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Author: BIDYUT PRAVA MISHRA × End date: 2024 × Start date: 2020 ×
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    ThesisItemOpen Access
    QUANTITATIVE IDENTIFICATION, CHARACTERIZATION AND VALIDATION OF SPECIES SPECIFIC ANIMAL DERIVED GELATIN BY CONVENTIONAL AND MULTIPLE REACTION MONITORING MASS SPECTROMETRY APPROACHES
    (SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502. (A.P.) INDIA, 2023-12) BIDYUT PRAVA MISHRA; ESWARA RAO .B (MAJOR); NAVEENA .B.M; NAGA MALLIKA .E; SRINIVASA RAO .T; NARENDRA NATH .D
    Comprehensive analysis of gelatin extracted from water buffalo hide, pig, sheep, broiler chicken and spent hen skins using different acid and/alkali pre-treatment followed by thermo-hydrolysis was carried out. Highest (P < 0.05) gelatin yield was obtained from sheep skin (SSG, 9.55%) and water buffalo hide (BHG, 9.47%) relative to chicken (CSG, 6.06%), pig (PSG, 5.98%) and spent hen (SHSG, 5.98%) skins, respectively. The SSG showed highest transmittance (%) among all the gelatin samples. Buffalo hide presented the highest collagen solubility (75.76%), followed by chicken (68.95%), sheep (59.58%), spent hen (11.74%) and pig (5.23%) skin, respectively. The PSG gels showed lower L*, a* and b* values and higher (P < 0.05) viscosity relative to other samples. The SSG had higher (P < 0.05) gel strength, foaming capacity and foaming stability as compared to others. Fourier transform infrared (FTIR) spectroscopy analysis of gelatin exhibited the presence of amide-I peaks for all samples in the lower frequency range (1629.9-1647.26 cm-1), whereas, only SSG, BHG and PSG revealed amide-II peaks at lower frequency range (1500.67-1523.82 cm-1) indicating NH deformation. Scanning electron microscopy analysis revealed that BHG gel had a consistent network of thicker strands and medium-sized holes whereas, PSG and SSG gel showed a sheet-like appearance with no voids. The SDS-PAGE fractionation of extracted gelatin powder followed by MALDI-TOF MS analysis has identified the collagen alpha 1 (I) chain as a predominant component responsible for gelatin stability and detected unique species-specific peptides. Current study has demonstrated the potential of using water buffalo hide and sheep skin as an alternative to pig skin/bovine hides or bones for industrial production of gelatin with superior quality attributes. In the second phase of experiment, the binary mixture samples were prepared by spiking chicken meat patties (CMP) with 0.1, 0.5, 1.0 and 2.5% (w/w) of in-house derived pig skin gelatin (PSG)/water buffalo hide gelatin (BHG). Gelatin was extracted from these binary mixture samples by using cold acetone and were subjected to LC-MS/MS multiple reaction monitoring (MRM) mass spectrometry for authentication of presence of bovine and or porcine gelatin in processed meat products. The current study has identified 2 unique bovine gelatin peptides of m/z 781.3364 (GETGPAGPAGPIGPVGAR) and m/z 852.7189 and 3 unique porcine gelatin peptides of m/z 774.570 (GETGPAGPAGPVGPVGAR), m/z 971.776 (VPAGECCPVCPEGEVSPTDQETTGVEGPK) and m/z 727.436 and the optimized MRM-MS method was successful in detecting BHG/PSG in chicken meat patties up to a 0.5% (w/w) of the total weight and these levels can be considered as the minimum level of detection (LOD) of BHG / PSG in gelatin containing processed products. By using calibration curves for bovine gelatin (certified reference material) peptide of m/z 781.3 (transition 781.3  127.1466) and porcine gelatin specific peptide of m/z 774. 570 (transition 774.570  70.1561), gelatin content of 10.03 nmol/ml and 45.88 nmol/ml were detected in gelatin samples extracted from cooked CMP spiked with 0.5% (w/w) of BHG and PSG, respectively. To screen the undeclared source of gelatin in commercial samples available in the market, 25 commercial food samples (marshmallows, commercial gelatin powder, jelly candies, chicken sausages, chicken meat balls, chewing gums and chocolate cookies) were procured from the local market of Hyderabad. The gelatin (if any) extracted using cold acetone and subjected to MRM mass spectrometry to authenticate the animal origin of gelatin in commercial samples. The findings revealed that 2 bovine gelatin peptides and 3 porcine gelatin peptides identified in the current study are highly unique and specific in detecting the species of gelatin and gelatin-containing foods and are able to identify the adulteration of processed foods with BHG and PSG. In the present study, out of 25 tested samples, 4 samples showed the presence of bovine gelatin and 6 samples showed the presence of porcine gelatin. The LC-MS/MS MRM method optimized in the present study was suitable in detecting the presence of animal derived gelatin in commercial samples.