CN107858391A - A kind of method prepared by milk macromolecules polypeptide based on gastro-intestinal digestion - Google Patents
A kind of method prepared by milk macromolecules polypeptide based on gastro-intestinal digestion Download PDFInfo
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- CN107858391A CN107858391A CN201711111381.0A CN201711111381A CN107858391A CN 107858391 A CN107858391 A CN 107858391A CN 201711111381 A CN201711111381 A CN 201711111381A CN 107858391 A CN107858391 A CN 107858391A
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- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
Method prepared by a kind of milk macromolecules polypeptide based on gastro-intestinal digestion of the present invention, is related to field of polypeptide preparation.Take milk protein, oil bath heating denaturation, after being cooled to room temperature, using energy gathering type ultrasound pretreating milk albumen.After pretreatment terminates, temperature is adjusted to 40~60 DEG C, neutral proteinase is added, enzyme deactivation after digesting to certain degree of hydrolysis, is spray-dried after cooling, is prepared into macromolecular milk polypeptide finished product.The present invention's is prepared into milk protein macromolecular milk polypeptide, can eliminate the uncomfortable reaction of human body；The digestive functions of human body are made full use of, macromolecular milk polypeptide is further degraded in vivo, biological activity significantly improves (using hypotensive activity as index)；Using thermal ultrasound pretreating milk albumen, the homogeneity of enzymolysis product is improved, compared with directly digesting, its milk protein amount not digested is remarkably decreased.
Description
Technical field
The present invention relates to field of polypeptide preparation, particularly relates to prepared by a kind of milk macromolecules polypeptide based on gastro-intestinal digestion
Method.
Background technology
Milk is optimal natural nutrient substance for calf.(account for total protein to contain rich in casein in milk
Amount 80%), especially αs1- casein, the cow's milk of high casein are adapted to the gastro-intestinal digestion absorption characteristics of calf, when milk enters
After calf abomasum, the quick solidification by the chymotrypsin enzyme effect secreted by abomasum, milk is set to be changed into grumeleuse from liquid, formation
Casein grumeleuse is gradually decomposed in a very long time after its feeding by rennin and pepsin, plays lasting supply albumen
The effect of matter.Our people's milk drink has long history, and milk has turned into the important nutraceutical in people's life, still
During nutritious supplementary pharmaceutical of the cow's milk rich in casein as people, particularly when heavy dose is drunk, often produce indigestion,
The problems such as digestive discomfort, main cause are that the casein in milk enters after human stomach, rapid flocculation in acid condition,
But the casein for similar to the chrymotrypsin secreted by calf abomasum, causing to flocculate is lacked in human gastrointestinal tract not by certainly
Pepsin rapid digestion that status is secreted (Zhang Lanwei, in human milk early stage milk protein, amino acid composition with cow's milk to score
Analysis, 1997), therefore it is unfavorable for own absorption, digestive discomfort is caused, while can also influence the absorption of other nutriments.
Milk protein is prepared into milk polypeptide can efficiently solve above mentioned problem, due to the change of protein structure, junket egg
Be decomposed into CPP in vain, be advantageous to its absorption, the method can not only eliminate casein digest slowly caused by not
Suitable reaction, can also improve its biological activity, including anti-lipid, immunological regulation, anti-inflammatory, hypotensive, anticancer, it is antimicrobial,
Antioxidation (Lotti Egger, Update on bioactive peptides after milk and cheese
digestion,2017).The preparation method of currently used milk polypeptide is mainly prepared into small molecule using enzymatic isolation method
Milk polypeptide or macromolecular milk polypeptide.The preparation of small molecule milk polypeptide therein does not consider that digestion of the human body to polypeptide is made
With using the internal basic norm that is directly absorbed as of micromolecule polypeptide (mainly dipeptides and tripeptides) (if to prepare two, tripeptides to be ultimate
Target, its average degree of hydrolysis is up to more than 33.3%), cause Degree of Enzymatic Hydrolysis too high, the link such as follow-up centrifugation, UF membrane and concentration
Also cause its preparation process complicated, cost is too high, and small molecule milk polypeptide can also by the protease digestion in intestines and stomach
The problem of producing excessive degradation (Katherina Fern á ndez, Simulated digestion of
proanthocyanidins in grape skin and seed extracts and the effects of
digestion on the angiotensin I-converting enzyme(ACE)inhibitory activity,
2013), for problem above, digestive functions can be made full use of by preparing macromolecular milk polypeptide, realize the complete of protein resource
Utilize (saving the links such as centrifugation, UF membrane and concentration).However, the preparation of macromolecular milk polypeptide still has part not at present
Foot, because the low enzymolysis time of degree of hydrolysis is short, causes hydrolysis degree inside and outside milk protein molecule aggregate to differ, i.e. aggregation outer egg
Excessively digested in vain, internal albumen is not digested, causes the uniformity of enzymolysis product low.The hydrolysis degree of usual albumen produces to it
The bioactivity of thing has significant impact.Therefore, the homogeneity raising macromolecular polypeptides product quality for improving enzymolysis product has
Significance.
Alkali protease and neutral proteinase are all the conventional protease for preparing polypeptide, are compared and alkali protease, in
Property protease prepare polypeptide bitter taste it is low, also, different from alkali protease (restriction enzyme site is mainly hydrophobic amino acid, with
Pepsin is similar), the protease in the restriction enzyme site and intestines and stomach of neutral proteinase is complementary, can more fully utilize
Macromolecular polypeptides are changed into micromolecule polypeptide by digestive functions.Also, research shows that protein pretreatment modification contributes to
The enzymolysis property and biological activity of albumen are improved, conventional pretreatment modification method includes physical modification and chemical modification, thing
Reason is modified because its expense is low, have no toxic side effect, action time is short and influences the advantages that smaller to product nutritional properties, and widely
Applied to the study on the modification of protein, for example, microwave radiation aids in papain hydrolysis soybean protein, reaction rate improves
More than 5 times (Zhang Qi, the research of microwave radiation auxiliary papain hydrolysis soybean protein, 2012), ultrasound pretreatment can be notable
Improve glycinin and beta-conglycinin enzymolysis efficiency and antioxidation activity (Xu Jing, is ultrasonically treated to soybean protein enzyme
The influence of solution thing structure and antioxidation activity, 2017).At present, high uniformity is prepared using physical modification method assistance enzymolysis method
The research of macromolecular milk polypeptide also there is no report.
The content of the invention
It is an object of the invention to provide a kind of preparation method of macromolecular polypeptides, specifically using heat and ultrasound pretreatment
Albumen, the method that the milk macromolecules polypeptide based on gastro-intestinal digestion is prepared using enzymatic isolation method.
In order to realize foregoing invention purpose, its specific technical scheme is as follows：
Take milk, oil bath heating denaturation, after being cooled to room temperature, using ultrasonic (cell crushing instrument) pretreating milk of energy gathering type
(protein concentration 34g/L), it is as follows to be ultrasonically treated parameter：5~60min of ultrasonic time, 40~500W/L of ultrasonic power.Pretreatment knot
Shu Hou, temperature is adjusted to 40~60 DEG C, adds 2%~10% (E/S) neutral proteinase, digest to certain degree of hydrolysis (2%~
20%) enzyme deactivation afterwards, dried after cooling, be prepared into macromolecular milk polypeptide finished product.
Advantages of the present invention：The present invention compared with prior art, has advantages below：Milk is prepared into macromolecular milk
Polypeptide, the uncomfortable reaction of human body can be eliminated；The digestive functions of human body are made full use of, make macromolecular milk polypeptide in vivo
Further degraded, biological activity (using hypotensive activity as index) improve 9.96%-23.52%；Big based on gastro-intestinal digestion
In terms of the preparation of molecular function polypeptide, neutral proteinase is because the protease in its restriction enzyme site and intestines and stomach is complementary, therefore its enzyme
Solution product is better than alkali protease by the biological activity after gastro-intestinal digestion；Using thermal ultrasound pretreating milk, show
The homogeneity for improving enzymolysis product is write, protein content declines 18.83%~47.56% (with direct enzyme in its milk not digested
Solution is compared).
Below by embodiment, technical scheme is described in further detail.
Embodiment
Used term in the present invention, unless otherwise specified, typically have those of ordinary skill in the art usual
The implication of understanding.The present invention is described in further detail with reference to specific embodiment, and with reference to data.It should be understood that these
Embodiment is that the present invention is further described, it is impossible to is interpreted as limiting the scope of the present invention, the skill in the field
Art engineer can make some nonessential modifications and adaptations to the present invention according to the content of foregoing invention.
Below in an example, the various processes and method not being described in detail are conventional methods as known in the art.
The source of agents useful for same, trade name and it is necessary to list its constituent person, indicates on the first appearance, thereafter phase used
With reagent unless otherwise specified, it is identical with the content indicated first.
The present embodiment and the protease of reference examples are purchased from Sigma companies.
The present embodiment and the milk of reference examples are that (protein content 3.4%, fat contains bright excellent times of pasteurize fresh milk
0%) amount, is purchased from Ou Shang supermarkets of industry.
The present embodiment and the degree of hydrolysis of reference examples are determined using pH-stat methods.
The present embodiment and the in-vitro simulated pipe intestinal digesting of reference examples are with the following method：Enzymolysis product is taken, according to 2%
(E/S) enzyme concentration adds pepsin, digests 2 hours, after enzymolysis terminates, enzyme deactivation, pancreatin is added according to 4% (E/S) enzyme concentration,
Enzymolysis 4 hours, after enzymolysis terminates, enzyme deactivation, centrifugation, the hypotensive activity of measure gastro-intestinal digestion product.
The present embodiment and the hypotensive activity of reference examples measure are with angiotensin converting enzyme (angiotensin
Converting enzyme, ACE) inhibiting rate index, with reference to Dingqing sesame, (impulse ultrasound assistance enzymolysis method prepares beautiful for concrete operations
The research of cream-coloured powder ACEI active peptides, 2008) method.
The protein assay method not digested in the present embodiment and reference examples is：Using trichloroacetic acid precipitation (three chloroethenes
Acid it is final concentration of 10%).
Reference examples 1
400mL milk is measured, by temperature adjustment to 50 DEG C, adds 680 μ L neutral proteinases, it is 32% to digest to degree of hydrolysis
When enzyme deactivation, enzymolysis product after gastro-intestinal digestion hypotensive activity be 44.61%, precipitation capacity 6.51g.
Reference examples 2
400mL milk is measured, by temperature adjustment to 50 DEG C, adds 680 μ L neutral proteinases, it is 18% to digest to degree of hydrolysis
When enzyme deactivation, hypotensive activity of the enzymolysis product after gastro-intestinal digestion be 52.30%, precipitation capacity 7.17g.
Reference examples 3
400mL milk is measured, using energy gathering type ultrasound pretreating milk, it is as follows to be ultrasonically treated parameter：Ultrasonic time
20min, ultrasonic power 300W/L.The pretreated milk of ultrasound is put into water-bath, by its temperature adjustment to 50 DEG C, added
680 μ L neutral proteinases, digest to degree of hydrolysis be 18% when enzyme deactivation, hypotensive activity of the enzymolysis product after gastro-intestinal digestion be
60.2%, precipitation capacity 6.83g.
Control group 4
400mL milk is measured, after heating 20min in 100 DEG C of oil baths, is cooled to room temperature.Pretreated milk is put
Enter in water-bath, by its temperature adjustment to 50 DEG C, add 680 μ L neutral proteinases, digest to degree of hydrolysis be 18% when enzyme deactivation, enzyme
It is 58.8% to solve hypotensive activity of the product after gastro-intestinal digestion, precipitation capacity 6.12g.
Control group 5
400mL milk is measured, 20min is heated in 100 DEG C of oil baths, after being cooled to room temperature, using the pre- place of energy gathering type ultrasound
Milk is managed, it is as follows to be ultrasonically treated parameter：Ultrasonic time 20min, ultrasonic power 300W/L.The pretreated milk of ultrasound is put into
In water-bath, by its temperature adjustment to 50 DEG C, add 680 μ L alkali proteases, digest to degree of hydrolysis be 18% when enzyme deactivation, enzymolysis
Hypotensive activity of the product after gastro-intestinal digestion is 56.4%, precipitation capacity 4.61g.
Embodiment 1
400mL milk is measured, 20min is heated in 100 DEG C of oil baths, after being cooled to room temperature, using the ultrasonic (cell of energy gathering type
Broken instrument) pretreating milk (protein concentration 34g/L), it is as follows to be ultrasonically treated parameter：Ultrasonic time 20min, ultrasonic power 300W/
L.The pretreated milk of ultrasound is put into water-bath, by its temperature adjustment to 50 DEG C, adds 680 μ L neutral proteinases, enzyme
Enzyme deactivation when solution to degree of hydrolysis is 18%, hypotensive activity of the enzymolysis product after gastro-intestinal digestion are 64.6%, and precipitation capacity is
3.90g。
Embodiment 2
400mL milk is measured, 20min is heated in 100 DEG C of oil baths, after being cooled to room temperature, using the ultrasonic (cell of energy gathering type
Broken instrument) pretreating milk (protein concentration 34g/L), it is as follows to be ultrasonically treated parameter：Ultrasonic time 5min, ultrasonic power 40W/L.
The pretreated milk of ultrasound is put into water-bath, by its temperature adjustment to 40 DEG C, adds 2.72 μ L neutral proteinases, enzymolysis
Enzyme deactivation when to degree of hydrolysis being 20%, hypotensive activity of the enzymolysis product after gastro-intestinal digestion is 62.3%, and precipitation capacity is
3.76g。
Embodiment 3
400mL milk is measured, 20min is heated in 100 DEG C of oil baths, after being cooled to room temperature, using the ultrasonic (cell of energy gathering type
Broken instrument) pretreating milk (protein concentration 34g/L), it is as follows to be ultrasonically treated parameter：Ultrasonic time 60min, ultrasonic power 500W/
L.The pretreated milk of ultrasound is put into water-bath, by its temperature adjustment to 60 DEG C, adds 1360 μ L neutral proteinases, enzyme
Enzyme deactivation when solution to degree of hydrolysis is 2%, hypotensive activity of the enzymolysis product after gastro-intestinal digestion are 57.51%, and precipitation capacity is
5.82g。
Claims (5)
1. a kind of method prepared by milk macromolecules polypeptide based on gastro-intestinal digestion, it is characterised in that carry out as steps described below：
Milk is taken, oil bath heating is denatured, dense using ultrasonic (cell crushing instrument) pretreating milk of energy gathering type, albumen after being cooled to room temperature
34g/L is spent, after pretreatment terminates, temperature is adjusted to 40~60 DEG C, neutral proteinase is added, is gone out after digesting to certain degree of hydrolysis
Enzyme, dried after cooling, be prepared into macromolecular milk polypeptide finished product.
2. a kind of method that according to claim 1 prepared by milk macromolecules polypeptide based on gastro-intestinal digestion, it is characterised in that：
5~60min of ultrasonic time.
3. a kind of method that according to claim 1 prepared by milk macromolecules polypeptide based on gastro-intestinal digestion, it is characterised in that：
The neutral proteinase, it is characterized in that：Addition is 2%~10% (E/S).
4. a kind of method that according to claim 1 prepared by milk macromolecules polypeptide based on gastro-intestinal digestion, it is characterised in that：
The degree of hydrolysis of albumen is 2%~20%.
5. a kind of method that according to claim 1 prepared by milk macromolecules polypeptide based on gastro-intestinal digestion, it is characterised in that：
40~500W/L of ultrasonic power.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711111381.0A CN107858391A (en) | 2017-11-13 | 2017-11-13 | A kind of method prepared by milk macromolecules polypeptide based on gastro-intestinal digestion |
| PCT/CN2018/114949 WO2019091471A1 (en) | 2017-11-13 | 2018-11-12 | Preparation of macromolecular polypeptide based on gastrointestinal digestion and method for in situ real-time monitoring process of same |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711111381.0A CN107858391A (en) | 2017-11-13 | 2017-11-13 | A kind of method prepared by milk macromolecules polypeptide based on gastro-intestinal digestion |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019091471A1 (en) * | 2017-11-13 | 2019-05-16 | 江苏大学 | Preparation of macromolecular polypeptide based on gastrointestinal digestion and method for in situ real-time monitoring process of same |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103275176A (en) * | 2013-06-18 | 2013-09-04 | 天津商业大学 | ACE inhibitory peptide and preparation method thereof |
| CN103695517A (en) * | 2013-12-26 | 2014-04-02 | 江苏大学 | Method for preparing functional polypeptide by utilizing sweep frequency ultrasonic wave preprocessed zein |
| CN104450848A (en) * | 2014-12-31 | 2015-03-25 | 临沂格瑞食品有限公司 | Milk micro-molecular biological active peptide and preparation method thereof |
-
2017
- 2017-11-13 CN CN201711111381.0A patent/CN107858391A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103275176A (en) * | 2013-06-18 | 2013-09-04 | 天津商业大学 | ACE inhibitory peptide and preparation method thereof |
| CN103695517A (en) * | 2013-12-26 | 2014-04-02 | 江苏大学 | Method for preparing functional polypeptide by utilizing sweep frequency ultrasonic wave preprocessed zein |
| CN104450848A (en) * | 2014-12-31 | 2015-03-25 | 临沂格瑞食品有限公司 | Milk micro-molecular biological active peptide and preparation method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019091471A1 (en) * | 2017-11-13 | 2019-05-16 | 江苏大学 | Preparation of macromolecular polypeptide based on gastrointestinal digestion and method for in situ real-time monitoring process of same |
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