Aquatic marssonia sp. And application thereof

Abstract

The invention belongs to the technical field of microorganisms, and in particular relates to a strain of aquatic source marssonia and application thereof. The name of the strain is CMLH375, the classification name of the strain is Massia suwonensis, the strain is preserved in the China General Microbiological Culture Collection Center on December 2, 2024, and the preservation number of the strain is CGMCC No.32864. The invention further discloses a preparation method of the strain. The strain can be used for preparing beta-glucosidase, converting glucoside type soy isoflavone into aglycone type soy isoflavone, reducing the content of mycotoxin in soybean meal, reducing the content of antinutritional factors in the soybean meal, improving the laying rate of poultry, improving the quality of poultry eggs, reducing the feed-egg ratio of poultry and reducing the dosage of soybean meal and corn in feed.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Suwon Marseillaris and application thereof. Background Art

[0002] The laying hen industry is an important part of the development of modern animal husbandry and the modernization of agriculture and rural areas. It is closely related to the safe and stable supply of agricultural products. Improving egg-laying performance and egg quality is the main goal of laying hen farming. As the laying age of laying hens increases, the physiological functions of the body weaken, and the body is prone to oxidative stress and immune stress, which affects the secretion of reproductive hormones in the body, and then affects egg-laying performance and egg quality, which greatly reduces the income of the farm. Therefore, improving the egg-laying performance and egg quality of laying hens at this stage is crucial to the production and economic benefits of farming.

[0003] In the new era of "antibiotics-free feed, antibiotic-reducing breeding, and antibiotic-free products", microecological preparations, Chinese herbal medicines, plant extracts, etc. are regarded as "safe, reliable, and environmentally friendly" alternatives to antibiotics and have been widely used in breeding production. Soybean Isoflavones (SIF), as a class of polyphenol compounds in leguminous plants, have multiple biological activities such as antioxidant, immunity enhancement, cancer inhibition, maintaining normal reproductive function of animals, and promoting egg-laying performance of poultry. Therefore, soy isoflavones have the potential to become a beneficial additive for improving egg-laying performance and egg quality in laying hens. However, soy isoflavones mostly exist in nature as bound glycosides with low activity, while the content of highly active free aglycones is extremely low, and conversion is required to improve their activity. Compared with general conversion methods such as acid hydrolysis, alkaline hydrolysis, and Smith hydrolysis, microbial solid-state fermentation can convert soy isoflavones from glycosides to aglycones, which has the advantages of no pollution, high hydrolase activity, and low cost.

[0004] Soybean meal is an industrial byproduct produced during the extraction of soybean oil. About 97% of the total amount is used for animal feed. Due to its rich nutrition and low water content, soybean meal is considered to be a good source of laying hen feed. According to existing reports, soybean meal contains rich soybean isoflavones, but the content of glycoside isoflavones that are not easily absorbed by the body far exceeds the content of free isoflavones. Therefore, the low-activity soybean isoflavones contained in it can be converted into highly active soybean isoflavone aglycones through microbial solid-state fermentation. In recent years, the domestic soybean resources are in short supply and the degree of dependence on imports is high, which has increased the feed cost of laying hen farming, seriously affecting the development of domestic laying hen farming. Therefore, soybean meal can be fermented by microorganisms to tap the potential of soybean meal and improve its digestibility and utilization to alleviate the increasingly scarce situation of soybean resources. At present, there are not many studies on improving feed quality by microbial solid-state fermentation at home and abroad. Most of them are to add exogenous substances rather than fully utilize the nutrients of the original feed. Therefore, it is of great academic and economic value to screen effective strains that efficiently convert soybean isoflavones to ferment soybean meal and apply it to laying hen farming. Summary of the invention

[0005] The first aspect of the present invention aims to provide a strain of Suwon Marseillaria.

[0006] The second aspect of the present invention aims to provide a culture.

[0007] The third aspect of the present invention aims to provide a method for preparing the culture of the second aspect of the present invention.

[0008] The fourth aspect of the present invention aims to provide a bacterial agent.

[0009] The fifth aspect of the present invention is to provide a fermented soybean meal.

[0010] The sixth aspect of the present invention aims to provide the use of the Suwon Massimo bacteria of the first aspect, the culture of the second aspect, the bacterial agent of the fourth aspect, and the fermented soybean meal of the fifth aspect.

[0011] The seventh aspect of the present invention aims to provide a product.

[0012] The eighth aspect of the present invention aims to provide a feed.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] The first aspect of the present invention provides a strain of Massilia suwonensis, named CMLH375, classified and named Massilia suwonensis, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on December 2, 2024, with a deposit number of CGMCC No.32864.

[0015] The second aspect of the present invention provides a culture obtained by culturing the Mizuhara Massimo Bacteria of the first aspect of the present invention.

[0016] The third aspect of the present invention provides a method for preparing the culture of the second aspect of the present invention, comprising inoculating the Suwon Marseillaris of the first aspect of the present invention into a culture medium and culturing the culture to obtain.

[0017] In some embodiments, the culture medium is at least one of R2A liquid culture medium, LAB liquid culture medium, and MRS liquid culture medium; further is MRS liquid culture medium.

[0018] In some embodiments, the culture temperature is 28-46°C; further 37-40°C.

[0019] In some embodiments, the culturing time is 16-20 hours.

[0020] A fourth aspect of the present invention provides a bacterial agent comprising: the Sugen Massimo bacteria of the first aspect of the present invention or the culture of the second aspect.

[0021] In some embodiments, the bacterial agent further comprises: an auxiliary material acceptable to the bacterial agent.

[0022] In some embodiments, the auxiliary material comprises at least one of a culture medium, an additive and a carrier.

[0023] A fifth aspect of the present invention provides a fermented soybean meal obtained by fermenting the soybean meal with the Suwon Massimo bacteria of the first aspect of the present invention, the culture of the second aspect, or the bacterial agent of the fourth aspect.

[0024] In some embodiments, the method for preparing the fermented soybean meal is: inoculating the culture of the second aspect of the present invention into a soybean meal solution, and fermenting to obtain.

[0025] In some embodiments, the soybean meal solution is a mixture of soybean meal and water.

[0026] In some embodiments, the mass ratio of soybean meal to water is 1:(0.2-1.8); further 1:(0.4-1.6); further 1:(0.6-1.6); further 1:(0.6-0.8).

[0027] In some embodiments, the soybean meal is 20-60 mesh soybean meal; further 30-50 mesh soybean meal; further 35-45 mesh soybean meal.

[0028] In some embodiments, the inoculation amount is 2% v / v-20% v / v; further 3%-18%; further 6%-12%; further 6%-9%.

[0029] In some embodiments, the OD of the culture is 600 The value is 1.56-1.75; further it is 1.70-1.75.

[0030] In some embodiments, the fermentation time is 12-144 h; further 20-120 h; further 22-26 h.

[0031] In some embodiments, the fermentation temperature is 26-48°C; further 28-46°C; further 38-42°C; further 39-40°C.

[0032] In some embodiments, the fermentation further comprises a drying step.

[0033] The sixth aspect of the present invention provides any one of A1)-A2) applications:

[0034] A1) Use of the waterborne Marseille bacteria of the first aspect, the culture of the second aspect, or the bacterial agent of the fourth aspect of the present invention in any one of a1) to a5);

[0035] a1) preparing β-glucosidase;

[0036] a2) converting glycoside-type soy isoflavones into aglycone-type soy isoflavones;

[0037] a3) Reducing the content of mycotoxins in soybean meal;

[0038] a4) Reducing the content of anti-nutritional factors in soybean meal;

[0039] a5) preparing a product, wherein the product is used in any one of a1) to a4);

[0040] A2) Use of the water-derived Massimo bacteria of the first aspect, the culture of the second aspect, the bacterial agent of the fourth aspect, or the fermented soybean meal of the fifth aspect of the present invention in any one of b1) to b5);

[0041] b1) Improve the egg production rate of poultry;

[0042] b2) Improve the quality of poultry eggs;

[0043] b3) Reduce the feed-to-egg ratio of poultry;

[0044] b4) Reduce the amount of soybean meal and corn in feed;

[0045] b5) preparing a product, wherein the product is used in any one of b1) to b4).

[0046] In some embodiments, the glycoside type soy isoflavones include at least one of daidzein, daidzein, and genistin.

[0047] In some embodiments, the aglycone-type soy isoflavones include at least one of daidzein, daidzein and genistein.

[0048] In some embodiments, the mycotoxin comprises deoxynivalenol.

[0049] In some embodiments, the antinutritional factor comprises β-conglycinin.

[0050] In some embodiments, the product described in a5) is a reagent.

[0051] In some embodiments, the poultry is chicken; further, laying hen.

[0052] In some embodiments, the egg quality comprises at least one of eggshell strength, egg weight, egg white height, Haugh unit, yolk color, yolk weight, eggshell weight, and egg white weight; and further comprises at least one of egg white height and Haugh unit.

[0053] In some embodiments, the product b5) is feed, medicine, microbial additive, or fermented feed.

[0054] A seventh aspect of the present invention provides a product comprising: the Sumigen Massimo bacteria of the first aspect of the present invention, the culture of the second aspect, the bacterial agent of the fourth aspect, or the fermented soybean meal of the fifth aspect.

[0055] In some embodiments,

[0056] Any product from c1) to c2):

[0057] c1) A product comprising: the waterborne Marseille bacteria of the first aspect, the culture of the second aspect, or the bacterial agent of the fourth aspect of the present invention;

[0058] c2) A product comprising: the Sumigen Massimo bacteria of the first aspect, the culture of the second aspect, the bacterial agent of the fourth aspect, or the fermented soybean meal of the fifth aspect.

[0059] In some embodiments, the product described in c1) is a reagent.

[0060] In some embodiments, the product described in c1) is used in any one of a1) to a4) in the sixth aspect of the present invention.

[0061] In some embodiments, the product described in c2) is feed, medicine, microbial additive, or fermented feed.

[0062] In some embodiments, the product described in c2) is used in any one of b1) to b4) in the sixth aspect of the present invention.

[0063] An eighth aspect of the present invention provides a feed comprising: a basal diet and the fermented soybean meal according to the fifth aspect of the present invention.

[0064] In some embodiments, the content of the fermented soybean meal in the feed is 3-15wt%; further 4.267-12.8wt%; further 7.8-8.6wt%.

[0065] In some embodiments, the basal diet is a corn-soybean meal-based basal diet.

[0066] In some embodiments, the corn-soybean meal-based basal diet comprises: corn, corn dry whole alcohol grains, soybean meal, soybean oil, rock powder, homemade semi-finished pre-SYL pre-powder F for laying hens at the peak of egg production, DL-methionine and L-lysine sulfate.

[0067] According to the screening target, physiological and biochemical characteristics and genetic characteristics, the present invention uses modern molecular biological methods to separate and screen a strain of Suwon Marseillaria CMLH375 from naturally fermented wheat bran through a large number of specific sortings and from wheat bran that has been naturally fermented for 7 days. The strain is an aerobic Gram-negative bacterium, which can form a large yellow circular colony with a bulge in the middle after being cultured on an MRS solid medium at 37°C for 24 hours. The 16S rRNA gene of the bacteria is amplified using a universal primer sequence to perform a PCR amplification reaction and sequence the same. The sequencing results are sequenced in the NCBI database to analyze the homology of the strains, and it is found that the 16SrRNA (SEQ ID NO: 3) thereof has the highest sequence homology with Suwon Marseillaria, with a similarity of 99.27%. The strain was inoculated in MRS liquid medium and cultured for 24 hours. The β-glucosidase activity of the bacterial supernatant was 3.12±0.44μmol / (g·min), and the bacterial enzyme activity was 8.75±0.19μmol / (g·min). The culture (fermentation liquid) of the strain was inoculated in 40-mesh soybean meal and fermented for 48 hours. The fermentation product was separated and purified and then detected by HPLC. The product was preliminarily speculated by comparing with the standard products of daidzein, daidzein, and genistein. Finally, it was determined that Suwon Marseilles could convert glycoside-type soybean isoflavones in soybean meal into aglycon-type soybean isoflavones. Aglycon-type soybean isoflavones have multiple biological activities such as anti-oxidation, improving immunity, inhibiting cancer, and maintaining normal reproductive function of animals. Therefore, Suwon Marseilles can become a beneficial fermented feed inoculant to improve egg production performance and egg quality in poultry production. The bacteria was fermented and inoculated into soybean meal to produce fermented soybean meal, which was then used in poultry feed. Poultry feeding experiments were conducted to study its probiotic properties. The results showed that Suwon Marseille bacteria can reduce the feed-egg ratio of poultry, increase poultry egg production, and improve the quality of poultry eggs. It can be used as a new and effective microorganism in the field of fermented feed production.

[0068] The beneficial effects of the present invention are:

[0069] The present invention provides a strain of Massilia suwonensis, named CMLH375, classified and named Massilia suwonensis, which was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration Committee on December 2, 2024, and the deposit number is CGMCC No. 32864. The strain can be used to prepare β-glucosidase, convert glycoside-type soybean isoflavones into aglycone-type soybean isoflavones, reduce the content of mycotoxins in soybean meal, reduce the content of anti-nutritional factors in soybean meal, increase poultry egg production rate, improve poultry egg quality, reduce poultry feed-egg ratio, and reduce the amount of soybean meal and corn in feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1The figures show the status of some strains on aesculin MRS medium during the initial screening of bacteria with high β-glucosidase production in Example 1.

[0071] Figure 2 A standard curve of p-nitrophenol (pNP) solution is shown.

[0072] Figure 3 The figure shows the growth status of Massilia subspecies CMLH375 on MRS solid medium.

[0073] Figure 4 The growth curve of Massimodium aquatica CMLH375 is shown.

[0074] Figure 5 The results show the effect of fermentation of soybean meal by Suwon Massimo Bacteria CMLH375 on the conversion of soybean isoflavones. DETAILED DESCRIPTION

[0075] definition

[0076] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0077] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0078] The present invention is further described in detail below through specific examples.

[0079] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0080] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The materials and reagents used in the examples are commercially available unless otherwise specified. The manufacturers of the reagents used are indicated, and similar products from other manufacturers are substitutes.

[0081] The composition of the culture medium in the embodiment of the present invention is as follows:

[0082] Cellobiose medium: yeast extract 2.5 g / L, cellobiose 2.5 g / L, peptone 2.5 g / L, ammonium sulfate 41 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.2 g / L, and the rest is water.

[0083] MRS solid medium: peptone 10 g / L, beef extract powder 5 g / L, yeast extract powder 4 g / L, glucose 20 g / L, potassium dihydrogen phosphate 2 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, agar 15 g / L, Tween 1 g / L, and the rest is water.

[0084] Esculin MRS medium: peptone 10 g / L, beef extract powder 5 g / L, yeast extract powder 4 g / L, glucose 20 g / L, potassium dihydrogen phosphate 2 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, agar 15 g / L, Tween 1 g / L, esculin 1 g / L, ferric ammonium citrate 3 g / L, and the rest is water.

[0085] MRS liquid medium: peptone 10 g / L, beef extract powder 8 g / L, yeast extract powder 4 g / L, glucose 20 g / L, potassium dihydrogen phosphate 2 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 1 g / L, and the rest is water.

[0086] The soybean meal powder required for fermentation is obtained by pulverizing conventional soybean meal and passing it through a 40-mesh sieve, and the wheat bran is not treated at all.

[0087] In the experiments of Examples 1-6 of the present invention, three replicates were set, and 27 replicates were set for the animal experiment in Example 7. The experimental data were statistically analyzed using SPSS 26.0 software, and one-way analysis of variance was used. P<0.05 indicates significant difference, and 0.05<P<0.1 indicates a significant trend.

[0088] Example 1: Screening of bacteria with high β-glucosidase production

[0089] A1) Primary screening of strains with high β-glucosidase production

[0090] Mix wheat bran and sterile distilled water at a mass ratio of 1:1, seal it, and end fermentation after 7 days of fermentation at 37°C with an acid fragrance. Take 2 g of the obtained naturally fermented wheat bran and add it to 50 mL of cellobiose medium, shake and culture for 24 h, then dilute it. Take 10 -3 、10 -4 、10 -5 dilution solutions and plate them on an esculin MRS plate. After culturing upside down for 24 h, if a degradation zone appears around the colony, it is the target strain producing β-glucosidase. Then use an inhibition zone measuring instrument to measure the ratio of the black circle diameter to the single colony diameter of the strain with an obvious black circle. Pictures of the measurement of the black degradation zone of some strains are shown in Figure 1 , and select strains with a larger ratio for the next rescreening.

[0091] A2) Rescreening of high-yielding β-glucosidase lactic acid bacteria

[0092] The colonies producing black circles on the culture medium were picked and placed on MRS solid culture medium, and the strains were purified by the four-zone streak method. The strains were cultured at 37°C for 12 h. The operation can be repeated until a pure strain is obtained.

[0093] The p-NP semi-quantitative colorimetric method was used to evaluate the glycosidase activity of the primary screening strains, and the final enzyme activity was calculated based on the standard curve. The specific enzyme activity measurement scheme is as follows:

[0094] 1) After the strain was cultured in MRS liquid medium at 37°C and 180 rpm for 72 h, 1 mL of the bacterial suspension was taken and centrifuged (8000 rpm, 4°C, 2 min) to obtain the bacterial cells and the bacterial supernatant. The bacterial supernatant was directly used for enzyme activity determination. The bacterial cells were washed twice with 0.85% NaCl solution and then suspended in 0.5 mL 0.85% NaCl solution for enzyme activity determination.

[0095] 2) Reaction system (3.5 mL): Take 1 mL of bacterial cells, wash them twice with 1 mL of 0.85% NaCl, suspend them in 0.5 mL of 0.85% NaCl solution, and then add 0.5 mL of CH 3 COOH-CH 3 COONa buffer (concentration 0.2 mol / L, pH 5.0) and 0.5 mL of p-nitrophenyl-β-glucoside (pH 5.0, pNPG concentration 5 mmol / L) were mixed and reacted at 45°C for 0.5 h. Then, twice the volume (2 mL) of 0.5 M Na 2 CO 3 The solution was centrifuged (8000 rpm, 4°C, 15 min), and 200 μL of the supernatant was pipetted onto a 96-well plate. The absorbance was then measured at 400 nm using a full-wavelength microplate reader.

[0096] 3) Prepare blank samples by using 0.85% NaCl buffer instead of bacterial suspension, and other treatments are the same as the samples.

[0097] 4) Take 0.5 mL of the bacterial supernatant and mix it directly with 0.5 mL of CH 3 COOH-CH 3 COONa buffer (concentration of 0.2 mol / L, pH 5.0) and p-nitrophenyl-β-glucoside (pH 5.0, pNPG concentration 5 mmol / L) were mixed, and MRS liquid culture medium was used instead of the bacterial supernatant to prepare a blank sample. Other treatments were the same as above.

[0098] 5) The standard curve was obtained by colorimetric analysis of p-nitrophenol (pNP) solutions of different concentrations (10-60 μmol / L) at 400 nm. Figure 2 The dry weight of the bacteria was calculated as follows: g of bacterial cells per liter = OD600 / 2.6168.

[0099] Enzyme activity definition: the amount of pNP produced per gram of bacteria (dry weight) per minute under the above reaction conditions. Enzyme activity unit: p-nitrophenol μmol / (g·min). The results of enzyme activity determination of some strains are shown in Table 1.

[0100] Table 1. Results of enzyme activity assay of some strains

[0101] Strain number Cell (bacteria) enzyme activity μmol / (g·min) Enzyme activity in bacterial supernatant μmol / (g·min) CMLH375 8.75±0.19 3.12±0.44 ZT1 7.34±3.35 5.81±0.09 XT4 7.12±0.94 1.67±0.2 RMF2 6.61±0.3 5.69±0.52 K25 5.99±3.99 5.8±0.33 SM1 5.6±0.66 5.99±0 ZD2 4.23±0.85 3.62±0.02 ZL12 3.28±0.25 3.42±0.05

[0102] Example 2: Identification of strain CMLH375 with strong enzyme activity

[0103] B1) Extract genomic DNA from strain CMLH375 according to the instructions of the kit (DNeasyR Blood & Tissue Kit, QIANGEN), and detect it by 0.8% agarose gel electrophoresis, without diffusion or tailing; and use a microplate reader to detect the quality and concentration of the extracted DNA. DNA samples with an A260 / A280 ratio of 1.8-2.0 and a concentration of not less than 20ng / μL are considered qualified samples.

[0104] B2) PCR amplification

[0105] The qualified strain DNA samples were subjected to PCR amplification reaction using universal primers for bacterial 16S rRNA gene amplification. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the specific sequences are as follows:

[0106] Upstream primer 27F: 5′-AGAGTTTGATCATGGCTCAG-3′, SEQ ID NO: 1;

[0107] Downstream primer 1492R: 5′-TAGGGTTACCTTGTTACGACTT-3′, SEQ ID NO:2.

[0108] Reaction system (30 μL): DNA template 1.5 μL, upstream primer 27F 1.5 μL, downstream primer 1492R 1.5 μL, Gold Mix 25.5 μL (Beijing Qingke Xinye Biotechnology Co., Ltd.).

[0109] The reaction procedure is shown in Table 2.

[0110] Table 2. PCR program

[0111]

[0112]

[0113] The 16S rRNA gene sequence of CMLH375 was compared with the EZ NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to analyze the homology of the strains. The bacteria with the highest homology to CMLH375 was Massilia suwonensis, with a similarity of 99.27%.

[0114] The strain CMLH375 is named CMLH375, classified as Massilia suwonensis, and was deposited in the General Microbiology Center of the China Culture Collection Administration on December 2, 2024, with the deposit number CGMCC No.32864. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0115] Example 3: Colony morphology and characteristics of Massilia suwonensis CMLH375

[0116] The best growth conditions for the isolated Suwon Marseillaria CMLH375 were 37°C and aerobic. After culturing on MRS solid medium for 48 h, large yellow round colonies were formed with smooth raised surfaces and neat colony edges ( Figure 3 ).

[0117] Example 4: Growth curve drawing of Massilia suwonensis CMLH375

[0118] The growth curve of Suwon Marseillaria CMLH375 was drawn at 37℃ and 180rpm for 26 hours ( Figure 4 ) It can be seen that strain CMLH375 entered the logarithmic growth phase at 4h, entered the plateau phase at 8h, and began to decline at 22h.

[0119] Example 5: Fermentation of soybean meal using Suwon Massimo Bacillus CMLH375 and optimization of fermentation conditions

[0120] C1) Test materials and methods

[0121] Raw materials: 40 mesh soybean meal, distilled water;

[0122] Preparation of fermentation broth: Take the above-mentioned water source Marseille bacteria and inoculate them into MRS liquid culture medium at a 2% v / v inoculation rate, and culture them at 37°C for 20 hours to reach OD 600 The value was about 1.72, and after activation three times, the fermentation liquid of Suwon Marseille CMLH375 (OD 600value is about 1.72);

[0123] C2) Optimization of soybean meal fermentation conditions: The effects of different factors on conversion were investigated, and single-factor tests were conducted on fermentation temperature, fermentation time, strain ratio, inoculation amount, and liquid-to-solid ratio, taking aglycones as the judgment standard;

[0124] C3) High performance liquid chromatography detection

[0125] After the fermentation of soybean meal is completed, 0.1 g of sample is accurately weighed and dissolved in 80% methanol solution, ultrasonically oscillated for 30 min, and the volume of 90% methanol solution is adjusted to 10 mL. The methanol solution is mixed using an oscillator, filtered through a filter membrane, and 2 mL of the filtrate is pipetted into a chromatographic bottle for later use.

[0126] Preparation of standard solutions: Weigh 4 mg of dried daidzein, daidzin, genistin, daidzein, daidzin and genistein accurately, and dilute to 10 mL with dimethyl sulfoxide solution to prepare standard stock solutions, with a concentration of 400 mg / L. Take 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL of the six standard stock solutions of daidzein, daidzin, genistin, daidzein, daidzin and genistein, respectively; add an equal volume of water, and dilute to 10 mL with 50% dimethyl sulfoxide solution to obtain a standard series with concentrations of 4.0 mg / L, 8.0 mg / L, 16.0 mg / L, 24.0 mg / L, 32.0 mg / L and 40.0 mg / L, respectively.

[0127] On-machine detection instrument conditions: chromatographic column: C18 ODS-3 (5μm, 4.6mm×250mm); column temperature: 30°C; mobile phase: mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution (pH=3); gradient elution, as shown in Table 3 below; flow rate: 1.0mL / min detection wavelength: 260nm.

[0128] The linear equations and correlation coefficients of the standard curves of daidzein, daidzein, genistin, daidzein, daidzein and genistein are shown in Table 4.

[0129] Table 3. HPLC mobile phase ratio

[0130] Time / min Mobile phase A / % v / v Mobile phase B / % v / v 0 12 88 10 18 82 23 24 76 30 30 70 50 30 70 55 80 20 56 12 88 60 12 88

[0131] Table 4. Standard curve linear equations and correlation coefficients for daidzein, daidzein, genistin, daidzein, daidzein and genistein

[0132]

[0133]

[0134] The changes in the isoflavone content of soybean meal at different inoculation amounts (v / v) at 37°C, a liquid-to-solid ratio of 1:1 (i.e., the mass ratio of distilled water to soybean meal is 1:1), and fermentation for 48 hours are shown in Table 5. The changes in the isoflavone content of soybean meal at different fermentation times at 37°C, an inoculation amount of 6%, a liquid-to-solid ratio of 1:1 are shown in Table 6. The changes in the isoflavone content of soybean meal at different fermentation temperatures at an inoculation amount of 6%, a liquid-to-solid ratio of 1:1, and fermentation for 48 hours are shown in Table 7. The changes in the isoflavone content of soybean meal at 37°C, an inoculation amount of 6%, a fermentation for 48 hours, and different liquid-to-solid ratios are shown in Table 8. It can be seen from Tables 5-8 that the optimal fermentation conditions are an inoculation amount of 6%, a liquid-to-solid ratio of 0.8:1 (although the aglycone yield of a liquid-to-solid ratio of 1.6:1 is higher than that of 0.8:1, there is no significant difference, and in actual production, the lower the moisture content, the better to prevent mold, so a liquid-to-solid ratio of 0.8:1 is set as the optimal fermentation condition), a temperature of 40°C, and a fermentation time of 24 hours.

[0135] Table 5. Changes in soybean isoflavone content in soybean meal with different inoculation amounts at 37°C, liquid-to-solid ratio of 1:1, and fermentation for 48 h

[0136]

[0137] Table 6. Changes in soybean isoflavone content in soybean meal at different fermentation times at 37°C, inoculation volume 6%, and liquid-to-solid ratio 1:1

[0138]

[0139]

[0140] Table 7. Changes in soybean isoflavone content in soybean meal at different fermentation temperatures with inoculation amount of 6%, liquid-to-solid ratio of 1:1, and fermentation for 48 hours

[0141]

[0142] Table 8. Changes in soybean isoflavone content at 37℃, 6% inoculation, 48h fermentation, and different liquid-to-solid ratios

[0143]

[0144]

[0145] Example 6: Fermentation of soybean meal using Suwon Marseillaria CMLH375 under optimal fermentation conditions

[0146] D1) Measurement indicators

[0147] The isoflavone contents of various types of soybean meal fermented under the optimal fermentation conditions were detected.

[0148] Since a high liquid-to-solid ratio in actual production can easily lead to moldy feed, the fermentation conditions (actual fermentation conditions) finally adopted in the present invention are as follows: inoculation amount 6%, liquid-to-solid ratio 0.6:1, temperature 37°C, fermentation time 72h, and the soybean content was measured under these conditions.

[0149] To detect the anti-nutritional factors and mycotoxins content of fermented soybean meal under the optimal fermentation conditions.

[0150] D2) High performance liquid chromatography test The raw materials and method are the same as those in step C1) of Example 5; the materials required for the detection of anti-nutritional factors and mycotoxins content are obtained by drying the fermented soybean meal at 70° C. for 24 h, grinding it with a pulverizer, and then passing it through a 60-mesh sieve.

[0151] D3) The HPLC detection step is the same as step C3) in Example 5; the activity of β-conglycinin before and after soybean meal fermentation is determined according to the kit operation method of Ruixin Biotechnology Co., Ltd., and the content of vomitoxin DON is determined according to the kit operation method of Jiangsu Enzyme Immunity Industrial Co., Ltd.

[0152] D4) Test results

[0153] It can be seen from Table 9 that under the optimal fermentation conditions (inoculum size 6%, liquid-to-solid ratio 0.8:1, temperature 40°C, fermentation time 24h), the soybean isoflavone aglycone content can be significantly increased to 1998.88±13.7μg / g. Under the actual fermentation conditions (inoculum size 6%, liquid-to-solid ratio 0.6:1, temperature 37°C, fermentation time 72h), the soybean meal aglycone content can also be increased from 39.57±6.31μg / g to 1631.19±37.24μg / g.

[0154] Table 9. Soybean isoflavone content of soybean meal fermented by Suwon Marseilla CMLH375 under different conditions and unfermented soybean meal ( Figure 5 )

[0155]

[0156] It can be seen from Table 10 that the use of the Suwon Massimo Bacillus CMLH375 of the present invention under the optimal fermentation conditions can significantly reduce the anti-nutritional factors and mycotoxin content of unfermented soybean meal.

[0157] Table 10. Changes in the content of vomitoxin and β-conglycinin in soybean meal fermented by CMLH375 under optimal conditions and actual conditions and unfermented soybean meal

[0158]

[0159] Example 7: Application of Suwon Marseillaria CMLH375 in the preparation of fermented feed for laying hens

[0160] Feeding experiment of Roman pink laying hens

[0161] E1) Experimental Animal Chicken Grouping

[0162] The experimental broilers were 1080 Roman pink laying hens (Sichuan Tieqi Lishi Industrial Co., Ltd.) in the late egg-laying period (48 weeks old), with an average egg production rate of 0.889±0.025. They were randomly divided into 4 groups: control group (CON), soybean isoflavone 40μg / g group (CMLH375 40μg / g), soybean isoflavone 80μg / g group (CMLH375 80μg / g), soybean isoflavone 120μg / g group (CMLH375 120μg / g), 10 replicates per group, and 27 chickens per replicate. The broilers were raised in H-type cages with free drinking water. To ensure the intake of a quantitative amount of aglycone-type soybean isoflavones, quantitative feeding was adopted. During the experiment, the immunization procedure was carried out according to the conventional breeding procedure of laying hens. The experimental period was 42 days.

[0163] The above-mentioned soy isoflavones 40 μg / g group (CMLH375 40 μg / g), soy isoflavones 80 μg / g group (CMLH37580 μg / g), and soy isoflavones 120 μg / g group (CMLH375 120 μg / g) were respectively added with 42.67 g / kg, 85.33 g / kg, and 128 g / kg of the fermented soybean meal prepared under the actual fermentation conditions in Example 6 in the feed (calculated as 1500 μg of aglycone-type soy isoflavones per gram of fermented soybean meal).

[0164] E2) Experimental diet

[0165] The experimental diet was prepared with reference to the Roman Pink Layer Feeding Manual as a corn-soybean meal-based basal diet and fed in the form of powder and pellets, and the nutritional level of each group was accurately calculated using feed formulation software (Table 11).

[0166] Table 11. Basic diet composition and nutrient levels

[0167]

[0168] E3) To ensure that the nutritional level of each group of laying hens is consistent, the feed amount for each chicken in the blank group is 110 g, and the daily feed mixing rules for different treatment groups (calculated as 270 chickens) are shown in Table 12.

[0169] Table 12. Daily mixing rules

[0170]

[0171]

[0172] E4) Determination of indicators

[0173] Production performance

[0174] Taking repetition as the unit, record the daily laying rate of marketable eggs (SER) of laying hens. Randomly select 30 eggs at the fourth week of the experiment to detect egg quality, and weigh the remaining feed after the experiment to calculate the feed conversion ratio (FCR) of each group.

[0175] Laying rate of marketable eggs (SER) = total number of marketable eggs / number of chickens in stock × 100%.

[0176] Egg quality detection indicators include: albumen height, Haugh unit.

[0177] Albumen height and Haugh unit were measured using an EMT-7300 multi-functional egg quality detector (Robotmation, Japan). The measurement method refers to NY / T 823-2020 "Nomenclature and Measurement Statistics of Poultry Production Performance".

[0178] Feed conversion ratio (FCR) = total feed consumption (kg) within a certain period / total egg production (kg) during the same period.

[0179] E5) Results

[0180] As can be seen from Table 13, at the 6th week of the experiment, compared with the control group, the laying rate of marketable eggs (SER) in the CMLH375 120 μg / g group was significantly increased (P < 0.05), and there was a tendency of difference at the 3rd and 4th weeks (0.05 < P < 0.1). During the whole experiment, the average laying rate of marketable eggs (SER) in the three CMLH375 treatment groups was significantly increased compared with the blank control group (P < 0.05).

[0181] As can be seen from Table 14, at the 4th week of the experiment, compared with the control group, the albumen height and Haugh unit in the three CMLH375 treatment groups were significantly different from those in the blank control group (P < 0.05), and the treatment groups significantly increased the albumen height and Haugh unit of eggs.

[0182] As can be seen from Table 15, feeding laying hens with the fermented soybean meal feed prepared by fermenting Massilia aquatica can reduce the feed conversion ratio of laying hens. Among them, the CMLH375 120 μg / g group has the best effect, and the feed conversion ratio can reach 2.005:1.

[0183] As can be seen from Table 11 and Table 15, feeding laying hens with the fermented soybean meal feed prepared by fermenting Massilia aquatica can reduce the usage of soybean meal and corn in the feed on the premise of reducing the feed conversion ratio.

[0184] In summary, adding soybean meal fermented by Suwon Massiella CMLH375 of the present invention to the laying hen diet can significantly improve the egg production rate of Roman pink laying hens in the late laying period, improve egg quality, and reduce the feed-egg ratio. Therefore, Suwon Massiella can be used as a feeding microbial additive in fermented feed, and the recommended addition amount of fermented soybean meal of Suwon Massiella CMLH375 of the present invention in laying hen diet is 80kg / t.

[0185] Table 13. Effect of soybean isoflavones transformed by Suwon Marseilla on the egg production rate of Roman Pink laying hens

[0186]

[0187] Table 14. Effect of soybean isoflavones transformed by Suwon Marseilla on the quality of Roman pink eggs

[0188]

[0189] Table 15. Effect of soybean isoflavones transformed by Suwon Marseillaria on the feed-to-egg ratio of Roman pink laying hens

[0190]

[0191] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A strain of Massilia suwonensis, named CMLH375, classified as Massilia suwonensis, was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on December 2, 2024, with the deposit number CGMCC NO.32864.

2. A culture obtained by culturing the Suwon Massimo bacteria according to claim 1.

3. A method for preparing the culture according to claim 2, comprising inoculating the Suwon Massimo Bacteria according to claim 1 into a culture medium and culturing the culture to obtain the culture.

4. A bacterial agent comprising: the Mizuhara Massimo bacteria according to claim 1 or the culture according to claim 2.

5. A fermented soybean meal obtained by fermenting soybean meal using the Suwon Massimo bacteria according to claim 1, the culture according to claim 2, or the bacterial agent according to claim 4.

6. The fermented soybean meal according to claim 5, characterized in that The method for preparing the fermented soybean meal is as follows: inoculating the culture according to claim 2 into a soybean meal solution, and fermenting to obtain; Preferably, the soybean meal solution is a mixture of soybean meal and water; Preferably, the mass ratio of the soybean meal to water is 1:(0.2-1.8); further 1:(0.4-1.6); Preferably, the inoculation amount is 2% v / v-20% v / v; further 3%-18%.

7. The fermented soybean meal according to claim 5 or 6, characterized in that The fermentation time is 12-144h; further 20-120h; Preferably, the fermentation temperature is 26-48°C; further 28-46°C; further 38-42°C.

8. Any application of A1)-A2): A1) Use of the waterborne Marseille bacteria according to claim 1, the culture according to claim 2, or the bacterial agent according to claim 4 in any one of a1) to a5); a1) preparing β-glucosidase; a2) converting glycoside-type soy isoflavones into aglycone-type soy isoflavones; a3) Reducing the content of mycotoxins in soybean meal; a4) Reducing the content of anti-nutritional factors in soybean meal; a5) preparing a product, wherein the product is used in any one of a1) to a4); A2) Use of the waterborne Marseille bacteria according to claim 1, the culture according to claim 2, the bacterial agent according to claim 4, or the fermented soybean meal according to any one of claims 5 to 7 in any one of b1) to b5); b1) Improve the egg production rate of poultry; b2) Improve the quality of poultry eggs; b3) Reduce the feed-to-egg ratio of poultry; b4) Reduce the amount of soybean meal and corn in feed; b5) preparing a product, wherein the product is used in any one of b1) to b4).

9. The use according to claim 8, characterized in that: The glycoside type soy isoflavones include at least one of daidzein, daidzein and genistin; Preferably, the aglycone type soybean isoflavones include at least one of daidzein, daidzein and genistein; Preferably, the mycotoxin comprises vomitoxin; Preferably, the anti-nutritional factor comprises β-conglycinin; Preferably, the product described in a5) is a reagent; Preferably, the poultry is chicken; Preferably, the egg quality comprises at least one of eggshell strength, egg weight, egg white height, Haugh unit, yolk color, yolk weight, eggshell weight, and egg white weight; Preferably, the product b5) is feed, medicine, microbial additive, or fermented feed.

10. A product comprising: the Sugenous Massimo bacteria according to claim 1, the culture according to claim 2, the bacterial agent according to claim 4, or the fermented soybean meal according to any one of claims 5 to 7.

11. A feed comprising: a basic diet and the fermented soybean meal according to any one of claims 5 to 7.

12. The feed according to claim 11, characterized in that The content of the fermented soybean meal in the feed is 3-15wt%; further 4.267-12.8wt%; Preferably, the basal diet is a corn-soybean meal type basal diet.

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