Bioresource Technology 100 (2009) 13661370

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Optimization of culture medium for the production of phenyllactic acid by Lactobacillus sp. SK007
Wanmeng Mu *,1, Chao Chen 1, Xingfeng Li, Tao Zhang, Bo Jiang
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China

a r t i c l e

i n f o

a b s t r a c t
Optimization of medium components for the production of PLA by the strain Lactobacillus sp. SK007, which was isolated from Chinese traditional pickles, was carried out using response surface methodology (RSM) based on a uniform design and a central composite rotatable design (CCRD). The nal concentration of the optimized medium was 30 g L1 glucose, 5 g L1 phenylpyruvic acid (PPA), 47 g L1 corn steep liquor, 3 g L1 K2HPO4, 3 g L1 CH3COONa, 30 g L1 yeast powder and 3 mL L1 Tween-80. A maximum PLA yield of 2.30 g L1 was achieved, which was signicantly higher than previously reported. 2008 Elsevier Ltd. All rights reserved.

Article history: Received 12 June 2008 Received in revised form 7 August 2008 Accepted 7 August 2008 Available online 14 September 2008 Keywords: Phenyllactic acid Response surface methodology Optimization Lactobacillus sp. SK007

1. Introduction Phenyllactic acid (PLA) is a novel antimicrobial compound, which was rst found in Geotrichum candidum and shown to inhibit the growth of Listeria monocytogens (Dieuleveux et al., 1998a). It is active against Gram-positive, Gram-negative bacteria and fungi (Dal Bello et al., 2007; Dieuleveux and Gueguen, 1998; Dieuleveux et al., 1998b; Lavermicocca et al., 2003; Schnurer and Magnusson, 2005; Yang and Clausen, 2005). In addition to its use as an antimicrobial agent in foods (Dal Bello et al., 2007; Lavermicocca et al., 2003, 2000), PLA has potential as a pharmaceutical agent since its analogue Danshensu from Chinese medicine is applied presently. Approaches to production of PLA that have been reported can be categorized mainly into chemical and biosynthetic. Chemical approaches include clemmemsen reduction of zinc amalgam and hydrochloric acid, and clemmensen reduction of azlactone (Bubl and Butts, 1951). PLA has also been obtained by catalytic hydrogenation in presence of Raney-Ni catalyst or PdC alloy (Morita and Mori, 1996) catalyst. A high yield and conversion were obtained using chemical method however this was at the expense of the environment and extreme reaction conditions. Contrary to chemical synthesis, biosynthesis was shown to be an environmentally friendly method, and the bioproduct was readily accepted by consumers (Branen et al., 2002). PLA biosynthesis utilizing various microorganisms has been reported. Kamata stated in a patent application in which mutants of Brevibacterium lactofermentum
* Corresponding author. Tel.: +86 510 85327859. E-mail address: wmmu@jiangnan.edu.cn (W. Mu). 1 These authors had equal contributions to this work. 0960-8524/$ - see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.08.010

(Kamata et al., 1986) produced PLA (1.94 g L1). Geotrichum candidum grown in TSBYE (trypticase soy broth with yeast extract) was found to produce about 0.6 to 1 g L1 PLA (Dieuleveux et al., 1998 a). Many lactic acid bacteria (LAB) have been used to produce PLA such as Lactobacillus plantarum (Lavermicocca et al., 2000; Strom et al., 2002; Valerio et al., 2004; Strom et al., 2005) and Lactobacillus coryniformis (Magnusson et al., 2003). In these studies, the maximum PLA production using LAB grown in MRS broth was 99 mg L1 (Valerio et al., 2004). This study focuses on improving the ability of LAB to produce PLA, given LAB has GRAS (generally recognized as safe) status. The optimal design of the culture medium is a very important aspect in the eld of food microbiology and fermentation (Kim et al., 2002, 2005). Statistical experimental design techniques are very useful tools for the screening of nutrient, as they can provide statistical models which help understand the interactions among the process parameters at varying levels and calculation of the optimal level of each parameter for a given target. The application of statistical experimental design techniques in fermentation process development can improve product yield, reduce process variability, as well as reduce development time and overall costs (Ren et al., 2008; Kammoun et al., 2008; Yu et al., 2008; Pan et al., 2008). Response surface methodology (RSM) is widely used to study the effect of several variables and to seek the optimum conditions for a multivariable system (Myers and Montgomery, 2002). Successful application of RSM to enhance fermentation products by optimizing the culture medium has been reported (Singh et al., 2008; Deepak et al., 2008; Senthikumar et al., 2008; Wang and Liu, 2008). On the other hand, there are no reports available on optimization of medium components for PLA production. There is little information on the use of corn steep liquor for the

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industrial production of PLA, and the metabolic pathway utilizing PPA by Lactobacillus sp. SK007 is not yet clearly presented. In this study, PLA was produced by the strain of LAB; Lactobacillus sp. SK007. RSM based on uniform design and central composite rotatable design (CCRD) was adopted to obtain the optimal medium. The metabolic pathway utilizing phenylpyruvic acid (PPA) was postulated. 2. Methods 2.1. Microorganism The PLA-producing strain Lactobacillus sp. SK007 (GenBank accession number: DQ534529) was isolated from Chinese traditional pickles and maintained by our laboratory (Li et al., 2007). 2.2. Culture media The medium used for seed activation was DeMan-RogosaSharpe (MRS) medium. For the production of PLA, a modied medium was used, which contained glucose, phenylpyruvic acid (PPA), corn steep liquor, yeast powder, triammonium citrate, K2HPO4, CH3COONa, Tween-80, MgSO4, and MnSO4. PPA was slightly soluble in the water, so aqueous ammonia was used for improving its solubility. Both media were nally corrected to pH 6.206.40, and then sterilized at 121 C for 15 min in an autoclave. 2.3. Reagents DL-phenyllactic acid (PLA) and PPA were purchased from Sigma (St. Louis, USA) and Fluka (Switzerland) respectively. HPLC grade methanol and other analytical grade chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai). 2.4. Inoculation and fermentation The seed culture was grown at 30 C for 12 h in 10 mL test tube without shaking. A 2% (v/v) inoculum was aseptically added to 10 mL of the modied medium. The fermentation was carried out in a 15 mL test tube at 30 C for 24 h without shaking. Culture media were centrifuged (10,000g for 15 min, 4 C) and the supernatant was ltered (0.45 lm pore-size membrane) for further analysis. 2.5. Experiment design 2.5.1. Uniform design The Uniform design is an efcient fractional factorial design. It was rst proposed by (Fang, 1980). Examples of successful applications of the uniform design method for improving technologies in various elds have been consistently reported (Yu et al., 2008; Liu and Fang, 2007). The uniform design was used in this research to select nutrients that signicantly inuenced PLA production to obtain a smaller, more manageable set. The growth nutrients were screened as seven variables at eleven levels. The variables (nutrients) were glucose, PPA, corn steep liquor, yeast powder, K2HPO4, CH3COONa, and Tween-80. Table 1 shows the coded and non-coded values of the experimental variables. Due to the uniform design, 11 experimental trials were performed with the different conditions array shown in Table 2. The experiments were carried out in duplicate. This was necessary for estimating the variability of measurements. The yields are reported as means of the duplicates. 2.5.2. Response surface methodology and central composite rotatable design (RSMCCRD) RSM and multivariate data analysis are currently applied as an integral part of data analysis in food process design, optimization

Table 1 Nutrient variables and their corresponding levels Independent variables Glucose (g L1) Corn steep liquor (g L1) Phenylphruvic acid (g L1) K2HPO4 (g L1 CH3COONa (g L1) Yeast powder (g L1) Tween-80 (mL L1) X1 X2 X3 X4 X5 X6 X7 Levels 1 10 10 1.0 1.0 1.0 10 1.0 2 12 13 1.4 1.2 1.4 12 1.2 3 14 16 1.8 1.4 1.8 14 1.4 4 16 19 2.2 1.6 2.2 16 1.6 5 18 22 2.6 1.8 2.6 18 1.8 6 20 25 3.0 2.0 3.0 20 2.0 7 22 28 3.4 2.2 3.4 22 2.2 8 24 31 3.8 2.4 3.8 24 2.4 9 26 34 4.2 2.6 4.2 26 2.6 10 28 37 4.6 2.8 4.6 28 2.8 11 30 40 5.0 3.0 5.0 30 3.0

Table 2 Uniform design with the observed responsea Run order Variable levels X1 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 X2 2 4 6 8 10 1 3 5 7 9 11 X3 3 6 9 1 4 7 10 2 5 8 11 X4 4 8 1 5 9 2 6 10 3 7 11 X5 5 10 4 9 3 8 2 7 1 6 11 X6 7 3 10 6 2 9 5 1 8 4 11 X7 10 9 8 7 6 5 4 3 2 1 11 0.76 0.01 0.93 0.02 1.18 0.02 0.87 0.04 1.10 0.02 1.23 0.01 1.55 0.03 1.29 0.05 1.55 0.02 1.11 0.04 2.10 0.03 PLA yield (g L1)

a X1 Glucose, X2 Corn steep liquor, X3 PPA, X4 K2HPO4, X5 CH3COONa, X6 Yeast powder, X7 Tween-80.

and data interpretation. A CCRD with three variables at ve levels was followed to determine the response pattern and also to determine the synergy of variables (Deepak et al., 2008; Onesmus and Qian, 2008). According to this design, 17 runs were conducted containing three replications at the central point for estimating the purely experimental uncertainty variance. The coded and actual values of the variables were presented in Table 3. The CCRD design of experiment was given in Table 4. The relationship of variables was determined by tting a second order polynomial equation to data obtained from the 17 runs. 2.6. Statistical analysis The uniform design and analysis of data were done using SAS Release 8.01 TS Level 01M0. The central composite rotatable design and analysis of data were conducted by Design-Expert Version 7.1.2 (State-Ease Inc., Minneapolis, MN). The response surface analysis was based on the multiple linear regressions taking into account the main, quadratic and interaction effects, according to the following equation

Table 3 Coded and uncoded values of the experimental variables Independent variables Coded values 1.68 Glucose (g L1) Corn steep liquor (g L1) Phenylphruvic acid (g L1) X1 X2 X3 19.91 13.18 3.32 1 24 20 4 0 30 30 5 1 36 40 6 1.68 40.09 46.82 6.68

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Table 4 Central composite rotatable design and experimental PLA yield Run order X1 Glucose 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 1 1 1 1 1 1 1 1.68 1.68 0 0 0 0 0 0 0 X2 Corn steep liquor 1 1 1 1 1 1 1 1 0 0 1.68 1.68 0 0 0 0 0 X3 Phenylphruvic acid 1 1 1 1 1 1 1 1 0 0 0 0 1.68 1.68 0 0 0 1.86 1.75 1.44 1.56 1.67 2.02 1.62 1.90 1.42 1.60 1.68 2.30 1.75 1.75 2.06 2.02 1.94 PLA yield (g L1)

plains the role of each variable and second order interactions in producing PLA. The mathematical model was reduced to

Y 1:86 0:017X 1 0:0025X 3 0:038X 7 0:000023X 2 1 0:000082X 1 X 3 0:00040X 2 3

2

Y b0 b1 A b2 B b3 C b11 A2 b22 B2 b33 C 2 b12 AB b13 AC b23 BC e 1

where b0 is the scaling constant while b1, b2, b3 ,. . ., b23 are the regression estimates and e is the error. 2.7. Analytical method PLA was determined using HPLC equipped with an Agilent Zorbax SB-C18 column (4.6 mm 150 mm, 5 lm). Linear gradient elution was used with methanol/0.05% triuoroacetic acid (solvent A) and water/0.05% triuoroacetic acid (solvent B) at 1 ml/min and A/ B ratios of 10:90, 100:0, 100:0, and 10:90, with run times of 0, 20, 23 and 25 min, respectively. PLA was detected at 210 nm (Li et al., 2007). 3. Results and discussion 3.1. Screening of signicant nutrients using a uniform design Since most industrial experiments usually involve many variables, a full factorial design entails a large number of experiments. To reduce the number of experiments to a practical level, only a small set from all the possibilities is selected. Based on the signal factor analysis, the general range of nutrients concentration was determined. The method of stepwise selection was then applied to select the nutrients that signicantly inuenced the PLA yield (Myers and Montgomery, 2002). The uniform design of experiment and the PLA yields of the experimental trials were shown in Table 2. The regression model was altered repeatedly by adding or removing a variable in accordance with a stepping criteria value of 0.15. After the process of stepping, X1, X2, X3 were nally selected into the model as the main variables. According to F-values, the sequence of importance was as follows; X1 (glucose) > X7 (Tween-80) > X3 (PPA). The signicance of the three variables can be explained as follows. Glucose was the main carbon source, which was an indispensable nutrient for the growth. Tween-80 addition into the medium can stimulate the growth of LAB, improve the growth rate, shorten the cultivation time as well as improve the permeability of cells, and hence enabled excretion of the product out of cells (Johnsson et al., 1995). PPA as the substrate was undoubtedly important for the fermentation. By applying multiple regression analysis on the experimental data a second order polynomial model was obtained which ex-

The model coefcient of determination (R = 0.95) was calculated to be indicating that 95% of the variability in the response could be explained by the model. From the equation, it can be seen that the value of X7 was independent of the value of X1 and X3 in the variation intervals. The yield of PLA was just linear with the value of X7 and the relationship was positive, so the maximum value of X7 (3.0 mL L1) was the optimum. The values of relatively insignicant variables, X4, X5, X6, were determined to be 3 g L1 K2HPO4, 3 g L1 CH3COONa and 30 g L1 yeast powder, respectively. Canonical analysis was performed to determine the shape of the tted response and determine if the estimated stationary point is a maximum, minimum, or saddle point. It was demonstrated to be a saddle point. Since analysis of the surface response revealed that the stationary point for PLA was a saddle, a ridge analysis was performed to predict the possible direction of optimal parameters that may produce the maximum response (Table 5). In this method, the saddle point was considered as the origin of the ridge (center point), and the optimal response values were estimated in a given radius sphere surface. Via this technique, the range for further optimization can be obtained (Liyana-Pathirana and Shahidi, 2005). On the basis of results analyzed above, a CCRDRSM was taken into account to further optimize X1 and X3. In addition, corn steep liquor (X2), a common industrial by-product, was considered as a main factor in the next step probably because its composition is complex and it is suitable for industrial use. 3.2. Further optimization of the nutrients using CCRD and RSM Glucose (X1), corn steep liquor (X2), PPA (X3) and their interactions were studied using the CCRDRSM design. To examine the combined effect of the three independent variables on PLA yield, a CCRD of 23 = 8 plus 3 center points and 2 3 = 6 star points leading to a total of 17 experiments were performed. The CCRD design of experiment and respective experimental yields were given in Table 4. A second order polynomial coefcient for each term of the equation was determined through multiple regression analysis. In the analysis of multiple regression, the effect of the corn steep liquor on PLA yield also was insignicant during the optimized intervals. This was proved by the response surface shown in Fig. 1. According to Fig. 1, the concentration of corn steep liquor was not signicantly related to the yield, and thus the mathematical model was simplied by the elimination of corn steep liquor. In

Table 5 Ridge analysisa Coded radius Estimated response Standard error Uncoded factor values X1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
a

X3 3.00 3.06 3.13 3.20 3.29 3.39 3.50 3.64 3.79 3.98 4.19

X7 2.00 2.06 2.13 2.20 2.27 2.35 2.43 2.50 2.57 2.64 2.70

1.26 1.32 1.38 1.44 1.50 1.55 1.61 1.66 1.71 1.76 1.82

0.10 0.11 0.12 0.12 0.13 0.14 0.14 0.14 0.14 0.14 0.13

20.00 20.74 21.42 22.03 22.57 23.03 23.41 23.70 23.89 24.00 24.02

X1 Glucose, X3 PPA, X7 Tween-80.

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Fig. 1. Response surface and contour plots for the effects of glucose (X1) and corn steep liquor (X2) on PLA yield.

Table 6 ANOVA for response surface reduced quadratic model (X1, Glucose; X3, PPA) Source Model X1 X3 X2 1 X2 3 X1 X3 Residual Lack of t Pure error Total Sum of squares 0.55 0.060 0.030 0.38 0.10 0.048 0.34 0.33 0.0075 0.89 Degree of freedom 5 1 1 1 1 1 11 9 2 16 Mean square 0.11 0.060 0.030 0.38 0.10 0.048 0.031 0.037 0.0037 F-value 3.57 1.94 0.98 12.31 3.26 1.56 9.83 P>F 0.037 0.191 0.344 0.005 0.098 0.237 0.096 Signicant

the next regression analysis, the main, quadratic and interaction effects of X1 and X3 were maintained. The ANOVA (analysis of variance) summary is shown in the Table 6. The model F-value of 3.57 implied that the model was signicant and there was only a 0.037 chance that a model F-value could have occurred due to noise. Since the model showed insignicant lack of t, the response was sufciently explained by the regression equation. The generated regression relationship is given in Eq. (3).

Y 1:998 0:068X 1 0:047X 3 0:17X 2 0:09X 2 0:08X 1 X 3 1 3

Not signicant

3
The relationship that transpired between X1 (glucose), X3 (PPA) and the PLA yield is illustrated as a three dimensional representation of the response surfaces and two-dimensional contour plots

Fig. 2. Response surface and contour plots for the effects of glucose (X1) and PPA (X3) on PLA yield.

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W. Mu et al. / Bioresource Technology 100 (2009) 13661370 Johnsson, T., Nikkila, P., Toivonen, L., 1995. Cellular fatty acid proles of Lactobacillus and Lactococcus strains in relation to the oleic acid content of the cultivation medium. Appl. Environ. Microbiol. 61, 44974499. Kamata, M., Toyomasu, R., Suzuki, E., Tanaka, T., 1986. Production of D-phenylactic acid through fermentation process. Japanese Patent No. JP61108396. Kammoun, R., Naili, B., Bejar, S., 2008. Application of a statistical design to the optimization of parameters and culture medium for a-amylase production by Aspergillus oryzae CBS 819.72 grown on gruel (wheat grinding by-product). Bioresour. Technol. 99, 56025609. Kim, J.H., Han, K.C., Koh, Y.H., Ryu, Y.W., Seo, J.H., 2002. Optimization of fed-batch fermentation for xylitol production by Candida tropicalis. J. Ind. Microbiol. Biotechnol. 29, 1619. Kim, H.O., Lim, J.M., Joo, J.H., Kim, S.W., Hwang, H.J., Choi, J.W., Yun, J.W., 2005. 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generated by the model in Fig. 2. It shows that the PLA yield increased with an increase in the amounts of the glucose up to an optimum (maximum PLA yield) beyond which there was a decrease. PPA had a similar trend to that of glucose. The interaction term X1X3 with a positive regression coefcient in Eq. (3) provided evidence that the PLA yield was favored by an increase in the factor interactions up to a value beyond which there was a decline. The optimized concentrations of glucose, PPA and corn steep liquor were 30, 5 and 47 g L1, respectively, and the maximum product of PLA (2.30 g L1) was achieved. In conclusion, the optimal medium for maximum production of PLA by Lactobacillus sp. SK007 was determined using a uniform design method and a CCRDRSM. PLA was produced using corn steep liquor as the main nitrogen source instead of peptone in MRS as the main nitrogen source giving a maximum yield of 2.30 g L1. This is a relatively high yield compared to previously reported results (Valerio et al., 2004). The optimized medium composition was 30 g L1 glucose, 5 g L1 PPA, 47 g L1 corn steep liquor, 3 g L1 K2HPO4, 3 g L1 CH3COONa, 30 g L1 yeast powder and 3 mL L1 Tween-80. With the optimized culture medium, 200-L fermentation of Lactobacillus sp. SK007 was performed. The PLA produced could reached approximately 2.43 g L1 after 32 h fermentation, which was corresponded with the results of RSM optimization. This work reports successfully use of corn steep liquor for synthesis of PLA from PPA. Use of corn steep liquor increases the feasibility of industrial PLA production due to decreased expenses. The metabolic pathway for PLA synthesis from PPA by Lactobacillus sp. SK007 is postulated to involve PPA as an intermediate in conversion of Phe to PLA (Li et al., 2008). This is because of trace amounts of Phe that were observed (data not shown). PLA was a side-product of phenylalanine metabolism pathways in LAB (Vermeulen et al., 2006), in which phenylalanine was transaminated to phenylpyruvic acid (PPA) rst, and PPA was further reduced to PLA. The transamination process was the rate-limiting step and the bottleneck in PLA formation when phenylalanine was initial material (Vermeulen et al., 2006; Li et al., 2007). Using PPA as substrate was helpful to increase the fermentation yield of PLA. Acknowledgements This work was supported by the grants from the National High Technology 863 Program of China (2006AA10Z334), the National Natural Science Foundation of China under Key Program, Project No. 20436020 and the Research Program of State Key Laboratory of Food Science and Technology, Jiangnan University, Project No. SKLF-MB-200804. References
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