CN106165819B - The method of phenylalanine in quick release rice protein - Google Patents
The method of phenylalanine in quick release rice protein Download PDFInfo
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- CN106165819B CN106165819B CN201610798071.XA CN201610798071A CN106165819B CN 106165819 B CN106165819 B CN 106165819B CN 201610798071 A CN201610798071 A CN 201610798071A CN 106165819 B CN106165819 B CN 106165819B
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- pronase
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- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 title claims abstract description 97
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 title claims abstract description 97
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 95
- 235000009566 rice Nutrition 0.000 title claims abstract description 95
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 67
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 61
- 240000007594 Oryza sativa Species 0.000 title 1
- 241000209094 Oryza Species 0.000 claims abstract description 94
- 108010059712 Pronase Proteins 0.000 claims abstract description 36
- 239000012460 protein solution Substances 0.000 claims abstract description 26
- 101000693530 Staphylococcus aureus Staphylokinase Proteins 0.000 claims abstract description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000006243 chemical reaction Methods 0.000 claims description 35
- 239000007788 liquid Substances 0.000 claims description 33
- 239000006228 supernatant Substances 0.000 claims description 23
- 102000004190 Enzymes Human genes 0.000 claims description 21
- 108090000790 Enzymes Proteins 0.000 claims description 21
- 108091005804 Peptidases Proteins 0.000 claims description 16
- 239000004365 Protease Substances 0.000 claims description 16
- 235000019419 proteases Nutrition 0.000 claims description 16
- 239000003643 water by type Substances 0.000 claims description 11
- 244000063299 Bacillus subtilis Species 0.000 claims description 3
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 3
- 241000187392 Streptomyces griseus Species 0.000 claims description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 1
- 238000002604 ultrasonography Methods 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 19
- 230000007071 enzymatic hydrolysis Effects 0.000 abstract description 14
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 abstract description 14
- 238000010298 pulverizing process Methods 0.000 abstract description 14
- 238000005457 optimization Methods 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 2
- 235000018102 proteins Nutrition 0.000 description 54
- 230000000694 effects Effects 0.000 description 29
- 229940088598 enzyme Drugs 0.000 description 18
- 102000035195 Peptidases Human genes 0.000 description 15
- 238000004321 preservation Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 108090000145 Bacillolysin Proteins 0.000 description 8
- 102000035092 Neutral proteases Human genes 0.000 description 8
- 108091005507 Neutral proteases Proteins 0.000 description 8
- 230000007062 hydrolysis Effects 0.000 description 8
- 238000006460 hydrolysis reaction Methods 0.000 description 8
- 238000005119 centrifugation Methods 0.000 description 7
- 239000000758 substrate Substances 0.000 description 5
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000001506 fluorescence spectroscopy Methods 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 239000005018 casein Substances 0.000 description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 3
- 235000021240 caseins Nutrition 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- VAJVDSVGBWFCLW-UHFFFAOYSA-N 3-Phenyl-1-propanol Chemical compound OCCCC1=CC=CC=C1 VAJVDSVGBWFCLW-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 108090000317 Chymotrypsin Proteins 0.000 description 2
- 201000011252 Phenylketonuria Diseases 0.000 description 2
- DYUQAZSOFZSPHD-UHFFFAOYSA-N Phenylpropyl alcohol Natural products CCC(O)C1=CC=CC=C1 DYUQAZSOFZSPHD-UHFFFAOYSA-N 0.000 description 2
- 229960002376 chymotrypsin Drugs 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 108010011485 Aspartame Proteins 0.000 description 1
- 102000000496 Carboxypeptidases A Human genes 0.000 description 1
- 108010080937 Carboxypeptidases A Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010068370 Glutens Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 208000036626 Mental retardation Diseases 0.000 description 1
- 108010069013 Phenylalanine Hydroxylase Proteins 0.000 description 1
- 102100038223 Phenylalanine-4-hydroxylase Human genes 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000000605 aspartame Substances 0.000 description 1
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
- 229960003438 aspartame Drugs 0.000 description 1
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- 235000008452 baby food Nutrition 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000023852 carbohydrate metabolic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 235000021312 gluten Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000001814 protein method Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The present invention provides a kind of method of phenylalanine in quick release rice protein, rice protein is configured to certain density rice protein solution with water, through supersonic cell pulverization process, then neutral proteinase and pronase stepwise discretization rice protein are utilized, with the phenylalanine in quick release rice protein.The present invention by carrying out multifactor optimization to enzymatic hydrolysis condition, finally establish it is a set of can in quick release rice protein phenylalanine method.This method is easy to operate, mild condition, is easy to industrialize phenylalanine in quick release rice protein, removes phenylalanine for distinct methods, and further separation of phenylalanine provides good technical support.Rice protein is handled using this method, 80% or more of the total phenylalanine content of free phenylalanine Zhan in rice protein.
Description
Technical field
The invention belongs to technical field of enzyme engineering, specifically, being related to phenylalanine in a kind of quick release rice protein
Method.
Background technique
Phenylalanine is a kind of essential amino acid, most of in vivo to aoxidize through phenylalanine hydroxylase catalytic action
At tyrosine, and important neurotransmitter and hormone are synthesized together with tyrosine, participate in body glycometabolism and fat metabolism.Together
When, phenylalanine is the primary raw material for producing new type of health type sweetener Aspartame.Phenylalanine cannot be by phenylketonuria
Patient's eubolism, and lead to the permanent damage of central nervous system.As long as being diagnosed and being given in time in neonatal period
Low-phenylalanine diet, can avoid infant Mental retardation.Food therapy is that treatment phenylketonuria is a kind of generally acknowledged, long-term hard
The method with remarkable result is held, i.e., phenylalanine intake is in control infant food to reach a kind of side that organism metabolism balances
Method.
The method of phenylalanine mainly has enzymatic hydrolysis absorption method in deproteination at present.Exist in enzymolysis process in research at present
The problems such as enzymolysis time is too long, enzymolysis process causes subsequent technique complicated be unsuitable for being mass produced.Cabrera-Padilla
Deng using phenylalanine in novel enzyme mebrane reactor deproteination, in enzymolysis process small molecular substance through hyperfiltration treatment, make anti-
It answers object and product to efficiently separate in system, reaction efficiency can be improved, reduce enzyme Competitive assays, but in the case, utilize
Chymotrypsin and carboxypeptidase-A enzymolysis lactoalbumin, enzymolysis time are still up to 18h；Zhou Zhiwei etc. is using a kind of purified crude
Microbial protease acts on casein, and enzymolysis time is controlled in 12h；Lopes etc. controls pH during protein extraction
9-12 needs desalting processing in the follow-up process, is unfavorable for actual industrial production.As it can be seen that establishing a kind of quick release phenylalanine
Method, select suitable pretreatment mode, the high enzyme class of efficiency of selection, the reaction condition in strict control enzymolysis process,
To in albumen phenylalanine quickly dissociate it is vital.
Summary of the invention
The object of the present invention is to provide a kind of methods of phenylalanine in quick release rice protein.
In order to achieve the object of the present invention, in quick release rice protein provided by the invention phenylalanine method, will be big
Rice gluten is configured to certain density rice protein solution with water, through supersonic cell pulverization process, then utilizes neutral protein
Enzyme and pronase stepwise discretization rice protein, with the phenylalanine in quick release rice protein.
The method specifically includes the following steps:
S1, rice protein is configured to certain density rice protein solution with water, after supersonic cell pulverization process,
Neutral proteinase is added to be digested, enzymolysis liquid I is obtained；
S2, pronase is added into enzymolysis liquid I, continues to digest, obtain enzymolysis liquid II；
S3, the protease in enzymolysis liquid II is inactivated, contains the free phenylalanine released in centrifugation gained supernatant.
In preceding method, the mass percentage concentration of the rice protein solution is 1-2%, preferably 1%.
In preceding method, the condition for carrying out supersonic cell pulverization process is as follows: power 200-400W, ultrasonic 4-6s,
Every 3-5s, amount to 4-6min.
Method above-mentioned, S1 concrete operations are as follows: rice protein is configured to certain density rice protein solution with water,
After supersonic cell pulverization process, neutral proteinase is added by the additional amount of every gram of rice protein 3200-6400U, in 40-60
DEG C reaction 1-5h, obtain enzymolysis liquid I.
It is preferred that the additional amount of neutral proteinase is 4000-5600U/g rice protein, more preferable 4800-5600U/g rice egg
It is white, most preferably 5600U/g rice protein；Preferable reaction temperature is 45-55 DEG C, more preferable 45-50 DEG C, most preferably 50 DEG C；It is preferred that
Reaction time is 2-4h, more preferable 2-3h, most preferably 2h.
Method above-mentioned, S2 concrete operations are as follows: adding into enzymolysis liquid I by the additional amount of every gram of rice protein 50-146U
Enter pronase, in 30-50 DEG C of reaction 1-5h, obtains enzymolysis liquid II.
It is preferred that the additional amount of pronase is 74-122U/g rice protein, more preferable 98U/g rice protein；It is preferred that anti-
Answering temperature is 35-45 DEG C, more preferable 35-40 DEG C, most preferably 37 DEG C；Preferred reaction time is 2-4h, more preferable 2h.
Method above-mentioned, S3 concrete operations are as follows: enzymolysis liquid II is placed into 10-20min in 90-95 DEG C of water bath with thermostatic control,
Inactivate protease, then 4000-4500r/min is centrifuged 20-30min and takes supernatant.
Preferably, in quick release rice protein of the present invention phenylalanine method the following steps are included:
S1, rice protein is configured to the rice protein solution that mass percentage concentration is 1% with water, through supersonic cell powder
After broken processing, neutral proteinase is added by the additional amount of every gram of rice protein 5600U and obtains enzymolysis liquid I in 50 DEG C of reaction 2h；
Carry out the condition of supersonic cell pulverization process are as follows: power 300W, ultrasonic 5s are spaced 4s, amount to 6min；
S2, the additional amount that every gram of rice protein 98U is pressed into enzymolysis liquid I are added pronase and obtain in 37 DEG C of reaction 2h
To enzymolysis liquid II；
S3, enzymolysis liquid II is placed into 10min in 90 DEG C of waters bath with thermostatic control, inactivates protease, then 4200r/min is centrifuged
20min takes supernatant.
Method above-mentioned further comprises the step of gained supernatant is dried.It is preferred that using freeze-drying.
Protein content >=75% in rice protein of the present invention, fat content≤1%, phenylalanine content >=
4.72%.
Neutral proteinase used in the present invention derives from bacillus subtilis, by the fermented generation of bacillus subtilis, such as
Through liquid deep layer fermenting, concentration extraction is refined and is made；Belong to single enzyme, no specific cleavage site.The neutral protein
Enzyme can be prepared by art methods, also can purchase commercial prod, such as purchased from Beijing Suo Laibao Science and Technology Ltd, commodity
Catalog number (Cat.No.): Z8030, enzyme activity are 6 × 104U/g。
Pronase used in the present invention derives from streptomyces griseus.Institute's pronase can be by art methods system
It is standby, it also can purchase commercial prod, such as purchased from Beijing Suo Laibao Science and Technology Ltd, cat. no: P8360, enzyme activity are
7.00×103U/g。
The present invention is to neutral proteinase activity is defined as: casein substrate is under given conditions through enzyme hydrolysis, per minute
Generation l μ g tyrosine is a unit of activity, is indicated with U.
The present invention is to pronase enzyme activity is defined as: casein substrate through enzyme hydrolysis, produces per minute under given conditions
Raw l μ g tyrosine is a unit of activity, is indicated with U.
The present invention by carrying out multifactor optimization to enzymatic hydrolysis condition, finally establish it is a set of being capable of quick release rice protein
The method of middle phenylalanine.This method is easy to operate, mild condition, is easy to industrialize phenylpropyl alcohol ammonia in quick release rice protein
Acid removes phenylalanine for distinct methods, and further separation of phenylalanine provides good technical support.Using this method pair
Rice protein is handled, 80% or more of the total phenylalanine content of free phenylalanine Zhan in rice protein.
Detailed description of the invention
Fig. 1, which is 1 neutral proteinase difference enzymolysis time of experimental example of the present invention, dissociates the influence of effect to phenylalanine；
Fig. 2, which is 2 neutral proteinase additional amount of experimental example of the present invention, dissociates the influence of effect to phenylalanine；
Fig. 3, which is 3 neutral protease enzymolysis temperature of experimental example of the present invention, dissociates the influence of effect to phenylalanine；
Fig. 4, which is 4 pronase enzymolysis time of experimental example of the present invention, dissociates the influence of effect to phenylalanine；
Fig. 5, which is 5 pronase additional amount of experimental example of the present invention, dissociates the influence of effect to phenylalanine；
Fig. 6, which is 6 pronase hydrolysis temperature of experimental example of the present invention, dissociates the influence of effect to phenylalanine.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
Neutral proteinase used in following embodiment is purchased from Beijing Suo Laibao Science and Technology Ltd, cat. no:
Z8030, enzyme activity are 6 × 104U/g；Pronase used is purchased from Beijing Suo Laibao Science and Technology Ltd, cat. no:
P8360, enzyme activity are 7.00 × 103U/g.Rice protein used, protein content >=75%, fat content≤1%, phenylpropyl alcohol
Histidine content 4.72%.Fluorescence spectrometry free phenylalanine content is used in following embodiment.
The method of phenylalanine in 1 quick release rice protein of embodiment
It the described method comprises the following steps:
(1) rice protein is configured to the rice protein solution that mass percentage concentration is 1% with water, through supersonic cell powder
After broken processing (power 300W, ultrasonic 5s are spaced 4s, amount to 6min), it is added by the additional amount of every gram of rice protein 5600U neutral
Protease obtains enzymolysis liquid I in 50 DEG C of reaction 2h；
(2) pronase is added by the additional amount of every gram of rice protein 98U into enzymolysis liquid I to obtain in 37 DEG C of reaction 2h
To enzymolysis liquid II；
(3) enzymolysis liquid II is placed into 10min in 90 DEG C of waters bath with thermostatic control, inactivates protease, then 4200r/min is centrifuged
20min contains the free phenylalanine largely released in the supernatant collected.Using fluorescence spectrometry supernatant middle reaches
From phenylalanine content, the 80.12% of the total phenylalanine content of free phenylalanine amount Zhan in rice protein.Then by the solution
Carry out freeze-drying preservation.
The method of phenylalanine in 2 quick release rice protein of embodiment
It the described method comprises the following steps:
(1) rice protein is configured to the rice protein solution that mass percentage concentration is 2% with water, through supersonic cell powder
After broken processing (power 400W, ultrasonic 6s are spaced 3s, amount to 4min), it is added by the additional amount of every gram of rice protein 6400U neutral
Protease obtains enzymolysis liquid I in 40 DEG C of reaction 1h；
(2) pronase is added by the additional amount of every gram of rice protein 146U into enzymolysis liquid I, in 30 DEG C of reaction 1h,
Obtain enzymolysis liquid II；
(3) enzymolysis liquid II is placed into 20min in 95 DEG C of waters bath with thermostatic control, inactivates protease, then 4500r/min is centrifuged
25min contains the free phenylalanine largely released in the supernatant collected.Using fluorescence spectrometry supernatant middle reaches
From phenylalanine content, the 66.52% of the total phenylalanine content of free phenylalanine amount Zhan in rice protein.Then by the solution
Carry out freeze-drying preservation.
The method of phenylalanine in 3 quick release rice protein of embodiment
It the described method comprises the following steps:
(1) rice protein is configured to the rice protein solution that mass percentage concentration is 1.5% with water, through supersonic cell
After pulverization process (power 200W, ultrasonic 4s are spaced 5s, amount to 5min), by the additional amount addition of every gram of rice protein 3200U
Property protease obtains enzymolysis liquid I in 60 DEG C of reaction 5h；
(2) pronase is added by the additional amount of every gram of rice protein 50U into enzymolysis liquid I to obtain in 50 DEG C of reaction 5h
To enzymolysis liquid II；
(3) enzymolysis liquid II is placed into 15min in 93 DEG C of waters bath with thermostatic control, inactivates protease, then 4000r/min is centrifuged
30min contains the free phenylalanine largely released in the supernatant collected.Using fluorescence spectrometry supernatant middle reaches
From phenylalanine content, the 65.76% of the total phenylalanine content of free phenylalanine amount Zhan in rice protein.Then by the solution
Carry out freeze-drying preservation.
The method of phenylalanine in 4 quick release rice protein of embodiment
Difference with embodiment 1, which is only that in step (1), is heated to 80 DEG C of water-baths for the rice protein solution that concentration is 1%
15min is shaken, instead of supersonic cell pulverization process.
Using the phenylalanine in above method release rice protein, the total phenylalanine of final free phenylalanine content Zhan
The 52.70% of content.
The method of phenylalanine in 5 quick release rice protein of embodiment
Difference with embodiment 1, which is only that in step (2), replaces pronase with chymotrypsin.
Using the phenylalanine in above method release rice protein, the total phenylalanine of final free phenylalanine content Zhan
The 61.35% of content.
The multifactor optimization of enzymatic hydrolysis condition:
The 1 neutral protease enzymolysis time of experimental example dissociates the influence of effect to phenylalanine
Step 1: rice protein is configured to the rice protein solution that mass percentage concentration is 1% with water, it is thin through ultrasonic wave
Born of the same parents' pulverization process (power 300W, ultrasonic 5s are spaced 4s, amount to 6min)；
Step 2: neutral proteinase (additional amount 5600U/g albumen) is added in rice protein solution after first step reaction,
Enzymatic hydrolysis 1h/2h/3h/4h/5h is carried out in 50 DEG C of thermostatic control oscillator vibration；
Step 3: second step takes out after reaction, it is put into 90 DEG C of waters bath with thermostatic control and keeps l0min, inactivate protease,
4200r/min centrifugation 20min takes supernatant；
Step 4: supernatant is the sufficiently free rice protein solution of phenylalanine, the freeze-dried preservation of the solution.
The neutral protease enzymolysis time dissociates the result is shown in Figure 1 of influential effect to phenylalanine.As seen from Figure 1: 1-
In 2h, free phenylalanine amount significantly increases, and in 2-5h, free phenylalanine amount is held essentially constant, this may be due to
2h or so, enzyme preparation have come into full contact with substrate, fully reacting, and phenylalanine is sufficiently free under the reaction conditions, therefore
Select best enzymolysis time of the 2h as pronase.
2 neutral proteinase additional amount of experimental example dissociates the influence of effect to phenylalanine
Step 1: rice protein is configured to the rice protein solution that mass percentage concentration is 1% with water, it is thin through ultrasonic wave
Born of the same parents' pulverization process (power 300W, ultrasonic 5s are spaced 4s, amount to 6min)；
Step 2: neutral proteinase (additional amount 3200/4000/4800/ is added in rice protein solution after first step reaction
5600/6400U/g albumen), enzymatic hydrolysis 2h is carried out in 50 DEG C of thermostatic control oscillator vibration；
Step 3: second step takes out after reaction, it is put into 90 DEG C of waters bath with thermostatic control and keeps l0min, inactivate protease,
4200r/min centrifugation 20min takes supernatant；
Step 4: supernatant is the sufficiently free rice protein solution of phenylalanine, the freeze-dried preservation of the solution.
Neutral proteinase additional amount is shown in Fig. 2 to the dissociate result of influential effect of phenylalanine.As seen from Figure 2: neutral
Protease additional amount 3200U/g albumen, 4000U/g albumen, 4800U/g albumen, 5600U/g albumen, 6400U/g albumen, are adding
Between enzyme amount 3200-5600U/g albumen, phenylalanine free amount is significantly increased, this may be since enzyme concentration is in substrate protein
It comes into full contact with, between 5600-6400U/g albumen, the free amount of phenylalanine is gradually decreased, this may be because of enzyme concentration mistake
Greatly, enzyme carries out self decomposition, leads to the free effect decline of phenylalanine.
3 neutral protease enzymolysis temperature of experimental example dissociates the influence of effect to phenylalanine
Step 1: rice protein is configured to the rice protein solution that mass percentage concentration is 1% with water, it is thin through ultrasonic wave
Born of the same parents' pulverization process (power 300W, ultrasonic 5s are spaced 4s, amount to 6min)；
Step 2: neutral proteinase (additional amount 5600U/g albumen) is added in rice protein solution after first step reaction,
Enzymatic hydrolysis 2h is carried out in 40/45/50/55/60 DEG C of thermostatic control oscillator vibration；
Step 3: second step takes out after reaction, it is put into 90 DEG C of waters bath with thermostatic control and keeps l0min, inactivate protease,
4200r/min centrifugation 20min takes supernatant；
Step 4: supernatant is the sufficiently free rice protein solution of phenylalanine, the freeze-dried preservation of the solution.
Neutral proteinase difference hydrolysis temperature is shown in Fig. 3 to the dissociate result of influential effect of phenylalanine.It can be seen by Fig. 3
Out: it is 50 DEG C that neutral proteinase, which recommends optimum temperature,.Between 45-50 DEG C, free phenylalanine amount is significantly increased, and temperature is 50
DEG C or more, free phenylalanine amount is on a declining curve after first tending towards stability, this may be since neutral proteinase activity is at 50 DEG C
Left and right reaches highest, but considers the saving of resources costs, selectes 50 DEG C and is used as peak enzymolysis-ability temperature.
4 pronase enzymolysis time of experimental example dissociates the influence of effect to phenylalanine
Step 1: rice protein is configured to the rice protein solution that mass percentage concentration is 1% with water, it is thin through ultrasonic wave
Born of the same parents' pulverization process (power 300W, ultrasonic 5s are spaced 4s, amount to 6min)；
Step 2: neutral proteinase (additional amount 5600U/g albumen) is added after first step reaction, in 50 DEG C of water bath with thermostatic control
Enzymatic hydrolysis 2h is carried out in oscillator；
Step 3: second step is after reaction, pronase (additional amount 100U/g albumen) is added and is uniformly mixed, 37
DEG C thermostatic control oscillator vibration in carry out enzymatic hydrolysis 1/2/3/4/5h；
Step 4: third step is taken out after reaction, it is put into 90 DEG C of waters bath with thermostatic control and keeps l0min, inactivate protease,
4200r/min centrifugation 20min takes supernatant up to rice protein enzymolysis liquid；
Step 5: supernatant is the sufficiently free rice protein solution of phenylalanine, the freeze-dried preservation of the solution.
Pronase difference enzymolysis time is shown in Fig. 4 to the dissociate result of influential effect of phenylalanine.It can be seen by Fig. 4
Out: pronase enzymatic hydrolysis 1-2h in, free phenylalanine amount gradually rises, in 2-3h, free phenylalanine amount gradually under
It drops, free phenylalanine amount is held essentially constant after 3h, this may be since in 3h or so, pronase enzyme-to-substrate is sufficiently connect
Touching, phenylalanine is sufficiently free under the reaction conditions, and after neutral protease enzymolysis, pronase is to rice protein water
It solves liquid and carries out specific cleavage.Therefore, best enzymolysis time of the 2h as pronase is selected.
5 pronase additional amount of experimental example dissociates the influence of effect to phenylalanine
Step 1: rice protein is configured to the rice protein solution that mass percentage concentration is 1% with water, it is thin through ultrasonic wave
Born of the same parents' pulverization process (power 300W, ultrasonic 5s are spaced 4s, amount to 6min)；
Step 2: neutral proteinase (additional amount 5600U/g albumen) is added after first step reaction, in 50 DEG C of water bath with thermostatic control
Enzymatic hydrolysis 2h is carried out in oscillator；
Step 3: second step is after reaction, it is added pronase (additional amount 50/74/98/122/146U/g albumen)
It is uniformly mixed, enzymatic hydrolysis 2h is carried out in 37 DEG C of thermostatic control oscillator vibration；
Step 4: third step is taken out after reaction, it is put into 90 DEG C of waters bath with thermostatic control and keeps l0min, inactivate protease,
4200r/min centrifugation 20min takes supernatant up to rice protein enzymolysis liquid；
Step 5: supernatant is the sufficiently free rice protein solution of phenylalanine, the freeze-dried preservation of the solution.
Pronase additional amount is shown in Fig. 5 to the dissociate result of influential effect of phenylalanine.As seen from Figure 5: in chain
Within the scope of 50-98U/g albumen, free phenylalanine amount significantly increases fungi protease additional amount, but if additional amount is higher than
98U/g albumen, free phenylalanine amount acutely decline.This may be that the restriction enzyme site that can be acted on due to pronase is abundant
Reaction, phenylalanine sufficiently dissociate from rice protein, and enzyme concentration can excessively make self decomposition in the reaction, lead to benzene
Alanine cannot sufficiently dissociate.Therefore, select 98U/g albumen as the optimal addn of pronase.
6 pronase hydrolysis temperature of experimental example dissociates the influence of effect to phenylalanine
Step 1: rice protein is configured to the rice protein solution that mass percentage concentration is 1% with water, it is thin through ultrasonic wave
Born of the same parents' pulverization process (power 300W, ultrasonic 5s are spaced 4s, amount to 6min)；
Step 2: neutral proteinase (additional amount 5600U/g albumen) is added after first step reaction, in 50 DEG C of water bath with thermostatic control
Enzymatic hydrolysis 2h is carried out in oscillator；
Step 3: second step is after reaction, pronase (additional amount 98U/g albumen) is added and is uniformly mixed, in perseverance
Enzymatic hydrolysis 2h is carried out in tepidarium oscillator, hydrolysis temperature is controlled at 30/35/40/45/50 DEG C；
Step 4: third step is taken out after reaction, it is put into 90 DEG C of waters bath with thermostatic control and keeps l0min, inactivate protease,
4200r/min centrifugation 20min takes supernatant up to rice protein enzymolysis liquid；
Step 5: supernatant is the sufficiently free rice protein solution of phenylalanine, the freeze-dried preservation of the solution.
Pronase hydrolysis temperature to phenylalanine dissociate influential effect as a result, seeing Fig. 6.As seen from Figure 6:
Within the scope of 30-50 DEG C, free phenylalanine amount first increases to tend towards stability afterwards, and for temperature at 40 DEG C or more, free phenylalanine amount is aobvious
Decline is write, this is because temperature is excessively high, enzyme activity is gradually reduced, and reaction efficiency is low, it is contemplated that economic benefit.Therefore, 35 DEG C are selected
As the best hydrolysis temperature of pronase.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (1)
1. a kind of method of phenylalanine in quick release rice protein, which is characterized in that the described method comprises the following steps:
(1) rice protein is configured to the rice protein solution that mass percentage concentration is 1% with water, at supersonic cell crushing
After reason, neutral proteinase is added by the additional amount of every gram of rice protein 5600U and obtains enzymolysis liquid I in 50 DEG C of reaction 2h；
Ultrasound condition: power 300W, ultrasonic 5s are spaced 4s, amount to 6min；
(2) pronase is added by the additional amount of every gram of rice protein 98U into enzymolysis liquid I and obtains enzyme in 37 DEG C of reaction 2h
Solve liquid II；
(3) enzymolysis liquid II is placed into 10min in 90 DEG C of waters bath with thermostatic control, inactivates protease, then 4200r/min is centrifuged
20min contains the free phenylalanine largely released in the supernatant collected；
Wherein, the neutral proteinase derives from bacillus subtilis, and the pronase derives from streptomyces griseus.
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| CN1140554A (en) * | 1995-05-25 | 1997-01-22 | 雀巢制品公司 | Enhanced procedures for preparing food hydrolysates |
| JP2007167017A (en) * | 2005-12-26 | 2007-07-05 | Advance Co Ltd | Fermentation extracted/activated extract derived from various mixing food material, and method for producing the same |
| CN104403017A (en) * | 2014-11-19 | 2015-03-11 | 中国农业科学院农产品加工研究所 | Preparation method of low-viscosity and high-purity yeast mannan |
| CN105876072A (en) * | 2016-04-15 | 2016-08-24 | 中国农业科学院农产品加工研究所 | Method for increasing solubility of rice protein |
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| CN1140554A (en) * | 1995-05-25 | 1997-01-22 | 雀巢制品公司 | Enhanced procedures for preparing food hydrolysates |
| JP2007167017A (en) * | 2005-12-26 | 2007-07-05 | Advance Co Ltd | Fermentation extracted/activated extract derived from various mixing food material, and method for producing the same |
| CN104403017A (en) * | 2014-11-19 | 2015-03-11 | 中国农业科学院农产品加工研究所 | Preparation method of low-viscosity and high-purity yeast mannan |
| CN105876072A (en) * | 2016-04-15 | 2016-08-24 | 中国农业科学院农产品加工研究所 | Method for increasing solubility of rice protein |
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