Chinese herbal medicine leavening agent for improving egg laying performance of hens in later egg laying period as well as preparation method and application of Chinese herbal medicine leavening agent

Abstract

The invention provides a Chinese herbal medicine leavening agent for improving the egg laying performance of hens in the later egg laying period as well as a preparation method and application of the Chinese herbal medicine leavening agent. The Chinese herbal medicine leavening agent is obtained by fermenting compound Chinese herbal medicines including motherwort, angelica sinensis, ligusticum wallichii, peach kernels, astragalus membranaceus and baked ginger with compound probiotics, namely bacillus subtilis, saccharomyces cerevisiae and lactobacillus plantarum. The compound probiotics are adopted for solid-state fermentation of the compound Chinese herbal medicine, strain compatibility is reasonable, the process is simple and efficient, synergistic fermentation is achieved, and release of active ingredients of the Chinese herbal medicine can be effectively promoted; the Chinese herbal medicine leavening agent is added into hen feed in the later egg laying period in proportion, the fat metabolism of hens can be adjusted, the content of intestinal digestive enzymes and reproductive hormone can be increased, the ovarian function of laying hens can be improved, the egg laying performance and the egg quality can be improved, and the Chinese herbal medicine leavening agent has good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine fermented feed technology, specifically relating to a traditional Chinese medicine fermentation agent, preparation method and application for improving the egg production performance of hens in the later stage of egg production. Background Technology

[0002] As laying hens enter the late laying period (average weekly laying rate drops below 80%, around 60-72 weeks of age), their reproductive system ages, their intestinal absorption of nutrients decreases, and their immunity weakens. This leads to a decline in laying performance and egg quality, severely hindering the healthy development of the laying hen industry and reducing the economic benefits of farming. Extending the peak laying period and improving egg quality have become crucial for achieving high-quality development in the laying hen industry.

[0003] Traditional Chinese medicine (TCM) possesses pharmacological characteristics such as being purely natural, residue-free, and free from drug resistance. It can regulate intestinal flora, improve feed utilization, and enhance animal immune function, leading to its widespread application in animal production. However, due to the complex composition, low content, poor palatability, unstable efficacy, and large dosage requirements of TCM, direct addition to feed can affect its efficacy, hindering the large-scale promotion and application of TCM feed additives. Fermenting TCM with probiotics is an effective measure to achieve efficient utilization of TCM. Probiotic fermentation of TCM not only enhances efficacy, promotes the effective release of active ingredients, degrades macromolecules to generate more easily absorbed small-molecule active ingredients and toxic byproducts, but also promotes the growth and reproduction of probiotics, improves intestinal flora and enhances immunity. This facilitates the synergistic effect of probiotics and TCM pharmacological actions, thereby promoting animal growth and development, shortening the feeding cycle, improving feed conversion ratio, enhancing efficacy, and reducing feeding costs. Its application prospects in livestock and poultry farming are broad.

[0004] Currently, traditional Chinese medicines commonly used in late-laying hens generally focus on warming and tonifying the spleen and kidneys, while also promoting blood circulation, clearing heat and detoxifying, and regulating antioxidant functions to maintain egg production performance, improve egg quality, and prolong the laying cycle. Commonly used probiotic strains in the fermentation of traditional Chinese medicines include lactic acid bacteria, Bacillus, yeast, and molds, with fermentation methods including single-strain fermentation and compound-strain fermentation. However, problems in actual production and application include: arbitrary selection and compatibility of probiotic strains; lack of scientific basis for fermentation product testing indicators; inaccurate production process parameters; reliance on experience for fermentation; long fermentation times; unstable fermentation quality; susceptibility to contamination; and unstable or no application effects. For example, patent publication number CN117562190A discloses a fermentation product obtained by fermenting a fermentation substrate containing compound traditional Chinese medicine in the presence of compound microbial agents and compound enzymes; the compound traditional Chinese medicine is Astragalus membranaceus, hawthorn, Poria cocos, Angelica sinensis, Leonurus japonicus, Alpinia oxyphylla, and Taraxacum mongolicum; the compound microbial agents are Lactobacillus plantarum, Saccharomyces cerevisiae, and Aspergillus niger; the compound enzymes are phytase, cellulase, xylanase, and pectinase; anaerobic fermentation is carried out at 37°C for 7 days, but the traditional Chinese medicine needs to be dried, soaked, and sterilized to obtain a liquid culture medium; patent publication number CN116650569A discloses a compound traditional Chinese medicine containing Angelica sinensis, Astragalus membranaceus, Dipsacus asper, Poria cocos, Cornus officinalis, Citrus reticulata, and Glycyrrhiza uralensis fermented with eucalyptus oil and Bacillus subtilis, but this patent requires adding the traditional Chinese medicine to wheat bran, corn flour, soybean meal, and maifanite, and fermenting at 30°C for 7 days.

[0005] To enhance the efficacy of compound probiotic fermentation of compound traditional Chinese medicine, fully leverage the synergistic benefits of probiotics and traditional Chinese medicine, optimize the fermentation process, simplify operating procedures, reduce the risk of contamination during fermentation, and, more importantly, enhance ovarian function in late-laying hens to improve egg production performance and egg quality, this invention employs an anaerobic gas-generating bag for compound solid-state fermentation of three probiotics. Parameters significantly affecting the viable count, flavonoid, total phenol, and polysaccharide content of the fermentation products, such as the compound ratio, feed-to-water ratio, fermentation temperature, and fermentation time, were optimized and screened. This provides a technology for compound probiotic solid-state fermentation of compound traditional Chinese medicine, which has been validated in late-laying hens, achieving the effects of promoting ovarian function, increasing egg production rate, and improving egg quality. Summary of the Invention

[0006] The purpose of this invention is to address the practical problems and production needs related to decreased egg production performance and weakened immunity in laying hens during the later stages of egg production, as well as the fermentation of compound herbal medicines with probiotics, by developing a herbal fermentation agent to improve egg production rate in laying hens. Another purpose of this invention is to provide a method for preparing the herbal fermentation agent. Yet another purpose of this invention is to provide applications of the aforementioned herbal fermentation agent.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A traditional Chinese medicine fermentation agent for improving the egg production performance of hens in the later stages of egg production, wherein the traditional Chinese medicine fermentation agent is obtained by fermenting traditional Chinese medicine with compound probiotics, wherein the traditional Chinese medicine is composed of the following substances in parts by weight: 40-44 parts of Leonurus japonicus, 34-38 parts of Angelica sinensis, 9-13 parts of Ligusticum chuanxiong, 9-13 parts of Prunus persica, 4-6 parts of Astragalus membranaceus, and 4-6 parts of Zingiber officinale; wherein the compound probiotics are composed of the following substances in parts by weight: 2-4 parts of Bacillus subtilis, 0.8-1.2 parts of Saccharomyces cerevisiae, and 0.8-1.2 parts of Lactobacillus plantarum.

[0008] Furthermore, the Lactobacillus plantarum is Lactobacillus plantarum JLP035, with the accession number CGMCC NO.32318.

[0009] Furthermore, egg production performance refers to improving egg production rate, freshness, and the rate of qualified eggs.

[0010] The herbal fermentation agent provided by this invention can also significantly reduce the rate of substandard eggs and the feed conversion ratio.

[0011] The herbal fermentation agent described above is preferably composed of the following herbal ingredients in parts by weight: 42 parts of Leonurus japonicus, 36 parts of Angelica sinensis, 11 parts of Ligusticum chuanxiong, 11 parts of Prunus persica, 5 parts of Astragalus membranaceus, and 5 parts of Zingiber officinale (processed); the compound probiotic is composed of the following herbal ingredients in parts by weight: 3 parts of Bacillus subtilis, 1 part of Saccharomyces cerevisiae, and 1 part of Lactobacillus plantarum JLP035.

[0012] In the above-mentioned herbal fermentation agent, preferably, the viable bacterial content of Bacillus subtilis is 4.0 × 10⁻⁶. 9 ~6.0×10 9 The CFU / g and viable count of brewer's yeast were 4.0 × 10⁻⁶. 9 ~6.0×10 9 The viable count of *Lactobacillus plantarum* JLP035 was 4.0 × 10⁻⁶ CFU / g. 9 ~6.0×10 9 CFU / g.

[0013] This invention also provides a method for preparing a traditional Chinese medicine fermentation agent, which uses Bacillus subtilis, Saccharomyces cerevisiae and Lactobacillus plantarum as fermentation strains, and uses compound traditional Chinese medicines such as Leonurus japonicus, Angelica sinensis, Ligusticum chuanxiong, Prunus persica, Astragalus membranaceus and Zingiber officinale as substrates for solid-state fermentation. After fermentation, the product is a probiotic fermented traditional Chinese medicine preparation.

[0014] The preparation method of the Chinese herbal medicine fermentation agent as described above may specifically include the following steps: (1) Place the compound Chinese herbal medicine components, including motherwort, angelica, chuanxiong, peach kernel, astragalus and dried ginger, into a grinder and grind them into powder, and pass them through a 50-mesh sieve for later use; (2) Weigh out the following compound Chinese herbal medicines by weight: 40-44 parts of Leonurus japonicus, 34-38 parts of Angelica sinensis, 9-13 parts of Ligusticum chuanxiong, 9-13 parts of Prunus persica, 4-6 parts of Astragalus membranaceus and 4-6 parts of Zingiber officinale, and compound probiotics: 3 parts of Bacillus subtilis, 1 part of Saccharomyces cerevisiae and 1 part of Lactobacillus plantarum, for later use; (3) According to the weight ratio described above, put the compound probiotics into 200-240 parts of water, dissolve them completely, then add the compound Chinese medicine and stir evenly; (4) Pack the well-mixed compound probiotics and compound Chinese medicine into a fermentation bag with a breathing valve and seal it; (5) Place the fermentation bag in a constant temperature, sealed and clean culture device and culture at 35~37℃ for 35~40h to obtain the compound Chinese medicine probiotic fermentation preparation.

[0015] In the preparation method described above, preferably, in step (2), the viable bacterial content of Bacillus subtilis is 4.0 × 10⁻⁶. 9 ~6.0×10 9 The CFU / g and viable count of brewer's yeast were 4.0 × 10⁻⁶. 9 ~6.0×10 9 The CFU / g concentration of *Lactobacillus plantarum* was 4.0 × 10⁻⁶. 9 ~6.0×10 9 CFU / g.

[0016] Furthermore, the preservation number of the Lactobacillus plantarum is CGMCC NO.32318.

[0017] In the preparation method described above, preferably, in step (4), the isothermal incubation is performed at 35°C for 38.8 hours.

[0018] The viable cell content in the fermentation preparation was calculated using the colony plate count method: 5g of the fermented compound Chinese medicine was added to 45ml of sterile water and mixed thoroughly. A tenfold serial dilution method was used, selecting three appropriate gradients, which were then spread onto LB agar plates, YPD agar plates, and MRS agar plates, with three replicates for each gradient. After incubation at 37 ℃ for 48 h, the total colony count was determined to obtain the number of Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum, respectively. The flavonoid, total phenol, and polysaccharide contents in the fermentation preparation were detected using kit methods: the flavonoid detection kit, total phenol detection kit, and polysaccharide detection kit (all purchased from Nanjing Jiancheng Bioengineering Institute) were used for detection, respectively.

[0019] This invention also provides the application of the herbal fermentation agent obtained by the above preparation method as an additive in the preparation of feed to improve the egg production performance of laying hens. The herbal fermentation agent can be directly added to the feed of laying hens in the later stages of egg production in a specified proportion.

[0020] As described above, preferably, the herbal fermentation agent is applied to laying hen feed, more preferably to the feed of laying hens in the later stages of egg production.

[0021] Furthermore, the application involves adding the herbal fermentation agent to the basal diet, wherein the amount of the herbal fermentation agent added is 0.5-1% of the basal diet.

[0022] The application of a compound traditional Chinese medicine and its fermented preparation in improving eggshell strength, wherein the compound traditional Chinese medicine includes 40-44 parts of Leonurus japonicus, 34-38 parts of Angelica sinensis, 9-13 parts of Ligusticum chuanxiong, 9-13 parts of Prunus persica, 4-6 parts of Astragalus membranaceus, and 4-6 parts of Zingiber officinale (processed). The fermented preparation is obtained by fermenting the compound traditional Chinese medicine with a compound probiotic consisting of 2-4 parts of Bacillus subtilis, 0.8-1.2 parts of Saccharomyces cerevisiae, and 0.8-1.2 parts of Lactobacillus plantarum.

[0023] This invention, based on the physiological characteristics of hens in the late laying period and the basic principles of traditional Chinese medicine formulation, uses a rational combination of herbs including Leonurus japonicus, Angelica sinensis, Ligusticum chuanxiong, Prunus persica, Astragalus membranaceus, and Zingiber officinale (processed). This combination can promote follicle development and increase egg production by activating ovarian function. Specifically, Leonurus japonicus clears heat and detoxifies, invigorates blood and regulates menstruation, promoting oviduct repair and ovarian development; Angelica sinensis nourishes blood, invigorates blood circulation, regulates menstruation and relieves pain, replenishing qi and blood, clearing the uterus, and improving ovarian function; Ligusticum chuanxiong invigorates blood circulation, dispels wind and relieves pain, improving blood stasis and promoting ovulation and reproductive hormone secretion; Prunus persica invigorates blood circulation, removes blood stasis, clears blood stasis, and improves ovarian and oviduct stasis; Astragalus membranaceus invigorates qi and raises yang, strengthens the spleen and benefits the lungs, enhancing the body's immune function; and Zingiber officinale (processed) warms the meridians, stops bleeding, and relieves pain, improving ovarian function and reducing blood-spotted eggs. The above prescription is reasonable and can effectively regulate the blood and qi of laying hens, improve ovarian function, and has a good therapeutic and preventive effect on the decline in egg production performance in the later stage of egg production.

[0024] The herbal fermentation agent provided by this invention selects Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum as the compound strains for fermenting traditional Chinese medicines, based on the functional and growth characteristics of the microorganisms. Bacillus subtilis exhibits strong biological stability and environmental resistance, and can secrete digestive enzymes such as proteases, amylases, and cellulases, which helps release and transform the effective components of traditional Chinese medicines, improving feed utilization. Saccharomyces cerevisiae can utilize the fiber, sugars, and proteins in medicinal materials to produce secondary metabolites such as organic acids and antibiotics, increasing the active ingredients of the traditional Chinese medicines. Lactobacillus plantarum possesses probiotic characteristics such as lactic acid production and improved immunity, and can also decompose macromolecules, increasing the release of active ingredients from traditional Chinese medicines. By rationally optimizing the ratio of the three probiotic strains and the fermentation process, the synergistic effect of probiotics and traditional Chinese medicines can be fully utilized. The fermentation product not only contains a large number of probiotics and their metabolites but also a large number of active ingredients from traditional Chinese medicines. The number of Bacillus subtilis in the obtained herbal fermentation agent can reach (2.47±0.03)×10⁻⁶. 7CFU / g, Saccharomyces cerevisiae count (3.47±0.01)×10 8 CFU / g, Lactobacillus plantarum count (2.85±0.02)×10 8 CFU / g, flavonoid content 4.26±0.04mg / g, total phenol content 17.23±0.06mg / g, polysaccharide content 56.84±0.22mg / g.

[0025] The beneficial effects of this invention are as follows: This invention provides a traditional Chinese medicine fermentation agent to improve the egg production performance of hens in the late laying stage. When added to hens in the late laying stage in a certain proportion, it can significantly increase the activity of digestive enzymes in the intestines and the content of reproductive hormones in the serum, significantly reduce the rate of substandard eggs and the feed conversion ratio, and improve the egg production rate, egg quality and freshness of hens in the late laying stage.

[0026] This invention provides a method for preparing a traditional Chinese medicine fermentation agent to improve the egg production performance of hens in the later stages of egg production. The method is simple to operate, has a short fermentation time, stable fermentation quality, and stable application effect. When added to laying hen feed as an additive, it can significantly increase eggshell strength, egg quality, and freshness. Attached Figure Description

[0027] Figure 1 The effect of the compound ratio on the number of three viable bacteria, flavonoids, total phenols and polysaccharides; Figure 2 The effect of the feed-to-water ratio on viable bacteria count, flavonoids, total phenols, and polysaccharides; Figure 3 The effect of fermentation temperature on the number of three viable bacteria, flavonoids, total phenols and polysaccharides; Figure 4 The effect of fermentation time on the number of three viable bacteria, flavonoids, total phenols and polysaccharides; Figure 5 The content of the compound herbal preparations under the fermentation conditions of Examples 2 and 3 is given. Detailed Implementation

[0028] This invention addresses the physiological characteristics and nutritional needs of laying hens in the late laying period (60 weeks of age to culling), providing a traditional Chinese medicine fermentation agent and its preparation method that can improve the laying performance and egg quality of laying hens in this stage. The fermentation agent utilizes a rational combination of compound probiotics and compound traditional Chinese medicines, employing an efficient fermentation process that leverages the probiotic advantages of Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum. The fermentation product contains a large number of probiotics and active ingredients from traditional Chinese medicines, achieving a synergistic effect between the two for better application results. The obtained traditional Chinese medicine fermentation agent has a Bacillus subtilis viable count of (2.47±0.03)×10⁻⁶. 8 CFU / g, viable count of Saccharomyces cerevisiae (3.47±0.01)×108 CFU / g, viable count of Lactobacillus plantarum (2.85±0.02)×10 8 The concentrations of CFU / g, flavonoids, total phenols, and polysaccharides were 4.26±0.04 mg / g and 17.23±0.06 mg / g, respectively. The addition of fermented herbal preparations significantly increased the activity of digestive enzymes in the intestines of laying hens and the levels of reproductive hormones in their serum, reduced the rate of defective eggs and the feed conversion ratio, and improved the egg production rate, egg quality, and freshness in the later stages of egg production.

[0029] It should be noted that the *Lactiplantibacillus plantarum* JLP035 used in this invention is a microbial strain permitted for use in feed production under Announcement No. 2045 (2013). It was isolated from the intestines of healthy laying hens by the Beijing Academy of Agricultural and Forestry Sciences and possesses excellent probiotic characteristics such as high lactic acid production, resistance to gastric acid and bile salts, strong adhesion, and good antibacterial properties. The strain has been identified and preserved by the China General Microbiological Culture Collection Center (CGMCC) as *Lactiplantibacillus plantarum* JLP035, with accession number CGMCC NO.32318 and preservation date of October 23, 2024. The *Bacillus subtilis* and *Saccharomyces cerevisiae* used can be commercially available products.

[0030] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the present invention without departing from its spirit and essence are within the scope of the present invention.

[0031] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents or raw materials used in the embodiments may be commercially available products.

[0032] Example 1: Optimization of process parameters for solid-state fermentation of compound probiotic traditional Chinese medicine The live bacteria content of Bacillus subtilis (available from Angel Yeast Co., Ltd.), Saccharomyces cerevisiae (available from Angel Yeast Co., Ltd.), and Lactobacillus plantarum JLP035 were adjusted to 5.0 × 10⁻⁶ cells / ml, respectively. 9A compound traditional Chinese medicine (TCM) powder (11.71% moisture content) was inoculated with CFU / g. A single-factor study was conducted to investigate the effects of fermentation parameters, including the inoculum ratio, substrate-to-water ratio, fermentation temperature, and fermentation time, on the viable cell count, flavonoid content, total phenolic content, and polysaccharide content of the fermented TCM. Only a few examples are presented below, showing the results of viable cell count (CFU / g), flavonoid content (mg / g), total phenolic content (mg / g), and polysaccharide content (mg / g) of the fermented TCM under different inoculum ratios, substrate-to-water ratios, fermentation temperatures, and fermentation times. The fermentation substrate consisted of TCM powder in the following proportions by weight: 42 parts Leonurus japonicus, 36 parts Angelica sinensis, 11 parts Ligusticum chuanxiong, 11 parts Prunus persica, 5 parts Astragalus membranaceus, and 5 parts Zingiber officinale (processed). The inoculum ratio (inoculation amount) was determined by inoculating 110 portions of compound traditional Chinese medicine with Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum in a weight ratio of 1:1:1, 2:1:1, 1:2:1, 1:1:2, 3:1:1, 1:3:1, and 1:1:3, respectively. The material-to-water ratio was set at 1:1.5, and the fermentation temperature was 37 ℃. The viable cell count (CFU / g), flavonoid content (mg / g), total phenolic content (mg / g), and polysaccharide content (mg / g) of the fermented herbs were measured after 24 h of fermentation. The results are shown in […]. Figure 1 The material-to-water ratio refers to the ratio of traditional Chinese medicine to water. Based on the optimal compound ratio of Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum, a fermentation temperature of 37℃ and a fermentation time of 24 hours were established. The effects of different material-to-water ratios (1:1.5, 1:1.75, 1:2, 1:2.5, 1:3) on the viable cell count (CFU / g), flavonoid content (mg / g), total phenolic content (mg / g), and polysaccharide content (mg / g) of the fermented traditional Chinese medicine were measured. The results are shown in […]. Figure 2 Based on the optimal compound ratio and material-to-water ratio, and with a fermentation time of 24 hours, the viable cell count (CFU / g), flavonoid content (mg / g), total phenolic content (mg / g), and polysaccharide content (mg / g) of the fermented Chinese herbal medicines were measured at different fermentation temperatures (31℃, 33℃, 35℃, 37℃, 39℃). The results are shown in [Figure number missing]. Figure 3 Based on the optimal compound ratio, material-to-water ratio, and fermentation temperature, the viable cell count (CFU / g), flavonoid content (mg / g), total phenolic content (mg / g), and polysaccharide content (mg / g) of the fermented Chinese herbal medicines were measured at different fermentation times (24h, 36h, 48h, 60h, 72h). The results are shown in the table below. Figure 4The viable cell content in the fermented preparation was calculated using the colony plate count method: 5g of the fermented compound Chinese medicine was added to 45ml of sterile water and mixed thoroughly. A tenfold serial dilution method was used, selecting three appropriate gradients, which were then spread onto LB agar plates, YPD agar plates, and MRS agar plates, with three replicates for each gradient. The plates were incubated at 37 ℃ for 48 h, and the total colony count was recorded to obtain the number of Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum, respectively. The flavonoid, total phenol, and polysaccharide contents in the fermented compound Chinese medicine were detected using kits: the flavonoid, total phenol, and polysaccharide detection kits (all purchased from Nanjing Jiancheng Bioengineering Institute) were used for detection according to their instructions.

[0033] Due to the diversity of the three probiotic strains (belonging to aerobic, facultative anaerobic, and anaerobic bacteria respectively), the fermentation efficiency during mixed fermentation is affected by factors such as inconsistent growth temperature, inconsistent time of metabolite production, and the influence of metabolites on growth rate. This can easily lead to low quality of fermented Chinese herbal medicines and even side effects. To fully utilize the synergistic effect and probiotic properties of the three probiotics in the mixed fermentation of Chinese herbal medicines, it is necessary to scientifically screen and optimize the fermentation process parameters for the mixed fermentation of Chinese herbal medicines with the three probiotics. Figure 1-4 It is evident that under different compound ratios (inoculation amount), substrate-to-water ratios, fermentation temperatures, and fermentation times, the effects of the three compound probiotics fermenting compound traditional Chinese medicine on the viable cell count (CFU / g), flavonoid content (mg / g), total phenolic content (mg / g), and polysaccharide content (mg / g) of the fermented traditional Chinese medicine varied. To obtain the optimal fermentation process parameters, principal component analysis was used based on single-factor screening to analyze the effects of the substrate-to-water ratio, fermentation temperature, and fermentation time on six indicators (viable cell count, flavonoid content, total phenolic content, and polysaccharide content) of the fermented traditional Chinese medicine. The optimal fermentation parameters were obtained as follows: a compound ratio of Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae at a weight ratio of 3:1:1 (g:g:g), a fermentation time of 38.8 h, a fermentation temperature of 35.3℃, and a substrate-to-water ratio of 1:2.05.

[0034] Example 2: Preparation of Compound Probiotic Solid-State Fermentation Compound Traditional Chinese Medicine Preparation An example of a method for preparing a compound probiotic solid-state fermentation compound herbal preparation according to the present invention includes the following steps: (1) Prepare the raw materials for fermenting compound Chinese herbal medicine by weight: Bacillus subtilis (live bacteria content of 5.0 × 10⁻⁶) 9 Three portions of (CFU / g) and brewer's yeast (live count 5.0 × 10⁻⁶) were added. 9 One serving of Lactobacillus plantarum JLP035 (cFU / g) with a live count of 5.0 × 10⁻⁶ CFU / g. 91 part of CFU / g), 220 parts of tap water, and 110 parts of compound Chinese herbal medicine (including a mixture of 42 parts of Leonurus japonicus, 36 parts of Angelica sinensis, 11 parts of Ligusticum chuanxiong, 11 parts of Prunus persica, 5 parts of Astragalus membranaceus and 5 parts of Zingiber officinale; each component of the Chinese herbal medicine was ground separately in a grinder and passed through a 50-mesh sieve before use).

[0035] (2) Dissolve Bacillus subtilis, Saccharomyces cerevisiae and Lactobacillus plantarum in tap water, then add the compound Chinese herbal medicine and stir. (3) Pack the above-mentioned well-mixed materials into a fermentation bag with a breathing valve and seal it; (4) Place the fermentation bag in a constant temperature, sealed and clean culture device and culture at 35.3℃ for 38.8h to obtain a compound probiotic solid fermentation compound Chinese herbal medicine preparation.

[0036] Example 3: Preparation of Compound Probiotic Solid-State Fermentation Compound Traditional Chinese Medicine Preparation Another example of the preparation method of the compound lactic acid bacteria solid-state fermented weaned piglet feed according to the present invention includes the following steps: (1) Prepare the raw materials for fermenting compound Chinese herbal medicine by weight: Bacillus subtilis (live bacteria content of 5.0 × 10⁻⁶) 9 Two samples of *Lactobacillus plantarum* (CFU / g) and *Lactobacillus plantarum* JLP035 (live count 5.0 × 10⁻⁶) were tested. 9 0.8 parts (CFU / g) and 5.0 × 10⁸ parts (live bacteria content of brewer's yeast). 9 0.8 parts (CFU / g), 200 parts tap water, and 100 parts compound Chinese herbal medicine (prepared according to the ratio of 40 parts motherwort, 34 parts angelica, 9 parts chuanxiong, 9 parts peach kernel, 4 parts astragalus and 4 parts dried ginger; each component of the Chinese herbal medicine should be ground separately in a grinder and passed through a 50-mesh sieve in advance for later use).

[0037] (2) Dissolve Bacillus subtilis, Lactobacillus plantarum and Saccharomyces cerevisiae in tap water, then add the compound Chinese herbal medicine and stir. (3) Pack the above-mentioned well-mixed materials into a fermentation bag with a breathing valve and seal it; (4) Place the fermentation bag in a constant temperature, sealed and clean culture device and culture at 34℃ for 35h to obtain a compound probiotic solid fermentation compound Chinese herbal medicine preparation.

[0038] Example 4: Changes in indicators of compound traditional Chinese medicine before and after fermentation Following the preparation methods in Examples 2 and 3, fresh samples of the compound probiotic fermented compound herbal medicine were obtained, corresponding to fermented feed 1 and fermented feed 2. Three parallel samples were prepared, and the viable bacteria count, flavonoid, total phenol, and polysaccharide content in the fresh samples were determined according to the method in Example 1. The results are shown below. Figure 5 .

[0039] Depend on Figure 5 It can be seen that, according to the preparation methods of Examples 2 and 3, the number of Bacillus subtilis in the fermented compound Chinese herbal medicine (fresh sample) was (2.47±0.03)×10⁻⁶. 7 CFU / g and (1.48±0.03)×10 7 The CFU / g and the number of Saccharomyces cerevisiae were (3.47±0.01)×10⁻⁶. 8 CFU / g and (2.44±0.04)×10 8 CFU / g and Lactobacillus plantarum count were (2.85±0.02)×10 8 CFU / g and (2.01±0.02)×10 8 The CFU / g and flavonoid contents were 4.26±0.04 mg / g and 3.60±0.02 mg / g, respectively; the total phenol contents were 17.23±0.06 mg / g and 12.72±0.04 mg / g, respectively; and the polysaccharide contents were 56.84±0.22 mg / g and 43.83±0.02 mg / g, respectively. The results show that fermentation with compound probiotics not only increases the probiotic content in the traditional Chinese medicine but also promotes the effective release of its active ingredients. This facilitates the synergistic effect of probiotics and traditional Chinese medicine, promoting the absorption of active ingredients in the intestines of hens in the late laying period, improving intestinal microecological balance, enhancing immunity, and ultimately improving egg production performance in hens during the late laying period.

[0040] The results also show that the quality of fermented traditional Chinese medicine (TCM) prepared with Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae is greatly affected by different compound ratios and fermentation parameters. The fermented TCM prepared according to the optimal ratio and fermentation parameters (Example 2) exhibited superior results in terms of the viable count of the three probiotics and active ingredients compared to fermented TCM prepared with other ratios and fermentation parameters (Example 3). This demonstrates that the fermentation strain and fermentation process are key factors influencing the quality and efficacy of fermented TCM prepared with compound probiotics. By scientifically and rationally combining the proportion (amount) of compound probiotics and the components of the TCM, and by screening and optimizing the optimal fermentation process parameters such as the material-to-water ratio, fermentation temperature, and fermentation time, a shorter fermentation time (less than 40 hours), simple and efficient operation can be achieved, thereby improving the quality and efficacy of fermented TCM and obtaining the best fermentation results.

[0041] Example 5: Application of Fermented Traditional Chinese Medicine in Laying Hens in the Late Laying Stage 1. Experimental Design Eight hundred healthy 60-week-old Dawu Jinfeng laying hens with similar weight and egg production rate were selected and randomly divided into four groups: control group, experimental group 1, experimental group 2, and experimental group 3. Each group had four replicates, with 50 hens per replicate. The control group was supplemented with only a basal diet. Experimental group 1 was supplemented with a basal diet plus 1% of a compound traditional Chinese medicine (42 parts of Leonurus japonicus, 36 parts of Angelica sinensis, 11 parts of Ligusticum chuanxiong, 11 parts of Prunus persica, 5 parts of Astragalus membranaceus, and 5 parts of Zingiber officinale). Experimental group 2 was supplemented with a basal diet plus 0.5% of the basal diet weight of the fermented compound traditional Chinese medicine (prepared according to Example 2). Experimental group 3 was supplemented with a basal diet plus 1% of the basal diet weight of the fermented compound traditional Chinese medicine (prepared according to Example 2). The basal diet was formulated according to the "Chicken Feeding Standard" (NY / T 33-2004). The experiment adopted a three-tiered cage rearing system with free access to feed and water. The chickens were fed according to the above groups for 56 days. Eggs were collected at 15:30 every day, and the flocking status was observed.

[0042] Table 1. Composition and nutrient levels of basal diet (air-dried basal) % Raw material composition content Nutritional level content corn 61 Metabolizable energy (MJ / kg) 11.25 bran 1.8 crude protein 16.1 soybean meal 24.5 calcium 3.9 stone powder 9 Available phosphorus 0.56 Soybean oil 0.7 Phytase 0.38 premix 3 Lysine 0.8 total 100 Methionine 0.37 Note: The premix provides the following per kilogram of diet: Vitamin A 12,000 IU, Vitamin B1 4 mg, Vitamin B2 12 mg, Vitamin B6 8 mg, Vitamin B12 0.05 mg, Vitamin D3 5,000 IU, Vitamin E 60 mg, Vitamin K 5 mg, Biotin 0.40 mg, Pantothenic Acid 12 mg, Folic Acid 1.80 mg, Nicotinamide 40 mg, Cu 9 mg, Zn 70 mg, Fe 80 mg, Se 0.20 mg, Mn 100 mg, and I 0.50 mg.

[0043] 2. Measurement Indicators Using replicates as a unit, the feed intake was recorded weekly, and the remaining feed was weighed at the end of each week to calculate the average daily feed intake. Using replicates as a unit, the number of experimental chickens, the number of eggs laid, the number of broken eggs, the number of soft-shelled eggs, the number of rough-shelled eggs, and the egg weight were recorded daily to calculate the average egg weight, laying rate, and defective egg rate (broken eggs + soft-shelled eggs + rough-shelled eggs). The calculation formulas are: Average egg weight (g) = Total egg weight laid per replicate per day / Number of eggs laid per replicate per day; Laying rate (%) = Number of eggs laid per replicate per day / Number of chickens per replicate per day × 100%; Defective egg rate (%) = Number of defective eggs / Total number of eggs laid × 100%; Average daily feed intake (g / d) = Total weekly feed consumption / 7 / Number of chickens; Feed conversion ratio = Total weekly feed consumption / Total weekly egg weight. Once a week, at the end of the experiment, 10 eggs were randomly selected from each replicate, and the eggshell strength was measured using an eggshell strength tester. The main operating procedure was as follows: place the egg with the blunt end facing up and the sharp end facing down on the eggshell strength tester, move the probe downwards, and the value when the eggshell shows the first crack is the eggshell strength value. Egg shape index and shell thickness were measured using vernier calipers. Eggshell thickness was measured at the blunt end, equator, and acute end of the egg, and the average of these measurements was taken as the shell thickness. The longitudinal and transverse diameters of the egg were measured using vernier calipers, and the egg shape index was calculated as longitudinal diameter / transverse diameter. The eggshell ratio (%) was calculated as eggshell weight / egg weight × 100%. Albumin height, yolk color, and Haugh units were measured using an automated egg quality analyzer. Five chickens were randomly selected for each replicate, and the digestive enzyme content of the duodenal contents was measured. Two chickens were randomly selected for each replicate, and blood was collected via the subwing vein method. Serum levels of total cholesterol, triglycerides, high-density lipoprotein, low-density lipoprotein, creatine kinase, and alkaline phosphatase were measured using an automated biochemical analyzer. Serum reproductive hormone levels were measured using an ELISA kit. Data were analyzed using one-way ANOVA with SPSS 25.0 software. Results are expressed as mean ± standard deviation.

[0044] 3. Results and Analysis 3.1 The results of the effect of compound probiotic fermented compound traditional Chinese medicine preparation on the egg production performance of laying hens are shown in Table 2.

[0045] Table 2. Effects of compound probiotic fermented traditional Chinese medicine preparations on egg production performance of laying hens. project control group Experiment 1 group Experimental Group 2 Experiment 3 groups Egg production rate (%) <![CDATA[79.92±1.56 b ]]> <![CDATA[81.52±2.38 b ]]> 85.08±.80ᵃ <![CDATA[81.94±1.35 ab ]]> Non-compliant egg rate (%) <![CDATA[3.82±10.29 a ]]> <![CDATA[2.59±0.22 ab ]]> <![CDATA[1.35±0.18 b ]]> <![CDATA[1.32±0.18 b ]]> Average daily feed intake (g / d) 108.90±0.95 109.25±2.06 110.55±0.40 108.57±0.40 Feed-to-egg ratio (F / G) 2.26±0.04ᵃ <![CDATA[2.22±0.05 a ]]> 2.15±0.02ᵇ <![CDATA[2.21±0.04 a ]]> Note: Data in the same row with no letter or containing the same letter above the header indicates that the difference is not significant. P >0.05), different lowercase letters indicate significant differences ( P <0.05). The same applies below.

[0046] As shown in Table 2, compared with the control group, the egg production rate of experimental group 2 increased by 6.46% ( P <0.05%, the rate of substandard eggs decreased by 64.66% ( P<0.05, the feed conversion ratio decreased by 4.87% ( P <0.05); Compared with group 1, the egg production rate of group 2 increased by 4.37% ( P <0.05, feed conversion ratio decreased by 3.15% ( P <0.05; compared with group 3, the feed conversion ratio of group 2 decreased by 2.71% ( P <0.05; compared with the control group, the rate of substandard eggs in experimental group 3 decreased by 53.93% ( P <0.05). It can be seen that as laying hens enter the later stages of egg production, the egg production rate decreases and the rate of defective eggs increases. Adding a certain dose of probiotic fermented compound traditional Chinese medicine preparation to the feed of laying hens can significantly improve the egg production rate, significantly reduce the rate of defective eggs and the feed conversion ratio, and improve the egg production performance of laying hens in the later stages of egg production.

[0047] 3.2 The results of the effect of compound probiotic fermented compound traditional Chinese medicine preparation on egg quality are shown in Table 3.

[0048] Table 3. Effects of compound probiotic fermented traditional Chinese medicine preparations on egg quality. As shown in Table 3, compared with the control group, the eggshell strength of experimental groups 1, 2, and 3 increased by 3.79%, 2.85%, and 7.92%, respectively. P <0.05); Compared with the control group, experimental group 1, and experimental group 2, the Hardy units in experimental group 3 increased by 2.34%, 1.95%, and 1.81%, respectively (<0.05); P <0.05). This indicates that adding a certain dose of probiotic-fermented compound traditional Chinese medicine preparation to the feed of laying hens in the later stages of egg production can improve eggshell strength and Haugh units, thus improving egg quality in the later stages of egg production. The significant increase in Haugh units in group 3 of the experiment indicates a significant improvement in egg freshness, resulting in a substantial improvement in protein quality.

[0049] 3.3 The effects of compound probiotic fermented compound traditional Chinese medicine preparations on digestive enzymes in the intestinal contents of laying hens are shown in Table 4.

[0050] Table 4. Effects of compound probiotic fermented traditional Chinese medicine preparations on digestive enzymes in the intestinal contents of laying hens (U) project control group Experiment 1 group Experimental Group 2 Experiment 3 groups Amylase (U) <![CDATA[360.41±12.84 b ]]> <![CDATA[472.33±11.29 a ]]> <![CDATA[518.68±18.42 a ]]> <![CDATA[488.86±16.46 a <!-- 8 -->]]> Trypsin (U) <![CDATA[4146.53±63.13 b ]]> <![CDATA[4485.63±65.22 a ]]> <![CDATA[4504.60±53.57 a ]]> <![CDATA[4574.29±46.81 a ]]> Lipase (U) 1.68±0.07 1.52±0.09 1.40±0.02 1.48±0.14 As shown in Table 4, compared with the control group, the amylase activities of experimental groups 1, 2, and 3 increased by 31.05%, 43.91%, and 35.64%, respectively. P <0.05%, and trypsin activity increased by 8.18%, 8.64%, and 10.32%, respectively. P<0.05). Increased amylase and trypsin activity can improve the degradation rate of carbohydrates and proteins in feed by laying hens, which is beneficial to the digestion and absorption of nutrients.

[0051] 3.4 The effects of compound probiotic fermented compound traditional Chinese medicine preparations on serum biochemistry and reproductive hormones in laying hens are shown in Table 5.

[0052] Table 5. Effects of compound probiotic fermented traditional Chinese medicine preparations on serum biochemistry and reproductive hormones in laying hens. project control group Experiment 1 group Experimental Group 2 Experiment 3 groups Total cholesterol (mmol / L) 3.13±0.18 3.33±0.29 3.27±0.18 2.69±0.20 Triglycerides (mmol / L) <![CDATA[14.16±0.45 a ]]> <![CDATA[13.63±1.02 a ]]> <![CDATA[12.29±1.49 a ]]> <![CDATA[9.41±0.85 b ]]> High-density lipoprotein (mmol / L) 0.93±0.05 0.95±0.07 1.10±0.12 0.98±0.06 Low-density lipoprotein (mmol / L) <![CDATA[1.23±0.06 a ]]> <![CDATA[1.12±0.05 a ]]> <![CDATA[1.03±0.07 a ]]> <![CDATA[0.91±0.05 b ]]> Creatine kinase (U / L) 4828.16±155.49 4851.52±174.25 4382.96±178.38 5072.59±169.41 Alkaline phosphatase (U / L) 302.48±28.39 272.88±26.33 275.78±27.72 193.17±12.56 Luteinizing hormone (IU / L) <![CDATA[7.69±1.00 b ]]> <![CDATA[8.59±0.66 ab ]]> <![CDATA[9.99±0.62 a ]]> <![CDATA[10.57±0.76 a ]]> Follicle-stimulating hormone (IU / L) 18.95±1.93 22.45±1.61 22.70±1.40 21.29±1.38 Progesterone (nmol / L) <![CDATA[9.87±0.90 b ]]> <![CDATA[12.33±0.67 a ]]> <![CDATA[11.52±0.76 a ]]> <![CDATA[13.34±0.70 a ]]> Estradiol (pmol / L) <![CDATA[37.02±2.05 b ]]> <![CDATA[44.67±2.98 a ]]> <![CDATA[44.07±3.42 a ]]> <![CDATA[46.23±1.62 a ]]> As shown in Table 5, compared with the control group, the triglyceride levels in experimental groups 1, 2, and 3 decreased by 3.74%, 13.21%, and 33.55%, respectively (P<0.05); the low-density lipoprotein levels decreased by 8.94%, 16.26%, and 26.02%, respectively (P<0.05); the progesterone levels increased by 24.92%, 16.72%, and 35.16%, respectively (P<0.05); and the estradiol levels increased by 20.66%, 19.04%, and 24.88%, respectively (P<0.05). Compared with the control group, the luteinizing hormone levels in experimental groups 2 and 3 increased by 29.91% and 37.45%, respectively (P<0.05). The results show that adding a certain dose of probiotic-fermented compound traditional Chinese medicine preparation to the feed of laying hens in the later stages of egg production can improve lipid metabolism and increase the levels of reproductive hormones, thereby promoting follicle growth, development, and maturation, and improving egg production performance. The significant reduction in low-density lipoprotein (LDL) in laying hens indicates that it can significantly improve the health of the hens themselves, reduce metabolic and cardiovascular risks, indirectly optimize egg quality, and improve the lipid composition of egg yolks. Furthermore, it can regulate the cholesterol content of egg yolks: although cholesterol in egg yolks is a natural component, lowering LDL can reduce the efficiency of cholesterol transport from the liver to the yolk, potentially leading to a slight decrease in yolk cholesterol content (this needs to be assessed in conjunction with comprehensive feed nutrition), which is more in line with consumer demand for low-cholesterol eggs. Improving the fatty acid ratio in egg yolks: Lower LDL levels are often associated with increased intake of unsaturated fatty acids (such as Omega-3 and Omega-6) in laying hen feed. These fatty acids enter the yolk through lipid metabolism, increasing the proportion of unsaturated fatty acids and decreasing the proportion of saturated fatty acids in the yolk, thus enhancing the nutritional value of eggs (e.g., easier absorption by the human body and reduced saturated fat intake). Maintaining normal yolk shape and stability: Improved liver health associated with lower LDL levels ensures the normal assembly of lipids in the yolk, reducing quality problems such as broken yolks and soft yolk texture caused by abnormal liver metabolism, and extending the fresh storage period of eggs.

Claims

1. A traditional Chinese medicine fermentation agent for improving the egg production performance of hens in the later stages of egg production, characterized in that, The herbal fermentation agent is obtained by fermenting herbal medicines with compound probiotics. The herbal medicines are composed of the following components in parts by weight: 40-44 parts of Leonurus japonicus, 34-38 parts of Angelica sinensis, 9-13 parts of Ligusticum chuanxiong, 9-13 parts of Prunus persica, 4-6 parts of Astragalus membranaceus, and 4-6 parts of Zingiber officinale (processed). The compound probiotics are composed of the following components in parts by weight: 2-4 parts of Bacillus subtilis, 0.8-1.2 parts of Saccharomyces cerevisiae, and 0.8-1.2 parts of Lactobacillus plantarum.

2. The herbal fermentation agent according to claim 1, characterized in that, Egg production performance refers to improving egg production rate, freshness, and the rate of qualified eggs.

3. The herbal fermentation agent according to claim 1, characterized in that, The herbal medicines are composed of the following components in parts by weight: 42 parts of Leonurus japonicus, 36 parts of Angelica sinensis, 11 parts of Ligusticum chuanxiong, 11 parts of Prunus persica, 5 parts of Astragalus membranaceus, and 5 parts of Zingiber officinale (processed); the compound probiotics are composed of the following components in parts by weight: 3 parts of Bacillus subtilis, 1 part of Saccharomyces cerevisiae, and 1 part of Lactobacillus plantarum.

4. A method for preparing a fermentation agent for traditional Chinese medicine, characterized in that, The preparation method uses Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum as fermentation strains, and solid-state fermentation is carried out using compound Chinese herbal medicines such as Leonurus japonicus, Angelica sinensis, Ligusticum chuanxiong, Prunus persica, Astragalus membranaceus, and Zingiber officinale as substrates. After fermentation, the product is a probiotic fermented Chinese herbal medicine preparation.

5. The preparation method according to claim 4, characterized in that, Includes the following steps: (1) Place the compound Chinese herbal medicine components, including motherwort, angelica, chuanxiong, peach kernel, astragalus and dried ginger, into a grinder and grind them into powder, and pass them through a 50-mesh sieve for later use; (2) Weigh out the following compound Chinese herbal medicines by weight: 40-44 parts of Leonurus japonicus, 34-38 parts of Angelica sinensis, 9-13 parts of Ligusticum chuanxiong, 9-13 parts of Prunus persica, 4-6 parts of Astragalus membranaceus and 4-6 parts of Zingiber officinale, and compound probiotics: 3 parts of Bacillus subtilis, 1 part of Saccharomyces cerevisiae and 1 part of Lactobacillus plantarum, for later use; (3) According to the weight ratio described above, put the compound probiotics into 200-240 parts of water, dissolve them completely, then add the compound Chinese medicine and stir evenly; (4) Pack the well-mixed compound probiotics and compound Chinese medicine into a fermentation bag with a breathing valve and seal it; (5) Place the fermentation bag in a constant temperature, sealed and clean culture device and culture at 34~36℃ for 35~40h to obtain the compound Chinese medicine probiotic fermentation preparation.

6. The preparation method according to claim 4, characterized in that, In step (1), the viable count of Bacillus subtilis is 4.0 × 10⁻⁶. 9 ~6.0×10 9 The CFU / g and viable count of brewer's yeast were 4.0 × 10⁻⁶. 9 ~6.0×10 9 The CFU / g concentration of *Lactobacillus plantarum* was 4.0 × 10⁻⁶. 9 ~6.0×10 9 CFU / g.

7. The preparation method according to claim 5, characterized in that, In step (4), the isothermal incubation is performed at 35°C for 38.8 hours.

8. The application of the herbal fermentation agent according to any one of claims 1-3 or the preparation method according to any one of claims 4-7 as an additive in the preparation of feed to improve the egg production performance of laying hens.

9. The application according to claim 8, characterized in that, The amount of the herbal fermentation agent added is 0.5-1% of the basal diet.

10. The application of a compound traditional Chinese medicine and its fermentation preparation in improving eggshell strength, characterized in that, The compound Chinese herbal medicine includes 40-44 parts of Leonurus japonicus, 34-38 parts of Angelica sinensis, 9-13 parts of Ligusticum chuanxiong, 9-13 parts of Prunus persica, 4-6 parts of Astragalus membranaceus, and 4-6 parts of Zingiber officinale (processed). The fermented preparation is obtained by fermenting the compound Chinese herbal medicine with compound probiotics consisting of 2-4 parts of Bacillus subtilis, 0.8-1.2 parts of Saccharomyces cerevisiae, and 0.8-1.2 parts of Lactobacillus plantarum.

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