CN106282252A - A kind of high density fermentation prepares culture medium and the fermentation process thereof of gamma-polyglutamic acid- - Google Patents
A kind of high density fermentation prepares culture medium and the fermentation process thereof of gamma-polyglutamic acid- Download PDFInfo
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- CN106282252A CN106282252A CN201510287786.4A CN201510287786A CN106282252A CN 106282252 A CN106282252 A CN 106282252A CN 201510287786 A CN201510287786 A CN 201510287786A CN 106282252 A CN106282252 A CN 106282252A
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Abstract
The invention discloses a kind of high density fermentation and prepare culture medium and the fermentation process thereof of gamma-polyglutamic acid-, belong to bioengineering field.The present invention selects bacillus subtilis Z-115 for producing strain, uses the strategy adding different fed-batch fermentation culture medium, and high density fermentation produces gamma-polyglutamic acid-.Preparation process include produce strain prepare, the preparation of culture medium, high density fermentation, product extract.Using high density fermentation culture medium and the bacillus subtilis of fermentation process cultivation thereof of the present invention, cell density relatively common fermentation significantly improves, and viable count is not less than 1010Cfu/ml, it is achieved that the high density fermentation of bacillus subtilis, such that it is able to reduce product separation expense, shortens the production cycle, reduces production cost, improve production efficiency.
Description
Technical field
The present invention relates to a kind of high density fermentation and prepare culture medium and the fermentation process thereof of gamma-polyglutamic acid-, belong to bioengineering field.
Background technology
Gamma-polyglutamic acid-is the outer amino acid polymer of a kind of born of the same parents that microorganism produces; there is excellent water solublity, superpower adsorptivity and biodegradability; catabolite is non-harmful glutamic acid; it it is a kind of excellent environment-friendly type macromolecule material; the biggest commercial value and social value can be all had in industries such as cosmetics, environmental conservation, food, medicine, agricultural, control of desert as water-retaining agent, adsorbent for heavy metal, flocculant, slow releasing agent and pharmaceutical carrier etc..
High density fermentation seeks in unit interval, unit reactor volume, on the basis of not affecting intracellular product yield, and accumulation cell concentration as much as possible.Culture medium in high cell density fermentation should fully meet the nutritional requirement of microorganism, and provides suitable environmental condition for the growth and breeding of cell.
Bacillus subtilis Z115 is the strain bacterial strain (Zhang Chao being separated by Shandong University Building biotech research center and being preserved, Luan Xingshe, Zhu Mingsheng, Sun Na, Yang Tongjian, Wang Shuyan. various aminoacid produce the facilitation of polyglutamic acid to bacillus subtilis. food industry [J], 2013,1(34): 119-122.), this bacterial strain belongs to bacillus.In above-mentioned article, author passes through experimentation various aminoacid and fermentation of bacillus subtilis produces the impacts of polyglutamic acid.But this technology also exists in actual applications, and product design is low, production efficiency is low, complex operation, amino acid media high in cost of production not enough, limits and is widely popularized and applies.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of product design is high, efficiency is high, easily operated, the practical industrialization culture medium of low cost and fermentation process thereof.
Concrete technical scheme is as follows:
(1) slant strains is cultivated: bacillus subtilis Z-115 is inoculated in slant medium, and heat insulating culture 48h at 37 DEG C；
(2) shaking flask spawn culture: after strain activates well on slant medium, every strain bacterium takes a full ring and is linked in 100 mL fermentation cultures (250 mL triangular flask), and 200r/min, 24 h cultivated by 37 DEG C of shaking tables；
(3) preparation of seed culture medium: be transferred in 500 mL fermentation cultures (1000 mL triangular flask) by the inoculum concentration of 10%, 200r/min, 48 h cultivated by 37 DEG C of shaking tables；
(4) fermentation: loading basal fermentation medium by the liquid amount of 75% in 5L fermentation tank, 5-10% accesses production strain and is stirred aerobic culture 48-50h the most by volume；The condition of culture of described fermentation tank is: speed of agitator is 200r/min, and temperature is 37 DEG C, and tank pressure is 0.01-0.03MPa；
(5) product extracts: fermentation liquid is centrifuged 10min in 10000r/min and removes thalline, supernatant adds 2.5 times of volume dehydrated alcohol, shake up, 10000r/min, 5min are so centrifugal that to precipitate, precipitation distilled water dissolves, dialysed overnight, dialysis solution adds 2 times of volume dehydrated alcohol, and the precipitate obtained is dried in 105 DEG C of baking ovens.
In said method, the slant culture based formulas in step (1) is: sucrose 20g/L, peptone 10g/L, sodium chloride 5g/L, sodium glutamate 20g/L, and g/L, pH7.0,121 DEG C of sterilizing 15 min is stand-by for agar 18.
In said method, the shake-flask culture based formulas in step (2) is: sucrose 20g/L, peptone 10g/L, sodium chloride 5g/L, and sodium glutamate 20g/L, pH7.0,121 DEG C sterilizing 15 min is stand-by.
The same Shake flask medium of seed culture based formulas in said method, in step (3).
In said method, the basal fermentation medium in step (4): sucrose 30-50g/L, peptone 20-30g/L, sodium chloride 10-16g/L, sodium glutamate 30-40g/L, pH7.0,121 DEG C sterilizing 15 min；Fed-batch fermentation culture medium A: sucrose 80-100g/L；Peptone 30-40g/L, pH7.0,121 DEG C sterilizing 15 min；Fed-batch fermentation culture medium B: sucrose 80-100g/L；Sodium glutamate 40-60g/L；Peptone 30-40g/L, pH7.0,121 DEG C sterilizing 15 min.
In said method, in step (4), after fermentation starts 15 h, starting to add fed-batch fermentation culture medium A, when fermentation liquid light absorption value under 600nm wavelength is not further added by, terminate adding fed-batch fermentation culture medium A, now, bacillus subtilis viable count reaches 1010cfu/ml.Hereafter, sweat maintains the concentration of reduced sugar in fermentation liquid at 40 g/L, when concentration of reduced sugar is less than setting value, start to add fed-batch fermentation culture medium B.
The present invention prepares the method for polyglutamic acid and has technique simply, reduces production cost, improves production efficiency, can carry out large-scale production.
The invention have the advantages that
(1) the invention provides and be suitable to the culture medium of gamma-polyglutamic acid-high density fermentation, improve cell density.
(2) present invention establishes the cultural method being suitable to gamma-polyglutamic acid-high density fermentation, reduces production cost, improves production efficiency.
(3)
During the fermentation, it not simply to carry out feed supplement, but two kinds of different supplemented mediums of the employing of novelty, a kind of fed-batch fermentation culture medium A is applicable to the growth of thalline, and a kind of fed-batch fermentation culture medium B is applicable to the accumulation of product.
Detailed description of the invention
Below by specific embodiment, the present invention will be further elaborated, it should be appreciated that, the description below is merely to explain the present invention, and is not limited thereof.If no special instructions, the percent occurred in following embodiment is percetage by weight.
Embodiment
1
1, slant strains is cultivated: bacillus subtilis Z-115 is inoculated in slant medium, and heat insulating culture 48h at 37 DEG C；Slant culture based formulas is: sucrose 20g/L, peptone 10g/L, sodium chloride 5g/L, sodium glutamate 20g/L, agar 18 g/L, pH7.0.
2, shaking flask spawn culture: after strain activates well on slant medium, every strain bacterium takes a full ring and is linked in 100 mL fermentation cultures (250 mL triangular flask), and 200r/min, 24 h cultivated by 37 DEG C of shaking tables；Shake-flask culture based formulas is: sucrose 20g/L, peptone 10g/L, sodium chloride 5g/L, sodium glutamate 20g/L, pH7.0.
3, the preparation of seed culture medium: be transferred in 500 mL fermentation cultures (1000 mL triangular flask) by the inoculum concentration of 10%, 200r/min, 48 h cultivated by 37 DEG C of shaking tables；The same Shake flask medium of seed culture based formulas.
4, fermentation: loading basal fermentation medium by the liquid amount of 75% in 5L fermentation tank, 5-10% accesses production strain and is stirred aerobic culture 48-50h the most by volume.Described basal fermentation medium: sucrose 50g/L, peptone 30g/L, sodium chloride 16g/L, sodium glutamate 40g/L, pH7.0；Fed-batch fermentation culture medium A: sucrose 100g/L；Peptone 40g/L, pH7.0；Fed-batch fermentation culture medium B: sucrose 100g/L；Sodium glutamate 60g/L；Peptone 40g/L, pH7.0.The condition of culture of described fermentation tank is: speed of agitator is 200r/min, and temperature is 37 DEG C, and tank pressure is 0.01-0.03MPa.Training method is: after fermentation starts 15 h, start to add fed-batch fermentation culture medium A, when fermentation liquid light absorption value under 600nm wavelength is not further added by, terminates adding fed-batch fermentation culture medium A, and now, bacillus subtilis viable count reaches 1010cfu/ml.Hereafter, sweat maintains the concentration of reduced sugar in fermentation liquid at 40 g/L, when concentration of reduced sugar is less than setting value, start to add fed-batch fermentation culture medium B.
5, product extracts: fermentation liquid is centrifuged 10min in 10000r/min and removes thalline, supernatant adds 2.5 times of volume dehydrated alcohol, shake up, 10000r/min, 5min are so centrifugal that to precipitate, and precipitation distilled water dissolves, dialysed overnight, dialysis solution adds 2 times of volume dehydrated alcohol, the precipitate obtained is dried in 105 DEG C of baking ovens, obtains gamma-polyglutamic acid-crude product, and yield is 37.6g/L.
Embodiment
2
The method using embodiment 1 prepares gamma-polyglutamic acid-, and except for the difference that, the process that step 4 uses is: load basal fermentation medium by the liquid amount of 75% in 5L fermentation tank, and 5% access the most by volume produces strain and is stirred aerobic culture 48-50h.Described basal fermentation medium: sucrose 30g/L, peptone 20g/L, sodium chloride 10g/L, sodium glutamate 30g/L, pH7.0；Fed-batch fermentation culture medium A: sucrose 80g/L；Peptone 30g/L, pH7.0；Fed-batch fermentation culture medium B: sucrose 80g/L；Sodium glutamate 40g/L；Peptone 30g/L, pH7.0.The ultimate output of gamma-polyglutamic acid-is 33.5g/L.
Embodiment
3
The method using embodiment 1 prepares gamma-polyglutamic acid-, and except for the difference that, the process that step 4 uses is: load basal fermentation medium by the liquid amount of 75% in 5L fermentation tank, and 7% access the most by volume produces strain and is stirred aerobic culture 48-50h.Described basal fermentation medium: sucrose 40g/L, peptone 25g/L, sodium chloride 14g/L, sodium glutamate 35g/L, pH7.0；Fed-batch fermentation culture medium A: sucrose 90g/L；Peptone 35g/L, pH7.0；Fed-batch fermentation culture medium B: sucrose 90g/L；Sodium glutamate 50g/L；Peptone 35g/L, pH7.0.The ultimate output of gamma-polyglutamic acid-is 36.5g/L.
Claims (6)
1. high density fermentation prepares culture medium and a fermentation process thereof for gamma-polyglutamic acid-, it is characterized in that comprising the following steps:
(1) slant strains is cultivated: bacillus subtilis Z-115 is inoculated in slant medium, and heat insulating culture 48h at 37 DEG C；
(2) shaking flask spawn culture: after strain activates well on slant medium, every strain bacterium takes a full ring and is linked in 100 mL fermentation cultures (250 mL triangular flask), and 200r/min, 24 h cultivated by 37 DEG C of shaking tables；
(3) preparation of seed culture medium: be transferred in 500 mL fermentation cultures (1000 mL triangular flask) by the inoculum concentration of 10%, 200r/min, 48 h cultivated by 37 DEG C of shaking tables；
(4) fermentation: loading basal fermentation medium by the liquid amount of 75% in 5L fermentation tank, 5-10% accesses production strain and is stirred aerobic culture 48-50h the most by volume；The condition of culture of described fermentation tank is: speed of agitator is 200r/min, and temperature is 37 DEG C, and tank pressure is 0.01-0.03MPa；
(5) product extracts: fermentation liquid is centrifuged 10min in 10000r/min and removes thalline, supernatant adds 2.5 times of volume dehydrated alcohol, shake up, 10000r/min, 5min are so centrifugal that to precipitate, precipitation distilled water dissolves, dialysed overnight, dialysis solution adds 2 times of volume dehydrated alcohol, and the precipitate obtained is dried in 105 DEG C of baking ovens.
A kind of high density fermentation the most according to claim 1 prepares culture medium and the fermentation process thereof of gamma-polyglutamic acid-, it is characterized in that the slant culture based formulas in step (1) is: sucrose 20g/L, peptone 10g/L, sodium chloride 5g/L, sodium glutamate 20g/L, g/L, pH7.0,121 DEG C of sterilizing 15 min is stand-by for agar 18.
A kind of high density fermentation the most according to claim 1 prepares culture medium and the fermentation process thereof of gamma-polyglutamic acid-, it is characterized in that the shake-flask culture based formulas in step (2) is: sucrose 20g/L, peptone 10g/L, sodium chloride 5g/L, sodium glutamate 20g/L, pH7.0,121 DEG C of sterilizing 15 min are stand-by.
A kind of high density fermentation the most according to claim 1 prepares culture medium and the fermentation process thereof of gamma-polyglutamic acid-, it is characterised in that the same Shake flask medium of seed culture based formulas in step (3).
A kind of high density fermentation the most according to claim 1 prepares culture medium and the fermentation process thereof of gamma-polyglutamic acid-, it is characterized in that basal fermentation medium in step (4): sucrose 30-50g/L, peptone 20-30g/L, sodium chloride 10-16g/L, sodium glutamate 30-40g/L, pH7.0,121 DEG C of sterilizing 15 min；Fed-batch fermentation culture medium A: sucrose 80-100g/L；Peptone 30-40g/L, pH7.0,121 DEG C sterilizing 15 min；Fed-batch fermentation culture medium B: sucrose 80-100g/L；Sodium glutamate 40-60g/L；Peptone 30-40g/L, pH7.0,121 DEG C sterilizing 15 min.
A kind of high density fermentation the most according to claim 1 prepares culture medium and the fermentation process thereof of gamma-polyglutamic acid-, it is characterized in that in step (4), after fermentation starts 15 h, start to add fed-batch fermentation culture medium A, when fermentation liquid light absorption value under 600nm wavelength is not further added by, terminating adding fed-batch fermentation culture medium A, now, bacillus subtilis viable count reaches 1010Cfu/ml, hereafter, maintains the concentration of reduced sugar in fermentation liquid at 40 g/L, when concentration of reduced sugar is less than setting value, starts to add fed-batch fermentation culture medium B in sweat.
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| CN107287254A (en) * | 2017-07-14 | 2017-10-24 | 武汉乐阳生物科技有限公司 | The zymotechnique of γ polyglutamic acids |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107287254A (en) * | 2017-07-14 | 2017-10-24 | 武汉乐阳生物科技有限公司 | The zymotechnique of γ polyglutamic acids |
| CN107287254B (en) * | 2017-07-14 | 2021-06-29 | 武汉光华时代生物科技有限公司 | Fermentation process of gamma-polyglutamic acid |
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