Fermented feed of lonicera japonica by-product, and preparation method and application thereof

Abstract

The present application belongs to the field of animal nutrition and feed science and technology, and particularly relates to a honeysuckle by-product fermented feed as well as a preparation method and application thereof. The present application uses mature branch and leaf honeysuckle by-products as raw materials, wheat bran as an auxiliary material, and adds a bacteria-enzyme fermenting agent compounded by specific composite enzymes and composite microorganisms to obtain a functional fermented feed through anaerobic solid-state sealed fermentation. The present application has simple process and controllable cost, can efficiently degrade raw material crude fiber, increase crude protein content, release active ingredients, and realize high-value utilization of honeysuckle by-products. When added into the diet of laying hens in the later stage of egg production at a suitable ratio, the present application can significantly improve the egg production rate, enhance the body's antioxidant and anti-inflammatory capacity, improve the intestinal barrier and microecology, delay ovarian aging, and prolong the egg production period, thereby providing a new technology for green and healthy breeding of laying hens.

Description

Technical Field

[0001] This invention belongs to the field of animal nutrition and feed science and technology, specifically relating to a fermented feed made from honeysuckle by-products, its preparation method, and its application. Background Technology

[0002] The egg-laying hen industry is a crucial pillar of animal husbandry and a core industry for ensuring a stable supply of poultry eggs and promoting agricultural and rural economic development. In the entire egg-laying hen breeding cycle, the late-laying stage (over 450 days old) directly determines the overall economic benefits of the entire breeding cycle. However, in actual large-scale production, late-laying hens commonly experience a series of physiological and performance decline problems, specifically manifested as a continuous decrease in egg production rate, ovarian function decline, impaired intestinal barrier function, increased oxidative stress, elevated systemic inflammation levels, and reduced cecal intestinal microbial diversity. These problems not only directly lead to increased feed conversion ratios, higher breeding costs, and a forced shortening of the laying cycle, but also reduce the disease resistance of hens, severely restricting the quality improvement, efficiency enhancement, and healthy sustainable development of the egg-laying hen industry. Developing safe, green, and efficient functional feed ingredients to address the series of physiological and performance decline problems in late-laying hens, and improving the health status of hens and delaying performance decline through nutritional regulation, is currently a research hotspot and core industry demand in the egg-laying hen breeding field.

[0003] Honeysuckle is a traditional Chinese medicinal and edible plant. Its flower buds are rich in chlorogenic acid, flavonoids, saponins, and other bioactive components, which have been proven to have good antibacterial, anti-inflammatory, antiviral, and immunomodulatory effects. Related research in livestock and poultry farming has also confirmed that the active components of honeysuckle have multiple physiological functions, including promoting the digestion and absorption of nutrients in animals, improving intestinal flora structure, enhancing the body's antioxidant capacity, and strengthening immunity, making it a potential development ingredient for functional feed for livestock and poultry. During the large-scale cultivation and processing of honeysuckle, a large amount of by-products are generated annually due to plant pruning, flower bud harvesting, and processing at the production site. These by-products mainly consist of mature honeysuckle branches and leaves. Taking Xiushan County, known as the "Hometown of Honeysuckle in my country," as an example, the annual output of mature branches and leaves by-products from honeysuckle cultivation and processing alone is nearly 100,000 tons, indicating a very large resource quantity. Relevant research data shows that mature branches and leaves of honeysuckle still contain 12.62 mg / g of chlorogenic acid, which is about 40% of that in honeysuckle buds, and has similar physiological effects to the buds. At the same time, compared with honeysuckle buds, which have high economic value and high application costs, its mature branches and leaves by-products have significant advantages such as wide availability, large yield and low cost, and have extremely high resource development and utilization value.

[0004] Currently, the development and utilization of honeysuckle mainly focuses on its flower buds, while a large amount of mature branches and leaves, as by-products, have not been effectively utilized for high-value purposes. Most by-products are directly discarded or incinerated, resulting not only in a significant waste of valuable biological resources but also in serious environmental pollution problems. This increases the environmental disposal costs of the honeysuckle planting and processing industry, hindering the overall improvement of the honeysuckle industry chain's comprehensive benefits. Therefore, developing a simple, cost-controllable, and scalable technology for the high-value utilization of honeysuckle by-products, transforming them into functional feed ingredients applicable to livestock and poultry farming, would possess significant economic, ecological, and social value.

[0005] Enzyme-microbe co-fermentation technology is a novel biological treatment technology that combines the efficient degradation effect of enzymes with the fermentation and transformation effect of microorganisms. Through the synergistic effect of enzymes and microorganisms, it can efficiently degrade anti-nutritional factors in raw materials, improve the nutrient composition and digestibility of raw materials, and enrich functional components such as probiotics and organic acids during fermentation. It is one of the core technologies for the current development of agricultural by-product feed. However, current technologies lack enzyme-microbe co-fermentation technology specifically developed for the characteristics of honeysuckle by-products. It is impossible to achieve efficient degradation of crude fiber and full release of functional active ingredients in honeysuckle by-products, nor can a mature technical solution be developed for the precise application of fermented honeysuckle by-products in late-laying egg production. It is also impossible to address the core pain points of declining production performance and health in late-laying egg production through nutritional regulation.

[0006] In summary, existing technologies cannot achieve the harmless, resource-based, and high-value utilization of honeysuckle by-products, nor can they address the core technological bottlenecks in late-laying egg production through the feed application of honeysuckle by-products. There is an urgent need to develop a fermentation technology adapted to the characteristics of honeysuckle by-products and apply the fermented products to late-laying egg production, thereby achieving high-value utilization of agricultural by-products and providing a new technical solution for green and healthy egg production in late-laying egg production. Summary of the Invention

[0007] The purpose of this invention is to provide a fermented feed made from honeysuckle by-products, its preparation method, and its application. This significantly reduces the environmental disposal costs of the honeysuckle planting and processing industry, improves the overall economic benefits of the entire industrial chain, avoids the environmental pollution problems caused by the arbitrary disposal of by-products, and provides the feed industry with a new type of functional raw material that is widely available, low in cost, and of stable quality.

[0008] The objective of this invention is achieved through the following technical solution: This invention provides a method for preparing fermented feed from honeysuckle by-products, comprising the following steps: (1) Raw material pretreatment: After harvesting the mature branches and leaves of honeysuckle by-products, crush them and add wheat bran as an auxiliary material to mix them evenly to obtain the fermentation substrate; (2) Addition of bacterial enzymes: Spray the bacterial enzyme fermentation agent into the fermentation substrate and stir evenly; the bacterial enzyme fermentation agent is prepared by dissolving a compound enzyme and compound microorganism in water at a mass ratio of (5–8):10; the compound enzyme is a mixture of acidic protease, cellulase and phytase in a mass ratio of 1:1:1; the compound microorganism is composed of brewer's yeast, Bacillus subtilis, Lactobacillus plantarum and Bifidobacterium in a mass ratio of (2–3):(2–3):(2–3):1. (3) Solid fermentation: The substrate with added bacterial enzyme fermentation agent is subjected to solid sealed fermentation. The fermentation temperature is controlled at 15–35℃, the fermentation time is 15–20 days, and the pH of the material at the end of fermentation is ≤4.5, thus obtaining fermented feed of honeysuckle by-product.

[0009] Furthermore, the amount of the bacterial enzyme fermentation agent added is 4–8 wt% of the total mass of the fermentation substrate.

[0010] Furthermore, the mass ratio of the honeysuckle by-product to wheat bran is (70–85):(15–30).

[0011] Furthermore, the moisture content of the fermentation substrate was adjusted to 50–58 wt% before solid-state fermentation.

[0012] Furthermore, the solid-state sealed fermentation adopts an anaerobic solid-state fermentation method using a plastic film or fermentation bag compacted and sealed; the particle size of the mature branches and leaves of the honeysuckle by-product after crushing is 2.0–5.0 cm.

[0013] The present invention also provides a fermented feed made from honeysuckle by-products, which is prepared by the aforementioned preparation method.

[0014] The present invention also provides an application of the fermented feed of honeysuckle by-products in the preparation of special feed for laying hens in the later stage of egg production.

[0015] The present invention also provides a special feed for laying hens in the late laying period, comprising, by weight percentage, 85–92% basal diet, 5–10% fermented feed of honeysuckle by-products, and 3–5% premix.

[0016] Furthermore, the premix contains compound vitamins, compound minerals, amino acids, and zeolite powder.

[0017] Furthermore, the fermented honeysuckle by-product feed is pulverized twice to 40-60 mesh before use, and the special feed is fed as powder feed.

[0018] The beneficial effects of this invention are as follows: This invention uses bio-fermentation technology to transform by-products that are usually discarded or incinerated into functional feed ingredients that can be directly applied to livestock and poultry farming. This significantly reduces the environmental disposal costs of the honeysuckle planting and processing industry, improves the overall economic benefits of the entire industrial chain, avoids the environmental pollution problems caused by the indiscriminate disposal of by-products, and provides the feed industry with new functional raw materials that are widely available, low in cost, and stable in quality. The fermentation process is simple to operate, the conditions are controllable, and the production cost is low. It does not require complex large-scale equipment and can be directly adapted to the needs of large-scale production.

[0019] This invention employs a synergistic fermentation technique combining complex enzymes and complex microorganisms. Addressing the characteristics of honeysuckle byproducts—high crude fiber content and functional components largely bound and difficult to release—an enzymatic hydrolysis system using a balanced blend of acidic protease, cellulase, and phytase efficiently degrades anti-nutritional factors such as cellulose, hemicellulose, and phytic acid, simultaneously releasing bound chlorogenic acid, flavonoids, and other functional active ingredients. Furthermore, a microbial system composed of brewer's yeast, Bacillus subtilis, Lactobacillus plantarum, and Bifidobacterium achieves a synergistic effect: rapid aerobic proliferation reduces the system's oxygen partial pressure, while Lactobacillus plantarum's dominant acid production lowers the system's pH. Ultimately, the pH of the fermentation product remains stable at 4.5 or below. The fermentation scheme of this invention not only increases the crude protein content of the raw material by more than 20% compared with that before fermentation, but also significantly reduces the levels of neutral detergent fiber, acid detergent fiber, and acid detergent lignin, greatly improving the feed value and nutrient digestibility of the raw material. At the same time, the fermentation product is rich in active probiotics and organic acids, with good palatability and antibacterial and anti-mold effects. It can achieve long-term stable storage without the need for additional preservatives. The combined use of wheat bran as an adjunct and the bacterial enzyme fermentation system also achieves a significant synergistic effect. The fermentation effect is far superior to the treatment method of fermenting honeysuckle branches and leaves alone or fermenting wheat bran alone, filling the industry gap in the existing technology of lacking a special fermentation technology for the characteristics of honeysuckle by-products.

[0020] The fermented honeysuckle by-product feed prepared by this invention is precisely tailored to the physiological characteristics and breeding needs of late-laying laying hens. Adding it to the basal diet of late-laying laying hens in an appropriate proportion can significantly improve their production performance. A 5% addition can increase the egg production rate of late-laying laying hens over 450 days old from 78.54% to 88.10%, with a simultaneous and significant increase in average daily egg production and a decreasing feed conversion ratio. It also maintains stable feed intake, effectively alleviating the common production pain points of continuously declining egg production rate and reduced breeding profits in late-laying laying hens, significantly slowing down the decline in laying performance and effectively extending the effective laying cycle. Furthermore, except for a slight decrease in eggshell thickness in the 5% addition group, the fermented product of this invention has no adverse effects on core commercial quality indicators such as egg weight, eggshell strength, albumen height, Haugh unit, yolk ratio, and eggshell ratio. While increasing breeding output, it fully protects the commercial value of eggs, directly bringing significant economic benefits to laying hen farmers.

[0021] The fermented honeysuckle by-product feed prepared by this invention can improve ovarian function in late-laying hens from a reproductive physiological perspective, delaying the ovarian aging process. Adding an appropriate proportion of the fermented product to the diet can significantly increase the number of dominant and large white follicles in late-laying hens. Simultaneously, it significantly increases the levels of key reproductive hormones such as progesterone, follicle-stimulating hormone, and luteinizing hormone in the serum and ovarian tissue of laying hens in a dose-dependent manner. It can also upregulate the gene expression level of corresponding hormone receptors in the ovary, enhancing the responsiveness of the laying hen to reproductive hormones. This effectively improves the core problems of ovarian function decline, accelerated follicle atresia, and reproductive endocrine disorders in late-laying hens, providing a solid physiological basis for increasing egg production and extending the laying cycle. It overcomes the technical bottleneck of existing nutritional regulation technologies, which struggle to fundamentally delay the aging of the reproductive system in laying hens.

[0022] The fermented honeysuckle by-product feed prepared by this invention can significantly enhance the antioxidant and anti-inflammatory capabilities of laying hens in the late laying period, effectively alleviating the common problems of increased oxidative stress and elevated levels of systemic chronic inflammation in laying hens during this period. Adding the fermented product to the diet can significantly upregulate the expression levels of antioxidant-related genes in the liver and ovarian tissues of laying hens, significantly increase the activity of glutathione peroxidase in ovarian tissue, and reduce the risk of lipid peroxidation damage. Simultaneously, it can significantly downregulate the gene expression levels of pro-inflammatory factors such as IL-1β, TNF-α, and IL-6 in the liver, ovary, and intestinal tissues such as the duodenum and jejunum of laying hens. This alleviates the chronic inflammatory state of the body at multiple tissue levels, improves the overall health of laying hens, and provides a health guarantee for the long-term stable production performance of laying hens.

[0023] The fermented honeysuckle by-product feed prepared by this invention can effectively improve the intestinal barrier function of laying hens in the late laying period, optimize the cecal microbiota, and build a healthy and stable intestinal micro-ecosystem. Adding the fermented product to the diet can upregulate the expression of genes related to tight junctions in the duodenum, jejunum, and ileum of laying hens, such as ZO-1 and CLDN1, enhancing the intestinal mechanical barrier function. Simultaneously, it can significantly increase the content of beneficial short-chain fatty acids such as acetic acid, propionic acid, butyric acid, and valeric acid in the cecal contents of laying hens, providing sufficient energy supply to intestinal epithelial cells and further maintaining the integrity of the intestinal barrier. A 5% addition can also significantly increase the Chao1 index, ACE index, Shannon index, and Simpson index of the cecal microbiota in laying hens, greatly improving the diversity and richness of the intestinal microbial community. It can also effectively regulate the phylum and genus-level bacterial community structure, optimizing the composition of the intestinal flora. A healthy intestinal microecology can further improve the body's efficiency in digesting and absorbing nutrients, inhibit the proliferation of harmful bacteria in the intestine, and form a virtuous cycle of intestinal health, overall health, and improved production performance. Attached Figure Description

[0024] 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.

[0025] Figure 1 This is a diagram showing the effect of fermented honeysuckle by-products of this invention on the expression of ovarian hormone receptor genes in late-laying laying hens. Figure 2 This is a diagram showing the effect of fermented honeysuckle by-products of this invention on the expression of liver inflammation-related genes in laying hens during the late laying period; Figure 3 This is a diagram showing the effect of fermented honeysuckle by-products of this invention on the expression of genes related to ovarian inflammation in laying hens during the late laying period. Figure 4 This is a diagram showing the effect of fermented honeysuckle by-products of this invention on the expression of genes related to small intestinal inflammation in laying hens during the late laying period; Figure 5 This is a diagram showing the effect of fermented honeysuckle by-products of this invention on the expression of antioxidant-related genes in the liver of laying hens in the late laying period; Figure 6 This is a diagram showing the effect of fermented honeysuckle by-products of this invention on the expression of antioxidant-related genes in the ovaries of laying hens in the late laying period; Figure 7 This diagram illustrates the effect of fermented honeysuckle byproducts from this invention on the expression of intestinal barrier-related genes in laying hens during the late laying period. Detailed Implementation

[0026] 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.

[0027] 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. Every smaller range between any stated value or intermediate value within a stated range, and 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.

[0028] 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.

[0029] 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 apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] 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.

[0031] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods, reagents, instruments and materials used in the following embodiments are all conventional experimental methods, commercially available feed-grade reagents, conventional testing instruments and commercially available qualified raw materials in the art; the bacterial enzyme preparations involved are all commercially available feed-grade products that meet national standards.

[0032] Example 1: Preparation of feed co-fermented with fungi and enzymes from honeysuckle by-products 1.1 Experimental Materials and Reagents The by-products of honeysuckle are freshly harvested mature branches and leaves of honeysuckle, sourced from a standardized honeysuckle planting base in Xiushan County, Chongqing. The auxiliary material is commercially available feed-grade wheat bran, with moisture content, total mold count, and other indicators meeting the hygiene standards for feed raw materials. The compound enzyme is a uniform mixture of acidic protease, cellulase, and phytase in a mass ratio of 1:1:1, all of which are feed-grade enzyme preparations that meet national standards. The compound microorganisms are a uniform mixture of brewer's yeast, Bacillus subtilis, Lactobacillus plantarum, and Bifidobacterium in a mass ratio of (2–3):(2–3):(2–3):1, all of which are feed-grade live bacteria preparations that meet national standards.

[0033] 1.2 Preparation of bacterial enzyme fermentation agent Accurately weigh the compound enzyme and compound microorganisms at a mass ratio of (5–8):10, add them to sterile water and stir thoroughly to dissolve, thus preparing a homogeneous bacterial enzyme fermentation agent, which is then refrigerated at 4°C for later use.

[0034] 1.3 Experimental Grouping and Fermentation Preparation Six test groups were set up, corresponding to mixture group 1, mixture group 2, mixture group 3, mixture group 4, mixture group 5, and mixture group 6, respectively. The preparation methods of materials for each group are as follows: Mixed feed group 1: Freshly harvested mature branches and leaves of honeysuckle were cut into 2.0–5.0 cm pieces using a forage shredder, and then mixed thoroughly with wheat bran in a mass ratio of 8:2 to obtain mixed feed group 1. Mixture Group 2: Take the above Mixture 1 and spray the prepared bacterial enzyme fermentation agent at 4-8 wt% of the total mass of the mixture, stirring while spraying to ensure that the material is mixed evenly, to obtain Mixture 2; Feed Group 3: Freshly harvested mature branches and leaves of honeysuckle were cut into 2.0–5.0 cm using a forage shredder to obtain Feed Group 3; Mixture No. 4: Take the above-mentioned material No. 3 and spray the prepared bacterial enzyme fermentation agent at 4-8 wt% of the total mass of the material while spraying and stirring to ensure that the material is mixed evenly to obtain Mixture No. 4; Feed Group 5: Use commercially available, qualified feed-grade wheat bran as feed group 5; Mixture No. 6: Take the above-mentioned material No. 5 and spray the prepared bacterial enzyme fermentation agent at 4-8 wt% of the total mass of the material while spraying and stirring to ensure that the material is mixed evenly, thus obtaining Mixture No. 6.

[0035] Fermentation condition control: Adjust the substrate moisture content of each of the above groups 1-6 to 50-58 wt%, then pack them into food-grade anaerobic fermentation bags, compact and degas them, and then vacuum seal them. Place the sealed materials in a clean fermentation workshop for anaerobic solid-state fermentation. During the fermentation process, control the ambient temperature at 15-35℃, the fermentation time at 15-20 days, and control the pH of the materials at the end of the fermentation process to ≤4.5. After the fermentation is completed, the corresponding fermented materials for each group are obtained.

[0036] 1.4 Experimental Results and Analysis The nutrient composition of each fermentation batch is shown in Table 1.

[0037] Table 1. Results of nutrient composition determination of each mixture after fermentation.

[0038] Note: Different lowercase letters in the superscript of data in the same column indicate significant differences (P < 0.05), while the same letter in the superscript or no letter indicates no significant differences (P > 0.05), and the same applies below.

[0039] Table 1 shows that there were highly significant differences in the nutritional composition of the fermented materials among the groups (P<0.0001). Among them, the No. 2 mixture group (honeysuckle branches and leaves: wheat bran = 8:2 + synergistic fermentation with microorganisms and enzymes) had the best fermentation effect, with its crude protein content significantly higher than other groups, and more than 20% higher than the unfermented No. 1 mixture group. The contents of neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were significantly lower than other groups, indicating that the cellulose, hemicellulose, and lignin in honeysuckle by-products were efficiently degraded after synergistic fermentation with microorganisms and enzymes. Hemicellulose was effectively decomposed into carbohydrates that can be utilized by animals, greatly improving the feed value of the material. At the same time, the experimental results confirmed that the combined addition of wheat bran and microorganisms and enzymes has a significant synergistic effect, and the fermentation effect is significantly better than fermenting honeysuckle branches and leaves alone (No. 4 mixture group) or fermenting wheat bran alone (No. 6 mixture group).

[0040] Example 2: In vitro biomimetic digestion-in vitro fermentation experiment of honeysuckle by-product fermented feed for chickens 2.1 Test Materials Fermentation substrate: Fermented feed of honeysuckle by-products prepared from mixture group 2 in Example 1 was pulverized into ultrafine powder, passed through a 0.3 mm sieve, sealed, dried and stored for later use; Experimental animals: 21-day-old healthy AA broilers were fed an antibiotic-free basal diet throughout the entire process to prepare cecal digesta inoculum; Experimental reagents: Sterile phosphate buffer (pH 6.8) preheated to 39°C, which met the sterility requirements for anaerobic fermentation experiments.

[0041] 2.2 Experimental Design and Methods 2.2.1 Preparation of cecal inoculation solution In a sterile anaerobic chamber, 10 healthy 21-day-old broilers were randomly selected, euthanized, and immediately aseptically dissected to collect complete cecal chyme. The cecal chyme from the 10 chickens was mixed in equal amounts and diluted with preheated sterile phosphate buffer (pH 6.8) at a ratio of 1:5 (w / v). The mixture was filtered through four layers of sterile gauze to remove residue, and the cecal inoculation solution was prepared. The entire process was carried out under a strictly anaerobic environment and a constant temperature of 39°C. The solution was prepared and used immediately.

[0042] 2.2.2 In vitro fermentation test The control group and the experimental group were set up, with 6 replicates in each group. The specific grouping is as follows: Control group: Take 20 mL of the cecal inoculum prepared above and add it into a sterile anaerobic digestion tube without adding any fermentation substrate; Experimental group: Accurately weigh 1g of the fermentation substrate prepared above, add it into a sterile anaerobic digestion tube, then add 20mL of the cecal inoculation solution prepared above, and shake thoroughly to mix.

[0043] After sealing each digestion tube, immediately place it in a constant temperature incubator for anaerobic in vitro fermentation. Strictly control the fermentation temperature at 41℃ and the fermentation time at 24h. After fermentation, immediately place the digestion tube in an ice-water bath for 10min to completely stop the fermentation. Aseptically collect the fermentation broth and freeze it at -20℃ for later testing.

[0044] 2.3 Detection Indicators The contents of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid in the fermentation broth of each group were determined by gas chromatography, and the total short-chain fatty acid content was calculated. Three replicates were set up for each sample, and the average value of the results was taken.

[0045] 2.4 Experimental Results and Analysis The results of the determination of short-chain fatty acids in the fermentation broth of each group are shown in Table 2.

[0046] Table 2. Short-chain fatty acid yield of honeysuckle by-products after in vitro fermentation.

[0047] As shown in Table 2, compared with the control group, the contents of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid in the fermentation broth of the experimental group were significantly increased (P < 0.05), and the total short-chain fatty acid content increased by 83.59% compared with the control group, with the difference reaching a highly significant level (P < 0.0001). The above results confirm that the fermented feed of honeysuckle by-products prepared by this invention can be efficiently degraded and utilized by cecal microorganisms of broilers, and can significantly promote the production of key short-chain fatty acids such as acetic acid, propionic acid, and butyric acid. As the main energy source for intestinal epithelial cells, short-chain fatty acids have the core role of improving intestinal energy supply, enhancing intestinal barrier function, and inhibiting the proliferation of harmful bacteria in the intestine, verifying that the fermented product of this invention has excellent potential for intestinal health regulation.

[0048] Example 3: Application test of fermented honeysuckle by-products in laying hens during the late laying period. 3.1 Test Materials Fermentation feed used in the experiment: fermented feed made from honeysuckle by-products prepared from mixture group 2 in Example 1; Experimental animals: 450-day-old healthy Luqin No. 7 laying hens. Before the experiment, laying hens with similar egg production rate, weight and good health were selected. Test reagents and instruments: serum and ovarian reproductive hormone ELISA assay kit, antioxidant index assay kit, real-time fluorescence quantitative PCR related reagents; fully automated biochemical analyzer, high-speed refrigerated centrifuge, real-time fluorescence quantitative PCR instrument, eggshell strength tester, egg quality tester, optical microscope and image analysis system, gas chromatograph, high-throughput sequencing platform and other conventional instruments and equipment in this field.

[0049] 3.2 Experimental Design Ninety-six 450-day-old Luqin No. 7 laying hens with no significant differences in body weight and egg production rate were selected for the experiment. A single-factor completely randomized experimental design was adopted, and the hens were randomly divided into four treatment groups, with four replicates in each group and six hens in each replicate. The pre-feeding period was one week, and the trial period was eight weeks.

[0050] The feed treatment protocols for each group are as follows: Control group (CON): fed a corn-soybean meal basal diet with 5.00% mulberry leaf powder added, and no fermented honeysuckle by-products added; 1%FLH group: 1.00% fermented honeysuckle by-product was added to the basal diet to replace the mulberry leaf powder in the basal diet in an equal proportion, that is, the amount of mulberry leaf powder added to the diet was 4.00%; 3%FLH group: 3.00% fermented honeysuckle by-product was added to the basal diet to replace mulberry leaf powder in the basal diet in an equal proportion, that is, the amount of mulberry leaf powder added to the diet was 2.00%; 5%FLH group: 5.00% fermented honeysuckle by-product was added to the basal diet to replace the mulberry leaf powder in the basal diet in an equal proportion, that is, no mulberry leaf powder was added to the diet.

[0051] During the experiment, the main nutritional indicators such as metabolizable energy, crude protein, calcium, available phosphorus, and amino acids of each group of diets remained consistent. The composition and nutrient content of each group of diets are shown in Table 3.

[0052] Table 3. Diet composition and nutrient content (air-dried basis)

[0053] Note: 1) Dry matter, crude protein, and crude fat are measured values, while the rest are calculated values; 2) Per kilogram of feed contains: VA, 15,000 IU; VD3, 3,450 IU; VE, 22.5 IU; VK, 2.25 mg; VB1, 2.7 mg; VB2, 8.4 mg; VB6, 4.86 mg; VB12, 0.03 mg; niacin, 44.55 mg; folic acid, 1.47 mg; biotin, 0.18 mg; pantothenic acid, 16.56 mg; Cu, 8.5 mg; Fe, 102 mg; Zn, 72.76 mg; Mn, 97.34 mg; I, 0.48 mg; and Se, 0.3 mg.

[0054] 3.3 Detection Indicators and Methods 3.3.1 Egg production performance During the experiment, the number of eggs laid, total egg weight, number of dead hens, and daily feed consumption were accurately recorded daily in replicates. After the experiment, the average daily feed intake, average egg weight, average daily egg production, laying rate, and feed conversion ratio of each group of laying hens were calculated using the following formulas: Egg production rate (%) = Total number of eggs produced / (Number of experimental chickens × Number of experimental days) × 100% Average daily egg production (g / bird) d) = Total egg weight / (Number of experimental chickens × Number of experimental days) Feed conversion ratio = Total feed consumption during the trial period / Total egg weight during the trial period 3.3.2 Egg quality On the day the experiment ended, 15 fresh, undamaged, and undeformed eggs were randomly selected from each replicate, stored at 4℃, and the egg quality was tested within 24 hours. The measured indicators included: egg weight, eggshell strength, albumen height, Haugh unit, eggshell weight, yolk weight, and eggshell thickness. The eggshell ratio and yolk ratio were calculated.

[0055] 3.3.3 Sample Collection After the experiment, six healthy laying hens were randomly selected from each group. After fasting for 12 hours, blood was collected from the wing vein. Approximately 5 mL of blood sample was collected using a coagulation-promoting tube. After standing at room temperature for 30 minutes to allow for initial stratification, the sample was centrifuged at 3000 r / min for 10 minutes. The upper serum layer was carefully separated, aliquoted into sterile EP tubes, and stored at -20℃ for the determination of serum biochemical and reproductive hormone indicators.

[0056] After blood collection, the experimental chickens were slaughtered by exsanguination through the carotid artery, and the following tissue samples were aseptically collected: Abdominal fat and liver: Abdominal fat and liver were completely removed, and the livers were accurately weighed to calculate the abdominal fat percentage and liver index. Two tissue samples were taken from the same part of the liver, placed in sterile cryovials, flash-frozen in liquid nitrogen, and then transferred to an ultra-low temperature freezer at -80°C for storage. These samples were used to determine antioxidant indicators and the expression of inflammation-related genes. Intestinal tissue: The duodenum, jejunum, and ileum were separated, and three equal tissue samples were taken from each segment. One sample was fixed in 4% paraformaldehyde fixative for morphological observation of the intestinal tissue; the other two samples were placed in sterile cryovials, flash-frozen in liquid nitrogen, and stored at -80°C for the determination of expression of intestinal inflammation-related genes and intestinal barrier-related genes. At the same time, cecal contents were aseptically collected, placed in sterile cryovials, flash-frozen in liquid nitrogen, and stored at -80°C for the determination of cecal short-chain fatty acid content and analysis of microbial community structure. Reproductive system tissues: The infundibulum, dilatation, isthmus, and shell gland of the fallopian tube were separated, and one equal volume of tissue sample from each was fixed in 4% paraformaldehyde fixative for morphological observation of the fallopian tube tissues; the ovary was completely removed, and follicles at each stage were separated and classified and counted according to their diameter (>8mm, 5-8mm, 2-5mm); three equal volume tissue samples from the ovary were placed in sterile cryovials, flash-frozen in liquid nitrogen, and stored at -80℃ for the determination of reproductive hormones, antioxidant indicators, inflammation-related genes, and hormone receptor gene expression.

[0057] 3.3.4 Reproductive hormone measurement The levels of progesterone (PROG), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in serum and in ovarian tissue were measured using an enzyme-linked immunosorbent assay (ELISA) kit. The operation was strictly performed in accordance with the kit instructions, and three replicates were set up for each sample.

[0058] 3.3.5 Determination of antioxidant index Using appropriate commercially available kits, the total antioxidant capacity (T-AOC), catalase (CAT), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) activities, and malondialdehyde (MDA) content in liver and ovarian tissues were determined. The operation was strictly performed in accordance with the kit instructions, and three replicates were set up for each sample.

[0059] 3.3.6 Gene Expression Assay The relative mRNA expression levels of inflammation-related genes, antioxidant-related genes, ovarian hormone receptor genes, and intestinal barrier-related genes in liver, ovary, and intestinal tissues were determined using real-time quantitative PCR (qRT-PCR). β-actin was used as an internal reference gene, and the relative expression level of the target gene was calculated using the 2-ΔΔCt method. The primers used were chicken-derived gene-specific primers, which met the requirements for real-time quantitative PCR amplification.

[0060] 3.3.7 Observation of organizational morphology Intestinal and fallopian tube tissue samples fixed in 4% paraformaldehyde were dehydrated, cleared, impregnated with paraffin, and embedded to prepare 4μm thick paraffin sections. After HE staining, the tissue morphology was observed using an optical microscope. The villus height and crypt depth of the duodenum, jejunum, and ileum were measured using a professional image analysis system, and the villus-crypt ratio was calculated. The villus length of the infundibulum, dilatation, isthmus, and decapitated gland of the fallopian tube was measured. Ten complete and clear fields of view were selected from each section for measurement, and the average value was taken.

[0061] 3.3.8 Determination of short-chain fatty acids in the cecum The contents of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, and hexanoic acid in cecal contents were determined by gas chromatography. Three replicates were set up for each sample, and the average value of the results was taken.

[0062] 3.3.9 Analysis of the cecal microbiota structure High-throughput sequencing technology was used to sequence the V3-V4 regions of the 16S rRNA gene of microorganisms in the cecal contents, and the Alpha diversity index (Chao1, ACE, Shannon, Simpson) of the microbial community, as well as the relative abundance of the community at the phylum and genus levels, were analyzed.

[0063] 3.4 Experimental Results and Analysis 3.4.1 Effects of fermented honeysuckle byproducts on egg production performance of laying hens in the late laying period The statistical results of egg production performance for each group are shown in Table 4.

[0064] Table 4. Effects of fermented honeysuckle by-products on egg production performance of laying hens in the late laying period.

[0065] Table 4 shows that, compared with the control group, adding different proportions of fermented honeysuckle by-products to the diet had no significant effect on the feed conversion ratio and average egg weight of laying hens in the late laying period (P > 0.05). With the increase in the proportion of fermented honeysuckle by-products, the average daily feed intake of laying hens in the late laying period showed a trend of first decreasing and then increasing, with significant differences between groups (P < 0.05). The average daily egg production and egg production rate of the 5% FLH group were significantly higher than those of the other groups (P < 0.05), with the egg production rate increasing from 78.54% in the control group to 88.10%, and the average daily egg production increasing from 44.15 g / hen in the control group. d increased to 47.91g / each d. The above results indicate that adding 5% fermented honeysuckle by-product to the diet can significantly improve the egg production rate and daily egg production of laying hens in the late laying period, and effectively delay the decline in the production performance of laying hens in the late laying period.

[0066] 3.4.2 Effects of fermented honeysuckle byproducts on egg quality in the later stages of laying. The results of the egg quality determination for each group are shown in Table 5.

[0067] Table 5. Effects of fermented honeysuckle by-products on egg quality in the later stages of egg production.

[0068] Table 5 shows that adding different proportions of fermented honeysuckle by-products to the diet had no significant effect on egg weight, eggshell strength, albumen height, Haugh unit, eggshell weight, yolk weight, eggshell ratio, or yolk ratio (P > 0.05). Compared with the control group, the eggshell thickness of the 1% FLH group and the 3% FLH group showed no significant change (P > 0.05), while the eggshell thickness of the 5% FLH group was significantly reduced (P < 0.05). These results indicate that the fermented honeysuckle by-products of this invention significantly improve the egg production performance of laying hens without adversely affecting the core commercial quality indicators of eggs, thus ensuring the market value of the eggs.

[0069] 3.4.3 Effects of fermented honeysuckle byproducts on organ indices and follicle count in laying hens during the late laying period The results of liver index and abdominal fat percentage measurements for each group are shown in Table 6, and the statistical results of follicle count for each group are shown in Table 7.

[0070] Table 6. Effects of fermented honeysuckle by-products on liver index and abdominal fat percentage in laying hens during late laying period.

[0071] Table 7. Effects of fermented honeysuckle byproducts on follicle count in laying hens during late laying period.

[0072] Note: Dominant follicles are those with a diameter > 8 mm, small yellow follicles are those with a diameter of 5-8 mm, and large white follicles are those with a diameter of 2-5 mm.

[0073] As shown in Table 6, compared with the control group, the addition of fermented honeysuckle by-products to the diet had no significant effect on the slaughter weight, liver weight, and liver index of laying hens in the late laying period (P>0.05); the abdominal fat weight and abdominal fat percentage of the 1%FLH group and the 5%FLH group were significantly higher than those of the control group (P<0.05).

[0074] Table 7 shows that, compared with the control group, the number of dominant follicles and large white follicles in the 3% FLH group and the 5% FLH group were significantly increased (P < 0.05); the number of small yellow follicles in the 1% FLH group was significantly lower than that in the control group (P < 0.05). These results indicate that adding 3%-5% fermented honeysuckle by-products to the diet can significantly increase the number of dominant follicles and large white follicles in laying hens during the late laying period, effectively improving ovarian reserve function and providing a core physiological basis for the sustained improvement of egg production.

[0075] 3.4.4 Effects of fermented honeysuckle byproducts on oviduct morphology in late-laying hens The results of the measurement of villus length in different parts of the fallopian tube in each group are shown in Table 8.

[0076] Table 8. Effects of fermented honeysuckle by-products on oviduct morphology in late-laying hens.

[0077] As shown in Table 8, compared with the control group, adding different proportions of fermented honeysuckle by-products to the diet had no significant effect on the villi length of the oviduct infundibulum, enlarged part, isthmus, and shell gland of laying hens in the late laying period (P>0.05). This indicates that the fermented honeysuckle by-products of the present invention will not have an adverse effect on the normal tissue structure and physiological function of the oviduct of laying hens.

[0078] 3.4.5 Effects of fermented honeysuckle byproducts on reproductive hormone levels in laying hens during the late laying period The results of the determination of reproductive hormone content in serum and ovarian tissue of each group are shown in Table 9.

[0079] Table 9. Effects of fermented honeysuckle by-products on serum and ovarian reproductive hormones in laying hens during late laying period.

[0080] Table 9 shows that, compared with the control group, the addition of fermented honeysuckle by-products to the diet significantly increased the levels of PROG, FSH, and LH in the serum and ovarian tissue of laying hens (P < 0.05), and the levels showed a dose-dependent relationship with increasing proportion of fermented honeysuckle by-products. The levels of all reproductive hormones in the 5% FLH group were significantly higher than in the other groups (P < 0.05). These results indicate that the fermented honeysuckle by-products of this invention can significantly increase the reproductive hormone levels in laying hens during the late laying period, effectively improve the reproductive endocrine status of laying hens, delay ovarian aging, and thus prolong the effective laying cycle of laying hens.

[0081] 3.4.6 Effects of fermented honeysuckle byproducts on the expression of ovarian hormone receptor genes in late-laying hens The results of the determination of the relative expression levels of ovarian hormone receptor gene mRNA in each group are as follows: Figure 1 As shown in the results, compared with the control group, the addition of 3% and 5% fermented honeysuckle by-products to the diet significantly increased the expression level of PROGR mRNA in the ovary (P < 0.05); the addition of 1% fermented honeysuckle by-products significantly increased the expression level of AR mRNA in the ovary (P < 0.05); and the addition of 1% and 5% fermented honeysuckle by-products showed a trend of increasing the expression level of ESRα mRNA in the ovary (P = 0.057). These results confirm that fermented honeysuckle by-products can enhance the response of laying hens to reproductive hormones by upregulating the expression of ovarian hormone receptor genes, further improving ovarian function and delaying the ovarian aging process.

[0082] 3.4.7 Effects of fermented honeysuckle byproducts on the expression of inflammation-related genes in laying hens during the late laying period The results of the determination of the relative expression levels of inflammation-related gene mRNAs in the liver, ovary, and small intestine tissues of each group are as follows: Figure 2 , Figure 3 , Figure 4 As shown.

[0083] The results are analyzed as follows: Liver tissue: Compared with the control group, the addition of fermented honeysuckle by-products to the diet significantly reduced the mRNA expression of IL-1β in the liver (P<0.05); the 1%FLH group and the 5%FLH group significantly reduced the mRNA expression of TNF-α in the liver (P<0.05).

[0084] Ovarian tissue: Compared with the control group, the addition of fermented honeysuckle by-products to the diet significantly reduced the mRNA expression levels of IL-1β and TNF-α in the ovaries (P < 0.05).

[0085] Small intestinal tissue: Compared with the control group, the 1%FLH group and the 5%FLH group significantly reduced the mRNA expression level of IL1-β in the duodenum (P<0.05), and the 1%FLH group significantly reduced the mRNA expression level of IL-6 in the duodenum (P<0.05); the addition of fermented honeysuckle by-products to the diet significantly reduced the mRNA expression level of IL-1β in the jejunum (P<0.05), and the expression level was negatively correlated with the amount added. The 3%FLH group and the 5%FLH group significantly reduced the mRNA expression level of IL-6 in the jejunum (P<0.05).

[0086] The above results indicate that the fermented honeysuckle by-product of the present invention can significantly downregulate the gene expression of pro-inflammatory factors in the liver, ovary, and intestinal tissues of laying hens in the late laying period, effectively alleviate the chronic inflammatory state of the body, and significantly enhance the body's anti-inflammatory ability.

[0087] 3.4.8 Effects of fermented honeysuckle byproducts on the antioxidant capacity of laying hens in the late laying period The results of antioxidant marker measurements in liver and ovarian tissues of each group are shown in Table 10. The results of relative expression levels of antioxidant-related gene mRNAs in liver and ovarian tissues of each group are shown in Table 10. Figure 5 , Figure 6 As shown.

[0088] Table 10. Effects of fermented honeysuckle by-products on antioxidant indices of liver and ovary in laying hens during late laying period.

[0089] As shown in Table 10, compared with the control group, the addition of different proportions of fermented honeysuckle by-products to the diet had no significant effect on the activities of T-AOC, CAT, T-SOD, GSH-Px and MDA content in the liver tissue of laying hens in the late laying period (P>0.05); the GSH-Px activity in the ovarian tissue of the 3%FLH group and the 5%FLH group was significantly higher than that in the control group (P<0.05), and showed an increasing trend with the increase of the addition ratio.

[0090] The results of liver antioxidant-related gene expression showed that, compared with the control group, the addition of fermented honeysuckle by-products had no significant effect on the mRNA expression levels of SOD1, SOD2, CAT, and KEAP1 in the liver (P>0.05), but significantly increased the mRNA expression level of GPX1 in the liver (P<0.05); the 3%FLH group and the 5%FLH group significantly increased the mRNA expression level of Nrf2 in the liver (P<0.05); the 1%FLH group significantly increased the mRNA expression level of HO-1 (P<0.05); and the 1%FLH group and the 5%FLH group significantly increased the mRNA expression level of NQO1 (P<0.05).

[0091] The results of ovarian antioxidant gene expression showed that, compared with the control group, the addition of fermented honeysuckle by-products had no significant effect on the mRNA expression levels of SOD1 and NQO1 in the ovary (P>0.05), but significantly increased the mRNA expression level of GPX1 in the ovary (P<0.05). The CAT mRNA expression level in the 1%FLH group was significantly higher than that in other groups (P<0.05), the Nrf2 mRNA expression level in the 3%FLH group was significantly higher than that in other groups (P<0.05), and the HO-1 mRNA expression level in the 5%FLH group was significantly higher than that in other groups (P<0.05). The KEAP1 mRNA expression level in the 1%FLH group and the 3%FLH group was significantly higher than that in the control group (P<0.05).

[0092] The above results indicate that the fermented honeysuckle by-product of the present invention can effectively enhance the antioxidant capacity of laying hens in the late laying period by upregulating the expression of antioxidant-related genes in the body, increasing the activity of antioxidant enzymes in ovarian tissue, alleviating oxidative stress damage to the ovary and the body, and delaying ovarian aging.

[0093] 3.4.9 Effects of fermented honeysuckle byproducts on intestinal morphology and barrier-related gene expression in late-laying hens The results of the small intestinal tissue morphology measurements for each group are shown in Table 11. The results of the relative expression levels of intestinal barrier-related gene mRNAs for each group are shown in Table 11. Figure 7 As shown.

[0094] Table 11 Effects of fermented honeysuckle byproducts on intestinal morphology of laying hens in the late laying period.

[0095] As shown in Table 11, compared with the control group, the addition of different levels of fermented honeysuckle by-products to the diet had no significant effect on the villus height, crypt depth and villus-crypt ratio of the duodenum, jejunum and ileum of laying hens in the late laying period (P>0.05), indicating that the fermented honeysuckle by-products of the present invention will not have an adverse effect on the normal tissue structure of the intestinal tract of laying hens.

[0096] The expression results of intestinal barrier-related genes showed that, compared with the control group, the 1%FLH group significantly increased the expression level of ZO-1 mRNA in the duodenum (P<0.05), the 3%FLH group and the 5%FLH group significantly decreased the expression level of OCLN mRNA in the duodenum (P<0.05), and the 3%FLH group significantly increased the expression level of CLDN1 mRNA in the duodenum (P<0.05). In the jejunum, the expression level of ZO-1 mRNA in the 1%FLH group was significantly higher than that in the other three groups (P<0.05). In the ileum, the expression level of ZO-1 mRNA in the 1%FLH group and the 5%FLH group was significantly higher than that in the control group (P<0.05), and the expression level of CLDN1 mRNA in the ileum of each experimental group was significantly higher than that in the control group (P<0.05).

[0097] The above results confirm that the fermented honeysuckle by-product of the present invention can effectively enhance the intestinal barrier function of laying hens in the late laying period and maintain intestinal health homeostasis by upregulating the expression of genes related to tight junctions in the gut.

[0098] 3.4.10 Effects of fermented honeysuckle byproducts on the content of short-chain fatty acids in the cecum of laying hens in the late laying period The results of the determination of short-chain fatty acid content in the cecal contents of each group are shown in Table 12.

[0099] Table 12 Effects of fermented honeysuckle byproducts on short-chain fatty acids in the cecal umbilicus of laying hens in the late laying period.

[0100] Table 12 shows that, compared with the control group, the addition of fermented honeysuckle by-products to the diet significantly increased the contents of acetic acid, propionic acid, butyric acid, and valeric acid in the cecal contents of laying hens in the late laying period (P < 0.05), while the contents of isobutyric acid, isovaleric acid, and hexanoic acid did not change significantly (P > 0.05). These results indicate that the fermented honeysuckle by-products of this invention can significantly promote the production of beneficial short-chain fatty acids in the cecum of laying hens, providing sufficient energy for intestinal epithelial cells, further maintaining intestinal barrier function, and regulating the intestinal microecological balance.

[0101] 3.4.11 Effects of fermented honeysuckle byproducts on the cecal microbiota structure of laying hens in the late laying period The results of the Alpha diversity index of the cecal microbiota in each group are shown in Table 13, and the results of the relative abundance of microbiota at the phylum and genus levels are shown in Tables 14 and 15, respectively.

[0102] Table 13. Effects of fermented honeysuckle by-products on Alpha diversity of cecal microbiota in late-laying laying hens.

[0103] Table 14. Effects of fermented honeysuckle by-products on the abundance of cecal microbiota in laying hens during the late laying period.

[0104] Table 15. Effects of fermented honeysuckle byproducts on the abundance of cecal microbiota in laying hens during the late laying period.

[0105] As shown in Table 13, compared with the control group, the Chao1 index, ACE index, Shannon index and Simpson index of the 5% FLH group were significantly increased (P<0.05), indicating that adding 5% fermented honeysuckle by-product to the diet can significantly improve the diversity and richness of the intestinal microbial community of laying hens in the late laying period, which is conducive to building a healthy and stable intestinal micro-ecosystem.

[0106] The results of phylum-level bacterial abundance analysis showed that, compared with the control group, the 1%FLH group and the 5%FLH group significantly reduced the relative abundance of Actinobacteria (P<0.05); the 5%FLH group significantly increased the relative abundance of Dethiobacterium and Algebraic Bacteria (P<0.05).

[0107] The genus-level abundance results showed that, compared with the control group, the 1%FLH group and the 5%FLH group significantly reduced the relative abundance of Bifidobacterium (P < 0.05); the 1%FLH group and the 3%FLH group significantly increased the relative abundance of UCG-001 (Prevotellaceae) (P < 0.05); the 5%FLH group significantly increased the relative abundance of Desulfovibrio (P < 0.05) and significantly decreased the relative abundance of Caproiciproducens (P < 0.05); and the 3%FLH group significantly increased the relative abundance of Micrococcus (Rare) (P < 0.05).

[0108] The above results confirm that the fermented honeysuckle by-product of the present invention can effectively regulate the intestinal microbial community structure of laying hens in the late laying period, optimize the community composition, enhance community diversity, promote the colonization of beneficial bacteria, regulate the abundance of harmful bacteria, and thus maintain intestinal health homeostasis, laying the foundation for the health and production performance of laying hens.

[0109] 3.5 Experiment Summary The results of this series of animal experiments show that adding the fermented honeysuckle by-product prepared in this invention to the diets of laying hens in the late laying period can significantly improve the egg production rate and daily egg output, improve ovarian function and reproductive endocrine status, enhance the body's antioxidant and anti-inflammatory capabilities, improve intestinal barrier function, optimize the cecal microbiota, effectively delay the decline in the production performance of laying hens in the late laying period, prolong the laying cycle, and significantly improve the overall breeding benefits. Based on the comprehensive test indicators and production performance data, the optimal addition level of the fermented honeysuckle by-product in the diets of laying hens in the late laying period is 5%.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing fermented feed from honeysuckle by-products, characterized in that, Includes the following steps: (1) Raw material pretreatment: After harvesting the mature branches and leaves of honeysuckle by-products, crush them and add wheat bran as an auxiliary material to mix them evenly to obtain the fermentation substrate; (2) Addition of bacterial enzymes: Spray the bacterial enzyme fermentation agent into the fermentation substrate and stir evenly; the bacterial enzyme fermentation agent is prepared by dissolving a compound enzyme and a compound microorganism in water at a mass ratio of (5–8):10; the compound enzyme is a mixture of acidic protease, cellulase and phytase in a mass ratio of 1:1:1; the compound microorganism is composed of brewer's yeast, Bacillus subtilis, Lactobacillus plantarum and Bifidobacterium in a mass ratio of (2–3):(2–3):(2–3):

1. (3) Solid fermentation: The substrate with added bacterial enzyme fermentation agent is subjected to solid sealed fermentation. The fermentation temperature is controlled at 15–35℃, the fermentation time is 15–20 days, and the pH of the material at the end of fermentation is ≤4.5, thus obtaining fermented feed of honeysuckle by-product.

2. The preparation method according to claim 1, characterized in that, The amount of the bacterial enzyme fermentation agent added is 4–8 wt% of the total mass of the fermentation substrate.

3. The preparation method according to claim 1, characterized in that, The mass ratio of honeysuckle by-products to wheat bran is (70–85):(15–30).

4. The preparation method according to claim 1, characterized in that, Before solid-state fermentation, the moisture content of the fermentation substrate is adjusted to 50–58 wt%.

5. The preparation method according to claim 1, characterized in that, The solid-state sealed fermentation adopts an anaerobic solid-state fermentation method using a plastic film or fermentation bag compacted and sealed; the particle size of the mature branches and leaves of honeysuckle by-product after crushing is 2.0–5.0 cm.

6. A fermented feed made from honeysuckle by-products, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 5.

7. The application of the fermented honeysuckle by-product feed according to claim 6 in the preparation of special feed for laying hens in the later stage of egg production.

8. A special feed for laying hens in the late laying period, characterized in that, By weight percentage, it comprises 85–92% basal diet, 5–10% fermented honeysuckle by-product feed as described in claim 6, and 3–5% premix.

9. The special feed for laying hens in the late laying period according to claim 8, characterized in that, The premix contains compound vitamins, compound minerals, amino acids, and zeolite powder.

10. The special feed for laying hens in the late laying period according to claim 8, characterized in that, The fermented honeysuckle by-product feed is pulverized twice to 40-60 mesh before use, and the special feed is fed as a powder feed.

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