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ThesisItem Open Access PHENOTYPIC AND GENOTYPIC CHARACTERISATION OF ANTIMICROBIAL RESISTANCE IN ENTEROCOCCUS SPECIES OF DAIRY ORIGIN(ICAR-NDRI, KARNAL, 2022) RAVIKANT VINAYAKRAO VINCHURKAR; DIWAS PRADHANPrevalence of antibiotic resistance among food-based enterococci is matter of concern because of its opportunistic pathogenicity as well as its ability to disseminate AMR genes to other bacteria in the food chain. The present study was undertaken to characterize antimicrobial resistance in Enterococcus species of dairy origin. A total of 64 dairy samples comprising of traditional Dahi and raw milk samples were collected from the districts of Karnal, Kaithal, Sirsa, Bhiwani and Jind of Haryana region. The sample were processed for isolation of enterococci by pour plate technique using selective media viz. Citrate Azide and Bile Esculin Agar and further identified by phenotypic and genotypic approach. A total of 235 isolates were initially shortlisted based on Gram’s staining and catalase test. The genotypic identification of isolates was done by using genus specific PCR. Finally, a total of 140 isolates were genotypically confirmed as enterococci. A total of 84 Enterococcus isolates were further tested for antimicrobial susceptibility against major antibiotic classes as per CLSI guidelines. The highest resistance in the enterococci isolates were observed against Cefuroxime (50%), Rifampicin (21.43%), Cefepime (19.05%), Erythromycin (15.48%), Fosfomycin (9.52%), Cefotaxime (8.33%), as well certain isolates showing resistance against Tetracycline, High Level Streptomycin, Gentamycin, Norfloxacin, Chloramphenicol and Levofloxacin. Extended Spectrum β Lactamases production was confirmed in 9 out of 24 isolates. PCR for antibiotic resistance genes in respective resistant isolates showed positive results for Aminoglycoside resistance genes such as aac(6’)-Ie- aph(2”)-Ia, aph(3’)-IIIa, ant(6)-Ia, Tetracyclines resistance genes [tet(M), tet(O)], Chloramphenicol resistance genes (catA8) and also certain isolates with Macrolide resistance genes such as ermA, ermB, ermC, ermF and msrA. Among the 43 resistant isolates, 69.76% of isolates were positive for multi-drug transporter gene (emeA), while 25.58% and 30.23% were positive for Transposons family, Tn-5397 (tndX) and Tn-916/Tn-1545 (Int-Tn), respectively. Further, 4 isolates also showed the presence of class-1 Integron gene, while 12 isolates were positive for class-3 Integron gene. A total of 43 resistance isolates were tested for virulence traits. Among these only 2 (4.6%) isolates demonstrated Gelatinase production (also positive for gelE gene), 17 (39.53%) for Hemolysin production (6 showed positive for cylA gene) and none for DNase production. No significant correlation between the virulence and antibiotic resistance traits were observed in the isolates. Further, the resistant isolates have been sent for 16S rRNA partial sequencing for species identification and the most resistant isolate B1(C) for Whole Genome Sequencing to study the organization of AMR genes in the isolate.ThesisItem Open Access EVALUATING THE IRON BIOAVAILABILITY FROM EXOPOLYSACCHARIDE KAR1- IRON COMPLEX AND ITS EFFECT ON ANAEMIC RAT MODEL(ICAR-NDRI, KARNAL, 2022) SHASHANK GOWDA B G; PRADIP V. BEHAREIron deficiency anaemia is the most common micronutrient deficiency disorder among people of different age groups, for which effective nutritional strategies are warranted to tackle its devastating effects on public health. The present study investigated the iron bioavailability from an EPSKar1-iron complex using Caco-2 cells and anaemic rats. Exopolysaccharide (EPSKar1) extracted from Lacticaseibacillus rhamnosus Kar1 had the carbohydrate and protein content of 78.33±0.86% and 3.58±0.27%, respectively. Upon purification by ion-exchange chromatography, the carbohydrate and protein concentrations were found to be 95.42±0.04% and 0.30±0.02%, respectively. The purified EPSkar1 was complexed with FeSO4 iron salt and the obtained 100 mg EPSKar1-iron complex contained 53.65±0.04 mg EPSKar1 and 44.26±0.14 mg iron. During simulated gastro-intestinal digestion of 100 mg EPSKar1-iron complex; 9.20±0.23 mg of free iron was released at the intestinal phase corresponding to 20.92±0.53% iron bio-accessibility. When the cytotoxicity of iron and EPSKar1 was evaluated on Caco-2 cells, no significant difference (p <0.05) in the loss of viability was found at the highest concentration of EPSKar1 (75 mg/mL) and FeSO4 (10 mg/mL). In iron bioavailability study using Caco-2 cells, there was a significant increase (p <0.05) in the iron uptake from the cells treated with EPSKar1-iron complex (61.27±1.96%) compared to FeSO4 salt (28.04±0.50%). The synthesis of ferritin by the Caco-2 cells was significantly increased (p <0.05) after absorbing iron from EPSKar1-iron complex (32.72±0.73 ng/mL) than from only FeSO4 salt (21.77±0.10 ng/mL). Further, the EPSKar1-iron complex was experimented on anaemic rats for a period of 20 days to evaluate iron bioavailability after inducing anaemia by feeding iron free diet for 62 days. The apparent digestibility coefficient (ADC), % iron balance, and % retention/intake was found to be significantly higher (p <0.05) in rats fed with the highest concentration of complex (50 mg/Kg BW), when compared to only FeSO4 fed rats and the rats fed with 25 mg EPSKar1-iron complex/Kg BW. These results were consistent throughout the experimental period of 20 days. There was a significant increase (p <0.05) in the blood haemoglobin content (12.76±0.19 g/dL) of rats fed with higher concentration of complex when compared to rats fed with low concentration of complex (11.91±0.29 g/dL) and only FeSO4 fed rats (10.54±0.21 g/dL). The iron absorption indicators like transferrin and ferritin were found to be significantly higher (p <0.05) in high concentration complex fed rats when compared with only FeSO4 and low concentration complex fed rats. Taken together, these findings suggest that the iron in EPSKar1 complex form has greater bioavailability than uncomplexed iron. Hence, the complex may serve as an ideal molecule for fortifying food products as a source of iron fortificant. Nevertheless, further human clinical trials are highly warranted in order to validate it as an effective nutraceutical for an anaemic group of population.ThesisItem Open Access SURVEILLANCE OF HEAT DESICCATED AND ACID COAGULATED DAIRY PRODUCTS FOR ANTIBIOTIC RESISTANT BACTERIAL PATHOGENS(ICAR-NDRI, KARNAL, 2022) AYANTIKA DAS; RAGHU, H. V.The goal of the current study was to determine the prevalence of four pathogenic microorganisms- Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Enterococci- in various traditional dairy products from the Karnal region. This was done by using conventional methods and polymerase chain reaction (PCR) to confirm the results from Ram Nagar, Kunjpura Road, and other areas of Karnal, one hundred samples of heat-desiccated (Khoa, peda, burfi, gulabjamun, and other Khoa-based sweets) and acid-coagulated (paneer, rasogulla) foods were collected. These samples underwent testing for bacterial pathogens (Coagulase positive S. aureus, E. coli, Listeria spp., and Enterococcus spp.). Using traditional method, E. coli was detected in 45 Khoa and Khoa-based sweets and 19 samples of paneer and Chhana-based sweets out of 100 samples. All E. coli samples were then genotypically analyzed, and 7 isolates, each from heat desiccated and heat acid coagulated dairy products were found to be E. coli. In the instance of Enterococcus spp., 13 isolates were further confirmed as Enterococcus spp. by PCR based methods, and 26 samples were determined to be positive by conventional based procedures. In the case of S. aureus, 59 samples tested positive for the organism, of which 18, also tested positive for coagulase. Only 6 isolates were proven to be coagulase positive S. aureus after genotypic identification.Out of 100 samples, 3 samplestwo of paneer and one each of Khoa/mawa—were positive for Listeria spp., albeit only one isolate was confirmed genotypically by PCR. According to CLSI recommendations, the disc diffusion assay was used to test the antibiotic sensitivity of all the verified isolates. All 45 E. coli isolates exhibited resistance to penicillin, oxacillin, clindamycin, and erythromycin; however, 12 and 10 isolates, respectively, were resistant to ampicillin and trimethoprim. An E. coli isolate had intermediate resistance to ampicillin, meropenem, and ertapenem, whereas 18 E. coli isolates had intermediate resistance to cefotaxime, ceftriaxone, and cefepime. Ceftazidime, meropenem, and ertapenem were effective against more than 90% of the E. coli isolates.Three isolates in the ESBL antibiotic case demonstrated resistance to cefotaxime and ceftriaxone and were identified as ESBL group of antibiotic resistant. The same three isolates were resistant to the antibiotics of the carbapenamase class. Twelve isolates of S. aureus with coagulase positive exhibited penicillin resistance, while eight isolates exhibited oxacillin and rifampicin resistance. Six isolates among those were resistant to methicillin, one isolate displayed intermediate sensitivity and eleven isolates had shown sensitivity to methicillin groups. Four S. aureus isolates demonstrated resistance to imipenem, but all other S. aureus isolates confirmed susceptibility to meropenem and ertapenem. For Enterococcus, 26 isolates exhibited penicillin and erythromycin resistance, 22 isolates exhibited nitrafurantoin and rifampicin resistance, and 4 isolates exhibited vancomycin resistance.Penicillin, rifampicin, erythromycin, and trimethoprim resistance was found in one Listeria spp. isolate verified by PCR. The confirmation of bacterial pathogens and the gene responsible for antibiotic resistance in various bacterial pathogens isolated from indigenous dairy products is currently the subject of further study.ThesisItem Open Access DEVELOPMENT OF PAPER-BASED ANALYTICAL DEVICE FOR RAPID DETECTION OF ß-LACTAM GROUP OF ANTIBIOTICS IN MILK(ICAR-NDRI, KARNAL, 2023) PRASHANT GOEL; NARESH KUMARIndiscriminate use of antibiotics and their presence especially the ß-lactam group in milk has immense public health and processing implications. These residues may develop antimicrobial resistance (AMR) among dairy pathogens which are widely reported in the literature. The current research project entitled “Development of Paper-Based Analytical Device for Rapid Detection of ß-lactam Group of Antibiotics in Milk” based on the enzyme induction principle. Initially, four different strains of Bacillus spp. were screened for the induction of ß-lactamase enzyme and one strain of Bacillus cereus showed induction of ß-lactamase enzyme in presence of ß- lactam group of antibiotic and was selected for further development of assay. For the development of strip based test, six Whatman filter papers (Grade 1, 2, 3, 6, 42 and 602H) were evaluated based on better colour development and intensity on the strip and a Grade-3 paper was selected. Subsequently, assay protocol for detection of ß- lactam group in milk was optimised on strip based on enzyme induction and enzyme-substrate reaction. The optimised parameters include spore volume (20 μL), O.D of the spores (0.8), substrate volume (10 μL), exposure time (30 min.), incubation time (30 min.) and temperature (37°C). The interference study with contaminants like non-ß-lactam antibiotics, pesticides, aflatoxin M1, heavy metals, and other chemical contaminants was carried out with no cross-reactivity in the working of the assay. The limit of detection (LODs) for various ß-lactam antibiotics, including amoxicillin, penicillin, ampicillin, carbenicillin, cloxacillin, nafcillin, oxacillin, cephalothin, cefalexin, cefoxitin, cefazolin, and cefuroxime were achieved in spiked milk at 1ppb, 1ppb, 1ppb, 10ppb, 10ppb, 10ppb, 20ppb, 10ppb 1000ppb, 10ppb 300ppb and 100ppb, respectively with invariably complying FSSAI /Codex MRL standards. The developed test was evaluated under field conditions with 200 raw milk and 105 pasteurised milk samples wherein 7 raw milk samples were found positive with the developed strip-based test. These samples were also tested with the AOAC-approved CHARM-ROSA test and a 100% correlation was achieved. The test kit components such as lyophilized spores and substrate functionalized paper strips were also evaluated for shelf stability at 4°C and -20°C respectively and found stable up to 9 months. The developed paper strip test was translated on pillar-based analytical device which was fabricated at the University of Southampton. The pillar-based chip was designed with AutoCAD software, having nine pillars of 4 mm diameter x 1 mm height on the pillar chip, and paring nine wells of 4.5 mm diameter x 2.4 mm deep on the chip. The dimensions of the chip were 20 x 20 x 3 mm (width x length x height). Pillar-based analytical device was fabricated on a poly (methyl methacrylate) (PMMA) sheet using a micro-milling machine. Filter papers (Whatman Grade 3) of 4 mm (diameter) were attached to the pillars with double-sided tape. The optimised assay parameters include sample volume (15 μL), substrate volume (3μL) and incubation temperature (37°C) for high throughput analysis. Trials with spiked milk and raw milk samples were carried out successfully with pillar-based analytical device. The developed spore-based strip test and pillar-based analytical device would be of immense use for screening of ß-lactam group in milk under field conditions. The developed test is robust, user-friendly, cost-effective, sensitive, and selective with the capability to cover most of the ß-lactam group antibiotics specified in regulatory standards.ThesisItem Open Access PRECLINICAL SAFETY ASSESSMENT OF INDIGENOUS PROBIOTIC Limosilactobacillus fermentum NCDC 400(ICAR-NDRI, KARNAL, 2023) BASAVAPRABHU H N; PRADIP V. BEHARERegardless of the long and safe use of lactic acid bacteria throughout human evolution, the safety assessment of potential probiotic strains has become a pragmatic regulatory obligation before clinical translation. The present study was undertaken to evaluate the pre-clinical safety and toxicology of Limosilactobacillus fermentum NCDC 400, an in-house industrially important and potential probiotic strain, using a battery of in vitro, in silico, and in vivo approaches. In vitro tests suggest that the strain was devoid of debilitating phenotypes/ activities of hemolysin, coagulase, urease, DNase, gelatinase, β-glucuronidase, β-glucosidase (harmful enzymes), platelet aggregation, mucin and phenylalanine degradation. Overproduction of adverse metabolites such as biogenic amines, D-lactate, ammonia, and indole was also not witnessed by this strain. The test strain was sensitive to the antagonistic property of human serum (> 3 log10 reductions at 3 h). Under the purview of regulatory guidelines, L. fermentum NCDC 400 was sensitive to all the antibiotics enlisted by EFSA guidelines for obligate heterofermentative lactobacilli. The targeted PCR assays using genomic and plasmid DNA of L. fermentum NCDC 400 against the genes conferring unusual antibiotic resistance viz. tetracycline, erythromycin, chloramphenicol, and beta-lactams were found absent. The virulence genes encoding coagulase (Coa), nuclease (nucA), and gelatinase (gelE) were also unseen. Further, an in-depth analysis of the whole genome of L. fermentum NCDC 400 using suitable bioinformatics pipelines and their subsequent validation through multiple databases inferred that this bacterium is non-pathogenic to humans as well as absence of potential antibiotic resistance genes (extrinsic resistance) bearing lateral transferability. In cell culture assays, L. fermentum NCDC 400 was found to be non-cytotoxic to Caco-2 cells even at a concentration as high as 1011 CFU/ mL as well as did not alter their cellular integrity when cultured on transwell inserts. In oral toxicity tests, the daily oral administration of L. fermentum NCDC 400 at 108 (low dose) and 1010 (high dose) cells/ mouse for 14 (acute), 28 (subacute), and 90 (subchronic) days did not induce treatment-related toxicity either in terms of physiological behaviors or clinical parameters (hematology, clinical biochemistry, histopathology, and immune status) of mice. Episodes of L. fermentum NCDC 400-mediated bacterial translocation to extra-intestinal organs were unnoticed in all three oral toxicity studies. Safety and efficacy evaluation of L. fermentum NCDC 400 in an immunocompromised mice model revealed no test strain related adverse effect on the physiological behaviors or clinical parameters of mice; rather it augmented the immune status of mice by immune stimulation (cytokine balance). L. fermentum NCDC 400 was well tolerated in the immunocompromised mice model and did not foster any adverse effects like genotoxicity even at a high dose (1010 CFU/mice/day). Owing to these findings, a dose of 1010 CFU/mice/day may be considered NOEL (No Observable Effect Level) in both healthy and immunocompromised mice. Moreover, NCDC 400 intervention resulted in enhancing the richness and diversity of gut bacteria by fostering beneficial saccharolytic microbes including potential next-generation probiotics (NGPs) while declining the pathobionts. Overall, the results that emerged from this study underscore the safe and non-toxic behavior of L. fermentum NCDC 400 and thereby call for and should facilitate further human clinical trials.ThesisItem Open Access STRIP BASED TEST FOR RAPID DETECTION OF β-LACTAM RESISTANT E. coli IN MASTITIC MILK(ICAR-NDRI, KARNAL, 2021) AVINASH JASWAL; NARESH KUMARResearch gap in the development of diagnosis for Antimicrobial Resistant (AMR) bacteria has been a huge challenge. As per the global guidelines by European food safety authority (EFSA), Codex Alimentarius Commission (CAC), Food and agriculture organization (FAO) and one health monitoring programs, E. coli has been declared as an indicator organism in the dairy animal’s surveillance for AMR. Currently, AMR testing before prescription of antibiotics is not being practiced and broad spectrum antibiotics are given directly to the animals without antimicrobial susceptibility test (AST).Hence, the current study was designed for development of “Rapid Antimicrobial susceptibility assay for detection of extended spectrum β-Lactamase (ESBL), Ampicillinase C -β-Lactamase (AmpC) and Carbapenem resistant E. coli”. Three colorimetric based approaches were followed, which included Iodometric method, Acidimetric method and Marker enzyme based assay. Out of these, marker enzyme based rapid AST assay was selected based on its direct detection of β-Lactam resistant E. coli from cell colonies without going for multiple enrichment and processing steps (culture washing, cell lysis and centrifugation). The developed assay was working on the principle of growth of β-lactam resistant E. coli in E. coli selective media (ECSM) and interaction of its marker enzyme with specific chromogenic substrate which indicates the presence or absence of β-lactam resistant E. coli.The assay primarily involves two steps viz. preparation of antibiotic disc functionalized with 15 μl ECSM and 15 μl chromogenic substrate and second step involved addition of functionalized disc in E. coli culture tube. The change in color from light yellow to bluish-green within 5 hours of incubation indicated the presence of β- lactam resistant E. coli whereas no change in color indicated the absence of β-lactam resistant E. coli. Further, Cross reactivity study with β-lactamase negative standard E. coli ATCC (American Type Culture Collection) 25922 strain was carried out to check the specificity of the developed assay. Subsequently, the protocol for detection of β-lactam resistant E. coli from reconstituted spiked milk was optimized and successfully linked with the developed rapid marker-enzyme based assay. Further, the developed assay was evaluated under field conditions with E. coli isolated from milk. Out of total 240 individual milk samples, 35 were found to be confirmatory positive for E. coli. Among 35 E. coli isolates, 7 isolates (NK-51,NK-62,NK- 95, NK-120,NK-137,NK-163 , NK-165) were found to be positive for ESBL production while one isolate (KF 7831) was found to be ESBL and Amp C co-producer by developed assay. Similar antibiotic resistant pattern was observed with conventional AST assay as well as rapid automated BD (Becton Dickinson) phoenix system. Likewise, all eight E. coli isolates were also confirmed as ESBL by targeting CTX-M (Cefotaxime), SHV (Sulfhydryl variable) and TEM (Temoniera) genes. Based on the above finding, the developed assay can be used for rapid diagnosis of E. coli in dairy, health, fisheries, poultry, environment etc. The novel characteristics of developed assay has been filed in the form of an Indian Patent with application no. 202111007462ThesisItem Open Access NMR BASED METABOLOMIC PROFILING OF MILK FROM CATTLE FOR EARLY DIAGNOSIS OF SUBCLINICAL MASTITIS(ICAR-NDRI, KARNAL, 2022) SHEFALI BHASIN; SUNITA GROVERMilk is an integral part of our food system. India has the largest population of cattle and buffaloes in the world and owing to this status, we also rank first in production of milk worldwide. High yielding dairy cattle are prone to a large number of production elated diseases like mastitis which causes huge economic loss to the dairy industry worldwide. Subclinical form is even more dangerous as it goes unnoticed and causes irreversible damage to the health of the milch animal before it can be detected and ultimately leading to culling of animal from the herd. The present study involved high yielding Karan Fries cattle from the Livestock research Centre of NDRI, Karnal. A total of 112 samples out of 185 were selected following the screening using CMT to study in detail for subclinical mastitis. On the basis of somatic cell counts (SCC), two groups were segregated viz. healthy with somatic cell count upto 2.5×105 cells/mL and 3.5×105 cells/mL for subclinical mastitis (SCM). Physicochemical components analysis involving, EC, pH, Lactose (%) and Fat (%) were found to be significantly correlated with SCC in the milk samples. Hotis test for detection of major disease causing pathogen indicated 10% of total studied samples totally negative and all SCM infected samples positive for Staphylococci. Microbiological culturing and further confirmation using PCR of samples revealed that 28% of SCM samples were positive for S. aureus culture. Multiplex PCR (mPCR) analysis confirmed the presence of S. aureus DNA in 52% of healthy and 83% of SCM samples. Out of total samples, 59% of SCM samples confirmed the presence of E. coli DNA whereas 45% were found positive for S. agalactiae, 64% positive for S. dysgalactiae and only 24% positive for S. uberis. NMR based metabolomics revealed and shortlisted a total of 22 metabolites for studying the metabolomic changes during the course of SCM. The univariate analysis identified 7 metabolites out of 22 in both the groups (Healthy vs SCM) as variable metabolites. Multivariate analysis (PCA, PLS-DA, OPLS-DA and sPLS-DA) revealed and shortlisted various metabolites as important i.e. nine metabolites (PLS-DA), nine metabolites (OPLS-DA) and 10 metabolites (sPLS-DA). ROC analysis confirmed that six metabolites have sufficient potential to work as biomarkers namely Fumaric Acid, Lactate, Acetate, Adenine, Glutamine and Glucose. A multivariate ROC derived analysis to check the authenticity of these potential markers gave a predictive accuracy above 68%. The above study concludes that EC, pH, Lactose (%) and Fat (%) can be used as important parameters for detecting SCM. S. aurues was found to be the major causative agent of SCM occurrence in Karan Fries cattle at Livestock centre of NDRI, Karnal. Six metabolites Fumaric acid, Lactate, Acetate, Adenine, Glutamine, Glucose have sufficient potential for development of diagnostic kits based on multi analytes for early detection of SCM.ThesisItem Open Access ANTI-OXIDATIVE AND ANTI-INFLAMMATORY EFFECTS OF LACTIC ACID BACTERIA DERIVED EXOPOLYSACCHARIDES ON MICE MODEL(ICAR-NDRI, KARNAL, 2022) MANORAMA KUMARI; PRADIP BEHAREThe present study was designed to screen EPS Kar1, Ram1, Ram12, and 399 derived from LAB, for anti-oxidative and anti-inflammatory potential and to investigate its alleviating potential in Dgalactose induced stress model. The crude EPS Kar1, Ram12, 399, and 400 (positive control in vitro) purified by anion-exchange, while EPS Ram1 by cation exchange chromatography, based on the negative and positive zeta potential value, respectively. Monosaccharide composition (mannose, rhamnose, and arabinose in EPSRam12; glucose, N-Acetyl-glucosamine and mannose in a molar ratio of 8:4:1 in EPSKar1; glucose and fructose in the molar ratio of 3:1in EPSRam1; glucose and N-Acetylglucosamine in the molar ratio of 8:1 in EPS399) analysis by UFLC showed that EPSs were heteropolysaccharides. The molecular weight of EPSKar1 was found to be 7.8x105 Da, whereas 2.6x106 Da for EPSRam12. FTIR analysis revealed that EPSs comprised of functional hydroxyl (O-H) and carboxyl (C=O) groups and glycosidic bonds. NMR analysis revealed that EPSRam12 is a branched chain polysaccharide with α-configuration, while EPS Ram1 and 399 are linear polysaccharides with α-configuration, and EPSKar1 is a branched chain polysaccharide with β-configuration. SEM micrograph revealed a homogeneous sheet with a microporous and rough structure of EPSKar1, coarse surfaces, resembling polysaccharide sheets overlaid like morphology of EPSRam12, a compact flake like structure made up of similar sizes morphology of EPSRam1, and a spider web like network structure with porous entanglement of EPS 399. EPS Ram12 showed better scavenging activities against DPPH, ABTS, hydroxyl, and superoxide radicals among four EPS but less than the positive control EPS400 and ascorbic acid, while the chelating activity on ferrous ion was less than EPSKar1 but greater than EPSRam1 and 399 in a concentration (0.125 to 5mg/mL)-dependent manner. EPSRam12 showed better immunostimulatory activity in vitro in terms of increasing the viability of murine macrophage cells (no cytotoxicity upto the concentration of 800 μg/mL) and enhancing the capability of macrophage cell phagocytosis, as well as promoting the secretion of anti-inflammatory cytokine IL-10 and decreasing the production of proinflammatory cytokines (TNF-α, IL-1β, IL-6) and NO in LPStreated macrophage cell, compared to EPS Kar1, Ram1and 399, but less than positive control EPS400, indicating its potential for use as an immunomodulator in functional food areas. The three different dose of D-galactose (D100, D300, and D500 mg/kg body weight) and duration (0th,15th, 30th, 45th, and 60th day) were used for developing an oxidative and inflammatory stressed mice model, revealed a significant decrease in the level of blood glucose, organ indices (spleen, thymus, liver, brain and kidney), serum antioxidants (SOD, CAT, GSH-Px and TAC), and antiinflammatory cytokine IL-10, whereas a significant increase in oxidants (MDA and NO) level, proinflammatory cytokines (TNF-α, IL-1β, and IL-6), ALT and AST in serum as well as LF in tissue (liver and brain) from 30th to 60th day of dissection, compared to the control. No significant difference observed in all the above parameters between 45th and 60th day among the D-galactose administered groups (D100, D300, and D500 mg/kg body weight). Histological examination reveals the presence of severe liver damage, typical brain tissue injury characteristics, and damaged normal epithelial architecture in colon of D-galactose (D100, D300, and D500) mice. The dose of 1% D-galactose (D100) for 45th day was selected to induce the oxidative and inflammatory stress with the intraperitoneal injection of D-galactose. EPSRam12 supplementation significantly alleviated the objectionable changes induced by D-galactose, strengthen the intestinal tight junctions (ZO-1, Claudin-1, Occludin) gene expression, inhibited the upregulation of NF-κB p65, COX-2 and iNOS expression, manipulated the gut microbiota in D-galactose induced mice, resulting in enhanced proportions of the phylum Bacteroidetes/ Firmicutes (B/F), promoting the proliferation of beneficial short chain producing bacteria while decreasing the number of harmful bacteria, enhanced the cecal acetate, butyrate and total short-chain fatty acid concentrations compared with the respective short chain fatty acid in the D-galactose group, which were correlated with the protective effects of EPSRam12.Therfore, the finding foster the potential application of EPSRam12 in food and drug as a novel antioxidant and anti-inflammatory agent, as well as the positive gut microbiota modulator, and short chain fatty acid enhancer molecule.ThesisItem Open Access EFFICACY OF PROBIOTIC LACTOBACILLI AGAINST LEAD INDUCED NEUROTOXICITY IN RAT MODEL(ICAR-NDRI, KARNAL, 2021) HEMLATA SINGH; CHAND RAMProbiotics are increasingly being employed as metal bio-adsorbents due to presence of negatively charged functional groups on their surfaces. Lead a common food contaminant is highly toxic and carcinogenic to humans. Present investigation was aimed to delineate the efficacy of probiotic lactobacilli against lead induced neurotoxicity in rat model. Forty-one Lactobacillus strains were evaluated for their lead bioadsorption potential at 37°C with 8 log CFU/mL of lactobacilli in presence of lead concentration @ 50 mg/L. Ten lactobacilli strains showing lead binding of >80% were selected for further investigations. Highest lead bioadsorption was observed by PS 121 (96.77±1.30%) and HD 51 (96.40±1.05%). Two lactobacilli strains viz., Lactobacillus plantarum HD 51 and L. plantarum MDD 56 were also evaluated for probiotic attributes (acid and bile salt tolerance, cell surface hydrophobicity, antimicrobial activity and antibiotic sensitivity testing) and identified as Lactobacillus plantarum. The selected ten lactobacilli were reconfirmed upto species level using 16S rRNA techniques. The selected lactobacilli strains were able to bind lead within 1 h at pH 5 with strong lead-probiotic complex as washing had no effect on its binding ability. These strains were able to tolerate lead upto 800 mg/L in MRS broth except L. plantarum CRD 7 and L. fermentum NCDC 214 which did not show any growth upto 48 h of incubation. L. plantarum HD 51 observations on antioxidant ability showed highest percent inhibition for DPPH (58.09±0.49) and ABTS (69.15±0.28), while L. rhamnosus PS 121 exhibited maximum percent inhibition for lipid peroxidation (48.86±2.04). Maximum lead bioaccessibility reduction was observed by L. plantarum HD 51 (87.32±4.60%) under simulated digestive tract conditions indicative of strong lead-probiotic complex formation. Based on principal component analysis L. plantarum HD 51was selected for its in-vivo efficacy evaluation. Scanning electron microscopy images also revealed lead adsorption ability of L. plantarum HD-51 on its surface. Lead dosage of 50 mg/Kg bw for 30 days was standardized for induction of neurotoxicity in wistar rats. Daily administration of probiotic L. plantarum HD 51 for 50 days regulated the neurotransmitter level and antioxidative enzymes in brain. Histopathological studies also revealed that L. plantarum HD 51 administration reduced neuronal degradation in cerebrum and cerebellum region along with decreased infiltration of inflammatory cells. Increase in δ- Aminolevulinic acid dehydratase activity with reduction in serum creatinine and plasma corticosterone biomarkers was observed in L. plantarum HD 51 fed group. The mRNA expression of intestinal tight junction proteins (Zona Occludin 1, Zona Occludin 2, Claudin, Claudin 5 and Occludin) were downregulated in lead and L. plantarum HIF 33 (non-metal binding bacterial group) fed group, while L. plantarum HD 51 fed groups showed upregulation of these genes. Similar observations were noticed for blood brain barrier tight junction proteins gene expression indicating disruption of blood brain barrier in lead and L. plantarum HIF 33 fed group. Further, L. plantarum HD 51 demonstrated lowering of lead body burden by excretion of lead in faeces. Quantitative estimation of short chain fatty acid in faeces revealed increased levels of acetic acid in L. plantarum HD 51 fed group whereas lead fed group indicated increased level of propionic and butyric acid. Thus, it is concluded that probiotic L. plantarum HD 51 has potent lead bioadsorption potential and it can be used as a prophylactic in lead induced neurotoxicity.
