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Application of Non-ionising Radiation Based Enzyme

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Nalawade S. A, Akanksha, H.Umesh Hebbar



Although many processing 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 before drying of fruits and vegetables. The main purpose 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 hotwater, steamand acid. The conventional blanching has many drawbacks such as loss of water soluble nutrients (Lavelli, Zanoni, & Zaniboni, 2007), quality deterioration (Gornicki & Kaleta, 2007) and environmental problems (Bomben, 1977). Bitter gourd is known for its medicinal properties and has been used in various Asian and African herbal medicine systems from a long time Grover 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 responsible for anti-diabetic property in bitter gourd is Charantin, a hypoglycemic compound which is a mixture of two compounds (1:1) 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 purpose of blanching is to inactivate enzymes such as Peroxidase (POD), Polyphenol oxidase (PPO) allowing stabilization and minimize 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 most heat resistant enzymes, therefore when POD is inactivated most of other enzymes present might not survive (Halpin & Lee, 1987). 90% reduction in POD activity 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, carbohydrates 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 effectiveness 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 better nutritional 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 using 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 Methods

Bitter gourd dark green (moisture content 92 ± 1.2%w.b) were purchased from a local market. All the fruits were washed with running water under tap to remove adhering dust and reduce the surface micro-flora and stored at 4 ± 1°C until further use. The bitter gourd fruits were procured from a single source and almost the same degree of maturity was maintained in order to minimize variation in raw material quality.

Sample preparation

Bitter 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 Systems

Water Blanching

Bitter gourd slices (100 g) were immersed in hot water (1:3) maintained at 90 ± 2°C for 2 min. The blanched samples were immediately cooled to room temperature (25°C) by dipping in water for 5 min. Surface excess moisture of slices was removed using filter paper and subjected for analysis.

Steam Blanching

Bitter 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.

About 100 g of bitter gourd slices spread uniformly on the stainless steel (AISI 304) conveyor of IR heater developed (Hebbar and Ramesh 2006) at the authors institute and exposed to IR radiation at a chamber air temperature 200°C 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 wire 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 Blanching

MW 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 extract preparation

Slices were homogenized with 0.1 M sodium phosphate buffer of pH 7 in the ratio of 1:3. Homogenate was filtered through muslin cloth and centrifuged at 5,100 g for 20 min at 4° C. Supernatant was collected and used for the assay.

Enzyme analysis.

POD Assay

The substrate solution was composed 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 µL of enzymatic extract were added and the increase in OD was recorded at 470 nm for 5 min using ultraviolet (UV) visible spectrophotometer (UV- 160A, Shimadzu, Japan). Enzyme activity was determined from the slope of the linear portion of the graph relating absorbance with time and expressed as ΔAbs470/min.g sample (Fujita et al. 1997).

PPO Assay

A spectrophotometric assay at 411 nm using 0.1 M catechol as substrate was used to quantify PPO activity (Weemaes et al. 1997).

Moisture content

Moisture content of raw and blanched bitter gourd was analyzed using the AOAC method. Values reported are the average of triplicate determinations

Estimation of Ascorbic acid

The ascorbic acid was determined by 2,6-dichlorophenol- indophenol visual titration methods,(Ranganna 1986) given as follows:

Standardization of dye

Ascorbic acid (100 mg of l-ascorbic acid) was dissolved in 3% HPO3 and volume 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 filled 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 pink color, which persisted for around 15 s. The dye factor was determined as milligram of ascorbic acid per milliliter of the dye, using the formula:

Sample 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, which persisted for at least 15 s. The vitamin C content was calculated as:

Percentage retention of ascorbic acid was calculated by,


A= amount of ascorbic acid present after processing

A0= amount of ascorbic acid present in fresh bitter gourd

Both values taken dry basis

Estimation of Chlorophyll

Estimation 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 supernatant 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 volumetric 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 equation


A=absorbance at specific wavelength

V= final volume of chlorophyll extracted in 80% acetone

W= weight of tissue extracted

The results were expressed as % on dry basis.

Percentage retention of ascorbic acid was calculated by,


A= amount of chlorophyll present after processing

A0= amount of chlorophyll present in fresh bitter gourd

Both values taken dry basis

Estimation of Charantin

Soxhlet extraction

About 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 obtain viscous crude extract and purified prior to the analysis with HPLC.

Sample purification

To purify the crude extract, the protocol as described in Chanchai (2002) was carried out. Briefly, 5 ml of 50:50 (v/v) methanol–water 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 precipitate. The precipitate was then added with 5 ml of 70:30 (v/v) methanol–water, 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 µl of 1:1 (v/v) chloroform–methanol 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 0.45µm nylon membrane filter (Millipore, USA) before being analyzed by an HPLC.


HPLC analysis was carried out for the quantification of Charantin present in the sample with C-18 Ascentis column (5µm particle, 4.6 mm × 250mm ID). The mobile phase used was 100:2 (v/v) methanol-water and flow rate was maintained at 1mL/min. The UV detector was set at the wavelength of 204nm and the sample injection volume was 20µL.

Percentage retention of charantin was calculated by,


A= amount of charantin present after processing

A0= amount of charantin present in fresh bitter gourd

Both values taken dry basis

Kinetics of enzyme inactivation

The reaction rate constant was determined using first order equation

Where 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 equation:


Enzyme Inactivation

The activity of POD and PPO in fresh samples were found POD - 4×103 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 95°C (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 period. The bitter gourd slices were blanched using IR radiation at 200°C 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 lost (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 200°C retained higher ascorbic acid (∼ 93%) compared with other two conditions.(table.1) The results showed that that the duration–temperature combination decided the retention of ascorbic acid, rather than temperature or duration alone. IR blanching removed nearly 10% moisture, which could be a favorable factor, if drying is the subsequent step.


The Author would like to thank UGC for the award of Junior Research Fellowship (RGNF). Authors wish to thanks Director, CFTRI for extending infrastructure & other facilities for carrying out this work.

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