TWI381049B - Liquid medium for bacillus subtilis and method for manufacturing of the liquid medium - Google Patents

Description

Liquid medium for cultivating Bacillus subtilis and preparation method thereof

The invention relates to a microbial liquid medium and a preparation method thereof, in particular to a microbial liquid medium for cultivating bacteria to grow rapidly and capable of increasing the amount of bacteria per unit volume, and a preparation method thereof, wherein the microbial liquid medium can be applied to A microorganism for microbial pesticides, such as Bacillus subtilis , is cultivated.

In general, crop cultivation is used to control pests and diseases. In addition to the application of general chemical pesticides, the concept of using microbial pesticides has also become popular. Microbial pesticides make products against microorganisms or metabolites of microorganisms against crop pathogens, pests, weed control or induced crops, and their microbial sources include bacteria, fungi, viruses and protozoa.

Among the microbial pesticides, Bacillus subtilis has the greatest potential for biological control. The application of Bacillus subtilis is extensive, including soil diseases, leaf surface diseases (such as bean rust), and storage period diseases (such as peach brown rot). In addition to disease, citrus blue mold, in addition to the root development of a variety of crops, there is also a considerable promotion.

A liquid medium for cultivating microorganisms in a general microbial pesticide contains a carbon source, a nitrogen source, an inorganic salt, and water. However, it is necessary to adjust a carbon source and a nitrogen source suitable for the microorganism according to different strains. Please refer to the Patent No. 302942 of the Republic of China Patent No. 302942, the use of its plant disease control agent and pest control agent, and the method for controlling the same, and the medium composition for cultivating the bacterium D747 in the patent case includes: 1% glucose, 2% starch, 0.5% polypeptone, 1% dry yeast, 1% defatted soybean, 0.2% potassium phosphate, 0.2% sodium chloride and 0.3% calcium carbonate. The culture of the bacterium D747 was slow to grow using the medium. After three days of cultivation, the microbial pesticide can be prepared by centrifugation, so that the amount of bacteria containing the bacterium D747 in the microbial pesticide reaches 10 ⁹ cfu/ml, and the microbial pesticide can be applied to the field. Please refer to Patent Publication No. 200724676, "Production Technology of Bacillus subtilis WG6-14 and Its Applicability". The medium composition of Bacillus subtilis WG6-14 contains fishmeal powder, soy flour, sugar, phosphorus and sulfur. Such mineral elements, yeast powder, seaweed powder, milk powder, bitter lime, etc., using the medium to culture the Bacillus subtilis WG6-14 for more than three days, the amount of bacteria can reach 10 ¹⁰ cfu / ml. The two patents have a complicated culture medium for cultivating Bacillus subtilis, and the cost of the composition is also high, and the growth rate of the bacteria is slow, and the amount of bacteria contained in the unit volume is not high in a certain period of time.

Please refer to Patent No. I280852, "Method for Producing Bacillus Subtilis Biocide or Soil Modifier". The microbial pesticide is prepared as follows: a medium containing: starch, protein, molasses, yeast powder, corn soaking solution , (NH ₄ ) ₂ SO ₄ , K ₂ HPO ₄ , MgSO ₄ ‧7H ₂ O, CaCl ₂ ‧2H ₂ O, and MnSO ₄ ‧H ₂ O, etc., after culturing a Bacillus subtilis for fermentation and enrichment It is concentrated in a vacuum and dried to obtain a Bacillus subtilis pesticide. Wherein, since the amount of bacteria obtained in the medium is insufficient for direct application, the bacterial liquid must be vacuum-concentrated in a vacuum concentrator, the degree of vacuum is at least 0.5 atm (atm), and the boiling point of the bacterial liquid falls below 60 ° C. The condition for concentrating the bacterial solution in a vacuum is preferably a vacuum of 0.01 atm and a boiling point of 35 ° C or less, and then adding the other additives (acid or lye) to the concentrated bacteria, or concentrating in a vacuum to be sprayed. The drying mechanism can completely remove the water and make the powder to achieve the amount of 10 ⁹ cfu/ml. This step requires more expensive equipment and a longer preparation time to concentrate the bacterial liquid contained in the medium. In order to improve the efficacy of the fungicide.

Therefore, in order to improve the problem of insufficient bacteria in the preparation of the microbial pesticide, the step of concentrating the microbial liquid of the microbial pesticide can be started. Therefore, the present invention improves the microbial liquid medium and the preparation method thereof to increase the growth rate of the microorganism and increase one unit. The amount of bacteria contained in the volume of the bacterial liquid can be used to improve the concentration of the bacterial liquid in the production of the microbial pesticide to reduce the cost and reduce the processing time.

The main object of the present invention is to provide a microorganism in a liquid medium culture medium capable of rapidly growing by the liquid medium of the microorganism, and the amount of bacteria per unit volume obtained is high in a certain period of time.

A secondary object of the present invention is to provide a method for preparing a microbial liquid medium, which is simple in operation, and the microbial liquid medium is convenient to manufacture.

In order to achieve the foregoing object, the technical means used in the present invention comprises: a microbial liquid medium comprising a carbon source of 6-16% by weight, a nitrogen source of 0.2-0.6%, a fatty acid of 0.3-0.7%, 0.3 ~0.7% rice bran powder and 82.0~93.2% rice bran water.

The invention relates to a method for preparing a microbial liquid medium, which comprises the steps of preparing a rice water, wherein the water is mixed with rice bran, wherein the rice bran accounts for 9.0 to 13.7% of the total weight of water and rice bran, and the rice bran residue is removed after heating and stirring for at least one hour. One meter of water; one mixing step, taking 6-16% by weight of carbon source, 0.2~0.6% of nitrogen source, 0.3~0.7% of fatty acid, 0.3~0.7% of rice bran powder and 82~93.2% After the rice bran water is uniformly mixed, a mixed liquid is obtained; and in a sterilization step, the mixed solution is subjected to a high temperature autoclave operation to obtain a microbial liquid medium.

The above and other objects, features, and advantages of the present invention will become more apparent from the description of the preferred embodiments of the invention. The microbial pesticide prepared by the microbial liquid medium, wherein the microbial liquid medium can promote the growth of the microorganism, and maintain a high concentration of the bacteria, and the preparation time of the microbial pesticide prepared by the microbial liquid medium is short and can reach a high level. The amount of bacteria in the concentration.

The microbial liquid medium of the present invention can be used for preparing a microbial liquid medium for microbial pesticides, and the strain having biological control ability, in particular, a Bacillus subtilis , is cultured in the microbial liquid medium of the present invention to obtain a bacterial liquid, and The microbial liquid culture medium of the invention can make the bacteria grow faster, and the amount of bacteria per unit volume is high in a certain period of time, and the bacteria volume is high when the culture is completed, and the bacterial liquid can be directly applied as a microbial pesticide, the microbial pesticide The lyophilization or decompression concentration instrument does not need to be concentrated to remove the bacterial liquid, which can save the production cost and process time of the microbial pesticide.

A microbial liquid culture medium of the present invention comprises a carbon source of 6 to 16% by weight, a nitrogen source of 0.2 to 0.6%, a fatty acid of 0.3 to 0.7%, a rice bran powder of 0.3 to 0.7%, and an 82. ~93.2% rice bran water is evenly mixed and prepared.

Please refer to Table 1, and further, the carbon source of the microbial liquid medium of the present embodiment is selected from the group consisting of Sucrose, Molasses and Dextrose as the main growth of microorganisms. The nutrient source is preferably 2 to 5% sucrose, 1 to 5% molasses and 3 to 6% glucose in this embodiment.

The nitrogen source of the microbial liquid medium of the present embodiment is selected from 0.2 to 0.6% beef extract or yeast extract to provide basic nutrients for the growth of microorganisms, and the best beef extract.

The fatty acid of the microbial liquid medium of the present embodiment is selected to be a 0.3-0.7% edible oil, and the fatty acid is also a basic nutrient for providing microbial growth, wherein the edible oil may be selected from bitter tea oil, flax oil, white. An edible oil which is commercially available, such as sesame oil, olive oil, canola oil, palm oil, peanut oil, sunflower oil, corn oil and coconut oil. For example, the edible oil of the present embodiment is preferably bitter tea oil or flax oil. .

In addition, the rice bran powder and the rice bran water system provided by the invention provide a trace element and a nutrient component required by a microorganism, and the basic essential nutrients during the growth of the microorganism are a carbon source and a nitrogen source, such as only a carbon source and a nitrogen source. The prepared microbial liquid culture medium can still grow microorganisms by culturing the microorganisms, but the medium prepared by adding the rice bran powder and the rice bran water according to the invention obviously has the effect of enhancing the growth of the bacteria, so as to improve the microorganisms. Growth rate, increase the amount of bacteria per unit volume.

Referring to FIG. 1, the method for preparing the microbial liquid medium of the present invention comprises: a rice water preparation step S1, a preparation step S2, and a sterilization step S3.

The rice bran water preparation step S1 is a mixture of water and rice bran, wherein the rice bran accounts for 9.0 to 13.7% of the total weight of water and rice bran, and after heating and stirring for at least one hour, the rice bran residue is removed to obtain one meter of water. For example, the rice bran water of the present embodiment is preferably taken with an appropriate volume of water, and the water is boiled and poured into a suitable amount of rice bran, wherein the ratio of the rice bran to water is 1:10, heating and stirring for at least After 1 hour, the rice bran residue was removed and one meter of water was obtained.

The mixing step S2 takes a carbon source of 6-16% by weight, a nitrogen source of 0.2-0.6%, a fatty acid of 0.3-0.7%, a rice bran powder of 0.3-0.7%, and an 82-92. After the rice bran water is uniformly mixed, a mixed liquid is obtained. For example, in this embodiment, the weight percentage is 2 to 5% sucrose, 1 to 5% molasses, 3 to 6% glucose, 0.2 to 0.6% beef extract, 0.3 to 0.7% rice bran powder, and 0.3 to 0.7. % edible oil is added to the rice bran water. The carbon source is sucrose, molasses and glucose; the nitrogen source is beef extract; the fatty acid is an edible oil, and one rice bran powder is added, and the above components are mixed and prepared with rice bran water. Wherein, the edible oil may be selected from the group consisting of bitter tea oil, flax oil, white sesame oil, olive oil, canola oil, palm oil, peanut oil, sunflower oil, corn oil and coconut oil, and the like, for example, The edible oil of the present embodiment is preferably bitter tea oil or flax oil.

In the sterilization step S3, the mixture is subjected to a high temperature autoclave operation to obtain a microbial liquid medium. In this embodiment, the temperature is set to 121 ° C in a high temperature autoclave, the pressure is 1.1 kg / cm ^ 2 (kg / cm ² ), and the sterilization time is at least 20 minutes. Wherein, the edible oil of the mixed liquid is not added at the time of the mixing step S2, and the remaining ingredients are autoclaved with the rice bran water and cooled to a suitable temperature (about 50 ° C), and the edible oil has a pore diameter of 0.45. The microporous (μm) filter is filtered and added to the sterilized mixture, and thus, the microbial liquid medium of the present invention is completed.

In order to confirm that the microbial liquid medium disclosed by the present invention does have an effect of increasing the amount of bacteria, a microorganism is cultured by the microbial liquid medium of the present invention and a conventional nutrient liquid medium (Nutrient broth; NB for short). For example, in this embodiment, a Bacillus subtilis is used for enrichment.

Referring to Table 3, when the Bacillus subtilis is cultured in the microbial liquid medium of the present invention, the amount of bacteria on the first day is 3.2×10 ⁹ cfu/ml, which is higher than the amount of bacteria cultured using the conventional nutrient liquid medium ( 8.7×10 ⁷ cfu/ml) is about 100 times higher, and the amount of bacteria cultured in the microbial liquid medium of the present invention reaches 7.3×10 ¹⁰ cfu/ml on the second day, and maintains a high bacterial amount until the third day. However, the amount of bacteria cultured in the conventional nutrient liquid medium decreased on the next day, and the amount of bacteria on the third day was nearly 10,000 times that of the microbial liquid medium of the present invention, and the microorganisms as the present invention could not be obtained. The high amount of bacteria that can be maintained by the liquid medium.

It can be seen that the microbial liquid medium of the present invention can enhance the growth of microorganisms, so that the first day of culture can exceed 100 times the amount of bacteria that can be grown by the conventional medium, and the amount of bacteria can be increased to 1000 on the next day. Times.

Further, the Bacillus subtilis is enriched by the microbial liquid culture medium of the present invention, and the Bacillus subtilis strain has a biological control ability, and the culture is carried out until the second day, that is, the Bacillus subtilis pesticide is applied in the field. The microbial pesticide prepared by using the microbial liquid medium of the present invention can be directly applied to the field, and even the microbial pesticide prepared by the microbial liquid medium of the present invention can be diluted to the conventional hay grass by 1000 times of the amount (about 10 ⁷ cfu/ml). The general application amount of bacillus pesticide (about 10 ⁷ cfu/ml) can reduce the cost of microbial pesticides applied by farmers, and if the microbial pesticide is applied to the soil, the microbial liquid culture medium of the experiment can provide nutrition for the crop. Fertilizer, no additional removal steps are required.

Therefore, the microbial liquid medium of the present invention is used to culture a strain having a biological control ability, and after the culture, a bacterial liquid is obtained, and the bacterial liquid is a microbial pesticide, and the microbial pesticide has a high concentration of bacteria, and no additional purchase is required. Vacuum concentrators, freeze dryers or other equipment for concentrating bacterial liquids to increase the amount of bacteria in the microbial pesticide.

Furthermore, a microbial pesticide is prepared by using the microbial liquid medium of the present invention and a manufacturing method thereof, wherein the microbial liquid medium of the present invention is used to culture a strain having a biological control ability, and the microbial liquid is a microbial pesticide. It has a high concentration of bacteria, can be applied after enrichment and culture, and does not need to concentrate the liquid or make other forms of preparation, and does not need to add an interfacial dispersion exhibiting agent, and the culture liquid is Can be applied directly.

Thereby, the microbial liquid medium of the present invention cultures a microorganism, so that the microorganism can achieve a high growth rate, and the biomass per unit volume can be obtained in a certain period of time; in addition, the microbial liquid medium of the present invention is applied to a microbial pesticide. The preparation can improve the growth rate of the bacteria in the microorganism pesticide, so as to increase the yield and reduce the cost.

The method for preparing the microbial liquid medium of the present invention has simple operation steps, thereby achieving the effects of low production cost and simple process.

While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims.

Fig. 1 is a flow chart showing a method for producing a microbial liquid medium of the present invention.

Claims (11)

A liquid medium for cultivating Bacillus subtilis comprises a carbon source of 6-16% by weight, a nitrogen source of 0.2-0.6%, a bitter tea oil of 0.3-0.7%, a rice bran powder of 0.3-0.7%, and 82.0 ~93.2% rice bran water; wherein, the rice bran water system is prepared by the following steps: the water is mixed with rice bran, wherein the rice bran accounts for 9.0 to 13.7% of the total weight of water and rice bran, and is heated and stirred for at least 1 hour, and then removed. Rice bran residue, get the rice water. A liquid medium for cultivating Bacillus subtilis, comprising a carbon source of 6-16% by weight, a nitrogen source of 0.2-0.6%, a flax oil of 0.3-0.7%, a rice bran powder of 0.3-0.7%, and 82.0-93.2 The rice bran water is prepared by the following steps: the water is mixed with the rice bran, wherein the rice bran accounts for 9.0 to 13.7% of the total weight of the water and the rice bran, and the rice bran residue is removed after heating and stirring for at least one hour. , get the rice water A liquid medium for culturing Bacillus subtilis according to claim 1 or 2, wherein the carbon source is selected from the group consisting of Sucrose, Molasses and Dextrose. A liquid medium for culturing Bacillus subtilis according to claim 1 or 2, wherein the nitrogen source is selected from 0.2 to 0.6% beef extract or yeast extract. A liquid medium for culturing Bacillus subtilis according to claim 1 or 2, wherein the liquid medium cultures a microorganism capable of biological control to prepare a microbial pesticide. A liquid medium for culturing Bacillus subtilis according to claim 1 or 2, wherein the liquid medium is cultured with Bacillus subtilis to prepare a microbial pesticide containing Bacillus subtilis. The invention relates to a method for preparing a liquid medium according to the first aspect of the present application, which comprises the steps of: preparing a rice water and mixing water with rice bran, wherein the rice bran accounts for 9.0 to 13.7% of the total weight of water and rice bran, and is heated and stirred. After at least 1 hour, remove the rice bran residue and obtain one meter of water; a mixing step, taking 6-16% by weight of carbon source, 0.2~0.6% of nitrogen source, 0.3~0.7% of bitter tea oil, 0.3~ 0.7% of rice bran powder and 82-93.2% of the rice bran water are uniformly mixed to obtain a mixed liquid; and a sterilization step, the mixture is subjected to high temperature and high pressure sterilization operation to obtain a microbial liquid medium. The invention relates to a method for preparing a liquid medium according to item 2 of the patent application of the present application, which comprises the steps of: preparing a rice water and mixing water with rice bran, wherein the rice bran accounts for 9.0 to 13.7% of the total weight of water and rice bran, and is heated and stirred. After at least 1 hour, the rice bran residue is removed to obtain one meter of water; a mixing step takes 6 to 16% by weight of carbon source, 0.2 to 0.6% of nitrogen source, 0.3 to 0.7% of saliva oil, 0.3 to 0.7%. The rice bran powder and the 82~93.2% of the rice bran water are uniformly mixed to obtain a mixed liquid; and in a sterilization step, the mixture is subjected to a high temperature and high pressure sterilization operation to obtain a microbial liquid medium. One of the methods described in claim 7 or 8 for culturing Bacillus subtilis The method for preparing a liquid medium, wherein the bitter tea oil or flax oil is added to the sterilized mixture after the sterilization step. A method for preparing a liquid medium for cultivating Bacillus subtilis according to the seventh or eighth aspect of the invention, wherein the carbon source is selected from the group consisting of Sucrose, Molasses and Dextrose. Cluster. A method for producing a microbial liquid medium according to claim 7 or 8, wherein the nitrogen source is selected from 0.2 to 0.6% beef extract or yeast extract.