Combination containing bacillus subtilis and lactobacillus casei and application of combination in soybean meal fermentation

Abstract

The invention discloses a compound preparation, the compound preparation contains bacillus subtilis and lactobacillus casei, the bacillus subtilis is bacillus subtilis, the strain number of the bacillus subtilis is YB-10, the bacillus subtilis is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.21103, and the lactobacillus casei is preserved in the China General Microbiological Culture Collection Center (CGMCC). The lactobacillus casei is lactobacillus casei, the strain number of the lactobacillus casei is TD01, the lactobacillus casei is preserved in the China Center for Type Culture Collection, and the preservation number of the lactobacillus casei is CCTCC NO: M 2022104. The bacillus subtilis YB-10 disclosed by the invention has a very high degradation rate on antigen protein in soybean meal, and can accumulate high-content acid soluble protein under the condition of solid state fermentation; the lactobacillus casei TD01 disclosed by the invention has a very good degradation effect on stachyose and raffinose in soybean meal; an intracellular metabolite obtained after autolysis of the lactobacillus casei TD01 cell has relatively high aminopeptidase activity, and bitter taste caused by accumulation of high acid soluble protein can be effectively removed.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a combination containing bacillus subtilis and lactobacillus casei and application of the combination in soybean meal fermentation. Background Art

[0002] Soybean meal is a plant protein raw material with high nutritional value. Its crude protein content is generally 43% to 46%, and it is widely used in livestock and poultry feed. Since soybean meal contains a variety of anti-nutritional factors such as soybean antigen protein, trypsin inhibitor, oligosaccharide and phytic acid, it affects the absorption and utilization of nutrients by the animal intestine, destroys the intestinal morphology and structure, and easily causes diarrhea in young animals.

[0003] Fermented soybean meal is a common process proposed to address the above-mentioned problems of soybean meal. It refers to the process of inoculating specific probiotics and enzyme preparations under artificially controllable conditions, so that high-molecular-weight proteins are converted into low-molecular-weight peptides under the synergistic action of bacteria and enzymes. At the same time, the content of antigenic proteins (such as soybean globulin and β-conglycinin) and undesirable oligosaccharides (such as stachyose and raffinose) is reduced to a certain extent, thereby modifying and increasing the value and promoting digestion and absorption in the gastrointestinal tract of livestock and poultry.

[0004] A high content of acid-soluble protein is an important indicator for measuring the degree of fermentation, but the existing technology for fermenting soybean meal generally has a low accumulation of acid-soluble protein, so its contribution to the elimination of antigenic proteins is very limited; in addition, although high-moisture enzymatic hydrolysis of soybean meal can achieve high-content acid-soluble protein accumulation, it will expose and release a large number of peptides with hydrophobic amino acid residues at the N-terminus (molecular weight between 500-1000Da). These hydrophobic amino acids have varying degrees of bitterness, affecting the palatability of the product.

[0005] Therefore, providing soybean meal fermentation strains and combination solutions that can achieve high acid-soluble protein, low bitter peptides, low antigenic protein and low undesirable oligosaccharide content is an urgent problem that technicians in this field need to solve. Summary of the invention

[0006] The invention first provides a lactobacillus casei. The lactobacillus casei is a lactobacillus casei (Lactobacillus casei), the strain number of which is TD01, and the lactobacillus casei is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO:M 2022104.

[0007] The present invention also provides a compound preparation, which contains Bacillus subtilis and the above-mentioned Lactobacillus casei;

[0008] In certain embodiments, the Bacillus subtilis is Bacillus subtilis, whose strain number is YB-10, which is deposited in the China General Microbiological Culture Collection Center with the deposit number: CGMCC NO.21103 (see CN 113151032A).

[0009] In certain embodiments, the viable bacterial count of Bacillus subtilis is 50 to 100 million cfu / g; the viable bacterial count of Lactobacillus casei is 10 to 50 million cfu / g.

[0010] In certain embodiments, the composition further comprises Saccharomyces cerevisiae.

[0011] In certain embodiments, the brewer's yeast is purchased from Angel Yeast Co., Ltd.'s high-temperature resistant brewer's high-activity dry yeast.

[0012] In certain embodiments, the viable count of Saccharomyces cerevisiae is 0.01 to 0.05 billion cfu / g.

[0013] The present invention also provides application of the compound preparation in soybean meal fermentation.

[0014] Finally, the present invention provides a soybean meal fermentation method, which comprises the step of inoculating the above-mentioned compound preparation.

[0015] In certain embodiments, the soybean meal fermentation adopts solid-state fermentation. After inoculation with the above-mentioned compound preparation, the moisture content is controlled at 50% to 60%, the temperature is 35° C. to 42° C., and static fermentation is performed for 3 to 5 days.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects:

[0017] 1. Bacillus subtilis YB-10 has a high degradation rate for antigenic proteins in soybean meal and can accumulate high levels of acid-soluble proteins under solid-state fermentation conditions;

[0018] 2. Lactobacillus casei TD01 has a good degradation effect on stachyose and raffinose in soybean meal; the intracellular metabolites of Lactobacillus casei TD01 cells after autolysis contain high aminopeptidase activity, which can effectively remove the bitterness caused by the accumulation of highly acid-soluble proteins.

[0019] Collection Instructions

[0020] The Bacillus subtilis YB-10 was isolated from cotton field soil and sent to the China General Microbiological Culture Collection Center for preservation on November 4, 2020. It is preserved in the China General Microbiological Culture Collection Center with the preservation number: CGMCC NO.21103. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0021] The Lactobacillus casei TD01 was isolated from natural cheese and sent to the China Center for Type Culture Collection for preservation on January 28, 2022. It was classified and named: Lactobacillus casei TD01, and was preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M 2022104. The address is: Wuhan University, Wuhan, Hubei, China. DETAILED DESCRIPTION

[0022] In order to make the technical problems to be solved, technical solutions and advantages of the present invention more clear, they are described in detail below in conjunction with specific embodiments.

[0023] Example 1: Effects of different Bacillus on the accumulation of acid-soluble proteins and the degradation of antigenic proteins in soybean meal

[0024] (1) Preparation of Bacillus suspension:

[0025] A loop was picked from a fresh slant of 15 different Bacillus species (including Bacillus subtilis, Bacillus licheniformis, Bacillus brevis, Bacillus megaterium, Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamese), inoculated into LB liquid culture medium, and cultured at 37°C and 200r / min for 12h as the inoculum, with a viable bacterial count of 600 million to 1 billion cfu / mL.

[0026] (2) Soybean meal treated with different Bacillus species:

[0027] Take different Bacillus suspensions, inoculate them according to the number of live bacteria per gram of soybean meal, add sterile water to a water content of 55% (w / w). Mix evenly, put them in fermentation bags, seal them with a sealing machine, leave a certain amount of air inside, and ferment them at 37°C for 3 days. Add an equal amount of sterile water under the same conditions to make a soybean meal sample as a blank control, and set 3 replicates for all groups.

[0028] After fermentation, the samples were dried at 60°C and crushed through a 60-mesh sieve. The acid-soluble protein content was tested according to GB / T22492-2008, and the globulin content was determined using the soybean globulin quantitative detection kit and the β-conglycinin quantitative detection kit of Tianjin Longke Ark Bioengineering Technology Co., Ltd.

[0029] Table 1 Effects of different bacterial agents on the accumulation of acid-soluble proteins and the degradation of antigenic proteins in soybean meal

[0030] Acid soluble protein (%) Soybean Globulin (mg / g) β-conglycinin (mg / g) Blank control 2.02 139.96 133.28 strain 1 7.99 69.54 90.25 strain 2 4.55 98.82 101.50 strain 3 6.34 70.76 87.63 strain 4 12.01 24.78 30.25 strain 5 6.16 72.06 89.94 strain 6 14.68 22.56 30.32 strain 7 12.28 33.75 28.61 strain 8 11.84 35.79 30.53 strain 9 10.31 43.48 55.35 strain 10 17.68 6.12 7.98 strain 11 19.25 1.34 3.08 strain 12 10.28 45.09 55.93 strain 13 9.05 46.45 66.02 strain 14 5.92 69.26 95.40 strain 15 8.48 48.98 66.56

[0031] Note: The data in the table are average values ​​after drying.

[0032] Table 1 shows that the acid-soluble protein in soybean meal after fermentation with strain 11 for 3 days reached 20.25%, which is much higher than other strains; while soybean globulin and β-conglycinin were 1.34% and 3.08%, respectively. Compared with the blank control, the degradation rates were 99.04% and 97.69%, respectively, and the degradation effect on antigenic protein was much better than other strains. Strain 11 is Bacillus subtilis YB-10, which is deposited in the China General Microbiological Culture Collection Center with the deposit number: CGMCC NO.21103.

[0033] Example 2: Degradation effects of different lactic acid bacteria on stachyose and raffinose

[0034] Preparation of lactic acid bacteria suspension:

[0035] MRS medium: peptone 10g / L, beef extract powder 5g / L, yeast extract powder 4g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, triammonium citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, Tween 80 1g / L, pH 6.2±0.2, sterilization at 121℃ for 15min.

[0036] A loop was picked from a fresh slant of 20 different lactic acid bacteria (including Lactobacillus plantarum, Pediococcus acidilactici, Pediococcus pentosaceus, Enterococcus faecalis, Enterococcus faecium, Bacillus coagulans, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus amylovora, Lactobacillus sakei, Lactobacillus fermentation, Lactobacillus brevis, Lactobacillus rhamnosus, and Lactobacillus helveticus), inoculated into MRS liquid culture medium, and cultured at 37°C for 24 hours as the inoculum, with a viable count of 800 million to 2 billion cfu / mL.

[0037] Comparison of the utilization ability of different lactic acid bacteria on stachyose and raffinose:

[0038] Lactic acid bacteria verification medium: peptone 10g / L, beef extract powder 5g / L, yeast extract powder 4g / L, stachyose 10g / L, raffinose 10g / L, dipotassium hydrogen phosphate 2g / L, triammonium citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, Tween 80 1g / L, pH 6.2±0.2, sterilization at 121℃ for 15min.

[0039] Take the bacterial suspension of different lactic acid bacteria and inoculate them into the lactic acid bacteria verification medium at 1% (v / v) respectively, and ferment them at 37°C for 24 hours. Add the same amount of sterile water as a blank control under the same conditions, and set 3 replicates for all groups.

[0040] The contents of stachyose and raffinose were detected by HPLC (the method refers to Journal of the Chinese Cereals and Oils Association, Vol. 29, No. 12, Dec. 2014). The results are shown in Table 2 below:

[0041] Table 2 Comparison of mannose metabolism ability of different lactic acid bacteria strains

[0042] pH Stachyose(g / L) Raffinose(g / L) Lactic acid (g / L) Blank control 6.22 11.245 10.378 0.428 strain 1 5.48 8.115 8.823 1.089 strain 2 6.02 10.672 10.005 0.605 strain 3 4.72 6.324 7.035 4.936 strain 4 4.94 6.891 6.935 4.260 strain 5 5.80 9.072 8.862 0.995 strain 6 4.07 5.024 5.235 9.030 strain 7 5.25 8.226 7.985 1.311 strain 8 5.76 9.103 8.605 0.998 strain 9 5.83 9.088 8.925 0.941 strain 10 3.94 5.002 4.651 10.150 strain 11 6.00 10.802 10.135 0.538 strain 12 5.54 8.242 8.808 1.083 strain 13 5.91 9.973 9.222 0.667 strain 14 4.49 6.239 6.381 6.303 strain 15 3.81 5.045 4.020 11.326 strain 16 4.87 6.642 7.005 4.338 strain 17 5.05 7.405 7.985 2.611

Claims

1. A Lactobacillus casei, characterized in that The Lactobacillus casei is Lactobacillus casei, the strain number of which is TD01, and which is preserved in China Center for Type Culture Collection with the preservation number: CCTCC NO: M2022104.

2. A compound preparation, characterized in that: The compound preparation contains the Lactobacillus casei and Bacillus subtilis according to claim 1, wherein the Bacillus subtilis is Bacillus subtilis, the strain number of which is YB-10, which is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC NO.21103.

3. The compound preparation according to claim 2, characterized in that: The number of live bacteria of the Bacillus subtilis is 50 to 100 million cfu / g; the number of live bacteria of the Lactobacillus casei is 10 to 50 million cfu / g.

4. The compound preparation according to claim 2, characterized in that: The compound preparation also contains brewer's yeast.

5. The compound preparation according to claim 4, characterized in that: The brewer's yeast was purchased from Angel Yeast Co., Ltd.'s high-temperature-resistant brewer's high-activity dry yeast.

6. The compound preparation according to claim 5, characterized in that: The live bacteria count of the brewer's yeast is 0.01 to 0.05 billion cfu / g.

7. Use of the compound preparation according to any one of claims 2 to 6 in soybean meal fermentation.

8. A soybean meal fermentation method, characterized in that: The fermentation method comprises the step of inoculating the compound preparation according to any one of claims 2-6.

9. The method according to claim 8, characterized in that The soybean meal fermentation adopts a solid-state fermentation method. After inoculating the compound preparation according to any one of claims 2-6, the moisture content is controlled at 50% to 60%, the temperature is 35° C. to 42° C., and the fermentation is performed statically for 3 to 5 days.