A strain of highly efficient tannin-degrading bacteria and its application in rapeseed meal fermentation

Abstract

This invention discloses *Bacillus licheniformis* strain LWY-37-2, which was deposited on February 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located in Beijing, with accession number CGMCC No. 26654. This invention also discloses a method for degrading condensed tannins using *Bacillus licheniformis* LWY-37-2, comprising the following steps: setting an inoculation viable count of 5 × 10⁻⁶ cells / year. 6 With a CFU / g content and a feed-to-water ratio of 1:0.8, the pH of the rapeseed meal fermentation substrate was adjusted to 7 without sterilization. Fermentation was carried out at 30℃ for 3 days, with the substrate turned over every 12 hours. The Bacillus licheniformis LWY-37-2 disclosed in this invention shows excellent application prospects in fermented plant-based feeds.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology and relates to the screening and application of condensation tannin degrading bacteria. Background Technology

[0002] Tannins, also known as plant polyphenols, are polyhydroxyphenols widely distributed in plants in nature. They are often present in high concentrations and are resistant to microbial decomposition. Based on their chemical structure, hydrolyzable tannins, condensed tannins, and brown algal polyphenols are considered the three main types of tannins. Hydrolyzable and condensed tannins are mainly found in terrestrial plants, while brown algal polyphenols are found only in brown seaweed.

[0003] Condensed tannins (CT), also known as proanthocyanidins, are complex macromolecular polyphenolic compounds. They are oligomers or polymers composed of flavan-3-ols, whose basic units are linked by C–C bonds, and a few by C–O–C bonds. The flavan-3-ol units have a typical C6–C3–C6 flavonoid skeleton. Studies have found that the flavan-3-ol subunits in condensed tannins vary, but common subunits include catechin, epicatechin, gallocatechin, and epigallocatechin. Proanthocyanidins are composed of catechin and epicatechin. Condensed tannins linked by C4–C8 or C4–C6 bonds are called type B condensed tannins, while those doubly linked by an additional ether bond at C2–O–C7 or C2–O–C5 are type A condensed tannins. Most natural plant condensed tannins are type B condensed tannins.

[0004] Condensed tannins are polymers composed of flavanols, commonly found in natural plants. They are widely present in the flowers, fruits, bark, and seeds of various plants and are one of the main anti-nutritional factors in plant-based feeds. Many agricultural wastes and oil processing byproducts, such as persimmon peel, lychee peel, pomegranate peel, mangosteen peel, rapeseed hull, and peanut skin, contain condensed tannins. Studies have found that peanut skin contains a condensed tannin trimer with the structure epicatechin–(4β→6)–epicatechin–(2β→O→7,4β→8)–catechin. Berries and fruits are the best sources of condensed tannins; blueberries, cranberries, black elderberries, blackcurrants, and blueberries are all rich in condensed tannins. For cereals, triticale straw also has a relatively high condensed tannin content, reaching 0.86%. Because of its unique physiological and biochemical properties of complexing proteins and amino acids, which affect the digestion and absorption of nutrients in animals, it is widely regarded as an anti-nutritional factor in the field of animal nutrition research. Its anti-nutritional effects are manifested in two aspects: (1) the complex formed by the interaction between condensed tannins and proteins in saliva produces an astringent taste, affecting the palatability of feed and reducing the animal's appetite and feed intake; (2) the polyphenolic hydroxyl groups of condensed tannins are easy to complex with proteins, polysaccharides, metal ions, etc. to form complexes, which hinder the digestion and absorption of nutrients.

[0005] Rapeseed meal is an important source of feed protein, second only to soybean meal. In 2022, global rapeseed meal production reached 44.74 million tons, with my country's production reaching 8.99 million tons, indicating significant potential for substitution with soybean meal. However, its use in feed, especially in monogastric animal diets, is limited by the presence of anti-nutritional factors such as condensed tannins. Studies have found that the total condensed tannin content in rapeseed bran is 0.5190%, with insoluble condensed tannins accounting for over 70% of the total condensed tannin content in both rapeseed and bran. Furthermore, the condensed tannin content in rapeseed bran varies depending on the type of rapeseed and its growing environment, ranging from 0.014% to 2.3%. Under alkaline conditions, the condensed tannins in rapeseed meal can form active substances (quinones and semiquinones). These substances can interact with proteins, potentially causing the rapeseed meal to darken in color and develop a bitter taste, which is a major reason for the decline in the quality of feed products containing rapeseed meal. How to detoxify anti-nutritional factors such as condensed tannins in rapeseed meal is the main challenge faced in the application of rapeseed meal.

[0006] Microbial fermentation is a promising and valuable detoxification method, capable of reducing anti-nutritional content and improving the palatability and nutritional value of rapeseed meal. Patent CN 201810586648.X discloses a tannin-degrading microbial agent, Klebsiella pneumoniae. Currently, most of the condensation-tannin-degrading bacteria screened by researchers are molds or other unapproved strains for food and feed, making them unsuitable for widespread application in fermented feed. Therefore, it is necessary to find safer strains capable of degrading condensation-tannins to develop rapeseed meal condensation-tannin fermentation detoxification technology. Summary of the Invention

[0007] This invention aims to domesticate and screen a strain of bacteria from nature that exhibits excellent degradation performance of plant-derived condensed tannins. Based on this, a degradation process for condensed tannins by this strain was established using rapeseed meal as raw material. The effects of fermentation on the nutritional value of rapeseed meal were evaluated using an in vitro biomimetic digestion method, and the application prospects of the target strain in fermented plant-derived feed were explored.

[0008] This invention screens natural microorganisms that can efficiently degrade condensed tannins from the surface soil of tea plantations rich in condensed tannins, raw Pu-erh tea, and bovine rumen fluid, and studies their degradation performance on rapeseed hull condensed tannins. The strains are identified through morphological observation, physiological and biochemical characteristics research, and molecular biological methods.

[0009] This invention obtained a condensed tannin-degrading bacterium, Bacillus licheniformis strain LWY-37-2, through screening. Using Bacillus licheniformis strain LWY-37-2 as the fermentation strain, the single-strain solid-state fermentation process of rapeseed meal was optimized. The changes in condensed tannin content and nutritional components before and after fermentation were investigated. Furthermore, using a monogastric animal biomimetic digestive system, broiler endogenous enzymes were used for in vitro biomimetic digestion to evaluate its nutritional value.

[0010] This invention discloses Bacillus licheniformis strain LWY-37-2, which was deposited on February 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located in Beijing, with accession number CGMCC No. 26654.

[0011] This invention also discloses a method for degrading condensed tannins using Bacillus licheniformis strain LWY-37-2, comprising the following steps: setting the inoculation viable count to 5 × 10⁻⁶. 6 CFU / g, material-to-water ratio 1:0.8, pH adjusted to 7 on a non-sterilized rapeseed meal fermentation substrate, fermented at 30℃ for 3 days, with the material turned over once every 12 hours.

[0012] This invention also discloses a method for producing feed ingredients by degrading condensed tannins using Bacillus licheniformis strain LWY-37-2, comprising the following steps: setting the inoculation viable count to 5 × 10⁻⁶. 6 CFU / g, material-to-water ratio 1:0.8, pH adjusted to 7 on a non-sterilized rapeseed meal fermentation substrate, fermented at 30℃ for 3 days, turning over once every 12 hours; dried; crushed and sieved.

[0013] The present invention also discloses a bacterial agent comprising a bacterial suspension of Bacillus licheniformis strain LWY-37-2.

[0014] This invention also discloses the application of Bacillus licheniformis strain LWY-37-2 in the degradation of condensed tannins.

[0015] This invention also discloses the application of Bacillus licheniformis strain LWY-37-2 in feed fermentation.

[0016] This invention also discloses a method for obtaining feed ingredients by using Bacillus licheniformis strain LWY-37-2 to degrade condensed tannins.

[0017] The present invention also discloses the application of a bacterial suspension including Bacillus licheniformis strain LWY-37-2 in rapeseed meal fermentation.

[0018] The present invention also discloses the application of feed ingredients obtained by using Bacillus licheniformis strain LWY-37-2 to degrade condensed tannins in feed.

[0019] The present invention also discloses a screening method for condensed tannin-degrading bacteria, comprising the following steps: 1) using condensed tannin as the sole carbon source, and gradually acclimating the bacteria to a culture medium with increasing condensed tannin concentration to screen for condensed tannin-degrading bacteria; 2) using bacterial, lactic acid bacteria, yeast, or fungal enrichment culture medium for isolation and purification.

[0020] Specifically, the present invention is as follows.

[0021] 1. Bacillus licheniformis strain LWY-37-2, which was deposited on February 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC), Beijing, with accession number CGMCC No. 26654.

[0022] 2. A method for degrading condensed tannins using Bacillus licheniformis strain LWY-37-2 as described in item 1, comprising the following steps:

[0023] Set the inoculation count to 5 × 10⁶ live bacteria. 6 CFU / g, material-to-water ratio 1:0.8, pH adjusted to 7 on a non-sterilized rapeseed meal fermentation substrate, fermented at 30℃ for 3 days, with the material turned over once every 12 hours.

[0024] 3. A method for producing feed ingredients by degrading condensed tannins using Bacillus licheniformis strain LWY-37-2 as described in item 1, comprising the following steps:

[0025] Set the inoculation count to 5 × 10⁶ live bacteria. 6 CFU / g, material-to-water ratio 1:0.8, pH adjusted to 7 on the basis of non-sterilized rapeseed meal fermentation substrate, fermentation time at 30℃ for 3 days, turning the material once every 12 hours;

[0026] drying;

[0027] Grind and sieve.

[0028] 4. A bacterial agent comprising a bacterial suspension of Bacillus licheniformis strain LWY-37-2 as described in item 1.

[0029] 5. The application of Bacillus licheniformis strain LWY-37-2 as described in item 1 in the degradation of condensed tannins.

[0030] 6. The application of Bacillus licheniformis strain LWY-37-2 as described in item 1 in feed fermentation.

[0031] 7. Feed ingredients obtained according to the method described in item 3.

[0032] 8. Application of the microbial agent described in item 4 in rapeseed meal fermentation.

[0033] 9. Application of the feed ingredients described in item 7 in feed.

[0034] 10. A method for screening condensation tannin-degrading bacteria, comprising the following steps:

[0035] 1) Using condensed tannins as the sole carbon source, acclimatize the bacteria step by step with increasing concentrations of condensed tannins in the culture medium and screen for condensed tannin-degrading bacteria.

[0036] 2) Use bacterial, lactic acid bacteria, yeast, or fungal enrichment media for isolation and purification.

[0037] This invention domesticates and screens a natural strain of bacteria that exhibits excellent degradation performance for condensed tannins from various plant sources. Based on this, using rapeseed meal as the feed ingredient, a degradation process for condensed tannins by this strain was established. The effects of fermentation on the nutritional value of rapeseed meal were evaluated using an in vitro biomimetic digestion method, demonstrating its promising application prospects in fermented plant-based feeds. Attached Figure Description

[0038] Figure 1 This is a growth curve of the strain of the present invention.

[0039] Figure 2 This is a growth curve of the strain of the present invention in tolerant culture media with different substrate concentrations.

[0040] Figure 3 The colony and morphological structure of the strain of the present invention are shown. Figure 3A represents the colony state of the strain of the present invention on LB agar plates. Figure 3 B represents the Gram staining result of the strain of this invention.

[0041] Figure 4 This is a graph showing the carbon source metabolism and chemical sensitivity of the strain of the present invention on GENⅢ microplates.

[0042] Figure 5 This indicates the influence of the fermentation substrate on fermentation parameters.

[0043] Figure 6 This indicates the effect of temperature on the degradation rate of condensed tannins in rapeseed meal.

[0044] Figure 7 This indicates the effect of pH on the degradation rate of condensed tannins in rapeseed meal.

[0045] Figure 8 This indicates the effect of the number of live bacteria inoculated on the degradation rate of condensed tannins in rapeseed meal.

[0046] Figure 9 This indicates the effect of the feed-to-water ratio on the degradation rate of condensed tannins in rapeseed meal.

[0047] Information on the preservation of biological materials

[0048] The Bacillus licheniformis strain LWY-37-2 provided by this invention was deposited on February 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located in Beijing, with the accession number CGMCC No. 26654. Detailed Implementation

[0049] To better understand this invention, the following embodiments are provided in conjunction with the accompanying drawings. It should be understood that the embodiments of this invention are for illustrative purposes only and not for limiting the invention; the scope of protection of this invention is defined solely by the claims. The embodiments provided are merely preferred embodiments and are not intended to limit the invention in any way. Those skilled in the art can make changes, substitutions, or modifications based on the content of this invention to form different implementation methods. However, any changes and modifications, or equivalent substitutions, made to the method of this invention without departing from the inventive concept are within the scope of protection of this invention.

[0050] Example 1: Test Materials and Test Methods

[0051] 1.1 Test Materials

[0052] The condensation tannin-degrading bacteria were screened from the surface soil of tea plantations, COFCO Yiwu old-tree Pu'er tea, and bovine rumen fluid. The rapeseed meal was from northern China. The rapeseed hull condensation tannins were crudely extracted in the laboratory, with a purity of approximately 45%. Based on the standard curves of molar and mass concentrations plotted using the vanillin method, the degree of polymerization of the extracted condensation tannins was calculated to be approximately 3.68.

[0053] Condensed tannin standards were purchased from Beijing Solarbio Science & Technology Co., Ltd. Catechin standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0054] 1.2 Test Methods

[0055] 1.2.1 Extraction of condensed tannins from rapeseed hulls

[0056] The crude extraction of rapeseed hull condensed tannins was improved based on previous methods, and the specific procedures are as follows:

[0057] (1) Weigh 10g of defatted rapeseed hull powder that has passed through a 40-mesh sieve (particle size 0.45mm), extract condensed tannins with 70% acidic ethanol at a material-to-liquid ratio of 1:12 for 20 minutes with ultrasonic assistance, centrifuge at 5000rpm / min for 10 minutes and collect the supernatant. Repeat the above operation 3 times.

[0058] (2) Wash the residue twice with 70% acidic ethanol, combine the extract and washing liquid, and concentrate the mixture in a rotary evaporator at 40°C to recover ethanol.

[0059] (3) Add an equal amount of ethyl acetate solution, stir with a magnetic stirrer for 15 min, centrifuge, collect the supernatant (ethyl acetate layer), extract three times and combine the extracts.

[0060] (4) Add 3 to 4 times the volume of petroleum ether to the above extract, stir thoroughly, let stand overnight at 4°C, filter and collect the precipitate.

[0061] (5) The precipitate was dried using a freeze dryer to obtain crude brown rapeseed hull condensed tannin powder.

[0062] 1.2.2 Determination of the degree of polymerization of condensed tannins

[0063] The method for determining the degree of polymerization of condensed tannins was improved based on Liu Jiwei's method, and the procedure is as follows:

[0064] A series of catechin standards and condensed tannin samples of a certain concentration were prepared using methanol and acetic acid respectively (using 2% to 6% methanol to promote the dissolution of condensed tannin acetic acid solution);

[0065] The standard curve of the molar concentration of catechin standard and the determination of the amount of substance n (mol / L) of condensed tannin sample: 1 mL of the sample solution to be tested was added to 5 mL of solution containing 4% hydrochloric acid and 0.5% vanillin acetic acid. After reacting in the dark at 20℃ for 5 min, the absorbance was measured at 500 nm.

[0066] The standard curve of catechin standard and the determination of the mass concentration m (g / L) of condensed tannin sample: 1 mL of the sample solution to be tested was added to 5 mL of methanol solution containing 4% hydrochloric acid and 0.5% vanillin, and the reaction was carried out at 30 °C in the dark for 30 minutes. The absorbance was detected at 500 nm.

[0067] The average degree of polymerization is calculated using the following formula.

[0068] DP = m / (n×M)

[0069] m: Mass concentration of the condensed tannin sample, m (g / L)

[0070] M: Catechin molecular weight 290.27

[0071] n: Amount of condensed tannin sample (mol / L)

[0072] 1.2.3 Determination of Condensed Tannin Content

[0073] The determination of condensed tannin content was based on previous research, and the traditional n-butanol-hydrochloric acid method was improved. The specific operation is as follows.

[0074] 1) Determination of soluble condensed tannin content: Weigh 20 mg of raw material that has passed through a 40-mesh sieve and extract it by sonication in 2 mL of 70% acidic acetone for 20 minutes. Centrifuge and collect the supernatant. Take 0.5 mL of the supernatant and 2 mL of reaction reagent containing n-butanol (acetone:n-butanol:12N concentrated HCl:water = 46.8:43:5:5.2) and 67 μL of ferric ammonium sulfate catalyst (2N HCl solution containing 2% FeNH4(SO4)2). Mix well and centrifuge. Take 200 μL of the supernatant and read the absorbance at 550 nm using an ELISA reader as the blank before the reaction. Vortex the remaining mixed reaction system again for 5 s and react in a water bath at 70℃ for 2.5 hours. Cool to room temperature and read the absorbance at 550 nm using an ELISA reader.

[0075] 2) Determination of insoluble condensed tannin content: Add 500 μL of 70% acidic acetone to the residue after determining the soluble condensed tannin content, then add 2 mL of the same reaction reagent as in step 1) and 67 μL of ferric ammonium sulfate catalyst, mix well and sonicate for 10 min. Centrifuge, take 200 μL of supernatant, and read the absorbance at 550 nm as a blank. Vortex the remaining mixed reaction system again for 5 s, react in a water bath at 70℃ for 2.5 hours, cool to room temperature, and read the absorbance at 550 nm using a microplate reader.

[0076] 1.2.4 Measurement Indicators and Methods

[0077] The viable count of Bacillus licheniformis was determined according to the national standard GB / T 26428-2010, "Detection of Bacillus subtilis in feed microbial preparations". For sensory evaluation, randomly selected fermentation samples from each group were evaluated according to the method of Luo Yuanqin et al. The polypeptide content was determined according to the method of Lu Wei et al., using the trichloroacetic acid precipitation method. The crude protein content of the diet was determined using a Dumas fully automated nitrogen analyzer; the contents of crude fiber, neutral detergent fiber, and acid detergent fiber were determined using the filter bag method; the crude fat content was determined using Soxhlet extraction; the amino acid content change was measured using an automated amino acid analyzer; and the crude ash content was determined according to the national standard GB / T6438-2007, "Determination of Crude Ash in Feed".

[0078] pH determination: Take two portions of each sample, 5g each, add 50mL of distilled water, stir well, let stand at 4℃ for 6 hours, filter, and measure the pH of the supernatant with a pH meter.

[0079] 1.2.5 Data Processing and Analysis

[0080] Excel software was used to organize the data on process optimization and biomimetic digestion. SPSS 22.0 software was used to perform one-way ANOVA on the results of the single-factor experiments, combined with Duncan's method for multiple comparisons. The results of the orthogonal experiments were analyzed using a general linear model, with P < 0.05 considered significant. Graphpad Prism 8.0 was used for plotting.

[0081] 1.2.6 Culture medium

[0082] Acclimation medium (g / L): sodium nitrate 2, dipotassium hydrogen phosphate 1, potassium chloride 0.5, magnesium sulfate 0.5, ferrous sulfate heptahydrate 0.01. Autoclave at 121℃ for 20 minutes, add condensed tannin filtered sterilized solution, and gradually increase the condensed tannin concentration from 1 g / L to 5 g / L.

[0083] Initial screening culture medium (g / L): sodium nitrate 2, dipotassium hydrogen phosphate 1, potassium chloride 0.5, magnesium sulfate 0.5, ferrous sulfate heptahydrate 0.01, autoclaved at 121℃ for 20 minutes, and an appropriate amount of condensed tannin filtered sterilization solution was added.

[0084] Secondary screening medium (g / L): sodium nitrate 2, dipotassium hydrogen phosphate 1, potassium chloride 0.5, magnesium sulfate 0.5, ferrous sulfate heptahydrate 0.01, autoclaved at 121℃ for 20 minutes, and an appropriate amount of rapeseed hull condensed tannin filtered sterilized solution was added.

[0085] Substrate tolerance medium (g / L): glucose 20, sodium nitrate 2, dipotassium hydrogen phosphate 1, potassium chloride 0.5, magnesium sulfate 0.5, ferrous sulfate heptahydrate 0.01, autoclaved at 115℃ for 30 minutes, and an appropriate amount of rapeseed hull condensed tannin filtered sterilized solution was added.

[0086] LB medium (g / L): peptone 10, yeast extract 5, sodium chloride 10, autoclaved at 121℃ for 20 min.

[0087] Bacterial enrichment medium (g / L): peptone 10, yeast extract 5, sodium chloride 10, agar 15, autoclave at 121℃ for 20 minutes, cool and solidify, add 1 ml of condensed tannin filter sterilization solution with a concentration of 1 g / L, and spread evenly.

[0088] MRS medium (g / L): peptone 10, yeast extract 5, beef extract 10, glucose 20, dipotassium hydrogen phosphate 2, sodium acetate 5, triammonium citrate 2, magnesium sulfate 0.1, manganese sulfate monohydrate 0.05, Tween 80 1, sterilized at 121℃ for 20 min.

[0089] Lactic acid bacteria enrichment medium (g / L): peptone 10, yeast extract 5, beef extract 10, glucose 20, dipotassium hydrogen phosphate 2, sodium acetate 5, triammonium citrate 2, magnesium sulfate 0.1, manganese sulfate monohydrate 0.05, Tween 80 1, agar 15. Sterilize at 121℃ for 20 minutes. After cooling and solidification, add 1 ml of condensed tannin filter sterilization solution with a concentration of 1 g / L and spread evenly.

[0090] YPD medium (g / L): peptone 20, yeast extract 10, glucose 20, sterilized at 121℃ for 20 min.

[0091] Yeast enrichment medium (g / L): peptone 20, yeast extract 10, glucose 20, agar 15, sterilized at 121℃ for 20 minutes, cooled and solidified, then added 1 ml of condensed tannin filter sterilized solution with a concentration of 1 g / L, and spread evenly.

[0092] PDB medium (g / L): Potato extract 4g, glucose 20g, sterilized at 121℃ for 20min.

[0093] Fungal enrichment medium (g / L): Potato extract 4, glucose 20, agar 15, sterilized at 121℃ for 20 minutes, cooled and solidified, then added 1 ml of condensed tannin filter sterilized solution with a concentration of 1 g / L, and spread evenly.

[0094] Example 2: Screening of condensed tannin degrading bacteria

[0095] 2.1 Enrichment and domestication of condensation tannin-degrading bacteria

[0096] Condensed tannins are rich in phenolic hydroxyl groups and readily form precipitates through complexation reactions with proteins and metal ions. They exhibit broad-spectrum antibacterial activity, preventing the growth of most bacterial species in enrichment solutions from various sources. Gradient acclimatization methods can effectively improve the survival rate of strains with condensed tannin degradation potential and enhance their degradation performance.

[0097] Using surface soil from tea plantations, raw Pu-erh tea, and bovine rumen fluid as bacterial sources, strain enrichment solutions were prepared for each sample using a dilution-mixing method. Accurately weigh 10g of each sample or measure 10mL of each sample, add it to a 90ml Erlenmeyer flask containing sterile physiological saline and glass beads, and shake at 200r / min for 20 minutes to thoroughly mix the sample and disperse the bacteria. Use a pipette to transfer the supernatant suspension to a sterile centrifuge tube for later use.

[0098] The above suspension was serially diluted with sterile physiological saline. A small amount of the liquid was then placed in an acclimatization medium and cultured at 30°C with shaking for 7 days. After standing, the supernatant was added to a medium with progressively increasing condensed tannin concentration. This process was repeated to gradually increase the condensed tannin concentration in the medium to 5 g / L.

[0099] To selectively screen for bacteria, lactic acid bacteria, yeasts, and fungi capable of degrading condensed tannins, after acclimatization, 1 mL of the culture solution was diluted and spread onto various enrichment media. The media were then incubated at a suitable temperature for 2–4 days. Single colonies of different sizes, morphologies, and colors were selected for isolation and purification. All treatments were performed in triplicate. The purified strains were stored in 50% glycerol at -20°C.

[0100] This invention uses condensed tannins as the sole carbon source and isolates a total of 105 strains through domestication and targeted screening of culture media. Among them, 45 strains were isolated by targeted screening of bacterial enrichment culture media, 36 strains were isolated by targeted screening of lactic acid bacteria enrichment culture media, 5 strains were isolated by targeted screening of yeast enrichment culture media, and 19 strains were isolated by targeted screening of fungal enrichment culture media.

[0101] 2.2 Initial screening of condensation tannin degrading bacteria

[0102] After activating single colonies of 105 strains isolated and purified from domestication and directional screening media twice, they were inoculated into primary screening media with a concentration of 1 g / L condensed tannins and cultured at a constant temperature of 28℃~37℃ with shaking for 48 hours. After centrifugation at 3000 rpm for 15 minutes, the supernatant was taken to determine the condensed tannin content, and the 10 strains with the best condensed tannin degradation effect were screened.

[0103] 2.3 Secondary screening of condensation tannin degrading bacteria

[0104] Ten strains obtained from the initial screening were activated and inoculated into secondary screening medium at an inoculum rate of 2%. They were cultured at a constant temperature of 28–37°C with shaking for 48 hours, centrifuged at 3000 rpm / min for 15 minutes, and the supernatant was collected to determine the rapeseed husk condensed tannin content. Three replicates were set for each strain. After fermentation, the supernatant was centrifuged again to determine the rapeseed husk condensed tannin degradation rate. The strain with the best rapeseed husk condensed tannin degradation effect, LWY-37-2, was selected as the target strain for subsequent experiments.

[0105] Example 3: Study on the biological characteristics of condensation tannin degrading bacteria

[0106] 3.1 Determination of growth curve of condensed tannin degrading bacteria

[0107] The target strains obtained from the secondary screening were streaked on plates. Single colonies were picked and inoculated into 10 mL of liquid LB medium. The culture was shaken at 37°C for 24 h to serve as the seed culture. 1% of the seed culture was inoculated into 150 mL of liquid LB medium and shaken at 37°C for 24 h. The bacterial culture was collected every 2 h at different time points, and the OD value (OD = 600 nm) was measured using a UV-Vis spectrophotometer to plot the growth curve.

[0108] Figure 1 The growth curve of the target strain LWY-37-2 shows that the spores generated within 16 to 24 hours rapidly multiply and grow, with the strain's OD value reaching its maximum at 22 hours.

[0109] 3.2 Assessment of substrate tolerance of condensation tannin-degrading bacteria

[0110] Centrifuge the activated bacterial seed culture at 5000 rpm for 10 minutes, remove the supernatant, and wash the bacterial precipitate twice with sterile 0.85% physiological saline to prepare a 5 mL volume solution with 10 viable cells. 8 CFU / mL bacterial suspension. The bacterial suspension was inoculated at 2% into substrate tolerance media with condensed tannin concentrations of 0.1 g / L, 0.5 g / L, 1 g / L, 3 g / L, and 5 g / L, respectively. The cultures were incubated at 37°C with shaking for 48 hours using a growth curve analyzer, and the OD was calculated. 600 The absorbance of the bacterial solution was measured every 2 hours, and growth curves of the strains under different concentrations of condensed tannin were plotted.

[0111] Figure 2The growth curves of strain LWY-37-2 in tolerant media with different substrate concentrations are shown. As the condensed tannin substrate concentration gradually increased from 0.1 g / L to 5 g / L, the growth trend of strain LWY-37-2 slowed significantly, with the maximum OD value decreasing from 0.80 to 0.21. The strain exhibited the most vigorous growth at a condensed tannin concentration of 0.1 g / L; the growth rate slowed at 0.5 g / L; at 1 g / L, the strain entered the decline phase after 24 hours; and when the condensed tannin concentration exceeded 3 g / L, the growth trend further declined, and growth became very slow.

[0112] Example 4: Identification of condensation tannin degrading bacteria

[0113] 4.1 Observation of colony and strain morphology

[0114] The target strain was inoculated onto LB solid medium using the streak plating method and incubated at 37°C for 24 hours. Colony morphology, color, edge regularity, and surface texture were observed under a magnifying glass. Simultaneously, the target strain was inoculated into LB liquid medium and incubated at 37°C with shaking for 22 hours. Gram staining was used for microscopic observation of the morphology of the target strain.

[0115] Figure 3 The colony and morphological structure of the strain of the present invention are shown. Figure 3 A represents the colony state of the strain of the present invention on LB agar plates. Figure 3 B represents the Gram staining result of the strain of this invention. The colony morphology was observed under a magnifying glass, as shown... Figure 3 As shown in Figure A: the colonies are light orange-white, 3–4 mm in diameter, with rough, irregular edges, opaque, and slightly raised and wrinkled in the center, similar in morphology to Bacillus spp. After culturing this strain in LB liquid medium with shaking for 22 hours, staining was performed according to the Gram staining kit instructions. The results are as follows: Figure 3 As shown in B: After staining, the strain appears as a short, purple rod with small spores around the bacterial cells. Therefore, it is preliminarily determined that strain LWY-37-2 is a Gram-positive spore-forming bacillus.

[0116] 4.2 Molecular biological identification of strain 16S rDNA

[0117] DNA from the target strain was extracted using a bacterial genomic DNA extraction kit. The 16S rDNA of the strain was amplified using universal 16S rDNA primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-CTACGGCTACCTTGTTACGA-3'). After amplification, 3 μL of the PCR product was subjected to 1% agarose gel electrophoresis to confirm the PCR amplified fragment. Sequence analysis of the amplified PCR products was performed by Shanghai Paisennuo Biotechnology Co., Ltd. The conserved sequence results were analyzed using Nucleotide BLAST on the NCBI website, revealing that the 16S rDNA gene sequence of strain LWY-37-2 showed high homology (100%) with both *Bacillus licheniformis* strain DSM 13 and strain Bacillus licheniformis strain BCRC11702.

[0118] The obtained sequence results were compared with BLAST by NCBI to obtain species information similar to the sequence of the target species. The sequences with high similarity were selected for phylogenetic analysis using the NJ nearest neighbor method. The phylogenetic tree was constructed using MEGA 7.0 software, and it was found that strain LWY-37-2 was on the same branch as Bacillus licheniformis. Based on the colony morphology observation results of the above strain, the strain was identified as Bacillus licheniformis.

[0119] 4.3 Study on the physiological and biochemical characteristics of the strain

[0120] To investigate the metabolic capacity of strain LWY-37-2 to different carbon sources and its tolerance to chemically sensitive substances, this invention utilizes the Biolog automated microbial identification system and GenIII microplates to study the physiological and biochemical phenotype of strain LWY-37-2.

[0121] The Biolog GenIII microplates (containing 71 carbon sources and 23 chemically sensitive reagents) were used to perform physiological and biochemical tests on the strains, study the target strains' ability to utilize different carbon sources and their sensitivity to different chemical substances, and further determine the strain species.

[0122] Using a sterile inoculation loop, the bacterial suspension of strain LWY-37-2 was streaked onto LB solid medium for isolation. After incubation at 37°C for 16 hours, it was streaked again for 16 hours. Before the experiment, the GenIII plate and IF-B inoculation solution were removed from the refrigerator and preheated to room temperature. In the laminar flow hood, a suitable amount of bacterial clumps were scraped from the plate using a sterile pipette tip and spread onto a dry 2mL sterile centrifuge tube. The clumps were then dispersed using a sterile pipette tip, and 2mL of IF-B inoculation solution was added to prepare a high-concentration bacterial suspension. The suspension was allowed to stand for 5 minutes to allow residual bacterial clumps to settle. A suitable amount of the bacterial suspension was then added to the IF-B inoculation solution, and the turbidity was adjusted to approximately 95%T using a turbidimeter.

[0123] Pour the prepared inoculum into a V-shaped sample loading well. Using an 8-channel pipette, aspirate the inoculum into the pipette tip and add 100 μL of inoculum to each well of a GenIII microplate. After loading, incubate the microplate in a 37°C OmniLog incubator for 22 hours, repeating twice. Data acquisition and analysis were performed using Microlog software.

[0124] The screening test data were compiled and calculated using Excel spreadsheets. One-way ANOVA was performed using SPSS 22.0 software to test the significance of differences between groups, with P < 0.05 considered significant.

[0125] Figure 4 This is a map of carbon source metabolism and chemical sensitivity of strain LWY-37-2 on GENⅢ microplates.

[0126] The available carbon sources for strain LWY-37-2 are shown in Table 1. As shown in Table 1, strain LWY-37-2 can utilize 22 of the 71 carbon sources contained in the GEN III microplate, with 11 carbon sources having a utilization level above 150, accounting for 50% of all available carbon sources. These are dextrin, D-trehalose, D-cellobiose, sucrose, D-minobiose, β-formyl-D-glucoside, D-salicylic acid, α-D-glucose, D-mannose, D-fructose, and L-malic acid, demonstrating the good carbon source adaptability of strain LWY-37-2. Among the carbon sources that the strain utilizes well, most are sugars, with only L-malic acid being an acid, indicating that Bacillus licheniformis LWY-37-2 has a broad metabolic capacity for sugars.

[0127] Table 1. Available carbon sources for strain LWY-37-2

[0128]

[0129] The tolerance of strain LWY-37-2 to chemically sensitive substances is shown in Table 2. As shown in Table 2, strain LWY-37-2 exhibits good acid and salt tolerance, and can grow and metabolize under conditions of pH 5 and 8% sodium chloride. It shows strong tolerance to antibiotics such as acetomycin, lincomycin, nalidixic acid, and aztreonam, but shows sensitivity and intolerance to other antibiotics such as rifamycin SV, clostridial acid, dimethylaminotetracycline, tetradecanoic acid, and vancomycin.

[0130] Table 2. Tolerance of strain LWY-37-2 to chemically sensitive substances

[0131]

[0132] Example 5: Optimization of Single-Strain Solid-State Fermentation Process for Rapeseed Meal

[0133] 5.1 Test Materials

[0134] The rapeseed meal selected was aged rapeseed meal stored at the Changping Nankou Pilot Base of the Chinese Academy of Agricultural Sciences. Its nutritional composition on a dry matter basis was: crude protein 41.17%, dry matter content 93.40%, crude fiber 25.41%, neutral detergent fiber content 58.15%, acid detergent fiber content 43.09%, crude fat 1.5%, polypeptides 3.48%, soluble condensed tannin content 0.1126%, insoluble condensed tannin content 0.2563%, and total condensed tannin content 0.3690%.

[0135] 5.2 Culture medium and fermentation inoculum

[0136] LB medium (g / L): peptone 10, yeast extract 5, sodium chloride 10, sterilized at 121℃ for 20 minutes, used for culturing Bacillus licheniformis;

[0137] Fermentation medium: 50g rapeseed meal (passed through a 40-mesh sieve), 50mL sterile water, natural pH.

[0138] Fermentation inoculum: Bacillus licheniformis LWY-37-2 plates were streaked and cultured at 37℃ for 24 hours. Single colonies were picked from the plates and placed in 50 mL of LB liquid medium. The culture was then incubated at 37℃ and 200 r / min for 22 hours to prepare a single-cell fermentation culture.

[0139] 5.3 Inoculation time and single-strain solid-state fermentation steps

[0140] Since Bacillus licheniformis LWY-37-2 reached the late logarithmic growth phase after 22 hours, the bacterial suspension was diluted and plated. At this point, the viable count was 4.2 × 10⁻⁶. 8 CFU / mL, therefore the inoculation time was determined to be 22 hours of incubation.

[0141] 5.4 Single-strain solid-state fermentation steps

[0142] Take 50g of rapeseed meal raw material and put it into a 250mL beaker. Mix the strain culture solution diluted to the corresponding number of viable bacteria with sterile water. Inoculate it into the fermentation substrate at a certain ratio, stir it evenly with a sterile glass rod, seal it with a breathing membrane, and let it ferment at a constant temperature. Turn the material over once every 12 hours. After fermentation, dry it at 45℃, pulverize it and pass it through a 40-mesh sieve for testing.

[0143] Material-to-water ratio calculation: mass of fermentation substrate / volume of sterile water (m / v).

[0144] 5.5 Preliminary Experiment of Rapeseed Meal Fermentation

[0145] To investigate the feasibility of using Bacillus licheniformis LWY-37-2 to degrade condensed tannins in rapeseed meal and to determine fermentation parameters, based on previous research results from our laboratory, an inoculum size of 1% (4.2 × 10⁻⁶) was used. 6 Rapeseed meal was fermented using a process involving a CFU / g concentration, a feed-to-water ratio of 1:1, a fermentation temperature of 37℃, and a fermentation time of 2 days. After fermentation, the product was dried at 45℃, pulverized, and passed through a 40-mesh sieve. The soluble and insoluble condensed tannin contents were measured to evaluate the fermentation effect of single-strain solid-state fermentation and determine the fermentation indicators.

[0146] The rapeseed meal produced in the preliminary fermentation experiment was soft in texture, produced white microbial cells, and had a slightly rancid odor characteristic of Bacillus licheniformis fermented feed, indicating vigorous growth of Bacillus licheniformis LWY-37-2 in the fermentation medium. The rapeseed meal contained 0.1052% soluble condensed tannins, 0.2394% insoluble condensed tannins, and 0.3446% total condensed tannins (the sum of soluble and insoluble condensed tannins). The degradation rate of soluble condensed tannins after fermentation was significantly higher than that of insoluble condensed tannins (P<0.05), with degradation rates of 41.69% and 2.90%, respectively. Due to the high degree of polymerization and complex structure of insoluble condensed tannins, microbial fermentation had an insignificant effect on their degradation. To simplify the experimental procedure, the degradation rate of soluble condensed tannins was selected as the fermentation indicator; in the following experiments, the degradation rate of soluble condensed tannins will be referred to simply as the condensed tannin degradation rate.

[0147] 5.6 Determination of Fermentation Substrate

[0148] To investigate whether sterilization of the fermentation substrate could improve fermentation efficiency, Bacillus licheniformis LWY-37-2 inoculum was inoculated at a rate of 1% (4.2 × 10⁻⁶). 6(CFU / g) was inoculated into both sterilized and non-sterilized fermentation media, with process parameters identical to the preliminary experiment. The media were incubated statically at 37℃ for 2 days to study the fermentation status. Immediately after fermentation, the viable count of *Bacillus licheniformis* was determined, and sensory evaluation was performed. The media were then dried at 45℃, pulverized, and passed through a 40-mesh sieve. The content of condensed tannins, polypeptides, and pH were measured. Two blank control groups were set up: one with sterilized substrate and the other with non-sterilized substrate. Neither group was inoculated with fermentation seed liquid, and both were incubated under the same conditions for 2 days.

[0149] Table 3 shows the effects of fermentation substrate on the sensory properties, viable cell count, and pH of single-strain fermented rapeseed meal. As shown in Table 3, sterilization treatment in the uninoculated control group had no significant effect on feed texture, Bacillus licheniformis viable cell count, or feed color. However, due to the fumigation effect of high-temperature steam, the sterilized rapeseed meal released a bitter taste. In the unsterilized control group, fermentation also occurred due to contaminants carried by the raw materials, resulting in a decrease in pH and a slightly sour and musty odor. In contrast, the fermentation medium inoculated with Bacillus licheniformis LWY-37-2 produced white bacteria on the feed surface, indicating vigorous growth of Bacillus licheniformis. Furthermore, the large-scale growth of Bacillus licheniformis caused clumping in the feed. However, the substrate-sterilized fermentation group and the substrate-unsterilized fermentation group had different odors. The sterilized fermentation group produced a sour, rancid odor with a pH of 5.30, while the unsterilized fermentation group produced a sour and ammonia-like odor with a pH of 4.81.

[0150] Table 3. Effects of fermentation substrate on sensory properties, viable cell count, and pH of single-strain fermented rapeseed meal.

[0151]

[0152] Further, the degradation rate of condensed tannins and the increase rate of polypeptides in rapeseed meal under different treatment groups were determined to study the effect of fermentation substrate on the fermentation effect of Bacillus licheniformis LWY-37-2.

[0153] Figure 5 This indicates the impact of the fermentation substrate on fermentation parameters. For example... Figure 5 As shown, the degradation rate of condensed tannins in the non-sterile fermentation group with inoculated bacterial solution was higher than that in the sterile fermentation group, and the increase rate of polypeptides was also significantly higher in the non-sterile fermentation group (P<0.05).

[0154] Based on the combined sensory evaluation and fermentation index results, it was found that the fermentation effect was best when the substrate was not sterilized; therefore, the fermentation substrate was chosen to be unsterilized.

[0155] 5.7 Single-factor experiment on solid-state fermentation of rapeseed meal by single strain

[0156] To determine the significant factors affecting the solid-state fermentation of rapeseed meal by a single strain, single-factor fermentation experiments were conducted on each fermentation factor. The fermentation substrate was not sterilized, the pH was left natural, and the bacterial culture was prepared to a viable count of 1×10⁻⁶. 6Inoculate the fermentation substrate with an inoculum of CFU / mL at an appropriate ratio (calculated based on the viable cell count designed for the experiment), seal with a breathing membrane, and incubate statically at 37°C for 2 days, turning and aerating every 12 hours as the basic fermentation conditions. Based on these basic fermentation conditions and referring to previous studies in our laboratory, we changed the temperature (27°C, 30°C, 33°C, 37°C, 40°C), the initial pH (4.0, 5.0, 6.0, 7.0, 8.0), and the inoculated viable cell count (1×10⁻⁶). 5 CFU / g, 5×10 5 CFU / g, 1×10 6 CFU / g, 5×10 6 CFU / g, 1×10 7 The fermentation effect was evaluated using the condensed tannin degradation rate (X%), with the material-to-water ratio (1:0.6, 1:0.8, 1:1, 1:1.2, 1:1.4) as the index. The appropriate level of each fermentation factor was determined, with three replicates for each level. The specific groupings are shown in Table 4.

[0157] Table 4. Factor Level Table for Single-Factor Experimental Design

[0158]

[0159] Figure 6 The effect of temperature on the degradation rate of condensed tannins in rapeseed meal is indicated by lowercase letters above the columns; different letters indicate significant differences (P < 0.05), while the same letters indicate no significant differences (P ≥ 0.05). Figure 6 As shown, the degradation rate of condensed tannins in rapeseed meal was 42.03% and 42.44% at 30℃ and 33℃, respectively, which were significantly higher than those at 27℃, 37℃ and 40℃ (P<0.05). Both excessively high and low temperatures reduced the degradation rate of condensed tannins.

[0160] Figure 7 The degradation rate of condensed tannins in rapeseed meal fermented by Bacillus licheniformis LWY-37-2 at different pH values ​​is represented by lowercase letters above the columns. Different values ​​indicate significant differences (P < 0.05), while the same values ​​indicate no significant differences (P ≥ 0.05). Figure 7 It was found that when the pH of the fermentation medium was adjusted to 7, the degradation rate of condensed tannins was 29.98%, which was significantly higher than that of other groups (P<0.05). Therefore, pH 7 was determined to be the optimal pH for single-strain solid-state fermentation.

[0161] Figure 8 The degradation rate of condensed tannins in rapeseed meal fermented by Bacillus licheniformis LWY-37-2 under different inoculation viable cell counts is represented by lowercase letters above the column. Different values ​​indicate significant differences (P < 0.05), while the same values ​​indicate no significant differences (P ≥ 0.05). Figure 8 It can be seen that the number of live bacteria inoculated is greater than 5 × 10⁻⁶. 6The degradation rate of condensed tannins was significantly increased at CFU / g (P<0.05), 5×10 6 The degradation rate of the CFU / g fermentation group was 41.43%, the same as that of the 1×10 group. 7 There was no significant difference in CFU / g among the fermentation groups (P>0.05). To save fermentation costs, the optimal inoculum count was determined to be 5×10⁻⁶. 6 CFU / g.

[0162] Figure 9 The degradation rate of condensed tannins in rapeseed meal fermented by Bacillus licheniformis LWY-37-2 under different feed-to-water ratios is represented by lowercase letters above the columns. Different values ​​indicate significant differences (P < 0.05), while the same values ​​indicate no significant differences (P ≥ 0.05). Figure 9 It can be seen that the degradation rate was 34.98% when the material-to-water ratio was 1:1, which showed the best fermentation effect. The degradation rate of condensed tannins was significantly higher than that of other groups (P<0.05). Therefore, the optimal material-to-water ratio was determined to be 1:1.

[0163] 5.8. Orthogonal Experiment to Optimize Single-Strain Fermentation Process of Rapeseed Meal

[0164] Based on the results of the single-factor experiment, under the premise that the fermentation substrate is not sterilized, the inoculation count should be 5 × 10⁶ viable cells. 6 Based on CFU / g and pH 7 as the basic fermentation conditions, fermentation time, fermentation temperature, and feed-to-water ratio were used as the three factors for orthogonal experimental optimization. Three levels were determined for each factor, and a three-factor, three-level L9(3) design was employed. 4 Orthogonal experiments were used to further optimize the process of single-strain solid-state fermentation of rapeseed meal. The table of levels for each factor and the orthogonal experimental design are shown in Table 5, with three replicates for each level.

[0165] Table 5, L9(3) 4 Orthogonal experimental design factor level table

[0166]

[0167] Using the degradation rate of condensed tannins (X%) and the increase rate of peptides (Y%) as evaluation indicators, a weighted method was used with the total change rate M as the evaluation indicator (total change rate M = 0.85 × degradation rate of condensed tannins X + 0.15 × increase rate of peptides Y) to conduct L9 (3) 4 The orthogonal experiment with three factors and three levels further optimized the process of single-strain solid-state fermentation of rapeseed meal. The results of the orthogonal experiment and the range analysis are shown in Table 6.

[0168] Table 6. Results of the orthogonal experiment and range analysis of single-strain solid-state fermentation

[0169]

[0170] Range analysis revealed that the optimal single-strain solid-state fermentation process was: fermentation temperature 30℃, material-to-water ratio 1:0.8, and fermentation time 3 days. The influence of each experimental factor on the total rate of change was ranked as follows: time > material-to-water ratio > temperature.

[0171] Table 7 shows the analysis of variance for the total rate of change at different factor levels. According to Table 7, level 1 had the highest total rate of change for temperature, which was not significantly different from level 2 (P>0.05), but significantly higher than level 3 (P<0.05). Level 2 had a higher total rate of change for temperature than level 3, but the difference was not significant (P>0.05). For the material-to-water ratio, the total rate of change was: level 1 > level 2 > level 3. The total rate of change for the material-to-water ratio at levels 1 and 2 was significantly higher than at level 3 (P<0.05), but there was no significant difference between them (P>0.05). The total rate of change for fermentation time at level 2 was greater than at level 1, but not significantly (P>0.05). The total rate of change for fermentation time at levels 1 and 2 was significantly greater than the total rate of change for fermentation time at level 3 (P<0.05).

[0172] Table 7. Analysis of variance on the total rate of change for different factor levels

[0173]

[0174] Note: Data in the same column are labeled with lowercase letters. Different letters indicate significant differences (P < 0.05), while the same letters indicate no significant differences (P ≥ 0.05).

[0175] The results of the ANOVA on the effects of different factor levels on the degradation rate of condensed tannins in rapeseed meal under single-strain solid-state fermentation are shown in Table 8. Table 8 shows that for different temperature levels, the degradation rate of condensed tannins was: Level 1 > Level 2 > Level 3, and the degradation rates of condensed tannins at levels 1 and 2 were significantly higher than at level 3 (P<0.05), but there was no significant difference between them (P>0.05). For different material-to-water ratios, the degradation rate of condensed tannins was: Level 2 > Level 1 > Level 3, and the degradation rates of condensed tannins at levels 1 and 2 were significantly higher than at level 3 (P<0.05), but there was no statistically significant difference between the material-to-water ratios at levels 1 and 2 (P>0.05). For different fermentation times, the degradation rate of condensed tannins was: Level 2 > Level 1 > Level 3, and the degradation rates of condensed tannins at fermentation times at levels 1 and 2 were significantly higher than at level 3 (P<0.05), but there was no statistically significant difference between levels 1 and 2 (P>0.05).

[0176] Table 8. Analysis of variance on the degradation rate of condensed tannins at different factor levels

[0177]

[0178] Note: Data in the same column are labeled with lowercase letters. Different letters indicate significant differences (P < 0.05), while the same letters indicate no significant differences (P ≥ 0.05).

[0179] The results of the analysis of variance on the effect of different factor levels on the increase rate of peptides in rapeseed meal fermented by single-strain fermentation are shown in Table 9. Table 9 shows that for different temperature levels, the peptide increase rate was: Level 3 > Level 2 > Level 1, and the peptide increase rate at Level 3 was significantly higher than that at Levels 1 and 2 (P<0.05), while there was no significant difference between Levels 1 and 2 (P>0.05). For different material-to-water ratios, the peptide increase rate at Level 1 was significantly higher than that at Levels 2 and 3 (P<0.05), and the peptide increase rate at Level 2 was significantly higher than that at Level 3 (P<0.05). For different fermentation times, the peptide increase rate was: Level 2 > Level 3 > Level 1, and the peptide increase rates at Levels 2 and 3 were significantly higher than that at Level 1 (P<0.05), while there was no statistically significant difference between Levels 2 and 3 (P>0.05).

[0180] Table 9. Analysis of variance on the effect of different factor levels on the increase rate of peptides

[0181]

[0182] Note: In the same column, lowercase letters above the data indicate significant differences (P < 0.05) if different letters are different, and no significant differences if the same letters are the same.

[0183] (P≥0.05).

[0184] 5.9 Single-strain solid-state fermentation of rapeseed meal under optimal process

[0185] Since the optimal fermentation process was not included in the orthogonal experimental group, the obtained optimal process was validated, and the changes in the nutrients in rapeseed meal before and after fermentation were investigated. The inoculation count was set at 5 × 10⁶ viable cells. 6 The pH of rapeseed meal was adjusted to 7 with a non-sterilized fermentation substrate. Under experimental conditions of 30℃, a material-to-water ratio of 1:0.8, and a fermentation time of 3 days, 500g of rapeseed meal was fermented in a fermentation bag equipped with a one-way breathing valve at a constant temperature. The material was turned over every 12 hours. After fermentation, the pH was measured, and the meal was then dried at 45℃, pulverized, and passed through a 40-mesh sieve for analysis. The results were compared with a control group (inoculated with sterile water) and unfermented rapeseed meal to analyze the changes in the content of soluble condensed tannins, insoluble condensed tannins, polypeptides, crude protein, amino acids, crude fat, crude fiber, neutral detergent fiber, and acid detergent fiber before and after fermentation.

[0186] After fermentation, the fermented rapeseed meal was loose and slightly clumped, yellowish-brown in color, with a sour and ammonia-like odor, and a pH of 5.10. Relevant indicators of the fermented rapeseed meal were measured, revealing a soluble condensed tannin degradation rate of 64.98%, an insoluble condensed tannin degradation rate of 4.47%, a total condensed tannin degradation rate of 22.96%, a polypeptide increase rate of 151.63%, and a total change rate of 77.97%. The highest total change rate in the orthogonal experimental group was 47.77%, indicating that the optimal fermentation process was significantly better than the highest group in the orthogonal experiment (P<0.05).

[0187] Table 10 shows the nutrient content (dry matter basis) of rapeseed meal after single-strain solid-state fermentation. As shown in Table 10, the crude protein content of the rapeseed meal after fermentation was 42.86%, an increase of 4.10% compared with the content before fermentation; the total energy was 18.34 MJ / kg, an increase of 2.97% compared with the content before fermentation; the crude fat content was 4 g / kg, a decrease of 73.33% compared with the content before fermentation; the crude ash content remained unchanged before and after fermentation; the crude fiber content was 27.60%, an increase of 8.62% compared with the content before fermentation; the neutral detergent fiber content was 56.90%, a decrease of 2.15% compared with the content before fermentation; and the acid detergent fiber content was 39.88%, a decrease of 7.45% compared with the content before fermentation.

[0188] Table 10. Nutrient content (dry matter basis) of rapeseed meal fermented by single-strain solid-state fermentation

[0189]

[0190] Table 11 shows the changes in amino acid content in rapeseed meal after fermentation using the optimal process. As shown in Table 11, solid-state fermentation with Bacillus licheniformis LWY-37-2 increased the total amino acid content in rapeseed meal by 3.21%. Methionine saw the largest increase in content, at 14.29%; cystine and serine were the second and third largest increases, at 7.41% and 6.12%, respectively. Unlike the increasing trend in total amino acid content, lysine and arginine contents both decreased.

[0191] Table 11. Changes in amino acid content of rapeseed meal from single-strain solid-state fermentation (dry matter basis)

[0192]

[0193] Example 6: Metabolomics Analysis of Fermented Rapeseed Meal

[0194] Metabolomics analysis was performed on rapeseed meal fermented using the optimal process, with unfermented rapeseed meal as a control. The analysis was commissioned to Wuhan Metawell Biotechnology Co., Ltd.

[0195] 6.1 Dry Sample Extraction Procedure

[0196] (1) Biological samples were placed in a freeze dryer (Scientz-100F) for vacuum freeze drying;

[0197] (2) Grind into powder using a grinder (MM 400, Retsch) (30Hz, 1.5 minutes);

[0198] (3) Weigh 50 mg of sample powder using an electronic balance (MS105DM) and add 1200 μL of 70% methanol-water internal standard extraction solution pre-cooled at -20℃;

[0199] (4) Vortex once every 30 minutes, each lasting 30 seconds, for a total of 6 vortices;

[0200] (5) After centrifugation (12000 rpm, 3 minutes), aspirate the supernatant, filter the sample with a microporous membrane (0.22 μm pore size), and store it in a sample vial for UPLC-MS / MS analysis.

[0201] 6.2 Chromatographic and Mass Spectrometry Acquisition Conditions

[0202] The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (ExionLC). TM AD) and tandem mass spectrometry (MS / MS).

[0203] Liquid phase conditions mainly include:

[0204] 1) Column: Agilent SB-C18 1.8μm, 2.1mm×100mm;

[0205] 2) Mobile phase: Phase A is ultrapure water (with 0.1% formic acid added), and Phase B is acetonitrile (with 0.1% formic acid added);

[0206] 3) Elution gradient: The proportion of phase B is 5% at 0.00 min, and increases linearly to 95% within 9.00 min, and is maintained at 95% for 1 min. From 10.00 to 11.10 min, the proportion of phase B decreases to 5% and is balanced at 5% until 14 min.

[0207] 4) Flow rate 0.35 mL / min; column temperature 40℃; injection volume 2 μL.

[0208] Mass spectrometry conditions mainly include:

[0209] Electrospray ionization (ESI) temperature was 500 °C; ion spray voltage (IS) was 5500 V (positive ion mode) / -4500 V (negative ion mode); ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 50, 60, and 25 psi, respectively, and collision-induced ionization parameters were set to high. QQQ scans were performed using MRM mode with the collision gas (nitrogen) set to medium. DP and CE for each MRM ion pair were optimized through further declustering potential (DP) and collision energy (CE). A specific set of MRM ion pairs was monitored at each epoch based on the metabolites eluted within each epoch.

[0210] 6.3 Qualitative and quantitative analysis of metabolites

[0211] Mass spectrometry data were processed using Analyst 1.6.3 software. Based on a local metabolic database, qualitative and quantitative mass spectrometry analyses of the sample metabolites were performed.

[0212] 6.4 Screening Results of Some Differential Metabolites

[0213] The degradation rates of some differentially metabolites are shown in Table 12.

[0214] Table 12. Degradation rates of some differentially expressed metabolites

[0215]

[0216] Example 7: Nutritional value assessment of fermented rapeseed meal

[0217] The in vitro biomimetic digestion experiment was conducted according to the operating manual of the monogastric animal biomimetic digestion system. Two g each of fermented and unfermented rapeseed meal (passed through a 60-mesh sieve) were weighed. Using endogenous chicken enzymes as digestive enzymes, the monogastric animal biomimetic digestion system (SDS-II) was used to simulate the digestion process of feed in the stomach, small intestine, and large intestine of broilers. The dry matter, energy, crude protein, amino acid digestibility, and metabolizable energy of the rapeseed meal in both groups were evaluated. Six replicates were performed for each treatment, with one digestion tube per replicate.

[0218] Calculation formula:

[0219]

[0220]

[0221]

[0222]

[0223] DMME(MJ / Kg)=(GE1-GE2) / (1000×DM1) (Formula 5)

[0224] In the formula: SDDM is the biomimetic digestibility of the dry matter of the tested feed; SDGE is the total energy digestibility of the tested feed; CPDMD is the dry-basis crude protein digestibility of the tested feed; AADMD is the dry-basis amino acid digestibility of the tested feed; DMME is the dry-basis apparent metabolizable energy of the tested feed; DM1 is the dry matter mass of the feed processed by the machine; DM2 is the dry matter mass of the biomimetic digestion residue; GE1 is the total energy value of the tested feed (J); GE2 is the total energy value of the biomimetic digestion residue of the tested feed (J); CP1 is the crude protein content of the tested feed processed by the machine (g); CP2 is the crude protein content of the biomimetic digestion residue of the tested feed (g); AAL1 is the amino acid content of the tested feed processed by the machine; AAL2 is the amino acid content in the biomimetic digestion residue of the tested feed.

[0225] Fermented rapeseed meal was digested in vitro using a monogastric animal biomimetic digestive system to investigate its nutritional value. Table 13 shows the dry matter, protein, and total energy digestibility and digestible energy (dry matter basis) of rapeseed meal and fermented rapeseed meal. As shown in Table 13, the dry matter digestibility, protein digestibility, total energy digestibility, and digestible energy of rapeseed meal fermented by Bacillus licheniformis LWY-37-2 were significantly greater than those of unfermented rapeseed meal (P<0.05). The dry matter digestibility of fermented rapeseed meal increased by 19.88%, protein digestibility by 27.03%, total energy digestibility by 25.15%, and digestible energy by 24.58%.

[0226] Table 13. Digestibility of dry matter, protein, and total energy of rapeseed meal and fermented rapeseed meal, and digestible energy (dry matter basis).

[0227]

[0228] 60 mg of rapeseed meal before and after fermentation were weighed separately. The rapeseed meal was hydrolyzed with 6 mol / L hydrochloric acid at 110℃ for 22 hours. After hydrolysis, the solution was cooled and filtered through a 0.22 μm membrane. 1 mL of the clear filtrate was concentrated in a vacuum concentrator at 60℃ to eliminate hydrochloric acid interference. The solution was then redissolved with 0.01 mol / L hydrochloric acid and filtered again through a 0.22 μm membrane. The amino acid content was measured using an automatic amino acid analyzer. The biomimetic digestibility of amino acids was calculated using a formula, and the results are shown in Table 14. Table 14 shows the amino acid digestibility (dry matter basis) of rapeseed meal and fermented rapeseed meal.

[0229] Table 14. Amino acid digestibility of rapeseed meal and fermented rapeseed meal (dry matter basis)

[0230]

[0231] As shown in Table 14, compared with the unfermented rapeseed meal group, the digestibility of all amino acids in the fermented rapeseed meal group was improved, with the total amino acid digestibility increasing by 25.32%. Among the amino acids, cystine showed the largest increase in digestibility, reaching 49.89%. Moreover, the biomimetic digestibility of lysine, valine, isoleucine, phenylalanine, aspartic acid, alanine, glycine, and proline all increased by more than 30%.

Claims

1. Bacillus licheniformis LWY-37-2, this strain was deposited on February 21, 2023 at the China General Microbiological Culture Collection Center (CGMCC), located in Beijing, with accession number CGMCC No. 26654.

2. A method for degrading condensed tannins in rapeseed meal using Bacillus licheniformis LWY-37-2 as described in claim 1, comprising the following steps: Set the inoculation count to 5 × 10⁶ live bacteria. 6 CFU / g, material-to-water ratio 1:0.8, pH adjusted to 7 on a non-sterilized rapeseed meal fermentation substrate, fermented at 30℃ for 3 days, with the material turned over once every 12 hours.

3. A method for producing feed ingredients by degrading rapeseed meal condensed tannins using Bacillus licheniformis LWY-37-2 as described in claim 1, comprising the following steps: Set the inoculation count to 5 × 10⁶ live bacteria. 6 CFU / g, material-to-water ratio 1:0.8, pH adjusted to 7 on the basis of non-sterilized rapeseed meal fermentation substrate, fermentation time at 30℃ for 3 days, turning the material once every 12 hours; drying; Grind and sieve.

4. A bacterial agent comprising a bacterial suspension of Bacillus licheniformis LWY-37-2 as described in claim 1.

5. The application of Bacillus licheniformis LWY-37-2 as described in claim 1 in the degradation of condensed tannins in rapeseed meal.

6. The feed ingredients obtained by the method according to claim 3.

7. The application of the microbial agent according to claim 4 in rapeseed meal fermentation.

8. The use of the feed ingredients according to claim 6 in the preparation of feed.

Images