CN107034165A - Enterococcus faecalis high density fermentation culture medium and its zymotechnique - Google Patents
Enterococcus faecalis high density fermentation culture medium and its zymotechnique Download PDFInfo
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Abstract
The present invention relates to a kind of high density fermentation culture medium of enterococcus faecalis and its zymotechnique, fermentation medium component and its content are as follows：20~30g/L of glycerine, 10~15g/L of dregs of beans, 1~3g/L of ammonium sulfate, three 2~3g/L of hypophosphite monohydrate hydrogen dipotassium, 3~8g/L of sodium acetate trihydrate, two 2~4g/L of citric acid monohydrate sodium, 0.3~0.8g/L of bitter salt, 0.1~0.3g/L of Manganous sulfate monohydrate.Zymotechnique:Use high dissolved oxygen (25%) and combine glycerine for carbon source through fermentation, with the constant zymotic fluid pH value 5.5 of sodium carbonate, and flow feeding controls glycerol concentration in 10g/L during the fermentation, OD (600nm) is more than 35 mark fermentation ends after cultivating 16 hours, and zymotic fluid viable count containing enterococcus faecalis is up to 8 × 1010More than CFU/mL.The present invention uses high dissolved oxygen combination glycerine to reduce the lactic acid concn in zymotic fluid for the fermentation method of carbon source, and zymotic fluid can reach 8 × 10 containing enterococcus faecalis10More than CFU/mL, reduces the separation expense of biomass, shortens the production cycle, reduction production cost, raising production efficiency.
Description
Technical field
The present invention relates to microbial technology field, and in particular to a kind of enterococcus faecalis high density fermentation culture medium and its fermentation
Technique, it is adaptable to the high density fermentation production of enterococcus faecalis.
Background technology
《Catalogue of feed additive varieties (2013)》The strain that enterococcus faecalis is defined as to be added in feed, its
Biofilm can be formed in animal intestinal tract to be attached on animal intestinal tract mucous membrane, and is developed, grows and bred.Enterococcus faecalis belongs to
Normal flora in people and animal intestinal tract, into enteron aisle after can effectively colonize.Enterococcus faecalis fermentation produces lactic acid, advantageously reduces
Intestinal environment pH, suppresses the growth of harmful bacteria.It can also produce hydrogen peroxide in enterococcus faecalis metabolic process, the material such as bacteriocin,
There is certain killing action to pathogenic bacteria, and natural antibiotics can be produced, be conducive to body health, at present should in aquaculture
With MRS culture mediums are selected than wide but traditional Enterococcus faecalis fermentation, expensive, the viable count after fermentation is low, in benefit more
In the case that the raw effective dosage of bacterium is fixed, low fermentation level adds cost equal to covert.The culture medium of low cost is high
The zymotechnique amplified of effect is for promoting the industrialization of probiotics fermention to have great guidance and practice significance.
The content of the invention
More can effectively ferment the high thalline life of enterococcus faecalis acquisition it is an object of the invention to provide one kind than prior art
The enterococcus faecalis high density fermentation culture medium of object amount.
Another object of the present invention is to use above-mentioned enterococcus faecalis high density fermentation culture medium there is provided one kind, and in excrement
Rationally addition feed supplement during enterococcus is fermented, so as to obtain the enterococcus faecalis high density fermentation of more high-biomass than prior art
Technique.
Third object of the present invention is that provide above-mentioned enterococcus faecalis high cell density fermentation gives birth in Enterococcus faecalis fermentation
Application in production.
The above-mentioned technical problem of the present invention is mainly what is be addressed by following technical proposals：The purpose of the present invention is logical
Cross what following technical scheme was realized：
The present invention provides a kind of enterococcus faecalis (Enterococcus faecalis) high density fermentation culture medium, the excrement intestines
The seed and fermentation medium of coccus high density fermentation are fluid nutrient medium, and its component and its content are as follows：20~30g/ of glycerine
L, 10~15g/L of dregs of beans, 1~3g/L of ammonium sulfate, three 2~3g/L of hypophosphite monohydrate hydrogen dipotassium, 3~8g/L of sodium acetate trihydrate, two
2~4g/L of citric acid monohydrate sodium, 0.3~0.8g/L of bitter salt, 0.1~0.3g/L of Manganous sulfate monohydrate.
Present invention also offers the zymotechnique of the enterococcus faecalis high density fermentation culture medium, key step is as follows：
(1) the enterococcus faecalis glycerine strain of freezen protective is lined on MRS agar mediums, places 32~39 DEG C of cultures
Cultivated in case to growing single bacterium colony；The full single bacterium colony of picking is inoculated in MRS liquid tubes in above-mentioned single bacterium colony, 32~39 DEG C,
After 50~200r/min Shaking cultures 10 hours；Transferred with 1% inoculum concentration in MRS fluid nutrient mediums, culture is made after 8~10 hours
For seed liquor；
(2) enterococcus faecalis high density fermentation culture medium is prepared, needed for culture medium is down to during temperature, dissolved oxygen electrode is connected, marked
It is 100% to determine dissolved oxygen；
(3) seed liquor obtained by step (1) is inoculated in the fermentation medium of step (2) with 2%~8% inoculum concentration
In；
(4) fermentation parameter is set as：32~39 DEG C of temperature；50~300r/min of rotating speed；0.5~1.0VVM of ventilation ratio, dimension
Hold dissolved oxygen 20~30%；It is 4.5~7.0 that zymotic fluid pH value is maintained in fermentation process, when pH is less than setting value, auto-feeding alkali
Property nertralizer (ammoniacal liquor, KOH, NaOH, CaCO3Or Na2CO3Any of) maintain setting value.Continuously add feed liquid (feed supplement
Culture medium：Glycerine 800g/L and ammonium sulfate 80g/L), keep glycerol concentration to maintain 10g/L or so；
(5) no longer reduce or when light absorption value of the zymotic fluid under 600nm wavelength is more than 35 when adding material liquid pH, terminate hair
Ferment, produces enterococcus faecalis viable count up to 8 × 1010More than CFU/mL zymotic fluid.
Wherein, it is described add feed liquid key component and its content be：Glycerine 800g/L, ammonium sulfate 80g/L.
Preferably, dissolved oxygen 20~30% is maintained to be more conducive to the growth that enterococcus faecalis is lived in fermentation parameter.
Preferably, pH value is 4.5~7.0 growths for being more conducive to enterococcus faecalis work in fermentation parameter.
The high density fermentation culture medium and its zymotechnique of the present invention is applied to enterococcus faecalis (Enterococcus
Faecalis), but to reach best invention effect, it is preferably applied to enterococcus faecalis (Enterococcus faecalis)
CICC 20419.Enterococcus faecalis (Enterococcus faecalis) CICC 20419 is bought in Chinese industrial microorganism fungus kind
Preservation administrative center.
The enterococcus faecalis high density fermentation culture medium and its zymotechnique of the present invention has advantages below：Present invention optimizes
Enterococcus faecalis high density fermentation culture medium, is carbon source by high dissolved oxygen combination glycerine, and feeds back control using pH during the fermentation
System and glycerine ammonium sulfate couple feed-batch process, reduce Lactic Acid from Fermentation Broth concentration, so as to release the feedback inhibition of lactic acid, use
The enterococcus faecalis of high density fermentation culture medium and its zymotechnique fermentation of the present invention, cell density is significantly carried compared with common fermentation
Height, viable count is 8 × 1010More than CFU/mL, this is the unapproachable fermentation standard of existing technology, is that existing use is mended in batches
Expect that fermentation method obtains 2.78 times of high density fermentation enterococcus faecalis viable count；More common MRS culture mediums fed batch fermentation training
Support bacterium number 2.2 × 109CFU/mL, improves more than 40 times, realizes the high density fermentation of enterococcus faecalis；Biomass can be reduced
Separation expense, shorten the production cycle so that reduce production cost, improve production efficiency.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the accompanying drawing used required in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can be with
Other accompanying drawings are obtained according to these accompanying drawings.
Fig. 1 is the block diagram of influence of the different carbon source to enterococcus faecalis high density fermentation in embodiments of the invention 1.
Fig. 2 is the column of influence of the different carbon source to enterococcus faecalis high density fermentation lactic acid producing in embodiments of the invention 1
Figure.
Fig. 3 is the block diagram of influences of the difference pH to enterococcus faecalis high density fermentation in embodiments of the invention 2.
Fig. 4 is the post of influence of the different alkaline pH nertralizers to enterococcus faecalis high density fermentation in embodiments of the invention 3
Shape figure.
Fig. 5 is the block diagram of influence of the constant dissolved oxygen to enterococcus faecalis high density fermentation in embodiments of the invention 4.
Fig. 6 is the block diagram in 5 glycerine of embodiments of the invention-ammonium sulfate coupling flow feeding batch fermentation experiment.
Fig. 7 is enterococcus faecalis high density fermentation in embodiments of the invention 6 and the line chart compared.
Embodiment
The preferred embodiments of the present invention are described in detail 1-7 below in conjunction with the accompanying drawings, so that advantages of the present invention and spy
Levying can be easier to be readily appreciated by one skilled in the art, and apparent clearly be defined so as to be made to protection scope of the present invention.
By the following example by the more specific description present invention, it should be understood that the embodiment is merely to illustrate this
Invention, rather than the scope of the present invention is limited in any way.
In the present embodiment, enterococcus faecalis derives from Chinese industrial Microbiological Culture Collection administrative center, and deposit number is
CICC 20419。
Embodiment 1 carries out enterococcus faecalis high density fermentation using different carbon source
Fermentation medium：Dregs of beans 10g/L, ammonium sulfate 2g/L, three hypophosphite monohydrate hydrogen dipotassium 2g/L, sodium acetate trihydrate 5g/
L, two citric acid monohydrate sodium 2g/L, bitter salt 0.3g/L, Manganous sulfate monohydrate 0.2g/L, glycerine/glucose/sucrose/
Starch 20g/L, pH 7.0 is adjusted to 25% ammoniacal liquor.
It is prepared by seed liquor：MRS (glucose 20g/L will be lined in the enterococcus faecalis glycerine strain of 80 DEG C of preservations of ﹣；Albumen
Peptone 10g/L；Dusty yeast 5g/L；Diammonium hydrogen citrate 2g/L；Sodium acetate 5g/L；Dipotassium hydrogen phosphate 2g/L；Magnesium sulfate 0.58g/L；
Manganese sulfate 0.25g/L) on agar medium, place and cultivated in 32 DEG C of incubators to growing single bacterium colony；The full single bacterium colony of picking
Access is equipped with MRS liquid tubes, and 37 DEG C, 50r/min Shaking cultures are after 10 hours；Transferred with 1% inoculum concentration in MRS Liquid Cultures
Base, 37 DEG C, 100r/min concussion and cultivates are used as seed liquor after 8 hours.
Ferment tank culture:121 DEG C in demarcation dissolved oxygen electrode, preparation fermentation tank culture medium 3L, addition 5L fermentation tanks,
15min autoclavings are stand-by.Pot temperature is adjusted to 37 DEG C, pH is adjusted to 7.0 using 25% ammoniacal liquor, and be inoculated with seed liquor
90ml, starts fermenting procedure.Fermentation parameter is set as：37 DEG C of temperature, initial speed 150r/min；Throughput and rotating speed are adjusted,
The constant dissolved oxygen maintained is respectively 5%, 10%, 15%, 20%；Analyze different carbon source (glycerine, glucose, sucrose, starch) right
The influence of Enterococcus faecalis fermentation tank fermented and cultured.
As a result：As shown in Figure 1 and Figure 2, given birth to when enterococcus faecalis uses glycerol as carbon source under conditions of dissolved oxygen control 20%
Preferably, S-type curve increases long status, and demurrage is in 0~3h, and 3h~14h increases in logarithm, and breeds after 14h slow
Slowly, plateau is initially entered.Glycerine reduces the concentration of Lactic Acid from Fermentation Broth as carbon source, releases the feedback inhibition of lactic acid,
With the dissolved oxygen content increase of maintenance, the efficiency that glycerine is converted into energy is improved, the further growth of thalline is promoted, improved
The viable count of zymotic fluid.In fermentation process 1.82 × 10 are reached in the viable count of enterococcus faecalis high density fermentation10CFU/mL, and sugarcane
The thalline weight in wet base that sugar and glucose can reach as carbon source is relatively low than glycerine, is 0.96 × 1010CFU/mL or so, but still
Realize more than 1.1 × 1010CFU/mL high density fermentation.
Embodiment 2 controls influences of the different pH to enterococcus faecalis high density fermentation
Fermentation medium：Dregs of beans 15g/L, glycerine 25g/L, ammonium sulfate 2g/L, three hypophosphite monohydrate hydrogen dipotassium 3g/L, three hydrations
Sodium acetate 5g/L, two citric acid monohydrate sodium 3g/L, bitter salt 0.5g/L, Manganous sulfate monohydrate 0.25g/L.
It is prepared by seed liquor：MRS (glucose 20g/L will be lined in the enterococcus faecalis glycerine strain of 80 DEG C of preservations of ﹣；Albumen
Peptone 10g/L；Dusty yeast 5g/L；Diammonium hydrogen citrate 2g/L；Sodium acetate 5g/L；Dipotassium hydrogen phosphate 2g/L；Magnesium sulfate 0.58g/L；
Manganese sulfate 0.25g/L) on agar medium, place and cultivated in 33 DEG C of incubators to growing single bacterium colony；The full single bacterium colony of picking
Access is equipped with MRS liquid tubes, and 32 DEG C, 150r/min Shaking cultures are after 10 hours；Transferred with 1% inoculum concentration in the training of MRS liquid
Base is supported, 37 DEG C, 100r/min concussion and cultivates are used as seed liquor after 8 hours.
Ferment tank culture:Fermentation tank culture medium 3L is prepared, 121 DEG C are added in 5L fermentation tanks, 15min autoclavings treat
With.Pot temperature is adjusted to 32 DEG C, pH to 7.0 is adjusted using 25% ammoniacal liquor；Seed liquor 240ml is inoculated with, starts fermenting procedure.Hair
Ferment parameter setting is：39 DEG C of fermentation temperature, rotating speed 300r/min；0.1~0.5VVM of ventilating ratio；Using 25% ammoniacal liquor as neutralization
Agent, the constant pH for selecting stream plus process maintenance is respectively 4.5,5.0,5.5,6.0,6.5,7.0, when pH is down to below setting value
When, automatic benefit alkali function, which automatically control flowing, to be added, and pH is maintained stopping fermentation after setting value, fermentation 16h.Every kind of constant pH
Condition does the parallel test of 3 batches.
Interpretation of result：Such as Fig. 3, constant pH is that fermentation viable count, apparently higher than under other constant pH, reaches under conditions of 5.5
2.3×1010CFU/mL。
The optimization of the most suitable alkaline pH nertralizer of embodiment 3
Fermentation medium：Dregs of beans 10g/L, glycerine 20g/L, ammonium sulfate 1g/L, three hypophosphite monohydrate hydrogen dipotassium 2g/L, three hydrations
Sodium acetate 8g/L, two citric acid monohydrate sodium 2g/L, bitter salt 0.4g/L, Manganous sulfate monohydrate 0.25g/L.
It is prepared by seed liquor：MRS (glucose 20g/L will be lined in the enterococcus faecalis glycerine strain of 80 DEG C of preservations of ﹣；Albumen
Peptone 10g/L；Dusty yeast 5g/L；Diammonium hydrogen citrate 2g/L；Sodium acetate 5g/L；Dipotassium hydrogen phosphate 2g/L；Magnesium sulfate 0.58g/L；
Manganese sulfate 0.25g/L) on agar medium, place and cultivated in 34 DEG C of incubators to growing single bacterium colony；The full single bacterium colony of picking
Access is equipped with MRS liquid tubes, and 39 DEG C, 200r/min Shaking cultures are after 10 hours；Transferred with 1% inoculum concentration in the training of MRS liquid
Base is supported, 37 DEG C, 100r/min concussion and cultivates are used as seed liquor after 10 hours.
Ferment tank culture:Fermentation tank culture medium 3L is prepared, 121 DEG C are added in 5L fermentation tanks, 15min autoclavings treat
With.Pot temperature is adjusted to 37 DEG C, and is inoculated with seed liquor 120ml, starts fermenting procedure.Fermentation parameter is set as：37 DEG C of temperature；
Rotating speed 50r/min；0.1~0.5VVM of ventilating ratio；Zymotic fluid pH value lower limit set is 5.5.Respectively selection ammoniacal liquor, KOH, NaOH,
Na2CO3As alkaline pH nertralizer, when pH is down to below setting value, carried out using the automatic benefit alkali systemic-function of fermentation system
Automatically control stream plus, pH is maintained setting value, control be used as using the batch fermentation that adds without nertralizer stream.After fermentation 16h
Stop fermentation.The parallel test of 3 batches is done under the conditions of every kind of alkaline pH nertralizer.
Interpretation of result：No matter which kind of nertralizer used, its viable bacteria of fermenting, which is all significantly higher than not flowing, adds alkaline pH nertralizer
Viable count.As shown in figure 4, with 30% Na2CO3As nertralizer, fermentation viable count is apparently higher than ammoniacal liquor group, KOH groups, NaOH
Group.It is thus determined that 30% Na2CO3Most suitable alkaline pH nertralizer.
The optimization of the most suitable constant dissolved oxygen of the fermentation parameter of embodiment 4
Fermentation medium：Dregs of beans 12g/L, glycerine 28g/L, ammonium sulfate 1.5g/L, three hypophosphite monohydrate hydrogen dipotassium 2g/L, three water
Close sodium acetate 6g/L, two citric acid monohydrate sodium 3g/L, bitter salt 0.8g/L, Manganous sulfate monohydrate 0.15g/L.
It is prepared by seed liquor：MRS (glucose 20g/L will be lined in the enterococcus faecalis glycerine strain of -80 DEG C of preservations；Albumen
Peptone 10g/L；Dusty yeast 5g/L；Diammonium hydrogen citrate 2g/L；Sodium acetate 5g/L；Dipotassium hydrogen phosphate 2g/L；Magnesium sulfate 0.58g/L；
Manganese sulfate 0.25g/L) on agar medium, place and cultivated in 39 DEG C of incubators to growing single bacterium colony；The full single bacterium colony of picking
Access is equipped with MRS liquid tubes, and 35 DEG C, 170r/min Shaking cultures are after 10 hours；Transferred with 1% inoculum concentration in the training of MRS liquid
Base is supported, 37 DEG C, 100r/min concussion and cultivates are used as seed liquor after 9 hours.
Ferment tank culture:Fermentation tank culture medium 3L is prepared, 121 DEG C are added in 5L fermentation tanks, 15min autoclavings treat
With.Pot temperature is adjusted to 37 DEG C, pH is adjusted to 6.5 using 25% ammoniacal liquor, and is inoculated with seed liquor 90ml, starts fermenting procedure.
Fermentation parameter is set as：37 DEG C of temperature, with 30% Na2CO3For nertralizer, when pH is down to 5.5, automatic alkali function of mending is carried out certainly
Dynamic controlling stream adds, and pH is maintained 5.5；Throughput and rotating speed are adjusted, the constant dissolved oxygen of maintenance is respectively 20%, 25%, 30%；
Stop fermentation after fermentation 16h.Every kind of controlled pH conditions do the parallel test of 3 batches.
Interpretation of result：As shown in figure 5, control dissolved oxygen ferments under conditions of 25%, viable count exists apparently higher than control dissolved oxygen
Under 20% or 30%, fermentation viable count reaches 4.8 × 1010CFU/mL。
5 glycerine of embodiment-ammonium sulfate coupling flow feeding batch fermentation experiment
1st, enterococcus faecalis high density fermentation culture medium component and its content are：
Dregs of beans 13g/L, glycerine 22g/L, ammonium sulfate 3g/L, three hypophosphite monohydrate hydrogen dipotassium 3g/L, sodium acetate trihydrate 7g/L,
Two citric acid monohydrate sodium 2g/L, bitter salt 0.6g/L, Manganous sulfate monohydrate 0.3g/L.
2nd, enterococcus faecalis high density fermentation key step is as follows
(1) the enterococcus faecalis glycerine strain of freezen protective is lined into MRS (glucose 20g/L；Peptone 10g/L；Yeast
Powder 5g/L；Diammonium hydrogen citrate 2g/L；Sodium acetate 5g/L；Dipotassium hydrogen phosphate 2g/L；Magnesium sulfate 0.58g/L；Manganese sulfate 0.25g/
L) on agar medium, place in 37 DEG C of incubators and cultivate to growing single bacterium colony；The full single bacterium colony of picking in above-mentioned single bacterium colony
Access is equipped with MRS liquid tubes, and 37 DEG C, 50r/min Shaking cultures are after 10 hours；Transferred with 1% inoculum concentration in MRS Liquid Cultures
Base, 37 DEG C, 100r/min concussion and cultivates are used as seed liquor after 8 hours；
(2) glycerine 800g and ammonium sulfate 80g are weighed, plus distilled water dissolves and is settled to 100mL, in 103.4kPa vapour pressures
Power, steam sterilizing 30min obtains feed supplement liquid at 110 DEG C；
(3) enterococcus faecalis high density fermentation culture medium is prepared, needed for culture medium is down to during temperature, dissolved oxygen electrode is connected, marked
It is 100% to determine dissolved oxygen；
(4) it is inoculated with, the seed liquor obtained by step (1) is inoculated in the fermentation medium of step (3) with 5% inoculum concentration
In；
(5) fermentation parameter is set as：37 DEG C of temperature, regulation throughput and rotating speed, dissolved oxygen maintain 25%；In fermentation process
It is 5.5 to maintain zymotic fluid pH value, when pH is less than setting value, 30% Na2CO3Solution maintains setting value；
A groups:Feed supplement flow acceleration is controlled in 10g/h, makes glycerol concentration in 2g/L or so.
B groups:Feed supplement flow acceleration 25g/h, glycerol concentration 15g/L or so.
C groups:Feed supplement flow acceleration is changed according to the density value of bacterial growth, feed supplement flow acceleration is controlled in 10~25g/h,
Glycerol concentration is in 10g/L or so
(6) fermentation is stopped after fermentation 16h.Every kind of controlled pH conditions do the parallel test of 3 batches.
3rd, interpretation of result：As shown in fig. 6, Enterococcus faecalis fermentation viable count ferments apparently higher than A groups and B groups under the conditions of C groups
Viable count reaches 7.3 × 1010CFU/mL。
The high density fermentation cultural method of the enterococcus faecalis of embodiment 6
1st, the composition and preparation of enterococcus faecalis high density fermentation seed and fermentation medium：
The main component and content of seed and fermentation medium be：Dregs of beans 10g/L, glycerine 30g/L, ammonium sulfate 2.5g/L,
Three hypophosphite monohydrate hydrogen dipotassium 2.5g/L, sodium acetate trihydrate 8g/L, two citric acid monohydrate sodium 4g/L, bitter salt 0.7g/
L, Manganous sulfate monohydrate 0.1g/L.
The main component and content of supplemented medium be：Glycerine 800g and ammonium sulfate 80g
Feed-batch culture basigamy compound method is as follows:
Culture medium each component is weighed, fully after dissolving, required volume is settled to, 110 DEG C sterilize 30 minutes.
Seed flask culture medium compound method is as follows：
Culture medium each component is weighed, fully after dissolving, adjusts pH to 7.0 with 25% ammonia spirit, is settled to required volume,
110 DEG C sterilize 30 minutes.
Fermentation tank culture medium compound method is as follows：
(1) 20g/L sodium hydroxide solutions are loaded in fermentation tank, liquid amount is 70%, 0.11-0.16Mpa sterilizing 1h, cooling
Tapping afterwards, fermentation tank is vented after rinsing fermentation tank 2 times with running water.
(2) each component is weighed respectively by above-mentioned content, dissolved with distilled water, be fitted into 0.14Mp in fermentation tank and sterilize 30 points
Clock.
(3) stir and evenly mix, 37 DEG C, 100rpm insulation 1h are adjusted after pH to 7.0,0.14Mpa sterilizings 40 with 25% ammonia spirit
Minute.When temperature stabilization is at 37 DEG C, correction dissolved oxygen is 100%.
(4) culture medium after sterilizing is in 37 DEG C, 50rpm, under the conditions of stuffiness before empty training to inoculation.
2. it is prepared by seed liquor
(1) the enterococcus faecalis inclined-plane of picking fresh cultured, is seeded in seed flask culture medium, 37 DEG C, 50r/min shaking flasks
Culture is used as one-level shake-flask seed liquid in 8 hours.
(2) secondary seed medium is prepared in 50L fermentation tanks, to secondary seed medium and one-level shaking flask kind before culture
Sub- liquid carries out microscopy, it is ensured that without living contaminants.
(3) 50L fermentation tank rotating speeds are adjusted to 100rpm before being inoculated with, and secondary seed medium is adjusted with 25% ammoniacal liquor of sterilizing
After pH to 7.0, primary seed solution is accessed in 50L fermentation tanks according to 2% inoculum concentration.
(4) fermentation parameter is set：37 DEG C of fermentation temperature；Rotating speed 200rpm；0.1~0.5VVM of ventilating ratio；；PH is not controlled not mend
Material, fermentation 8h terminates as secondary seed solution.
High density fermentation under the conditions of 3.2 tons of fermentation tank scales
(1) microscopy is carried out to fermentation medium and secondary seed solution before fermenting, it is ensured that pollution-free.
(2) 2 tons of fermentation tank rotating speeds are adjusted to 100rpm before being inoculated with, and adjusted with 25% ammoniacal liquor of sterilizing after pH to 6.8, by two
Level seed liquor, which is all forwarded in 2 tons of fermentation tanks, is fermented.
(3) fermentation parameter is set as：37 DEG C of fermentation temperature；Throughput and rotating speed are adjusted, dissolved oxygen is maintained 25% or so；Hair
It is 5.5 that zymotic fluid pH value is maintained during ferment, and when pH is less than setting value, 30% sodium carbonate liquor maintains setting value；It is logical
Excessively stream adds supplemented medium, and feed supplement flow acceleration is controlled in 10~25g/h, glycerol concentration is maintained 10g/L or so；
(4) no longer reduce or when light absorption value of the zymotic fluid under 600nm wavelength is not further added by when adding material liquid pH, terminate hair
Ferment, produces enterococcus faecalis viable count up to 8.9 × 1010More than CFU/mL zymotic fluid.
4. contrast test
(1) main component and content of tradition MRS culture mediums are：
Glucose：20g/L, peptone：10g/L, beef extract：5g/L, dusty yeast：4g/L, three hypophosphite monohydrate hydrogen dipotassiums：
2g/L, sodium acetate trihydrate：5g/L, diammonium hydrogen citrate：2g/L, bitter salt：0.5g/L, Manganous sulfate monohydrate：
0.25g/L, Tween 80：1ml/L.
(2) seed flask culture medium collocation method is as follows
Culture medium each component is weighed, fully after dissolving, adjusts pH to 6.2 with glacial acetic acid, is settled to required volume, 121 DEG C
Sterilizing 15 minutes.
(3) fermentation tank culture medium compound method is as follows：
Load 20g/L sodium hydroxide solutions in fermentation tank, liquid amount is 60%, 0.11-0.16Mpa sterilizing 1h, after cooling
Tapping, fermentation tank is vented after rinsing fermentation tank 2 times with running water.Weigh culture medium each component, add appropriate running water stir to
Fully dissolving, adds running water to the 60% of fermentation tank cumulative volume.37 DEG C, 100rpm insulation 1h adjust pH to 6.2 with glacial acetic acid
Afterwards, 0.12Mpa sterilizes 30 minutes.When temperature stabilization is at 37 DEG C, correction dissolved oxygen is 100%.Culture medium after sterilizing at 38 DEG C,
50rpm, empty training is to before being inoculated with the conditions of stuffiness.
The fermentation of (4) 2 tons of fermentation tanks
The enterococcus faecalis inclined-plane of picking fresh cultured, is seeded in seed flask culture medium, 37 DEG C of quiescent culture 10h conducts
Primary seed solution.Secondary seed medium is prepared in 50L fermentation tanks, to secondary seed medium and primary seed solution before culture
Carry out microscopy, it is ensured that without living contaminants.Microscopy is carried out to fermentation medium and secondary seed solution before fermentation, it is ensured that pollution-free.
2 tons of fermentation tank rotating speeds are adjusted to 100rpm before inoculation, and pH to 6.2 is adjusted with 40% sodium hydroxide solution of sterilizing
Afterwards, by secondary seed solution all access fermentation tank.
Fermentation parameter is set as：37 DEG C of temperature；Rotating speed 100r/min；Stuffiness；Zymotic fluid pH value is maintained in fermentation process
For 6.2, when pH exceedes setting value, feed liquid (supplemented medium is added:Glucose 800g/L and ammonium sulfate 80g/L)；When pH is low
When setting value, auto-feeding antalkali (20% sodium hydroxide solution of sterilizing) maintains setting value.
(4) no longer reduce or when light absorption value of the zymotic fluid under 600nm wavelength is not further added by when adding material liquid pH, terminate hair
Ferment, produces enterococcus faecalis viable count up to 1.8 × 1010More than CFU/mL zymotic fluid.
5 interpretations of result
As shown in fig. 7, the culture medium and its zymotechnique of the application present invention, can obtain enterococcus faecalis viable count up to 8.9
×1010Cfu/ml zymotic fluid, and the maximum viable count of the existing enterococcus faecalis for using fed batch fermentation mode to obtain for 3.2 ×
1010CFU/mL, the high density fermentation enterococcus faecalis viable count that culture medium and its zymotechnique of the invention is obtained is prior art
2.78 times, fully met enterococcus faecalis as the production requirement of feeding lactobacillus.
This is not limited to, any change or replacement expected without creative work should all cover the guarantor in the present invention
Within the scope of shield.Therefore, protection scope of the present invention should be determined by the scope of protection defined in the claims.
Claims (6)
1. a kind of enterococcus faecalis high density fermentation culture medium, it is characterised in that the seed and hair of the enterococcus faecalis high density fermentation
Ferment culture medium is fluid nutrient medium, and its component and its content are as follows：20~30g/L of glycerine, 10~15g/L of dregs of beans, ammonium sulfate 1~
3g/L, three 2~3g/L of hypophosphite monohydrate hydrogen dipotassium, 3~8g/L of sodium acetate trihydrate, two 2~4g/L of citric acid monohydrate sodium, seven hydrations
0.3~0.8g/L of magnesium sulfate, 0.1~0.3g/L of Manganous sulfate monohydrate.
2. the present invention provides the zymotechnique of the enterococcus faecalis high density fermentation culture medium described in claim 1, key step is such as
Under：
(1) the enterococcus faecalis glycerine strain of freezen protective is lined on MRS agar mediums, placed in 32~39 DEG C of incubators
Culture is to growing single bacterium colony；The full single bacterium colony of picking is inoculated in MRS liquid tubes in above-mentioned single bacterium colony, 32~39 DEG C, 50~
After 200r/min Shaking cultures 10 hours；Transferred with 1% inoculum concentration in MRS fluid nutrient mediums, 32~39 DEG C, 50~200r/min
Culture is used as seed liquor after 8~10 hours；
(2) enterococcus faecalis high density fermentation culture medium is prepared, needed for culture medium is down to during temperature, dissolved oxygen electrode is connected；
(3) seed liquor obtained by step (1) is inoculated in the fermentation medium of step (2) with 2%~8% inoculum concentration；
(4) fermentation parameter is set as：32~39 DEG C of temperature；50~300r/min of rotating speed；0.5~1.0VVM of ventilation ratio, is maintained molten
Oxygen 20~30%；It is 4.5~7.0 that zymotic fluid pH value is maintained in fermentation process, when pH is less than setting value, in auto-feeding alkalescence
And agent, setting value is maintained, feed liquid is continuously added, keeps glycerol concentration to maintain 10g/L or so；
(5) no longer reduce or when light absorption value of the zymotic fluid under 600nm wavelength is more than 35 when adding material liquid pH, terminate fermentation, i.e.,
Enterococcus faecalis viable count is obtained up to 8 × 1010More than CFU/mL zymotic fluid.
3. the zymotechnique of enterococcus faecalis high density fermentation culture medium according to claim 2, the master for adding feed liquid
Component and its content is wanted to be：Glycerine 800g/L, ammonium sulfate 80g/L.
4. remain molten in the zymotechnique of enterococcus faecalis high density fermentation culture medium according to claim 2, fermentation parameter
Oxygen is 20~30%.
5. pH value is in the zymotechnique of enterococcus faecalis high density fermentation culture medium according to claim 2, fermentation parameter
4.5~7.0.
6. the zymotechnique of enterococcus faecalis high density fermentation culture medium according to claim 2, the antalkali is
Ammoniacal liquor, KOH, NaOH, CaCO3 or Na2Any of CO3.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108179130A (en) * | 2018-03-21 | 2018-06-19 | 广州同心源生物科技有限公司 | A kind of preparation method of high activity Enterococcus faecalis microorganisms preparation dry powder |
| CN109294960A (en) * | 2018-11-15 | 2019-02-01 | 湖北华扬科技发展有限公司 | A kind of fermentation medium and fermentation process for enterococcus faecalis |
| CN112011483A (en) * | 2020-08-13 | 2020-12-01 | 山东天润和生物工程有限公司 | Culture method suitable for enterococcus faecalis enrichment |
| CN114181872A (en) * | 2022-01-07 | 2022-03-15 | 北京青蓝伟业科技有限公司 | High-density fermentation culture method for enterococcus faecium |
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| CN105087420A (en) * | 2015-03-30 | 2015-11-25 | 北京伟嘉人生物技术有限公司 | High-density fermentation medium and fermentation technology for forage-use enterococcus faecium |
| CN106148212A (en) * | 2015-03-16 | 2016-11-23 | 江西科诺生物科技有限公司 | A kind of feeding enterococcus faecalis high density fermentation culture medium and fermentation process thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106148212A (en) * | 2015-03-16 | 2016-11-23 | 江西科诺生物科技有限公司 | A kind of feeding enterococcus faecalis high density fermentation culture medium and fermentation process thereof |
| CN105087420A (en) * | 2015-03-30 | 2015-11-25 | 北京伟嘉人生物技术有限公司 | High-density fermentation medium and fermentation technology for forage-use enterococcus faecium |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108179130A (en) * | 2018-03-21 | 2018-06-19 | 广州同心源生物科技有限公司 | A kind of preparation method of high activity Enterococcus faecalis microorganisms preparation dry powder |
| CN109294960A (en) * | 2018-11-15 | 2019-02-01 | 湖北华扬科技发展有限公司 | A kind of fermentation medium and fermentation process for enterococcus faecalis |
| CN112011483A (en) * | 2020-08-13 | 2020-12-01 | 山东天润和生物工程有限公司 | Culture method suitable for enterococcus faecalis enrichment |
| CN112011483B (en) * | 2020-08-13 | 2022-07-19 | 山东天润和生物工程有限公司 | Culture method suitable for enterococcus faecalis enrichment |
| CN114181872A (en) * | 2022-01-07 | 2022-03-15 | 北京青蓝伟业科技有限公司 | High-density fermentation culture method for enterococcus faecium |
| CN114181872B (en) * | 2022-01-07 | 2023-11-24 | 北京大北农科技集团股份有限公司 | Enterococcus faecium high-density fermentation culture method |
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