Isolation and Characterization of Exopolysaccharides FromYeast Isolates

 

Priyanka Prakash Kharat, Sarita RamsaranYadav, Mangala Lakshmi Ragavan, Nilanjana Das*

Bioremediation Laboratory, School of Bio Sciences and Technology, VIT University,

Vellore-632014, Tamil Nadu, India.

 

ABSTRACT:

Exopolysaccharides (EPS) are high molecular weight polymers which are long chain composed of sugar residues and secreted by microbes into the surrounding environment. Sugar compositions of exopolysaccharides are dependent to culture medium composition and different physical conditions.Bacteria and fungi are most common EPS producers . In this study, we aimed to isolate the yeasts from different sources for maximum EPS production using optimized conditions. Yeast isolates PS1 and PS2wereanalyzed for maximum EPS production on modified EPS media and optimized physical conditions such as carbon source, temperature and incubation duration. The maximum EPS (0.2 g/L) production was noted in PS1 and PS2.Characterization of purified EPS was done by HPLC, FT-IR and viscometer. PS1 (0.33mg/ml) and PS2 (0.28 mg/ml) showed increased carbohydrate content andsimilarly protein content also quantified for PS1(0.29µg/ml) and PS2 (0.180µg/ml) which indicates glycoprotein quantity on EPS. PS1showed 3% viscosity than PS2. We demonstrate the biological activities of purified EPS viz. Surfactant activity, flocculant activity and antioxidant activities.All these activities are observed in PS1; incaseof PS2 it is not observed. In conclusion, yeast (PS1) exopolysaccharideshas a potential role in the textile, pharmaceutical and cosmetics industries.

 

 

INTRODUCTION:

Exopolysaccharides arelong-chain polysaccharides consisting of branched sugar derivatives andnon-carbohydrate components¹.Among the microbial EPS producers, bacteria and fungi are most common. Bacterial EPSs have been studied extensively by researchers². EPS produc­tion from fungi has been studied adequately over the last two decades. Different EPS production by fungi including Ganodermalucidum, Agaricusblazi, Cordycepssp., Lentinusedodes, and Grifolafron­dosathrough submerged cultures had been reported, all of which have different and interesting biological activities³.

 

Exopolysaccharide has rapidly emerged as anew and industrially important source of polymericmaterials, which are gradually becoming economically competitive⁴. Microorganisms belonging to Bacillus sp. have been known to produce extra cellular polysaccharides such as cellulose. Microbial EPSs have many applications in the food and pharmaceutical industries⁵. In this work we aimed to isolate yeast from different sources for maximum EPS production under optimized conditions. To evaluate their biological activities for commercial purposes.

 

MATERIALS AND METHODS:

Sample collection and isolation of yeast:

Yeast was isolated from different sources and yeast isolate PS1 and PS2 were maintained on YEPD media (Glucose-10gm, Yeast extract 0.08gm, Peptone 0.12gm, MgSO₄.7H2O, -0.1gm, KH₂PO₄-1gm, Distilled water- 200ml, NaCl-0.2gm)⁶.

 

 

EPS production:

The cultures were streaked onto YEPD agar plates and incubated for 24 hrs at 37°C and the strains, which produced slimy colonies, were recorded as capable of producing EPS. The selected strains were inoculated to 100 ml of EPS producing media comprised sucrose (50 g/l), yeast extract (0.4 g/l), peptone (0.6 g/l), K₂HPO₄ (5 g/l), MgSO₄.7H2O (0.5 g/l), and NaCl (1 g/l). The culture was then incubated for 15 days with continuous agitation of 150 rpm at room temperature. The culture was then centrifuged at 10,000 rpm for 20 minutes at 4°C and supernatant was transferred to fresh tube, and twice the volume of ice-cold n-propanol was added, and EPS was allowed to precipitate overnight. It was centrifuged at 12,000 rpm for 30 minutes. The pellet was precipitated with isopropanol for decolourization. Then, the pellet was dissolved in 1 ml of sterile distilled water. A standard graph was plotted using different concentrations of glucose from stock solution (1 mg/ml)⁷.

 

SEM analysis:

Scanning electron microscopy (SEM) was done to study the surface morphology of EPS producing yeast isolates. 1.5ml of late log phase cultures were centrifuged at 10,000rpm for 10min. The pellet obtained was washed twice with phosphate buffered saline and fixed in 2.5% glutaraldehyde in double distilled water (DDW) for 2h. Fixed cells were washed twice with DDW and were subsequently dehydrated with series of ethanol from 25% to 100% for 5min each and left to dry overnight in a desiccator. The specimens were mounted onto cover slip and stained by alcian blue for 1 min then washed with DW for twice. After drying slides are examined under SEM analysis⁸.

 

Optimization studies:

The exopolysaccharide production was optimized using three parameters i.e. time, carbon source with varying concentration and temperature. Glucose, Fructose, Sucrose served as a carbon sources at different concentrations (2%, 6%, 8%).The growth of yeast isolates was observed at different temperatures (25˚C, 30˚C, 38˚C). Isolated yeast strains were inoculated in basal media with minor modifications (Glucose-10gm, Yeast extract 0.08gm, Peptone 0.12gm, MgSO₄.7H2O,-0.1gm, KH₂PO₄-1gm, Distilledwater- 200ml, NaCl-0.2gm) and incubatedat 30˚C over a period of for 30 days⁹.

 

EPS extraction and purification:

For evaluation for EPS production, the selected samples PS1 and PS2 were collected from 15^th and 30^th-day incubation. The EPS was obtained by the addition of equal volume of Ethanol and n-propanol. The mixture was vortexed for 5 min. The precipitate left overnight at R.T, then thesupernatant was centrifuged for 20 min at 4000 rpm and precipitation was gathered in Petri-plates and dried in alyophilizer at 80˚C⁹

 

Chemical analysis:

Estimation of carbohydrates in EPS:

To test the presence of simple sugars Oligosaccharides and Polysaccharides, Estimation of carbohydrates was done by DuBois method (DuBois et al., 1956) 1ml of PS1 and PS2 samples and 1ml of phenol solution were added in the 5ml of concentrated sulphuric acid. Glucose was used as the standard in the range of 20-100µg.The solution was incubated for 30 min at R.T and the O.D was read at 490nm¹⁰.

 

Estimation of protein in EPS:

Proteins are the major components which may be found covalently bounded to a polysaccharide material. To test the presence and quantity of protein content in the EPS extracted from Saccharomyces cerevisiae, the ultraviolet absorption technique adopted by Warburg and Christian (1941) was used¹⁰.

 

Infra Red Spectroscopy Analysis of Basal EPS:

The molecular structure and group composition of the polymer was investigated using Perkin Elmer infra red spectrophotometer. The analysis enables a partial identification of the polymers by comparing with known standard polysaccharides. The method, practically, involves making of small salt discs mixture composed of 1 mg of the tested material and 300 mg of pure dry KBr, which then pressed mechanically into a disk. The disk was then measured at an infra red spectra range from 500 to 4000 nm as described by Lloyd and Dodgson; Robyte and White¹⁰.

 

HPLC:

Separation and identification of sugars from the EPS sample done by HPLC analysis,which was equippedwith Aqueous GPC start up the kitsegment and eluted with refined water at a stream rate of 1000µl/min at 20˚C¹⁰. The separated component of samples was monitored by a refractive record indicator. Before this Exopolysaccharides were hydrolyzed and dissolvedin CH₃OH. The columnwas adjusted with various molecularmass standard and a standard bend was set up.

 

Antioxidant assay:

Totalantioxidant capacity of yeast autolysates was assayed by the phosphor molybdenum method as described by Kumaran and Karunakaran as follows: Known volumes (0.1-0.3 ml) of each yeast autolysate were added to test tube then completed to a constant volume (0.3 ml) with DW. 3.0 ml of reagent solution (0.6 Msulphuric acid, 28.0 mM sodium phosphate and 4.0 mM ammonium molybdate) were added to each tube and mixed well then incubated at 95°C for 90 min. Blank was prepared by the same procedure without yeast autolysate. Ascorbic acid solution (0.03%, w/v) was used as positive control. After cooling to room, the absorbance of the solution was measured at 695 nm. Increased absorbance of the reaction mixture indicated increased total antioxidant capacity¹¹.

Equation

            A_control - A_sample

I% =------------------------- X 100

                    A_sample

 

DPPH activity:

The antioxidant activity of yeast autolysates, based on the scavenging activity of the stable DPPH free radical, was determined by the method described by Lee et al. [33] as follows: Known volumes (50-100 µl) of yeast autolysate were individually added to test tubes then completed to a known volume (1.0 ml) by DW. 1.0 ml of DPPH solution (0.2 mM in ethanol) was added to each tube then mixed well and incubated at room temperature for 30 min. Control was prepared by the same procedure without yeast autolysate. Ascorbic acid solution (0.03%, w/v) was used as a positive control. The absorbance (A) of the solution was measured at 517 nm. Inhibition of DPPH free radical in percent (I%) was calculated from the following equation¹¹:

                                  Control OD - Sample OD

Radical Scavenging =------------------------------ X 100

Activity %                        Control OD

                    

VISCOSITY:

To study the relation between the EPS concentrations and the viscosity of the samples,the viscosity behavior of EPS solutions extracted from the yeast isolates wereanalyzedat constant temperature 25˚C and constant shear rate 122.3 1/sec using Oswald’s viscometer. The Concentration of EPS solutions ranges from 1 to 3% ¹².

 

Biofloculant activity-:

The flocculating activity was measured according to the method of Kurane et al using a suspension of kaolin clay as test material with minor modifications. Kaolin was suspended in distilled water at a concentration of 5000 mg l−1 (kaolin suspension). In a test tube, 4.5 ml of kaolin suspension was added and mixed with 0.25 ml of CaCl2 solution (90 mM). To this mixture, 100µl of 10 mg/L concentration of the EPS ofthe test bioflocculant was added and vortexed for 30s and left for 5 min at room temperature. The absorbance of the upper phase at 550 nm (A) was measured with a spectrophotometer¹². A control experiment in which 100µl of distilled water, instead of the bioflocculant, added to the suspension was performed in the same manner, and the absorbance was measured (B). Flocculating activity (%) was defined and calculated as

                                B - A

Flocculation  =--------------------- X 100

Activity %               B

 

Biosurfactant activity:

Extracted EPS were analysed for surfactant activity by used oil spread method. 24 h old inoculum grown in YEPD was used Petriplate base was filled with 50ml of distilled water .On this water, twenty microliter of crude oil was layered uniformly. Further, ten microliter of culture was added on different spots on the crude oil which is coated on water surface. Occurance of clear zone was an indication of a biosurfactant producer¹³.

 

RESULTS AND DISCUSSION:

Two yeast strains were isolated from different sources such as milk and fruit. Isolated yeast was morphologically identified by light microscope followed by simple staining (Fig.1). Initially, yeast isolates PS1 and PS2 were screened for EPS production on basal media. Exopolysaccharide production was observed by SEM analysis using alcian blue staining method (Fig.2).

 

Fig.1: Identification of yeast by light microscope

 

 

Fig.2: SEM analysis for EPS production

 

Optimization studies:

EPS production is directly proportional to the cell growth and cell density. Production of EPS is related to the secondary metabolism of yeast, and their structure and physical properties depend on many factors, which include the composition of the basal medium, temperature and oxygen concentration¹⁴. Basal media with 6 % glucose showed maximum growth of yeast isolates (Fig 3a). Both isolates showed increased growth at 30˚C (optimum temperature) over a period of 15 d, prolonged incubation showed the maximum EPS production (Fig 3c).

 

Fig.3: Optimization studies 3a) Carbon source 3b) Temperature and 3c) Incubation period

 

Extraction and purification of EPS:

PS1 and PS2 culture was centrifuged for 20 min at 4000 rpm. The supernatantwas transferred into afresh tube and yeast lysate was discarded. EPS were precipitated by addition of 1:2 ratio of ethanol and n-propanol respectively. PS1, PS 2 showed 0.2 mg and 0.12 mg of dry weight respectively using n-propanol precipitation (Fig 4b). Incase of ethanol precipitation 0.4 mg was decreased on both the isolates (Fig 4a). EPS precipitate was further filtered by dialysis membrane for 2-3 days at 4˚C. The resulting precipitate was recovered by lyophilisation at 83˚C for 7 h. Extracted EPS were stored at -20˚C.

Biochemical characterization:

Purified EPS showed 0.33mg/ml of carbohydrate inPS1and 0.28 mg/ml of carbohydrates in PS2.The quantity of Carbohydrates in PS1 was higher than the PS2 by 0.5% (Fig 5a). The higher density of protein estimation was 0.29µg/ml for PS1 and 0.180µg/ml forPS2 (Fig 5b). The density of PS2 was higher than the PS1 by 110 %. Viscosity analysis was carried out at different concentrations of 1%, 2% and 3% for extracted EPS at constant shear rate 122.1 /sec. PS1 showed more viscosity at 3 % than PS2 (Fig5c).

 

 

Fig.4: Precipitation of EPS a) by Ethanol b) by n-propanol

 

 

Fig. 5: Biochemical characterization of EPS a) Protein estimation b) Carbohydrate estimation and c) Viscosity analysis

Instrumental analysis:

The FTIR spectrum of EPS exhibited many peaks and the absorption at 3331.07, 2788.20, 1635.64 and 1507.11 cm^-1 is assigned to –OH stretching, aldehyde stretching, CO stretching and NH secondary amides groups, respectively (Fig.6). However, while absorption at 1635.64cm^-1 is due to stretching vibration of the carboxylate group.

 

 

Fig.6: FT-IR analysis of EPS extracted from a) PS1 and b) PS2

 

The sugar content of extracted EPS was analysed by high-performance liquid chromatography (HPLC) system.PS 1 showed more sugar content such as glucose, fructose, sucrose, maltose, and lactose. Incase of PS2 glucose and fructose only observed (Fig.7).

 

 

Biological activities of yeast isolates

The total antioxidant activity analysed by calorimetrically at 695nm and results showed PS1 has more efficiency than PS 2 (Fig.8a). Scavenging activity was analysed by DPPH (Fig.8b) and the result showed PS1 has 95 % activity and PS2 showed 90%.

 

Fig.7: HPLC analysis of EPS extracted from PS1and PS2

 

Fig. 8: Antioxidant activityof PS1 and PS2 a) Total antioxidant activity and b) DPPH scavenging activity

 

The bio-flocculating activity of the EPS (PS1and PS2) was measured by using a Kaolin solution.250 µl of 90mM CaCl₂ was added in 20 ml of 0.1 g/l kaolin solution. Optical density was measured by spectrophotometer at 550 nm. Results showed PS1 has increased absorbance 0.08 and PS2 showed decreased activity 0.02 (Fig.9a).Mustard oil was utilized by PS1 isolates and showed clear zone formation this indicates biosurfactant activity (Fig.9b).

 

 

Fig. 9: Applications of EPS a) Biosurfactant activity and b) Bioflocculant activity

 

CONCLUSION:

Two yeasts strains were isolated from different sources namely PS1 and PS2. In this study we aimed to produce maximum EPS production from newly isolated yeast under optimized conditions. PS1 showed maximum EPS production (2 g/L) under favourable conditions. Biochemical analysis showed increased carbohydrate content of PS1(0.33mg/ml) and PS2 (0.28 mg/ml) and adequate amount of protein content of PS1(0.29µg/ml) and PS2(0.180µg/ml) PS1 has highest viscosity at 3 % of EPS than other concentrations. Yeast Exopolysaccharides has significant biological activities viz. biosurfactant and bioflocculant activity, which is only noted in PS1. Increased antioxidant activity and free radical scavenging activity (95 %) was observed in PS1 isolate. This study suggests that, exopolysaccharides produced from yeast isolates has high thermal ability and insoluble in non-polar solvents.Yeast EPS shows high flocculation activity up to 85 %. In conclusion PS1 exopolysaccharides can be used in various field of cosmetics, pharmaceuticals and food industries.

 

ACKNOWLEDGEMENTS:

We thank VIT University, Vellore for providing financial assistance and laboratory facilities.

 

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Accepted on 05.08.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(2):537-542.