Food Chemistry 319 (2020) 126554

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

Application of bamboo leaves extract to Chinese yellow rice wine brewing T

for ethyl carbamate regulation and its mitigation mechanism
⁎
Wanyi Zhou, Jingjin Hu, Xinglin Zhang, Qihe Chen
Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, China

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

Keywords: Bamboo leaves extract (BLE) contains various effective ingredients, including phenolic compounds. In this study,
Ethyl carbamate the effect of BLE on ethyl carbamate (EC) formation was investigated in Chinese yellow rice wine brewing with
Chinese yellow rice wine three different fermentation starters (Saccharomyces cerevisiae, Saccharomyces cerevisiae and Lactobacillus brevis,
Bamboo leaves extract and Chinese yeast). As a result, BLE showed significant inhibition effect on EC in multi-microbial fermented rice
Saccharomyces cerevisiae
wine, by preventing the reactions between urea/citrulline and ethanol. We found that BLE had influence on
Lactobacillus brevis
arginine transport (GAP1, CAN1, ALP1, and VBA2 gene) in Saccharomyces cerevisiae (S. cerevisiae), which sig-
nificantly up-regulated arginine uptake gene expression in vacuole (VBA2 gene) so that inhibited arginine
metabolism. Besides, the presence of BLE could improve the overall quality of Chinese yellow rice wine.
Consequently, it was worthwhile applying BLE to Chinese rice wine fermentation, especially the wine brewing
with S. cerevisiae and Lactobacillus brevis starter.

1. Introduction
Chinese yellow rice wine has been widely consumed and appre-
ciated in China for thousands of years. Traditional Chinese yellow rice
wine is fermented from cooked glutinous rice using Chinese yeast (the
traditional fermentation starter). The complex microbial community in
Chinese yeast can create unique flavor. However, ethyl carbamate (EC)
has been detected in Chinese yellow rice wine, which is significantly
higher than other alcoholic beverages (rice cooking wine, white spirit,
wine, beer) (Wu, Pan, Wang, Shen, & Yang, 2012). EC can induce
malignant tumors which firstly found in lungs of mice (Nettleship &
Henshaw, 1943). Plenty of researches have proved that EC can cause
multiple-site tumors in several species (Mirvish, 1968; Salaman & Roe,
1953; Tannenbaum, 1964; Thorgeirsson, Dalgard, Reeves, & Adamson,
1994). Thus, EC is now classified as a “probable human carcinogen” by
the IARC (Thorgeirsson et al., 1994).
There have been many studies about EC reduction during fermen-
tation, focusing on modification of fermentation methods and starters,
application of suitable enzymes, and genetic alteration of EC precursors
and their pathways (Chiva, Baiges, Mas, & Guillamon, 2009; Fang,
Dong, Xu, He, & Chen, 2013; Jiao et al., 2014). In alcoholic beverages,
EC is usually formed by the spontaneously reaction between ethanol
and its precursors, including urea and citrulline, which are mainly
produced by arginine catabolism (Hasnip, Caputi, Crews, & Brereton,
2004; Li et al., 2015; Liu, Pritchard, Hardman, & Pilone, 1995; Uthurry,
Lepe, Lombardero, & Del Hierro, 2006; Wu et al., 2014; Zhao et al.,
2013). In general, urea and citrulline are primarily accumulated by
urea cycle process and arginine deiminase (ADI) pathway, respectively
(Davis, 1986; Liu & Pilone, 1998; Liu et al., 1995). EC formation can be
greatly decreased by fermentation starters variation, using Sacchar-
omyces cerevisiae (S. cerevisiae) rather than the Chinese yeast (Fang,
Dong, Li, & Chen, 2015; Zhou, Fang, & Chen, 2017). EC formation can
be further decreased by co-cultivation with S. cerevisiae and Lactoba-
cillus brevis (L. brevis) (Fang, Zhou, & Chen, 2019). Notably, gallic acids
can exert negative effects on arginine metabolism via ADI enzyme in-
hibition (Alberto, Manca de Nadra, & Arena, 2012). Recently, it has
been reported that the presence of gallic acids can reduce EC formation
in Chinese yellow rice wine, using S. cerevisiae starter (Zhou et al.,
2017). Actually, the abundance of phenolic compounds in glutinous
rice and wheat materials, especially the gallic acid, make it a natural
inhibitor of EC during brewing (Que, Mao, & Pan, 2006; Xie, Xu, & Fan,
2011). Phenolic compounds are also effective ingredients of bamboo
leaves extract (BLE) which have been applied to food and medicine for
thousands of years in Asia. BLE has a plenty of biological functions, like
anti-oxidant and anti-tumor activities (Koide, Collier, Berry, & Panee,
2011; Kweon, Hwang, & Sung, 2001; Park & Jhon, 2010; Park, Lim,
Kim, Choi, & Lee, 2007).
In this work, we comparatively investigate the metabolic and

⁎
Corresponding author at: Yuhangtang Rd. 866, Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, China.
E-mail addresses: xinglinzhang@zju.edu.cn (X. Zhang), chenqh@zju.edu.cn (Q. Chen).

https://doi.org/10.1016/j.foodchem.2020.126554
Received 17 September 2019; Received in revised form 26 February 2020; Accepted 2 March 2020
Available online 03 March 2020
0308-8146/ © 2020 Elsevier Ltd. All rights reserved.
W. Zhou, et al.
transcriptional regulatory effects of BLE on EC metabolism during
Chinese yellow rice wine brewing with three different fermentation
starters, S. cerevisiae, S. cerevisiae and L. brevis, and Chinese yeast. In
addition, the influences of BLE addition on general quality of rice wine
are identified by ethanol content, amino acids, volatile flavor com-
pounds, and sensory evaluation analysis.
2. Materials and methods
2.1. Chemicals and materials
L-citrulline (at least 98%) and L-arginine (at least 98.5%) were ob-
tained from Sangon Biotech Corporation (Shanghai, China). Bamboo
leaves extract (contains at least 30% total flavonoids) was purchased
from Zhejiang Shengshi Biological Technology Ltd. Corporation
(Zhejiang, China). 9-xanthydrol (98%) and EC (at least 99%) were
purchased from Sigma-Aldrich Chemical Corporation (St Louis, MO,
USA). Methanol alcohol (HPLC grade) was obtained from Tianjin Shield
Specialty Chemical Ltd. Corporation (Tianjin, China). Besides, other
reagents used in this work were of analytical grade. Chinese koji and
Chinese yeast were obtained from Wuzhanmao Yellow rice wine
Industry (Zhejiang, China).
2.2. Culture media
Basal culture medium (BM): 5 g/L peptone, 5 g/L L-arginine, 10 beef
extract and 20 g/L glucose (pH 6.0). Yeast Extract Peptone Dextrose
Medium (YPD): 1% yeast extract, 2% peptone and 2% glucose. MRS
medium: 10 g/L peptone, 5 g/L beef extract, 20 g/L glucose, 1 mL/L
Tween 80, 2 g/L K2HPO4·3H2O, 5 g/L sodium acetate, 2 g/L citric acid
diamine, 0.58 g/L MgSO4·7H2O and 0.25 g/L MnSO4·4H2O (pH
6.2–6.6).
2.3. Chinese yellow rice wine fermentation
The fermentation process of Chinese yellow rice wine was carried
out by the method from our earlier study (Zhou et al., 2017) with minor
modification. 1.25 kg raw glutinous rice was dipped in 1.5 L water for
two days at 28 °C and then steamed for 20 min. The fermentation
starter (2.5 g), Chinese koji (225 g) and water (1.5 L) were mixed with
prepared cooked rice for pre-fermentation which lasted 4–5 days at
28 °C. Then it was set at 18 °C for post-fermentation. In addition,
Bamboo leaves extract was added at the third day of brewing process.
At the tenth day of brewing process, L. brevis (5 × 109 cfu/L) was
supplemented in the culture broth.
Notably, three kinds of fermentation starters were used in this test:
yeast fermentation system brewing by S. cerevisiae (designated as SC
system), double-microbial fermentation system brewing by S. cerevisiae
and L. brevis (designated as SL system), traditional fermentation system
brewing by Chinese yeast (designated as CY system). According to the
differences of bamboo leaves extract addition, each fermentation
system was further divided into a total of three subgroups which per-
formed with 0 mg/L (designated as SC, SL and CY), 250 mg/L (desig-
nated as SC250, SL250 and CY250) and 500 mg/L(designated as SC500,
SL500 and CY500), respectively.
2.4. Chinese yellow rice wine fermentation simulation with S. cerevisiae and
L. brevis
Before simulation experiment, initial microbial suspensions were
prepared as follows: S. cerevisiae was grown in YPD for 24 h (28 °C). L.
brevis was grown by growing in MRS for 24 h (37 °C). And then, S.
cerevisiae and L. brevis suspension were mixed in a volume ratio of 2:1 to
prepare an initial double-microbial suspension.
In simulation experiment, there existed three kinds of simulated
culture systems: system inoculated with initial S. cerevisiae suspension
2
Food Chemistry 319 (2020) 126554
(designated as SC), system inoculated with initial L. brevis suspension
(designated as LB), double-microbial culture system inoculated with
initial double-microbial suspensions (designated as SL). In this test,
initial microbial suspensions were used to inoculate BM without
shaking. System SC and SL were cultured for 36 h at 28 °C to
OD600 = 0.7–0.9, while system LB were cultured for 48 h at 37 °C to
OD600 = 0.5–0.6. Then, ethanol was added to all these systems to a
final concentration of 15%(v/v) which normally present in Chinese
yellow rice wine. In the mean time, bamboo leaves extract was further
supplemented. According to the differences of bamboo leaves extract
addition, simulated culture system was further divided into two sub-
groups which performed with 0 mg/L (designated as SCK, SLCK and
LCK) and 500 mg/L (designated as SE, SLE and LE), respectively.
Fermented samples were obtained by timing sampling and stored at
−80 °C for subsequent metabolic and transcriptional analyses.
2.5. Ethyl carbamate determination
The concentration of EC was analyzed by HPLC-FLD method (Fu
et al., 2010). The samples were concentrated to one tenth of the original
volume at 55 °C. Then derivative reactions were conducted between
1 mL concentrated samples (or standard EC) and 0.2 mL 9-xanthydrol
(0.01 M), in which 0.1 mL HCl (1.5 M) was added before reacting. All
the reaction mixtures had to react for 30 min without light after fully
mixing and finally put into chromatographic vials.
EC concentration was analyzed by HPLC-FLD method with a
Shimadzu RF-20A fluorescence detector. Chromatographic conditions
were as follows: Mobile phase A and B were methanol and water. The
wavelength of excitation and emission were kept at 233 nm and
600 nm, respectively. Chromatographic column was operated at 35 °C
and injection volume was 20 μL.
2.6. Urea and citrulline determination
Urea and citrulline concentration were determined by the method
described by Fang et al (Fang et al., 2015). In short, urea and citrulline
could react with acidic diacetyl monoxime in the presence of thiose-
micarbazide under different conditions. The reaction mixture was he-
ated by boiling water-bath for 15 min and then measured at the wa-
velength of 526 nm. The reaction mixture of citrulline was heated by
boiling water-bath for 5 min and measured at the wavelength of
530 nm.
2.7. Intracellular ornithine transcarbamylase (OTCase) activity
determination
Intracellular ornithine transcarbamylase (OTCase) activity was
measured using the method reported previously (Carrasco, Perez-Ortin,
& del Olmo, 2003). The crude extract of OTCase enzyme was prepared
by 15 min ultrasound treatment. OTCase activity was activated by
MnCl2 (10 mM) at 55 °C for 20 min. Subsequently, OTCase enzyme
reacted with its substrate (0.1 M arginine) for 55 min at 37 °C. The
enzyme-substrate reaction would break down arginine to produce or-
nithine and was ended by adding glacial acetic acid. To detect ornithine
concentration, ornithine reacted with ninhydrin in the presence of
phosphoric acid for 30 min at 100 °C. The content of ornithine was
finally determined by comparing with an ornithine standard solution
(0.6 mM). One unit activity of OTCase was defined as the molar amount
of ornithine produced per minute per microgram of protein in this
study.
2.8. Amino acids and volatile flavor compounds analysis
The amino acids concentration was analyzed by automatic amino
acid analyser (S 7130; Sykam Company, Munich, Germany). The vo-
latile flavor compounds were detected by gas chromatography-mass
W. Zhou, et al.
spectrometry (GC–MS) (7890B, 5977A, Agilent Technologies Inc.,
California, USA). However, the volatile flavor compounds needed to be
extracted before detection by headspace solid phase microextraction
(HS-SPME) with divinylbenzene/carboxen/poly (dimethylsiloxane)
(DVB/CAR/PDMS) coated fiber. The method of HS-SPME extraction
and GC–MS were referred to the literature by Fang et al (Fang et al.,
2015). The comparative effects of volatile flavors and amino acids
regulated by bamboo leaves extract were also elucidated using heat
maps.
2.9. Sensory evaluation
Sensory characters of Chinese yellow rice wine samples were eval-
uated by 11 panelists (5 males and 6 females) from Zhejiang University
with sensory evaluator experience. Four main sensory attributes of
appearance, aroma, taste and style were chosen to characterize the
sensory properties of Chinese rice wine samples (GB/T 13662-2008).
There were four reference scales to illustrate slight, weak, moderate and
strong intensity of each attribute, prepared as described in Table S1.
The samples were provided at a random order in clear cups to the pa-
nelists. Drinking water was serviced between every samples to gargle.
All evaluations were conducted individually. The results of sensory
profile evaluation were plotted on radar maps.
2.10. Ethanol content determination
Ethanol content was determined by a China official method (GB/T
13662-2008).
2.11. Real Time fluorescence-based quantitative PCR (RT-qPCR)
RNA from S. cerevisiae was extracted using Yeast RNA Kit (Omega
Bio-tek Inc., Norcross, Georgia, USA). 500 ng of RNA was used to
synthesize cDNA using PrimeScriptTMRT reagent Kit with gDNA Eraser
(Perfect Real Time) (Takara Bio Inc., Shiga, Japan). All primers for RT-
qPCR were designed by Primer Premier 6.0 software (PREMIER Bio soft
International, Palo Alto, USA) and the sequences were shown in Table
S2. ACT1 was performed as a reference gene. RT-qPCR was prepared
using SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (Takara Bio Inc., Shiga,
Japan). The amplification process was performed in a CFX96 Real-Time
PCR Detection System (Bio-Rad Laboratories, Inc., Berkeley, California,
USA). These results were analyzed by the 2-ΔΔCT method.
2.12. Statistical analysis
In this study, statistical analysis was carried out by one-way analysis
of variance (ANOVA) using Origin Pro 9.0 software. All the data of
results were expressed as means ± standard deviations (SD) of at least
three determinations. Significant differences were determined by Tukey
HSD test. P < 0.05 is considered to be statistically significant.
3. Results and discussion
3.1. Effects of BLE addition on Chinese yellow rice wine fermentation
3.1.1. EC reduction by BLE addition in three different fermentation systems
As well known, EC, a group 2A carcinogen, can be produced during
the fermentation of Chinese yellow rice wine (Lachenmeier, Przybylski,
& Rehm, 2012; Thorgeirsson et al., 1994). In this study, the regulatory
effects of BLE on EC formation in three different fermentation systems
were investigated. The results of EC concentration in multi-microbial
fermentation systems (SL and CY system) were shown in Fig. 1, which
revealed that BLE could dramatically inhibit EC formation in multi-
microbial fermented Chinese yellow rice wine. As in contrast to the
control, EC levels decreased significantly in experimental groups during
fermentation process, especially in group SL500 and CY500. After
3
Food Chemistry 319 (2020) 126554
Fig. 1. Effect of bamboo leaves extract (BLE) on EC formation in Chinese yellow
rice wine brewing with different fermentation starters. (CY system fermented
by Chinese yeast, CY: contain no additive; CY250: contain 250 mg/L BLE;
CY500: contain 500 mg/L BLE. SL system fermented by S. cerevisiae and L.
brevis, SL: contain no additive; SL250: contain 250 mg/L BLE; SL500: contain
500 mg/L BLE).
33 days of brewing, EC concentration could be finally reduced by
10.58%, 31.25%, 21.67%, and 34.69% in group SL250, SL500, CY250,
and CY500, respectively. Besides, EC level was dramatically lower in
experimental groups of SL system than CY system. These results made it
clear that EC concentration could be mostly reduced in the double-
microbial fermentation system and the inhibition effect of BLE on EC
formation was dose-dependent. However, the concentrations of EC in
SC system were shown in Fig. S1, which revealed that BLE did little
effect on EC reduction in S. cerevisiae fermented Chinese yellow rice
wine. Thus, in the following study, we focused on the influence of BLE
addition in multi-microbial fermentation systems.
3.1.2. Contributions of BLE addition to EC precursors
In multi-microbial fermentation systems, the concentrations of the
main EC precursors, urea and citrulline, were shown in Fig. 2(a, b). In
contrast to the control, urea and citrulline concentrations were higher
in experimental groups, except group SL250. High levels of urea and
citrulline in Chinese yellow rice wine meant that the amounts of pre-
cursors used to produce EC were lower. So that EC concentration re-
duced as determined above. Thus, the possible mechanism was that the
reactions between urea/citrulline and ethanol were inhibited by BLE
addition, especially the addition of 500 mg/L BLE.
By the way, the slightly decrease of EC did not lead to high pre-
cursors concentrations in group SL250, suggesting that BLE might
promote precursors catabolism or inhibit arginine metabolism.
However, low concentration of BLE influenced more on precursors
catabolism or arginine metabolism than their reactions between ethanol
in the double-microbial fermentation system.
3.1.3. Influence of BLE addition on OTCase activity
The main EC precursors, urea and citrulline, are metabolites of ar-
ginine metabolism which involves two key enzymes, arginine deimi-
nase (ADI) and catabolic ornithine transcarbamylase (OTCase) (Davis,
1986; Liu & Pilone, 1998; Liu et al., 1995). In this study, the regulatory
effect of BLE on OTCase activity in different groups was investigated
and presented in Fig. S2. There was little difference on OTCase activity
between experimental groups and the control, except the group SL500.
The results implied that BLE could not reduce EC formation by in-
creasing OTCase activity during brewing in those groups. However,
higher OTCase activity did not lead to citrulline reduction in group
SL500, indicating that high concentration of BLE might influence less
on citrulline catabolism than the reaction between citrulline and
W. Zhou, et al.
Fig. 2. Variation and comparison of urea and citrulline content in the process of Chinese yellow rice wine under different disposals. (a) Variation and comparison of
urea content. (b) Variation and comparison of citrulline content. (CY system fermented by Chinese yeast, CY: contain no additive; CY250: contain 250 mg/L BLE;
CY500: contain 500 mg/L BLE. SL system fermented by S. cerevisiae and L. brevis, SL: contain no additive; SL250: contain 250 mg/L BLE; SL500: contain 500 mg/L
BLE).
ethanol. Therefore, BLE addition could not cut down EC concentration
by OTCase activity regulation.
3.2. General quality analysis of Chinese yellow rice wine fermentation with
BLE addition
In this study, comparative analyses of ethanol content, amino acids,
volatile flavor compounds, and sensory profile in different groups were
made to evaluate the impact of BLE on the general quality of Chinese
yellow rice wine in multi-microbial fermentation systems. BLE did good
for ethanol content of Chinese yellow rice wine, to some degree. The
China official standard requires Chinese yellow rice wine to have an
ethanol content of more than 8% (v/v) (GB/T 13662-2008). Obviously,
ethanol content reached the requirement in all groups (Fig. S3). Actu-
ally, the ethanol content in commercially available Chinese yellow rice
wine is generally 15% (v/v). Obviously, ethanol contents in CY system
were all higher than 15% (v/v) and only existed some minor differences
between experimental and control groups. By supplying BLE, ethanol
content increased in SL system and finally reached to 14%(v/v) in
group SL500. These results revealed that BLE addition had a little
beneficial effect on ethanol formation in SL system.
There are plenty of amino acids in Chinese yellow rice wine, which
primarily produced by hydrolysis of protein and performed as nitrogen
source during brewing (Zhu, Li, Liu, & Zheng, 2008). As precursors for
aroma components, amino acids can directly contribute diverse taste
characteristics to Chinese yellow rice wine, which enables the wine to
be mellow, rich, soft, smooth, harmonious, multifragrant, and so on (Li,
Zeng, Liu, Zhu Ge, & Yu, 2010; Shen et al., 2010). The concentrations of
amino acids were shown in Fig. S4. In each amino acid we detected,
there only existed some minor differences between experimental and
control groups in each fermentation systems. Besides, no dramatic
differences of total amino acids contents were observed, except the
group SL500. The total amino acids in group SL500 were significantly
higher than the control. Therefore, BLE addition had no dramatic effect
on amino acids in CY system, but a little beneficial effect in SL system,
during the brewing of Chinese yellow rice wine.
In Chinese yellow rice wine, aroma is a crucial determinant of the
hedonic quality, which related to numbers of volatile flavor substances
(including higher alcohols, esters and volatile acids) produced during
fermentation. Among them, isoamyl alcohol, phenylethyl alcohol and
ethyl acetate were quantitatively the most crucial odor-active com-
pounds and had great influence on aroma profile of Chinese yellow rice
wine (Chen, Xu, & Qian, 2013). In this study, over thirty volatile flavor
compounds were identified by GC–MS and shown in Fig. 3. It could be
seen that the main flavor substances in the rice wine were isoamyl al-
cohol, phenylethyl alcohol, ethyl acetate, ethyl lactate, isoamyl acetate,
4
Food Chemistry 319 (2020) 126554
ethyl caprylate, phenylethyl acetate, ethyl decanoate, ethyl palmitate
and benzaldehyde; most volatile flavors compounds were present in
both the experimental and control groups, but there were still some
differences. The higher alcohols were lower in all experimental groups
than that in the control. As for each ester we detected, there existed
some differences between control and experimental groups. In short,
esters were more abundant in control group in SL system, but more
abundant in experimental groups in CY system. Besides, benzaldehyde
with bitter almond aroma was moderately higher in all experimental
groups. In conclusion, BLE addition had no dramatic effect on volatile
flavor compounds in CY system, but a little disadvantage effects in SL
system.
BLE addition had a beneficial effect on sensory profile of Chinese
yellow rice wine. The sensory profile of Chinese yellow rice wine
samples was assessed and compared in comprehensive consideration of
individual preference. The results of sensory profile evaluation were
presented in Fig. 4(a, b). Comparing with two control groups, the wines
exhibited a little bit higher levels of appearance, aroma, taste, style and
final total mouthfeel attribute scores in all experimental groups, except
CY500 which had a slightly lower aroma, taste attribute and final total
mouthfeel attribute scores. However, the sensory profile of experi-
mental samples was better than that of the control while taking overall
analysis. In conclusion, the results indicated that the addition of BLE
had beneficial effects on general quality of Chinese yellow rice wine in
multi-microbial fermentation systems.
3.3. Metabolic and transcriptional regulatory effects of BLE on S. cerevisiae
and L. brevis in BM broth
According to the results of above fermentation experiment, EC
concentration could be reduced using the double-microbial fermenta-
tion starter (S. cerevisiae and L. brevis), and the addition of 500 mg/L
BLE could dramatically inhibit the formation of EC in SL fermentation
system. Therefore, in three different Chinese yellow rice wine simulated
culture systems, the metabolic and transcriptional regulatory effects of
500 mg/L BLE on S. cerevisiae and L. brevis were investigated, which
were respectively presented in Fig. 5 (a, b, c, d) and Fig. 6 (a, b).
The results of cell growth in different groups were determined by
OD600 and shown in Fig. 5 (a). Upon BLE addition, OD600 increased in
group SE, increased slightly in group SLE, and had no significant change
in group LE, which indicated that BLE promoted the growth of S. cer-
evisiae but had little influence on L. brevis growth. Besides, the varia-
tions of OTCase activity in different culture systems were shown in
Fig. 5 (b). There were no remarkable differences between experimental
groups and the control in all three different simulated culture systems,
which implied that BLE had little influence on OTCase activity. Further,
W. Zhou, et al.
the concentrations of urea and citrulline were presented in Fig. 5 (c, d).
Upon BLE supplementation, urea and citrulline concentrations de-
creased in group SLE, decreased slightly in group SE, and had little
change in group LE, which confirmed that the urea and citrulline me-
tabolism of S. cerevisiae were influenced by BLE and then reduced the
concentrations of extracellular urea and citrulline.
Transportation and metabolism of arginine in S. cerevisiae are shown
in Fig. 6 (a). Urea is a primary precursor of EC in Chinese yellow rice
wine. Intracellular urea is usually produced by arginine degradation in
S. cerevisiae through arginase (CAR1 gene) and ornithine amino-
transferase (CAR2 gene) (Cooper, 1982; Steinle, Bergander, &
Steinbuchel, 2009). Intracellular arginine derives from protein de-
gradation and uptake from fermentation broth. Extracellular arginine is
firstly transferred into cytoplasm by arginine permeases (ALP1 and
CAN1 gene) and general amino acid permease (GAP1 gene), and then
transferred into vacuole by vacuolar basic amino acid transporter 2
(VBA2 gene) (Zhang et al., 2016). Overexpression of Can1p and Alp1p
can increase the efficiency of arginine uptake (Zhang et al., 2016).
However, arginine in vacuole will retain as a nitrogen reserve, even
though external arginine is depleted (Jiao et al., 2014).
Urea metabolism in S. cerevisiae involves in two key enzymes, urea
5
Food Chemistry 319 (2020) 126554
Fig. 3. Relative content (%) of volatile flavor com-
pounds in the process of Chinese yellow rice wine
under different disposals. (CY system fermented by
Chinese yeast, CY: contain no additive; CY250:
contain 250 mg/L BLE; CY500: contain 500 mg/L
BLE. SL system fermented by S. cerevisiae and L.
brevis, SL: contain no additive; SL250: contain
250 mg/L BLE; SL500: contain 500 mg/L BLE).
carboxylase and allophanate hydrolase, which are activated by urea
amidolyase (DUR1,2 gene) (Genbauffe & Cooper, 1986; Whitney &
Cooper, 1972; Zhao et al., 2013). In addition, the accumulation and
excretion of urea to the extracellular refer to a complex system (An &
Ough, 1993). In Chinese yellow rice wine fermentation, extracellular
urea can be reabsorbed and reutilized through a sodium–urea sym-
porter (DUR3 gene) in S. cerevisiae (Cooper & Sumrada, 1975; Monteiro
& Bisson, 1991). DUR1,2 and DUR3 expression are regulated by two
transcription factors (DAL81 and DAL82 gene) (Elberry, Majumdar,
Cunningham, Sumrada, & Cooper, 1993). Citrulline in S. cerevisiae can
be degraded by argininosuccinate synthetase (ARG1 gene) (Steinle
et al., 2009).
Thus, the transcriptional expression levels of these genes in S. cer-
evisiae were assayed by RT-qPCR and the altered expression results
were shown in Fig. 6. Compared to the control without BLE, GAP1
(1.67-fold), CAN1 (1.20-fold) and VBA2 (3.10-fold) showed up-reg-
ulation, while ALP1 (0.66-fold) showed down-regulation. These find-
ings reflected that BLE might influence arginine transport and change
the distribution of arginine in cytoplasm and vacuole, so that influence
the arginine metabolism in S. cerevisiae. As for arginine, urea and ci-
trulline metabolism, there was not much fold change on CAR1, CAR2,
Fig. 4. Comparison of the sensory profile
in the process of Chinese yellow rice wine
fermentation under different disposals.
Sensory characters were evaluated by 11
panelists. The values are shown as the
means. (CY system fermented by Chinese
yeast, CY: contain no additive; CY250:
contain 250 mg/L BLE; CY500: contain
500 mg/L BLE. SL system fermented by S.
cerevisiae and L. brevis, SL: contain no ad-
ditive; SL250: contain 250 mg/L BLE;
SL500: contain 500 mg/L BLE).
W. Zhou, et al. Food Chemistry 319 (2020) 126554

Fig. 5. Metabolic regulatory effects of

bamboo leaves extract (BLE) on S.
cerevisiae and L. brevis in simulated
culture systems under different dis-
posals. (a) Effects of bamboo leaves
extract on cell growth. (b) Variation
and comparison of OTCase activity.
(c) Variation and comparison of urea
content. (d) Variation and compar-
ison of citrulline content. (SCK: S.
cerevisiae with no additive; LCK: L.
brevis with no additive; SLCK: double-
microbial system with no additive;
SE: S. cerevisiae with 500 mg/L BLE;
LE: L. brevis with 500 mg/L BLE. SLE:
double-microbial system with
500 mg/L BLE).

DUR1,2, DUR3 and ARG1. The present results implied that BLE had
little transcriptional regulatory effects on citrulline, urea and arginine
catabolism in S. cerevisiae. Besides, DAL82 (1.45-fold) showed up-reg-
ulation, while DAL81 (0.46-fold) showed down-regulation. The results
indicated that BLE had some influence on these two transcription fac-
tors. Herein, we presumed that BLE might significantly increase argi-
nine uptake in vacuole and decrease cytoplasmic arginine level in S.
cerevisiae in order to regulate arginine metabolism and finally reduce
the concentration of extracellular urea and citrulline.
4. Conclusion
was dose-dependent. In general, there were two possible mechanisms
for EC reduction: on the one hand, BLE prevented the reactions between
urea/citrulline and ethanol; on the other hand, BLE might significantly
influence arginine transport in S. cerevisiae, which increased arginine
uptake in vacuole and decreased cytoplasmic arginine content by up-
regulating arginine uptake gene in vacuole (VBA2 gene). Therefore,
arginine metabolism was inhibited so that finally reduced the con-
centrations of extracellular urea and citrulline. Besides, we found that
the supplementation of BLE could improve the general quality of
Chinese yellow rice wine. Thus, it was worthwhile applying BLE to
Chinese yellow rice wine during mixed-fermentation with S. cerevisiae
and L. brevis.

To sum up, EC concentration could be reduced by using appropriate

fermentation starter, especially the S. cerevisiae and L. brevis starter. CRediT authorship contribution statement
However, BLE could only show significant inhibition effect on EC for-
mation in multi-microbial fermented Chinese yellow rice wine, which Wanyi Zhou: Methodology, Validation, Formal analysis,

Fig. 6. Transcriptional regulation ef-

fects of bamboo leaves extract on S.
cerevisiae in simulated culture systems
under different disposals. (a)
Transportation and metabolism of ar-
ginine in S. cerevisiae. (b) Real Time
fluorescence-based quantitative PCR
of arginine transportation and meta-
bolism genes in S. cerevisiae (GAP1,
CAN1, VBA2, ALP1, CAR1, CAR2,
DUR1,2, DUR3, ARG1, DAL81, and
DAL82 genes). The left y-axis indicates
relative gene expression levels de-
termined by RT-qPCR. Relative gene
expressions were normalized by com-
parison with the expression of ACT1
and analyzed using the 2−ΔΔCT
Method.

6
W. Zhou, et al.
Investigation, Data curation, Writing - original draft, Visualization.
Jingjin Hu: Conceptualization, Methodology, Validation, Formal ana-
lysis, Visualization. Xinglin Zhang: Resources, Writing - review &
editing, Supervision. Qihe Chen: Conceptualization, Methodology,
Resources, Supervision, Project administration, Funding acquisition.
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 work was financially supported by the Natural Science
Foundation of China (31871904, 31171734).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2020.126554.
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