CN103320484A - Method for improving the fermentation yield of hyaluronic acid (HA) - Google Patents
Method for improving the fermentation yield of hyaluronic acid (HA) Download PDFInfo
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Abstract
The invention discloses a method for improving the fermentation yield of hyaluronic acid (HA), and aims to solve the problems that the yield of the HA is restricted under the influence of the physiological metabolic properties of streptococcus zooepidemicus serving as production strains, while the yield of the HA produced by a microbial fermentation method is required to be improved and the production cost is required to be reduced imperatively at present. The method comprises the following steps: slant culture, seed culture, fermentation culture, first-stage culture and second-stage culture. By utilizing the method, the fermentation yield of the HA can be further improved and the fermentation comprehensive cost is reduced in the mode of combining fed-batch culture and intermittent pH stress, so that the application of the HA in the field of medicaments, makeup and food healthcare is promoted. By utilizing the method, the conventional fermentation culture mode is subverted; the problem that the yield cannot be further improved in the method for producing the HA is effectively solved; the method has important significance to improve the fermentation yield of the HA, reduce the production cost of enterprises and promote the application of the HA.
Description
Technical field
The present invention relates to biological field, especially a kind of method that improves hyaluronic acid fermentation output, it combines the hyaluronic fermentation yield of Effective Raise by adopting batch feeding cultivation and intermittence pH to coerce in utilizing streptococcus zooepidemicus fermentative production hyaluronic acid process.
Background technology
Hyaluronic acid referred to as HA, is by β-(1 → 3) and acidic mucopolysaccharide that β-(1 → 4) glycosidic link is formed by connecting by glucuronic acid and acetylglucosamine.It has the advantageous properties such as humectant, visco-elasticity and biocompatibility, especially has unique performance of keeping humidity, has been widely used at present the fields such as medicine, daily use chemicals, food, is one of domestic and international biochemical industry circle focus product.
In recent years, microbe fermentation method has replaced animal tissues's extraction method, becomes the main production method of HA.Yet, in recent years, along with the rise of the prices of raw and semifnished materials and the raising of corresponding production cost, cause the cost of Production by Microorganism Fermentation HA further to improve, HA has consisted of huge challenge to Production by Microorganism Fermentation, and then has limited the widespread use of HA in medicine, makeup and health care of food field.Therefore, how further to improve the output of Production by Microorganism Fermentation HA, become the key of restriction HA industry sustainable development.Because production bacterial strain streptococcus zooepidemicus (
S.
Zooepidemicus) self physiological metabolism characteristic, so that the raising of HA output is subject to the restriction of many factors, as: high fermentation viscosity, low mass transfer pass oxygen, thalli growth and HA biosynthesizing to the competition of carbon source and energy etc.Therefore, how to improve the output of Production by Microorganism Fermentation HA, reduce production costs, become the urgent problems of concern of people.
Summary of the invention
Goal of the invention of the present invention is: for the physiological metabolism properties influence of being produced bacterial strain streptococcus zooepidemicus self, so that the raising of HA output is restricted, and at present in the urgent need to improving the output of Production by Microorganism Fermentation HA, the problem that reduces production costs provides a kind of method that improves hyaluronic acid fermentation output.The present invention can further improve hyaluronic fermentation yield by adopting batch feeding cultivation and intermittence pH to coerce the mode that combines, and reduces the fermentation comprehensive cost, and then promotes HA in the application in medicine, cosmetic and health care of food field.The present invention overturns the traditional zymotic training method, efficiently solve and adopt when producing bacterial strain streptococcus zooepidemicus production HA, the problem that output can't further improve, the present invention is for improving hyaluronic fermentation yield, reduce enterprise's production cost, promote that the application of HA is significant.
To achieve these goals, the present invention adopts following technical scheme:
A kind of method that improves hyaluronic acid fermentation output comprises the steps:
(1) slant culture: on slant medium, place constant incubator to cultivate 12-16h slant medium the streptococcus zooepidemicus bacterial classification inoculation, the temperature in the constant incubator is 35-40 ℃, gets the inclined-plane seed;
(2) seed culture: the inclined-plane seed that step (1) is cultivated is inoculated on the seed culture medium to be cultivated, and culture temperature is 35-40 ℃, and incubation time is 6-16h, when the pH of seed culture medium value is reduced to 5.6-6.0, gets seed liquor;
(3) fermentation culture: the seed liquor access is equipped with in the fermentor tank of fermention medium, mixing speed 120 r/min, air flow 100 vvm, tank pressure 0.03 Mpa, then 37 ℃ of temperature are carried out the fs and are cultivated;
(4) fs cultivates: the online automatic control feed supplement of batch feeding utilization tank carries out feed supplement, controls the pH value of solution in the online automatic control feed supplement tank for neutral, when the carbon source concentration in the feed supplement tank is 20-25g/L, carries out subordinate phase and cultivates:
(5) subordinate phase is cultivated: add basic solution in online automatic control feed supplement tank, pH value to 7.5 in coercing the online automatic control feed supplement of Timing tank ~ 9.0, after the time of coercing finishes, enter stationary time, pH value to 7.0 in stationary time is regulated online automatic control feed supplement tank, repeat successively the time of coercing and stationary time, to fermentation ends, must contain hyaluronic fermented liquid;
In the described slant medium, contain the 34g heart and brain in every liter of slant medium and soak powder, 10g glucose, 8g yeast powder, 17g agar powder;
In the described seed culture medium, contain 18g sucrose, 18g yeast powder, 1.8g magnesium sulfate heptahydrate, 0.08g four water manganous sulfates, 1.8g potassium primary phosphate, 18 calcium carbonate, 1mL trace element solution, 37mL damping fluid in every liter of seed culture medium;
In the described fermention medium, contain 18g yeast powder, 5.9g Sodium phosphate dibasic, 1.2g vitriolate of tartar, 67g sucrose, 2.0g magnesium sulfate heptahydrate, 1mL trace element solution in every liter of fermention medium;
In the described trace element solution, every liter of trace element solution contains 2.0g calcium chloride, 0.046g zinc chloride, 0.019g cupric sulfate pentahydrate;
In the described damping fluid, every liter of damping fluid contains 36.76g Sodium phosphate dibasic, 15.98g SODIUM PHOSPHATE, MONOBASIC, 12.5g sodium bicarbonate;
In the described step (4), the online automatic control feed supplement of batch feeding utilization tank carries out feed supplement, and the initial sugar concentration of feed supplement is 10 g/L, begins to carry out feed supplement after 2h is carried out in fermentation, and the stream rate of acceleration of feed supplement is calculated according to following formula:
In the formula
XBe cell concn in the fermentor tank (g/L),
SBe concentration of substrate in the fermentor tank (g/L),
μBe thalline specific growth rate (h
-1), v is fermentating liquid volume (L),
FBe bottoms stream rate of acceleration (g/L*h),
S F Be the concentration (g/L) of adding substrate,
Y X/S Be the yield coefficients (g/g) of cell to substrate, (
VX)
0Be the initial cell amount (g) of culture system, t is the time;
In the described step (5), the time of coercing is 0.5-2h, and described stationary time is 0.5-2h.
In the described step (1), place constant incubator to cultivate 12-16h slant medium, the temperature in the constant incubator is 37 ℃.
In the described step (5), described basic solution is sodium hydroxide solution.
In the described step (5), in online automatic control feed supplement tank, add basic solution, after pH value to 8.5 in coercing the online automatic control feed supplement of the Timing tank time of coercing finishes, enter stationary time, pH value to 7.0 in stationary time is regulated online automatic control feed supplement tank, repeat successively the time of coercing and stationary time, to fermentation ends, must contain hyaluronic fermented liquid.
In the described step (5), the time of coercing is 1h, and described stationary time is 1h.
Existing Production by Microorganism Fermentation HA adopts one time to produce substratum or single batch fermentation to produce HA substantially, and the present invention has overturned the fermentation culture pattern of this routine.The present invention coerces the mode that combines by the batch culture pattern with intermittent pH, Effective Raise the hyaluronic fermentation yield in the final fermented liquid.Compare with existing microbe fermentation method, the present invention can the Effective Raise fermented liquid in the output of HA to more than the 6.0-6.5g/L, improve at least 20%-30% than the 5g/L of present normal fermentation method.
The present invention can obtain higher carbon source to the transformation efficiency of HA by the batch culture pattern, can obtain higher thalline specific growth rate and adopt batch feeding to cultivate.Wherein, batch feeding is cultivated and is divided into two stages.Wherein, the first cultivation stage adopts the index feeding culture, and the time of fs is 0-6h, when fermentation proceeds to 6h, when the carbon source concentration in the feed supplement tank is 25g/L, carries out subordinate phase and cultivates.Wherein, subordinate phase is cultivated and is the batch feeding cultivation, best performance is: namely coerce the time at 6-7h() control pH value is 8.5, then be stationary time at 7-8h() control pH value to 7.0, namely coerce the time at 8-9h(again) control pH value is 8.5, then be stationary time at 9-10h() control pH value to 7.0, so repeatedly, until fermentation ends.Adopt aforementioned best training method, the output of HA is 8.0g/L in the fermented liquid, has significant progressive meaning.
The applicant finds in practice, because the most suitable growth pH value of cell is 7.0, in fermenting process, a certain period of cell is controlled its pH value not in the most suitable growth pH value district, even cell is in a kind of inferior the most suitable growth environment within certain period, may force cell to change the production capacity approach, to improve production capacity efficient, to accelerate the HA synthesis rate, with helper cell opposing adverse environment.Intermittent pH coerces to reach and changes cell production capacity approach, improves cell production capacity efficient, the fermentation yield of Effective Raise HA.
Embodiment
Disclosed all features in this specification sheets, or the step in disclosed all methods or the process except mutually exclusive feature and/or step, all can make up by any way.
Disclosed arbitrary feature in this specification sheets is unless special narration all can be replaced by other equivalences or the alternative features with similar purpose.That is, unless special narration, each feature is an example in a series of equivalences or the similar characteristics.
Embodiment 1
One, prepares
1) preparation damping fluid
Take by weighing each component by following ratio of weight and number respectively: 36.76 parts of Sodium phosphate dibasics, 15.98 parts of SODIUM PHOSPHATE, MONOBASIC, 12.5 parts of sodium bicarbonates, each component is mixed, be prepared into damping fluid.In every liter of damping fluid, contain 36.76g Sodium phosphate dibasic, 15.98g SODIUM PHOSPHATE, MONOBASIC, 12.5g sodium bicarbonate.
2) preparation trace element solution
Take by weighing each component by following ratio of weight and number respectively: 2.0 parts of calcium chloride, 0.046 part of zinc chloride, 0.019 part of cupric sulfate pentahydrate, each component is mixed with water, be prepared into trace element solution.In every liter of trace element solution, contain 2.0g calcium chloride, 0.046g zinc chloride, 0.019g cupric sulfate pentahydrate.
3) preparation slant medium
Take by weighing each component by following ratio of weight and number respectively: 34 parts of heart and brain soak powder, 10 parts of glucose, 8 parts of yeast powders, 17 parts of agar powders, and each component is mixed with water, are prepared into slant medium, and the pH value of solution is 7.0.In every liter of slant medium, contain the 34g heart and brain and soak powder, 10g glucose, 8g yeast powder, 17g agar powder.
4) preparation seed culture medium
Get respectively 18g sucrose, 18g yeast powder, 1.8g magnesium sulfate heptahydrate, 0.08g four water manganous sulfates, 1.8g potassium primary phosphate, 18g calcium carbonate, 1mL trace element solution, 37mL damping fluid, be prepared into 1 liter of seed culture medium, its pH value is 7.0.As required, can be made into 50 liters or 100 liters of seed culture mediums, the component of seed culture medium is prepared by aforementioned ratio, and wherein trace element solution, damping fluid are prepared by the present embodiment abovementioned steps respectively.
5) preparation fermention medium
Get respectively 18g yeast powder, 5.9g Sodium phosphate dibasic, 1.2g vitriolate of tartar, 67g sucrose, 2.0g magnesium sulfate heptahydrate, 1mL trace element solution, be prepared into 1 liter of fermention medium, the pH value of this fermention medium is 7.0.As required, can prepare 100 or 200 liters of fermention mediums, the component of fermention medium is prepared by aforementioned ratio, and wherein, trace element solution is prepared by the present embodiment abovementioned steps.
Two, preparation
(1) slant culture: on slant medium, place constant incubator to cultivate 14h slant medium the streptococcus zooepidemicus bacterial classification inoculation, the temperature in the constant incubator is 37 ℃, gets the inclined-plane seed.
(2) seed culture: the inclined-plane seed of preparation is seeded to upper cultivation the in the 2L triangular flask that the 500mL seed culture medium is housed, culture temperature is 37 ℃, and incubation time is 8h, and shaking speed is 200 r/min, when the pH of seed culture medium value is reduced to 5.6-6.0, get seed liquor.
(3) fermentation culture: the seed liquor access is equipped with in the fermentor tank of fermention medium, and mixing speed is 120 r/min, air flow 100 vvm, and tank pressure 0.03 Mpa, then 37 ℃ of temperature are carried out the fs and are cultivated.
(4) fs cultivates: the online automatic control feed supplement of batch feeding utilization tank carries out feed supplement, control the pH value of solution in the online automatic control feed supplement tank for neutral, pH adopts the pH electrode to detect online, and dissolved oxygen electrode detects oxyty online, once detects analysis every the 1h sampling.When fermentation proceeds to 6h, when the carbon source concentration in the raising feed supplement tank is 23g/L, carries out subordinate phase and cultivate.
Wherein, (0-6h) carries out index feeding culture (namely adding fermention medium in online automatic control feed supplement tank) within the fs, the initial sugar concentration of index fed batch cultivation is 10 g/L, carry out index stream since exponential phase of growth (2h) and add (namely adding fermention medium in online automatic control feed supplement tank), the stream rate of acceleration is calculated according to following formula:
In the formula
XBe cell concn in the fermentor tank (g/L),
SBe concentration of substrate in the fermentor tank (g/L),
μBe thalline specific growth rate (h
-1), v is fermentating liquid volume (L),
FBe bottoms stream rate of acceleration (g/L*h),
S F Be the concentration (g/L) of adding substrate,
Y X/S Be the yield coefficients (g/g) of cell to substrate, (
VX)
0Be the initial cell amount (g) of culture system, t is the time.
(5) subordinate phase is cultivated: add mass percent in the online automatic control feed supplement tank and be 20% NaOH solution and carry out pH regulator, make it satisfy pH and coerce requirement.Namely coerce the time at 6-7h() regulate the pH value to 8.3 in the online automatic control feed supplement tank, after the time of coercing finishes, enter stationary time, be stationary time at 7-8h() regulate pH value in the online automatic control feed supplement tank to neutral value to 7.0, repeat successively the time of coercing and stationary time, so circulation to the 14h fermentation ends, must contain hyaluronic fermented liquid.
After measured, in the prepared fermented liquid of the present embodiment, the output of HA is to contain the 6.5g hyaluronic acid in every liter of fermented liquid.
Embodiment 2
One, prepares
The preparation of damping fluid, preparation trace element solution, slant medium, seed culture medium, fermention medium is all identical with embodiment 1.
Two, preparation
(1) slant culture: on slant medium, place constant incubator to cultivate 12h slant medium the streptococcus zooepidemicus bacterial classification inoculation, the temperature in the constant incubator is 40 ℃, gets the inclined-plane seed.
(2) seed culture: the inclined-plane seed of preparation is seeded to upper cultivation the in the 2L triangular flask that the 500mL seed culture medium is housed, culture temperature is 35 ℃, and incubation time is 12h, and shaking speed is 200 r/min, when the pH of seed culture medium value is reduced to 5.6-6.0, get seed liquor.
(3) fermentation culture: the seed liquor access is equipped with in the fermentor tank of fermention medium, and mixing speed is 120 r/min, air flow 100 vvm, and tank pressure 0.03 Mpa, then 37 ℃ of temperature are carried out the fs and are cultivated.
(4) fs cultivates: the online automatic control feed supplement of batch feeding utilization tank carries out feed supplement, control the pH value of solution in the online automatic control feed supplement tank for neutral, pH adopts the pH electrode to detect online, and dissolved oxygen electrode detects oxyty online, once detects analysis every the 1h sampling.When fermentation proceeds to 6h, when the carbon source concentration in the raising feed supplement tank is 25g/L, carries out subordinate phase and cultivate.
Wherein, (0-6h) carries out index feeding culture (namely adding fermention medium in online automatic control feed supplement tank) within the fs, the initial sugar concentration of index fed batch cultivation is 10 g/L, carry out index stream since exponential phase of growth (2h) and add (namely adding fermention medium in online automatic control feed supplement tank), the stream rate of acceleration is calculated according to following formula:
In the formula
XBe cell concn in the fermentor tank (g/L),
SBe concentration of substrate in the fermentor tank (g/L),
μBe thalline specific growth rate (h
-1), v is fermentating liquid volume (L),
FBe bottoms stream rate of acceleration (g/L*h),
S F Be the concentration (g/L) of adding substrate,
Y X/S Be the yield coefficients (g/g) of cell to substrate, (
VX)
0Be the initial cell amount (g) of culture system, t is the time.
(5) subordinate phase is cultivated: add mass percent in the online automatic control feed supplement tank and be 15% NaOH solution and carry out pH regulator, make it satisfy pH and coerce requirement.Namely coerce the time at 6-6.5h() regulate the pH value to 8.1 in the online automatic control feed supplement tank, after the time of coercing finishes, enter stationary time, be stationary time at 7-8h() regulate pH value in the online automatic control feed supplement tank to neutral value to 7.0, repeat successively the time of coercing and stationary time, so circulation to the 13h fermentation ends, must contain hyaluronic fermented liquid.
After measured, in the prepared fermented liquid of the present embodiment, the output of HA is to contain the 6.8g hyaluronic acid in every liter of fermented liquid.
Embodiment 3
One, prepares
The preparation of damping fluid, preparation trace element solution, slant medium, seed culture medium, fermention medium is all identical with embodiment 1.
Two, preparation
(1) slant culture: on slant medium, place constant incubator to cultivate 16h slant medium the streptococcus zooepidemicus bacterial classification inoculation, the temperature in the constant incubator is 35 ℃, gets the inclined-plane seed.
(2) seed culture: the inclined-plane seed of preparation is seeded to upper cultivation the in the 2L triangular flask that the 500mL seed culture medium is housed, culture temperature is 35 ℃, and incubation time is 10h, and shaking speed is 200 r/min, when the pH of seed culture medium value is reduced to 5.6-6.0, get seed liquor.
(3) fermentation culture: the seed liquor access is equipped with in the fermentor tank of fermention medium, and mixing speed is 120 r/min, air flow 100 vvm, and tank pressure 0.03 Mpa, then 37 ℃ of temperature are carried out the fs and are cultivated.
(4) fs cultivates: the online automatic control feed supplement of batch feeding utilization tank carries out feed supplement, control the pH value of solution in the online automatic control feed supplement tank for neutral, pH adopts the pH electrode to detect online, and dissolved oxygen electrode detects oxyty online, once detects analysis every the 1h sampling.When fermentation proceeds to 6h, when the carbon source concentration in the raising feed supplement tank is 20g/L, carries out subordinate phase and cultivate.
Wherein, (0-6h) carries out index feeding culture (namely adding fermention medium in online automatic control feed supplement tank) within the fs, the initial sugar concentration of index fed batch cultivation is 10 g/L, carry out index stream since exponential phase of growth (2h) and add (namely adding fermention medium in online automatic control feed supplement tank), the stream rate of acceleration is calculated according to following formula:
，
In the formula
XBe cell concn in the fermentor tank (g/L),
SBe concentration of substrate in the fermentor tank (g/L),
μBe thalline specific growth rate (h
-1), v is fermentating liquid volume (L),
FBe bottoms stream rate of acceleration (g/L*h),
S F Be the concentration (g/L) of adding substrate,
Y X/S Be the yield coefficients (g/g) of cell to substrate, (
VX)
0Be the initial cell amount (g) of culture system, t is the time.
(5) subordinate phase is cultivated: add mass percent in the online automatic control feed supplement tank and be 20% NaOH solution and carry out pH regulator, make it satisfy pH and coerce requirement.Namely coerce the time at 6-7.5h() regulate the pH value to 8.1 in the online automatic control feed supplement tank, after the time of coercing finishes, enter stationary time, be stationary time at 7.5-9h() regulate pH value in the online automatic control feed supplement tank to neutral value to 7.0, repeat successively the time of coercing and stationary time, so circulation to the 18h fermentation ends, must contain hyaluronic fermented liquid.
After measured, in the prepared fermented liquid of the present embodiment, the output of HA is to contain the 8g hyaluronic acid in every liter of fermented liquid.
Embodiment 4
One, prepares
The preparation of damping fluid, preparation trace element solution, slant medium, seed culture medium, fermention medium is all identical with embodiment 1.
Two, preparation
(1) slant culture: on slant medium, place constant incubator to cultivate 14h slant medium the streptococcus zooepidemicus bacterial classification inoculation, the temperature in the constant incubator is 36 ℃, gets the inclined-plane seed.
(2) seed culture: the inclined-plane seed of preparation is seeded to upper cultivation the in the 2L triangular flask that the 500mL seed culture medium is housed, culture temperature is 37 ℃, and incubation time is 6h, and shaking speed is 200 r/min, when the pH of seed culture medium value is reduced to 5.6-6.0, get seed liquor.
(3) fermentation culture: the seed liquor access is equipped with in the fermentor tank of fermention medium, and mixing speed is 120 r/min, air flow 100 vvm, and tank pressure 0.03 Mpa, then 37 ℃ of temperature are carried out the fs and are cultivated.
(4) fs cultivates: the online automatic control feed supplement of batch feeding utilization tank carries out feed supplement, control the pH value of solution in the online automatic control feed supplement tank for neutral, pH adopts the pH electrode to detect online, and dissolved oxygen electrode detects oxyty online, once detects analysis every the 1h sampling.When fermentation proceeds to 6h, when the carbon source concentration in the raising feed supplement tank is 22g/L, carries out subordinate phase and cultivate.
Wherein, (0-6h) carries out index feeding culture (namely adding fermention medium in online automatic control feed supplement tank) within the fs, the initial sugar concentration of index fed batch cultivation is 10 g/L, carry out index stream since exponential phase of growth (2h) and add (namely adding fermention medium in online automatic control feed supplement tank), the stream rate of acceleration is calculated according to following formula:
In the formula
XBe cell concn in the fermentor tank (g/L),
SBe concentration of substrate in the fermentor tank (g/L),
μBe thalline specific growth rate (h
-1), v is fermentating liquid volume (L),
FBe bottoms stream rate of acceleration (g/L*h),
S F Be the concentration (g/L) of adding substrate,
Y X/S Be the yield coefficients (g/g) of cell to substrate, (
VX)
0Be the initial cell amount (g) of culture system, t is the time.
(5) subordinate phase is cultivated: add mass percent in the online automatic control feed supplement tank and be 10% NaOH solution and carry out pH regulator, make it satisfy pH and coerce requirement.Namely coerce the time at 6-8h() regulate the pH value to 8.1 in the online automatic control feed supplement tank, after the time of coercing finishes, enter stationary time, be stationary time at 8-10h() regulate pH value in the online automatic control feed supplement tank to neutral value to 7.0, repeat successively the time of coercing and stationary time, so circulation to the 22h fermentation ends, must contain hyaluronic fermented liquid.
After measured, in the prepared fermented liquid of the present embodiment, the output of HA is to contain the 7.2g hyaluronic acid in every liter of fermented liquid.
Embodiment 5
One, prepares
The preparation of damping fluid, preparation trace element solution, slant medium, seed culture medium, fermention medium is all identical with embodiment 1.
Two, preparation
(1) slant culture: on slant medium, place constant incubator to cultivate 12h slant medium the streptococcus zooepidemicus bacterial classification inoculation, the temperature in the constant incubator is 37 ℃, gets the inclined-plane seed.
(2) seed culture: the inclined-plane seed of preparation is seeded to upper cultivation the in the 2L triangular flask that the 500mL seed culture medium is housed, culture temperature is 37 ℃, and incubation time is 10h, and shaking speed is 200 r/min, when the pH of seed culture medium value is reduced to 5.6-6.0, get seed liquor.
(3) fermentation culture: the seed liquor access is equipped with in the fermentor tank of fermention medium, and mixing speed is 120 r/min, air flow 100 vvm, and tank pressure 0.03 Mpa, then 37 ℃ of temperature are carried out the fs and are cultivated.
(4) fs cultivates: the online automatic control feed supplement of batch feeding utilization tank carries out feed supplement, control the pH value of solution in the online automatic control feed supplement tank for neutral, pH adopts the pH electrode to detect online, and dissolved oxygen electrode detects oxyty online, once detects analysis every the 1h sampling.When fermentation proceeds to 6h, when the carbon source concentration in the raising feed supplement tank is 23g/L, carries out subordinate phase and cultivate.
Wherein, (0-6h) carries out index feeding culture (namely adding fermention medium in online automatic control feed supplement tank) within the fs, the initial sugar concentration of index fed batch cultivation is 10 g/L, carry out index stream since exponential phase of growth (2h) and add (namely adding fermention medium in online automatic control feed supplement tank), the stream rate of acceleration is calculated according to following formula:
In the formula
XBe cell concn in the fermentor tank (g/L),
SBe concentration of substrate in the fermentor tank (g/L),
μBe thalline specific growth rate (h
-1), v is fermentating liquid volume (L),
FBe bottoms stream rate of acceleration (g/L*h),
S F Be the concentration (g/L) of adding substrate,
Y X/S Be the yield coefficients (g/g) of cell to substrate, (
VX)
0Be the initial cell amount (g) of culture system, t is the time.
(5) subordinate phase is cultivated: add mass percent in the online automatic control feed supplement tank and be 20% NaOH solution and carry out pH regulator, make it satisfy pH and coerce requirement.Namely coerce the time at 6-7h() regulate the pH value to 8.2 in the online automatic control feed supplement tank, after the time of coercing finishes, enter stationary time, be stationary time at 7-8h() regulate pH value in the online automatic control feed supplement tank to neutral value to 7.0, repeat successively the time of coercing and stationary time, so circulation to the 15h fermentation ends, must contain hyaluronic fermented liquid.
After measured, in the prepared fermented liquid of the present embodiment, the output of HA is to contain the 7.6g hyaluronic acid in every liter of fermented liquid.
The present invention is not limited to aforesaid embodiment.The present invention expands to any new feature or any new combination that discloses in this manual, and the arbitrary new method that discloses or step or any new combination of process.
Claims (6)
1. a method that improves hyaluronic acid fermentation output is characterized in that, comprises the steps:
(1) slant culture: on slant medium, place constant incubator to cultivate 12-16h slant medium the streptococcus zooepidemicus bacterial classification inoculation, the temperature in the constant incubator is 35-40 ℃, gets the inclined-plane seed;
(2) seed culture: the inclined-plane seed that step (1) is cultivated is inoculated on the seed culture medium to be cultivated, and culture temperature is 35-40 ℃, and incubation time is 6-16h, when the pH of seed culture medium value is reduced to 5.6-6.0, gets seed liquor;
(3) fermentation culture: the seed liquor access is equipped with in the fermentor tank of fermention medium, mixing speed 120 r/min, air flow 100 vvm, tank pressure 0.03 Mpa, then 37 ℃ of temperature are carried out the fs and are cultivated;
(4) fs cultivates: the online automatic control feed supplement of batch feeding utilization tank carries out feed supplement, controls the pH value of solution in the online automatic control feed supplement tank for neutral, when the carbon source concentration in the feed supplement tank is 20-25g/L, carries out subordinate phase and cultivates:
(5) subordinate phase is cultivated: add basic solution in online automatic control feed supplement tank, pH value to 7.5 in coercing the online automatic control feed supplement of Timing tank ~ 9.0, after the time of coercing finishes, enter stationary time, pH value to 7.0 in stationary time is regulated online automatic control feed supplement tank, repeat successively the time of coercing and stationary time, to fermentation ends, must contain hyaluronic fermented liquid;
In the described slant medium, contain the 34g heart and brain in every liter of slant medium and soak powder, 10g glucose, 8g yeast powder, 17g agar powder;
In the described seed culture medium, contain 18g sucrose, 18g yeast powder, 1.8g magnesium sulfate heptahydrate, 0.08g four water manganous sulfates, 1.8g potassium primary phosphate, 18 calcium carbonate, 1mL trace element solution, 37mL damping fluid in every liter of seed culture medium;
In the described fermention medium, contain 18g yeast powder, 5.9g Sodium phosphate dibasic, 1.2g vitriolate of tartar, 67g sucrose, 2.0g magnesium sulfate heptahydrate, 1mL trace element solution in every liter of fermention medium;
In the described trace element solution, every liter of trace element solution contains 2.0g calcium chloride, 0.046g zinc chloride, 0.019g cupric sulfate pentahydrate;
In the described damping fluid, every liter of damping fluid contains 36.76g Sodium phosphate dibasic, 15.98g SODIUM PHOSPHATE, MONOBASIC, 12.5g sodium bicarbonate;
In the described step (4), the online automatic control feed supplement of batch feeding utilization tank carries out feed supplement, and the initial sugar concentration of feed supplement is 10 g/L, begins to carry out feed supplement after 2h is carried out in fermentation, and the stream rate of acceleration of feed supplement is calculated according to following formula:
In the formula
XBe cell concn in the fermentor tank (g/L),
SBe concentration of substrate in the fermentor tank (g/L),
μBe thalline specific growth rate (h
-1), v is fermentating liquid volume (L),
FBe bottoms stream rate of acceleration (g/L*h),
S F Be the concentration (g/L) of adding substrate,
Y X/S Be the yield coefficients (g/g) of cell to substrate, (
VX)
0Be the initial cell amount (g) of culture system, t is the time;
In the described step (5), the time of coercing is 0.5-2h, and described stationary time is 0.5-2h.
2. the method for described raising hyaluronic acid fermentation output according to claim 1 is characterized in that, in the described step (1), place constant incubator to cultivate 12-16h slant medium, the temperature in the constant incubator is 37 ℃.
3. the method for described raising hyaluronic acid fermentation output according to claim 1 is characterized in that, in the described step (5), described basic solution is sodium hydroxide solution.
4. the method for each described raising hyaluronic acid fermentation output according to claim 1-3, it is characterized in that, in the described step (5), in online automatic control feed supplement tank, add basic solution, after the time of coercing of the pH value to 8.5 in coercing the online automatic control feed supplement of Timing tank finishes, enter stationary time, pH value to 7.0 in stationary time is regulated online automatic control feed supplement tank, repeat successively the time of coercing and stationary time, to fermentation ends, must contain hyaluronic fermented liquid.
5. the method for each described raising hyaluronic acid fermentation output is characterized in that according to claim 1-4, and in the described step (5), the time of coercing is 1h, and described stationary time is 1h.
6. the method for described raising hyaluronic acid fermentation output according to claim 4 is characterized in that, in the described step (5), the time of coercing is 1h, and described stationary time is 1h.
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