Application of kaempferol in repairing imbalance of fish intestinal microflora

Abstract

The invention discloses application of kaempferol in repairing imbalance of fish intestinal microflora. Researches show that exposure of tetramethyl bisphenol A (TMBPA) affects richness and diversity of fish enteric microorganisms, the richness and diversity of the fish enteric microorganisms are dynamically changed during exposure, relative proportion of harmful bacteria in the fish enteric microorganisms is increased, and unbalance of the fish enteric microorganisms is caused; by adopting kaempferol administration treatment, the proportion of harmful bacteria in the intestinal tracts of the fishes can be reduced, the proportion of beneficial bacteria in the intestinal tracts of the fishes can be increased, and unbalanced intestinal microflora of the fishes can be helped to recover to a healthy state. The result shows that kaempferol can repair fish intestinal microflora imbalance caused by TMBPA and restore fish intestinal health.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, and more specifically, relates to the application of kaempferol in repairing the imbalance of the intestinal microbial community in fish. Background Technology

[0002] Tetramethyl bisphenol A (TMBPA) is an important organic chemical raw material, belonging to the bisphenol class of compounds. It is a derivative formed by replacing the four hydroxyl groups of bisphenol A with methyl groups, and its chemical formula is C6H2O. 21 H 24 O2. Tetramethylbisphenol A (TMBPA) possesses high thermal stability, chemical stability, and UV resistance, thus finding wide application in various industrial sectors. Due to its strong stability and resistance to degradation, it easily enters aquatic environments through wastewater discharge and landfill leachate, becoming a typical endocrine disruptor (EDC). TMBPA's health effects on fish involve multiple levels, including reproduction, development, endocrine function, and immunity, and it is characterized by "significant effects at low doses."

[0003] The gut microbiota (or gut microbiota for short) is considered a vast and complex ecosystem within the body, and its composition and variations are closely related to environmental factors and the individual host. The gut microbiota plays a crucial role in promoting intestinal development, maintaining intestinal health, resisting pathogen invasion, and regulating lipid metabolism and energy absorption. The "balance" of the gut microbiota depends on its diversity (species richness) and stable proportions (beneficial bacteria dominate, harmful bacteria are suppressed). Imbalance manifests as: a decrease in the number of beneficial bacteria; excessive proliferation of harmful bacteria; and a decline in gut microbiota diversity and richness.

[0004] This study shows that TMBPA exposure causes an imbalance in the gut microbiota of fish. Kaempferol, a natural dietary flavonoid, is widely found in tea, cruciferous vegetables (such as broccoli and Brussels sprouts), grapefruit, and various medicinal plants (such as delphinium, witch hazel, and kaempferol). Patent application CN119015272A discloses the application of kaempferol in treating liver damage in fish; patent application CN118948835A discloses the role of kaempferol in improving impaired spawning function in fish. However, there are currently no reports on the use of kaempferol for the repair and treatment of gut microbiota imbalance in fish. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of kaempferol in the preparation of products that repair the imbalance of the intestinal microbial community in fish.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution: This study shows that TMBPA exposure affects the diversity and richness of the gut microbiota in marine medaka, as well as the proportion of each bacterial group, with a large number of harmful or opportunistic pathogens (such as...) Mycobacterium , Vibrio An increased proportion of harmful bacteria (such as kaempferol) leads to an imbalance in the gut microbiota of marine killifish. Kaempferol intervention can reduce the proportion of harmful bacteria in the fish's gut and increase the proportion of beneficial bacteria (such as kaempferol, etc.). Akkermansia , Lactobacillus and Ligilactobacillus The proportion of (etc.) can help restore the imbalanced gut microbiota to a state similar to the control group. This indicates that kaempferol can repair the imbalance of fish gut microbiota caused by TMBPA and restore fish gut health.

[0007] Therefore, this invention provides the application of kaempferol in the preparation of products that repair the imbalance of the intestinal microbial community in fish.

[0008] Furthermore, this study explores the application of kaempferol in the preparation of products that repair the imbalance of the gut microbiota in fish caused by tetramethylbisphenol A exposure.

[0009] Furthermore, the method involves adding Artemia containing kaempferol to the fish farming water to feed them, thereby repairing the imbalance of the fish's intestinal microbial community.

[0010] Therefore, the present invention also provides the application of Artemia containing kaempferol in the preparation of products that repair the imbalance of the intestinal microbial community in fish.

[0011] Furthermore, the application of Artemia containing kaempferol in the preparation of products for repairing the imbalance of the gut microbiota in fish caused by tetramethylbisphenol A exposure.

[0012] Specifically, the product helps restore the imbalanced gut microbiota of fish to a healthy state by reducing the proportion of harmful bacteria and increasing the proportion of beneficial bacteria in the gut.

[0013] Furthermore, the method for preparing the Artemia containing kaempferol involves hatching Artemia in artificial seawater containing kaempferol, thereby enriching the insect body with kaempferol.

[0014] Furthermore, the concentration of kaempferol added is 25-35 mg / L (preferably 30 mg / L).

[0015] Furthermore, the incubation is carried out at a constant temperature of 24-28°C for 20-30 hours (preferably at a constant temperature of 26°C for 24 hours).

[0016] Furthermore, the salinity of the artificial seawater is 30‰.

[0017] Furthermore, the fish is a killifish. Preferably, it is a marine killifish, which is a model fish with wide salinity adaptability, short sexual maturity cycle, stable reproductive capacity, and excellent traits, and is often used in scientific research.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of kaempferol in repairing the imbalance of the intestinal microbiota in fish. The study shows that TMBPA exposure affects the richness and diversity of the intestinal microbiota in fish, with dynamic changes occurring during the exposure period. The relative proportion of harmful bacteria in the fish intestinal microbiota increases, leading to an imbalance. Treatment with kaempferol can reduce the proportion of harmful bacteria and increase the proportion of beneficial bacteria in the fish gut, helping the imbalanced intestinal microbiota to return to a healthy state. This indicates that kaempferol can repair the TMBPA-induced imbalance of the fish intestinal microbiota and restore intestinal health. Attached Figure Description

[0019] Figure 1 A timeline diagram showing the sampling points for the kaempferol recovery experiment.

[0020] Figure 2 This is a distribution map of the number of OUT cells in the gut microbiota of marine medaka.

[0021] Figure 3 Venn diagram analysis of the gut microbiota of marine medaka.

[0022] Figure 4 Shannon index analysis of gut microbiota in marine medaka.

[0023] Figure 5 Analysis of the Chao1 index in the gut microbiota of marine medaka.

[0024] Figure 6 PCoA analysis of the gut microbiota of marine medaka.

[0025] Figure 7 This is a diagram showing the phylum-level composition and distribution of the gut microbiota of marine medaka.

[0026] Figure 8 This is a map showing the genus-level composition and distribution of the gut microbiota of marine medaka. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0028] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0029] Ocean medaka ( Oryzias melastigma It is a model fish with wide salinity adaptability, short sexual maturity cycle, stable reproductive capacity and excellent traits, and is often used in scientific research.

[0030] Artemia ( Artemia salina Brine shrimp, also known as brine shrimp, is rich in protein, amino acids and fat, and is an indispensable food organism in aquaculture.

[0031] Tetramethylbisphenol A (TMBPA), CAS No.: 5613-46-7, purchased from Sigma-Aldrich (Shanghai).

[0032] Kaempferol, CAS No.:520-18-3, structure shown below, purchased from MACKLIN, lot number: C15941008, catalog number K812226-5g.

[0033] Example 1: Effects of TMBPA exposure on gut microbiota homeostasis in seawater medaka and the restorative effect of kaempferol. 1. Method (1) Experimental design for the recovery of kaempferol Experimental Groups and Treatments: The experiment was set up with four groups: (1) Control group (NC), which was cultured in normal artificial seawater throughout the entire process; (2) TMBPA exposure group (TT), which was continuously exposed to 50 µg / L (detection concentration: 19.38 µg / L) TMBPA for 7 days; (3) Natural recovery group (NR), which was transferred to normal artificial seawater after the exposure to observe natural recovery; and (4) Drug-induced recovery group (KR), which was transferred to normal artificial seawater after the exposure to feed Artemia containing kaempferol to evaluate the intervention effect. This design clarified whether kaempferol promoted the recovery of medaka by comparing the NR and KR groups, and verified the initial toxic effect of TMBPA with the NC group as the baseline.

[0034] Timeframes and dynamic monitoring: The experimental period covered the exposure period (7 days) and the recovery period (28 days). During the exposure phase, dynamic toxicity monitoring was conducted on the TT group at 24 hours, 72 hours, and 7 days; during the recovery phase, samples were taken from the NC, NR, and KR groups at 14 days and 28 days to analyze the recovery of physiological function and the long-term intervention effect of kaempferol. The exposure design and sampling timeframes are as follows: Figure 1 As shown.

[0035] Preparation of Artemia containing kaempferol: Kaempferol at a concentration of 30 mg / L was added to artificial seawater (salinity 30‰) to hatch Artemia, allowing the larvae to accumulate kaempferol. The hatching process lasted 24 hours under constant temperature conditions of 26 ℃. After hatching, the Artemia were rinsed with running water to remove any residual kaempferol from their surface. These kaempferol-treated Artemia were used as KR feed and fed twice daily. The remaining groups were fed normally hatched Artemia twice daily.

[0036] (2) Analysis of gut microbiota diversity in marine medaka Total genomic DNA was extracted from gut samples of marine medaka (killifish) using the TGuide S96 magnetic bead soil / feces DNA extraction kit (Beijing Tiangen Biotech Co., Ltd.), following the manufacturer's instructions. The quality and quantity of the extracted DNA were checked by 1.0% agarose gel electrophoresis, and the concentration and purity of the DNA were determined using a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific, Wilmington, USA). The variable regions V3-V4 of the bacterial 16S rRNA gene were amplified using primers. The amplified products were purified using the Omega DNA purification kit (Omega Corporation) and quantified using Qsep-400 (BiOptic Corporation). The amplicon libraries were subjected to paired-end sequencing (2 × 250 bp) on the Illumina novaseq 6000 platform (Beijing Bomei Biotechnology Co., Ltd.).

[0037] Using USEARCH (version 10.0), high-quality sequences with similarity exceeding 97% were grouped into the same operational taxonomic unit (OTU). Taxonomic annotation of OTUs / ASVs was based on a Naive Bayes classifier in QIIME2, using the SILVA database (release 138.1) with a confidence threshold of 70%. Alpha diversity analysis was performed using QIIME2 software to identify the complexity of species diversity in each sample. Beta diversity was calculated using principal coordinate analysis (PCoA) to assess the diversity of species complexity in the samples. One-way ANOVA was used to compare bacterial abundance and diversity. Linear discriminant analysis (LDA) combined with effect size (LEfSe) was applied to assess taxa with different abundances. Sequencing data analysis was performed on the BMKCloud online platform (https: / / www.biocloud.net).

[0038] 2. Results (1) High-throughput sequencing was performed on each group of samples in this study. A total of 6,962,633 raw reads were obtained using Illumina novaseq6000. After quality control assembly, a total of 6,381,090 clean reads were obtained. The OTUs in each sample were obtained by OTU / ASV noise reduction processing on the final clean reads. The results are as follows: Figure 2 As shown, after TMBPA exposure, the number of intestinal OTUs in female, male, and mixed-sex fish in the 24h exposure group (TT24h), 72h exposure group (TT72h), and 7d exposure group (TT7d) all showed a trend of first decreasing and then increasing, and were all lower than the control group (NC7d). This indicates that TMBPA affects the survival of some intestinal microorganisms in marine killifish. After 14 days of recovery from TMBPA exposure, the number of OTUs in female, male, and mixed-sex fish was as follows: kaempferol-treated recovery group (KR14d) > natural recovery group (NR14d) > control group (NC14d). After 28 days of recovery, the number of OTUs in the gut of female and mixed-sex fish showed the following order: kaempferol-treated recovery group (KR28d) > control group (NC28d) > natural recovery group (NR28d); however, the number of OTUs in the gut of male fish showed the following order: natural recovery group (NR28d) > control group (NC28d) > kaempferol-treated recovery group (KR28d). This suggests that there may be sex differences in the persistent effects after cessation of exposure.

[0039] (2) The Venn diagram results of the OUT feature sequence are as follows: Figure 3 As shown, the number of OUTs common to the gut at three different exposure time points (TT24h, TT72h, and TT7d) was 74 in females, 48 ​​in males, and 114 in a mixed analysis of both sexes. At recovery time 14d, the number of OUTs specific to the gut of females in the KR14d group was 10881, the number of OUTs specific to the gut of males was 10041, and the number of OUTs specific to the gut of both females and males was 20121. The number of OUTs specific to the gut of the KR14d group was the highest among the three groups. This suggests that kaempferol may be exerting a therapeutic effect, increasing the number of beneficial microorganisms in the gut. At recovery time 28d, the number of OUTs specific to the gut of females in the KR28d group was 927, the number of OUTs specific to the gut of males was 803, and the number of OUTs specific to the gut of both sexes was 1653.

[0040] (3) In this study, the dilution curves of each group gradually flattened out, and the Coverage index of each group was greater than or equal to 99.97%, proving that most of the microbial information and sequence data were reliable, the sequencing depth was sufficient, and subsequent data analysis could be performed. Alpha diversity can reflect the species diversity and species richness of the samples. Among them, the Shannon index is used to measure species diversity, and the Chao1 index is used to measure species richness. In this study, TMBPA affected the diversity and richness of the gut microbiota of sea medaka to some extent.

[0041] The results of Shannon index analysis of the gut microbiota of sea medaka are as follows: Figure 4 As shown, in the Shannon index of female fish, the exposure groups (TT24h, TT72h, and TT7d) at all three time points were significantly lower than the control group (NC7d); the KR14d group was significantly higher than the NC14d and NR14d groups; the NR14d group was significantly higher than the NC14d group; and the average values ​​of KR28d and NC28d were similar. In the Shannon index of male fish, the TT24h, TT72h, and TT7d groups were significantly lower than the NC7d group; the TT72h group was significantly lower than the TT24h and TT7d groups; the NR14d group was significantly lower than the KR14d group; and the KR28d index was closer to the NR28d and NC28d indexes. In the comprehensive analysis of the Shannon index of female and male fish, the TT24h, TT72h and TT7d groups were significantly lower than the NC7d group; the TT72h group was significantly lower than the TT24h and TT7d groups; the KR14d group was significantly higher than the NC14d and NR14d groups; and the KR28d group was closer to the NC28d group than the NR28d group.

[0042] The results of the Chao1 index analysis of the gut microbiota of marine medaka are as follows: Figure 5As shown, in the Chao1 index of female fish, the TT24h, TT72h, and TT7d groups were all lower than the NC7d group, with the TT24h and TT72h groups being significantly lower than the NC7d group; the KR14d group was significantly higher than the NC14d and NR14d groups, with the NR14d group being significantly higher than the NC14d group; the KR28d index was closer to the NC28d index than the NR28d index. In the Chao1 index of male fish, the TT24h, TT72h, and TT7d groups were all lower than the NC7d group, with the TT72h group being significantly lower than the control group (NC7d) and other time-point exposure groups; the NC14d group was significantly lower than the NR14d and KR14d groups, with the KR14d group being significantly higher than the NC14d group; the KR28d index was closer to the NC28d index than the NR28d index. In the comprehensive analysis of the Chao1 index of male and female fish, the TT24h, TT72h, and TT7d groups were significantly lower than the NC7d group; the TT72h group was significantly lower than the TT24h and TT7d groups; the NC14d group was significantly lower than the NR14d and KR14d groups, while the KR14d group was significantly higher than the NC14d group; the KR28d index was closer to the NR28d and NC28d indexes. Overall, TMBPA affected the richness and diversity of the gut microbiota in marine killifish, and these factors underwent dynamic changes during the exposure period; kaempferol may have increased beneficial gut bacteria, helping the gut to recover to a state close to the control group.

[0043] (5) This study used principal coordinates analysis (PCoA) to conduct Beta diversity analysis. This analysis can classify multiple samples, further show the differences in species diversity among samples, and reflect the similarity of species types within and between groups. The closer the distance, the higher the similarity of the bacterial community among the samples and the smaller the degree of difference.

[0044] like Figure 6 As shown, during the 7-day TMBPA exposure period, the exposed groups and the control group showed certain differences in PCoA distribution at each time point. At the 14-day recovery time after kaempferol administration, the NC14d, NR14d, and KR14d groups showed some differences in PCoA distribution. At the 28-day recovery time after kaempferol administration, the KR28d and NC28d groups showed good convergence in PCoA distribution, while the NR28d group still showed some differences from these two groups. These results further illustrate that TMBPA exposure alters the diversity and richness of the gut microbiota in sea medaka; and that kaempferol can help the gut microbiota of exposed sea medaka recover to a healthy state.

[0045] (6) Using SILVA as a reference database, a Naive Bayes classifier was used to perform taxonomic annotation on the feature sequences, which yielded the species classification information corresponding to each feature. Subsequently, the community composition of each sample was statistically analyzed at each level (phylum, class, order, family, genus, species). This study mainly explored the effects of experimental treatments on the gut microbial community composition of marine medaka at the phylum and genus levels, and selected the top 15 microbial communities with the highest relative abundance at the phylum level and the top 20 microbial communities with the highest relative abundance at the genus level for mapping.

[0046] like Figure 7 As shown, at the phylum level, the top three microbial communities with the highest relative abundance in the NC7d group in female fish are: Firmicutes (29.51%) Bacteroidota (23.01%) Proteobacteria (21.99%). The top three microbial communities in terms of relative abundance in the TT24h, TT72h, and TT7d groups were respectively Proteobacteria , Actinobacteriota and Firmicutes Three groups Proteobacteria The relative abundance percentages were 51.29%, 39.04%, and 36.76%, respectively. Actinobacteriota The relative abundance percentages were 22.86%, 31.70%, and 27.09%, respectively. Firmicutes The relative abundance percentages were 13.72%, 14.19%, and 15.41%, respectively. The top three microbial communities in terms of relative abundance in the NC14d, NR14d, and KR14d groups were all... Firmicutes , Bacteroidota and Proteobacteria Three groups Firmicutes The relative abundance percentages were 32.06%, 29.17%, and 30.54%, respectively. Bacteroidota The relative abundance percentages were 24.52%, 24.67%, and 25.32%, respectively. Proteobacteria The relative abundance percentages were 18.36%, 17.33%, and 15.39%, respectively. The top three microbial communities in terms of relative abundance in the NC28d, NR28d, and KR28d groups were all... Firmicutes , Bacteroidota and Proteobacteria Three groups Firmicutes The relative abundance percentages were 36.70%, 30.85%, and 37.04%, respectively. Bacteroidota The relative abundance percentages were 31.73%, 26.07%, and 31.07%, respectively. Proteobacteria The relative abundance percentages were 9.58%, 25.31%, and 13.59%, respectively. Figure 7 A1-C1).

[0047] The top three microbial communities in terms of relative abundance in the NC7d group of male fish were: Firmicutes (29.65%) Bacteroidota (22.59%) Proteobacteria (20.19%). The top three most abundant microbial communities in both the TT24h and TT7d groups were... Firmicutes (31.00% and 29.31%) Proteobacteria (28.61% and 24.94%) and Bacteroidota (15.52% and 20.13%); the top three microbial communities in terms of relative abundance in the TT72h group were Proteobacteria (60.33%) Actinobacteriota (16.79%) and Fusobacteriota (14.40%). The top three most abundant microbial communities in the NC14d, NR14d, and KR14d groups were all... Proteobacteria , Firmicutes and Bacteroidota Three groups Proteobacteria The relative abundance percentages were 48.31%, 17.72%, and 17.29%, respectively. Firmicutes The relative abundance percentages were 20.33%, 29.86%, and 28.74%, respectively. Bacteroidota The relative abundance percentages were 13.05%, 25.64%, and 25.17%, respectively. The top three microbial communities in terms of relative abundance in the NC28d, NR28d, and KR28d groups were all... Firmicutes , Bacteroidota and Proteobacteria Three groups Firmicutes The relative abundance percentages were 36.56%, 32.62%, and 34.72%, respectively. Bacteroidota The relative abundance percentages were 27.50%, 31.33%, and 33.48%, respectively. Proteobacteria The relative abundance percentages were 15.18%, 13.59%, and 12.75%, respectively. Figure 7 A2-C2).

[0048] Overall, TMBPA exposure affected the dominant community of gut microbiota at the phylum level in marine killifish, for example, in different exposure groups of male and female fish. Proteobacteria The proportions of all of them have increased relatively. Bacteroidota The proportions of both decreased relatively. After recovery from kaempferol administration, the dominant community at the phylum level in the female KR14d group was similar to that in the control group (NC14d), while the dominant community at the phylum level in the male KR28d group was similar to that in the control group (NC28d). This suggests that there may be a difference in the recovery speed between male and female fish, with female fish possibly recovering to normal levels more quickly.

[0049] like Figure 8 As shown, at the genus level, the dominant bacterial genus in the NC7d group of female fish is...Vibrio (9.51%) Bacteroides (4.60%) and unclassified_Muribaculaceae (4.00%); the dominant bacterial genera in the TT24h, TT72h, and TT7d groups were Mycobacterium , Vibrio , Ruegeria , ZOR0006 and Cetobacterium The dominant bacterial genus in the NC7d group of male fish was... unclassified_Muribaculaceae (4.08%) Bacteroides (3.52%) and unclassified_Bacteria (3.64%) Figure 8 A1); The dominant bacterial genera in the male fish TT24h, TT72h, and TT7d groups were: Vibrio , Cetobacterium , Ruegeria , Mycobacterium ( Figure 8 A2). Dominant bacterial genera in these exposure groups at different time points. Mycobacterium , Vibrio , Ruegeria and Cetobacterium The TMBPA exposure contained a large number of harmful bacteria, which was higher than that in the control group, indicating that TMBPA exposure leads to an increase in the relative proportion of harmful bacteria in the gut microbiota of marine medaka.

[0050] After 14 days of recovery, the dominant bacterial genera in the female fish of the NC14d, NR14d, and KR14d groups were similar, namely... unclassified_Muribaculaceae , unclassified_Bacteria , Bacteroides and Lactobacillus wait( Figures 5 - 8 B1). The dominant bacterial genus in the NC14d group of male fish was... Vibrio , Shewanella and ZOR0006 The dominant bacterial genera in the NR14d and KR14d groups are similar, being... unclassified_Muribaculaceae , unclassified_Bacteria , Bacteroides and Lactobacillus ( Figure 8 B2).

[0051] After 28 days of recovery, in the female fish of the NR28d group... Vibrio The highest percentage was 11.83% in the KR28d group. Vibrio The proportion was relatively low at 2.33% in the control group (NC28d). Vibrio It accounts for only 0.02%. Some species of Vibrio are important pathogens of acute gastroenteritis. This suggests that kaempferol can help reduce the proportion of harmful bacteria in the gut of sea medaka. LactobacillusIt is a beneficial bacteria genus, accounting for 0.95% in the NC28d group, 0.73% in the NR28d group, but 1.50% in the KR28d group. Lactobacillus The relative proportion is more than twice that of the NR28d group. Figure 8 C1).

[0052] In male fish, the dominant bacterial genera were similarly distributed in the NC28d, NR28d, and KR28d groups, respectively. Bacteroides , Paraprevotella , Blautia and unclassified_Muribaculaceae However, some conditionally pathogenic or potentially harmful bacteria in the NR28d group, such as Vibrio , Streptococcus The proportion of bacteria in the KR28d group was relatively high; while some beneficial bacteria, such as... Akkermansia , Lactobacillus and Ligilactobacillus The relative proportion of these groups was higher than that of the NR28d group. Figure 8 C2).

[0053] This indicates that kaempferol can increase the proportion of beneficial bacteria in the gut of sea medaka, maintain gut health, and help repair damage.

[0054] The above research results of this invention show that TMBPA affects the diversity and richness of the gut microbiota of marine medaka, as well as the proportion of each bacterial group, with a large number of harmful bacteria or opportunistic pathogens (such as... Mycobacterium , Vibrio The proportion of beneficial bacteria (such as...) increased. Kaempferol helped restore the imbalanced gut microbiota to a state similar to the control group, and some of these beneficial bacteria (such as...) Akkermansia , Lactobacillus and Ligilactobacillus The increased proportion of (etc.) indicates that kaempferol can repair the imbalance of gut microbiota in fish caused by TMBPA and restore gut health.

Claims

1. Application of kaempferol in the preparation of products that repair the imbalance of intestinal microbiota in fish.

2. The application according to claim 1, characterized in that, Application of kaempferol in the preparation of products for repairing the imbalance of fish gut microbiota caused by tetramethylbisphenol A exposure.

3. The application according to claim 1, characterized in that, Artemia containing kaempferol was added to the water for feeding fish.

4. Application of Artemia containing kaempferol in the preparation of products that repair the imbalance of gut microbiota in fish.

5. The application according to claim 4, characterized in that, This study investigates the application of Artemia containing kaempferol in the preparation of products that repair the imbalance of the intestinal microbiota in fish caused by tetramethylbisphenol A exposure.

6. The application according to any one of claims 1 to 5, characterized in that, The product helps restore the imbalanced gut microbiota of fish to a healthy state by reducing the proportion of harmful bacteria and increasing the proportion of beneficial bacteria in the gut.

7. The application according to any one of claims 3 to 5, characterized in that, The method for preparing the brine shrimp containing kaempferol is to hatch brine shrimp in artificial seawater with added kaempferol, thereby enriching the insect body with kaempferol.

8. The application according to claim 7, characterized in that, The concentration of kaempferol added is 25–35 mg / L.

9. The application according to claim 7, characterized in that, The incubation process involves constant temperature incubation at 24–28°C for 20–30 hours.

10. The application according to any one of claims 1 to 5, characterized in that, The fish in question is the medaka.

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