CN109609576A - A kind of extracting method of hickory nut polypeptide - Google Patents

A kind of extracting method of hickory nut polypeptide Download PDF

Info

Publication number
CN109609576A
CN109609576A CN201811639785.1A CN201811639785A CN109609576A CN 109609576 A CN109609576 A CN 109609576A CN 201811639785 A CN201811639785 A CN 201811639785A CN 109609576 A CN109609576 A CN 109609576A
Authority
CN
China
Prior art keywords
hickory nut
hickory
polypeptide
powder
ball
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201811639785.1A
Other languages
Chinese (zh)
Inventor
许文灏
王鲁黔
苏为科
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang University of Technology ZJUT
Original Assignee
Zhejiang University of Technology ZJUT
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang University of Technology ZJUT filed Critical Zhejiang University of Technology ZJUT
Priority to CN201811639785.1A priority Critical patent/CN109609576A/en
Publication of CN109609576A publication Critical patent/CN109609576A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Water Supply & Treatment (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a kind of extracting method of hickory nut polypeptide, the methods are as follows: by degreasing hickory nut powder, is added in solid-phase reagent and mixes, mechanical ball mill mixes under conditions of 5~30Hz, obtains ball-milled powder；Ball-milled powder addition buffer solution is sufficiently mixed, pH value is adjusted to 8~9, alkali protease is added, after digesting completely under conditions of 45~55 DEG C, the solution after enzyme deactivation is centrifuged by enzyme deactivation, collects supernatant, and dialysis obtains hickory nut polypeptide.Present invention employs mechanochemistry combination zymolysis techniques to prepare hickory nut polypeptide, compared with tradition enzymatic hydrolysis, degreasing hickory nut powder and solid-phase reagent carry out being co-mulled and made into crushing, plant cell wall ruptures under the effect of mechanical stress repeatedly, the dissolution for being conducive to the macromoleculars such as protein, not only shortens enzymolysis time, improves hydrolysis result, and compared with common enzymatic hydrolysis, hickory nut polypeptide yield can be improved 64.9%.

Description

A kind of extracting method of hickory nut polypeptide
Technical field
The invention belongs to food or health food processing technique fields, and in particular to a kind of to utilize mechanochemistry ancillary technique The method for preparing hickory nut polypeptide and primary purification.
Background technique
Hickory nut (Carya cathayensis) is that Juglandaceae (Juglandaceae) hickory (Carya) fallen leaves are tall Wood, alias Chinese walnut, numb walnut, the plant whole world of hickory share 18 kinds, 2 subspecies, China be its source area it One.Hickory nut is distributed in China north and south, Yunnan, Hunan, Guizhou, the main producing region that zhejiang and other places area is China hickory nut.Mountain Walnut is sweet in flavor, mild-natured, first recorded in 3rd century of Christian era Shanxi for written by Zhang Hua when " natural science will ", " Mingyi Bielu " of rear Tao Hongjing and Lee Precious Compendium of Material Medica is also on the books.It is with invigorating qi and benefiting blood, the dry resolving sputum of tune, temperature compensation kidney lung, removing toxicity for detumescence and other effects (when Lee Beijing precious Compendium of Materia Medica [M]: Chinese archives publishing house, 2002:89-93).Hickory nut whole body is all treasured, and hickory kernel can be grown Blood-nourishing arteries and veins improves a poor appetite, pitch-black beard and hair, adjusts neurasthenia, amnesia；Fresh root skin decoction is embathed, and foot hemorrhoid are controlled；Fresh exocarp It smashes and takes juice, diseases such as skin tinea etc. (" Tianmu Mountains of Zhejiang Province medicine plants will " Beijing [M]: Science Press, 2001:88-91) is controlled in wiping.It is existing Generation studies have shown that hickory nut have the effects that it is antitumor, antibacterial, anti-oxidant.
Protein rich in hickory kernel, Zhang Tingzhou (nutrition of Zhang Tingzhou hickory nut, biological characteristics and are opened Hair utilize status [J] food and fermentation industries, 2006,3218 (6): 90-93) detection discovery hickory nut protein content be 7.8%-9.6%, amino acid content are up to 25%, Wang Jiping (Major Nutrient in Wang Jiping, Li Yanan, Ma Jianwei hickory kernel Research [J] Food Science of ingredient, 1998,19 (4): 44-46) etc. have detected the protein content of hickory nut dried product and be 28.1%, 17 kinds of amino acid are contained in hickory nut, total amount reaches 27.198%.Its value is similar to English walnut, and wherein with glutamic acid Content highest accounts for the 20.19% of total amount, and arginine content takes second place, and accounts for the 13.22% of total amount.Glutamic acid has brain strengthening function, energy Brain cell is promoted to be breathed, the ammonia be conducive in brain tissue excludes, and arginine is for senile chronics such as cardiovascular and cerebrovascular diseases Disease has significant health-care effect.Essential amino acid also rich in hickory nut, ratio reach 49.41%, it is abundant must Amino acid is needed to show that hickory nut protein has nutritive value abundant.
The discovery of scientific research in recent years, human consumption's protein is after digesting enzyme effect, and the non-principal shape with amino acid Formula absorbs, but is absorbed in the form of peptide, and nutriment needed for some peptides can not only provide growth in humans, development has simultaneously There are many physiological functions, such as improve minerals transport and absorption, antibacterial and virus, improve immunity, is anti-oxidant, drop gallbladder Sterol, antitumaous effect remove free radical anti-aging and anti-hypertension and hyperlipidemia etc..
Currently, the extracting method of biologically active peptide has acid-hydrolysis method, alkali hydrolysis method and enzymatic isolation method.When acid, basic hydrolysis polypeptide, The segment length that hydrolysis obtains peptide is not easy to control, and is destroyed partial amino-acid, polluted protein.And enzyme digestion reaction item Part is more mild, and hydrolysis degree is easy to control, destroys to amino acid low with environmental, and enzyme has high efficiency and single-minded Property, aminosal obtains the molecular weight distribution of product than more uniform.Therefore, polypeptide is extracted at present generally use enzyme hydrolysis method (preparation of Chen Yonghao walnut protein peptide and de- hardship progress [J] Agriculture of Anhui science, 2012,40 (24): 12219- 12221).The research that enzyme process prepares walnut polypeptide is relatively more (CN 101228918A), which passes through ultrasonic extraction method walnut Protein digests walnut protein, and centrifugation takes supernatant, supernatant is dialysed, removes impurity and macromolecular Object is closed, then obtained dialyzate is concentrated, is finally dried in vacuo again, polypeptide powder is made.However, walnut protein Common enzymatic hydrolysis haves the shortcomings that enzymolysis time is longer, yield is lower.
In recent years, application of the mechanochemistry in extraction is more and more wider, this is attributed to the fact that mechanochemistry breaks plant cell wall, The recovery rate for improving effective component, can achieve simplified technique, reduces cost, the purpose to reduce environmental pollution.
Currently, hickory nut is partially squeezed for grease using other than direct processing and eating, and the mountain after squeezing The comprehensive utilizating research report of walnut grouts is very few.A large amount of degreasing hickory nut slag is attached by as feed and fertilizer or discarding It is value added lower.To make full use of the hickory nut resource in China and improving its economic value, deep processing research is carried out to it to seem It is particularly important.The present invention studies the preparation of hickory nut polypeptide simultaneously using hickory nut grouts as raw material, is the synthesis of hickory nut grouts Technical basis is provided using with new product development.
Summary of the invention
The object of the present invention is to provide a kind of extracting methods of hickory nut polypeptide, try in high energy mechanical power and assistant chemical Under the action of agent, plant cell wall granularity under physics and chemical action reduces and generates crack, and it is big to be conducive to protein etc. The dissolution of molecular substance；Without using a large amount of acid-base reagent, it is environmentally protective and can under the conditions of comparatively gentle efficiently quickly Hickory nut polypeptide is extracted, it is made to shorten cumbersome step in production technology, cost is reduced, reaches the advantages that efficient, environmentally friendly.
To achieve the goals above, the following technical solutions are proposed by the present invention:
The present invention provides a kind of extracting method of hickory nut polypeptide, and the method carries out as follows:
(1) hickory kernel is dry, smashing, then refluxing extraction (preferably repeats to mention three times, every time at 69 DEG C with n-hexane 1 hour), vacuum filtration, filtration cakes torrefaction obtains degreasing hickory nut powder.
(2) the degreasing hickory nut powder for obtaining step (1) is added in solid-phase reagent and mixes, in 5~30Hz (preferably 15 ~25Hz) under conditions of mechanical ball mill mix, obtain ball-milled powder；The solid-phase reagent is one of following: sodium hydroxide, hydrogen-oxygen Change potassium, boric acid, citric acid, silica gel, calcium carbonate, sodium carbonate, sodium bicarbonate, potassium chloride, sodium chloride, sodium dihydrogen phosphate, phosphoric acid hydrogen The mixture of one or more of disodium, lauryl sodium sulfate or borax arbitrary proportion；
(3) buffer solution is added in the ball-milled powder that step (2) obtain to be sufficiently mixed, adjusts pH value to 8~9, alkali is added Property protease, under conditions of 45~55 DEG C digest completely after, enzyme deactivation, by after enzyme deactivation solution be centrifuged (preferably in 7500r/min 10min is centrifuged on centrifuge), supernatant is collected, the dialysis membrane for being 0.3kDa with molecular cut off is dialysed (excellent in deionized water Select 3 days), trapped fluid is collected, freeze-drying obtains hickory nut polypeptide.
Further, hickory kernel described in step (1) includes that fresh hickory kernel, hickory kernel be cooked or hickory nut Benevolence oil expression grouts.
Further, step (1) the n-hexane volumetric usage is calculated as 5ml/g with hickory kernel weight.
Further, 40 meshes are crossed after step (1) filtration cakes torrefaction, obtain degreasing hickory nut powder.
Further, step (2) solid-phase reagent is one of following: sodium hydroxide, citric acid, disodium hydrogen phosphate, phosphoric acid Sodium dihydrogen, potassium hydroxide, calcium carbonate, sodium hydroxide and calcium carbonate mass ratio 1:1 mixing, sodium hydroxide and sodium carbonate mass ratio The mixing of 1:1, the mixing of sodium hydroxide and disodium hydrogen phosphate mass ratio 1:1, sodium hydroxide and sodium dihydrogen phosphate mass ratio 1:1 Mixing.
Further, step (2) described ball milling is carried out under the zirconium oxide ball milling pearl effect of partial size 8mm, and number is 6~20 It is a, preferably 8~14.
Further, step (2) the mechanical ball mill method are as follows: ball milling 10min, interval 5min are a cycle, are co-mulled and made into 1 ~10 periods, preferably 3~8 periods.
Further, step (2) solid-phase reagent and degreasing hickory nut powder mass ratio are 0.01~0.1:1, preferably 0.02~0.06:1.
Further, step (3) the buffer solution additional amount is calculated as 10~100ml/g with ball-milled powder weight, preferably 20~60ml/g；The buffer solution is the phosphate or boric acid-borax buffer solution of pH 9.
Further, the preferred lichens bacillus protein enzyme of step (3) described alkali protease, enzyme activity 200U/mg, the alkali Property protease and ball-milled powder mass ratio be 1~6:100, preferably 1.5~3:100.
Further, step (3) enzymatic hydrolysis condition be 45~55 DEG C, revolving speed be 170rpm shaking table in shaking enzymatic hydrolysis 30 ~90min, preferably 60~90min.
Further, step (3) enzyme deactivation is enzyme deactivation 10min in boiling water bath.
The present invention has the following advantages and effects with respect to the prior art:
(1) present invention employs mechanochemistry combination zymolysis techniques to prepare hickory nut polypeptide, compared with tradition enzymatic hydrolysis, degreasing Hickory nut powder and solid-phase reagent carry out being co-mulled and made into crushing, and plant cell wall ruptures under the effect of mechanical stress repeatedly, are conducive to egg The dissolution of the macromoleculars such as white matter, not only shortens enzymolysis time, improves hydrolysis result, and compared with common enzymatic hydrolysis, mountain core Peach polypeptide yield can be improved 64.9%.
(2) hickory nut comprehensive utilization of resources, albumen of the hickory nut rich in high-quality fat and high quality, the present invention are realized Using hickory nut as raw material, the preparation of hickory nut polypeptide is studied, the comprehensive utilization of hickory nut resource is realized.
(3) present invention process is easy to operate, economic and environment-friendly, mild condition, and it is shallowly high-quality to obtain product color.
Detailed description of the invention
Fig. 1 is degreasing hickory nut powder scanning electron microscope (SEM) photograph (2000 times, 20 μm), and A is before mechanochemistry is handled, and B is mechanization After processing.
Specific embodiment
The present invention is described further combined with specific embodiments below, and but the scope of the present invention is not limited thereto.
The measurement of product content of peptides described in following embodiment is using reduced glutathione as standard items, using biuret Method measures content of peptides.
Using absorbance value as ordinate, reduced glutathione mass concentration is abscissa, draws polypeptide standard curve, is returned Return equation:
Y=0.0939x+0.0034, R2=0.9994
The assay of degreasing hickory nut powder protein is referring to GB 5009.5-2016, using micro triumphant formula nitriding, egg White matter conversion coefficient is 6.25.
The calculation formula of polypeptide yield:
Polypeptide yield (%)=soluble polypeptide quality (g)/[gross mass (g) of degreasing hickory nut powder × degreasing hickory nut The content of amyloid proteins matter] × 100%.
The alkali protease used in the present invention is bacillus licheniformis protease, and enzyme activity 200U/mg is purchased from Shanghai source leaf Biotechnology Co., Ltd.The enzyme deactivation refers to enzyme deactivation 10min in boiling water bath.
Embodiment 1
(1) hickory kernel oil expression grouts are dry, smash, then with the amount of every g powder addition 5ml n-hexane be added just oneself Alkane, refluxing extraction 1 hour at 69 DEG C, after repetition mentions three times, vacuum filtration, filtration cakes torrefaction crosses 40 meshes, obtains degreasing hickory nut Powder (content 48.34% of protein).Scanning electron microscope (SEM) photograph is control with hickory kernel as shown in Figure 1.Pass through scanning electron microscope (SEM) microstructure of degreasing hickory nut powder is observed, it can be seen that mechanochemistry treated degreasing hickory nut powder, granularity Apparent decline, granulation uniformity improve.Microstructure shows that untreated degreasing walnut powder shows that comparison is closely large stretch of Layer structure, and treated that degreasing walnut powder is then loose little particle state for mechanochemistry, surface area significantly increases.Cause This, the contact area of mechanochemistry treated degreasing walnut powder and enzyme increases, and is more advantageous to the progress of enzymatic hydrolysis.
(2) degreasing hickory nut powder 1.2g, NaOH 0.03g, CaCO are added into zirconia ball grinding jar30.03g, diameter 10, the zirconium oxide ball milling pearl of 8mm, grinds at 15Hz, and 10min is a period, interval 5min, 3 periods of ball milling, acquisition ball Milling end；
(3) ball-milled powder 0.84g is weighed, 40mL pH=9 buffer solution, gained mixed liquor 1mol/L are added thereto HCL aqueous solution adjust pH to 8, be added alkali protease 0.02g, digest 1h under conditions of 55 DEG C, 170rpm；Enzyme deactivation, will Solution after enzyme deactivation is centrifuged 10min on 7500r/min centrifuge, and collects supernatant, is 0.3kDa's with molecular cut off Dialysis membrane dialysis treatment 3 days in deionized water, collect the liquid in bag filter, and freeze-drying obtains hickory nut polypeptide 0.1965g, calculating polypeptide yield is 50.81%.
Embodiment 2
(1) degreasing hickory nut powder is the same as embodiment 1.
(2) degreasing hickory nut powder 1.2g, NaOH 0.06g, the zirconia ball of diameter 8mm are added into zirconia ball grinding jar 10, pearl, mill, grinds at 20Hz, and 10min is a period, interval 5min, 3 periods of ball milling, acquisition ball-milled powder.
(3) ball-milled powder 0.84g is weighed, 40mL pH=9 buffer solution, gained mixed liquor 1mol/L are added thereto HCL aqueous solution adjust pH to 9, be added alkali protease 0.02g, digest 1h under conditions of 55 DEG C, 170rpm；Enzyme deactivation, will Solution after enzyme deactivation is centrifuged 10min on 7500r/min centrifuge, and collects supernatant, is 0.3kDa's with molecular cut off Dialysis membrane dialysis treatment 3 days in deionized water, collect the liquid in bag filter, and freeze-drying obtains hickory nut polypeptide 0.2567g, calculating polypeptide yield is 66.38%.
Embodiment 3
(1) degreasing hickory nut powder is the same as embodiment 1.
(2) degreasing hickory nut powder 1.2g, citric acid 0.06g, the zirconia ball of diameter 8mm are added into zirconia ball grinding jar 8, pearl, mill, grinds at 20Hz, and 10min is a period, interval 5min, 3 periods of ball milling, acquisition ball-milled powder.
(3) ball-milled powder 0.84g is weighed, 40mL pH=9 buffer solution, gained mixed liquor 3mol/L are added thereto NaOH aqueous solution adjust pH to 8.5, be added alkali protease 0.02g, digest 1h under conditions of 55 DEG C, 170rpm；Enzyme deactivation, Solution after enzyme deactivation is centrifuged 10min on 7500r/min centrifuge, and collects supernatant, is 0.3kDa with molecular cut off Dialysis membrane dialysis treatment 3 days in deionized water, collect the liquid in bag filter, freeze-drying obtains hickory nut polypeptide 0.2024g, calculating polypeptide yield is 52.34%.
Embodiment 4
(1) fresh hickory kernel is dry, smashes, then the amount addition n-hexane of 5ml n-hexane is added with every g powder, and 69 DEG C Lower refluxing extraction 1 hour, after repetition mentions three times, vacuum filtration, filtration cakes torrefaction crosses 40 meshes, obtains degreasing hickory nut powder (albumen The content 23.87% of matter).
(2) degreasing hickory nut powder 1.2g, NaOH 0.03g, NaCO are added into zirconia ball grinding jar30.03g, diameter 9, the zirconium oxide ball milling pearl of 8mm, grinds at 25Hz, and 10min is a period, interval 5min, 3 periods of ball milling, acquisition ball milling Powder.
(3) ball-milled powder 0.84g is weighed, 40mL pH=9 buffer solution, gained mixed liquor 1mol/L are added thereto HCL aqueous solution adjust pH to 9, be added alkali protease 0.02g, digest 1h under conditions of 55 DEG C, 170rpm；Enzyme deactivation, will Solution after enzyme deactivation is centrifuged 10min on 7500r/min centrifuge, and collects supernatant, is 0.3kDa's with molecular cut off Dialysis membrane dialysis treatment 3 days in deionized water, collect the liquid in bag filter, and freeze-drying obtains hickory nut polypeptide 0.1074g, calculating polypeptide yield is 56.24%.
Embodiment 5
(1) degreasing hickory nut powder is the same as embodiment 4.
(2) the zirconium oxide ball milling of degreasing hickory nut powder 1.2g, KOH 0.06g, diameter 8mm are added into zirconia ball grinding jar It 7, pearl, is ground at 20Hz, 10min is a period, interval 5min, 4 periods of ball milling, acquisition ball-milled powder.
(3) ball-milled powder 0.84g is weighed, 40mL pH=9 buffer solution, gained mixed liquor 1mol/L are added thereto HCL aqueous solution adjust pH to 9, be added alkali protease 0.02g, digest 1h under conditions of 55 DEG C, 170rpm；Enzyme deactivation, will Solution after enzyme deactivation is centrifuged 10min on 7500r/min centrifuge, and collects supernatant, is 0.3kDa's with molecular cut off Dialysis membrane dialysis treatment 3 days in deionized water, collect the liquid in bag filter, and freeze-drying obtains hickory nut polypeptide 0.1098g, calculating polypeptide yield is 57.50%.
Embodiment 6
(1) degreasing hickory nut powder is the same as embodiment 4.
(2) degreasing hickory nut powder 1.2g, Na are added into zirconia ball grinding jar2HPO40.06g, the zirconium oxide of diameter 8mm It 8, ball milling pearl, is ground at 15Hz, 10min is a period, interval 5min, 3 periods of ball milling, acquisition ball-milled powder.
(3) ball-milled powder 0.84g is weighed, 40mL pH=9 buffer solution, gained mixed liquor 1mol/L are added thereto HCL aqueous solution adjust pH to 8, be added alkali protease 0.02g, digest 1h under conditions of 50 DEG C, 170rpm；Enzyme deactivation, will Solution after enzyme deactivation is centrifuged 10min on 7500r/min centrifuge, and collects supernatant, is 0.3kDa's with molecular cut off Dialysis membrane dialysis treatment 3 days in deionized water, collect the liquid in bag filter, and freeze-drying obtains hickory nut polypeptide 0.1036g, calculating polypeptide yield is 54.25%.
Embodiment 7
(1) the cooked drying of hickory kernel, smashing, then with every g powder addition 5ml n-hexane amount be added n-hexane, 69 Refluxing extraction 1 hour at DEG C, after repetition mentions three times, vacuum filtration, filtration cakes torrefaction crosses 40 meshes, obtains degreasing hickory nut powder (egg The content 30.25% of white matter).
(2) degreasing hickory nut powder 1.2g, NaOH 0.03g, Na are added into zirconia ball grinding jar2HPO40.03g, diameter 8, the zirconium oxide ball milling pearl of 8mm, grinds at 15Hz, and 10min is a period, interval 5min, 3 periods of ball milling, acquisition ball milling Powder.
(3) ball-milled powder 0.84g is weighed, 40mL pH=9 buffer solution, gained mixed liquor 1mol/L are added thereto HCL aqueous solution adjust pH to 8, be added alkali protease 0.024g, digest 1h under conditions of 55 DEG C, 170rpm；Enzyme deactivation, will Solution after enzyme deactivation is centrifuged 10min on 7500r/min centrifuge, and collects supernatant, is 0.3kDa's with molecular cut off Dialysis membrane dialysis treatment 3 days in deionized water, collect the liquid in bag filter, and freeze-drying obtains hickory nut polypeptide 0.1387g, calculating polypeptide yield is 57.31%.
Embodiment 8
(1) degreasing hickory nut powder is the same as embodiment 7.
(2) degreasing hickory nut powder 1.2g, CaCO are added into zirconia ball grinding jar30.06g, the zirconia ball of diameter 8mm 10, pearl, mill, grinds at 20Hz, and 10min is a period, interval 5min, 2 periods of ball milling, acquisition ball-milled powder.
(3) ball-milled powder 0.84g is weighed, 40mL pH=9 buffer solution, gained mixed liquor 1mol/L are added thereto HCL aqueous solution adjust pH to 9, be added alkali protease 0.02g, digest 1h under conditions of 55 DEG C, 170rpm；Enzyme deactivation, will Solution after enzyme deactivation is centrifuged 10min on 7500r/min centrifuge, and collects supernatant, is 0.3kDa's with molecular cut off Dialysis membrane dialysis treatment 3 days in deionized water, collect the liquid in bag filter, and freeze-drying obtains hickory nut polypeptide 0.1293g, calculating polypeptide yield is 53.43%.
Embodiment 9
(1) degreasing hickory nut powder is the same as embodiment 7.
(2) degreasing hickory nut powder 1.2g, NaH are added into zirconia ball grinding jar2PO40.06g, the zirconium oxide of diameter 8mm It 7, ball milling pearl, is ground at 15Hz, 10min is a period, interval 5min, 4 periods of ball milling, acquisition ball-milled powder.
(3) ball-milled powder 0.84g is weighed, 20mL pH=9 buffer solution, gained mixed liquor 1mol/L are added thereto NaOH aqueous solution adjust pH to 9, be added alkali protease 0.02g, digest 1h under conditions of 45 DEG C, 170rpm；Enzyme deactivation, will Solution after enzyme deactivation is centrifuged 10min on 7500r/min centrifuge, and collects supernatant, is 0.3kDa's with molecular cut off Dialysis membrane dialysis treatment 3 days in deionized water, collect the liquid in bag filter, and freeze-drying obtains hickory nut polypeptide 0.1177g, calculating polypeptide yield is 48.64%.
Embodiment 10
(1) degreasing hickory nut powder is the same as embodiment 7.
(2) degreasing hickory nut powder 1.2g, NaOH 0.03g, NaH are added into zirconia ball grinding jar2PO40.03g, diameter 10, the zirconium oxide ball milling pearl of 8mm, grinds at 15Hz, and 10min is a period, interval 5min, 3 periods of ball milling, acquisition ball Milling end.
(3) ball-milled powder 0.84g is weighed, 30mL pH=9 buffer solution, gained mixed liquor 1mol/L are added thereto HCL aqueous solution adjust pH to 9, be added alkali protease 0.02g, digest 1h under conditions of 55 DEG C, 170rpm；Enzyme deactivation, will Solution after enzyme deactivation is centrifuged 10min on 7500r/min centrifuge, and collects supernatant, is 0.3kDa's with molecular cut off Dialysis membrane dialysis treatment 3 days in deionized water, collect the liquid in bag filter, and freeze-drying obtains hickory nut polypeptide 0.1352g, calculating polypeptide yield is 55.87%.
Influence of the 1 ball milling enzymatic hydrolysis condition of table to polypeptide yield

Claims (10)

1. a kind of extracting method of hickory nut polypeptide, it is characterised in that the method carries out as follows:
(1) hickory kernel is dry, smashing, then refluxing extraction, vacuum filtration, filtration cakes torrefaction obtain at 69 DEG C with n-hexane Degreasing hickory nut powder；
(2) the degreasing hickory nut powder for obtaining step (1) is added in solid-phase reagent and mixes, mechanical ball under conditions of 5~30Hz Mill mixes, and obtains ball-milled powder；The solid-phase reagent is one of following: sodium hydroxide, citric acid, disodium hydrogen phosphate, di(2-ethylhexyl)phosphate Hydrogen sodium, potassium hydroxide, calcium carbonate, sodium carbonate one or more arbitrary proportion mixture；
(3) buffer solution is added in the ball-milled powder that step (2) obtain to be sufficiently mixed, adjusts pH value to 8~9, alkaline egg is added White enzyme, after digesting completely under conditions of 45~55 DEG C, the solution after enzyme deactivation is centrifuged by enzyme deactivation, collects supernatant, with retention point The dialysis membrane that son amount is 0.3kDa is dialysed in deionized water, collects trapped fluid, and freeze-drying obtains hickory nut polypeptide.
2. the extracting method of hickory nut polypeptide as described in claim 1, it is characterised in that hickory kernel described in step (1) includes Raw fresh hickory kernel, hickory kernel be cooked or hickory kernel extracts oil grouts.
3. the extracting method of hickory nut polypeptide as described in claim 1, it is characterised in that step (1) the n-hexane volumetric usage 5ml/g is calculated as with hickory kernel weight.
4. the extracting method of hickory nut polypeptide as described in claim 1, it is characterised in that cross 40 mesh after step (1) filtration cakes torrefaction Sieve obtains degreasing hickory nut powder.
5. the extracting method of hickory nut polypeptide as described in claim 1, it is characterised in that step (2) solid-phase reagent is following One of: sodium hydroxide, citric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydroxide, calcium carbonate, sodium hydroxide and carbonic acid are calcareous Measure mixing than 1:1, the mixing of sodium hydroxide and sodium carbonate mass ratio 1:1, sodium hydroxide and disodium hydrogen phosphate mass ratio 1:1 Mixing, the mixing of sodium hydroxide and sodium dihydrogen phosphate mass ratio 1:1.
6. the extracting method of hickory nut polypeptide as described in claim 1, it is characterised in that step (2) described mechanical ball mill is in grain The zirconium oxide ball milling pearl effect of diameter 8mm is lower to be carried out, and number is 6~20.
7. the extracting method of hickory nut polypeptide as claimed in claim 6, it is characterised in that step (2) the mechanical ball mill method Are as follows: ball milling 10min, interval 5min are a cycle, are co-mulled and made into 1~10 period.
8. the extracting method of hickory nut polypeptide as described in claim 1, it is characterised in that step (2) solid-phase reagent and degreasing Hickory nut powder mass ratio is 0.01~0.1:1.
9. the extracting method of hickory nut polypeptide as described in claim 1, it is characterised in that step (3) the buffer solution additional amount 10~100ml/g is calculated as with ball-milled powder weight；The buffer solution is the phosphate or boric acid-borax buffer solution of pH9.
10. the extracting method of hickory nut polypeptide as described in claim 1, it is characterised in that step (3) alkali protease with Ball-milled powder mass ratio is 1~6:100.
CN201811639785.1A 2018-12-29 2018-12-29 A kind of extracting method of hickory nut polypeptide Pending CN109609576A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811639785.1A CN109609576A (en) 2018-12-29 2018-12-29 A kind of extracting method of hickory nut polypeptide

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811639785.1A CN109609576A (en) 2018-12-29 2018-12-29 A kind of extracting method of hickory nut polypeptide

Publications (1)

Publication Number Publication Date
CN109609576A true CN109609576A (en) 2019-04-12

Family

ID=66015823

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811639785.1A Pending CN109609576A (en) 2018-12-29 2018-12-29 A kind of extracting method of hickory nut polypeptide

Country Status (1)

Country Link
CN (1) CN109609576A (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103468773A (en) * 2013-09-06 2013-12-25 江苏丘陵地区镇江农业科学研究所 Extraction method of walnut kernel polypeptide
CN104719609A (en) * 2015-04-08 2015-06-24 北京市农林科学院 Walnut protein concentrate and preparing method thereof
CN106561813A (en) * 2016-10-31 2017-04-19 李业清 Fermented yoghurt walnut juice for relaxing muscles and stimulating blood circulation
CN107858393A (en) * 2017-12-13 2018-03-30 云南省林业科学院 A kind of method that polypeptide is extracted from walnut dregs
CN108553556A (en) * 2018-04-16 2018-09-21 湖州耕香生物科技有限公司 A method of preparing the total glycosides of west safflower from west safflower

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103468773A (en) * 2013-09-06 2013-12-25 江苏丘陵地区镇江农业科学研究所 Extraction method of walnut kernel polypeptide
CN104719609A (en) * 2015-04-08 2015-06-24 北京市农林科学院 Walnut protein concentrate and preparing method thereof
CN106561813A (en) * 2016-10-31 2017-04-19 李业清 Fermented yoghurt walnut juice for relaxing muscles and stimulating blood circulation
CN107858393A (en) * 2017-12-13 2018-03-30 云南省林业科学院 A kind of method that polypeptide is extracted from walnut dregs
CN108553556A (en) * 2018-04-16 2018-09-21 湖州耕香生物科技有限公司 A method of preparing the total glycosides of west safflower from west safflower

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
RAHELEH JAHANBANI，等: "Antioxidant and Anticancer Activities of Walnut (Juglans regia L.) Protein Hydrolysates Using Different Proteases", 《PLANT FOODS HUMAN NUTRITION》 *
李建杰，等: "碱性蛋白酶对核桃蛋白水解条件的优化研究", 《中国食品学报》 *

Similar Documents

Publication Publication Date Title
CN101367881B (en) High-efficiency preparation method for glossy ganoderma polysaccharide
CN101348529B (en) Extraction and separation method of medlar polysaccharide
CN105249086B (en) A kind of preparation process of the red date concentrated juice of high cAMP contents
CN104206641B (en) A kind of giant salamander polypeptide powder and preparation method thereof
CN102805350A (en) Method for preparing dietary fibers from wastes and byproducts generated in tangerine processing
CN109793856B (en) Preparation method of dendrobium officinale powder
CN105062990A (en) Complex enzyme preparation for extracting effective plant components and method for preparing complex enzyme preparation
CN105154511A (en) Method for extracting amino acids from mulberry leaf
CN103039937A (en) Plant salt and preparation method thereof
CN102512353A (en) Dictyophora indusiata water extract and preparation method and application thereof
CN106832021A (en) A kind of preparation method of Chinese cassia tree slag Thick many candies
CN105595334B (en) A kind of maca composite sheet and preparation method thereof
CN101899120B (en) Method for refining jujube polysaccharide
CN106892994A (en) A kind of preparation method of blueberry residue Thick many candies
CN109006179A (en) A kind of production method of mushroom cultivation substrate
CN109609576A (en) A kind of extracting method of hickory nut polypeptide
CN106892989A (en) A kind of blackberry, blueberry preparation method
CN105367678A (en) Lycium barbarum polysaccharide extraction method
CN107411068B (en) Red date cyclic adenosine monophosphate chewable tablet and preparation method thereof
CN106928314A (en) A kind of method that earthworm polypeptide is extracted from earthworm
CN106689641A (en) Production technology of walnut peptide powder
CN109364110A (en) A method of flavones is extracted from U.S. rattan shell
CN105532350B (en) A kind of preparation method of hainan holly leaf ginkgo biloba extract compound tea
CN107319527A (en) Giant salamander nutrient powder product is prepared using radix polygonati officinalis
CN106035869A (en) Preparation method of siraitia grosvenorii and isatis root extract compound tea

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20190412