Mulberry endophyte producing 1-deoxynojirimycin, separation and purification method thereof and application

Abstract

The present invention belongs to the technical field of fermentation engineering, and discloses a mulberry endophyte producing 1-deoxynojirimycin, a separation and purification method and application thereof. The strain is Methylobacterium zasneri, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on August 29, 2022. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 25606. The strain of the present invention is separated from the mulberry endophyte by a simple separation and purification method, and is applied to fermentation engineering. By optimizing the fermentation conditions, DNJ can be quickly and highly produced, with a yield of up to 51.6 μg / mL. DNJ is used in the field of industrial production of products with hypoglycemic function after extraction and purification.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial utilization and relates to Methylobacterium zasneri, specifically to a mulberry endophyte producing 1-deoxynojirimycin, and a separation and purification method and application thereof. Background Art

[0002] Mulberry is an important tree species widely planted in China and even around the world, playing an important role in agriculture, textiles, gardening and landscape. Mulberry is a plant with both medicinal and edible properties. Its branches, leaves and fruits are rich in polysaccharides, alkaloids and other bioactive ingredients, which are of great research value.

[0003] Research has found that mulberry is the plant with the highest content of 1-deoxynojirimycin (DNJ). DNJ is a piperidine polyhydroxy alkaloid with a chemical name of 3,4,5-trihydroxy-2-hydroxymethyltetrahydropyridine. As an effective α-glucosidase inhibitor, it has a significant hypoglycemic effect. In recent years, with the improvement of living standards, the number of diabetic patients has increased dramatically, resulting in an increase in the demand for hypoglycemic substances year by year. It is necessary to increase the production of upstream raw materials for hypoglycemic drugs such as DNJ.

[0004] The Chinese invention patent application with application number 202010269030.8 discloses a method for preparing high-purity 1-deoxynojirimycin. This method directly extracts DNJ from mulberry leaves and enriches DNJ through ammonium cation exchange resin. This method is limited by the DNJ content in mulberry leaves, the yield of DNJ obtained is limited, and the operation steps are cumbersome and the production cycle is long. With the advancement of fermentation engineering technology, finding strains that can produce high DNJ and efficiently producing DNJ through fermentation tanks has become a key solution to the problem of DNJ yield. Summary of the invention

[0005] The purpose of the present invention is to provide a mulberry endophyte that produces 1-deoxynojirimycin, a separation and purification method and application thereof. The strain is separated from the mulberry endophyte by a simple separation and purification method and applied to a fermentation project to achieve the purpose of rapid and high yield of DNJ; the fermentation liquid obtained in the fermentation project is extracted and purified to achieve the purpose of producing a product with hypoglycemic function.

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

[0007] A mulberry endophyte that produces 1-deoxynojirimycin, the strain is Methylobacterium zatmanii, deposited in the General Microbiology Center of the China Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is August 29, 2022, the deposit number is CGMCC No. 25606, the Latin name is Methylobacterium sp., and it is further identified as Methylobacterium zatmanii.

[0008] As a limitation of the present invention, the 16S rDNA sequence of the mulberry endophyte producing 1-deoxynojirimycin is 1438 bp in length, and the specific sequence is shown in SEQ ID NO.1, which is:

[0009]

[0010] The present invention also provides a method for separating and purifying mulberry endophytes producing 1-deoxynojirimycin, which comprises washing, drying and sterilizing mulberry leaves, sticking them on the surface of a solid culture medium, culturing them at 28°C for 72 hours, and separating and purifying the colonies by a plate streaking method.

[0011] As a limitation, the solid culture medium is potato dextrose agar medium.

[0012] The present invention also provides an application of the mulberry endophyte producing 1-deoxynojirimycin, which comprises inoculating the mulberry endophyte producing 1-deoxynojirimycin into a modified fermentation medium, and fermenting the medium at a temperature of 24-32°C and a rotation speed of 130-170 r / min to obtain a fermentation liquid containing 1-deoxynojirimycin.

[0013] As a limitation of the present invention, the fermentation time is 60-78 hours.

[0014] As another limitation of the present invention, the raw materials for making the effective components of the improved fermentation medium include, per 100 parts by weight, 18-25 parts of potatoes, 1-5 parts of carbon sources, 1-4 parts of nitrogen sources, 0.1-0.3 parts of inorganic salts and the balance of water. The initial pH value of the improved fermentation medium is 7.2-7.4.

[0015] As a further limitation of the present invention, the carbon source is glucose, maltose, lactose, sucrose, mannitol or soluble starch;

[0016] The nitrogen source is beef extract, peptone, soybean cake powder or yeast extract powder;

[0017] The inorganic salt is magnesium sulfate, calcium chloride, potassium dihydrogen phosphate, sodium chloride or calcium carbonate.

[0018] The present invention also provides an application, which is to use the fermentation liquid containing 1-deoxynojirimycin for producing a product with blood sugar lowering function after extraction and purification.

[0019] Due to the adoption of the above technical solution, the present invention has the following technical advances compared with the prior art:

[0020] The present invention obtains a mulberry tree endophyte with high 1-deoxynojirimycin production through a simple and efficient separation and purification method, and uses a molecular biological method to identify the bacterium. Through sequence comparison and construction of a phylogenetic tree, it is confirmed that the bacterium is a 1-deoxynojirimycin-producing Methylobacterium zanseri that has not been reported, and has been deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC.25606.

[0021] The present invention adopts improved fermentation medium and optimized fermentation conditions, significantly promotes the DNJ production of mulberry endophytes producing 1-deoxynojirimycin, reduces fermentation costs, and can make the DNJ production in the fermentation liquid reach 51.6 μg / mL.

[0022] The mulberry endophyte producing 1-deoxynojirimycin provided by the present invention has high industrial application potential, and can be particularly applied to the technical field of producing blood sugar-lowering functional products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram of the initial screening results of the SY2 strain in Example 1 of the present invention;

[0024] Figure 2 is a characteristic diagram of the colony morphology of the SY2 strain in Example 1 of the present invention;

[0025] Figure 3 is a Gram staining result diagram of the SY2 strain in Example 1 of the present invention;

[0026] Figure 4 This is a graph showing the PCR amplification results of 16SrDNA of the SY2 strain in Example 1 of the present invention;

[0027] Figure 5 is a phylogenetic tree diagram of the SY2 strain in Example 1 of the present invention;

[0028] Figure 6 is a chromatogram of the DNJ standard solution in Example 1 of the present invention;

[0029] Figure 7 is a chromatogram of the culture fluid of the SY2 strain in Example 1 of the present invention;

[0030] Figure 8 This is a graph showing the yield of DNJ obtained by fermentation using different carbon sources in Example 8 of the present invention;

[0031] Fig. 9 This is a graph showing the yield of DNJ obtained by fermentation using glucose as a carbon source in Example 8 of the present invention;

[0032] Fig.10 This is a graph showing the DNJ yields obtained by fermentation using different nitrogen sources in Example 9 of the present invention;

[0033] Fig.11 This is a graph showing the yield of DNJ obtained by fermentation using beef extract as a nitrogen source in Example 9 of the present invention;

[0034] Fig.12 This is a graph showing the yield of DNJ obtained by fermentation with different inorganic salts in Example 10 of the present invention;

[0035] Fig.13This is a graph showing the yield of DNJ obtained by fermentation using magnesium sulfate as an inorganic salt in Example 10 of the present invention;

[0036] Fig.14 This is a graph showing the yield of DNJ produced by fermentation at different rotation speeds in Example 12 of the present invention;

[0037] Fig.15 This is a graph showing the yield of DNJ produced by fermentation at different fermentation temperatures in Example 13 of the present invention;

[0038] Fig.16 This is a graph showing the yield of DNJ produced by fermentation at different fermentation times in Example 14 of the present invention;

[0039] Fig.17 This is a graph showing the effect of the interaction between fermentation time and fermentation temperature on DNJ yield in Example 15 of the present invention;

[0040] Fig.18 This is a graph showing the effect of the interaction between fermentation time and rotation speed on DNJ yield in Example 15 of the present invention;

[0041] Fig.19 This is a graph showing the effect of the interaction between fermentation temperature and rotation speed on DNJ yield in Example 15 of the present invention. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below through specific examples. It should be understood that the described examples are only used to explain the present invention, but not to limit the present invention.

[0043] Example 1 Isolation, purification and identification of mulberry endophytes producing 1-deoxynojirimycin

[0044] (1) Isolation and purification of strains

[0045] Healthy mulberry leaf tissues from Botou City, Cangzhou City, Hebei Province were taken, rinsed with tap water, dried, and surface disinfected: soaked in 75% ethanol by volume for 5 minutes, taken out and soaked in 4% sodium hypochlorite solution by mass for 5 minutes, and finally rinsed with sterile water for 3 times;

[0046] Cut the surface-sterilized mulberry leaf tissue into 1 cm × 1 cm cubes, adhere the cut surface to the surface of potato dextrose agar medium (i.e., PDA solid medium), seal it, and culture it in a 28°C incubator for 72 hours. After the colonies grow out, separate and purify the single colonies by the plate streak method to obtain a single strain, which is then stored in a 4°C refrigerator for later use.

[0047] The above single strains were screened for mulberry endophytes that may produce DNJ by the color change circle method:

[0048] (I) DNJ screening medium formula and pH: KNO3 0.1%, K2HPO4 0.05%, MgSO4·7H2O 0.05%, NaCl 0.05%, FeSO4 0.001%, soluble starch 2% and agar 2%, adjusted to pH 7.3.

[0049] (II) Screening method: Use a cork puncher to punch holes on the DNJ screening medium, inject 500 μl of culture medium containing each single strain into the hole, then add agar solution containing 6 U of glucoamylase to the surface of the DNJ screening medium until the surface of the medium is filled, place it in a 28°C incubator for 1 hour, then add iodine solution to the surface of the medium and observe the color change. If a blue ring appears, it means that the single strain is a mulberry endophyte that may produce DNJ.

[0050] The results are as follows Figure 1 , a blue ring appeared around the culture fluid of the single strain SY2, indicating that the SY2 strain was a mulberry endophyte that could produce DNJ.

[0051] (2) Identification of strains

[0052] 1) Morphological identification:

[0053] The SY2 strain was inoculated onto a PDA solid culture medium plate, streaked and inverted at 28°C for 72 hours, and the colony color, morphology, edge characteristics, glossiness, and transparency were observed; Gram staining was performed and its state was observed under a microscope.

[0054] The observation and staining results are as follows: Figure 2 As shown in the figure, the colonies are pink, round, with regular edges, smooth and moist surface, and opaque; under an optical microscope, the strain is rod-shaped; Gram staining results are as follows Figure 3 As shown, the result was negative.

[0055] 2) Molecular identification:

[0056] Molecular identification of SY2 strain: The genomic DNA of endophytic bacteria SY2 strain was used as template and amplified with bacterial 16SrDNA universal primers, such as Figure 4 , a 1438 bp fragment was obtained, and after testing, the specific sequence was shown as SEQ ID NO.1.

[0057] Through analysis and comparison, the homology between SEQ ID NO.1 and the Methylobacterium of accession number AF531770.1 was as high as 100%. The phylogenetic analysis results based on 16SrDNA showed that the strain was identified as Methylobacterium, presumably Methylobacterium zatmanii or Methylobacterium extorquens; its taxonomic units are: Bacteria; Proteobacteria; Alphaproteobacteria; Rhizobiales; Methylobacteriaceae; Methylorubrum. Figure 5 As shown, according to the phylogenetic tree comparison, the SY2 strain is more closely related to Methylobacterium zatmanii.

[0058] Based on the 16S rDNA sequence alignment results, physiological and biochemical characteristics, and morphological characteristics of the SY2 strain, the strain was identified as Methylobacterium zatmanii, and the strain was deposited in the General Microbiology Center of China Culture Collection Administration, the storage address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the storage date is August 29, 2022, the storage number is CGMCC No. 25606, and the Latin name is Methylobacterium zatmanii.

[0059] 3) Identification of physiological and biochemical characteristics:

[0060] (3.1) The physiological and biochemical characteristics of the SY2 strain were determined according to the Manual of Identification of Common Bacterial Systems. The results showed that the SY2 strain was negative for H2S production, while the starch hydrolysis test, indole test, gelatin liquefaction test and methyl red reaction results were all positive.

[0061] (3.2) Identification of DNJ production characteristics:

[0062] For SY2 strain, DNJ content in culture medium was determined by high performance liquid chromatography: the inoculation concentration was 10 7 cfu / mL of SY2 strain was added to liquid PDA medium and cultured at 28°C for 48h to obtain the culture fluid for high performance liquid chromatography determination:

[0063] Preparation of DNJ standard solution: weigh 10 mg of DNJ standard solution into a 100 mL volumetric flask, add ultrapure water to make up to volume to obtain a DNJ standard solution with a concentration of 100 μg / mL, dilute the 100 μg / mL DNJ standard solution with ultrapure water, prepare standard solutions with concentrations of 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL and 50 μg / mL, respectively, and place in a 4 °C refrigerator for use.

[0064] Derivatization treatment: 200 μL of SY2 strain culture medium and DNJ standard solution were respectively taken into two 1.5 mL centrifuge tubes, and centrifuged at 8000 r / min for 20 min. 100 μL of supernatant was taken into a 1.5 mL centrifuge tube, and 100 μL of 0.4 mol / L potassium borate buffer at pH 8.5 and 100 μL of 5 mmol / L Fmoc-CL solution (dissolved in 50% acetonitrile) were added. After mixing, the mixture was reacted at room temperature for 20 min. Then, 100 μL of 1 mol / L glycine solution and 100 μL of 0.1% acetic acid aqueous solution were added, and the mixture was mixed thoroughly. The mixture was allowed to react at room temperature for 2 h before testing.

[0065] HPLC determination conditions: the chromatographic column used was a C18 column, the mobile phase was acetonitrile-0.1% acetic acid aqueous solution in a volume ratio of 55:45, the detection wavelength was 254 nm, the column temperature was 30°C, the flow rate was 1.0 mL / min, and the injection volume was 20 μL.

[0066] The results of HPLC determination, such as Figure 6 and Figure 7 ,According to the HPLC analysis, 3.6min was the peak time of DNJ derivatives, and the DNJ content in the culture medium of SY2 strain was 8.92μg / mL.

[0067] The results showed that strain SY2 was an endophyte of mulberry that produced DNJ.

[0068] Example 2-7 Application of mulberry endophytes producing 1-deoxynojirimycin

[0069] Examples 2-7 are respectively a method for producing DNJ by fermentation using DNJ-producing mulberry endophytes, specifically comprising: weighing potato flour, carbon source, nitrogen source, inorganic salt, and distilled water according to Table 1, mixing, adjusting the pH value to 7.2-7.4, and sterilizing at 121°C for 25 minutes to prepare an improved fermentation medium; inoculating the culture at a concentration of 10% by volume with a strain of 5% by volume; 7 cfu / mL of SY2 strain were added to the corresponding improved fermentation medium and fermented for 60-78h at a temperature of 24-32°C and a rotation speed of 130-170r / min; the only difference between them is the culture medium, initial pH value and fermentation process parameters. The specific differences are shown in Table 1:

[0070] Table 1 List of parameters of fermentation method of mulberry endophyte producing 1-deoxynojirimycin in Example 2-7

[0071]

[0072] The fermentation time refers to the culture time of the strain in the fermentation medium during the fermentation process.

[0073] According to common knowledge in the art, the fermentation broth containing 1-deoxynojirimycin can be used to produce drugs and other products with hypoglycemic function after extraction and purification.

[0074] Example 8 Comparison of DNJ yields produced by fermentation using modified culture medium with different carbon sources

[0075] This example uses a modified culture medium with different carbon sources, and the inoculation concentration is 10 at a volume fraction of 5%. 7 cfu / mL of the SY2 strain, the fermentation process in Example 2 was used, that is, at a temperature of 28°C and a rotation speed of 150r / min, the fermentation was carried out for 72h to produce DNJ. At 72h of fermentation, the DNJ yield was detected by the high performance liquid chromatography method in Example 1.

[0076] Among them, equal amounts of maltose, glucose, lactose, sucrose, yeast extract, mannitol, soluble starch and bran were used as carbon sources, and the contents of potato flour and distilled water were kept the same, but no nitrogen source and inorganic salt were added. The dosage ratio was referenced to Example 2, and improved culture media with different carbon sources were prepared respectively. Equal amounts of SY2 strains were added respectively. When each fermentation was performed for 72 hours, the DNJ yield was detected. Figure 8 Compared with yeast extract and bran, the use of glucose, maltose, lactose, sucrose, mannitol or soluble starch disclosed in the present invention as a carbon source can achieve a higher DNJ yield, all of which are above 4.5 μg / ml.

[0077] In a total of 100 kg of culture medium, 1%, 2%, 3%, 4% and 5% by weight of glucose were used as the carbon source, and other components remained unchanged to prepare improved fermentation medium with different carbon sources. Equal amounts of SY2 strains were added to each medium. After fermentation for 72 hours, the DNJ production was detected. Fig. 9 , DNJ production was above 8μg / ml.

[0078] Example 9 Comparison of DNJ yields produced by fermentation using modified culture medium with different nitrogen sources

[0079] This example uses a modified culture medium with different nitrogen sources, and the inoculation concentration is 10 7cfu / mL of the SY2 strain, the fermentation process in Example 2 was used, that is, at a temperature of 28°C and a rotation speed of 150r / min, the fermentation was carried out for 72h to produce DNJ. At 72h of fermentation, the DNJ yield was detected by the high performance liquid chromatography method in Example 1.

[0080] Among them, equal amounts of beef extract, peptone, soybean cake powder, yeast extract powder, urea, sodium nitrate, ammonium sulfate and ammonium chloride were used as nitrogen sources, and according to the conclusion of Example 8, 3% glucose was selected as the carbon source, and the contents of potato flour and distilled water were kept the same, but no inorganic salt was added. The dosage ratio was referenced to Example 2 to prepare improved culture media with different nitrogen sources, and equal amounts of SY2 strains were added. When each fermentation was performed for 72 hours, the DNJ yield was detected. Fig.10 Compared with urea, sodium nitrate, ammonium sulfate and ammonium chloride, the beef extract, peptone, soybean cake powder or yeast extract powder disclosed in the present invention as a nitrogen source can achieve a higher DNJ yield, all of which are above 20 μg / ml.

[0081] In a total of 100 kg of culture medium, 0.50%, 1%, 2%, 3% and 4% of beef extract were used as nitrogen sources, respectively, and other components remained unchanged to prepare improved culture media with different nitrogen sources. Equal amounts of SY2 strains were added to each culture medium. After fermentation for 72 hours, the DNJ production was detected. Fig.11 , DNJ production was above 25μg / ml.

[0082] Example 10 Comparison of DNJ yields produced by fermentation using improved fermentation medium with different inorganic salts

[0083] This example uses a modified fermentation medium with different inorganic salts, and the inoculation concentration is 10 7 cfu / mL of the SY2 strain, the fermentation process in Example 2 was used, that is, at a temperature of 28°C and a rotation speed of 150r / min, the fermentation was carried out for 72h to produce DNJ. At 72h of fermentation, the DNJ yield was detected by the high performance liquid chromatography method in Example 1.

[0084] Among them, equal amounts of magnesium sulfate, calcium chloride, potassium dihydrogen phosphate, sodium chloride and calcium carbonate were used as inorganic salts, respectively. According to the conclusions of Examples 8 and 9, 3% glucose was selected as the carbon source, 1% beef extract was selected as the nitrogen source, the contents of potato flour and distilled water were kept the same, and the dosage ratio was referenced to Example 2 to prepare improved fermentation medium with different inorganic salts. Equal amounts of SY2 strains were added respectively. When each fermentation was performed for 72 hours, the DNJ yield was detected. Fig.12 By using the magnesium sulfate, calcium chloride, potassium dihydrogen phosphate, sodium chloride and calcium carbonate disclosed in the present invention as inorganic salts, a DNJ yield of 25 μg / ml can be obtained.

[0085] In a total amount of 100 kg of improved fermentation medium, 0.1%, 0.15%, 0.20%, 0.25% and 0.30% by weight of magnesium sulfate were used as inorganic salts, and other components remained unchanged to prepare improved fermentation medium with different inorganic salts. Equal amounts of SY2 strains were added to each medium. After fermentation for 72 hours, the DNJ production was detected. Fig.13 , DNJ production was above 30μg / ml.

[0086] Example 11 Optimization experiment of improved fermentation medium

[0087] Based on the Box-Behnken sampling principle, three factors that affect DNJ content were selected: carbon source addition, nitrogen source addition and inorganic salt addition, for a three-factor three-level response surface analysis experiment.

[0088] The response surface optimization of the fermentation medium of the SY2-producing strain was carried out by the regression model designed by Design Expert 11 software, and the optimal addition amounts of glucose, beef extract and magnesium sulfate were 3.52%, 1.00% and 0.26%, respectively. According to the software prediction results, actual experiments were carried out to verify the feasibility: with a total amount of 100 kg, 3.50% glucose, 1.00% beef extract and 0.26% magnesium sulfate were taken by mass percentage, and other conditions were kept the same as in Example 2 to prepare the improved fermentation medium for fermentation and detect the DNJ production. The experiment was repeated 3 times and the results were averaged.

[0089] The results showed that the average DNJ yield was 41.13 μg / mL after fermentation using a modified fermentation medium containing 3.52% glucose, 1.00% beef extract and 0.26% magnesium sulfate, which significantly improved the DNJ yield of the SY2 strain. At the same time, it was shown that the response surface optimization method using a regression model can be used to optimize the fermentation conditions of the strain.

[0090] Example 12 Comparison of DNJ yields produced by fermentation at different speeds

[0091] In this example, SY2 strain was used to ferment DNJ under different speed conditions. The inoculation concentration was 10 7 cfu / mL of SY2 strain, the medium used was the improved fermentation medium of Example 11, and other parameters were the same as those of Example 2, that is, the temperature was 28°C and the fermentation was performed for 72 hours. At 72 hours of fermentation, the DNJ yield was detected by high performance liquid chromatography in Example 1.

[0092] The shaking speeds were 130 r / min, 140 r / min, 150 r / min, 160 r / min and 170 r / min, and the DNJ yield was detected after 72 h of fermentation. Fig.14When the fermentation speed was 130-170r / min, the DNJ production was above 40μg / ml. When the speed was 150r / min and 160r / min, the DNJ production was higher, but the difference was not significant.

[0093] Example 13 Comparison of DNJ yields produced by fermentation at different fermentation temperatures

[0094] In this example, SY2 strain was used to produce DNJ under different fermentation temperature conditions. The culture medium used was the improved culture medium of Example 11. The inoculation concentration was 10% by volume and 5% by volume. 7 cfu / mL of SY2 strain, and other parameters were the same as those in Example 2. At 72 h of fermentation, the DNJ yield was detected by the high performance liquid chromatography method in Example 1.

[0095] Among them, the fermentation temperatures were 24°C, 26°C, 28°C, 30°C and 32°C, and the DNJ production was detected at 72h of each fermentation. Fig.15 , the fermentation temperature was 24-32℃, and the DNJ production was above 35μg / ml. With the increase of fermentation temperature, the DNJ production gradually increased. When the fermentation temperature reached 30℃, the DNJ production no longer increased. Thereafter, with the increase of fermentation temperature, the DNJ production gradually decreased.

[0096] Example 14 Comparison of DNJ yields produced by fermentation at different fermentation times

[0097] This example adopts the improved culture medium of Example 11, and the inoculation concentration is 10 at a volume fraction of 5%. 7 cfu / mL of SY2 strain, and at 56h, 60h, 66h, 72h and 78h of fermentation, the DNJ production was detected by high performance liquid chromatography in Example 1, and the other fermentation process parameters were the same as those in Example 2.

[0098] The results are as follows Fig.16 As the fermentation time increased, the DNJ production gradually increased. When the fermentation time reached 72h, the DNJ production reached 50μg / ml. Thereafter, as the fermentation time increased, the DNJ production gradually decreased.

[0099] Example 15 Optimization experiment of fermentation process

[0100] Based on the Box-Behnken sampling principle, three fermentation process factors that affect DNJ content were selected: fermentation time (A), fermentation temperature (B) and rotation speed (C) for a three-factor three-level response surface analysis experiment. 7cfu / mL of SY2 strain was used to determine the interactive effect of fermentation time, fermentation temperature and rotation speed, so as to better optimize the fermentation process.

[0101] (S1) Design the factor level table of Box-Behnken test design, as shown in Table 2.

[0102] Table 2 Box-Behnken test design factor level table

[0103]

[0104] (S2) Response surface experimental design

[0105] 17 groups of experiments were designed by Design Expert 11 software. The DNJ yield (Y) was used as the response value. A three-factor three-level experiment was conducted to investigate the effects of fermentation time, fermentation temperature and rotation speed on DNJ yield. The SY2 strain culture medium used was the improved culture medium of Example 11. When the fermentation was 72 hours, the DNJ yield was detected by the high performance liquid chromatography method in Example 1. Each group of experiments was repeated 3 times, and the average value was taken for the analysis data. The experiment was conducted according to the Box-Behnken table. The DNJ yield was detected by the high performance liquid chromatography method in Example 1. The experimental design and results are shown in Table 3.

[0106] Table 3 Box-Behnken test design and results

[0107]

[0108] As shown in Table 3, a DNJ yield of 51.6 μg / ml can be obtained by using a fermentation process with a fermentation time of 72 h, a fermentation temperature of 30°C and a rotation speed of 160 r / min.

[0109] (S3) Regression equation and variance analysis

[0110] Using Design Expert 11 software for regression analysis, we obtained the quadratic multiple regression equation with DNJ production as the objective function:

[0111] Y=51.48+0.2125A-0.025B-0.0125C+0.275AB+0.1AC+0.075BC-0.315A 2 -1.14B 2 -0.615C 2 The data in Table 3 were subjected to variance analysis, and the results are shown in Table 4.

[0112] Table 4 Analysis of variance of response surface experimental design results

[0113]

[0114] Note: “*” indicates significant difference (P<0.05); “**” indicates extremely significant difference (P<0.01).

[0115] As shown in Table 4, the F value can be used to test the significance of the effect of each variable on the response value: the larger the F value, the more significant the corresponding variable. When the model significance test P<0.05, it means that the model is statistically significant.

[0116] As can be seen from Table 4, the order of the influence of fermentation process on DNJ yield is: A>B>C, that is, culture time>culture temperature>rotation speed; culture time (A) has a very significant effect on DNJ yield (P < 0.01), the equation interaction term AB has a very significant effect (P < 0.01), and the quadratic term A 2 , B 2 , C 2 The influence is extremely significant (P<0.01); the P value of the model is less than 0.0001, which is much less than 0.01, indicating that the model is extremely significant, and the P value of the lack-of-fit term is 0.0751, which is greater than 0.05, indicating that the lack-of-fit term is not significant, indicating that there is no significant difference between the model and the actual experimental values, and the experimental error is small; therefore, this regression equation can be used instead of the actual experimental values ​​to analyze the experimental results.

[0117] (S4) Credibility analysis of regression model

[0118] The correlation coefficient of the regression model variance analysis is shown in Table 5. As can be seen from the table, the negative correlation coefficient R 2 =98.44%, the corrected negative correlation coefficient Adj R 2 =96.43%, indicating that the model can reflect 96.43% of the changes in the response values, and only 3.57% may produce variations; therefore, the model fitting degree is relatively accurate, the experimental error is small, and it well reflects the relationship between DNJ production and culture time, culture temperature and rotation speed. 2 =92.34%, this coefficient is reasonably consistent with the corrected negative correlation coefficient, indicating that the model designed for this response surface experiment has good predictive ability; the signal-to-noise ratio measured by AdeqPrecision is 18.107, which is greater than 4, indicating that the signal of this experimental model is sufficient and has resolution; the coefficient of variation cv=0.28%<10%, indicating that the experimental model has good reproducibility and high reliability.

[0119] Table 5 Correlation coefficients of variance analysis

[0120]

[0121] (S5) Analysis of the interaction of various factors

[0122] from Fig.17 It can be seen that the interaction of fermentation time and fermentation temperature on DNJ yield presents a parabolic distribution, and the longitudinal span of the parabola is large and the contour lines present a significant ellipse, indicating that the interaction between the two has a significant effect on DNJ yield; when the fermentation time is constant, with the increase of fermentation temperature, DNJ yield first increases and then decreases; similarly, when the fermentation temperature remains unchanged, with the increase of fermentation time, DNJ content also increases first and then decreases; considering only the effect of the two factors, the optimal process combination is: fermentation time is 72-78h, and fermentation temperature is near the horizontal combination of 29-31℃.

[0123] Fig.18 The vertical span of the interaction surface shown is small, and the contour lines are quasi-circular, indicating that the interaction between the two has no significant effect on DNJ production. DNJ production increases first and then decreases with the increase of fermentation time and rotation speed. When the fermentation time is 72-78h and the rotation speed is around 155-165r / min, the DNJ production under this combination is the highest. The actual process control of this condition is more favorable to the result. Fig.19 As shown in the figure, the fluctuation range of the fermentation temperature direction curve is large, and its influence on DNJ production is greater than that of the rotation speed. When the fermentation temperature is 29-31℃ and the rotation speed is 155-165r / min, the DNJ production is higher. This condition should be controlled to obtain the optimized process parameters.

[0124] Taking the maximum DNJ yield as the optimization goal, according to the running results of Design Expert 11 software, the optimal conditions for DNJ yield under the combined influence of fermentation time, fermentation temperature and rotation speed are: fermentation time is 74.135h, fermentation temperature is 30.065℃, and rotation speed is 160.206r / min. Under this condition, the DNJ yield predicted by the model is 51.517μg / mL.

[0125] It should be noted that the above embodiments are only preferred embodiments of the present invention and are not other forms of limitation on the present invention. Any technician familiar with the profession may use the above technical content as inspiration to change or modify it into an equivalent embodiment with equivalent changes. However, as long as it does not deviate from the technical essence of the claims of the present invention, simple modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of protection of the claims of the present invention.

Claims

1. A mulberry endophyte producing 1-deoxynojirimycin, characterized in that: The strain is Methylobacterium zatmanii, which is deposited in the General Microbiology Center of the China Culture Collection Administration. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is August 29, 2022. The deposit number is CGMCC No. 25606, and the Latin name is Methylobacterium zatmanii.

2. An application of mulberry endophyte producing 1-deoxynojirimycin, characterized in that: The method comprises inoculating the mulberry endophyte producing 1-deoxynojirimycin according to claim 1 into a modified fermentation medium, and fermenting the medium at a temperature of 24-32° C. and a rotation speed of 130-170 r / min to obtain a fermentation liquid containing 1-deoxynojirimycin; Among them, the raw materials for making the effective ingredients of the improved fermentation medium include, per 100 parts by weight: 18-25 parts of potatoes, 1-5 parts of glucose, maltose, lactose, sucrose, mannitol or soluble starch, 1-4 parts of beef extract, peptone, soybean cake powder or yeast extract powder, 0.1-0.3 parts of magnesium sulfate, calcium chloride, potassium dihydrogen phosphate, sodium chloride or calcium carbonate, and the remainder of water.

3. The use of mulberry endophyte producing 1-deoxynojirimycin according to claim 2, characterized in that: The fermentation time is 60-78h.

4. The use of mulberry endophyte producing 1-deoxynojirimycin according to claim 2 or 3, characterized in that: The fermentation liquid containing 1-deoxynojirimycin is used for producing a drug with a blood sugar lowering function after extraction and purification.

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