CN115725466B - Staphylococcus equi and application thereof in fish culture - Google Patents
Staphylococcus equi and application thereof in fish culture Download PDFInfo
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Abstract
The invention discloses staphylococcus equine and application thereof in fish culture, and relates to a strain and application thereof. The staphylococcus equi Staphylococcus equorum HL-27 of the invention is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No:24992. the staphylococcus equine Staphylococcus equorum HL-27 strain has the capability of producing siderophores and DDP-IV inhibitors, and the HL-27 strain is added into fish feed for fish culture, so that iron element which is more beneficial to absorption is provided for fish, the problem of waste 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 in the cultivation environment polluted by the organic phosphorus.
Description
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 materials in fish baits, and the proper addition of the saccharides in the fish baits can play roles in saving the bait cost, reducing protein energy supply, improving protein deposition and promoting the growth of fish. However, the fish has low sugar utilization capability, and when the sugar level in the feed exceeds a certain limit, the fish has low disease resistance, slow growth, high death rate and other symptoms, and the fish has limited capability of relieving the glucose load, so that the sugar utilization capability is low, and the large amount of the sugar in the fish feed is limited.
Along with the development of fish culture technology, research on the requirements of fish on trace elements is increasingly paid attention to, the trace elements are mineral elements, the content of the trace elements is small, but the trace elements are indispensable nutrient substances, and trace elements which have positive important effects on the growth and development of fish at present are iron, copper, manganese, zinc, iodine, selenium and the like. Wherein iron is a component of heme and myoglobin, takes part in the transportation of oxygen and carbon dioxide by the identity of a carrier, and takes part in the construction of systems such as cytochrome and peroxidase of aquatic animals, and the physiological and biochemical processes such as respiration and biological oxidation in the aquatic animals are closely related. Proper amount of iron element exists in aquatic animal body, so that normal metabolism can be maintained, and disease preventing capability of the aquatic animal body is improved. The fish body lacks iron element, which can reduce the hematopoietic function of the organism tissue and even cause anemia in severe cases. At present, the problem of iron deficiency is solved by adding ferrous salt into the feed, but the absorption and utilization rate is low, and the waste of the feed is easily caused.
Disclosure of Invention
The invention provides a staphylococcus equine strain and application thereof.
The staphylococcus equine disclosed by the invention is staphylococcus equine Staphylococcus equorum HL-27, which is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No:24992.
the invention relates to application of staphylococcus equi in fish culture.
The staphylococcus equi of the invention is added into fish feed for fish culture.
The staphylococcus equine Staphylococcus equorum HL-27 colony of the invention is spherical or elliptic (slightly irregular), has a diameter of about 1-4mm, is milk white convex, is opaque, has moist and glossy surface, has no flagella, can not move, and is gram-positive.
Staphylococcus equine Staphylococcus equorum HL-27 was inoculated onto CAS medium and after a period of incubation, a distinct color-changing ring was formed around the colonies, indicating that the HL-27 strain had a strong siderophore production capacity, with a Su value of 48% at 37 ℃. Staphylococcus equine Staphylococcus equorum HL-27 sequesters iron ions by a high affinity siderophore and immobilizes them to form elemental iron. The staphylococcus equi Staphylococcus equorum HL-27 can be added into fish feed to directly provide iron element for fish, so that the iron element can be absorbed by fish conveniently, normal metabolism of fish can be maintained, and disease prevention capability of the organism can be improved.
Staphylococcus equi Staphylococcus equorum HL-27 forms an obvious hydrolysis ring around a colony after culturing for a period of time on a SKM culture medium, the diameter D of the hydrolysis ring of HL-27 is 19.74mm, the diameter D of the colony is 5.40mm, and the D/D is 3.66; the productivity of the DDP-IV inhibitor is up to 55.61% at 37 ℃, and the HL-27 strain has stronger ability of producing the DDP-IV inhibitor. Fish have three sources of glucagon and GLP-1 (incretin glucagon-like peptide-1), namely pancreas, intestine and brain, respectively, and DDP-IV (dipeptidyl peptidase-1) inhibitor can indirectly achieve the aim of reducing blood sugar of fish by prolonging half-life of GLP-1. The staphylococcus equine Staphylococcus equorum HL-27 is added into the fish feed to improve the digestion utilization rate of the feed and the growth performance of the fish.
The staphylococcus equi Staphylococcus equorum HL-27 strain is inoculated on Meng Jinna organophosphorus medium, obvious organophosphorus dissolving ring is formed around bacterial colony after culturing for a period of time, the diameter D of the organophosphorus dissolving ring of HL-27 is 13.66mm, the diameter D of bacterial colony is 3.29mm, and the D/D is 4.15, which shows that the HL-27 strain has stronger organophosphorus dissolving function. The HL-27 strain has higher tolerance to pH and bile salts. The HL-27 strain is subjected to a hemolysis experiment, and the result is negative, so that the strain disclosed by the invention has no risk of human and animal diseases and is high in use safety.
The staphylococcus equine Staphylococcus equorum HL-27 strain of the present invention has the ability to produce siderophores and DDP-IV inhibitors. The HL-27 strain is added into fish feed for fish culture, can replace ferrous salt, provides iron element which is more favorable for absorption for fish, has no waste problem, reduces blood sugar of fish, and improves the digestion utilization rate of feed. The HL-27 strain also has the function of decomposing organic phosphorus, and can effectively eliminate the toxic action of the organic phosphorus in the cultivation environment polluted by the organic phosphorus.
The staphylococcus equine Staphylococcus equorum HL-27 is staphylococcus equine and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation address of Beijing Chaoyang area North Xicilu No. 1 and No. 3. The collection number is CGMCC No.24992, and the collection date is 2022, 5 months and 30 days.
Drawings
FIG. 1 shows the screening results of the siderobotics of Staphylococcus equi Staphylococcus equorum HL-27;
FIG. 2 shows a milk proteolytic loop on a media of 2% nonfat dry milk of Staphylococcus equi Staphylococcus equorum HL-27%;
FIG. 3 is a phylogenetic tree constructed from Staphylococcus equi Staphylococcus equorum HL-27;
FIG. 4 shows a phosphate solubilizing loop on an organophosphorus solid medium of Staphylococcus equi Staphylococcus equorum HL-27.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
The first embodiment is as follows: the staphylococcus equine of the present embodiment is staphylococcus equine Staphylococcus equorum HL-27, which is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms), and has a preservation number of CGMCC No:24992, the preservation date is 2022, 5 and 30.
The current application of staphylococcus equi (Staphylococcus equorum) mainly focuses on degrading heavy metal ions, preventing gray mold of fruits and vegetables and prolonging the shelf life of meat products, and reports of staphylococcus equi (Staphylococcus equorum) with siderophores and DDP-IV inhibitors are still the first.
The staphylococcus equine Staphylococcus equorum HL-27 strain of the present invention has the ability to produce siderophores and DDP-IV inhibitors. The HL-27 strain is added into fish feed for fish culture, can replace ferrous salt, provides iron element which is more favorable for absorption for fish, has no waste problem, reduces blood sugar of fish, and improves the digestion utilization rate of feed. The HL-27 strain also has the function of decomposing organic phosphorus, and the current organic phosphorus pesticide is easy to enter underground water, rivers and lakes through surface permeation and field water body flow in the use process, and finally causes water body pollution. The HL-27 strain has the function of decomposing organic phosphorus, is used as feed for fish culture, can effectively eliminate the toxic action of the organic phosphorus in a culture environment polluted by the organic phosphorus, and improves the yield of the fish.
The method for obtaining the staphylococcus equine of the present embodiment:
1. separating and purifying strains: 2021, selecting carp population cultivated in paddy field in Dali area of Panjin city of Liaoning, randomly selecting 3 healthy 1 year old carps (150+ -10 g), anaesthetizing with MS-222 anaesthetic (250 mg/L), wiping the surface of the carps with absolute ethyl alcohol, dissecting with sterilized scissors and forceps in an ultra clean bench, taking out the whole intestinal tract of the carps, lightly extruding the content, placing into conical flask containing glass beads and 50mL sterile water, oscillating at 180r/min at room temperature for 30min, performing gradient dilution, and concentrating 10 -3 、10 -4 、10 -5 100 mu L of the gradient is coated on an LB solid culture medium plate, each gradient is repeated for 3 times, and the mixture is placed at 28 ℃ for culturing for 24-48 hours. After 48h of culture, strains with different shapes are selected for separation.
2. Identification of bacterial siderophores
2.1 reactivation of the separated and purified strain, transferring the strain to an LB plate for culturing for 24 hours at 28 ℃, picking a single colony by using a sterilized toothpick, transferring the single colony to a solid detection culture medium of chrome azure S (Chromeazurol S, CAS), culturing for 2-3 days at 37 ℃ in an inverted mode, and observing the size of a color-changing ring around the colony.
After a period of culture, obvious color-changing rings are formed around colonies, and strains with the capacity of producing siderophores are selected, wherein the diameter D of the color-changing rings of the HL-27 strain in LB culture medium is 9.66mm, the diameter D of the colonies is 3.47mm, the D/D of the colonies is 2.78, and the HL-27 strain has the capacity of producing siderophores with high capacities (shown in figure 1).
2.2 further experiments on the strain HL-27, which shows a distinct color circle:
(1) Inoculating the activated lawn into an iron-limiting SA liquid culture medium, and performing shake culture at 37 ℃ for 48 hours 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 for 15 minutes at 13000rpm, and removing sediment to leave a supernatant;
(3) Transferring the supernatant into a test tube treated by concentrated hydrochloric acid, adding a certain amount of freshly prepared CAS detection solution to make the volume ratio of the supernatant to the detection solution be 1:1, fully mixing uniformly, and standing at room temperature for 1h;
(4) Measuring an absorbance value (A) at a wavelength of 630nm, taking double distilled water as a control for zeroing, taking the absorbance value (Ar) at the wavelength of 630nm after the unvaccinated SA limited iron culture medium and the detection liquid which are measured by the same method are mixed as a reference value, and expressing the activity unit of the siderophore by the following formula:
Su≈(Ar-As)/Ar×100；
wherein: su is the siderophore content; ar is the OD value of the SA limited iron culture medium and the supernatant of the detection liquid which are not inoculated; as is the OD of the culture supernatant.
When the unit of siderophore activity is less than 10, it is generally considered negative, and the mixture of siderophores and the detection liquid has no color change.
The Su value of the siderophore produced by the strain HL-27 at 37 ℃ is 48%, which indicates that the strain has stronger siderophore producing capacity.
3. Identification and assay of DDP-IV inhibitor-producing strains
3.1 bacterial strain HL-27 was inoculated on SKM medium and after 48h of cultivation at 28℃a distinct hydrolytic circle was formed around the colony, the hydrolytic circle diameter D of HL-27 was 19.74mm, the colony diameter D was 5.40mm and D/D was 3.66 (as shown in FIG. 2). The HL-27 strain has the ability to produce DDP-IV inhibitors.
3.2 inoculating HL-27 into a 96-well plate, accurately dripping 25 mu L, 1.6mmol/L glycyl-prolyl-p-nitroaniline and 25 mu L CFS or CFE into each well; the reaction was carried out at 37℃for 15 minutes, 50. Mu.L of DDP-IV (dipeptidyl peptidase IV) was further added thereto at 0.01U/mL, the reaction was continued at 37℃for 1 hour, and then 100. Mu.L of sodium acetate buffer solution (pH=4.0) was further added thereto to terminate the reaction, and the absorbance of the reaction solution was measured at 405nm using an enzyme-labeled instrument to obtain the inhibition ratio of the strain DDP-IV.
Wherein, the formula of the DDP-IV inhibition rate is:
A sample to be measured : 25. Mu.L of sample +25. Mu.LGly-pro-phy +50. Mu.LDDP-IV + 100. Mu.L of sodium acetate;
A sample blank : 25. Mu.L of sample +50. Mu.L of LTris-HCl +25. Mu.L of LGly-pro-phy + 100. Mu.L of sodium acetate;
A negative control :25 mu LTris-HCl+25 mu LGly-pro-phy+50 mu LDDP-IV+100 mu L sodium acetate;
A negative blank :75 mu LTris-HCl+25 mu LGly-pro-phy+100 mu L sodium acetate.
The productivity of the DDP-IV inhibitor of the HL-27 strain reaches 55.61% at 37 ℃, which shows that the HL-27 strain has stronger ability of producing the DDP-IV inhibitor.
4. Identification of Strain HL-27
4.1 physiological and biochemical identification: bacterial strain HL-27 was streaked on solid LB medium plates in three sections, single colonies were isolated and their morphology was described, and gram staining and physiological and biochemical identification of the strain were performed according to the handbook of the commonly used bacterial systems identification.
The colony characteristics of HL-27 on LB solid medium are as follows: the colony is spherical or elliptic (slightly irregular), has a diameter of about 1-4mm, is milky convex, is opaque, has moist, glossy and flagellum-free surface, cannot move, and is gram-positive. The physiological and biochemical indicators of part of HL-27 are shown in Table 1. According to the description of the physiological characteristics of Bacillus in the Bojie's Manual of bacteria identification, HL-27 has the same characteristics as the physiological biochemistry of the species of Staphylococcus equi (Staphylococcus equorum), from which it is deduced that the strain HL-27 may be Staphylococcus equi (Staphylococcus equorum).
TABLE 1 physiological and biochemical results of strain HL-27
4.2 16S rRNA identification: and (3) selecting a bacterial genome DNA extraction kit of Beijing Soxhaust biological technology company, and extracting, separating and purifying the strain DNA. The bacterial universal primer 27F/1492R is adopted for PCR amplification, and the PCR amplification system is a 25 mu L system: 10 Xbuffer 2.5. Mu.L, taq enzyme 0.5. Mu.L, primer 27F 0.5. Mu.L, primer 1492R 0.5. Mu.L, 1. Mu.L of DNA template, ddH 2 O20. Mu.L. The reaction procedure is set to 95 ℃ for 5min of pre-denaturation; denaturation at 94℃for 50s, annealing at 56℃for 30s, extension at 72℃for 1.5min, cycle times for 30 times, extension at 72℃for 10min again, and preservation at 4 ℃. The PCR amplified product was sent to RuiBiotech company for sequencing. And comparing sequencing results of the strain 16S rRNA through NCBI database, and constructing a phylogenetic tree.
After sequencing the 16S rRNA sequence, BLAST alignment in NCBI revealed that the 16S rRNA gene sequence of the HL-27 strain was 99% similar to Staphylococcus equi (Staphylococcus equorum). The phylogenetic tree is shown in FIG. 3, and the minimum branch of the HL-27 strain and the staphylococcus equi (Staphylococcus equorum) (NR_ 027520.1) can be seen through the phylogenetic tree, the evolutionary distance is relatively short, and the comprehensive physiological and biochemical indexes identify the HL-27 strain as the staphylococcus equi (Staphylococcus equorum).
The second embodiment is as follows: the application of the staphylococcus equi in fish culture in the specific embodiment is provided.
And a third specific embodiment: the second difference between this embodiment and the second embodiment is that: the staphylococcus equi is added into fish feed for fish culture. The other is the same as in the second embodiment.
The specific embodiment IV is as follows: in this embodiment, a staphylococcus equi Staphylococcus equorum HL-27 bacterial liquid was prepared.
The staphylococcus equi Staphylococcus equorum HL-27 bacterial liquid culture medium consists of 1000mL 10% bean sprout juice and 10.48g 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); wherein 100g of soybean sprouts is added with 1000mL of water, boiled for 1h, filtered and supplemented with water to 1L, and the soybean sprouts are subjected to damp-heat sterilization at 121 ℃ and stored for standby, namely 10% of soybean sprout juice by mass fraction.
Inoculating Staphylococcus equorum HL-27 seed solution into Staphylococcus equorum HL-27 bacterial solution culture medium at 37deg.C and 180r/min to obtain viable count of 24h,Staphylococcus equorum HL-27 bacterial solution of 1.65X10 9 cfu/mL (whereas horse stomach grape ball)The viable count of the seed liquid of the fungus HL-27 is 6.51X10 8 cfu/mL, staphylococcus equi HL-27 seed liquid is prepared by inoculating in LB liquid culture medium).
Example 1
The staphylococcus equine (Staphylococcus equorum) HL-27 is respectively inoculated on a flat plate of Meng Jinna organophosphorus bacteria culture medium by using a sterilizing toothpick, is placed at 28 ℃ for culturing for 24-48 hours, forms obvious organophosphorus dissolving rings around colonies, adopts a crisscross method to measure the diameters D of the colonies and the organophosphorus dissolving rings, and calculates D/D; the phosphate solubilizing ring on the Staphylococcus equorum HL-27 organophosphorus solid medium is shown in FIG. 4, the diameter D of the phosphate solubilizing ring of 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 stronger function of solubilizing organophosphorus.
EXAMPLE 2 detection of stress resistance of Staphylococcus equi Staphylococcus equorum HL-27
1. Acid resistance detection: the spore count was 2X 10 8 cfu/mL of Staphylococcus equi HL-27 was inoculated into LB liquid medium at pH 2.0, 3.0 and 4.0 in an inoculum size of 2% (v/v), the bacterial liquid was aspirated after 3 hours, the viable count was measured after 24 hours of culture at 37℃and the survival rate of the strain was calculated.
2. And (3) detecting the bile salt resistance: the activated spore number is 2 multiplied by 10 8 cfu/mL of Staphylococcus equi HL-27 bacteria liquid 1mL of the diluted liquid was sucked into a sterilized dish, and then the plate was poured with LB solid medium containing 0.5%, 1.0% and 1.5% sodium taurocholate, while MRS solid medium containing no sodium taurocholate was used as a control group, cultured at 37℃for 48 hours, colony counting was performed, and the survival rate of the strain was calculated.
As can be seen from Table 2, the effect on the number of spores of Staphylococcus equi HL-27 was small at pH 3.0 and 4.0, and the number of spores of HL-27 was significantly decreased at pH 2.0; and when the concentration of bile salts is increased, the change of the number of spores of the staphylococcus equi HL-27 is smaller. Staphylococcus equi HL-27 has high tolerance to pH and bile salts.
TABLE 2 acid and bile salt resistance test of Staphylococcus equi HL-27
3. Hemolysis experiment: hemolysis is classified into alpha hemolysis (incomplete hemolysis, producing a grass green hemolytic ring), beta hemolysis (complete hemolysis, in the form of a well-defined, colorless transparent hemolytic ring), and non-hemolysis.
Each fresh blood plate was streaked with Staphylococcus equi HL-27 and was observed after culturing at 30℃for 48 hours. The test result of staphylococcus equine HL-27 shows negative, which indicates that staphylococcus equine HL-27 has no risk of human and animal diseases.
Example 3
1. Preparing staphylococcus equi Staphylococcus equorum HL-27 bacterial liquid
Inoculating the seed solution of Staphylococcus equi Staphylococcus equorum HL-27 to the culture medium of Staphylococcus equi Staphylococcus equorum HL-27 at 37deg.C and 180r/min for 24 hr to obtain a viable count of Staphylococcus equi HL-27 of 1.65X10 9 cfu/mL (while the viable count of the seed liquid of the staphylococcus equi HL-27 is 6.51X10) 8 cfu/mL, staphylococcus equi HL-27 seed liquid is prepared by inoculating in LB liquid culture medium). Wherein the culture medium of the strain of staphylococcus equi Staphylococcus equorum HL-27 is prepared from 1000mL 10% bean sprout juice (soybean sprout 100g added with 1000mL water, boiled for 1h, filtered to supplement water to 1L, sterilized at 121deg.C under moist heat, and stored for use), 10.48g 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) cultivation test is carried out in a circulating water tank of a laboratory of the institute of aquatic products of Heilongjiang, china, and the carp used for the test is purchased from Jing Bo farm of Dandong, liaoning province. 200 carps with initial weight of 66g and breeding in the current year are selected and temporarily cultured in a cement pond with the weight of 4.3m multiplied by 1.5m multiplied by 1.2m for 1 week to adapt to the test environment, and a control group feed (Tongwei feed 181) is fed during the temporary culture period. After temporary rearing, 120 carps with similar weight, better activity and intact body surface are selected and randomly divided into 2 groups (a control group and a test group), 3 groups are parallel, 20 parallel carps are placed in 6 circulating water rearing boxes with the size 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 more than 24 hours of aeration is used as test water body, gas stone is adopted for 24 hours of aeration during the test, and the daily consumption is 08:00 and 16:00 feeding, namely feeding a control group feed (the feed-liquid ratio is 1:0.18) mixed with staphylococcus equi (Staphylococcus equorum) HL-27 bacterial liquid into a test group, and feeding the control group feed into the control group; feeding for 2 times a day, apparent feeding, sucking at the bottom of 1 time a day, changing water for 1 time a day, changing water amount about 1/2, water temperature (23+ -1) deg.C during test, dissolved oxygen (6+ -0.5) mg/L, pH (7.1+ -0.5), and cultivation 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, and is stripped of viscera and mesenteric fat and is weighed to calculate the growth index.
Weight gain rate (WGR,%) =100× (average terminal body mass-average initial body mass)/average initial body mass Feed Coefficient (FCR) =average feed delivery rate/(terminal average body mass-initial average body mass)
Full of fertilizer (CF, g/cm) 3 ) =100×terminal body mass/length 3
Survival rate (SR,%) =100× (number of final individuals/number of initial individuals)
Intestinal lipid ratio (ISI,%) =100×intestinal lipid mass/mass
Dirty volume ratio (VSI,%) =100×visceral mass/mass
As can be seen from Table 3, the survival rate of carp was not significantly different from group to group (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 all other indexes are increased to a certain extent, so that the feed utilization rate of HL-27 can be obviously improved.
TABLE 3 influence of HL-27 on carp growth Properties
Claims (2)
1. A staphylococcus equine is characterized in that the staphylococcus equine isStaphylococcus equorum HL-27, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No:24992.
2. use of staphylococcus equi in fish farming according to claim 1, wherein said use does not include disease treatment.
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| CN114507628A (en) * | 2022-03-30 | 2022-05-17 | 昆明理工大学 | Staphylococcus equinus FS310 and application thereof |
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