Sequential Heating and Solvent Precipitation For Effective Pullulan Recovery From Jaggery Based Fermentation Broths

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1 Research Article ISSN: V S Rama Krishna Ganduri et al. / Journal of Pharmacy Research,(5),9-3 Available online through Sequential Heating and Solvent Precipitation For Effective Pullulan Recovery From Jaggery Based Fermentation Broths V S Rama Krishna Ganduri,, Srinivasulu K, Usha Kiranmayi M 3, Bodaiah B, Y V V Aswani Kumar, Vijaya Lakshmi M 3, Sudhakar Poda, * Department of Biotechnology, K. L. University, Green Fields, Vaddeswaram- 55, Guntur (Dist), A.P., India. Department of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur- 55, A.P., India. 3 Department of Microbiology and Botany, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur- 55, A.P., India. *Corresponding author. Dr. Sudhakar. Poda Assistant Professor Acharya Nagarjuna University Nagarjunanagar Guntur- 55, A.P., India. Received on:-3-; Revised on: 7--; Accepted on: -5- ABSTRACT Background: Cost-effective downstream processing is very important aspect in successful fermentative production of pullulan biopolymer. Objective: To study the heat treatment method for removal of proteins in cell free supernatant and to optimize the pullulan concentration using several solvents in terms of type, solvent ratio and precipitation time for their efficacy. Materials and methods: Fermentation broths obtained from sucrose and jaggery based shake flasks fermentations were exposed to heat at 5 to C for 5 to min to remove proteins and precipitated with organic solvents like ethanol, methanol, acetone, isopropanol, chloroform and diethyl ether. Various effects such as supernatant: solvent ratio, precipitation time were analyzed. Results: For jaggery (5 g/l) fermentation broth, the optimum heating temperature and time were found at C and 5- min, respectively. Addition of Isopropanol, solvent ratio of :3 and hours of precipitation time at C were observed for maximum pullulan concentration from precipitation. Conclusion: These sequential steps may enhance efficiency of pullulan recovery and would become effective downstream processing method. KEYWORDS: Pullulan, recovery, heat treatment, solvent ratio, precipitation time, jaggery. INTRODUCTION Pullulan, an exo-, homo-, linear- polysaccharide produced by a yeastlike fungi called Aureobasidium pullulans and was first reported by Bauer in 93. It is the one of best most potential, biocompatible polymer consists of maltotriose subunits, linked by both α-, and α-, glycosidic linkages. Pullulan possess excellent properties viz., color-, odor-, taste-less powder, edible, biodegradable and non-toxic nature due to its structure, that prompts the use of pullulan as potential candidate for its application in food, pharma, cosmetic and biomedical industries,3. A. pullulans is a polymorphic fungus belongs to Ascomycota phylogeny and shows morphological characteristics like young and swollen blastospores, mycelia and chlamydospores. Jaggery (also known as Panela or gur), a traditional non-centrifuged, unpurified and concentrated sugar cane product contains 75-5% sucrose, was used as low-cost carbon substrate for the production of pullulan 5-7. Commercial production of pullulan from fermentation process is limited due to cost-effective recovery of pullulan in its Journal of Pharmacy Research Vol. Issue 5 May downstream processing from cell free fermentation broth. Mostly, pullulan was extracted using precipitation technique by organic solvents using various ways -3. In the present study, we have attempted to remove the extracellular proteins by heating cell free fermentation broth and studied with different effects for maximizing the pullulan concentration from solvent precipitation. MATERIALS AND METHODS MATERIALS Aureobasidium pullulans MTCC 95 used in this study was procured from Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh, India. The organism was maintained on Potato Dextrose Agar (PDA) plates at C and subcultured prior to each experimental run. Jaggery was purchased from local market and it was mainly composed of sucrose. Biochemicals such as PDA, Yeast Extract were obtained from M/s. Qualigens, India and other medium chemicals were obtained from M/s. Himedia, Mumbai, India. Solvents such as acetone, ethanol, methanol, isopropanol, chloroform, diethyl ether used in pullulan recovery were obtained from M/s. Merck India Ltd., Mumbai, India. 9-3

2 METHODS V S Rama Krishna Ganduri et al. / Journal of Pharmacy Research,(5),9-3 Inoculum development A loop-full of freshly grown cultures from PDA slants were aseptically transferred to a 5 ml conical flask containing in 5 ml of sucrose based medium (sucrose: 5. g/l; yeast extract: 3. g/l; KH PO : 5. g/l; KCl,.5; MgSO 7H O:. g/l; NaCl:. g/l in double distilled water). Media ph (initial) was adjusted to 5. and sterilized at C for 5 min. Similarly, jaggery based medium (replacing sucrose with jaggery) was also used for shake flask fermentations 7. Shake flask fermentation Shake flask fermentations were carried out with (sterilized) ml of both sucrose and jaggery mediums in 5 ml Erlenmeyer flasks, at an initial carbon substrate (sucrose and jaggery) concentrations of 5, 75 and g/l, individually with an inoculum concentration of 5% (v/v). These flasks were incubated for 9 hours at 3 C and 5 rpm on a rotary shaker 7. Harvesting After 9 hours, fermentation broth was centrifuged at, rpm for minutes, at ambient temperature, to remove the cell biomass and heated to inactivate the extracellular enzymes synthesized by A. pullulans. The supernatant obtained was precipitated with twice the volume of ice cold solvent and kept overnight at C. The precipitated exopolysaccharide (EPS) was separated by centrifugation at, rpm for minutes and the precipitate was dried to constant weight at C 7. The concentration of pullulan was expressed as g/l. Effects of heating temperature and time Fermentation broths obtained from shake flask studies (5, 75 and g/l concentration of sucrose and jaggery) were centrifuged at, rpm for minutes to remove the cell biomass. Supernatants containing desired pullulan and extracellular enzymes were subjected to heating at different temperatures from 5 C to C for constant minutes and cooled. Twice volumes of ice cold isopropanol were added to these heated supernatants and incubated at C and dry weights of pullulan were estimated. Protein concentration in the supernatants was assayed as per Lowry O h et al. The heating temperature at which maximum proteins were denatured and degraded were also studied. To study the effect of heating time, the broth supernatant was heated for varying time from 5 to min at 5 C and subsequently protein and pullulan concentrations were also analyzed. methanol, chloroform and diethyl ether were screened for pullulan precipitation from cell free broths of different sucrose and jaggery (5, 75 and g/l) fermentations. In each case, cell free broth was mixed with -volumes of each organic solvent and pullulan concentration was estimated as per the method described above. All the experiments were carried out in triplicate and the average values of data obtained are reported. Effect of solvent ratio In this case, cell free supernatant was added with varying organic solvent ratios from : to :9 (v/v), to find the probable volume of solvent for maximum pullulan precipitation. Effect of precipitation time To study the influence of contact time for pullulan precipitation, cell free supernatant was mixed with solvent and incubated up to hours at C and analyzed at every hours interval time. RESULTS AND DISCUSSION Fermentations of yeast-like fungus, Aureobasidium pullulans MTCC 95 using sucrose and jaggery as carbon substrates, released some extracellular proteins in addition to exopolysaccharide, pullulan. Proteins produced during fermentation, pose great problems during downstream processing of pullulan recovery and need to be removed from broth. The decomposition of pullulan was greater than proteins and by heating the broth was led to degrade the proteins and pure pullulan was obtained. Figure shows the precipitation of pullulan by the addition of two volumes of ice cold isopropanol to broth in a centrifuge tube. Screening of organic solvents Six different organic solvents, namely ethanol, acetone, isopropanol, Figure. Precipitation of pullulan by isopropanol addition (A) front view, (B) side view. Journal of Pharmacy Research Vol. Issue 5 May 9-3

3 V S Rama Krishna Ganduri et al. / Journal of Pharmacy Research,(5),9-3 Proteins removal with heat treatment Effect of heat treatment to denature the proteins in fermentation broth was investigated for the precipitation of thermos-sensitive proteins. Fig. shows that maximum proteins were denatured at C and hence it was employed in further studies. Pullulan concentration was not much influenced by heating temperatures. To study the influence of heating time at this temperature ( C), cell free supernatants were kept for different time limits (5 to min.) and 5 to min was the optimum time for maximum protein precipitation (Fig. 3). Results obtained are in good agreement with data obtained by many researchers,. EPS Protein precipitation of pullulan. Of the remaining organic solvents, isopropanol had shown much precipitated pullulan concentration compared to acetone and ethanol. Figures and 5 shows the similar trends of precipitation were observed in both sucrose and jaggery fermentation broths, respectively. Hence, isopropanol was used in further experiments Sucrose_5 g/l Sucrose_75 g/l Sucrose_ g/l Ethanol Actone Methanol Isopropanol Chloroform Diethyl Ether Figure. Effect of organic solvents on pullulan precipitation in sucrose fermentation broths Temperature, C Figure. Effect of heating temperature on protein denaturation and pullulan precipitation. EPS Heating Time, min. Protein Figure 3. Effect of heating time on protein denaturation and pullulan precipitation. Screening of organic solvents and supernatant to solvent ratio Among the six different organic solvents employed for pullulan precipitation from cell free fermentation broth, addition of methanol, chloroform and diethyl ether to heated broth did not give significant Jaggery_5g/L Jaggery_75g/L Jaggery_g/L Ethanol Actone Methanol Isopropanol Chloroform Diethyl Ether Figure 5. Effect of organic solvents on pullulan precipitation in jaggery fermentation broths. The ratios of heated cell free broth to isopropanol were varied from : to :9 (v/v) to study the effect of solvent ratio on maximum pullulan precipitation. It was observed that from :3 to : ratios were resulted maximum pullulan concentration (Fig. ). Recent studies have reported that addition of isopropanol to supernatant in three to eight volumes 5, five volumes, two volumes and one volume yielded maximum pullulan concentration. In another study by Kachhawa D K et al 7 and Sugumaran K R & Ponnusami V, 5 also reported that two volumes of ethanol precipitated more amount of pullulan. Journal of Pharmacy Research Vol. Issue 5 May 9-3

4 V S Rama Krishna Ganduri et al. / Journal of Pharmacy Research,(5),9-3 Sucrose Jaggery : : :3 : :5 : :7 : :9 Ratio (culture supernatant : Isopropanol), volume : volume Figure. Effect of culture supernatant: isopropanol on pullulan precipitation. Effect of precipitation time Figure 7 shows the maximum pullulan concentration was obtained at hours of precipitation time and thereafter constant pullulan concentration was recovered. Earlier studies by Chi Z & Zhao S, 3 also reported that optimum precipitation time was hours. Sucrose Jaggery Precipitation Time, hr Figure 7. Effect of precipitation time on pullulan recovery. CONCLUSION An appropriate downstream processing of pullulan fermentation broth is required to accomplish one precipitation step using suitable organic solvent. But, in this study cell free supernatant was subjected to heat treatment to remove most of the proteins without affecting the pullulan recovery. The optimum heating temperature and time were found at C and 5- min, respectively. To see which organic solvent affects the pullulan precipitation, different solvents were tested and Isopropanol with supernatant: solvent ratio of :3 and precipitation time of hours yielded maximum pullulan concentration. This concludes a simple one-step downstream processing for pullulan recovery at its optimized conditions. ACKNOWLEDGEMENTS All the authors express their deep thanks to the management of Journal of Pharmacy Research Vol. Issue 5 May K L University, Green Fields, Vaddeswaram, Guntur-District, Andhra Pradesh, India, for their kind support and encouragement. CONFLICT OF INTEREST All the authors declare no conflict of interests. REFERENCES. Jakovljevic D, Miroslav M V, Radulovic M, Hranisavljevnic- Jakovljevic M, Fine structural analysis of the fungal polysaccharide pullulan elaborated by Aureobasidium pullulans, CH- strain, J Serb Chem Soc,, (), Singh R S, Saini G K, Kennedy J F, Pullulan: Microbial sources, production and applications, Carbohydr Polym,, 73(), Shingel K I, Current knowledge on biosynthesis, biological activity, and chemical modification of the exopolysaccharide, pullulan, Carbohydr Res.,, 339: 7.. Ronen M, Guterman H, Shabtai Y, Monitoring and control of pullulan production using vision sensor, J Biochem Biophys Methods.,, 3, Vijayendra S V N, Bansal D, Prasad M S, Nand K, Jaggery: a novel substrate for pullulan production by Aureobasidium pullulans CFR-77, Proc Biochem.,, 37, Ananya M, Prasad G S, Choudhury A R, Cost effective production of pullulan from agri-industrial residues using response surface methodology, Int J of Biological Macromolecules,,, Rama Krishna Ganduri V S, Sambasiva Rao K R S, Usha Kiranmayi M, Vijaya Lakshmi M, Sudhakar P, Production of Pullulan using Jaggery as substrate by Aureobasidium pullulans MTCC 95, Curr Trends in Biotech and Pharm,, (), Shengjun Wu, Zhengyu Jin, Jin Moon Kim, Qunyi Tong, Hanqing Chen, Downstream processing of pullulan from fermentation broth, Carbohydr polym, 9, 77, Singh R S, Saini G K, Kennedy J F, Downstream processing and characterization of pullulan from a novel colour variant strain of Aureobasidium pullulans FB-, Carbohydr polym, 9, 7, Choudhury A R, Bhattacharjee P, Prasad G S, Development of Suitable Solvent System for Downstream Processing of Biopolymer Pullulan Using Response Surface Methodology, 3, PLoS ONE (), e777.. Bishwambhar M, Suneetha V, A study on downstream processing for the production of pullulan by Aureobasidium pullulans SB- from the fermentation broth, Research Journal of Recent Sciences, 3, vol.(isc-),

5 V S Rama Krishna Ganduri et al. / Journal of Pharmacy Research,(5),9-3. Sugumaran K R, Durgalakshmi R, Ponnusami V, Downstream processing of pullulan recovery from palm kernel hydrolysate, International Journal of ChemTech Research,, (5), Suneetha V, Purification of pullulan from microorganisms for food and biomedical applications, Research Journal of Pharmaceutical, Biological and Chemical Sciences, 5, (), 3-.. Lowry O h, Nira j, Rosebrough a, Lewis farr., Rose j, Randall., Protein measurement with the folin phenol reagent., Sugumaran K R, Ponnusami V, Downstream processing studies for pullulan recovery in solid state fermentation using asian palmyra palm kernel- inexpensive substrate, Biotechnology-An Indian Journal.,, 9(), 79.. Pollock T J, Thorne L, Armentrout R W, Isolation of new Aureobasidium strains that produce high-molecular-weight pullulan with reduced pigmentation, Applied and Environmental Microbiology, 99, 5, Kachhawa D K, Bhattacharjee P, Singhal R S, Studies on downstream processing of pullulan. Carbohydrate Polymers., 3, 5, 5.. Chi Z, Zhao S, Optimization of medium and new cultivation conditions for pullulan production by a new pullulanproducing yeast strain, Enzyme and Microbial Technology, 3, 33,. Source of support: Nil, Conflict of interest: None Journal of Pharmacy Research Vol. Issue 5 May 9-3