Fermentative Production of Melanin by the Fungus Auricularia auricula Using Wheat Bran Extract as Major Nutrient Source

Abstract

Melanin is a natural pigment with great development potential as a healthful food colorant. Low cost fermentation medium using wheat bran extract as a major nutrient source, was evaluated for production of melanin from the fungus Auricularia auricula in submerged culture. Effects of wheat bran extract, l-tyrosine and CuSO₄ on tyrosinase activity and melanin yield were investigated. Results showed that wheat bran extract, l-tyrosine and CuSO₄ concentrations influenced tyrosinase activity and increased melanin yield. Box-Behnken design indicated the following optimal medium composition: wheat bran extract 26.80% (v/v), l-tyrosine 1.59 g/L and CuSO₄ 0.11 g/L. Under these conditions, the highest melanin yield (519.54 mg/L) was obtained. The present study avoids the use of purified tyrosinase, expensive chemical methods or the cumbersome extraction of melanin from animal or plant tissues. These results might provide a reference for the development of a cost-effective medium for commercial production of melanin used in food industry.

Introduction

Melanin is a dark-colored polyphenolic pigment produced from oxidative polymerization of phenolic or indolic compounds by tyrosinase (Riley, 1997). These natural pigments are synthesized by some fungi, plants, animals and several bacterial species (Dalfard et al., 2006). Melanins from different sources possess similar physicochemical properties which include strong light absorbance, unusual solubility and remarkable redox properties. In addition, melanin has a number of healthful functions, such as antioxidation (Hung et al., 2002; Liu et al., 2011; Róźanowska et al., 1999; Tu et al., 2009; Wu et al., 2008), anti-HIV activity (Manning et al., 2003; Montefiori and Zhou, 1991), and immunomodulatory activity (Sava et al., 2001). These functions promise natural melanin with great development potential as a healthful food colorant.

Auricularia auricula, a non-toxic macro-fungus, has been used as food and drug in China for a long time (Zhang et al., 1995). A. auricula fruit bodies, a kind of edible black-brown mushroom, are rich in melanin and are increasingly popular as a “black food” in China (Chen et al., 2008; Tu et al., 2009; Wang et al., 2006). However, the extraction process of melanin from tissues of A. auricula fruit bodies is tedious and expensive. In addition, A. auricula fruit bodies grow on solid media, the time to complete the fruit bodies is also long and the product quality control is difficult (Wu et al., 2006).

Melanin production through fermentation has been believed to be a convenient and efficient method, which has many advantages, including short fermentation period, low producing cost, high product output and easy downstream processing (Wu et al., 2006). Melanin synthesis by bacteria through adding l-tyrosine has been reported recently (Lagunas-Muñoz et al., 2006; Lin et al., 2005; Ruan et al., 2004; Santos and Stephanopoulos, 2008). Escherichia coli is used for producing melanin firstly, but many hidden troubles and dangers existed by E. coli fermentation. Some shiga toxin-producing E. coli are primarily food-borne pathogens that can cause severe and potentially fatal human disease (Vu-khac and Cornick, 2008). Thus, melanin produced by these bacteria can not be used in food industry.

A large amount of melanin can also be produced by A. auricula through submerged culture and the production of melanin is mediated primarily via tyrosinase (EC 1.14.18.1). Therefore, promoting tyrosinase activity is helpful in stimulating melanin production (Van Gelder et al., 1997). At present, a major concern in the production of melanin is the reduction of cost of raw materials which accounted for 30 – 40% of total production cost of industrial productions (Joo et al., 2002). Utilization of cheap carbon sources is considered as an effective approach. Many agricultural by-products and waste materials have been used as carbon sources to produce valuable compounds such as lactic acid, succinic acid and xylanase (Gao et al., 2008; Walia et al., 2013; Zheng et al., 2009). For the production of these compounds, wheat bran has been used as one of major substrates, but their application in melanin production is little explored.

In the present study, the production of melanin by A. auricula was carried out using low cost fermentative substrate (wheat bran extract). The effects of other parameters (l-tyrosine and CuSO₄) on tyrosinase activity and melanin yield were also investigated. Response surface methodology (RSM) was employed to optimize medium composition (wheat bran extract, l-tyrosine and CuSO₄) in order to obtain the maximal melanin yield.

Materials and Methods

Materials    Wheat bran was generously supplied by Jiangnan Co., Ltd. (Jiangsu Province, China), pulverized and sifted through a sieve (opening 0.43 mm), respectively. Nutrients from the powder were extracted with deionized water at a ratio of 4 mL/g (water/raw materials) at 60°C for 5 h, followed by incubation at 100°C for 0.5 h. The homogenate was centrifuged at 2,683×g for 5 min and the supernatant was used as the major nutrient source in fermentation medium (wheat bran extract). Synthetic melanin and l-tyrosine were purchased from Sigma Chemicals Co. (St. Louis, USA). All the other chemicals and reagents used in the experiment were of analytical grade.

Microorganism and maintenance    The fungal strain A. auricula RF201 was purchased from the Institute of Edible Mushroom of Jiangsu Academy of Agricultural Sciences (Jiangsu Province, China). Stock cultures were maintained on potato dextrose agar (PDA) slants and subcultured every two months. Slants were inoculated with mycelia and incubated at 25°C for 7 d.

Inoculum preparation and flask culture    For production of the inoculum, the strain was transferred to seed medium (potato dextrose broth medium) with 6 mm diameter discs from PDA plates. Four discs were inoculated to 50 mL liquid medium in an Erlenmeyer flask (250 mL) and then incubated at 25°C on a reciprocating shaker (100 rpm) for 5 d. The inoculum (10%, v/v) was transferred into an Erlenmeyer flask (250 mL) containing 50 mL of fermentation medium. The basal medium composition was 40% (v/v) wheat bran extract, 2 g/L l-tyrosine, 0.1 g/L CuSO₄, 1 g/L MgSO₄, 1 g/L KH₂PO₄ and 0.1 g/L vitamin B₁. The values of medium composition were varied based on the experimental design. All media were cultivated at 25°C for 5 d at the pH 8.0 and rotation time of 100 rpm.

Effects of wheat bran extract, l-tyrosine and CuSO₄ on tyrosinase activity and melanin production    In order to determine the proper scope of wheat bran extract, l-tyrosine and CuSO₄ for tyrosinase activity and melanin production from the fungus A. auricula in submerged culture, different culture media were prepared as follows: Various wheat bran extract concentrations ranging from 10% to 60% (v/v), l-tyrosine at a range of concentrations from 0.5 to 10 g/L, CuSO₄ at different concentrations ranging from 0.01 to 0.2 g/L. The desirable concentration scope of wheat bran extract, l-tyrosine and CuSO₄ resulted in higher melanin yield was used to optimize melanin production by A. auricula using response surface methodology.

Optimization of medium composition for melanin production    The Box-Behnken experimental design with three factors and three levels was employed to optimize the medium composition in order to obtain the highest melanin yield. Wheat bran extract (X₁), l-tyrosine (X₂) and CuSO₄ (X₃) were chosen as independent variables in this design. Based on the single-factor experiments, X₁ (20%, 30% and 40%), X₂ (1, 1.5 and 2 g/L) and X₃ (0.05, 0.1 and 0.15 g/L) were determined as critical levels with significant effect on melanin production. The complete design consisted of seventeen combinations including five replicates of the center point (Table 1).

Table 1. Box-Behnken design and experimental melanin yield.

Run	X1	X2	X3	Melanin yield (mg/L)
	Wheat bran extract (%)	l-Tyrosine (g/L)	CuSO4 (g/L)	Observed value	Predicted value
1	30	1.5	0.10 (0)	513.17±23.67	508.44
2	40	2.0	0.10 (0)	384.89±12.80	375.30
3	20	2.0	0.10 (0)	461.66±22.49	456.90
4	30	1.0	0.05 (−1)	362.93±23.76	363.90
5	40	1.5	0.15 (1)	356.65±17.85	369.18
6	30	1.5	0.10 (0)	502.24±32.51	508.44
7	20	1.0	0.10 (0)	411.31±12.83	425.88
8	20	1.5	0.05 (−1)	451.01±23.37	437.43
9	30	1.5	0.10 (0)	505.14±21.98	508.44
10	20	1.5	0.15 (1)	447.02±26.60	450.78
11	40	1.5	0.05 (−1)	358.54±21.69	355.83
12	30	1.5	0.10 (0)	504.68±29.08	508.44
13	30	2.0	0.15 (1)	409.25±20.86	408.28
14	30	1.0	0.15 (1)	466.63±19.16	451.31
15	30	1.5	0.10 (0)	516.97±31.68	508.44
16	40	1.0	0.10 (0)	344.50±12.73	344.27
17	30	2.0	0.05 (−1)	453.67±19.66	468.99

The experimental results were analysed by quadratic stepwise regression to fit the second-order eq. (1):   

where Y stands for melanin yield, X₁, X₂, X₃ for independent variables, β₀ for the model intercept and B_i, B_ii, B_ij for regression coefficients of variables for intercept, linear, quadratic and interaction terms, respectively. The software Design-Expert 7.0.0 Trial (State-Ease Inc., Minneapolis, USA) was used to obtain the coefficients of the quadratic polynomial model.

Assay of tyrosinase activity    Fermentation medium was centrifuged at 2,683×g for 20 min at 4°C, and then the supernatant was used for enzyme activity measurement. Tyrosinase activity was determined quantitatively by measuring the oxidation products of l-tyrosine at 37°C in a reaction mixture containing 2 mM l-tyrosine and 0.1 M phosphate buffer (pH 6.8). The changes of absorbance in presence of the enzyme were monitored for 30 min at 475 nm. One unit of tyrosinase activity was defined as the amount of enzyme that catalyzes the appearance of 1 µmol of l-dopachrome (molar extinction coefficient ɛ = 3700 mol⁻¹ cm⁻¹) per min under the assay conditions (Dalfard et al., 2006).

Determination of melanin    Determination of melanin was performed as described by Zou et al. (2010) with proper modification. Fermentation medium was centrifuged (2,683×g, 5 min) and filtered to remove mycelia and insoluble particles. The supernatant was adjusted to pH 2.0 with 3 M HCl to precipitate melanin, followed by centrifugation at 16,770×g for 20 min and the supernatant was discarded. The precipitated melanin was washed with chloroform, ethyl acetate and ethanol and then dissolved in 0.01 M NaOH. For the quantification of melanin, the optical densities of solution containing melanin at 400 nm were determined with a UV-2802 diode array spectrophotometer (Unico Instrument Co. Ltd., Princeton, NJ, USA) and compared to a synthetic melanin.

Statistical analysis    The experimental results obtained were expressed as means ± SD of triplicates. Statistical analysis was performed using Fisher's F-test. P < 0.05 was regarded as significant and P < 0.01 as very significant.

Results and Discussion

Effects of wheat bran extract on tyrosinase activity and melanin yield in fermentation medium    The effects of increasing wheat bran extract concentrations on tyrosinase activity and melanin yield in A. auricula fermentation medium are shown in Fig. 1. Both melanin yield and tyrosinase activity increased firstly and then decreased. When wheat bran extract concentration in fermentation medium was 30%, the highest melanin yield (471.1 mg/L) and the maximal tyrosinase activity (16.2 U/mL) were obtained.

Fig. 1.

Effects of wheat bran extract concentration on tyrosinase activity and melanin yield.

The nutrient of wheat bran was enwrapped in cellulose and was difficult to be utilized by A. auricula. Therefore, wheat bran extract was prepared and used as carbon sources for tyrosinase synthesis and melanin production by this fungus. The higher tyrosinase activity and melanin yield using wheat bran extract as carbon sources might be due to higher content of carbohydrate, particularly cellulose and hemicellulose which could promote tyrosinase synthesis and melanin production of A. auricula (Gao et al., 2008; Hemery et al., 2009; Rouanet et al., 1989). More importantly, wheat bran was an abundant and very cheap agricultural residue (Xu et al., 2005) and its utilization was to reduce the cost of fermentation medium. However, tyrosinase activity and melanin yield slowly decreased when wheat bran extract concentration exceeded 30%. This was probably because that higher sugar concentration (above 0.5%) or other unknown factors inhibited tyrosinase synthesis and melanin production (Lagunas-Muñoz et al., 2006; Wang et al., 2008).

Effects of l-tyrosine on tyrosinase activity and melanin yield in fermentation medium    As expected, l-tyrosine addition effectively increased melanin yield and enhanced tyrosinase activity, as shown in Fig. 2. Melanin yield increased rapidly with increase of l-tyrosine concentration, but it gradually decreased when the concentration of l-tyrosine was above 1.5 g/L. Tyrosinase activity was also enhanced with l-tyrosine addition, but it reached a constant value when l-tyrosine concentration was between 1.5 and 10 g/L.

Fig. 2.

Effects of l-tyrosine concentration on tyrosinase activity and melanin yield.

The induction by l-tyrosine on tyrosinase synthesis has also been reported (Chandel and Zzmi, 2009; Rhee et al., 2002). Elevating l-tyrosine levels can also stimulate melanin production (Santos and Stephanopoulos, 2008). These increases of melanin in the experiment were probably caused by increases in the substrate in the vicinity of the cytosolic tyrosinase, which suggested that tyrosinase activity and melanin yield were regulated by l-tyrosine addition. However, at a higher l-tyrosine concentration (above1.5 g/L), synthesis of tyrosinase was inhibited and production of melanin gradually decreased. At higher concentration, l-tyrosine remained insoluble in fermentation medium (Chandel and Zzmi, 2009). The extremely low solubility of l-tyrosine resulted in considerable carryover of it into the mycelium. This indicated that higher l-tyrosine concentration had negative effect on tyrosinase synthesis and melanin production. Thus, comparatively lower l-tyrosine concentration should be used for the production of melanin to avoid its precipitation.

Effects of CuSO₄ on tyrosinase activity and melanin yield in fermentation medium    The effects of CuSO₄ addition on tyrosinase activity and melanin yield in A. auricula fermentation medium are presented in Fig. 3. There was a gradual increase in melanin yield with increasing CuSO₄ concentration, but a slight decrease occurred when CuSO₄ concentration exceeded 0.1 g/L. Melanin yield was 462.69 mg/L when CuSO₄ concentration in fermentation medium was 0.1 g/L. Tyrosinase activity increased gradually by addition of CuSO₄ at the concentration rang of 0.01 – 0.2 g/L.

Fig. 3.

Effects of CuSO₄ concentration on tyrosinase activity and melanin yield.

Copper provided by CuSO₄, was an essential constituent of tyrosinase. When copper was transfered to the apotyrosinase, it resulted in incorporation of two copper ions and released the activated tyrosinase (Claus and Decker, 2006; Gruhn and Miller-Ji, 1991). Therefore, copper was one of the primary contributors to enhancement of total tyrosinase activity. However, the concentration of substrate l-tyrosine in fermentation medium was constant. Hence, the increase of tyrosinase activity would not further enhance the melanin yield. This finding makes the whole process of melanin production economically more feasible and efficient in the potential application in food industry.

Analysis of Box-Behnken experiment for melanin production    The medium components including wheat bran extract, l-tyrosine and CuSO₄ as independent variables were further optimized for the maximum melanin yield. The Box-Behnken design and the corresponding response values are shown in Table 1. A second-order polynomial model describing the correlation between melanin yield and the three variables in this study was obtained in eq. (2) below:   

The statistical significance of eq. (2) was checked by F-test, and the results of analysis of variance (ANOVA) are shown in Table 2. The model F-value of 55.73 obtained by ANOVA indicated that the model was very significant (P < 0.01). Meanwhile, the lack of fit F value of 5.86 and the associated P-value of 0.0558 indicated that the lack of fit was not significant. For the model fitted, the coefficient of determination (R²), which could check the quality of a model, was 0.9775 implying that the sample variation of 97.75% for the melanin yield was attributed to the independent variables, and only 2.25% of the total variation could not be explained by the model. Adjusted coefficient of determination (adjusted R²) is a modified measure of the coefficient of determination that takes into account the number of independent variables included in the regression equation and the sample size. The closer adjusted R² is to 1, the better does the developed model fit the actual data. ANOVA showed that the model designed in this study had a satisfactory value of adjusted R² (0.9599). These results suggested that the developed model could adequately represent the real relationship among the parameters chosen.

Table 2. Analysis of variance (ANOVA) of the regression model.

Source	Sum of squares	Degree of freedom	Mean squares	F-value	P-value
Model	57193.62	7	8170.52	55.73	< 0.0001
X1	13319.57	1	13319.57	90.85	< 0.0001
X2	1925.41	1	1925.41	13.13	0.0055
X3	356.44	1	356.44	2.43	0.1534
X12	17156.83	1	17156.83	117.03	< 0.0001
X22	8158.53	1	8158.53	55.65	< 0.0001
X32	7182.29	1	7182.29	48.99	< 0.0001
X1 X2	24.75	1	24.75	0.13	0.7252
X1 X3	1.10	1	1.10	0.01	0.9406
X2 X3	5484.88	1	5484.88	37.41	0.0002
Residual	1319.45	9	146.61
Lack of Fit	1160.85	5	232.17	5.86	0.0558
Pure Error	158.60	4	39.65
Corrected Total	58513.08	16

R² = 0.9775, adjusted R² = 0.9599.

Effects of wheat bran extract, l-tyrosine and CuSO₄ on melanin production    Response surfaces plots were used to illustrate the interactive effects of wheat bran extract, l-tyrosine and CuSO₄ concentrations on melanin production. Response surface plots for melanin yield are presented in Figs. 4–6.

Fig. 4.

Response surface plot showing the effect of wheat bran extract and l-tyrosine on melanin yield. CuSO₄ concentration was constant at 0.1 g/L.

Fig. 5.

Response surface plot showing the effect of wheat bran extract and CuSO₄ on melanin yield. l-Tyrosine concentration was constant at 1.5 g/L.

Fig. 6.

Response surface plot showing the effect of l-tyrosine and CuSO₄ on melanin yield. Wheat bran extract concentration was constant at 30%.

Fig. 4 shows the effect of wheat bran extract and l-tyrosine on melanin yield in A. auricula fermentation medium at a CuSO₄ concentration of 0.1 g/L. Wheat bran extract and l-tyrosine had very significant linear and quadratic effects (P < 0.01) on melanin yield. However, wheat bran extract and l-tyrosine did not interact significantly (P > 0.05) (Table 2). When wheat bran extract concentration was set, the melanin yield increased when l-tyrosine concentration reached a certain value (approximately 1.6 g/L), and then leveled off. At a fixed l-tyrosine concentration, melanin yield increased when wheat bran extract concentration was extended from 20% to 27% but rapidly decreased when wheat bran extract concentration was further elevated. This indicated that wheat bran extract was the principal factor affecting the melanin production. To avoid the decrease of melanin yield, wheat bran extract concentration should not exceed 27%.

Fig. 5 shows the effect of wheat bran extract and CuSO₄ on melanin yield in A. auricula fermentation medium at a constant l-tyrosine concentration of 1.5 g/L. CuSO₄ had very significant quadratic effect (P < 0.01). However, wheat bran extract and CuSO₄ did not interact significantly (P > 0.05) (Table 2). The melanin yield increased when CuSO₄ addition increased from 0.05 to 0.11 g/L; thereafter it decreased when CuSO₄ concentration was above 0.11 g/L. At a fixed CuSO₄ concentration, the melanin yield sharply increased with wheat bran extract addition at the beginning, but decreased rapidly when wheat bran extract concentration was raised from 27% to 40%, which also implied that melanin yield was significantly influenced by wheat bran extract.

Fig. 6 shows the effect of l-tyrosine and CuSO₄ on melanin yield in A. auricula fermentation medium at a constant wheat bran extract concentration of 30%. The interaction between l-tyrosine and CuSO₄ on melanin production was very significant (P < 0.01) (Table 2). At a fixed CuSO₄ concentration, l-tyrosine addition led to a gradual increase of melanin yield but it declined later. When l-tyrosine concentration was about 1.6 g/L, melanin yield reached its peak, indicating that too much l-tyrosine would decrease melanin yield. When l-tyrosine concentration was set, melanin yield gradually increased with the addition of CuSO₄ and the optimal concentration was about 0.11 g/L. Adding CuSO₄ to fermentation medium might stimulate tyrosinase activity and promote melanin production by A. auricula. Our results also confirmed that melanin synthesis was related to Cu2+, which was also reported in previous papers (Babitskaya et al., 2000; Santos and Stephanopoulos, 2008).

Optimization of medium composition and Verification of the model    According to the RSM test results, the optimal medium components to obtain the highest melanin yield were determined as follows: wheat bran extract 26.80%, l-tyrosine 1.59 g/L and CuSO₄ 0.11 g/L. The melanin yield was 519.54±23.16 mg/L and this value was not significantly different (P > 0.05) from the predicted value of 516.33 mg/L. These data proved that the model designed in this study was valid.

Conclusions

The effects of wheat bran extract, l-tyrosine and CuSO₄ on tyrosinase activity and melanin yield in A. auricula fermentation medium were investigated. The results revealed that wheat bran extract, l-tyrosine and CuSO₄ concentrations influenced tyrosinase activity and increased melanin yield. These factors were chosen to further optimize medium composition by Box-Behnken experiment design. ANOVA analysis in RSM indicated that the model could be employed to optimize medium composition for melanin production. The best combinations of medium components were 26.80%wheat bran extract, 1.59 g/L l-tyrosine and 0.11 g/L CuSO₄. Under the optimised conditions, the highest melanin yield (519.54 mg/L) was obtained. The present study is the first to report the statistical optimization of medium composition for production of melanin by A. auricula using cheaper wheat bran extract as a major nutrient source. These results might provide a reference for the development of a cost-effective medium for commercial production of melanin used in food industry.

Acknowledgments    This work was supported by Program for Liaoning Excellent Talents in University (No. LJQ2015031) and Fundamental Research Funds for the Central Universities (No. DC201501080).

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