Screening and identification of Monacus strain with high TMP production and statistical optimization of its culture medium composition and liquid state fermentation conditions using response surface methodology (RSM)

ABSTRACT Teramethylpyrazine (TMP) is a pyrazine alkaloid with a variety of pharmacological effects and widely used in clinical practice. In this study, Monascus strain M-3 with high TMP and low citrinin productions was screened from red yeast rice collected from different areas of Fujian province in China. Monascus strain M-3 was further identified through molecular biological methods based on the sequencing of different gene regions. Results showed that primer set of β-tubulin-F/β-tubulin-R targeting the β-tubulin gene was more suitable for the identification of Monascus strain M-3. Based on comparative analysis of the results obtained by different primer sets, Monacus strain M-3 was finally identified as Monascus purpureus. Then, the composition of culture medium and liquid state fermentation conditions of Monacus strain M-3 were optimized by single-factor experiment and central composite design of response surface methodology, with the purpose of optimizing the TMP productions. The optimum composition of culture medium and liquid state fermentation conditions are as follows: starch 5.1%, peptone 3.51%, Mg2SO4 0.1%, NaNO3 0.2%, KH2PO4 0.25%, fermentation temperature of 28.23 °C, fermentation time of 9 d, shaker speed of 180 r/min, inoculum size of 7.21% and loading volume of 100 mL/250 mL triangular flask. Under the optimum composition of culture medium and liquid state fermentation conditions, the TMP production of Monacus strain M-3 can reach 13.49 µg/mL. This study is expected to provide a new approach of TMP production through biosynthesis by Monascus strain, and promote the further development of functional foods or medicines from Monascus fermentation products.


Introduction
Tetramethylpyrazine (also called 2,3,5,6-Tetramethylpyrazine (TMP), as shown in Figure 1) is one of the main active ingredients of Rhizoma Chuanxiong, Zingiberaceae Curcuma rhizomes and Euphorbiaceae Jatropha stems [1]. Previous studies have evidenced that TMP has the pharmacological activity of anti-platelet aggregation and the physiological functions of softening the blood vessels, increasing coronary blood flow, improving microcirculation and scavenging free radicals [2][3][4][5]. It has been used in clinical applications as a medicinal agent with high security for more than 30 years, especially in the treatment of patients with cerebral ischemic diseases, because of its neuroprotective effects and global cerebral ischemia [6,7].
Currently, the method of preparation of TMP includes chemical synthesis [8], extraction from Chinese traditional medicinal plants (Rhizoma Chuanxiong, Z. Curcuma rhizomes and E. Jatropha stems) [9] and biosynthesis [10,11]. However, chemical synthesis has great limitations because of its high chemical residues, low security, environmental destruction and other factors. Extraction from medicinal plants has great limitations due to the large raw material wastage, time-consuming of extraction process and the needs of large quantities of organic solvents. Therefore, both of chemical synthesis and extraction from medicinal plants still cannot well meet the needs of scientific research and the pharmaceutical industry. Compared with chemical synthesis and extraction from medicinal plants, biosynthesis has obvious advantages, especially for health foods and drugs production. It has been reported that some micro-organisms (such as Bacillus subtilis [12] and Sulfitobacter pontiacus [13]) can produce a certain amount of TMP. The application of Monascus species in foods, medicine and industry can be traced back to a thousand years ago. The secondary metabolism of Monascus produces various valuable components, including pigments, monacolin K, g-aminobutyric acid, ergosterol, etc. [14][15][16]. In the former work of our laboratory, we had discovered Monascus also produces a certain amount of TMP. Compared with B. subtilis and S. pontiacus, the production of TMP from Monascus sp. is relatively low. The TMP production is closely dependent on Monascus strain, medium composition (carbon source, nitrogen source and other nutritional factors) and environmental factors (oxygen and temperature, etc.). Therefore, it is necessary to further optimize the fermentation process of Monascus sp. for the higher production of TMP.
There are a variety of fermentation methods in the batch production of micro-organisms, including solid state fermentation, liquid state fermentation, immobilized cells fermentation and so on. They have different applications in the fermental cultivation of Monascus sp., such as red pigment production through immobilized Monascus fermentation, lovastatin production by solid state fermentation. Compared with solid state fermentation, liquid state fermentation has many advantages [17]: (1) the substrates, products and other matters in the liquid are easy to spread and the distribution is uniform, which is beneficial to the growth of micro-organism; (2) liquid culture has the characteristics of convenient operation and control; (3) the separation and purification of the active ingredient in fermented product is more convenient.
To date, no report is available on the optimum liquid state fermentation conditions of Monascus for the production of TMP. In this study, Monascus strain M-3 with high TMP and low citrinin productions was screened from red yeast rice collected from different areas of Fujian province in China, and further identified by molecular biological method based on the sequencing of different gene regions. Furthermore, the composition of culture medium and liquid state fermentation conditions of TMP production by Monacus strain were optimized by means of single-factor experiment and central composite design (CCD). The results would provide a new approach of TMP production through biosynthesis by Monascus strain, and promote the further development of functional foods or medicines from Monascus fermentation products.

Sample collection
Twenty samples of dry commercial traditional red yeast rice were obtained from the small-scale factories located in the northern, southern and western areas of Fujian province in China. Samples were stored at 4 C immediately after collection prior to testing. Ten grams of powdered wine fermentation starter sample were homogenized in 90 mL of 0.85% w/v sterile physiological saline. Then a series of decimal dilutions were made. One millilitre each from the dilutions of 10 ¡4 , 10 ¡5 , 10 ¡6 and 10 ¡7 was poured onto Czapek-Dox agar (Difco, Detroit, MI, USA) and potato dextrose agar (Difco) supplemented with ampicillin (100 ng/mL, Merck, Darmstadt, Germany) and incubated at 28 C for 3 d. All the colonies isolated were identified with traditional methods including a macroscopic level (surface colour, reverse side colour, spores and diameter) and microscopic characteristics such as conidiophores, conidia and fertile hyphae. Monascus strains were screened from the isolates and subcultured on new Czapek-Dox agar plates (Difco) and purified by repeated streaking. The purified isolates were obtained and maintained on potato dextrose agar (Difco) slants at 4 C until use.

Screening of Monascus strains with high TMP production
Pure cultures were incubated in 250 mL Erlenmeyer flasks containing 50 mL of fermentation medium (10 g/L glycerinumautoclaved separately at 121 C for 15 min, 10 g/L peptone, 1 g/L Mg 2 SO 4 , 2 g/L KH 2 PO 4 at pH 7.0) at 30 C for 5 d with shaking at 150 r/min. The fermentation broth was used for TMP production measurement by high-performance liquid chromatography (HPLC) method [18].
Identification of Monascus strain with the highest TMP production DNA extractions for Monascus strain were carried out using a benzyl chloride method [19]. DNA extract was dissolved in 50 mL TE containing RNase. The yields and fragmentation of the DNA were determined by ethidium bromide-UV detection on 1% (w/v) agarose gel. Nucleic acid extracts from each sample were also analysed spectrophotometrically at 260 and 280 nm by using a DU800 spectrophotometer (Beckman Coulter, Fullerton, CA, USA). The DNA extracts were stored in a freezer at ¡80 C until analysis. Different gene regions were amplified using different fungal primers (Supplementary  material Table S1) under the conditions according to relevant literature [20][21][22][23]. Sequencing in both directions was performed by the Sangon Biotech co., Ltd. (Shanghai, China) and then manually corrected for ambiguities using BioEdit v.7.0. A sequence alignment program (BLAST) was used for strain identification according to pairwise similarity with sequences in public databases (http://www.ncbi.nlm.nih.gov/BLAST/) [24].

HPLC analysis of citrinin
Fermentation products (0.1 g of weight) were extracted with 3 mL 95% ethanol in 60 C for 30 min. The extracted solution was filtrated using 0.45 mm filter. The concentration of secondary metabolites was measured by HPLC method using an Agilent 1260 Series LC system equipped with a photodiode array detector (Waters 2996; Waters Ltd., Milford, MA, USA). Chromatographic separation was conducted on Phenomenex® Luna 5u C18 (2) (250 £ 4.6 mm) column. Solvent A (1 L acetonitrile with 0.05% TFA) and solvent B (distilled water with 0.05% TFA) were used as the mobile phase. Solvent A/ solvent B: 75/25 was eluted for 30 min. The eluent was pumped at a flow rate of 0.5 mL/min. The concentration of citrinin was determined by fluorescence detector (emission: 330 nm; emission: 500 nm). The citrinin was identified by comparison of their retention time values and spectra with known standards [25].

Optimization for culture medium for higher TMP productions
The effects of different carbon source, nitrogen sources and their proportions on the TMP production were studied. The fermentation conditions were as follows: inoculum size 10%, shaker speed 150 r/min, fermentation temperature 30 C; the content of TMP in the fermented products was determined after fermentation of 5 days.

Optimization of liquid state fermentation conditions for higher TMP productions
Flask experiments were performed in 250 mL Erlenmeyer flasks containing 50 mL of modified optimum medium (51 g/L starchautoclaved separately at 121 C for 15 min, 35.1 g/L peptone, 1 g/L Mg 2 SO 4 , 2 g/L KH 2 PO 4 ). First, the best fermentation time was selected by examining the TMP producing ability at different fermentation time by HPLC. The experiment conditions were: 10.0% inoculum size of in modified optimum medium at pH 7.0, 30 C, shaking at 150 r/min. The selected fermentation time was applied in the study of other conditions.
The culture medium and fermentation conditions of Monascus strains with high TMP production were optimized by the test design. First, the factors that affect the yield of TMP were selected by single-factor experiment. Then, the selected factors were further optimized using central composite experimental design of response surface methodology (RSM).

Statistical analyses
The central composite experimental design of RSM was used to study the variables independently for their interactions and quadratic effects. The experimental data were analysed by Design Expert© 9.0 software. The two degree polynomial of the relationship between the response variable and the independent variable was obtained. Model can be described as The multiple repeat of centre point provided a sufficient degree of freedom for test error estimation. The experimental design references were carried out, and the experimental data were analysed by Design Expert© 9.0 software.

Results and discussion
Characteristics and screening of Monascus strain with high TMP production Twenty strains were isolated from red yeast rice collected from different areas of Fujian province in China. Their colonial morphology is shown in Figure 2. The TMP productions of different Monascus strains were measured by HPLC method. Results showed that the TMP productions of different Monascus strains were significantly different (as shown in Figure 3) (p < 0.01). The TMP yields of vast majority of strains were relatively low (<5.0 mg/mL). It is noteworthy that the TMP yields of Monacus strain M-3, M-9 and M-10 were higher than 8.0 mg/mL. Parallel tests also indicated that the TMP productions of Monacus strain M-3, M-9 and M-10 were relatively stable (data not shown). What is of importance, Monascus-fermented products may be contaminated by citrinin, initially named monascidin A, which could damage the kidneys and liver. Previous studies by others have pointed out that most of Monascus sp. excretes certain amount of citrinin [26]. In this study, the amount of citrinin produced by the twenty strains isolated from red yeast rice were also determined through HPLC-FD method. Results showed that a lower amount of citrinin produced by Monacus strain M-3 compared to Monascus strains M-9 and M-10. Therefore, Monacus strain M-3 was selected for further study.

Identification of Monacus strain M-3 through sequencing of different gene regions
The extracted genomic DNA of Monacus strain M-3 is shown in Figure 4(a). The electrophoretic patterns of PCR amplification products with four primer sets are shown in Figure 4(b). In all cases, sequence similarities approached 99% with publicly available sequences at the GenBank (as shown in Table 1 [28]. Comparative sequence analyses based on sequence divergence of specific genes have been conducted in our previous research work, which indicated that the resolution of 18S rRNA gene sequences and ITS-5.8S rRNA gene sequences were insufficient for the identification of Monascus strains at the species level  Table 1). Based on the above identification through four primer sets and their comparative analysis, Monacus strain M-3 can be finally identified as M. purpureus.

Optimization for culture medium for higher TMP productions
Single-factor tests for carbon and nitrogen source Carbon is the main component of microbial cells and their metabolites. The carbon source provides the necessary energy for the fermentation process, and is one of the main components of the medium [29]. Effects of  several different carbon sources on the yield of TMP are shown in Figure 5(a). When starch was used as carbon source, Monacus strain M-3 had the highest TMP yield. Therefore, starch was used as carbon source in the following experiment. In order to determine the optimum amount of starch, the effect of different starch amount on the yield of TMP was determined. As shown in Figure 6(a), the TMP production reached the highest point when adding 5% starch. Too much or too little starch addition is not beneficial for the synthesis of TMP. Too little carbon source addition would lead to nutritional deficiencies in cell growth and product synthesis process, and finally resulting in cell death. Too much carbon source addition also affects the composition of the medium, causing pH decrease. Nitrogen source is mainly used to constitute the cell material (amino acids, proteins, nucleic acids) and nitrogen metabolites [30]. The common nitrogen sources can be divided into organic nitrogen source and inorganic nitrogen source. The effects of different nitrogen sources on the fermentation were investigated in this study. As shown in Figure 5(b), the TMP productions were relatively higher when using peptone, L-glutamic acid or asparagine as nitrogen source. Peptone is the ideal nitrogen source for micro-organisms, because it contains proteins, peptides, free amino acids and also contains fat, growth factors, inorganic salt and other materials. When using peptone as the nitrogen source during fermentation, the growth status of Monacus strain was better than individual amino acid as the nitrogen source. Therefore, peptone was regarded as the optimal nitrogen source for the TMP productions of Monacus strain M-3. In order to determine the optimum amount of peptone, the effects of different peptone amount on the yields of TMP were also determined. As shown in Figure 6(b), the TMP yields reached the highest point when adding 3% peptone. Too much or too little peptone addition was not conducive to TMP synthesis.

Central composite experimental design
The results obtained from the run of 13 experimental sets, suggested by CCD models of RSM were analysed by Design Expert© 9.0. Analysis of variance (ANOVA), regression coefficients and polynomial regression equation were obtained. Table 2 shows the relationship between TMP production and independent variables, its ANOVA results are tabulated in Table 3. The model has a 'F-value' of 60.14, which is large enough and implies that the model is significant. The 'Coefficient of Determination' (R 2 ) value of the study was 0.9773. The high R 2 value suggests that strong correlation exists between the observed values and the values predicted by the CCD model [31]. The 'adjusted R-squared' value was 0.9610. The quadratic polynomial equation obtained by multiple regression analysis depicting the effect of parameters (X1 and X2) and their interactions in the response (TMP production) is described in the following equation: Y ¼ 13:71 À 1:09 Â 1 À 1:13 Â 2 À 2:61 Â 12 À 3:04 Â 22 þ 0:56 Â 1 Â X2: (1) Figure 8(a) shows the 2D contour plots and 3D surface plots of the RSM study, which disclose the mutual interaction in between parameters and response. The validation of RSM result was done by the numerical optimization of CCD model. The predicted optimum values for the parameters are shown in Table 2. An experiment was performed using parameters suggested by numerical optimization, then TMP production was measured manually in triplicates with the help of Archimedes principle. The maximum value of TMP production observed was 13.48 § 0.94 mg/mL, which is very close to the value 13.06 § 1.31 mg/mL predicted by RSM. This maximum TMP production was found against the 5.1% starch, 3.51% peptone. This experiment validates that the RSM method is applicable for the parameter optimization to medium composition.

Optimization of liquid state fermentation conditions for higher TMP productions
Single-factor tests for liquid state fermentation conditions After determining the optimum amount of carbon source and nitrogen source in the culture medium, the effects of fermentation time, fermentation temperature, shaker speed and inoculation amount on the yield of TMP were further investigated. As shown in Figure 7(a), the TMP production of Monacus strain M-3 reached maximum value (12.31 mg/mL) after 9 d of fermentation. As shown in Figure 7(b), the TMP production increased with the increase of temperature, and reached a maximum of 11.93mg/mL at 28 C. However, when the temperature continued to increase, the TMP production appeared to decline, indicating that the optimum fermentation temperature was 28 C. Besides, the TMP production increased with the increase of shaking speed, reaching a maximum of 10.38 mg/mL at 180 r/min (Figure 7(c)), and then remained at comparable levels (210 r/min). This result indicated that oxygen supply is positively associated with TMP production. The amount of TMP production increased with an increase in inoculated volume from 5% (7.85 g/L) to 7% (11.36 g/L) (Figure 7(d)). However, when the inoculum continued to increase, the TMP production appeared to decline, indicating that the optimum fermentation inoculum size of was 5%.

Central composite experimental design
According to the single-factor experiment, temperature and inoculation amount (the two main factors affecting the yield of TMP) were chosen to do the response surface test design of two factors and five levels. The results by central composite experimental design (CCD models of RSM) were analysed by Design Expert© 9. ANOVA, regression coefficients and polynomial regression equation were obtained. Table 4 shows the relationship   Figure 7. Effects of different fermentation conditions on TMP production.
between TMP yeilds and independent variables, its ANOVA results were tabulated in Table 5. The model has an 'F-value' of 54.35, which is large enough and implies that the model is significant. The 'Coefficient of Determination' (R 2 ) value of the study was 0.9749. The high R 2 value suggests that strong correlation exists between the observed values and the values predicted by the CCD model the 'adjusted R-squared' value of 0.9570. The quadratic polynomial equation obtained by multiple regression analysis depicting the effect of parameters (X1 and X2) and their interactions in the response (TMP production), and is described in the following equation: Y ¼ 13:71 À 1:09 Â 1 À 1:13 Â 2 À 2:61 Â 12 À 3:04 Â 22 þ 0:56 Â 1 Â X2: (2) Figure 8(b) shows the 2D contour plots and 3D surface plots of the RSM study, which disclose the mutual interaction in between parameters and response. The predicted optimum values for the parameters are shown in Table 4. The optimized value of TMP productions from the second-order quadratic model defined by Equation (2) was 13.71 § 1.01 mg/mL under the optimum composition of culture medium and liquid state fermentation conditions: starch 5.1%, peptone 3.51%, Mg 2 SO 4 0.1%, NaNO 3 0.2%, KH 2 PO 4 0.25%, fermentation temperature 28.23 C, fermentation time of 9 d, shaker speed of 180 r/min, inoculum size of 7.21% and loading volume of 100 mL /250 mL triangular flask. The experiment was carried out under the above-predicted optimum conditions in order to check the validity of the model, and a yield of 14.32 § 1.01 mg/mL was obtained. This demonstrated a good match between the experimental and predicted values, thus substantiating the proposed model. The results validate that the RSM method is applicable for the parameter optimization to culture medium composition and liquid state fermentation conditions.

Conclusions
In summary, Monascus strain M-3 with the highest TMP production ability was screened from red yeast rice collected from different areas of Fujian province in China. The target Monacus strain M-3 was identified as   M. purpureus by molecular biological method. The liquid state fermentation conditions of Monacus strain M-3 were further determined by the single-factor test and CCD of RSM: starch 4.4%, peptone 3.51%, Mg 2 SO 4 0.1%, NaNO 3 0.2%, KH 2 PO 4 0.25%, culture temperature of 28.23 C, fermentation time of 9 d, shaker speed of 180 r/min, inoculum size of 7.21% and loading volume of 100 mL/250 mL triangular flask. Under the optimum composition of culture medium and liquid state fermentation conditions, the TMP production could be as high as 13.49 mg/mL. The study is expected to provide a new approach of TMP production through biosynthesis by Monascus strain. The results would promote the further development of functional foods or medicines from Monascus fermentation products, and provide reference for China's traditional food industry. The final yield of TMP by Monacus strain M-3 is still lower compared to Bacillus sp. strains reported by others. Optimization to higher TMP production is required, which could be achieved by selecting and breeding the excellent strains through physical or chemical mutagenesis. In addition, further studies also need to be focused on the specific metabolic pathways of TMP production by Monascus.

Disclosure statement
No potential conflict of interest was reported by the authors.