CN109880786A - A method of promoting Bacillus coagulans spore formation - Google Patents
A method of promoting Bacillus coagulans spore formation Download PDFInfo
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Abstract
The present invention relates to a kind of methods that promotion Bacillus coagulans spore is formed, belong to technical field of biological fermentation, the method be bacillus coagulans fermented and cultured logarithmic growth initial stage into culture medium successively between flow blocking add glucose solution and calcium carbonate, promote the formation of gemma.Compared with prior art, the supplement of the method carbon source can promote the increase of viable count, improve cell density, accelerate the formation of gemma, and the addition of calcium carbonate can effectively shorten the sporulation time, and the tolerance of gemma is increased while improving Number of spores.
Description
Technical field
The invention belongs to technical field of biological fermentation, and in particular, to a kind of that Bacillus coagulans spore is promoted to be formed
Method.
Background technique
Bacillus coagulans (Bacillus coagulans) are gram-positive bacteria (G+).The bacterium can generate lactic acid,
The resting spore body with strong tolerance can be generated again；Therefore, while there is the advantage of lactic acid bacteria and bacillus.Condense gemma
Bacillus can form a large amount of metabolite during the growth process, such as coaguin (Coagulin), Pfansteihl and lactosporin.
With the quantity for increasing beneficial bacteria of intestinal tract, treatment diarrhea enhancing is immune, improves irritable bowel syndrome, reduces cholesterol, inhibits to cause
The probiotic properties such as the growth of germ；And have broad spectrum antibacterial, can substitute antibiotics effectively inhibit in aquatic products and livestock-raising
Harmful bacteria breeding, improve host intestine micro-ecological environment；Some enzymes and nutriment can also be generated simultaneously, promote place
Host growth development metabolism is accelerated in the main absorption to dietary intake nutrition.
As a kind of lactic acid bacillus, bacillus coagulans have been widely used in food, drug, health care and herding
Marine industry, corresponding product emerge one after another, and demand is continuously increased.Because gemma have resistance strong, high temperature resistant, acid and alkali-resistance,
The advantages that survival rate is high, is sprouted in cold storage, on the market about the product form of bacillus coagulans from the form of nutrition body cell
It is shifted to spore form.Although recent domestic is about bacillus coagulans High Density Cultivation and the research of rush gemma culture
It very much, is all to be optimized from bacillus coagulans culture medium and training method, but High Density Cultivation can be taken into account but also mentioned
High gemma is rarely reported at the research of rate.At present about generally existing spore forming rate and product in bacillus industrialized production
The problems such as unstable quality, complicated medium component, the general temperature using in change fermentation process, dissolved oxygen, speed of agitator come
Gemma yield is improved, fermentation time is shortened, but above method is cumbersome in actual operation, energy consumption is big, increases and is produced into
This.
Summary of the invention
In order to solve deficiency in the prior art, the purpose of the present invention is to provide a kind of promotion Bacillus coagulans spores
The method of formation.The method refers to during bacillus coagulans bacillus cultivation and fermentation, is successively spaced benefit in certain time
Adding glucose and calcium carbonate, the supplement of carbon source can promote the increase of viable count, and cell density is improved, the formation of gemma is accelerated,
And the addition of calcium carbonate can effectively shorten the sporulation time, and the tolerance of gemma is increased while improving Number of spores
Property.
To achieve the goals above, the present invention use the specific scheme is that
A method of promote Bacillus coagulans spore formation, bacillus coagulans fermented and cultured logarithmic growth initial stage to
In culture medium successively between flow blocking add carbon source and calcium carbonate.
It is advanced optimized as to above scheme, the carbon source is glucose.
As the further optimization to above scheme, the method specifically includes the following steps:
Step 1: bacillus coagulans original strain is inoculated in first cell culture medium according to the inoculum concentration that volume ratio is 1:15 ~ 25
Middle carry out activation culture, condition of culture are as follows: 200~220rpm of shaking speed, 35~38 DEG C of temperature, 20~22h of time, culture knot
Beam obtains first class inoculum；The first cell culture medium is by mass percentage, composed of the following components: glucose 1.6~1.8%, albumen
Peptone 1.0~1.4%, yeast powder 0.6~1.0%, magnesium sulfate 0.4~0.6%, surplus are water；PH 7.8~8.2；
It is carried out Step 2: first class inoculum obtained by step 1 is inoculated in secondary medium according to the inoculum concentration that volume ratio is 1:30
Fermented and cultured, condition of culture are as follows: 35~38 DEG C of 200~240rpm of shaking speed, temperature, time 40-50h；When culture to the 8th
Disposably fill into glucose solution when~10h, the amount of filling into of the glucose solution be secondary medium total volume 1.2~
1.6%, the concentration of the glucose solution is 1.0g/mL；Final concentration is disposably filled into when continuing cultivation and fermentation to 14~18h
For the calcium carbonate solid of 40~80g/L；
The secondary medium is by mass percentage, composed of the following components: glucose 1.2~1.6%, peptone 1.2~
1.6%, yeast powder 0.8~1.2%, magnesium sulfate 0.4~0.6%, surplus are water；PH 7.8~8.2.
As the further optimization to above scheme, the first class inoculum activated through step 1 is inoculated in secondary medium
It is middle expanded at least once culture after carry out fermented and cultured described in step 2 again；The condition of culture for expanding culture are as follows: inoculation
Amount volume ratio is 1:30,200~240rpm of shaking speed, 35~38 DEG C of temperature, 16~20h of time.
The utility model has the advantages that
1, the present invention adds glucose and calcium carbonate by flowing on the basis of basal medium, is to improve bacillus coagulans in work
The yield of gemma, shortening production life cycle, raising gemma resistance and extension gemma shelf life provide necessity in industry production
Condition, add glucose after, viable count significantly increases about 10 times；After adding calcium carbonate, gemma maturation time is obviously shortened,
And spore production is stablized 108CFU/mL significantly improves the gemma productivity of bacillus coagulans.
2, the present invention promotes sporulation to mention firstly, the supplement of carbon source can promote the increase of viable count by two-step method
High cell density accelerates the formation of gemma, adds grape using batch feeding stream in bacillus coagulans early growth period, the application
Viable count is increased to 1.0 × 10 by the method for sugar9CFU/mL.The addition of calcium carbonate can effectively shorten the sporulation time,
The tolerance of gemma is increased while improving Number of spores.The shortening of sporulation time can effectively drop in industrial production
Energy consumption and production cost in low production, and the medium component in the application is simple and easy to get, significantly reduces production cost.
3, the application is had found by investigation and comparison, and bacillus coagulans do not add the feelings of calcium carbonate when cultivating the 16th hour
Under condition, gemma is not formed；And when cultivating the 16th hour, after adding calcium carbonate, gemma can be observed within the 18th hour in culture.
The additive amount of calcium carbonate of the present invention is 40~80g/L, and the addition of calcium carbonate can quickly neutralize bacillus coagulans fermentation process
The lactic acid of middle formation makes thallus quickly enter the environment of suitable sporulation, shortens the sporulation time；And calcium carbonate is a kind of
Raw materials for production cheap and easy to get are easier to separate with product compared to other solid materials in the industrial production.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described.
Embodiment 1
A method of promoting Bacillus coagulans spore formation, the specific steps are as follows:
1) bacillus coagulans original strain is connected in first cell culture medium and is activated, inoculum concentration volume ratio is 1:15, fills liquid
Amount is 30 mL/250mL, and condition of culture is that shaking speed is 200rpm, and cultivation temperature is 36 DEG C, and incubation time is 22 hours, training
Supporting terminates to obtain first class inoculum.The first cell culture medium is composed of the following components according to mass percent: glucose 1.8%, albumen
Peptone 1.2%, yeast powder 0.6%, magnesium sulfate 0.3%, surplus are water；pH 7.8.
2) the bacillus coagulans first class inoculum after activating in step 1) is inoculated in secondary liquid culture medium and is expanded
Big culture, inoculum concentration volume ratio are 1:30mL, and liquid amount is 30 mL/250mL；Condition of culture is that shaking speed is 230rpm/
Min, cultivation temperature are 37 DEG C, and incubation time is 16 hours, and culture terminates to obtain second class inoculum.
3) the bacillus coagulans second class inoculum handled through step 2 is inoculated in secondary medium and carries out fermentation training
It supports, inoculum concentration volume ratio is 1:30mL, and liquid amount is 30 mL/250mL；Condition of culture is that shaking speed is 230rpm/min, training
Supporting temperature is 37 DEG C；Sterile dextrose solution, the addition of the glucose solution are added into culture medium when cultivating small to the 8th
Amount is the 1.5% of culture volume, and the concentration of glucose solution is 1.0g/mL；Continue culture and adds sterile carbonic acid to the 16th hour
Calcium solid so that in culture medium calcium carbonate final concentration of 60g/L；Cultivation cycle is 40~45 hours, is collected after culture
Culture solution obtains the bacillus coagulans liquid product of high gemma rate, detects to obtain viable count 4.5 using dilution spread flat band method
×109CFU/mL, 80 DEG C of heating water bath 10min inactivate nutrition body cell, and it is 3.2 × 10 that dilution spread, which obtains gemma number,8 CFU/
mL。
The secondary medium is by mass percentage, composed of the following components: glucose 1.2~1.6%, peptone 1.2
~1.6%, yeast powder 0.8~1.2%, magnesium sulfate 0.4~0.6%, surplus are water；PH 7.8~8.2.
The first cell culture medium and secondary medium need sterilization treatment, sterilising conditions before use are as follows: and 115~121 DEG C,
Sterilize 20~30min.
Embodiment 2
A method of promoting Bacillus coagulans spore formation, specific steps:
1) bacillus coagulans original strain is connected in first cell culture medium and is activated, inoculum concentration volume ratio is 1:25, fills liquid
Amount is 50 mL/250mL, and condition of culture is that shaking speed is 210rpm/min, and cultivation temperature is 37 DEG C, and incubation time is 20 small
When, culture terminates to obtain first class inoculum.The first cell culture medium is composed of the following components according to mass percent: glucose
1.6%, peptone 1.4%, yeast powder 1.0%, magnesium sulfate 0.4%, surplus are water, pH 8.0.
2) the bacillus coagulans first class inoculum after step 1) activates is inoculated in secondary medium, inoculum concentration volume
Than for 1:25mL, liquid amount 50mL/250mL；Condition of culture is that shaking speed is 220rpm/min, cultivation temperature 36.5
DEG C, incubation time is 18 hours, and culture terminates to obtain second class inoculum.
3) the bacillus coagulans second class inoculum handled through step 2 is inoculated in secondary medium and carries out fermentation training
It supports, inoculum concentration volume ratio is 1:50mL, liquid amount 50mL/250mL；Condition of culture is that shaking speed is 240rpm/min, training
Supporting temperature is 38 DEG C；Sterile dextrose solution, the addition of the glucose solution are added into culture medium when cultivating small to the 8th
Amount is the 1.4% of culture volume, and the concentration of glucose solution is 1.0g/mL；Continue culture and adds sterile carbonic acid to the 16th hour
Calcium solid so that in culture medium calcium carbonate final concentration of 60g/L；It is maximum to gemma rate to continue culture, is collected after culture
Culture solution obtains the bacillus coagulans liquid product of high gemma rate, detects to obtain viable count 8.0 using dilution spread flat band method
×109CFU/mL, 80 DEG C of heating water bath 10min inactivate nutrition body cell, and it is 5.38 × 10 that dilution spread, which obtains gemma rate,8 CFU/
mL。
The secondary medium is by mass percentage, composed of the following components: glucose 1.2~1.6%, peptone 1.2
~1.6%, yeast powder 0.8~1.2%, magnesium sulfate 0.4~0.6%, surplus are water；PH 7.8~8.2.
The first cell culture medium and secondary medium need sterilization treatment, sterilising conditions before use are as follows: and 115~121 DEG C,
Sterilize 20~30min.
Embodiment 3
A method of promoting Bacillus coagulans spore formation, the specific steps are as follows:
1) original strain is connected in first cell culture medium and is activated, inoculum concentration volume ratio is 1:25, and liquid amount is 50 mL/
250mL, condition of culture are that shaking speed is 210rpm/min, and cultivation temperature is 37.5 DEG C, and incubation time is 18 hours, culture knot
Beam obtains first class inoculum.The first cell culture medium is composed of the following components according to mass percent: glucose 1.7%, peptone
1.3%, yeast powder 0.9%, magnesium sulfate 0.5%, surplus are water, pH 8.2.
2) the bacillus coagulans first class inoculum activated through step 1) is inoculated in secondary medium, inoculum concentration volume ratio
For 1:30mL, liquid amount 30mL/250mL；Condition of culture is that shaking speed is 230rpm/min, and cultivation temperature is 36.5 DEG C,
Incubation time is 16 hours, and culture terminates to obtain second class inoculum.
3) the bacillus coagulans second class inoculum handled through step 2 is inoculated in secondary medium, inoculum concentration volume
Than for 1:30mL, liquid amount 30mL/250mL；Condition of culture is that shaking speed is 200rpm/min, and cultivation temperature is 38 DEG C；
1.5% glucose solution is added when cultivating small to the 8th, the additive amount of the glucose solution is the 1.5% of culture volume, Portugal
The concentration of grape sugar juice is 1.0g/mL；Continue culture to the 16th hour addition calcium carbonate solid, so that calcium carbonate in culture medium
Final concentration of 50g/L；It is maximum to gemma rate to continue culture, culture solution is collected after culture and obtains the condensation gemma of high gemma rate
Bacillus liquid product detects to obtain viable count 7.10 × 10 using dilution spread flat band method9CFU/mL, 80 DEG C of heating water baths
10min inactivates nutrition body cell, and dilution spread obtains gemma rate 2.79 × 108 CFU/mL。
The secondary medium is by mass percentage, composed of the following components: glucose 1.2~1.6%, peptone 1.2
~1.6%, yeast powder 0.8~1.2%, magnesium sulfate 0.4~0.6%, surplus are water；PH 7.8~8.2.
The first cell culture medium and secondary medium need sterilization treatment, sterilising conditions before use are as follows: and 115~121 DEG C,
Sterilize 20~30min.
Embodiment 4
A method of promoting Bacillus coagulans spore formation, the specific steps are as follows:
1) original strain is connected in first cell culture medium and is activated, inoculum concentration volume ratio is 1:15, and liquid amount is 30 mL/
250mL, condition of culture are that shaking speed is 220rpm, and cultivation temperature is 36.5 DEG C, and incubation time is 18 hours, and culture terminates
First class inoculum.The first cell culture medium is composed of the following components according to mass percent: glucose 1.6%, peptone 1.4%,
Yeast powder 1.0%, magnesium sulfate 0.4%, surplus are water.
2) the bacillus coagulans first class inoculum after activating in step 1) is inoculated in secondary medium and carries out expansion training
It supports, inoculum concentration volume ratio is 1:30mL, and liquid amount is 30 mL/250mL；Condition of culture is that shaking speed is 230rpm/min, training
Supporting temperature is 37 DEG C；Glucose solution is added into culture medium when cultivating small to the 8th, the additive amount of the glucose solution is
The 1.5% of culture volume, the concentration of glucose solution are 1.0g/mL；Continue to cultivate to the 16th hour addition calcium carbonate solid,
So that in culture medium calcium carbonate final concentration of 40g/L；50h is cultivated, culture solution is collected after culture and obtains high gemma rate
Bacillus coagulans liquid product detects to obtain viable count 5.39 × 10 using dilution spread flat band method9CFU/mL, 80 DEG C of water-baths
It heats 10min and inactivates nutrition body cell, it is 3.04 × 10 that dilution spread, which obtains gemma number,8 CFU/mL。
The secondary medium is composed of the following components according to mass percent: glucose 1.5%, peptone 1.5%,
Yeast powder 1.0%, magnesium sulfate 0.5%, surplus are water.
Reference examples 5
As control, substantially the same manner as Example 4 to the processing method of bacillus coagulans in reference examples 5, difference only exists
In: reference examples 5 added calcium carbonate solid in Shi Weixiang culture medium to the 16th hour in fermented and cultured, and culture is collected after culture
Liquid obtains bacillus coagulans liquid product, detects to obtain viable count 6.12 × 10 using dilution spread flat band method9CFU/mL, 80
Then DEG C heating water bath 10min inactivation nutrition body cell is detected, fail to detect gemma.
To further illustrate the characteristic using gemma obtained by the method for the present invention, by following several tests to gemma tolerance
It is detected.
Taking the gemma as obtained by 1 the method for embodiment is test group；Because in the prior art sometimes in fermentation medium
Middle addition calcium carbonate is to promote cell Proliferation, therefore, take sporulation culture medium formed gemma resistance as a control group, to carbon
Sour calcium current, which adds, forms gemma (CaCO under stimulation3) gemma (Control) resistance for being formed of resistance and sporulation culture medium
Compare, detection scheme is as follows:
One, influence of the temperature to 9951 gemma of Bacillus coagulans CGMCC, as a result as shown in table 1 below.
Table 1: influence of the temperature to 9951 gemma of Bacillus coagulans CGMCC
Two, influence of the cholate to 9951 gemma of Bacillus coagulans CGMCC, as a result as shown in table 2 below.
Table 2: influence of the cholate to 9951 gemma of Bacillus coagulans CGMCC
Three, influence of the pH to 9951 gemma of Bacillus coagulans CGMCC, as a result as shown in table 3 below.
Influence of the table 3: pH to 9951 gemma of Bacillus coagulans CGMCC
It is found by table 1, though the gemma that calcium carbonate stimulates the gemma heat resistance to be formed to be formed slightly poorer to gemma culture medium, due to raising
It is quenched very short with the pelletization time in material granulation, it is usually no more than 3min, while pelleting temperature is usually no more than 100 DEG C of conducts
Temperature upper limit, therefore the gemma that application calcium carbonate generates is little to its survival rate during feed granulating.Test result
As shown in table 2, calcium carbonate, which stimulates, to form gemma 2h survival rate under 0.3% gallbladder salinity and is up to 99.79%；In 0.7% cholate
2h survival rate is handled under concentration still up to 87.22%, the gemma of gemma culture medium formation is above, due to bacterium depositing in enteron aisle
Living and proliferation allows for the cholate environment of tolerance 0.3%, thus the gemma that generates of calcium carbonate stimulation enter enteron aisle after can be smooth
It survives and breeds.The gastric acid of general animal is pH2.0-3.0, and the residence time of food under one's belt is 1-2h, and therefore, this patent is set
Setting pH is 1.5,2.0,3.0,4.0, and the processing time is 1h, 2h.Table 3 shows that calcium carbonate stimulates to form gemma in the processing of pH 1.5
The survival rate of 1h is only 7.14, and slightly above gemma culture medium forms gemma；However this patent is found, and in pH >=3, gemma survival
Rate is all larger than 100%, illustrates that gemma can rapidly and efficiently be sprouted and be played prebiotic reaching enteron aisle after the processing of Gastric pH >=3
Effect.In conclusion calcium carbonate stimulates and to form the acidproof of gemma, bile tolerance performance and can preferably play the probiotic properties of the bacterium.
It should be noted that embodiment described above is interpreted as illustrative, to be not intended to limit the present invention protection
Range, protection scope of the present invention are subject to claims.To those skilled in the art, without departing substantially from of the invention real
Under the premise of matter and range, some nonessential modifications and adaptations made to the present invention still fall within protection scope of the present invention.
Claims (4)
1. a kind of method for promoting Bacillus coagulans spore to be formed, it is characterised in that: in bacillus coagulans fermented and cultured
Logarithmic growth initial stage into culture medium successively between flow blocking add carbon source and calcium carbonate.
2. a kind of method for promoting Bacillus coagulans spore to be formed as described in claim 1, it is characterised in that: the carbon source
For glucose.
3. a kind of method for promoting Bacillus coagulans spore to be formed as claimed in claim 2, it is characterised in that: including following
Step:
Step 1: bacillus coagulans original strain is inoculated in first cell culture medium according to the inoculum concentration that volume ratio is 1:15 ~ 25
Middle carry out activation culture, condition of culture are as follows: 200~220rpm of shaking speed, 35~38 DEG C of temperature, 20~22h of time, culture knot
Beam obtains first class inoculum；The first cell culture medium is by mass percentage, composed of the following components: glucose 1.6~1.8%, albumen
Peptone 1.0~1.4%, yeast powder 0.6~1.0%, magnesium sulfate 0.4~0.6%, surplus are water；PH 7.8~8.2；
It is carried out Step 2: first class inoculum obtained by step 1 is inoculated in secondary medium according to the inoculum concentration that volume ratio is 1:30
Fermented and cultured, condition of culture are as follows: 35~38 DEG C of 200~240rpm of shaking speed, temperature, time 40-50h；When culture to the 8th
Disposably fill into glucose solution when~10h, the amount of filling into of the glucose solution be secondary medium total volume 1.2~
1.6%, the concentration of the glucose solution is 1.0g/mL；Final concentration is disposably filled into when continuing cultivation and fermentation to 14~18h
For the calcium carbonate solid of 40~80g/L；
The secondary medium is by mass percentage, composed of the following components: glucose 1.2~1.6%, peptone 1.2~
1.6%, yeast powder 0.8~1.2%, magnesium sulfate 0.4~0.6%, surplus are water；PH 7.8~8.2.
4. a kind of method for promoting Bacillus coagulans spore to be formed as claimed in claim 3, it is characterised in that: will be through step
One activation first class inoculum be inoculated in secondary medium expanded at least once culture after carry out again described in step 2 ferment training
It supports；The condition of culture for expanding culture are as follows: inoculum concentration volume ratio is 1:30,200~240rpm of shaking speed, temperature 35~38
DEG C, 16~20h of time.
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| CN110734873A (en) * | 2019-10-10 | 2020-01-31 | 华南理工大学 | method for increasing number of bacillus coagulans spores and application |
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| CN112852680A (en) * | 2021-03-22 | 2021-05-28 | 四川润格生物科技有限公司 | Liquid fermentation method of bacillus coagulans with high spore number |
| CN112941005A (en) * | 2021-01-22 | 2021-06-11 | 武汉微康益生菌研究院有限公司 | Method for rapidly producing spores by bacillus coagulans |
| CN113244274A (en) * | 2021-04-28 | 2021-08-13 | 河南科技大学 | Application of bacillus coagulans in relieving toxicity of micro-plastics |
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| CN112852680A (en) * | 2021-03-22 | 2021-05-28 | 四川润格生物科技有限公司 | Liquid fermentation method of bacillus coagulans with high spore number |
| CN113244274A (en) * | 2021-04-28 | 2021-08-13 | 河南科技大学 | Application of bacillus coagulans in relieving toxicity of micro-plastics |
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