CN109371083A - A kind of preparation method of Japanese croaker flesh of fish immunomodulatory peptides - Google Patents
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Abstract
The present invention relates to a kind of preparation methods of Japanese croaker flesh of fish immunomodulatory peptides, using Japanese croaker meat as raw material, using mouse macrophage RAW264.7 opposite proliferation rate as index, using the extraction process of response phase method optimization Japanese croaker meat immune-active peptides.On the basis of single factor experiment, using solid-liquid ratio, pH, hydrolysis temperature and enzymolysis time as investigation factor, four factors, three hydraulic test design is carried out using Box-Behnken method, using Japanese croaker meat polypeptide to mouse macrophage RAW264.7 opposite proliferation rate as response, carry out the condition optimizing of enzymolysis process.The present invention is that Japanese croaker flesh of fish extraction immune-active peptides lay the foundation, and the preparation process for further research Japanese croaker meat immune peptide provides reference.
Description
Technical field
The present invention relates to a kind of preparation methods of Japanese croaker flesh of fish immunomodulatory peptides, belong to immune class biology system
Product technical field.
Background technique
Immune system is most important to our existence, has the function of defending pathogen, but immune system is vulnerable to more
The influence of kind factor, including pressure, unhealthy habit, pathogen and antigen etc..What is had found is several with adjusting people
The drug of body immune response has a cyclosporin, tacrolimus, glucocorticoid, phytol, aristolochic acid, plumbagin and left-handed
Imidazoles etc..However, since the toxic side effect of these drugs and high cost limit its use in patients, and most of exempt from
Epidemic disease adjusts drug and is not suitable for chronic or preventative purposes.Studies have shown that using food-borne albumen as raw material, through being commercialized protease
Oligopeptides, polypeptide and some hydrolyzate of protease hybrid peptides for hydrolyzing and obtaining after isolating and purifying are shown in animal or test cell line
Significant immunomodulatiory effects out.
In recent years, researcher extracts biologically active peptide from marine organisms and largely grind to immunoloregulation function
Study carefully.Contain collagen abundant in the Japanese croaker flesh of fish, is the good raw material for extracting biologically active peptide.
Marine active peptides are because it is with immunological regulation, vasodilator, antitumor, antifatigue, anti-oxidant, anti-inflammatory and anti-true
The physiological activity such as bacterium become one of the research emphasis of current maritime functional component.Enzymatic isolation method is that extraction prepares marine active peptide
A kind of common method, prepare the hot spot that marine active peptide has become Recent study using enzymatic isolation method extraction.Ye Shengwang etc.
Polypeptide is prepared from clam by pepsin, verify it has immunoregulation effect in vitro in vivo.Vo Thanh-Sang
Deng[4]It is extracted from spirulina with enzymatic isolation method and has purified anti-inflammatory polypeptide.Wang Yukai etc. extracts oligopeptides from oyster, effect
In mouse, discovery has significant immunization.Ahn Chang Bum etc. uses pepsin using salmon pectoral fin by-product as raw material
Enzymatic hydrolysis is prepared for anti-inflammatory polypeptide.Hou Hu etc., which is extracted from gadus skeleton with trypsase, has immunocompetent polypeptide.
Japanese croaker (Nibea japonica), is commonly called as black wool Chang, is subordinate to Perciformes, Sciaenidae, Nibea, main
It is distributed in East China Sea and South Japan sea.Japanese croaker delicious flavour, full of nutrition, fish glue is built up one's health by taking tonic in rare
Product.There is collagen abundant in fish-skin, Yu Fangmiao etc. extracts Japanese croaker collagen simultaneously using pepsin
Study its physicochemical property.Tang Yunping etc. has studied the biocompatibility of Japanese croaker collagen.Japanese Huang aunt
Protein content is higher in the flesh of fish, is easy to be digested, but it prepares immunocompetence for extracting as albumen source
The research of peptide is fresh for report.
Summary of the invention
It is an object of the invention to overcome the shortcomings of the prior art, and a kind of pass through is provided and digests acquisition protein peptides,
And reaction conditions are mild, the reaction time is short, high-efficient, reaction process is easy to control, and albumen peptide yield is high, to immunological regulation function
The preparation method of apparent Japanese croaker flesh of fish immunomodulatory peptides can be acted on.
To achieve the goals above, the invention adopts the following technical scheme:
A kind of preparation method of Japanese croaker flesh of fish immunomodulatory peptides, the preparation method comprises the following steps:
A) pre-process: Japanese croaker is cleaned after removing fish-skin, fin and internal organ, rubs, several with alcohol degreasing after rubbing
It is secondary；Japanese croaker flesh of fish centrifugation after degreasing, collect precipitating be washed till after no ethanol flavor freeze it is spare；
B) it digesting: pure water and protease being added in the Japanese croaker flesh of fish that step a) is obtained, enzyme deactivation is living after digesting 2-10h,
It is centrifuged after being cooled to room temperature, obtains supernatant；
C) post-process: the supernatant for taking step b) to obtain, freeze-drying obtain Japanese croaker flesh of fish immunomodulatory peptides；
D) screen: it is 100 μ g/mL solution that the enzymolysis product of freeze-drying, which is made into concentration, detects 5 groups of enzymolysis products pair using mtt assay
The influence of 264.7 cell Proliferation of RAW, and opposite proliferation rate is calculated, the protease most preferably digested is filtered out, using response phase method
Optimize the extraction process of Japanese croaker meat immune-active peptides.
Preferably, meat 95% ethyl alcohol of Japanese croaker rubbed in step a), solid-liquid ratio 1: 6 (m/v), degreasing temperature
Degree is 50 DEG C, degreasing time 1h, and degreasing number is 2 times, and the Japanese croaker meat after degreasing is pre-chilled in 4 DEG C, 12000r/min
Centrifuge be centrifuged 15min.
Preferably, the solid-liquid ratio of the Japanese croaker flesh of fish and pure water is 1: 10 in step b), unit g/mL；Protease
Additive amount be 1500U/g.
Preferably, protease is papain, trypsase, neutral proteinase, alkali protease and pepsin
One or more of.
Preferably, papain enzymolysis condition is 60 DEG C, pH 6.0；Trypsin digestion condition is 37 DEG C, pH
8.0；Neutral protease enzymolysis condition is 45 DEG C, pH 7.0；Alkali protease enzymatic hydrolysis condition is 45 DEG C, pH 10.0；Pepsin
Enzymatic hydrolysis condition is 37 DEG C, pH 2.0.
Preferably, 12000r/min is centrifuged 15min after cooling in step b).
Preferably, in step d)
The beneficial effects of the present invention are: protein peptides are obtained by enzymatic hydrolysis, the mild, reaction time with reaction conditions
The features such as short, high-efficient, reaction process is easy to control, and albumen peptide yield is high, obvious to immunoloregulation function effect；It is real by verifying
It tests gained actual value and model predication value is close, it was demonstrated that optimizing extraction Japanese croaker meat polypeptide using response phase method is accurately may be used
Capable, immune-active peptides are extracted for the Japanese croaker flesh of fish and are laid the foundation, and are mentioned further to study its immunoregulation effect mechanism
For reference.
Detailed description of the invention
Fig. 1 is influence of the different protease to RAW264.7 cell with respect to appreciation rate.
Fig. 2 is influence of the enzyme additive amount to RAW264.7 cell opposite proliferation rate.
Fig. 3 is influence of the solid-liquid ratio to RAW264.7 cell opposite proliferation rate.
Fig. 4 is influence of the hydrolysis temperature to RAW264.7 cell opposite proliferation rate.
Fig. 5 is the influence of pH value RAW264.7 cell opposite proliferation rate.
Fig. 6 is influence of the enzymolysis time to RAW264.7 cell opposite proliferation rate.
Fig. 7 is interactive surface chart between each factor.
Specific embodiment
Below in conjunction with specific embodiments and the drawings, the present invention will be further explained.
Sample and reagent
Japanese croaker, Zhejiang Prov. Inst. of Marine Products provide；Trypsase, alkali protease, pepsin, Papain
Enzyme and neutral proteinase, Beijing Asia-Pacific Heng Xin Biotechnology Co., Ltd；DMEM culture medium, Gibco company；3- (4,5- diformazans
Base thiazole -2) -2,5- diphenyltetrazolium bromide bromide (MTT), dimethyl sulfoxide (DMSO), Sigma company.Remaining reagent is domestic
It analyzes pure.
Cell
Mouse monokaryon macrophage RAW264.7 is purchased from Shanghai cell institute of the Chinese Academy of Sciences, by the culture of this laboratory passage.
Key instrument and reagent
JJ-2 organizes pulper: Shanghai is than bright Instrument Ltd.；DK-S24 water-bath: the gloomy limited public affairs of letter laboratory apparatus in Shanghai
Department；CKX41 inverted microscope: Japanese OLYMPUS company；CF16RX II type high speed low temperature centrifugal machine: Hitachi, Japan；
ZHJH-C1209C type superclean bench: Shanghai Zhi Cheng analysis instrument manufacturing company；Forma3111 CO2Incubator: the U.S.
Thermo company；WRO-70 type ultrapure water instrument: Millipore company of the U.S.；Microplate reader: Bio-Rad company of the U.S.；BSA124S
Type electronic balance: German SatoriusAG company；ALPHA 1-4/LDplus type freeze drier: German CHRIST company.
Embodiment
Japanese croaker meat polypeptide process flow
Japanese croaker meat → rubbing is at meat gruel → alcohol degreasing → is washed to no ethanol flavor → enzymatic hydrolysis → enzyme deactivation and live → is cooled to
Room temperature → centrifuging and taking supernatant → freeze-drying → Japanese croaker meat polypeptide
The pretreatment of Japanese croaker meat
With reference to degreasing methods such as the great great mansions of fourth, a certain amount of Japanese croaker is taken, removes fish-skin, the organs such as fin and internal organ, cleaning
Completely.It is rubbed with pulverizer.Meat 95% ethyl alcohol of the Japanese croaker of rubbing, solid-liquid ratio 1: 6 (m/v), skimming temp 50
DEG C, degreasing time 1h, degreasing number is 2 times.By the Japanese croaker meat after degreasing 4 DEG C, 12000r/min be pre-chilled from
Scheming is centrifuged 15min, collects precipitating.Precipitating is washed till no ethanol flavor with pure water, it is spare that -20 DEG C of freezings are put into after freeze-drying.
The screening of best enzyme
The pretreated Japanese croaker meat of 5 parts of 10.0g is weighed, pure water, every kind of protease are added with solid-liquid ratio 1: 10 (g/mL)
Enzyme additive amount be 1500U/g, after mixing respectively every kind of protease recommend optimal temperature and pH under the conditions of: Papain
Enzyme enzymatic hydrolysis condition is 60 DEG C, pH 6.0；Trypsin digestion condition is 37 DEG C, pH 8.0；Neutral protease enzymolysis condition is 45
DEG C, pH 7.0；Alkali protease enzymatic hydrolysis condition is 45 DEG C, pH 10.0；Pepsin enzymatic hydrolysis condition is 37 DEG C, pH 2.0, enzymatic hydrolysis
6h, enzyme deactivation is lived 10min in boiling water after the completion of enzymatic hydrolysis, is cooled to room temperature 12000r/min centrifugation 15min, is taken supernatant freezing dry
It is dry.It is 100 μ g/mL solution that the enzymolysis product of freeze-drying, which is made into concentration, detects 5 groups of enzymolysis products to RAW264.7 using mtt assay
The influence of cell Proliferation, and opposite proliferation rate is calculated, filter out the protease most preferably digested.
Experiment of single factor
In the case where 1.3.3 filters out most suitable protease, influence Japanese croaker meat enzymatic hydrolysis cause be known as solid-liquid ratio, enzyme add
Amount, pH, hydrolysis temperature and enzymolysis time.Other experimental conditions are fixed, respectively investigate solid-liquid ratio (1: 6,1: 8,1: 10,1: 12,1:
14g/mL), the additive amount of enzymolysis time (2,4,6,8,10h), hydrolysis temperature (50,55,60,65,70 DEG C), most suitable protease
The influence of (500,1000,1500,2000,2500U/g) and pH (4.0,5.0,6.0,7.0,8.0) to hydrolysis result, mtt assay inspection
Survey opposite proliferation rate.
Response surface experiments design
Under the premise of single factor experiment result, using hydrolysis temperature, enzymolysis time, solid-liquid ratio and pH as technological parameter, design four
The horizontal Box-Behnken response surface experiments of factor three, with Japanese croaker meat polypeptide to the opposite proliferation rate of 264.7 cell of RAW
For index, the test of 5 central points and 29 various combinations is designed, experimental factor is horizontal as shown in table 1.
1 response surface analysis factor of table and level
Measurement of the Japanese croaker meat polypeptide to the opposite proliferation rate of 264.7 cell of RAW
Opposite proliferation rate method is measured with reference to Ye Shengwang etc., a certain amount of Japanese croaker meat polypeptide is weighed, uses cell culture fluid
It is made into the concentration of 100 μ g/mL.Its influence to RAW264.7 cell Proliferation is detected using mtt assay.Opposite increase is calculated by formula (1)
Grow rate.
5 kinds of protease enzymolysis products are as shown in Figure 1 to the opposite proliferation rate situation of 264.7 cell of RAW.As seen from the figure, pawpaw egg
White enzyme enzymolysis product is up to 53.60% to the opposite proliferation rate of 264.7 cell of RAW, is followed successively by neutral proteinase, alkali thereafter
Property protease and pepsin, trypsin digestion product are minimum to the opposite proliferation rate of 264.7 cell of RAW, and digest production
Object would also vary from the opposite proliferation rate height of 264.7 cell of RAW.Therefore papain is selected to mention for this experiment is optimal
Take enzyme.
Fig. 2 show influence of the enzyme concentration to 264.7 cell opposite proliferation rate of RAW.With the increase of enzyme additive amount, day
This spotted maigre meat polypeptide persistently increases RAW264.7 cell opposite proliferation rate, this is because enzyme additive amount increases, substrate reactions
It is more thorough, when enzyme additive amount reaches 2000U/g, to 264.7 cell opposite proliferation rate highest of RAW.When enzyme additive amount continues to increase
Added-time, substrate sufficiently digest, and spotted maigre meat polypeptide is to 264.7 cell opposite proliferation rate of RAW no longer with enzyme additive amount
Increase and increase, slightly decline instead, this may be because the polypeptide generated caused by digesting again.Thus most suitable enzyme addition
Amount is in 2000U/g.
Fig. 3 show influence of the solid-liquid ratio to 264.7 cell opposite proliferation rate of RAW.When solid-liquid ratio is 1: 10, yellow aunt
Flesh of fish polypeptide reaches maximum to 264.7 cell opposite proliferation rate of RAW.In 1: 6~1: 14 range of solid-liquid ratio, with solid-liquid ratio
Increase, spotted maigre meat polypeptide first increases RAW264.7 cell opposite proliferation rate and reduces afterwards.Therefore, best solid-liquid ratio is 1:
10。
Fig. 4 show influence of the hydrolysis temperature to 264.7 cell opposite proliferation rate of RAW.As the temperature rises, it digests
Effect is better, and spotted maigre meat polypeptide is higher to 264.7 cell opposite proliferation rate of RAW.When hydrolysis temperature is more than 60 DEG C, yellow aunt
Flesh of fish polypeptide declines 264.7 cell opposite proliferation rate of RAW.Therefore, best hydrolysis temperature is 60 DEG C.
Fig. 5 show influence of the pH to RAW264.7 cell opposite proliferation rate.When pH is 6.0, Japanese croaker meat is more
Peptide reaches 58.44% to 264.7 cell opposite proliferation rate of RAW.In the range of pH4.0~8.0, with the raising of pH, yellow aunt
Flesh of fish polypeptide first gradually rises RAW264.7 cell opposite proliferation rate and gradually decreases afterwards.Therefore, Optimal pH 6.0.
Fig. 6 show influence of the enzymolysis time to 264.7 cell opposite proliferation rate of RAW.With the extension of enzymolysis time,
Japanese croaker meat polypeptide is higher and higher to 264.7 cell opposite proliferation rate of RAW, and hydrolysis result is better.When enzymolysis time reaches
When 6h, spotted maigre meat polypeptide reaches highest to RAW264.7 cell opposite proliferation rate, then reduces.Therefore select enzymolysis time for 6h
Left and right.
Response surface experiments
Model foundation and significance test
Table 2Box-Behnken experimental design and result
On the basis of experiment of single factor result, chooses solid-liquid ratio, pH, hydrolysis temperature, 4 factor of enzymolysis time, 3 level and rung
Answer face Optimum Experiment, using Japanese croaker meat polypeptide to the opposite proliferation rate of mouse macrophage RAW 264.7 as response, point
Analyse the optimum process index of Japanese croaker meat enzymatic hydrolysis.
The experimental design of response surface and it the results are shown in Table 2.Each factor obtains solid-liquid ratio, pH, enzymatic hydrolysis temperature after regression fit
The secondary multinomial regression equation of degree, enzymolysis time are as follows: opposite proliferation rate=60.42-1.29A+4.64B+0.52C+5.16D-
2.16AB+1.88AC-2.70AD-2.66BC-3.71BD-2.31CD-6.37A2-20.68B2-3.46C2-5.09D2。
Significance analysis is carried out to model, as shown in Table 3, P model < 0.0001 shows that model is extremely significant；Opposite proliferation
Item P=0.0553 is intended in the mistake of rate, shows to lose quasi- not significant；Coefficient R2=0.9521 illustrates have between predicted value and measured value
There is higher correlation；RAdj 2=0.9043, illustrate that selected quadratic regression model is appropriate.By to regression equation
Significance analysis can be seen that C2Influence to response is significant, B, D, A2、B2And D2It is in extremely significant property that the F of item, which is examined, thus may be used
Know that test factor is not simple linear relationship to response, and first order B, D difference is extremely significant, shows pH and solid-liquid ratio pair
The opposite proliferation rate of mouse macrophage RAW 264.7 has a significant impact.
3 regression model of table and variance analysis
Note: * significant difference (P < 0.05)；* difference is extremely significant (P < 0.01).Similarly hereinafter.
Response surface pattern analysis
Response surface figure is the pictute of regression equation, and response surface curved surface can more intuitively reflect each factor and the friendship of factor two-by-two
Influence of the interaction to response.The response surface figure curved surface gradient is precipitous, the intensive ovalisation of contour indicates two factors interaction shadow
It rings greatly, and gentle gradient, contour are rounded, indicate that reciprocal effect is small between two factors.
For more intuitive 2 factors of performance influence to mouse macrophage RAW264.7 opposite proliferation rate simultaneously.
If Fig. 7 draws the response surface figure of two factors, can visually see by figure, the reciprocation of each experimental factor is not significant.Test
Solid-liquid ratio has a significant impact the proliferation rate of 264.7 cell of RAW in factor, and pH takes second place.It is soft through Design Expert 8.05b
It is solid-liquid ratio 1: 11.2g/mL, pH 6.1, enzymolysis time 5.4h, enzymatic hydrolysis temperature that part, which analyzes the optimum enzymolysis condition that the model obtains,
58.8 DEG C of degree.It with this condition, is 62.3% to the proliferation rate of mouse macrophage RAW264.7 cell.
Analysis according to embodiments of the present invention determines that extracting protease used in Japanese croaker meat polypeptide is Papain
Enzyme.PH, solid-liquid ratio, enzyme concentration, reaction temperature and enzymolysis time have been investigated to mouse macrophage RAW264.7 opposite proliferation rate
Influence.It designs to have obtained the optimal procedure parameters of Japanese croaker meat polypeptide extraction by Box-Behnken response surface experiments
For solid-liquid ratio 1: 11.2g/mL, pH 6.1, enzymolysis time 5.4h, 58.8 DEG C of hydrolysis temperature.Actual value and mould as obtained by verifying
Type predicted value is close, it was demonstrated that it is accurate feasible for optimizing extraction Japanese croaker meat polypeptide using response phase method, is the yellow aunt of Japan
The fish flesh of fish extracts immune-active peptides and lays the foundation, and provides reference further to study its immunoregulation effect mechanism.
Claims (7)
1. a kind of preparation method of Japanese croaker flesh of fish immunomodulatory peptides, which is characterized in that the preparation method includes following
Step:
A) pre-process: Japanese croaker is cleaned after removing fish-skin, fin and internal organ, rubs, several with alcohol degreasing after rubbing
It is secondary；Japanese croaker flesh of fish centrifugation after degreasing, collect precipitating be washed till after no ethanol flavor freeze it is spare；
B) it digesting: pure water and protease being added in the Japanese croaker flesh of fish that step a) is obtained, enzyme deactivation is living after digesting 2-10h,
It is centrifuged after being cooled to room temperature, obtains supernatant；
C) post-process: the supernatant for taking step b) to obtain, freeze-drying obtain Japanese croaker flesh of fish immunomodulatory peptides；
D) screen: it is 100 μ g/mL solution that the enzymolysis product of freeze-drying, which is made into concentration, detects 5 groups of enzymolysis products pair using mtt assay
The influence of 264.7 cell Proliferation of RAW, and opposite proliferation rate is calculated, the protease most preferably digested is filtered out, using response phase method
Optimize the extraction process of Japanese croaker meat immune-active peptides.
2. a kind of preparation method of Japanese croaker flesh of fish immunomodulatory peptides according to claim 1, which is characterized in that step
Rapid a) middle meat 95% ethyl alcohol of Japanese croaker rubbed, solid-liquid ratio 1: 6 (m/v), skimming temp is 50 DEG C, and degreasing time is
1h, degreasing number are 2 times, and the Japanese croaker meat after degreasing is centrifuged 15min in the centrifuge that 4 DEG C, 12000r/min are pre-chilled.
3. a kind of preparation method of Japanese croaker flesh of fish immunomodulatory peptides according to claim 1, which is characterized in that step
It is rapid b) in the solid-liquid ratio of the Japanese croaker flesh of fish and pure water be 1: 10, unit g/mL；The additive amount of protease is 1500U/g.
4. a kind of preparation method of Japanese croaker flesh of fish immunomodulatory peptides according to claim 1 or 3, feature exist
In protease is one or more of papain, trypsase, neutral proteinase, alkali protease and pepsin.
5. a kind of preparation method of Japanese croaker flesh of fish immunomodulatory peptides according to claim 4, which is characterized in that wood
Melon protease hydrolyzed condition is 60 DEG C, pH 6.0；Trypsin digestion condition is 37 DEG C, pH 8.0；Neutral protease enzymolysis item
Part is 45 DEG C, pH 7.0；Alkali protease enzymatic hydrolysis condition is 45 DEG C, pH 10.0；Pepsin enzymatic hydrolysis condition is 37 DEG C, pH
2.0。
6. a kind of preparation method of Japanese croaker flesh of fish immunomodulatory peptides according to claim 1, which is characterized in that step
It is rapid b) in it is cooling after 12000r/min be centrifuged 15min.
7. a kind of preparation method of Japanese croaker flesh of fish immunomodulatory peptides according to claim 1, which is characterized in that step
It is rapid d) in
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| 胡旭阳: "日本黄姑鱼肉活性肽的制备及其免疫调节作用研究", 《中国优秀硕士学位论文全文数据库（电子期刊） 工程科技I辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111635923A (en) * | 2020-06-12 | 2020-09-08 | 福建农林大学 | Eel immunomodulatory glycopeptide and efficient preparation method thereof |
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