CN103992971B - A kind of Bacillus cereus MBRH3 bacterial strain and screening technique thereof and application - Google Patents
A kind of Bacillus cereus MBRH3 bacterial strain and screening technique thereof and application Download PDFInfo
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- CN103992971B CN103992971B CN201410205714.6A CN201410205714A CN103992971B CN 103992971 B CN103992971 B CN 103992971B CN 201410205714 A CN201410205714 A CN 201410205714A CN 103992971 B CN103992971 B CN 103992971B
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Abstract
The present invention relates to a kind of Bacillus cereus MBRH3 bacterial strain and screening technique thereof and application, belong to technical field of bioengineering.How the problem solved is from screening a kind of new bacterial strain from sea mud, and has the ability producing SOD.A kind of Bacillus cereus MBRH3 bacterial strain and screening technique thereof and application are provided.By sea mud being added in the culture medium with Thallus Laminariae (Thallus Eckloniae) as nutritional labeling, cultivate under the conditions of 30 DEG C～40 DEG C, obtain culture, after culture is diluted, it is seeded on the culture medium flat plate with Thallus Laminariae (Thallus Eckloniae) as nutritional labeling, cultivate under the conditions of 30 DEG C～40 DEG C, then picking individual colonies is rule to transfer on the culture medium flat plate with Thallus Laminariae (Thallus Eckloniae) as nutritional labeling and cultivated, it is thus achieved that pure culture Bacillus cereus MBRH3 bacterial strain.It is high that this bacterial strain has product SOD, and can be with Thallus Laminariae (Thallus Eckloniae) as nutritional labeling, raw materials used abundant and low cost.
Description
Technical field
The present invention relates to a kind of Bacillus cereus MBRH3 bacterial strain and screening technique thereof and application, belong to biological work
Journey technical field.
Background technology
Superoxide dismutase (EC1.15.1.1, Superoxide dismutase, SOD) is the single-minded of living cells generation
Ultra-oxygen anion free radical (O in property catalysis biological body- 2) occur dismutation reaction to generate hydrogen peroxide and the metalloenzyme of oxygen, its dimension
Hold generation and the dynamic equilibrium of removing of biological interior free yl, biology is had protection, health care and the effect of curing the disease.SOD is gold
Belong to enzyme, Fe-SOD, Mn-SOD and Cu Zn-SOD can be divided into according to the different metal ion that it combines.Prokaryotic cell and part are planted
Thing mainly contains Fe-SOD, in yellow.Containing Mn-SOD in eukaryotic cell mitochondrion and prokaryotic cell, in purple.The leaf of plant
Green body, peroxisome and eukaryotic cell cytoplasm are contained within more Cu Zn-SOD, in aeruginous.In recent years, at chain
Mould Pseudomonas is found that Ni-SOD and Fe Zn-SOD, Hepar Bovis seu Bubali is found that Co Zn-SOD.Visible, SOD kind is day by day
Abundant so that it is to apply the most extensive.As at field of medicaments, SOD can treat because of O2 -The disease caused, such as arthritis and class wind
Wet arthritis etc.；At field of food, SOD can as the additive of health food, have resisting fatigue, defying age, radioprotective and
Antiinflammation, it may also be used for fruit freshness preserving；In household chemicals field, SOD has sun-proof, crease-resistant, speckle dispelling, antiinflammatory action；Agricultural and
Plant protection field, SOD has raising anti-adversity and toleration etc..
SOD mainly obtains by extracting or screening from animal, plant and the microbial cell etc. lived, and different bios
The strength difference of SOD is very big, and the difference of the different parts SOD of same organism is the biggest.If pluck and blood were once SOD
Main source, but, derive from the SOD of animal and easily cause the adverse consequences such as cross infection and anaphylaxis, application is by very
Big restriction；In plant, particularly Semen sojae atricolor, Semen Maydis, Folium Mori, Bulbus Allii and Radix et Caulis Opuntiae Dillenii equal size are of a relatively high, but its extraction process is multiple
Miscellaneous, so that production cost is higher and can not produce in a large number；And fermentable acquisition SOD is current most common method, utilize
Fermentable produces SOD and has the significant advantages such as yield is high, technique is simple, is the main method obtaining SOD, in particular with
The engineering bacteria of the product SOD of the structure of molecular biotechnology now, makes to obtain SOD by fermentation more efficient.Such as Wang Suilou, Wang Su
Virtue et al. obtains SOD Producing Strain by screening, and being lived by fermentation SOD enzyme reaches 600U/g wet thallus and respectively than 3048U/mg alive,
But it carries out screening mainly by soil effect raw material and obtains, and it mainly uses the conventional nutrients such as glucose as cultivation
Base.And the microorganism that is richly stored with in ocean, therefore, from sea mud, how to screen new bacterial strain, and there is the energy producing SOD
Power, has higher researching value.
And Thallus Laminariae (Thallus Eckloniae) is perennial macro, China's Thallus Laminariae (Thallus Eckloniae) yield about 900,000 tons/year, is the half of Gross World Product, row
First.Thallus Laminariae (Thallus Eckloniae) not only has edibility, or the raw material of industry of a kind of high added value.The main component of Thallus Laminariae (Thallus Eckloniae) is Brown algae
Glue, simultaneously rich in the mineral element such as iodine, calcium and several amino acids and vitamin, kelp nourishing enriches, the Thallus Laminariae (Thallus Eckloniae) that its content is most
Polysaccharide have suppression tumor growth, improve renal failure, reduce blood fat, the pharmacological action such as reduce blood pressure, and is natural health care
Food, the people by the many countries of China and Southeast Asia are liked.Though China is Thallus Laminariae (Thallus Eckloniae) yield big country and kelp processing big country, but
It is kelp processing level also segment distance compared with World Developed Countries, and there is presently no employing with Thallus Laminariae (Thallus Eckloniae) with nutritional labeling
Making the culture medium of state's microbe to screen, therefore, carrying out Thallus Laminariae (Thallus Eckloniae) application potential research in a deep going way is to improve the effective way of Thallus Laminariae (Thallus Eckloniae) value
Footpath, has significantly more efficient application prospect.
Summary of the invention
The present invention is directed to the problem in the presence of above-mentioned prior art, the present invention provides a kind of Bacillus cereus
MBRH3 bacterial strain and screening technique thereof and application, the problem of solution is to realize providing a kind of new bacterial strain, and described bacterial strain has high yield
The effect of SOD.
An object of the present invention technical scheme is that, a kind of Bacillus cereus
MBRH3 bacterial strain, described Bacillus cereus MBRH3 bacterial strain at the deposit number of China typical culture collection center is
CCTCC NO.M2013581。
The bacterial strain of the present invention is to screen to obtain from bottom silt, is a kind of new bacterial strain, belongs to gram positive bacteria, institute
State Bacillus cereus MBRH3 bacterial strain and be preserved in China typical culture collection center on November 19th, 2013, its
Deposit number is CCTCC NO.M2013581, and preservation place is Wuhan, China Wuhan University.
The Bacillus cereus MBRH3 bacterial strain bacterium colony of the present invention is rounded, smooth surface, neat in edge, milky
Opaque, thalline thickness is difficult to provoke；Thalline length about 1-2.5 μm, diameter about 0.5-1.0 μm；Shaft-like chaining.Physiology and biochemistry is real
Testing result to show, this bacterium is gram positive bacteria；All can grow in 5 DEG C to 45 DEG C temperature ranges, optimum growth temperature is 35
DEG C, all can grow when pH is 3.5-8.5, optimum growh pH is 6.5, facultative aerobic.The bacterial strain of the present invention can utilize the Thallus Laminariae (Thallus Eckloniae) to be
Nutritional labeling, and there is the advantage that SOD yield is high.
As preferably, the 16S rRNA sequence such as SEQ.NO1 institute of Bacillus cereus MBRH3 bacterial strain described above
Show.
The purpose of the present invention two technical scheme is that, a kind of Bacillus cereus
The screening technique of MBRH3 bacterial strain, the method comprises the following steps:
A, take sea mud sample and add in culture medium with Thallus Laminariae (Thallus Eckloniae) as nutritional labeling, train under the conditions of 30 DEG C～40 DEG C
Supporting, make aimed strain be preserved, non-targeted microorganism is eliminated, and obtains culture；
B, take culture obtained above dilution after, be seeded on the culture medium flat plate with Thallus Laminariae (Thallus Eckloniae) as nutritional labeling, control
After temperature is cultivated under the conditions of 30 DEG C～40 DEG C, then picking individual colonies is put down in the new culture medium with Thallus Laminariae (Thallus Eckloniae) as nutritional labeling
Lining out switching is cultivated, it is thus achieved that single bacterium colony pure culture Bacillus cereus MBRH3 bacterial strain.
The screening technique of the Bacillus cereus MBRH3 bacterial strain of the present invention, employing sea mud is sample, and makees with Thallus Laminariae (Thallus Eckloniae)
For the culture medium of sole nutrition composition, the bacterial strain obtained after being screened by cultivation, there is the ability height producing SOD, with the present invention's
The SOD function that Bacillus cereus MBRH3 bacterial strain produces is identical, by selecting Thallus Laminariae (Thallus Eckloniae) as nutritional labeling or as producing
The raw material of SOD more can play its advantage, it is possible to increase the selectivity of screening and directionality, enhances the screening of dominant strain,
Make more effectively to screen Bacillus cereus MBRH3 bacterial strain and for producing SOD；And use Thallus Laminariae (Thallus Eckloniae) as raw material, have
Abundant raw material and the advantage of low cost, be more beneficial for industrialization and produce.
In the screening technique of above-mentioned Bacillus cereus MBRH3 bacterial strain, as preferably, with dried seaweed material
In culture medium with Thallus Laminariae (Thallus Eckloniae) as nutritional labeling described in weight meter, step A and step B the percetage by weight of Thallus Laminariae (Thallus Eckloniae) as 2wt%～
5wt%.Thallus Laminariae (Thallus Eckloniae) is by commercial, after Thallus Laminariae (Thallus Eckloniae) is used water soaking, and pulverizing after stripping and slicing, it is deployed into and containing dried seaweed material is
The kelp paste of 2wt%～5wt%.As excellent the most preferred, step A becomes for nutrition with Thallus Laminariae (Thallus Eckloniae) with described in step B
In the culture medium divided, the percetage by weight of Thallus Laminariae (Thallus Eckloniae) is 3wt%～4wt%.
In the screening technique of above-mentioned Bacillus cereus MBRH3 bacterial strain, as preferably, described sea mud is seabed
Mud.
The purpose of the present invention three technical scheme is that, a kind of Bacillus cereus
The application of MBRH3 bacterial strain, described bacterial strain is used for producing SOD.
As preferably, Bacillus cereus MBRH3 inoculation is cultivated to the culture medium containing Thallus Laminariae (Thallus Eckloniae),
When strain OD value reaches 2.20, take seed and be inoculated in culture medium, control temperature and carry out under the conditions of 30 DEG C～40 DEG C
After fermentation culture 48 hours, obtain SOD.
In the application of above-mentioned Bacillus cereus MBRH3 bacterial strain, as preferably, culture medium described above
Weight proportion including following component:
MnSO4·7H2O:0.3g/L～0.7g/L, KH2PO4: 0.5g/L～0.8g/L, K2HPO4: 0.2g/L～0.5g/L,
Glucose: 1.0g/L～3.0g/L, Semen Maydis starch: 1.0g/L～3.0g/L, Carnis Bovis seu Bubali cream: 1.0g/L～3.0g/L, kelp paste
1.5ml/L～3.0ml/L, pH7.0.
In sum, the present invention compared with prior art has the advantage that
The Bacillus cereus MBRH3 bacterial strain of the present invention, is a kind of new bacterial strain, it is possible to utilize Thallus Laminariae (Thallus Eckloniae) to become for nutrition
Point, there is higher product SOD ability, and the screening technique of the present invention using Thallus Laminariae (Thallus Eckloniae) for nutritional labeling as cultivation, there is raw material rich
The rich advantage with low cost, and selectivity and the directionality of screening can be improve, enhance the screening of dominant strain.
Figure of description
Fig. 1 is the colonial morphology figure of the Bacillus cereus MBRH3 bacterial strain of the present invention.
Fig. 2 is the displaing micro photo figure of this bright Bacillus cereus MBRH3 bacterial strain.
Fig. 3 is the graph of a relation between observation and the predictive value that this bright Bacillus cereus MBRH3 bacterial strain enzyme is lived.
Fig. 4 be the present invention culture medium in Semen Maydis starch and kelp paste SOD activity affected graphics.
Fig. 5 be the present invention culture medium in Semen Maydis starch and glucose SOD activity affected graphics.
Fig. 6 be the present invention culture medium in Semen Maydis starch and manganese sulfate SOD activity affected graphics.
Fig. 7 be the present invention culture medium in kelp paste and glucose SOD activity affected graphics.
Fig. 8 is that produce kelp paste and the manganese sulfate during enzyme is cultivated of the present invention affect graphics to SOD activity.
Fig. 9 is that produce glucose and the manganese sulfate during enzyme is cultivated of the present invention affect graphics to SOD activity.
Detailed description of the invention
Below by specific embodiments and the drawings, technical scheme is described in further detail, but this
Invention is not limited to these embodiments.
In above example, sea mud uses bottom silt, and described bottom silt collection is from sea, Jian Men port, City of Taizhou
The bottom silt in district (28 ° of 1 ' 57 ' ' N, 121 ° of 36 ' 38 ' ' E), through drying for standby.
The culture medium being sole nutrition composition with Thallus Laminariae (Thallus Eckloniae) described in following example, described Thallus Laminariae (Thallus Eckloniae) is to select to buy on market
Thallus Laminariae (Thallus Eckloniae).Preferably, described Thallus Laminariae (Thallus Eckloniae) is first configured to kelp paste, uses pure water by after kelp soaking 24 hours, makes after stripping and slicing
After fully pulverizing by soy bean milk making machine, the kelp paste using pure water to be deployed into containing dried seaweed substance weight is 2wt%～5wt% is standby
With.
Embodiment 1
The screening of Bacillus cereus MBRH3 bacterial strain
Take 2g bottom silt sample add with Thallus Laminariae (Thallus Eckloniae) for the culture medium of sole nutrition composition in, Thallus Laminariae (Thallus Eckloniae) described in culture medium
Weight is 5wt% (with the dry matter weight gauge of Thallus Laminariae (Thallus Eckloniae)), control temperature 35 DEG C, rotating speed be 200rpm under conditions of cultivate
After fermenting 108 hours, making aimed strain be preserved, non-targeted microorganism is eliminated, and obtains fermentation liquid；
Take in 5mL fermentation liquid addition 50mL sterilized water and be sufficiently mixed, be diluted to 10 successively1～106Times, then, take dilution
104With 105The each 0.2mL of sample of two extension rates is coated in the flat board still only having the culture medium that Thallus Laminariae (Thallus Eckloniae) is nutritional labeling respectively again
On, the percetage by weight of Thallus Laminariae (Thallus Eckloniae) is 5wt% (with the dry matter weight gauge of Thallus Laminariae (Thallus Eckloniae)), controls temperature and trains under conditions of 37 DEG C
Support 72 hours, then picking individual colonies is rule switching on the culture medium flat plate that new Thallus Laminariae (Thallus Eckloniae) is nutritional labeling, the weight hundred of Thallus Laminariae (Thallus Eckloniae)
Mark is 5wt% (with the dry matter weight gauge of Thallus Laminariae (Thallus Eckloniae)), controls temperature and cultivates at 35 DEG C, it is thus achieved that single bacterium colony pure culture
Bacillus cereus MBRH3 bacterial strain.
Embodiment 2
The screening of Bacillus cereus MBRH3 bacterial strain
Take 2g bottom silt sample add with Thallus Laminariae (Thallus Eckloniae) for the culture medium of sole nutrition composition in, Thallus Laminariae (Thallus Eckloniae) described in culture medium
Weight is 2wt% (with the dry matter weight gauge of Thallus Laminariae (Thallus Eckloniae)), control temperature 30 DEG C, rotating speed be 200rpm under conditions of cultivate
After fermenting 108 hours, making aimed strain be preserved, non-targeted microorganism is eliminated, and obtains fermentation liquid；
Take in 5mL fermentation liquid addition 50mL sterilized water and be sufficiently mixed, be diluted to 10 successively1～106Times, then, take dilution
104With 105The each 0.2mL of sample of two extension rates is coated in the flat board still only having the culture medium that Thallus Laminariae (Thallus Eckloniae) is nutritional labeling respectively again
On, the percetage by weight of Thallus Laminariae (Thallus Eckloniae) is 2wt% (with the dry matter weight gauge of Thallus Laminariae (Thallus Eckloniae)), controls temperature and trains under conditions of 35 DEG C
Support 72 hours, then picking individual colonies is rule switching on the culture medium flat plate that new Thallus Laminariae (Thallus Eckloniae) is nutritional labeling, the weight hundred of Thallus Laminariae (Thallus Eckloniae)
Mark is 2wt% (with the dry matter weight gauge of Thallus Laminariae (Thallus Eckloniae)), controls temperature and cultivates at 35 DEG C, it is thus achieved that single bacterium colony pure culture
Bacillus cereus MBRH3 bacterial strain.
Embodiment 3
The screening of Bacillus cereus MBRH3 bacterial strain
Take 2g bottom silt sample add with Thallus Laminariae (Thallus Eckloniae) for the culture medium of sole nutrition composition in, Thallus Laminariae (Thallus Eckloniae) described in culture medium
Weight is 3wt% (with the dry matter weight gauge of Thallus Laminariae (Thallus Eckloniae)), control temperature 40 DEG C, rotating speed be 200rpm under conditions of cultivate
After fermenting 108 hours, making aimed strain be preserved, non-targeted microorganism is eliminated, and obtains fermentation liquid；
Take in 5mL fermentation liquid addition 50mL sterilized water and be sufficiently mixed, be diluted to 10 successively1～106Times, then, take dilution
104With 105The each 0.2mL of sample of two extension rates is coated in the flat board still only having the culture medium that Thallus Laminariae (Thallus Eckloniae) is nutritional labeling respectively again
On, the dry matter weight of Thallus Laminariae (Thallus Eckloniae) is 3wt% (with the dry matter weight gauge of Thallus Laminariae (Thallus Eckloniae)), controls temperature and trains under conditions of 40 DEG C
Support 84 hours, then picking individual colonies is rule switching on the culture medium flat plate that new Thallus Laminariae (Thallus Eckloniae) is nutritional labeling, the weight hundred of Thallus Laminariae (Thallus Eckloniae)
Mark is 3wt% (with the dry matter weight gauge of Thallus Laminariae (Thallus Eckloniae)), controls temperature and cultivates at 35 DEG C, it is thus achieved that single bacterium colony pure culture
Bacillus cereus MBRH3 bacterial strain.
Embodiment 4
The screening of Bacillus cereus MBRH3 bacterial strain
Take 2g bottom silt sample add with Thallus Laminariae (Thallus Eckloniae) for the culture medium of sole nutrition composition in, Thallus Laminariae (Thallus Eckloniae) described in culture medium
Dry matter weight is 4wt%, control temperature 40 DEG C, rotating speed be 200rpm under conditions of carry out cultivation and fermentation 108 hours after, make
Aimed strain is preserved, and non-targeted microorganism is eliminated, and obtains fermentation liquid；
Take in 5mL fermentation liquid addition 50mL sterilized water and be sufficiently mixed, be diluted to 10 successively1～106Times, then, take dilution
104With 105The each 0.2mL of sample of two extension rates is coated in the flat board still only having the culture medium that Thallus Laminariae (Thallus Eckloniae) is nutritional labeling respectively again
On, the dry matter weight of Thallus Laminariae (Thallus Eckloniae) is 4wt%, controls temperature and carries out cultivating under conditions of 40 DEG C 84 hours, then picking individual colonies
Line switching on the culture medium flat plate that new Thallus Laminariae (Thallus Eckloniae) is nutritional labeling, the percetage by weight of Thallus Laminariae (Thallus Eckloniae) is 4wt% (doing with Thallus Laminariae (Thallus Eckloniae)
Substance weight meter), control temperature and cultivate at 35 DEG C, it is thus achieved that single bacterium colony pure culture Bacillus cereus MBRH3 bacterium
Strain.
Application Example 1
Culture medium described in this application embodiment includes the weight proportion of following component:
MnSO4·7H2O:0.3g/L, KH2PO4: 0.5g/L, K2HPO4: 0.2g/L, glucose: 1.0g/L, Semen Maydis starch:
1.0g/L, Carnis Bovis seu Bubali cream: 1.0g/L, kelp paste 1.5ml/L, pH7.0；In described kelp paste, the mass percent of Thallus Laminariae (Thallus Eckloniae) is 5wt%.
Embodiment 1 will obtain single bacterium colony pure culture Bacillus cereus MBRH3 inoculation to containing Thallus Laminariae (Thallus Eckloniae)
Culture medium in cultivate, inoculum concentration is 2%, use 600nm detect its growing state, when strain OD value reaches 2.20,
Take 1mL seed to be inoculated in culture medium, control temperature 30 DEG C～40 DEG C, rotating speed be 200rpm under conditions of cultivate
48 hours, obtain the culture fluid containing SOD.Taking sample detection enzyme to live, each sample carries out 3 parallel laboratory tests, and aquesterilisa compares,
The work of SOD enzyme is the meansigma methods of 3 equality experiments.
Application Example 2
The concrete grammar of this application embodiment is substantially consistent with Application Example 1, and it differs only in described product enzyme and cultivates
Base is different, and culture medium described in this application embodiment includes the weight proportion of following component:
MnSO4·7H2O:0.7g/L, KH2PO4: 0.8g/L, K2HPO4: 0.5g/L, glucose: 3.0g/L, Semen Maydis starch:
3.0g/L, Carnis Bovis seu Bubali cream: 3.0g/L, kelp paste 3.0ml/L, pH7.0；In described kelp paste, the mass percent of Thallus Laminariae (Thallus Eckloniae) is 3wt%.
Application Example 3
The concrete grammar of this application embodiment is substantially consistent with Application Example 1, and it differs only in described product enzyme and cultivates
Base is different, and culture medium described in this application embodiment includes the weight proportion of following component:
MnSO4·7H2O:0.5g/L, KH2PO4: 0.7g/L, K2HPO4: 0.3g/L, glucose: 2.0g/L, Semen Maydis starch:
2.0g/L, Carnis Bovis seu Bubali cream: 2.0g/L, kelp paste 2.0ml/L, pH7.0；In described kelp paste, the mass percent of Thallus Laminariae (Thallus Eckloniae) is 4wt%.
Application Example 4
The concrete grammar of this application embodiment is substantially consistent with Application Example 1, and it differs only in described product enzyme and cultivates
Base is different, and culture medium described in this application embodiment includes the weight proportion of following component:
MnSO4·7H2O:0.4g/L, KH2PO4: 0.6g/L, K2HPO4: 0.4g/L, glucose: 1.5g/L, Semen Maydis starch:
1.0g/L, Carnis Bovis seu Bubali cream: 2.5g/L, kelp paste 2.5ml/L, pH7.0；In described kelp paste, the mass percent of Thallus Laminariae (Thallus Eckloniae) is 2wt%.
Randomly select the Bacillus cereus MBRH3 bacterial strain that above-described embodiment 1 obtains identify and analyze, specifically
Qualification and the method for analysis and corresponding qualification result as follows.
Bacillus cereus MBRH3 strain morphology and bio-chemical characteristics are with reference to industrial microorganism experimental technique handbook
(Zhu Gejian, Wang Zhengxiang. industrial microorganism experimental technique handbook [M]. China Light Industry Press, 1994) and primary Jie Shi antibacterial mirror
Determine handbook carry out (R.E. Buchanan, N.E. Ji this this. the outstanding Bacteria Identification handbook [M] of uncle. Science Press, Beijing, 1984).
The assay method that SOD enzyme is lived:
(with reference to Zheng Tie once, painting proposed the spy of pyrogallol autoxidation method in female .SOD enzyme activity determination to the assay method that SOD enzyme is lived
Beg for [J]. Tianjin microorganism, 1995, (1): l-5.) use the Autoxidation Method of trace 1,2,3,-thrihydroxy-benzene to measure, take culture fluid 1mL to be measured
Take the Tris-HCl buffer (containing EDTA1mM, pH8.2) of supernatant 0.1mL and 3mL0.1mol/L after Li Xin, 0.1mL connects benzene three
Phenol solution (preparation of 0.05mol/L, 0.1mol/L hydrochloric acid) forms total system, and above-mentioned buffer preheats at 35 DEG C～37 DEG C.
Under wavelength 325nm, survey absorbance, make blank inactivateing equivalent amounts of enzyme liquid.Described enzyme is lived and is defined: under conditions of 37 DEG C,
In the reactant liquor of 1mL, enzyme amount when suppression rosette like growth speed per minute reaches 50% is defined as a unit of activity
(U)。
SOD enzyme (U/mL)=2 alive (ΔA325-Δ’A325)/ΔA325×3.2×n/V
In above-mentioned formula, Δ A325 is A325 difference of reading during rosette like growth, Δ ' A325 is for adding A325 after enzyme liquid
Difference of reading, 3.2 is the reactant liquor volume measured, and n is enzyme liquid extension rate, and V is long-pending for the enzyme liquid of reaction.
Above-mentioned analysis and qualification result show:
The Physiology and biochemistry of the Bacillus cereus MBRH3 bacterial strain of the present invention and morphological feature, such as Fig. 1 and Fig. 2 institute
Showing, the bacterium colony of the Bacillus cereus MBRH3 bacterial strain of the present invention is rounded, and smooth surface, neat in edge, milky is impermeable
Bright, thalline thickness is difficult to provoke；Thalline length about 1～2.5 μm, diameter about 0.5～1.0 μm；Shaft-like chaining.Bio-chemical characteristics
Result shows, the bacterial strain of the present invention is gram positive bacteria；All can grow in 5 DEG C to 45 DEG C temperature ranges, the most suitable growth temperature
Degree is 35 DEG C, all can grow when pH is 3.5～8.5, and optimum growh pH is 6.5, facultative aerobic.The carbon source that can utilize has: sugarcane
Sugar, 2-Acetamido-2-deoxy-D-glucose, 4-O-(2-Amino-2-deoxy-.beta.-D-glucosyl)-D-glucosamine., galactose, glucose, fructose, inulin, maltose, mannose, arabinose, wood
Sugar etc.；Secernent enzyme is as follows: catalase, galactase, beta-glucosidase, urine enzyme, saccharase, maltase, shallow lake
Powder enzyme etc..
The PCR amplification of the Bacillus cereus MBRH3 bacterial strain 16SrRNA of the present invention uses conventional method,
In the present invention, the DNA extraction in PCR amplification uses the bacterial genomes of Shanghai SBS Genetech gene technology company limited to extract reagent
Box, concrete extracting method is carried out according to the method for test kit, and the primer that amplification uses is universal primer, by Shanghai SBS Genetech base
Because company limited synthesizes, primer is:
5’-GAGTTTGATCCTGGCTCA-3’；
5’-CGGTTACCTTGTTACGACTT-3’；
Pcr amplification reaction condition is: 94 DEG C of denaturations 4min, enters and circulates, 94 DEG C of degeneration 50s, 55 DEG C of annealing 50s, 72
DEG C extending 2min, 35 circulations, 72 DEG C extend 4 DEG C of states of preservation after 10min.PCR primer UltraClean PCR Clean-
Up kit purification, and completed order-checking by Shanghai biological engineering company limited, sequencing result is as shown in SEQ.NO1.By the present invention's
Bacillus cereus MBRH3 bacterial strain 16SrRNA gene order carries out homology analysis, and result shows, with Bacillus
Cereus P2, Max Ident reaches 99%.Select representational bacterial strain to use software Mega3.1 to utilize and be bordered by method
(Neighbor-Joinning) phylogenetic tree construction is verified, again shows that the Bacillus cereus of the present invention
The genetic distance that MBRH3 bacterial strain belongs to Bacillus is closest.Table is identified from morphology, Physiology and biochemistry and molecular biological characteristics
Bright, this Pseudomonas in Bacillaceae (Bacillaceae), Bacillus (Bacillus), Bacillus cereus (cereus),
Named Bacillus cereus MBRH3.Document report the most can not met with Thallus Laminariae (Thallus Eckloniae) for raw material production SOD for this bacterium
Road.
The optimization of product SOD culture medium:
Use center combination test (Central Composite Design, CCD) method, use software Dsign-
Expert carries out design and analysis of experiment, and utilizing experiment of single factor to filter out has 4 kinds of culture medium of potential impact to become product SOD
Point, i.e. Semen Maydis starch, kelp paste, glucose and 4 kinds of medium components of manganese sulfate, further determine that the optimal of each composition of culture medium
Content is also verified.Then, use center combination design method (CCD) Optimal Medium, center combination design level, encode and test
The results are shown in Table shown in 1.
By the result of above-mentioned table 1, software matching gained quadratic polynomial equation is: Y=193.78+4.30X1+
30.76X2+10.71X3+5.34X4+11.82X1X2+1.04X1X3+8.18X1X4+6.50X2X3-0.70X2X4-11.00X3X4-
19.70X2 1-14.19X2 2-25.49X2 3-19.53X2 4
Central combination design variance and regression analysis is as shown in table 2, as seen from table, and model p=0.0054 < 0.05,
Illustrate that this model is significant under the probability level of 99.5%.And lose matching not significantly (p=0.0517 > 0.05), mould is described
Without being re-introduced into other factors in type.Coefficient of determination R2=0.9857, illustrate that 98.57% experiment situation of change can use this mould
Type is explained, R2Adj=0.9357, with R2It is consistent, shows that SOD condition optimizing fermentation model is reasonable.Shown in Fig. 3,
It can be seen that its observation and predictive value are all distributed near same straight line, further illustrate this model and be consistent with the fact.
Fermentation culture conditions is: temperature 35 DEG C, rotating speed 200rpm；Liquid amount: 50mL/250mL；Inoculum concentration: 2%；Cultivate
Time: 48 hours.Data take the meansigma methods of three parallel laboratory tests；Significance analysis uses t-inspection, and P < 0.05 is considered as significantly, P <
0.01 is that difference is the most notable.Table 1 below is center combination level, coding and experimental result.Table 2 be center combination design variance and
Interpretation of result.
Table 1:
From above-mentioned software matching gained quadratic polynomial equation it can be seen that the value of described quadratic power coefficient be respectively-
19.69 ,-14.17 ,-25.49 and-19.53, parabola is described, and Open Side Down, can obtain maximum.Regression equation is asked
Lead, in conjunction with Fig. 4, the three-dimensional response surface of Fig. 5, Fig. 6, Fig. 7 and Fig. 8, try to achieve the maximum of this model, X1=1, X2=0.74, X3=
0.2 and X4=0.61, the actual value (g/L) of corresponding Semen Maydis starch, kelp paste, glucose and manganese sulfate is respectively 4.50,
4.11,2.10 and 0.62, the maximum response of this model is, 203.38U/ml.The Bacillus of the experimental result display present invention
During SOD is produced in cereus MBRH3 fermentation, the effect of Thallus Laminariae (Thallus Eckloniae) is significant (P2=0.002 < 0.05), this with Thallus Laminariae (Thallus Eckloniae) is
The aimed strain of sole nutrition screening substances is consistent, also illustrates that this bacterial strain has certain specificity, and it can be good at profit
With Thallus Laminariae (Thallus Eckloniae), and glucose is the energy substance that microorganism can directly utilize, and this experiment displays that the generation of SOD is affected by glucose
Significantly (P2=0.002 < 0.0165).
Table 2:
In conjunction with the relation reflected shown in Fig. 4 between Semen Maydis pulp and two kinds of nutrition of Thallus Laminariae (Thallus Eckloniae) and on the impact producing SOD, significantly
Property inspection display P (0.0968) ＞ 0.05, illustrate that the reciprocal action between them is inapparent, say, that no matter they
Value is in high level or low-level, and its enzymatic productivity all can not reach maximum.ANOVA analyzes display Semen Maydis starch and Thallus Laminariae (Thallus Eckloniae)
Dependency between slurry, between Semen Maydis starch and glucose, between Semen Maydis starch and manganese sulfate and between kelp paste and glucose
The most not notable, but, from Fig. 5, Fig. 6, Fig. 7 and Fig. 8 it can be seen that various trophic factors all meets normal state to the impact producing SOD
Distribution, illustrates that such material is met universal law as the raw material producing SOD by microorganism utilization.As produce SOD several mainly
Nutrient substance, the only significant interaction (p=0.035 < 0.05) between glucose and manganese sulfate, change them and cultivating
Enzymatic productivity can be made when concentration in base reaches certain level to reach maximum.During producing SOD, glucose and sulphuric acid
The reason significantly that influences each other between manganese is probably glucose when being utilized, and can produce acid, cause the pH value of yeasting to change,
The pH value of fermentation system changes the structure absorbing or changing SOD that also can affect manganese sulfate, and bacterial strain is utilized again by this change
Glucose produces impact, and this is also in close relations with manganese from this SOD of side illustration, belongs to Mn-SOD.
Specific embodiment described in the present invention is only to present invention spirit explanation for example.Technology neck belonging to the present invention
Described specific embodiment can be made various amendment or supplements or use similar mode to replace by the technical staff in territory
Generation, but without departing from the spirit of the present invention or surmount scope defined in appended claims.
Although the present invention has been made a detailed description and has quoted some specific embodiments as proof, but skilled to this area
For technical staff, as long as it is obvious for can making various changes without departing from the spirit and scope of the present invention or revise.
Claims (4)
1. a Bacillus cereus MBRH3 bacterial strain, it is characterised in that described Bacillus cereus MBRH3 bacterial strain
Deposit number in China typical culture collection center is CCTCC NO.M2013581, and the 16S rRNA sequence of described bacterial strain
Row are as shown in SEQ.NO 1.
2. the application of a Bacillus cereus MBRH3 bacterial strain as claimed in claim 1, it is characterised in that described
Bacillus cereus MBRH3 bacterial strain is used for producing SOD.
The application of Bacillus cereus MBRH3 bacterial strain the most according to claim 2, it is characterised in that by Bacillus
Cereus MBRH3 inoculation is cultivated to the culture medium containing Thallus Laminariae (Thallus Eckloniae), when strain OD value reaches 2.20, takes seed
It is inoculated in culture medium, after control temperature is cultivated under the conditions of 30 DEG C～40 DEG C, obtains SOD.
The application of Bacillus cereus MBRH3 bacterial strain the most according to claim 3, it is characterised in that described product enzyme is trained
Foster base includes the weight proportion of following component:
MnSO4·7H2O:0.3g/L～0.7g/L, KH2PO4: 0.5g/L～0.8g/L, K2HPO4: 0.2g/L～0.5g/L, Fructus Vitis viniferae
Sugar: 1.0g/L～3.0g/L, Semen Maydis starch: 1.0g/L～3.0g/L, Carnis Bovis seu Bubali cream: 1.0g/L～3.0g/L, kelp paste: 1.5mL/
L～3.0mL/L, pH:7.0.
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