Process for the synthesis of adenosine-containing compositions by microbial fermentation and adenosine-containing compositions

Abstract

This invention provides a method for synthesizing adenosine-containing compositions via microbial fermentation, as well as the adenosine-containing compositions. By introducing a trace amount of Bacillus subtilis, which is capable of producing subtilis lipopeptides, into the later stages of fermentation, which is prone to problems such as low dissolved oxygen and low glycoside production, the subtilis lipopeptides produced by Bacillus subtilis act as a super biosurfactant, continuously emulsifying the culture medium and significantly reducing the surface tension of the culture medium. This prevents the crystallization of high concentrations of adenosine in the culture medium and increases its dissolved oxygen level, thus greatly improving the yield of adenosine. Furthermore, the resulting compositions can be directly used as cosmetic raw materials.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation synthesis technology, and specifically relates to a method for synthesizing adenosine-containing compositions by microbial fermentation and the adenosine-containing compositions. Background Technology

[0002] Adenosine, also known as adenosine nucleoside, is an endogenous purine nucleoside found throughout human cells and possessing important physiological functions. It has physiological effects on the cardiovascular system and other systems and tissues of the body. In cosmetics, adenosine is used for its anti-aging, firming, soothing, repairing, hair loss prevention, hair growth, moisturizing, and brightening effects. Therefore, it has received considerable attention in recent years, becoming a gentle yet highly effective active ingredient in anti-aging, repairing, and hair loss prevention cosmetics.

[0003] Currently, adenosine production methods are mainly divided into chemical synthesis and biosynthesis. Biosynthesis utilizes adenosine-producing bacteria, such as auxotrophic strains of Bacillus subtilis, as the production strain. After slant culture and seed tank expansion, the culture is inoculated into a fermenter for fermentation to produce a fermentation broth. The fermentation broth is then filtered through membrane filtration, concentration, crystallization, centrifugation, recrystallization, centrifugation, drying, and pulverization to obtain the final product. However, in the process of microbial fermentation synthesis of adenosine, especially when using medium or large fermenters, several challenges arise. First, Bacillus subtilis, being an aerobic bacterium, requires a high dissolved oxygen level to support efficient metabolism and adenosine synthesis. Second, the low solubility of adenosine leads to increased adenosine concentration and crystallization in the later stages of fermentation. The crystals encapsulate the bacterial cells, hindering mass transfer and oxygen supply, thus triggering a vicious cycle of low dissolved oxygen and low adenosine production. The aforementioned problems are not very obvious or even difficult to detect in the laboratory (using micro-fermenters of a few liters) or pilot-scale (using small fermenters of tens of liters), but become increasingly apparent in pilot-scale (using medium-sized fermenters of 100 to several hundred liters) or mass production (using large fermenters of 1000 liters or more), becoming one of the technical problems that urgently need to be solved in the process of industrialization. The inventors once tried to solve this problem by increasing the stirring rate in the later stage of fermentation, but it had almost no effect, and excessively high stirring rates would reduce dissolved oxygen efficiency and easily damage the cells. Summary of the Invention

[0004] In view of the above-mentioned problems existing in the prior art, the purpose of the present invention is to provide a method for synthesizing adenosine-containing compositions by microbial fermentation. In the later stage of fermentation, which is prone to problems such as low dissolved oxygen and low glycoside production, a trace amount of Bacillus subtilis, which can produce subtilis lipopeptides, is introduced. The subtilis lipopeptides produced by Bacillus subtilis act as a super biosurfactant, continuously emulsifying the culture medium and significantly reducing the surface tension of the culture medium. This prevents the crystallization of high concentrations of adenosine in the culture medium and increases its dissolved oxygen level, thus greatly improving the yield of adenosine. The resulting composition can be directly used as a cosmetic raw material.

[0005] To achieve the above objectives, in a first aspect of the present invention, a method for synthesizing an adenosine-containing composition by microbial fermentation is provided, comprising the following steps:

[0006] S1: The first culture medium containing adenosine-producing Bacillus subtilis and a substrate suitable for the growth of the adenosine-producing Bacillus subtilis is introduced into the fermenter and fermented at the first fermentation temperature. During fermentation, sterile oxygen or sterile air is introduced into the fermenter, the first culture medium is stirred and / or shaken, and the viscosity of the first culture medium is monitored in real time.

[0007] S2: When the viscosity of the first culture medium reaches the first preset value, a second culture medium containing Bacillus subtilis lipopeptide and a substrate suitable for the growth of Bacillus subtilis lipopeptide is introduced into the first culture medium to form a mixed culture medium. Fermentation is carried out at the second fermentation temperature (during fermentation, sterile oxygen or sterile air is continuously introduced into the fermenter, the mixed culture medium is continuously stirred and / or shaken, and the viscosity of the mixed culture medium is monitored in real time).

[0008] S3: When the viscosity of the mixed culture medium reaches the second preset value, stop the fermentation, adjust the pH of the mixed culture medium to approximately neutral, and obtain the crude product containing the adenosine composition.

[0009] Furthermore, the adenosine-producing Bacillus subtilis in S1 is selected from one or more of the following Bacillus subtilis preservation numbers: CGMCC No. 16753 (strain number ACA301), CGMCC No. 1304 (strain number JSIM-1025), CGMCC No. 4484 (strain number A409), CGMCC No. 23448 (strain number HDCC112-21027), CGMCC No. 0733 (strain number HW9801), or their mutant strains.

[0010] Furthermore, the substrates suitable for the growth of the adenosine-producing Bacillus subtilis in S1 include carbon sources, nitrogen sources, and metal salts. The carbon source is selected from one or more of glucose, glycerol, sucrose, and corn steep liquor; the nitrogen source is selected from one or more of peptone, beef extract, fish meal, corn steep liquor, yeast extract, monosodium glutamate, urea, and ammonium salts; the metal salts include potassium salts, sodium salts, magnesium salts, manganese salts, and ferrous salts; wherein, the ammonium salt is selected from one or two of (NH4)2SO4 and NH4Cl; the potassium salt is selected from one or more of KH2PO4, K2HPO4, K2SO4, and KCl; the sodium salt is selected from one or more of Na2SO4, NaCl, and NaNO3; the magnesium salt is selected from one or more of MgSO4, MgCl2, and Mg(NO3)2; the manganese salt is selected from one or more of MnSO4, MnCl2, and Mn(NO3)2; and the ferrous salt is selected from one or more of FeSO4, FeCl2, and Fe(NO3)2.

[0011] Furthermore, the first fermentation temperature in S1 is 28-38℃, the initial dissolved oxygen in the first culture medium is greater than or equal to 10%, preferably greater than or equal to 20%, and the tank pressure of the fermenter is 0.02~0.06MPa (gauge pressure relative to atmospheric pressure).

[0012] Furthermore, the oxygen content in the sterile air in S1 is approximately 19.5-21%; the oxygen content in the sterile oxygen is higher than 21%, for example, 30%, 40%, 50% or even higher. Bacillus subtilis, as an aerobic bacterium, requires a high dissolved oxygen level in its culture medium, which is related to factors such as temperature, tank pressure, stirring, and aeration. However, the inventors discovered that in the microbial fermentation system for adenosine synthesis of this invention, when the fermenter is increased to 100 liters or more, the dissolved oxygen level in the later stages of fermentation significantly decreases, significantly affecting the adenosine yield. This problem was not observed in the laboratory (using micro-fermenters of a few liters) and the pilot-scale test (using small fermenters of tens of liters). In addition, the inventors also found that simply increasing the oxygen content in the sterile oxygen or increasing the flow rate of the sterile air in the later stages of fermentation is insufficient to maintain a high dissolved oxygen level.

[0013] Furthermore, in S1, the stirring of the first culture medium is achieved by mechanical stirring (e.g., paddle, turbine, propeller, anchor, frame, ribbon stirring) and / or airflow stirring (e.g., bubbling).

[0014] Furthermore, the shaking of the first culture medium in S1 is achieved by a reciprocating shaker or a rotary shaker.

[0015] Furthermore, in S1, the viscosity of the first culture medium is monitored in real time using an online rotational viscometer, an online vibrational viscometer, an online ultrasonic viscometer, an online optical viscometer, or an online rheological viscometer.

[0016] Furthermore, the first preset value in S2 is 10-13 mPa·s, preferably 11-12 mPa·s. The inventors discovered that in the microbial fermentation system for adenosine synthesis of the present invention, when the viscosity of the culture medium reaches 10-11 mPa·s, the dissolved oxygen level begins to decrease significantly, and even adjusting factors such as temperature, tank pressure, stirring, and aeration has no significant effect. When the viscosity of the culture medium reaches 12-13 mPa·s, the growth of the strain and glycoside production essentially cease, because the OD value of the culture medium peaks and the residual sugar hardly decreases further.

[0017] Furthermore, the lipopeptide-producing Bacillus subtilis in S2 is selected from one or more of the following Bacillus subtilis preservation numbers: CGMCC No. 2947 (strain number BIT09S1), CGMCC No. 14375 (strain number LPB-3), CGMCC No. 8925 (strain number Z-5), CGMCC No. 22013 (strain number 50499), CGMCC No. 28191 (strain number ME11), CGMCC No. 34998 (strain number 2KR-18.1.13-26), or their mutant strains.

[0018] Further, the substrates suitable for the growth of the lipopeptide-producing Bacillus subtilis in S2 include a carbon source, a nitrogen source, and a metal salt. The carbon source is selected from one or more of glucose, glycerol, sucrose, and corn steep liquor; the nitrogen source is selected from one or more of peptone, beef extract, fish meal, corn steep liquor, yeast extract, monosodium glutamate, urea, and ammonium salts; the metal salts include potassium salts, sodium salts, calcium salts, magnesium salts, manganese salts, ferrous salts, and copper salts; wherein the ammonium salt is selected from one or two of (NH4)2SO4 and NH4Cl; the potassium salt is selected from one or more of KH2PO4, K2HPO4, K2SO4, and KCl; and the sodium salt is selected from Na2SO4, N... One or more of aCl and NaNO3; the calcium salt is selected from one or two of CaCl2 and Ca(NO3)2; the magnesium salt is selected from one or more of MgSO4, MgCl2, and Mg(NO3)2; the manganese salt is selected from one or more of MnSO4, MnCl2, and Mn(NO3)2; the ferrous salt is selected from one or more of FeSO4, FeCl2, and Fe(NO3)2; the copper salt is selected from one or more of CuSO4, CuCl2, and Cu(NO3)2. The substrates and fermentation conditions required for the growth of lipopeptide-producing Bacillus subtilis and adenosine-producing Bacillus subtilis are similar, and the substrates required for their growth are basically interchangeable.

[0019] Furthermore, when the second culture medium was introduced into the fermenter in S2, the ratio of CFU to viable cells of *Bacillus subtilis* producing lipopeptide in the second culture medium to the ratio of CFU to viable cells of *Bacillus subtilis* producing adenosine-producing in the first culture medium in the fermenter was 0.003-0.008%. It is important to note that the introduction of *Bacillus subtilis* producing lipopeptide significantly inhibited the growth of *Bacillus subtilis* producing adenosine-producing. Although both require similar substrates and fermentation conditions, they cannot co-grow for extended periods. However, the inventors also discovered that if only a trace amount (within the above range) of *Bacillus subtilis* producing lipopeptide was introduced in the later stages of fermentation, its inhibitory effect on the growth of *Bacillus subtilis* producing adenosine-producing was negligible. However, the subtilis lipopeptides produced by *Bacillus subtilis*, as a super-strong biosurfactant, could continuously emulsify the culture medium and significantly reduce its surface tension, thereby preventing high concentrations of adenosine crystallization and increasing dissolved oxygen levels. Therefore, this greatly improved the adenosine yield of *Bacillus subtilis* producing adenosine.

[0020] Furthermore, the second fermentation temperature in S2 is 36-38℃, preferably 37℃.

[0021] Furthermore, the second preset value in S3 is 15-18 mPa·s, preferably 16-17 mPa·s. The inventors discovered that in the microbial fermentation method for synthesizing adenosine according to the present invention, when the viscosity of the mixed culture medium reaches 17-18 mPa·s, the growth of the strain and glycoside production essentially cease, which is equivalent to extending the time for strain growth and glycoside production, thus significantly improving the yield of adenosine.

[0022] Furthermore, in S3, the pH of the mixed culture medium is adjusted to approximately neutral by adding a dilute sodium hydroxide solution (0.1-0.5M, used when the pH is too low) and / or a dilute hydrochloric acid solution (0.1-0.5M, used when the pH is too high). Approximately neutral means that the pH of the mixed culture medium is 6.8-7.2 after stirring evenly.

[0023] Furthermore, the above-mentioned method for synthesizing adenosine-containing compositions by microbial fermentation also includes the following steps:

[0024] S4: The crude product containing adenosine obtained in S3 is purified to obtain the refined product containing adenosine.

[0025] Furthermore, the purification operation in S4 sequentially includes membrane filtration, concentration, crystallization, centrifugation, recrystallization, and centrifugation. This purification operation is a mature existing technology, and the number of recrystallization and centrifugation steps can be increased or decreased according to the actual requirements of the product. It should be noted that after the above purification operation, the purified product containing adenosine is in the form of a paste, which contains a certain amount of surfactin sodium. Surfactin sodium is also a safe and mild cosmetic raw material. On the one hand, it is a super surfactant with high stability and efficient emulsification; on the other hand, it also has broad-spectrum antibacterial and multi-effect skin care (soothing and anti-inflammatory, oil control and anti-acne, barrier repair, antioxidant, and promoting the penetration and absorption of other functional ingredients). Therefore, the purified product containing adenosine does not need to purify and separate the surfactin sodium and can be used directly as a cosmetic or cosmetic raw material.

[0026] Furthermore, the above purification process includes membrane filtration, concentration, crystallization, centrifugation, recrystallization, and centrifugation, followed by drying and pulverization. After drying and pulverization, a mixture of granular adenosine and a small amount of sodium subtilisin is obtained, which can be sold as a cosmetic ingredient.

[0027] In a second aspect of the invention, an adenosine-containing composition is provided, comprising a crude product of an adenosine-containing composition or a refined product of an adenosine-containing composition obtained by the above-described method of synthesizing an adenosine-containing composition by microbial fermentation.

[0028] Furthermore, in the purified product containing the adenosine composition, the adenosine content is greater than 99% by weight, more preferably greater than 99.5%; and the content of sodium subtilis lipopeptide is 0.01%-0.5%, more preferably 0.1%-0.5%.

[0029] The beneficial technical effects of the present invention are manifested in at least the following aspects:

[0030] (1) The present invention introduces lipopeptide-producing Bacillus subtilis in a timely and appropriate manner only in the later stage of fermentation. This can avoid the significant inhibitory effect of Bacillus subtilis on the growth and production of adenosine-producing Bacillus subtilis. At the same time, the Bacillus subtilis lipopeptide produced by Bacillus subtilis continuously emulsifies the culture medium and significantly reduces the surface tension of the culture medium, thereby preventing the high concentration of adenosine crystallization in the culture medium and increasing its dissolved oxygen level. Therefore, it greatly improves the yield of adenosine produced by adenosine-producing Bacillus subtilis in the later stage of fermentation.

[0031] (2) The refined product containing adenosine obtained in this invention contains a certain amount of sodium subtilis lipopeptide. Since sodium subtilis lipopeptide is also a safe and mild cosmetic raw material with broad-spectrum antibacterial and multi-effect skin care, there is no need to purify and separate the sodium subtilis lipopeptide in it. It can be used directly as a cosmetic or cosmetic raw material. Therefore, it does not increase the purification and separation cost of the product, but increases the efficacy of the product as a cosmetic or cosmetic raw material. Detailed Implementation

[0032] The embodiments of the present invention will be described in detail below. The embodiments described below are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the embodiments described below.

[0033] In the following preferred embodiments of the present invention, two different adenosine-producing Bacillus subtilis and two different lipopeptide-producing Bacillus subtilis were selected to implement and verify the method and effect of the microbial fermentation method for synthesizing adenosine-containing compositions of the present invention.

[0034] Example 1

[0035] In Example 1, the adenosine-producing Bacillus subtilis was Bacillus subtilis ACA301, with the strain preservation number CGMCC No. 16753, provided by Jiangsu Aochuang Biotechnology Co., Ltd.; the lipopeptide-producing Bacillus subtilis was Bacillus subtilis 2KR-18.1.13-26, with the strain preservation number CGMCC No. 34998, provided by Shanghai Baiyindi Biotechnology Co., Ltd.

[0036] The preparatory work for the microbial fermentation synthesis in Example 1 is as follows:

[0037] (1) Activation of bacterial strains: Take the preserved Bacillus subtilis ACA301 and Bacillus subtilis 2KR-18.1.13-26 strains and inoculate them into sterile activation medium (purchased from Guangdong Huankai Microbiology) and incubate at 34℃ for 21 hours until fresh bacterial cells grow. These are the activated bacterial strains.

[0038] (2) Preparation of the first culture medium: The first fermentation medium was sterilized, cooled to 40°C, and the pH was adjusted to 7. The activated Bacillus subtilis ACA301 strain was inoculated into the seed first fermentation medium for pre-culture for 2-3 hours, which is the first culture medium. The strain culture conditions were: temperature 34°C, pH 7, dissolved oxygen 12%, and tank pressure 0.03 MPa.

[0039] The substrates in the first fermentation medium include: glucose 20 g / L, corn steep liquor 30 g / L, yeast powder 10 g / L, ammonium sulfate 2 g / L, dipotassium hydrogen phosphate 5 g / L, sodium chloride 5 g / L, magnesium sulfate 1.0 g / L, manganese chloride 0.1 g / L, and ferrous chloride 1.0 g / L.

[0040] (3) Preparation of the second culture medium: The second fermentation medium was sterilized, cooled to 40℃, and the pH was adjusted to 7. The activated Bacillus subtilis 2KR-18.1.13-26 strain was inoculated into the second fermentation medium for pre-culture for 1-2 hours, which is the second culture medium. The strain culture conditions were: temperature 37℃, pH 7, dissolved oxygen 12%, and tank pressure 0.03MPa.

[0041] The substrates in the second fermentation medium include: glucose 20 g / L, corn steep liquor 30 g / L, yeast powder 10 g / L, ammonium sulfate 2 g / L, dipotassium hydrogen phosphate 5 g / L, sodium chloride 5 g / L, calcium chloride 5 g / L, magnesium sulfate 1.0 g / L, manganese chloride 0.1 g / L, ferrous chloride 1.0 g / L, and copper chloride 0.1 g / L.

[0042] Both the first and second culture media are freshly prepared and pre-cultured before being introduced into the fermenter. The first culture media is prepared according to the actual usage, generally about 70-80% of the fermenter volume. The second culture media is prepared with a slight surplus. Before being introduced into the fermenter, the usage is calculated based on the number of CFU / live bacteria in the sampled first and second culture media, and then introduced into the fermenter.

[0043] The steps of the method for synthesizing adenosine-containing compositions by microbial fermentation in Example 1 are as follows:

[0044] S1: 150L of the first culture medium is introduced into a medium-sized fermenter with a capacity of 200L and fermented at the first fermentation temperature of 34℃. During fermentation, sterile air (initial dissolved oxygen 12%, tank pressure 0.06MPa) is introduced into the fermenter, the first culture medium is stirred, and the viscosity of the first culture medium is monitored in real time.

[0045] S2: When the viscosity of the first culture medium reaches the first preset value of 11 mPa·s, an appropriate amount of the second culture medium (the ratio of the number of live bacteria in the first culture medium and the second culture medium is 100000:5, that is, the percentage of the number of live bacteria in the second culture medium to the number of live bacteria in the mixed culture medium is about 0.005%) is introduced into the first culture medium in the fermenter to form a mixed culture medium. Fermentation is carried out at the second fermentation temperature of 37°C. During the fermentation, sterile air is continuously introduced into the fermenter, the mixed culture medium is continuously stirred, and the viscosity of the mixed culture medium is continuously monitored in real time.

[0046] The calculation method for the amount of the second culture medium introduced into the fermenter is as follows: Before introducing the second culture medium into the fermenter, samples are taken from both the second culture medium and the first culture medium in the fermenter at that time, and the CFU / viable cell counts are measured separately to calculate the amount of the second culture medium that should be introduced into the fermenter. For example, using SYTO9 / PI double staining + flow cytometry, the viable cell count in the culture medium can be measured in real time (about 10 minutes), and the ratio of viable cell counts is 100,000:(3~8), which is a very narrow range. If too much lipopeptide-producing Bacillus subtilis is added, on the one hand, it will inhibit the growth of adenosine-producing Bacillus subtilis, affecting the adenosine yield; on the other hand, too much lipopeptide produced will result in too low surface tension of the culture medium, excessive foaming, and a decrease in dissolved oxygen. If too little lipopeptide-producing Bacillus subtilis is added, due to the inhibitory effect of adenosine-producing Bacillus subtilis, it can hardly grow, and therefore cannot produce enough lipopeptides to reduce the surface tension of the culture medium, thereby improving the dissolved oxygen and adenosine crystallization problems.

[0047] S3: When the viscosity of the mixed culture medium reaches the second preset value of 16 mPa·s, stop the fermentation, adjust the pH of the mixed culture medium to 7.0, and obtain the crude product containing the adenosine composition.

[0048] S4: The crude product containing adenosine obtained in S3 is subjected to membrane filtration, concentration, crystallization, centrifugation, recrystallization, centrifugation, drying, and pulverization processes in sequence to obtain the refined product containing adenosine.

[0049] The above purification steps are mature existing technologies and can be flexibly adjusted according to the requirements of product purity. In this embodiment, the culture medium obtained in S3 is filtered through a membrane filter to obtain a primary filtrate. The primary filtrate is then filtered through a continuous series ion exchange column and an ultrafiltration membrane to obtain an ultrafiltrate. The ultrafiltrate is introduced into a rotary evaporator for concentration to obtain a concentrated solution. After crystallization in a crystallizer, centrifugation, recrystallization, centrifugation, drying, and pulverization, a refined product containing an adenosine composition is obtained.

[0050] The purified product of the adenosine-containing composition obtained in Example 1 was analyzed by liquid chromatography-mass spectrometry (LC-MS). Adenosine and lipopeptides were separated sequentially by liquid chromatography. The adenosine content was approximately 99.72 wt%, and the lipopeptide content was approximately 0.11 wt%. The characteristic peaks of both were very obvious in their respective mass spectra. (m / z) = 268.1, 290.1 ​​represent adenosine [M+H] + [M+Na] + The characteristic peaks, and the clustered peaks at m / z = 1008.6, 1022.7, 1036.7, and 1050.7, are attributed to the [M+H] homologues of C13, C14, C15, and C16 lipopeptides, respectively. +As can be seen, the purified product of the adenosine-containing composition obtained in Example 1 is mainly composed of adenosine, but it does contain a small amount of lipopeptides.

[0051] Examples 2-5

[0052] The strains used in Examples 2-5, the preparatory work before microbial fermentation synthesis, and the methods for synthesizing adenosine-containing compositions by microbial fermentation are basically the same as in Example 1. The only difference is that the amount of the second culture medium introduced in step S2 of Examples 2-5 is different from that in Example 1, that is, the ratio of the number of viable bacteria in the first culture medium and the second culture medium is different, as shown in the table below:

[0053] Example Ratio of viable bacteria in the first and second culture media Percentage of viable bacteria in the second culture medium 2 100000:1 0.001% 3 100000:3 0.003% 4 100000:8 0.008% 5 100000:10 0.01%

[0054] Example 6

[0055] The adenosine-producing Bacillus subtilis used in Example 6 was Bacillus subtilis HDCC12-21027, with the strain preservation number CGMCC No. 23448, provided by Hangzhou Sino-American East China Pharmaceutical Co., Ltd.; the lipopeptide-producing Bacillus subtilis used was Bacillus subtilis 50499, with the strain preservation number CGMCC No. 22013, provided by Professor Long Xuwei's research group at Nanjing University of Science and Technology.

[0056] The preparatory work for the microbial fermentation synthesis in Example 6 is as follows:

[0057] (1) Activation of strains: Take the preserved Bacillus subtilis HDCC12-21027 and Bacillus subtilis 50499 strains and inoculate them into sterile activation medium (purchased from Guangdong Huankai Microbiology). Incubate at 34℃ for 21 hours until fresh cells grow, which are the activated strains.

[0058] (2) Preparation of the first culture medium: The first fermentation medium was sterilized, cooled to 40℃, and the pH was adjusted to 7. The activated Bacillus subtilis HDCC12-21027 inoculum was inoculated into the seed first fermentation medium for pre-culture for 2-3 hours, which is the first culture medium. Culture conditions: temperature 35℃, pH 7, dissolved oxygen 15%, tank pressure 0.03MPa.

[0059] The substrates in the first fermentation medium include: glucose 20 g / L, corn steep liquor 20 g / L, yeast powder 10 g / L, ammonium chloride 2 g / L, urea 1.0 g / L, dipotassium hydrogen phosphate 1 g / L, calcium chloride 5 g / L, magnesium sulfate 1.0 g / L, and manganese sulfate 0.1 g / L.

[0060] (3) Preparation of the second culture medium: The second fermentation medium was sterilized, cooled to 40°C, and the pH was adjusted to 7. The activated Bacillus subtilis 50499 strain was inoculated into the second fermentation medium for pre-culture for 1-2 hours, which is the second culture medium. The strain culture conditions were: temperature 37°C, pH 7, dissolved oxygen 15%, and tank pressure 0.03 MPa.

[0061] The substrates in the second fermentation medium include: glucose 20 g / L, corn steep liquor 20 g / L, yeast powder 10 g / L, ammonium chloride 2 g / L, urea 1.0 g / L, dipotassium hydrogen phosphate 5 g / L, calcium chloride 5 g / L, magnesium sulfate 1.0 g / L, manganese sulfate 0.1 g / L, ferrous chloride 1.0 g / L, and copper chloride 0.1 g / L.

[0062] The steps of the method for synthesizing adenosine-containing compositions by microbial fermentation in Example 6 are as follows:

[0063] S1: 150L of the first culture medium is introduced into a medium-sized fermenter with a capacity of 200L and fermented at the first fermentation temperature of 35℃. During fermentation, sterile air (initial dissolved oxygen 15%, tank pressure 0.06MPa) is introduced into the fermenter, the first culture medium is stirred, and the viscosity of the first culture medium is monitored in real time.

[0064] S2: When the viscosity of the first culture medium reaches the first preset value of 12 mPa·s, an appropriate amount of the second culture medium (the ratio of the number of live bacteria in the first culture medium and the second culture medium is 100000:5, that is, the percentage of the number of live bacteria in the second culture medium to the number of live bacteria in the mixed culture medium is about 0.005%) is introduced into the first culture medium in the fermenter to form a mixed culture medium. Fermentation is carried out at the second fermentation temperature of 37°C. During the fermentation, sterile air is continuously introduced into the fermenter, the mixed culture medium is continuously stirred, and the viscosity of the mixed culture medium is continuously monitored in real time.

[0065] S3: When the viscosity of the mixed culture medium reaches the second preset value of 17 mPa·s, stop the fermentation, adjust the pH of the mixed culture medium to 7.0, and obtain the crude product containing the adenosine composition.

[0066] S4: The crude product containing adenosine obtained in S3 is subjected to membrane filtration, concentration, crystallization, centrifugation, recrystallization, centrifugation, drying, and pulverization processes in sequence to obtain the refined product containing adenosine.

[0067] Examples 7-10

[0068] The strains used in Examples 7-10, the preparatory work before microbial fermentation synthesis, and the methods for synthesizing adenosine-containing compositions by microbial fermentation are basically the same as in Example 6. The only difference is that the amount of the second culture medium introduced in step S2 of Examples 7-10 is different from that in Example 1, that is, the ratio of the number of viable bacteria in the first culture medium and the second culture medium is different, as shown in the table below:

[0069] Example Ratio of viable bacteria in the first and second culture media Percentage of viable bacteria in the second culture medium 7 100000:1 0.001% 8 100000:3 0.003% 9 100000:8 0.008% 10 100000:10 0.01%

[0070] Comparative Example 1

[0071] The adenosine-producing Bacillus subtilis used in Comparative Example 1 was Bacillus subtilis ACA301, with the strain preservation number CGMCC No.16753, provided by Jiangsu Aochuang Biotechnology Co., Ltd. The main difference between Comparative Example 1 and Example 1 is that a second culture medium containing lipopeptide-producing Bacillus subtilis was not added.

[0072] The preparations for the microbial fermentation synthesis in Comparative Example 1 are as follows:

[0073] (1) Activation of strain: Take the preserved Bacillus subtilis ACA301 and inoculate it into sterile activation medium (purchased from Guangdong Huankai Microbiology). Incubate at 34℃ for 21 hours until fresh cells grow, which is the activated strain.

[0074] (2) Preparation of the first culture medium: The first fermentation medium was sterilized, cooled to 40°C, and the pH was adjusted to 7. The activated Bacillus subtilis ACA301 strain was inoculated into the seed first fermentation medium for pre-culture for 2-3 hours, which is the first culture medium. The strain culture conditions were: temperature 34°C, pH 7, dissolved oxygen 12%, and tank pressure 0.03 MPa.

[0075] The substrates in the first fermentation medium include: glucose 20 g / L, corn steep liquor 30 g / L, yeast powder 10 g / L, ammonium sulfate 2 g / L, dipotassium hydrogen phosphate 5 g / L, sodium chloride 5 g / L, magnesium sulfate 1.0 g / L, manganese chloride 0.1 g / L, and ferrous chloride 1.0 g / L.

[0076] The steps of the microbial fermentation method for synthesizing the adenosine-containing composition in Comparative Example 1 are as follows:

[0077] S1: 150L of the first culture medium is introduced into a medium-sized fermenter with a capacity of 200L and fermented at the first fermentation temperature of 34℃. During fermentation, sterile air (initial dissolved oxygen 12%, tank pressure 0.06MPa) is introduced into the fermenter, the first culture medium is stirred, and the viscosity of the first culture medium is monitored in real time.

[0078] S2: When the viscosity of the first culture medium reaches the first preset value of 11 mPa·s, fermentation is carried out at the second fermentation temperature of 37°C. During fermentation, sterile air is continuously introduced into the fermenter, the mixed culture medium is continuously stirred, and the viscosity of the mixed culture medium is continuously monitored in real time.

[0079] S3: When the viscosity of the mixed culture medium reaches the second preset value of 16 mPa·s, stop the fermentation, adjust the pH of the mixed culture medium to 7.0, and obtain the crude product containing the adenosine composition.

[0080] S4: The crude product containing adenosine obtained in S3 is subjected to membrane filtration, concentration, crystallization, centrifugation, recrystallization, centrifugation, drying, and pulverization processes in sequence to obtain the refined product containing adenosine.

[0081] Comparative Example 2

[0082] Comparative Example 2 used adenosine-producing Bacillus subtilis HDCC12-21027, with the strain preservation number CGMCC No.23448, provided by Hangzhou Sino-American East China Pharmaceutical Co., Ltd. The main difference between Comparative Example 2 and Example 6 is that a second culture medium containing lipopeptide-producing Bacillus subtilis was not added.

[0083] The preparations for the microbial fermentation synthesis in Comparative Example 2 are as follows:

[0084] (1) Activation of strain: Take the preserved Bacillus subtilis HDCC12-21027 and inoculate it into sterile activation medium (purchased from Guangdong Huankai Microbiology). Incubate at 34℃ for 21 hours until fresh cells grow, which is the activated strain.

[0085] (2) Preparation of the first culture medium: The first fermentation medium was sterilized, cooled to 40℃, and the pH was adjusted to 7. The activated Bacillus subtilis HDCC12-21027 inoculum was inoculated into the seed first fermentation medium for pre-culture for 2-3 hours, which is the first culture medium. Culture conditions: temperature 35℃, pH 7, dissolved oxygen 15%, tank pressure 0.03MPa.

[0086] The substrates in the first fermentation medium include: glucose 20 g / L, corn steep liquor 20 g / L, yeast powder 10 g / L, ammonium chloride 2 g / L, urea 1.0 g / L, dipotassium hydrogen phosphate 1 g / L, calcium chloride 5 g / L, magnesium sulfate 1.0 g / L, and manganese sulfate 0.1 g / L.

[0087] The steps of the microbial fermentation method for synthesizing the adenosine-containing composition in Comparative Example 2 are as follows:

[0088] S1: 150L of the first culture medium is introduced into a medium-sized fermenter with a capacity of 200L and fermented at the first fermentation temperature of 35℃. During fermentation, sterile air (initial dissolved oxygen 15%, tank pressure 0.06MPa) is introduced into the fermenter, the first culture medium is stirred, and the viscosity of the first culture medium is monitored in real time.

[0089] S2: When the viscosity of the first culture medium reaches the first preset value of 12 mPa·s, fermentation is carried out at the second fermentation temperature of 37°C. During fermentation, sterile air is continuously introduced into the fermenter, the mixed culture medium is continuously stirred, and the viscosity of the mixed culture medium is continuously monitored in real time.

[0090] S3: When the viscosity of the mixed culture medium reaches the second preset value of 17 mPa·s, stop the fermentation, adjust the pH of the mixed culture medium to 7.0, and obtain the crude product containing the adenosine composition.

[0091] S4: The crude product containing adenosine obtained in S3 is subjected to membrane filtration, concentration, crystallization, centrifugation, recrystallization, centrifugation, drying, and pulverization processes in sequence to obtain the refined product containing adenosine.

[0092] Comparative Example 3

[0093] Comparative Example 3 used adenosine-producing Bacillus subtilis, Bacillus subtilis ACA301, strain preservation number CGMCC No.16753, provided by Jiangsu Aochuang Biotechnology Co., Ltd. The main difference between Comparative Example 3 and Example 1 is that a second culture medium containing lipopeptide-producing Bacillus subtilis was not added, but instead, surfactin Na was directly added, purchased from Guangdong Best Biochemical Technology Co., Ltd.

[0094] The preparations for the microbial fermentation synthesis in Comparative Example 3 are as follows:

[0095] (1) Activation of strain: Take the preserved Bacillus subtilis ACA301 and inoculate it into sterile activation medium (purchased from Guangdong Huankai Microbiology). Incubate at 34℃ for 21 hours until fresh cells grow, which is the activated strain.

[0096] (2) Preparation of the first culture medium: The first fermentation medium was sterilized, cooled to 40°C, and the pH was adjusted to 7. The activated Bacillus subtilis ACA301 strain was inoculated into the seed first fermentation medium for pre-culture for 2-3 hours, which is the first culture medium. The strain culture conditions were: temperature 34°C, pH 7, dissolved oxygen 12%, and tank pressure 0.03 MPa.

[0097] The substrates in the first fermentation medium include: glucose 20 g / L, corn steep liquor 30 g / L, yeast powder 10 g / L, ammonium sulfate 2 g / L, dipotassium hydrogen phosphate 5 g / L, sodium chloride 5 g / L, magnesium sulfate 1.0 g / L, manganese chloride 0.1 g / L, and ferrous chloride 1.0 g / L.

[0098] (3) Preparation of the second culture medium: Sterilize the second fermentation medium, cool it to 40°C, adjust the pH value to 7, add 2.0 g / L of Bacillus subtilis lipopeptide sodium to the second fermentation medium, stir and mix evenly, and maintain the temperature at 34°C to obtain the second culture medium.

[0099] The substrates in the second fermentation medium include: glucose 20 g / L, corn steep liquor 30 g / L, yeast powder 10 g / L, ammonium sulfate 2 g / L, dipotassium hydrogen phosphate 5 g / L, sodium chloride 5 g / L, calcium chloride 5 g / L, magnesium sulfate 1.0 g / L, manganese chloride 0.1 g / L, ferrous chloride 1.0 g / L, and copper chloride 0.1 g / L.

[0100] The steps of the microbial fermentation method for synthesizing the adenosine-containing composition in Comparative Example 3 are as follows:

[0101] S1: 150L of the first culture medium is introduced into a medium-sized fermenter with a capacity of 200L and fermented at the first fermentation temperature of 34℃. During fermentation, sterile air (initial dissolved oxygen 12%, tank pressure 0.06MPa) is introduced into the fermenter, the first culture medium is stirred, and the viscosity of the first culture medium is monitored in real time.

[0102] S2: When the viscosity of the first culture medium reaches the first preset value of 11 mPa·s, the second culture medium is continuously added dropwise to the first culture medium in the fermenter (the dropwise addition rate is 10 mL / min) to form a mixed culture medium. Fermentation is carried out at the second fermentation temperature of 37℃. During the fermentation, sterile air is continuously introduced into the fermenter, the mixed culture medium is continuously stirred, and the viscosity of the mixed culture medium is continuously monitored in real time.

[0103] S3: When the viscosity of the mixed culture medium reaches the second preset value of 16 mPa·s, stop adding the second culture medium and stop fermentation. Adjust the pH of the mixed culture medium to 7.0 to obtain the crude product containing the adenosine composition.

[0104] S4: The crude product containing adenosine obtained in S3 is subjected to membrane filtration, concentration, crystallization, centrifugation, recrystallization, centrifugation, drying, and pulverization processes in sequence to obtain the refined product containing adenosine.

[0105] Comparative Example 4

[0106] Comparative Example 4 used adenosine-producing Bacillus subtilis, namely Bacillus subtilis HDCC12-21027, with the strain preservation number CGMCC No. 23448, provided by Hangzhou Sino-American East China Pharmaceutical Co., Ltd. The main difference between Comparative Example 4 and Example 6 is that a second culture medium containing lipopeptide-producing Bacillus subtilis was not added; instead, Surfactin Na was directly added, purchased from Guangdong Best Biochemical Technology Co., Ltd.

[0107] The preparations for the microbial fermentation synthesis in Comparative Example 4 are as follows:

[0108] (1) Activation of strain: Take the preserved Bacillus subtilis HDCC12-21027 and inoculate it into sterile activation medium (purchased from Guangdong Huankai Microbiology). Incubate at 34℃ for 21 hours until fresh cells grow, which is the activated strain.

[0109] (2) Preparation of the first culture medium: The first fermentation medium was sterilized, cooled to 40℃, and the pH was adjusted to 7. The activated Bacillus subtilis HDCC12-21027 inoculum was inoculated into the seed first fermentation medium for pre-culture for 2-3 hours, which is the first culture medium. Culture conditions: temperature 35℃, pH 7, dissolved oxygen 15%, tank pressure 0.03MPa.

[0110] The substrates in the first fermentation medium include: glucose 20 g / L, corn steep liquor 20 g / L, yeast powder 10 g / L, ammonium chloride 2 g / L, urea 1.0 g / L, dipotassium hydrogen phosphate 1 g / L, calcium chloride 5 g / L, magnesium sulfate 1.0 g / L, and manganese sulfate 0.1 g / L.

[0111] (3) Preparation of the second culture medium: Sterilize the second fermentation medium, cool it to 40°C, adjust the pH value to 7, add 2.0 g / L of Bacillus subtilis lipopeptide sodium to the second fermentation medium, stir and mix evenly, and maintain the temperature at 35°C to obtain the second culture medium.

[0112] The substrates in the second fermentation medium include: glucose 20 g / L, corn steep liquor 20 g / L, yeast powder 10 g / L, ammonium chloride 2 g / L, urea 1.0 g / L, dipotassium hydrogen phosphate 5 g / L, calcium chloride 5 g / L, magnesium sulfate 1.0 g / L, manganese sulfate 0.1 g / L, ferrous chloride 1.0 g / L, and copper chloride 0.1 g / L.

[0113] The steps of the microbial fermentation method for synthesizing the adenosine-containing composition in Comparative Example 4 are as follows:

[0114] S1: 150L of the first culture medium is introduced into a medium-sized fermenter with a capacity of 200L and fermented at the first fermentation temperature of 35℃. During fermentation, sterile air (initial dissolved oxygen 15%, tank pressure 0.06MPa) is introduced into the fermenter, the first culture medium is stirred, and the viscosity of the first culture medium is monitored in real time.

[0115] S2: When the viscosity of the first culture medium reaches the first preset value of 12 mPa·s, the second culture medium is continuously added dropwise to the first culture medium in the fermenter (the dropwise addition rate is 10 mL / min) to form a mixed culture medium. Fermentation is carried out at the second fermentation temperature of 37℃. During the fermentation, sterile air is continuously introduced into the fermenter, the mixed culture medium is continuously stirred, and the viscosity of the mixed culture medium is continuously monitored in real time.

[0116] S3: When the viscosity of the mixed culture medium reaches the second preset value of 17 mPa·s, stop adding the second culture medium and stop fermentation. Adjust the pH of the mixed culture medium to 7.0 to obtain the crude product containing the adenosine composition.

[0117] S4: The crude product containing adenosine obtained in S3 is subjected to membrane filtration, concentration, crystallization, centrifugation, recrystallization, centrifugation, drying, and pulverization processes in sequence to obtain the refined product containing adenosine.

[0118] The yields of the adenosine-containing compositions obtained in the above embodiments and comparative examples are shown in the table below:

[0119] Examples / Comparative Examples Yield (g / L) Examples / Comparative Examples Yield (g / L) Example 1 52.68 Example 2 43.25 Example 3 51.93 Example 4 50.22 Example 5 42.57 Example 6 36.41 Example 7 28.49 Example 8 33.16 Example 9 35.04 Example 10 29.78 Comparative Example 1 38.45 Comparative Example 2 26.55 Comparative Example 3 40.06 Comparative Example 4 27.31

[0120] As can be seen from the above results:

[0121] (1) The present invention introduces an appropriate amount (0.003-0.008%) of lipopeptide-producing Bacillus subtilis only in the later stage of fermentation. This can avoid the significant inhibitory effect on the growth and production of adenosine-producing Bacillus subtilis. At the same time, the subtilis lipopeptide produced by Bacillus subtilis continuously emulsifies the culture medium and significantly reduces the surface tension of the culture medium, thereby preventing the crystallization of high concentrations of adenosine in the culture medium and increasing its dissolved oxygen level. Therefore, compared with Comparative Examples 1 and 2 without the addition of lipopeptide-producing Bacillus subtilis, or compared with Comparative Examples 3 and 4 with the direct addition of sodium subtilis lipopeptide, the yield of adenosine produced by adenosine-producing Bacillus subtilis in the later stage of fermentation is significantly improved.

[0122] (2) The yield comparisons of Examples 1-5 and Examples 6-10 show that introducing an appropriate amount (0.003-0.008%) of lipopeptide-producing Bacillus subtilis is crucial for increasing yield. If too much lipopeptide-producing Bacillus subtilis is introduced, it will inhibit the growth of adenosine-producing Bacillus subtilis, affecting adenosine yield; on the other hand, too much lipopeptide produced will result in excessively low surface tension of the culture medium, excessive foaming, and reduced dissolved oxygen. If too little lipopeptide-producing Bacillus subtilis is added, it will hardly grow due to the inhibitory effect of adenosine-producing Bacillus subtilis, thus failing to produce enough lipopeptides to reduce the surface tension of the culture medium and improve dissolved oxygen and adenosine crystallization.

[0123] (3) The yield comparisons of Examples 1, 1, and 3, and Examples 6, 2, and 4 show that the continuous addition of an appropriate amount of sodium subtilis lipopeptide in the later stages of fermentation can slightly increase the yield of adenosine. This is because sodium subtilis lipopeptide, as a super-strong biosurfactant, can emulsify the culture medium and significantly reduce its surface tension, thereby preventing the crystallization of high-concentration adenosine in the culture medium and increasing its dissolved oxygen level. However, the effect of directly adding sodium subtilis lipopeptide is not as good as adding lipopeptide-producing Bacillus subtilis. This is because as Bacillus subtilis grows in the mixed culture medium, the amount of sodium subtilis lipopeptide it produces gradually increases, which coincides with the trend that the culture medium with gradually increasing viscosity requires an increasing amount of sodium subtilis lipopeptide. The two form a synergistic effect to a certain extent.

[0124] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for synthesizing an adenosine-containing composition by microbial fermentation, characterized in that, Includes the following steps: S1: A first culture medium containing adenosine-producing Bacillus subtilis and a substrate suitable for the growth of the adenosine-producing Bacillus subtilis is introduced into a fermenter and fermented at a first fermentation temperature. During fermentation, sterile oxygen or sterile air is introduced into the fermenter, the first culture medium is stirred and / or shaken, and the viscosity of the first culture medium is monitored in real time. S2: When the viscosity of the first culture medium reaches a first preset value, a second culture medium containing Bacillus subtilis lipopeptide and a substrate suitable for the growth of Bacillus subtilis lipopeptide is introduced into the first culture medium to form a mixed culture medium, and fermentation is carried out at a second fermentation temperature. S3: When the viscosity of the mixed culture medium reaches the second preset value, fermentation is stopped, and the pH value of the mixed culture medium is adjusted to approximately neutral to obtain a crude product containing adenosine composition.

2. The method for synthesizing adenosine-containing compositions by microbial fermentation according to claim 1, characterized in that, The adenosine-producing Bacillus subtilis mentioned in S1 is selected from one or more of the following Bacillus subtilis preservation numbers: CGMCC No. 16753, CGMCC No. 1304, CGMCC No. 4484, CGMCC No. 23448, CGMCC No. 0733, or their mutant strains; the lipopeptide-producing Bacillus subtilis mentioned in S2 is selected from one or more of the following Bacillus subtilis preservation numbers: CGMCC No. 2947, CGMCC No. 14375, CGMCC No. 8925, CGMCC No. 22013, CGMCC No. 28191, CGMCC No. 34998, or their mutant strains.

3. The method for synthesizing adenosine-containing compositions by microbial fermentation according to claim 1, characterized in that, The substrates suitable for the growth of the adenosine-producing Bacillus subtilis in S1 include a carbon source, a nitrogen source, and a metal salt; wherein, The carbon source is selected from one or more of glucose, glycerol, sucrose, and corn steep liquor; The nitrogen source is selected from one or more of the following: peptone, beef extract, fish meal, corn steep liquor, yeast extract, monosodium glutamate, urea, and ammonium salts. The metal salts include potassium salts, sodium salts, magnesium salts, manganese salts, and ferrous salts; wherein, The ammonium salt is selected from one or two of (NH4)2SO4 and NH4Cl; The potassium salt is selected from one or more of KH2PO4, K2HPO4, K2SO4, and KCl; The sodium salt is selected from one or more of Na2SO4, NaCl, and NaNO3; The magnesium salt is selected from one or more of MgSO4, MgCl2, and Mg(NO3)2; The manganese salt is selected from one or more of MnSO4, MnCl2, and Mn(NO3)2; The ferrous salt is selected from one or more of FeSO4, FeCl2, and Fe(NO3)2.

4. The method for synthesizing adenosine-containing compositions by microbial fermentation according to claim 1, characterized in that, In S1, the first fermentation temperature is 28-38℃, the initial dissolved oxygen in the first culture medium is greater than or equal to 10%, and the pressure of the fermenter is 0.02~0.06MPa; in S2, the second fermentation temperature is 36-38℃.

5. The method for synthesizing an adenosine-containing composition by microbial fermentation according to claim 1, characterized in that, In S1, the viscosity of the first culture medium is monitored in real time by means of an online rotational viscometer, an online vibrational viscometer, an online ultrasonic viscometer, an online optical viscometer, or an online rheological viscometer.

6. The method for synthesizing an adenosine-containing composition by microbial fermentation according to claim 1, characterized in that, In S2, the first preset value is 10-13 mPa·s; in S3, the second preset value is 15-18 mPa·s.

7. The method for synthesizing adenosine-containing compositions by microbial fermentation according to claim 1, characterized in that, When the second culture medium is introduced into the fermenter in S2, the ratio of the number of CFUs / live bacteria of the lipopeptide-producing Bacillus subtilis in the second culture medium to the number of CFUs / live bacteria of the adenosine-producing Bacillus subtilis in the first culture medium in the fermenter at that time is 0.003-0.008%.

8. The method for synthesizing an adenosine-containing composition by microbial fermentation according to claim 1, characterized in that, The method further includes the following steps: S4: The crude product of the adenosine-containing composition obtained in S3 is purified to obtain the refined product of the adenosine-containing composition.

9. The method for synthesizing an adenosine-containing composition by microbial fermentation according to claim 15, characterized in that, The purification operation described in S4 includes, in sequence, membrane filtration, concentration, crystallization, centrifugation, recrystallization, centrifugation, drying, and pulverization.

10. A composition containing adenosine, characterized in that, The adenosine-containing composition comprises a crude product of an adenosine-containing composition obtained by the method of any one of claims 1-7 or a refined product of an adenosine-containing composition obtained by the method of any one of claims 8-9.