Bacillus velezensis and protease and application thereof in meal fermentation

Abstract

The invention relates to bacillus velezensis and protease as well as application of the bacillus velezensis and the protease in meal fermentation. The name of the strain is bacillus velezensis GLY6-6, the preservation number is CGMCC No.33263, the strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on December 31, 2024, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China. The bacillus velezensis provided by the invention can be applied to meal fermentation, is particularly suitable for fermentation of soybean meal and rapeseed meal, and has the advantages of high enzyme yield, high enzyme activity, good fermentation effect and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to a Bacillus velezensis and a protease and application thereof in fermented meal. BACKGROUND

[0002] As an important industrial strain, Bacillus velezensis can be widely applied in the fields of food industry, agriculture and environmental protection, biological control and the like. However, different strains and different production processes have important influences on the types and yields of enzymes produced, and also affect the further application of the enzymes. At present, the use of Bacillus velezensis to produce enzymes has the problems of low enzyme production efficiency and the like.

[0003] Soybean meal is a by-product after soybean oil is pressed, and has a high protein content. However, the anti-nutritional factors contained in the soybean meal limit the further application. The anti-nutritional factors include protease inhibitors, plant lectins, flatulence factors, antigenic proteins and the like. The antigenic proteins, such as glycinin and beta-conglycinin, are allergens, which can cause intestinal allergic reactions and allergic symptoms, thereby causing intestinal damage and affecting nutrient absorption.

[0004] Rapeseed meal is a main by-product after rapeseed oil is pressed, and is rich in protein (35%-45%), and is a potential high-quality protein source. However, the protein in the rapeseed meal is mostly in the form of a macromolecular complex, and part of the protein is combined with structural carbohydrates such as cellulose and lignin to form a wrapped structure, so that it is difficult for digestive enzymes to directly contact and decompose, and the digestibility is relatively low. Acid-soluble protein generally refers to protein or polypeptide that can be dissolved under acidic conditions, and is applied in multiple fields such as feed, food, and preparation of bioactive peptides. If a suitable microbial fermentation technology or a suitable enzyme is selected to decompose the indigestible macromolecular protein in the rapeseed meal into soluble components such as small molecular peptides and free amino acids, it will be beneficial to break the compact structure of the rapeseed meal protein and the bondage of the anti-nutritional factors, improve the solubility and bioactivity of the product, and thus improve the utilization rate of the rapeseed meal protein. However, the key of the technology is how to select a suitable strain and a protease. SUMMARY

[0005] In view of the problems existing in the prior art, the present application provides a Bacillus velezensis and a protease and application thereof in fermented meal.

[0006] The technical solution for solving the above technical problem is as follows: The present application provides a Bacillus velezensis, Bacillus velezensis GLY6-6, with a preservation number of CGMCC No. 33263.

[0007] The present invention provides a bacterial agent, comprising the above-mentioned Bacillus Velez subtilis or the fermentation liquid of the above-mentioned Bacillus Velez subtilis.

[0008] The invention provides a protease, which is obtained by fermenting Bacillus velezensis.

[0009] The invention provides a method for producing protease, comprising the following steps: inoculating Bacillus velezensis into a culture medium and performing fermentation culture.

[0010] Furthermore, the method further comprises the steps of centrifuging and collecting the enzymatic hydrolysate after the fermentation culture.

[0011] The present invention provides the use of the above-mentioned Bacillus Velez subtilis in enzyme production. The enzyme may be a protease. The above-mentioned Bacillus Velez subtilis can be used to prepare an enzyme preparation.

[0012] The present invention provides application of the above-mentioned Bacillus Velez, bacterial agent or protease in fermentation or enzymatic hydrolysis of meal.

[0013] The invention provides a method for fermenting or enzymatically hydrolyzing meal, comprising the following steps: utilizing the above-mentioned Bacillus Velez subtilis, bacterial agent or protease to ferment or enzymatically hydrolyze meal.

[0014] The meals include soybean meal and / or rapeseed meal.

[0015] The Bacillus Velez GLY6-6 provided by the present invention has the strongest protease production ability when the inoculation amount is 3%, the temperature is 37°C, the culture medium pH is 8, and the culture time is 12 hours. The produced protease is a neutral mesophilic protease, the optimal enzyme reaction temperature is 55°C, and the optimal enzyme reaction pH is 7. After culture under these conditions and reaction under the optimal reaction conditions, the enzyme activity is as high as 94.74 U / mL. The bacterial liquid is centrifuged and the supernatant is used to enzymatically hydrolyze rapeseed meal. Through process optimization, the absolute value of acid-soluble protein in the rapeseed meal enzymatically hydrolyzed under the optimal process can reach 19.48%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the phylogenetic tree of GLY6-6 16S rRNA gene sequences.

[0017] Figure 2 Construct a phylogenetic tree for GLY6-6 and its related species.

[0018] Figure 3 The colony morphology characteristics of strain GLY6-6, wherein A is a Gram staining photo of strain GLY6-6, B is a spore staining photo of strain GLY6-6, and C is a colony morphology photo of strain GLY6-6.

[0019] Figure 4 This is the growth curve of GLY6-6.

[0020] Figure 5 These are the results of the transparent zone test on the skimmed milk powder solid culture medium plate.

[0021] Figure 6 To screen the experimental results of the effects of different times on enzyme production capacity.

[0022] Figure 7 To screen the experimental results of the effect of GLY6-6 on protease production ability at different temperatures.

[0023] Figure 8 To screen the experimental results of the effect of different inoculation amounts of GLY6-6 on protease production capacity.

[0024] Figure 9 The experimental results are to screen the effect of GLY6-6 on protease production ability in different pH culture media.

[0025] Figure 10 The results are for testing the optimal reaction temperature of protease.

[0026] Figure 11 This is the experimental result of detecting the optimal reaction pH of protease.

[0027] Figure 12 The results of the experiment were used to test the temperature tolerance of protease.

[0028] Figure 13 The results are for testing the tolerance of protease to pH value.

[0029] Figure 14 These are the experimental results for detecting the effect of NaCl concentration on protease activity.

[0030] Figure 15 These are the experimental results for detecting the effects of metal ions on protease activity.

[0031] Figure 16 These are the results of a single-factor experiment on enzymatic hydrolysis of rapeseed meal using crude GLY6-6 enzyme solution. DETAILED DESCRIPTION

[0032] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0033] In the examples, soybean meal and rapeseed meal were purchased from Yunnan Xinanhong Feed Co., Ltd., item number 202412081708.

[0034] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, natural pH, prepared with water.

[0035] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder, natural pH, prepared with water.

[0036] Skim milk powder solid culture medium plates can be prepared according to the following proportions: weigh 8 g of skim milk powder, add 200 mL of distilled water, stir to dissolve, and sterilize in a 65°C water bath for 40 min (low-temperature sterilization); weigh 6 g of agar, add 200 mL of distilled water, and sterilize at 121°C for 15 min. Mix the sterilized skim milk powder and cooled agar, shake gently, and immediately pour into a sterile culture dish. After solidification at room temperature, store at 4°C for later use.

[0037] In the examples, where specific techniques or conditions are not specified, all methods are conventional methods or those described in the literature in the field, or according to the product instructions. Reagents and instruments used, where the manufacturer is not specified, are conventional products available through regular channels or can be prepared according to conventional methods in the field.

[0038] Unless otherwise specified, the solutions of the present invention are prepared using water as the solvent.

[0039] The following is an introduction through specific embodiments. Example 1

[0040] In October 2018, rumen fluid samples were collected from Holstein cows with fistulas. The collection method included: cleaning the skin around the fistula and opening the fistula plug; discarding the first 50 mL of liquid that flowed out; and extracting approximately 100-200 mL of rumen fluid from different depths (50-80 cm), injecting it into sterile bottles pre-filled with CO2, and quickly sealing it.

[0041] Rumen fluid samples were separated and screened. The experimental method included the following steps: After collecting rumen fluid, filter it through four layers of gauze. The filtrate was quickly frozen with liquid nitrogen and stored in a -80°C freezer. Under a sterile operating table, the rumen filtrate was thawed at room temperature. Using a sterile pipette, 1 mL of rumen fluid was mixed with 9 mL of normal saline in a shaker and thoroughly shaken. After thorough mixing, the mixture was incubated in an 80°C water bath for 20 minutes to kill non-spore-forming bacteria. The mixture was then centrifuged at 3000 rpm for 2 minutes at room temperature. The supernatant was diluted 10-fold and serially diluted to obtain rumen dilutions of 10⁻³ and 10⁻⁷, respectively. 50 μL of each of the 10⁻³ and 10⁻⁷ dilutions was spread onto skim milk solid culture plates and incubated in a 37°C incubator for 24 hours. After the incubation period, the largest hydrolysis zone was selected for further streaking. The target strain was obtained using the above method and named GLY6-6. Example 2

[0042] (1) 16S rRNA gene analysis of strain GLY6-6 The 16S rRNA gene sequence was determined using bacterial 16S rRNA gene universal primers 27F and 1492R, and a 1453 bp gene fragment was obtained.

[0043] 27F: 5′-GTTTGATCMTGGCTCAG-3′ (SEQ ID NO: 1); 1492R: 5′-TACGGYTACCTTGTTACGACTT-3′ (SEQ ID NO: 2).

[0044] The 16S rRNA gene sequence of strain GLY6-6 is shown in SEQ ID NO: 3:

[0045] Comparison with known model bacteria on the EzbioCloud website (http: / / www.ezbiocloud.net / eztaxon / ) showed that strain GLY6-6 had the highest similarity with the known model strains Bacillus siamensis KCTC 13613T and Bacillus velezensis CR-502T, which were 100% and 99.93%, respectively. A phylogenetic tree of strain GLY6-6 and related groups based on 16S rRNA gene sequences was constructed, as shown in the following figure: Figure 1 shown.

[0046] (2) Sequence analysis of the gyrB gene of strain GLY6-6 The conserved gyrB gene was detected using the universal primers up-1 and up-2r for bacterial gyrB gene to obtain the gyrB gene sequence.

[0047] up-1: 5′-GAAGTCATCATGACC GTTCTGCAYGCNGGNGGNAARTTYGA-3′ (SEQ ID NO: 4); up-2r: 5′-AGCAGGATACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3′ (SEQ ID NO: 5).

[0048] The gyrB gene sequence of strain GLY6-6 is shown in SEQ ID NO:6:

[0049] Comparison with NCBI showed that strain GLY6-6 had the highest similarity with the known model bacterium Bacillus velezensis BCRC 17467T, which was 98.88%. A phylogenetic tree was constructed for strain GLY6-6 and its related species, as shown in Figure 2. Figure 2 As shown, strain GLY6-6 and Bacillus velezensis BCRC 17467T clustered into the same branch, and finally GLY6-6 was identified as Bacillus velezensis.

[0050] (3) Colony morphology characteristics of strain GLY6-6 like Figure 3 As shown, the bacteria were positive for GLY6-6 Gram staining (A), with rod-shaped cells and endospores (B). After culturing in TSA medium at 30°C for 24 h, the colonies were milky white, round, smooth, and slightly convex (C).

[0051] (4) Physiological and biochemical characteristics of strain GLY6-6 The fatty acid composition of strain GLY6-6 was detected by the microbial fatty acid rapid identification system (MIDI). The results showed that the main fatty acids of strain GLY6-6 were C15:0 anteiso, C15:0 iso, C16:0, C17:0 anteiso and C17:0iso, with contents of 39.82%, 17.20%, 16.69%, 6.83% and 7.43%, respectively.

[0052] API 50CH was used for analysis, and the test results were as follows: positive reactions included glucose, fructose, mannitol, α-methyl-D-glucoside, N-acetyl-glucosamine, esculin, cellobiose, maltose, sucrose, starch, and glycogen; weak positive reactions included glycerol, L-arabinose, ribose, D-xylose, mannose, inositol, sorbitol, amygdalin, arbutin, salvinol, lactose, trehalose, inulin, raffinose, and erucose; Negative reactions were observed with erythrose, D-arabinose, L-xylose, adonitol, β-methyl-D-xyloside, galactose, sorbose, rhamnose, dulcitol, α-methyl-D-mannosylglycoside, melibiose, melezitose, xylitol, D-turanose, D-lyxose, D-tagatose, D-rock sugar, L-rock sugar, D-arabitol, L-arabitol, gluconate, 2-keto-gluconate, and 5-keto-gluconate.

[0053] (5) Determination of growth curve The strain GLY6-6 was inoculated in LB liquid medium at an inoculation amount of 2% (volume percentage), and cultured at 37°C, 200 rpm for 24 h. Every 2 h, OD600 was detected by using an enzyme-labeled instrument, and a growth curve was drawn. The experimental results are shown in Figure 4 It can be seen from the figure that GLY6-6 reached the first plateau at 8 h, continued to grow at 12 h, and reached the second plateau at 21 h.

[0054] (6) Defatted milk powder solid medium plate transparent circle experiment The strain GLY6-6 was inoculated in defatted milk powder solid medium plate, and cultured at 37°C, static condition for 21 h. The experimental results are shown in Figure 5 After the strain GLY6-6 was inoculated in the defatted milk powder solid medium plate, an obvious transparent circle was produced, and the diameter of the transparent circle could reach 2.47 cm.

[0055] Based on the above 16S rRNA gene, gyrB conservative gene, and colony morphology and physiological and biochemical characteristics (whole cell fatty acid and API 50CH) analysis, the strain GLY6-6 was identified as Bacillus velezensis.

[0056] On December 31, 2024, the strain GLY6-6 was preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation name was Bacillus velezensis GLY6-6. The recommended classification name was Bacillus velezensis, the preservation number was CGMCC No. 33263, and the preservation address was No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

[0057] Example 3 The GLY6-6 was used to produce protease, including the following steps: (1) Activation of the strain, including the following steps: using an inoculation ring to streak the GLY6-6 strain preserved in a glycerol tube in an LB solid medium plate, pH natural, 37°C static culture for 21 h to obtain a single colony (first generation).

[0058] (2) The single colony in the LB solid medium plate was inoculated in 10 mL LB liquid medium, pH natural, 37°C, 200 rpm culture for 21 h, and the bacterial liquid was collected, which was the second generation bacterial liquid.

[0059] (3) The second generation of bacteria liquid was inoculated into LB liquid medium (pH 8) at an inoculation amount of 3% (volume percentage), and cultured at 37°C and 200 rpm for 12 h, and the bacteria liquid was collected, which was the third generation of bacteria liquid.

[0060] (4) The third generation of bacteria liquid was centrifuged at 12000 rpm and 4°C for 20 min, and the supernatant was collected (which was the GLY6-6 crude enzyme liquid).

[0061] Example 4: Screening of GLY6-6 protease production conditions (1) Effect of different culture times on enzyme production capacity Referring to the method of Example 3, the culture temperature of step (3) of Example 3 was set to 37°C, the pH of LB liquid medium was adjusted to 7, the inoculation amount of GLY6-6 was set to 2% (volume percentage), and the culture time was 24 h. 1 mL of bacteria liquid was taken every 3 h, centrifuged at 12000 rpm and 4°C for 20 min, and the supernatant was taken as the crude enzyme liquid. The Folin method (GB / T 28715-2012) was used to determine the protease activity of the crude enzyme liquid at a temperature of 40°C and a pH of 7. The rest was the same as Example 3.

[0062] The experimental results are shown in Table 1. Figure 6 As shown in Table 1, when the culture time was 12 h, the protease activity of strain GLY6-6 was the highest, which was 57.26 U / mL.

[0063] (2) Effect of different culture temperatures on enzyme production capacity Referring to the method of Example 3, the culture time of step (3) of Example 3 was set to 12 h, the pH of LB liquid medium was adjusted to 7, the inoculation amount of GLY6-6 was set to 2% (volume percentage), and the culture temperature was set to 31°C, 34°C, 37°C and 40°C, respectively. 1 mL of bacteria liquid cultured at different temperatures was taken, centrifuged at 12000 rpm and 4°C for 20 min, and the supernatant was taken. The rest was the same as Example 3. The detection method of protease activity was the same as Example 4(1).

[0064] The experimental results are shown in Table 2. Figure 7 As shown in Table 2, when the culture temperature was 37°C, the protease activity of strain GLY6-6 was the highest, which was 56.97 U / mL.

[0065] (3) Effect of different inoculation amounts on enzyme production capacity Referring to the method of Example 3, the incubation time of step (3) of Example 3 was set to 12 h, the incubation temperature was set to 37°C, the pH of the LB liquid medium was adjusted to 7, and the inoculum amount of GLY6-6 was set to 1%, 2%, 3%, 4%, 5%, and 6%, respectively, all expressed as volume percentages. 1 mL of bacterial solution was centrifuged at 12,000 rpm and 4°C for 20 min, and the supernatant was collected. The rest of the process was the same as in Example 3. The method for detecting protease activity was the same as in Example 4 (1).

[0066] The experimental results are as follows Figure 8 As shown, when the inoculation amount was 3%, the protease production capacity of strain GLY6-6 was the highest, which was 64.90 U / mL.

[0067] (4) Effect of different pH on enzyme production capacity Referring to the method of Example 3, the culture time of step (3) of Example 3 was set to 12 h, the culture temperature was set to 37°C, the inoculum amount of GLY6-6 was set to 2% (volume percentage), and the pH of the LB liquid culture medium was adjusted to 6, 7, 8, 9, and 10, respectively. 1 mL of bacterial solution was taken and centrifuged at 12,000 rpm and 4°C for 20 min. The supernatant was taken. The rest was the same as in Example 3. The method for detecting protease activity was the same as in Example 4 (1).

[0068] The experimental results are as follows Figure 9 As shown, when the pH of the culture medium was 8, the protease production capacity of strain GLY6-6 was the highest, which was 59.41 U / mL.

[0069] By screening the protease production conditions of GLY6-6, the optimal conditions for protease production were determined to be: inoculation amount 3% (volume percentage), culture temperature 37℃, culture medium pH 8, and culture time 12 h.

[0070] Example 5 Study on the enzymatic properties of GLY6-6 protease The crude GLY6-6 enzyme solution was prepared using the method of Example 3.

[0071] (1) Detecting the optimal reaction temperature of the enzyme. The experimental method includes the following steps: adding 1 mL of GLY6-6 crude enzyme solution, adding 1 mL of substrate (the substrate is 1% casein solution, mass percentage), adding 1 mL of phosphate buffer solution to a test tube, mixing them evenly, and performing the enzyme reaction under the conditions of pH 7 and NaCl concentration of 0. The reaction time is 10 min, and the enzyme reaction temperature is set to 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C, respectively. The rest is the same as the method for determining protease activity in Example 4.

[0072] The experimental results of detecting the optimal reaction temperature of the enzyme are as follows Figure 10As shown in Table 5, when the reaction temperature was 55℃, the highest protease activity was 93.93 U / mL.

[0073] (2) The optimal reaction pH of the enzyme was detected, and the experimental method included the following steps: based on Example 5(1), the enzyme reaction temperature was 40℃, and the pH value of the enzyme reaction system was adjusted to 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0, respectively. The rest was the same as Example 5(1).

[0074] The experimental results of detecting the optimal reaction pH of the protease are shown in Table 6. Figure 11 As shown in Table 6, when the pH was 7, the protease activity was the highest, which was 84.91 U / mL, and therefore, the optimal pH of the enzyme was 7.

[0075] (3) The temperature tolerance of the enzyme was detected, and the experimental method included the following steps: the GLY6-6 crude enzyme solution was placed in a water bath at 40℃, 50℃, 60℃, 70℃, and 80℃, respectively, and incubated for 100 min. The relative protease activity was measured every 20 min. The calculation formula of the relative protease activity was: protease activity after incubation / protease activity without incubation x 100%. When detecting the protease activity, the temperature of the enzyme reaction was 55℃, the pH value of the enzyme reaction was 7, and the rest was the same as Example 5(1).

[0076] The experimental results of detecting the temperature tolerance of the enzyme are shown in Table 7. Figure 12 As shown in Table 7, when incubated at 40-50℃, the enzyme activity was relatively stable.

[0077] (4) The pH tolerance of the enzyme was detected, and the experimental method included the following steps: based on Example 5(3), the GLY6-6 crude enzyme solution was placed in a condition with pH of 6.0, 7.0, 8.0, 9.0, and 10.0 for 100 min, and the relative protease activity was measured every 20 min. The rest was the same as Example 5(3).

[0078] The experimental results of detecting the pH tolerance of the enzyme are shown in Table 8. Figure 13 As shown in Table 8, when the pH was 6.0-8.0, the enzyme activity was stable.

[0079] (5) The effect of salt concentration on the protease activity was detected The effect of salt concentration on protease activity was detected, and the experimental method included the following steps: four portions of GLY6-6 crude enzyme solution were taken, different concentrations of NaCl were added into the GLY6-6 crude enzyme solution, the final concentration of NaCl in the crude enzyme solution was 2%, 4%, 6%, and 8% respectively, all in mass percentage. The temperature was kept at 40°C for 2 hours. Then, the enzyme reaction was carried out according to the method of Example 5(3), the enzyme reaction temperature was 55°C, the pH value of the enzyme reaction was 7, the relative protease activity was determined, and the detection method was the same as that of Example 5(3).

[0080] The experimental results of the effect of NaCl concentration on enzyme activity are shown in Table 3. Figure 14 As shown in Table 3, when the content of NaCl was between 0% and 4%, the protease activity did not decrease significantly.

[0081] (6) Detection of the effect of metal ions on protease activity The effect of metal ions on protease activity was detected, and the experimental method included the following steps: a control group and an experimental group were set. The control group was GLY6-6 crude enzyme solution (without adding metal ions); the experimental group was GLY6-6 crude enzyme solution to which different metal ions were added, including FeSO4, NaCl, MgCl2, ZnCl2, CuSO4, MnSO4, and CaCl2, and the final concentration of the metal ions in the crude enzyme solution was 2.5 mmol / L. The control group and the experimental group were kept at 40°C for 2 hours. Then, the enzyme reaction was carried out according to the method of Example 5(3), the enzyme reaction temperature was 55°C, the pH value of the enzyme reaction was 7, the relative protease activity was determined, and the rest was the same as that of Example 5(3).

[0082] The experimental results of the effect of metal ions on enzyme activity are shown in Table 4. Figure 15 As shown in Table 4, when Fe 2＋ , Na ＋ , Mg 2＋ , Zn 2＋ , Cu 2＋ , Mn 2＋ , Ca 2＋ , and K ＋ metal ions were added into GLY6-6 crude enzyme solution, Ca 2＋ and Cu 2＋ had an activating effect on the protease, and Mg 2 ＋ , K ＋ , Fe 2＋ had an inhibitory effect on the protease.

[0083] From the above experimental results, it can be seen that the optimal temperature for the protease prepared by GLY6-6 to function is 50-60°C (preferably 55°C), the pH is 6-8 (preferably pH 7), the protease belongs to neutral medium temperature protease, and Ca 2＋ and Cu2 ＋ Activates proteases, Mg 2＋ , K ＋ 、Fe 2＋ It has an inhibitory effect on proteases.

[0084] Example 6 Application of GLY6-6 Protease in Enzymatic Hydrolysis of Rapeseed Meal The crude GLY6-6 enzyme solution was prepared using the method of Example 3.

[0085] 6.1 Single-factor experiment on enzymatic hydrolysis of rapeseed meal with GLY6-6 crude enzyme solution The enzymatic hydrolysis temperature, water content, crude enzyme solution dosage and enzymatic hydrolysis time were controlled as single variables to explore the effects of different conditions on the acid-soluble protein content (absolute value) of enzymatic hydrolyzed rapeseed meal. The experimental results of single factors are as follows: Figure 16 shown.

[0086] Moisture content = water mass / (rapeseed meal mass + water mass) × 100%.

[0087] The crude enzyme solution dosage is the ratio of the volume of GLY6-6 crude enzyme solution (mL) to the mass of rapeseed meal (g).

[0088] (1) GLY6-6 crude enzyme solution was mixed with rapeseed meal. The enzymatic hydrolysis temperatures were 35°C, 40°C, 45°C, 50°C, and 55°C, respectively. The moisture content of the rapeseed meal was adjusted to 50%. The volume-to-mass ratio of GLY6-6 crude enzyme solution to rapeseed meal was 1 mL / g, and the enzymatic hydrolysis time was 48 h. After the reaction, 10 g of enzymatically hydrolyzed rapeseed meal was taken and the acid-soluble protein content was determined using the Kjeldahl method (NY / T 3801-2020). The experimental results showed that the best effect was achieved when the enzymatic hydrolysis temperature was 45°C, and the acid-soluble protein content was the highest, at 18.78%.

[0089] (2) Referring to the method in 6.1 (1), the enzymatic hydrolysis temperature was 45°C, and the moisture content of rapeseed meal was set to 30%, 40%, 50%, and 60%, respectively. The rest were the same as in 6.1 (1). The experimental results showed that when the moisture content was 50%, the acid-soluble protein content was the highest, at 19.42%.

[0090] (3) Referring to the method in 6.1 (1), the enzymatic hydrolysis temperature was 45°C, and the volume-to-mass ratios of crude GLY6-6 enzyme solution to rapeseed meal were 0.4 mL / g, 0.6 mL / g, 0.8 mL / g, 1.0 mL / g, and 1.2 mL / g, respectively. The rest were the same as in 6.1 (1). The experimental results showed that when the volume-to-mass ratio of crude GLY6-6 enzyme solution to rapeseed meal was 0.4 mL / g, the acid-soluble protein content was the highest, at 18.46%.

[0091] (4) Refer to the method of 6.1(1), the enzymolysis temperature is 45℃, and the enzymolysis time is set to be 24 h, 36 h, 48 h, 60 h and 72 h respectively, and the rest is the same as 6.1(1). The experimental results show that when the enzymolysis time is 24 h, the acid-soluble protein content reaches 18.34%, and continuing to enzymolysis has no effect on the acid-soluble protein content.

[0092] 6.2 Orthogonal experiment of GLY6-6 crude enzyme solution enzymolysis of rapeseed meal According to the single factor experiment results, L93 4 Orthogonal experiment table, the optimal combination is determined by the acid-soluble protein content (absolute value) as an index after enzymolysis. The acid-soluble protein content detection method is the same as 6.1(1).

[0093] The experimental results are shown in Table 1, and it can be seen that the optimal combination is: temperature 45℃, water content of rapeseed meal 45%, enzyme solution dosage (volume-mass ratio of GLY6-6 crude enzyme solution to rapeseed meal) 0.4 mL / g, and enzymolysis time 24 h.

[0094] Table 1 Orthogonal experiment

[0095] In summary, through single factor experiment and orthogonal experiment, the optimal enzymolysis process is obtained as follows: temperature 36-50℃ (preferably 45℃), water content 40-50% (preferably 45%), enzyme solution dosage 0.2-0.6 mL / g (preferably 0.4 mL / g), and enzymolysis time 12-48 h (preferably 24 h). When the optimal process is used to enzymolysis rapeseed meal, the acid-soluble protein content is 19.48%.

[0096] Example 7 Comparison of the effects of GLY6-6 crude enzyme solution and commercial protease preparation on enzymolysis of rapeseed meal.

[0097] The GLY6-6 crude enzyme solution is prepared by the method of Example 3, and the neutral protease activity is detected by Folin method under the conditions of enzymolysis temperature 55℃ and enzymolysis pH 7, and the detection method refers to Example 4(1), The neutral protease activity of the crude enzyme solution is 90 U / mL.

[0098] The commercial protease preparation is purchased from Challenge Group. The neutral protease activity of the commercial protease preparation is 100000 U / g.

[0099] The enzyme solution group and the enzyme preparation group were set. The enzyme solution group: 20 mL of crude enzyme solution + 20 g of rapeseed meal; the enzyme preparation group: 20 mL of an aqueous solution of a commercially available protease preparation (containing 0.018 g of a commercially available protease preparation) + 20 g of rapeseed meal. The enzyme activity of the enzyme solution group was the same as that of the enzyme preparation group. In each group, the solid-liquid ratio was 1 g:1 mL, the enzyme hydrolysis temperature was 45°C, and the enzyme hydrolysis time was 48 h. The Kjeldahl method was used to detect the acid-soluble protein content (absolute value), and the detection method was the same as that in Example 6.1 (1).

[0100] The experimental results are shown in Table 2. When the enzyme activity, temperature, and enzyme hydrolysis time are the same, the GLY6-6 crude enzyme solution provided by the application has better effect on the enzyme hydrolysis of rapeseed meal, and the absolute content of acid-soluble protein can reach 19.11%. It is shown that the crude enzyme solution prepared by fermentation of the strain GLY6-6 has a significant effect on the enzyme hydrolysis of rapeseed meal.

[0101] Table 2 Comparison of acid-soluble protein contents of the enzyme solution group and the enzyme preparation group

[0102] Example 8 Application of Bacillus velezensis GLY6-6 in fermentation of soybean meal The GLY6-6 bacterial solution was the second generation bacterial solution prepared by the method of Example 3.

[0103] Fermentation of soybean meal by GLY6-6: GLY6-6 bacterial solution was inoculated into soybean meal at an inoculation amount of 4% (i.e. 4 mL of GLY6-6 bacterial solution was inoculated into 100 g of soybean meal), the fermentation temperature was 37°C, and the fermentation time was 24 h.

[0104] The Kjeldahl method was used to detect the acid-soluble protein content (absolute value) in the fermentation product, and the detection method was the same as that in Example 6.1 (1).

[0105] The Kjeldahl method (GB / T 6432-2018) was used to determine the crude protein content.

[0106] The competitive enzyme-linked adsorption method was used to determine the content of soybean globulin. The detection method included the following steps: soybean globulin antigen was pre-coated on the micro-pore strip of the enzyme-labeled plate, the soybean globulin in the sample and the pre-coated antigen competed for the soybean globulin antibody, after the addition of enzyme-labeled secondary antibody, TMB substrate was used for color development, the absorbance value of the sample was negatively correlated with the content of soybean globulin contained therein, and the content of soybean globulin in the sample could be obtained by comparison with the standard curve.

[0107] The content of conglycinin is determined by a competitive enzyme-linked adsorption method, and the detection method comprises the following steps: pre-coating conglycinin antigen on the microwell strip of an enzyme label plate, the conglycinin in the sample and the pre-coated antigen compete for the conglycinin antibody, after adding the enzyme label secondary antibody, developing color with TMB substrate, the absorbance value of the sample is negatively correlated with the content of conglycinin contained therein, and the content of conglycinin in the sample can be obtained by comparing with the standard curve.

[0108] The experimental results are shown in Table 3, and it can be seen that, by using GLY6-6 bacterial liquid to ferment soybean meal, the acid-soluble protein content in the soybean meal can be significantly increased, the contents of glycinin and conglycinin can be reduced, the degradation rate of glycinin can reach 92.9%, and the degradation rate of conglycinin can reach 85.5%.

[0109] Table 3 Effect of fermentation of Bacillus velezensis GLY6-6 on the contents of acid-soluble protein and antigen protein in soybean meal

[0110] Example 9 Comparison of GLY6-6 and other Bacillus velezensis The GLY6-6 crude enzyme liquid is prepared by the method of Example 3. Under the conditions of the optimal reaction temperature (55℃) and pH 7, the neutral protease is detected by the Folin method, and the detection method is referred to Example 4 (1). The results show that, in the GLY6-6 crude enzyme liquid prepared by the application, the neutral protease activity is 94.74 U / mL (i.e. Each milliliter of GLY6-6 crude enzyme liquid contains 94.74 U of neutral protease).

[0111] Bacillus velezensis (CGMCC No. 20920 2020.10.20) is purchased from China General Microbiological Culture Collection Center. The crude enzyme liquid is prepared by using the above-mentioned Bacillus velezensis, and the experimental method is referred to Example 3. Under the conditions of the optimal reaction temperature and pH, the protease activity detection method is the same as that in Example 4, and the results show that, in the crude enzyme liquid prepared by Bacillus velezensis (CGMCC No. 20920), the neutral protease activity is 49.07 U / mL (i.e. each milliliter of crude enzyme liquid contains 49.07 U of neutral protease).

[0112] The above results show that, compared with other Bacillus velezensis, the crude enzyme liquid prepared by GLY6-6 of the application contains higher neutral protease activity.

[0113] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A Bacillus Velezii, characterized in that The strain is named Bacillus velezinoffii ( Bacillus velezensis ) GLY6-6, deposit number is CGMCC No. 33263.

2. A bacterial agent, characterized in that The invention comprises the Bacillus Velez subtilis according to claim 1 or the fermentation broth of the Bacillus Velez subtilis according to claim 1.

3. A protease, characterized in that The product is obtained by fermentation with the Bacillus Velez subtilis described in claim 1.

4. A method for producing protease, characterized in that: The following steps are involved: The Bacillus Velez subtilis described in claim 1 is inoculated into a culture medium and fermented.

5. The method for producing protease according to claim 4, characterized in that: The method further comprises the steps of centrifuging and collecting the enzymatic hydrolysate after the fermentation culture.

6. Use of the Bacillus Velez subtilis according to claim 1 in enzyme production.

7. The application according to claim 6, characterized in that Used to prepare enzyme preparations.

8. Use of any one of the Bacillus Velez subtilis according to claim 1, the bacterial agent according to claim 2, and the protease according to claim 3 in fermentation or enzymatic hydrolysis of meal.

9. The application according to claim 8, characterized in that: The meals include soybean meal and / or rapeseed meal.

10. A method for fermenting or enzymatically hydrolyzing meal, characterized in that: The method comprises the following steps: fermenting or enzymolyzing meal by using the Bacillus Velez subtilis described in claim 1, the bacterial agent described in claim 2 or the protease described in claim 3.

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