Prussella sp. And application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to Prussella sp. And application thereof. The preservation number of the Princisteria sp. Is CGMCC (China General Microbiological Culture Collection Center) No. 34408. The application comprises degradation of glucosinolate or improvement of rapeseed meal quality. According to the invention, a strain of Plesteria sp. With glucosinolate degradation capability is obtained through screening, and the Plesteria sp. Can effectively reduce the content of antinutritional factors in the rapeseed dregs and improve the quality of the rapeseed dregs, so that the Plesteria sp. Can be better applied to feeds. In addition, the rapeseed meal fermented by the Presteria sp. Has the effect of improving the oxidation resistance of fish organisms, and has important value in the field of feed development.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to a Priestia sp. and application thereof. BACKGROUND

[0002] With the significant improvement of living standards, people's demand for animal protein continues to increase, which promotes the rapid development of livestock breeding industry. However, in this process, the supply and demand contradiction of high-quality protein feed resources is also increasingly prominent. High-quality protein feed is the cornerstone of modern livestock breeding industry, and its stability and quality directly affect the growth performance, health status and breeding efficiency of livestock and poultry.

[0003] Under such circumstances, it is particularly important to develop new unconventional protein feed resources, which has far-reaching strategic significance. The development of unconventional protein feed resources not only can alleviate the pressure of traditional protein feed resources, but also can provide more diversified and sustainable feed choices for the breeding industry, which helps to promote the green and efficient development of livestock breeding industry.

[0004] Rapeseed meal, as a plant protein source with great development potential, has attracted widespread attention. It is the main by-product after oil extraction from rapeseed, and has high economic value and nutritional value. The crude protein content of rapeseed meal is as high as 35%-40%, compared with traditional protein feed such as soybean meal, its price is more market competitive, which can effectively reduce the breeding cost, especially in the current market environment of large fluctuations in feed prices, the economic advantage of rapeseed meal is more obvious.

[0005] However, the application of rapeseed meal also faces some challenges. Its main problem is that it contains a variety of anti-nutritional factors, such as glucosinolate, tannin and phytic acid, etc. These anti-nutritional factors will have adverse effects on the growth and health of animals. Among them, glucosinolate is one of the main anti-nutritional factors in rapeseed meal. Glucosinolate can produce toxic substances such as isothiocyanate in the animal body after metabolism, which can interfere with the function of thyroid gland, cause goiter, and thus affect the growth and development and production performance of animals. SUMMARY

[0006] In order to solve the problems existing in the prior art, the present application provides a Priestia sp. and application thereof.

[0007] In the first aspect, the present application provides a Priestia sp. CP71, the preservation number of which is CGMCC No. 34408.

[0008] The present application screens a Priestia sp. with glucosinolate degradation ability by glucosinolate degradation experiment combined with ultraviolet mutagenesis, and preserves it, and the preservation information is as follows: Deposit accession number: CGMCC No. 34408; classification name: Pseudomonas sp. Priestia sp. Deposit unit: China General Microbiological Culture Collection Center; deposit address: No. 1, Yikhinxi Lu, Chaoyang Qu, Beijing, China Institute of Microbiology; deposit date: April 30, 2025.

[0009] In a second aspect, the present application provides a microbial agent, comprising the aforementioned Pseudomonas sp. CP71 or a fermentation product thereof.

[0010] Further, the microbial agent is a solid microbial agent, a liquid microbial agent or a microbial agent, and the total viable count of the Pseudomonas sp. CP71 in the microbial agent is 1 x 10 7-10 cfu / g.

[0011] In a third aspect, the present application provides a preparation method of the aforementioned microbial agent, comprising: Placing all microorganisms in a fermentation system for fermentation culture.

[0012] Further, the fermentation substrate of the fermentation system is rapeseed meal, and the culture conditions include: temperature 35~40℃, static fermentation.

[0013] In a fourth aspect, the present application provides a product, comprising the aforementioned Pseudomonas sp. CP71 or the aforementioned microbial agent; the product is a feed additive, a desulfurizer, a fertilizer or a plant growth promoter.

[0014] In a fifth aspect, the present application provides the aforementioned Pseudomonas sp. CP71 or the aforementioned microbial agent for use in any one of the following: (1) degrading glucosinolates; (2) preparing a reagent for degrading glucosinolates.

[0015] In a sixth aspect, the present application provides the aforementioned Pseudomonas sp. CP71 or the aforementioned microbial agent for use in any one of the following: (1) improving the quality of rapeseed meal; (2) preparing a reagent for improving the quality of rapeseed meal.

[0016] Further, the improvement of the quality of rapeseed meal includes: increasing the contents of crude protein, crude fat, neutral detergent fiber and acid detergent fiber, and reducing the content of glucosinolates.

[0017] In a seventh aspect, the present application provides the aforementioned Pseudomonas sp. CP71 or the aforementioned microbial agent for use in any one of the following: (1) improving the antioxidant capacity of fish organisms; (2) preparing a reagent for improving the antioxidant capacity of fish organisms.

[0018] The present application has the following advantages: The present application is based on a strain of Pristinamycete CP71 with the ability to degrade glucosinolates, which is obtained through screening and ultraviolet mutagenesis. The addition of Pristinamycete CP71 in rapeseed meal fermentation can effectively reduce the content of glucosinolates. The Pristinamycete CP71 provided by the present application can significantly improve the quality of fermented rapeseed meal, and provides a technical basis for realizing soybean meal reduction. In addition, the rapeseed meal fermented by Pristinamycete CP71 can improve the antioxidant capacity of fish organisms, and has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0020] Figure 1 is the macroscopic morphology of the strain CP71 provided by the present application embodiment 2.

[0021] Figure 2 is the microscopic morphology of the strain CP71 provided by the present application embodiment 2.

[0022] Figure 3 is the 16S rDNA sequence comparison result of the strain CP71 provided by the present application embodiment 2. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present application more clear, the technical solutions in the present application will be described clearly and completely in the following combined with the drawings in the present application. Obviously, the described embodiments are some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0024] The experimental methods involved in the following embodiments are all conventional methods in the art, for example, the experimental manual in the art can be referred to, or the conditions suggested in the manufacturer's instruction can be followed, if not specifically mentioned.

[0025] The experimental materials and reagents involved in the following embodiments can be obtained from commercial channels, if not specifically mentioned.

[0026] Embodiment 1 The present embodiment provides a method for screening glucosinolate degrading bacteria, comprising: 1. Crude extraction of glucosinolate glucoside in rapeseed meal An appropriate amount of rapeseed meal was weighed into a pulverizer, crushed, and then passed through a 60-mesh sieve. The crushed rapeseed meal was placed in a constant-temperature drying oven at 110°C and dried for 2 h to inactivate the myrosinase. The dried rapeseed meal was placed in a conical flask, and n-hexane solution was added at a ratio of 1:6 (m / v). The flask was sealed and incubated at 40°C and 160 r / min for 6 h to extract the lipid material from the rapeseed meal. After the extraction, the mixture was filtered, and the filtrate was collected for drying.

[0027] The defatted rapeseed meal was added with the prepared 70% ethanol solution at a ratio of 1:4 (m / v) and ultrasonically extracted at a power of 150 W for 20 min. After the ultrasonic extraction, the mixture was centrifuged at 5000 r / min for 10 min, and the supernatant was collected in a beaker. The precipitate was subjected to the ultrasonic extraction for 3 times. The extract was concentrated in a rotary evaporator at 45°C. The concentrated solution was added with 0.2 mol / L lead acetate-zinc acetate solution until no flocculent precipitate was observed. The supernatant was collected by low-speed centrifugation, and the protein precipitate was washed with a small amount of 70% ethanol and centrifuged again.

[0028] The obtained supernatant was pre-cooled in a -80°C refrigerator for 2 h and then vacuum freeze-dried for 2 days to obtain a yellow hygroscopic solid. The solid was quickly scraped and stored in a -20°C refrigerator in a sealed state for later use (crude glucosinolate extract from rapeseed meal).

[0029] 2. Preparation of glucosinolate screening medium 1.0 g of ammonium chloride, 5.0 g of crude glucosinolate extract from rapeseed meal, 3.0 g of potassium dihydrogen phosphate, 0.478 g of magnesium sulfate, 0.011 g of calcium chloride, 6.78 g of disodium hydrogen phosphate, and 20.0 g of agar were placed in a conical flask, and 1 L of distilled water was added. The mixture was stirred until completely dissolved, and the pH value was adjusted to about 7.0 using 1 mol / L sodium hydroxide solution. The mixture was autoclaved at 121°C for 20 min to obtain the glucosinolate screening medium.

[0030] 3. Screening of glucosinolate-degrading bacterial strains The bacteria in the laboratory strain library were activated and inoculated onto the glucosinolate screening medium by plate streaking. The inoculated medium was placed in a 37°C constant-temperature incubator and incubated upside down. The growth of bacteria on the glucosinolate screening medium was observed at 24 h, 36 h, and 48 h of incubation. The strains with fast growth, large colonies, and large number were selected for subsequent experiments.

[0031] Using the inoculation loop after burning sterilization, an appropriate amount of strain colonies obtained by preliminary screening were picked and inoculated into a shaking tube containing 3 mL of liquid medium. The shaking tube was placed in a 37°C constant temperature incubator and cultured overnight at 180 r / min shaking, to obtain a first-stage seed liquid.

[0032] 1 mL of the first-stage seed liquid was inoculated into a conical flask containing 50 mL of liquid medium, and was also cultured for 24 h under the condition of constant temperature and shaking at 180 r / min at 37°C, to obtain a second-stage seed liquid.

[0033] The OD value of the second-stage seed liquid at a wavelength of 600 nm was determined with un-inoculated liquid medium as a blank control. The OD600 of the second-stage seed liquid was controlled to be between 0.4 and 0.8.

[0034] The prepared seed liquid with an OD600 of about 0.6 was inoculated into the rapeseed meal solid fermentation medium at an inoculation amount (m / v) of 10%. The fermentation was carried out at 37°C for 7 d, and the rapeseed meal fermentation medium was stirred every 12 h during the fermentation.

[0035] After 7 d of fermentation, the fermented rapeseed meal was placed in a 55°C oven and dried to a constant weight (the weight difference was less than 0.3 mg after two consecutive drying), crushed, and stored in a sealed bag.

[0036] 4. Determination of glucosinolate content in rapeseed meal The glucosinolate content in the rapeseed meal before and after fermentation was determined, and the strain with the highest glucosinolate degradation rate was selected as the target strain, including the following procedures: 4.1 The color developing solution of palladium chloride (PdCl2), 0.1% carboxymethyl cellulose sodium (CMC-Na) solution and 0.03 mol / L HCL solution were prepared.

[0037] 4.2 The water bath was preheated before the test. 100 mg of crushed fermented rapeseed meal was weighed and placed in a 20 mL glass test tube, 3 replicates were set for each group, and the labels were marked. The glass tube was placed in the preheated water bath and boiled for 10-15 min. After the end, 10 mL of distilled water at about 90°C was added to the glass tube, and it was boiled in water bath for 25 min. After the end, the test tube was taken out and cooled at room temperature, diluted with distilled water and mixed well, and stood for 20 min. After standing, the solution was centrifuged at 5000 r / min for 10 min, and the precipitate was discarded.

[0038] 4.3 The reagents were mixed according to the following method: (1) Using 5 mL pipette to take 2 mL of filtrate and place it in a 10 mL cuvette, add 4 mL of CMC-Na solution, then add 2 mL of PdCl2 color developing solution, mix well up and down, place at room temperature for 1 h, measure the absorbance value, and the value obtained after subtracting the blank control is E1; (2) Using 5 mL pipette to take 2 mL of filtrate and place it in a 10 mL cuvette, add 4 mL of CMC-Na solution, then add 2 mL of 0.03 mol / L HCL solution, mix well up and down, place at room temperature for 2 h, measure the absorbance value, and the value obtained after subtracting the blank control is E2.

[0039] (3) Use distilled water instead of filtrate as a blank control to measure the absorbance to eliminate the influence of the reagent itself.

[0040] (4) The obtained data is processed according to the following formula: (i) E = E1 - E2.

[0041] (ii) Sulfur glycoside content (μmol / g) = 0.2 + 185.2E.

[0042] (iii) Sulfur glycoside degradation rate (%) = (sulfur glycoside content before treatment - sulfur glycoside content after treatment) / sulfur glycoside content before treatment x 100%.

[0043] 5. Ultraviolet mutagenesis Select 13 strains with a sulfur glycoside degradation rate greater than 50%, activate the third generation sample and inoculate into the corresponding liquid medium, incubate at 37°C, 180 rpm / min in a constant temperature shaker for 16 h, centrifuge at 8000 rpm for 10 min, discard the supernatant, resuspend with the same volume of sterile physiological saline, wash and centrifuge twice at 8000 rpm for 10 min, then add the same volume of sterile physiological saline, vortex and mix well to form a bacterial suspension.

[0044] Preheat the ultraviolet lamp on the super-clean workbench for 30 min, place a sterile glass culture dish with a diameter of 9 cm on the magnetic stirrer, adjust the distance from the ultraviolet lamp to 30 cm, take 8 mL of bacterial suspension in the culture dish, open the lid when the speed is stable, turn on the ultraviolet lamp, and start timing.

[0045] Irradiation time: 0s, 10s, 15s, 20s, 25s, 30s, 40s, 60s, 80s, 100s, 120s, 150s, 200s, 250s The bacterial suspension after irradiation is immediately stored in the dark, and the mutagenized bacterial suspension is placed under an infrared lamp at a distance of 10 cm from the lamp. -5Dilution, take 100 μL evenly coated on the corresponding solid medium, placed in an incubator at 37°C overnight culture 16h, the same conditions do three parallel experiments, take the average of the normal value for the final colony count, calculate the mortality rate.

[0046] Take the mortality rate of 70-80% of the plate to keep, pick up the colony or morphological mutations in the obvious species of culture, according to the above method to determine the degradation rate of glucosinolate, obtain the degradation rate of glucosinolate of 78.09% of the strain 1, named CP71.

[0047] Example 2 This example is aimed at the strain CP71 screened in example 1 to carry out physical and chemical property identification and function verification, including the following flow: 1, morphological observation.

[0048] Macroscopic morphology: as shown in Figure 1 , CP71 in TSA medium at 37°C for 24h, the colony was light yellow, round, surface wet, opaque, edge neat.

[0049] Microscopic morphology: as shown in Figure 2 , CP71 in TSA medium at 37°C for 24h, the cell was rod-shaped, 1.3-1.6 μm x 12.9-4.4 μm, single, pair or chain arrangement, gram-positive.

[0050] 2, physiological and biochemical detection.

[0051] Physiological and biochemical characteristics analysis showed that CP71 presented positive reaction to a variety of enzyme substances, including: phenylalanine arylamine enzyme, α-galactosidase, alanine-phenylalanine-proline arylamine enzyme, pyruvate, alanine arylamine enzyme, maltotriose, β-glucosidase, α-glucosidase, D-glucose, etc. The results showed that CP71 had a relatively wide carbohydrate metabolism ability and proteolytic activity, which may be closely related to its adaptability and functionality in complex environment. The detailed physiological and biochemical characteristics detection results are shown in the table below, which provides an important theoretical basis for the potential application of the strain in agriculture, food and industry and other fields.

[0052] Table 1 physiological and biochemical characteristics of CP71 strain

[0053] After 16S rDNA sequencing of the strain CP71, its gene sequence was obtained, and NCBI was used for BLAST sequence comparison. After comparison, CP71 was a bacteria of the genus Pristinamylum (as shown in Figure 3 ).

[0054] 3, function verification.

[0055] The application further selects the aforementioned bacteria of the genus of Pristinamycete (Pristinia) Priestia sp. ) CP71 to perform fermentation experiments.

[0056] Before the experiment, the strain is activated and cultured to the logarithmic growth phase, the concentration of the bacterial liquid is adjusted to 1×10 8 CFU / mL, and a liquid bacterial agent is prepared. Rapeseed meal that has passed through a 40-mesh sieve is used as the fermentation substrate, and after high-pressure sterilization at 121℃ for 20 minutes, sterile water is added at a material-to-water ratio of 1:1 (w / v) and mixed uniformly, and then 10% (v / w) of the liquid bacterial agent is inoculated. The fermentation system is placed in a 37℃ constant-temperature incubator and left to ferment for 7 days, and the fermentation is stirred once a day to ensure uniform fermentation.

[0057] After the fermentation is completed, the sample is transferred to a 55℃ oven and dried to a constant weight, and the sample is turned regularly during the drying process to ensure uniform drying. After the sample is dried, the contents of crude protein, crude fat, neutral detergent fiber, acid detergent fiber, and glucosinolate are determined. Three repetitions are set for the experiment, and sterilized rapeseed meal that is not inoculated with the bacterial agent is set as a blank control. The results show that the crude protein content of the fermentation treatment group is increased by 6.18%, the crude fat is increased by 28.71%, the neutral detergent fiber is increased by 10.81%, the acid detergent fiber is increased by 12.91%, and the glucosinolate degradation rate is 81.81%.

[0058] Table 2 Comparison of the nutritional value of rapeseed meal before and after fermentation (on a dry basis)

[0059] 4. Breeding effect verification.

[0060] The application further applies the strain CP71 involved in Example 1 to aquaculture, including the following process: 54 selected brocade carp are randomly divided into 2 test groups by a single-factor random grouping design, each group has 3 repetitions, and each repetition has 9 brocade carp. The control group of brocade carp is fed with a daily ration added with 30% rapeseed meal, and the brocade carp is fed twice a day. The test group uses the aforementioned rapeseed meal fermented by the bacteria of the genus of Pristinamycete CP71 to replace the rapeseed meal to feed the brocade carp. After 45 days of breeding, 6 brocade carp are randomly selected from each group and placed in water containing 40 mg / L eugenol for anesthesia. Blood is collected from the tail vein of the brocade carp, centrifuged in a 4℃ centrifuge at a speed of 8000 rpm for 15 min, and the supernatant is taken. The superoxide dismutase, glutathione-peroxidase, glutamic transaminase, and glutamic-pyruvic transaminase in the serum are determined by colorimetry.

[0061] Table 3 Comparison of the biochemical indexes of brocade carp serum

[0062] It can be seen from this that rapeseed meal fermented by Priesteria bacteria CP71 can be used in special aquatic feed. Compared with unfermented rapeseed meal, rapeseed meal fermented by CP71 can increase the activity of superoxide dismutase and glutathione peroxidase in the serum of koi, reduce the activity of serum aspartate aminotransferase and alanine aminotransferase, improve the antioxidant capacity of fish, and promote liver health.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A Pristerophora (Pseudofish) CP71 characterized in that, Priestia sp. ) CP71, characterized in that, The preservation number of the Priestia sp. CP71 is CGMCC No. 34408.

2. An inoculant characterized in that, The product comprises the Priestia sp. CP71 of claim 1, or the microbial inoculum of any one of claims 2-3; and the product is a feed additive, a desulfurizer, a fertilizer, or a plant growth promoter.

3. The bacterial agent of claim 2, wherein The bacterial agent is a solid bacterial agent, a liquid bacterial agent, or a microbial bacterial agent, wherein the total number of viable bacteria of the P. pentosaceus CP71 is 1 x 10 7-10 cfu / g.

4. Process for the preparation of the bacterial agent according to any one of claims 2-3, characterized in that, The product comprises the Priestia sp. CP71 of claim 1, or the microbial inoculum of any one of claims 2-3; and the product is a feed additive, a desulfurizer, a fertilizer, or a plant growth promoter. The product comprises the Priestia sp. CP71 of claim 1, or the microbial inoculum of any one of claims 2-3; and the product is a feed additive, a desulfurizer, a fertilizer, or a plant growth promoter.

5. The preparation method according to claim 4, characterized in that 7. The use of the Priestia sp. CP71 of claim 1, or the microbial inoculum of any one of claims 2-3 in any one of: (1) degrading glucosinolates; (2) preparing a reagent for degrading glucosinolates.

6. A product characterized by, 8. The use of the Priestia sp. CP71 of claim 1, or the microbial inoculum of any one of claims 2-3 in any one of: (1) improving the quality of rapeseed meal; (2) preparing a reagent for improving the quality of rapeseed meal. The improvement of the quality of rapeseed meal comprises increasing the contents of crude protein, crude fat, neutral detergent fiber, and acid detergent fiber, and decreasing the content of glucosinolates.

10. The use of the Priestia sp. CP71 of claim 1, or the microbial inoculum of any one of claims 2-3 in any one of: (1) improving the antioxidant capacity of fish organisms; (2) preparing a reagent for improving the antioxidant capacity of fish organisms.

9. Use according to claim 8, characterized in that, ​ ​

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