Fermented total mixed ration containing moringa and method of making same

Abstract

The present application relates to the field of feed, and discloses a fermented total mixed ration containing moringa and a preparation method thereof, the preparation method comprising: anaerobically fermenting fermentation raw materials including whole corn, rape straw, moringa leaves, mixed concentrates and premix at room temperature to obtain the fermented total mixed ration, and the amount of the moringa leaves is 1-10 wt.% (calculated based on dry matter) based on the total amount of the fermentation raw materials; the fermented total mixed ration contains crude protein and organic acid, and the amount of the crude protein is 130-142 g / kg and the amount of the organic acid is 38-45 g / kg based on dry matter. The feed prepared by the present application adds a certain amount of moringa leaves, can improve the antibacterial activity of the feed, can inhibit the proliferation of harmful microorganisms in the aerobic exposure stage, and can maintain the content of crude protein, organic acid and the like in the feed stable.

Description

Technical Field

[0001] This invention relates to the field of feed, specifically to a fermented total mixed diet containing moringa and its preparation method. Background Technology

[0002] Compared to traditional feeds, fermented total mixed diets (FTMRs) have garnered significant attention in ruminant nutrition due to their superior nutrient retention, palatability, and extended shelf life. However, despite these advantages, the aerobic stability of FTMRs, which are rich in carbohydrates, proteins, and lactic acid, remains a key challenge. Exposure to air during feeding or storage often triggers harmful microbial activity, leading to nutrient loss and mycotoxin formation. Therefore, maintaining the fermentation quality of FTMRs while improving their aerobic stability is crucial for optimizing feed utilization and animal health. Summary of the Invention

[0003] This invention aims to overcome the shortcomings of existing technologies and solve the problem of insufficient aerobic stability of FTMR when exposed to air, providing a fermented total mixed diet containing Moringa and its preparation method. The fermented total mixed diet (hereinafter referred to as feed) prepared using the method provided by this invention contains abundant protein, organic acids, and other nutrients, providing rich nutrition for ruminants. Furthermore, even when exposed to air during storage and feeding, the nutrients in the feed remain stable, and the crude protein content may even increase, further enhancing the nutritional value of the feed.

[0004] To achieve the above objectives, the first aspect of the present invention provides a method for preparing a fermented total mixed diet containing moringa, wherein the preparation method includes: anaerobic fermentation of fermentation raw materials including whole corn, rapeseed straw, and moringa leaves at room temperature to obtain a fermented total mixed diet, wherein, based on the total amount of the fermentation raw materials, the amount of moringa leaves is 1-10 wt.% (dry matter).

[0005] The fermented total mixed diet contains crude protein and organic acids, wherein, on a dry matter basis, the amount of crude protein is 130-142 g / kg and the amount of organic acids is 30-45 g / kg.

[0006] This invention uses whole corn, rapeseed straw, moringa leaves and other raw materials for anaerobic fermentation, especially with the addition of a certain amount of moringa leaves. The resulting feed is rich in a variety of highly nutritious and beneficial components, including abundant easily digestible and absorbable crude protein with high bioavailability. Organic acids such as acetic acid, propionic acid and butyric acid can not only provide energy for ruminants, but also maintain the acidity of the feed, which is beneficial to improving the oxidative stability of the feed.

[0007] Moringa leaves are rich in protein, vitamins, flavonoids, phenolic acids, and other secondary metabolites. The protein content is approximately 220-350 mg / g, which can further enhance the nutritional value of feed. The total flavonoid content is approximately 120-150 mg / g, and the total polyphenol content is approximately 43-47 mg / g. These flavonoids and polyphenols can improve the antioxidant and antibacterial activity of feed, inhibiting the proliferation of harmful microorganisms during fermentation and aerobic exposure, thus maintaining stable levels of crude protein and organic acids. In particular, this application found that adding a certain amount of moringa leaves can unexpectedly increase the crude protein content during aerobic exposure, further enhancing the nutritional value of the feed. Currently available feeds, even if they produce relatively rich nutrients during fermentation, generally experience a decline in nutrient content during storage and feeding due to oxygen exposure, resulting in an actual nutritional value lower than the claimed value. This application overcomes these shortcomings, especially by being rich in organic acids, which helps maintain a low pH value in the feed and improves its storage stability.

[0008] A second aspect of the present invention provides a fermented total mixed diet containing moringa, wherein the diet is prepared according to the preparation method described in the first aspect of the present invention. Attached Figure Description

[0009] Figure 1 The relative abundance of horizontal bacteria during aerobic exposure;

[0010] Figure 2 Changes in lactate levels and the abundance of genes in related metabolic pathways during aerobic exposure;

[0011] Figure 3 Temperature changes in feed during aerobic exposure;

[0012] Figure 4 The relative abundance of antimicrobial peptide genes PmrF and rosB in feed during aerobic exposure. Detailed Implementation

[0013] The specific embodiments of this application will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this application.

[0014] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0015] Furthermore, the term "and / or" in the specification and claims is used to describe the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0016] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0017] In this application, "whole plant maize" refers to the entire maize plant, including kernels, cob, stalk, leaves, husks, etc.

[0018] The first aspect of this invention provides a method for preparing a fermented total mixed diet (TMD) containing Moringa, wherein the preparation method includes: anaerobic fermentation of fermentation raw materials including whole corn, rapeseed straw, and Moringa leaves at room temperature to obtain a fermented TMD, wherein, based on the total amount of the fermentation raw materials, the amount of Moringa leaves is 1-10 wt.% (dry matter).

[0019] The fermented total mixed diet contains crude protein and organic acids, wherein, on a dry matter basis, the amount of crude protein is 130-142 g / kg and the amount of organic acids is 30-45 g / kg.

[0020] This invention uses whole corn, rapeseed straw, moringa leaves and other raw materials for anaerobic fermentation, especially with the addition of a certain amount of moringa leaves. The resulting feed is rich in a variety of highly nutritious and beneficial components. The crude protein and water-soluble carbohydrates are easy to digest and absorb, with high bioavailability. Organic acids such as acetic acid, propionic acid and butyric acid can not only provide energy for ruminants, but also maintain the acidity of the feed, which is beneficial to improving the oxidative stability of the feed.

[0021] Moringa leaves are rich in protein, vitamins, flavonoids, phenolic acids, and other secondary metabolites. The protein content is approximately 220-350 mg / g, which can further enhance the nutritional value of feed. The total flavonoid content is approximately 120-150 mg / g, and the total polyphenol content is approximately 43-47 mg / g. These flavonoids and polyphenols can improve the antioxidant and antibacterial activity of feed, inhibiting the proliferation of harmful microorganisms during fermentation and aerobic exposure, thus maintaining stable levels of crude protein and organic acids in the feed. In particular, it has been discovered that adding a certain amount of moringa leaves can even unexpectedly increase the crude protein content during aerobic exposure, further enhancing the nutritional value of the feed. Currently available feeds, even if they produce relatively rich nutrients during fermentation, generally experience a decline in nutrient content during storage and feeding due to oxygen exposure, resulting in an actual nutritional value lower than the claimed value.

[0022] Preferably, based on the total amount of the fermentation raw materials, the amount of Moringa leaves is 4-8 wt.% on a dry matter basis.

[0023] Preferably, based on the total amount of the fermentation raw materials, the amount of Moringa leaves is 5-7 wt.% on a dry matter basis.

[0024] The amount of Moringa leaves added is crucial in determining whether various nutrients in the feed can remain stable under aerobic exposure, which in turn affects the actual value of the feed that ultimately enters the ruminant's body. The amount of Moringa leaves added can be 1 wt.%, 2 wt.%, 4 wt.%, 5 wt.%, 6 wt.%, 7 wt.%, 8 wt.%, 10 wt.%, or any value between any two of these values.

[0025] Fermentation time is generally no less than 25 days to further improve the aerobic exposure stability of the feed.

[0026] Preferably, the amount of crude protein is 135-142 g / kg, and the organic acid includes lactic acid, the amount of which is 34-38 g / kg.

[0027] Preferably, the amount of crude protein is 140-142 g / kg, the amount of lactic acid is 35-36 g / kg, and the pH of the fermented total mixed diet is 4-5.

[0028] The final feed can contain crude protein of 130 g / kg, 135 g / kg, 140 g / kg, 142 g / kg, or any value between any two of these; water-soluble carbohydrates of 170 g / kg, 175 g / kg, 180 g / kg, 185 g / kg, 190 g / kg, or any value between any two of these; organic acids of 38 g / kg, 40 g / kg, 42 g / kg, 45 g / kg, or any value between any two of these; lactic acid of 34 g / kg, 35 g / kg, 36 g / kg, 37 g / kg, 38 g / kg, or any value between any two of these; and the pH of the feed can be 4, 4.5, 5, etc.

[0029] Preferably, the fermented total mixed diet further contains structured carbohydrates and ammonia nitrogen, wherein the structured carbohydrates include neutral detergent fiber and acid detergent fiber, and the amount of structured carbohydrates is 590-750 g / kg on a dry matter basis, and the amount of ammonia nitrogen does not exceed 60 g / kg.

[0030] Preferably, the amount of the structural carbohydrate is 590-650 g / kg, and the amount of the ammonia nitrogen does not exceed 52 g / kg.

[0031] Neutral detergent fiber (NDF) mainly includes cellulose, hemicellulose, and lignin, while acid detergent fiber (ADF) mainly includes cellulose and lignin. These are the primary energy sources for rumen microorganisms, which ferment them to produce volatile fatty acids (acetic acid, propionic acid, and butyric acid). These provide 60-70% of the energy for ruminants. However, not all detergent fibers are digestible; digestion is slow, and the final digestibility depends on the degree of lignification. Therefore, appropriate NDF and ADF content is an important component of feed nutritional value. The NDF and ADF content of the feed prepared by this invention can be 590 g / kg, 600 g / kg, 620 g / kg, 650 g / kg, 670 g / kg, 700 g / kg, 750 g / kg, or any value between any two of these values. A suitable NDF to ADF ratio can provide sufficient energy for ruminants. Especially after adding Moringa leaves, the NDF and ADF content of the feed will slightly decrease under aerobic exposure, indicating that the fiber degradation rate increases under aerobic exposure, which is beneficial for improving digestibility.

[0032] Ammonia nitrogen originates from the degradation of proteins, and its content is positively correlated with the pH value of silage. The higher the content, the higher the pH of the silage, resulting in poorer storage stability and quality. The feed prepared by this invention has a very low ammonia nitrogen content because the added moringa leaves can inhibit the activity of proteins, thereby reducing the ammonia nitrogen content.

[0033] Preferably, based on the total amount of the fermentation raw materials, on a dry matter basis, the amount of whole corn is 30-40 wt%, and the amount of rapeseed straw is 8-12 wt.%. Based on the total amount of the fermentation raw materials, the amount of whole corn can be 30 wt%, 32 wt%, 35 wt%, 37 wt%, 40 wt%, or any value between any two of these, and the amount of rapeseed straw can be 8 wt.%, 9 wt.%, 10 wt.%, 11 wt.%, 12 wt.%, or any value between any two of these.

[0034] Preferably, the fermentation raw materials further include corn flour, soybean meal, wheat bran, and cottonseed meal. Based on the total amount of the fermentation raw materials, on a dry matter basis, the amount of corn flour is 25-30 wt.%, the amount of soybean meal is 5-6 wt.%, the amount of wheat bran is 8-10 wt.%, and the amount of cottonseed meal is 5-6 wt.%. Based on the total amount of the fermentation raw materials, the amount of corn flour can be 25 wt.%, 26 wt.%, 27 wt.%, 28 wt.%, 29 wt.%, 30 wt.%, or any value between any two of these; the amount of wheat bran can be 8 wt.%, 9 wt.%, 10 wt.%, or any value between any two of these; and the amount of cottonseed meal can be 5 wt.%, 5.2 wt.%, 5.5 wt.%, 5.7 wt.%, 6 wt.%, or any value between any two of these.

[0035] To further supplement minerals and vitamins, the fermentation raw materials may further include salt, copper sulfate pentahydrate, ferrous sulfate, zinc sulfate monohydrate, manganese sulfate monohydrate, lysine, choline chloride, calcium hydrogen phosphate, limestone powder, and a premixed compound vitamin feed for beef cattle. The amount of salt, based on the total amount of the fermentation raw materials, is 0.3-1 wt.%, such as 0.3 wt.%, 0.5 wt.%, 0.7 wt.%, 1 wt.%, or any value between any two of these values. Other minerals and vitamins can be added as needed; there are no special requirements, as long as they meet the growth needs of beef cattle.

[0036] Preferably, the anaerobic fermentation time is 25-35 days, and more preferably 30 days.

[0037] Preferably, the moringa leaves are fresh moringa leaves.

[0038] A second aspect of the present invention provides a fermented total mixed diet containing moringa, wherein the diet is prepared according to the preparation method described in the first aspect of the present invention.

[0039] Compared with existing technologies, the present invention has the following significant beneficial effects: By selecting suitable raw materials and adding an appropriate amount of Moringa leaves, the present invention can effectively inhibit the reproduction of aerobic putrefactive bacteria while maintaining the aerobic stability of the feed, maintain the stability of the microbial community structure, and delay the aerobic putrefaction process without affecting the composition of the dominant lactic acid bacteria community and the stability of lactic acid content. As a result, the feed produced is rich in a variety of highly nutritious and beneficial components such as crude protein, water-soluble carbohydrates, and organic acids. In particular, the added Moringa leaves can keep the various nutrients of the feed stable even when exposed to oxygen, and even increase the crude protein content and decrease the content of structural carbohydrates, further improving the application value and digestibility of the feed.

[0040] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the test methods and testing equipment used in the following embodiments are conventional test methods and testing equipment in the art.

[0041] Fresh moringa leaves were harvested from Guizhou Moringa Qinglong Company and chopped into 2-5 cm pieces;

[0042] The mixed concentrate includes corn flour, soybean meal, wheat bran, cottonseed meal, salt, and premix. Based on the total amount of fermented raw materials, the composition is: corn flour 27 wt.%, soybean meal 5.44 wt.%, wheat bran 9.58 wt.%, cottonseed meal 5.48 wt.%, salt 0.50 wt.%, and premix 2 wt.%. The premix is ​​a beef cattle mixed premix, including: feed-grade copper sulfate pentahydrate, ferrous sulfate, zinc sulfate monohydrate, manganese sulfate monohydrate, lysine, choline chloride, beef cattle compound vitamin supplement, and phosphate. Calcium hydroxide and limestone powder are used in beef cattle premix to provide various essential minerals and vitamins. Based on 1000g of beef cattle premix, the main nutrients include vitamin A (40,000-250,000 IU), vitamin D3 (10,000-100,000 IU), vitamin E (≥100 IU), phosphorus (≥1 wt.%), calcium (5-9 wt.%), iron (800-1000 mg), zinc (800-3000 mg), and copper (800-3000 mg). All raw materials were purchased from Henan Dabeinong Co., Ltd.

[0043] Example 1

[0044] On a dry matter basis, 380 kg of whole corn, 100 kg of rapeseed straw, 500 kg of mixed concentrate (including 20 kg of premix) and 20 kg of moringa leaves (total raw materials of 1000 kg) were mixed evenly, then packaged into silage bags (about 300 g / bag), vacuum sealed, and anaerobic fermented at room temperature for 30 days to obtain fermented total mixed ration.

[0045] Example 2

[0046] On a dry matter basis, 360 kg of whole corn, 100 kg of rapeseed straw, 500 kg of mixed concentrate (including 20 kg of premix) and 40 kg of moringa leaves were mixed evenly, then divided into silage bags (about 300 g / bag), vacuum sealed, and anaerobic fermented at room temperature for 30 days to obtain fermented total mixed ration.

[0047] Example 3

[0048] On a dry matter basis, 340 kg of whole corn, 100 kg of rapeseed straw, 500 kg of mixed concentrate (including 20 kg of premix) and 60 kg of moringa leaves were mixed evenly, then divided into silage bags (about 300 g / bag), vacuum sealed, and anaerobic fermented at room temperature for 30 days to obtain fermented total mixed ration.

[0049] Example 4

[0050] On a dry matter basis, 320 kg of whole corn, 100 kg of rapeseed straw, 500 kg of mixed concentrate (including 20 kg of premix) and 80 kg of moringa leaves were mixed evenly, then divided into silage bags (about 300 g / bag), vacuum sealed, and anaerobic fermented at room temperature for 30 days to obtain fermented total mixed ration.

[0051] Example 5

[0052] The process was carried out in accordance with Example 1, except that the fermentation time was 20 days.

[0053] Comparative Example 1

[0054] On a dry matter basis, 400 kg of whole corn, 100 kg of rapeseed straw, and 500 kg of mixed concentrate (including 20 kg of premix) were mixed evenly, then packaged into silage bags (about 300 g / bag), vacuum-packed and powdered, and then anaerobic fermented at room temperature for 30 days to obtain fermented total mixed ration.

[0055] Comparative Example 2

[0056] The procedure was carried out in accordance with Example 1, except that an equal amount of Moringa branches were used instead of Moringa leaves.

[0057] Comparative Example 3

[0058] The procedure was carried out in accordance with Example 1, except that an equal amount of forage was used instead of whole corn plants.

[0059] Comparative Example 4

[0060] The procedure was carried out in accordance with Example 1, except that the whole corn plant was replaced with an equal amount of rapeseed straw.

[0061] Test Example 1

[0062] The nutritional quality of the feed was tested and its stability after aerobic exposure.

[0063] Using the feeds prepared in Examples 1-3 and Comparative Examples 1-5 as test subjects, the crude protein, water-soluble carbohydrates, lactic acid, neutral detergent fiber, acid detergent fiber, ammonia nitrogen and pH value of the feeds were tested respectively. After the feeds were aerobically exposed for 7 days (during which the feeds were gently turned once a day to ensure uniform oxygen distribution), their nutritional quality was tested again.

[0064] Test method:

[0065] Approximately 200 g of sample was weighed and dried to constant weight in a 65 ℃ oven (WGL-125B, Tianjin Tester Instrument Co., Ltd.) for calculating dry matter (DM). The dried sample was then pulverized using a small high-speed grinder (FW100, Tianjin Tester Instrument Co., Ltd.) and passed through a 1 mm sieve. Crude protein (CP) content was determined using a Dumas nitrogen analyzer (Rapid N exceed, Germany). Neutral detergent fiber (NDF) and acid detergent fiber (ADF) content were determined using an automated fiber analyzer (ANKOM-2 000i, USA). Water-soluble carbohydrate (WSC) content was determined using the sulfuric acid-anthrone colorimetric method.

[0066] Take 10 g of the prepared feed or feed that has been aerobically exposed for 7 days and mix it with 90 mL of sterile water. Extract at 4 ℃ for 24 h. Filter the extract through four layers of gauze and Xinhua filter paper (Hangzhou Xinhua Co., Ltd.), and immediately measure the pH using a glass electrode pH meter (PHSJ-3F, Shanghai Instrument & Electronics Scientific Instruments Co., Ltd.). Centrifuge 5 mL of the extract at 4 ℃ and 12000×g for 10 min. Filter the supernatant through a 0.22 μm filter membrane for organic acid analysis. Lactic acid content was primarily analyzed using a high-performance liquid chromatography system (Vanquish Core HPLC, Thermo Fisher Scientific, USA) equipped with a differential detector for quantification. Ammonia nitrogen (NH3-N) was determined using the phenol-hypochlorite method.

[0067] The test results are shown in Table 1.

[0068] Table 1

[0069] name Aerobic exposure time CP WSC NDF ADF lactic acid ammonia nitrogen pH <![CDATA[g kg -1 DM]]> <![CDATA[g kg -1 DM]]> <![CDATA[g kg -1 DM]]> <![CDATA[g kg -1 DM]]> <![CDATA[g kg -1 DM]]> <![CDATA[g kg -1 TN]]> Example 1 0 days 130.41 176.21 499.59 236.67 34.77 59.06 4.66 7 days 144.53 152.91 483.80 197.97 38.39 49.02 4.68 Example 2 0 days 135.87 187.87 432.85 198.96 36.02 50.64 4.69 7 days 141.83 160.82 486.37 205.45 37.24 48.68 4.67 Example 3 0 days 141.01 183.67 418.98 184.75 35.27 45.43 4.68 7 days 143.37 144.46 431.92 174.90 38.35 42.09 4.64 Example 4 0 days 141.17 174.46 443.85 186.80 34.13 46.84 4.65 7 days 151.70 157.58 450.23 185.59 33.65 40.93 5.43 Example 5 0 days 134.25 170.05 507.65 243.34 33.78 57.45 4.71 7 days 149.75 149.38 492.83 205.27 36.12 46.32 4.77 Comparative Example 1 0 days 142.83 180.59 464.34 226.62 37.47 62.6 4.70 7 days 134.43 147.87 496.68 212.04 11.80 58.18 5.66 Comparative Example 2 0 days 129.98 175.40 515.67 249.34 30.11 61.45 5.35 7 days 136.56 154.32 507.76 209.58 34.04 53.98 5.22 Comparative Example 3 0 days 137.95 178.31 536.79 287.24 29.86 66.04 4.91 7 days 142.08 144.16 511.47 260.56 25.67 59.34 5.47 Comparative Example 4 0 days 123.31 142.51 620.18 386.44 17.21 82.13 5.67 7 days 119.45 109.45 590.37 378.57 13.76 84.78 6.78

[0070] Note: g and kg in the table -1 DM refers to the corresponding substance on a dry matter basis, with units of g / kg. -1 g kg -1 TN refers to the substance as total nitrogen.

[0071] As can be seen from Table 1, compared with Comparative Example 1, the CP content of the material prepared by the method provided by this invention not only did not decrease after aerobic exposure, but actually increased. This indicates that the nutritional value of the feed not only did not decrease after aerobic exposure like traditional feed, but actually increased, while the CP content of the feed prepared in Comparative Example 1 decreased after aerobic exposure. NDF and ADF, on the other hand, remained basically stable or decreased after aerobic exposure (only the total amount of NDF and ADF in Example 2 increased slightly, mainly because under aerobic exposure conditions, other easily aerobic nutrients in this group of feeds were preferentially consumed over NDF and ADF, leading to an increase in the relative content of NDF and ADF in dry matter, which also indirectly indicates...). In contrast, the aerobic nutrients in the feeds of other groups were better preserved, resulting in NDF and ADF remaining relatively stable or decreasing after aerobic exposure. However, the total NDF and ADF levels in the feed of Comparative Example 1 increased significantly after aerobic exposure, indicating a greater decrease in other aerobic nutrients and a reduction in NDF and ADF content, thus decreasing the feed's digestibility. Furthermore, the lactic acid content in the feed prepared by this invention remained stable or even increased during aerobic exposure, while the lactic acid content in Comparative Example 1 decreased significantly after aerobic exposure. This not only reduced the nutritional value of the feed but also hindered its preservation. Adding Moringa leaves also helped maintain the pH value of the feed during aerobic exposure, preventing pH increases and spoilage. Additionally, this application found that the ammonia nitrogen content in the feed prepared by this invention was significantly lower than that in Comparative Example 1, indicating that adding Moringa can reduce protein hydrolysis, prevent the production of ammonia nitrogen, and improve the nutritional value of the feed.

[0072] As can be seen from Example 1 and Comparative Example 2, when Moringa branches are used instead of Moringa leaves, the total amount of ADF and NDF in the obtained feed increases, its digestibility decreases, the lactic acid content in the feed decreases, the ammonia nitrogen content increases, and the pH value of the feed increases after aerobic exposure, which is not conducive to maintaining storage stability. This may be because the content of antioxidant substances contained in Moringa branches is not as high as that in fresh Moringa leaves, the proliferation of Lactobacillus during feed fermentation is not as high as in this application, and the degradation of protein increases, resulting in a decrease in the antioxidant capacity of the feed.

[0073] As can be seen from Example 1 and Comparative Example 3, when forage is used to replace whole corn, the easily digestible nutrients CP and WSC of the obtained feed are basically the same. As can be seen from Example 1 and Comparative Example 4, when rapeseed straw is used to replace whole corn, the easily digestible nutrients CP and WSC of the obtained feed decrease. However, the feeds in Comparative Examples 3 and 4 show a significant decrease after 7 days of aerobic exposure. This fully demonstrates that the feed prepared by fermentation of Moringa leaves and the raw materials provided in this application has a better synergistic effect and is more conducive to maintaining the aerobic stability of the feed.

[0074] Test Example 2

[0075] Metagenomic sequencing

[0076] Using Example 1 and Comparative Example 1 as test subjects, total genomic DNA of the microbial community was extracted according to the EZNA® soil DNA kit (Omega Bio-tek, Norcross, GA, US) instructions. After DNA extraction, DNA concentration and purity were measured, and DNA integrity was checked using 1% agarose gel electrophoresis. DNA fragmentation was performed using a Covaris M220 (Genetron Health, China), and fragments of approximately 350 bp were selected for constructing PE libraries. Metagenomic sequencing was performed using the Illumina NovaSeq™ XPlus (Illumina, USA) sequencing platform (Shanghai Meiji Biopharmaceutical Technology Co., Ltd.). Metagenomic data analysis was performed on the Meiji Bio Cloud Platform (Han et al., 2024). FastP (Chen et al., 2018) (https: / / github.com / OpenGene / fastp, version 0.20.0) was used to cut the adapter sequences at the 3' and 5' ends of reads, removing reads shorter than 50 bp and with an average base quality value below 20, retaining high-quality sequences for data quality control. The optimized sequences were assembled using MEGAHIT (Li et al., 2015) (https: / / github.com / voutcn / megahit, version 1.1.2). Contigs ≥300 bp were selected from the assembled results as the final assembly. ORFs were predicted for the contigs in the assembled results using Prodigal (Hyatt et al., 2010) (https: / / github.com / hyattpd / Prodigal, version 2.6.3). Genes with a nucleic acid length of 100 bp or more were selected and translated into amino acid sequences.

[0077] The predicted gene sequences of all samples were clustered using CD-HIT (Fu et al., 2012) (http: / / weizhongli-lab.org / cd-hit / , version 4.7) with parameters of 90% identity and 90% coverage. The longest gene in each cluster was selected as the representative sequence to construct a non-redundant gene set. The SOAP aligner (Li et al., 2008) software (https: / / github.com / ShujiaHuang / SOAPaligner, version soap2.21 release) was used to align the high-quality reads of each sample with the non-redundant gene set (95% identity), and the abundance information of the genes in the corresponding samples was statistically analyzed. Using Diamond (Buchfink et al., 2015) (https: / / github.com / bbuchfink / diamond, version 2.0.13), the amino acid sequences of the non-redundant gene set were aligned with the NR, KEGG, and CARD databases respectively (BLASTP alignment parameters were set with an expected e-value of 1e-5). Species annotations were obtained from the taxonomic information database corresponding to the NR database, and then the abundance of the species was calculated using the sum of gene abundance corresponding to the species. KEGG functions corresponding to genes were obtained from the KEGG database. The abundance of the corresponding functional category was calculated using the sum of gene abundance corresponding to EC. Antibiotic resistance function annotation information was obtained from the CARD database, and then the abundance of the antibiotic resistance function was calculated using the sum of gene abundance corresponding to the antibiotic resistance function.

[0078] Test results are as follows Figure 1 As shown in the figure, T0-0 represents the feed without moringa leaves (from Comparative Example 1) with 0 days of aerobic exposure. Similarly, T0-3 and T0-7 represent the feed of Comparative Example 1 with 3 days and 7 days of aerobic exposure, respectively. T2-0 represents the feed with moringa leaves (from Example 1). Similarly, T2-3 and T2-7 represent the feed of Example 1 with 3 days and 7 days of aerobic exposure, respectively. The figure shows that aerobic exposure significantly reduced the relative abundance of *Lactiplantibacillus* in Comparative Example 1 to 19.07%, while *Oceanobacillus* significantly increased, reaching a relative abundance of 34.48%. The decrease in lactic acid content and the increase in pH in the feed prepared in Comparative Example 1 are related to the decrease in *Lactiplantibacillus* microorganisms and the increase in *Oceanobacillus* microorganisms.

[0079] The feed prepared by this invention, after the addition of Moringa leaves, still showed that Lactiplantibacillus remained the dominant bacteria after aerobic exposure. Compared with before exposure, its abundance was very stable and even slightly increased. It can be seen that the addition of Moringa leaves can significantly improve the activity of lactobacilli in the feed during aerobic exposure, which is beneficial to maintaining the fermentation stability of the feed. This is consistent with the previous conclusion that the pH remained stable or even decreased after aerobic exposure, and the lactic acid content increased.

[0080] The activities of lactate-metabolizing enzymes in the feeds prepared in Example 1 and Comparative Example 1 during aerobic exposure were determined, and the results are as follows: Figure 2 As shown. Figure 2 This is a metabolic pathway diagram of lactate, drawn based on the KEGG database, showing the main conversion pathways of lactate through glycolysis and the tricarboxylic acid cycle. In the diagram, T0-0 represents data from 0 days of aerobic exposure in the feed without moringa leaves (the feed of Comparative Example 1), T0-2 represents data from 3 days of aerobic exposure in the feed without moringa leaves, T0-7 represents data from 7 days of aerobic exposure in the feed without moringa leaves, T2-0 represents data from 0 days of aerobic exposure in the feed with 2 wt.% moringa leaves (the feed of Example 1), T2-2 represents data from 3 days of aerobic exposure in the feed with 2 wt.% moringa leaves, and T2-7 represents data from 7 days of aerobic exposure in the feed with 2 wt.% moringa leaves. According to the arrow order, in the first subplot, Lactate represents lactate content, and 1.1.1.27, 1.2.4.1, 1.8.1.4, 2.3.1.14, etc., represent various enzymes involved in the metabolic process, and Oxaloacetate represents oxaloacetate. As shown in the figure, during aerobic exposure, the gene expression levels of a series of enzymes in Comparative Example 1, including citrate synthase (EC 2.3.3.1), aconitase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), α-ketoglutarate dehydrogenase complex (EC 1.2.4.2), succinate dehydrogenase (EC 1.3.5.1), and malate dehydrogenase (EC 1.1.1.37), were significantly higher than those in Example 1 (P<0.05), while the individual enzymes in Example 1 remained relatively stable. This is consistent with the fact that lactic acid levels in the tested feed groups remained stable or even increased.

[0081] Temperature changes in the feeds prepared in each embodiment and Comparative Example 1 during aerobic exposure were recorded, and the results are as follows: Figure 3As shown, it can be seen that with the increase of aerobic exposure time, the temperature of the feed in Comparative Example 1 first rises significantly, then falls back and then continues to rise, and it always remains at a high level (average temperature exceeds 28°C, and the highest temperature reaches 32°C). However, the temperature of the feed prepared by this invention remains at a relatively low level throughout the entire aerobic exposure process (not exceeding 27°C, and the temperature does not change significantly). It is evident that adding Moringa leaves can keep the temperature of the feed stable during aerobic exposure, which is beneficial for maintaining the applied substances in the feed at a stable level.

[0082] Antimicrobial peptides are a class of polypeptide molecules derived from the innate immune system. They possess broad-spectrum antimicrobial activity and can effectively target and kill various pathogens such as bacteria, fungi, and viruses. The relative abundance of the antimicrobial peptide genes PmrF and rosB in the feeds prepared according to Example 1 and Comparative Example 1 during aerobic exposure was determined. The test results are as follows: Figure 4 As shown. Figure 4 (a) represents the relative abundance of the antimicrobial peptide gene PmrF. Figure 4 (b) shows the relative abundance of the antimicrobial peptide gene rosB. It can be seen that, compared with the control group 1 without the addition of Moringa leaves, the feed of the present invention showed a highly consistent trend in the changes of the antimicrobial peptide genes PmrF and rosB during aerobic exposure. Both remained at a very stable high level, which indicates that the addition of Moringa leaves can keep the antimicrobial peptide genes PmrF and rosB stable.

[0083] In summary, the addition of Moringa leaves helps maintain a high level of Lactiplantibacillus in feed under aerobic exposure and inhibits Oceanobacillus. It also helps suppress microbial thermogenic activity, slows down the aerobic putrefaction process, and thus maintains stable temperature during feed storage.

[0084] In addition, under aerobic conditions, the addition of Moringa leaves has the ability to maintain the stability of antimicrobial peptide gene abundance. This may be because it effectively alleviates the negative effects of aerobic stress by providing antioxidants, thereby supporting the stability of antimicrobial peptide gene abundance. This helps to improve the feed's resistance to spoilage in an aerobic environment.

[0085] Furthermore, no molds, yeasts, or Escherichia coli were detected in any of the feeds containing Moringa leaves before or after aerobic exposure, indicating that adding Moringa leaves can inhibit the growth of harmful bacteria.

[0086] The above-mentioned multiple effects ultimately manifest as Moringa leaves promoting the stable survival of lactic acid bacteria in feed under aerobic exposure, maintaining the stability of lactic acid catabolism enzyme gene abundance (such as citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase complex, etc.), and keeping the abundance of antimicrobial peptide genes related to aerobic putrefaction (such as Pmr and rosB) stable. Ultimately, this results in increased CP content, reduced ammonia nitrogen content, inhibited lactic acid consumption, maintained pH stability, and significantly reduced loss of nutrients in feed after aerobic exposure.

[0087] The contents not described in detail in this specification are existing technologies known to those skilled in the art, and will not be elaborated upon here.

[0088] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for preparing a fermented total mixed diet containing moringa, characterized in that, The preparation method includes: anaerobic fermentation of fermentation raw materials comprising whole corn plants, rapeseed straw, and moringa leaves at room temperature to obtain a fermented total mixed diet, wherein, based on the total amount of the fermentation raw materials, the amount of moringa leaves is 4-8 wt.% (dry matter). The fermented total mixed diet contains crude protein and organic acids. On a dry matter basis, the amount of crude protein is 130-142 g / kg, the amount of organic acids is 38-45 g / kg, and the organic acids include lactic acid, with the amount of lactic acid being 34-38 g / kg. Based on the total amount of the fermentation raw materials, on a dry matter basis, the amount of whole corn plant is 30-40 wt%, and the amount of rapeseed straw is 8-12 wt%. The fermentation raw materials also include corn flour, soybean meal, wheat bran and cottonseed meal. Based on the total amount of the fermentation raw materials, on a dry matter basis, the amount of corn flour is 25-30 wt.%, the amount of soybean meal is 5-6 wt.%, the amount of wheat bran is 8-10 wt.%, and the amount of cottonseed meal is 5-6 wt.

2. The preparation method according to claim 1, wherein, On a dry matter basis, based on the total amount of the fermentation raw materials, the amount of Moringa leaves is 5-7 wt.%.

3. The preparation method according to claim 1 or 2, wherein, The amount of crude protein is 135-142 g / kg.

4. The preparation method according to claim 3, wherein, The crude protein content is 140-142 g / kg, the lactic acid content is 35-36 g / kg, and the pH of the fermented total mixed diet is 4-5.

5. The preparation method according to claim 1 or 2, wherein, The fermented total mixed diet also contains structured carbohydrates and ammonia nitrogen. The structured carbohydrates include neutral detergent fiber and acid detergent fiber. On a dry matter basis, the amount of structured carbohydrates is 590-750 g / kg, and the amount of ammonia nitrogen is no more than 60 g / kg.

6. The preparation method according to claim 5, wherein, The amount of the structural carbohydrate is 590-650 g / kg, and the amount of the ammonia nitrogen does not exceed 52 g / kg.

7. The preparation method according to claim 1, wherein, The anaerobic fermentation time is 25-35 days; And / or, the moringa leaves are fresh moringa leaves.

8. A fermented total mixed diet containing moringa, characterized in that, Prepared according to the preparation method according to any one of claims 1-7.

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