A strain of Streptomyces noursei and its application

Abstract

The present invention belongs to the technical field of food fermentation production, and specifically relates to a strain of Streptomyces noursei and its application in the preparation of ε-polylysine. Streptomyces noursei QPL-9 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on November 27, 2024, with a deposit number of CGMCC No.32818, and was classified as Streptomyces noursei Streptomyces noursei The deposit address is Beijing, China. The Streptomyces noursei of the present invention is used to ferment and prepare ε-polylysine using mannitol as a carbon source and lysine fermentation waste liquid as a nitrogen source, and the yield of ε-polylysine exceeds 80 g / L.

Description

Technical Field

[0001] The invention belongs to the technical field of food fermentation production, and specifically relates to a strain of Streptomyces noursei and application thereof in the preparation of epsilon-polylysine. Background Art

[0002] ε-Polylysine is a bioactive substance produced by microbial fermentation, usually produced by the polymerization of 25 to 35 L-lysines. ε-Polylysine has strong antibacterial activity, a broad antibacterial spectrum, and high safety. It has broad application prospects in the fields of food preservation, medicine, and materials.

[0003] The main production method of ε-polylysine is microbial fermentation, and the microorganism commonly used for fermentation is Streptomyces parvum ( Streptomyces albulus ), Chisso Corporation of Japan has achieved industrial production of thousands of tons per year using mutant strains of Streptomyces parvum, and the breeding of high-yield strains in China is also progressing and has achieved certain results. However, the current fermentation production process based on Streptomyces parvum mainly uses glucose or glycerol as a carbon source and yeast powder as a nitrogen source, which has problems such as high fermentation costs.

[0004] Streptomyces noursei is an actinomycete isolated from soil. Chinese patent application CN202410228970.0 discloses a strain of Streptomyces noursei ( Streptomyces noursei )NN-Q01-01, which was isolated from the rhizosphere soil of healthy passion fruit, has an antagonistic effect on passion fruit stem rot, especially on passion fruit stem rot caused by Fusarium solani. Chinese patent CN202311325780.2 discloses a strain of Streptomyces noursei NK27 that produces glutathione and its application in plant disease prevention and control. None of the above application fields of Streptomyces noursei involve ε-polylysine. Summary of the invention

[0005] The invention aims to provide a strain of Streptomyces noursei, and utilize the strain to efficiently produce epsilon-polylysine by taking mannitol as a carbon source and lysine fermentation waste liquid as a nitrogen source.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A strain of Streptomyces noursei QPL-9 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 27, 2024, with the deposit number CGMCC No.32818, and was classified as Streptomyces noursei Streptomyces noursei , the storage address is Beijing, China.

[0008] A use of the Streptomyces noursei in preparing ε-polylysine.

[0009] A method for producing epsilon-polylysine comprises utilizing the Streptomyces noursei, taking mannitol as a carbon source, and using waste liquid generated by industrial fermentation of lysine as a nitrogen source, and fermenting and culturing the strain to obtain epsilon-polylysine.

[0010] Furthermore, the Streptomyces noursei is cultured in a culture medium with mannitol as a carbon source and lysine fermentation waste liquid as a nitrogen source, and then ε-polylysine is obtained by fermentation.

[0011] Furthermore, the Streptomyces noursei is inoculated in a solid culture medium containing 1.5-2.0 g / 100 ml of agar, the culture temperature is 28-32° C., and the culture time is 5-7 days; after the culture, the culture temperature is 28-32° C. in a seed culture medium, and the shaking culture is carried out for 24-48 hours to obtain a seed liquid; after the culture, the seed liquid is inoculated into a fermentation medium at a volume ratio of 10%, 28-32° C., and the air intake is 1-3 vvm (the ratio of the ventilation volume per minute to the actual liquid volume of the tank body), and the dissolved oxygen is controlled to 20%-40% by adjusting the stirring speed. When the pH of the fermentation liquid naturally drops to 4.0, the pH is controlled to 4.0, and the mannitol concentration is controlled to 0.1-10 g / L and the ammonia nitrogen concentration is controlled to 0.1-1 g / L. The fermentation is carried out for 5-8 days to obtain an ε-polylysine fermentation liquid.

[0012] The solid culture medium contains 20 grams of mannitol, 20 grams of soybean powder, and 20 grams of agar powder per liter of water, and the pH is 7.2-7.4;

[0013] The seed culture medium contains 30 grams of mannitol and 20 grams of lysine fermentation waste liquid per liter of water, and the pH is 6.5-7.0;

[0014] The fermentation medium contains 30-50 grams of mannitol and 30-70 grams of lysine fermentation waste liquid per liter of water, and the pH value is 6.5-7.0.

[0015] The concentration of amino nitrogen in the above lysine fermentation waste liquid is 40-60 g / L, and the concentration of L-lysine is 80-160 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure is a curve showing the concentration variation of mannitol and ε-polylysine in the preparation process of the present invention. DETAILED DESCRIPTION

[0017] The present invention is further described below with reference to examples. It should be noted that the specific implementation methods described herein are only for illustrating and explaining the present invention and are not intended to limit the present invention.

[0018] In the following examples, the lysine fermentation waste liquid comes from Fufeng Group, and its main components include bacterial protein, ammonium sulfate and L-lysine. It has been determined that the amino nitrogen concentration in the lysine fermentation waste liquid is 40-60 g / L, and the L-lysine concentration is 80-160 g / L. All experimental raw materials not specially marked can be obtained commercially. Example 1

[0019] Streptomyces noursei ( Streptomyces noursei )

[0020] 1. Separation

[0021] Soil samples were obtained from a university in Laishan District, Yantai City. 50 ml of ML culture medium (30 g / L mannitol, 20 g / L lysine fermentation waste liquid (wherein the concentration of amino nitrogen was 1 g / L and the concentration of L-lysine was 2 g / L), pH 7.0) was added to each gram of soil sample and enriched and cultured at 30°C for 48 hours. Then the samples were diluted and spread on a culture dish containing ML solid culture medium (30 g / L mannitol, 20 g / L lysine fermentation waste liquid (wherein the concentration of amino nitrogen was 1 g / L and the concentration of L-lysine was 2 g / L), 15 g / L agar powder, pH 7.0) and cultured at 30°C for 7 days. After a single colony was grown, 260 colonies of Streptomyces were selected and inoculated into fermentation medium (50 g / L mannitol, 30 g / L lysine fermentation waste liquid (of which the concentration of amino nitrogen was 1.5 g / L and the concentration of L-lysine was 3 g / L), pH 7.0), and cultured in a shaking incubator at 30°C for 72 hours. The production of ε-polylysine was measured, and the strain with the highest production was selected for the next step of UV mutagenesis.

[0022] 2. Mutagenesis

[0023] The preliminarily selected strains were inoculated into the above fermentation medium, cultured in a shaking incubator at 30°C until the mid-logarithmic phase, washed with physiological saline and suspended, and adjusted to 10 8 bacteria / ml, take 5 ml into a 9 cm culture dish, and induce mutation for 15 seconds, 30 seconds, 1 minute and 5 minutes respectively at a distance of 30 cm from the UV lamp, take 100 μl of the mutagenic bacterial solution obtained under different mutagenesis conditions, spread it on the above-mentioned ML solid culture medium, culture at 30°C in the dark for 7 to 8 days, select a single colony and inoculate it into the fermentation medium, culture at 30°C in a shaking incubator for 72 hours, measure the production of ε-polylysine, and select the strain with the highest production for the next step of evolution.

[0024] 3. Evolution

[0025] The strain obtained by mutagenesis screening was inoculated into evolution medium No. 1 (80 g / L mannitol, 40 g / L lysine fermentation waste liquid (wherein the concentration of amino nitrogen was 2 g / L and the concentration of L-lysine was 4 g / L), pH 7.0), and cultured in a shaking incubator at 30°C for 48 hours. Then, the inoculation amount was transferred to fresh evolution medium No. 1 at 1%, and cultured in a shaking incubator at 30°C for another 48 hours. After repeating this process 5 times, the bacterial solution obtained above was transferred to evolution medium No. 2 (90 g / L mannitol, 45 g / L lysine fermentation waste liquid (wherein the concentration of amino nitrogen was 2.2 g / L and the concentration of L-lysine was 4.5 g / L), pH 7.0), and the operation was repeated 5 times again. The bacterial solution obtained above was transferred to evolution medium No. 3 (mannitol 90 g / L The above bacterial solution was transferred to the evolution medium No. 4 (110 g / L mannitol, 55 g / L lysine fermentation waste liquid (wherein the concentration of amino nitrogen was 2.7 g / L and the concentration of L-lysine was 5.5 g / L), pH 7.0), and the operation was repeated 5 times; the above bacterial solution was transferred to the evolution medium No. 5 (120 g / L mannitol, 60 g / L lysine fermentation waste liquid (wherein the concentration of amino nitrogen was 3 g / L and the concentration of L-lysine was 6 g / L), pH 7.0), and the operation was repeated 5 times. The final bacterial solution was spread on the culture dish containing the above ML solid medium and cultured at 30°C for 7 days. After a single colony was grown, the colony was selected and inoculated into the above fermentation medium and cultured in a shaking incubator at 30° C. for 72 hours. The yield of ε-polylysine was measured and the strain with the highest yield was selected and named QPL-9.

[0026] 4. Identification

[0027] The genome of strain QPL-9 was extracted and its 16S rDNA sequence was amplified using the 27F / 1492R primer pair, where the sequence of the 27F primer was 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1) and the sequence of the 1492R primer was 5'-TACGGTTACCTTGTTACGACTT-3' (SEQ ID NO.2). The obtained DNA fragment was sequenced and then submitted to NCBI for Blast analysis. The results showed that the 16S rDNA sequence of QPL-9 (SEQ ID NO.3) was similar to that of Streptomyces noursei ( Streptomyces noursei ) The 16S rDNA sequences of IFO14147 are completely identical.

[0028] 16S rDNA sequence

[0029]

[0030] Based on the above characteristics, the strain with high yield of ε-polylysine was identified as Streptomyces noursei, and was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on November 27, 2024, with the deposit number CGMCC No.32818, and the classification name was Streptomyces noursei Streptomyces noursei , the storage address is Beijing, China. Example 2

[0031] Fermentation production of ε-polylysine by Streptomyces noursei QPL-9

[0032] Streptomyces noursei QPL-9 stored in -80℃ ultra-low temperature freezer was streaked onto MS solid medium (20 g / L mannitol, 20 g / L soybean powder, 20 g / L agar powder, pH 7.2), cultured at 30℃ for 5-7 days, and after the surface of the medium was covered with black spores, spores were collected from the plate and inoculated into a conical flask containing 50 ml seed medium (30 g mannitol, 20 g lysine fermentation waste liquid, pH 6.7), and cultured in a shaking incubator at 30℃ and 220 rpm. Cultivate for 24 hours to obtain seed liquid; inoculate the cultivated seed culture liquid into a fermentation tank filled with fresh fermentation medium (50 g mannitol, 50 g lysine fermentation waste liquid, pH 6.5) at an inoculation rate of 10% (volume ratio), control the fermentation temperature to 30°C, the air intake to 3vvm (the ratio of the minute ventilation volume to the actual volume of the tank liquid), and control the dissolved oxygen to 30% by adjusting the stirring speed. The pH is not controlled in the early stage of fermentation. When the pH of the fermentation liquid naturally drops to 4.0, open ammonia water to control the pH at 4.0. When the mannitol concentration is lower than 5 g / L, start to add mannitol to control the mannitol concentration in the fermentation liquid to 5-10 g / L; when the ammonia nitrogen concentration is lower than 0.5 g / L, start to flow lysine fermentation waste liquid to control the ammonia nitrogen concentration in the fermentation liquid to 0.5-1 g / L. Detect the content of ε-polylysine and mannitol in the fermentation liquid every 24 hours (see Figure 1 ), after 9 days of fermentation, the yield of ε-polylysine was 82.6 g / L.

Claims

1. A strain of Streptomyces noursei, characterized in that: The Streptomyces noursei was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on November 27, 2024, with a deposit number of CGMCC No.32818 and a classification name of Streptomyces noursei Streptomyces noursei , the storage address is Beijing, China.

2. Use of the Streptomyces noursei according to claim 1 in the preparation of ε-polylysine.

3. A method for producing ε-polylysine, characterized in that: By using the Streptomyces noursei described in claim 1, taking mannitol as a carbon source and the waste liquid produced by industrial fermentation of lysine as a nitrogen source, the strain can be fermented and cultured to obtain ε-polylysine.

4. The method for producing ε-polylysine according to claim 3, characterized in that: The method comprises culturing the Streptomyces noursei described in claim 1 in a culture medium with mannitol as a carbon source and lysine fermentation waste liquid as a nitrogen source, and then fermenting to obtain ε-polylysine.

5. The method for producing ε-polylysine according to claim 4, characterized in that: The Streptomyces noursei described in claim 1 is inoculated into a solid culture medium containing 1.5-2.0 g / 100 ml of agar, the culture temperature is 28-32° C., and the culture time is 5-7 days; after the culture, the culture temperature is 28-32° C. in a seed culture medium, and the shaking culture is carried out for 24-48 hours to obtain a seed liquid; after the culture, the seed liquid is inoculated into a fermentation medium at a volume ratio of 10%, the temperature is 28-32° C., the air flow rate is 1-3 vvm, and the dissolved oxygen is controlled to 20%-40% by adjusting the stirring speed. When the pH of the fermentation liquid naturally drops to 4.0, the pH is controlled to 4.0, and the mannitol concentration is controlled to 0.1-10 g / L and the ammonia nitrogen concentration is controlled to 0.1-1 g / L, and the fermentation is carried out for 5-8 days to obtain an ε-polylysine fermentation liquid.

6. The method for producing ε-polylysine according to claim 5, characterized in that: The solid culture medium contains 20 grams of mannitol, 20 grams of soybean powder, and 20 grams of agar powder per liter of water, and the pH is 7.2-7.4; The seed culture medium contains 30 grams of mannitol and 20 grams of lysine fermentation waste liquid per liter of water, and the pH is 6.5-7.0; The fermentation medium contains 30-50 grams of mannitol and 30-70 grams of lysine fermentation waste liquid per liter of water, and the pH value is 6.5-7.0.

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