaerobically incubated at 37oC for 72 hours (Dimitris and Robert, 2009).
Isolation of lactic acid bacteria from human milk
Probiotic bacteria can be isolated from human milk. Samples were collected from healthy volunteer mothers. Isolation was carried out by usual plate technique and transferred to sterile broth medium (Rowaida, 2007; Magdalena and Marian, 2005).
Six lactic acid bacterial strains were isolated from Romanian fermented vegetables. Leuconostoc citreum 344 and Lactobacillus brevis 183 were selected on the basis of antagonistic property against Listeria monocytogenes ATCC 1911, Leuconostoc mesenteroides 348 and Lactobacillus brevis 312 against Escherichia coli ATCC 25922 and Lactobacillus plantarum 327 and Lactobacillus brevis 380 against Salmonella enterica ATCC 14028. The cultures were stocked in MRS broth at -75oC in the presence of 25% (v/v) of glycerol as cryoprotectant (Grosu-Tudor and Zamfir, 2012).
Kimoto et al., (2004) used 54 fermented vegetables, 9 silages and 4 grasses for the isolation of lactic acid bacteria. MRS broth was used in addition to 1.6% (w/v) of agar and 0.8% (w/v) CaCO3. 1 gm of each sample was mixed homogeneously with 9 ml of 0.85% (w/v) sterilized NaCl solution. Serial dilutions were made from the suspensions in NaCl solution and plated on the surface of MRS agar by spreading technique. After incubation at 30oC for 48 hours anaerobically, those colonies were isolated which form clear zones around their own colonies. Each colony was inoculated and incubated at 30oC into the broth for testing catalase reaction. Catalase negative strains were preserved in MRS broth with 15% (v/v) glycerol at -80oC.
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Lactic acid bacteria were isolated from industrial sausages produced without the addition of starter cultures. The lactic acid bacteria were counted by microbiological analysis (MRS agar; according to De Man, Rogosa and Sharpe). The isolates morphologically identified as gram-positive bacilli and biochemically identified as catalase-negative using API 50 CHL (BioMerieux) test and the computer program APILAB Plus. All 50 isolates were considered as excellent which were confirmed by the API test (Fleck et al., 2012).
331 lactic acid bacteria were isolated from kantong production sites of Northern region of Ghana. All the samples were collected aseptically from kantong production at different stages of production viz. 0 h, 24 h, 48 h and final product. Samples were serially diluted, cultured on MRS agar and incubated at 30oC for 48 hours. Each colony was identified phenotypically, biochemically and genotypically at the UDS/DANIDA MICROBIOLOGY LAB, Novrongo Ghana. Cultures were preserved on MRS agar at 4oC (Kpikpi et al., 2010).
Raw milk from Sunhwa Dairy Farm, Korea was collected and stored at 5oC. It was cultured in Lactobacillus MRS broth and then streaked onto Bromocrezol Purple agar at 37oC for 48 hours either aerobically or anaerobically. Sub-culturing was done in specific medium , dissolved in skim milk solution containing 20% glycerol and stored at -70oC (Hyun jue et al., 2006).
Lactobacilli were isolated from crop, gizzard, ileum and caecum of 20 adult healthy chicks. Every sample was mixed separately and homogeneously with sterilized phosphate buffer saline (PBS) (Ashraf et al., 2009). Similarly, Awan and Rahman (2005) obtained 20 conventional yogurt samples in the sterile plastic bags from the local market. Each sample was mixed homogeneously by dissolving in 100 ml of sterilized phosphate buffer saline. Both samples were serially diluted by 10-fold and inoculated on MRS agar plates by pour plate technique. All agar plates were incubated anaerobically at 37oC for 48 hours. Morphologically distinct colonies were selected and transferred by streaking technique on the surface of new agar plates and then pure colonies were obtained.
Safety of probiotics
The use of lactic acid bacteria as a probiotic requires a thorough safety assessment. The probiotic strain should be well studied and documented for its functional attributes (Holzapfel et al., 2000). Qualified Presumption of Safety (QPS) approach was developed by EFSA (European Food Safety Authority) and used it as a tool for the safety assessment of probiotic microorganisms in food. Generally Recognizes As safe (GRAS) system was developed by US for the assessment of microorganisms. For example- Probiotic strains viz. Lactobacillus reuteri DSM 17938, Bifidobacterium lactis BB12, Lactobacillus rhamnosus GG has been accepted by US as GRAS (Jankovic et al., 2010). FAO/WHO has formulated guidelines about the safety assessment of the probiotic strains. Parameters include susceptibility patterns of antibiotics, production of toxin along with the hemolytic potential, metabolic activities and infectivity in immunocompromised animals (FAO/WHO, 2002).
US FDA has established safety criteria of the probiotics in one of the following ways:-
1. FDA should approve the ingredient as a food additive
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2. Either FDA or a panel of qualified scientific experts should evaluate the ingredient as GRAS
3. FDA approved the ingredient prior to September 6, 1958
GRAS status of probiotic lactic acid bacteria should be considered on the following evidences:-
1. Human trials – 143 clinical trials were performed during 1961 to 1968 on 7526 humans. It was estimated that oral administration of probiotic lactic acid bacteria is well tolerated by the subjects. It was considered safe for the use of human as no adverse effects were reported.
2. Animal models – Administration of probiotic lactic acid bacteria in experimental animals in vivo such as guinea pigs, rats, mice etc. No toxicity, bacteremia or pyrogenicity was observed. It helps in prolong the survival of the experimental animals.
3. In vitro studies – Probiotic lactic acid bacteria are found to be non-invasive when experiments are done for microbial adhesion (with human intestinal epithelial cells) or for inducing immunomodulatory effects (with human lymphoid carcinoma cell lines).
4. Surveillance of probiotic market – Some points should be considered for marketing probiotic-containing products:
i) People believe in preventing the disease than curing
ii) People are more aware towards health and nutrition
iii) Functional foods give a low-cost substitute for maintaining good health
iv) People want to get rid of environmental hazards such as pollution, pathogen microbes and chemicals in food and water
v) Scientific studies provide support for efficacy of probiotics to maintain health (Sanders, 1998)
EFSA mentioned some protocols for determination and evaluation of minimum inhibitory concentrations (MICs) for the pertinent antibiotics (EFSA, 2004). Hemolytic activity is also determined for the safety assessment of the probiotic microorganisms (FAO/WHO, 2002).
Complete hydrolysis, partial hydrolysis or no hydrolysis on blood agar plates describe the hemolytic activity (Pisano et al., 2014).
In vitro tests for pathogenicity traits: Strains should be tested for the
i) Ability of bacteria to bind to human cells such as platelets (Harty et al., 1993)
ii) Ability to produce enzymes such as glycosidases, gelatinases and proteases (Oakey et al., 1995; Bernardeau et al., 2006)
iii) Production of known human toxins such as cytolysins (Tan et al., 2013)
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Figure 3. Safety efficacy
33
Literature (including toxicology)
Genus/species/strain viability contamination
Viability during storage
Tolerance of food/feed additives
Stability during processing
Stability during storage
Tolerance to low pH/bile/intestinal enzymes/juices
Resistance to antibodies
Intestinal tissue adherence
Antimicrobial activity Microbial adhesion-interference
Overall Probiotic-Spectrum
EXCELLENT- Plan in vivo studies
iv) Ability to produce positive decarboxylase activity (Amino acids are decarboxylated to biogenic amines by substrate-specific decarboxylases of bacteria) (Bover-Cid and Holzapfel, 1999)
For the safety assessment of probiotics, it is important to conduct population-based surveillance for the isolation of probiotic bacteria from diseased patients (Salminen et al., 2006; Swenson et al., 2009).
Prebiotics
The term ‘prebiotic’ was used by Gibson and Roberfroid in 1995 (Gibson and Roberfroid, 1995). Prebiotics are “indigestible fermented food products which helps in stimulating the growth, composition and activity of gut microflora and improves the health and well-being of the host” (Roberfroid, 2000). Prebiotic are intermediate between simple sugars and polysaccharides and of low molecular weight compounds (Solange et al., 2007).
50 years ago, Lactulose was used as a prebiotic supplement (Macgillivray et al., 1959). Compounds which have gut resistant properties and ability of selective fermeability by gut microorganisms are developed as prebiotics (Gibson and Fuller, 2000; Ooi and Liong, 2010) such as oligosaccharides (Isomaltooligosaccharides, glucooligosaccharides, lactosucrose and xylooligosaccharides), polysaccharides (Starch, resistant starch and modified starch) and sugar alcohols (Cummings et al., 2001). Prebiotics are produced by extraction from natural sources or by chemical synthesis (Nugent, 2005; Mussato and Mancilha, 2007).
According to Wang, (2009), examples of prebiotics are inulin-type fructans, trans-galactooligosaccharides, lactulose, lactosucrose, soybean-oligosaccharides, isomaltose, xylo-oligosaccharides, gluco-oligosaccharides, fructo-oligosaccharides, galacto-oligosaccharides.
Sources: - Traditional sources of prebiotics are soybeans, inulin sources (Artichoke, chicory root, jerusalem and jicama), raw oats, unrefined wheat, unrefined barley and yacon (Anandharaj et al., 2014). Breast milk is also a good source of prebiotics as it contain natural oligosaccharides. Breast feeding infants have more Lactobacilli and Bifidobacteria in their gut, which acts as a baby’s defence against pathogens (Newburg, 2005; Morais and Jacob, 2006).
Effective dose:- 5-10g/ day for healthy adults.
Table 7. Health benefits of prebiotics (Macfarlane et al., 2008)
Prebiotic substrate
Type of study Dosage Effect on microflora
Placebo-controlled study (12 subjects)
10 g prebiotic daily for 8 weeks
Counts of Bifidobacteria and Lactobacilli increased in fecal samples
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GOS Feeding study (12 subjects)
2.5 g prebiotic daily for 3 weeks
Increased count of Bifidobacteria in fecal samples, reduction in number of Clostridia and Bacteroides
Placebo-controlled study (30 subjects)
8.1 g GOS syrup, 8.1 g GOS plus 3 X 1010
Bifidobacterium lactis Bb-12 or 3 X 1010
Bifidobacterium lactis without GOS for 3 weeks
Bifidobacteria increase seen with GOS alone. GOS plus Bifidobacterium lactis and Bifidobacterium lactis on its
own resulted in faecal excretion of the organism and reduced numbers of Bifidobacterium longum.
TOS Feeding study (8 volunteers)
10 g prebiotic daily for 3 weeks
Increased fecal count in Bifidobacteria, no effect seen in Enterobacteria
Parallel study (40 subjects)
7.5 g or 15 g prebiotic per day for 3 weeks
Lactobacilli count increased in 15 g/day in fecal sample, small reduction in Enterobacteria and no effect seen in Clostridia
GOS/FOS Placebo-controlled study (90 term infants)
4 or 8 g/l low molecular weight GOS and high molecular weight FOS daily for 28 days
Significantly increase in Lactobacilli and Bifidobacteria counts
GOS/FOS (9:1)
Double-blinded, randomized controlled trial (20 infants, age 28-90 days)
0.8 g/100 ml GOS/FOS daily for 6 weeks
Bifidobacteria count increased in stool sample.
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Synbiotics
The synergistic combination of probiotics and prebiotics is known as synbiotics. This combination helps in enhancing the survival and activity of the gut microbiota. (Palaria et al., 2012). Synbiotics also helps in survival, implantation and growth of newly added probiotic strains (Sridevi-Sivakami and Subhashree, 2011). Synbiotics helps in the prevention of gastrointestinal diseases in humans and animals (Palaria et al., 2012)
For example- FOS in combination with Bifidobacterium strain or lactitol in combination with Lactobacillus strain (Gibson and Roberfroid, 1995).
Applications
1. Fermented foods
Fermentation is a process of achieving a desired biochemical change by modifying the food with the help of microorganisms or enzymes. Fermentation is used for preservation of foods in many countries (Hull et al., 1992). Lactobacillus, Bifidobacterium and Streptococcus thermophilus are mostly used for the production of fermented foods. Lactic acid bacteria are either used alone or in conjunction with molds, microcooci and yeasts (Hammes and Tichaczek, 1994). Dairy foods, beverages and cereals comprise fermented foods, used in terms of total production and consumption (Campbell-Platt, 1994). Approximately 80 bifid-containing products are in the global market which includes buttermilk, sour cream, fortified milk, frozen desserts, powdered milk and yogurt (Modler et al., 1990).
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Figure 4. Antimicrobials from lactic acid bacteria
2. Supplemented foods
For the preparation of probiotic products, Lactobacillus bulgaricus, Lactobacillus lactis, Lactobacillus salivarius, Lactobacillus plantarum, Streptococcus thermophilus, Enterococcus faecium, Enterococcus faecalis and Bifidobacterium species are used. In Japan and Europe, Bifidobacteria is used for the preparation of probiotic food (Ishibashi and Shimamura, 1993). In 1971, the first bifidus product was developed by Morinaga Milk Industry Company in Japan. This product is low-fat fresh milk containing Lactobacillus acidophilus and Bifidobacterium longum. Its full scale production as started in 1977 (Ishibashi and Shimamura, 1993).
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ANTIMICROBIALSLactic acid, acetic acid, hydrogen peroxide, ethanol, formic acid, fatty acids, diacetyl, acetaldehyde, acetoin, reuterin,
reutericyclin, bacteriocins, bacteriocin-like compounds and other low molecular mass compounds with antimicrobial
activity
Lactic acid bacteria
Food products
Fermentedfood
Food biopreservatives
Functional food
Starter Non-starter cultures Probioticcultures (adjunct, protective) bacteria
Pharmaceuticals
Probiotics as living drugs
Healthy effects including protection against pathogens
Table 8. Lactic acid bacteria-supplemented foods available in market (Tamime et al., 1995)
Origin Trade name Lactic acid bacteria culture
Denmark A B milk products Lactobacillus acidophilus and Bifidobacterium bifidum
Cultura® Lactobacillus acidophilus and Bifidobacterium bifidum
Germany Acidophilus bifidus yogurt Lactobacillus delbrueckii spp.bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Bifidobacterium bifidum or Bifidobacterium longum
Bifidus milk Bifidobacterium bifidum or Bifidobacterium longum
Bifighurt® Bifidobacterium bifidum or Bifidobacterium longum
Biograde® Lactobacillus acidophilus, Bifidobacterium bifidum and Streptococcus thermophilus
Bioghurt® Lactobacillus acidophilus, Bifidobacterium bifidum and Streptococcus thermophilus
Biomild® Lactobacillus acidophilus and Bifidobacterium sp.
Sweet bifidus milk Bifidobacterium sp.
France B A® Bifidobacterium longumDiphilus milk® Lactobacillus acidophilus and
Bifidobacterium bifidumOfilus® Streptococcus thermophilus, Lactobacillus
acidophilus, Bifidobacterium bifidum or Lactococcus lactis ssp. cremoris, Lactobacillus acidophilus and Bifidobacterium bifidum
Japan Mil-Mil® Bifidobacterium bifidum, Bifidobacterium breve and Lactobacillus acidophilus
Sweet acidophilus bifidus milk
Lactobacillus longum and Bifidobacterium longum
Sweet bifidus milk Bifidobacterium sp.India Prolife® Lactobacillus acidophilus Czechoslovakia Biokys® Bifidobacterium bifidum, Lactobacillus
acidophilus and Pediococcus acidilactici Chile Progurt® Lactococcus lactis biovar diacetilactis,
Bifidobacterium bifidum, Lactococcus lactis ssp. cremoris and Lactobacillus acidophilus
UK Bifidus milk with yogurt flavor
Bifidobacterium bifidum, Bifidobacterium longum and Bifidobacterium infantis
USSR Bifilakt® or Bifilact® Lactobacillus sp. and Bifidobacterium sp.
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3. Pharmaceutical products
These preparations helps in achieving the colonization of the microorganisms in the gut during the treatment of various gastrointestinal disorders such as chronic constipation, duodenitis, liver diseases, peptic ulcers and post-antibiotic therapy (Tamime et al., 1995).
Table 9. Pharmaceutical probiotics available in market (Reddy, 2007)
Product name CompositionAcidophilas (Wakunaga probiotics) Lactobacillus acidophilus, lipase, protease, amylase
and lactase enzymesBifa 15 (Eden foods) Bifodobacterium longumBifilac (Tablets) Streptococcus faecalis, Clostridium butyricum,
Bacillus mesentericus and Lactobacillus sprogenesKyo-Dophilus capsules (Wakunaga probiotics)
Lactobacillus acidophilus, Bifidobacterium bifidum and Bifidobacterium longum
Kyo-Dophilus tablets (Wakunaga probiotics)
Lactobacillus acidophilus
Pre Pro (Fourrts) Streptococcus faecalis, Clostridium butyricum, Bacillus mesentericus, Lactobacillus acidophilus, FOS (Fructo-oligosaccharides
Probiota tablets Lactobacillus acidophilus Replenish (Innercleanse 2000) Lactobacillus acidophilus, Lactobacillus plantarum,
Lactobacillus bifidus, Lactobacillus bulgaricus, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus brevis, FOS (Fructo-oligosaccharides)
Sporlac (Powder) Lactobacillus sporogenesTH1 Probiotics (Jarrow Formulas) Bifidobacterium longum, Saccharomyces boulardii,
Lactobacillus casei and Lactobacillus plantarumVSL 5310 cells/g of three strains of Bifidobacterium, four
strains of lactobacilli and one strain of Streptococcus salivarius ssp. thermophilus
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MATERIALS AND METHODS
1. Collection of different traditional food samples viz. Yogurt, Jalebi batter, seera, dough,
kefir, pickles, soy milk, olive, saukerkraut. All samples will be collected in clean and
sterilized polythene bags and will be stored in refrigerator for further use.
2. Isolation of Lactic Acid Bacteria
a. The samples in solid form will be crushed properly in a clean sterilized pestle mortar by
adding distilled water and then homogenized for 15 minutes on vortex mixture.
b. Liquid samples will be taken as such for isolation.
c. Stock will be made from the samples by adding 1 ml of sample in 9 ml of distilled water.
d. All samples will be serially diluted by serial dilution range of 10-1 to 10-9.
e. The samples (0.1ml each) from each dilution will be mounted by spread plate method on
sterilized petri plates containing MRS agar medium (de Man et al., 1960) for isolation of
bacterial colonies.
3. Purification of isolated colonies
a. Individual colonies will be selected and purified using streak plate technique on MRS
medium.
4. Purified strain will be identified on the basis of its colonial morphology (By Gram’s
staining) and biochemical tests.
a. Phenotypic characterization- Color, form, margin, elevation of selected isolates will be
identified after their anaerobic growth on MRS agar
b. Biochemical characterization- Biochemical tests like catalase test, citrate utilization test,
gas production from glucose, casein hydrolysis, H2S production and sugar fermentation will
be performed on screened isolates.
5. Effect of temperature on the growth of screened LAB – Viability of isolates will be
examined by inoculating the strains in MRS broth and incubate at 20, 25, 30, 35, 40 and
45oC. Thermotolerance of isolates will be examined after 24 h.
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6. Screening of isolates for Antimicrobial activity by Bit/ Disc preparation of bacterial isolate
(Barefoot and Klaenhammer, 1983) and by Well diffusion method (Kimura et al., 1998) –
The isolated bacteria will be inoculated on MRS agar medium for 72 h at 37oC. Borer and
wells will be cut out of MRS agar plates and bits of bacterial isolates will be kept on lawn
culture of indicator bacteria and will be kept for incubation at 35oC for 24h.
7. Identification of screened isolates by genotypic characterization by isolation of genomic
DNA by Agarose gel electrophoresis and PCR.
8. Toxigenicity and antagonistic assessment of screened lactic acid bacteria
a. Acid tolerance – 108 CFU/ml of isolated strain will be inoculated into the modified MRS
broth and incubated at 35oC for 24h. After centrifugation, pellet will be diluted in phosphate
buffer saline of different pH. After plating it on modified MRS agar, acid tolerance will be
determined by comparing the final plate count after 3h with the initial plate count at 0h
(Liong and Shah, 2005).
b. Bile tolerance – 108 CFU/ml of isolated strain will be inoculated into the modified MRS
broth with bile salt and incubated at 35oC for 8h. Growth will be monitored by measuring
absorbance at 620 nm for 8h at hourly intervals (Walker and Gilliland, 1993).
c. Hemolytic activity – Bacterial cultures will be grown on Sheep Blood Agar plates and
incubate at 35oC for 24-48 h (Harrigan, 1998).
d. DNAse production – Isolates will be inoculated on DNAse agar medium and incubate at
35oC for 24-48 h (Gupta and Malik, 2007).
e. Gelatinase production – Isolates will be inoculated on MRS agar supplemented with 3%
gelatin and incubated for 35oC for 24- 48 h (Harrigan and McCance, 1990).
f. Autoaggregation – Isolates will be inoculated in MRS broth at 35oC for 24 h and after
centrifugation, absorbance of upper suspension will be measured at 600nm after every hour
(Del Re et al., 2000).
g. Bacterial adhesion to solvents – Isolates will be inoculated in MRS broth at 35oC for 24 h.
Cell suspension will be put in contact with hydrocarbons and after centrifugation, absorbance
of aqueous phase will be measured at 600nm at 0 h and 2 h (Rosenberg et al., 1980).
41
h. Bacterial adhesion to mucin – Bacterial cells will be inoculated in MRS broth at 35oC for
24 h and will be added to mucin pre-coated micro titre plate. After incubation, absorbance
will be measured at 620 nm (Vesterlund et al., 2005).
i. Bacteriocin activity – Bacterial isolates will be grown on TGY broth. After incubation,
supernatant of bacterial isolates will be poured into the wells of indicator medium and
incubate at 35oC for 24 h. Zones of inhibition around the wells will be measured (Papagianni
and Anastasiadou, 2009).
j. Estimation of lactic acid production by HPLC.
k. H2O2 production - – Bacterial cells will be inoculated in MRS broth at 35oC for 24 h.
Diluted H2SO4 will be added and suspension will be titrated against 0.1 N KMnO4 (AOAC,
1995).
l. Exopolysaccharide production – Isolates will be inoculated in ruthenium red milk agar at
35oC for 24 h. Overnight cultured will be observed for EPS production (Mora et al., 2002).
9. Preparation of functional foods using probiotic isolates
a. Preparation of fermented blue berry probiotic jam
b. Preparation of fermented peach probiotic candies.
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