Culture medium and culture method for improving AVG synthesized by streptomyces PL11

Abstract

The invention discloses a culture medium and a culture method for improving AVG synthesized by streptomyces PL11, and belongs to the technical field of biology. Aiming at the obvious defects existing in the optimization research on the production of AVG from streptomyces PL11, through systematic substance combination screening and concentration optimization, a YEPEG culture medium is used as a basic culture medium, and ammonium ferrous sulfate hexahydrate is added, so that the AVG produced by streptomyces PL11 is obtained. The culture medium prepared by taking the carbon source, the nitrogen source, the homoserine, the aspartic acid and the lysine as core components provides sufficient raw material supply for synthesizing the AVG through strain metabolism, and the AVG yield of the streptomyces PL11 is increased. By accurately controlling the culture time (56h) and the shaker condition (28 DEG C, 200rpm), the problem that the AVG synthesis peak value is missed or the synthesized AVG is decomposed by the strain in the later stage is avoided, and the bottleneck of AVG industrial application is broken through.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a culture medium and culture method for improving the synthesis of AVG by Streptomyces PL11. Background Technology

[0002] Streptomyces is a Gram-positive bacterium widely distributed in the natural environment and capable of producing a variety of bioactive metabolites. Aminoethoxyvinyl glycine hydrochloride (AVG) is a natural amino acid that can be obtained from Streptomyces fermentation broth. It competitively inhibits the enzyme activity catalyzing the conversion of S-adenosylmethionine to 1-aminocyclopropanecarboxylic acid (ACC) in the ethylene synthesis pathway, thus inhibiting ethylene production in plant tissues. Studies have found that spraying AVG on fruits such as apples, pears, and peaches reduces ethylene content and extends shelf life.

[0003] Our research group previously isolated a Streptomyces strain PL11 from soil that can produce AVG; however, its AVG yield was low, with an AVG yield of approximately 1.4 μg mg⁻¹ (dry wt) after 48 h of culture in MM medium. -1 After culturing in YEME medium for 72 h, the AVG yield was approximately 0.6 μg mg⁻¹ (dry wt). -1 Although the yield of AVG increased by 30% after chemical mutagenesis, it is still difficult to carry out industrial application. Therefore, it is necessary to explore new methods to further increase the yield of AVG from Streptomyces PL11. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a culture medium and culture method for improving the synthesis of AVG by Streptomyces PL11. By optimizing the composition of the culture medium and the culture conditions, this invention significantly improves the yield of AVG synthesized by Streptomyces PL11, overcoming the shortcomings of existing Streptomyces PL11 AVG synthesis methods which have low yields and cannot meet the needs of industrialization.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a culture medium for improving the synthesis of AVG by Streptomyces PL11. The culture medium is prepared by using YEPEG medium as the basal medium and adding 10-12 g / L carbon source, 5-7 g / L nitrogen source, 0.03-0.05 g / L ferrous ammonium sulfate hexahydrate, 1-2 g / L homoserine, 2-4 g / L aspartic acid, and 5-7 g / L essential amino acids.

[0006] The essential amino acid is either lysine or methionine.

[0007] The carbon source is one or more of glycerol, maltose, soluble starch, sucrose, or glucose; the nitrogen source is one or more of yeast extract, yeast extract, or bacterial peptone.

[0008] Preferably, the culture medium is prepared by adding 10 g / L glucose, 5 g / L yeast extract, 0.03 g / L Fe(NH4)2(SO4)2•6H2O, 1 g / L homoserine, 2 g / L aspartic acid, and 5 g / L lysine to YEPEG medium as the basal medium.

[0009] Preferably, the pH of the culture medium is 7.

[0010] The biological preservation number of the PL11 Streptomyces strain is CGMCC NO. 24263, and this strain is recorded in Chinese patent CN114891696 B.

[0011] In a second aspect, the present invention provides a method for improving AVG synthesis by Streptomyces PL11, comprising the following steps: (1) Activation of strain: The preserved PL11 Streptomyces was inoculated into TSB medium, cultured on a shaker for 5 days, and then transferred to fresh TSB medium for 2 days to activate the strain. (2) Liquid fermentation: The activated bacterial solution was inoculated into the above culture medium at an inoculation rate of 3-4%, and cultured on a shaker at 28℃ and 200rpm for 56 hours.

[0012] The TSB culture medium is prepared as follows: 16-18 g tryptone, 2-4 g plant peptone, 4-6 g NaCl, 2-3 g K2HPO4, 2-3 g glucose, and 1 L distilled water. The mixture is then autoclaved at 121°C for 15-20 min.

[0013] In a third aspect, this invention provides a method for accurately determining AVG in the fermentation broth of Streptomyces PL11: HPLC and LC-MS. The combination of the two methods is complementary, with HPLC ensuring quantitative efficiency and LC-MS ensuring qualitative accuracy, providing dual support for subsequent experimental conclusions.

[0014] The beneficial effects of this invention are: This invention addresses significant shortcomings in the optimization of AVG production by *Streptomyces PL11*. Through systematic screening and concentration optimization of material combinations, using YEPEG medium as the basal medium and adding ferrous ammonium sulfate hexahydrate, along with carbon sources, nitrogen sources, homoserine, aspartic acid, and essential amino acids as core components, the prepared medium provides ample raw material for the strain's metabolic synthesis of AVG, increasing AVG production by more than 20 times compared to existing technologies. Precise control of the culture time (56 h) and shaking conditions (28℃, 200 rpm) avoids missing the AVG synthesis peak or the subsequent decomposition of synthesized AVG by the strain. Improved assay methods—HPLC and LC-MS—allow for efficient separation and quantification of AVG, while LC-MS further confirms the target substance through mass spectrometry specificity, avoiding interference from impurities. The method for improving AVG synthesis by *Streptomyces PL11* provided by this invention fully meets the needs of industrial production, overcoming the bottleneck in the industrial application of AVG. Attached Figure Description

[0015] Figure 1: Effect of different culture times on the AVG production of Streptomyces PL11.

[0016] Figure 2: Effect of different pH values ​​on the yield of Streptomyces AVG.

[0017] Figure 3: Liquid chromatography-mass spectrometry (LC-MS) chromatogram of the sample.

[0018] Figure 4 : The first-order mass spectrum of the sample being tested.

[0019] Figure 5 : Secondary mass spectrum of the sample to be tested.

[0020] Figure 6 : High performance liquid chromatogram of the sample to be tested. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0023] TSB medium: 17 g tryptone (or trypsin), 3 g plant peptone (or soybean peptone), 5 g NaCl, 2.5 g K2HPO4, 2.5 g glucose, 1 L distilled water, autoclaved at 121°C for 20 min.

[0024] Manufacturer of AVG standard products: Shanghai Macklin Biochemical Co.; CAS No.: 5720-26-8.

[0025] Test strain: Streptomyces ( Streptomyces sp. PL11 was isolated and screened by the Fruit and Vegetable Postharvest Storage and Preservation Team of the College of Food Science and Technology, Shandong Agricultural University. The biological preservation number of this strain is CGMCC NO. 24263, and this strain is recorded in Chinese patent CN114891696 B.

[0026] Example 1: Effect of different culture times on Streptomyces AVG yield YEPEG medium was supplemented with 10 g / L glucose, 5 g / L yeast extract, 0.03 g / L Fe(NH4)2(SO4)2•6H2O, 1 g / L homoserine, 2 g / L aspartic acid, and 5 g / L lysine. Different culture times were set, i.e., 0-96 h. The culture was carried out under the conditions of 4% inoculum, 28℃, and 200 r / min shaking speed. Each treatment group was set with 3 replicates.

[0027] Centrifuge 2 mL of the cultured bacterial solution, discard the supernatant, and prepare the bacterial culture. Take 0.5 mL of the supernatant from the prepared bacterial culture and add 0.25 mL of triethylamine-acetonitrile solution (1:1 v / v) and 0.25 mL of phenyl isothiocyanate-acetonitrile solution (1:1 v / v). Vortex to mix, and let stand at room temperature for 1 h. Add 0.05 mL of 20% acetic acid solution and mix well. Add 1 mL of n-hexane to the derivatized solution and vortex for 1 min. Allow to separate into layers, discard the upper n-hexane layer. Carefully aspirate the lower layer using a syringe, filter through a 0.45 μL organic filter into a vial for liquid chromatography analysis.

[0028] The results showed that AVG production increased with increasing culture time. The highest AVG production of Streptomyces PL11 was achieved at a culture time of 56 h. After 56 h, AVG was absorbed and decomposed into other substances by Streptomyces PL11, and its content in the fermentation broth gradually decreased.

[0029] Example 2: Effect of different pH values ​​on Streptomyces AVG yield YEPEG medium was supplemented with 10 g / L glucose, 5 g / L yeast extract, 0.03 g / L Fe(NH4)2(SO4)2•6H2O, 1 g / L homoserine, 2 g / L aspartic acid, and 5 g / L lysine; the pH of the medium was adjusted to 3, 5, 7, 9, and 11, respectively. The medium was cultured for 56 h at an inoculum size of 4%, a culture temperature of 28℃, and a shaking speed of 200 r / min.

[0030] Centrifuge 2 mL of the cultured bacterial solution, discard the supernatant, and prepare the bacterial culture. Take 0.5 mL of the supernatant from the prepared bacterial culture and add 0.25 mL of triethylamine-acetonitrile solution (1:1 v / v) and 0.25 mL of phenyl isothiocyanate-acetonitrile solution (1:1 v / v). Vortex to mix, and let stand at room temperature for 1 h. Add 0.05 mL of 20% acetic acid solution and mix well. Add 1 mL of n-hexane to the derivatized solution and vortex for 1 min. Allow to separate into layers, discard the upper n-hexane layer. Carefully aspirate the lower layer using a syringe, filter through a 0.45 μL organic filter into a vial for liquid chromatography analysis.

[0031] The results showed that, among the adjusted pH values, AVG production was highest at pH = 7. An acidic environment inhibited cell growth and key enzyme activity, while a strongly alkaline environment damaged cell structure and affected nutrient absorption, both of which significantly reduced AVG production.

[0032] Example 3: Determination of Streptomyces AVG Yield by High Performance Liquid Chromatography Combined with High Performance Liquid Chromatography-Tandem Mass Spectrometry 1. Qualitative detection of AVG production by Streptomyces PL11 using high performance liquid chromatography-tandem mass spectrometry (1) Preparation of the test solution YEPEG medium was supplemented with 10 g / L glucose, 5 g / L yeast extract, 0.03 g / L Fe(NH4)2(SO4)2•6H2O, 1 g / L homoserine, 2 g / L aspartic acid, and 5 g / L lysine. Different culture times were set, i.e., 0-96 h. The culture was carried out with shaking at an inoculum size of 4%, a culture temperature of 28℃, and a shaking speed of 200 r / min. Each treatment group was set with 3 replicates.

[0033] Centrifuge 2 mL of the cultured bacterial solution, discard the supernatant, and prepare the bacterial culture. Using a syringe, draw 0.5 mL of the supernatant from the prepared bacterial culture and filter it through a 0.45 μL organic filter into a vial.

[0034] (2) High performance liquid chromatography-tandem mass spectrometry Column: C 18Chromatographic column; mobile phase A: formic acid in water (0.1%); mobile phase B: acetonitrile (0.1% formic acid); injection volume: 10 μL; column temperature: 35℃; elution conditions and mass spectrometry conditions are shown in the table below.

[0035] Table 1: Chromatographic elution procedures for high performance liquid chromatography-tandem mass spectrometry Table 2: Mass Spectrometry Conditions for High Performance Liquid Chromatography-Tandem Mass Spectrometry (3) Preparation of standard solutions Accurately weigh 1 g of AVG standard and prepare a 1 mg / mL standard stock solution using Wahaha water. Pipette 0.5 mL of the supernatant and add sequentially 0.25 mL of triethylamine-acetonitrile solution (1:1 v / v) and 0.25 mL of phenyl isothiocyanate-acetonitrile solution (1:1 v / v). Vortex to mix and let stand at room temperature for 1 h. Add 0.05 mL of 20% acetic acid solution and mix well. Add 1 mL of n-hexane to the derivatized solution and vortex for 1 min. Allow to separate into layers and discard the upper n-hexane layer. Carefully aspirate the lower layer using a syringe and filter through a 0.45 μL organic filter into a vial to prepare the standard working solution.

[0036] (4) Inject 1 mg / mL standard working solution into the liquid chromatograph and record the chromatogram. The main peak retention time is used for qualitative analysis. Then, inject the test solution into the liquid chromatography-mass spectrometry instrument and compare to confirm the presence of AVG in PL11 Streptomyces.

[0037] 2. Quantitative determination of AVG production by Streptomyces PL11 using high performance liquid chromatography. (1) Preparation of the test solution YEPEG medium was supplemented with 10 g / L glucose, 5 g / L yeast extract, 0.03 g / L Fe(NH4)2(SO4)2•6H2O, 1 g / L homoserine, 2 g / L aspartic acid, and 5 g / L lysine. Different culture times were set, i.e., 0-96 h. The culture was carried out with shaking at an inoculum size of 4%, a culture temperature of 28℃, and a shaking speed of 200 r / min. Each treatment group was set with 3 replicates.

[0038] Centrifuge 2 mL of the cultured bacterial solution, discard the supernatant, and prepare the bacterial culture. Take 0.5 mL of the supernatant from the prepared bacterial culture and add 0.25 mL of triethylamine-acetonitrile solution (1:1 v / v) and 0.25 mL of phenyl isothiocyanate-acetonitrile solution (1:1 v / v). Vortex to mix, and let stand at room temperature for 1 h. Add 0.05 mL of 20% acetic acid solution and mix well. Add 1 mL of n-hexane to the derivatized solution and vortex for 1 min. Allow to separate into layers, discard the upper n-hexane layer. Carefully aspirate the lower layer using a syringe and filter through a 0.45 μL organic filter into a vial.

[0039] (2) High performance liquid chromatography conditions Column: C 18 Chromatographic column; Mobile phase A: sodium acetate; Mobile phase B: methanol; Elution program: 0.5 mL / min, 36% methanol; Injection volume: 5 μL; Detector: UV detector (deuterium lamp); Detection wavelength: 254 nm; Column temperature: 40℃ LC time program: 60 min.

[0040] (3) Preparation of standard solutions Accurately weigh 1 g of AVG standard and prepare a 1 mg / mL standard stock solution using Wahaha water. Then, prepare a series of working solutions at concentrations of 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1 mg / mL using a serial dilution method. Take 0.5 mL of the supernatant and add 0.25 mL of triethylamine-acetonitrile solution (1:1 v / v) and 0.25 mL of phenyl isothiocyanate-acetonitrile solution (1:1 v / v) respectively. Vortex to mix and let stand at room temperature for 1 h. Add 0.05 mL of 20% acetic acid solution and mix well. Add 1 mL of n-hexane to the derivatized solution and vortex for 1 min. Allow to separate into layers and discard the upper n-hexane layer. Carefully aspirate the lower layer using a syringe and filter through a 0.45 μL organic filter into a vial to prepare the series of standard working solutions.

[0041] (4) Take 5 μL of each of the series of standard working solutions and inject them into the liquid chromatograph according to the chromatographic conditions of technical scheme (2) to record the chromatogram. In the concentration range of 0~1 mg / L, the concentration and peak area of ​​AVG have a good linear relationship. The regression equation of the standard curve is calculated with the concentration of AVG as the abscissa (X) and the peak area as the ordinate (Y). The equation is Y=7×10 7 X-10 6 (R²=0.997). Qualitative analysis was performed based on the retention time of the main peak in the standard curve solution. The concentration of AVG in the test solution was determined using the external standard method of the standard curve, and the corresponding content was calculated.

[0042] Example 4: A method to improve AVG synthesis by Streptomyces PL11 (1) Preparation of culture medium: 10 g / L glucose, 5 g / L yeast extract, 0.03 g / L Fe(NH4)2(SO4)2•6H2O, 1 g / L homoserine, 2 g / L aspartic acid and 5 g / L lysine were added to YEPEG culture medium; the pH of the culture medium was adjusted to 7.

[0043] (2) Activation of strain: The preserved PL11 Streptomyces was inoculated into TSB medium, cultured on a shaker for 5 days, and then transferred to fresh TSB medium for 2 days to activate the strain; (3) Liquid fermentation: The activated bacterial solution was inoculated into the culture medium at an inoculation rate of 4%, and cultured on a shaker at 28°C and 200 rpm for 56 hours.

[0044] Comparative Example 1: Methods to improve AVG synthesis in Streptomyces PL11 The only difference between Comparative Example 1 and Example 4 is the culture medium used. The culture medium used in Comparative Example 1 was prepared as follows: 10 g / L glucose, 5 g / L yeast extract, and 0.03 g / L Fe(NH4)2(SO4)2•6H2O were added to YEPEG medium; the pH of the culture medium was adjusted to 7.

[0045] Comparative Example 2: Methods to improve AVG synthesis in Streptomyces PL11 The only difference between Comparative Example 2 and Example 4 is the culture medium used. The culture medium used in Comparative Example 2 was prepared as follows: 10 g / L glucose, 5 g / L yeast extract, 0.03 g / L Fe(NH4)2(SO4)2•6H2O, and 2 g / L aspartic acid were added to YEPEG medium; the pH of the culture medium was adjusted to 7.

[0046] Comparative Example 3: Methods to improve AVG synthesis in Streptomyces PL11 The only difference between Comparative Example 3 and Example 4 is the culture medium used. The culture medium used in Comparative Example 3 was prepared as follows: 10 g / L glucose, 5 g / L yeast extract, 0.03 g / L Fe(NH4)2(SO4)2•6H2O, and 1 g / L homoserine were added to YEPEG medium; the pH of the culture medium was adjusted to 7.

[0047] Comparative Example 4: Methods to improve AVG synthesis in Streptomyces PL11 The only difference between Comparative Example 4 and Example 4 is the culture medium used. The culture medium used in Comparative Example 4 was prepared as follows: 10 g / L glucose, 5 g / L yeast extract, 0.03 g / L Fe(NH4)2(SO4)2•6H2O, and 5 g / L lysine were added to YEPEG medium; the pH of the culture medium was adjusted to 7.

[0048] Comparative Example 5: Methods to improve AVG synthesis in Streptomyces PL11 The only difference between Comparative Example 5 and Example 4 is the culture medium used. The culture medium used in Comparative Example 5 was prepared as follows: 10 g / L glucose, 5 g / L yeast extract, 0.03 g / L Fe(NH4)2(SO4)2•6H2O, and 5 g / L methionine were added to YEPEG medium; the pH of the culture medium was adjusted to 7.

[0049] Experimental Example 1: Determination of the effect of different amino acid additions on Streptomyces AVG yield To verify the effect of adding different amino acids to the culture medium on the AVG yield of Streptomyces, this experiment used liquid chromatography to determine the AVG yield of Streptomyces cultured in Example 4 and Comparative Examples 1-5. The statistical results are shown in Table 3.

[0050] Table 3: AVG Production Measurement The test results showed that while adding methionine to the culture medium increased the yield of Streptomyces AVG compared to the medium without amino acids, the effect was minimal. Adding aspartic acid, homoserine, and lysine to the culture medium all increased the yield of Streptomyces AVG to varying degrees compared to the medium without amino acids. The combination of aspartic acid, homoserine, and lysine showed the most significant promoting effect on Streptomyces AVG yield.

[0051] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications made within the spirit and principles of this application are not permitted. Equivalent substitutions and improvements should all be included within the scope of protection of this application.

Claims

1. A culture medium for enhancing AVG synthesis in Streptomyces PL11, characterized in that, The culture medium is prepared by using YEPEG medium as the basal medium and adding 10-12 g / L carbon source, 5-7 g / L nitrogen source, 0.03-0.05 g / L ferrous ammonium sulfate hexahydrate, 1-2 g / L homoserine, 2-4 g / L aspartic acid, and 5-7 g / L essential amino acids.

2. The culture medium according to claim 1, characterized in that, The essential amino acid is either lysine or methionine.

3. The culture medium according to claim 1, characterized in that, The carbon source is one or more of glycerol, maltose, soluble starch, sucrose, or glucose; the nitrogen source is one or more of yeast extract, yeast extract, or bacterial peptone.

4. The culture medium according to claim 1, characterized in that, The culture medium was prepared by adding 10 g / L glucose, 5 g / L yeast extract, 0.03 g / L Fe(NH4)2(SO4)2•6H2O, 1 g / L homoserine, 2 g / L aspartic acid, and 5 g / L lysine to YEPEG medium as the basal medium.

5. The culture medium according to any one of claims 1 or 4, characterized in that, The pH of the culture medium is 7.

6. The culture medium according to any one of claims 1 or 4, characterized in that, The biological preservation number of the PL11 Streptomyces is: CGMCC NO. 24263.

7. A method for improving the synthesis of AVG by Streptomyces PL11, characterized in that, Includes the following steps: (1) Activation of strain: The preserved PL11 Streptomyces was inoculated into TSB medium, cultured on a shaker for 5 days, and then transferred to fresh TSB medium for 2 days to activate the strain. (2) Liquid fermentation: The activated bacterial solution is inoculated into the culture medium described in claim 1 at an inoculation rate of 3-4%, and cultured on a shaker at 28°C and 200 rpm for 56 hours.

8. The method according to claim 7, characterized in that, The TSB culture medium is prepared as follows: 16-18 g tryptone, 2-4 g plant peptone, 4-6 g NaCl, 2-3 g K2HPO4, 2-3 g glucose, and 1 L distilled water. The mixture is then autoclaved at 121°C for 15-20 min.

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