Food Chemistry 135 (2012) 2490–2497 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Physicochemical characteristics and antioxidant activity of arginine-modified melanin from Lachnum YM-346 Ming Ye ⇑, Yan Wang, Geng-yi Guo, Yun-long He, Ying Lu, Ying-wang Ye, Qing-hua Yang, Pei-zhou Yang College of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, Anhui 230009, China a r t i c l e i n f o Article history: Received 15 March 2012 Received in revised form 20 June 2012 Accepted 21 June 2012 Available online 14 July 2012 Keywords: Lachnum Melanin Arginine modification Water solubility Antioxidant activity a b s t r a c t Seven kinds of amino acids were used to modify the non-water-soluble extracellular melanin (LEM346) from Lachnum YM-346. It was found that arginine–melanin (ALEM346) had the highest solubility, being 4.55% (g g 1) in 30 °C distilled water. Elemental analysis, infrared spectrum and mass spectrum analysis revealed that LEM346 molecule contained indole quinone structure, its molecular formula speculated to be C18H8O6N2. Infrared spectrum analysis showed that ALEM346 had characteristic absorption peaks at 1672.346 and 1637.679 cm 1. Mass spectrum analysis indicated that ALEM346 contained three types of arginine–melanin molecules. When the ALEM346 concentration was 500 mg L 1, its total antioxidant capacity was equivalent to a-tocopherol of 46.65 mmol L 1, the DPPH and O2  scavenging rates and the Fe2+ chelating rate were 89.05%, 93.81% and 80.18%, respectively, suggesting that the antioxidant activity of ALEM346 was stronger than that of LEM346. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Melanins are biological macromolecules composed of various types of phenolic or indolic monomer (Butler & Day, 1998). Melanins are produced by animals, plants, and microbes (Gómez-Marín & Sánchez, 2010; Tu, Sun, Tian, Xie, & Chen, 2009; Wang, Pan, Tang, & Huang, 2006), and can be used as light protectant, anti-radiation agent, chelating agent, immunostimulating agent, liver-protecting agent, natural antioxidant and biological semiconductor material (Bettinger, Bruggeman, Misra, Borenstein, & Langer, 2009; Sava, Galkin, Hong, Yang, & Huang, 2001; Sava, Hung, Blagodarsky, Hong, & Huang, 2003; Szpoganicz, Gidanian, Kong, & Farmer, 2002; Tu et al., 2009). Some animals and plants have melanin in their bodies, but resources are limited. Using microbial fermentation to produce melanin has the advantages of not being restricted by seasons, being low cost, being easy to operate, and requiring mild reaction conditions. Bacillus subtilis, Laetiporus sulphureus, Hypoxylon archeri and Lachnum can produce melanin (Gómez-Marín & Sánchez, 2010; Olennikov, Agafonova, Stolbikova, & Rokhin, 2011; Wu, Shan, Yang, & Ma, 2008; Ye, Xu, Chen, Yang, & Lin, 2010), but they are only soluble in alkaline solution, and insoluble in water generally, which has substantially limited their application in industry, agriculture and medicine. However, it was reported that methylation of tyrosine melanin (non-water-soluble) could be used to make it soluble in water (Wilczok, Bilinska, Buszman, & Kopera, 1984) and Monas⇑ Corresponding author. Tel.: +86 5512901505x8614; fax: +86 5512919368. E-mail address: yeming123@sina.com (M. Ye). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.06.120 cus pigment modified by amino acids could also soluble in water (Gan, Jian, Guan, Liu, & Yang, 2008). Lachnum is a class of saprophytic fungi. For many years, domestic and foreign scholars have studied its biodiversity (Chen, Ye, & Tan 2008). In recent years, we have found that some species of Lachnum, under submerged culture conditions, can produce a great amount of melanin, which was stable to temperature, light, food additives, reducing agents, etc., but insoluble in water (Ye, Wang, Qian, Chen, & Hu, 2011; Ye, Xu, Chen, Liang, & Sun, 2009; Ye et al., 2009; Ye et al., 2010). This study is intended to obtain extracellular melanin (LEM346) by fermentation of Lachnum YM-346, modify its molecule with different amino acids, and reveal its physicochemical characteristics and evaluate its antioxidant activity. 2. Materials and methods 2.1. Strain Lachnum YM-346 was isolated and preserved in the Laboratory of Microbial Resources and Application of Hefei University of Technology. 2.2. Preparation and solubility of LEM346 The extracellular melanin of Lachnum YM-346 (LEM346) was fermented, extracted and purified according to the method of Ye et al. (2010). Fermentation medium was as follows: potato extract M. Ye et al. / Food Chemistry 135 (2012) 2490–2497 20%; glucose 20.0 g L 1; yeast extract 5.0 g L 1; tyrosine 0.02 g L 1; magnesium sulfate 0.01 M and pH 8.0. LEM346 (10 mg) was added to 20 mL of water, aqueous acid, alkali, or common organic solvents (such as chloroform, ethyl acetate, ethanol, acetic acid, ether, hexane and acetone), shaken for 30 min and centrifuged at 5000 rpm for 10 min. The absorbance values of supernatants were recorded at 500 nm in a TU-1810PC UV–Vis spectrophotometer (Shanghai, China) to measure the solubility of melanin, as reported by Tu et al. (2009). 2.3. Amino acid modification of LEM346 Aspartic acid, threonine, lysine, histidine, arginine, tryptophan and glycine (0.05 g of each) were dissolved in 10 mL distilled water to prepare solutions of 5 g L 1 added with 0.1 g LEM346, shaken for 30 min and centrifuged at 5000 rpm for 10 min. The supernatants obtained were diluted 20 times with distilled water, and the absorbance values at 500 nm were measured. Sample with no added amino acids was used as blank control. The greater the absorbance value is, the higher the water solubility of the amino acid-melanin is. Arginine-melanin (ALEM346) with the greatest absorbance value was used for the following experiments. Arginine at 0.025, 0.05, 0.075, 0.1, 0.125 and 0.15 g was dissolved in 10 mL distilled water, added with 0.1 g LEM346, shaken till full dissolution, and after centrifugation and dilution, the absorbance values of the solutions were measured. The greatest absorbance value was used to determine the addition amount of arginine. ALEM346 was dialysed with distilled water for 48 h and freeze dried for the following experiments. 2.4. Preparation of melanin solutions ALEM346 (1 g) was taken and dissolved in 100 mL distilled water, and diluted to the desired concentration. LEM346 of 1 g was dissolved in 100 mL ammonia water (5%). After being heated in a 50 °C water bath for 1 h, NH3 was removed by evaporation to obtain melanin solution with pH 7–7.5, which was diluted to the desired concentration. The above two solutions were prepared for using in the following experiments. 2.5. Physicochemical characteristics 2.5.1. Determination of the water solubility of ALEM346 Distilled water (exactly 10 mL) was put in a dry beaker, added with a certain amount of ALEM346, and stirred in a 30 °C water bath. After 30 min, there was still melanin solid not dissolved. The sample was centrifuged at 5000 rpm for 10 min, and the supernatant was discarded. The remaining melanin solid was dried and weighed. 2.5.2. Determination of the colour value Method of Wang et al. (2006) with minor modification was used for the determination. ALEM346 solution and LEM346 solutions (1 mL; 50 mg L 1) were prepared, and their absorbance values at their respective maximum absorption wavelengths were measured. The colour value E1%1 cm = AB/M, where A is the absorbance value, B is the dilution times, and M is the mass of melanin. 2.5.3. SEM images of LEM346 According to the method of Gómez-Marín and Sánchez (2010) with minor modification, LEM346 was scanned and imaged for different amplified times using JSM-6490LV scanning electron microscope with a working voltage of 20 kV and a working distance of 6.0 mm. 2491 2.5.4. Elemental analysis of LEM346 Elemental analyser (Elementar Vario EL elemental analyser, Hanau, Germany) was adopted to determine the percentage contents of C, H, O and N in LEM346. 2.5.5. Ultraviolet–visible absorption spectra TU-1810PC UV–Vis spectrophotometer (Shanghai, China) was used to determine the maximum absorption wavelengths of LEM346 and ALEM346 between 190 and 800 nm. 2.5.6. Infrared spectrum LEM346 and ALEM346 (2 mg of each) were evenly mixed with 400 mg KBr, and pressed into tablets. The samples were scanned by a 6700 FT-IR spectrometer between 4000 and 500 cm 1 (Ye et al., 2011). 2.5.7. Mass spectrum Liquid Chromatography-Time of Flight Mass Spectrometer (ACQUITY UPLC-LCT Premier XE; Waters, Milford, MA) was used to determine the main peaks and molecular weights of LEM346 and ALEM346. For the mass spectrometer, the following parameters were used: ion source (ESI), MS scan (positive V optics), MS range (m/z 100–1000), capillary voltage (2400 kV), cone voltage (60 V), desolvation temperature (350 °C), source temperature (110 °C), desolvation gas flow (600 L h 1), cone gas flow (40 L h 1). 2.5.8. 1H NMR measurement Bruker Avance AV-400 spectrometer (Bruker BioSpin GmbH; Rheinstetten, Germany) was used for 1H NMR analysis with a frequency of 500.13 MHz, a temperature of 298 K and a delay time of 1 s. The samples were dissolved in D2O/NaOD, and chemical shifts are given in ppm. 2.6. Antioxidant activity 2.6.1. Measurement of total antioxidant capacity Method of Bougatef et al. (2009) with minor modification was adopted. ALEM346 solutions (1 mL) of different mass concentrations (50–500 mg L 1, the same hereinafter) were evenly mixed with 10 mL of reagents (containing H2SO4, Na3PO4 and ammonium molybdate with the final concentrations being 0.6, 28 and 4 mM, respectively), and placed in a water bath at 80 °C for 90 min. After cooling to room temperature with running water, the absorbance values were measured at 695 nm. LEM346 was used as the control. The total antioxidant capacity was expressed by the a-tocopherol equivalents: A = 0.011C + 0.0049 (R2 = 0.987), where A was the absorbance value at 695 nm, and C was the equivalent concentration of a-tocopherol (mM). 2.6.2. DPPH scavenging activity assay DPPH ethanol solution (0.1 mM; 1 mL) was evenly mixed with ALEM346 solutions of different concentrations (3 mL for each); the mixtures were shaken vigorously and left to stand for 30 min in the dark, and then the absorbance values (Asample) of the mixtures at 5l7 nm were measured (Turkoglu, Duru, Nazime, Kivika, & Gezer, 2007). The ethanol solution was used instead of the sample solution as blank control (Ablank), LEM346 and BHT (2,6-di-tertbutyl-4-methylphenol) were used as positive control. DPPH
scavenging rate (%) = 100  [(Ablank Asample)/Ablank]. 2.6.3. O2  scavenging activity assay Pyrogallol oxidation method was used (Xu & Guo, 2008). ALEM346 solutions (1 mL) of different concentrations were evenly mixed with 3 mL 50 mM Tris–HCl buffer (pH = 8.2). The mixtures were left standing at 25 °C for 20 min, then added with 0.3 mL pyrogallol solution (7 mM) that had been preheated at 25 °C. After 2492 M. Ye et al. / Food Chemistry 135 (2012) 2490–2497 shaking and reaction for 4 min, 1 mL 10 mM HCl was added to terminate the reaction. The absorbance values (Asample) at wavelength 318 nm were measured. LEM346 was used as the control. O2  scavenging rate (%) = 100  [1 (Asample Asample0 )/Ablank], where Asample0 indicates the absorbance value using water instead of reagents; Ablank indicates the absorbance value using water instead of sample solutions. 2.6.4. Fe2+ chelating activity assay ALEM346 solutions (1 mL) of different concentrations were evenly mixed with 0.05 mL FeCl2 (2 mM), and added with 0.2 mL ferrozine solution (5 mM). The mixtures were shaken and left standing at room temperature for 20 min, the absorbance values (Asample) of the mixtures were measured at 562 nm. Distilled water was used instead of the sample solution as blank control (Ablank), LEM346 and EDTA-2Na were used as positive control. Fe2+ chelating rate (%) = 100  [(Ablank Asample)/Ablank] (Wang, Zhang, Zhang, & Li, 2008). 3. Results and discussions 1:1 (weight of arginine being 0.1 g). When the arginine amount was greater than 0.1 g, the absorbance value of the solution almost remained in the range of 0.402–0.403. Accordingly, the optimum mass ratio of arginine and LEM346 was 1:1. 3.3. Physicochemical characteristics 3.3.1. The water solubility of ALEM346 Similarly, ALEM346 was insoluble in common organic solvents, redissolved only in alkaline solution and precipitated in acidic aqueous solution. Interesting, ALEM346 was soluble in water, and its solubility in 30 °C distilled water was 4.55% (g g 1). Therefore, ALEM346 was a water-soluble melanin. 3.3.2. The colour value Low colour value is an important factor to inhibit the industrial production of pigment by liquid fermentation (Yi, Shen, Han, Fan, & Gu, 2005). The colour values (E1%1 cm) of ALEM346 and LEM346 were 194.6 and 221.0, respectively, both higher than that of melanin isolated from Osmanthus fragrans seeds (E1%1 cm = 60.24) (Wang et al., 2006). 3.1. LEM346 and its solubility The extracellular melanin extracted from the fermentation broth of Lachnum YM-346 was frozen and dried under vacuum after alkaline extraction and acid hydrolysis and organic solvent (chloroform, ethyl acetate and ethanol) treatment to obtain LEM346, and its yield was 1.805 g L 1. The solubility experiments indicated that LEM346 was insoluble in water and common organic solvents (such as chloroform, ethyl acetate, ethanol, acetic acid, ether, hexane and acetone), redissolved only in alkaline solution and precipitated in acidic aqueous solution. 3.2. Amino acid modification of LEM346 The absorbance values of LEM346 solutions under the effect of 7 different amino acids were greatly different from each other; i.e., there was great difference in the solubility between them. The arginine-melanin (ALEM346) solution had the highest absorbance value (0.357 ± 0.005), followed by the lysine-melanin and histidine-melanin, and the addition of aspartic acid, threonine, tryptophan and glycine hardly increased the solubility of the melanin in distilled water. Arginine was the best modifying amino acid. When the weight of arginine was in the range of 0–0.1 g, the absorbance value of the solution increased with the increase of the arginine amount. The absorbance value reached a maximum (0.403 ± 0.008) when the mass ratio of arginine and LEM346 was 3.3.4. Elemental analysis of LEM346 The percentages of C, H, O and N in LEM346 were 62.1 ± 0.25, 2.3 ± 0.014, 27.6 ± 0.13 and 8.0 ± 0.041, respectively. The C/H in LEM346 was high, indicating that there are many aromatic structures in the LEM346 molecule. The N content in LEM346 was between 6% and 9%, and there was no sulfur contained, indicating that LEM346 is an indole-type eumelanin, which agrees with the literature (Butler & Day, 1998). 3.3.5. Ultraviolet–visible absorption spectra LEM346 and ALEM346 had maximum absorption peaks at 211 and 202 nm (Fig. 1), respectively, which was similar to the maximum absorption wavelengths of L. singerianum melanin and Lachnum YM-223 melanin (Ye et al., 2009; Ye et al., 2011). The UV–Vis spectrum showed an exponential decay without distinguishing characteristics in the region between 202–800 nm. There was no absorption peak between 260–280 nm in the UV spectra, indicating that LEM346 and ALEM346 do not contain protein and nucleic acid. b 1.4 1.4 1.2 1.0 1.0 0.8 0.8 A 1.2 A a 3.3.3. SEM images of LEM346 SEM images of LEM346 showed that it had a blocky, crystal structure with an irregular surface, which was similar to that of the melanin from Lachnum singerianum (Ye, Chen, Li, Guo, & Yang, 2011). 0.6 0.6 0.4 0.4 0.2 0.2 0.0 200 0.0 300 400 500 600 700 800 200 300 400 500 /nm /nm Fig. 1. UV spectra of LEM346 (a) and ALEM346 (b). 600 700 800 M. Ye et al. / Food Chemistry 135 (2012) 2490–2497 2493 Fig. 2. Mass spectra of LEM346 (a) and ALEM346 (b). 3.3.6. Mass spectrum analysis Mass spectrometry is an effective method to study the molecular structure of organic compounds. It speculates the molecular weight and possible molecular structure of the unknown compound via the information obtained by the ion fragments. The molecular weight of LEM346 can be determined to be 348 based on the quasi-molecular ion peaks of [M]+ and [M+H]+ in its mass spectrum (Fig. 2a). Combined with the elemental analysis results, we speculate that its molecular formula is C18H8O6N2 (meeting the nitrogen rule), with the unsaturation degree being 15. Melanin is formed by polymerisation of indole quinone, which is produced through a series of chemical reactions of tyrosine (Víctor et al., 2010). Based on the molecular formula, unsaturation degree, infrared spectrum and mass spectrum of LEM346, its proposed structural formula is shown in Fig. 3. The mass-to-charge ratios (m/z) of ALEM346 (348, 522, 696, 870) have a difference of 174 between adjacent values (Fig. 2b), which is equal to the molecular weight of arginine. Accordingly, 522, 696 and 870 are weights of the arginine-melanin molecules formed by combination of one LEM346 molecule with one, two and three arginine molecules, respectively. 2494 M. Ye et al. / Food Chemistry 135 (2012) 2490–2497 O N COOH O O N O CH3 Fig. 3. Possible structural formula of LEM346. 3.3.7. Infrared spectrum analysis Infrared spectroscopy has been used in study of the chemical structure of many melanins such as the melanin from the muscles of Taihe black-bone silky fowl, the melanin from bacillus thuringiensis and the melanin of L. sulphureus, etc. (Aghajanyan et al., 2005; Olennikov et al., 2011; Tu et al., 2009). Fungal melanin has characteristic absorption peaks near 3333 and 1639 cm 1 because it has C@O and O(N)–H groups (Bilinska, 1996). Infrared spectrum of LEM346 (Fig. 4a) showed absorption maxima at 3218.802 cm 1, caused by oscillation of O–H groups linked by N–H groups of indole ring; 2923.404 and 2811.457 cm 1, caused by stretching vibration of C–H neighbouring to quinone; 1614.815 cm 1, caused by stretching vibration of C@O of quinone neighbouring to the carboxyl group and influenced by another C@O adjacent to it; 1400.067–1386.526 cm 1 were due to the presence of amide and amine groups and deformational changes in NH groups of secondary amines, CH2 groups of aliphatic radicals, CH groups adjacent to COOH and OH groups; 1280.502–1236.474 cm 1 were caused by stretching vibration of C@O groups of acid (Babitskaya, Scherba, Filiminova, & Grigorchuk, 2000). These results indicated that LEM346 contains carboxyl-substituted indole quinone and methyl-substituted indole quinone structure. The absorption peaks of the infrared spectrum of ALEM346 were different from that of LEM346 (Fig. 4b). ALEM346 had two absorption peaks between 1700 and 1600 cm 1 (at 1672.346 and 1637.679 cm 1), which were the characteristic absorption peaks formed by binding of the carboxyl group in melanin and the amino group in arginine. In addition, ALEM346 had the absorption peaks of C@N stretching vibration of guanidino (1496.45–1574.906 cm 1) and C–H bending vibration of (CH2)3 (1463.706 and 1126.225–1315.214 cm 1) in arginine. a 100 3.4.1. Total antioxidant capacity a-Tocopherol is currently one of the most widely used antioxidants. The total antioxidant capacity of the active substance can be measured by comparison with a-tocopherol (Bougatef et al., 2009). The total antioxidant capacities of both ALEM346 and LEM346 increased with the increase of the concentration (Fig. 6a), and exhibited a good dose–effect relationship (ALEM346: y = 0.0832x + 5.0453, R2 = 0.9826; LEM346: y = 0.0788x + 2.776, R2 = 0.9802). ALEM346 and LEM346 at 500 mg L 1 were equivalent to a-tocopherol of 46.65 and 42.18 mM, respectively. The total antioxidant capacities of ALEM346 were higher than that of LEM346. The total antioxidant capacities of ALEM346 and LEM346 were both lower than that of BHA, but were both significantly higher than that of the extracellular melanin of Lachnum YM-223 (Ye et al., 2010). 3.4.2. DPPH scavenging activity DPPH has a single electron, becomes purple in alcohol solution, and has strong absorption at 517 nm. After adding antioxidants into the DPPH reaction system, the antioxidant can pair with the single electron of DPPH and reduce the amount of DPPH, gradually weakening the solution colour (Kim, Lee, Lee, & Lee, 2002), so DPPH scavenging activity of the antioxidant is measured at 517 nm. The DPPH scavenging activities of both ALEM346 and LEM346 increased with the increase of the concentration 831.051 653.422 20 3500 3000 2500 2000 1500 Wavenumbers (cm-1) 1000 500 20 4000 3500 1672.346 1637.679 1574.915 1408.015 2928.890 40 3376.585 1236.474 1088.863 1614.815 1539.075 1386.526 2811.457 3218.802 40 60 3137.729 %Transmittance 80 60 2923.404 %Transmittance 3.4. Antioxidant activity b 100 80 4000 3.3.8. NMR analysis 1 H NMR spectrum of LEM346 solution presented a series of broad peaks (Fig. 5a), in the 2.2–2.5 ppm region assigned to the CH3 group that is connected to the indole groups. In the 2.5– 3.5 ppm region peaks were assigned to NH group that is connected to the indole groups. The resonance at 3.5–4.0 ppm was assigned to the CH group that is connected to NH group. Peaks in the 7.0– 7.5 ppm region may correspond to the indole or other aromatic heterocyclic ring of the melanin polymer chain (Jalmi, Bodke, Wahidullah, & Raghukumar, 2012; Ye et al., 2011). Comparing the 1H NMR spectrum of ALEM346 (Fig. 5b) with that of LEM346, the peak at 2.5–3.5 ppm region has disappeared, showing that the NH group of LEM346 was connected to the OH group of arginine. A new peak at 1.4–2.0 ppm was assigned to CH2 group of arginine backbone chain. The resonance at 3.5–4.3 was assigned to CH group that is connected to NH2 group and COOH group in arginine (Shriner, Hermann, Morrill, Curtin, & Fuson, 2007, chap. 6 and 7; Xu, Liu, Jiang, Liu, & Wang, 2006). 3000 2500 2000 1500 Wavenumbers (cm-1) Fig. 4. Infrared spectra of LEM346 (a) and ALEM346 (b). 1000 500 M. Ye et al. / Food Chemistry 135 (2012) 2490–2497 2495 Fig. 5. 1H NMR spectra of LEM346 (a) and ALEM346 (b). (Fig. 6b), and exhibited a good dose–effect relationship (ALEM346: y = 0.1702x + 10.044, R2 = 0.9930; LEM346: y = 0.1738x + 5.0153, R2 = 0.9948). When the concentration was 500 mg L 1, the DPPH scavenging rates of ALEM346 and LEM346 reached 89.05% and 87.25%, respectively, both slightly lower than that of BHT (91.02%). The DPPH scavenging activity of ALEM346 was higher than that of LEM346, and both significantly higher than that (47.7%) of black sesame melanin (Shan, Xu, Jin, Liu, & Wang, 2008). 2496 M. Ye et al. / Food Chemistry 135 (2012) 2490–2497 ALEM346 a LEM346 BHA b Scavenging rate of DPPH• /% α -tocopherol equivalen concentration/mmol •L-1 160 140 120 100 80 60 40 20 0 50 100 150 200 250 300 350 400 450 500 ALEM346 50 ALEM346 LEM346 Concentration/mg L Vc d Chelating rate of Fe2+ /% - Scavenging rate of O2 •/% 100 90 80 70 60 50 40 30 20 10 0 50 BHT 100 150 200 250 300 350 400 450 500 Concentration/mg L c LEM346 100 90 80 70 60 50 40 30 20 10 0 100 150 200 250 300 350 400 450 500 ALEM346 LEM346 EDTA-2Na 100 90 80 70 60 50 40 30 20 10 0 50 100 150 200 250 300 350 400 450 500 Concentration/mg L Concentration/mg L Fig. 6. Antioxidant activities of ALEM346 and LEM346. (a) Total antioxidant capacity of ALEM346 and LEM346; (b) DPPH scavenging assay; (c) O2 chelating assay. 3.4.3. O2  scavenging activity Pyrogallol can oxidise fast under alkali conditions, generating a series of coloured intermediates with absorption wavelength near 318 nm, and releasing O2  at the same time, so the O2 scavenging ability can be measured by colorimetry. The O2  scavenging activities of ALEM346 and LEM346 were similar, both increasing with the increase of the concentration. When the concentration was 500 mg L 1, the O2  scavenging rates of ALEM346 and LEM346 reached 93.81% and 86.27%, respectively, with the IC50 values being 223.47 and 250.78 mg L 1, respectively (Fig. 6c). The O2  scavenging activity of ALEM346 was slightly higher than those of LEM346 and Vc (with an IC50 value of 235.95 mg L 1), and significantly higher than that of melanin from Taihe silky fowl (with IC50 value of 1879 mg L 1) (Tu et al., 2009). 3.4.4. Fe2+ chelating activity The metal-ion chelating ability is an important property of antioxidants (Guo, Hou, Wei, Sun, & Fan, 2010). Ferrozine combines quantitatively with Fe2+ to form a red complex. When melanin is present, the melanin will compete with ferrozine to combine Fe2+, weakening the red colour of the complex, so the Fe2+ chelating activity of the melanin can be determined by the change of colour, namely, the change of the absorbance value. The Fe2+ chelating activities of ALEM346 and LEM346 both increased with increasing concentration (Fig. 6d). When the concentration was 500 mg L 1, the Fe2+ chelating rates of ALEM346 and LEM346 reached 80.18% and 72.15%, respectively, with the IC50 values being 203.41 and 217.75 mg L 1, respectively, significantly lower than that of EDTA-2Na (42.26 mg L 1). At present, scholars are seeking new molecules as chelators that can chelate transition metal ions (Fe2+, Cu2+), to prevent the transition metal ion-catalysed production of OH in vivo, killing erythrocytes, degrading DNA, etc. (Perez, Wei, & Guo, 2009). ALEM346 and LEM346 have the ability to chelate Fe2+, which may be because the carboxyl or hydroxyl group with high charge density in the mela-  scavenging assay; (d) Fe2+ nin molecule interacts with Fe2+, creating a new chelator. The Fe2+ chelating activity of ALEM346 is higher than that of LEM346, and can be used as a Fe2+ chelator. 4. Conclusions The yield of the extracellular melanin from Lachnum YM346(LEM346) was 1.805 g L 1. Among the modified amino acidmelanins, arginine-melanin (ALEM346) had the highest solubility, being 4.55% (g g 1) in 30 °C distilled water. The maximum absorption wavelength and colour value E1%1 cm of LEM346 were both higher than those of ALEM346. Elemental analysis, infrared spectrum, mass spectrum and 1H NMR spectrum analysis indicated that LEM346 molecular contained indole quinone structure, with its molecular formula speculated to be C18H8O6N2. ALEM346 had characteristic absorption peaks formed by combination of the carbonyl group in melanin and the amino group in arginine at 1672.346 and 1637.679 cm 1. Mass spectrum indicated that ALEM346 contained three types of arginine-melanin molecules, which were formed by the addition reaction of one melanin molecule with one, two and three arginine molecules. The antioxidant activity of ALEM346 was higher than that of non-water-soluble LEM346, suggesting that ALEM346 can be used in the industry of food, medicine and cosmetics as a new antioxidant with stronger activity. This study provided a new method to allow melanin to dissolve in water. In future studies, we will modify melanin with other amino acids or other modifiers to improve its water-solubility and bioactivities further. 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