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20171
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Subject: Biochemistry × Author: Bajaj, Sonali × End date: 2019 × Start date: 2010 ×
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    ThesisItemOpen Access
    Biochemical studies on synthesis, hydrolysis, characterization and storage of pearl millet lipids
    (CCSHAU, 2017) Bajaj, Sonali; Chugh, L.K.
    The present investigation is the first ever attempt for studying synthesis, hydrolysis, physico-chemical characterization and storage stability of pearl millet lipids. A field experiment on high-lipids hybrid HHB 94 raised under irrigated and water deficit condition was carried out during kharif-2014 to correlate deposition of lipids and activity of acetyl CoA carboxylase (ACCase) in developing grains. Activity of ACCase could not be determined and correlated with higher amount of deposition of lipids in the grains under irrigated condition due to very high labile nature of the enzyme. For studying hydrolysis of lipids, flour of two R-lines (male parents) viz., fast lipids hydrolyzing (FLH) G73-107 and slow lipids hydrolyzing (SLH) HBL11and two A-Lines (female parents) viz., ICMA 97111 (FLH) and ICMA 95222 (SLH), F1 hybrids: ICMA 97111A × G73-107, ICMA 97111A × HBL 11, ICMA 95222 × HBL 11and ICMA 95222 × G73-107 along with that of one population WHC 901-445, seven check hybrids, two check varieties and thirty designated B-lines was stored at 37°C and RH 40-50% for 10 days. Lipids content, rate of hydrolysis of lipids determined as periodic increase of fat acidity (FA) in flour during storage, total hydrolysis of lipids measured as total buildup of FA on day 10, level of in vitro and in situ activities of lipolytic enzymes recorded periodically and changes manifested in the water soluble fraction of flour reflected as changes in pH were determined. Fresh flour (0 day storage) served as control. Crossing of either of SLH A-line (ICMA 95222) or R-line (HBL 11) with any of FLH A-line (ICMA 97111) or R-line (G73-107) did not result in development of F1 hybrids those were SLH. No correlation was found between lipids content and either total FA or rate of development of FA or activities of esterase and lipase of R-lines, A-lines, F1 hybrids, populations, check varieties, check hybrids, or designated B-lines. Strong correlation coefficients between rate of increase in FA and activities of lipolytic enzymes (r=0.855** for in vitro esterase, r=0.773** for in situ lipase and r=0.718** for in situ esterase) in stored flour of the designated B-lines indicated that development of FA is controlled by the level of activities of lipolytic enzymes and not by lipids content. No correlation was found between decrease in pH of water extract of flour and any of the other parameters associated with hydrolysis of lipids which indicated chemical changes taking place in water soluble fraction of flour of designated B-lines are independent of fat content and activities of lipolytic enzymes. To further investigate role of lipolytic enzymes in hydrolysis of lipids in pearl millet flour, one p-nitophenyl butyrate (p-NPB) dependent esterase was partially purified by ammonium sulphate precipitation (30-60% saturation), gel filtration through Sephadex G-75. Molecular weight of the purified enzyme was approx 60 kDa. The enzyme exhibited Michaelis-Menten kinetics for its substrates, p-NPB. The Km of purified enzyme was 0.65 mM. The purified preparation exhibited optimum activity at 45°C. The enzyme was stable up to 60°C for 20 min. Studies on stored flour showed that the activities of esterase (as well as lipase) did not change for 10 days of storage. This indicated in situ stability of lipolytic enzyme at 37°C. The purified preparation showed maximum activity at pH 8.2 and substantial activity at pH between 6 and 7 (which prevails in pearl millet flour) which explains presence of highly active esterase in situ. DTT and EDTA were potent inhibitors of the purified esterase followed by PMSF and ascorbic acid. Inhibition of the enzyme by ascorbic acid might have implications for developing processing technologies for improving shelf life of flour by arresting hydrolysis of lipids. The released variety HC 20 was found to be SLH because of low activities of lipolytic enzymes compared to the population WHC 901-445. Physico-chemical properties i.e. specific gravity, saponification value, unsaponifiable matter and refractive index of lipids extracted from bran and flour of both the genotypes were near identical. Total tocopherol content in bran and flour lipids of HC 20 was higher than that of WHC 901-445. In terms of hydrolysis and oxidative changes, lipids of HC 20 were comparatively more stable during storage compared to that of WHC 901-445.