Bacillus velezensis TYF-QPY-E98 for producing saccharifying enzyme, microbial inoculum and application of microbial inoculum

Abstract

The invention provides bacillus velezensis TYF-QPY-E98 for producing saccharifying enzyme, a bacterial agent and application of the bacterial agent, and belongs to the technical field of microorganisms. The bacillus velezensis TYF-QPY-E98 provided by the invention is classified and named as Bacillus velezensis, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.30424, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation date is April 24, 2024. The saccharifying enzyme activity of the bacillus velezensis TYF-QPY-E98 provided by the invention can reach 2181 U / g, the saccharifying power can reach 713.12 mg / g.h, the liquefying power can reach 1.62, and the prepared enhanced yeast for making hard liquor is stronger in flavor.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Bacillus Velezii TYF-QPY-E98 producing saccharifying enzyme, a bacterial agent and application thereof. Background Art

[0002] Glucoamylase, also known as glucoamylase (EC3.2.1-3, abbreviated as GA), is an exoamylase that can hydrolyze α-1,4 glycosidic bonds from the non-reducing ends of starch or oligosaccharides in sequence. It can also hydrolyze α-1,3 bonds and α-1,6 bonds, and hydrolyze starch into glucose units. Therefore, it is widely used in industries such as alcohol, liquor, antibiotics, and organic acids. It is the enzyme product with the largest output in my country.

[0003] Daqu, a fermentation agent for edible vinegar, is made by the traditional natural inoculation method. It contains a large number of microorganisms and the enzymes they secrete. During the vinegar brewing process, the enzymes secreted by the microorganisms in Daqu are involved in starch liquefaction, starch saccharification, carbohydrate fermentation and the formation of flavor substances in the vinegar brewing process. Therefore, the quality of Daqu has a huge impact on the vinegar yield and quality.

[0004] In the vinegar brewing process, Daqu needs to provide microorganisms to transform the raw materials. The more important step is the saccharification of starch. After saccharification, starch will be converted into glucose to provide raw materials for subsequent fermentation. In the process of starch saccharification, saccharifying enzymes are the most important enzymes. In this process, the saccharifying enzymes secreted by the microorganisms in Daqu saccharify starch into glucose, and also provide nutrients for these microorganisms, which is beneficial to the growth and metabolism of microorganisms. Therefore, the activity of saccharifying enzymes is one of the important indicators for evaluating the quality of Daqu. The higher the enzyme activity of saccharifying enzymes, the more conducive it is to the utilization of raw materials and the improvement of vinegar yield. Therefore, screening saccharifying enzyme strains is of great significance for improving the activity of Daqu saccharifying enzymes. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a saccharifying enzyme-producing Bacillus Velez subtilis TYF-QPY-E98, a bacterial agent and an application thereof. The saccharifying enzyme-producing Bacillus Velez subtilis TYF-QPY-E98 provided by the present invention has high saccharifying enzyme activity and liquefaction power, and the prepared enhanced Daququ has a stronger aroma.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A saccharifying enzyme-producing Bacillus velezensis TYF-QPY-E98, wherein the Bacillus velezensis TYF-QPY-E98 is classified and named Bacillus velezensis, and is deposited in the General Microbiology Center of China Microorganism Culture Collection Committee, with a deposit number of CGMCC NO.30424, a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit date of April 24, 2024.

[0008] In certain embodiments, the Bacillus velez TYF-QPY-E98 is isolated from vinegar mash fermented using a traditional Shanxi aged vinegar process.

[0009] In certain embodiments, the effective sequence length of 16S rDNA of the Bacillus velez TYF-QPY-E98 is 1486 bp, and the sequence is shown in SEQ ID NO.1.

[0010] In certain embodiments, the culture characteristics of the Bacillus velez TYF-QPY-E98 are: the surface of the colony is moist, and is an opaque milky white convex hill, and the edge of the colony is irregular, showing typical bacterial morphological characteristics.

[0011] The present invention also provides a bacterial agent, wherein the bacterial agent comprises the Bacillus Velezii TYF-QPY-E98 described in the above technical solution.

[0012] The present invention also provides an application of the Bacillus Velez TYF-QPY-E98 described in the above technical solution or the bacterial agent described in the above technical solution in vinegar brewing.

[0013] The present invention also provides an application of the Bacillus Velez TYF-QPY-E98 described in the above technical solution or the bacterial agent described in the above technical solution in edible vinegar Daqu.

[0014] The present invention also provides a method for strengthening vinegar Daqu, comprising the following steps:

[0015] S1. Soaking the crushed bran in water, and pressing the soaked bran into a rectangular curved block; the water contains a bacterial suspension of Bacillus Velez TYF-QPY-E98 described in the above technical solution or the bacterial agent described in the above technical solution;

[0016] S2. Place the koji blocks in a fermentation room for cultivation to obtain fortified vinegar koji.

[0017] In certain embodiments, the soaking in S1 is such that the moisture content of the pulverized bran is preferably 38%.

[0018] In certain embodiments, the v / w ratio of the bacterial suspension in S1 to water is preferably 1%, and the concentration of Bacillus velezensis TYF-QPY-E98 in the bacterial suspension is preferably 1.0×10 6 CFU / mL.

[0019] In certain embodiments, the culture conditions in S2 are preferably: 0d to 4d, 42°C, relative humidity >90%; 5d to 13d, 55°C to 60°C, relative humidity >90%; 14d to 24d, 45°C to 50°C, relative humidity <80%; 25d to 35d, room temperature, Daqu moisture content less than 14%.

[0020] In certain embodiments, the saccharifying enzyme activity of the enhanced Daqu can reach 2181 U / g, the saccharifying power can reach 713.12 mg / gh, and the liquefaction power can reach 1.62.

[0021] Beneficial technical effects: The present invention provides a strain of Bacillus velezensis TYF-QPY-E98 producing saccharifying enzyme, a bacterial agent and its application. The classification name of the Bacillus velezensis TYF-QPY-E98 is Bacillus velezensis, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, with a deposit number of CGMCC NO.30424, a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit date of April 24, 2024. The saccharifying enzyme activity of the Bacillus velezensis TYF-QPY-E98 provided by the present invention can reach 2181U / g, the saccharifying power can reach 713.12mg / gh, the liquefaction power can reach 1.62, and the prepared Daququ has a stronger aroma. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the transparent circle picture of TYF-QPY-E98;

[0023] Figure 2 is the morphological characterization diagram of TYF-QPY-E98;

[0024] Figure 3 It is the agarose gel electrophoresis diagram of the 16S rDNA gene amplification product of TYF-QPY-E98;

[0025] Figure 4 This is the phylogenetic tree of TYF-QPY-E98. DETAILED DESCRIPTION

[0026] In order to better understand the present invention, the content of the present invention is further explained in conjunction with the following examples, but the content of the present invention is not limited to the following examples. The materials, reagents, etc. used in the examples and test examples of the present invention, unless otherwise specified, can be obtained from commercial channels; the methods used in the examples and test examples of the present invention, unless otherwise specified, are conventional methods.

[0027] Instruments and Materials

[0028] (1) Culture medium:

[0029] Slant culture medium (g / L): 10% tryptone, 10% sodium chloride, 5% yeast extract, 20% agar powder, pH 7.0; sterilize at 121°C, high pressure steam for 20 min.

[0030] LB medium (g / L): 10% tryptone, 10% sodium chloride, 5% yeast extract, pH 7.0, 121°C, high-pressure steam sterilization for 20 min.

[0031] Beef extract peptone medium (g / L): beef extract 3, peptone 5, sodium chloride 10, agar powder 20, pH 7.0-7.2, 121℃, high pressure steam sterilization for 20min.

[0032] Glucoamylase production screening medium (g / L): peptone 10, beef powder 5, NaCl 5, soluble starch 10, agar powder 20, pH = 7.0, 121 ° C, high-pressure steam sterilization for 20 min.

[0033] Liquid fermentation medium for saccharifying enzyme producing strain (g / L): peptone 20, soluble starch 20, disodium hydrogen phosphate 5, magnesium sulfate 0.1, sodium chloride 0.1, pH 7.0, 121°C, high pressure steam sterilization for 20 min.

[0034] (2) Main experimental instruments:

[0035] Constant temperature biochemical incubator, high-speed refrigerated centrifuge, constant temperature shaker, clean workbench, vertical pressure steam sterilizer, full wavelength microplate reader, PCR instrument, electrophoresis instrument, etc.

[0036] Example 1 Screening and identification of saccharifying enzyme producing strains

[0037] (1) Take 5g of vinegar mash (from Shanxi Laifu Laochen Vinegar Factory) and put it in 45mL of sterile water. After shaking thoroughly, let it stand at room temperature. Take the supernatant and dilute it tenfold. Then spread it on the saccharifying enzyme screening medium to isolate the saccharifying enzyme producing strain. After culturing for a certain period of time, use the spot grafting method to inoculate a single colony on the saccharifying enzyme screening medium with a sterilized gun tip. After inverted cultivation at 40℃ for 24h, add iodine solution, measure the diameter of the transparent circle and the diameter of the colony respectively, and calculate the ratio of the two. Screen the colonies with a ratio greater than or equal to 2.7 and inoculate them on the slant of the test tube (slant medium). Do 3 replicates for each strain.

[0038] (2) Rescreening of saccharifying enzyme-producing strains

[0039] The strains obtained from the initial screening were inoculated on the test tube slant and activated and cultured at 40°C for 24 hours: 1 ring of the activated strains was picked and inoculated into the liquid fermentation medium of the saccharifying enzyme producing strains, and cultured with shaking at 150r / min for 24 hours; the activated seed liquid was inoculated into the liquid fermentation medium at an inoculum of 3% (V / V), and cultured with shaking at 40°C and 150r / min for 48 hours. The fermentation liquid was centrifuged at 8000r / min for 10 minutes, and the activity of saccharifying enzyme in the fermentation supernatant was determined by the DNS method. The strain with the highest saccharifying enzyme activity was isolated as the target strain. Each strain was repeated 3 times in parallel.

[0040] After measurement, the D / d value of strain TYF-QPY-E98 was the largest, which was 4.17 ( Figure 1 ), and the saccharifying enzyme activity was also the highest, which was 2181U / g, and strain TYF-QPY-E98 was selected as the target strain.

[0041] (3) Identification of strain TYF-QPY-E98

[0042] Morphological identification: Streak strain TYF-QPY-E98 on beef extract peptone plates and culture at 37°C for 1 day to observe the morphological characteristics of the colonies. Figure 2 The surface of the bacterial colony is moist, opaque milky white convex hills, and the edge of the colony is irregular, showing typical bacterial morphological characteristics.

[0043] Molecular identification: The purified strain was inoculated into LB medium, cultured at 120 r / min and 30°C for more than 12 h, the bacterial solution was used as a template, and the universal primer pair 27F / 1492R (upstream primer was 27F (SEQ ID NO.2): 5'-AGAGTTTGATCCTGGCTCAG-3', downstream primer was 1492R (SEQ ID NO.3): 5'-TACGGCTACCTTGTACGACTT-3') was selected as the primer. The reaction conditions for PCR amplification were: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 90 s, 30 cycles; extension at 72°C for 5 min to 10 min, and storage at 4°C for 15 min.

[0044] The PCR amplification products were subjected to agarose gel electrophoresis. Figure 3 As shown by Figure 3 It can be seen that the amplified product band at about 1500 bp is brighter and there are no other miscellaneous bands.

[0045] The 16S rDNA product obtained by PCR amplification was entrusted to Sangon Biotech Co., Ltd. for first generation sequencing. The sequencing result is shown in SEQ ID NO.1. The obtained sequencing results were compared with the NCBI database, and the strains with the closest species relationship to the tested strains were selected to construct a phylogenetic tree ( Figure 4 ). The results showed that strain TYF-QPY-E98 and Bacillus velezensis were clustered in the same branch. Combined with the morphological characteristics, strain TYF-QPY-E98 was identified as Bacillus velezensis. Strain TYF-QPY-E98 was deposited in the General Microbiology Center of China National Microbiological Culture Collection on April 24, 2024. The deposit location is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30424.

[0046] Sequence SEQ ID NO.1:

[0047]

[0048] Example 2 Strengthening Daqu Preparation

[0049] The crushed bran was mixed with water to a moisture content of about 38%, wherein the moisture contained 1% (v / w) of bacterial suspension (strain TYF-QPY-E98 1.0×10 6 CFU / mL), the soaked bran was tightly pressed into a rectangular block, and a blank control group was taken without inoculation of TYF-QPY-E98; then, the blocks were stacked layer by layer in a fermentation room (constant temperature and humidity incubator) for culture, and the temperature and humidity during the culture process were 0d-4d, 42°C, relative humidity>90%; 5d-13d, 55°C-60°C, relative humidity>90%; 14d-24d, 45°C-50°C, relative humidity<80%; 25d-35d, room temperature, and the moisture content of the Daqu was less than 14%, thus obtaining the fortified Daqu.

[0050] The above-mentioned fortified Daqu was used as the experimental group, and the Daqu obtained without inoculation of TYF-QPY-E98 was used as the control group. The physical and chemical indicators (saccharification power, liquefaction power, fermentation power, esterification power) of the two Daqu were tested, and the quality of Daqu was analyzed by sensory evaluation. The methods of physical and chemical indicators and sensory evaluation are as follows:

[0051] Saccharification and liquefaction power: refer to the method described in QB / T 4257-2011. They respectively indicate the content of starch liquefied and glucose generated per hour by 1g of absolute dry koji in acetic acid-sodium acetate buffer solution (35℃, pH4.6). The former is titrated by the Fehling method, and the latter is based on the characteristic reaction of starch and iodine.

[0052] Fermentation capacity: refer to the same method as above. At 30℃, microorganisms in 0.5g vinegar koji use sugar to ferment to produce CO2 and alcohol. The quality of CO2 produced within 72 hours is used to measure the strength of fermentation capacity.

[0053] Esterification power: refer to the same method as above. It refers to the ability of 25g of absolute dry koji to catalyze a certain amount of ethanol and hexanoic acid into ethyl hexanoate within 7 days at 35℃.

[0054] Sensory evaluation: Observe and record the appearance, color, and smell of the control group and the test group. Good quality bran koji should have uniform mycelium inside and outside, no agglomeration, no strange smell, and have a good aroma.

[0055] Table 1 Physical and chemical indicators of enhanced Daqu

[0056]

[0057] Table 2 Sensory evaluation table of enhanced Daqu

[0058]

[0059] The physical and chemical indicators of Daqu are shown in Table 1, and the sensory evaluation is shown in Table 2. It can be seen from Tables 1 and 2 that compared with the blank control group without inoculation of strains, the saccharification power and liquefaction power of the enhanced Daqu in the experimental group are higher than those in the control group. When the inoculation ratio is 1%, the saccharification power of Daqu is 713.12 mg / gh, which is 259.83 mg / gh higher than that in the control group; the liquefaction power is increased from 0.79 to 1.62. And the flavor of the enhanced Daqu in the experimental group is stronger.

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A saccharifying enzyme-producing Bacillus Velezii TYF-QPY-E98, characterized in that: The classification name of the Bacillus velezensis TYF-QPY-E98 is Bacillus velezensis, and it is preserved in the General Microbiology Center of the China Culture Collection Administration, with the preservation number CGMCCNO.30424. The preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation date is April 24, 2024.

2. The Bacillus Velezii TYF-QPY-E98 according to claim 1, characterized in that The Bacillus Velez TYF-QPY-E98 is isolated from vinegar mash fermented by using the traditional Shanxi aged vinegar process.

3. A bacterial agent, characterized in that: The bacterial agent contains the Bacillus Velezii TYF-QPY-E98 according to claim 1 or 2.

4. Use of the Bacillus Velez TYF-QPY-E98 described in claim 1 or 2 or the bacterial agent described in claim 3 in vinegar brewing.

5. Use of the Bacillus Velezii TYF-QPY-E98 described in claim 1 or 2 or the bacterial agent described in claim 3 in fortifying edible vinegar Daqu.

6. A method for strengthening vinegar Daqu, characterized in that: The following steps are involved: S1. Soaking the crushed bran in water and pressing the soaked bran into a rectangular curved block; the water contains a bacterial suspension of Bacillus Velez TYF-QPY-E98 according to claim 1 or 2 or the bacterial agent according to claim 3; S2. Place the koji blocks in a fermentation room for cultivation to obtain fortified vinegar koji.

7. The method according to claim 6, characterized in that The soaking in S1 is performed so that the moisture content of the crushed bran is 38%.

8. The method according to claim 6, characterized in that The v / w ratio of the bacterial suspension in S1 to water is 1%, and the concentration of Bacillus Velezii TYF-QPY-E98 in the bacterial suspension is 1.0×10 6 CFU / mL.

9. The method according to claim 6, characterized in that The culture conditions in S2 are: 0d-4d, 42°C, relative humidity>90%; 5d-13d, 55°C-60°C, relative humidity>90%; 14d-24d, 45°C-50°C, relative humidity<80%; 25d-35d, room temperature, Daqu moisture content less than 14%.

10. The method for strengthening vinegar Daqu according to any one of claims 6 to 9, characterized in that: The saccharifying enzyme activity of the enhanced Daqu can reach 2181U / g, the saccharifying power can reach 713.12mg / gh, and the liquefaction power can reach 1.62.

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