Chrysanthemum stem and leaf fermented feed additive with function of regulating immunity and application of chrysanthemum stem and leaf fermented feed additive

Abstract

The invention relates to the technical field of feed additives, in particular to a chrysanthemum stem and leaf fermented feed additive with an immunity regulating effect and application thereof. According to the method, the chrysanthemum stems and leaves are fermented by lactobacillus plantarum, so that the contents of total flavonoids and total phenolic acids are remarkably increased, and an optimal fermentation system is established by optimizing the fermentation process conditions of the chrysanthemum stems and leaves; through DSS mouse model and related index determination, the fermented chrysanthemum stems and leaves can effectively improve mouse colitis symptoms and effectively improve immunity, and the effect is obviously better than that of unfermented chrysanthemum stems and leaves. The feed additive can be applied to the livestock and poultry industry as a novel feed additive, promotes application of medicinal and edible medicinal materials in the field of feed, promotes development of green animal husbandry, and has remarkable economic and ecological benefits.

Description

Technical Field

[0001] The invention relates to the technical field of feed additives, and in particular to a chrysanthemum stem and leaf fermented feed additive with immune regulating function and application thereof. Background Art

[0002] Traditional Chinese medicine (TCM) fermentation technology can generate a variety of bioactive substances through microbial metabolism, enhancing efficacy while reducing toxicity. This technology is widely used in medicinal material processing and functional feed development. Studies have shown that fermented TCM can significantly improve the growth performance and health of livestock and poultry by regulating the animal's intestinal microbiome, enhancing immune responses, and promoting nutrient absorption. Compared to antibiotics, fermented TCM poses no risk of drug resistance, exhibits fewer toxic side effects, and utilizes TCM byproducts, reducing resource waste and environmental pollution, thus meeting the needs of green farming. Furthermore, modern TCM processing technologies, such as microbial enzymatic hydrolysis and ultrafine grinding, can further release active ingredients, enhancing nutrient absorption and feed conversion.

[0003] Chrysanthemum morifolium Ramat., a traditional medicinal plant, is rich in active ingredients such as flavonoids, polysaccharides, and volatile oils. It exhibits a variety of biological activities, including antioxidant, immune-enhancing, antibacterial, antiviral, anti-inflammatory, and antihypertensive properties. Its capitulum is used as a medicinal herb, widely used in traditional Chinese medicine preparations, health teas, and food additives. However, its stems and leaves are underutilized and often discarded as waste, resulting in a significant waste of resources. Fermentation of chrysanthemum stems and leaves effectively promotes the dissolution of active ingredients such as polysaccharides and flavonoids, increasing their bioavailability. The fermented products not only enhance the immune function and disease resistance of livestock and poultry, but also possess low toxicity and resistance to drug resistance, meeting the requirements of environmentally friendly farming.

[0004] In summary, the development of new fermented feed additives using chrysanthemum stems and leaves as raw materials can not only realize the resource utilization of agricultural by-products, but also provide a sustainable solution for improving animal health and production performance. Summary of the Invention

[0005] Purpose of the Invention

[0006] The invention aims to provide a chrysanthemum stem and leaf fermented feed additive capable of improving immunity.

[0007] Technical Solution

[0008] The present invention provides a feed additive, which is prepared by fermentation of lactic acid bacteria;

[0009] The lactic acid bacteria are selected from Lactobacillus plantarum

[0010] The present invention aims to provide the feed additive for regulating immune function

[0011] The present invention provides a fermentation broth prepared from a fermented chrysanthemum stem and leaf feed additive having an immune-regulating effect, which is prepared by the following preparation method:

[0012] (1) Grinding and sieving chrysanthemum stems and leaves to obtain fermentation material

[0013] (2) Add water to the fermentation material to obtain a fermentation medium

[0014] (3) Inoculating plant lactobacillus liquid into the fermentation medium for shaking fermentation to obtain chrysanthemum stem and leaf fermented feed additive

[0015] As a preferred specific implementation method of the present invention, the sieving in step (1) is through a 20-80 mesh sieve. Preferably, the sieving in step (1) is through a 60 mesh sieve.

[0016] As a preferred specific implementation method of the present invention, the volume ratio of the fermentation material to water in step (2) is 1:(6.25-100), preferably 1:25.

[0017] As a preferred specific implementation method of the present invention, in step (3), 5% to 35% of Lactobacillus plantarum fermentation liquid is added to the fermentation medium.

[0018] Preferably it is 5%.

[0019] In step (3), the initial pH value of the fermentation medium after inoculating Lactobacillus plantarum is controlled to be 5.0-9.0. Preferably, the initial pH value of the fermentation medium after inoculating Lactobacillus plantarum is controlled to be 6.8-7.2.

[0020] As a preferred embodiment of the present invention, the fermentation time in step (3) is 1-5 days. Preferably, the fermentation time in step (3) is 2 days.

[0021] As a preferred embodiment of the present invention, the volume ratio of the fermentation material to water in step (2) is 1:50, the fermentation time in step (3) is 1 day, and the inoculation amount is 5%.

[0022] By establishing a DSS mouse model for 14 days and measuring relevant indicators, oral administration of the feed additive of the present invention can significantly improve the colitis condition of mice and enhance the immune function, and the oral administration effect is significantly better than that of the control group. Therefore, the feed additive provided by the present invention can enhance the immune function.

[0023] Beneficial effects

[0024] The present invention ferments chrysanthemum stems and leaves with plant lactobacillus, significantly improving the content of total flavonoids and total phenolic acids, and establishing an optimal fermentation system by optimizing the fermentation process conditions of chrysanthemum stems and leaves; through the determination of DSS mouse model and related indicators, the fermented chrysanthemum stems and leaves can effectively improve the symptoms of colitis in mice and effectively improve the immune function, and its effect is significantly better than that of unfermented chrysanthemum stems and leaves. Since zoonotic pathogens (such as Campylobacter jejuni and Yersinia) may cause acute enteritis, long-term infection or immune abnormality may develop into chronic colitis; or antibiotics are often used to promote animal growth or prevent infection, which may cause antibiotic residues to enter the human body through the food chain, destroy the balance of intestinal flora, and increase the risk of colitis. Therefore, the present invention can be used as a novel feed additive in livestock and poultry farming, promote the application of medicinal and edible medicinal materials in the field of feed, promote the development of green animal husbandry, and have significant economic and ecological benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The effect of different material-liquid ratios on the content of total phenols and total flavonoids in fermented feed of chrysanthemum stems and leaves;

[0026] Figure 2 To study the effects of different inoculation amounts on the contents of total phenols and total flavonoids in fermented feeds of chrysanthemum stems and leaves;

[0027] Figure 3 To study the effects of different fermentation times on the contents of total phenols and total flavonoids in fermented feeds of chrysanthemum stems and leaves;

[0028] Figure 4 is the relative body weight change of mice;

[0029] Figure 5 is the change in the length of the mouse colon;

[0030] Figure 6 CD11b+Ly6C+ monocytes (A), CD11b+Ly6G+ neutrophils (B) and CD11b+F4 / 80+

[0031] Changes in macrophages (C).

[0032] Where: * and # both represent p-values, # represents the comparison with the control group, and * represents the comparison with the dss group. *: P ≤ 0.05, indicating that the result is significant at the 0.05 level; **: P ≤ 0.01, indicating that the result is significant at the 0.01 level; ***: P ≤ 0.001, indicating that the result is significant at the 0.001 level. DETAILED DESCRIPTION

[0033] The technical solutions of the present invention will be described in detail below through specific examples. The examples are only partial examples of the present invention, not all embodiments. Based on the examples in the present invention, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present invention.

[0034] Example 1 Optimization of fermentation process conditions

[0035] 1 Materials and Methods

[0036] 1.1 Materials and Reagents

[0037] Chrysanthemum stems and leaves were provided by the Chrysanthemum Medicinal Materials Cooperation Demonstration Base of Nanjing University of Chinese Medicine; Lactobacillus plantarum (CICC 21809) was provided by the China Industrial Microbiological Culture Collection Center; MRS broth (batch number: HB0384-1) was provided by Qingdao Haibo Biological.

[0038] 1.2 Instruments and Equipment

[0039] Clean bench (Suzhou Purification Equipment Co., Ltd.); GR85DA fully automatic autoclave (Zhiwei Instrument Co., Ltd.); SPX-150B

[0040] Type III biochemical incubator (Tianjin Test Instrument Co., Ltd.); ZQZY-C8E shaking incubator (Shanghai Zhichu Instrument Co., Ltd.); Q ultrapure water preparation instrument (Millipore, USA); Su-PerMax 3000FA multifunctional microplate reader (Shanghai Shanpu Biotechnology Co., Ltd.); MicroCL17R high-speed refrigerated centrifuge (Thermo Fisher, USA); crusher (DFY-300): Wenling Linda Machinery Co., Ltd.

[0041] 1.3 Experimental methods

[0042] 1.3.1 Preparation of chrysanthemum stem and leaf powder

[0043] The fresh chrysanthemum stems and leaves were dried in the shade for 7 days and then crushed and passed through a 60-mesh sieve.

[0044] 1.3.2 Preparation of fermentation broth

[0045] The Lactobacillus plantarum strain (CICC 21809) was streaked on MRS agar medium for recovery and cultured at 37°C for 48 hours. The activated single colony was picked and placed in a 37°C MRS liquid medium environment for culture for 24 hours. 1% of the inoculum amount of the bacterial solution was taken for second-generation amplification culture and cultured for 8 hours to obtain the fermentation bacterial solution.

[0046] 1.3.3 Fermentation

[0047] 5% of the fermentation liquid obtained in 1.3.2 was inoculated into chrysanthemum stems and leaves at a material-liquid ratio of 1:50 and fermented at 37°C, 180 rpm, for 1 day.

[0048] 2. Single-factor experiment

[0049] Single-factor optimization was performed on the three influencing factors of solid-liquid ratio, inoculation amount, and fermentation time. The initial fermentation conditions were: solid-liquid ratio of 1:20, inoculation amount of 1%, and fermentation time of 24 hours. The solid-liquid ratios in the single-factor optimization experiment were 1:100, 1:50, 1:25, 1:12.5, and 1:6.25 respectively.

[0050] The inoculation rates were 1%, 5%, 15%, 25%, and 35%, respectively; the fermentation times were 1 day, 2 days, 3 days, 4 days, and 5 days, respectively. Each experiment was repeated 3 times.

[0051] 3. Orthogonal experiment to optimize fermentation conditions

[0052] Based on the single factor results, three levels were selected, and the membership comprehensive scoring method was used as the optimization index for the total phenolic and total flavonoid contents. A three-factor three-level orthogonal experiment was designed and conducted, as shown in Table 1.

[0053] Table 1 Orthogonal test factors and levels

[0054]

[0055] 4. Determination of active ingredients

[0056] After the fermentation was completed, the mixture was centrifuged (8000 rpm, 15 min) and the supernatant was collected to obtain the fermentation liquid.

[0057] Determination of total flavonoids content: The total flavonoids content was determined by the NaNO2-Al(NO3)3-NaOH method. A rutin standard solution with a mass concentration of 1.05 mg / ml was prepared, and 0, 100 ul, 200 ul, 300 ul, 400 ul, 500 ul, and 600 ul of the rutin standard solution were respectively drawn into 1.5 ml centrifuge tubes. After adding 70% methanol to 600 ul of the centrifuge tube, 50 ul of 5% NaNO2 was added, the solution was shaken and placed at room temperature for 6 min, 50 ul of 10% Al(NO3)3 was added, the solution was shaken and placed at room temperature for 6 min, 400 ul of 4% NaOH was added, the solution was shaken and placed at room temperature for 15 min, the absorbance at 510 nm was measured, and a standard curve was drawn.

[0058] Determination of total phenol content: The total phenol extraction rate was determined by the Folin-Ciocalteu method. A gallic acid standard solution with a mass concentration of 1.01 mg / ml was prepared. 0, 10, 20, 30, 40, 50, and 60 μl of the standard solution were respectively drawn into a 1.5 ml centrifuge tube, and distilled water was added to 60 μl. 250 μl of FC reagent was added to each tube and mixed. The tube was placed in a dark place for 4 min. After adding 250 μl of 10% Na2CO3, distilled water was added to 1 ml. The tube was kept in a constant temperature water bath at 45°C for 15 min. The absorbance at 765 nm was measured and a standard curve was drawn.

[0059] 5. Data statistics and analysis

[0060] All experiments were repeated three times. Data were processed using Microsoft Excel 2010, and plotted using Origin 2019. Data were analyzed using SPSS 26 professional statistical software, and data are presented as mean ± standard deviation. Before performing the T-test for significance, a one-way ANOVA test for homogeneity of variance was performed to ensure that the variances of the independent samples were consistent. The results of the significance analysis were considered as follows: when P > 0.05, the difference between the groups was not statistically significant; when P < 0.05, the difference between the groups was statistically significant.

[0061] 6. Experimental Results

[0062] 6.1 Effect of material-liquid ratio on fermentation

[0063] The results are as follows Figure 1 As shown, as the water ratio increases, the total phenol and total flavonoid contents after fermentation show a trend of first increasing and then decreasing. At a solid-liquid ratio of 1:25, the total phenol and total flavonoid contents reach their highest levels, then decrease as the water ratio increases. Increasing the water ratio provides a suitable growth environment for the fermentation bacteria, but excessively high water ratios can lead to the growth of other bacteria, thereby affecting the growth of the fermentation bacteria. Therefore, the preferred solid-liquid ratio for chrysanthemum stem and leaf fermented feed additives is 1:25.

[0064] 6.2 Effect of inoculation amount on fermentation

[0065] The results are as follows Figure 2 As shown: when the inoculation amount of the strain is 15%, the content of its effective ingredients total phenols and total flavonoids is the highest. When the inoculation amount is small, the secondary metabolites produced by the metabolism of the fermentation substrate are small, and the function of promoting the release of total flavonoids and total phenolic components is weak, so the content is low. When the inoculation amount is too much and exceeds the appropriate range, there will be intensified competition for the utilization of substrates, which limits the normal metabolism of the fermentation bacteria. Excessive microorganisms will cause the rapid accumulation of metabolites (such as lactic acid and ethanol), drastic changes in pH value, changes in the environment, inhibition of bacterial activity or destruction of substrate structure. Therefore, the preferred inoculation amount of chrysanthemum stem and leaf fermented feed additive is 15%.

[0066] 6.3 Effect of fermentation time on fermentation

[0067] The results are as follows Figure 3 As shown, with increasing fermentation time, the total phenolic and flavonoid contents initially increase and then decrease, reaching their highest levels on the second day of fermentation. In the early stages of fermentation, microorganisms are in the adaptation and proliferation phase, and substrate breakdown is slow. Under optimal fermentation conditions, bacterial metabolism is active, maximizing the release of active ingredients. However, if fermentation is prolonged, substrate depletion leads to further decomposition or conversion of metabolites, increasing contamination with harmful bacteria and fermentation costs. Therefore, the optimal fermentation time for chrysanthemum stem and leaf fermented feed additives is two days.

[0068] 7. Orthogonal experiment results

[0069] Based on the results of the single-factor experiment, a comprehensive membership scoring method was used as the optimization metric for total phenolic and total flavonoid contents. A three-factor, three-level orthogonal experiment was designed and conducted. The experimental results are shown in the table, along with the variance analysis results. The table shows that the order of influence of each factor on the comprehensive score is A > B > C, i.e., material-to-liquid ratio > fermentation time > inoculation amount. Taking all factors into consideration, the optimal fermentation process was determined to be A1B3C1. Under these conditions, the total flavonoid content of chrysanthemum stems and leaves after fermentation was verified to be 9.416 mg / g, the total phenolic content was 24.740 mg / g, and the comprehensive score was 0.987, indicating good stability. The fermentation product of chrysanthemum stems and leaves obtained using the optimal process was used for subsequent experiments.

[0070] Table 2 Orthogonal experimental results of chrysanthemum stem and leaf fermentation process

[0071]

[0072] Table 3 Results of variance analysis of orthogonal experiment on chrysanthemum stem and leaf fermentation process

[0073]

[0074] Example 2 Therapeutic effects of chrysanthemum stems and leaves before and after fermentation on DSS-induced acute ulcerative colitis in mice

[0075] 1. Animal Experiment Design

[0076] The experimental design divided 45 mice into 9 groups, with 5 mice in each group, including control group, DSS group, 5-ASA group, low, medium and high dose groups before fermentation of chrysanthemum stems and leaves (CSLL; 100 mg / kg, CSLM; 200 mg / kg, CSLH400 mg / kg), and low, medium and high dose groups after fermentation of chrysanthemum stems and leaves (FCSLL; 100 mg / kg, FCSLM200 mg / kg, FCSLH400 mg / kg).

[0077] The animals were fed adaptively at 25°C under a 12-hour light / 12-hour dark cycle for 7 days. Starting on day 8, the acute ulcerative colitis model was established in all eight groups except the control group, who were treated with 2.5% (w / v) DSS for 7 days. The control group drank normal water. Subsequently, the 5-ASA, CSLL, CSLM, CSLH, FCSLL, FCSLM, and FCSLH groups were gavaged daily with 0.1 ml / 10 g of the corresponding drug for 7 days. The control and DSS groups were gavaged daily with 0.9% saline.

[0078] 5-ASA (5-aminosalicylic acid) served as a positive control. It is a first-line treatment for inflammatory bowel disease (IBD), particularly ulcerative colitis (UC), and is also used in some patients with Crohn's disease (CD). It relieves intestinal inflammation and promotes mucosal healing through local anti-inflammatory effects.

[0079] 2. Determination of Mouse Weight

[0080] The body weight of mice was recorded every day.

[0081] Calculate relative body weight = (daily body weight / initial body weight)

[0082] result:

[0083] Depend on Figure 4 As can be seen, the control mice gradually gained weight, had shiny fur, and had normal diet and behavior. Mice in the DSS group lost weight and became listless. Compared with the DSS group, mice in the CSL and FCSL groups gained significantly more weight, with the FCSL group showing a greater effect than the CSL group. These results suggest that FCSL mitigates weight loss caused by DSS-induced acute ulcerative colitis, with the FCSLH group showing comparable efficacy to the positive drug group.

[0084] 3. Determination of mouse colon length

[0085] The colon length of mice in each group was measured;

[0086] result:

[0087] Depend on Figure 5 As can be seen, compared with the control group, the colon length of the DSS group was significantly shortened, while the colon length of the CSL and FCSL groups was significantly increased compared to the DSS group, with the FCSL group showing a superior effect to the CSL group. These results indicate that FCSL can improve the symptoms of colon shortening caused by DSS-induced acute ulcerative colitis in mice and maintain intestinal health, with the FCSLH group showing comparable effects to the positive drug group.

[0088] 4. Determination of mouse immune cells

[0089] The colon tissue of mice was obtained and minced, digested, washed, centrifuged, filtered, and resuspended. The cell suspension was then stained with CD45APC, Ly-6CPE-Cy7, CD11b PE, Ly-6G BV421, and F4-80 FITC antibodies, respectively, and cell analysis was performed using flow cytometry.

[0090] result:

[0091] Depend on Figure 6 As can be seen, compared with the control group, the DSS group had a significant increase in CD11b+Ly6C+ monocytes, CD11b+Ly6G+ neutrophils, and CD11b+F4 / 80+ macrophages. Compared with the DSS group, both the FCSL and CSL groups reduced CD11b+Ly6C+ monocytes, CD11b+Ly6G+ neutrophils, and CD11b+F4 / 80+ macrophages, with the FCSL group showing a superior effect to the CSL group. These results indicate that FCSL can effectively inhibit DSS-induced inflammatory responses and has a role in regulating intestinal immune cells.

[0092] It can be seen from the above experimental examples that the present invention provides a chrysanthemum stem and leaf fermented feed additive with an immune-regulating effect. The present invention uses plant lactobacillus for fermentation to provide an additive for chrysanthemum stem and leaf feed, and is used to prepare chrysanthemum stem and leaf fermented feed. The fermented feed prepared by the additive significantly increases the total phenol and total flavonoid content of the chrysanthemum stem and leaf feed, promotes the enhancement of intestinal immune function, and improves animal health and production performance. The present invention promotes the innovative application of medicinal and edible medicinal materials in the field of feed, realizes the efficient utilization of agricultural by-products, and provides broad application prospects for the sustainable development of green animal husbandry.

Claims

1. A fermented chrysanthemum stem and leaf feed additive with immune regulation effect, characterized in that: It is obtained by the following steps: (1) Grinding and sieving the chrysanthemum stems and leaves to obtain fermentation material; (2) adding water to the fermentation material to obtain a fermentation medium; (3) The bacterial liquid of Lactobacillus plantarum strain CICC 21809 was inoculated into the fermentation medium and shaker fermented to obtain chrysanthemum stem and leaf fermented feed additive.

2. The fermented chrysanthemum stem and leaf feed additive according to claim 1, characterized in that The sieving in step (1) is 20-80 mesh; and the volume ratio of the fermentation material to water in step (2) is 1:6.25-100.

3. The fermented chrysanthemum stem and leaf feed additive according to claim 1, characterized in that In step (3), 1% to 35% of Lactobacillus plantarum fermentation liquid is added to the fermentation medium.

4. The fermented chrysanthemum stem and leaf feed additive according to claim 1, characterized in that In step (3), the initial pH value of the fermentation medium inoculated with plant lactobacillus is controlled to be 5.0-9.

0.

5. The fermented chrysanthemum stem and leaf feed additive according to claim 1, characterized in that The fermentation temperature in step (3) is 24-42°C.

6. The chrysanthemum stem and leaf fermented feed additive according to claim 1, characterized in that The fermentation time in step (3) is 1-5 days; the shaking fermentation in step (3) is carried out in a constant temperature shaking incubator with a rotation speed of 120-210 r / min.

7. The chrysanthemum stem and leaf fermented feed additive according to claim 1, characterized in that In step (2), the volume ratio of the fermentation material to water is 1:50, and in step (3), the fermentation time is 1 day and the inoculation amount is 5%.

8. A fermentation broth, characterized in that The fermentation liquid is obtained by separating the fermented product from the chrysanthemum stem and leaf fermented feed additive according to any one of claims 1 to 7.

9. Use of the fermented chrysanthemum stem and leaf feed additive according to any one of claims 1 to 7 or the fermentation liquid according to claim 8 in feed.

10. Use of the fermented chrysanthemum stem and leaf feed additive according to any one of claims 1 to 7 or the fermentation liquid according to claim 8 in the preparation of a drug for treating ulcerative colitis.

Images