TWI430750B - Production method for fermentation product of bacillus amyloliquefaciens with property of anti-ultraviolet activity - Google Patents

Description

Method for producing fermentation product of liquefied Bacillus amyloliquefaciens with anti-ultraviolet activity

The invention relates to a method for producing a fermentation product of a liquefied Bacillus amyloliquefaciens having anti-ultraviolet activity, in particular to a method for producing a UV-resistant effect after a specific liquefied Bacillus amyloliquefaciens is subjected to a formulation culture step.

According to the US EPA (U.S. Environmental Protection Agency), many types of Bacillus spp. microbial pesticide products have been approved for marketing, as an agent for controlling plant diseases and insect pests, in addition to the commonly known Bacillus subtilis. In addition, it includes Bacillus subtilis var. amyloliquefaciens, Bacillus cereus, Bacillus licheniformis, Bacillus pumilus, Bacillus thuringiensis, Japanese beetle spores. Bacillus popilliae and Bacillus sphaericus. Spore Bacillus subtilis has been developed and applied, and there are Bacillus subtilis, Bacillus amyloliquefaciens, Cactus bacillus, etc. in the prevention and treatment of diseases; Suribacterium, Bacillus cerevisiae and Bacillus sphaericus in pest control.

Many important plant diseases are caused by soil or rhizosphere fungi. Researchers use the antagonism between soil microorganisms to develop biological control agents that inhibit fungal growth and apply them to field control. Bacillus subtilis is Gram-positive, aerobic rod-shaped bacteria with perennial flagella and endospores as its main morphological features. These bacteria are common in soil and plant surface, in food, feed additives, enzymes, The development of biotechnology industry such as seed protection agents has been used for many years, and it is a beneficial microbial species that is generally considered to be safe. Recently, it has been applied to soil roots and pathogens to compete for the nutrients in the root system to become the dominant species, thereby reducing the harm of pathogenic bacteria; it can also directly spray plant leaves to protect leaf fungal diseases, such as bean rust; it can also be applied to soil or Seed dressing treatment to prevent soil diseases; it has a very obvious promoting effect on the growth of various crops, especially the development of roots; it can also be used as an antifungal agent to prevent spoilage after harvesting fruits and vegetables, such as peach brown rot and Citrus Penicillium, etc.

The liquefied Bacillus amyloliquefaciens was discovered in 1943 by Japanese scholar Fukomoto, which produces a large amount of α-amylase and protease. In the beginning, Bacillus amyloliquefaciens was listed as one of the subspecies of Bacillus subtilis because the appearance and performance characteristics of this strain were very similar to those of Bacillus subtilis. However, in 1967, Welker and Compbell used DNA hybridization to find that the gene similarity between Bacillus subtilis and Bacillus aeruginosa was only between 14.7 and 15.4%, and the DNA guanine-plus-cytosine component (G+C%) of Bacillus subtilis was 41.5~ 43.5%, and the G+C% of Bacillus algolyticus is 43.5~44. 9% showed that Bacillus subtilis and Bacillus aeruginosa were different varieties.

However, it can be known that Bacillus aeruginosa can also be used, which is applied to the range of biological control agents for inhibiting fungal growth of crops, and when used against the defense range of culture, the strains need to act in response to crops in sunlight, however, in sunlight. Ultraviolet rays have lethality. Therefore, the survival rate of strains in sunlight is not high, and it is not effective in producing biological control effects relative to crop cultivation and defense.

In view of this, the inventor has been in the process of researching and improving the existing structures and defects by providing rich experience in design and development and actual production of the relevant industries for many years, and providing a production method of fermentation products containing Bacillus amyloliquefaciens having anti-ultraviolet activity. In order to achieve better practical value for the purpose.

The purpose and efficacy of a method for producing a fermentation product containing Bacillus amyloliquefaciens having anti-ultraviolet activity is achieved by the following technique: a strain of Bacillus amyloliquefaciens (contained as BF-1 [registered number: BCRC910498] 】, named BA [registration number: BCRC910395] and the novel strain named YCMA1 [registration number: BCRC910509] as the initial inoculation source, the initial inoculation source was added to the basic culture NB medium shake flask with carbon source and nitrogen. The culture medium of the source is cultured, and the inoculation source of the batch culture is obtained, and then transferred to a stirred liquid fermentation tank to add a medium having a carbon source and a nitrogen source and an anti-ultraviolet additive for secondary culture, thereby obtaining ultraviolet protection. Fermentation product of active function.

Compared with the CK (LB) medium, the fermentation product prepared by the invention can increase the cell concentration by more than 15 times, and the cells are irradiated with UV-B (6). After 30 minutes at 30 μW/cm 2 ), the survival rate was significantly improved. Therefore, the fermentation product of the present invention can be applied to a biological pesticide composition to inhibit the active growth of crop fungi and bacterial diseases, and can also be used with biocides for fungicides, insecticides, herbicides and Suri strains. .

The inoculum concentration of the batch culture of the present invention is 10 ⁹ cfu/ml or more, and preferably 10 ¹⁰ cfu/ml. The resulting fermentation product contains at least 10 ¹¹ cfu/ml spores.

The carbon source medium of the present invention is further one of 0.1% to 1% maltodextrin, 0.1% to 1% glucose, 0.1% to 1% molasses, 0.1% to 1% sucrose, 0.1% to 1% soluble starch. The above mixture; and the nitrogen source medium is further 0.1% to 1.5% soy protein isolate, 0.1% to 1% yeast powder, 0.1 to 1% yeast extract, 0.1 to 1% malt extract, 0.1 %~1% corn extract one or more mixtures; UV-resistant additives further 0.01%~0.5% deacetylated 80% chitosan, 0.01%~0.5% molasses, 0.01%~0.5% water A mixture of one or more of salicylic acid, 0.01% to 0.5% humic acid.

Another object of the present invention is to provide a strain of Bacillus amyloliquefaciens (including a novel strain designated as BF-1 [registered number: BCRC910498], named BA [registered number: BCRC910395], and named YCMA1 [registered number: BCRC910509]] The resulting fermentation product is used to inhibit the growth of at least one fungal genus belonging to the following group consisting of: guava, Myxosporium psidii Sawada et Kurosawa, Rhizoctonia solani, flax Leaf blight B Ipolaris oryzae (Breda deHaan) Shoemaker, Colletotrichum gloeosporioides (Penz.) Sacc. Colletotrichum gloeosporioides, Colletotrichum gloeosporioides (Penz. & Sace.), Botryodiplodia theobromae Pat. (Mango), Papaya anthrax Colletotrichum gloeosporioides Penzig, Papaya rot, Botyodiplodia theobromae Pat. (Papaya), Phytophthora palmivora (Butler) Butler. , Verticillium dahliae Fusarium sp. , Onionium oxysporum f. Sp. Cepae, Pestalotiopsis euginae, Pestalotiopsis psidii (Pat.) Mordue Pestalotia psidii, Alternaria brassicae (BERK) Sacc. .

Furthermore, the strain of Bacillus amyloliquefaciens of the present invention comprises a novel strain designated as BF-1 [registered number: BCRC910498], named BA [registered number: BCRC910395] and named YCMA1 [registered number: BCRC910509]. Fermentation product for the inhibition of crop bacterial diseases At least one kind of bacteria includes: bacterial spots (Q), citrus canker (R), and cucurbit bacterial fruit spots (S).

Continued, the fermentation product of the Bacillus amyloliquefaciens strain of the invention (including a novel strain named BF-1 having a unique gene sequence of 16S rRNA, YyaR, ItuA, ItuB, ItuC, ItuD) for use in combination with fungicidal The type of agent includes: 1. Aliphatic nitrogens; 2. Amidoxime; Aminic acid; 4. Benzoguanamine; 5. Benzoquinone aniline; Sulfonamides; 7. Antibiotics; 8. Phytosinins; 9. Aromatic; 10. Benzimidazoles; 11. Benzimidazole precursors; Amino formates; Imidazoles; Triazoles; Copper; 16. Dichlorophenyldimethylimine imines; Dithioaminoformate; 18. Polymeric dithiocarbamate salts; Imidazoles; 20. Organic phosphorus; Pyridines; 22. Pyrimidines; 23. Quinoline; 24.醌 class; 25. Thiazoles; 26. Urea; 27. Triazole benzothiazoles.

The fermentation product prepared by the strain of Bacillus amyloliquefaciens of the present invention (including a novel strain named BF-1 having a unique gene sequence of 16S rRNA, YyaR, ItuA, ItuB, ItuC, ItuD) is used to match at least one insecticide The type of agent, including: 1. Three nitrogen well system; 2. Organic nitrogen and heterocyclics; 3. Ammonia methanate; 4. Pyridine organic thiophosphates; Biphenyls; 6. Organochlorines; 7. Antibiotics; 8. New nicotine; 9. 10. Organic phosphorus; 11. Linear amine organothiophosphates; Pyrazoles; Azole.

The liquefied Bacillus amyloliques strain of the invention comprises a novel strain named BF-1 having a unique gene sequence of 16S rRNA, YyaR, ItuA, ItuB, ItuC, ItuD The obtained fermentation product is used to match at least one herbicide species, comprising: 1. Phenoxy acid system; 2. Hypophosphorous acid system; 3. Indoleamines; 4. Diphenyl ether system; Dinitroaniline; 6. Tertiary amines.

First Figure: Flow chart of the steps of the present invention

Second figure: Comparison of UV resistance of Bacillus amyloliquefaciens (BF-1 strain) of the present invention

Figure 3: Comparison of UV resistance of Bacillus amyloliquefaciens (BA strain) of the present invention

Figure 4: Comparison of UV resistance of Bacillus amyloliquefaciens (YCMA1 strain) of the present invention

Figure 5: Survival data of Bacillus amyloliquefaciens (BF-1 strain) with S. cerevisiae

For a more complete and clear disclosure of the technical content, the purpose of the invention and the effects thereof achieved by the present invention, the following is a detailed description, and please refer to the drawings and drawings: First, please refer to The first figure shows a method for producing a fermentation product of Bacillus amyloliquefaciens having anti-ultraviolet activity according to the present invention, and the steps are as follows: (1) Obtaining a bacterial inoculation source - obtaining a strain of Bacillus amyloliquefaciens (including a name of BF- 1 [registration number: BCRC91049 8], named BA [registration number: BCRC910395] and a novel strain named YCMA1 [registration number: BCRC910509] as the initial inoculation source; (2) preparation of batch culture inoculation source - 1 % ratio inoculation is used for inoculation source amplification culture, and the initial inoculation source is cultured in a medium containing a carbon source and a nitrogen source in a shake flask of NB medium, and cultured for 1 to 3 days, wherein the culture condition is a temperature of 30-35 ° C, a stirring rate. 100-250 rpm, the aeration rate is 0.25-1.5 vvm, the pH value is 5.0-7.0, in order to obtain the inoculation source of the batch culture, the concentration of the inoculation source of the batch culture is 10 ⁹ cfu/ml or more; (3) the liquid culture for mass production - will be Taken at a concentration of 10 ⁹ cfu / ml or with culture medium inoculated from a liquid mass production fermenter stirred added nitrogen source and a carbon source comprising UV additives, for 3 to 6 days in liquid culture, wherein the culture conditions Temperature 30-35 ° C, stirring rate 100-250 rpm, aeration rate 0.25-1.5 vvm, pH value 5.0-7.0; (4) obtaining fermentation products - after 3 to 5 days of cultivation through the above steps, by agitated liquid fermentation tank The fermentation product is collected as a fermentation product having a function of anti-UV activity.

In addition, a medium containing a carbon source and a nitrogen source may be added to the step (3) in the step (3) of the fermentation process for 2 to 5 days. In the continuation, the strain is added to the carbon source, nitrogen source medium or antibiotic through the above-mentioned production culture process. The ultraviolet additive may be selected from the following materials: 0.1% to 1% maltodextrin, 0.1% to 1% glucose, 0.1% to 1% molasses, 0.1% to 1% sucrose, 0.1% to 1 One or more mixtures of % soluble starch; and the nitrogen source medium is 0.1%~1.5% soy protein isolate, 0.1%~1% yeast powder, 0.1~1% yeast extract, 0.1~1% malt a mixture of 0.1% to 1% corn extract or one or more of the extracts; wherein the UV-resistant additive is 0.01% to 0.5% deacetylated 80% chitosan, 0.01% to 0.5% molasses, A mixture of 0.01% to 0.5% salicylic acid, 0.01% to 0.5% humic acid, or a mixture of more than one.

The fermentation product obtained by the formula culture was tested for survival rate in the chemical pesticide: a fermentation colony was selected on the LA medium plate, inoculated into 3 ml LB, and cultured at 28 ° C for 24 hours under constant temperature shaking, using a spectrometer ( Spectrophotometer ultrospec2000, pharmacia biotech, USA) Adjust the absorption value (A620) to adjust the bacterial concentration to 10 ⁸ colony-forming units (cfu/ml), take 100 μl of bacterial solution, 100 μl of 10X stock chemical agent and 800 μl of ddH ₂ O was added to the mixture and shaken evenly, and placed on the table for 10 minutes. In the control group, 100 μl of the bacterial solution was mixed with 900 μl of ddH ₂ O. After 10 minutes, 100 μl was serially diluted, and then diluted to 10 ^-3 to 10 ^{- 5} Each 100 μl was plated on the LA plate, and then placed in a 28 ° C incubator for 24 hours. After 24 hours, the number of colonies was counted, and the survival rate was reversed according to the following formula. The chemical pesticides used in the present invention include fungicides, bactericides, insecticides, herbicides and spreading agents of different dilution ratios, and the medicaments used are listed in Table 1, Table 2 and Table 3, respectively. And the dilution factor of the drug is the recommended concentration.

In the continuation, the anti-ultraviolet formula was tested in a culture method of Bacillus licheniformis named BF-1 [registered number: BCRC910498], the UVB test time was 30 minutes; the data sheet under the treatment of UVB irradiation for 30 minutes [please Referring to the second figure, the chart shows that CK(LB) is the reaction state of the UVB irradiation performed before the cultivation of the Bacillus amyloliquefaciens strain, and the step (2) [formulation one] and the step are carried out with respect to the strain. (3) The reaction of UVB irradiation after [Formulation 2] was made to make a comparison chart, respectively. It can be seen from the graph that the fermentation product cultivated by the method of the present invention has a higher survival rate than the former two after 30 minutes of UVB irradiation. Therefore, the fermentation product cultivated by the process of the present invention has a good anti-ultraviolet effect.

In the group of liquefied Bacillus amyloliquefaciens, the strain named BA [registered number: BCRC910395] (please refer to the third figure) and YCMA1 [registered number: BCRC910509] [see the fourth figure] strain can be applied to the present invention. The cultivation process is carried out in the process step, and the fermented product which is cultivated has the same anti-ultraviolet effect.

In addition, the liquefied Bacillus amyloliquefaciens can be used in combination with S. cerevisiae to form a bio-pesticide composition, as shown in the fifth figure, which is shown in the liquefied Bacillus licheniformis (here named BF-1 liquefied starch) Bacillus is used to carry out the addition step (2) [Formulation 1] and Step (3) [Formulation 2] followed by the survival rate table of S. cerevisiae (Bt); in the graph, CK (LB) is simply liquefied Survival status after Bacillus amyloliquefaciens strain and Bacillus amyloliquefaciens + S. cerevisiae, and the survival reaction after step (2) [formulation 1] and step (3) [formulation 2] are carried out with respect to the state of the above two strains, The comparison chart is separately prepared. It can be seen from the chart that the fermentation product cultivated by the method of the present invention has higher survival rate than the former two after the strain of S. cerevisiae. Therefore, the fermentation product cultivated by the process of the present invention is matched with S. After that, it will not be suppressed by Suri.

The above-mentioned embodiments or the drawings are not intended to limit the structure or the dimensions of the present invention, and any suitable changes or modifications may be made without departing from the scope of the invention.

In summary, the embodiments of the present invention can achieve the expected use efficiency, and the specific structure disclosed therein has not been seen in similar products. It has not been disclosed before the application. Cheng has fully complied with the requirements and requirements of the Patent Law. He has filed an application for an invention patent in accordance with the law, and he is pleased to review it and grant a patent.

Claims (8)

A method for producing a fermentation product of Bacillus amyloliquefaciens having anti-ultraviolet activity, the steps of which are as follows: (1) obtaining a fungal inoculation source - obtaining a strain of Bacillus amyloliquefaciens as an initial inoculation source; and (2) preparing an inoculation source for batch culture - inoculation at a ratio of 1% for inoculation source amplification culture, the initial inoculation source is added to a medium containing a carbon source and a nitrogen source in a shake flask of NB medium, and cultured for 1 to 3 days, wherein the culture condition is a temperature of 30-35 ° C. The stirring rate is 100-250 rpm, the aeration rate is 0.25-1.5 vvm, and the pH value is 5.0-7.0, so as to obtain the inoculation source of the batch culture, the concentration of the inoculation source of the batch culture is 10 ⁹ cfu/ml or more; (3) Production liquid culture - The culture concentration of 10 ⁹ cfu / ml or more is used to inoculate a medium containing a carbon source and a nitrogen source and an anti-ultraviolet additive in a stirred liquid fermentation tank for mass production, and liquid culture is carried out 3 to 6 Day, while the culture conditions are temperature 30-35 ° C, stirring rate 100-250 rpm, aeration rate 0.25-1.5 vvm, pH 5.0-7.0; wherein the UV-resistant additive comprises at least one of the following: 0.01% ~0.5% deacetylated 80% chitosan 0.01%~0.5% molasses, 0.01%~0.5% salicylic acid, 0.01%~0.5% humic acid; (4) Obtaining fermentation products - cultivating 3~5 through the above steps After the day, the fermentation product is collected by the agitated liquid fermentation tank as a fermentation product having ultraviolet and anti-living substances. A method for producing a fermentation product of Bacillus aeruginosa having ultraviolet-resistance activity according to claim 1, wherein the carbon source medium comprises at least one of the following: 0.1% to 1% maltodextrin, 0.1 %~1% glucose, 0.1%~1% molasses, 0.1%~1% sucrose, 0.1%~1% soluble starch. A method for producing a fermentation product of Bacillus aeruginosa having ultraviolet-resistance activity according to claim 1 or 2, wherein the nitrogen source medium comprises at least one of the following: 0.1% to 1.5% soy protein isolate, 0.1%~1% yeast powder, 0.1~1% yeast extract, 0.1~1% malt extract, 0.1%~1% corn extract. A method for producing a fermentation product of Bacillus licheniformis having anti-ultraviolet activity as described in claim 1, wherein the concentration of the inoculum of the batch culture is 10 ¹⁰ cfu/ml. A method for producing a fermentation product of Bacillus licheniformis having anti-UV activity according to claim 1, wherein the fermentation product contains at least 10 ¹¹ cfu/ml spore. A method for producing a fermentation product of Bacillus aeruginosa having ultraviolet-resistance activity according to claim 1, wherein the concentration of the anti-living substance iturin A in the step (4) is 10 mg/L or more. A method for producing a fermentation product of Bacillus aeruginosa having ultraviolet-resistance activity according to claim 6, wherein the concentration of the anti-living substance surfactin in the step (4) is allowed to stand at room temperature for 2 to 7 days. Up to 10mg / L or more. A method for producing a fermentation product of Bacillus licheniformis having anti-ultraviolet activity according to the first aspect of the invention, wherein the cultivation of the carbon source and the nitrogen source may be added in the fermentation process in the step (3) for 2 to 5 days. Based on step (3).