CN102406050B - Walnut low molecular weight polypeptide and preparation method thereof - Google Patents
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Abstract
The invention discloses a walnut low molecular weight polypeptide and a preparation method thereof. The preparation method comprises the following steps: hydrolyzing walnut albumen powder through a high static pressure technique combined with a biological enzyme; decoloring and adsorbing to remove bitter substances in the walnut low molecular weight polypeptide, and then preparing the walnut low molecular weight polypeptide with high purity, good quality and high low molecular weight peptide proportion; and directly packaging or carrying out freeze drying to obtain the finished walnut low molecular weight polypeptide product. The walnut low molecular weight polypeptide disclosed by the invention can be widely applied to medicine raw materials, medicaments, food, health care products, food additives and the like. By using the method disclosed by the invention, the hydrolysis degree of protein is improved so that the proportion of the walnut low molecular weight polypeptide is improved, thereby being more beneficial to the absorption and utilization of walnut polypeptide, enhancing the nutrition and health care effects of walnut and improving the product additional value of walnut. Thus, the walnut low molecular weight polypeptide has a good market application prospect.
Description
Technical field
The present invention relates to a kind of walnut micromolecule polypeptide and preparation method thereof.
Background technology
Walnut another name English walnut is one of world's " four is big " dry fruit, also is the traditional economy crop that China has cultivation history in several thousand.Walnut kernel contains the indispensable trace elements of human body such as multiple nutritional components such as protein, aliphatic acid, sugar, fiber, vitamin and zinc, manganese, chromium, has the characteristic of " integration of drinking and medicinal herbs ".Phosphatide in the walnut has excellent health functions to cranial nerve.Frequent edible walnut can the body-building body, again can be anti-ageing.According to surveying and determination, every hectogram walnut contains protein 14.9 grams, fat 58.8 grams.Lipase 37 in the walnut 1% is linoleic acid, and 12% is leukotrienes, carbohydrate 9.6 grams, dietary fiber 9.6 grams, carrotene 30 micrograms, 43.21 milligrams of vitamin Es, 3.44 milligrams in potassium, 56 milligrams of calcium, 294 milligrams in phosphorus, 2.7 milligrams of iron, selenium 4.62 micrograms, 2.17 milligrams on zinc.With regard to nutritional labeling, walnut nutritious value is 8.5 times of soybean, 6 times of peanut, and 12 times of egg, 25 times of milk, 10 times of meat, 1 jin of walnut kernel is equivalent to the nutritive value of 5 jin of eggs or 9 jin of milk.18 seed amino acid A wide selection of colours and designs in the walnut protein, and 8 kinds of essential amino acids content of needed by human body are reasonable, and it is all higher that effect has glutamic acid, aspartic acid, the arginine content of critical function to Human Physiology.Institute's walnut protein is a kind of good protein sources.The nourishing of walnut and medical value have clearly record in the ancient medical book of China, put down in writing walnut " benefiting qi and nourishing blood, moistening dryness and resolving phlegm, the beneficial gate of vitality, sharp three warmers; warm lung ease constipation; control the cold of insufficiency type and breath with cough ... " in the Compendium of Material Medica, the traditional Chinese medical science thinks that walnut has the effect of temperature compensation lung kidney, is a kind of good tonic, cerebral nerve is had help greatly, is brain boosting and supplementing and the neurasthenic good merchantable brand for the treatment of.Modern medicine study shows, that walnut has is anti-ageing, reduce cholesterol, keep cardiovascular health to play an important role.
In recent years, the walnut product of exploitation increases day by day, also there is patent of invention to relate to technology such as milling process and microwave-assistant sol agent extraction method and prepares walnut oil in the walnut, use and all can produce the walnut protein cake that accounts for walnut kernel weight 40%～60% after various extractive techniques prepare walnut oil, because the walnut protein cake is rich in rich in protein, have good value and wide development prospect, also cause people's attention gradually.Walnut polypeptide generally is made of 3～20 amino acid, its physiological active functions comprise promote nutrient absorption, improve immunity, regulate neural, anti-oxidant, hypotensive and antibiotic etc.The method that generally prepares micromolecule polypeptide now comprises chemical hydrolysis and enzyme hydrolysis, and chemical hydrolysis is to utilize strong acid and strong base protein hydrolysate, cheap and simple, but reaction condition is violent, and amino acid is impaired serious in the production process; Enzyme hydrolysis method is the method that applying biological enzyme hydrolysis split protein produces specific peptide under the condition of gentleness, but hydrolysis time is long, degree of hydrolysis is on the low side, and the ratio of small-molecular peptides is lower after the hydrolysis.According to the shortcoming of the various method for hydrolysis of addressing previously, develop the method for preparing walnut polypeptide that walnut micromolecule polypeptide content is high in a kind of reaction condition gentleness and the product and be significant.
Summary of the invention
The purpose of this invention is to provide a kind of walnut micromolecule polypeptide and preparation method thereof.
Walnut micromolecule polypeptide provided by the present invention is to prepare according to the method that comprises the steps: be that 8.0～8.5 cushioning liquid is mixed with walnut protein solution with the walnut protein powder with the pH value, and carry out enzyme digestion reaction to wherein adding papain; Reaction is handled 10～30min after finishing again under 300～600MPa ultra-high pressure condition, obtain walnut micromolecule polypeptide solution.
Among the present invention, described walnut protein powder can obtain by commercial sources, also can prepare according to following method: adding water in the degreasing walnut grouts grinds, and obtains the walnut protein slurries; Regulate pH value to 8.5～9.5 of described walnut protein slurries with alkali, behind insulation vibration 70～90min under 50～60 ℃ of conditions, leave standstill 20～30min then, filter, obtain supernatant; The pH value of regulating described supernatant with acid is to there being precipitation to separate out again, and the centrifugation precipitation is centrifugal again after washing precipitation with water 2～3 times, carries out freeze drying or spray-drying again after gained walnut protein precipitate with deionized water is dissolved, and namely gets the walnut protein powder.
Wherein, the mass ratio of described degreasing walnut grouts and water is 1: 8～1: 10; Also need soak before carrying out described grinding, the time of immersion is 1～2h; Described grinding adopts colloid mill to grind; Described pH value to 3.5～4.5 of regulating described supernatant with acid; Described alkali is the sodium hydroxide solution of concentration 2～3mol/L; Described acid is the hydrochloric acid solution of concentration 2～3mol/L.
Among the present invention, the condition of described enzyme digestion reaction is: the mass concentration of walnut protein solution is 35～50g/L, and the ratio of papain and walnut protein is 60～80U/g, and reaction temperature is 45～55 ℃, and the reaction time is 4～5h.Used cushioning liquid specifically can be phosphate buffer.
The condition of preferred enzyme digestion reaction is: the mass concentration of walnut protein solution is 35g/L, and the ratio of papain and walnut protein is 80U/g, and reaction temperature is 50 ℃, and the reaction time is 5h.
In order to make enzyme digestion reaction more abundant, in enzymolysis process, need reaction system is shaken, oscillation rate can be 90～120rpm.
In order to obtain the more excellent walnut micromolecule polypeptide product of quality and purity, described method also comprises the step that walnut micromolecule polypeptide solution is decoloured, goes bitter taste.Concrete grammar is as follows: add that active carbon stirs that 1～2h decolours in the described walnut micromolecule polypeptide solution and after absorption removes bitter taste, filter and collect filtrate, obtain the walnut micromolecule polypeptide solution of purifying; Wherein, the addition of described active carbon is 5～7% of walnut protein quality.
The walnut micromolecule polypeptide solution of above-mentioned purifying directly can obtains the walnut polypeptide liquid product.
Certainly convenient and be applicable to more multi-form for accumulating, also its further freeze drying can be made walnut micromolecule polypeptide powder.
The walnut micromolecule polypeptide for preparing according to the method described above, its form mainly be molecular weight less than the micromolecule polypeptide of 2000Da, especially molecular weight is at the micromolecule polypeptide of 300～1500Da.Compare with the walnut polypeptide of simple zymolysis technique preparation, the composition showed increased of micromolecule polypeptide in the walnut micromolecule polypeptide that the inventive method obtains, ratio obviously increases; And have characteristics such as degree of hydrolysis is moderate, purity is high, solubility is good, biological function is strong.Described biological function comprise promote nutrient absorption, improve immunity, regulate neural, anti-oxidant, hypotensive and antibiotic etc.Therefore, this walnut micromolecule polypeptide can be widely used in medical material, medicine, food, health products and food additives etc.
The present invention is raw material with the degreasing walnut grouts, obtains the walnut protein powder after handling through soda acid; Adopt high static pressure to assist biologically active enzyme preparation hydrolysis walnut protein, make the micromolecule polypeptide of molecular weight below 2000Da; And adopt and to have stronger adsorbing active carbon bitter substance contained in the walnut micromolecule polypeptide is adsorbed, effectively reduce the bitter taste of product, improved purity and the quality of walnut micromolecule polypeptide.
The present invention improves the hydrolysis degree of protein on the basis of super-pressure auxiliary enzymes aminosal, walnut micromolecule polypeptide ratio is improved, more be conducive to absorption and the utilization of walnut polypeptide, strengthen the nutrition and health care effect of walnut, improve the value-added content of product of walnut, have good market application prospect.
Description of drawings
Fig. 1 is preparation technology's flow chart of walnut micromolecule polypeptide of the present invention.
Fig. 2 is the molecular weight distribution of the walnut micromolecule polypeptide of traditional enzymatic isolation method preparation.
Fig. 3 is the molecular weight distribution of the walnut micromolecule polypeptide of the embodiment of the invention 1 preparation.
The specific embodiment
Below by specific embodiment method of the present invention is described, but the present invention is not limited thereto.
Experimental technique described in the following embodiment if no special instructions, is conventional method; Described reagent and material if no special instructions, all can obtain from commercial channels.
Used degreasing walnut grouts are Shijiazhuang, Hebei ten thousand brilliant dry fruit Co., Ltd cold press walnut grouts among the following embodiment.Papain is the P3250 of Beijing Baeyer enlightening Bioisystech Co., Ltd, and enzyme activity is 2U/mg.
Embodiment 1, preparation walnut micromolecule polypeptide
(1) extraction of walnut protein:
Accurately take by weighing walnut protein cake (degreasing walnut grouts) 100kg after the oil expression, stir after adding 800kg water logging bubble 2h, open colloid mill circulation defibrination to walnut protein slurries and be fine and smooth uniform state; Use rare alkali lye (concentration is the NaOH solution of 2.0mol/L) that slurry pH value is transferred to 9.0 then, then after insulation under 60 ℃ of conditions and constantly stirring vibration 80min, leave standstill 30min; Use the flame filter press filtration and obtain supernatant, supernatant is adjusted pH to 4.5 with hydrochloric acid namely has precipitation to separate out, the centrifugation precipitation, centrifugal again after washing precipitation with water 2 times, freeze drying is carried out in the dissolving of gained walnut protein precipitate with deionized water again, obtained the walnut protein powder, standby.
(2) preparation of micromolecule polypeptide:
Be the walnut protein liquid that the PBS of 8.0 0.1mol/L is mixed with the walnut protein powder 35g/L with pH, be that the ratio of 60U/g adds papain (enzyme live 2U/mg) behind insulation vibration 5h (oscillation rate is 90rpm) under 50 ℃ of conditions according to the concentration ratio of enzyme-to-substrate, with 600MPa ultra high pressure treatment 10min, obtain walnut micromolecule polypeptide solution.
(3) decolour, go bitter taste:
In walnut micromolecule polypeptide solution, add the active carbon account for walnut protein opaque amount 5% and stir that 1～2h decolours and bitter taste is removed in absorption, filtration obtains the higher walnut protein micromolecule polypeptide of purity solution after obtaining removing filter residue, and directly can obtains walnut micromolecule polypeptide liquid.
(1) extraction of walnut protein:
Accurately take by weighing walnut protein cake (degreasing walnut grouts) 100kg after the oil expression, stir after adding 800kg water logging bubble 2h, open colloid mill circulation defibrination to walnut protein slurries and be fine and smooth uniform state, with rare alkali lye slurries pH is transferred to 9.0 then, then after insulation under 50 ℃ of conditions and constantly stirring vibration 80min, leave standstill 30min, use the flame filter press filtration and obtain supernatant, supernatant is adjusted pH to 4.5 with hydrochloric acid namely has precipitation to separate out, centrifugation precipitation, centrifugal again after washing precipitation with water 3 times, freeze drying is carried out in the dissolving of gained walnut protein precipitate with deionized water again, obtain the walnut protein powder, standby.
(2) preparation of micromolecule polypeptide:
Be the walnut protein liquid that the PBS of 8.0 0.1mol/L is mixed with the walnut protein powder 35g/L with pH, be that the ratio of 80U/g adds papain behind insulation vibration 5h (oscillation rate is 120rpm) under 50 ℃ of conditions according to the ratio of enzyme-to-substrate concentration, with 300MPa ultra high pressure treatment 10min, obtain the walnut micromolecule polypeptide.
(3) decolour, go bitter taste:
The active carbon stirring 1～2h of the walnut micromolecule polypeptide adding 5% after the process hydrolysis process decolours and bitter taste is removed in absorption, obtains the higher walnut protein micromolecule polypeptide of purity solution after filtration obtains removing filter residue.
(4) freeze drying: walnut micromolecule polypeptide liquid is put into temperature and is solid for-20 ℃ of refrigerator 5h freeze to the polypeptide liquid cooling, carry out freeze drying (the material baffle temperature is 12 ℃, and vacuum is 20Pa, and condenser temperature is-45 ℃) again, obtain Powdered walnut micromolecule polypeptide powder, obtain finished product after the packing.
Walnut micromolecule polypeptide powder yield is 75～85% (in the walnut protein content).Walnut protein content adopts the Kjeldahl among the GB GB/T 5009.5-2003 to measure.
Gained walnut micromolecule polypeptide powder is a kind of milky or pale yellow powder, the polypeptide degree of hydrolysis is more than 30%, and the mensuration of polypeptide degree of hydrolysis adopts ninhydrin method in conjunction with high performance liquid chromatography-ground substance assistant laser desorption ionization flight time mass spectrum (HPLC-MALDI-TOF-MS) method.Adopt high performance liquid chromatography-ground substance assistant laser desorption ionization flight time mass spectrum (HPLC-MALDI-TOF-MS) method to measure the molecular weight distribution of products obtained therefrom, calculating molecular weight is more than 90% less than the content of peptides of 2000Da.
Claims (8)
1. method for preparing the walnut micromolecule polypeptide comprises the steps: with the pH value to be that 8.0～8.5 cushioning liquid is mixed with walnut protein solution with the walnut protein powder, and carries out enzyme digestion reaction to wherein adding papain; Reaction is handled 10～30min after finishing again under the 300-600MPa ultra-high pressure condition, obtain walnut micromolecule polypeptide solution;
The condition of described enzyme digestion reaction is: the mass concentration of walnut protein solution is 35～50g/L, and the ratio of papain and walnut protein is 60～80U/g, and reaction temperature is 45～55 ℃, and the reaction time is 4～5h;
Need vibrate in the process of described enzyme digestion reaction, oscillation rate is 90～120rpm.
2. method according to claim 1, it is characterized in that: the condition of described enzyme digestion reaction is: the mass concentration of walnut protein solution is 35g/L, and the ratio of papain and walnut protein is 80U/g, and reaction temperature is 50 ℃, and the reaction time is 5h.
3. method according to claim 1 and 2, it is characterized in that: described walnut protein powder prepares according to following method: adding water in the degreasing walnut grouts grinds, and obtains the walnut protein slurries; Regulate pH value to 8.5～9.5 of described walnut protein slurries with alkali, behind insulation vibration 70～90min under 50～60 ℃ of conditions, leave standstill 20～30min then, filter, obtain supernatant; The pH value of regulating described supernatant with acid is to there being precipitation to separate out again, and the centrifugation precipitation will precipitate and carry out freeze drying or spray-drying again after washing 2～3 times and namely get the walnut protein powder.
4. method according to claim 3, it is characterized in that: the mass ratio of described degreasing walnut grouts and water is 1:8～1:10; Also need soak before carrying out described grinding, the time of immersion is 1～2h; Described grinding adopts colloid mill to grind; Described pH value to 3.5～4.5 of regulating described supernatant with acid; Described alkali is the sodium hydroxide solution of concentration 2～3mol/L; Described acid is the hydrochloric acid solution of concentration 2～3mol/L.
5. method according to claim 4, it is characterized in that: described method also comprises the step that described walnut micromolecule polypeptide solution is decoloured, goes bitter taste, specific as follows: as to add in the described walnut micromolecule polypeptide solution after active carbon stirs 1-2h, filter and collect filtrate, obtain the walnut micromolecule polypeptide solution of purifying; Wherein, the addition of described active carbon is 5～7% of walnut protein quality.
6. method according to claim 5, it is characterized in that: described method comprises that also the walnut micromolecule polypeptide solution to purifying carries out the step that freeze drying obtains walnut micromolecule polypeptide powder.
7. the walnut micromolecule polypeptide that each described method prepares among the claim 1-6.
8. walnut micromolecule polypeptide according to claim 7, it is characterized in that: the molecular weight of described walnut micromolecule polypeptide is less than 2000Da.
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| CN102630804A (en) * | 2012-04-27 | 2012-08-15 | 湖北智慧果林业科技有限公司 | Production process of walnut peptide powder |
| CN102696858A (en) * | 2012-05-25 | 2012-10-03 | 淄博十里香食品有限公司 | Preparation method for high-purity walnut protein powder |
| CN102696767B (en) * | 2012-06-15 | 2016-02-17 | 江南大学 | A kind of deep working method fully utilizing walnut kernel production low fat Walnut Milk, active peptide, albumen powder |
| CN103109971B (en) * | 2013-03-04 | 2014-09-10 | 四川省均易润泽食品有限公司 | Method for preparing homoarginine walnut peptide |
| 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 |
| CN103484518B (en) * | 2013-09-11 | 2016-03-30 | 邹远东 | A kind of walnut protein peptide and preparation method thereof and its application |
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| CN104031968B (en) * | 2014-06-27 | 2016-10-19 | 广西壮族自治区中国科学院广西植物研究所 | The application of walnut polypeptide |
| CN105002247A (en) * | 2015-07-30 | 2015-10-28 | 北京天肽生物科技有限公司 | Micromolecule walnut peptide and preparation method thereof |
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| CN106333056A (en) * | 2016-08-23 | 2017-01-18 | 慈晓丽 | Method for preparing walnut oil and walnut peptide synchronously |
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| CN111109608A (en) * | 2019-12-28 | 2020-05-08 | 江苏天美健大自然生物工程有限公司 | Camel milk walnut protein powder containing various substances and preparation method thereof |
| CN111802507B (en) * | 2020-07-23 | 2022-08-30 | 青岛农业大学 | Method for removing bitter walnut peptide by using sugar alcohol liposome precursor |
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Effective date of registration: 20200507 Address after: 361116, Fujian, Xiamen, Tong Wo District, No. three road, No. 336, No. five, building on the east side of the building B Patentee after: XIAMEN YUANZHIDAO BIOTECH Co.,Ltd. Address before: 100193 Beijing Old Summer Palace West Road, Haidian District, No. 2 Patentee before: CHINA AGRICULTURAL University |