Padma Ambalam

Isolation and characterization of antimicrobial proteins produced by a potential probiotic strain of human Lactobacillus rhamnosus 231 and its effect on selected human pathogens and food spoilage organisms

Abstract Objective: To study in vitro properties of potential probiotics and the antimicrobial activity of Lactobacillus rhamnosus 231 isolated from human faeces. Methods and Results: Lact. rhamnosus 231 isolated from human faeces tolerated bile salt (4%), phenol (0.5%), and NaCl (4%) and retained viability at low pH (2.5). The cell-free culture (CFC) filtrate and extracellular protein concentrate (EPC) of Lact. rhamnosus 231 contained antimicrobial substances active against Pseudomonas aeruginosa, Escherichia coli, Enterobacter aerogenes, Staphylococcus aureus, Salmonella spp., Helicobacter pylori, Campylobacter jejuni, Bacillus cereus, Bacillus megaterium, and Listeria monocytogenes. EPC contained a mixture of low molecular weight antimicrobial proteins, produced during log and stationary phases of growth against the test organisms. Thermostability of the antimicrobial proteins and their sensitivity to proteinase K was observed to be test organism specific. The antimicrobial activity was observed in the pH range 4.5–9 except against Ps. aeruginosa and Ent. aerogenes. These antimicrobial proteins are low molecular weight (4 kDa) anionic peptides as determined by tricine-SDS-PAGE and 2D gel. Periodic acid-Schiffs (PAS) staining of gel confirmed the presence of carbohydrate moiety with low molecular weight peptides. The antimicrobial activity of the partially purified protein was determined against Staph. aureus 74B, H. pylori 33, H. pylori 17874, and C. jejuni CJE 33566. Conclusion: Human Lact. rhamnosus 231 exhibits in vitro properties of potential probiotic. CFC filtrate and EPC of Lact. rhamnosus 231 exhibit antimicrobial activity against potential human pathogens and food spoilage organisms. Antimicrobial proteins in EPC were partially purified and characterized. In vitro properties of potential probiotic and antimicrobial properties of Lact. rhamnosus 231 could be useful as food additive against human pathogens and removal of food contaminants in the target environment.

A novel multi-strain probiotic and synbiotic supplement for prevention of Clostridium difficile infection in a murine model.

The protective effect of a multi‐strain probiotic and synbiotic formulation was evaluated in C57BL/6 mice infected with Clostridium difficile (CD) NAP1/027. Antibiotic‐treated mice were divided into the following four groups: Group 1, fed with a synbiotic formulation consisting of Lactobacillus plantarum F44, L. paracasei F8, Bifidobacterium breve 46, B. lactis 8:8, galacto‐oligosaccharides, isomalto‐oligosaccharides, and resistant starch; Group 2, fed with the same four probiotic strains as Group 1; Group 3, fed with the same prebiotic supplements as Group 1 for 7 days before CD infection; and Group 4 (control group) antibiotic treated and infected with NAP1/027 strain. Feces and cecal contents were collected for microbial cell viability, quantitative PCR (qPCR), toxin analyses and histopathology. Synbiotics‐ and probiotics‐fed mice showed a significant increase in total bifidobacteria (P < 0.05). The total lactobacilli count was increased in Group 1. Tests for cecal toxins were negative in Group 2 mice, whereas one sample each from Group 1 and 3 was positive. qPCR of cecal contents showed significant reduction in NAP1/027 DNA copies in Groups 1 and 2 and significantly higher numbers of B. breve 46, L. plantarum F44, and L. paracasei F8 in Groups 1 and 2 (P < 0.05); these changes were much less pronounced in Groups 3 and 4. Our findings indicate that the newly developed synbiotic or multi‐strain probiotic formulation confers protection against NAP1/027 infection in C57BL/6 mice. This holds promise for performing human studies.

Antimicrobial Profile of Lactic Acid Bacteria Isolated from Vegetables and Indigenous Fermented Foods of India against Clinical Pathogens Using Microdilution Method.

In dairy and food industries lactic acid bacteria(LAB)have been used in form of starter culture that plays vital role in fermentation;as flavouring and texturizing or as preservative agents.There is increasing evidence that lactobacilli which inhabit the gastrointestinal tract develop antimicrobial activities and participatein the host’s defence system[1].During fermentation,most of the LAB produces a number of different compounds like organic acids,hydrogen peroxide,diacetyl,acetaldehyde,carbon dioxide,polysaccharides,and proteinaceous compounds called bacteriocins or bacteriocinogenic peptides[2-3].Such metabolites exert antimicrobial activity and are

Probiotics and Colorectal Cancer

Probiotics are living microbes that when taken in an adequate amount confers a health benefit to the host. Predominant probiotic microorganisms include Lactobacillus spp, Bifidobacterium spp., and Enterococcus spp. The yeast, Saccharomyces boulardii, and other bacterial species like Bacillus and Clostridium butyricum have been studied extensively. Several mechanisms have been proposed for probiosis, mainly attributed to their abilities to strengthen the intestinal barrier, to modulate the host immune system, and to produce antimicrobial compounds. The main therapeutic and health benefits of probiotics include prevention of diarrhea, hypercholesterolemia, and inflammatory bowel disease and improvement in lactose utilization and mineral absorption, anti-Helicobacter pylori activity, and anticancerous activity (ACA). The ACA of probiotic is mainly accredited to the (1) inactivation of cancerogenic compounds, (2) lowering of intestinal pH, (3) modulation and enhancement of the host’s innate immunity through the secretion of anti-inflammatory molecules, (4) alteration of the intestinal microflora, (5) antiproliferative effects via regulation of apoptosis and cell differentiation, and (6) inhibition of tyrosine kinase signaling pathways. Faecalibacterium prausnitzii anti-inflammatory commensal could be used as a potential probiotic against colitis and CRC.

Short-Chain Fatty Acids

Short-chain fatty acids are formed in the colon as a result of microbial fermentation (Bifidobacterium and Lactobacillus) of undigested bioactive carbohydrates, including prebiotics and dietary fiber, and their significant role in orchestrating colon carcinogenesis is of current interest among researchers. Acetate, propionate, and butyrate are the major by-products of SCFA formation. Propionate and butyrate are extensively involved in the cell differentiation, growth arrest, and apoptosis of cancer cells. The anticancer effects of acetate, propionate, and butyrate (molar ratio of approximately 3:1:1) is supported immensely by epidemiological, in vitro and in vivo, studies. The role of lactate has also been considered by several researchers. Diet and dietary habits alter the composition of the gut microflora. An inverse relationship between dietary fiber intake and the incidence of human colon cancer has been observed. Prebiotics and dietary fibers curtail the risk of colon cancer through diverse mechanisms. The interplay between propionate and butyrate, and gastrointestinal epithelial cells (colonocytes) in reducing colon carcinogenesis is a current topic of interest for many researchers. About 70–90 % butyrate is metabolized by the colonocytes. SCFA also modulate colonic and intracellular pH, cell volume, ion transport and regulate cell proliferation, differentiation, and gene expression. Propionate inhibits cell growth and activates apoptosis in colorectal carcinoma cells. Butyrate inhibits histone deacetylase resulting in histone hyperacetylation and growth inhibition in the colonic epithelial cells. This link between histone hyperacetylation, hyperacetylation-induced transcriptional regulation and growth inhibition has been considered as the foremost factors in preventing colon cancer. However, “butyrate paradox,” another concept, has not been confirmed till date. The function of SCFA in the suppression of inflammation and immunomodulation is discussed.

Prebiotic preferences of human lactobacilli strains in co-culture with bifidobacteria and antimicrobial activity against Clostridium difficile.

To evaluate robustness, prebiotic utilization of Lactobacillus paracasei F8 and Lactobacillus plantarum F44 in mono‐ and co‐cultures with Bifidobacterium breve 46 and Bifidobacterium animalis sub sp. lactis 8 : 8 and antimicrobial activity of co‐culture against Clostridium difficile.

Bioactive Carbohydrate: Prebiotics and Colorectal Cancer

Prebiotics are defined as the indigestible food components that are selectively fermented by the intestinal microbes and promote changes in the gut environment, gut microbial community structure, and their metabolism. Prebiotics have been reported to avert colorectal cancer development by altering the composition and activity of the gut microflora. Administration of prebiotics has been reported to reduce colon cancer and its biomarkers. Oligosaccharides such as inulin, fructo-oligosaccharide, galacto-oligosaccharide, lactulose, soy-oligosaccharide, xylo-oligosaccharide, lactitol, resistant starch, etc., are some of the commercially available prebiotics. Short-chain fructo-oligosaccharides thwart colon carcinogenesis possibly by modulating the colonic ecosystem. Galacto-oligosaccharides, lactulose, and fructo-oligosaccharides are observed to increase the intestinal concentration of lactate, stool frequency, and weight; and decrease fecal concentration of secondary bile acids, fecal pH, and nitroreductase and β-glucuronidase activities, thus preventing colon carcinogenesis. Lactulose is reported to reduce the proliferation and occurrence of adenoma. Resistant starch prevents cancer by modulating gene expression and DNA methylation. Prebiotics undergo fermentation by gut microbes leading to the formation of short-chain fatty acids and enhance the immunity of the host. Butyrate has been observed to have direct effects in preventing colon cancer. Prebiotics are observed to modulate the colonic gut environment and aid in the growth and development of Bifidobacteria spp.

Probiotics and Bioactive Carbohydrates in Colon Cancer Management

The gastrointestinal (GI) system is an important part of the human body involved in digestion. Animals and humans, live in harmony with microbes and establish a symbiotic relationship with them. The human microbiota contains as many as 10 14 bacterial cells, which is ten-fold higher than the number of human cells present in our body. With the development of highthroughput sequencing technique, it is possible to study the gut microbiota and its role in health and disease. It performs a number of essential protective, structural and metabolic functions for host health, including food processing, digestion of complex host-indigestible polysaccharides, pathogen displacement, and synthesis of vitamins. The gut microbiota may exert benefi cial functions beyond the gut such as controlling obesity, gastrointestinal disorders, infl ammatory bowel disease, and irritable bowel syndrome. Also, the altered microbiota has been linked to neuropsychological disorders (depression). The gut microbiota has also been implicated in the development of colorectal cancer (CRC). CRC is one of the major health problems in the world, and the risk factors associated with it are genetic as well as environmental. It evolves through a stepwise accumulation of genetic and epigenetic alterations, leading to the transformation of normal colonic mucosa into invasive cancer. In this chapter, the role of the gut microbiota in health and disease, CRC and its types, prevalence and mechanism are briefl y discussed.

Synbiotics and Colorectal Cancer

Synbiotics are defined as the combination of appropriate probiotics and prebiotics, where the latter form the substrate for the growth and development of selective indigenous or introduce beneficial bacteria in the colon. The prebiotics commonly in use are inulin, fructo-oligosaccharide, galacto-oligosaccharide, lactose, etc., and the probiotics investigated include Bifidobacterium and Lactobacillus spp. Prebiotics form the food for the growth of probiotics. The mechanism of action of synbiotics is a collective effort of pro- and prebiotics. Administration of synbiotics could prevent the initiation or early stage of cancer and also treat the existing tumors. Synbiotic interventions bring about significant alterations in the composition of the colonic microbiome leading to the altered metabolic activity of the organ. It reduces the exposure of cytotoxic agents, including mutagens and carcinogens, to the intestinal lining; decreases cell proliferation in the colonic tissue; and develops mucosal structure. It is reported that administration of synbiotics results in noteworthy changes in the fecal flora, with an increase in Bifidobacterium and Lactobacillus and a reduction in the harmful pathogens such as Clostridium perfringens. These also inhibit the fecal water to cause necrosis in the colonic cells and improve epithelial barrier functions. Synbiotics are also reported to show immunomodulatory effects.

Preventive and Therapeutic Effects of Dietary Fibers Against Cardiovascular Diseases

Abstract Cardiovascular diseases (CVDs) and coronary diseases are leading causes of mortality and morbidity in developed countries. In recent years, although the incidences of these have declined among European countries and United States, it still accounts for almost half (48%) of all deaths in Europe and a third (32.8%) in US. CVD is an inflammatory disease associated with hypercholesterolemia and hypertension. Etiological studies have suggested that modified lipoproteins (e.g., oxidized) present in the arterial subendothelium plays a key role in CVD and coronary heart disease (CHD). Dietary fibers of terrestrial and marine origin are the cornerstones for CVD treatment. Cellulose, hemicellulose, gums, mucilages, pectins, oligosaccharides, lignins, etc. were reported to lower atherogenic lipoprotein levels and degree of oxidation, blood pressure, thrombogenesis, and concentrations of some relevant factors (homocysteine), thus averting CVD and CHD. In this chapter, the emphasis is specified on the therapeutic role of dietary fibers and its components in the prevention of cardiovascular and other relevant diseases. Importance is given to in vitro, in vivo, and clinical studies to bring forth the significance of dietary fibers as a nutraceutical.