CN108783461A - Ultrasonic wave added/combined-enzyme method synchronizes the method for extraction coix seed active constituent and its preparation method and application of microemulsion - Google Patents
Ultrasonic wave added/combined-enzyme method synchronizes the method for extraction coix seed active constituent and its preparation method and application of microemulsion Download PDFInfo
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- CN108783461A CN108783461A CN201810495480.1A CN201810495480A CN108783461A CN 108783461 A CN108783461 A CN 108783461A CN 201810495480 A CN201810495480 A CN 201810495480A CN 108783461 A CN108783461 A CN 108783461A
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Classifications
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Health & Medical Sciences (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
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- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
Ultrasonic wave added/combined-enzyme method synchronizes the method for extraction coix seed active constituent and its preparation method and application of microemulsion, belongs to biomedicine technical field.The extracting method of the coix seed active constituent is made by following steps：Distilled water mixing is added in extracting degreasing Job's tears powder drying and crushing, sieving, and amylase is added, and alkali protease, ultrasound-assisted extraction is added in ultrasound-assisted extraction, and enzyme deactivation is centrifuged after being rapidly cooled to room temperature, collects supernatant and obtains coix seed activated protein；Ethyl alcohol is added in centrifugation, and coix seed active polysaccharide is obtained through precipitation filtering, centrifugation, concentrate drying；Coix seed active polysaccharide is handled using gel chromatography column, elution, and eluent is concentrated under reduced pressure through ultrafiltration membrane UF membrane, obtains coix seed polysaccharide extraction concentrate.The present invention prepares the method that the ultrasonic wave added/combined-enzyme method provided synchronizes extraction, and the total content of Thick many candies is 10-15%（Phenol sulfuric acid procedure）, reduce the viscosity of Aqueous extracts.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to ultrasonic wave added/combined-enzyme method synchronizes extraction coix seed and lives
The property method of ingredient and its preparation method and application of microemulsion.
Background technology
Job's tears（Coix lacroyma-jobi L. var.ma-yuen Stapf）For grass family annual plant, also cry
Jobstears, meter Ren.Its seed decapsidate and kind skin are coix seed (coix lachryma-jobi kernel), are traditional medicines
Eat homologous food.Five kinds of constituent contents of K, Ca, Na, Mg, P are higher in coix seed, to helping human body to maintain acid-base balance, promoting people
The activity of internal certain enzymes is cooperateed with protein and is played an important role in maintenance body osmotic pressure, is a kind of good nutrition
The cereal of balance.Unsaturated fatty acid content is higher in coix seed oil, also contains polysaccharide, thiamine, riboflavin, niacin, dimension
Raw element E etc..
Coixan（Coxian）For the heteromeric polysaccharide of non-sulfuric acid, contains acetylamino in molecule, have in the solution
Preferable dispersibility.Coixan has significant bioactivity and healthcare function, such as antitumor, hypoglycemic, norcholesterol,
The various actives functions such as bacterial-infection resisting and enhancing are immune.Meanwhile coixan can inhibit muscle glycogen glycolysis, hepatic glycogen point
Solution inhibits gluconeogenesis, and then achievees the purpose that make blood glucose level reduction.Meanwhile coixan can be improved effectively
Internal superoxide dismutase activity (SOD), sweet peptide peroxidase (GSH-Px) activity of paddy flesh and reduced form in red blood cell
The sweet peptide of paddy flesh (GSH) concentration, reduces the oxidative stress of body, prevents the peroxidization of lipid, reduces liver malonaldehyde
(MDA) content realizes its internal antioxidation, reduces and repair the damage suffered by liver cell.Its water extract is carried out
Separation screening, obtain Polysaccharide A, B, C separately constitute for：Polysaccharide A (rhamnose：Arabinose：Xylose：Sweet sugar dew sugar：Gala
Sugar) by (1：1：1：11：10) it forms；Polysaccharide B (rhamnose:Arabinose：Xylose：Mannose：Galactolipin：Glucose) by (3：
18：13：3：10：5) it forms；Polysaccharide C is a kind of glucan.
In China, diabetes B is multiple-factor inheritance and different substantiality disease, and illness rate increases year by year.China's adult blood drop,
Dyslipidemia illness rate is 18.6%, has become serious social concern；There are about ten thousand people of 120-160 to die of painstaking effort every year in the whole world
Pipe disease and headstroke.The remedy measures of hypertension are comprehensive, and the conditioning of diet is an importance for the treatment of, is naturally being produced
Found in object diabetes, hypertension supplementary therapy functional food, related drugs and preventative functional food are developed, to subtract
Few side effects of pharmaceutical drugs simultaneously improve symptom, improve the quality of living.Have no that combined-enzyme method ultrasonic wave added synchronizes extraction coix seed at present
Polysaccharide is used for the exploitation of lower hyperlipidemia, hypertension, hyperglycemia functional product.
Invention content
In view of the problems of the existing technology, it is an object of the invention to design to provide a kind of ultrasonic wave added/combined-enzyme method
The technical solution of the method for synchronous extraction coix seed active constituent and its preparation method and application of microemulsion.The present invention uses nothing
It is malicious, harmless, noresidue is dangerous, with supercritical CO2By-product-degreasing Job's tears powder is to process raw material after extraction coixenolide, is passed through
Ultrasonic wave added adjusts feed liquid, and alpha-amylase is added, and adjusts pH value addition alkali protease and is digested, enzyme deactivation after enzymolysis is fast
Speed is centrifuged after being cooled to room temperature, and collects supernatant and constant volume, measures protein content.With the protein extract of extraction
For raw material, by 1：5 are added absolute ethyl alcohol, and polysaccharide active matter, the Job's tears of acquisition are obtained through precipitation filtering, centrifugation, concentrate drying
Benevolence active polysaccharide component.
The ultrasonic wave added/combined-enzyme method synchronizes extraction coix seed albumen, active polysaccharide component, prepares coixan micro emulsion
Liquid, by animal experiments show that with serum total cholesterol index, serum triglyceride index, blood markers, sky is substantially reduced
The resistance to index of abdomen blood glucose, blood, can be used for the fields such as food, health products, medicine, biochemical industry, be particularly suitable for exploitation drop " three high " work(
It can property food.The yield for effectively inhibiting raising polysaccharide, reduces the viscosity of Aqueous extracts.While the application of complex enzyme, ultrasonic wave added can
Effectively to reduce the generation of product denaturation, the problems such as having a poor flavour.Coix seed added value is greatly improved, product matter can be effectively improved
Amount and utilization rate.
The method that the ultrasonic wave added/combined-enzyme method synchronizes extraction coix seed active constituent, it is characterised in that by following
Step is made：
1）Extracting degreasing Job's tears powder drying and crushing crosses 40-100 mesh sieve, distilled water mixing is added, and amylase, ultrasonic wave auxiliary is added
Alkali protease, ultrasound-assisted extraction is added in extraction, and enzyme deactivation is centrifuged after being rapidly cooled to room temperature, collects supernatant
Liquid obtains coix seed activated protein；
2）Take step 1）Ethyl alcohol is added in obtained centrifugation, and coix seed activity is obtained through precipitation filtering, centrifugation, concentrate drying
Polysaccharide；
3）By step 2）The coix seed active polysaccharide of gained is respectively adopted DEAE-52, at Sephadex G-100 gel chromatography columns
Reason, with 5-10 times of column volume water elution, eluent is concentrated under reduced pressure through ultrafiltration membrane UF membrane, obtains coix seed polysaccharide extraction
Concentrate.
The method that the ultrasonic wave added/combined-enzyme method synchronizes extraction coix seed active constituent, it is characterised in that described
Step 1）By weight 1 in middle Job's tears powder：Distilled water is added in the solid-liquid ratio of 5-20.
The method that the ultrasonic wave added/combined-enzyme method synchronizes extraction coix seed active constituent, it is characterised in that described
Step 1）Middle amylase enzymolysis condition is：Amylase addition is the 0.5-1%, pH 5-7, temperature 40-50 of Job's tears powder weight
℃。
The method that the ultrasonic wave added/combined-enzyme method synchronizes extraction coix seed active constituent, it is characterised in that described
Step 1）Middle ultrasound-assisted extraction condition is：Ultrasonic power is 400-800W, extraction time 20-100 minutes.
The method that the ultrasonic wave added/combined-enzyme method synchronizes extraction coix seed active constituent, it is characterised in that described
Step 1）Neutral and alkali protease hydrolyzed condition is：Alkali protease addition is 0.5-1%, the pH 8-12 of Job's tears powder weight, temperature
Degree is 50-70 DEG C.
The method that the ultrasonic wave added/combined-enzyme method synchronizes extraction coix seed active constituent, it is characterised in that described
Step 2）Middle precipitation and the weight ratio of absolute ethyl alcohol are 1：0.5.
The method that obtained coix seed polysaccharide extraction concentrate prepares coixan microemulsion, it is characterised in that packet
Include following steps：
Take coix seed polysaccharide extraction concentrate 5-20 parts by weight, pectin 30-60 parts by weight, sodium alginate 10-20 parts by weight,
Vitamin E 1-5 parts by weight, vitamin C 1-10 parts by weight, it is high-pressure homogeneous at 30-50 DEG C of temperature, obtain dispersion phase average
The stabilization coixan microemulsion of 300-500 μm of grain size.
The method, it is characterised in that take coix seed polysaccharide extraction concentrate 8-15 parts by weight, pectin 35-55 weights
Measure part, sodium alginate 12-18 parts by weight, vitamin E 2-4 parts by weight, vitamin C 2-8 parts by weight.
The method, it is characterised in that pill, granule or tablet is made in coixan microemulsion.
Application of the coixan microemulsion in preparing reducing blood sugar and blood fat functional food.
The application, feature is in the 0.02- that the coixan microemulsion additive amount is functional food
30%。
The present invention has the advantages that with the prior art：
1, the extracting method of existing coix seed functional active components cannot synchronize extraction coix seed albumen and coixan.This hair
Bright purpose is to provide a kind of ultrasonic wave added/combined-enzyme method and synchronizes extraction coix seed albumen, active polysaccharide component, prepares coix seed
Polysaccharide microemulsion.
2, coixan microemulsion prepared by the present invention, by animal experiments show that solid with the total courage of serum is substantially reduced
Alcohol index, serum triglyceride index, blood markers, fasting blood-glucose, the resistance to index of blood, can be used for food, health products, medicine, life
The fields such as materialization work are particularly suitable for exploitation drop " three high " functional food.
3, the present invention prepares the method that the ultrasonic wave added/combined-enzyme method provided synchronizes extraction, and the total content of Thick many candies is 10-
15%（Phenol sulfuric acid procedure）, reduce the viscosity of Aqueous extracts.The application of complex enzyme, ultrasonic wave added simultaneously can effectively reduce product change
The generation of property, the problems such as having a poor flavour.Coix seed added value is greatly improved, product quality and utilization rate can be effectively improved.
Description of the drawings
Fig. 1 is the morphologic variation diagram of diabetic mice islet tissue.
Specific implementation mode
It further illustrates the present invention with reference to embodiments.
Embodiment 1：
Extracting degreasing Job's tears powder drying and crushing crosses 50 mesh sieve, according to 1：Distilled water is added in 10 solid-liquid ratio, and the dosage of enzyme is Job's tears powder
0.5%, the pH 3 of weight under the conditions of temperature 45 C, with ultrasound-assisted extraction 50 minutes, extracts albumin.Adjusting pH value is
10, alkali protease is added, the dosage of enzyme is the 0.5% of Job's tears powder weight, under the conditions of temperature 50 C, uses ultrasound-assisted extraction
It is digested within 50 minutes, enzyme deactivation after enzymolysis is centrifuged after being rapidly cooled to room temperature, collects supernatant measurement protein and contains
Amount is 15%.
It is precipitated as raw material, according to 1：Distilled water is added in 10 solid-liquid ratio, and it is 7 to adjust pH value, under the conditions of 95 DEG C of temperature, uses
After ultrasound-assisted extraction 50 minutes, it is centrifuged.1 is pressed in extracting solution：0.5 be added absolute ethyl alcohol, through precipitation filtering, from
The heart, concentrate drying obtain polysaccharide active matter, and the coixan active component of acquisition is 15%.
By 20 parts by weight of coixan active component concentrate, 40 parts by weight of pectin, 10 parts by weight of sodium alginate, dimension life
5 parts by weight of plain E, 5 parts by weight of vitamin C, it is high-pressure homogeneous at 40 DEG C of temperature, obtain the steady of 450 μm of particle diameter of dispersing phase
Determine coixan microemulsion, contains coixan 20%, encapsulation rate 50%.
Embodiment 2
Extracting degreasing Job's tears powder drying and crushing crosses 50 mesh sieve, according to 1：Distilled water is added in 20 solid-liquid ratio, and the dosage of enzyme is Job's tears powder
1%, the pH 5 of weight under the conditions of 30 DEG C of temperature, with ultrasound-assisted extraction 100 minutes, extracts albumin.It is 9 to adjust pH value,
Alkali protease is added, the dosage of enzyme is the 1% of Job's tears powder weight, under the conditions of temperature 70 C, is divided with ultrasound-assisted extraction 100
Clock is digested, and enzyme deactivation after enzymolysis is centrifuged after being rapidly cooled to room temperature, is collected supernatant measurement protein content and is
17%。
It is precipitated as raw material, according to 1：Distilled water is added in 20 solid-liquid ratio, and it is 7 to adjust pH value, under the conditions of 80 DEG C of temperature, uses
After ultrasound-assisted extraction 100 minutes, it is centrifuged.1 is pressed in extracting solution：0.5 is added absolute ethyl alcohol, filtered through precipitation,
Centrifugation, concentrate drying obtain polysaccharide active matter, and the coixan active component of acquisition is 17%.
By 10 parts by weight of coixan active component concentrate, 20 parts by weight of pectin, 10 parts by weight of sodium alginate, dimension life
2 parts by weight of plain E, 2 parts by weight of vitamin C, it is high-pressure homogeneous at 30 DEG C of temperature, obtain the steady of 400 μm of particle diameter of dispersing phase
Determine coixan microemulsion, contains coixan 15%, encapsulation rate 42%.
3 function assessment evaluation experimental of embodiment
One, animal experiment
1, given the test agent：Implement 1 art method coixan microemulsion according to the present invention, the used time is made into various concentration polysaccharide
C1, polysaccharide C2, polysaccharide C3 applications.
2, animal subject：SPF grades of ICR mouse, male, week old are 6-8 weeks, weight 20g-22g（Shanghai Si Laike is dynamic
Object experimental animal Co., Ltd provides）.
3, experiment packet：Six groups of experiment point：Normal group, model group, positive group（Melbine）, tested group of three agent
Amount group（Polysaccharide C1, polysaccharide C2, polysaccharide C3）, every group of 10 animals.
4, feeding environment：According to standard animal care, 22-25 DEG C of indoor temperature, relative humidity 50%-60% is provided with feed,
Free water.
Two, experimental method reference《Health food detects and assessment technique specification》The middle auxiliary hyperglycemic function method of inspection with
Operation carries out.
1, the diabetes mice model of SZT inductions
1.1 sodium citrate buffer solutions are prepared：2.10g citric acids addition distilled water 100ml is made into citric acid mother liquor, referred to as A liquid；
2.94g trisodium citrates addition distilled water 100ml is made into sodium citrate mother liquor, referred to as B liquid；A, B liquid are pressed 1:1.32 ratio
Mixing, pH meter measure pH value, set up pH value of solution=4.0, are the required 0.1mol/L sodium citrate buffer solutions for preparing STZ.
The preparation of 1.2 STZ solution：STZ is dissolved in 0.1mol/L sodium citrate buffer solutions, Fresh is at 10 mg/
0.22 μm of bacteria filter filtration sterilization is used in combination in the STZ solution of mL concentration.Pay attention to being protected from light preparation, matching while using.
1.3 modeling method：Preoperative 12h fasting, model group mouse carry out abdominal cavity note according to the STZ of 120mg/kg dosage
It penetrates, control group gives the sodium citrate buffer solution of same dose.After STZ injects 3d, fasting blood-glucose is surveyed, blood glucose is selected to be higher than
16.7mmol/L is included in formal experiment.
1.4 continuous gavages 15 days detect the weight and amount of drinking water of each group mouse daily, and empty insulin tolerance is surveyed after 15 days
OGTT plucks eyeball blood sampling, is stored at room temperature 2h, after in 4 DEG C, 3000r centrifuges 10 minutes extraction serum, is put into -80 refrigerators and freezes.
Pancreas islet is taken simultaneously, and liver, lungs, kidney is for pathology and molecular Biological Detection.
Three, experimental result
1. observing the diabetes mice modeling result of SZT inductions：Model group blood glucose value is 19.26 ± 0.689（mmol/L）.
2. a pair high fat diet SZT induction glycosuria sugar ICR mouse have carried out insulin tolerance OGTT（Such as table 1）, triglycerides,
The measurement of the indexs such as total cholesterol and weight in serum.SZT induces glycosuria sugar ICR mouse after continuous gavage 15 days, fasting blood
Sugar, insulin tolerance are close to melbine control group（0.25mg/w/body）.Total cholesterol and weight in triglycerides, serum
It is significantly better than model group.
Table 1 SZT induction glycosuria sugar ICR mouse insulin tolerance OGTT after continuous gavage 15 days
。
3, the morphologic variation of diabetic mice islet tissue
As shown in Figure 1, visible Normal group Mice Islet Cells marshalling is dyed through HE, fine and close, clear in structure, cell
Nuclear chromatin is evenly distributed, and extracellular matrix is less, and microvessel structure is normal, it is seen that a small amount of fibroblast, interstitial is without apparent
Inflammatory cell infiltration；Diabetic model group Mice Islet Cells are disorganized, and space between cells increases, and structure is unintelligible, has
Nuclear membrane shrinkage, and have inflammatory cell infiltration, iuntercellular also shows fibroblast infiltration.Polysaccharide group cell arrangement neatly connects
Nearly Normal group.
Claims (10)
1. the method that ultrasonic wave added/combined-enzyme method synchronizes extraction coix seed active constituent, it is characterised in that be made by following steps：
1）Extracting degreasing Job's tears powder drying and crushing crosses 40-100 mesh sieve, distilled water mixing is added, and amylase, ultrasonic wave auxiliary is added
Alkali protease, ultrasound-assisted extraction is added in extraction, and enzyme deactivation is centrifuged after being rapidly cooled to room temperature, collects supernatant
Liquid obtains coix seed activated protein；
2）Take step 1）Ethyl alcohol is added in obtained centrifugation, and coix seed activity is obtained through precipitation filtering, centrifugation, concentrate drying
Polysaccharide；
3）By step 2）The coix seed active polysaccharide of gained is respectively adopted DEAE-52, at Sephadex G-100 gel chromatography columns
Reason, with 5-10 times of column volume water elution, eluent is concentrated under reduced pressure through ultrafiltration membrane UF membrane, obtains coix seed polysaccharide extraction
Concentrate.
2. the method that ultrasonic wave added as described in claim 1/combined-enzyme method synchronizes extraction coix seed active constituent, feature exist
In the step 1）By weight 1 in middle Job's tears powder：Distilled water is added in the solid-liquid ratio of 5-20.
3. the method that ultrasonic wave added as described in claim 1/combined-enzyme method synchronizes extraction coix seed active constituent, feature exist
In the step 1）Middle amylase enzymolysis condition is：Amylase addition is 0.5-1%, the pH 5-7 of Job's tears powder weight, temperature
Degree is 40-50 DEG C.
4. the method that ultrasonic wave added as described in claim 1/combined-enzyme method synchronizes extraction coix seed active constituent, feature exist
In the step 1）Middle ultrasound-assisted extraction condition is：Ultrasonic power is 400-800W, extraction time 20-100 minutes.
5. the method that ultrasonic wave added as described in claim 1/combined-enzyme method synchronizes extraction coix seed active constituent, feature exist
In the step 1）Neutral and alkali protease hydrolyzed condition is：Alkali protease addition is the 0.5-1%, pH of Job's tears powder weight
For 8-12, temperature is 50-70 DEG C.
6. the method that ultrasonic wave added/combined-enzyme method as described in claim 1 synchronizes extraction coix seed active constituent, feature exist
In the step 2）Middle precipitation and the weight ratio of absolute ethyl alcohol are 1：0.5.
7. the method that the coix seed active constituent obtained using claim 1 prepares coixan microemulsion, it is characterised in that
Include the following steps：
Take coix seed polysaccharide extraction concentrate 5-20 parts by weight, pectin 30-60 parts by weight, sodium alginate 10-20 parts by weight,
Vitamin E 1-5 parts by weight, vitamin C 1-10 parts by weight, it is high-pressure homogeneous at 30-50 DEG C of temperature, obtain dispersion phase average
The stabilization coixan microemulsion of 300-500 μm of grain size.
8. the method for claim 7, it is characterised in that take coix seed polysaccharide extraction concentrate 8-15 parts by weight, fruit
Glue 35-55 parts by weight, sodium alginate 12-18 parts by weight, vitamin E 2-4 parts by weight, vitamin C 2-8 parts by weight.
9. the method for claim 7, it is characterised in that pill, granule or piece is made in coixan microemulsion
Agent.
10. the coixan microemulsion being prepared by claim 7 is in preparing reducing blood sugar and blood fat functional food
Application.
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