阅读说明：本技术 一种利用纳他霉素发酵副产物提高纳他霉素发酵产量的方法 (Method for improving natamycin fermentation yield by using natamycin fermentation by-products ) 是由 宗工理 曹广祥 付加芳 张佩佩 陈曦 于 2021-07-28 设计创作，主要内容包括：本发明涉及一种利用纳他霉素发酵副产物提高纳他霉素发酵产量的方法。将发酵结束后的纳他霉素发酵液通过离心收集上清液,得到纳他霉素发酵副产物；然后在纳他霉素发酵0～48h后,按照发酵培养基体积的2.5～7.5％,将纳他霉素发酵副产物添加至发酵培养基中,继续发酵,得纳他霉素。经本发明的方法得到的纳他霉素摇瓶发酵产量最高为2.40±0.17g/L。采取扩大生产后,在1L发酵体系中,发酵24h后添加5％的纳他霉素发酵副产物,其纳他霉素产量为8.83～8.86g/L,显著高于未添加纳他霉素发酵副产物的6.70～6.72g/L。(The invention relates to a method for improving natamycin fermentation yield by using natamycin fermentation by-products. Centrifuging the natamycin fermentation liquor after fermentation is finished, and collecting supernatant to obtain a natamycin fermentation by-product; and then after the natamycin is fermented for 0-48 h, adding the natamycin fermentation by-product into the fermentation medium according to 2.5-7.5% of the volume of the fermentation medium, and continuing to ferment to obtain the natamycin. The natamycin shake flask fermentation yield obtained by the method of the invention is 2.40 +/-0.17 g/L at most. After the expanded production is adopted, 5% of natamycin fermentation byproducts are added after fermentation is carried out for 24 hours in a 1L fermentation system, the natamycin yield is 8.83-8.86 g/L and is obviously higher than 6.70-6.72 g/L of natamycin fermentation byproducts which are not added.)

1. A method for improving natamycin fermentation yield by using natamycin fermentation byproducts is characterized by comprising the following steps: centrifuging the natamycin fermentation liquor after fermentation is finished, and collecting supernatant to obtain a natamycin fermentation by-product; and then after the natamycin is fermented for 0-48 h, adding the natamycin fermentation by-product into the fermentation medium according to 2.5-7.5% of the volume of the fermentation medium, and continuing to ferment to obtain the natamycin.

2. The method for improving natamycin fermentation yield by using natamycin fermentation by-products as claimed in claim 1, wherein the conditions of centrifugal collection are 9000-11000 rpm for 12-18 min.

3. The method for improving natamycin fermentation yield using natamycin fermentation by-product of claim 1, wherein the enzyme activity of cholesterol oxidase in the natamycin fermentation by-product is greater than or equal to 1U/mL; more preferably 1.3 to 1.5U/mL.

4. The method for improving natamycin fermentation yield using natamycin fermentation by-product according to claim 1, wherein the strain used for natamycin fermentation production is Streptomyces fuscoporia (Streptomyces gilvosporus), Streptomyces natalensis (Streptomyces natalensis), Streptomyces chattanoga (Streptomyces chattanoogensis) or Streptomyces lydicus (Streptomyces lydicus).

5. The method for improving natamycin fermentation yield using natamycin fermentation by-product as claimed in claim 1, wherein natamycin fermentation by-product is added after 12-36 h of natamycin fermentation production; more preferably after 24 hours.

6. The method for improving natamycin fermentation yield using natamycin fermentation by-product of claim 1, wherein the total natamycin fermentation time is 100-150 h; further preferably 120 h.

7. The method for improving natamycin fermentation yield using a natamycin fermentation by-product according to claim 1 wherein the natamycin fermentation by-product is added in an amount of 5% by volume of the fermentation medium.

8. The method for improving natamycin fermentation yield using natamycin fermentation by-product of claim 1, comprising in particular the steps of:

(1) centrifuging the natamycin fermentation liquor after fermentation is finished, and collecting supernatant to obtain a natamycin fermentation by-product;

(2) under the aseptic condition, coating natamycin producing strain spores in an agar slant culture medium for activation, scraping the activated spores into a seed culture medium for shake culture to obtain a seed solution;

(3) inoculating the seed solution obtained in the step (2) into a fermentation culture medium according to the inoculation amount of 5-10%, performing fermentation culture, after fermenting for 0-48 h, adding the natamycin fermentation by-product collected in the step (1) into the fermentation culture medium according to 2.5-7.5% of the volume of the fermentation culture medium, and continuing to ferment for 72-120 h to obtain the natamycin.

9. The method for improving natamycin fermentation yield by using natamycin fermentation by-products according to claim 8, wherein in step (2), the formula of the agar slant culture medium is 5.0-15.0 g/L of glucose, 1.0-5.0 g/L of malt extract powder, 1.0-5.0 g/L of yeast extract, 3.0-10.0 g/L of peptone, 10.0-20.0 g/L of agar powder, and pH is 7.0-7.2;

the formula of the seed culture medium is as follows: 10.0-30.0 g/L of glucose, 3.0-10.0 g/L of yeast extract, 30.0-50.0 g/L of soybean cake powder and 7.0-7.2 of pH;

the conditions of shaking table culture are that the rotating speed is 150-200 rpm, the temperature is 26-30 ℃, and the culture time is 36-48 h.

10. The method for improving natamycin fermentation yield using natamycin fermentation by-product according to claim 8 wherein in step (3) the fermentation medium has the formula: soybean peptone 20g/L, yeast extract 4.5g/L, sodium chloride 2g/L, crystalline magnesium sulfate 1g/L, glucose 60g/L, pH 7.5.

Technical Field

The invention relates to a method for improving natamycin fermentation yield by using natamycin fermentation byproducts, belonging to the technical field of natamycin preparation.

Background

Natamycin (Pimaricin) is a natural, broad-spectrum and high-efficiency polyene macrolide antifungal agent with the molecular formula of C₃₃H₄₇NO_l3Molecular weight is 665.75, and melting point is 280 ℃. The molecular structure of natamycin contains a lactone ring skeleton structure consisting of 26 carbon atoms, wherein 4 conjugated double bonds form a polyene chromophore, and a trehalose is connected outside the inner ester ring. The natamycin finished product is white or cream-colored odorless tasteless crystalline powder, and trimolecular water is combined on natamycin molecules in the form of crystal water. The natamycin molecule contains a basic group and an acid group, and the isoelectric point is 6.5. Due to the characteristics of stable property, low dosage, high efficiency and safety, the antibacterial agent not only has good antibacterial effect on the aspects of food raw material preservation and finished product preservation, but also is widely applied to the aspects of medicine, silage, grain storage, poultry cultivation and the like.

Chinese patent document CN104946709A discloses a method for producing natamycin by fermentation, which comprises the step of feeding reducing sugar and propionate simultaneously during the fermentation process. The highest fermentation unit of the natamycin synthesized by the method can reach 8.13 g/L. But the yield of natamycin in the prior art can still be further improved.

A cholesterol oxidase gene (sgnE/pimE/scnE/slnE) exists in a natamycin biosynthesis gene cluster, and the encoded cholesterol oxidase does not participate in the synthesis and condensation reaction of a mother nucleus and a side chain of natamycin. However, in recent years, domestic and foreign studies prove that cholesterol oxidase is indispensable in the biosynthesis of natamycin. Cholesterol oxidase may be involved in the synthesis and accumulation of natamycin as a signaling protein [ Mendes MV, Recio E, Anton N, Guerra SM, Santos-Abertura J, Martin JF, Aparicio JF. Cholesterol oxidases act as signaling proteins for the biosynthesis of the polyethylene macrolactone pimaricin. chemistry and Biology,2007,14:279-290 ]. Further related studies indicate that addition of finished cholesterol oxidase at the early stage of fermentation can increase the production of natamycin [ Wang M, Wang SH, Zong GL, Hou ZW, Liu F, Liao DJ, Zhu XQ.Improvement of natamycin production by cholesterol oxidase in Streptomyces gilvosporus, Journal of Microbiology and Biotechnology,2016,26 (2).).

During natamycin fermentation, cholesterol oxidase can be secreted into the fermentation medium, and therefore, a large amount of cholesterol oxidase capable of improving natamycin yield exists in natamycin fermentation byproducts. However, in the prior natamycin production, extraction and purification process, the cholesterol oxidase is treated as waste liquid along with other fermentation byproducts, so that the phenomenon that the valuable byproducts are wasted is caused.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for improving the natamycin fermentation yield by using natamycin fermentation byproducts. The inventor firstly discovers that the natamycin fermentation by-products can promote the improvement of the natamycin fermentation yield, and reasonably utilizes the natamycin fermentation by-products while improving the natamycin fermentation yield, thereby changing waste into valuable.

The technical scheme of the invention is as follows:

a method for improving natamycin fermentation yield by using natamycin fermentation byproducts comprises the following steps:

centrifuging the natamycin fermentation liquor after fermentation is finished, and collecting supernatant to obtain a natamycin fermentation by-product; and then after the natamycin is fermented for 0-48 h, adding the natamycin fermentation by-product into the fermentation medium for continuous fermentation according to 2.5-7.5% of the volume of the fermentation medium to obtain the natamycin.

The natamycin fermentation liquor after fermentation is fermentation liquor after normal fermentation for 108-120 h, because natamycin is slightly soluble, after centrifugation, the obtained precipitate is mainly a natamycin product, the natamycin product is recovered and then is added into the fermentation liquor to be extracted for natamycin extraction, and the supernatant is a natamycin fermentation byproduct and is added into a fermentation medium in the natamycin fermentation process for increasing the natamycin yield.

According to the invention, the centrifugal collection conditions are preferably 9000-11000 rpm for 12-18 min.

According to the invention, preferably, the enzyme activity of the cholesterol oxidase in the natamycin fermentation by-product is more than or equal to 1U/mL; more preferably 1.3 to 1.5U/mL.

According to a preferred embodiment of the invention, the strain used for the fermentative production of natamycin is Streptomyces fuscoporus (Streptomyces gilvosporus), Streptomyces natalensis (Streptomyces natalensis), Streptomyces chattanoogensis (Streptomyces chattanoogensis) or Streptomyces lydicus (Streptomyces lydicus).

According to the optimization of the method, natamycin fermentation byproducts are added after natamycin fermentation production is carried out for 12-36 h; more preferably after 24 hours.

According to the optimization of the method, the total fermentation time of the natamycin is 100-150 h; further preferably 120 h.

According to a preferred embodiment of the present invention, the natamycin fermentation by-product is added in an amount of 5% by volume of the fermentation medium.

A method for improving natamycin fermentation yield by using natamycin fermentation byproducts comprises the following steps:

(1) centrifuging the natamycin fermentation liquor after fermentation is finished, and collecting supernatant to obtain a natamycin fermentation by-product;

(2) under the aseptic condition, coating natamycin producing strain spores in an agar slant culture medium for activation, scraping the activated spores into a seed culture medium for shake culture to obtain a seed solution;

(3) inoculating the seed solution obtained in the step (2) into a fermentation culture medium according to the inoculation amount of 5-10% for fermentation culture, after fermenting for 0-48 h, adding the natamycin fermentation byproducts collected in the step (1) into the fermentation culture medium according to 2.5-7.5% of the volume of the fermentation culture medium, and continuing to ferment for 100-150 h to obtain the natamycin.

Preferably, in step (2), the agar slant culture medium has the following formula: 5.0-15.0 g/L of glucose, 1.0-5.0 g/L of malt extract powder, 1.0-5.0 g/L of yeast extract, 3.0-10.0 g/L of peptone, 10.0-20.0 g/L of agar powder and pH 7.0-7.2;

the formula of the seed culture medium is as follows: 10.0-30.0 g/L of glucose, 3.0-10.0 g/L of yeast extract, 30.0-50.0 g/L of soybean cake powder and 7.0-7.2 of pH.

According to the invention, in the step (2), the rotation speed is 150-200 rpm, the temperature is 26-30 ℃, and the culture time is 36-48 h.

Preferably, in step (3), the fermentation medium has a formula of: soybean peptone 20g/L, yeast extract 4.5g/L, sodium chloride 2g/L, crystalline magnesium sulfate 1g/L, glucose 60g/L, pH 7.5.

The experimental procedures not specified in the present invention, especially the natamycin fermentation process, were performed according to the methods described in the prior art.

The invention has the following beneficial effects:

1. due to the extremely low solubility (50-100 mg/L) of natamycin in water, the natamycin fermentation broth, natamycin and thalli after fermentation can be separated by sufficient centrifugation, and a natamycin fermentation byproduct containing cholesterol oxidase is obtained. And then the natamycin fermentation by-product is added into the fermentation medium, so that the characteristic that the cholesterol oxidase can improve the yield of the natamycin can be effectively utilized, and the yield of the natamycin production strain can be obviously improved. The natamycin shake flask fermentation yield obtained by the method of the invention is 2.40 +/-0.17 g/L at most. After the expanded production is adopted, 5% of natamycin fermentation byproducts are added after fermentation is carried out for 24 hours in a 1L fermentation system, the natamycin yield is 8.83-8.86 g/L and is obviously higher than 6.70-6.72 g/L of the natamycin fermentation byproducts which are not added, and the natamycin yield is improved by 31.84%. Compared with the natamycin yield of 7.15-7.69 g/L obtained by directly adding commercial cholesterol oxidase into the fermentation medium, the natamycin yield of the method is improved by 23.49%.

2. According to the invention, the natamycin fermentation byproduct containing the cholesterol oxidase is added into the fermentation culture medium, so that the cholesterol oxidase can be provided for the production and preparation of natamycin, and the yield of natamycin is increased; on the other hand, the natamycin fermentation medium adopts commercialized components such as glucose, peptone, yeast powder and inorganic salt, and the carbon source which is continuously supplemented in a longer fermentation process often remains a part which is not consumed after the production is finished.

Detailed Description

The following examples and test examples are intended to illustrate the present invention, but are not intended to limit the scope of the present invention.

The materials involved in the invention are all common commercial products.

Preparing an agar slant culture medium: 10g/L of glucose, 3g/L of malt extract powder, 3g/L of yeast extract, 5g/L of peptone, 15g/L of agar powder, pH7.0, sterilizing at 121 ℃ for 20min, and cooling for later use.

Preparing a shake flask seed culture medium: 20g/L of glucose, 5g/L of yeast extract, 40g/L of soybean cake powder, pH 7.2, sterilization at 121 ℃ for 20min, and cooling for later use.

Preparation of a natamycin liquid seed culture medium in a fermentation tank: 10g/L of glucose, 3g/L of yeast extract, 5g/L of soybean peptone and 3g/L of malt extract powder; 1.40g/L of defoaming agent; sterilizing at the temperature of 121 ℃ for 20min at the pH of 7.0-7.2, and cooling for later use.

Preparation of a fermentation medium: soybean peptone 20g/L, yeast extract 4.5g/L, sodium chloride 2g/L, crystalline magnesium sulfate 1g/L, glucose 60g/L, pH 7.5; sterilizing at 121 deg.C for 20min, and cooling.

Measurement of pH: direct measurements were performed using a mettler pH meter.

Example 1: preparation of natamycin fermentation by-products

Activating streptomyces natalensis on an agar slant culture medium, scraping streptomyces natalensis spores from the agar slant culture medium after activation, and inoculating the spores into a shake flask seed culture medium; shaking and culturing at 30 deg.C and 200rpm in shaking table for 24h to obtain seed solution; transferring the seed liquid into a fermentation medium according to the inoculation amount of 5%, fermenting for 120h at 200rpm and 28 ℃, and obtaining natamycin fermentation liquid after the fermentation is finished.

And centrifuging the natamycin fermentation liquor obtained in the step (10000 rpm) for 15min, and collecting supernatant to obtain a natamycin fermentation byproduct. And (3) measuring the enzyme activity of the cholesterol oxidase of the natamycin fermentation by-product, wherein the enzyme activity reaches more than 1U/mL, and the qualified fermentation supernatant is obtained.

The specific determination method of the enzyme activity of the cholesterol oxidase comprises the following steps:

preparing a solution A: 4-aminoantipyrine, 1 mmol/L; phenol, 6 mmol/L; peroxidase, 7000U/L; potassium phosphate buffer (pH 7.5), 25 mmol/L;

preparing a solution B: cholesterol, 8.26 g/L; triton X-100, 4.26%; isopropanol is used as a solvent;

and (3) putting 3mL of the solution A into a test tube, preserving the heat at 37 ℃ for 3min, adding different amounts of hydrogen peroxide solutions to enable the final concentration to be 0.02-0.10 mmol/L, reacting for 5min, measuring the absorbance at 500nm, and establishing a standard curve. And (3) putting 3mL of the solution A and 150 mu L of the solution B in a 5mL EP tube, preserving the heat at 37 ℃ for 3min, adding 50 mu L of fermentation broth supernatant, accurately reacting for 5min, boiling in a boiling water bath for 3min, cooling in an ice bath, and measuring the absorbance at 500 nm.

The enzyme activity calculation formula is as follows:

enzyme activity (U/mL) ═ Cx (V)₁/V₂)×N÷T，

C, calculating the concentration of the hydrogen peroxide according to a standard curve; v₁The total volume of the reaction solution; v₂Volume of enzyme solution; n, dilution times; t, reaction time.

The calculation result shows that the enzyme activity of the cholesterol oxidase of the natamycin fermentation by-product is 1.5 +/-0.07U/mL.

Example 2

A method for improving natamycin fermentation yield by using natamycin fermentation byproducts comprises the following steps:

1) under the aseptic condition, taking the streptomyces natalensis to activate on an agar slant culture medium, scraping a streptomyces natalensis spore from the agar slant culture medium after activation, and inoculating the streptomyces natalensis spore into a shake flask seed culture medium; shaking and culturing at 30 deg.C and 200rpm in shaking table for 24h to obtain seed solution;

2) transferring the seed liquid into a fermentation medium according to the inoculation amount of 5%, wherein the fermentation medium is contained in 500mL triangular shake flasks, the volume of each 500mL triangular shake flask-contained fermentation medium is 50mL, 6 bottles are contained in each experimental group, the natamycin fermentation by-product in the embodiment 1 is added while the seed liquid is inoculated, the seed liquid is fermented at 200rpm and 28 ℃ for 120h, and the natamycin fermentation liquid is obtained after the fermentation is finished; wherein the addition amounts of natamycin fermentation by-products are 0% (control group), 2.5% (experimental group 1), 5% (experimental group 2), 7.5% (experimental group 3) and 10% (experimental group 4) of the volume of the fermentation medium, respectively.

The natamycin content was determined for the above 5 groups and the results are shown in table 1.

The method for measuring the content of the natamycin is carried out according to the method specified in the national standard GB 25532-2010.

TABLE 1 Effect of different amounts of natamycin fermentation by-products on natamycin production

Group of	By-product addition (volume ratio)	By-product cholesterol oxidase enzyme activity (U/mL)	Natamycin yield (g/L)
				Control group	0	1.5±0.07	1.13±0.06
Experimental group 1	2.5	1.5±0.07	1.32±0.14
				Experimental group 2	5	1.5±0.07	1.90±0.15
Experimental group 3	7.5	1.5±0.07	1.748±0.17
				Experimental group 4	10	1.5±0.07	0.962±0.13

As can be seen from the results in Table 1, the addition of natamycin fermentation by-product containing cholesterol oxidase to the shake flask fermentation medium can effectively increase the yield of natamycin. Compared with the control group, the yield of natamycin in the experimental groups 1-3 is increased by 16.81-68.14%. Compared with the control group, the yield of the natamycin in the experimental group 1 is improved by 16.81 percent. Compared with the control group, the yield of the natamycin in the experimental group 2 is improved by 68.14 percent. Compared with the control group in the experimental group 3, the yield of natamycin is improved by 54.69%. The yield of natamycin was slightly reduced in the experimental group 4 compared to the control group. According to the above data results, the amount of natamycin fermentation by-product added is preferably 5% in the examples hereinafter of the invention.

Example 3

A method for improving natamycin fermentation yield by using natamycin fermentation byproducts comprises the following steps:

1) under the aseptic condition, taking the streptomyces natalensis to activate on an agar slant culture medium, scraping a streptomyces natalensis spore from the agar slant culture medium after activation, and inoculating the streptomyces natalensis spore into a shake flask seed culture medium; shaking and culturing at 30 deg.C and 200rpm in shaking table for 24h to obtain seed solution;

2) transferring the seed liquid into a fermentation medium according to the inoculation amount of 5%, wherein the fermentation medium is contained in a 500mL triangular shake flask, the volume of the fermentation medium contained in each 500mL triangular shake flask is 50mL, each experimental group comprises 6 bottles, the fermentation culture is carried out at 200rpm and the temperature of 28 ℃, the natamycin fermentation by-product in the embodiment 1 is added in the fermentation culture process, the addition amount is 5% of the volume of the fermentation medium, the fermentation is continued, the total fermentation time is 120h, and the fermentation is finished, so that natamycin fermentation liquid is obtained; wherein the addition time of the natamycin fermentation by-products is respectively 0h after fermentation culture (control group), 12h after fermentation culture (experimental group 1), 24h after fermentation culture (experimental group 2), 36h after fermentation culture (experimental group 3), 48h after fermentation culture (experimental group 4) and 60h after fermentation culture (experimental group 5).

The natamycin content was determined for the above 6 groups and the results are shown in table 2.

The method for measuring the content of the natamycin is carried out according to the method specified in the national standard GB 25532-2010.

TABLE 2 Effect of different natamycin fermentation by-product addition times on natamycin yield

The results in table 2 show that the natamycin yields of the experimental groups 1 to 5 are significantly improved and the improvement range is 27.5 to 120.2% compared with the control group. However, the addition of the natamycin fermentation by-product containing cholesterol oxidase at different time of strain fermentation affects the natamycin yield, the natamycin yield of the experimental group 2 is increased by 94.9% compared with the natamycin yield of the control group, is increased by 14.2% compared with the experimental group 1, the natamycin yield of the experimental group 3 is increased by 120.2% compared with the natamycin yield of the control group, is increased by 29.1% compared with the experimental group 1, and the natamycin yield of the experimental group 4 and the experimental group 5 is decreased compared with the experimental group 1, but the natamycin yield is still increased by more than 27.5% compared with the natamycin yield of the control group. Based on the above data results, the addition time of natamycin fermentation by-product in the examples hereinafter of the invention is preferably 24 h.

Example 4

A method for improving natamycin fermentation yield by using natamycin fermentation byproducts comprises the following steps:

1) under the aseptic condition, taking streptomyces natalensis to activate on an agar slant culture medium, scraping streptomyces natalensis spores from the agar slant culture medium after activation, inoculating the streptomyces natalensis spores into a fermentation tank liquid seed culture medium, and stirring and culturing for 48 hours under the conditions of 29 ℃ and 300rpm to obtain seed liquid;

2) inoculating the seed solution into a fermentation medium according to the inoculation amount of 10%, wherein the volume of the fermentation medium before inoculation is 0.8L, putting the fermentation medium into a 1L full-automatic fermentation tank, fermenting at 29 ℃, controlling the dissolved oxygen DO value to be 30-40% in the fermentation process, feeding 20% NaOH with concentration of 20% when the pH value naturally drops to 6.00 +/-0.10 from the initial pH value in the initial culture stage, and maintaining the pH value to be 6.00 +/-0.20 until the fermentation is finished; adding a glucose solution with the mass percentage concentration of 40% in the fermentation process, controlling the glucose content to be 15.00 +/-5.00 g/L all the time, adding the natamycin fermentation by-product in the embodiment 1 after fermenting for 24 hours, wherein the addition amount is 5% of the volume of the fermentation medium, and continuously fermenting for 96 hours to obtain natamycin; setting a control group without adding natamycin fermentation by-products

The natamycin content was determined for group 2 above, and the results are shown in table 3.

The method for measuring the content of the natamycin is carried out according to the method specified in the national standard GB 25532-2010.

TABLE 3 influence of different amounts of natamycin fermentation by-products on natamycin production in 1L fermentation system

As can be seen from Table 3, the natamycin yield of the experimental group is 8.86g/L due to the addition of 5% by volume of the natamycin fermentation by-product containing cholesterol oxidase after 24 hours of fermentation, which is significantly higher than the natamycin yield of the control group without addition of the natamycin fermentation by-product of 6.72g/L, and the yield is increased by 31.84%. The verification result of the 1L fermentation system shows that the addition of the natamycin fermentation by-product containing cholesterol oxidase with the volume of 5 percent can obviously improve the yield of natamycin.

Example 5

A method for improving natamycin fermentation yield by using natamycin fermentation byproducts comprises the following steps:

1) under aseptic condition, activating streptomyces on an agar slant culture medium, scraping streptomyces donovani spores from the agar slant culture medium after activation, inoculating the streptomyces donovani spores into a fermentation tank liquid seed culture medium, and stirring and culturing for 48 hours under the conditions of 29 ℃ and 300rpm to obtain a seed liquid;

2) inoculating the seed solution into a fermentation medium according to the inoculation amount of 10%, wherein the volume of the fermentation medium before inoculation is 0.8L, putting the fermentation medium into a 1L full-automatic fermentation tank, fermenting at 29 ℃, controlling the dissolved oxygen DO value to be 30-40% in the fermentation process, feeding 20% NaOH with concentration of 20% when the pH value naturally drops to 6.00 +/-0.10 from the initial pH value in the initial culture stage, and maintaining the pH value to be 6.00 +/-0.20 until the fermentation is finished; adding a glucose solution with the mass percentage concentration of 40% in the fermentation process, controlling the glucose content to be 15.00 +/-5.00 g/L all the time, adding the natamycin fermentation by-product in the example 1 after the experiment group is fermented for 24 hours, wherein the addition amount is 5% of the volume of the fermentation medium, and continuously fermenting for 96 hours to obtain the natamycin; setting a control group, wherein the control group 1 is not added with exogenous substances; control group 2 was supplemented with an equivalent amount of purified commercially available cholesterol oxidase derived from Brevibacterium (Brevibacterium) as that of the experimental group; in control group 3, a commercially available cholesterol oxidase derived from Streptomyces sp was added in an amount equivalent to that in the experimental group.

The natamycin content was determined for the above 4 groups and the results are shown in table 4.

The method for measuring the content of the natamycin is carried out according to the method specified in the national standard GB 25532-2010.

TABLE 4 Effect of different cholesterol oxidases on natamycin production in 1L fermentation System

As can be seen from Table 4, since the test group was fermented for 24 hours and then 5% by volume of the natamycin fermentation by-product containing cholesterol oxidase was added, and the control groups 2 and 3 were fermented for 24 hours and then pure products of cholesterol oxidase derived from Brevibacterium and Streptomyces were added, the natamycin yields were all higher than 6.70g/L, 8.83g/L, 7.15g/L and 7.69g/L, respectively, of the natamycin of the control group 1, and the yields were increased to 31.79%, 6.7% and 14.77%. Due to different sources of cholesterol oxidase, compared with a control group added with pure cholesterol oxidase from brevibacterium and streptomyces, the natamycin fermentation yield of the experimental group is improved by 23.49% compared with that of the control group 2, and is improved by 14.82% compared with that of the control group 3, and the yield reaches the highest. Therefore, the verification result of the 1L fermentation system of this example shows that the addition of 5% by volume of natamycin fermentation by-product containing cholesterol oxidase can significantly improve the yield of natamycin.