Physicochemical Properties of Melanin from A. auricula Fruiting Bodies

The physicochemical properties of melanin from Auricularia auricula fruiting bodies were studied. The result showed that A. auricula fruiting bodies melanin powder was dark with a little red and yellow colored (L* = 41.03, a* = 2.26, b* = 3.78). A. auricula fruiting bodies melanin was insoluble in both water and common organic solvents. It dissolved only in alkali aqueous solution and precipitated acidic aqueous solution (pH<3). A. auricula fruiting bodies melanin was gradually oxidative bleached by oxidant and was stable to reducer. It exhibited strong optical absorbance in a wide UV-VIS spectral range. Furthermore, these physicochemical properties were very similar to those of the melanin reported in the previous studies and to those of synthetic melanin. A. auricula fruiting bodies melanin could potentially be used in the food industry as a natural colorant.


INTRODUCTION
Melanin is a dark-colored polyphenolic pigment produced from oxidative polymerization of phenolic or indolic compounds by tyrosinase (Chen et al., 2008).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, including strong light absorbance, unusual solubility and remarkable redox properties.In addition, melanin has a number of healthful functions, such as antioxidation (Tu et al., 2009), anti-HIV activity (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 is a precious macro-fungus distributed in the Northeast Provinces of China, which is a Tremellales fungus belonging to the Basidiomycotina and has been used as food and drug in China for a long time (Zhang et al., 1995).A. auricula fruiting bodies, a kind of edible black-brown mushroom, are rich in melanin and are increasingly popular as a "black food" in China.Melanin is considered to be one of the most important functional components in these "black food".However, there is little information available about physicochemical properties of melanin from A. auricula fruit-bodies in current reports.In this study, melanin was isolated and purified from A. auricula dried fruit-bodies and its physicochemical properties were investigated.

MATERIALS AND METHODS
Materials: Dried fruiting bodies of A. auricula were purchased from a local market in Dongning City (Heilongjiang Province, China), pulverized and sifted through a 40-mesh sieve.The powder (moisture content 12-15% on dry basis) stored in dark bags to prevent from moisture and light.All the chemicals and reagents used in the experiment were of analytical grade.

Isolation and purification of melanin:
A. auricula fruiting bodies powder was washed with running water at a ratio of 30 mL/g (water/raw materials) for 5 min, followed by centrifugation at 4,000 g for 5 min.The precipitate was immersed into water at a liquid-solid ratio 50 mL/g and the initial pH was adjusted to 12.0 with 1 M NaOH.Then, the mixture was sonicated at 50°C for 30 min by an ultrasound cleaning bath (KQ250-DB, Jiangsu Province, China) working at an ultrasound input power of 250 W. Afterward, the sample was centrifuged at 4,000 g for 5 min and the supernatant containing melanin was obtained.
Purification of melanin was performed as described by Wu et al. (2008) with proper modification.Melanin extract was first adjusted to pH 2.0 with 3 M HCl to precipitate melanin, followed by centrifugation at 10,000 g for 20 min and the was collected.The pellet was washed with chloroform, ethyl acetate and ethanol for three times.Finally, the purified melanin was lyophilized and stored at -20°C.
Visual color of melanin: Visual color of melanin powder was measured using a Minolta colorimeter (CR-400, Minolta Camera Co. Ltd., Osaka, Japan) with the Hunter Lab color system.The color values were expressed as L* (whiteness or brightness/darkness), a* (redness/greenness) and b* (yellowness/blueness).

Solubility of melanin:
The melanin (100 mg) was added to 10 mL of water, aqueous acid, alkali, or common organic solvents (such as ethanol, methanol, chloroform, acetone, aether, petroleum ether, benzene, ethyl acetate, butanol, etc.) under stirring for 1 h and stood for 0.5 h and then filtered.The absorbances of solutions were recorded at 400 nm in a UV-2802 diode array spectrophotometer (Unico Instrument Co. Ltd., Princeton, NJ, USA) to attain the solubility of melanin (Wang et al., 2006).
Redox properties of melanin: Redox properties of melanin were measured according to the basic procedure designed with minor modifications (Wang et al., 2006).Ten milliliter of 100 mg/L melanin solutions and 50 mL of different concentrations of KMnO 4 , K 2 Cr 2 O 7 , H 2 O 2 , NaOCl and Na 2 SO 3 were mixed and then the homogenate absorbance was determined at 400 nm.
UV-VIS absorption spectra of melanin: Melanin was separately dissolved in alkaline distilled water (pH 10.0) at final concentration of 20 mg/L.The UV-VIS absorption spectra of the melanin solution was scanned using a UV-2802 diode array spectrophotometer (Unico Instrument Co. Ltd., Princeton, NJ, USA) at wavelengths ranging from 200 to 800 nm.

RESULTS AND DISCUSSION
Visual color: Color values of A. auricula fruiting bodies melanin are shown in Table 1.Results from the colorimeter indicated that melanin presented lower L* value (41.03), a* value (2.26) and b* value (3.78) in Hunter Lab color system.This might be caused by high conjugation degree of melanin which resulted weak spectral absorbance.

Solubility:
As was shown in Table 2, the solubility experiments indicated that A. auricula fruiting bodies melanin was insoluble in both water and all common organic solvents (such as ethanol, methanol, chloroform, acetone, aether, petroleum ether, benzene, ethyl acetate, butanol, etc.).It dissolved only in alkali aqueous solution and precipitated in acidic aqueous solution (pH<3).The solubility of A. auricula fruiting bodies melanin was very similar to those of natural melanin previously reported and synthetic melanin (Babitskaya et al., 2000;Tu et al., 2009).

Redox properties:
The experimental results showed that A. auricula fruiting bodies melanin exhibited marked redox properties (Table 2).They were gradually However, the absorbance of the melanin remained almost unchanged in Na 2 SO 3 , indicating that melanin was stable to reducer.These results revealed that A. auricula fruiting bodies melanin presented the same redox properties of natural melanins previously reported (Babitskaya et al., 2000;Chen et al., 2008).

UV-VIS absorption spectra:
The UV-VIS spectra (200-800 nm) of A. auricula fruiting bodies melanin is shown in Fig. 1.Melanin absorbed strongly in the UV region and progressively less as the wavelength increased.According to a previous report (Riley, 1997), strong optical absorbance in a wide spectral range was one of the most conspicuous properties of melanin due to the high degree of conjugation in the molecule.However, A. auricula fruiting bodies melanin exhibited an additional shoulder at wavelength 260-280 nm.This property was similar to those of the melanins isolated from plant or animal, such as black tea (Sava et al., 2001), Osmanthus fragrans' seeds (Wang et al., 2006) and black-bone silky fowl (Tu et al., 2009).It was well known that normal proteins had an absorption maximum at about 280 nm.Therefore, A. auricula fruiting bodies melanin might contain a certain amount of protein.

Table 1 :
Color values of A. auricula fruiting bodies melanin