Screening
and Optimization of High-Yield Diastase-Producing Strains from Shedian Baijiu
Fermented Mash
Yanbo Liu1,2,3, Zhijun Zhao1,2,3, Wenjuan Zhang1,2,3, Wenning Gu1,2,3, Xian Wang4, Xiyu Sun1,2,5 and Chunmei Pan1,2,3*
1College of Food and Biological Engineering (Liquor
Industry College), Henan University of Animal Husbandry and Economy, Zhengzhou,
Henan 450046, China
2Henan Liquor Style Engineering Technology Research
Center, Henan University of Animal Husbandry and Economy, Zhengzhou, Henan
450046, China
3Zhengzhou Key Laboratory of Liquor Brewing
Microbial Technology, Henan University of Animal Husbandry and Economy,
Zhengzhou, Henan 450046, China
4SheDianLaoJiu Co. Ltd., Sheqi, Henan 473300, China
5ZhangGongLaoJiu Wine Co. Ltd., Mingling,
Henan476733, China
*For correspondence: sige518888@163.com
Received 28 August 2020;
Accepted 22 September 2020; Published 10 December 2020
Abstract
Diastase-producing
bacteria were isolated from the fermented mash of Shedian baijiu (stilled
liquors). Then the bacteria were preliminarily screened with a transparent ring
method and diastatic activity was detected for secondary screening. Finally, 1 high-yield diastase-producing strain was
obtained and identified as Bacillus velezensis by morphologic
observation and 16S rDNA molecular biological identification. The
enzyme-producing conditions of this strain were studied via single-factor experiments and optimized through response
surface method. On basis of liquid fermentation
medium, the optimal diastasic conditions of this strain were: soluble starch as
the carbon source, peptone as the nitrogen source, initial pH 4.0, inoculation
amount of 10%, and fermentation time of 3 d. The enzymatic activity after
optimization was up to 120.87 ± 1.37 U/mL, or 4.06 times the initial activity. © 2021 Friends Science
Publishers
Keywords: Shedian liquor fermented mash;
High-yield diastase-producing strain; Isolated identification; Response surface
methodology; Enzyme production condition optimization
Introduction
Luzhou-flavor baijiu is distilled
liquor resulting from natural fermentation of grains (Long et al. 2018; Zou et
al. 2018). Fermented mash is the material
produced from microbiologic blending and fermentation of liquor materials (Yang
et al. 2018a) and consists of diverse
bacteria, including actinomycetales and saccharomycetes. The metabolites of
these microorganisms are an important material basis accounting for the unique
flavour and mouth feel of baijiu (Wang et
al. 2008; Fang et al. 2019).
During the
fermentation of baijiu, the microorganisms contained in fermented mash secrete
various enzymes that can decompose macromolecules (e.g., proteins,
lipids, glucoses) in the raw materials into micromolecules (e.g., amino
acids, fatty acids, oligosaccharides) (Guo et
al. 2014). In particular, diastases are an important enzymatic system in
the saccharification stage of baijiu fermentation (Yu et al. 2015; Finley 2018). α-diastase can rapidly hydrolyze
the glycosidic bonds of α-1,4 glucose and break huge starch molecules into
micromolecules, fast decreasing the starch slurry viscosity and forming dextrin
and minor oligosaccharides and maltoses, which are involved in the next step of
fermentation (Perry et al. 2007).
Hence, the effects of diastases on baijiu production cannot be ignored. The
fermented mash is rich in diastases, so screening high-yield diastase-producing
strains from the fermented mash and applying them into the liquor-making
industry will largely improve the utilization rate and distillation yield of
raw materials, and the functional bacteria screened out can also be applied
into other fields.
In recent
years, many efforts have been made to screen high-yield diastase-producing
strains from baijiu Daqu (the yeast for making hard liquor) (Effront and Biodin
1916; Sun et al. 2019; Yao et al. 2019), but there is little
research about screening high-yield diastase-producing strains from the
fermented mash of baijiu. Only one study has focused on high-yield
diastase-producing strains from the fermented mash of mild-fragrant liquors.
Hence, our study is targeted at the fermented mash from Luzhou-flavor Shedian
liquors and at screening high-yield diastasic strains from the fermented mash.
Moreover, the enzyme-producing conditions were investigated. The findings will
theoretically underlie the culture of favorable bacteria from fermented mash.
Materials
and Methods
Materials
The main material was the fermented
mash collected from Shedian Laojiu Co., Ltd. (Henan Province, China).
Culture media
TruSeqTM DNA Sample Prep Kit、FastDNA
SPIN Kit for Soil Kit, American MP
Biomedicals Com.; AxyPrepDNA Gel Extraction Kit, American Axygen Biosciences
Com.; QuantiFluor™-ST, American
Promega Com.; DNA Polymerase AP221-02、Trans DNA 15KMarker, Beijing Quan shijin Biotechnology Co., Ltd.
The culture
media were: a starch agar screening medium (1% peptone, 0.5% beef extract, 0.5%
NaCl, 0.2% soluble starch, 2% agar powder, 121°C, sterilization for 30 min); a
seed liquid medium (0.5% beef extract, 0.5% NaCl, 1% peptone, pH 7.0, 121°C,
sterilization for 30 min); a liquid fermentation medium (20 g of soluble
starch, 20 g of peptone, 5 g of Na2HPO4, 0.1 g of MgSO4,
0.1 g of NaCl); an agar slant medium (10 g of peptone, 5 g of beef extract, 5 g
of NaCl, 20 g of agarose, 1 L of water, pH 7.0, 121°C, sterilization for 30
min).
Instruments
The instruments included a ZQLY-300S thermostatic
vibration incubator (Shanghai Zhichu Instrument Co., Ltd.), an LDZM-60KCS
upright steam sterilizer (Shanghai Shenan Medical Instrument); an SW-CJ-2F
double two-sided purification workbench (Suzhou Purification Equipment Co.,
Ltd.); an LQ-C3002 electronic balance (Shanghai Yaoxin Electronic Technology
Co., Ltd.); an HH-6 thermostat water bath (Changzhou Fangke Instrument Co.,
Ltd.); a 101-2AS electrothermal blowing dry box (Beijing Kewei Yongxing
Instrument Co., Ltd.); a DNP-9272BS-III electro-heating thermostatic cultivator
(Shanghai CIMO Medical Instrument Manufacturing Ltd. Co.).
Preliminary screening
Fermented mash (10 g) was added
into a triangular flask containing 90 mL of sterile water at 37°C and then
oscillated at 150 r/min for 2 h. Then 1 mL of the solution was sucked using a
pipette and diluted to 10-6. From the diluent, 100 μL
was sucked to paint a tablet, which was then placed into the thermostatic
incubator for 24 h of cultivation at 37°C (Luo and Xie 2012). After that, the
plate was colored with solid iodine fumigation. Specifically, iodine particles
(about 1 g) were uniformly laid on a white paper, area of which should be
smaller than that of a culture dish. Then the culture dish containing strains
was backed-off onto the iodine particles and was then rotated for 40 s to make
the coloring uniform. After the medium turned blue, the sizes of the
transparent rings around the strains were observed, and the diastase-producing
strains were preliminarily screened (Li et
al. 2009). The strains with representative fungal colonies as-selected were
purified on a beef extract peptone medium, and freeze-preserved in glycerole.
Afterwards,
the strains were separately dripped onto the starch agar screening medium and
cultured for 24 h in the thermostatic incubator, followed by solid iodine
fumigation. Then the transparent rings around the diastase-producing fungal
colonies were observed, and the ratio of transparent ring diameter (D) to
colony diameter (d) was determined with a ruler. The strains from large D/d
were selected and purified so as to preliminarily screen out the diastase-producing
strains (Mao et al. 2015).
Secondary screening
Acquisition of rude enzyme liquids:
From the slant medium, the single bacterial colonies
were picked off and inoculated into the liquid seed culture medium, followed by
culture for 24 h in a shaking table at 150 r/min and 37°C. After that, the seed
medium was inoculated at a concentration of 10% onto a liquid fermentation
medium, followed by 24 h of shaking culture at 150 r/min and 37°C (Wang et al. 2017a). After the fermentation
liquids were centrifuged (8000 r/min, 10 min), the supernate was collected and
used as the enzyme liquids for measurement of diastase activity (Liu et al. 2010).
Detection of enzymatic activity: Enzymatic activity was detected using a modified YOO method (Wang and
Tang 1995; Shi and Jiang 1996). Specifically,
5 mL of a 0.5% (mass fraction) soluble starch solution was collected and
preheated in a water bath at 40°C
for 10 min; then the rude enzyme solution (0.5 mL) after appropriate dilution
was added, followed by 5 min of accurate reaction in the water bath at 40°C. The reaction was terminated by adding
5 mL of 0.l mol/L H2SO4. Then 0.5 mL of the resulting
solution was mixed with 5 mL of a sparse iodine solution for coloration,
followed by measurement of absorbance at 620 nm. Also 0.5 mL of water instead
of 0.5 mL of the reaction system was used as a blank, and a tube without adding
the enzyme solution (the same volume of water instead) was used as a control.
Unit of
enzymatic activity: 1 activity unit (U) was defined as the quantity of the
enzyme needed to hydrolyze 1 mg of 0.5% starch at 40°C within 5 min.
Enzymatic activity
Where Rt is the
absorbance of the control; R is he absorbance of the reaction solution; D
is the dilution times of the enzyme solution and was adjusted during the
experiments to make (Rt-R)/Rt fall
within 0.2 and 0.7. From the tested results, the smallest absorbance
corresponded to the strain with the strongest enzymatic activity.
Identification of strains
Morphologic observation: The strains screened out from the plate culture were observed in terms of
colonial morphology and mobility; then single bacterial colonies after the
plate culture were selected and divided by Gram's staining into Gram-positive
bacteria (G+) and Gram-negative bacteria (G-).
Molecular biological identification
Total DNA was extracted from the
strains using a Tiangen bacterial genomic DNA extraction kit and sent to
polymerase chain reaction (PCR). The forward and reverse primers were the
universal primers used in bacterial 16S rDNA amplification: 27F
(5′-AGAGTTTGATAGAGTTTGATC-3′) /1492R (5′-GGTTACCTTGTTACGACTT-3′).
The PCR conditions were: predenaturation at 94°C for 4 min; denaturation
at 94°C for 1 min, annealing at 55°C for 60 s, extension at 72°C for 2 min, 30
cycles; extension at 72°C for 10 min. After that, the PCR products (5 μL)
were processed by 1% agarose electrophoretic analysis to detect yield and
specificity in Shanghai Sangon Biotech Co., Ltd. The sequencing results were
compared, on Blast, with the data from National Center of Biotechnology
Information (NCBI), and the approximate sequences were sent to phylogenetic
analysis.
Optimization of enzyme-producing conditions
Single-factor assays: During single-factor
analysis, the effects of carbon source (bran, corn flour, glucose, sucrose,
soluble starch), nitrogen source (ammonium sulfate, urea, soybean meal powder,
ammonium nitrate, peptone), initial pH (3.0, 4.0, 5.0, 6.0, 7.0), inoculation
amount (4%, 6%, 8%, 10%, 12%), and fermentation time (1, 2, 3, 4, 5 d) on
enzyme yields were investigated. The target strains after activation were
inoculated at the amount of 10% into triangular flasks, followed by shaken
culture at 150 r/min and 37°C prior to enzymatic activity measurement. Each
group was repeated 3 times and the average value was used. Then the optimal
enzyme-producing conditions were determined.
Response surface methodology (RSM) optimization
With diastatic activity as the response target, the
experimental data were analyzed with BoxBehnken Design from RSM and with Design
Expert 8.0.6. The optimal test conditions were determined and validated (Salehi
et al. 2017).
Results
Screening of high-yield diastase-producing strains from
fermented mash
The samples of Shedian Baijiu fermented mash were
isolated, purified and cultured and the resulting single bacterial colonies
were inoculated onto the starch agar screening medium for 1 d of culture at
37°C, followed by solid iodine fumigation. The results were illustrated in Fig.
1. After preliminary screening, over 90 diastase-producing strains were
obtained. After measurement of transparent ring diameter D and strain diameter
d, 7 strains with D/d > 3 were selected and sent to secondary screening
(Table 1).
From the 7 strains with large
transparent rings as-screened, the diastase activity of each strain was
detected using the modified YOO method. Results showed the enzymatic activity
of strain 5 was up to 29.78 U/mL and was larger than the other 6 strains (Table
1). Hence, strain 5 with the strongest diastase-producing ability was selected
as the test strain.
Identification of high-yield
diastase-producing strains from fermented mash
Morphologic observation:
The strain 5 was inoculated onto the starch agar screening medium for 1 h
of culture at 37°C. Then the colonial morphology was observed. It was found the
strain was generally round-shaped and milk white, while its middle was bulged
up and its surface was dry and folded. The results were shown in Fig. 2a.
Gram's staining showed this strain was purple and Gram-positive (Fig. 2b).
Molecular biological
identification: Strain 5 was analyzed via
16S rDNA sequencing, and the PCR products were detected by agarose gel
electrophoresis (Fig. 3). The PCR fragments were in size of 1500 bp. The
amplified gene sequences as-determined were compared with NCBI and a
phylogenetic tree (Fig. 4) was built on Mega 6.0. Strain 5 was identified to be
Bacillus velezensis.
Results of single-factor
optimization
Effects of carbon source
on enzyme yield of diastase-producing strain: The effects
of carbon source on enzyme yield of the diastase-producing strain were
illustrated in Fig. 5. The enzymatic activity of the strain maximized to 61.34
U/mL when the carbon source was soluble starch. Hence, the optimal carbon
source for strain 5 was soluble starch.
Effects of nitrogen
source on enzyme yield of strain 5: The effects of nitrogen source on
enzyme yield of the diastase-producing strain were plotted in Fig. 6. The
enzymatic activity of the strain maximized to 58.36 U/mL when the nitrogen source
was peptone. Thus, the optimal nitrogen source for strain 5 was peptone.
Effects of initial pH on
enzyme yield of strain 5: The effects of initial pH on enzyme yield of the
diastase-
Table 1: Results of strain screening
No. |
D/d-value |
Enzymatic activity (U/mL) |
5 |
3.33 |
29.78 |
8 |
3.50 |
13.31 |
9 |
3.78 |
15.23 |
13 |
3.56 |
13.75 |
19 |
3.03 |
12.95 |
23 |
3.06 |
12.43 |
2-13 |
3.27 |
12.87 |
Fig. 1: Transparent rings of
diastase-producing strains
Fig. 2: Colonial morphology and Gram's staining of strain 5
Fig. 3: PCR amplification and electrophoretic analysis
producing strain were illustrated in Fig. 7. The diastase
yield of strain 5 increased with the rise of pH within 3.0 - 4.0, but then
decreased with further rise at pH > 4.0. The diastase yield maximized to
73.01 U/mL at initial pH 4.0, indicating the optimal initial pH for strain 5
was 4.0.
Effects of inoculation
amount on enzyme yield of strain 5: The
effects of inoculation amount on enzyme yield of the diastase-producing strain
were illustrated in Fig. 8.
Fig. 4: Phylogenetic tree analysis
Fig. 5: Effects of carbon source on enzyme yield of strain 5
Fig. 6: Effects of nitrogen sources on enzyme yield of strain 5
Fig. 7: Effects of initial pH on enzyme yield of strain 5
The diastase yield of strain 5 increased with the
rise of inoculation quantity within 4% - 10%, but then decreased with further
rise of inoculation quantity at > 10%. The diastase yield maximized to 29.01
U/mL at the inoculation amount of 10%, indicating the optimal inoculation
amount for strain 5 was 10%.
Effects of fermentation
time on enzyme yield of strain 5: The effects of fermentation time on
enzyme yield of the diastase-producing strain were illustrated in Fig. 9. The
diastase yield of strain 5 increased with the prolonging of fermentation time
within 1 - 3 d, but then decreased with further rise of fermentation time at
> 3 d. The diastase yield maximized to 94.37 U/mL at fermentation time of 3
d, indicating the optimal fermentation time for strain 5 was 3 d.
RSM optimization of enzyme-producing conditions
Box-Behnken design: Based on single-factor tests, we conducted
Box-Behnken design on Design Expert 8.0.6. The enzyme-producing conditions of
strain 5 were analyzed via 3-factor
3-level assays. The RSM factors and levels were listed in Table 2 and the
results were shown in Table 3.
With enzymatic activity (Y) as the
response value, the test results were analyzed via Box-Behnken Design. Then the data in Table 3 were analyzed on
Design Expert 8.0.6. The regression equation of enzymatic activity with
fermentation time (A), initial pH (B) and inoculation amount (C) was:
Y=121.23-0.70A+2.49B+1.09C+1.29AB+1.07AC+1.38BC-26.53A2-20.37B2-8.36C2
Then the confidence and variance of
the equation were analyzed (Table 4). The results of F=617.26 and P < 0.0001 indicate it reached the
extreme significant level; the lack-of-fit as the variance was at P=0.2427>0.05, indicating lack-of-fit
was insignificant and no lack-of-fit factor existed, which reflect the actual
situations and the equation are suitable. The coefficient of determination was
R2 =0.9956, suggesting the test results of this equation well fitted
the results of model prediction and were reliable. Hence, the results are
reliable, and this equation can be used to analyze and predict the enzymatic
activity of this strain.
RSM and contour analysis
of enzymatic activity: The RSM
curves and contour curves showing the between-two interactive effects of
fermentation time, initial pH and inoculation amount on enzymatic activity were
plotted in Fig. 10–12.
Determination of optimal
fermentation conditions and verification
The effects of B, A2, B2 and C2
were all extremely significant (P <
0.01) and the effects ranked as B>C>A, or namely initial pH >
inoculation amount > fermentation time (Table 4). The optimal culture
conditions were found to be: fermentation time of 2.99 d, initial pH at 4.06,
and inoculation amount of 10.14%. The
enzymatic activity under the optimal conditions was predicted to be 121.35
U/mL.
Fig. 8: Effects of inoculation amount
on enzyme yield of strain 5
Fig. 9: Effects of fermentation time on
enzyme yield of strain 5
Fig. 10: The RSM curves and contour curves showing the interactive effects of
fermentation time and initial pH on enzymatic activity
To validate the effectiveness of RSM and considering the
convenience of actual operations, we modified these factors to be fermentation
time of 3 d, initial pH at 4 and inoculation amount of 10%. Then 3 parallel
tests were conducted under these conditions, and the actual enzymatic activity
was 120.87 ± 1.37 U/mL, which was basically close to the predicted
value.
Discussion
Table 2: RSM factors and levels
Level |
Factor |
||
A-Fermentation time/d |
B-Initial pH |
C-Inoculation amount/% |
|
-1 |
2 |
3 |
8 |
0 |
3 |
4 |
10 |
1 |
4 |
5 |
12 |
Table 3: Response surface test design and results
No. |
A-Fermentation time/d |
B-Initial pH |
C-Inoculation amount/% |
Enzymatic
activity U/mL |
1 |
4 |
3 |
10 |
71.6 |
2 |
3 |
4 |
10 |
120.45 |
3 |
4 |
5 |
10 |
78.23 |
4 |
3 |
4 |
10 |
119.41 |
5 |
3 |
3 |
8 |
89.9 |
6 |
4 |
4 |
12 |
86.58 |
7 |
2 |
4 |
8 |
88.24 |
8 |
2 |
3 |
10 |
73.01 |
9 |
3 |
5 |
8 |
93.05 |
10 |
3 |
4 |
10 |
122.5 |
11 |
2 |
5 |
10 |
74.48 |
12 |
4 |
4 |
8 |
82.11 |
13 |
3 |
4 |
10 |
123.14 |
14 |
2 |
4 |
12 |
88.41 |
15 |
3 |
5 |
12 |
97.85 |
16 |
3 |
4 |
10 |
120.65 |
17 |
3 |
3 |
12 |
89.2 |
Table 4: Analysis of variance of regression equation
Sources |
SS |
DF |
MS |
F-value |
P-value |
S |
|
Model |
5555.37 |
9 |
617.26 |
617.26 |
176.17 |
< 0.0001 |
Extremely significant |
A |
3.95 |
1 |
3.95 |
3.95 |
1.13 |
0.3237 |
|
B |
49.50 |
1 |
49.50 |
49.50 |
14.13 |
0.0071 |
Extremely significant |
C |
9.55 |
1 |
9.55 |
9.55 |
2.73 |
0.1428 |
|
AB |
6.66 |
1 |
6.66 |
6.66 |
1.90 |
0.2105 |
|
AC |
4.62 |
1 |
4.62 |
4.62 |
1.32 |
0.2885 |
|
BC |
7.56 |
1 |
7.56 |
7.56 |
2.16 |
0.1853 |
|
A2 |
2964.10 |
1 |
2964.10 |
2964.10 |
845.95 |
< 0.0001 |
Extremely significant |
B2 |
1746.67 |
1 |
1746.67 |
1746.67 |
498.50 |
< 0.0001 |
Extremely significant |
C2 |
294.45 |
1 |
294.45 |
294.45 |
84.03 |
< 0.0001 |
Extremely significant |
Residual |
24.53 |
7 |
3.50 |
3.50 |
|
|
|
Lack of fit |
15.01 |
3 |
5.00 |
5.00 |
2.10 |
0.2427 |
Not significant |
Pure error |
9.52 |
4 |
2.38 |
2.38 |
|
|
|
Total |
5579.90 |
16 |
|
|
|
|
|
Many efforts have been made to screen high-yield
diastase-producing strains from baijiu Daqu or oceans, but little research has focused
on the fermented mash of baijiu. The diastase-generating yeast screened out from the fermented mash of mild fragrant baijiu
had the enzymatic activity up to 118 U/mL (Yu et al. 2015). The α-diastase-producing bacterium isolated from oceans had the
enzymatic activity of 77.44 U/mL (Wang et
al. 2017b). The
diastase-producing bacterium isolated from lakes had the enzymatic activity of
147.53 U/mL (Henipigul et al. 2017). Thus, the
high-yield diastase-producing strain screened out in this study is of high
ability. If this strain can be further mutagenized or gene-recombined, its
enzymatic activity will be improved.
At present, a number of bacilli have
been applied into agriculture, industry and environmental protection and have
made great contribution to humans and the society (Guo et al. 2007; Pérez-García et
al. 2011; Panda et al. 2014; Sumi
et al. 2015). Bacilli also play very
important roles in the field of baijiu making (Luo et al. 2019). For instance, the bacilli identified in fermented
mash can produce multiple enzymes, including diastase, protease and cellulose (Gashaw
and Gessesse 1997; Wang et al. 2009). In particular, diastases
are a critical part and appropriate control of diastase-producing bacilli
during baijiu fermentation can improve the utilization rate and distillation
yield of starch raw materials. As reported,
bacilli can produce higher alcohols, higher ketones and other fragrant
compounds, which are at trace levels in baijiu and play the roles of fragrance
appending, assisting and seasoning in Luzhou-flavor liquors (Yang et al. 2018b). As reported, the fragrance of Luzhou-flavor
liquors mainly originates from the esterified fragrance-enhancing phase at late
fermentation, and bacilli bloom at this phase and become the predominant
bacterial colonies of fermented mash, thus playing an important role in
fragrant enhancement (Hu et al.
2014). Thus, in addition to the
enzyme-producing ability, bacilli can provide baijiu with intense fragrance. The bacillus screened out in our study
has broad application prospects in baijiu production.
Fig. 11: The RSM curves and contour curves showing the interactive effects of
fermentation time and inoculation amount on enzymatic activity.
Fig. 12: The RSM curves and contour curves showing the interactive effects of
inoculation amount and initial pH on enzymatic activity
Conclusion
The strains were screened preliminarily with the
transparent ring method and secondly with diastatic activity measurement.
Finally, 1 high-yield diastase-producing strain was screened out from Shedian
baijiu fermented mash and its initial enzymatic activity was 29.78 U/mL.
Morphologic observation and 16S rDNA molecular biological analysis identified
it as Bacillus velezensis. The enzyme-producing conditions were then
studied through single-factor tests and optimized by RSM to be: soluble starch
as carbon source, peptone as nitrogen source, initial pH at 4, inoculation
amount of 10%, and fermentation time of 3 d. Under these conditions, the
enzymatic activity was up to 120.87 ± 1.37 U/mL, which increased by 4.06 times
from the initial level, indicating this strain has potential industrial
application values.
Acknowledgments
This work supported by the Major Science and Technology
Projects of Henan Province of China (181100211400), Key Technologies Research
and Development Program of Henan Province of China (202102110130), Scientific
Research Foundation for Docotors of Henan University of Animal Husbandry and
Economy (2018HNUAHEDF011) and Key Subject Projects of Henan University of
Animal Husbandry and Economy.
Yanbo
Liu, Zhijun Zhao and Chunmei Pan planned the experiments, Wenjuan Zhang,
Wenning Gu and Xian Wang interpreted the results, Yanbo Liu and Chunmei Pan
made the write up and Xiyu Sun statistically analyzed the data and made
illustrations
References
Effront J, A Boidin (1916). Manufacture
of pressed yeast, Vol. 528, pp:3‒21. US Patent 1
Fang C, H Du, W Jia (2019). Compositional differences and similarities
between typical Chinese baijiu and western liquor as revealed by mass
spectrometry-based metabolomics. Metabolites 9; Article 2
Finley JW (2018). Beer and Wine, pp:483‒510. Principles of
Food Chemistry, Springer, Cham, Switzerland
Gashaw M, A Gessesse (1997). Thermostable amylase production by
immobilized thermophilic Bacillus
spp. Biotechnol Technol 11:447-450
Guo CS, TB
Chui, Y Guo (2007). Developments in alkaline cellulase from alkaliphilic bacillus.
Amino Acids Biotic Resour 2007; Article 35
Guo JH, HW
Guo, CY Jiang (2014). Gray relational grade analysis of enzyme activities and
esters production during fermentation of liqour fermented grains. Food Industr
35:236‒239
Henipigul M, M
Emerjan, B Ayan, M Dilmurat, T Dilbar (2017).
Isolation of an α= amylase producing Bacillus
and optimization of its fermentation conditions. Acta Sci Nat Univ Sunyat
56:126
Hu YH, N Cai, Y Dai (2014). Analysis of volatile compounds in stacking
fermented grains of Nong-xiang Liquor by GC-MS. Liquor Mak Sci Technol 2014:93‒96
Long T, ZS Xu, Q Wang, HX Zhang (2018). Comparative analysis of traditional
Baijiu brewing and industrial alcohol fermentation in China. Chin Brew
37:7‒11
Li L, TY Yang,
XY Dai (2009). Fast screening of amylase-producing bacteria from liquor daqu. J
Henan Inst Sci Technol 37:42‒44
Liu YQ, RN
Wu, JH Duan (2010). Optimization of the fermentation condition for
acid-resistant α-amylase producing strain. J Anhui Agric Sci 38:19888‒19890
Luo JC, H Xie (2012). Screening of Bacillus
spp. from Daqu and study of its metabolites. Liquor Mak Sci Technol
5:35‒40
Luo L, X Li, X Chang (2019). Research progress in the application of Bacillus and its mechanism in Baijiu
production. Liquor Mak Sci Technol 2019:99‒104
Mao X, D
Huang, CP Shen (2015). Isolation, identification and fermentation
characteristics of amylase-producing strain for isolation, identification and
fermentation characteristics of amylase-producing strain from Moutai-flavor
Daqu. Chin Brew 34:24‒27
Panda AK, BS
Singh, DM Surajit, KN Senthil, G Gurusubramanian, PA Kumar (2014). Brevibacillus as a biological tool: A
short review. Anton Leeuwen 105:623‒639
Pérez-García A, D Romero, A De Vicente (2011). Plant protection and growth
stimulation by microorganisms: Biotechnological applications of Bacilli in agriculture. Curr Opin
Biotechnol 22:187‒193
Perry GH, NJ Dominy, KG Claw (2007). Diet and the evolution of human
amylase gene copy number variation. Nat Genet 39:1256-1260
Salehi K, A Bahmani, B Shahmoradi (2017). Response surface methodology
(RSM) optimization approach for degradation of Direct Blue 71 dye using CuO-ZnO
nanocomposite. Intl J Environ Sci Technol 14:2067‒2076
Shi YC, YM
Jiang (1996). Comparison of five methods for the assaying of α-amylase
activity. Microbiol Chin 23:371‒373
Sumi CD, BW Yang, IC Yeo, YT Hahm (2015). Antimicrobial peptides of
the genus Bacillus: A new era for
antibiotics. Can J Microbiol 61:93‒103
Sun SJ, L Zhai, XB Bai (2019). Optimization of enzyme production
conditions and enzymatic properties of a high amylase-production Saccharomycopsis fibuligera. Food Ferment
Industr 45:31‒37
Wang C, D Shi, G Gong (2008) Microorganisms in Daqu: A starter culture of
Chinese Maotai-flavor liquor. World J Microbiol Biotechnol 24:2183‒2190
Wang FR, JC
Tang (1995). Studies on the method for determining high temperature
α-amylase’s activity. Food Ferment Industr 2:27‒30
Wang L, JR Han, JL Zhao (2009). Isolation of thermotolerant protease
producing Bacillus from Fen Daqu and
fermented grains. Chin Brew 01:67‒69
Wang XX, X
Geng, ZL Wu (2017a). Isolation and identification of a bacteria strain with
high yield of amylase from Congtai Daqu. Liquor Mak Sci Technol 1:30‒32
Wang MD, GZ Wang, XY Ye (2017b). Screening of marine microorganisms
produced α-amylase and it's enzymatic properties. J Chin Inst Food Sci
Technol 17:77‒84
Yang P, P Hu,
M Fan (2018a). Analysis of the volatile components from Jiuqu and fermented
grains in the pits of the sauce-flavor Baijiu. Chin Brew 37:166‒171
Yang B, J Zhou, HM Ming (2018b). Screening & identification of a Bacillus strain with high fermenting
power in Nongxiang Daqu. LiquorMak Sci Technol 05:17‒22
Yao C, GY Li,
GB Zhang (2019). Isolation and identification of thermophilic fungi from moderate/high-temperature
Daqu and determination of enzyme activity. Liquor Mak Sci Technol 46:32‒35
Yu CW, DS
Xue, W Guo (2015). Isolation of amylase-producing yeast from fermented grains
and study on optimization of fermentation conditions. Liquor Mak Sci Technol 42:71‒75
Zou W, G Ye, K Zhang (2018). Diversity, function, and application of
Clostridium in Chinese strong flavor baijiu ecosystem: a review. J Food Sci
83:1193‒1199