Journal of Food Processing and Preservation ISSN 1745-4549

FERMENTATIVE PRODUCTION OF MELANIN BY

AURICULARIA AURICULA
YU ZOU1 and MIXIA TIAN
College of Life Science, Dalian Nationalities University, Dalian 116600, China

1
Corresponding author. ABSTRACT
TEL. 1 86-411-87656215;
FAX: 1 86-411-87656215; Low cost fermentation medium using wheat bran extract as a major nutrient
EMAIL: yuzou126@126.com source, was evaluated for production of melanin from the fungus Auricularia
auricula in submerged culture. Effects of wheat bran extract, L-tyrosine and
Received for Publication October 10, 2015
CuSO4 on tyrosinase activity and melanin yield were investigated. Results showed
Accepted for Publication February 3, 2016
that wheat bran extract, L-tyrosine and CuSO4 concentrations influenced tyrosin-
doi:10.1111/jfpp.12909 ase activity and increased melanin yield. Box–Behnken design indicated the fol-
lowing 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 mela-
nin yield (519.54 mg/L) was obtained. Results from present study indicated
fermentative production was an economical and efficient preparation method of
melanin.

PRACTICAL APPLICATIONS
Melanin is a natural pigment with great development potential as a healthful food
colorant. However, the extraction process of melanin from animal or plant tissues
is tedious and expensive. This paper presents an alternative for melanin produc-
tion through A. auricula fermentation using low cost medium. Experimental
result confirmed that fermentative production was a convenient and efficient
preparation method of melanin, which might have great potential for being
scaled-up to a commercially production of melanin.

INTRODUCTION fruit bodies is tedious and expensive. In addition, A. auric-

Melanin is a dark-colored polyphenolic pigment produced
from oxidative polymerization of phenolic or indolic com-
pounds by tyrosinase (Riley 1997). These natural pigments
are synthesized by some fungi, plants, animals and several
bacterial species. Melanins from different sources possess
similar healthful functions, such as antioxidation (Wu et al.
2008; Tu et al. 2009; Liu et al. 2011), anti-HIV activity
(Montefiori and Zhou 1991; Manning et al. 2003), and
immunomodulatory activity (Sava et al. 2001). These func-
tions 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. 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). However,
the extraction process of melanin from tissues of A. auricula
ula 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. A large amount of melanin can also be produced
by A. auricula through submerged culture and the produc-
tion of melanin is mediated primarily via tyrosinase (EC
1.14.18.1). Therefore, promoting tyrosinase activity is help-
ful 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

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PRODUCTION OF AURICULARIA AURICULA MELANIN Y. ZOU and M. TIAN

considered as an effective approach. Many agricultural by- TABLE 1. BOX-BEHNKEN DESIGN AND EXPERIMENTAL MELANIN
products and waste materials have been used as carbon YIELD
sources to produce valuable compounds such as lactic acid, X1 X2 X3
succinic acid and xylanase (Gao et al. 2008; Zheng et al.
Wheat bran L-Tyrosine CuSO4 Melanin
2009; Walia et al. 2013). For the production of these com- Run extract (%) (g/L) (g/L) yield (mg/L)
pounds, wheat bran has been used as one of major sub-
1 30 1.5 0.10 513.17 6 23.67
strates, but its application in melanin production is little
2 40 2.0 0.10 384.89 6 12.80
explored. 3 20 2.0 0.10 461.66 6 22.49
In the present study, the production of melanin by 4 30 1.0 0.05 362.93 6 23.76
A. auricula was carried out using low cost fermentative 5 40 1.5 0.15 356.65 6 17.85
substrate (wheat bran extract). The effects of other 6 30 1.5 0.10 502.24 6 32.51
parameters (L-tyrosine and CuSO4) on tyrosinase activ- 7 20 1.0 0.10 411.31 6 12.83
8 20 1.5 0.05 451.01 6 23.37
ity and melanin yield were also investigated. Response
9 30 1.5 0.10 505.14 6 21.98
surface methodology (RSM) was employed to optimize 10 20 1.5 0.15 447.02 6 26.60
medium composition (wheat bran extract, L-tyrosine 11 40 1.5 0.05 358.54 6 21.69
and CuSO4) in order to obtain the maximum melanin 12 30 1.5 0.10 504.68 6 29.08
yield. 13 30 2.0 0.15 409.25 6 20.86
14 30 1.0 0.15 466.63 6 19.16
15 30 1.5 0.10 516.97 6 31.68
MATERIALS AND METHODS 16 40 1.0 0.10 344.50 6 12.73
17 30 2.0 0.05 453.67 6 19.66
Materials
Wheat bran was generously supplied by Jiangnan Co., Ltd. Effects of Medium Components on
(Jiangsu Province, China), pulverized and sifted through a Tyrosinase Activity and Melanin Production
0.42 mm sieve, respectively. Nutrients from the powder were In order to determine the proper scope of medium compo-
extracted with deionized water at a ratio of 4 mL/g (water/ nents for tyrosinase activity and melanin production from
raw materials) at 60C for 5 h, followed by incubation at the fungus A. auricula in submerged culture, different cul-
100C for 0.5 h. The homogenate was centrifuged at ture media were prepared as follows: Various wheat bran
4,000 rpm for 5 min and the supernatant was used as the extract concentrations ranging from 10% to 60% (v/v), L-
major nutrient source in fermentation medium (wheat bran tyrosine at a range of concentrations from 0.5 to 10 g/L,
extract). Synthetic melanin and L-tyrosine were purchased CuSO4 at different concentrations ranging from 0.01 to
from Sigma Chemicals Co. (St. Louis, USA). All the other 0.2 g/L. The desirable concentration scope of medium com-
chemicals and reagents used in the experiment were of ana- ponents resulted in higher melanin yield was used to opti-
lytical grade. mize melanin production by A. auricula using RSM.

Microorganism and Flask Culture Response Surface Optimization of Medium

Composition for Melanin Production
The fungal strain A. auricula RF201 was purchased from
the Institute of Edible Mushroom of Jiangsu Academy of Wheat bran extract (X1), L-tyrosine (X2) and CuSO4 (X3)
Agricultural Sciences (Jiangsu Province, China). For pro- were chosen as independent variables in Box-Behnken
duction of the inoculum, A. auricula was inoculated to experimental design. Based on the single-factor experi-
seed medium (potato dextrose broth medium) from ments, X1 (20%, 30% and 40%), X2 (1, 1.5 and 2 g/L) and
potato dextrose agar slant and then incubated at 25C on a X3 (0.05, 0.1 and 0.15 g/L) were determined as critical levels
reciprocating shaker (100 rpm) for 5 d. The inoculum with significant effect on melanin production (Table 1).
(10%, v/v) was transferred into an Erlenmeyer flask The experimental results were analysed by quadratic step-
(250 mL) containing 50 mL of fermentation medium. The wise regression to fit the second-order Eq. (1):
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/ X
3 X3 X
3
Y 5b0 1 Bi Xi 1 Bii Xi 2 1 Bij Xi Xj (1)
L KH2PO4 and 0.1 g/L vitamin B1. The values of medium i51 i51 i51
composition were varied based on the experimental
design. All media were cultivated at 25C for 5 d at the where Y stands for melanin yield, X1, X2, X3 for independent
pH 8.0 and rotation speed of 100 rpm. variables, b0 for the model intercept and Bi, Bii, Bij for

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Y. ZOU and M. TIAN
FIG. 1. EFFECTS OF WHEAT BRAN EXTRACT CONCENTRATION ON
TYROSINASE ACTIVITY AND MELANIN YIELD
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 4,000 rpm for 20
minutes at 4C, and then the supernatant was used for
enzyme activity measurement. Tyrosinase activity was deter-
mined by measuring the oxidation products of L-tyrosine at
37C 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 minutes at
475 nm. One unit of tyrosinase activity was defined as the
amount of enzyme that catalyzes the appearance of 1 lmol
of L-dopachrome (molar extinction coefficient
e 5 3,700 M21 cm21) 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 (4,000 rpm, 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 10,000 rpm for 20 minutes 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 mela-
nin 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.
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PRODUCTION OF AURICULARIA AURICULA MELANIN
FIG. 2. EFFECTS OF L-TYROSINE CONCENTRATION ON TYROSINASE
ACTIVITY AND MELANIN YIELD
Statistical Analysis
The experimental results obtained were expressed as
means 6 SD of triplicates. Statistical analysis was performed
using Fisher’s F-test and p < 0.05 was regarded as
significant.
RESULTS AND DISCUSSION
Effects of Wheat Bran Extract on Tyrosinase
Activity and Melanin Yield
The effects of increasing wheat bran extract concentrations
on tyrosinase activity and melanin yield in A. auricula fer-
mentation 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.
The nutrient of wheat bran was enwrapped in cellu-
lose 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 pro-
duction 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 pro-
mote tyrosinase synthesis and melanin production of
A. auricula (Rouanet et al. 1989; Gao et al. 2008;
Hemery et al. 2009). More importantly, wheat bran was
an abundant and very cheap agricultural residue 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
3
PRODUCTION OF AURICULARIA AURICULA MELANIN Y. ZOU and M. TIAN

ably caused by increases in the substrate in the vicinity

FIG. 3. EFFECTS OF CUSO4 CONCENTRATION ON TYROSINASE
ACTIVITY AND MELANIN YIELD
of the cytosolic tyrosinase, which suggested that
tyrosinase activity and melanin yield were regulated by
L-tyrosine addition. However, at higher L-tyrosine con-
centration (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 Azmi
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 nega-
tive effect on tyrosinase synthesis and melanin produc-
tion. Thus, comparatively lower L-tyrosine concentration
should be used for the production of melanin to avoid
its precipitation.

sugar and phytic acid concentration inhibited tyrosinase
synthesis and melanin production (Lagunas-Mu~ noz
et al. 2006; Wang et al. 2008).
Effects of L-Tyrosine on Tyrosinase Activity
and Melanin Yield
As expected, L-tyrosine addition effectively increased mela-
nin 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. Tyrosin-
ase 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.
The induction by L-tyrosine of tyrosinase synthesis has
also been reported (Rhee et al. 2002; Chandel and Azmi
2009). Elevating L-tyrosine levels can also stimulate mel-
anin production (Santos and Stephanopoulos 2008).
These increases of melanin in the experiment were prob-
Effects of CuSO4 on Tyrosinase Activity and
Melanin Yield
The effects of CuSO4 addition on tyrosinase activity and
melanin yield in A. auricula fermentation medium are pre-
sented 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 con-
centration in fermentation medium was 0.1 g/L. Tyrosinase
activity increased gradually by addition of CuSO4 at the
concentration range of 0.01–0.2 g/L.
Copper provided by CuSO4, was an essential constituent
of tyrosinase. When copper was transfered to the apotyrosi-
nase, it resulted in incorporation of two copper ions and
released the activated tyrosinase (Gruhn and Miller-Ji 1991;
Claus and Decker 2006). 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

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
X1X2 24.75 1 24.75 0.13 0.7252
X1X3 1.10 1 1.10 0.01 0.9406
X2X3 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 5 0.9775

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Y. ZOU and M. TIAN
of tyrosinase activity would not further enhance the melanin
yield. This finding makes the whole process of melanin pro-
duction 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:
Y 52787:09134:22X1 1707:37X2 20:64X12 2176:08X22
216520:51X32 21481:21X2 X3
(2)
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 significant (p < 0.05).
Meanwhile, the lack of fit P-value of 0.0558 indicated that
the lack of fit was not significant. For the model fitted, the
coefficient of determination (R2) was 0.9775 implying that
the sample variation of 97.75% for the melanin yield was
attributed to the independent variables. These results sug-
gested that the developed model could adequately represent
the real relationship among the parameters chosen.
Optimization of Medium Composition and
Verification of Model
According to the RSM test results, the optimal medium
components to obtain the highest melanin yield were deter-
mined as follows: wheat bran extract 26.80%, L-tyrosine
1.59 g/L and CuSO4 0.11 g/L. The verification of the model
was performed by the method previously reported by
Derringer and Suich (1980). Under the optimal conditions,
the melanin yield was 519.54 6 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
Wheat bran extract, L-tyrosine and CuSO4 concentrations
influenced tyrosinase activity and increased melanin yield.
The best combinations of medium components were
26.80% wheat bran extract, 1.59 g/L L-tyrosine and 0.11 g/L
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PRODUCTION OF AURICULARIA AURICULA MELANIN
CuSO4. Under the optimized conditions, the highest mela-
nin yield (519.54 mg/L) was obtained. The present study
might provide a reference for the development of a cost-
effective medium for commercial production of melanin
used in food industry.
NOMENCLATURE
ANOVA Analysis of variance
RSM Response surface methodology
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