ISOLATION AND CHARACTERIZATION OF AMYLASE PRODUCING BACTERIA FROM ORANGE AND POMEGRANATE PEEL

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1 Page7111 Indo American Journal of Pharmaceutical Research, 2016 ISSN NO: ISOLATION AND CHARACTERIZATION OF AMYLASE PRODUCING BACTERIA FROM ORANGE AND POMEGRANATE PEEL G.Prameela 1, K. Priya Dharshini 2, M.Chidanandappa 1.K.Kamala 1 * 1* Department of Biotechnology, Rayalaseema University, Kurnool. 2 Departnment of Chemistry, S.V University, Tirupati. ARTICLE INFO Article history Received 08/11/2016 Available online 03/12/2016 Keywords Amylase, Bacillus Sps, Orange Peel And Pomegranate Peel. ABSTRACT The enzymes from microbial sources are more stable and obtained cheaply. Amylases are among the most important enzymes and are of great significance in present day industry. Starch degrading bacteria are most important for industries such as food, fermentation, textile and paper. Thus isolating and manipulating pure culture from various soils has manifold importance for various biotechnology industries. Amylase production from bacteria is economical as the enzyme production rate is higher in bacteria as compared to other microorganism. The aim of the current study was to isolate amylase producing bacteria from the soil samples collected. The isolation was done by serial dilution and plating method. Characteristic feature of the strains indicates them as Bacillus sps. Bacterial isolates were screened for amylolytic activity by starch agar plate method. The optimum ph for production was 7, whereas maximum growth was observed at 1gm Orange Peel and pomegranate Powder. The concentration of enzyme increases with increase in substrate concentration. The ph range was found to be 7 and incubation time 48hrs with 1ml as inoculum for optimum growth. Corresponding author Dr. Kamala Department of Biotechnology, Rayalaseema University, Kurnool. Please cite this article in press as Dr.Kamala et al. Isolation and Characterization of Amylase Producing Bacteria from Orange and Pomegranate Peel. Indo American Journal of Pharmaceutical Research.2016:6(11). Copy right 2016 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Page7112 INTDROUCTION The first to be commercially produced enzymes are amylases. In food industry amylases are required for the production of glucose syrup, and crystalline glucose. The potential of use of microorganisms as sources of relevant enzymes has stimulated in recent years [1-3], in industries like food, feed, leather, textile, and paper, these enzymes can be used in a large number [4-7]. Bacillus species are heterogeneous forms and gram positive bacteria usually found in soil, known to be good producers of alpha amylases and widely used for commercial production of enzyme. An amylase catalyses hydrolysis of starch in to sugars. Amylases present in the plants, animals, and microorganisms, but industrial demands are reached by the microbial sources [8]. Foods contains huge amounts of starch with little sugar, such as rice and potatoes, may obtain somewhat sweet taste as they are chewed for the reason that amylase degrades starch into sugar. As diastase, amylase enzyme to be first discovered and isolated. In medicinal and clinical the use of amylase relevance has widened [9], and the application of amylase in starch saccharification, brewing industries, food and baking. Also Amylases have shown a significant role in textile and detergent industries [10-12]. In enzyme production, the nature of the moistening agent and the ratio of substrate are crucial factors [13]. Citrus fruits comprise an important group of fruit crops manufactured worldwide. In the fruit processing industry large amounts of waste materials are produced, in the form of peel, pulp, seeds, etc. The waste material present significant disposal difficulties, and when not used in any way it cause odor and soil pollution. Since the 1980s the worldwide production of citrus has increased drastically. Estimations show that in 2010 the orange production will reach 66.4 million metric tons, which is an increase with 14% compared with that of Almost half, 30.1 million metric tons of the produced orange will be manufactured to yield juice, essential oils and other by products. The outer part or the skin of an orange is called orange peel. In our country the orange peel is a waste material. Its proper utilization may help to have a friendly environment as well as it may help to add our economic development. Orange peel may be used as a source of cellulose for isolation of its cellulosic material. The derivatization of isolated cellulosic substance may remove the waste disposal problem and earn economic significance. The cellulose acetate has versatile commercial uses. Cost of substrates on enzyme producing microbes can be cultivated has always been an essential factor in production. Generally Complex lignocelluloytic residues are considered as the best substrates [14-16]. Only the initial ph was maintained the fermentation medium ph was not monitored after autoclaving and. The enzyme was consequently harvested and amylase activity [17]. Worrying about that the citrus fruit processing industries generate vast amounts of waste material, which cause important disposal difficulties [8]. Previous studies have reported the successful hydrolysis of citrus peel waste into sugars and their subsequent conversion into ethanol [5] In the dried citrus peels the fat content is low and rich in cellulose, hemicelluloses, proteins and pectin. Citrus peels are the major solid by-product and comprise around 50% of the fresh fruit weight in the citrus processing industry. The effect of nitrogen source and moistening agents on amylase activity was also determined under the optimized conditions of incubation period, temperature, initial ph, and substrate [18]. In the same way the waste peel of pomegranate also having nutritional values, the present study deal with the production of amylase enzymes. The present study mainly focused on isolation and characterization of amylase producing bacteria from orange and pomegranate peel waste. MATERIALS AND METHODS Isolation of Amylase producing bacteria: The soil sample from surrounding Rayalaseema University, Kurnool. The soil sample were collected and dried under shade. One gram soil from each collected sample was added in first test tube containing 10 ml saline distilled water and mixed well using vortex mixer to get parental culture suspension. Then subjected to serial dilution 0.5ml suspension was taken from each test tube, spread over agar plates and incubated at 37 o C for 24 hrs. The colonies with smooth surface margins, circular elevated and colorless were isolated and sub- cultured for pure cultures. The selected pure bacterial colonies are tested for amylase enzyme production on Starch Agar Medium. Screening for Amylase Activity (Starch Iodine Test) Isolated colonies were picked up from each plate containing pure culture and streaked over starch agar plates with starch as the only carbon source. After incubation at 37ºC for hrs., individual plates were flooded with Gram s iodine (Gram s iodine- 250 mg iodine crystals added to 2.5gm potassium iodide solution, and 125ml of water, stored at room temperature) to produce a deep blue colored starch-iodine complex. In the zone of degradation no blue colour forms, which is the basis of the detection and screening of an amylolytic strain. The colonies which were showing zone of clearance in starch agar plates were maintained on to nutrient agar slants. Morphological and Biochemical Characteristics: Gram staining, methyl red, Vogues Proskauer's, citrate utilization, were carried out. Gram staining Preparation of the glass microscopic slide Grease or oil free slides are essential for the preparation of microbial smears. Grease or oil from the fingers on the slides is removed by washing the slides with soap and water. Wipe the slides with spirit or alcohol. After cleaning, dry the slides and place them on laboratory towels until ready for use.

3 Page7113 Methyl red and Voges-Proskauer test The isolate was inoculated separately into a test tube containing MRVP medium. (Composition of methylredvogusproskauer s medium is glucose phosphate, water, distilled water, ph 7.5). And it was incubated at 37deg.C for hrs. After incubation add 1-2 drops of methyl red indicator, and 2-3 drops of reagent was added and shaken thoroughly. It was then allowed to react for minutes. The colour change to red indicates positive for methyl red and colorless indicates positive for vogesproskauer test. Methyl red indicator is Methyl red, Absolute Alcohol, dist. Water. If the test is positive the medium with culture turns red which infers that the organism is a mixed acid fermented. Vogus proskauer test The isolate was incubated separately into test tubes containing MRVP medium. The dextrose, Di-potassium hydrogen phosphate distilled Water, at a ph of 7.5. Then they are incubated at 37 deg. C for 48 hrs. To the inoculate 40% KOH and of 5% Alpha-naphthol (Barrod s reagent) is added and shaken well, allowed it to stand for 2 minutes. A positive test involves production of pink colour in the medium, which indicates that the organism has fermented glucose and produce acetone. Citrate utilization test One agar slants of Kosser s citrate agar medium were prepared. (Kosser s medium consist of sodium ammonium phosphate, Dipotassium hydrogen phosphate, Bromomethyl Blue, Sodium citrate, dist. Water. The isolate was inoculated on to slant and incubated at 37 o C for 24 hrs. Citrate utilization is indicated by growth and change in colour of the medium from green to blue. Enzyme production medium: Production medium contained (g/l) Trypticase 10gm, peptone 5gm, (NH4) 2 SO4 3gm, K 2 HPO 4 2gm, L-Cysteine HCl 0.5gm, MgSO 4 0.2gm.10 ml of medium was taken in a 100 ml conical flask. The flasks were sterilized in autoclave at 121 o C for 15 min and after cooling the flask was inoculated with overnight grown bacterial culture. The inoculated medium was incubated at 37 o C in shaker incubator for 24 hr. At the end of the fermentation period, the culture medium was centrifuged at 5000 rpm for 15 min to obtain the crude extract, which served as enzyme source. Amylase activity assay: The α-amylase activity was assayed by the dinitrosalicylic acid (DNS) procedure following the method of Bernfeld (1955) [19], using 1% soluble starch (Merck, product number 1257, Darmstadt, Germany) as substrate. 10 microliters of the enzyme was incubated for 30 min at 35 C with 500 µl universal buffer and 40 µl soluble star0ch. The reaction was stopped by addition of 100 µl DNS and heated in boiling water for 10 min. Then absorbance was read at 540 nm after cooling in ice for 5 min. One unit of α- amylase activity was defined as the amount of enzyme required to produce 1 mg maltose in 30 min at 35 C. A standard curve of absorbance against amount of maltose released was constructed to enable calculation of the amount of maltose released during α- amylase assays. Serial dilutions of maltose in the universal buffer at ph 6.5 were made to give following range of concentrations of 2, 1, 0.5, 0.25, mg ml 1. A blank without substrate but with α-amylase extract and a control containing no α-amylase extract but with substrate were run simultaneously with the reaction mixture. All assays were performed in duplicate and each assay repeated at least three times. Effect of ph The effect of various ph ranges on production of amylase was investigated. Production medium was prepared with Lactose as substrate and distributed 25 ml each into 10 different flasks. Then the each flask Ph is adjusted 6, 7, 8 and 9 respectively and labeled they were then autoclaved. 5% of overnight grown culture was inoculated into the each flask. All the flasks were then incubated at 37 0 C in incubator for 48 hrs. After 48 hrs amylase activity was assayed for each ph value by Nessler s assay. RESULTS BACTERIAL IDENTIFICATION AND ENZYME CHARACTERIZATION The bacterial isolates were collected and processed by serial dilution and spread plate method for the isolation of amylase producing bacteria (Figure 1). The inoculated samples on agar plates exhibited various types of microbial growth on incubation. The bacterial isolates hydrolysed the starch on starch agar, orange and pomegranate peel power (Figure 1&3).. The strain showing maximum inhibition zone by enzyme in starch hydrolysis test were selected and further characterized by morphological and biochemical tests.

4 Page7114 Identification of bacterial cultures Figure 1: Photograph showing growth of Bacillus culture. Figure 2: Photograph showing growth of Gram s staining. Methyl Red Test: VP showed positive result as red colour was produced immediately. Citrate test: Table 1: Results of soil bacteria biochemical tests for identification. Source Starch hydrolysis Soil +ve V. P. +ve Citrate Bacterium identified +ve Bacillus sps Citrate Test showed positive result as the colour changes from Green to Blue (Left tube in the figure is the Sample and the Right tube is a demonstration of Negative result in a reference sample.the bacterial isolates from the soil given positive results to starch hydrolysis, Voges-Proskauer, citrate hydrolysis tests. It clearly indicates that the bacterial isolates present in the soil were identified as Bacillus species. Screening of Amylase Producing Bacteria: The bacteria isolated from soil were screened for amylase production on starch agar medium.

5 Page7115 Effect ph on amylase activity: Figure 3: Photograph showing growth of Bacillus culture. Table 2: Effect of ph on amylase activity. ph Concentration of enzyme produced (µg/min/ml) Changes in amylase enzyme production by bacillus species under different ph condition are presented in Table 7. Bacterial isolates were allowed to grow in media of different ph ranging from 4.0 to 8.0. Maximum enzyme activity was observed in medium of ph 7.0. However majority of microorganisms producing alkaline amylases show growth and enzyme production under alkaline condition. Effect of carbon source on enzyme activity Concentration of enzyme production by Orange Peel Powder as carbon sources Table 3: Effect of Orange Peel Powder as carbon source on enzyme activity. Sample Concentration of orange powder mg/l Amylase activity µg/min/ml Orange Peel Powder Orange Peel Powder Orange Peel Powder Orange Peel Powder Orange Peel Powder

6 Page7116 Concentration of enzyme production by as Pomegranate Peel Powder carbon sources. Table 4: Effect of Pomegranate Peel Powder as carbon source on enzyme activity. Sample Concentration mg/l Amylase activity µg/min/ml Pomegranate Peel Powder Pomegranate Peel Powder Pomegranate Peel Powder Pomegranate Peel Powder Pomegranate Peel Powder Carbon source: Various sources of Carbon such as orange and pomegranate peel powder were used to original carbon source in growth media. Results obtained showed that, orange and pomegranate peel powder brought the amylase production at 1000mg/ml. at 24 hr incubation in bacillus sp (Fig 4). Previous studies reported that the different carbon sources have varied influence on the extracellular enzymes especially amylase strains. Results obtained showed that increase in concentration of various substrates increases the amylase production more (Fig 5). Amylase activity on NAM containing Orange peel as carbon source Figure 4&5: Amylase activity on NAM containing Orange peel as carbon source, showed positive result with a clear transparent zone around the colony.

7 Page7117 DISCUSSION Microorganisms are the most important sources for enzyme production. Selection of the right organism plays a key role in high yield of desirable enzymes. For production of enzymes for industrial use, isolation and characterization of new promising strains using cheap carbon and nitrogen source is a continuous process. Gangadharan et al., [20] worked on the surface methodology for the optimization of α-amylase production by Bacillus amyloliquefaciens. Fooladi and Sajjadian [21] analyzed the produced enzyme activity and found at maximum level of activity at 70 C in the presence of soluble starch (1%) at ph 6. With addition of calcium (10 mm) and peptone (1%) to the mineral medium, shortened the lag period and improved the growth and α-amylase synthesis. Amylases are obtained from various origins like plant, animal, bacterial and fungal. Several researchers produces amylase enzyme using Bacillus sps. The present showed that the hydrolysed peel powder of orange and pomegranate showed significant growth of bacterium. Microorganisms have become increasingly important as producer of industrial enzymes. Due to their biochemical diversity and the ease with which enzyme concentrations may be increased by environmental and genetic manipulation, attempts are now being made to replace enzymes, which traditionally have been isolated from complex eukaryotes. Starch degrading amylolytic enzymes are most important in the biotechnology industries with huge application in food, fermentation, textile and paper. Present study is focussed on the isolation and characterization of amylase producing bacteria from the soil samples. Verma et al [22] they worked on purification and production of amylase from bacteria isolated from a waste potato. India. Behal et al [23] studied thermostable amylase producing Bacillus sp that revealed an optimum enzyme activity at ph 8.0 whereas in other species the optimum activity was at ph 7.0. In this study, in submerged fermentation the maximum amount of α-amylase production (1.88 µg/min/ml) was obtained with orange peel as carbon source, ph 7 at 37 C for 24 h. In Submerged fermentation highest amylase production (1.04µg/min/ml) was obtained with Pomegranate peel powder as carbon source, ph 7 at 37 C for 24 h. Summary and Conclusion In the present study, an attempt has been made to isolate amylase producing bacteria from soil sample. The bacterial cultures were isolated from the collected soil samples by serial dilution method and maintained on nutrient agar medium at 4 o C. Biochemical characterization of the selected bacterial isolates was done; the isolates showed positive results with Methyl red, Citrate test, Voges proskauer test, Starch hydrolysis. 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