CN101829316B - Lactalbumin hydrolysate and application thereof in preparing glucose-lowering medicament - Google Patents
Lactalbumin hydrolysate and application thereof in preparing glucose-lowering medicament Download PDFInfo
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- CN101829316B CN101829316B CN2010101666399A CN201010166639A CN101829316B CN 101829316 B CN101829316 B CN 101829316B CN 2010101666399 A CN2010101666399 A CN 2010101666399A CN 201010166639 A CN201010166639 A CN 201010166639A CN 101829316 B CN101829316 B CN 101829316B
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- lactalbumin
- lactalbumin hydrolysate
- hydrolysate
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Abstract
The invention discloses a lactalbumin hydrolysate. The lactalbumin hydrolysate is concentrated solution obtained by performing heat treatment with lactalbumin, performing hydrolysis with alkaline protease and performing ultrafilteration according to cut-off molecular weight of 10,000. The prepared lactalbumin hydrolysate comprises 18 kinds of amino acids which account for over 70 percent. Animal experiments prove that the prepared lactalbumin hydrolysate can obviously reduce glucose tolerance of mice, stimulate insulin secretion, obviously increase insulin content in the mice, and obviously reduce random blood glucose in the mice. The prepared lactalbumin hydrolysate can be used for preparing the glucose-lowering medicament, can obtain obvious effects, and has a great application prospect and a bright market prospect.
Description
Technical field
The present invention relates to a kind of lactalbumin hydrolysate, specifically a kind of lactalbumin hydrolysate that utilizes hot water treatment and hydrolysis by novo to obtain, and the purposes in the preparation hypoglycemic medicine.
Background technology
Existing method for hydrolysis adopts alkaline protease, papain etc. to carry out the hydrolysis of substrate more.Lyophilizing after the direct desalination of product that obtains in the existing hydrolyzed whey protein research wherein has many residual enzymes, has a strong impact on the quality and the bio-safety of product.
And previously find that the multiple hydrolyzate that different hydrolysising conditions obtains has effects such as antiallergic, antioxidation, blood pressure lowering in the various researchs, Shang Weiyou research report lactalbumin hydrolysate has hypoglycemic effect.
Summary of the invention
In order to remedy the single of defective and function on the prior art lactalbumin hydrolysate method for preparing, first purpose of the present invention provides the improve one's methods lactalbumin hydrolysate of preparation of a kind of utilization.
A purpose more of the present invention is, the new purposes of the lactalbumin hydrolysate that method for preparing obtains is provided.
To achieve these goals, invention thinking of the present invention is:
In order to eliminate the residual of enzyme in the hydrolysis process previously, can consider method with ultrafiltration, utilize the difference of the molecular weight of itself and product to be removed, to improve its safety.
Mostly lactalbumin hydrolysate is macromolecular substances such as small peptide class or even aminoacid; Some receptors bind in the macromolecular complex mass-energy of discovering the small peptide class and the body is arranged and bring into play corresponding biological effect; Therefore the present invention attempts carrying out effect of lowering blood sugar research with lactalbumin hydrolysate, in the hope of the physiological function of finding that it is new.
Concrete technical scheme of the present invention is:
A kind of lactalbumin hydrolysate of the present invention is prepared from following method:
(1) lactalbumin is formulated as 5% (g/L) solution in 35 ℃～45 ℃ distilled water, is heated to 85 ℃, aquation 10～15min;
(2) be cooled to 50 ℃～55 ℃, transfer pH to 8.0;
(3) temperature maintenance adds alkaline protease at 50 ℃～55 ℃ ℃, and final concentration is controlled at 3%, in hydrolytic process, regulates pH value and maintains 8.0, till pH no longer changes;
(4) be heated to 85 ℃～90 ℃, continue 10～15min deactivation alkaline protease;
(5) 4 ℃, 3000rpm, 15～20min is centrifugal, gets the supernatant hydrolyzed solution;
(6) with the ultrafiltration of hydrolyzed solution process ultrafilter membrane, molecular cut off is 10000, and it is residual to remove alkaline protease;
(7) collect filter liquor, and concentrate down for 60 ℃ with vacuum rotary evaporator;
(8) with concentrated solution-40 ℃ extremely-50 ℃ of lyophilizing.
Said lactalbumin can be selected commercially available concentrated lactoalbumin WPC etc. for use.
Said ultrafilter membrane is Millipore (NMWL:10000), adopts Millipore 8400 type ultrafiltration cups to carry out ultrafiltration.
Polypeptide molecular weight is 900～1900 in the lactalbumin hydrolysate of method for preparing, and most of polypeptide molecular weight is 1800～1900.
The lactalbumin hydrolysate of the present invention's preparation detects nitrogen content 67.9% through Kjeldahl, and the response rate is 102.5%, and it is 6.7ml that formaldehyde method detects alkali consumption, and degree of hydrolysis is 14.8%.
The new purposes of the lactalbumin hydrolysate of method for preparing is its application in the preparation hypoglycemic medicine.
Advantage of the present invention and benefit:
The present invention prepares and contains aminoacid in 18 in the lactalbumin hydrolysate, and amino acid content surpasses 70%.Zoopery confirms that the lactalbumin hydrolysate that the present invention prepares can significantly reduce the mice carbohydrate tolerance, and stimulates insulin secretion, and significantly promotes the mouse islets cellulose content, significantly reduces mice random blood sugar content.The lactalbumin hydrolysate of the present invention's preparation can be used for the preparation of hypoglycemic medicine, and can obtain remarkable result, has good application prospects and market prospect.
Description of drawings
Describe embodiment of the present invention in detail below in conjunction with accompanying drawing:
The hydrolysate peptide chromatography colored graph that Fig. 1 measures embodiment 1 preparation for the DNS-Cl staining is (up: reagent blank; Middle row: lactalbumin chromatography colored graph; Descending: lactalbumin hydrolysate chromatography colored graph.)；
Fig. 2 is for adopting Tianjin, island CS9301pc thin layer fluorescent scanning appearance scanning hydrolysate chromatography figure (redness: reagent blank; Black: lactalbumin tomographic map; Blue: the lactalbumin hydrolysate tomographic map);
Fig. 3 is that the hydrolysate aminoacid of embodiment 1 preparation detects chromatogram;
Fig. 4 is the lactalbumin hydrolysate mass spectrum;
Fig. 5 is that lactalbumin hydrolysate is to normal mouse blood sugar influence figure;
Fig. 6 is the influence of lactalbumin hydrolysate to the normal mouse random blood sugar;
Fig. 7 is the influence of lactalbumin hydrolysate to the anti-sugar amount of diabetes KKay mice;
Fig. 8 is the influence of lactalbumin hydrolysate to diabetes KKay mice random blood sugar;
Fig. 9 is that lactalbumin hydrolysate influences diabetes KKay mice fasting glucose;
Figure 10 is that lactalbumin hydrolysate is to the excretory influence of diabetes kkay mice blood insulin;
Figure 11 is that the milk surum hydrolysate is to DDP-IV enzyme body outer suppressioning experiment.
The specific embodiment
Raw material of the present invention source:
Lactalbumin sample: U.S. Agri-Mark company 80% lactalbumin powder.
Alkaline protease: precious along company (3 * 10 available from Beijing letter profit
5The U/g food stage).
Ultrafiltration apparatus: the Model 8400 ultrafiltration cups of Millipore company and Millipore (NMWL:10000) ultrafilter membrane.
The preparation and the purification of embodiment 1 lactalbumin hydrolysate
1. get lactalbumin sample 50g, be dissolved in 40 ℃ of distilled water of 1000ml, be heated to 85 ℃, aquation 15 minutes.This stage solution colour is by the faint yellow milky that becomes, and milk fragrance is arranged.
2. be cooled to 55 ℃, regulate pH8.0 with 1N NaOH.
3. temperature maintenance is at 55 ℃, add rapidly alkaline protease 3g (6%, W/W), in hydrolytic process, constantly regulate pH value and maintain 8.0 with 1N NaOH, till pH no longer changed, on average alkali consumption was the 90ml/50g sample, about 5～6h of response time.
4. be heated to 90 ℃, continue 15 minutes deactivation alkaline proteases.
5. be cooled to room temperature, measure salinity.
6. adopt 4 ℃ in low-temperature and high-speed centrifuge, 3000rpm, 20min gets supernatant.
With hydrolyzed solution pack into Model 8400 ultrafiltration cups through the NMWL:10000 ultrafilter membrane with the high pure nitrogen ultrafiltration of pressurizeing, collect ultrafiltrate and concentrated down with 60 ℃ of vacuum rotary evaporators.
8. concentrated solution is adopted vacuum freeze drier-40 ℃ lyophilizing, obtain pale yellow powder,, place moisture barrier bag ,-20 ℃ of freezing preservations for the purification thing.
The lactalbumin hydrolysate nitrogen content of embodiment 2 embodiment 1 preparation and the mensuration of the response rate
Each several part product in hydrolysis and the purge process is roasting to weight in 80 ℃ of baking boxs respectively 1..
2. the according to the form below weighing adopts Kjeldahl to detect nitrogen content and calculate recovery rate respectively, and the result sees table 1.
Table 1
Can know that through table 1 it is 67.9% that the lactalbumin hydrolysate of the embodiment of the invention 1 preparation detects nitrogen content through Kjeldahl, the response rate is 102.5%.
The lactalbumin hydrolysate alkali consumption and the degree of hydrolysis of embodiment 3 embodiment 1 preparation are measured (formaldehyde method)
1. the neutralization of formaldehyde: formalin postpone of a specified duration meeting partial oxidation formic acid, getting before the use and using 0.1mol/L NaOH standard solution titration after one times of the original-pack formaldehyde upper solution adding distil water dilution is 7.0 subsequent use to pH.
2. the protein hydrolyzate 8ml that gets embodiment 1 preparation adds distilled water 60ml; Magnetic agitation and use the indication of accurate pH meter use down 0.05mol/L NaOH titration to pH be 7.0 o'clock; The formalin 10ml that becomes reconciled in adding, the volume V1 (ml) of the 0.1mol/L NaOH solution that is consumed when writing down its pH titration to 9.2 gets not hydrolyzed protein solution 8ml of same concentrations simultaneously; Do blank experiment by above-mentioned steps, the NaOH standard solution consumer product V2 (ml) in record burette this moment.
3. the calculating of degree of hydrolysis (DH):
The degree of hydrolysis definition is the percentage ratio that the range of hydrolysed peptides bond number accounts for the peptide bond sum, and computing formula is following:
DH％＝M(V1-V2)(CwoV/1000)(1/htot)×100
Wherein: htot=(1 * Pro%)/110
Cwo is a lactoalbumin soln protein quality concentration (g/L);
V is that hydrolyzed solution is taken volume (ml);
Htot is contained total peptide bond number (mmol/g raw material) in the material protein
Pro% is a lactalbumin material protein content.
Under this experiment condition, it is 6.7ml that titration V1-V2 on average consumes alkaline error, and calculating degree of hydrolysis is 14.8%.
1. the DNS-Cl of sample dyeing;
2. get lactalbumin sample, hydrolysis dialysis afterproduct and each 5mg of purified product and be dissolved in the 1ml distilled water, be added in the band frosted lid 2ml teat glass according to the following table sampling respectively.Addition is seen table 2
Table 2
3. regulate with 0.1mol/L NaOH and respectively manage between the pH value 9.0～9.5, in room temperature lucifuge reaction 4h or 40 ℃ of baking ovens, react 2h, reaction is kept in Dark Place and 4 ℃ of refrigerators after finishing.
4. exhibition layer (polyamide film chromatography).
5. employing polyamide film, developing agent is a formic acid: water: Methanamide=1: 20: 1
Adopt the CS9301pc thin layer fluorescent scanning appearance scanning of Tianjin, island, result such as Fig. 1 and shown in Figure 2, Fig. 1 measures the hydrolysate peptide chromatography colored graph that embodiment 1 prepares for the DNS-Cl staining, and is up: reagent blank; Middle row: lactalbumin chromatography colored graph; Descending: lactalbumin hydrolysate chromatography colored graph.Fig. 2 is for adopting Tianjin, island CS9301pc thin layer fluorescent scanning appearance scanning hydrolysate chromatography figure (redness: reagent blank; Black: lactalbumin tomographic map; Blue: the lactalbumin hydrolysate tomographic map).
Table 3 is the amino acid content testing result of national drug and the report of metabolite analysis center, because be acid-hydrolysis method, tryptophan does not detect.
Table 3
Numbering aminoacid lactalbumin hydrolysate lactalbumin
(g/100g) (g/100g)
1 Aspartic Acid 7.58 8.51
2 threonine 5.02 5.42
3 serines 3.76 3.87
4 glutamic acid 11.93 13.33
5 glycine 1.36 1.47
6 alanine 3.72 4.01
7 cystine 1.50 2.11
8 valines 3.95 4.66
9 methionine 1.92 1.82
10 isoleucine 4.18 5.11
11 leucines 7.89 8.55
12 tyrosine 2.19 2.44
13 phenylalanine 2.35 2.69
14 lysines 6.13 6.98
15 histidine 1.46 1.55
16 arginine 1.80 2.04
17 proline-4 .26 4.89
18 tryptophans do not detect and detect
Add up to 71.0 79.4
It is as shown in Figure 3 that aminoacid detects chromatogram.The result is visible, and the various amino acid contents and the lactalbumin stock yard of lactalbumin hydrolysate do not have significant change basically, and aminoacid is not lost.
Fig. 4 is the lactalbumin hydrolysate mass spectrum.See that from mass spectrometry results the molecular weight distribution of polypeptide product is 900～1900 in the hydrolysate of embodiment 1 preparation, major part is the polypeptide of molecular weight 1800～1900.
Following examples are lactalbumin hydrolysate influences zoopery to glycometabolic
Embodiment 7 lactalbumin hydrolysates are to the influence of mouse blood sugar
1. lactalbumin hydrolysate influences normal mouse blood sugar
1.1 carbohydrate tolerance
Select kunming mice for use, survey body weight and fasting glucose behind the 6h on an empty stomach, be divided into experimental group and matched group by randomly assigne.Experimental group is irritated stomach lactalbumin hydrolysate and each 2.0g/kg of glucose, irritates stomach dosage 0.01ml/g; Matched group is irritated stomach glucose 2.0g/kg, irritates stomach dosage 0.01ml/g, adopts the blood glucose of Johnson & Johnson steady blood glucose meter detection its 0.5h, 1h and 2h after irritating stomach, result such as table 4 and Fig. 5:
Table 4
*P＜0.05，**P＜0.01。
Table 4 and Fig. 5 explain that lactalbumin hydrolysate can effectively improve the normal mouse carbohydrate tolerance.
1.2 random blood sugar
Select kunming mice for use, survey body weight and blood glucose behind the 12h that takes food at random, be divided into experimental group and matched group by randomly assigne.Experimental group is irritated stomach lactalbumin hydrolysate 2.0g/kg, irritates stomach dosage 0.01ml/g; Matched group is irritated the stomach distilled water, irritates stomach dosage 0.01ml/g, adopts the steady blood glucose meter of Johnson & Johnson to detect its 0.5h and 1h after irritating stomach, the blood glucose of 2h, and result such as table 5 and Fig. 6:
Table 5
*P＜0.05，**P＜0.01
Table 5 and Fig. 6 explain that lactalbumin hydrolysate can effectively reduce the random blood sugar of normal mouse 0.5h, 1h and 2h.
2. lactalbumin hydrolysate is to the influence of diabetes KKay mouse blood sugar
2.1 carbohydrate tolerance
Select KKay mice (type 2 diabetes mellitus mice) for use, survey body weight and fasting glucose behind the 6h on an empty stomach, be divided into experimental group and matched group by randomly assigne.Experimental group is irritated stomach lactalbumin hydrolysate and each 2.0g/kg of glucose, irritates stomach dosage 0.01ml/g; Matched group is irritated stomach glucose 2.0g/kg, irritates stomach dosage 0.01ml/g, adopts the blood glucose of Johnson & Johnson steady blood glucose meter detection its 0.5h, 1h and 2h after irritating stomach, result such as table 6 and Fig. 7:
Table 6
*P＜0.05，**P＜0.01。
Table 6 and Fig. 7 explain that lactalbumin hydrolysate can effectively improve type 2 diabetes mellitus mice carbohydrate tolerance.
2.2 random blood sugar
Select KKay mice (type 2 diabetes mellitus mice) for use, survey body weight and blood glucose behind the ad lib 6h, be divided into experimental group and matched group by randomly assigne.Experimental group is irritated stomach lactalbumin hydrolysate 2.0g/kg, irritates stomach dosage 0.01ml/g; Matched group is irritated the stomach distilled water, irritates stomach dosage 0.01ml/g, adopts the steady blood glucose meter of Johnson & Johnson to detect the blood glucose of its 1h and 2h after irritating stomach, result such as table 7 and shown in Figure 8.
Table 7
*P＜0.05，**P＜0.01。
Table 7 and Fig. 8 explain that lactalbumin hydrolysate can effectively reduce the random blood sugar of type 2 diabetes mellitus mice 1h and 2h.
3.2 fasting glucose
Select KKay mice (type 2 diabetes mellitus mice) for use, survey body weight and blood glucose behind the 6h on an empty stomach, be divided into experimental group and matched group by randomly assigne.Experimental group is irritated stomach lactalbumin hydrolysate 2.0g/kg, irritates stomach dosage 0.01ml/g; Matched group is irritated the stomach distilled water, irritates stomach dosage 0.01ml/g, adopts the steady blood glucose meter of Johnson & Johnson to detect the blood glucose of its 1h and 2h after irritating stomach, result such as table 8 and shown in Figure 9.
Table 8
Table 8 and Fig. 9 explain that lactalbumin hydrolysate can effectively reduce the fasting glucose of type 2 diabetes mellitus mice 1h and 2h.
Select KKay mice (type 2 diabetes mellitus mice) for use, survey body weight and blood glucose behind the 12h on an empty stomach, be divided into experimental group and matched group by randomly assigne.Experimental group is irritated stomach lactalbumin hydrolysate 2.0g/kg, irritates stomach dosage 0.01ml/g; Matched group is irritated the stomach distilled water, irritates stomach dosage 0.01ml/g, and the 0.5h blood sampling detects plasma insulin, result such as table 9 and shown in Figure 10 after irritating stomach.
Table 9
Visible from table 9 and Figure 10, the lactalbumin hydrolysate of embodiment 1 preparation can stimulate the plain secretion of diabetes kkay mouse islets.
Embodiment 9 lactalbumin hydrolysates are to the active influence of DPP-IV
DPP-IV (dipeptides acyl carboxylic peptidyl enzyme) is the hydrolytic enzyme of incretin GPL-1, and the activity that suppresses this enzyme can improve GLP-1 level in the blood plasma, and GLP-1 has the effect of intense stimulus insulin secretion.Shown in figure 11, the lactalbumin hydrolysate of embodiment 1 preparation can suppress the activity of DPP-IV.
Claims (5)
1. a lactalbumin hydrolysate is characterized in that, said lactalbumin hydrolysate is that by the concentrated solution that molecular cut off 10000 obtains, said lactalbumin hydrolysate is prepared from following method with after adding hydrolysis by novo behind the lactalbumin heat treatment:
(1) lactalbumin is formulated as 5% solution in 35 ℃～45 ℃ distilled water, is heated to 85 ℃, aquation 10～15min;
(2) be cooled to 50 ℃～55 ℃, transfer pH to 8.0;
(3) temperature maintenance adds alkaline protease at 50 ℃～55 ℃, and final concentration is controlled at 3%, in hydrolytic process, regulates pH value and is maintained 8.0, till pH no longer changes;
(4) be heated to 85 ℃～90 ℃, continue 10～15min deactivation alkaline protease;
(5) 4 ℃, 3000rpm, 15～20min is centrifugal, gets the supernatant hydrolyzed solution;
(6) hydrolyzed solution is crossed the ultrafilter membrane dialysis, molecular cut off is 10000, and it is residual to remove alkaline protease;
(7) collect filter liquor, and concentrate down for 60 ℃ with vacuum rotary evaporator;
(8) with concentrated solution-40 ℃ extremely-50 ℃ of lyophilizing;
Polypeptide molecular weight is 900～1900 in the said lactalbumin hydrolysate;
Said lactalbumin hydrolysate detects nitrogen content 67.9% through Kjeldahl, and the response rate is 102.5%, and it is 6.7ml that formaldehyde method detects alkali consumption, and degree of hydrolysis is 14.8%.
2. lactalbumin hydrolysate according to claim 1 is characterized in that, polypeptide molecular weight is 1800～1900 in the said lactalbumin hydrolysate.
3. claim 1 or 2 described lactalbumin hydrolysates are in the purposes of preparation in the hypoglycemic medicine.
4. the described purposes of claim 3 is characterized in that, acceptable auxiliary is processed peroral dosage form on said lactalbumin hydrolysate and the pharmaceutics.
5. the described purposes of claim 4 is characterized in that, said peroral dosage form is tablet, capsule, oral liquid or granule.
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| RU2550798C2 (en) * | 2011-05-12 | 2015-05-10 | Василий Иосифович Зоря | Method of stimulating insulin secretion |
| CN103695518B (en) * | 2013-12-30 | 2016-04-06 | 东北农业大学 | Whey protein peptide is utilized to reduce product and the preparation method of serum cholesterol level |
| CN114736943B (en) * | 2022-03-14 | 2023-06-20 | 厦门元之道生物科技有限公司 | Functional whey polypeptide and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0799577A1 (en) * | 1994-10-14 | 1997-10-08 | Morinaga Milk Industry Co., Ltd. | Peptide mixture and products thereof |
| CN1474656A (en) * | 2000-09-11 | 2004-02-11 | Ŧ������Ʒ�� | Improved bioactive whey protein hydrolysate |
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| AU2167901A (en) * | 1999-12-17 | 2001-06-25 | N.V. Nutricia | Use of modified foodstuff ingredients for preventing the development of a diabetes mellitus (iddm) with an existing epidemiologically established risk |
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| EP0799577A1 (en) * | 1994-10-14 | 1997-10-08 | Morinaga Milk Industry Co., Ltd. | Peptide mixture and products thereof |
| CN1474656A (en) * | 2000-09-11 | 2004-02-11 | Ŧ������Ʒ�� | Improved bioactive whey protein hydrolysate |
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