CN115725466A - Staphylococcus equisimilis and application thereof in fish culture - Google Patents

Abstract

The invention discloses staphylococcus equine gastrus and application thereof in fish culture, and relates to a strain and application thereof. The Staphylococcus equisimilis strain equirum HL-27 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No:24992. the Staphylococcus equi Staphylococcus aureus equivalent HL-27 strain has the capability of producing an iron carrier and a DDP-IV inhibitor, and the HL-27 strain is added into fish feed for fish culture, so that iron elements which are more beneficial to absorption are provided for the fish, the waste problem is avoided, the blood sugar of the fish is reduced, and the digestion utilization rate of the feed is improved. The HL-27 strain also has the function of decomposing organic phosphorus, and can effectively eliminate the toxic action of the organic phosphorus when being used in the culture environment polluted by the organic phosphorus.

Description

Staphylococcus equine and application thereof in fish culture

Technical Field

The invention relates to a strain and application thereof.

Background

The saccharides are organic matters which are widely distributed in nature, are the cheapest energy supply substances in the fish baits, and can play the roles of saving bait cost, reducing protein energy supply, improving protein deposition and promoting fish growth by properly adding the saccharides into the fish baits. However, the utilization capacity of the fish to sugar is low, the sugar level in the feed exceeds a certain limit, which causes the symptoms of low disease resistance, slow growth, high mortality and the like of the fish, the capacity of the fish for relieving the glucose load is limited, which shows that the utilization capacity to the sugar is low, and further, the large-scale use of the sugar in the feed for the fish is limited.

With the development of fish culture technology, research on the requirements of fish on trace elements is increasingly emphasized, the trace elements are mineral elements, the trace elements are small in content but indispensable nutrient substances, and the trace elements which are determined to play an important role in the growth and development of the fish at present comprise iron, copper, manganese, zinc, iodine, selenium and the like. Wherein, iron is a component of heme and myoglobin, participates in the transportation of oxygen and carbon dioxide by the identity of a carrier, and the iron element also participates in the construction of systems of cytochrome, peroxidase and the like of aquatic animals, and the physiological and biochemical processes of respiration, biological oxidation and the like in the aquatic animals are closely related. The aquatic animal has proper amount of iron element, and can maintain its normal metabolism and raise the disease preventing capacity of the body. The lack of iron in fish can reduce the hematopoietic function of organism tissues, and even lead to anemia in severe cases. At present, the problem of iron element deficiency is solved by adding ferrite into the feed, but the iron element is low in absorption and utilization rate, so that the feed is easy to waste.

Disclosure of Invention

The invention provides staphylococcus equi and application thereof.

The Staphylococcus equi disclosed by the invention is Staphylococcus equi Staphylococcus aureus HL-27, which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No:24992.

the invention also discloses application of the staphylococcus equi in fish culture.

The staphylococcus equi is added into fish feed for fish culture.

The Staphylococcus equi Staphylococcus aureus equivalent HL-27 colony is spherical or elliptical (slightly irregular), has the diameter of about 1-4mm, is milky convex, is opaque, has a wet surface, is glossy, has no flagellum, cannot move and is a gram-positive coccus by gram staining.

Staphylococcus aureus HL-27 is inoculated on a CAS culture medium, and after a period of culture, an obvious color-changing ring is formed around a colony, which indicates that the HL-27 strain has stronger siderophore production capacity, and the Su value of siderophore production at 37 ℃ is 48%. Staphylococcus equinus equirum HL-27 chelates iron ions through a high-affinity iron carrier and fixes the iron ions to form an iron element. The Staphylococcus equinus equivalent HL-27 can directly provide iron element for fishes, is convenient for the fishes to absorb, maintains the normal metabolism of the fishes, and improves the disease defense capability of organisms when being added into the fish feed.

Culturing Staphylococcus aureus HL-27 on SKM culture medium for a period of time to form an obvious hydrolysis ring around the colony, wherein the diameter D of the hydrolysis ring of the HL-27 is 19.74mm, the diameter D of the colony is 5.40mm, and the D/D is 3.66; the yield of the DDP-IV inhibitor is as high as 55.61% at 37 ℃, and the HL-27 strain has stronger ability of producing the DDP-IV inhibitor. The fish has three sources of glucagon and GLP-1 (incretin insulin glucagon blood sugar sample peptide-1), namely pancreas, intestine and brain, and the DDP-IV (dipeptidyl peptidase-1) inhibitor can indirectly achieve the purpose of reducing the blood sugar of the fish by prolonging the half-life period of the GLP-1. The addition of Staphylococcus equi gastrococcus equi equorum HL-27 in the fish feed can improve the digestion utilization rate of the feed and the growth performance of fish.

Staphylococcus aureus Staphylococcus equiorum HL-27 strain is inoculated on a Monkina organophosphorus culture medium, an obvious organophosphorus dissolving ring is formed around a bacterial colony after the culture for a period of time, the diameter D of an inorganic phosphorus dissolving ring of the HL-27 is 13.66mm, the diameter D of the bacterial colony is 3.29mm, and the D/D is 4.15, which shows that the HL-27 strain has a strong organophosphorus dissolving function. The HL-27 strain has higher tolerance to pH and bile salt. The HL-27 strain is subjected to hemolysis experiment, the result is negative, the strain has no risk of causing diseases to human and livestock, and the use safety is high.

The Staphylococcus equi Staphylococcus equorum HL-27 strain has the capability of producing siderophores and DDP-IV inhibitors. The HL-27 bacterial strain is added into fish feed for fish culture, can replace ferrous salt, provides iron element which is more beneficial to absorption for fish, has no waste problem, reduces the blood sugar of fish and improves the digestion utilization rate of the feed. The HL-27 strain also has the function of decomposing organic phosphorus, and can effectively eliminate the toxic action of the organic phosphorus when being used in the culture environment polluted by the organic phosphorus.

The Staphylococcus equisimilis Staphylococcus equivus HL-27 is Staphylococcus equivus, is preserved in China general microbiological culture Collection center (CGMCC), and the preservation address is No. 3 of Xilu No. 1 Beijing of Chaoyang district in Beijing. The microbial research institute of Chinese academy of sciences has a preservation number of CGMCC No.24992 and a preservation date of 2022, 5 months and 30 days.

Drawings

FIG. 1 shows the result of screening Staphylococcus equi Staphylococcus aureus HL-27 siderophore;

FIG. 2 is a milk protein hydrolysis loop on Staphylococcus equisimilis equivus equivalent HL-27% skim milk powder medium;

FIG. 3 is a phylogenetic tree constructed by Staphylococcus equi Staphylococcus equorum HL-27;

FIG. 4 shows the phosphate solubilizing ring on Staphylococcus equi equum HL-27 organophosphorus solid culture medium.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive efforts based on the embodiments of the present invention, shall fall within the scope of protection of the present invention.

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

The first specific implementation way is as follows: the Staphylococcus equi according to the embodiment is Staphylococcus equi Staphylococcus equum HL-27, which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No:24992 and the preservation date is 2022, 5 months and 30 days.

At present, the application of Staphylococcus equi (Staphylococcus equi) is mainly focused on degrading heavy metal ions, preventing gray mold of fruits and vegetables and prolonging the shelf life of meat products, and the Staphylococcus equi (Staphylococcus equi) with an iron carrier and a DDP-IV inhibitor is reported for the first time.

The Staphylococcus equi Staphylococcus equorum HL-27 strain has the capability of producing siderophores and DDP-IV inhibitors. The HL-27 bacterial strain is added into fish feed for fish culture, can replace ferrous salt, provides iron elements which are more beneficial to absorption for the fish, does not have the problem of waste, and simultaneously reduces the blood sugar of the fish and improves the digestion utilization rate of the feed. The HL-27 bacterial strain also has the function of decomposing organic phosphorus, and the prior organic phosphorus pesticide is easy to permeate through the ground surface and flow into underground water, rivers and lakes through field water during the use process, thereby finally causing water pollution. The HL-27 strain has the function of decomposing organic phosphorus, can be used for fish culture as feed, can effectively eliminate the toxic action of the organic phosphorus in the culture environment polluted by the organic phosphorus, and improves the yield of the fish.

A method of obtaining staphylococcus equine gastri according to the present embodiment:

1. separating and purifying strains: selecting carp colony cultured in paddy field in large depression area of Panjin City in Liaoning 9 months in 2021 years, randomly selecting 3 healthy 1-year-old carps (150 +/-10 g), anesthetizing with MS-222 anesthetic (250 mg/L), wiping the surface of the carps with absolute ethyl alcohol, dissecting with sterilized scissors and tweezers in a super clean bench, taking out the whole intestinal tract of the carps, slightly extruding the content, placing in a conical flask containing glass beads and 50mL of sterile water, oscillating at room temperature for 30min at the rotating speed of 180r/min, performing gradient dilution, and diluting 10 th day ^-3 、10 ^-4 、10 ^-5 And coating 100 mu L of the gradient on an LB solid medium plate, repeating each gradient for 3 times, and culturing at 28 ℃ for 24-48 h. After culturing for 48h, selecting strains with different shapes for separation.

2. Identification of bacterial siderophore

2.1 reactivating the separated and purified bacterial strain, transferring the bacterial strain to an LB plate to be cultured for 24 hours at 28 ℃, selecting a single bacterial colony by using a sterilized toothpick to be connected to a Chromeazurol S (CAS) solid detection medium, carrying out inverted culture for 2-3 days at 37 ℃, and observing the size of a color-changing ring around the bacterial colony.

After a period of culture, obvious color change circles are formed around colonies, and strains capable of producing siderophores are selected, wherein the color change circle diameter D of the HL-27 strain in an LB culture medium is 9.66mm, the colony diameter D is 3.47mm, the D/D is 2.78, and the strain HL-27 has strong siderophores producing capability (shown in figure 1).

2.2 further tests on the strain HL-27 which showed a clear color circle:

(1) Inoculating the activated lawn in an iron-limiting SA liquid culture medium, and performing shake culture at 37 ℃ for 48h to obtain HL-27 bacterial liquid;

(2) Transferring the HL-27 bacterial liquid to be detected, which grows for 48 hours, into a sterilized 10mL centrifuge tube, centrifuging at 13000rpm for 15min, removing the precipitate and reserving the supernatant;

(3) Transferring the supernatant into a test tube treated by concentrated hydrochloric acid, adding a certain amount of the CAS detection solution prepared in situ to ensure that the volume ratio of the supernatant to the detection solution is 1;

(4) Measuring absorbance value (A) at the wavelength of 630nm, taking double distilled water as a control for zero adjustment, taking the absorbance value at the wavelength of 630nm after mixing the uninoculated SA iron-limiting culture medium and the detection liquid which are measured by the same method as a reference value (Ar), and expressing the activity unit of the siderophore by the following formula:

Su≈(Ar-As)/Ar×100；

in the formula: su is the siderophore content; ar is the OD value of the detected uninoculated SA iron-limiting culture medium and the detected liquid supernatant; as is the OD of the supernatant of the medium tested.

When the activity unit of the siderophore is less than 10, the siderophore is considered to be negative, and the mixture of the siderophore and the detection liquid has no color change.

The Su value of the strain HL-27 for producing the siderophore at 37 ℃ is 48 percent, which indicates that the strain has stronger siderophore production capability.

3. Identification and content determination of strain producing DDP-IV inhibitor

3.1 the strain HL-27 is inoculated on an SKM culture medium, after 48 hours of culture at 28 ℃, an obvious hydrolysis ring is formed around a colony, the diameter D of the hydrolysis ring of the HL-27 is 19.74mm, the diameter D of the colony is 5.40mm, and the D/D is 3.66 (shown in figure 2). The HL-27 strain has the capability of producing a DDP-IV inhibitor.

3.2 inoculating HL-27 to a 96-pore plate, and accurately dripping 25 mu L, 1.6mmol/L glycyl-prolyl-p-nitroaniline and 25 mu L CFS or CFE into each pore; reacting at 37 ℃ for 15min, adding 50 mu L of 0.01U/mL DDP-IV (dipeptidyl peptidase IV), continuing the reaction at 37 ℃ for 1h, adding 100 mu L of 1mol/L sodium acetate buffer solution (pH = 4.0) to stop the reaction, and detecting the absorbance of the reaction solution at 405nm by using a microplate reader to obtain the DDP-IV inhibition rate of the strain.

Wherein, the DDP-IV inhibition rate calculation formula is as follows:

A _{sample to be tested} :25 μ L of sample +25 μ L of LGly-pro-phy +50 μ L of LDDP-IV +100 μ L of sodium acetate;

A _{sample blank} :25 μ L of sample +50 μ L of LTris-HCl +25 μ L of LGly-pro-phy +100 μ L of sodium acetate;

A _{negative control} :25 μ L of Tris-HCl +25 μ L of LGly-pro-phy +50 μ L of LDDP-IV +100 μ L of sodium acetate;

A _{negative blank} :75 μ L of Tris-HCl +25 μ L of LGly-pro-phy +100 μ L of sodium acetate.

The yield of the DDP-IV inhibitor of the HL-27 strain at 37 ℃ is as high as 55.61 percent, which shows that the HL-27 strain has stronger capability of producing the DDP-IV inhibitor.

4. Identification of Strain HL-27

4.1 physiological and biochemical identification: the strain HL-27 is streaked in three zones on a solid LB culture medium plate, a single colony is separated, the form of the single colony is described, and gram staining and physiological and biochemical identification are carried out on the strain according to a manual for identifying a common bacterial system.

The colony characteristics of HL-27 on LB solid medium are as follows: the colony morphology is spherical or elliptical (slightly irregular), the diameter is about 1-4mm, the colony is milky-white and convex, the colony is opaque, the surface is wet, glossy, has no flagellum and cannot move, and gram stain is gram-positive cocci. Some physiological and biochemical indicators of HL-27 are shown in Table 1. According to the description of Bacillus physiological characteristics in Bergey's Manual of identification of bacteria, HL-27 has the same characteristics with the physiological and biochemical characteristics of Staphylococcus equi (Staphylococcus equi) model species, and the strain HL-27 is deduced to be Staphylococcus equi (Staphylococcus equi) from all the physiological and biochemical characteristics.

TABLE 1 physiological and biochemical results of Strain HL-27

4.2 16S rRNA identification: a bacterial genome DNA extraction kit of Beijing Soilebao Biotechnology company is selected to extract separated and purified bacterial strain DNA. Carrying out PCR amplification by using a bacterial universal primer 27F/1492R, wherein the PCR amplification system is a 25 mu L system: 2.5 μ L of 10 Xbuffer, 0.5 μ L of Taq enzyme, 0.5 μ L of primer 27F, 0.5 μ L of primer 1492R, 1 μ L of DNA template, ddH ₂ O20. Mu.L. The reaction program is set as pre-denaturation at 95 ℃ for 5min; denaturation at 94 ℃ for 50s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 1.5min, cycle times for 30 times, re-extension at 72 ℃ for 10min, and storage at 4 ℃. The PCR amplification product was sent to RuiBiotech for sequencing. And comparing sequencing results of the 16S rRNA of the strain through an NCBI database, and constructing a phylogenetic tree.

After sequencing the 16S rRNA sequence, BLAST alignment in NCBI found that the 16S rRNA gene sequence of HL-27 strain has 99% similarity with Staphylococcus equi (Staphylococcus equi). Phylogenetic Tree As shown in FIG. 3, it can be seen that one branch of HL-27 strain, which is the smallest in common with Staphylococcus aureus (NR _ 027520.1), is closer in evolutionary distance, and HL-27 strain is identified as Staphylococcus aureus (Staphylococcus aureus) by comprehensive physiological and biochemical indicators.

The second embodiment is as follows: the use of staphylococcus equi-gastric in fish farming according to embodiment one.

The third concrete implementation mode: the second embodiment is different from the first embodiment in that: staphylococcus equine is added to fish feed for fish culture. The rest is the same as the second embodiment.

The fourth concrete implementation mode is as follows: in this embodiment, a bacterial solution of Staphylococcus equisimilis strain HL-27 was prepared.

The Staphylococcus equinus Staphylococcus equiorum HL-27 bacterial liquid culture medium consists of 1000mL of 10% bean sprout juice and 10.48g of beta-C ₁₂ H ₂₂ O ₁₁ ·H ₂ O (maltose), 2.36g casein peptone, 1.91g NaNO ₃ (sodium nitrate) and 0.73gFeCl ₃ (ferric chloride); wherein 100g of soybean sprout is added with 1000mL of water, boiled for 1h, filtered, complemented with water to 1L, and stored for standby after moist heat sterilization at 121 ℃, namely the soybean sprout juice with the mass fraction of 10%.

Inoculating 0.3% of the Staphyloccocus equiorum HL-27 seed liquid into a Staphyloccocus equiorum HL-27 bacterial liquid culture medium, culturing at 37 ℃ and 180r/min for 24h, wherein the viable count of the Staphyloccocus equiorum HL-27 bacterial liquid reaches 1.65 multiplied by 10 ⁹ cfu/mL (viable count of staphylococcus equi HL-27 seed liquid is 6.51 multiplied by 10) ⁸ cfu/mL, staphylococcus equi HL-27 seed liquid was inoculated in LB liquid medium).

Example 1

Respectively inoculating Staphylococcus aureus (Staphylococcus aureus) HL-27 on a flat plate of a Monkina organophosphorus bacteria culture medium by using a sterilized toothpick, culturing at 28 ℃ for 24-48h to form an obvious organophosphorus dissolving ring around the colony, measuring the diameter D of the colony and the diameter D of the organophosphorus dissolving ring by adopting a cross method, and calculating D/D; the phosphate solubilizing ring on the Staphyloccocus equiorum HL-27 organophosphorus solid culture medium is shown in figure 4, the diameter D of the inorganic phosphate dissolving ring of the HL-27 is 13.66mm, the diameter D of a bacterial colony is 3.29mm, the D/D is 4.15, and the HL-27 strain has a strong organophosphorus solubilizing function.

Example 2 stress resistance test of Staphylococcus equisimilis equirum HL-27

1. And (3) detecting acid resistance: the number of spores was 2X 10 ⁸ The method comprises the steps of inoculating 2% (v/v) of cfu/mL staphylococcus equine HL-27 into LB liquid culture medium with pH of 2.0, 3.0 and 4.0, sucking bacterial liquid after 3h for plating, culturing at 37 ℃ for 24h, measuring the number of viable bacteria, and calculating the survival rate of the strain.

2. And (3) detecting bile salt resistance: the number of activated spores is 2 × 10 ⁸ 1mL of diluent is sucked by cfu/mL staphylococcus equi HL-27 bacterial liquid and put into a sterilized plate, then the LB solid culture medium containing 0.5 percent, 1.0 percent and 1.5 percent sodium taurocholate is used for pouring the plate, meanwhile, MRS solid culture medium containing no sodium taurocholate is used as a control group, the plate is cultured for 48 hours at 37 ℃, colony counting is carried out, and the survival rate of the bacterial strain is calculated.

As can be seen from Table 2, the influence on the number of HL-27 spores of Staphylococcus equi at pH 3.0 and 4.0 was small, and the number of HL-27 spores was significantly reduced at pH 2.0; and when the concentration of bile salts is increased, the number of HL-27 spores of the staphylococcus equi is slightly changed. Staphylococcus equinus HL-27 has high tolerance to pH and bile salt.

TABLE 2 Staphylococcus equi HL-27 test for acid and bile salt resistance

3. Hemolysis experiment: hemolysis is classified into alpha hemolysis (incomplete hemolysis, producing a grass green hemolytic cycle), beta hemolysis (complete hemolysis, a well-defined, colorless and transparent hemolytic cycle) and non-hemolysis.

Staphylococcus aureus HL-27 was streaked on each fresh blood plate, and observed after culturing at 30 ℃ for 48 hours. The experimental result of staphylococcus equi HL-27 shows that the staphylococcus equi HL-27 is negative, which indicates that the staphylococcus equi HL-27 has no risk of causing diseases to people and livestock.

Example 3

1. Preparing Staphylococcus equi Staphylococcus equorum HL-27 bacterial liquid

Inoculating 0.3% Staphylococcus equi aureus HL-27 seed solution to Staphylococcus equi aureus HL-27 culture medium, culturing at 37 deg.C and 180r/min for 24h until the viable count of Staphylococcus equi HL-27 reaches 1.65 × 10 ⁹ cfu/mL (viable count of staphylococcus equi HL-27 seed liquid is 6.51 multiplied by 10) ⁸ cfu/mL, staphylococcus equi HL-27 seed liquid was inoculated in LB liquid medium). Wherein the Staphylococcus equi Staphylococcus aureus equivalent HL-27 bacteria liquid culture medium comprises 1000mL of bean sprout juice with the mass fraction of 10% (100 g of soybean sprout is added with 1000mL of water, boiled for 1h, filtered, and then water is supplemented to 1L, and the bean sprout juice with the mass fraction of 10% is stored for later use after moist heat sterilization at 121 ℃ and 10.48g of beta-C ₁₂ H ₂₂ O ₁₁ ·H ₂ O (maltose), 2.36g casein peptone, 1.91g NaNO ₃ (sodium nitrate) and 0.73g FeCl ₃ (ferric chloride).

2. The carp (Cyprinus carpio) breeding test is carried out in a circulating water pond of the laboratory of the aquatic research institute of Heilongjiang, china academy of aquatic sciences, and the carp used in the test is purchased from a Jingbo farm in Dandong, liaoning province. 200 carps bred in the current year and with the initial weight of about 66g are selected and temporarily cultured in a cement pond with the length of 4.3m multiplied by 1.5m multiplied by 1.2m for 1 week to adapt to the test environment, and the control group feed (Tongwei feed 181) is fed during the temporary culture period. After temporary rearing, selecting 120 carps with similar weight, good vitality and no damage to body surfaces, randomly dividing the carps into 2 groups (a control group and a test group), wherein each group is 3 parallel, 20 parallel carps are placed in 6 circulating water rearing boxes with the length of 1.3m multiplied by 0.8m multiplied by 0.7m, the water body covers more than 80 percent of the rearing boxes, tap water with the aeration time of more than 24h is used as a test water body, and 24h of air inflation is adopted during the test period, 08:00 and 16: feeding at 00, wherein a test group is fed with a control group feed (the feed-liquid ratio is 1; feeding for 2 times a day, apparent satiety, bottom absorption for 1 time a day, water change amount about 1/2, water temperature (23 + -1) DEG C, dissolved oxygen of (6 + -0.5) mg/L, pH of (7.1 + -0.5) during the test period, and culture period of 6 weeks (42 days). Before sampling, the test fish stops eating for 24 hours, is put into ice water for cold shock, is weighed to measure the body length and the body weight, strips off the viscera and the mesentery fat and is weighed, and the growth index of the test fish is calculated.

Weight gain ratio (WGR,%) =100 × (average final body mass-average initial body mass)/average initial body mass Feed Coefficient (FCR) = average bait dosage/(final average body mass-initial average body mass)

Fullness (CF, g/cm) ³ ) =100 × terminal body mass/body length ³

Survival rate (SR,%) =100 × (number of final individuals/number of initial individuals)

Gut-lipid ratio (ISI,%) =100 × gut-lipid mass/body mass

Visceral body ratio (VSI,%) =100 x visceral mass/body mass

As can be seen from Table 3, the survival rate of Cyprinus Carpio was not significantly different between the groups (P > 0.05). The feed coefficient of the test group is obviously lower than that of the control group, the weight gain rate of the treatment group is obviously higher than that of the control group, and other indexes are increased to a certain degree, which shows that the utilization rate of the feed can be obviously improved by HL-27.

TABLE 3 Effect of HL-27 on carp growth Performance

Claims (2)

1. The Staphylococcus equi-gastric is characterized by being Staphylococcus equi-gastric Staphylococcus equi equum HL-27, being preserved in China general microbiological culture Collection center with the preservation number of CGMCC No:24992.

2. use of staphylococcus equi-gastric according to claim 1 in fish farming.

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