CN103146613A - Fermentation culture method for bacillus subtilis PTS-394 - Google Patents
Fermentation culture method for bacillus subtilis PTS-394 Download PDFInfo
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Abstract
The invention relates to a fermentation culture method for bacillus subtilis PTS-394. The fermentation culture method comprises the following steps of: inoculating a bacillus subtilis PTS-394 seed solution in a fermentation culture medium and carrying out fermentation culture for two days on a shaking table. The fermentation culture method is characterized in that a carbon source in the fermentation culture medium is corn starch, and a nitrogen source is soybean powder; three key factors affecting bacterial content predicated value are corn starch, soybean powder and yeast powder; the content of the three key factors in the fermentation culture medium is 11.48g/L, 13.35g/L and 0.90g/L, respectively; three key factors affecting the bacterial content and the inhibition zone diameter in the fermentation culture conditions are temperature, an initial pH value and liquid filling amount; and in the fermentation culture process, the temperature is 30.84 DEG C, the initial pH value is 6.93 and the liquid filling amount is 65.15mL/250mL.
Description
Technical field
The present invention relates to the fermentation culture method of a kind of subtilis PTS-394, especially a kind of Plackett-Burman method and Response Surface Method utilized optimized the cultural method of realizing after fermention medium and culture condition subtilis PTS-394.
Background technology
Along with the adjustment of crop mix, the facilities vegetable cultivated area increases year by year, and the facilities vegetable planting structure distribution of high yield, continuous cropping provides abundant nutrition and Reproduction Conditions for disease and pest.Facility solanaceous vegetables soil-borne disease (bacterial wilt, epidemic disease, root knot nematode etc.) is frequently broken out in recent years.Bacterial wilt is that a kind of crushing soil passes Micobial Disease, still keeps green though the their early stage over-ground part shows as the vertical blade that withers, therefore named " blue or green withered ".Bacterial wilt is to endanger in the world maximum, the widest, one of the most serious Plant diseases that causes damage of distribution, is called as " cancer " of plant.Be subjected to the harm of bacterial wilt the most serious with plants of Solanaceae such as tomato, capsicums in China staple crops.The sickness rate of general field is 10%-15%, and the sickness rate in grave illness field is up to 80%-100%.
At present, in production, facility for prevention and control solanaceous vegetables bacterial wilt mainly relies on chemical pesticide.This not only causes the ecotope severe contamination, and directly increases the residual of toxic chemical substance in vegetables, and human health is brought serious harm.In order to solve vegetable crop and quality contradiction, reach the purpose of the usage quantity of controlling plant diseases, minimizing chemical pesticide, the method for using biological control to control Plant diseases more and more receives national governments and the people's concern.
The screening of Institute of Plant Protection, academy of agricultural sciences, Jiangsu Province is that obtain, subtilis PTS-394 that the solanaceous vegetables bacterial wilt is had fine preventive effect, and has applied for that on November 1st, 2011 patent, publication number are CN102550607A.Wherein disclosed morphological specificity is recited as: the bacterial strain nourishing body is shaft-like, and two ends are oval, peritrichous.Size be (0.7-0.8) micron * (2.0-3.0) micron.Without pod membrane.Give birth in gemma, ellipse, size be (0.6-0.9) micron * (1.0-1.5) micron.When subtilis PTS-394 cultivates on beef broth peptone agar plate, the colony edge waviness, cream-coloured, there is gauffer on the surface.Quality is opaque shape, not chromogenesis.Gram-reaction is positive.H
2O
2Enzyme reaction is positive, the V.P. reacting positive.L-arabinose, D-Glucose, D-wood sugar, PEARLITOL 25C are utilized as the positive.Starch Hydrolysis, gelatine liquefication, Trisodium Citrate utilization, otan form and react also all positive.In addition, subtilis PTS-394 also has many premium propertiess, as: wider antimicrobial spectrum is arranged, the various plants pathogenic bacteria is had very strong restraining effect; Fast growth, accommodative ability of environment is strong, can surely grow on the soil and plant surface, and form dominant population; Induce the host to produce disease resistance; Secretion produces multiple antimicrobial substance.At present, the biological company limited in the green source of Jiangsu Province's Yangzhou has produced
20,000,000,000Subtilis PTS-394 viable bacteria aqua.
Bacterial content in subtilis PTS-394 active bacteria formulation is important technology and the economic target of product.Improve bacterial content in fermented liquid, just must be optimized its fermentation culture based component and proportioning, culture condition parameters.
In recent years, the researchist utilizes Plackett-Burman (PB) experimental design method can filter out fast the key factor that target value is had the greatest impact from influence factor, and can point out direction and change step to be widely used in the screening of fermentation culture medium for microbe composition and processing parameter for the climbing experiment.Response Surface Method (Response Surface Method, RSM) is the strong and simple and easy reliable statistics experimental design optimization method of a kind of suitability, is a kind of complex art of optimizing bioprocess.Domestic existing many reports about adopting Response Surface Method that fermentation process is optimized.
At present, utilize that Plackett-Burman method and Response Surface Method optimization obtains in subtilis PTS-394 zymocyte liquid, the viable bacteria amount as fermention medium and the culture condition of purpose, is not seen in report to improve.
Summary of the invention
The object of the invention is to: for the lower practical problems of bacterial content in present subtilis PTS-394 active bacteria formulation, provide a kind of based on traditional subtilis PTS-394 fermentation culture method, wherein fermention medium and culture condition is optimized, improves bacterial content in fermented liquid.
the object of the present invention is achieved like this: the fermentation culture method of a kind of subtilis PTS-394, be included in inoculation subtilis PTS-394 seed liquor in fermention medium, cultivate 2d in the shaking table top fermentation, it is characterized in that: the carbon source in fermention medium is W-Gum, nitrogenous source is soyflour, three key factors that affect the bacterial content predictor are W-Gum, soyflour and yeast powder, three key factor content in fermention medium are followed successively by 11.48 g/L, 13.35 g/L and 0.90 g/L, three key factors that affect bacterial content and antibacterial circle diameter in fermentation culture conditions are temperature, initial p H value and liquid amount, they are selected in the fermentation culture process: 30.84 ℃ of temperature, initial p H 6.93, liquid amount 65.15 mL/250 mL.。
In the present invention: the component of fermention medium is: W-Gum 11.48 g/L, soyflour 13.35 g/L and yeast powder 0.90 g/L, CaCO3 3 g, NaCl 0.5 g, fish meal 10 g; The liquid amount of fermention medium is 65.15 mL/250 mL, and in fermention medium, after the seed liquor of inoculation subtilis PTS-394, initial pH 6.93 is placed in 30.84 ℃, and shaker fermentation is cultivated.
In the present invention: fermentation culture 2d, the bacterial content in fermented liquid are (6.38 ± 0.16) E+10 cfu/mL, are 32.12 ± 0.08 mm to the ralstonia solanacearum antibacterial circle diameter.
The invention has the advantages that: be important technology and the economic target of product due to the effective bacterial content in subtilis PTS-394 active bacteria formulation.The present invention utilizes Optimal Medium component and proportioning and the culture condition after improvement, improves bacterial content in subtilis PTS-394 zymocyte liquid, for subtilis PTS-394 high-efficiency prevention and control bacterial wilt provides theoretical foundation.
Embodiment
Embodiment 1 determines culture medium carbon source, nitrogenous source
The basic fermention medium of subtilis PTS-394 (being that in the patent application of CN102550607A, embodiment 3 is open by publication number): Semen Maydis powder 20 g, soybean cake powder 10 g, soyflour 5 g, yeast powder 1.5 g, CaCO
33 g, NaCl 0.5 g, fish meal 10 g, moisturizing to 1000 mL, pH 7.5.
With 20 g/L Semen Maydis powder of above-mentioned basic fermention medium with sucrose, Zulkovsky starch, W-Gum, maltose, lactose, 6 kinds of different carbon source equivalent of wood sugar (20 g/L) are replaced, and are made into the substratum of different carbon sources.With 10 g/L soybean cake powder of basic fermention medium with soyflour, Tryptones, yeast soaks powder, extractum carnis, peptone, 6 kinds of different nitrogen sources equivalent of fish meal (10 g/L) are replaced, and are made into the substratum of different nitrogen sources.
The medium pH of different carbon and nitrogen sources is transferred neutral rear packing, and liquid amount is 100 mL/250 mL triangular flasks, sterilization.After 2% inoculum size inoculation subtilis PTS-394 seed liquor (bacterial content is as 2.11E+10 cfu/mL), be placed in 28 ℃, shake training 48 h in the shaking table of 150 rpm, adopt plate dilution method to detect the fermented liquid bacterial content, 3 repetitions are established in each processing, average.Carbon source, nitrogenous source shaker test the results are shown in Table 1.
The impact on PTS-394 fermented liquid bacterial content of the different carbon sources of table 1, nitrogenous source
Result shows: use W-Gum to be carbon source, the fermented liquid bacterial content is maximum, so W-Gum is the optimum carbon source of subtilis PTS-394 fermentation culture.Use soyflour to be only nitrogen source, the fermented liquid bacterial content is maximum, so soyflour is the optimum nitrogen source of subtilis PTS-394 fermentation culture.
The basic fermention medium of PTS-394 is adjusted into W-Gum 20g, soyflour 15g, yeast powder 1.5g, CaCO
33 g, NaCl 0.5g, fish meal 10g, moisturizing is to 1000mL, and pH7.5 is defined as initial fermention medium (abbreviation initial medium).
Embodiment 2 determines the key factor of fermentation culture based component
Adopt the Plackett-Burman experimental design, subtilis PTS-394 medium component (W-Gum, soyflour, yeast powder, CaCO from embodiment 1
3, NaCl, fish meal) and filter out key factor in 6 factors.The PB experimental design is as shown in table 2:
The Plackett-Burman experimental design of the table 2 medium component factor and result
Annotate: in experiment, each factor is got two levels, and high level " 1 " represents that this component content is 1.5 times of initial fermention medium; Low-level " 1 " represents that this component content is 0.5 times of initial fermention medium.
His-and-hers watches 2 data are carried out regression analysis, and the equation of gained Main Factors coding is:
Y 0 =9.47+0.12A+0.055B-0.032C+0.020D+9.095E-003E-0.012F
In formula:
Y 0 Be the predictor (cfu/mL) (lower same) of bacterial content, the dependency of this equation is
R 0 2 =0.9440.
Culture factors is carried out the Plackett-Burman variance analysis, the results are shown in Table 3.6 factors of medium component have W-Gum (A) significantly on α=0.05 level, soyflour (B), and conspicuous level is respectively 99.95% and 98.34%.Illustrate that being changed significantly of these 2 factors affects subtilis PTS-394 fermented liquid bacterial content.The conspicuous level of yeast powder is 90.70%, and the fermented liquid bacterial content is also had impact more significantly.Therefore determine that W-Gum, soyflour, 3 factors of yeast powder are to affect the key factor of bacterial content in medium component.
The variance analysis of each factor of table 3 substratum Plackett-Burman experimental design
The optimal proportion of embodiment 3 fermention medium key factors
Analyze the PB experimental result as can be known, W-Gum, 3 factors of soyflour and yeast powder are for affecting the key factor of subtilis PTS-394 bacterial content, according to fit equation
Y 0 Each variation coefficient is determined climbing direction and the step-length of key factor, carries out steepest climbing experiment.Climbing experimental design and the results are shown in Table 4:
The climbing experimental design of table 4 medium component key factor steepest and result
As can be seen from Table 4, in the variation of 3 factors combination, under the combination of numbering 4, bacterial content is maximum, therefore selects this combination as the central value of response curved surface experiment, carries out next step RSM experimental design.
Determined each horizontal value of Response Surface Method experimental design according to steepest climbing experimental result (table 4), the Response Surface Method experimental design the results are shown in Table 5:
Table 5 Response Surface Method is determined the best proportioning of medium component key factor
Annotate: in experiment, each factor is got two levels, and high level " 1 " represents that this component content is 1.5 times of No. 4 combinations in table 4 (central value of response curved surface experiment); Low-level " 1 " represents that this component content is 0.5 times of No. 4 combinations in table 4 (central value of response curved surface experiment).
Adopt Design-Expert 8.0 softwares to carry out variance analysis, carry out the multinomial regression fit of secondary, the dynamic response model that draws bacterial content and antibacterial circle diameter is respectively:
Y 1 =9.61+0.13A+0.046B-0.025C-0.007AB+0.013AC+0.011BC-0.16A
2-0.044B
2-0.084C
2
The dependency of this equation is
R 1 2 =0.9709.
Y 2 =8.18+0.45A-0.092B-0.22C-0.16AB+0.30AC+0.065BC-1.06A
2-0.39B
2-0.48C
2
The dependency of this equation is
R 2 2 =0.9786.
In formula,
Y 1 For same under bacterial content cfu/mL(),
Y 2 For same under antibacterial circle diameter mm().
Respond the curved surface canonical parse as can be known, there is maximum value in regression model: as W-Gum 11.48 g/L, soyflour 13.35 g/L and yeast powder 0.90 g/L.This moment, the bacterial content predictor was 4.43 E+10 cfu/mL, and antibacterial circle diameter is 26.20 mm.Therefore, the optimization fermention medium of subtilis PTS-394 (abbreviation Optimal Medium) optimal proportion is: W-Gum 11.48 g/L, soyflour 13.35g/L and yeast powder 0.90g/L, CaCO
33g, NaCl 0.5g, fish meal 10g.
The compliance test result of embodiment 4 Optimal Mediums
Carry out respectively the shake flask fermentation experiment with the initial medium of embodiment 1 acquisition and the Optimal Medium of embodiment 3 model predictions, method steps is all the part of embodiment 1 in the patent application of CN102550607A with reference to publication number, result is table 6, shown by table 6, initial medium and Optimal Medium fermented liquid bacterial content and antibacterial circle diameter measured value and model predication value all without significant difference, have verified that this model has higher degree of fitting on α=0.05 level; Optimal Medium is compared with initial medium, and its bacterial content and bacteriostatic activity have improved respectively 104.7% and 29.2%, has shown effect of optimization preferably.
The confirmatory experiment of table 6 Optimal Medium
Annotate: different letter representations significant difference on 0.05 level after each row of data.
Embodiment 5 determines the key factor of subtilis PTS-394 fermentation culture conditions
According to the normal fermentation condition, set the span of the culture condition factor: temperature (28 ℃-35 ℃), pH(6.5-8.0), inoculum size (0.1%-3%), liquid amount (30-100mL/250mL), incubation time (24-60h), rotating speed (130-180rpm), cell age (12-36h).Adopt the PB experimental design to screen from above-mentioned 7 factors bacterial strain PTS-394 is grown and have the Factors of remarkably influenced.In experiment, each factor is got two levels, i.e. high level " 1 " and low-level " 1 ".Experimental design and result are as shown in table 7, and his-and-hers watches 7 data are carried out regression analysis, and the gained equation is:
Y 0 =9.68+0.054A-0.020B-0.015E, the dependency of this equation is
R 0 2 =0.9755.
The results of analysis of variance (table 8) shows: in 7 factors on α=0.05 level the significant factor culture temperature (A) is arranged, initial pH(B) and liquid amount (E), its conspicuous level is respectively 0.0003,0.0141 and 0.0328, illustrates that being changed significantly of these 3 factors affects zymophyte content.
The Plackett-Burman experimental design of the table 7 culture condition factor and result
Annotate: in experiment, each factor is got two levels, and high level " 1 " represents the maximum in this factor span; Low-level " 1 " represents the Schwellenwert in this factor span.
The variance analysis of table 8 culture condition factor Plackett-Burman experimental design
Embodiment 6 determines subtilis PTS-394 optimal culture condition
Take the PB experiment analysis results as foundation, the experiment of climbing, climbing experimental design and the results are shown in Table 9, by finding out in table, the bacterial content during conditional combination that numbering 3 is processed is maximum, and therefore selecting this is the central value of response curved surface experiment, carries out next step experiment.
The climbing experimental design of table 9 culture condition key factor steepest and result
Determined each horizontal value of Response Surface Method experimental design according to steepest climbing experimental result, the Response Surface Method experimental design the results are shown in Table 10.Adopt Design-Expert 8.0 softwares to carry out variance analysis, carry out the multinomial regression fit of secondary, the dynamic response model that draws bacterial content and antibacterial circle diameter is respectively:
Y 1 (bacterial content)=9.79+0.040A-0.021B-0.016C+0.011AB+5.87E-003AC-4.45E-003B C-
0.058A
2-0.029B
2-6.84E-003C
2
The dependency of this equation is
R 1 2 =0.9853.
Y 2 (antibacterial circle diameter)=8.18+0.45A-0.092B-0.22C-0.16AB+0.30AC+0.065BC-1.06A
2-0.39B
2-0.48C
2
The dependency of this equation is
R 2 2 =0.9691.
Through response curved surface canonical parse as can be known, there is maximum value in regression model.When 3 key factors are respectively 30.84 ℃ of temperature, pH 6.93 and liquid amount 65.15 mL/250mL, other factors are: 2% inoculum size, 150 rpm, when incubation time is 48 h, the predictor of fermented liquid bacterial content and antibacterial circle diameter is 6.32 E+10 cfu/mL and 32.24 mm.
Table 10 Response Surface Method is determined the numerical value of culture condition key factor
Embodiment 7 Optimal Mediums are in the shake flask fermentation checking of optimizing under fermentation culture conditions
the Optimal Medium of the model prediction that provides with embodiment 3 carries out grouping experiment, one group is adopted initial fermentation culture conditions, its method steps is the part of embodiment 1 in the patent application of CN102550607A with reference to publication number, another group is for optimizing fermentation culture conditions, in the patent application of its method steps take publication number as CN102550607A, embodiment 1 part is the basis, only change 3 key factor temperature wherein, pH value and liquid amount, be 30.84 ℃ of temperature, pH 6.93, liquid amount 65.15 mL/250mL, two groups are carried out respectively the shake flask fermentation experiment, result is table 11, shown by table 11, initial fermentation culture conditions and optimize fermentation culture conditions dual mode fermented liquid bacterial content and bacteriostatic activity measured value and model predication value on α=0.05 level all without significant difference, verified that this model has higher degree of fitting, fermentation culture conditions after optimization is compared with initial fermentation culture conditions, and its bacterial content and antibacterial circle diameter have improved respectively 39.2% and 20.9%, has shown effect of optimization preferably.
The confirmatory experiment of table 11 optimum culture condition
Annotate: different letter representations significant difference on 0.05 level after each row of data.
Above each embodiment is not to concrete restriction of the present invention; as long as in the fermentation culture process of subtilis PTS-394; fermention medium adopts three the key factor restricted portions that affect the bacterial content predictor that relate in claim; and adopt three the key factor restricted portions that affect bacterial content and antibacterial circle diameter that relate in claim in fermentation culture conditions, all belong to protection scope of the present invention.
Claims (3)
1. the fermentation culture method of subtilis PTS-394, be included in inoculation subtilis PTS-394 seed liquor in fermention medium, cultivate 2d in the shaking table top fermentation, it is characterized in that: the carbon source in fermention medium is W-Gum, nitrogenous source is soyflour, three key factors that affect the bacterial content predictor are W-Gum, soyflour and yeast powder, three key factor content in fermention medium are followed successively by 11.48 g/L, 13.35 g/L and 0.90 g/L, three key factors that affect bacterial content and antibacterial circle diameter in fermentation culture conditions are temperature, initial p H value and liquid amount, they are selected in the fermentation culture process: 30.84 ℃ of temperature, initial p H 6.93, liquid amount 65.15 mL/250 mL.
2. the fermentation culture method of subtilis PTS-394 according to claim 1, it is characterized in that: the component of fermention medium is: W-Gum 11.48 g/L, soyflour 13.35 g/L and yeast powder 0.90 g/L, CaCO
33 g, NaCl 0.5 g, fish meal 10 g; The liquid amount of fermention medium is 65.15 mL/250 mL, and in fermention medium, after the seed liquor of inoculation subtilis PTS-394, initial pH 6.93 is placed in 30.84 ℃, and shaker fermentation is cultivated.
3. the fermentation culture method of subtilis PTS-394 according to claim 1 and 2, it is characterized in that: fermentation culture 2d, bacterial content in fermented liquid is (6.38 ± 0.16) E+10 cfu/mL, is 32.12 ± 0.08 mm to the ralstonia solanacearum antibacterial circle diameter.
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