CN105018389A - Bacillus sp. CAMT22370 strain and application thereof - Google Patents
Bacillus sp. CAMT22370 strain and application thereof Download PDFInfo
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- CN105018389A CN105018389A CN201510468214.6A CN201510468214A CN105018389A CN 105018389 A CN105018389 A CN 105018389A CN 201510468214 A CN201510468214 A CN 201510468214A CN 105018389 A CN105018389 A CN 105018389A
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Abstract
The invention discloses a Bacillus sp. CAMT22370 strain which is collected by China Center for Type Culture Collection, and the collection number is CCTCC NO:M2015247. The Bacillus sp. CAMT22370 strain can produce low-temperature glucose oxidase at high yield, and thus, has favorable preservation effect, thereby being suitable for physical distribution preservation of prawns and aquatic products. The invention also discloses a method for producing glucose oxidase by fermenting the strain. The method is simple and easy to implement, and greatly enhances the yield and production efficiency.
Description
Technical field
The present invention relates to a kind of microbe to screen and fermentation arts, be specifically related to
bacillus spthe method of CAMT22370 and liquid fermenting malaga carbohydrate oxidase thereof.
Background technology
Glucose oxidase (Glucose oxidase, GOD; EC 1.1.3.4.) utilize oxygen molecule as electron acceptor(EA), in specific manner β-D-Glucose is oxidized to gluconic acid and hydrogen peroxide, thus effectively oxygen is removed.Food-processing, medicine and biological field is widely used in due to its Nantural non-toxic side effect.Based on the deoxidation effect of its uniqueness and the freshening effect produced, at present in field widespread uses such as fruit, flour products, drinks, dairy products.It is fresh-keeping that horse Qinghe etc. tentatively attempt commercial glucose oxidase being applied to prawn, and result shows that comparatively contrast extends the freshness date of about 1 day under the refrigerated condition of 4 DEG C, imply that glucose oxidase will produce good effect in the fresh-keeping field of prawn.
The suitability for industrialized production of current glucose oxidase mainly adopts Lu Sheng aspergillus niger or mould solid or liquid state fermentation, but there is the defect that expression amount is lower and foreign protein is more during the fermentation, very large difficulty is brought to separation and purification, causing product cost to increase, is the limiting factor of this enzyme of restriction in field of food widespread use.The glucose oxidase industrial enzyme preparation purity that current China produces is general lower, dependence on import for a long time, and the substitute products therefore finding glucose oxidase import zymin have important practical significance.Meanwhile, the many temperature ranges at 30-40 DEG C of the Lu Sheng bacterium source glucose oxidase optimum temperuture reported, and vigor is relatively low at the Food Cold Chain temperature of 0-10 DEG C, fresh-keeping effect is not good.
Summary of the invention
Produce that product enzyme activity existing for bacterial strain Lu Sheng source is lower based on current glucose oxidase, activity is not strong under cold condition, and the limitation that tunning foreign protein content is higher, the invention provides and a kind ofly from oceanic resources, be separated the method for low temperature glucose oxidase superior strain and the application of malaga carbohydrate oxidase thereof.
One of the object of the invention there is provided a kind of marine bacteria strain producing low temperature glucose oxidase.
Two of the object of the invention there is provided a kind of application utilizing aimed strain to produce low temperature glucose oxidase.
For this reason, the invention provides a bacillus (
bacillus sp) CAMT22370, be preserved in China typical culture collection center, deposit number is CCTCC NO:M2015247.Genus bacillus of the present invention
bacillus sp.cAMT22370 is the deep-sea bacterial strain can producing low temperature glucose oxidase, this bacterium is preserved in China typical culture collection center on April 27th, 2015, this preservation center is referred to as CCTCC, depositary institution address is Luo Jia Shan, wuchang, wuhan Wuhan University, and deposit number is CCTCC No:M2015247.
Genus bacillus CCTCC NO:M2015247 of the present invention can adopt the substratum containing peptone and yeast extract paste to cultivate, such as: peptone 5g, and yeast extract paste 1g, ferrous sulfate 0.1g, natural sea-water 1L, pH7.6-7.8.
Present invention also offers the application of above-mentioned genus bacillus in fermentative production glucose oxidase.
Preferably, described glucose oxidase vigor under low temperature (0 ~ 10 DEG C) is greater than 4U/mL.
Present invention also offers a kind of preparation method of glucose oxidase, it adopts genus bacillus
bacillus sp.cAMT22370 fermentation obtains.Preferably, described fermentation is liquid fermenting.
The present invention by single factor experiment to genus bacillus
bacillus sp.the condition of CAMT22370 liquid fermenting malaga carbohydrate oxidase is optimized.
The invention discloses genus bacillus
bacillus sp.the condition of CAMT22370 malaga carbohydrate oxidase, it is cultivated, fermentation condition is simple, stable hereditary property.This bacterium source is in deep-sea, and the suitableeest carbon source of its fermentation is maltose, and nitrogenous source spectrum is wide, but is good with organonitrogen, and customary inorganic salts kind is not remarkable on the impact of product enzyme activity, to add the CaCl of 0.05% mass ratio
2comparatively suitable.Such as: maltose 5g, yeast powder 10g, calcium chloride 2.8g, sodium-chlor 1g, tween-80 1.5mL, deionized water 1L, pH7.5.Fermentation condition can be: inoculum size 2 ~ 10% volume fraction, produces enzyme temperature 22 ~ 37 DEG C, fermentation time 36 ~ 84h, rotating speed 0 ~ 250rpm; More preferably, inoculum size is 6 ~ 8%(volume fraction), produce enzyme temperature 30 ~ 35 DEG C, fermentation time 40 ~ 72h, rotating speed 150 ~ 250rpm.Most preferably, being suitable for inoculum size during fermentation is 7%(volume fraction), producing enzyme temperature is 31 DEG C, and suitable fermentation time is 60h, and suitable rotating speed is 200rpm, and now enzyme activity reaches more than 13U/mL.
The invention has the beneficial effects as follows:
Genus bacillus provided by the invention
bacillus sp.cAMT22370 can produce low temperature glucose oxidase, the glucose oxidase that the enzyme work of this enzyme at the Food Cold Chain temperature of 0 ~ 10 DEG C is originated relative to traditional aspergillus niger etc. significantly improves, thus the fresh-keeping effect significantly improved at low temperatures, the logistics being extremely suitable for the aquatic foods such as the preservation by low temperature of fishery products, particularly prawn is fresh-keeping.Also can be applicable to the fresh-keeping of the fields such as fruit, flour products, drinks, dairy products.For time fresh-keeping, available low temperature glucose oxidase of the present invention replaces traditional glucose oxidase and prepares preservation agent, after fishing in cleaning process by immersion 5 ~ 15min process prawn, then drain containing enzyme preservation agent, all the other preservation techniques and means can remain unchanged.
Moreover, genus bacillus provided by the invention
bacillus sp.cAMT22370 bacterial strain can also high-yield of low-temperature glucose oxidase.
Present stage, commercial glucose oxidase was all produced by mold fermentation, there is the defect of producing the low and separate complex of enzyme activity.With genus bacillus
bacillus sp.cAMT2237 ferments, and not only enzyme activity is high for the low temperature glucose oxidase of generation, and is separated easily.
Genus bacillus provided by the invention
bacillus sp.cAMT22370 genetic stability is good, stably can keep the good character of its high-yield of low-temperature glucose oxidase.
Present invention also offers a kind of preparation method of glucose oxidase, by the technique of traditional zymotic malaga carbohydrate oxidase, adopt genus bacillus
bacillus sp.cAMT22370 ferments, can a large amount of low temperature glucose oxidase of high yield.Fermenting process is simple, and output and production efficiency all obtain and significantly improves simultaneously.
Accompanying drawing explanation
Fig. 1 is carbon source kind pair
bacillus sp.the effect diagram of CAMT22370 malaga carbohydrate oxidase vigor;
Fig. 2 is nitrogenous source kind pair
bacillus sp.the effect diagram of CAMT22370 malaga carbohydrate oxidase vigor;
Fig. 3 is Inorganic Salts pair
bacillus sp.the effect diagram of CAMT22370 malaga carbohydrate oxidase vigor;
Fig. 4 is liquid amount pair
bacillus sp.the effect diagram of CAMT22370 malaga carbohydrate oxidase;
Fig. 5 is inoculum size pair
bacillus sp.the effect diagram of CAMT22370 malaga carbohydrate oxidase;
Fig. 6 is leavening temperature pair
bacillus sp.the effect diagram of CAMT22370 malaga carbohydrate oxidase;
Fig. 7 is fermentation time pair
bacillus sp.the effect diagram of CAMT22370 malaga carbohydrate oxidase;
Fig. 8 is rotating speed pair
bacillus sp.the effect diagram of CAMT22370 malaga carbohydrate oxidase;
Fig. 9 is to the effect diagram of change process black in prawn cold storage procedure containing enzymic fermentation liquid;
Figure 10 is to the effect diagram of microbial reproduction speed in prawn cold storage procedure containing enzymic fermentation liquid.
Embodiment
In the present invention, enzyme activity is defined as per minute catalysis and produces 1.0 μ g H
2o
2amount be an enzyme activity unit.Remove under specific background or special instruction, enzyme activity all measures under room temperature (25 DEG C).
The substratum that following examples adopt is:
enrichment and seed culture medium: peptone 5g, yeast extract paste 1g, ferrous sulfate 0.1g, natural sea-water 1L, pH7.6-7.8.
fermention medium:maltose 5g, yeast powder 10g, calcium chloride 2.8g, sodium-chlor 1g, tween-80 1.5mL, deionized water 1L, pH7.5.
embodiment 1:the screening of malaga carbohydrate oxidase bacterial strain
The screening method of genus bacillus CAMT22370 of the present invention comprises the following steps:
(1)
the enrichment of bacterium: take 1g ooze and shake evenly in the antiseptic sea water test tube of 9ml, leave standstill, get supernatant liquor stepwise dilution to 10
-2, 10
-3, l0
-4doubly, the sample liquid respectively getting 1mL different concns after concussion is evenly seeded in each substratum respectively, is put in 30 DEG C of incubators to be inverted to cultivate.
(2)
primary dcreening operation: by enrichment mycelium dilution to about 10
4/ mL concentration, according to product H
2o
2methylene blue can be oxidized to colourless, the color developing culture medium of design containing methylene blue concentration gradient, method of dilution butteron on plate is adopted to be inoculated in color developing culture medium dilution bacteria suspension, according to the size preliminary judgement malaga carbohydrate oxidase vigor of water white transparency circle around thalline height cultivate 48h at 30 DEG C after, select some bacterial strains and carry out follow-up shake flask fermentation and sieve again.
(3)
multiple sieve: the main microplate reader that adopts of multiple sieve compares screening fast to the glucose oxidase enzyme activity in the fermented liquid of primary dcreening operation bacterial strain.Utilize the H that glucose oxidase 37 DEG C of catalysis glucose produce
2o
2indigo carmine can be made to fade, its fading extent within the specific limits with produce H
2o
2amount is proportionate, and calculates catalysate H accordingly by typical curve
2o
2amount, evaluates enzyme activity height.Enzyme activity is defined as per minute catalysis and produces 1.0 μ g H
2o
2amount be an enzyme activity unit.Soluble proteins measures and adopts Coomassie brilliant G-250 method to measure.
embodiment 2the qualification of malaga carbohydrate oxidase bacterial strain
To the bacterial strain that embodiment 1 filters out, morphology and molecular biology method is adopted to identify it.
Identification of morphology mainly adopts colonial morphology and microscopic morphology to observe, the method that Physiology and biochemistry qualification adopts glycolysis-test, Starch Hydrolysis test, V-P test, methyl red test, oxidase test, triple sugar iron agar test and hydrogen sulfide indole-power agar to test according to the metabolic fate of bacterial strain is carried out, and molecular biology identification adopts 16SrDNA order-checking to identify with the method for comparison.Comprehensively bacterial strain ownership is carried out to form, Physiology and biochemistry and biomolecular science qualification result.
Wherein, first molecular biology identification adopts DNA of bacteria to extract test kit and extracts genomic dna, then universal primer is adopted to carry out pcr amplification, amplification system and program operating parameters routinely, the 16S rDNA gene fragment length completing mensuration after PCR primer purifying is 1492bp, through comparison, with
bacillus spsimilarity is the highest, and homology reaches 100%.
Result shows, and screen the bacterial strain obtained and have following characteristics: (1) colonial morphology: white, diameter is less, rounded, smooth surface, low projection, more transparent, neat in edge; (2) cellular form: shaft-like, single arrangement, single-ended raw flagellum, Gram-positive; (3): physiological and biochemical property: growth temperature is 4-40 DEG C, aerobic, can 7%(wt be tolerated) sodium-chlor, energy gelatin hydrolysate, starch, casein, ammonium sulfate, ammonium nitrate, D-glucosamine can be utilized, glycogen, trisaccharide maltose, D-trehalose, D-ribose can be utilized to be carbon source, inulin, D-MANNOSE, L-rhamnosyl, D-semi-lactosi, N.F,USP MANNITOL and wood sugar can not be utilized, indole reaction is negative, methyl red test, catalase, oxydase and β-NAG glycosides enzyme test are positive, produce catalase, do not produce hydrogen sulfide.
This bacterial strain is identified through 16S rDNA, and recording gene fragment length is 1492bp.By the gene order input Genbank obtained, gene order in the gene order of acquisition and database contrasts by application Blast program.Result shows, with genus bacillus
bacillus spthe homology of 16S rDNA sequence be 100%.By this result in conjunction with strain morphology and Physiology and biochemistry identification mark, determine that this bacterium is genus bacillus
bacillus sp, be preserved in China typical culture collection center, deposit number is CCTCC NO:M2015247.
embodiment 3genus bacillus CAMT22370 liquid fermenting malaga carbohydrate oxidase condition optimizing
This experiment is optimized the condition of enzyme production screening the genus bacillus CAMT22370 obtained.Specifically, mainly the substratum of liquid fermenting malaga carbohydrate oxidase and culture condition are optimized.Unless stated otherwise, following percentage ratio is weight percentage.
the screening of carbon source kind: at 0.3% extractum carnis, on the basis of 0.5% sodium-chlor, add glucose respectively with the addition of 1%, lactose, sucrose, maltose, sorbyl alcohol, seminose, make only containing the substratum of a certain carbon source.Fermentation inoculum size is 5%(volume ratio), rotating speed 150rpm, 28 DEG C of ferment at constant temperature cultivate 72h, and measuring enzyme activity, take enzyme activity as criterion, and the maximum carbon source of yield of enzyme is best fermenting carbon source, the results are shown in Figure 1.Result shows that the maltose of 1% is optimum carbon source.
the screening of nitrogenous source kind: at 1% maltose, on the basis of 0.5% sodium-chlor, the addition with 0.5% adds multivalent protein peptone respectively, yeast extract paste, extractum carnis, peptone, Tryptones, fish peptone, urea, Secondary ammonium phosphate and ammonium nitrate, makes only containing the substratum of single nitrogenous source.Fermentation inoculum size is 5%(volume ratio), rotating speed 150rpm, 28 DEG C of ferment at constant temperature cultivate 72h, and measuring enzyme activity, take enzyme activity as criterion, and the maximum nitrogenous source of yield of enzyme is best fermentation nitrogen source, the results are shown in Figure 2.Result shows that organonitrogen is better than inorganic nitrogen, little on the impact of product enzyme between organonitrogen, considers with the peptone of 0.5% for suitable nitrogen source.
the screening of Inorganic Salts: at 1% maltose, on the basis of 0.5% peptone, add CaCl respectively with the addition of 0.05%
2, MgSO
4, KCl, MnCl
2, ZnCl
2, FeSO
4, make only containing the liquid nutrient medium of a certain inorganic salt.Fermentation inoculum size is 5%(volume ratio), rotating speed 150rpm, 28 DEG C of ferment at constant temperature cultivate 72h, and measuring enzyme activity, take enzyme activity as criterion, and the maximum inorganic salt of yield of enzyme are optimal inorganic salts, the results are shown in Figure 3.Result shows that Inorganic Salts is little on the impact of product enzyme activity, considers the CaCl adding 0.05%
2comparatively suitable.
the optimization of shaking flask liquid amount: form at substratum on the basis determined, adopt 250mL Erlenmeyer flask to carry out shake flask fermentation, liquid amount is respectively 10%, 20%, 30%, 40%, 50%, 60% of cumulative volume, at 35 DEG C, 150rpm condition bottom fermentation 72h, measure glucose oxidase enzyme in fermented liquid and live, result is as Fig. 4.When result shows that liquid amount is 20%, enzyme is lived the highest, and along with the increase of liquid amount, glucose oxidase enzyme is lived and reduced rapidly.
the optimization of inoculum size: at in-built 20% fermention medium of 250mL Erlenmeyer flask, inoculate the seed liquor of 2%, 4%, 6%, 8%, 10% respectively, 35 DEG C, 150rpm condition bottom fermentation 72h, measure glucose oxidase enzyme in fermented liquid and live, result is as Fig. 5.Result shows that the inoculum size of 6% is comparatively suitable.
the optimization of leavening temperature: at postvaccinal triangular flask is placed in 22 DEG C, 25 DEG C, 28 DEG C, 31 DEG C, 34 DEG C, 37 DEG C respectively, 150rpm, liquid amount is 20%, fermentation culture 72h.Survey glucose oxidase enzyme in its fermented liquid to live, result is as Fig. 6.It is higher that result shows to produce enzyme activity at 31 DEG C of bottom fermentations.
fermentation time is optimized: be 20% by liquid amount after inoculation, the triangular flask of inoculum size 6% is placed in 31 DEG C, 150rpm respectively, cultivates 84h, and every 12h sampling, survey its glucose oxidase enzyme and live, result is as Fig. 7.Produce enzyme activity when result shows fermentation 60h and reach maximum, enzyme is lived in having and is declined more by a small margin afterwards.
the optimization of rotating speed: be 20% by liquid amount after inoculation, the triangular flask of inoculum size 6% is placed in 31 DEG C respectively and cultivates 60h, and rotating speed is set as leaving standstill respectively, 50rpm, 100rpm, 150rpm, 200rpm, 250rpm, investigates rotating speed to the impact of producing enzyme activity, the results are shown in Figure 8.Produce enzyme activity when result shows 200rpm and reach maximum.
embodiment 4:the inheritance stability Journal of Sex Research of genus bacillus CAMT22370
Genus bacillus CAMT22370 continuous passage is cultivated, and it is alive to survey its enzyme, using enzyme activity as the index evaluating genetic stability.Result is as shown in table 1, and go down to posterity 8 times, glucose oxidase enzyme activity remains on about 13U/mL, and standard deviation is 0.75, shows that bacterial strain has good genetic stability.
The mitotic stability of table 1 genus bacillus CAMT22370
embodiment 5:a kind of preparation method of low temperature glucose oxidase
Prepare low temperature glucose oxidase in accordance with the following methods:
(1) fermention medium is prepared: maltose 5g, yeast powder 10g, calcium chloride 2.8g, sodium-chlor 1g, tween-80 1.5mL, deionized water 1L, pH7.5.
(2) with ordinary method activation genus bacillus CAMT22370, and prepare seed liquor, wherein seed culture medium is: peptone 5g, yeast extract paste 1g, ferrous sulfate 0.1g, natural sea-water 1L, pH7.6-7.8.
(3) in fermention medium, access 6%(volume fraction) seed liquor, cultivate 60h, rotating speed 200rpm in 28 DEG C of shaking tables.
(4) collect fermented liquid, centrifuging and taking supernatant, measure the vigor of glucose oxidase.
embodiment 6: the enzyme activity change under genus bacillus CAMT22370 fermentation malaga carbohydrate oxidase low temperature
Example 5 genus bacillus CAMT22370 ferment institute's malaga carbohydrate oxidase and the identical lower aspergillus niger of condition (
a.niger) CICC3357 produce glucose oxidase, respectively measure the enzyme activity of two kinds of enzymes at 0 ~ 10 DEG C (n=3), aspergillus niger culture medium is conventional potato sucrose substratum, and culture condition is with genus bacillus CAMT22370.Result is as shown in table 2.
Table 2 genus bacillus CAMT22370 compares (U/mL) with enzyme activity under aspergillus niger CICC3357 low temperature
From table 2, the enzyme activity of glucose oxidase under 0 ~ 10 DEG C of low temperature that aspergillus niger (A.niger) CICC3357 produces is no more than 4U/mL substantially, the glucose oxidase that the present invention produces enzyme activity at this low temperature, more than 5U/mL, significantly improves relative to the glucose oxidase enzyme activity of Aspergillus niger origin.
embodiment 7:genus bacillus CAMT22370 compares with aspergillus niger CICC3357 malaga carbohydrate oxidase ability
The method identical with embodiment 6 is adopted to produce glucose oxidase, measure the enzyme activity of genus bacillus CAMT22370 and aspergillus niger CICC3357 at fermentation 24,36,48,60,72 and 84h time institute malaga carbohydrate oxidase respectively, and compare, result is as shown in table 3.
Table 3 genus bacillus CAMT22370 and aspergillus niger CICC3357 malaga carbohydrate oxidase vigour changes (U/mL)
From table 3, glucose oxidase enzymatic productivity in 24 ~ 84h that aspergillus niger CICC3357 produces is no more than 11U/mL substantially, enzymatic productivity can close to 15U/mL in this fermentation time for the glucose oxidase that the present invention produces, therefore, for the glucose oxidase of Aspergillus niger origin, enzymatic productivity of the present invention significantly improves.
embodiment 8:a kind of preparation method of low temperature glucose oxidase
Prepare low temperature glucose oxidase in accordance with the following methods:
(1) fermention medium is prepared: maltose 5g, yeast powder 10g, calcium chloride 2.8g, sodium-chlor 1g, tween-80 1.5mL, deionized water 1L, pH7.5.
(2) with ordinary method activation genus bacillus CAMT22370, and prepare seed liquor, wherein seed culture medium is: peptone 5g, yeast extract paste 1g, ferrous sulfate 0.1g, natural sea-water 1L, pH7.6-7.8.
(3) in fermention medium, access 7%(volume fraction) seed liquor, cultivate 60h, rotating speed 200rpm in 31 DEG C of shaking tables.
(4) collect fermented liquid, centrifuging and taking supernatant, measure the vigor of glucose oxidase.
Result shows, and in fermented liquid, the vigor of glucose oxidase under 4 DEG C of low temperature is 4.21U/mL, output 13.89 U/mL.
embodiment 9.low temperature glucose oxidase is used to carry out fresh-keeping
Rear size will be fished for homogeneous, healthy Penaeus vannamei adopts the method keep-alive of oxygenation keep-alive to transport laboratory back, support temporarily and reject death after 3 days, mutilation, the abnormal individuals such as variable color, with aseptic water washing, putting into trash ice makes shrimp body suffer a shock, Penaeus vannamei after shock (is comprised the glucose oxidase prepared in above-described embodiment 8 of 0.1% weight with containing enzyme complex biological preservative, the tea-polyphenol of 1% weight, 1% water-soluble chitosan, all the other are water) soak 5min, then pull out and drain the unnecessary fermented liquid in shrimp surface, cold wind dries up and loads in sterilized antistaling bag, prawn after pack is positioned in 4 DEG C of refrigerators and refrigerates.Simultaneously using distilled water immersion group as negative control, using the fresh treasured of commercially available preservation agent shrimp as positive control, its using method is carried out to specifications.
Colour-difference meter tracing detection three groups of Penaeus vannameis are adopted to change at the color and luster of During Cold Storage, color difference measurement adopts the micro-process colour-difference meter of WSC-I type, diaphragm diameter 30mm, condensor diameter 30mm, with standard ceramic plate (X=91.295, Y=94.295, Z=107.045) as working standard, measure the reflected light of prawn surface in test sample box, calculate L value by tristimulus values (X, Y, Z).Wherein L represents color clarity, and L=0 is black, and L=100 is white, and L value is less, represents that black change is heavier, the index using brightness value L as the black range degree of prawn, and often group numerical value repeats survey and averages for three times, the results are shown in Figure 9.As seen from Figure 9, compared with the control, the preservation agent containing glucose oxidase fermented liquid of the present invention effectively can slow down the black change process of prawn at During Cold Storage.
Total number of bacterial colony measures and adopts GB/T 4789.2-2010 " food microbiological analysis general provisions " to carry out, and Penaeus vannamei sample is put into the abundant homogenate of ultrasound homogenizer, homogenate is taken on Bechtop 1g and add 9mL sterilized water, become 10 after stirring
-1gradient bacteria suspension, dilution series bacteria suspension is prepared in operation successively.Liquid-transfering gun pipettes the different dilution bacteria suspension of 1mL, be inoculated in nutrient agar medium sterile petri dish with tilt-pour process, be inverted in 37 DEG C of constant incubators after quick mixing and cultivate 2-3 days, then enumeration is carried out according to standard program, relatively different treatment is on the impact of Penaeus vannamei microbial reproduction, the results are shown in Figure 10.
As seen from Figure 10, different treatment is remarkable to bacterial reproduction rate, distilled water immersion group bacterium Fast-propagation, 4th day more than 7.0, lose commodity value, shrimp fresh treasured group bacterial reproduction speed comparatively distilled water group obviously declines, but still be in slow propradation, still microorganism quality can be better kept to when the 5th day in refrigeration, different from the first two process, fermented liquid group total number of bacterial colonies containing glucose oxidase of the present invention is in the lower state lower than 5.0 always, inhibits microbial reproduction preferably.
Claims (7)
1. a genus bacillus (
bacillus sp) CAMT22370, be preserved in China typical culture collection center, deposit number is CCTCC NO:M2015247.
2. the application of genus bacillus CAMT22370 in fermentative production glucose oxidase of claim 1.
3. application according to claim 2, wherein said glucose oxidase vigor at 0 ~ 10 DEG C is greater than 4U/mL.
4. a preparation method for glucose oxidase, is characterized in that: it adopts genus bacillus CAMT22370 fermentation to obtain.
5. the preparation method of a kind of glucose oxidase according to claim 4, is characterized in that: described fermentation is liquid fermenting, and the substratum of fermentation adopts maltose as carbon source, using organonitrogen as nitrogenous source, and is added with CaCl
2.
6. the preparation method of a kind of glucose oxidase according to claim 4, is characterized in that: fermentation condition is: inoculum size 2 ~ 10% volume, produces enzyme temperature 22 ~ 37 DEG C, fermentation time 36 ~ 84h, rotating speed 0 ~ 250rpm.
7. the preparation method of a kind of glucose oxidase according to claim 4, is characterized in that: fermentation condition is: inoculum size 6 ~ 8% volume, produces enzyme temperature 30 ~ 35 DEG C, fermentation time 40 ~ 72h, rotating speed 150 ~ 250rpm.
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| WANG ZHIXIN ET AL.: "studies on the properties of glucose oxidase from aspergillus niger A9", 《JOURNAL OF AGRICULTURAL UNIVERSITY OF HEBEI》 * |
| 徐德峰等: "Bacillus sp.CAMT22370源葡萄糖氧化酶对南美白对虾贮藏性能的影响", 《食品科学》 * |
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