An Enterococcus faecalis strain producing cholic acid and its application

Abstract

The present invention belongs to the technical field of microbiology, and particularly relates to an Enterococcus faecalis strain producing cholic acid and its application. The purpose of the present invention is to provide a new option for increasing the content of HUFA in the muscle of farmed animals. The technical solution of the present invention is an Enterococcus faecalis DZNEF-13-021, with the preservation number of CGMCC No. 29592. The present invention also provides the application of the Enterococcus faecalis DZNEF-13-021 in increasing the content of highly unsaturated fatty acids in farmed animals. The present invention screened an Enterococcus faecalis strain with high cholic acid production. This strain is suitable for long-term use in farmed animals in the form of feed, bacterial agents, bacterial powders, etc.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a strain of Enterococcus faecalis producing cholic acid and its application. Background Art

[0002] During the cultivation process of large yellow croaker, the fat content level often determines the plumpness of the edible part of the large yellow croaker. An appropriate fat content can not only enable consumers to better feel the fat fragrance during consumption, but also make the fish body surface more round and the weight greater. During the detection of the muscle quality of large yellow croaker cultured in enclosures and cages, it was found that the total amino acids, crude protein, and total PUFA of large yellow croaker cultured in enclosures were significantly higher than those of large yellow croaker cultured in cages. Especially the content of HUFA (highly unsaturated fatty acids) with a carbon chain length greater than 20, the difference between wild large yellow croaker and cultured large yellow croaker is extremely significant. In addition, HUFA is also the fatty acid with the highest content in wild large yellow croaker, because the HUFA content is an important factor affecting the taste and market price of large yellow croaker.

[0003] At present, the research on the HUFA content of large yellow croaker mainly focuses on nutrition and feed. Enriched rotifers rich in HUFA are used to feed large yellow croaker larvae, and a concentration of 0.15 g / L is more suitable for the growth of large yellow croaker larvae. Generally, the HUFA content of large yellow croaker is proportional to the HUFA content in the fed feed, and the relative contents of DHA and EPA in the liver increase with the increase of the HUFA content in the feed. However, 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 with 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 the feed has become an important issue.

[0004] Cholic acid (hydroxy-5beta-cholanic acid) is an important component of bile acids. The cholic acid molecule contains both hydrophilic hydroxyl and carboxyl groups, as well as hydrophobic methyl and hydrocarbon nuclei. Therefore, the main configuration of cholic acid has two sides, hydrophilic and hydrophobic, making the molecule have the characteristics of an interfacial active molecule, which can reduce the surface tension between the oil and water phases and promote lipid emulsification. Fat is dissolved in the center of the cholic acid molecular cluster and is wrapped by the cholic acid molecules, reducing the surface tension of the fat, emulsifying the fat into microdroplets, and dispersing them in the intestinal cavity. In this way, the action area of pancreatic lipase is increased, and its action of decomposing fat substances is accelerated. The absorption efficiency of lipid substances in the intestine is improved, and the utilization efficiency of HUFA (highly unsaturated fatty acids) in the large yellow croaker body is increased. Because whether the content of cholic acid in the intestine can be increased has become an important basis for improving the HUFA absorption efficiency of large yellow croaker. Summary of the Invention

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

[0006] The technical solution of the present invention is an Enterococcus faecalis DZNEF-13-021 with a 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 cholanic acid.

[0009] Furthermore, the farmed animal is an aquatic animal.

[0010] Even further, the aquatic animal is a fish.

[0011] In particular, the fish is a large yellow croaker.

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

[0013] In particular, the preparation method of the bacterial powder includes the following steps: collecting the fermentation broth of the Enterococcus faecalis DZNEF-13-021, centrifuging, collecting the bacterial sludge, and redissolving it with the centrifuged supernatant to form a viscous state; mixing the bacterial sludge with a carrier evenly; granulating and drying; obtaining the bacterial powder.

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

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

[0016] Furthermore, the weight ratio of the bacterial sludge, corncob powder and montmorillonite powder is 55:30:15.

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

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

[0019] Specifically, the preparation method of the feed comprises the following steps: collecting the fermentation broth of Enterococcus faecalis DZNEF-13-021, centrifuging, collecting the bacterial sludge, and redissolving it with the centrifuged supernatant to form a viscous state; mixing the bacterial sludge evenly with a carrier; granulating and drying to obtain a bacterial powder; and mixing the bacterial powder with a basal feed.

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

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

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

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

[0024] Preferably, the addition amount of Enterococcus faecalis in the feed is 0.1 - 1 Kg per ton of basal feed.

[0025] Preferably, the addition amount of Enterococcus faecalis in the feed is 0.5 - 1 Kg per ton of basal feed.

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

[0027] The present invention also provides an Enterococcus faecalis bacterial powder, which is composed of Enterococcus faecalis DZNEF-13-021 bacterial sludge and a carrier with a weight ratio of 55:45.

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

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

[0030] Furthermore, the weight ratio of the bacterial sludge, corncob powder and montmorillonite powder is 55:30:15.

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

[0032] Preferably, the addition amount of Enterococcus faecalis in the feed is 0.1 - 1 Kg per ton of basal feed.

[0033] Preferably, the addition amount of Enterococcus faecalis in the feed is 0.5 - 1 Kg per ton of basal feed.

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

[0035] The present invention also provides a method for cultivating animals with a 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 cholic acid.

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

[0038] Particularly, the method for preparing the bacterial powder comprises the following steps: collecting the fermentation broth of Enterococcus faecalis DZNEF-13-021, centrifuging, collecting the bacterial sludge, re-dissolving it with the centrifuged supernatant to form a viscous state; mixing the bacterial sludge evenly with a carrier; granulating and drying; obtaining the bacterial powder.

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

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

[0041] Further, the weight ratio of the bacterial sludge, corncob powder and montmorillonite powder is 55:30:15.

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

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

[0044] Particularly, the method for preparing the feed comprises the following steps: collecting the fermentation broth of Enterococcus faecalis DZNEF-13-021, centrifuging, collecting the bacterial sludge, re-dissolving it with the centrifuged supernatant to form a viscous state; mixing the bacterial sludge evenly with a carrier; granulating and drying; obtaining the bacterial powder; mixing the bacterial powder with a basal feed.

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

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

[0047] Further, the weight ratio of the bacterial sludge, corncob powder and montmorillonite powder is 55:30:15.

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

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

[0050] Preferably, the addition amount of Enterococcus faecalis in the feed is 0.1 - 1 Kg per ton of basic feed.

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

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

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

[0054] Even further, the aquatic animal is a fish.

[0055] Especially, the fish is Pseudosciaena crocea.

[0056] Advantages of the present invention: A strain of Enterococcus faecalis with high cholic acid production was screened in the present invention. This strain is suitable for long-term use in farmed animals in the form of feed, microbial agents, bacterial powder, etc., can improve the absorption efficiency of lipids in farmed animals, increase the content of HUFA (highly unsaturated fatty acids) in muscle tissue, improve the meat quality, and has broad application prospects.

[0057] Enterococcus faecalis DZNEF - 13 - 021 of the present invention is deposited in the China Center for Type Culture Collection (abbreviated as CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number is CGMCC No. 29592, and the deposit date is January 15, 2024. Description of the Drawings

[0058] Figure 1 It is a microscopic examination picture of Enterococcus faecalis, an oil immersion objective lens of 1600 times, and the scale converted is 1500:1.

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

[0060] Figure 3 It is that a variety of strains are isolated from the breeding environment.

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

[0062] Figure 5 It is a standard curve for detecting cholic acid by high performance liquid chromatography.

[0063] Figure 6 It is the cholic acid content in the bacterial powder detected by high performance liquid chromatography. Detailed Embodiments

[0064] By chance, the inventor isolated a variety of strains from the feces of farm animals. After separating and purifying each type of strain, the fermentation broth of each strain was prepared in the laboratory. After the fermentation ended, the high-performance liquid chromatography was used to detect the cholic acid content in the fermentation broth of different media, and a to-be-determined strain with high cholic acid production was obtained. Through means such as biological characteristic detection, physiological and biochemical identification, and 16srDNA sequencing analysis, it was finally identified as Enterococcus faecalis. The specific application method of this Enterococcus faecalis was studied and prepared into bacterial powder for application in the feed addition of large yellow croaker, which has a significant ability to increase the content of long-chain unsaturated fat HUFA in the muscle tissue of large yellow croaker.

[0065] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following will, in conjunction with the attached drawings and preferred embodiments, detail the specific implementation manners, structures, features, and their effects according to the present invention as follows.

[0066] Example 1 Screening of Strains with High Cholic Acid Production

[0067] 1). Screening and Verification

[0068] A variety of strains were isolated from the feces of animals in the farm, and the single colonies were isolated by streaking on culture dishes. The medium components (g / L): corn flour 20g, soybean meal 10g, glucose 5g, yeast extract 5g, sodium chloride 5g, K2HPO4 3g, KH2PO4 3g, MnSO4 0.3g, agarose 17g, adjust the pH to 7.0. Sterilize at 121 °C. After cooling to 55 °C, pour the plates, the fermentation temperature is 36 °C, put them into an anaerobic tank, add an oxygen scavenger, and statically culture. Subculture the single colonies ( Figure 3 ), for standby.

[0069] 2). Liquid Fermentation

[0070] Each isolated bacterium was inoculated into a liquid medium. The medium components: medium (g / L): corn steep liquor powder 20g, soybean meal 10g, glucose 5g, yeast extract 5g, sodium chloride 5g, KH2PO4 3g, adjust the pH to 8.5. Sterilize at 121 °C. The fermentation temperature is 36 °C, and statically culture to obtain the fermentation broth of different strains ( Figure 4 ).

[0071] 3). Detection of Cholic Acid Content

[0072] 3.1 Instrument and Sample Preparation:

[0073] 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 environments with up to 100% aqueous phase and as low as pH 1.0, column volume is about 2.49 mL); vacuum pump and suction filtration device. Electronic balance (sensitivity 0.0001 g). Prepare 1L volumetric flask, 2mL injection vial, 1L glass bottle, 10mL centrifuge tube, 2mL syringe, 0.22μm microporous filter membrane (organic phase & aqueous phase), pipette and corresponding tips (200μL, 1mL, 5mL), etc. Place the volumetric flask, injection vial, centrifuge tube, etc. in an ultrasonic cleaner, ultrasonically clean with a washing powder solution for 1h, and rinse thoroughly with tap water; place them in the cleaner again and ultrasonically clean with tap water for 0.5h - 1h, and rinse thoroughly with tap water; place them in the cleaner for the third time and ultrasonically clean with ultrapure water for 0.5h - 1h, and rinse thoroughly with ultrapure water. Invert the volumetric flask and let it dry naturally, and place other materials in the oven to dry.

[0074] Reagents: formic acid (chromatographic grade), acetonitrile, methanol. Unless otherwise specified, the water used in this experiment is all chromatographic grade ultrapure water.

[0075] Bile acid standards: including five standards of cholic acid, deoxycholic acid, hyodeoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid (Note: cholic acid, deoxycholic acid, hyodeoxycholic acid, ursodeoxycholic acid are purchased from the National Institutes for Food and Drug Control; deoxycholic acid - sigma is purchased from sigma; hyodeoxycholic acid is purchased from Macklin). Precisely weigh about 0.020 g of bile acid standards with a ten-thousandth balance, place them in a 10mL volumetric flask, dissolve with the mobile phase and make up to the mark, shake well, and prepare a standard stock solution with a concentration of 2 mg / mL. The above stock solution is stored in a 4℃ refrigerator, and the storage time of the stock solution is 3 months. Dilute the stock solution to the required concentration when in use. (Note: If the sample is not easily soluble, it can be placed in an ultrasonic instrument and ultrasonically treated for 10 min until completely dissolved.) Gradient dilute with the mobile phase based on the stock solution. Precisely weigh 125μL, 250μL, 500μL, 1000μL, 1500μL, 2000μL, 2500μL appropriately and place them in 7 10mL volumetric flasks respectively. Dilute with the mobile phase and make up to 25μg / mL, 50μg / mL, 100μg / mL, 200μg / mL, 300μg / mL, 400μg / mL, 500μg / mL standard solutions, filter through a 0.22μm microporous filter membrane, and add to the injection vial. That is, a standard gradient series solution is obtained (the ratio table is shown in Table 1).

[0076] Table 1 Ratio table of series gradients

[0077]

[0078] Mobile phase preparation:

[0079] (I)Filter water using a 0.45 μm microporous filter membrane. During the filtration process, the first 100 mL - 200 mL of water should be discarded, and then collection can start. Then transfer it into a 1 L glass bottle, cover the lid, and set aside for later use.

[0080] (II)Prepare 0.2% formic acid aqueous solution: Take 2 mL of formic acid and make up the volume to 1 L in a volumetric flask. Filter the above 0.2% formic acid aqueous solution using the method in (I), then transfer it into a 1 L glass bottle, cover the lid, and set aside for later use.

[0081] 3.2 Chromatographic conditions

[0082] The chromatographic conditions are as follows: For the ELSD detector, the nitrogen gas flow rate is 1.6 L / min, the drift tube temperature is 110 °C (evaporator temperature 60 °C; nebulizer temperature 60 °C); the mobile phase passing time (Smoothing) is 30 seconds; mobile phase A is 0.2% formic acid solution, mobile phase C is acetonitrile, and the gradient elution is: 0 - 7 min, 5% C; 7 - 20 min, 5% → 80% C, 20.1 - 25 min: 5% C, and then return to the initial mobile phase at later times; the flow rate is 1.0 mL / min; the injection volume is 10 μL; the column temperature is 25 °C.

[0083] 3.3 Sample preparation

[0084] Reference solution: Weigh accurately about 0.020 g of cholic acid reference standard, place it in a 10 mL volumetric flask, dissolve it with methanol and make up the volume to the mark, shake well to obtain a stock solution with a concentration of 2 mg / mL. Then dilute it according to a series of gradients.

[0085] Test sample solution: Use a pipette to aspirate 25 μL of the fermentation broth, add it to a stoppered conical flask, tightly add 50 mL of methanol, weigh it, ultrasonically treat it (200 w, frequency 40 kHz) for 20 min, make up the lost weight with methanol, filter, and you will get the solution.

[0086] 3.4 Liquid phase detection results

[0087] Taking the reference standard concentration (X, μg / mL) as the abscissa and the chromatographic peak area Y as the ordinate, plot the standard working curve and perform linear regression calculation. The standard curve is as Figure 5As shown, the regression curve equation of cholic acid (from the National Institutes for Food and Drug Control) is: y = 32.923x - 967.19, R2 = 0.9951. The results show that there is a good linear relationship between the chromatogram and the peak area of cholic acid (from the National Institutes for Food and Drug Control) in the range of 25 - 400 μg / mL. The peak emergence time of cholic acid is 17.428 - 17.488 min. The peak emergence conditions of each standard are shown in Table 2.

[0088] Table 2 Relationship between the content and peak area of each standard

[0089]

[0090] The determination results of the high-performance liquid chromatography content of cholic acid in the fermentation samples of different single colony strains are shown in Table 3. Among them, for strain No. 5 (designated as DZNEF-13-021), the cholic acid content in the obtained fermentation broth is 1.584%, which has nutritional value and can be further developed.

[0091] Table 3 Determination results of the high-performance liquid chromatography content of cholic acid in different fermentation samples

[0092]

[0093] 4) Identification of the above-mentioned strain No. 5

[0094] Based on the colony morphology and MRS anaerobic culture method in the previous cultivation, it was determined that strain No. 5 is a lactic acid bacterium. Microscopic examination showed that it is coccus, completely different from bacillus, and it was basically determined to be Enterococcus faecalis. Further physiological and biochemical identification and molecular identification were carried out.

[0095] Through the purchased Enterococcus faecalis biochemical identification strip (Qingdao Haibo Biotech), the identification was carried out according to the specified steps. As shown in Table 4: The morphological characteristics of this Enterococcus faecalis are: the cells are elliptical, with round ends at both ends, and the size is generally 0.8 - 1.2 μm × 1.5 - 2.0 μm, Gram-positive ( Figure 1 and Figure 2 ).

[0096] Table 4 Results of physiological and biochemical reactions

[0097]

[0098] DNA extraction of Enterococcus faecalis and sequencing identification of PCR-amplified 16s rDNA fragment:

[0099] Add 400 µL of sterile pure water into a 1.5 mL centrifuge tube after sterilization. At the same time, pick up a full loop of the strain to be tested from the culture dish in the laminar flow hood and add it into the centrifuge tube. Place the centrifuge tube on an oscillator to mix evenly, insert the float, and place it in an ultrasonic instrument at 40 KHZ and a power of 600 w for 5 minutes. Take it out and mix evenly again, and then ultrasonicate for another 5 minutes. In total, ultrasonicate 3 times, 5 minutes each time. After that, put the bacterial solution after ultrasonic disruption into the refrigerator for use as a DNA template.

[0100] During the experiment, it was found that for a small number of strains, whether it was ultrasonic disruption or high-temperature heating to denature the cells, it was difficult to effectively release the DNA inside the cells. Therefore, for such strains, a DNA extraction kit was used to extract the bacterial DNA as a template and then perform PCR amplification. The kit used in this process was the Bacterial Genomic DNA Rapid Extraction Kit (Sangon Biotech, Product Number: B518225). The standard operating procedure was as follows: Take 1 mL of the overnight cultured bacterial solution and add it into a 1.5 mL centrifuge tube. Centrifuge at 8,000 rpm for 1 min at room temperature, discard the supernatant, and collect the bacterial cells. Add 500 µL of Buffer Digestion and mix evenly by shaking. Incubate in a water bath at 65 °C for 2 h 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 when the lysis is complete. If the solution does not become clear, it means the sample is not completely lysed, and the water bath time should be appropriately extended. Add 200 µL of Buffer PB, mix well by inverting thoroughly, and place on ice for 5 min. Centrifuge at 10,000 rpm for 5 min at room temperature, and transfer the supernatant (500 - 550 µL) to a new 1.5 mL centrifuge tube. Add an equal volume of isopropanol, invert 5 - 8 times to mix well, and let it 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 wash for 1 - 3 min, centrifuge at 10,000 rpm for 2 min, and discard the supernatant (twice). Open the lid and invert at room temperature for 5 - 10 min until the residual ethanol completely evaporates. Dissolve the obtained DNA in 50 - 100 µL of TE Buffer. The extracted DNA can be immediately used for the next experiment or stored at -20 °C.

[0101] PCR system (25 µL): 16 µL of purified water, 2.5 µL of PCR buffer, 0.5 µL of dNTP, 1 µL each of the upstream and downstream primers (27F and 1492R), 2 µL of template, 0.5 µL of Taq enzyme, Mg 2+1.5 µL. The PCR process parameters were set as follows: 94 °C for 10 minutes (to increase DNA release); 94 °C for 30 s, 55 °C for 30 s, 72 °C for 90 s, for 30 cycles; extension at 72 °C for 10 min; preservation at 10 °C. The PCR products were subjected to gel electrophoresis using 1% agarose. During the gel process, the color reagent (Sangon Biotech, 4s green color reagent) was added at 1 µL / 100 mL. 2 µL of the PCR products were mixed with 1 µL of loading buffer and loaded into the agarose gel electrophoresis tank. The voltage was set at 170 V for 20 minutes. The amplified products were recovered and sent for sequencing.

[0102] Copy out the base sequence in the nucleic acid sequence txt file of the 16sDNA fragment sequence sent back after Sangon sequencing. At the same time, log in to the NCBI gene database, enter the Blast alignment mode, select the nucleic acid alignment database, input the sequence of the seq file of the sequencing into the alignment window, adjust the alignment parameters, and select the strains with a sequence similarity of more than 90% as the reference strains for identification.

[0103] The value of the DNA concentration was detected by a nucleic acid analyzer. Therefore, the method of gradient dilution was adopted and explored through experiments, diluted 10-fold, 100-fold, 150-fold, and 200-fold respectively. Finally, it was detected that the DNA template concentration diluted 10-fold was suitable for the current PCR system. From the blast alignment results, the 16s sequence of this strain reached 98% similarity with various Enterococcus faecalis strains. Therefore, it was determined that this strain belongs to Enterococcus faecalis. At the same time, it was sent to the China General Microbiological Culture Collection Center in Beijing for preservation, and the preservation number was CGMCC No. 29592.

[0104] SEQ ID No.1 16s rDNA

[0105]

[0106] Example 2 Preparation of Microecological Products by Drying and Crushing Enterococcus faecalis Fermentation Broth

[0107] The concentrated bacterial liquid was obtained by centrifugation with a disc centrifuge, and then the bacterial sludge was obtained by centrifugation with a tubular centrifuge. The bacterial sludge was redissolved with a small amount of centrifuged supernatant to make it uniformly viscous and easy to mix evenly. The bacterial sludge was mixed evenly with carriers (corn cob powder and montmorillonite powder) (55% Enterococcus faecalis bacterial sludge, 15% montmorillonite (feed grade), 30% corn cob powder) using a high-speed mixer. Then, the mixed wet bacterial powder was prepared into uniform granules through a granulator and placed in a fluidized bed dryer for drying. During fluidized bed drying, the material temperature was controlled below 35 °C, so that the humidity of the dried bacterial powder was below 10%. The single-batch processing time was about 30 - 60 min. After that, the cholic acid content in the bacterial powder product was efficiently detected to be 1.882% (see Figure 6 ).

[0108] Example 3 Results of Field Clinical Animal Experiments

[0109] The juvenile fish used in the experiment were all from Ningde Jinling Aquatic Science and Technology Co., Ltd., and all breeding experiments were also carried out in the nursery of this company. Before the experiment started, the juvenile large yellow croakers taken from the sea-cage were first temporarily raised in an indoor cement pool of 3 m × 3 m × 1.5 m for 1 week, and were fed with commercial large yellow croaker feed at 8:30 and 15:30 every day for one week. After domestication, the formal experiment was carried out.

[0110] Before selecting the experimental fish, they were starved for 24 h and anesthetized with clove oil cement (Shanghai Medical Instrument Co., Ltd.) at a ratio of 1:10000 to avoid stress response and injury of large yellow croakers during the selection process. The water and mucus on the surface of the test fish body were wiped dry with a gauze. On the premise of confirming that the fish body had no injury, normal and healthy large yellow croakers were selected for the determination of initial weight and body length. According to different weight ranges, grouped experiments were carried out. During the experiment period, the feed added with Enterococcus faecalis bacterial powder was fed once at 8:30 and 15:30 every day. The addition amounts of Enterococcus faecalis bacterial powder were 100 g, 500 g, and 1 kg per ton of basic feed respectively. Before feeding, the feed should be taken out of the freezer in advance, moistened with a small amount of water for 20 min and then fed until the large yellow croakers showed apparent satiety. The entire breeding experiment lasted for 70 d. During the experiment process, the seawater temperature was 25 ± 2 °C, the seawater salinity was 30 - 35‰, and the dissolved oxygen was maintained above 5 mg / L.

[0111] 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 determined by taking the dorsal muscle of each fish. 160 healthy large yellow croakers were selected, and the dorsal muscle of the large yellow croaker was taken. The total lipids were extracted by the chloroform-methanol method (1:2). Referring to GB 5009.168—2016, the experimental parameters were set. After the fatty acids in the muscle were treated with a methanol solution containing 15% KOH at 20 °C for 1 h, the fatty acids in the sample were methylated. The lipid esterification was maintained at 85 °C 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 a gas chromatograph (Hewlett-Packard HP-5890) equipped with a cross-linked 5% phenylmethyl silicone gum column (L = 25 m, ID = 0.32 min, DF = 0.25 μmol / L, HP-Ultra2, using nitrogen as the carrier gas). Nonadecenoic acid at 50 g / L was used as the internal standard, and the dosage was 10% of the total amount of fatty acids. Detection was carried out with a flame ionization detector, and the temperatures of the nozzle and the detector were 190 °C and 250 °C, respectively. The column temperature was set to rise from 190 °C to 260 °C (2 °C / min) and maintained for 5 min. The phenotypic data were sorted in descending order, and the high and low phenotypic values were regarded as extreme phenotypes. Through the Shapiro-Wilk test, the HUFA phenotypic data followed a normal distribution (P>0.05). In this experiment, the absolute quantification method (mg / 100 g sample wet weight) was used for data analysis and representation. The results are shown in Table 5 and Table 6: This Enterococcus faecalis had little effect on the long-chain unsaturated fatty acids of C12, C16, and C18, but had a greater impact on HUFA (highly unsaturated fatty acids). Compared with the control group, it could increase the content by nearly 100%, which could significantly improve the flavor and nutritional value of large yellow croakers and was helpful for the green and high-economic-value sustainable aquaculture industry.

[0112] Table 5 Changes in fatty acids in the muscle of 160 samples (4 groups) on the 0th day (unit: % dry matter)

[0113]

[0114] Table 6 Changes in fatty acids in the muscle of 176 samples (4 groups) on the 70th day (unit: % dry matter)

[0115]

[0116] The above are only the preferred embodiments of the present invention and do not impose any formal restrictions on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments of equivalent changes within the scope of the technical solution of the present invention by using the technical content disclosed above. However, as long as it does not depart from the content of the technical solution of the present invention, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

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

2. Use of the Enterococcus faecalis DZNEF-13-021 according to 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 DZNEF-13-021 according to claim 1 is prepared into feed, bacterial powder or bacterial agent for use.

4. The application according to claim 3, wherein: The preparation method of the bacterial powder includes the following steps: collecting the fermentation broth of the Enterococcus faecalis DZNEF-13-021 according to claim 1, centrifuging, collecting the bacterial sludge, and redissolving it with the centrifuged supernatant to form a viscous state; mixing the bacterial sludge with a carrier evenly; granulating and drying; obtaining the bacterial powder; the carrier is corncob powder, montmorillonite powder and / or medical stone; the weight ratio of the bacterial sludge to the carrier is 55:

45.

5. The application according to claim 3, wherein: The preparation method of the feed includes the following steps: collecting the fermentation broth of the Enterococcus faecalis DZNEF-13-021 according to claim 1, centrifuging, collecting the bacterial sludge, and redissolving it with the centrifuged supernatant to form a viscous state; mixing the bacterial sludge with a carrier evenly; granulating and drying; obtaining the bacterial powder; mixing the bacterial powder with the basal feed; the carrier is corncob powder, montmorillonite powder or / and medical stone; the weight ratio of the bacterial sludge to the carrier is 55:

45.

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

7. The application according to claim 5, wherein: The addition amount of the Enterococcus faecalis DZNEF-13-021 bacterial powder in the feed is 0.1 - 1 Kg / ton of basal feed.

8. An Enterococcus faecalis bacterial powder, characterized in that: It is composed of the Enterococcus faecalis DZNEF-13-021 bacterial sludge and a carrier with a weight ratio of 55:45, and the carrier is corncob powder, montmorillonite powder or / and medical stone.

9. A feed containing Enterococcus faecalis, characterized in that: The Enterococcus faecalis DZNEF-13-021 according to claim 1 is prepared into bacterial powder and then mixed with the basal feed; the addition amount of the Enterococcus faecalis DZNEF-13-021 bacterial powder is 0.1 - 1 Kg / ton of basal feed.

10. A method for culturing large yellow croaker with a high content of highly unsaturated fatty acids, characterized in that: It includes the following steps: feeding the large yellow croaker with the Enterococcus faecalis DZNEF-13-021 according to claim 1 and / or the feed containing the Enterococcus faecalis DZNEF-13-021 according to claim 1.

Images