CN117487689A - Bacteroides fragilis for producing arachidonic acid and application thereof in improving fatty liver hemorrhagic syndrome of laying hens - Google Patents
Bacteroides fragilis for producing arachidonic acid and application thereof in improving fatty liver hemorrhagic syndrome of laying hens Download PDFInfo
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- CN117487689A CN117487689A CN202311196101.6A CN202311196101A CN117487689A CN 117487689 A CN117487689 A CN 117487689A CN 202311196101 A CN202311196101 A CN 202311196101A CN 117487689 A CN117487689 A CN 117487689A
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- Prior art keywords
- bacteroides fragilis
- arachidonic acid
- bacteroides
- fatty liver
- cgmcc
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
The invention provides a bacteroides fragilis (Bacteroides fragilis) which is preserved in China general microbiological culture collection center with a preservation number of CGMCC No.28150. The bacteroides fragilis disclosed by the invention can improve liver steatosis caused by HELP diet. The bacterium has a certain tolerance to acid and bile salts, so that the bacterium can colonise the intestinal tract through the gastrointestinal tract environment. The strain can change intestinal flora, remarkably improve the abundance of bacteroides and produce arachidonic acid to improve fatty liver hemorrhagic syndrome of laying hens. This was confirmed in chicken LMH cell experiments, i.e. the addition of arachidonic acid reversed hepatic cell steatosis induced by oleic and palmitic acids. Compared with other probiotics for improving the fatty liver of the laying hen, the bacteroides fragilis which can be used for improving the fatty liver of the laying hen has a clearer action mode, and the beneficial effect of the bacteroides fragilis through the metabolites is verified.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to an arachidonic acid-producing bacteroides fragilis and application thereof in improving the fatty liver hemorrhagic syndrome of laying hens, in particular to application in improving the fatty liver hemorrhagic syndrome of laying hens induced by high-fat diet.
Background
The Fatty Liver Hemorrhagic Syndrome (FLHS) of the laying hen is a metabolic disease characterized by lipid metabolism disorder of the laying hen, and the direct cause of the disease is that Triglyceride (TG) is excessively accumulated in liver cells, and the main pathology is liver steatosis, and liver hemorrhage is broken when serious, so that the laying hen dies. The incidence rate of FLHS of the cage-raised laying hens is up to 30%, and liver steatosis is generated in the initial incidence period, so that the laying rate is reduced, and the egg laying peak period is shortened; with the progress of the disease, liver lesions deepen, rupture and bleeding cause sudden death of the laying hens, and the death rate is about 5%, which is the primary cause of death of the laying hens due to non-infectious diseases. In conclusion, the disease causes great economic loss for the laying hen breeding.
At present, the clinical medicines for preventing and treating the FLHS of the laying hens mainly comprise choline chloride, vitamin E, vitamin B, inositol and the like, but have certain side effects (hepatotoxicity, heavy metal accumulation and the like) and have longer administration time.
Numerous reports have been presented to demonstrate the important improvement effect of intestinal symbiotic bacteroides on liver lipid metabolism, for example, a strain of folic acid-producing bacteroides xylanisolvens is found in the institute of China academy of sciences microorganism group Liu Hongwei/Liu Shuangjiang, the strain can significantly improve the non-alcoholic fatty liver of rats, and a strain of succinic acid-producing parabacteroides diri is also found, and the strain can significantly improve the glycolipid metabolism of organisms. Bacteroides as a 'new generation' of probiotics which have great probiotics potential and are safe and nontoxic are rarely reported and applied to improving the FLHS of laying hens.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a strain of bacteroides fragilis (Bactoidesfragilis) for producing arachidonic acid, which is preserved in China general microbiological culture collection center (CGMCC) for 8 months and 10 days in 2023, and the preservation address is North Chen Xiu No. 1 and No. 3 in the Chaoyang area of Beijing city, and the preservation number is CGMCC No.28150. And the bacterium is proved to play a role in resisting liver steatosis through the metabolite arachidonic acid, so that the bacterium is used for improving the fatty liver hemorrhagic syndrome of the laying hen induced by high-fat diet. The invention solves the problem of toxic and side effects of the current clinical application of the layer chicken FLHS, and aims to prevent and treat the layer chicken FLHS and improve lipid metabolism disorder of the layer chicken liver and organism.
The inventor of the application finds that compared with healthy laying hens, the bacteroides abundance in the intestinal tract of the laying hens suffering from FLHS and the arachidonic acid content in the liver are obviously reduced, and correlation analysis finds that the bacteroides abundance and the arachidonic acid content are highly correlated. Thus, the inventors of the present application speculate that the decrease in bacteroides abundance in the gut is a significant cause of the decrease in arachidonic acid content in the liver. Subsequently, we screened from the genus caecum of laying hens for Bacteroides fragilis capable of secreting arachidonic acid and performed a series of experiments to verify our hypothesis.
The invention provides a bacteroides fragilis (bacteroides fragilis), which is preserved in China general microbiological culture collection center (CGMCC) with a preservation number of CGMCC NO.28150.
The invention also provides a bacteroides fragilis bacterial liquid, which is obtained by carrying out amplification culture on the bacteroides fragilis bacterial strain.
Preferably, the expansion culture is specifically: inoculating the bacteroides fragilis strain into a BHI liquid culture medium for activating and increasing bacteria, and performing anaerobic culture for 24-48 hours at 37+/-1 ℃.
The invention also provides application of the bacteroides fragilis or bacteroides fragilis bacterial liquid in preparation of products of the fatty liver hemorrhagic syndrome of the laying hens.
Preferably, the concentration of the Bacteroides fragilis bacterial liquid is 1×10 8 -1×10 10 CFU/mL。
Preferably, the concentration of the Bacteroides fragilis bacterial liquid is 1×10 9 CFU/mL。
Preferably, the bacteroides fragilis is applied to the preparation of products for improving fatty liver hemorrhagic syndrome of laying hens induced by high-fat diet.
Preferably, the bacteroides fragilis improves the fatty liver hemorrhagic syndrome of the laying hen induced by high fat diet by producing arachidonic acid.
The invention also provides a medicine, and the effective component of the medicine is the bacteroides fragilis or the bacteroides fragilis bacterial liquid.
The invention also provides a method for producing arachidonic acid, which comprises inoculating the bacteroides fragilis strain into BHI liquid culture medium, and anaerobic culturing at 37+ -1deg.C for 24-48 h.
The bacteroides fragilis disclosed by the invention can improve liver steatosis caused by HELP diet. The bacterium has a certain tolerance to acid and bile salts, so that the bacterium can colonise the intestinal tract through the gastrointestinal tract environment. The strain can change intestinal flora, remarkably improve the abundance of bacteroides and produce arachidonic acid to improve fatty liver hemorrhagic syndrome of laying hens. This was confirmed in chicken LMH cell experiments, i.e. the addition of arachidonic acid reversed hepatic cell steatosis induced by oleic and palmitic acids. Compared with other probiotics for improving the fatty liver of the laying hen, the bacteroides fragilis which can be used for improving the fatty liver of the laying hen has a clearer action mode, and the beneficial effect of the bacteroides fragilis through the metabolites is verified.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 shows the arachidonic acid content produced by Bacteroides fragilis according to the present invention.
FIG. 2 shows the effect of bacteroides fragilis CGMCCNO.28150 on blood lipid of LMH cells.
FIG. 3 shows the effect of arachidonic acid on blood lipid in LMH cells.
Fig. 4 shows that bacteroides fragilis CGMCC NO.28150 reduces the weight and liver weight of the laying hen.
Fig. 5 shows the effect of bacteroides fragilis CGMCC NO.28150 on liver steatosis of the laying hen.
FIG. 6 shows the effect of bacteroides fragilis CGMCC NO.28150 on the blood lipid and liver function of the laying hen.
FIG. 7 shows the effect of Bacteroides fragilis CGMCC NO.28150 on intestinal flora structure.
FIG. 8 shows the effect of Bacteroides fragilis CGMCC NO.28150 on intestinal flora composition.
FIG. 9 shows the hepatic arachidonic acid content.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagents. The quantitative tests in the following examples were each set up for at least three replicates and the results averaged.
Example 1
1 materials and methods
1.1 materials and reagents
Screening bacteroides:
healthy laying hen cecum and its contents; brain heart extract Broth (BHI) GB standard, bacteroides-bile-esculin (BBE) agar, buchner agar, agar powder, hemin, vitamin K1, antibiotics: vancomycin, gentamicin, kanamycin (a maritime organism, china); sterile defibrinated sheep blood (nanjingsen Bei Ga biotechnology, china).
Determining the content of arachidonic acid:
arachidonic acid ELISA kit (Shanghai Jiang Lai Biotechnology Co., ltd., china).
Cell assay:
chicken liver cancer (LMH) cells (given away by beijing institute of agriculture and forestry); D-MEM/F-12 (gibco, U.S.A.); fetal bovine serum; arachidonic acid (soribao, china); oil red O (Solarbio, china); triglyceride (TG) kit, total cholesterol (T-CHO) kit, low density lipoprotein (LDL-C) kit, high density lipoprotein (HDL-C) kit (Nanjing institute of biological engineering, china).
1.2 test methods
1.2.1 screening of Bacteroides:
(1) Preparation of a culture medium: bacteroides-bile-esculin (BBE) agar: 66.7g BBE was weighed out and dissolved in 1L distilled water and autoclaved at 121℃for 15min.
Brain heart extract Broth (BHI) GB standard 0: 38.5g of BHI was weighed and dissolved in 1L of distilled water containing 0.2g of hemin and 0.2g of vitamin K1, and autoclaved at 121℃for 15min.
Buchner agar (LKV): 43.1g of Brookfield agar is weighed and dissolved in 1L of distilled water, the mixture is autoclaved for 15min at 121 ℃, and after the temperature is reduced to about 50 ℃, 0.01 percent by weight of vancomycin, 0.01 percent by weight of kanamycin and 50mL of sheep blood are added.
(2) Collecting cecum content of healthy layer chicken (Hebei province), suspending with PBS, respectively coating on prepared LKV and BBE plates, and anaerobic culturing at 37deg.C for 48 hr.
(3) After the cultivation is finished, single colonies of the suspected bacteroides are picked and purified for two generations.
(4) The single colony after purification is picked up, activated and enriched in BHI liquid culture medium with pH=7.0-7.4, and anaerobic culture is carried out for 24 hours at 37 ℃.
(5) And (3) extracting DNA of the activated bacterial liquid, sequencing by a 16S rDNA technology, comparing sequencing results in an NCBI database, and identifying strain types.
1.2.2 identification and yield determination of arachidonic acid-producing Bacteroides fragilis
The method is carried out according to the steps in the instruction book of the arachidonic acid ELISA kit.
1.2.3 acid and bile salts resistance
BHI broth ph=3 was adjusted with dilute hydrochloric acid. Centrifuging the activated preserved bacteroides fragilis CGMCC No.281503000 ×g for 10min, discarding the supernatant, re-suspending by using an equal volume of BHI culture solution with pH=3, counting viable bacteria on a flat plate, culturing at 37 ℃ for 3h, and counting the viable bacteria after the culturing is finished.
Bacterial viability = number of bacteria after end/number of bacteria after resuspension x 100%.
The bile salt concentration of the BHI broth was adjusted to 0% (blank), 0.1% (0.1 g bile salt was added to 100mL of the broth), 0.3% (0.3 g bile salt was added to 100mL of the broth), and 0.5% (0.5 g bile salt was added to 100mL of the broth). Respectively inoculating 2% (100 mL culture solution is inoculated with 2mL activated bacterial solution), and performing anaerobic culture at 37 ℃ for 12h. The absorbance of the bacterial liquid at od=600 was measured.
Calculating the bile salt resistance: OD (optical density) Bile salts /OD Blank space ×100％。
1.2.4 cell experiments
Cell modeling: oleic acid and palmitic acid were used to induce steatosis in chicken liver cancer cells (LMH).
Cell test of bacterial liquid supernatant: the bacterial liquid is added while the lipolysis of chicken liver cancer cells (LMH) is induced. The suspension of the bacteroides fragilis CGMCC NO.28150 with OD600 = 0.5 is selected and respectively added into cells in different volume ratios (2 percent and 5 percent), and the cells are incubated for 24 hours by adopting a DMEM high-sugar culture medium.
Arachidonic acid cell assay: arachidonic acid acts on fatty liver model cells at various concentrations of 20, 40, 60, 80 μm, respectively.
1.2.5 animal test
In the invention, 72 healthy Beijing powder laying hens with the age of 30 weeks are used together. Before the start of the experiment, the laying hens were randomly divided into 6 groups of 12 laying hens each. The blank group is continuously fed with basic ration and is fed with carrier solution (PBS), and the rest five groups are fed with high-energy low-protein ration (HELP) to induce the fatty liver of the laying hens. When HELP feed is fed from the beginning, the five groups are also respectively fed with carrier solution (PBS), low-dose bacteroides fragilis CGMCC No.28150 suspension (L-BF or LBF for short), medium-dose bacteroides fragilis CGMCC No.28150 suspension (M-BF or MBF for short), high-dose bacteroides fragilis CGMCC No.28150 suspension (H-BF or HBF for short) and feed added with 0.3% of arachidonic acid. The formulation of the basic ration formula accords with the chicken raising standard (NY/T33-2004) in the agricultural industry standard of the people's republic of China. The induction of fatty liver by HELP feed has been demonstrated in our previous studies. The dosage of basal ration arachidonic acid was determined according to earlier reports.
Wherein, the bacteroides fragilis CGMCC NO.28150 suspension is obtained: activating the bacteroides fragilis CGMCC NO.28150 strain by using a BHI culture solution, centrifuging, discarding culture medium supernatant, and re-suspending the bacterial solution by using PBS, wherein the concentration of the high-medium low-dose bacteroides fragilis CGMCC NO.28150 suspension is as follows: 1X 10 10 CFU/mL，1×10 9 CFU/mL and 1X 10 8 CFU/mL。
All layers were placed in a room with a double cage design, with 2 chickens per individual cage. One week before chicken entry, the room and coop were sterilized according to conventional procedures. The room temperature is maintained at 20 ℃, so that the laying hens can receive 16 hours of illumination every day. The feeding experiment lasted 10 weeks in total, during which time the hens had a free diet, with the first two weeks being the adaptive feeding phase and the next 8 weeks being the formal experiment.
Biological sample collection: the chicken body weight was weighed weekly and recorded. All layers were fasted for 12h before slaughtering at the end of the experiment, and were free to drink water. Subsequently, a blood sample was collected from the subwinged vein and placed on ice. After collection is completed, a serum sample is obtained from these blood by centrifugation at 3000 rpm for 10 minutes. The layers were then euthanized under anesthesia to collect the tissue. The liver of the laying hen was collected, weighed, and the relative weight was calculated, relative weight = tissue organ weight (g)/body weight (kg). The left lobe portion of the liver was fixed with 10% formalin and used to make paraffin sections of the liver, HE stained and oil red o stained. The two ends of cecum are ligated, and the tissue and the content in the middle section of cecum are taken and respectively placed in sterile and aseptic freezing tubes for analysis of intestinal flora. The tissue samples except the samples for analysis are all placed at-80 ℃ and stored until the next analysis.
Biochemical analysis: triglyceride (TG), cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) in cells were all detected using commercial kits. TC, TG, HDL-C, LDL-C, AST, ALT in serum were all detected using a biochemical blood analyzer.
Intestinal flora detection: bacterial DNA was extracted from the cecum content using a bacterial genomic DNA extraction kit (Tiangen Biotechnology Co., beijing, china) according to the instructions of use. Primers 341F (5 '-ACTCCTACGGGRSGCAGAG-3') and 806R (5 '-GGACTACVVGGGTATCTAATC-3') were used for PCR amplification of the 16S rRNA gene. The purified PCR products were sequenced on-machine PE 250 using NovaSeq 6000. The reads of the samples were stitched using FLASH (Version 1.2.11) to yield Raw Tags, which were then quality controlled using fastp software (Version 0.23.1) to yield Clean Tags. Clear Tags were removed by aligning the chimeric sequences with the Silva database. The obtained data are subjected to ASVs denoising, species annotation and the like by using QIIME2 software, and alpha diversity, beta diversity and principal coordinate analysis are analyzed by using the data.
Liver arachidonic acid detection: detection was performed by LC-MS/MS method.
Statistical analysis: analysis was performed using GraphPad Prism 9.5.1 software. The comparison between the groups uses Tukey's test in one-way anova.
1.3 test results
1.3.1 Bacteroides screening:
by screening of Bacteroides fragilis (Bacteroides fragilis) n6 in combination with identification of 16S rDNA.
The screened bacteroides fragilis bacterial liquid is then tested for arachidonic acid production, and the result shows that the bacteroides fragilis can produce arachidonic acid, and the result is shown in figure 1.
FIG. 1 shows the arachidonic acid content produced by Bacteroides fragilis according to the present invention.
The screened bacteroides fragilis (bacteroides fragilis) n6 is preserved in China general microbiological culture collection center (CGMCC) for 8 months and 10 days in 2023, wherein the preservation address is the North Chen Xili No. 1, 3 of the Chaoyang district of Beijing, and the preservation number is CGMCC No.28150.
1.3.2 acid and bile salt resistance results
Acid resistance: the survival rate of the bacteroides fragilis CGMCC NO.28150 bacteria is 60.24 percent.
Bile salt resistance: the survival rate of the bacteroides fragilis CGMCC NO.28150 is 150.81 percent when the concentration of sodium taurocholate is 0.1 percent; the survival rate is 90.55% when the concentration of sodium taurocholate is 0.3%; the survival rate is 59.27% when the concentration of sodium taurodeoxycholate is 0.5%.
1.3.3 cell assay results
Bacteroides fragilis CGMCC NO.28150 bacterial liquid: from FIG. 2, it is evident that the results of the indexes such as TG and LDL-C of LMH cells are remarkably increased by the induction of palmitic acid and oleic acid, and the successful induction of the steatosis of the LMH cells is proved. As shown in figure 2, after the action of the bacteroides fragilis CGMCC NO.28150 bacterial liquid, the TG and LDL-C of LMH cells are reduced to different degrees, and the HDL-C level is increased, which proves that the bacteroides fragilis CGMCC NO.28150 bacterial liquid has obvious improvement effect on the fat model of the LMH cells.
FIG. 2 shows the effect of bacteroides fragilis CGMCC NO.28150 on blood lipid of LMH cells.
Arachidonic acid: as shown in fig. 3, under the action of arachidonic acid, the steatosis of LMH cells has a remarkable improving effect.
FIG. 3 shows the effect of arachidonic acid on blood lipid in LMH cells.
1.3.4 animal experiment results
(1) Weight and liver weight changes
The body weight of the hens increased significantly at week 7 of HELP diet feeding compared to Control group. And at weeks 5, 7 and 8, the intervention of medium-dose bacteroides fragilis CGMCC No.28150 significantly reduced the increase in body weight compared to the HELP group. High and low doses of bacteroides fragilis CGMCC No.28150 intervention did not significantly reduce body weight compared to HELP, but there was still a clear trend (fig. 4). Accordingly, three doses of bacteroides fragilis CGMCC No.28150 were able to reduce liver weight and liver index compared to HELP (fig. 4).
Fig. 4 shows that bacteroides fragilis CGMCC No.28150 reduces the weight and liver weight of the laying hen.
(2) Influence of bacteroides fragilis CGMCC NO.28150 on liver steatosis of laying hens
The general anatomical diagram of the laying hen shows that the liver of the HELP group shows obvious steatosis, and under the action of the bacteroides fragilis CGMCC No.28150, the liver steatosis is obviously improved, and the improvement of the M-BF group is particularly obvious. In contrast, liver H & E staining and oil red O staining showed that HELP diet induced steatosis, hepatocyte ballooning and lipid deposition were improved by bacteroides fragilis CGMCC No.28150 compared to Control group. From the semi-quantitative results of the oil red o staining, the improvement in the M-BF group was very significant (fig. 5).
FIG. 5 shows the effect of bacteroides fragilis CGMCC No.28150 on liver steatosis of the layer chicken.
The intervention of the bacteroides fragilis CGMCC NO.28150 obviously improves liver injury and obviously reduces the contents of AST and ALT in serum. Long-term HELP diet resulted in increased blood lipid levels in the layers. Compared with the Control group, the HELP group TG, TC and LDL-C levels were significantly increased, HDL-C levels were significantly decreased, and this trend was significantly reversed after supplementation with Bacteroides fragilis CGMCC No.28150 (FIG. 6).
FIG. 6 shows the effect of bacteroides fragilis CGMCC No.28150 on the blood fat and liver function of the laying hen.
(3) Influence of bacteroides fragilis CGMCC NO.28150 on intestinal flora structure composition
The influence of the bacteroides fragilis CGMCC NO.28150 on the intestinal flora structure of the laying hens is researched by adopting a cecum content sample 16S rRNA sequencing method. To determine whether the sequencing depth and sample size of the study was adequate, a species dilution curve and a species accumulation box plot based on the shannon index were used to determine. As the amount of sequencing data increases, the diversity index curve tends to flatten, indicating that the amount of sequencing data is reasonable. As the sample size increases, the species abundance increases and decreases, the box plot position flattens out, and the sample size for the study is sufficient. Based on statistical results (fig. 7a, b), HELP diets did not alter the alpha diversity of the intestinal flora. Compared with the HELP group, the bacteroides fragilis CGMCC NO.28150 improves dominance index (P= 0.0674), which has a positive effect on the uniformity of the cecum flora to a certain extent, but is not significant. In addition, according to the index results of chao1, simpson, shannon and the like, the bacteroides fragilis CGMCC NO.28150 has no effect of changing the alpha diversity of the cecum flora. The effect of HELP diet on intestinal flora beta diversity is similar. PCoA and NMDS results based on jaccard index showed that the intestinal flora of HELP group and Control group clustered separately, and MBF also exhibited a different flora structure than HELP group (FIGS. 7C, D). The jaccard index-based PCoA analysis only considers species and not species abundance, so we have found by the unweighteduniferac-based NMDS analysis that the regional systems of the three groups of cecum bacteria are not significantly distinguished. To determine the rationality of the group, intra-group and inter-group differences were analyzed by Analysis ofsimilarities (ANOSIM) based on the Bray-Curtis distance. According to the results, R-value of the comparison between the three groups is greater than 0, indicating that the difference between the groups is greater than the difference in the groups. In general terms. The bacteroides fragilis CGMCC No.28150 has a certain effect on the structure of cecum flora, but is not obvious (figure 7).
FIG. 7 shows the effect of Bacteroides fragilis CGMCC No.28150 on the structure of intestinal flora.
Next, the composition between species of the cecum flora was further analyzed. At the portal level, the cecal bacilloidota and Firmicutes and take up absolute advantage, with a ratio of about 90%, followed by Euryarchaeota. HELP diet resulted in a decrease in Bactoidota and an increase in Firmics, whereas feeding Bacteroides fragilis CGMCC No.28150 intervenes in this process and showed the opposite result. Firmics and Bactoidota are closely related to body lipid metabolism, and in general, the ratio (F/B) of these two is positively related to NAFLD. The intervention of bacteroides fragilis CGMCC No.28150 reduces F/B but is not significant. In addition, the interference of the bacteroides fragilis CGMCC NO.28150 obviously reduces the relative abundance of the Proteibacteria. At the genus level, the bacterial composition of the MBF group is closer to the Control group. HELP diets changed the relative abundance of bacterial species, bactoides, monoglobus, clostridium was decreased, succinatimmons and Faecalicocus were increased, and MBF groups showed the opposite trend. From FIG. 8, the relative abundance of Bacteroides is much greater than that of other bacteria, occupying the dominant position of intestinal flora. In combination with the Lefse analysis, both bacterioidota and bacterioides are significantly enriched in MBF, a marked differential bacteria. Metastat analysis also demonstrated the degree of contribution of Bactoides (P < 0.01). In conclusion, the addition of Bacteroides fragilis CGMCC No.28150 reversed the intestinal flora changes caused by HELP diet and enriched bacterides, which had a positive effect on the improvement of liver lipid metabolism (fig. 8).
FIG. 8 shows the effect of Bacteroides fragilis CGMCC No.28150 on the composition of intestinal flora.
Liver arachidonic acid: through detection, the content of arachidonic acid in the liver of the laying hens fed with the bacteroides fragilis CGMCC No.28150 group is obviously increased compared with that of the HELP group (figure 9).
FIG. 9 shows the hepatic arachidonic acid content.
In conclusion, the bacteroides fragilis CGMCC NO.28150 can improve liver steatosis caused by HELP diet. The bacterium has a certain tolerance to acid and bile salts, so that the bacterium can colonise the intestinal tract through the gastrointestinal tract environment. The strain can change intestinal flora, remarkably improve the abundance of bacteroides and produce arachidonic acid to improve fatty liver hemorrhagic syndrome of laying hens. This was confirmed in chicken LMH cell experiments, i.e. the addition of arachidonic acid reversed hepatic cell steatosis induced by oleic and palmitic acids. Compared with other probiotics for improving the fatty liver of the laying hen, the bacteroides fragilis CGMCC NO.28150 which can be used for improving the fatty liver of the laying hen has a clearer action mode, and the beneficial effect exerted by the metabolites is verified.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A strain of bacteroides fragilis (bacteroides fragilis), characterized in that: the bacteroides fragilis is preserved in China general microbiological culture collection center (CGMCC) No.28150.
2. The bacteroides fragilis bacterial liquid is characterized in that: is obtained by culturing the bacteroides fragilis strain of claim 1 in an expanded manner.
3. The bacteroides fragilis bacteria solution according to claim 2, wherein: the expansion culture is specifically as follows: inoculating the bacteroides fragilis strain into a BHI liquid culture medium for activating and increasing bacteria, and performing anaerobic culture for 24-48 hours at 37+/-1 ℃.
4. Use of bacteroides fragilis as defined in claim 1 or bacteroides fragilis bacterial liquid as defined in claim 2 or 3 for preparing a product of a layer fatty liver hemorrhagic syndrome.
5. The use according to claim 4, characterized in that: the concentration of the bacteroides fragilis bacterial liquid is 1 multiplied by 10 8 ～1×10 10 CFU/mL。
6. The use according to claim 5, characterized in that: the concentration of the bacteroides fragilis bacterial liquid is 1 multiplied by 10 9 CFU/mL。
7. Use according to any one of claims 4-6, characterized in that: the application of the bacteroides fragilis in preparing products for improving fatty liver hemorrhagic syndrome of laying hens induced by high-fat diet.
8. The use according to claim 7, characterized in that: the bacteroides fragilis improves the fatty liver hemorrhagic syndrome of the laying hens induced by high-fat diet by producing arachidonic acid.
9. A medicament, characterized in that: the active ingredient of the medicine is the bacteroides fragilis of claim 1 or the bacteroides fragilis bacterial liquid of claim 2 or 3.
10. A method for producing arachidonic acid, characterized by: inoculating the bacteroides fragilis strain into a BHI liquid culture medium, and performing anaerobic culture at 37+/-1 ℃ for 24-48 hours.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311196101.6A CN117487689A (en) | 2023-09-18 | 2023-09-18 | Bacteroides fragilis for producing arachidonic acid and application thereof in improving fatty liver hemorrhagic syndrome of laying hens |
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| CN202311196101.6A CN117487689A (en) | 2023-09-18 | 2023-09-18 | Bacteroides fragilis for producing arachidonic acid and application thereof in improving fatty liver hemorrhagic syndrome of laying hens |
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