Cottonseed meal fermented feed, preparation method and application of cottonseed meal fermented feed in fattening pigs

Abstract

The invention discloses a cottonseed meal fermented feed, a preparation method and application of the cottonseed meal fermented feed in fattening pigs, and relates to the technical field of fermented feeds. The cottonseed fermented feed is prepared from a fermentation product which is obtained by carrying out combined fermentation on cottonseed cakes or cottonseed meal by virtue of lactobacillus pentosus and a compound enzyme, and a fermentation product which is obtained by carrying out combined fermentation on the cottonseed cakes or the cottonseed meal by virtue of the lactobacillus pentosus and the compound enzyme, the lactobacillus pentosus is preserved in Wuhan University in China, and the preservation number is CCTCC NO: M 20231547. According to the invention, theoretical basis and reference are provided for soybean meal reduction of the cottonseed fermented feed in livestock production, and the prepared cottonseed meal fermented feed has good application prospects in improvement of production performance of poultry, aquatic products and / or livestock animals.

Description

Technical Field

[0001] This invention relates to the field of fermented feed technology, and more specifically, to a cottonseed meal fermented feed, its preparation method, and its application in fattening pigs. Background Technology

[0002] Enzymatic hydrolysis is divided into single-enzyme hydrolysis and compound-enzyme hydrolysis techniques. Single-enzyme hydrolysis involves adding a specific enzyme to simply break down large molecules. It is highly specific and can increase the content of small peptides, but it can only catalyze or decompose one type of compound, thus its application range is relatively limited, its efficiency is low, and it cannot meet diverse needs. Compound enzymes are composed of multiple enzymes, which can more effectively and comprehensively degrade large molecules into smaller molecules, break down indigestible fibers in raw materials, release nutrients, degrade carbohydrates into smaller molecules such as glucose, and promote digestion and absorption in animals (Chen Qinghua, 2024).

[0003] There are currently no studies evaluating the energy efficiency of FCSM (fermented cottonseed meal) in the Chinese Swine Nutrition Requirements and NRC (National Academy of Sciences) catalogues. Furthermore, the nutritional levels of CSM (cottonseed meal) vary greatly depending on the cotton variety, growing conditions, processing methods, and source, particularly in crude protein and crude fiber. Changes in nutrient composition directly affect the consistency of digestible energy (DE) and metabolizable energy (ME). Assessing the energy requirements of animals for FCSM provides an important foundation for precision farming. Previous studies have only evaluated the energy efficiency of CSM in sows and the net energy prediction and digestible energy assessment of growing pigs. However, they have not evaluated the energy efficiency of FCSM in growing-finishing pigs and its impact on animal growth performance. Studies have found that, after comparing the effects of fermented / unfermented CSM, finishing pigs with the same proportion of FCSM showed significantly higher average daily weight gain than the unfermented group. However, the effects of fermented products produced by different fermentation strains and processes vary, requiring specific evaluation of feeding effects.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a fermented cottonseed meal feed, its preparation method, and its application in fattening pigs to improve the nutritional quality of fermented cottonseed meal. This invention utilizes a compound enzyme for synergistic fermentation and explores the effects of different formulations of fermented cottonseed meal feed on nutritional indicators such as total energy, dry matter, crude fat, crude protein, acid-soluble protein, crude fiber, neutral detergent fiber, acid detergent fiber, lactic acid, acetic acid, propionic acid, butyric acid, and amino acids. The resulting feed addition ratio for fattening pigs is also provided. The fermented cottonseed meal feed provided by this invention shows promising application prospects in improving the production performance of poultry, aquatic animals, and / or livestock.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides a cottonseed fermented feed, comprising: a fermentation product obtained by co-fermentation of cottonseed cake or cottonseed meal by Lactiplantibacillus pentosus and a compound enzyme; Lactiplantibacillus pentosus is deposited at Wuhan University with accession number CCTCC NO:M 20231547.

[0008] Secondly, the present invention provides a method for preparing cottonseed fermented feed, which includes the following steps: mixing and fermenting the above-mentioned Lactobacillus pentosus, compound enzyme, and cottonseed cake or cottonseed meal.

[0009] Thirdly, the present invention provides a method for fattening pigs, wherein the above-mentioned cottonseed fermented feed or the cottonseed fermented feed prepared by the above-mentioned method is fed to the pigs.

[0010] Fourthly, the present invention provides the application of cottonseed fermented feed or cottonseed fermented feed prepared by the above-mentioned method for improving the production performance of poultry, aquatic and / or livestock.

[0011] The present invention has the following beneficial effects:

[0012] This invention utilizes a newly isolated strain of *Lactiplantibacillus pentosus* for cottonseed meal fermentation. By co-fermenting cottonseed meal with *Lactiplantibacillus pentosus* and a compound enzyme, the nutritional content of the fermented cottonseed meal can be improved, increasing various nutritional indicators such as crude protein, acid-soluble protein, lactic acid, acetic acid, propionic acid, butyric acid, and amino acids, while effectively reducing the content of crude fiber, neutral detergent fiber, and acid detergent fiber. Based on this, feeding the prepared fermented cottonseed meal to finishing pigs can significantly improve their growth performance, increase nutrient digestibility, and improve their body weight and average daily weight gain; reduce their feed conversion ratio; lower serum uric acid and ammonia levels in growing-finishing pigs; increase growth hormone and insulin-like growth factor levels; increase pepsin activity in growing-finishing pigs; and increase lipase and trypsin activity in the jejunum. Therefore, this invention has promising applications in improving the production performance of poultry, aquatic animals, and / or livestock.

[0013] Furthermore, the present invention provides a theoretical basis and reference for reducing soybean meal usage in livestock production using cottonseed meal fermented feed. The cottonseed meal fermented feed provided by the present invention can be used as a substitute for soybean meal. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0015] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0016] In the first aspect, this invention provides a cottonseed meal fermented feed, comprising: a fermentation product obtained by co-fermentation of cottonseed meal with Lactiplantibacillus pentosus and a complex enzyme; Lactiplantibacillus pentosus is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, on August 31, 2023, with accession number CCTCC NO:M 20231547, and is identified as viable, with the proposed classification name: Lactiplantibacillus pentosus.

[0017] The *Lactobacillus pentosus* is a gossypol-detoxifying strain first screened from cotton field humus soil by the inventors. After multiple streak culture, a single strain was obtained. Then, by increasing the content of the acetate-gossypol solution in the culture medium, the strain was re-screened and a highly efficient gossypol-detoxifying strain was obtained. After testing, the strain provided by this invention has a high detoxification rate of free gossypol and can be fermented anaerobicly, facultatively, or aerobically.

[0018] Co-fermentation of cottonseed meal using *Lactobacillus pentosus* and a compound enzyme can improve various nutritional indicators of fermented cottonseed meal, including crude protein, acid-soluble protein, lactic acid, acetic acid, propionic acid, butyric acid, and amino acids, while effectively reducing the content of crude fiber, neutral detergent fiber, and acid detergent fiber. Based on this, feeding the prepared fermented cottonseed meal to finishing pigs can significantly improve their growth performance, increase nutrient digestibility, and improve their body weight and average daily weight gain; reduce their feed conversion ratio; lower serum uric acid and ammonia levels in growing-finishing pigs; increase growth hormone and insulin-like growth factor levels; increase pepsin activity in growing-finishing pigs; and increase lipase and trypsin activity in the jejunum. This invention has promising applications in improving the production performance of poultry, aquatic animals, and / or livestock.

[0019] The present invention provides a theoretical basis and reference for reducing soybean meal usage in livestock production by using cottonseed meal fermented feed. The cottonseed meal fermented feed provided by the present invention can be used as a substitute for soybean meal.

[0020] In a preferred embodiment of the present invention, the amount of compound enzyme added accounts for 0.15-0.25% of the total mass of cottonseed meal. For example, the amount of compound enzyme added accounts for 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24%, or 0.25% of the total mass of cottonseed meal.

[0021] After extensive and long-term screening, the inventors discovered that adding 0.15-0.25% of a compound enzyme can significantly increase the crude protein content of cottonseed meal, and fermentation time and dosage significantly affect the acid-soluble protein content. With prolonged enzymatic hydrolysis time, the content of small peptides significantly increased, and adding 0.15%-0.25% of the compound enzyme significantly improved the acid-soluble protein content in fermented cottonseed meal. The group with 0.25% compound enzyme showed the highest relative content of small peptides in fermented cottonseed meal.

[0022] The nutrient content of FCSM can be significantly increased through synergistic fermentation of bacteria and enzymes, including significantly increased crude protein content, significantly enriched lactic acid, acetic acid, propionic acid, and butyric acid content, and increased content of essential amino acids, non-essential amino acids, and total amino acids.

[0023] In a preferred embodiment of the present invention, each g of the complex enzyme comprises the following enzyme-active raw materials: 30,000 U of xylanase, 10,000 U of acidic protease, 10,000 U of neutral protease, 20,000 U of alkaline protease, 5,000 U of β-glucanase, 3,000 U of pectinase, 2,000 U of cellulase, and 100 U of α-galactosidase.

[0024] In other embodiments, the complex enzyme can be selected from any commercially available complex enzyme.

[0025] In a preferred embodiment of the present invention, the co-fermentation is anaerobic, facultative anaerobic, or aerobic fermentation. The fermentation conditions are as follows: the water content of the fermentation broth is controlled at 45-55%, the inoculum size of *Lactobacillus pentosus* is 8-12%, the fermentation time is 11-13 days, and the fermentation temperature is 37℃±0.5℃. Under the above fermentation conditions, a high free gossypol detoxification effect is achieved. For example, the water content of the fermentation broth is 40%, 45%, 50%, or 55%, the inoculum size of *Lactobacillus pentosus* is 8%, 9%, 10%, 11%, or 12%, and the fermentation time is at least 7 days, for example, 11 days, 12 days, or 13 days.

[0026] Secondly, the present invention provides a method for preparing cottonseed meal fermented feed, which includes the following steps: mixing and fermenting the above-mentioned Lactobacillus pentosus, compound enzyme and cottonseed cake or cottonseed meal.

[0027] In a preferred embodiment of the present invention, the amount of compound enzyme added accounts for 0.15-0.25% of the total mass of the fermentation raw materials. For example, the amount of compound enzyme added accounts for 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24%, or 0.25% of the total mass of cottonseed cake or cottonseed meal.

[0028] In a preferred embodiment of the present invention, the fermentation is anaerobic, facultative anaerobic, or aerobic fermentation. The fermentation conditions are as follows: the water content of the fermentation broth is controlled at 45-55%, the inoculum size of *Lactobacillus pentosus* is 8-12%, the fermentation time is 11-13 days, and the fermentation temperature is 37℃±0.5℃. Under the above fermentation conditions, a high detoxification effect of free gossypol is achieved.

[0029] In one embodiment, the product of mixed fermentation can be centrifuged, the fermentation precipitate can be dried to obtain cottonseed meal fermented feed, or all fermentation liquid can be used to prepare cottonseed fermented feed.

[0030] Thirdly, the present invention provides a method for fattening pigs, wherein the above-mentioned cottonseed meal fermented feed or the cottonseed meal fermented feed prepared by the above-mentioned method is fed to pigs.

[0031] In a preferred embodiment of the present invention, the amount of fermented cottonseed meal added accounts for 2.4-9.6% of the daily ration by weight, such as 2.4%, 4.5%, 4.8%, 7.2%, and 9.6%.

[0032] In a preferred embodiment of the present invention, the amount of cottonseed meal fermented feed added accounts for 7.2%-9.6% of the daily ration by weight.

[0033] In a preferred embodiment of the present invention, the amount of cottonseed meal fermented feed added accounts for 7.2% of the mass of the daily ration.

[0034] Fourthly, the present invention provides the application of cottonseed meal fermented feed or cottonseed meal fermented feed prepared by the above-mentioned method in improving the production performance of poultry, aquatic and / or livestock animals.

[0035] In a preferred embodiment of the present invention, the poultry is selected from: chicken, duck, goose or pigeon;

[0036] Livestock are selected from: pigs, cattle, sheep, horses, donkeys, deer, camels, or alpacas; aquatic products are selected from: fish or shrimp.

[0037] The aquaculture species are selected from any one or more of the following: grass carp, mandarin fish, largemouth bass, yellow catfish, catfish, crucian carp, common carp, silver carp, bighead carp, black carp, yellow eel, snakehead, eel, tilapia, redfin pufferfish, yellow pufferfish, and zebrafish.

[0038] In a preferred embodiment of the present invention, the application includes at least one of the following application methods:

[0039] (1) Increase the weight and average daily weight gain of fattening pigs;

[0040] (2) Reduce the feed conversion ratio for fattening pigs;

[0041] (3) Dry matter digestibility is significantly increased;

[0042] (4) Increased digestibility of neutral detergent fibers and acid detergent fibers;

[0043] (5) Reduce serum uric acid and blood ammonia levels in growing-finishing pigs;

[0044] (6) Increase the levels of growth hormone and insulin-like growth factor in growing-finishing pigs;

[0045] (7) Improve the activity of pepsin in growing-finishing pigs;

[0046] (8) Increase the activity of lipase and trypsin in the jejunum;

[0047] (9) Cottonseed fermented feed is used as a substitute for soybean meal.

[0048] In summary, this invention has promising applications in improving the production performance of poultry, aquatic animals, and / or livestock.

[0049] This invention employs a total manure collection and nesting algorithm to determine the efficacy of FCSM in growing pigs. Through an ileal terminal amino acid digestibility assay, the CP (content of FCSM) and the apparent ileal digestibility (AID) and standard ileal digestibility (SID) values ​​of amino acids in growing pigs were determined, providing data reference for further experimental research and practical application in growing-finishing pigs. Based on previous data, the application effects of adding different proportions of FCSM to replace soybean meal in growing-finishing pigs were investigated to verify the influence of FCSM on the production performance, nutrient digestibility, serum biochemistry, and digestive enzyme activity of growing-finishing pigs, and to screen the optimal proportion of FCSM to replace soybean meal, providing a theoretical basis and reference for reducing soybean meal usage in livestock production.

[0050] Example 1

[0051] Lactiplantibacillus pentosus, named L. pentosus YL10, with accession number CCTCC NO:M 20231547.

[0052] This embodiment evaluated the effects of different amounts of compound enzymes added on crude protein, acid-soluble protein, and nutrients in the cottonseed meal microbial-enzyme co-fermentation products.

[0053] 1. Cottonseed meal fermentation test materials

[0054] CSM was purchased from Beijing Zhongcheng Tianli Biotechnology Co., Ltd., and pulverized for use in CSM fermentation experiments. The fermentation strain was the laboratory-screened and preserved strain L. pentosus YL10. The compound enzyme was provided by Yiduoli Company, and its components were xylan (30000U / g), acidic protease (10000U / g), neutral protease (10000U / g), alkaline protease (20000U / g), β-glucanase (5000U / g), pectinase (3000U / g), cellulase (2000U / g), and α-galactosidase (100U / g).

[0055] Lactic acid bacteria culture medium (MRS) was purchased from Qingdao Haibo Biotechnology Co., Ltd. The appropriate weight was weighed according to the kit requirements, and ultrapure water was added before autoclaving. Adding 2% agar powder to the liquid medium resulted in solid culture.

[0056] 2. Cottonseed meal fermentation with microorganisms and enzymes.

[0057] Based on the optimal fermentation conditions selected in the preliminary screening, the moisture content of the fermentation broth was set at 45%, the fermentation time at 11 days, and different proportions of compound enzymes (as a percentage of cottonseed meal by mass) were added: 0.1%, 0.15%, 0.2%, and 0.25%. 100g of feed (fermentation product) was collected on days 1, 3, 5, 7, and 9 of enzymatic hydrolysis, dried at 65℃, and the CP and acid-soluble protein content were measured. After determining the proportion of compound enzymes, the fermentation broth was prepared by adding 8% bacterial inoculum (i.e., the inoculum amount, as a percentage of the total material to be fermented), with a moisture content of 45%, a fermentation time of 11 days, and a fermentation temperature of 37℃. After thorough stirring, anaerobic fermentation was carried out, followed by drying for testing.

[0058] The following indicators of anaerobic fermentation products with different ratios of compound enzymes were tested: total energy, dry matter, crude fat, crude protein, acid-soluble protein, crude fiber, neutral detergent fiber, acid detergent fiber, lactic acid, acetic acid, propionic acid, and butyric acid.

[0059] On days 1, 3, 5, 7 and 9 of the strain's fermentation, 1.00 g of fresh sample was weighed into a 15 mL centrifuge tube, 9 mL of ultrapure water was added, and the mixture was shaken and mixed using a vortex mixer. The pH value of the supernatant was then measured and recorded using a calibrated pH meter.

[0060] The specific testing indicators and methods are as follows:

[0061] (1) Determination of dry matter

[0062] Refer to the People's Republic of China National Standard GB / T 6435-2006, Determination of Moisture in Feed. First, weigh 1.00 g of the sample into a constant-weight aluminum box, dry it in an oven at 103℃ for 4 hours, weigh it again after constant weight, and then calculate the result.

[0063] (2) Measurement of energy

[0064] The total energy of the sample was determined using a semi-automatic calorimeter (SDACM3100, Hunan Sande Technology Co., Ltd.). Approximately 1.00g of sample was weighed, the iron wire was fixed in place, and the sample was placed into the instrument according to the instrument's requirements. The data was recorded after the instrument finished measuring.

[0065] (3) Determination of crude protein

[0066] The determination of crude protein in feed was performed according to the national standard GB / T 6432-2018. The Kjeldahl method was used. Approximately 0.50 g of sample was weighed and vertically placed into a digestion tube. Copper sulfate and potassium sulfate pentahydrate were then added to the digestion tube as catalysts to promote the subsequent chemical reaction. Next, 10 mL of concentrated sulfuric acid was slowly added, and the digestion tube was placed in the apparatus for digestion for 3.5 hours. After cooling to room temperature, the crude protein content of ammonium sulfate was determined using an automated Kjeldahl nitrogen analyzer. Once the result was accurate, the crude protein content in the sample was determined, and the hydrochloric acid consumption was recorded.

[0067] (4) Determination of acid-soluble proteins

[0068] Weigh approximately 3.00 g of sample and vertically place it into a 50 mL centrifuge tube. Add 25 mL of 15% trichloroacetic acid solution, mix thoroughly, and let stand for 30 minutes. Then, transfer the solution to a centrifuge tube and centrifuge at 4000 rpm for 10 minutes. Pass the solution through filter paper to further remove impurities. Accurately pipette 10 mL of the filtrate into a digestion tube, digest according to the method for determining crude protein, and bring the volume to 100 mL. Subsequently, transfer 10 mL of the digestion solution for distillation to determine its CP content. A blank reagent is also tested simultaneously to ensure the accuracy of the experimental results.

[0069] (5) Determination of crude fat

[0070] The determination of crude fat in feed was performed according to the national standard GB / T 6433-2006. The Soxhlet extraction method was used. 0.50 g of sample (accurate to four decimal places) was weighed, folded, and marked with a pencil. The sample was dried at 105℃ to constant weight, with a weight difference of less than 0.0008 g. The sample was then placed in an extraction tube, anhydrous ether was added, and the apparatus was connected and heated in a 53℃ water bath to allow the ether to reflux. After the extraction, the sample was placed in a fume hood to allow residual ether to fully evaporate before being dried in an oven to constant weight.

[0071] (6) Determination of crude fiber

[0072] The determination of crude fiber in feed was performed according to the national standard GB / T 6433-2006. Approximately 1.00 g of sample was weighed, pre-degreased to remove carbonates, connected to a filter pot, and 150 mL of sulfuric acid was added. After boiling for 30 min, heating was stopped. The sulfuric acid was filtered out under vacuum using the filter pot. The remaining residue was washed with 30 mL of hot water, dried, and then degreased again. The equipment was connected, 150 mL of potassium hydroxide was added, and after boiling for 30 min, the sample was filtered again. The filter pot was placed in an ashing dish and dried at 130℃ for 2 h. The sample was then carbonized at low temperature on an electric furnace until smokeless. It was then transferred to a muffle furnace and calcined at 550±20℃ for 4 h. After cooling in air for approximately 1 min, the sample was placed in a desiccator and cooled for 30 min. The two weighings were both less than 0.002 g.

[0073] (7) Determination of neutral detergent fibers

[0074] Weigh 0.50g of sample into a filter bag, seal it with a sealer, and mark it with a solvent-resistant pen. Perform a blank test at the same time. Place the sample into the equipment. Add 20g of anhydrous sodium sulfite and 4mL of α-amylase to the neutral detergent solution and dissolve it completely. Add 2000mL of the solution to the equipment to ensure that the sample is completely submerged. After digestion, add 2000mL (80-90℃) of distilled water and 4mL of α-amylase. Then remove the filter bag, gently squeeze out the extra water, and place it in a beaker. Add acetone and soak for 5min. Then dry it in a fume hood and then put it in a 102℃ oven. After 4h, remove it, cool it to room temperature in a desiccator, and weigh it again.

[0075] (8) Determination of acid detergent fibers

[0076] Take the sample of neutral detergent fiber tested in 4.1.5.7 and put it into the equipment. Add 2000mL of acid detergent fiber solution. After digestion, drain the liquid and add 2000mL of distilled water (80-90℃). Wash three times and remove the water. Place the filter bag in a beaker, add acetone and soak for 5min. Then dry it in a fume hood and put it in a 102℃ oven. After 4h, take it out and cool it to room temperature in the desiccator. Weigh it again.

[0077] (9) Determination of lactic acid

[0078] According to the instructions of the Nanjing Jiancheng reagent kit, weigh approximately 1.00 g of sample into a 2 mL centrifuge tube, add 1 mL of distilled water, mix well, and then transfer 20 μL of the supernatant into another 2 mL centrifuge tube. Add 20 μL of distilled water to each blank well, and then add 1 mL of enzyme working solution to each well. Next, add 200 μL of colorimetric solution to each reaction tube, mix thoroughly, and incubate at 37°C for 10 min. Finally, add 2 mL of stop solution to each tube, mix well, and then measure the absorbance at 530 nm using a microplate reader.

[0079] (10) Determination of short-chain fatty acids

[0080] Accurately weigh 1.00 g of fresh sample and record the weight of each sample. Vertically place the sample into a 10 mL centrifuge tube, add 5 mL of ultrapure water, vortex, incubate overnight at 4 °C, shake to mix for 15 min, centrifuge at 10000 rpm for 10 min, collect the supernatant, add 4 mL of ultrapure water to the precipitate, mix well, centrifuge again, and combine the supernatant with the precipitate in a 10 mL colorimetric tube to a final volume. Transfer the final volume to a 10 mL centrifuge tube, centrifuge at 10000 rpm for 15 min, then transfer the supernatant. Add the supernatant to a 1.5 mL centrifuge tube at a ratio of v:v = 9:1 (900 μL supernatant + 100 μL 25% metaphosphoric acid), mix well, and let stand at room temperature for 3–4 h. After centrifugation, filter through a 45 μm microporous membrane (nylon-based) into a sample vial (600 μL or more) for analysis.

[0081] (11) Determination of amino acids

[0082] The determination of amino acids in feed was performed according to the People's Republic of China National Standard GB / T 18246-2019. 0.10 g of the sample was weighed into a 20 mL ampoule, and 10 mL of hydrochloric acid hydrolysis solution was pipetted into the sample. The sample was placed in a constant temperature drying oven for 22 h. After cooling, the sample was mixed thoroughly, and an appropriate amount of the filtrate was dried in a rotary evaporator. This process was repeated twice. Then, 3 mL of lemon yogurt solution was added to reconstitute the solution, and the mixture was shaken well. After centrifugation, the supernatant was collected, filtered, and then tested.

[0083] (12) Determination of TiO2

[0084] Weigh 0.10 g of freeze-dried food paste and 0.15 g of feed sample into a porcelain crucible. Carbonize the sample over a low flame on an electric furnace until no smoke is produced. Then, ignite the sample in a muffle furnace at 580 °C for 14 h. After cooling, add 5 mL of sulfuric acid solution (7.40 mol / L) and heat over a low flame on an electric furnace until the sample is completely dissolved. After cooling, transfer approximately 15 mL of distilled water to a 50 mL volumetric flask containing 10 mL of 30% H₂O₂. Dilute the flask to 50 mL with distilled water. Filter the solution through qualitative filter paper before analysis.

[0085] (13) Calculation formula

[0086] Digestible energy and metabolizable energy were calculated using the formula of Adeola (2001), and net energy was calculated using the formula of Noblet (1994).

[0087] Apparent digestible energy of diet (MJ / kg) = (Geochemical intake – Fecal Geochemical intake) / Dietary intake

[0088] Apparent metabolizable energy of diet (MJ / kg) = (Geoenergy from food intake - Geoenergy from feces - Geoenergy from urine) / Food intake

[0089] FCSM apparent digestible energy or metabolizable energy (MJ / kg) = [Experimental diet apparent digestible energy or metabolizable energy - (100% - X%) × basal diet apparent digestible energy or metabolizable energy] / X%

[0090] FCSM apparent metabolizable energy (MJ / kg) = [Experimental diet apparent metabolizable energy - (100% - X%) × basal diet apparent metabolizable energy] / X%

[0091] Net energy (MJ / kg) = 0.7 × metabolizable energy + 1.61 × crude fat + 0.48 × starch - 0.91 × crude protein - 0.87 × acid detergent fiber

[0092] Corrected digestible energy of basal diet (MJ / kg) = Digestible energy of basal diet / Proportion of corn added to basal diet

[0093] Basal diet corrected metabolizable energy (MJ / kg) = basal diet metabolizable energy / basal diet corn addition ratio X% is the percentage of the tested raw material in the diet, which is 20%.

[0094] The apparent and standard terminal ileal digestibility were calculated using the formula proposed by Stein et al. (1998), where DM (Drymatter) represents dry matter content and DMI represents dry matter intake.

[0095] Amino Acid AID (%) = 100 - [TiO2 concentration of the feed to be tested (g / kg DM) × amino acid content of the digesta (g / kg DM) / TiO2 concentration of the digesta (g / kg DM) × amino acid content of the diet to be tested (g / kg DM)] × 100

[0096] Ileal endogenous amino acid flow (g / kg DMI) = Digestive amino acid content (g / kg DM) × Feed TiO2 (g / kg DM) / Digestive TiO2 concentration (g / kg DM)

[0097] Amino acid SID (%) = Amino acid AID (%) + [Ileal endogenous amino acid flow (g / kg DMI) / Amino acid content of the diet to be tested (g / kg DM)] × 100.

[0098] (14) Statistical Analysis

[0099] Data were organized using Excel 2021. One-way ANOVA was performed using SPSS 26.0 software to compare different compound enzyme addition ratios, with Duncan's method used for multiple comparisons between groups. The t-test was used to compare unfermented CSM and FCSM data. Data plotting was performed using GraphPad Prism 9.3.0 software. Each feed sample was tested in triplicate. Data are expressed as mean ± standard error. Different superscript letters and P < 0.05 indicate statistical significance, and P < 0.001 indicate highly statistical significance.

[0100] 3. Results

[0101] (1) The analysis of crude protein and acid-soluble protein in FCSM by enzymatic hydrolysis is shown in Table 1. The results showed that the proportion of different compound enzymes added significantly affected the crude protein content of FCSM. The crude protein content of the original CSM was 45.95%, and the crude protein content on day 3 after adding 0.1% compound enzyme was 46.08%. Compared with 0.1% compound enzyme, the addition of 0.15%, 0.2%, and 0.25% compound enzyme significantly increased the crude protein content of FCSM, to 47.52%, 47.63%, and 47.80%, respectively. In addition, fermentation time and addition amount significantly affected the acid-soluble protein content. With the extension of enzymatic hydrolysis time, the content of small peptides increased significantly, and tended to stabilize after 5 days of enzymatic hydrolysis. The acid-soluble protein content of the original CSM was 4.58%, and on day 3 of enzymatic hydrolysis, the acid-soluble protein content of FCSM with 0.1% compound enzyme was 7.71%. Compared with 0.1% compound enzyme, the addition of 0.15%, 0.2%, and 0.25% compound enzyme significantly increased the content of acid-soluble proteins in FCSM. On days 5 and 7 of enzymatic hydrolysis, the 0.25% compound enzyme group showed the highest relative content of small peptides in FCSM compared to other groups, at 15.83% and 16.74%, respectively. Therefore, the 0.25% compound enzyme ratio was selected for further experimental research.

[0102] Table 1. Effects of different compound enzyme addition ratios on crude protein and acid-soluble protein in FCSM.

[0103]

[0104] (2) The nutritional components were analyzed by selecting a compound enzyme addition ratio of 0.25%.

[0105] The chemical composition of FCSM synergistically fermented with bacteria and enzymes is shown in Table 2. Compared with unfermented CSM, FCSM significantly increased the crude protein content from 45.95% to 51.21%, the acid-soluble protein content from 4.58% to 23.78%, and significantly enriched lactic acid, acetic acid, propionic acid, and butyric acid. Meanwhile, crude fiber decreased from 15.90% to 14.30%, neutral detergent fiber from 34.55% to 29.92%, and acid detergent fiber from 21.02% to 20.38%. The changes in amino acid composition are shown in Table 3. Compared with the original CSM, synergistic fermentation significantly increased the essential amino acid content from 14.03% to 20.31%, the non-essential amino acid content from 15.86% to 23.68%, and the total amino acid content from 29.84% to 43.99%.

[0106] Table 2. Changes in nutritional composition of FCSM

[0107]

[0108]

[0109] Table 3. Changes in amino acid content in FCSM

[0110]

[0111] Example 2

[0112] FCSM energy test and terminal ileal amino acid digestibility test.

[0113] Laboratory Animals and Feeding Management

[0114] (1) Effective energy test

[0115] The effective energy experiment of growing pigs was conducted in the central metabolic laboratory of the training building at Hunan Agricultural University. Castrated three-way crossbred boars with an initial weight of 25.19 ± 1.72 kg were selected and placed in metabolic cages (1.4 × 0.7 × 0.6 m²). 3 After 5 days of acclimatization, pigs were randomly divided into two treatments, with eight replicates per treatment and one pig per replicate. The experimental diet consisted of a corn-based diet and FCSM (fermented microbial solution) replacing 20% ​​of the corn in the basal diet (see Table 4). The experiment lasted 12 days, including a 7-day pre-feeding period and a 5-day fecal and urine sample collection period. During the experiment, feed intake was measured at 4% of body weight, divided into two equal portions, and fed at 8:30 AM and 4:00 PM daily. Remaining feed and spilled feed were accurately collected daily, dried, weighed, and recorded to calculate feed intake.

[0116] (2) Amino acid digestibility test

[0117] The digestibility of amino acids in the terminal ileum was tested at the same location as the metabolic cages. Fourteen healthy castrated three-way crossbred boars with an initial weight of 30.25 ± 1.45 kg were selected and, after 5 days of acclimatization, randomly divided into two treatments, with seven replicates per treatment and one pig per replicate. The experimental pigs underwent T-tube surgery and care according to the method of Stein et al. (1998), with a postoperative recovery period of 10 days. The experimental period lasted 7 days, including a 5-day pre-feeding period and a 2-day digesta collection period. 0.30% titanium dioxide (TiO2) was added to the experimental diet as an indicator, and a nitrogen-free diet was used to correct for endogenous nitrogen loss. The FCSM diet was the sole source of protein and amino acids. The composition of the experimental diets is shown in Table 5. During the experiment, the feed intake was set at 4% of the pig's body weight, and feeding was conducted twice daily at 08:00 AM and 16:00 PM. The temperature in the experimental barn was maintained at 22–26℃ to ensure the comfort of the pigs. After feeding each afternoon, the pigsty is cleaned to keep the ground hygienic and tidy.

[0118] The digestible and metabolizable energy results of FCSM are shown in Table 4. On a dry matter basis, the digestible and metabolizable energy were 12.40 MJ / kg and 11.48 MJ / kg, respectively, with a net energy of 8.33 MJ / kg. The net energy value was used to calculate the net energy of the feed formulation. Compared to unfermented CSM, FCSM has higher digestible and metabolizable energy under similar nutritional value.

[0119] Table 4. Energy values ​​of FCSM in growing pigs

[0120]

[0121] Table 5. Composition of the experimental diet

[0122] Table 5. Dietary composition and nutrient levels (% of feeding basal level)

[0123]

[0124]

[0125] Note: 1 The premix provides per kilogram of feed: Vitamin A, 6000 IU; Vitamin D 3, 2000 IU; Vitamin E, 12 IU; Vitamin K3, 2 mg; Vitamin B1, 2.5 mg; Vitamin B2, 3 mg; Vitamin B6, 2 mg; Vitamin B... 120.035mg; Vitamin B5, 10mg; Pantothenic acid, 5mg; Folic acid, 1mg; Biotin, 0.15mg; Nicotinamide, 15mg; 0.15mg Selenium (Na2SeO3); 0.14mg Iodine (KI); 30mg Zinc (ZnSO4); 20mg Copper (CuSO4.5H2O); 30mg Iron (FeSO4.H2O); 1mg Manganese (MnSO4).

[0126] As shown in Table 6, the AID of crude protein in FCSM was 78.16%, and the SID was 89.06%. The mean AID of essential amino acids ranged from 60.42% to 84.53%, with lysine, methionine, threonine, and tryptophan values ​​of 64.43%, 72.67%, 60.42%, and 67.96%, respectively. The SID ranged from 71.83% to 89.61%, with lysine, methionine, threonine, and tryptophan values ​​of 73.74%, 81.84%, 71.83%, and 77.12%, respectively. The mean AID of non-essential amino acids ranged from 50.65% to 80.86%, and the mean SID ranged from 57.16% to 90.19%. A review of the "Nutritional Requirements of Chinese Swine" (2020) and the "Table of Feed Composition and Nutritional Value in China (33rd Edition, 2022)" revealed that FCSM not only improved the nutritional quality of feed but also increased the SID values ​​of various amino acids and crude protein, with a relative increase in crude protein digestibility of 15.67%.

[0127] Table 6. Amino acid terminal ileal digestibility (%) in FCSM of growing pigs

[0128]

[0129]

[0130] Example 3

[0131] This embodiment tests the effects of adding different proportions of FCSM to replace soybean meal on the production performance of growing-finishing pigs.

[0132] 1. Experimental methods for growing-finishing pigs

[0133] Sixty healthy three-way crossbred (Duroc × Landrace × Large White) growing-finishing pigs with an initial body weight of 67.75 ± 0.40 kg were selected for the experiment and randomly divided into 6 treatments, with 10 replicates per treatment (1 pig per replicate), for a period of 60 days. The control group was fed a corn-soybean meal basal diet, while the experimental groups were fed a differentially substituted diet of 2.4%, 4.8%, 7.2%, and 9.6% FCSM and 9.6% unfermented CSM, respectively, to replace soybean meal. The experimental diets (isoenergetic and isonitrogenous) and their composition are detailed in Table 5-1. The nutrient composition and amino acid levels of the diets in both the control and experimental groups met the recommended standards of the Chinese Nutrition Requirements for Swine (2020). In the last week of the experiment, 0.3% TiO2 was added to the diets as an indicator to determine nutrient digestibility. The experiment was conducted at the Kelikang Pig Farm in Puji Town, Liuyang City. All experimental pigs were housed individually in metabolic cages with free access to feed and water. The temperature in the pigsty was controlled at 25-31℃, the humidity at 50-70%, and ventilation was maintained. The pigsty was also regularly disinfected, and the feed intake and health status of the experimental pigs were recorded daily.

[0134] During the final week of the experiment, pigs were fed a diet containing 0.3% TiO2. Three days before the end of the experiment, fresh fecal samples (excluding feed impurities) were collected from all experimental pigs and placed in resealable bags. After nitrogen fixation with dilute sulfuric acid, the samples were temporarily stored in dry ice. On the last day of the experiment, seven pigs from each group, close to their average body weight and fasted for 12 hours, were slaughtered and blood was collected. Blood samples were collected in blood collection tubes, allowed to stand for 1 hour, centrifuged at 3000 rpm / min for 8 minutes, and the supernatant was aliquoted into 1.5 mL centrifuge tubes and rapidly frozen in liquid nitrogen for subsequent analysis. From the isolated digestive organs, gastric and jejunal chyme were rapidly collected and preserved in liquid nitrogen. Jejunum and ileum were dissected, and ileum, cecum, and colonic chyme were collected and rapidly frozen in liquid nitrogen for subsequent analysis.

[0135] The method for determining TiO2 is the same as step (12) in Example 1.

[0136] 2. Growth performance testing methods

[0137] At the beginning and end of the experiment, the feed and experimental pigs were weighed, and the initial and final body weights were recorded. Additionally, uneaten feed and spilled feed were recorded before each feeding, and the average daily weight gain, average daily feed intake, and feed conversion ratio were calculated based on the feed usage.

[0138] Total feed intake during the trial period (kg / head) = Average daily feed intake (kg / d) × Number of trial days (d)

[0139] Total weight gain during the trial period (kg) = Final body weight (kg) - Initial body weight (kg)

[0140] 3. Apparent digestibility of nutrients

[0141] After the experiment, the product was dried in an oven at 65℃ for 72 hours, then pulverized and bagged for subsequent testing of apparent digestibility and other indicators. The methods for detecting and calculating dry matter, total energy, crude protein, crude fat, neutral detergent fiber, and acid detergent fiber were consistent with the methods for detecting the chemical composition of FCSM in Example 1 above.

[0142] 3. Results

[0143] (1) The effects of FCSM on the growth performance of growing pigs are shown in Table 7. The final body weight and average daily weight gain of growing-finishing pigs in the FCSM-7.2% treatment group were significantly higher than those in the control group, the FCSM-2.4% group, and the FCSM-4.8% group (P<0.05). The feed conversion ratio (FCR) of growing-finishing pigs showed a quadratic trend with the amount of FCSM added, and the FCR of the FCSM-7.2% treatment group was significantly lower than that of the control group (FCSM-9.6% treatment group) (P<0.05). Furthermore, the FCR of growing-finishing pigs showed a quadratic linear trend with the amount of FCSM added (P<0.05). All production performance indicators of the group with the same proportion of FCSM were higher than those of the unfermented CSM group, but the differences were not significant (P>0.05).

[0144] (2) Effects of adding different proportions of FCSM to replace soybean meal on the apparent nutrient digestibility of growing and finishing pigs.

[0145] The effects of FCSM on the nutrient digestibility of growing pigs are shown in Table 8. Compared with the control group, dry matter digestibility and total energy digestibility showed a significant quadratic linear relationship with increasing FCSM addition level (P<0.05). The dry matter digestibility of the FCSM-7.2% group was significantly higher than that of other treatment groups, and the total energy digestibility was significantly higher than that of the control group. Protein digestibility showed a significant linear relationship with the amount of FCSM added (P<0.05). The FCSM-4.8% and 7.2% groups significantly improved the digestibility of neutral detergent fiber and acid detergent fiber, showing a highly significant linear and quadratic functional relationship with the amount of FCSM added (P<0.05). The digestibility of neutral and acid detergent fiber in the FCSM-9.6% group was significantly higher than that in the FCSM-9.6% group (P<0.05).

[0146] Table 7. Effects of different FCSM ratios on soybean meal replacement on growth performance of growing-finishing pigs.

[0147]

[0148]

[0149] Note: a and b represent statistically significant differences among the five groups (Control, FCSM-2.4%, 4.8%, 7.2%, and 9.6%), P < 0.05.

[0150] Table 8. Effects of different FCSM ratios on apparent nutrient digestibility in growing-finishing pigs.

[0151]

[0152]

[0153] Note: a and b represent significant differences among the five groups (Control, FCSM-2.4%, 4.8%, 7.2%, and 9.6%), P < 0.05.

[0154] Example 4

[0155] Effects of adding FCSM-7.2% to replace soybean meal on serum biochemical and hormone levels in growing-finishing pigs.

[0156] Serum parameters

[0157] Serum biochemical indicators, including total protein (g / L), blood urea nitrogen (μg / mL), and uric acid (mmol / L), were measured using a fully automated biochemical analyzer (KHB450, Shanghai Kehua Bioengineering Co., Ltd.). Blood ammonia (μmol / L) was measured according to the blood ammonia reagent kit method from Beijing Box Biotechnology Co., Ltd.

[0158] Serum-related hormone markers: Adiponectin and leptin were detected according to the reagent kits and methods of Wuhan Huamei Biotechnology Co., Ltd. Growth hormone (ng / mL) and insulin-like growth factor (ng / mL) were detected according to the ELISA kit instructions of Shanghai ZCIBIO Technology Co., Ltd.

[0159] Based on comprehensive growth performance indicators, the FCSM-7.2% experimental group showed the best results. Therefore, further analysis was conducted on the FCSM-7.2% experimental group and the control group. The effects of the FCSM-7.2% experimental group on serum biochemical and hormonal indicators of growing pigs are shown in Table 9. The results showed that, compared with the control group, the FCSM-7.2% group significantly reduced serum uric acid and blood ammonia levels in growing-finishing pigs (P<0.05) and increased growth hormone and insulin-like growth factor levels (P<0.05).

[0160] Table 9. Effects of FCSM-7.2% soybean meal substitution on serum biochemical and hormone levels in growing-finishing pigs.

[0161]

[0162] Example 5

[0163] Determination of digestive enzyme activity in gastric and jejunal chyme.

[0164] Before testing, the samples were thawed at 4°C. Next, 0.20 g of the chyme was accurately weighed and transferred to a 2 mL centrifuge tube. Then, physiological saline was added at a ratio of 1:9 (g:mL), and the mixture was thoroughly homogenized for 30 seconds. The homogenate was then centrifuged at 4°C (4000 rpm). The supernatant was used to detect the activities of pepsin, lipase, α-amylase, and trypsin according to the assay methods of the kits developed by Beijing Boxbio and Nanjing Jiancheng.

[0165] The effects of adding 7.2% FCSM on pepsin activity and jejunal digestive enzyme activity in growing pigs are shown in Table 10. The results showed that the addition of 7.2% fermented cottonseed meal tended to increase pepsin activity in growing-finishing pigs (P = 0.059) and significantly increased the activities of lipase and trypsin in the jejunum (P < 0.05).

[0166] Table 10 Effects of FCSM-7.2% soybean meal substitution on pepsin activity and jejunal contents digestive enzyme activity in growing-finishing pigs.

[0167]

[0168] In summary, this invention utilizes a newly isolated strain of *Lactobacillus pentosus* for cottonseed fermentation. By co-fermenting cottonseed cake or cottonseed meal with *Lactobacillus pentosus* and a compound enzyme, the nutritional content of fermented cottonseed meal can be improved, increasing various nutritional indicators such as crude protein, acid-soluble protein, lactic acid, acetic acid, propionic acid, butyric acid, and amino acids, while effectively reducing the content of crude fiber, neutral detergent fiber, and acid detergent fiber. Based on this, feeding the prepared fermented cottonseed meal to finishing pigs can significantly improve their growth performance, increase nutrient digestibility, and improve their body weight and average daily weight gain; reduce their feed conversion ratio; lower serum uric acid and ammonia levels in growing-finishing pigs; increase growth hormone and insulin-like growth factor levels; increase pepsin activity in growing-finishing pigs; and increase lipase and trypsin activity in the jejunum. Therefore, this invention has promising applications in improving the production performance of poultry, aquatic animals, and / or livestock.

[0169] Furthermore, the present invention provides a theoretical basis and reference for reducing soybean meal usage in livestock production using cottonseed meal fermented feed. The cottonseed meal fermented feed provided by the present invention can be used as a substitute for soybean meal.

[0170] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fermented cottonseed feed, characterized in that, It includes: fermentation products of cottonseed cake or cottonseed meal after co-fermentation of cottonseed cake or cottonseed meal by Lactiplantibacillus pentosus and a complex enzyme; the Lactiplantibacillus pentosus is deposited at Wuhan University with accession number CCTCC NO:M 20231547.

2. The cottonseed fermented feed according to claim 1, characterized in that, The amount of the compound enzyme added accounts for 0.15-0.25% of the total mass of cottonseed cake or cottonseed meal; Preferably, each g of the complex enzyme comprises the following enzyme-active raw materials: 30,000 U xylanase, 10,000 U acidic protease, 10,000 U neutral protease, 20,000 U alkaline protease, 5,000 U β-glucanase, 3,000 U pectinase, 2,000 U cellulase, and 100 U α-galactosidase.

3. The cottonseed fermented feed according to claim 1, characterized in that, The combined fermentation is anaerobic, facultative anaerobic, or aerobic fermentation. The fermentation conditions are as follows: the water content of the fermentation broth is controlled at 45-55%, the inoculum amount of Lactobacillus pentosus is 8-12%, the fermentation time is 11-13 days, and the fermentation temperature is 37℃±0.5℃.

4. A method for preparing fermented cottonseed feed, characterized in that, It includes the following steps: mixing and fermenting the Pentosacchari lactobacillus and the compound enzyme as described in claim 1 with cottonseed cake or cottonseed meal.

5. The method for preparing fermented cottonseed feed according to claim 4, characterized in that, The amount of the compound enzyme added accounts for 0.15-0.25% of the total mass of cottonseed cake or cottonseed meal; Preferably, the fermentation is anaerobic, facultative anaerobic, or aerobic fermentation, and the fermentation conditions are as follows: the water content of the fermentation broth is controlled at 45-55%, the inoculum amount of Lactobacillus pentosus is 8-12%, the fermentation time is 11-13 days, and the fermentation temperature is 37℃±0.5℃.

6. A method for fattening pigs, characterized in that, The cottonseed fermented feed prepared by the method of any one of claims 1-3 or claims 4-5 is fed to pigs.

7. The method for fattening pigs according to claim 6, characterized in that, The amount of fermented cottonseed feed added accounts for 2.4-9.6% of the daily ration by weight.

8. The method for fattening pigs according to claim 7, characterized in that, The amount of fermented cottonseed feed added should account for 7.2-9.6% of the daily ration by weight. Preferably, the amount of fermented cottonseed feed added accounts for 7.2% of the daily ration by weight.

9. The application of cottonseed fermented feed prepared by the method of preparing cottonseed fermented feed according to any one of claims 1-3 or any one of claims 4-5 in improving the production performance of poultry, aquatic and / or livestock animals.

10. The application according to claim 9, characterized in that, The poultry are selected from: chickens, ducks, geese, or pigeons; The livestock are selected from: pigs, cattle, sheep, horses, donkeys or deer; the aquatic products are selected from: fish or shrimp; Preferably, the application includes at least one of the following application methods: (1) Increase the weight and average daily weight gain of fattening pigs; (2) Reduce the feed conversion ratio for fattening pigs; (3) Dry matter digestibility is significantly increased; (4) Increased digestibility of neutral detergent fibers and acid detergent fibers; (5) Reduce serum uric acid and blood ammonia levels in growing-finishing pigs; (6) Increase the levels of growth hormone and insulin-like growth factor in growing-finishing pigs; (7) Improve the activity of pepsin in growing-finishing pigs; (8) Increase the activity of lipase and trypsin in the jejunum; (9) The cottonseed fermented feed is used as a substitute for soybean meal.

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