CN106319005B

CN106319005B - Fermentation medium capable of increasing output of nosiheptide and culture method

Info

Publication number: CN106319005B
Application number: CN201510400650.XA
Authority: CN
Inventors: 周贤龙; 石怀月; 刘静
Original assignee: Mudanjiang Bioseen Biological Co ltd
Current assignee: Mudanjiang Bioseen Biological Co ltd
Priority date: 2015-07-09
Filing date: 2015-07-09
Publication date: 2020-10-27
Anticipated expiration: 2035-07-09
Also published as: CN106319005A

Abstract

The invention discloses a fermentation medium capable of improving the yield of nosiheptide, which comprises the following components: KNO₃0.5‑1g/L、(NH₄)₂SO₄0.8-1.5g/L, NaCl 3-5g/L, 35-45g/L soybean powder, 1-2.5g/L yeast powder, 5-6g/L glucose, 0.01-0.02g/L amylase, 40-50g/L starch, 3.5-5 g/L light calcium carbonate and 0.1-0.5mg/L serine; is prepared with water. The invention also improves the fermentation method for improving the output of the nosiheptide by inoculating the active streptomyces CGMCC NO.8732 with the culture medium, and the method can obviously improve the output of the nosiheptide, obtain objective economic benefit and have wide application prospect.

Description

Fermentation medium capable of increasing output of nosiheptide and culture method

Technical Field

The invention relates to the field of microbial fermentation, in particular to a fermentation medium capable of improving the yield of nosiheptide and a culture method.

Background

The Naxitin is a novel non-absorbable animal feed additive, and the meat yield of poultry can be greatly improved by adding a small amount of Naxitin into animal feed. Because it does not remain in the animal body, it does not have a negative effect on human beings. The action mechanism is as follows: inhibiting the synthesis of protein can inhibit the growth of bacteria. At present, the fermentation level of nosiheptide is low, the cost is high, and the application is limited.

Disclosure of Invention

The first purpose of the invention is to provide an optimized culture medium for improving the yield of nosiheptide, and in order to achieve the purpose, the invention adopts the following technical means:

a fermentation medium for increasing production of nosiheptide, comprising: KNO₃0.5-1g/L、(NH₄)₂SO₄0.8-1.5g/L, NaCl 3-5g/L, 35-45g/L soybean powder, 1-2.5g/L yeast powder, 5-6g/L glucose, 0.01-0.02g/L amylase, 40-50g/L starch, 3.5-5 g/L light calcium carbonate and 0.1-0.5mg/L serine; is prepared with water.

Wherein, preferably, the fermentation medium comprises: KNO₃1g/L、(NH₄)₂SO₄1.5g/L, NaCl 4g/L, soybean powder 40g/L, yeast powder 2g/L, glucose 5g/L, amylase 0.01g/L, corn starch 50g/L, light calcium carbonate 5g/L, serine 0.2-0.4mg/L, and water.

Wherein the soybean powder has a particle size range of 60 +/-5 meshes and can be purchased from Wuchang Kangshengwang food and grain processing factories.

Wherein the yeast powder can be purchased from Huawei bioengineering GmbH of agriculture in Hubei.

Wherein the amylase is alpha-amylase available from Shandong Colt enzyme preparation, Inc.

The pH of the culture medium of the present invention is preferably controlled to be in the range of 6.5 to 7, and the specific control is known to those skilled in the art, and the present invention is not particularly limited thereto.

The media traditionally available for the fermentative production of Naxi peptide consist of (one of the most commonly used): glucose 4%, soybean powder 3%, starch 4%, sodium sulfate 0.1%, sodium chloride 0.25%, calcium carbonate 0.5%, and elemental solution 5ml/L, but this culture medium found that the yield of the nosiheptide is not ideal in practical application. The invention obtains a new culture medium which can obviously improve the yield of nosiheptide by optimizing the formula of the traditional culture medium for fermenting and culturing nosiheptide (especially adding serine).

The invention also provides a method for preparing nosiheptide by fermenting the culture medium, which comprises the following steps:

(1) inoculating streptomyces livelii CGMCC NO.8732 to the culture medium;

(2) culturing the inoculated culture medium at 220-250rpm/min and 28-30 ℃ for 67-75 hours, and separating the fermentation product to obtain the nosiheptide.

The microorganism provided by the invention is active streptomyces (Streptomyces)streptomyces actuosus) BJX004, which has been deposited in China general microbiological culture Collection center (CGMCC for short), No. 3 of West Lu 1 of the North Chen of the Korean-Yang district of Beijing, institute of microbiology of China academy of sciences, zip code 100101, preservation number CGMCC NO.8732, preservation date 2014, 1 month and 17 days, the classification name of the strain is: active streptomyces (streptomyces actuosus）BJX004。

Wherein the Streptomyces actiosus CGMCC NO.8732 is preferably provided in the form of seed liquid, and the inoculation amount is 4-6% (v/v), preferably 5% (v/v).

Preferably, the culture conditions are at 230-.

And the separation of the fermentation product is to leach the fermentation liquor with dichloromethane, wash dichloromethane extract with water, concentrate the solution into a crude product, wash the crude product with n-heptane, and dry the crude product at room temperature to obtain a refined product. The specific operation is known to those skilled in the art, and the present invention is not particularly limited thereto. By adopting the separation means, the purity and the yield of the product of the nosiheptide can be further ensured.

By adopting the fermentation conditions, the yield of the nosiheptide can reach 5600ug/mL to the maximum after the fermentation is finished, and compared with the nosiheptide obtained by fermentation culture in the common culture medium (see the 8 th section of the invention content) under the same culture conditions, the yield of the nosiheptide is improved by more than 12%.

Detailed Description

In the following examples, all the raw materials used are known commercial products, and soybean flour was purchased from Wuchang Kangshengwang grain and oil food processing factories; the yeast powder is purchased from the biological engineering company Limited of China warfarin Hubei; the amylase is an alpha-amylase, available from Shandong Longkott enzyme preparations, Inc.

Example 1

This example provides a medium useful for increasing the production of nosiheptide, the medium having the following formulation:

KNO₃1g/L、(NH₄)₂SO₄1.5g/L, NaCl 4g/L, 40g/L of soybean meal, 2g/L of yeast powder, 5g/L of glucose, 0.01g/L of amylase, 50g/L of corn starch, 5g/L of light calcium carbonate and 0.4mg/L of serine; is prepared with water.

Example 2

KNO₃0.5g/L、(NH₄)₂SO₄0.8g/L, NaCl 3g/L, 35g/L soybean meal, 1g/L yeast powder, 5g/L glucose, 0.01g/L amylase, 40g/L starch, 3.5g/L light calcium carbonate and 0.1mg/L serine; with waterAnd (4) preparing.

Example 3

KNO₃0.5g/L、(NH₄)₂SO₄0.8g/L, NaCl 5g/L, 45g/L of soybean meal, 2.5g/L of yeast powder, 6g/L of glucose, 0.02g/L of amylase, 45g/L of starch, 4g/L of light calcium carbonate and 0.5mg/L of serine; is prepared with water.

Example 4

KNO₃1g/L、(NH₄)₂SO₄1.5g/L, NaCl 5g/L, soybean flour 45g/L, yeast powder 2.5g/L, glucose 6g/L, amylase 0.02g/L, starch 50g/L, light calcium carbonate 5g/L, serine 0.5 mg/L; is prepared with water.

Example 5

This example provides a method for producing nosiheptide by fermentation, comprising the following steps:

(1) inoculating the seed solution of streptomyces livelii CGMCC NO.8732 to the culture medium prepared in the example 1, wherein the inoculation amount is 5% (v/v);

(2) culturing the inoculated culture medium at 220rpm/min and 28 ℃ for 72 hours, separating the fermentation product (extracting the fermentation liquid by dichloromethane, then washing dichloromethane extract, concentrating the fermentation liquid, washing the crude product by n-heptane, and drying at room temperature to obtain the refined nosiheptide).

In this example, the yield of nosiheptide was 5600 ug/mL.

Example 6

This example also provides a method for producing nosiheptide by fermentation, which is different from example 5 only in that the culture medium prepared in example 2 is used in the present example, the inoculation amount is 4% (v/v), the fermentation culture conditions are 220rpm/min, the temperature is 30 ℃ for 75 hours, and the yield of nosiheptide is 5450 ug/mL.

Example 7

This example also provides a method for producing nosiheptide by fermentation, which is different from example 5 only in that the culture medium prepared in example 3 is used in this example, the inoculation amount is 6% (v/v), the fermentation culture conditions are 250rpm/min, 29 ℃ for 67 hours, and the yield of nosiheptide is 5370 ug/mL.

Comparative example 1

The comparative example differs from example 5 only in that no serine was added to the medium used for the fermentation culture.

In this comparative example, the yield of nosiheptide was 5000 ug/mL.

Comparative example 2

The only difference in this comparative example compared to example 6 is that the specific conditions for the fermentation culture were 72 hours at 25 ℃ at 180 rpm/min.

In this comparative example, the yield of nosiheptide was 5230 ug/mL.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. A method for preparing nosiheptide by fermentation, which is characterized in that: the method comprises the following steps:

(1) inoculating Streptomyces actuosus (Streptomyces actuosus) CGMCC NO.8732 to a fermentation medium; the fermentation medium had the following composition: KNO₃0.5-1g/L、(NH₄)₂SO₄0.8-1.5g/L, NaCl 3-5g/L, 35-45g/L soybean powder, 1-2.5g/L yeast powder, 5-6g/L glucose, 0.01-0.02g/L amylase, 40-50g/L starch, 3.5-5 g/L light calcium carbonate and 0.1-0.5mg/L serine; prepared by water, wherein the pH value range of the culture medium is 6.5-7;

2. The method of claim 1, wherein the fermentation medium has the following composition: KNO₃1g/L、(NH₄)₂SO₄1.5g/L, NaCl 4g/L, soybean powder 40g/L, yeast powder 2g/L, glucose 5g/L, amylase 0.01g/L, corn starch 50g/L, light calcium carbonate 5g/L, serine 0.2-0.4mg/L, and water.

3. The method of claim 1, wherein: the particle size range of the soybean meal is 60 +/-5 meshes.

4. The method according to any one of claims 1 to 3, wherein: the Streptomyces actuosus (CGMCC NO. 8732) is provided in the form of seed liquid, and the inoculation amount is 4-6% (v/v).

5. The method of claim 4, wherein: the Streptomyces actuosus (CGMCC NO. 8732) is provided in the form of seed liquid, and the inoculation amount is 5% (v/v).

6. The method according to any one of claims 1 to 3, wherein: the culture conditions are that the culture is carried out at 230-240rpm/min and 28-30 ℃ for 72-75 hours.

7. The method according to any one of claims 1 to 3, wherein: the separation of the fermentation product is specifically as follows: leaching the fermentation liquor with dichloromethane, then washing dichloromethane extract with water, concentrating to obtain crude product, washing the crude product with n-heptane, and drying at room temperature to obtain refined product.

8. The method of claim 4, wherein: the separation of the fermentation product is specifically as follows: leaching the fermentation liquor with dichloromethane, then washing dichloromethane extract with water, concentrating to obtain crude product, washing the crude product with n-heptane, and drying at room temperature to obtain refined product.