EFFECT OF DIFFERENT FACTORS ON BIOSYNTHESIS OF EXOPOLYSACCHARIDE FROM ANTARCTIC YEAST

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1 EFFECT OF DIFFERENT FACTORS ON BIOSYNTHESIS OF EXOPOLYSACCHARIDE FROM ANTARCTIC YEAST S. Rusinova-Videva 1, K. Pavlova 1, I. Panchev, K. Georgieva 1, M. Kuncheva 1 Institute of Microbiology, Bulgarian Academy of Sciences, Department of Microbial Biosynthesis and Biotechnology, Plovdiv,Bulgaria University of Food Technologies, Plovdiv,Bulgaria Correspondence to: Snezhana Rusinova-Videva jrusinova@abv.bg ABSTRACT Psychrophilic yeast Isolate 100, was selected as a producer of exopolysaccharide. A laboratory scheme was created for obtaining of the biopolymer. Biotechnological and physicochemical factors influencing its biosynthesis were investigated. It was determined that inocolum quantity from 6-10%, cultivated for 8 hours at C, is suitable for maximum synthesis. The exopolysaccharide that was synthessed in the culture liquid was thermostable at 0 C to 70 C and it degrades at 80 C. The quantity of the sedimentary polymer, that was synthesis on environment with arabinose and mannose from Isolate 100, after incubation in ethanol at C for 10 hours staying at the time of 10 hours in ethanol at C, was increased with 0%. The influence of the temperature over drying of the exopolysaccharide at 6 C and 10 C was studied. At the low temperature of drying the polymer contained near 0% humidity. The effect of the experimental conditions (temperature, time, concentration of %, %, %, %) on the apparent viscosity values of the cultural liquid was studied. It was found to reveal a behaviour of non-newtonian liquid. The power law equation of Oswald- de Waale describing the change of the apparent viscosity was derived. Keywords: Antarctic yeast, Isolate 100, exopolysaccharide, production Introduction Water-soluble polymers are widely used as thickeners and gelling agents in a broad range of cosmetic and personal care products today. Examples include shampoos and conditioners, hair and body gels, skin creams, liquid and cream foundations, toothpaste, sunscreen lotions and sprays. The natural polymers are mainly polysaccharides and their chemical derivatives. Polysaccharide thikeners include guar gum plus its derivatives, locust bean gum, cellulosic and xanthan gum. Gelling agents include alginate, carrageenan, pectin and gellan gum. A great number of microorganisms bacteria, fungi and yeasts, produce polysaccharides with different texture, 07 structure and interesting physicochemical property and functional characteristics. Xanthan, gellan, dextran, hyaluronan, alginate, curdlan were synthesizied from bacterial producers (9) pollulan is produced by strains of the yeast-like fungus Aureobasidium pullulans (8). The polysaccharides glucans, mannans, glucomannans, galactomannans and phosphomannans have been obtained by yeast belonging to the following genera: Candida (, ); Cryptococcus (1, 17); Rhodotorula (10, 1); and Sporobolomyces (1, 6). Psychrophilic yeast strains were investigated for exopolysaccharides biosynthesis. The strains Sporobolomyces salmonicolor AL 1 and Cryptococcus flavus AL 1 produced exopolysaccharides with basic monosaccharide composition of glucose and mannose in correlation 1, :1 and 1:,1 respectively (11, 1). There is an increasing trend, which is driven by

2 environmental concerns and consumer awareness, for the use of natural materials in cosmetic formulations. The system consists of a combination of two naturally occurring polymers, xanthan gum and konjac mannan. The two polymers interact synergistically and will form fluid gels under the appropriate processing conditions. Xanthan gum and konjac mannan were used for obtaining of shampoo, body lotion, hair gel and sun lotion (18). The glucomannan synthesized from Sp. salmonicolor AL 1 had a distinct emulsifying power and it was experimented for the receiving of creams emulsion (7). Microbial exopolysaccharides are related to the secondary metabolites and their structure and properties depends on many different factors, which may include medium composition, type of carbon and energy source, microbe system used, and other fermentation conditions such as ph, temperature, and oxygen concentration. The amount of carbon substrate converted by the microbial cells to polymer depends on the composition of growth medium. Generaly, media containing a high carbon to limiting nutrient ratio are favored for polysaccharide production (9). In addition, the composition of the medium nutritious and cultivation condition, the obtaining of polysaccharides by contemporary biotechnology in industrial scale, it requires specification on every stage from technological scheme like location the condition of separation of the supernatant of biomass, determination of regime temperature of concentration, specifying the conditions of precipitation and staying at low temperature of precipitational polysaccharide. This article reports on the development of the laboratory scheme for obtaining of biopolymer from psychrophilic yeasts and effect of biotechnological and physicochemical factors influencing its biosynthesis. Materials and Methods Antarctic yeast The antarctic yeast were isolated from Antarctic soil and moss. The isolated colonies were reinoculated several times for purity, maintained on malt slant agar and stored at C. The Isolate 100 is part of collection of antarctic yeasts in the summer Exopolysaccharide (EPS) production The fermentation medium contained (g l -1 ): sucrose, 0; (NH ) SO,.; KH PO, 1.0; MgSO.7H O, 0.; NaCl, 0.1; CaCl.H O, 0.1; yeast extract, 1,0. Which carbon sourse are investigated and monosaccharide arabinose and mannose in concentration 1% for synthesis of EPS from Isolate 100. The initial ph was adjusted to ph.. The inoculum from psychrophilic isolates were obtained in 100 ml Erlenmeyer flasks containing 0 ml of Sabouraud medium (Merck, Germany), on a rotary shaker at 0 rpm at C for 8 h. The fermentation medium was inoculated with.0%,.0%, 6.0%, 8.0%, 10.0% w/v inoculum. The cultivation was performed at C for 10 h with shaking at 0 rpm. Cells were collected by centrifugation. The exopolysaccharides in the supernatant were precipitated with two volumes of cold absolute ethanol, held at C overnight and then centrifuged at 6000 g for 0 min, washed with ethanol and dried at 6 C and 10 C. The cultural liquid with exopolyssacharide was thermal manipulated at 0 C, 60 C, 70 C, 80 C for investigation of thermal stability of the biopolymer. Analyses The yield of exopolysaccharides and the dry biomass were determined by the weight method after drying to constant mass at 10 C. The viscosity was determined by Rheotest -, Prufgerate- Werk Medingen sitz Freital, Germany. Results and Discussion The amount of inoculum is one of the factors which determines the course of the biosynthetic process. The influence of the inoculum quantity of Isolate 100 at C was studied (Fig. ). The culture was obtained in a medium of Saburou for 8 hours. It was clarified beforehand that the most suitable for the biosynthetic process is % sucrose in the fermentation medium. Experiments were conducted with %, %, 6%, 8% and 10% culture. The best results were obtained with 8% and 10% inoculum and the amount of the obtained biopolymer varies from, g.l -1 to 6, g.l -1. With %, % and 6% inoculum the respective quantity obtained was,8 g.l -1,,7 g.l -1 and,8 g.l -1. Essential differences in the quantity of EPS with 8% and 10% inoculum were not observed. 8% and 10% inoculum could be used in subsequent fermentation processes. There is a proportional correlation between the amount of culture and the increase of EPS. In attitude to biomass the data shows that %, % and 6% inoculum were accumulated 1, g.l -1, 1,6 g.l -1 and,8 g.l -1 respectively. Higher cell growth from,6 g.l -1 to, g.l -1 respectively, was observed with 8% and 10% inoculum. 08

3 With obtaining of exopolysaccharide, the biomass was showed like waste product, which can be used as fodder for animals because it has high content of protein and other biological active compounds. Fatty acids, phospholipids, tocopherols and sterols can be extracted from biomass. The thermostability of the biopolymer in the liquid culture was examined according to the laboratory scheme for obtaining of exopolysaccharide (Fig. 1). ANTARCTIC YEAST STRAINS Inoculum (0,8%-1.0%), at C for 8 h, rotary shaker at 0 rpm Fermentation, at C,10h organic solvent during precipitation are reduced. Liquid culture with no cells in it was heated to 0 C, 60 C, 70 C and 80 C for 10, 0 and 0 minutes respectively.(fig. ) The heat treatment of liquid culture at 0 C showed that the amount of EPS increases with 8% for the time interval from 10 to 0 min. Similar result was observed during the heat treatment at 60 C and 70 C with the increase of the time interval, the amount of the polymer in the liquid culture increases from,6 g.l -1 to,0 g.l -1 and from,0 g.l -1 to,0 g.l -1 respectively. It is assumed that with the increase of temperature, the polymerization process was induced and an increase in the molecular weight of the polymer was observed. The thermostability of liquid culture at 80 C shows an inverse correlation to the other temperatures in the experiment - the amount of the biopolymer in the liquid culture decreases, most probably due to the depolymerization processes. Centrifugation 000 g, 0 min SUPERNATANT Concentration - times,at 60 C Precipitation with volumes 96% ethanol BIOMASS Lipid products EPS g.l -1 ; Biomass g.l EPS g.l -1 Biomass g.l -1 Contact of organic liquid and polymer(h, C) Centrifugation 000 g, 10 min Inoculum (%) Draying at 6 C Partial puryfication Fig.. The effect of inoculum from Isolate 100 on quantity of bioplymer (black) and cells grow (grey), at С Draying ( 6 C) EXOPOLYSACCHARIDE Fig. 1. Scheme for production of exopolysaccharide from Antarctic yeast strains (Isolate 100) It was made optimization for the stage of concentration from laboratorial scheme by it study of thermostability of the biopolymer in cultural liquid. An experiment in which the liquid culture was concentrated at different temperatures, at which the biopolymer to retained. The consumption of During the investigation of the monosaccharide composition of EPS, it was found that arabinose and mannose are basic monosaccharide components. 10 hour fermentations with 1% arabinose and mannose were carried out. It was established that, Isolate 100 metabolizes arabinose and mannose as carbon sources, and uses them for accumulation of biomass and biopolymer. The next stage in the laboratory scheme separation and precipitation follows the termination of the fermentation. Two volumes of ethanol were used for the precipitation of the liquid culture and the influence of arabinose and mannose on biosynthesis of EPS as carbon sourse was also studied. The dynamics of 09

4 obtaining of the EPS in ethanol at C was studied in the interval from to 10 hours (Fig. ). According to the data from the fermentation process of arabinose there is an increase in the amount of precipitating EPS from,8 g.l -1 analysed on the th hour of precipitation to,0 g.l -1 analysed on the 10 th hour. Similar experiment was done also with 1% mannose. This carbon sourse was found suitable for biosynthesis of exopolysaccharide and biomass. The results shown in Fig. represent the increase in the amount of accumulated biopolymer in ethanol from, g.l -1 on the th hour to, g.l -1 on the 10 th hour. EPS g.l o C 60 o C 70 o C 80 o C Time (min) Fig.. Thermostability of cultural liquid containing biopolymer, from Isolate 100, at 0 С (closed circle), at 60 С (open circle), at 70 С (closed triangle), at 80 С (open triangle) It is obvious that higher quantities of EPS are accumulated during the experiments with arabinose. The last stage in the laboratory scheme is drying the polymer, which is related to the residual moisture. It was found that temperature of drying at 10 C the EPS retains 11% of the moisture. This can be explained with the constitutive moisture in the polymeric molecule. Moreover, the influence of different factors on biosynthesis of exopolysaccharide, it was made characterization of cultural liquid with the biopolymer, trough study of dependence between concentration of sucrose, time of fermentation and production of exopolysaccharide. The influence of concentration of cultural liquid x 1 (%) and the time of biosyntes x (%) on the production y of biopolymer at temperature C. The effect of concentration of the cultural liquid x 1 (%) and the time for biosynthesis x (h) on the yield of the biopolymer (Y) at C was studied. The experimental data was manipulated by the least square method (), using a software for multiple regression of the type Y=f(x 1,x ). The following equation was achieved (1): (1) Y= -0,6 +,086x 1 -, x + 1, x 1.x, x 1-11, x We used the power law model τ= K. γ n for the prediction of the flow properties of biopolymer solutions. This equation for non- Newtonian flow behoviour is known as the Oswaldde Waale where n is the power law exponent, K is the apparent viscosity or consistency index (the dimension of K is Pa.s -n ). A Newtonian fluid would of course have an n value of 1,0 and K would equal its viscosity. For pseudoplastic fluid n will be between 0 and 1,0 while for dilatant liquids the value will be greater than 1. In our investigation of solutions of the biopolymer 0,1 n 0,8 and 0,00 K 0,099 what shows that its solutions have behavior of pseudoplastic fluids (). Arabinose g.l -1 ; Mannose g.l Arabinose g.l -1 Mannose g.l Time (h) Fig.. Time course of quantity biopolymer synthesized from Isolate 100 on arabinose (closed circle) and mannose (open circle), after precipitation and staying at С Conclusions From investigation factors were obtained results, which may have application for optimization of technological process at production of exopolysaccharide from Antarctic yeast. 10

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