CN107201389A - A kind of peanut protein polypeptide and its application - Google Patents
A kind of peanut protein polypeptide and its application Download PDFInfo
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- CN107201389A CN107201389A CN201710414031.5A CN201710414031A CN107201389A CN 107201389 A CN107201389 A CN 107201389A CN 201710414031 A CN201710414031 A CN 201710414031A CN 107201389 A CN107201389 A CN 107201389A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/04—Preparation of other alcoholic beverages by mixing, e.g. for preparation of liqueurs
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Abstract
The invention discloses a kind of peanut protein polypeptide and its application, the peanut protein polypeptide is peanut lipid-loweringing peptide of the molecular weight between 3000 6000Da, and the peanut lipid-loweringing peptide is prepared using microwave radiation technology enzymolysis technology, and the preparation method comprises the following steps：The step of the step of the step of pre-treatment, protein-modified step, primary enzymolysis, secondary enzymolysis, go out enzyme the step of, the step of of isolating and purifying, high pressure steam sterilization the step of, be concentrated and dried the step of.By improving the enzymolysis process and technology of peanut protein polypeptide, using super ripple assistance enzymolysis technology, the polypeptide with Lipid-lowering activities is prepared, the peptide yield of peanut lipid-loweringing peptide prepared by the present invention is high, and purity is high.
Description
Technical field
The present invention relates to a kind of biotechnology, more particularly to a kind of peanut protein polypeptide and its application.
Background technology
Protein content in peanut is about 25%-36%, higher than sesame and rape, is only second to soybean.Peanut protein is
A kind of nutritive value very high adequate proteins, belongs to food plant protein, and its biological value (BV) is 58, and efficiency value is 117, is contained
There are 8 kinds of essential amino acids of needed by human body, be especially enriched in arginine (9%), and arginine has Immune enhancement function, to safeguarding
Health and early children development play an important roll.Research finds that protein has more more preferable than protein through the obtained peptide of hydrolysis
Nutritional properties, experiment confirms that the absorptivity of peptide is higher than amino acid, is more easy to, is faster absorbed by the body by small intestinal mucosa than amino acid
Utilize.Peanut protein active peptides have a variety of remarkable physiological functions in nutrition and health care field, and one is that polypeptide can be by enteron aisle not
Through degraded directly absorption, infiltration rate and absorptivity ratio protein and amino acid are all high, thus can be used as enteral nutrition agent and stream
The form of matter food is supplied to the crowd under special health；Two be that it not only provides human body necessary nutrient protein,
The content of Blood Cholesterol and triglyceride can also be effectively reduced, accelerates power consumption in vivo, burn fat, resistance is fat；Three are
Peanut protein active peptides can provide abundant amino acid for human body, promote protein synthesis, inhibitory RNA enzymatic activity
Decline, remove the free radical and heavy metal in human body, improve cell metabolism, be that immune system preparation resists to antibacterium and infection
Body, improves immune function of human body.
Peanut medical value itself is high, and high protein is a kind of potential plant protein resource, but at present to peanut protein
Research be concentrated mainly in terms of peanut protein antioxygenic property, immunologic function, and to the peanut protein with lipid-reducing function
Research but report it is still few.Therefore deep development is carried out to peanut lipoprotein reducing using modern food biotechnology, preparation is provided
There is the functional food of health-care effect, can not only improve the utilization rate of existing resource, one is provided for the Depth Study of peanut protein
New approach, increases the economic benefit of peasant；And scientific and technological content and the market of peanut cultivation and food processing enterprises can be strengthened
Competitiveness, has important society to improving China's aquatic products and modernizing comprehensive processing technology level and develop healthy food industry
Meaning and economic benefit.
The information for being disclosed in the background section is merely intended to understanding of the increase to the general background of the present invention, without answering
When be considered as recognizing or imply in any form the information structure oneself be persons skilled in the art well known to prior art.
The content of the invention
In order to overcome the deficiencies in the prior art, an object of the present invention is to provide a kind of peanut protein polypeptide.Pass through
The enzymolysis process and technology of peanut protein polypeptide are improved, using super ripple assistance enzymolysis technology, the polypeptide with Lipid-lowering activities is prepared,
The peptide yield of peanut lipid-loweringing peptide prepared by the present invention is high, and purity is high.
The second object of the present invention is a kind of application of peanut protein polypeptide in compound peanut protein polypeptide wine.This hair
Bright compound peanut protein polypeptide using peanut protein polypeptide as the lipid-loweringing composition mainly added, hawthorn for auxiliary addition lipid-loweringing into
Point, both use cooperatively, and can play a part of Synergistic.
An object of the present invention adopts the following technical scheme that realization：
A kind of peanut protein polypeptide, is peanut lipid-loweringing peptide of the molecular weight between 3000-6000Da, and the peanut lipid-loweringing peptide is used
Microwave radiation technology enzymolysis technology is prepared, and the preparation method comprises the following steps：
The step of pre-treatment：
Peanut is added water wetting, peanut mud is made in the boiling 15min at 121 DEG C, cooling, crushing, stirring；
Protein-modified step：
Peanut mud is weighed, according to solid-to-liquid ratio 1:10 add alkali lye, then adjust pH value to 8.2-8.5, micro- at 45-55 DEG C
Ripple is handled 10 minutes, and 20min is incubated afterwards, and pH value is maintained at less than 8.5, and centrifugation takes supernatant liquor, adds acid solution, adjusts pH value
To 4-4.5, microwave treatment 10 minutes is incubated 20min afterwards, and pH value is maintained at less than 4.5, and centrifugation takes lower sediment, dissolves,
Obtain modified peanut protein solution；
The step of primary enzymolysis：
It is molten for substrate with modified peanut protein, adjust pH value to 7.5 with hydrochloric acid, be 0.5- according to substrate mass percent
0.8%, which adds self-control protease, mixes thoroughly, then at 55 DEG C, microwave treatment 20 minutes, and pH value is maintained at less than 7.5；
The step of secondary enzymolysis：
After first time enzymolysis processing, first time enzymolysis liquid is down to normal temperature, pH value is adjusted again to 7.5, the bottom of according to
Amount of substance percentage is that 0.8-1.2% addition self-control protease is mixed thoroughly, at 55 DEG C, microwave treatment 20 minutes, and pH value is maintained at
Less than 7.5, enzymolysis terminates；
Go out enzyme the step of：
Enzymolysis is again started up microwave hyperthermia to 90 DEG C after terminating, and maintains 10min, obtains the enzyme hydrolyzate that goes out；
The step of isolating and purifying：
The hydrolyzate for enzyme activity of going out is down to room temperature, enzyme activity of going out is isolated and purified using Sephadex G-15 gel filtration chromatographies
Hydrolyzate；
The step of high pressure steam sterilization：
Hydrolyzate through isolating and purifying is 121 DEG C in temperature, and pressure is high pressure steam sterilization under conditions of 0.1MPa
15min；
The step of concentrate drying：
Adjust pH value to 7.0 the hydrolyzate after high pressure steam sterilization, carry out concentrating low-temperature vacuum drying, produce peanut lipid-loweringing
Peptide.
Further, in protein-modified step, the alkali lye is 3mol/L NaOH；The acid solution is 3mol/L
Citric acid.
Further, in the step of first and second digests, the self-control protease is the culture medium based on peanut meal
Induce the compound protease of aspergillus oryzae secretion.
Further, in the step of first and second digests, the manufacturing process of the compound protease is as follows：Cross 80 mesh sieves
Wheat bran 20g, cross 80 mesh peanut meal powder 8.0g, flour 2.5g, calcium chloride 0.028g and be mixed into 30g pure water, load triangular flask,
Tying jump a queue after the 30min that sterilized in moist heat sterilization pot at 121 DEG C, scattered culture medium clinker, natural cooling is shaken after taking-up while hot
To room temperature；Then be inoculated into from aspergillus oryzae test tube strains in triangular flask culture medium, 30 DEG C are incubated, daily shaking flask once,
54h terminates culture；Use pH value to stir 1h at 40 DEG C for 7.0 0.2M phosphate buffers and extract protease；Centrifuged at 4 DEG C
30min, collects supernatant, and concentrated frozen produces compound protease after drying.
Further, in the step of first and second digests, the vigor of the self-control protease is 4.5 × 105U/g。
Further, the step of protein-modified step, primary enzymolysis, secondary enzymolysis microwave described in the step of
Frequency is 1800MHz, and power is 10KW.
Further, it is described specific as follows the step of isolate and purify：Use deionized water balance gel chromatography column, column dimensions
For 1.6cm × 70cm, filler is sephadex resin Sephadex G-15, and flow velocity is 1.0mL/min；By the water for enzyme activity of going out
Solution liquid is dissolved in the solution that deionized water is configured to 20mg/mL, and 2ml upper props are eluted, elution flow rate is 2mL/ with deionized water
Min, and eluent is collected with Fraction collection device, 4mL/ pipes, in detecting light absorption value under 214nm, will belong to the collection liquid at same peak
Mixing, is then concentrated.
The second object of the present invention adopts the following technical scheme that realization：
A kind of preparation method of compound peanut protein polypeptide wine, is brewed by peanut protein polypeptide as described above and formed.Bag
Include following steps：
The step of auxiliary material medicinal plant extraction：
Hawthorn is taken, the pure water immersion 1h of 10 times of 80 DEG C of amounts is added, boils 1.5h, filter, filter residue adds 8 times of amount pure water
1h is boiled again, and thick paste is concentrated under reduced pressure into Rotary Evaporators, it is standby；
The step of blending：
Using concentration be 35% rice wine as base fluid, add 40-45% base liquor weight the peanut protein polypeptide and 5-8%
The hawthorn thick paste of base liquor weight, stirs, and sealed storage 2 months；
The step of super ripple of double frequency is aged：
Surpassed ripple ripening in turn under the conditions of two kinds of super wave frequency rates, the first super ripple condition：300W, 35kHz's
Super ripple 15min；Second surpass ripple condition：300W, 45kHz super ripple 15min；Common ultrasound 2h；
The step of allotment is examined：
Adjust alcohol concentration and pol, storage, it is ensured that wine body is limpid transparent；Then it is filling after the assay was approved, produce compound
Peanut protein polypeptide wine.
Compared with prior art, the beneficial effects of the present invention are：
(1) by improving the enzymolysis process and technology of peanut protein polypeptide, using super ripple assistance enzymolysis technology, preparation has
The polypeptide of Lipid-lowering activities, the peptide yield of peanut lipid-loweringing peptide prepared by the present invention is high, and purity is high, can be used as additive or active drug
Thing is added to a variety of industries and fields such as human food, medicine, health products, undoubtedly to extension loach industrial chain, is improved comprehensively
Loach industry comprehensive benefit, realizes that growth of agricultural efficiency increasing peasant income rural area is flourishing, to promoting the Belt and Road construction to be of great importance.
(2) the whole technological process of the present invention is short, the time is short, and energy consumption is low, pollute less, without using toxic reagent, contamination-free
Discharge, reaches clean manufacturing target.
(3) selection of enzyme is crucial during controlled enzymatic hydrolysis, and it not only influences the physiological function of final product, raw material profit
With rate, reaction speed, and directly affect the local flavor and physicochemical characteristics of product.The selection of enzyme, the control of enzymolysis process
It is the key technology that enzymatic isolation method prepares active peptide.The present invention is using the culture medium induction aspergillus oryzae secretion based on peanut meal
Compound protease digest while coordinate microwave to carry out assistance enzymolysis, make the degree of hydrolysis and peptide yield of peanut hydrolysate all compared with
Height, and the molecular weight of Peanut Polypeptide that enzymolysis is obtained is between 3000-6000Da, with effect for reducing fat.
(4) obtained peanut protein polypeptide is isolated and purified by Sephadex G-15 gel chromatography filtering chromatograms of the present invention
Molecular weight is between 3000-6000Da, and the peanut protein polypeptide component effect for reducing fat is strong, and research finds that not peptide molecular weight is smaller
Or bigger its Lipid-lowering activities are stronger, the activity of peptide also constituted by its amino acid, sort or peptide the factor such as space structure shadow
Ring, its Lipid-lowering activities is most strong between 3000-6000Da peanut protein polypeptides for the molecular weight obtained by present invention purifying.In addition, this
The separation condition of invention is gentle, sample recovery rate is high, experimental result reappearance is high, simple equipments economy.
Embodiment
Below, with reference to embodiment, the present invention is described further, it is necessary to which explanation is, what is do not collided
Under the premise of, new embodiment can be formed between various embodiments described below or between each technical characteristic in any combination.
In the present invention, if not refering in particular to, all part, percentage are unit of weight, equipment and raw material for being used etc.
It is commercially available or commonly used in the art.Method in following embodiments, is the normal of this area unless otherwise instructed
Rule method.
A kind of peanut protein polypeptide, is peanut lipid-loweringing peptide of the molecular weight between 3000-6000Da, and the peanut lipid-loweringing peptide is used
Microwave radiation technology enzymolysis technology is prepared, and the preparation method comprises the following steps：
The step of pre-treatment：
Peanut is added water wetting, peanut mud is made in the boiling 15min at 121 DEG C, cooling, crushing, stirring；
Protein-modified step：
Peanut mud is weighed, according to solid-to-liquid ratio 1:10 add alkali lye, then adjust pH value to 8.2-8.5, micro- at 45-55 DEG C
Ripple is handled 10 minutes, and 20min is incubated afterwards, and pH value is maintained at less than 8.5, and centrifugation takes supernatant liquor, adds acid solution, adjusts pH value
To 4-4.5, microwave treatment 10 minutes is incubated 20min afterwards, and pH value is maintained at less than 4.5, and centrifugation takes lower sediment, dissolves,
Obtain modified peanut protein solution；Alkali lye is 3mol/L NaOH；Acid solution is 3mol/L citric acids.The frequency of microwave is
1800MHz, power is 10KW.
The step of primary enzymolysis：
It is molten for substrate with modified peanut protein, adjust pH value to 7.5 with hydrochloric acid, be 0.5- according to substrate mass percent
0.8%, which adds self-control protease, mixes thoroughly, then at 55 DEG C, microwave treatment 20 minutes, and pH value is maintained at less than 7.5；Microwave
Frequency is 1800MHz, and power is 10KW.
The step of secondary enzymolysis：
After first time enzymolysis processing, first time enzymolysis liquid is down to normal temperature, pH value is adjusted again to 7.5, the bottom of according to
Amount of substance percentage is that 0.8-1.2% addition self-control protease is mixed thoroughly, at 55 DEG C, microwave treatment 20 minutes, and pH value is maintained at
Less than 7.5, enzymolysis terminates；The frequency of microwave is 1800MHz, and power is 10KW.
In the step of first and second digests, self-control protease is the culture medium induction aspergillus oryzae point based on peanut meal
The compound protease secreted.The vigor for making protease by oneself is 4.5 × 105U/g.The manufacturing process of compound protease is as follows：Cross 80 mesh
The wheat bran 20g of sieve, crosses 80 mesh peanut meal powder 8.0g, flour 2.5g, calcium chloride 0.028g and is mixed into 30g pure water, load triangle
Bottle, tying of jumping a queue shakes scattered culture medium clinker while hot after the 30min that sterilized in moist heat sterilization pot at 121 DEG C after taking-up, naturally cold
But to room temperature；Then it is inoculated into from aspergillus oryzae test tube strains in triangular flask culture medium, 30 DEG C incubated, daily shaking flask one
Secondary, 54h terminates culture；Use pH value to stir 1h at 40 DEG C for 7.0 0.2M phosphate buffers and extract protease；At 4 DEG C
30min is centrifuged, supernatant is collected, concentrated frozen produces compound protease after drying.
Protein digestion refers to the process of that protein is degraded into peptide or amino acid in the presence of protease.Enzymolysis is to change
Protein is made, one of the most effective approach of protein value is improved.But different albumen enzyme effect identical protein sources, acquisition
The different from those of protein enzymatic hydrolyzate product is very remote, because different protease has different catalyst mechanisms and protease work(
Can group.
Peanut contains substantial amounts of carbohydrate, wherein there is part aqueous polysaccharide, these polysaccharide can be sent out with peanut protein
Raw albumen-polysaccharide graft reaction, generates the contact probability of polymer, reduction protease and protein, reduces enzymolysis efficiency；Simultaneously
Water-insoluble fiber easily absorbs expansion, reduces the solvent in hydrolyzation system, increases viscosity, except that can reduce protease and albumen
The contact probability of matter, can also because viscosity is too high and oxygen content in reduction system, increase the possibility of microbial infection, so as to influence
Hydrolysis result.
The present invention is during self-control protease is prepared, and peanut meal promotes the growth of aspergillus oryzae, aspergillus oryzae as nitrogen source
The protein in peanut meal can not directly be utilized, it is necessary to secret out of protease the degradation of proteins into small-molecular peptides or amino acid
It can absorb, in aspergillus oryzae growth course, in order to obtain nutrition from culture medium, aspergillus oryzae is except meeting extracellular proteinase
Outside, a series of other enzymes, such as carbohydrase and cellulase can also be secreted.
During peanut is hydrolyzed, protease, carbohydrase and cellulase can be incorporated into many syrup above albumen
Solution is separated into oligosaccharides or monose from protein, and the cellulose in system can be also hydrolyzed into polysaccharide or oligosaccharides, reduces body
The viscosity of system, accelerates the progress of protein hydrolysis.It can be seen that, the present invention is using the culture medium induction aspergillus oryzae based on peanut meal
The compound protease of secretion digest while cooperation microwave progress assistance enzymolysis, obtains the degree of hydrolysis and peptide of peanut hydrolysate
Rate is all higher, and the molecular weight of Peanut Polypeptide that enzymolysis is obtained is between 3000-6000Da, with effect for reducing fat.
Go out enzyme the step of：
Enzymolysis is again started up microwave hyperthermia to 90 DEG C after terminating, and maintains 10min, obtains the enzyme hydrolyzate that goes out；
The step of isolating and purifying：
The hydrolyzate for enzyme activity of going out is down to room temperature, enzyme activity of going out is isolated and purified using Sephadex G-15 gel filtration chromatographies
Hydrolyzate；The step of isolating and purifying is specific as follows：Use deionized water balance gel chromatography column, column dimensions be 1.6cm ×
70cm, filler is sephadex resin Sephadex G-15, and flow velocity is 1.0mL/min；The hydrolyzate for enzyme activity of going out is dissolved in
Ionized water is configured to 20mg/mL solution, and 2ml upper props are eluted with deionized water, and elution flow rate is 2mL/min, and with point
Walk collector and collect eluent, 4mL/ pipes, in detecting light absorption value under 214nm, the collection liquid for belonging to same peak are mixed, Ran Houjin
Row concentration.
Gel filtration chromatography is the molecular sieve effect using the gel for having network structure, according to the molecular size range of separated object
Difference is separated.When mixing peptide solution by gel column, because gel contains a large amount of micropores, only allow buffer solution and phase
Less to molecular mass peptide enters wherein, and the larger peptide of relative molecular mass by exclusion outside gel particle, filling out
Expect to flow in gap, peptide more less than relative molecular mass is eluted earlier, peptide composition to be separated just presses different phases
Molecular mass is sized and come.
The molecule of obtained peanut protein polypeptide is isolated and purified by Sephadex G-15 gel chromatography filtering chromatograms of the present invention
Amount is between 3000-6000Da, and the peanut protein polypeptide component effect for reducing fat is strong, and research is found, not peptide molecular weight is smaller or gets over
Its big Lipid-lowering activities are stronger, the activity of peptide also by the constituting of its amino acid, sort or the factor such as space structure of peptide is influenceed, by
Its Lipid-lowering activities is most strong between 3000-6000Da peanut protein polypeptides for the molecular weight that present invention purifying is obtained.In addition, the present invention
Separation condition is gentle, sample recovery rate is high, experimental result reappearance high, simple equipments economy.
The step of high pressure steam sterilization：
Hydrolyzate through isolating and purifying is 121 DEG C in temperature, and pressure is high pressure steam sterilization under conditions of 0.1MPa
15min；Sterilization processing is the key link of peptide matters in process, and appropriate process for sterilizing can not only kill micro- life
The activity of thing, inactive enzyme, extends the shelf lives of peptide matters, in addition, some special active materials can also be generated, to lipid-loweringing
Effect has certain booster action.
The step of concentrate drying：
Adjust pH value to 7.0 the hydrolyzate after high pressure steam sterilization, carry out concentrating low-temperature vacuum drying, produce peanut lipid-loweringing
Peptide.
A kind of preparation method of compound peanut protein polypeptide wine, is brewed by peanut protein polypeptide as above and formed.Including such as
Lower step：
The step of auxiliary material medicinal plant extraction：
Hawthorn is taken, the pure water immersion 1h of 10 times of 80 DEG C of amounts is added, boils 1.5h, filter, filter residue adds 8 times of amount pure water
1h is boiled again, and thick paste is concentrated under reduced pressure into Rotary Evaporators, it is standby；
The step of blending：
Using concentration be 35% rice wine as base fluid, add 40-45% base liquor weight peanut protein polypeptide and 5-8% base liquors
The hawthorn thick paste of weight, stirs, and sealed storage 2 months；
The step of super ripple of double frequency is aged：
Surpassed ripple ripening in turn under the conditions of two kinds of super wave frequency rates, the first super ripple condition：300W, 35kHz's
Super ripple 15min；Second surpass ripple condition：300W, 45kHz super ripple 15min；Common ultrasound 2h；
The step of allotment is examined：
Adjust alcohol concentration and pol, storage, it is ensured that wine body is limpid transparent；Then it is filling after the assay was approved, produce compound
Peanut protein polypeptide wine.
The Testing index of the polypeptide wine of the present invention includes enterprise's mark sense organ and physical and chemical index, such as table 1-2：
The organoleptic requirements of table 1
Table 2 examines physical and chemical index
Hawthorn is the dry mature fruit of rosaceous plant hawthorn.It is sour sweet, slightly warm in nature.Fructus Crataegi contains crataegolic acid, apple
Tartaric acid, citric acid, caffeic acid, lactones, fat, Hyperoside, lipolytic enzyme, tannin, protein, Quercetin, riboflavin, carrot
The Multiple components such as element, carbohydrate and vitamins.Through clinical research confirmation, hawthorn can significantly decrease blood fat and cholesterol, resist
Lipid peroxidation, effectively prevents and treats atherosclerosis.Peanut protein polypeptide plays the role of lipid-loweringing with hawthorn, and the present invention is with flower
Raw albumen polypeptide is the lipid-loweringing composition mainly added, and hawthorn is the lipid-loweringing composition of auxiliary addition, and both use cooperatively, and can play
The effect of Synergistic.
In addition, super ripple ageing under the conditions of double frequency using being aged, because the ultrasonic wave of bifrequency makes appearance inside wine liquid
The hard point and decrease point of two kinds of ripples, at the decrease point of two kinds of different ripples, are formed because its relative pressure is smaller, therefore easily
Different small bubbles, and when the hard point of these bubble motions to two kinds of different ripples, because pressure is larger, and make these gas
Follicular rupture.Two kinds of huge energy are generated in the generation of bubble and rupture process, each component molecules in wine liquid is accelerated and is good for
Fracture and generation, improve intermolecular effective collision rate, enhance the affinity between polar molecule, not only increase alcohol
Degree of association between molecule and hydrone, and the association group being likely to form between bigger and firm polar molecule, so that wine
Alcohols, aldehydes, acids and esters obtain huge energy in liquid, accelerate occur polymerization, hydroformylation, acidifying, esterification and cracking
Deng a series of complex reaction, the esters molecule in system is made to produce, the composition such as some esters and acids may also be given to be formed with this
It is gregarious, accelerate the curing of wine liquid, make wine can be superior in fragrance, mouthfeel.
Embodiment 1
A kind of peanut protein polypeptide, is peanut lipid-loweringing peptide of the molecular weight between 3000-6000Da, and the peanut lipid-loweringing peptide is used
Microwave radiation technology enzymolysis technology is prepared, and the preparation method comprises the following steps：
The step of pre-treatment：
Peanut is added water wetting, peanut mud is made in the boiling 15min at 121 DEG C, cooling, crushing, stirring；
Protein-modified step：
Peanut mud is weighed, according to solid-to-liquid ratio 1:10 add alkali lye, pH value are then adjusted to 8.3, at 45-55 DEG C, at microwave
Reason 10 minutes, is incubated 20min afterwards, and pH value is maintained at less than 8.5, and centrifugation takes supernatant liquor, adds acid solution, adjusts pH value extremely
4.3, microwave treatment 10 minutes is incubated 20min afterwards, and pH value is maintained at less than 4.5, and centrifugation takes lower sediment, dissolves, obtains
Modified peanut protein solution；Alkali lye is 3mol/LNaOH；Acid solution is 3mol/L citric acids.The frequency of microwave is 1800MHz, power
For 10KW.
The step of primary enzymolysis：
It is molten for substrate with modified peanut protein, adjust pH value to 7.5 with hydrochloric acid, be 0.5- according to substrate mass percent
0.8%, which adds self-control protease, mixes thoroughly, then at 55 DEG C, microwave treatment 20 minutes, and pH value is maintained at less than 7.5；Microwave
Frequency is 1800MHz, and power is 10KW.
The step of secondary enzymolysis：
After first time enzymolysis processing, first time enzymolysis liquid is down to normal temperature, pH value is adjusted again to 7.5, the bottom of according to
Amount of substance percentage is that 0.8-1.2% addition self-control protease is mixed thoroughly, at 55 DEG C, microwave treatment 20 minutes, and pH value is maintained at
Less than 7.5, enzymolysis terminates；The frequency of microwave is 1800MHz, and power is 10KW.
In the step of first and second digests, self-control protease is the culture medium induction aspergillus oryzae point based on peanut meal
The compound protease secreted.The vigor for making protease by oneself is 4.5 × 105U/g.The manufacturing process of compound protease is as follows：Cross 80 mesh
The wheat bran 20g of sieve, crosses 80 mesh peanut meal powder 8.0g, flour 2.5g, calcium chloride 0.028g and is mixed into 30g pure water, load triangle
Bottle, tying of jumping a queue shakes scattered culture medium clinker while hot after the 30min that sterilized in moist heat sterilization pot at 121 DEG C after taking-up, naturally cold
But to room temperature；Then it is inoculated into from aspergillus oryzae test tube strains in triangular flask culture medium, 30 DEG C incubated, daily shaking flask one
Secondary, 54h terminates culture；Use pH value to stir 1h at 40 DEG C for 7.0 0.2M phosphate buffers and extract protease；At 4 DEG C
30min is centrifuged, supernatant is collected, concentrated frozen produces compound protease after drying.
Go out enzyme the step of：
Enzymolysis is again started up microwave hyperthermia to 90 DEG C after terminating, and maintains 10min, obtains the enzyme hydrolyzate that goes out；
The step of isolating and purifying：
The hydrolyzate for enzyme activity of going out is down to room temperature, enzyme activity of going out is isolated and purified using Sephadex G-15 gel filtration chromatographies
Hydrolyzate；The step of isolating and purifying is specific as follows：Use deionized water balance gel chromatography column, column dimensions be 1.6cm ×
70cm, filler is sephadex resin Sephadex G-15, and flow velocity is 1.0mL/min；The hydrolyzate for enzyme activity of going out is dissolved in
Ionized water is configured to 20mg/mL solution, and 2ml upper props are eluted with deionized water, and elution flow rate is 2mL/min, and with point
Walk collector and collect eluent, 4mL/ pipes, in detecting light absorption value under 214nm, the collection liquid for belonging to same peak are mixed, Ran Houjin
Row concentration.
The step of high pressure steam sterilization：
Hydrolyzate through isolating and purifying is 121 DEG C in temperature, and pressure is high pressure steam sterilization under conditions of 0.1MPa
15min；
The step of concentrate drying：
Adjust pH value to 7.0 the hydrolyzate after high pressure steam sterilization, carry out concentrating low-temperature vacuum drying, produce peanut lipid-loweringing
Peptide.
Embodiment 2
A kind of peanut protein polypeptide, is peanut lipid-loweringing peptide of the molecular weight between 3000-6000Da, and the peanut lipid-loweringing peptide is used
Microwave radiation technology enzymolysis technology is prepared, and the preparation method comprises the following steps：
The step of pre-treatment：
Peanut is added water wetting, peanut mud is made in the boiling 15min at 121 DEG C, cooling, crushing, stirring；
Protein-modified step：
Peanut mud is weighed, according to solid-to-liquid ratio 1:10 add alkali lye, pH value are then adjusted to 8.2, at 45-55 DEG C, at microwave
Reason 10 minutes, is incubated 20min afterwards, and pH value is maintained at less than 8.5, and centrifugation takes supernatant liquor, adds acid solution, adjusts pH value to 4,
Microwave treatment 10 minutes, is incubated 20min afterwards, and pH value is maintained at less than 4.5, and centrifugation takes lower sediment, dissolves, is modified
Peanut protein solution；
The step of primary enzymolysis：
It is molten for substrate with modified peanut protein, adjust pH value to 7.5 with hydrochloric acid, be 0.5- according to substrate mass percent
0.8%, which adds self-control protease, mixes thoroughly, then at 55 DEG C, microwave treatment 20 minutes, and pH value is maintained at less than 7.5；
The step of secondary enzymolysis：
After first time enzymolysis processing, first time enzymolysis liquid is down to normal temperature, pH value is adjusted again to 7.5, the bottom of according to
Amount of substance percentage is that 0.8-1.2% addition self-control protease is mixed thoroughly, at 55 DEG C, microwave treatment 20 minutes, and pH value is maintained at
7.5 hereinafter, enzymolysis terminates；
Go out enzyme the step of：
Enzymolysis is again started up microwave hyperthermia to 90 DEG C after terminating, and maintains 10min, obtains the enzyme hydrolyzate that goes out；
The step of isolating and purifying：
The hydrolyzate for enzyme activity of going out is down to room temperature, pH value is adjusted to 7.5, using Sephadex G-15 gel filtration chromatographies point
The hydrolyzate for enzyme activity of being gone out from purifying；
The step of high pressure steam sterilization：
Hydrolyzate through isolating and purifying is 121 DEG C in temperature, and pressure is high pressure steam sterilization under conditions of 0.1MPa
15min；
The step of concentrate drying：
Adjust pH value to 7.0 the hydrolyzate after high pressure steam sterilization, carry out concentrating low-temperature vacuum drying, produce peanut lipid-loweringing
Peptide.
Embodiment 3
A kind of peanut protein polypeptide, is peanut lipid-loweringing peptide of the molecular weight between 3000-6000Da, and the peanut lipid-loweringing peptide is used
Microwave radiation technology enzymolysis technology is prepared, and the preparation method comprises the following steps：
The step of pre-treatment：
Peanut is added water wetting, peanut mud is made in the boiling 15min at 121 DEG C, cooling, crushing, stirring；
Protein-modified step：
Peanut mud is weighed, according to solid-to-liquid ratio 1:10 add alkali lye, pH value are then adjusted to 8.5, at 45-55 DEG C, at microwave
Reason 10 minutes, is incubated 20min afterwards, and pH value is maintained at less than 8.5, and centrifugation takes supernatant liquor, adds acid solution, adjusts pH value extremely
4.5, microwave treatment 10 minutes is incubated 20min afterwards, and pH value is maintained at less than 4.5, and centrifugation takes lower sediment, dissolves, obtains
Modified peanut protein solution；
The step of primary enzymolysis：
It is molten for substrate with modified peanut protein, adjust pH value to 7.5 with hydrochloric acid, be 0.5- according to substrate mass percent
0.8%, which adds self-control protease, mixes thoroughly, then at 55 DEG C, microwave treatment 20 minutes, and pH value is maintained at less than 7.5；
The step of secondary enzymolysis：
After first time enzymolysis processing, first time enzymolysis liquid is down to normal temperature, pH value is adjusted again to 7.5, the bottom of according to
Amount of substance percentage is that 0.8-1.2% addition self-control protease is mixed thoroughly, at 55 DEG C, microwave treatment 20 minutes, and pH value is maintained at
Less than 7.5, enzymolysis terminates；
Go out enzyme the step of：
Enzymolysis is again started up microwave hyperthermia to 90 DEG C after terminating, and maintains 10min, obtains the enzyme hydrolyzate that goes out；
The step of isolating and purifying：
The hydrolyzate for enzyme activity of going out is down to room temperature, pH value is adjusted to 8.0, using Sephadex G-15 gel filtration chromatographies point
The hydrolyzate for enzyme activity of being gone out from purifying；
The step of high pressure steam sterilization：
Hydrolyzate through isolating and purifying is 121 DEG C in temperature, and pressure is high pressure steam sterilization under conditions of 0.1MPa
15min；
The step of concentrate drying：
Adjust pH value to 7.0 the hydrolyzate after high pressure steam sterilization, carry out concentrating low-temperature vacuum drying, produce peanut lipid-loweringing
Peptide.
Comparative example 1
Difference with embodiment 1 is in first and second digests, and the self-control protease of the present invention is not used, and selects existing
Some papains, the vigor of papain is 4.5 × 105U/g。
Comparative example 2
Difference with embodiment 1 is the step of removal is isolated and purified, and remaining step is identical with parameter.
Embodiment 4
A kind of compound peanut protein polypeptide wine, is made up of following steps：
The step of auxiliary material medicinal plant extraction：
Hawthorn is taken, the pure water immersion 1h of 10 times of 80 DEG C of amounts is added, boils 1.5h, filter, filter residue adds 8 times of amount pure water
1h is boiled again, and thick paste is concentrated under reduced pressure into Rotary Evaporators, it is standby；
The step of blending：
Using concentration be 35% rice wine as base fluid, add 40-45% base liquor weight peanut protein polypeptide and 5-8% base liquors
The hawthorn thick paste of weight, stirs, and sealed storage 2 months；
The step of super ripple of double frequency is aged：
Surpassed ripple ripening in turn under the conditions of two kinds of super wave frequency rates, the first super ripple condition：300W, 35kHz's
Super ripple 15min；Second surpass ripple condition：300W, 45kHz super ripple 15min；Common ultrasound 2h；
The step of allotment is examined：
Adjust alcohol concentration and pol, storage, it is ensured that wine body is limpid transparent；Then it is filling after the assay was approved, produce compound
Peanut protein polypeptide wine.
Comparative example 3
The step of difference with embodiment 4 is to remove auxiliary material medicinal plant extraction, without hawthorn thick paste, remaining step with
Parameter is identical.
Comparative example 4
Difference with embodiment 4 is the step of removal super ripple of double frequency is aged, and remaining step is identical with parameter.
Below, peptide yield, purity and the effect for reducing fat of the peanut lipid-loweringing peptide prepared to the present invention are measured and verified.
1st, the measure of peptide yield
It is combined and is measured with formol titration using trichloroacetic acid (TCA) precipitation method.Take 10mL enzymolysis liquids plus 10mL
20% (g/g) TCA, centrifuges 10min, the supernatant of gained is denoted as supernatant after stirring evenly standing half an hour under 4800r/minTCA,
The total nitrogen content and ammonia nitrogen content in supernatant are determined with Kjeldahl's method and formol titration respectively, then peptide nitrogen quantity and peptide are obtained
Rate is calculated as the following formula, as a result such as table 3：
The total peptide nitrogen quantity=supernatant of supernatantTCANitrogen pool-supernatantTCATotal ammoniacal nitrogen amount,
The total peptide nitrogen content of peptide yield=supernatant/raw material total protein nitrogen content × 100%.
2nd, the measure of purity
With bovine serum albumin(BSA) (BSA) for standard items, the BSA solution of various concentrations is made.It is separately added into 7 test tubes
5mL coomassie brilliant blue staining solutions dye 5min.Using the solution in the 1st test tube as blank control, determine in remaining 6 test tube
Absorbance of the solution at 595nm.With protein quality concentration (gL-1) it is abscissa, absorbance is ordinate, is carried out linear
Regression analysis.Obtained equation of linear regression is Y=8.714X+0.025 (r=0.998), and the range of linearity is 0-100 μ g.As a result
Show, linear relationship is good, can be for carrying out the measure of protein content.Peanut egg obtained by difference extraction embodiment 1-3
White polypeptide determines absorbance as test sample at 595nm, and calculates protein content according to standard curve, calculates protein
Mass fraction/%, as a result such as table 3.
3rd, the checking of effect for reducing fat
Cholesterol is one of important composition of blood fat in blood of human body, using cholesterol-lowering activity as index, the observation present invention
Influence of the peanut lipid-loweringing peptide to three kinds of cholates, higher to the inhibiting rate of three kinds of cholates, cholesterol-lowering activity is higher, as a result such as
Table 3.
This experiment is using complexing of the peanut lipid-loweringing peptide in furfural determination of color course of reaction to cholate, with peanut
Lipid-loweringing peptide is index to the adsorption capacity of cholate, three kinds of cholates of dexycholate and taurocholate, evaluates peanut drop
The cholesterol-lowering activity of lipopeptid.
Using the content of three kinds of cholates of furfural determination of color, three kinds of cholate standard liquids are taken respectively, add 40%
Sulfuric acid solution, vibration adds 1% furfuryl aldehyde solution 1mL, mixes, 60 DEG C of incubation 40min, after temperature drops to room temperature, in 620nm
Light absorption value is surveyed at wavelength, using concentration as abscissa, light absorption value is ordinate, draw three standard curves.Example 1-6 system
Product 2ml, its absorbance is surveyed according to furfural development process, and three kinds of cholic acid salinity in supernatant are calculated by standard curve.Calculation formula
It is as follows：
Inhibiting rate (%)=(P1-P2)/P1 × 100%
In formula：P1 is respectively the initial mass concentration (mg/ml) of three kinds of cholate solutions
P2 is the mass concentration (mg/ml) of three kinds of cholate solutions after absorption
Peptide yield, purity and the lipid-lowering effect of the peanut lipid-loweringing peptide of table 3
The peptide yield of peanut lipid-loweringing peptide prepared by 1-3 of the embodiment of the present invention is high, and purity is high, can be used as additive or activity
Medicine is added to a variety of industries and fields such as human food, medicine, health products, undoubtedly to extension peanut industry chain, Quan Mianti
High peanut industry comprehensive benefit, realizes that growth of agricultural efficiency increasing peasant income rural area is flourishing, to promoting the Belt and Road construction to be of great importance.
And the peptide yield of peanut lipid-loweringing peptide prepared by comparative example 1-2 has larger difference compared with the present invention, illustrate the self-control of the present invention
Protease and the step of isolate and purify it is high to whole peptide yield, purity height influence is larger.
The data explanation of embodiment 4 and 5, both peanut protein polypeptide of the present invention and hawthorn use cooperatively, and can play association
With the effect of synergy.Without hawthorn, its inhibiting rate can decline nearly more than 15%, that is to say, that its lipid-lowering effect is also under meeting
Drop；In addition, the data explanation of embodiment 4 and 6, the synergistic function of both this peanut protein polypeptide and hawthorn is with the super ripple of double frequency
The step of ageing, is relevant, removes the step of super ripple of double frequency is aged, its lipid-lowering effect can also decline.
Above-mentioned embodiment is only the preferred embodiment of the present invention, it is impossible to limit the scope of protection of the invention with this,
The change and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention
Claimed scope.
Claims (9)
1. a kind of peanut protein polypeptide, it is characterised in that be molecular weight between 3000-6000Da peanut lipid-loweringing peptide, the peanut
Lipid-loweringing peptide is prepared using microwave radiation technology enzymolysis technology, and the preparation method comprises the following steps：
The step of pre-treatment：
Peanut is added water wetting, peanut mud is made in the boiling 15min at 121 DEG C, cooling, crushing, stirring；
Protein-modified step：
Peanut mud is weighed, according to solid-to-liquid ratio 1:10 add alkali lye, pH value are then adjusted to 8.2-8.5, at 45-55 DEG C, at microwave
Reason 10 minutes, is incubated 20min afterwards, and pH value is maintained at less than 8.5, and centrifugation takes supernatant liquor, adds acid solution, adjusts pH value to 4-
4.5, microwave treatment 10 minutes is incubated 20min afterwards, and pH value is maintained at less than 4.5, and centrifugation takes lower sediment, dissolves, obtains
Modified peanut protein solution；
The step of primary enzymolysis：
It is molten for substrate with modified peanut protein, adjust pH value to 7.5 with hydrochloric acid, add according to substrate mass percent for 0.5-0.8%
Enter to make protease by oneself and mix thoroughly, then at 55 DEG C, microwave treatment 20 minutes, pH value is maintained at less than 7.5；
The step of secondary enzymolysis：
After first time enzymolysis processing, first time enzymolysis liquid is down to normal temperature, pH value is adjusted again to 7.5, according to substrate matter
Amount percentage be 0.8-1.2% add self-control protease mix thoroughly, at 55 DEG C, microwave treatment 20 minutes, pH value be maintained at 7.5 with
Under, enzymolysis terminates；
Go out enzyme the step of：
Enzymolysis is again started up microwave hyperthermia to 90 DEG C after terminating, and maintains 10min, obtains the enzyme hydrolyzate that goes out；
The step of isolating and purifying：
The hydrolyzate for enzyme activity of going out is down to room temperature, pH value is adjusted to 7.5-8.0, using Sephadex G-15 gel filtration chromatographies point
The hydrolyzate for enzyme activity of being gone out from purifying；
The step of high pressure steam sterilization：
Hydrolyzate through isolating and purifying is 121 DEG C in temperature, and pressure is high pressure steam sterilization 15min under conditions of 0.1MPa；
The step of concentrate drying：
Adjust pH value to 7.0 the hydrolyzate after high pressure steam sterilization, carry out concentrating low-temperature vacuum drying, produce peanut lipid-loweringing peptide.
2. peanut protein polypeptide as claimed in claim 1, it is characterised in that in protein-modified step, the alkali lye
For 3mol/L NaOH；The acid solution is 3mol/L citric acids.
3. peanut protein polypeptide as claimed in claim 1, it is characterised in that in the step of first and second digests, the self-control
Protease is the compound protease of the culture medium induction aspergillus oryzae secretion based on peanut meal.
4. peanut protein polypeptide as claimed in claim 3, it is characterised in that in the step of first and second digests, described compound
The manufacturing process of protease is as follows：The wheat bran 20g of 80 mesh sieves is crossed, 80 mesh peanut meal powder 8.0g, flour 2.5g, calcium chloride are crossed
0.028g is mixed into 30g pure water, loads triangular flask, and tying of jumping a queue sterilizes 30min after in moist heat sterilization pot at 121 DEG C,
Scattered culture medium clinker is shaken after taking-up while hot, room temperature is naturally cooled to；Then triangular flask training is inoculated into from aspergillus oryzae test tube strains
Support in base, 30 DEG C incubated, once, 54h terminates culture to daily shaking flask；Use pH value for 7.0 0.2M phosphate buffers
1h is stirred at 40 DEG C and extracts protease；30min is centrifuged at 4 DEG C, supernatant is collected, concentrated frozen produces compound protein after drying
Enzyme.
5. peanut protein polypeptide as claimed in claim 3, it is characterised in that in the step of first and second digests, the self-control
The vigor of protease is 4.5 × 105U/g。
6. peanut protein polypeptide as claimed in claim 1, it is characterised in that in protein-modified step, primary enzymolysis
The frequency of microwave described in the step of step, secondary enzymolysis is 1800MHz, and power is 10KW.
7. peanut protein polypeptide as claimed in claim 1, it is characterised in that described specific as follows the step of isolate and purify：With
Deionized water balance gel chromatography column, column dimensions are 1.6cm × 70cm, and filler is sephadex resin Sephadex G-
15, flow velocity is 1.0mL/min；The hydrolyzate for enzyme activity of going out is dissolved in the solution that deionized water is configured to 20mg/mL, 2ml upper props are used
Deionized water is eluted, and elution flow rate is 2mL/min, and collects eluent, 4mL/ pipes, under 214nm with Fraction collection device
Light absorption value is detected, the collection liquid for belonging to same peak is mixed, then concentrated.
8. a kind of compound peanut protein polypeptide wine, it is characterised in that many as the peanut protein as described in claim any one of 1-7
Peptide is brewed and formed.
9. compound peanut protein polypeptide wine as claimed in claim 8, it is characterised in that be prepared from by following steps：
The step of auxiliary material medicinal plant extraction：
Hawthorn is taken, the pure water immersion 1h of 10 times of 80 DEG C of amounts is added, boils 1.5h, filter, filter residue adds 8 times of amount pure water again
1h is boiled, thick paste is concentrated under reduced pressure into Rotary Evaporators, it is standby；
The step of blending：
Using concentration be 35% rice wine as base fluid, add 40-45% base liquor weight the peanut protein polypeptide and 5-8% base liquors
The hawthorn thick paste of weight, stirs, and sealed storage 2 months；
The step of super ripple of double frequency is aged：
Surpassed ripple ripening in turn under the conditions of two kinds of super wave frequency rates, the first super ripple condition：300W, 35kHz super ripple
15min；Second surpass ripple condition：300W, 45kHz super ripple 15min；Common ultrasound 2h；
The step of allotment is examined：
Adjust alcohol concentration and pol, storage, it is ensured that wine body is limpid transparent；Then it is filling after the assay was approved, produce compound peanut
Polypeptide wine.
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| CN109777849A (en) * | 2019-03-07 | 2019-05-21 | 中南林业科技大学 | A kind of preparation method of debittered peach kernel extracting proteolytic polypeptide |
| CN112626155A (en) * | 2020-06-09 | 2021-04-09 | 南京泛成生物科技有限公司 | Preparation method of pea peptide |
| CN115581295A (en) * | 2022-08-24 | 2023-01-10 | 华南理工大学 | Soybean flavoring base material with freshness enhancing effect and preparation method and application thereof |
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| CN109777849A (en) * | 2019-03-07 | 2019-05-21 | 中南林业科技大学 | A kind of preparation method of debittered peach kernel extracting proteolytic polypeptide |
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| CN115581295A (en) * | 2022-08-24 | 2023-01-10 | 华南理工大学 | Soybean flavoring base material with freshness enhancing effect and preparation method and application thereof |
| CN115947778A (en) * | 2022-09-07 | 2023-04-11 | 南京中医药大学 | A small peptide with lipid-lowering activity, preparation method and application thereof |
| CN115947778B (en) * | 2022-09-07 | 2023-12-08 | 南京中医药大学 | A small peptide with lipid-lowering activity, preparation method and application thereof |
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