CN102433279B - Method for preparing directed vat set bacillus coagulans starter - Google Patents
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Abstract
The invention relates to a method for preparing a directed vat set bacillus coagulans starter. The method comprises the following steps of: concentrating fermentation liquor of bacillus coagulans, wherein the concentration conditions of the fermentation liquor are that: the centrifugal rotating speed is 6,000r/min, the centrifugal time is 20min, and the centrifugal temperature is 4 DEG C; adding a protective agent, wherein the adopted protective agent is an aqueous solution which contains 5 percent m/m sucrose and 10 percent m/m maltodextrin; adding bacterial sludge into the aqueous solution of the protective agent; and drying by a vacuum drying method at the drying temperature of 60 DEG C under the drying pressure of 12,000Pa. The viable count of the obtained starter is 3.24*10<10>cfu/g; the sporation rate reaches 90.3 percent; and ideal liquid and solid fermentation effects can be achieved according to one thousandth of inoculation amount.
Description
Technical field
The invention belongs to the biotechnological formulation field, is a kind of preparation method of direct-throwing Bacillus coagulans starter.
Background technology
Bacillus coagulans is one type of facultative Bacteroides nodosus with terminal spore structure, is the normal mikrobe useful to the host.It is extensively added in various food and the feed, edible after in vivo breeding and field planting ability strong, viable count is high, and is favourable to the HUMAN HEALTH ten minutes.Bacillus coagulans to the health-care effect of human body mainly through two kinds of approach: the one, the effect of Bacillus coagulans thalline itself, it can produce a large amount of useful meta-bolitess, suppresses the growth of spoilage organism, keeps body intestinal microflora balance; The 2nd, its meta-bolites can promote intestinal peristalsis, strengthens the vigor of enzyme, helps body to digest and assimilate.
Throw type leaven (directed vat set DVS) is meant that a series of height concentrate and standardized dry starter culture, can directly add heat treated raw material ferment and need not to its carry out activation, other pre-treatment work such as spread cultivation.Direct-throwing Bacillus coagulans starter does not have product as yet both at home and abroad at present.Direct-throwing Bacillus coagulans preparation has the following advantages: viable bacteria content is high, and inoculum size is little, and production cost is low; Preservation term is long; Convenient transportation, purchase; Can prevent effectively that bacterial classification from polluting and degeneration, improve product quality; Need not enlarged culturing, effectively shorten the production cycle, raise labour productivity.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, provide that a kind of cost is low, simple to operate, the preparation method of direct-throwing Bacillus coagulans starter with unique advantage.
The present invention realizes that the technical scheme of purpose is following:
A kind of preparation method of direct-throwing Bacillus coagulans starter concentrates the Bacillus coagulans fermented liquid, and fermented liquid concentrates condition and is: centrifugal rotational speed 6000r/min; Centrifugal 20min, centrifuging temperature are 4 ℃, add protective material; The protective material that is adopted is the aqueous solution that contains 5%m/m sucrose and 10%m/m maltodextrin, and bacterium mud is joined in protectant aqueous solution, adopts boulton process dry; Drying temperature is elected 60 ℃ as, and drying pressure is elected 12000Pa as.
And; The name of said Bacillus coagulans is called TQ33; Specific name Bacillus coagulans, deposit number is: CGMCC No.5233, preservation date: on September 9th, 2011; The preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center.
And said Bacillus coagulans liquid seed culture medium is g/L: peptone 20.0, yeast extract paste 10.0, glucose 20.0, CaCO
3Be 10.0, pH7.2-7.4; Culture condition is: cultivate 20h for 37 ℃.
And, the volume ratio 1 of said bacterium mud and protectant aqueous solution: 2-5.
And said Bacillus coagulans vacuum-drying survival rate is 90.3%, and viable count reaches 3.24 * 10
10Cfu/g.
And said Bacillus coagulans starter is applied to liquid state fermentation by 1 ‰ inoculum size, and the highest viable count is 8.1 * 10
9Cfu/mL.
And said Bacillus coagulans starter is applied to solid state fermentation by 1 ‰ inoculum size, and the highest viable count is 1.0 * 10
10Cfu/g.
Advantage of the present invention and positively effect:
1, Bacillus coagulans TQ33 bacterial strain adopts boulton process to prepare the thalline starter through after the high-density amplification culture among the present invention.Using the survival rate of the direct-throwing Bacillus coagulans starter of present method production is 90.3%, and viable count reaches 3.56 * 10
10Cfu/g, the inoculum size according to 1 ‰ just can reach the liquid and solid state fermentation effect of ideal.Developed a kind of novel high-activity preparation method of direct-throwing Bacillus coagulans starter cheaply based on this bacterial strain.
2, the present invention adopts boulton process to prepare direct-throwing Bacillus coagulans starter first, compares with former liquid seed inoculation, and it is little that starter has inoculum size, directly uses, and labor savings shortens the production cycle; Can prevent effectively that bacterial classification from polluting and degeneration; Preserve convenient management, advantages such as product with stable quality.
3, the present invention has confirmed that Bacillus coagulans adopts centrifugal rotational speed 6000r/min in concentration process, 4 ℃ of following centrifugal 20min, and centrifugal yield is 93.27%.
4, the present invention has confirmed that protective material is the aqueous solution that contains 5%m/m sucrose and 10%m/m maltodextrin in the thalline process of vacuum drying, and final Bacillus coagulans survival rate is 90.3%, and viable count is 3.24 * 10
10Cfu/g.
5, be applied to liquid state fermentation with the direct-throwing Bacillus coagulans starter of the inventive method preparation by 1 ‰ inoculum size, the highest viable count is 8.1 * 10
9Cfu/mL.
6, be applied to solid state fermentation with the direct-throwing Bacillus coagulans starter of the inventive method preparation by 1 ‰ inoculum size, the highest viable count is 1.0 * 10
10Cfu/g.
Description of drawings
Fig. 1 is a 200L fermentor tank growth curve of the present invention;
Fig. 2 is the influence of the single protective material of the present invention to Bacillus coagulans vacuum-drying survival rate; Wherein, each protective material concentration of 1 level is: sucrose 3%, lactose 3%, trehalose 3%, Sodium Glutamate 3%, maltodextrin 3%, skim-milk 3%; Each protective material concentration of 2 levels is: sucrose 5%, lactose 5%, trehalose 5%, Sodium Glutamate 5%, maltodextrin 5%, skim-milk 5%;
Fig. 3 is the influence of composite protectant of the present invention to Bacillus coagulans vacuum-drying survival rate, wherein, and No. 1 5% sucrose; No. 2 5% sucrose+10% skim-milks; No. 3 5% sucrose+10% skim-milk+3% Sodium Glutamates; No. 4 5% sucrose+10% maltodextrins; No. 5 5% sucrose+10% skim-milk+5% Sodium Glutamates;
Fig. 4 is the influence of temperature of the present invention to Bacillus coagulans vacuum-drying survival rate;
Fig. 5 is the influence of pressure of the present invention to Bacillus coagulans vacuum-drying survival rate;
Fig. 6 compares for direct-throwing Bacillus coagulans starter liquid state fermentation effect of inoculation of the present invention;
Fig. 7 compares for direct-throwing Bacillus coagulans starter solid state fermentation effect of inoculation of the present invention.
Embodiment
Below in conjunction with embodiment, the present invention is further specified, following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
The bacterial classification that the present invention uses is Bacillus coagulans TQ33; Specific name Bacillus coagulans; Deposit number is: CGMCC No.5233; Preservation date: on September 9th, 2011, the preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center.
1. seed and fermention medium:
Liquid seed culture medium (g/L): peptone 20.0, yeast extract paste 10.0, glucose 20.0, CaCO
310.0, pH7.2-7.4.
Liquid state fermentation substratum (g/L): peptone 10.0, yeast extract paste 10.0, glucose 6.0, pH7.0.
2. thalline activation:
The bacterial classification of glycerine pipe cold storage is received the 250mL that the 50mL liquid seed culture medium is housed shake in the bottle, 37 ℃ of shake-flask culture 20 hours.
3. the thalline seed fermentation is cultivated:
It was that the 500mL of 100mL shakes in the bottle that the bacterial classification that activation is good is received liquid amount with 5% inoculum size, in 37 ± 1 ℃ of shake-flask culture 20 hours.
4. the high-density amplification culture of thalline:
(1) 50L seed tank culture
Inoculum size with 8% is inoculated in Bacillus coagulans in the seeding tank that initial liquid amount is 30L, and leavening temperature is 37 ℃, initial mixing speed 320r/min, and ventilation is 1: 0.7, tank pressure is 0.05MPa.Cultivated 20 hours.
(2) 200L fermentor cultivation
Inoculum size with 4.0% is inoculated in the 200L jar with Bacillus coagulans and carries out fermentation culture, and liquid amount is 60% of a tank volume, and leavening temperature is 37 ℃, initial mixing speed 220r/min, and ventilation is 1: 0.5, cultivates 20 hours.
5. spinning:
Adopt centrifuging concentrating and separating thalline, separation condition is: the centrifugal 20min of 6000r/min, centrifuging temperature are 4 ℃.
Centrifugal effect measuring:
6. the preparation of bacteria suspension:
After centrifugal, abandoning supernatant is added the composite protectant that configures with the bacterium mud after concentrating and is processed bacterium suspension-s, and bacterium mud and composite protectant volume ratio are 1: 3.
7. vacuum-drying:
To add protectant bacterial classification through vacuum-drying 10 hours, make high vigor Bacillus coagulans starter.
The vacuum-drying survival rate is measured
Get the dry bacterium powder of testing sample in Bechtop, add the 10mL SPSS, leave standstill 20min and make its rehydration, adopt then and pour into counting process mensuration viable count, survival rate is calculated as follows:
9. the mensuration of cell concentration:
Dull and stereotyped tilt-pour process counting is got 1mL bacterium liquid with transfer pipet and is added in the 9mL SPSS test tube, mixes; Take turns doing 10 times of gradient dilutions; Get 1mL dilution back bacterium liquid and inject flat board, each extent of dilution do three parallel, the counting substratum after will melting is again poured flat board into; With bacterium liquid and substratum mixing, solidify back 37 ℃ and cultivate 48h.
Below be confirming to direct-throwing Bacillus coagulans TQ33 starter preparation condition; Comprise confirming of the righttest thalline harvest time, centrifugation time, cf-, vacuum-drying protective material, temperature, pressure and other parameters, and the mensuration of the effect of direct-throwing Bacillus coagulans starter.
One, confirming of the righttest thalline harvest time:
Liquid state fermentation is carried out from fermentor tank, taking a sample behind the 6h, and dull and stereotyped tilt-pour process counting is surveyed viable count constantly.
Recording viable count is: 1.5 * 10
8Cfu/mL.
Liquid state fermentation is carried out from fermentor tank, taking a sample behind the 16h, and dull and stereotyped tilt-pour process counting is surveyed viable count constantly.
Recording viable count is: 8.2 * 10
8Cfu/mL.
The result
Can know (accompanying drawing 1) from the fermenting process Bacillus coagulans growth curve of drawing, fermentation is carried out 16h left and right sides viable count and is reached maximum, is about 8.2 * 10
8Cfu/mL gets into stationary phase, and viable count remains unchanged basically, and therefore the righttest thalline harvest time is 16h-20h.
Two, spinning:
1, fermented liquid pH is to the influence of separating effect
Get the bacterium liquid of cultivating 16h in the fermentor tank, do not regulate its pH, under the condition of 6000r/min, 4 ℃ of centrifugal 20min, collect supernatant and throw out, carry out live bacterial count respectively, calculate centrifugal yield.
Recording centrifugal deposition thing viable count is 3.8 * 10
10Cfu/g; The supernatant viable count is 2.4 * 10
4Cfu/g; Centrifugal yield is 82.1%.
Get the bacterium liquid of cultivating 16h in the fermentor tank and regulate pH to 6.8, under the condition of 6000r/min, 4 ℃ of centrifugal 20min, collect supernatant and throw out, carry out live bacterial count respectively, calculate centrifugal yield with NaOH.
Record: centrifugal deposition thing viable count is 3.9 * 10
10Cfu/g; The supernatant viable count is 2.23 * 10
4Cfu/g; Centrifugal yield is 84.9%.
The result
Fermented liquid pH influences not significantly (subordinate list 1) to the centrifugal yield of Bacillus coagulans, and therefore, pH does not adjust to fermented liquid, can save labour intensity, and the technology that simplifies the operation, centrifugal yield are 82.1%, and the settling viable count is 3.8 * 10
10Cfu/g.
2, centrifugation time is to the influence of separating effect
When cf-is 4297g (5000r/min), behind 4 ℃ of centrifugal 15min, collect supernatant and throw out, carry out live bacterial count respectively, calculate centrifugal yield.
Record: centrifugal deposition thing viable count is 3.78 * 10
10Cfu/g; The supernatant viable count is 2.31 * 10
4Cfu/g; Centrifugal yield is 85.67%.
When cf-is 5157g (6000r/min), behind 4 ℃ of centrifugal 15min, collect supernatant and throw out, carry out live bacterial count respectively, calculate centrifugal yield.
Record: centrifugal deposition thing viable count is 3.79 * 10
10Cfu/g; The supernatant viable count is 1.47 * 10
4Cfu/g; Centrifugal yield is 89.20%.
When cf-is 5157g (6000r/min), behind 4 ℃ of centrifugal 20min, collect supernatant and throw out, carry out live bacterial count respectively, calculate centrifugal yield.
Record: centrifugal deposition thing viable count is 3.63 * 10
10Cfu/g; The supernatant viable count is 1.16 * 10
4Cfu/g; Centrifugal yield is 93.92%.
The result
Centrifugal 15min-20min, centrifugal yield is higher, but with 6000r/min, and during centrifugal 20min, centrifugal yield is up to 93.92% (subordinate list 2), and and 5000r/min, the centrifugal yield significant difference of centrifugal 15min is so centrifugation time is decided to be 20min.
3, cf-is to the influence of separating effect
When cf-is 4297g (5000r/min), behind 4 ℃ of centrifugal 20min, collect supernatant and throw out, carry out live bacterial count respectively, calculate centrifugal yield.
Record: centrifugal deposition thing viable count is 3.71 * 10
10Cfu/g; The supernatant viable count is 2.4 * 10
3Cfu/g; Centrifugal yield is 89.48%.
When cf-is 5157g (6000r/min), behind 4 ℃ of centrifugal 20min, collect supernatant and throw out, carry out live bacterial count respectively, calculate centrifugal yield.
Record: centrifugal deposition thing viable count is 3.63 * 10
10Cfu/g; The supernatant viable count is 1.16 * 10
4Cfu/g; Centrifugal yield is 93.92%.
When cf-is 6451g (8000r/min), behind 4 ℃ of centrifugal 20min, collect supernatant and throw out, carry out live bacterial count respectively, calculate centrifugal yield.
Record: centrifugal deposition thing viable count is 3.14 * 10
10Cfu/g; The supernatant viable count is 6.5 * 10
3Cfu/g; Centrifugal yield is 83.44%.
The result
Along with the increase of cf-, centrifugal yield reduces gradually, with the centrifugal 20min of 5000r/min; Centrifugal yield is 89.48%; Centrifugal 20min under other cf-conditions, centrifugal yield is all lower, wherein the centrifugal 20min of 7000r/min; Centrifugal yield is reduced to 90.70% (subordinate list 3), shows that the centrifugal 20min of 7000r/min is the critical condition of centrifugal yield.So select the centrifugal 20min of 6000r/min.
4, centrifuging temperature is to the influence of separating effect
When cf-is 4348g (5000r/min), behind 20 ℃ of centrifugal 20min, collect supernatant and throw out, carry out live bacterial count respectively, calculate centrifugal yield.
Record: centrifugal deposition thing viable count is 3.04 * 10
10Cfu/g; The supernatant viable count is 3.46 * 10
4Cfu/g; Centrifugal yield is 78.27%.
When cf-is 5257g (6000r/min), behind 20 ℃ of centrifugal 20min, collect supernatant and throw out, carry out live bacterial count respectively, calculate centrifugal yield.
Record: centrifugal deposition thing viable count is 3.79 * 10
10Cfu/g; The supernatant viable count is 2.64 * 10
4Cfu/g; Centrifugal yield is 86.30%.
The result
Cf-and centrifugation time are constant, and temperature increases, and the centrifugal yield of Bacillus coagulans reduces (subordinate list 4).Under the same terms, 20 ℃ of centrifugal yield of 4 ℃ of ratios improve about 4%-10%.So it is centrifugal under being chosen in 4 ℃.
Three, vacuum-drying is protectant confirms
Protectant concentration of below mentioning is the weight percent concentration of the aqueous solution of tie substance.
1, single protectant definite:
Collect the bacterium mud after centrifugal, with weight percent be 3% aqueous sucrose solution as protective material, be 3: 1 mixed by the volume ratio of protective material and bacterium mud.Measure the viable count of vacuum-drying front and back, calculate the thalline survival rate.
Recording survival rate is 12.6%.
Collect the bacterium mud after centrifugal, with weight percent be 3% trehalose as protective material, be 3: 1 mixed by the volume ratio of protective material and bacterium mud.Measure the viable count of vacuum-drying front and back, calculate the thalline survival rate.
Recording survival rate is 20.8%.
Collect the bacterium mud after centrifugal, with weight percent be 5% sucrose as protective material, be 3: 1 mixed by the volume ratio of protective material and bacterium mud.Measure the viable count of vacuum-drying front and back, calculate the thalline survival rate.
Recording survival rate is 17.8%.
The result
Sucrose provide protection effect is (accompanying drawing 2) significantly, and massfraction is that 3% sucrose Bacillus coagulans survival rate is 12.6%, adds 5% sucrose and can make its survival rate reach 17.8%.
2, confirming of composite protectant:
Protectant concentration of below mentioning is the weight percent concentration of the aqueous solution of tie substance.
Sucrose with 5% is the basis, adds 10% skim-milk as protective material, is 3: 1 mixed by the volume ratio of protective material and bacterium mud.Measure the viable count of vacuum-drying front and back, calculate the thalline survival rate.
Recording survival rate is 40.5%.
Sucrose with 5% is the basis, adds 10% maltodextrin as protective material, is 3: 1 mixed by the volume ratio of protective material and bacterium mud, measures the viable count before and after the vacuum-drying, calculates the thalline survival rate.
Recording survival rate is 90.3%.
Sucrose with 5% is the basis, adds 10% skim-milk and 3% Sodium Glutamate as protective material, is 3: 1 mixed by the volume ratio of protective material and bacterium mud, measures the viable count before and after the vacuum-drying, calculates the thalline survival rate.
Recording survival rate is 80.7%.
The result
Other composite protectant effects are seen accompanying drawing 3, and when being protective material with the aqueous solution that contains 5% sucrose and 10% maltodextrin, the Bacillus coagulans survival rate reaches and is up to 90.3%.Therefore, composite protectant is 5% sucrose and the 10% maltodextrin aqueous solution.
All the other protections of specifically not listing are please with reference to the description of drawings of Fig. 3 and Fig. 3.
Four, confirming of vacuum-drying condition
1, Heating temperature confirms
At drying chamber pressure is that 3000Pa and inventory are under the situation of 40g, when drying temperature is 40 ℃, measures the viable count of vacuum-drying front and back, calculates the thalline survival rate.
Recording survival rate is 95%.
At drying chamber pressure is that 3000Pa and inventory are under the situation of 40g, when drying temperature is 60 ℃, measures the viable count of vacuum-drying front and back, calculates the thalline survival rate.
Recording survival rate is 93.7%.
The result
When drying temperature was 60 ℃, the thalline survival rate was high, compared survival rate with 50 ℃ and did not almost have difference with 40 ℃, and can shorten time of drying 60 ℃ the time.Therefore, drying temperature is elected 60 ℃ (accompanying drawings 4) as.
2, drying pressure confirms
Being 60 ℃ in the kiln temperature is under the situation of 40g with inventory, and drying pressure is 800Pa, measures the viable count of vacuum-drying front and back, calculates the thalline survival rate.
Recording survival rate is 94.5%.
Being 60 ℃ in the kiln temperature is under the situation of 40g with inventory, and drying pressure is 3000Pa, measures the viable count of vacuum-drying front and back, calculates the thalline survival rate.
Recording survival rate is 93%.
The result
When drying pressure was 12000Pa, the thalline survival rate was high, and time of drying is short.Therefore, drying pressure is elected 12000Pa (accompanying drawing 5) as.
Five, the application of direct-throwing Bacillus coagulans starter in liquid state fermentation
The Bacillus coagulans starter is added in sterilized 0.85% saline water, and normal temperature rehydration 20min inserts the liquid state fermentation substratum of the bacterium of going out then by 0.1% inoculum size, form (g/L): peptone 10.0; Yeast extract paste 10.0; Glucose 10.0.In 37 ± 1 ℃ of shake-flask culture 16h, survey viable count.
Recording viable count is: 6.0 * 10
9Cfu/mL.
Bacillus coagulans that will be after liquid activation inserts the liquid state fermentation substratum of the bacterium of going out by 5% inoculum size, (g/L): peptone 10.0; Yeast extract paste 10.0; Glucose 10.0.In 37 ± 1 ℃ of shake-flask culture 16h, survey viable count.Recording viable count is: 7.2 * 10
9Cfu/mL.
The result
Use direct-throwing Bacillus coagulans starter to contrast in the liquid state fermentation substratum by inoculum size 0.1% and inoculum size 5% liquid inoculation, the highest viable count is respectively 8.1 * 10
9Cfu/mL and 7.8 * 10
9Cfu/mL (accompanying drawing 6).Obviously reduce inoculum size, and can reach better ferment effect.
Six, the application of direct-throwing Bacillus coagulans starter in solid state fermentation
The Bacillus coagulans starter is added in sterilized 0.85% saline water, normal temperature rehydration 20min, inserting what went out bacterium by 0.1% inoculum size then is main solid-state fermentation culture medium with the dregs of beans, forms (%): dregs of beans 60, glucose 20, peptone 20.Add entry 150ml according to every 100g substratum then.In 37 ± 1 ℃ of aerobic cultivation 26h, survey viable count.
Recording viable count is: 7.9 * 10
9Cfu/g.
It is main solid-state fermentation culture medium with the dregs of beans that Bacillus coagulans that will be after liquid activation inserts what went out bacterium by the inoculum size of 10% (v/w), forms (%): dregs of beans 60, glucose 20, peptone 20.Add entry 150ml according to every 100g substratum then.In 37 ± 1 ℃ of aerobic cultivation 26h, survey viable count.
Recording viable count is: 7.3 * 10
9Cfu/g.
The result
Use direct-throwing Bacillus coagulans starter to contrast in solid-state fermentation culture medium by inoculum size 0.1% and inoculum size 10% liquid inoculation, the highest viable count is respectively 10.0 * 10
9Cfu/g and 9.7 * 10
9Cfu/g (accompanying drawing 7).There is the lag phase of about 14h in starter inoculation back thalline, and growth then is quick, and final ferment effect is desirable, can obviously reduce inoculum size.
Conclusion
Through to direct-throwing Bacillus coagulans starter Research on Process, Bacillus coagulans concentrates and adopts final Bacillus coagulans survival rate to reach 90.3%, and the viable count of starter is 3.24 * 10
10Cfu/g, the inoculum size by 1 ‰ just can reach ideal liquid state or solid state fermentation effect, and the highest viable count is respectively 8.1 * 10
9Cfu/mL, 1.0 * 10
10Cfu/g.
Table 1 fermented liquid pH is to the influence of separating effect
Table 2 centrifugation time is to the influence of separating effect
Table 3 cf-is to the influence of separating effect
Table 4 centrifuging temperature is to the influence of separating effect
Claims (1)
1. the preparation method of a direct-throwing Bacillus coagulans starter is characterized in that: the Bacillus coagulans fermented liquid is concentrated obtain bacterium mud, fermented liquid concentrates condition and is: centrifugal rotational speed 6000r/min; Centrifugal 20min, centrifuging temperature are 4 ℃, and bacterium mud is joined in protectant aqueous solution; The protective material that is adopted is the aqueous solution that contains 5% m/m sucrose and 10% m/m maltodextrin; The employing boulton process is dry, and drying temperature is elected 60 ℃ as, and drying pressure is elected 12000 Pa as; The name of said Bacillus coagulans is called TQ33, and deposit number is: CGMCC No. 5233, specific name Bacillus coagulans, said bacterium mud and protectant volume ratio 1:2-5.
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