CN101434927A - A strain of bacillus subtilis for producing lab ferment - Google Patents

Abstract

The invention relates to a strain of Bacillus subtilis producing rennet. The bacterial strain is isolated from cheese and has been preserved in China Center for Type Culture Collection (CCTCC) with the preservation number M208023 and the name is Bacillus subtilis QL-2 (Bacillus subtilis QL-2). Its fermented liquid is rich in rennet and has good curdling effect. It has the potential to be used in the industrial production of chymosin.

Description

A strain of Bacillus subtilis producing chymosin

technical field

The present invention relates to a rennet-producing Bacillus subtilis QL-2 (Bacillus subtilis QL-2), which is isolated from cheese and has been preserved in the China Center for Type Culture Collection (CCTCC), with a preservation number of M208023. The fermented liquid is rich in rennet and has good curdling effect, and has the potential to be used in the industrial production of rennet.

Background technique

Cheese is rich in a variety of nutrients, easy to digest and absorb, and not easy to cause fat, so it is regarded as an ideal food by nutritionists, known as "milk gold", ^[1] . Rennet is a key enzyme preparation for coagulating milk in cheese production ^[2] , and it is an acidic protease. The curdling process can be divided into two steps: the first step is to specifically hydrolyze the peptide bond between 105-106 phenylalanine and methionine in the κ-casein polypeptide chain in milk to form a stable paraκ-casein and hydrophilic glycomacropeptide; the second step is when the total κ-casein is hydrolyzed about 85%, in the presence of Ca ⁺ to form coagulation through the chemical bonds formed between casein micelles Block or coagulated milk, so that the changed rennet and casein micelles precipitate ^[3,4] .

Rennet has a wide range of sources and can be obtained from animals, plants and microorganisms. The traditional rennet is derived from the rennet extracted from the calf’s abomasum (the fourth stomach) ^[5] , but with the continuous development of the world’s cheese industry, it is obvious that a large number of calves are slaughtered every year to obtain rennet. It is extremely incompatible with the development of modern industry; plant-based chymosin can be extracted from papaya, pineapple, fig, safflower and other plants ^[6] , but its proteolytic ability is strong, and it is affected by time, region, and long growth cycle. Restricted by other conditions, it is difficult to develop; more than 40 kinds of microorganisms have been found to produce chymosin with a certain activity, and these microorganisms are mainly actinomycetes, bacteria and fungi ^[7] . Since Arima ^[8] discovered in 1964 that Mucor pusillus can produce high-activity chymosin, many scientists have carried out a lot of research on it. Qian Shijun et al ^{. [9]} from the Institute of Microbiology, Chinese Academy of Sciences screened a strain of Mucor micromyces from 19 strains of Mucor, and completed the strain cultivation, enzyme extraction and purification. The ratio of hydrolytic activity is greatly improved, and there is no cellulose activity at all. The chymotrypsin produced by it was purified and its enzymatic properties were studied. Guo Guangyuan et al ^[10] screened 2,502 strains of actinomycetes, fungi, and bacteria isolated from actinomycetes, fungi, and bacteria during the investigation of actinomycetes areas and resources in different regions of Yunnan, and found that actinomycetes Streptomyces (Streptomyces), Micromonas (Micromonospora), Madura Actinomyces (Actinomadura), etc., 9.8% of fungi and 8.8% of bacteria have certain enzyme-producing activities. At present, the microbial rennet in the world is mainly produced by Mucor miehei ^[11] .

A strain of Bacillus subtilis QL-2 involved in the present invention, its fermented liquid is rich in rennet, and the curdling activity is high, and the growth cycle is short, limited by climate, region and time, it is used to produce curd Compared with the previous chymosin-producing microorganisms, the enzyme has lower cost, convenient enzyme extraction and high economic benefits. Bacillus subtilis is a common non-pathogenic bacteria, and the rennet produced by its fermentation is suitable for industrial cheese production.

references:

[1] Du Kun, Zhang Yaning. The nutritional value and research trends of cheese [J]. Chinese Food and Nutrition, 2005, 10: 45-46.

[2] Satyendra K Garg, Bhavdish N johri. Food Reviews International [J]. 1994.10(3): 313-355.

[3] Jing Legang. Rennet and its application in cheese production [J]. China Livestock and Food, 1997, 2: 78-79.

[4] Guo Benheng. Dairy Chemistry [M]. Beijing: China Light Industry Press, 2001.

[5] Liu Zhenmin, Liu Hui, etc. Screening of rennet strains in wine medicine and research on enzyme production conditions [J]. Food and Fermentation Industry, 2000, 27(5): 8-11.

[6] Zhang Hongmei, Liu Zhongbin. Research progress of chymosin [J]. Journal of Tongji University (Medical Edition), 2004, 25(3): 254-257.

[7] Zhou Junqing, Lin Qinlu, Zhao Mouming. Research progress of microbial rennet [J]. China Food Additives, 2004, 2:6-9.

[8] Arima, K, and Iwasaki, S. Milk Coagulating Enzyme "Microbial Rennet" and Method of Preparation Thereof. United Stated Paptent, 3, 151, 039.

[9] Qian Shijun, Zhang Chunqing, Jiao Qinghua, etc. Purification and properties of Mucor microbiome chymosin [J]. Microbiology Journal, 1989: 272-277.

[10] Guo Guangyuan, Jiang Chenglin, Ma Jun. Research on microbial chymosin I. Screening, fermentation, preparation and toxicity of strains [J]. Microbiology Bulletin, 1988, 15(5): 207-210.

[11] G A Tucker, L J Wood (English). Translated by Li Yanqun and Xiao Gongnian. Application of enzymes in food processing [M]. China Light Industry Press, 2001.

Contents of the invention

The rennet-producing bacterium involved in the present invention is Bacillus subtilis QL-2 (Bacillus subtilis QL-2), which is a bacterial strain isolated from cheese. It has been preserved in the China Center for Type Culture Collection (CCTCC) with the preservation number M208023.

The cultivation method of the Bacillus subtilis QL-2 producing rennet of the present invention is as follows:

Liquid culture: inoculate the strain in NA (3g beef extract, 5g peptone, 10g glucose, 1g yeast extract, 1000ml distilled water, pH7.0) liquid medium, shake at 28-30°C with a rotation speed of 120r/m Culture on bed for 28-32h;

Solid culture: Add 1.0-1.5% agar powder to NA (beef extract 3g, peptone 5g, glucose 10g, yeast extract 1g, distilled water 1000ml, pH7.0) liquid medium to make a plate, inoculate the strain, and inoculate at 28- Cultured in a 30°C incubator for 2 days.

The characteristics of the Bacillus subtilis QL-2 producing chymosin of the present invention are as follows:

(1). Morphological characteristics:

The bacteria are rod-shaped, with perinatal flagella; a few spore-forming, middle-growing, oval-shaped cysts are enlarged; Gram staining is positive.

(2).Cultivation characteristics:

The strain was cultured on NA medium at 30°C for 2 days. The colony was milky white, round, with neat edges, and the center was raised like a steamed bun. It was smooth and not easy to stir up. It did not produce soluble pigments and had peculiar smell.

(3). Biochemical characteristics of the strain:

Catalase positive, facultative aerobic growth, VP reaction positive, acid produced in VP solution with pH value less than 6.0, casein hydrolyzed, sucrose produced reducing sugar, fructan produced, starch and gelatin hydrolyzed, tyrosine not hydrolyzed.

(4). Physiological characteristics of the strain:

Glucose can be used to produce acid without gas, sucrose, maltose, inositol, mannitol, soluble starch, sodium acetate can be used, and galactose is not used. The optimum growth pH is between 8 and 9, it can grow in 10% sodium chloride, and the optimum growth temperature is 37°C.

(5). Genetic characteristics:

Amplification and sequencing primers:

27f: 5’-AGA GTT TGA TCC TGG CTC AG-3’

1492r: 5’-CGG TTA CCT TGT TAC GAC TT-3’

517f: 5'-CCA GCA GCC GCG GTA AT-3'

The total DNA of the QL-2 bacterial strain was extracted, and the 16S rDNA gene of the bacterial strain was amplified by PCR with primers 27f and 1492r, and sequenced with primers 27f, 1492r, and 517f. The 16S rDNA gene sequence of the bacterial strain was:

1 AAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGAGGACAGGTGAGTAACA

61 CGTGGGTAACCTGCCTGTAAGACTGGTAATAACTCCGGGAAACCGGGGCTAATACCGGAT

121 GGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGG

181 ACCCGCGGCGCATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCAACGATGCGTAGCC

241 GACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGG

301 CAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGA

361 TGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGG

421 GCGGTACCTTGACGGTACCTAACCAGAAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGG

481 TAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTT

541 TCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAA

601 CTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTG

661 GAGGAACACCAGTGGCCCCAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAG

721 CGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAA

781 GTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGG

841 AGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGC

901 ATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAAT

961 CCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAG

1021 CTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGC

1081 CAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGAT

1141 GACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAAC

1201 AAAGGGCAGCGAAACCGCGAGGTTAAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCG

1261 CAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGC

1321 GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACATTCTACTAAAAGTTTGTAAC

1381 ACCCCGAAGTTTTCCGGTTGCAAGG

(6). The fermentation broth of Bacillus subtilis QL-2 is rich in rennet and has good curdling effect. It has the potential to be used in the industrial production of chymosin.

Detailed ways

Example 1 Isolate Bacillus subtilis QL-2 from cheese with dilution plate method:

(1) Preparation of cheese diluent: Weigh 5g of cheese, quickly pour it into 45ml of sterile water with glass beads, shake it on a shaker for 20min, let it stand for clarification, and become a suspension of 10 ⁻¹ . Aseptically draw 0.5ml of its supernatant and add 4.5ml of sterile water to obtain a 10 ^-2 dilution. Repeat this and dilute with sterile water to 7 concentrations of 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 .

(2) Cultivation: Add sterilized casein hydrolysis plates cooled to 45-50°C for each dilution (take 5g of skim milk powder and add it to 50mL of distilled water or use 50mL of skim milk, otherwise known as 1.5g of agar, dissolve it in 50mL of distilled water. The two liquids are sterilized separately. When it is cooled to 45-50°C, mix the two liquids and pour them into a flat plate to serve), and mix them immediately to make a flat plate. Incubate at 30°C in an inverted incubator.

(3) Pick a single colony: After 2 to 3 days, after the colony grows on the plate, pick a single colony with a transparent circle around it and put it in 30ml sterilized NA (beef extract 3.0g, peptone 5.0g, glucose 10g, Yeast extract 1.0g, water 1000ml, pH 7.0) in liquid medium, shake culture at 30 ℃ constant temperature on the shaker.

(4) Purification: the plate streak separation method was used for purification, and the shake flask culture was streaked on the NA solid medium for separation. Smear plate test until a pure culture is obtained to obtain Bacillus subtilis QL-2. The bacterial culture was milky white on the NA solid medium, the colony was round, the center was raised, the edge was smooth, and the center was moist and easy to pick.

(5) Preservation: the purified Bacillus subtilis QL-2 was stored in glycerol at -20°C.

Example 2 Characterization of Bacillus subtilis QL-2

(1). Bacterial morphology observation and staining reaction

Bacillus subtilis QL-2 was inoculated in NA solid medium and cultured at a constant temperature for a suitable time, then stained for microscopic examination. Including simple staining, Gram staining (crystal shoxenamide staining method), flagella staining (silver nitrate staining method), capsule staining (wet ink method), spore staining (modified Schaeffer-Fulton's staining method).

Staining results: Bacillus subtilis QL-2 is a rod-shaped bacterium, G ⁺ , the flagella grows around, and each bacterium has 8 to 16 flagella, and its length is about 1 to 2 times that of the bacterium; it has a capsule ; A small number of spore-producing, mesozoic, with enlarged sporangia.

(2). Effects of Environmental Factors on Bacillus subtilis QL-2

Effect of O ₂ on Bacillus subtilis QL-2

Use the deep layer agar method to measure the effect of _O2 on the growth of Bacillus subtilis QL-2, insert Bacillus subtilis QL-2 into the test tube containing glucose beef extract peptone agar deep layer medium (pH7.0), and keep the temperature at 28°C After 48 hours of static cultivation in the box, the growth status was continuously observed until the result was clear. The results showed that Bacillus subtilis QL-2 grew on the surface and close to the surface, and was an aerobic or facultative anaerobic bacteria.

Effect of temperature on Bacillus subtilis QL-2

Prepare NA agar plates so that the thickness of the solidified medium is 1.5 to 2 times that of ordinary medium. Spread Bacillus subtilis QL-2 on plates, take two sets of plates and place them upside down at 4°C, 15°C, 30°C, 37°C, 45°C, 50°C, 55°C and 60°C for 48 hours to observe their growth status. It can be seen from the table below that the optimum growth temperature of Bacillus subtilis QL-2 is 37°C, and it can still grow normally at a temperature above 45°C, which is a high-temperature microorganism.

Table 1 Effect of temperature on the growth of QL-2

temperature(℃) 4 15 30 37 45 50 55 60 Growth Status - - ++ +++ ++ + + -

"—" means no growth, "+" means poor growth, "++" means normal growth, and "+++" means good growth.

Effect of Osmotic Pressure on Bacillus subtilis QL-2

Plates of beef extract peptone agar medium containing 0.85%, 5%, 10%, 15%, 20% and 25% NaCl were prepared. Spread Bacillus subtilis QL-2 on a flat plate and culture it in an incubator at 28°C. After 4 days, observe and record the growth of Bacillus subtilis QL-2 on plates containing different concentrations of NaCl. It can be seen from the table below that Bacillus subtilis QL-2 cannot grow at a salt concentration of 15%, and it does not belong to halophilic bacteria.

Table 2 The effect of osmotic pressure on the growth of QL-2

NaCl concentration% 0.85 5 10 15 20 25 Growth Status +++ ++ + - - -

"—" means no growth, "+" means poor growth, "++" means normal growth, and "+++" means good growth.

Effect of pH on Bacillus subtilis QL-2

Prepare NA liquid media with pH 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. The same amount of overnight culture of Bacillus subtilis QL-2 was inoculated by aseptic operation to ensure that the concentration of the bacterial solution inserted into each culture bottle was consistent. 130rpm/min, shake culture at 30°C for 24h, measure the OD600 value of the culture. It can be seen from the table below that the optimum pH for the growth of Bacillus subtilis QL-2 is between 8 and 9, and its growth condition in an alkaline environment is obviously better than that in an acidic environment.

Table 3 The effect of pH on the growth of Bacillus subtilis QL-2

pH 3 4 5 6 7 8 9 10 11 12 OD600 0 0 0.424 0.664 0.620 0.797 0.676 0.474 0.323 0.160

3. Physiological and biochemical reactions of Bacillus subtilis QL-2

The biochemical characteristics were identified with the automatic microbial identification system VITEK32 and the supporting aerobic bacillus identification card (BAC card), and a total of 31 biochemical indicators were detected.

Table 4 Biochemical identification results of Bacillus subtilis QL-2

Note: For the full name of the biochemical index abbreviation, refer to the instructions for use of the VITEK Aerobic Bacillus Identification Card (BAC Card).

Supplementary H ₂ O ₂ reaction was positive. It is obviously consistent with the characteristics of Bacillus subtilis.

4. 16S rDNA oligonucleotide sequence analysis:

Amplification and sequencing primers:

27f: 5’-AGA GTT TGA TCC TGG CTC AG-3’

1492r: 5’-CGG TTA CCT TGT TAC GAC TT-3’

517f: 5'-CCA GCA GCC GCG GTA AT-3'

The total DNA of the QL-2 bacterial strain was extracted, and the 16S rDNA gene of the bacterial strain was amplified by PCR with primers 27f and 1492r, and sequenced with primers 27f, 1492r, and 517f. The 16S rDNA gene sequence of the bacterial strain was:

1 AAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGAGGACAGGTGAGTAACA

61 CGTGGGTAACCTGCCTGTAAGACTGGTAATAACTCCGGGAAACCGGGGCTAATACCGGAT

121 GGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGG

181 ACCCGCGGCGCATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCAACGATGCGTAGCC

241 GACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGG

301 CAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGA

361 TGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGG

421 GCGGTACCTTGACGGTACCTAACCAGAAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGG

481 TAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTT

541 TCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAA

601 CTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTG

661 GAGGAACACCAGTGGCCCCAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAG

721 CGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAA

781 GTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGG

841 AGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGC

901 ATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAAT

961 CCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAG

1021 CTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGC

1081 CAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGAT

1141 GACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAAC

1201 AAAGGGCAGCGAAACCGCGAGGTTAAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCG

1261 CAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGC

1321 GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACATTCTACTAAAAGTTTGTAAC

1381 ACCCCGAAGTTTTCCGGTTGCAAGG

Through the Internet (http://www.ncbi.nlm.nihgov/blast/blast.cgi), the 16S rDNA sequence of Bacillus subtilis QL-2 was compared with the known sequence in GenBank with BLAST software. The results showed that the sequence homology between Bacillus subtilis QL-2 and Bacillus subtilis reached 99%,

Embodiment 3 Bacillus subtilis QL-2 produces the activity of chymosin and the curdling activity of its fermented liquid

(1).Activity of Bacillus subtilis QL-2 to produce chymosin

The culture of Bacillus subtilis QL-2 shaken at 30°C and 120rpm for 24h was inoculated into NA liquid medium (30ml/250ml Erlenmeyer flask, the inoculum size was 1:30), and shaken at 30°C and 120rpm on a shaker. After culturing for 24 hours, the supernatant was centrifuged to measure the chymosin activity, and the chymosin activity of the fermentation liquid reached the highest 44.4SU.

2. Content of rennet in Bacillus subtilis QL-2 fermentation broth

Using Maxiren chymosin as a standard, make a standard curve of rennet concentration and rennet activity. According to the chymosin activity of the fermentation broth when Bacillus subtilis QL-2 was cultured for 30 hours, the content of chymosin was calculated to be 5100 mg/L.

Claims (3)

[Claim 1] The present invention is a rennet-producing Bacillus subtilis QL-2 (Bacillus subtilisQL-2), which is isolated from cheese, and its fermented liquid has good curdling effect, and has been preserved in China Typical Culture Collection Center (CCTCC), the preservation number is M208023; [Claim 2] Bacillus subtilis QL-2 according to claim 1, characterized in that: (1) Morphological characteristics: The bacteria are rod-shaped, with perinatal flagella; a few spore-forming, mesophytic, oval, and enlarged cysts; Gram staining is positive; (2) Culture characteristics: The strain was cultured on NA medium at 30°C for 2 days, and the colony was milky white, round, with neat edges, and the center was raised like a steamed bun, smooth, not easy to stir up, no soluble pigment, and had an odor; (3) Biochemical characteristics of the strain: Catalase positive, facultative aerobic growth, VP reaction positive, acid produced in VP solution with pH value less than 6.0, casein hydrolyzed, sucrose produced reducing sugar, fructan produced, starch and gelatin hydrolyzed, tyrosine not hydrolyzed; (4) Physiological characteristics of the strain: Can use glucose to produce acid without producing gas, can use sucrose, maltose, inositol, mannitol, soluble starch, sodium acetate, do not use galactose, the optimum growth pH is between 8 and 9, in 10% sodium chloride Can grow, the optimum growth temperature is 37°C; (5) The 16S rDNA gene sequence of the strain is: 1 AAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGAGGACAGGTGAGTAACA 61 CGTGGGTAACCTGCCTGTAAGACTGGTAATAACTCCGGGAAACCGGGGCTAATACCGGAT 121 GGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGG 181 ACCCGCGGCGCATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCAACGATGCGTAGCC 241 GACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGG 301 CAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGA 361 TGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGG 421 GCGGTACCTTGACGGTACCTAACCAGAAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGG 481 TAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTT 541 TCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAA 601 CTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTG 661 GAGGAACACCAGTGGCCCCAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAG 721 CGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAA 781 GTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGG 841 AGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGC 901 ATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAAT 961 CCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAG 1021 CTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGC 1081 CAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGAT 1141 GACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAAC 1201 AAAGGGCAGCGAAACCGCGAGGTTAAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCG 1261 CAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGC 1321 GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACATTCTACTAAAAGTTTGTAAC 1381 ACCCCGAAGTTTTCCGGTTGCAAGG [Claim 3] A strain of Bacillus subtilis QL-2 according to claims 1 and 2, characterized in that the fermentation broth of the bacterial strain is rich in rennet and has good curdling effect, and has potential for industrial production.