International Journal of Current Research and Review
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IJCRR - 4(16), August, 2012

Pages: 134-145

Date of Publication: 28-Aug-2012

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Author: Yogesh Saini, Ramavtar Sharma, Mukesh Kumar, Sumer Singh, Pawan Kumar

Category: General Sciences

Abstract:Various types of fungal species have been reported to be employed for production of pectinases. As extracellular pectinases are easier to harvest and thus the scale up is cheaper and simpler. Thus in the present study, kinnow waste was used to generate extracellular fungal pectinases from natural sources along withextraction of pectin. Morphological examination of screened isolates revealed that Isolate#1 and Isolate#3 could be of Rhizopusgenus and Isolate#2 could of Aspergillus genus. Aspergillusoryzaevaroryzaewas used as standard fungi in the present. Quantitative estimation of total protein was made to know total enzymatic activities generated from isolates. It was done via SmF as well as SSF and pure pectin resulted in maximum activity among substrates, followed by peel powder and finally pomace. In SmF, among all isolates, Isolate #3 was found to produce maximum total protein and all other enzymatic activities(Total protein-8.69mg/ml/10min, Pectinase-763.82 nmoles/ml/60min, Polygalacturonase activity-99.83 nmoles/ml/10min andPL-1038.68nmoles/ml/15min). Extraction of pectin resulted in 20% yield from kinnow peel powder, 80% Ethanol was found to be suitable for extraction of pectin and 7.3% calcium pectate was obtained.

Keywords: Kinnowpeels; Polygalacturonase; Aspergillusoryzaevaroryzae; Pectinase; Extracellular fungal

Full Text:

Kinnow is one of the most important citrus crops of northern India especially of Punjab, which produces about 0.180 MMT of kinnow, accounting over 50% of the national produce (Hindustan Times; May 7, 2011). Kinnow is highly relished as a fresh fruit owing to its rich organoleptic and thirst quenching properties and has a high appeal. Steps are already underway to harness the immense potential ofthis cropandtap the market, by attacking the major problem of bitterness in extracted kinnow juice. The attempts have been mainly based on curative approaches and of late, preventive approach is also being worked out. Processing and utilization of kinnow into various products eventually leads to generation of waste in form of peels and pomace. Kinnow waste is conventionally biotransformed anaerobically into humus, although many valuable byproducts can be produced from the rich waste. In other words wealth can be derived from this waste by value addition and products such as pectin, peel oil, pomace powder, dietary fibers and predominantly pectinases can be easily harnessed. Of these products, pectin and pectinases have a wide global market. Pectinases accounts for 10% of global industrial enzymes produced and their market is increasing day by day (Stutzenberger, 1992). Pectinases are the group of enzymes, which cause degradation of pectin that are chain molecules with a rhamnogalacturonan backbone, associated with other polymers and carbohydrates. These pectinases have wide applications in fruit juice industry and wine industry. In fruit juice industry, it is used for clarification, where reduction in viscosity is caused which ultimately leads to formation of clear juice. They increase the yield of juices by enzymatic liquefaction of pulps; these pectinases also helps in formation of pulpy products by macerating the organized tissue into suspension of intact cells. In wine industry pectinases are mainly used for decreasing astringency by solubilizing anthocyanins without leaching out procyadin polyphenols, and pectinases also increase pigmentation by extracting more anthocyanins(Tucker and Woods, 1991) Pectinases can be produced by both submerged and solid state fermentation (SSF). Submerged fermentation is cultivation of microorganisms on liquid broth. It requires high volumes of water, continuous agitation and generates lot of effluents. SSF incorporates microbial growth and product formation on or within particles of a solid substrate (Mudgett, 1986) under aerobic conditions, in the absence or near absence of free water, and does not generally require aseptic conditions for enzyme production. Many filamentous fungi like Aspergillusniger, Aspergillus. awamori, Penicilliumrestrictum, Trichodermaviride, Mucorpiriformis and Yarrowialipolytica.etc are used in both submerged as well as solid state fermentation for production of various industrially important products such as citric acid, ethanol etc. Fungi like Aspergillusniger, Aspergillusoryzae, Penicilliumexpansum, which are generally regarded as safe (GRAS) by United States Food and Drugs Administration (USFDA) are employed in food industry (Pariza and Foster, 1983).Some bacteria (Bacillus licheniformis, Aeromonascavi, Lactobacillus etc), yeasts like Saccharomyces, Candida and Actinomycetes like Streptomycetes are also used. Amongst these, the filamentous fungi are most commonly employed (Pandeyet al, 1999). Fungi can produce both intracellular as well as extracellular enzymes. All fungi are hetrotrophic, and rely on carbon compounds synthesized by other living organisms. Small molecules like mono disaccharides fatty acids and amino acids can easily pass through but for breaking down of larger complex compounds like pectin, fungi secrete extra cellular enzymes. It is well known that as compared to intracellular enzymes, the extra cellular enzymes are easier to be extracted. Intracellular enzymes require more time and costly chemicals for extraction (Hankin and Anagnostakis, 1975).Till date, Substrates used for solid-state fermentation are materials of plant origin like grains rice, corn, root, tubers, and legumes. Apart from these, pomace, mango peels, orange waste like peels and other fruit and vegetable industry waste are also being in much use (Smith and Aidoo, 1988) Kinnow waste also holds a promising substrate because of rich pectin content in peels. Sudhakar and Miani (1992) have reported 18.3% pectin in mandarin peels, which is quite close to the pectin content of apple pomace and is utilized for commercial extraction of pectin (Girdharilal et al, 1998). As Pectin is the ideal substrate for production of Pectinases; it was thought that attempts should be made to extract pectinase from kinnow waste after isolation of potential pectinolytic fungi from natural sources. Till now, no report isavailable on pectinase production using kinnow waste as substrate. Also, pectin itself can be extracted from kinnow waste (mainly from peels) as a commercially important by-product. Apple, lemon, orange, mango, tomato, beet, carrots etc are the commonly used sources for extraction of pectin. Of these, mango peels, apple pomace, lemon pulp and orange pulp are most commonly used. Pectin finds wide application in the manufacture of many fruit products like jams, jellies, marmalades, preservatives etc and thus are indispensable to the fruit juice industry. It is also used as a thickening agent for sauces, ketchups, flavored syrups and as a texturing agent in fruit flavored milk desserts (Girdharilalet al, 1998). In view of the above-mentioned points, the present investigation was undertaken and attempts were made to produce pectinases from the isolated fungi from natural sources and to extract pectin from kinnow Waste as substrate.

Material procurement
Kinnow as natural source of pectinolytic fungi Rotten and fungus infected kinnow fruits were collected from different fruit shops/ vendors in Bikaner in the month of January 2012.

Kinnow peels and Kinnowpomace powder
Kinnow peels and Kinnowpomace, which is juice extracted kinnow fruit, were collected from different fruit shops/vendors in Bikaner and also from Plant Biotechnology Cente, Swami Keshwanand Rajasthan Agariculture University ,Bikaner , where another project on kinnow fruit was going on during the course of present study. Kinnow peel powder and pomace powder was made by followingprocedure - Peels and pomace were first washed many a times with water to remove all adhering substances. Small pieces of peels were made using knife and these were then dried in tray drier at 500C for 24 hr with intermittent shaking. Similarly washed pomace was well spread uniformly and dried. The dried kinnow peels and pomace were then made to powder using a mechanical grinder (Philips India limited, Kolkata).

Aspergillusoryzaevaroryzae (as standard)
Freeze dried culture of Aspergillusoryzaevaroryzae (MTCC 3567) were collected from Microbial Type Culture Collection and Gene Bank(MTCC), Institute of Microbial Technology (IMTECH), Chandigarh. The culture was activated according to the following method mentioned as by suppliers: 1. Firstly, a mark was made on the ampoule near the middle of cotton wool with a sharp file. The surface around the mark was then disinfected with 100% alcohol. 2. The ampoule was then wrapped with thick cotton wool and marked area was broken, cotton plug was then carefully removed and 0.4ml sterile water was added to make a suspension. 3. The suspension was allowed to stand for 20 minutes, after 20 min, few drops of suspension were streaked onto Medium 117 (Annexure I) in number of petri plates and slants. 4. Rest of the suspension was transferred to 5 ml of Medium 117(broth) in a test tube. 5. The plates were then incubated in BOD at 250C for 5 days. 6. After the growth of fungi, fungal culture was maintained by subculturing the fungi on the same medium throughout the experimental period. Chemicals, media and standard enzyme All the chemicals used in the present investigation were of Analytical grade (AR grade). Dgalacturonic acid and Disodium hydrogen arsenate were procured from Sigma, Aldrich Corp, MO, USA. Polygalacturonic acid, Neocuproine, pectin, Congo red dye was procured from Himedia Laboratories Limited, Mumbai. Standard enzyme- Pectinase was procured from Himedia Laboratories Limited, Mumbai. All the experiments for isolation, screening and pectinolytic enzyme production were done under sterile conditions and adequate safety measures were undertaken (Annexure IX)

Isolation of Pectinolytic fungi
Rotten and fungus infected kinnows were swabbed in 0.8% saline in an autoclaved stomacher bags. Dilutions upto10-8 was made and pour plating of higher dilutions namely 10-5, 10-6, 10-7, and 10-8 was done using melted CzapekAgar (Annexure II). The above dilutions were plated in duplicates. The plates were then incubated at 300C for 12 days. The colonies thus isolated were then subcultured3- 4 times on Czapek agar till active growth of isolates were obtained, and thereafter maintained on the same media for further experimental work.

Screening of isolates having pectinolytic activity
Qualitative test for screening of isolates having pectinolytic activity For screening purpose, Czapek agar having1% pure pectin was used as sole carbon source and Congo red (@150mg/l) was also added in medium, so as to clearly visualize the formed clear zones. Simultaneously, control plate was set having Congo red but without pectin or any other carbon source. Following methodology was adopted for screening of potential isolates -Using a flamed and cooled cork borer, one disc of fungal hyphae from leading edge of actively growing colonies was cut on petri plate. With a flamed and cooled transfer needle, discs were then transferred to Czapek agar media having pectin as sole carbon source. Plates were then incubated at 300C for 12 days. Selection was done on the basis of formation of clear zones and the corresponding diameters were noted during that span of time. Morphological examination of isolates was done using Image Analyser having compound microscope. Quantitative estimation of pectinolytic activity of screenedpectinolytic isolates Quantitative estimation of pectinolytic activity screened isolates was done on submerged as well as solid state fermentation. Submerged Fermentation In order to undertake submerged fermentation firstly pure pectin was used as substrate, subsequently it was replaced by kinnow peel powder and then finally by kinnowpomace powder. For each isolate, 2 flasks were prepared; one was treated as test flask and the other as control flask. In every sterile flask, 1 disc of respective fungal isolate was added and the flasks were properly plugged. The flasks were then incubated in incubator shaker for 12 days at 300C at 250 rpm. Aliquots were withdrawn every 0, 3, 6, 9, 12 day for carrying out assays namely: Total protein, Pectinase assay, Polygalacturonase assay and Pectin Lyase assay according to methods explained in section 3.5. Experiments were also conducted to know the effect of number of fungal discs (1, 2 and 3) as well as amount of pectin 1% and 2% on total protein using the above methodology. Solid State Fermentation Solid State Fermentation was carried out in sterile250ml flasks. Experiments were performed using only kinnow peel powder as sole carbon source. For every isolate and standard fungus, experiments were set up using 5 sterile flasks. Aliquots were drawn after adequate dilution (10 times) on every 0, 3rd, 6th, 9th and 12thday.

Solid State Fermentation was carried as follows:
15g of substrate was taken and 8ml of sterile water was added. One disc of respective fungal hyphae was mixed to 5ml of sterile water and a suspension was made. From this suspension, 1ml was withdrawn and inoculated into each of the 5 flasks. The flasks were then incubated at 300C for respective period of time. For proper aeration, flasks were intermittently shaken. In order to estimate the Total Protein, Pectinase, Polygalacturonase and Pectin Lyase, 10ml ofSterile water was added and then properly mixed; the mixture thus obtained was gently shaken and was filtered using coarse filter paper.The obtained filtrate was then centrifuged and resulting supernatant was used for conducting the assays.

In order to quantify total protein and pectinolytic enzymes produced via submerged and solid state fermentation, following experiments were conducted: Total protein content (BIURET METHOD) 5mg albumin/ml was used as protein standard Reagents used: Biuret reagent (Annexure III) Firstly standard curve with different concentrations of bovine albuminserum (BSA) namely-100l, 150l, 200l, 250l 300l was made. For analysis of total protein, 3ml of biuret reagent was added to 2ml of test protein solution in a sterile test tube and the mixture was properly mixed. The tubes were then warmed at 370C for 10 min with shaking and finally the tubes were cooled and absorbance was noted at 540nm.

Pectinase assay
WBC Home manual/protocol index methodology was followed to estimate the pectinase activity. Reagents used:
I) 0.1 M Phosphate buffer pH 5.0
ii) Color reagent A (Annexure IV)
Iii) Color reagent B (Annexure V)
IV) D-galacturonic acid-1mg/ml
V) 0.5%Polygalacturonic acid substrate (Annexure

1. In one test tube 6ml of substrate and 1ml of buffer was added and thenproperly mixed (reagent blank).

2. In second test tube 6ml substrate and 1ml of enzyme solution 0.1mg/mlwas added (test sample).

3. In third test tube 6ml buffer and 1ml enzyme sample at 1mg/ml (sampleblank) was taken.

4. For standard D- galacturonic acid, different concentration 0, 20, 40, 60, 80, 100, 120 ?g of D-galacturonic acid was used.

5. The above three reaction tubes along with standard tubes were incubated at 370C water bath with shaking for 60min±1min. After incubation the tubes were immediately placed into ice water to stop reaction.100µl aliquot was drawn from each tube and was pipetted into another set of tubes also placed on ice water.

6. To each reaction tube and standard tube. 2ml color reagent A and 2ml color reagent B was added, and the mixture was mixed properly by inversion. The tubes were then placed into boiling water bath for 13min±1min. The tubes were then cooled and then 2ml water was added. Again proper mixing was done by inversion. The absorbance was noted at 450nm using water blank and disposable cuvettes were used throughout the assay owing to sticking of formed orange color in cuvettes.

Polygalacturonase (PG)
PG assay was carried by method as described Baldwin and Pressey(1989).

1. In a test tube, 2ml of 0.1M Sodium acetate, .025ml of 0.15 M Sodiumchloride, 0.5ml of 1% polygalacturonic acid and .05ml of enzymesolution was taken. Test tubes were then incubated at 370C for 15 minin water bath with shaking.

2. The above solution was then analyzed for reducing groups following theArsenomolybdate method given by Nelson Somogyi (1952). Reagents usedAlkaline copper tartarate (Annexure VII) Arsenomolybdate reagent (Annexure VII)

i) Aliquots of .1 or .2 ml of above solution was pipetted out in test tube.

ii)0.2,0.4,0.6,0.8,1.0 ml of standard solution (100?g/ml glucosesolution) were taken into series of tubes.

iii) The volume in both sample and standard tubes was made to 2ml with distilled water

IV) 2ml distilled water was pipetted out in separate test tubes to set up ablank. v) 1ml of alkaline copper tartarate reagent was added to each tube

vi) The tubes were then placed in boiling water bath for 10 minutes andthen cooled; and 1ml of arsenomolybdolic reagent was added to alltest tubes taking full precautions

vii) The volume in each tube was then made to 10ml with water

viii) The absorbance of blue color was then determined at 620 nm after10 min.

PectinLyase Assay
Pectin lyase was assayed by measuring the increase in absorbance at 235nm according to method described by Albersheim and Killias (1962). In a properly washed and cleaned test tube 0.2ml of 0.1M Sodium acetate (pH 5.5), 25ml of 0.12M sodium chloride, 0.5ml of 1% pectin (pH 5.5) and 0.05ml of enzyme solution was pippeted out and mixed. The tubes were then incubated at 370C for 15 min in water bath with shaking. 5ml of water was then added to the above reaction mixture and absorbance was then determined at 235nm.

Extraction and estimation of pectin
Pectin was extracted by method given by Rao and Maini, (1999) as shown in Figure 2.1. 15 g of peel powder was weighed and was taken in 150ml flask; to it 30ml of dilute acid .05N Hydrochloric acid was added. Extraction was done by boiling the above mixture at 1000C for 60 min. The marc was then separated from the extract after filtration. The extraction was performed2 Times. Filtrates obtained were then combined and then cooled. Two volumes of absolute alcohol were added to precipitate pectin. Pectin was also precipitated using different strengths of alcohol namely 90%, 80%, 70%, 60%, and 50%. Yield of pectin was calculated by the following formula:

                               Amount of pectin obtained x 100
Yield of pectin = --------------------------------------------
                                Amount of peel powder

Estimation of pectin (Ranganna, 2000)
The procedure followed was as follows: 1.200mg of dried pectin was weighed into a 1-litre beaker and it was wetted with 2 or 3 ml of 100% alcohol. 2.400ml of water was added with stirring to it. 3. The solution was then boiled and then cooled immediately. 4. Then it was transferred into a 500ml volumetric flask and volume was made up. 5. 200ml aliquot was pipetted out each into two 1l beakers. 6. 250ml water was added and solution was neutralized with 1N Sodium hydroxide using phenolphthalein as indicator Afterwards, 10ml of 1N Sodium hydroxide was pipette out in excess with constant stirring and was allowed to stand overnight. 7. Then next day 50ml of 1N Acetic acid was added and after 5minutes25ml of 1N calcium chloride was added with stirring. 8. Solution was allowed to stand for 1 hour, and was then boiled for 2 min. 9. The solution was filtered using previously prepared filter paper (filter paper was made wet in hot water, dried in oven at 1020C for 2 hours, cooled in a dessicator and weighed in a covered dish) precipitate obtained was washed thoroughly with boiling water. 10. In an originally weighed dish, the filter paper containing calcium pectat was transferred and was dried overnight at 1000C. 11. Weighing dish was cooled in dessicator and then it was finally weighed. % Calcium pectate was calculated using following formula:

                                 Wt of calcium pectate x 500 x 100
% Calcium Pectate = -----------------------------------------------
                                       Ml of filtrate taken x wt of
                                       Sample taken for estimation

The present investigation was undertaken in order to harness the valuable products namely fungal pectinases as well as pectin from waste obtained from processing/ utilization of kinnow fruits. As it is well reported that certain sporulating strains like Aspergillusniger, Aspergillusoryzae, Rhizopusoryzaeetc have pectinolytic activity, the purpose was to screen similar isolates from nature and employee them for the production of pectinolytic enzymes. Only extracellular pectinases were targeted because in comparison tointracellular pectinases, extracellular are easier to harvest and scaling up work can be more easily attempted. Furthermore, as the isolated strains were to thrive only on pectin, pectin content of kinnow fruit was determined. The results of experiments performed to quantify enzymatic activity as well as amount of pectin are being systematically represented below.

Isolation of pectinolytic fungi
The collected rotten and fungus infected fruits were used as source of natural pectinolytic fungi and such fruits were collected in the month of January 2012. From the collected samples, eight potential fungi were screened for pectinolytic activity on selective media (Czapek media having pure pectin as the only carbon source) in duplicates, by taking higher dilutions from 10-5 to10-8 . Plates having Czapek media without any carbon source were treated as control, which showed no sign of growth. Based on visual observance, only three isolates gave clear zones of diameter 2.3mm to 4.1mm as shown in Plate 4.1 -4.3. In the first isolate, clear zone of diameter 2.9mm was observed on 4th day of its incubation at 300C, for the second isolate clear zone of diameter 2.2mm was observed on 6th day of its incubation, and for isolate#3 zone of 4.1mm was observed on 4th day; thereafter excessive growth was seen in each plate thus covering entire plate by mycelia. On the basis of literature review, Aspergillusoryzaevaroryzae and Rhizopusoryzae were treated as standard. Freeze dried culture of R. oryzae failed in being activated even after performing repeated trials. Thus onlyAspergillusoryzaevaroryzae was treated asstandard in the present study.

Morphological examination of the screened isolates
Morphological examination of screened isolates was performed using Image Analyser with compound microscope. Isolate # 1 and Isolate # 3 showed clear aerial hyphae, and black colored spores were also found by macroscopicalexamination microscopic examination showed presence of sporangiospores in sporangia at the tip of fertile hyphae, the sporangiophore. Sporangiophoreswere arising from the nodeand sporangia were present at the end of sporangiophores. The swollen tip of sporangiophorecolumella, projecting into sporangia, was also seen. Rhizoid like structures were also found arising from node as shown in Plate .4 (a and b) and in Plate 3.6 (a and b). As these characters belong to Rhizopus genus from this observation it can be inferred that Isolate # 1 and Isolate # 3 belongtoRhizopus genus. Isolate #2


showed definite zones of growth and mycelia were greenish. Spore bearing heads were seen as large and globular and were tightly packed.Chains of conidia were also seen. Conidia weregreenish in color. From these observations, it can beinferred that Isolate#2 might be belonging to Aspergilus genus (Plate3.5 a and b).


I Medium 117 Composition of Czapek yeast extract agar (CYA) Czapekcocncentrate 10.0ml K2HPO4 1.0g Yeast extract 5.0g Sucrose 30.0g Agar 15.0g Disttiled water 1.0L Czapek Concentrate

NaNO3 30.0g KCl 5.0g MgSO4.7H2O 5.0g FeSO4.7H2O 0.1g Distilled water 100.0ml

Czapek agar Stock solution A50.0ml Stock solution C50.0ml Sucrose 30.0g Zinc solution 1ml Copper solution 1.0ml Agar 20.0g Distilled water 1 litre Stock solution A NaNO3 4.0g KCl 1.0g MgSO4.7H2O 20.0mg Distilled water 100.0ml Zinc solution ZnSO4.7H20 1.0g Distilled water 100.0ml Stock solution C K2HPO4 2.0g Distilled water 100.0ml Copper solution CuSO4.5H2O 0 .5g Distilled water 100.0 ml

Biuret reagent Copper sulphate 3.0g Sodium potassium tartarate 9.0g (in 500ml of 0.2M/L Sodium hydroxide) Sodium iodide 5.0g Make up to 1L with 0.2mol/litre Sodium hydroxide

Color reagent A Sodium carbonate 40.0g (in 600ml water) Glycine 16.0g Copper sulphatepentahydrate Make up to 1L with Color reagent B Neocuprine-HCl 0 .450g

1.2g Make up to 1 L with water and store at 40C in a brown bottle

.5% Polygalacturonic acid substrate Heated 500ml Phosphate buffer on hot plate. While heating slowly 2.5g polygalacturonic acid is slowly added. Heated and stirred until dissolved, cooled and stored at 40C.

Alkaline Copper tartarate (A) Anhydrous sodium carbonate 2.5g Sodium bicarbonate 2.0g Potassium sodium tartarate 2.5g Anhydrous sodium sulphate 20.0g Dissolved in 80 ml water and make up to 100 ml (B) Copper sulphate 15g. Dissolved in small amount of water added 1 drop of Sulphuric acid and make up to 100ml. Mix 4mlof B and 96 ml of A.

Arsenomolybdate reagent Ammonium molybdate 2.5g Sulphuric acid 2.5ml Disodium hydrogen arsenate 0.3g(dissolved in 25ml water) Mixed well and incubated at 370C for 24 to 48 hours

Biosafety precaution while performing experiments

1. Hand gloves and mouth covers were worn while performing all fungal experiments in sporulating rooms.

2. Sporulating room was properly fumigated before performing experiments.

3. Discarded the fungi by properly autoclaving the flasks after performing experiments. Precautions taken during Nelson Somogyi method

1. Mouth covers and hand gloves were worn while handling dihydrogensodium arsenate, as it is very carcinogenic.

2. Mouth pippeting was completely avoide

I thank the almighty whose blessings have enabled me to accomplish my dissertation work successfully. It is my pride and privilege to express my sincere thanks and deep sense of gratitude to Dr. Ramavtarsharma, Associate Professor Plant Biotechnology Centre, Swami keshwanad rajasthan agriculture university, Bikaner for her valuable advice, splendid supervision and constant patience through which this work was able to take the shape in which it has been presented. It was her valuable discussions and endless endeavors through which I have gained a lot. Her constant encouragement and confidence-imbibing attitude has always been a moral support for me.


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34. Ranganna.S.2000. Pectin. Handbook of Analysis and Quality Control for fruit and vegetable products, Tata McGraw Hill Publication, New Delhi, Second Edition., 40- 42.

35. Rao, S.D.V. and Maini, S.B. 1999.Manufacture of Pectins from Mango Peels. Beverage and Food world., 17-17-18.

36. Rombouts, F. M. andPilnik, W. 1980. Pectic Enzymes. Economic Microbiology (A. H. Rose, ed.), Vol.5, Academic Press, London.,227-282.

37. Sandhu, K.S.; B.S.; Shukla, F.C. 1985. Physicochemical changes during storage of kinnow mandarin oranges and pineapples juice concentrates. J. Food Sci. and Technol., 22, 342-345.

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39. Singh, S.A.; Plattner, H. and Diekmann, H.1999a.Exopolygalacturonaselyase from thermophilicBacillus sp. Enzyme Microb Technol., 25,420-424.

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Dr. Pramod Kumar Manjhi joined Editor-in-Chief since July 2021 onwards

COPE guidelines for Reviewers

SCOPUS indexing: 2014, 2019 to 2021

Awards, Research and Publication incentive Schemes by IJCRR

Best Article Award: 

One article from every issue is selected for the ‘Best Article Award’. Authors of selected ‘Best Article’ are rewarded with a certificate. IJCRR Editorial Board members select one ‘Best Article’ from the published issue based on originality, novelty, social usefulness of the work. The corresponding author of selected ‘Best Article Award’ is communicated and information of award is displayed on IJCRR’s website. Drop a mail to for more details.

Women Researcher Award:

This award is instituted to encourage women researchers to publish her work in IJCRR. Women researcher, who intends to publish her research work in IJCRR as the first author is eligible to apply for this award. Editorial Board members decide on the selection of women researchers based on the originality, novelty, and social contribution of the research work. The corresponding author of the selected manuscript is communicated and information is displayed on IJCRR’s website. Under this award selected women, the author is eligible for publication incentives. Drop a mail to for more details.

Emerging Researcher Award:

‘Emerging Researcher Award’ is instituted to encourage student researchers to publish their work in IJCRR. Student researchers, who intend to publish their research or review work in IJCRR as the first author are eligible to apply for this award. Editorial Board members decide on the selection of student researchers for the said award based on originality, novelty, and social applicability of the research work. Under this award selected student researcher is eligible for publication incentives. Drop a mail to for more details.

Best Article Award

A study by Dorothy Ebere Adimora et al. entitled \"Remediation for Effects of Domestic Violence on Psychological well-being, Depression and Suicide among Women During COVID-19 Pandemic: A Cross-cultural Study of Nigeria and Spain\" is awarded Best Article of Vol 14 issue 23
A study by Muhas C. et al. entitled \"Study on Knowledge & Awareness About Pharmacovigilance Among Pharmacists in South India\" is awarded Best article for Vol 14 issue 22
A study by Saurabh Suvidha entitled \"A Case of Mucoid Degeneration of Uterine Fibroid with Hydrosalphinx and Ovarian Cyst\" is awarded Best article of Vol 14 issue 21
A study by Alice Alice entitled \"Strengthening of Human Milk Banking across South Asian Countries: A Next Step Forward\" is awarded Best article of Vol 14 issue 20
A study by Sathyanarayanan AR et al. entitled \"The on-task Attention of Individuals with Autism Spectrum Disorder-An Eye Tracker Study Using Auticare\" is awarded Best article of Vol 14 issue 19
A study by Gupta P. et al. entitled \"A Short Review on \"A Novel Approach in Fast Dissolving Film & their Evaluation Studies\" is awarded Best Article of Vol 14 issue 18.
A study by Shafaque M. et al. entitled \"A Case-Control Study Performed in Karachi on Inflammatory Markers by Ciprofloxacin and CoAmoxicillin in Patients with Chronic Suppurative Otitis Media\" is awarded Best Article of Vol 14 issue 17
A study by Ali Nawaz et al. entitled \"A Comparative Study of Tubeless versus Standard Percutaneous Nephrolithotomy (PCNL) \? A Randomized Controlled Study\" is awarded Best Article for Vol 14 issue 16.
A study by Singh R. et al. entitled \"A Prospective Study to Find the Association of Astigmatism in Patients of Vernal Keratoconjunctivitis (VKC) in a Tertiary Health Care Centre in India (Vindhya Region MP)\" is awarded Best Article for Vol 14 issue 15
A Study by Humaira Tahir et al. entitled "Comparison of First Analgesic Demand after Major Surgeries of Obstetrics and Gynecology between Pre-Emptive Versus Intra-Operative Groups by Using Intravenous Paracetamol: A Cross-Sectional Study" is awarded Best Article for Vol 14 issue 14
A Study by Monica K. entitled "Risk Predictors for Lymphoma Development in Sjogren Syndrome - A Systematic Review" is awarded Best Article for Vol 14 issue 13
A Study by Mokhtar M Sh et al. entitled "Prevalence of Hospital Mortality of Critically Ill Elderly Patients" is awarded Best Article for Vol 14 issue 12
A Study by Vidya S. Bhat et al. entitled "Effect of an Indigenous Cleanser on the Microbial Biofilm on Acrylic Denture Base - A Pilot Study" is awarded Best Article for Vol 14 issue 11
A Study by Pandya S. et al. entitled "Acute and 28-Day Repeated Dose Subacute Toxicological Evaluation of Coroprotect Tablet in Rodents" is awarded Best Article for Vol 14 issue 10
A Study by Muhammad Zaki et al. entitled "Effect of Hemoglobin Level on the Severity of Acute Bronchiolitis in Children: A Case-Control Study" is awarded Best Article for Vol 14 issue 09
A Study by Vinita S & Ayushi S entitled "Role of Colour Doppler and Transvaginal Sonography for diagnosis of endometrial pathology in women presenting with Abnormal Uterine Bleeding" is awarded Best Article for Vol 14 issue 08
A Study by Prabhu A et al. entitled "Awareness of Common Eye Conditions among the ASHA (Accredited Social Health Activist) Workers in the Rural Communities of Udupi District- A Pilot Study" is awarded Best Article for Vol 14 issue 07
A Study by Divya MP et al. entitled "Non-Echoplanar Diffusion-Weighted Imaging and 3D Fiesta Magnetic Resonance Imaging Sequences with High Resolution Computed Tomography Temporal Bone in Assessment and Predicting the Outcome of Chronic Suppurative Otitis Media with Cholesteatoma" is awarded Best Article for Vol 14 issue 06
A Study by Zahoor Illahi Soomro et al. entitled "Functional Outcomes of Fracture Distal Radius after Fixation with Two Different Plates: A Retrospective Comparative Study" is awarded Best Article for Vol 14 issue 05
A Study by Ajai KG & Athira KN entitled "Patients’ Gratification Towards Service Delivery Among Government Hospitals with Particular Orientation Towards Primary Health Centres" is awarded Best Article for Vol 14 issue 04
A Study by Mbungu Mulaila AP et al. entitled "Ovarian Pregnancy in Kindu City, D.R. Congo - A Case Report" is awarded Best Article for Vol 14 issue 03
A Study by Maryam MJ et al. entitled "Evaluation Serum Chemerin and Visfatin Levels with Rheumatoid Arthritis: Possible Diagnostic Biomarkers" is awarded Best Article for Vol 14 issue 02
A Study by Shanthan KR et al. entitled "Comparison of Ultrasound Guided Versus Nerve Stimulator Guided Technique of Supraclavicular Brachial Plexus Block in Patients Undergoing Upper Limb Surgeries" is awarded Best Article for Vol 14 issue 01
A Study by Amol Sanap et al. entitled "The Outcome of Coxofemoral Bypass Using Cemented Bipolar Hemiarthroplasty in the Treatment of Unstable Intertrochanteric Fracture of Femur in a Rural Setup" is awarded Best Article Award of Vol 13 issue 24
A Study by Manoj KP et al. entitled "A Randomized Comparative Clinical Trial to Know the Efficacy of Ultrasound-Guided Transversus Abdominis Plane Block Against Multimodal Analgesia for Postoperative Analgesia Following Caesarean Section" is awarded Best Article Award of Vol 13 issue 23
A Study by Karimova II et al. entitled "Changes in the Activity of Intestinal Carbohydrases in Alloxan-Induced Diabetic Rats and Their Correction with Prenalon" is awarded Best Article of Vol 13 issue 22
A Study by Ashish B Roge et al. entitled "Development, Validation of RP-HPLC Method and GC MS Analysis of Desloratadine HCL and It’s Degradation Products" is awarded Best Article of Vol 13 issue 21
A Study by Isha Gaurav et al. entitled "Association of ABO Blood Group with Oral Cancer and Precancer – A Case-control Study" is awarded Best Article for Vol 13 issue 20
A Study by Amr Y. Zakaria et al. entitled "Single Nucleotide Polymorphisms of ATP-Binding Cassette Gene(ABCC3 rs4793665) affect High Dose Methotrexate-Induced Nephrotoxicity in Children with Osteosarcoma" is awarded Best Article for Vol 13 issue 19
A Study by Kholis Ernawati et al. entitled "The Utilization of Mobile-Based Information Technology in the Management of Dengue Fever in the Community Year 2019-2020: Systematic Review" is awarded Best Article for Vol 13 issue 18
A Study by Bhat Asifa et al. entitled "Efficacy of Modified Carbapenem Inactivation Method for Carbapenemase Detection and Comparative Evaluation with Polymerase Chain Reaction for the Identification of Carbapenemase Producing Klebsiella pneumonia Isolates" is awarded Best Article for Vol 13 issue 17
A Study by Gupta R. et al. entitled "A Clinical Study of Paediatric Tracheostomy: Our Experience in a Tertiary Care Hospital in North India" is awarded Best Article for Vol 13 issue 16
A Study by Chandran Anand et al. entitled "A Prospective Study on Assessment of Quality of Life of Patients Receiving Sorafenib for Hepatocellular Carcinoma" is awarded Best article for Vol 13 issue 15
A Study by Rosa PS et al. entitled "Emotional State Due to the Covid – 19 Pandemic in People Residing in a Vulnerable Area in North Lima" is awarded Best Article for Vol 13 issue 14
A Study by Suvarna Sunder J et al. entitled "Endodontic Revascularization of Necrotic Permanent Anterior Tooth with Platelet Rich Fibrin, Platelet Rich Plasma, and Blood Clot - A Comparative Study" is awarded Best Article for Vol 13 issue 13
A Study by Mona Isam Eldin Osman et al. entitled "Psychological Impact and Risk Factors of Sexual Abuse on Sudanese Children in Khartoum State" is awarded Best Article for Vol 13 issue 12
A Study by Khaw Ming Sheng & Sathiapriya Ramiah entitled "Web Based Suicide Prevention Application for Patients Suffering from Depression" is awarded Best Article for Vol 13 issue 11
A Study by Purushottam S. G. et al. entitled "Development of Fenofibrate Solid Dispersions for the Plausible Aqueous Solubility Augmentation of this BCS Class-II Drug" is awarded Best article for Vol 13 issue 10
A Study by Kumar S. et al. entitled "A Study on Clinical Spectrum, Laboratory Profile, Complications and Outcome of Pediatric Scrub Typhus Patients Admitted to an Intensive Care Unit from a Tertiary Care Hospital from Eastern India" is awarded Best Article for Vol 13 issue 09
A Study by Mardhiah Kamaruddin et al. entitled "The Pattern of Creatinine Clearance in Gestational and Chronic Hypertension Women from the Third Trimester to 12 Weeks Postpartum" is awarded Best Article for Vol 13 issue 08
A Study by Sarmila G. B. et al. entitled "Study to Compare the Efficacy of Orally Administered Melatonin and Clonidine for Attenuation of Hemodynamic Response During Laryngoscopy and Endotracheal Intubation in Gastrointestinal Surgeries" is awarded Best Article for Vol 13 issue 07
A Study by M. Muthu Uma Maheswari et al. entitled "A Study on C-reactive Protein and Liver Function Tests in Laboratory RT-PCR Positive Covid-19 Patients in a Tertiary Care Centre – A Retrospective Study" is awarded Best Article of Vol 13 issue 06 Special issue Modern approaches for diagnosis of COVID-19 and current status of awareness
A Study by Gainneos PD et al. entitled "A Comparative Evaluation of the Levels of Salivary IgA in HIV Affected Children and the Children of the General Population within the Age Group of 9 – 12 Years – A Cross-Sectional Study" is awarded Best Article of Vol 13 issue 05 Special issue on Recent Advances in Dentistry for better Oral Health
A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" is awarded Best Article of Vol 13 issue 06
A Study by Chen YY and Ghazali SRB entitled "Lifetime Trauma, posttraumatic stress disorder Symptoms and Early Adolescence Risk Factors for Poor Physical Health Outcome Among Malaysian Adolescents" is awarded Best Article of Vol 13 issue 04 Special issue on Current Updates in Plant Biology to Medicine to Healthcare Awareness in Malaysia
A Study by Kumari PM et al. entitled "Study to Evaluate the Adverse Drug Reactions in a Tertiary Care Teaching Hospital in Tamilnadu - A Cross-Sectional Study" is awarded Best Article for Vol 13 issue 05
A Study by Anu et al. entitled "Effectiveness of Cytological Scoring Systems for Evaluation of Breast Lesion Cytology with its Histopathological Correlation" is awarded Best Article of Vol 13 issue 04
A Study by Sharipov R. Kh. et al. entitled "Interaction of Correction of Lipid Peroxidation Disorders with Oxibral" is awarded Best Article of Vol 13 issue 03
A Study by Tarek Elwakil et al. entitled "Led Light Photobiomodulation Effect on Wound Healing Combined with Phenytoin in Mice Model" is awarded Best Article of Vol 13 issue 02
A Study by Mohita Ray et al. entitled "Accuracy of Intra-Operative Frozen Section Consultation of Gastrointestinal Biopsy Samples in Correlation with the Final Histopathological Diagnosis" is awarded Best Article for Vol 13 issue 01
A Study by Badritdinova MN et al. entitled "Peculiarities of a Pain in Patients with Ischemic Heart Disease in the Presence of Individual Combines of the Metabolic Syndrome" is awarded Best Article for Vol 12 issue 24
A Study by Sindhu Priya E S et al. entitled "Neuroprotective activity of Pyrazolone Derivatives Against Paraquat-induced Oxidative Stress and Locomotor Impairment in Drosophila melanogaster" is awarded Best Article for Vol 12 issue 23
A Study by Habiba Suhail et al. entitled "Effect of Majoon Murmakki in Dysmenorrhoea (Usre Tams): A Standard Controlled Clinical Study" is awarded Best Article for Vol 12 issue 22
A Study by Ghaffar UB et al. entitled "Correlation between Height and Foot Length in Saudi Population in Majmaah, Saudi Arabia" is awarded Best Article for Vol 12 issue 21
A Study by Siti Sarah Binti Maidin entitled "Sleep Well: Mobile Application to Address Sleeping Problems" is awarded Best Article for Vol 12 issue 20
A Study by Avijit Singh"Comparison of Post Operative Clinical Outcomes Between “Made in India” TTK Chitra Mechanical Heart Valve Versus St Jude Mechanical Heart Valve in Valve Replacement Surgery" is awarded Best Article for Vol 12 issue 19
A Study by Sonali Banerjee and Mary Mathews N. entitled "Exploring Quality of Life and Perceived Experiences Among Couples Undergoing Fertility Treatment in Western India: A Mixed Methodology" is awarded Best Article for Vol 12 issue 18
A Study by Jabbar Desai et al. entitled "Prevalence of Obstructive Airway Disease in Patients with Ischemic Heart Disease and Hypertension" is awarded Best Article for Vol 12 issue 17
A Study by Juna Byun et al. entitled "Study on Difference in Coronavirus-19 Related Anxiety between Face-to-face and Non-face-to-face Classes among University Students in South Korea" is awarded Best Article for Vol 12 issue 16
A Study by Sudha Ramachandra & Vinay Chavan entitled "Enhanced-Hybrid-Age Layered Population Structure (E-Hybrid-ALPS): A Genetic Algorithm with Adaptive Crossover for Molecular Docking Studies of Drug Discovery Process" is awarded Best article for Vol 12 issue 15
A Study by Varsha M. Shindhe et al. entitled "A Study on Effect of Smokeless Tobacco on Pulmonary Function Tests in Class IV Workers of USM-KLE (Universiti Sains Malaysia-Karnataka Lingayat Education Society) International Medical Programme, Belagavi" is awarded Best article of Vol 12 issue 14, July 2020
A study by Amruta Choudhary et al. entitled "Family Planning Knowledge, Attitude and Practice Among Women of Reproductive Age from Rural Area of Central India" is awarded Best Article for special issue "Modern Therapeutics Applications"
A study by Raunak Das entitled "Study of Cardiovascular Dysfunctions in Interstitial Lung Diseas epatients by Correlating the Levels of Serum NT PRO BNP and Microalbuminuria (Biomarkers of Cardiovascular Dysfunction) with Echocardiographic, Bronchoscopic and HighResolution Computed Tomography Findings of These ILD Patients" is awarded Best Article of Vol 12 issue 13 
A Study by Kannamani Ramasamy et al. entitled "COVID-19 Situation at Chennai City – Forecasting for the Better Pandemic Management" is awarded best article for  Vol 12 issue 12
A Study by Muhammet Lutfi SELCUK and Fatma entitled "Distinction of Gray and White Matter for Some Histological Staining Methods in New Zealand Rabbit's Brain" is awarded best article for  Vol 12 issue 11
A Study by Anamul Haq et al. entitled "Etiology of Abnormal Uterine Bleeding in Adolescents – Emphasis Upon Polycystic Ovarian Syndrome" is awarded best article for  Vol 12 issue 10
A Study by entitled "Estimation of Reference Interval of Serum Progesterone During Three Trimesters of Normal Pregnancy in a Tertiary Care Hospital of Kolkata" is awarded best article for  Vol 12 issue 09
A Study by Ilona Gracie De Souza & Pavan Kumar G. entitled "Effect of Releasing Myofascial Chain in Patients with Patellofemoral Pain Syndrome - A Randomized Clinical Trial" is awarded best article for  Vol 12 issue 08
A Study by Virendra Atam et. al. entitled "Clinical Profile and Short - Term Mortality Predictors in Acute Stroke with Emphasis on Stress Hyperglycemia and THRIVE Score : An Observational Study" is awarded best article for  Vol 12 issue 07
A Study by K. Krupashree et. al. entitled "Protective Effects of Picrorhizakurroa Against Fumonisin B1 Induced Hepatotoxicity in Mice" is awarded best article for issue Vol 10 issue 20
A study by Mithun K.P. et al "Larvicidal Activity of Crude Solanum Nigrum Leaf and Berries Extract Against Dengue Vector-Aedesaegypti" is awarded Best Article for Vol 10 issue 14 of IJCRR
A study by Asha Menon "Women in Child Care and Early Education: Truly Nontraditional Work" is awarded Best Article for Vol 10 issue 13
A study by Deep J. M. "Prevalence of Molar-Incisor Hypomineralization in 7-13 Years Old Children of Biratnagar, Nepal: A Cross Sectional Study" is awarded Best Article for Vol 10 issue 11 of IJCRR
A review by Chitra et al to analyse relation between Obesity and Type 2 diabetes is awarded 'Best Article' for Vol 10 issue 10 by IJCRR. 
A study by Karanpreet et al "Pregnancy Induced Hypertension: A Study on Its Multisystem Involvement" is given Best Paper Award for Vol 10 issue 09

List of Awardees

A Study by Ese Anibor et al. "Evaluation of Temporomandibular Joint Disorders Among Delta State University Students in Abraka, Nigeria" from Vol 13 issue 16 received Emerging Researcher Award

A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" from Vol 13 issue 06 received Emerging Researcher Award

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IJCRR Code of Conduct: To achieve a high standard of publication, we adopt Good Publishing Practices (updated in 2022) which are inspired by guidelines provided by Committee on Publication Ethics (COPE), Open Access Scholarly Publishers Association (OASPA) and International Committee of Medical Journal Editors (ICMJE)

Disclaimer: International Journal of Current Research and Review (IJCRR) provides platform for researchers to publish and discuss their original research and review work. IJCRR can not be held responsible for views, opinions and written statements of researchers published in this journal.


International Journal of Current Research and Review (IJCRR) provides platform for researchers to publish and discuss their original research and review work. IJCRR can not be held responsible for views, opinions and written statements of researchers published in this journal


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