Effect of Irradiation Insecticidal Technology on Micro-ecology, Physical and Chemical Properties of Strong-flavor Baijiu Daqu

doi:10.21203/rs.3.rs-3734251/v1

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Effect of Irradiation Insecticidal Technology on Micro-ecology, Physical and Chemical Properties of Strong-flavor Baijiu Daqu

https://doi.org/10.21203/rs.3.rs-3734251/v1

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In this paper, the influence of high-energy electron beam irradiation technology on the microecology of strong-flavor Baijiu Daqu (SBD) was studied. The effects of different irradiation doses (0-3.55 kGy) on the microecology and physicochemical properties of SBD were determined. The interaction between bacteria, fungi, and physicochemical factors in SBD was examined. The potential of 0-1.40 kGy irradiation for pests eradication was analyzed. Under the irradiation dose range of 0.27–1.40 kGy, the micro-ecology of SBD could almost return to the level observed in non-irradiated samples within 3 months, ensuring a storage period of up to 120 d without infestation. Liquefying Power, Saccharifying Power, Acidity and Moisture all meet the industry standards of SBD and can be used for liquor making. The total bacterial count in SBD was approximately 1.4 x 10⁴ CFU/g, while the count of fungi and yeast was about 4.4 x 10⁴ CFU/g. This study provides an experimental reference for the application of electron beam irradiation technology in the insecticidal treatment of SBD.

High-energy electron beam irradiation

Strong-flavor Baijiu Daqu

Storage period

Microbial communities

Physiochemical properties

China Baijiu is one of the oldest alcoholic beverages in China (Jin et al., 2017). The production of Strong-flavor Baijiu accounts for over 70% of the total Baijiu production in China, with about 9.1 million tons is produced annually (Zou et al., 2018). Various fermented foods are commonly produced using starter cultures. In the production of Chinese traditional Baijiu, Daqu, a mixed culture starter, plays a crucial role in the saccharification and fermentation processes (Zheng et al., 2011). The traditional manufacturing technique of Strong-flavor Baijiu Daqu (SBD)involves around one month of fermentation and over three months of storage maturation. However, due to the uncontrolled nature of the solid-state fermentation process, the traditional manufacturing technique of SBD leads to inconsistent product quality and a significant number of failed goods.

pests have a direct impact on agricultural food production and storage, resulting in a 20%-30% loss of productivity (Balwinder et al.,2017). As grains are used as raw materials in the brewing of SBD, there is extensive insect infestation in the Baijiu sector during the storage period of SBD (Singh et al., 2018). Various chemical and non-chemical treatments can be applied to store grain to control pests (Moiranthem et al., 2021). However, these methods may cause insects to develop resistance, produce harmful residues, or not be suitable for temperature-sensitive foods, or the technology may be expensive and difficult to implement on a large scale. (Aktar et al., 2009. Birla et al., 2008. Li et al., 2015). In more than 60 countries, radiation has been permitted to use to minimize microbiological contamination and prevent insect infestation in wheat, whole wheat flour, and dehydrated seasonings (Li et al., 2015; Zhang et al.,2020). However, there are little research that used radiation technology to control insect pests in the process of Daqu-making.

X-rays and gamma rays are two common types of irradiation therapies. The technology of electron beam irradiation is based on particle ionizing radiation. It does not use radioactive materials that are harmful to the environment, and at the same time it has a higher dose rate.It can be turned off when it is not in use, and it does not involve radioactive isotopes.These inspire users' confidence (Ahn et al., 2013; Fan et al.,2017; Zhang et al.,2018; Hallman,2011).

In conclusion, the Daqu-making process of Baijiu is susceptible to pests infestations, thereby emphasizing the necessity of addressing this concern. Microorganisms, on the other hand, play a vital role in the Daqu-making process. Although irradiation is expected to be a solution to the pests problem, its impact on microbial ecology remains unclear. Therefore, this study aims to explore the appropriate dosage of SBD irradiation insecticidal technology and its effects on the micro-ecology, as well as physical and chemical properties of SBD. This will provide theoretical support for the application of irradiation technology in the Daqu-making industry.

2.1 Sample collection and preparation

Samples were taken from the Daqu-making factory of a brewery in Mianyang, Sichuan. In April 2022, a batch of medium and high-temperature SBD, which was put into storage on the same day and stored in different places was randomly selected for the experiment. The production process of the distillery's SBD involves pressing wheat with varying degrees of crushing, without adding any other starter-making materials. These samples were treated by high-energy electron beam at the doses of 0 kGy, 0.20 kGy, 1.00 kGy, 2.00 kGy, 2.5 kGy, and 3 kGy. The actual irradiation dose was determined by the measured value of the dosimeter. To prevent contamination and ensure that the detected microorganisms were the original ones affected by the irradiation treatment, samples was stored in a cool and ventilated food-grade translucent plastic box. Strict aseptic procedures were followed during the subsequent sampling. At intervals of 0, 25, 60, and 90 d after irradiation, three blocks of SBD were randomly sampled from each irradiated SBD block, then crushed and mixed evenly. Eight to ten grams samples were transferred to sterile 15 ml centrifuge tubes, with three samples collected from each sample. Finally, the samples were stored in a refrigerator at -80°C for further analysis.

In order to test the killing effect of high-energy electron beam on insect pests in SBD, the SBD blocks were irradiated at the doses of 0 kGy, 0.15 kGy, 0.25 kGy, 0.50 kGy, and 1.50 kGy, respectively, according to the results of physical and chemical properties and microecological tests in the early stage. The actual irradiation dose was determined through dosimeter measurement. Experiments were conducted during early summer. Another copy of the same batch of bricks was taken and not treated, serving as a blank control. Samples were stored separately in a ventilated and cool food-grade translucent plastic container, covered with gauze. It was also treated with pests control measures to prevent the escape of SBD pests. After a period of time, the SBD pests in different containers were counted to compare the killing effects of different irradiation doses.

The daily average temperature and humidity were recorded using a specialized laboratory thermometer. The temperature and humidity of each node environment were determined by taking 15 d as a node and finding the average temperature and humidity for each node.

2.2 Determination of D10 values of different microorganisms

After irradiation with various doses, the number of microbial colonies in SBD was determined by traditional culturable technique. SPSS software was used to calculate the D10 values of different microorganisms through linear fitting.

2.3 Determination and analysis of physical and chemical properties

The total titratable acidity is determined by potentiometric titration with pH indicating the end point (Mao et al., 2022). To determine the moisture content, a constant weight is reached at 105°C (Mao et al., 2022). In the analysis of SBD, the glucoamylase activity and liquefaction enzyme activity were determined using dinitrosalicylic acid (Mao et al., 2022).

2.4 Microbial culture and enumeration

The total bacteria culture medium used in this study is PCA (Plate Count Agar), autoclaved at 121°C for 15 min. The pH of PCA is adjusted to 7.0 ± 0.2. The medium for the fungus is Bengal red agar medium, which is sterilized at 121°C for 15 min. The natural pH of the Bengal red agar medium is maintained. To prepare the SBD sample, 10 g of SBD powder were homogenized in 90 mL of sterile 0.9% sodium chloride solution in a shaking incubator at 180 rpm for 30 minutes. The homogenized samples (1.0 mL) was then continuously diluted 10 times in sterile 0.9% sodium chloride solution. Subsequently, an aliquot of the diluted sample (0.1 mL) was coated on a plate. For the culture of bacteria, the plates were incubated at 38°C for 48 h. As for the fungi, they were cultured at 28°C for 48–72 h. Finally, SBD microorganisms were cultured on a regular basis.

2.5 Metagenomic DNA extraction, amplification and sequencing

Microbial DNA was extracted from SBD samples using the E.Z.N.A soil DNA kit (Omega Bio-tek, Norcross, GA, U.S.), following the manufacturer's plan. The DNA concentration and purification were determined using a Nano Drop 2000 UV-Vis spectrophotometer (Thermo Scientific, Wilmington, USA), and the DNA quality was checked through 1% agarose gel electrophoresis. The v3-v4 hypervariable region of the bacterial 16S rRNA gene was amplified using primers 338f (5-actcctacgggaggcagcag-3) and 806r (5-ggactachvgggtwtctaat-3). Additionally, the fungus ITS1 was amplified using primers ITS1F (5-cttgttcatttagagagtaa-3) and ITS2R (5ʹ-GCTGCGTTC), and TTcatcgatgc-3 was amplified using a thermal cycle PCR system (GeneAmp 9700, ABI, USA). Subsequently, the purified amplicons were combined and sequenced (2×300) on the Illumina MiSeq platform (Illumina, San Diego, USA) using the standard protocols. For data analysis, the PCR reaction program was set according to the manufacturer's plan and the "Atacama Soil Microbiology Tutorial" of Qiime2docs. The analysis involved identifying bacteria and fungi with different abundances in samples and groups using the linear discriminant analysis effect (LEfSe), as well as calculating the characteristic level α diversity index to estimate a single sample. To study the structural changes of microbial communities among samples, the β diversity distance measurement (weighted UniFrac) was conducted, and the results were visualized through principal coordinate analysis (PCoA). The sequencing scheme of this study is consistent with that of our team in the previous period (Zeng et al., 2022).

3.1 Effect of high-energy electron beam irradiation on daqu microorganisms during the storage period

Samples of SBD was irradiated with a high-energy electron beam in the warehouse. The actual irradiation doses were measured using dosimeters and found to be 0 kGy, 0.37 kGy, 1.40 kGy, 2.80 kGy, 3.10 kGy, and 3.55 kGy. The effect of these different irradiation dosages on SBD yeast, mold, and total bacteria during storage was investigated by using the dilution coating plate counting method. The results of the counting are displayed in Tables 1 and 2.

The influence of irradiation dose on the number of yeast, mold, and total bacteria in SBD samples after 0 d of storage can be observed in Tables 1 and 2. The data demonstrates that the number of microorganisms decreases as the irradiation dose increases. However, the effect of irradiation diminishes over time. After 40 d of storage, SBD samples irradiated with 1.40 kGy or less can restore the total number of microorganisms, mold and yeast to similar levels as samples without irradiation. The number of microorganisms exhibits a comparable order of magnitude.

The D10 values of each bacterium were calculated by linear fitting using the data in Table 1–2. The analysis revealed that the D10 value for fungi was 2.84 kGy, while the D10 value for total bacteria was 1.13 kGy. Figure 1 and Table 3 provide a summary of the relevant findings.

Through investigation in a Daqu-making factory, it was discovered that in recent years there has been an excessive presence of acid-producing bacteria in SBD. In order to examine the impact of irradiation on acid-producing bacteria, a traditional culturable counting method was employed. It was observed that an irradiation dose of 0.23 kGy was able to eliminate 20% of acid-producing bacteria in SBD samples. Additionally, it was noted that within a 90-day period following irradiation, there was a decline in the number of acid-producing bacteria as the irradiation dose increased at each tested point. However, as the storage time progressed, the impact of irradiation on the quantity of acid-producing bacteria gradually diminished. In the later stages of storage, the impact of environmental factors on the number of acid-producing bacteria in SBD samples exceeded that of irradiation. The experimental findings are detailed in Annex 1.

Table 1

Total number of yeast and mold (CFU/ g) during storage of Daqu with different irradiation doses.
	0 kGy	0.37 kGy	1.4 kGy	2.8 kGy	3.1 kGy	3.55 kGy
0 d	3.8×10⁴	3.00×10⁴	2\(.\)00×10⁴	1.60×10⁴	9.00×10³	2.00×10³
5 d	1.20×10⁴	1.80\(\)×10⁴	5.60×10³	1.40×10³	1.40×10³	6.8\(0\)×10²
10 d	2.40×10⁴	1.40×10⁴	1.30×10⁴	2.40×10³	3.00×10³	4.50×10²
15 d	1.6\(0\)×10⁴	7.4\(0\)×10³	9.3\(0\)×10³	5.80×10³	7.8\(0\)×10³	2.20\(\)×10³
25 d	2.05 ×10⁴	3.00×10⁴	1.57×10⁴	1.16×10³	2.02×10³	1.50\(\)×10²
40 d	1.46 ×10⁴	1.48×10⁴	1.07×10⁴	3.04×10⁴	7.00×10³	1.18×10³
60 d	8.8\(0\)×10³	2.61×10⁴	5.70×10³	2.90×10³	4.40×10³	2.30×10²
90 d	1.42×10⁴	3.34\(\)×10⁴	9.40×10³	3.60×10³	2.74×10³	3.66×10²

Table 2

Total number of bacteria (CFU/ g) during storage of Daqu with different irradiation doses.
	0 kGy	0.37 kGy	1.4 kGy	2.8 kGy	3.1 kGy	3.55 kGy
0 d	6.00×10⁸	5.20×10⁸	3.40×10⁶	2.40×10⁶	1.40×10⁶	7.40×10⁵
5 d	4.30\(\)×10⁸	1.6\(0\)×10⁸	1.30×10⁶	7.80×10⁶	3.90×10⁶	8.50×10⁵
10 d	2.37×10⁶	2.60×10⁶	6.00×10⁵	2.40×10⁵	4.50×10⁵	3.00×10⁴
15 d	2.10\(\)×10⁵	2.00×10⁵	3.30\(\)×10⁴	3.19\(\)×10⁴	5.00\(\)×10⁴	2.10\(\)×10⁴
25 d	4.30\(\)×10⁴	5.30×10⁴	2.30×10³	4.00\(\)×10⁴	2.00\(\)×10⁴	4.00×10³
40 d	2.59\(\)×10⁴	2.80×10⁴	2.15×10⁴	8.70×10³	8.90×10³	2.30×10³
60 d	2.70\(\)×10⁴	3.00\(\)×10⁴	2.66×10⁴	1.25\(\)×10⁴	1.22\(\)×10⁴	7.20×10²
90 d	4.69\(\)×10⁴	4.62\(\)×10⁴	3.34\(\)×10⁴	1.34 ×10³	9.24\(\)×10³	1.10×10³

Table 3

D10 values of total bacteria, yeast and mold
Types of microorganisms	Regression equation	D₁₀
Total bacteria	Y=-0.8666X + 8.85888, R² = 0.992	1.15 kGy
Mold and yeast	Y=-0.26836X + 4.63684, R² = 0.744	3.73 kGy

3.2 Microbial community composition

The top four bacterial genera, accounting for a total proportion of 91.01%-98.64% in Fig. 2, are Acetobacter (34.44% − 51.52%), Saccharopolyspora (33.64% − 48.44%), Streptomyces (5.35% − 11.08%), and Gluconobacter (2.72% − 8.08%). Among the fungal genera, Thermoascus has a ratio of 32.35–76.66%, Thermomyces has a ratio of 10.40–29.78%, and Aspergillus has a ratio of 5.57–14%. These three fungal genera have a total ratio of 93.31–99.00%.

As shown in Fig. 2(a), the microbial ecological structure of bacteria in SBD remains relatively stable compared to fungi within the period in 0–90 days after irradiation. The predominant bacteria in SBD are Acetobacter, Saccharopolyspora, and Streptomyces, which account for over 90% of the population. The proportion of Acetobacter exhibits a decreasing trend followed by an increasing trend after 25 and 60 d of storage, corresponding to different level of irradiation. Specifically, at radiation doses of 0 kGy and 2.80 kGy, the proportion of Acetobacter reached its peak and decreased significantly at radiation doses of 1.40 kGy and 3.55 kGy. After 90 d of storage, the proportion of Acetobacter initially increased and then decreased with the increase of irradiation dose. The highest number of Acetobacter was observed under the treatment of 2.80 kGy. The proportion of Saccharopolyspora exhibits an inverse relationship with Acetobacter, whereas the proportion of Streptomyces opposes the combined proportion of Saccharopolyspora and Acetobacter.

In the time range of 0–90 d, Fig. 2(b) illustrates an overall upward trend in the proportion of Thermoascus and a general downward trend in the proportion of other fungi in each sample. However, as the storage time extended, the differences among the samples gradually narrowed. On the 25th day, compared to the samples without irradiation, the proportion of Thermoascus decreased more, while the proportion of unclassified fungi increased more under the irradiation of 0.37 kGy to 2.80 kGy. At 60 d, the proportion of Thermoascus initially increased and then decreased with the increase in irradiation dose, peaking at 1.40 kGy. Conversely, the proportions of unclassified fungi, Thermomyces, and Aspergillus showed a trend of initially decreasing and then increasing to some extent. Finally, at 90 d, the proportion of Thermoascus initially decreased and then increased with the increase in radiation dose. In contrast, the proportions of unclassified fungi, Thermomyces, and Aspergillus initially increased and then decreased to some extent.

3.3. Microbial abundance and diversity

The effect of irradiation treatment on the microbial ecology of SBD increased at first and then gradually decreased over time, as depicted in Fig. 3. Furthermore, the difference between the irradiated samples decreased as the storage time extended. Specifically, upon observing the circled points in the figure, it becomes evident that the microbial ecology of SBD bacteria treated with irradiation doses of 0 kGy, 0.37 kGy, and 1.40 kGy after 90 d of storage is similar to that of the samples treated with 0 kGy for 90 d of storage. Similarly, the microbial ecology of SBD fungi treated with 0.37 kGy, 1.40 kGy, and 2.80 kGy irradiation after 60 d of storage is comparable to that of the samples treated with 0 kGy for the same duration. The microbial ecology of SBD fungi irradiated with 0 kGy- kGy, and 3.55 kGy after 90 d of storage was similar to that of the samples stored for 90 d after being treated with 0 kGy.

3.4 Effect of electron beam irradiation on physicochemical properties of daqu

Under the irradiation treatment of 0 kGy − 3.55 kGy, it can be observed from Fig. 4(a) that with the passage of time, both the irradiated and non-irradiated samples exhibited a similar fluctuation pattern in moisture content during each period. Acidity, saccharifying power, and liquefaction power, fluctuated in the initial 15 d after irradiation and gradually returned to their unirradiated levels thereafter. Figure 4(b) shows that by the 90th day, the acidity of all samples was higher compared to the 0 d. The irradiated samples exhibited relatively lower acidity than the untreated control group, with only minimal changes. Furthermore, according to Fig. 4(c) and 4(d), both saccharifying power and liquefaction power fluctuated during the early stages of sample storage. However, on the 15th day, these enzyme activities tended to stabilize, with little difference observed among the different samples.

3.5 Relationship between physicochemical properties and genus-level microorganisms

Figure 5(a) demonstrates a positive correlation between Acetobacter and Saccharopolyspora in relation to the liquefaction of SBD. Additionally, Streptomyces has a relatively strong positive correlation with saccharifying power. Moving on to Figure 5(b), Thermoascus exhibits a relatively strong positive correlation with the acidity of SBD. Similarly, Byssochlamys shows a relatively strong positive correlation with the moisture content.

3.6 Study on treatment of Daqu insect pests by electron beam irradiation technology

From Table 4 and Fig. 2, it is evident that when the radiation dose is 0 kGy, pests in Daqu can be observed roughly within 30 d, with the pests body becoming prominent after 60 d. At the radiation dose of 0.18 kGy, no pests are found within approximately 60 d of storage, and only a few pests are observed between 75 and 90 d.. However, for the radiation dose of 0.27 kGy or higher, no pests are found after 90 d of storage.

Table 4

environmental temperature and humidity records
Storage time(d)	0 ~ 15	16 ~ 30	31 ~ 45	46 ~ 60	61 ~ 75	76 ~ 90	91 ~ 105	106 ~ 120
Average Temperature(℃)	20.8	20.8	23.6	25.9	29.2	28.7	30.9	34.2
Average Humidity(%)	62.8	72.6	77.4	73.9	65.6	66.9	61.8	47.0

Table 5

Effects of different irradiation doses on the growth of Daqu pests during storage
Radiation dose(kGy)	0	0.18	0.27	0.50	1.4
15 d of infestation	-	-	-	-	-
30 d of infestation	+	-	-	-	-
45 d of infestation	++	-	-	-	-
60 d of infestation	+++	-	-	-	-
75 d of infestation	++++	+	-	-	-
90 d of infestation	++++	+	-	-	-
105 d of infestation	++++	+	-	-	-
120 d of infestation	++++	++	-	-	-
“-” No pests,

“+”Star to grow pests (less than 30 can be observed),

“++”A certain number of pests can be observed (30–100 pests can be observed),

“+++”The number of pests is high (100–300 pests can be observed),

“++++”Infestation of pests, the number of uncontrolled (can be observed more than 300 pests).

Under the condition of ensuring the number of microorganisms in SBD, it can be observed from Table 1 and Table 2 that the total bacteria, yeast, and fungi in SBD can be restored to the same level of magnitude as the samples without irradiation treatment when the irradiation dose of SBD is below 1.40 kGy after 90 d of storage. Therefore, when using high-energy electron beam for pests control, the irradiation dose should not exceed 1.40 kGy. It can be observed from Table 1 and Table 2 that the D10 values of fungi and yeast are significantly higher than the D10 values of the total colony count. This indicates that fungi and yeast are more resistant to radiation under the same conditions. This phenomenon might arise from the relatively dense cell membrane structure of fungi and yeast, some of which have similar flagella-like structures. After irradiation, these cells may be more easily repaired, resulting in their relatively higher D10 values. Similar results have been reported in a number of studies (Taghinejad et al., 2009).

Under the irradiation treatment of 2.80 kGy, a significant decrease in the total number of bacteria was observed, compared to the group treated with 1.40 kGy irradiation. However, the number of Acetobacter showed only a slight decrease relative to the total number of bacteria. This suggests that the irradiation treatment of 2.80 kGy had a greater impact on certain bacteria in SBD. This is evident from Fig. 2(a), where the microecological structure fluctuates. Additionally, Fig. 3(a) and (b) demonstrate a negative correlation between the change in bacterial diversity in SBD and the proportion of Acetobacter. This correlation is also supported by information provided in Table 2 and Annex 1's Tables 1. Furthermore, Fig. 2(b) indicates that the change in microecological structure can be attributed to the increase in temperature in the SBD storage environment during the summer. Thermoascus, which contains thermotolerant fungi (Hosoya et al., 2014), is more suitable for growth in summer conditions. This leads to an increase in the proportion of Thermoascus in SBD samples, giving it a population advantage and ultimately affecting the microecological structure of SBD. This change may also be responsible for the observed fluctuations in Fig. 3(c).

If SBD is to be irradiated, it is recommended that the irradiation dose should not exceed 1.40 kGy, as shown in Fig. 4. This is because the influence of irradiation treatment below 1.40 kGy on the micro-ecological structure is eliminated after 90 d. Additionally, saccharifying power and liquefaction power mainly represent α-amylase and β-amylase activity, as depicted in Fig. 5. Previous studies have indicated that a radiation treatment of 10 kGy has minimal impact on protein content (Mollakhalili-Meybodi et al., 2022), which may explain the observed unaffected saccharifying power and liquefaction power under the radiation treatment employed in this study.

The findings presented in Fig. 6(a) differ from previous studies that have shown a positive correlation between the proportion of Acetobacter and acidity. In light of this inconsistency, we propose three potential explanations for this phenomenon. Firstly, the data from standard plate counting indicates a significant decrease in the number of Acetobacter over time in storage, suggesting a corresponding decline in their impact on acidity. Secondly, it is possible that the increased acidity of SBD samples under high-dose irradiation is the result of the death of SBD microorganisms, the dissociation of inorganic phosphoric acid and deoxyribose, and the damage to the fluorine-carbon heterocyclic ring, leading to an increase in H + concentration (Acua-Argüelles et al., 1994). Furthermore, a previous study reported a positive correlation between thermophilic ascomycetes and total acid content (Hang et al., 2023). Although the total number of fungi measured by the traditional plate counting method remained relatively stable during the storage period, high-throughput sequencing revealed a noticeable increase in the proportion of Thermoascus, suggesting a potential influence on acidity. Taken together, these three points collectively explain the contradiction of the negative correlation between Acetobacter and acidity.

From Table 6, it can be observed that the pests in SBD can be effectively controlled within 120 d when the irradiation dose reaches 0.27 kGy or above. Therefore, it is generally believed that SBD should be stored for 3–4 months before it is put into production. Consequently, the irradiation pesticide dose of SBD should not be less than 0.27 kGy.

Different types of Daqu used in Baijiu-making have different dominant strains. This is because the composition of microorganisms in SBD is closely related to its maturity, storage time, Daqu-making season, Daqu-making area, and the surrounding environment (Gou et al., 2015). As a starter, SBD itself contains a variety of microorganisms. It is precisely because of the different microbial composition of different SBD that different Strong-flavor Baijiu brands with distinct characteristics are produced. The dominant strains found in this study are Streptomyces, Acetobacter, and Wickerhamomyces, which are also commonly found in other medium and high temperature Daqu (Singh et al., 2018; Zhang et al., 2014). Thermostable microorganisms detected in our research, such as Saccharopolyspora, Aspergillus, Thermoascus, and Thermophilus, form dominant populations in other high-temperature Daqu. These microorganisms are capable of producing enzymes such as α-amylase, glucoamylase, and protease, which play a vital role in fermentation and saccharification (Zhu et al., 2022). Therefore, the high saccharifying power and liquefaction power of Daqu used in our research can be attributed to these thermostable microorganisms. It's worth mentioning that the Daqu used in our research originated from Sichuan, China, where Thermophilic Ascomycetes are known to form a dominant population (Gou et al., 2015). This factor can explain the regional differences in Daqu flavor characteristics.

In addition, the presence of other dominant microbial populations in SBD that also influence its quality. Rhizopus in SBD is abundant in developed systematic amylase and protease, enabling the complete degradation of macromolecular components in wheat bran. This degradation provides nutrients for the growth and reproduction of strains, including cellulase, xylanase, protease, and lipase (Ezeilo et al., 2022; Negi et al., 2020). Gluconobacter, a unique non-model microorganism, acts as a biocatalyst in Daqu, facilitating the quantitative production of oxidation products in industrial-related reactions. These oxidation products are released into the culture medium and can be transformed into excellent candidates for biotransformation (Ripoll et al., 2023). Consequently, Gluconobacter has been widely utilized in various applications over the past 20 years due to its distinctive ability in the incomplete oxidation of sugars and alcohols. To reduce the abundance of Acetobacter in SBD, which loses ability to germinate in low oxygen conditions, starter-making plants often prolong the storage period of Daqu. However, recent investigations have revealed an excessive presence of Acetobacter in starter-making plants, highlighting a common issue in the production of SBD that aligns with the results of the high-throughput test conducted in this study.

In this paper, the micro-ecological detection, the analysis of physical and chemical properties and the observation of pests after irradiation were carried out, and it was finally concluded that the irradiation dose of high-energy electron beam should be 0.27 kGy-1.40 kGy to achieve better pests control effect on the premise that the micro-ecological and physical and chemical properties of SBD after irradiation and storage for three months were not affected much. This indicates that high energy electron beam irradiation technology is expected to be a good processing method to control insect pests in the process of making SBD.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence.

Acknowledgements

We would like to thank Sichuan Forgood Distillery Co., Ltd., Runxiang Irradiation Co., Ltd., Sichuan Institute of Atomic Energy Physics and Shenzhen Weikemeng Technology Group Co., Ltd. for their technical support.

Funding

This work was supported by[Open Project Program of Irradiation Preservation Technology Key Laboratory of Sichuan Province, Sichuan Institute of Atomic Energy] (Grant number FZBC2020010), and [Doctoral Foundation of Southwest University of Science and Technology] (Grant number 18ZX7157).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author contributions

All authors contributed to the study conception and design. Material preparation,investigation, experiment, formal analysis, validation, visualisation, data collection and analysis were performed by [YW], [JX], [YQ] and [XZ]. The first draft of the manuscript was written by [YW] and all authors commented on previous versions of the manuscript, [YZ] contributed equally to this work. methodology, supervision, writing (review and editing), project administration, funding acquisition, methodology, methodology, supervision, writing (review and editing), project administration, funding acquisition, methodology were performed by [DW], [XC], [YZ]. methodology, formal analysis, validation by [PG], [LD], [FX], [XX], [LQ]. All authors read and approved the final manuscript.

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Annex1EffectofirradiationonAcetobacter.docx

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Effect of Irradiation Insecticidal Technology on Micro-ecology, Physical and Chemical Properties of Strong-flavor Baijiu Daqu

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Abstract

Figures

1 Introduction

2 Materials and methods

2.1 Sample collection and preparation

2.2 Determination of D10 values of different microorganisms

2.3 Determination and analysis of physical and chemical properties

2.4 Microbial culture and enumeration

2.5 Metagenomic DNA extraction, amplification and sequencing

3 Results

3.1 Effect of high-energy electron beam irradiation on daqu microorganisms during the storage period

3.2 Microbial community composition

3.3. Microbial abundance and diversity

3.4 Effect of electron beam irradiation on physicochemical properties of daqu

3.5 Relationship between physicochemical properties and genus-level microorganisms

3.6 Study on treatment of Daqu insect pests by electron beam irradiation technology

4 Discussion

5 Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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