CN109439565A - One plant of Pediococcus pentosaceus bacterial strain, its probiotics and preparation method thereof - Google Patents

Abstract

One plant of Pediococcus pentosaceus bacterial strain, its probiotics and preparation method thereof, the bacterial strain are preserved in China typical culture collection center, and culture presevation number is CCTCC NO:M 2018109, classification naming are as follows: Pediococcus pentosaceus SL001.After being mixed in feed the probiotics that the bacterial strain is applicable in aquaculture made of correlation method; feeding is carried out to freshwater fish; infection of the fish-pathogenic bacterias such as Aeromonas hydrophila to freshwater fishes such as grass carp, crucians can quickly be inhibited; its protective rate >=70%, while can also improve freshwater fish intestinal microflora, the growth for promoting freshwater fish, the immunity for improving freshwater fish.

Description

One plant of Pediococcus pentosaceus bacterial strain, its probiotics and preparation method thereof

Technical field

The present invention relates to aquatic microorganisms technical fields, and in particular to one plant of Pediococcus pentosaceus bacterial strain, its probiotics And preparation method thereof.

Background technique

China's Cultivated water area is vast, and the demand with people to protein resource food increases, aquaculture industry Development is increasingly taken seriously.It is aobvious according to " investigation of Chinese Fishery cultivation market and development prospect research report (version in 2018) " Show, China's aquaculture is based on freshwater aquiculture, and yield accounting is more than 60%, and freshwater aquiculture accounts for freshwater aquiculture based on fish 88% or more of total output.In recent years, the attention with country to agriculture environmental protection, aquatic products intensive fish pond area extend rapidly, The disease outburst of corresponding intensive fish pond is increasingly severe, becomes the technical bottleneck for restricting fish aquaculture sustainable development.

Currently, reported freshwater fish disease mainly based on bacterial disease, is particularly acute, thermophilic aqueous vapor unit cell Fungus diseases cause fish underproduction loss serious, break out within nearly 2 years rate up to 50% or more, fish underproduction loss late is up to 20%~30%, often Direct economic loss caused by year is more than 10,000,000,000 yuan.

The Control Technology measure of existing cultured freshwater fish disease, mainly using antibiotic, chemicals, probiotics and Vaccine, wherein be the main means of fish diseases prevention and control using chemicals, although there is certain effect, chemicals Long-term a large amount of uses will lead to pathogen and generate drug resistance, influence ecological environment and lead to aquatic products pesticide residue.Probiotics is A major class in microorganism, growth and breeding speed is fast, and the period is short, and can generate some metabolites during the growth process, These metabolites can inhibit the breeding of pathogen and pathogen to reach certain ecology and microecological balance, while certain metabolism Product can promote fish growth.Vaccine effect in aquiculture disease prevention and treatment is also preferable, and the most of China are to go out at present How live vaccine the preparation cost of vaccine and is immunized aquatic livestock at this stage, is always the bottleneck that large-scale popularization uses.

Currently, the common probiotics probiotics of aquaculture industry has bacillus, lactic acid bacteria, Bifidobacterium, ferment Female bacterium etc., since the brood cell of bacillus has the advantages such as high temperature resistant, acid and alkali-resistance, so that bacillus processing is more convenient, application Range is wider.Lactic acid bacteria can generate organic acid, hydrogen peroxide, biacetyl, bacteriocin etc., be the natural bacteriostatic of fish-pathogenic bacteria Product, while lactic acid bacteria can also generate prebiotics, promote animal body growth and development.As beneficial bacteria of intestinal tract, it can determine lactic acid bacteria It grows in animal intestinal wall, restores and enhancing enteron aisle stable state plays a role, maintain host immune stable state to play, help host short of money Resisting pathogenic microbes and promotion host reach promotion growth of animal to the absorption of nutriment, the mesh of disease preventing and treating 's.

Pediococcus pentosaceus (Pediococcus pentosaceus) one kind as lactic acid bacteria, belong to Lactobacillaceae, piece ball Pseudomonas is a kind of typical lactic acid bacteria, can produce the substances such as acid and IIa class pediocin with sugar fermentation, have and kill pathogen And the function of putrefactive microorganisms.In recent years, lactic acid bacteria class Pediococcus pentosaceus cultivated in the world as probiotics field by To great attention.2013, No. 2045 bulletin " catalogue of feed additive varieties (2013) " of The Ministry of Agriculture of the People's Republic of China, MOA Middle regulation, Pediococcus pentosaceus are the feed level microbe additive strains that can be used as probiotics Direct-fed cultivated animals.

102132788 B of CN discloses preparation and the application side of a kind of myxococcus fulvus probiotics used for aquiculture Method, comprising the following steps: (1) take one plant of orange myxobacter JCH-04, deposit number CGMCCNo.2893, bacterial strain is cultivated in CY It is cultivated 3 days, in the fermentation medium 30 DEG C, the culture of 180rpm shaking flask 5 days for 30 DEG C on base；(2) from one ring strain of inclined-plane picking It is inoculated in CY fluid nutrient medium and cultivates 3 days for 30 DEG C of test tube, then will be inoculated in equipped with 30 DEG C of cultures 3 in fermentation medium test tube It；(3) by the liquid spawn of step (2) be inoculated in equipped with 30 DEG C in fermentation medium triangular flask, 180rpm shaking flask culture 5 It；(4) the shaking flask culture bacterium solution of step (3) is condensed into 50,000,000,000 CFU/mL bacterium solutions with 0.2 μm of micro-filtrate membrane filtration, acquisition Concentration bacterium solution is probiotics, is not referred to the stability of the probiotics.

Existing probiotics easy in inactivation during transporting, using and saving, stability is poor, so that it is living to reduce its biology Property effect；After some bacterial strains enter alimentary canal, it is not highly resistant to the effect of hydrochloric acid, bile acid etc., to can not determine for a long time Enteron aisle is planted to function；Probiotics preventive effect is better than therapeutic effect, but acts on slowly, needs to be used continuously for a long time, cost Unfavorable factors and the limitation of the technology such as it increase accordingly.

Summary of the invention

The technical problem to be solved by the present invention is to overcome drawbacks described above of the existing technology, provide one plant of pentose piece Coccus new strains, Pediococcus pentosaceus bacterial strain amphimicrobian growth, have preferable tolerance, into after alimentary canal, energy to high temperature It is effective against the effect of hydrochloric acid, bile acid etc., enteron aisle can be colonized the long period and play effect.

The further technical problems to be solved of the present invention are to provide a kind of pair of fish-pathogenic bacteria good antimicrobial effect, can improve Fish immunity power and promotion fish growth, and bacterial strain heat resistance is strong, and stability is good, after being absorbed by fish body, can inhibit rapidly The growth of fish-pathogenic bacteria, it is especially significant to the anti-infected by Aeromonas hydrophila control efficiency of the freshwater fishes such as grass carp, crucian, have Conducive to the probiotics for being suitable for the prevention and treatment of aquaculture fish disease of microbial inoculum transport and Shelf-life.

The further technical problems to be solved of the present invention are to provide a kind of preparation method of probiotics.

The technical solution used to solve the technical problems of the present invention is that one plant of Pediococcus pentosaceus bacterial strain, on March 24th, 2018 It is preserved in China typical culture collection center (abbreviation CCTCC；Address: Wuhan, China Wuhan University), culture presevation number is CCTCC NO:M 2018109, classification naming are as follows: Pediococcus pentosaceus SL001；Latin name:Pediococcus pentosaceus SL001。

It is as follows that the present invention further solves technical solution used by its technical problem: a kind of suitable for the micro- of aquaculture Ecological agent is made of the Pediococcus pentosaceus SL001 fermentation that culture presevation number is CCTCC NO:M 2018109.Suppression therein Bacterium active material is insensitive to protease, stablizes in high temperature, not easy in inactivation during transporting, using and saving, stability It is good；It can inhibit the growth of freshwater fish pathogen, fish intestines Bacterial community can be improved, have facilitation to fish growth, and The immunity of fish can be improved.

Further, the Pediococcus pentosaceus SL001 probiotics are liquid preparation or solid pharmaceutical preparation.

Further, the animal of the aquaculture is freshwater fish.

Further, the freshwater fish is grass carp, crucian or carp.

The present invention further solves technical solution used by its technical problem: a kind of suitable for the micro- of aquaculture The preparation method of ecological agent, comprising the following steps:

(1) inoculation activation: Pediococcus pentosaceus SL001 preservation slant strains being transferred on activated inclined plane culture medium and are activated overnight, Obtain activated seed liquor；The activated inclined plane culture medium is MRS fluid nutrient medium, the activation culture condition: temperature 30 ~40 DEG C, liquid amount is the 10~30% of container volume, 12~16 h of anaerobism stationary culture；

(2) expand culture for the first time: step (1) the activated seed liquor is inoculated into culture medium by 1~2% inoculum concentration It carries out expanding culture for the first time in the 50 L fermentors that liquid amount is 65~70%；The first time expands the fermented and cultured of culture Based formulas: 15~20 g/L of glucose, 3~5 g/L of fish meal protein peptone, 1~4 g/L of soy peptone, beef extract 5~10 G/L, yeast powder 10~15 g/L, MgSO₄•7H₂O 0.3~0.5 g/L, MnSO₄•5H₂0.1~0.2 g/L of O, ammonium citrate 1~2 g/L, 2~3 g/L of sodium acetate, 1~2 mL/L of Tween-80；The first time expands the condition of culture of culture: initial PH is 6.2~6.4, controlled at 30~40 DEG C, cultivates 12~15h, entire incubation is stirred without ventilation every 6~8 h It mixes once, every time 2~5 min, OD600 is 3.0~5.0；

(3) expand culture second: step (2) first time being expanded into the seed liquor that culture obtains and is connect by 8~10% inoculum concentrations In the 500 L fermentors that kind is 70~75% to culture medium liquid amount, the same step of fermentative medium formula of second of expansion culture (2) first time expands the fermentative medium formula of culture, expands the condition of culture of culture for second: initial pH is 6.2~ 6.4, controlled at 30~40 DEG C, entire incubation stirs once, every time 2~5 min without ventilation every 6-8 h, OD600 is 3.0~5.0, until terminating fermentation in fermentor when pH=3.4~4.0；

(4) concentration collect: after putting tank, be concentrated, collect, obtain Tiny ecosystem liquid preparation or it is spray-dried after obtain Tiny ecosystem Solid pharmaceutical preparation.

The present invention using Pediococcus pentosaceus SL001 prepare probiotics admix in feed feed freshwater fish when, by 1 ×10⁹~1 × 10¹¹Cfu/g ratio, which is admixed or sprayed, to be added in feed or feed adding according to the 1%~3% of fish weight Add feed.

The invention has the advantages that: (1) to identify that the bacterial strain is pentose piece ball by 16S rRNA gene and genome sequencing Bacterium new strains (culture presevation number: CCTCC NO:M 2018109, classification naming are as follows: Pediococcus pentosaceus SL001, Classification system:Pediococcus pentosaceus SL001), with the Pediococcus pentosaceus sequence homology highest announced in ncbi database Up to 99%；(2) have using the probiotics of Pediococcus pentosaceus preparation and improve fish immunity power, good anti-bacterial effect, fish can be promoted The features such as class growth and strong resistance；(3) can be determined in fish intestines using the probiotics of Pediococcus pentosaceus SL001 preparation It grows, fish intestines Bacterial community can be improved, adjust microecological balance, latent pathogen determines in Tiny ecosystem site reduction enteron aisle It grows；(4) preparation method using the probiotics of Pediococcus pentosaceus SL001 preparation is simple, and low production cost can scale Production can partially replace the use of antibiotic, and toxic side effect will not be generated by feeding freshwater fish, be had a good application prospect.

Biomaterial preservation situation explanation

The Pediococcus pentosaceus bacterial strain of the present invention is preserved in China typical culture collection center on March 24th, 2018 (referred to as CCTCC；Address: Wuhan, China Wuhan University), culture presevation number is CCTCC NO:M 2018109, classification naming are as follows: penta Sugar-tablet coccus SL001；Latin name:Pediococcus pentosaceus SL001。

Detailed description of the invention

Fig. 1 is Pediococcus pentosaceus SL001 have been announced through 16S rRNA gene and genome sequencing, and with ncbi database Pediococcus pentosaceus sequence result figure is compared；

Fig. 2 Pediococcus pentosaceus SL001 fermented supernatant fluid In Vitro Bacteriostasis test result figure, a in figure: antibacterial plate observation；B: antibacterial Enclose size；

Fig. 3 Pediococcus pentosaceus SL001 scanning electron microscopic observation figure；

Fig. 4 Pediococcus pentosaceus SL001 heat resistance test result figure；

Fig. 5 Pediococcus pentosaceus SL001 Substance analysis of physical and chemical property result figure；

Fig. 6 Pediococcus pentosaceus SL001 fermented supernatant fluid high performance liquid chromatography separation characteristic pattern；

Fig. 7 Pediococcus pentosaceus SL001 fermented supernatant fluid high performance liquid chromatography separation active material bacteriostatic test result figure；

Fig. 8 fluorescence quantitative PCR detection influences grass carp muscle cell growth related gene expression result chart, in figure: PA is first Group, DB are second group；

Impact analysis result figure of the probiotics of Fig. 9 Pediococcus pentosaceus SL001 preparation to grass carp intestinal microorganism group；

The impact analysis result figure that the probiotics of Figure 10 Pediococcus pentosaceus SL001 preparation express grass carp immune factor.

Specific embodiment

Below with reference to embodiment and attached drawing, the invention will be further described.

Culture medium used in the embodiment of the present invention can be commercially available or according to " microbiological culture media handbook " The record of (Microbiology Culture Media Manual) is prepared, and agents useful for same passes through routine business approach It obtains.

The Pediococcus pentosaceus new strains of the present invention, are preserved in China typical culture collection center, and culture presevation number is CCTCC NO:M 2018109, classification naming are as follows: Pediococcus pentosaceus SL001；Latin name Pediococcus pentosaceus SL001。

Pediococcus pentosaceus SL001 bacterial strain amphimicrobian growth of the present invention, there is certain tolerance, bacterial strain to high temperature Substance in fermentation liquid is insensitive to protease, stablizes in high temperature, surveys through 16S rRNA gene and full-length genome Sequence, and the Pediococcus pentosaceus sequence announced with ncbi database is compared, as the result is shown highest similarity be 99%(referring to Fig. 1).

It is applicable in the probiotics of aquaculture described in the present embodiment, is CCTCC NO:M by culture presevation number 2018109, Pediococcus pentosaceus SL001 fermentation are made；The probiotics can inhibit the growth of freshwater fish pathogen, can improve Fish intestines Bacterial community has facilitation to fish growth, and can improve the immunity of fish.

The Pediococcus pentosaceus SL001 separation identification of the present invention and research method are as follows:

(1) screening of Pediococcus pentosaceus new strains and the bacteriostatic test to fish-pathogenic bacteria

Pedotheque is acquired from seabeach near Hainan Province, China Dadong River, and dilution is coated on MRS plate through gradient dilution On, after 37 DEG C of 24 h of Anaerobic culturel, pick from the plate 13 plants of bacteriums carry out scribing line be further purified, later will be purified Bacterium access MRS meat soup in 37 DEG C of 48 h of anaerobic fermentation culture, fermented supernatant fluid is collected by centrifugation, select 6 plants of fish-pathogenic bacterias for Indicator bacteria, be respectively as follows: Aeromonas hydrophila (Aeromonas hydrophila G1), Aeromonas veronii (Aeromonas veronii X005), Aeromonas sobria (Aeromonas sobriaB002), Edwardsiella tarda (Edwardsiella tarda EIB202), format galactococcus (Lactococcus garvieaeB010), Plesiomonas shigelloides (Plesiomonas ShigelloideB008), above-mentioned pathogen is carried out bacteriostatic test (referring to fig. 2) with Odontothrips loti, is sent out Existing one plant of bacterium has good fungistatic effect to 6 kinds of fish-pathogenic bacterias, therefore this plant of bacterium is named as SL001.

(2) it is identified using 16S rRNA gene

Strain inoculated is in MRS broth bouillon, and 37 DEG C of 12 h of anaerobism stationary culture culture, thalline were collected by centrifugation, utilizes bacterium Genome extraction kit (production of Shanghai Sheng Gong Bioisystech Co., Ltd) extracts bacterial genomes, utilizes 16S rRNA gene Amplimer.

27F:5 '-AGAGTTTGATCCTGGCTCAG-3 '；

1492R:5 '-ACGGCTACCTTGTTACGACTT-3 '.

With above-mentioned primer amplification 16S rRNA gene, the expected length of sequence is about 1500 bp.

PCR reaction system (20 μ L): 2 μ L Ex Taq Buffer(10 ×), 1.6 μ L dNTP Mixture(2.5 Mmol/L), 0.6 μ L(10 μm ol/L of upstream primer), 0.6 μ L(10 μm ol/L of downstream primer), template 1 μ L, 0.2 μ LTaKaRa Ex Taq(5 U/ μ L), 14 μ L H2O.

PCR response procedures: 95 DEG C of 5 min of initial denaturation；30 circulations: 95 DEG C, 45 sec；55 DEG C, 45 sec；72 DEG C, 1.5 min；72 DEG C, 10 min.

16S rRNA gene PCR product is detected and is recycled through 1.0 % agarose gel electrophoresis.Then by PCR after purification Product is attached with pMD-18T vector, is transformed into bacillus coli DH 5 alpha competent cell, and ammonia benzyl resistance screening is carried out, Plasmid is extracted after picking positive transformant serves the sequencing of Hai Shenggong Bioisystech Co., Ltd.

(3) strain cell morphologic observation, physiological and biochemical test and bacterium Heat-tolerance Determination

The milky that bacterial strain bacterium colony on MRS plate is rounded, surface wettability, edge are neat, passes through scanning electron microscopic observation, bacterium Body shows as bigeminy or tetrad is spherical (referring to Fig. 3).Referring to " primary Jie Shi Bacteria Identification handbook ", sugared hair has been carried out to the bacterial strain Ferment test, gelatin liquefaction test, catalase test, nitrate reduction test, mobility test etc., as a result such as table 1.

The Bacterial Physiological biochemical test of 1 Pediococcus pentosaceus SL001 of table

Note: "+" indicates positive in table, and "-" indicates negative.

Bacterial strain 37 DEG C of Anaerobic culturels in MRS meat soup dispense bacterium solution to several 1.5 to logarithmic growth phase (about 12 h) In mL centrifuge tube, respectively in 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C of 15 min of water-bath, with After be coated in MRS plate, after 37 DEG C of 24 h of Anaerobic culturel count clump count (referring to fig. 4), as the result is shown 80 DEG C it Before, there are also good activity for thallus.

(4) physicochemical property and separation of Pediococcus pentosaceus SL001 Substance

By 48 h fermented supernatant fluid of Pediococcus pentosaceus SL001 bacterial strain (12000 rpm, 30 min centrifugation) respectively through 30 DEG C, 60 DEG C, 80 DEG C, 90 DEG C, 100 DEG C, after 121 DEG C and 50 mg/mL pepsin, 30 min, respectively take 100 μ L for antibacterial It tests (referring to Fig. 5), utilizes the PBS solution (PL of identical pH；Lactic acid is adjusted) and the conduct control of MRS meat soup.

48 h fermented supernatant fluid of Pediococcus pentosaceus SL001 bacterial strain (12000 rpm, 30 min centrifugation) is separately taken, through 1.5 times of bodies Product acetonitrile overnight precipitation, takes supernatant, is lyophilized in freeze drier, is redissolved with water after centrifugation, ultra high efficiency liquid phase is utilized after filtering Chromatography (1290 Infinity of Agilent；Pillar be the mm of ZORBAX SB-C18(4.6 × 150,5 μm)) initial gross separation (referring to Fig. 6).Mobile phase is ddH2O and acetonitrile, 1 mL/min of flow velocity, chromatography eluant program are as follows: 0~4 min acetonitrile 0%, 4~5 min Acetonitrile 0%~4%, 5~6 min acetonitriles 4%, 6~6.5 min acetonitriles 4%~5%, 6.5~7 min acetonitriles 5%, 7~7.5 min second Nitrile 5%~6%, 7.5~9 min acetonitriles 6%, 9~9.5 min acetonitriles 6%~8%, 9.5~11 min acetonitriles 8%, 11~11.5 min Acetonitrile 8%~10%, 11.5~15 min acetonitriles 10%, 15~30 min acetonitriles 10%~100%, Detection wavelength is respectively 215 nm.The component being collected into is lyophilized in vacuum concentration instrument, acetonitrile is removed, ddH is added₂O dissolution, then carries out bacteriostatic activity inspection It surveys (referring to Fig. 7).

The method that the present embodiment prepares probiotics using Pediococcus pentosaceus SL001, comprising the following steps:

(1) inoculation activation: Pediococcus pentosaceus SL001 preservation slant strains being transferred on activated inclined plane culture medium and are activated overnight, Obtain activated seed liquor；The activated inclined plane culture medium is MRS fluid nutrient medium, and the activation culture condition: temperature is 37 DEG C, culture medium liquid amount is the 15% of container volume, anaerobism stationary culture 12h；

(2) expand culture for the first time: step (1) the activated seed liquor is inoculated into culture medium dress liquid by 2% inoculum concentration Amount expands culture to carry out first time in 70% 50 L fermentors；The first time expands the fermentative medium formula of culture are as follows: 20 g/L of glucose, 5 g/L of fish meal protein peptone, 3 g/L of soy peptone, 7 g/L of beef extract, 13 g/L of yeast powder, MgSO₄•7H₂O 0.4 g/L、MnSO₄•5H₂0.2 g/L of O, 1 g/L of ammonium citrate, 3 g/L of sodium acetate, 2 mL/ of Tween-80 L；The first time expands the condition of culture of culture: initial pH is 6.3, controlled at 37 DEG C, cultivates 12h, entirely cultivated Cheng Wuxu ventilation is stirred once, every time 2 min, OD every 6 h₆₀₀It is 4.0；

(3) expand culture second: step (2) first time being expanded into the seed liquor that culture obtains and is inoculated with by 10% inoculum concentration Expand culture, the fermented and cultured of second of expansion culture second of progress in the 500 L fermentors for being 70% to culture medium liquid amount The same step of based formulas (2) first time expands the fermentative medium formula of culture, the culture item of second of the expansion culture Part: initial pH is 6.3, controlled at 37 DEG C, cultivates 12h, and entire incubation is primary every 6 h stirring without ventilating, often Secondary 2 min, OD₆₀₀It is 4.0, until terminating fermentation in fermentor when pH=3.7；

(4) concentration is collected: after putting tank, being concentrated, is collected, obtain Tiny ecosystem liquid preparation, by obtaining Tiny ecosystem after spray drying Solid pharmaceutical preparation.

Application example 1: right after the probiotics spice feeding grass carp of the new bacterium SL001 preparation of above-mentioned Pediococcus pentosaceus The impact analysis of Growth of Grass Carps Ctenopharyngodon Idellus:

(1) healthy grass carp (32.1 ± 16 g of weight) is randomly divided into 2 groups, every group 50.Test uses 72 h of aeration originally Water, water temperature are controlled at 20 DEG C~23 DEG C, and grass carp was temporarily supported before on-test and is stopped eating 48 h, and when on-test weighs the initial of grass carp Weight.

(2) first groups: 1 × 10 will be added with⁹The feed of the probiotics of cfu/g Pediococcus pentosaceus SL001 preparation is thrown Graze fish, daily feeding 2 times, continuously feeds 30 days according to weight 1% to food every time；Second group: by the feed feeding grass of sterilizing Fish, daily feeding 2 times are continuously fed 30 days every time according to 1% feeding of weight.

(3) at the end of to be tested, the weight of grass carp is weighed, formula is utilized: WGR (%)=100 × (Wt-W0)/ W0；SGR (%)=100 × (lnWt lnW0)/T calculates Growth of Grass Carps Ctenopharyngodon Idellus performance indicator, WGR in formula: weight gain； SGR: logarithm proportion of weight to height；Wt: fish average weight when off-test；W0: fish average weight when on-test.It the results are shown in Table 2.

The probiotics spice of the new bacterium SL001 preparation of 2 Pediococcus pentosaceus of table feeds the influence to Growth of Grass Carps Ctenopharyngodon Idellus performance

Note: PA in table: test group；DB: control group.

(4) 5 are then selected at random to be dissected, take its musculature, influence growth using fluorescence quantitative PCR detection Expression conditions (referring to Fig. 8), it may be assumed that MSTN-1, MSTN-2(myostatin gene) and the increasing of IGF-1, IGF-2(insulin The long factor).

Application example 2: right after the probiotics spice feeding grass carp of the new bacterium SL001 preparation of above-mentioned Pediococcus pentosaceus The impact analysis of grass carp intestinal microorganism group:

(1) healthy grass carp (29.8 ± 11 g of weight) is randomly divided into 2 groups, every group 40.Test uses 72 h of aeration originally Water, water temperature are controlled at 20 DEG C~23 DEG C, and grass carp was temporarily supported before on-test and is stopped eating 48 h.

(2) first groups: 1 × 10 will be added with⁹ The feeding of the probiotics of the new bacterium SL001 preparation of cfu/g Pediococcus pentosaceus Material feeds grass carp, and daily feeding 2 times is continuously fed 30 days according to weight 1% to food every time；Second group: the feed of sterilizing is thrown Graze fish, daily feeding 2 times, continuously feeds 30 days according to weight 1% to food every time.

(3) at the end of to be tested, 6 are selected at random for every group and dissected, take its enteron aisle, carry out high-flux sequence, as a result It has been shown that, the new bacteria microorganism preparation of the Pediococcus pentosaceus prepared through the foregoing embodiment can quickly inhibit Aeromonas and vibrios to exist It is bred in enteron aisle, potential probiotics is promoted to increase significant (referring to Fig. 9).

Application example 3: right after the probiotics spice feeding grass carp of the new bacterium SL001 preparation of above-mentioned Pediococcus pentosaceus The impact analysis of grass carp immune effect:

(1) healthy grass carp (30.1 ± 10 g of weight) is randomly divided into 2 groups, every group 20.Test uses 72 h of aeration originally Water, water temperature are controlled at 20 DEG C~23 DEG C, and grass carp was temporarily supported before on-test and is stopped eating 48 h.

(2) first groups: 1 × 10 will be added with⁹The feeding of the probiotics of the new bacterium SL001 preparation of cfu/g Pediococcus pentosaceus Expect feeding grass carp, daily feeding 2 times is continuously fed 30 days according to weight 1% to food every time；Second group: the feed of sterilizing is fed Grass carp is eaten, daily feeding 2 times is continuously fed 30 days according to weight 1% to food every time.

(3) at the end of to be tested, 3 are selected at random for every group and are dissected, take its liver and spleen tissue, extract total serum IgE, Using fluorescence quantitative PCR detection test group (PA) and the immune factor expression quantity of control group (DB), discovery is exempted from test group Epidemic disease factor expression amount is significantly higher than control group fish (referring to Figure 10).

Application example 4: right after the probiotics spice feeding grass carp of the new bacterium SL001 preparation of above-mentioned Pediococcus pentosaceus Infection analysis of the anti-microbial pathogen Aeromonas hydrophila to grass carp:

(1) healthy grass carp (50.5 ± 12 g of weight) is randomly divided into 2 groups, every group 50.Test uses 72 h of aeration originally Water, water temperature are controlled at 20 DEG C~23 DEG C, and grass carp was temporarily supported before on-test and is stopped eating 48 h.

(2) first groups: 1 × 10 will be added with⁹The feeding of the probiotics of the new bacterium SL001 preparation of cfu/g Pediococcus pentosaceus Expect feeding grass carp, daily feeding 2 times is continuously fed 30 days according to weight 1% to food every time；Second group: the feed of sterilizing is fed Grass carp is eaten, daily feeding 2 times is continuously fed 30 days according to weight 1% to food every time.

(3) after feeding 30 days, 0.1 mL injected to every grass carp, 1 × 10⁶The pathogen Aeromonas hydrophila of cfu/mL, It is observed continuously 10 days, statistics cumulative mortality is shown in Table 3, the new bacterium of the Pediococcus pentosaceus prepared through the foregoing embodiment as known from Table 3 Microorganism formulation can quickly inhibit the breeding of pathogen Aeromonas hydrophila, reach 80%~90% to the protection of grass carp.

Grass carp protection is tested after the probiotics spice feeding grass carp of the new bacterium SL001 preparation of 3 Pediococcus pentosaceus of table As a result

Note: data are cumulative mortality in table.

Sequence table

<110>Hunan Normal University

The preparation method and application of<120>one plants of Pediococcus pentosaceus new strains, its probiotics

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catcatgccc cttatgacct gggctacaca cgtgctacaa tggatggtac aacgagtcgc 1260

gagaccgcga ggttaagcta atctcttaaa accattctca gttcggactg taggctgcaa 1320

ctcgcctaca cgaagtcgga atcgctagta atcgcggatc agcatgccgc ggtgaatacg 1380

ttcccgggcc ttgtacacac cgcccgtcac accatgagag tttgtaacac ccaaagccgg 1440

tggggtaacc ttttaggagc tagccgtcta atgtgaaaag ttatttta 1488

Claims (6)

1. one plant of Pediococcus pentosaceus bacterial strain, which is characterized in that be preserved in China typical culture collection center, culture presevation number is CCTCC NO:M 2018109, classification naming are as follows: Pediococcus pentosaceus SL001；Latin name:Pediococcus pentosaceus SL001。

2. a kind of probiotics suitable for aquaculture, which is characterized in that the culture presevation number as described in claim 1 is The Pediococcus pentosaceus SL001 fermentation of CCTCC NO:M 2018109 is made.

3. being suitable for the probiotics of aquaculture according to claim 2, which is characterized in that the probiotics are Liquid preparation or solid pharmaceutical preparation.

4. being suitable for the probiotics of aquaculture according to Claims 2 or 3, which is characterized in that the aquaculture Animal be freshwater fish.

5. being suitable for the probiotics of aquaculture according to claim 4, which is characterized in that the fresh-water fishes are grass Fish, crucian or carp.

6. a kind of preparation method as described in Claims 2 or 3 suitable for the probiotics of aquaculture, which is characterized in that The following steps are included:

(1) inoculation activation: Pediococcus pentosaceus SL001 preservation slant strains being transferred on activated inclined plane culture medium and are activated overnight, Obtain activated seed liquor；The activated inclined plane culture medium is MRS fluid nutrient medium, the activation culture condition: temperature 30 ~40 DEG C, liquid amount is the 10~30% of container volume, 12~16 h of anaerobism stationary culture；

(2) expand culture for the first time: step (1) the activated seed liquor is inoculated into culture medium by 1~2% inoculum concentration It carries out expanding culture for the first time in the 50 L fermentors that liquid amount is 65~70%；The first time expands the fermented and cultured of culture Based formulas: 15~20 g/L of glucose, 3~5 g/L of fish meal protein peptone, 1~4 g/L of soy peptone, beef extract 5~10 G/L, yeast powder 10~15 g/L, MgSO₄•7H₂O 0.3~0.5 g/L, MnSO₄•5H₂0.1~0.2 g/L of O, ammonium citrate 1~2 g/L, 2~3 g/L of sodium acetate, 1~2 mL/L of Tween-80；The first time expands the condition of culture of culture: initial PH is 6.2~6.4, controlled at 30~40 DEG C, cultivates 12~15h, entire incubation is stirred without ventilation every 6~8 h It mixes once, every time 2~5 min, OD₆₀₀It is 3.0~5.0；

(3) expand culture second: step (2) first time being expanded into the seed liquor that culture obtains and is connect by 8~10% inoculum concentrations In the 500 L fermentors that kind is 70~75% to culture medium liquid amount, the same step of fermentative medium formula of second of expansion culture (2) first time expands the fermentative medium formula of culture, expands the condition of culture of culture for second: initial pH is 6.2~ 6.4, controlled at 30~40 DEG C, entire incubation stirs once, every time 2~5 min without ventilation every 6-8 h, OD₆₀₀It is 3.0~5.0, until terminating fermentation in fermentor when pH=3.4~4.0；

(4) concentration collect: after putting tank, be concentrated, collect, obtain Tiny ecosystem liquid preparation or it is spray-dried after obtain Tiny ecosystem Solid pharmaceutical preparation.