High-yield protease bacillus and application thereof in rice wine koji

Abstract

The bacillus is classified and named as bacillus velezensis SAU-MC-3X, the bacillus velezensis SAU-MC-3X is preserved in the China General Microbiological Culture Collection Center (CGMCC), the address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC No.34098, and the preservation time is April 3, 2025. The invention also discloses a screening method of the bacillus, a strain fermentation characteristic and a protease production characteristic in application of rice wine koji. The bacillus velezensis obtained by the invention has huge application potential in rice wine koji.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and specifically relates to the screening of a Bacillus strain and the fermentation characteristics of the strain, as well as the study of its protease production characteristics in nutrient broth medium and fermented rice wine koji. Background Art

[0002] Traditional Chinese rice wine is an alcoholic beverage made from glutinous rice, fermented with koji (dice yeast). Also known as sweet wine, sweet yeast wine, milk wine, or fermented glutinous rice, rice wine is beloved for its simple brewing process and sweet, mellow flavor. The proteases in rice wine koji, typically produced by bacteria such as Bacillus, break down proteins into amino acids and alcohols, improving raw material utilization and providing the material basis for ethanol fermentation and the production of aroma compounds during the liquor production process. This significantly impacts the yield and aroma of the liquor. However, the complex and uncontrollable microbial composition of traditional koji results in unstable protease activity and low fermentation efficiency, leading to significant fluctuations in rice wine quality and long production cycles.

[0003] Bacteria are important bacterial groups that produce proteases, and common bacteria include Bacillus, lactic acid bacteria, acetic acid bacteria, etc. Bacillus produces spores, has special resistance, and can withstand adverse conditions such as high temperature and high acid. In order to improve the directional regulation ability of proteases in koji, researchers have tried to optimize the microbial composition of koji through functional strain screening. For example, Zhu Guoxing et al. screened 6 strains of Bacillus velez, such as Bacillus velez, with high protease production from Xuanjiu high-temperature koji. Among them, Bacillus velez had the highest enzyme production. After it was made into bran koji, the protease activity reached 256.72 U / g, which greatly improved the quality of sesame-flavored liquor. In the present invention, Bacillus velez ( Bacillus velezensis ) SAU-MC-3X was used to make koji, which was then used in rice wine fermentation. The protease activity was as high as 669.08 U / mL, which proved that inoculating the protease-producing strain SAU-MC-3X could improve the protease activity of koji. Summary of the Invention

[0004] The present invention aims to screen and provide a kind of Bacillus velezine with the ability to produce protease ( Bacillus velezensi s) SAU-MC-3X, and used this Bacillus to make koji to ferment rice wine, providing a microbial resource for protease production in the rice wine brewing process.

[0005] The strain provided by the present invention is Bacillus velezinoffii ( Bacillus velezensis) SAU-MC-3X, which was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 34098 and the deposit date April 3, 2025.

[0006] The Bacillus velezensis SAU-MC-3X was isolated from rice wine koji and, after primary screening, secondary screening, purification, and identification, was found to have good protease production. Its storage conditions are as follows: a single colony was picked from a well-growing plate and transferred into nutrient broth. The culture was incubated at 37°C for 48 hours. 600 μL of the culture was then transferred into a storage tube containing 600 μL of 60% glycerol and stored in a -20°C freezer.

[0007] The PCA medium formula is 5 g tryptone, 2.5 g yeast extract powder, 1 g glucose, 1000 mL distilled water, pH 7.2±0.2. If a solid medium is required, add 2% agar.

[0008] The formula of the protease production screening culture medium is 3 g beef extract, 10 g soy peptone, 5 g sodium chloride, 15 g skim milk powder, 1000 mL distilled water, natural pH, and if a solid culture medium is required, 2% agar can be added; skim milk should be sterilized separately at 115°C for 15 min and then mixed with the sterilized beef extract peptone culture medium.

[0009] The nutrient broth medium formula is 3 g beef extract powder, 10 g peptone, 5 g sodium chloride, 1000 mL distilled water, pH 7.2±0.2. If a solid medium is required, add 2% agar.

[0010] The culture characteristics of the Bacillus Velez SAU-MC-3X are as follows: after culturing in nutrient broth solid medium at 37°C for 48 hours, the colonies are round, wrinkled, and easily lifted, with a membranous texture and a pale yellow color. Under an oil immersion lens (100×10), the Bacillus Velez SAU-MC-3X bacteria appear rod-shaped. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 (a) is the colony morphology of Bacillus velez SAU-MC-3X, and (b) is the cell morphology of Bacillus velez SAU-MC-3X under a 100×10 microscope.

[0012] Figure 2 This is the phylogenetic tree of Bacillus velez SAU-MC-3X.

[0013] Figure 3 This is the growth curve of Bacillus velez SAU-MC-3X.

[0014] Figure 4 (A) is a graph showing the growth of Bacillus Velez SAU-MC-3X at different pH values, (B) is a graph showing the growth of Bacillus Velez SAU-MC-3X at different glucose contents, (C) is a graph showing the growth of Bacillus Velez SAU-MC-3X at different temperatures, and (D) is a graph showing the growth of Bacillus Velez SAU-MC-3X at different alcohol concentrations. DETAILED DESCRIPTION

[0015] Unless otherwise specified, the experimental methods described in the following examples are all conventional methods; the reagents and biological materials described are all commercially available unless otherwise specified.

[0016] In the following examples, the percentages are by mass unless otherwise specified.

[0017] The following examples further illustrate the specific implementation of the present invention, but are not limited to these examples.

[0018] Case Study 1: Screening and Identification of Protease-Producing Strains

[0019] (1) Isolation and purification of protease-producing strains from koji: Grind and mix the koji sample, weigh 25 g of the sample and add it to 225 mL of sterile saline, and use the 10-fold gradient dilution method to prepare 10 -2 -10 -7 Select an appropriate dilution and apply 1 mL of the solution to PCA solid medium. Incubate in an inverted incubator at 37°C for 48 hours. Pick individual colonies based on their morphological characteristics and repeatedly streak them onto PCA plates until a pure strain free of contaminants is obtained. Transfer the purified strain to PCA slant medium and store at 4°C until ready for use.

[0020] (2) Screening of protease-producing strains: Single colonies after purification and culture were selected and inoculated onto a protease-producing screening medium. The culture was inverted at 37°C for 48 h. Single colonies that produced a transparent hydrolysis zone on the casein plate were selected. The ratio of the hydrolysis zone to the colony diameter was used as the screening standard. Among them, SAU-MC-3X had the largest ratio of hydrolysis zone diameter to colony diameter, which was 5.00.

[0021] (3) Rescreening of protease-producing strains: The strains initially screened were inoculated into nutrient broth and cultured at 37°C for 48 h. After 48 h of fermentation, the fermentation broth was centrifuged at 8000 r / min for 10 min, and the supernatant was aspirated to measure the protease activity, thereby screening the protease-producing strains with the highest enzyme activity. Among them, SAU-MC-3X had the highest protease activity, at 27.55 U / mL, which was higher than the high-yielding protease strain screened by Zhao Hua et al., further demonstrating that this strain has a strong protease production capacity.

[0022] (4) Strain identification

[0023] Morphological identification: After activating with PCA liquid medium for 48 hours, use a sterile inoculation loop to pick up a loop of bacterial liquid and streak it on a PCA plate. After incubating at 37℃ for 48 hours, record the characteristics of the colony according to size, color, texture, etc.; wash the colony with sterile saline and observe the single cell morphology under a 100x microscope. The colony and cell morphology of the strain SAU-MC-3X are as follows: Figure 1 shown.

[0024] Physiological and biochemical identification: Refer to the Manual of Common Bacterial Systematic Identification. Strain SAU-MC-3X can ferment glucose, sucrose, and fructose, produces catalase, and is positive in gelatin hydrolysis, starch hydrolysis, and VP tests. Combined with strain morphology and physiological and biochemical tests, SAU-MC-3X was preliminarily identified as a Bacillus species.

[0025] Molecular identification: The strain slant was taken and sent to Qingke Biotechnology Co., Ltd. for universal 27F / 1492R bacterial sequencing. The strain SAU-MC-3X was sequenced and the sequence obtained was as follows:

[0026] After splicing, the sequencing results were compared by BLAST on the NCBI website for homology, and the strain sequences were imported into MEGA6.0 to construct a phylogenetic tree, such as Figure 2 As shown, the results show that SAU-MC-3X and Bacillus velezensis Combined with morphological and physiological and biochemical identification, the strain SAU-MC-3X was identified as Bacillus velezinis ( Bacillus velezensis ). It was deposited in the General Microbiology Center of China Culture Collection of Microorganisms (CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No. 34098, and the deposit date is April 3, 2025.

[0027] Case study 2: Fermentation performance determination of strain SAU-MC-3X

[0028] (1) Determination of growth curve of protease-producing strains: The activated SAU-MC-3X strain seed liquid was inoculated into nutrient broth liquid culture medium at a 2% inoculum (v / v), cultured at 37°C for 48 h, and the OD was measured every 2 h. 600nm .like Figure 3 As shown in the figure, the growth is slow in the early stage of culture (0-6 h), and enters the logarithmic phase at 4-12 h with the development of culture time. 600 It showed exponential growth, with a small increase in 12-16 hours, and the subsequent trend basically tended to be flat and in a stable period.

[0029] (2) pH tolerance test: The activated SAU-MC-3X strain seed liquid was inoculated at a 2% inoculum (v / v) into nutrient broth liquid culture medium with pH values of 3.0, 3.5, 4.0, 4.5, and 5.0, respectively. The OD values were measured after culturing at 37°C for 48 h. 600nm , draw the pH growth tolerance curve of Bacillus velez. Figure 4 As shown in (A), the OD value showed an upward trend with the increase of pH and reached the maximum at pH 5.0.

[0030] (3) Glucose tolerance test: The activated SAU-MC-3X strain seed liquid was inoculated with 2% inoculum (v / v) into nutrient broth liquid culture medium with glucose content of 16%, 20%, 24%, 28%, and 32%, respectively. The OD values were measured after culturing at 37°C for 48 h. 600nm , draw the sugar tolerance curve of Bacillus Velez. Figure 4As shown in (B), when the sugar content is less than 20%, the cell growth increases with the increase of sugar content, but when it is greater than 20%, the cell growth decreases with the increase of sugar content, indicating that high sugar content will inhibit the growth of the strain.

[0031] (4) Temperature tolerance test: The purified Bacillus velezensis SAU-MC-3X strain was activated and inoculated into nutrient broth liquid culture medium at a 2% inoculum (v / v). After constant temperature cultivation at 24°C, 28°C, 32°C, 36°C, and 40°C for 48 h, the uninoculated nutrient broth liquid culture medium was used as the blank zero parameter to measure the OD 600nm , draw the temperature growth tolerance curve of Bacillus velez. Figure 4 As shown in (C), the OD value of Bacillus velezensis SAU-MC-3X strain first increased and then decreased, and the OD value was the largest at 36°C, indicating that the strain grew best at 36°C.

[0032] (5) Alcohol tolerance test: The activated SAU-MC-3X strain seed liquid was inoculated with 2% inoculum (v / v) into nutrient broth liquid culture medium with alcohol content (v / v) of 3%, 6%, 9%, 12%, and 15%, respectively. The OD was measured after culturing at 37°C for 48 h. 600nm , draw the alcohol tolerance curve of Bacillus Velez. Figure 4 As shown in (D), the absorbance of SAU-MC-3X was the highest when the ethanol addition amount was 3%. Its OD value showed a downward trend with the increase of ethanol addition amount, and decreased significantly when the ethanol addition amount was 12%.

[0033] Case Study 3: Safety Testing of Strain SAU-MC-3X

[0034] (1) Hemolytic activity of the strain

[0035] A loopful of seed liquid was streaked onto Columbia wool blood agar plates and incubated at 37°C for 48 hours to observe for the formation of a hemolytic ring. The results showed that SAU-MC-3X did not form a hemolytic ring on the blood agar plates, thus being considered γ-hemolytic, i.e., non-hemolytic.

[0036] (2) Antibiotic sensitivity of strains

[0037] The antibiotic susceptibility test was performed using the disc diffusion method. 100 μL of the selected Bacillus bacteria, activated and cultured for 48 hours, was evenly spread on a solid nutrient broth plate. The plate was allowed to stand for 10 minutes until the solution was absorbed. Then, susceptibility test strips were placed at equal distances from the plate and incubated at 37°C for 48 hours. The diameter of the transparent zone was measured. The antibiotic susceptibility of the SAU-MC-3X strain is shown in Table 1. As shown, SAU-MC-3X was sensitive to eight antibiotics, including erythromycin (E) and ceftriaxone (CTR).

[0038] Table 1 Antibiotic susceptibility test of strain SAU-MC-3X

[0039] Table 1 Antibiotic sensitivity test of strain SAU-MC-3X

[0040] Case Study 4: Determination of Protease Activity in Rice Wine Koji Produced by Strain SAU-MC-3X

[0041] Prepare bacterial suspension: pick bacteria SAU-MC-3X and inoculate into nutrient broth liquid culture medium, culture at 37℃ for 48 h, and adjust the seed liquid to 1×10 7 CFU / mL, centrifuge at 8000 r / min for 10 min, discard the supernatant, resuspend the bacteria in an equal amount of sterile saline and mix well.

[0042] To prepare the koji: Prepare 500 g of glutinous rice flour, 60 g of Polygonum hydropiper, 20 g of Poria cocos, and 20 g of Licorice root. Grind the Polygonum hydropiper, Poria cocos, and Licorice root into a powder in a blender. Pass the powder through an 80-mesh sieve and mix thoroughly with the glutinous rice flour. Add 500 g of water in small batches. Add an appropriate amount of bacterial suspension, adding 0.8% mold, 0.03% yeast, and 0.06% bacteria, along with 0.6% commercial koji. Knead the mixture into a koji ball approximately 2 cm in diameter and cover with plastic wrap. A layer of white mycelium will develop. Dense white hairs, approximately 3-4 mm long and devoid of color, will form on the koji, indicating normal growth of the sweet wine koji mold. Finally, dry the koji at 45°C and store it in a vacuum bag at 4°C. A koji without the SAU-MC-3X bacterial suspension was also used as a control.

[0043] Rice wine koji protease activity assay: The protease activity of rice wine koji was determined according to Shang Hailin's method. The results showed that the protease activity of the control group was 508.50 U / mL, while that of the group containing the protease-producing strain SAU-MC-3X was significantly increased, reaching 669.08 U / mL. This demonstrates that inoculation with the protease-producing strain SAU-MC-3X increases the protease activity of rice wine koji.

Claims

1. A strain of Bacillus velezinoffii ( Bacillus velezensis ) SAU-MC-3X, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 34098 and the deposit date April 3, 2025.

2. according to claim 1 described Bacillus Velez( Bacillus velezensis ) SAU-MC-3X, characterized by: The colonies are round, with wrinkles on the surface, a membranous texture, easy to pick up, and light yellow in color, providing a good source of bacteria for the production of protease in rice wine.

3. A method for preparing distiller's yeast, comprising adding the Bacillus Velez subtilis according to claim 1 ( Bacillus velezensis ) The rice wine koji produced by SAU-MC-3X is characterized by: It improves the color and aroma of rice wine koji, effectively improves the quality of rice wine koji, and plays a positive role in improving the rice wine fermentation process.

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