Bacillus velezensis FSUXF-717 capable of promoting diastatic fermentation and improving flavor of fermented wine and application of bacillus velezensis FSUXF-717

Abstract

The invention discloses bacillus velezensis FSUXF-717 capable of promoting diastatic fermentation and improving flavor of fermented wine and application of the bacillus velezensis FSUXF-717, and relates to the technical field of microorganisms. The preservation number of the bacillus velezensis FSUXF-717 provided by the invention is GDMCC No: 64133, the fermentation liquor and microbial inoculum of the strain can promote the saccharification process of the wine, and meanwhile, the strain can enhance the fermentation to significantly increase the total amino acid content (plt: 0.05) and increase the variety and content of flavor substances such as esters and organic acids, and has a promoting effect on the flavor formation of the wine body. The acquisition of the strain provides a strain resource for the development of wine brewing and fermented food, has important practical significance for promoting high-efficiency and high-quality wine brewing, and also provides a theoretical basis and technical support for the directional regulation and control of the quality of traditional fermented food.

Description

TECHNICAL FIELD

[0001] The present application relates to a bacillus velezensis FSUXF-717 capable of promoting saccharification and fermentation and improving the flavor of fermented wine and its application, and belongs to the field of food microorganisms. BACKGROUND

[0002] The brewing and flavor of wine are closely related to the microbial flora and its composition. Bacteria are the core flora of wine flavor formation, which can produce esters and other flavor substances through metabolism, thereby affecting the formation of wine flavor. Organic acids are important aroma and taste substances in wine. Studies have shown that organic acids can enhance and buffer the aroma and taste of wine, reduce the pungency and bitterness, and promote the aging of new wine and prolong the aftertaste. Free amino acids are important flavor precursors and nutritional ingredients in fermented wine, which can participate in Maillard reaction, esterification reaction, etc., and affect the color, aroma and nutritional value of the wine. Ester mainly provides fruit aroma, floral aroma, sweetness and milk taste, etc. in wine. The types and concentrations of esters in different wines give the product distinct flavor characteristics. Increasing the content of ester substances helps to enrich the aroma and flavor of wine, and also improves the taste and roundness. Ethyl octanoate, ethyl nonanoate, phenethyl acetate and other ester compounds have fruit aroma, floral aroma, honey aroma and other flavor characteristics, and increasing these substances helps to provide a rich flavor and improve the quality of fermented wine. Ethyl heptadecanoate, ethyl octadecenoate and other long-chain fatty acid ethyl esters help to reduce the pungency of wine and have a positive impact on the taste of the wine body. Heptanoic acid, octanoic acid, 1-octen-3-ol, 1-propanol, phenylacetaldehyde and other substances are the irritant substances in wine. Heptanoic acid and octanoic acid have a cellar smell, acid smell or oil smell, and enols such as 1-octen-3-ol usually have an oil smell and green smell. Reducing these substances helps to reduce the irritability of wine and improve the taste and quality.

[0003] Mold is the core flora in the saccharification stage. Aspergillus, Rhizopus and other molds can produce amylase, cellulase and other enzymes to carry out saccharification, thereby decomposing starch, cellulose and other macromolecules in the raw materials into small molecules of sugar that can be directly utilized by yeast, affecting the fermentation efficiency and liquor yield. Existing technologies show that Bacillus velezensis can be applied in the brewing process. However, there are few studies on Bacillus velezensis that can promote saccharification. If Bacillus velezensis that can promote saccharification and fermentation and improve the flavor of fermented wine can be screened, it will have certain practical application value in the brewing industry. SUMMARY

[0004] In this study, Bacillus Velez-like FSUXF-717 was applied to the fermentation process. Compared with the blank control (CK) group, the experimental group (enhanced fermentation with Bacillus Velez-like FSUXF-717) showed a 43.04% increase in the activity of liquefaction enzymes in the mash (1721.15±19.66 U / g), a 20.64% increase in the activity of saccharifying enzymes (1492.49±14.87 U / g), and a 79.44% increase in the activity of proteases (1230.77±40.15 U / g) after 24 hours of saccharification. Furthermore, the saccharification coverage of the mash surface increased, and the mash hardness decreased by 96.23%. After saccharification, water was added to the fermentation at a solid-liquid ratio of 1:1.5, resulting in a reducing sugar content of 6.29±0.003 g / L, 21 times that of the control group.

[0005] After enhanced fermentation with Bacillus Velez-F17, the total free amino acid content in the mash increased by 67.50%, with umami amino acids increasing by 65.66% and sweet amino acids by 77.70%. The total organic acid content in the experimental mash peaked at 861.80 μg / mL on the 15th day, a 17.80% increase compared to the control mash at the same time. The levels of organic acids such as succinic acid and acetic acid also increased in the experimental group compared to the control group. On the 5th, 10th, and 15th days of fermentation, the total number of flavor compounds in the mash of the experimental group was 208, 205, and 211, respectively, with total contents of 13,030.13, 14,766.98, and 18,688.29 mg / kg, respectively, representing increases of 11.81%, 3.28%, and 7.10% compared to the control group. The levels of esters that influence the flavor of the wine, such as octyl acetate, ethyl isohexanoate, ethyl arachidate, ethyl heptadecanoate, and ethyl palmitate, all increased. Furthermore, the wine yield of the experimental group was 81.07%, a slight increase from the control group's 78.25%.

[0006] In vitro safety testing of Bacillus velezensis FSUXF-717 demonstrated that the strain exhibited γ-hemolytic activity, meaning it did not undergo hemolysis. Virulence gene PCR testing revealed that the strain contained no cytotoxins or vomitoxins, and its enterotoxin T was inactive. Based on these results, the strain was considered safe.

[0007] The present invention provides a strain of Bacillus velezine (Bacillus velezine) 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 velezensis FSUXF-717 was deposited in the Guangdong Provincial Microbial Culture Collection Center on December 8, 2023, with the deposit number GDMCC No: 64133.

[0008] The Bacillus velezensis FSUXF-717 colony of the present invention is milky white, translucent, irregularly round, convex in the middle, smooth on the surface, and wrinkled on the edge; Gram staining is positive, the bacteria are rod-shaped, and arranged individually; spores can be formed under adverse conditions; glucose, mannose, sucrose, raffinose, and esculin can be utilized, glucose gas production is positive, and starch can be hydrolyzed.

[0009] The present invention provides the use of Bacillus velez FSUXF-717 in increasing the activity of fermented mash enzymes and / or improving the quality of wine, wherein the enzyme activities include liquefaction enzyme, saccharification enzyme and / or protease; and the improvement of wine quality includes increasing the free amino acid content, increasing the organic acid content and / or improving the wine flavor.

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

[0011] In one embodiment, improving the flavor of wine comprises 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 velez FSUXF-717 is mixed with distiller's yeast and inoculated into brewing raw materials for fermentation.

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

[0017] In one embodiment, after inoculation, saccharification and fermentation are carried out in a constant temperature box at 28-32° C. for 24-48 hours, and then water is added and fermented for 15 days.

[0018] The present invention also provides a method for producing saccharifying enzyme, which comprises fermenting Bacillus velez FSUXF-717 in brewing raw materials or culture medium, wherein the deposit number of the Bacillus velez FSUXF-717 is GDMCC No: 64133.

[0019] The present invention also provides a method for reducing the hardness of fermented mash. The method comprises inoculating Bacillus velez FSUXF-717 and distiller's yeast into brewing raw materials for fermentation. The deposit number of the Bacillus velez FSUXF-717 is GDMCC No: 64133.

[0020] The present invention also provides a method for producing ethyl isocaproate, phenylethyl isovalerate, ethyl tridecanoate and / or ethyl cyclohexanecarboxylate, wherein Bacillus velez FSUXF-717 and distiller's yeast are co-inoculated into winemaking raw materials for fermentation, and the preservation number of the Bacillus velez FSUXF-717 is GDMCC No: 64133.

[0021] The present invention also provides application of Bacillus Velez subtilis FSUXF-717 in winemaking.

[0022] Beneficial effects: The present invention discloses a strain of Bacillus velez FSUXF-717 that can promote saccharification and fermentation and improve the flavor of fermented wine and its application, relating to the field of microbial technology. The Bacillus velez FSUXF-717 provided by the present invention has a deposit number of GDMCC No. 64133. The fermentation liquid and bacterial agent of the strain can promote the saccharification process of wine. At the same time, the strain can significantly increase the total amino acid content ( p <0.05), increasing the types and content of flavor compounds such as esters and organic acids, promoting the formation of flavor in wine. The acquisition of this strain provides a bacterial resource for the development of winemaking and fermented foods, which is of great practical significance for promoting efficient and high-quality winemaking. It also provides a theoretical basis and technical support for the targeted regulation of the quality of traditional fermented foods.

[0023] Biomaterial Deposit A strain of Bacillus velezinis ( Bacillus velezensis ) FSUXF-717, classified as Bacillus velezensis , was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 8, 2023, with the deposit number GDMCCNo: 64133, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a diagram of the state of the mash after saccharification for 24 hours according to the present invention; the left is the control group and the right is the experimental group.

[0025] Figure 2 The figure shows the change of the hardness of the mash after enhanced fermentation by Bacillus Velez subtilis FSUXF-717 of the present invention with saccharification time.

[0026] Figure 3The figure shows the changes in total sugar and reducing sugar in the mash after enhanced fermentation with Bacillus Velez subtilis FSUXF-717 of the present invention, where day 0 represents the end of the saccharification stage and the beginning of the fermentation stage.

[0027] Figure 4 This is the change in the content of flavor amino acids in the mash after enhanced fermentation with Bacillus Velez subtilis FSUXF-717 of the present invention.

[0028] Figure 5 This is the change in the organic acid content in the mash after enhanced fermentation by Bacillus Velez subtilis FSUXF-717 of the present invention.

[0029] Figure 6 This is the change in the number of flavor substances in the mash after enhanced fermentation with Bacillus Velez subtilis FSUXF-717 of the present invention.

[0030] Figure 7 The figure shows the changes in the content of flavor substances in the mash after enhanced fermentation with Bacillus Velez subtilis FSUXF-717 of the present invention.

[0031] Figure 8 The figure shows a comparison of the saccharifying enzyme activities in the mash of the present invention's Bacillus Velez FSUXF-717 and other strains of the same genus.

[0032] Figure 9 The figure shows a comparison of the liquefaction enzyme activities in the mash of the Bacillus Velez subtilis FSUXF-717 of the present invention and other strains of the same genus. DETAILED DESCRIPTION

[0033] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0034] The distiller's yeast involved in the following embodiments was purchased from Yongzhou Yada Technology Industrial Co., Ltd. in Hunan Province with the item number 4300779133967773830.

[0035] The definition and calculation method of enzyme activity involved in the following examples are: Definition of liquefaction enzyme (α-amylase) activity: 1 enzyme activity unit (U) is the amount of enzyme required to catalyze the decomposition of starch to produce 1 umol reducing sugar (calculated as glucose) within 1 minute at a specific temperature (60°C) and pH (6.0).

[0036] Definition of saccharifying enzyme (glucoamylase) activity: The amount of enzyme required to catalyze the decomposition of soluble starch to produce 1 mg of glucose within 1 hour at a specific temperature (60°C) and pH (4.5) is defined as 1 enzyme activity unit (U).

[0037] Definition of protease activity: 1 enzyme activity unit (U) is the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine within 1 minute at a specific temperature (40°C) and pH (7.5).

[0038] Enzyme activity determination and calculation methods: DNS method, Folin-phenol method.

[0039] Example 1 Screening of Bacillus velezinis FSUXF-717 Guangdong glutinous rice wine mash was used as the sample. After pretreatment, 0.5 mL of the sample was mixed and added to 4.5 mL of 0.9% saline for gradient dilution. The appropriate gradient dilution solution was spread on LB solid medium and cultured at 37°C for 48 hours. Typical colonies were picked and streaked on LB plates for purification. Single colonies were picked and transferred to LB liquid medium for culture for 24 hours. After centrifugation, the resulting bacteria were preserved with 50% glycerol to obtain Bacillus velezensis ( Bacillus velezensis ) FSUXF-717, the Bacillus velezensis FSUXF-717 is deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCCNo: 64133.

[0040] Example 2 Determination of enzyme activity of mash after enhanced fermentation by Bacillus velez FSUXF-717 The winemaking process for enhanced fermentation with Bacillus Velez FSUXF-717 is as follows: Weigh 1000 g of glutinous rice per portion, 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, remove it, and let it cool to 35°C. Add 6 g of koji and 150 mL of the second-generation bacterial culture of Bacillus Velez FSUXF-717 (with a bacterial cell concentration of 1.37 × 10 7 The brewing process for the control group was identical to that described above, except that Bacillus velezensis FSUXF-717 was not added. The raw materials from the experimental and control groups were mixed thoroughly, placed into fermentation vats, sealed with gauze, and placed in a 30°C incubator for saccharification and fermentation for 24 hours. Samples were collected at 0, 6, 12, 18, and 24 hours and stored in a -20°C refrigerator until further use. After saccharification, water was added at 1.5 times the weight of the rice and fermented for 15 days. The experiment was concluded after 15 days, and subsequent testing for physical and chemical parameters and volatile and non-volatile substances was performed.

[0041] (1) Determination of liquefaction enzyme activity of fermented grains: 10 g of fermented grains was weighed into a 250 mL beaker, 100 mL of pH 6.0 phosphate buffer was added, and it was placed in a 40°C water bath for 1 h (stir every 10 min), then filtered through qualitative filter paper to obtain the liquefaction enzyme solution. The DNS colorimetric method was used to determine the liquefaction enzyme activity of the fermented grains samples at 0, 12, and 24 h of saccharification. The steps of the determination method are shown in Table 1.

[0042] Table 1 Steps of enzyme activity determination method

[0043] Results: As shown in Table 2, the liquefaction enzyme activity of the fermented grains in the experimental group (added Bacillus velezensis FSUXF-717 for intensified brewing) was 1721.15 ± 19.66 U / g at 24 h of saccharification, which was 43.04% higher than that of the control group at the same time.

[0044] Table 2 Liquefaction enzyme activity of fermented grains

[0045] (2) Determination of saccharifying enzyme activity of fermented grains: 10 g of fermented grains was weighed into a 250 mL beaker, 100 mL of pH 4.6 acetic acid-sodium acetate buffer was added, and it was placed in a 30°C water bath for 1 h (stir every 10 min), then filtered through qualitative filter paper, and the obtained filtrate was the saccharifying enzyme solution. The DNS colorimetric method was used to determine the saccharifying enzyme activity.

[0046] Results: As shown in Table 3, the saccharifying enzyme activity of the fermented grains in the experimental group was 1492.49 ± 14.87 U / g at 24 h of saccharification, which was 20.64% higher than that of the control group at the same time.

[0047] Table 3 Saccharifying enzyme activity of fermented grains

[0048] (3) Determination of protease activity of fermented grains: Preparation of fermented grains sample: 5 g of fermented grains sample was weighed, 20 mL of pre-cooled phosphate buffer solution (pH 7.5) was added, and the sample was ground in an ice bath, then centrifuged at 4°C and 5000 rpm for 15 min to obtain the protease solution.

[0049] Transfer 1 mL of protease solution to a test tube and incubate in a constant-temperature water bath at 40°C for 5 minutes. Add 2 mL of 2% casein solution, mix thoroughly, and incubate in a constant-temperature water bath at 40°C for 15 minutes. Add 3 mL of 10% trichloroacetic acid, let stand at room temperature for 20 minutes, and then centrifuge at 4500 rpm for 20 minutes at 4°C. Aspirate 1 mL of the supernatant, add 5 mL of 0.55 mol / L sodium carbonate solution and 0.5 mL of Folin-phenol reagent, and incubate in a constant-temperature water bath at 40°C for 20 minutes to develop color. Measure absorbance at 680 nm, and calculate the protease activity.

[0050] Results: As shown in Table 4, when the saccharification and fermentation reached 12 h, the protease activity of the mash in the experimental group was 824.79±53.42 U / g, which was 56.91% higher than that of the control group at the same time; when the saccharification and fermentation reached 24 h, the protease activity of the mash in the experimental group was 1230.77±40.15 U / g, which was 79.44% higher than that of the control group at the same time.

[0051] Table 4 Protease activity in fermented mash

[0052] Based on the above results, it was found that the addition of Bacillus Velezii FSUXF-717 to strengthen the fermentation significantly increased the enzyme activities of liquefaction enzyme, saccharification enzyme and protease in the mash ( p <0.05). Liquefaction and saccharification enzymes break down starch in grain raw materials into small reducing sugars, while proteases break down gluten, releasing encapsulated starch granules and other macromolecular sugars, facilitating the complete breakdown and conversion of sugars. Furthermore, amino acids are important precursors for the formation of complex flavor compounds (such as esters) in fermented wines. Proteases break down proteins into small amino acids, facilitating the formation of flavor compounds. Therefore, Bacillus velezensis FSUXF-717 effectively promotes saccharification and fermentation, as well as the formation of amino acids and flavor compounds.

[0053] Example 3 Determination of Hardness Changes of Fermented Grains 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.

[0054] Table 5 Changes in mash hardness during saccharification

[0055] Saccharification breaks down sugars in the raw materials, reducing the hardness of the mash. At the 24th hour of saccharification, the mash hardness decreased by 96.23% after the addition of Bacillus Velez-F717 compared to the blank control. Similarly, for the group supplemented with Bacillus Velez-F717, the mash hardness decreased by 96.13% at 24 hours compared to the initial 0 hour of saccharification. These results suggest that the addition of Bacillus Velez-F717 to intensify fermentation can effectively promote saccharification and fermentation, improve saccharification efficiency, and shorten saccharification and fermentation time.

[0056] Example 4 Determination of Changes in Reducing Sugar and Total Sugar Contents in Fermented Grains (1) Determination of reducing sugar content in fermented grains: Draw the standard curve: Take 7 25 mL test tubes, add 0, 0.2, 0.6, 1.0, 1.4, 1.8, 2.0 mL of glucose standard solution (1 g / L) respectively, make up to 2 mL with distilled water, add 2 mL of DNS reagent, boil for 5 min. After cooling, dilute to 25 mL with distilled water, and 540 nm Measure the absorbance value and draw a standard curve.

[0057] Determination: Take 1 g of fermented grains and add 8 mL of distilled water, mix well, and centrifuge at 8°C and 5000 rpm for 10 min. Take 2 mL of the supernatant, add 2 mL of DNS reagent, and boil in boiling water for 5 min. After cooling, dilute to 25 mL with distilled water. 540 nm The absorbance value was measured at 400 nm and the reducing sugar content in the mash was calculated after being substituted into the standard curve.

[0058] (2) Determination of total sugar content in mash: Directly measure using a handheld sugar meter.

[0059] Results: As Figure 3 As shown in the figure, the total sugar content of the two groups of mash was basically the same, and the reducing sugar content of the experimental group showed a slight increase and then a downward trend as the fermentation progressed. On the 0th day of the fermentation stage (i.e., the end of the saccharification stage), the reducing sugar content of the experimental group was significantly higher than that of the control group ( p <0.05), indicating that Bacillus Velez FSUXF-717 effectively promoted the production of reducing sugars in the fermentation system during the saccharification stage. Throughout the fermentation process, the reducing sugar content in the experimental group was generally higher than that in the control group, indicating that the enhanced fermentation with the addition of Bacillus Velez FSUXF-717 resulted in a higher conversion rate of reducing sugars.

[0060] Example 5 Determination of free amino acid content in fermented grains Weigh 1 g of fermented grains sample, add 5 mL of water, sonicate at 25°C for 10 minutes, and centrifuge at 10,000 rpm for 10 minutes. Add 5 mL of 10% sulfosalicylic acid to the supernatant, mix thoroughly, refrigerate overnight at 4°C, and centrifuge at 10,000 rpm for 15 minutes. The supernatant was filtered through a 0.22 μm aqueous filter membrane and the amino acid content was determined using an automated amino acid analyzer.

[0061] 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 reached 176.90 mg / mL, a 67.50% increase compared to the control group. The total umami amino acid content in the experimental group reached 21.68 mg / mL, a 65.62% increase compared to the control group. The total sweet amino acid content in the experimental group reached 54.50 μg / mL, a 77.70% increase compared to the control group. Throughout the fermentation process, the total bitter amino acid content in the mash of both groups also increased. However, research has shown that the presence of bitter amino acids such as lysine and phenylalanine does not necessarily indicate a strong bitter taste; rather, they can enrich the body of the wine to a certain extent. In summary, the addition of Bacillus velezensis FSUXF-717 to enhance fermentation can increase the free amino acid content in wine.

[0062] Table 6 Changes in free amino acid content of mash samples at different fermentation stages

[0063] Note: Different lowercase letters indicate significant differences between different samples of the same amino acid ( p <0.05).

[0064] Example 6 Determination of organic acid content in fermented grains This experiment used liquid chromatography to determine the content of eight common organic acids in fermented mash. A 1 mL sample was added to 2 mL of 0.1% aqueous phosphoric acid solution. Ultrasonic extraction was performed for 40 minutes, followed by centrifugation at 5000 rpm for 5 minutes. The supernatant was filtered through a 0.45 μm aqueous filter and then analyzed. The elution procedure was 100% 25 mM / L potassium dihydrogen phosphate (pH 2.5), with a run time of 30 minutes, a column temperature of 35°C, a flow rate of 1.0 mL / min, a detection wavelength of 210 nm, and a 10 μL injection volume.

[0065] The results, as shown in Table 7, indicate that the total organic acid content in the experimental group increased throughout the fermentation process, reaching a peak of 861.80 μg / mL on day 15 of fermentation, a 17.80% increase compared to 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 mash of the experimental group were all higher than those in the control group. Succinic acid is an important organic acid that enhances the freshness of rice wine, while acetic acid, a volatile organic acid with a high content in rice wine, imparts a refreshing sourness to the wine. Appropriate organic acids can create rich layers and significantly influence the flavor and mouthfeel of the wine. This analysis suggests that the addition of Bacillus velezensis FSUXF-717 to intensify fermentation significantly increases the organic acid content in the wine, enriching its flavor.

[0066] Table 7 Changes in organic acid content of mash samples at different fermentation stages

[0067] Example 7 Determination of Volatile Flavor Substances in Fermented Grains A 200 mg sample of fermented grains was placed in a 20 mL headspace vial. 10 μL of a 10 mg / mL internal standard solution of 2-octanol was added. In another headspace vial, 10 μL of a 10 mg / L standard mixture of normal alkanes was added. The volatile components in the fermented grains were analyzed using HS-SPME-GC-MS.

[0068] Gas chromatography conditions: DB-Wax column (30 m × 250 μm × 0.25 μm), helium carrier gas, extraction at 60 °C for 30 min, desorption time of 4 min, flow rate of 1 mL / min, purge flow rate of 3 mL / min, no split.

[0069] Column oven heating program: initial temperature 40 °C, hold for 4 min, increase to 245 °C at 5 °C / min, hold for 5 min.

[0070] Mass spectrometry conditions: EI ion source (-70 eV), temperature (front inlet / transfer line 250°C, ion source 230°C, quadrupole 150°C), full scan acquisition, m / z: 20-400, solvent delay 2.37 min.

[0071] The relative content of each component was calculated using the internal standard method. The calculation formula is as follows: (1); Where: C is the relative content of a single component, mg / kg; A1 is the peak area of ​​a single component; A2 is the internal standard peak area; C2 is the internal standard concentration, mg / mL; V2 is the internal standard volume, uL; M is the sample mass, g.

[0072] Results: As Figure 6 、 7 As shown in Table 8, on fermentation days 5, 10, and 15, the total number of flavor compounds in the mash of the experimental group was 208, 205, and 211, respectively, with total contents of 13,030.13, 14,766.98, and 18,688.29 mg / kg, respectively, representing increases of 11.81%, 3.28%, and 7.10% compared to the control group (CK group). On fermentation day 15, the total ester content in the experimental group was 6,774.70 mg / kg, an increase of 11.65% compared to the control group at the same time (Table 8). These experimental results indicate that enhanced fermentation with Bacillus velezensis FSUXF-717 increased the number and content of flavor compounds, including esters, to a certain extent, improving the flavor quality of the fermented wine.

[0073] The main ester substances in the mash, such as ethyl octanoate and ethyl decanoate, have a fruity and sweet aroma that can enhance the richness of the wine; ethyl heptanoate and ethyl nonanoate have honey and fruity aromas, providing a mellow taste to the fermented wine; ethyl benzoate and phenylethyl acetate have rose and floral aromas; and ethyl palmitate has fruity and creamy aromas. The content of these flavor substances in the experimental group was significantly increased compared with the control group ( p <0.05), which have low sensory thresholds, allowing changes in relatively low concentrations to impact the wine's flavor. Flavor compounds like ethyl isohexanoate, ethyl arachidate, and ethyl heptadecanoate, absent in the control group, were produced after enhanced fermentation in the experimental group containing Bacillus velezensis FSUXF-717. These flavor compounds contribute fruity and rose-like aromas to the wine. Furthermore, long-chain fatty acid ethyl esters like ethyl heptadecanoate and ethyl octadecenoate help mitigate the wine's pungency, positively impacting the wine's mouthfeel and further enhancing the richness of the fermented wine's quality.

[0074] Table 8 Changes in ester flavor compound content in mash samples at different fermentation stages

[0075] Note: nd means not detected.

[0076] As shown in Table 9, VIP≥1, p<0.05 As a screening condition, the part of flavor substances with significant difference changes were screened, and analysis showed that compared with the control group, n-hexanol, 1-octene-3-ol, 1-propanol, phenylacetaldehyde, valeric acid, heptanoic acid and octanoic acid and other substances gradually decreased during the fermentation process, and 3-methyl-2-butene-1-alcohol and other substances were even not detected at last. In the sensory threshold evaluation, n-hexanol has a bitter taste; 1-octene-3-ol and 3-methyl-2-butene-1-alcohol and other enols usually have an oil taste, green taste; valeric acid, heptanoic acid and octanoic acid and other substances have a pit cellar taste, acid smell or oil taste. These substances are common irritating flavor substances in fermented wine, and reducing their content can help improve the taste and quality of the wine. The results show that the intensified fermentation of Bacillus velezensis FSUXF-717 can reduce the production of irritating substances to a certain extent during the fermentation process of the wine body, reduce the pungent taste of the wine body, and maintain the mellow and softness of the wine body.

[0077] Table 9 Changes in the contents of part of flavor substances in fermented grains at different fermentation stages

[0078] Note: nd means not detected.

[0079] Example 8 Calculation of the yield of intensified fermentation The yield of the finished product was calculated. The wine was collected by segmented distillation, and the alcohol content was measured by alcohol meter.

[0080] The actual yield was converted into standard alcohol degree (50 degrees) and calculated according to the following formula: Standard alcohol degree yield = actual alcohol yield × actual alcohol content ÷ standard alcohol content; Yield = (standard alcohol degree yield ÷ raw material dosage) × 100%.

[0081] Results: Table 10 Calculation method of yield

[0082] Experimental group: (87.00 × 74.50 + 855.00 × 58.50 + 1556.80 × 28.80) ÷ 50 ÷ 2500.00 × 100% = 81.07%.

[0083] Control group: (176.60 × 71.40 + 709.90 × 58.50 + 1621.70 × 26.80) ÷ 50 ÷ 2500.00 × 100% = 78.25%.

[0084] The above results show that after adding Bacillus velezensis FSUXF-717 for intensified fermentation, the production of high-grade liquor is improved, and the overall yield is slightly improved.

[0085] Example 9 Production of saccharifying enzyme by fermentation of Bacillus velez FSUXF-717 alone Liquid fermentation medium for saccharifying enzyme-producing strains (g / L): 20g of peptone, 20g of soluble starch, 5g of disodium hydrogen phosphate, 0.1g of magnesium sulfate, 0.1g of sodium chloride, pH=7.0; sterilize with high-pressure steam at 121°C for 20 min.

[0086] A single colony of activated Bacillus velezensis FSUXF-717 was picked and inoculated into the liquid fermentation medium of the saccharifying enzyme-producing strain, and cultured at 37°C and 160 r / min for 24 h. The seed liquid was inoculated at an inoculum rate of 3% (V / V) into the liquid fermentation medium of the saccharifying enzyme-producing strain, and cultured at 37°C and 160 r / min 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 by the DNS method.

[0087] Results: The equation of glucose standard curve is y=1.18022*x+0.0625(R 2 =0.9991>0.995).

[0088] Substituting the measurement results into the standard curve, the glucose concentration was calculated to be 0.85746 mg / mL.

[0089] Calculation formula of saccharifying enzyme activity: .

[0090] (X is the saccharifying enzyme activity (U / g); c is the glucose concentration (mg / mL); n is the enzyme solution dilution factor; m is the sample weight (g)) Substituting into the calculation formula, the saccharifying enzyme activity was 2057.41 U / g.

[0091] The saccharifying enzyme activity of the fermentation broth of Bacillus velezensis FSUXF-717 discovered in the present invention is 2057.41 U / g.

[0092] Comparative Example 1 According to the method for determining enzyme activity in Example 2, the liquefaction enzyme and saccharification enzyme of fermented mash of other Bacillus Velez subsp. of the same species were determined.

[0093] The results are as follows Figure 8 and Figure 9 As shown in the figure, the liquefaction enzyme activity and saccharification enzyme activity of the mash after the enhanced fermentation of Bacillus Velez FSUXF-717 were significantly higher than those of the mash after the enhanced fermentation of Bacillus Velez B1~B6 ( p<0.05), indicating that the Bacillus velezensis FSUXF-717 discovered in the present invention has a stronger ability to promote saccharification and fermentation.

[0094] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Application of Bacillus velez FSUXF-717 in increasing fermented mash enzyme activity and / or improving wine quality, characterized in that: The enzyme activity includes liquefaction enzyme, saccharification enzyme and / or protease; the improvement of wine quality includes increasing the free amino acid content, increasing the organic acid content and / or improving the wine flavor; The Bacillus velezensis FSUXF-717 has been deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No: 64133.

2. The use according to claim 1, characterized in that The improvement of wine flavor includes increasing the content of ethyl octanoate, phenylethyl acetate, ethyl isohexanoate and / or ethyl heptadecanoate in the wine, and / or reducing the content of n-hexanol, 1-octen-3-ol, phenylacetaldehyde and / or heptanoic acid.

3. The use according to claim 1, characterized in that The organic acids include succinic acid, oxalic acid, and acetic acid; the free amino acids include sweet amino acids; and the wine includes rice wine.

4. The use according to claim 3, characterized in that The Bacillus velezensis FSUXF-717 was mixed with koji and inoculated into the brewing raw materials for fermentation.

5. The use according to claim 4, characterized in that The inoculation amount of the Velez Bacillus FSUXF-717 in the brewing raw materials is (1×10 6 )~(1×10 7 ) CFU / mL.

6. The use according to claim 5, characterized in that After inoculation, saccharification and fermentation were carried out in a constant temperature box at 28-32℃ for 24-48 hours, and then water was added and fermented for 15 days.

7. A method for producing saccharifying enzyme, characterized in that: The Bacillus velez FSUXF-717 is fermented in brewing raw materials or culture medium, and the preservation number of the Bacillus velez FSUXF-717 is GDMCC No: 64133.

8. A method for reducing the hardness of fermented grains, characterized in that: Bacillus velez FSUXF-717 and distiller's yeast are co-inoculated into brewing raw materials for fermentation. The preservation number of the Bacillus velez FSUXF-717 is GDMCC No: 64133.

9. A method for producing ethyl octanoate, phenylethyl acetate, ethyl isohexanoate and / or ethyl heptadecanoate, characterized in that: Bacillus velez FSUXF-717 and distiller's yeast are co-inoculated into brewing raw materials for fermentation. The preservation number of the Bacillus velez FSUXF-717 is GDMCC No: 64133.

10. Application of Bacillus velez FSUXF-717 in winemaking, characterized in that: The deposit number of the Bacillus velezensis FSUXF-717 is GDMCC No: 64133.

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