CN1137269C - Process for preparing extrac of small marine fish - Google Patents
Process for preparing extrac of small marine fish Download PDFInfo
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- CN1137269C CN1137269C CNB001308238A CN00130823A CN1137269C CN 1137269 C CN1137269 C CN 1137269C CN B001308238 A CNB001308238 A CN B001308238A CN 00130823 A CN00130823 A CN 00130823A CN 1137269 C CN1137269 C CN 1137269C
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Abstract
The present invention provides a preparation method for extracting polypeptide mixture products by using small marine rough fish as raw material, which comprises the technological steps of proteinase enzymolysis, ultrafiltration, ion exchange separation, elution, concentration, precipitation, mixing, etc. The content of the total protein of the obtained extraction products is larger than 75%, wherein the content of free amino acid is larger than 15%; the molecular weight of the contained polypeptide mixture is smaller than 50kDa; the content of the polypeptide is larger than 50%. The polypeptide mixture prepared by the method can be used for nutrient health care products and medicines for treating anemia. The present invention can realize scientific deep processing of small marine rough fish having the advantages of large resources and low cost and is suitable for industrial production, and the economic value of the products is high.
Description
Technical field
The present invention relates to a kind of method of from ocean fish, extracting the polypeptide mixture goods.
Background technology
Small fish of marine products has a huge resource of catching in that China is coastal, at present to the utilization of this resource be mostly as the low price fish in the market direct marketing or directly become fish meal as feed ingredient as feed or roughing, its resource value is not fully utilized.
Summary of the invention
The objective of the invention is to propose a kind of big suitability for industrialized production that is suitable for, from small fish of marine products, extract the preparation method of polypeptide mixture goods.Realization improves its utilization of resources greatly and is worth the deep processing and utilization of small fish of marine products with huge resource.
The present invention with the small fish of marine products be preparation method that raw material extracts the polypeptide mixture goods comprise protease hydrolyzed, ultrafiltration, ion-exchange separate and concentrate, processing step such as precipitation, specifically:
The first step: enzymolysis
Fresh small fish of marine products is clean with tap water, deboner is got meat, pulverizes the adult fish gruel then.Get a fish gruel, add the deionized water of 1～5 times of weight in proportion, stirring is warming up to 50～70 ℃, weighs 0.5～3.0% ratio in fish again and adds proteolytic enzyme, is incubated to be warming up to 90～100 ℃ and keep some minutes after 1～5 hour rapidly.
Second step: ultrafiltration
The mixture that above-mentioned reaction finishes is centrifugal, and extracting centrifugal liquid is used the press filtration of middling speed filter paper again, collects filtrate, is the ultra-filtration membrane separation filtration of 10kDa～50kDa with the intercepting molecular weight, collects its filtration fraction.
The 3rd step: ion-exchange
Among the liquid of above-mentioned filtration fraction, add mixed phosphate, regulator solution is the buffer system of pH6～8, phosphatic concentration is 0.01～0.1M in the solution, dosage by 1.5～2.5 kilograms of every liter of liquid adds anionite-exchange resin, under room temperature, intermittently stirred 0.5～3 hour, centrifugal then, get centrifugate A.The dosage of pressing 300～500 milliliters of per kilogram resins again adds the phosphate buffer soln of 0.01～0.1M pH6～8, and is centrifugal after 0.5～1 hour with the mixed with resin stirring, gets centrifugate B.
The 4th step: concentrating and precipitating
A is mixed with B, and vacuum decompression is concentrated into 1/5～1/7 of original volume.Concentrated solution is cooled to 5～10 ℃, adds the cold acetone of 3～5 times of concentrated solution volumes again, stir fast, left standstill then 30～120 minutes, then in 4000 rev/mins centrifugal, taking precipitate is dry under vacuum-10 ℃～30 ℃ temperature, yellow powder powder solid I.
The 5th step: wash-out
The dosage of pressing 0.5～1.0 liter of per kilogram resin in above-mentioned resin after centrifugal adds elutriant, contains the NaCl of 0.1～0.4M in this elutriant, and adjust pH is 2～7, makes it have certain ionic strength and suitable pH value.Slowly stirred 1～5 hour under the room temperature, centrifugal then, get centrifugate C.Use dosage to add above-mentioned elutriant again by 300～500 milliliters of per kilogram resins, centrifugal after 0.5～1 hour with the mixed with resin stirring, get centrifugate D.
The 6th step: concentrating and precipitating
C and D are mixed, and vacuum decompression is concentrated into 1/8～1/10 of original volume.Concentrated solution is cooled to 5～10 ℃, removes by filter the salt crystal of being separated out, filtrate continuation is concentrated into 1/12～1/15 of original volume, is cooled to 5～10 ℃ and remove by filter the salt crystal of being separated out again, presses V then
2: V
1=3～5 volume adds cold acetone, left standstill 30～120 minutes behind the stirring and evenly mixing fast, then in 4000 rev/mins centrifugal, get be deposited under the vacuum-10 ℃～30 ℃ temperature dry, yellow powder powder solid II.
The 7th step: mix
Solid I and solid II are mixed promptly.
The present invention is extract obtained to be yellow powder powder solid, is dissolved in water and is the yellowish brown clear liquid.The total protein content of this product is greater than 75%, and wherein free aminoacid content is greater than 15%, and contained polypeptide mixture molecular weight is less than 50kDa, content of peptides greater than the polypeptide molecular weight more than 50%, 90% between 2k～8kDa.Product oral dosage every day 1～5g of the present invention can once take or part vic.Experimental results show that the effect that the polypeptide mixture of this method preparation has significant rising body oxyphorase and erythrocyte quantity.Can be used as nutritive health-care food and treatment for anemia medicine.The present invention can realize the science deep processing to resource small fish of marine products big, with low cost, and product economy is worth high, is suitable for suitability for industrialized production.
Embodiment
Embodiment one
(1) fresh small fish of marine products with tap water clean, deboner gets meat, pulverizes the adult fish gruel then.Get a fish gruel, add the deionized water of 1 times of weight, stirring is warming up to 50 ℃, weighs the papoid of ratio adding vigor 80～900,000 units of 0.5% again by fish, is incubated to be warming up to 90 ℃ and keep some minutes after 5 hours rapidly.
(2) mixture that above-mentioned reaction is finished is centrifugal, and extracting centrifugal liquid is used the press filtration of middling speed filter paper again, collects filtrate, is the ultra-filtration membrane separation filtration of 50kDa with the intercepting molecular weight, collects the filtration fraction of molecular weight below 50kDa.
(3) among whenever going up the liquid of stating ultra filtration, add 0.44 gram Na
2HPO
412H
2O and 1.37 gram NaH
2PO
42H
2O, regulator solution pH are 6, phosphatic concentration is 0.01M in the solution, and the dosage by 1.5 kilograms of every liter of liquid adds anionite-exchange resin again, intermittently stirs 0.5 hour under room temperature, and is centrifugal then, centrifugate A.The dosage of pressing 300 milliliters of per kilogram resins again adds the phosphate buffer soln of 0.01M pH6, and is centrifugal after 0.5 hour with the mixed with resin stirring, gets centrifugate B.
(4) A is mixed with B, vacuum decompression is concentrated into 1/5 of original volume.Concentrated solution is cooled to 5 ℃, adds the cold acetone of 3 times of volumes, stir fast, left standstill then 30 minutes, then in 4000 rev/mins centrifugal, taking precipitate is drying to obtain yellow powder powder solid I under 25 ℃ of temperature of vacuum.
(5) dosage of pressing 0.5 liter of per kilogram resin in above-mentioned resin after centrifugal adds elutriant, the NaCl of 0.2M concentration and the HAC of 0.04M concentration in containing in this elutriant.Slowly stirred 1 hour under the room temperature, centrifugal then, get centrifugate C.Use dosage to add above-mentioned elutriant again by 300 milliliters of per kilogram resins, centrifugal after 0.5 hour with the mixed with resin stirring, get centrifugate D.
(6) C and D are mixed, vacuum decompression is concentrated into 1/8 of original volume.Concentrated solution is cooled to 5 ℃, remove by filter the salt crystal of being separated out, filtrate continuation is concentrated into 1/12 of original volume, be cooled to 5 ℃ and remove by filter the salt crystal of being separated out again, the cold acetone that adds 3 times of volumes left standstill 30 minutes behind the stirring and evenly mixing fast, and is centrifugal in 4000 rev/mins then, get and be deposited in drying under 25 ℃ of temperature of vacuum, get yellow powder powder solid II.
(7) solid I and solid II are mixed promptly.
Embodiment two
(1) fresh small fish of marine products with tap water clean, deboner gets meat, pulverizes the adult fish gruel then.Get a fish gruel, add the deionized water of 3 times of weight, stirring is warming up to 60 ℃, weighs the papoid of ratio adding vigor 80～900,000 units of 1.5% again by fish, is incubated to be warming up to 90～100 ℃ and keep some minutes after 3 hours rapidly.
(2) mixture that above-mentioned reaction is finished is centrifugal, and extracting centrifugal liquid is used the press filtration of middling speed filter paper again, collects filtrate, is the ultra-filtration membrane separation filtration of 40kDa with the intercepting molecular weight, collects filtration fraction.
(3) in whenever going up the liquid of stating ultra filtration, add 10.92 gram Na
2HPO
412H
2O and 3.04 gram NaH
2PO
42H
2O, regulator solution pH are 7, phosphatic concentration is 0.05M in the solution, and the dosage by 2.0 kilograms of every liter of liquid adds anionite-exchange resin again, intermittently stirs 1.5 hours under room temperature, and is centrifugal then, centrifugate A.The dosage of pressing 400 milliliters of per kilogram resins again adds the phosphate buffer soln of 0.05M pH7, and is centrifugal after 45 minutes with the mixed with resin stirring, gets centrifugate B.
(4) A is mixed with B, vacuum decompression is concentrated into 1/6 of original volume.Concentrated solution is cooled to 8 ℃, adds the cold acetone of 4 times of volumes, left standstill 90 minutes after stirring fast, then in 4000 rev/mins centrifugal, taking precipitate is drying to obtain yellow powder powder solid I under 10 ℃ of temperature of vacuum.
(5) dosage of pressing 0.75 liter of per kilogram resin in above-mentioned resin after centrifugal adds elutriant, contains the NaCl of 0.4M concentration and the HAC of 0.08M concentration in this elutriant.Slowly stirred 3 hours under the room temperature, centrifugal then, get centrifugate C.Use dosage to add above-mentioned elutriant again by 400 milliliters of per kilogram resins, centrifugal after 45 minutes with the mixed with resin stirring, get centrifugate D.
(6) C and D are mixed, vacuum decompression is concentrated into 1/9 of original volume.Concentrated solution is cooled to 10 ℃, remove by filter the salt crystal of being separated out, filtrate continuation is concentrated into 1/14 of original volume, be cooled to 10 ℃ and remove by filter the salt crystal of being separated out again, the cold acetone that adds 4 times of volumes left standstill 90 minutes behind the stirring and evenly mixing fast, and is centrifugal in 4000 rev/mins then, get and be deposited in drying under 10 ℃ of temperature of vacuum, get yellow powder powder solid II.
(7) solid I and solid II are mixed promptly.
Embodiment three
(1) fresh small fish of marine products with tap water clean, deboner gets meat, pulverizes the adult fish gruel then.Get a fish gruel, add the deionized water of 5 times of weight in proportion, stirring is warming up to 70 ℃, weighs the papoid that 3.0% ratio adds vigor 80～900,000 units in fish again, is incubated to be warming up to 90～100 ℃ and keep some minutes after 1 hour rapidly.
(2) mixture that above-mentioned reaction is finished is centrifugal, and extracting centrifugal liquid is used the press filtration of middling speed filter paper again, collects filtrate, is the ultra-filtration membrane separation filtration of 10kDa with the intercepting molecular weight, collects the filtration fraction of molecular weight below 10kDa.
(3) add 33.92 gram Na in the filtered solution whenever going up to state
2HPO
412H
2O and 0.83 gram NaH
2PO
42H
2O, regulator solution pH are 8, phosphatic concentration is 0.1M in the solution, and the dosage by 2.5 kilograms of every liter of liquid adds anionite-exchange resin again, intermittently stirs 3 hours under room temperature, and is centrifugal then, centrifugate A.The dosage of pressing 500 milliliters of per kilogram resins again adds the phosphate buffer soln of 0.1M pH8, and is centrifugal after 1 hour with the mixed with resin stirring, emits centrifugate B.
(4) A is mixed with B, vacuum decompression is concentrated into 1/7 of original volume.Concentrated solution is cooled to 10 ℃, adds the cold acetone of 5 times of volumes, stir fast, left standstill then 120 minutes, then in 4000 rev/mins centrifugal, taking precipitate is drying to obtain yellow powder powder solid I under vacuum-10 ℃ temperature.
(5) dosage of pressing 1.0 liters of per kilogram resins in above-mentioned resin after centrifugal adds elutriant, contains the NaCl of 0.3M and 0.05M pH value in this elutriant and be 7 phosphate buffer soln.Slowly stirred 5 hours under the room temperature, centrifugal then, get centrifugate C.Use dosage to add above-mentioned elutriant again by 500 milliliters of per kilogram resins, centrifugal after 1 hour with the mixed with resin stirring, get centrifugate D.
(6) C and D are mixed, vacuum decompression is concentrated into 1/10 of original volume.Concentrated solution is cooled to 8 ℃, remove by filter the salt crystal of being separated out, filtrate continuation is concentrated into 1/15 of original volume, be cooled to 8 ℃ and remove by filter the salt crystal of being separated out again, the cold acetone that adds 5 times of volumes left standstill 120 minutes behind the stirring and evenly mixing fast, and is centrifugal in 4000 rev/mins then, get and be deposited in drying under the vacuum-10 ℃ temperature, get yellow powder powder solid II.
(7) solid I and solid II are mixed promptly.
Claims (1)
1. the making method of an extrac of small marine fish is characterized in that adopting following steps to make:
(1) flesh of fish of fresh small fish of marine products is made the fish gruel, get a fish gruel, the deionized water that adds 1～5 times of weight in proportion, stir and be warming up to 50～70 ℃, weigh 0.5～3.0% ratio in the flesh of fish again and add papoid, be incubated and be warming up to 90～100 ℃ and keep some minutes after 1～5 hour rapidly;
(2) mixture that above-mentioned reaction is finished is centrifugal, and extracting centrifugal liquid is used the press filtration of middling speed filter paper again, collects filtrate, is the ultra-filtration membrane separation filtration of 10kDa～50kDa with the intercepting molecular weight, collects its filtration fraction;
(3) among the liquid of above-mentioned filtration fraction, add mixed phosphate, regulator solution is the buffer system of pH6～8, phosphatic concentration is 0.01～0.1M in the solution, dosage by 1.5～2.5 kilograms of every liter of liquid adds anionite-exchange resin, under room temperature, intermittently stirred 0.5～3 hour, centrifugal then, get centrifugate A, the dosage of pressing 300～500 milliliters of per kilogram resins again adds 0.01～0.1M, the phosphate buffer soln of pH6～8, centrifugal after 0.5～1 hour with the mixed with resin stirring, get centrifugate B;
(4) A is mixed with B, vacuum decompression is concentrated into 1/5～1/7 of original volume, concentrated solution is cooled to 5～10 ℃, the cold acetone that adds 3～5 times of concentrated solution volumes again, stir fast, leave standstill 30～120 minutes then, centrifugal, taking precipitate is dry under vacuum-10 ℃～30 ℃ temperature, yellow powder powder solid I;
(5) dosage by 0.5～1.0 liter of per kilogram resin adds the NaCl that contains 0.1～0.4M in above-mentioned resin after centrifugal, and adjust pH is 2～7 elutriant, slowly stirred 1～5 hour under the room temperature, centrifugal then, get centrifugate C, use dosage to add above-mentioned elutriant again by 300～500 milliliters of per kilogram resins, centrifugal after 0.5～1 hour with the mixed with resin stirring, get centrifugate D;
(6) C and D are mixed, vacuum decompression is concentrated into 1/8～1/10 of original volume, and concentrated solution is cooled to 5～10 ℃, remove by filter the salt crystal of being separated out, filtrate continuation is concentrated into 1/12～1/15 of original volume, is cooled to 5～10 ℃ and remove by filter the salt crystal of being separated out again, presses V then
2: V
1=3～5 volume adds cold acetone, left standstill 30～120 minutes behind the stirring and evenly mixing fast, then in 4000 rev/mins centrifugal, get be deposited under the vacuum-10 ℃～30 ℃ temperature dry, yellow powder powder solid II;
(7) solid I and solid II are mixed promptly.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001308238A CN1137269C (en) | 2000-12-01 | 2000-12-01 | Process for preparing extrac of small marine fish |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001308238A CN1137269C (en) | 2000-12-01 | 2000-12-01 | Process for preparing extrac of small marine fish |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1356393A CN1356393A (en) | 2002-07-03 |
| CN1137269C true CN1137269C (en) | 2004-02-04 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB001308238A Expired - Fee Related CN1137269C (en) | 2000-12-01 | 2000-12-01 | Process for preparing extrac of small marine fish |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2003236156A1 (en) * | 2003-04-24 | 2005-01-04 | Shin-Jen Shiao | Pharmaceutical compositions used for immune disease treatment and improvement |
| CN100386035C (en) * | 2003-09-19 | 2008-05-07 | 长沙巨鲸科技开发有限公司 | Production process of nutritious fish liquor and the hydrolase formula thereof |
| CN101326958B (en) * | 2008-07-22 | 2011-10-26 | 浙江大学 | Peptide feedstuff additive for regulating immunity function of sea water fish |
| CN102286582B (en) * | 2011-06-21 | 2013-07-24 | 华南理工大学 | Method for preparing memory-improving bioactive peptide from deep sea fish |
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2000
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