A fermentation medium for producing natamycin

Abstract

The present invention discloses a fermentation medium for producing natamycin, relating to the field of microbial fermentation. In addition to a carbon source, a nitrogen source, and a phosphorus source, the fermentation medium contains threonine and biotin. The combined action of threonine and biotin increases the content of propionyl-CoA, a precursor of natamycin. Glutamic acid is added to inhibit the utilization of acetyl-CoA in the tricarboxylic acid cycle, thereby increasing the content of acetyl-CoA, a precursor of natamycin, and promoting the synthesis of natamycin. The present invention is the first to utilize the combined action of threonine and biotin in a natamycin fermentation medium to increase the content of propionyl-CoA, a precursor of natamycin, while simultaneously adding glutamate to inhibit the utilization of acetyl-CoA in the tricarboxylic acid cycle, thereby increasing the content of acetyl-CoA, a precursor of natamycin. These combined actions enhance the metabolic flux of natamycin synthesis, thereby increasing the fermentation yield.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation culture, relates to the preparation of a fermentation culture medium, and particularly relates to a fermentation culture medium for producing natamycin. Background Art

[0002] Natamycin (Nαtαmycin) is a natural antifungal compound fermented by Streptomyces chrysoporus. It belongs to the polyene macrolide class and is widely and effectively inhibiting the growth of various molds and yeasts, as well as the production of mycotoxins. Its mechanism of action is through its lactone ring structure interacting with sterol compounds on the fungal cell membrane to form antibiotic-sterol compounds, which disrupt the fungal cytoplasmic membrane structure. The hydrophilic portion (polyol portion) of the macrolide forms pores in the membrane, impairing cell membrane permeability, leading to the leakage of amino acids, electrolytes, and other substances from the bacteria, resulting in bacterial death. In June 1982, the US Food and Drug Administration officially approved natamycin as a food preservative. In 1996, the China Food Additive Standardization Chemical Technical Committee officially approved natamycin as a food preservative, allowing for widespread use in food preservation and antifungal treatment. However, natamycin production is plagued by low yields and complex fermentation formulas. Chinese patent CN109943610A discloses a method for adding saturated fatty acids. However, saturated fatty acids are mostly insoluble in water, resulting in low microbial utilization. Furthermore, excessive amounts of saturated fatty acids affect the fluid properties of the fermentation broth, making material transfer more difficult. The resulting natamycin content after 120 hours of fermentation is only 7.5 g / L. Chinese patent CN106755216A discloses a method for adding valine and threonine during fermentation, while also adding soybean oil. This complex process results in a final natamycin content of only approximately 4 g / L. Summary of the Invention

[0003] In order to solve the above-mentioned problems, the present invention provides a high-efficiency, simple and low-cost natamycin fermentation medium capable of increasing the natamycin content.

[0004] To achieve the purpose, the inventors proposed a technical solution: a fermentation medium for producing natamycin, wherein the fermentation medium contains threonine, biotin and glutamic acid.

[0005] A further technical solution is that the content of threonine is 0.1-0.3 g / L, the content of biotin is 0.1 g / L, and the content of glutamic acid is 0.2-0.4 g / L.

[0006] A further technical solution is that the fermentation medium further comprises: corn starch, peptone, yeast powder, potassium dihydrogen phosphate, and α-amylase.

[0007] A further technical solution is that the components and content of the fermentation medium are: corn starch 60g / L, peptone 20g / L, yeast powder 10g / L, potassium dihydrogen phosphate 0.2g / L, α-amylase 0.06g / L, threonine 0.1-0.3 g / L, biotin 0.1g / L, and glutamic acid 0.2-0.4g / L.

[0008] A further technical solution is that the components and content of the fermentation medium are: corn starch 60g / L, peptone 20g / L, yeast powder 10g / L, potassium dihydrogen phosphate 0.2g / L, α-amylase 0.06g / L, threonine 0.2g / L, biotin 0.1g / L, and glutamic acid 0.3g / L.

[0009] The above technical solution achieves the following beneficial effects: Based on the biosynthetic pathway of natamycin and incorporating the metabolic regulation mechanism associated with natamycin biosynthesis in Streptomyces chrysoporus, the present invention creatively proposes adding threonine, biotin, and glutamate to the fermentation medium. Through the combined regulatory action of these three elements, the accumulation of natamycin synthesis precursors is achieved, thereby improving natamycin fermentation levels. This method significantly improves natamycin fermentation levels, with the highest natamycin fermentation level reaching 12.312 g / L in shake flasks after 120 hours of fermentation, a 129.73% increase compared to the control. This further reduces natamycin production costs and promotes the promotion and application of natamycin as a safe biopreservative.

[0010] The mechanism by which threonine, biotin, and glutamate work together to increase natamycin content can be explained by three points: First, threonine undergoes deamination to form α-ketobutyrate, which, under the catalysis of the pyruvate dehydrogenase complex, forms propionyl-CoA. Second, biotin is a key coenzyme for carboxyltransferase and carboxylase, and carboxylase can convert acetyl-CoA into propionyl-CoA. The combined action of threonine and biotin can significantly increase the concentration of propionyl-CoA. Third, glutamate is converted to α-ketobutyrate in vivo through deamination. α-ketoglutarate inhibits the absorption and utilization of acetyl-CoA by the tricarboxylic acid cycle, increasing its concentration. Simultaneously, with the assistance of the coenzyme biotin, carboxyltransferase converts acetyl-CoA into propionyl-CoA, providing a precursor for the synthesis of natamycin and increasing its content. Excessive use of any one of threonine, biotin and glutamic acid will affect the normal metabolic growth of the bacteria. Therefore, in order to increase the content of propionyl-CoA, the precursor of natamycin, the three need to be used in combination to increase the content of propionyl-CoA in different ways, thereby promoting the synthesis of natamycin. DETAILED DESCRIPTION

[0011] The present invention aims to solve the problem of low natamycin fermentation content in the prior art and provides a fermentation medium that can significantly increase the natamycin content. The present invention is described in detail below with reference to specific examples.

[0012] Since the slant, seed liquid culture medium and culture conditions all adopt the technology disclosed in the industry, the slant and seed liquid culture in the comparative examples and examples described below all adopt unified culture conditions. The specific culture method is: take a glycerol tube of Streptomyces chrysoporus preserved by the applicant, inoculate the slant culture medium with bacteria, and after the slant matures, dig out part of the colony and the slant culture medium and inoculate the seed culture medium together. After the seed liquid matures, take part of the seed liquid and inoculate the fermentation medium. The fermentation culture temperature is 30°C, the rotation speed is 220 rpm, and the culture is cultured for 120 hours.

[0013] Comparative Example 1

[0014] The fermentation medium consisted of 60 g / L corn starch, 20 g / L peptone, 10 g / L yeast extract, 0.2 g / L potassium dihydrogen phosphate, and 0.001 g α-amylase per gram of corn starch. Appropriate amounts of raw materials were weighed and dissolved in water, then the volume was adjusted to 250 mL. The mixture was evenly distributed into five 500 mL Erlenmeyer flasks (50 mL per flask). Sterilized at 121°C for 30 minutes, the mixture was cooled to room temperature, and the mature seed solution was inoculated into sterile fermentation medium at a 6% (v / v) inoculation rate. The culture temperature was 30°C and the rotation speed was 220 rpm. After 120 hours of incubation, the natamycin content in the fermentation flasks was determined.

[0015] Example 1

[0016] The difference from Comparative Example 1 is that, in addition to adding corn starch, peptone, yeast powder, potassium dihydrogen phosphate, and α-amylase in the same proportions to the fermentation medium, 0.1 g / L of threonine is additionally added.

[0017] Example 2

[0018] The difference from Comparative Example 1 is that, in addition to adding corn starch, peptone, yeast powder, potassium dihydrogen phosphate, and α-amylase in the same proportions to the fermentation medium, 0.1 g / L of biotin is additionally added.

[0019] Example 3

[0020] The difference from Comparative Example 1 is that, in addition to adding corn starch, peptone, yeast powder, potassium dihydrogen phosphate, and α-amylase in the same proportions to the fermentation medium, 0.2 g / L of glutamic acid is additionally added.

[0021] Example 4

[0022] The difference from Comparative Example 1 is that, in addition to adding corn starch, peptone, yeast powder, potassium dihydrogen phosphate, and α-amylase in the same proportions to the fermentation medium, 0.1 g / L threonine and 0.1 g / L biotin are additionally added.

[0023] Example 5

[0024] The difference from Comparative Example 1 is that, in addition to adding corn starch, peptone, yeast powder, potassium dihydrogen phosphate, and α-amylase in the same proportions to the fermentation medium, 0.1 g / L threonine and 0.2 g / L glutamic acid were additionally added.

[0025] Example 6

[0026] The difference from Comparative Example 1 is that, in addition to adding corn starch, peptone, yeast powder, potassium dihydrogen phosphate, and α-amylase in the same proportions to the fermentation medium, 0.1 g / L of biotin and 0.2 g / L of glutamic acid were additionally added.

[0027] Example 7

[0028] The difference from Comparative Example 1 is that, in addition to adding corn starch, peptone, yeast powder, potassium dihydrogen phosphate, and α-amylase in the same proportions to the fermentation medium, 0.1 g / L threonine, 0.1 g / L biotin, and 0.2 g / L glutamic acid are additionally added.

[0029] Example 8

[0030] The difference from Comparative Example 1 is that, in addition to adding corn starch, peptone, yeast powder, potassium dihydrogen phosphate, and α-amylase in the same proportions to the fermentation medium, 0.2 g / L threonine, 0.1 g / L biotin, and 0.3 g / L glutamic acid are additionally added.

[0031] Example 9

[0032] The difference from Comparative Example 1 is that, in addition to adding corn starch, peptone, yeast powder, potassium dihydrogen phosphate, and α-amylase in the same proportions to the fermentation medium, 0.3 g / L threonine, 0.1 g / L biotin, and 0.4 g / L glutamic acid are additionally added.

[0033] Example 10

[0034] The difference from Comparative Example 1 is that, in addition to adding corn starch, peptone, yeast powder, potassium dihydrogen phosphate, and α-amylase in the same proportions to the fermentation medium, 0.4 g / L threonine, 0.1 g / L biotin, and 0.3 g / L glutamic acid are additionally added.

[0035] Example 11

[0036] The difference from Comparative Example 1 is that, in addition to adding corn starch, peptone, yeast powder, potassium dihydrogen phosphate, and α-amylase in the same proportions to the fermentation medium, 0.2 g / L threonine, 0.2 g / L biotin, and 0.3 g / L glutamic acid are additionally added.

[0037] Example 12

[0038] The difference from Comparative Example 1 is that, in addition to adding corn starch, peptone, yeast powder, potassium dihydrogen phosphate, and α-amylase in the same proportions to the fermentation medium, 0.2 g / L threonine, 0.1 g / L biotin, and 0.5 g / L glutamic acid are additionally added.

[0039] The natamycin content results obtained in Comparative Example 1 and Examples 1-12 are shown in Table 1.

[0040] Table 1 Natamycin content obtained in different examples

[0041]

[0042] The comparative culture medium is a conventional fermentation medium, while Examples 7, 8, and 9 are fermentation media of the present invention. The results of Examples 1-12 and Comparative Example 1 (Table 1) clearly demonstrate that the fermentation medium of the present invention produces 129.73% higher natamycin content than the conventional fermentation medium. Further increasing the content of any one of threonine, biotin, and glutamate results in a decrease in natamycin content. This is primarily due to the synergistic effects of these three components on intracellular metabolism. Excessively high levels can disrupt intracellular metabolism, while lower levels fail to achieve the desired effect of increasing the natamycin synthesis rate.

[0043] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A fermentation medium for producing natamycin, characterized in that: The components and contents of the fermentation medium are: 60 g / L corn starch, 20 g / L peptone, 10 g / L yeast powder, 0.06 g / L α-amylase, 0.2 g / L potassium dihydrogen phosphate, 0.1-0.3 g / L threonine, 0.1 g / L biotin, and 0.2-0.4 g / L glutamic acid.

2. The fermentation medium for producing natamycin according to claim 1, characterized in that: The components and contents of the fermentation medium are: 60 g / L corn starch, 20 g / L peptone, 10 g / L yeast powder, 0.2 g / L potassium dihydrogen phosphate, 0.06 g / L α-amylase, 0.2 g / L threonine, 0.1 g / L biotin, and 0.3 g / L glutamic acid.

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