Stable liquid alkaline protease preparation as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly provides a stable liquid alkaline protease preparation which comprises liquid alkaline protease, NaCl, polysaccharide and histidine. Based on the mass of the liquid alkaline protease, the addition amount of NaCl is 5%-10%, the addition amount of polysaccharide is 0.1%-0.5%, and the addition amount of histidine is 0.02%-0.1%. According to the stable liquid alkaline protease preparation provided by the invention, NaCl, polysaccharide and histidine are added into alkaline protease, filtration and sterilization are performed after pH is adjusted, the stability of the liquid alkaline protease is obviously improved, heat preservation is performed for 6 months at the temperature of 45 DEG C, and the enzyme activity is basically free of loss. The preparation has simple components and is especially suitable for feed and food industries.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a stable liquid alkaline protease preparation, its preparation method, and its application. Background Technology

[0002] Alkaline proteases are a class of enzymes that can degrade proteins to produce polymorphic amino acids, and are widely used in the feed, food, and pharmaceutical industries. Compared with conventional acid and alkaline hydrolysis of proteins, using proteases to hydrolyze proteins has advantages such as mild reaction conditions, high hydrolysis specificity, and no need for pH adjustment or desalting after hydrolysis.

[0003] In the use of proteases, liquid proteases have advantages such as not producing dust and being able to mix quickly with proteins, making them applicable in far more scenarios than solid products. However, due to their ability to hydrolyze themselves, liquid proteases suffer from poor product stability, rapid decline in enzyme activity, and a much shorter shelf life than solid products.

[0004] Existing methods for improving the stability of alkaline proteases mainly include adding polyols, inorganic salts, polysaccharides, protease inhibitors, and surfactants. The drawback is that the amount of polyols added is large, often exceeding 20% ​​of the enzyme preparation, which limits the final enzyme activity. Furthermore, using protease inhibitors or surfactants poses certain risks to the application of alkaline proteases in the food industry. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of poor stability of liquid proteases and the influence of additives on product performance in the prior art.

[0006] Therefore, the present invention provides a stable liquid alkaline protease preparation, comprising liquid alkaline protease, NaCl, polysaccharide and histidine; based on the mass of liquid alkaline protease, the amount of NaCl added is 5%-10%, the amount of polysaccharide added is 0.1%-0.5%, and the amount of histidine added is 0.02%-0.1%.

[0007] Specifically, the enzyme activity in the above-mentioned liquid alkaline protease is 100,000-500,000 U / mL.

[0008] Specifically, the polysaccharides mentioned above include one or more of soluble soybean polysaccharides, chitosan, trehalose, and sodium carboxymethyl cellulose.

[0009] The present invention also provides a method for preparing the above-mentioned stable liquid alkaline protease preparation, comprising the following steps: filtering the microbial fermentation broth that produces alkaline protease, centrifuging or allowing it to stand and decanting to obtain a supernatant containing alkaline protease, adding NaCl, polysaccharide and histidine to it according to the mass ratio, mixing evenly and dissolving, adjusting the pH and filtering to remove bacteria to obtain a stable liquid alkaline protease preparation.

[0010] Specifically, the aforementioned microorganisms include Bacillus or recombinant engineered bacteria; the recombinant engineered bacteria contain a target gene derived from Bacillus that expresses an alkaline protease.

[0011] Specifically, the aforementioned Bacillus species include any one of Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus amyloliquefaciens, and Bacillus belye.

[0012] Specifically, the polysaccharides mentioned above include one or more of soluble soybean polysaccharides, chitosan, trehalose, and sodium carboxymethyl cellulose;

[0013] Specifically, the pH is adjusted to 4.5-5.5 before filtration for sterilization.

[0014] Specifically, the above-mentioned process involves adjusting the pH to 4.5-5.5 with acid before filtration for sterilization; the acid includes any one of HCl, H2SO4, H3PO4, formic acid, acetic acid, and citric acid.

[0015] The present invention also provides the application of the above-mentioned stable liquid alkaline protease preparation in the feed, food and pharmaceutical industries.

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

[0017] The stable liquid alkaline protease formulation provided by this invention significantly improves the stability of the liquid alkaline protease by adding NaCl, polysaccharides, and histidine to the alkaline protease, adjusting the pH, and then filtering for sterilization. After incubation at 45°C for 6 months, the enzyme activity remains virtually unaffected. Furthermore, this formulation has a simple composition, making it particularly suitable for the feed and food industries.

[0018] The present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0019] Figure 1 These are the enzyme activity results of stable liquid alkaline protease preparations prepared in different embodiments after being stored at 45°C for 180 days.

[0020] Figure 2 These are the enzyme activity results prepared in different comparative examples and stored at 45°C for 180 days. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0022] This invention provides a stable liquid alkaline protease preparation, comprising liquid alkaline protease, NaCl, polysaccharide, and histidine; based on the mass of liquid alkaline protease, the amount of NaCl added is 5%-10%, the amount of polysaccharide added is 0.1%-0.5%, and the amount of histidine added is 0.02%-0.1%.

[0023] The liquid alkaline protease is preferably an alkaline protease fermentation supernatant with an enzyme activity of 100,000-500,000 U / mL. The polysaccharide includes one or more of soluble soybean polysaccharide, chitosan, trehalose, and sodium carboxymethyl cellulose.

[0024] The present invention also provides a method for preparing the above-mentioned stable liquid alkaline protease preparation, comprising the following steps: filtering the microbial fermentation broth that produces alkaline protease, centrifuging or allowing it to stand and decanting to obtain a supernatant containing alkaline protease, or further concentrating the supernatant to obtain a concentrated solution, adding NaCl, polysaccharide and histidine to it according to the mass ratio, mixing evenly and dissolving, preferably adjusting the pH to 4.5-5.5 with acid and then filtering to remove bacteria, to obtain a stable liquid alkaline protease preparation.

[0025] The microorganisms include Bacillus or recombinant engineered bacteria; the recombinant engineered bacteria contain a target gene derived from Bacillus that expresses an alkaline protease. Bacillus includes any one of Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus amyloliquefaciens, and Bacillus bellelesii; the recombinant engineered bacteria include Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Aspergillus niger, etc. The acids include any one of HCl, H₂SO₄, H₃PO₄, acetic acid, and citric acid.

[0026] The effects of the stable liquid alkaline protease formulation of the present invention will be studied through specific embodiments below.

[0027] Example 1: This example provides a stable liquid alkaline protease preparation, comprising liquid alkaline protease, NaCl, polysaccharide, and histidine; the enzyme activity and the amounts of NaCl, polysaccharide, and histidine added are shown in Table 1.

[0028] This embodiment also provides the above-mentioned stable liquid alkaline protease preparation, including the following steps:

[0029] (1) Bacillus subtilis was inoculated into the culture medium and cultured at 37℃ for 48 h to obtain Bacillus subtilis fermentation broth. Culture medium formula: glucose 1.5%, soybean meal 10%, corn steep liquor 1%, Na2HPO4 0.4%, pH adjusted to 7.0 with NaOH and sterilized at 121℃ for 30 min.

[0030] (2) The fermentation broth of Bacillus subtilis producing alkaline protease was filtered and centrifuged to obtain a supernatant containing alkaline protease. NaCl, polysaccharide and histidine were added to it according to the mass ratio, mixed evenly and dissolved. The pH was adjusted to 4.5 with dilute hydrochloric acid and then filtered through a 0.22 μm filter membrane to remove bacteria, thus obtaining a stable liquid alkaline protease preparation.

[0031] The prepared stable liquid alkaline protease preparation was stored at 45°C, and enzyme activity was tested and residual rate was calculated every 30 days. The results are as follows: Figure 1 As shown.

[0032] Example 2: This example provides a stable liquid alkaline protease preparation, comprising liquid alkaline protease, NaCl, polysaccharide, and histidine; the enzyme activity and the amounts of NaCl, polysaccharide, and histidine added are shown in Table 1.

[0033] This embodiment also provides the above-mentioned stable liquid alkaline protease preparation, including the following steps:

[0034] (1) Bacillus subtilis was inoculated into the culture medium and cultured at 37℃ for 72h to obtain Bacillus subtilis fermentation broth. Culture medium formula: glucose 1.5%, soybean meal 10%, corn steep liquor 1%, Na2HPO4 0.4%, pH adjusted to 7.0 with NaOH and sterilized at 121℃ for 30min.

[0035] (2) The fermentation broth of Bacillus subtilis producing alkaline protease was filtered and centrifuged to obtain a supernatant containing alkaline protease. NaCl, polysaccharide and histidine were added to it according to the mass ratio, mixed evenly and dissolved. The pH was adjusted to 5.5 with dilute phosphoric acid and then filtered through a 0.22 μm filter membrane to remove bacteria, thus obtaining a stable liquid alkaline protease preparation.

[0036] The prepared stable liquid alkaline protease preparation was stored at 45°C, and enzyme activity was tested and residual rate was calculated every 30 days. The results are as follows: Figure 1 As shown.

[0037] Example 3: This example provides a stable liquid alkaline protease preparation, comprising liquid alkaline protease, NaCl, polysaccharide, and histidine; the enzyme activity and the amounts of NaCl, polysaccharide, and histidine added are shown in Table 1.

[0038] This embodiment also provides the above-mentioned stable liquid alkaline protease preparation, including the following steps:

[0039] (1) Bacillus subtilis was inoculated into the culture medium and cultured at 37℃ for 96 h to obtain Bacillus subtilis fermentation broth. Culture medium formula: glucose 1.5%, soybean meal 10%, corn steep liquor 1%, Na2HPO4 0.4%, pH adjusted to 7.0 with NaOH and sterilized at 121℃ for 30 min.

[0040] (2) The fermentation broth of Bacillus subtilis producing alkaline protease was filtered and centrifuged to obtain a supernatant containing alkaline protease. NaCl, polysaccharide and histidine were added to it according to the mass ratio, mixed evenly and dissolved. After adjusting the pH to 5.0 with citric acid, it was filtered through a 0.22 μm filter membrane for sterilization to obtain a stable liquid alkaline protease preparation.

[0041] The prepared stable liquid alkaline protease preparation was stored at 45°C, and enzyme activity was tested and residual rate was calculated every 30 days. The results are as follows: Figure 1 As shown.

[0042] Example 4: This example provides a stable liquid alkaline protease preparation, comprising liquid alkaline protease, NaCl, polysaccharide, and histidine; the enzyme activity and the amounts of NaCl, polysaccharide, and histidine added are shown in Table 1.

[0043] This embodiment also provides the above-mentioned stable liquid alkaline protease preparation, including the following steps:

[0044] (1) Bacillus subtilis was inoculated into the culture medium and cultured at 37℃ for 48h to obtain Bacillus subtilis fermentation broth. Culture medium formula: glucose 1.5%, soybean meal 10%, corn steep liquor 1%, Na2HPO4 0.4%, pH adjusted to 7.0 with NaOH and sterilized at 121℃ for 30min.

[0045] (2) The fermentation broth of Bacillus subtilis producing alkaline protease was filtered and centrifuged to obtain a supernatant containing alkaline protease. NaCl, polysaccharide and histidine were added to it according to the mass ratio, mixed evenly and dissolved. The pH was adjusted to 4.5 with dilute acetic acid and then filtered through a 0.22 μm filter membrane to remove bacteria, thus obtaining a stable liquid alkaline protease preparation.

[0046] The prepared stable liquid alkaline protease preparation was stored at 45°C, and enzyme activity was tested and residual rate was calculated every 30 days. The results are as follows: Figure 1 As shown.

[0047] Example 5: This example provides a stable liquid alkaline protease preparation, comprising liquid alkaline protease, NaCl, polysaccharide, and histidine; the enzyme activity and the amounts of NaCl, polysaccharide, and histidine added are shown in Table 1.

[0048] This embodiment also provides the above-mentioned stable liquid alkaline protease preparation, including the following steps:

[0049] (1) Bacillus subtilis was inoculated into the culture medium and cultured at 37℃ for 48h to obtain Bacillus subtilis fermentation broth. Culture medium formula: glucose 1.5%, soybean meal 10%, corn steep liquor 1%, Na2HPO4 0.4%, pH adjusted to 7.0 with NaOH and sterilized at 121℃ for 30min.

[0050] (2) The fermentation broth of Bacillus subtilis producing alkaline protease was filtered and centrifuged to obtain a supernatant containing alkaline protease. NaCl, polysaccharide and histidine were added to it according to the mass ratio, mixed evenly and dissolved. The pH was adjusted to 5.0 with dilute sulfuric acid and then filtered through a 0.22 μm filter membrane to remove bacteria, thus obtaining a stable liquid alkaline protease preparation.

[0051] The prepared stable liquid alkaline protease preparation was stored at 45°C, and enzyme activity was tested and residual rate was calculated every 30 days. The results are as follows: Figure 1 As shown.

[0052] Example 6: This example provides a stable liquid alkaline protease preparation, comprising liquid alkaline protease, NaCl, polysaccharide, and histidine; the enzyme activity and the amounts of NaCl, polysaccharide, and histidine added are shown in Table 1.

[0053] This embodiment also provides the above-mentioned stable liquid alkaline protease preparation, including the following steps:

[0054] (1) Bacillus subtilis was inoculated into the culture medium and cultured at 37°C for 72 hours to obtain Bacillus subtilis fermentation broth.

[0055] (2) The fermentation broth of Bacillus subtilis producing alkaline protease was filtered and centrifuged to obtain a supernatant containing alkaline protease. NaCl, polysaccharide and histidine were added to it according to the mass ratio, mixed evenly and dissolved. The pH was adjusted to 5.5 with dilute sulfuric acid and then filtered through a 0.22 μm filter membrane to remove bacteria, thus obtaining a stable liquid alkaline protease preparation.

[0056] The prepared stable liquid alkaline protease preparation was stored at 45°C, and enzyme activity was tested and residual rate was calculated every 30 days. The results are as follows: Figure 1 As shown.

[0057] Example 7: This example provides a stable liquid alkaline protease preparation, comprising liquid alkaline protease, NaCl, polysaccharide, and histidine; the enzyme activity and the amounts of NaCl, polysaccharide, and histidine added are shown in Table 1.

[0058] This embodiment also provides the above-mentioned stable liquid alkaline protease preparation, including the following steps:

[0059] (1) Bacillus subtilis was inoculated into the culture medium and cultured at 37℃ for 72h to obtain Bacillus subtilis fermentation broth. Culture medium formula: glucose 1.5%, soybean meal 10%, corn steep liquor 1%, Na2HPO4 0.4%, pH adjusted to 7.0 with NaOH and sterilized at 121℃ for 30min.

[0060] (2) The fermentation broth of Bacillus subtilis producing alkaline protease was filtered and centrifuged to obtain a supernatant containing alkaline protease. NaCl, polysaccharide and histidine were added to it according to the mass ratio, mixed evenly and dissolved. After adjusting the pH to 5.0 with citric acid, it was filtered through a 0.22 μm filter membrane for sterilization to obtain a stable liquid alkaline protease preparation.

[0061] The prepared stable liquid alkaline protease preparation was stored at 45°C, and enzyme activity was tested and residual rate was calculated every 30 days. The results are as follows: Figure 1 As shown.

[0062] Example 8: This example provides a stable liquid alkaline protease preparation, comprising liquid alkaline protease, NaCl, polysaccharide, and histidine; the enzyme activity and the amounts of NaCl, polysaccharide, and histidine added are shown in Table 1.

[0063] This embodiment also provides the above-mentioned stable liquid alkaline protease preparation, including the following steps:

[0064] (1) Bacillus subtilis was inoculated into the culture medium and cultured at 37℃ for 96 h to obtain Bacillus subtilis fermentation broth. Culture medium formula: glucose 1.5%, soybean meal 10%, corn steep liquor 1%, Na2HPO4 0.4%, pH adjusted to 7.0 with NaOH and sterilized at 121℃ for 30 min.

[0065] (2) The fermentation broth of Bacillus subtilis producing alkaline protease was filtered and centrifuged to obtain a supernatant containing alkaline protease. NaCl, polysaccharide and histidine were added to it according to the mass ratio, mixed evenly and dissolved. The pH was adjusted to 5.5 with dilute phosphoric acid and then filtered through a 0.22 μm filter membrane to remove bacteria, thus obtaining a stable liquid alkaline protease preparation.

[0066] The prepared stable liquid alkaline protease preparation was stored at 45°C, and enzyme activity was tested and residual rate was calculated every 30 days. The results are as follows: Figure 1 As shown.

[0067] Example 9: This example provides a stable liquid alkaline protease preparation, comprising liquid alkaline protease, NaCl, polysaccharide, and histidine; the enzyme activity and the amounts of NaCl, polysaccharide, and histidine added are shown in Table 1.

[0068] This embodiment also provides the above-mentioned stable liquid alkaline protease preparation, including the following steps:

[0069] (1) Bacillus subtilis was inoculated into the culture medium and cultured at 37℃ for 48h to obtain Bacillus subtilis fermentation broth. Culture medium formula: glucose 1.5%, soybean meal 10%, corn steep liquor 1%, Na2HPO4 0.4%, pH adjusted to 7.0 with NaOH and sterilized at 121℃ for 30min.

[0070] (2) The fermentation broth of Bacillus subtilis producing alkaline protease was filtered and centrifuged to obtain a supernatant containing alkaline protease. NaCl, polysaccharide and histidine were added to it according to the mass ratio, mixed evenly and dissolved. The pH was adjusted to 4.5 with dilute hydrochloric acid and then filtered through a 0.22 μm filter membrane to remove bacteria, thus obtaining a stable liquid alkaline protease preparation.

[0071] The prepared stable liquid alkaline protease preparation was stored at 45°C, and enzyme activity was tested and residual rate was calculated every 30 days. The results are as follows: Figure 1 As shown.

[0072] Table 1 Initial enzyme activity and types and amounts of each component in different embodiments

[0073]

[0074] Depend on Figure 1 It is known that the stable liquid alkaline protease preparation provided by the present invention has virtually no loss of enzyme activity when stored at 45°C for 6 months.

[0075] Comparative Example 1:

[0076] This comparative example provides a liquid alkaline protease formulation, which differs from Example 1 only in that it does not contain histidine. The liquid alkaline protease formulation was stored at 45°C, and enzyme activity was measured and residual rate calculated every 30 days. The results are as follows: Figure 2 As shown.

[0077] Comparative Example 2:

[0078] This comparative example provides a liquid alkaline protease formulation, which differs from Example 2 only in that it does not contain histidine. The liquid alkaline protease formulation was stored at 45°C, and enzyme activity was measured and residual rate calculated every 30 days. The results are as follows: Figure 2 As shown.

[0079] Comparative Example 3:

[0080] This comparative example provides a liquid alkaline protease preparation, which differs from Example 3 only in that it does not contain histidine. The liquid alkaline protease preparation was stored at 45°C, and enzyme activity was measured and residual rate calculated every 30 days. The results are as follows: Figure 2 As shown.

[0081] Comparative Example 4:

[0082] This comparative example provides a liquid alkaline protease formulation, which differs from Example 4 only in that it does not contain histidine and instead contains 20% glycerol. The liquid alkaline protease formulation was stored at 45°C, and enzyme activity was measured and residual rate calculated every 30 days. The results are as follows: Figure 2 As shown.

[0083] Comparative Example 5:

[0084] This comparative example provides a liquid alkaline protease preparation, which differs from Example 5 only in that it does not contain histidine and instead contains 20% glycerol. The liquid alkaline protease preparation was stored at 45°C, and enzyme activity was measured and residual rate calculated every 30 days. The results are as follows: Figure 2 As shown.

[0085] Comparative Example 6:

[0086] This comparative example provides a liquid alkaline protease formulation, which differs from Example 6 only in that it does not contain histidine and instead contains 20% glycerol. The liquid alkaline protease formulation was stored at 45°C, and enzyme activity was measured and residual rate calculated every 30 days. The results are as follows: Figure 2 As shown.

[0087] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.

Claims

1. A stable liquid alkaline protease preparation, characterized in that: It includes liquid alkaline protease, NaCl, polysaccharides, and histidine; based on the mass of liquid alkaline protease, the amount of NaCl added is 5%-10%, the amount of polysaccharides added is 0.1%-0.5%, and the amount of histidine added is 0.02%-0.1%.

2. The stable liquid alkaline protease preparation as described in claim 1, characterized in that: The enzyme activity of the liquid alkaline protease is 1×10⁻⁶. 5 -5×10 5 U / mL.

3. The stable liquid alkaline protease preparation as described in claim 1, characterized in that: The polysaccharide includes one or more of soluble soybean polysaccharide, chitosan, trehalose, and sodium carboxymethyl cellulose.

4. The method for preparing the stable liquid alkaline protease preparation according to any one of claims 1-3, characterized in that, The process includes the following steps: filtering the microbial fermentation broth that produces alkaline protease, centrifuging or allowing it to stand and decanting to obtain a supernatant containing alkaline protease, adding NaCl, polysaccharide and histidine to it in a mass ratio, mixing and dissolving it, adjusting the pH and filtering to remove bacteria to obtain a stable liquid alkaline protease preparation.

5. The method for preparing the stable liquid alkaline protease preparation as described in claim 4, characterized in that: The microorganisms include Bacillus or recombinant engineered bacteria; the recombinant engineered bacteria contain a target gene derived from Bacillus that expresses an alkaline protease.

6. The method for preparing the stable liquid alkaline protease preparation as described in claim 5, characterized in that: The Bacillus species include any one of Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus amyloliquefaciens, and Bacillus belesii.

7. The method for preparing the stable liquid alkaline protease preparation as described in claim 4, characterized in that: After adjusting the pH to 4.5-5.5, filter to sterilize.

8. The method for preparing the stable liquid alkaline protease preparation as described in claim 7, characterized in that: After adjusting the pH to 4.5-5.5 with acid, the solution is filtered for sterilization; the acid includes any one of HCl, H2SO4, H3PO4, formic acid, acetic acid, and citric acid.

9. The application of the stable liquid alkaline protease preparation as described in any one of claims 1-3 in the feed, food, and pharmaceutical industries.

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