CN110196333A - A kind of extracting method and application of Harengula zunasi glycoprotein - Google Patents
A kind of extracting method and application of Harengula zunasi glycoprotein Download PDFInfo
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Abstract
The present invention relates to technological field of biochemistry, disclose the extracting method and application of a kind of Harengula zunasi glycoprotein, the extracting method includes pretreatment, ultrasonic treatment+hot water extraction, alcohol precipitation is concentrated, washing purifying, dialysis freeze-drying and etc., comparison optimization has been carried out by extraction process of the experiment of single factor combination orthogonal experiment method to Harengula zunasi glycoprotein, obtain Harengula zunasi glycoprotein optimum extraction process parameter, the extracting method of Harengula zunasi glycoprotein of the invention has design reasonable, simple process, it is easy to operate, product recovery rate is high, purity is high, without largely using toxic solvent, Product Safety is good, preparation cost is low, the advantages that application easy to spread；The present invention also illustrates that Harengula zunasi glycoprotein has remarkable effect in terms of Culture in vitro by cell experiment verifying, and the research and development for being applied to drug, food for Harengula zunasi glycoprotein provide reference frame.
Description
Technical field
The present invention relates to technological field of biochemistry, more particularly to a kind of extracting method of Harengula zunasi glycoprotein and
Using.
Background technique
Glycoprotein is oligonucleotide chain and polypeptide is covalently attached in a variety of forms and biologically active substance, glycoprotein are made a living
One of important biomolecule macromolecular, is widely present in animals and plants and microorganism in object, or even in single-celled organisms and virus
In be also found, in a variety of manners, Species distributing in the fluid of inside and outside cell and tissue of organism, constitute biology it is intracorporal more
Kind active material.Non-natural glycoprotein has the physiological activity such as anti-oxidant, antitumor, raising immunity.Glycoprotein sends out growth
Educate, information transmitting, immune system, a variety of vital movements such as nervous system play an important role, while with a variety of diseases (as cardiovascular
Disease, liver-kidney diseases, diabetes and tumor and cancer etc.) generation, development it is related.It studies with the development of science and technology and both at home and abroad
The attention that scholar utilizes marine resources development, the glycoprotein research derived from marine animal are increasingly becoming hot spot, attract many
The worker of subject such as chemistry, biology, biochemistry, immunology, cell biology, medicine and pharmacology and Food Science is engaged in this
The further investigation in one field.
Harengula zunasi, also known as green peel, green plate be beautiful, green squama, willow leaf blueness etc., is clupeidae animal, be distributed in China's Bohai and Yellow Seas,
The East Sea and the South Sea, Harengula zunasi protein rich in and the minerals such as amino acid needed by human, vitamin and calcium, iron, phosphorus
Element is a kind of fish full of nutrition, with different physiological roles and potentiality to be exploited.Harengula zunasi is widely used, can also be used as medicine,
It is imitated with the efficacy of removing toxicity for detumescence, cures mainly sea snake and bite, had to the poisoning symptoms such as expiratory dyspnea, purple infantile malnutrition due to digestive disturbances or intestinalparasites, teeth clenched, exhausting are alleviated
Special efficacy.But so far there are no about Harengula zunasi glycoprotein extracting method and its research and report to immune system effect
Road.
In conclusion research and develop a kind of Harengula zunasi glycoprotein extracting method and application it is actually necessary.
Summary of the invention
(deficiency) in order to overcome at least one of the drawbacks of the prior art described above, the present invention provide a kind of Harengula zunasi glycoprotein
Extracting method and application, the extracting method of Harengula zunasi glycoprotein of the invention have design rationally, simple process, operation side
Just, product recovery rate is high, with high purity, property is stable, and without largely using toxic solvent, preparation cost is low, application easy to spread
The advantages that.
To achieve the above object, The technical solution adopted by the invention is as follows:
A kind of extracting method of Harengula zunasi glycoprotein, comprising the following steps:
1. pretreatment: fresh Harengula zunasi is scaled clean, drained away the water, taken flesh of fish stripping and slicing, be placed in meat grinder and rub,
Harengula zunasi muddy flesh is obtained, is weighed；
2. ultrasonic treatment+hot water extraction: 1. Harengula zunasi muddy flesh and distilled water that step is obtained is by 1:20-40 (g/mL)
Solid-liquid ratio be uniformly mixed, be ultrasonically treated 20-40min, obtain Harengula zunasi mixture merging water-bath, heat under the conditions of 80-100 DEG C
Flooding 2-4h, filtering, gained filtrate is spare, and gained filter residue surpasses after mixing with distilled water by the solid-liquid ratio of 1:20-40 (g/mL)
Sonication 20-40min, then at 80-100 DEG C of hot water extraction 1h, filtering merges filtrate twice, and it is mixed to obtain Harengula zunasi glycoprotein
Close solution；
3. alcohol precipitation is concentrated: rotary evaporator is added in 2. mixed solution that step is obtained, and 50-70 DEG C is concentrated by evaporation to substance
The dehydrated alcohol of 3 times of volumes is added in concentrate, 12-18h is stood under the conditions of 4 DEG C, alcohol precipitation is carried out, by alcohol precipitation for long-pending 1/5
Object is collected after centrifugation sediment, then repeats alcohol precipitation to supernatant and operate to no Precipitation, what when supernatant each alcohol precipitation was added
Dehydrated alcohol amount is its 3 times of volumes, merges sediment after alcohol precipitation；
4. removing floating preteins: 3. sediment that step obtains successively is used dehydrated alcohol, acetone washing, dehydrated alcohol and third
The dosage of ketone is subject to immersion precipitation object, is completely dissolved sediment with distilled water after washing, and acquired solution uses Sevage method
Removal floating preteins obtain Harengula zunasi glycoprotein solution；
5. dialysis freeze-drying: being packed into bag filter to 4. Harengula zunasi glycoprotein solution that step obtains, be placed in distilled water, 4 DEG C
Lower dialysis 48h, by the freeze-drying of dialysis trapped substance to get Harengula zunasi glycoprotein powder, weighing.
Further, the step 4. in, using Sevage method removal floating preteins specific steps are as follows: it is molten in sediment
It solves and Sevage reagent is added in acquired solution, under the conditions of 4 DEG C, be placed in magnetic stirring apparatus and vibrate 1h, then be placed in a centrifuge, with
The revolving speed of 12000r/min is centrifuged 15-20min, and solution is followed successively by water layer, albumin layer, organic layer from top to bottom after centrifugation, utilizes
Upper solution is sucked out pipette, repeats the above steps, until when albumin layer no longer occurs in solution after centrifugation, i.e., it is believed that solution
In protein eliminate substantially, collect supernatant, rotary evaporation remove organic solvent, obtain Harengula zunasi glycoprotein solution；Its
In, the dosage of the Sevage reagent is 1/4 volume of the sediment solution, and the Sevage reagent is by n-butanol and chlorine
It is imitative to be mixed in the ratio of 1:4.
Further, the step 2. in, the condition of the ultrasonic treatment are as follows: 25-30 DEG C of temperature, ultrasonic power 40W,
The time of ultrasonic treatment is 30min, is ultrasonically treated the equipment used as supersonic wave cleaning machine.
Further, the step 2. in, in Harengula zunasi mixture and filter residue hot water leaching process, the solid-liquid ratio of extraction is equal
For 1:20 (g/mL), the temperature of hot water extraction is 100 DEG C.
Further, the step 3. in, when repeating alcohol precipitation operation to supernatant, the dehydrated alcohol amount that is added every time
It is equivalent to 3 times of volumes of supernatant volume.
Further, the step 5. in, the molecular cut off of bag filter used is 5kDa, every 6 hours in dialysis procedure
Replace a dialyzate.
Harengula zunasi glycoprotein recovery rate=step of the invention 5. gained Harengula zunasi glycoprotein powder weight/step 1. gained
Harengula zunasi muddy flesh weight * 100%.
The invention also discloses a kind of Harengula zunasi glycoprotein being prepared such as above-mentioned extracting method in Culture in vitro
In application, being experimentally confirmed Harengula zunasi glycoprotein, (RAW264.7 is thin to mouse source mononuclear macrophage leukaemia cell
Born of the same parents) proliferation, phagocytic activity and cell secretion NO, IL-6 and TNF-α amount influence, to illustrate that Harengula zunasi glycoprotein has
Culture in vitro effect.
Compared with prior art, the beneficial effect of technical solution of the present invention is:
In Harengula zunasi glycoprotein extracting method of the invention, ultrasonic treatment is the strong cavitation for utilizing ultrasonic wave, Qiang Zhen
Dynamic and high acceleration characteristic, accelerates the dissolution of Harengula zunasi glycoprotein, to improve glycoprotein recovery rate；Using hot water extraction
Mode extracts Harengula zunasi glycoprotein, and simple process is easy to operate, highly-safe without using toxic solvent；Second is used after concentration
Alcohol carries out alcohol precipitation to glycoprotein solution, the glycoprotein in different molecular weight stage can be collected extraction, ensure that and produce sugar
The integrality of protein classes, while preparation efficiency is improved, it is nontoxic；Floating preteins are effectively removed using Sevage method, are avoided
Interference of the floating preteins to product recovery rate and purity, improves the purity of product；Again using the salinity in dialysis removal product
And small molecular weight impurity, it is ensured that product purity, dialysis trapped substance are freeze-dried, and can not only prevent protein denaturation, but also be able to maintain
Proper constituent in protein, finally obtained glycoprotein have many advantages, such as that loose, solubility is good, stable structure.
In short, the extracting method design of Harengula zunasi glycoprotein of the invention rationally, it is simple process, easy to operate feasible, slightly
Product is easy to separate and purify, and product recovery rate is high, with high purity；Without largely using toxic solvent, product safety in extraction process
Property is good；Preparation condition is mild, and gained Harengula zunasi glycoprotein structure is stablized, while also ensuring the activity of glycoprotein；Extracting method
Used in instrument reagent it is simple and easy to get, therefore preparation cost is low, and extracting method of the invention has good repetition again
Existing property, application easy to spread.
Detailed description of the invention
Fig. 1 is influence of the ultrasonic time to Harengula zunasi glycoprotein recovery rate；
Fig. 2 is influence of the extraction time to Harengula zunasi glycoprotein recovery rate；
Fig. 3 is influence of the Extracting temperature to Harengula zunasi glycoprotein recovery rate；
Fig. 4 is influence of the solid-liquid ratio to Harengula zunasi glycoprotein recovery rate；
Fig. 5 is that phend-sulphuric acid measures sugared content standard curve and regression equation；
Fig. 6 is influence of the Harengula zunasi glycoprotein to RAW264.7 cell Proliferation；
Fig. 7 is influence of the Harengula zunasi glycoprotein to RAW264.7 cell phagocytic activity；
Fig. 8 is the influence that Harengula zunasi glycoprotein secretes NO to RAW264.7 cell；
Fig. 9 is the influence that Harengula zunasi glycoprotein secretes IL-6 to RAW264.7 cell；
Figure 10 is influence of the Harengula zunasi glycoprotein to RAW264.7 cell TNF secretion-α.
Specific embodiment
Further illustrate that the present invention, following embodiment are the preferable embodiment party of the present invention below by specific embodiment
Formula, but embodiments of the present invention are not limited by following embodiments, therefore the scope of protection of present invention is not limited to
In described.
Embodiment 1:
A kind of extracting method of Harengula zunasi glycoprotein, comprising the following steps:
1. pretreatment: fresh Harengula zunasi is scaled clean, drained away the water, taken flesh of fish stripping and slicing, be placed in meat grinder and rub,
Harengula zunasi muddy flesh is obtained, is weighed；
2. ultrasonic treatment+hot water extraction: 1. Harengula zunasi muddy flesh and distilled water that step is obtained by 1:20 (g/mL) material
Liquor ratio is uniformly mixed, and is ultrasonically treated 30min, obtains Harengula zunasi mixture merging water-bath, hot water extraction 3h under the conditions of 100 DEG C,
Filtering, gained filtrate is spare, and gained filter residue is ultrasonically treated 30min after mixing with distilled water by the solid-liquid ratio of 1:20 (g/mL), then
In 100 DEG C of hot water extraction 1h, filtering merges filtrate twice, obtains Harengula zunasi glycoprotein mixed solution；
3. alcohol precipitation is concentrated: rotary evaporator is added in 2. mixed solution that step is obtained, and 50 DEG C are concentrated by evaporation to original volume
1/5, the dehydrated alcohol of 3 times of volumes is added in concentrate, 12h is stood under the conditions of 4 DEG C, carries out alcohol precipitation, by alcohol hypostasis from
It collects sediment after the heart, then alcohol precipitation is repeated to supernatant and is operated to no Precipitation, when supernatant each alcohol precipitation is added anhydrous
Amount of alcohol is its 3 times of volumes, merges sediment after alcohol precipitation；
4. removing floating preteins: 3. sediment that step obtains successively is used dehydrated alcohol, acetone washing, dehydrated alcohol and third
The dosage of ketone is subject to immersion precipitation object, is completely dissolved sediment with distilled water after washing, and acquired solution uses Sevage method
Removal floating preteins obtain Harengula zunasi glycoprotein solution；
5. dialysis freeze-drying: being packed into the dialysis that molecular cut off is 5kDa to 4. Harengula zunasi glycoprotein solution that step obtains
Bag, is placed in distilled water, dialyse 48h at 4 DEG C, and the dialyzate of replacement in every 6 hours, the trapped substance that will dialyse are cold in dialysis procedure
It is lyophilized dry to get Harengula zunasi glycoprotein powder, weighing.
Wherein, the step 2. in, the condition of the ultrasonic treatment are as follows: 28 DEG C of temperature, ultrasonic power 40W, ultrasound at
The equipment of reason is SB-4200DTD supersonic wave cleaning machine.
Wherein, the step 4. in, using Sevage method removal floating preteins specific steps are as follows: sediment dissolve institute
It obtains and Sevage reagent is added in solution, under the conditions of 4 DEG C, be placed in magnetic stirring apparatus and vibrate 1h, then be placed in a centrifuge, with
The revolving speed of 12000r/min is centrifuged 15min, and solution is followed successively by water layer, albumin layer, organic layer from top to bottom after centrifugation, utilizes shifting
Upper solution is sucked out liquid pipe, repeats the above steps, until when albumin layer no longer occurs in solution after centrifugation, i.e., it is believed that in solution
Protein eliminate substantially, collect supernatant, rotary evaporation remove organic solvent, obtain Harengula zunasi glycoprotein solution；Wherein,
The dosage of the Sevage reagent is 1/4 volume of the sediment solution, and the Sevage reagent is by n-butanol and chloroform
It is mixed in the ratio of 1:4.
Embodiment 2:
Embodiment 2 and the operating procedure of embodiment 1 are essentially identical, and difference is:
The step is 2. in ultrasonic treatment+hot water extraction: 1. Harengula zunasi muddy flesh and distilled water that step is obtained is by 1:30
(g/mL) solid-liquid ratio is uniformly mixed, 25 DEG C of ultrasonic treatment 40min, obtains Harengula zunasi mixture merging water-bath, 90 DEG C of conditions
Lower hot water extraction 2h, filtering, gained filtrate is spare, ultrasound after gained filter residue is mixed with distilled water by the solid-liquid ratio of 1:30 (g/mL)
40min is handled, then at 90 DEG C of hot water extraction 1h, filtering merges filtrate twice, obtains Harengula zunasi glycoprotein mixed solution；
3. the step is concentrated in alcohol precipitation: rotary evaporator is added in 2. mixed solution that step is obtained, and 60 DEG C of evaporations are dense
It is reduced to the 1/5 of original volume, the dehydrated alcohol of 3 times of volumes is added in concentrate, 16h is stood under the conditions of 4 DEG C, carries out alcohol precipitation,
Alcohol hypostasis is collected after centrifugation sediment, then alcohol precipitation is repeated to supernatant and is operated to no Precipitation, when each alcohol precipitation of supernatant
The dehydrated alcohol amount of addition is its 3 times of volumes, merges sediment after alcohol precipitation；
4. the step is removed in floating preteins: Sevage reagent, 4 DEG C of conditions are added in sediment dissolution acquired solution
Under, after vibrating 1h, 12min is centrifuged with the revolving speed of 12000r/min, remaining condition is identical with operation.
Embodiment 3:
Embodiment 3 and the operating procedure of embodiment 1 are essentially identical, and difference is:
The step is 2. in ultrasonic treatment+hot water extraction: 1. Harengula zunasi muddy flesh and distilled water that step is obtained is by 1:40
(g/mL) solid-liquid ratio is uniformly mixed, and is ultrasonically treated 20min at 30 DEG C, obtains Harengula zunasi mixture merging water-bath, 80 DEG C of items
Hot water extraction 4h under part, filtering, gained filtrate is spare, and gained filter residue surpasses after mixing with distilled water by the solid-liquid ratio of 1:40 (g/mL)
Sonication 20min, then at 80 DEG C of hot water extraction 1h, filtering merges filtrate twice, obtains Harengula zunasi glycoprotein mixed solution；
3. the step is concentrated in alcohol precipitation: rotary evaporator is added in 2. mixed solution that step is obtained, and 70 DEG C of evaporations are dense
It is reduced to the 1/5 of original volume, the dehydrated alcohol of 3 times of volumes is added in concentrate, 18h is stood under the conditions of 4 DEG C, carries out alcohol precipitation,
Alcohol hypostasis is collected after centrifugation sediment, then alcohol precipitation is repeated to supernatant and is operated to no Precipitation, when each alcohol precipitation of supernatant
The dehydrated alcohol amount of addition is its 3 times of volumes, merges sediment after alcohol precipitation；
4. the step is removed in floating preteins: Sevage reagent, 4 DEG C of conditions are added in sediment dissolution acquired solution
Under, after vibrating 1h, 20min is centrifuged with the revolving speed of 12000r/min, remaining condition is identical with operation.
One, comparative experiments:
Optimize Harengula zunasi glycoprotein extracting method of the invention most in conjunction with to compare by experiment of single factor and orthogonal experiment
Good extraction conditions, determine influence Harengula zunasi glycoprotein recovery rate principal element are as follows: ultrasonic time, extraction time, Extracting temperature,
Solid-liquid ratio.
The ultrasonic time refers to the time being ultrasonically treated to Harengula zunasi muddy flesh (or extracting gained filter residue for the first time).
The extraction time, Extracting temperature refer to time and the temperature of hot water extraction.
During the solid-liquid ratio refers to ultrasonic treatment+hot water extraction, Harengula zunasi muddy flesh (or gained filter is extracted for the first time
Slag) with the mass volume ratio of distilled water, indicated with g/mL.
1. experiment of single factor
Single factor test is carried out with ultrasonic time, extraction time, Extracting temperature, the solid-liquid ratio in Harengula zunasi glycoprotein extraction process
Experiment.
1. influence of the ultrasonic time to Harengula zunasi glycoprotein recovery rate
Harengula zunasi muddy flesh 100g is weighed every time, is 90 DEG C, extraction time 3h in Extracting temperature, solid-liquid ratio is 1:30 (g/
ML), remaining extraction step studies different ultrasonic times (respectively 20min, 30min, 40min) to blueness in the case where being consistent
The influence of squama fish glycoprotein recovery rate.Experimental result is as shown in table 1 and attached drawing 1:
Influence of 1 ultrasonic time of table to Harengula zunasi glycoprotein recovery rate
By table 1 and attached drawing 1 as it can be seen that being 3h between upon extracting, Extracting temperature is 90 DEG C, when solid-liquid ratio is 1:30 (g/mL),
With the extension of ultrasonic time, the trend of reduction after first increase is presented in the recovery rate of Harengula zunasi glycoprotein, when ultrasonic time is
When 30min, recovery rate is up to 0.052%.Through analyzing, under the action of the ultrasonic wave of certain time, since ultrasonic wave has
There is the characteristic of strong cavitation, strong vibration and high acceleration, the dissolution of glycoprotein can be accelerated, to reach higher extraction
Rate；But when ultrasonic time is too long, glycoprotein is easy to occur to destroy under the cavitation of ultrasonic wave and decompose again, to make sugared egg
White recovery rate reduces.The experimental results showed that when other conditions are constant, when ultrasonic time is 30min, the extraction of Harengula zunasi glycoprotein
Rate highest.
2. influence of the extraction time to Harengula zunasi glycoprotein recovery rate
Harengula zunasi muddy flesh 100g is weighed every time, is 30min in ultrasonic time, Extracting temperature is 90 DEG C, solid-liquid ratio 1:30
(g/mL), remaining extraction step studies different extraction times (respectively 2h, 3h, 4h) to Harengula zunasi sugar in the case where being consistent
The influence of protein extracting ratio.Experimental result is as shown in table 2 and attached drawing 2:
Influence of 2 extraction time of table to Harengula zunasi glycoprotein recovery rate
By table 2 and attached drawing 2 as it can be seen that when ultrasonic time is 30min, Extracting temperature is 90 DEG C, and solid-liquid ratio is 1:30 (g/mL)
When, with the extension of extraction time, the recovery rate of Harengula zunasi glycoprotein is presented first increase after reduction trend, upon extracting between be
When 3h, glycoprotein recovery rate is up to 0.052%.Through analyzing, within the regular hour, Harengula zunasi glycoprotein is dissolved in water
In, and as the dissolution rate for extending its glycoprotein of extraction time is higher；But as extraction time extends again, in 90 DEG C of extraction
At a temperature of, the glycoprotein in extracting solution is easy to happen oxygenolysis, and the glycoprotein concentration in extracting solution is caused to reduce.Test table
It is bright, when other conditions are constant, and extraction time is 3h, the recovery rate highest of Harengula zunasi glycoprotein.
3. influence of the Extracting temperature to Harengula zunasi glycoprotein recovery rate
Harengula zunasi muddy flesh 100g is weighed every time, is 30min in ultrasonic time, extraction time 3h, solid-liquid ratio is 1:30 (g/
ML), remaining extraction step studies different Extracting temperatures (respectively 80 DEG C, 90 DEG C, 100 DEG C) to green squama in the case where being consistent
The influence of fish glycoprotein recovery rate.Experimental result is as shown in table 3 and attached drawing 3:
Influence of 3 Extracting temperature of table to Harengula zunasi glycoprotein recovery rate
By table 3 and attached drawing 3 as it can be seen that when ultrasonic time is 30min, extraction time 3h, when solid-liquid ratio is 1:30 (g/mL),
With the rise of extracting temperature, the recovery rate of Harengula zunasi glycoprotein constantly increases, its glycoprotein when Extracting temperature reaches 100 DEG C
Yield is up to 0.068%.Through analyzing, due to the continuous raising with Extracting temperature, glycoprotein molecule movement velocity is accelerated,
To make mass transfer movement velocity of the glycoprotein components between water and Harengula zunasi cell tissue also constantly speed, while with temperature liter
Height may increase the extent of the destruction to Harengula zunasi cell, to improve the recovery rate of glycoprotein.Experiment shows when other
Part is constant, when Extracting temperature is 100 DEG C, the recovery rate highest of Harengula zunasi glycoprotein.
4. influence of the solid-liquid ratio to Harengula zunasi glycoprotein recovery rate
Harengula zunasi muddy flesh 100g is weighed every time, is 30min, extraction time 3h, Extracting temperature 100 in ultrasonic time
DEG C, research different feed liquid ratio (respectively 1:20,1:30,1:40) is to Harengula zunasi sugar in the case that remaining extraction step is consistent
The influence of protein extracting ratio.Experimental result is as shown in table 4 and attached drawing 4:
Influence of 4 solid-liquid ratio of table to Harengula zunasi glycoprotein recovery rate
By table 4 and attached drawing 4 as it can be seen that when ultrasonic time is 30min, extraction time 3h, when Extracting temperature is 90 DEG C, with
Solid-liquid ratio increases, and the extraction of Harengula zunasi glycoprotein takes the lead in reducing after increasing, and when solid-liquid ratio is 30mL/g, glycoprotein yield is most
A height of 0.052%.It is analyzed, is primarily due to when feed liquid is smaller, system large viscosity, mobility is poor, Harengula zunasi sugar egg
It is white to fail to be completely dissolved in water, and when solid-liquid ratio is excessive, the recovery rate of glycoprotein slightly reduces instead.Experiment show when
Other conditions are constant, and when solid-liquid ratio is 1:30 (g/mL), the recovery rate of Harengula zunasi glycoprotein is higher.
2. orthogonal experiment
Based on experiment of single factor as a result, by orthogonal experiment verifying optimum extraction process parameter, selection ultrasonic time (A),
4 extraction time (B), Extracting temperature (C), solid-liquid ratio (D) factors are empirical factor, are to refer to Harengula zunasi glycoprotein recovery rate
Mark carries out L9(34) orthogonal, it studies influence of each factor level to Harengula zunasi glycoprotein recovery rate and content and mentions
The optimum process condition taken, factor level design are as shown in table 5:
5 orthogonal experiment factor level table of table
Orthogonal experiment is carried out according to the design of orthogonal experiment factor level table 5, Harengula zunasi glycoprotein Orthogonal experiment results are such as
Shown in table 6:
6 Orthogonal experiment results table of table
Note: in table 6, K1Indicate the summation for the recovery rate that each factor is tested every time in 1 level；K2Indicate each factor 2
The summation for the recovery rate tested every time when horizontal；K3Indicate the summation for the recovery rate that each factor is tested every time in 3 level；k1Table
Show the average value for the recovery rate that each factor is tested every time in 1 level；k2Indicate the extraction that each factor is tested every time in 2 level
The average value of rate；k3Indicate the average value for the recovery rate that each factor is tested every time in 3 level；Very poor R refers to same factor pair
The k answered1、k2、k3The difference of middle maxima and minima.
As shown in Table 6, RC>RB>RA>RD, so this four factors are suitable to the conspicuousness of Harengula zunasi glycoprotein extraction rate impact
Sequence is followed successively by Extracting temperature > extraction time > ultrasonic time > solid-liquid ratio, wherein the influence degree of Extracting temperature and extraction time compared with
Greatly, the influence degree of ultrasonic time and solid-liquid ratio is smaller.It follows that Harengula zunasi glycoprotein extraction influence factor optimal combination is
A2B2C3D1, i.e. ultrasonic time is 30min, and extraction time 3h, Extracting temperature is 100 DEG C, and solid-liquid ratio is that 1:20 (g/mL) is green
The optimum process condition that squama fish glycoprotein extracts, the recovery rate of Harengula zunasi glycoprotein is 0.057% with this condition.
Two, Harengula zunasi glycoprotein purity confirmatory experiment:
Harengula zunasi glycoprotein purity prepared by the verifying embodiment of the present invention 1 is determined by experiment.Glycoprotein is macromolecular
Compound, purity rubric cannot be measured with common small molecule compound purity rubric, because of even glycoprotein sterling,
It is microcosmic be also it is inhomogenous, therefore, Harengula zunasi glycoprotein purity characteristic manner prepared by the present invention are as follows: Harengula zunasi glycoprotein is pure
Degree=sugared content+protein content.
1. sugared content measures
Measuring method: concentration-absorbance standard is formulated with the absorbance of phend-sulphuric acid measurement different glucose
Curve, then Harengula zunasi glycoprotein sample is made into certain density solution and surveys its absorbance by operation under standard curve item, it substitutes into
Concentration-absorbance standard curve equation calculates the sugared content in gained glycoprotein.
The preparation of standard curve: accurately weighing anhydrous grape saccharide 1mg, adds appropriate distilled water to dissolve, is held with 10mL
Measuring bottle is settled to graduation mark, shakes up, and is configured to the standard solution of 0.1mg/mL.
0.0,0.2,0.4,0.6,0.8,1.0,2.0,4.0 mL of glucose standards solution is drawn respectively in 10mL volumetric flask
In, distilled water is added and is settled to graduation mark, shakes up, is made into a series of glucose standards solution.
200 μ L series standard solution are drawn respectively in 2mL centrifuge tube, then be separately added into 100 μ L now with 5% phenol
Reagent adds the 500 μ L concentrated sulfuric acids, mixed liquor is shaken up after shaking up, measure the extinction of solution after placement 20min at 490nm
Value draws standard curve and establishes regression equation, and standard curve and regression equation are as shown in Fig. 5.
10mg Harengula zunasi glycoprotein accurately is weighed, distilled water is added to dissolve, is settled to 10mL, the final concentration of 1mg/mL of sample；
It is accurate to draw each sample solution 0.1mL, standard curve making method is then pressed, its light absorption value is measured.According to the light absorption value measured
Sugared content with the regression equation calculation glycoprotein sample of foundation is 15.2%.
2. protein content determination
During preventing protein determination, partially protein degradation causes experimental error, and spy passes through measurement amino acid
Method come measure characterization Harengula zunasi glycoprotein in protein content, measuring method are as follows: weigh 20mg Harengula zunasi glycoprotein in water
Then Xie Guanzhong is added the hydrochloric acid solution that 18mL concentration is 6mol/L, is filled with nitrogen 2min, vacuumizes tube sealing, be placed in oven
For 24 hours, the hydrolyzate after the completion of hydrolyzing is placed in 100 DEG C of water-baths, so that hydrochloric acid is sufficiently volatilized, to slough hydrolysis for 110 DEG C of hydrolysis
Acid in liquid is washed 3 times with distilled water, continues to evaporate remaining hydrochloric acid, is then settled to 5mL with distilled water, then micro- with 0.45 μm
Hole membrane filtration, filtrate carry out Contents of Amino Acids analysis with amino-acid analyzer, and measurement result is as shown in table 7:
7 amino acid content testing result of table
As can be seen from Table 7, the amino acid content in Harengula zunasi glycoprotein prepared by the present invention is 59.74%, that is, is indicated
Protein content in Harengula zunasi glycoprotein is 59.74%,
In conclusion Harengula zunasi glycoprotein purity prepared by the present invention=sugared content+protein content, i.e. Harengula zunasi sugar egg
Bai Chundu is 74.94%.
Three, the immunological regulation experiment of Harengula zunasi glycoprotein:
The Culture in vitro of the Harengula zunasi glycoprotein as made from the experimental verification embodiment of the present invention 1 acts on, experiment side
Method and result are as follows:
1. experimental subjects: mouse source mononuclear macrophage leukaemia cell (264.7 cell of RAW).
2. main agents:
| Main agents | Specification | Manufacturer |
| Thiazolyl blue (MTT) | 100mg | Sigma company |
| Lipopolysaccharides (LPS) | 10mg | Shanghai source leaf biology Co., Ltd |
| Determination of nitric oxide kit | 500T | Shanghai green skies biology Co., Ltd |
| Mouse TNF-α ELISA kit | 96T | Shenzhen Xin Bosheng Biotechnology Co., Ltd |
| Mouse IL-6ELISA kit | 96T | Shenzhen Xin Bosheng Biotechnology Co., Ltd |
3. the culture and passage of cell
The culture of 264.7 cell of RAW: existed with the complete medium containing the dual anti-DEME of 10% fetal calf serum and 1%
Containing 5% (V/V) CO2, cultivate in 37 DEG C of constant incubators；
It is passed on when cell sticks culture bottle bottom, with the pancreatin digestive juice vitellophag containing 0.25%EDTA.
The cell is adherent more loose, and the film enzymic digestion time is unsuitable too long, should handle with care in an experiment.
4. influence of the Harengula zunasi glycoprotein to RAW264.7 cell Proliferation
Using mtt assay measurement Harengula zunasi glycoprotein on RAW264.7 macrophage proliferation ability to influence.Take logarithmic growth
264.7 cell of phase RAW, blood counting chamber count, adjustment concentration of cell suspension to 3 × 104Cells/mL is seeded to 96 orifice plates,
Every 100 μ L of hole, 37 DEG C of cultures are for 24 hours；Supernatant is abandoned, the Harengula zunasi glycoprotein sample solution of various concentration (10-120 μ g/mL) is added,
3 multiple holes are arranged in each concentration, and culture solution is as blank control, and 37 DEG C of cultures are for 24 hours；10 μ of MTT solution is added in every hole after for 24 hours
L continues to be placed in incubator and cultivates 4h；Supernatant is sopped up later, and 100 μ L dimethyl sulfoxides are added in every hole, and oscillation mixes, enzyme mark
Instrument detects the light absorption value at 570nm, and experimental result is as shown in Fig. 6.
By attached drawing 6 as it can be seen that Harengula zunasi glycoprotein can promote RAW264.7 macrophage in 10-120 μ g/mL concentration range
The proliferation of cell, and in 10-80 μ g/mL concentration range, with the increase of Harengula zunasi glycoprotein concentration, facilitation is brighter
It is aobvious, and although have promotion cel l proliferation in 80-120 μ g/mL, but effect is lower than 80 μ g/mL.It follows that Harengula zunasi is sugared
Albumen promotes the effect of cell Proliferation most obvious in 80 μ g/mL.
5. influence of the Harengula zunasi glycoprotein to RAW264.7 cell phagocytic activity
Influence of the experiment detection Harengula zunasi glycoprotein to RAW264.7 macrophage phagocytosis is swallowed using dimethyl diaminophenazine chloride.It takes
Logarithmic growth phase cell, is counted with blood counting chamber, adjustment concentration of cell suspension to 3 × 104Cells/mL is seeded to 96 holes
Plate, every 100 μ L of hole, 37 DEG C of cultures are for 24 hours；Supernatant is abandoned, the Harengula zunasi glycoprotein sample that various concentration (10-120 μ g/mL) is added is molten
6 multiple holes are arranged in liquid, each concentration, and culture solution is as blank control, and the LPS solution of 2 μ g/mL is as positive control, 37 DEG C of trainings
It supports for 24 hours；Abandon supernatant afterwards for 24 hours, 100 μ L neutral red solutions (0.5mg/mL) are added in every hole, continue to cultivate 1h；Supernatant is abandoned after 1h,
PBS solution is washed 3 times；100 μ L cell pyrolysis liquid (glacial acetic acids are added in every hole；Ethyl alcohol=1:1 (v/v)), incubation at room temperature overnight, is shaken
It swings, microplate reader detects light absorption value at 540nm, and experimental result is as shown in Fig. 7.
In fig. 7: Black represents blank control group, containing only culture solution；CK represents negative control group, containing culture solution+carefully
Born of the same parents；LPS represents positive controls, the LPS solution of+2 μ g/mL containing culture solution+cell.
By attached drawing 7 as it can be seen that Harengula zunasi glycoprotein can promote RAW264.7 macrophage in 10-120 μ g/mL concentration range
The phagocytic activity of cell, and in 10-80 μ g/mL concentration range, with the increase of Harengula zunasi glycoprotein concentration, facilitation is more
Obviously, although there are promotion phagocytosis, but effect and the no significant difference in 80 μ g/mL in 80-120 μ g/mL.Thus
Out, Harengula zunasi glycoprotein promotes cytophagy most obvious in 80 μ g/mL.
6. influence of the Harengula zunasi glycoprotein to RAW264.7 cell secretion NO, IL-6 and TNF-α
When glycoprotein acts on RAW264.7 cell, it can induce the generation of various kinds of cell immune response, pass through rush
The mode of proliferation and generation cell factor into cell realizes immunoregulation effect.Wherein, NO is the intracorporal active material of biology,
The burst size of NO is to judge that macrophage immunity adjusts the important indicator whether activity enhances；RAW264.7 cell is by glycoprotein
Stimulation, secrete cytokines, such as tumor necrosis factor (TNF) and interleukins (IL), these factor energy can be promoted
Regulate and control the immune function of body to a certain extent, and plays a significant role in immune response；Lipopolysaccharides (LPS) is leather
Main component in gram-negative bacteria cell wall, has toxicity to host, generates immune response with inducing cell
Effect is commonly used for the positive control of immunoregulatory activity evaluation.
Logarithmic growth phase cell counts, culture medium diluting cells suspension, and adjustment concentration of cell suspension is extremely
5x105Cells/mL is seeded to 24 orifice plates, every 100 μ L of hole, and 37 DEG C of cultures for 24 hours, abandon supernatant, and various concentration is added in experimental group
4 multiple holes, negative control group (CK)=culture is arranged in the Harengula zunasi glycoprotein sample solution of (10-120 μ g/mL), each concentration
Liquid+cell, positive controls (LPS)=LPS solution (2 μ g/mL)+culture solution+cell, group of cells are cultivated for 24 hours in 37 DEG C.
1. the influence using Griess reagent detection Harengula zunasi glycoprotein to RAW264.7 cell secretion NO content.It draws
50 μ L Griess reagent I are added in 50 μ L of cell culture supernatant, add 50 μ L Griess reagent II solution, and oscillation mixes,
Using microplate reader detection 540nm locate light absorption value, while will use the diluted series of concentrations of DMEM culture solution (1,2,5,10,40,60,
100nmol/L)NaNO2Solution draws standard curve, regression equation is established, according to regression equation calculation cell as standard items
The content of NO in supernatant, experimental result is as shown in attached drawing 8.
It by attached drawing 8 as it can be seen that Harengula zunasi glycoprotein can remarkably promote RAW264.7 cell secretion NO, and is in dose dependent,
When Harengula zunasi glycoprotein concentration reaches 60 μ g/mL, the burst size of NO is about 35 times of blank control group.
2. the influence using ELISA reagent method detection Harengula zunasi glycoprotein to RAW264.7 cell secretion IL-6 content.It inhales
100 μ L of cell culture supernatant is taken, using IL-6ELISA reagent according on the detection cell of detection method described in kit specification
The content of IL-6 in clear liquid, experimental result such as 9 institute of attached drawing that Harengula zunasi glycoprotein influences RAW264.7 cell secretion IL-6
Show.
By attached drawing 9 as it can be seen that compared with positive controls, Harengula zunasi glycoprotein promotes RAW264.7 cell secretion IL-6's
Effect is less significant, but as the increase of dosage, the secretory volume of IL-6 are also in increased trend, facilitation is gradually significant.When
When Harengula zunasi glycoprotein concentration reaches 60 μ g/mL, the secretory volume of IL-6 improves 4.75 times than blank control group.
3. using ELISA reagent method detection Harengula zunasi glycoprotein to the influence of RAW264.7 cell TNF secretion-alpha content.
100 μ L of cell culture supernatant is drawn, is detected carefully using TNF-α ELISA reagent according to detection method described in kit specification
The content of TNF-α in born of the same parents' supernatant, the experimental result such as attached drawing that Harengula zunasi glycoprotein influences RAW264.7 cell TNF secretion-α
Shown in 10.
By attached drawing 10 as it can be seen that compared with blank control group, Harengula zunasi glycoprotein can be shown in 5-60 μ g/mL concentration range
It writes and promotes RAW264.7 macrophages secrete TNF-α, and be in dose-dependence, when Harengula zunasi glycoprotein concentration reaches 60 μ g/
When mL, the secretory volume of TNF-α is 21 times of blank control group.
In conclusion the Harengula zunasi glycoprotein of low dosage can significantly improve the proliferation to RAW264.7 cell, phagocytic activity,
And RAW264.7 cell secretion NO, IL-6 and TNF-α are remarkably promoted, to illustrate that Harengula zunasi glycoprotein is made with immunological regulation
With being applied to immunological regulation drug to Harengula zunasi, the research and development of health food and Harengula zunasi maximize development and utilization and provide ginseng
Examine foundation.
The above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be to the present invention
Embodiment restriction.For those of ordinary skill in the art, it can also be made on the basis of the above description
Its various forms of variation or variation, there is no need and unable to be exhaustive to all embodiments.It is all in spirit of the invention
With any modifications, equivalent replacements, and improvements made within principle etc., the protection scope of the claims in the present invention should be included in
Within.
Claims (10)
1. a kind of extracting method of Harengula zunasi glycoprotein, which comprises the following steps:
1. pretreatment: fresh Harengula zunasi being scaled clean, drained away the water, taken flesh of fish stripping and slicing and rub, obtain Harengula zunasi muddy flesh；
2. ultrasonic treatment+hot water extraction: 1. Harengula zunasi muddy flesh and distilled water that step is obtained by 1:20-40 (g/mL) feed liquid
Than being uniformly mixed, it is ultrasonically treated 20-40min, obtains Harengula zunasi mixture merging water-bath, hot water extraction under the conditions of 80-100 DEG C
2-4h, filtering, gained filtrate is spare, and gained filter residue repeats ultrasonic treatment and hot water extraction extracts, and then filters, and merges two
Secondary filtrate obtains Harengula zunasi glycoprotein mixed solution；
3. alcohol precipitation is concentrated: rotary evaporator is added in 2. mixed solution that step is obtained, and 50-70 DEG C is concentrated by evaporation to original volume
1/5, the dehydrated alcohol of 3 times of volumes is added in gained concentrate, 12-18h is stood under the conditions of 4 DEG C, alcohol precipitation is carried out, by alcohol hypostasis
Sediment is collected after centrifugation, then alcohol precipitation is repeated to supernatant and is operated to no Precipitation, merges sediment；
4. removing floating preteins: 3. sediment that step obtains successively being used dehydrated alcohol, acetone washing, then will be precipitated with distilled water
Object dissolution, acquired solution remove floating preteins using Sevage method, obtain Harengula zunasi glycoprotein solution；
5. dialysis freeze-drying: bag filter is packed into 4. Harengula zunasi glycoprotein solution that step obtains, is placed in distilled water, dialyse 48h,
Dialysis trapped substance is freeze-dried to get Harengula zunasi glycoprotein powder.
2. the extracting method of Harengula zunasi glycoprotein according to claim 1, which is characterized in that the step 4. in, use
The specific steps of Sevage method removal floating preteins are as follows: Sevage reagent is added in sediment dissolution acquired solution, in 4 DEG C of items
Under part, 1h is vibrated, 15-20min is then centrifuged with the revolving speed of 12000r/min, supernatant is collected, repeats the step to no egg
White is precipitated.
3. the extracting method of Harengula zunasi glycoprotein according to claim 2, which is characterized in that the step 4. in, use
The condition of Sevage method removal floating preteins are as follows: the dosage of the Sevage reagent is 1/4 volume of the sediment solution,
The Sevage reagent is mixed by n-butanol and chloroform in the ratio of 1:4.
4. the extracting method of Harengula zunasi glycoprotein according to claim 1, which is characterized in that the step 2. in, it is described
The time of ultrasonic treatment is 30min.
5. the extracting method of Harengula zunasi glycoprotein according to claim 1, which is characterized in that the step 2. in, green squama
The extraction solid-liquid ratio of fish meat puree and distilled water is 1:20 (g/mL), and the temperature of hot water extraction is 100 DEG C, extraction time 3h.
6. the extracting method of Harengula zunasi glycoprotein according to claim 1, which is characterized in that the step 2. in, gained
It when filter residue repeats to extract, is mixed with distilled water by the solid-liquid ratio of 1:20-40 (g/mL), 20-40min is ultrasonically treated, then at 80-
100 DEG C of hot water extraction 1h.
7. the extracting method of Harengula zunasi glycoprotein according to claim 1, which is characterized in that the step 2. in, it is described
The condition of ultrasonic treatment are as follows: 25-30 DEG C of temperature, ultrasonic power 40W.
8. the extracting method of Harengula zunasi glycoprotein according to claim 1, which is characterized in that the step 3. in, to upper
When clear liquid repeats alcohol precipitation operation, the dehydrated alcohol amount being added every time is equivalent to 3 times of volumes of supernatant.
9. the extracting method of Harengula zunasi glycoprotein according to claim 1, which is characterized in that the step 5. in, dialysis
Operation carries out at 4 DEG C, and the molecular cut off of bag filter used is 5kDa, the dialyzate of replacement in every 6 hours in dialysis procedure.
10. a kind of Harengula zunasi glycoprotein obtained such as the described in any item extracting methods of claim 1-9 is in Culture in vitro
In application.
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| CN112552370A (en) * | 2020-12-11 | 2021-03-26 | 中国药科大学 | Method for extracting water-soluble protein from shrimp meat |
| CN114369138A (en) * | 2022-02-21 | 2022-04-19 | 福建农林大学 | Method for preparing glycoprotein by utilizing hairtail processing by-products |
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| CN114369138A (en) * | 2022-02-21 | 2022-04-19 | 福建农林大学 | Method for preparing glycoprotein by utilizing hairtail processing by-products |
| CN114369138B (en) * | 2022-02-21 | 2024-01-12 | 福建农林大学 | Method for preparing glycoprotein by using hairtail processing byproducts |
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