Compound protease for feed and application thereof

Abstract

The application relates to a compound protease for feed and application thereof, and belongs to the technical field of animal feed. The application provides a compound protease for feed, which is composed of alkaline protease, neutral protease and a carrier, wherein the alkaline protease and the neutral protease are respectively obtained through fermentation production of bacillus licheniformis and bacillus subtilis. The compound protease has good effects in terms of heat resistance, acid resistance, endogenous enzyme resistance and enzymolysis effect, thereby showing a good improvement effect on animal production performance. Compared with the feed additive protease product in the prior art, the compound enzyme for feed provided by the application has obvious improvement in yield increase effect after being added into animal feed.

Description

Technical Field

[0001] This application relates to a compound protease for feed and its application, belonging to the field of animal feed technology. Background Technology

[0002] Proteases are a class of enzymes that specifically hydrolyze proteins. They are among the world's largest-volume and most widely marketed industrial enzyme products, and are widely used in food, detergents, and other fields. They are also gradually being extended to the feed industry, but their application there is still in its early stages, with few truly successful case studies. As protein is the second largest component of feed, maximizing its utilization is crucial, making the development of proteases, which are key to improving protein digestibility, extremely important.

[0003] Proteases are widely recognized for their role in supplementing deficiencies in endogenous enzymes in animals, improving feed utilization, reducing feed costs, promoting livestock growth, and reducing environmental pollution. In the livestock industry, proteases are primarily added to feed, which is then extruded at high temperatures to form pellets. These pellets are ingested by animals and, after passing through the stomach, play a major role in protein digestion in the intestines, thereby improving protein digestibility. However, the current application of proteases in feed is not optimistic and still faces many challenges. For example, proteases are proteins, and it remains to be seen whether they can withstand the damage caused by high temperature, high humidity, and high pressure during the high-temperature pelleting process. The presence of hydrochloric acid and pepsin in animal gastric juice creates a very low pH, which may degrade exogenous proteases. Can exogenous proteases withstand this gastric environment? Given their ability to coexist with endogenous proteases, can exogenous proteases perform functions beyond those of endogenous proteases, i.e., can they work synergistically and complementaryly with endogenous proteases? Under normal circumstances, endogenous proteases are effective at degrading proteins in protein-rich feeds such as soybean meal, but have little effect on proteins in grains such as corn. Adding exogenous proteases can better break down large protein molecules in raw materials, thereby improving protein digestibility. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a compound protease for feed, comprising an alkaline protease, a neutral protease, and a carrier. The alkaline and neutral proteases are obtained through fermentation by *Bacillus licheniformis* and *Bacillus subtilis*, respectively. The compound protease of this invention exhibits excellent effects in terms of heat resistance, acid resistance, endogenous enzyme resistance, and enzymatic hydrolysis efficiency, thus demonstrating a significant improvement in animal production performance.

[0005] According to one aspect of this application, a complex protease for feed is provided, the complex protease comprising alkaline protease and neutral protease.

[0006] Optionally, the complex protease may also include a carrier.

[0007] Optionally, the weight ratio of the alkaline protease is 20-40%, and the weight ratio of the neutral protease is 20-50%.

[0008] Optionally, the weight ratio of the alkaline protease is selected from 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or any value between them.

[0009] Optionally, the weight percentage of the neutral protease is selected from 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or any value between these values.

[0010] Optionally, the weight ratio of the alkaline protease is 30-40%, and the weight ratio of the neutral protease is 30-35%.

[0011] Optionally, the alkaline protease has a weight ratio of 30%, the neutral protease has a weight ratio of 30%, and the carrier has a weight ratio of 40%.

[0012] Optionally, the carrier is selected from one or more of talc, zeolite, sodium sulfate, corn starch, rice husk powder, and maifanite powder.

[0013] Optionally, the carrier is talc.

[0014] Optionally, the alkaline protease is derived from Bacillus licheniformis, and the neutral protease is derived from Bacillus amyloliquefaciens.

[0015] Optionally, the amino acid sequence of the alkaline protease is shown in SEQ ID NO: 1, and the amino acid sequence of the neutral protease is shown in SEQ ID NO: 2.

[0016] The amino acid sequence of SEQ ID NO: 1 is as follows:

[0017] MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYAKGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ。

[0018] The amino acid sequence of SEQ ID NO: 2 is as follows:

[0019] VGLGKKLSVAVAASFMSLTISLPGVQAAENPQLKENLTNFVPKHSLVQSELPSVSDKAIKQYLKQNGKVFKGNPSERLKLIDHTTDDLGYKHFRYVPVVNGVPVKDSQVIIHVDKSNNVYAINGELNNDASAKTANSKKLSANQALDHAFKAIGKSPEAVSNGNVANKNKAELKAAATKDGKYRLAYDVTIRYIEPEPANWEVTVDAETGKVLKKQNKVEHAAATGTGTTLKGKTVSLNISSESGKYVMRDLSKPTGTQIITYDLQNRQYNLPGTLVSSTTNQFTTSSQRAAVDAHYNLGKVYDYFYQTFKRNSYDNKGGKIVSSVHYGSKYNNAAWIGDQMIYGDGDGSFFSPLSGSMDVTAHEMTHGVTQETANLNYENQPGALNESFSDVFGYFNDTEDWDIGEDITVSQPALRSLSNPTKYGQPDHYKNYRNLPNTDAGDYGGVHTNSGIPNKAAYNTITKIGVKKAEQIYYRALTVYLTPSSSFKDAKAALIQSARDLYGSQDAASVEAAWNAVGL。

[0020] According to another aspect of this application, the use of any of the above-mentioned feed-grade complex proteases in animal feed is provided.

[0021] Optionally, the amount of the feed-grade compound protease added is 300g / t-500g / t.

[0022] According to the last aspect of this application, a feed additive and feed comprising any of the above-mentioned feed complex proteases are provided.

[0023] The beneficial effects of this application include, but are not limited to:

[0024] 1. The feed compound protease according to this application has good heat resistance and acid resistance, and can achieve good stability, thus having excellent proteolytic effect.

[0025] 2. The compound protease for feed according to this application has a good in vitro proteolytic effect on soybean meal, corn gluten meal, and complete feed, and is suitable as a feed additive.

[0026] 3. The feed compound protease according to this application, compared with the protease products of the prior art, has a significant improvement in production performance when applied to broiler breeding production, which can increase the production income in the breeding industry and has important production and economic value. Attached Figure Description

[0027] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0028] Figure 1 This is a diagram showing the degradation results of antigenic proteins after enzymatic hydrolysis of soybean meal, as described in the embodiments of this application. Detailed Implementation

[0029] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present application are all purchased through commercial channels.

[0030] Example 1: Comparative Analysis of the Heat Resistance of Different Protease Samples

[0031] Reagents include: 0.25M sodium acetate solution (pH 5.5);

[0032] Experimental Methods: The composite protease (composed of alkaline protease and neutral protease) sample of the present invention and commercially available protease DP (alkaline protease, produced by Bacillus licheniformis), protease BB (composed of acidic protease, neutral protease and alkaline protease), protease KP (alkaline protease, produced by Pichia pastoris), protease LP (alkaline protease, produced by Bacillus licheniformis), protease NP (neutral protease, produced by Bacillus subtilis), and protease AP (acidic protease, produced by Aspergillus niger) were dissolved and diluted with distilled water to a certain enzyme activity (5000 U / mL). 1 mL of the diluted enzyme solution was added to 9 mL of pH 5.5 sodium acetate buffer and mixed thoroughly to ensure an enzyme activity of 500 U / mL during treatment. 5 mL of the mixed enzyme solution was placed in a water bath at different temperatures (75℃, 80℃) for 3 min. After incubation, the solution was cooled to room temperature in an ice-water bath. The enzyme activity was determined according to Appendix B of GB / T23527-2009 (Folin method). The enzyme activity of the compound protease of this invention is U_temperature-resistant, while the enzyme activity of other commercially available proteases is U_0. Then, the enzyme activity retention rate at different treatment temperatures is calculated using the following formula: Enzyme activity retention rate (%) = (U_temperature-resistant / U_0) × 100%.

[0033] The experimental results are shown in Table 1.

[0034] Table 1 Results of enzyme activity retention rates after water bath heat resistance of different proteases

[0035] Sample Name Enzyme activity retention rate at 75℃ Enzyme activity retention rate at 80℃ Complex protease 78％ 58.72％ DP protein 27.54％ 0.00％ Protease BB 77.73％ 5.90％ proteinase KP 66.80％ 0.49％ Protease LP 68.54％ 7.85％ Protease NP 0.00％ 0.00％ Protease AP 0.00％ 0.00％

[0036] Example 2: Comparative Analysis of Acid Resistance and Endogenous Enzyme Tolerance of Different Protease Samples

[0037] Reagents include: hydrochloric acid solution (1 mol / L), dissolve 90 mL of concentrated hydrochloric acid in 800 mL of distilled water, stir until homogeneous, and bring the volume to 1000 mL; sodium hydroxide solution (2 mol / L), weigh 80 g of solid sodium hydroxide into 800 mL of distilled water, stir until dissolved, and bring the volume to 1000 mL; pH 3.0 gastric buffer solution: Sodium chloride 5.18g, potassium chloride 0.50g, anhydrous sodium dihydrogen phosphate 12g (sodium dihydrogen phosphate dihydrate 15.60g), dissolved in 800mL distilled water, pH adjusted to 3.0 at 39℃ with hydrochloric acid or sodium hydroxide solution, then brought to a final volume of 1000mL with distilled water and refrigerated at 4℃; Simulated gastric juice (pepsin concentration 215U / mL): Weigh 0.0304g pepsin (Sigma P7000), add to 80mL of the corresponding pH gastric buffer solution, stir with a magnetic stirrer until dissolved (approximately 15min), then bring to a final volume of 100mL with pH 3.0 gastric buffer solution. Prepare fresh before use.

[0038] The experimental methods include:

[0039] A) Stability in gastric acid

[0040] The compound protease of the present invention (composed of alkaline protease and neutral protease) and commercially available protease DP (alkaline protease, produced by Bacillus licheniformis), protease BB (composed of acidic protease, neutral protease and alkaline protease), protease KP (alkaline protease, produced by Pichia pastoris), protease LP (alkaline protease, produced by Bacillus licheniformis), protease NP (neutral protease, produced by Bacillus subtilis), and protease AP (acidic protease, produced by Aspergillus niger) were diluted with distilled water to 5000 U / mL respectively.

[0041] Control group: Take 1 mL of diluted enzyme solution into a test tube, add 9 mL of distilled water, mix well, and immediately take 1 mL from the test tube to dilute with the corresponding enzyme activity detection buffer for enzyme activity detection. The enzyme activity is U0.

[0042] Experimental group: Pipette 1 mL of diluted enzyme solution into a test tube, add 9 mL of pH 3.0 buffer, mix well, and place the test tube in a 39℃ water bath shaker for 2 hours (180 rpm). After the water bath, take 1 mL from the test tube and dilute it with the corresponding enzyme activity detection buffer for enzyme activity detection. The enzyme activity is U / U0. Then calculate the enzyme activity retention rate. The calculation formula is as follows: Enzyme activity retention rate after gastric acid (%) = U / U0 × 100%.

[0043] B) Stability in endogenous enzymes

[0044] The compound protease of the present invention (composed of alkaline protease and neutral protease) and commercially available protease DP (alkaline protease, produced by Bacillus licheniformis), protease BB (composed of acidic protease, neutral protease and alkaline protease), protease KP (alkaline protease, produced by Pichia pastoris), protease LP (alkaline protease, produced by Bacillus licheniformis), protease NP (neutral protease, produced by Bacillus subtilis), and protease AP (acidic protease, produced by Aspergillus niger) were diluted with distilled water to 5000 U / mL respectively.

[0045] Control group: Take 1 mL of diluted enzyme solution into a test tube, add 9 mL of distilled water, mix well, and immediately take 1 mL from the test tube to dilute with the corresponding enzyme activity detection buffer for enzyme activity detection. The enzyme activity is U0.

[0046] Experimental group: Take 1 mL of diluted enzyme solution into a test tube, add 9 mL of simulated gastric juice, mix well, and place the test tube in a 39℃ water bath constant temperature shaker for 2 hours (180 rpm). After the water bath, take 1 mL from the test tube and dilute it with the corresponding enzyme activity detection buffer for enzyme activity detection. The enzyme activity is U gastric juice. Then calculate the enzyme activity retention rate. The calculation formula is as follows: Endogenous enzyme activity retention rate (%) = U gastric juice / U0 × 100%.

[0047] The experimental results are shown in Table 2.

[0048] Table 2. Results of enzyme activity retention in simulated gastric acid.

[0049] Sample Name Gastric acid retention rate Gastric retention rate (including endogenous enzymes) Complex protease 93.74％ 90.47％ DP protein 0.20％ 0％ Protease BB 0％ 0％ proteinase KP 84.06％ 5.64％ Protease LP 0％ 0％ Protease NP 99.56％ 0％

[0050] Example 3: Comparison of in vitro enzymatic hydrolysis effects of proteases with different ratios

[0051] Materials and Methods: Materials included proteases (alkaline protease, neutral protease, and carrier) and complete feed.

[0052] Reagent preparation: Hydrochloric acid solution (1 mol / L): Dissolve 90 mL of concentrated hydrochloric acid in 800 mL of distilled water, stir well, and bring the volume to 1000 mL; Sodium hydroxide solution (2 mol / L): Weigh 80 g of solid sodium hydroxide into 800 mL of distilled water, stir until dissolved, and bring the volume to 1000 mL; pH 2.80 gastric buffer: Dissolve 5.18 g of sodium chloride, 0.50 g of potassium chloride, 12 g of anhydrous sodium dihydrogen phosphate (15.60 g of sodium dihydrogen phosphate dihydrate), and 5 mL of BC10 solution in 800 mL of distilled water. Adjust the pH to 2.80 at 41°C with hydrochloric acid or sodium hydroxide solution, bring the volume to 1000 mL with distilled water, and refrigerate at 4°C; pH 6.91 intestinal buffer: 13.68 g anhydrous disodium hydrogen phosphate (34.47 g disodium hydrogen phosphate dodecahydrate), 38.80 g anhydrous sodium dihydrogen phosphate (50.44 g sodium dihydrogen phosphate dihydrate), dissolved in 800 mL distilled water, adjusted to pH 6.91 at 41°C with hydrochloric acid or sodium hydroxide solution, and brought to a final volume of 1000 mL with distilled water. Incubate at room temperature. Simulated gastric fluid (pepsin concentration 1722 U / mL): Weigh 0.2428 g pepsin (Sigma P7000), add to 80 mL of the corresponding pH gastric buffer solution, stir with a magnetic stirrer until dissolved (approximately 15 min), and bring to a final volume of 100 mL with pH 2.80 gastric buffer. Prepare fresh before use. Simulated intestinal fluid (trypsin concentration 543 U / mL): Weigh 0.3017 g trypsin (Amresco). Add 20 mL of distilled water to 0458, stir with a magnetic stirrer until dissolved (about 15 min), and bring the volume to 25 mL with distilled water. Prepare and use immediately.

[0053] During the reaction, the effective concentration of pepsin was approximately 1550 U / mL, and the effective concentration of trypsin was approximately 49.4 U / mL.

[0054] The experimental methods include:

[0055] A) Weighing centrifuge tubes: As needed, number the 50mL capped centrifuge tubes and dry them at 105℃ to constant weight. Place them in a desiccator to cool, and weigh them immediately with an electronic analytical balance after taking them out.

[0056] B) Mixed feed: Select different feed ingredients according to the experimental requirements and record the sample weight of the feed ingredients;

[0057] C) Enzyme solution dilution: The amount of enzyme solution added during the enzymatic hydrolysis process is 1 mL. Calculate the dilution factor of the enzyme based on the pre-set addition amount and dilute the enzyme with water.

[0058] D) In ​​vitro enzymatic hydrolysis process: stomach (41℃ incubation for 4 hours), then to intestine (41℃ incubation for 15 hours), the total reaction incubation time is 19 hours;

[0059] d1) Weigh 1.0000g (±0.0010) of mixed feed into a 50mL centrifuge tube, add 9mL of simulated gastric juice, mix well, add 1mL of enzyme solution diluted to the corresponding enzyme activity, mix well again, and then incubate in a shaking water bath at 41℃ and 180rpm for 4h. For the blank control, replace 1mL of enzyme solution with 1mL of water.

[0060] d2) After incubation for 4 hours, quickly add 6 mL of intestinal buffer, mix well, then add 1.6 mL of simulated intestinal fluid, and continue incubation at 41°C with shaking for 15 hours. Then centrifuge at 4000 rpm for 10 minutes and collect the supernatant for later use. Wash the centrifuge tube containing the residue with water and centrifuge three times, then wash three times with 95% alcohol (the residue must be mixed with water or alcohol before each centrifugation). Then place the residue in a 60°C constant temperature drying oven for 12 hours, and then transfer it to a 105°C constant temperature drying oven to dry to constant weight. Weigh it with an electronic analytical balance.

[0061] E) Detection index - small peptide content

[0062] After enzymatic hydrolysis, the sample was centrifuged at 4000 rpm for 10 min, and the supernatant was collected. The supernatant was then precipitated with 30% trichloroacetic acid solution at a 1:1 ratio to precipitate the large molecular weight protein. The supernatant was centrifuged at 4000 rpm for 10 min, and the supernatant was diluted with water to the appropriate factor. 1.0 mL of the supernatant was placed in a 10×150 mm test tube, and 5.0 mL of basic copper reagent was added to each tube. The mixture was vortexed for 30 s and allowed to stand at room temperature for 10 min. Then, 0.5 mL of Folin-Ciocalteu reagent was added sequentially, and the mixture was vortexed for 30 s and allowed to stand at room temperature for 30 min. The absorbance A was measured at 660 nm using a 1 cm cuvette. The corresponding concentration was determined from the standard curve, and the small peptide content C and the small peptide increment ΔC were calculated.

[0063] The results are shown in Table 3. Data on the increase in small peptides indicate that the formulations containing 30% alkaline protease, 30% neutral protease, and 40% talc as the carrier exhibited the best in vitro enzymatic hydrolysis performance. Among different carriers, talc had a slightly better impact on product performance than other carriers; therefore, talc was selected for sample preparation in subsequent trials.

[0064] Table 3 Comparison of in vitro enzymatic hydrolysis effects of different protease formulations

[0065]

[0066] Example 4: Evaluation of the in vitro enzymatic hydrolysis effect of different protease samples

[0067] 1) Experimental materials

[0068] Proteases: Protease No. 1 (commercially available, composed of 60% neutral-alkaline protease and 40% carrier), Protease No. 2 (commercially available, composed of 20% neutral protease, 25% acidic protease, 15% alkaline protease and 40% carrier), Protease No. 3 (commercially available, composed of 20% neutral protease, 5% acidic protease, 35% alkaline protease and 40% carrier), Protease No. 4 (the complex protease product of this invention, composed of 30% alkaline protease, 30% neutral protease and 40% carrier);

[0069] Feed ingredients: Soybean meal, corn gluten meal, and complete broiler feed were used as experimental substrates for verification;

[0070] Reagent preparation: Hydrochloric acid solution (1 mol / L): Dissolve 90 mL of concentrated hydrochloric acid in 800 mL of distilled water, stir well, and bring the volume to 1000 mL; Sodium hydroxide solution (2 mol / L): Weigh 80 g of solid sodium hydroxide into 800 mL of distilled water, stir until dissolved, and bring the volume to 1000 mL; pH 2.80 gastric buffer: Dissolve 5.18 g of sodium chloride, 0.50 g of potassium chloride, 12 g of anhydrous sodium dihydrogen phosphate (15.60 g of sodium dihydrogen phosphate dihydrate), and 5 mL of BC10 solution in 800 mL of distilled water. Adjust the pH to 2.80 at 41°C with hydrochloric acid or sodium hydroxide solution, bring the volume to 1000 mL with distilled water, and refrigerate at 4°C; pH 6.91 intestinal buffer: 13.68 g anhydrous disodium hydrogen phosphate (34.47 g disodium hydrogen phosphate dodecahydrate), 38.80 g anhydrous sodium dihydrogen phosphate (50.44 g sodium dihydrogen phosphate dihydrate), dissolved in 800 mL distilled water, adjusted to pH 6.91 at 41°C with hydrochloric acid or sodium hydroxide solution, and brought to a final volume of 1000 mL with distilled water. Incubate at room temperature. Simulated gastric fluid (pepsin concentration 1722 U / mL): Weigh 0.2428 g pepsin (Sigma P7000), add to 80 mL of the corresponding pH gastric buffer solution, stir with a magnetic stirrer until dissolved (approximately 15 min), and bring to a final volume of 100 mL with pH 2.80 gastric buffer. Prepare fresh before use. Simulated intestinal fluid (trypsin concentration 543 U / mL): Weigh 0.3017 g trypsin (Amresco). Add 20 mL of distilled water to 0458, stir with a magnetic stirrer until dissolved (about 15 min), and bring the volume to 25 mL with distilled water. Prepare and use immediately.

[0071] During the reaction, the effective concentration of pepsin was approximately 1550 U / mL, and the effective concentration of trypsin was approximately 49.4 U / mL.

[0072] 2) Experimental methods

[0073] A) Weighing centrifuge tubes: As needed, number the 50mL capped centrifuge tubes and dry them at 105℃ to constant weight. Place them in a desiccator to cool, and weigh them immediately with an electronic analytical balance after removing them from the desiccator.

[0074] B) Mixed feed: Select different feed ingredients according to the experimental requirements and record the sample weight of the feed ingredients.

[0075] C) Enzyme solution dilution: The amount of enzyme solution added during the enzymatic hydrolysis process is 1 mL. Calculate the dilution factor of the enzyme according to the pre-set addition amount and dilute the enzyme with water.

[0076] D) In ​​vitro enzymatic hydrolysis process: stomach (41℃ incubation for 4 hours) to intestine (41℃ incubation for 15 hours), the total reaction incubation time is 19 hours;

[0077] d1) Weigh 1.0000±0.0010g of mixed feed into a 50mL centrifuge tube, add 9mL of simulated gastric juice, mix well, add 1mL of enzyme solution diluted to the corresponding enzyme activity, mix well again, and then incubate in a shaking water bath at 41℃ and 180rpm for 4h. For the blank control, replace 1mL of enzyme solution with 1mL of water.

[0078] d2) After incubation for 4 hours, quickly add 6 mL of intestinal buffer, mix well, then add 1.6 mL of simulated intestinal fluid, and continue incubation at 41°C with shaking for 15 hours. Then centrifuge at 4000 rpm for 10 minutes and collect the supernatant for later use. Wash the centrifuge tube containing the residue with water and centrifuge three times, then wash three times with 95% alcohol (the residue must be mixed with water or alcohol before each centrifugation). Then place the residue in a 60°C constant temperature drying oven for 12 hours, and then transfer it to a 105°C constant temperature drying oven to dry to constant weight. Weigh it with an electronic analytical balance.

[0079] E) Detection index - small peptide content

[0080] After enzymatic hydrolysis, the sample was centrifuged at 4000 r / min for 10 min, and the supernatant was collected. The supernatant was precipitated with 30% trichloroacetic acid solution at a ratio of 1:1, and centrifuged at 4000 r / min for 10 min. The supernatant was diluted with water to the appropriate factor, and 1.0 mL was taken into a 10×150 mm test tube. 5.0 mL of basic copper reagent was added to each tube, vortexed for 30 s, and placed at room temperature for 10 min. Then, 0.5 mL of Folin-Ciocalteu reagent was added sequentially, vortexed for 30 s, and placed at room temperature for 30 min. The absorbance value A was detected at 660 nm using a 1 cm cuvette. The corresponding concentration was determined on the standard curve, and the small peptide content C and the small peptide increment ΔC were calculated.

[0081] Table 4 Experimental Design

[0082] Group deal with Comparison No enzymes added Sample 1 Protease No. 1 500g / t Sample 2 Protease No. 2 500g / t Sample 3 500g / t of protease No. 3 Sample 4 500g / t of protease No. 4

[0083] The experimental conditions are shown in Table 4, and the results are shown in Table 5. It can be seen that after in vitro enzymatic hydrolysis, sample 4 showed the best enzymatic hydrolysis effect on soybean meal substrate, with a 12.94 mg / g increase in small peptide content compared to the blank control group. Sample 1 was the second best, while samples 2 and 3 showed almost no increase in small peptide content. Sample 4 also showed the best enzymatic hydrolysis effect on corn gluten meal, with a 7.50 mg / g increase in small peptide content compared to the blank control group. In the enzymatic hydrolysis of complete feed substrates, samples 1 and 4 showed comparable effects, with increases of 2.90 mg / g and 2.47 mg / g in small peptide content, respectively, compared to the blank control group.

[0084] Table 5. In vitro enzymatic digestion results of different protease samples

[0085]

[0086] The degradation of antigenic proteins after enzymatic hydrolysis of soybean meal, as shown in the following results. Figure 1 As shown, after in vitro enzymatic hydrolysis, the antigen protein detection results of enzymatically hydrolyzed soybean meal showed that samples 4 and 1 had relatively fewer large molecular proteins and more small molecules decomposed, while the antigen protein degradation ability of protease group 3 was slightly lower than that of 4 and 1, but better than that of 2.

[0087] Example 5: Effects of different protease formulations on broiler production performance

[0088] Experimental materials: The experimental animals were 8 healthy AA broiler chicks of similar weight;

[0089] Experimental Design and Treatment: 4368 one-day-old AA broilers were selected and randomly divided into 7 groups, with 8 replicates in each group and 78 birds in each replicate. The experimental grouping is shown in Table 6. The experiment started from one day old and lasted for 42 days. The specific experimental design is shown in Table 6.

[0090] Table 6 Experimental Design

[0091]

[0092] Experimental diet: The experiment used a corn and soybean meal basal diet, which was pelleted. The experimental diet was formulated by the experimental plant according to the general formula of the experimental feed. The formula composition and nutritional level were the same as in Table 7. The experimental feed was pelleted.

[0093] Table 7. Composition and Nutritional Indicators of Dietary Formulas

[0094]

[0095]

[0096]

[0097] The experimental results are shown in Table 8. Significant differences (P<0.05) were observed among the broiler groups in terms of slaughter weight, average daily weight gain, feed conversion ratio, and European index. Groups G and E showed the best performance in slaughter weight, average daily weight gain, feed conversion ratio, and European index. Compared to the control group, slaughter weight increased by 81g and 71g in groups G and E, respectively, while average daily weight gain increased by 1.95g / day and 1.70g / day, respectively. Group G had the lowest feed conversion ratio, followed by group E. Compared to the control group, groups G and E improved their feed conversion ratio by 0.047 and 0.041, respectively. The trend in the European index was consistent with that of weight, daily weight gain, and feed conversion ratio, with group G showing the best performance, increasing its European index by 27 compared to the control group. Group E also showed an increase of 22 compared to the control group.

[0098] Table 8 Broiler production performance (1-42 days old)

[0099]

[0100]

[0101] Example 6: Effects of different proteases on the growth performance of broiler chickens

[0102] Materials and Methods: 3744 healthy AA broiler chicks of similar weight were used; the proteases included protease 1 (commercially available, composed of 60% neutral-alkaline protease and 40% carrier), protease 2 (commercially available, composed of 20% neutral protease, 25% acidic protease, 15% alkaline protease and 40% carrier), protease 3 (commercially available, composed of 20% neutral protease, 5% acidic protease, 35% alkaline protease and 40% carrier), and protease 4 (the composite protease product of this invention, composed of 30% alkaline protease, 30% neutral protease and 40% carrier);

[0103] Experimental method: 3744 one-day-old AA broilers were randomly divided into 6 groups, with 8 replicates in each group and 78 birds in each replicate. The experiment started from one day old and lasted for 42 days. The specific experimental design is shown in Table 9.

[0104] Table 9 Experimental Design

[0105] coding Group Daily food feed form A control group PC Basic diet (no additional samples needed) Granular material B Negative control NC Basal diet (reduced by 1% soybean meal, reduced by 3% ± amino acids) Granular material C Experimental group 1 NC diet + 500g / t Protease No. 1 Granular material D Experimental group 2 NC diet + 500g / t Protease No. 2 Granular material E Experimental group 3 NC diet + 500g / t Protease No. 3 Granular material F Experimental group 4 NC diet + 500g / t of protease No. 4 Granular material

[0106] The experiment used a corn and soybean meal-based basal diet, which was pelleted. The experimental diet was formulated by the experimental plant according to the general formula of the experimental feed. The formula composition and nutritional level were the same as those in Table 10. The experimental feed was pelleted.

[0107] Table 10. Dietary Formula Composition and Nutritional Indicators

[0108]

[0109]

[0110]

[0111] Uniform feeding management and immunization programs were implemented. During the experiment, chickens were artificially fed and had free access to water. Cumulative feed intake from day 1 to 42 was recorded in replicates to calculate the average daily feed intake during the experiment. The entire flock was weighed at 42 days of age in replicates to calculate average body weight and feed conversion ratio at each stage, and to ultimately assess the economic benefits of broiler farming. The health status of the flock was observed daily during the experiment, and the number of dead chickens was recorded and weighed in replicates to calculate the mortality rate.

[0112] As shown in Table 11, statistically significant differences were observed among the broiler groups in terms of slaughter weight and average daily weight gain. Group B (negative control group) had the lowest scores in weight, daily weight gain, and European index, indicating that the reduction in dietary protein levels had a significant adverse effect on the normal growth and development of broilers. Compared with Group B, Group F (compound protease of this invention) and Group E (protease No. 3) showed significantly higher slaughter weight and average daily weight gain, by 32.9g and 0.80g, and 27.5g and 0.6g, respectively. The feed conversion ratio was 7g (P<0.05), and the overall level was close to that of the positive control group, with no statistical difference (P>0.05). As for the feed conversion ratio, there was no significant difference among the groups (P>0.05), but the lowest was group A (positive control), and the highest was group B. Compared with group B, the best feed conversion ratio was also group F and group E, which reduced the feed conversion ratio by 0.013 and 0.012, respectively. Although there was no significant difference in survival rate and European index (P>0.05), group F showed the best results.

[0113] Table 11 Broiler production performance (1-42 days old)

[0114]

[0115]

[0116] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A compound protease for feed, characterized in that, The complex protease includes an alkaline protease and a neutral protease, wherein the weight ratio of the alkaline protease is 30-40% and the weight ratio of the neutral protease is 30-35%. The alkaline protease is derived from Bacillus licheniformis, and the neutral protease is derived from Bacillus amyloliquefaciens. The amino acid sequence of the alkaline protease is shown in SEQ ID NO: 1, and the amino acid sequence of the neutral protease is shown in SEQ ID NO:

2. The complex protease also includes a carrier, which is selected from one or more of talc, zeolite, sodium sulfate, corn starch, rice husk powder, and maifanite powder.

2. The feed-grade compound protease according to claim 1, characterized in that, The alkaline protease has a weight ratio of 30%, the neutral protease has a weight ratio of 30%, and the carrier has a weight ratio of 40%.

3. The application of the feed-grade complex protease as described in claim 1 or 2 in animal feed.

4. Feed additives and feed products containing the feed-grade complex protease as described in claim 1 or 2.

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