Laxmi Ananthanarayan

Glucose oxidase - an overview.

Glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase; EC 1.1.2.3.4) catalyzes the oxidation of beta-D-glucose to gluconic acid, by utilizing molecular oxygen as an electron acceptor with simultaneous production of hydrogen peroxide. Microbial glucose oxidase is currently receiving much attention due to its wide applications in chemical, pharmaceutical, food, beverage, clinical chemistry, biotechnology and other industries. Novel applications of glucose oxidase in biosensors have increased the demand in recent years. Present review discusses the production, recovery, characterization, immobilization and applications of glucose oxidase. Production of glucose oxidase by fermentation is detailed, along with recombinant methods. Various purification techniques for higher recovery of glucose oxidase are described here. Issues of enzyme kinetics, stability studies and characterization are addressed. Immobilized preparations of glucose oxidase are also discussed. Applications of glucose oxidase in various industries and as analytical enzymes are having an increasing impact on bioprocessing.

Antioxidant activity of selected foodstuffs

The growing interest in the substitution of synthetic food antioxidants by natural antioxidants and in the health implications of antioxidants as nutraceuticals has fostered research on vegetable sources and the screening of raw materials for identifying antioxidants. Plant and plant products have been used as a source of medicine for a long time. Among the more important constituents of edible plant products, low molecular weight antioxidants are the most important species. It is known that consumption of fruits and vegetables is essential for normal health of human beings. The aim of the present work was to evaluate the total antioxidant activity of selected natural food materials by an in vitro method involving the measurement of oxidation of linoleic acid by fluorimetry. Among the food materials chosen for the present study, pomegranate peel gave the maximum antioxidant activity due to the presence of its high polyphenolic content. At a concentration of 60 ppm, pomegranate peel powder reduced lipid peroxidation by 65% in an in vitro assay.

Purification of a bifunctional amylase/protease inhibitor from ragi (Eleusine coracana) by chromatography and its use as an affinity ligand

An ammonium sulphate fraction (20-60%) of bifunctional amylase/protease inhibitor from ragi (Eleusine coracana) was purified by affinity chromatography to give 6.59-fold purity with 81.48% yield. The same ammonium sulphate fraction was also subjected to ion exchange chromatography and was purified 4.28-fold with 75.95% yield. The ion exchange fraction was subjected to gel filtration and the inhibitor was purified to 6.67-fold with 67.36% yield. Further sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to check the homogeneity of purified amylase/trypsin inhibitor obtained through affinity, ion exchange and gel chromatography. The molecular weight of the inhibitor was found to be 14 kDa. This purified inhibitor was used as affinity ligand for the purification of a commercial preparation of pancreatic amylase.

Use of the backslopping method for accelerated and nutritionally enriched idli fermentation.

BACKGROUND Idli is a cereal-legume-based fermented food, widely consumed in India. It is popular for its nutritional content, sour taste and appetising aroma. The fermentation time of idli batter varies from 12 to 14 h. A reduction in fermentation time of idli batter is of great significance for large-scale idli production units since it will reduce the batch time and will lead to a greater output. Accelerated fermentation can be potentially achieved by using the backslopping method. The inoculum for backslopping was optimally fermented (12 h) idli batter, dried at room temperature. RESULTS Backslopping reduced the fermentation time of idli batter from the conventional 12 h to 3 h while successfully maintaining sensory attributes of the product. In the idlis prepared by backslopped expedited batter, thiamine was found to be 50% higher; while reduction of anti-nutrients phytate (11%) and trypsin inhibitor (16%) was higher than the conventionally fermented (12 h) idli batter. CONCLUSION Backslopping not only accelerated idli batter fermentation but also enhanced its nutritional value. A similar process can be designed for other fermented foods, to expedite fermentation thus reducing the time requirement.

Fermentative Production, Purification and Characterization of Nisin

Bacteriocins are bactericidal or bacteriostatic in action and active against closely related species. Nisin is the bacteriocin produced by the lactic acid bacteria (LAB). Nisin is a small (3353 Da), cationic, hydrophobic, and 34-amino acid peptide. It is used in products such as pasteurized processed cheese, salad dressing, and liquid whole eggs to inhibit the growth of Gram-positive microorganisms including Listeria monocytogenes. The objective of the present work was to study production, purification and characterization of nisin. The production of nisin was carried out using Lactococcus lactis subsp lactis MTCC 440 by one-factor-at-a-time method and statistical design (Orthogonal array). Purification was carried out using ammonium sulphate precipitation followed by hydrophobic interaction and gel chromatography. Characterization was done for pH and temperature stability. The activity of nisin after one factor at a time optimization was found to be 5120 AU/ml. The activity of the nisin increased to 6800 AU/ml after optimization by Orthogonal Array design. Ammonium sulphate precipitation gives good yield of nisin with 60 to 80% saturation with 2.52 fold purity. The overall purification by hydrophobic interaction and gel filtration chromatography was 10.87 fold with 50.84% yield and 8.8 fold with 49.65% yield as compared to crude broth respectively.

Identification of putative and potential cross-reactive chickpea (Cicer arietinum) allergens through an in silico approach

BACKGROUND Allergy has become a key cause of morbidity worldwide. Although many legumes (plants in the Fabaceae family) are healthy foods, they may have a number of allergenic proteins. A number of allergens have been identified and characterized in Fabaceae family, such as soybean and peanut, on the basis of biochemical and molecular biological approaches. However, our understanding of the allergens from chickpea (Cicer arietinum L.), belonging to this family, is very limited. OBJECTIVE In this study, we aimed to identify putative and cross-reactive allergens from Chickpea (C. arietinum) by means of in silico analysis of the chickpea protein sequences and allergens sequences from Fabaceae family. METHODS We retrieved known allergen sequences in Fabaceae family from the IUIS Allergen Nomenclature Database. We performed a protein BLAST (BLASTp) on these sequences to retrieve the similar sequences from chickpea. We further analyzed the retrieved chickpea sequences using a combination of in silico tools, to assess them for their allergenicity potential. Following this, we built structure models using FUGUE: Sequence-structure homology; these models generated by the recognition tool were viewed in Swiss-PDB viewer. RESULTS Through this in silico approach, we identified seven novel putative allergens from chickpea proteome sequences on the basis of similarity of sequence, structure and physicochemical properties with the known reported legume allergens. Four out of seven putative allergens may also show cross reactivity with reported allergens since potential allergens had common sequence and structural features with the reported allergens. CONCLUSION The in silico proteomic identification of the allergen proteins in chickpea provides a basis for future research on developing hypoallergenic foods containing chickpea. Such bioinformatics approaches, combined with experimental methodology, will help delineate an efficient and comprehensive approach to assess allergenicity and pave the way for a better understanding of the biological and medical basis of the same.

Co-immobilization of glucose oxidase-catalase: Optimization of immobilization parameters to improve the immobilization yield

Co-immobilization of glucose oxidase (EC 1.1.3.4) and catalase (EC 1. 11.1.6) on non-porus glass surfaces using ?-aminopropyltriethoxysilane and polyethyleneimine as an activator and gluteraldehyde as cross linking agent has been carried out for its potential use. Polyethyleneimine was found to be a superior immobilization activator than ?-aminopropyltriethoxysilane. In present study, the effects of rough and smooth beads and optimization of the ratio of enzyme concentrations, activator material and concentration of the cross linking agent were investigated using response surface technology. With optimized concentration of glucose oxidase to catalase ratio (0.97), polyethyleneimine (150 mg/l) and gluteraldehyde (15 ml), the effect of glass bead concentration for maximum immobilization yield was investigated. Central composite optimization strategy with 16 experiments increased immobilization yield from 82.63% to 92.74%. It was observed that 0.3 ml of beads per 120 U of glucose oxidase were necessary for higher immobilization yield.

Delayed post-harvest ripening-associated changes in Manilkara zapota L. var. Kalipatti with composite edible coating

BACKGROUND There has been limited research on extending the shelf-life of sapota (Manilkara zapota L. var. Kalipatti) fruit. An edible coating made up of methyl cellulose (MC) and palm oil (PO) was applied to study the extension in shelf-life. Changes in physical and chemical properties of fruit were studied along with peroxidase (POD), polyphenol oxidase (PPO) and pectin methylesterase (PME) activities during post-harvest ripening of sapota. RESULTS The fruits coated with 15 g L-1 MC and 11.25 g L-1 PO showed significant (P < 0.05) delay in physiological weight loss, decrease in fruit firmness losses as well as slower fruit darkening. The coating on the fruits resulted in better retention of ascorbic acid, delayed the loss of total phenolic content, and delayed the increase in total soluble solids and total reducing sugars as compared to control fruits. The coating either delayed or reduced the enzyme activities of POD, PPO and PME of the fruit. CONCLUSION The results suggest that edible coating made up of MC-PO has potential to maintain the quality of sapota fruit. The edible coating extended the shelf-life of sapota fruit by 3 days preserving fruit quality up to 7 days at 24 ± 1 °C and 65 ± 5 %RH.

Partial purification, characterization and thermal inactivation kinetics of peroxidase and polyphenol oxidase isolated from Kalipatti sapota (Manilkara zapota)

BACKGROUND The extraction, purification, and characterisation of peroxidase (POD) and polyphenol oxidase (PPO) were studied for Kalipatti sapota fruit. The crude enzyme extract was partially purified by ammonium sulfate precipitation followed by BioGel P100 size exclusion and Unosphere Q anion-exchange chromatography. RESULTS Molecular weights of 20 kDa (POD) and 24 kDa (PPO) were indicated by SDS-PAGE. A single band was observed on SDS-PAGE with a fold purity of 10.38 and 7.42 for POD and PPO, respectively. Michaelis-Menten constants for POD and PPO were 22.3 and 23.0 mmol L-1 using guaiacol and catechol as substrates. Thermal inactivation kinetics was studied in the temperature range of 60-95 °C. The crude extract of POD and PPO showed D-values of 2.2-60.2 and 1.0-35.2 min; Z-values of 18.7 ± 0.4 and 16.0 ± 0.3 °C; and activation energies (Ea ) of 128.6 and 151.0 kJ mol-1 , respectively. CONCLUSION POD and PPO showed good stability over a wide range of pH and temperature. As reflected by Z and Ea values, the fruit matrix had no significant influence towards enzyme stability. Designing of thermal process should take into consideration D- and Z-values of the enzymes along with D- and Z-values of microorganisms to obtain a product with better shelf life.

Effect of extrusion on thermal, textural and rheological properties of legume based snack

Attempts have been made to improve dough handling properties and quality of legume based snack by incorporating extruded black gram (EBG) flour as partial substitute for raw black gram (RBG) flour. In present work overall quality improvement was achieved by analyzing (a) thermal properties of RBG and EBG flour (b) rheological properties (shear stress, shear rate, storage modulus, loss modulus, deflection angle and complex viscosity) of legume based snack dough and (c) post frying characteristics (colour and texture) of legume based snack. Three different legume based snack samples with different flour formulations (RBG flour, RBG flour incorporated with 25% EBG flour and RBG flour incorporated with 50% EBG flour) were prepared, characterized and compared with standard market sample. Dough exhibited shear thinning behaviour and G′ and G″ showed rising behaviour with angular frequency whereas, complex viscosity showed decreasing behaviour. Herschel–Bulkley model was best fitted. Significant changes were observed in values of onset, peak and endset gelatinization temperatures on extraction of black gram flour which improved dough handling properties during papad processing and enhanced organoleptic profile of end product.