The liquor brewing process is the crystallization of the wisdom of the ancient working people in China and has a long history. As the saying goes, "koji is the bone of liquor", which fully illustrates the important role of koji in liquor. Daqu is made of single wheat or a mixture of wheat, barley and peas as raw materials, crushed, mixed with water, pressed into brick-like koji blocks, and cultivated under artificial control under a certain temperature and humidity. Before being used for liquor, Daqu needs to be stored for a period of time, which can purify the bacteria, which is conducive to controlling the slow fermentation process at a lower temperature, thereby improving the quality of the liquor.
Zhang Liang et al. found that the saccharification power and fermentation power of medium and high temperature Daqu changed little after 3 to 6 months of storage, and combined with the change in the number of microorganisms, it was concluded that the best effect of koji medicine after 4 months of storage. Liu Xue et al. concluded that the microbial indexes and physical and chemical indexes in medium-high temperature Daqu tended to be stable after 3 months of storage, and with the extension of the storage period, acid-producing bacteria such as bacteria gradually decreased, and beneficial microorganisms such as bacillus increased, and it was suggested that Daqu should be stored for 3 months at least before it could be put into production and use. Shi Si et al. found that during the storage of medium and high temperature Daqu for 5 months, the fungal community structure was continuously adjusted, and the fermentation capacity of Daqu increased slowly, while the saccharification capacity generally decreased. Some researchers have also studied the flavor components of Daqu during the storage period. For example, Xing Gang et al. used headspace-solid phase micro extraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS) to study the volatile flavor components during the storage of Daqu at medium and high temperatures, and found that the content of most flavor compounds decreased during the storage of Daqu for 5 20 days, and tended to be stable at 15 days of storage. At present, there are many studies on the physicochemical and biochemical indexes and microbial changes of high-temperature Daqu during storage, but there are few studies on the effect of storage time on the flavor substances of medium-high temperature Daqu, and the optimal storage time of Daqu has not yet been determined. Therefore, exploring the optimal storage time of medium and high temperature Daqu in order to guide the production of Daqu more scientifically has become an urgent problem for brewing enterprises.
In this paper, the physicochemical and biochemical index values, sensory scores, and the types and contents of flavor substances in the storage process of Daqu were analyzed, and the effect of storage time on the quality of Daqu was analyzed, which further provided a reference for determining the optimal storage time and production and use of Daqu.
1 Materials and methods
1.1 Materials and Reagents
Experimental sample: medium and high temperature finished Daqu, a liquor production enterprise in Sichuan.
Experimental reagents: formaldehyde, caproic acid, glucose, absolute ethanol, iodine, methylene blue, potassium sodium tartrate, all of which are analytically pure, Chengdu Kelong Chemical Reagent Factory.
1.2 Instruments and equipment
DHG-9013A Electric Drying Oven, Shanghai Shanzhi Instrument & Equipment***AR224CN Analytical Balance, Shanghai Tianping Instrument Co., Ltd.; PHS-3C acidity meter, Shanghai INESA Scientific Instrument Co., Ltd. ***LS-50HJ vertical pressure steam autoclave, Suzhou Aopu laboratory equipment ***SW-CJ-1FD ultra-clean workbench, Shanghai right instrument *** solid phase micro extraction head (2 cm 50 30 M DVB CAR PDMS), SUPELCO company, USA; Agligent 6890N-5975B Gas Chromatography-Mass Spectrometer, Ansett**, USA.
1.3 Experimental Methods
1.3.1 Sampling Method.
The medium and high temperature finished Daqu fermented for 30 days and stored for 60 days was taken as the sampling object. The outbound time of qualified finished koji (i.e., a total of 90 days from Anqu to the delivery time) is the starting point of the study, which is recorded as 0 days of storage, and follow-up sampling is carried out every 30 days, and a total of 5 groups of samples are taken d). Each time, 3 parallel samples were collected from the original load of the 3 new brewing platforms of the koji warehouse, and the sampling method was to select 1 large koji from the four corners and the center position of the curve layer in the center of the qu pile. Each piece of koji was crushed through a 20 mesh sieve, and each koji powder was mixed evenly and concentrated to 200 g by quartering method as 1 sample, a total of 15 samples, 4 Refrigerator for later use.
1.3.2. Determination of physical and chemical indicators.
References for the determination of Daqu moisture, acidity, wine-making power, esterification power, and amino acid nitrogen indexes.
1.3.3 Daqu sensory evaluation method.
The sensory evaluation of the outer surface, cross-section, skin, and aroma of Daqu was carried out by 10 koji-making technicians and reference [12], as shown in Table 1.
1.3.4 Determination of volatile flavor compounds in Daqu.
Take 13.1 Daqu sample passed through a 40 mesh sieve, mixed evenly, took 3 g of Daqu and put it into a 20 ml headspace bottle, and then added 30 l of 2-octanol internal standard solution (mass concentration of 6933 mg L), 60 equilibration for 20 min, headspace adsorption for 30 min, GC-MS separation and identification after 250 resolution for 5 min, and the heating program references.
Qualitative analysis method: The unknown compounds were compared with the standard spectral library at the same time as computer search, and 90% of the components were retained.
Quantitative analysis method: 2-octanol was used as the internal standard for semi-quantification, and the concentration of volatile flavor substances identified in Daqu samples was calculated.
1.3.5 Data Processing and Analysis.
Statistical software such as Excel 2007, Origin Pro 2018C, IBM SPSS Statistics 23 were used for data processing and analysis.
2 Results and Analysis
2.1 Analysis of the differences in physicochemical and biochemical index values and sensory scores of Daqu at different storage times
One-way ANOVA was performed on the physicochemical and biochemical indexes and sensory scores of Daqu at different storage times, and the results are shown in Table 2.
It can be seen from Table 2 that there were no significant differences in acidity, esterification power, amino acid nitrogen content and sensory score in different storage times, which may be due to the low moisture and temperature of koji billet during storage, which inhibited the growth and metabolism of microorganisms, and various enzyme activities were also passivated, so that there was no obvious effect on microbial acid production and ester production. There was a significant difference in moisture between the finished Daqu after 0 days of storage and 60 days of storage, which may be due to the evaporation of capillary water inside the koji blank, resulting in a significant decrease in water content. There was no significant difference in the moisture content of Daqu within 60 120 days of storage, which may be due to the equilibrium between the water content of the original carrier billet and the relative humidity of the ambient air at this stage, which affected the evaporation of koji billet water. Liquor is a comprehensive evaluation of Daqu's liquefaction power, saccharification power and microbial liquor production ability. There was a significant difference in the wine-producing power between the finished Daqu after 0 days of storage and 30 days of storage, and the number of alcohol-producing yeasts may be reduced due to the changes of moisture and temperature at this stage, and the wine-making power value was significantly reduced.
2.2 Analysis of volatile flavor compounds in Daqu at different storage times
A total of 48 Daqu flavor compounds were identified by GC-MS analysis of Daqu samples with different storage times, including 18 esters, 5 aldehydes and ketones, 5 alcohols, 8 aromatics, 6 alkanes, and 6 pyrazines, and the total ion chromatograms of volatile components are shown in Figure 1, and the Wayne diagrams of Daqu flavor substances at different storage times are shown in Figure 2.
According to the content of 48 kinds of Daqu flavor substances, the cluster heat map analysis was carried out, as shown in Figure 3. The heat map can visually show the differences and changes in the content of flavor substances in Daqu at different storage times, and the darker the color, the more the content of the substance.
It can be seen from Figures 2 and 3 that there were 31 common flavor compounds in Daqu at different storage times, including ethyl oleate, ethyl linoleate, 2,3,5,6-tetramethylpyrazine, etc., among which ethyl oleate and ethyl linoleate were higher in Daqu at each storage time, which may be related to the synthesis of fatty acids and alcohols generated by lipid degradation. 2-phenylcrotonaldehyde and 2,6-dimethylpyrazine were unique in Daqu after 0 days of storage, and 2,5-dimethylpyrazine was unique in Daqu after 90 days of storage. 2-Phenylcrotonaldehyde is a flavor substance containing unsaturated aldehyde group, showing floral and sweet aroma, and it has been reported that linoleic acid and linolenic acid in grain raw materials are first oxidized by lipoxygenase to produce hydroperoxide, and then further decomposed to form unsaturated aldehydes. 2,6-Dimethylpyrazine and 2,5-Dimethylpyrazine have baking and nutty aromas, and are considered to be important aroma substances in medium and high temperature Daqu, and the formation of these pyrazines is related to the Maillard reaction and the metabolism of microorganisms such as thermostable Bacillus.
As can be seen from Figure 3, the five kinds of Daqu samples can be well clustered into three categories: the storage d finished Daqu is clustered into one category, and the storage D finished Daqu is a separate category. Sixteen substances, including ethyl dodecanoate, ethyl pentadecanate, and 2,3,5,6-tetramethylpyrazine, were found to have higher contents in Daqu, which was stored for 0 days. The contents of 1-en-3-octanol and 2,5-dimethylpyrazine were higher in Daqu, the finished product after 90 days of storage. Eight substances, including ethyl oleate, ethyl linoleate and 1-pentadene, were found to have high contents in Daqu, the finished product after 120 days of storage. In general, with the extension of storage time, the color of the heat map became lighter and lighter, and the content of flavor substances decreased as a whole, among which the content of 39 flavor substances, such as amyl acetate, ethyl acetate and 2,3,5,6-tetramethylpyrazine, decreased after 30 days of storage, which may be related to the volatilization, oxidative decomposition and microbial transformation of the substances.
2.3 Analysis of the differences of the main flavor substances of Daqu at different storage times
From the Venn diagram and flavor clustering heat map, it can be seen that there are differences in the number and content of flavor compounds in Daqu at different storage times, and the composition structure of 48 flavor compounds in Daqu at different storage times is studied by combining the relative percentage diagram of flavor substance content in Daqu at each storage time, as shown in Figure 4. Make use of the software SPSS 220 Principal component analysis (PCA) was performed on 48 flavor compounds of Daqu with different storage times, and the characteristic values and contribution rates of principal components are shown in Table 3.
As can be seen from Table 3, the cumulative contribution rate of the components of the principal component eigenvalues "1" extracted from Daqu samples with different storage times is more than 85%, which can better represent the information reflected in the original data. The absolute value of the principal component load factor of the Daqu sample is 08 flavor substances, combined with the literature, it was determined that there were 8 important flavor substances (amyl acetate, ethyl nonanoate, ethyl acetate, ethyl octamate, phenethyl alcohol, ethyl caprate, 2,3,5-trimethylpyrazine, 2,3,5,6-tetramethylpyrazine), and the relative percentages of important flavor substances in Daqu at different storage times are shown in Figure 5.
It can be seen from Figure 4 that the total content of volatile flavor compounds in Daqu decreased with the extension of storage time, and the number of flavor compounds gradually decreased, with 47 types of flavor compounds in Daqu samples stored for 0 days and 37 types in Daqu samples stored for 120 days. The ratios of esters, alkanes and aldehydes and ketones to the total flavor compounds in the Daqu samples stored on the first three were the top three, and the sum of these three types of substances in the samples at these two time points reached more than 90%, indicating that the composition of flavor compounds in the two Daqu samples was similar. The ratios of esters, aldehydes and ketones to the total flavor compounds in the Daqu samples stored on the first three were the top three, and the sum of these three types of substances in the samples at these two time points reached more than 96%, indicating that the composition of flavor compounds in the two Daqu samples was similar. The ratios of esters, alkanes and aromatic compounds in the Daqu samples stored for 120 days to the original content of the total flavor compounds in the new brewing platform ranked the top three, respectively, indicating that the composition of flavor compounds in Daqu after 120 days of storage was not similar to that of Daqu samples stored for 120 days. The ester content was dominant in the Daqu samples with different storage times, and its content accounted for 72 percent of the total flavor substance content70%～85.04%。The ester content of Daqu was 6 when it was stored for 0 days68 g g, and the ester content was 3 after 30 days of storage62 g g, a decrease of 4579%。The content of alkanes increased to a certain extent during storage, which was consistent with related studies. The content of aldehydes and ketones was 0 when Daqu was stored for 30 days88 g g, the content of aldehydes and ketones was 0 after 120 days of storage13 g g, a decrease of 8524%。There was no significant difference in the proportions of aromatic and pyrazine compounds in Daqu samples with different storage times, which were 427%～6.33% and 080%～2.88%。
As can be seen from Figure 5, the ratios of ethyl nonanoate, ethyl octamate, and phenylethanol content to the content of total important flavor substances in Daqu samples stored for 0 days were the top three, respectively, and the sum of these three substances was 57about 89%; The ratios of phenylethanol, ethyl nonanoate and ethyl acetate to the content of total important flavor substances in Daqu samples stored on storage day were the top three, respectively, and the sum of these three substances accounted for more than 60%. The ratios of ethyl acetate, phenylethyl alcohol and amyl acetate to the content of total important flavor substances in Daqu samples stored for 60 days were the top three, and the sum of these three substances was 72about 43%; The ratios of ethyl acetate, phenethyl ethanol and ethyl nonanoate to the content of total important flavor substances in Daqu samples stored for 120 days were the top three, and the sum of these three substances was 7561% or so. The ratio of phenylethanol content to important flavor substances in Daqu at different storage times was 1410%～40.60%, accounting for the largest proportion; The ratio of 2,3,5-trimethylpyrazine content to important flavor substance content was 115%～2.24%, the smallest proportion. The contents of ethyl octamate, 2,3,5,6-tetramethylpyrazine, respectively, were stored in Daqu for 0 days. The contents of ethyl octadecate and 2,3,5,6-tetramethylpyrazine after 120 g of storage were 16 g g, respectively. 03 g g, respectively, decreased. 25%；The ethyl nonanoate content was 0 when Daqu was stored for 0 days27 g g, and the ethyl nonanoate content was 0 after 30 days of storage09 g g, a decrease of 6679%；The ethyl acetate content of Daqu was 0 when it was stored for 30 days08 g g, and the ethyl acetate content was 0 after 60 days of storage20 g g, an increase of 60%.
2.4 Analysis of important influencing factors of Daqu quality at different storage times
In order to further improve the important influencing factors of the quality of Daqu with different storage times, 31 common flavor components of 5 samples of Daqu with different storage times were selected, and their contents, physicochemical and biochemical index values and sensory scores of Daqu were PCA using Origin Pro 2018C software, and the results are shown in Figure 6.
Fig.6 Pcafig6 PCA of Daqu Samples with Different Storage Time Note: 1-2-ethylhexanol; 2-2,3-butanediol; 3-1-en-3-octanol; 4-acetaldehyde; 5-secoctanone; Tetraethyl 6-silicate; ethyl 7-heptanoate; Ethyl 8-caprylate; Ethyl 9-dodecanate; Ethyl 10-caprate; Ethyl 11-octamate; 12-ethyl oleate; Ethyl 13-nonanoate; Ethyl 14-n-hexanoate; Ethyl 15-tetradecanoate; Ethyl 16-acetate; Ethyl 17-linoleate; Amyl 18-acetate; Ethyl 19-hexadenoate; Ethyl 20-pentadecanate; Ethyl 21-9-hexadecenoate; 22-phenylethanol; 23-naphthalene; 24-styrene; 25-benzaldehyde; 26-2,6-Di-tert-butyl-4-methylphenol; 27-1-pentadene; 28-n-dodecane; 29-n-tetradecane; 30-2,3,5,6-tetramethylpyrazine; 31-2,3,5-trimethylpyrazine.
As shown in Figure 6, the first .
The contribution rates of the first and second principal components are as follows: 51%, which can better reflect the original information of Daqu samples with different storage times. The arrows represent different physicochemical and biochemical indexes, sensory scores and flavor compounds, and the direction represents the influence orientation of the index on the Daqu samples with different storage times, and the length represents the influence on the physical and chemical index values, sensory scores and flavor compound content of Daqu at different storage times, and the absolute value of the principal component loading coefficient is 08. Correlation analysis of more than physicochemical and biochemical indicators, sensory scores and flavor compounds. In Fig. 6, 10 flavor substances, including benzaldehyde (25), ethyl tetradecanoate (15), ethyl nonanoate, and 3,5,6-tetramethylpyrazine (30), were positively correlated with Daqu, and tetraethyl silicate (6) were negatively correlated. After 30 days and 60 days of storage, the finished products were distributed in the second quadrant, and the esterification capacity index and acetaldehyde (4) were positively correlated with it, and ethyl hexanoate (19) was negatively correlated with it. After 90 days of storage, Daqu was distributed in the third quadrant, and tetraethyl silicate (6) was positively correlated with it, and 10 flavor substances such as benzaldehyde (25), ethyl tetradecanoate (15) and ethyl nonanoate (13) were negatively correlated. After 120 days of storage, Daqu was distributed in the fourth quadrant, and six flavor substances, including amino acid nitrogen and ethyl caprate, 3,5-trimethylpyrazine (31) and styrene (24), were positively correlated, and esterification ability index and acetaldehyde (4) were negatively correlated.
In summary, from the analysis of physicochemical and biochemical indexes and sensory scores of Daqu at different storage times, compared with Daqu samples on 60 d and 0 d, there was no significant change in other indexes except for its moisture content, and the esterification capacity and sensory score increased respectively. 03%；However, the ester content of Daqu in the five Daqu samples stored for 60 days was higher in the total flavor compounds, which was 8392%, and the 60-day Daqu sample contained phenylethanol, 2,3,5,6-tetramethylpyrazine, 2,3,5-trimethylpyrazine and other important flavor compounds. Considering the economic benefits of warehouse turnover and capital backlog, it is suggested that the optimal storage time of finished Daqu is 60 days, that is, the actual storage time is 120 days from the end of Daqu fermentation.
3 Conclusion
The analysis of physicochemical and biochemical indexes and sensory scores of Daqu at different storage times showed that due to the influence of moisture and temperature, there was no significant change in the other indexes except for the significant changes in moisture content and liquorification power of Daqu at different storage times in Anqu in autumn.
There were 31 common flavor compounds in the 5 Daqu samples, and esters were the main esters in the finished Daqu samples at different storage times, accounting for 7270%～85.04%, and phenylethanol accounted for a relatively large proportion of the important flavor substances, and its content accounted for 1410%～40.60%。The contents of 39 flavor compounds, including amyl acetate, ethyl acetate and 2,3,5,6-tetramethylpyrazine, decreased after 30 days of storage, and the contents of ethyl octadecate and 2,3,5,6-tetramethylpyrazine decreased after 120 days of storage. 25%, which may be related to the volatilization, oxidative decomposition and microbial transformation of substances. According to PCA, the correlation between Daqu and its physicochemical and biochemical indexes, sensory scores and flavor substances was different at different storage times, among which ethyl nonanoate was mainly associated with 0 d storage and 90 d storage, and 120 d Daqu was mainly associated with ethyl caprate and amino acid nitrogen indexes.
Based on the comprehensive analysis of the physicochemical and biochemical indexes, sensory scores and flavor substances of Daqu after 60 days of storage, and considering the influence of storage time on economic benefits, it was suggested that the storage time of the finished Daqu was 60 days, that is, the actual storage time was 120 days from the end of Daqu fermentation. Due to the inconsistency of the quality of Daqu at different storage times, it is suggested that Daqu with different storage periods can be used in combination with each other, which is conducive to controlling the fermentation parameters of the cellar and improving the quality of liquor.
Growing flavor in 2024 This study preliminarily explored the effects of storage time on the physicochemical and biochemical indexes, sensory scores and flavor substances of Daqu, and suggested the optimal storage time of Daqu on this basis, but due to the limitation of the number of samples and the influence of various factors in the detection of flavor substances, it needs to be further explored and applied in actual production. In addition, the metabolic activity of microorganisms and the microenvironment of koji-making are also important factors influencing the quality of Daqu, combined with high-throughput sequencing and other technologies, the change law of microorganisms during the storage period of Daqu and the monitoring of environmental temperature, humidity and other indicators during storage will be the focus of future research.