A biosynthetic method of dipeptidin B

Abstract

The present invention belongs to the field of biotechnology, and specifically discloses a biosynthesis method of dipeptidin B, which comprises the following steps: inoculating bovine bacillus (Paenibacillusbovis) CGMCCNo.8333 in a skim milk starch medium for fermentation. The method uses the method of bovine bacillus CGMCCNo.8333 fermenting a skim milk starch medium to complete the biosynthesis of dipeptidin B for the first time, discloses that bovine bacillus CGMCCNo.8333 has a new use of fermenting a skim milk starch medium to synthesize dipeptidin B, broadens the source of dipeptidin B, and has a simple composition and a natural source of the culture medium, so that the synthesized dipeptidin B has higher safety in use.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a biosynthetic method of dipeptidin B Background Art

[0002] Type 2 diabetes is a common endocrine and metabolic disorder that occurs frequently in the elderly. As my country's social structure continues to age, the incidence of type 2 diabetes is also increasing, which has brought a huge economic burden to families and society. Studies have shown that long-term or improper use of traditional hypoglycemic drugs can cause irreversible damage to the function of insulin β cells, which in turn makes it difficult to control blood sugar levels in patients. The British Diabetes Prospective Trial showed that once pancreatic β cell function fails, traditional drugs are difficult to control. Therefore, it is crucial whether hypoglycemic drugs can effectively lower blood sugar while reducing damage to or protecting pancreatic β cells.

[0003] In recent years, dipeptidyl peptidase-4 (DPP-4) has become a new target for the development of hypoglycemic drugs. DPP-4 inhibitors inhibit the activity of DPP-4 in the body, increase the concentration of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulin-releasing polypeptide (GIP) in the body, and then promote insulin secretion and inhibit glucagon secretion to play a hypoglycemic effect, and can improve the function of pancreatic α and β cells. Therefore, the use of DPP-4 inhibitors will not lead to side effects such as hypoglycemia caused by insulin or some hypoglycemic drugs.

[0004] At present, there are many DPP-4 inhibitors of natural origin, however, only a few of them are recognized worldwide. Diprotin A and B are two recognized DPP-4 inhibitors, but a review of reports related to Diprotin found that most of them are related to Diprotin A, while there are almost no studies involving Diprotin B, and even few articles reporting its natural synthesis method have been published. Therefore, finding a biosynthetic method for Diprotin B and completely solving the source problem of Diprotin B is the primary problem that technicians in this field need to solve. Summary of the invention

[0005] Based on the above technical problems, the present invention provides a biosynthetic method of dipeptidin B. The dipeptidin B (Diprotin B) is a known compound, and its molecular formula is C 16 H 29 N3O4.

[0006] Specifically, the biosynthesis method of dipeptidin B comprises the following steps: fermenting Paenibacillus bovis CGMCC No.8333 in a culture medium to obtain a fermentation liquid containing dipeptidin B.

[0007] Preferably, the method further comprises inoculating Paenibacillus bovis CGMCC No. 8333 into the culture medium.

[0008] Preferably, the culture medium is skim milk starch medium.

[0009] Preferably, the inoculum size of Bacillus bovis CGMCC No.8333 is 1.6 x 10 6 ~8x10 6 cfu / mL.

[0010] Preferably, the skim milk starch culture medium comprises skim milk powder, starch and water, wherein the skim milk powder accounts for 0.5-4.5% by mass of the skim milk starch culture medium, the starch accounts for 0.5-4.5% by mass of the skim milk starch culture medium, and the rest is water.

[0011] Preferably, the fermentation is a shaking culture with a shaking speed of 100 to 300 rpm.

[0012] Preferably, the fermentation temperature is 25°C to 35°C.

[0013] Preferably, the fermentation time is 3 to 14 days.

[0014] Furthermore, the content of dipeptidyl peptidase B in the fermentation broth is greater than 1 mg / mL when the fermentation is terminated.

[0015] The present invention also provides dipeptidyl peptidase B obtained by the above biosynthesis method.

[0016] The above technical solution uses the method of fermenting skim milk starch culture medium with Paenibacillus bovis CGMCC No. 8333 for the first time to complete the biosynthesis of dipeptidin B, and discloses that Paenibacillus bovis CGMCC No. 8333 has a new use of fermenting skim milk starch culture medium to synthesize dipeptidin B, which broadens the source of dipeptidin B. At the same time, the culture medium with simple composition and natural source makes the synthesized dipeptidin B safer to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the UHPLC-Q-TOF MS secondary mirror matching image of dipeptidin B. DETAILED DESCRIPTION

[0018] In order to more clearly explain the technical solution of the present invention, the technical solution of the present invention will be further explained below in conjunction with specific implementation methods:

[0019] In a specific embodiment, a method for biosynthesis of dipeptidin B is provided, comprising the following steps: inoculating Paenibacillus bovis CGMCC No.8333 into a skim milk starch medium for fermentation to obtain a fermentation broth containing dipeptidin B.

[0020] The preparation method in the above technical scheme uses Paenibacillus bovis CGMCC No. 8333 to ferment skim milk starch culture medium for the first time to complete the biosynthesis of dipeptidin B, discloses that Paenibacillus bovis CGMCC No. 8333 has a new use of fermenting skim milk starch culture medium to synthesize dipeptidin B, and broadens the source of dipeptidin B. At the same time, the culture medium with simple composition and natural source makes the synthesized dipeptidin B safer to use.

[0021] In addition, the fermentation medium used in biosynthesis is widely available, low-cost, and naturally safe, avoiding the use of chemical synthetic medium; plus the fermentation bacteria use a single strain of bovine Bacillus. The combination of the above raw materials and strains is conducive to the standardization of the synthesis process and cost control of industrial large-scale production.

[0022] Preferably, the inoculum size of Bacillus bovis CGMCC No.8333 is 1.6 x 10 6 ~8x10 6 cfu / mL; preferably 3.2x10 6 ~6.4x10 6 cfu / mL, preferably 4.8x10 6 cfu / mL.

[0023] Preferably, the skim milk starch culture medium comprises skim milk powder, starch and water, wherein the skim milk powder accounts for 0.5-4.5% by mass of the skim milk starch culture medium, the starch accounts for 0.5-4.5% by mass of the skim milk starch culture medium, and the rest is water. The preparation method may comprise the following steps: adding skim milk powder and starch to distilled water, mixing well, sterilizing at 95-125° C. for 5-20 minutes, and cooling to obtain the product.

[0024] Preferably, the fermentation method is shaking culture, and the shaking speed is 100-300 rpm; more preferably 150-250 rpm; more preferably 200 rpm.

[0025] Preferably, the fermentation temperature is 25°C to 35°C; more preferably 28°C to 32°C; more preferably 30°C.

[0026] Preferably, the fermentation time is 3 to 14 days; more preferably 5 to 10 days; more preferably 7 days.

[0027] It can also be seen from Comparative Example 1 that when the preferred fermentation parameters are outside the range, the efficiency of bovine Bacillus CGMCC No. 8333 in fermenting skim milk starch medium to synthesize dipeptidin B is significantly reduced. Within the preferred range, the inoculation amount, oscillation speed, fermentation temperature and time affect each other, making the bovine Bacillus CGMCC No. 8333 more capable of fermenting and synthesizing dipeptidin B.

[0028] Furthermore, the content of dipeptidyl peptidase B in the fermentation broth is greater than 1 mg / mL when the fermentation is terminated.

[0029] The above specific implementation methods are further illustrated by examples below, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples that do not specify specific conditions are selected according to conventional methods and conditions, or according to the product instructions. The reagents used in the examples, if not specified, are all analytically pure reagents purchased from Sinopharm Group. Other test instruments, reagents, and strains, if not specifically specified, can be purchased directly through commercial channels.

[0030] Example 1

[0031] 1. Materials and methods

[0032] (a) Preparation of seeds (fermentation strains): The lyophilized powder of Bacillus bovis CGMCC No. 8333 (for the source of the strain, please refer to the Chinese patent publication number CN 103740618A) was dissolved in a small amount of sterile distilled water, and a loop of the solid culture medium (purchased from OXOID Co., UK) was streaked with an inoculating loop, and aerobic culture was carried out at 30° C. for 48 h. A single colony was picked up with an inoculating loop and placed in 10 mL of TYC liquid culture medium (purchased from OXOID Co., UK). The colony was evenly dispersed in the liquid culture medium using a vortex oscillator, and cultured at 30° C. and 180 rpm for 24 h. The colony was taken out and inoculated into TYC liquid culture medium (purchased from OXOID Co., UK) at an inoculum size of 2% (v / v). Co., UK), after shaking culture at 30°C and 180 rpm for 24 h, the culture was centrifuged at 15,000 rpm for 10 min, the supernatant was discarded, the bacteria were washed twice with sterile distilled water, and then suspended with sterile distilled water of the original culture volume to obtain seeds for fermentation. The bacterial concentration of the seed liquid was 1.6 x 10 8 cfu / mL.

[0033] (b) Preparation of skim milk starch medium: 1% by mass of skim milk powder, 4% by mass of starch and distilled water were thoroughly mixed, sterilized at 125° C. for 5 min, and cooled to room temperature to obtain a skim milk starch medium of the required concentration.

[0034] (c) Structural identification of dipeptidin B in fermentation samples: ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometry (UHPLC-Q-TOF MS).

[0035] Preparation of the sample to be tested: Add pre-cooled methanol / acetonitrile / water solution (2:2:1, v / v) to an appropriate amount of fermentation sample, vortex mix, low-temperature ultrasonic for 30 minutes, stand at -20℃ for 10 minutes, centrifuge at 14,000g and 4℃ for 20 minutes, and vacuum dry the supernatant. For mass spectrometry analysis, add 100μL acetonitrile aqueous solution (acetonitrile: water = 1:1, v / v) for re-dissolution, vortex, centrifuge at 14,000g and 4℃ for 15 minutes, and take the supernatant to obtain the sample to be tested.

[0036] Chromatographic conditions: ultra-high performance liquid chromatography system Agilent 1290 Infinity LC (Agilent, USA), chromatographic column ACQUITY UPLC BEHAmide HILIC (Waters, USA), injection volume 2 μL, column temperature 25°C, flow rate 0.5 mL / min, mobile phase A is water + 25 mM ammonium acetate + 25 mM ammonia water, and B is acetonitrile. The gradient elution program is shown in Table 1:

[0037] Table 1 Liquid chromatography gradient elution program

[0038] Time (min) ％A ％B Flow rate (mL / min) 0.0 5％ 95％ 0.5 0.5 5％ 95％ 0.5 7.0 35％ 65％ 0.5 8.0 60％ 40％ 0.5 9.0 60％ 40％ 0.5 9.1 5％ 95％ 0.5 12.0 5％ 95％ 0.5

[0039] Mass spectrometry conditions: mass spectrometer AB Triple TOF 6600 (SCIEX, USA), detection mode electrospray ionization (ESI) positive and negative ion modes, ESI source setting parameters are as follows: nebulizer gas auxiliary heating gas 1 (Gas1): 60, auxiliary heating gas 2 (Gas2): 60, curtain gas (CUR): 30psi, ion source temperature: 600℃, spray voltage (ISVF) ±5500V (positive and negative modes), primary mass-to-charge ratio detection range: 60-1000Da, secondary product ion mass-to-charge ratio detection range: Range: 25-1000Da, primary mass spectrometry scanning accumulation time: 0.20s / spectra, secondary mass spectrometry scanning accumulation time: 0.05s / spectra, the secondary mass spectrum was acquired in data-dependent acquisition mode (IDA), and the peak intensity value screening mode was adopted, declustering voltage (DP): ±60V (positive and negative modes), collision energy: 35±15eV, IDA settings are as follows: dynamic exclusion of isotope ions range: 4Da, 10 fragment spectra were collected in each scan.

[0040] Data analysis: The raw data in Wiff format were converted into .mzXML format by ProteoWizard, then analyzed by XCMS software, and secondary mirror matching was performed with standard substances in the database to finally determine the metabolites.

[0041] (d) Quantitative detection of dipeptidyl peptidase B in fermentation samples: liquid chromatography tandem mass spectrometry (HPLC-MS / MS).

[0042] Preparation of the sample to be tested: centrifuge the fermentation broth (15,000 g, 15 min) to obtain the supernatant, add three times the volume of anhydrous ethanol to the supernatant, centrifuge again (15,000 g, 15 min), obtain the supernatant, rotary evaporate to remove ethanol, freeze-dry, and dissolve in deionized water with the original volume of the fermentation broth to obtain the sample to be tested.

[0043] Preparation of standard samples: Peptideprotin B was prepared with deionized water into 10 standard samples with concentration gradients of 1, 2, 5, 10, 20, 50, 100, 200, 500 and 1000 ppb.

[0044] Chromatographic conditions: Chromatographic column: Acquity uplc HSS T3 1.8 um 2.1×100mm, injection volume: 4ul, column temperature 35°C, flow rate 0.25mL / min, mobile phase: A is water + 0.1% formic acid, B is 0.1% acetonitrile, gradient elution program see Table 2.

[0045] Table 2 Liquid chromatography gradient elution program

[0046] Time (min) ％A ％B Flow rate (mL / min) 0.0 98.0 2.0 0.25 1.0 98.0 2.0 0.25 9.0 50.0 50.0 0.25 12.0 2.0 98.0 0.25 13.5 2.0 98.0 0.25 14.0 98.0 2.0 0.25 20.0 98.0 2.0 0.25

[0047] Mass spectrometry conditions: ionization mode: electrospray ionization (ESI); positive ion mode; scanning mode: Full-MS-ddMS 2; spray voltage: 3,500 V; temperature: 350°C; sheath gas flow rate: 30 psi; sweep gas flow rate: 0 psi; auxiliary gas flow rate: 10 psi.

[0048] Data analysis: The above standard samples of dipeptidin B were loaded onto the liquid chromatography-tandem mass spectrometry system in sequence, and a standard curve of the relationship between the chromatographic peak area and the concentration of the standard samples was obtained by fitting. Subsequently, the sample to be tested was loaded, and its concentration was calculated based on the chromatographic peak area of ​​dipeptidin B.

[0049] 2. Biosynthesis of dipeptidin B

[0050] The seeds of Paenibacillus bovis CGMCC No.8333 were aseptically inoculated at an inoculum amount of 3% (v / v) into a skim milk starch medium containing 1% (w / w) skim milk powder and 4% (w / w) starch, and cultured at 30°C and 200 rpm for 7 days to obtain a fermentation liquid containing dipeptidin B.

[0051] 3. Structural identification and quantitative determination of dipeptidyl peptidase B in fermentation samples

[0052] The fermentation samples were tested by the above-mentioned UHPLC-Q-TOF MS method and analyzed by XCMS software. It was found that the substance numbered M328T385 in the metabolite completely matched the mass spectrometry ion peak characteristic signal of the dipeptidin B standard in the database, so the substance was determined to be dipeptidin B. The results are as follows Figure 1 The fermentation sample was tested by the above HPLC-MS / MS method, and the content of dipeptidin B was determined to be 2.69 mg / mL.

[0053] Example 2

[0054] 1. Materials and methods

[0055] (a) Preparation of seeds (fermentation strains): same as in Example 1.

[0056] (b) Preparation of skim milk starch medium: 0.5% by mass of skim milk powder, 4.5% by mass of starch and distilled water were thoroughly mixed, sterilized at 95° C. for 20 min, and cooled to room temperature to obtain a skim milk starch medium of the required concentration.

[0057] (c) Structural identification of dipeptidase B in fermentation samples: Same as Example 1.

[0058] (d) Quantitative detection of dipeptidase B in fermentation samples: Same as Example 1.

[0059] 2. Biosynthesis of dipeptidin B

[0060] The seeds of Paenibacillus bovis CGMCC No.8333 were aseptically inoculated at an inoculum amount of 5% (v / v) into a skim milk starch medium containing 0.5% (w / w) skim milk powder and 4.5% (w / w) starch, and cultured at 32° C. and 300 rpm for 3 days to obtain a fermentation liquid containing dipeptidin B.

[0061] 3. Structural identification and quantitative determination of dipeptidyl peptidase B in fermentation samples

[0062] The fermentation sample was tested by the above UHPLC-Q-TOF MS method and analyzed by XCMS software, and the results were the same as those in Example 1. The fermentation sample was tested by the above HPLC-MS / MS method, and the content of dipeptidin B was determined to be 1.05 mg / mL.

[0063] Example 3

[0064] 1. Materials and methods

[0065] (a) Preparation of seeds (fermentation strains): same as in Example 1.

[0066] (b) Preparation of skim milk starch medium: 4.5% by mass of skim milk powder, 0.5% by mass of starch and distilled water were thoroughly mixed, sterilized at 100° C. for 15 min, and cooled to room temperature to obtain a skim milk starch medium of the required concentration.

[0067] (c) Structural identification of dipeptidase B in fermentation samples: Same as Example 1.

[0068] (d) Quantitative detection of dipeptidase B in fermentation samples: Same as Example 1.

[0069] 2. Biosynthesis of dipeptidin B

[0070] The seeds of Paenibacillus bovis CGMCC No.8333 were aseptically inoculated at an inoculum amount of 1% (v / v) into a skim milk starch medium containing 4.5% (w / w) skim milk powder and 0.5% (w / w) starch, and cultured at 28°C and 100 rpm for 14 days to obtain a fermentation liquid containing dipeptidin B.

[0071] 3. Structural identification and quantitative determination of dipeptidyl peptidase B in fermentation samples

[0072] The fermentation sample was tested by the above UHPLC-Q-TOF MS method and analyzed by XCMS software, and the results were the same as those in Example 1. The fermentation sample was tested by the above HPLC-MS / MS method, and the content of dipeptidin B was determined to be 1.93 mg / mL.

[0073] Example 4

[0074] 1. Materials and methods

[0075] (a) Preparation of seeds (fermentation strains): same as in Example 1.

[0076] (b) Preparation of skim milk starch medium: 2.5% by mass of skim milk powder, 2.5% by mass of starch and distilled water were thoroughly mixed, sterilized at 120° C. for 10 min, and cooled to room temperature to obtain a skim milk starch medium of the required concentration.

[0077] (c) Structural identification of dipeptidase B in fermentation samples: Same as Example 1.

[0078] (d) Quantitative detection of dipeptidase B in fermentation samples: Same as Example 1.

[0079] 2. Biosynthesis of dipeptidin B

[0080] The seeds of Paenibacillus bovis CGMCC No.8333 were aseptically inoculated at an inoculum amount of 2% (v / v) into a skim milk starch medium containing 2.5% (w / w) skim milk powder and 2.5% (w / w) starch, and cultured at 25°C and 250 rpm for 5 days to obtain a fermentation liquid containing dipeptidin B.

[0081] 3. Structural identification and quantitative determination of dipeptidyl peptidase B in fermentation samples

[0082] The fermentation sample was tested by the above UHPLC-Q-TOF MS method and analyzed by XCMS software, and the results were the same as those in Example 1. The fermentation sample was tested by the above HPLC-MS / MS method, and the content of dipeptidin B was determined to be 1.63 mg / mL.

[0083] Example 5

[0084] 1. Materials and methods

[0085] (a) Preparation of seeds (fermentation strains): same as in Example 1.

[0086] (b) Preparation of skim milk starch medium: 3% by mass of skim milk powder, 2% by mass of starch and distilled water were thoroughly mixed, sterilized at 110° C. for 12 min, and cooled to room temperature to obtain a skim milk starch medium of the required concentration.

[0087] (c) Structural identification of dipeptidase B in fermentation samples: Same as Example 1.

[0088] (d) Quantitative detection of dipeptidase B in fermentation samples: Same as Example 1.

[0089] 2. Biosynthesis of dipeptidin B

[0090] The seeds of Paenibacillus bovis CGMCC No.8333 were aseptically inoculated at an inoculum amount of 4% (v / v) into a skim milk starch medium containing 3% (w / w) skim milk powder and 2% (w / w) starch, and cultured at 35°C and 150 rpm for 10 days to obtain a fermentation liquid containing dipeptidin B.

[0091] 3. Structural identification and quantitative determination of dipeptidyl peptidase B in fermentation samples

[0092] The fermentation sample was tested by the above UHPLC-Q-TOF MS method and analyzed by XCMS software, and the results were the same as those in Example 1. The fermentation sample was tested by the above HPLC-MS / MS method, and the content of dipeptidin B was determined to be 1.88 mg / mL.

[0093] Comparative Example 1

[0094] The inoculation amount, fermentation temperature, fermentation time and fermentation oscillation speed in Example 1 were adjusted one by one to obtain the following group of fermentation broths prepared by different methods. The quantitative determination of dipeptidyl peptidase B was performed on the fermentation broths obtained in each group, and the results are shown in Table 3.

[0095] Table 3 The content of dipeptidyl peptidase B in the fermentation broth prepared by different methods

[0096]

[0097]

[0098] It can be concluded from the results shown in Table 3 that when the inoculation amount, fermentation temperature, fermentation time and fermentation oscillation speed in the biosynthesis method of dipeptidin B are adjusted outside the preferred range, bovine Bacillus CGMCC No. 8333 can still synthesize dipeptidin B, but its yield is significantly reduced.

[0099] Comparative Example 2

[0100] Referring to the method described in Example 1, the content of dipeptidyl peptidase B in the fermentation broth prepared from different culture media was compared. The specific operation was as follows:

[0101] 1. Materials and methods

[0102] (a) Preparation of seeds (fermentation strains): same as in Example 1.

[0103] (b) Preparation of skim milk starch medium: 0.5% by mass of skim milk powder, 4.5% by mass of starch and distilled water were thoroughly mixed, sterilized at 95° C. for 20 min, and cooled to room temperature to obtain a skim milk starch medium of the required concentration.

[0104] Preparation of skim milk culture medium: 5% by mass of skim milk powder is thoroughly mixed with distilled water, sterilized at 95°C for 20 minutes, and cooled to room temperature to obtain skim milk culture medium of the required concentration.

[0105] Preparation of starch culture medium: Mix 5% starch by mass with distilled water, sterilize at 95°C for 20 minutes, and cool to room temperature to obtain starch culture medium of required concentration.

[0106] (c) Structural identification of dipeptidase B in fermentation samples: Same as Example 1.

[0107] (d) Quantitative detection of dipeptidase B in fermentation samples: Same as Example 1.

[0108] 2. Biosynthesis of dipeptidin B

[0109] The seeds of Paenibacillus bovis CGMCC No.8333 were aseptically inoculated into the above three culture media at an inoculation rate of 3% (v / v), and cultured at 30°C and 200 rpm for 7 days to obtain a fermentation liquid containing dipeptidin B.

[0110] 3. Structural identification and quantitative determination of dipeptidyl peptidase B in fermentation samples

[0111] The above three fermentation samples were tested by the above UHPLC-Q-TOF MS method and analyzed by XCMS software, and the results were the same as those in Example 1. The fermentation samples were tested by the above HPLC-MS / MS method, and the results are shown in the following table.

[0112] Table 4 Content of dipeptidyl peptidase B in fermentation broth prepared from different culture media

[0113]

[0114] From the results shown in Table 4, it can be concluded that when Paenibacillus bovis CGMCC No.8333 was inoculated in three culture media with the same mass percentage, although Diprotin B could be fermented and synthesized, the Diprotin B yield of the strain in pure skim milk medium or starch medium was significantly reduced compared with the mixed medium of Example 1.

[0115] Comparative Example 3

[0116] The method described in Reference Example 1 was used to compare the content of dipeptidin B in fermentation broths prepared by Bacillus bovis CGMCC No.8333, Lactobacillus casei ATCC 393 (purchased from ATCC), Lactobacillus bulgaricus LB340 (provided by Danisco), and Streptococcus thermophilus ST-BODY-3 (provided by Chr. Hansen). The specific operation was as follows:

[0117] 1. Materials and methods

[0118] (a) Preparation of seeds (fermentation strains):

[0119] Preparation of bovine Bacillus CGMCC No.8333 seeds: same as Example 1.

[0120] Preparation of Lactobacillus casei and Lactobacillus bulgaricus seeds: the lyophilized powders of Lactobacillus casei ATCC 393 and Lactobacillus bulgaricus LB340 were dissolved with a small amount of sterile distilled water respectively, a ring was streaked on MRS solid medium (purchased from Merck Co. Germany) with an inoculating loop respectively, and taken out after 37 ℃ of anaerobic culture for 24 hours, and a single bacterium colony was picked up with an inoculating loop and put into 1mMRS liquid (purchased from Merck Co. Germany), and a vortex oscillator was used to evenly disperse the bacterium colony in the liquid medium, and taken out after 37 ℃ of anaerobic culture for 24 hours, and inoculated into 50mMRS liquid with 2% (v / v) inoculum size, and after 37 ℃ of cultivation for 24 hours, the culture was centrifuged at 9,000rpm for 10 minutes, and the supernatant was discarded. After the thalline was washed twice with sterile distilled water, the sterile distilled water suspension of the original culture volume was used to obtain the corresponding fermentation seeds.

[0121] Preparation of thermophilic Streptococcus seeds: The freeze-dried powder of thermophilic Streptococcus ST-BODY-3 was dissolved in a small amount of sterile distilled water, and a ring was taken with an inoculating loop and streaked on M17 solid culture medium (purchased from Merck Co., Germany), and anaerobically cultured at 40°C for 24 hours, and a single colony was picked up with an inoculating loop and placed in 1 mL of M17 liquid (purchased from Merck Co., Germany). The colony was evenly dispersed in the liquid culture medium using a vortex oscillator, and anaerobically cultured at 40°C for 24 hours, and the colony was taken out and inoculated in 50 mL of M17 liquid with an inoculum of 2% (v / v). After cultured at 40°C for 24 hours, the culture was centrifuged at 9,000 rpm for 10 minutes, the supernatant was discarded, the bacteria were washed twice with sterile distilled water, and then suspended with sterile distilled water of the original culture volume to obtain seeds for fermentation.

[0122] (b) Preparation of skim milk starch culture medium: same as in Example 1.

[0123] (c) Quantitative detection of dipeptidase B in fermentation samples: Same as Example 1.

[0124] 2. Fermentation of skim milk starch medium

[0125] Each strain was aseptically inoculated at an inoculum size of 3% (v / v) into a skim milk starch medium containing 1% (w / w) skim milk powder and 4% (w / w) starch, and cultured (anaerobic culture at 37°C for Lactobacillus bulgaricus and Lactobacillus casei, anaerobic culture at 40°C for Streptococcus thermophilus, and shaking culture at 30°C, 200rpm for Paenibacillus bovis) for 7 h to obtain the corresponding fermentation broth.

[0126] 3. Quantitative determination of dipeptidyl peptidase B in fermentation samples

[0127] The fermentation samples were tested by the above HPLC-MS / MS method, and the results are shown in Table 5:

[0128] Table 5 The content of dipeptidyl peptidase B in the fermentation broth prepared by different strains

[0129]

[0130] As shown in Table 5, except for Paenibacillus bovis CGMCC No. 8333, no dipeptidin B was detected in the fermentation broth of other conventional fermentation strains, indicating that other conventional fermentation strains do not have the ability to produce dipeptidin B by fermenting skim milk starch medium.

[0131] The above is a detailed introduction to the biosynthesis method of dipeptidin B provided by the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the present invention, and the description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, the present invention can also be improved and modified in several ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A biosynthetic method of dipeptidin B, characterized in that: The following steps are involved: Fermenting Paenibacillus bovis CGMCC No.8333 in a culture medium to obtain a fermentation liquid containing dipeptidin B; The culture medium is a skim milk starch culture medium, which includes skim milk powder, starch and water. The skim milk powder accounts for 0.5-4.5% of the skim milk starch culture medium by mass, the starch accounts for 0.5-4.5% of the skim milk starch culture medium by mass, and the rest is water.

2. The biosynthetic method of dipeptidase B according to claim 1, characterized in that: The method further comprises inoculating the bovine Bacillus CGMCC No.8333 into the culture medium at an inoculation amount of 1.6×10 6 ~8×10 6 cfu / mL.

3. The biosynthetic method of dipeptidase B according to claim 1, characterized in that: The fermentation is a shaking culture with a shaking speed of 100 to 300 rpm.

4. The biosynthetic method of dipeptidase B according to claim 1, characterized in that: The fermentation temperature is 25°C to 35°C.

5. The biosynthetic method of dipeptidase B according to claim 1, characterized in that: The fermentation time is 3 to 14 days.

6. The biosynthetic method of dipeptidase B according to claim 1, characterized in that: When the fermentation is terminated, the content of dipeptidyl peptidase B in the fermentation broth is greater than 1 mg / mL.

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