Optimal conditions for exopolysaccharide production by lactobacillus plantarum t10 - Tran Bao Khanh

Journal of Science and Technology 54 (4A) (2016) 40-47 OPTIMAL CONDITIONS FOR EXOPOLYSACCHARIDE PRODUCTION BY Lactobacillus plantarum T10 Tran Bao Khanh 1, 2 , Do Thi Bich Thuy 2 , Doan Thi Thanh Thao 2 1 Faculty of Chemistry, Hue University of Science, 77 Nguyen Hue street, Hue city 2 Faculty of Engineering and Food Technology, Hue University of Agriculture and Forestry, 102 Phung Hung street, Hue city * Email: tranbaokhanh@huaf.edu.vn Received: 15 August 2016, Accepted for publication: 5 October 2016 ABSTRACT Exopolysaccharide (EPS) production ability of Lactobacillus plantarumT10 was studied. The supplement of some sugars (lactose, saccharose, and glucose) gave the positive effects on EPS production of L. plantarum T10, in which the addition of lactose 4 % resulted in the most efficiency for EPS yield (274.83 µg/mL). The addition of 0.4 % of yeast extract into culture medium with 4 % lactose provided the highest EPS yields compared to other nitrogen sources (peptone, beef extract), which were 378.32 mg/mL. The optimal conditions for EPS production of L. plantarum T10 in MRS broth with 4 % of lactose and 0.4 % yeast extract supplement were also studied. The results indicated that the highest EPS yield (417.11 mg/L) was obtained in the conditions of 10 6 CFU/ml initial cell density, temperature of 35 o C, pH 5.5 and 48 h incubation. Keywords: exopolysaccharide, cultivation condition, Lactobacillus plantarum, sugar. 1. INTRODUCTION Exopolysaccharide (EPS) are the biopolymer which is widely used in agriculture, medicine and food industry. In food industry, EPSs are used as food additives such as gelling agents, viscosifiers, stabilizers, emulsifiers, and water binders to improve food texture. In addition, there has been an increasing interest in exploiting the EPS-producing lactic acid bacteria for their possible biological activities including antitumor, immunostimulatory, cholesterol-lowering and antioxidant activities. Therefore, EPSs from lactic acid bacteria have potential for development as food additives or functional food ingredients with both health and economic benefit [1]. EPSs produced by lactic acid bacteria are influenced by many factors, e.g. medium components, pH, temperature, and fermentation time course. Many studies showed that the best carbon sources for EPS production of lactic acid bacteria are lactose, glucose, and saccharose. Furthermore, nitrogen sources also an important medium component determining the EPS yield [2, 3, 4, 5, 6, 7]. The present work was carried out to understand the effect of different cultural conditions on EPS production of L.plantarum T10. Results obtained are the basis for further researches on Optimal conditions for exopolysaccharide production by Lactobacillus plantarum T10 41 extraction and purification of EPS, and on determination of EPS structure produced by L. plantarumT10. 2. MATERIALS AND METHODS 2.1. Materials L.plantarumT10 was isolated from Hue traditional fermented foods (unpublished). 2.2. Methods 2.2.1. Isolation of EPS Batch cultures of L. plantarumT10 were performed using 100 ml flask of MRS medium with different studied fermentation conditions.EPS was then isolated from the fermented sample according to Yang et al. (1999) with some modifications. Briefly, the samples were heated at 100 °C for 15 min to inactivate enzymes which are capable of polymer degradation. Then, they were cooled down to room temperature. Cool samples were then added trichloro-acetic acid with a final concentration of 30 % (w/v). The precipitated proteins and bacterial cells were removed after centrifugation (13000 ×g for 10 min at 4 °C). The supernatants containing EPS were mixed with a double volume of ethanol and then stored at 4 °C for 24 h. The precipitated EPS was collected by centrifugation (10000 ×g for 10 min at 4 °C) (repeat two times), and then dissolved in distilled water for further analysis [3, 8]. 2.2.2. Determination of EPS content The amount of EPS (μg/ml) was estimated by the phenol-sulfuric acid method. In this method, the concentrated sulfuric acid breaks down EPS (polysaccharides/oligosaccharides) to monosaccharides. Pentoses are then dehydrated to furfural, and hexoses to hydroxymethyl furfural. These compounds then react with phenol to produce a yellow-gold color. The absorption of the reaction solution is measured at 490 nm. The EPS content is determined based on a glucose standard curve [9]. 2.2.3. Effect of carbon sources (sucrose, lactose, or glucose) and nitrogen sources (peptone, beef extract, or yeast extract) on EPS production of L. plantarum T10 - Effect of carbon sources on EPS production The separated supplement of 3 sugars (sucrose, lactose, glucose) with different concentrations into MRS broth was used to investigate the effect of carbon sources on EPS production of L. Plantarum T10 (Table 1). - Effect of nitrogen sources on EPS production Three different components including peptone, beef extract and yeast extract were separately supplemented into MRS broth to determine the effect of nitrogen sources on EPS production of L. plantarum T10 (Table 2). 2.2.4. Effect of cultivation conditions for the EPS production Tran BaoKhanh, Do Thi Bich Thuy, Doan Thi Thanh Thao 42 The influence of different factors (initial cell density, initial pH of medium, incubation temperature, and time course) on the EPS production of L. plantarumT10 was studied. The strain was incubated at various initial cell density (10 4 , 10 5 , 10 6 , 10 7 ,10 8 cfu/mL), pH values (4; 4.5; 5; 5.5; 6; 6.5), temperature (30 o C, 35 o C, 40 o C, 45 o C) and incubation time (12 h, 24 h, 36 h, 48 h, 60 h, 72 h) in MRS broth.After incubation, the samples were taken for EPS determination. 2.3. Statistical analysis Each test was carried out in triplicate. The data was analyzed by SPSS 22.0 software. Duncan’s multiple range test was used to determine the significant difference (p < 0.05) among the samples. 3. RESULTS AND DISCUSSION 3.1. Effect of carbon source on EPS production of L.plantarumT10 The increase in sugar concentrations resulted in the increase in the EPS yields for all of three carbon sources (glucose, lactose, and sucrose) (Table 1). However, the EPS yields started to decrease at a certain sugar supplement concentration, which was 6 %, 5 % and 5 % for glucose, lactose and sucrose, respectively. Furthermore, different types of sugars had different impact on the EPS production of the strain. The highest EPS level was obtained when 4 % lactose was supplemented (274.83 mg/L), following by 5 % glucose supplement (251.01 mg/L) and 4 % sucrose supplement (236.74 mg/L). Table1. Effect of carbon source on EPS production of L. plantarum T10 (mg/L). Supplement conc. (%) Carbon sources 0 2 3 4 5 6 Glucose 140.44 e 167.47 d 195.76 c 222,51 b 251.01a 224.22 b Lactose 140.44 e 177.15 d 206.70 c 274.83 a 243.24 b 231.01 b Sucrose 140.44 d 176.58 c 203.89 b 236.74 a 174.67 c 112.23 e Our results were in agreement with other studies. According to the study of Yuksekdag and Aslim [8], glucose is the most effective carbon source for the EPS production of L. fertility. delbrueckii subsp. bulgaricus (B3, G12) and Str. thermophilus (W22).In their study, the highest EPS yield was obtained when the glucose concentration was 30 g/L. L. fermentum TDS030603 cultivated with the supplement of glucose, lactose or fructose was able to produce EPS higher than with the supplement of saccharose. In the study of Pham et al (06/2000), lactose stimulated L. rhamnosus R to produce EPS better than glucose did [7]. Emanuel Vamanu et al. [10] also studied the effect of three types of sugars (glucose, lactose, and saccharose) on the EPS production of L. paracasei IL2 and L. plantarum IL3; the results indicated that saccharose was the most appropriate carbon source for L. paracasei IL2 to produce EPS. Both saccharose and glucose had similar impact on EPS production of L. plantarum IL3. Li Zhang et al. [1] found Optimal conditions for exopolysaccharide production by Lactobacillus plantarum T10 43 that L. plantarum C88 was able to produce 69 mg/L EPS in MRS medium supplemented with 2 % glucose. In other study, lactose was chosen to investigate the EPS production of L. plantarum MTCC 9510 [11]. In general, lactose and glucose provided the most effective effect on the EPS production of L. plantarum T10. Hence, we chose the lactose supplement of 4 % to prepare the medium for further experiments. 3.1 Effect of nitrogen sources on EPS production of L.plantarum T10 Table2.Effect of nitrogensources on EPS production of L. plantarum T10. Peptone Beef extract Yeast extract Supplement concentrations (%) Yield of EPS (mg/L) Supplement concentrations (%) Yield of EPS (mg/L) Supplement concentrations (%) Yield of EPS (mg/L) 0.2 184.06 b 0.2 238.32 c 0.1 272.96 c 0.4 172.23 bc 0.4 275.03 b 0.2 315.40 b 0.6 169.54 bc 0.6 286.50 b 0.3 318.45 b 0.8 164.79 bc 0.8 310.28 a 0.4 378.32 a 1.0 156.01 c 1.0 318.08 a 0.5 365.52 a Similar to the effect of carbon sources, the addition of each type of nitrogen source (peptone, beef extract, and yeast extract) also stimulated the EPS production ofL.plantarum T10 to a certain level. From the results, yeast extract was the best nitrogen source for the EPS production in comparison to the others. The highest EPS yield was obtained when 0.4 % of yeast extract was supplemented (378.32 mg/L), following by beef extract and peptone. In fact, no polymer in yeast extract culture without strain was detected by phenol-sulfuric acid method in our study. Therefore, EPS amount found in yeast extract culture mainly come from the EPS production activity of L. plantarum T10.These nitrogen sources (peptone, beef extract, and yeast extract) were also found to enhance the EPS production of L. confusus TISTR 1498 [4]. Yeast extract was the appropriate nitrogen source for EPS production of L. plantarum 70810 [12] and of L. plantarum [13]. The supplement of different nitrogen sources may provide the positive or negative effect on the EPS production of bacteria. The effect may depend on the strain. In this study, all three types of nitrogen source gave positive impact on the EPS yield of L. plantarumT10. Yeast extract with the supplement of 0.4 % provided the highest EPS yield of 378.32 mg/L. Thus, this concentration of yeast extract supplement (0.4 %) was chosen to prepare the medium for further study. 3.2. Effect of initial cell density on EPS production of L. plantarum T10 EPS yields varied depending on the initial cell density (Fig. 1). They increased from 240.68 to 391.29 mg/L when the initial cell was inoculated from 10 4 to 10 6 CFU/mL, while they decreased from 391.29 to 256.25 mg/L when the initial cell density was from 10 6 to 10 8 CFU/mL. It could be seen that the more initial cell density inoculated the more EPS was produced. However, when the initial cell density increased to a certain level, the EPS production was gradually inhibited because of the competition in the cell development. Tran BaoKhanh, Do Thi Bich Thuy, Doan Thi Thanh Thao 44 In the study of Ismail and Nampoothiri [11], the initial cell density of 10 9 CFU/mL was the best condition for L.plantarum MTCC 9510 to synthesize EPS. In our study, the appropriate initial cell density was at 10 6 CFU/mL. Figure 1.Effect of the initial cell density on EPS production of L. plantarum T10. 3.3. Effect of pH on EPS production of L.plantarum T10 Figure 2.Effect of initial pH on EPS production of L. plantarum T10. pH had a great impact on the EPS producing ability of L.plantarum T10. More and more EPS was produced when the pH of medium increased from 4 to 5.5. The EPS yield reached the highest level at pH 5.5 (397.72 mg/L). However, the amount of EPS trended to reduce when the pH increased above 5.5 (Fig. 2). The influence of pH medium on EPS production of lactic acid bacteria was also examined in some studies. L. delbrueckiissp. Bulgaricus obtained the highest EPS yield at the pH value of 6.5 [8], while the optimal pH for L. casei CRL 87 was at 6.0 [14]. L. fermentum F6 produced the best EPS yield at pH 6.5 [15]. In this study, pH medium of 5.5 was chosen for further experiment. 3.2 Effect of incubation temperature on EPS production of L. plantarum T10 The EPS yields produced by L. plantarum T10 at different temperatures were showed in Figure 3. Obviously, the optimal temperature for EPS production was at 35 o C (410.44 mg/L). At higher temperatures, the EPS yields were reduced. 0 100 200 300 400 4 5 6 7 8 240.69e 354.96b 391.30a 323.41c 256.26d E P S ( m g /L ) Initial cell density (log10 cfu/mL ) 0 100 200 300 400 4 4.5 5 5.5 6 6.5 104.22f 141.94e 289.87d 397.72a 388.45b 309.34c E P S ( m g /L ) Initial pH Optimal conditions for exopolysaccharide production by Lactobacillus plantarum T10 45 Figure 3.Effect of incubation temperature on EPS production of L. plantarum T10. Each strain has its own optimal temperature for cell development and EPS production. S. thermophiles BN1 synthesized EPS highest at temperature of 37 °C [16]. However, in the study of L. De Vuysta et al. [17], the optimal temperature for EPS production of S. thermophiles was at 42 °C. 3.3 Effect of incubation time on EPS production of L.plantarumT10 The incubation time also determined the amount of EPS produced by L. plantarum T10 (Figure 4). Particularly, L. plantarum T10 produced more and more EPS when the incubation time increased gradually from 12 h to 48 h. The highest EPS yield was reached at 48 h (417.11 mg/L). After 48 h incubation, the amount of EPS started to decrease. Our results were in agreement with the study of Fukuda et al. [6]. In the MRS medium supplemented with 1 % glucose, galactose, or sucrose, the amount of EPS synthesized by L. fermentum TDS030603 was highest after 48 h incubation. Similarly, in their study, the EPS level started to fall down after 72 h. It could be explained that there were polymer-degrading enzymes appeared at the end stages of fermentation. These enzymes may reduce the amount of EPS produced by lactic acid bacteria. Figure 4.Effect of incubation time on EPS production of L. plantarum T10. 4. CONCLUSIONS Optimal condition for EPS production by L. plantarum T10 has been found in this study. The highest EPS yield (417.11 mg/L) was obtained in the medium with the supplement of 4% lactose, 0.4 % yeast extract and the initial cell density of 10 6 CFU/ml, temperature of 35 o C, pH 5.5, and 48h incubation time course. 0 200 400 600 30 35 40 45 355.72b 410.44a 350.15b 318.85c E P S ( m g /L ) Temperature (oC) 326,25 372,51 382,80 417.11 386,37 380,03 0 50 100 150 200 250 300 350 400 450 12 24 36 48 60 72 E P S ( m g /L ) Time (h) Tran BaoKhanh, Do Thi Bich Thuy, Doan Thi Thanh Thao 46 Acknowledgements.The authors gratefully acknowledge to VLIR Network Vietnam for financial support. REFERENCES 1. 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TÓM TẮT ĐIỀU KIỆN TỐI ƯU CHO SỰ TỔNG HỢP EXOPOLYSACCHARIDE BỞI CHỦNG LACTOBACILLUS PLANTARUM T10 Trần Bảo Khánh1, 2, *, Đỗ Thị Bích Thủy1, Đoàn Thị Thanh Thảo1 1Khoa Hóa, Trường Đại học Khoa học Huế, 77 Nguyễn Huệ, Huế 2Khoa Cơ khí công nghệ, Trường Đại học Nông Lâm Huế, 102 Phùng Hưng, Huế * Email: tranbaokhanh@huaf.edu.vn Khả năng sinh tổng hợp exopolysaccharide bởi chủng Lactobacillus plantarumT10 đã được nghiên cứu. Việc bổ sung một số loại đường đơn vào môi trường nuôi cấy đã có tác dụng tích cực đến quá trình sinh tổng hợp EPS bởi chủng L. plantarumT10, theo đó khi bổ sung 4 % lactose thì cho hàm lượng EPS cao hơn so với các loại đường còn lại (274,83 mg/L). Khi bổ sung 0,4 % dịch chiết nấm men vào môi trường nuôi cấy có 4 % lactose thì lượng EPS thu được cao nhất so với các nguồn nitơ bổ sung khác (gồm peptone và dịch chiết thịt) với hàm lượng là 378,32 mg/L. Điều kiện nuôi cấy thích hợp cho chủng L. plantarum T10 sinh tổng hợp EPS trong môi trường MRS có bổ sung 4 % lactose và 0,4 % dịch chiết nấm men cũng đã được khảo sát. Kết quả cho thấy lượng EPS thu được cao nhất là 417,11 mg/L khi nuôi cấy với mật độ tế bào ban đầu 106 CFU/ml ở 35 oC, pH 5,5 thời gian nuôi cấy là 48 giờ. Từkhóa: exopolysaccharide, điều kiện nuôi cấy, đường, Lactobacillus plantarum.