CN104946713A - Preparation method and application of glucose-lowering lipid-lowering peptide based on sea cucumber protein - Google Patents
Preparation method and application of glucose-lowering lipid-lowering peptide based on sea cucumber protein Download PDFInfo
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Abstract
The invention discloses a preparation method and application of glucose-lowering lipid-lowering peptide based on sea cucumber protein. The preparation method comprises the following steps: taking sea cucumber powder, adding deionized water, and heating in a water bath; homogenizing to obtain a sea cucumber homogenate solution; adjusting the pH value to be 7.0-10.0, adding protease I, performing enzymolysis reaction for 2-4 hours, then adding protease II into the reaction system, performing enzymolysis continuously for 2-4 hours, and ending the reaction to obtain sea cucumber protein hydrolysate; inactivating the protease I and the protease II in the sea cucumber protein hydrolysate; cooling, then centrifuging to remove impurities, and keeping the supernatant; and adding 95% ethanol, standing, then centrifuging, taking the supernatant, adjusting the pH value of the supernatant to be 6.0-8.0, and passing the supernatant through an ultrafiltration membrane with the cutoff molecular weight of 3000-10000Da, and collecting an ultra-filtrate to obtain the peptide. The sea cucumber protein peptide prepared by the method disclosed by the invention can achieve good effects of lowering glucose and lipid of diabetes and hyperlipemia patients, and is high in edible safety.
Description
Technical field
The present invention relates to fishery products deep processing and biological technical field, particularly a kind of hypoglycemic peptide preparation method based on sea cucumber albumen and application thereof.
Background technology
Along with social progress and Economic development, the mode of life of people there occurs very large change, and the sickness rate of the metabolic disease such as diabetes and hyperlipidaemia raises year by year.According to IDF (International Diabetes Federation, IDF) statistics, end 2013, global diabetes number has reached 3.82 hundred million.Expect 2035, the puzzlement that the whole world will have 5.92 hundred million people to be subject to diabetes.At present, China has become the maximum country of diabetic subject's number, and patient numbers is close to 100,000,000.Long term hyperglycemia can bring out body fat metabolism disorder, causes hyperlipemia, increases the risk of cardiovascular disorder.According to the display of the World Health Organization (World Health Organization, WHO) publish data, within 2008, nearly 1,730 ten thousand people in the whole world die from cardiovascular disorder.End 2012, China's cardiovascular patient number is 2.9 hundred million, about has 3,500,000 people to die from cardiovascular disorder every year.At present, the method for the treatment of diabetes comprises insulin injection and orally-taken blood sugar reducing medicine, but these methods for the treatment of easily cause the untoward reactions such as hypoglycemia, hepar damnification, lactic acidosis and gastrointestinal discomfort.For the treatment of hyperlipidaemia, conventional stanin fat-reducing medicament, long-term taking also can produce toxic side effect to human body.Therefore, the functions of reducing sugar and reducing fat composition in active research food, exploitation safe, natural, the efficient hypoglycemic peptide product derived from food has become the integral part of the metabolic disease such as diabetes and hyperlipidaemia non-drug therapy from now on.Such as Chinese patent 201210462662.1, Wu Chengye etc. utilize neutral proteinase hydrolysis laver, and preparation has the marine alga polypeptide of hypoglycemic and hypotensive activity.
Sea cucumber is food important in ocean and drug resource.Wall of sea cucumber Stichopus japonicus is that sea cucumber mainly eats (medicine) use position, and containing abundant collagen protein, mucopolysaccharide and saponin(e, wherein sea cucumber protein content accounts for its body wall dry weight more than 80%.Sea cucumber albumen is rich in multiple bioactive sequences, can discharge under suitable proteolytic enzyme effect, becomes the peptide section with biologic activity such as anti-oxidant, immunomodulatory, hypotensive and hypoglycemics.In recent years, the activity of sea cucumber protein hydrolysate obtains and studies widely.Li Wei etc. disclose the sea cucumber nectar with immunomodulatory, improving water flood and radiation-resisting functional in Chinese patent 201310215842.4; Liu Kai etc. utilize neutral proteinase hydrolysis sea cucumber albumen to prepare to have and promote that postoperative wound heals the sea cucumber protein peptide of effect in Chinese patent 201410114474.9.
At present, the patented technology about enzyme hydrolytic sea cucumber albumen adopts the digestion pattern of single enzyme or prozyme one one-step hydrolysis mostly, and this digestion pattern is unfavorable for the hydrolysis of sea cucumber albumen, is also unsuitable for the release of bioactive sequences in sea cucumber albumen.Although king is contained in Chinese patent 201310278840.X utilize Sumizyme MP and immobilized enzyme (comprising neutral protease and papoid) fractional hydrolysis sea cucumber albumen, but in order to improve the enzymolysis efficiency of immobilized enzyme, need cross the insolubles filtered in Sumizyme MP initial hydrolysis liquid, this process can lose the unhydrolysed sea cucumber albumen of part.In addition, due to use and the inherent fishy smell of marine organisms of alkali lye in enzymolysis process, the sea cucumber protein peptide obtained can be made to have unpleasant odor.Wang Qiukuan etc. utilize resin anion(R.A) to carry out de-raw meat to sea cucumber protein peptide in Chinese patent 201210053085.0, this method complex operation and consuming time, efficiency is low, is unfavorable for suitability for industrialized production; Wu Qinglin utilizes gac to de-taste process to sea cucumber protein peptide in Chinese patent 201110449621.4, and this treatment process can be lost the functional component in protein polypeptide and reduce its biologic activity.And adopt microencapsulation both can cover the unpleasant odor of sea cucumber protein peptide, the functional component in polypeptide can not be lost again.
Adopt two enzyme fractional hydrolysis pattern to improve the degree of hydrolysis of sea cucumber albumen, preparation has the sea cucumber protein peptide of hypoglycemic activity and utilizes microencapsulation to promote its product Quality Research not yet to find.Therefore, develop that a kind of controllable enzyme process prepares high reactivity, the method for sea cucumber albumen hypoglycemic peptide of excellent in flavor has important theory significance and using value.
Summary of the invention
For above-mentioned the deficiencies in the prior art, the invention provides a kind of hypoglycemic peptide preparation method based on sea cucumber albumen and application thereof, adopt that the hypoglycemic peptide activity prepared of the method is high, excellent in flavor.
Technical scheme of the present invention is such realization:
Based on a hypoglycemic peptide preparation method for sea cucumber albumen, comprise the following steps:
1) get fresh sea cucumber to clean, gill, pulverize after lyophilize, obtain Stichopus japonicus powder; Get Stichopus japonicus powder, add deionized water by 1:20 ~ 1:30 part by weight, then in 75 ~ 95 DEG C of water-baths, heat 15 ~ 30min.Under homogenate power 1500 ~ 3500r/min condition, homogenate 5 ~ 10min, obtains sea cucumber homogenate;
2) regulating step 1) pH value to 7.0 ~ 10.0 of described sea cucumber homogenate, add proteolytic enzyme I, make sea cucumber albumen and proteolytic enzyme I carry out enzyme digestion reaction under 40 ~ 60 DEG C of conditions; Described proteolytic enzyme I is Sumizyme MP or trypsinase;
3) step 2) enzyme digestion reaction carries out after 2 ~ 4h, keeps the temperature of reaction system of described enzyme digestion reaction constant, in described reaction system, add proteinase II, and continue enzymolysis 2 ~ 4h, termination reaction, obtains sea cucumber protein hydrolyte;
4) by step 3) the sea cucumber protein hydrolyte of gained is warming up to 75 ~ 95 DEG C, keeps 15 ~ 30min, deactivation proteolytic enzyme I wherein and proteinase II; The centrifugal removal of impurity after cooling, retains supernatant liquor; Described proteinase II is any one in compound protease, papoid and neutral protease;
5) to step 4) add 95% ethanol in gained supernatant liquor, its final volume is made to reach 60 ~ 80% of total system, centrifuging and taking supernatant liquor after 4 DEG C of standing 10 ~ 15h, regulate described supernatant liquor pH value to 6.0 ~ 8.0, and be the ultra-filtration membrane of 3000 ~ 10000Da by the molecular weight that dams by described supernatant liquor, collect ultrafiltrated, obtain sea cucumber albumen hypoglycemic peptide.
Based on a hypoglycemic peptide preparation method for sea cucumber albumen, also comprise the step of following microencapsulation process:
6) by soluble in water for microencapsulation wall material, wall material solution is made;
7) by step 6) the wall material solution of gained joins step 5 by a certain percentage) in the sea cucumber albumen hypoglycemic peptide that obtains, stirred at ambient temperature 0.5 ~ 2h, obtains mixing solutions;
8) to step 7) mixing solutions of gained carries out spraying dry, obtains microencapsulated protein polypeptide.
Described step 2) in the add-on of proteolytic enzyme I be that every 100g Stichopus japonicus powder adds 200 ~ 800KU proteolytic enzyme I.Wherein, 1U is defined as the amount that per minute caseinhydrolysate at 40 DEG C produces 1 μ g tyrosine, and KU is 1000U.
Described step 3) in the add-on of proteinase II be that every 100g Stichopus japonicus powder adds 200 ~ 800KU proteinase II.Wherein, 1U is defined as the amount that per minute caseinhydrolysate at 40 DEG C produces 1 μ g tyrosine, and KU is 1000U.
Described step 2) in adjust ph alkali lye used be any one in food grade sodium carbonate, sodium bicarbonate, Sodium phosphate dibasic, salt of wormwood, saleratus, dipotassium hydrogen phosphate or potassium hydroxide solution; Adjust ph acid solution used is any one in food grade lactic acid or citric acid solution.
Described step 7) in wall material be the condensed whey protein of protein content >=80%.
Described step 7) in wall material be the mixture of the condensed whey protein of sodium alginate and protein content >=80%, condensed whey protein and sodium alginate mixed weight are than being 1:10 ~ 1:35.
Described step 7) in the mass ratio of sea cucumber albumen hypoglycemic peptide and wall material be 1:1 ~ 1:5.
The mass concentration of described wall material solution mesospore material can according to the quality of polypeptide in protein polypeptide solution, and the dry matter content of mixing solutions is determined.
A kind of application of hypoglycemic peptide in food source property hypoglycemic protective foods of the sea cucumber albumen obtained based on the hypoglycemic peptide preparation method of sea cucumber albumen.
Beneficial effect of the present invention:
1. the present invention adopts the digestion pattern of two enzyme fractional hydrolysis, effectively can improve the degree of hydrolysis of sea cucumber albumen, simple for process, is convenient to suitability for industrialized production;
2. the microencapsulation adopted in the present invention, can cover the unpleasant odor of sea cucumber protein peptide effectively, and does not affect primary formation and the biologic activity of polypeptide;
3. adopt the sea cucumber protein peptide edible safety prepared of the inventive method high, have no side effect, good absorbing effect, good hypoglycemic lipid-lowering effect can be played to diabetes and hyperlipidemia patient, can play a role in health care to healthy population.
Embodiment
Be clearly and completely described the technical scheme in the embodiment of the present invention below, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making other embodiments all obtained under creative work prerequisite, belong to the scope of protection of the invention.
Based on a hypoglycemic peptide preparation method for sea cucumber albumen, comprise the following steps:
1) get fresh sea cucumber to clean, gill, pulverize after lyophilize, obtain Stichopus japonicus powder; Get Stichopus japonicus powder, add deionized water by 1:20 ~ 1:30 part by weight, then in 75 ~ 95 DEG C of water-baths, heat 15 ~ 30min.Under homogenate power 1500 ~ 3500r/min condition, homogenate 5 ~ 10min, obtains sea cucumber homogenate;
2) regulating step 1) pH value to 7.0 ~ 10.0 of described sea cucumber homogenate, add proteolytic enzyme I, make sea cucumber albumen and proteolytic enzyme I carry out enzyme digestion reaction under 40 ~ 60 DEG C of conditions; Described proteolytic enzyme I is Sumizyme MP or trypsinase;
3) step 2) enzyme digestion reaction carries out after 2 ~ 4h, keeps the temperature of reaction system of described enzyme digestion reaction constant, in described reaction system, add proteinase II, and continue enzymolysis 2 ~ 4h, termination reaction, obtains sea cucumber protein hydrolyte;
4) by step 3) the sea cucumber protein hydrolyte of gained is warming up to 75 ~ 95 DEG C, keeps 15 ~ 30min, deactivation proteolytic enzyme I wherein and proteinase II; The centrifugal removal of impurity after cooling, retains supernatant liquor; Described proteinase II is any one in compound protease, papoid and neutral protease;
5) to step 4) add 95% ethanol in gained supernatant liquor, its final volume is made to reach 60 ~ 80% of total system, centrifuging and taking supernatant liquor after 4 DEG C of standing 10 ~ 15h, regulate described supernatant liquor pH value to 6.0 ~ 8.0, and be the ultra-filtration membrane of 3000 ~ 10000Da by the molecular weight that dams by described supernatant liquor, collect ultrafiltrated, obtain sea cucumber albumen hypoglycemic peptide.
6) by soluble in water for microencapsulation wall material, wall material solution is made;
7) by step 6) the wall material solution of gained joins step 5 by a certain percentage) in the sea cucumber albumen hypoglycemic peptide that obtains, stirred at ambient temperature 0.5 ~ 2h, obtains mixing solutions;
8) to step 7) mixing solutions of gained carries out spraying dry, obtains microencapsulated protein polypeptide.
Embodiment 1
The present embodiment provides a kind of hypoglycemic peptide preparation method based on sea cucumber albumen, comprises the following steps:
1) get fresh sea cucumber to clean, gill, pulverize after lyophilize, obtain Stichopus japonicus powder; Take 10g Stichopus japonicus powder, add 200mL deionized water, in the water-bath of 90 DEG C, heat 20min.Homogenate power is homogenate 5min under 2000r/min condition, regulates pH to 9.0 with sodium carbonate solution.Add Sumizyme MP 20KU, under 60 DEG C of water bath condition, hydrolysis 3h, then, directly adds papoid 30KU, and continue hydrolysis 3h, measuring now enzymolysis solution degree of hydrolysis is 45.2%.The sea cucumber protein enzymatic hydrolyzate of gained is warming up to 85 DEG C, and keep 15min, go out enzyme.Get supernatant liquor after the centrifugal 15min of 4000r/min, add 95% ethanol and make its final volume be 80% (volume ratio), 4 DEG C of standing 10h.Centrifuging and taking supernatant liquor, regulates supernatant liquor pH to 7.0, and is the ultra-filtration membrane of 10000Da by the molecular weight that dams by supernatant liquor, and collect ultrafiltrated, obtain sea cucumber albumen hypoglycemic peptide solution 15g, wherein content of peptides is 6.8g.
2) take 13.6g condensed whey protein, add 150mL water, make it dissolve completely.Then, joined in described sea cucumber albumen hypoglycemic peptide solution, stirring at room temperature 2h, spray-dried obtained microencapsulation polypeptide powder.
Degree of hydrolysis can measure by the following method:
Draw 0.5mL to have gone out the sample of enzyme, cool and be transferred in 25mL volumetric flask, with 1% sodium lauryl sulphate (SDS) solution constant volume, standing.Get SDS solution and the 1mL phosphoric acid buffer (pH 8.2 of 0.125mL enzymolysis solution, 0.2mol/L) and 1mL 0.1% trinitro-benzene-sulfonic acid method (TNBS) solution vibration mixing, in 50 DEG C of water-bath lucifuge reaction 60min, react complete after, by the HCl termination reaction immediately of 2mL 0.1mol/L, room temperature measures its absorbance after placing 30min under 420nm.0 ~ 5.0 × 10-3mol/L L-Leu does typical curve.Degree of hydrolysis following formula calculates:
In formula, h: to represent in hydrolyzate every gram of cleaved peptide bond mmole number (meq/g protein);
Htot: represent peptide bond mmole number total in every gram of material protein.
Embodiment 2
1) take 10g Stichopus japonicus powder, add 250mL deionized water, in the water-bath of 85 DEG C, heat 30min.Homogenate power is homogenate 8min under 1500r/min condition, regulates pH to 7.0 with lactic acid solution.Add trypsinase 80KU, under 40 DEG C of water bath condition, hydrolysis 3h, then, directly adds papoid 30KU, and continue hydrolysis 3h, measuring now enzymolysis solution degree of hydrolysis is 42.5%.The sea cucumber protein enzymatic hydrolyzate of gained is warming up to 85 DEG C, and keep 15min, go out enzyme.Get supernatant liquor after the centrifugal 15min of 4000r/min, add 95% ethanol and make its final volume be 60% (volume ratio), 4 DEG C of standing 12h.Centrifuging and taking supernatant liquor, regulates supernatant liquor pH to 7.0, and is the ultra-filtration membrane of 3000Da by the molecular weight that dams by supernatant liquor, and collect ultrafiltrated, obtain sea cucumber albumen hypoglycemic peptide 13.16g, wherein content of peptides is 6.4g.
2) take 12.8g condensed whey protein and 0.36g sodium alginate, add 150mL water, make it dissolve completely.Then, joined in described sea cucumber albumen hypoglycemic peptide solution, stirring at room temperature 2h, spray-dried obtained microencapsulation polypeptide powder.Degree of hydrolysis measuring method is with embodiment 1.
Embodiment 3
1) take 10g Stichopus japonicus powder, add 250mL deionized water, in the water-bath of 75 DEG C, heat 30min.Homogenate power is homogenate 5min under 2000r/min condition, regulates pH to 10.0 with sodium hydrogen carbonate solution.Add Sumizyme MP 40KU, under 50 DEG C of water bath condition, hydrolysis 3h, then, directly adds compound protease 80KU, and continue hydrolysis 3h, measuring now enzymolysis solution degree of hydrolysis is 41.8%.The sea cucumber protein enzymatic hydrolyzate of gained is warming up to 75 DEG C, and keep 30min, go out enzyme.Get supernatant liquor after the centrifugal 15min of 4000r/min, add 95% ethanol and make its final volume be 70% (volume ratio), 4 DEG C of standing 10h.Centrifuging and taking supernatant liquor, regulates supernatant liquor pH to 8.0, and is the ultra-filtration membrane of 8000Da by the molecular weight that dams by supernatant liquor, and collect ultrafiltrated, obtain sea cucumber albumen hypoglycemic peptide 15g, wherein content of peptides is 6.5g.
2) take 75.0g condensed whey protein, add 150mL water, make it dissolve completely.Then, joined in described sea cucumber albumen hypoglycemic peptide solution, stirring at room temperature 0.5h, spray-dried obtained microencapsulation polypeptide powder.Degree of hydrolysis measuring method is with embodiment 1.
Embodiment 4
1) take 10g Stichopus japonicus powder, add 250mL deionized water, in the water-bath of 95 DEG C, heat 20min.Homogenate power is homogenate 8min under 1500r/min condition, regulates pH to 8.0 with solution of potassium carbonate.Add trypsinase 30KU, under 40 DEG C of water bath condition, hydrolysis 2h, then, directly adds neutral protease 40KU, and continue hydrolysis 4h, measuring now enzymolysis solution degree of hydrolysis is 39.8%.The sea cucumber protein enzymatic hydrolyzate of gained is warming up to 85 DEG C, and keep 15min, go out enzyme.Get supernatant liquor after the centrifugal 15min of 4000r/min, add 95% ethanol and make its final volume be 60% (volume ratio), 4 DEG C of standing 15h.Centrifuging and taking supernatant liquor, regulates supernatant liquor pH to 7.0, and is the ultra-filtration membrane of 10000Da by the molecular weight that dams by supernatant liquor, and collect ultrafiltrated, obtain sea cucumber albumen hypoglycemic peptide 15.2g, wherein content of peptides is 6.5g.
2) take 13.6g condensed whey protein, add 100mL water, make it dissolve completely.Then, joined in described sea cucumber albumen hypoglycemic peptide solution, stirring at room temperature 1.5h, spray-dried obtained microencapsulation polypeptide powder.Degree of hydrolysis measuring method is with embodiment 1.
Embodiment 5
1) take 10g Stichopus japonicus powder, add 250mL deionized water, in the water-bath of 95 DEG C, heat 15min.Homogenate power is homogenate 10min under 1500r/min condition, regulates pH to 8.0 with potassium bicarbonate solution.Add trypsinase 30KU, under 40 DEG C of water bath condition, hydrolysis 2h, then, directly adds compound protease 20KU, and continue hydrolysis 4h, measuring now enzymolysis solution degree of hydrolysis is 40.5%.The sea cucumber protein enzymatic hydrolyzate of gained is warming up to 85 DEG C, and keep 15min, go out enzyme.Get supernatant liquor after the centrifugal 15min of 4000r/min, add 95% ethanol and make its final volume be 60% (volume ratio), 4 DEG C of standing 15h.Centrifuging and taking supernatant liquor, regulates supernatant liquor pH to 7.0, and is the ultra-filtration membrane of 6000Da by the molecular weight that dams by supernatant liquor, and collect ultrafiltrated, obtain sea cucumber albumen hypoglycemic peptide 15.6g, wherein content of peptides is 6.4g.
2) take 16.2g condensed whey protein, add 250mL water, make it dissolve completely.Then, joined in described sea cucumber albumen hypoglycemic peptide solution, stirring at room temperature 1h, spray-dried obtained microencapsulation polypeptide powder.Degree of hydrolysis measuring method is with embodiment 1.
Embodiment 6
1) take 10g Stichopus japonicus powder, add 300mL deionized water, in the water-bath of 85 DEG C, heat 30min.Homogenate power is homogenate 5min under 3500r/min condition, regulates pH to 10.0 with potassium hydroxide solution.Add Sumizyme MP 40KU, under 50 DEG C of water bath condition, hydrolysis 4h, then, directly adds neutral protease 50KU, and continue hydrolysis 2h, measuring now enzymolysis solution degree of hydrolysis is 40.8%.The sea cucumber protein enzymatic hydrolyzate of gained is warming up to 90 DEG C, and keep 15min, go out enzyme.Get supernatant liquor after the centrifugal 15min of 4000r/min, add 95% ethanol and make its final volume be 60% (volume ratio), 4 DEG C of standing 12h.Centrifuging and taking supernatant liquor, regulates supernatant liquor pH to 6.0, and is the ultra-filtration membrane of 8000Da by the molecular weight that dams by supernatant liquor, and collect ultrafiltrated, obtain sea cucumber albumen hypoglycemic peptide 15.6g, wherein content of peptides is 6.5g.
2) take 14.6g condensed whey protein and 1.46g sodium alginate, add 200mL water, make it dissolve completely.Then, joined in described sea cucumber albumen hypoglycemic peptide solution, stirring at room temperature 1.5h, spray-dried obtained microencapsulation polypeptide powder.Degree of hydrolysis measuring method is with embodiment 1.
In above-described embodiment, adjust ph alkali lye used is any one in food grade sodium carbonate, sodium bicarbonate, Sodium phosphate dibasic, salt of wormwood, saleratus, dipotassium hydrogen phosphate or potassium hydroxide solution; Adjust ph acid solution used is any one in food grade lactic acid or citric acid solution.
Comparative example 1
Take 10g Stichopus japonicus powder, add 200mL deionized water, in the water-bath of 90 DEG C, heat 20min.Homogenate power is homogenate 5min under 2000r/min condition, regulates pH to 9.0.Add Sumizyme MP 30KU, under 55 DEG C of water bath condition, hydrolysis 6h, constantly add in hydrolytic process alkali lye maintain reaction system pH constant, measure now enzymolysis solution and be hydrolyzed to 25.8%; The sea cucumber protein enzymatic hydrolyzate of gained is warming up to 85 DEG C, and keep 15min, go out enzyme.Get supernatant liquor after the centrifugal 15min of 4000r/min, add 95% ethanol and make its final volume be 60% (volume ratio), 4 DEG C of standing 10h.Centrifuging and taking supernatant liquor, regulates supernatant liquor pH to 7.0, and is the ultra-filtration membrane of 10000Da by the molecular weight that dams by supernatant liquor, collects ultrafiltrated, obtains sea cucumber albumen hypoglycemic peptide.Degree of hydrolysis measuring method is with embodiment 1.
By above-described embodiment and comparative example known, method of the present invention effectively can improve the degree of hydrolysis of sea cucumber albumen, and can cover the bad flavor of sea cucumber protein peptide.Simple for process described in the inventive method, be beneficial to suitability for industrialized production.
Test example 1
The present embodiment provides the application of a kind of hypoglycemic peptide of the sea cucumber albumen obtained based on the hypoglycemic peptide preparation method of sea cucumber albumen in food source property hypoglycemic protective foods.
1. experiment material and method
1.1 experiment material
1) sea cucumber protein peptide
The sea cucumber protein peptide that embodiment 1 is obtained.
2) laboratory animal
C57BL/6 mouse, male, 10 ~ 12 week age, 50.
3) key instrument and reagent
Blood glucose meter, total cholesterol test kit, Triglyceride Reagent box, high density lipoprotein cholesterol test kit and low density lipoprotein cholesterol test kit etc.
4) other materials
High lipid food (composition comprises 73% basal feed, 10% lard, 10% yolk powder, 1% cholesterol and 6% milk powder).
1.2 experimental technique
1) foundation of obese model
After 50 C57BL/6 mouse feed 1 week with basal feed adaptability, carry out random packet according to body weight.40 C57BL/6 mouse are fed high lipid food 12 weeks continuously, set up fat C57BL/6 mouse model.10 C57BL/6 mouse are fed chow diet, as Normal group.
2) experimental design
By 40 C57BL/6 obesity mices, be divided into 4 groups, often organize 10, be set to dosage group and sea cucumber protein peptide high dose group in obese model control group, sea cucumber protein peptide low dose group, sea cucumber protein peptide respectively.Specific experiment animal grouping and dosage as described in Table 1.
The grouping of table 1 laboratory animal and dosage
| Group | Feed for nursing | Gavage medicine | Dosage (mg/kg b.w.) |
| Normal group | Chow diet | Distilled water | —— |
| Obese model control group | High lipid food | Distilled water | —— |
| Sea cucumber protein peptide low dose group | High lipid food | Sea cucumber protein peptide | 100 |
| Dosage group in sea cucumber protein peptide | High lipid food | Sea cucumber protein peptide | 200 |
| Sea cucumber protein peptide high dose group | High lipid food | Sea cucumber protein peptide | 400 |
3) feeding manner
Often organizing 10 C57BL/6 mouse is that 1 mouse cage is raised, and 5 groups of experiments are carried out simultaneously, to feed DIFFERENT FEED, normally feed water according to the design of table 1.
4) administering mode
Measure rat body weight before administration every day, according to the different dosing dosage of different group, be dissolved in the distilled water of same volume, with the mode administration of gavage.Timing every day gastric infusion once.Test continuous 6 weeks, experimental session mouse freely drinks water and ingests.
5) index determining
After experiment terminates, fasting 12h, measures body weight.After anesthesia, broken end gets blood, collect serum, measure fasting plasma glucose, triglyceride level (TC), total cholesterol (TG), high density lipoprotein cholesterol (HDL-c) and low density lipoprotein cholesterol (LDL-c) in serum.
2. experimental result and analysis
Different treatment group C57BL/6 Mouse Weight and the every biochemical indicator of blood, as described in Table 2.
Table 2 different treatment group C57BL/6 Mouse Weight and the every biochemical indicator of blood
Note: numerical value represents with the form of mean+SD; Different letter represents significant difference, p<0.05.
Each dosage group Mice Body weight average is significantly lower than hyperlipidemia model group (p<0.05).Middle dosage group and high dose group Mouse Weight and Normal group Mouse Weight difference are not significantly (p>0.05).
Compared with hyperlipidemia model group, the fasting plasma glucose of middle dosage group and high dose group mouse, TC content are significantly lower than hyperlipidemia model group (p<0.05); Between middle dosage group and high dose group, difference is not significantly (p>0.05).
Hyperlipidemia model group and each dosage group mouse TG content are significantly higher than Normal group (p<0.05); Each dosage group mouse TG content is significantly lower than hyperlipidemia model group (p<0.05); Between each dosage group, difference is not significantly (p>0.05).
Middle dosage group and high dose group mouse HDL-c content are significantly higher than hyperlipidemia model group (p<0.05), and difference remarkable (p>0.05) between Normal group; Between middle dosage group and high dose group, difference is not significantly (p>0.05).
Hyperlipidemia model group and each dosage group mouse LDL-c content are significantly higher than Normal group (p<0.05), and middle dosage group and high dose group mouse LDL-c content are significantly lower than hyperlipidemia model group (p<0.05); Between middle dosage group and high dose group, difference is not significantly (p>0.05).
3. experiment conclusion
The sea cucumber protein peptide of 200mg/kg b.w. dosage, can the increase of effective obesity controlling C57BL/6 Mouse Weight, reduce the triglyceride level in its fasting plasma glucose and blood, total cholesterol and low density lipoprotein cholesterol content, raise the high density lipoprotein cholesterol content in its blood.Visible, long-term taking sea cucumber of the present invention protein peptide has certain regulating effect to body blood sugar and blood fat, has certain auxiliary curative effect to treatment hyperglycemia and hyperlipidaemia.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1., based on a hypoglycemic peptide preparation method for sea cucumber albumen, it is characterized in that comprising the following steps:
1) get fresh sea cucumber to clean, gill, pulverize after lyophilize, obtain Stichopus japonicus powder; Get Stichopus japonicus powder, add deionized water by 1:20 ~ 1:30 part by weight, then in 75 ~ 95 DEG C of water-baths, heat 15 ~ 30min; Under homogenate power 1500 ~ 3500r/min condition, homogenate 5 ~ 10min, obtains sea cucumber homogenate;
2) regulating step 1) pH value to 7.0 ~ 10.0 of described sea cucumber homogenate, add proteolytic enzyme I, make sea cucumber albumen and proteolytic enzyme I carry out enzyme digestion reaction under 40 ~ 60 DEG C of conditions; Described proteolytic enzyme I is Sumizyme MP or trypsinase;
3) step 2) enzyme digestion reaction carries out after 2 ~ 4h, keeps the temperature of reaction system of described enzyme digestion reaction constant, in described reaction system, add proteinase II, and continue enzymolysis 2 ~ 4h, termination reaction, obtains sea cucumber protein hydrolyte;
4) the sea cucumber protein hydrolyte of step 3) gained is warming up to 75 ~ 95 DEG C, keeps 15 ~ 30min, deactivation proteolytic enzyme I wherein and proteinase II; The centrifugal removal of impurity after cooling, retains supernatant liquor; Described proteinase II is any one in compound protease, papoid and neutral protease;
5) in step 4) gained supernatant liquor, 95% ethanol is added, its final volume is made to reach 60 ~ 80% of total system, centrifuging and taking supernatant liquor after 4 DEG C of standing 10 ~ 15h, regulate described supernatant liquor pH value to 6.0 ~ 8.0, and be the ultra-filtration membrane of 3000 ~ 10000Da by the molecular weight that dams by described supernatant liquor, collect ultrafiltrated, obtain sea cucumber albumen hypoglycemic peptide.
2. a kind of hypoglycemic peptide preparation method based on sea cucumber albumen according to claim 1, characterized by further comprising the step of following microencapsulation process:
6) by soluble in water for microencapsulation wall material, wall material solution is made;
7) joined by a certain percentage by the wall material solution of step 6) gained in the sea cucumber albumen hypoglycemic peptide that step 5) obtains, stirred at ambient temperature 0.5 ~ 2h, obtains mixing solutions;
8) spraying dry is carried out to the mixing solutions of step 7) gained, obtain microencapsulated protein polypeptide.
3. a kind of hypoglycemic peptide preparation method based on sea cucumber albumen according to claim 1, is characterized in that, described step 2) in the add-on of proteolytic enzyme I be that every 100g Stichopus japonicus powder adds 200 ~ 800KU proteolytic enzyme I.
4. a kind of hypoglycemic peptide preparation method based on sea cucumber albumen according to claim 1, is characterized in that, in described step 3), the add-on of proteinase II is that every 100g Stichopus japonicus powder adds 200 ~ 800KU proteinase II.
5. a kind of hypoglycemic peptide preparation method based on sea cucumber albumen according to claim 1, it is characterized in that, described step 2) in adjust ph alkali lye used be any one in food grade sodium carbonate, sodium bicarbonate, Sodium phosphate dibasic, salt of wormwood, saleratus, dipotassium hydrogen phosphate or potassium hydroxide solution; Adjust ph acid solution used is any one in food grade lactic acid or citric acid solution.
6. a kind of hypoglycemic peptide preparation method based on sea cucumber albumen according to claim 2, it is characterized in that, the wall material in described step 7) is the condensed whey protein of protein content >=80%.
7. a kind of hypoglycemic peptide preparation method based on sea cucumber albumen according to claim 2, it is characterized in that, wall material in described step 7) is the mixture of the condensed whey protein of sodium alginate and protein content >=80%, and condensed whey protein and sodium alginate mixed weight are than being 1:10 ~ 1:35.
8. a kind of hypoglycemic peptide preparation method based on sea cucumber albumen according to claim 2, it is characterized in that, in described step 7), the mass ratio of sea cucumber albumen hypoglycemic peptide and wall material is 1:1 ~ 1:5.
9. the application of hypoglycemic peptide in food source property hypoglycemic protective foods of the sea cucumber albumen obtained based on the hypoglycemic peptide preparation method of sea cucumber albumen described in any one of claim 1 ~ 8.
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