CN101972004A - Preparation method and application of oyster zymolyte - Google Patents
Preparation method and application of oyster zymolyte Download PDFInfo
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Abstract
The invention discloses a method for preparing oyster zymolyte with antioxidation and antiaging activity and application thereof. The method includes the following steps: dry oyster powder is taken as raw material, aqueous solution with protein mass fraction of 2% is prepared, water bath is carried out for 30min at 52.5-55 DEG C, then combinative protease is used for successive or simultaneous hydrolysis; enzyme adding amount of each protease is 6000-8000U/g substrate protein, pH is regulated to be 6.0-7.0, hydrolysis is carried out for 2 hours at the temperature of 52.5-55 DEG C, enzymatic hydrolysate is centrifuged, and then supernate is subject to ultrafilteration by virtue of ultrafilteration membrane with the cut-off molecular weight of 1kDa; and retentate is subject to spray drying, thus obtaining oyster zymolyte. The oyster zymolyte can be used for preparing food additive with antioxidation activity or nutritional food with antiaging activity. The invention has the advantages of available raw materials, simple preparation process, high efficiency and no pollution; and zymolyte has strong antioxidation activity and obvious antiaging effect.
Description
Technical field
The present invention relates to prepare a kind of method that possesses the active oyster protein zymolyte that delays senility, can be used as anti-oxidant, delay senility nutrient or medicine interpolation, belong to the marine biotechnology application.
Background technology
Oyster is commonly called as oyster, oyster, is first in the world cultivated shellfish, also is one of China's four big cultivated shellfishes, is distributed widely in ground such as China Liaoning, Shandong, Fujian and Guangdong is coastal.Oyster meat is tender delicious, and dietotherapeutic in China's history of existing centuries, have the laudatory title of " marine milk ", is that China classifies one of health care remedy diet of integration of drinking and medicinal herbs in the first batch as.The protein content of oyster can be up to 50%, and amino acid is formed fully.Also contain abundant glycogen, DHA (DHA) and eicosapentaenoic acid unrighted acids such as (EPA), vitamin and mineral matters such as selenium and zinc in the oyster meat.Over past ten years, enzyme preparation has obtained general application in the seashells recourse processing process, and the research of shellfish protein enzymolysis technology and zymolyte effect thereof constantly makes progress.At present, zymolysis technique become that the seashells protein resource is high-valued, the important means of resource, ecology development and use.But, how to use for reference and break through traditional zymolysis technique and technology, to the green high-valued exploitation of seashells protein resource, be still important field of research in China's shellfish comprehensive utilization of resources.
Summary of the invention
The present invention is raw material with the ostreae testa pulverata, adopts the combination zymolysis technique to obtain the oyster zymolyte, significantly is better than the effect of various single enzyme hydrolysis.Prove conclusively oyster combination zymolyte by various anti-oxidant detection architecture and had obvious antioxidation activity; And adopting the mensuration of index such as lipid peroxide content and activities of antioxidant enzymes in drosophila survival experiment, the murine liver tissue, conclusive evidence oyster combination zymolyte has the activity that delays senility significantly.The notable biological activity that the zymolyte of selecting this new technology to prepare has can be realized the higher value application of oyster.
The present invention realizes by following technological means, the combination enzymolysis process: being made into the albumen quality mark with ostreae testa pulverata in water is 2% the aqueous solution, 52.5 ℃ of-55 ℃ of water-baths were soaked 30 minutes in the reactor, then successively or add the combined protein enzyme simultaneously and be hydrolyzed; The enzyme concentration of each protease is the 6000-8000U/g substrate protein, regulates pH 6.0-7.0 with phosphate buffer, and reaction temperature is hydrolysis 2 hours under 52.5-55 ℃ the condition, boils the 10min enzyme that goes out; Centrifugal each enzymolysis liquid, getting the supernatant molecular cut off is the milipore filter ultrafiltration of 1kDa, trapped fluid carries out spray-drying, obtains the oyster zymolyte; Wherein said combined protein enzyme is neutral proteinase and composite flavor enzyme.Preferably 55 ℃ of temperature, pH6.0, neutral proteinase and composite flavor enzyme that to add two kinds of enzyme amounts simultaneously be the 6000U/g substrate protein.
One of application of above-mentioned oyster zymolyte, removing DPPH, OH, ultra-oxygen anion free radical effect and reducing power thereof with it is foundation, measure in conjunction with the mouse liver even slurry detection architecture, proved that the oyster zymolyte has stronger antioxidation activity in vitro, can prepare food, food ingredient or health products with antioxidation activity.
Two of the application of above-mentioned oyster zymolyte, through the evaluation that delays senility, measuring three aspects with existence test, lipid peroxide assay and the antioxidase activity of fruit bat is evaluation index, has proved conclusively the oyster zymolyte and has improved average life span and the maximum life span of fruit bat; Improve SOD activity in the mouse body, reduce the content of MDA in the mouse body, have function in delaying senility, can prepare nutraceutical with delaying senility function.
Beneficial effect of the present invention: ostreae testa pulverata is behind combined protein enzyme enzymolysis, and its peptide yield and antioxidation activity all are improved, and possesses tangible function in delaying senility.
The present invention has advantages such as raw material sources are extensive, cost of manufacture is low, preparation technology is simple, efficient is high, pollution-free, meets the large-scale production requirement, implements easily.Made oyster zymolyte both can be used as that to have the nutraceutical that delays senility directly edible, also can be used as food ingredient or health products.
Description of drawings
Fig. 1 is a process chart of the present invention.
The specific embodiment
Below in conjunction with embodiment the present invention is further elaborated:
The protease that uses among the present invention comprises: neutral proteinase (enzyme activity is 16.0 ten thousand U/g), alkali protease (enzyme activity is 154.64 ten thousand U/mL), composite flavor enzyme (enzyme activity is 6.9 ten thousand U/g) can enzyme preparation Co., Ltd of section provide by the Wuxi is outstanding; Trypsase (enzyme activity is 26.26 ten thousand U/g), papain (enzyme activity is 4.96 ten thousand U/g) and pepsin (enzyme activity is 1.6 ten thousand U/g) are provided by Shanghai chemical reagents corporation.
Comparative Examples 1
The preparation of oyster neutral protease enzymolysis thing
Accurately take by weighing dry ostreae testa pulverata, adding water, to be made into the albumen quality mark be 2% the aqueous solution, puts into reactor, and 55 ℃ of water-baths were soaked 30 minutes, add neutral proteinase according to the enzyme-to-substrate albumen quality than amount, with phosphate buffer regulator solution pH to 7.0 for 8000U/g; Placed 55 ℃ of following enzymolysis of thermostat water bath 2 hours, 10 minutes enzymes that go out are boiled in the zymolyte heating, centrifugal 15 minutes of 6000r/min, and supernatant milipore filter ultrafiltration, the desalination of molecular cut off 1kDa, trapped fluid concentrates, spray-drying, obtains the oyster zymolyte.The peptide yield of hydrolysis ostreae testa pulverata is 46.8%, and the hydroxy radical clearance rate is 90.9%.
Comparative Examples 2
The preparation of oyster composite flavor enzyme zymolyte
Accurately take by weighing dry ostreae testa pulverata, adding water, to be made into the albumen quality mark be 2% the aqueous solution, puts into reactor, and 50 ℃ of water-baths were soaked 30 minutes, add the composite flavor enzyme according to the enzyme-to-substrate albumen quality than amount, with phosphate buffer regulator solution pH to 6.0 for 8000U/g; Placed 50 ℃ of following enzymolysis of thermostat water bath 2 hours, 10 minutes enzymes that go out are boiled in the enzymolysis liquid heating, centrifugal 15 minutes of 6000r/min, and supernatant milipore filter ultrafiltration, the desalination of molecular cut off 1kDa, trapped fluid concentrates, spray-drying, obtains the oyster zymolyte.The peptide yield of hydrolysis ostreae testa pulverata is 42.2%, and the hydroxy radical clearance rate is 88.6%.
Embodiment 3
The preparation of oyster combination zymolyte
Accurately take by weighing dry ostreae testa pulverata, adding water, to be made into albumen quality concentration be 2% aqueous solution, and 55 ℃ of water-baths were soaked 30 minutes, press earlier the enzyme-to-substrate albumen quality than being that 8000U/g adds neutral proteinase, regulate pH 7.0 with phosphate buffer, place 55 ℃ of following enzymolysis hydrolysis 120min of thermostat water bath; The composite flavor enzyme that adds 8000U/g again is at 50 ℃ of temperature, pH 6.0, hydrolysis 120min.10 minutes enzymes that go out are boiled in the zymolyte heating, centrifugal 15 minutes of 6000r/min, and supernatant milipore filter ultrafiltration, the desalination of molecular cut off 1kDa, trapped fluid concentrates, spray-drying, obtains the oyster zymolyte.The peptide yield of hydrolysis ostreae testa pulverata is 53.6%, and the hydroxy radical clearance rate is 98.2%.
Embodiment 4
The preparation of oyster combination zymolyte
Accurately take by weighing dry ostreae testa pulverata, adding water, to be made into albumen quality concentration be 2% aqueous solution, and 55 ℃ of water-baths were soaked 30 minutes, press earlier the enzyme-to-substrate albumen quality than being that 8000U/g adds the composite flavor enzyme, regulate pH 6.0 with phosphate buffer, place 50 ℃ of following enzymolysis hydrolysis 120min of thermostat water bath; The neutral proteinase that adds 8000U/g again is at 55 ℃ of temperature, pH 7.0, hydrolysis 120min.10 minutes enzymes that go out are boiled in the zymolyte heating, centrifugal 15 minutes of 6000r/min, and supernatant milipore filter ultrafiltration, the desalination of molecular cut off 1kDa, trapped fluid concentrates, spray-drying, obtains the oyster zymolyte.The peptide yield of hydrolysis ostreae testa pulverata is 49.1%, and the hydroxy radical clearance rate is 98.4%.
Embodiment 5
The preparation of oyster combination zymolyte
Accurately take by weighing dry ostreae testa pulverata, add water and be made into albumen quality concentration 2% aqueous solution, put into reactor, 52.5 ℃ water-bath was soaked 30 minutes, press every kind of enzyme-to-substrate albumen quality than being the enzyme concentration of 6000U/g substrate protein, add neutral proteinase and composite flavor enzyme simultaneously, with phosphate buffer regulator solution pH to 6.0; Placed 52.5 ℃ of following enzymolysis of thermostat water bath 2 hours, zymolyte boils the enzyme that went out in 10 minutes lives, centrifugal 15 minutes of 6000r/min, and supernatant milipore filter ultrafiltration, the desalination of molecular cut off 1kDa, trapped fluid concentrates, spray-drying, obtains the oyster zymolyte.The peptide yield of hydrolysis ostreae testa pulverata is 92.7%, and the hydroxy radical clearance rate is 99.8%.
Embodiment 6
The preparation of oyster combination zymolyte
Accurately take by weighing dry ostreae testa pulverata, add water and be made into concentration 2% aqueous solution, put into reactor, 52.5 ℃ water-bath was soaked 30 minutes, press every kind of enzyme-to-substrate albumen quality than being the enzyme concentration of 6000U/g substrate protein, add neutral proteinase and composite flavor enzyme simultaneously, with phosphate buffer regulator solution pH to 7.0; Placed 52.5 ℃ of following enzymolysis of thermostat water bath 2 hours, zymolyte boils the enzyme that went out in 10 minutes lives, centrifugal 15 minutes of 6000r/min, and supernatant milipore filter ultrafiltration, the desalination of molecular cut off 1kDa, trapped fluid concentrates, spray-drying, obtains the oyster zymolyte.The peptide yield of hydrolysis ostreae testa pulverata is 90.3%, and the hydroxy radical clearance rate is 91.8%.
The zymolyte activity test is (is example to measure the material for preparing among the embodiment 5) for example.
Test 1: remove DPPH free radical (DPPH) test
Get embodiment 5 preparation-obtained oyster zymolytes, compound concentration is the solution of 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 6.0mg/mL, and the DPPH solution concentration is 0.04g/L, and solvent is an absolute ethyl alcohol, and the detection wavelength is 517nm.The clearance rate computing formula is:
K＝[1-(A
i-A
j)/A
0]×100
In the formula: K is the clearance rate to the DPPH free radical, %; A
0The light absorption value that adds the 2mL absolute ethyl alcohol for the DPPH ethanol solution of 2mL; A
iThe light absorption value that adds 2mL protein hydrolysate solution for the DPPH ethanol solution of 2mL; A
jThe light absorption value that adds 2mL protein hydrolysate solution for the 2mL absolute ethyl alcohol.Each sample replication is averaged for three times.
By result of the test (seeing Table 1) as can be known, when oyster zymolyte concentration was 6.0mg/mL, its clearance rate to DPPH had reached 92.3%, illustrated that zymolyte has stronger removing ability to the DPPH free radical.
Test 2: the test of removing hydroxy radical (OH)
Get embodiment 5 preparation-obtained oyster zymolytes, compound concentration is respectively 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 6.0mg/mL.Respectively get the hydrating solution of the above-mentioned concentration of 2mL, add the FeSO of 2mL 6mmol/L successively
4, 2mL 6mmol/L H
2O
2, leave standstill 10min behind the mixing, add 2mL 6mmol/L salicylic acid again, mixing leaves standstill 30min, surveys its light absorption value at the 510nm place and is designated as A
i, the light absorption value when replacing salicylic acid with distilled water is designated as A
jThe blank group replaces protein hydrolysate solution with distilled water, and light absorption value is designated as A
0Calculate the clearance rate K of protein hydrolysate according to following formula to hydroxy radical:
K＝[1-(A
i-A
j)/A
0]×100
As shown in Table 1, the oyster zymolyte has the ability of stronger removing hydroxy radical, and its maximal clearance to the OH free radical can reach 95.4%.
Test 3: remove superoxide anion (O
2 -) test
Get embodiment 5 preparation-obtained oyster zymolytes, compound concentration is respectively the sample solution of 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 6.0mg/mL.The Tris-HCl cushioning liquid (pH8.2) of getting 4.50ml concentration and be 0.1mol/L is in test tube, preheating 20min in 25 ℃ of water-baths, add the 1ml testing sample solution, 0.5mL concentration is the pyrogallol solution of 2.5mmol/L, behind the mixing, in 25 ℃ of water-baths, behind the accurate response 4min, add the HCl solution 0.1mL cessation reaction of 10mol/L immediately, with the Tris-HCl buffer solution zeroing of pH 8.2, under wavelength 325nm, measure light absorption value.Control group replaces pyrogallol solution with 0.5mL distilled water, sets up the reagent blank pipe simultaneously.The clearance rate computing formula is:
In the formula: A
0The light absorption value of-blank; The light absorption value of Ai-sample
The background light absorption value of the sample when Aj-distilled water replaces pyrogallol
Result's (seeing Table 1): the oyster zymolyte has the ability of stronger removing superoxide anion, and its maximal clearance has reached 72.3%.
Table 1 oyster zymolyte is to the removing ability of various free radicals
Test 4: reducing power test
Get embodiment 5 preparation-obtained oyster zymolytes, compound concentration is respectively the sample solution of 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 6.0mg/mL.The pre-temperature of oyster sample solution of each concentration that the FRAP working solution of the fresh configuration of 2.5ml is added 1ml reads absorbance to 37 ℃ in 593nm.Light absorption value substitution calibration curve equation is tried to achieve corresponding Fe
2+Concentration.
The TAC measurement result of oyster enzymolysis liquid sees Table 2.Enzymolysis liquid is along with the increase of concentration, Fe
2+Concentration big more, the expression Fe
3+Be reduced to Fe
2+Ability strong more, show that the oyster zymolyte has stronger reduction.
Test 5: the lipid peroxidation inhibitory action test in the mouse liver even slurry system
Get embodiment 5 preparation-obtained oyster zymolytes, compound concentration is respectively the sample solution of 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 6.0mg/mL.The oyster sample liquid of 0.1ml LH, 0.2ml, the H of 0.1ml 10mmol/L get in the liver tissue homogenate of preparation 10% (w/v)
2O
2Solution and 0.1ml Tris-HCl buffer solution (pH7.4) mix, behind 37 ℃ of placement 1h, add the thiobarbituricacid (TBA) of 1ml 0.67% (w/v) and the trichloroacetic acid (TCA) of 1ml 10%, and in 100 ℃ of water-baths, boiled 15 minutes, put ice bath then and cool off centrifugal (6000rpm after 10 minutes, 10min), get supernatant and survey light absorption value in the 532nm place, calculate the lipid peroxidation inhibiting rate.Blank group replaces sample with the Tris-HCl buffer solution.All samples is done three repetitions.
Lipid peroxidation inhibiting rate (%)=(A
Sample-A
Blank) * 100/A
Blank
H when as can be seen from Table 2, the oyster zymolyte is to LH system incubation
2O
2The generation of bringing out lipid peroxidation has certain inhibitory action, when the concentration of 6.0mg/ml, zymolyte to the lipid peroxidation inhibiting rate up to 40%.
The reduction of table 2 oyster zymolyte and lipid peroxidation inhibitory action
Through above-mentioned evidence, have significant anti-oxidation efficacy according to method provided by the invention than the oyster zymolyte that conventional method obtains, can be used to prepare corresponding product, be applied to aspects such as food, health products.
Test 6: drosophila survival test
(1) chooses female, the male drosophila of the not mating of hatching in the 10h, use etherization, be divided into control group, oyster zymolyte interpolation high dose group (6mg/mL) and oyster zymolyte at random by sex then and add low dose group (1mg/mL), each 100 of every group of male and female, place temperature (25 ± 1) ℃, cultivate in the incubator that the illumination in 12 hours of humidity 55-65% replace.Every pipe is put the 2ml culture medium, seals with double gauze.
(2) the last fortnight is all fed basal medium, and each test group begins to add the oyster zymolyte after 14 days.
(3) dead fly number is regularly observed and write down to every morning, changed a subculture in 4 days, observes till the extremely complete death of fruit bat.Reject the fruit bat number that flies away accidentally in improper former cause death and the operating process in the experimentation.
(4) statistics is respectively organized fruit bat average life span and MaLS at last.
Table 3 oyster zymolyte is to the influence of life span of drosophila melanogaster (x ± s)
As can be seen from Table 3: average life span and maximum life span that the oyster zymolyte can the significant prolongation fruit bat.
Test 7: to the influence test of murine liver tissue MDA and SOD
Set up D-galactolipin mouse aging model, set up sampling test group, model group and control group at random, test group is irritated stomach oyster zymolyte, and (6mg/ml, 1mg/ml), 8 weeks back preparation, 10% LH carries out MDA content and SOD determination of activity in the hepatic tissue.
Table 4 is respectively organized SOD in Mice, MDA measurement result (x ± s)
As can be seen from Table 4, add the oyster zymolyte SOD vigor in the mouse body that can significantly raise, reduce the content of MDA in the mouse body.
Through above-mentioned evidence, oyster zymolyte prepared according to the methods of the invention has significant function of delaying senility, can be used to prepare corresponding product, is applied in food and the health products.
Claims (4)
1. the preparation method of oyster zymolyte, it is characterized in that adopting the combination enzymolysis process, may further comprise the steps: being made into the albumen quality mark with ostreae testa pulverata in water is 2% the aqueous solution, 52.5 ℃ of-55 ℃ of water-baths were soaked 30 minutes in the reactor, then successively or add the combined protein enzyme simultaneously and be hydrolyzed; The enzyme concentration of each protease is the 6000-8000U/g substrate protein, regulates pH 6.0-7.0 with phosphate buffer, and reaction temperature is hydrolysis 2 hours under 52.5-55 ℃ the condition, boils the 10min enzyme that goes out; Centrifugal each enzymolysis liquid, getting the supernatant molecular cut off is the milipore filter ultrafiltration of 1kDa, trapped fluid carries out spray-drying, obtains the oyster zymolyte; Wherein said combined protein enzyme is neutral proteinase and composite flavor enzyme.
2. the preparation method of the described oyster zymolyte of claim 1 is characterized in that 55 ℃ of temperature, pH6.0, and neutral proteinase and composite flavor enzyme that to add two kinds of enzyme amounts simultaneously be the 6000U/g substrate protein carry out enzymolysis.
3. the application of claim 1 or 2 described oyster zymolytes can prepare food, food ingredient or health products with antioxidation activity.
4. the application of claim 1 or 2 described oyster zymolytes can prepare and has nutraceutical or the health products that delay senility.
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| CN104878061A (en) * | 2015-05-15 | 2015-09-02 | 海南大学 | Double-enzyme preparation method of oyster meat nutrient solution with antioxidation function |
| CN105200105A (en) * | 2015-09-10 | 2015-12-30 | 烟台源力德海洋生物有限公司 | Preparation method of oyster protein liver protecting peptide preparation |
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