CN108143776A - A kind of preparation method of Larimichthys crocea non-specific immunostimulating agents - Google Patents

A kind of preparation method of Larimichthys crocea non-specific immunostimulating agents Download PDF

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CN108143776A
CN108143776A CN201711348831.8A CN201711348831A CN108143776A CN 108143776 A CN108143776 A CN 108143776A CN 201711348831 A CN201711348831 A CN 201711348831A CN 108143776 A CN108143776 A CN 108143776A
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曾霖
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Zhejiang Ocean University ZJOU
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Abstract

The invention discloses a kind of preparation methods of Larimichthys crocea non-specific immunostimulating agents, 10 ~ 20 parts of β 1 are added in 3 ~ 5 parts of Leuconostoc mesenteroides bacterium powders, 3 glucans, 1 ~ 2h is stirred at a temperature of 20 ~ 25 DEG C, 1 ~ 3 part of astragalus root extract, 1 ~ 3 part of RADIX CODONOPSIS PILOSULAE from Shanxi of China's extract, 1 ~ 3 part of RADIX POLYGONI MULTIFLORI PREPARATA extract, 1 ~ 3 part of cistanche extracts and 3 ~ 5 parts of Gotu Kola P.Es are added, freeze-drying after mixing obtains immunostimulant.It has the beneficial effect that：Immunostimulant physiological activity prepared by the method for the present invention is low, safety, transfection are good, it is in delivery cell by active antigen, and enhance the phagocytic activity and bactericidal activity of macrophage and neutrophil, the decline of cellular immune function is delayed to improve the nospecific immunity of Larimichthys crocea simultaneously, so as to substantially reduce Larimichthys crocea to the infectious agents resistance such as bacterium and virus, the survival rate of Larimichthys crocea is improved.

Description

A kind of preparation method of Larimichthys crocea non-specific immunostimulating agents
Technical field
The present invention relates to aquaculture field, the preparation method of specifically a kind of Larimichthys crocea non-specific immunostimulating agents.
Background technology
((immunologic stimulant) is to refer to adjust animal immune system and activate immune to immunostimulant Function, enhancing body is to a kind of substance of the infectious agents resistance such as bacterium and virus.In recent years, home and abroad is carried out Immunostimulant is used for the research of aquiculture animal communicable disease prevention, main purpose to be in order to by immunostimulation Agent is difficult to the virus and bacteriosis of the aquiculture animal to prove effective for prevention using chemicals.
It is exactly body pair that the immunologic mechanism of fish, which can be divided into specificity and nospecific immunity, so-called nospecific immunity, The defense mechanism of unspecific pathogen, wherein fish epidermal mucus is distributed in, the lysozyme in blood and kidney and other organs (lysozyme), it is exactly a kind of under the synergistic effect of complement (complement), can is to deposit by the enzyme complement of bacterolysis The histone matter being in fish mucus and blood, activated channel have two:First, it can be activated by antigen antibody complex Classical pathway, second is that can be gathered by the bacteria lipopolysaccharide (lipopolysaccharide, LPS) unrelated with antibody and peptide The alternative route of the activation such as sugar.No matter the complement of which kind of pathway activation can dissolve thalline.
Invention content
The purpose of the present invention is to provide a kind of Leuconostoc mesenteroides growth reproduction speed is fast, bacterium powder harvest yield is big The preparation method of Larimichthys crocea non-specific immunostimulating agents.The immunostimulant safety that is prepared, transfection are good, can be apparent Improve the nospecific immunity of Larimichthys crocea.
The problem of present invention in background technology for mentioning, the technical solution taken is：NTM048 bacterial strains were purchased from positioned at day The independent rows politics and law of Mu Geng Jinshi City of this Chiba county (2-5-8Kazusakamatari, Kisarazu-shi, Chiba, Japan) People's product assessment technique fundamental mechanism patent Organism Depositary, preserving number are NITE BP-1519, and Classification And Nomenclature is intestines Film leukonid (Leuconostoc mesenteroides).
A kind of preparation method of Larimichthys crocea non-specific immunostimulating agents, includes the following steps：
1）Slant activation culture：By the Leuconostoc mesenteroides (Leuconostoc that preserving number is NITE BP-1519 Mesenteroides it) is inoculated on slant medium and carries out activation culture, pH is 6.0 ~ 6.5, and temperature is 20 ~ 25 DEG C, activation training Support the time for 12 ~ for 24 hours, add in 1 ~ 2 times of sterile water in the medium, be uniformly mixing to obtain seed liquor.Inclined-plane culture based component And its parts by weight are：60 ~ 70 portions of sucrose, 20 ~ 30 portions of maltose, 5 ~ 6 parts of yeast extracts, 5 ~ 6 parts of soy peptones, 0.005 ~ 0.008 part of biotin, 0.01 ~ 0.02 part of FeSO4·7H2O, 0.2 ~ 0.3 part of MgSO4·7H2O, 0.01 ~ 0.02 part of MnSO4· H2O, 3.2 ~ 4.8 parts of K2HPO4·3H2O, 0.5 ~ 0.9 part of KH2PO4, 0.04 ~ 0.06 part of CaCl2·6H2O, 0.2 ~ 0.5 part of NaCl, 0.1 ~ 0.2 part of Tween-20,10 ~ 20 parts of agar and 1000 ~ 1200 parts of water；
2）Level-one shaking flask culture：Seed liquor is transferred to by 3 ~ 10% inoculum concentration in level-one kind shaking flask, constant-temperature table culture, pH It is 6.0 ~ 6.5, temperature is 20 ~ 25 DEG C, and shaking speed is 100 ~ 150r/min, and incubation time is 12 ~ 36h；
3）Second-level shake flask culture：The bacterium solution that level-one shaking flask culture obtains is transferred to by 3 ~ 10% inoculum concentration in two level kind shaking flask, Constant-temperature shaking culture is carried out, pH is 6.0 ~ 6.5, and temperature is 20 ~ 25 DEG C, and shaking speed is 100 ~ 200r/min, and incubation time is 12~36h；
4）Fermentation tank culture：Bacterium solution after two level kind shaking flask culture is inoculated into the hair that sterilization crosses by 3 ~ 10% inoculum concentration In fermentation tank, fermentation tank culture, pH is 6.0 ~ 6.5, and temperature is 25 ~ 30 DEG C, and shaking speed is 150 ~ 250r/min.Fermentation medium Ingredient and its parts by weight are：100 ~ 120 portions of sucrose, 50 ~ 60 portions of maltose, 5 ~ 6 parts of yeast extracts, 5 ~ 6 parts of soy peptones, 0.001 ~ 0.003 part（S）- 4- benzyl -2- oxazolidones, 0.01 ~ 0.02 part of FeSO4·7H2O, 0.2 ~ 0.3 part of MgSO4·7H2O、 0.01 ~ 0.02 part of MnSO4·H2O, 3.2 ~ 4.8 parts of K2HPO4·3H2O, 0.5 ~ 0.9 part of KH2PO4, 0.04 ~ 0.06 part of CaCl2· 6H2O, 0.04 ~ 0.06 part of NaCl, 0.1 ~ 0.2 part of Tween-20 and 1000 ~ 1200 parts of water.Supplementing culture medium in fermentation process, obtains To zymotic fluid.Supplementing culture medium during fermentation adds fermentation medium after 12 ~ 12.5h, and adds 18 ~ 22% glycerine for the first time, Fermentation medium is added after 20 ~ 20.5h for the second time, fermentation medium is added for the third time after 24 ~ 26h, is fermented to 46 ~ 50h.Strain Flush, growth and breeding speed is fast, strain fast-growth after being inoculated into fermentation tank, and quickly enters logarithmic phase, ferments 16 ~ 20h enters stationary phase.During fermentation,（S）Other compositions have coupling in -4- benzyl -2- oxazolidones and culture medium, Strain division growth is promoted to form neoblast, nitrogen element content improves in chitosan structure in the cell wall of generation, and chitosan is lived Property functional group be modified, reduce the physiological activity of chitosan, be greatly reduced its danger；
5）It is prepared by bacterium powder：By centrifuging or filtering or Magneto separate zymotic fluid obtains wet Leuconostoc mesenteroides, washed with sterile water or PBS After washing, ultrasonication processing, freeze-drying obtains Leuconostoc mesenteroides bacterium powder.Ultrasonication condition is：Ultrasonication power is 430 ~ 460W crushes the time as 17 ~ 23min, time interval 10：8s/s；
6）It is prepared by immunostimulant：10 ~ 20 parts of β -1,3- glucans, 20 ~ 25 are added in 3 ~ 5 parts of Leuconostoc mesenteroides bacterium powders At a temperature of DEG C stir 1 ~ 2h, add 1 ~ 3 part of astragalus root extract, 1 ~ 3 part of RADIX CODONOPSIS PILOSULAE from Shanxi of China's extract, 1 ~ 3 part of RADIX POLYGONI MULTIFLORI PREPARATA extract, 1 ~ 3 part of cistanche extracts and 3 ~ 5 parts of Gotu Kola P.Es, after mixing freeze-drying obtain immunostimulant.It is above-mentioned to exempt from Epidemic disease stimulant can active antigen in delivery cell, and the activity of lysozyme and complement can be improved, increase the C3 ingredients of complement in body; The phagocytic activity of macrophage and neutrophil can not only be enhanced, but also the sterilization that can improve these cells is lived Property;Natural killer cells (NK) is activated, enhances it and kills outside the activity of foreign matter cell, moreover it is possible to promote macrophage interleukins One 2 (interleukin-2, IL-2).Leuconostoc mesenteroides bacterium powder, beta-1,3-dextran and Chinese herbal medicine extract（Astragalus root carries Take object, RADIX CODONOPSIS PILOSULAE from Shanxi of China's extract, RADIX POLYGONI MULTIFLORI PREPARATA extract, cistanche extracts and Gotu Kola P.E）There is coupling, can inhibit P65 subunits nuclear translocation caused by LPS.By inhibiting NF- κ B transcriptional activities, so as to inhibit downstream inflammatory factor IL-6, TNF- The release of α and play anti-inflammatory effect.At the same time, also promote the generation of IL-10, maintain proinflammatory factor and anti-inflammatory factors it Between balance.And the combination of NF- κ B and IL-2 promoters can also be enhanced by activating the nuclear translocation of senile cell NF- κ B, Improve IL-2 expression, the decline of anti-aging cellular immune function.In conclusion above-mentioned immunostimulant is presented by active antigen Cell, and enhance the phagocytic activity and bactericidal activity of macrophage and neutrophil, while the cellular immunity work(that slows down aging Can decline improve the nospecific immunity of Larimichthys crocea, so as to substantially reduce Larimichthys crocea to the infectiousness cause of disease such as bacterium and virus Body resistance improves the survival rate of Larimichthys crocea.
Preferably, level-one kind Shake flask medium ingredient and its parts by weight are：90 ~ 100 portions of sucrose, 30 ~ 40 portions of maltose, 5 ~ 6 parts of yeast extracts, 5 ~ 6 parts of soy peptones, 5 ~ 8 parts of dibasic ammonium citrates, 5 ~ 8 parts of sodium acetates, 0.01 ~ 0.02 part of FeSO4· 7H2O, 0.2 ~ 0.3 part of MgSO4·7H2O, 0.01 ~ 0.02 part of MnSO4·H2O, 3.2 ~ 4.8 parts of K2HPO4·3H2O, 0.5 ~ 0.9 part KH2PO4, 0.04 ~ 0.06 part of CaCl2·6H2O, 0.2 ~ 0.5 part of NaCl, 0.1 ~ 0.2 part of Tween-20 and 1000 ~ 1200 parts of water.
Preferably, two level kind Shake flask medium ingredient and its parts by weight are：100 ~ 120 portions of sucrose, 50 ~ 60 parts of malt Sugar, 5 ~ 6 parts of yeast extracts, 5 ~ 6 parts of soy peptones, 5 ~ 8 parts of dibasic ammonium citrates, 5 ~ 8 parts of sodium acetates, 0.01 ~ 0.02 part of FeSO4· 7H2O, 0.2 ~ 0.3 part of MgSO4·7H2O, 0.01 ~ 0.02 part of MnSO4·H2O, 3.2 ~ 4.8 parts of K2HPO4·3H2O, 0.5 ~ 0.9 part KH2PO4, 0.04 ~ 0.06 part of CaCl2·6H2O, 0.2 ~ 0.5 part of NaCl, 0.1 ~ 0.2 part of Tween-20 and 1000 ~ 1200 parts of water.On Nutriment abundance in level-one kind Shake flask medium and two level kind Shake flask medium is stated, Leuconostoc mesenteroides growth and breeding can be met Demand, the bacterium cell cultivated in pair or short catenation, cell wall thickness higher than MS medium cultures goldbeater's skin it is bright Beading bacterium cell wall.
Compared with prior art, the advantage of the invention is that：Immunostimulant physiological activity prepared by the method for the present invention is low, Safety, transfection are good, by active antigen in delivery cell, and enhance macrophage and neutrophil phagocytic activity and Bactericidal activity, while the decline of cellular immune function is delayed to improve the nospecific immunity of Larimichthys crocea, it is big so as to substantially reduce Yellow croaker improves the survival rate of Larimichthys crocea to the infectious agents resistance such as bacterium and virus.
Specific embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
A kind of preparation method of Larimichthys crocea non-specific immunostimulating agents, includes the following steps：
1）Slant activation culture：By the Leuconostoc mesenteroides (Leuconostoc that preserving number is NITE BP-1519 Mesenteroides it) is inoculated on slant medium and carries out activation culture, pH 6.5, temperature is 22 DEG C, the activation culture time For 12h, 1.5 times of sterile water is added in the medium, is uniformly mixing to obtain seed liquor.Inclined-plane culture based component and its parts by weight For：65 portions of sucrose, 25 portions of maltose, 5.5 parts of yeast extracts, 5.5 parts of soy peptones, 0.007 part of biotin, 0.02 part FeSO4·7H2O, 0.26 part of MgSO4·7H2O, 0.01 part of MnSO4·H2O, 4.2 parts of K2HPO4·3H2O, 0.8 part of KH2PO4、0.05 Part CaCl2·6H2O, 0.4 part of NaCl, 0.1 part of Tween-20,15 parts of agar and 1000 parts of water；
2）Level-one shaking flask culture：Seed liquor is transferred to by 3 ~ 10% inoculum concentration in level-one kind shaking flask, constant-temperature table culture, pH It is 6.5, temperature is 22 DEG C, shaking speed 100r/min, and incubation time is for 24 hours.Level-one kind Shake flask medium ingredient and its again Measuring part is：100 portions of sucrose, 35 portions of maltose, 5.5 parts of yeast extracts, 5.5 parts of soy peptones, 6 parts of dibasic ammonium citrates, 7 parts of acetic acid Sodium, 0.01 part of FeSO4·7H2O, 0.27 part of MgSO4·7H2O, 0.01 part of MnSO4·H2O, 4.3 parts of K2HPO4·3H2O, 0.7 part KH2PO4, 0.05 part of CaCl2·6H2O, 0.4 part of NaCl, 0.1 part of Tween-20 and 1000 parts of water；
3）Second-level shake flask culture：The bacterium solution that level-one shaking flask culture obtains is transferred to by 3 ~ 10% inoculum concentration in two level kind shaking flask, Constant-temperature shaking culture, pH 6.5 are carried out, temperature is 22 DEG C, shaking speed 150r/min, and incubation time is for 24 hours.Two level kind is shaken Bottle medium component and its parts by weight are：100 portions of sucrose, 55 portions of maltose, 5.5 parts of yeast extracts, 5.5 parts of soy peptones, 6 parts Dibasic ammonium citrate, 6 parts of sodium acetates, 0.01 part of FeSO4·7H2O, 0.2 part of MgSO4·7H2O, 0.01 part of MnSO4·H2O, 4.3 parts K2HPO4·3H2O, 0.7 part of KH2PO4, 0.05 part of CaCl2·6H2O, 0.4 part of NaCl, 0.1 part of Tween-20 and 1000 parts of water；
4）Fermentation tank culture：Bacterium solution after two level kind shaking flask culture is inoculated into the fermentation tank that sterilization crosses by 5% inoculum concentration In, fermentation tank culture, pH 6.5, temperature is 28 DEG C, shaking speed 200r/min.Fermentation medium components and its parts by weight For：110 portions of sucrose, 55 portions of maltose, 5.7 parts of yeast extracts, 5.6 parts of soy peptones, 0.002 part（S）- 4- benzyls -2- Oxazolidone, 0.01 part of FeSO4·7H2O, 0.27 part of MgSO4·7H2O, 0.01 part of MnSO4·H2O, 4.1 parts of K2HPO4·3H2O、 0.8 part of KH2PO4, 0.05 part of CaCl2·6H2O, 0.05 part of NaCl, 0.1 part of Tween-20 and 1000 parts of water.It is supplemented in fermentation process Culture medium obtains zymotic fluid.Supplementing culture medium during fermentation adds fermentation medium after 12h, and adds 20% glycerine for the first time, Fermentation medium is added after 20h for the second time, for 24 hours after add fermentation medium for the third time, ferment to 48h；
5）It is prepared by bacterium powder：Wet Leuconostoc mesenteroides is obtained by centrifuging zymotic fluid, after being washed with PBS, ultrasonication processing, freezing It is dried to obtain Leuconostoc mesenteroides bacterium powder.Ultrasonication condition is：Ultrasonication power is 450W, and it is 20min to crush the time, when Between between be divided into 10：8s/s；
6）It is prepared by immunostimulant：16 parts of β -1,3- glucans, at a temperature of 22 DEG C are added in 4 parts of Leuconostoc mesenteroides bacterium powders 1.5h is stirred, adds 2 parts of astragalus root extracts, 2 parts of RADIX CODONOPSIS PILOSULAE from Shanxi of China's extracts, 2 parts of RADIX POLYGONI MULTIFLORI PREPARATA extracts, 2 parts of Herba Cistanches extractions Object and 4 parts of Gotu Kola P.Es, after mixing freeze-drying obtain immunostimulant.
Embodiment 2：
A kind of preparation method of Larimichthys crocea non-specific immunostimulating agents, includes the following steps：
1）Slant activation culture：By the Leuconostoc mesenteroides (Leuconostoc that preserving number is NITE BP-1519 Mesenteroides it) is inoculated on slant medium and carries out activation culture, pH 6.0, temperature is 25 DEG C, the activation culture time For 12h, 1.5 times of sterile water is added in the medium, is uniformly mixing to obtain seed liquor.Inclined-plane culture based component and its parts by weight For：65 portions of sucrose, 25 portions of maltose, 5.5 parts of yeast extracts, 5.5 parts of soy peptones, 0.007 part of biotin, 0.02 part FeSO4·7H2O, 0.25 part of MgSO4·7H2O, 0.02 part of MnSO4·H2O, 4.2 parts of K2HPO4·3H2O, 0.8 part of KH2PO4、0.05 Part CaCl2·6H2O, 0.4 part of NaCl, 0.1 part of Tween-20,15 parts of agar and 1000 parts of water；
2）Level-one shaking flask culture：Seed liquor is transferred to by 3 ~ 10% inoculum concentration in level-one kind shaking flask, constant-temperature table culture, pH It is 6.0, temperature is 25 DEG C, shaking speed 100r/min, and incubation time is for 24 hours.Level-one kind Shake flask medium ingredient and its again Measuring part is：100 portions of sucrose, 35 portions of maltose, 5.5 parts of yeast extracts, 5.5 parts of soy peptones, 5.5 parts of dibasic ammonium citrates, 7 parts of second Sour sodium, 0.02 part of FeSO4·7H2O, 0.27 part of MgSO4·7H2O, 0.02 part of MnSO4·H2O, 4.6 parts of K2HPO4·3H2O、0.7 Part KH2PO4, 0.05 part of CaCl2·6H2O, 0.4 part of NaCl, 0.1 part of Tween-20 and 1000 parts of water；
3）Second-level shake flask culture：The bacterium solution that level-one shaking flask culture obtains is transferred to by 3 ~ 10% inoculum concentration in two level kind shaking flask, Constant-temperature shaking culture, pH 6.0 are carried out, temperature is 25 DEG C, shaking speed 150r/min, and incubation time is for 24 hours.Two level kind is shaken Bottle medium component and its parts by weight are：100 portions of sucrose, 55 portions of maltose, 5.5 parts of yeast extracts, 5.5 parts of soy peptones, 5.5 Part dibasic ammonium citrate, 5.5 parts of sodium acetates, 0.02 part of FeSO4·7H2O, 0.2 part of MgSO4·7H2O, 0.02 part of MnSO4·H2O、 4.6 parts of K2HPO4·3H2O, 0.7 part of KH2PO4, 0.05 part of CaCl2·6H2O, 0.4 part of NaCl, 0.1 part of Tween-20 and 1000 parts Water；
4）Fermentation tank culture：Bacterium solution after two level kind shaking flask culture is inoculated into the fermentation tank that sterilization crosses by 5% inoculum concentration In, fermentation tank culture, pH 6.0, temperature is 28 DEG C, shaking speed 200r/min.Fermentation medium components and its parts by weight For：110 portions of sucrose, 55 portions of maltose, 5.7 parts of yeast extracts, 5.5.5 parts of soy peptones, 0.002 part（S）- 4- benzyls- 2- oxazolidones, 0.02 part of FeSO4·7H2O, 0.27 part of MgSO4·7H2O, 0.02 part of MnSO4·H2O, 4.1 parts of K2HPO4· 3H2O, 0.8 part of KH2PO4, 0.05 part of CaCl2·6H2O, 0.05 part of NaCl, 0.1 part of Tween-20 and 1000 parts of water.In fermentation process Supplementing culture medium obtains zymotic fluid.Supplementing culture medium during fermentation adds fermentation medium after 12h, and adds 20% for the first time Glycerine adds fermentation medium after 20h for the second time, for 24 hours after add fermentation medium for the third time, ferment to 48h；
5）It is prepared by bacterium powder：Wet Leuconostoc mesenteroides is obtained by filtering fermentating liquid, after sterile water washing, ultrasonication processing, Freeze-drying obtains Leuconostoc mesenteroides bacterium powder.Ultrasonication condition is：Ultrasonication power is 450W, and the broken time is 23min, time interval 10：8s/s；
6）It is prepared by immunostimulant：20 parts of β -1,3- glucans, at a temperature of 20 DEG C are added in 5 parts of Leuconostoc mesenteroides bacterium powders 2h is stirred, adds 1 part of astragalus root extract, 3 parts of RADIX CODONOPSIS PILOSULAE from Shanxi of China's extracts, 2 parts of RADIX POLYGONI MULTIFLORI PREPARATA extracts, 2 parts of cistanche extracts With 5 parts of Gotu Kola P.Es, freeze-drying after mixing obtains immunostimulant.
Embodiment 3：
A kind of preparation method of Larimichthys crocea non-specific immunostimulating agents, includes the following steps：
1）Slant activation culture：By the Leuconostoc mesenteroides (Leuconostoc that preserving number is NITE BP-1519 Mesenteroides it) is inoculated on slant medium and carries out activation culture, pH 6.2, temperature is 24 DEG C, the activation culture time For 24 hours, to add in 1.5 times of sterile water in the medium, being uniformly mixing to obtain seed liquor.Inclined-plane culture based component and its parts by weight For：65 portions of sucrose, 24 portions of maltose, 5.5 parts of yeast extracts, 5.5 parts of soy peptones, 0.007 part of biotin, 0.02 part FeSO4·7H2O, 0.25 part of MgSO4·7H2O, 0.02 part of MnSO4·H2O, 4.7 parts of K2HPO4·3H2O, 0.8 part of KH2PO4、 0.045 part of CaCl2·6H2O, 0.4 part of NaCl, 0.1 part of Tween-20,16 parts of agar and 1000 parts of water；
2）Level-one shaking flask culture：Seed liquor is transferred to by 6% inoculum concentration in level-one kind shaking flask, constant-temperature table culture, pH is 6.2, temperature is 24 DEG C, shaking speed 100r/min, and incubation time is for 24 hours.Level-one kind Shake flask medium ingredient and its weight Part it is：100 portions of sucrose, 35 portions of maltose, 5.5 parts of yeast extracts, 5.5 parts of soy peptones, 5.5 parts of dibasic ammonium citrates, 7 parts of acetic acid Sodium, 0.02 part of FeSO4·7H2O, 0.27 part of MgSO4·7H2O, 0.02 part of MnSO4·H2O, 4.7 parts of K2HPO4·3H2O, 0.7 part KH2PO4, 0.045 part of CaCl2·6H2O, 0.4 part of NaCl, 0.1 part of Tween-20 and 1000 parts of water；
3）Second-level shake flask culture：The bacterium solution that level-one shaking flask culture obtains is transferred to by 6% inoculum concentration in two level kind shaking flask, into Row constant-temperature shaking culture, pH 6.2, temperature are 24 DEG C, shaking speed 150r/min, and incubation time is for 24 hours.Two level kind shaking flask Medium component and its parts by weight are：100 portions of sucrose, 55 portions of maltose, 5.5 parts of yeast extracts, 5.5 parts of soy peptones, 5.5 parts Dibasic ammonium citrate, 5.5 parts of sodium acetates, 0.02 part of FeSO4·7H2O, 0.2 part of MgSO4·7H2O, 0.02 part of MnSO4·H2O、4.7 Part K2HPO4·3H2O, 0.7 part of KH2PO4, 0.045 part of CaCl2·6H2O, 0.4 part of NaCl, 0.1 part of Tween-20 and 1000 parts of water；
4）Fermentation tank culture：Bacterium solution after two level kind shaking flask culture is inoculated into the fermentation tank that sterilization crosses by 5% inoculum concentration In, fermentation tank culture, pH 6.2, temperature is 28 DEG C, shaking speed 200r/min.Fermentation medium components and its parts by weight For：110 portions of sucrose, 55 portions of maltose, 5.7 parts of yeast extracts, 5.5.5 parts of soy peptones, 0.002 part（S）- 4- benzyls- 2- oxazolidones, 0.02 part of FeSO4·7H2O, 0.27 part of MgSO4·7H2O, 0.02 part of MnSO4·H2O, 4.1 parts of K2HPO4· 3H2O, 0.8 part of KH2PO4, 0.045 part of CaCl2·6H2O, 0.045 part of NaCl, 0.1 part of Tween-20 and 1000 parts of water.Fermentation process Middle supplementing culture medium, obtains zymotic fluid.Supplementing culture medium during fermentation, for 24 hours after add fermentation medium for the first time, and add 20% glycerine adds fermentation medium after 20h for the second time, for 24 hours after add fermentation medium for the third time, ferment to 48h；
5）It is prepared by bacterium powder：Wet Leuconostoc mesenteroides is obtained by Magneto separate zymotic fluid, after sterile water washing, at ultrasonication Reason, freeze-drying obtain Leuconostoc mesenteroides bacterium powder.Ultrasonication condition is：Ultrasonication power is 450W, and the broken time is 23min, time interval 10：8s/s；
6）It is prepared by immunostimulant：20 parts of β -1,3- glucans, at a temperature of 20 DEG C are added in 5 parts of Leuconostoc mesenteroides bacterium powders 2h is stirred, adds 1 part of astragalus root extract, 3 parts of RADIX CODONOPSIS PILOSULAE from Shanxi of China's extracts, 2 parts of RADIX POLYGONI MULTIFLORI PREPARATA extracts, 3 parts of cistanche extracts With 5 parts of Gotu Kola P.Es, freeze-drying after mixing obtains immunostimulant.
Embodiment 4：
Experiment is carried out in June to September, cultured large yellow croaker net cage is divided into 8 groups, 5 networking casees are test group, and 3 groups are control group, are made With the feed containing immunostimulant（Per kilogram feed addition immunostimulant 50mg）It feeds, continuously feeds 8 weeks, statistics nature hair The sick death rate calculates relative immunity protective rate（Relative Percent Survival, RPS）, RPS=（1- immune groups are dead Rate/control group the death rate）×100%.The results show that the disease incidence of experimental group Larimichthys crocea is remarkably decreased, average survival carries High by 37.39%, relative immunity protective rate RPS shows that Larimichthys crocea can be enhanced in cultivation to disease by feeding immunostimulant up to 84.40% The resistance of bacterium infection（Table 1）；
The 1 disease-resistant result of the test of rheum officinale fish culture of table
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme of the present invention is described in detail in embodiment described above, it should be understood that the above is only For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in the spirit of the present invention, Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.

Claims (8)

1. a kind of preparation method of Larimichthys crocea non-specific immunostimulating agents, it is characterised in that include the following steps：
1）It is prepared by bacterium powder：By the Leuconostoc mesenteroides (Leuconostoc that preserving number is NITE BP-1519 Mesenteroides) slant activation culture, the culture of level-one shaking flask, second-level shake flask culture, fermentation tank culture obtain sufficient bacterium Kind, strain ultrasonication is handled, freeze-drying obtains Leuconostoc mesenteroides bacterium powder；
2）It is prepared by immunostimulant：10 ~ 20 parts of β -1,3- glucans, 20 ~ 25 are added in 3 ~ 5 parts of Leuconostoc mesenteroides bacterium powders At a temperature of DEG C stir 1 ~ 2h, add 1 ~ 3 part of astragalus root extract, 1 ~ 3 part of RADIX CODONOPSIS PILOSULAE from Shanxi of China's extract, 1 ~ 3 part of RADIX POLYGONI MULTIFLORI PREPARATA extract, 1 ~ 3 part of cistanche extracts and 3 ~ 5 parts of Gotu Kola P.Es, after mixing freeze-drying obtain immunostimulant.
2. a kind of preparation method of Larimichthys crocea non-specific immunostimulating agents according to claim 1, it is characterised in that： The inclined-plane culture based component and its parts by weight are：60 ~ 70 portions of sucrose, 20 ~ 30 portions of maltose, 5 ~ 6 parts of yeast extracts, 5 ~ 6 parts Soy peptone, 0.005 ~ 0.008 part of biotin, 0.01 ~ 0.02 part of FeSO4·7H2O, 0.2 ~ 0.3 part of MgSO4·7H2O、 0.01 ~ 0.02 part of MnSO4·H2O, 3.2 ~ 4.8 parts of K2HPO4·3H2O, 0.5 ~ 0.9 part of KH2PO4, 0.04 ~ 0.06 part of CaCl2· 6H2O, 0.2 ~ 0.5 part of NaCl, 0.1 ~ 0.2 part of Tween-20,10 ~ 20 parts of agar and 1000 ~ 1200 parts of water.
3. a kind of preparation method of Larimichthys crocea non-specific immunostimulating agents according to claim 1, it is characterised in that： The level-one kind Shake flask medium ingredient and its parts by weight are：90 ~ 100 portions of sucrose, 30 ~ 40 portions of maltose, 5 ~ 6 parts of yeast Cream, 5 ~ 6 parts of soy peptones, 5 ~ 8 parts of dibasic ammonium citrates, 5 ~ 8 parts of sodium acetates, 0.01 ~ 0.02 part of FeSO4·7H2O、0.2~0.3 Part MgSO4·7H2O, 0.01 ~ 0.02 part of MnSO4·H2O, 3.2 ~ 4.8 parts of K2HPO4·3H2O, 0.5 ~ 0.9 part of KH2PO4、0.04~ 0.06 part of CaCl2·6H2O, 0.2 ~ 0.5 part of NaCl, 0.1 ~ 0.2 part of Tween-20 and 1000 ~ 1200 parts of water.
4. a kind of preparation method of Larimichthys crocea non-specific immunostimulating agents according to claim 1, it is characterised in that： The two level kind Shake flask medium ingredient and its parts by weight are：100 ~ 120 portions of sucrose, 50 ~ 60 portions of maltose, 5 ~ 6 parts of yeast Cream, 5 ~ 6 parts of soy peptones, 5 ~ 8 parts of dibasic ammonium citrates, 5 ~ 8 parts of sodium acetates, 0.01 ~ 0.02 part of FeSO4·7H2O、0.2~0.3 Part MgSO4·7H2O, 0.01 ~ 0.02 part of MnSO4·H2O, 3.2 ~ 4.8 parts of K2HPO4·3H2O, 0.5 ~ 0.9 part of KH2PO4、0.04~ 0.06 part of CaCl2·6H2O, 0.2 ~ 0.5 part of NaCl, 0.1 ~ 0.2 part of Tween-20 and 1000 ~ 1200 parts of water.
5. a kind of preparation method of Larimichthys crocea non-specific immunostimulating agents according to claim 1, it is characterised in that： The fermentation medium components and its parts by weight are：100 ~ 120 portions of sucrose, 50 ~ 60 portions of maltose, 5 ~ 6 parts of yeast extracts, 5 ~ 6 parts of soy peptones, 0.001 ~ 0.003 part（S）- 4- benzyl -2- oxazolidones, 0.01 ~ 0.02 part of FeSO4·7H2O、0.2~ 0.3 part of MgSO4·7H2O, 0.01 ~ 0.02 part of MnSO4·H2O, 3.2 ~ 4.8 parts of K2HPO4·3H2O, 0.5 ~ 0.9 part of KH2PO4、 0.04 ~ 0.06 part of CaCl2·6H2O, 0.04 ~ 0.06 part of NaCl, 0.1 ~ 0.2 part of Tween-20 and 1000 ~ 1200 parts of water.
6. a kind of preparation method of Larimichthys crocea non-specific immunostimulating agents according to claim 1, it is characterised in that：Institute Supplementing culture medium during the fermentation stated adds fermentation medium after 12 ~ 12.5h, and adds 18 ~ 22% glycerine for the first time, 20 ~ Fermentation medium is added after 20.5h for the second time, fermentation medium is added for the third time after 24 ~ 26h, is fermented to 46 ~ 50h.
7. a kind of preparation method of Larimichthys crocea non-specific immunostimulating agents according to claim 1, it is characterised in that： The ultrasonication condition is：Ultrasonication power is 430 ~ 460W, crushes the time as 17 ~ 23min, time interval 10： 8s/s 。
8. a kind of preparation method of Larimichthys crocea non-specific immunostimulating agents according to claim 1, it is characterised in that：Institute The slant activation culture stated, pH are 6.0 ~ 6.5, and temperature is 20 ~ 25 DEG C, the activation culture time for 12 ~ for 24 hours；Level-one shaking flask culture, PH is 6.0 ~ 6.5, and temperature is 20 ~ 25 DEG C, and shaking speed is 100 ~ 150r/min, and shaking flask incubation time is 12 ~ 36h；Two level is shaken Bottle culture, pH are 6.0 ~ 6.5, and temperature is 20 ~ 25 DEG C, and shaking speed is 100 ~ 200r/min, and shaking flask incubation time is 12 ~ 36h； Fermentation tank culture, pH are 6.0 ~ 6.5, and temperature is 25 ~ 30 DEG C, and shaking speed is 150 ~ 250r/min.
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Application publication date: 20180612