A process for preparing a biological composition

Abstract

The present invention relates to the biological field, and in particular to a biological composition preparation process, an enzyme and an application thereof, wherein the composition is a milk-derived active peptide, and the enzyme comprises a sequence shown in any one of SEQ ID NOs: 2-5. In particular, the enzyme having an amino acid sequence as shown in SEQ ID NOs: 4 or 5 has significantly improved hydrolysis activity when preparing the biological composition milk-derived active peptide, and the milk-derived active peptide has good anti-fatigue activity. Therefore, it has a good market prospect.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a biological composition preparation process, an enzyme and an application thereof. Background Art

[0002] Many biological activities of milk-derived active peptides have been identified, including immunomodulatory, antibacterial, analgesic, anti-fatigue, anti-inflammatory, anti-cancer, antiviral, antioxidant, anti-thrombotic, anti-obesity, anti-cytotoxic, anti-hypertensive and antiseptic properties. Milk-derived active peptides have been identified as potential ingredients for functional foods that promote health. These bioactive peptides have multifunctional properties against diet-related chronic diseases, especially non-communicable diseases, namely obesity, cardiovascular disease and diabetes. Milk contains approximately 3.5% protein, of which approximately 80% is casein and 20% is whey protein. Summary of the invention

[0003] In one aspect of the present invention, a process for preparing a biological composition is provided, wherein the process comprises the following steps:

[0004] Take skimmed milk powder, evenly disperse it in water, and use ultrafiltration membrane to ultrafilter to obtain milk protein liquid; use buffer to adjust pH to 7-9, add mutant of subtilisin enzyme to 0.5-6.0 mg / mL, and hydrolyze at 45-65°C.

[0005] In some embodiments of the present invention, a process for preparing a biological composition is provided, characterized in that the preparation process comprises the following steps:

[0006] Take skimmed milk powder, evenly disperse it in water, and use ultrafiltration membrane to obtain milk protein liquid; use buffer to adjust the pH to 7-9, add enzyme with amino acid sequence as shown in SEQ ID NO: 4 or 5 to 0.5-6.0 mg / mL, and hydrolyze at 45-65°C.

[0007] In some embodiments of the present invention, the mutant of the Subtilisin enzyme comprises a sequence shown in any one of SEQ ID NOs: 2-5.

[0008] In some embodiments of the present invention, the amino acid sequence of the mutant of the Subtilisin enzyme is shown in any one of SEQ ID NOs: 2-5.

[0009] In some embodiments of the present invention, the amino acid sequence of the mutant of the Subtilisin enzyme is shown in SEQ ID NO: 4 or 5.

[0010] In some embodiments of the present invention, the amino acid sequence of the mutant of the Subtilisin enzyme is shown in SEQ ID NO:2.

[0011] In some embodiments of the present invention, the amino acid sequence of the mutant of the Subtilisin enzyme is shown in SEQ ID NO:3.

[0012] In some embodiments of the present invention, the amino acid sequence of the mutant of the Subtilisin enzyme is shown in SEQ ID NO:4.

[0013] In some embodiments of the present invention, the amino acid sequence of the mutant of the Subtilisin enzyme is shown in SEQ ID NO:5.

[0014] In some embodiments of the present invention, the skim milk powder is uniformly dispersed in 5 times the weight of water.

[0015] In some embodiments of the present invention, ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to remove small-molecule lactose and obtain a macromolecular milk protein solution.

[0016] In some embodiments of the invention, a buffer is used to adjust the pH to 8.5.

[0017] In some embodiments of the invention, tripotassium citrate is used to adjust the pH to 8.5.

[0018] In some embodiments of the present invention, the mutant of the Subtilisin enzyme is added to 5.0 mg / mL.

[0019] In some embodiments of the present invention, the hydrolysis reaction temperature is 50°C.

[0020] In some embodiments of the present invention, the hydrolysis reaction time is 0.5 h.

[0021] In some embodiments of the present invention, the hydrolyzate is diluted with 3 times the amount of water, ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 6 kDa to obtain a small molecule peptide solution, and freeze-dried to obtain milk-derived active peptides.

[0022] In some embodiments of the present invention, the preparation process includes the steps described in Example 3.

[0023] In some embodiments of the present invention, the preparation process includes the steps described in Example 4.

[0024] In some embodiments of the present invention, the preparation process comprises the following steps:

[0025] Take skimmed milk powder, evenly disperse it in 5 times the weight of water, use an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to ultrafilter, and obtain a macromolecular milk protein liquid; use tripotassium citrate to adjust the pH to 8.5, add a mutant of subtilisin enzyme with an amino acid sequence as shown in SEQ ID NO:4 or 5 to 5.0 mg / mL; carry out a hydrolysis reaction at 50°C for 0.5h to obtain a hydrolyzate; then dilute the hydrolyzate with 3 times the amount of water, use an ultrafiltration membrane with a molecular weight cutoff of 6 kDa to remove macromolecules, and obtain a small molecular peptide liquid; freeze-dry, and obtain milk-derived active peptides.

[0026] In some embodiments of the present invention, the composition is a milk-derived active peptide. In some embodiments of the present invention, the composition is a milk-derived active peptide obtained by hydrolyzing milk powder.

[0027] In some embodiments of the present invention, the steps of the preparation process are as follows:

[0028] Take skimmed milk powder, evenly disperse it in 5 times the weight of water, use an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to ultrafilter, and obtain a macromolecular milk protein liquid; use tripotassium citrate to adjust the pH to 8.5, add a mutant of subtilisin enzyme with an amino acid sequence as shown in SEQ ID NO:4 or 5 to 5.0 mg / mL; carry out a hydrolysis reaction at 50°C for 0.5h to obtain a hydrolyzate; then dilute the hydrolyzate with 3 times the amount of water, use an ultrafiltration membrane with a molecular weight cutoff of 6 kDa to remove macromolecules, and obtain a small molecular peptide liquid; freeze-dry, and obtain milk-derived active peptides.

[0029] In some embodiments of the present invention, the steps of the preparation process are as follows:

[0030] Take skimmed milk powder, evenly disperse it in 5 times the weight of water, and ultrafilter it using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain a macromolecular milk protein liquid; use tripotassium citrate to adjust the pH to 8.5, add a mutant of the subtilisin enzyme with an amino acid sequence as shown in SEQ ID NO: 4 or 5 to 5.0 mg / mL; hydrolyze at 50° C. for 0.5 h to obtain a hydrolyzate; then dilute the hydrolyzate with 3 times the amount of water, and ultrafilter it through an ultrafiltration membrane with a molecular weight cutoff of 6 kDa to remove macromolecules to obtain a small molecular peptide liquid; and freeze-dry to obtain.

[0031] In some embodiments of the present invention, the steps of the preparation process are as follows:

[0032] Take skimmed milk powder, evenly disperse it in 5 times the weight of water, use an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to ultrafilter, and obtain a macromolecular milk protein liquid; use tripotassium citrate to adjust the pH to 8.5, add a mutant of Subtilisin enzyme with an amino acid sequence as shown in SEQ ID NO:4 to 5.0 mg / mL; carry out a hydrolysis reaction at 50°C for 0.5h to obtain a hydrolyzate; then dilute the hydrolyzate with 3 times the amount of water, use an ultrafiltration membrane with a molecular weight cutoff of 6 kDa to remove macromolecules, and obtain a small molecular peptide liquid; freeze-dry, and obtain milk-derived active peptides.

[0033] In some embodiments of the present invention, the steps of the preparation process are as follows:

[0034] Take skimmed milk powder, evenly disperse it in 5 times the weight of water, use an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to ultrafilter, and obtain a macromolecular milk protein liquid; use tripotassium citrate to adjust the pH to 8.5, add a mutant of Subtilisin enzyme with an amino acid sequence as shown in SEQ ID NO:5 to 5.0 mg / mL; carry out a hydrolysis reaction at 50°C for 0.5h to obtain a hydrolyzate; then dilute the hydrolyzate with 3 times the amount of water, use an ultrafiltration membrane with a molecular weight cutoff of 6 kDa to remove macromolecules, and obtain a small molecular peptide liquid; freeze-dry, and obtain milk-derived active peptides.

[0035] In another aspect of the present invention, a preparation process of a composition for relieving fatigue is provided, wherein the composition is a milk-derived active peptide, and the steps of the preparation process are as follows:

[0036] Take skimmed milk powder, evenly disperse it in 5 times the weight of water, use an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to ultrafilter, and obtain a macromolecular milk protein liquid; use tripotassium citrate to adjust the pH to 8.5, add a mutant of subtilisin enzyme with an amino acid sequence as shown in SEQ ID NO:4 or 5 to 5.0 mg / mL; carry out a hydrolysis reaction at 50°C for 0.5h to obtain a hydrolyzate; then dilute the hydrolyzate with 3 times the amount of water, use an ultrafiltration membrane with a molecular weight cutoff of 6 kDa to remove macromolecules, and obtain a small molecular peptide liquid; freeze-dry, and obtain milk-derived active peptides.

[0037] In another aspect of the present invention, an enzyme is provided, characterized in that the amino acid sequence of the enzyme comprises any one of the sequences shown in SEQ ID NO: 2-5.

[0038] In some embodiments of the present invention, the amino acid sequence of the enzyme is shown in any one of SEQ ID NOs: 2-5. In some embodiments of the present invention, the amino acid sequence of the enzyme is shown in SEQ ID NOs: 4 or 5. In some embodiments of the present invention, the amino acid sequence of the enzyme is shown in SEQ ID NO: 2. In some embodiments of the present invention, the amino acid sequence of the enzyme is shown in SEQ ID NO: 3. In some embodiments of the present invention, the amino acid sequence of the enzyme is shown in SEQ ID NO: 4. In some embodiments of the present invention, the amino acid sequence of the enzyme is shown in SEQ ID NO: 5.

[0039] In another aspect of the present invention, a nucleic acid is provided, characterized in that the nucleic acid encodes the enzyme described in the present invention.

[0040] In another aspect of the present invention, there is provided use of the enzyme according to the present invention in preparing a biological composition.

[0041] In some embodiments of the present invention, the biological composition is a milk-derived active peptide.

[0042] In some embodiments of the invention, the biological composition has fatigue reducing activity.

[0043] In some embodiments of the present invention, the biological composition is a milk-derived active peptide that can reduce fatigue.

[0044] The technical effect of the technical solution of the present invention is as follows: the Subtilisin mutant of the present invention can be used to prepare a biological composition milk-derived active peptide, the mutant has significantly improved hydrolysis activity, and the milk-derived active peptide has good anti-fatigue activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The amino acid residues near the catalytic active center of subtilisin are given as an example, assuming that the expected mature peptide is substantially consistent with the catalytic active center of subtilisin. DETAILED DESCRIPTION

[0046] Example 1: Mutant Design

[0047] Based on the alkaline protease Subtilisin (SEQ ID NO: 1) from Bacillus subtilis, the inventors first truncated the expected mature peptide and then designed a series of mutants based on the following assumptions.

[0048] Without being bound by theory, the hypothesis is: it is assumed that replacing the amino acid residues near the catalytic active center with small-volume amino acid residues may reduce the steric hindrance effect of binding to the substrate and enhance the activity of the alkaline protease; the proteins with the highest content in bovine milk are usually mainly four caseins and two whey proteins, and the amino acid residues with the highest content are mainly E, K, L, and P, and the alkaline protease usually has low specificity in the catalytic site. Therefore, it is assumed that after mutating the amino acid residues near the catalytic active center of the alkaline protease to residues R / D / F / W that have stronger interactions with E / K / L / P, the catalytic activity can be improved by enhancing the stability of the enzyme's catalytic intermediate conformation.

[0049] The designed mutants are BS-MU1 (SEQ ID NO: 2), BS-MU2 (SEQ ID NO: 3), BS-MU3 (SEQ ID NO: 4), and BS-MU4 (SEQ ID NO: 5), which are then tested and screened.

[0050] Example 2. Preparation of mutants

[0051] The target gene fragments were synthesized outsourced according to the reference method, i.e., the coding DNA fragments of the enzymes and mutants shown in SEQ ID NOs: 1-5 with codon optimization (ExpOptimizer) for Bacillus subtilis, and the ends contained restriction endonuclease cleavage sites of the corresponding restriction endonucleases.

[0052] The original constitutive promoter P of the pMA5 plasmid HpaII Replaced with maltose-inducible promoter P malA , and obtained pMA5-MalA plasmid (SEQ ID NO: 6).

[0053] The target gene fragments were cloned into the pMA5-MalA plasmid through the NdeI and BamHI restriction sites. The ligation products were transformed into Escherichia coli DH5α competent cells, and the transformed bacterial solution was spread on an LB plate containing ampicillin to screen positive clones to complete the construction of the recombinant plasmids of the enzyme and mutants. For example, the recombinant plasmid sequence of BS-MU3 (SEQ ID NO: 4) is shown in SEQ ID NO: 7.

[0054] The recombinant plasmid was transformed into Bacillus subtilis (Lianzu, catalog number LZ-JZ0654) using the Spizizen chemical transformation method, and positive transformants were verified using colony PCR. The cells were cultured for 20 h in LB agar plates and LB liquid medium containing the corresponding antibiotics for activation. The cells were inoculated into 2×SR fermentation medium (yeast powder 50 g / L, peptone 30 g / L, K 2 HPO 4 6 g / L, maltose 30 g / L) and cultured at 37°C for 50 h.

[0055] The cells were collected by centrifugation, lysate was added and ultrasonically disrupted, and the supernatant was centrifuged again. The supernatant was filtered through a 0.45 um filter membrane, desalted using a HiPrep 26 / 10 desalting column, purified using ion chromatography and RP-HPLC, and the eluate was freeze-dried to obtain Subtilisin enzyme (SEQ ID NO: 1) with a purity of 96.8%-97.9% and its mutants BS-MU1 (SEQ ID NO: 2), BS-MU2 (SEQ ID NO: 3), BS-MU3 (SEQ ID NO: 4), and BS-MU4 (SEQ ID NO: 5).

[0056] Example 3. Optimal hydrolysis conditions for mutants

[0057] Skim milk powder (Sigma, item number 70166) was uniformly dispersed in water at a specific ratio (200 mg / mL), and ultrafiltration was performed using an ultrafiltration membrane (Merck) with a molecular weight cutoff of 10 kDa to remove small-molecule lactose to obtain a large-molecule milk protein solution. The pH was adjusted to 7-9 using a buffer solution, and the subtilisin enzyme or any mutant thereof prepared in Example 2 was added to 0.5-6.0 mg / mL, respectively. The mixture was hydrolyzed at 45-65°C.

[0058] Amine nitrogen was determined by potentiometric titration with formaldehyde and total nitrogen was determined by Kjeldahl method. The degree of hydrolysis was calculated.

[0059] Degree of hydrolysis (DH) % = amino nitrogen / total nitrogen × 100%.

[0060] By exploring the reaction conditions within the above parameter range, it was found that the optimal hydrolysis conditions of Subtilisin enzyme and its mutants were basically the same, namely about 50°C and pH 8.5.

[0061] Example 4. Preparation and purification of milk-derived active peptides

[0062] Take skim milk powder (Sigma, item number 70166), evenly disperse it in 5 times the weight of water (200 mg milk powder / 1 mL water), and use an ultrafiltration membrane (Merck) with a molecular weight cutoff of 10 kDa to ultrafilter (remove small molecular lactose and other components) to obtain a macromolecular milk protein solution. Use tripotassium citrate to adjust the pH to 8.5, and add the Subtilisin enzyme or any mutant thereof prepared in Example 2 to 5.0 mg / mL. Hydrolyze the reaction at 50 ° C for 0.5 h to obtain a hydrolyzate. Then dilute the hydrolyzate with 3 times the amount of water, and ultrafilter through an ultrafiltration membrane with a molecular weight cutoff of 6 kDa to remove macromolecules to obtain a small molecule peptide solution. Freeze-dry to obtain milk-derived active peptides.

[0063] The hydrolyzate was subjected to the formaldehyde potentiometric titration method to determine the amino nitrogen, and the Kjeldahl method to determine the total nitrogen. The degree of hydrolysis of the hydrolyzate was calculated. Degree of hydrolysis (DH) % = amino nitrogen / total nitrogen × 100%. The test was repeated 5 times, and the results are shown in Table 1, expressed as mean ± standard deviation.

[0064] Table 1. Milk protein hydrolysis test results

[0065] Enzymes Degree of hydrolysis (%) Subtilisin 12.5±3.9 BS-MU1 7.3±2.0 BS-MU2 3.6±3.8 BS-MU3 39.4±6.2 BS-MU4 27.7±5.1

[0066] The hydrolysis activities of BS-MU3 and BS-MU4 were significantly better than those of Subtilisin, BS-MU1 and BS-MU1, all reaching statistically significant differences (P<0.01).

[0067] Example 5. Anti-fatigue activity test of milk-derived active peptides

[0068] Adult healthy male white mice of Kunming species (weight 21-26g) were randomly divided into groups, 10 mice in each group, and normal diet and drinking water were taken every day. The milk-derived active peptides obtained by hydrolysis of Subtilisin enzyme or its mutants were dissolved in sterile water to prepare a 10mg / mL solution, and 0.2mL was given to mice by gavage every day. The negative control group was gavaged with 0.2mL sterile water every day. It was given continuously for 30 days. On the 31st day, the root of the mouse tail was loaded with 5% lead sheet of body weight, placed in a swimming box with a water depth of 50cm and a water temperature of 25°C to swim, and the time from the start of swimming to the death of the mouse was recorded, which was the weight-bearing swimming time of the mouse. The results are shown in Table 2, expressed by average value. The weighted swimming time of mice in the BS-MU1 and BS-MU4 groups was comparable to that of the Subtilisin group (P>0.05), but was better than that of the negative control group (P<0.01). Surprisingly, the weighted swimming time of mice in the BS-MU3 group was better than that of the Subtilisin group, with a statistically significant difference (P<0.01), which proved that the anti-fatigue activity of the milk-derived active peptides obtained by hydrolysis of BS-MU3 was better than that of the Subtilisin hydrolysate.

[0069] Table 2. Anti-fatigue activity test results of milk-derived active peptides

[0070] Grouping Mouse weight-bearing swimming time (s) Subtilisin hydrolyzed milk-derived active peptides 691 Milk-derived active peptides obtained by hydrolysis of BS-MU1 685 Milk-derived active peptides obtained by hydrolysis of BS-MU3 857 Milk-derived active peptides obtained by hydrolysis of BS-MU4 713 Negative control group 602

[0071] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A process for preparing a biological composition, characterized in that: The preparation process steps are as follows: Take skimmed milk powder, evenly disperse it in 5 times the weight of water, and ultrafilter it using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain a large-molecule milk protein liquid; use tripotassium citrate to adjust the pH to 8.5, and add a mutant of the subtilisin enzyme with an amino acid sequence as shown in SEQ ID NO:4 to 5.0 mg / mL; hydrolyze at 50°C for 0.5h to obtain a hydrolyzate; then dilute the hydrolyzate with 3 times the amount of water, and ultrafilter it through an ultrafiltration membrane with a molecular weight cutoff of 6 kDa to remove large molecules to obtain a small-molecule peptide liquid; freeze-dry to obtain.

2. An enzyme, characterized in that The amino acid sequence of the enzyme is shown in SEQ ID NO:

4.

3. A nucleic acid, characterized in that The nucleic acid encodes the enzyme according to claim 2.

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