CN112795601B - Fermentation method for increasing L-hydroxyproline yield - Google Patents
Fermentation method for increasing L-hydroxyproline yield Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 155
- 230000004151 fermentation Effects 0.000 title claims abstract description 155
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 title claims abstract description 51
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 title claims abstract description 50
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- MUNWAHDYFVYIKH-PHDIDXHHSA-N cis-4-hydroxy-D-proline betaine Chemical compound C[N+]1(C)C[C@H](O)C[C@@H]1C([O-])=O MUNWAHDYFVYIKH-PHDIDXHHSA-N 0.000 description 1
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- 125000001841 imino group Chemical group [H]N=* 0.000 description 1
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- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical class [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/24—Proline; Hydroxyproline; Histidine
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a fermentation method for improving the yield of L-hydroxyproline, which comprises the following steps: (1) Inoculating the seed liquid of the L-hydroxyproline production strain into a sterilized fermentation tank filled with a fermentation culture medium for fermentation at an initial stage; fermentation conditions are as follows: the temperature is 32-33 ℃, the air volume is 3-4L/min, the stirring speed is 200rpm, and the tank pressure is 0.02-0.04MPa; when the strain is in the logarithmic phase, slowly increasing the stirring speed to 540rpm; (2) When the residual sugar in the fermentation liquor is reduced to 0-0.1g/L, performing the middle and later stages of fermentation; fermentation conditions are as follows: the temperature is 32-33 ℃, the pH is 7.0 +/-0.1, the rotating speed is 750rpm, the tank pressure is 0.02MPa, the air quantity is 6-7L/min, and when the OD600 of 200 times of diluted fermentation liquor reaches 0.6-0.7, the fermentation is finished. The method can improve the fermentation yield of the L-hydroxyproline and the conversion rate of the saccharic acid.
Description
Technical Field
The invention relates to the technical field of L-hydroxyproline production, in particular to a fermentation method for increasing the yield of L-hydroxyproline.
Background
Hydroxyproline (Hydroxyproline) is one of the imino acids, usually with a hydroxyl group at the fourth position, but sometimes also at the 3 rd position. There are 4 stereoisomers due to the two asymmetric carbon atoms. The animal glue and the ossein contain L-hydroxyproline, and D-hydroxyprolic acid does not exist in nature. L-allohydroxyproline is a component of the toxic polypeptide phalloidin (pharoids) obtained from Amanita pharoids.
Such amino acids are not present in typical proteins. Among the methyl derivatives of hydroxyproline in nature are levo-stachydrine (betanicine), dextro-stachydrine (turicine) and gamma-hydroxy archaeal acid (gamma-A hydroxygraphic acid). Hydroxyproline is synthesized in vivo from proline, but is not in a free state but is hydroxylated in a peptide chain. Its decomposition is similar to proline, but proceeds with hydroxyl groups. The molecular formula is as follows: c 5 H 9 NO 3 Molecular weight 131.13. Has unique sweet taste in bitter taste, and can improve flavor of fruit juice beverage, cold beverage, etc. Has special flavor, and can be used as fragrant raw material. The application is as follows: flavoring agent, nutrition enhancer, and flavoring agent, and is mainly used for fruit juice, cold beverage, nutritional beverage, etc.; used as a biochemical reagent.
The existing hydroxyproline production methods comprise a chemical decomposition method and a microbial fermentation method. The chemical method is to hydrolyze proteins such as gelatin, bone glue, casein, soybean epidermis and the like by hydrochloric acid, extract imide acid by a nitrosation method, and then refine and crystallize the imide acid after resin chromatography, the yield is about 7.0 percent, and the defects of limited raw material sources, high production cost, environmental pollution and the like exist, so that the industrial production is difficult to realize.
The microbial fermentation method has the advantages of low raw material cost, mild reaction conditions, easy realization of large-scale production and the like. With the intensive research on L-hydroxyproline fermentation, microbial fermentation has become the most important method for producing L-hydroxyproline. According to related reports, the fermentation yield level of domestic L-hydroxyproline is about 50g/L, the international level is about 70g/L, and the existing L-hydroxyproline fermentation method still has the problems of low acid yield and low saccharic acid conversion rate. Therefore, how to optimize the fermentation process of the L-hydroxyproline is a problem which needs to be solved urgently at present.
Disclosure of Invention
In view of the above prior art, the present invention aims to provide a fermentation method for increasing the yield of L-hydroxyproline.
In order to realize the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a fermentation method for increasing the yield of L-hydroxyproline, comprising the steps of:
(1) Inoculating the seed liquid of the L-hydroxyproline production strain into a sterilized fermentation tank filled with a fermentation culture medium for fermentation at an initial stage; fermentation conditions are as follows: the temperature is 32-33 ℃, the air volume is 3-4L/min, the stirring speed is 200rpm, and the tank pressure is 0.02-0.04MPa; when the strain is in the logarithmic phase, slowly increasing the stirring speed to 540rpm, detecting the sugar content in the fermentation liquor in real time, and supplementing sugar when the sugar content is lower than 2g/L, so as to maintain the sugar content in the fermentation liquor at 1.9-2.1g/L; when the OD600 of the 100-time diluted fermentation liquor reaches 0.400, adjusting the air quantity to 4L/min, increasing the stirring rotating speed to 600rpm, and stopping sugar supplement;
(2) When the residual sugar in the fermentation liquor is reduced to 0-0.1g/L, performing the later stage in the fermentation; conditions of fermentation: the temperature is 32-33 ℃, the pH is 7.0 +/-0.1, the rotating speed is 750rpm, the tank pressure is 0.02MPa, the air quantity is 6-7L/min, and glucose is fed in to control the residual sugar content in the fermentation liquor to be 0.05-0.1g/L; and when the OD600 of the 200-time diluted fermentation liquor reaches 0.6-0.7 after 52-62 hours, ending the fermentation.
Preferably, the fermentation medium comprises the following components: glucose 20g/L, (NH) 4 ) 2 SO 4 1g/L, 3g/L of yeast powder, 2g/L of citric acid and KH 2 PO 4 5g/L、K 2 HPO 4 5g/L、MgSO 4 ·7H 2 O 1.5g/L、FeSO 4 ·7H 2 O 0.1g/L、MnSO 4 ·H 2 O 9mg/L、Co(NO 3 ) 2 ·6H 2 O 9.8mg/L、VB 1 0.3mg/L、VB 3 0.3mg/L and 100mg/L of ampicillin; pH7.0. + -. 0.1.
Preferably, in step (1), the inoculation amount of the seed solution is 8%.
Preferably, the seed solution of the L-hydroxyproline production strain is obtained by culturing the L-hydroxyproline production strain through a first-stage seed bottle and a second-stage seed tank.
Preferably, the culture medium for the first-class seed bottle culture is LB liquid culture medium.
Preferably, after the strains in the first-stage seed bottle are cultured to a logarithmic phase, the bacterial liquid in the first-stage seed bottle is inoculated to the second-stage seed tank for culture, and the inoculation amount is 5-15%.
Preferably, the medium components of the secondary seeding tank culture are as follows: glucose 30g/L, (NH) 4 ) 2 SO 4 1g/L, 6g/L yeast powder, lemon2g/L of citric acid and KH 2 PO 4 2.5g/L、K 2 HPO 4 2.5g/L、MgSO 4 ·7H 2 O 1g/L、MnSO 4 ·H 2 O 9mg/L、ZnSO 4 12.8mg/L、Co(NO 3 ) 2 ·6H 2 O 9.8mg/L、VB 1 1mg/L、VB 3 1mg/L and 100mg/L of ampicillin; pH 7.0. + -. 0.1.
Preferably, the conditions for secondary seeding tank culture are as follows: the temperature is 32-33 ℃, the pH value is 7.0, the air quantity is 3-4L/min, the stirring speed is 200rpm, and the tank pressure is 0.02-0.04MPa.
Preferably, in the step (1), when the fermentation liquid is subjected to sugar supplement, the glucose solution is added to the fermentation liquid by a peristaltic pump.
The number of the strains is increased sharply in a logarithmic phase, a large amount of glucose is consumed to ensure sufficient energy for splitting, the content of the sugar in the fermentation liquor is reduced sharply at the stage, and the sugar needs to be controlled to be about 2 g/L.
Preferably, the pH is controlled by feeding ammonia water with a mass concentration of 20-30% during fermentation.
In a second aspect of the present invention, there is provided L-hydroxyproline prepared by the above-mentioned method.
The invention has the beneficial effects that:
1. the method has the advantages of simple process, low production cost, and great improvement on the yield of the L-hydroxyproline and the conversion rate of the saccharic acid, and is particularly suitable for industrial production.
2. According to the invention, the fermentation yield of L-hydroxyproline and the saccharic acid conversion rate are improved through anaerobic catalytic fermentation, wherein the yield of L-hydroxyproline exceeds 100g/L, and the saccharic acid conversion rate is more than 30%.
3. The invention further optimizes the fermentation process of the L-hydroxyproline, controls the content of residual sugar in the fermentation liquor to be 0-0.1g/L by feeding glucose, does not form substrate limitation, does not cause glucose effect, and exerts the production capacity of the strain to the maximum extent.
4. The fermentation method can be automatically controlled through the fermentation tank, has simple and convenient process operation, low equipment requirement, short fermentation time, low production cost and obvious economic benefit, and is particularly suitable for industrialized large-scale production.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background section, the prior L-hydroxyproline fermentation method still has the problems of low acid yield and low sugar-acid conversion rate, so that further technical challenges are still needed to improve the fermentation yield of L-hydroxyproline.
Based on the method, the hydroxyproline production strain is inoculated into a fermentation medium containing the hydroxyproline for fermentation to obtain the L-hydroxyproline.
Whether the culture conditions are optimized or not directly influences the change of metabolic modes and metabolic flux, thereby influencing the yield of the target product. Wherein, the composition and the proportion of the fermentation medium have great influence on the growth of strains, the formation of L-hydroxyproline, the quality and the yield of products and the like. The invention optimizes the composition of the fermentation medium, and the optimized fermentation medium comprises the following components:
glucose 20g/L, (NH) 4 ) 2 SO 4 1g/L, 3g/L of yeast powder, 2g/L of citric acid and KH 2 PO 4 5g/L、K 2 HPO 4 5g/L、MgSO 4 ·7H 2 O 1.5g/L、FeSO 4 ·7H 2 O 0.1g/L、MnSO 4 ·H 2 O 9mg/L、Co(NO 3 ) 2 ·6H 2 O 9.8mg/L、VB 1 0.3mg/L、VB 3 0.3mg/L and 100mg/L of ampicillin (added after sterilization); pH 7.0. + -. 0.1.
In the fermentation medium of the invention, KH is selected 2 PO 4 And K 2 HPO 4 The function of the compound is important as an acid-base buffer pair, and strains are inhibited by negative feedback regulation under low pH, namely excessive inhibition generation of productsThanks to the activities, KH is added 2 PO 4 And K 2 HPO 4 The pH value of the fermentation medium is kept in a stable state, which is beneficial to improving the utilization rate of the strains; the yeast powder and the citric acid are necessary components, the strain is an auxotroph engineering strain, and if the strain does not grow without the yeast powder and the citric acid; the invention discovers Fe 2+ 、Mg 2+ 、Mn 2 + 、Zn 2+ 、Co 2+ 、Cu 2+ Is an important ion influencing the growth of the thalli of the engineering bacteria constructed by the invention, and the invention researches Fe by a method combining absorption spectrum and thin-layer chromatography 2+ 、Mg 2+ 、Mn 2+ 、Zn 2+ 、Co 2+ 、Cu 2+ The influence on the growth of engineering bacteria and the synthesis of amino acid is found out, and Fe with proper concentration is found 2+ 、Mg 2+ 、Mn 2+ 、Co 2+ Can promote the increase of the biomass of the thalli and the total amino acid synthesis amount, and then determines Fe through orthogonal experiments 2+ 、Mg 2+ 、Mn 2+ 、Co 2+ The action relationship and the dosage of various trace elements; the fermentation medium is added with VB1 and VB3, because the production strain is engineering bacteria, and proper amount of added vitamin can promote the biomass increase of the engineering bacteria; the fermentation medium of the invention is added with a proper amount of citric acid, so that the enzyme activity of the strain citrate synthase is artificially reduced, the normal running of the citric acid cycle of the thalli is ensured by adding a proper amount of citric acid, and enough energy is provided to ensure the normal running of the biosynthesis of hydroxyproline.
The research of the invention finds that after a proper amount of ampicillin is added into a fermentation medium, the probability of bacterial infection of a fermentation tank is close to 0 percent, the possibility of bacterial infection caused by misoperation is reduced, and simultaneously, the screening effect can be achieved, and engineering bacteria with lost plasmids cannot continue to survive due to multiple passages, and cannot compete with engineering bacteria with high acid production for resources.
In conclusion, the invention optimizes the composition and the dosage of the fermentation culture medium, so that the fermentation culture medium is more favorable for the growth of strains and the formation of L-hydroxyproline.
The invention also provides key process control of fermentation, which comprises the following steps:
and controlling the glucose concentration in the fermented liquid by adopting a segmented sugar supply mode. The initial fermentation stage is the stage of the strain in the lag phase and the early-middle logarithmic phase on the growth curve, when the strain is in the late lag phase, the increase of the number of the strains is not obvious, and at this time, the initial fermentation stage is defined as the end of the initial fermentation stage, and the initial conditions are maintained: the fermentation temperature is 32-33 ℃, the air volume is 3-4L/min, the stirring speed is 200rpm, and the tank pressure is 0.02-0.04MPa; the initial condition is to reduce the time of the strain in the lag phase, and the strain can enter the logarithmic phase at the fastest speed under the condition through research.
When the strain is in the logarithmic phase, slowly increasing the stirring speed to 540rpm, detecting the sugar content in the fermentation liquor in real time, and when the sugar content is lower than 2g/L, slowly supplementing sugar by using a peristaltic pump; the lower limit value of the sugar content is determined for better controlling the sugar content in the next stage without affecting the increase of the number of the bacterial cells.
When the OD600 of the 100-time diluted fermentation liquor reaches 0.400, adjusting the air quantity to 4L/min, and increasing the stirring rotating speed to 600rpm; according to the one-step growth curve of the engineering bacteria, the strains are in the last logarithmic phase to the stationary phase, the rotating speed is increased so as to ensure that the distribution of nutrient substances in the culture medium is more balanced, and the air volume is increased so as to provide sufficient oxygen and increase the biomass of the bacteria.
When the residual sugar in the fermentation liquor is reduced to 0-0.1g/L, the fermentation is started, and the fermentation conditions are as follows: the temperature is 32-33 ℃, the pH value is 7.0 +/-0.1, the rotating speed is 750rpm, the tank pressure is 0.02MPa, the air quantity is 6-7L/min, and glucose is fed in a flowing manner, so that the content of residual sugar in the fermentation liquor is controlled to be 0.05-0.1g/L; and when the OD600 of the 200-time diluted fermentation liquor reaches 0.6-0.7 after 52-62 hours, ending the fermentation. The parameters can maximize the acid production capacity of the engineering bacteria, and the fermentation method is the most suitable for the engineering bacteria.
In the fermentation process, in order to ensure the smooth reaction, the method also comprises the steps of supplementing the defoaming agent, ammonia water and sugar. The sugar is supplied to the fermentation tank through a sugar supply tank under the condition that the peristaltic pump controls the flow rate. The residual sugar content in the fermentation liquor is controlled to be 0.05-0.1g/L by feeding glucose, thus not forming substrate limitation, not causing glucose effect and giving full play to the production capacity of the strain.
The ammonia water is supplemented by adding ammonia water with certain concentration, such as 20-30% by mass concentration into the fermentation tank under the control of peristaltic pump, so that pH of the fermentation liquor is maintained at 7.0, and acid production is affected when pH is high or low.
The defoaming agent is fed according to the foam condition in the fermentation tank.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available.
The yeast powder is OXOIDLP0021.
The detection method of the related indexes in the fermentation process comprises the following steps:
OD value determination: after the fermentation broth was diluted to 1/50, 1/100, and 1/200 with distilled water, the OD value was measured at 600nm with a spectrophotometer.
2.pH: and (5) measuring by an acidimeter.
3. Dissolving oxygen: the dissolved oxygen was measured on-line with an electrode, with the dissolved oxygen level of the dissolved oxygen electrode in air being set at 100% and the dissolved oxygen in a saturated sodium sulfite solution being 0.
4. Residual sugar content, sugar content in fermentation broth: the measurement was carried out by an M100 glucose meter and a titration method, respectively.
5.L-hydroxyproline content: the chromatography method on paper is adopted.
The microbial strain preservation adopted by the invention adopts a glycerin tube for ultra-low temperature preservation. The fermentation process adopts a secondary fermentation tank for fermentation. The fermentation tank used in the experiment of the invention is a 35L fermentation tank.
Example 1: fermentation method for increasing L-hydroxyproline yield
The method comprises the following steps: (1) Inoculating the seed liquid of the L-hydroxyproline production strain into a sterilized fermentation tank filled with a fermentation culture medium according to the inoculation amount of 8% for fermentation initial stage; fermentation conditions are as follows: the temperature is 32-33 ℃, the air volume is 3-4L/min, the stirring speed is 200rpm, and the tank pressure is 0.02-0.04MPa; when the strain is in the logarithmic phase, slowly increasing the stirring speed to 540rpm, detecting the sugar content in the fermentation liquor in real time, and when the sugar content is lower than 2g/L, slowly supplementing sugar by using a peristaltic pump, and maintaining the sugar content in the fermentation liquor at 1.9-2.1g/L; when the OD600 of the 100-time diluted fermentation liquor reaches 0.400, adjusting the air quantity to 4L/min, increasing the stirring rotating speed to 600rpm, and stopping sugar supplement;
the fermentation medium comprises the following components: glucose 20g/L, (NH) 4 ) 2 SO 4 1g/L, 3g/L of yeast powder, 2g/L of citric acid and KH 2 PO 4 5g/L、K 2 HPO 4 5g/L、MgSO 4 ·7H 2 O 1.5g/L、FeSO 4 ·7H 2 O 0.1g/L、MnSO 4 ·H 2 O 9mg/L、Co(NO 3 ) 2 ·6H 2 O 9.8mg/L、VB 1 0.3mg/L、VB 3 0.3mg/L and 100mg/L of ampicillin (added after sterilization); pH7.0 + -0.1;
(2) When the residual sugar in the fermentation liquor is reduced to 0-0.1g/L, performing the middle and later stages of fermentation; conditions of fermentation: the temperature is 32-33 ℃, the pH is 7.0, the rotating speed is 750rpm, the tank pressure is 0.02MPa, the air quantity is 6-7L/min, and glucose is fed in to control the residual sugar content in the fermentation liquor to be 0.05-0.1g/L; after 48 hours, the content of sugar in the fermentation liquor is difficult to control, and the acid production of hydroxyproline reaches more than 80 g/L; at 62h, when the OD600 of the 200-time diluted fermentation liquor reaches 0.7, ending the fermentation; and starting to put and kill the cans after 63 h.
The seed liquid of the L-hydroxyproline production strain is obtained by culturing the L-hydroxyproline production strain through a first-stage seed bottle and a second-stage seed tank. After the strain in the first-stage seed bottle is cultured to a logarithmic phase (the seed solution is cultured for about 10 hours, and the OD600nm of the 50-fold diluted seed solution is about 0.300-0.330), the bacterial liquid in the first-stage seed bottle is inoculated to a second-stage seed tank for culture, and the inoculation amount is 5-15%.
The culture medium for the first-stage seed bottle culture is LB liquid culture medium; second stageThe culture medium for seeding tank culture comprises the following components: glucose 30g/L, (NH) 4 ) 2 SO 4 1g/L, 6g/L of yeast powder, 2g/L of citric acid and KH 2 PO 4 2.5g/L、K 2 HPO 4 2.5g/L、MgSO 4 ·7H 2 O 1g/L、MnSO 4 ·H 2 O 9mg/L、ZnSO 4 12.8mg/L、Co(NO 3 ) 2 ·6H 2 O 9.8mg/L、VB 1 1mg/L、VB 3 1mg/L and 100mg/L of ampicillin (added after the sterilization is finished); pH 7.0. + -. 0.1.
The conditions for secondary seeding tank culture are as follows: the temperature is 32-33 ℃, the pH value is 7.0, the air quantity is 3-4L/min, the stirring speed is 200rpm, and the tank pressure is 0.02-0.04MPa.
In the fermentation process, ammonia water with the mass concentration of 25% is added through a peristaltic pump in a flowing mode, so that the pH value of the fermentation liquor is maintained to be about 7.0; and adding a defoaming agent in a flowing manner for defoaming according to the foam condition in the fermentation tank.
Detection shows that by adopting the fermentation process of the embodiment, the tank volume is 22.92L, the hydroxyproline yield is 2476g, the acid yield is 108g/L, the actual sugar consumption is 6112g, the killed sugar content is 96g/L, and the sugar-acid conversion rate is as follows: (96 × 22.92)/6112 × 100% =36%.
Example 2: fermentation method for increasing L-hydroxyproline yield
The method comprises the following steps:
(1) Inoculating the seed liquid of the L-hydroxyproline production strain into a sterilized fermentation tank filled with a fermentation culture medium according to the inoculation amount of 8% for fermentation initial stage; fermentation conditions are as follows: the temperature is 32-33 ℃, the air volume is 3-4L/min, the stirring speed is 200rpm, and the tank pressure is 0.02-0.04MPa; when the strain is in the logarithmic phase, slowly increasing the stirring speed to 540rpm, detecting the sugar content in the fermentation liquor in real time, and when the sugar content is lower than 2g/L, slowly supplementing sugar by using a peristaltic pump, and maintaining the sugar content in the fermentation liquor at 1.9-2.1g/L; when the OD600 of the 100-time diluted fermentation liquor reaches 0.400, adjusting the air quantity to 4L/min, increasing the stirring rotation speed to 600rpm, and stopping sugar supplement;
the fermentation medium comprises the following components: glucose 20g/L, (NH) 4 ) 2 SO 4 1g/L, 3g/L of yeast powder, 2g/L of citric acid and KH 2 PO 4 5g/L、K 2 HPO 4 5g/L、MgSO 4 ·7H 2 O 1.5g/L、FeSO 4 ·7H 2 O 0.1g/L、MnSO 4 ·H 2 O 9mg/L、Co(NO 3 ) 2 ·6H 2 O 9.8mg/L、VB 1 0.3mg/L、VB 3 0.3mg/L and 100mg/L of ampicillin (added after the sterilization is finished); pH7.0 + -0.1;
(2) When the residual sugar in the fermentation liquor is reduced to 0-0.1g/L, performing the later stage in the fermentation; conditions of fermentation: the temperature is 32-33 ℃, the pH value is 7.0, the rotation speed is 750rpm, the tank pressure is 0.02MPa, the air quantity is 6-7L/min, glucose is fed in to control the residual sugar content in the fermentation liquor to be 0.05-0.1g/L, and when the residual sugar content in the fermentation liquor is 38 hours, the glucose content in the fermentation liquor is 0.11g/L, and sugar supplement is stopped; when the time is 40 hours, the glucose content in the fermentation liquor is reduced, and sugar supplement is started again; after 52 hours, the content of sugar in the fermentation liquor is difficult to control, and the acid production of hydroxyproline reaches more than 80 g/L; at 62h, when the OD600 of the 200-time diluted fermentation liquor reaches 0.66, the fermentation is finished; and (3) starting can discharge and killing the cans after 63 h.
The seed solution of the L-hydroxyproline production strain is obtained by performing primary seed bottle culture and secondary seed tank culture on the L-hydroxyproline production strain. After the strain in the first-stage seed bottle is cultured to a logarithmic phase (the seed solution is cultured for about 10 hours, and the OD600nm of the seed solution diluted by 50 times is about 0.300-0.330), the bacterial liquid in the first-stage seed bottle is inoculated to a second-stage seed tank for culture, and the inoculation amount is 5-15%.
The culture medium for the first-stage seed bottle culture is an LB liquid culture medium; the culture medium for the secondary seeding tank culture comprises the following components: glucose 30g/L, (NH) 4 ) 2 SO 4 1g/L, 6g/L of yeast powder, 2g/L of citric acid and KH 2 PO 4 2.5g/L、K 2 HPO 4 2.5g/L、MgSO 4 ·7H 2 O 1g/L、MnSO 4 ·H 2 O 9mg/L、ZnSO 4 12.8mg/L、Co(NO 3 ) 2 ·6H 2 O 9.8mg/L、VB 1 1mg/L、VB 3 1mg/L and 100mg/L of ampicillin (added after sterilization); pH 7.0. + -. 0.1.
The conditions for secondary seeding tank culture are as follows: the temperature is 32-33 ℃, the pH value is 7.0, the air quantity is 3-4L/min, the stirring speed is 200rpm, and the tank pressure is 0.02-0.04MPa.
In the fermentation process, ammonia water with the mass concentration of 25% is added through a peristaltic pump in a flowing mode, so that the pH value of the fermentation liquor is maintained to be about 7.0; and adding a defoaming agent in a flowing manner for defoaming according to the foam condition in the fermentation tank.
Through detection, by adopting the fermentation process of the embodiment, the tank placing volume is 22.7L, the hydroxyproline production is 2338g, the acid production amount is 103.5g/L, the actual sugar consumption is 6732g, the post-killing sugar content is 91.15g/L, and the sugar-acid conversion rate is as follows: (91.15 × 22.7)/6732 × 100% =30.7%.
Comparative example 1
In comparison with example 1, in step (2), the fermentation conditions at the latter stage in the fermentation were not subjected to residual sugar content control. As a result, compared with example 1, the sugar consumption was increased by about 15%, the acid production was decreased by about 20%, and other secondary metabolites were increased, which seriously increased the difficulty of the late-stage extraction.
Comparative example 2
Compared with example 1, the components of the fermentation medium are changed, and the components of the fermentation medium do not contain Co (NO) 3 ) 2 ·6H 2 O、VB 1 And VB 3 . As a result, the OD600 was less than 0.400 at 20 hours in the fermentor, and the number of cells was insufficient, and acid was hardly produced in the latter stage.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (1)
1. A fermentation method for improving the yield of L-hydroxyproline is characterized by comprising the following steps:
inoculating the seed liquid of the L-hydroxyproline production strain into a 35L sterilized fermentation tank filled with a fermentation culture medium for fermentation at an initial stage; fermentation conditions are as follows: the temperature is 32-33 ℃, the air volume is 3-4L/min, the stirring speed is 200rpm, and the tank pressure is 0.02-0.04MPa; when the strain is in the logarithmic phase, slowly increasing the stirring speed to 540rpm, detecting the sugar content in the fermentation liquor in real time, and supplementing sugar when the sugar content is lower than 2g/L, so as to maintain the sugar content in the fermentation liquor at 1.9-2.1g/L; when the OD600 of the 100-time diluted fermentation liquor reaches 0.400, adjusting the air quantity to 4L/min, increasing the stirring rotating speed to 600rpm, and stopping sugar supplement; when the residual sugar in the fermentation liquor is reduced to 0-0.1g/L, performing the middle and later stages of fermentation; and (3) fermentation conditions: the temperature is 32-33 ℃, the pH value is 7.0, the rotation speed is 750rpm, the tank pressure is 0.02MPa, the air quantity is 6-7L/min, and the glucose is fed to control the residual sugar content in the fermentation liquor to be 0.05-0.1g/L; at 62h, when the OD600 of the 200-time diluted fermentation liquor reaches 0.7, ending the fermentation;
the fermentation medium comprises the following components: glucose 20g/L, (NH) 4 ) 2 SO 4 1g/L, 3g/L of yeast powder, 2g/L of citric acid and KH 2 PO 4 5g/L、K 2 HPO 4 5g/L、MgSO 4 ·7H 2 O 1.5g/L、FeSO 4 ·7H 2 O 0.1g/L、MnSO 4 ·H 2 O 9mg/L、Co (NO 3 ) 2 ·6H 2 O 9.8mg/L、VB 1 0.3mg/L、VB 3 0.3mg/L and 100mg/L of ampicillin; pH7.0 + -0.1;
the seed liquid of the L-hydroxyproline production strain is obtained by culturing the L-hydroxyproline production strain through a first-stage seed bottle and a second-stage seed tank;
the culture medium for the secondary seeding tank culture comprises the following components: glucose 30g/L, (NH) 4 ) 2 SO 4 1g/L, 6g/L of yeast powder, 2g/L of citric acid and KH 2 PO 4 2.5g/L、K 2 HPO 4 2.5g/L、MgSO 4 ·7H 2 O 1g/L、MnSO 4 ·H 2 O 9mg/L、ZnSO 4 12.8mg/L、Co(NO 3 ) 2 ·6H 2 O 9.8mg/L、VB 1 1mg/L、VB 3 1mg/L and 100mg/L of ampicillin; pH7.0 + -0.1; the culture conditions are as follows: the temperature is 32-33 ℃, the pH value is 7.0, the air quantity is 3-4L/min, the stirring speed is 200rpm, and the tank pressure is 0.02-0.04MPa;
in the step (1), the inoculation amount of the seed liquid is 8%;
the culture medium for the first-stage seed bottle culture is LB liquid culture medium;
after the strains in the first-stage seed bottle are cultured to a logarithmic phase, inoculating the bacterial liquid in the first-stage seed bottle to a second-stage seed tank for culture, wherein the inoculation amount is 5-15%;
in the step (1), when sugar is supplemented to the fermentation liquor, a glucose solution is added into the fermentation liquor by a peristaltic pump.
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