Research article Institute of Brewing & Distilling

Received: 15 January 2016 Revised: 20 September 2016 Accepted: 20 September 2016 Published online in Wiley Online Library: 2 November 2016

(wileyonlinelibrary.com) DOI 10.1002/jib.384

Optimization of fermentation conditions for

Chinese bayberry wine by response surface
methodology and its qualities
Jing Du,1,2 Fei Han,3 Peibin Yu,4 Jieying Li2 and Liuping Fan1,2*
Colour and flavour are both important challenges for bayberry wine production because of a shortage of selected strains. The
yeast Saccharomyces cerevisiae strain YF152, separated from a natural Chinese bayberry fermentation mash [alcohol tolerance
capacity (18.0%), SO2 (200 mg/L), sugar content (40%) and pH (2.50)] was employed to brew Chinese bayberry wine. Response
surface methodology was used to simultaneously analyse the effects of the fermentation conditions on Chinese bayberry wine.
The optimum conditions were found to be a temperature of 26.5 °C, an initial sugar content of 22.0°Brix, an inoculum size of 4.0%
and an initial pH of 2.90. Under these conditions, the final alcohol content of the bayberry wine was 13.4%, the anthocyanin
content was 77 mg/L and the residual sugar content was 1 g/L. These numbers agreed well with the predicted values. The major
characteristic flavour components of the wine were 3-methyl-1-butanol, 2-phenylethanol, ethyl acetate, 2-methylbutyl acetate
and acetic acid. Copyright © 2016 The Institute of Brewing & Distilling

Keywords: Chinese bayberry wine; fermentation condition; response surface methodology; anthocyanin

Introduction winemaking (20). At present, there are only a few special yeast
strains used for brewing Chinese bayberry wine. Currently,
Chinese bayberry (Myrica rubra Sieb. & Zucc.), an important Chinese bayberry wine produced in China almost always uses
economic Asian fruit crop, belongs to the family Myricaceae (1). commercial yeasts and wine active dry yeasts. However, the
Chinese bayberry fruit, with a sweet/sour taste, an exquisite flavour Chinese bayberry wine produced often fails to meet the needs
and an attractive purple, red or dark red colour, is popular with of the consumer owing to the lower alcohol and poor flavour.
local people (2). Unfortunately, the Chinese bayberry fruit is highly In addition, these yeasts usually ferment at pH 3.30–3.60,
susceptible to mechanical injury and microbiological decay, which leading to a Chinese bayberry wine with a faded colour. Thus,
limits its postharvest life to 2 days at 20 °C or 5 days at 0 °C. The it was deemed necessary to develop a special yeast strain for
antioxidant capacity of the fruit decreases rapidly during storage brewing Chinese bayberry wine of good quality.
(2,3). Therefore, there is a demand for alternative processing In this work, a wild yeast strain was selected after screening
methods to extend the storage time and achieve a longer (YF152) and studied regarding the effect of fermentation
consumption period (4). conditions such as temperature, initial sugar, inoculum size
Winemaking is a promising way of processing fruit. Increas- and initial pH on alcohol content, anthocyanin content and
ingly, researchers have been directing their attention at fruity the residual sugar content of the Chinese bayberry wine. The
wines such as apple (5,6), papaya (7), raspberry (8), strawberry fermentation conditions were then optimized by response
(9), guava (10), pineapples (11), mango (12), bael (13), cagaita surface methodology (RSM) and the flavour analysed by gas
(14) and purple sweet potato (15). However, there is very little chromatography–mass spectrometry (GC–MS). The study
research about Chinese bayberry wine because of its short provides a reference for the industrial production of Chinese
shelf-life and unstable quality. bayberry wine with a higher alcohol content, favourable colour
Colour instability is one of the important challenges for bayberry and satisfactory flavour.
products. The colour of Chinese bayberry is mainly related to the
presence of anthocyanins (16). The major anthocyanin in bayberry
fruit has been identified as a cyanidin 3-glucoside, which
represents >95% of the total pigments (17); however, it is more
unstable than other kinds of anthocyanins and usually decreases * Correspondence to: Liuping Fan, School of Food Science and Technology,
sharply during fermentation, leading to wine with a poor colour Jiangnan University, Wuxi 214122, China. E-mail: fanliuping@jiangnan.edu.cn
(15). The stabilities of anthocyanins of Chinese bayberry juice 1
Institute of Food Research, Hezhou University, Guangxi, 542899, China
(2,16,18,19) have been studied and the results can be used to
improve the stability and content of anthocyanins of Chinese 2
The State Key Laboratory of Food Science and Technology, School of Food
bayberry products. Science and Technology, Jiangnan University, Wuxi 214122, China
The pH value of fresh bayberry ranges from 2.80 to 3.20, 3
Academy of state administration of grain, Beijing 100037, China
over which range the anthocyanin shows better stability and
the colour appears more attractive. Saccharomyces cerevisiae 4
National Engineering Laboratory for Cereal Fermentation Technology,
is the core microorganism in fermentation, especially in Jiangnan University, Wuxi 214122, China
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Materials and methods with increasing concentrations of alcohol (10.0, 12.0, 14.0, 16.0,
18.0 and 20.0%), sugar (20, 30, 40 and 50%), pH (1.50, 2.00, 2.50
Bayberry must and 3.00) and SO2 (100, 150, 200 and 250 mg/L) (22). The yeasts
were inoculated at 5.0% into 25 mL tubes containing 10 mL liquid
Chinese bayberry, hand-harvested at a mature stage from an
YPD and were inoculated at 28 °C for 4 days. The Durham tubes
orchard in Wuxi, Jiangsu province, China, was transported to our
were assessed every 24 h.
laboratory within 1 h and then frozen stored at
20 °C before
use. The Chinese bayberry juice was obtained using a juice extrac-
tor and was filtered using four layers of sterile cheesecloth after Single factor experiment
first naturally thawing the juice at 20 °C. The composition of the
The following four main influencing factors were investigated in
fresh Chinese bayberry juice was analysed with contents as
this study: temperature (16.0, 20.0, 24.0 and 28.0 °C), initial sugar
follows: total sugar of 92 g/L, titratable acidity of 0.86 g/100 mL
concentration (20.0, 22.0, 24.0, 26.0 and 28.0°Brix), inoculum size
(expressed as citric acid), pH of 2.95, total soluble solid content
(0.2, 1.0, 5.0 and 9.0%) and initial pH (2.50, 3.00, 3.50 and 4.00).
(TSS) of 11.2°Brix and an anthocyanin content of 290 g/L. Sucrose
The alcohol content, anthocyanin content and residual sugar
was added into the juice to ameliorate the TSS and the original
content were measured after the fermentation. In addition to the
pH was adjusted using calcium carbonate or citric acid. Equal
studied factors, other conditions were fixed at a temperature of
volumes of 120 mL of juice, with different TSS and pH levels, were
24 °C, an initial sugar of 24.0°Brix, an inoculum size of 5.0% and
placed into separate 250 mL sterilized Erlenmeyer flasks,
an initial pH of 3.00. After being inoculated with varied inoculum
pasteurized at 100 °C for 5 min, and immediately cooled to 20 °C.
sizes, all flasks were fitted with a fermentation bolt and
fermentation took place under stationary conditions at different
Yeast strain temperatures for 8–14 days until the weight loss was <0.2 g/day.
Each treatment was performed in triplicate.
The yeast strain used in this study was a wild strain, designated
YF152, isolated from a natural bayberry fermentation mash and
identified as Saccharomyces cerevisiae, cultivated by the China Optimization experimental design
Centre for Type Culture Collection, Wuhan University, with
Based on the single factor experiment, a central composite
accession number CCTCC M 2015627. The pure culture of the
experimental design (CCD) was employed to study the effects of
strain was conserved on a YPD agar slant (1.0% yeast extract,
linear, interaction and squared terms of the independent variables,
2.0% peptone, 2.0% dextrose, adding 2.0% agar when required) at
namely temperature (X1,°C), initial sugar (X2,°Brix), inoculum size
4 °C. The inoculation steps were as follows. A tricyclic pure culture
[X3, % (v/v)] and initial pH (X4), on the dependent variables using
from the slant was placed into 50 mL of the liquid YPD medium
Design Expert 7.1.6 (Stat-Ease Inc., USA) software. The levels of
and stirred at 150 rpm in an incubator shaker for 12 h, then
the independent variables and their coded values are provided in
inoculated as 10% (v/v) of the culture with 100 mL of the liquid
Table 1. A full-factorial CCD with 30 experiments was employed
YPD medium and incubated in the shaker under the same
(Table 2) to optimize the fermentation conditions. The experimen-
conditions for 12 h. The yeast concentration was determined
tal data from the CCD were analysed using a response surface
with a haemocytometer and adjusted to 2.00 × 108 cell/mL with
regression and fitted to a quadratic model (eqn 1):
sterile distilled water.
The strains RV171, BV818 and RW (Angel Yeast Co. Ltd, China),
4 4 4
which are commercial yeast strains, were employed to brew Y i ¼ b0 þ ∑ bi X i þ ∑ bij X i X j þ ∑ bii X i 2 (1)
Chinese bayberry wine, and a comparison was made with the i¼1 i;j¼1ði≠j Þ i¼1

experimental yeast strain YF152. The fermentation conditions were

as follows: initial sugar content of 20°Brix, inoculum of 0.2%, initial The dependent variables (Y) were the alcohol content [Y1, %
pH of 3.5 and fermentation at 28 °C for 8–14 days (until the weight (v/v)], the anthocyanin content (Y2, mg/L) and the residual sugar
loss was <0.2 g/day). content (Y3, g/L) of the Chinese bayberry wine; b0 is the constant
coefficient; bi is the linear effects; bij is the interaction term; bii is
the squared effects; and Xi and Xj are the coded values of variables
The tolerance capability of the yeast
i and j, respectively. The significance of all terms in the polynomial
The tolerance capacity of YF152 for alcohol, sugar, pH and SO2 was functions was evaluated statistically using an F-value at a
studied by determining the inflated conditions of the inverted probability (P) of 0.01 and 0.05. All samples were fermented using
tube (Durham) used for collecting the CO2 that was liberated stationary conditions for ~8–14 days until the weight loss was
through fermentation (21). The media were liquid YPD, modified <0.2 g/day.

Table 1. Independent variables and their coded values used for optimization of condition for fermentation of Chinese bayberry wine

Independent Units Symbol Coded levels

variable
-2
1 0 +1 +2
Temperature °C X1 20.0 22.0 24.0 26.0 28.0
Initial sugar °Brix X2 22.0 23.0 24.0 25.0 26.0
Inoculum size % X3 1.0 3.0 5.0 7.0 9.0
Initial pH X4 2.50 2.75 3.00 3.25 3.50
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Table 2. Central composite experimental design (CCD) and experiment results for fermentation of Chinese bayberry wine

Standard Coded values of the Independent variables Dependent variables

order
X1 X2 X3 X4 Y1 Y2 Y3
1
1
1
1
1 11.1 91 51
2 1
1
1
1 13.0 86 22
3
1 1
1
1 10.8 79 79
4 1 1
1
1 12.0 77 61
5
1
1 1
1 11.2 87 52
6 1
1 1
1 12.6 86 27
7
1 1 1
1 11.0 72 76
8 1 1 1
1 12.2 69 56
9
1
1
1 1 13.0 58 19
10 1
1
1 1 14.0 57 4
11
1 1
1 1 13.4 52 34
12 1 1
1 1 13.6 48 30
13
1
1 1 1 13.8 57 11
14 1
1 1 1 14.2 55 2
15
1 1 1 1 13.5 48 32
16 1 1 1 1 13.6 46 28
17
2 0 0 0 11.4 73 56
18 2 0 0 0 13.1 56 24
19 0
2 0 0 12.8 70 8
20 0 2 0 0 12.9 53 49
21 0 0
2 0 13.0 74 26
22 0 0 2 0 13.1 57 25
23 0 0 0
2 9.9 109 79
24 0 0 0 2 14.1 35 14
25 0 0 0 0 13.7 62 21
26 0 0 0 0 13.4 67 25
27 0 0 0 0 13.6 64 22
28 0 0 0 0 13.4 68 25
29 0 0 0 0 13.5 65 24
30 0 0 0 0 13.8 61 18
X1, temperature (°C); X2, initial sugar (°Brix); X3, inoculum size (%); X4, initial pH; Y1, alcohol content (%); Y2, anthocyanin content (mg/L);
Y3, residual sugar content ( g/L).

Component analysis (carboxen/polydimethysiloxane fibre, Supelco, Belfonte, PA, USA)

with an extraction temperature of 50 °C for 30 min, allowing the
The anthocyanin content of Chinese bayberry wine was
volatiles to reach equilibrium in the headspace. After the
determined using the differential pH method (23). The absorbance
extraction, the fibre was inserted immediately into an injection
was measured at 510 and 700 nm, using a UV-1800 spectropho-
port of a GC–MS (Scion SQ 456, Bruker, USA) and desorbed for
tometer [Mapada Instruments (Shanghai) Ltd, China]. The alcohol
8 min at 250 °C. The chromatography was performed using a
content was determined by the method described by Kumar
DB-Wax column (30 m × 0.25 mm × 0.25 μm) helium as a carrier
(12). The pH was determined using a Toledo FE-20 K pH-meter
gas at 0.8 mL/min (constant flow). The oven temperature
[Mettler-Toledo Instruments (Shanghai) Ltd, China] (19). The total
programme was as follows: 40 °C for 3 min; 90 °C at 5 °C/min;
sugar was first hydrolysed into reducing sugars using 20 mL of
90–230 °C at 10 °C/min; and finally maintained at 230 °C for
6 mol/L HCl in a 68 °C water bath for 15 min, was then neutralized
7 min. The electron impact ionization mass spectrometer was
with 200 g/L NaOH, and finally determined using the
operated as follows: ionization voltage, 70 eV; ion source tempera-
dinitrosalicylic acid method (25). The titratable acidity was
ture, 200 °C; and injection port temperature, 250 °C. Compounds
estimated according to the official methods of AOAC (26). The
were identified by comparison with the reference spectra of the
TSS (°Brix) content was determined using a hand refractometer
National Institute for Standards and Technology (Gaithersburg,
(Erma, Japan) (27).
MD, USA), the Wiley MS library and the relative peak area (28).

Volatile organic compound analysis

Statistical analysis
Solid-phase microextraction (SPME)–GC–MS was used to analyse
the volatile organic compounds (VOCs) of the Chinese bayberry All sample analyses were performed in triplicate. The data of the
wine. Aliquots of wine samples (8 mL) were placed in each SPME single factor experiment were expressed as the means ± standard
vial (15 mL). The extraction was performed using SPME fibre deviation, subjected to a statistical analysis with the software
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 J. Du et al.
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SPSS/PC version 13.0 (SPSS Inc., Chicago, IL, USA) and graphically because the higher temperature leads to a rapid release of CO2,
plotted using Origin Lab (Origin Pro, version 8.0). Significant removing some of the alcohol at the same time. The alcohol con-
differences among the mean values were analysed by a one-way tent of the bayberry wine fermented at 24 °C was the highest
analysis of variance (ANOVA) with the application of Duncan’s test (13.6%). The anthocyanin content decreased significantly from 77
at a level of p < 0.05. to 55 mg/L when the temperature varied between 20 and 28 °C,
illustrating that temperature is an important factor in destabilizing
the molecular structure of anthocyanins (30), and higher tempera-
Results and discussion tures will accelerate the destruction of anthocyanins (31,32). The
residual sugar showed an opposite trend compared with the
The tolerance capacity of the yeast
alcohol content, and the lowest content was 23 g/L fermented at
The yeast YF152 showed an excellent tolerance capacity by rapidly 24 °C. In summary, 24 °C was chosen as the optimum fermentation
inflating the Durham tubes within 24 h under the following temperature in the following experiment.
conditions: 18.0% alcohol content, 200 mg/L of SO2, 40% sugar
content and a pH of 2.50, which was very suitable to brew Chinese
bayberry wine. Osho (29) studied the alcohol and sugar tolerance Effect of initial sugar on fermentation of Chinese bayberry
of wine yeasts isolated from fermenting cashew apple juice and wine
found that the best four yeasts were able to grow in alcohol Figure 1(b) shows that the initial sugar content had a significant
concentrations of 9.0, 10.0, 11.0 and 12.0% (29); however, their influence on the alcohol content, the anthocyanin content and
alcohol tolerances were below that of YF152. the residual sugar content (p < 0.05). The data illustrated that,
when the initial sugar content reached 20.0–24.0°Brix, the alcohol
content began to increase gradually from 11.1 to 13.6%; however,
Effect of temperature on fermentation of Chinese bayberry
when the sugar content exceeded 24.0°Brix, the alcohol content
wine
began to decrease. It is posited that the biological activity of the
As shown in Fig. 1(a), the temperature significantly affected the yeast was inhibited by the higher sugar content (20), leading to a
alcohol content, anthocyanin content and residual sugar content higher residual sugar content and a lower alcohol content in the
(p < 0.05). It was found that, when the temperature was <24 °C, Chinese bayberry wine. There was no difference in the
the alcohol content increased significantly with the rise in anthocyanin content between the initial sugar contents of 20.0
temperature but decreased obviously at 28 °C, which may be and 22.0°Brix (p > 0.05); however, when the content exceeded

Figure 1. Effect of (a) temperature, (b) initial sugar, (c) inoculum size and (d) initial pH on alcohol content, anthocyanin content and residual sugar content of Chinese bayberry
766

wine (error bar shows standard deviation; different letters indicate values are different at the 0.05 level of significance).

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22.0°Brix, the anthocyanin content decreased significantly from 68 adsorbed anthocyanin during the fermentation (35,36). As the
to 46 mg/L (p < 0.05); the higher sugar content possibly had a inoculum size increased, the anthocyanin content decreased. How-
negative effect on the stability of the anthocyanins (33). The lowest ever, the inoculum size also influenced the period of fermentation;
amount of initial sugar produced the lowest residual sugar content, therefore, a higher inoculum size could shorten the fermentation
and the residual sugar content of the bayberry wine, fermented at time, but an inoculum size of 5.0% appeared satisfactory.
20.0 and 22.0°Brix, was <12 g/L. The anthocyanin content of
bayberry wine at an initial sugar content of 24.0°Brix decreased
by 6.0% compared with that of 22.0°Brix; however, the alcohol con-
Effect of initial pH on fermentation of Chinese bayberry wine
tent increased by 7.1%. Therefore, the initial sugar content had a
greater effect on the alcohol content than on the anthocyanin As shown in Fig. 1(d), the initial pH influenced the alcohol content
content, and 24.0°Brix was the desired initial sugar content. significantly in the pH range of 2.50–3.50 (p < 0.05), with an
increase in the alcohol content from 9.9 to 14.1%; however, when
the pH exceeded 3.50, the alcohol content decreased to 13.6% at a
pH of 4.00; therefore, the optimum fermentation pH of YF152 was
Effect of inoculum size on fermentation of Chinese bayberry
3.50. Sevda and Rodrigues (10) compared the alcohol production
wine
of guava wine using two different strains at different pH levels with
According to Fig. 1(c), the inoculum size played an important role in a sugar content of 22.0°Brix. For one type of yeast, the maximum
the alcohol, anthocyanin and residual sugar contents. When the alcohol production was 7.3% (v/v) at a pH of 3.50, which was
inoculum size was 0.2%, the alcohol content was <12%, and the re- 6.9% lower than YF152 at the same pH. For the other type of yeast,
sidual sugar could not be fully utilized (34). Between the inoculum the maximum alcohol production was 8.4% (v/v) at a pH of 4.00
sizes of 0.2 and 5.0%, the alcohol content increased with increases (10), which was lower than YF152 at a pH of 2.50 at 9.9%. Therefore,
in the inoculum size, and the residual sugar content decreased at YF152 proved to be more suitable for brewing at a low pH. The
the same time. However, the alcohol content of the Chinese bay- initial pH had a greater effect on the anthocyanin content than
berry wine with an inoculum size of 9.0% was lower than that with temperature, initial sugar and inoculum size, with a dramatic
an inoculum size of 5.0%; considering that, when the inoculum size decrease in the anthocyanin content from 109 to 23 mg/L, with
was too high, most of the nutrition of the bayberry juice was an increase in pH from 2.50 to 4.00. A lower pH led to a higher
utilized for the growth and reproduction of yeast, the substrate of content of anthocyanin, since anthocyanins are more stable at a
the metabolic reaction was greatly reduced (34) and the alcohol lower pH (16), and they are easily destroyed at a high pH. The
decreased with the concurrent rapid release of CO2. In addition, effect of the initial pH on the residual sugar content was the
the inoculum size also significantly influenced the colour of the opposite of that of the alcohol content, and the least residual sugar
bayberry wine (p < 0.05) because the cell walls of the yeast strains content was obtained at a pH 3.50 at 14 g/L. The anthocyanin

Table 3. Analysis of variance for the experimental results of the CCD

Source d.f. Y1 Y2 Y3
F-Value p-Value F-Value p-Value F-Value p-Value
Model 14 49.51 <0.0001** 30.60 <0.0001** 112.47 <0.0001**
X1 1 94.88 <0.0001** 7.55 0.0149* 174.51 <0.0001**
X2 1 5.50 0.0332* 37.31 <0.0001** 415.24 <0.0001**
X3 1 1.59 0.2260 9.96 0.0065** 1.60 0.2252
X4 1 453.06 <0.0001** 362.37 <0.0001** 766.47 <0.0001**
X1X2 1 4.88 0.0431* 0.016 0.9024 7.58 0.0148*
X1X3 1 1.76 0.2048 0.062 0.8065 0.47 0.5017
X1X4 1 19.52 0.0005** 0.016 0.9024 26.66 0.0001**
X2X3 1 0.049 0.8281 0.76 0.3966 0.47 0.5017
X2X4 1 1.22 0.2867 1.55 0.2316 7.58 0.0148*
X3X4 1 1.22 0.2867 0.39 0.5424 1.07 0.3181
X21 1 49.54 <0.0001** 0.036 0.8515 68.90 <0.0001**
X22 1 12.73 0.0028** 0.62 0.4424 9.72 0.0071**
X23 1 5.81 0.0292* 0.27 0.6127 3.12 0.0979
X24 1 71.99 <0.0001** 6.96 0.0186* 126.12 <0.0001**
Residual 15
Lack of fit 10 2.38 0.1752 2.32 0.1408 1.19 0.4503
Pure error 5
Total 29
* Significant at 0.05 level.
** Significant at 0.01 level.
X1, temperature (°C); X2, initial sugar (°Brix); X3, inoculum size (%); X4, initial pH; Y1, alcohol content (%); Y2, anthocyanin content (mg/L);
Y3, residual sugar content ( g/L).
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content at a pH of 2.50 was the highest at ~109 mg/L, but the of Chinese bayberry wine during fermentation (p < 0.05). The
alcohol content only reached 10%, and the residual sugar was full-factorial CCD with 30 experiments is shown in Table 2.
more than 75 g/L. Although the alcohol content at a pH of 3.50 The fermentation runs were performed in a standard order. The
was the highest and the residual sugar content was the lowest, calculated regression equation for the optimization of fermenta-
the anthocyanin content only reached 34 mg/L, making the tion conditions showed that the alcohol content [Y1, % (v/v)], an-
Chinese bayberry wine appear shallow. Therefore, a pH of 3.00 thocyanin content (Y2, mg/L) and residual sugar content (Y3, g/L)
was the desired value. were a function of the temperature (X1,°C), initial sugar (X2,°Brix),
inoculum size [X3, %(v/v) and initial pH (X4). By applying a multiple
regression analysis to fit the response function (Yi), the following
Model prediction and optimization
second-order polynomial equations were found to adequately
The single-factor experiment showed that the temperature, initial represent the alcohol content, anthocyanin content and residual
sugar, inoculum size and initial pH significantly affected the quality sugar content.

Figure 2. Response surface graph of (a) temperature vs initial sugar and (b) temperature vs initial pH on alcohol content; (c) initial sugar vs initial pH on anthocyanin content; (d)
768

temperature vs initial sugar; (e) temperature vs initial pH; and (f ) initial sugar vs initial pH on residual sugar content (the other two factors were fixed at zero level).

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Y 1 ¼ 13:57 þ 0:45X 1
0:11X 2 þ 0:058X 3 þ 0:98X 4 (2) Figure 2(c) shows the effect of the initial sugar and pH on the

0:12X 1 X 2
0:075X 1 X 3
0:25X 1 X 4
0:012X 2 X 3 anthocyanin content (temperature and inoculum size were fixed
þ0:063X 2 X 4 þ 0:063X 3 X 4
0:30X 1 2
0:15X 2 2 at zero). The highest anthocyanin content was obtained when

0:10X 3 2
0:37X 4 2 the variables were both at the minimum point within the range
studied.
Y 2 ¼ 64:50
2:25X 1 -5:00X 2
2:58X 3
15:58X 4 (3)

0:12X 1 X 2 þ 0:25X 1 X 3 þ 0:13X 1 X 4
0:87X 2 X 3 Residual sugar content
þ1:25X 2 X 4 þ 0:63X 3 X 4 þ 0:15X 1 2
0:60X 2 2 The residual sugar content reflects the ability of the yeast to utilize
þ0:40X 3 2 þ 2:02X 4 2 sugar and is responsible for imparting sweetness to the final wine
(39). In this experiment, the desired residual sugar content was
Y 3 ¼ 22:50
7:83X 1 þ 12:08X 2
0:75X 3
16:42X 4 (4) <4 g/L. As shown in Table 3, the model terms X1, X2, X4, X1X4, X21,
þ2:00X 1 X 2 þ 0:50X 1 X 3 þ 3:75X 1 X 4
0:50X 2 X 3 X22 and X24 were significant at p < 0.01, and model terms X1X2,

2:00X 2 X 4
0:75X 3 X 4 þ 4:60X 1 2 þ 1:73X 2 2 X1X4 had significant effects at p < 0.05, whereas the other model
þ0:98X 3 2 þ 6:23X 4 2 terms were not significant.
Figure 2(d) shows the effect of temperature and initial sugar on
the residual sugar content (inoculum size and initial pH were
The experimental results and an ANOVA for the alcohol content, fixed at zero). The lowest residual sugar content was obtained
anthocyanin content and residual sugar content are presented in with a temperature of 24–28 °C and an initial sugar content of
Tables 2 and 3, respectively. The model F-values of 49.51, 30.60 22.0–23.0°Brix. The effects of temperature and initial pH on the
and 112.47 implied that the three models were significant. There residual sugar content (initial sugar and inoculum size were fixed
was only a 0.01% chance that a ‘model F-value’ this large could oc- at zero level) are shown in Fig. 2(e). The minimum response plot
cur owing to noise. The ‘lack of fit F-values’ of 2.38, 2.72 and 1.19 was obtained with a temperature of 24–26 °C and an initial pH of
implied that the ‘lack of fit’ of the three models was not significant 3.20–3.40, which is opposite to the effect on the alcohol content.
relative to the pure error. In addition, R2 values for all the response In contrast, Fig. 2(f ) shows the effect of initial sugar and initial pH
variables were >0.96, indicating that the regression model ex- (temperature and inoculum size were fixed at zero) on the residual
plained the reaction well. sugar content. The lowest residual sugar content was obtained
with an initial sugar content of 22.0–23.0°Brix and an initial pH of
3.00–3.50.
Alcohol content
Wine fermentation is the result of many interactions and depends
not only on the strains but also on the physicochemical factors of The optimum fermentation conditions
the medium, sugars, acidity, temperature and others (37). Values The independent variables, namely fermentation temperature,
of ‘Prob > F′ <0.05 indicated the model terms were significant. initial sugar, inoculum size and initial pH, were optimized using
Among the model terms, X1, X4, X1X4, X21, X22 and X24 were the statistical software Design Expert 7.1.6. For convenient
significant with p < 0.01, and the model terms X2, X1X2, and X23 practical operation, the optimum combination of factors was set
had significant effects with p < 0.05, whereas the X3, X1X3, X2X3, as follows: temperature (26.5 °C), initial sugar (22.0°Brix), inoculum
X2X4 and X3X4 model terms were not significant. size (4.0%) and initial pH (2.90). The responses predicted from the
Figure 2(a, b) shows the three-dimensional response surfaces mode were 13.3%, 78 mg/L and 1 g/L. Repeated experiments
that are constructed to show the effects of the variables of bay- were performed to verify the predicted model. The actual
berry wine fermentation on the alcohol content (Y1). Figure 2(a) alcohol content, anthocyanin content and residual sugar
shows the effect of temperature and initial sugar on the alcohol content were 13.4%, 77 mg/L and 1 g/L, respectively. Verification
content (inoculum size and initial pH were fixed at zero). The of the model indicated no difference between the predicted
alcohol content increased with the increase in temperature in and actual values.
the range of 20–26 °C and initial sugar of 22.0–23.3°Brix, and it
peaked with a temperature of 25–27 °C and the initial sugar
content of 23.0–24.0°Brix. Figure 2(b) shows the effects of
temperature and the initial pH on the alcohol content (initial sugar
and inoculum size were fixed at zero), which demonstrated that
the alcohol content peaked at a temperature of 23–26 °C and an
initial pH of 3.20–3.40.

Anthocyanin content
The stability of anthocyanins, during the fermentation of purple
sweet potato wine (15) and red wine (38), has been studied, but
little research was related to the stability of anthocyanins in
Chinese bayberry wine. The major anthocyanin was different from
that in purple sweet potato and grape. Among the model terms,
only X1, X2, X3, X4 and X24 had significant effects on the anthocyanin
content (p < 0.05), especially the initial sugar (X2) and pH (X4), with Figure 3. The GC–MS photograph of volatile organic compounds of Chinese bay-
p-values <0.0001.
769

berry wine.

J. Inst. Brew. 2016; 122: 763–771 Copyright © 2016 The Institute of Brewing & Distilling wileyonlinelibrary.com/journal/jib
 J. Du et al.
Institute of Brewing & Distilling

Table 4. Comparison of different yeast strains for the fermentation of fruit wine

Yeast strains Fermentation conditions Fermentation indicators

X1 X2 X3 X4 Y1 Y2 Y3
Saccharomyces cerevisiae YF152a 26.5 22.0 4.0 2.90 13.4 77 1
S. cerevisiae RV171a 28.0 20.0 0.2 3.50 6.3 23 90
S. cerevisiae BV818a 28.0 20.0 0.2 3.50 7.1 30 76
S. cerevisiae RWa 28.0 20.0 0.2 3.50 10.0 21 32
S. cerevisiae (var. bayanus)b 22.5 20.0 11.9 3.80 10.0 — —
S. cerevisiae CFTRI 101c 31.4 22.0 0.5 3.20 12.46 — —
a
Used for brewing Chinese bayberry wine.
b
Used for brewing mango wine.
c
Used for brewing mulberry fruit wine.
—, Not detected.
X1, temperature (°C); X2, initial sugar (°Brix); X3, inoculum size (%); X4, initial pH; Y1, alcohol content (%); Y2, anthocyanin content (mg/L);
Y3, residual sugar content ( g/L).

VOCs of bayberry wine good yeast strain for brewing Chinese bayberry wine with a
better fermentation performance than many other yeast strains,
The VOCs of wines are a unique mixture of volatile compounds
especially at a lower pH.
that originate from fruits (varietal aromas), secondary products
formed during the wine fermentation (fermentative aromas) and
aging (post-fermentative aromas) (40). The complexity of the VOCs
can dramatically increase during alcoholic fermentation (41). Acknowledgements
Figure 3 displays the GC–MS photograph of VOCs in Chinese The authors acknowledge the financial support of the Special Fund
bayberry wine. Higher levels of alcohols, esters and acids are for Agro-scientific Research in the Public Interest (201303073-01),
the main group of compounds that form the fermentation Grain Research in the Public Interest (201313011-6-4), the
bouquet (42), taking up 34.3, 49.1 and 13.3%, respectively. The Fundamental Research Funds for the Central Universities
compounds 3-methyl-1-butanol (69.8%) and 2-phenylethanol ( JUSRP51501), Jiangsu Province Science and Technology Infra-
(26.1%) were the major higher alcohols produced by YF152, structure Construction plan (BM2014051/004), China National
Natural Science Foundation (31371812, 31871878), National Key
contributing a fruity, apple brandy aroma (28) and a sweet, rose,
Research and Development Program (2016YFD0401404), Jiangsu
floral flavour (43), respectively. The sum content of ethyl acetate Province Science and Technology Project (SBN2014010290,
and 2-methylbutyl acetate accounted for 59.4% of the total esters, BN2014058), and Qing Lan Project which has enabled us to carry
mainly synthesized by the reaction between alcohols and out this study.
acetyl-CoA during alcoholic fermentation, imparting a floral and
fruity flavour (28). Acetic acid, which comprised 7.8% of the total
VOCs, was the largest acid component. All of these substances
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