CN105018389B - A kind of bacillus sp. CAMT22370 and its application - Google Patents
A kind of bacillus sp. CAMT22370 and its application Download PDFInfo
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Abstract
The invention discloses a kind of bacillus（Bacillus sp）CAMT22370, is preserved in China typical culture collection center, and deposit number is CCTCC NO:M2015247.The bacillus can high-yield of low-temperature glucose oxidase, so as to have a good fresh-keeping effect, the logistics suitable for aquatic products such as prawns is fresh-keeping.The invention also discloses the method that fermenting and producing glucose oxidase is carried out using the bacterial strain.This method is simple and easy, while yield and production efficiency are all significantly improved.
Description
Technical field
The present invention relates to a kind of microbe to screen and fermentation arts, and in particular toBacillus spCAMT22370 and its
The method of liquid fermentation malaga carbohydrate oxidase.
Background technology
Glucose oxidase（Glucose oxidase, GOD；EC 1.1.3.4.）By the use of oxygen molecule as electron acceptor,
β-D-Glucose is oxidized to gluconic acid and hydrogen peroxide in specific manner, so as to effectively remove oxygen.Due to its natural nothing
Toxic side effect and be widely used in food processing, medicine and biological field.The caused guarantor based on its unique deoxidation effect
Fresh effect, at present in the field extensive use such as fruit, Flour product, drinks, dairy products.Tentatively attempt commercialization in horse Qinghe etc.
Glucose oxidase to be applied to prawn fresh-keeping, the results showed that extend about 1 day fresh-keeping compared with control under 4 DEG C of refrigerated condition
Phase, it imply that glucose oxidase will produce good effect in prawn preservation field.
The industrialized production of glucose oxidase mainly uses terrestrial aspergillus niger or mould solid or liquid state fermentation at present, but
The defects of expression quantity is relatively low and foreign protein is more during the fermentation be present, bring very big difficulty to isolating and purifying, cause to produce
Product cost increases, and is to restrict the enzyme in the wide variety of limiting factor of field of food.The glucose oxidase of China's production at present
Industrial enzyme preparation purity is generally relatively low, relies on import for a long time, therefore find the replacement of glucose oxidase import enzyme preparation
Product has important practical significance.Meanwhile the terrestrial bacterium source glucose oxidase optimum temperature reported is more at 30-40 DEG C
Temperature range, and vigor is relatively low at a temperature of 0-10 DEG C of Food Cold Chain, and fresh-keeping effect is bad.
The content of the invention
Based on the producing enzyme vigor present in current glucose oxidase production bacterial strain terrestrial source is relatively low, under cryogenic conditions it is living
Property is not strong, and the limitation that tunning foreign protein content is higher, is separated the invention provides one kind from marine resources low
The method of warm glucose oxidase superior strain and its application of malaga carbohydrate oxidase.
One of the object of the invention there is provided a kind of marine bacteria strain for producing low temperature glucose oxidase.
The two of the object of the invention there is provided a kind of application that low temperature glucose oxidase is produced using aimed strain.
Therefore, the invention provides a bacillus（Bacillus sp）CAMT22370, it is preserved in Chinese Typical Representative training
Thing collection is supported, deposit number is CCTCC NO：M2015247.The bacillus of the present inventionBacillus sp.CAMT22370 is the deep-sea bacterial strain that can produce low temperature glucose oxidase, and the bacterium was preserved in China on April 27th, 2015
Type Tissue Collection, the collection are referred to as CCTCC, and depositary institution address is that wuchang, wuhan Luo Jia Shan Wuhan is big
Learn, deposit number is CCTCC No: M2015247.
The bacillus CCTCC NO of the present invention:M2015247 can be used and carried out containing the culture medium of peptone and yeast extract
Culture, such as：Peptone 5g, yeast extract 1g, ferrous sulfate 0.1g, natural sea-water 1L, pH7.6-7.8.
Present invention also offers application of the above-mentioned bacillus in fermenting and producing glucose oxidase.
Preferably, the glucose oxidase is in low temperature（0~10℃）Lower vigor is more than 4U/mL.
Present invention also offers a kind of preparation method of glucose oxidase, it uses bacillusBacillus sp.
CAMT22370 fermentations are made.Preferably, the fermentation is liquid fermentation.
The present invention is by single factor experiment to bacillusBacillus sp. CAMT22370 liquid fermentations malaga sugar
The condition of oxidizing ferment is optimized.
The invention discloses bacillusBacillus sp. The condition of CAMT22370 malaga carbohydrate oxidases, it is trained
Support, fermentation condition it is simple, stable hereditary property.The bacterium source is in deep-sea, and its most suitable carbon source of fermenting is maltose, and nitrogen source is composed
Extensively, but with organic nitrogen it is preferred, customary inorganic salts species is not notable to producing enzyme effect of vigor, to add the CaCl of 0.05% mass ratio2
It is more suitable.Such as：Maltose 5g, dusty yeast 10g, calcium chloride 2.8g, sodium chloride 1g, Tween-80 1.5mL, deionized water
1L, pH7.5.Fermentation condition can be：The volume fraction of inoculum concentration 2 ~ 10%, 22 ~ 37 DEG C, 36 ~ 84h of fermentation time of producing enzyme temperature, turn
0 ~ 250rpm of speed；It is highly preferred that inoculum concentration is 6 ~ 8%（Volume fraction）, 30 ~ 35 DEG C, 40 ~ 72h of fermentation time of producing enzyme temperature, turn
150 ~ 250rpm of speed.Most preferably, suitable inoculum concentration is 7% during fermentation（Volume fraction）, producing enzyme temperature is 31 DEG C, suitable fermentation
Time is 60h, and suitable rotating speed is 200rpm, and now enzyme activity reaches more than 13U/mL.
The beneficial effects of the invention are as follows：
Bacillus provided by the inventionBacillus sp. CAMT22370 can produce low temperature glucose oxidase, should
Enzyme activity of the enzyme at a temperature of 0 ~ 10 DEG C of Food Cold Chain significantly improves relative to the glucose oxidase in the sources such as traditional aspergillus niger,
So as to greatly improve fresh-keeping effect at low temperature, the aquatic products such as the preservation by low temperature for aquatic products, particularly prawn are very suitable for
The logistics of food is fresh-keeping.It can also be applied to the fresh-keeping of the fields such as fruit, Flour product, drinks, dairy products.For it is fresh-keeping when, can use
The low temperature glucose oxidase of the present invention substitutes traditional glucose oxidase to prepare antistaling agent, leads to after fishing in cleaning process
Immersion 5 ~ 15min processing prawns are crossed, then drain antistaling agent containing enzyme, remaining preservation technology and means can keep constant.
Moreover, bacillus provided by the inventionBacillus sp. CAMT22370 bacterial strains can also high-yield of low-temperature
Glucose oxidase.
The glucose oxidase of commercialization at this stage is produced by mold fermentation, there is that producing enzyme vigor is low and separation is complicated
The defects of.With bacillusBacillus sp. CAMT2237 is fermented, caused low temperature glucose oxidase not only enzyme activity
Power is high, and separates easily.
Bacillus provided by the inventionBacillus sp. CAMT22370 genetic stabilities are good, can stably keep
The merit of its high-yield of low-temperature glucose oxidase.
Present invention also offers a kind of preparation method of glucose oxidase, by traditional zymotic malaga carbohydrate oxidase
Technique in, using bacillusBacillus sp. CAMT22370 is fermented, being capable of the substantial amounts of low temperature grape of high yield
Carbohydrate oxidase.Fermentation process is simple and easy, while yield and production efficiency are all significantly improved.
Brief description of the drawings
Fig. 1 is carbon source kind pairBacillus sp.The influence figure of CAMT22370 malaga carbohydrate oxidase vigor；
Fig. 2 is nitrogen source species pairBacillus sp.The influence figure of CAMT22370 malaga carbohydrate oxidase vigor；
Fig. 3 is Inorganic Salts pairBacillus sp.The influence figure of CAMT22370 malaga carbohydrate oxidase vigor；
Fig. 4 is liquid amount pairBacillus sp.The influence figure of CAMT22370 malaga carbohydrate oxidases；
Fig. 5 is inoculum concentration pairBacillus sp.The influence figure of CAMT22370 malaga carbohydrate oxidases；
Fig. 6 is fermentation temperature pairBacillus sp.The influence figure of CAMT22370 malaga carbohydrate oxidases；
Fig. 7 is fermentation time pairBacillus sp.The influence figure of CAMT22370 malaga carbohydrate oxidases；
Fig. 8 is rotating speed pairBacillus sp.The influence figure of CAMT22370 malaga carbohydrate oxidases；
Fig. 9 is influence figure of the liquid containing enzyme fermentation to blacking process in prawn cold storage procedure；
Figure 10 is influence figure of the liquid containing enzyme fermentation to microbial reproduction speed in prawn cold storage procedure.
Embodiment
In the present invention, the definition of enzyme activity produces 1.0 μ g H for catalysis per minute2O2Amount be an enzyme activity list
Position.Except under specific background or illustrating, enzyme activity is in room temperature（25℃）Lower measure.
The culture medium that following examples use for：
Enrichment and seed culture medium：Peptone 5g, yeast extract 1g, ferrous sulfate 0.1g, natural sea-water 1L, pH7.6-7.8.
Fermentation medium：Maltose 5g, dusty yeast 10g, calcium chloride 2.8g, sodium chloride 1g, Tween-80 1.5mL, go from
Sub- water 1L, pH7.5.
Embodiment 1：The screening of malaga carbohydrate oxidase bacterial strain
Bacillus CAMT22370 of the present invention screening technique comprises the following steps：
（1）The enrichment of bacterium：Weigh 1g oozes to shake in 9ml antiseptic sea water test tube uniformly, stand, take supernatant
10 are diluted to step by step-2, 10-3, l0-4Times, respectively take the sample liquid of 1mL various concentrations to be seeded to respectively in each culture medium after concussion uniformly,
It is put in 30 DEG C of incubators and is inverted culture.
（2）Primary dcreening operation：Mycelium dilution will be enriched with to about 104/ mL concentration, according to product H2O2Methylene blue can be oxidized to nothing
Color, the chromogenic culture medium containing methylene blue concentration gradient is designed, dilution bacteria suspension is inoculated in colour developing using method of dilution butteron on plate
Culture medium, the glycoxidative enzyme activity of size preliminary judgement malaga at 30 DEG C after culture 48h according to water white transparency circle around thalline
Power height, select some bacterial strains and carry out follow-up shake flask fermentation secondary screening.
（3）Secondary screening：Secondary screening is mainly carried out using ELIASA to the glucose oxidase vigor in the zymotic fluid of primary dcreening operation bacterial strain
It is quick relatively to screen.Utilize H caused by 37 DEG C of catalysis glucose of glucose oxidase2O2Indigo carmine can be made to fade, it fades
Degree within the specific limits with caused H2O2Amount is proportionate, and catalysate H is calculated accordingly by standard curve2O2Amount, is commented
Valency enzyme activity height.The definition of enzyme activity produces 1.0 μ g H for catalysis per minute2O2Amount be an enzyme activity unit.Can
Dissolubility protein determination is determined using Coomassie brilliant G-250 method.
The identification of the malaga carbohydrate oxidase bacterial strain of embodiment 2
The bacterial strain filtered out to embodiment 1, is identified it using morphology and molecular biology method.
Morphological Identification is mainly observed using colonial morphology and microscopic morphology, and Physiology and biochemistry identifies the metabolism according to bacterial strain
Course is using glycolysis experiment, Starch Hydrolysis experiment, V-P experiments, methyl red test, oxidase test, triple sugar iron agar experiment
Carried out with the method for hydrogen sulfide indole-power agar experiment, the side with comparing is sequenced using 16SrDNA for molecular biology identification
Method is identified.It is comprehensive that bacterial strain ownership is carried out to form, Physiology and biochemistry and biomolecular science qualification result.
Wherein, molecular biology identification is first using DNA of bacteria extracts kit extraction genomic DNA, then using logical
Enter performing PCR with primer to expand, routinely operating parameter, PCR primer complete the 16S of measure after purification for amplification system and program
RDNA genes fragment length is 1492bp, through comparing, withBacillus spSimilitude highest, homology is up to 100%.
As a result show, the bacterial strain for screening to obtain has following characteristics：（1）Colonial morphology：White, diameter is smaller, rounded,
Surface is smooth, low projection, relatively transparent, neat in edge；（2）Cellular morphology：It is shaft-like, single arrangement, single-ended raw flagellum, gram sun
Property；（3）：Physiological and biochemical property：Growth temperature is 4-40 DEG C, aerobic, is resistant to 7%（wt）Sodium chloride, can gelatin hydrolysate, starch,
Casein, ammonium sulfate, ammonium nitrate, D- Glucosamines can be utilized, is carbon using glycogen, maltotriose, D- trehaloses, D-ribose
Source, it is impossible to which, using inulin, D-MANNOSE, L- rhamnoses, D- galactolipins, mannitol and xylose, indole reaction is negative, methyl red
Experiment, catalase, oxidizing ferment and β-NAG glycosides enzyme test are the positive, produce catalase, do not produce hydrogen sulfide.
The bacterial strain identifies that it is 1492bp to measure genetic fragment length through 16S rDNA.The gene order of acquisition is inputted
Genbank, gene order in the gene order of acquisition and database is contrasted using Blast programs.As a result show, with bud
Spore bacillusBacillus sp16S rDNA sequences homology be 100%.This result combination strain morphology and physiology are given birth to
Change identification mark, it is bacillus to determine the bacteriumBacillus sp, China typical culture collection center is preserved in, is protected
It is CCTCC NO to hide numbering: M2015247.
The bacillus CAMT22370 liquid fermentation malaga carbohydrate oxidase condition optimizings of embodiment 3
The condition of enzyme production for the bacillus CAMT22370 that this experiment obtains to screening is optimized.Specifically, mainly
It is that the culture medium and condition of culture of liquid fermentation malaga carbohydrate oxidase are optimized.Unless stated otherwise, following percentage
For percetage by weight.
The screening of carbon source kind：On the basis of 0.3% beef extract, 0.5% sodium chloride, Portugal is separately added into 1% addition
Grape sugar, lactose, sucrose, maltose, sorbierite, mannose, is made the culture medium containing only a certain carbon source.Inoculum concentration of fermenting is 5%
（Volume ratio）, rotating speed 150rpm, 28 DEG C of ferment at constant temperature culture 72h, determine enzyme activity, using enzyme activity as criterion, yield of enzyme
Maximum carbon source is optimal fermenting carbon source, as a result sees Fig. 1.As a result the maltose for showing 1% is optimum carbon source.
The screening of nitrogen source species：On the basis of 1% maltose, 0.5% sodium chloride, it is separately added into 0.5% addition more
Valency peptone, yeast extract, beef extract, peptone, tryptone, fish peptone, urea, diammonium hydrogen phosphate and ammonium nitrate, system
Into the culture medium containing only single nitrogen source.Inoculum concentration of fermenting is 5%（Volume ratio）, rotating speed 150rpm, 28 DEG C of ferment at constant temperature culture 72h,
Enzyme activity is determined, using enzyme activity as criterion, the maximum nitrogen source of yield of enzyme is optimal fermentation nitrogen source, as a result sees Fig. 2.As a result
Show that organic nitrogen is better than inorganic nitrogen, producing enzyme is influenceed less, to consider using 0.5% peptone as suitable nitrogen between organic nitrogen
Source.
The screening of Inorganic Salts：On the basis of 1% maltose, 0.5% peptone, added respectively with 0.05% addition
Enter CaCl2、MgSO4、KCl、MnCl2、ZnCl2、FeSO4, the fluid nutrient medium containing only a certain inorganic salts is made.Fermentation inoculum concentration be
5%（Volume ratio）, rotating speed 150rpm, 28 DEG C of ferment at constant temperature culture 72h, determine enzyme activity, using enzyme activity as criterion, producing enzyme
The maximum inorganic salts of amount are optimal inorganic salts, as a result see Fig. 3.As a result show that Inorganic Salts are little to producing enzyme effect of vigor,
Consider to add 0.05% CaCl2It is more suitable.
The optimization of shaking flask liquid amount：On the basis of culture medium composition determines, shaking flask hair is carried out using 250mL conical flasks
Ferment, liquid amount are respectively 10%, 20%, 30%, 40%, 50%, the 60% of cumulative volume, and at 35 DEG C, ferment 72h under the conditions of 150rpm, are surveyed
Glucose oxidase enzyme activity in zymotic fluid is determined, as a result such as Fig. 4.As a result enzyme activity highest when liquid amount is 20% is shown, with liquid amount
Increase, glucose oxidase enzyme activity reduces rapidly.
The optimization of inoculum concentration：In built-in 20% fermentation medium of 250mL conical flasks, 2%, 4%, 6%, 8%, 10% is inoculated with respectively
Seed liquor, 35 DEG C, ferment 72h under the conditions of 150rpm, determines glucose oxidase enzyme activity in zymotic fluid, as a result such as Fig. 5.As a result table
Bright 6% inoculum concentration is more suitable.
The optimization of fermentation temperature：Triangular flask after inoculation is respectively placed in 22 DEG C, 25 DEG C, 28 DEG C, 31 DEG C, 34 DEG C, 37 DEG C
Under, 150rpm, liquid amount 20%, fermented and cultured 72h.Glucose oxidase enzyme activity in its zymotic fluid is surveyed, as a result such as Fig. 6.As a result
Show that enzymatic production vigor is higher at 31 DEG C.
Fermentation time optimizes：Liquid amount is 20% after being inoculated with, and the triangular flask of inoculum concentration 6% is respectively placed in 31 DEG C, 150rpm,
84h is cultivated, is sampled every 12h, surveys its glucose oxidase enzyme activity, as a result such as Fig. 7.As a result producing enzyme vigor during fermentation 60h is shown
Reach maximum, enzyme activity has afterwards declines more by a small margin.
The optimization of rotating speed：Liquid amount is 20% after being inoculated with, and the triangular flask of inoculum concentration 6% is respectively placed in 31 DEG C of culture 60h, turns
Speed is respectively set as standing, 50rpm, 100rpm, 150rpm, 200rpm, 250rpm, investigates influence of the rotating speed to producing enzyme vigor,
As a result Fig. 8 is seen.As a result show that producing enzyme vigor reaches maximum during 200rpm.
Embodiment 4：Bacillus CAMT22370 inheritance stability Journal of Sex Research
By bacillus CAMT22370 continuous passage cultures, and its enzyme activity is surveyed, evaluation genetic stability is used as using enzyme activity
Index.As a result as shown in table 1, passing on 8 times, glucose oxidase vigor is maintained at 13U/mL or so, standard deviation 0.75,
Show that bacterial strain has preferable genetic stability.
The bacillus CAMT22370 of table 1 mitotic stability
Embodiment 5：A kind of preparation method of low temperature glucose oxidase
Low temperature glucose oxidase is prepared in accordance with the following methods：
（1）Prepare fermentation medium：Maltose 5g, dusty yeast 10g, calcium chloride 2.8g, sodium chloride 1g, Tween-80
1.5mL, deionized water 1L, pH7.5.
（2）Bacillus CAMT22370 is activated with conventional method, and prepares seed liquor, wherein seed culture medium is：Albumen
Peptone 5g, yeast extract 1g, ferrous sulfate 0.1g, natural sea-water 1L, pH7.6-7.8.
（3）6% is accessed into fermentation medium（Volume fraction）Seed liquor, in 28 DEG C of shaking table culture 60h, rotating speed
200rpm。
（4）Zymotic fluid is collected, centrifuging and taking supernatant, determines the vigor of glucose oxidase.
Embodiment 6：Enzyme activity change under bacillus CAMT22370 fermentation malaga carbohydrate oxidase low temperature
The bacillus CAMT22370 of Example 5 fermentation institute's malaga carbohydrate oxidases and the identical lower aspergillus niger of condition
（A.niger）Glucose oxidase caused by CICC3357, enzyme activity of two kinds of enzymes at 0 ~ 10 DEG C is determined respectively（n=3）, it is black
Aspergillus culture medium is conventional potato sucrose culture medium, and condition of culture is the same as bacillus CAMT22370.As a result such as the institute of table 2
Show.
The bacillus CAMT22370 of table 2 and enzyme activity under aspergillus niger CICC3357 low temperature comparison（U/mL）
From table 2, aspergillus niger（A.niger）Enzyme of the glucose oxidase caused by CICC3357 under 0 ~ 10 DEG C of low temperature
Vigor is substantially no more than 4U/mL, and the enzyme activity of glucose oxidase at this low temperature caused by the present invention is more than 5U/mL, relatively
Greatly improved in the glucose oxidase vigor of Aspergillus niger origin.
Embodiment 7：Bacillus CAMT22370 is compared with aspergillus niger CICC3357 malaga carbohydrate oxidase abilities
Glucose oxidase is produced using method same as Example 6, determines bacillus CAMT22370 and black respectively
Aspergillus CICC3357 is compared in the enzyme activity of 24,36,48,60,72 and 84h when institutes malaga carbohydrate oxidase that ferments, and is tied
Fruit is as shown in table 3.
The bacillus CAMT22370 of table 3 and aspergillus niger CICC3357 malaga carbohydrate oxidase vigour changes（U/mL）
From table 3, the enzymatic productivity in 24 ~ 84h of glucose oxidase caused by aspergillus niger CICC3357 does not surpass substantially
11U/mL is crossed, the enzymatic productivity in this fermentation time of glucose oxidase caused by the present invention is close to 15U/mL, therefore relatively
For the glucose oxidase of Aspergillus niger origin, enzymatic productivity of the present invention greatly improves.
Embodiment 8：A kind of preparation method of low temperature glucose oxidase
Low temperature glucose oxidase is prepared in accordance with the following methods：
（1）Prepare fermentation medium：Maltose 5g, dusty yeast 10g, calcium chloride 2.8g, sodium chloride 1g, Tween-80
1.5mL, deionized water 1L, pH7.5.
（2）Bacillus CAMT22370 is activated with conventional method, and prepares seed liquor, wherein seed culture medium is：Albumen
Peptone 5g, yeast extract 1g, ferrous sulfate 0.1g, natural sea-water 1L, pH7.6-7.8.
（3）7% is accessed into fermentation medium（Volume fraction）Seed liquor, in 31 DEG C of shaking table culture 60h, rotating speed
200rpm。
（4）Zymotic fluid is collected, centrifuging and taking supernatant, determines the vigor of glucose oxidase.
As a result show, vigor of the glucose oxidase under 4 DEG C of low temperature is 4.21U/mL, the U/ of yield 13.89 in zymotic fluid
mL。
Embodiment 9. is carried out fresh-keeping using low temperature glucose oxidase
Homogeneous, the healthy Penaeus Vannmei of size after fishing is transported back laboratory using the method keep-alive of oxygenation keep-alive, temporarily
The abnormal individuals such as death, mutilation, discoloration are rejected after supporting 3 days, with aseptic water washing, being put into trash ice makes shrimp body suffer a shock, will
Penaeus Vannmei complex biological preservative containing enzyme after shock（The Portugal prepared in above-described embodiment 8 comprising 0.1% weight
Grape carbohydrate oxidase, the Tea Polyphenols of 1% weight, 1% water soluble chitosan, remaining is water）5min is soaked, then pulls out and drains shrimp body surface
The unnecessary zymotic fluid in face, cold wind are dried up and loaded in sterilized antistaling bag, and the prawn after pack is positioned in 4 DEG C of refrigerators and refrigerated.
Simultaneously using distilled water immersion group as negative control, using the fresh treasured of commercially available antistaling agent shrimp as positive control, its application method according to
Specification is carried out.
Changed using color and luster of the three groups of Penaeus Vannmeis of colour difference meter tracing detection during refrigeration, color difference measurement uses
WSC-I type microprocessor colour difference meters, diaphragm diameter 30mm, condenser diameter 30mm, with standard ceramic plate（X=91.295, Y=
94.295 Z=107.045）As working stamndard, reflected light of the measurement prawn surface in test sample box, by tristimulus values（X、Y、
Z）Calculate L values.Wherein L represents color clarity, and L=0 is black, and L=100 is white, and L values are smaller, represents that blacking is heavier,
Index using brightness value L as prawn blacking degree, every group of numerical value are repeated to survey and averaged three times, as a result see Fig. 9.Can by Fig. 9
To find out, compared with the control, the antistaling agent containing glucose oxidase zymotic fluid of the present invention can effectively slow down prawn during refrigeration
Blacking process.
Total plate count measure uses GB/T 4789.2-2010《Food microbiological examination general provisions》Carry out, South America is right in vain
Shrimp sample is put into ultrasound homogenizer and is fully homogenized, and homogenate is weighed into 1g on superclean bench adds 9mL sterilized waters, and stirring is equal
Into 10 after even-1Gradient bacteria suspension, successively operation preparation dilution series bacteria suspension.Liquid-transfering gun pipettes the bacterium of 1mL difference dilution factors
Suspension, it is inoculated in tilt-pour process in nutrient agar sterile petri dish, 37 DEG C of constant incubator culture 2-3 is inverted in after quick mixing
My god, bacterium colony counting then is carried out according to standardization program, compares influence of the different disposal to Penaeus Vannmei microbial reproduction, as a result
See Figure 10.
As seen from Figure 10, different disposal influences notable on bacterial reproduction speed, and distilled water immersion group bacterium is quickly numerous
To grow, more than 7.0, lose commodity value within the 4th day, the fresh precious group bacterial reproduction speed of shrimp is decreased obviously compared with distilled water group, but still in
Slow propradation, remain to preferably keep microorganism quality when refrigeration was to the 5th day, it is different from the first two processing, containing the present invention
The zymotic fluid group total number of bacterial colonies of glucose oxidase is constantly in the relatively low state less than 5.0, preferably inhibits micro- life
Thing is bred.
Claims (7)
1. a kind of bacillus (Bacillus sp) CAMT22370, is preserved in China typical culture collection center, preservation is compiled
Number it is CCTCC NO:M2015247.
2. applications of the bacillus CAMT22370 of claim 1 in fermenting and producing glucose oxidase.
3. application according to claim 2, wherein glucose oxidase vigor at 0~10 DEG C is more than 4U/mL.
A kind of 4. preparation method of glucose oxidase, it is characterised in that：It uses the bacillus described in claim 1
CAMT22370 fermentations are made.
A kind of 5. preparation method of glucose oxidase according to claim 4, it is characterised in that：The fermentation is liquid
Fermentation, the culture medium of fermentation, as carbon source, using organic nitrogen as nitrogen source, and are added with CaCl2 using maltose.
A kind of 6. preparation method of glucose oxidase according to claim 4, it is characterised in that：Fermentation condition is：Connect
Kind 2~10% volumes of amount, 22~37 DEG C, 36~84h of fermentation time, 0~250rpm of rotating speed of producing enzyme temperature.
A kind of 7. preparation method of glucose oxidase according to claim 4, it is characterised in that：Fermentation condition is：Connect
Kind 6~8% volumes of amount, 30~35 DEG C, 40~72h of fermentation time, 150~250rpm of rotating speed of producing enzyme temperature.
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| Bacillus sp.CAMT22370源葡萄糖氧化酶对南美白对虾贮藏性能的影响;徐德峰等;《食品科学》;20161130;1-8 * |
| studies on the properties of glucose oxidase from aspergillus niger A9;wang zhixin et al.;《journal of agricultural university of hebei》;20060430;摘要 * |
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