Kuppamuthu Kumaresan

Extraction process optimization of polyphenols from Indian Citrus sinensis – as novel antiglycative agents in the management of diabetes mellitus

BackgroundDiabetes mellitus is a chronic metabolic disorder characterized by increased blood glucose level. It has become an epidemic disease in the 21st century where, India leads the world with largest number of diabetic subjects. Non-enzymatic glycosylation (glycation) is severe form of diabetes, occurs between reducing sugar and proteins which results in the formation of advanced glycation end products (AGEs) that leads to the other complicated secondary disorders. In this context, Mangifera indica (Mango), Syzygium cumini (Jambul), Vitis vinifera (Grapes), Citrus sinensis (Orange), Artocarpus heterophyllus (Jackfruit), Manilkara zapota (Sapodilla) seeds were evaluated for their antiglyation activity. Attempts were made to isolate the polyphenols in the seeds that have recorded the maximum activity.MethodsDifferent extraction methods (shake flask, centrifugation and pressurized hot water) using various extractants (organic solvents, hot water and pressurized hot water) were adopted to investigate the in vitro antiglycation activity. Central composite (CCD) design based Response Surface Methodology (RSM) was espoused to optimize the extraction process of polyphenols from the fruit seeds that have recorded poor antiglycation activity. The PTLC analysis was performed to isolate the polyphenols (Flavonoids and phenolic acids) and LC-PDA-MS analysis was done for structure prediction.ResultsPressurized hot water extraction of Artocarpus heterophyllus (87.52%) and Citrus sinensis seeds (74.79%) was found to possess high and low antiglycation activity, respectively. The RSM mediated optimization process adopted for the Citrus sinensis seeds have revealed that 1:15 solvent ratio (hexane to heptane), 6 minutes and 1:20 solid to liquid ratio as the optimal conditions for the extraction of polyphenols with a maximum antiglycation activity (89.79%). The LC-PDA-MS analysis of preparative thin layer chromatography (PTLC) eluates of Artocarpus heterophyllus seed has showed the presence of compounds like quercetin (301.2), 4-hydroxy phenyl acetic acid (149.0), rhamnosyl-di-hexosyl quercetin sulphate (857.6), quercetin-3-O-xyloside (428.2), rutin (613.4), diosmetin (298.1) and luteolin (283.0).ConclusionThe Artocarpus heterophyllus was observed to possess a significant antiglycation activity and the activity of Citrus sinensis was improved after the optimization process, which proved that both the seeds may be used as a traditional medicine in the management of chronic diabetes mellitus.

An insight into microbial lipases and their environmental facet

Lipases are serine hydrolases that catalyze the hydrolysis and synthesis of esters formed from glycerol and long-chain fatty acids, by acting at the oil–water interface. Lipases from microbial sources have received heightened attention for an array of industrial applications, and these enzymes have been well exploited in the environmental sector as well. In this article, we present an overview of microbial lipase, including the microorganisms from which it could be produced; the application of recombinant DNA technology tools to produce lipase with enhanced properties, the effective use of waste materials as substrates for lipase production; the usage of statistical tools to efficiently optimize the production medium; lipase purification strategies; and the immobilization of the enzyme on a variety of support materials. The next section of the article provides a gist of its application in diversified spheres and focusses exclusively on the environmentally relevant ones. Lipase-catalyzed esterification, transesterification, and interesterification reactions, an emerging area of green chemistry; lipase-mediated in vitro biopolymer synthesis and degradation; and the application of lipase for remediating fat and oil constituents in wastewater are dealt with in-depth. When its full potential is harnessed, the enzyme could play a pivotal role in environmental management.

Pretreatment of coconut mill effluent using celite‐immobilized hydrolytic enzyme preparation from Staphylococcus pasteuri and its impact on anaerobic digestion

Biological treatment of oil and grease (O&G)‐containing industrial effluents has long been a challenging issue. Practically feasible avenues to bring down their O&G load and enhance treatability are desired. In one such endeavour, the partially purified lipase from Staphylococcus pasteuri COM‐4A was immobilized on celite carrier and applied for the enzymatic hydrolysis of unsterilized coconut oil mill effluent. In batch hydrolysis experiments, optimum conditions of 1% (w/v) immobilized lipase beads, one in four effluent dilution, and a contact time of 30 h resulted in 46% and 24% increase in volatile fatty acids and long‐chain fatty acids and a concomitant 52% and 32% decrease in O&G and chemical oxygen demand (COD) levels, respectively. Batch anaerobic biodegradation trials with this prehydrolyzed effluent showed 89%, 91%, and 90% decrease in COD, proteins, and reducing sugars, respectively. These results were validated in a hybrid stirred tank—upflow anaerobic sludge blanket reactor. Average COD and O&G reductions effected by the hybrid reactor were found to be 89% and 88%, whereas that by the control reactor without enzymatic hydrolysis were only 60% and 47%, respectively. A maximum of 0.86 L methane gas was generated by the hybrid reactor per gram of VS added. Hence, this celite‐immobilized crude lipase, sourced from a native laboratory isolate, seems to be a workable alternative to commercial enzyme preparations for the management of lipid‐rich industrial effluents.

Hydrolytic Enzyme Profiling of Bacillus Subtilis COM6B and Its Application in the Bioremediation of Groundnut Oil Mill Effluent

Industrial effluents rich in oil and grease pose hindrance to the functioning of wastewater treatment units and also affect the quality of receiving water bodies. Pretreatment of such wastewaters to bring about lipid hydrolysis makes them more amenable to conventional biological treatment and hydrolytic enzymes, especially lipases, find promising applications in this sector. In our study, the bacterial strain Bacillus subtilis COM6B isolated from groundnut mill effluent was cultivated in minimal media based on residual oil waste from the extraction process, in which it produced lipase and other extracellular hydrolytic enzymes such as protease and amylase. Applying response surface methodology led to a 1.8-fold increase in oil waste removal by the isolate. As a further study, the effluent discharged from the oil mill was treated in batch mode using pure cultures of the isolate and the effects of incubation time, inoculum size and effluent dilution on the treatment process were investigated. A maximum of 95, 93 and 98 % reduction in biochemical oxygen demand, chemical oxygen demand and oil and grease respectively, were achieved after treatment with COM6B. Hence, the isolate could serve as a potential candidate for remediating the fat and oil contaminants and reducing the organic load of wastewaters.

Gene cloning, expression, and characterization of the Bacillus amyloliquefaciens PS35 lipase

Abstract Lipases are enzymes of immense industrial relevance, and, therefore, are being intensely investigated. In an attempt to characterize lipases at molecular level from novel sources, a lipase gene from Bacillus amyloliquefaciens PS35 was cloned, heterologously expressed in Escherichia coli DH5α cells and sequenced. It showed up to 98% homology with other lipase sequences in the NCBI database. The recombinant enzyme was then purified from E. coli culture, resulting in a 19.41-fold purification with 9.7% yield. It displayed a preference for long-chain para-nitrophenyl esters, a characteristic that is typical of true lipases. Its optimum pH and temperature were determined to be 8.0 and 40 °C, respectively. The half-lives were 2.0, 1.0 and 0.5 h at 50 °C, 60 °C and 70 °C, respectively. The metal ions K+ and Fe3+ enhanced the enzyme activity. The enzyme displayed substantial residual activity in the presence of various tested chemical modifiers, and interestingly, the organic solvents, such as n-hexane and toluene, also favored the enzyme activity. Thus, this study involves characterization of B. amyloliquefaciens lipase at molecular level. The key outcomes are novelty of the bacterial source and purification of the enzyme with desirable properties for industrial applications.

Effect of Pleurotus djamor var. roseus, an edible mushroom on neutrophil functions

Immune stimulation is an effective as well as protective approach against emerging infectious diseases. Mushroom has been valued throughout the world as both food and medicine for thousands of years. The immunomodulatory activities of methanolic extract of Pleurotus djamor var. roseus (MEPDR, 500 mg/kg/body weight for 48 days) were assessed by testing the various neutrophil functions like adherence, phagocytosis (phagocytic index), avidity index and nitroblue tetrazolium reduction in albino rats. Rats were randomly divided into four groups namely, MEPDR treated and corresponding immunised rats were used. Sheep red blood cells (SRBCs) were used for immunisation. The immunised rats were inoculated with SRBCs. The studied neutrophil functions were significantly stimulated in MEPDR-treated groups.

Evaluation of In vitro Antiglycation Activity of Selected Indian Vegetable Sources-Awareness in the Management of Diabetes Mellitus

Abstract: Ethnobotanical report on vegetables such as Raphanus sativus (Radish), Trichosanthes cucumerina (Snake gourd), Solanum melongena (Brinjal), Brassica oleracea var. capitata (Cabbage) and Brassica oleracea var. botrytis (Cauliflower) has revealed significant medicinal properties. In this background, an attempt was made to explore the antiglycation activity of the selected vegetables. The hot water extracts of Brassica oleracea var. botrytis and Solanum melongena has recorded high (85.45±1.96 %) and low (58.86 ± 0.26 %) antiglycation activity, respectively. An improved antiglycation activity (85.37 %, 1.45 fold increase) through central composite design (CCD) based response surface methodology (RSM) was observed in Solanum melongena. Two strong spots belong to flavonoid (yellow color) and phenolic acid (blue color) group of compounds, isolated from Brassica oleracea var. botrytis has recorded 84.35 ±1.25 % antiglycation activity. The IC values of DPPH (diphenyl dipicryl hydrazine) and FRAP (ferric ion reducing antioxidant power) assays adopted for 2D PTLC eluate of Brassica oleracea var. botrytis was found to be 269.37 µg/ ml (r2=0.9677 and p=0.0025) and 51.61 µg/ ml (r2=0.9307 and p=0.0079), respectively. The LC-MS analysis of 2D PTLC eluate of Brassica oleracea var. botrytis has revealed the presence of polyphenols such as chlorogenic acid, methyl apigenin, catechin and one unknown compound.