Food Chemistry 308 (2020) 125665

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analysis of factors related to browning of Dangshan pear (Pyrus spp.) wine T

a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d
Hua Yang , Tiantian Tian , Hong Gu , Xiaomin Li , Guolin Cai ,
⁎
Junyong Suna,b,c,d, Dianhui Wua,b,c,d, Jian Lua,b,c,d,
a
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China
b
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, PR China
c
Jiangsu Provincial Research Center for Bioactive Product Processing Technology, 1800 Lihu Road, Wuxi 214122, PR China
d
School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, PR China

A R T I C LE I N FO A B S T R A C T

Keywords: The effects of different dissolved oxygen concentrations (DOC) on the browning degree, amino acids, total
Analysis phenols, reducing sugars, polyphenoloxidase (PPO), peroxidase (POD), phenylalanine ammonia-lyase (PAL) of
Factors pear wine, and the relationship between various quality indicators and browning degree were investigated.
Browning Dynamic model fitting analysis of the changes of physiochemical indicators of pear wine in the storage process
Pear wine
were performed. The importance of the physiochemical indicators effect on the browning of pear wine during
the storage process was analyzed by OPLS (orthogonal partial least squares discriminant analysis), and the effect
of dissolved oxygen on the browning of pear wine was systematically revealed. The results showed that dissolved
oxygen, total phenols and amino acids had the greatest influence on the browning degree of pear wine. It
provided a theoretical basis for revealing the browning mechanism and inhibiting the browning of pear wine.

1. Introduction
Pears are rich in nutrients, phenolic substances and have anti-
oxidant properties (Jennings, MacGregor, Spector, & Cassidy, 2017;
Mangels & Mohler III, 2017). Processing pears into pear wine not only
increases the added value of pears, but also enriches the variety of the
wine market to provide consumers with more choices. Browning is a
thorny problem in the production and processing of pear wine, because
browning can cause irreversible defects in the quality of pear wine. Due
to the lack of in-depth study on the browning mechanism of pear wine,
the development of an effective solution to the browning problem of
pear wine has been limited. At present, the most in-depth and extensive
research on the browning of fruit wines is about white wine. Oxidation
played an important role in the composition and sensory properties of
white wines (Coetzee, Van Wyngaard, Suklje, Silva Ferreira, & Du Toit,
2016). Some research showed that wine oxidation was a complex
process involving oxygen, phenols, sugars, amino acids and various
enzymes (Li, Guo, & Wang, 2008). Among the above factors that cause
wine browning, the presence of oxygen, especially the presence of
dissolved oxygen, was a key factor for browning. Proper exposure to
oxygen might be beneficial to the quality of the wine, while too low or
too high exposure levels might be detrimental to sensory properties
such as the color and odor of wine (Panero, Motta, Petrozziello, Guaita,
& Bosso, 2015). White wines and rose wines were accidentally exposed
to the air and their quality will be compromised (Carrascón, Bueno,
Fernandez-Zurbano, & Ferreira, 2017). Dissolved oxygen in white wine
could be consumed by reaction with wine ingredients, unless there was
a sufficient concentration of preservative to cause harmful changes
(Bradshaw, Barril, Clark, Prenzler, & Scollary, 2011). The ability of
different wines to interact with oxygen was highly dependent on the
composition of the wine (Ferreira, Carrascon, Bueno, Ugliano, &
Fernandez-Zurbano, 2015). The oxidative browning of wine was di-
vided into enzymatic browning and non-enzymatic browning. PPO,
POD and PAL were associated with enzymatic browning. Amino acids
and sugars were primarily involved in non-enzymatic browning. Phe-
nolic substances were involved in both enzymatic browning and non-
enzymatic browning (Gao et al., 2015) (Oliveira, Barros, Ferreira, &
Silva, 2015). The main oxidoreductases responsible for browning
during grape processing were PPO and POD. Caftaric acid or p-cou-
maric acid (belong to phenols) was oxidized by PPO to produce caf-
feoyltartaric acid o-quinones (CTAQ), which are powerful oxidants and
able to oxidize other compounds in wine, and POD enhance the de-
gradation of phenols when coexisting with PPO (Li et al., 2008). The
existence of PAL might play an important indirect role in the browning
of pear wine. In addition, amino acid and sugar composition also had an
impact on the browning of wine (Pérez-Bernal, Villar-Navarro, Morales,

⁎
Corresponding author at: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, PR China.
E-mail address: jlu@jiangnan.edu.cn (J. Lu).

https://doi.org/10.1016/j.foodchem.2019.125665
Received 24 June 2019; Received in revised form 6 October 2019; Accepted 6 October 2019
Available online 17 October 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
H. Yang, et al.
Ubeda, & Callejón, 2017).
In this study, the pear wines after the main fermentation were
bottled and stored, their dissolved oxygen content was adjusted into
three groups of high, medium and low. The effects of different con-
centrations of dissolved oxygen on the browning index, PPO, POD, PAL,
total phenols, total amino acids and reducing sugars of pear wine and
the changes of these physiochemical indexes were investigated.
Through the correlation analysis and OPLS analysis, the key factors
affecting the browning of pear wine were found out, and the browning
mechanism of pear wine was revealed macroscopically. The results
provided understanding of browning formation as impacted by intrinsic
and extrinsic parameters, such as wine composition or storage condi-
tions. And the research was a theoretical basis for solving the browning
problem of pear wine.
2. Materials and methods
2.1. Materials and solvents
Dangshan pears were bought from the supermarket in Wuxi, China
(produced in Dangshan County, Anhui Province, China in 2017), the
pears had a total sugar content (expressed as reducing sugars) of
65.36 g/L and a total acid content (expressed as tartaric acid) of 4.47 g/
L. S. cerevisiae strains SY was purchased from Angel Yeast Co. Ltd. All
the chemicals used in the analysis were of analysis grade.
2.2. Wine samples
The fermentation process of Dangshan pear wine was as follows:
Dangshan pears were washed clean and had their nucleus removed.
Dangshan pear juice was produced by crushing and filtering, which was
supplemented with water (ratio of 20%). The total sugar content (cal-
culated based on glucose) of Dangshan pear juice was adjusted to
220 g/L with sucrose, treated with sulphur dioxide (70 mg/L) before
fermentation, then Dangshan pear musts were inoculated with 0.3‰ of
yeast. Bioreactors containing 2 L of Dangshan pear musts were main-
tained at 20 ± 1 °C until the CO2 weight loss decreases below 0.2 g.
2.3. Storage of pear wine samples with different DOC
Preparation of low-dissolved oxygen pear wine sample (LDOW):
200 mL pear wine was filled into a transparent glass bottle with a vo-
lume of 200 mL, and no top air body was present. Blowing the bottom
of the glass bottle with nitrogen first when filling.
Preparation of middle-dissolved oxygen pear wine sample (MDOW):
150 mL pear wine was filled into a transparent glass bottle of 200 mL
with a headspace of 50 mL. Blowing the bottom of the glass bottle with
nitrogen first when filling.
Preparation of high-dissolved oxygen pear wine sample (HDOW):
100 mL pear wine was filled into a transparent glass bottle of 200 mL
with a headspace of 100 mL. Blowing the bottom of the glass bottle with
nitrogen first when filling. The glass bottles had high molecular density,
good sealing and high barrier property, these glass bottles were all
sealed with the tinplate spiral cover (with inner liner), so the package
had good airtightness. At the first measurement, the dissolved oxygen
concentration of all pear wines was the same, and then the headspace
oxygen reserved in the bottle was used to form HDOW, MDOW and
LDOW.
The pear wines were stored at a constant temperature of 25 °C, and
the physical and chemical indicators of pear wines were measured
weekly for 15 weeks. To prevent sample loss during the test, the test
sample was prepared in six copies. Three samples were tested during
the experiment.
2
Food Chemistry 308 (2020) 125665
2.4. Determination of DOC
The dissolved oxygen concentration of pear wine was determined by
AZ 8403 Oxygen Detector under a nitrogen atmosphere in a hand-held
air bag. The pear wine was not shaken before and after the measure-
ment.
2.5. Determination of browning
Refer to the method of Dusan and Katherine and modify it slightly to
determine the absorbance value of pear wine at 420 nm as the browning
index (BI). Pear wine was centrifuged at 12,000 rmp for 5 min, then
95% ethanol solution was added with a volume ratio of 1:1, and vor-
texed for 10 min. The solution was centrifuged at 12,000 rmp for 5 min,
and then the absorbance A420 at 420 nm was measured with spectro-
photometer, and distilled water and 95% ethanol were mixed at a vo-
lume ratio of 1:1 as a blank control (Tomic, Grigorakis, Loupassaki, &
Makris, 2017).
2.6. Determination of enzyme activity
PPO: Added 2 mL of 0.05 mol/L phosphate buffer (pH 7.8, con-
taining 1% PVPP) and 2 mL of 0.04 mol/L catechol solution to the test
tube, placed water bath at 25 °C for 5 min, and then added 5 mL pear
wine sample using the phosphate buffer as a blank control, the change
of absorbance at wavelength of 425 nm was measured by the ultra-
violet–visible spectrophotometer (de Ávila et al., 2019; Thaisakornphun
& Tongchitpakdee, 2017).
POD: Added 0.5 mL of guaiacol, 1 mL of 30% H2O2, 3 mL of 0.1 mol/
L phosphate buffer (pH 6.0) and 0.5 mL pear wine sample to the test
tube. The reaction system without the pear wine sample was used as a
blank control, and the change of absorbance at 470 nm was measured
by ultraviolet–visible spectrophotometer (de Ávila et al., 2019;
Thaisakornphun & Tongchitpakdee, 2017).
PAL: Refer to the method of Jiangli and Is¸ıl Gürsul. Added 3.5 mL of
borate buffer (pH 8.8), 0.5 mL of 0.02 mol/L L-phenylalanine solution
and 1.0 mL of pear wine sample to the test tube, placed water bath at
40 °C for 1 h, then added 0.2 mL 6 mol/L HCl. The reaction system
without the pear wine sample was used as a blank control, and the
change of absorbance at 290 nm was measured by ultraviolet–visible
spectrophotometer (Li, Yuan, Zhang, & Jiang, 2006; Gürsul, Gueven,
Grohmann, & Knorr, 2016).
The absorbance values obtained were plotted against time and the
initial slope value of the curve was used to calculate both PPO, POD and
PAL activities. The change of 0.01 optical density (OD) per minute was
an enzyme activity unit, expressed in U/ml.
2.7. Determination of total phenols content
The total phenols content in pear wine was determined by Folin-
Ciocalteu colorimetry (Yang, Gadi, Paulino, & Thomson, 2010).
2.8. Determination of total amino acids content
Improved the method of Barry et al. Added 5 mL of pear wine
samples into two 100 mL triangle flasks, respectively. Added 2 drops of
neutral red indicator to a triangular flask and titrated to amber with
0.1 M NaOH, the volume of consumed NaOH was recorded as V1.
Added 2 drops of thymolphthalein indicator and 5 mL of neutral for-
maldehyde to another flask, after 1 min, titrated with 0.1 M NaOH until
light blue, the volume of consumed NaOH was recorded as V2.
Amino acid content (%) = M*(V2 − V1)*0.014*100/V; M was the
molar concentration of NaOH and V was the volume of pear wine
sample (Gump, Zoecklein, Fugelsang, & Whiton, 2002).
H. Yang, et al.
2.9. Determination of reducing sugars content
The amount of reducing sugars produced was determined using the
Di-Ntro Salicylic Acid (DNS) method (Nwachukwu et al., 2008).
2.10. Dynamic model fitting analysis of the changes of physiochemical
indicators
Kinetic model: It was necessary to establish the kinetic model to
obtain basic kinetic information of the system in order to describe the
reaction rate as a function of experimental variables, to predict the
changes of physiochemical indicators of pear wines during the storage.
Some literatures showed that the reaction kinetic models related to food
quality changes mainly included zero-order (Eq. (1)), first-order (Eq.
(2)), parabolic model (Eq. (3)), fractional conversion model (Eq. (4))
(Vaikousi, Koutsoumanis, & Biliaderis, 2008; Gonçalves, Pinheiro,
Abreu, Brandão, & Silva, 2010; Maskan, 2001).
A = A 0 + kt
A = A 0 ∗ exp(kt)
A = ( A0 + kt )2
A = A∞ + (A 0 − A∞) ∗ exp(kt)
2.11. Statistical analysis of data
OPLS analysis and correlation analysis were performed by SIMCA
14.1. Comprehensively deal with the response information of multiple
variables, systematically analyzed the influence of a series of physical
and chemical indicators on the browning of pear wine and screen out
the variables with significant response, which was necessary to study
the browning mechanism of pear wine.
3. Results and discussion
3.1. Changes in DOC of pear wines during storage
The DOC changes of HDOW, MDOW and LDOW as shown in Fig. 1 a.
The DOC in the three pear wine samples showed a downward trend
during storage. The rate of decline in DOC at the beginning of storage
was significantly higher than the rate of decline at the end of storage.
This trend was same as the study of Yuan, in wine storage process, the
DOC dropped rapidly at the beginning and thereafter remained at a
lower level (Gao et al., 2015). In 0-7th week, the DOC in HDOW de-
creased by 36.94%, and the DOC in MDOW and LDOW decreased by
57.23% and 66.39%, respectively. It was showed that a large amount of
dissolved oxygen was consumed in the initial stage of storage. In some
wines, the initial rate of oxygen consumption was more than 70 times
higher than the subsequent test values (Ferreira, Carrascon, Bueno,
Ugliano, & Fernandez-Zurbano, 2015). Danielwicz et al. pointed out
that this phenomenon was caused by the oxidation of epicatechin and
gallic acid molecules to produce highly reducing quinones and thus
consumed a large amount of oxygen (Danilewicz, 2011).
The DOC change trend in MDOW was similar to the change trend in
LDOW. The DOC in HDOW was significantly higher than the DOC in
LDOW and MDOW at the end of storage. The headspace oxygen in the
HDOW was continuously dissolved into the pear wine, dissolved oxygen
was consumed and a certain amount of supplement was also obtained.
The data of DOC varied with storage time were fitted by common
function associated with food quality changes. The reaction kinetic
parameters were shown in Table 1. The results shown that the DOC
changes in MDOW and LDOW were consistent with the fractional
conversion model. (R2adj values were 0.980 and 0.974, respectively).
According to the model, it was estimated that the initial DOC in MDOW
was 2.22 mg/L, and the DOC was 0.56 mg/L at the end of storage. The
Food Chemistry 308 (2020) 125665
initial DOC in the LDOW was 2.13 mg/L, and the DOC was 0.47 mg/L at
the end of storage. These values were very close to the actual mea-
surements. It shown that the DOC changes of the two pear wine samples
could be described successfully by fractional conversion model. Com-
paring the k values, it could be found that the DOC drop rate in the
LDOW was slightly faster than the MDOW. The change of DOC in
HDOW accorded with the zero-order reaction kinetic equation, R2adj
values was 0.895。Similarly, Scheling et al. found that the fractional
conversion model described well the decreasing trend of DOC in the
orange juice storage, R2adj = 0.97 (Wibowo et al., 2015).
3.2. Changes in total phenols of pear wines during storage
In the different pear wine samples, the decline trend of total phenols
content was consistent. When the storage time was 15 weeks, the total
phenols content in HDOW was the least and in LDOW was the highest
(Fig. 1 b). The initial content of total phenols in the three pear wine
samples were 269.78 μg/L. By the end of storage, the total phenols
(1)
content in HDOW, MDOW and LDOW were 123.53 μg/L, 143.29 μg/L
(2) and 162.26 μg/L, respectively. In HDOW, the total phenols decreased
by 54.21%, and in MDOW and LDOW decreased by 46.89% and
(3) 39.85%, respectively. The results indicated that the higher the DOC
during storage, the more total phenols were consumed. The changes of
(4)
total phenols content in different DOC pear wine samples were con-
sistent with the zero-order reaction kinetics equation, and R2adj was all
greater than 90%, the fitting result was very good. Phenols were the key
component of fruit wines, the overall quality of red wine was sig-
nificantly affected by various phenolic compounds (Setford, Jeffery,
Grbin, & Muhlack, 2017). On the one hand, phenolic substances were
good natural antioxidants (Melo et al., 2015). On the other hand,
phenolic substances caused browning of white wine. In white wine,
hydroxycinnamic acids and flavonols were the main compounds in-
volved in the reactions that caused oxidative browning. And another
study had shown that browning of white wine was caused by the pre-
sence of flavan-3-ol (Ma & Waterhouse, 2018). Phenols in wine were
rapidly oxidized in the presence of oxygen, the oxidation of o-dihy-
droxyphenols and trihydric phenols leaded to the formation of quinones
and hydrogen peroxide (Petrozziello et al., 2018). The polymerization
of quinones produced brown matter, which made the color of white
wine difficult to be accepted by consumers. Correlation analysis showed
that there was a high correlation between DOC change and total phe-
nols content in the three pear wine samples during the storage, the
correlation coefficients were 0.954, 0.943, and 0.922, respectively. The
content of phenolic compounds in HDOW was greatly reduced, prob-
ably because HDOW provided sufficient reactive oxygen to react with
phenolic substances and cause browning.
3.3. Changes in amino acids content of pear wines during storage
The changes in amino acids content during storage of different DOC
pear wine samples were shown in Fig. 1c, the trend of decreasing the
amino acids content of the three pear wine samples were consistent.
During storage, the total amino acids content in HDOW, MDOW and
LDOW decreased by 65.53%, 59.47% and 68.75%, respectively. At the
end of storage, the total amino acids content in different pear wine
samples was 391.30 mg/L, 460.11 mg/L and 354.74 mg/L, the differ-
ence was not significant. The changes of total amino acids content in
different DOC pear wine samples were consistent with the zero-order
reaction kinetics equation (Table 1). In the fitting equation of HDOW
and LDOW, R2adj were 0.925, 0.974, respectively; and in the MDOW,
R2adj = 0.898. The correlation analysis between the change of DOC and
the change of total amino acids content showed that their correlation
was high in HDOW, the correlation coefficient r was 0.969; in MDOW
and LDOW, the values were 0.835, 0.889, respectively. It indicated that
the amino acids content in pear wine had a strong correlation with
DOC. General research suggested that amino acids were closely related
3
H. Yang, et al.
Fig. 1. Changes of physiochemical indicators of three pear wines during storage. a: Changes of DOC. b: Changes of total phenols content. c: Changes of total amino
acids content. d: Changes of reducing sugars content. e: Changes of POD activity. f: Changes of browning degree.
to the browning of wine, it is involved in non-enzymatic browning
(Millard reaction). The Maillard reaction was that reducing sugars and
amino compounds (amino acids or proteins) undergo a complex reac-
tion process to form brown or even black macromolecular substances.
Volatile compounds responsible for typical aroma or aging aromas in
some sweet natural wines be linked with Maillard reactions (Oliveira,
Ferreira, De Freitas, & Silva, 2011). In wine, methionine might act as
one of the antioxidants, cysteine could act as both antioxidant and as
reactive nucleophile reacted with quinones to form brown polymers,
phenylalanine also participated in certain reaction processes, while the
mechanism of action was not clear (Carrascón et al., 2018; Oliveira
et al., 2011). Maillard reaction also contributed to the formation of
color during the fermentation of mango wine (Reddy & Reddy, 2005).
The total amino acids content in different DOC pear wine samples de-
creased during storage, probably the amino acids participating in the
Maillard reaction and causing browning of pear wine.
4
Food Chemistry 308 (2020) 125665
3.4. Changes in reducing sugars content of pear wines during storage
The content of reducing sugars plays a key balancing role in the
taste of pear wine. However, in addition to the Maillard reaction with
amino acids, the reducing sugars in wine can also cause browning
through caramelization. A study had shown that the increase in color
intensity during storage of white wine was related to the caramelization
and Maillard reaction in which sugars and amino acids were involved
(Moreira, Lopes, Ferreira, Cabral, & de Pinho, 2018). However, the
storage environment of wine was complicated, and it was related to
temperature, pH and dissolved oxygen. Whether the caramelization
reaction was one of the main causes of browning of white wine and
other wines was still in dispute. Some scholars believed that the car-
amelization reaction of sugars generally depended on high temperature
conditions, so the caramelization reaction was more likely to occur only
in the pasteurization process of sweet wines (Oliveira et al., 2011). As
shown in Fig. 1 d, the initial reducing sugars content of the three pear
wine samples were 0.790 mg/L. At the 5th week, the reducing sugars
H. Yang, et al. Food Chemistry 308 (2020) 125665

Table 1
Estimated kinetic parameters describing the changes during storage of quality parameters linked to DOC change of pear wines.
Fitting project Samples Storage time Kinetic model Kinetic parameters

A0 k A∞ R2adj

DOC HDOW 0-15w zero-order 2.08 ± 0.06 −0.0813 ± 0.0072 0.895

MDOW 0-15w fractional conversion 2.22 ± 0.06 −0.2375 ± 0.02 0.56 ± 0.05 0.980
LDOW 0-15w fractional conversion 2.13 ± 0.07 −0.2532 ± 0.0282 0.47 ± 0.05 0.974

Browning degree HDOW 0-15w parabolic model 0.07 ± 0.00 0.0043 ± 3.9375E-4 0.889
MDOW 0-15w zero-order 0.06 ± 0.00 0.0027 ± 2.607E-4 0.874
LDOW 0-15w zero-order 0.06 ± 0.00 0.0022 ± 2.3274E-4 0.850

Total phenols HDOW 0-15w zero-order 236.82 ± 5.27 −7.3717 ± 0.5983 0.910
MDOW 0-15w zero-order 251.08 ± 3.44 −6.8585 ± 0.3908 0.953
LDOW 0-15w zero-order 260.67 ± 2.14 −6.6847 ± 0.2429 0.981

Amino acids HDOW 0-15w zero-order 1094.31 ± 27.44 −42.3426 ± 3.1165 0.925
MDOW 0-15w zero-order 1082.79 ± 28.68 −37.4727 ± 3.2576 0.898
LDOW 0-15w zero-order 1125.18 ± 17.43 −47.0132 ± 1.9798 0.974

Reducing sugars HDOW 0-8w zero-order 0.80 ± 0.02 0.0152 ± 0.0034 0.701
MDOW 0-8w zero-order 0.80 ± 0.01 0.0200 ± 0.0030 0.842
8-15w zero-order 1.14 ± 0.09 −0.0302 ± 0.0077 0.674
LDOW 0-7w first-order 0.78 ± 0.01 0.0291 ± 0.0032 0.920
7-15w fractional conversion 0.90 ± 0.23 −0.2210 ± 0.0784 0.40 ± 0.02 0.949

POD HDOW 0-5w zero-order 426.53 ± 31.31 −36.4000 ± 10.3428 0.695

5-15w zero-order 164.86 ± 8.90 4.0812 ± 0.8491 0.689
MDOW 0-5w zero-order 411.60 ± 23.05 −36.5867 ± 7.6142 0.815
5-15w zero-order 142.88 ± 19.89 7.7467 ± 1.8964 0.611
LDOW 0-5w zero-order 415.78 ± 28.38 −34.5867 ± 9.3741 0.716
5-15w zero-order 162.48 ± 15.86 6.1430 ± 1.5119 0.608

Table 2 content was 0.803 mg/L, and the total content of reducing sugars in-
The Constant and variable (X) coefficient of OPLS model of browning index. creased by 1.65% during storage. The reducing sugars in pear wine was
Item HDOW MDOW LDOW
mainly composed of glucose and fructose. The changes in reducing
sugars content were fluctuating during storage of different DOC pear
Constant 4.509 6.129 7.290 wine samples. In the early stage of storage, the reducing sugars content
DOC −0.367 −0.279 −0.279 was increased, and the content was gradually reduced in the later stage
Total phenols −0.332 −0.285 −0.271
Total amino acids −0.421 −0.258 −0.265
of storage. It was because sucrose in pear wine could be broken down
Reducing sugars 0.073 −0.056 0.052 into glucose and fructose, while glucose and fructose might be con-
sumed and involved in some reactions. The consumption and supple-
mentation of glucose and fructose resulted in fluctuations in the redu-
content was 0.905 mg/L in HDOW, and the reducing sugars content was cing sugars content. Since the reducing sugars content in the stored
0.786 mg/L at the end of storage. There was almost no change in the process was fluctuating, the fitting result of the kinetic equation was
total content of reducing sugars before and after storage. At the 6th segmented (Table 1). In 0-8th week, the change of reducing sugars
week, the maximum content of reducing sugars in MDOW was content in HDOW accorded with the zero-order reaction kinetic equa-
0.950 mg/L. At the end of storage, the reducing sugars content was tion, R2adj = 0.701; In the 8-15th week, there was no suitable fitting
0.761 mg/L, and the total content of reducing sugars during storage equation for the change of reducing sugars content of HDOW. During
decreased by 3.67%. At the 7th week, the reducing sugars content in the storage period of 0-8th week and 8-15th week, the change of re-
LDOW was 0.939 mg/L. At the end of storage, the reducing sugars ducing sugars content of MDOW accorded with the zero-order reaction

Fig. 2. Principal component analysis of browning degree changes of three pear wines; a: In HDOW; b: In MDOW; c: In LDOW.

5
H. Yang, et al. Food Chemistry 308 (2020) 125665

Table 3
Correlation analysis between various quality indicators in different DOC pear wine samples.
HDOW

DOC Total phenols Amino acids Reducing sugars POD Browning degree

DOC 1 0.954** 0.969** 0.371 0.691** −0.970**

Total phenols 1 0.948** 0.316 0.746** −0.958**
Amino acids 1 0.513* 0.591* −0.981**
Reducing sugars 1 −0.009 −0.385
POD 1 −0.620**
Browning degree 1
MDOW

DOC Total phenols Amino acids Reducing sugars POD Browning degree

DOC 1 0.943** 0.835** 0.129 0.790** −0.941**

Total phenols 1 0.958** 0.378 0.626** −0.960**
Amino acids 1 0.589* 0.415 −0.870**
Reducing sugars 1 −0.396 −0.190
POD 1 −0.766**
Browning degree 1

LDOW
DOC Total phenols Amino acids Reducing sugars POD Browning degree

DOC 1 0.922** 0.889** −0.246 0.844** −0.979**

Total phenols 1 0.984** 0.069 0.671** −0.952**
Amino acids 1 0.137 0.616* −0.930**
Reducing sugars 1 −0.634** 0.183
POD 1 −0.813**
Browning degree 1

Note: * indicates significant at P < 0.05, and ** indicates significant at P < 0.01.

Fig. 3. The comparison scatter plot of the measured value and the predicted value of browning degree; a: In HDOW; b: In MDOW; c: In LDOW.

kinetics equation, and the values of R2adj were 0.842 and 0.674, re-
spectively. Within 0–7 weeks, the change of reducing sugars content in
LDOW pear wine samples accorded with the first-order reaction kinetics
equation, R2adj = 0.920; within 7–15 weeks, it accorded with the frac-
tional conversion model, R2adj = 0.949. Correlation analysis between
DOC and reducing sugars showed that the correlation coefficients were
0.371, 0.129, and −0.246 in HDOW, MDOW and LDOW, respectively,
indicating that the correlation between reducing sugars content and
DOC in the three pear wine samples was not significant.
3.5. Changes in PPO, POD and PAL activity of pear wines during storage
PPO and POD were considered to be the most critical enzymes as-
sociated with browning in grape and wine, and when POD and PPO
coexist, phenol degradation was increased. PPO was a copper-con-
taining oxidoreductase, which catalyzed the formation of corre-
sponding anthraquinones by phenols or polyphenol oxides under the
action of molecular oxygen, and further reacted to form dark brown
substances (Kaintz, Mauracher, & Rompel, 2014). POD was an iron-
containing oxidoreductase, which was considered to be highly

6
H. Yang, et al.
Fig. 4. The double-labeled map obtained by OPLS regression analysis; a: In HDOW; b: In MDOW; c: In LDOW.
Fig. 5. VIP diagram of the influence of quality indicators on the browning degree; a: In HDOW; b: In MDOW; c: In LDOW.
correlated with enzymatic browning. Some researchers believed that
the oxidation of POD to phenolic substances is the key mechanism for
the enzymatic browning of pears. POD generally used H2O2 as an
electron acceptor to catalyze the reaction of phenolic substrates to form
pigments. Especially when coexisted with PPO, its interaction with
phenolic substances would be significantly enhanced, playing a key role
in the process of enzymatic browning. PAL was a kind of enzyme closely
related to the metabolism of phenolic substances, which indirectly ef-
fect the synthesis of phenolic substances in plant tissues and played an
important role in the enzymatic browning of fruits and vegetables
(Chidtragool, Ketsa, Bowen, Ferguson, & van Doorn, 2011). The re-
search of Yudou found that the expression pattern of the genes PAL1,
PAL2 of PAL and the genes PbPPO1 of PPO was correlated with changes
in core browning of ‘Yali’ pears (Cheng et al., 2015). So, the browning
of Dangshan pear wine was likely related to PPO, POD and PAL.
This study examined the changes in PPO, POD and PAL activity
during the storage of pear wine with different DOC. The results showed
7
Food Chemistry 308 (2020) 125665
that no enzyme activity of PPO and PAL was detected during the sto-
rage process of 15 weeks. The enzyme activity of POD fluctuated greatly
during storage (Fig. 1e). At 0-5th week, the POD activity of pear sam-
ples with different DOC showed a rapid decline, while at 5-15th week,
the POD activity increased slowly. The POD activity of the three pear
wine samples was in the range of 0-5th week and 5-15th week, which
met the zero-order reaction kinetics equation. They were poor fitted the
equation with R2adj values only above 0.6. Correlation analysis between
POD and DOC showed that the correlation coefficients were 0.691,
0.790 and 0.844 in HDOW, MDOW and LDOW, respectively. It showed
that the change of POD activity had a certain correlation with DOC in
pear wine storage process.
3.6. Browning degree change of pear wines during storage
Color is an important sensory indicator of fruit wine. At present, the
research on the browning of fruit wine involves white wine, cider and
H. Yang, et al.
litchi wine, among which the research on browning mechanism and
browning inhibition of white wine is the most (Ricci, Parpinello, &
Versari, 2017; Feng, Lin, & Qin, 2011; Millet, Poupard, Guilois-Dubois,
Zanchi, & Guyot, 2019; de Lerma, Peinado, Moreno, & Peinado, 2010).
Different fruit wines have different browning mechanisms due to the
different nature of the raw materials, and the browning mechanism of
fruit wine is the key to inhibit browning of fruit wine. In the storage
process of the pear wines, their color was gradually deepening, which
mean that the browning degree of pear wine was gradually strength-
ened. The browning degree of the different DOC pear wine samples
changed with time during storage was as shown in Fig. 1f. The results
shown that in the initial stage of storage, the browning in HDOW was
slow, and after 10 weeks, the browning speed was accelerated. At the
end of storage, the color change of HDOW was the most, and the color
was the darkest at the end of the storage. At the 15th week, the A420 of
the HDOW was 0.137, the color of MDOW and LDOW was lighter and
the browning degree was relatively lower, A420 were 0.110 and 0.096
respectively. It could also be seen from the Fig. 1f that the higher the
DOC of the pear wine sample, the greater the browning degree of the
pear wine. The correlation analysis between the browning degree and
DOC, total phenols, amino acid, reducing sugars and POD were shown
in Table 2. It could be seen that the browning degree had a high cor-
relation with DOC, total phenols and amino acids in different pear wine
samples. The correlation coefficient between DOC, total phenols and
browning degree were greater than 0.9, indicating DOC and total
phenols were the most critical factor affecting browning. The fitting
result of the kinetic equation shown that in HDOW, the change of
browning degree satisfied the parabolic model, R2adj values was 0.889.
In MDOW and LDOW, The zero-order reaction kinetic equation could be
used adequately with R2adj values were 0.874 and 0.850, respectively.
3.7. Establishment of OPLS regression model for pear wine browning
3.7.1. PCA analysis of browning degree of pear wine
It was well known that it was necessary to perform PCA analysis
before doing the OPLS regression model, to investigate the objective
distribution of the data. Took the browning degree of pear wine as the
dependent variable Y, the DOC, total phenols content, total amino acids
content, total reducing sugars content and POD activity of the pear wine
were the independent variable X, and PCA analysis was performed on
browning changes of different DOC pear wine samples. The PCA ana-
lysis results were shown in Fig. 2. It could be seen from the PCA scores
that the browning degree of 0-15w of different DOC pear wine samples
had a trend change. As the first principal component migrated to the
left, the browning degree of the pear wine sample gradually deepened,
and in the Fig. 2, the darker the color toward the left point. This in-
dicated that the change of the quality index of pear wine and the
change of browning degree were related, and the data could be further
used to establish the OPLS regression model of pear wine browning
degree.
3.7.2. OPLS regression model of browning degree of pear wine
Took the browning degree of pear wine as the dependent variable Y
and the quality index of pear wine as the independent variable X, the
OPLS regression model of different DOC pear wine samples were es-
tablished by SIMCA 14.1. The model consisted of a linear combination
of constants and respective variables, the constant and independent
coefficients were shown in Table 3. The OPLS prediction model was
verified. The comparison scatter plot of the measured value and the
predicted value was shown in Fig. 3. In the prediction equations of
HDOW, MDOW and LDOW, R2 was 0.977, 0.946 and 0.976, respec-
tively. It showed that the OPLS model accurately predicted the
browning degree according to the quality index of the pear wine
sample, and the predicted result was reliable.
Fig. 4 was the double-labeled map obtained by OPLS regression
analysis. The relationship between the independent variable and the
8
Food Chemistry 308 (2020) 125665
dependent variable could be seen more intuitively. When the in-
dependent variable and the dependent variable were in the same di-
rection, indicated that they were positively correlated, and the depen-
dent variable increased as the independent variable increased. Fig. 5
were the VIP diagram of the influence of quality indicators on the
browning degree in different DOC pear wine samples, from which the
importance of each quality index on browning degree can be seen. In
HDOW, the greatest impact on browning was the total amino acids
content, followed by DOC and total phenols content. In MDOW, the
greatest impact on browning was the total phenols content, followed by
DOC and total amino acids content. In LDOW, the most influential in-
dicator of browning was DOC, followed by total phenols and total
amino acids content. Considered the VIP score map and correlation
analysis of different DOC pear wine samples, it could be concluded that
the DOC had the greatest influence on the browning in the pear wine
sample, the second was the total phenols content, and the third was the
total amino acids content. Combined with the reference, it could be
inferred that the main cause of browning of pear wine was the oxidative
polymerization of phenolic substances and the Maillard reaction in
which amino acids participated.
4. Conclusions
Through the dynamic monitoring of browning degree, DOC, total
phenols, total amino acids, reducing sugars content, POD activity and
OPLS regression model analysis of different DOC pear wine samples in
storage process, it was concluded that in the storage process of pear
wine, DOC was the first factor affecting browning of pear wine, total
phenols was the second factor, and followed by total amino acids.
Combined with published studies, it could be inferred that browning in
pear wine was dominated by non-enzymatic browning, mainly by oxi-
dative polymerization of phenolic substances and Maillard reaction
involving amino acids. Therefore, in the future, the research on the
inhibition of browning in pear wine should be based on the inhibition of
oxidative polymerization of phenolic substances and Maillard reaction.
They are the key to solving the browning problem of pear wine.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This study is supported by the National Natural Science Foundation
of China (31701730 and 31701588), the Program of Introducing
Talents of Discipline to Universities (111 project) (111-2-06), the
Priority Academic Program Development of Jiangsu Higher Education
Institutions (PAPD), the Collaborative Innovation Center of Jiangsu
Modern Industrial Fermentation, the Jiangnan University Postgraduate
Overseas Research Project, and the Fundamental Research Funds for
the Central Universities (JUSRP21914).
Compliance with ethical standards
The authors declare that they have no conflict of interests in per-
forming this work. This article does not contain any experiments on
human participants or animals performed by any of the authors.
Informed consent was obtained from all participants included in the
study.
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