A Rhodobacter capsulatus water dispersible granule and its preparation and application

Abstract

The present invention discloses a Rhodobacter capsulatus water dispersible granule, its preparation and application. The Rhodobacter capsulatus water dispersible granule comprises a Rhodobacter capsulatus concentrated fermentation broth, a wetting agent, a dispersing agent and a filler. The preparation of the Rhodobacter capsulatus water dispersible granule includes: placing the dispersing agent, the wetting agent and the filler in a powder mixing tank, mixing them evenly, and then performing ultrafine grinding in a jet mill; slowly adding the Rhodobacter capsulatus concentrated fermentation broth to the mixture of the dispersing agent and the ultrafinely ground material, kneading in a stirrer, extruding and granulating through a 2 mm sieve mesh in a swing granulator after the material just agglomerates into blocks, drying in an oven at 50 °C, and finally screening through a 2 mm vibrating screen. After passing the inspection, the Rhodobacter capsulatus water dispersible granule is obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of biological agents, and particularly relates to a Rhodobacter capsulatus water dispersible granule and its preparation and application. Background Art

[0002] Photosynthetic bacteria are a special group of microorganisms that can grow and reproduce using solar energy. They can carry out photosynthesis using hydrogen sulfide, carbon dioxide, etc., and can also synthesize nutrients required by plants using small molecule organic substances, and produce growth promoting factors to activate the activity of plant cells and improve the photosynthesis ability of plants. Therefore, photosynthetic bacteria have broad application prospects in agriculture.

[0003] Photosynthetic bacteria can enhance the photosynthesis of crops, increase the chlorophyll content, improve the photosynthetic efficiency, and thus promote the growth and material accumulation of crops; moreover, photosynthetic bacteria can promote the growth of beneficial microorganisms in the soil, inhibit the reproduction of harmful microorganisms, improve the physical and chemical properties of the soil, and increase the soil organic matter and nitrogen and phosphorus contents; they can also degrade residual pesticides and toxic compounds in the soil, reduce soil pollution, and improve soil fertility. The photosynthetic bacteria fermentation broth contains various physiologically active substances such as chlorophyll, vitamins, and amino acids, and these substances can promote seed germination, improve the germination rate and emergence rate. Photosynthetic bacteria contain antibacterial and antiviral substances, which can inactivate the pathogenicity of pathogens and inhibit the occurrence and spread of diseases. They can also promote the reproduction of beneficial microorganisms and inhibit the growth of harmful flora, thereby improving the disease resistance of crops.

[0004] The existing photosynthetic bacteria dosage forms seen in the market are roughly liquid preparations, concentrated solutions, solid preparations, freeze-dried powders, etc. Since photosynthetic bacteria have high requirements for the production process, and the survival rate of the bacterial strains is relatively low during storage, transportation, etc., the annual registration volume of photosynthetic bacteria agents in China is relatively small. Relatively speaking, liquid preparations are easier to culture and produce in large quantities, and the activity of the cultures is stronger, the metabolite content of photosynthetic bacteria is rich, and the application effect is also better. Currently, it is the most widely used dosage form among various dosage forms. This form has problems such as easy contamination, loss, difficult preservation, and inconvenient transportation of the bacterial cells, which is not conducive to the storage and quality stability of the product, and the transportation cost is extremely high. In addition, due to the limitations of its ecological characteristics and growth conditions, the shelf life of the bacterial agent is relatively short. Once expired, even if there is still a certain amount of bacteria, the effect will be greatly reduced. In addition, photosynthetic bacteria freeze-dried powder needs to be stored in a low-temperature environment, usually recommended to be stored in a refrigerator to ensure its biological activity. Because it is sensitive to temperature, the unopened freeze-dried powder should be stored in a refrigerator at 2-8 degrees Celsius, and it needs to be used up within three days after opening, otherwise the activity will drop rapidly, especially in high-temperature situations. Therefore, special attention needs to be paid to temperature control during transportation, which increases the transportation cost and difficulty. Summary of the Invention

[0005] To solve the above technical problems, the object of the present invention is to provide a Rhodobacter capsulatus water dispersible granule and its preparation and application.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] A Rhodobacter capsulatus A12, the classification and naming of Rhodobacter capsulatus is Rhodobacter capsulatus.A12. It was deposited in the China Center for Type Culture Collection on September 7, 2018, and the deposit number is CCTCC NO: M2018604.

[0008] A Rhodobacter capsulatus water dispersible granule, comprising a Rhodobacter capsulatus concentrated fermentation broth, a wetting agent, a dispersing agent and a filler.

[0009] Furthermore, the mass percentage of the Rhodobacter capsulatus concentrated fermentation broth is 40% - 50%;

[0010] The wetting agent uses carboxymethyl cellulose with a mass percentage of 0.1%;

[0011] The dispersing agent uses lignin dispersant Ufoxane 3A with a mass percentage of 4%;

[0012] The filler is attapulgite, and the mass ratio of the attapulgite to the total mass ratio of the Rhodobacter capsulatus concentrated fermentation broth, the wetting agent, and the dispersing and disintegrating agent is 100%.

[0013] The preparation of a Rhodobacter capsulatus water dispersible granule includes:

[0014] A. Mix 4% of lignin dispersant Ufoxane 3A with 0.1 of carboxymethyl cellulose and attapulgite, and ultra-finely pulverize to become an auxiliary agent;

[0015] B. Add 40% - 50% of the Rhodobacter capsulatus concentrated fermentation broth to the ultra-finely pulverized auxiliary agent mixture, mix it evenly and then stir and knead to form agglomerates;

[0016] C. Extrude and granulate the agglomerated materials through a 2 mm mesh screen in a granulator; D. Dry the extruded granules in an oven at 50 °C;

[0017] E. Screen the dried granules through a 2 mm screen and conduct a viable bacteria count test. If qualified, the Rhodobacter capsulatus water dispersible granule is obtained; otherwise, perform step B.

[0018] Furthermore, the bacterial content in the Rhodobacter capsulatus concentrated fermentation broth is 5×10 10 cfu / g.

[0019] Further, step C is carried out for drying at a temperature of 50°C ± 2°C.

[0020] Further, the Rhodobacter capsulatus water dispersible granule is obtained by screening through a standard sieve with a pore diameter of 2 mm.

[0021] Further, the carboxymethyl cellulose, the lignin dispersant Ufoxane 3A, and attapulgite are subjected to ultrafine grinding under a pressure of 0.8 kg and a grinding pressure of 0.6 kg.

[0022] Application of a Rhodobacter capsulatus water dispersible granule in preventing and controlling diseases of crops, promoting growth, and increasing production.

[0023] Compared with the prior art, one or more embodiments of the present invention may have the following advantages:

[0024] The Rhodobacter capsulatus water dispersible granule provided by the present invention is beneficial to maintaining high activity of the product, effectively prolonging the shelf life of the product, facilitating storage and transportation, and significantly improving in preventing and controlling crop viruses and increasing production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a flow chart of the preparation method of the Rhodobacter capsulatus water dispersible granule;

[0026] Figure 2 is a diagram of the disintegration process of the Rhodobacter capsulatus water dispersible granule;

[0027] Figure 3 is a diagram of the finished product of the Rhodobacter capsulatus water dispersible granule. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings.

[0029] Example 1

[0030] The Rhodobacter capsulatus water dispersible granule includes a Rhodobacter capsulatus concentrated fermentation broth, a wetting agent, a dispersing and disintegrating agent, and attapulgite.

[0031] The mass percentage of the Rhodobacter capsulatus concentrated fermentation broth is 40% - 50%;

[0032] The wetting agent uses carboxymethyl cellulose with a mass percentage of 0.1%;

[0033] The dispersing and disintegrating agent uses the lignin dispersant Ufoxane 3A with a mass percentage of 4%;

[0034] The filler is attapulgite, and the mass ratio of the attapulgite to the total mass ratio of the Rhodobacter capsulatus concentrated fermentation broth, wetting agent, and dispersing and disintegrating agent is 100%.

[0035] Example 2

[0036] As Figure 1 shown, this example also provides a preparation method of Rhodobacter capsulatus water dispersible granules, including:

[0037] A. Mix 4% by mass of lignin dispersant Ufoxane 3A and 0.1% by mass of carboxymethyl cellulose with attapulgite, and pulverize;

[0038] B. Add 40% - 50% by mass of Rhodobacter capsulatus concentrated fermentation broth to the ultra - pulverized auxiliary mixture, mix evenly, and then stir and knead in a blender to form agglomerates;

[0039] C. Extrude and granulate the agglomerated materials through a 20 - mesh sieve in a swing granulator;

[0040] D. Dry the extruded granules in an oven at about 50°C;

[0041] E. Screen the dried granules through a 2 - mm sieve, and conduct a viable bacteria count test. If qualified, the Rhodobacter capsulatus water dispersible granules are obtained; otherwise, perform step B.

[0042] The bacterial content in the above - mentioned Rhodobacter capsulatus concentrated fermentation broth is 5×10 10 cfu / g; step C is carried out for drying at a temperature of 50°C ± 2°C; the Rhodobacter capsulatus water dispersible granules are obtained by screening through a standard sieve with a 2 - mm pore size; the lignin dispersant Ufoxane 3A and attapulgite are ultra - pulverized under a pressure of 0.8 kg and a pulverizing pressure of 0.6 kg.

[0043] Example 3

[0044] This example also provides a method for determining the viable bacteria count in the above - prepared Rhodobacter capsulatus water dispersible granules, including: using the plate colony counting method (A) or the most probable number method (MPN method) (B) to determine the effective viable bacteria quantity. Select three dilution gradients of 10 -8 、10 -9 、10 -10 for determination. Take 20 sterilized screw - cap test tubes filled with 9 mL of culture medium, and mark them in groups of 3 tubes as 10 -8 、10 -9 、10 -10, CK. Using a 1 mL sterile pipette tip, aspirate 1 mL of the dilution solution for each concentration gradient according to the requirements of aseptic operation and place it into the corresponding numbered test tubes. Screw on the test tube caps tightly. Add 1 mL of sterile water to the 3 test tubes labeled CK as a control. Place them in a constant temperature incubator at 30 ± 1 °C and irradiate and culture them under an incandescent lamp with an illumination of 2500 lx - 3000 lx for 7 d - 10 d. Count the number of test tubes that turn completely red among the three test tubes for each concentration. Correlate the distribution pattern of the number of red tubes at the three concentrations to the MPN table to obtain the corresponding MPN value. MPN value × 10 n is the concentration of viable bacteria in the sample.

[0045] Example 4

[0046] The screening of attapulgite as a filler in the water dispersible granule includes:

[0047] Using the direct mixing method, spray mix a certain weight of different fillers such as corn starch, rice bran, attapulgite, kaolin, talc powder, activated carbon, diatomaceous earth, bentonite, etc. with the Rhodobacter capsulatus concentrated liquid (content 5 × 1010 cfu / g). Granulate through a rotary extrusion granulator, dry in an electrothermal constant temperature drying oven at (50 °C ± 2 °C) until the water content is 17% - 20%, crush and sieve, and then calculate the content of Rhodobacter capsulatus in each gram of sample through the algorithm in Example 3 above. The experiment is repeated 3 times. Through the experiment, the filler with good adsorption activity for Rhodobacter capsulatus is screened out as attapulgite, as shown in Table 1:

[0048] Table 1

[0049]

[0050] The screening of the mixing method of photosynthetic bacteria and additives, i.e., dispersants:

[0051] Mix the dispersants that have passed the safety screening: lignin dispersant REAX910, lignin dispersant Ufoxane 3A, carboxylate dispersant D - 1008, carboxylate copolymer SD - 816, naphthalene sulfonate condensate Morwet D - 425, sodium dodecyl sulfate K12, etc. with attapulgite at a dosage of 4% respectively. Add the Rhodobacter capsulatus concentrated fermentation liquid (bacterial content 5 × 10 10 cfu / g) to the mixture and mix and stir by spraying. Then add the mixed sample to a swing granulator to prepare different samples, and screen the mixing method by measuring the viable bacteria count and disintegration time. The dispersibility is determined according to the pesticide dispersibility determination method of GB T 32775 - 2016. The experiment shows that under the condition of equivalent viable bacteria count, the disintegration time of the spray mixing method is shorter (as shown in Table 2):

[0052] Table 2

[0053]

[0054] As can be seen from the table, the lignin dispersant Ufoxane 3A, the carboxylate dispersant D-1008, and the carboxylate copolymer SD-816 have a high suspension rate, a short disintegration time, and a high viable bacteria count, which can reach 10 9 ~10 10 cfu / g (see Table 2 for the screening of the mixing method and dispersant of Rhodobacter capsulatus A12 water dispersible granules). As Figure 2 shown is the disintegration process of Rhodobacter capsulatus A12 water dispersible granules.

[0055] Screening of wetting agents for water dispersible granules:

[0056] Using the graduated cylinder test method, add 250 mL of 342 mg / L hard water to a 250 mL graduated cylinder; quickly pour Rhodobacter capsulatus water dispersible granules into the graduated cylinder with weighing paper, without stirring, immediately start the stopwatch, and record the time when 99% of the sample sinks to the bottom of the cylinder. When carboxymethyl cellulose is used as the wetting agent, when the dosage is 1‰, the wetting time is 53 s, and the effective viable bacteria count is 4*10 9 cfu / g; when the dosage is increased to 2% and 4% respectively, the wetting times are 48 s and 45 s respectively, and the effective viable bacteria counts are 5*10 8 and 4*10 8 , and the difference is not obvious. When calcium alkylbenzene sulfonate is used as the wetting agent, the viable bacteria count and wetting time at dosages of 2% and 4% are not very different, but the suspension performance is not good. Considering comprehensively, the dosage of the wetting agent is 1‰ carboxymethyl cellulose (see Table 3 for the screening of wetting agents for Rhodobacter capsulatus A12 water dispersible granules):

[0057] Table 3

[0058]

[0059] Through the above formula screening test, combined with the safety of viable bacteria by additives, the optimal formula of Rhodobacter capsulatus water dispersible granules was determined as follows: 40%-50% of the concentrated fermentation broth of Rhodobacter capsulatus; wetting agent: 0.1% of carboxymethyl cellulose; dispersing and disintegrating agent: 4% of Ufoxane 3A; attapulgite supplemented to 100%, stirred until it forms a mass and does not stick together when put into the machine; the sample is as Figure 3 shown.

[0060] This example also tests the thermal stability and storage stability of Rhodobacter capsulatus water dispersible granules:

[0061] Prepare 6 batches of samples according to the formula provided in this example. Put 200 g of the sample into a foil bag and seal it under vacuum with a vacuum sealer. Seal at least 3 bottles for each batch and weigh them separately. Place the sealed vacuum sample bags in an incubator at 45 ± 2 °C for 14 d. Take them out and cool to room temperature. Wipe the glass bottles clean and conduct tests within 24 h. The results show that the bacterial counts of the Rhodobacter capsulatus water dispersible granules after heat storage are all above 2.0×10 8 cfu / g, and the heat storage stability is good, indicating that the formulation of the preparation is stable (as shown in Table 4):

[0062] Table 4

[0063]

[0064] In the experiment, the Rhodobacter capsulatus in the water dispersible granules was replaced with photosynthetic bacteria with GFP tags. The samples in vacuum-sealed packages were stored at 20 - 25 °C. Take out one bag every 30 d to observe and calculate the number of effective viable bacteria. The results show that the number of viable bacteria is more than 80% of the initial number of viable bacteria.

[0065] Example 5 This example also provides the application of Rhodobacter capsulatus water dispersible granules in preventing and controlling crop diseases, promoting growth and increasing production:

[0066] 1) Indoor growth-promoting activity of Rhodobacter capsulatus A12 water dispersible granules on pepper seedlings:

[0067] A total of 4 treatments were set in the experiment:

[0068] A: A12 WG 100 g / acre;

[0069] B: A12 WG 50 g / acre;

[0070] C: A12 SC 170 ml / acre;

[0071] D: CK (clear water).

[0072] Spray the preparation on the pepper seedlings for the first time at the 3-leaf stage, and then spray it once every week for a total of 3 times. The growth-promoting effect was statistically analyzed 15 days after the third spraying (as shown in Table 5):

[0073] Table 5

[0074]

[0075] 2) Indoor growth-promoting experiment of Rhodobacter capsulatus A12 WG on tobacco

[0076] A total of 5 treatments were set in the experiment:

[0077] A: A12 WG 1 g / ml;

[0078] B: A12 WG 1g / ml + 5% amino-oligosaccharide;

[0079] C: A12 SC 200-fold dilution;

[0080] D: A12 SC 200-fold dilution + 5% amino-oligosaccharide;

[0081] E: CK (clear water).

[0082] After the tobacco was transplanted and survived, the preparation was sprayed for the first time, and then once every week for a total of 3 times. The growth-promoting effect was counted on the 15th day after the third spraying (as shown in Table 6):

[0083] Table 6

[0084]

[0085] 3) Field growth-promoting activity of Rhodobacter capsulatus A12 water dispersible granule on cabbage

[0086] A total of 5 treatments were set up in the experiment:

[0087] A: A12 WG 100 g / mu;

[0088] B: A12 WG 50 g / mu;

[0089] C: A12 SC 170 ml / mu;

[0090] D: CK (clear water);

[0091] After the cabbage was transplanted and survived, the preparation was sprayed for the first time, and then once every week for a total of 3 times. The growth-promoting effect was counted on the 15th day after the third spraying (as shown in Table 7):

[0092] Table 7

[0093]

[0094] 4) Field yield-increasing effect of Rhodobacter capsulatus A12 WG on watermelon

[0095] A total of 6 treatments were set up in the experiment, with 4 replicates for each treatment:

[0096] A: A12 WG 100 g / mu

[0097] B: A12 WG 100 g / mu + 5% amino-oligosaccharide

[0098] C: A12 SC 200-fold dilution

[0099] D: CK (clear water)

[0100] Spray the preparation for the first time in small plots during the watermelon seedling stage, and then spray it once every week for a total of 3 times. On the 15th day after the third spraying, the fruit weight per plant was statistically analyzed, and the watermelon yield during the experiment was calculated (as shown in Table 8):

[0101] Table 8

[0102]

[0103] 5) Field yield increase effect of Rhodobacter capsulatus A12 WG on chili peppers

[0104] A total of 6 treatments were set up in the experiment, with 4 replicates for each treatment:

[0105] A: A12 WG 100g / mu + 5% amino-oligosaccharide

[0106] B: A12 WG 100g / mu

[0107] C: A12 WG 75g / mu

[0108] D: CK (clear water)

[0109] Spray the preparation for the first time during the chili pepper seedling stage, and then spray it once every week for a total of 3 times. On the 15th day after the third spraying, the fruit weight per plant was statistically analyzed, and the chili pepper yield during the experiment was calculated (as shown in Table 9):

[0110] Table 9

[0111] Processing Number of first crop results (pcs) Yield per mu converted to kg Yield increase rate % A 36.3 2498 61.58 B 25.6 2076 48.84 C 32.6 2301 34.41 D 9.6 1546 -

[0112] 6) Field control effect of Rhodobacter capsulatus A12 WG on cucumber bacterial angular leaf spot

[0113] A total of 6 treatments were set up in the experiment, with 4 replicates for each treatment:

[0114] A: A12 WG 100g / mu

[0115] B: A12 WG 100g / mu + 5% amino-oligosaccharide

[0116] C: A12 SC 200-fold dilution

[0117] D: A12 SC 200-fold dilution + 5% amino-oligosaccharide

[0118] E: 3% thiabendazole wettable powder

[0119] F: CK (clear water)

[0120] Spray the preparation for the first time at the initial stage of cucumber bacterial angular leaf spot, and then spray it once every week for a total of 3 times. The disease incidence was statistically analyzed before each spraying and on the 10th day after the third spraying, and the cucumber yield during the experiment was calculated (as shown in Table 10):

[0121] Table 10

[0122]

[0123] Although the embodiments disclosed in the present invention are as above, the above content is only an embodiment adopted for the convenience of understanding the present invention and is not intended to limit the present invention. Any person skilled in the art within the technical field to which the present invention pertains may make any modifications and variations in the form of implementation and details without departing from the spirit and scope disclosed by the present invention. However, the scope of patent protection of the present invention shall still be subject to the scope defined by the appended claims.

Claims

1. A Rhodobacter capsulatus A12 water dispersible granule, characterized in that, The Rhodobacter capsulatus is classified and named as Rhodobacter capsulatus.A12, which has been deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M2018604; The water dispersible granule of Rhodobacter capsulatus comprises a concentrated fermentation broth of Rhodobacter capsulatus, a wetting agent, a dispersing agent and a filler; The mass percentage of the concentrated fermentation broth of Rhodobacter capsulatus is 40% - 50%; The wetting agent used is carboxymethyl cellulose with a mass percentage of 0.1%; The dispersing agent used is lignin dispersant Ufoxane 3A with a mass percentage of 4%; The filler is attapulgite, and the mass ratio of the attapulgite to the total mass ratio of the concentrated fermentation broth of Rhodobacter capsulatus, the wetting agent and the dispersing agent is 100%.

2. The preparation method of the Rhodobacter capsulatus A12 water dispersible granule according to claim 1, characterized in that, It includes: A. Mix lignin dispersant Ufoxane 3A with a mass percentage of 4% and carboxymethyl cellulose with a mass percentage of 0.1% with attapulgite, and perform ultrafine grinding to form an auxiliary agent; B. Add the concentrated fermentation broth of Rhodobacter capsulatus with a mass percentage of 40% - 50% to the ultrafinely ground auxiliary agent mixture, mix it evenly and then stir and knead to agglomerate into a mass; C. Extrude and granulate the agglomerated material through a 2 mm sieve in a granulator; D. Dry the extruded granules in an oven at 50°C ± 2°C; E. Screen the dried granules through a 2 mm sieve and conduct a viable bacteria count test. If qualified, the water dispersible granule of Rhodobacter capsulatus is obtained; otherwise, perform step B.

3. The preparation method of the Rhodobacter capsulatus A12 water dispersible granule according to claim 2, wherein, The bacterial content in the fermented liquid of the Rhodobacter capsulatus concentrated liquid is 5×10 10 cfu / g.

4. The preparation method of the Rhodobacter capsulatus A12 water dispersible granule according to claim 2, characterized in that The carboxymethyl cellulose, lignin dispersant Ufoxane 3A and attapulgite are subjected to ultrafine grinding under a pressure of 0.8 kg and a grinding pressure of 0.6 kg.

5. Application of the water dispersible granule of Rhodobacter capsulatus A12 as claimed in claim 1 in the prevention and control of bacterial angular leaf spot of cucumber, wherein the Rhodobacter capsulatus is classified and named as Rhodobacter capsulatus.A12, which has been deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M2018604.

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