Asparagus straw fermented feed, its production method and application

Abstract

The application discloses asparagus straw fermented feed and a production method and application thereof, and belongs to the field of animal feed. The application uses bacillus subtilis, paracasei lactobacillus and candida utilis as fermentation agents, and prepares asparagus straw fermented feed with increased protein content and reduced crude fiber content through solid-state fermentation of asparagus straw by means of synergistic action of bacteria and enzymes. Experimental results show that the asparagus straw fermented feed provided by the application added to a basic daily ration can significantly improve the growth performance, slaughtering performance, apparent digestibility of nutrients and meat quality of Wan Yue black pigs, increase the content of nutritional components and umami amino acids in the muscle, and significantly expand the breeding benefits of the Wan Yue black pigs. Feeding the asparagus straw feed can also significantly improve the richness of cecal microorganisms, and through improvement of intestinal microflora, the method is helpful to maintaining the body health of the Wan Yue black pigs.

Description

Technical Field

[0001] This invention relates to the field of animal feed, and in particular to a fermented asparagus straw feed, its production method, and its application. Background Technology

[0002] The sustained and rapid development of the economy and society has improved the quality of life for the people, boosting consumer demand for livestock and poultry products such as meat, eggs, and milk. This has led to the continuous expansion of the livestock and poultry farming industry, and consequently, increased market demand for feed. However, influenced by a combination of factors, the prices of corn and soybean meal, as core feed ingredients, have continued to rise, not only limiting the healthy development of the feed industry but also significantly increasing livestock farming costs. The rational development and efficient utilization of unconventional feed resources can be a key measure to ensure the sustainable, stable, and rapid development of both the feed and livestock industries. This measure can alleviate the resource conflict between humans and livestock over food, reduce input costs in livestock production, and play a significant role in optimizing the microenvironment of livestock and poultry pens.

[0003] The edible portion of asparagus consists only of the tender stems, accounting for only 23.5% of the total yield. The remaining 76.5% is non-edible stalks. Asparagus growers generate a huge amount of waste stalks during harvesting, and indiscriminate disposal leads to resource waste and environmental pollution. However, asparagus stalks have a high water content, making them difficult to transport and preserve, hindering their resource utilization. Currently, they are mostly disposed of through natural decomposition or on-site burning. This method not only causes serious environmental pollution but also exacerbates asparagus stem blight.

[0004] Fermenting feed using microbial solid-state fermentation can extend its shelf life, improve its palatability and nutritional value, and offers advantages over methods such as drying, including easier process control, simpler operation, lower energy consumption, and lower cost. Currently, research on asparagus straw fermented feed is scarce and lacks a comprehensive framework, particularly regarding technical solutions for asparagus straw fermented pig feed. Therefore, systematic and standardized research is urgently needed to provide a suitable microbial fermented feed for pig farming. Summary of the Invention

[0005] The purpose of this invention is to provide a fermented asparagus straw feed, its production method, and its application, in order to solve the problems existing in the prior art. This invention prepares fermented asparagus straw feed with increased protein content and reduced crude fiber content through solid-state fermentation of asparagus straw by bacteria and enzymes, which helps to expand the breeding benefits of Wanyue black pigs and maintain the health of black pigs.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for producing fermented asparagus straw feed, comprising the following steps: Bacillus subtilis, Lactobacillus paracasei, and Candida utilis were mixed to prepare a fermentation agent; The asparagus stalks are crushed, mixed with wheat bran, and cellulase is added. The mixture is then inoculated with the fermentation agent and fermented to obtain the fermented asparagus stalk feed.

[0007] Furthermore, the ratio of the viable counts of Bacillus subtilis, Lactobacillus paracasei, and Candida utilis is 1:2:2.

[0008] Furthermore, the inoculum size of the fermentation agent is 10. 7 CFU / g.

[0009] Furthermore, the fermentation temperature is 33°C and the time is 4 days.

[0010] Furthermore, the mass ratio of the asparagus stalks to the wheat bran is 3:1.

[0011] Furthermore, the amount of cellulase added is 2 mg / kg.

[0012] The present invention also provides a fermented asparagus straw feed obtained according to the above-described production method.

[0013] The present invention also provides an application of the above-mentioned fermented asparagus straw feed in the preparation of pig feed.

[0014] The present invention also provides a pig feed, which consists of the above-mentioned fermented asparagus straw feed and a basal diet.

[0015] Furthermore, the mass fraction of the fermented asparagus straw feed is 4%.

[0016] The present invention discloses the following technical effects: This invention uses Bacillus subtilis, Lactobacillus paracasei, and Candida utilis as fermentation agents to prepare fermented asparagus straw through synergistic solid-state fermentation of asparagus straw, resulting in a feed with increased protein content and reduced crude fiber content. Experimental results show that adding the fermented asparagus straw feed provided by this invention to the basal diet significantly improves the growth performance, slaughter performance, apparent nutrient digestibility, and meat quality of Wanyue Black pigs, increases the content of nutrients and umami amino acids in the muscle, and significantly expands the breeding benefits of Wanyue Black pigs. Feeding this asparagus straw feed also significantly increases the richness of cecal microbiota, which helps maintain the health of Wanyue Black pigs by improving the intestinal microbiota. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A graph showing the cumulative curve results of the microbial species effects of fermented asparagus on the cecum of black pigs. Figure 2 A Venn diagram showing the effects of fermented asparagus on the cecal microbiota of black pigs. Figure 3 To investigate the effects of fermented asparagus on the cecal microbiota of black pigs Alpha Analysis results chart; Figure 4 To investigate the effects of fermented asparagus on the cecal microbiota of black pigs PCoA Analysis results chart; Figure 5 To investigate the effects of fermented asparagus on the cecal microbiota of black pigs LEfSe Analysis results chart. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] This invention explores the use of a mixture of three bacteria—Lactobacillus paracasei, Bacillus subtilis, and Candida utilis—to ferment asparagus straw using a synergistic bacterial-enzyme approach. The fermentation parameters of asparagus straw were optimized, and the nutritional value and microbial community changes of the fermented asparagus straw feed before and after fermentation were analyzed. The study discovered the optimal fermentation process parameters for the synergistic solid-state fermentation of asparagus straw, transforming this agricultural processing waste into high-quality biological feed through fermentation, thus achieving efficient resource utilization of waste.

[0025] Example 1 1. Materials and Methods 1.1 Test Materials The asparagus stalks used in this invention are produced and supplied by Anhui Xinzu Asparagus Co., Ltd. Using the asparagus stalks as a substrate, a plant fermentation material is produced by adding microorganisms stored in the Animal Nutrition and Feed Laboratory of Anhui Agricultural University: Lactobacillus paracasei (purchased from Guangzhou Haoxiang Fine Chemical), Bacillus subtilis (purchased from Guangzhou Haoxiang Fine Chemical), Candida utilis (purchased from the online store Junchang Biological Laboratory), and cellulase (purchased from Shandong Longket Enzyme Preparation Co., Ltd.).

[0026] 1.2 Substrate Treatment Asparagus stalks were pulverized using a pulverizer and passed through a 40-mesh sieve. Based on previous experimental results, asparagus stalks were mixed with wheat bran at a mass ratio of 3:1, and 2 mg / kg cellulase was added. Bacillus subtilis, Lactobacillus paracasei, and Candida utilis were diluted to a concentration of 1×10⁻⁶. 7 CFU / g is available for subsequent testing.

[0027] 1.3 Determination of nutrient composition of fermented asparagus straw feed The laboratory determined the content of its routine nutritional indicators. The samples to be tested were placed in a 65℃ oven and dried for 24 hours. After drying, they were removed, rehydrated, and placed in resealable bags for later use. The determination of crude protein, crude fat, acid detergent fiber, and neutral detergent fiber in the dried samples followed Zhang Liying's method.

[0028] 1.4 Single-factor fermentation experiment of mixed strains Take the prepared substrate and set up five ratios, with the following mixing ratios (based on viable cell count): Bacillus subtilis: Lactobacillus paracasei: Candida utilis 1:1:1, 1:2:1, 1:1:2, 2:1:2, and 1:2:2. The fermentation moisture content is 55%, and the inoculum size is 1×10⁻⁶. 7 The fermentation temperature was 33℃, and the fermentation time was 6 days. After fermentation, the pH value, crude fiber content, and crude protein content of the feed were measured. Each experiment was repeated three times.

[0029] 1.5 Fermentation Moisture Single-Factor Fermentation Experiment Take the prepared substrate and set five moisture gradients with moisture content of 45%, 50%, 55%, 60%, and 65%. Other fermentation conditions are as follows: inoculum size is 1×10⁻⁶. 7 The fermentation temperature was 33℃, and the fermentation time was 6 days. After fermentation, the pH value, crude fiber content, and crude protein content of the feed were measured. Each experiment was repeated three times.

[0030] 1.6 Fermentation Time Single-Factor Fermentation Experiment Take the prepared substrate and set five time gradients for 4 days, 5 days, 6 days, 7 days, and 8 days. Other fermentation conditions are as follows: inoculum size 1×10⁻⁶. 7 The fermentation conditions were: CFU / g, fermentation moisture content 55%, fermentation temperature 33℃. After fermentation, the pH value, crude fiber content, and crude protein content of the feed were measured. Each group of experiments was set up in triplicate.

[0031] 1.7 Fermentation Temperature Single-Factor Fermentation Experiment Take the prepared substrate and set five temperature gradients: 29℃, 31℃, 33℃, 35℃, and 37℃. Other fermentation conditions are: inoculum size 1×10⁻⁶. 7 CFU / g, fermentation moisture 55%, fermentation time 6 days, after fermentation, the pH value, crude fiber content, crude protein content and acid-soluble protein content of the feed were measured, and three replicates were set up for each group of experiments.

[0032] 1.8 Single-factor fermentation experiment on inoculum size Take the prepared substrate, set up five different gradients, and inoculate at a rate of 10. 6 CFU / g, 5×10 6 CFU / g, 10 7 CFU / g, 5×10 7 CFU / g, 10 8 CFU / g, other inoculation conditions were: fermentation time 6 days, fermentation moisture 55%, fermentation temperature 33℃, after fermentation, the pH value, crude fiber content and crude protein content of the feed were measured, and three replicates were set up for each group of experiments.

[0033] 1.9 Orthogonal Experiment Based on the optimization results of the single-factor experiments, fermentation temperature, substrate moisture, inoculum amount and fermentation time were selected as optimization factors. The substrate was a 3:1 mixture of asparagus and wheat bran. The ratio of mixed microbial agents was the result of the single-factor fermentation experiment of mixed microbial agents. A four-factor three-level orthogonal experiment was used for optimization design to obtain the best fermentation combination.

[0034] 2. Results and Analysis 2.1 Results of single-factor fermentation experiment with mixed strains As shown in Table 1, when the ratio of Bacillus subtilis, Lactobacillus paracasei, and Candida utilis was 1:2:2, the protein content in fermented asparagus straw increased significantly. P <0.05), crude fiber content decreased ( P <0.05), pH value is 3.77.

[0035] Table 1 Single-factor experiment on the proportion of mixed bacterial strains 2.2 Results of Single-Factor Fermentation Experiment on Fermentation Moisture Content As shown in Table 2, when the fermentation moisture content was 55%, the protein content in the fermented asparagus straw increased significantly ( P <0.05), crude fiber content decreased ( P <0.05), pH value is 3.66.

[0036] Table 2 Single-factor experiment on fermentation moisture content 2.3 Results of Single-Factor Experiment on Fermentation Time Table 3 shows that when the fermentation time was 4 days, the protein content in the fermented asparagus straw increased significantly ( P <0.05), crude fiber content decreased ( P <0.05), pH value is 3.72.

[0037] Table 3 Single-factor experiment on fermentation time 2.4 Results of Single-Factor Fermentation Experiment Based on Fermentation Temperature Table 4 shows that when the fermentation time is 33℃, the protein content in the fermented asparagus straw increases significantly ( P <0.05), crude fiber content decreased ( P <0.05), pH value is 3.74.

[0038] Table 4 Single-factor experiment on fermentation temperature 2.5 Results of Single-Factor Fermentation Experiment with Inoculum Size As shown in Table 5, the inoculum size is 10. 7 At CFU / g, the protein content in fermented asparagus straw increased significantly ( P <0.05), crude fiber content decreased ( P <0.05), pH value is 3.78.

[0039] Table 5 Single-factor experiment on inoculum size 2.6 Results of Orthogonal Experiments Orthogonal experimental design: To explore the optimal parameters for asparagus straw fermentation, establish a fermentation process suitable for the characteristics of asparagus by-products, and achieve a dual improvement in nutrition and function, this invention conducts orthogonal experiments based on fermentation parameters obtained from single-factor experiments.

[0040] Table 6 Orthogonal Experimental Design The optimal parameters for asparagus straw fermentation can be obtained from the orthogonal experiment results: Asparagus stalks were crushed and mixed with wheat bran at a mass ratio of 3:1, and 0.02% cellulase was added. Fermentation was carried out through a synergistic fermentation process using bacteria and enzymes. The specific conditions were: a ratio of Bacillus subtilis: Lactobacillus paracasei: Candida utilis of 1:2:2; a fermentation temperature of 33℃; a moisture content of 55%; a fermentation time of 4 days; and an inoculum size of 10. 7 Protein content increased significantly at CFU / g ( P <0.05), crude fiber content decreased ( P <0.05), pH stable.

[0041] 2.7 Determination of Nutritional Components in Fermented Asparagus Straw Feed As shown in Table 7, fermentation of asparagus straw successfully increased the protein content by 16.45% ( P <0.05), crude fiber content decreased significantly by 50.21% ( P <0.05), pH maintained, and the fermented feed has good shape and flavor, proving its feasibility as a conventional feed.

[0042] Table 7 Comparison of nutritional components between fermented asparagus straw and fresh asparagus feed Example 2 1. Materials and Methods 1.1 Experimental Design Eighteen healthy black pigs aged 120 days and of similar weight were randomly divided into two groups: the CON group and the AF group, with three replicates in each group and three pigs in each replicate. The CON group was fed a basal diet (specific formula and nutrient composition are shown in Table 8), while the AF group had 4% (by mass fraction) of the basal diet replaced by fermented asparagus straw feed. All black pigs were housed in the same pigpen and had free access to feed and water during the experiment. They received routine immunizations, and the experiment lasted for 91 days.

[0043] Table 8. Nutritional levels of basal diets (air-dried basal diet) 1.2 Feeding and Management This invention was conducted at Fengchao Ecological Breeding Co., Ltd. in Huaining County, Anqing City, Anhui Province. Black pigs were kept in enclosed pigsties, with three pigs per pen. Before the experiment began, the pigsties and feed troughs were cleaned, disinfected, and sterilized. During the experiment, the black pigs in each pen had free access to feed and water. Throughout the experiment, consistent feeding and management conditions were maintained for the pig herd, and the health and feed intake of the pigs were checked daily. Immunization and disinfection procedures were strictly implemented according to the farm's disease prevention measures.

[0044] 1.3 Sample Collection and Processing On the morning of the first day of the experiment, each pig was weighed on an empty stomach, and its initial weight was recorded. After the start of the experiment, the amount of feed given to each pen and the amount of leftover feed were weighed and recorded. The pigs in each pen were weighed on an empty stomach every 28 days. After slaughter, 500 g of the longissimus dorsi muscle was taken from each pig and divided into three portions. The first muscle sample was used for on-site meat quality (45 min) index determination. The second muscle sample was stored at -20℃ for meat quality (24 h) index determination. The third muscle sample was wrapped in aluminum foil and stored in a liquid nitrogen tank for muscle nutrient analysis. During the slaughter experiment, the cecum was separated, and the cecal chyme was removed, aliquoted into 5 mL centrifuge tubes, and stored in a liquid nitrogen tank.

[0045] 1.4 Growth Performance Measurement Weigh each pig on an empty stomach and record its weekly feed intake. Calculate average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (F / G).

[0046] 1.5 Digestibility determination Hydrochloric acid insoluble ash (AIA) was used as an endogenous indicator to evaluate nutrient digestibility in pigs. The specific procedure was as follows: Three days before the end of the experiment, fecal samples were collected using the total fecal collection method after feeding, with approximately 50 g samples collected each time. These samples were then treated with 10% dilute hydrochloric acid and toluene for nitrogen fixation and preservation, and frozen at -20℃ for later testing. The formula for calculating nutrient digestibility is: Nutrient digestibility (%) = [1 - (Nutrients in feed AIA / AIA in feces × Nutrients in feces) / Nutrients in feed] × 100.

[0047] 1.6 Carcass performance testing At the end of the experiment, all experimental pigs were slaughtered, and their heads, hooves, tails, and most of their internal organs were removed, while the back fat and kidneys were retained. The back fat was weighed, and the carcass weight was measured to calculate the back fat ratio and dressing percentage. The backfat thickness at the thickest point of the shoulder, the last rib, and the loin-sacral junction on the left side of the carcass was measured using calipers, and the average of these three values ​​was taken as the backfat thickness. The outline of the cross-section at the junction of the last thoracic vertebra and the first lumbar vertebra of the longissimus dorsi muscle on the left side of the carcass was drawn using graph paper, and the area was measured as the eye muscle area. The lean meat percentage was determined according to the method described by Walstra (1996).

[0048] Y = 61.21920 - 0.77665×X1+ 0.15239×X2; Where Y = estimated lean meat percentage; X1 = pork thickness (mm) including skin, measured at a point 7 cm from the midline between the last third and fourth ribs; X2 = tenderloin thickness (mm), measured at a point 7 cm from the midline between the last third and fourth ribs. To minimize measurement error, the same person was consistently used for all measurements.

[0049] 1.7 Meat quality determination 1.7.1 pH value Take a piece of meat sample from the longissimus dorsi muscle at the thorax-lobe junction. The width and thickness of the sample should both be greater than 3.0 cm. The pH value measured 45 minutes after slaughter is recorded as pH1, and the pH value measured 24 hours after slaughter is recorded as pH2. 24 Follow the pH meter instructions (the pH meter needs to be set according to the meat temperature). Insert the electrode directly into the puncture hole in the middle of the dissected longissimus dorsi muscle, to a depth of at least 1 cm, ensuring the electrode tip is completely embedded in the meat sample. Wait for the reading to stabilize for at least 5 seconds before reading the pH (accurate to 0.01). Store the meat sample in a refrigerator at 0-4℃ for 24 hours to obtain the pH value. 24 .

[0050] 1.7.2 Flesh Color Determination Measurements were taken within 45-60 minutes post-slaughter. Fresh cross-sections of the longissimus dorsi muscle (i.e., the oculomotor muscle) at the thoracolumbar junction were analyzed using a colorimeter (660 lx). The L value (brightness), a value (redness), and b value (yellowness) were recorded. Three measurements were taken for each value, and the arithmetic mean was calculated, i.e., L*. 45min a* 45min and b* 45min The same procedure was performed 24 hours after slaughter, and L* was measured. 24h a*24h and b* 24h .

[0051] Note: L value is the brightness of the muscle, with 0 for black and 100 for white. L>53 indicates PSE meat; a value measures the change of muscle from red (ideal color) to green, and the higher the a value, the better the meat quality; b value measures the change of muscle from yellow (ideal color) to blue, and the lower the b value, the better the meat quality.

[0052] 1.7.3 Measurement of dripping water loss Within 45-60 minutes of the pig's respiratory arrest, the eye muscle from the 3rd-4th intercostal space from the bottom was harvested. The meat sample was cut into 2cm thick slices along the fiber direction, and then trimmed into strips 5cm long and 3cm wide. The peripheral muscle membrane was removed. The weight of each strip before hanging (W1) was measured using a balance. One end of each strip was then hung with a hook and placed in a numbered sample bag, with half of the hook protruding from the bag. Nitrogen gas was introduced to fill the bag, suspending the strip in the center to prevent contact between the meat sample and the bag. The bag opening was tied tightly to the hook with cotton thread and hung on a rack. The rack was removed, and the bag was stored at 2-4℃ for 24 hours. The sample bag was then opened, the strips removed, and the surface moisture absorbed with filter paper. Finally, the weight of each strip after hanging (W2) was measured.

[0053] The calculation formula is: Drip loss rate = (Weight before hanging W1 - Weight after hanging W2) / Weight before hanging W1 × 100%.

[0054] 1.7.4 Shear Force Measurement Take the mid-segment of the longissimus dorsi muscle from the first to fourth lumbar vertebrae, with a length, width, and height of at least 6×3×5 cm. Remove the surface fat, place the sample in a plastic bag, seal it tightly, and remove as much air as possible. Store at 4℃ for 72 hours. Remove the cooked sample and let it sit at room temperature for 1 hour. Then, open the plastic bag, insert a thermometer into the center of the muscle, reseal the sample, keep the bag opening upwards, and place it in an 80℃ constant temperature water bath. Cover and continue heating until the center temperature of the muscle reaches 70℃. Remove the sample and cool the center temperature to 0-4℃. Using a 1.27 cm diameter circular sampler, drill and cut the sample parallel to the muscle fibers, with a hole length of at least 2.5 cm. The sampling position should be at least 5 mm from the edge of the sample, and the distance between the edges of two samples should be at least 5 mm. Discard any samples with obvious defects. Measure at least three samples immediately after sampling. Determine the shear force using a tenderness tester. Perform nine replicates for each sample and take the average value.

[0055] 1.8 Measurement of intramuscular fat content Weigh a fresh sample of the longissimus dorsi muscle and record the weight. Place the fresh meat sample in a freeze dryer to dry and pulverize. Weigh an appropriate amount of the freeze-dried pulverized muscle sample onto filter paper, fold it into a filter paper packet, and mark the sample number with a pencil. Place the fat packet in a 105℃ oven to dry for 2 hours, then cool it in a desiccator for 30 minutes and weigh it. Repeat this step until constant weight is achieved. Place the fat packet in a Soxhlet extractor and extract with petroleum ether. After extraction, place the fat packet in a fume hood to allow the residual petroleum ether to evaporate, then dry it in a 105℃ oven for 2 hours. Remove it and cool it in a desiccator for 30 minutes, then weigh it. Repeat this step until constant weight is achieved. Calculate the intramuscular fat content using the formula.

[0056] 1.9 Determination of muscle amino acid content Free amino acids from muscle were released using acid hydrolysis, and the amino acid content in the hydrolysate was further determined using an automated amino acid analyzer.

[0057] 1.10 Intestinal Microbiome Assay Fresh cecal chyme samples were weighed and their information recorded. The fresh chyme was then flash-frozen and ground in liquid nitrogen. An appropriate amount of the ground chyme was weighed into a centrifuge tube, and DNA was extracted. The sample number was labeled with a barcode. The DNA solution was tested in a nucleic acid analyzer for 2 hours, then stored in a refrigerator for 30 minutes before quantification. This process was repeated until the samples were deemed acceptable. The DNA solution was then placed in a sequencer for library construction using third-generation sequencing technology. After library construction, the library was placed on a sequencing platform, and after filtering out low-quality sequences, it was analyzed in a bioinformatics workflow for 2 hours. Afterward, it was calibrated in statistical software for 30 minutes before plotting. This process was repeated until all species were annotated. The abundance of gut microbiota was calculated using an algorithm.

[0058] 1.11 Data Processing and Analysis This invention uses Excel to organize experimental data, SPSS 26.0 software to perform statistical analysis, and one-way ANOVA combined with Duncan's method for multiple comparisons. P <0.05 indicates a significant difference.

[0059] 2. Results and Analysis 2.1 Effects of fermented asparagus feed on the growth performance and digestibility of Wanyue black pigs As shown in Table 9, compared with the CON group, the material weight ratio of the AF group decreased significantly by 4.54% after the experiment. P <0.05, while pre-slaughter weight, average daily weight gain, and daily feed intake showed no significant changes ( P >0.05).

[0060] As shown in Table 10, compared with the CON group, the crude protein digestibility of the AF group was significantly increased by 4.17% after the experiment.P <0.05), and the digestibility of crude fat increased significantly by 3.05% ( P <0.05), and the digestibility of crude fiber increased significantly by 4.17% ( P <0.05); while the digestibility of acid-insoluble ash content, phosphorus, and calcium in pig feces showed no significant changes ( P >0.05).

[0061] Table 9. Effects of fermented asparagus feed on the growth performance of black pigs. Table 10. Effects of fermented asparagus straw feed on the digestibility of black pigs. 2.2 Effects of fermented asparagus straw feed on slaughter performance and meat quality of Wan Yue black pigs As shown in Table 11, compared with the CON group, the extraocular muscle area of ​​the AF group increased significantly by 7.4% after the trial. P <0.05), the lean meat percentage increased significantly by 4.22% ( P <0.05), backfat thickness at the 6th-7th ribs decreased significantly by 3.04% ( P <0.05); while carcass weight and carcass length showed no significant changes ( P >0.05).

[0062] As shown in Table 12, compared with the CON group, the flesh color score of the AF group increased significantly by 8.4% after the trial. P <0.05), a* 45min Significantly increased by 9.80% ( P <0.05), a* 24h Significantly increased by 8.65% ( P <0.05); L* 45min Significantly reduced by 4.41% ( P <0.05), L* 24h Significantly reduced by 3.90% ( P <0.05), b* 45min Significantly reduced by 8.09% ( P <0.05), b* 24h Significantly reduced by 4.00% ( P <0.05), intramuscular fat content decreased significantly by 16.62% ( P <0.05), drip loss was significantly reduced by 13.20% ( P <0.05); while pH1, pH 24 There was no significant difference in shear force. P >0.05).

[0063] Table 11 Effects of fermented asparagus straw feed on slaughter performance of black pigs Table 12 Effects of fermented asparagus straw feed on the quality of black pork 2.3 Effects of fermented asparagus straw feed on amino acids in the muscle of black pigs As shown in Table 13, compared with the CON group, the glutamate (Glu) content in the AF group was significantly increased by 16.92% after the experiment. P <0.05), and the level of umami amino acids (DAA) increased significantly by 7.48% ( P <0.05), the proportion of umami amino acids increased significantly by 19.95% ( P <0.05); glycine (Gly) decreased significantly by 11.48% ( P <0.05), tyrosine (Tyr) decreased significantly by 14.44% ( P <0.05); while the contents of aspartic acid (ASP), threonine (Thr), serine (ser), alanine (Ala), valine (Val), methionine (Met), isoleucine (Ile), phenylalanine (Phe), lysine (Lys), and arginine (Arg) showed no significant differences. P >0.05).

[0064] Table 13 Effects of fermented asparagus straw feed on amino acid content in the muscle of black pigs 2.4 Effects of fermented asparagus straw feed on gut microbiota in black pigs Depend on Figure 1 The genus-level species accumulation curve shows that as the sample size increases, more than 180 genera were detected. The number of samples reached a plateau at point 10, indicating that the sample size was sufficient to cover the real species pool. Furthermore, the core species shared by all samples remained stable at 58 genera, accounting for 31% of the total, indicating the existence of a stable core microbial community framework. More than 120 genera were variable microbial communities that were individual / treatment related.

[0065] Depend on Figure 2 Venn diagrams revealed that both groups shared 1041 core OTUs, representing a common framework and corresponding to 58 core genera in the cumulative curves. The CON group had only 27 unique OTUs, indicating no systematic enrichment and random individual differences. The AF group had 166 unique OTUs, approximately six times that of the CON group, suggesting that the AF intervention either introduced or systematically enriched a large number of functional species. The alpha diversity of fermented asparagus on the cecal microbiota of fattening pigs is shown in [the figure]. Figure 4As shown, the ACE index and Chao1 index were used to evaluate community richness. Compared with the CON group, the ACE index and Chao1 index of the AF group were significantly higher ( P <0.05), indicating that asparagus addition improved the richness of the cecal microbiota in black pigs. Principal coordinate analysis (PCoA) was used to assess the differences in microbiota structure between the two groups. Figure 3 The PCoA analysis results showed that PCoA1 and PCoA2 explained 48.73% and 14.66% of the variation, respectively. The PCoA results showed a certain degree of segregation between the CON group and the AF group, indicating that the addition of asparagus altered the cecal microbial community structure of black pigs.

[0066] Depend on Figure 5 LEfSe analysis (LDA>3.5) revealed significantly different biomarker bacteria in the gut microbiota between the two groups. Compared to the CON group, the AF group was mainly enriched in Oscillospiraceae, Rikenellaceae_RC9_gut_group, Verrucomicrobiota, and related bacteria, while the CON group was mainly enriched in Parabacteroides, Tannerellaceae, and Porphyromonadaceae-related bacteria. Combined with ALpha diversity analysis, the experimental group showed significantly increased ACE and Chao1 indices, suggesting that 4% asparagus replacement diet promoted increased gut microbiota richness and may have altered the gut microecological composition by enriching fiber-utilizing bacteria.

[0067] 2.5 Summary After replacing part of the basal diet with fermented asparagus straw feed, the AF group showed the following changes compared to the CON group: (1) In terms of production performance, compared with the CON group, the material weight ratio of the AF group decreased significantly by 4.54% after the test.

[0068] (2) In terms of digestibility, compared with the CON group, the AF group showed a significant increase in crude protein digestibility by 4.17%, crude fat digestibility by 3.05%, and crude fiber digestibility by 4.17% after the experiment.

[0069] (3) In terms of slaughter performance, compared with the CON group, the AF group showed a significant increase of 7.4% in eye muscle area, a significant increase of 4.22% in lean meat percentage, and a significant decrease of 3.04% in back fat thickness at the 6th-7th ribs after the experiment.

[0070] (4) Regarding pork quality, compared with the CON group, the meat color score of the AF group was significantly higher by 8.4% after the experiment, a* 45min Significantly increased by 9.80%, a* 24hSignificantly increased by 8.65%; L* 45min Significantly reduced by 4.41%, L* 24h Significantly reduced by 3.90%, b* 45min Significantly reduced by 8.09%, b* 24h Significantly reduced by 4.00%.

[0071] (5) In terms of intramuscular amino acids, compared with the CON group, the AF group showed a significant increase in glutamic acid content by 16.92%, a significant increase in umami amino acid content by 7.48%, a significant increase in the proportion of umami amino acids by 19.95%, a significant decrease in glycine content by 11.48%, and a significant decrease in tyrosine content by 14.44%.

[0072] (6) Regarding gut microbiota, cumulative curves confirmed sufficient sample size and revealed the ecological hierarchy of 58 core skeletal genera and over 120 variable genera. Venn diagrams showed 166 unique OTUs in the AF group. Alpha diversity analysis showed significantly increased ACE and Chao1 indices in the AF group. PCoA results indicated a certain degree of segregation between the CON and AF groups, suggesting that asparagus addition altered the cecal microbiota structure of black pigs. LEfSe analysis (LDA>3.5) revealed significantly different biomarker bacteria in the two gut microbiota groups. Compared to the CON group, the AF group was mainly enriched in Oscillospiraceae, Rikenellaceae_RC9_gut_group, Verrucomicrobiota, and related bacteria, while the CON group was mainly enriched in Parabacteroides, Tannerellaceae, and Porphyromonadaceae-related bacteria.

[0073] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for producing fermented asparagus straw feed, characterized in that, Includes the following steps: Bacillus subtilis, Lactobacillus paracasei, and Candida utilis were mixed to prepare a fermentation agent; The asparagus stalks are crushed, mixed with wheat bran, and cellulase is added. The mixture is then inoculated with the fermentation agent and fermented to obtain the fermented asparagus stalk feed.

2. The production method according to claim 1, characterized in that, The ratio of viable Bacillus subtilis, Lactobacillus paracasei, and Candida utilis is 1:2:

2.

3. The production method according to claim 1, characterized in that, The inoculum size of the fermentation agent is 10. 7 CFU / g.

4. The production method according to claim 1, characterized in that, The fermentation was carried out at a temperature of 33°C for 4 days.

5. The production method according to claim 1, characterized in that, The mass ratio of the asparagus stalks to the wheat bran is 3:

1.

6. The production method according to claim 1, characterized in that, The amount of cellulase added is 2 mg / kg.

7. A fermented asparagus straw feed obtained by the production method according to any one of claims 1-6.

8. The application of the asparagus straw fermented feed as described in claim 7 in the preparation of pig feed.

9. A type of pig feed, characterized in that, It consists of the asparagus straw fermented feed and the basic diet as described in claim 7.

10. The pig feed as described in claim 9, characterized in that, The mass fraction of the fermented asparagus straw feed is 4%.

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