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The human gut microbiota play a significant role in nutritional processes. The concept of probiotics has led to widespread consumption of food preparations containing probiotic microbes such as curd and yogurt. Curd prepared at home is consumed every day in most homes in southern India. In this study the home-made curd was evaluated for lactic acid bacteria (LAB) with probiotic potential.
Fifteen LAB (12 lactobacilli, 1 Lactococcus, 2 Leuconostoc) and one yeast isolated from home-made curd were evaluated for resistance to acid, pepsin, pancreatin and bile salts; antimicrobial resistance; intrinsic antimicrobial activity; adherence to Caco-2 epithelial cells; ability to block pathogen adherence to Caco-2 cells; ability to inhibit interleukin (IL)-8 secretion from HT-29 epithelial cells in response to Vibrio cholerae; and ability to induce anti-inflammatory cytokine expression in THP-1 monocyte cells.
Lactobacillus abundance in fermenting curd peaked sharply at 12 h. Nine of the strains survived exposure to acid (pH 3.0) for at least one hour, and all strains survived in the presence of pancreatin or bile salts for 3 h. None showed haemolytic activity. All were resistant to most antimicrobials tested, but were sensitive to imipenem. Most strains inhibited the growth of Salmonella Typhimurium while five inhibited growth of V. cholerae O139. Seven strains showed adherence to Caco-2 cells ranging from 20-104 per cent of adherence of an adherent strain of Escherichia coli, but all inhibited V. cholerae adherence to Caco-2 cells by 20-100 per cent. They inhibited interleukin-8 secretion from HT-29 cells, in response to V. cholerae, by 50-80 per cent. Two strains induced IL-10 and IL-12 messenger ribonucleic acid (mRNA) expression in THP-1 cells.
LAB in curd had properties consistent with probiotic potential, but these were not consistent across species. LAB abundance in curd increased rapidly at 12 h of fermentation at room temperature and declined thereafter.
Keywords: Antimicrobial resistance, cytokines, fermented milk, gastrointestinal pathogen, lactobacilli, yogurt
The lumen of the gut contains a complex consortium of microbes. The gut microbiome, that these microbes collectively constitute, profoundly influences nutritional, physiologic and protective processes in the human host. Their activity includes the fermentation of unabsorbed dietary carbohydrate with production of short chain fatty acids, production of vitamins like biotin and vitamin K, mediation of immune responses and oral tolerance, and protection of the host against invasion by pathogenic microbes 1 . In healthy individuals, there is a balance between beneficial and pathogenic constituents of the microbiota. Alterations in this balance can lead to a state of dysbiosis that contributes to illness and morbidity 2 ,3 . Metchnikoff was the first to associate lactic acid bacteria (LAB) in fermented yogurt with health and longevity of certain Balkan communities 4 . The concept of probiotic microbes that he introduced has led to the widespread consumption of food preparations containing LAB and/or bifidobacteria, with the expectation that they will confer health benefits including reduction of cholesterol levels, improvement of immune function, resistance to infectious diseases, and prevention of colon cancer 5 . The Food and Agricultural Organization (FAO) defines probiotics as live microorganisms, which when administered in adequate amounts, confer a health benefit on the host and suggests that a number of in vitro characteristics may be used to predict whether a specific microbe will have probiotic properties, before subjecting them to more rigorous tests to prove that they provide a health benefit 6 . These are echoed in regional and societal guidelines 7 ,8 .
Fermented foods containing LAB are traditionally used every day as food in many cultures. Curd, prepared by fermentation of milk with an inoculum of previously made curd, is used in most households in southern India where it constitutes a significant part of the daily diet. The LAB that ferment the milk are likely to differ slightly in each household as there is no standardized starter culture used to prepare curd. Although curd is believed to have probiotic properties 9 ,10 , there is little documentation of this in the literature. This study was, therefore, undertaken to evaluate the LAB from home-made curd in southern India for probiotic properties.
Bacterial strains and source: Clinical isolates of a pathogenic Escherichia coli, Vibrio cholerae O139, Salmonella Typhimurium strain and Shigella flexneri were kindly donated by the Department of Clinical Microbiology, Christian Medical College and Hospital (CMCH), Vellore, Tamil Nadu, India.
Growth medium: Vibrio was grown in alkaline peptone water while the remaining pathogenic strains were grown in brain heart infusion broth (Hi-media, Mumbai, India) at 37°C. Total viable counts of the LAB isolates were determined using de Man Rogosa Sharpe (MRS) agar at 37°C. Shigella and Salmonella were enumerated on Salmonella Shigella (SS) agar (Hi-media, Mumbai, India), E. coli on MacConkey agar (Hi-media, Mumbai, India) and V. cholerae on thiosulphate-citrate-bile salts-sucrose (TCBS) agar (Hi-media, Mumbai, India) after 18 h at 37°C.
Isolation of LAB and their identification by API 50CH: The study was carried out in the department of Gastrointestinal Sciences, CMCH, Vellore, Tamil Nadu, India between 2008 and 2010. Samples of freshly made curd were obtained from 16 households within a 50 km radius of Vellore and transported immediately to the laboratory. The households chosen belonged to laboratory researchers who made curd each day at home using a small portion of curd to inoculate milk which was then left covered overnight at room temperature. Curd samples were transported to the laboratory in the morning and were plated on MRS agar and incubated overnight at 37 ° C. Microbial colonies that grew out in culture were identified by Gram stain, catalase test and biochemical characterization 11 . API 50 CH was used in conjunction with API 50 CHL (bioMerieux, Chennai, India) medium for the identification of LAB. Strains identified by the API 50CH system were subjected to growth curve analysis to determine the time period during which exponential growth was seen. LAB were cultured on Lactobacillus MRS broth (Himedia, Mumbai, India) for 16 h at 37°C with 10 per cent CO2 under anaerobic conditions and used for testing.
Growth kinetics of LAB: The profile of LAB growth during curd formation was explored through time course studies involving laboratory fermentation of milk using curd as starter culture. Inocula of fresh curd were used to seed three (two pasteurized, one unpasteurized) 150 ml aliquots of milk at 1 per cent (w/v) concentration. The milk was allowed to ferment for 48 h at room temperature and a 1.5 ml aliquot removed every 4 h for culture and for deoxyribonucleic acid (DNA) isolation. The samples were streaked on MRS agar, MacConkey agar and blood agar plates. MRS agar plates were incubated for 24 h at 37°C in anaerobic conditions with 10 per cent CO2, while MacConkey agar and blood agar plates were incubated for 48 h at 37°C. After incubation, colonies were enumerated and variations in colony morphology at different durations were noted. DNA was isolated from the curd samples 12 and subjected to real time polymerase chain reaction (PCR) using primers targeted at 16S rRNA gene sequences specific to Lactobacillus genus 13 ,14 . Amplification of lactobacillus sequences was expressed relative to the amplification of sequences conserved for domain bacteria (universal).
Studies done with LAB: The following experiments were carried out when the strains were in early stationary phase. The isolates were screened for attributes consistent with probiotic properties. These included resistance to acid and pepsin, bile salt and pancreatin tolerance, antimicrobial resistance, intrinsic antimicrobial production, adhesion to (and inhibition of pathogen adhesion to) Caco-2 intestinal epithelial cells, modulation of IL-8 response in HT-29 colonic epithelial cells and modulation of cytokine expression in THP-1 monocyte cells.
Tolerance to acid, pepsin, bile and pancreatin and intrinsic haemolytic activity: The ability of the organisms to survive adverse conditions was assessed by incubating the organisms in MRS broth culture in presence of these adverse influences, and removing aliquots at specified times for plating on MRS agar plates to ascertain growth. The ability of the isolates to survive in the presence of hydrochloric acid (pH 1.0 and pH 3.0), pepsin (3 mg/ml, pH 2.0), pancreatin (1 mg/ml, pH 8.0) and bile salts (0.3% w/v Ox Gall) was measured. The reagents for these tests were obtained from Sigma Chemical Co., USA. Intrinsic haemolytic activity was determined by culture on Columbia blood agar plates for 48 h and observing haemolysis around the colonies.
Antimicrobial susceptibility and activity: Antimicrobial sensitivity to ampicillin, co-amoxyclav, gentamicin, chloramphenicol, rifampicin, norfloxacin, vancomycin and imipenem were determined using the Kirby-Bauer disc diffusion method 15 (Himedia, Mumbai, India) with standard definitions for antimicrobial resistance or sensitivity 16 . The discs contained the following amounts per disc (in µg): Ampicillin (10), co-amoxyclav (30), gentamicin (10). chloramphenicol (30), rifampicin (5), norfloxacin (10), vancomycin (30), imipenem (10), tetracycline (30).
Intrinsic antimicrobial activity against E. coli, S. Typhimurium, V. cholerae and S. flexneri was determined by well-diffusion in BHI soft agar plates and zones of inhibition were measured.
Adherence to Caco-2 cells: The ability of the bacteria to adhere to Caco-2 cells 17 , a colon cancer cell line with small intestinal differentiation was tested. Caco-2 cells were grown in Dulbecco's modified Eagle medium (DMEM) (Sigma Chemical Co., USA), supplemented (v/v) with 10 per cent foetal calf serum inactivated at 56°C for 30 min, 1 per cent non-essential amino acids, 1 per cent glutamine and 20 µg/ml of streptomycin and penicillin, at 37°C in 10 per cent CO2/90 per cent air. The cells were seeded into 24-well tissue culture plates (BD, Gurgaon, India) at a concentration of 1 million cells per ml, and incubated for seven days with change of medium every two days. Two ml volumes of early stationary growth phase broth cultures were pelleted, washed and suspended in non-supplemented DMEM to a concentration corresponding to McFarland standard 3. One ml of suspension containing 10 8 bacteria was overlaid onto the Caco-2 monolayers, and plates incubated at 37°C in 10 per cent CO2/90 per cent air for 90 min. The microbial suspension was aspirated, the monolayers washed twice, and 1 ml of 0.1 per cent Triton X-100 added for ten minutes to detach adherent microbial cells. The cells were plated on MRS agar at 1:100 and 1:10000 dilutions and incubated at 37°C in 10 per cent CO2/90 per cent air for 24 h, and colonies enumerated 18 . E. coli was used as a positive control and the adhesion of the test strains was expressed relative to the E. coli adhesion value.
Inhibition of pathogen adhesion: The ability of the strains to inhibit pathogen adhesion to Caco-2 cells was also tested in vitro 19 . Briefly, Caco-2 monolayers in 24 well plates were overlaid with the test strains for 90 min, after which the bacteria were removed and the cell monolayer washed thrice with DMEM. One ml of DMEM containing 10 8 V. cholerae or S. Typhimurium cells was overlaid on the monolayers, incubated for 90 min, and adherent V. cholera or S. Typhimurium enumerated using the procedure described above.
Relative expression of interleukin (IL)-8, IL-10, IL-12, and tumour necrosis factor (TNF)-α by real time PCR and ELISA: The bacteria were tested for modulation of cytokine expression or release from immune cells. Modulation of interleukin-8 release from HT-29 cells in response to V. cholerae was used as a test system to determine putative probiotic properties in the isolated lactic acid bacteria 20 . Pre-exposure to the test isolates (10 8 /ml) for 90 min was followed by exposure of the cell monolayers to 10 8 V. cholerae cells for four h, after which supernatant and cells were separately saved for analysis. IL-8 concentrations in the supernatant were estimated by ELISA (Opt EIA Set, BD Biosciences, USA). Modulation of the cytokine response in THP-1 monocyte-macrophage cell lines was used to investigate the LAB for potential probiotic properties. THP-1 is a human mononuclear leukaemia cell line that can secrete the anti-inflammatory cytokine IL-10 and was grown in flasks, dissociated, washed and resuspended in medium prior to the experiments. THP-1 cell suspension (1×10 6 cells/ml/well) was placed in 24-well tissue culture plates (Falcon Milliwell, BD, USA), and incubated for one hour. Bacterial cells suspended in non-supplemented RPM1 (10 8 cells/ml) were added and incubated at 37°C in 10 per cent CO2/90 per cent air for two h. The microbial suspension was aspirated from the wells, one ml of fresh RPMI medium containing 20 µg gentamicin added to each well, and incubated at 37°C in 10 per cent CO2/90 per cent air for 4 h. The wells were aspirated and the suspensions were centrifuged at 7500 × g for 10 min at 4°C. To the pellets, 300 µl of TRI reagent was added and stored at -80°C for RNA isolation. RNA was isolated in batches using TRI reagent and converted to cDNA using a reverse transcriptase core kit (Eurogentec, Liege, Belgium). Expression of the housekeeping gene actin was monitored to confirm cDNA conversion. Real-time PCR using SYBR green 20 was carried out to quantitate IL-10, IL-12 and TNF-α gene expression relative to expression of β-actin, using appropriate primers ( Table I ).
Primers used in real time PCR