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. Samples Test for amylolytic activity of required quantity were taken according to the standard methods. The fruit Petri plates with bacterial colonies were flooded with Gram’s iodine reagent (0.01 waste was collected after peeling and crushing operations, only after which they M I2-KI solutions) and observed for zone of starch degradation as revealed by lose their utility. Rotten fruits were also included in the collection. Before forming clear and transparent zones. Among all colonies, the colony having preparation of the composite sample, hand sorting was emphasized to segregate highest degradation of starch was selected for isolation of pure culture, physico- and to remove unwanted materials like straw, plastic covers, packaging materials chemical characterization and partial purification of the enzyme. etc.

Casein agar medium Isolation of Bacteria from the soil sample of Mango Fruit processing Milk agar was prepared by adding 1% sterile skimmed milk (autoclaved at 10 lbs Industries for 10 min) to the nutrient agar medium and cooled at 45°C. The prepared plates Bacteria were isolated from the compost sample collected from mango fruit were inoculated with single colony of each isolate and incubated for 2 days at processing industrial area by serial dilution and plating techniques. Sample was 37°C. Hydrolysis of milk protein was seen as a clear zone around colony. inoculated into Nutrient Agar medium and incubated for 48 hours at 37°C. The pectinolytic property of all the 16 isolates was studied by puncturing the medium Identification of selected isolate on CPA medium, a quantitative test for pectin degrading bacteria. Identification and characterization of efficient pectinolytic bacterial isolates: Identification of the bacterial isolate was carried out by determining morphological, Screening of the isolates for pectin degrading bacteria cultural and biochemical characteristics. It was done according to the Bergey’s One of the efficient bacterial isolate is selected according to their highest Manual of Systemic Bacteriology [11, 12]. pectinolytic activity on the basis of their growth and formation of clearing zones on Citrus Pectin Agar (CPA) medium by using iodine-potassium Iodide solution (I-KI Sugar utilization test solution). The 16 bacterial isolates were able to grown on medium containing The ability of the isolates to ferment various carbohydrates was tested. Different citrus pectin as a source of carbon. The strain is tested for pectin hydrolysis by carbohydrates used in the test were monosaccharide like glucose, galactose, plate assay, at pH 6.0. The strain is classified as very good producer of pectin fructose and arabinose; disaccharides like lactose, sucrose and maltose; depolymerising enzymes when showed clear zones around colonies of 15 mm polysaccharides like soluble starch and glycogen. Peptone water was when the zones were at least 10 mm weak producers when the zones were at supplemented with 0.5% of each sugar Andrade’s indicator (0.005%) was added least 5 mm and poor producers when no pectinolytic activity and no clear zones to the medium and Durham tubes were inserted into the medium tubes. Medium were observed. Good pectinolytic activity was exhibited by the isolate. The isolate was sterilized at 15 lbs pressure for 15 minutes. The tubes were inoculated with is designated and numbered as ‘mango pectinolytic bacteria’ as mpb2. Based on the isolates and incubated at 37°C for 24-48 hours and acid/gas production was the morphological, biochemical and physiological tests, one of the isolate was recorded. initially identified as Bacillus sp. It was characterized according to the guidelines of Bergey’s Manual of Systemic Bacteriology (Volume- III) [11] and Manual of IMVIC tests Medical Microbiology [12]. Indole test The tryptone broth tubes are inoculated with the isolated organism and Kovac’s Identification of selected bacterial isolates reagent of 0.5ml was added to 48 hrs of incubation to test the ability of the isolates At all stages of growth, the cells were found as Gram positive Bacilli.

International Journal of Agriculture Sciences ISSN: 0975-3710&E-ISSN: 0975-9107, Volume 10, Issue 13, 2018 || Bioinfo Publications || 6553 Sridevi K., Venkatesh G., Sumanth M. and Vijayalakshmi K.

Table-1 Morphological, physiological and biochemical characteristics of and of and Utilization of sugars Bacillus megaterium 1 Cellobiose - SN Characteristics B. megaterium 2 Dextrose + Morphological Characteristics - 3 Fructose + Colony morphology 4 Galactose + 1 Configuration Irregular 5 Lactose + 2 Margin Undulate 6 Maltose + 3 Elevation Flat 7 Sucrose + 4 Surface Smooth 8 Mellibiose + 5 Colony color Cream 9 Mannitol + 6 Opacity Opaque 10 Sorbitol - 7 Gram’s reaction +ve 11 Xylose - 8 Cell shape Long-rods 12 Raffinose + 9 Spore + 13 Mannose + 10 Endospore + 14. Salicin + 11 Position Central 15 Adonitol - 12 Spore Shape Oval 16 Trehalose + 13 Sporangia bulging Non-bulging 17 Ionositol + 14 Motility Motile 18 Mannose + 15 Fluorescence - 19 Mannitol - Physiological characteristics 20 Raffinose - Temperature range Growth at temperature Utilization of Nitrogen sources - Amino acids 1 4°C - 1 L-Aspargine + 2 12°C - 2 L-Alanine + 3 15°C - 3 L-Histidine + 4 25°C - 4 L-Arginine + 5 30°C + 6 37°C + Carbon, Nitrogen utilization pattern, morphological and other biochemical tests 7 42°C + were performed according to standard methods of the bacteria. B. megaterium is 8 50°C + Gram positive, motile, endospore producing, aerotolerant and non-lactose 9 55oC + 10 60oC + fermentor. The physiological conditions for growth are temperature 37°C, pH 7 Optimum temperature 42oC and 1% NaCl concentration. It can tolerate even up to 12% of NaCl concentration. pH Range Growth at pH It is a Catalase positive, Oxidase positive, Urease positive organism. It can 1 pH 5.0 + ferment sugars like Dextrose, Fructose, Millibiose, Mannitol, Raffinose, Mannose, 2 pH 5.5 + Trehalose and Ionositol. Biochemical tests like Indole negative, Methyl Red 3 pH 6.0 + negative, Voges-proskauer test negative, Citrate positive, Nitrate Reductase 4 pH 6.5 + 5 pH 7.0 + positive, H2S production negative. It can hydrolyze cellulose, starch, pectin, 6 pH 8.0 + Gelatin, Arginine, Tween 60, casein and Urea (Fig below). 7 pH 9.0 + 8 pH 10.0 + 9 pH 11.0 - 10 Optimum pH 6.0 NaCl range (%) 1-12% 1 1.0 + 2 2.0 + 3 4.0 + 4 6.0 + 5 8.0 + 6 10.0 + 7 12.0 + 8 Optimum NaCl conc. 1% Biochemical Test - Degradation/Hydrolysis of 1 Casein + 2 Cellulose + 3 Starch + 4 Gelatin + 5 Tween 60 - Fig-1 Screening for pectinolytic activity of mango pectinolytic bacteria (mpb2) using 6 Urea + Citrus Pectin Agar 7 Esculin + Enzymatic activity – Enzyme 1 Catalase activity + Characterization of the isolates 2 Oxidase activity + Based on the morphological, biochemical and physiological tests performed the 3 Urease activity + mpb was initially identified in the laboratory as Bacillus sp., which were further Biochemicaltests confirmed at species level as Bacillus megaterium MTCC 10773 (mpb2) by the IMVIC Tests characterization with the assistance of IMTECH, Chandigarh, India. 1 Indole test - 2 Methyl Red test - 3 Voges-proskauer test - Discussion 4 Citrate Utilization + One of the agro-wastes currently causing pollution problems is the mango peel 5 Hydrogen sulphide Test - from fruit processing industries, which poses considerable disposal problems and 6 Nitrate reduction + ultimately leads to environmental pollution. Mango fruit compost samples were 7 Arginine dihydrolase + collected from fruit processing industries.

International Journal of Agriculture Sciences ISSN: 0975-3710&E-ISSN: 0975-9107, Volume 10, Issue 13, 2018 || Bioinfo Publications || 6554 Isolation and Screening of Pectinolytic Bacteria (Bacillus megaterium) from the Mango Fruit

Fig-2 Identification of Bacillus megaterium, A) Colony morphology; B) Catalase production; C) Sugar fermentation test D) Biochemical tests and Hydrolysis of E) Cellulose, F) Protease and G) Starch of the bacterial isolate mpb2

The efficient bacterial isolate was selected according to their highest pectinolytic Research Category: Screening of pectinolytic bacteria activity on the basis of their growth and formation of clearing zones on Citrus Abbreviation Pectin Agar (CPA) medium by using iodine potassium iodide solution [13]. Among CMC- Carboxy Methyl Cellulose 16 bacteria strains screened on the specific medium, one of the bacterial strains GPP- Glucose Phosphate Peptone showed clear zones and found as pectinase producers with the assistance of CPA- Citrus Pectin Agar IMTECH, Chandigarh. The bacterial colony showing maximum zone diameter was selected as the best strain. The isolate was designated and numbered as ‘mango Acknowledgement / Funding: Author thankful to SGS Arts College, Sri pectinolytic bacteria’ mpb2, the morphological examination, biochemical and Venkateswara University, Tirupati, 517502, Andhra Pradesh, India physiological tests of screened isolate were performed and mpb2 isolate was initially identified as Bacillus megaterium (MTCC 10773). *Research Guide or Chairperson of research: Dr K. Vijayalakshmi University: Sri Padmavati Mahila Visvavidyalayam, Tirupati, 517502, Andhra Conclusion Pradesh, India This study revealed bacterial isolates were found associated with compost, Research project name or number: PhD Thesis collected from fruit processing industries around Tirupati, Chittoor district, Andhra Pradesh. Abiotic factors such as temperature, pH and saline concentration are Author Contributions: All author equally contributed also influenced the growth of B. megaterium. Author statement: All authors read, reviewed, agree and approved the final Application of research: Finally concluded that based on the morphological manuscript examination, biochemical and physiological tests of the isolate resemble as Conflict of Interest: None declared Bacillus megaterium and is suitable for the production of pectinase enzyme useful for the pectin degradation. Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors.

International Journal of Agriculture Sciences ISSN: 0975-3710&E-ISSN: 0975-9107, Volume 10, Issue 13, 2018 || Bioinfo Publications || 6555 Sridevi K., Venkatesh G., Sumanth M. and Vijayalakshmi K.

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