MATERIALS AND METHODS

Chemicals: Folin-Ciocalteu reagent, gallic acid, lutin, gallotannic acid and ascorbic acid were obtained from Sinopharm Chemical Reagent (Shanghai, China). 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) and 2, 4, 6-Tris (2-pyridyl)-1 and 3, 5-triazine (TPTZ) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other general chemicals were of analytical grade and were obtained from local suppliers.

Mulberry samples and winemaking: Mulberry fruits (Morus spp.) were collected in May of 2016 in the Central-Eastern of China. The fruits in this study had a °Brix of 18±0.26, a titratable acidity of 7.79±0.01 g L^–1 as citric acid and a pH of 3.69±0.22. Mulberries were crushed into mash and then treated at 16°C for 24 h with pectinase (0.05 g kg^–1, Laffort, Sydney, Austrilia) and potassium metabisulphite (70 mg kg^–1). The mashes were then adjusted to 20°Brix with food-grade pure sucrose and inoculated with S. cerevisiae (Zymaflore F15, Laffort, Sydney, Austrilia) at 0.2 g kg^–1. Alcoholic fermentation was carried out at 25°C and ended when the residual sugar content was below about 4.0 g L^–1. Separation of the wine pomace was performed at the end of alcoholic fermentation. Potassium metabisulphite (40 mg kg^–1) was added and then the wine samples were aged at 16°C for 3 months.

Total phenolic content (TPC): The TPC was determined by the Folin-Ciocalteu method¹³. The absorbance of each sample was determined at 765 nm. The results were expressed as mg gallic acid equivalents (GAE) per one liter of wine (mg GAE L^–1).

Total anthocyanin content (TAC): The TAC was estimated using the pH differential method¹⁴. Aliquots of each sample were diluted with pH 1.0 or 4.5 buffers to the same dilution. The absorbance was measured at 510 and 700 nm in both pH 1.0 and 4.5 buffers. The TAC was calculated using Eq. 1:

(1)

The results were expressed as mg cyanidin-3-glucoside (C3G) equivalents per one liter of wine (mg C3G L^–1).

Total flavonoid content (TFC): The TFC was measured using a colorimetric assay adapted from Mahmood et al.¹⁵. The absorbance was measured at 510 nm. The results were expressed as mg rutin equivalents (RE) per one liter of wine (mg RE L^–1).

Total tannin content (TTC): The TTC was measured by the Folin-Denis method¹⁶. The absorbance was measured at 700 nm. The equation obtained for the calibration curve of gallotannic acid (0.2-130 mg L^–1) was Y = 0.0236x+0.65 (r = 0.9995). The results were expressed as mg gallotannic acid equivalent per one liter of wine.

Free radical scavenging capacity (DPPH): The DPPH free radical-scavenging capacity was estimated by adding 2.95 μL of 0.1 mM DPPH methanolic solution to 50 μL of the sample extracts. The solution was thoroughly mixed and placed in the dark for 30 min. The absorbance was measured at 517 nm. The results were expressed in mg VC equivalent antioxidative capacity per one liter of wine (mg VC L^–1).

Ferric reducing antioxidant power (FRAP) assay: The FRAP assay was adopted from that described by Si et al.¹⁷. The FRAP reagent was prepared fresh daily in acetate buffer (adjusted to pH 3.6 by acetic acid) by mixing TPTZ solution (10 mM in 40 mM HCl) and 20 mM iron chloride solution in a proportion of 10:1:1, respectively. Each sample (90 μL) was mixed with 3.0 mL of the FRAP reagent and incubated for 10 min at 37°C. The absorbance was read at 593 nm. The results were expressed as mmol ferrous ion per one liter of wine (mmol Fe²⁺ L^–1).

Reducing power assay (RP): The RP was determined by the methods described by Infante et al.¹⁶. The absorbance was read at 700 nm after standing for 2 min, the final result was expressed as mg RP equivalent per one liter of wine (mg RP L^–1).

Statistical analysis: Data were expressed as the means±standard deviation (SD) of triplicate determinations. Mean differences at p<0.05 level were determined by one-way ANOVA followed by Duncan’s range test using Prism™ v6.0 software.

RESULTS

Bioactive components of mulberry wines during fermentation: The total contents of anthocyanin (TAC), phenolic (TPC), total tannin (TTC) and flavonoid (TFC) were measured in mulberry wine over 4 days of fermentation (Fig. 1). The TAC of mulberry wine peaked on day 1 (911.73 mg L^–1) and remained slightly decreased from day 2-4 (Fig. 1a).

Fig. 1(a-d):	Changes in the contents of (a) Total anthocyanins content (TAC), (b) Total phenolics content (TPC), (c) Total tannins content (TTC) and (d) Total flavonoids content (TFC) during fermentation of mulberry wine
	Different lower cases represent significant differences (p<0.05), data were expressed as the Mean± Standard Deviation (SD)

Fig. 2(a-c):	Changes in (a) Free radical scavenging capacity (DPPH), (b) Ferric reducing antioxidant power (FRAP) and (c) Reducing power (RP) in mulberry wine during fermentation
	Different lower cases represent significant differences (p<0.05), data were expressed as the Mean±Standard Deviation (SD)

Increases in TPC were observed on each day of fermentation (Fig. 1b). Changes in the TTC (Fig. 1c) and TFC (Fig. 1d) during fermentation were similar to those observed for the TPC (Fig. 1b). The maximal observed levels of TPC (2482.61 mg L^–1), TTC (1279.71 mg L^–1) and TFC (664.20 mg L^–1) were all on day 4.

Antioxidant activities of mulberry wines during fermentation: The levels of DPPH, FRAP and RP in mulberry wine were assayed during 4 days of alcoholic fermentation (Fig. 2). Each activity increased in mulberry wine during alcoholic fermentation, from day 0-4 and reached their maximum values at day 4.

Bioactive components of mulberry wines during aging: The total contents of anthocyanin (TAC), phenolic (TPC), tannin (TTC) and flavonoids (TFC) were measured in mulberry wine over 90 days of aging (Fig. 3). The TAC, TPC, TTC and TFC decreased during aging. The TAC (Fig. 3a) ranged from 719.39-158.80 mg L^–1, while the levels of TPC (Fig. 3b) decreased from 2492.61-1936.52 mg L^–1.

Antioxidant activities of mulberry wines during aging: Antioxidant assays (DPPH, FRAP and RP) were performed to evaluate the antioxidant activity of mulberry wine during aging (Fig. 4). The changes in antioxidant activities were consistent with the changes in the TAC, TPC, TTC and TFC in mulberry wines, which decreased during aging.

Fig. 3(a-d):	Changes in the (a) Total anthocyanins content (TAC), (b) Total phenolics content (TPC), (c) Total flavonoids content (TTC) and (d) Total flavonoids content (TFC) in mulberry wine during aging for 90 days
	Different lower cases represent significant differences (p<0.05), data were expressed as the Mean±Standard Deviation (SD)

DISCUSSION

Mulberry fruits are rich in phenolic compounds, anthocyanins and other flavonoid compounds¹⁸. A previous study indicated that anthocyanins were transferred into mulberry juice via maceration prior to wine fermentation¹⁹. In this study, the levels of anthocyanins in mulberry wine increased from day 0-1 during fermentation, but decreased from day 2-4, indicating that the rate of anthocyanin degradation might be greater than the initial rate of anthocyanin dissolution during the 1st day²⁰. The changes in TPC and TFC during alcoholic fermentation of mulberry juice were consistent with red wine of Di Egidio et al.²¹.

As reported by Lim et al.²² and Zhang et al.²³, changes in TPC and antioxidant capacity correlate during the wine making process. In this study, the antioxidant activities (Fig. 2a-c) and phenolics content (Fig. 1b) of mulberry wine continually increased during fermentation.

Anthocyanins have a low stability and their degradation is influenced by pH, light, temperature and oxygen²⁴. In this study, the total anthocyanin contents decreased during mulberry wine aging. Since most phenolic substances are not sensitive to light, heat or oxygen²⁵, the phenolics content decreased more slowly during the aging period. It has been reported that phenolics are responsible for antioxidant capacity^26,27. Furthermore, Ramful et al.²⁸ reported that the phenolics content of mauritian citrus fruit pulp extracts correlated strongly with the antioxidant activities as determined by the FRAP assays.

Fig. 4(a-c):	Changes in (a) Free radical scavenging capacity (DPPH), (b) Ferric reducing antioxidant power (FRAP) and (c) Reducing power (RP) of mulberry wine during aging over 90 days
	Different lower cases represent significant differences (p<0.05), data were expressed as the Mean±Standard Deviation (SD)

CONCLUSION

Mulberry juice was fermented and aged so that the total contents of anthocyanins (TAC), phenolics (TPC), flavonoids (TFC), tannins (TTC) as well as the antioxidant activities could be analyzed during the wine-making process. Mulberry wine has high TAC, TPC, TFC and TTC contents and antioxidant activity. The changes in the antioxidant activity were consistent with the changes in the TAC, TPC, TFC and TTC during fermentation and aging. These results indicate that changes in the timing of the mulberry wine-making process may more effectively deliver the antioxidant capacities of the mulberry fruit to the consumer.

SIGNIFICANCE STATEMENT

This study showed that the changes in antioxidant activities of mulberry wines were consistent with the levels of phenolic compounds in mulberry.

ACKNOWLEDGMENTS

This study was supported by the Natural Science Fund Project of Colleges and Universities in Anhui province (KJ2016A544), the visiting training program in China and abroad of outstanding young talents of higher education in Anhui Province (gxfx2017222) and the Anhui Province district Key Project (15czz03101).