International Journal of Advances in Science and Technology, Vol. 3, No.

6, 2011

Production and Assay of Alpha-amylase by Lactobacillus plantarum (A6) using Natural Media
Mani Ramakrishnan1*, Lingaiah Rajanna2, Ankeeta Upadhyay1 and Sounak Bera 1
1

PG Department of Biotechnology, CMR Institute of Management Studies (Autonomous), C. A. #2, 3rd Cross, 6th A Main, HRBR Layout, 2nd Block, Kalyana Nagar, Bangalore 560 043. 2 Department of Botany, Bangalore University, Jnana Bharathi campus, Bangalore 560 056. *maniramiyer@yahoo.com

Abstract
Alpha-amylase is one of the most widely used enzymes commercially, produced at a large scale in many industries like food processing, feed, detergents, therapeutics and breweries. The conventional method of producing alpha-amylases in most of the industries is by solid-state fermentation using different species of fungi and bacteria. In the present exploration, the production of alpha-amylase was studied using Lactobacillus plantarum (A6) in the fruit pulp (mango) and oil seed cake (groundnut) as the natural media. In the partially purified enzyme, highest activity produced was 330 IU/ml using mango pulp substrate followed by oil seed cake (230 IU/ml). The amount of glucose units released from starch by the action of the enzyme alpha-amylase was estimated. Present study was focused on the significance of production of -amylase using L. plantarum over other conventional substrates and the organisms used.

Keywords: Lactobacillus, amylase, natural substrate, oil seed cake and mango pulp extract

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Introduction
Amylases are among the most important enzymes and are of great significance in biotechnology and are commercially important in various starch processing industries [1]. The thermo stable alpha-amylases from Bacillus spp. play important role in textile and paper industries, starch liquefaction, food, adhesive and sugar production, and various other industries and therefore are of considerable commercial interest [2]. Several studies have been under-taken to define ideal culturing and nutritional conditions for obtaining higher yields of the enzyme and various carbohydrate sources have also been tested as enzyme production-increasing agents for different strains. Complex fermentation media which give optimum production of alpha amylase have been reported, but no successful attempt to develop cheap substrates for optimum production of this enzyme with industrial relevance. The regular use of peptone-beef extract-based fermentation media is not commercially viable for industries. L. plantarum is a widespread member of the genus Lactobacillus, is first isolated from human saliva, commonly found in fermented food products as well as anaerobic plant matter. It has the ability to liquefy gelatin. L. plantarum has the largest genome known among the lactic acid bacteria and a very flexible and versatile species. Its a facultative, hetero-fermentative, gram-positive, aero-tolerant bacterium grows at 15 C (59 F) and produces both isomers of lactic acid (dextro and levo). L. plantarum and related lactobacilli respire oxygen but have no respiratory chain or cytochromes in the consumed oxygen ultimately and ends up as hydrogen peroxide. The peroxide acts as a weapon to exclude competing bacteria from the food source. In place of the protective enzyme superoxide dismutase present in almost all other oxygen-tolerant cells, this organism accumulates millimolar quantities of manganese. Because of this L. plantarum cannot be used to produce active enzymes that require a heme complex such as true catalases. L. plantarum, like many lactobacillus species, can be cultured using MRS (Man, Rogosa and Sharpe) media. The entire genome has recently been sequenced, and promoter libraries have been developed for both conditional and constitutive gene expression, adding to the utility of L. plantarum. It is also commonly employed as the indicative organism in niacin bioassay experiments as it is a niacin auxotroph. L. plantarum A6, isolated from fermented cassava, can break down raw starch that has not been subjected to preliminary physicochemical treatment. When the pH was kept at 6, the microorganism cultured in a bioreactor excreted a high alpha-amylase activity (60 U/ml) [3]. L. plantarum is a lactic acid bacterium common in numerous natural fermentation processes, such as those of silage, cabbage, cucumber, olive, cassava, etc. This was the first description of an L. plantarum amylolytic strain, and it was found that it synthesizes large amounts of extracellular aamylase. A investigation of this enzymatic activity was carried out due to the original features of the microorganism. However, all of these studies were performed with soluble starch, whereas in nature, starch is found in a crystalline insoluble form which makes it less available to enzymatic hydrolysis. Work was therefore carried out to determine the capacity of the strain L. plantarum A6 to break down raw starch. This information would be useful in evaluating the potential for the utilization of L. plantarum A6 as a starter in certain traditional fermentation system. Sudaporn and Saovanee (2010) [4] cultivated Lactobacillus plantarum strain KV1 in whey-containing medium for use as starter in vegetable fermentation. Recently, Bertrand et al. (2011) [5] reported the production of raw starch degrading highly thermostable amylase and lactic acid using Lactobacillus fermentum strain 04BBA19. For efficient commercial production, a continuous effort is essential to find cheaper substrate sources. Available carbon and nitrogen sources are the decisive factors in the optimum production of enzymes and these differ very much from substrate to substrate. Several low price oilseed cakes like from groundnut are abundantly available and are used as cattle feed, fertilizer and some after processing edible for humans as well. The options of cheap raw materials like the various oilseed cakes that increase the yield of alpha amylases have not been attempted. In the present study, an attempt is made to find the effect of oilseed cakes and fresh fruit juice for extracellular alpha-amylase production by L. plantarum (A6).

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Materials and Methods Organism used L. plantarum (A6) isolate was isolated from Cassava fermented for one week [6] and the pure culture was used in the study for production of amylase using different natural media. Media Media used for this purpose was MRS media developed by de Man, Rogosa and Sharpe to replace the tomato juice medium and the meat extract tomato juice medium supporting growth of lactobacilli [7]. MRS media composition (g/l) Ingredients Universal peptone Meat extracts Yeast extract D (+)-Glucose Dipotassium hydrogen phosphate Diammonium hydrogen citrate Sodium acetate Magnesium sulphate Manganous sulphate Agar pH 6.5 37C

10.0 5.0 5.0 20.0 2.0 2.0 5.0 0.1 0.05 12.0

The pure culture was maintained in petriplate, growth was observed every 24 hours for a week.. For enzyme production broth was used with the 20% v/v mango pulp extract (MPE) as the carbon source and in the other groundnut oil seed cake (GSC) 20% w/v with a control added with D (+)-glucose. Aliquots were autoclaved and inoculated the test organism (L. plantarum) by streak plate method under aseptic condition. Inoculated plates were incubated at 37C. Enzyme activity was assayed every 24 hours by calorimetric method [8]. Reagents Dinitrosalicylic acid (DNS) reagent: Dissolved 1g of DNS, 200 mg crystalline phenol and 50 mg sodium sulphite in 100 ml of 1% NaOH. Other chemicals such as 70% ethanol, phosphate buffer, starch solution, glucose and distilled water [8]. ESTIMATION OF AMYLASE ACTIVITY BY DNS METHOD Extraction of -amylase Overnight grown culture broth was filtered through a sterilised funnel with glass wool and centrifuged at 5000rpm for 30 min. Supernatant was precipitated overnight with two volume of chilled acetone, precipitate was separated by centrifugation at 5000rpm for 30 min, re-suspended in 5 ml of ice cold 10mM calcium chloride solution, kept overnight at 4oC, then centrifuged at 10000 rpm for 20 min. The supernatant was used as enzyme source [1]. Amylase activity To one ml of enzyme solution one ml of starch solution added and incubated at 37oC for 15 min. The reaction was stopped by the addition of 2 ml of DNS reagent and kept in boiling water bath for five min. One ml of potassium sodium tartrate was added, cooled in running tap water then made the volume 10 ml and red the absorbance at 560 nm. Maltose was used for the preparation of standard graph for enzyme activity estimation. A unit of -amylase is expressed as mg of maltose produced per min incubation with 1% starch [8].

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Result and discussion The pure culture of L. plantarum (A6) isolated from fermented cassava was tested for production of -amylase in the natural media. The pure culture showed normal growth in MRS media plated by quadrant streak. L. plantarum (A6) was tested gram positive rods, catalase and oxidase were also positive (Figure 1). The MRS culture media contain polysorbate 0.1%, acetate, magnesium and manganese which are known to act as special growth factors for Lactobacilli as well as a rich nutrient base. These substances can be used at varying concentrations and combinations, but inevitably a compromise has to be reached between selectivity and productivity of the organism sought.

Figure 1. A. Isolated pure culture of Lactobacillus plantarum (A6), B. Microscopic view (Oil immersion, 100 x) of Gram Positive Lactobacillus plantarum (A6), C. Catalase positive test of the study organism and D. Oxidase positive test of the study organism. In the present study, production of -amylase was achieved by using L. plantarum (A6) in different natural media such as mango pulp extract and groundnut oil seed cake including the control with 5 % D + Glucose (Table 1). Amylolytic enzymes play important role in the degradation of starch and are produced in bulk from microorganisms and represent about 25% to 33% of the world enzyme market [9]. Significant increase in the production of -amylase was achieved over control with 5% glucose. Highest yield was achieved at 24 h incubated culture. Broth incubated for 48 h and 72 h shown decreased amylase activity. Highest activity (330 IU/ml) recorded among the two natural media supplement used was from Mango pulp extract. Ground nut oil seed cake added MRS broth yielded higher than (230 IU/ml) control and lesser than mango pulp extract (Figure 2). Control yielded maximum of 156 IU/ml. Increased enzyme activity is indicated by the fact that the concentration of glucose is found to be higher when the enzyme extracted from natural media is made to act on starch. Amylase activity was considered as more concentration of glucose, higher activity of the alphaamylase. The amount of glucose was estimated in both the natural media which will interfere in the amylase activity determination by DNS method [8].

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Table 1 Assay of -amylase production with L. plantarum in MRS media supplemented with different natural substrates and D (+)-glucose as control. -amylase production (IU/ml) Incubation time (h) 24 48 72 Control (D + Glucose 5 %) 158 0.21 138 0.33 135 0.28 Mango pulp extract 330 0.42 222 0.76 145 0.98 Groundnut oil seed cake 230 0.56 144 0.50 90 0.84

*Values are mean of three replicates Standard deviation Since, alpha- amylase is one of the most widely used enzymes in many industries it is commercially produced at a large scale. The conventional method of producing alpha-amylases in most of the industries is by solid-state fermentation method which utilizes strains of Aspergillus oryzae for stationary culture with wheat bran and submerged aerated and agitated. The wheat bran and the strains of A.niger have been employed extensively for the production of fungal amylase. For this process, the wheat bran spread in relatively thin layer is moistened with water or dilute acid, sterilized and inoculated with spores of a fungus such as Aspergillus niger [10]. In the present investigation, the production of alpha-amylase was estimated using L. plantarum (A6) and there was a remarkable increase in the activity of the enzyme (330 g/ml) produced in the natural substrate, mango pulp extract (Table 1). This was inferred due to the release of more glucose units from starch by the action of the enzyme alpha amylase. It is mandatory that the amount of glucose present in the natural media was calculated which will add up the enzyme yield. Swetha et. al., (2007) [11] reported amylase production from the wheat bran extract using Aspergillus oryzae at 70 h incubation at 30oC. It shows that compared to the natural substrates used for the production of amylase using fungi as a source of micro organism mango pulp extract using L. plantarum (A6) yielded commendable activity (300 IU / ml) in the present study at 34oC incubation for 24 h. It also indicates that the enzyme could have higher thermostability. Alpha-amylase has an entire plethora of applications in commercial sectors. Alpha-amylase is used in ethanol production to break starches in grains into fermentable sugars. The amylases are employed commercially for the preparation of sizing agents and removal of starch sizing from woven cloth. They are used for the liquefaction of heavy starch process which forms during the heating steps in manufacture of corn and chocolate syrup. It is employed as a replacement of for malt preparation, hydrolysis of starch in brewing. It is widely used in the detergent industry as a de-staining agent for removing the stains arising due to starch and starch products [12-13]. Apart, from the industrial usage, amylase is also used to pre- digest the feed meant for the cattle [14-15]. In the developing countries like India, the incorporation of amylase in the food of malnourished children has shown a rise of 3% rise in the uptake of the nutrition of food as shown by recent studies [16]. As India is the third largest oil- producing country in the world, there is a large amount of oil-seed cake that is produced once the oil is extracted from the oil seed. This oil seed cake is then mainly used as a fertilizer, to increase soil fertility. Hence, oil seed cakes can be used to produce the enzyme alpha-amylase of a higher enzyme activity that can bring about a revolution in the industrial sectors. Alternatively, fruit pulp extracts can also be supplemented to obtain the enzyme of higher activity. The first step in the production of highfructose corn syrup is the treatment of cornstarch with alpha-amylase, producing shorter chains of sugars called oligosaccharides. L. plantarum is the most common bacterium used in silage inoculants. During the anaerobic conditions of ensilage, these organisms quickly dominate the microbial population, and, within 48 hours, they begin to produce lactic and acetic acids via the Embden-Meyerhof- Paranas Pathway, further diminishing their competition. Under these conditions, L. plantarum strains producing high levels of heterologous proteins have been found to remain highly competitive. This quality could allow this species isolated in the present study could be utilized as an effective biological pre-treatment for lingo-cellulosic biomass. L. plantarum is commonly found in many fermented food products including

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sauerkraut, pickles, brined olives, Korean kimchi, Nigerian ogi, sourdough and other fermented plant material, and also some cheeses fermented sausages and stockfish. The high level of this organism in food also makes it an ideal candidate for the development of probiotics. Frias et al. (2008) [17] reported that L. plantarum has been applied to reduce the allergenicity of soy flour. The result showed that, compared to other microbes, L. plantarum-fermented soy flour showed the highest reduction in IgE immunoreactivity (9699%), depending upon the sensitivity of the plasma used. Above all, the ability of L. plantarum to survive in the human gastro-intestinal tract makes it a possible in vivo delivery vehicle for therapeutic compounds or proteins. The ability of L. plantarum to produce antimicrobial substances that helps them to survive in the gastro intestinal tract of human.

Figure 2. Graphical representation of -amylase production with L. plantarum (A6) in MRS media with different natural substrates. A-C -amylase production in MRS media with Mango Pulp extract, Ground nut oil cake and D (+)-glucose as control, respectively.

References
[1] Bernfeld, P. Amylases, Methods Enzymol, vol. no. 1, pp.149-158, 1955 [2] Griffin, P. J., W. M. Fogarty, Preliminary observation on the starch degrading system elaborated by Bacillus polymyxa. Biochemical Society Transactions, Vol. 1, pp. 397-400, 1973. [3] Giraud, E., Allain, C., R. Maurice, Degradation of raw starch by a wild amylolytic strain of Lactobacillus plantarum, Applied and Environmental Microbiology, vol. 60, no. 12, pp. 4319-4323, 1994. [4] Sudaporn L., C. D. Saovanee, Cultivation of Lactobacillus plantarum KV1 in Wheycontaining medium for use as starter in vegetable fermentation, International Journal of Biotechnology and Biochemistry, Vol. 6, No. 6, pp. 877888, 2010. [5] Bertrand Tatsinkou Fossi1, Frederic Tavea, Celine Jiwoua and Robert Ndjouenkeu, Simultaneous production of raw starch degrading highly thermostable alpha-amylase and lactic acid by Lactobacillus fermentum 04BBA19, African Journal of Biotechnology Vol. 10, no. 34, pp. 6564-6574, 2011.

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[6] Aneja, K. R., Experiments in microbiology, plant pathology and biotechnology. 4th Edition. New Age International Publishers, India, 2003. [7] De Man, J.C., M. Rogosa, M. Elisabeth Sharpe, "A Medium for the Cultivation of Lactobacilli". Journal of Applied Bacteriology, vol. 23, 130-135, 1960. [8] Sadasivam, S., A. Manickam, Biochemical Methods, 2nd Edition, New Age International Publishers, India, 1996. [9] Asoodeh, A., J. Chamani, M. Lagziana, A novel thermostable, acidophilic a-amylase from a new thermophilic Bacillus sp. Ferdowsicous isolated from Ferdows hot mineral spring in Iran: Purification and biochemical characterization, International Journal of Biological Macromolecules, vol. 46, pp. 289-297, 2010. [10] Kuo, M. J., P. A. Hartman, Isolation of amylolytic strains of Thermoactinomyces vulgaris and production of thermophilic actinomycete amylases. Journal of Bacteriology, vol. 92, 723-726, 1966. [11] Swetha, S., Dhanya G., K. M. Nampoothiri, R. S. Carlos, P. Ashok, Alpha amylase production by Aspergillus oryzae employing solid-state fermentation, Journal of Scientific and Industrial Research, vol. 66, pp. 621-626, 2007. [12] Krishna, C., M. Chandrasekaran, Banana waste as substrate for a-amylase production by Bacillus subtilis (CBTK 106) under solid-state fermentation. Applied Microbiology and Biotechnology, vol. 46, no. 2, 106-111, 1996. [13] Ramakrishnan, M. 1997. Exploitation of litter fungus for production of ethanol and hard wood craft pulping and biobleaching. M. Phil. Thesis, Bharathiar University, Coimbatore, India. [14] Norton, C. L., H. D. Eaton, Dry calf starters for dairy calves, Cornell Agricultural University Experiment Station Bulletin, vol. 835, no. 32, 1946. [15] Lander, P. E. The feeding of farm animals in India, Macmillan and Co. Ltd., London. appendices I and IV, 1949. [16] Kuppuswamy, S., M. Srinivasan, V. Subrahmanyan. Proteins in foods. Indian Council of Medical Research, Special report series. New Delhi Wesley Press, Mysore City, India. Special report series 33, 81, 87, pp 90-93, 1958. [17] Frias Juana, Young Soo Song, Cristina Martnez-Villaluenga, Elvira Gonz Alez De Mejia, Concepcion Vidal-Valverde, Immunoreactivity and Amino Acid Content of Fermented Soybean Products, Journal of Agricultural Food Chemistry, vol. 56, pp. 99105, 2008.

Authors Profile
Dr. Mani Ramakrishnan received his Ph. D degree in Botany from Bangalore University, Bangalore, India in 2011. This author received Summer Research Fellow- Teachers (2011) from Indian Academy of Sciences, Bangalore. He is having 13 years of experience in teaching and research in the field of Biotechnology. He published 5 research paper in National and International Peer Review Journals and presented 14 research papers in National and International conferences. Author is a member Indian Society for Technical Education, Society of Cytologists and Geneticists and Indian Association for Angiosperm Taxonomy.

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Dr. Lingaiah Rajanna received his Ph.D degree from Mysore University, Mysore, India in the year 2000. This author is Excellent Researcher Award from Bangaore University. He is JRF and SRF awardee from CSIR. A life member of Indian Science congress, Society of Cytologists and Geneticists and Indian Remote Sensing Society. He is also the member of Delhi Botanical University Association, Swamy botanical Club. He is currently working as a Professor in Department of Botany, Bangalore University. He is having overall teaching experience of 15 years including professional colleges. He published 23 research papers in National and International Peer Review Journals and presented 50 research papers in National and International conferences.

Ms. Ankeeta Upadhyay completed her Master degree in Biotechnology from PG Department of Biotechnology, CMR Institute of Management Studies (Autonomous), affiliated to Bangalore University, Bangalore, India in the year 2011. Currently she is associated with Ernst & young, Bangalore. Mr. Sounak Bera Completed his Master degree in Biotechnology from PG Department of Biotechnology, CMR Institute of Management Studies (Autonomous), affiliated to Bangalore University, Bangalore, India in the year 2011. He is engaged in clearing National Eligibility Tests for Higher studies.

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