Nghiên cứu sự thay đổi của vi sinh vật và yếu tố lí hóa trong quá trình sản xuất bánh men rượu phần

Tạp chí Khoa học và Công nghệ 52 (2) (2014) 167-176 
MICROBIAL AND PHYSIOCHEMICAL CHANGES DURING 
THE INCUBATION OF FEN-DAQU 
Le Van Diep1, *, Han Bei-Zhong2 
1Faculty of Chemistry, Vinh University, 182, Le Duan Street, Vinh City, Nghe An province 
2College of Food Science and Nutritional Engineering, China Agricultural University, China 
*Email: levandiep@vinhuni.edu.vn 
Received: 19 October 2012; Accepted for publication: 24 February 2014 
 ABSTRACT 
Fen liquor is typical of Chinese light-flavor liquor, which is fermented from sorghum with 
Fen-Daqu powder. Fen-Daqu is a saccharifying agent and fermentation starter obtained by 
natural solid-state fermentation under non-sterile conditions. The standard plate count and the 
online measurement methods were used to enumerate the surviving microorganisms, and 
measure the physiochemical in Fen-Daqu during the incubation. Total counts of mesophilic 
aerobic bacteria (30 °C and 55 °C), bacterial endospore (30 °C and 55 °C), lactic acid bacteria, 
enterobacteriaceae and fungi starting with minimum level around 106, <104, <105, <105, 105, < 
104 and 105 cfu/g and attaining maximum around 1011, 109, 109, 109, 107, 105 and 108 cfu/g, 
respectively. During the incubation of Daqu the microorganisms increased from Woqu to 
Liangmei periods and gradually decreased during the later phases. The pH in Daqu was 
increased over time during the incubation. The total acidity in Daqu increased and reaches to 
maximum at Shangmei phase (around 4.5 g lactic acid per kg Daqu) and then gradually 
decreased over the time. The relative humidity in incubation room was reduced from around 
100 % to around 20 %. Temperature in incubation room was increased over time from the first 
to middle period and decreased in the Yangqu phase. Temperature in Daqu inner was rapidly 
increased from around 20 to 40 ºC at Shangmei phase, dropped to 30 ºC at Liangmei phase, and 
then increased gradually until reached to maximal 52 ºC at Dahuo phase, finally decreased to 
original temperature (25 ºC). The moisture was decreased from around 45 % to around 10 % 
during successive phases of incubation. Based on these results, a microbiological regulation for 
the production of Fen-Hongxin Daqu is proposed. 
Keywords: Chinese liquor starter; Chinese liquor; traditional fermented; food microbial. 
1. INTRODUCTION 
Fen-Daqu is a natural fermentation starter, especially for distilled Chinese Fen liquor and 
traditional Chinese Fen vinegar production. Fen-Daqu is prepared from barley and peas by five 
Le Van Diep, Han Bei-Zhong 
167 
steps: (i) Ingredients formulation; (ii) Grinding and mixing; (iii) Shaping; (iv) Incubation (about 
1 month) and (v) Maturation (about 6 months). The incubation step is divided into seven phases: 
Woqu, Shangmei, Liangmei, Chaohuo, Dahuo, Houhuo and Yangqu, as described previously [1]. 
The production of Fen-Daqu is still the constitution of the traditional fermentation 
technology without artificial added microorganisms. It has been reported all microorganisms 
related to saccharification and fermentation in the starter are derived from materials and from 
environment [2]. Other reported showed that the microbial distribution on the surface of Fen-
Daqu were among of the bacteria, Lactobacillales, Actinomycetales, while among the fungi such 
as Saccharomycopsis and Issatchenkia were found in both the surface layer and the interior of 
Daqu [3]. Fen-Daqu also contain various enzymes, including amylase, protease, lipase, cellulose 
[4] and other metabolites, degradation products, and important flavor compounds [5]. 
Temperature plays an important role in the production of Daqu. The production of Daqu 
involves specific time-temperature control schemes resulting in a succession of microorganisms 
and natural result of metabolism. But until now, most of Daqu production still relies on workers’ 
experience. During the production of Fen-Daqu almost physicochemical parameters such as 
temperature, relative humidity, and moisture are detected by workers’ experience, such as “hand 
like a thermometer”[6]. 
We hypothesize that there was a converging relationship between the physicochemical 
change and microbial amount in Fen-Daqu during the incubation and they could be reflected the 
specific fermentation events and also relative to the quality of Daqu. But up to now, no 
microbial and physicochemical characteristics of Fen-Daqu during its phases of incubation have 
been reported. 
The objective of this research was to determine microbiological and physicochemical 
changes during the incubation of Fen-Daqu and also to assess whether these parameters could be 
used to control the quality of Fen-Daqu intermediate products. 
2. MATERIALS AND METHODS 
2.1. Sampling 
Fen-Daqu samples were obtained from Xinghuacun Fenjiu Group, Shanxi province, China. 
Daqu is fermented and matured in stacked layers. Samples were collect ... phases, and attaining maximum level (> 
8 log cfu/g) at Shangmei phase and then gradually decreased over the later phases. It could be 
explained that in the Shangmei phase reached to the optimum temperature, relative humidity and 
0
1
2
3
4
5
6
7
8
9
WQ SM LM CH DH HH YQ
Lo
g 
(cf
u
/g
) Lactic Acid Bacteria
Enterobacteriaceae
Fungi
0
2
4
6
8
10
12
WQ SM LM CH DH HH YQ
Lo
g 
(cf
u
/g
)
Total viable count (30°C)
Total viable count (55°C)
Bacterial spores (30°C)
Bacterial spores (55°C)
Le Van Diep, Han Bei-Zhong 
171 
moisture (as showed in figures 4 and 7) for fungal growth, therefore highest number were found. 
It was reported that the non-Saccharomyces yeasts represented most of the total yeasts 
population in Fen-Daqu [3], the non-Saccharomyces yeasts, such as Trichosporon asahii, 
Debaryomyces hansenii, Hanseniaspora guilliermondii were found in other Daqu [11]. They 
produce secondary metabolites, which can contribute to the final taste and flavor of wine [12]. 
Other reported showed that three types of mold (Thermomyces, Penicillium and Aspergillus) 
were found in Fen-Daqu. Among of them, Thermomyces were abundant in the interior Daqu [3]. 
That could be due to a higher temperature in the inner Daqu (as showed in figures 5 and 6). 
Figures 1 and 2 showed that at Woqu phase, the total viable counts of bacteria, fungal and 
LBA counts were quite high (range of 5 - 9 Log cfu/g). It could be explained that most of them 
are derived from materials or environment [2]. In addition the incubation room often used for 
several batches of Daqu making without sterilization, therefore the spores accumulated in 
environment and bring to this phase. 
It also observed that the level of fungi and LAB were lower than bacteria, which imply the 
dominant group of microorganism in Daqu is bacteria rather than fungi or LAB. That showed a 
positive correlation with the composition of microorganisms in Daqu [4, 13]. 
3.2. Physicochemical changes during the incubation 
Figure 3. Change of pH and total acidity during the incubation of Fen-Daqu 
(WQ: Woqu; SM: Shangmei; LM: Liangmei; CH: Chaohuo; DH: Dahuo; HH: Houhuo; YQ: Yangqu). 
Figure 3 showed that the pH increased over time during the incubation of Daqu. The rate of 
pH increase was slower in the first three phases (Woqu, Shangmei and Liangmei), and then 
become significant faster until Dahuo phase and finally keep in a steady level. 
Figure 3 showed that the total acidity increased at first phases, attaining to the maximum 
level (near 5 g/kg) at Shangmei phase, after that decreased during the middle phases and then 
gradually increased again during the later phases. The total acidity in Daqu is derived from acid 
producing microbial species, which mainly produce acetic acid, lactic acid, or the degradation of 
lipid and protein, etc [14]. 
It was observed that the total acidity attain maximum level at Shangmei phase, while the 
maximal level of LAB occurred in Liangmei phase. It could be explained that other bacteria such 
as acetic acid bacteria also present with high number in Daqu, they produce acetic acid and will 
0
1
2
3
4
5
6
7
8
WQ SM LM CH DH HH YQ
pH
0.0
1.0
2.0
3.0
4.0
5.0
6.0
Ac
id
ity
(g/
kg
)pH
Total acidity
Microbial and physiochemical changes during the incubation of Fen-Daqu 
172 
0
5
10
15
20
25
30
35
40
45
50
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Time (day)
Te
m
pe
ra
tu
re
(°C
)
0
10
20
30
40
50
60
70
80
90
100
RH
(%
)
Room temperature
RH
WQ CH DH HH YQ SM LM 
lead increase of titratable acidity. 
Figure 4 showed the changes of relative humidity and temperature in incubation room 
during the incubation of Fen-Daqu. The relative humidity was increased during the first phase 
and then decreased during later phases of the incubation. It attain at maximum level at Shangmei 
phase, this can be explained as that during this phase the temperature increased quickly as well 
as growth of microorganisms and the vapor released to the environment without artificial air 
ventilation. In other phases, since natural ventilation through turn of Daqu and open air windows 
and doors the relative humidity was reduced from 100 % to 20 %. It was also observed that the 
temperature increased rapidly in Shangmei phase from 15 ºC up to 40 ºC and Chaohuo, Dahuo 
phases attain maximum 45 ºC. That showed a positive correlation with the change of microbial 
count during these phases (see figures 1 and 2). In Liangmei phase, due to the good ventilation 
the heat was released to the surroundings at a lower temperature about 20 ºC, that in order to 
prevent damage overheating. 
Figure 4. Change of temperature and RH in incubation room during the incubation of Fen-Daqu 
(WQ: Woqu; SM: Shangmei; LM: Liangmei; CH: Chaohuo; DH: Dahuo; HH: Houhuo; YQ: Yangqu). 
Figure 5 showed that the temperature in surface of Daqu was increased from the end of 
Woqu phase to the end of Dahuo phase, and then reduced during the later phase. It could be 
related to the growth of microbial during these phases. 
Figure 6 showed that the room temperature increased over time during production of Daqu. 
Exception of Yangqu phase, the inner temperature of Daqu was higher than room temperature, 
that’s mainly because of microbial growth in Daqu. 
During the incubation of Daqu, at the beginning of Shangmei phase, the inner temperature 
decreased from 25 ºC to 18 ºC, and then rapidly increased to a higher level of above 40 ºC. After 
that, it dropped to 33 ºC at the end of Shangmei phase. During Liangmei phase, the temperature 
decreased again to 30 ºC and increased gradually until reached to maximal 52 ºC at the 
beginning of Dahuo phase. From that level, the temperature started to decrease slowly and 
finally back to original temperature (25 ºC). The aim of Shangmei to Liangmei phase is to 
activate initial microbial growth and to allow the temperature to increase gradually, attaining 30-
40 ºC in 3-5 d. The initial 24-48 h is considered as a crucial time for establishing the structure of 
Daqu’s microbial community, and hence the pioneer microorganisms such as fungi start to 
Le Van Diep, Han Bei-Zhong 
173 
10 
15 
20 
25 
30 
35 
40 
45 
50 
55 
60 
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 
Time (day)
Tempertature (°C) Daqu inner temperature 
Room temperature
WQ CH
 DH 
 HH
 YQ
SM LM
0
5
10
15
20
25
30
35
40
45
50
WQ SM LM CH DH HH YQ
Te
m
p.
(ºC
)
colonize and mycelium will spread over the surface of Daqu [1]. 
Figure 5. Change of temperature in incubation room 
(WQ: Woqu, SM: Shangmei, LM: Liangmei, CH: Chaohuo, DH: Dahuo, HH: Houhuo, YQ: Yangqu). 
Figure 6. Change of temperature in Daqu inner and incubation room during the incubation of Fen-Daqu. 
(WQ: Woqu; SM: Shangmei; LM: Liangmei; CH: Chaohuo; DH: Dahuo; HH: Houhuo; YQ: Yangqu). 
High temperature during the incubation phase (Chaohuo, Dahuo and Houhuo) could 
enhanced proteolysis and accumulation of amino acids [15, 16], and help to produce more 
volatile compounds such as pyrazines that could be formed through the Maillard reaction 
between saccharides and amino residues [17, 18]. 
The room temperature measured by IR thermometer (figure 5) was significant different 
with the data obtained with ibutton (figure 6), however the general trend is quite similar. Since 
Microbial and physiochemical changes during the incubation of Fen-Daqu 
174 
IR thermometer was placed in the space between Daqu blocks, and the distance to Daqu is quite 
short, therefore the measurements easily can be influenced by the activity inside of Daqu, 
especially the growth of different microorganisms. The ibutton was placed on the wall, which 
gives more accurate results and reflect the changes of room temperature. 
Figure 7 showed that the moisture in Fen-Daqu samples was decreased from around 45 % 
to around 10 % during successive phases of incubation. During these phases the moisture rather 
rapid decreased from Shangmei to Houhuo phase, due to the increased temperature and 
decreased of relative humidity in incubation room (as showed in figure 4), and good ventilated. 
Figure 7. Change of moisture in Fen-Daqu during the incubation. 
(WQ: Woqu; SM: Shangmei; LM: Liangmei; CH: Chaohuo; DH: Dahuo; HH: Houhuo; YQ: Yangqu). 
The moisture in Woqu phase showed a positive correlation with the percentage of water was 
added to the grinding and mixing stage about 40 %. There was a gradual decrease in the 
moisture content of the samples from Houho to Yangqu phase. That showed a positive 
correlation with the aim of this phase is to allow the equilibration of moisture, acidy and enzyme 
activity [16]. 
4. CONCLUSION 
The microbial and physiochemical changes in Fen-Daqu during the incubation were 
determined in this study. It also revealed a strong correlation between microbial and 
physiochemical measurements. This could help Daqu producers to monitor the progress of the 
Daqu manufacturing process by measuring the physiochemical parameters, in order to regulate 
the functional strains. 
Acknowledgment. We thank Shanxi Xinghuacun Fenjiu Group Company for Daqu sampling assistance 
and advice. This study is funded by National Natural Science Foundation of China (No. 31071592) and 
KNAW-China Joint Research Project (No. 07CDP015) from the Royal Netherlands Academy of Arts and 
Sciences. 
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10
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M
o
ist
u
re
(%
)
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175 
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Microbial and physiochemical changes during the incubation of Fen-Daqu 
176 
TÓM TẮT 
NGHIÊN CỨU SỰ THAY ĐỔI CỦA VI SINH VẬT VÀ YẾU TỐ LÍ HÓA TRONG QUÁ 
TRÌNH SẢN XUẤT BÁNH MEN RƯỢU PHẦN 
Lê Văn Điệp1, *, Han Bei-Zhong2 
1Khoa Hóa học, Trường Đại học Vinh, 182 Lê Duẫn, TP. Vinh, Nghệ An. 
2 Khoa Công nghệ Thực phẩm, Trường Đại học Nông Nghiệp Trung Quốc. 
*Email: levandiep@vinhuni.edu.vn 
Bánh men rượu Phần (Fen-Daqu) vừa là tác nhân đường hóa, vừa là tác nhân lên men trong 
sản xuất rượu Phần, một loại rượu trắng có hương thanh nhẹ nổi tiếng ở tỉnh Sơn Tây, Trung 
Quốc. Nó được sản xuất bởi quá trình lên men tự nhiên từ nguồn nguyên liệu đại mạch và đậu 
Hà lan. Phương pháp nuôi cấy vi sinh vật truyền thống và các kĩ thuật phân tích trực tuyến được 
sử dụng trong phân tích sự thay đổi của các nhóm vi sinh vật và các yếu tố lí hóa. 
Sự thay đổi của vi sinh vật phân biệt theo từng nhóm hiếu khí (30 °C), vi sinh vật ưa nhiệt 
(55 °C), bào tử vi khuẩn (30 °C và 55 °C), vi khuẩn lactic, enterobacteriaceae và nấm từ mức 
thấp nhất khoảng 106, 104, 105, 105, 105, 104 and 105 cfu/g và đạt cực đại ở khoảng 1011, 109, 109, 
109, 107, 105 and 108 cfu/g. Trong quá trình ủ vi sinh vật thay đổi theo xu thế tăng dần từ Woqu 
đến Liangmei và sau đó giảm dần theo thời gian. pH tăng dần, còn độ ẩm phòng giảm dần từ 
khoảng 100 % xuống còn 20 % và thủy phần bánh men giảm dần từ khoảng 45 % xuống còn 
khoảng 10 % theo thời gian quá trình ủ. Nhiệt độ phòng ủ tăng dần từ pha đầu cho đến pha giữa 
và giảm nhẹ ở pha sau. Nhiệt độ bên trong bánh men tăng nhanh ở giai đoạn Shangmei đạt 40 ºC 
sau đó giảm xuống còn 30 ºC ở giai đoạn Liangmei, tiếp đó tăng và đạt đến cực đại 52 ºC tại 
Dahuo, cuối cùng giảm dần đến nhiệt độ phòng (25 ºC) ở pha sau. 
Từ các kết quả trên và qua phân tích sự tác động qua lại giữa chúng có thể ứng dụng vào 
quá trình điều chỉnh sự phát triển của vi sinh vật trong quá trình sản xuất bánh men. 
Từ khóa: bánh men rượu, rượu trắng, lên men truyền thống, vi sinh vật.