WO2023016387A1 - Bacillus amyloliquefaciens and use thereof in preparation of 1-deoxynojirimycin - Google Patents

Bacillus amyloliquefaciens and use thereof in preparation of 1-deoxynojirimycin Download PDF

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WO2023016387A1
WO2023016387A1 PCT/CN2022/110772 CN2022110772W WO2023016387A1 WO 2023016387 A1 WO2023016387 A1 WO 2023016387A1 CN 2022110772 W CN2022110772 W CN 2022110772W WO 2023016387 A1 WO2023016387 A1 WO 2023016387A1
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bacillus amyloliquefaciens
fermentation
preparation
culture
medium
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彭湘屏
郑玲辉
朱进伟
孙琼
高祥
石磊
张敏
陈世敏
汪超
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浙江珲达生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring

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  • the invention belongs to the technical field of industrial biology, and in particular relates to a strain of Bacillus amyloliquefaciens capable of producing 1-deoxynojirimycin, and a method and application for fermenting and producing 1-deoxynojirimycin using the strain.
  • 1-Deoxynojirimycin (1-Deoxynojirimycin, DNJ) is a piperidine alkaloid, the chemical name is 3,4,5-trihydroxy-2-hydroxymethyltetrahydropyridine, which is present in plants, microorganisms
  • the natural sugar analogues in silkworm body the highest content in mulberry tree in nature, is a powerful sugar metabolism enzyme inhibitor (such as ⁇ -glucosidase, hexokinase, glucuronidase and glycogen phosphatase, etc.), It can significantly delay the degradation process of polysaccharides, reduce the peak of postprandial blood sugar, and stabilize fasting blood sugar.
  • DNJ can also be used in the food field. Foods made from mulberry leaves, as functional hypoglycemic products, have been allowed to be sold in the market in Japan and other East Asian countries, showing the broad prospects of DNJ in the field of health food.
  • DNJ used in medicine is basically extracted from mulberry leaves.
  • the content of DNJ in natural products is relatively low; .
  • the synthesis of artificial DNJ is difficult and costly, and it is not suitable for large-scale production.
  • microorganisms that can produce DNJ have been reported at home and abroad as Streptomyces lavendulae, Monascus purpureus, Bacillus subtilis, Escherichia coli, and Bacillus amyloliquefaciens. .
  • Yohji Ezure et al. (1985) obtained the highest yield of Streptomyces lavender DNJ by mutation, which can reach 4-5g/L.
  • CN105296565A discloses that the purity of DNJ obtained by solid-state fermentation of Bacillus subtilis is relatively low. Gu Chengchen (2016) reported that the fermentation level of Monascus purple DNJ was 0.0279g/L. The fermentation level of Escherichia coli DNJ reported by KR1020190041680 was 0.264g/L. Kenji Yamagishi et al. (2016) and CN201810125101.X reported a strain of Bacillus amyloliquefaciens producing DNJ at a level of about 1.1 g/L.
  • the microorganisms that can produce 1-deoxynojirimycin reported in the prior art generally have the disadvantages of low yield of 1-deoxynojirimycin and long fermentation time. Therefore, it is of great significance to screen high-yield 1-deoxynojirimycin strains.
  • one of the objects of the present invention is to provide a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HDCC00252 with a high yield of 1-deoxynojirimycin, which is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee (CGMCC), the deposit number is CGMCC NO.22781, and the deposit date is June 25, 2021.
  • CGMCC General Microorganism Center of China Microbiological Culture Collection Management Committee
  • the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HDCC00252 of the present invention is obtained by screening the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HDCC00031 through NTG mutagenesis and ARTP-ultraviolet compound mutagenesis.
  • Another object of the present invention is to provide the application of the Bacillus amyloliquefaciens HDCC00252 or its fermentation liquid in the preparation of 1-deoxynojirimycin.
  • Another object of the present invention is to provide a method for preparing 1-deoxynojirimycin, which is prepared by fermentation with the Bacillus amyloliquefaciens described in claim 1.
  • the fermentation process includes performing aerobic fermentation in a fermentation medium containing assimilable carbon source and/or nitrogen source.
  • the carbon source is selected from glucose, glycerol, sucrose, fructose, lactose, maltose, dextrin, starch, mannitol, sorbitol; preferably glucose, sucrose, lactose or any combination thereof;
  • the nitrogen source is selected from corn steep liquor (powder), yeast extract, yeast extract powder, yeast peptone, soybean peptone, bovine bone peptone, meat peptone, fish powder peptone, nitrate, ammonium salt; preferably Yeast extract powder, nitrates, ammonium salts or any combination thereof.
  • the fermentation medium further includes inorganic salts, preferably sulfates, phosphates, ferrous salts, more preferably ammonium sulfate, dipotassium hydrogen phosphate, and ammonium ferrous sulfate.
  • inorganic salts preferably sulfates, phosphates, ferrous salts, more preferably ammonium sulfate, dipotassium hydrogen phosphate, and ammonium ferrous sulfate.
  • the fermentation medium contains 0.1-2% of glucose, 0.5-4% of sucrose, 0.5-10% of lactose, 2-5% of yeast extract powder, 0-4% of ammonium sulfate, and 0-4% of sodium nitrate. 3%, ferrous ammonium sulfate 0.1-0.8%, dipotassium hydrogen phosphate 0.2-0.6%.
  • the fermentation temperature is 28-40° C.
  • the pH of the culture medium is 5.0-9.0
  • the culture time is 12-100 hours.
  • the Bacillus amyloliquefaciens is fermented by inoculating seed liquid into the fermentation medium;
  • the seed liquid is obtained by culturing Bacillus amyloliquefaciens HDCC00252 in a seed medium.
  • the conditions of the seed cultivation are as follows: the temperature of the seed cultivation is 28-40° C., the pH of the medium is 5.0-9.0; the cultivation time is 4-24 hours;
  • the seed medium contains 0.2-4% of glucose, 0.2-1% of yeast extract powder, 0.2-2% of sodium chloride and 0.2-4% of peptone.
  • Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HDCC00252 of the present invention has high production capacity, and its ability to produce 1-deoxynojirimycin has been greatly improved compared with other strains in the prior art.
  • the titer can reach 5.31g/L. And the fermentation cycle is short, which is beneficial to realize industrial production.
  • Fig. 1 is the HPLC collection of illustrative plates of departure bacterial strain fermented liquid
  • Fig. 2 is the LCMS collection of illustrative plates of departure bacterial strain fermented liquid
  • Fig. 3 is the morphological figure of bacterial colonies starting from
  • Fig. 4 is the microscopic examination picture of the starting strain.
  • Embodiment 1 departure bacterial strain source
  • the original DNJ-producing strain was isolated from soil samples of mulberry roots in Xiniuqiao Village, Tongxiang City, Zhejiang province.
  • the composition of the liquid primary screening medium is: lactose 2.5%, ammonium sulfate 0.4%, the pH is adjusted to 7.5 before disinfection, and the disinfection conditions are 121-123°C for 30min.
  • Embodiment 2 DNJ original production strain (HDCC00031) morphological examination and physiological and biochemical tests
  • the colonies are irregular round or oval, white, with rough surface and bulges.
  • the colony morphology is shown in Figure 3, and the microscope photo is shown in Figure 4.
  • the measured 16S rDNA sequence (SEQ ID NO: 1) of the bacterial strain (HDCC00031) is compared with the sequences of related species and genus in the GenBank database by BLAST after proofreading, as shown in Table 5 (only the homologous It was found that the classification parameters of this strain were very close to those of Bacillus amyloliquefaciens. Therefore, the strain HDCC00031 was identified as a strain of Bacillus amyloliquefaciens.
  • Embodiment 4 DNJ high-yield strain (HDCC00252) mutagenesis screening
  • the original strain (HDCC00031) as the starting strain, collect its fresh bacterial lawn, wash it with sterile physiological saline, add glass beads to oscillate to disperse, and obtain a bacterial suspension.
  • the bacterial suspension was mixed with 500 ⁇ g/ml NTG mother solution 1:1 (v/v), placed on a shaker at 30°C for 30 minutes, centrifuged at 14,000 rpm, resuspended with normal saline, and washed repeatedly for 3 times before gradient dilution.
  • aqueous acetic acid solution 1.5mL 0.1% (V/V) at last, carry out liquid chromatography analysis after filtering through 0.45 ⁇ m microporous membrane, select titer to be higher than the bacterial strain more than 100% of contrast and carry out double screening, confirm that production capacity is stable and reliable Repeated strains were used as starting strains for further mutagenesis screening.
  • the composition of the liquid primary screening medium is: lactose 2.5%, ammonium sulfate 0.4%, the pH is adjusted to 7.5 before disinfection, and the disinfection conditions are 121-123°C for 30min.
  • the strain with the highest DNJ content was selected and preserved after three consecutive rounds of streaking, isolation and purification, and the preservation number was HDCC00252. (The strain was then deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on June 25, 2021, with a preservation number of CGMCC NO.22781).
  • Bacteria recovery and activation Take the original strain HDCC00031, thaw it at room temperature, inoculate 0.1ml of the bacterial suspension onto the LB solid plate, spread it evenly, and culture it in a 37°C incubator for 24 hours to obtain activated and recovered bacteria. moss.
  • the liquid seed culture medium consists of 1.5% glucose, 0.5% yeast extract powder, 1% peptone and 1% sodium chloride. Adjust the pH to 7.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
  • Fermentation culture take the liquid seeds qualified for cultivation, inoculate them into a 250ml Erlenmeyer flask with 30ml optimized liquid fermentation medium at a ratio of 4% (V/W), wrap them up and place them on a shaker at 37°C and 220rpm The shaking culture was completed after 4 days.
  • the formula of the liquid fermentation medium is as follows: 0.4% of glucose, 2% of sucrose, 2% of lactose, 3% of yeast extract powder, 0.1% of ammonium sulfate, 0.05% of sodium nitrate, 0.25% of ferrous ammonium sulfate, and 0.28% of dipotassium hydrogen phosphate. Adjust the pH to 8.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
  • Chromatographic column Kromasil C18 analytical column (4.6mm ⁇ 250mm, 5 ⁇ m)
  • Detection conditions fluorescence detector, excitation wavelength 254nm, emission wavelength 322nm
  • the sample derivatized in the same way as the known concentration DNJ reference substance as a standard calculate the sample concentration according to the standard concentration, peak area, and sample peak area, and the result confirms that the DNJ content in the fermented liquid obtained by this embodiment is 0.12g/L.
  • Bacterial recovery and activation take the bacterial strain HDCC00252, thaw it at room temperature, inoculate 0.1ml of the bacterial suspension onto the LB solid plate, spread it evenly, and culture it in a 37°C incubator for 24 hours to obtain an activated and recovered bacterial lawn .
  • the liquid seed culture medium consists of 1.5% glucose, 0.5% yeast extract powder, 1% peptone and 1% sodium chloride. Adjust the pH to 7.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
  • Fermentation culture take the liquid seeds qualified for cultivation, inoculate them into a 250ml Erlenmeyer flask with 30ml optimized liquid fermentation medium at a ratio of 4% (V/W), wrap them up and place them on a shaker at 37°C and 220rpm The shaking culture was completed after 4 days.
  • the formula of the liquid fermentation medium is as follows: 0.4% of glucose, 2% of sucrose, 2% of lactose, 3% of yeast extract powder, 0.1% of ammonium sulfate, 0.05% of sodium nitrate, 0.25% of ferrous ammonium sulfate, and 0.28% of dipotassium hydrogen phosphate. Adjust the pH to 8.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
  • Chromatographic column Kromasil C18 analytical column (4.6mm ⁇ 250mm, 5 ⁇ m)
  • Detection conditions fluorescence detector, excitation wavelength 254nm, emission wavelength 322nm
  • Bacterial recovery and activation take the bacterial strain HDCC00252, thaw it at room temperature, inoculate 0.1ml of the bacterial suspension onto the LB solid plate, spread it evenly, and culture it in a 37°C incubator for 24 hours to obtain an activated and recovered bacterial lawn .
  • the liquid seed medium consists of 4% glucose, 1% yeast extract powder, 4% peptone and 2% sodium chloride. Adjust the pH to 5.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
  • Fermentation culture take the liquid seeds qualified for cultivation, inoculate them into a 250ml Erlenmeyer flask with 30ml optimized liquid fermentation medium at a ratio of 4% (V/W), wrap them up and place them on a shaker at 28°C and 220rpm The shaking culture was completed after 4 days.
  • the liquid fermentation medium formula consists of: 0.1% of glucose, 0.5% of sucrose, 10% of lactose, 5% of yeast extract powder, 4% of ammonium sulfate, 0.1% of ferrous ammonium sulfate, and 0.6% of dipotassium hydrogen phosphate. Adjust the pH to 9.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
  • Chromatographic column Kromasil C18 analytical column (4.6mm ⁇ 250mm, 5 ⁇ m)
  • Detection conditions fluorescence detector, excitation wavelength 254nm, emission wavelength 322nm
  • the sample derivatized by the same method as the known concentration DNJ reference substance as a standard calculate the sample concentration according to the standard concentration, peak area, and sample peak area, and the result confirms that the DNJ content in the fermented liquid obtained by this embodiment is 5.01g/L.
  • Bacterial recovery and activation take the bacterial strain HDCC00252, thaw it at room temperature, inoculate 0.1ml of the bacterial suspension onto the LB solid plate, spread it evenly, and culture it in a 37°C incubator for 24 hours to obtain an activated and recovered bacterial lawn .
  • the liquid seed medium consists of 0.2% glucose, 0.2% yeast extract powder, 0.2% peptone and 0.2% sodium chloride. Adjust the pH to 9.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
  • Fermentation culture take the liquid seeds qualified for cultivation, inoculate them into a 250ml Erlenmeyer flask with 30ml optimized liquid fermentation medium at a ratio of 4% (V/W), wrap them up and place them on a shaker at 37°C and 220rpm The shaking culture was completed after 4 days.
  • the formula of the liquid fermentation medium is as follows: 2% of glucose, 4% of sucrose, 0.5% of lactose, 2% of yeast extract powder, 3% of sodium nitrate, 0.8% of ferrous ammonium sulfate, and 0.2% of dipotassium hydrogen phosphate. Adjust the pH to 5.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
  • Chromatographic column Kromasil C18 analytical column (4.6mm ⁇ 250mm, 5 ⁇ m)
  • Detection conditions fluorescence detector, excitation wavelength 254nm, emission wavelength 322nm
  • the sample derivatized by the same method as the known concentration DNJ reference substance as a standard calculate the sample concentration according to the standard concentration, peak area, and sample peak area, and the result confirms that the DNJ content in the fermented liquid obtained by this embodiment is 4.98g/L.

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Abstract

Provided is a Bacillus amyloliquefaciens HDCC00252, the deposit number thereof being CGMCC No. 22781. Also provided is a method for producing 1-deoxynojirimycin by means of fermentation culture of the Bacillus amyloliquefaciens HDCC00252.

Description

一种解淀粉芽孢杆菌及其在制备1-脱氧野尻霉素中的应用A kind of Bacillus amyloliquefaciens and its application in the preparation of 1-deoxynojirimycin 技术领域technical field
本发明属于工业生物技术领域，具体涉及一株能够生产1-脱氧野尻霉素的解淀粉芽孢杆菌，以及利用该菌种发酵生产1-脱氧野尻霉素的方法及应用。The invention belongs to the technical field of industrial biology, and in particular relates to a strain of Bacillus amyloliquefaciens capable of producing 1-deoxynojirimycin, and a method and application for fermenting and producing 1-deoxynojirimycin using the strain.
背景技术Background technique
1-脱氧野尻霉素(1-Deoxynojirimycin,DNJ)是一种哌啶生物碱，化学名称为3,4,5-三羟基-2-羟甲基四氢吡啶，是一种存在于植物、微生物及蚕体中的天然糖类似物，自然界以桑树中含量最高，为强效的糖代谢酶抑制剂(比如α－葡萄糖苷酶、己糖激酶、葡萄糖醛酸酶和糖原磷酸酶等)，可显著延缓多糖的降解过程，降低餐后血糖的峰值，稳定空腹血糖。并且，还有减肥及增加胰岛敏感性、抗病毒、抗肿瘤转移等作用，在医药和保健品有广泛的应用。此外，DNJ也可用于食品领域，以桑叶为原料的食品，作为功能性降糖的产品在日本等东亚国家已经允许在市场上销售，显示出DNJ在保健食品领域的广阔前景。1-Deoxynojirimycin (1-Deoxynojirimycin, DNJ) is a piperidine alkaloid, the chemical name is 3,4,5-trihydroxy-2-hydroxymethyltetrahydropyridine, which is present in plants, microorganisms And the natural sugar analogues in silkworm body, the highest content in mulberry tree in nature, is a powerful sugar metabolism enzyme inhibitor (such as α-glucosidase, hexokinase, glucuronidase and glycogen phosphatase, etc.), It can significantly delay the degradation process of polysaccharides, reduce the peak of postprandial blood sugar, and stabilize fasting blood sugar. Moreover, it also has the effects of losing weight, increasing the sensitivity of islets, anti-virus, and anti-tumor metastasis, and is widely used in medicine and health care products. In addition, DNJ can also be used in the food field. Foods made from mulberry leaves, as functional hypoglycemic products, have been allowed to be sold in the market in Japan and other East Asian countries, showing the broad prospects of DNJ in the field of health food.
目前，医药上所用的DNJ基本上都是从桑叶提取的，一方面天然产物中DNJ含量较低，另一方面，DNJ的分离纯化较为复杂，再加上提取过程中的损失，产量很低。而人工DNJ合成难度较大，成本高，不适于大规模的生产。目前，国内外报道可以产生DNJ的微生物有淡紫色链霉菌(Streptomyces lavendulae)、紫红曲霉(Monascus  purpureus)、枯草芽孢杆菌(Bacillus subtilis)、大肠杆菌(Escherichia coli)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。其中，Yohji Ezure等人(1985)突变获得淡紫色链霉菌DNJ产量最高，可以达到4～5g/L，但是该菌种为丝状菌，发酵过程动力消耗大，发酵周期长。CN105296565A公开了使用枯草芽孢杆菌固态发酵得到DNJ纯度较低。顾澄琛(2016)报道的紫红曲霉DNJ的发酵水平为0.0279g/L。KR1020190041680报道的大肠杆菌DNJ的发酵水平为0.264g/L。Kenji Yamagishi等人(2016)以及CN201810125101.X报道了一株产DNJ的解淀粉芽孢杆菌，其水平约为1.1g/L。综上，现有技术所报道的可以产1-脱氧野尻霉素的微生物，其普遍存在1-脱氧野尻霉素产量低、发酵时间长等缺点。因此，筛选高产1-脱氧野尻霉素的菌株具有重要意义。At present, DNJ used in medicine is basically extracted from mulberry leaves. On the one hand, the content of DNJ in natural products is relatively low; . However, the synthesis of artificial DNJ is difficult and costly, and it is not suitable for large-scale production. At present, microorganisms that can produce DNJ have been reported at home and abroad as Streptomyces lavendulae, Monascus purpureus, Bacillus subtilis, Escherichia coli, and Bacillus amyloliquefaciens. . Among them, Yohji Ezure et al. (1985) obtained the highest yield of Streptomyces lavender DNJ by mutation, which can reach 4-5g/L. However, this strain is a filamentous fungus, and the fermentation process consumes a lot of power and the fermentation cycle is long. CN105296565A discloses that the purity of DNJ obtained by solid-state fermentation of Bacillus subtilis is relatively low. Gu Chengchen (2016) reported that the fermentation level of Monascus purple DNJ was 0.0279g/L. The fermentation level of Escherichia coli DNJ reported by KR1020190041680 was 0.264g/L. Kenji Yamagishi et al. (2016) and CN201810125101.X reported a strain of Bacillus amyloliquefaciens producing DNJ at a level of about 1.1 g/L. In summary, the microorganisms that can produce 1-deoxynojirimycin reported in the prior art generally have the disadvantages of low yield of 1-deoxynojirimycin and long fermentation time. Therefore, it is of great significance to screen high-yield 1-deoxynojirimycin strains.
发明内容Contents of the invention
为解决现有技术存在的不足，本发明的目的之一在于提供了一株高产1-脱氧野尻霉素的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)HDCC00252，保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号为CGMCC NO.22781，保藏日期为2021年06月25日。In order to solve the deficiencies in the prior art, one of the objects of the present invention is to provide a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HDCC00252 with a high yield of 1-deoxynojirimycin, which is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee (CGMCC), the deposit number is CGMCC NO.22781, and the deposit date is June 25, 2021.
本发明所述的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)HDCC00252是以解淀粉芽孢杆菌(Bacillus amyloliquefaciens)HDCC00031为出发菌株，通过NTG诱变、ARTP-紫外复合诱变筛选得到。The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HDCC00252 of the present invention is obtained by screening the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HDCC00031 through NTG mutagenesis and ARTP-ultraviolet compound mutagenesis.
本发明的另一目的在于提供了所述的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)HDCC00252或其发酵液在制备1-脱氧野尻霉素中的应用。Another object of the present invention is to provide the application of the Bacillus amyloliquefaciens HDCC00252 or its fermentation liquid in the preparation of 1-deoxynojirimycin.
本发明的再一目的在于提供了一种1-脱氧野尻霉素的制备方法，所述的方法采用权利要求1所述的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)进行发酵制备。Another object of the present invention is to provide a method for preparing 1-deoxynojirimycin, which is prepared by fermentation with the Bacillus amyloliquefaciens described in claim 1.
具体的，所述的发酵过程包括在含有可同化的碳源和/或氮源的发酵培养基里，进行有氧发酵。Specifically, the fermentation process includes performing aerobic fermentation in a fermentation medium containing assimilable carbon source and/or nitrogen source.
作为一种实施方式，所述的碳源选自葡萄糖、甘油、蔗糖、果糖、乳糖、麦芽糖、糊精、淀粉、甘露醇、山梨醇；优选为葡萄糖、蔗糖、乳糖或其任一组合；As an embodiment, the carbon source is selected from glucose, glycerol, sucrose, fructose, lactose, maltose, dextrin, starch, mannitol, sorbitol; preferably glucose, sucrose, lactose or any combination thereof;
作为一种实施方式，所述的氮源选自玉米浆(粉)、酵母膏、酵母浸出粉、酵母蛋白胨、大豆蛋白胨、牛骨蛋白胨、肉胨、鱼粉蛋白胨、硝酸盐、铵盐；优选为酵母浸出粉、硝酸盐、铵盐或其任意几种的组合。As an embodiment, the nitrogen source is selected from corn steep liquor (powder), yeast extract, yeast extract powder, yeast peptone, soybean peptone, bovine bone peptone, meat peptone, fish powder peptone, nitrate, ammonium salt; preferably Yeast extract powder, nitrates, ammonium salts or any combination thereof.
作为一种实施方式，所述的发酵培养基还包括无机盐，优选为硫酸盐、磷酸盐、亚铁盐，更优选为硫酸铵、磷酸氢二钾、硫酸亚铁铵。As an embodiment, the fermentation medium further includes inorganic salts, preferably sulfates, phosphates, ferrous salts, more preferably ammonium sulfate, dipotassium hydrogen phosphate, and ammonium ferrous sulfate.
作为一种实施方式，所述的发酵培养基含有葡萄糖0.1～2％，蔗糖0.5～4％，乳糖0.5～10％，酵母浸出粉2～5％，硫酸铵0～4％，硝酸钠0～3％，硫酸亚铁铵0.1～0.8％，磷酸氢二钾0.2～0.6％。As an embodiment, the fermentation medium contains 0.1-2% of glucose, 0.5-4% of sucrose, 0.5-10% of lactose, 2-5% of yeast extract powder, 0-4% of ammonium sulfate, and 0-4% of sodium nitrate. 3%, ferrous ammonium sulfate 0.1-0.8%, dipotassium hydrogen phosphate 0.2-0.6%.
作为一种实施方式，所述发酵温度为28-40℃，所述培养基pH为5.0-9.0；培养时间为12-100小时。As an embodiment, the fermentation temperature is 28-40° C., the pH of the culture medium is 5.0-9.0, and the culture time is 12-100 hours.
作为一种实施方式，所述解淀粉芽孢杆菌是通过种子液接种至所述发酵培养基中进行发酵培养的；As an embodiment, the Bacillus amyloliquefaciens is fermented by inoculating seed liquid into the fermentation medium;
其中，所述种子液是将解淀粉芽孢杆菌(Bacillus amyloliquefaciens)HDCC00252在种子培养基里进行种子培养得到的。Wherein, the seed liquid is obtained by culturing Bacillus amyloliquefaciens HDCC00252 in a seed medium.
所述种子培养的条件为：种子培养的温度为28-40℃，所述培养基pH为5.0-9.0；培养时间为4-24小时；The conditions of the seed cultivation are as follows: the temperature of the seed cultivation is 28-40° C., the pH of the medium is 5.0-9.0; the cultivation time is 4-24 hours;
所述的种子培养基含葡萄糖0.2～4％，酵母浸出粉0.2～1％，氯化钠0.2～2％，蛋白胨0.2～4％。The seed medium contains 0.2-4% of glucose, 0.2-1% of yeast extract powder, 0.2-2% of sodium chloride and 0.2-4% of peptone.
与现有技术相比，本发明的有益效果在于：Compared with prior art, the beneficial effect of the present invention is:
本发明所述的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)HDCC00252生产能力高，其产1-脱氧野尻霉素的能力比现有技术中其他菌种有了大幅度的提高，1-脱氧野尻霉素的效价可达到在5.31g/L。且发酵周期短，有利于实现工业化生产。Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HDCC00252 of the present invention has high production capacity, and its ability to produce 1-deoxynojirimycin has been greatly improved compared with other strains in the prior art. The titer can reach 5.31g/L. And the fermentation cycle is short, which is beneficial to realize industrial production.
附图说明Description of drawings
图1为出发菌株发酵液的HPLC图谱；Fig. 1 is the HPLC collection of illustrative plates of departure bacterial strain fermented liquid;
图2为出发菌株发酵液的LCMS图谱；Fig. 2 is the LCMS collection of illustrative plates of departure bacterial strain fermented liquid;
图3为出发菌株菌落形态图；Fig. 3 is the morphological figure of bacterial colonies starting from;
图4为出发菌株镜检图。Fig. 4 is the microscopic examination picture of the starting strain.
具体实施方式Detailed ways
下述实施例中采用的材料、试剂等如无特殊说明，皆为普通市售品，皆可于市场购得。Unless otherwise specified, the materials and reagents used in the following examples are commercially available and can be purchased in the market.
以下结合具体实施例，对本发明作进一步说明，应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention but not to limit the scope of the present invention.
实施例1出发菌株来源 Embodiment 1 departure bacterial strain source
从浙江桐乡市西牛桥村桑树根系土壤样品中分离到了产DNJ的原始菌种。The original DNJ-producing strain was isolated from soil samples of mulberry roots in Xiniuqiao Village, Tongxiang City, Zhejiang Province.
采集浙江桐乡市西牛桥村不同地点桑树根际3～10cm深层土壤样品20g，装入无菌样品袋，编号并记录取样地点、日期。所有土样分别过60目筛网，每个土样各取1g加入到带有玻璃珠的无菌生理盐水中，充分振荡后于80℃水浴40min，静置10min后取上清液进行梯度稀释，取10-2～10-6梯度稀释液0.1mL分别接种涂布到营养琼脂培养基上，37℃倒置培养2～5天，每天观察。挑选白色产襥的菌落，经两轮划线传代纯化后作为待选菌株，取待选菌株斜面，用不锈钢铲刮取少量菌苔，接种到液体初筛培养基中并搅散，置于37℃摇床上220rpm振荡培养5天，获得初筛发酵液。取发酵液1ml，10000rpm高速离心10min，去除菌体，上清液保存于2～8℃冰箱中待检。(液体初筛培养基配方组成为：乳糖2.5％，硫酸铵0.4％，消毒前调pH至7.5，消毒条件为121～123℃、30min。)Collect 20g of mulberry rhizosphere 3-10cm deep soil samples from different locations in Xiniuqiao Village, Tongxiang City, Zhejiang Province, put them into sterile sample bags, number them, and record the sampling locations and dates. All soil samples were passed through a 60-mesh sieve, and 1 g of each soil sample was added to sterile saline with glass beads. After fully shaking, put it in a water bath at 80°C for 40 minutes, and after standing for 10 minutes, take the supernatant for gradient dilution. , take 0.1mL of 10-2-10-6 gradient dilution solution to inoculate and spread on nutrient agar medium respectively, incubate upside down at 37°C for 2-5 days, and observe every day. Select the white bacterial colony, and after two rounds of streaking and passage purification, it will be used as the strain to be selected. Take the slant of the strain to be selected, scrape a small amount of bacterial lawn with a stainless steel shovel, inoculate it into the liquid primary screening medium and stir it up, and place it at 37 Cultivate with shaking at 220rpm on a shaking table for 5 days to obtain a primary screening fermentation broth. Take 1ml of fermentation broth, centrifuge at 10000rpm for 10min at a high speed, remove bacteria, and store the supernatant in a refrigerator at 2-8°C until testing. (The composition of the liquid primary screening medium is: lactose 2.5%, ammonium sulfate 0.4%, the pH is adjusted to 7.5 before disinfection, and the disinfection conditions are 121-123°C for 30min.)
以纯水当作参比对照，连同每个样品取上清液0.1mL与0.8mL水及0.1mL蔗糖酶震荡均匀后加入2mL 10％的蔗糖溶液，放入25℃水浴反应1h。反应后各取0.6m L反应液加入1mLDNS试剂，沸水浴5min，冷水冷却至室温，加8.4mL水稀释至10mL，混匀后各吸取1ml加入到96孔UV板，在520nm波长下测吸光值，如果样品管吸 光值低于参比管吸光值，则可认为该发酵液中存在蔗糖酶抑制剂，且抑制剂相对含量可以用吸光值来进行表征。With pure water as the reference control, together with each sample, take 0.1mL supernatant, 0.8mL water and 0.1mL sucrase, shake evenly, add 2mL 10% sucrose solution, and put it in a 25°C water bath for 1h. After the reaction, take 0.6mL reaction solution and add 1mL DNS reagent, boil water bath for 5min, cool to room temperature with cold water, add 8.4mL water to dilute to 10mL, absorb 1ml each after mixing and add to 96-well UV plate, measure the absorbance value at 520nm wavelength , if the absorbance value of the sample tube is lower than the absorbance value of the reference tube, it can be considered that there is a sucrase inhibitor in the fermentation broth, and the relative content of the inhibitor can be characterized by the absorbance value.
挑选吸光值远低于参比的样品上清液2ml，加入等体积乙醇混匀后10000rpm高速离心10min。取上清液20μL置于1.5mL的离心管中，加入0.4mol/L的硼酸盐缓冲液(pH＝8.5)20μL及2mmol/L的FMOC-Cl的乙腈溶液40μL，振荡混匀，于25℃水浴中反应20min，加入1mol/L的甘氨酸20μL，静置5min，让多余的衍生化试剂反应完。最后加入1.5mL 0.1％(V/V)的醋酸水溶液，经0.45μm微孔滤膜滤过后进行液相色谱分析，查找与DNJ标准品有相同保留时间的样品，最后用LCMS进行分子量测定，以确定是否为DNJ。Select 2ml of the sample supernatant whose absorbance value is much lower than that of the reference, add an equal volume of ethanol to mix, and then centrifuge at 10000rpm for 10min at high speed. Take 20 μL of the supernatant and place it in a 1.5 mL centrifuge tube, add 20 μL of 0.4 mol/L borate buffer (pH=8.5) and 40 μL of 2 mmol/L FMOC-Cl in acetonitrile solution, vortex and mix well, and place at 25 React in a water bath at ℃ for 20 minutes, add 20 μL of 1 mol/L glycine, and let stand for 5 minutes to allow the excess derivatization reagent to react. Add 1.5mL of 0.1% (V/V) acetic acid aqueous solution at last, carry out liquid chromatography analysis after filtering through 0.45 μm microporous membrane, look for the sample that has identical retention time with DNJ standard substance, carry out molecular weight measurement with LCMS at last, with Determine if it is DNJ.
实验结果：本实验共分离1164株菌落，成功从编号为25#的浙江桐乡市西牛桥村桑树根系土壤样品中，分离得到了47株产DNJ的菌株，该47株菌株再次经过两轮划线分离纯化后，进行发酵验证，DNJ的含量最高为0.12g/L，其HPLC图谱和LCMS图谱如图1和图2所示，挑出该单株编号为HDCC00031。Experimental results: A total of 1164 colonies were isolated in this experiment, and 47 DNJ-producing strains were successfully isolated from the mulberry root soil sample numbered 25# in Xiniuqiao Village, Tongxiang City, Zhejiang Province. After line separation and purification, fermentation verification was carried out, and the content of DNJ was up to 0.12g/L. Its HPLC spectrum and LCMS spectrum are shown in Figure 1 and Figure 2, and the number of the selected single plant was HDCC00031.
实施例2DNJ原始生产菌种(HDCC00031)形态学检查及生理生化试验 Embodiment 2 DNJ original production strain (HDCC00031) morphological examination and physiological and biochemical tests
在营养琼脂平板上划线接种菌株(HDCC00031)，置于37℃培养，记录菌落形态及颜色等特征，革兰氏染色后光学显微镜观察。生理生化特征，分别对目的菌株进行葡萄糖发酵实验、淀粉水解、V-P测定、吲哚试验、明胶水解、过氧化氢酶试验、硝酸盐还原试验、产气实验、厌氧琼脂实验。Streak inoculation of strain (HDCC00031) on nutrient agar plate, culture at 37°C, record the characteristics of colony shape and color, and observe with optical microscope after Gram staining. Physiological and biochemical characteristics, glucose fermentation test, starch hydrolysis, V-P determination, indole test, gelatin hydrolysis, catalase test, nitrate reduction test, gas production test, anaerobic agar test were carried out on the target strain respectively.
形态特征：菌落不规则圆形或椭圆，白色，表面粗糙，有***。菌落形态如图3所示，镜检照片如图4所示。Morphological characteristics: The colonies are irregular round or oval, white, with rough surface and bulges. The colony morphology is shown in Figure 3, and the microscope photo is shown in Figure 4.
生理生化特征：详见表1～表4。Physiological and biochemical characteristics: see Table 1 to Table 4 for details.
表1原始菌株(HDCC00031)的碳源和氮源利用情况Table 1 The carbon source and nitrogen source utilization of the original strain (HDCC00031)
Figure PCTCN2022110772-appb-000001
Figure PCTCN2022110772-appb-000001
表2原始菌株(HDCC00031)主要的生理生化特征Table 2 The main physiological and biochemical characteristics of the original strain (HDCC00031)
试验项目Pilot projects 结果result 试验项目Pilot projects 结果result
明胶液化gelatin liquefaction ++ 牛奶胨化peptonization of milk ++
淀粉水解starch hydrolysis ++++ 硝酸盐还原Nitrate reduction --
精氨酸水解Arginine hydrolysis -- 吲哚indole --
氧化酶Oxidase ++ V.P实验V.P experiment --
过氧化氢酶Catalase ++ M.R实验M.R. experiment --
β-半乳糖苷酶β-galactosidase ++ // //
表3原始菌株(HDCC00031)生长pH试验Table 3 Original strain (HDCC00031) growth pH test
pHpH 2.02.0 3.03.0 4.04.0 5.05.0 6.06.0 7.07.0 8.08.0 9.09.0
生长情况growing situation 00 00 11 22 33 44 44 44
表4原始菌株(HDCC00031)生长温度试验Table 4 original strain (HDCC00031) growth temperature test
温度(℃)temperature(℃) 1010 2020 2525 2828 3737 4545
生长情况growing situation 00 00 22 33 44 00
*注：0，无生长；1，生长很弱；2，能生长；3，生长良好；4，生长最好；+，阳性；-，阴性；w，弱。*Note: 0, no growth; 1, very weak growth; 2, able to grow; 3, good growth; 4, best growth; +, positive; -, negative; w, weak.
实施例3DNJ原始生产菌种(HDCC00031)16S rDNA鉴定Embodiment 3DNJ original production strain (HDCC00031) 16S rDNA identification
取原始菌种(HDCC00031)斜面，收集新鲜菌苔，采用上海生工的SK8255试剂盒提取DNA基因组，采用通用引物(27F和1492R)进行16S rRNA基因扩增，PCR产物经检测纯化后，直接进行序列测定，测序由浙江工业大学生物工程研究所进行。菌株(HDCC00031)所测的16S rDNA序列(SEQ ID NO:1)经校对后与GenBank数据库中相关种、属的序列进行同源序列BLAST比较，如表5所示(表中只列出同源性较高的模式菌株)，发现该菌株和解淀粉芽孢杆菌(Bacillus amyloliquefaciens.)分类相关参数非常接近，故将菌株HDCC00031鉴定为解淀粉芽孢杆菌属(Bacillus amyloliquefaciens)菌株。Take the slant of the original strain (HDCC00031), collect the fresh bacterial lawn, use Shanghai Sangon’s SK8255 kit to extract the DNA genome, and use universal primers (27F and 1492R) to amplify the 16S rRNA gene. After the PCR product is detected and purified, it is directly processed Sequencing was performed by the Institute of Bioengineering, Zhejiang University of Technology. The measured 16S rDNA sequence (SEQ ID NO: 1) of the bacterial strain (HDCC00031) is compared with the sequences of related species and genus in the GenBank database by BLAST after proofreading, as shown in Table 5 (only the homologous It was found that the classification parameters of this strain were very close to those of Bacillus amyloliquefaciens. Therefore, the strain HDCC00031 was identified as a strain of Bacillus amyloliquefaciens.
表5菌株(HDCC00031)和典型模式菌株的同源性Homology of Table 5 strain (HDCC00031) and typical type strain
Figure PCTCN2022110772-appb-000002
Figure PCTCN2022110772-appb-000002
Figure PCTCN2022110772-appb-000003
Figure PCTCN2022110772-appb-000003
实施例4 DNJ高产菌种(HDCC00252)诱变筛选 Embodiment 4 DNJ high-yield strain (HDCC00252) mutagenesis screening
1.NTG诱变筛选1. NTG mutagenesis screening
以原始菌种(HDCC00031)为出发菌株，收集其新鲜菌苔，用无菌生理盐水洗下，加玻璃珠振荡打散，获得菌悬液。菌悬液与500μg/ml的NTG母液1：1(v/v)混合，置于30℃摇床上诱变处理30min，14000rpm高速离心，用生理盐水重悬，如此反复洗涤3次后梯度稀释，涂布到营养琼脂平板上，置于37℃培养24h，用不锈钢铲刮取少量菌体，接种到液体初筛培养基中并搅散，置于37℃摇床上220rpm振荡培养5天，获得初筛发酵液。取发酵液1ml，加入等体积乙醇混匀后超声处理20min，混匀，14000rpm高速离心10min。再取上清液20μL置于1.5mL的离心管中，加入0.4mol/L的硼酸盐缓冲液(pH＝8.5)20μL及2mmol/L的FMOC-Cl的乙腈溶液40μL，振荡混匀，于25℃水浴中反应20min，加入1mol/L的甘氨酸20μL，静置5min，让多余的衍生化试剂反应完。最后加入1.5mL 0.1％(V/V)的醋酸水溶液，经0.45μm微孔滤膜滤过后进行液相色谱分析，挑选效价高于对照100％以上的菌株进行复筛，确定生产能力稳定可重复的菌株作为继续诱变筛选的出发菌株。(液体初筛培养基配方组成为：乳糖2.5％，硫酸铵0.4％，消毒前调pH至7.5，消毒条件为121～123℃、30min。)Using the original strain (HDCC00031) as the starting strain, collect its fresh bacterial lawn, wash it with sterile physiological saline, add glass beads to oscillate to disperse, and obtain a bacterial suspension. The bacterial suspension was mixed with 500 μg/ml NTG mother solution 1:1 (v/v), placed on a shaker at 30°C for 30 minutes, centrifuged at 14,000 rpm, resuspended with normal saline, and washed repeatedly for 3 times before gradient dilution. Spread it on a nutrient agar plate, culture it at 37°C for 24 hours, scrape a small amount of bacteria with a stainless steel shovel, inoculate it into the liquid primary screening medium and stir it up, place it on a shaking table at 37°C at 220rpm and shake it for 5 days to obtain the primary Sift the broth. Take 1ml of fermentation broth, add an equal volume of ethanol and mix well, then sonicate for 20min, mix well, and centrifuge at 14000rpm for 10min at high speed. Then take 20 μL of the supernatant and place it in a 1.5 mL centrifuge tube, add 20 μL of 0.4 mol/L borate buffer (pH=8.5) and 40 μL of 2 mmol/L FMOC-Cl in acetonitrile solution, shake and mix well, and put in React in a water bath at 25°C for 20 minutes, add 20 μL of 1 mol/L glycine, and let stand for 5 minutes to allow the excess derivatization reagent to react. Add the aqueous acetic acid solution of 1.5mL 0.1% (V/V) at last, carry out liquid chromatography analysis after filtering through 0.45 μm microporous membrane, select titer to be higher than the bacterial strain more than 100% of contrast and carry out double screening, confirm that production capacity is stable and reliable Repeated strains were used as starting strains for further mutagenesis screening. (The composition of the liquid primary screening medium is: lactose 2.5%, ammonium sulfate 0.4%, the pH is adjusted to 7.5 before disinfection, and the disinfection conditions are 121-123°C for 30min.)
2.ARTP-紫外复合诱变筛选2. ARTP-ultraviolet compound mutagenesis screening
取NTG诱变筛选获得的高产菌株作为出发菌株，收集其新鲜菌苔，用无菌生理盐水洗下，加玻璃珠振荡打散，离心收集菌体，加入少量无菌生理盐水重悬，获得浓缩菌悬液。取浓缩菌悬液10μL，涂于ARTP(ARTP-IIS)载片表面，放入到处理仓，设定功率100W，气体流量10SLM，照射时长8S。将载片取下，放入到1ml无菌生理盐水中，振荡将菌体洗下，然后进行梯度稀释至10 -1～10 -4，每个梯度稀释液0.1ml涂布到营养琼脂平板上。将接种好的平板，置于15W紫外灯下方约30cm处，打开平皿盖，开启紫外灯进行紫外诱变处理，处理时长10S，盖上平皿盖，包裹好置于37℃培养24h。用不锈钢铲刮取少量菌体，接种到液体初筛培养基中并搅散，置于37℃摇床上220rpm振荡培养5天，获得初筛发酵液。取发酵液1ml，加入等体积乙醇混匀后超声处理20min，混匀，14000rpm高速离心10min。再取上清液20μL置于1.5mL的离心管中，加入0.4mol/L的硼酸盐缓冲液(pH＝8.5)20μL及2mmol/L的FMOC-Cl的乙腈溶液40μL，振荡混匀，于25℃水浴中反应20min，加入1mol/L的甘氨酸20μL，静置5min，让多余的衍生化试剂反应完。最后加入1.5mL 0.1％(V/V)的醋酸水溶液，经0.45μm微孔滤膜滤过后进行液相色谱分析。(液体初筛培养基配方组成为：乳糖2.5％，硫酸铵0.4％，消毒前调pH至7.5，消毒条件为121～123℃、30min。) Take the high-yield strain obtained by NTG mutagenesis screening as the starting strain, collect its fresh bacterial lawn, wash it with sterile normal saline, add glass beads to shake and disperse, collect the bacteria by centrifugation, add a small amount of sterile normal saline to resuspend, and obtain concentrated bacterial suspension. Take 10 μL of the concentrated bacterial suspension, apply it on the surface of the ARTP (ARTP-IIS) slide, put it into the processing chamber, set the power to 100W, the gas flow rate to 10SLM, and the irradiation time to 8S. Remove the slide, put it into 1ml of sterile physiological saline, shake to wash the bacteria, and then perform gradient dilution to 10 -1 ~ 10 -4 , spread 0.1ml of each gradient dilution on the nutrient agar plate . Place the inoculated plate about 30cm below the 15W UV lamp, open the lid of the plate, turn on the UV lamp for ultraviolet mutagenesis treatment, the treatment time is 10 seconds, cover the plate, wrap it and place it at 37°C for 24 hours. Scrape a small amount of bacteria with a stainless steel shovel, inoculate into the liquid primary screening medium and stir to disperse, place on a shaker at 37°C at 220rpm and vibrate for 5 days to obtain the primary screening fermentation broth. Take 1ml of the fermentation broth, add an equal volume of ethanol and mix well, then sonicate for 20min, mix well, and centrifuge at 14000rpm for 10min at high speed. Then take 20 μL of the supernatant and place it in a 1.5 mL centrifuge tube, add 20 μL of 0.4 mol/L borate buffer (pH=8.5) and 40 μL of 2 mmol/L FMOC-Cl in acetonitrile solution, shake and mix well, and put in React in a water bath at 25°C for 20 minutes, add 20 μL of 1 mol/L glycine, and let stand for 5 minutes to allow the excess derivatization reagent to react. Finally, 1.5 mL of 0.1% (V/V) acetic acid aqueous solution was added, filtered through a 0.45 μm microporous membrane, and analyzed by liquid chromatography. (The composition of the liquid primary screening medium is: 2.5% lactose, 0.4% ammonium sulfate, adjust the pH to 7.5 before disinfection, and the disinfection conditions are 121-123°C, 30min.)
挑选出DNJ含量最高的的菌株，经连续三轮划线分离纯化后进行保藏，保藏编号为HDCC00252。(该菌种之后于2021年06月25 日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.22781)。The strain with the highest DNJ content was selected and preserved after three consecutive rounds of streaking, isolation and purification, and the preservation number was HDCC00252. (The strain was then deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on June 25, 2021, with a preservation number of CGMCC NO.22781).
实施例5原始菌种发酵制备1-脱氧野尻霉素Example 5 Fermentative preparation of 1-deoxynojirimycin by original strain
(1)菌种复苏活化：取原始菌种HDCC00031，在室温下解冻，吸取0.1ml菌悬液接种到LB固体平板，涂布均匀，置于37℃培养箱内培养24h，得到活化复苏的菌苔。(1) Bacteria recovery and activation: Take the original strain HDCC00031, thaw it at room temperature, inoculate 0.1ml of the bacterial suspension onto the LB solid plate, spread it evenly, and culture it in a 37°C incubator for 24 hours to obtain activated and recovered bacteria. moss.
(2)液体种子制备：取活化复苏的菌苔，用接种环刮取一环，接种到盛有100ml液体种子培养基的500ml三角瓶中，包扎好置于37℃、220rpm的摇床上振荡培养16h，控制种子液OD值≥5.0。(2) Preparation of liquid seeds: Take the activated and revived bacterial lawn, scrape a loop with an inoculation loop, inoculate it into a 500ml triangular flask containing 100ml liquid seed medium, wrap it up and place it on a shaking table at 37°C and 220rpm for shaking culture 16h, control the OD value of the seed solution ≥ 5.0.
液体种子培养基组成为：葡萄糖1.5％，酵母浸出粉0.5％，蛋白胨1％，氯化钠1％。消毒前调pH至7.0，消毒条件为121～123℃、30min。The liquid seed culture medium consists of 1.5% glucose, 0.5% yeast extract powder, 1% peptone and 1% sodium chloride. Adjust the pH to 7.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
(3)发酵培养：取培养合格的液体种子，以4％(V/W)的比例接种到装有30ml优化液体发酵培养基的250ml三角瓶中，包扎好置于37℃、220rpm的摇床上振荡培养4天结束。(3) Fermentation culture: take the liquid seeds qualified for cultivation, inoculate them into a 250ml Erlenmeyer flask with 30ml optimized liquid fermentation medium at a ratio of 4% (V/W), wrap them up and place them on a shaker at 37°C and 220rpm The shaking culture was completed after 4 days.
液体发酵培养基配方组成为：葡萄糖0.4％，蔗糖2％，乳糖2％，酵母浸出粉3％，硫酸铵0.1％，硝酸钠0.05％，硫酸亚铁铵0.25％，磷酸氢二钾0.28％。消毒前调pH至8.0，消毒条件为121～123℃、30min。The formula of the liquid fermentation medium is as follows: 0.4% of glucose, 2% of sucrose, 2% of lactose, 3% of yeast extract powder, 0.1% of ammonium sulfate, 0.05% of sodium nitrate, 0.25% of ferrous ammonium sulfate, and 0.28% of dipotassium hydrogen phosphate. Adjust the pH to 8.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
(4)样品处理：发酵结束后，取发酵液2ml，10000rpm高速离心10min去除菌体，然后取上清液，加入等体积乙醇混匀后10000rpm高速离心10min。取上清液20μL置于1.5mL的离心管中，加入0.4mol /L的硼酸盐缓冲液(pH＝8.5)20μL及2mmol/L的FMOC-Cl的乙腈溶液40μL，振荡混匀，于25℃水浴中反应20min，加入1mol/L的甘氨酸20μL，静置5min，让多余的衍生化试剂反应完。最后加入1.5mL0.1％(V/V)的醋酸水溶液，经0.45μm微孔滤膜滤过后进行液相色谱分析。(4) Sample treatment: After the fermentation, take 2ml of the fermentation broth, and centrifuge at 10,000rpm for 10min to remove bacteria, then take the supernatant, add an equal volume of ethanol to mix, and then centrifuge at 10,000rpm for 10min. Take 20 μL of the supernatant and place it in a 1.5 mL centrifuge tube, add 20 μL of 0.4 mol/L borate buffer (pH=8.5) and 40 μL of 2 mmol/L FMOC-Cl in acetonitrile, shake and mix well, and place at 25 React in a water bath at ℃ for 20 minutes, add 20 μL of 1 mol/L glycine, and let stand for 5 minutes to allow the excess derivatization reagent to react. Finally, 1.5 mL of 0.1% (V/V) acetic acid aqueous solution was added, filtered through a 0.45 μm microporous membrane, and analyzed by liquid chromatography.
(5)液相分析方法：(5) Liquid phase analysis method:
色谱柱：Kromasil C18分析柱(4.6mm×250mm，5μm)Chromatographic column: Kromasil C18 analytical column (4.6mm×250mm, 5μm)
流动相：乙腈-0.1％冰醋酸(50:50，V/V)Mobile phase: acetonitrile-0.1% glacial acetic acid (50:50, V/V)
流速：1.0mL/minFlow rate: 1.0mL/min
柱温：30℃Column temperature: 30°C
检测条件：荧光检测器，激发波长254nm，发射波长322nmDetection conditions: fluorescence detector, excitation wavelength 254nm, emission wavelength 322nm
进样体积：10μLInjection volume: 10μL
(6)实验结果：(6) Experimental results:
以已知浓度DNJ对照品采用同样方法衍生处理的样品作为标准，根据标准浓度、峰面积、样品峰面积进行计算得到样品浓度，结果确认该实施方案所得发酵液中DNJ含量为0.12g/L。Using the sample derivatized in the same way as the known concentration DNJ reference substance as a standard, calculate the sample concentration according to the standard concentration, peak area, and sample peak area, and the result confirms that the DNJ content in the fermented liquid obtained by this embodiment is 0.12g/L.
实施例6 1-脱氧野尻霉素高效制备Example 6 High-efficiency preparation of 1-deoxynojirimycin
(1)菌种复苏活化：取菌种HDCC00252，在室温下解冻，吸取0.1ml菌悬液接种到LB固体平板，涂布均匀，置于37℃培养箱内培养24h，得到活化复苏的菌苔。(1) Bacterial recovery and activation: take the bacterial strain HDCC00252, thaw it at room temperature, inoculate 0.1ml of the bacterial suspension onto the LB solid plate, spread it evenly, and culture it in a 37°C incubator for 24 hours to obtain an activated and recovered bacterial lawn .
(2)液体种子制备：取活化复苏的菌苔，用接种环刮取一环，接种到盛有100ml液体种子培养基的500ml三角瓶中，包扎好置于 37℃、220rpm的摇床上振荡培养16h，控制种子液OD值≥5.0。(2) Preparation of liquid seeds: Take the activated and revived bacterial lawn, scrape a loop with an inoculation loop, inoculate it into a 500ml triangular flask containing 100ml liquid seed medium, wrap it up and place it on a shaking table at 37°C and 220rpm for shaking culture 16h, control the OD value of the seed solution ≥ 5.0.
液体种子培养基组成为：葡萄糖1.5％，酵母浸出粉0.5％，蛋白胨1％，氯化钠1％。消毒前调pH至7.0，消毒条件为121～123℃、30min。The liquid seed culture medium consists of 1.5% glucose, 0.5% yeast extract powder, 1% peptone and 1% sodium chloride. Adjust the pH to 7.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
(3)发酵培养：取培养合格的液体种子，以4％(V/W)的比例接种到装有30ml优化液体发酵培养基的250ml三角瓶中，包扎好置于37℃、220rpm的摇床上振荡培养4天结束。(3) Fermentation culture: take the liquid seeds qualified for cultivation, inoculate them into a 250ml Erlenmeyer flask with 30ml optimized liquid fermentation medium at a ratio of 4% (V/W), wrap them up and place them on a shaker at 37°C and 220rpm The shaking culture was completed after 4 days.
液体发酵培养基配方组成为：葡萄糖0.4％，蔗糖2％，乳糖2％，酵母浸出粉3％，硫酸铵0.1％，硝酸钠0.05％，硫酸亚铁铵0.25％，磷酸氢二钾0.28％。消毒前调pH至8.0，消毒条件为121～123℃、30min。The formula of the liquid fermentation medium is as follows: 0.4% of glucose, 2% of sucrose, 2% of lactose, 3% of yeast extract powder, 0.1% of ammonium sulfate, 0.05% of sodium nitrate, 0.25% of ferrous ammonium sulfate, and 0.28% of dipotassium hydrogen phosphate. Adjust the pH to 8.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
(4)样品处理：发酵结束后，取发酵液2ml，10000rpm高速离心10min去除菌体，然后取上清液，加入等体积乙醇混匀后10000rpm高速离心10min。取上清液20μL置于1.5mL的离心管中，加入0.4mol/L的硼酸盐缓冲液(pH＝8.5)20μL及2mmol/L的FMOC-Cl的乙腈溶液40μL，振荡混匀，于25℃水浴中反应20min，加入1mol/L的甘氨酸20μL，静置5min，让多余的衍生化试剂反应完。最后加入1.5mL0.1％(V/V)的醋酸水溶液，经0.45μm微孔滤膜滤过后进行液相色谱分析。(4) Sample treatment: After the fermentation, take 2ml of the fermentation broth, and centrifuge at 10,000rpm for 10min to remove bacteria, then take the supernatant, add an equal volume of ethanol to mix, and then centrifuge at 10,000rpm for 10min. Take 20 μL of the supernatant and place it in a 1.5 mL centrifuge tube, add 20 μL of 0.4 mol/L borate buffer (pH=8.5) and 40 μL of 2 mmol/L FMOC-Cl in acetonitrile solution, vortex and mix well, and place at 25 React in a water bath at ℃ for 20 minutes, add 20 μL of 1 mol/L glycine, and let stand for 5 minutes to allow the excess derivatization reagent to react. Finally, 1.5 mL of 0.1% (V/V) acetic acid aqueous solution was added, filtered through a 0.45 μm microporous membrane, and analyzed by liquid chromatography.
(5)液相分析方法：(5) Liquid phase analysis method:
色谱柱：Kromasil C18分析柱(4.6mm×250mm，5μm)Chromatographic column: Kromasil C18 analytical column (4.6mm×250mm, 5μm)
流动相：乙腈-0.1％冰醋酸(50:50，V/V)Mobile phase: acetonitrile-0.1% glacial acetic acid (50:50, V/V)
流速：1.0mL/minFlow rate: 1.0mL/min
柱温：30℃Column temperature: 30°C
检测条件：荧光检测器，激发波长254nm，发射波长322nmDetection conditions: fluorescence detector, excitation wavelength 254nm, emission wavelength 322nm
进样体积：10μLInjection volume: 10μL
(6)实验结果：(6) Experimental results:
以已知浓度DNJ对照品采用同样方法衍生处理的样品作为标准，根据标准浓度、峰面积、样品峰面积进行计算得到样品浓度，结果确认该实施方案所得发酵液中DNJ含量为5.31g/L，相对于原始菌株提升了44倍。Adopt the sample of known concentration DNJ reference substance to adopt the same method derivation process as standard, calculate and obtain sample concentration according to standard concentration, peak area, sample peak area, the result confirms that DNJ content is 5.31g/L in the fermented liquid obtained by this embodiment, Compared with the original strain, it has been improved by 44 times.
为了评估该菌种遗传稳定性，采用相同的条件，连续进行4轮分离菌落鉴定，每一代的摇瓶发酵水平汇总如下表6所示。结果表明该菌种连续传代4代仍遗传稳定。In order to evaluate the genetic stability of the strain, the same conditions were used to carry out 4 consecutive rounds of isolated colony identification, and the shake flask fermentation level of each generation is summarized in Table 6 below. The results showed that the strain was still genetically stable after 4 consecutive generations.
表6CGMCC NO.22781菌株遗传稳定性Table 6 CGMCC NO.22781 strain genetic stability
代时Times F1F1 F2F2 F3F3 F4F4
鉴定效价identification titer 5.12g/L5.12g/L 5.29g/L5.29g/L 5.08g/L5.08g/L 5.20g/L5.20g/L
实施例7 1-脱氧野尻霉素发酵制备Example 7 1-deoxynojirimycin fermentation preparation
(1)菌种复苏活化：取菌种HDCC00252，在室温下解冻，吸取0.1ml菌悬液接种到LB固体平板，涂布均匀，置于37℃培养箱内培养24h，得到活化复苏的菌苔。(1) Bacterial recovery and activation: take the bacterial strain HDCC00252, thaw it at room temperature, inoculate 0.1ml of the bacterial suspension onto the LB solid plate, spread it evenly, and culture it in a 37°C incubator for 24 hours to obtain an activated and recovered bacterial lawn .
(2)液体种子制备：取活化复苏的菌苔，用接种环刮取一环，接种到盛有100ml液体种子培养基的500ml三角瓶中，包扎好置于28℃、220rpm的摇床上振荡培养24h，控制种子液OD值≥5.0。(2) Preparation of liquid seeds: Take the activated and revived bacterial lawn, scrape a ring with an inoculation loop, inoculate it into a 500ml triangular flask containing 100ml liquid seed medium, wrap it up and place it on a shaking table at 28°C and 220rpm for shaking culture 24h, control the seed solution OD value ≥ 5.0.
液体种子培养基组成为：葡萄糖4％，酵母浸出粉1％，蛋白胨4％，氯化钠2％。消毒前调pH至5.0，消毒条件为121～123℃、30min。The liquid seed medium consists of 4% glucose, 1% yeast extract powder, 4% peptone and 2% sodium chloride. Adjust the pH to 5.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
(3)发酵培养：取培养合格的液体种子，以4％(V/W)的比例接种到装有30ml优化液体发酵培养基的250ml三角瓶中，包扎好置于28℃、220rpm的摇床上振荡培养4天结束。(3) Fermentation culture: take the liquid seeds qualified for cultivation, inoculate them into a 250ml Erlenmeyer flask with 30ml optimized liquid fermentation medium at a ratio of 4% (V/W), wrap them up and place them on a shaker at 28°C and 220rpm The shaking culture was completed after 4 days.
液体发酵培养基配方组成为：葡萄糖0.1％，蔗糖0.5％，乳糖10％，酵母浸出粉5％，硫酸铵4％，硫酸亚铁铵0.1％，磷酸氢二钾0.6％。消毒前调pH至9.0，消毒条件为121～123℃、30min。The liquid fermentation medium formula consists of: 0.1% of glucose, 0.5% of sucrose, 10% of lactose, 5% of yeast extract powder, 4% of ammonium sulfate, 0.1% of ferrous ammonium sulfate, and 0.6% of dipotassium hydrogen phosphate. Adjust the pH to 9.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
(4)样品处理：发酵结束后，取发酵液2ml，10000rpm高速离心10min去除菌体，然后取上清液，加入等体积乙醇混匀后10000rpm高速离心10min。取上清液20μL置于1.5mL的离心管中，加入0.4mol/L的硼酸盐缓冲液(pH＝8.5)20μL及2mmol/L的FMOC-Cl的乙腈溶液40μL，振荡混匀，于25℃水浴中反应20min，加入1mol/L的甘氨酸20μL，静置5min，让多余的衍生化试剂反应完。最后加入1.5mL0.1％(V/V)的醋酸水溶液，经0.45μm微孔滤膜滤过后进行液相色谱分析。(4) Sample treatment: After the fermentation, take 2ml of the fermentation broth, and centrifuge at 10,000rpm for 10min to remove bacteria, then take the supernatant, add an equal volume of ethanol to mix, and then centrifuge at 10,000rpm for 10min. Take 20 μL of the supernatant and place it in a 1.5 mL centrifuge tube, add 20 μL of 0.4 mol/L borate buffer (pH=8.5) and 40 μL of 2 mmol/L FMOC-Cl in acetonitrile solution, vortex and mix well, and place at 25 React in a water bath at ℃ for 20 minutes, add 20 μL of 1 mol/L glycine, and let stand for 5 minutes to allow the excess derivatization reagent to react. Finally, 1.5 mL of 0.1% (V/V) acetic acid aqueous solution was added, filtered through a 0.45 μm microporous membrane, and analyzed by liquid chromatography.
(5)液相分析方法：(5) Liquid phase analysis method:
色谱柱：Kromasil C18分析柱(4.6mm×250mm，5μm)Chromatographic column: Kromasil C18 analytical column (4.6mm×250mm, 5μm)
流动相：乙腈-0.1％冰醋酸(50:50，V/V)Mobile phase: acetonitrile-0.1% glacial acetic acid (50:50, V/V)
流速：1.0mL/minFlow rate: 1.0mL/min
柱温：30℃Column temperature: 30°C
检测条件：荧光检测器，激发波长254nm，发射波长322nmDetection conditions: fluorescence detector, excitation wavelength 254nm, emission wavelength 322nm
进样体积：10μLInjection volume: 10μL
(6)实验结果：(6) Experimental results:
以已知浓度DNJ对照品采用同样方法衍生处理的样品作为标准，根据标准浓度、峰面积、样品峰面积进行计算得到样品浓度，结果确认该实施方案所得发酵液中DNJ含量为5.01g/L。Using the sample derivatized by the same method as the known concentration DNJ reference substance as a standard, calculate the sample concentration according to the standard concentration, peak area, and sample peak area, and the result confirms that the DNJ content in the fermented liquid obtained by this embodiment is 5.01g/L.
实施例8 1-脱氧野尻霉素发酵制备Example 8 1-deoxynojirimycin fermentation preparation
(1)菌种复苏活化：取菌种HDCC00252，在室温下解冻，吸取0.1ml菌悬液接种到LB固体平板，涂布均匀，置于37℃培养箱内培养24h，得到活化复苏的菌苔。(1) Bacterial recovery and activation: take the bacterial strain HDCC00252, thaw it at room temperature, inoculate 0.1ml of the bacterial suspension onto the LB solid plate, spread it evenly, and culture it in a 37°C incubator for 24 hours to obtain an activated and recovered bacterial lawn .
(2)液体种子制备：取活化复苏的菌苔，用接种环刮取一环，接种到盛有100ml液体种子培养基的500ml三角瓶中，包扎好置于40℃、220rpm的摇床上振荡培养4h。(2) Preparation of liquid seeds: Take the activated and revived bacterial lawn, scrape a ring with an inoculation loop, inoculate it into a 500ml triangular flask containing 100ml liquid seed medium, wrap it up and place it on a shaking table at 40°C and 220rpm for shaking culture 4h.
液体种子培养基组成为：葡萄糖0.2％，酵母浸出粉0.2％，蛋白胨0.2％，氯化钠0.2％。消毒前调pH至9.0，消毒条件为121～123℃、30min。The liquid seed medium consists of 0.2% glucose, 0.2% yeast extract powder, 0.2% peptone and 0.2% sodium chloride. Adjust the pH to 9.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
(3)发酵培养：取培养合格的液体种子，以4％(V/W)的比例接种到装有30ml优化液体发酵培养基的250ml三角瓶中，包扎好置于37℃、220rpm的摇床上振荡培养4天结束。(3) Fermentation culture: take the liquid seeds qualified for cultivation, inoculate them into a 250ml Erlenmeyer flask with 30ml optimized liquid fermentation medium at a ratio of 4% (V/W), wrap them up and place them on a shaker at 37°C and 220rpm The shaking culture was completed after 4 days.
液体发酵培养基配方组成为：葡萄糖2％，蔗糖4％，乳糖0.5％，酵母浸出粉2％，硝酸钠3％，硫酸亚铁铵0.8％，磷酸氢二钾0.2％。消毒前调pH至5.0，消毒条件为121～123℃、30min。The formula of the liquid fermentation medium is as follows: 2% of glucose, 4% of sucrose, 0.5% of lactose, 2% of yeast extract powder, 3% of sodium nitrate, 0.8% of ferrous ammonium sulfate, and 0.2% of dipotassium hydrogen phosphate. Adjust the pH to 5.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.
(4)样品处理：发酵结束后，取发酵液2ml，10000rpm高速离 心10min去除菌体，然后取上清液，加入等体积乙醇混匀后10000rpm高速离心10min。取上清液20μL置于1.5mL的离心管中，加入0.4mol/L的硼酸盐缓冲液(pH＝8.5)20μL及2mmol/L的FMOC-Cl的乙腈溶液40μL，振荡混匀，于25℃水浴中反应20min，加入1mol/L的甘氨酸20μL，静置5min，让多余的衍生化试剂反应完。最后加入1.5mL0.1％(V/V)的醋酸水溶液，经0.45μm微孔滤膜滤过后进行液相色谱分析。(4) Sample treatment: After the fermentation, take 2ml of fermentation broth, and centrifuge at 10,000rpm for 10min to remove bacteria, then take the supernatant, add an equal volume of ethanol to mix, and then centrifuge at 10,000rpm for 10min. Take 20 μL of the supernatant and place it in a 1.5 mL centrifuge tube, add 20 μL of 0.4 mol/L borate buffer (pH=8.5) and 40 μL of 2 mmol/L FMOC-Cl in acetonitrile solution, vortex and mix well, and place at 25 React in a water bath at ℃ for 20 minutes, add 20 μL of 1 mol/L glycine, and let stand for 5 minutes to allow the excess derivatization reagent to react. Finally, 1.5 mL of 0.1% (V/V) acetic acid aqueous solution was added, filtered through a 0.45 μm microporous membrane, and analyzed by liquid chromatography.
(5)液相分析方法：(5) Liquid phase analysis method:
色谱柱：Kromasil C18分析柱(4.6mm×250mm，5μm)Chromatographic column: Kromasil C18 analytical column (4.6mm×250mm, 5μm)
流动相：乙腈-0.1％冰醋酸(50:50，V/V)Mobile phase: acetonitrile-0.1% glacial acetic acid (50:50, V/V)
流速：1.0mL/minFlow rate: 1.0mL/min
柱温：30℃Column temperature: 30°C
检测条件：荧光检测器，激发波长254nm，发射波长322nmDetection conditions: fluorescence detector, excitation wavelength 254nm, emission wavelength 322nm
进样体积：10μLInjection volume: 10μL
(6)实验结果：(6) Experimental results:
以已知浓度DNJ对照品采用同样方法衍生处理的样品作为标准，根据标准浓度、峰面积、样品峰面积进行计算得到样品浓度，结果确认该实施方案所得发酵液中DNJ含量为4.98g/L。Using the sample derivatized by the same method as the known concentration DNJ reference substance as a standard, calculate the sample concentration according to the standard concentration, peak area, and sample peak area, and the result confirms that the DNJ content in the fermented liquid obtained by this embodiment is 4.98g/L.

Claims (10)

  1. 一种解淀粉芽孢杆菌(Bacillus amyloliquefaciens)HDCC00252，保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号为CGMCC NO.22781，保藏日期为2021年06月25日。A Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HDCC00252, preserved in the General Microorganism Center (CGMCC) of China Microbiological Culture Collection Management Committee (CGMCC), the preservation number is CGMCC NO.22781, and the preservation date is June 25, 2021.
  2. 一种含权利要求1所述的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)的发酵液。A fermented liquid containing the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) described in claim 1.
  3. 根据权利要求1所述的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)或其发酵液在制备1-脱氧野尻霉素中的应用。The application of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) or its fermented liquid according to claim 1 in the preparation of 1-deoxynojirimycin.
  4. 一种1-脱氧野尻霉素的制备方法，其特征在于：采用权利要求1所述的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)进行发酵制备。A preparation method of 1-deoxynojirimycin, characterized in that: the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) described in claim 1 is used for fermentation preparation.
  5. 根据权利要求4所述的制备方法，其特征在于：所述的发酵过程包括在含有可同化的碳源和/或氮源的发酵培养基里，进行有氧发酵。The preparation method according to claim 4, characterized in that: the fermentation process includes performing aerobic fermentation in a fermentation medium containing assimilable carbon source and/or nitrogen source.
  6. 如权利要求5所述的制备方法，其特征在于：所述的碳源选自葡萄糖、甘油、蔗糖、果糖、乳糖、麦芽糖、糊精、淀粉、甘露醇、山梨醇；优选为葡萄糖、蔗糖、乳糖或其任意几种的组合；The preparation method according to claim 5, characterized in that: the carbon source is selected from glucose, glycerol, sucrose, fructose, lactose, maltose, dextrin, starch, mannitol, sorbitol; preferably glucose, sucrose, Lactose or any combination thereof;
    和/或所述的氮源选自玉米浆(粉)、酵母膏、酵母浸出粉、酵母蛋白胨、大豆蛋白胨、牛骨蛋白胨、肉胨、鱼粉蛋白胨、硝酸盐、铵盐；优选为酵母浸出粉、硝酸盐、铵盐或其任意几种的组合。And/or the nitrogen source is selected from corn steep liquor (powder), yeast extract, yeast extract powder, yeast peptone, soybean peptone, bovine bone peptone, meat peptone, fish powder peptone, nitrate, ammonium salt; preferably yeast extract powder , nitrate, ammonium salt or any combination thereof.
  7. 根据权利要求5所述的制备方法，其特征在于：所述的发酵培养基还包括无机盐，优选为硫酸盐、磷酸盐、亚铁盐，更优选为硫酸铵、磷酸氢二钾、硫酸亚铁铵。The preparation method according to claim 5, characterized in that: the fermentation medium also includes inorganic salts, preferably sulfate, phosphate, ferrous salt, more preferably ammonium sulfate, dipotassium hydrogen phosphate, sulfurous acid ferric ammonium.
  8. 根据权利要求5所述的制备方法，其特征在于：所述的发酵培养基含有葡萄糖0.1～2％，蔗糖0.5～4％，乳糖0.5～10％，酵母浸出粉2～5％，硫酸铵0～4％，硝酸钠0～3％，硫酸亚铁铵0.1～0.8％，磷酸氢二钾0.2～0.6％，余量为水。The preparation method according to claim 5, characterized in that: the fermentation medium contains 0.1-2% of glucose, 0.5-4% of sucrose, 0.5-10% of lactose, 2-5% of yeast extract powder, and 0% ammonium sulfate. ~4%, sodium nitrate 0~3%, ferrous ammonium sulfate 0.1~0.8%, dipotassium hydrogen phosphate 0.2~0.6%, and the balance is water.
  9. 根据权利要求5所述的制备方法，其特征在于：所述发酵温度为28-40℃，所述培养基pH为5.0-9.0；培养时间为12-100小时。The preparation method according to claim 5, characterized in that: the fermentation temperature is 28-40°C, the pH of the culture medium is 5.0-9.0; the culture time is 12-100 hours.
  10. 根据权利要求5所述的制备方法，其特征在于：所述解淀粉芽孢杆菌是通过种子液接种至所述发酵培养基中进行发酵培养的；The preparation method according to claim 5, characterized in that: the Bacillus amyloliquefaciens is inoculated into the fermentation medium by seed liquid to carry out fermentation culture;
    其中，所述种子液是将解淀粉芽孢杆菌(Bacillus amyloliquefaciens)HDCC00252在种子培养基里进行种子培养得到的；Wherein, the seed liquid is obtained by culturing Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HDCC00252 in the seed medium;
    和/或所述种子培养的条件为：种子培养的温度为28-40℃，所述培养基pH为5.0-9.0；培养时间为4-24小时；And/or the conditions of the seed culture are: the temperature of the seed culture is 28-40°C, the pH of the medium is 5.0-9.0; the culture time is 4-24 hours;
    和/或所述的种子培养基含葡萄糖0.2～4％，酵母浸出粉0.2～1％，氯化钠0.2～2％，蛋白胨0.2～4％。And/or the seed medium contains 0.2-4% of glucose, 0.2-1% of yeast extract powder, 0.2-2% of sodium chloride and 0.2-4% of peptone.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20140047992A (en) * 2012-10-15 2014-04-23 한국식품연구원 Bacillus amyloliquefaciens 335n producing 1-deoxynojirimycin and method for producing fermented soybean having anti-diabetic activity using thereof
KR20140047993A (en) * 2012-10-15 2014-04-23 한국식품연구원 Bacillus amyloliquefaciens 26n producing 1-deoxynojirimycin and method for producing fermented soybean having anti-diabetic activity using thereof
CN108070548A (en) * 2018-02-07 2018-05-25 华中农业大学 One plant height produces the bacillus amyloliquefaciens engineering bacteria and fermentation process of 1-DNJ
CN112094789A (en) * 2020-11-10 2020-12-18 中国科学院天津工业生物技术研究所 Bacillus amyloliquefaciens and application thereof in preparation of 1-deoxynojirimycin
CN113637605A (en) * 2021-08-09 2021-11-12 浙江珲达生物科技有限公司 Bacillus amyloliquefaciens and application thereof in preparation of 1-deoxynojirimycin
CN113774002A (en) * 2021-11-02 2021-12-10 浙江珲达生物科技有限公司 Bacillus amyloliquefaciens culture medium and application thereof

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2907190A1 (en) * 1979-02-23 1980-08-28 Bayer Ag 1-DESOXYNOJIRIMYCIN PRODUCTION
JP5526416B2 (en) * 2010-09-14 2014-06-18 独立行政法人国際農林水産業研究センター Food having α-glucosidase inhibitory activity
KR101474301B1 (en) * 2012-10-15 2014-12-18 한국식품연구원 Bacillus amyloliquefaciens 140N producing 1-Deoxynojirimycin and method for producing fermented soybean having anti-diabetic activity using thereof
CN104593267B (en) * 2014-11-13 2017-07-25 浙江工业大学 Monascus purpureus and its application in 1 DNJ is prepared
CN110747156B (en) * 2019-11-16 2021-03-30 湖北大学 Application of knock-out bacillus amyloliquefaciens nanR gene in improvement of yield of 1-deoxynojirimycin

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20140047992A (en) * 2012-10-15 2014-04-23 한국식품연구원 Bacillus amyloliquefaciens 335n producing 1-deoxynojirimycin and method for producing fermented soybean having anti-diabetic activity using thereof
KR20140047993A (en) * 2012-10-15 2014-04-23 한국식품연구원 Bacillus amyloliquefaciens 26n producing 1-deoxynojirimycin and method for producing fermented soybean having anti-diabetic activity using thereof
CN108070548A (en) * 2018-02-07 2018-05-25 华中农业大学 One plant height produces the bacillus amyloliquefaciens engineering bacteria and fermentation process of 1-DNJ
CN112094789A (en) * 2020-11-10 2020-12-18 中国科学院天津工业生物技术研究所 Bacillus amyloliquefaciens and application thereof in preparation of 1-deoxynojirimycin
CN113637605A (en) * 2021-08-09 2021-11-12 浙江珲达生物科技有限公司 Bacillus amyloliquefaciens and application thereof in preparation of 1-deoxynojirimycin
CN113774002A (en) * 2021-11-02 2021-12-10 浙江珲达生物科技有限公司 Bacillus amyloliquefaciens culture medium and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BAI DOU, LONG LING; ZHU NAISHUO: "Mutation Breeding of a Isolated Bacillus subtilis var. niger for Producing the 1-Deoxynojirimycin", FUDAN XUEBAO - JOURNAL OF FUDAN UNIVERSITY (NATURAL SCIENCE), vol. 55, no. 1, 15 February 2016 (2016-02-15), pages 104 - 111, XP093035052, ISSN: 0427-7104, DOI: 10.15943/j.cnki.fdxb-jns.2016.01.014 *
CAI DONGBO; LIU MENGJIE; WEI XUETUAN; LI XINMIAO; WANG QIN; NOMURA CHRISTOPHER T.; CHEN SHOUWEN: "Use ofBacillus amyloliquefaciensHZ-12 for High-Level Production of the Blood Glucose Lowering Compound, 1-Deoxynojirimycin (DNJ), and Nutraceutical Enriched Soybeans via Fermentation", APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, HUMANA PRESS INC, NEW YORK, vol. 181, no. 3, 24 October 2016 (2016-10-24), New York , pages 1108 - 1122, XP036185389, ISSN: 0273-2289, DOI: 10.1007/s12010-016-2272-8 *

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