M. aquatica and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a Massilia suwonensis strain and application thereof. The strain is named CMLH375, classified and named as Massilia suwonensis, and preserved in the China General Microbiological Culture Collection Center on December 2, 2024, with a preservation number of CGMCC No. 32864. The strain can be used to prepare beta-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, improve the egg production rate of poultry, improve the egg quality of poultry, reduce the feed-egg ratio of poultry, and reduce the use amount of soybean meal and corn in feed.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of *Massezia spp.* and its applications. Background Technology

[0002] The egg-laying hen industry is an important component of modern animal husbandry development and agricultural and rural modernization, and is closely related to the safe and stable supply of agricultural products. Improving egg production performance and egg quality is the main goal of egg-laying hen farming. As the laying age of hens increases, various physiological functions of the body weaken, and the body is prone to oxidative stress and immune stress, affecting the secretion of reproductive hormones, which in turn affects egg production performance and egg quality, significantly reducing the profitability of farms. Therefore, improving the egg production performance and egg quality of hens at this stage is crucial to the production and economic benefits of farming.

[0003] In the new era of "antibiotic-free feed, reduced antibiotic use in livestock farming, and antibiotic-free products," microecological preparations, traditional Chinese medicine, and plant extracts are considered "safe, reliable, and environmentally friendly" antibiotic alternatives and have been widely used in livestock production. Soybean isoflavones (SIF), a class of polyphenolic compounds from legumes, possess various bioactive functions, including antioxidant activity, immune enhancement, anticancer properties, maintenance of normal reproductive function in animals, and promotion of egg production performance in poultry. Therefore, soy isoflavones have the potential to become a beneficial additive in egg production to improve egg production performance and egg quality. However, soy isoflavones in nature mostly exist in the form of bound glycosides with low activity, while the content of highly active free aglycones is extremely low, requiring conversion to increase 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, offering advantages such as no pollution, high hydrolytic enzyme activity, and low cost.

[0004] Soybean meal is an industrial byproduct of soybean oil extraction, with approximately 97% of its total production used in animal feed. Due to its rich nutritional content and low moisture content, soybean meal is considered a good source of feed for laying hens. According to existing reports, soybean meal contains abundant soy isoflavones; however, the content of glycoside isoflavones, which are not easily absorbed by the body, far exceeds that of free isoflavones. Therefore, microbial solid-state fermentation can convert the low-activity soy isoflavones into highly active soy isoflavone aglycones. Furthermore, in recent years, the scarcity of domestic soybean resources and high reliance on imports have increased feed costs for laying hens, severely impacting the development of the domestic laying hen industry. Therefore, microbial fermentation of soybean meal can tap its potential, improve its digestibility and utilization, and alleviate the increasingly scarce soybean resources. Currently, there is limited research both domestically and internationally on using microbial solid-state fermentation to improve feed quality; most studies focus on adding exogenous substances rather than fully utilizing the original feed's nutrients. Therefore, screening for highly efficient strains that convert soy isoflavones for fermenting soybean meal and applying it to laying hen farming has significant academic and economic value. Summary of the Invention

[0005] The first objective of this invention is to provide a strain of *Morseia sapiens*.

[0006] A second aspect of the present invention is to provide a culture.

[0007] The third objective of this invention is to provide a method for preparing the culture of the second aspect of this invention.

[0008] The fourth aspect of this invention is to provide a microbial agent.

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

[0010] The sixth aspect of this invention aims to provide the application of the *Massezi sacchariformis* of the first aspect, the culture of the second aspect, the microbial agent of the fourth aspect, and the fermented soybean meal of the fifth aspect.

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

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

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] In a first aspect, a strain of Massimo suwonensis, named CMLH375 and classified as Massimo suwonensis, is provided. It was deposited on December 2, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32864.

[0015] A second aspect of the present invention provides a culture obtained by culturing *Massezia spp.*, a bacterium of the first aspect of the present invention.

[0016] A third aspect of the present invention provides a method for preparing a culture of the second aspect of the present invention, wherein *Miscanthus sinensis* of the first aspect of the present invention is inoculated into a culture medium and cultured to obtain the culture.

[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; more specifically, it is MRS liquid culture medium.

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

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

[0020] A fourth aspect of the present invention provides a microbial agent comprising: *Morgiella jejuni* of the first aspect of the present invention or a culture of the second aspect of the present invention.

[0021] In some embodiments, the microbial agent further comprises: excipients acceptable to the microbial agent.

[0022] In some embodiments, the excipient comprises at least one of culture medium, additives, and carrier.

[0023] A fifth aspect of the present invention provides a fermented soybean meal, obtained by fermenting soybean meal using the *Morseella sacchariformis* of the first aspect of the present invention, the culture of the second aspect, or the microbial agent of the fourth aspect.

[0024] In some embodiments, the fermented soybean meal is prepared by inoculating the culture of the second aspect of the present invention into a soybean meal solution and fermenting it to obtain the product.

[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); even further, 1:(0.6-1.6); and still further, 1:(0.6-0.8).

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

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

[0029] In some embodiments, the OD of the culture 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 hours; further, 20-120 hours; and even further, 22-26 hours.

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

[0032] In some embodiments, the fermentation process is followed by a drying step.

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

[0034] A1) The use of the *Massezi sericulture* of the first aspect of the present invention, the culture of the second aspect, or the bacterial agent of the fourth aspect in any one of a1)-a5);

[0035] a1) Preparation of β-glucosidase;

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

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

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

[0039] a5) Prepare a product, said product being used in any one of a1)-a4);

[0040] A2) The use of the *Massezi* of the first aspect of the present invention, the culture of the second aspect, the microbial agent of the fourth aspect, or the fermented soybean meal of the fifth aspect in any one of b1)-b5);

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

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

[0043] b3) Reduce the feed conversion ratio in poultry;

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

[0045] b5) Prepare a product, which is used in any one of b1)-b4).

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

[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 vomitoxin.

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

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

[0051] In some embodiments, the poultry is a chicken; more specifically, a laying hen.

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

[0053] In some embodiments, the product described in b5) is feed, medicine, microbial additive, or feed fermentation material.

[0054] A seventh aspect of the present invention provides a product comprising: *Morgiella shuigensis* of the first aspect of the present invention, a culture of the second aspect, a microbial agent of the fourth aspect, or fermented soybean meal of the fifth aspect.

[0055] In some implementations...

[0056] Any one of the products c1)-c2):

[0057] c1) A product comprising: *Morgiella salina* of the first aspect of the present invention, a culture of the second aspect, or a bacterial agent of the fourth aspect;

[0058] c2) A product comprising: *Aquaticus simonii* of the first aspect of the present invention, a culture of the second aspect, a microbial agent of the fourth aspect, or fermented soybean meal of the fifth aspect.

[0059] In some embodiments, c1) is a reagent.

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

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

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

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

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

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

[0066] In some embodiments, the corn-soybean meal basal diet comprises: corn, corn dry ethanol grains, soybean meal, soybean oil, limestone powder, self-made semi-finished pre-SYL pre-powdered feed F for laying hens during peak egg production, DL-methionine, and L-lysine sulfate.

[0067] Based on the screening targets, physiological and biochemical characteristics, and genetic characteristics, this invention utilizes modern molecular biology methods to isolate and screen a strain of *Masses suis* CMLH375 from naturally fermented wheat bran after a large number of specific sorting processes, following 7 days of natural fermentation. This strain is an aerobic, Gram-negative bacterium that forms large, yellow, circular colonies with a raised center after culturing on MRS solid medium at 37°C for 24 hours. PCR amplification of the bacterial 16S rRNA gene was performed using universal primers, followed by sequencing. The sequencing results were compared with the NCBI database to analyze the homology of the strain. The analysis revealed that its 16S rRNA (SEQ ID NO: 3) showed the highest sequence homology with *Masses suis*, with a similarity of 99.27%. After inoculating the strain in MRS liquid medium and culturing for 24 hours, the supernatant of the bacterial culture was used to measure the β-glucosidase activity, which was 3.12 ± 0.44 μmol / (g·min), and the bacterial cell activity was 8.75 ± 0.19 μmol / (g·min). The culture of this strain (fermentation broth) was then inoculated into 40-mesh soybean meal and fermented for 48 hours. The fermentation product was separated and purified, and analyzed by HPLC. Preliminary analysis of the product was performed by comparing it with standards of daidzein, genistein, and genistein. Ultimately, it was determined that *M. sugarcane* can convert glycoside-type soybean isoflavones in soybean meal into aglycone-type soybean isoflavones. Aglycone-type soybean isoflavones possess various biological activities, including antioxidant activity, immune enhancement, anticancer effects, and maintenance of normal reproductive function in animals. Therefore, *M. sugarcane* can be used as a beneficial fermented feed inoculant for improving egg production performance and egg quality in poultry production. The bacterium was fermented and inoculated into soybean meal to produce fermented soybean meal. This fermented soybean meal was then applied to poultry feed, and a feeding trial was conducted to study its probiotic properties. The results showed that *Massezia sericata* could reduce the feed conversion ratio in poultry, increase egg production rate, and improve egg quality. It can be considered a novel and effective microorganism for use in the production of fermented feed.

[0068] The beneficial effects of this invention are:

[0069] This invention provides a strain of *Massilia suwonensis*, named CMLH375, which was deposited on December 2, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32864. This strain can be used to prepare β-glucosidase, convert glycoside-type soybean isoflavones to 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 the feed conversion ratio in poultry, and reduce the amount of soybean meal and corn used in feed. Attached Figure Description

[0070] Figure 1The image shows the status of some strains on aesculin MRS medium during the initial screening of bacteria producing high β-glucosidase in Example 1.

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

[0072] Figure 3 The growth status of *Massezia suspensa* CMLH375 on MRS solid medium is shown.

[0073] Figure 4 The growth curve of *Miscanthus sinensis* CMLH375 is shown.

[0074] Figure 5 The effect of fermented soybean meal containing *Miscanthus sinensis* CMLH375 on the conversion of soybean isoflavones was demonstrated. Detailed Implementation

[0075] definition

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

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

[0078] The present invention will be further described in detail below through specific embodiments.

[0079] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0080] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available. For reagents whose manufacturers are listed, similar products from other manufacturers are substituted.

[0081] The composition of the culture medium in this embodiment of the 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, the remainder being 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, 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, 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, the rest is water.

[0086] The soybean meal powder required for fermentation is obtained by screening the conventional soybean meal through a 40-mesh sieve after pulverization, and the wheat bran is not treated.

[0087] In the experiments of Examples 1-6 of the present invention, three replicates were set, and 27 replicates were set in the animal experiment of Example 7. The experimental data was statistically analyzed using SPSS 26.0 software, and one-way ANOVA 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 high β-glucosidase-producing strains

[0090] Mix wheat bran and sterile distilled water in a mass ratio of 1:1, seal, and ferment at 37°C for 7 days until there is an acid fragrance smell to end the fermentation. Take 2 g of the obtained naturally fermented wheat bran, add it to 50 mL of cellobiose medium, shake and culture for 24 h, then dilute. Take 10 -3 、10 -4 、10 -5 dilution solutions and plate them on an esculin MRS plate. After incubating 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 some strains during the measurement of the black degradation zone are shown in Figure 1 , and select the strains with a larger ratio for the next rescreening.

[0091] A2) Secondary screening of high-β-glucosidase-producing lactic acid bacteria

[0092] Colonies producing black circles on the culture medium are picked and transferred to MRS solid medium. The strain is purified using the four-zone streak method and incubated at 37°C for 12 hours. This process can be repeated until a pure strain is obtained.

[0093] The glycosidase activity of the initially screened strains was assessed using the p-NP semi-quantitative colorimetric method, and the final enzyme activity was calculated based on the standard curve. The specific enzyme activity measurement protocol is as follows:

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

[0095] 2) Reaction system (3.5 mL): Take 1 mL of bacterial cells, wash twice with 1 mL of 0.85% NaCl, and then suspend the bacterial cells in 0.5 mL of 0.85% NaCl solution. Mix this with 0.5 mL of CH3COOH-CH3COONa buffer (concentration 0.2 mol / L, pH 5.0) and 0.5 mL of p-nitrophenyl-β-glucosinolate (pH 5.0, pNPG concentration 5 mmol / L). Incubate at 45℃ for 0.5 h, then immediately add twice the volume (2 mL) of 0.5 M Na2CO3 solution to terminate the reaction. Centrifuge (8000 rpm, 4℃, 15 min), and aspirate 200 μL of the supernatant into each well of a 96-well plate. Then, measure the absorbance at 400 nm using a full-wavelength microplate reader.

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

[0097] 4) Take 0.5 mL of bacterial supernatant and mix it directly with 0.5 mL of CH3COOH-CH3COONa buffer (concentration 0.2 mol / L, pH 5.0) and p-nitrophenyl-β-glucosinolate (pH 5.0, pNPG concentration 5 mmol / L). Use MRS liquid medium instead of bacterial supernatant to prepare blank samples. Other treatments are the same as above.

[0098] 5) The standard curve was obtained by colorimetric determination 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 is defined as the amount of pNP produced per gram of bacteria (dry weight) per minute under the above reaction conditions. Enzyme activity units are expressed as μmol / (g·min) of p-nitrophenol. Table 1 shows the enzyme activity results for some strains.

[0100] Table 1. Enzyme activity assay results of some strains

[0101] strain number Cellular (bacterial) 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) Genomic DNA was extracted from strain CMLH375 according to the instructions of the kit (DNeasyR Blood & Tissue Kit, QIANGEN). The DNA was detected by 0.8% agarose gel electrophoresis, and no diffusion or tailing was observed. The extracted DNA was then analyzed for quality and concentration using a microplate reader. DNA samples with an A260 / A280 ratio between 1.8 and 2.0 and a concentration not less than 20 ng / μL were considered acceptable.

[0104] B2) PCR amplification

[0105] PCR amplification was performed on qualified bacterial strain DNA samples using universal primers for 16S rRNA gene amplification. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and their 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 Xinyue Biotechnology Co., Ltd.).

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

[0110] Table 2. PCR Procedure

[0111]

[0112]

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

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

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

[0116] The optimal growth conditions for the isolated *Masses hygroscopicus* CMLH375 were 37°C and aerobic conditions. After 48 hours of cultivation on MRS solid medium, it formed large, yellow, round colonies with a smooth, raised surface and regular edges. Figure 3 ).

[0117] Example 4: Plotting the growth curve of Massilia suwonensis CMLH375

[0118] Plot the growth curve of *Morse bacillus suis* CMLH375 after 26 hours of incubation at 37°C and 180 rpm. Figure 4 It can be seen that strain CMLH375 enters the logarithmic growth phase at 4h, enters the plateau phase at 8h, and begins to decline at 22h.

[0119] Example 5: Fermentation of soybean meal using *Miscanthus sinensis* CMLH375, with optimization of fermentation conditions.

[0120] C1) Test materials and methods

[0121] Ingredients: 40-mesh soybean meal, distilled water;

[0122] Fermentation broth preparation: Take the preserved *Aquaticus simonii* strain and inoculate it into MRS liquid medium at a 2% v / v inoculum rate. Incubate at 37°C for 20 hours to reach OD500. 600 The OD value was approximately 1.72. After three activations, the above-mentioned fermentation broth of *Miscanthus sinensis* CMLH375 was obtained. 600(Value approximately 1.72);

[0123] C2) Optimization of soybean meal fermentation conditions: The effects of different factors on the conversion were investigated. Using aglycone as the criterion, single-factor experiments were conducted on fermentation temperature, fermentation time, strain ratio, inoculum size, and liquid-to-solid ratio.

[0124] C3) High-performance liquid chromatography detection

[0125] After the soybean meal fermentation is complete, accurately weigh 0.1g of sample, dissolve it in 80% methanol solution, sonicate for 30min, and dilute to 10mL with 90% methanol solution. Mix the methanol solution with a shaker, filter through a filter membrane, and then transfer 2mL of the filtrate into a chromatographic bottle for later use.

[0126] Preparation of standard solutions: Accurately weigh 4 mg each of dried daidzein, daidzin, genistein, daidzein, daidzein, and genistein, and dilute to 10 mL with dimethyl sulfoxide solution to obtain standard stock solutions with a concentration of 400 mg / L. Pipette 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL from the six standard stock solutions, 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.

[0127] Instrument conditions for instrumentation: Column: C18 ODS-3 (5μm, 4.6mm×250mm); Column temperature: 30℃; Mobile phase: Mobile phase A is acetonitrile, mobile phase B is aqueous phosphoric acid 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 for daidzein, daidzein, genistein, daidzein, daidzein and genistein are shown in Table 4.

[0129] Table 3. Mobile phase ratios for high performance liquid chromatography

[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. Linear equations and correlation coefficients of standard curves for daidzein, genistein, daidzein, and genistein.

[0132]

[0133]

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

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

[0136]

[0137] Table 6. Changes in soybean isoflavone content in soybean meal at different fermentation times under conditions of 37℃, 6% inoculum, and a liquid-to-solid ratio of 1:1.

[0138]

[0139]

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

[0141]

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

[0143]

[0144]

[0145] Example 6: Fermentation of soybean meal using *Miscanthus sinensis* CMLH375 under optimal fermentation conditions.

[0146] D1) Measurement Indicators

[0147] The isoflavone content of different types of soybeans in fermented soybean meal was detected under optimal fermentation conditions.

[0148] Since an excessively high liquid-to-feed ratio can easily lead to feed spoilage in actual production, the fermentation conditions (actual fermentation conditions) ultimately adopted in this invention are as follows: inoculum amount 6%, liquid-to-feed ratio 0.6:1, temperature 37℃, fermentation time 72h, and the soybean content was measured under these conditions.

[0149] The content of anti-nutritional factors and mycotoxins in fermented soybean meal under optimal fermentation conditions was detected.

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

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

[0152] D4) Test Results

[0153] As shown in Table 9, under the optimal fermentation conditions (6% inoculum, 0.8:1 liquid-to-material ratio, 40℃ temperature, and 24h fermentation time), the content of soybean isoflavone aglycones can be significantly increased to 1998.88±13.7μg / g. Under the actual fermentation conditions (6% inoculum, 0.6:1 liquid-to-material ratio, 37℃ temperature, and 72h fermentation time), the content of soybean meal isoflavone aglycones can also be increased from 39.57±6.31μg / g to 1631.19±37.24μg / g.

[0154] Table 9. Isoflavone content of soybean meal fermented and unfermented soybean meal by *Morseella asiatica* CMLH375 under different conditions ( Figure 5 )

[0155]

[0156] As shown in Table 10, under optimal fermentation conditions, the use of the *Aquaticus simonii* CMLH375 of this invention can significantly reduce the content of anti-nutritional factors and mycotoxins in unfermented soybean meal.

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

[0158]

[0159] Example 7: Application of *Morgiformis suis* CMLH375 in the preparation of fermented feed for laying hens

[0160] Lohmann Pink Hen Feeding Trial

[0161] E1) Experimental animal chicken grouping

[0162] The experiment used 1080 Lohmann Pink laying hens (Sichuan Tieqilishi Industry Co., Ltd.) in the late laying period (48 weeks of age), with an average laying rate of 0.889±0.025. They were randomly divided into four groups: control group (CON), a group with 40 μg / g soy isoflavones (CMLH375 40 μg / g), a group with 80 μg / g soy isoflavones (CMLH375 80 μg / g), and a group with 120 μg / g soy isoflavones (CMLH375 120 μg / g), with 10 replicates per group and 27 hens per replicate. They were housed in H-shaped cages with free access to water. To ensure adequate intake of aglycone-type soy isoflavones, they were fed a fixed amount of feed. The immunization program followed the standard laying hen rearing procedure during the experiment. The experiment lasted 42 days.

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

[0164] E2) Experimental Diets

[0165] The experimental diets were formulated according to the Lohmann Powder Chicken Feeding Manual, and were fed in powder form. The nutrient levels of each group were accurately calculated using feed formulation software (Table 11).

[0166] Table 11. Composition and Nutritional Levels of Basal Diets

[0167]

[0168] E3) To ensure that the nutritional intake of each group of laying hens is consistent, with the feed intake per hen in the blank group being 110g, the daily feed mixing rules for different treatment groups (calculated as 270 hens) are shown in Table 12.

[0169] Table 12. Daily Mixing Rules

[0170]

[0171]

[0172] E4) Measurement Indicators

[0173] Production performance

[0174] Taking repetition as the unit, record the daily genuine egg production rate (SER) of laying hens. Randomly select 30 eggs in 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] Genuine egg production rate (SER) = total number of genuine eggs / number of chickens in stock × 100%.

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

[0177] Albumen height and Haugh unit are measured by 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) in a certain period / total egg production (kg) in the same period.

[0179] E5) Results

[0180] As can be seen from Table 13, in the 6th week of the experiment, compared with the control group, the genuine egg production rate (SER) of the CMLH375 120 μg / g group was significantly increased (P < 0.05), and there was a tendency of difference in the third and fourth weeks (0.05 < P < 0.1). During the whole experiment period, the average genuine egg production rate (SER) of 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, in the 4th week of the experiment, compared with the control group, the albumen height and Haugh unit of the three CMLH375 treatment groups were significantly different from those of 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 fermented soybean meal feed prepared by fermenting with Marica shimizui 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 fermented soybean meal feed prepared by fermenting with Marica shimizui 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 with *Miscanthus sinensis* CMLH375 of this invention to the diet of laying hens can significantly improve the rate of high-quality eggs in the later stages of laying in Lohmann pluperch hens, and can also improve egg quality and reduce the feed conversion ratio. Therefore, *Miscanthus sinensis* can be used as a feed microbial additive in fermented feed, and the recommended addition amount of fermented soybean meal fermented with *Miscanthus sinensis* CMLH375 of this invention to the diet of laying hens is 80 kg / t.

[0185] Table 13. Effects of soybean isoflavone conversion by *Massezia suihara* on egg production rate of standard Lohmann pink laying hens.

[0186]

[0187] Table 14. Effects of soybean isoflavone conversion by *Massezia sucogenensis* on egg quality of Lohmann pink eggs

[0188]

[0189] Table 15. Effect of soybean isoflavone conversion by *Massezia suihara* on feed conversion ratio in Lohmann powder-treated laying hens.

[0190]

[0191] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A strain of *Mortisus suis*, named CMLH375, is classified as *Mortisus suis*. Massilia suwonensis It was deposited on December 2, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.32864.

2. A culture obtained by culturing the *Miscanthus sinensis* as described in claim 1.

3. The method for preparing the culture according to claim 2, wherein the *Miscanthus sinensis* of claim 1 is inoculated into a culture medium and cultured to obtain the culture.

4. A microbial agent comprising: the *Miscanthus sinensis* of claim 1 or the culture of claim 2.

5. A fermented soybean meal, obtained by fermenting soybean meal using the *Morseella asiatica* of claim 1, the culture of claim 2, or the microbial agent of 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: the culture described in claim 2 is inoculated into a soybean meal solution and fermented to obtain the product.

7. The fermented soybean meal according to claim 6, characterized in that, The soybean meal solution is a mixture of soybean meal and water.

8. The fermented soybean meal according to claim 7, characterized in that, The mass ratio of soybean meal to water is 1:(0.2-1.8).

9. The fermented soybean meal according to claim 8, characterized in that, The mass ratio of soybean meal to water is 1:(0.4-1.6).

10. The fermented soybean meal according to claim 6, characterized in that, The inoculation amount is 2% v / v - 20% v / v.

11. The fermented soybean meal according to claim 10, characterized in that, The inoculation amount is 3%-18%.

12. The fermented soybean meal according to any one of claims 5-11, characterized in that, The fermentation time is 12-144 hours.

13. The fermented soybean meal according to claim 12, characterized in that, The fermentation time is 20-120 hours.

14. The fermented soybean meal according to any one of claims 5-11, characterized in that, The fermentation temperature is 26-48℃.

15. The fermented soybean meal according to claim 14, characterized in that, The fermentation temperature is 28-46℃.

16. The fermented soybean meal according to claim 15, characterized in that, The fermentation temperature is 38-42℃.

17. Any one of the following applications: A1)-A2) A1) The use of the *Massezia sericata* of claim 1, the culture of claim 2, or the bacterial agent of claim 4 in any one of a1)-a5); a1) Preparation of β-glucosidase; a2) Converting glycoside-type soy isoflavones into aglycone-type soy isoflavones; a3) Reduce the content of mycotoxins in soybean meal; a4) Reduce the content of anti-nutritional factors in soybean meal; a5) Prepare a product, said product being used in any one of a1)-a4); The product described in a5) is a reagent; A2) The use of the *Massezia suspensa* of claim 1, the culture of claim 2, the microbial agent of claim 4, or the fermented soybean meal of any one of claims 5-16 in any one of b1)-b5); b1) Increase the egg production rate of poultry; b2) Improve the quality of poultry eggs; b3) Reduce the feed conversion ratio in poultry; b4) Reduce the amount of soybean meal and corn used in feed; b5) Prepare a product, said product being used in any one of b1)-b4); The glycoside-type soy isoflavones are at least one of daidzein, daidzein, and genistein. The aglycone-type soy isoflavones are at least one of daidzein, daidzein and genistein; The mycotoxin is vomitoxin; The anti-nutritional factor is β-conglycinin; The products described in b5) are feed, microbial additives, or fermented feed.

18. The application according to claim 17, characterized in that, The poultry mentioned is chicken; or The egg quality includes at least one of the following: eggshell strength, egg weight, albumen height, Haugh unit, yolk color, yolk weight, eggshell weight, and egg white weight.

19. A product comprising: the *Morgiella simonii* of claim 1, the culture of claim 2, the microbial agent of claim 4, or the fermented soybean meal of any one of claims 5-16; The products are reagents, feed, microbial additives, or fermented feed for animal feed.

20. A feed comprising: a basal diet and fermented soybean meal as described in any one of claims 5-16.

21. The feed according to claim 20, characterized in that, The fermented soybean meal content in the feed is 3-15 wt%.

22. The feed according to claim 21, characterized in that, The fermented soybean meal content in the feed is 4.267-12.8 wt%.

23. The feed according to any one of claims 20-22, characterized in that, The basal diet is a corn-soybean meal type basal diet.

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