Research Article ISOLATION and SCREENING of PECTINOLYTIC BACTERIA (Bacillus Megaterium) from the MANGO FRUIT YARDS
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International Journal of Agriculture Sciences ISSN: 0975-3710 & E-ISSN: 0975-9107, Volume 10, Issue 13, 2018, pp.-6552-6556. Available online at https://www.bioinfopublication.org/jouarchive.php?opt=&jouid=BPJ0000217 Research Article ISOLATION AND SCREENING OF PECTINOLYTIC BACTERIA (Bacillus megaterium) FROM THE MANGO FRUIT YARDS SRIDEVI K.1*, VENKATESH G.2, SUMANTH M.3 AND VIJAYALAKSHMI K.4 1SGS Arts College, Sri Venkateswara University, Tirupati, 517502, Andhra Pradesh, India 2Contractual Teacher, College of Horticulture, Anantharajupeta, Dr Y.S.R. Horticultural University, Venkataramannagudem, 534101, Andhra Pradesh, India 3Shanmugha Arts, Science, Technology & Research Academy, Thirumalaisamudram, Thanjavur, 613401, Tamil Nadu, India 4Retd Prof in Microbiology, Sri Padmavati Mahila Visvavidyalayam, Tirupati, 517502, Andhra Pradesh, India *Corresponding Author: Email - [email protected], [email protected] Received: June 27, 2018; Revised: July 10, 2018; Accepted: July 11, 2018; Published: July 15, 2018 Abstract: As the population and the improved life style of people increasing day by day, results in the increased generation of wastes the country. Especially the food and agricultural industries produce large amount of wastes, causing a serious disposal problem. And it is becoming more problematic in countries like India where the economy is mainly based on agriculture and where the farming practice is very expensive. The past decade, the concept of sustainable development has become entrenched in our social conscience inextricably linking the growth of global economy to the protection of resources and our environment over the other types of waste treatment methods. One of the agro- wastes currently causing pollution problems is the mango peel from fruit processing industries. Microorganisms play an important role in the degradation of pectin which is major component of mango peel. The action of different extracellular enzymes on mango peel leads to the biodegradation of pectin. The type of enzymes is known as pectinases which hydrolyses pectic substances readily. Therefore, the main objective of the present study was to isolate, screen and to study the effect of various factors on pectinolytic activities of Bacillus strain from soils of decomposed fruit waste from mango processing industries Keywords: Agro waste, Biodegradation, Mango peel, Pectinases, Isolation, Screening, Microorganisms Citation: Sridevi K., et al., (2018) Isolation and Screening of Pectinolytic Bacteria (Bacillus megaterium) from the Mango Fruit Yards. International Journal of Agriculture Sciences, ISSN: 0975-3710 & E-ISSN: 0975-9107, Volume 10, Issue 13, pp.- 6552-6556. Copyright: Copyright©2018 Sridevi K., et al., This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Introduction Mango is one of the most delicious and widely cultivated fruits of the tropical decomposed fruit waste from mango processing industries in Chittoor district, world. It is processed extensively, thereby generating huge quantity of solid and Andhra Pradesh, India. liquid waste. This constitute about 40-50 % of total waste. Around 52 mango fruit processing units are established in Chittoor district of Andhra Pradesh. These Materials and Methods units process the mangos of 60,000 tonnes per anum, of which 40,000 tonnes Isolation of Pectinolytic bacteria after processing are exported. Typical factory will process 5 tonnes of Totapuri Collection of soil samples from mango fruit processing industries mangoes (a popular variety of this region) per hour and peel would be Soil samples were collected from 12 different locations at a depth of 15cm from accumulating as waste. Pectin is the majorly available organic resources in mango waste dumping yards of Vinsari and Varsha mango fruit processing industries peels where the amount of pectin can be very high i.e.,12.85% when compared to around Tirupati, Chittoor district of Andhra Pradesh [5]. The soil samples were lemon peels (15-20 %) and apple peels (10%). Many of the workers suggested transported to the laboratory in sterile polythene bags, air-dried and mixed the use of mango peels as a source of pectin [1]. Pectin is used to improve the thoroughly to make a composite sample [6]. The soil samples were serially diluted quality of many food and pharmaceutical products. The antioxidative activity of and plated on to the nutrient agar medium plates and incubated at 37°C for 24 mango peel fibre has been found to have better invitro properties than lemon hours. fibres and used as a substrate for mushroom cultivation. Many experiments resulted that pectinases are produced from different carbon sources. Meanwhile, Screening of pectinolytic bacteria from fruit waste many reports have showed the optimization of various factors like fermentation, Citrus Pectin agar medium (g/L) preparation microbiological parameters and different fermentation strategies to production of All morphological colonies were purified by repeated streaking on to the 1% citrus pectinases [2]. The microorganism’s bacteria, yeast, fungi have capacity produce pectin Agar with 67% of metoxilation, 0.14% (NH4)2 SO4, 0.2% K2HPO4, 0.02% pectinases [3]. The advantage of using micro-organisms for the production of MgSO4.7H2O and 0.10% nutrient solution (5mg /L FeSO4.7H2O, 1.6 mg/L MnSO4, enzymes is that these are not influenced by climatic and seasonal factors, and can H2O, 1.4 mg/L ZnSO4.7H2O, 2.0 mg/L COCl2 with pH 6.0. be subjected to genetic and environmental manipulations to increase the yield. The pectinases activity is also influenced by the various physical and chemical Plate assay of depolymerized pectin: parameters which are vital for yield increase [4]. Hence, the present investigation The same medium used for isolation of cultures, supplemented with 2% agar agar. was to study the pectinolytic activities of Bacillus strains isolated from soils of Pure cultures were inoculated by making puncture in the medium and incubated International Journal of Agriculture Sciences ISSN: 0975-3710&E-ISSN: 0975-9107, Volume 10, Issue 13, 2018 || Bioinfo Publications || 6552 Isolation and Screening of Pectinolytic Bacteria (Bacillus megaterium) from the Mango Fruit Yards for 48h at 30°C. After the colonies reached around 3 mm, iodine-potassium iodide to convert the tryptophan to indole. The production of red colour at the junction of solution (1.0 gm Iodine, 5g of KI and 330 mL H2O) was added to detect clearance reagent and broth indicates a positive indole reaction. zones [7]. Methyl Red (MR) test Test for potent isolates for cellulolytic, proteolytic and amylolytic activity The ability of the isolates to ferment glucose and acid production was determined The potent pectinolytic isolates were checked for cellulolytic, proteolytic and using GPP medium. 2-3 drops of methyl red indicator was added to the 48 hrs amylolytic activities by using CMC agar medium, casein agar medium and starch culture tubes and results were recorded immediately. Appearance of bright red agar medium through getting clear zones. colour in the medium indicated a positive reaction. E.coli and K.pneumoniae were used as positive and negative controls respectively for MR and VP tests. CMC agar medium The CMC agar medium contains 1.0 % peptone, 1.0 % CMC, 0.2 % K2HPO4, 1 % Voges-Proskauer (VP) test agar, 0.03 % MgSO4.7H2O, 0.25 % (NH4)2SO4 and 0.2 % gelatin at pH7 [8]. Voges-Proskauer (VP) test was used to test the ability of the isolates to produce Acetoin as the end product can be detected by adding 5% solution of α-napthol in Test for cellulolytic activity ethanol and 0.2 ml of 40% KOH to 1 ml of GPP broth cultures inoculated with the Pure cultures of bacterial isolates were transferred individually onto CMC agar isolates incubated at 37oC for 48 hrs. Appearance of eosin pink colour indicates a plates. After 48 hours of incubation, the CMC agar plates were flooded with 1 % positive reaction of Voges-Proskauer (VP) test. congo red and allowed for 15 min at room temperature. The plates were counterstained by 1M NaCl. The cellulose hydrolysis can be indicated by Citrate utilization test appearance of clear zones around growing bacterial colonies incubated for 48 The ability of the organism to utilize citrate as carbon and energy source for its hours at 30oC [9]. The bacterial colonies having the largest clear zones were growth and an ammonium salt as a source of nitrogen was determined by selected for identification. streaking isolates on Simmons’s citrate agar slant (pH 6.8) and incubating for 24 hrs at 37°C. A positive test indicates the change in the colour of the medium from Tarch agar medium: green to blue and appearance of a streak of growth. K.pneumoniae and E.coli Starch agar medium of 100 ml was prepared by 0.1 g of dextrose, 0.7g of were used as positive and negative controls. dipotassium phosphate, 0.2 g of monopotassium phosphate, 0.01 g magnesium sulphate, and 0.05 g of sodium citrate, 0.1 g of ammonium sulphate, 2% starch Results and Discussion and 1.5 g of agar in 100 ml of distilled water. The medium was sterilized, cooled, Collection of soil sample from the mango fruit processing industries poured in to individual petri dishes and allowed for solidification [10]. Mango fruit compost samples were collected from Vinsari and Varsha fruit processing industries around Tirupati, Chittoor district, Andhra Pradesh.