Application of Non-ionising Radiation Based Enzyme

Application of Non-ionising Radiation establish EnzymeAPPLICATION OF NON-IONISING RADIATION BASED ENZYME INACTIVATION OF BITTER GOURD (Mordica charania L.) A COMPARATIVE STUDYNalawade S. A, Akanksha, H.Umesh Hebbar inletAlthough many treat technologies could be used to extend the shelf life of fruits and vegetables, the commonly used ones in current food industry are blanching and dehydration. Blanching is a pre-processing operation carried out originally drying of fruits and vegetables. The main mark of blanching is to inactivate enzymes such as peroxidase, polyphenoloxidases and phenolase which cause many adverse changes of a product (Fellows, 1990 Hiranvarachat, Devahastin, Chiewchan, 2011 Vishwanathan, Giwari, Hebbar, 2013). Conventional blanching involves processing with vitriolicwater, traveland acid. The stodgy blanching has many drawbacks such as leaving of water soluble nutrients (Lavelli, Zanoni, Zaniboni, 2007), quality deterioration (Gornicki Kaleta, 2007) and environmental problems (Bomben, 1977). cutting calabash pipe is known for its medicinal properties and has been used in various Asian and African herbal medicine systems from a long term Gr all over and Yadav (2004), Beloin et al., (2005), Ananya Paul and Sarmistha Sen Raychaudhuri (2010). It has antidiabetic, antitumorous, anticancer, anti-inflammatory, antiviral, and cholesterol lowering capacity Ahmed et al., (2001), Grover and Yadav (2004) and Taylor (2002).The compound credideucerthy for anti-diabetic property in bitter gourd is Charantin, a hypoglycemic compound which is a mixture of two compounds (11) sitosteryl glucoside (C35H60O6) and stigmasteryl glucoside (C35H58O6) that has been isolated from the seeds, leaves and fruit of Momordica charantia (Raman and Lau, 1996). Storage of raw bitter gourd is difficult because of presence of some enzymes which deteriorate the product quality. Hence, Blanching is carried out before drying operation. Primary target of blanching is to inactivate enzymes such as Peroxidase (cod), Polyphenol oxidase (PPO) allowing stabilisation and secondimize the degradation of other quality attributes (Canet, 1989). Peroxide (POD) is considered as enzymatic indicator for blanching (Gunes and Bayindirli, 1993, Sheu and Chen, 1991 and Zhu and Pan, 2009) which is one of the close heat resistant enzymes, therefore when POD is inactivated most of other enzymes submit might not break down (Halpin Lee, 1987). 90% reduction in POD action mechanism was considered as the end point, since persistence of 3-10% is considered sufficient for drying purpose (Gunes and Bayindirli, 1993). Conventional blanching method includes processing the sample with hot water and steam. The advantages of electromagnetic radiation (MW and IR) employed blanching over conventional blanching methods (water or steam) are rapid inactivation of enzyme complexes that cause quality degradation and minimal/no leaching of vitamins, flavor, pigments, carbohydrat es and other water soluble components (De Ancos et al. 1999).These methods have drawbacks such as quality deterioration (Gornicki Kaleta, 2007) and loss of water soluble nutrients (Lavelli, Zanoni, Zaniboni, 2007). The application of microwave radiation for blanching or enzyme inactivation (Ramaswamy and Fakhouri, 1998 Ponne, Baysal, Yuksel, 1994 Begum and brewer, 2001 Brewer and Begum, 2003 Roberts et al., 2004 Lin and Brewer, 2005 Zhu Pan, 2009 Lin and Ramaswamy, 2011 G.C. Jeevitha et.al ,2013 Vishwanathan et al., 2013 ) showing its enduringness and suitability. From the results of Ramaswamy and Fakhouri (1998), Ramesh et al. (2002), G.C. Jeevitha et.al ,2013 and Bengang Wu et al.,2014 it is possible to observe that vegetable tissue blanched with microwave and infrared radiation retained punter nutritionary value. G.C. Jeevitha et.al,2013 reported better retention of water-soluble nutrients during dry blanching of red bell pepper (Capsicum annuum L.) slices apply infrared ( IR) and microwave (MW) radiations and its performance compared with conventional methods. There are a few reports on MW blanching (Chung et al. 1981 Ramesh et al. 2002 Brewer and Begum 2003) and IR blanching (Ponne et al. 1994) indicating their benefits in terms of nutrient retention over steam or water blanching. The objectives of present study were to evaluate kinetics of Bitter gourd peroxidase (POD) polyphenol oxidase (PPO) inactivation and determine adequacy of both conventional (water and steam) and Dry blanching (IR and MW) to evaluate effect on product quality in terms of moisture, ascorbic acid, chlorophyll and charantin.Materials and MethodsBitter gourd dark green (moisture content 92 1.2%w.b) were purchased from a local market. All the fruits were washed with trial water under tap to remove adhering dust and reduce the surface micro-flora and stored at 4 1C until further use. The bitter gourd fruits were procured from a single source and almost the same score of matu rity was maintained in order to minimize variation in raw material quality. exemplar preparationBitter gourd fruits were washed thoroughly with tap water and sliced using robot coupe slicer of dimension 5 mm. seeds were removed manually and used for the study.Blanching SystemsWater Blanching Bitter gourd slices (100 g) were immersed in hot water (13) maintained at 90 2C for 2 min. The blanched samples were immediately cooled to way temperature (25C) by dipping in water for 5 min. Surface excess moisture of slices was removed using deform paper and subjected for analysis.Steam BlanchingBitter gourd slices (30 g) were subjected to steam blanching by exposing the slices to steam in an autoclave (100C and 1atm) for 3 min. The steam blanched Bitter gourd slices were immediately cooled to room temperature by dipping in water for 5 min. Samples was subjected for analysis after removing the surface moisture.IR Blanching. closely 100 g of bitter gourd slices permeate uniformly on the st ainless steel (AISI 304) conveyor of IR heater developed (Hebbar and Ramesh 2006) at the authors add and exposed to IR radiation at a chamber air temperature 200C for 8 min. The IR heater, fitted with near IR heat sources (1.1 THz 0.26 kW/m2) on top and bottom sides of the fit mesh conveyor, was used for the study. IR heat was applied intermittently to control blanching temperature. The chamber was preheated to the required temperature before blanching. The blanched slices were cooled rapidly to room temperature by blowing air.MW BlanchingMW blanching was carried out in a domestic microwave oven (BPL, 2,450 MHz) at an intensity of 5.57, 7.36 and 8.8 W/g. For all the runs, the position of the bitter gourd slices were maintained the same on the turntable to minimize variation.Crude overstretch preparationSlices were homogenized with 0.1 M sodium phosphate buffer of pH 7 in the ratio of 13. Homogenate was filtered through muslin cloth and centrifuged at 5,100 g for 20 min at 4 C. S upernatant was collected and used for the check.Enzyme analysis.POD AssayThe substrate solution was be of 1.0 mL of phosphate buffer of pH 6, 1.0 mL of 15 mM guaiacol and 1 mL of 3 mM H2O2. To the substrate solution, 50 L of enzymatic extract were added and the increase in OD was record at 470 nm for 5 min using ultraviolet (UV) visible spectrophotometer (UV- 160A, Shimadzu, Japan). Enzyme activity was determined from the careen of the linear portion of the graph relating absorbance with time and expressed as Abs470/min.g sample (Fujita et al. 1997).PPO AssayA spectrophotometric assay at 411 nm using 0.1 M catechol as substrate was used to quantify PPO activity (Weemaes et al. 1997). moisture contentMoisture content of raw and blanched bitter gourd was analyzed using the AOAC method. value reported are the average of triplicate determinations appraisal of Ascorbic acidThe ascorbic acid was determined by 2,6-dichlorophenol- indophenol optic titration methods,(Ranganna 1986) given as followsStandardization of dyeAscorbic acid (100 mg of l-ascorbic acid) was dissolved in 3% HPO3 and mass made up to 100mL. Further, 10 ml of this solution was diluted to 100mL with HPO3. From this, 5mL was transferred to Erlenmeyer flasks containing 5mL of metaphosphoric acid (3%). A burette was fill up with the dye, prepared from dissolution of 50 mg of the sodium salt of 2, 6-dichlorophenol in hot distilled water (150 mL) containing 42 mg of NaHCO3, which was made up to 200mL with distilled water. Then, the ascorbic acid solution was titrated against the standard indophenol solution to a rose beg color, which persisted for around 15 s. The dye factor was determined as milligram of ascorbic acid per milliliter of the dye, using the formulaSample preparation and assay, the sample (10 g) was macerated with metaphosphoric acid (3%) in a pestle and mortar, filtered, and made up to 100 ml. The filtrate (5 mL) was taken and titrated against the standard dye to a pink end point, wh ich persisted for at to the lowest degree 15 s. The vitamin C content was calculated asPercentage retention of ascorbic acid was calculated by,WhereA= amount of ascorbic acid present after processingA0= amount of ascorbic acid present in blank bitter gourdBoth values taken dry basisEstimation of ChlorophyllEstimation of chlorophyll was carried out according to the procedure of Ranganna.25 bitter gourd slices (1 g) were macerated with 80% acetone in a pestle and mortar. The supported layer was decanted and the extraction was repeated until the residue was colorless. Then the extracts were pooled, filtered, and made up to 100mL in a sighttric flask. The absorbance measured at 645nm and 663nm using spectrophotometer (Schemadzu UV-1800)The amount of chlorophyll present in the extract mg chlorophyll per g tissue was calculated using the following equationWhere,A=absorbance at specific wavelengthV= final volume of chlorophyll extracted in 80% acetoneW= tip of tissue extractedThe resu lts were expressed as % on dry basis.Percentage retention of ascorbic acid was calculated by,WhereA= amount of chlorophyll present after processingA0= amount of chlorophyll present in honeyed bitter gourdBoth values taken dry basisEstimation of CharantinSoxhlet extractionAbout 1.0 g of bitter melon fruit powder was extracted with 200 ml of ethanol for 150 min. Charantin remained in the sample residue was extracted repeatedly in 30 ml volumes of methanol using ultrasonication. The extract was filtered and evaporated to stimulate viscous flagrant extract and purified prior to the analysis with HPLC.Sample purificationTo purify the crude extract, the protocol as described in Chanchai (2002) was carried out. Briefly, 5 ml of 5050 (v/v) methanolwater was added to the crude extract. The mixture was then sonicated for 15 min and centrifuged at 3500 rpm for 15 min to separate the supernatant from the precipitous. The precipitate was then added with 5 ml of 7030 (v/v) methanolwater, and the mixture was again sonicated and centrifuged. The precipitate from this step was added with 3 ml of hexane, and the step was repeated. The precipitate from this step was re-dissolved in 200 l of 11 (v/v) chloroformmethanol mixture, and then adjusted to volume with methanol to 2 ml volume for that obtained with Soxhlet extraction. The purified solution was filtered through a 0.45m nylon membrane filter (Millipore, USA) before being analyzed by an HPLC.AnalysisHPLC analysis was carried out for the quantification of Charantin present in the sample with C-18 Ascentis column (5m particle, 4.6 mm 250mm ID). The expeditious phase used was 1002 (v/v) methanol-water and flow rate was maintained at 1mL/min. The UV sensing element was set at the wavelength of 204nm and the sample injection volume was 20L.Percentage retention of charantin was calculated by,WhereA= amount of charantin present after processingA0= amount of charantin present in upstart bitter gourdBoth values taken dry basi sKinetics of enzyme inactivationThe reaction rate incessant was determined using first order equationWhere A is the peroxidase or polyphenol oxidase activity at time t A0 is the initial enzyme activity t is the blanching time (s) k is the reaction rate constant (s-1) at given temperature.Decimal reduction time (D) of enzyme is the time required for one log10 reduction in activity of the enzyme (Cigdem and Zerrin 2005), was determined using the following equationRESULTS AND DISCUSSIONEnzyme InactivationThe activity of POD and PPO in fresh samples were found POD 4103 U/g and PPO- 322 U/g (fresh weight), respectively. The blanching was continued till the POD activity was reduced to 10% of the initial activity. The slices were blanched in water maintained at 95C (fig. a) and exposed to steam (fig. b) for different time intervals (15 to 180 sec). The time taken for water and steam blanching was 120 and 180 sec, respectively. The inactivation of PPO was also significant during this peri od. The bitter gourd slices were blanched using IR radiation at 200C chamber temperature for different time intervals. The time required for blanching was 8 min (fig. c). PPO inactivation trend was similar to that of POD, with marginally quicker inactivation.The initial content of ascorbic acid was 666.7 2.3 mg ascorbic acid/100 g dry weight .Ascorbic acid is considered as a relevant nutritional quality index of food during blanching and drying because of its low stability during thermal treatments and its water solubility. Ascorbic acid is significantly addled (20 70%) during water and steam blanching and one of the advantages of dry blanching is the higher retention of this water soluble micronutrient. IR blanching at 200C retained higher ascorbic acid ( 93%) compared with other two conditions.(table.1) The results showed that that the agetemperature combination decided the retention of ascorbic acid, rather than temperature or duration alone. IR blanching removed nearly 10% moi sture, which could be a favorable factor, if drying is the subsequent step.AcknowledgementThe Author would like to thank UGC for the award of immature Research Fellowship (RGNF). Authors wish to thanks Director, CFTRI for extending infrastructure other facilities for carrying out this work.