CN1057127C - All-hydrolytic method for fixed composite exopeptidase and bitter-removing method thereof - Google Patents
All-hydrolytic method for fixed composite exopeptidase and bitter-removing method thereof Download PDFInfo
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- CN1057127C CN1057127C CN93103782A CN93103782A CN1057127C CN 1057127 C CN1057127 C CN 1057127C CN 93103782 A CN93103782 A CN 93103782A CN 93103782 A CN93103782 A CN 93103782A CN 1057127 C CN1057127 C CN 1057127C
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- exopeptidase
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Abstract
The present invention relates to a method for making protein complete hydrolysate or no-bitterness protein hydrolysate. The method adopts a mixture of various kinds of exopeptidase comprising carboxyl peptidase and aminopeptidase or composite exopeptidase directly extracted from a pancreas or a kidney to make immobilized composite peptidase combined with other kinds of endopeptidase or immobilized endopeptidase, and then the immobilized composite peptidase is used for completely hydrolyzing protein or removing the bitterness of protein hydrolysate so that protein complete hydrolysate or no-bitterness protein hydrolysate can be made. The method has the advantages of simplicity, convenient operation and high efficiency. The method can be used for making food, health care products and medicine which contain the protein complete hydrolysate or the no-bitterness protein hydrolysate.
Description
Under technical field: the invention belongs to biotechnology enzyme engineering field, relate to that can be used for food, healthcare products and medicine manufacturing a kind of adopts immobilization compound exopeptidase complete hydrolysis protein or the partial hydrolystate and remove the method for its hydrolysis bitter taste of further degrading.
Prior art: amino acid and protein hydrolystate are the staple products that is widely used in food, health protection product, pharmacy and other light industry industry, and amino acid can be produced with fermentation method and protein complete hydrolysis method.Fermentation method is to produce one or more amino acid separately, and being unsuitable for nutrition is the manufacturing of multiple amino acids mixture of purpose.The protein complete hydrolysis is a kind of effective ways of production multiple amino acids mixture.The protein complete hydrolysis mainly is to adopt acid system or alkaline process at present, promptly under high temperature, the condition of high voltage, with acid or base catalysis proteolysis, produces free amino acid mixture.The efficient of acid-base method is higher, but when adopting acid as catalyzer, has in hydrolytic process that several seed amino acids are destroyed to be fallen, and can not obtain proteinic complete hydrolysis thing.Adopt alkali as catalyzer, have partial amino-acid to be broken down in hydrolytic process too, but also racemization can take place, a large amount of amino acid are lost its biological activity.Can not obtain real proteinic complete hydrolysis thing, certainly neither ideal protein complete hydrolysis method.
Hill, R.L.and Schmidt, W.R., J.Biol.Chem.237,389-395 (1961) reported and used papoid, and was used in combination carboxypeptidase, leucine aminopeptidase(LAP) and pepx etc. carry out complete hydrolysis to protein method.The composition and the theoretical value of the amino acid mixing liquid that obtains are basic identical.But carboxypeptidase and aminopeptidase all are to participate in reaction enzymes with the free state form can't reclaim in this method, can not reuse, and cost is higher, is difficult to industrial applications.
The protein portion hydrolyzate has important purposes in food and pharmaceutical industry, adopt the polypeptide product of enzymatic hydrolysis preparation the characteristic bitter taste often to be arranged and limited its purposes.Vmetsu, H.etal.J.Agric.Food Chem.31,50-56 (1983): Minagama, H..J, Food Sci.54.1225-1229 (1989) has reported for work respectively and has adopted carboxypeptidase and the further degraded of aminopeptidase to have the polypeptide of bitter taste, so that remove the method for protein hydrolystate bitter taste.Thibault, P.A.and Momsan, P.F.FR2625651 (1988) disclose a kind of method of utilizing carboxypeptidase on the yeast cells wall and bitter peptides reaction to eliminate bitter taste.These methods all are to utilize exopeptidase further to degrade to contain the polypeptide of bitter taste, destroy the bitter taste polypeptides structure to eliminate the bitter taste of protein hydrolystate.But exopeptidase comprises that the source of carboxypeptidase and aminopeptidase is very limited, and price is higher, though effectively, also can't drop into industrial applications.Because list is planted the narrow spectrum restriction of exopeptidase, the de-bittering effect of complete hydrolysis and hydrolysate is often not very good yet in addition.
Goal of the invention: the present invention utilizes immobilization compound exopeptidase (comprising carboxypeptidase and aminopeptidase) protein hydrolysate, or further degrade proteins partial hydrolystate, makes protein complete hydrolysis thing and also sloughs its bitter taste.The objective of the invention is to utilize the synergy between the compound exopeptidase, more effectively carry out the protein complete hydrolysis and slough its bitter taste.Owing to adopt enzyme immobilization technology, reduced use cost, present technique is expected to be used for the industrialization purpose.
The present invention includes following content and scheme:
A kind of compound exopeptidase of preparation comprises carboxypeptidase and aminopeptidase earlier.Method can adopt and extract single exopeptidase earlier, after mix by a certain percentage; Also can directly from animal, plant and microbial material, extract the mixture of multiple exopeptidase.In that above-mentioned exopeptidase mixture is immobilized on a kind of carrier, make the immobilization compound exopeptidase.Can be used for immobilized carrier sepharose, gac, silica gel, sintered glass, ion-exchange cellulose, ion exchange resin and other natural macromolecular material are arranged.Can be used for immobilized method absorption method, coupling method and crosslinking are arranged.
And then, be used in combination endopeptidase or immobilization endopeptidase and pepx with immobilization compound exopeptidase protein hydrolysate or protein portion hydrolyzate, can discharge total free aminoacids from the polypeptide end.Separated free amino acid, and circulation immobilization compound exopeptidase processing, but with regard to complete hydrolysis protein.
Handle the protein hydrolystate that bitter taste is arranged with the immobilization compound exopeptidase, can remove the bitter taste of protein hydrolystate.The reaction of immobilization compound exopeptidase can be adopted stirred reactor, also can adopt the post bed bioreactor.
Major advantage of the present invention and effect:
(1) adopt compound exopeptidase because between the exopeptidase and and endopeptidase between synergy, protein all-hydrolytic efficient is higher.
(2) owing to adopt compound exopeptidase, can directly utilize natural compound exopeptidase source, the preparation method of enzyme is simple, and the cost of enzyme reduces.
(3) adopt enzyme immobilization technology, enzyme can reuse, and running cost obviously reduces, and the proteinic industrialization of enzyme process complete hydrolysis becomes possibility.
(4) the immobilization compound exopeptidase is used for the debitterize of protein hydrolyzate, and effect is very good, and this will enlarge the purposes of protein hydrolyzate in food and pharmaceutical industry.
Embodiment 1
Fresh bovine or Pancreas Sus domestica 1Kg add water 4-10 liter, smash back its self-dissolving 8-24 hour that allow under 10-25 ℃ to pieces.The self-dissolving pancreatic secretion descended centrifugal 15-45 minute at 3000-10000 rev/min.Collect supernatant liquor, filter.Filtrate adds ammonium sulfate to 25-70% saturation ratio, collecting precipitation.To precipitate water-soluble, behind the dialysis desalting compound exopeptidase solution.
Shrimp shell chitin 50-100g adds 30-50%NaOH solution 200-600 milliliter, makes the 20-80% chitosan at 30-70 ℃ of following deacetylation.Add 0.5-2% formaldehyde solution again in above-mentioned chitosan, regulate the pH value to 4-8,0-4 ℃ was reacted 8-24 hour down.Unreacted formaldehyde is removed in washing, adds the 100-300 milliliter, and 0.5-2% compound exopeptidase solution allows its reaction 8-24 hour under 0-4 ℃.Remove unreacted compound exopeptidase then, make the immobilization compound exopeptidase.
Preparation casein solution (1-10%) is regulated the pH value to 6-8.5.With endopeptidase (trypsinase, Chymotrypsin, papoid, bromeline, ficin, aspergillus oryzae protease or subtilisin) or immobilization endopeptidase partial hydrolysis casein, make the casein partial hydrolystate.Also can prepare the casein partial hydrolystate with the mixture or the immobilization endopeptidase mixture caseinhydrolysate of above-mentioned endopeptidase.
With above-mentioned immobilization compound exopeptidase, the further above-mentioned casein partial hydrolystate of hydrolysis in column type reactor or stirring reactor.Reaction conditions is pH5-8,30 ℃-60 ℃ of temperature.Can make no bitter taste casein hydrolysate, separate with ion-exchanger again and remove the not peptide of complete hydrolysis, prepare casein all-hydrolytic thing.
Add sugar on above-mentioned no bitter taste casein hydrolysate or casein all-hydrolytic thing basis, inorganic salt and VITAMIN are made into healthcare products, food or medicine.
Embodiment 2
Fresh pig or Ren Bovis seu Bubali 200g add after 1000 ml waters smash to pieces, filter down at 0-4 ℃, and filtrate adds ammonium sulfate, collects the precipitation of gained between the 30-80% saturation ratio.The gained precipitation is water-soluble, and dialysis desalting gets compound exopeptidase solution.
All the other are identical with embodiment 1.
Embodiment 3
Carboxypeptidase A, protaminase, carboxypeptidase y and leucine aminopeptidase(LAP) are pressed 1: 0.5-1: 0.1-0.4: the 0.4-0.8 mixed is made into compound exopeptidase solution (0.5-2%) again.
All the other are identical with embodiment 1.
Embodiment 4
The compound exopeptidase solution manufacturing method is identical with embodiment 1,2,3
Part chitosan preparation method is with embodiment 1.50-100g part chitosan, add 100-300 milliliter compound exopeptidase solution (0.5-2%), regulate the pH value to 6-8, placed 8-24 hour down at 0-4 ℃, adding glutaraldehyde to final concn again is 0.5-2%, reacted 8-24 hour down at 0-4 ℃, unreacted enzyme and glutaraldehyde are removed in washing, make the immobilization compound exopeptidase.
All the other are identical with embodiment 1.
Embodiment 5
With chitin instead of part chitosan, do not remove the ethanoyl on the chitin, directly carry out the immobilization of compound exopeptidase, preparation immobilization compound exopeptidase with the method for embodiment 1 or embodiment 4.
All the other are identical with embodiment 1.
Embodiment 6
Direct usefulness removes the chitin behind the ethanoyl fully, and promptly chitin carries out the immobilization of compound exopeptidase.Process for fixation is identical with embodiment 1 or embodiment 4 described methods, preparation immobilization compound exopeptidase.
All the other are identical with embodiment 1.
Embodiment 7
Replace shrimp shell chitin with crab shell chitin, surplus all identical with embodiment 6.
Embodiment 8
Replace casein with soybean protein, all the other are identical with embodiment 1.
Claims (8)
1. immobilization compound exopeptidase protein all-hydrolytic and debittering method is characterized in that described method comprises:
1) fresh bovine or Pancreas Sus domestica 1Kg add water 4-10 liter, smash back its self-dissolving 8-24 hour that allow under 10-25 ℃ to pieces, and the self-dissolving pancreatic secretion descended centrifugal 15-45 minute at 3000-10000 rev/min, collected supernatant liquor, filtered; Filtrate adds ammonium sulfate to the 25-70% saturation ratio, collecting precipitation, will precipitate water-soluble, behind the dialysis desalting compound exopeptidase solution,
2) shrimp shell chitin 50-100g, add 30-50%NaOH solution 200-600 milliliter, make the 20-80% chitosan, in above-mentioned chitosan, add 0.5-2% formaldehyde solution again at 30-70 ℃ of following deacetylation, regulate the pH value to 4-8,0-4 ℃ was reacted 8-24 hour down; Unreacted formaldehyde is removed in washing, adds the 100-300 milliliter, and 0.5-2% compound exopeptidase solution allows its reaction 8-24 hour under 0-4 ℃, removes unreacted compound exopeptidase then, makes the immobilization compound exopeptidase,
3) preparation casein solution 1-10%, regulate the pH value to 6-8.5,, make the casein partial hydrolystate with endopeptidase or immobilization endopeptidase partial hydrolysis casein, also can prepare the casein partial hydrolystate with the mixture or the immobilization endopeptidase mixture caseinhydrolysate of above-mentioned endopeptidase
4) with above-mentioned immobilization compound exopeptidase, the further above-mentioned casein partial hydrolystate of hydrolysis in column type reactor or stirring reactor, reaction conditions is pH5-8,30 ℃-60 ℃ of temperature, can make no bitter taste casein hydrolysate, separate with ion-exchanger again and remove the not peptide of complete hydrolysis, prepare casein all-hydrolytic thing.
2. according to the method for claim 1; it is characterized in that; wherein the described compound exopeptidase solution of step 1) also can substitute pancreas with the kidney of fresh bovine or pig and prepare, and with the kidney 200g of fresh bovine or pig, adds after 1000 ml waters smash to pieces; filter down at 0-4 ℃; filtrate adds ammonium sulfate, collects the precipitation of gained between the 30-80% saturation ratio, and the gained precipitation is water-soluble; dialysis desalting gets compound exopeptidase solution.
3. according to the method for claim 1, it is characterized in that, wherein the described compound exopeptidase solution of step 1) also can use Carboxypeptidase A, protaminase, carboxypeptidase y and leucine aminopeptidase(LAP) by 1: 0.5-1: 0.1-0.4: the 0.4-0.8 mixed is made into compound exopeptidase solution 0.5-2% again.
4. according to the method for claim 1, it is characterized in that, wherein step 2) described immobilization compound exopeptidase solution also can prepare in order to lower section chitosan method; 50-100g part chitosan, add 100-300 milliliter compound exopeptidase solution 0.5-2%, regulate the pH value to 6-8, placed 8-24 hour down at 0-4 ℃, adding glutaraldehyde to final concn again is 0.5-2%, reacted 8-24 hour down at 0-4 ℃, unreacted enzyme and glutaraldehyde are removed in washing, make the immobilization compound exopeptidase.
5. according to each method of claim 1-4, it is characterized in that, can use chitin instead of part chitosan.
6. according to each method of claim 1-4, it is characterized in that, can be directly with the chitin that removes fully behind the ethanoyl, promptly chitin carries out the immobilization of compound exopeptidase in order to preparation immobilization compound exopeptidase.
7. according to each method of claim 1-4, it is characterized in that, can replace shrimp shell chitin with crab shell chitin.
8. according to the method for claim 1, it is characterized in that wherein step 3) also can prepare the protein portion hydrolyzate with soy-protein.
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| Application Number | Priority Date | Filing Date | Title |
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| CN93103782A CN1057127C (en) | 1993-04-23 | 1993-04-23 | All-hydrolytic method for fixed composite exopeptidase and bitter-removing method thereof |
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| CN93103782A CN1057127C (en) | 1993-04-23 | 1993-04-23 | All-hydrolytic method for fixed composite exopeptidase and bitter-removing method thereof |
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| CN1078495A CN1078495A (en) | 1993-11-17 |
| CN1057127C true CN1057127C (en) | 2000-10-04 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104046673A (en) * | 2013-03-14 | 2014-09-17 | 中国食品发酵工业研究院 | Industrial manufacturing method and use of CPPs-containing hypoallergenic casein peptide whole-powder |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK0946106T3 (en) * | 1996-12-23 | 2002-09-16 | Dsm Nv | Process for preparing a protein hydrolyzate |
| ATE414775T1 (en) * | 1997-05-16 | 2008-12-15 | Novozymes Inc | POLYPEPTIDES WITH PROLYLDIPEPTIDYLAMINOPEPTIDASE ACTIVITY AND NUCLEIC ACIDS CODING THEREFOR |
| US6800467B1 (en) * | 1997-05-16 | 2004-10-05 | Novozymes Biotech, Inc. | Polypeptides having aminopeptidase activity and nucleic acids encoding same |
| AU2008325032A1 (en) * | 2007-11-07 | 2009-05-14 | Mead Johnson Nutrition Company | Method for decreasing bitterness and improving taste of protein-free and hydrolyzed infant formulas |
| CN102379359B (en) * | 2010-08-30 | 2013-09-25 | 内蒙古伊利实业集团股份有限公司 | Casein enzymatic hydrolysis method and hydrolysis product |
| CN104352609A (en) * | 2014-10-16 | 2015-02-18 | 陕西天谷生物科技集团有限公司 | Preparation method of water-soluble immature bitter orange extract with bitterness removed |
| CN107058278B (en) * | 2017-04-11 | 2020-06-05 | 北京工商大学 | Preparation method of carboxypeptidase A immobilized enzyme |
| CN111132556A (en) | 2017-06-09 | 2020-05-08 | 诺维信公司 | Polypeptides, uses and methods for hydrolyzing proteins |
| CN112063676B (en) * | 2020-09-02 | 2021-11-12 | 杭州汉库医药科技有限公司 | Casein purification method and preparation method of iron protein succinate |
| CN113261643A (en) * | 2021-04-22 | 2021-08-17 | 诺利如一（安阳）生物科技有限公司 | Method for removing bitterness of soybean peptide |
| CN115669792B (en) * | 2021-07-30 | 2024-06-18 | 中国海洋大学 | Method for preparing debitterized low-molecular mucin by using sturgeon tendons |
| CN113584005B (en) * | 2021-08-27 | 2024-03-01 | 江南大学 | Preparation of aminopeptidase and application of aminopeptidase in protein debittering |
| CN116268174B (en) * | 2023-03-10 | 2024-04-12 | 华南理工大学 | Low-bitter casein zymolyte and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86108557A (en) * | 1986-12-20 | 1988-07-13 | 同济大学 | With proteinaceous waste material is material, enzyme method method of extracting protein hydrolystate and products thereof |
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- 1993-04-23 CN CN93103782A patent/CN1057127C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86108557A (en) * | 1986-12-20 | 1988-07-13 | 同济大学 | With proteinaceous waste material is material, enzyme method method of extracting protein hydrolystate and products thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104046673A (en) * | 2013-03-14 | 2014-09-17 | 中国食品发酵工业研究院 | Industrial manufacturing method and use of CPPs-containing hypoallergenic casein peptide whole-powder |
| CN104046673B (en) * | 2013-03-14 | 2020-07-21 | 中国食品发酵工业研究院 | Industrial manufacturing method and application of low-sensitization casein peptide whole powder containing CPPs |
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| CN1078495A (en) | 1993-11-17 |
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