Enterococcus faecalis for producing cholic acid and application of enterococcus faecalis

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to enterococcus faecalis for producing cholic acid and application of the enterococcus faecalis. The invention aims to provide a new choice for improving the HUFA content in the muscle of the bred animal. According to the technical scheme, the enterococcus faecalis DZNEF-13-021 is obtained, and the preservation number of the enterococcus faecalis DZNEF-13-021 is CGMCC (China General Microbiological Culture Collection Center) No. 29592. The invention also provides an application of the enterococcus faecalis DZNEF-13-021 in the improvement of the content of highly unsaturated fatty acids in cultured animals, and the application of the enterococcus faecalis DZNEF-13-021 in the improvement of the content of highly unsaturated fatty acids in the cultured animals. The enterococcus faecalis with high yield of cholic acid is screened out. The strain is suitable for being used by bred animals for a long time in the modes of feed, fungicide, bacterial powder and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a bile acid-producing Enterococcus faecalis and application thereof. Background Art

[0002] During the breeding process of yellow croaker, the fat content level often determines the fullness of the edible part of the yellow croaker. The appropriate fat content can not only allow consumers to better feel the fat aroma during consumption, but also make the fish's body surface more rounded and heavier. During the muscle quality testing of yellow croaker farmed in nets and cages, it was found that the total amino acid, crude protein, and PUFA of yellow croaker farmed in nets were significantly higher than those of yellow croaker farmed in cages. In particular, the content of HUFA (highly unsaturated fatty acids) with a carbon chain length greater than 20, the difference between wild yellow croaker and farmed yellow croaker is extremely significant. In addition, HUFA is also the fatty acid with the highest content in wild yellow croaker, because the HUFA content is an important factor affecting the taste and market price of yellow croaker.

[0003] At present, the research on the HUFA content of large yellow croaker is mainly focused on nutrition and feed. HUFA-rich fortified rotifers are used to feed large yellow croaker fry, among which 0.15g / L concentration is more suitable for the growth of large yellow croaker fry. In general, the HUFA content of large yellow croaker is proportional to the HUFA content in the feed, and the relative content of liver DHA and EPA increases with the increase of HUFA content in the feed. But it is not a simple linear relationship. When the n-3 HUFA level in the feed is 0.98%, the growth traits are significantly enhanced compared with the ordinary group of 0.15% HUFA (P<0.05). Within a certain range, the HUFA content in the muscle tissue of large yellow croaker is proportional to the HUFA content in the feed. At the same time, how to improve the absorption and utilization efficiency of HUFA in feed becomes an important issue.

[0004] Bile acid (hydroxy-5beta-cholanic acid) is an important component of bile acid. The bile acid molecule contains both hydrophilic hydroxyl and carboxyl groups, as well as hydrophobic methyl and hydrocarbon cores. Therefore, the main configuration of bile acid has two sides, hydrophilic and hydrophobic, which makes the molecule have the characteristics of interfacial active molecules, can reduce the surface tension between oil and water phases, and promote lipid emulsification. Fat dissolves in the center of the bile acid molecular group and is wrapped by the bile acid molecules, which reduces the surface tension of fat, emulsifies fat into droplets, and disperses in the intestinal cavity, thus increasing the action area of ​​pancreatic lipase and accelerating its decomposition of fat substances. Improve the absorption efficiency of lipid substances in the intestine and increase the utilization efficiency of HUFA (highly unsaturated fatty acids) in large yellow croaker. Because whether the content of bile acid in the intestine can be increased becomes an important basis for improving the absorption efficiency of HUFA in large yellow croaker. Summary of the invention

[0005] The purpose of the present invention is to provide a new option for increasing the content of HUFA in the muscles of farmed animals.

[0006] The technical solution of the present invention is Enterococcus faecalis DZNEF-13-021, with the preservation number of CGMCC No.29592.

[0007] The present invention also provides the use of the Enterococcus faecalis DZNEF-13-021 in increasing the content of highly unsaturated fatty acids in farmed animals.

[0008] Specifically, the highly unsaturated fatty acid is bile acid.

[0009] Furthermore, the farmed animals are aquatic animals.

[0010] Furthermore, the aquatic animal is a fish.

[0011] Particularly, the fish is large yellow croaker.

[0012] Preferably, in the application, the Enterococcus faecalis DZNEF-13-021 is prepared into feed, bacterial powder or bacterial agent for use.

[0013] Particularly, the preparation method of the bacterial powder comprises the following steps: collecting the fermentation liquid of the Enterococcus faecalis DZNEF-13-021, centrifuging, collecting bacterial mud, and re-dissolving the bacterial mud with the centrifuged supernatant into a viscous state; uniformly mixing the bacterial mud with a carrier; granulating, drying; and obtaining bacterial powder.

[0014] Specifically, the carrier is an inorganic salt carrier, such as corn cob powder, montmorillonite powder, medical stone and / or stone powder.

[0015] Specifically, the weight ratio of the bacterial mud to the carrier is 55:45.

[0016] Furthermore, the weight ratio of the mushroom mud, corn cob powder and montmorillonite powder is 55:30:15.

[0017] Wherein, the drying temperature is controlled below 35°C.

[0018] Specifically, the humidity of the bacterial powder is below 10%.

[0019] Particularly, the preparation method of the feed comprises the following steps: collecting the fermentation liquid of the Enterococcus faecalis DZNEF-13-021, centrifuging, collecting bacterial sludge, and re-dissolving the bacterial sludge with the centrifugal supernatant into a viscous state; uniformly mixing the bacterial sludge with a carrier; granulating and drying; obtaining bacterial powder; and mixing the bacterial powder with a basic feed.

[0020] Specifically, the carrier is corn cob powder, montmorillonite powder, medical stone and / or stone powder.

[0021] Specifically, the weight ratio of the bacterial mud to the carrier is 1:1.

[0022] Wherein, the drying temperature is controlled below 35°C.

[0023] Specifically, the humidity of the bacterial powder is below 10%.

[0024] Preferably, the amount of Enterococcus faecalis added to the feed is 0.1-1 kg / ton of basic feed.

[0025] Preferably, the addition amount of Enterococcus faecalis in the feed is 0.5-1 kg / ton of basic feed.

[0026] More preferably, the addition amount of Enterococcus faecalis in the feed is 1 Kg / ton of basic feed.

[0027] The invention also provides an Enterococcus faecalis powder, which comprises Enterococcus faecalis DZNEF-13-021 bacterial mud and a carrier in a weight ratio of 55:45.

[0028] Specifically, the carrier is corn cob powder, montmorillonite powder, medical stone and / or stone powder.

[0029] Specifically, the weight ratio of the bacterial mud to the carrier is 55:45.

[0030] Furthermore, the weight ratio of the mushroom mud, corn cob powder and montmorillonite powder is 55:30:15.

[0031] The present invention also provides a feed containing Enterococcus faecalis. The Enterococcus faecalis is prepared into bacterial powder and then mixed with a basic feed.

[0032] Preferably, the amount of Enterococcus faecalis added to the feed is 0.1-1 kg / ton of basic feed.

[0033] Preferably, the addition amount of Enterococcus faecalis in the feed is 0.5-1 kg / ton of basic feed.

[0034] More preferably, the addition amount of Enterococcus faecalis in the feed is 1 Kg / ton of basic feed.

[0035] The present invention also provides a breeding method for animals with high content of highly unsaturated fatty acids, comprising the following steps: feeding Enterococcus faecalis DZNEF-13-021 to the animals.

[0036] Specifically, the highly unsaturated fatty acid is bile acid.

[0037] Specifically, Enterococcus faecalis DZNEF-13-021 is prepared into fermentation liquid, bacterial powder, bacterial agent or feed containing Enterococcus faecalis DZNEF-13-021.

[0038] Particularly, the preparation method of the bacterial powder comprises the following steps: collecting the fermentation liquid of the Enterococcus faecalis DZNEF-13-021, centrifuging, collecting bacterial mud, and re-dissolving the bacterial mud with the centrifuged supernatant into a viscous state; uniformly mixing the bacterial mud with a carrier; granulating, drying; and obtaining bacterial powder.

[0039] Specifically, the carrier is corn cob powder, montmorillonite powder, medical stone and / or stone powder.

[0040] Specifically, the weight ratio of the bacterial mud to the carrier is 55:45.

[0041] Furthermore, the weight ratio of the mushroom mud, corn cob powder and montmorillonite powder is 55:30:15.

[0042] Wherein, the drying temperature is controlled below 35°C.

[0043] Specifically, the humidity of the bacterial powder is below 10%.

[0044] Particularly, the preparation method of the feed comprises the following steps: collecting the fermentation liquid of the Enterococcus faecalis DZNEF-13-021, centrifuging, collecting bacterial sludge, and re-dissolving the bacterial sludge with the centrifugal supernatant into a viscous state; uniformly mixing the bacterial sludge with a carrier; granulating and drying; obtaining bacterial powder; and mixing the bacterial powder with a basic feed.

[0045] Specifically, the carrier is corn cob powder, montmorillonite powder, medical stone and / or stone powder.

[0046] Specifically, the weight ratio of the bacterial mud to the carrier is 55:45.

[0047] Furthermore, the weight ratio of the mushroom mud, corn cob powder and montmorillonite powder is 55:30:15.

[0048] Wherein, the drying temperature is controlled below 35°C.

[0049] Specifically, the humidity of the bacterial powder is below 10%.

[0050] Preferably, the amount of Enterococcus faecalis added to the feed is 0.1-1 kg / ton of basic feed.

[0051] Preferably, the addition amount of Enterococcus faecalis in the feed is 0.5-1 kg / ton of basic feed.

[0052] More preferably, the addition amount of Enterococcus faecalis in the feed is 1 Kg / ton of basic feed.

[0053] Furthermore, the animal is an aquatic animal.

[0054] Furthermore, the aquatic animal is a fish.

[0055] Particularly, the fish is large yellow croaker.

[0056] Beneficial effects of the present invention: The present invention screened a strain of Enterococcus faecalis that produces high bile acid. The strain is suitable for long-term use in the form of feed, bacterial agents, bacterial powder, etc. for farmed animals, can improve the absorption efficiency of lipids in farmed animals, increase the HUFA (highly unsaturated fatty acids) content in muscle tissue, improve meat quality, and has broad application prospects.

[0057] The Enterococcus faecalis DZNEF-13-021 of the present invention is deposited in the China Center for Type Culture Collection (CGMCC for short), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 29592 and the deposit date January 15, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a microscopic picture of Enterococcus faecalis, taken with a 1600x oil objective, which converts to a scale of 1500:1.

[0059] Figure 2 This is a picture of the colony morphology of Enterococcus faecalis.

[0060] Figure 3 Various strains are isolated from the breeding environment.

[0061] Figure 4 It is the state of the fermentation broth of each strain.

[0062] Figure 5 This is the standard curve for high performance liquid chromatography detection of bile acid.

[0063] Figure 6 It is a high performance liquid chromatography method to detect the bile acid content in bacterial powder. DETAILED DESCRIPTION

[0064] By chance, the inventor isolated a variety of strains from animal feces in a farm, separated and purified each type of strain, and prepared fermentation broth of each strain in the laboratory. After the fermentation was completed, the bile acid content in the fermentation broth of different culture media was detected by high-performance liquid chromatography, and a strain with high bile acid production was obtained. After biological characteristic detection, physiological and biochemical identification, 16srDNA sequencing analysis and other means, it was finally identified as Enterococcus faecalis. The specific application of this Enterococcus faecalis was studied, and the bacterial powder was prepared and applied in the feed addition of large yellow croaker. It has a significant ability to increase the content of long-chain unsaturated fat HUFA in the muscle tissue of large yellow croaker.

[0065] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0066] Example 1 Screening of high bile acid producing strains 1) Screening and verification A variety of strains were isolated from animal feces in the breeding farm, and single colonies were isolated by streaking in a culture dish. Culture medium ingredients (g / L): 20g cornmeal, 10g soybean meal, 5g glucose, 5g yeast extract, 5g sodium chloride, 3g K2HPO4, 3g KH2PO4, 0.3g MnSO4, 17g agarose, pH adjusted to 7.0. Sterilize at 121°C, and after cooling to 55°C, pour the plate, ferment at 36°C, place in an anaerobic tank, add oxygen remover, and culture. Subculture single colony ( Figure 3 ),spare.

[0067] 2) Liquid fermentation Each isolated strain was inoculated into a liquid culture medium. The culture medium components were as follows: culture medium (g / L): 20 g corn syrup powder, 10 g soybean meal, 5 g glucose, 5 g yeast extract, 5 g sodium chloride, 3 g KH2PO4, pH adjusted to 8.5. Sterilized at 121°C. Fermentation temperature was 36°C. The fermentation broth of different strains was obtained ( Figure 4 ).

[0068] 3) Bile acid content detection 3.1 Instrument and sample preparation: Agilent 1260 II ELSD high performance liquid chromatograph, ELSD detector; Agilent ZORBAX SB-Aq (5μm 4.6mm×150mm) chromatographic column (good polarity, high stability, can adapt to up to 100% aqueous phase and as low as pH1.0 environment, column volume is about 2.49mL); vacuum pump and filtration device. Electronic balance (sensitivity 0.0001g). Prepare 1L volumetric flask, 2mL injection bottle, 1L glass bottle, 10mL centrifuge tube, 2mL syringe, 0.22μm microporous filter membrane (organic phase & aqueous phase), pipette and corresponding gun tip (200μL, 1mL, 5mL), etc. Place the volumetric flask, injection bottle, centrifuge tube, etc. in an ultrasonic cleaner, use a detergent solution to ultrasonically clean them for 1 hour, and rinse them with tap water; place them in the cleaner again and use tap water to ultrasonically clean them for 0.5h~1h, and rinse them with tap water; place them in the cleaner for the third time and use ultrapure water to ultrasonically clean them for 0.5h~1h, and rinse them with ultrapure water. Turn the volumetric flask upside down to dry naturally, and place the other materials in an oven to dry.

[0069] Reagents: formic acid (chromatographic grade), acetonitrile, methanol. Unless otherwise specified, the water used in this experiment is chromatographic grade ultrapure water.

[0070] Bile acid standards: including five standards: bile acid, deoxycholic acid, hyodeoxycholic acid, ursodeoxycholic acid, and chenodeoxycholic acid (Note: bile acid, deoxycholic acid, hyodeoxycholic acid, ursodeoxycholic acid, and chenodeoxycholic acid were purchased from the China Food and Drug Administration; deoxycholic acid-sigma was purchased from sigma; hyodeoxycholic acid was purchased from McLean). Use a 1 / 10,000 balance to accurately weigh about 0.020g of bile acid standard, place it in a 10mL volumetric flask, add mobile phase to dissolve and dilute to the scale, shake well, and prepare a standard stock solution (stock solution) with a concentration of 2 mg / mL. The above stock solutions are stored in a refrigerator at 4°C, and the mother solution can be stored for 3 months. Dilute the stock solution to the required concentration when it is used. (Note: If the sample is not easy to dissolve, it can be placed in an ultrasonic instrument for 10 minutes until it is completely dissolved.) Use the mobile phase for gradient dilution based on the mother solution. Accurately weigh 125μL, 250μL, 500μL, 1000μL, 1500μL, 2000μL, and 2500μL and place them in 7 10mL volumetric flasks. Dilute with mobile phase and make up to 25μg / mL, 50μg / mL, 100μg / mL, 200μg / mL, 300μg / mL, 400μg / mL, and 500μg / mL standard solutions, filter through 0.22μm microporous membrane, and add to injection bottle. The standard gradient series solutions are obtained (see Table 1 for the ratio table).

[0071] Table 1 Series gradient ratio table Mobile phase preparation: (I) Use a 0.45 μm microporous filter membrane to filter the water. During the filtration process, the first 100 mL to 200 mL of water should be discarded, and then the water should be collected and placed in a 1 L glass bottle. Cover the bottle with a lid and set aside.

[0072] (II) Prepare 0.2% formic acid aqueous solution: Take 2 mL of formic acid and dilute to 1 L volumetric flask. Filter the 0.2% formic acid aqueous solution using the method in (I), then put it into a 1 L glass bottle, cover it with a lid and set aside.

[0073] 3.2 Chromatographic conditions The chromatographic conditions were as follows: ELSD detector nitrogen flow rate of 1.6 L / min, drift tube temperature of 110°C (evaporator temperature of 60°C; nebulizer temperature of 60°C); mobile phase smoothing time of 30 seconds); mobile phase A was 0.2% formic acid solution, mobile phase C was acetonitrile, gradient elution was: 0-7min, 5%C; 7-20min, 5%→80%C, 20.1-25min: 5%C, and then returned to the initial mobile phase; flow rate of 1.0mL / min; injection volume of 10μL; column temperature of 25°C.

[0074] 3.3 Sample preparation Reference solution: Accurately weigh about 0.020 g of bile acid standard, place in a 10 mL volumetric flask, add methanol to dissolve and dilute to scale, shake well, and prepare a 2 mg / mL standard stock solution (stock solution). Then dilute according to a series of gradients.

[0075] Detection sample solution: use a pipette to draw 25 μL of fermentation liquid, add it into a stoppered conical flask, add 50 mL of methanol tightly, weigh the weight, treat it with ultrasound (200w, frequency 40kHz) for 20 minutes, make up the loss in weight with methanol, filter, and obtain.

[0076] 3.4 Liquid phase test results With the concentration of the reference substance (X, μg / mL) as the horizontal axis and the peak area of ​​the chromatographic peak Y as the vertical axis, a standard working curve is drawn and a linear regression calculation is performed. Figure 5 As shown, the regression curve equation of bile acid (China Inspection Institute) is: y=32.923x-967.19, R2 =0.9951. The results show that in the range of 25~400μg / mL, the chromatogram and peak area of ​​bile acid (China Inspection Institute) have a good linear relationship. Bile acid peak time: 17.428~17.488min. The peak conditions of each standard are shown in Table 2.

[0077] Table 2 Relationship between the content and peak area of ​​each standard The results of the high-performance liquid phase determination of bile acid content in fermentation samples of different single-colony strains are shown in Table 3. Among them, the bile acid content in the fermentation broth of strain No. 5 (numbered as DZNEF-13-021) was 1.584%, which has nutritional value and can be further developed.

[0078] Table 3 Results of HPLC determination of bile acid content in different fermentation samples 4) Identification of the above strain No. 5 Based on the colony morphology of the previous culture and the MRS anaerobic culture method, strain No. 5 was determined to be lactic acid bacteria. Microscopic examination showed that it was a coccus, which was completely different from a bacillus, and was basically confirmed to be Enterococcus faecalis. Further physiological and biochemical identification and molecular identification were carried out.

[0079] The purchased biochemical identification strips of Enterococcus faecalis (Qingdao Haibo Biological) were used for identification according to the prescribed steps, as shown in Table 4: The morphological characteristics of the short Enterococcus faecalis are: the bacteria are oval, with rounded ends, and the size is generally 0.8~1.2μm×1.5~2.0μm, Gram-positive ( Figure 1 and Figure 2 ).

[0080] Table 4 Physiological and biochemical reaction results DNA extraction and PCR amplification of Enterococcus faecalis 16s rDNA fragment sequencing and identification: Add 400µL of sterile pure water to the sterilized 1.5mL centrifuge tube. At the same time, pick up a full loop of the strain to be tested from the culture dish in the clean bench and add it to the centrifuge tube. Place the centrifuge tube on an oscillator for oscillation and mixing. Insert it into the float and place it in an ultrasonic instrument. Ultrasonicate at 40KHZ and 600w for 5 minutes. Take it out and shake it again. Ultrasonicate it for 5 minutes again. Ultrasonicate it 3 times in total, each time for 5 minutes. Then put the bacterial solution after ultrasonic disruption into the refrigerator as a DNA template for standby use.

[0081] During the experiment, it was found that a small number of strains were difficult to effectively release the DNA in the cells, whether they were ultrasonically broken or heated to denature the cells. Therefore, for such strains, a DNA extraction kit was used to extract the DNA of the bacteria, which was used as a template for PCR amplification. The kit used in this process is the Bacterial Genomic DNA Rapid Extraction Kit (Shanghai Shenggong, product number: B518225). The standard operating steps are as follows: Take 1 mL of overnight cultured bacterial culture, add it to a 1.5 mL centrifuge tube, centrifuge at 8,000 rpm for 1 min at room temperature, discard the supernatant, and collect the bacteria. Add 500 µL Buffer Digestion and shake to mix. Incubate at 65℃ for 2 hours until the cells are completely lysed. During the water bath, invert and mix once every 10 minutes to promote sample lysis. The mixture becomes clear and transparent, indicating that the lysis is complete. If the solution does not become clear, it means that the sample is not completely lysed, and the water bath time should be appropriately extended. Add 200 µL Buffer PB, invert and mix thoroughly, and place on ice for 5 minutes. Centrifuge at 10,000 rpm for 5 min at room temperature, transfer the supernatant (500~550µL) to a new 1.5 mL centrifuge tube. Add an equal volume of isopropanol, invert 5~8 times to mix thoroughly, and let stand at room temperature for 2~3 min. Centrifuge at 10,000 rpm for 5 min at room temperature, discard the supernatant. Add 1 mL of 75% ethanol, invert and rinse for 1~3 min, centrifuge at 10,000 rpm for 2 min, and discard the supernatant (twice). Invert at room temperature with the lid open for 5~10 min until the residual ethanol is completely evaporated. Dissolve the obtained DNA with 50~100 µL TE Buffer. The extracted DNA can be used for the next experiment immediately or stored at -20℃.

[0082] PCR system (25µL): 16µL purified water, 2.5µL PCR buffer, 0.5µL dNTP, 1µL each of upstream and downstream primers (27F and 1492R), 2µL template, 0.5µL Taq enzyme, Mg 2+ 1.5µL. The PCR process parameters were set as follows: 94℃ for 10 minutes (to increase the amount of DNA released); 94℃ for 30s, 55℃ for 30s, 72℃ for 90s, 30 cycles; 72℃ for 10min extension; 10℃ storage. The PCR product was electrophoresed using 1% agarose gel. During the gel process, the color developer (Shanghai Bioengineering, 4s green color developer) was added at 1µL / 100mL. 2µL of the PCR product was mixed with 1µL of loading buffer and applied to the agarose gel electrophoresis tank. The voltage was set to 170V for 20 minutes. The amplified product was recovered and sent for sequencing.

[0083] Copy the 16sDNA fragment sequence and the base sequence in the nucleic acid sequence txt file sent back by Bioengineering after sequencing. At the same time, log in to the NCBI gene database, enter the Blast comparison mode, select the nucleic acid comparison database, enter the sequenced seq file sequence into the comparison window, adjust the comparison parameters, and select the strain with a sequence similarity of more than 90% as the reference strain for identification.

[0084] The value of DNA concentration depends on the nucleic acid analyzer for detection, so the gradient dilution method is adopted to explore through experiments, diluting 10 times, 100 times, 150 times and 200 times respectively. Finally, it is detected that the concentration of DNA template diluted 10 times is suitable for the current PCR system. The blast comparison results show that the 16s sequence of this strain has a similarity of 98% with a variety of Enterococcus faecalis strains, so it is determined that this strain belongs to Enterococcus faecalis. At the same time, it was sent to the China Microbiological Culture Collection Center in Beijing for preservation, with the preservation number CGMCC No.29592.

[0085] SEQ ID No.1 16s rDNA

[0086] Example 2 Preparation of microecological products by drying and crushing Enterococcus faecalis fermentation liquid The concentrated bacterial liquid was obtained by centrifugation in a disc centrifuge, and then the bacterial sludge was obtained by centrifugation in a tubular centrifuge. The bacterial sludge was re-dissolved with a small amount of centrifugal supernatant to make it uniform and viscous, which was easy to mix evenly. The bacterial sludge and carrier (corn cob powder and montmorillonite powder) were mixed evenly with a high-speed mixer (55% Enterococcus faecalis bacterial sludge, 15% montmorillonite (feed grade), and 30% corn cob powder). The mixed wet bacterial powder was then prepared into uniform granules by a granulator and placed in a boiling dryer for drying. During boiling drying, the material temperature was controlled below 35°C, so that the humidity of the bacterial powder after drying was below 10%. The processing time for a single batch was about 30 to 60 min. After that, the bile acid content in the bacterial powder product was efficiently detected to be 1.882% (see Figure 6 ).

[0087] Example 3 Results of clinical animal experiments in Waitang The juveniles used in the experiment were from Ningde Jinling Aquatic Technology Co., Ltd., and all breeding experiments were also carried out in the company's nursery. Before the experiment began, the yellow croaker juveniles taken from the sea area net cages were temporarily kept in a 3 m×3 m×1.5 m indoor cement pool for 1 week, and fed with yellow croaker commercial feed at 8:30 and 15:30 every day for one week, and then the formal experiment was carried out after acclimation.

[0088] Before the experimental fish were selected, they were starved for 24 h and anesthetized with clove oil cement (Shanghai Medical Device Co., Ltd.) at a dilution of 1:10000 to avoid stress and damage to the large yellow croaker during the selection process. The water and mucus on the surface of the test fish were wiped dry with gauze. On the premise of confirming that the fish body was not damaged, normal and healthy large yellow croaker were selected for initial weight and length measurement. The group experiment was carried out according to different weight ranges. During the experiment, feed with Enterococcus faecalis powder was fed once at 8:30 and 15:30 every day. The amount of Enterococcus faecalis powder added was 100g, 500g, and 1kg mixed with 1 ton of basic feed. Before feeding, the feed should be taken out of the freezer in advance, soaked in a small amount of water for 20 minutes, and then fed until the large yellow croaker was apparently full. The entire breeding experiment lasted for 70 days. During the experiment, the temperature of the seawater was 25 ± 2 ℃, the salinity of the seawater was 30~35‰, and the dissolved oxygen was maintained above 5mg / L.

[0089] The muscle n-3 HUFA (unsaturated fatty acid) content of 160 large yellow croakers was measured after feeding. The muscle n-3 HUFA content was measured by taking the back muscle of each fish. 160 healthy large yellow croakers were selected. The back muscle of large yellow croaker was selected and the total lipid was extracted by chloroform-methanol method (1:2). The experimental parameters were set according to GB 5009.168-2016. The fatty acids in the muscle were treated with 15% KOH methanol solution at 20℃ for 1 h to methylate the fatty acids in the sample. The lipids were esterified at 85℃ for 15 minutes with 6.5% boron trifluoride (BF3) methanol solution (Morita, Osaka, Japan). After mixing in hexane (25 mg / mL), the fatty acid methyl ester preparation was analyzed by gas chromatography (GC). The obtained fatty acid methyl esters were analyzed and quantified by gas chromatograph (Hewlett-Packard HP-5890) equipped with a cross-linked 5% phenylmethyl silicone gel column (L=25m, ID=0.32min, DF=0.25 μmol / L, HP-Ultra2, with nitrogen as carrier gas). 50 g / L of nonadecanoic acid was used as the internal standard, and the amount used was 10% of the total fatty acids. The detection was carried out by hydrogen flame ionization detector, and the nozzle and detector temperatures were 190℃ and 250℃, respectively. The column temperature was set from 190℃ to 260℃ (2℃ / min) and maintained for 5 min. The phenotypic data were sorted in descending order, and high and low phenotypic values ​​were regarded as extreme phenotypes. The HUFA phenotypic data followed a normal distribution by Shapiro-Wilk test (P>0.05). The absolute quantification method (mg / 100 g sample wet weight) was used for data analysis and presentation in this experiment. The results are shown in Tables 5 and 6: The faecal enterococci have little effect on long-chain unsaturated fatty acids of C12, C16 and C18, but have a greater effect on HUFA (highly unsaturated fatty acids), increasing the content by nearly 100% compared with the control group, which can significantly improve the flavor and nutritional value of large yellow croaker, and is helpful for green, high-economic-value sustainable aquaculture.

[0090] Table 5 Changes in fatty acids in muscle of 160 samples (4 groups) on day 0 (unit: % dry matter) Table 6 Changes in fatty acids in muscle of 176 samples (4 groups) on day 70 (unit: % dry matter) The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. An Enterococcus faecalis DZNEF-13-021, with a deposit number of CGMCC No.29592.

2. Use of the Enterococcus faecalis described in claim 1 in increasing the content of highly unsaturated fatty acids in large yellow croaker.

3. The application according to claim 2, characterized in that: The Enterococcus faecalis described in claim 1 is prepared into feed, bacterial powder or bacterial agent for use.

4. The application according to claim 3, characterized in that: The preparation method of the bacterial powder comprises the following steps: collecting the fermentation liquid of Enterococcus faecalis according to claim 1, centrifuging, collecting bacterial mud, and re-dissolving it into a viscous state with the centrifugal supernatant; uniformly mixing the bacterial mud with a carrier; granulating, and drying; obtaining the bacterial powder; the carrier is corn cob powder, montmorillonite powder, medical stone and / or stone powder; the weight ratio of the bacterial mud to the carrier is 55:

45.

5. The application according to claim 3, characterized in that: The preparation method of the feed comprises the following steps: collecting the fermentation liquid of Enterococcus faecalis according to claim 1, centrifuging, collecting bacterial sludge, and re-dissolving it into a viscous state with the centrifugal supernatant; uniformly mixing the bacterial sludge with a carrier; granulating and drying; obtaining bacterial powder; mixing the bacterial powder with a basic feed; the carrier is corn cob powder, montmorillonite powder, medical stone or / and stone powder; the weight ratio of the bacterial sludge to the carrier is 55:

45.

6. The use according to claim 4 or 5, characterized in that: The drying temperature is controlled below 35° C.; the moisture content of the bacterial powder is below 10%.

7. The use according to claim 6, characterized in that: The added amount of Enterococcus faecalis in the feed is 0.1-1 kg / ton of basic feed.

8. An Enterococcus faecalis powder, characterized in that: Its components are the Enterococcus faecalis sludge as claimed in claim 1 and a carrier in a weight ratio of 55:45, and the carrier is corn cob powder, montmorillonite powder, medical stone and / or stone powder.

9. A feed containing Enterococcus faecalis, characterized in that: The Enterococcus faecalis described in claim 1 is prepared into bacterial powder and then mixed with basic feed; the addition amount of the Enterococcus faecalis is 0.1-1 kg / ton of basic feed.

10. A method for cultivating large yellow croaker with high content of highly unsaturated fatty acids, characterized by: The method comprises the following steps: feeding the Enterococcus faecalis described in claim 1 and / or feed containing the Enterococcus faecalis described in claim 1 to large yellow croaker.

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