Method for preparing 25 (OH) VD3 through microbial fermentation and application of 25 (OH) VD3

Abstract

The invention discloses a method for preparing 25 (OH) VD3 through microbial fermentation and application of the 25 (OH) VD3. The method comprises the following steps that microbial strains are placed in a liquid culture medium for shake cultivation; then adding vitamin D3 and a substrate additive solution, and continuously carrying out shake cultivation to obtain fermentation liquor; extracting the fermentation liquor by adopting an organic solvent, and carrying out vacuum concentration on the obtained organic phase to obtain 25 (OH) VD3; the substrate additive solution comprises a lauryl sodium sulfate aqueous solution, a polyethylene glycol 400 aqueous solution and a bovine serum albumin aqueous solution; according to the microbial fermentation method, the lauryl sodium sulfate, the polyethylene glycol and the bovine serum albumin are used as substrate auxiliaries, the synthesis efficiency of biologically converting the vitamin D into the 25 (OH) VD3 is remarkably improved, a solution is provided for the problems of high production cost and low utilization rate of the vitamin D in the prior art, and the method has remarkable economic benefits and application values.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology and specifically relates to a method for preparing 25(OH)VD by microbial fermentation. 3 methods and their applications. Background Art

[0002] Vitamin D plays an important role in the growth and development of livestock and poultry. In fact, in practical applications, vitamin D supplementation is achieved by taking 25(OH)VD 3 As an exogenous source for nutritional regulation, especially as feed for livestock and poultry production, 25(OH)VD 3 The introduction of 25(OH)VD can not only significantly increase egg production and egg quality, but also play an important role in improving pig reproductive capacity. Microbial conversion technology has become a popular choice for 25(OH)VD because of its mild reaction conditions, strong selection specificity and low cost. 3 It is an emerging technology approach for synthetic preparation, but the application effect and production efficiency of existing technologies still need to be improved. Vitamin D can be converted by selecting appropriate microbial strains and optimizing microbial fermentation. 3 Process for the synthesis of high-yield 25(OH)VD 3 It is of great significance. Summary of the invention

[0003] In order to improve the above technical problems, the present invention provides a method for preparing 25(OH)VD by microbial fermentation. 3 methods and their applications.

[0004] To achieve the above objectives, the present invention is implemented through the following technical solutions: A method for preparing 25(OH)VD by microbial fermentation 3 The method comprises the following steps: placing the microbial strain in a liquid culture medium for shaking culture; then adding vitamin D 3 The substrate auxiliary agent solution is continuously shaken to obtain a fermentation broth; the fermentation broth is extracted with an organic solvent, and the obtained organic phase is vacuum concentrated to obtain 25(OH)VD 3 ; The substrate auxiliary solution includes sodium dodecyl sulfate (SDS) aqueous solution, polyethylene glycol 400 (PEG400) aqueous solution and bovine serum albumin (BSA) aqueous solution.

[0005] Preferably, the volume ratio of the sodium dodecyl sulfate aqueous solution, the polyethylene glycol 400 aqueous solution and the bovine serum albumin aqueous solution is (1-10): (10-80): (10-100); the amount of the sodium dodecyl sulfate aqueous solution added is 0.01-0.1% of the volume of the liquid culture medium. The volume ratio of the sodium dodecyl sulfate aqueous solution, the polyethylene glycol 400 aqueous solution and the bovine serum albumin aqueous solution can be specifically 1:10:10, 1:50:50, 1:80:100, 10:80:100. Among them, the sodium dodecyl sulfate aqueous solution is prepared from sodium dodecyl sulfate (SDS) and deionized water; the polyethylene glycol 400 aqueous solution is prepared from polyethylene glycol 400 (PEG400) and deionized water; and the bovine serum albumin aqueous solution is prepared from bovine serum albumin (BSA) and deionized water.

[0006] Preferably, the concentration of the sodium dodecyl sulfate aqueous solution is 100-150 mg / L; the concentration of the polyethylene glycol 400 aqueous solution is 100-150 mg / L; and the concentration of the bovine serum albumin aqueous solution is 100-150 mg / L.

[0007] Preferably, the microbial strain is at least one of Saccharomyces cerevisiae, Escherichia coli, Candida albicans, Bacillus subtilis, Aspergillus niger, Rhodotorula glutinis and Pichia pastoris.

[0008] Preferably, the microbial strain is Saccharomyces cerevisiae.

[0009] Preferably, the liquid culture medium comprises: 5-10 g / L yeast powder, 10-15 g / L peptone, 10-15 g / L NaCl, and a pH value of 7.0-8.0. The pH value is preferably 7.2-7.8, specifically 7.0, 7.2, 7.5, 7.6, 7.8, 8.0.

[0010] Preferably, the vitamin D 3 The mass volume ratio of the feed amount to the liquid culture medium is 1~1.5mg:1mL.

[0011] Preferably, vitamin D is added 3The shaking culture time of the substrate auxiliary solution is 12 to 96 hours, preferably 36 to 72 hours, and specifically 36 hours, 42 hours, 60 hours, or 72 hours.

[0012] As a general inventive concept, the present invention also provides the above-mentioned microbial fermentation to prepare 25 (OH) VD 3 25(OH)VD prepared by the method 3 Application in poultry feed.

[0013] Preferably, the poultry feed is pig feed.

[0014] Beneficial effects of the present invention: (1) The present invention uses SDS, PEG400 and BSA as substrate adjuvants to increase the substrate vitamin D 3 The solubility of 25(OH)VD 3 The synthesis efficiency can be as high as 16.7 μg / mL, which is 3.2 times higher than that without substrate auxiliary.

[0015] (2) The addition of PEG400 and BSA in the present invention effectively alleviates the toxicity of SDS to microorganisms, ensures the normal growth and metabolism of microorganisms, and avoids the negative effects caused by excessive use of SDS.

[0016] (3) Compared with the traditional chemical synthesis method, the microbial fermentation method of the present invention is more environmentally friendly and has a lower production cost. The reaction system of the present invention has good operability and scalability, is suitable for industrial production, and can maintain high production capacity during large-scale fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 25(OH)VD prepared in Example 1 3 Actual picture of the product powder.

[0018] Figure 2 25(OH)VD 3 ELISA kit standard curve chart.

[0019] Figure 3 Preparation of 25(OH)VD for Example 1 and Comparative Examples 1-3 3 Comparison chart of synthesis efficiency. DETAILED DESCRIPTION

[0020] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0021] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0022] Example 1 A method for preparing 25(OH)VD by microbial fermentation 3 The method is as follows: 1. Streak the Saccharomyces cerevisiae INVSc1 glycerol strain on a YPD medium plate and culture it in a 30°C incubator for 24-48 hours until a clearly visible single colony grows; the Saccharomyces cerevisiae INVSc1 glycerol strain ( Saccharomyces cerevisiae INVSc1 Yeast Glycerol Stock) was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd., product number: D0432.

[0023] 2. Pick a single colony and inoculate it into 5 mL YPD liquid culture medium. Culture it in a shaker at 30°C and 200 rpm for 12-16 hours until the OD600 reaches 0.8-1.0 to obtain seed solution.

[0024] 3. Add 10 mL of liquid culture medium to a 50 mL conical flask, inoculate at a 2% inoculation rate (200 μL of seed solution when OD600 = 1.0), and culture on a shaking table at 30 °C for 24 h at a shaking speed of 200 r / min. The liquid culture medium contains 5 g / L yeast powder, 10 g / L peptone, and 10 g / L NaCl. The initial pH value of the liquid culture medium is 7.5.

[0025] 4. Take 10 mg of vitamin D 3 The substrate was put into a conical flask, and then the SDS aqueous solution, PEG400 aqueous solution and BSA aqueous solution were put into the conical flask, and the fermentation culture was carried out on a shaking table at 30 °C for 72 h, and the shaking speed was 200 r / min to obtain the fermentation liquid. Among them, the volume ratio of the SDS aqueous solution, the PEG400 aqueous solution and the BSA aqueous solution was 10:80:100; the amount of the SDS aqueous solution added was 0.1% of the volume of the liquid culture medium, the concentration of the SDS aqueous solution was 100 mg / L, the concentration of the PEG400 aqueous solution was 100 mg / L, and the concentration of the BSA aqueous solution was 100 mg / L.

[0026] 5. Use dichloromethane as the extraction solvent and mix the fermentation liquid with it for extraction three times.

[0027] 6. Use a vacuum rotary evaporator to concentrate the organic phase after the extraction to obtain the target product 25(OH)VD 3 , the actual picture is as follows Figure 1 shown.

[0028] Example 2 A method for preparing 25(OH)VD by microbial fermentation 3 25(OH)VD prepared by the method 3 For application in poultry feed, the steps are as follows: 1. The 25(OH)VD obtained by microbial fermentation in Example 1 3 Mix with pig feed at a ratio of 0.3% (w / w). Pig feed raw material addition mass ratio: corn flour: 60%; soybean meal: 20%; wheat flour: 10%; vegetable oil: 4%; mineral additives: 2%; other vitamins and trace elements premix: 4%. The various raw materials of the above feed are crushed and sieved for subsequent mixing and processing.

[0029] 2. Add the above ingredients into the mixer and mix thoroughly. The mixing time is usually 20-30 minutes to ensure that the nutrients are evenly distributed.

[0030] 3. The mixed feed is processed into pellets by a pellet machine. The particle size can be adjusted according to the eating habits and digestive characteristics of livestock and poultry, usually between 1 and 3 mm.

[0031] 4. The pellet feed is dried in a dryer to ensure that the moisture content is between 8 and 12%.

[0032] Comparative Example 1 The method is basically the same as Example 1, except that no substrate auxiliary solution (SDS aqueous solution, PEG400 aqueous solution and BSA aqueous solution) is added.

[0033] Comparative Example 2 The method is basically the same as Example 1, except that only the SDS aqueous solution is added, and the PEG400 aqueous solution and the BSA aqueous solution are not added; the amount of the SDS aqueous solution added is 0.1% of the volume of the liquid culture medium; and the concentration of the SDS aqueous solution is 100 mg / L.

[0034] Comparative Example 3 The method is basically the same as Example 1, except that only SDS aqueous solution and PEG400 aqueous solution are added, and BSA aqueous solution is not added; the volume ratio of SDS aqueous solution to PEG400 aqueous solution is 10:80, and the amount of SDS aqueous solution added is 0.1% of the volume of the liquid culture medium; the concentration of SDS aqueous solution is 100 mg / L, and the concentration of PEG400 aqueous solution is 100 mg / L.

[0035] The sample 25(OH)VD 3 Dissolved in PBS buffer solution, using commercial 25(OH)VD 3 ELISA kit for yield concentration test, 25(OH)VD 3 ELISA kit (D751005) was purchased from Sangon Biotech (Shanghai) Co., Ltd. First, 25(OH)VD 3 The standard product of the ELISA kit was used to establish the standard curve, and the standard product 25(OH)VD was established using an ELISA reader. 3 Relationship with absorbance OD value. 25(OH)VD 3 The data for establishing the standard curve of the ELISA kit are shown in Table 1.

[0036] Table 1:

[0037] Based on the above data, a curve of the relationship between the concentration of the standard and the absorbance OD value of the microplate reader was drawn to obtain 25(OH)VD 3 ELISA kit standard curve diagram, such as Figure 2 shown.

[0038] The target product 25(OH)VD prepared in Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3 3 The OD value test data is shown in Table 2.

[0039] Table 2:

[0040] According to 25(OH)VD 3 ELISA kit standard curve diagram, the target product 25 (OH) VD in Example 1 and Comparative Examples 1 to 3 3 The OD value was converted into the yield concentration value (μg / mL). The specific test results are shown in Table 3.

[0041] Table 3:

[0042] Figure 3 Preparation of 25(OH)VD for Example 1 and Comparative Examples 1-3 3 The yield concentration value (yield) comparison chart. By combining SDS, PEG400 and BSA as substrate additives, Example 1 significantly increased the yield of 25(OH)VD 3The yield concentration value (yield) can be as high as 16.7 μg / mL, which is 3.2 times higher than the yield concentration value (yield) of comparative example 1 without substrate additive. This shows that the microbial fermentation based on the combination of SDS, PEG400 and BSA provided by the present invention can produce 25(OH)VD 3 The reaction system can effectively improve the synthesis efficiency of the product and has good prospects for industrial application.

[0043] From the comparison between Example 1 and Comparative Examples 2-3, it can be seen that if the substrate auxiliary agent lacks PEG400 or lacks both PEG400 and BSA, 25(OH)VD 3 The synthesis efficiency will be significantly reduced.

[0044] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing 25(OH)VD3 by microbial fermentation, characterized in that: The following steps are involved: The microbial strain is placed in a liquid culture medium for shaking culture; then vitamin D3 and a substrate auxiliary solution are added and the shaking culture is continued to obtain a fermentation liquid; the fermentation liquid is extracted with an organic solvent, and the obtained organic phase is vacuum concentrated to obtain 25(OH)VD3; The substrate auxiliary solution includes a sodium dodecyl sulfate aqueous solution, a polyethylene glycol 400 aqueous solution and a bovine serum albumin aqueous solution.

2. The method for preparing 25(OH)VD3 by microbial fermentation according to claim 1, characterized in that: The volume ratio of the sodium dodecyl sulfate aqueous solution, the polyethylene glycol 400 aqueous solution and the bovine serum albumin aqueous solution is (1-10): (10-80): (10-100); the addition amount of the sodium dodecyl sulfate aqueous solution is 0.01-0.1% of the volume of the liquid culture medium.

3. The method for preparing 25(OH)VD3 by microbial fermentation according to claim 1, characterized in that: The concentration of the sodium dodecyl sulfate aqueous solution is 100-150 mg / L; the concentration of the polyethylene glycol 400 aqueous solution is 100-150 mg / L; and the concentration of the bovine serum albumin aqueous solution is 100-150 mg / L.

4. The method for preparing 25(OH)VD3 by microbial fermentation according to any one of claims 1 to 3, characterized in that: The microbial strain is at least one of saccharomyces cerevisiae, Escherichia coli, Candida albicans, Bacillus subtilis, Aspergillus niger, red yeast and Pichia pastoris.

5. The method for preparing 25(OH)VD3 by microbial fermentation according to claim 4, characterized in that: The microbial strain is Saccharomyces cerevisiae.

6. The method for preparing 25(OH)VD3 by microbial fermentation according to any one of claims 1 to 3, characterized in that: The components of the liquid culture medium include: 5-10 g / L yeast powder, 10-15 g / L peptone, and 10-15 g / L NaCl; and the pH value is 7.0-8.

0.

7. The method for preparing 25(OH)VD3 by microbial fermentation according to any one of claims 1 to 3, characterized in that: The mass volume ratio of the vitamin D3 feeding amount to the liquid culture medium is 1-1.5 mg:1 mL.

8. The method for preparing 25(OH)VD3 by microbial fermentation according to any one of claims 1 to 3, characterized in that: Vitamin D3 and substrate auxiliary solution were added and the shaking culture was continued for 12 to 96 hours.

9. Use of 25(OH)VD3 prepared by the method for preparing 25(OH)VD3 by microbial fermentation according to any one of claims 1 to 8 in poultry feed.

10. The use according to claim 9, characterized in that: The poultry feed is pig feed.

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