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Efficient Production of Pullulan by Aureobasidium Pullulans Grown On

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Efficient Production of Pullulan by Aureobasidium Pullulans Grown On b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 180–185 ht tp://www.bjmicrobiol.com.br/ Biotechnology and Industrial Microbiology Efficient production of pullulan by Aureobasidium pullulans grown on mixtures of potato starch hydrolysate and sucrose 1 1 ∗ Chao An , Sai-jian Ma , Fan Chang, Wen-jiao Xue Microbiology Institute of Shaanxi, Xi’an, PR China a r t i c l e i n f o a b s t r a c t Article history: Pullulan is a natural exopolysaccharide with many useful characteristics. However, pullu- Received 1 June 2015 lan is more costly than other exopolysaccharides, which limits its effective application. The Accepted 16 August 2016 purpose of this study was to adopt a novel mixed-sugar strategy for maximizing pullulan Available online 19 November 2016 production, mainly using potato starch hydrolysate as a low-cost substrate for liquid-state Associate Editor: Gisele Monteiro de fermentation by Aureobasidium pullulans. Based on fermentation kinetics evaluation of pullu- Souza lan production by A. pullulans 201253, the pullulan production rate of A. pullulans with mix- tures of potato starch hydrolysate and sucrose (potato starch hydrolysate:sucrose = 80:20) −1 Keywords: was 0.212 h , which was significantly higher than those of potato starch hydrolysate alone −1 Mixed sugar (0.146 h ) and mixtures of potato starch hydrolysate, glucose, and fructose (potato starch −1 −1 Aureobasidium pullulans hydrolysate:glucose:fructose = 80:10:10, 0.166 h ) with 100 g L total carbon source. The Pullulan results suggest that mixtures of potato starch hydrolysate and sucrose could promote Potato starch hydrolysate pullulan synthesis and possibly that a small amount of sucrose stimulated the enzyme Batch kinetics responsible for pullulan synthesis and promoted effective potato starch hydrolysate conver- sion effectively. Thus, mixed sugars in potato starch hydrolysate and sucrose fermentation might be a promising alternative for the economical production of pullulan. © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). usually biosynthesized by strains of the yeast-like polymor- Introduction 3–5 phic fungus Aureobasidium pullulans. Because of its strictly linear structure, pullulan has been used in applications such Pullulan is an extracellular water-soluble homo-polysaccha- as blood plasma substitutes and as an additive for food, adhe- 1,6 ride composed of linear maltotriose-units interconnected via sives, and cosmetics. Despite these applications, pullulan is ␣ −1 -1,6-glycosidic linkages. These linkages endow pullulan with more costly ($25 kg ) than other exopolysaccharides, which 1,2 7 structural flexibility and high aqueous solubility. Pullulan is is a major limiting factor for its effective application. ∗ Corresponding author at: Shaanxi Institute of Microbiology, No. 76 Xiying Rd, Xi’an, Shaanxi Province 710043, PR China. E-mail: [email protected] (W. Xue). 1 Chao An and Sai-jian Ma contributed equally to this work. http://dx.doi.org/10.1016/j.bjm.2016.11.001 1517-8382/© 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 180–185 181 A primary strategy to solve the problem of high production Preparation of potato starch hydrolysate costs is to search for cheap carbon and nitrogen sources, which are nutritionally rich enough to support growth of the microor- Potato starch was purchased from a local agricultural mar- 8 ganism and the production of pullulan. Leathers concluded ket. The pH of a slurry of potato starch at a concentration of ◦ that an agricultural waste product, maize residue, can be used 40% (w/v) was adjusted to pH 6.5 and instantly heated to 70 C 1 as a carbon source for pullulan production by A. pullulans. within 3 min in the presence of 0.1% ␣-amylase. After lique- ◦ Other waste products from the agriculture and food indus- faction, the temperature was quickly reduced to 55 C within tries, such as deproteinized whey, cassava starch residue, 1 min, and the pH was adjusted to 5.0. Amyloglucosidase was beet molasses, sugar cane juice, and sweet potato and peat then added at a concentration of 0.3% (w/w) and incubated for hydrolysates, have also been considered as economical sub- prolonged hydrolysis. After hydrolysis, the hydrolysates were 8,9 8 strates for pullulan production. filtered. Potato, a cheap and available agricultural product, con- tains a large amount of starch, which is a suitable feedstock Analytical methods for industrial fermentation. Potato is also a major crop in Shaanxi Province of China. However, low productivity of pul- The culture was centrifuged at 10 000 × g for 10 min to remove lulan fermentation using potato starch as a substrate is a the microorganism. The biomass (mycelia and yeast-like cells) 10 drawback that has not yet been solved. Available data dry weight (BDW) was determined by washing the sediment ◦ show that both the amount of exopolysaccharide produced with distilled water and drying at 105 C overnight. Fifteen and the rate of carbohydrate source consumption are clearly milliliters of the supernatant was transferred into a test tube, influenced by the particular carbohydrate used, and Duan and then 30 mL of cold ethanol was added to the test tube and ◦ proposed that high pullulan yield is related to high activi- mixed thoroughly. The solution was then held at 4 C for 12 h ␣ ties of -phosphoglucose mutase, UDPG-pyrophosphorylase, to precipitate the exopolysaccharide, which was separated by and glucosyltransferase in A. pullulans Y68 grown on dif- centrifugation at 8000 × g for 10 min. The precipitated mate- 11 ◦ ferent sugars. Based on these considerations, mixed-sugar rial was dried at 80 C, and the final weight determined as fermentation might be an efficient fermentation strategy that of exopolysaccharide. The total concentration of reduc- that leads to high productivity by providing more than one ing sugar was determined by the dinitrosalicylic acid (DNS) substrate. 13 method. FTIR Fourier transform infrared spectroscopy (FTIR) In our previous research, sucrose was demonstrated to spectra of pullulan samples for composition analysis were be superior to other sugars, based on pullulan yield, and a directly acquired using the Smart-iTR setup without further mixture of sucrose and potato starch hydrolysate (PSH) was preparation.14 found to significantly enhance the pullulan formation rate of A. pullulans 201253 when using a carbon source in a flask. Batch fermentation of pullulan in a 10-L bioreactor Finally, based on fermentation kinetics evaluation for pullu- lan formation by A. pullulans 201253, the model parameters Batch fermentation was carried out in a 10-L bioreactor associated with polysaccharide formation (m, n, and (dP/dt)/X) (Yangge Bioengineering Equipment Co., Ltd, Shanghai, China) and substrate consumption ˛, ˇ and (dS/dt)/X were assayed to ◦ containing 7.0 L fermentation medium at 28 C with agitation elaborate the fermentation kinetic processes using a mixture of 500 rpm. The inoculum volume was 5.0% (v/v). Samples of potato starch hydrolysate and sucrose. were taken periodically to determine the concentration of pul- lulan, biomass, and reducing sugar. Material and methods Determination of parameters during the fermentation Microorganism and culture process A. pullulans 201253 was obtained from the American Type Cul- Biomass formation ture Collection (Rockville, MD, USA) and was maintained at The logistic equation was used to fit the biomass curves. A ◦ 4 C on potato dextrose agar (PDA) slants and sub-cultured common autonomous rate equation is the differential form of every 2 weeks. For long-term storage, cultures were main- the logistic equation: ◦ tained at −80 C in a 20% glycerol solution. For inoculum ◦ X X preparation, A. pullulans was grown at 28 C for 48 h in a d = X 1 − , (1) dt max X medium containing 50 g glucose, 2.5 g yeast extract, 0.6 g max (NH4)2SO4, 1 g NaCl, 5 g K2HPO4, and 0.2 g MgSO4·7H2O per liter where and X are the initial specific growth rate and of deionized water, at an initial pH of 6.5 with agitation at max max the maximum biomass concentration, respectively. The inte- 230 rpm. grated form using X = X(t = o) gives a sigmoidal variation X(t) Basic fermentation medium consisted of 50 g of car- 0 that may empirically represent both an exponential and a sta- bon source, 2.5 g yeast extract, 0.6 g (NH4)2SO4, 1 g NaCl, tionary phase: 5 g K2HPO4, and 0.2 g MgSO4·7H2O per liter of deionized 12 water, at an initial pH of 6.5 with agitation at 230 rpm. To t X e max evaluate the effects of mixed carbon sources on pullulan pro- 0 X(t) = . (2) maxt − X /X − e duction, the carbon source was varied in this study. 1 ( 0 max)(1 ) 182 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 180–185 Substrate consumption Specific growth rate -1 0.8 Specific production rate The substrate balance for polysaccharide formation may be Spec ific substrate consumption rate written as follows (modification of Luedeking and Piret): -1 0.7 ) S X P -1 0.6 d = 1 d 1 d − − − keX (h (5) t Yx/s t YP/s t µ d d d 0.5 rate, where Yx/s and Yp/s are the yield coefficients for the biomass , 0.4 and product, respectively, and ke is the specific maintenance growth 0.3 rate (i.e., the substrate used to support cell activity even in the ecific absence of growth).
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