CN104745554B - Bacillus produces the fermentation medium and fermentation process of protease and gemma - Google Patents
Bacillus produces the fermentation medium and fermentation process of protease and gemma Download PDFInfo
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- CN104745554B CN104745554B CN201510180738.5A CN201510180738A CN104745554B CN 104745554 B CN104745554 B CN 104745554B CN 201510180738 A CN201510180738 A CN 201510180738A CN 104745554 B CN104745554 B CN 104745554B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12N3/00—Spore forming or isolating processes
Abstract
Bacillus produces the fermentation medium and fermentation process of protease and gemma, is related to protease and fermentation medium.The composition of raw materials of the fermentation medium of bacillus production protease and gemma is：Peptone 7g/L, yeast extract 7g/L, NaCl20g/L, CaCl20.2g/L, pH 9.0.Fermentation process is as follows：The bacillus production protease of sterilization and the fermentation medium of gemma are packed into conical flask, the seed liquor of bacillus A3440 is seeded in conical flask again and carries out fermented and cultured, that is, completes the fermentation for the beneficial bacteria of intestinal tract of abalone culture using the fermentation medium of bacillus production protease and gemma.By optimization of fermentation conditions, the gemma rate and prolease activity of bacterium solution are improved.Gemma rate is 94.3% with optimal conditions, and prolease activity 294.44U/mL is conducive to the culture of bacillus, reduces the cost of follow-up work.
Description
Technical field
The present invention relates to protease and fermentation medium, the fermentation that protease and gemma are produced more particularly, to bacillus is trained
Support base and fermentation process.
Background technology
It is supported with the fast development of aquaculture, the raising of intensive degree and the worsening of coastal water quality, aquatic products
It is increasing to grow animal burst disease.Though aquatic products is alleviated and is controlled in the application of antibiotic and chemicals to a certain extent
The generation and sprawling of biological disease, but it is following the drawbacks of also increasingly appear.It not only runs counter to the health that people are advocated and supports
Objective is grown, while also disturbs breeding environment and aquatic animal microecological balance, causes aquatic livestock to the susceptible of pathogenic microorganism
Property, the more seriously drug resistance enhancing of pathogen so that aquiculture disease preventing and controlling are all the more difficult.In addition, antibiotic
In the in vivo residual of aquatic animal, enrichment so that aquatic product quality reduces, and becomes aquatic products sales volume and export trade amount
Limiting factor.
1986, Kozasa etc. for the first time using bud pole bacterium processing Japanese eel reduce due to infection tarda and
After caused death, probiotics is rapidly developed in terms of aquatic products research.Probiotics refer to " when take in it is a certain amount of when to host
The effective living microorganisms of health " have many advantages, such as to have no toxic side effect, noresidue, have no drug resistance.Probiotics is in water at this stage
Application in production cultivation is mainly reflected in by improving the microbiologic population inside and outside cultivation subject, and increase cultivation object is to raising
The grazing rate of material enhances its nutritive value, while improves the immunocompetence of cultivation object, and the yield of cultivation object is improved with this,
Positive effect is all played in the cultivation of fish and shellfish.In recent years, the theory to probiotics preparation in terms of aquaculture
Worldwide upsurge has been increasingly becoming with application study.The Ministry of Agriculture of China discloses 5 kinds and can be applied to animal-breeding
Microorganism, including Bacillus acidi lactici, bacillus, saccharomycete, streptococcus fecalis and Bifidobacterium.
Bacillus belongs to bacillus more, and Gram-positive is mostly aerobic or amphimicrobian.It, which has, divides
Solution conversion and adaptable, metabolizable generation protease, amylase, a variety of enzymes such as esterase improve cultivation object to feed
Absorption and conversion, promote growth of animal, while it makes it as the micro- life of feed to cultivation object and the features such as the harmless mankind
All have broad application prospects on state additive and water body microecologic regulator.Mechanism of action table of the bacillus as probiotics
(1) reduces levels of toxic substances in breeding water body now, improves water environment；(2) improve the immunity of organisms of animal, reduce disease
Occurrence probability；(3) improve animal digestion enzyme activity, promote fast-growth；(4) microecological balance etc. in animal body is adjusted.
Therefore, the digestive enzyme activity of probiotics is improved, may advantageously facilitate host health growth.A key character for bacillus is,
When environment harshness or nutrition supply deficiency, an approximate circle endospore can be formed in bacillus, becomes gemma.
At this point, the metabolism of bacterium almost stops, there is extremely strong repellence to unfavorable condition, there is high temperature resistant, resistance to drying, resistance to
The characteristics of soda acid.Therefore bacillus preparation is easy to preserve and transport, and does not influence its quality still under severe conditions, can after activation
To continue to come into operation.The Number of spores that bacillus is formed in probiotics is a weight for judging probiotics quality
Want index.
At present, two aspects are included to the research of prebiotic bacillus：First, screening strain excellent, builds huge benefit
Raw bacterium resources bank.Second, for known beneficial bacterial strain, optimize its condition of culture by studying, reduce and cultivate cost, improve bacterium
Strain yield and activity, have important practical significance.The fermented and cultured of bacillus is industrialization large-scale production bacillus
The premise of preparation.
A kind of the applicant's intestinal probiotic powder for abalone culture disclosed in Chinese patent 201210016016.2 and
Its preparation method, which discloses bacillus (B.stratosphericus) A3440, which protected on 09 17th, 2011
China Committee for Culture Collection of Microorganisms's common micro-organisms center is hidden in, deposit number is CGMCC No.5253.
The content of the invention
It is an object of the invention to provide a kind of bacillus production protease and the fermentation medium of gemma.
Another object of the present invention is to provide a kind of fermentation medium use that protease and gemma are produced using bacillus
In the fermentation process of the beneficial bacteria of intestinal tract of abalone culture.
The composition of raw materials of the fermentation medium of bacillus production protease and gemma is：Peptone 7g/L, yeast extract
7g/L、NaCl20g/L、CaCl20.2g/L, pH 9.0；The bacillus is bacillus (B.stratosphericus)
A3440, the bacterium were preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 09 17th, 2011, protected
It is CGMCC No.5253 to hide number (referring to the first Chinese patent application 201210016016.2 of the applicant).
It is described that hair of the fermentation medium of protease and gemma for the beneficial bacteria of intestinal tract of abalone culture is produced using bacillus
Fermenting process is as follows：The bacillus production protease of sterilization and the fermentation medium of gemma are packed into conical flask, then will
The seed liquor of bacillus (B.stratosphericus) A3440, which is seeded in conical flask, carries out fermented and cultured, that is, completes to use
Bacillus produces fermentation of the fermentation medium of protease and gemma for the beneficial bacteria of intestinal tract of abalone culture.
250mL conical flasks can be used in the conical flask, and the addition of fermentation medium can be 30mL；The sterilization
Temperature can be 120 DEG C, and the time of sterilization can be 30min；The inoculum concentration of the inoculation can be gemma bar by mass percentage
Bacterium produces the 8% of the fermentation medium of protease and gemma；The condition of the fermented and cultured, which can be placed on constant-temperature table, ferments
Culture, 30 DEG C of cultivation temperature, incubation time is for 24 hours.
The present invention improves the gemma rate and prolease activity of bacterium solution by optimization of fermentation conditions.Through experiment, in optimization item
Gemma rate is 94.3% under part, and the culture medium that relatively sets out improves 8.77%；Prolease activity is 294.44U/mL, and relatively set out culture medium
Improve 76.67%.Be conducive to the culture of bacillus, reduce the cost of follow-up work.
Present invention optimizes the condition of culture of bacillus (B.stratosphericus) A3440, are bacillus preparation
Industrialized production theoretical foundation and technical support are provided.
Description of the drawings
Fig. 1 is casein standard curve in the present invention.
Fig. 2 is the growth curve of bacillus in the present invention (B.stratosphericus) A3440.
Fig. 3 is influence of the different carbon source to bacillus (B.stratosphericus) A3440 production protease in the present invention
Figure.
Fig. 4 be in the present invention different carbon source on the sporiferous influences of bacillus (B.stratosphericus) A3440
Figure.
Fig. 5 is influence of the different nitrogen sources to bacillus (B.stratosphericus) A3440 production protease in the present invention
Figure.
Fig. 6 be in the present invention different nitrogen sources on the sporiferous influences of bacillus (B.stratosphericus) A3440
Figure.
Fig. 7 is the shadow that different inorganic salts produce bacillus (B.stratosphericus) A3440 in protease in the present invention
Ring figure.
Fig. 8 be in the present invention different inorganic salts on the sporiferous influences of bacillus (B.stratosphericus) A3440
Figure.
Fig. 9 is that different metal ions produce protease to bacillus (B.stratosphericus) A3440 in the present invention
Influence figure.
Figure 10 is that different metal ions are sporiferous to bacillus (B.stratosphericus) A3440 in the present invention
Influence figure.
Figure 11 is the shadow that different temperatures produces bacillus (B.stratosphericus) A3440 in protease in the present invention
Ring figure.
Figure 12 be in the present invention different temperatures on the sporiferous influences of bacillus (B.stratosphericus) A3440
Figure.
Figure 13 is that different vaccination amount produces protease to bacillus (B.stratosphericus) A3440 in the present invention
Influence figure.
Figure 14 be in the present invention different vaccination amount to the sporiferous shadows of bacillus (B.stratosphericus) A3440
Ring figure.
Figure 15 is that different liquid amounts produce protease to bacillus (B.stratosphericus) A3440 in the present invention
Influence figure.
Figure 16 be in the present invention different liquid amounts to the sporiferous shadows of bacillus (B.stratosphericus) A3440
Ring figure.
Figure 17 is that different initial pH values produce protease to bacillus (B.stratosphericus) A3440 in the present invention
Influence figure.
Figure 18 is that different initial pH values are sporiferous to bacillus (B.stratosphericus) A3440 in the present invention
Influence figure.
Specific embodiment
Following embodiment will the present invention is further illustrated with reference to attached drawing.
Bacillus (B.stratosphericus) A3440 was preserved in Chinese microorganism strain on 09 17th, 2011
Preservation administration committee common micro-organisms center, deposit number are (special referring to the first China of the applicant for CGMCC No.5253
Profit application is 201210016016.2).
1 materials and methods
1.1 bacterial strain
Bacillus (B.stratosphericus) A3440 separates preservation by Xiamen University ocean with earth institute.
1.2 basal fermentation medium
The fermentation medium based on 2216E fluid nutrient mediums：Peptone 5g/L, yeast extract 1g/L, high ferric phosphate
0.01g/L, Chen Haishui 1L.
1.3 test method
1.3.1 seed liquor culture
Bacillus (B.stratosphericus) A3440 of -80 DEG C of preservations is inoculated in 2216E solids with oese
On culture medium, 30 DEG C of culture 48h are activated.After activation 2 times, from one ring monoclonal A3440 of picking on 2216E tablets, connect
Kind is spare after the 250mL conical flasks equipped with 100mL 2216E fluid nutrient mediums, 200r/min, 30 DEG C of shaking table culture 20h.
1.3.2 the measure of growth curve
Above-mentioned seed liquor is inoculated in the 250mL conical flasks equipped with 100mL basal fermentation mediums, inoculum concentration 1%,
200r/min, 30 DEG C of shaking table cultures.It is sampled every 2h, using basal fermentation medium as blank, measures zymotic fluid in 630nm conditions
Under absorbance.Using incubation time as abscissa, OD630For ordinate, the growth curve of drafting bacillus A3440.
1.3.3 the measure of gemma rate
Using dilution plate counting method, 10 are chosen6、107、108Three concentration gradients, 3 tablets of each gradient coating, 30
DEG C culture 48h after counted.Dilution plate after 80 DEG C of water-bath 10min of bacterium solution is counted, gemma rate=80 DEG C water-bath counting/hair
Zymotic fluid viable count.
1.3.4 the measure of prolease activity
Prepare standard serial solution with the casein standard solution of 100 μ g/mL, casein final concentration 0,10,20,30,40,
50 μ g/mL are handled using raw work modified form lowey method determination of protein concentration kit, its OD are measured in microplate reader respectively630,
Draw standard curve.Using distilled water as blank control.Standard curve is shown in Fig. 1.
Sample assay method：Zymocyte liquid 1ml is taken, 15min is centrifuged in 8000rpm, 0.2ml is taken to add in 2 1.5mL respectively
Centrifuge tube, labeled as experimental group and blank group.40 DEG C of water-bath 2min, blank group add in 0.4mol/L trichloroacetic acids 0.4ml.Each group
Add in 1% casein 0.8ml, 40 DEG C of heat preservation 20min.Experimental group adds in 0.4ml 0.4mol/L trichloroacetic acids, keeps the temperature 20min.
Centrifuging and taking supernatant dilutes 100 times.20ul is taken in ELISA Plate, using 20ul distilled water as control.Use raw work modified form lowey
The processing of method determination of protein concentration kit, measures its OD in microplate reader630。
Calculation formula：
Enzyme activity (U/mL)=△ OD*1ml*N/ (k*20min)
Wherein △ OD are experimental group and blank group OD630Difference；N is the extension rate of enzyme solution；K is slope of standard curve.
1.3.5 different carbon source is to the shadow of bacillus (B.stratosphericus) A3440 prolease activities and gemma rate
It rings
Peptone in basal fermentation medium is replaced with to starch, lactose, glucose, sucrose, the maltose of equivalent, with
Be not added with the culture medium of carbon source as negative control, using basal fermentation medium as positive control.Seed is added in 1% inoculum concentration
Liquid shakes training for 24 hours under the conditions of 30 DEG C, 200r/min.Zymotic fluid prolease activity and gemma rate are measured, compares different carbon source to bud
Spore bacillus (B.stratosphericus) A3440 prolease activities and the influence of gemma rate.
1.3.6 different nitrogen sources are to the shadow of bacillus (B.stratosphericus) A3440 prolease activities and gemma rate
It rings
Yeast extract in basal fermentation medium is replaced with to beef extract, ammonium sulfate, ammonium nitrate, the ammonium acetate of equivalent, with
The optimal carbon source of above-mentioned experiment replaces peptone in basal fermentation medium, right as feminine gender to be not added with the culture medium of nitrogen source
According to, using basal fermentation medium as positive control.Seed liquor is added in 1% inoculum concentration, training is shaken under the conditions of 30 DEG C, 200rpm
24h.Zymotic fluid prolease activity and gemma rate are measured, compares different nitrogen sources to bacillus (B.stratosphericus)
A3440 prolease activities and the influence of gemma rate.
1.3.7 different inorganic salts are to bacillus (B.stratosphericus) A3440 prolease activities and gemma rate
It influences
Chen Haishui in basal fermentation medium is replaced with into 10 ‰ NaCl, 10 ‰ KCl, 10 ‰ MgSO4,10 ‰ CaCl2
Add single steaming water, with the optimal carbon source and nitrogen source of above-mentioned experiment instead of the peptone in basal fermentation medium and yeast extract, with not
Add the culture mediums of inorganic salts as negative control, using basal fermentation medium as positive control.Seed is added in 1% inoculum concentration
Liquid shakes training for 24 hours under the conditions of 30 DEG C, 200rpm.Zymotic fluid prolease activity and gemma rate are measured, more different inorganic salts are to bud
Spore bacillus (B.stratosphericus) A3440 prolease activities and the influence of gemma rate.
1.3.8 different metal ions are to bacillus (B.stratosphericus) A3440 prolease activities and gemma rate
Influence
High ferric phosphate in basal fermentation medium is replaced with into equivalent Mg2+、Ca2+、Zn2+、Cu2+, with above-mentioned experiment most
Excellent carbon source, nitrogen source and inorganic salts replace peptone, yeast extract and Chen Haishui in basal fermentation medium, to be not added with metal ion
Culture medium as negative control, using basal fermentation medium as positive control.Seed liquor is added in 1% inoculum concentration, 30
DEG C, shake training for 24 hours under the conditions of 200rpm.Zymotic fluid prolease activity and gemma rate are measured, compares different metal ions to A3440 eggs
White enzyme activity and the influence of gemma rate.
1.3.9 the orthogonal optimization of medium component
According to above experimental result, select optimal carbon source, optimal nitrogen source, optimal inorganic salts, optimal metal ion four because
Plain four level design orthogonal experiments.Specific experiment design is as shown in table 1.
1 bacillus of table (B.stratosphericus) A3440 shaking flask Orthogonal Experiment and Designs
1.3.10 influence of the temperature to bacillus (B.stratosphericus) A3440 prolease activities and gemma rate
Fermentation medium is prepared according to orthogonal experiments.With 1% inoculum concentration add in seed liquor, respectively 20 DEG C, 25 DEG C,
30 DEG C, 35 DEG C, 40 DEG C of five kinds of temperature cultures shake training for 24 hours under the conditions of 200rpm.Measure zymotic fluid prolease activity and gemma
Rate compares influence of the different temperatures to bacillus (B.stratosphericus) A3440 prolease activities and gemma rate.
1.3.11 inoculum concentration is to the shadow of bacillus (B.stratosphericus) A3440 prolease activities and gemma rate
It rings
Fermentation medium is prepared according to orthogonal experiments.Kind is added in 1%, 3%, 5%, 8%, 10% inoculum concentration respectively
Sub- liquid shakes training for 24 hours under the conditions of Optimal Temperature, 200rpm.Zymotic fluid prolease activity and gemma rate are measured, compares different vaccination
Measure the influence to bacillus (B.stratosphericus) A3440 prolease activities and gemma rate.
1.3.12 liquid amount is to the shadow of bacillus (B.stratosphericus) A3440 prolease activities and gemma rate
It rings
Fermentation medium is prepared according to orthogonal experiments.Be separately added into 250mL conical flasks 20mL, 30mL, 40mL,
50mL, 60mL, 70mL culture medium add in seed liquor with optimal inoculum concentration, training are shaken under the conditions of Optimal Temperature, 200rpm for 24 hours.It surveys
Determine zymotic fluid prolease activity and gemma rate, more different liquid amounts are to bacillus (B.stratosphericus) A3440 eggs
White enzyme activity and the influence of gemma rate.
1.3.13 initial pH value is to bacillus (B.stratosphericus) A3440 prolease activities and gemma rate
It influences
Fermentation medium is prepared according to orthogonal experiments.With optimal inoculum concentration add in seed liquor, Optimal Temperature,
Training is shaken under the conditions of 200rpm for 24 hours.Zymotic fluid prolease activity and gemma rate are measured, more different initial pH values are to bacillus
(B.stratosphericus) A3440 prolease activities and the influence of gemma rate.
2. result and analysis
2.1 bacillus (B.stratosphericus) A3440 growth curves (see Fig. 2)
In 0~10h, thalli growth speed is slow；In 10~28h, thalli growth speed is accelerated；Since 28h, thalline life
Long speed is gradually reduced.Therefore select to be in the thalline of the vigorous logarithmic phase of vitality as seed liquor, select 24~26h as
Kind age.
2.2 training systern
2.2.1 different carbon source is to the shadow of bacillus (B.stratosphericus) A3440 prolease activities and gemma rate
It rings (see Fig. 3 and 4)
The result shows that using peptone, sucrose, lactose as carbon source, gemma rate is larger, all more than 85%.But peptone
As carbon source, biomass and prolease activity are all higher by other groups, therefore select peptone as optimal carbon source.
2.2.2 different nitrogen sources are to the shadow of bacillus (B.stratosphericus) A3440 prolease activities and gemma rate
It rings (see Figures 5 and 6)
The result shows that using yeast extract, ammonium acetate, beef extract as nitrogen source, gemma rate is larger, all more than 80%.But ferment
Female cream is all higher by other groups as nitrogen source, biomass and prolease activity, therefore selects yeast extract as optimal nitrogen source.
2.2.3 different inorganic salts are to bacillus (B.stratosphericus) A3440 prolease activities and gemma rate
It influences (see Fig. 7 and 8)
The result shows that each experimental group gemma rate and no significant difference, using NaCl and KCl as inorganic salts, biomass is larger,
And NaCl as inorganic salts when, the prolease activity of zymotic fluid is most strong, therefore selects NaCl as optimal inorganic salts.
2.2.4 different metal ions are to bacillus (B.stratosphericus) A3440 prolease activities and gemma rate
Influence (see Fig. 9, Figure 10)
The result shows that with Fe3+And Ca2+During as metal ion, zymotic fluid prolease activity is higher.With Ca2+As metal
When ion is as metal ion, there are highest biomass and gemma rate, therefore select Ca2+As optimal metal ion.
2.2.5 bacillus (B.stratosphericus) A3440 bacterial strain shaking flask orthogonal experiments (being shown in Table 2)
As shown in Table 2, very poor value RPeptone>RNaCl>RYeast extract>RCaCl2, it is known that peptone is as main influence factor, NaCl
Take second place.The optimal A3B3C4D4 that is combined as, i.e. 7 ‰ peptones, 7 ‰ yeast extracts, 20 ‰ NaCl, 0.2 ‰ are drawn by average K
CaCl2Combination carries out the optimization of fermentation condition.
2.2.6 different temperatures is to the shadow of bacillus (B.stratosphericus) A3440 prolease activities and gemma rate
It rings (see Figure 11 and 12)
The result shows that in 30 DEG C and 35 DEG C, the prolease activity highest of zymotic fluid, but with the rise of temperature, fermentation
The gemma rate of liquid constantly reduces, therefore selects 30 DEG C of optimum temperatures the most.
2.2.7 different vaccination amount is to bacillus (B.stratosphericus) A3440 prolease activities and gemma rate
It influences (see Figure 13 and 14)
The result shows that inoculum concentration is at 3%~8%, prolease activity highest, but when inoculum concentration is 5%, biomass is inclined
It is low, when inoculum concentration is 8%, there is higher biomass and gemma rate, therefore select 8% as most suitable inoculum concentration.
2 bacillus of table (B.stratosphericus) A3440 shaking flask orthogonal experiments
2.2.8 different liquid amounts are to bacillus (B.stratosphericus) A3440 prolease activities and gemma rate
It influences (see Figure 15 and 16)
The result shows that when liquid amount is 20~40mL, the prolease activity of zymotic fluid is higher, is 30mL in liquid amount
When, there is highest biomass and gemma rate, therefore select 30nL as most suitable liquid amount.
2.2.9 different initial pH values are to bacillus (B.stratosphericus) A3440 prolease activities and gemma rate
Influence (see Figure 17 and 18)
The result shows that when pH value is 9.0, zymotic fluid has highest prolease activity and gemma rate, therefore selects most suitable
PH value is 9.0.
Microbial fermentation is a complicated physiological and biochemical procedure, can direct shadow when thalline is in Different Nutrition condition
Ring the physiological metabolism of thalline.Improve prolease activity and bud in bacillus (B.stratosphericus) A3440 zymotic fluids
Spore rate, it is necessary to be optimized to fermentation condition.The present invention is to bacillus (B.stratosphericus) A3440 fermentation conditions
It optimizes, the fermentation medium of its optimal fermentation production protease and gemma is determined by single factor test and Orthogonal Optimization, it should
The composition of raw materials of fermentation medium is：Peptone 7g/L, yeast extract 7g/L, NaCl20g/L, CaCl20.2g/L, pH 9.0.Training
The condition of supporting is that the seed liquor of bacillus (B.stratosphericus) A3440 is seeded to 250, L tapers by 8% inoculum concentration
In bottle, the fermentation medium equipped with 30mL sterilizations in the conical flask；The conical flask is placed on constant-temperature table afterwards
Fermented and cultured is carried out, 30 DEG C of cultivation temperature, incubation time are for 24 hours.
Claims (5)
1. the fermentation process of the beneficial bacteria of intestinal tract of abalone culture is used for using the fermentation medium of bacillus production protease and gemma,
It is characterized in that specific method is as follows：The bacillus production protease of sterilization and the fermentation of gemma are packed into conical flask
Culture medium, then the seed liquor of bacillus (B.stratosphericus) A3440 is seeded in conical flask and carries out fermentation training
It supports, that is, completes the fermentation for the beneficial bacteria of intestinal tract of abalone culture using the fermentation medium of bacillus production protease and gemma；
The composition of raw materials of the fermentation medium of bacillus production protease and gemma is：Peptone 7g/L, yeast extract 7g/L,
NaCl20g/L、CaCl20.2g/L, pH 9.0；The bacillus is bacillus (B.stratosphericus) A3440.
2. as described in claim 1 using the fermentation medium of bacillus production protease and gemma for the enteron aisle benefit of abalone culture
The fermentation process of raw bacterium, it is characterised in that the conical flask uses 250mL conical flasks, and the addition of fermentation medium is 30mL.
3. as described in claim 1 using the fermentation medium of bacillus production protease and gemma for the enteron aisle benefit of abalone culture
The fermentation process of raw bacterium, it is characterised in that the temperature of the sterilization is 120 DEG C, and the time of sterilization is 30min.
4. as described in claim 1 using the fermentation medium of bacillus production protease and gemma for the enteron aisle benefit of abalone culture
The fermentation process of raw bacterium, it is characterised in that the inoculum concentration of the inoculation produces protease and gemma for bacillus by mass percentage
Fermentation medium 8%.
5. as described in claim 1 using the fermentation medium of bacillus production protease and gemma for the enteron aisle benefit of abalone culture
The fermentation process of raw bacterium, it is characterised in that the condition of the fermented and cultured is placed in carrying out fermented and cultured on constant-temperature table, culture
30 DEG C of temperature, incubation time is for 24 hours.
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| CN111187744A (en) * | 2020-03-16 | 2020-05-22 | 厦门大学 | High-density industrial fermentation medium for stratospheric bacillus and fermentation method thereof |
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| CN102578402A (en) * | 2012-01-18 | 2012-07-18 | 厦门大学 | Intestinal probiotic powder for abalone culture and preparation method thereof |
| CN103805535A (en) * | 2013-11-28 | 2014-05-21 | 中国农业科学院农业资源与农业区划研究所 | Isothermal layer bacillus, microbial agent and application of isothermal layer bacillus and microbial agent |
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| CN102578402A (en) * | 2012-01-18 | 2012-07-18 | 厦门大学 | Intestinal probiotic powder for abalone culture and preparation method thereof |
| CN103805535A (en) * | 2013-11-28 | 2014-05-21 | 中国农业科学院农业资源与农业区划研究所 | Isothermal layer bacillus, microbial agent and application of isothermal layer bacillus and microbial agent |
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