CN1513481A - Method for preparing non-specific immunity activator - Google Patents
Method for preparing non-specific immunity activator Download PDFInfo
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- CN1513481A CN1513481A CNA031329020A CN03132902A CN1513481A CN 1513481 A CN1513481 A CN 1513481A CN A031329020 A CNA031329020 A CN A031329020A CN 03132902 A CN03132902 A CN 03132902A CN 1513481 A CN1513481 A CN 1513481A
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Abstract
A non-specific immunoactivator as the feed additive for fish, shrimp and shellfish is prepared from the bifidobacterium DSM 20210, ATCC25866, ATCC25867, or ATCC25525 through collecting the bifidodacteria, schizolyzing cells, washing 4 times, centrifugal removal of schizolyzing agent, hydrolyzing, and enzymolyzing twice. It can improve the non-specific immunity, promote growth, and increase the conversion rate of feed.
Description
(1) technical field
The present invention relates to the improvement of anaerobe application technology, specifically is a kind of biological preparation---the preparation method of nonspecific immunity activator, it belongs to technical field of bioengineering.
(2) background technology
In current aquaculture, the phenomenon of quality is excessively devoted exclusive attention to output, ignored to ubiquity.The result causes cultivation density excessive, and water pollution increases the weight of, thereby causes disease to take place often, and particularly in recent years, virosis was broken out on a large scale in the aquatic animal, has caused massive losses for the Aquatic product aquaculture.Therefore the generation of control disease is significant to the culture fishery sustainable development.In Aquatic product disease control field, be to adopt antibiosis usually to prevent and treat and control the generation of disease traditionally.There are many drawbacks in such traditional method, and cause antibiotic to pollute as abuse of antibiotics, and then disease biological is developed immunity to drugs, perhaps because Drug abuse, and negative effect such as cause that drug residue exceeds standard.Simple be not have effect by antibiotic medicine to preventing and treating virosis at all.Owing to the autoimmune non-specific characteristics of aquatic animal, particularly the shrimp shellfish is all invertebrates again, does not therefore have the specific immunity system, can not produce antibody at specific cause of disease.Thereby in the Aquatic product disease control, almost can not use the virosis that vaccine is prevented and treated aquatic animal.Prevent and treat virosis with existing technical method and have defective.In aquaculture, about the research and the application of immunoactivator, all also be in the starting stage in the world at present, many technology are immature, and lack the effect that the otherwise effective technique index is estimated its application.As Japanese National Federation of Agricultural Co-Operative Associations (JP) 8-3, Otemachi disclosed " feedstuff of crustacean and feed additive " (ZL95104855.4), it mainly utilizes the compatibility between bacterial peptide polysaccharide, vitamin E and the vitamin C, insert the three in the feedstuff by a certain percentage, discovery can improve the anti-infection ability to pathogenic microorganism of crustacean, mainly show the vigor of engulfing of hemocyte, and improve the survival rate under the vibrio infection condition.Itself also has above-mentioned action function actual vitamin.When carrying out the antiviral experiment, find that there are not marked difference (P＞0.05) in experimental group and matched group mortality rate, show that its action effect is undesirable under the viral infection condition with above-mentioned prescription.Moreover, the ZL95104855.4 document has only been introduced the information that makes of bacterial peptide polysaccharide generally, information extraction for the bacterial peptide polysaccharide only discloses generally: " will carry out sonication by the bacterial cell of separating in the culture broth, and use the surfactant solution purification.”
According to the literature: it is Peptidoglycan that procaryotic cell wall such as antibacterial contains the macromolecule polyalcohol that a class is made up of polysaccharide chains, peptide subunit and a peptide bridge (interpeptide bridge).Complete Peptidoglycan is made up of amino sugar skeleton and peptide chain in the bacteria cell wall.Its primary structure is: N-acetyl-glucosamine (G) and-acetylmuramic acid (M) residue alternately are connected to form the polysaccharide thigh with β-1,4 key, per sharely contain 10-65 disaccharide unit.3-O-.alpha.-carboxyethyl-D-glucosamine. is the lactoyl ether (lactyether) of glucosamine, its lactyl part link to each other with the peptide chain of being made up of 4 amino acid moleculars (Yang Xiushan 1982).Be connected in the Peptidoglycan between the peptide chain on the-acetylmuramic acid, generally be the 3rd amino acid whose free amino group by the 4th amino acid whose carboxyl of the chain chain adjacent with another with peptide bond crosslinked (a peptide bridge), a peptide bridge is many to be made up of the residue of 1-6 amino acid molecular.The amino acid no that occurs in the different Peptidoglycans is limited, inferior peptide unit and between the composition of peptide bridge upper amino acid and the difference of arranging, determined the Peptidoglycan kind multiformity (with cause in 1992).The composition of the Peptidoglycan in the different prokaryotes, its cell wall has specificity, and after Peptidoglycan was hydrolyzed, its active regular meeting increased.Do not see more detailed bibliographical information about the Peptidoglycan extracting method.
(3) summary of the invention:
The purpose of this invention is to provide a kind of nonspecific immunity activator (hereinafter to be referred as: NSI) and preparation method thereof, it can be by oral or manual injection's method, be able to effectively improve widely the nonspecific immunity power of aquatic animal, strengthen biological premunition, promote the effect of healthy growth.This nonspecific immunity activator all can effectively improve the nonspecific immunity of Fish, shrimps and shellfish, enhancing is to the resistance of all kinds of cause of diseases, specifically be to improve antiviral property disease, bacterial disease, Mycophyta disease and parasitic infection, improve the breed survival rate of aquatic animal, reduce antibiotic dosage, improve aquatic product quality.Preserve the ecological environment and people ' s health.The nonspecific immunity activator product of making is pure biological product, safe in utilization, biology and environment without any pollution, are added in the breeding feed after mixing or are sprayed on the bait surface, also can be used in the seedling water of aquatic animal, can with other feed additive or compatibility of drugs.
The objective of the invention is to finish by following technical scheme, developed a kind of preparation method of nonspecific immunity activator, comprise through anaerobic fermentation and cultivating that separated and collected thalline from fermentation liquid again obtains the process that active nonspecific immunity in the somatic cells wall activates preparation.The strain of described fermentation culture is bacillus bifidus DSM20210, or bacillus bifidus ATCC25866, or bacillus bifidus ATCC25867, or bacillus bifidus ATCC25525; The process that the described active nonspecific immunity that obtains in the somatic cells wall activates preparation is:
(1). collect thalline: get the bacterium liquid that fermentation is finished, through 4000-8000g (centrifugal force F unit english abbreviation), 4 ℃ centrifugal treating 20-30 minute, abandoning supernatant keeps this bacterial sediment of collecting, and reuse 0.8% normal saline washing to this thalline be white;
(2). cell lysis: in the ratio of 1: 4 (g/ml), this thalline of white is mixed with the lysis agent, and insert in 95 ℃ of water-baths, constant temperature is handled and continuous stirring 1 hour, is cooled to room temperature more immediately, and thoroughly washs with distilled water;
(3). four centrifugal decomposition agents of removing of washing: the methanol aqueous solution of using Concentraton gradient is to warp (2). the thalline that the step handles, carry out three washings, centrifugal treating respectively, wherein centrifugal is with 5000-6000g, presses the Concentraton gradient carrying out washing treatment step by step; Use washing with acetone afterwards, and through the centrifugal treating of 4000-5000g, this bacterial sediment that will collect again carries out 0～4 ℃ cold drying to be handled;
(4). hydrolysis pretreatment: with (3). in the desciccate that obtains, add the ratio of pH6.2 phosphate buffer and the 0.3gEDTA liquid of 1 L in this product of every 500g, the preparation mixed liquor keeps constant temperature to reach 4 hours for 80 ℃, is cooled to 37 ℃ again;
(5). enzymolysis processing: to (4). in add the lysozyme of an amount of 1-5g/L in the refrigerative mixed liquor, keep the effect 24 hours down of 37 ℃ of constant temperature; Add the lysozyme of 1-3g/L afterwards, handled 12 hours down for 37 ℃;
(6). enzymolysis processing once more: the protease with an amount of 2-5g/L joins in this mixed liquor of bacteriolyze, under 50 ℃ of temperature, handled 24 hours, promptly to obtain containing effective active concentration be the oligose peptide class of 0.4-0.8% and contain alanine: glutamic acid: lysine: the mole ratio of glycine=3: 1: 1: 1 compositions, said composition are the yellowish-brown liquid with light fragrant and sweet abnormal smells from the patient---nonspecific immunity activator major ingredient preparation;
Maybe this yellowish-brown liquid dried is handled, promptly obtain character and have special fragrant and sweet abnormal smells from the patient, auburn dope---contain effective concentration and be 4-8% the oligose peptide active component and contain alanine: glutamic acid: lysine: the mole ratio of glycine=3: 1: 1: 1 compositions---nonspecific immunity activator crude product preparation.
Described (2). the lysis agent in the step, it is a nonionic surfactant.
Described nonionic surfactant, itself or 0.5%TritonX100, or 2%SDS.
Described (3). the methanol aqueous solution of Concentraton gradient in the step, its gradient concentration are 30%, 70%, 100%, or are 20%, 60%, 100%.
Described (6). in protease, itself or papain, or pronase.
The using method of described nonspecific immunity activator major ingredient preparation is: described nonspecific immunity activator major ingredient preparation is directly mixed with adjuvant by following weight ratio, afterwards, according to the production demand, the said mixture that adds an amount of concentration is in feedstuff, through feed processor, be processed into the oral breeding feed of aquatic animal:
Components by weight
Nonspecific immunity activator major ingredient preparation 100---1000 parts;
Short absorbent 0.5---2000 parts;
Immunity synergist 0.5---500 parts;
Phagostimulant 1---3000 parts.
Described short absorbent is: NaTDC (NaDC) is or/and be cephalin (CE); Described immune synergist is: or be sodium selenite, or/and be vitamin A, or/and be vitamin C; Described phagostimulant, it is glycosides propylhomoserin front three third fat, or is betanin.
The another kind of preparation method that nonspecific immunity of the present invention activates preparation also is: the active nonspecific immunity that obtains in the somatic cells wall activates the preparation composition, and its process is:
(1). collect thalline: get the bacterium liquid that fermentation is finished, through 5000-8000g, 4 ℃ centrifugal treating 20-30 minute, abandoning supernatant keeps this bacterial sediment of collecting, reuse 0.8% normal saline washs and with 6000～8000g centrifugal treating, obtains the wet thallus of white;
(2). water bath with thermostatic control: this wet thallus 100g that will collect is in 65 ℃ of water-baths, and constant temperature was handled 40 minutes, was cooled to room temperature afterwards;
(3). cell lysis: in the ratio of 1: 4 (g/ml), this wet thallus that is cooled to room temperature is mixed with the lysis agent, and insert 95 ℃ of water-baths 1 hour, continuous stirring is cooled to room temperature afterwards immediately, and thoroughly washs with distilled water;
(4). four washings, the centrifugal decomposition agent of removing: with the methanol aqueous solution of Concentraton gradient to through (3). this wet thallus that the step handles, wash respectively, the centrifugal substep gradient concentration of 5000-6000g handles; Use washing with acetone afterwards, through the centrifugal treating of 4000-5000g, this bacterial sediment that will collect again carries out 0～4 ℃ cold drying processing;
(5). enzymolysis removes nucleic acid: with (4). in Tris-hydrochloric acid (TrisHCl) buffer of the pH7.2 that contains deoxyribonuclease (DNase), ribonuclease (RNase) of this bacterial sediment adding of drying 300ml of obtaining; Under 37 ℃ of constant temperature, hatched 24 hours; Through 5000-6000g, 4 ℃ of centrifugal treating 40 minutes, collect this bacterial sediment again;
(6). enzymolysis removes foreign protein: Tris-hydrochloric acid (TrisHCl) buffer 300ml of the trypsin of usefulness content 2mg/ml and the Chymetin of content 0.5mg/ml, join (5). in this bacterial sediment of middle processing, and under 37 ℃ of constant temperature, digested 24 hours; Again with 5000-6000g, 4 ℃ of centrifugal treating 40 minutes, this bacterial sediment of collection;
(7). secondary enzymolysis is removed foreigh protein removing: with the pepsic 0.01M hydrochloric acid solution that contains 5mg/ml of 150ml, processing (6). this bacterial sediment of middle collection, after 5000-6000g, 4 ℃ of centrifugal treating 40 minutes, this bacterial sediment of collection; Tris-hydrochloric acid of the protease E of the content 5mg/ml of reuse 300ml (TrisHCl of pH7.4) buffer, digestion process is 24 hours under 37 ℃ of constant temperature; With 5000-6000g, 4 ℃ of centrifugal treating 40 minutes, collect this bacterial sediment at last;
(8). three step defats:, handle through following three steps again with this thalline of collection that above-mentioned protease digestion is crossed:
1.. add 300ml methanol, with 5000g, 4 ℃ of centrifugal treating 40 minutes, abandoning supernatant;
2.. add 300ml methanol: the mixed liquor of chloroform (1: 1 V/V), with 5000g, 4 ℃ of centrifugal treating 40 minutes, abandoning supernatant;
3.. add 300ml chloroform washing, again with 5000g, 4 ℃ of centrifugal treating 40 minutes, abandoning supernatant;
(9). dialysis treatment: with above-mentioned (8). in 3. the step extract 0～4 ℃ of following dried, Tris-hydrochloric acid of the protease E of the content 1mg/ml of reuse 200ml (TrisHCl of pH7.4) buffer is handled exsiccant extract; Under 37 ℃ of constant temperature, handled 24 hours; In this extract that constant temperature is handled is packed bag filter into again, dialysed 50 hours continuously with distilled water;
(10). desalination dialysis treatment: the product in the above-mentioned bag filter is added: the hydrochloric acid of 50ml, 0.05M, and under 85 ℃, handled 5 minutes, be cooled to room temperature afterwards immediately; With 10000-12000g, 4 ℃ of centrifugal treating 30 minutes, collect this bacterial sediment again; Reuse water dialysis 5 days is preserved this bacterial sediment lyophilizing afterwards;
(11). glycoprotein hydrolysis: with (10). the lyophilized products of middle acquisition, in product weight/buffer volume is 1: 5 ratio, the phosphate buffer that adds pH6.2 adds lysozyme and the 0.1g/L EDTA liquid of an amount of 1-3g/L again, handles 24 hours under 37 ℃ of constant temperature; Use the pronase of 1-5g/L afterwards, or with refining carase, 50 ℃ of hydrolysis process 24 hours;
(12). water dialysis dried: the said hydrolyzed treatment fluid was dialysed in distilled water 50 hours, 0 ℃ of following cold drying, promptly get injection nonspecific immunity activator dry powder formulations, the character of this dry powder: contain active ingredient oligose peptide class greater than 95%, sugar content 40-49%, the dry powder formulations product outward appearance powder that is white in color, the aqueous solution muddiness.
In the another kind of preparation method of described nonspecific immunity activator, described (3). the lysis agent is the TritonX100 of 0.5% concentration in the step, or 2%SDS; Described (4). the methanol aqueous solution of Concentraton gradient in the step, its Concentraton gradient are 30%, 70%, 100%, or are 20%, 60%, 100%.
The using method of the another kind of injection dry powder formulations of described nonspecific immunity activator is: with described injection dry powder formulations is with 1000 times (W/V) of normal saline dilution, sterilizes 15 minutes down at 121 ℃, deposits in-20 ℃ of following refrigerators; Injection volume by per kilogram fish body weight is: 50---150 μ l/Kg.
The invention has the advantages that:
A) by using this nonspecific immunity activator major ingredient preparation or coarse fodder preparation feed additive as fish, shrimp, shellfish, be used to activate the nonspecific immunity power of aquaculture organism, thereby reach the appeal of promotion to the disease-resistant protozoa (antibacterial, virus, parasite) of fish, shrimp, shellfish, promoted the growth promoter of fish, shrimp, shellfish, improve feed conversion rate, strengthened the abnormal survival rate of nursery stages such as shellfish, shrimp.Concrete effect is as follows:
(1) the short prawn viral diseases culture experiment result of immune activation agent formulation of the present invention sees Table 1-table 3:
After table 1 Penaeus vannamei is ingested and is added the bait 30 of immune activation agent formulation,
Viral infection resisting situation (the long 6-9cm of prawn body):
| NSI addition (gram/ton feedstuff) | The prawn number | Culture the survival rate (%) that begins after 30 days to infect after 20 days |
| ????20 | ????100 | ????28 |
| ????100 | ????100 | ????43 |
| ????500 | ????100 | ????70 |
| ????0 | ????100 | ????0 |
(2) the short Chinese prawn growth experiment result of immune activation agent formulation of the present invention:
Table 2 was cultured Chinese prawn after 30 days, the long rate of increase of prawn body (the long 4-6cm of prawn body):
| NSI addition (gram/ton feedstuff) | The prawn number | The long rate of increase of body after 30 days (%) |
| ????20 | ????100 | ????18 |
| ????100 | ????100 | ????25 |
| ????500 | ????100 | ????40 |
| ????0 | ????100 | ????15 |
(3) the short japonicus nonspecific immunity factor vigor experimental result of immunoactivator:
Table 3 was cultured japonicus after 30 days, phenol oxidase in the prawn body fluid, alkali phosphatase,
Acid phosphatase vigor and coagulation vigor (the long 9-11cm of prawn body)
| NSI addition (gram/ton feedstuff) | The phenol oxidase vigor | Alkaline phosphatase activity (King unit/100ml) | Acid phosphatase work (King unit/100ml) | The coagulation vigor |
| ????20 | ????100 | ????15.1 | ????5.2 | ????1∶64 |
| ????100 | ????320 | ????20.6 | ????7.9 | ????1∶128 |
| ????500 | ????502 | ????35.0 | ????10.4 | ????1∶256 |
| ????0 | ????64 | ????8.4 | ????43 | ????1∶64 |
B) the another kind of injection dry powder formulations of nonspecific immunity activator (NSI) of the present invention's preparation is as aquatic vertebrate injection activator such as Fish, Trionyx sinensis Wiegmanns.Its (NSI) has effects such as the Fish of enhancing head-kidney cytophagy vigor, antalzyme activity.Especially this injection activator injection is used for Trionyx sinensis Wiegmann, and its effect has the phagocytic function of the macrophage that strengthens Trionyx sinensis Wiegmann significantly, increases the Trionyx sinensis Wiegmann speed of growth.Also have this injection activator injection to be used for tilapia, its effect has and strengthens the resistance of tilapia to tarda significantly.This injection activator also can strengthen the anti-vibrio infection power of Fish such as rainbow trout, snapper.See Fig. 1: behind the nonspecific immunity activator of the various dose injection Luo Fei catfish, carry out infection experiment with tarda after, survival rate of each group in 8 days.
(4) accompanying drawing and embodiment
Embodiments of the invention can not limit the scope of the present invention, but can support the scope of the present invention.
Fig. 1 injects the survival rate curve chart of culturing after fish infects tarda again for the nonspecific immunity activator.
Strain used in the present invention is the laboratory collection by the applicant, is a kind of bacillus bifidus DSM20210, also is applicable to bacillus bifidus ATCC25866 and is applicable to bacillus bifidus ATCC25867, ATCC25525.
Anaerobic bacteria fermentation condition of the present invention is as follows:
Culture medium: glucose 10g, lactose 10g, tryptone 10g, yeast extract 6g, Carnis Bovis seu Bubali cream 6g, MgSO
42H
2O 0.2g, Na
2HPO
412H
2O 10g, KH
2PO
46g, adding distil water transfer pH to 7.0 to 1000ml with NaOH; Sterilize 116 ℃ 20min; Fermentation time 20 hours, pH=4.2-4.6 when fermentation is finished, bacterial concentration is about 5 * 10
8～9 * 10
9Cfu/ml.
The process that active nonspecific immunity in the extraction somatic cells wall of the present invention activates preparation is:
(1). collect thalline: get the bacterium liquid that fermentation is finished, through 4000-8000g, 4 ℃ centrifugal treating 20-30 minute, abandoning supernatant keeps this bacterial sediment of collecting, and reuse 0.8% normal saline washing to this thalline be white;
(2). cell lysis: in the ratio of 1: 4 (g/ml), TritonX100 in this thalline and lysis agent with white---the nonionic surfactant mixes, and inserts in 95 ℃ of water-baths, constant temperature processing and continuous stirring 1 hour, be cooled to room temperature more immediately, and thoroughly wash with distilled water;
(3). wash centrifugal removal decomposition agent four times: be 30%, 70%, 100% methanol aqueous solution with Concentraton gradient to through (2). this thalline that the step handles, carry out three washings, centrifugal treating, wherein centrifugal is with 5000-6000g, and substep is pressed the Concentraton gradient carrying out washing treatment; Use washing with acetone afterwards, and through the centrifugal treating of 4000-5000g, this bacterial sediment that will collect again carries out 0～4 ℃ cold drying to be handled;
(4). hydrolysis pretreatment: with (3). in the desciccate that obtains, get the ratio that this product of every 500g adds pH6.2 phosphate buffer and the 0.3gEDTA liquid of 1L, preparation mixed liquor, and keep 80 ℃ of constant temperature to reach 4 hours is cooled to 37 ℃ again;
(5). enzymolysis processing: in (4). in add the lysozyme of an amount of 1-5g/L in refrigerative this mixed liquor, and constant temperature was handled 24 hours down for 37 ℃; The lysozyme that adds 1-3g/L was afterwards handled 24 hours down for 37 ℃;
(6). enzymolysis processing once more: with protease (or the papain of an amount of 2-5g/L, or pronase) joins in this mixed liquor of bacteriolyze, under 50 ℃ of temperature, handled 24 hours, promptly obtain containing the active oligose peptide class of 0.4-0.8% and containing alanine: glutamic acid: lysine: the mole ratio of glycine=3: 1: 1: 1 compositions, said composition are the yellowish-brown liquid with light fragrant and sweet abnormal smells from the patient---nonspecific immunity activator major ingredient preparation;
Maybe this yellowish-brown liquid dried is handled, promptly obtain having special fragrant and sweet abnormal smells from the patient, the dope of dark brown character---contain 4-8% the oligose peptide active component and contain alanine: glutamic acid: lysine: the mole ratio of glycine=3: 1: 1: 1 compositions---nonspecific immunity activator crude product preparation.
In nonspecific immunity activator of the present invention, the material of its biologically active mainly is from the Peptidoglycan in the bacteria cell wall, and the size of this Peptidoglycan molecular weight and its biological activity are closely related.Adopt the biochemical extractive technique described in the present invention, can separation and purification go out the complete Peptidoglycan of antibacterial.Integrated peptidoglycan mainly is made up of N-acetyl glucose ammonia (NAG),-acetylmuramic acid (NAM) and specific peptide chain, analyzes above-mentioned three's content, can calculate the content of Peptidoglycan.The method of detection of peptides polysaccharide of the present invention is: at first, separate complete Peptidoglycan in the purification bacteria cell wall; Measure the percentage composition of-acetylmuramic acid (NAM) and the percentage composition of N-acetyl glucose ammonia (NAG) afterwards; Measure aminoacid composition and percentage composition in the peptide chain with liquid chromatography technology once more.
Wherein,-acetylmuramic acid (NAM) Determination on content method is as follows: get the 100mg sample through 6N HCI, 105 ℃ of hydrolysis 24 hours, filter, with equivalent NaOH neutralization.Sample solution, 10ug/ml fat corrected milk(FCM) acid solution, each 1ml of blank (distilled water) after water intaking is separated add 0.5ml 20%CuSO respectively
4Solution, mixing.Accurately be settled to 5.0ml with distilled water, each adds 0.2g CaO powder, carefully stirs evenly with thin splash bar, places 30min, 2500rpm, and 15min, careful each 1ml of sucking-off supernatant adds 1 20%CuSO respectively
4Solution, mixing drips the dense H of 6ml
2SO
4Solution, fully behind the mixing, boiling water bath 5min is after the tap water cooling, add 0.2ml right-xenol reagent, mixing, 30 ℃ of water bath with thermostatic control 30min make it to form color, in boiling water bath, boil then and boil 90s, cooling rapidly in wavelength 560nm place colorimetric, is tried to achieve the content of NAM according to the standard curve of drawing.
Wherein, N-acetyl glucose ammonia (NAG) Determination on content method is as follows: preparation N-acetylaminohexose standard solution 80ug/ml, and the accurate respectively standard solution 0,0.05,0.1,0.15,0.20 of drawing, 0.25ml, moisturizing is to 0.25ml.Sample filters through 6N HCI, 105 ℃ of hydrolysis 24 hours, and with equivalent NaOH neutralization, every 0.6ml adds saturated NaHCO
30.2ml, 5% (V/V) acetic anhydride 0.2ml of new preparation, room temperature 10min, with the glucosamine acetylation, boiling water bath 3min is to remove unnecessary anhydride, cooling.Add 0.1ml K to 0.25ml sample and each concentration standard solution
2B
4O
7Solution (0.8M, pH transfers to 9.1 with NaOH solution), boiling water bath 3min adds 3ml DMAB (dimethylaminobenzaldehyde, 0.01g/ml) solution, rapid mixing, 37 ℃ of water-bath 20min, its absorbance A of cooling back survey
544nm,, calculate the content of NAG according to the standard curve of drawing.
Nonspecific immunity activator major ingredient preparation of the present invention is as the prescription of oral formulations in feedstuff:
Table 4 adds oral immunity activator pharmaceutical formulation (unit: Kg) in the feedstuff aquatic feeds per ton
Nonspecific immunity activator of the present invention (NSI) is as a kind of dry powder formulations that is used for aquatic vertebrate injections such as Fish, Trionyx sinensis Wiegmann, and its preparation method also is the active nonspecific immunity activator composition that extracts in the somatic cells wall, and its process is:
(1). collect thalline: get the bacterium liquid that fermentation is finished, through 5000-8000g, 4 ℃ centrifugal treating 20-30 minute, abandoning supernatant keeps this bacterial sediment of collecting, reuse 0.8% normal saline washs and with 6000～8000g centrifugal treating, obtains the wet thallus of white;
(2). water bath with thermostatic control: this wet thallus 100g that will collect is in 65 ℃ of water-baths, and constant temperature was handled 40 minutes, was cooled to room temperature afterwards;
(3). cell lysis: in the ratio of 1: 4 (g/ml), this wet thallus that is cooled to room temperature is mixed with lysis agent (TritonX100 of 0.5% concentration or 2% SDS), and insert 95 ℃ of water-baths 1 hour, continuous stirring, be cooled to room temperature afterwards immediately, and thoroughly wash with distilled water;
(4). four washings, centrifugal: be 30%, 70%, 100% methanol aqueous solution with Concentraton gradient to through (3). this wet thallus that the step handles, wash respectively, the centrifugal substep gradient concentration processing of 5000-6000g; Use washing with acetone afterwards, through the centrifugal treating of 4000-5000g, this bacterial sediment that will collect again carries out 0～4 ℃ cold drying processing;
(5). enzymolysis removes nucleic acid: with (4). in Tris-hydrochloric acid (TrisHCl) buffer of the pH7.2 that contains deoxyribonuclease (DNase), ribonuclease (RNase) of this bacterial sediment adding of drying 300ml of obtaining; Under 37 ℃ of constant temperature, hatched 24 hours; Through 5000-6000g, 4 ℃ of centrifugal treating 40 minutes, collect this bacterial sediment again;
(6). enzymolysis removes foreign protein: Tris-hydrochloric acid (TrisHCl) buffer 300ml of the trypsin of usefulness content 2mg/ml and the Chymetin of content 0.5mg/ml, join (5). in this bacterial sediment of middle processing, and under 37 ℃ of constant temperature, digested 24 hours; Again with 5000-6000g, 4 ℃ of centrifugal treating 40 minutes, this bacterial sediment of collection;
(7). secondary enzymolysis is removed foreign protein: with the pepsic 0.01M hydrochloric acid solution of the content 5mg/ml of 150ml, handle (6). this bacterial sediment of middle collection, after 5000-6000g, 4 ℃ of centrifugal treating 40 minutes, this bacterial sediment of collection; Tris-hydrochloric acid of the protease E of the content 5mg/ml of reuse 300ml (TrisHCl of pH7.4) buffer, digestion process is 24 hours under 37 ℃ of constant temperature; With 5000-6000g, 4 ℃ of centrifugal treating 40 minutes, collect this bacterial sediment at last;
(8). three step defats:, handle through following three steps again with this thalline of collection that above-mentioned protease digestion is crossed:
1.. add 300ml methanol, with 5000g, 4 ℃ of centrifugal treating 40 minutes, abandoning supernatant;
2.. add 300ml methanol: the mixed liquor of chloroform (1: 1V/V), with 5000g, 4 ℃ of centrifugal treating 40 minutes, abandoning supernatant;
3.. add 300ml chloroform washing, again with 5000g, 4 ℃ of centrifugal treating 40 minutes, abandoning supernatant;
(9). dialysis treatment: with above-mentioned (8). in 3. the step extract 0～4 ℃ of following dried, Tris-hydrochloric acid of the protease E of the content 1mg/ml of reuse 200ml (TrisHCl of pH7.4) buffer is handled exsiccant extract; Under 37 ℃ of constant temperature, handled 24 hours; In this extract that constant temperature is handled is packed bag filter into again, dialysed 50 hours continuously with distilled water;
(10). desalination dialysis treatment: the product in the above-mentioned bag filter is added: the hydrochloric acid of 50ml, 0.05M, and under 85 ℃, handled 5 minutes, be cooled to room temperature afterwards immediately; With 10000-12000g, 4 ℃ of centrifugal treating 30 minutes, collect this bacterial sediment again; Reuse water dialysis 5 days is preserved this bacterial sediment lyophilizing afterwards;
(11). glycoprotein hydrolysis: with (10). the lyophilized products of middle acquisition, in product weight/buffer volume is 1: 5 ratio, the phosphate buffer that adds pH6.2 adds lysozyme and the 0.1g/L EDTA liquid of an amount of 1g/L again, handles 24 hours under 37 ℃ of constant temperature; Use the pronase of 1g/L afterwards, 50 ℃ of hydrolysis process 24 hours;
(12). water dialysis dried: the said hydrolyzed treatment fluid was dialysed in distilled water 50 hours, 0 ℃ of following cold drying, promptly get injection nonspecific immunity activator dry powder formulations, the character of this dry powder: contain active ingredient oligose peptide class greater than 95%, sugar content 40-49%, the product shows white powder, the aqueous solution muddiness.
The active ingredient oligose peptide class content assaying method of injection nonspecific immunity activator dry powder formulations of the present invention is with the method for the above-mentioned detection of peptides polysaccharide that adopts: at first, and the percentage composition of-acetylmuramic acid in the working sample (NAM) and N-acetyl glucose ammonia (NAG); Measure aminoacid composition and percentage composition in the peptide chain with liquid chromatography technology once more.
The described injection dry powder formulations of using method that the another kind of injection of described nonspecific immunity activator is used for powder preparation is with 1000 times (W/V) of normal saline dilution, sterilizes 15 minutes down at 121 ℃, deposits in-20 ℃ of following refrigerators; Injection volume by per kilogram fish body weight is: 50---150 μ l/Kg.The concrete application example of this dry powder formulations that uses is as follows, sees Table 5: table 5 is pressed per kilogram fish body weight injection various dose NSI influences experimental result to different Fish head-kidney cytophagy rates
Claims (10)
1, a kind of preparation method of nonspecific immunity activator, comprise through anaerobic fermentation and cultivating, separated and collected thalline from fermentation liquid again, obtain the process of the active nonspecific immunity activation preparation in the somatic cells wall, it is characterized in that: the strain of described fermentation culture is bacillus bifidus DSM 20210, or bacillus bifidus ATCC25866, or bacillus bifidus ATCC25867, or bacillus bifidus ATCC25525; The process that the described active nonspecific immunity that obtains in the somatic cells wall activates preparation is:
(1). collect thalline: get the bacterium liquid that fermentation is finished, through 4000-8000g, 4 ℃ centrifugal treating 20-30 minute, abandoning supernatant keeps this bacterial sediment of collecting, and reuse 0.8% normal saline washing to this thalline be white;
(2). cell lysis: in the ratio of 1: 4 (g/ml), this thalline of white is mixed with the lysis agent, and insert in 95 ℃ of water-baths, constant temperature is handled and continuous stirring 1 hour, is cooled to room temperature more immediately, and thoroughly washs with distilled water;
(3). four centrifugal decomposition agents of removing of washing: the methanol aqueous solution of using Concentraton gradient is to warp (2). the thalline that the step handles, carry out three washings, centrifugal treating respectively, wherein centrifugal is with 5000-6000g, presses the Concentraton gradient carrying out washing treatment step by step; Use washing with acetone afterwards, and through the centrifugal treating of 4000-5000g, this bacterial sediment that will collect again carries out 0～4 ℃ cold drying to be handled;
(4). hydrolysis pretreatment: with (3). in the desciccate that obtains, add the ratio of pH6.2 phosphate buffer and the 0.3gEDTA liquid of 1L in this product of every 500g, the preparation mixed liquor keeps constant temperature to reach 4 hours for 80 ℃, is cooled to 37 ℃ again;
(5). enzymolysis processing: to (4). in add the lysozyme of an amount of 1-5g/L in the refrigerative mixed liquor, keep the effect 24 hours down of 37 ℃ of constant temperature; Add the lysozyme of 1-3g/L afterwards, handled 12 hours down for 37 ℃;
(6). enzymolysis processing once more: the protease with an amount of 2-5g/L joins in this mixed liquor of bacteriolyze, under 50 ℃ of temperature, handled 24 hours, promptly to obtain containing effective active concentration be the oligose peptide class of 0.4-0.8% and contain alanine: glutamic acid: lysine: the mole ratio of glycine=3: 1: 1: 1 compositions, said composition are the yellowish-brown liquid with light fragrant and sweet abnormal smells from the patient---nonspecific immunity activator major ingredient preparation;
Maybe this yellowish-brown liquid dried is handled, promptly obtain character and have special fragrant and sweet abnormal smells from the patient, auburn dope---contain effective concentration and be 4-8% the oligose peptide active component and contain alanine: glutamic acid: lysine: the mole ratio of glycine=3: 1: 1: 1 compositions---nonspecific immunity activator crude product preparation.
2, according to the preparation method of the described nonspecific immunity activator of claim 1, it is characterized in that: described (2). the lysis agent in the step, it is a nonionic surfactant.
3, according to the preparation method of the described nonspecific immunity activator of claim 1, it is characterized in that: described nonionic surfactant, itself or 0.5%TritonX100, or 2%SDS.
4, according to the preparation method of the described nonspecific immunity activator of claim 1, it is characterized in that: described (3). the methanol aqueous solution of Concentraton gradient in the step, its gradient concentration are 30%, 70%, 100%, or are 20%, 60%, 100%.
5, according to the preparation method of the described nonspecific immunity activator of claim 1, it is characterized in that: described (6). in protease, itself or papain, or pronase.
6, the using method according to the described nonspecific immunity activator of claim 1 major ingredient preparation is: described nonspecific immunity activator major ingredient preparation is directly mixed with adjuvant by following weight ratio, afterwards, according to the production demand, the said mixture that adds an amount of concentration is in feedstuff, through feed processor, be processed into the oral breeding feed of aquatic animal:
Components by weight
Nonspecific immunity activator major ingredient preparation 100---1000 parts;
Short absorbent 0.5---2000 parts;
Immunity synergist 0.5---500 parts;
Phagostimulant 1---3000 parts.
7, according to the using method of the described nonspecific immunity activator of claim 6 major ingredient preparation, it is characterized in that: described short absorbent is: NaTDC (NaDC) is or/and be cephalin (CE); Described immune synergist is: or be sodium selenite, or/and be vitamin A, or/and be vitamin C; Described phagostimulant, it is glycosides propylhomoserin front three third fat or betanin.
8, activate the another kind of preparation method of preparation according to the described nonspecific immunity of claim 1, it is characterized in that: the described active nonspecific immunity that obtains in the somatic cells wall activates the preparation composition, and its process is:
(1). collect thalline: get the bacterium liquid that fermentation is finished, through 5000-8000g, 4 ℃ centrifugal treating 20-30 minute, abandoning supernatant keeps this bacterial sediment of collecting, reuse 0.8% normal saline washs and with 6000～8000g centrifugal treating, obtains the wet thallus of white;
(2). water bath with thermostatic control: this wet thallus 100g that will collect is in 65 ℃ of water-baths, and constant temperature was handled 40 minutes, was cooled to room temperature afterwards;
(3). cell lysis: in the ratio of 1: 4 (g/ml), this wet thallus that is cooled to room temperature is mixed with the lysis agent, and insert 95 ℃ of water-baths 1 hour, continuous stirring is cooled to room temperature afterwards immediately, and thoroughly washs with distilled water;
(4). four washings, the centrifugal decomposition agent of removing: with the methanol aqueous solution of Concentraton gradient to through (3). this wet thallus that the step handles, wash respectively, the centrifugal substep gradient concentration of 5000-6000g handles; Use washing with acetone afterwards, through the centrifugal treating of 4000-5000g, this bacterial sediment that will collect again carries out 0～4 ℃ cold drying processing;
(5). enzymolysis removes nucleic acid: with (4). in Tris-hydrochloric acid (TrisHCl) buffer of the pH7.2 that contains deoxyribonuclease (DNase), ribonuclease (RNase) of this bacterial sediment adding of drying 300ml of obtaining; Under 37 ℃ of constant temperature, hatched 24 hours; Through 5000-6000g, 4 ℃ of centrifugal treating 40 minutes, collect this bacterial sediment again;
(6). enzymolysis removes foreign protein: Tris-hydrochloric acid (TrisHCl) buffer 300ml of the trypsin of usefulness content 2mg/ml and the Chymetin of content 0.5mg/ml, join (5). in this bacterial sediment of middle processing, and under 37 ℃ of constant temperature, digested 24 hours; Again with 5000-6000g, 4 ℃ of centrifugal treating 40 minutes, this bacterial sediment of collection;
(7). secondary enzymolysis is removed foreigh protein removing: with the pepsic 0.01M hydrochloric acid solution that contains 5mg/ml of 150ml, processing (6). this bacterial sediment of middle collection, after 5000-6000g, 4 ℃ of centrifugal treating 40 minutes, this bacterial sediment of collection; Tris-hydrochloric acid of the protease E of the content 5mg/ml of reuse 300ml (TrisHCl of pH7.4) buffer, digestion process is 24 hours under 37 ℃ of constant temperature; With 5000-6000g, 4 ℃ of centrifugal treating 40 minutes, collect this bacterial sediment at last;
(8). three step defats:, handle through following three steps again with this thalline of collection that above-mentioned protease digestion is crossed:
1.. add 300ml methanol, with 5000g, 4 ℃ of centrifugal treating 40 minutes, abandoning supernatant;
2.. add 300ml methanol: the mixed liquor of chloroform (1: 1V/V), with 5000g, 4 ℃ of centrifugal treating 40 minutes, abandoning supernatant;
3.. add 300ml chloroform washing, again with 5000g, 4 ℃ of centrifugal treating 40 minutes, abandoning supernatant;
(9). dialysis treatment: with above-mentioned (8). in 3. the step extract 0～4 ℃ of following dried, Tris-hydrochloric acid of the protease E of the content 1mg/ml of reuse 200ml (TrisHCl of pH7.4) buffer is handled exsiccant extract; Under 37 ℃ of constant temperature, handled 24 hours; In this extract that constant temperature is handled is packed bag filter into again, dialysed 50 hours continuously with distilled water;
(10). desalination dialysis treatment: the product in the above-mentioned bag filter is added: the hydrochloric acid of 50ml, 0.05M, and under 85 ℃, handled 5 minutes, be cooled to room temperature afterwards immediately; With 10000-12000g, 4 ℃ of centrifugal treating 30 minutes, collect this bacterial sediment again; Reuse water dialysis 5 days is preserved this bacterial sediment lyophilizing afterwards;
(11). glycoprotein hydrolysis: with (10). the lyophilized products of middle acquisition, in product weight/buffer volume is 1: 5 ratio, the phosphate buffer that adds pH6.2 adds lysozyme and the 0.1g/L EDTA liquid of an amount of 1-3g/L again, handles 24 hours under 37 ℃ of constant temperature; Use the pronase of 1-5g/L afterwards, or with refining carase, 50 ℃ of hydrolysis process 24 hours;
(12). water dialysis dried: the said hydrolyzed treatment fluid was dialysed in distilled water 50 hours, 0 ℃ of following cold drying, promptly get injection nonspecific immunity activator dry powder formulations, the character of this dry powder: contain active ingredient oligose peptide class greater than 95%, sugar content 40-49%, the dry powder formulations product outward appearance powder that is white in color, the aqueous solution muddiness.
9, the another kind of preparation method of described according to Claim 8 nonspecific immunity activator is characterized in that: described (3). the lysis agent is the TritonX100 of 0.5% concentration in the step, or 2%SDS; Described (4). the methanol aqueous solution of Concentraton gradient in the step, its Concentraton gradient are 30%, 70%, 100%, or are 20%, 60%, 100%.
10, the using method of the another kind of injection dry powder formulations of described according to Claim 8 nonspecific immunity activator, it is characterized in that: described injection dry powder formulations is with 1000 times (W/V) of normal saline dilution, sterilized 15 minutes down at 121 ℃, deposit in-20 ℃ of following refrigerators; Injection volume by per kilogram fish body weight is: 50---150 μ l/Kg.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1321685C (en) * | 2005-08-31 | 2007-06-20 | 中国水产科学研究院黄海水产研究所 | Peptidoglycan immunopotentiator and its production process |
| CN101380288B (en) * | 2007-09-04 | 2013-01-16 | 欧莱雅 | Use of a Bifidobacterium species lysate for treating sensitive skin |
| CN111212575A (en) * | 2017-10-31 | 2020-05-29 | 森永乳业株式会社 | Composition for muscle building |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1321685C (en) * | 2005-08-31 | 2007-06-20 | 中国水产科学研究院黄海水产研究所 | Peptidoglycan immunopotentiator and its production process |
| CN101380288B (en) * | 2007-09-04 | 2013-01-16 | 欧莱雅 | Use of a Bifidobacterium species lysate for treating sensitive skin |
| CN111212575A (en) * | 2017-10-31 | 2020-05-29 | 森永乳业株式会社 | Composition for muscle building |
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