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ThesisItem Open Access ATTENUATED TOTAL REFLECTANCE-FOURIER TRANSFORM INFRARED SPECTROSCOPY COUPLED WITH CHEMOMETRICS TO DETECT SELECTED ANIMAL BODY FATS, VEGETABLE OILS AND THEIR ADMIXTURE IN GHEE(ICAR-NDRI, KARNAL, 2022) VIVEK SONVANSHI; KAMAL GANDHIGhee is a popular traditional dairy product of India. Due to its high demand and insufficient supply during lean season, it becomes prone to adulteration by unscrupulous traders in the market. The present study was conducted to develop suitable models using ATR-FTIR coupled with chemometrics for detection of selected animal body fats, vegetable oils and their admixture in ghee. Milk samples were procured and ghee samples were prepared using creamery butter method. Vegetable oils from five reputable brands and animal body adipose tissues were purchased from local market of Karnal. Animal body fats were extracted from the respective adipose tissues of the animal using dry rendering process. GC-analysis revealed that the short chain fatty acids were only present in pure ghee and not in adulterants targeted in the study. Linoleic acid concentration was significantly higher in soybean oil as compared to that in other oils and fat studied. Palmitic acid was the major fatty acid in vanaspati and trans fatty acids (eladic acid) was only present in vanaspati. Goat body fat contained higher amount of stearic acid as compared to that in other oils/ fats studied. Oleic acid concentration was higher in palmolein oil and pig body fat as compared to that in other oils and fats studied. Prepared cow and buffalo ghee were mixed in equal proportions to obtained pure mixed ghee (PMG). Adulterants, viz. soybean oil (SO), vanaspati (VG), palmolein oil (PO), goat body fat (GBF), pig body fat (PBF), sheep body fat (SBF) were added individually at 1, 3, 5, 10, 15 and 20 as well as in combinations, viz. SO+GBF, VG+GBF, PO+GBF, SO+PBF, VG+PBF, PO+PBF, SO+SBF, VG+SBF and PO+SBF in PMG in the ratios of 1:2.3, 2:4.6, 3:7, 4:9.3 and 5:11.6, respectively. FTIR spectra analysis of all the samples were performed and the data obtained was subjected to chemometric analysis. Functional group regions of the mixed ghee and adulterants were almost similar while slight differences in their finger print regions were observed. Wavenumber regions which were found useful for detecting adulteration of ghee with SO, VG, PO, GBF, PBF and SBF were 727-702, 1120-1080 and 985-955, 1167-1137, 1760-1730, 1190-1140 and 1100-970, 1190-1140 and 1120-970 and 732-710 cm-1, respectively. Wavenumber regions which were found useful for detecting adulteration of ghee with admixture of SO+GBF, VG+GBF, PO+GBF, SO+PBF, VG+PBF, PO+PBF, SO+SBF, VG+SBF and PO+SBF were 1180-1140 and 1120-1098, 1170-1145 and 1120-1087, 1175-1135 and 1125-1080, 1170-1140 and 1130-1090, 1180-1140 and 1120-1090, 1200-1130 and 1123-1093, 730-710, 1125-1085 and 740-700 cm-1, respectively. PCA applied in the selected regions showed separate clusters from PMG even for the lowest level of spiking of each adulterant and their admixture and as the level of spiking of adulterants increased, clusters shifted towards pure adulterants. PLS and PCR models applied in the selected regions of the FTIR spectra were equally efficient in detecting the selected adulterants in ghee as indicated from the R2, RMSEC, RMSEV and Bias values. Calibration curves between the actual and predicted levels of all the individual adulterants and their admixture in ghee were linear with a slope of 45° and no x or y intercepts indicating the suitability of the models in detecting them in ghee. SIMCA approach in conjunction with the established PLS models applied in the selected wavenumber regions showed the classification efficiency for pure mixed ghee, pure body fats and pure vegetable oils as 100% indicating that models were effectively developed for their detection in ghee. Classification efficiencies for ghee samples containing individual adulterants and their admixture at all the levels studied were never fell down below 85 and 73%, respectively. Using ATR-FTIR, we can detect up to 1% level of selected individual adulterants and 3.3% level of admixture of animal body fat and vegetable oil in ghee. Rapid, non-destructive, low cost and accurate analytical protocol involving a combined use of FTIR spectroscopy and chemometrics for rapid and accurate determination of soybean oil, vanaspati, palmolein oil, goat, pig, sheep body fat and admixture of SO+GBF, VG+GBF, PO+GBF, SO+PBF, VG+PBF, PO+PBF, SO+SBF, VG+SBF and PO+SBF in ghee is now available which the industries can adopt for regular testing of their samples.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 Process optimization for incorporation of oat bran and barley grain for production of shrikhand(NDRI (SRS), Bengaluru, 2016) Karpurapu, Uma; Balasubramanyam, B VThesisItem Open Access Lemongrass flavoured paneer process optimization utilization and evaluation of shelf life(NDRI (SRS), Bengaluru, 2016) Joseph, Krupa; Rao, K Jayaraj
