CN109652342B - Method for preparing Bt liquid fermentation culture medium by using vegetable wastes as raw materials - Google Patents
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Abstract
The invention relates to a method for preparing Bt liquid fermentation medium by using vegetable wastes as raw materials, which comprises the steps of selecting vegetable wastes and sterilizingBacteria, preparation of a fermentation medium and optimization of fermentation conditions. The invention takes the mixture of kidney bean juice and cabbage juice with the weight ratio of 2: 1 as the raw material of the fermentation medium of the bacillus thuringiensis, selects proper carbon source, nitrogen source additive and optimal metal ion by adding the nitrogen source and the carbon source and single-factor test, and determines the optimal concentration combination of the additive by utilizing orthogonal test to determine the soluble starch (6 g/L), the soybean cake powder (6 g/L), the Al and the like3+（0.4 g/L）、Fe2+(0.4 g/L) as an optimum exogenous additive. The optimized fermentation conditions are as follows: pH (7.0), temperature (35 ℃), and ventilation volume (80 mL/250 mL), and the biological activity measurement result shows that the prepared Bt strain has good insecticidal effect.
Description
Technical Field
The invention relates to a method for preparing a Bt liquid fermentation culture medium, in particular to a method for preparing the Bt liquid fermentation culture medium by taking vegetable wastes as raw materials, and belongs to the technical field of biological pesticides.
Background
Bacillus thuringiensis (B.thuringiensis) (B.thuringiensis)Bacillus thuringiensisBt) is currently a highly effective, large-dosage, safe biopesticide, which is expensive due to its relatively high cost of production. By selecting low cost fermentation raw materialAnd the culture medium is optimized by a method of biometry, so that the fermentation capacity is improved to reach or approach the effect of the original expensive culture medium.
Because the Bt culture medium has obvious influence on the toxicity of the bacterial strain, the research on the Bt fermentation culture medium is carried out all the time, and meanwhile, the wide use and popularization of the Bt bacterial strain are limited by the cost, and the research on the low-cost culture medium which ensures the normal growth of the bacterial strain and has better insecticidal effect is also emphasized.
In the research of Bt biopesticide production by monosodium glutamate wastewater in Yangjian, the supernatant of pretreated monosodium glutamate wastewater is used for preparing a culture medium, 3% of corn starch, 1% of peptone and 3% of soybean meal are added, the initial p H is 8.0 before sterilization, and shaking culture is carried out at the temperature of 30 ℃ and the speed of 200 rpm. Research on Bt liquid fermentation of Wuliyun by using beer waste as raw material comprises using 1/2 beer raw wastewater and 1/2 acidified wastewater as solutions, and adding 0.2% of glucose and (NH)4)2SO4 0.1%、ZnSO40.05%, Tween-800.15%, NaCl 0.6%, N substance 0.4%, yeast liquid 30% (V/V), KH2PO40.12 percent. The Bacillus thuringiensis produced by respectively synthesizing various culture media by utilizing hair hydrolysate, biogas slurry, municipal sludge and the like in the Tangling cloud also obviously reduces the cost of industrial raw materials and ensures the insecticidal effect of the Bacillus thuringiensis. The Wangzhui utilizes the waste liquor of vinasse to culture Bt, Zhongchong Yong, Guoailan and the like utilize industrial waste water to produce Bt, and blue encourage and the like culture Bt by fermenting alcohol with corn. The researches fully show the feasibility of preparing the Bt culture medium by utilizing waste, simultaneously can fully reduce the cost of industrial production of Bt biological pesticides, greatly improve the universality of Bt pesticides, indirectly treat waste generated in natural environment, life and industrial production operation, and effectively reduce the pollution to the environment. However, it is still the goal of market economy to further reduce the production cost of Bt biopesticides while maintaining the original biological activity of Bt.
Disclosure of Invention
The invention aims to provide a method for preparing a Bt liquid fermentation culture medium by using vegetable wastes as raw materials, which can reduce the production cost in the Bt fermentation process, and the adopted production raw materials are vegetable wastes of farmer markets.
In order to achieve the purpose of the invention, the following technical scheme is adopted.
The invention discloses a method for preparing a Bt liquid fermentation medium by using vegetable wastes as raw materials, which is characterized by comprising the following preparation steps:
(1) selecting vegetable wastes: collecting vegetable wastes of farmer market, sorting, selecting vegetable wastes of Chinese cabbage and kidney bean, cleaning, squeezing and filtering with a juicer to obtain viscous juice, centrifuging, and collecting supernatant to obtain kidney bean juice and Chinese cabbage juice;
(2) and (3) sterilization: sterilizing the kidney bean juice and the Chinese cabbage juice obtained in the step (1) at 121 ℃;
(3) preparing a fermentation medium: mixing the sterilized kidney bean juice and Chinese cabbage juice of step (2) at a mass ratio of 2: 1, adding 6 g/L soluble starch and 6 g/L, Al g soybean cake powder3+ 0.4 g/L、Fe2+ 0.4 g/L;
(4) Optimizing fermentation conditions: pH 7.0, temperature 35 ℃, aeration 80 mL/250 mL loading.
Wherein, the centrifugation in the step (1) has the centrifugation strength of 10000 r/min and the centrifugation time of 10 min.
And (3) centrifuging in the step (1) for 5 times.
The sterilization time in the step (2) is 20 min.
The invention has the advantages and benefits that:
1. the invention aims to reduce the cost of fermentation raw materials of Bt biological pesticides, and proper vegetable wastes are selected to replace the raw materials to provide partial nutrients required by normal physiological metabolism of the Bt biological pesticides.
2. Considering the importance of different carbon sources, nitrogen sources and mineral elements on the growth of the strain and the synthesis of bioactive substances, the selection of additives is comprehensively analyzed.
3. The fermentation level and toxicity of Bt are improved again by optimizing the culture medium, and technical support is provided for the mass and cheap culture of Bt strains on the premise of ensuring the insecticidal effect of Bt strains.
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FIG. 1 is a schematic illustration of the effect of different vegetable waste content on Bt growth;
FIG. 2 is a schematic diagram showing the effect of different carbon and nitrogen sources on the growth of Bt;
FIG. 3 is a schematic diagram showing the effect of different metal ions on the growth of Bt;
FIG. 4 is a schematic diagram of a visual analysis of an orthogonal test;
FIG. 5 is a schematic illustration of the effect of pH on Bt growth;
FIG. 6 is a schematic representation of the effect of temperature and media loading on Bt growth.
Detailed Description
In order to further illustrate the invention, but not to limit it, reference is made to the following examples. The experimental procedures described in the following examples are conventional unless otherwise specified.
Example 1: fermentation effect of different vegetable wastes on Bt
Collecting different kinds of vegetable wastes (tomato, Chinese cabbage, kidney bean, cabbage and spinach) in Fuzhou farmer market, sorting, cleaning, squeezing and filtering with a juicer to obtain viscous juice, centrifuging for 5 times with centrifugation strength of 10000 rpm for 10min to obtain supernatant, and sterilizing the sample at 121 deg.C for 20 min. Taking a standard strain Bt 8010, inoculating the standard strain Bt 8010 into the juice and LB liquid culture medium according to the volume ratio of 2%, performing shake culture in an incubator at 30 ℃ and 200rpm, and sampling and microscopic examination at intervals until the spore shedding amount and the content of parasporal crystals are both 50%, so as to obtain the Bt fermentation liquor. In figure 1, a) shows that kidney beans and Chinese cabbage can better promote the growth of Bt strains compared with tomatoes, spinach and cabbage, and the effect of cabbage and spinach is basically equivalent, and the tomato effect is not good enough and simultaneously more precipitates are generated by combining the experimental phenomenon, so that Chinese cabbage and kidney beans are selected in the subsequent experiment. As can be seen from b) in FIG. 1, Bt strain grows better when the ratio of kidney bean to Chinese cabbage juice is 2: 1, and the culture medium is prepared in this ratio in order to make the waste source wider.
Example 2 screening of various carbon, nitrogen and Metal ion additives
4 carbon sources (glucose, sucrose, soluble starch and mannitol) and 4 nitrogen sources (tryptone, corn flour, urea and soybean cake powder) are respectively added into the pretreated vegetable waste culture medium, the concentration is 6 g/L, and the concentrations are respectively compared with the LB culture medium group. The results in FIG. 2 show that, for carbon source additives, mannitol can not obviously promote the growth of the strains, the other three differences are relatively small, meanwhile, the promotion effect of soluble starch on the growth of the strains is optimal, and soluble starch, namely corn starch, is selected as the carbon source additive; the nitrogen source additive is small in promoting effect of urea, which is probably related to the inorganic nitrogen source, the tryptone has the best effect, and then the soybean cake powder is small in difference and has a good effect of promoting the growth of the strain. In addition, it was found that it is difficult to achieve a comparable or superior promoting effect by adding a carbon source or a nitrogen source alone as compared with a control group fermented in an LB medium, and therefore, it is necessary to consider that the effect of co-promoting the growth of the strain is increased by adding carbon source and nitrogen source additives simultaneously.
Adding 4 kinds of common metal ions (Mg) into the pretreated vegetable waste culture medium respectively on the basis of adding a carbon source and a nitrogen source2+、Fe2+、Al3+、Na+) The amounts added were 1.2 g/L, and they were compared with LB medium group. The results in FIG. 3 show that: the strain growth effect of the culture medium added with ferrous ions is optimal, the fermentation level is close to that of the LB culture medium, the promotion effect of the strain is obviously better than that of the rest metal ions, and aluminum ions are added, so the ferrous ions and the aluminum ions are selected as the metal ions.
Example 3 optimal combination of carbon, nitrogen, Metal ion additions (orthogonal test)
On the basis of an initial culture medium (including exogenous additives), in order to improve the fermentation level again, the culture medium needs to be optimized again, and a four-factor three-level orthogonal test is utilized to add a carbon source (soluble starch), a nitrogen source (soybean cake powder) and metal ions (ferrous ions and aluminum ions) into the pretreated vegetable waste culture medium according to different proportions, so as to screen out the optimal combination of the carbon source (soluble starch), the nitrogen source (soybean cake powder) and the metal ions.
Table 1 shows the design of the concentration levels of carbon source, nitrogen source, metal ions, and their orthogonal experiments were designed using the statistical software SPSS17, as shown in table 2. The results show that: analysis of variance showed soluble starch and soy cake flour (sig value)<0.05), which shows that the influence of the soluble starch and the soybean cake powder is obvious. Using Duncan's test and visual analysis (FIG. 4), the results show that the optimal combination selected for the four factors is A3B3C2D3(glucose 6 g/L, soybean cake powder 6 g/L, ferrous ion 0.4 g/L, aluminum ion 0.4 g/L). Meanwhile, the screened optimal culture medium and LB culture medium have equivalent fermentation levels, and compared with other 3 culture media selected randomly, the optimal culture medium has the function of obviously promoting the growth of the strain.
TABLE 1 orthogonal test factors and horizontal design
TABLE 2 results of orthogonal experimental design of media additives
Example 4 Effect of different fermentation conditions on Bt growth
Because the pH has obvious influence on the growth and metabolism of the microorganisms, the indirect influence of the pH of the culture medium on the growth of the microorganisms is controlled by adjusting the initial pH value of the culture medium on the basis of the pH adjustment of the microorganisms to the environment within a certain range. The experimental medium was set at different initial pH values, and the optimal initial pH was determined by its growth conditions to better promote the fermentative growth of the microorganisms. FIG. 5 shows that the growth of the bacteria is inhibited by the acidic and alkaline living environment with the optimal initial pH range of 7.0-7.5, especially under the fermentation conditions with the initial pH lower than 6.5 and higher than 8.0.
Meanwhile, the fermentation temperature in a certain range plays a decisive role in the metabolic growth speed of microorganisms, the synthesis of substances and the energy generation, so that the growth and metabolic speed of the microorganisms can be regulated by controlling the fermentation temperature, and meanwhile, the toxicity of the microorganisms can be influenced. Too low or too high a temperature not only results in low fermentation levels but may also cause damage or death of the microorganisms. FIG. 6 shows that the growth rate of bacteria is fastest at 35 ℃, and the growth rate is accelerated at 25-35 ℃ compared with other temperatures.
In addition, Bt bacteria are aerobic bacteria, the ventilation of the culture medium can ensure the normal growth and metabolism of the Bt bacteria, and the influence of the ventilation on the growth of the bacterial strains is researched by controlling the loading of the bacterial liquid in a 250 mL conical flask. FIG. 6 shows that too low ventilation has an inhibitory effect on the growth of the strain, since the strain grows better when the charge is 80 mL/250 mL due to the presence of sufficient oxygen.
Example 5 results of bioactivity assay
Through biological activity determination (table 3), the Bt 8010 strain fermented by the vegetable waste culture medium has good insecticidal effect, the result after 48 hours shows that the insecticidal rates of the original solution and the bacterial solution with the dilution multiple of 5 reach 100%, the insecticidal effects of the Bt strain fermented by comparing the laboratory standard culture medium (10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of sodium chloride, and pH = 7.0) are equivalent, the bacterial solution of the vegetable waste culture medium still has good insecticidal effect (80%) after being diluted to 20 times, and the mortality reaches 56.7% when the dilution multiple reaches 50 times. Bacterial liquid LC50=16.783 μ L/mL of the standard LB culture medium, and bacterial liquid LC50=17.420 μ L/mL of the vegetable waste show that the insecticidal effects of the two are close to each other.
TABLE 3 bioassay results for Cotton bollworm incipient hatch larvae
Claims (4)
1. A method for preparing a Bt liquid fermentation culture medium by using vegetable wastes as raw materials is characterized by comprising the following preparation steps:
(1) selecting vegetable wastes: collecting vegetable wastes of farmer market, sorting, selecting vegetable wastes of Chinese cabbage and kidney bean, cleaning, squeezing and filtering with a juicer to obtain viscous juice, centrifuging, and collecting supernatant to obtain kidney bean juice and Chinese cabbage juice;
(2) and (3) sterilization: sterilizing the kidney bean juice and the Chinese cabbage juice obtained in the step (1) at 121 ℃;
(3) preparing a fermentation medium: mixing the sterilized kidney bean juice and Chinese cabbage juice of step (2) at a mass ratio of 2: 1, adding 6 g/L soluble starch and 6 g/L, Al g soybean cake powder3+ 0.4 g/L、Fe2+ 0.4 g/L；
(4) Optimizing fermentation conditions: pH 7.0, temperature 35 ℃, and ventilation volume 80 mL/250 mL;
bt is bacillus thuringiensisBacillus thuringiensis。
2. The method for preparing Bt liquid fermentation medium by using vegetable waste as raw material according to claim 1, wherein the centrifugation in the step (1) is performed, the centrifugation strength is 10000 r/min, and the centrifugation time is 10 min.
3. The method for preparing the Bt liquid fermentation medium by using the vegetable wastes as the raw materials according to the claim 1, wherein the centrifugation in the step (1) is repeated for 5 times.
4. The method for preparing Bt liquid fermentation medium by using vegetable waste as raw material according to claim 1, wherein the sterilization time in the step (2) is 20 min.
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