CN109536554A - A kind of ultrasonic synergistic preparation method of arrowhead resistant starch and functional polypeptide - Google Patents
A kind of ultrasonic synergistic preparation method of arrowhead resistant starch and functional polypeptide Download PDFInfo
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- CN109536554A CN109536554A CN201811510460.3A CN201811510460A CN109536554A CN 109536554 A CN109536554 A CN 109536554A CN 201811510460 A CN201811510460 A CN 201811510460A CN 109536554 A CN109536554 A CN 109536554A
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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
The invention discloses the ultrasonic synergistic preparation methods of a kind of arrowhead resistant starch and functional polypeptide, are related to the efficient preparation technical field of arrowhead resistant starch and functional polypeptide.It carries out as steps described below: arrowhead raw material is cleaned up into the water, and carry out peeling impurity elimination processing；Arrowhead raw material is weighed, water is added to be beaten, extracts arrowhead starch；Add water to arrowhead slurry after extracting arrowhead starch, Protease Treatment is then added, boil enzyme deactivation after enzymatic hydrolysis, enzymatic hydrolysis while carries out adverse current impulse ultrasound assistance enzymolysis；Centrifugal treating finally is carried out to enzymolysis liquid, supernatant is collected and just obtains polypeptide liquid, spray drying or freeze-drying obtain polypeptide powder.The present invention takes ultrasonic wave added enzymatic treatment partition method to prepare arrowhead slag anti-oxidation peptide, not only can reduce the pressure of arrowhead slurry environmental pollution, also can produce significant economic benefit and social benefit.
Description
Technical field
The present invention relates to the efficient preparation technical fields of arrowhead resistant starch and functional polypeptide, specifically utilize the side of enzymatic hydrolysis
Albumen in residue after arrowhead extraction starch is transformed into the peptide material with antioxidant activity by method, belongs to biotechnology.
Background technique
Arrowhead is also referred to as Herba Passiflorae Cupiformis, it is a kind of aquatic herbaceous plant, and planting patterns is low-lying paddy field.Fresh arrowhead is in
Now long oval or ellipse, length are about 2.2-4.5cm, and diameter is about 1.8-3.2cm.Compendium of Material Medica records kind, I
State is the source area of arrowhead, mainly lives in low-lying paddy field area.Arrowhead is to be distributed in world wide, in China, court
Arrowhead is all edible in the area such as fresh, Japanese, India, Korea.In China, whenever winter-spring season, vegetable supply not in time,
Arrowhead can serve as one of the main vegetables in supply dull season.It is edible that arrowhead acts not only as a kind of vegetables, and its medicine
It is also very high with health value.From the perspective of traditional Chinese medicine, arrowhead have the characteristics that it is sweet in flavor, slightly cold, nontoxic edible,
Has effects that the efficacy of a drug of cool blood, cough-relieving, hemostasis, removing toxic substances, at the same time, the method for eating arrowhead is also more and more.Due to arrowhead
Not only can be as drug, but also can be as food, therefore receive more concerns.Meanwhile medicinal grind is eaten about arrowhead
Studying carefully also becomes more.But so far, we about the processing of arrowhead still based on the preliminary working of the place of production, i.e., directly it is in bulk on
City, or be vacuum-packed and sell after arrowhead is cleaned, the research report of the deep processing about arrowhead is still less.It is rich in arrowhead
Starch, protein, the ingredients such as inorganic elements, the deep processing of arrowhead starch at present is mainly to extract based on arrowhead starch, still
In arrowhead process, extract arrowhead starch after arrowhead raffinate, if drying if cost it is excessively high, increase business burden, usually make
Make burial processing for feed or as waste residue, but will lead to the pollution of soil and groundwater, while producing level is lower.It extracts kind
Contain a large amount of protein in arrowhead raffinate after aunt's starch, how " eating dry squeeze only ", which makes full use of money, is accomplished to arrowhead raw material
Source?
Compared with protein, active peptide has good processing performance.As it has good physicochemical property and hydrability
Matter has good solubility and " three-lows " performance, i.e., viscosity is low, gelation is low, foaminess is low.As basic nutriment,
The bioavilability of active peptide is high, is easier to absorb than amino acid, while having effects that adjust human physiological functions, is former albumen
Not available for matter and its composed amino acid.The polypeptide of some specific structures has antioxidant activity, can eliminate freedom
Base, suppresses or eliminates and slows down oxidation reaction, antioxidation mechanism include: to antioxidase provide hydrogen, buffered physiologic pH value,
Chelated metal ions and capture free radical etc..
It is past, most although what is used in many situations of the preparation of polypeptide is also the byproduct of processing of farm products
Be be easy to get, wheat embryo best in quality, maize yellow-powder, rice protein etc., and the quality as arrowhead residue is very poor, difficult
Protein resource with high-value-use is the emphasis that scientific worker is concerned about.
Summary of the invention
It is an object of the invention to overcome arrowhead for developing low value product, cost is too high；High level product is developed, lacks and closes
The practical problems such as suitable technology provide the ultrasonic synergistic preparation method of a kind of arrowhead resistant starch and functional polypeptide.The present invention
With enzyme treatment technique, Sonication assisted treatment technology is with the High water cut arrowhead slurry generated from arrowhead starch production process
Raw material carries out the preparation of high added value anti-oxidation peptide.
To achieve the above object, production technology of the invention includes the following steps:
The ultrasonic synergistic preparation method of a kind of arrowhead resistant starch and functional polypeptide carries out as steps described below:
(1) arrowhead raw material is cleaned up into the water, and carries out peeling impurity elimination processing；
(2) arrowhead raw material is weighed, adds water to be beaten by 1: 1 mass ratio, arrowhead homogenate is put into different double-frequency ultrasound items
Ultrasonic pretreatment is carried out under part；Centrifugally operated is then carried out, lower sediment is taken after being centrifuged 20min, is put into clean culture dish
In be placed in baking oven dry 48h, be ground up, sieved, arrowhead starch；Wherein ultrasound is carried out under the different ultrasound conditions
Wave pretreatment refers to: ultrasonic time 20min, and ultrasonic power density is 180W/150mL, double-frequency ultrasound frequency are as follows: 20/
28kHz, 20/35kHz, 20/40kHz, 28/40kHz, 20/60kHz, 40/60kHz；
(3) add water to arrowhead slurry after extracting arrowhead starch, make concentration of substrate 4%-8%, unit in arrowhead slurry
For g/ml；
(4) Protease Treatment is then added, enzyme concentration 2%-8% adjusts pH and temperature, enzyme digestion reaction 40-100min；
10min is boiled after enzymatic hydrolysis, is cooled to 20-30 DEG C, adjusts material liquid pH to 7.0-7.3.Wherein the enzyme is Papain
Enzyme, food flavor enzyme, neutral proteinase, alkali protease, trypsase, pepsin are carried out according to recommendation optimal pH and temperature
Reaction.
(5) adverse current impulse ultrasound assistance enzymolysis is carried out while enzymatic hydrolysis, wherein the adverse current impulse ultrasound assistance enzymolysis,
Design parameter is 2~10s of pulse width, 1~5s of pulse spacing, ultrasonic power 80W/L, supersonic frequency 28kHz, and feed liquid is with adverse current
The mode of circulation passes through ultrasonic probe, 40~90min of ultrasonic time；
(6) centrifugal treating is carried out to enzymolysis liquid, collects supernatant and just obtain polypeptide liquid, spray drying or freeze-drying obtains
Polypeptide powder can be made into capsule.
It wherein carries out ultrasonic pretreatment under ultrasound condition different described in step (2) to refer to: double-frequency ultrasound frequency
Preferably 40/60kHz.
In above-mentioned steps (4), alkali protease is screened most preferably to use enzyme, peak enzymolysis-ability process conditions are as follows: substrate is dense
Spend 8.0%, pH9.0, temperature 50 C, enzyme concentration 8%, enzymolysis time 90min.
In above-mentioned steps (5), the adverse current impulse ultrasound assistance enzymolysis, design parameter is between pulse width 10s, pulse
Every 5s, ultrasonic power 80W/L, supersonic frequency 28kHz, feed liquid passes through ultrasonic probe, ultrasonic time in a manner of circulated in countercurrent
90min。
The advantages of above-mentioned processing step includes:
1. the residue that arrowhead Starch Production generates to be processed into the anti-oxidation peptide of high added value under mild conditions, not only
It can reduce the pressure of arrowhead slurry environmental pollution, also can produce significant economic benefit and social benefit；
2. using arrowhead slurry as raw material can not need that raw material is dried, directly progress enzyme digestion reaction, meet low-carbon
The criterion of processing.
3. counter current ultrasonic wave is applied with during biological enzyme enzymatic hydrolysis, in ultrasonic wave added enzymolysis process, when shortening enzymatic hydrolysis
Between,
Hydrolysis result is improved, the usage amount of enzyme is reduced, improves the activity of enzymolysis product.
Detailed description of the invention
Fig. 1 is multi-mode ultrasonic wave biological processing equipment structure chart of the invention, wherein 1,2,3 be ultrasonic vibrating plate, 4 be Sheng
Liquid device, 5 be water-bath, and 6 be temp probe, and 7 be circulating pump, and 8 be Computerized controller, and 9,10,11 be ultrasonic controller.
Fig. 2 is adverse current ultrasound processing equipment structural schematic diagram, and 1 pops one's head in for energy-gathered ultrasonic, and 2 be ultrasonic wave controller, 3
It is feed inlet for ultrasonic cup type chamber, 4,5 be circulating pump, and 6 be liquid holding device, and 7 be discharge port.
Specific embodiment
Enzyme used in the present invention, comprising:
Alcalase2.4LFG protease (1.325 × 105U/g, optimum temperature, 50 DEG C；PH, 8.0), Novi believes biological skill
Art Co., Ltd；Cellulase: 8 000U/g of enzyme activity, neutral proteinase (enzyme activity is 16.0 ten thousand U/g), flavor protease (enzyme
Vigor is 6.9 ten thousand U/g) it is provided by Wuxi enzyme preparation Co., Ltd, Jie Neng section.
Fig. 1 is multi-mode ultrasonic wave biological processing equipment of the invention, which is furnished with a Computerized controller 8,
Ultrasound works parameter (ultrasonic power density, frequency, pulse working time, intermittent time and processing total time) can be set to control respectively
Three ultrasonic controllers 9,10,11 are made, are separately connected the ultrasonic vibrating plate 1,2,3 of three different frequencies, it can be achieved that single-frequency/two
A frequency/tri- frequency ultrasonic wave processing；Solution to be treated is put into and carries out single-frequency/double frequency/multiple frequency ultrasonic in liquid holding device 4
Assistance enzymolysis test, starting circulating pump 7 recycle solution.Oneself of solution temperature is realized by water-bath 5 and temp probe 6
Dynamic control.
Fig. 2 is adverse current ultrasound processing equipment of the invention, which is furnished with ultrasonic wave controller 2, controls ultrasound parameter, is gathered
The material that energy formula ultrasonic probe 1 enters ultrasonic cup type chamber 3 to feed inlet 4 is handled, and it is right to start circulating pump 5 while ultrasonic
Feed liquid is recycled, and 6 be liquid holding device, and 7 be discharge port.
Plant anti-oxidation (activity) peptide can eliminate free radical, suppress or eliminate and slow down oxidation reaction.Its is anti-oxidant
Mechanism includes: to provide hydrogen, buffered physiologic pH value, chelated metal ions and capture free radical etc. to antioxidase.Lead in the present invention
Measurement enzymolysis product is crossed to the antioxidant activity of the clearance rate evaluation enzymolysis product of DPPH free radical.It is diluted after enzymolysis product enzyme deactivation
To uniform concentration, the clearance rate of DPPH free radical is measured.
The measuring method of DPPH free radical scavenging activity: enzymatic hydrolysis supernatant is diluted to different concentration gradients, takes 2mL respectively
The enzymatic hydrolysis supernatant of various concentration is added the DPPH solution that 2mL concentration is 0.04mg/ml, is uniformly mixed in test tube, reaction
After 20min, it is centrifuged 10min in 3500rpm, taking supernatant to survey its light absorption value at 517nm is Ai；Separately respectively take the above-mentioned concentration of 2mL
Supernatant soln is separately added into dehydrated alcohol 2mL in test tube, after reacting 20min, is centrifugated 10min in 3500rpm, takes
Supernatant surveys its light absorption value at 517nm and is denoted as Aj；It is reacted with 2mL 0.04mg/mL DPPH and 2mL dehydrated alcohol as ginseng
Than light absorption value is denoted as A0。
EDPPH=[1- (Ai-Aj)/A0] × 100%
In formula: clearance rate (%) of the EDPPH-enzymatic hydrolysis supernatant to DPPH free radical；
A0The light absorption value of -2mL DPPH solution addition 2mL dehydrated alcohol；
AiThe light absorption value of 2mL enzymatic hydrolysis supernatant is added in -2mL DPPH solution；
AjThe light absorption value of 2mL enzymatic hydrolysis supernatant is added in -2mL dehydrated alcohol.
EC50Calculating: EC50It is the concentration when DPPH clearance rate of enzymolysis product reaches 50%.Sample is diluted to first
A series of concentration measure each concentration samples to the clearance rate of DPPH, draw the relation curve of clearance rate and concentration, look on curve
The EC of sample out50Value.
Specific implementation of the invention is described further below with reference to embodiment.
Embodiment 1
(1) arrowhead raw material is cleaned up into the water, and carries out peeling impurity elimination processing.Then arrowhead raw material 75g is weighed,
Add water to be beaten by 1: 1 mass ratio, arrowhead homogenate is put under different ultrasound conditions and carries out ultrasonic pretreatment；Ultrasound parameter
It is 20min for ultrasonic time, ultrasonic power density is 180W/150mL, the synchronous multifrequency of ultrasound works model selection；Double-frequency ultrasound
Frequency are as follows: 40/60kHz；Centrifugally operated is then carried out, lower sediment is taken after being centrifuged 20min, is put into juxtaposition in clean culture dish
Dry 48h, obtains arrowhead starch in baking oven.
(2) arrowhead slurry then is taken, water is added to adjust concentration of substrate 4%, be added under the conditions of pH7.5, temperature 60 C neutral
Protease carries out enzyme digestion reaction, enzyme concentration 2%；Digest 10min.
(3) adverse current impulse ultrasound assistance enzymolysis is carried out while enzymatic hydrolysis, wherein the adverse current impulse ultrasound assistance enzymolysis,
Design parameter is pulse width 2s, pulse spacing 1s, ultrasonic power 80W/L, supersonic frequency 28kHz, and feed liquid is with circulated in countercurrent
Mode passes through ultrasonic probe, ultrasonic time 40min；
(4) pH value is adjusted after digesting to 7.1, boils 10min, is cooled to 21 DEG C of temperature, 3500rpm is centrifuged 10min,
Clear liquid is collected, measurement DPPH free radical scavenging activity is 30.9% after supernatant is diluted 20 times.
Embodiment 2
(1) arrowhead raw material is cleaned up into the water, and carries out peeling impurity elimination processing.Then arrowhead raw material 75g is weighed,
Add water to be beaten by 1: 1 mass ratio, arrowhead homogenate is put under different ultrasound conditions and carries out ultrasonic pretreatment；Ultrasound parameter
It is 20min for ultrasonic time, ultrasonic power density is 180W/150mL, the synchronous multifrequency of ultrasound works model selection；Double-frequency ultrasound
Frequency are as follows: 20/60kHz；Centrifugally operated is then carried out, lower sediment is taken after being centrifuged 20min, is put into juxtaposition in clean culture dish
Dry 48h, obtains arrowhead starch in baking oven.
(2) arrowhead slurry then is taken, adds water to adjust concentration of substrate 8%, is added under the conditions of pH9.0, temperature 50 C
Alcalase alkali protease carries out enzyme digestion reaction, enzyme concentration 8%；Digest 100min.
(3) adverse current impulse ultrasound assistance enzymolysis is carried out while enzymatic hydrolysis, wherein the adverse current impulse ultrasound assistance enzymolysis,
Design parameter is pulse width 10s, pulse spacing 5s, ultrasonic power 80W/L, supersonic frequency 28kHz, and feed liquid is with circulated in countercurrent
Mode passes through ultrasonic probe, ultrasonic time 90min；
(4) pH value is adjusted after digesting to 7.1, boils 10min, is cooled to 21 DEG C of temperature, 3500rpm is centrifuged 10min,
Clear liquid is collected, measurement DPPH free radical scavenging activity is 46.9% after supernatant is diluted 20 times.
Comparative example 1
(1) arrowhead raw material is cleaned up into the water, and carries out peeling impurity elimination processing.Then arrowhead raw material 75g is weighed,
Add water to be beaten by 1: 1 mass ratio, arrowhead homogenate is put into be stirred and extracts 20min；Then carry out centrifugally operated, centrifugation
Lower sediment is taken after 20min, is put into clean culture dish and is placed in baking oven dry 48h, obtains arrowhead starch.
(2) arrowhead slurry then is taken, adds water to adjust concentration of substrate 8%, is added under the conditions of pH9.0, temperature 50 C
Alcalase alkali protease carries out enzyme digestion reaction, enzyme concentration 8%；Digest 100min.
(3) pH value is adjusted after digesting to 7.1, boils 10min, is cooled to 21 DEG C of temperature, 3500rpm is centrifuged 10min,
Clear liquid is collected, measurement DPPH free radical scavenging activity is 18.1% after supernatant is diluted 20 times.
Claims (4)
1. the ultrasonic synergistic preparation method of a kind of arrowhead resistant starch and functional polypeptide, it is characterised in that as steps described below into
Row:
(1) arrowhead raw material is cleaned up into the water, and carries out peeling impurity elimination processing；
(2) arrowhead raw material is weighed, adds water to be beaten by 1: 1 mass ratio, arrowhead homogenate is put under the conditions of different double-frequency ultrasounds
Carry out ultrasonic pretreatment；Centrifugally operated is then carried out, lower sediment is taken after being centrifuged 20min, is put into clean culture dish simultaneously
It is placed in baking oven dry 48h, is ground up, sieved, obtains arrowhead starch；It is pre- that ultrasonic wave wherein is carried out under the different ultrasound conditions
Processing refers to: ultrasonic time 20min, and ultrasonic power density is 180W/150mL, double-frequency ultrasound frequency are as follows: 20/28kHz, 20/
35kHz, 20/40kHz, 28/40kHz, 20/60kHz, 40/60kHz；
(3) add water to arrowhead slurry after extracting arrowhead starch, make concentration of substrate 4%-8% in arrowhead slurry, unit g/
ml；
(4) Protease Treatment is then added, enzyme concentration 2%-8% adjusts pH and temperature, enzyme digestion reaction 40-100min；Enzymatic hydrolysis
After boil 10min, be cooled to 20-30 DEG C, adjust material liquid pH to 7.0-7.3；Wherein the enzyme is papain, wind
Taste enzyme, neutral proteinase, alkali protease, trypsase, pepsin are reacted according to recommendation optimal pH and temperature；
(5) adverse current impulse ultrasound assistance enzymolysis is carried out while enzymatic hydrolysis, wherein the adverse current impulse ultrasound assistance enzymolysis, specifically
Parameter is 2~10s of pulse width, 1~5s of pulse spacing, ultrasonic power 80W/L, supersonic frequency 28kHz, and feed liquid is with circulated in countercurrent
Mode by ultrasonic probe, 40~90min of ultrasonic time；
(6) centrifugal treating is carried out to enzymolysis liquid, collects supernatant and just obtain polypeptide liquid, spray drying or freeze-drying obtains polypeptide
Powder can be made into capsule.
2. the ultrasonic synergistic preparation method of a kind of arrowhead resistant starch according to claim 1 and functional polypeptide, special
Sign is that ultrasonic pretreatment is carried out under ultrasound condition wherein different described in step (2) to be referred to: double-frequency ultrasound frequency is excellent
It is selected as 40/60kHz.
3. the ultrasonic synergistic preparation method of a kind of arrowhead resistant starch according to claim 1 and functional polypeptide, special
Sign is in above-mentioned steps (4) that alkali protease is screened most preferably to use enzyme, peak enzymolysis-ability process conditions are as follows: concentration of substrate
8.0%, pH9.0, temperature 50 C, enzyme concentration 8%, enzymolysis time 90min.
4. the ultrasonic synergistic preparation method of a kind of arrowhead resistant starch according to claim 1 and functional polypeptide, special
Sign is in above-mentioned steps (5) that the adverse current impulse ultrasound assistance enzymolysis, design parameter is pulse width 10s, pulse spacing
5s, ultrasonic power 80W/L, supersonic frequency 28kHz, feed liquid pass through ultrasonic probe, ultrasonic time in a manner of circulated in countercurrent
90min。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923282A (en) * | 2019-12-12 | 2020-03-27 | 江苏大学 | Method for preparing resistant starch by using ultrasonic-assisted amylase |
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| CN102273543A (en) * | 2011-08-16 | 2011-12-14 | 山东省花生研究所 | Method for preparing peanut antioxidant peptide through ultrasonic-assisted enzymolysis |
| CN106418054A (en) * | 2016-11-30 | 2017-02-22 | 江苏大学 | Method for preparing natural arrowhead juice by frequency-sweeping pulsed ultrasound assisted bienzyme hydrolysis |
| CN107337740A (en) * | 2017-07-24 | 2017-11-10 | 江苏大学 | A kind of method of arrowhead starch double-frequency ultrasound wavefront modifier |
| CN108713750A (en) * | 2018-03-07 | 2018-10-30 | 扬州宝应湖西岛有机农场有限公司 | A kind of processing method of arrowhead extract |
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2018
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| CN102273543A (en) * | 2011-08-16 | 2011-12-14 | 山东省花生研究所 | Method for preparing peanut antioxidant peptide through ultrasonic-assisted enzymolysis |
| CN106418054A (en) * | 2016-11-30 | 2017-02-22 | 江苏大学 | Method for preparing natural arrowhead juice by frequency-sweeping pulsed ultrasound assisted bienzyme hydrolysis |
| CN107337740A (en) * | 2017-07-24 | 2017-11-10 | 江苏大学 | A kind of method of arrowhead starch double-frequency ultrasound wavefront modifier |
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Application publication date: 20190329 |