Radix puerariae and flos chrysanthemi ferment preparation and application thereof in improving immunity and antioxidant capacity of apis cerana

Abstract

This invention discloses a fermented preparation of kudzu root and chrysanthemum and its application in improving the immune function and antioxidant capacity of Chinese honeybees. The fermented preparation is made from kudzu root, chrysanthemum, and water as main raw materials, fermented by Bacillus subtilis. In use, the resulting fermented liquid is mixed with a 50% sucrose solution at a volume ratio of 1:1 and then fed to Chinese honeybees. This invention promotes the release of flavonoid active ingredients from kudzu root and chrysanthemum through microbial fermentation, increases the total flavonoid content of the fermented liquid, and improves some antioxidant activity indicators. Feeding experiments with Chinese honeybees show that this fermented preparation can regulate malondialdehyde content in honeybees and promote the expression levels of genes related to immune regulation, antioxidant defense, and physiological metabolism. The raw materials for this preparation are widely available, the preparation process is simple, and no complex extraction process is required. It is safe, green, and suitable for functional supplementary feeding of Chinese honeybees.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine microbial fermentation and bee nutrition regulation technology, specifically involving a fermentation preparation made from kudzu root and chrysanthemum, and the application of this preparation in improving the immune function and antioxidant capacity of Chinese honeybees. Background Technology

[0002] Chinese honeybee ( Apis cerana cerana Bees are an important native bee species in my country. Under conditions of insufficient honey and pollen, temperature fluctuations, migration, and intensive breeding, bee colonies are prone to problems such as insufficient nutrient supply and increased oxidative stress, which in turn affect the condition of worker bees and the maintenance of the colony. In addition to ensuring adequate sugar and protein sources, supplementing with functional components that have antioxidant and physiological regulatory effects is a feasible approach to bee colony nutrition management.

[0003] Current supplemental feeding methods primarily utilize sucrose solutions, pollen, or pollen substitutes. Sugar solutions mainly provide energy, while pollen substitutes focus on protein and basic nutrients, with relatively limited effective components for antioxidant and stress regulation. Traditional Chinese medicine ingredients contain various flavonoids, phenolic acids, and other active substances; however, when directly ground and added to feed, the release and utilization of some components are limited by the plant tissue structure.

[0004] Kudzu root contains isoflavones such as puerarin and daidzein; chrysanthemum contains flavonoids such as luteolin and apigenin. Both raw materials can be used as plant-derived functional ingredients. When using them as bee feed, one of the problems that needs to be solved in formulation preparation is how to improve the release of active ingredients in the aqueous phase without using complex organic solvent extraction processes.

[0005] Bacillus subtilis ( Bacillus subtilis Fermentation can produce various extracellular enzymes. Using these enzymes to ferment kudzu root and chrysanthemum can alter the tissue structure of the raw materials and the composition of active ingredients in the aqueous phase. Existing research has shown that fermenting a combination of kudzu root and chrysanthemum directly applies the resulting product to the Chinese honeybee (Apis cerana). Apis cerana cerana There are still few technical solutions for functional supplementary feeding, so it is necessary to conduct further research on raw material ratios, fermentation conditions and feeding effects. Summary of the Invention

[0006] This invention provides a kudzu and chrysanthemum fermentation preparation. The fermentation preparation is made by fermenting kudzu and chrysanthemum with Bacillus subtilis to form a fermentation liquid that can be directly mixed with sugar solution for feeding. The kudzu and chrysanthemum fermentation preparation uses water as the fermentation medium. 1-5g of kudzu and 1-5g of chrysanthemum are added to every 100mL of water. The mixture is sterilized at 121℃ for 20 minutes, cooled to 30-37℃, and then inoculated with Bacillus subtilis seed liquid at an inoculation amount of 5% (v / v). Fermentation is carried out at 37℃ for 45-50 hours.

[0007] Another objective of this invention is to apply kudzu and chrysanthemum fermentation preparations in the preparation of bee supplementary feeds for improving the immune function and antioxidant capacity of Chinese honeybees.

[0008] When using, mix with a 50% sucrose solution at a volume ratio of 1:1 for supplementary feeding of Chinese honeybees.

[0009] This invention is based on Bacillus subtilis ( Bacillus subtilis Purchased from the China Center for Type Culture Collection, accession number CCTCC AB 2025223.

[0010] Advantages and technical effects of the present invention: 1. This invention promotes the release of flavonoid active ingredients in kudzu root and chrysanthemum through microbial fermentation, increases the total flavonoid content of the fermentation broth, and improves some antioxidant activity indicators; 2. Feeding experiments with Chinese honeybees showed that the fermented preparation could regulate the malondialdehyde content in honeybees and promote the expression levels of genes related to immune regulation, antioxidant defense and physiological metabolism, such as L(2)efl, SOD, Vg, TPS and CAT. 3. The raw materials for the formulation of this invention are widely available, the preparation process is simple, no complicated extraction process is required, and it is safe, green and suitable for functional supplementary feeding of Chinese honeybees. Attached Figure Description

[0011] Figure 1 This is a picture of the fermented kudzu root and chrysanthemum mixture. Figure 2 Results of total flavonoid content detection in fermentation broths with different ratios; Figure 3 Results of DPPH free radical scavenging capacity tests for fermentation broths with different ratios; Figure 4 Results of ABTS free radical scavenging ability tests in fermentation broths with different ratios; Figure 5 This represents the relative content of malondialdehyde (MDA). Figure 6 The results show the relative expression levels of the L(2)efl gene. Figure 7 The results show the relative expression levels of the SOD gene. Figure 8 The results show the relative expression levels of the Vg gene. Figure 9 The results show the relative expression levels of the TPS gene. Figure 10 The results show the relative expression levels of the CAT gene. Detailed Implementation

[0012] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings. Unless otherwise stated, the reagents and instruments used can be used under conventional conditions in the art. The following embodiments are used to illustrate the implementation of the present invention and are not intended to limit the scope of protection of the present invention.

[0013] Example 1: Preparation of Kudzu and Chrysanthemum Fermentation Broth 1. Weigh out kudzu root and chrysanthemum according to Table 1, add them to 100mL of reverse osmosis purified water (RO) respectively, mix thoroughly, and prepare five initial fermentation substrates G1-G5. The amount of kudzu root and chrysanthemum added to each group increases synchronously. Table 1

[0014] 2. Sterilization treatment Place the container containing the initial fermentation substrate in a high-pressure steam sterilizer and sterilize at 121°C for 20 minutes; after sterilization, remove it and allow it to cool naturally to 30-37°C. 3. Inoculation and Fermentation Under aseptic conditions, 5% (v / v) Bacillus subtilis was inoculated into the cooled fermentation substrate. Bacillus subtilis Seed solution, the concentration of which should be adjusted to OD before inoculation. 600 =1.0, after inoculation, mix well, and ferment at 37℃ and 180 rpm for 48 hours. The appearance of the fermented preparation is as follows. Figure 1 As shown.

[0015] 4. Preparation of feeding solution After fermentation, the fermentation broth was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected. The supernatant was then mixed with a 50% sucrose solution at a volume ratio of 1:1 and used to feed the Chinese honeybee. Apis cerana cerana Meanwhile, a control group was set up and fed Chinese honeybees directly with 50% sucrose solution (by weight). Apis cerana cerana Record relevant physiological indicators of bees.

[0016] Total flavonoid content in fermentation broth Unfermented and fermented samples of different ratios from G1 to G5 were taken respectively, and the total flavonoid content in each group of samples was detected using a total flavonoid assay kit (Suzhou Grace Biotechnology Co., Ltd.). The results are as follows: Figure 2 As shown in the figure. The results showed that, compared with the corresponding unfermented samples, the total flavonoid content of each group increased after fermentation. Among them, the total flavonoid content of groups G2, G3, G4 and G5 was significantly higher than that of the unfermented group (as shown in the figure). p<0.05), while although the G1 group showed an increasing trend, it did not reach a significant difference level ( p >0.05). With increasing amounts of kudzu root and chrysanthemum, the total flavonoid content in the fermentation broth generally increased, with group G5 showing the highest total flavonoid content after fermentation, and the most significant difference compared to the unfermented group. These results indicate that Bacillus subtilis fermentation can promote the release of flavonoids from kudzu root and chrysanthemum, increasing the total flavonoid level in the fermentation broth.

[0017] DPPH free radical scavenging ability of fermentation broth Unfermented and fermented samples from groups G1-G5 were taken separately and tested using a DPPH free radical scavenging ability kit (Suzhou Grace Biotechnology Co., Ltd.). The results are as follows: Figure 3 As shown in the figure. The results showed that the DPPH free radical scavenging capacity of different experimental groups after fermentation treatment differed. Among them, the DPPH free radical scavenging capacity of G1 and G4 groups after fermentation was higher than that of the corresponding unfermented groups, and the difference reached a significant level ( p <0.05); the DPPH free radical scavenging capacity of groups G2 and G3 after fermentation was lower than that of the corresponding unfermented groups, but still showed statistical differences (p<0.05); the DPPH free radical scavenging capacity of group G5 after fermentation was slightly higher than that of the unfermented group, but did not reach the level of statistical significance (p<0.05). p >0.05). Among the fermentation treatment groups, group G4 showed higher DPPH radical scavenging ability. This result indicates that the effect of fermentation on DPPH radical scavenging ability is related to the feed ratio.

[0018] ABTS free radical scavenging ability in fermentation broth Unfermented and fermented samples from experimental groups G1-G5 were taken and tested using the ABTS free radical scavenging ability kit (Suzhou Grace Biotechnology Co., Ltd.). The results are as follows: Figure 4 As shown in the figure. The results showed that there were differences in the ABTS free radical scavenging ability of different experimental groups after fermentation treatment. Among them, the ABTS free radical scavenging ability of the G2 group after fermentation treatment was significantly higher than that of the corresponding unfermented group ( p= 0.0169); The ABTS free radical scavenging capacity of the G1 group increased slightly after fermentation, but the difference did not reach a statistically significant level. p= 0.9995). The ABTS free radical scavenging abilities of groups G3, G4, and G5 all changed to some extent after fermentation treatment, but the differences did not reach a statistically significant level. p >0.05). The above results indicate that different ratios of kudzu root and chrysanthemum can affect the ABTS free radical scavenging capacity of the fermentation broth, with the G2 ratio having a more significant promoting effect.

[0019] Example 2: Feeding Trial of Chinese Honeybees and Detection of Related Physiological Indicators 1. Grouping and feeding of bee colonies Healthy honeybee colonies with similar colony strength were selected, and five fermentation treatment groups (G1-G5) were set up, with 3 hives in each group and 5 frames of bees in each hive. Groups G1-G5 were fed 2 mL of a mixture of the corresponding fermentation solution and 50% sucrose solution at a 1:1 volume ratio daily, while the CK group was fed an equal amount of 50% sucrose solution. The feeding was continued for 7 days.

[0020] Malondialdehyde content determination After feeding different ratios of kudzu root and chrysanthemum fermentation broth for 7 days, intestinal tissue of Chinese honeybees was collected. The MDA content was detected using a malondialdehyde (MDA) content reagent kit (Suzhou Grace Biotechnology Co., Ltd.). The results are as follows: Figure 5 As shown in the figure. The results indicated that there were significant differences in MDA content in the intestinal tissue of *Apis cerana* among the different treatment groups. Compared with the CK group, the MDA content in the G1 group was significantly lower (…). p <0.05), and significantly lower than in groups G2 and G3; MDA content was significantly higher in groups G2 and G3 ( p The levels of MDA in the G4 and G5 groups were <0.05, indicating relatively high levels. There was no significant difference in MDA content between the G4 and G5 groups and the CK group. These results suggest that different fermentation broth ratios have different regulatory effects on lipid peroxidation levels in honeybees, with the G1 treatment reducing MDA accumulation.

[0021] Detection of relative gene expression levels in bees To ensure the accuracy of gene expression analysis, this invention strictly adhered to specifications during the primer design stage: using the stably expressed internal reference gene β-actin in *Apis cerana* as a reference, specific amplification primers were designed using PrimerPremier 5.0 software targeting the target gene sequence. Design parameters included primer length of 18–24 bp, GC content of 40%–60%, similar annealing temperature (Tm) (approximately 60°C), and amplification product length of 100–300 bp. Primer dimers and hairpin structures were excluded based on software evaluation. All candidate primers underwent online homology comparison using the NCBI Primer-BLAST tool to confirm no significant homology with other sequences in the *Apis cerana* genome, ensuring species specificity. The final primers were synthesized by Shanghai Sangon Biotech Co., Ltd. using the solid-phase phosphoramidite method and purified by PAGE. They were provided in dry powder form and, after brief centrifugation before use, were prepared into a 100 μM stock solution with TE buffer and stored at -20°C for later use. Specific primer sequences are detailed in Table 2.

[0022] Table 2 ; After 7 days of feeding, 5 worker bees were taken from each group, their intestinal tissues were separated and ground, and total RNA was extracted and reverse transcribed to obtain cDNA. The relative expression levels of L(2)efl, SOD, Vg, TPS and CAT genes were detected by real-time quantitative PCR. Each group was compared with CK as a control. RNA was extracted using the TRIzol method, and the RNA was identified by spectrophotometry and electrophoresis. Using RNA as a template, cDNA was synthesized by reverse transcription using a cDNA synthesis kit that removes genomic DNA. The reverse transcription system was: 5´All-in-one qRTSuperMix 4µL, Enzyme Mix 1µL, template RNA xµL, RNase-free ddH2O 15-x µL, total volume 20µL; A 20 μL reaction system was prepared according to the reagent instructions of Taq Pro Universal SYBR qPCR Master Mix from Novizan Biotechnology Co., Ltd. (Table 3). The relative expression levels of L(2)efl, Vg, TPS, SOD and CAT genes were detected on a CFX96 real-time quantitative PCR instrument. The amplification experiment was carried out under the conventional amplification conditions (Table 4).

[0023] Table 3 PCR reaction system ; Table 4 PCR amplification steps

[0024] 4. Data Statistical Analysis Experimental data were statistically analyzed using GraphPad Prism 10.6.0 software. All data are expressed as mean ± standard deviation. Figures 2-4 The results of activity index detection in fermentation broths with different ratios are shown. Two-way ANOVA was used to evaluate the effects of fermentation treatment and raw material ratio on the detection indexes, and multiple comparisons between groups were performed. Figures 5-10 The results of the detection of relevant indicators of the Chinese honeybee shown were compared using one-way ANOVA to compare differences between different treatment groups, and significance analysis was performed. p <0.05 is used as the criterion for judging whether the difference is statistically significant. Different letters or asterisks in the figure indicate that there is a significant difference between the groups.

[0025] The relative expression levels of the L(2)efl gene in different treatment groups of *Apis cerana* were detected by real-time quantitative PCR. The results are as follows: Figure 6 As shown in the figure. The results showed that the expression level of L(2)efl gene was the highest in group G1, which was significantly higher than that in group G4. p<0.05), showing an increasing trend compared to other treatment groups; the G4 group had the lowest expression level, which was significantly different from the G1 group. No significant differences were observed among the CK, G2, G3, and G5 groups. p >0.05). This result indicates that different ratios of kudzu and chrysanthemum fermentation broth have a differential regulatory effect on the expression of the L(2)efl gene.

[0026] The relative expression levels of the SOD gene in different treatment groups of *Apis cerana* were detected by real-time quantitative PCR, and the results are as follows: Figure 7 As shown; the results indicated that the SOD gene expression levels in the G1 and G2 groups were significantly higher than those in the CK group ( p <0.001), and both were at a high expression level; although the expression levels of groups G3, G4, and G5 were higher than or close to those of the CK group, the differences compared with the CK group were not statistically significant. The results indicate that certain fermentation broth ratios of kudzu and chrysanthemum can promote the expression of SOD, an antioxidant-related gene in honeybees.

[0027] The relative expression levels of the Vg gene in different treatment groups of *Apis cerana* were detected by real-time quantitative PCR. The results are as follows: Figure 8 As shown in the figure. The results showed that the expression level of Vg gene in the G1 group was significantly higher than that in the CK group and other treatment groups ( p< The expression level of the G1-ratio kudzu and chrysanthemum fermentation broth significantly promoted the expression of the Vg gene in honeybees (G1 ratio 0.05%), showing a clear promoting effect. No significant differences were observed in the expression levels of the G2, G3, G4, and G5 groups compared to the CK group. These results indicate that the G1 ratio of kudzu and chrysanthemum fermentation broth significantly promoted the expression of the Vg gene in honeybees.

[0028] The relative expression levels of the TPS gene in different treatment groups of *Apis cerana* were detected by real-time quantitative PCR, and the results are as follows: Figure 9 As shown, the results indicated that the expression level of the TPS gene in the G1 group was significantly higher than that in the CK group and other partial treatment groups. p =0.0003); the expression level in group G3 was higher than that in group CK, but the difference was not statistically significant; groups G2 and G5 showed an increasing trend; group G4 showed no significant increase compared with group CK. These results indicate that different ratios of kudzu and chrysanthemum fermentation broth have different regulatory effects on TPS gene expression, with group G1 showing a more significant promoting effect.

[0029] The relative expression levels of the CAT gene in different treatment groups of *Apis cerana* were detected by real-time quantitative PCR, and the results are as follows: Figure 10 As shown in the figure. The results showed that the CAT gene expression level was highest in the G1 group, and significantly higher than that in the CK group and other treatment groups. p< 0.05); there were no significant differences among the CK, G2, G3, G4 and G5 groups. P >0.05). The results showed that the G1 ratio of kudzu and chrysanthemum fermentation broth could significantly promote the expression of the CAT gene in honeybees and improve the response level of antioxidant defense-related genes.

[0030] In summary, fermentation of kudzu root and chrysanthemum with Bacillus subtilis can effectively increase the total flavonoid content in the fermentation broth and regulate the scavenging capacity of DPPH and ABTS free radicals, indicating that the fermentation process is beneficial to the release of plant-derived active ingredients and the improvement of antioxidant activity. Further feeding experiments with Chinese honeybees revealed that the fermented kudzu root and chrysanthemum broth can regulate the MDA content in honeybees and promote the expression of genes related to immunity, antioxidant activity, and physiological regulation, such as L(2)efl, SOD, Vg, TPS, and CAT. Among these, the G1 ratio showed better regulatory effects in multiple indicators, suggesting that this fermented kudzu root and chrysanthemum preparation has the potential to improve the antioxidant capacity of Chinese honeybees and enhance their immune regulation level, and can be used as a functional supplementary feed preparation for Chinese honeybees.

Claims

1. A fermented preparation of kudzu root and chrysanthemum, characterized in that: Add 1-5g of kudzu root and 1-5g of chrysanthemum to every 100 mL of water, and inoculate with Bacillus subtilis after sterilization. Bacillus subtilis It is fermented to obtain the product.

2. The fermented kudzu and chrysanthemum preparation according to claim 1, characterized in that: Mix kudzu root and chrysanthemum with purified water, sterilize at 121℃ for 20 minutes, cool to 30-37℃, and then inoculate with OD. 600 The seed culture of Bacillus subtilis with a concentration of 1.0 was fermented in a shaker at 37°C for 45-50 hours to obtain the fermented broth of kudzu root and chrysanthemum.

3. The application of the fermented kudzu and chrysanthemum preparation of claim 1 in the preparation of a bee supplement feed for improving the immune function and antioxidant capacity of Chinese honeybees.

4. The application according to claim 3, characterized in that: When using this product, mix the fermented kudzu and chrysanthemum preparation with a 50% sucrose solution at a volume ratio of 1:1, and then feed it to Chinese honeybees.

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