Method for producing ectoine by adopting vibrio alginolyticus
By using Vibrio Alginolyticus ATCC 33787 fermentation strain, a single carbon source such as soluble starch is used to produce tetramidine, which solves the problem of high production costs in existing technology, and achieves high -efficiency and low -cost tetraidine production.
Patent Information
- Application Number
- CN202410894486.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-04
- Publication Date
- 2025-01-28
AI Technical Summary
In the existing tetrahydropyrimidine production methods, high salt concentration, high equipment corrosion, genetic instability, antibiotics need to be added, and a combination of multiple substances is required as a carbon source, resulting in high production costs.
The Vibrio Alginolyticus ATCC 33787 is used as a fermented bacterial strain. It is used as a single carbon source in a single carbon source in glucose, sucrose and soluble starch. There is no need to add expensive auxiliary carbon sources.
The production of tetramidine at a low -priced single carbon source is the only carbon source, which shortens the fermentation time, improves production efficiency, and reduces production costs.
Smart Images
Figure CN119372266A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biological fermentation, and in particular to a method for producing ectoine by using Vibrio alginolyticus. Background Art
[0002] Ectothiorhodospira halochloris DSM1059 was first discovered and its structure identified in 1985. As an amino acid derivative, it is stable and not easy to decompose. It is soluble in water, glycerol, methanol, ethanol and propylene glycol, but insoluble in dimethyl sulfoxide and chloroform. It is a compatible solute widely found in halophilic archaea, halophilic bacteria, fungi and salt algae. It has the functions of maintaining intracellular and extracellular osmotic pressure, enhancing enzyme activity, stabilizing DNA structure and protein structure stability. Therefore, ectohydropyrimidine is widely used in biomedical fields such as digestive tract diseases, dry eye syndrome, rhinitis, allergic rhinitis, organ transplantation and organ preservation, skin trauma and health care and beauty, and develops products such as digestive tract drugs, eye drops, nasal sprays, respiratory tract permeants, skin trauma drugs and new biological cosmetics.
[0003] At present, there are two methods for obtaining ectoine: chemical synthesis and biosynthesis. However, since ectoine contains a chiral carbon, it is difficult to synthesize it chemically. Therefore, ectoine is currently mainly obtained through biosynthesis. There are two main biosynthesis methods: the first is to screen out wild strains that can synthesize ectoine, such as Halorhodospirilla, Chromobacterium, Halomonas, Vibrio and Alkaliphilic Bacteria. The model strains widely used in the industrial-scale production of ectoine are mainly concentrated in the γ-Proteobacteria Halomonas, especially Halomonas and Chromobacterium. By utilizing the environment in which they synthesize ectoine and providing them with corresponding culture conditions, high yields of ectoine can be achieved through bacterial milking, fed-batch fermentation and resting bacterial fermentation. The second method is to transfer the ectABC gene cluster for ectoine synthesis into heterologous bacteria such as Escherichia coli and Corynebacterium glutamicum through genetic engineering, construct recombinant engineered bacteria, and utilize the non-existent pathway for degrading ectoine to achieve high production of ectoine through bacterial milking, fed-batch fermentation and resting bacterial fermentation.
[0004] The above two methods of producing ectoine by biosynthesis have the disadvantages of high salt concentration, large corrosion to equipment, genetic instability and the need to add antibiotics during the fermentation production process. At the same time, it is generally necessary to use a combination of multiple substances as carbon sources for fermentation to achieve efficient production of ectoine. Glucose is usually used as the main carbon source for fermentation, and some glycerol, L-aspartic acid and yeast extract are added as auxiliary carbon sources. Due to the high prices of glycerol, L-aspartic acid and yeast extract, the existing methods for producing ectoine are difficult to apply to industrial production. Therefore, by finding new strains that can directly use a low-priced single carbon source for production, the production of ectoine using a low-priced carbon source as the only carbon source will help reduce the production cost of ectoine.
[0005] Vibrio alginolyticus is a Gram-negative facultative anaerobic bacterium that is mainly distributed in waters at the mouths of oceans and rivers. Its number ranks first among marine Vibrio. It has been reported that Vibrio alginolyticus has an extremely fast growth rate. Summary of the invention
[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to propose a method for producing ectoine using Vibrio alginolyticus, which can utilize one of glucose, sucrose and soluble starch as a single carbon source to ferment and produce ectoine without adding expensive auxiliary carbon sources, which is conducive to industrial production and solves the problem of high cost of producing ectoine in traditional processes.
[0007] To achieve this object, the present invention adopts the following technical solutions:
[0008] A method for producing ectoine by using Vibrio alginolyticus ATCC 33787 is provided. The ectoine is produced by fermenting and culturing the Vibrio alginolyticus ATCC 33787. The carbon source used in the fermentation and culturing process is any one of starch, sucrose and glucose. The fermentation time is 8 to 16 hours.
[0009] Preferably, the concentration of the carbon source in the fermentation medium used in the fermentation culture process is 10-50 g / L.
[0010] Preferably, the carbon source is soluble starch.
[0011] Preferably, the fermentation temperature during the fermentation culture is 20 to 42°C.
[0012] Preferably, a nitrogen source is also added to the fermentation medium during the fermentation culture, and the nitrogen source is any one of ammonium sulfate, ammonium oxalate, ammonium acetate, ammonium chloride and ammonium phosphate;
[0013] The concentration of the nitrogen source in the fermentation medium is 1-2 g / L.
[0014] Preferably, the components of the fermentation medium used in the fermentation culture include: KH2PO4 12-15 g / L, KOH 4-5 g / L, MgSO4·7H2O 0.18-0.3 g / L, nitrogen source 1-2 g / L, FeSO4·7H2O 0.0008-0.0015 g / L, NaCl 20-40 g / L and carbon source 10-50 g / L.
[0015] Preferably, the method for producing ectoine using Vibrio alginolyticus comprises the following steps:
[0016] (1) inoculating the alginolytic Vibrio strain into a solid culture medium for activation to obtain activated bacteria;
[0017] (2) inoculating the activated bacterial cells into a seed culture medium for seed culture to obtain a seed solution;
[0018] (3) inoculating the seed liquid into a fermentation medium for fermentation culture to obtain a fermentation liquid containing ectoine;
[0019] (4) After separating and purifying the fermentation bacterial liquid containing ectoine, ectoine is obtained.
[0020] Preferably, in step (3), the seed liquid is inoculated into the fermentation medium at an inoculation rate of 1 to 10% for fermentation culture.
[0021] Preferably, in step (3), after the seed liquid is inoculated into the fermentation medium, it is placed on a shaker and fermented at 160 to 200 rpm for 8 to 16 hours to obtain a fermentation liquid containing ectoine.
[0022] Preferably, in step (2), the components of the seed culture medium include: KH2PO4 12-15 g / L, KOH 4-5 g / L, MgSO4·7H2O 0.18-0.3 g / L, nitrogen source 0-2 g / L, FeSO4·7H2O 0.0008-0.0015 g / L and NaCl 20-40 g / L.
[0023] The technical solution provided by the embodiments of the present application may have the following beneficial effects:
[0024] 1. This technical solution uses Vibrio alginolyticus ATCC 33787 as a fermentation strain to produce ectoine. Vibrio alginolyticus ATCC 33787 grows faster and can use one of glucose, sucrose and soluble starch as a single carbon source to ferment and produce ectoine. In addition, this technical solution uses Vibrio alginolyticus ATCC 33787 as a fermentation bacteria to produce ectoine, which can shorten the fermentation time. A high yield of ectoine can be obtained by fermenting for 8 to 16 hours. Compared with the traditional method that requires 24 hours of fermentation, the fermentation time of this technical solution is shorter, which can effectively improve the production efficiency of ectoine.
[0025] 2. Compared with existing strains, Vibrio alginolyticus ATCC 33787 can directly use low-cost starch as a carbon source to ferment and produce ectoine. Compared with glucose and sucrose, starch has a wide range of sources, making its price generally lower than that of glucose and sucrose. Starch contains soluble starch, so soluble starch is used as a single carbon source to produce ectoine, paving the way for the subsequent use of starch as a carbon source. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 Metabolic pathway diagram for the production of ectoine by Vibrio alginolyticus ATCC 33787;
[0027] Figure 2 is the growth curve of Vibrio alginolyticus ATCC 33787 in Example 1 of the present invention using glucose as a carbon source;
[0028] Figure 3 The content of ectoine in the supernatant of the fermentation of Vibrio alginolyticus ATCC 33787 with different carbon sources for 12 hours;
[0029] Figure 4 is the HPLC chromatogram of ectoine standard;
[0030] Figure 5 HPLC chromatogram of ectoine obtained by the method of Example 2 of the present invention (production by fermentation of soluble starch by Vibrio alginolyticus ATCC 33787); DETAILED DESCRIPTION
[0031] In order to facilitate the understanding of the present invention, the present invention is described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.
[0032] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. Raw materials used without specifying the manufacturer are all conventional products that can be obtained through commercial purchase.
[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.
[0034] The technical solution of the present invention is further explained below in conjunction with specific implementation methods.
[0035] A method for producing ectoine by using Vibrio alginolyticus ATCC 33787 is provided. The ectoine is produced by fermenting and culturing the Vibrio alginolyticus ATCC 33787. The carbon source used in the fermentation and culturing process is any one of starch, sucrose and glucose. The fermentation time is 8 to 16 hours.
[0036] It is worth noting that this technical solution discloses for the first time that Vibrio alginolyticus ATCC33787 is used as a base bacteria to produce ectoine. This technical solution uses Vibrio alginolyticus ATCC33787 (hereinafter referred to as: Vibrio alginolyticus ATCC 33787) as a fermentation strain to produce ectoine. Vibrio alginolyticus ATCC33787 can use one of glucose, sucrose and soluble starch as a single carbon source to ferment and produce ectoine. Moreover, compared with existing strains, Vibrio alginolyticus ATCC 33787 can directly use low-priced starch as a carbon source to ferment and produce ectoine. Compared with glucose and sucrose, the source of starch is wide, so the price of starch is generally lower than that of glucose and sucrose, and starch contains soluble starch. Therefore, soluble starch is used as a single carbon source to produce ectoine, paving the way for the subsequent use of starch as a carbon source. At the same time, the technical solution uses Vibrio alginolyticus ATCC 33787 as a fermentation bacterium to produce ectoine, which can shorten the fermentation time. A high yield of ectoine can be obtained by fermenting for 8 to 16 hours. Compared with the traditional method which requires fermentation for 24 hours or longer, the fermentation time of the technical solution is shorter, which can effectively improve the production efficiency of ectoine.
[0037] Specifically, the Vibrio alginolyticus ATCC 33787 used in this technical solution is deposited in the Shanghai Collection Center for Microorganisms, with the strain deposit number of SHBCC D10832 and the deposit address of Nanxiang Precision Medical Industrial Park, Nanxiang Town, Jiading District, Shanghai. The Vibrio alginolyticus ATCC 33787 used in this technical solution was purchased from the Shanghai Collection Center for Microorganisms.
[0038] Further explanation, literature data show that Vibrio alginolyticus strains are widely distributed in seawater and marine animals in estuaries, bays and coastal areas. They are usually bent into sharp arcs or straight rods, have long flagella, and have a short doubling time. Therefore, Vibrio alginolyticus has the potential to be used in industrial production.
[0039] To further illustrate, the concentration of the carbon source in the fermentation medium used in the fermentation culture process is 10-50 g / L.
[0040] It is worth noting that the concentration of the carbon source in the fermentation medium in the present technical solution is 10-50 g / L. Within this content range, efficient production of ectoine can be achieved.
[0041] Preferably, the concentration of the carbon source in the fermentation medium is 30 g / L. At this concentration, the yield of ectoine obtained is high and waste of carbon source and increase in cost can be avoided.
[0042] In further explanation, the carbon source is soluble starch.
[0043] In one embodiment of the present invention, the carbon source used in the fermentation process is soluble starch. The price of soluble starch is generally lower than that of sucrose and glucose. Using soluble starch as a carbon source can effectively reduce production costs. Moreover, using soluble starch as a carbon source is more conducive to the growth and reproduction of Vibrio alginolyticus ATCC 33787, and the yield of tetrahydropyrimidine produced is also higher.
[0044] Preferably, the starch is soluble starch, which is a starch derivative obtained by treating starch with an oxidant, acid, glycerol, enzyme or other methods. Soluble starch contains no reducing substances and has stable chemical properties.
[0045] To further explain, the fermentation temperature during the fermentation culture is 20 to 42°C.
[0046] Specifically, the fermentation temperature of the present technical solution is 20-42°C, which is conducive to improving the production efficiency and yield of ectoine. If the temperature is too high or too low, the growth rate of the bacteria will slow down, thereby reducing the production efficiency and the yield of ectoine.
[0047] More preferably, the fermentation temperature is 23-30° C. Within this temperature range, the yield of ectoine produced is higher.
[0048] Further description, a nitrogen source is also added to the fermentation medium during the fermentation culture, and the nitrogen source is any one of ammonium sulfate, ammonium oxalate, ammonium acetate, ammonium chloride and ammonium phosphate;
[0049] The concentration of the nitrogen source in the fermentation medium is 1-2 g / L.
[0050] It is worth noting that, by screening the NCBI gene library, this technical solution found that Vibrio alginolyticus ATCC 33787 can produce ectoine, and through further research, it was found that it can use glucose, sucrose and starch as carbon sources, and ammonium sulfate, ammonium oxalate, ammonium acetate, ammonium chloride and ammonium phosphate as nitrogen sources to produce ectoine.
[0051] In the technical solution, the concentration of the nitrogen source in the fermentation medium is 1-2 g / L, which can improve the yield and production efficiency of ectoine.
[0052] Preferably, the nitrogen source is ammonium sulfate. When ammonium sulfate is used as the nitrogen source during the fermentation process, a higher yield of ectoine is obtained.
[0053] To further illustrate, the components of the fermentation medium used in the fermentation culture include: KH2PO4 12-15 g / L, KOH 4-5 g / L, MgSO4·7H2O 0.18-0.3 g / L, nitrogen source 1-2 g / L, FeSO4·7H2O 0.0008-0.0015 g / L, NaCl 20-40 g / L and carbon source 10-50 g / L.
[0054] It is worth noting that the technical solution is used for the fermentation medium for producing ectoine by Vibrio alginolyticus ATCC 33787, which contains the carbon source and nitrogen source required for producing ectoine, and contains MgSO4·7H2O. MgSO4·7H2O is introduced by adding MgSO4·7H2O. 2+ ,Mg 2+ It is an important activator of various enzymes such as TCA pathway and EMP pathway. The fermentation medium of this technical solution can provide a high-salt environment for synthesizing ectoine, stimulate the bacteria to synthesize ectoine, and maintain osmotic pressure balance; during the fermentation process, the nitrogen source is converted into ammonium, and the ammonium is converted into L-aspartic acid to enter the ectoine synthesis pathway. L-aspartic acid is a precursor of ectoine and can be quickly converted into ectoine, thereby increasing the yield of ectoine.
[0055] Specifically, the technical solution optimizes and adjusts the carbon source content, nitrogen source content and sodium chloride content of the fermentation medium to obtain a medium for efficiently producing ectoine. The fermentation medium of the technical solution is used to ferment and produce ectoine, which can achieve efficient production of ectoine. In addition, the technical solution optimizes and adjusts the fermentation medium for Vibrio alginolyticus ATCC 33787, and finally achieves the production of ectoine using soluble starch as a carbon source.
[0056] Preferably, the pH value of the fermentation medium is 6 to 9. More preferably, the pH value of the fermentation medium is 6 to 7, which is conducive to maintaining the pH stability of the medium. If the pH value is too high or too low, it is easy to cause a large change in pH after high temperature and high pressure sterilization.
[0057] Preferably, the components of the fermentation medium used in the fermentation culture process include: KH2PO4 13.61 g / L, KOH 4.21 g / L, MgSO4·7H2O 0.25 g / L, nitrogen source 2 g / L, FeSO4·7H2O 0.0011 g / L, NaCl 30 g / L and carbon source 30 g / L.
[0058] Further explanation, the method for producing ectoine using Vibrio alginolyticus comprises the following steps:
[0059] (1) inoculating the alginolytic Vibrio strain into a solid culture medium for activation to obtain activated bacteria;
[0060] (2) inoculating the activated bacterial cells into a seed culture medium for seed culture to obtain a seed solution;
[0061] (3) inoculating the seed liquid into a fermentation medium for fermentation culture to obtain a fermentation liquid containing ectoine;
[0062] (4) After separating and purifying the fermentation bacterial liquid containing ectoine, ectoine is obtained.
[0063] Preferably, step (1) of the bacterial strain activation of the present technical solution comprises the following steps: picking bacterial strains and streaking them onto LBS solid culture medium, placing them in a constant temperature incubator, and culturing them at 30° C. for 12 to 24 hours, until a single clone grows, and an activated bacterial cell is obtained;
[0064] Preferably, the preparation of the seed solution of the technical solution (2) comprises the following steps: picking activated bacteria and inoculating them into a large test tube containing a seed culture medium, sealing the tube with a rubber stopper, placing the tube on a shaker at 30°C, and shaking and culturing the tube at 160-200 rpm for 12-24 hours to obtain the seed solution. Preferably, the OD value of the seed solution is 0.300-0.400.
[0065] Preferably, the solid culture medium used in step (1) is LBS solid culture medium, and more preferably, the LBS solid culture medium is 2216E agar solid base culture medium. The composition of 2216E agar solid base culture medium is as follows: 10 g / L peptone, 5 g / L yeast powder, 30 g / L sodium chloride and 30 g / L agar powder.
[0066] It is worth noting that the method for separating and purifying the fermentation broth containing ectoine in step (4) of the present technical solution is well known and can be carried out by conventional methods in the art.
[0067] Preferably, in one embodiment of the present invention, the method for separating and purifying the fermentation broth containing ectoine in step (4) is as follows: taking the fermentation broth containing ectoine after fermentation, centrifuging it at 12000 rpm for 5 minutes, filtering and taking the supernatant, adding acetonitrile to the supernatant, the volume ratio of the supernatant to acetonitrile is 49:1, and then filtering with a 0.22 μm filter membrane to obtain ectoine.
[0068] As a further explanation, in step (3), the seed liquid is inoculated into the fermentation medium at an inoculation amount of 1 to 10% for fermentation culture.
[0069] Specifically, the inoculation amount of the seed solution in the present technical solution is 1-10%, which is beneficial to improving the fermentation effect, the generation of ectoine and the increase of the yield of ectoine.
[0070] Preferably, the seed liquid is inoculated into the fermentation medium at an inoculation rate of 2% for fermentation culture.
[0071] Further explanation, in step (3), after the seed liquid is inoculated into the fermentation medium, it is placed on a shaker and fermented at 160-200 rpm for 8-16 hours to obtain a fermentation liquid containing ectoine.
[0072] The fermentation time of the technical solution is 8 to 16 hours, which is short and can effectively improve the production efficiency of ectoine, thereby further reducing the fermentation cost of ectoine.
[0073] This technical solution can realize the laboratory shake flask fermentation production of ectoine by optimizing the fermentation medium and fermentation conditions.
[0074] Further explanation, in step (2), the components of the seed culture medium include: KH2PO4 12-15 g / L, KOH 4-5 g / L, MgSO4·7H2O 0.18-0.3 g / L, glucose 30 g / L, nitrogen source 0-2 g / L, FeSO4·7H2O 0.0008-0.0015 g / L and NaCl 20-40 g / L.
[0075] Specifically, the seed culture medium of the present technical solution is M63 culture medium, and the components of M63 culture medium include: KH2PO4 12-15 g / L, KOH 4-5 g / L, MgSO4·7H2O 0.18-0.3 g / L, nitrogen source 0-2 g / L, FeSO4·7H2O 0.0008-0.0015 g / L and NaCl 20-40 g / L.
[0076] The technical solution of the present invention is further described below in conjunction with embodiments.
[0077] Example 1
[0078] This example relates to the biochemical identification of carbon source utilization by Vibrio alginolyticus ATCC 33787 (hereinafter referred to as Vibrio alginolyticus ATCC 33787), comprising the following steps:
[0079] (1) Take an inoculation loop and streak Vibrio alginolyticus ATCC 33787 onto LBS solid medium on a clean bench, activate at 30°C for 12 h, and wait for a single clone to grow to obtain activated bacteria;
[0080] (2) In an ultra-clean workbench, a monoclonal colony of step (1) was picked up with a sterile inoculation loop, inoculated into a 20 mL test tube containing 10 mL of seed culture medium, and cultured at 30°C and 200 rpm for 12 h. Subsequently, 200 μl of the colony was taken with a pipette and inoculated into a 20 mL test tube containing 10 mL of seed culture medium, and cultured at 30°C and 200 rpm for 12 h to obtain a seed solution; wherein the components of the seed culture medium are as follows: carbon source (soluble starch) 30 g / L, KH2PO4 13.61 g / L, KOH 4.21 g / L, MgSO4·7H2O 0.25 g / L, nitrogen source (ammonium sulfate) 2 g / L, FeSO4·7H2O 0.0011 g / L and NaCl 30 g / L;
[0081] (3) Use a pipette to add the seed solution at a 2% inoculum volume to 100 mL of M63 medium containing: 30 g / L carbon source (glucose, sucrose, soluble starch), 13.61 g / L KH2PO4, 4.21 g / L KOH, 0.25 g / L MgSO4·7H2O, 2 g / L nitrogen source (ammonium sulfate), 0.0011 g / L FeSO4·7H2O, and 30 g / L NaCl; The biomass (OD600) of the culture cultured with glucose as the sole carbon source was measured using a Thermo microplate reader at 2 h, 4 h, 6 h, 8 h, 12 h, 16 h, 24 h, 36 h, and 48 h of culture. The experiment was repeated three times, and the test results are shown in Table 1. Figure 2The content of ectoine in the fermentation supernatant was detected after 12 h of culture of M63 with glucose, sucrose, and soluble starch as the single carbon source and ammonium sulfate as the single nitrogen source. The experiment was repeated three times. The test results are shown in Figure 3 ;
[0082] Wherein, the composition of the fermentation medium ① in the above step (3) is as follows: KH2PO4 13.61 g / L, KOH 4.21 g / L, MgSO4·7H2O 0.25 g / L, nitrogen source (ammonium sulfate) 2 g / L, FeSO4·7H2O 0.0011 g / L, NaCl 30 g / L and carbon source (glucose) 30 g / L; the content of each component in fermentation medium ②, fermentation medium ③, fermentation medium ④ and fermentation medium ⑤ are the same as those in fermentation medium ①, except that the carbon source of fermentation medium ② is sucrose, the carbon source of fermentation medium ③ is soluble starch, the carbon source of fermentation medium ④ is lactose, and the carbon source of fermentation medium ⑤ is glycerol.
[0083] Specifically, OD600 is the optical density value measured at a wavelength of 600 nm. The OD600 value is proportional to the cell density of the microorganisms in the bacterial solution. The higher the cell density, the greater the OD600 value. Figure 2 It can be seen that when glucose was used as the only carbon source, there was a significant increase in biomass (OD600 value), indicating that Vibrio alginolyticus ATCC 33787 can grow using glucose alone and can be used for related fermentation research.
[0084] from Figure 3 It can be seen that when the carbon source is sucrose, the content of ectoine in the fermentation supernatant is the highest, followed by soluble starch and glucose. When the carbon source is soluble starch and sucrose, the difference in the content of ectoine in the fermentation supernatant is less than 0.05 g / L.
[0085] Example 2
[0086] A method for producing ectoine using Vibrio alginolyticus, comprising the following steps:
[0087] (1) Take an inoculation loop and streak Vibrio alginolyticus ATCC 33787 onto an LBS plate on a clean bench. Activate the plate at 30°C for 12 h until a single clone grows to obtain activated bacteria.
[0088] (2) In an ultra-clean workbench, a monoclonal colony of step (1) was picked up with a sterile inoculation loop, inoculated into a 20 mL test tube containing 10 mL of seed culture medium, and cultured at 30°C and 200 rpm for 12 h. Subsequently, 200 μl of the colony was taken with a pipette and inoculated into a 20 mL test tube containing 10 mL of seed culture medium, and cultured at 30°C and 200 rpm for 12 h to obtain a seed solution; wherein the components of the seed culture medium are as follows: KH2PO4 13.61 g / L, KOH 4.21 g / L, MgSO4·7H2O 0.25 g / L, nitrogen source (ammonium sulfate) 2 g / L, FeSO4·7H2O 0.0011 g / L, and NaCl 30 g / L;
[0089] (3) Inoculate the seed liquid with an inoculum amount of 2% into a 500 mL conical flask containing 100 mL fermentation medium using a pipette, the pH of the fermentation medium is 7.0, and culture at 30° C. and 200 rpm for 12 h to obtain a fermentation broth containing tetrahydropyrimidine; wherein the components of the fermentation medium of this embodiment are as follows: KH2PO4 13.61 g / L, KOH 4.21 g / L, MgSO4·7H2O 0.25 g / L, nitrogen source ((NH4)2SO4) 2 g / L, FeSO4·7H2O 0.0011 g / L, NaCl 30 g / L and soluble starch 30 g / L, and adjust the pH to 7 with KOH;
[0090] (4) After separating and purifying the fermentation bacterial liquid containing ectoine, ectoine is obtained.
[0091] Specifically, the content of ectoine obtained in this example was determined by taking a fermentation broth containing ectoine, centrifuging it at 12000 rpm for 5 min, then taking 196 μl of the supernatant, adding 4 μl of acetonitrile, filtering it with a 0.22 μm filter membrane, and completing the sample processing. The content of ectoine was determined by high performance liquid chromatography (HPLC), using a C18 chromatographic column; the mobile phase was acetonitrile (liquid A) and pure water (liquid B) and liquid A: liquid B = 2:98; the injection volume was 10 μl; the flow rate was 0.5 ml / min; the detection wavelength was 210 nm.
[0092] Figure 4 is the chromatogram of ectoine standard. Figure 5 This is the chromatogram of ectoine obtained by the method of Example 2. By comparison, it can be seen that the two are the same, which proves that ectoine can be produced by using soluble starch as a carbon source and Vibrio alginolyticus ATCC33787 as a fermentation bacterium.
[0093] Example 3
[0095] The method for producing ectoine by using Vibrio alginolyticus in this embodiment is the same as that in Example 2, except that the carbon source used in the fermentation medium in step (3) of this embodiment is 30 g / L glucose, and ectoine is produced by the method of this embodiment.
[0096] Example 4
[0097] The method for producing ectoine by using Vibrio alginolyticus in this embodiment is the same as that in Example 2, except that the carbon source used in the fermentation medium in step (3) of this embodiment is 30 g / L sucrose, and ectoine is produced by the method of this embodiment.
[0098] Comparative Example 1
[0099] The method for producing ectoine by using Vibrio alginolyticus in this comparative example is the same as that in Example 2, except that the carbon source used in the fermentation medium in step (3) of this comparative example is 30 g / L glycerol, and ectoine is produced by the method of this example.
[0100] The contents of ectoine obtained in Examples 1-4 and Comparative Example 1 were respectively detected, and the detection results are shown in Table 1 below.
[0101] Table 1 The content of tetrahydropyrimidine produced by shake flask fermentation under different carbon source conditions
[0102] Ectoin(g / L) Example 2 (soluble starch) 0.423 Example 3 (Glucose) 0.257 Example 4 (sucrose) 0.468 Comparative Example 1 (Glycerol) 0
[0103] Specifically, it can be seen from Table 1 that the ectoine yield of Example 4 using sucrose as the carbon source is the highest, and the ectoine yield of Example 2 using soluble starch as the carbon source is close to that of Example 4, which is 0.423 g / L; followed by Example 3 using glucose as the carbon source, with an ectoine yield of 0.257. From the test results of Comparative Example 1, it can be seen that the yield of ectoine produced by Vibrio alginolyticus ATCC 33787 in 8-16 hours with glycerol as the only carbon source is 0, which may be because the speed of producing ectoine using glycerol as the carbon source in Vibrio alginolyticus ATCC 33787 is extremely slow.
[0104] Example 5
[0105] The method for producing ectoine by using Vibrio alginolyticus in this embodiment is the same as that in Example 3, except that the nitrogen source used in the fermentation medium in step (3) of this embodiment is ammonium phosphate, and ectoine is produced by the method of this embodiment.
[0106] Example 6
[0107] The method for producing ectoine by using Vibrio alginolyticus in this embodiment is the same as that in Example 3, except that the nitrogen source used in the fermentation medium in step (3) of this embodiment is ammonium oxalate, and ectoine is produced by the method of this embodiment.
[0108] Example 7
[0109] The method for producing ectoine by using Vibrio alginolyticus in this embodiment is the same as that in Example 3, except that the nitrogen source used in the fermentation medium in step (3) of this embodiment is ammonium acetate, and ectoine is produced by the method of this embodiment.
[0110] Example 8
[0111] The method for producing ectoine by using Vibrio alginolyticus in this embodiment is the same as that in Example 3, except that the nitrogen source used in the fermentation medium in step (3) of this embodiment is ammonium chloride, and ectoine is produced by the method of this embodiment.
[0112] The contents of ectoine obtained by the methods of Examples 5-8 were respectively detected and compared with those of Example 3. The detection results are shown in Table 2 below.
[0113] Table 2 The content of tetrahydropyrimidine produced by shake flask fermentation under different nitrogen sources
[0114] Ectoin(g / L) Example 3 (ammonium sulfate) 0.257 Example 5 (ammonium phosphate) 0.220 Example 6 (ammonium oxalate) 0.148 Example 7 (Ammonium Acetate) 0.206 Example 8 (ammonium chloride) 0.158
[0115] Specifically, it can be seen from the inspection results in Table 2 that, compared with other nitrogen sources, Example 3 uses Vibrio alginolyticus ATCC33787 as the fermentation bacteria and uses ammonium sulfate as the nitrogen source to produce the highest yield of ectoine. Using ammonium phosphate and ammonium acetate as nitrogen sources, the yield of ectoine produced is close to that of Example 3, which are 0.220 g / L and 0.206 g / L, respectively, followed by Example 8 and finally Example 6.
[0116] Example 9
[0117] The method for producing ectoine by using Vibrio alginolyticus in this embodiment is the same as that in Example 3, except that the fermentation temperature in step (3) of this embodiment is 23° C., and ectoine is produced by the method of this embodiment.
[0118] Example 10
[0119] The method for producing ectoine by using Vibrio alginolyticus in this embodiment is the same as that in Example 3, except that the fermentation temperature in step (3) of this embodiment is 37° C., and ectoine is produced by the method of this embodiment.
[0120] The contents of ectoine obtained by the methods of Examples 9 and 10 were respectively detected and compared with those of Example 3. The detection results are shown in Table 3 below.
[0121] Table 3 The content of tetrahydropyrimidine produced by shake flask fermentation at different fermentation temperatures
[0122] Ectoin(g / L) Example 3 (30°C) 0.257 Example 9 (23°C) 1.323 Example 10 (37°C) 0.124
[0123] As can be seen from Table 3, in Example 9, when the fermentation temperature is 23°C, the content of ectoine produced by fermentation is the highest, which is 1.323 g / L; followed by Example 3, when the fermentation temperature is 30°C, the content of ectoine is 0.257 g / L; and finally, Example 10, when the fermentation temperature is 37°C, the content of ectoine is 0.124 g / L.
[0124] The technical principle of the present invention is described above in conjunction with specific embodiments. These descriptions are only for explaining the principle of the present invention and cannot be interpreted as limiting the scope of protection of the present invention in any way. Based on the explanations herein, those skilled in the art can associate other specific implementations of the present invention without paying creative labor, and these methods will fall within the scope of protection of the present invention.
Claims
1. A method for producing ectoine using Vibrio alginolyticus, characterized in that: Vibrio alginolyticus ATCC 33787 is used for fermentation culture to produce ectoine. The carbon source used in the fermentation culture process is any one of starch, sucrose and glucose. The fermentation time is 8 to 16 hours.
2. The method for producing ectoine using Vibrio alginolyticus according to claim 1, characterized in that: The concentration of the carbon source in the fermentation medium used in the fermentation culture process is 10-50 g / L.
3. The method for producing ectoine using Vibrio alginolyticus according to claim 1, characterized in that: The carbon source is soluble starch.
4. The method for producing ectoine using Vibrio alginolyticus according to claim 1, characterized in that: The fermentation temperature during the fermentation culture is 20 to 42°C.
5. The method for producing ectoine using Vibrio alginolyticus according to claim 1, characterized in that: In the fermentation culture, a nitrogen source is also added to the fermentation medium, wherein the nitrogen source is any one of ammonium sulfate, ammonium oxalate, ammonium acetate, ammonium chloride and ammonium phosphate; The concentration of the nitrogen source in the fermentation medium is 1-2 g / L.
6. The method for producing ectoine using Vibrio alginolyticus according to claim 5, characterized in that: The components of the fermentation medium used in the fermentation culture include: KH2PO4 12-15g / L, KOH 4-5g / L, MgSO4·7H2O 0.18-0.3g / L, nitrogen source 1-2g / L, FeSO4·7H2O 0.0008-0.0015g / L, NaCl 20-40g / L and carbon source 10-50g / L.
7. The method for producing ectoine using Vibrio alginolyticus according to claim 6, characterized in that: The steps include: (1) inoculating the alginolytic Vibrio strain into a solid culture medium for activation to obtain activated bacteria; (2) inoculating the activated bacterial cells into a seed culture medium for seed culture to obtain a seed solution; (3) inoculating the seed liquid into a fermentation medium for fermentation culture to obtain a fermentation liquid containing ectoine; (4) After separating and purifying the fermentation broth containing ectoine, ectoine is obtained.
8. The method for producing ectoine using Vibrio alginolyticus according to claim 7, characterized in that: In step (3), the seed liquid is inoculated into the fermentation medium at an inoculation amount of 1 to 10% for fermentation culture.
9. The method for producing ectoine using Vibrio alginolyticus according to claim 7, characterized in that: In step (3), the seed liquid is inoculated into the fermentation medium, placed on a shaker, and fermented at 160 to 200 rpm for 8 to 16 hours to obtain a fermentation liquid containing ectoine.
10. The method for producing ectoine using Vibrio alginolyticus according to claim 7, characterized in that: In step (2), the components of the seed culture medium include: KH2PO4 12-15 g / L, KOH 4-5 g / L, MgSO4·7H2O 0.18-0.3 g / L, nitrogen source 0-2 g / L, FeSO4·7H2O 0.0008-0.0015 g / L and NaCl 20-40 g / L.