CN103992971A - Bacillus cereus MBRH3 strain as well as screening method and application thereof - Google Patents
Bacillus cereus MBRH3 strain as well as screening method and application thereof Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The invention relates to a bacillus cereus MBRH3 strain as well as a screening method and application thereof, belongs to the field of bioengineering technology, and solves the problem of how to screen a new strain with SOD (superoxide dismutase) producing capability from sea mud. The screening method comprises the following steps: adding the sea mud into a culture medium with kelp as nutrients, culturing at 30-40 DEG C to obtain a cultured material, diluting the cultured material, inoculating the diluted material to a flat plate of the culture medium with the kelp as the nutrients, culturing at 30-40 DEG C, scribing the flat plate of the culture medium with the kelp as the nutrients, and culturing single colony to obtain a pure cultured material, namely the bacillus cereus MBRH3 strain. The strain is high in SOD producing rate and can take the kelp as the nutrients; the used raw materials are rich and low in cost.
Description
Technical field
The present invention relates to a kind of Bacillus cereus MBRH3 bacterial strain and screening method and application, belong to technical field of bioengineering.
Background technology
Superoxide-dismutase (EC1.15.1.1, Superoxide dismutase, SOD) is the interior ultra-oxygen anion free radical (O of specificity catalysis biological body that viable cell produces
- 2) there is the metalloenzyme of disproportionation reaction Hydrogen Peroxide and oxygen, it maintains the generation of biological interior free yl and the running balance of removing, and biology is had to protection, health care and the effect of curing the disease.SOD is metalloenzyme, can be divided into Fe-SOD, Mn-SOD and CuZn-SOD according to the different metal ion of its combination.Prokaryotic cell prokaryocyte and Activities of Some Plants mainly contain Fe-SOD, are yellow.In eukaryotic cell plastosome and prokaryotic cell prokaryocyte, contain Mn-SOD, be purple.Chloroplast(id), peroxysome and the eukaryotic cell cytoplasm of plant contain more CuZn-SOD, are blue-greenish colour.In recent years, in streptomyces, find Ni-SOD and FeZn-SOD, in beef liver, found CoZn-SOD.Visible, becoming increasingly abundant of SOD kind, makes its application also more extensive.As at field of medicaments, SOD can treat because of O
2 -the disease causing, as sacroiliitis and rheumatoid arthritis etc.; At field of food, SOD can be used as the additive of protective foods, has antifatigue, anti-ageing, radioprotective and antiinflammation, also can be used for fruit freshness preserving; In household chemicals field, that SOD has is sun-proof, crease-resistant, nti-freckle, anti-inflammatory action; In agricultural and plant protection field, SOD has the anti-adversity of raising and tolerance etc.
SOD mainly obtains by extracting or screening from animal, plant and the microorganism cells etc. of living, and the strength difference of different bio SOD is very large, and the difference of the different sites SOD of same organism is also very large.If pluck and blood were once the main sources of SOD, still, the SOD that derives from animal easily causes the adverse consequences such as cross infection and anaphylaxis, and application is very limited; In plant, particularly soybean, corn, mulberry leaf, garlic and Root and stem of Cholla equal size are relatively high, but its extraction process complexity, so that production cost is compared with high and can not produce in a large number; And microorganism fermentation to obtain SOD be the most frequently used method at present, utilize microorganism fermentative production SOD to there is the significant advantages such as output is high, technique is simple, it is the main method of obtaining SOD, particularly utilize the engineering bacteria of the product SOD of the structure of present molecular biotechnology, make to obtain SOD by fermentation more effective.The people such as Ru Wangsui building, Wang Sufang obtain SOD Producing Strain by screening, live and reach respectively 600U/g wet thallus and specific activity 3048U/mg by fermentation SOD enzyme, but it is mainly to utilize soil effect raw material to screen to obtain, and it mainly adopts the conventional nutritive ingredients such as glucose as substratum.And how therefore the microorganism that is richly stored with in ocean, screen new bacterial strain from ooze, and there is the ability of producing SOD, there is higher researching value.
And sea-tangle is perennial macro, approximately 900,000 tons/year of China's sea-tangle output, are the half of Gross World Product, ranked first position.Sea-tangle not only has edibleness, or a kind of industrial raw material of high added value.The main component of sea-tangle is algin, be rich in the mineral element such as iodine, calcium and multiple amino acids and VITAMIN simultaneously, kelp nourishing is abundant, the laminarin that its content is maximum has the tumor growth of inhibition, improves renal failure, reduces blood fat, the pharmacological action such as reduce blood pressure, natural protective foods, by the people of the many countries of China and South East Asia are liked.Though China is sea-tangle output big country and sea-tangle processing big country, but sea-tangle level of processing also has a segment distance compared with World Developed Countries, and also do not adopt the substratum of making state's microbe to screen with sea-tangle with nutritive ingredient at present, therefore, carrying out the research of sea-tangle application potential in a deep going way is to improve sea-tangle utility value effective way, has more effectively application prospect.
Summary of the invention
The present invention is directed to existing problem in above-mentioned prior art, the invention provides a kind of Bacillus cereus MBRH3 bacterial strain and screening method and application, the problem of solution is to realize providing a kind of new bacterial strain, and described bacterial strain has the effect of high yield SOD.
One of object of the present invention is achieved by the following technical programs, a kind of Bacillus cereus MBRH3 bacterial strain, and described Bacillus cereus MBRH3 bacterial strain is CCTCC NO.M2013581 at the deposit number at Chinese Typical Representative culture collection center.
Bacterial strain of the present invention is to screen and obtain from bottom silt, it is a kind of new bacterial strain, belong to gram-positive microorganism, described Bacillus cereus MBRH3 bacterial strain has been preserved in Chinese Typical Representative culture collection center on November 19th, 2013, its deposit number is CCTCC NO.M2013581, and preservation place is Wuhan, China Wuhan University.
Bacillus cereus MBRH3 bacterial strain bacterium colony of the present invention is rounded, smooth surface, and neat in edge, oyster white is opaque, and thalline thickness is difficult for provoking; The about 1-2.5 μ of thalline length m, diameter 0.5-1.0 μ m; Shaft-like chaining.Physiology and biochemistry experimental result shows, this bacterium is gram-positive microorganism; In 5 DEG C to 45 DEG C temperature ranges, all can grow, optimum growth temperature is 35 DEG C, when pH is 3.5-8.5, all can grow, and optimum growh pH is 6.5, facultative aerobic.Bacterial strain of the present invention can utilize sea-tangle for nutritive ingredient, and has advantages of that SOD output is high.
As preferably, the 16S rRNA sequence of Bacillus cereus MBRH3 bacterial strain described above is as shown in SEQ.NO1.
Two of object of the present invention is achieved by the following technical programs, a kind of screening method of Bacillus cereus MBRH3 bacterial strain, and the method comprises the following steps:
A, get ooze sample and add in the substratum taking sea-tangle as nutritive ingredient, under 30 DEG C～40 DEG C conditions, cultivate, aimed strain is preserved, non-target microorganism is eliminated, and obtains culture;
B, get after culture obtained above dilution, be seeded on the culture medium flat plate taking sea-tangle as nutritive ingredient, after control temperature is cultivated under 30 DEG C～40 DEG C conditions, select again single bacterium colony on the new culture medium flat plate taking sea-tangle as nutritive ingredient, rule switching cultivate, obtain single bacterium colony pure growth Bacillus cereus MBRH3 bacterial strain.
The screening method of Bacillus cereus MBRH3 bacterial strain of the present invention, employing ooze is sample, and substratum using sea-tangle as unique nutritive ingredient, the bacterial strain obtaining after screening by cultivation, the ability with product SOD is high, the SOD function producing with Bacillus cereus MBRH3 bacterial strain of the present invention is identical, more can bring into play its advantage by selecting sea-tangle as nutritive ingredient or as the raw material of producing SOD, can improve selectivity and the directional property of screening, strengthen the screening of dominant strain, make more effective screening Bacillus cereus MBRH3 bacterial strain and for the production of SOD, and adopt sea-tangle as raw material, have advantages of that abundant raw material and cost are low, be more conducive to industrialization and produce.
In the screening method of above-mentioned Bacillus cereus MBRH3 bacterial strain, as preferably, with the weighing scale of dried seaweed material, in the substratum taking sea-tangle as nutritive ingredient described in steps A and step B, the weight percentage of sea-tangle is 2wt%～5wt%.Sea-tangle by commercial, adopts sea-tangle after water soaking, after stripping and slicing, pulverizes, the kelp paste that to be deployed into containing dried seaweed material be 2wt%～5wt%.As excellent further preferred, in the substratum taking sea-tangle as nutritive ingredient described in steps A and step B, the weight percentage of sea-tangle is 3wt%～4wt%.
In the screening method of above-mentioned Bacillus cereus MBRH3 bacterial strain, as preferably, described ooze is bottom silt.
Three of object of the present invention is achieved by the following technical programs, a kind of application of Bacillus cereus MBRH3 bacterial strain, and described bacterial strain is for the production of SOD.
As preferably, Bacillus cereus MBRH3 inoculation is cultivated to the substratum that contains sea-tangle, in the time that bacterial classification OD value reaches 2.20, get seed and be inoculated in culture medium, control temperature and carry out fermentation culture after 48 hours under 30 DEG C～40 DEG C conditions, obtain SOD.
In the application of above-mentioned Bacillus cereus MBRH3 bacterial strain, as preferably, culture medium described above comprises the weight proportion of following composition:
MnSO
47H
2o:0.3g/L～0.7g/L, KH
2pO
4: 0.5g/L～0.8g/L, K
2hPO
4: 0.2g/L～0.5g/L, glucose: 1.0g/L～3.0g/L, corn starch: 1.0g/L～3.0g/L, extractum carnis: 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 following advantages:
Bacillus cereus MBRH3 bacterial strain of the present invention, it is a kind of new bacterial strain, can utilize sea-tangle for nutritive ingredient, there is higher product SOD ability, and screening method of the present invention is using sea-tangle as nutritive ingredient is as cultivation, have advantages of that abundant raw material and cost are low, and can improve selectivity and the directional property of screening, strengthened the screening of dominant strain.
Figure of description
Fig. 1 is the colonial morphology figure of 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 this bright Bacillus cereus MBRH3 bacterial strain enzyme observed value and predictor alive.
Fig. 4 is corn starch and the affect three-dimensional plot of kelp paste on SOD activity in culture medium of the present invention.
Fig. 5 is corn starch and the affect three-dimensional plot of glucose on SOD activity in culture medium of the present invention.
Fig. 6 is corn starch and the affect three-dimensional plot of manganous sulfate on SOD activity in culture medium of the present invention.
Fig. 7 is kelp paste and the affect three-dimensional plot of glucose on SOD activity in culture medium of the present invention.
Fig. 8 is kelp paste and the affect three-dimensional plot of manganous sulfate on SOD activity during product enzyme of the present invention is cultivated.
Fig. 9 is glucose and the affect three-dimensional plot of manganous sulfate on SOD activity during product enzyme of the present invention is cultivated.
Embodiment
Below by specific embodiments and the drawings, technical scheme of the present invention is described in further detail, but the present invention is not limited to these embodiment.
In above embodiment, ooze adopts bottom silt, and described bottom silt collection from sea area, Jian Men port, City of Taizhou the bottom silt of (28 ° 1 ' 57 ' ' N, 121 ° 36 ' 38 ' ' E), through drying for standby.
Substratum taking sea-tangle as unique nutritive ingredient described in following examples, described sea-tangle is to select the sea-tangle of buying on market.Preferably, described sea-tangle is first mixed with kelp paste, and employing pure water is by kelp soaking after 24 hours, and after using soybean milk maker fully to pulverize after stripping and slicing, the kelp paste that it is 2wt%～5wt% that employing pure water is deployed into containing dried seaweed substance weight is for subsequent use.
Embodiment 1
The screening of Bacillus cereus MBRH3 bacterial strain
Getting 2g bottom silt sample adds in the substratum taking sea-tangle as unique nutritive ingredient, the weight of sea-tangle described in substratum is 5wt% (in the dry matter weight of sea-tangle), under the condition that to control temperature be 200rpm at 35 DEG C, rotating speed, carry out cultivation and fermentation after 108 hours, aimed strain is preserved, non-target microorganism is eliminated, and obtains fermented liquid;
Get 5mL fermented liquid and add fully mixing in 50mL sterilized water, be diluted to successively 10
1～10
6doubly, then, get dilution 10
4with 10
5doubly the each 0.2mL of sample of two extension rates is coated in respectively that still only to have sea-tangle be on the flat board of substratum of nutritive ingredient, the weight percentage of sea-tangle is 5wt% (in the dry matter weight of sea-tangle), control temperature cultivates 72 hours under the condition of 37 DEG C, line switching on the culture medium flat plate that to select single bacterium colony be nutritive ingredient in new sea-tangle again, the weight percentage of sea-tangle is 5wt% (in the dry matter weight of sea-tangle), control temperature and cultivate at 35 DEG C, obtain single bacterium colony pure growth Bacillus cereus MBRH3 bacterial strain.
Embodiment 2
The screening of Bacillus cereus MBRH3 bacterial strain
Getting 2g bottom silt sample adds in the substratum taking sea-tangle as unique nutritive ingredient, the weight of sea-tangle described in substratum is 2wt% (in the dry matter weight of sea-tangle), under the condition that to control temperature be 200rpm at 30 DEG C, rotating speed, carry out cultivation and fermentation after 108 hours, aimed strain is preserved, non-target microorganism is eliminated, and obtains fermented liquid;
Get 5mL fermented liquid and add fully mixing in 50mL sterilized water, be diluted to successively 10
1～10
6doubly, then, get dilution 10
4with 10
5doubly the each 0.2mL of sample of two extension rates is coated in respectively that still only to have sea-tangle be on the flat board of substratum of nutritive ingredient, the weight percentage of sea-tangle is 2wt% (in the dry matter weight of sea-tangle), control temperature cultivates 72 hours under the condition of 35 DEG C, line switching on the culture medium flat plate that to select single bacterium colony be nutritive ingredient in new sea-tangle again, the weight percentage of sea-tangle is 2wt% (in the dry matter weight of sea-tangle), control temperature and cultivate at 35 DEG C, obtain single bacterium colony pure growth Bacillus cereus MBRH3 bacterial strain.
Embodiment 3
The screening of Bacillus cereus MBRH3 bacterial strain
Getting 2g bottom silt sample adds in the substratum taking sea-tangle as unique nutritive ingredient, the weight of sea-tangle described in substratum is 3wt% (in the dry matter weight of sea-tangle), under the condition that to control temperature be 200rpm at 40 DEG C, rotating speed, carry out cultivation and fermentation after 108 hours, aimed strain is preserved, non-target microorganism is eliminated, and obtains fermented liquid;
Get 5mL fermented liquid and add fully mixing in 50mL sterilized water, be diluted to successively 10
1～10
6doubly, then, get dilution 10
4with 10
5doubly the each 0.2mL of sample of two extension rates is coated in respectively that still only to have sea-tangle be on the flat board of substratum of nutritive ingredient, the dry matter weight of sea-tangle is 3wt% (in the dry matter weight of sea-tangle), control temperature cultivates 84 hours under the condition of 40 DEG C, line switching on the culture medium flat plate that to select single bacterium colony be nutritive ingredient in new sea-tangle again, the weight percentage of sea-tangle is 3wt% (in the dry matter weight of sea-tangle), control temperature and cultivate at 35 DEG C, obtain single bacterium colony pure growth Bacillus cereus MBRH3 bacterial strain.
Embodiment 4
The screening of Bacillus cereus MBRH3 bacterial strain
Getting 2g bottom silt sample adds in the substratum taking sea-tangle as unique nutritive ingredient, the dry matter weight of sea-tangle described in substratum is 4wt%, under the condition that to control temperature be 200rpm at 40 DEG C, rotating speed, carry out cultivation and fermentation after 108 hours, aimed strain is preserved, non-target microorganism is eliminated, and obtains fermented liquid;
Get 5mL fermented liquid and add fully mixing in 50mL sterilized water, be diluted to successively 10
1～10
6doubly, then, get dilution 10
4with 10
5doubly the each 0.2mL of sample of two extension rates is coated in respectively that still only to have sea-tangle be on the flat board of substratum of nutritive ingredient, the dry matter weight of sea-tangle is 4wt%, control temperature cultivates 84 hours under the condition of 40 DEG C, line switching on the culture medium flat plate that to select single bacterium colony be nutritive ingredient in new sea-tangle again, the weight percentage of sea-tangle is 4wt% (in the dry matter weight of sea-tangle), control temperature and cultivate at 35 DEG C, obtain single bacterium colony pure growth Bacillus cereus MBRH3 bacterial strain.
Application Example 1
Culture medium described in this application embodiment comprises the weight proportion of following composition:
MnSO
47H
2o:0.3g/L, KH
2pO
4: 0.5g/L, K
2hPO
4: 0.2g/L, glucose: 1.0g/L, corn starch: 1.0g/L, extractum carnis: 1.0g/L, kelp paste 1.5ml/L, pH7.0; In described kelp paste, the mass percent of sea-tangle is 5wt%.
Cultivate to the substratum that contains sea-tangle obtaining single bacterium colony pure growth Bacillus cereus MBRH3 inoculation in embodiment 1, inoculum size is 2%, adopt 600nm to detect its growing state, in the time that bacterial classification OD value reaches 2.20, getting 1mL seed is inoculated in culture medium, under the condition that to control temperature be 200rpm at 30 DEG C～40 DEG C, rotating speed, cultivate 48 hours, obtain the nutrient solution containing SOD.Sample thief detects enzyme and lives, and each sample carries out 3 parallel laboratory tests, and aqua sterilisa contrasts, and the work of SOD enzyme is the mean value of 3 equality experiments.
Application Example 2
The concrete grammar of this application embodiment is substantially consistent with Application Example 1, and its difference is only described culture medium difference, and culture medium described in this application embodiment comprises the weight proportion of following composition:
MnSO
47H
2o:0.7g/L, KH
2pO
4: 0.8g/L, K
2hPO
4: 0.5g/L, glucose: 3.0g/L, corn starch: 3.0g/L, extractum carnis: 3.0g/L, kelp paste 3.0ml/L, pH7.0; In described kelp paste, the mass percent of sea-tangle is 3wt%.
Application Example 3
The concrete grammar of this application embodiment is substantially consistent with Application Example 1, and its difference is only described culture medium difference, and culture medium described in this application embodiment comprises the weight proportion of following composition:
MnSO
47H
2o:0.5g/L, KH
2pO
4: 0.7g/L, K
2hPO
4: 0.3g/L, glucose: 2.0g/L, corn starch: 2.0g/L, extractum carnis: 2.0g/L, kelp paste 2.0ml/L, pH7.0; In described kelp paste, the mass percent of sea-tangle is 4wt%.
Application Example 4
The concrete grammar of this application embodiment is substantially consistent with Application Example 1, and its difference is only described culture medium difference, and culture medium described in this application embodiment comprises the weight proportion of following composition:
MnSO
47H
2o:0.4g/L, KH
2pO
4: 0.6g/L, K
2hPO
4: 0.4g/L, glucose: 1.5g/L, corn starch: 1.0g/L, extractum carnis: 2.5g/L, kelp paste 2.5ml/L, pH7.0; In described kelp paste, the mass percent of sea-tangle is 2wt%.
Choose at random the Bacillus cereus MBRH3 bacterial strain that above-described embodiment 1 obtains and identify and analyze, concrete qualification and analytical procedure and corresponding qualification result are as follows.
Bacillus cereus MBRH3 strain morphology and Physiology and biochemistry are tested with reference to industrial microorganism experimental technique handbook (Zhu Gejian, Wang Zhengxiang. industrial microorganism experimental technique handbook [M]. China Light Industry Press, 1994) and the primary Jie Shi Bacteria Identification handbook carries out (R.E. Buchanan, N.E. Ji this this. the outstanding Bacteria Identification handbook of uncle [M]. Science Press, Beijing, 1984).
The measuring method that SOD enzyme is lived:
The measuring method that SOD enzyme is lived (with reference to Zheng Tie once, the discussion [J] of pyrogallol autoxidation method in female .SOD enzyme activity determination is proposed in painting. Tianjin microorganism, 1995, (1): l-5.) adopt the Autoxidation Method of micro-pyrogallol to measure, get Tris-HCl damping fluid that nutrient solution 1mL to be measured gets supernatant liquor 0.1mL and 3mL0.1mol/L after centrifugal (containing EDTA1mM, pH8.2), 0.1mL pyrogallol solution (0.05mol/L, the preparation of 0.1mol/L hydrochloric acid) form total system, above-mentioned damping fluid carries out preheating at 35 DEG C～37 DEG C.Under wavelength 325nm, survey absorbancy, make blank with deactivation equivalent amounts of enzyme liquid.Described enzyme is lived and is defined: under the condition of 37 DEG C, in the reaction solution of 1mL, enzyme amount when per minute suppresses pyrogallol autoxidation rate 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, A325 difference of reading when Δ A325 is pyrogallol autoxidation, Δ ' A325 is for adding A325 difference of reading after enzyme liquid, 3.2 reaction solution volumes for measuring, n is enzyme liquid extension rate, V is that the enzyme liquid for reacting is long-pending.
Above-mentioned analysis and identification result shows:
Physiology and biochemistry and the morphological feature of Bacillus cereus MBRH3 bacterial strain of the present invention, as depicted in figs. 1 and 2, the bacterium colony of Bacillus cereus MBRH3 bacterial strain of the present invention is rounded, smooth surface, neat in edge, oyster white is opaque, and thalline thickness is difficult for provoking; Thalline length approximately 1～2.5 μ m, diameter 0.5～1.0 μ m; Shaft-like chaining.Physiology and biochemistry experimental result shows, bacterial strain of the present invention is gram-positive microorganism; In 5 DEG C to 45 DEG C temperature ranges, all can grow, optimum growth temperature is 35 DEG C, and pH can grow for 3.5～8.5 o'clock, and optimum growh pH is 6.5, facultative aerobic.The carbon source that can utilize has: sucrose, 2-Acetamido-2-deoxy-D-glucose, chitobiose, semi-lactosi, glucose, fructose, inulin, maltose, seminose, pectinose, wood sugar etc.; Secernent enzyme is as follows: catalase, galactase, beta-glucosidase, urine enzyme, sucrase, maltin, amylase etc.
The pcr amplification of Bacillus cereus MBRH3 bacterial strain 16SrRNA of the present invention adopts conventional method, DNA extraction in the present invention in pcr amplification adopts the bacterial genomes of match Parkson, Shanghai gene engineering company limited to extract test kit, concrete extracting method carries out according to the method for test kit, the primer that amplification adopts is universal primer, synthetic by match Parkson, Shanghai gene company limited, primer is:
5’-GAGTTTGATCCTGGCTCA-3’；
5’-CGGTTACCTTGTTACGACTT-3’；
Pcr amplification reaction condition is: 94 DEG C of denaturation 4min, enter circulation, and 94 DEG C of sex change 50s, 55 DEG C of annealing 50s, 72 DEG C are extended 2min, and 35 circulations are preserved 4 DEG C of states after 72 DEG C of extension 10min.PCR product is purified with UltraClean PCR Clean-up kit, and completes order-checking by Shanghai biotechnology company limited, and sequencing result is as shown in SEQ.NO1.Bacillus cereus MBRH3 bacterial strain 16SrRNA gene order of the present invention is carried out to homology analysis, result demonstration, with Bacillus cereus P2, Max Ident reaches 99%.Select representational bacterial strain to use software Mega3.1 utilization to face connection (Neighbor-Joinning) phylogenetic tree construction and verify, show that equally the genetic distance of Bacillus cereus MBRH3 bacterial strain of the present invention and Bacillus genus is the most approaching.Show from morphology, Physiology and biochemistry and molecular biological characteristics qualification, this Pseudomonas is in Bacillaceae (Bacillaceae), Bacillus (Bacillus), Bacillus cereus (cereus), called after Bacillus cereus MBRH3.Can also not meet bibliographical information taking sea-tangle as raw material production SOD for this bacterium.
Produce the optimization of SOD substratum:
Adopt center combination test (Central Composite Design, CCD) method, use software Dsign-Expert to carry out design and analysis of experiment, utilize experiment of single factor to filter out 4 kinds of medium components that have potential impact to producing SOD, be corn starch, kelp paste, glucose and 4 kinds of medium components of manganous sulfate, further determine optimum content the checking of the each composition of substratum.Then, use center combination design method (CCD) Optimal Medium, center combination design level, coding and experimental result are shown in Table 1.
By the result of above-mentioned table 1, software matching gained quadratic polynomial equation is: Y=193.78+4.30X
1+ 30.76X
2+ 10.71X
3+ 5.34X
4+ 11.82X
1x
2+ 1.04X
1x
3+ 8.18X
1x
4+ 6.50X
2x
3-0.70X
2x
4-11.00X
3x
4-19.70X
2 1-14.19X
2 2-25.49X
2 3-19.53X
2 4
Center combination experimental design variance and regression analysis is as shown in table 2, and as seen from table, model p=0.0054<0.05, illustrates that this model is significant under 99.5% probability level.And lose not remarkable (p=0.0517>0.05) of matching, illustrate in model without introducing again other factors.Coefficient of determination R
2=0.9857, illustrate 98.57% experiment changing conditions can use this model explanation, R
2adj=0.9357, with R
2conform to, show that SOD condition optimizing fermentation model is reasonable.Shown in Fig. 3, can find out, its observed value and predictor are all distributed near same straight line, further illustrate this model and conform to true.
Fermentation culture conditions is: 35 DEG C of temperature, rotating speed 200rpm; Liquid amount: 50mL/250mL; Inoculum size: 2%; Incubation time: 48 hours.Data are got the mean value of three parallel laboratory tests; Significance analysis adopts t-inspection, and P<0.05 is considered as significantly, and P<0.01 is that difference is extremely remarkable.Following table 1 is center combination level, coding and experimental result.Table 2 is center combination experimental variance and interpretation of result.
Table 1:
Can find out from above-mentioned software matching gained quadratic polynomial equation, the value of described quadratic power coefficient is respectively-19.69 ,-14.17 ,-25.49 and-19.53, and para-curve is described, and Open Side Down, can obtain maximum value.Regression equation is carried out to differentiate, and in conjunction with Fig. 4, the three-dimensional response surface of Fig. 5, Fig. 6, Fig. 7 and Fig. 8, tries to achieve the maximum value of this model, X
1=1, X
2=0.74, X
3=0.2 and X
4=0.61, the actual value (g/L) of corresponding corn starch, kelp paste, glucose and manganous sulfate is respectively 4.50,4.11,2.10 and 0.62, and the maximum response of this model is, 203.38U/ml.Experimental result shows that in the process of Bacillus cereus MBRH3 fermentation product SOD of the present invention, the effect of sea-tangle is significant (P
2=0.002<0.05), this is consistent with the aimed strain of the screening taking sea-tangle as unique nutritive substance, also illustrate that this bacterial strain has certain specificity, it can be good at utilizing sea-tangle, and glucose is the energy substance that microorganism can directly utilize, this experiment also shows the exert an influence significantly (P of glucose to SOD
2=0.002<0.0165).
Table 2:
In conjunction with having reflected the relation between two kinds of nutrition of corn steep liquor and sea-tangle and the impact on product SOD shown in Fig. 4, test of significance shows P (0.0968) > 0.05, illustrate that the interaction between them is inapparent, that is to say no matter they value in high level or low-level, its enzymatic productivity all can not reach maximum value.ANOVA analyze show between corn starch and kelp paste, dependency is not remarkable between corn starch and glucose, between corn starch and manganous sulfate and between kelp paste and glucose, but, can find out from Fig. 5, Fig. 6, Fig. 7 and Fig. 8, various nutritional factor all meet normal distribution to the impact of producing SOD, illustrate that such material is utilized and meets universal law by microorganism as the raw material that produces SOD.As several Major Nutrient materials that produce SOD, only have significant interaction (p=0.035<0.05) between glucose and manganous sulfate, change when their concentration in substratum makes it reach certain level and can make enzymatic productivity reach maximum value.In product SOD process, the reason significantly that influences each other between glucose and manganous sulfate may be that glucose is in the time being utilized, can produce acid, cause the pH value of yeasting to change, the pH value of fermentation system changes the structure that also can affect the absorption of manganous sulfate or change SOD, this change utilizes glucose to exert an influence to bacterial strain again, and this also illustrates that this SOD and manganese are in close relations, belong to Mn-SOD from the side.
Specific embodiment described in the present invention is only to the explanation for example of the present invention's spirit.Those skilled in the art can make various amendments or supplement or adopt similar mode to substitute described specific embodiment, but can't depart from spirit of the present invention or surmount the defined scope of appended claims.
Although the present invention has been made a detailed description and has quoted as proof some specific embodiments, to those skilled in the art, only otherwise it is obvious leaving that the spirit and scope of the present invention can make various changes or revise.
Claims (9)
1. a Bacillus cereus MBRH3 bacterial strain, is characterized in that, described Bacillus cereus MBRH3 bacterial strain is CCTCC NO.M2013581 at the deposit number at Chinese Typical Representative culture collection center.
2. Bacillus cereus MBRH3 bacterial strain according to claim 1, is characterized in that utilizing laminaria production SOD, and the 16SrRNA sequence of described Bacillus cereus MBRH3 bacterial strain is as shown in SEQ.NO1.
3. a screening method for Bacillus cereus MBRH3 bacterial strain, is characterized in that, the method comprises the following steps:
A, get ooze sample and add in the substratum taking sea-tangle as nutritive ingredient, under 30 DEG C～40 DEG C conditions, cultivate, aimed strain is preserved, non-target microorganism is eliminated, and obtains culture;
B, get after culture obtained above dilution, be seeded on the culture medium flat plate taking sea-tangle as nutritive ingredient, after control temperature is cultivated under 30 DEG C～40 DEG C conditions, select again single bacterium colony on the new culture medium flat plate taking sea-tangle as nutritive ingredient, rule switching cultivate, obtain single bacterium colony pure growth Bacillus cereus MBRH3 bacterial strain.
4. the screening method of Bacillus cereus MBRH3 bacterial strain according to claim 3, it is characterized in that, with the weighing scale of dried seaweed material, in the substratum taking sea-tangle as nutritive ingredient described in steps A and step B, the weight percentage of sea-tangle is 2wt%～5wt%.
5. the screening method of Bacillus cereus MBRH3 bacterial strain according to claim 4, it is characterized in that, with the weighing scale of dried seaweed material, in the substratum taking sea-tangle as nutritive ingredient described in steps A and step B, the weight percentage of sea-tangle is 3wt%～4wt%.
6. according to the screening method of Bacillus cereus MBRH3 bacterial strain described in claim 3-5 any one, it is characterized in that, described ooze is bottom silt.
7. an application for Bacillus cereus MBRH3 bacterial strain as claimed in claim 1, is characterized in that, described Bacillus cereus MBRH3 bacterial strain is for the production of SOD.
8. the application of Bacillus cereus MBRH3 bacterial strain according to claim 7, it is characterized in that, Bacillus cereus MBRH3 inoculation is cultivated to the substratum that contains sea-tangle, in the time that bacterial classification OD value reaches 2.20, getting seed is inoculated in culture medium, control temperature obtains SOD after cultivating under 30 DEG C～40 DEG C conditions.
9. the application of Bacillus cereus MBRH3 bacterial strain according to claim 8, is characterized in that, described culture medium comprises the weight proportion of following composition:
MnSO
47H
2o:0.3g/L～0.7g/L, KH
2pO
4: 0.5g/L～0.8g/L, K
2hPO
4: 0.2g/L～0.5g/L, glucose: 1.0g/L～3.0g/L, corn starch: 1.0g/L～3.0g/L, extractum carnis: 1.0g/L～3.0g/L, kelp paste 1.5ml/L～3.0ml/L, pH7.0.
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| CN105670957A (en) * | 2015-01-28 | 2016-06-15 | 大韩民国(管理部署:国立水产科学院) | Sea tangle medium and manufacturing method and diagnostic method for Pseudoalteromonas sp.infection |
| RU2801527C1 (en) * | 2023-03-08 | 2023-08-10 | Иван Владимирович Матвеев | Set of oligodeoxyribonucleotide primers and fluorescently labelled probe for identification of dna of agent causing meat spoilage in vacuum packaging |
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| CN105670957A (en) * | 2015-01-28 | 2016-06-15 | 大韩民国(管理部署:国立水产科学院) | Sea tangle medium and manufacturing method and diagnostic method for Pseudoalteromonas sp.infection |
| RU2801527C1 (en) * | 2023-03-08 | 2023-08-10 | Иван Владимирович Матвеев | Set of oligodeoxyribonucleotide primers and fluorescently labelled probe for identification of dna of agent causing meat spoilage in vacuum packaging |
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