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Screening of Levan Producing Bacteria from Soil Collected from Jaggery Field Nb

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Screening of Levan Producing Bacteria from Soil Collected from Jaggery Field Nb IJPCBS 2017, 7(3), 202-210 Laddha et al. ISSN: 2249-9504 INTERNATIONAL JOURNAL OF PHARMACEUTICAL, CHEMICAL AND BIOLOGICAL SCIENCES Available online at www.ijpcbs.com Research Article SCREENING OF LEVAN PRODUCING BACTERIA FROM SOIL COLLECTED FROM JAGGERY FIELD NB. Laddha1* and MP. Chitanand2 1Research Centre in Microbiology, Netaji Subhashchandra Bose College, Nanded-431 602, Maharashtra, India. 2Department of Microbiology, Netaji Subhashchandra Bose College, Nanded- 431 602, Maharashtra, India. ABSTRACT Levan is an extracellular polysaccharide composed predominantly of D-fructose residues joined by glycosidic bonds. At present, there are many polysaccharides in the market primarily of plant origin. But microbial levans are more preferred as dietary polysaccharide due to its mult- idimensional application as anti-lipidemic, anti-cholesterimic, anti-cancerous and as prebiotic. In this context, in present study twelve levan producing isolates were screened out from jaggery field. Iosolate SJ8 was the highest levan producer which was studied for cultural, morphological and biochemical characterization. It was identified as Bacillus subtilis (KC243314). The optimized fermentation conditions for levan production were pH 7, temperature 300 C,100rpm and 48hrs fermentation period. At these optimized conditions the maximum levan production was 30.6mg/ml. Sugar composition of the polymer was confirmed by analytical methods and TLC. Result proved the presence of fructose as the main sugar in the polymer. Keywords : Levan, Bacillus subtilis (KC243314), polymer, fructose. INTRODUCTION plasma substitute, drug activity prolonger5, anti- Levan is a biopolymer formed by β-(2,6) linkage obesity and lipid-lowering agent6, having β-(1,2) linkages on its branch points and osmoregulator, cryoprotector, or antitumor contains long chain of fructose units in its agent7. The application potential of this chemical structure . It develops in the course of biopolymer in the entire sectors is stimulating a trans-glycosylation reaction with the an intense and constant search aiming for a participation of the levansucrase [EC 2.4.1.10]1. better new options so that we can get better Studies to re-search levan producers are related alternative. In this context the present work was to its multifaceted applications in the food, aimed to screen levan producers from soil, cosmetic, pharmaceutical and medicine which is the rich source of various bacterial industries. Levan can be used as an emulsifier, species. formulation aid, stabilizing thickener, surface- finishing agent, encapsulating agent as well as a MATERIALS AND METHODS carrier for colours and flavours in the food Sample collection industry2. In pharmaceuticals levan is a non- Levan producers grow very well in sucrose digestible food fibre3 and, therefore, it can be containing medium, therefore soil from jaggery treated as a component of prebiotic nature. preparation fields were selected as samples for Levan can also be utilised to manufacture DFA this study. Soil samples were collected from the lV[di-D-fructose dianhydride]. DFA lV is different areas where jaggery was prepared characterised by half the sweetness of from sugarcane. saccharose, cyclical structure which guarantees its stability and, therefore, similarly to other Enrichment of bacteria DFAs, it can find application as a sweetener for Enrichment of bacteria was carried in sucrose diabetics4. In medicine, it can be used as a broth containing 5% sucrose. For this 1% soil 202 IJPCBS 2017, 7(3), 202-210 Laddha et al. ISSN: 2249-9504 sample was inoculated to 10ml sucrose biochemical media, sugar fermentation media containing broth. Tubes were incubated at 30o C and enzyme production media. for 48 hrs. Identification by 16S rRNA sequencing Screening of Levan producing bacteria The high levan producing strain was identified Ten fold serial dilutions of the tube showing by 16S rRNA sequencing from ARI Pune. turbidity were prepared. 1ml of diluted sample Genomic DNA of isolate was isolated using Gene was spread inoculated on 5% and 10% sucrose Elute Genomic DNA Isolation Kit (Sigma, USA) agar plates. Plates were incubated at 300C for 48 used as template for PCR. Each reaction mixture hrs. Organisms producing mucoid colonies were contained approximately 10ng of DNA; 2.5mM selected as levan producer. Cultural and MgCl2; 1x PCR buffer [Bangalore Genei, morphological characters of colonies were Bangalore, India]; 200µM each dCTP, dGTP, studied and colonies were sub-cultured on dAPT, and dTTP; 2 pmol of each, forward and nutrient agar slants and preserved for further reverse primer; and 1 U of Taq DNA studies. polymerase[(Bangalore Genei, Bangalore, India] in a final volume of 20µl. FDD2 and RPP2 Levan production primers were used to amplify almost entire 16S Inoculum preparation rRNA gene, as described previously [Rawlings Inoculum preparation was carried by 1995]. The PCR was performed using the inoculating active bacterial culture in 5 ml of Eppendorf Gradient Mastercycler system with a inoculum medium containing [g/L] sucrose 100, cycle of 940 C for 5 min; 30 cycles of 940 C, 600C 0 yeast extract 2, (NH4)2SO4 1, KH2PO4 2, MgSO4 and 72 C for 1min each; and final extension at 0.5. The inoculated medium was incubated at 720 C for 10min, and the mixture was held at 40 300C for 24 hr. C. The PCR product was precipitated using polyethylene glycol (PEG 6000, 8.5%), washed Production medium thrice using 70% ethanol and dissolved in Tris- 5% of inoculum was transferred aseptically to HCL [10mM, pH 8]. Samples were run on ABI 50ml production medium in 250ml Erlenmeyer Prism 3100 Genetic Analyser. The sequencing flask. The production medium had following output was analyzed using the accompanying composition [g/L] sucrose 100, yeast extract 2, DNA Sequence Analyzer computer software (NH4)2SO4 3, KH2PO4 1, MgSO4 0.6, MnSO4 0.2. [Applied Biosystems]. The sequence was The pH was adjusted to 7 before autoclaving. compared with NCBI GeneBank entries by using The inoculated production medium was the BLAST algorithm. incubated at 300C for 48 h on rotary shaker at 100rpm. Analytical studies The levan powder obtained was dissolved in Extraction of Levan deionized water.Total carbohydrate estimation After incubation the 10ml of fermentation broth in levan solution was carried by phenol was taken and centrifuged at 10000 rpm for 10 sulphuric acid method10. 2ml of 0.1N HCL was min. Cell pellet obtained was dried for biomass mixed with 2ml of levan solution and boiled at calculation and the clear supernatant was used 1000C for 60min. The acid hydrolyzate was for levan recovery. analyzed for fructose and glucose estimation. Fructose estimation was carried by Resorcinol Recovery and Determination of Levan method11 and glucose was estimated by The supernatant obtained was then used to Somogyi method12. precipitate levan by adding alcohol. Four volumes of chilled aqueous ethanol [70%] was TLC of levan added to it. The tube was inverted several times Hydrolyzed samples were applied to Silica Gel for proper mixing. The precipitate formed was plates saturated with solvent 1 Butanol: 2 allowed to settle down at 40C for 24hrs. The propanol: water: acetic acid [7:5:4:2]. Standards supernatant was discarded and precipitate was of glucose [Hi media Pvt. Ltd.] and fructose were collected. The precipitate was washed twice run and plates were sprayed with 5% sulfuric with aqueous ethanol and transferred to vial. It acid in methanol, air dried and then heated at was dried at 600C8,9 and dry weight was 1100 C until spots appeared. measured. Optimization of fermentation conditions on Biochemical characterization levan production Highest levan producer was characterized Highest levan producer was used for biochemically by inoculating in different optimization of fermentation conditions for 203 IJPCBS 2017, 7(3), 202-210 Laddha et al. ISSN: 2249-9504 levan production. The parameters studied were identified by biochemical characterization and pH, temperature, agitation and incubation 16S rRNA gene sequencing. period. Analytical studies Effect of pH on levan production Total carbohydrate content of levan produced Levan production was carried at pH 4, 5, 6, 7, 8, [Fig. 2] by majority of the isolates was more and 9. pH of the medium was adjusted by using than 400(µg/ml). Maximum carbohydrate 0.1N HCl and 0.1N NaOH. The 50ml of content was found in levan [650 µg/ml] production medium was inoculated with 24 hrs produced by isolate SJ8. old culture and incubated at 300C for 48hrs. Levan produced by 12 isolates was hydrolyzed Levan and biomass was measured. and hydrolyzate was analyzed for fructose and glucose content. Hydrolyzate from isolate SJ8 Effect of temperature on levan production showed maximum fructose concentration i.e. 50ml production medium was inoculated and 460µg/ml. Hydrolyzate from 8 isolates showed incubated at different temperature for 48hrs. fructose content above 200µg/ml [Fig. 2].Only The temperatures used were 10 0C, 20 0C, 30 0C, the presence of fructose and negligible presence 40 0C, 50 0C and 60 0C. After this the levan was of glucose confirmed the presence of fructose precipitated, dried and weighed. Similarly the polymer. effect of temperature on biomass was studied. The organism was caesinase, gelatinase, catalase and oxidase positive [Table 2]. It fermented Effect of agitation sugars glucose, arabinose and xylose with acid 24hrs old inoculum was inoculated in 50ml production only. The organism showed production medium and incubated at 300C for temperature tolerance upto 800 C [Table 3]. 48hrs at agitation speed of 50rpm, 100rpm, 150rpm, 200rpm and 250rpm, one flask was TLC of levan kept at stationary. After completion of Sugar components of levan were identified by fermentation levan was precipitated, dried till TLC analysis levan hydrolyzate. The Rf value of constant weight. Also the effect of agitation on acid hydrolyzed levan from Bacillus subtilis biomass was studied. (KC243314) was identical to that of fructose under laboratory conditions. This result Effect of fermentation period indicated that levans were composed solely of 24hrs old culture of inoculum was inoculated in fructose.
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