Peroxidase from pearl millet hybrid HHB 94 was purified stepwise by (NH4)2SO4 fractionation, gel filtration and ion exchange chromatography using Sephadex G-100 and DEAE-cellulose to near homogeneity. The protocol yielded 41% of peroxidase with 33.2-fold purification. The purified enzyme preparation has molecular masses of 31 kDa, as determined by gel filtration through Sephadex G-100, and 30.4 kDa, as determined by SDS-PAGE, suggesting that the enzyme is a monomer. The enzyme exhibited maximum activity at pH 5.6 and 30C. It was significantly more stable in the pH range of 6.0-6.8 and at temperature below 35C. Peroxidase showed Km values of 0.10 and 11 mM for o-dianisidine and H2O2, respectively. The activity of peroxidase was highly inhibited by DTT, b-mercaptoethanol and hydrazine, and moderately by sodium azide, sodium borohydride, oxalic acid, EDTA, DTNB, Mn 2+ , Na + and K + . The enzyme activity was enhanced by Ca 2+ and Fe 3+ . PRACTICAL APPLICATIONSPeroxidase is considered to have an empirical relationship to off-flavors and odors in foods. Earlier we have shown that peroxidase activity varied in different genotypes, i.e., HHB 94 (high) and 94222A ¥ 78/711 (low), which appeared to be responsible for odor generation in stored pearl millet meal. In 94222A ¥ 78/711, less off-odor was observed. The enzyme purified from mature pearl millet grains is strongly inhibited by DTT. This new information has been used to assay the enzyme in situ. We have found that in comparison to extractable activity, in situ activity of the enzyme is more strongly correlated with the development of offodor in stored flour. Thus, in situ assay is being used to screen germplasm for selecting contrasting genotypes (with respect to off-odor) that will be used to develop pearl millet hybrids with improved shelf life of flour. bs_bs_banner Journal of Food Biochemistry

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The present investigation was conducted to study the effect of storage period on flour quality of wheat, maize and pearl millet. Their flour were stored at 15 °C and 82 % relative humidity RH for one month and changes in fat acidity (FA), activities of peroxidase (POX), lipoxygenase (LOX) and polyphenol oxidase (PPO) in control and stored flour along with change in pH of water extract of flour were monitored. Increase in FA (mg KOH/100 g d.m.) and decrease in pH of flour was maximal in pearl millet (287.5 mg KOH/100 g d.m. and 0.4) followed by maize (151 mg KOH/100 g d.m. and 0.23) and wheat (61 mg KOH/100 g d.m. and 0.1) respectively. A decline in activity of POX, LOX and PPO was observed during storage. Pearl millet flour possessed almost double activity of POX (413 Units/g d.m.) in comparison to wheat (198 Units/g d.m.) and 1.3 fold higher than that of maize (153 Units/g d.m.) and even after decrease activity of POX was higher in flour of pearl millet compared to the other two. However, activity of LOX and PPO was found to be comparable in fresh flour of pearl millet, maize and wheat. Since pearl millet has poor shelf life and increase in FA, decrease in pH and activity of POX was found to be very high in pearl millet (poor shelf life) as compared to wheat and maize, therefore, these selected parameters might be used as biochemical markers to determine shelf life of flour.

This study describes the partial purification and characterization of lipoxygenase (LOX) from pearl millet mature grains of inbred HBL 0843-2. Two isoforms of LOX, i.e., LOX 1 and LOX 2, were purified using ammonium sulphate fractionation, gel filtration chromatography and ion exchange chromatography near homogeneity to 56 and 40 folds with yield of 28 and 24%, respectively. LOX 1 and LOX 2 having molecular masses of approximately 85 and 79 kDa, respectively were purified. LOX 1 and LOX 2 exhibited maximum activity at pH 4.5 and 4.8, respectively at 25°C temperature. Both the isoforms, which showed thermostability up to 35°C when incubated for 30 min, were stable at a pH range of 7-7.8. LOX 1 and LOX 2 had apparent K m value of 0.86 and 0.57 lM, respectively. Ascorbic acid and vitamin E inhibited 66-78 and 61-69% activity of LOX 1 and LOX 2, respectively but Na ? , Zn 2? and K ? strongly inhibited the activity of these isozymes. The present information about lipoxygenase enzyme might be valuable in drafting the strategies for its inactivation, which in turn can obstruct the LOX damaging effects on food products during processing and storage.

Shelf life of pearl millet flour is very short because of rapid development of rancidity. This investigation was carried out in view of generating breeding material for development of low rancid pearl millet hybrids/varieties. Flour of twenty-one genotypes; seven hybrids, seven CMS lines, five inbreds and two composites stored in covered aluminium boxes at 37 °C for 30 days along with respective fresh flour was analysed for shelf life indicators/determinants. Crude fat content and fat acidity (FA) of fresh flour of the genotypes varied from 3.8 to 7.2% and 11 to 75 mg KOH/100 g d.m., respectively. FA in stored flour ranged between 180 and 330 mg KOH/100 g d.m. After storage, magnitude of decrease in pH of water extract of flour of the genotypes varied from 0.15 to 0.44. Activity of peroxidase (POX) varied from 378 to 588 units in control flour and irrespective of the genotypes decreased upon storage. Increase in FA (difference between FA of fresh and stored flour) rather total build up of FA was positively associated with crude fat content (r = 0.440*) indicated comparatively more prominent role of lipolytic enzymes. Chemical changes taking place in water soluble fraction of flour were independent of fat content as no correlation was discerned between fat content and decrease in pH. Among the hybrids, HHB 197 had lowest crude fat content (4.7%), lowest total build up FA (212 mg KOH/100 g d.m.), slowest increase in FA (191 mg KOH/100 g d.m.), least decrease in pH (0.31) of water soluble fraction flour during storage and lowest activity of POX in fresh flour (377 units/g d.m). Among all the tested CMS lines, inbreds and composites, HBL 11 showed pattern of quantitative changes in FA, pH and POX activity similar to the hybrid HHB 197 and was identified a promising inbred for developing low-rancid pearl millet variety or hybrid.