Bacillus velezensis FSUXF-717 with the functions of promoting saccharification and fermentation and improving flavor of fermented wine and application thereof

Abstract

The application discloses a bacillus velezensis FSUXF-717 with the functions of promoting saccharification and fermentation and improving the flavor of fermented wine and an application thereof, and relates to the technical field of microorganisms. The bacillus velezensis FSUXF-717 provided by the application has the preservation number of GDMCC No: 64133, the fermentation liquor and the bacterial agent of the strain can promote the saccharification process of wine, meanwhile, the strain can significantly increase the total amino acid content (TAA) of the wine, p <0.05), increase the types and contents of flavor substances such as esters and organic acids, and promote the formation of the flavor of the wine body. The acquisition of the strain provides a strain resource for the development of wine and fermented food, has important practical significance for promoting high-efficiency and high-quality wine making, and provides a theoretical basis and technical support for the directional regulation of the quality of traditional fermented food.

Description

Technical Field

[0001] This invention relates to a strain of Bacillus berberis FSUXF-717 that promotes saccharification and fermentation and improves the flavor of fermented wine, and its applications, belonging to the field of food microbiology. Background Technology

[0002] The brewing and flavor of wine are closely related to the microbial community and its composition. Bacteria are the core microbial community in wine flavor formation, producing flavor compounds such as esters through metabolism, thus influencing the formation of wine flavor. Organic acids are important aroma and flavor compounds in wine. Studies have shown that organic acids enhance and buffer the aroma and taste of wine, reduce spiciness and bitterness, promote the aging of young wines, and prolong the aftertaste. Free amino acids are important flavor precursors and nutrients in fermented wines, participating in Maillard reactions, esterification reactions, etc., affecting the color, aroma, and nutritional value of the wine. Esters mainly provide wine with fruity, floral, sweet, and creamy aromas. The types and concentrations of esters in different wines give the products distinct flavor characteristics. Increasing the content of esters helps to enrich the aroma and flavor of wine, while also improving the mouthfeel and roundness. Ester compounds such as ethyl octanoate, ethyl nonanoate, and phenylethyl acetate possess flavor characteristics such as fruity, floral, and honey aromas. Increasing these substances helps to provide a fuller mouthfeel and richer flavor in wine, improving the quality of fermented wines. Long-chain fatty acid ethyl esters such as ethyl heptadecanate and ethyl octadecenoate help to reduce the spiciness of wine, positively impacting its taste. Heptanoic acid, octanoic acid, 1-octen-3-ol, 1-propanol, and phenylacetaldehyde are irritating substances in wine. Heptanoic acid and octanoic acid, for example, have earthy, sour, or oily odors, while enols such as 1-octen-3-ol typically have oily or raw, grassy odors. Reducing these substances helps to decrease the irritation of wine, improving its taste and quality.

[0003] Molds are the core microbial community in the saccharification stage. Molds such as Aspergillus and Rhizopus can perform saccharification by producing enzyme systems such as amylase and cellulase, thereby breaking down large molecules such as starch and cellulose in brewing raw materials into smaller sugar molecules that can be directly utilized by yeast, thus affecting fermentation efficiency and alcohol yield. Current technology indicates that Bacillus belliesi can be used in the brewing process; however, research on its ability to promote saccharification is limited. If a Bacillus belliesi strain can be screened that both promotes saccharification and fermentation and improves the flavor of fermented alcoholic beverages, it will have significant practical application value in the brewing industry. Summary of the Invention

[0004] This study applied Bacillus belyceta FSUXF-717 to the fermentation process of winemaking. Compared with the blank control group (CK group), the experimental group (fermentation enhanced with Bacillus belyceta FSUXF-717) showed the following improvements after 24 hours of saccharification: liquefying enzyme activity increased by 43.04% to 1721.15±19.66 U / g; saccharifying enzyme activity increased by 20.64% to 1492.49±14.87 U / g; protease activity increased by 79.44% to 1230.77±40.15 U / g; the saccharification coverage area on the mash surface increased; and the mash hardness decreased by 96.23%. After saccharification, water was added at a ratio of 1:1.5 for fermentation. At this point, the reducing sugar content was 6.29±0.003 g / L, which was 21 times that of the control group.

[0005] After adding Bacillus belyceta FSUXF-717 to enhance fermentation, the total free amino acid content in the mash increased by 67.50%, with umami amino acid content increasing by 65.66% and sweet amino acid content increasing by 77.70%. The total organic acid content in the experimental group mash peaked at 861.80 μg / mL on day 15, an increase of 17.80% compared to the control group at the same time. The contents of organic acids such as succinic acid and acetic acid in the experimental group were also increased compared to the control group. At the fermentation stages of days 5, 10, and 15, the total number of flavor compounds in the experimental group mash were 208, 205, and 211, respectively, with total contents of 13030.13, 14766.98, and 18688.29 mg / kg, respectively, representing increases of 11.81%, 3.28%, and 7.10% compared to the control group. The levels of esters that affect the flavor of the wine, such as octyl acetate, ethyl isohexanoate, ethyl arachidate, ethyl heptadecanate, and ethyl palmitate, were all increased. Furthermore, the yield of the experimental group was 81.07%, slightly higher than the 78.25% of the control group.

[0006] In vitro safety tests on Bacillus belyceta FSUXF-717 showed that the bacterium exhibited γ-hemolysis, meaning it did not cause hemolysis. Virulence gene PCR detection revealed that this strain did not contain cytotoxins, vomiting toxins, or enterotoxin T, and was inactive. Based on these results, this strain is considered safe.

[0007] This invention provides a strain of Bacillus vesiculosus that can promote saccharification and fermentation and improve the flavor of fermented wine. Bacillus velezensis FSUXF-717 and its application in wine fermentation: The Bacillus belye FSUXF-717 was deposited at the Guangdong Provincial Microbial Culture Collection Center on December 8, 2023, with accession number GDMCC No: 64133.

[0008] The *Bacillus belyssus* FSUXF-717 colonies of this invention are milky white, semi-transparent, irregularly round, with a raised center, smooth surface, and wrinkled edges; they are Gram-positive, rod-shaped, and arranged singly; they can form spores under adverse conditions; they can utilize glucose, mannose, sucrose, raffinose, and aescin; they are positive for glucose gas production and can hydrolyze starch.

[0009] This invention provides the application of Bacillus vesiculosus FSUXF-717 in improving the enzyme activity of fermented mash and / or improving the quality of wine, wherein the enzyme activity includes liquefying enzymes, saccharifying enzymes, and / or proteases; and the improvement of wine quality includes increasing the content of free amino acids, increasing the content of organic acids, and / or improving the flavor of wine.

[0010] In one embodiment, the improvement of wine flavor includes increasing the content of ethyl octanoate, phenylethyl acetate, ethyl isohexanoate, and / or ethyl heptadecanate in the wine, and / or decreasing the content of n-hexanol, 1-octen-3-ol, phenylacetaldehyde, and / or heptanoic acid.

[0011] In one embodiment, improving the flavor of the wine includes increasing the content of total esters in the wine.

[0012] In one embodiment, the organic acid includes, but is not limited to, succinic acid, oxalic acid, and acetic acid.

[0013] In one embodiment, the free amino acids include, but are not limited to, sweet amino acids.

[0014] In one embodiment, the wine includes, but is not limited to, rice wine.

[0015] In one embodiment, Bacillus vesiculosus FSUXF-717 is mixed with yeast and inoculated into the brewing raw materials for fermentation.

[0016] In one embodiment, the inoculum amount of Bacillus belyi FSUXF-717 in the brewing raw materials is (1×10⁻⁶). 6 )~(1×10 7 CFU / mL.

[0017] In one embodiment, after inoculation, the mixture is saccharified and fermented in a constant temperature incubator at 28~32℃ for 24h~48h, and then fermented with water for 15 days.

[0018] The present invention also provides a method for producing saccharifying enzymes, wherein Bacillus belyes FSUXF-717 is fermented in brewing raw materials or culture medium, wherein the preservation number of Bacillus belyes FSUXF-717 is GDMCC No: 64133.

[0019] The present invention also provides a method for reducing the hardness of brewing mash, wherein Bacillus vesicularis FSUXF-717 and yeast are co-inoculated into the brewing raw materials for fermentation, and the preservation number of Bacillus vesicularis FSUXF-717 is GDMCC No: 64133.

[0020] This invention also provides a method for producing ethyl isohexanoate, phenylethyl isovalerate, ethyl tridecanoate and / or ethyl cyclohexane, wherein Bacillus vesicularis FSUXF-717 is co-inoculated with yeast into brewing raw materials for fermentation, wherein the preservation number of Bacillus vesicularis FSUXF-717 is GDMCC No: 64133.

[0021] This invention also provides the application of Bacillus berberis FSUXF-717 in brewing.

[0022] Beneficial Effects: This invention discloses a strain of Bacillus vesicularis FSUXF-717 that can promote saccharification and fermentation and improve the flavor of fermented wine, and its applications, relating to the field of microbial technology. The Bacillus vesicularis FSUXF-717 provided by this invention has the preservation number GDMCC No. 64133. The fermentation broth and inoculant of this strain can promote the saccharification process of wine, and at the same time, the enhanced fermentation by this strain can significantly increase the total amino acid content (…). p The concentration of this strain (<0.05) increases the variety and content of flavor compounds such as esters and organic acids, thus promoting the flavor formation of the wine. The acquisition of this strain provides a microbial resource for the development of brewing and fermented foods, which is of significant practical importance for promoting efficient and high-quality brewing. It also provides a theoretical basis and technical support for the targeted regulation of the quality of traditional fermented foods.

[0023] Preservation of biological materials

[0024] A strain of Bacillus belye ( Bacillus velezensis FSUXF-717, classified as Bacillus velezensis It was deposited on December 8, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNo: 64133, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0025] Figure 1 This is a diagram showing the state of the mash after 24 hours of saccharification according to the present invention; the left side is the control group, and the right side is the experimental group.

[0026] Figure 2 This invention illustrates the change in hardness of fermented mash after enhanced fermentation with Bacillus vesiculus FSUXF-717 as a function of saccharification time.

[0027] Figure 3This diagram shows the changes in total sugar and reducing sugar in the mash after enhanced fermentation with Bacillus vesiculus FSUXF-717 according to the present invention. Day 0 represents the end of the saccharification stage and the beginning of the fermentation stage.

[0028] Figure 4 This paper describes the changes in the content of flavor amino acids in the fermented mash after enhanced fermentation with Bacillus vesiculus FSUXF-717 according to the present invention.

[0029] Figure 5 This invention illustrates the changes in organic acid content in fermented mash after enhanced fermentation with Bacillus vesiculus FSUXF-717.

[0030] Figure 6 This paper describes the changes in the number of flavor compounds in the mash after enhanced fermentation with Bacillus vesiculus FSUXF-717 according to the present invention.

[0031] Figure 7 This paper describes the changes in the content of flavor compounds in the mash after enhanced fermentation with Bacillus vesiculus FSUXF-717 according to the present invention.

[0032] Figure 8 This is a comparison of the saccharifying enzyme activity in fermented mash between Bacillus belyssus FSUXF-717 of this invention and other strains of the same genus.

[0033] Figure 9 This is a comparison of the liquefying enzyme activity in fermented mash between Bacillus FSUXF-717 of the present invention and other strains of the same genus. Detailed Implementation

[0034] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. These embodiments are further illustrations of the invention, but not limitations thereof.

[0035] The yeast used in the following examples was purchased from Yada Technology Industry Co., Ltd. in Yongzhou City, Hunan Province, with product number 4300779133967773830.

[0036] The following examples illustrate the definition and calculation methods of enzyme activity:

[0037] The enzyme activity of liquefying enzyme (α-amylase) is defined as the amount of enzyme required to catalyze the decomposition of starch into 1 μmol of reducing sugar (calculated as glucose) within 1 minute at a specific temperature (60℃) and pH (6.0), which is defined as 1 unit of enzyme activity (U).

[0038] The enzyme activity of glucoamylase is defined as the amount of enzyme required to catalyze the decomposition of soluble starch into 1 mg of glucose within 1 hour at a specific temperature (60℃) and pH (4.5), which is defined as 1 unit of enzyme activity (U).

[0039] The definition of protease activity is the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine within 1 minute at a specific temperature (40℃) and pH (7.5), which is defined as 1 unit of enzyme activity (U).

[0040] Enzyme activity assay calculation methods: DNS method, Folin-phenol method.

[0041] Example 1: Screening of Bacillus belye FSUXF-717

[0042] Using Guangdong glutinous rice wine mash as a sample, after pretreatment, 0.5 mL of the sample was added to 4.5 mL of 0.9% physiological saline for serial dilution. Appropriate serial dilutions were then plated onto LB solid medium and incubated at 37℃ for 48 h. Typical colonies were picked and streaked onto LB plates for purification. Single colonies were then transferred to LB liquid medium and incubated for 24 h. After centrifugation, the resulting bacterial cells were preserved with 50% glycerol, which is *Bacillus belye* (B. belye). Bacillus velezensis FSUXF-717, the Bacillus belyes FSUXF-717 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNo: 64133.

[0043] Example 2: Determination of enzyme activity in saccharification-promoting fermented mash after enhanced fermentation with Bacillus belye FSUXF-717

[0044] Brewing process using Bacillus belyeis FSUXF-717 for enhanced fermentation: Weigh 1000 g of glutinous rice, add 700 g of water, stir well, and let it soak for 12-15 hours. Steam the soaked glutinous rice in a rice steamer for 30 minutes, then remove and cool to 35℃. Add 6 g of yeast starter and 150 mL of second-generation Bacillus belyeis FSUXF-717 bacterial culture (bacterial concentration of 1.37 × 10⁻⁶). 7 The control group (CFU / mL) was designated as the FSUXF-717 group (i.e., the experimental group). The brewing process was the same as described above, except that Bacillus belye FSUXF-717 was not added. The raw materials for both the experimental and control groups were mixed thoroughly, placed in fermentation tanks, sealed with gauze, and placed in a 30℃ incubator for saccharification and fermentation for 24 h. Samples were taken at 0, 6, 12, 18, and 24 h and stored at -20℃ for later use. After saccharification, water was added at 1.5 times the weight of rice for fermentation. The experiment was completed after 15 days of fermentation, followed by testing of physicochemical indicators and volatile / non-volatile substances.

[0045] (1) Determination of liquefying enzyme activity in mash: Weigh 10 g of mash and place it in a 250 mL beaker. Add 100 mL of phosphate buffer solution with pH=6.0 and incubate in a water bath at 40℃ for 1 h (stirring once every 10 min). Filter the solution through qualitative filter paper to obtain the liquefying enzyme solution. The liquefying enzyme activity in mash samples after saccharification for 0, 12, and 24 h was determined using the DNS colorimetric method. The determination method and steps are shown in Table 1.

[0046] Table 1. Procedure for Enzyme Activity Assay

[0047]

[0048] Results: As shown in Table 2, when the experimental group (the brewing group enhanced with Bacillus vesiculosus FSUXF-717) was saccharified and fermented for 24 h, the liquefaction enzyme activity of the mash was 1721.15±19.66 U / g, which was 43.04% higher than that of the control group at the same time.

[0049] Table 2. Enzyme activity of liquefying enzymes in fermented mash

[0050]

[0051] (2) Determination of saccharifying enzyme activity in fermented mash: Weigh 10 g of fermented mash and place it in a 250 mL beaker. Add 100 mL of acetate-sodium acetate buffer solution with pH=4.6 and incubate in a water bath at 30℃ for 1 h (stirring once every 10 min). Filter through qualitative filter paper, and the resulting filtrate is the saccharifying enzyme solution. Determine the saccharifying enzyme activity using the DNS colorimetric method.

[0052] Results: As shown in Table 3, when the saccharification fermentation of the experimental group reached 24 h, the activity of saccharifying enzyme was 1492.49±14.87 U / g, which was 20.64% higher than that of the control group at the same time.

[0053] Table 3. Saccharifying enzyme activity in fermented mash

[0054]

[0055] (3) Determination of protease activity in fermented mash:

[0056] Pretreatment of fermented mash samples: Weigh 5 g of fermented mash sample, add 20 mL of pre-cooled phosphate buffer solution (pH=7.5), grind in an ice bath, and centrifuge the ground sample at 4℃ and 5000 rpm for 15 min to obtain protease solution.

[0057] Transfer 1 mL of protease solution to a test tube and incubate at 40°C for 5 min. Add 2 mL of 2% casein solution, mix well, and incubate at 40°C for 15 min. Add 3 mL of 10% trichloroacetic acid, let stand at room temperature for 20 min, and then centrifuge at 4500 rpm for 20 min at 4°C. Take 1 mL of the supernatant, add 5 mL of 0.55 mol / L sodium carbonate solution and 0.5 mL of Folin-Ciocalteu reagent, and incubate at 40°C for 20 min for color development. Measure the absorbance at 680 nm and calculate the protease activity.

[0058] Results: As shown in Table 4, when the experimental group was saccharified and fermented for 12 h, the activity of protease in the mash was 824.79±53.42 U / g, which was 56.91% higher than that of the control group at the same time. When the experimental group was saccharified and fermented for 24 h, the activity of protease in the mash was 1230.77±40.15 U / g, which was 79.44% higher than that of the control group at the same time.

[0059] Table 4. Enzyme activity of protease in fermented mash

[0060]

[0061] In summary, the analysis suggests that the addition of Bacillus belye FSUXF-717 to enhance fermentation significantly increased the enzyme activities of liquefying enzymes, saccharifying enzymes, and proteases in the mash. p <0.05). Liquefying enzymes and saccharifying enzymes break down starch in grain raw materials into small-molecule reducing sugars, while proteases break down glutenin, releasing large-molecule sugars such as encapsulated starch granules, which helps in the complete decomposition and conversion of sugars. Simultaneously, amino acids are important precursors for the formation of complex flavor compounds (such as esters) in fermented wines; proteases break down proteins into small-molecule amino acids, which is beneficial for the formation of flavor compounds. Therefore, *Bacillus belyssioides* FSUXF-717 can effectively promote saccharification, fermentation, and the formation of amino acids and flavor compounds.

[0062] Example 3: Determination of changes in the hardness of fermented mash

[0063] The hardness changes of the mash at different time points during the saccharification stage were measured using a texture analyzer, and the results are shown in Table 5.

[0064] Table 5. Changes in mash hardness during the saccharification stage

[0065]

[0066] Saccharification breaks down sugars in the raw materials, reducing the hardness of the mash. At 24 hours of saccharification, compared to the control group, the mash hardness decreased by 96.23% after adding *Bacillus belyssiensis* FSUXF-717 for enhanced fermentation. Similarly, in the group with *Bacillus belyssiensis* FSUXF-717, the mash hardness decreased by 96.13% at 24 hours of saccharification compared to 0 hours at the start of saccharification. In conclusion, the results suggest that adding *Bacillus belyssiensis* FSUXF-717 for enhanced fermentation effectively promotes saccharification and fermentation, improves saccharification efficiency, and shortens the saccharification and fermentation time.

[0067] Example 4: Determination of changes in reducing sugar and total sugar content in fermented mash

[0068] (1) Determination of reducing sugar content in fermented mash:

[0069] To construct a standard curve: Take seven 25 mL test tubes and add 0, 0.2, 0.6, 1.0, 1.4, 1.8, and 2.0 mL of glucose standard solution (1 g / L) respectively. Make up the volume to 2 mL with distilled water, add 2 mL of DNS reagent, and boil for 5 min. After cooling, bring the volume to 25 mL with distilled water. Analyze the standard curve using an OD curve. 540 nm Measure the absorbance value and plot the standard curve.

[0070] Determination: Take 1 g of fermented mash, add 8 mL of distilled water, mix well, and centrifuge at 8℃ and 5000 rpm for 10 min. Take 2 mL of the supernatant, add 2 mL of DNS reagent, and boil in water for 5 min. After cooling, dilute to 25 mL with distilled water. (OD value is missing from original text.) 540 nm The absorbance value was measured and substituted into the standard curve to calculate the reducing sugar content in the mash.

[0071] (2) Determination of total sugar content in mash: The total sugar content was determined directly using a handheld saccharimeter.

[0072] Result: As Figure 3 As shown, the total sugar content of the two groups of mash showed a basically consistent trend, with the reducing sugar content in the experimental group showing a slight increase followed by a decrease as fermentation progressed. At day 0 of fermentation (i.e., the end of the saccharification stage), the reducing sugar content in the experimental group was significantly higher than that in the control group. p The concentration of reducing sugars in the experimental group was <0.05%, indicating that Bacillus belyssioides FSUXF-717 effectively promoted the production of reducing sugars in the fermentation system during the saccharification stage. Throughout the fermentation process, the overall trend of reducing sugar content in the experimental group was higher than that in the control group, indicating that the conversion rate of reducing sugars was higher after adding Bacillus belyssioides FSUXF-717 to enhance fermentation.

[0073] Example 5: Determination of Free Amino Acid Content in Distilled Grains

[0074] Weigh 1 g of fermented mash sample, add 5 mL of water, sonicate at 25℃ for 10 min, and centrifuge at 10000 rpm for 10 min. Take 5 mL of the supernatant, add 5 mL of 10% sulfosalicylic acid, mix well, refrigerate at 4℃ overnight, and then centrifuge at 10000 rpm for 15 min. After centrifugation, filter the supernatant through a 0.22 μm aqueous filter membrane and determine the amino acid content using a fully automated amino acid analyzer.

[0075] Results: As shown in Table 6 and Figure 4 As shown, the free amino acid content in both the experimental and control groups increased during fermentation. On day 15 of fermentation, the total free amino acid content in the experimental group was 176.90 mg / mL, an increase of 67.50% compared to the control group; the total umami amino acid content in the experimental group was 21.68 mg / mL, an increase of 65.62% compared to the control group; and the total sweet amino acid content in the experimental group was 54.50 ug / mL, an increase of 77.70% compared to the control group. Throughout the fermentation process, the total bitter amino acid content in both groups of mash also showed an increasing trend. However, research indicates that the presence of bitter amino acids such as lysine and phenylalanine does not equate to a strong bitter taste; they can enrich the flavor of the wine to some extent. In conclusion, the addition of Bacillus belye FSUXF-717 to enhance fermentation can increase the free amino acid content in the wine.

[0076] Table 6. Changes in free amino acid content of mash samples at different fermentation stages

[0077]

[0078] Note: Different lowercase letters indicate significant differences between different samples of the same amino acid. p <0.05).

[0079] Example 6: Determination of Organic Acid Content in Distilled Grains

[0080] This experiment used liquid chromatography (HPLC) to determine the content of eight common organic acids in fermented mash. 1 mL of sample was added to 2 mL of 0.1% phosphoric acid aqueous solution, and the mixture was ultrasonically extracted for 40 min, followed by centrifugation at 5000 rpm for 5 min. The supernatant was filtered through a 0.45 μm aqueous membrane before being analyzed. The elution program was: 100% 25 mM / L potassium dihydrogen phosphate (pH=2.5), run time 30 min, column temperature 35℃, flow rate 1.0 mL / min, detection wavelength 210 nm, and injection volume 10 μL.

[0081] The results are shown in Table 7. Throughout the fermentation process, the total organic acid content in the experimental group showed an increasing trend, reaching a peak of 861.80 ug / mL on day 15 of fermentation, which was 17.80% higher than that in the control group at the same time. On day 15 of fermentation, the total contents of succinic acid, citric acid, acetic acid, oxalic acid, malic acid, and pyruvic acid in the experimental group mash were all higher than those in the control group. Succinic acid is an important organic acid for improving the freshness of the wine, and acetic acid is one of the more abundant volatile organic acids in rice wine, giving the wine a "refreshing" sour taste. Appropriate organic acids can form rich layers and have an important influence on the flavor and taste of the wine. The analysis shows that the addition of Bacillus belye FSUXF-717 to enhance fermentation can increase the content of organic acids in the wine to a certain extent, thus enriching the flavor of the wine.

[0082] Table 7. Changes in organic acid content of mash samples at different fermentation stages

[0083]

[0084] Example 7: Determination of volatile flavor compounds in fermented mash

[0085] Take 200 mg of fermented mash sample and place it in a 20 mL headspace vial. Add 10 μL of 10 mg / mL 2-octanol internal standard solution. Take another headspace vial and add 10 μL of 10 mg / L n-alkane mixed standard. Detect the volatile components in the fermented mash using HS-SPME-GC-MS.

[0086] Gas chromatography conditions: DB-Wax column (30 m × 250 μm × 0.25 μm), helium as carrier gas, extraction at 60℃ for 30 min, resolution time of 4 min, flow rate of 1 mL / min, purge flow rate of 3 mL / min, splitless.

[0087] Column oven heating program: Initial temperature 40℃, hold for 4 min, increase to 245℃ at 5℃ / min, hold for 5 min.

[0088] Mass spectrometry conditions: EI ion source (-70 eV), temperature (sample inlet / transfer line 250℃, ion source 230℃, quadrupole 150℃), Scan full scan acquisition information, m / z: 20-400, solvent delay 2.37 min.

[0089] The internal standard method is used to calculate the relative content of each component. The calculation formula is shown below:

[0090] (1);

[0091] In the formula:

[0092] C represents the relative content of a single component, in mg / kg;

[0093] A1 represents the peak area of ​​a single component;

[0094] A2 represents the area of ​​the internal standard peak;

[0095] C2 is the internal standard concentration, mg / mL;

[0096] V2 is the internal standard volume, in μL;

[0097] M represents the sample mass, in grams.

[0098] Result: As Figure 6 , 7 As shown, on days 5, 10, and 15 of fermentation, the total number of flavor compounds in the experimental group mash were 208, 205, and 211, respectively, with total contents of 13030.13, 14766.98, and 18688.29 mg / kg, representing increases of 11.81%, 3.28%, and 7.10% compared to the control group (CK group). Specifically, on day 15 of fermentation, the total ester content in the experimental group was 6774.70 mg / kg, an increase of 11.65% compared to the control group at the same time point (Table 8). Based on the experimental results, it is evident that the enhanced fermentation using *Bacillus belye* FSUXF-717 significantly improved the variety, quantity, and content of flavor compounds such as esters, thereby enhancing the flavor quality of the fermented wine.

[0099] The main esters in the fermented mash, such as ethyl octanoate and ethyl decanoate, have fruity and sweet aromas, enhancing the richness of the liquor; ethyl heptanoate and ethyl nonanoate have honey and fruity aromas, providing a mellow taste to the fermented liquor; ethyl benzoate and phenylethyl acetate have rose and floral aromas; and ethyl palmitate has fruity and creamy aromas. The experimental group showed a significant increase in the content of these flavor compounds compared to the control group. p (<0.05), these substances have low sensory thresholds, meaning even small changes in concentration can affect the flavor of the wine. Flavor compounds such as ethyl isohexanoate, ethyl arachidate, and ethyl heptadecanate, which were absent in the control group, were additionally produced in the experimental group after enhanced fermentation with the addition of Bacillus beleibrio FSUXF-717. These flavor compounds contribute fruity and rose aromas to the wine, and long-chain fatty acid ethyl esters such as ethyl heptadecanate and ethyl octadecanoate help to reduce the spiciness of the wine, positively impacting its mouthfeel and further enhancing the richness of the fermented wine's quality.

[0100] Table 8. Changes in the content of ester flavor compounds in mash samples at different fermentation stages.

[0101]

[0102] Note: nd indicates not detected.

[0103] As shown in Table 9, using VIP≥1, p Using a threshold of <0.05 as a screening criterion, flavor compounds exhibiting significant differences were selected. Analysis showed that, compared to the control group, the levels of hexanol, 1-octen-3-ol, 1-propanol, phenylacetaldehyde, valeric acid, heptanoic acid, and octanoic acid gradually decreased during fermentation in the experimental group supplemented with *Bacillus bereaves* FSUXF-717. Substances such as 3-methyl-2-buten-1-ol were even undetectable at the end. In sensory threshold evaluation, hexanol had a bitter taste; enols such as 1-octen-3-ol and 3-methyl-2-buten-1-ol typically had an oily or raw, grassy taste; valeric acid, heptanoic acid, and octanoic acid had a muddy, sour, or oily taste. These substances are common irritating flavor compounds in fermented wines, and reducing their content helps improve the taste and quality of the wine. The results indicate that the enhanced fermentation with *Bacillus bereaves* FSUXF-717 can, to some extent, reduce the production of irritating substances during wine fermentation, decrease the spiciness of the wine, and maintain a mellow and smooth body.

[0104] Table 9. Changes in the content of some flavor compounds in mash samples at different fermentation stages.

[0105]

[0106] Note: nd indicates not detected.

[0107] Example 8: Calculation of alcohol yield from enhanced fermentation

[0108] The yield of the finished wine was calculated. The wine was collected using a fractional distillation method, and the alcohol content was measured using an alcohol meter.

[0109] The actual output is converted to the standard alcohol content (50 degrees) and then calculated using the following formula:

[0110] Standard alcohol yield = Actual alcohol production × Actual alcohol content ÷ Standard alcohol content;

[0111] Alcohol yield = (Standard alcohol content yield ÷ Raw material usage) × 100%.

[0112] result:

[0113] Table 10 Calculation Method of Alcohol Yield

[0114]

[0115] Experimental group: (87.00×74.50+855.00×58.50+1556.80×28.80)÷50÷2500.00×100%=81.07%.

[0116] Control group: (176.60×71.40+709.90×58.50+1621.70×26.80)÷50÷2500.00×100%=78.25%.

[0117] The above results indicate that adding Bacillus vesiculosus FSUXF-717 to enhance fermentation increases the production of high-proof spirits and slightly improves the overall alcohol yield.

[0118] Example 9: Production of saccharifying enzymes by fermentation of Bacillus belye FSUXF-717 alone

[0119] Liquid fermentation medium (g / L) for saccharifying enzyme-producing strains: peptone 20, soluble starch 20, disodium hydrogen phosphate 5, magnesium sulfate 0.1, sodium chloride 0.1, pH=7.0; autoclaved at 121℃ for 20 min.

[0120] Single colonies of activated Bacillus belye FSUXF-717 were picked and inoculated into the liquid fermentation medium of the saccharifying enzyme-producing strain, and cultured at 37℃ and 160 r / min for 24 h with shaking. Seed culture was inoculated into the liquid fermentation medium of the saccharifying enzyme-producing strain at an inoculation rate of 3% (V / V), and cultured at 37℃ and 160 r / min with shaking for 48 h. The fermentation broth was centrifuged at 5000 rpm for 10 min, and the saccharifying enzyme activity of the fermentation supernatant was determined using the DNS method.

[0121] Results: The standard curve equation for glucose is y = 1.18022*x + 0.0625 (R²). 2 =0.9991>0.995).

[0122] Substituting the measurement results into the standard curve, the glucose concentration was calculated to be 0.85746 mg / mL.

[0123] Formula for calculating glucoamylase activity:

[0124] .

[0125] (X is the activity of saccharifying enzyme (U / g); c is the glucose concentration (mg / mL); n is the enzyme dilution factor; m is the sample weight (g))

[0126] Substituting into the calculation formula, the activity of the saccharifying enzyme is 2057.41 U / g.

[0127] The saccharifying enzyme activity of the Bacillus belysin FSUXF-717 fermentation broth discovered in this invention is 2057.41 U / g.

[0128] Comparative Example 1

[0129] Following the method for determining enzyme activity in Example 2, the liquefying enzymes and saccharifying enzymes of other Bacillus species of the same genus were measured in fermented mash.

[0130] The results are as follows Figure 8 and Figure 9 As shown, the liquefying enzyme activity and saccharifying enzyme activity of the mash after enhanced fermentation with Bacillus belye FSUXF-717 were significantly higher than those of the mash after enhanced fermentation with Bacillus belye B1~B6. p The value <0.05 indicates that the Bacillus FSUXF-717 discovered in this invention has a stronger ability to promote saccharification and fermentation.

[0131] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The application of Bacillus belye FSUXF-717 in improving enzyme activity in fermented glutinous rice mash, characterized in that, The enzyme is a liquefying enzyme, a saccharifying enzyme, and / or a protease; The Bacillus belyssus FSUXF-717 has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 64133.

2. The application as described in claim 1, characterized in that, Bacillus Fissuxif-717 was mixed with yeast and inoculated into the brewing raw materials for fermentation.

3. The application as described in claim 2, characterized in that, The inoculation amount of Bacillus vesiculus FSUXF-717 in the brewing raw materials was (1×10⁻⁶). 6 )~(1×10 7 CFU / mL.

4. The application as described in claim 3, characterized in that, After inoculation, the cells were saccharified and fermented in a constant temperature incubator at 28-32℃ for 24-48 hours, and then fermented with water for 15 days.

5. A method for producing saccharifying enzyme, characterized in that, Bacillus berleis FSUXF-717 was fermented in brewing raw materials or culture media, and the preservation number of Bacillus berleis FSUXF-717 was GDMCC No: 64133.

6. A method for reducing the hardness of fermented glutinous rice wine mash, characterized in that, Bacillus vesiculus FSUXF-717 was co-inoculated into the brewing raw materials with yeast for fermentation. The preservation number of Bacillus vesiculus FSUXF-717 is GDMCC No: 64133.

7. A method for increasing the content of ethyl octanoate, phenylethyl acetate, ethyl isohexanoate, and / or ethyl heptadecanate in fermented glutinous rice wine, characterized in that, Bacillus vesiculus FSUXF-717 was co-inoculated into the brewing raw materials with yeast for fermentation. The preservation number of Bacillus vesiculus FSUXF-717 is GDMCC No: 64133.

8. The application of Bacillus belye FSUXF-717 in brewing, characterized in that, The preservation number of the Bacillus belyssus FSUXF-717 is GDMCC No: 64133; the wine is a fermented glutinous rice wine.

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