Bacillus amylophilus strain and application thereof

Abstract

The present application provides a bacillus licheniformis strain HSCY3018, which is preserved in China Center for Type Culture Collection, and the preservation number is CCTCCM20221831, and the preservation date is November 28, 2022. The present application also provides the application of the HSCY3018 strain and a microbial inoculum thereof in food vinegar brewing. The HSCY3018 has high amylase and protease activities in food vinegar brewing, and can significantly improve the raw material utilization rate of food vinegar brewing. Compared with a control group, the starch utilization rate is increased by 58.50%, the lactic acid content in organic acid is increased by 72.95%, the total amount of amino acid is increased by 66.18%, the reducing sugar content is increased by 43.96%, and the total ester content is increased by 275.74%.

Description

Technical Field

[0001] This invention relates to the field of traditional vinegar brewing, specifically to a strain of Bacillus licheniformis that is highly tolerant and can produce a rich enzyme system and nutrients, thereby significantly improving the sensory quality of vinegar, and also to the application of the Bacillus licheniformis and its microbial agents in vinegar brewing. Background Technology

[0002] Traditionally, vinegar is brewed primarily using starchy ingredients such as glutinous rice and sorghum, supplemented with wheat bran and rice husks, through a multi-strain fermentation process. Currently, the vinegar brewing process suffers from low raw material utilization, resulting in a weak aroma and a harsh taste in the finished product, thus affecting its value.

[0003] Currently, industrial production often employs methods such as crushing raw materials, secondary fermentation of vinegar residue, high-temperature rinsing and smoking of the mash, adding compound enzyme preparations, and seasoning and flavor enhancement to improve the utilization rate of raw materials and enhance flavor in vinegar brewing. Although the flavor has been improved, the production cost has increased significantly, which is not conducive to its widespread application in production.

[0004] With the improvement of living standards, consumers' demand for high-quality vinegar is becoming more and more urgent. Brewing high-quality vinegar with unique flavors is now imperative for my country's vinegar industry, and the entire industry urgently needs to transform and upgrade.

[0005] Bacillus is a type of highly resistant bacteria with rapid growth and relatively high safety; some strains exhibit strong resistance to acetic acid and high temperatures. Therefore, screening for Bacillus licheniformis strains with excellent stress resistance suitable for the specific brewing environment of vinegar and applying them to the vinegar production process is of great significance for stabilizing and improving product quality. While there are reports of isolating Bacillus licheniformis from vinegar mash, research on utilizing it to produce abundant organic acids, amino acids, reducing sugars, ester aroma substances, and enhance flavor and taste in vinegar is still lacking. Therefore, improving the overall flavor and quality of traditional vinegar by innovating isolation and cultivation methods and relying on novel Bacillus licheniformis strains that can well adapt to the harsh environment of vinegar brewing has promising application prospects. Summary of the Invention

[0006] To solve the above-mentioned technical problems, the first aspect of this invention provides a Bacillus licheniformis, named HSCY3018, which is deposited at the China Center for Type Culture Collection with accession number CCTCC M 20221831 and deposit date of November 28, 2022.

[0007] A second aspect of the present invention provides a microbial agent comprising Bacillus licheniformis as described in the first aspect of the present invention.

[0008] In some embodiments, the preparation of the microbial agent includes mixing the strain described in the first aspect of the present invention with a sterile protectant, subjecting the mixture to heat shock, and then freeze-drying it under vacuum.

[0009] In some embodiments, the heat shock is performed in an incubator at 35-45°C for 5-25 seconds. Preferably, the heat shock is performed in an incubator at 40°C for 10 seconds.

[0010] In some embodiments, the sterile protectant protects components A and B. Preferably, component A is prepared by: 10g of skim milk powder, 100mL of distilled water, sterilized at 121°C for 20min; component A is prepared by: 0.5g of glycerol, 3g of lactose, 4g of mannitol, 5g of trehalose, 100mL of distilled water, sterilized at 121°C for 20min.

[0011] In some embodiments, after component A and component B are sterilized separately, they are cooled to room temperature and then mixed to obtain a sterile protectant.

[0012] The second aspect of this invention provides the application of Bacillus licheniformis described in the first aspect of this invention and the microbial agent described in the second aspect of this invention in vinegar brewing.

[0013] In some embodiments, the vinegar is balsamic vinegar, aged vinegar, or rice vinegar.

[0014] The third aspect of the present invention provides a method for brewing vinegar, comprising the following steps: (1) preparing a bacterial seed liquid or a microbial agent using Bacillus licheniformis as described in the first aspect of the present invention; (2) inoculating the bacterial seed liquid or microbial agent obtained in step (1) for fermentation.

[0015] In some embodiments, the vinegar is balsamic vinegar, aged vinegar, or rice vinegar.

[0016] In some embodiments, the preparation of the microbial agent includes mixing a Bacillus licheniformis strain with a sterile protectant, subjecting the mixture to heat shock, and then freeze-drying it under vacuum.

[0017] In some embodiments, the heat shock is performed in an incubator at 35-45°C for 5-25 seconds. Preferably, the heat shock is performed in an incubator at 40°C for 10 seconds.

[0018] In some embodiments, the sterile protectant protects components A and B. Preferably, component A is prepared by: 10g of skim milk powder, 100mL of distilled water, sterilized at 121°C for 20min; component A is prepared by: 0.5g of glycerol, 3g of lactose, 4g of mannitol, 5g of trehalose, 100mL of distilled water, sterilized at 121°C for 20min.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) This invention isolates and screens a strain of Bacillus licheniformis HSCY 3018 from the vinegar mash produced in the Zhenjiang vinegar production process. Applied to the food industry, this strain exhibits high amylase and protease activity, which can improve the utilization rate of starch and protein in raw materials. The amylase activity of this strain can reach 843.95 U / mL fermentation broth, and the protease activity can reach 256.18 U / mg fermentation broth.

[0021] (2) By using only a single strain of the present invention in traditional solid vinegar brewing, the content of organic acids, amino acids, reducing sugars, total esters and other substances in the product can be significantly increased, which has the beneficial effect of significantly improving the overall flavor and taste quality of the product and increasing the product yield.

[0022] (3) By using this strain in the brewing of Zhenjiang vinegar, the lactic acid content in the organic acids of the finished vinegar can be increased by 72.95% compared with the control group, the total amino acid content can be increased by 66.18%, the reducing sugar content can be increased by 43.96%, the total ester content can be increased by 275.74%, and the product yield can be increased by 12.96%, which significantly improves the taste and aroma of the product and the overall sensory quality is high.

[0023] (4) When this strain is used in vinegar brewing, it makes the vinegar more nutritious, has a mellow, smooth and non-irritating taste, and a rich ester aroma. Its sensory quality is significantly better than that of the control group, which can meet consumers' health and high-quality needs and has stronger market adaptability.

[0024] (5) This strain can also be made into microbial inoculants. The strain can be made into bacterial liquid or microbial inoculants for vinegar brewing, which can improve the flavor, taste and product quality. Attached Figure Description

[0025] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0026] Figure 1 Colony morphology of Bacillus licheniformis HSCY 3018. Detailed Implementation

[0027] The applicant will now provide a detailed description of the preparation and application processes of the product of this invention, using specific embodiments, to facilitate a clear understanding of the invention by those skilled in the art. However, it should be understood that the following embodiments should not be construed as limiting the scope of protection claimed in this application.

[0028] In this embodiment of the invention, the LB culture medium consisted of: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, 0.5 g disodium hydrogen phosphate, 0.5 g magnesium sulfate, 1000 mL distilled water, pH 7.0, and sterilized at 121°C for 20 min.

[0029] LB solid medium: 10.0g tryptone, 5.0g yeast extract, 10.0g sodium chloride, 0.5g disodium hydrogen phosphate, 20.0g agar, 0.5g magnesium sulfate, 1000mL distilled water, pH 7.0, sterilized at 121℃ for 20min.

[0030] Selection medium for protease production: 5.0g beef extract, 30.0g casein, 5.0g sodium chloride, 20.0g agar, 1000mL distilled water, natural pH, sterilized at 121℃ for 20min.

[0031] Amylase production screening medium: peptone 10.0g, urea 15.0g, yeast extract 5.0g, maltose 150.0g, corn starch 20.0g, glucose 1.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 5.0g, manganese sulfate 4.0g, Tween 80 1.0mL, sodium chloride 0.5g, distilled water 1000mL, pH 7.0, sterilized at 121℃ for 20min.

[0032] High-temperature α-amylase and saccharifying enzyme: purchased from Shandong Longket Enzyme Preparation Co., Ltd.

[0033] Example 1: Isolation and Identification of Bacillus licheniformis HSCY3018

[0034] (1) Strain isolation

[0035] Weigh 15g of Zhenjiang vinegar mash sample and place it in 85mL of sterilized LB medium. Incubate at 37℃ and 180r / min for 20min with shaking, then heat in a 90℃ water bath for 10min. Perform 10-fold serial dilutions on the sample, selecting a dilution gradient of 10... -3 10 -4 10 -5 and 10 -6 The diluted solution was applied in 100 μL portions to LB solid culture plates and incubated upside down at 37°C for 24 h.

[0036] (2) Strain purification

[0037] Select a single colony, stain it with Gram, and examine it under a microscope. For Gram-positive, rod-shaped bacteria, streak them three times on agar plates and preserve the single colony.

[0038] (3) Strain screening

[0039] The purified test strain was inoculated onto protease selection medium using the spot inoculation method. After incubation at 37°C for 24 hours, plates with 30-300 colonies were selected, and colonies with good growth and obvious clear zones were chosen; these were the protease-producing strains. The ratio of the clear zone diameter to the colony diameter (H / d) was measured, and 16 strains with a ratio greater than 4.0 were selected, indicating strains with strong protease production capacity. These 16 strains were then transferred to amylase selection medium and incubated at 40°C for 36 hours. 5 mL of iodine solution was added to each plate, evenly covering it. The strain with the largest clear zone was selected using the previous method, thus obtaining a strain that produces both high levels of protease and amylase. Its colony morphology is as follows: slightly moist surface, irregular shape, pale yellow, opaque, convex in the center, and wrinkled at the edges. Figure 1 As shown.

[0040] (4) Strain identification

[0041] For the purified and screened bacteria, fresh bacterial culture in the exponential growth phase was collected by centrifugation, and genomic DNA was extracted using a genomic DNA extraction kit. The full-length 16S rDNA sequence was amplified using universal bacterial primers P0-P6 (China Master's Thesis Full-text Database, Zhu Qihan, "Microbial Isolation and Acid Production Characteristics during Zhenjiang Vinegar Fermentation", 2008). Sequencing of the PCR amplification products was performed by Shanghai Sangon Biotech Co., Ltd. The obtained 16S rDNA sequence was analyzed by BLAST comparison in the NCBI database to determine its species. The full-length 16S rDNA sequence of this strain is 1468 bp, and the strain with the highest homology is Bacillus licheniformis, with a similarity of 99.86%. The 16S rDNA sequence is shown in SEQ ID NO: 1. Based on its characteristics, the strain of this invention was named Bacillus licheniformis HSCY3018.

[0042] (5) Determination of protease production capacity of strains

[0043] The protease activity of this strain was determined using the Folin-phenol colorimetric method, revealing an activity of 256.18 U / mg fermentation broth. As a control, the protease activity of Bacillus licheniformis in patent publication CN111718871A was measured using the same method, yielding a result of 34.61 U / mg.

[0044] (5) Determination of the strain's amylase production capacity

[0045] The 3,5-dinitrosalicylic acid (DNS) method revealed that the amylase activity of this strain reached 843.95 U / mL of fermentation broth. As a control, the amylase activity of a thermoresistant amylase-producing Bacillus licheniformis isolated from Daqu (a type of Chinese liquor) by You Yong et al. (You Yong et al., "Isolation and Identification of a Thermoresistant Amylase-Producing Bacillus licheniformis from Daqu of Special Aroma Baijiu," Brewing Technology, 2022, Vol. 3) was 53.42 U / mg.

[0046] Bacillus licheniformis HSCY 3018 was deposited on November 28, 2022, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC M 20221831.

[0047] Example 2: Application of HSCY3018 in Zhenjiang Vinegar Brewing

[0048] 1. Large-scale culture of the strain

[0049] Preparation of primary seed culture: Purified Bacillus licheniformis HSCY 3018 bacterial culture in the logarithmic growth phase was inoculated into LB medium at an inoculation rate of 4% (v / v) and cultured at 37℃ and 200 r / min for 24 h.

[0050] Preparation of secondary seed culture: A 50L liquid self-priming fermenter was used. 35L of water was added, and the stirring speed was 120 rpm. The water was heated to 90℃, and 10kg of corn starch (crushed and sieved through a 100-mesh sieve) was added. 3.0mL of 20,000 U / mL high-temperature α-amylase was added, and the mixture was kept warm for 35 minutes to obtain the mash. The mash temperature was then lowered to 45℃, and 0.50g of 50,000 U / g saccharifying enzyme was added. The mixture was kept warm for 30 minutes to obtain the saccharified liquid. 1.0% (w / v) peptone and 1.0% (w / v) yeast extract were added to the prepared saccharified liquid, and the mixture was sterilized at 121℃ for 20 minutes. After cooling to 37℃, the primary seed culture was inoculated at an inoculation rate of 4.0%, the aeration rate was adjusted to 0.15 vvm, the stirring speed was 150 rpm, and the mixture was fermented at 37℃ under pressure for 24 hours until the viable cell count reached 10⁻⁶. 10 cfu / mL.

[0051] 2. Preparation of mash

[0052] Take six 500kg vats and soak 80kg of high-quality glutinous rice in water overnight in each vat. Steam the glutinous rice until cooked, then rinse it with cold water until the temperature reaches about 40℃. Add 0.4kg of yeast starter, mix well, and pour the mixture into the vat, forming a funnel shape. When a certain amount of alcohol appears in the funnel, add 5.0kg of wheat koji to each vat, then add 250kg of water and stir well. During the alcoholic fermentation process, stir regularly, controlling the temperature at around 30℃. Fermentation is completed in 7 days. After fermentation, mix the mash from the six vats thoroughly and set aside.

[0053] 3. Add ingredients in groups to make mash.

[0054] Adding Bacillus licheniformis HSCY 3018 group: Take 3 large 500kg vats, add 200kg of the above-mentioned mash to each vat, then add the secondary seed fermentation liquid of the strain HSCY 3018 of this invention, the amount added is 3.0% of the mash mass, add 76kg of wheat bran and 35kg of rice bran, mix the mash and grains evenly, inoculate with 5.0% of the total weight of raw materials of seed mash (vinegar mash fermented to day 7), and then carry out solid-state layered fermentation with seed and turning mash according to the Zhenjiang vinegar brewing process until the end of fermentation.

[0055] Control group 1: Three large vats were selected, and distilled water was used to replace the secondary seed fermentation broth of strain HSCY 3018. The same amount and method of addition were used as in the group with added Bacillus licheniformis HSCY 3018.

[0056] 4. Fermentation and sealing of the mash

[0057] Following the traditional "solid-state layered fermentation technique" of Zhenjiang vinegar, the mash is turned layer by layer. Once the total acidity of the mash no longer increases, it is sealed and compacted, then salt is added and the mash is sealed for 7-10 days. After sealing, the mash is added to a vinegar-draining vat, and then roasted rice is added for vinegar extraction. The steps in Control Group 1 are consistent with those in Example 2.

[0058] 5. Finished Product Preparation

[0059] After the fermentation process is completed, the vinegar mash is added to a vinegar-draining vat, and then 6.0% roasted rice starch is added for vinegar draining. After simmering, aging, sterilization, and bottling, the finished product is obtained.

[0060] 6. Detection of total acid, reducing sugar and total ester content

[0061] The finished vinegar obtained in step 5 was tested. The total acid content was determined according to the method of GB 18187-2000, and the total acid was calculated as acetic acid; the reducing sugar content was determined according to the method of GB 5009.7-2016, and the reducing sugar was calculated as glucose; the total ester content was determined according to the method of GB / T 19777-2013, and the total ester was calculated as ethyl acetate.

[0062] Table 1 Comparison of total acid, reducing sugar and total ester content in finished vinegar

[0063]

[0064] Compared with control group 1, Example 2 using the strain of the present invention showed that the total acid content in the finished vinegar increased by 13.89%, the reducing sugar content increased by 43.96%, the total ester content increased by 275.74%, and the product yield increased by 12.96%. The significant increase in total ester content indicates that the Bacillus licheniformis HSCY 3018 used in this invention is conducive to the formation of esters, resulting in a vinegar with a rich ester aroma and unique flavor.

[0065] 7. Organic acid content detection

[0066] The content of nine organic acids in the finished vinegar was analyzed by HPLC, and the results are shown in Table 2. After fermentation, the organic acids in the vinegar brine were mainly acetic acid and lactic acid, with contents of 52.08 mg / mL and 22.12 mg / mL, respectively, in Example 2, which were 13.66% and 72.95% higher than those in Control Group 1. The increased organic acid content makes the vinegar smoother on the palate and has a longer aftertaste. This indicates that the Bacillus licheniformis HSCY 3018 used in this invention can metabolize and produce a relatively rich amount of organic acids. On the one hand, this can buffer the strong, pungent sour taste of acetic acid; on the other hand, it can esterify with alcohols to form esters, which can enrich the flavor and taste of vinegar and improve product quality.

[0067] Table 2 Organic acid content of Bacillus licheniformis HSCY3018 in Zhenjiang vinegar brewing

[0068]

[0069] 8. Starch content detection

[0070] Starch content was determined using the enzymatic hydrolysis method according to GB 5009.9-2016. Starch content was measured in the vinegar mash collected at the start and end of fermentation, and the results are shown in Table 3. Compared to control group 1, the starch utilization rate of Example 2 increased by 58.50%. This demonstrates that adding Bacillus licheniformis during the acetic acid fermentation stage significantly improved the starch utilization rate of the raw materials and reduced waste.

[0071] Table 3. Starch utilization rate of Bacillus licheniformis HSCY3018 in Zhenjiang vinegar brewing

[0072]

[0073] 9. Amino acid content detection

[0074] The content of 17 amino acids in the finished vinegar was analyzed by HPLC, and the results are shown in Table 4 below. At the end of fermentation, the total amino acid content in Example 2 reached 1639.07 mg / 100 mL, which was 66.18% higher than that in Control Group 1 (986.33 mg / 100 mL). This indicates that the Bacillus licheniformis HSCY3018 used in this invention provides abundant protease, thereby improving the protein utilization efficiency in the raw materials. The small molecule amino acids formed by decomposition are more conducive to enriching the taste, making the brewed Zhenjiang vinegar more mellow. The content of various amino acids in Example 2 was also generally higher than that in Control Group 1.

[0075] Table 4. Amino acid content of Bacillus licheniformis HSCY 3018 in Zhenjiang vinegar brewing

[0076]

[0077] 10. Sensory index analysis of finished vinegar

[0078] Thirty experienced sensory evaluators were selected to conduct sensory evaluation analysis on the products of Example 2 and Control Group 1. The results are shown in Table 5. A three-point test revealed that 28 out of the 30 evaluators indicated a significant difference between the products of Example 2 and Control Group 1. Descriptive analysis showed that the product of Example 2 had a brighter red color, clearer texture, richer ester aroma, milder taste, and lower irritation. A comprehensive preference-based scoring system showed that the product of Example 2 scored significantly higher than Control Group 1 in color, texture, aroma, and taste, indicating that the addition of Bacillus licheniformis during the acetic acid fermentation stage significantly improved the product's flavor and mouthfeel, thereby enhancing its sensory quality.

[0079] Table 5 Sensory index analysis of finished vinegar produced by Bacillus licheniformis HSCY 3018 in Zhenjiang vinegar brewing

[0080] Example 2 9.3 9.2 9.8 9.8 Control group 1 8.2 7.1 6.7 7.1

[0081] Example 3: Application of Bacillus licheniformis HSCY 3018 in Shanxi Aged Vinegar

[0082] 1. Large-scale culture of the strain

[0083] Preparation of primary seed culture: Purified Bacillus licheniformis HSCY 3018 bacterial culture in the logarithmic growth phase was inoculated into LB medium at an inoculation rate of 6% (v / v) and cultured at 40℃ and 180 r / min for 24 h.

[0084] Preparation of secondary seed culture: A 50L self-priming liquid fermenter was used. 35L of water was added, and the stirring speed was 140 rpm. The water was heated to 95℃, and 12kg of corn starch (crushed and passed through a 100-mesh sieve) was added. 3.5mL of 20,000 U / mL high-temperature α-amylase was added, and the mixture was kept at this temperature for 40 minutes to obtain the mash. The mash temperature was then lowered to 55℃, and 1.5g of 50,000 U / g saccharifying enzyme was added. The mixture was kept at this temperature for 40 minutes to obtain the saccharified liquid. 3.0% (w / v) peptone and 3.0% (w / v) yeast extract were added to the prepared saccharified liquid. The mixture was sterilized at 121℃ for 15 minutes. After cooling to 37℃, the primary seed culture was inoculated at a rate of 6% (v / v). The aeration rate was adjusted to 0.3 vvm, the stirring speed was 120 rpm, and the mixture was fermented at 40℃ under pressure for 30 hours until the viable cell count reached 10⁻⁶. 10 cfu / mL.

[0085] 2. Preparation of mash

[0086] Take six 500kg vats, and in each vat, add 100kg of sorghum crushed into four to six pieces, along with 50kg of water to moisten for 15 hours. Steam the sorghum until cooked, then add 200kg of water, stir, and cool to 30℃. Next, add 62kg of Daqu (a type of starter culture) and 0.1kg of active dry yeast, and stir well. During the alcoholic fermentation process, stir regularly, maintaining the temperature at around 30℃. Fermentation is completed in 13 days.

[0087] 3. Adding ingredients to make mash

[0088] Adding Bacillus licheniformis HSCY 3018 group: After the alcoholic fermentation is completed, select 3 large vats, add 150kg of mash, 60kg of wheat bran, and 75kg of rice bran to each vat, and inoculate 6.0% of the weight of the mash with the above-mentioned secondary seed fermentation liquid of Bacillus licheniformis HSCY 3018. After the bacterial liquid is thoroughly mixed with the added raw materials, inoculate 10% of the total weight of raw materials with fire mash (vinegar mash that has been fermenting vigorously for 2-3 days), and then carry out fermentation, smoking, vinegar leaching, and aging according to the solid-state fermentation process of Shanxi aged vinegar.

[0089] Control group 2: The remaining 3 large tanks were operated with distilled water instead of the secondary seed fermentation broth of strain HSCY 3018, following the same addition amount and method as the group with added Bacillus licheniformis HSCY 3018.

[0090] 4. Total acid, reducing sugar, and total ester content

[0091] Table 6 Comparison of total acid, reducing sugar and total ester content in finished vinegar

[0092]

[0093] Compared with control group 2, Example 3 using the strain of the present invention showed that the total acid content in the finished vinegar increased by 12.70%, the reducing sugar content increased by 32.55%, the total ester content increased by 224.81%, and the product yield increased by 11.02%. The significant increase in both reducing sugar and total ester content also indicates that the Bacillus licheniformis HSCY3018 used in this invention is conducive to the formation of esters, resulting in a vinegar with a rich ester aroma and a smooth, non-irritating taste.

[0094] 5. Organic acid content

[0095] The content of nine organic acids in the finished vinegar was analyzed by HPLC, and the results are shown in Table 7. After fermentation, the organic acids in the vinegar brine were mainly acetic acid and lactic acid, with contents of 39.06 mg / mL and 15.93 mg / mL, respectively, in Example 3, which were 13.66% and 41.50% higher than those in Control Group 1. This also indicates that the Bacillus licheniformis HSCY 3018 used in this invention can metabolize and produce a relatively rich amount of organic acids, reducing the irritation of the brewed vinegar.

[0096] Table 7 Organic acid content of Bacillus licheniformis HSCY3018 in Shanxi aged vinegar brewing

[0097]

[0098] 6. Starch content

[0099] Starch content was determined using the enzymatic hydrolysis method according to GB 5009.9-2016. Starch content was measured in the vinegar mash collected at the start and end of fermentation, and the results are shown in Table 8. Compared to control group 2, the starch utilization rate of Example 3 increased by 46.14%. This demonstrates that adding Bacillus licheniformis during the acetic acid fermentation stage significantly improved the starch utilization rate of the raw materials and reduced waste.

[0100] Table 8. Starch utilization rate of Bacillus licheniformis HSCY 3018 in Shanxi aged vinegar brewing

[0101]

[0102] 7. Amino acid content

[0103] The content of 17 amino acids in the finished vinegar was analyzed by HPLC, and the results are shown in Table 9 below. At the end of fermentation, the total amino acid content of Example 3 was 1106.50 mg / 100 mL, which was 55.53% higher than that of control group 2 (711.43 mg / 100 mL).

[0104] Table 9. Amino acid content of Bacillus licheniformis HSCY3018 in Shanxi aged vinegar brewing

[0105]

[0106] 8. Sensory index analysis of finished vinegar

[0107] Thirty experienced sensory evaluators were selected to conduct sensory evaluation analysis on the products of Example 3 and Control Group 2. The results are shown in Table 10. A three-point test revealed that 27 out of the 30 evaluators indicated a significant difference between the products of Example 3 and Control Group 2. Descriptive analysis showed that the product of Example 3 had a brighter red color, clearer appearance, richer ester aroma, smoother taste, lower irritation, and a more mellow overall flavor. A comprehensive preference-based scoring system showed that the product of Example 3 scored significantly higher than Control Group 2 in terms of color, appearance, aroma, and taste, indicating that the addition of Bacillus licheniformis during the acetic acid fermentation stage significantly improved product quality.

[0108] Table 10 Sensory index analysis of finished vinegar produced by Bacillus licheniformis HSCY 3018 in Shanxi aged vinegar brewing

[0109] Example 3 9.3 8.9 9.7 9.6 Control group 2 8.5 6.8 7.6 7.1

[0110] Example 4: Application of Bacillus licheniformis HSCY3018 in liquid rice vinegar brewing

[0111] 1. Preparation of Bacillus licheniformis inoculant

[0112] Bacillus licheniformis was inoculated into LB medium at a 5% inoculum and cultured at 37°C and 180 rpm for 36 hours. The bacterial cells were collected by centrifugation, and a sterile protectant was uniformly mixed with the bacterial cells and heat-shocked at 40°C for 10 seconds. The resulting bacterial agent was then freeze-dried under vacuum. The viable count of Bacillus licheniformis was approximately 10⁻⁶. 10 The cfu / g inoculant has a shelf life of 1 year under cool, dry conditions.

[0113] Preparation of sterile preservative: The above preservative is prepared as follows: A: 10g skim milk powder, 100mL distilled water, sterilized at 121℃ for 20min; B: 0.5g glycerol, 3g lactose, 4g mannitol, 5g trehalose, 100mL distilled water, sterilized at 121℃ for 20min; After sterilization of A and B respectively, they are cooled to room temperature and mixed to obtain the preservative.

[0114] Vacuum freeze-drying process parameters: cold trap temperature is -45℃, plate temperature is 28℃, initial vacuum degree is 30Pa, and final vacuum degree is 45Pa.

[0115] 2. Inoculation and fermentation

[0116] Two 500L fermenters were selected as Example 4 and Control Group 3. 100L of rice wine with an alcohol content of 9-10% vol was added to each. Acetobacter pasteurella, purchased from CGMCC (strain preservation number CGMCC1.2269), was inoculated at a 10% inoculum. The temperature was 30℃, the aeration rate was adjusted to 0.30 vvm, and the stirring speed was 200 r / min for acetic acid fermentation. When the alcohol content dropped below 5% vol, 3g of activated Bacillus licheniformis was added to the Example 4 fermenter, while no activation was added to the Control Group 3 fermenter. Acetic acid fermentation continued. The activation method was as follows: 1g of the inoculum was dissolved in 10mL of sterile water and activated at 37℃ for 30min before use.

[0117] 3. Detection of total acid, total ester and lactic acid content

[0118] After fermentation, the total acid, total ester, and lactic acid contents in the brewed rice vinegar were tested, as shown in Table 11. Total acid was calculated as acetic acid and determined by acid-base titration. Total ester was calculated as ethyl acetate, and its content was determined according to the method in GB / T 19777-2013. Lactic acid was determined by high-performance liquid chromatography, referring to the method in Appendix B of GB / T18623-2011 Geographical Indication Product Zhenjiang Vinegar.

[0119] Table 11. Total acid, total ester, and lactic acid content of Bacillus licheniformis HSCY 3018 in rice vinegar brewing.

[0120] Example 4 9.83±0.07 2.56±0.03 0.27±0.01 Control group 3 9.20±0.05 0.35±0.01 0.19±0.01

[0121] 4. Amino acid content

[0122] The content of 17 amino acids in the finished vinegar was analyzed by HPLC, and the results are shown in Table 12 below. At the end of fermentation, the total amino acid content of Example 3 was 736.47 mg / 100 mL, which was 46.95% higher than that of Control Group 3 (501.17 mg / 100 mL).

[0123] Table 12 Amino acid content of Bacillus licheniformis HSCY3018 in rice vinegar brewing

[0124]

[0125] 8. Sensory index analysis of finished vinegar

[0126] Thirty experienced sensory evaluators were selected to conduct sensory evaluation analysis on the products of Example 4 and Control Group 3. The results are shown in Table 13. A three-point test revealed that 26 out of the 30 evaluators indicated a significant difference between the products of Example 4 and Control Group 3. Descriptive analysis showed that the product of Example 4 had a brighter color, clearer appearance, richer ester aroma, milder taste, and lower irritation. A comprehensive preference-based scoring system showed that the product of Example 4 scored significantly higher than Control Group 3 in color, appearance, aroma, and taste, indicating that the addition of Bacillus licheniformis during the acetic acid fermentation stage significantly improved product quality.

[0127] Table 13 Sensory index analysis of finished vinegar produced by Bacillus licheniformis HSCY 3018 in rice vinegar brewing

[0128] Example 4 9.6 8.9 9.5 9.4 Control group 3 7.7 7.3 6.8 6.5

[0129] Control group 4:

[0130] Based on Example 2, the difference is that the Bacillus licheniformis used was commercially available, the product source was CICC, and the strain preservation number was CICC 10037. The following properties were tested on the finished vinegar brewed using Bacillus licheniformis, as shown in Tables 14 to 18.

[0131] Table 14. Total acid, reducing sugar, and total ester content in the finished vinegar of control group 4

[0132]

[0133] Table 15 Organic acid content of Bacillus licheniformis in control group 4 during Zhenjiang vinegar brewing

[0134]

[0135] Table 16 Starch utilization rate of Bacillus licheniformis in Zhenjiang vinegar brewing in control group 4

[0136]

[0137] Table 17. Amino acid content of Bacillus licheniformis in control group 4 during Zhenjiang vinegar brewing.

[0138]

[0139] Table 18 Sensory index analysis of finished vinegar from control group 4 in Zhenjiang vinegar brewing using Bacillus licheniformis.

[0140] Control group 4 8.2 7.9 6.9 6.4

[0141] As can be seen from the data in Tables 15-18, the finished vinegar produced using commercially available Bacillus licheniformis through the Zhenjiang vinegar brewing process has significantly lower starch utilization rate, reducing sugar, total esters, organic acid, amino acid content, and sensory indicators compared to Example 2. This indicates that the application of Bacillus licheniformis of the present invention in the field of vinegar brewing can significantly improve the utilization rate of raw materials, and can also produce abundant organic acids, amino acids, and reducing sugars to improve the mellowness of the vinegar's flavor. At the same time, it can also increase the amount of esters with good aroma. The resulting fermented vinegar has high nutritional value, a mellow taste, a pleasant sweet and sour flavor, and a rich ester aroma. This solves the problem that currently available pure brewed vinegar has a relatively pungent taste and a thin aroma, which can meet consumers' health and high-quality needs and has stronger market adaptability.

[0142] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the scope of the invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0143] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This description is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A strain of Bacillus licheniformis, characterized in that, The Bacillus licheniformis was named Bacillus licheniformis HSCY 3018 is deposited at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 20221831 and deposit date of November 28, 2022.

2. A microbial inoculant, characterized in that, The microbial agent comprises Bacillus licheniformis as described in claim 1.

3. The application of Bacillus licheniformis as described in claim 1 and the microbial agent as described in claim 2 in vinegar brewing.

4. The application according to claim 3, characterized in that, The vinegar mentioned is balsamic vinegar, aged vinegar, or rice vinegar.

5. A method for brewing vinegar, characterized in that, Includes the following steps: (1) Prepare seed liquid of strain or prepare microbial agent using Bacillus licheniformis as described in claim 1; (2) Ferment the strain seed liquid or microbial agent obtained in step (1) by inoculation.

6. The brewing method according to claim 5, characterized in that, The vinegar mentioned is balsamic vinegar, aged vinegar, or rice vinegar.

7. The method according to claim 5, characterized in that, The preparation of the microbial agent includes mixing a strain of Bacillus licheniformis with a sterile protectant, subjecting it to heat shock, and then freeze-drying it under vacuum.

8. The method according to claim 7, characterized in that, The heat shock was performed by heat shocking the incubator at 40°C for 10 seconds.

9. The method according to claim 7, characterized in that, The preparation method of the sterile protectant includes: 1) Preparation of component A: 10g of skim milk powder, 100mL of distilled water, sterilized at 121℃ for 20min; 2) Preparation of component B: 0.5g of glycerol, 3g of lactose, 4g of mannitol, 5g of trehalose, 100mL of distilled water, sterilized at 121℃ for 20min; 3) After sterilizing components A and B respectively, they are cooled to room temperature and then mixed.

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