A method for improving the yield of natamycin fermentation

Abstract

The present application relates to natamycin production technical field, specifically to a method for improving natamycin fermentation yield, comprising the following steps: shake flask seed culture: under sterile conditions, brown yellow spore streptomyces spores are coated on agar medium, and placed in a culture box for culture, then the cultured brown yellow spore streptomyces is prepared into spore suspension, and the spore suspension is taken and added into a triangular flask of seed culture medium for culture; fermentation tank fermentation culture: the cultured seed liquid is inoculated into a fermentation culture medium comprising proteose peptone, yeast powder, sodium chloride, magnesium sulfate, glucose, betaine, ampicillin, sodium succinate for stirring culture, and glucose is added during the culture until the fermentation is completed, and natamycin is obtained. The present application significantly improves the yield of brown yellow spore streptomyces producing natamycin by adding betaine, ampicillin and sodium succinate in the fermentation culture medium; the production method is simple to operate and is conducive to large-scale industrialization.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natamycin production, and particularly relates to a method for improving the fermentation yield of natamycin. BACKGROUND

[0002] Natamycin is a high-efficiency, safe and broad-spectrum antifungal biological preservative, which can effectively inhibit the growth of yeast and mold, prevent the formation of aflatoxin in filamentous fungi, and reduce the harm of pathogenic fungi to human body. Natamycin has very low solubility, and can be used to treat the surface of food to increase the shelf life of food without affecting the flavor and taste.

[0003] Sodium acetate is a precursor of natamycin synthesis. The acetate unit can form a polyketide by polymerization with propionic acid as a unit, which is conducive to the formation of lactone ring, further promotes the metabolism of the bacterial body, increases the flux of tricarboxylic acid cycle, and is conducive to increasing the yield of natamycin. Natamycin is a macrolide antibiotic. In the biosynthesis of macrolide antibiotics, some substances are involved in the synthesis and metabolic regulation of substances, and are the hub connecting primary metabolism and secondary metabolism. Some organic substances, such as vitamins and antibiotics, generally act as components or functions of coenzymes, which are conducive to increasing the yield of natamycin.

[0004] At present, natamycin is used in the production and preservation of dairy products, meat products, fruit juice beverages, grape wine and the like. However, the fermentation yield of natamycin is low, which leads to high price of natamycin and limits the application prospect. Therefore, it is of great significance to conduct in-depth research and development on natamycin fermentation. SUMMARY

[0005] In view of the technical problem of low fermentation yield of natamycin, the present application provides a method for improving the fermentation yield of natamycin.

[0006] A method for improving the fermentation yield of natamycin, comprising the following steps:

[0007] (1) Shake flask seed culture: under sterile conditions, spores of Streptomyces tanashiensis are coated on agar medium and cultured in a culture box. The cultured Streptomyces tanashiensis is prepared into a spore suspension, and the spore suspension is taken and added to a triangular flask containing seed culture medium for culture.

[0008] (2) Fermentation tank fermentation culture: the cultured seed liquid is inoculated into a fermentation culture medium containing proteose peptone, yeast powder, sodium chloride, magnesium sulfate, glucose, betaine, ampicillin and sodium succinate, and is subjected to stirring culture. Glucose is added during the culture until the fermentation is completed, and natamycin is obtained.

[0009] Furthermore, the concentration ratio of betaine, ampicillin, and sodium succinate is 10-20:1-2:40-60.

[0010] Furthermore, the fermentation inoculum size is 10%.

[0011] Furthermore, the fermentation temperature was 30℃ and the fermentation time was 110h.

[0012] Furthermore, the stirring speed in step (2) is 350-500 rpm.

[0013] Furthermore, the glucose concentration during fermentation should not exceed 20 g / L.

[0014] Furthermore, the fermentation volume is 8-10L.

[0015] Furthermore, after fermentation, the concentration of natamycin was measured to be 14.69-15.82 g / L.

[0016] The beneficial effects of this invention are as follows: adding betaine, ampicillin, and sodium succinate to the fermentation medium significantly increases the yield of natamycin produced by Streptomyces thallus; the production method provided by this invention is simple to operate and is conducive to large-scale industrialization. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a graph showing the relationship between natamycin content and fermentation time in Example 1.

[0019] Figure 2 This is a graph showing the natamycin content and fermentation time in Example 2.

[0020] Figure 3 This is a graph showing the relationship between natamycin content and fermentation time in Example 3.

[0021] Figure 4 This is a graph showing the natamycin content and fermentation time in Example 4.

[0022] Figure 5 This is a graph showing the relationship between natamycin content and fermentation time in Comparative Example 1. Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0024] Example 1

[0025] (1) Shake-flask seed culture: Under aseptic conditions, the spores of Streptomyces chrysosporium were spread on agar medium and cultured in an incubator at 30°C. The cultured Streptomyces chrysosporium was then prepared into a spore suspension. 1 ml of the spore suspension was added to an Erlenmeyer flask containing seed culture medium (10 g / L glucose, 6 g / L yeast powder, 6 g / L peptone, 5 g / L sodium chloride, pH 7.4). The mixture was cultured at 30°C for 20 h at a speed of 250 rpm.

[0026] (2) Fermentation culture in a 10L fermenter: The cultured seed culture was inoculated at a rate of 10% into the fermentation medium (20 g / L glucose, 6 g / L yeast extract, 20 g / L peptone, 2 g / L sodium chloride, 1 g / L magnesium sulfate, 0.5 g / L betaine, 0.1 g / L ampicillin, 2 g / L sodium succinate, pH 7.4), with a volume of 8L. The stirring speed was 350 rpm, and the culture temperature was 30℃. Glucose was added continuously during the process until the end of fermentation. The glucose concentration in the fermentation broth did not exceed 20 g / L. Fermentation was completed after 110 h. The natamycin content in the fermentation broth was determined by HPLC to be 15.82 g / L. The natamycin content versus fermentation time curve in the fermenter is shown in the figure below. Figure 1 .

[0027] Example 2

[0028] (1) Shake-flask seed culture: Under aseptic conditions, the spores of Streptomyces chrysosporium were spread on agar medium and cultured in an incubator at 30°C. The cultured Streptomyces chrysosporium was then prepared into a spore suspension. 1 ml of the spore suspension was added to an Erlenmeyer flask containing seed culture medium (10 g / L glucose, 6 g / L yeast powder, 6 g / L peptone, 5 g / L sodium chloride, pH 7.4). The mixture was cultured at 30°C for 20 h at a speed of 250 rpm.

[0029] (2) Fermentation culture in a 10L fermenter: The cultured seed culture was inoculated at a rate of 10% into the fermentation medium (20 g / L glucose, 6 g / L yeast extract, 20 g / L peptone, 2 g / L sodium chloride, 1 g / L magnesium sulfate, 1 g / L betaine, 0.05 g / L ampicillin, 3 g / L sodium succinate, pH 7.4), with a volume of 8L. The stirring speed was 400 rpm, and the culture temperature was 30℃. Glucose was added continuously during the process until the end of fermentation. The glucose concentration in the fermentation broth did not exceed 20 g / L. Fermentation was completed after 110 h. The natamycin content in the fermentation broth was determined by HPLC to be 15.04 g / L. The natamycin content versus fermentation time curve in the fermenter is shown in the figure below. Figure 2 .

[0030] Example 3

[0031] (1) Shake-flask seed culture: Under aseptic conditions, the spores of Streptomyces chrysosporium were spread on agar medium and cultured in an incubator at 30°C. The cultured Streptomyces chrysosporium was then prepared into a spore suspension. 1 ml of the spore suspension was added to an Erlenmeyer flask containing seed culture medium (10 g / L glucose, 6 g / L yeast powder, 6 g / L peptone, 5 g / L sodium chloride, pH 7.4). The mixture was cultured at 30°C for 20 h at a speed of 250 rpm.

[0032] (2) Fermentation culture in a 10L fermenter: The cultured seed culture was inoculated at a rate of 10% into the fermentation medium (20 g / L glucose, 6 g / L yeast extract, 20 g / L peptone, 2 g / L sodium chloride, 1 g / L magnesium sulfate, 0.5 g / L betaine, 0.05 g / L ampicillin, 2 g / L sodium succinate, pH 7.4), with a volume of 8L. The stirring speed was 450 rpm, and the culture temperature was 30℃. Glucose was added continuously during the process until the end of fermentation. The glucose concentration in the fermentation broth did not exceed 20 g / L. Fermentation was completed after 110 h. The natamycin content in the fermentation broth was determined by HPLC to be 15.15 g / L. The natamycin content versus fermentation time curve in the fermenter is shown in the figure. Figure 3 .

[0033] Example 4

[0034] (1) Shake-flask seed culture: Under aseptic conditions, the spores of Streptomyces chrysosporium were spread on agar medium and cultured in an incubator at 30°C. The cultured Streptomyces chrysosporium was then prepared into a spore suspension. 1 ml of this spore suspension was added to an Erlenmeyer flask containing seed culture medium (10 g / L glucose, 6 g / L yeast powder, 6 g / L peptone, 5 g / L sodium chloride, pH 7.4). The mixture was cultured at 30°C for 20 h at a speed of 250 rpm.

[0035] (2) Fermentation culture in a 10L fermenter: The cultured seed liquid was inoculated into the fermentation medium (20g / L glucose, 6g / L yeast extract, 20g / L peptone, 2g / L sodium chloride, 1g / L magnesium sulfate, 1g / L betaine, 0.1g / L ampicillin, 3g / L sodium succinate, pH 7.4) at a 10% inoculation rate, with a liquid volume of 8L. The stirring speed was 500rpm, and the culture temperature was 30℃. Glucose was added continuously during the process until the end of fermentation. The glucose concentration in the fermentation broth did not exceed 20g / L. Fermentation was completed after 110h. The natamycin content in the fermentation broth was determined by HPLC to be 14.69g / L. The natamycin content versus fermentation time curve in the fermenter is shown in the figure. Figure 4 .

[0036] Comparative Example 1

[0037] (1) Shake-flask seed culture: Under aseptic conditions, the spores of Streptomyces chrysosporium were spread on agar medium and cultured in an incubator at 30°C. The cultured Streptomyces chrysosporium was then prepared into a spore suspension. 1 ml of this spore suspension was added to an Erlenmeyer flask containing seed culture medium (10 g / L glucose, 6 g / L yeast powder, 6 g / L peptone, 5 g / L sodium chloride, pH 7.4). The mixture was cultured at 30°C for 20 h at a speed of 250 rpm.

[0038] (2) Fermentation culture in a 10L fermenter: The cultured seed culture was inoculated at a rate of 10% into the fermentation medium (20 g / L glucose, 6 g / L yeast extract, 20 g / L peptone, 2 g / L sodium chloride, 1 g / L magnesium sulfate, pH 7.4), with a volume of 8L. The stirring speed was 350 rpm, and the culture temperature was 30℃. Glucose was added continuously during the process until fermentation was completed. The glucose concentration in the fermentation broth did not exceed 20 g / L. Fermentation was completed after 110 h. The natamycin content in the fermentation broth was determined by HPLC to be 11.36 g / L. The natamycin content versus fermentation time curve in the fermenter is shown in the figure below. Figure 5 .

[0039] Compared with Comparative Example 1, Examples 1-4 showed that adding betaine, ampicillin, and sodium succinate to the fermentation medium significantly increased the yield of natamycin produced by Streptomyces thallus.

[0040] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A method for increasing natamycin fermentation yield, characterized in that, Includes the following steps: (1) Shake flask seed culture: Under aseptic conditions, the spores of Streptomyces chrysogenum were spread on agar medium and placed in an incubator for culture. The cultured Streptomyces chrysogenum was then prepared into a spore suspension. The spore suspension was then added to an Erlenmeyer flask in the seed culture medium for culture. (2) Fermentation culture in a fermenter: The cultured seed liquid was inoculated into a fermentation medium containing 20 g / L glucose, 6 g / L yeast powder, 20 g / L peptone, 2 g / L sodium chloride, 1 g / L magnesium sulfate, 0.5 or 1 g / L betaine, 0.05 or 0.1 g / L ampicillin, 2 or 3 g / L sodium succinate, and pH 7.

4. The medium was stirred and cultured, with glucose added during the culture process until fermentation was completed, and natamycin was obtained. The glucose concentration during fermentation should not exceed 20 g / L; The fermentation volume is 8-10L.

2. The method for increasing natamycin fermentation yield as described in claim 1, characterized in that, The fermentation inoculum size was 10%.

3. The method for increasing natamycin fermentation yield as described in claim 1, characterized in that, The fermentation temperature was 30℃ and the fermentation time was 110h.

4. The method for increasing natamycin fermentation yield as described in claim 1, characterized in that, The stirring speed in step (2) is 350-500 rpm.

5. The method for increasing natamycin fermentation yield as described in claim 1, characterized in that, After fermentation, the concentration of natamycin was measured to be 14.69-15.82 g / L.

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