Phenolic Compounds Content and Antioxidant Activity of Mulberry Wine

During Fermentation and Aging

1XiaoHua Xie, 2Lingli Zhang and 2Xueling Gao

1Chuzhou Vocational and Technical College, 2188 Fengle Road, Chuzhou, Anhui, China 2School of
Tea and Food Science, Anhui Agricultural University, 130 West Changjiang Road, Hefei, Anhui,
China

Abstract
Mulberry is a delicious and highly nutritious fruit. However, Mulberry's nutritional content can
change if processed into wine. This study aims to determine phenolic changes and antioxidant activity
of mulberry wine. Materials and Methods: Total anthocyanins (TAC), total phenolics (TPC), total
tannins (TTC) and total flavonoids (TFC) and antioxidant activity measured in mulberry wine during
alcoholic fermentation and aging. Data were analyzed by one-way ANOVA followed by Duncan range
test using Prism ™. Results: Overall, fermentation increases TPC, TTC, and TFC. TAC reaches a
maximum (911.73 mg LG1) on the first day of fermentation and reaches a minimum (158.80 mg
LG1) for 90 days (p <0.05). During the wine making process, the antioxidant capacity increases
during fermentation and decreases during aging. Conclusion: Mulberry wine has a higher TAC, TPC,
TTC, TFC and antioxidant activity and changes in TAC, TPC, TTC and TFC. In changes in
antioxidant activity during fermentation and aging, stability occurs.
Keywords: Mulberry, mulberry wine, aging, polyphenols, antioxidants

INTRODUCTION
Mulberry (Morus spp.) Is widespread throughout the world, it has a sweet taste and contains many
health benefits. Mulberry is widely processed into jams, pies, and liquor, because of the short
storage period. Besides the taste and benefits of mulberry fruit, Asian people like mulberry wine
because of its bioactive potential. Fermentation of mulberry fruit produces abundant phenolic
compounds.
The objective of this study was to determine the total changes in anthocyanins, total
phenolic, total tannins and total flavonoid contents during alcoholic fermentation and aging of
mulberry wine after their antioxidant activity. So that it can provide a better understanding of the
type and level of bioactive compounds and overall antioxidant activity in mulberry wine.

MATERIALS AND METHODS

Chemicals: Folin-Ciocalteu reagent, gallic acid, lutin, gallotannic acid and ascorbic acid were
obtained from Sinopharm Chemical Reagent (Shanghai, China). 2, 2Diphenyl-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 common chemicals are analytical classes and are obtained from local
suppliers.
Mulberry samples and wine making: Mulberry fruits (Morus spp.) Were collected in May 2016 at
the CentralEastern of China. The fruit in this study had EBrix 18 ± 0.26, titratable acidity 7.79 ± 0.01
g LG1 as citric acid and pH 3.69 ± 0.22. Mulberry was crushed into mash and then treated with 16EC
for 24 hours with pectinase (0.05 g kgG1, Laffort, Sydney, Austrila) and metabisulfite potassium (70
mg kgG1). Mashes were then adjusted to 20EBrix with food-grade pure sucrose and inoculated with
S. cerevisiae (Zymaflore F15, Laffort, Sydney, Austrilia) at 0.2 g kgG1. Alcohol fermentation is
carried out at 25EC until the sugar content remains below about 4.0 g LG1. Separation of pomace
wine is done after alcoholic fermentation. Potassium metabisulfite (40 mg kgG1) is added and then
sample the wine aged at 16EC for 3 months.
Total phenolic content (TPC): TPC is determined by the Folin-Ciocalteu method13. The absorbance
of each sample is determined at 765 nm. The results were expressed as mg gallic acid equivalent
(GAE) per one liter of wine (mg GAE LG1).
Total anthocyanin content (TAC): TAC is measured using a differential pH14 method. Aliquots of
each sample were diluted with a pH of 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.
Total flavonoid content (TFC): TFC was measured using a colorimetric test adapted from Mahmood
et al. 15. Absorbance is measured at 510 nm. The results were expressed as routine equivalents of mg
(RE) per one liter of wine (mg RE LG1).
Total tannin content (TTC): TTC was measured by the Folin-Denis16 method. The absorbance is
measured at 700 nm. The equation obtained for the gallotannic acid calibration curve (0.2-130 mg
LG1) is Y = 0.0236x + 0.65 (r = 0.9995). The results were expressed as mg equivalent of gallotannic
acid per one liter of wine.
Free radical scavenging capacity (DPPH): DPPH free radical scavenging capacity is estimated by
adding 2.95 μL 0.1 mM DPPH methanol solution to 50 µL sample extract. mixed and placed in the
dark for 30 minutes. The absorbance is measured at 517 nm. The results were expressed in mg are
equivalent to the antioxidant capacity per liter of wine (mg of VC LG1).
Ferric reduced antioxidant strength (FRAP) assay: The FRAP assay was adopted from that
described by Si et al. 17. 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
the proportion of 10: 1: 1, respectively. Each sample (90µL) was mixed with 3.0 mL FRAP reagent
and incubated for 10 minutes at 37EC. The absorbance is read at 593 nm. The results were expressed
as mmol ferrous ions per one liter of wine (mmol Fe2 + LG1).
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 LG1).
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 grapes during fermentation: Total contents of anthocyanins
(TAC), phenolic (TPC), total tannins (TTC) and flavonoids (TFC) measured in mulberry wine for 4
days of fermentation of TAC from mulberry grape peaked on day 1 and remained slightly decreased
from days 2-4.
TPC increases were observed on each day of fermentation. Changes in TTC and TFC during
fermentation were similar to those observed for TPC. The maximum observed level is TPC, TTC and
TFC all on day 4.
Antioxidant activity of mulberry grapes during fermentation: DPPH, FRAP and RP levels in
mulberry wine were tested for 4 days of alcohol fermentation. Every activity increases in mulberry
wine during alcoholic fermentation, from day 0-4 and reaches its maximum value on day 4.
The bioactive components of mulberry grapes during the aging process: The total content of
anthocyanins (TAC), phenolic (TPC), tannins (TTC) and flavonoids (TFC) are measured in mulberry
wine for more than 90 days of aging. TAC, TPC, TTC, and TFC decrease during aging. TACs range
from 719.39-158.80 mg LG1, while TPC levels decrease from 2492.61-1936.52 mg LG1.
Antioxidant activity of mulberry grapes during aging: Antioxidant assays (DPPH, FRAP and RP)
were performed to evaluate the antioxidant activity of mulberry wine during aging. The changes in
antioxidant activities were consistent with the changes in the TAC, TPC, TTC and TFC in mulberry
wines, which decreased during aging.

DISCUSSION
Mulberry fruit is rich in phenolic compounds, anthocyanins and other flavonoid compounds. In this
study, the level of anthocyanins in mulberry grape increased from day 0-1 during fermentation, but
decreased from days 2-4, the rate of anthocyanin degradation was greater than the initial rate of
anthocyanin dissolution during day 120.
 Changes in TPC and antioxidant capacity correlate
during the wine making process. In this study, the antioxidant activities and phenolics content
of mulberry wine continually increased during fermentation.
Anthocyanins have low stability and their degradation is influenced by pH, light, temperature
and oxygen. In this study, the total anthocyanin content decreased during aging of mulberry wine.
Because most phenolic substances are not sensitive to light, heat or oxygen, phenolic content
decreases more slowly during the aging period. The phenolic content of fruit extracts of mauritian
grapefruit correlates strongly with antioxidant activity as determined by the FRAP test.