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Enzyme and Microbial Technology 46 (2010) 9–13

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Enzyme and Microbial Technology

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ffect of fermentation ensilaging on recovery of oil from fresh water fish viscera

mit Kumar Raia, H.C. Swapnaa, N. Bhaskara, P.M. Halamib, N.M. Sachindraa,∗

Department of Meat, Fish & Poultry Technology, Central Food Technological Research Institute (Council of Scientific and Industrial Research), Mysore-570020, IndiaDepartment of Food Microbiology, Central Food Technological Research Institute (Council of Scientific and Industrial Research), Mysore-570020, India

r t i c l e i n f o

rticle history:eceived 27 May 2009eceived in revised form 6 September 2009ccepted 10 September 2009

a b s t r a c t

Fish viscera are an important source for biomolecules such as protein and lipids. Studies were carriedout to assess fermentation ensilaging as a method for recovery of oil from fresh water fish viscera. Thetotal lipid content in the viscera ranged from 19% to 21% and upto 85% of this could be recovered by

eywords:ish visceraermentationroteaseish oil

fermentation. Fermentation using added lactic cultures (Enterococcus faecium HAB01 and Pediococcusacidilactici K7) did not show any advantage over natural fermentation with respect to recovery of oil andno differences were observed in fatty acid composition of oil recovered by fermentation using differentcultures. Activity of acidic, neutral and alkaline proteases decreased during fermentation. Eventhoughdegree of protein hydrolysis increased during fermentation with highest (62.3%) being in fermentationusing Pediococcus acidilactici K7 no differences were found in oil recovery. With decrease in protease

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ipase activity the rate of chang

. Introduction

Fish processing generates considerable quantity of waste in theorm of edible and non-edible by-products. Considering 45% of theive weight to be the waste, it can be estimated that nearly 63.6

illion metric tonnes (MMT) of waste is generated globally (totalsh production of 141.4 MMT) out of which 2.8 MMT India (totalsh production of 6.3 MMT) [1]. The major non-edible by-productsrising out of fish processing include viscera, skin, scales, bonesnd bone frames (in case of surimi production). Most of the Inlandsheries sector is unorganized and have problem of disposing fishaste generated after processing fish for domestic consumption.

he fish waste generated is collected and dumped in waste siteshich sometimes are not controlled. These fish wastes are an

mportant source of proteins and lipids and efforts are being madeo recover these biomolecules [2]. The lipid-based compounds thatan be recovered are oils, omega-3 fatty acids, phospholipids, squa-ene, vitamins, cholesterol, etc. The lipids are the major factoresponsible for the offensive odors associated with fish and fishaste due to oxidation of unsaturated fatty acids present in these

ipids. The protein hydrolysates, surimi, peptides and amino acids,ollagen and gelatin, enzymes are the protein based components

hat can be recovered from fish waste. The recovery of componentsith potential biological activities and functionalities provides aeans for value addition to the fish processing waste and also add

o plant economy.

∗ Corresponding author. Tel.: +91 821 2514840; fax: +91 821 2517233.E-mail address: sachiprathi@yahoo.com (N.M. Sachindra).

141-0229/$ – see front matter © 2009 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2009.09.007

egree of hydrolysis also decreased.© 2009 Elsevier Inc. All rights reserved.

Today silage technology is recognized as being most useful forsolving the waste problem in the fish farming industry. Fish silageconcentrate is also a highly digested protein hydrolysate which isconvenient as a protein supply. Fish silage can be prepared eitherby acid treatment (organic acid) or by fermentation with bacterialculture and sugar [3]. Fermentation is a biological method whereinmicroorganisms in the form of lactic acid bacteria (LAB) are usedto generate acid in situ for preservation of waste [4] or for recoveryof by-product [5–9]. In addition to acid, some of the lactobacilliproduce antimicrobial compounds which increase the preserva-tion effect [10] and is also considered to prevent oxidation of fat[11]

Enzymatic hydrolysis of fish viscera has been done for obtain-ing protein hydrolysate with higher degree of hydrolysis [12]. Fewreports are available on recovery of lipids by enzymatic hydroly-sis of fish viscera [13,14]. Conventional method for recovery of fishoil involves physical treatments such as heating and separation ofoil by centrifugation. In conventional method the residue after oilrecovery is used as animal feed ingredient. Whereas by adoptingfermentation approach for recovery of oil from fish waste, it wouldalso be possible to recover other functional ingredients such asprotein hydrolysate and collagen which have numerous biomed-ical applications. Further, the conventional method is an energyintensive process compared to fermentation approach. Lactic acidfermentation has been used for recovery of bioactive molecules

from protein rich source such as shrimp waste and leather indus-try waste [15,16]. The objective of this work was to study theeffect of lactic acid fermentation ensilaging on the recovery of oilfrom fresh water fish viscera and to evaluate the quality of suchoil.

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of fermentation (Fig. 1). Decreases in pH were correlated with thegrowth of LAB in silage as it produces organic acid on fermentation.Studies have been done earlier by Ahmed et al. [24] on the changesin microbial population during natural fermentation of fish viscera.Large number of microorganisms associated with fresh water fish

0 A.K. Rai et al. / Enzyme and M

. Materials and methods

.1. Materials

Visceral mass obtained by dressing of freshly harvested Indian major carps (Rohund Catla) was procured from local market and transported to laboratory under icedonditions. Known LAB isolates namely Pediococcus acidilactici K7 and Enterococcusaecium HAB01 were obtained from the Institute culture collection. All microbi-logical media were procured from Hi-Media (Hi-Media, Mumbai, India). All theolvents and chemicals were of analytical grade. All the cultures were maintainedn Mann–Rogosa–Sharpe (MRS) agar (Hi-Media, Mumbai, India) slants, stored at◦C and sub-cultured periodically.

.2. Preparation of silage

The visceral mass without air bladder was homogenized in a blender (Stephanill; Stephan UM, Germany) for 5 min. The homogenized mass was mixed with 10%

extrose and 2% salt (w/w of viscera) by stirring. In case of fermentation with addedulture, 10% inoculum (24 h) was added to the homogenized viscera containing dex-rose (10%) and salt (2%). The LAB cultures (E. faecium HAB01-CLF2 and P. acidilactici7) were grown in 100 ml of MRS-Broth (Hi-Media, India) for 24 h at 37 ± 1 ◦C inshaking incubator (Technico Ltd., India) set at 100 rpm. The cells were harvestedy centrifuging (C31 Cooling centrifuge, Remi-India, India) at 3000 × g for 10 min.he harvested cells were washed twice with sterile physiological saline and resus-ended in physiological saline (100 ml). The biomass in the inoculum, after serialilution, was assayed by counting colony forming units (cfu) on MRS agar (Hi-Media,

ndia) plates. The counts in the inoculum were in the range of 108–109 cells/ml. Theilage mix was placed in airtight container and incubated for 72 h at 37 ◦C. pH andotal titrable acidity (TTA) were recorded at 0, 24, 48 and 72 h. pH was determinedsing a digital pH meter (Cyberscan 1001, Eutech, Singapore) by directly immersinghe combined glass calomel electrode in to the sample. TTA was estimated as perhe method described in [5] by determining the volume of 0.1N sodium hydrox-de (NaOH) required for increasing the pH of one gram of fermented mass to 8.0.itrogen measurements in the samples were carried out by Kjeldahl method [17]sing Kjeltec protein analyzer (Foss Analytica AB, Sweden). Aliquots of fresh viscerand fermented silage during fermentation were analyzed for proteolytic activitynd the oil recovered on fermentation were evaluated for its quality. Microbiologi-al quality of fresh viscera and silage samples during fermentation was assessed bynumeration of total plate count and LAB count during fermentation.

.3. Yield and quality of recovered lipid

Oil in the fermentation mixture (50 g) was recovered by centrifugation and vol-me of oil recovered was noted and oil recovery was determined as % of total lipidontent in fresh waste. Total lipid content in the fresh waste was determined by theethod of Bligh and Dyer [18].

ield (%) = Volume of oil recoveredTotal fat content

× 100

The quality of oil was evaluated with respect to iodine value, acid value andaponification value [17], and conjugated diene [19]. In order to prevent the inter-erence of the acids produced during fermentation the recovered oil was repeatedlyashed with water and used for determination of quality. For determination of

hanges in conjugated diene content, the oil was suitably diluted with ethanol andhe absorbance was measured at 234 nm.

For determination of fatty acid composition in the oil recovered, fatty acidethyl esters (FAME) were prepared and analyzed using gas chromatography (GC).

AME dissolved in hexane was analyzed by GC using OmegawaxTM 320 fused silicaapillary column (30 m × 0.32 mm × 0.25 �m). The conditions used for GC analysisas injection temperature of 250 ◦C, detector (FID) temperature of 260 ◦C and col-mn temperature of 200 ◦C for 60 min. The peaks were identified by comparing withuthentic standards.

.4. Activity of proteases, lipase and estimation of degree of hydrolysis

.4.1. Extraction of enzymesHomogenized visceral mass (10 gm) was extracted with cold water at 1:10 (w/v)

y homogenizing in a homogenizer (Polytron, Switzerland) for 2 min at 5000 rpm.he homogenate was allowed to stand for 10 min at 4 ◦C followed by centrifugationt 4 ◦C for 15 min at 7600 g. The supernatant was filtered and made up to a knownolume with cold water and was referred as crude extract (CE)

.4.2. Assay of proteasesThe activity of different protease classes (acidic, alkaline and neutral) in the

nzyme extract was determined by the method of Heu et al. [20]. Briefly, for alkalineroteases the assay mixture consisted of 1.25 ml of 0.05 M phosphate buffer of pH.0, 0.5 ml of 1% casein and 0.25 ml of enzyme extract. The reaction was stoppedy adding 5 ml of 5% TCA (w/v) after incubating the reaction mixture at 37 ◦C for0 min. The solution was filtered using Whatman no. 41 filter paper and the TCAoluble peptides in the filterate was determined by Lowry’s method [21] and specific

l Technology 46 (2010) 9–13

activity of proteases is expressed as mg tyrosine liberated/mg protein/min. Activityof the neutral proteases was determined by using the same method as above, butby using 0.05 M phosphate buffer of pH 7. Activity of acidic proteases was assayedusing 0.05 M acetate buffer of pH 4.0 with 2% (w/v) hemoglobin as a substrate

2.4.3. Assay of lipase activityLipase activity was assayed based on measurement of free fatty acid release

due to enzymatic hydrolysis of triglycerides in stabilized emulsion of vegetable oil[22]. Stabilized emulsion was prepared by homogenizing 50 ml of sunflower oil with50 ml of 2% bovine serum albumin and 3.5 ml of Tween 80. Assay was carried out byaddition of 1 ml of crude enzyme to 1 ml of stabilized lipase substrate in 1.5 ml of0.1 M Tris–HCl buffer at pH 8.0. Mixture was incubated for 6 h at 37 ◦C, after whichhydrolysis was stopped by addition of 3 ml of 95% ethyl alcohol. The mixture wasthen titrated with 0.01N NaOH using 1% phenolphthalein in ethanol as indicator.Blank determination was conducted in a similar manner except the crude enzymeextract was introduced into the assay system after addition of ethyl alcohol at theend of incubation period. A unit of lipase activity was defined as the volume of 0.01NNaOH required to neutralize the FFA formed on hydrolysis of oil per milligram ofprotein in extract.

2.4.4. Degree of hydrolysisDH of the hydrolyzed protein resulting from fermentation of fish waste was

estimated as per the methodology described by [5] and was computed as

DH (%) = 10% TCA soluble N2 in the sampleTotal N2 in the sample

× 100

2.5. Statistical analysis

The experiments were repeated three times and the statistical differencesbetween treatments were analyzed by using ANOVA and mean separation wasaccomplished by Duncan’s multiple range test using [23].

3. Results and discussion

Fresh visceral homogenate had a pH of 6.1 ± 0.3. pH of the massreduced significantly (p ≤ 0.001) to around 4.5 by the 3rd day offermentation. With reduction in pH, TTA increased from 456.7 �lto 1104.6 �l of 0.1N NaOH/g. However, the difference betweenthe three treatments (natural fermentation and fermentation withtwo added lactic cultures) was negligible (p ≥ 0.05), indicating thatthe in situ lactic acid bacteria are sufficient to cause fermentation.Reduction in pH of fermented fish waste signifies the utilizationof sugar and subsequent production of organic acids, which helpsto prevent the growth of spoilage causing organisms apart fromdegradation of the proteins in the material. The LAB count in theinitial silage mix was in the range of 6.0–7.5 log cfu/g and increasedto 9.0–10.0 log cfu/g on second day reduced marginally by third day

Fig. 1. Changes in total plate count and LAB count during fermentation of fish viscera(N—natural, P—Pediococcus, E—Enterococcus) (n = 3).

A.K. Rai et al. / Enzyme and Microbia

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ig. 2. Oil recovery (% of total lipids in fresh waste) and degree of protein hydrolysisuring fermentation (N—natural, P—Pediococcus, E—Enterococcus) (n = 3).

iscera decreases during fermentation except yeast and mouldsith increase in lactic acid bacteria.

The total lipid content in the visceral waste was 19–21%. Theield of oil recovered increased during fermentation and maxi-um oil recovery was observed on the 3rd day (Fig. 2). Even afterday of fermentation nearly 75% of the total lipid present in the

isceral waste was recovered by centrifugation of the fermentedass, which increased to nearly 85% by 3rd day of fermentation.

ut no significant (p ≥ 0.05) difference was observed between dif-erent treatments with respect to recovery of oil indicating thatven natural fermentation would be sufficient for maximum oilecovery from fresh water fish viscera. However, it is necessary tochieve an increased recovery of oil in shortest duration possibleo avoid the deterioration of lipids during fermentation. This cane achieved by screening different LAB isolates having proteolyticbility, so that faster hydrolysis of protein could be achieved, result-ng in increased yield of oil. Studies on isolation of proteolytic LABaving antimicrobial properties has to be carried out to select a bet-er starter culture for the fermentation of fish viscera, with respecto recovery of oil.

The quality of oil recovered with respect to acid value, saponi-cation value, iodine value and conjugated diene content isresented in Table 1. Acid value (mg KOH/g oil) increased from an

nitial value of 30.5 to 108.5–121.0 on 3rd day. The increase wasigher on the first day which can be due to the contaminationf acids formed on fermentation of lactic acid bacteria as well asipase activity. However, no significant difference was observed

able 1uality of oil recovered by fermentation (n = 3).

0 day 1 day 2 days 3 days

Acid valueNatural 30.5 ± 6.1 95.9 ± 2.1 107.1 ± 2.6 120.9 ± 4.8Pediococcus 30.5 ± 6.1 98.92 ± 3.9 107.1 ± 0.7 115.5 ± 3.3Enterococcus 30.5 ± 6.1 95.43 ± 2.1 102.9 ± 0.6 108.5 ± 2.7

Saponification valueNatural 260.7 ± 4.6 256.1 ± 0.9 254.1 ± 4.1 249.7 ± 1.7Pediococcus 260.7 ± 4.6 247.5 ± 9.5 250.1 ± 0.9 256.3 ± 0.8Enterococcus 260.7 ± 4.5 253.8 ± 2.5 254.62 ± 4.1 260.3 ± 1.1

Iodine valueNatural 94.8 ± 2.6 107.4 ± 4.6 110.9 ± 6.7 113.3 ± 6.6Pediococcus 94.8 ± 2.6 105.1 ± 9.9 116.9 ± 4.9 117.8 ± 3.2Enterococcus 94.8 ± 2.6 108.4 ± 6.6 113.9 ± 1.8 117.8 ± 0.8

Conjugated diene (A230)Natural 0.71 ± 0.02 0.70 ± 0.02 0.71 ± 0.05 0.73 ± 0.04Pediococcus 0.71 ± 0.02 0.69 ± 0.02 0.69 ± 0.02 0.71 ± 0.03Enterococcus 0.71 ± 0.02 0.70 ± 0.02 0.72 ± 0.04 0.73 ± 0.04

l Technology 46 (2010) 9–13 11

between three different treatments. High acid value in fish visceraand its increase during fermentation has been observed [25]. Vis-ceral wastes from fish are known to be rich source of lipases [26,27].Any delay in processing of waste results in increased acid value andoil recovered from such wastes need further refining. Fermentationmay also result in an increase in acid value due to the activity ofbacterial lipases. There were no significant (p ≥ 0.05) changes insaponification value during fermentation and without any majordifferences between natural fermentation and fermentation usingadded cultures. Iodine number was in the range of 95–118 andno significant (p ≥ 0.05) differences were observed between threetreatments.

Oxidation of lipids recovered during fermentation is studied interms of conjugated dienes. There was no significant difference inconjugated diene content in recovered oil during fermentation offish viscera. Changes in peroxide value, an indicator of lipid oxi-dation, during fermentation of fish viscera has been reported [25],where slight increase was observed in peroxide value which laterreduced due to breakdown of oxidized products. Further Ahmedand Mahendrakar [25] suggested the use of antioxidants to preventoxidation of lipids and development of rancidity during storage offermented fish viscera. Fermentation may also result in formationof peptides possessing antioxidant activity which can prevent lipidoxidation. Antioxidant activity of fermented shrimp waste [16] andleather fleshings [15] has been reported. Low content of highlyunsaturated fatty acids such as EPA and DHA in the lipid extractof fish waste (Table 2) may be one of the other factors for low levelof oxidation.

Fish oil consists virtually of pure triacylglycerol comprisingmore than 50 different type fatty acids [28]. Fatty acids in fishesare derived from two main sources, namely, biosynthesis and diet[29–31]. The chain length varies from C14 to C24 of varying degreeof unsaturation, from saturated to polyunsaturated. Fatty acid pro-file of total lipids from fresh water fish viscera indicated equaldistribution of saturated and unsaturated fatty acids (Table 2).The major fatty acids in viscera were palmitic (C14:0), palmitoic(C14:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2) and linolenicacid (C18:3). Oleic was the dominant whereas linoleic and linolenicacids were equally distributed among the other unsaturated fattyacid. Palmitic acid was dominant among saturated fatty acids thecontent of n-3 fatty acid was less in the lipid from fresh wateritself. Similar observation of higher amount of palmitic acid andoleic acid has been reported in catfish viscera [32]. EPA and DHA infresh viscera was 1.79% and 2.84% respectively and no change wasobserved upon fermentation, the concentration in the oil formedin fermented mass had 1.41–1.74% and 2.54–3.02% respectively(Table 2). There was only reduction in palmitoleic acid on fer-mentation whereas concentration of other unsaturated fatty acidremained similar on fermentation.

Proteolytic enzymes which have been widely investigated infish and aquatic invertebrate include gastric proteinases, intesti-nal proteinases and hepatoproteinases [27]. In case of lactic acidfermentation of visceral waste both intestinal as well as bacterialproteases are active which results in hydrolysis of tissue proteinresulting in release of oil. Proteases from viscera of fresh watercarps have been studied [33,34]. Acid ensilaging of fresh water fishviscera was shown to have negative effect on visceral proteases[35]. However, no reports are available on effect of fermentationensilaging on the activity of proteases in fish waste. Activity of allthe three proteases reduced considerably during fermentation andwas almost negligible on 3rd day of fermentation. Acidic protease

activity reduced from an initial value 11.5 units to less than 2.5 unitsby 2nd day of fermentation (Fig. 3). Similarly, the activity of neutraland alkaline proteases reduced from an initial value of 41.8 and 37.4units respectively to less than 4.0 units by 2nd day of fermentation(Fig. 3). Degree of protein hydrolysis increased during fermenta-

12 A.K. Rai et al. / Enzyme and Microbial Technology 46 (2010) 9–13

Table 2Fatty acid composition (% of total lipids) lipid extract during fermentation (n = 2).

FFA Fresh Natural P. acidilactici K7 E. faecium HAB01

Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3

C14:0 5.11 5.02 5.35 4.95 5.03 5.19 4.94 5.08 5.07 4.98C16:0 28.83 28.15 29.65 27.31 28.00 28.70 27.52 28.11 28.18 27.76C16:1 9.38 8.94 9.14 8.68 9.22 9.163 9.09 9.29 9.26 9.13C18:0 4.86 5.1 5.3 5.48 5.04 5.13 5.01 4.97 5.17 5.12C18:1n-9 20.9 21.75 22.25 21.57 22.2 21.58 21.97 21.93 22.11 22.87C18:1n-7 2.73 3.07 3.12 3.28 3.01 4.22 3.02 2.97 3.03 2.69C18:2n-6 4.20 4.14 4.14 4.37 4.25 4.22 4.3 4.31 4.22 4.14C18:3n-3 5.93 5.49 5.39 5.85 5.77 5.68 5.9 5.85 5.47 5.55C20:0 1.70 2.02 1.97 2.14 2.02 1.56 2.08 1.98 2.02 1.87C20:5n-3 1.79 1.50 1.41 1.57 1.59 1.57 1.67 1.60 1.43 1.58C22:5n-3 1.10 1.96 1.34 0.98 1.36 1.14 1.25 1.19 1.14 1.16C22:6n-3 2.84 2.94 2.60 2.92 3.02 2.81 2.94 2.73 2.54 2.86

Total 89.37 90.08 91.66 89.10 90.51 90.96 89.69 90.01 89.64 89.71SFA 40.50 40.29 42.27 39.88 40.09 40.58 39.55 40.14 40.44 39.73

50.42 50.38 50.14 49.87 49.20 49.989.49 9.04 10.31 9.99 10.36 10.29

A fatty acid. N: natural, K7: Pediococcus acidilactici K7, CLF2: Enterococcus faecium HAB 01.

tEbDt

Fa

USFA 48.87 49.79 49.39 49.22Unidentified 10.63 9.92 8.34 10.9

ll the values are mean of two values. SFA: saturated fatty acid, USFA: Unsaturated

ion and was highest for fermentation using Pediococcus (62.3%).

ven though, significant difference (p ≤ 0.001) was observed in DHetween three different treatments there was marginal increase inH with days of fermentation (Fig. 2). Protease activity was found

o be highest on first day of fermentation and as the activity of pro-

ig. 3. Acidic, neutral and alkaline proteases activity (specificctivity—mg tyrosine liberated/mg protein/min) during fermentation (n = 3).

Fig. 4. Lipase activity (specific activity—�l KOH consumed by the FFA released bymg enzyme protein/h) during fermentation (n = 3).

teases was reduced the rate of increase in DH also reduced. DH wasfound to be higher in fermentation with Pediococcus, which maybe attributed to the activity of proteases from the organisms. Withincrease in degree of hydrolysis the recovery of oil also increasedindicating that lipids present in waste tissues are released duringfermentation by hydrolysis of proteinaceous material in the waste.

Lipase activity (Fig. 4) decreased during fermentation from aninitial value of 181.2 units to less than 70 units on 3rd day. Thefermentation was carried out at 37 ◦C and decrease in pH duringfermentation influenced activity of lipases. As lipases are responsi-ble for the hydrolysis of lipids, resulting in increased FFA content,the reduction in lipase activity by fermentation is advantageous.With decrease in lipase activity the rate of increase in acid valuealso decreased which was maximum on the first day (Table 1).

4. Conclusion

The study indicated the usefulness of fermentation ensilagingof fresh water fish viscera for recovery of oil. More than 85% of oilpresent in fish viscera was recovered using fermentation and addi-tion of external starter culture did not show any addition benefit

over natural fermentation. During fermentation degree of proteinhydrolysis increased with a corresponding increase in oil yield.Activity of proteases and lipase reduced during fermentation. Thequality of recovered oil indicated the need for refining of the oilrecovered from fresh water fish viscera. There is a need for scale

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A.K. Rai et al. / Enzyme and Mi

tudies to assess the benefit of fermentation technique for recov-ry of oil from fish waste. We have already attempted recovery ofil in 1 kg batches and achieved >80% recovery. Additional scalep studies contemplated involve optimization of conditions forigher yield of oil from fermented mass and recovery of proteinydrolysate and collagen along with the oil from the fermentedaterial. The scale up studies will be taken up as an integrated

pproach and the details of which will be reported as a separateanuscript.

cknowledgement

This work was supported by funding from Department ofiotechnology, Govt. of India. Authors thank Dr. V. Prakash, Direc-or, CFTRI, for encouragement and permission to publish the work.

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