Comprehensive utilization of edible mushroom Auricularia auricula waste residue—Extraction, physicochemical properties of melanin and its antioxidant activity

Abstract In order to promote the comprehensive utilization of the Auricularia auricula waste residue, the extraction process and the physicochemical properties of melanin from A. auricula waste residue were studied. Furthermore, the chemical antioxidant activity of waste residue melanin and its protective effect on cell oxidative injury induced by H2O2 were investigated. The results indicated that the ultrasonic‐assisted extraction process could be used to extract the melanin from A. auricula waste residue. Melanin had a good solubility in alkali solution and exhibited a certain stability to thermal. There was no significant difference between A. auricula melanin control group and waste residue melanin on ABTS, DPPH, and hydroxyl radical scavenging activity. Waste residue melanin significantly inhibited the cell death caused by H2O2, and the cell viability was restored to 98.09 ± 5.97% when the melanin concentration was 1.6 mg/ml. Cell morphology observation confirmed that the melanin ameliorated the morphological changes of cells induced by oxidative stress.

. However, large amount of A. auricula waste residue will be produced during the extraction process of polysaccharides, and there is no good way to deal with these waste residues. Due to the special properties of melanin that are insoluble in water and organic solvents (Madhusudhan, Mazhari, Dastager, Mazhari, Dastager, & Agsar, 2014;Sun, Zhang, Chen, Zhang, Chen, Zhang, & Zhu, 2016), melanin still remains in the waste residues of A. auricula fruit body after extracting polysaccharides and other active substances. It has been reported that A. auricula melanin has good hepatoprotective (Hou et al., 2019) and antimicrobial activities (Bin, Wei, Xiaohong, Wei, Xiaohong, Mei, & Mingsheng, 2012). Thus, extracting the melanin from the waste residues of A. auricula can promote the comprehensive utilization and expand the application value of A. auricula.
In order to make the best use of the various active ingredients of A. auricula, the waste residue of A. auricula fruiting body after extraction of polysaccharide was selected as raw material to study the extraction process of melanin. Furthermore, the physical and chemical properties and antioxidant activity of the melanin were explored.
This work is of certain significance to the comprehensive utilization of A. auricula and also provides a reference for the utilization of the other edible fungus waste residue resources.

| Materials and chemicals
Auricularia auricula waste residue powder was obtained by drying the powder of fruiting body after the polysaccharide was extracted.
Human L02 hepatocyte cells were purchased from Fuheng Biological Technology Co.. ABTS radical and DPPH radical were purchased from Sigma-Aldrich. Chemical reagents such as sodium hydroxide, hydrochloric acid, ethyl acetate, chloroform, ethanol, salicylic acid, ferrous sulfate, and hydrogen peroxide were purchased from Sinopharm Co..

| Extraction procedure of melanin from Auricularia auricula waste residue
Auricularia auricula waste residue was added into a NaOH solution and placed in an ultrasonic bath with temperature and power control (PS-G60, JieKang Ultrasonic Equipment) to investigate the effects of various factors on the yield of melanin. After the extraction process was completed, the supernatant was obtained by centrifugation, and the melanin was purified according to the method previous reports Zou, Hu, Ma, Hu, Ma, & Tian, 2015).
The pH of the supernatant was adjusted to 1.5 with 6 mol/L HCl and kept in a 80°C water bath for 10 hr. The precipitate was collected by centrifugation, and the precipitate was washed with deionized water until neutral to obtain crude melanin. The crude melanin was redissolved in a 1.5 mol/L NaOH solution, and the insoluble impurities were removed by centrifugation. The pH of the supernatant was adjusted to 1.5 with 6 mol/L HCl and stored at 4°C for 5 hr. The precipitate was collected by centrifugation and washed to neutral with deionized water. Pure melanin was obtained by rinsing successively with chloroform, dichloromethane, ethyl acetate, absolute ethanol and deionized water, and then freeze-drying. The maximum absorption wavelength of the purified waste residue melanin was determined by an ultraviolet-visible spectrophotometer to be 215 nm. By measuring the absorbance of the melanin at 215 nm, a standard curve corresponding to the relationship between the mass concentration and the absorbance was plotted. The concentration of the melanin was calculated from the standard curve, and the melanin yield under each extraction condition was calculated according to Equation (1), the weight of the melanin extract is represented by w 0 , and the weight of A. auricula waste powder is represented by M.

| Experimental design
The effects of solid-liquid ratio (1:10 ~ 1:60), ultrasonic power (200 ~ 500 W), ultrasonic time (10 ~ 80 min), ultrasonic temperature (20 ~ 80°C), and NaOH concentration (0.125 ~ 0.750 mol/L) on the yield of melanin were investigated by single-factor experiments. Factors other than variables were kept unchanged in the experiment. The RSM experiment was carried out based on the factors that had greater influence on the yield of melanin in the single-factor experiment.
According to the principle of Box-Behnken design (BBD), the experiment was designed by Design-Expert 8.0.6 software to optimize the process parameters of ultrasonic-assisted extraction of waste residue melanin. Independent variables and their encoding levels are shown in Table 1. In order to evaluate the effect of independent variables on the yield of melanin, the independent variables A (Solid-liquid ratio), B (Ultrasonic power), and C (NaOH concentration) were designed and 17 experiments were carried out. The model of a second-order polynomial was expressed to predict the optimal point according to Equation (2), where Y represents the response variables, β 0 is the model constant, β i , β ii , and β ij are the coefficient of the linear effect, coefficient of the quadratic effect, and cross-product coefficients, respectively, X i and X j are the independent variables affecting the response. (1)

| Determination of chemical antioxidant activities of waste residue melanin
The water-soluble melanin was prepared based on previously reported methods (Yang et al., 2015) and used for further studies.
Waste residue melanin was dissolved in a 0.1 M NaOH solution and then adjusted pH to 7 with 0.1 m HCL solution under strong sonication. Then, water-soluble melanin was obtained by dialysis and freeze-drying.
The scavenging activity of A. auricula waste residue melanin to DPPH radical was measured on the basis of previously described methods . The melanin solution and 0.2 mM DPPH absolute ethanol solution were thoroughly mixed. The solution was placed in darkness for 30 min and measure the absorbance at 517 nm (A 1 ). The same volume of absolute alcohol was substituted for the DPPH solution, and the absorbance (A 2 ) was determined.
The same volume of distilled water was substituted for the melanin solution, and the absorbance (A 0 ) was determined. The scavenging capacity of DPPH radical was calculated according to Equation (3).
The ABTS radical cation (ABTS + ) test was conducted based on previously described methods . A 7 mmol/L ABTS solution and 2.45 mmol/L potassium persulfate solution were mixed, and the mixture was left in the dark for 16 hr. Then, the ABTS + solution was diluted with ethanol to an absorbance at 734 nm of 0.70 ± 0.02. Different concentrations of melanin solution were mixed with ABTS + solution and incubated in darkness for 60 min, and the absorbance was measured at 734 nm (A 1 ). Instead of the ABTS + working solution with an equal volume of distilled water, the absorbance was measured (A 2 ). Instead of the A. auricula melanin solution with an equal volume of distilled water, the absorbance was measured (A 0 ). The ability of ABTS radical scavenging rate was calculated according to Equation (3).
The hydroxyl radical scavenging rate was determined according to the procedure previously described (Ye et al., 2015). One milliliter of 9 mmol/L salicylic acid-ethanol solution, 9 mmol/L FeSO 4 solu-

| Protective effects of waste residue melanin on cell oxidative damage induced by H 2 O 2
The cell experiment was performed according to the method previously described (Yan et al., 2018). Human L02 hepatocytes were cultured with RPMI-1640 medium containing 10% FBS in a 37°C, 5% CO 2 incubator. The cells in logarithmic growth stage were inoculated into a 96-well plate at a density of 1 × 10 4 cells/well and cultured for 24 hr. The cells were pretreated with the melanin for 1 hr and then co-incubated with H 2 O 2 for 24 hr. The medium containing 10% FBS was changed to medium containing 0.5% FBS at 12 hr before melanin treatment to reduce the effect of serum.
MTT assay was used to detect cell viability. After the cells were well treated, the previous culture medium was removed, and 90 µl of fresh culture medium and 10 µl of 5 mg/ml of MTT were added.
After the cells were cultured at 37°C for 4 hr, the medium was removed and 150 µl of dimethyl sulfoxide (DMSO) was added to each well. The absorbance was measured at 490 nm by microplate reader. The morphology of L02 cells was observed by inverted microscope to study the effect of waste residue melanin on the morphology of cells.

| Statistical analysis
Design-Expert software 8.0.6 (Stat-Ease Inc.) and SPSS 13.0 (SPSS Inc.) were used to analyze the experimental data. Experiments were repeated at least in triplicate. The result values are presented as the mean ± standard deviation, p < .05 indicates a statistically significant difference, and p < .01 indicates a highly statistically significant difference.

| Single-factor experiment analysis
As shown in Figure 1a, the yield of melanin gradually increased when the solid-liquid ratio was from 1:10 to 1:40. Hence, 1:40 is chosen as the center of further experiments. As shown in Figure 1b, the yield of melanin increased when the ultrasonic power increased from 200 to 450 W. When the power reached 500 W, the yield began to decrease. So, 400 ~ 500 W was selected for the further experiments.
As shown in Figure 1c, the yield of melanin increased gradually when the ultrasonic time was from 10 to 50 min. After this, the yield of melanin was no longer increased. Therefore, 50 min was selected in the later RSM experiment. As shown in Figure 1d, the extraction rate of melanin increased with the increase in temperature before 70°C. However, when the temperature was higher than 70°C, the extraction rate remained unchanged. So, 70°C is a suitable temperature. As shown in Figure 1e, when the concentration of NaOH was 0.625 mol/L, the yield reached the maximum. After this, the yield began to decline. Therefore, 0.50 ~ 0.75 mol/L NaOH was chosen for the next experiment. According to the single-factor experiment, it can be concluded that the extraction rate of melanin remained unchanged when the ultrasonic time and temperature increased to a certain extent. Therefore, in the subsequent experiment, the ultrasonic time and the ultrasonic temperature were kept constant, and the optimum solid-liquid ratio, ultrasonic power, and NaOH concentration for extracting melanin from A. auricula waste residue were investigated.

| RSM model for waste residue melanin extraction process
According to the results of single-factor experiment, the ultrasonic  Table 2. The relationship between the melanin yield (Y) and three variables in coding factors was described by fitting second-order polynomial equation with linear multivariate regression method were as follows: The effectiveness of the fitted model was evaluated by analysis of variance (ANOVA). The results are shown in Table 3. The model F-value of 91.59 and the associated lower p-value (p < .0001) mean that the generated model is meaningful. The determination of the coefficient value (R 2 ) was 0.9916, and the adjusted R 2 value (Adj. R 2 ) was 0.9808 indicated a good correlation between the responses and independent variables. In addition, the low Fvalue (0.35) and associated high p-value (.5209) of "lack of fit" indicated the model is suitable for accurate prediction of variation (Quanhong & Caili, 2005).
The response surface curves were described by the regression model constructed, and corresponding three-dimensional response surfaces curves are shown in Figure 2. Each response surface plot illustrates the influence of two independent variables at an optimal level of the third variable. The optimal extraction conditions obtained by quadratic polynomial regression model were as follows: NaOH concentration was 0.58 mol/L, solid-liquid ratio was 1:44.01, and ultrasonic power was 461.82 W. Under optimum conditions, the predicted yield of melanin was 11.80%. Combined with the feasibility of practical operation, NaOH concentration of 0.58 mol/L, solid-liquid ratio of 1:44, and ultrasound power of 450 W were selected for verification experiments, and the yield of melanin was 11.99 ± 0.13%, which was in good agreement with the predicted value.
Ultrasound could produce strong vibration, cavitation effect, and agitation effect, thus promoting the dissolution of active components.
In comparison with traditional extraction methods, ultrasonic-assisted extraction can shorten the extraction time, improve the extraction efficiency, and reduce environmental pollution (Zhi, Xiaoxiang, & Jianrong, 2011). In this section, the ultrasound-assisted extraction conditions of melanin from A. auricula waste residues were optimized by single-factor experiment and RSM model, and the optimum process parameters were obtained to maximize the extraction rate of melanin. The results provide a reference for the comprehensive utilization of A. auricula.

| Physicochemical properties of melanin from Auricularia auricula waste residue
Melanin has typical characteristic absorption peaks in infrared spectra. The absorption peaks at about 3,300/cm correspond to the N-H   . The results showed that the melanin produced by A. auricula in different growth states was similar in structure.

TA B L E 2 Design and results of RSM
As shown in Table 4 and Figure 4a, the melanin has good solubility in alkaline solution (such as NaOH solution), but low solubility in distilled water and other organic solvents. The solubility of waste residue melanin was similar to the melanin that from other microorganisms (Kimura, Fukuda, Sanada, Fukuda, Sanada, & Imanaka, 2015;Madhusudhan et al., 2014). As shown in Figure 4b, the temperature had little effect on the stability of waste residue melanin.
With the prolongation of time, the absorbance of melanin changed little in different temperature. As shown in Figure 4c, the stability of  (Wu, Zhang, Yang, Zhang, Yang, Zhou, & Yang, 2018). As shown in Figure 4d and e, the melanin had a certain stability in reductants such as Na 2 SO 3 , but the stability was poor in oxidants such as H 2 O 2 . The melanin also reacted with oxidants such as KMnO 4 , causing the discoloration of the solution.

| Antioxidant activity of melanin from Auricularia auricula waste residue
The oxidation of ABTS produces relatively stable blue-green ABTS water-soluble free radicals. The reaction of antioxidants with ABTS radicals will cause the solution fade and the decrease of characteristic absorbance, the more obvious of the solution discoloration, the stronger of the total antioxidant ability of the tested substance (Zhao et al., 2008). DPPH is a stable-free radical in organic solvents. The alcohol solution of DPPH with a single electron appears purple, so it could accept one electron or hydrogen ion and have maximum absorption at a wavelength of 517 nm.
In the presence of free radical scavenger, the single electron of DPPH is captured, which caused the discoloration of the solution Hydroxyl radical is one of the important active free radicals causing tissue cell damage. The human body produces hydroxyl radicals through metabolism in normal life activities. Hydroxyl radicals are thought to be the most toxic free radicals, which can cause membrane peroxidation, protein cross-linking denaturation, and nucleic acid damage (Cacciuttolo, Trinh, Lumpkin, Trinh, Lumpkin, & Rao, 1993). As shown in Figure 5, A. auricula waste residue melanin has good antioxidant capacity, it has good scavenging activities to ABTS, DPPH, and hydroxyl radical, and its free radical scavenging activities were not significantly different from those of normal A. auricula melanin control group.

| Protective effect of waste residue melanin on H 2 O 2 -induced cell oxidative damage
The protective effect of melanin on H 2 O 2 -induced cell oxidative damage was studied by MTT assay and morphology of the cells.
First, different concentrations of H 2 O 2 were given to human normal liver L02 cells. As shown in Figure 6a,

| CON CLUS IONS
Auricularia auricula is a popular edible and medicinal fungus in Eastern countries. Polysaccharides and melanin are two active components in A. auricula. However, most of the studies and related products about A. auricula are focused on its polysaccharides.
After the extraction of A. auricula polysaccharides, the waste residue still contains melanin. Natural melanin can not only be used as a colorant, but also has many biological activities. Extracting melanin from the waste residue of A. auricula can make maximum use of A. auricula and reduce the waste of resources. In this study, the ultrasonic-assisted extraction process of melanin from A. auricula waste residue was optimized, and the physicochemical properties and antioxidant activity of melanin were also studied. The results indicated that the optimal extraction parameters of melanin from the waste residue of A. auricula were the NaOH concentra-

ACK N OWLED G M ENTS
This work was financially supported by the National Key R&D Program of China (2018YFD0400200).

CO N FLI C T O F I NTE R E S T
We declare that we have no conflict of interest.

E TH I C A L A PPROVA L
The uses of either humans or animals were not applicable in this study.