CN105039476A - Method for preparing black bean antioxidant polypepetide powder through compound enzyme method - Google Patents
Method for preparing black bean antioxidant polypepetide powder through compound enzyme method Download PDFInfo
- Publication number
- CN105039476A CN105039476A CN201510399925.2A CN201510399925A CN105039476A CN 105039476 A CN105039476 A CN 105039476A CN 201510399925 A CN201510399925 A CN 201510399925A CN 105039476 A CN105039476 A CN 105039476A
- Authority
- CN
- China
- Prior art keywords
- powder
- black
- soya bean
- black soya
- bean
- 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
Links
Landscapes
- Medicines Containing Plant Substances (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for preparing black bean antioxidant polypepetide powder through a compound enzyme method. The method includes the following steps: step one, separating black bean protein, namely preparing black bean powder, de-fatting the black bean powder, performing enzymolysis on de-fatted black beam powder, and finally centrifuging, freezing and drying to obtain black bean protein powder; and step two, preparing the black beam polypeptide powder. By the method, protein extraction efficiency is improved, enzyme consumption is low, and the cost is lowered. The black bean antioxidant polypeptide powder is safer and more nutrient to eat and worthy of popularization.
Description
Technical field
The invention belongs to biological technical field, relate to antioxidation polypeptide powder preparation field, be specifically related to a kind of method that prozyme prepares black soya bean antioxidation polypeptide powder.
Background technology
China's soybean resource enriches, and wherein black soya bean is called as " kings in beans ", and black soya bean is a kind of green food with high protein, feature low in calories.Black soya bean is exactly a kind of health care food materials of dietotherapeutic from ancient times, and theory of traditional Chinese medical science thinks that the sweet property of black soya bean taste is put down, the effect such as have promoting blood circulation and removing blood stasis, black invention order, promote longevity.Modern study shows, Semen sojae atricolor contains rich in protein, fat and micro-, also containing the functional factor such as saponin(e, flavones, its high-quality bean protein is higher than common soya bean by 24.5%, and indispensable amino acid composition structure level is totally better than soya bean, compared with soya bean, black soya bean Methionin exceeds 15.5%, Threonine exceeds 15.3%, and methionine(Met) exceeds 29%, is suitable for the Application and Development of anti-oxidant protein peptide series products.
Black soya bean polypeptide refers to the polypeptide mixture low to molecular weight that black soya bean protein obtains through the process such as enzymic hydrolysis, separation and purification, its water-soluble very high (soluble nitrogen NSI is about 99%), can be used as new healing potion, function food additive or fodder additives to use.Modern nutriology research find, polypeptide have faster than Amino Acid Absorption, specific absorption good, biological value high, is the active substance with different physiological roles.In recent years, the biotechnology of develop rapidly and separating and purifying technology, made it is found that the polypeptide be present in organism reached tens thousand of kinds, and had different physiological functions.In black soya bean, protein content enriches, and can obtain functional polypeptide by enzymolysis.It is reported; black soya bean polypeptide has adjustment human body physiological function; easily absorb as having and hypoallergenic, reduce blood fat and cholesterol, reduce blood pressure, promote mineral absorption and metabolism of fat, enhancing athlete's physique, helping to set up, promoting the absorption of calcium phosphorus and other trace elements, promoting brain development, raising hypermnesia immunologic function, protecting epidermic cell, prevent the multiple biological functions such as melanocyte precipitation.Therefore, take black soya bean as raw material, prepare the concern that the peptide matters with anti-oxidant activity is just being subject to albumen producer, functional black soya bean polypeptide has vast potential for future development.
Summary of the invention
For the deficiencies in the prior art, the object of the present invention is to provide a kind of prozyme to prepare the method for black soya bean antioxidation polypeptide powder, gained black soya bean antioxidation polypeptide powder reduces cost.
For solving prior art problem, the technical scheme that the present invention takes is:
Prozyme prepares a method for black soya bean antioxidation polypeptide powder, comprises the following steps:
The first step, the separation of black soya bean protein
Step 1, selects black soya bean, and removing impurity is placed in 4-10 DEG C of water and soaks 6-10h removal Testa sojae atricolor, dries 5-6h, makes its water content lower than 10%, then the peeling black soya bean after drying is ground the black bean powder that sieves to obtain at being placed in baking oven 50-60 DEG C;
Step 2, be 1:10-15g/mL mix after in 40-50 DEG C at stir with degreasing solvent according to mass volume ratio by black bean powder, incline supernatant liquor, then add sherwood oil and continue to stir, and leave standstill more than 5h, incline supernatant liquor, by air-dry for throw out degreasing black bean powder;
Step 3, extracting degreasing black bean powder adds distilled water according to 1:8-10g/mL solid-to-liquid ratio and stirs, pH to 4.5-5.5 is adjusted with lactic acid, add the cellulase of-0.5-0.9% black bean powder weight again, the hemicellulase of 0.5-0.7% and the polygalacturonase of 0.1-0.4%, shake ovum at 35-45 DEG C after stirring and educate 30-50min, be warming up to 50-55 DEG C again, add the Alcalase of 1.0% black bean powder weight again, the sodium carbonate solution of 5% adjusts pH to 7.5-8.0, continue concussion 1-2h, be heated to 85-95 DEG C, and be incubated 10-20min, the centrifugal 15-20min of 4000-5000rpm, collect supernatant liquor, precipitation after centrifugal adds deionized water according to the amount of degreasing black bean powder solid-to-liquid ratio 1:3-5g/mL, again according to first time enzyme amount 30% add cellulase successively, polygalacturonase and Alcalase, centrifugal supernatant liquor, be incorporated to first time centrifuged supernatant mixing,
Step 4, the pH to 5.0 of supernatant liquor is regulated with lactic acid, centrifugal collecting precipitation, then regulate supernatant liquor pH to 4.7 to leave standstill 30min, centrifugal collecting precipitation, last adjustment pH to 4.4, centrifugal collecting precipitation after leaving standstill, mixes three precipitations, obtains throw out with centrifugal after deionized water wash, after precooling treatment at throw out 4 DEG C, lyophilize obtains black bean protein powder;
Second step, the preparation of black soya bean polypeptide powder
The black bean protein powder getting the first step gained adds in 4-10 DEG C of water, black soya bean protein liquid mass concentration is made to be 3-6%, after stirring, adjust pH is 7.5-8.5, be heated to 90-95 DEG C, sterilizing 15-20min, be cooled to 50-55 DEG C again, successively through hydrolysis by novo, flavor protease hydrolysis and neutral proteinase hydrolysis, adjust ph to 4.5, by black soya bean protein liquid through microfiltration membrane pre-treatment, ultra-filtration and separation purifying again, regulate pH to 4.5, according to gac: black soya bean protein liquid is that the amount of 0.15-0.3g/L adds gac, 45-55 DEG C concussion 1-2h after leave standstill and centrifugal, collect supernatant liquor, being concentrated into concentration is at 4 DEG C, carry out chance deepfreeze after 25-50%, lyophilize obtains black soya bean antioxidation polypeptide powder product again.
As preferably, the first step, black bean powder at least 80 order in step 1.
As preferably, the first step, in step 2, degreasing solvent is ethanol and petroleum ether mixtures, and wherein volume fraction of ethanol is 15%.
As preferably, the first step, in step 3, the concentration of lactic acid is 2mol/L; The cellulase added, hemicellulase and polygalacturonase content are respectively 0.7%, 0.6% and 0.3% of black bean powder weight; Cellulase activity is greater than 100,000 u/g, and hemicellulase activity is greater than 50,000 u/g, and pectinase activity is greater than 10,000 u/g; It is 40 DEG C that concussion ovum educates temperature.
As preferably, second step, ultrafiltration flow velocity 15-40mL/min, temperature range 4-10 DEG C, feed pressure is 0.21MPa, top hole pressure 0.12MPa.
As preferably, second step, the Sumizyme MP of hydrolysis by novo is 2% times of black bean protein powder quality, and initial adjustment temperature is 40-60 DEG C, pH is 7.5-8.0, and the reaction times is 30-40min, regulates temperature to be 95 DEG C, insulation 10min; The flavor protease of flavor protease hydrolysis is 1.5% times of black bean protein powder quality, and initial adjustment temperature is 45-55 DEG C, pH is 7.0-7.5, reaction times 20min, regulates temperature to be 95 DEG C, insulation 10min; The neutral protease of neutral proteinase hydrolysis is 1.5% times of black bean protein powder quality, and regulate temperature to be 50 DEG C, pH is 6.5-7.5, reaction times 20min, regulates temperature to be 95 DEG C, insulation 10min.
As preferably, second step, lyophilize is-20 DEG C of refrigerator overnight, and freeze temperature is-25 DEG C, vacuum tightness 0.21bar.
beneficial effect
(1) in black soya bean protein extraction process, have employed combined-enzyme method, effective protein extraction efficiency reaches 87.6%.
(2) have employed prozyme multistage hydrolysis black soya bean albumen, the black soya bean macro-molecular protein for different qualities carries out enzymolysis stage by stage, improves the percent hydrolysis of protein and the utilization ratio of enzyme.
(3) according to the enzymatic property of hydrolysising protease, the pH value of Function protein solution, hydrolysis temperature, hydrolysis time, the hydrolysis environment hydrolysis of control by stages enzyme.Adopt this kind of method to improve the hydrolysis vigor of enzyme, decrease enzyme dosage, shorten enzymolysis time, energy efficient.
(4) the black soya bean polypeptide that prepared by the present invention is separated display through SephadexG-25, and molecular weight accounts for about 60% at the black soya bean polypeptide of below 10000Da, and functional polypeptide content is high.
(5) the black soya bean polypeptide that the present invention obtains has strong Scavenging ability significantly, when concentration 3.2mg/mL, and clearance rate about 85%.Meanwhile, within the scope of 8-32mg/mL, black soya bean polypeptide reducing power is comparatively strong, close to the resistance of oxidation of Vc.When concentration 40.00mg/mL, the inhibiting rate of black soya bean polypeptide to hydroxyl radical free radical reaches 85%.
(6) the present invention has that black soya bean polypeptide powder that is simple to operate, preparation is low without bitter taste, production cost, product return rate high, not only can directly as high protein nutritious supplementary, also as vegetable protein beverage, sports food, military food etc., can have a good application prospect.
(7) the present invention eliminates antinutritional factor, the Flatulent factors in black soya bean by enzymolysis, improves edible safety and the trophicity of black soya bean polypeptide.
Accompanying drawing explanation
The elution curve on SephadexG-25 gel of Fig. 1 black soya bean polypeptide;
Fig. 2 black soya bean polypeptide and Vc are to the scavenging(action) of DPPH;
The reducing power of Fig. 3 black soya bean polypeptide and Vc;
Fig. 4 black soya bean polypeptide and Vc hydroxyl radical free radical suppress.
Embodiment
The following examples can make the present invention of those skilled in the art comprehend, but do not limit the present invention in any way.
embodiment 1the separation of black soya bean protein
Step 1, selected high-quality black soya bean, and after removing impurity, be immersed in 6-10h in water, water temperature remains between 4-10 DEG C; The black soya bean expanded is rubbed, and removes Testa sojae atricolor.Peeling black soya bean is placed in 50-60 DEG C of baking oven, dries 5-6h, makes its water content below 10%.Then by the black soya bean abrasive dust after oven dry, mesh-of-grind will reach 80 orders and cross more than 80%, obtains black bean powder.
Step 2, black bean powder after pulverizing and degreasing solvent are according to 1:15(g/mL) ratio stirs 3h after mixing in 40-50 DEG C of digital display constant temperature blender with magnetic force, wherein, degreasing solvent is ethanol and petroleum ether mixtures, it is 15% that ethanol accounts for degreasing solvent volume fraction, the supernatant liquor that inclines is (yellow, containing sherwood oil, water and fat etc.), add young oil ether again to continue to stir 3h, then more than 5h is left standstill, incline supernatant liquor, and obtain air-dry for sediment fraction the powder that color and luster is white, this powder is degreasing black bean powder.
Step 3, takes the degreasing black bean powder of certain mass, is 1:8-10(g/mL according to solid-to-liquid ratio) add distilled water, mix with distilled water with digital display constant temperature blender with magnetic force being stirred to degreasing black bean powder under normal temperature.The lactic acid solution adjust pH of 2mol/L is 4.5-5.5, adds the cellulase (1.5 × 10 of 0.7% of black bean powder dry weight
3u/g), the hemicellulase (5 × 10 of 0.6%
3the polygalacturonase (1.5 × 10 of U/g) and 0.3%
3u/g).After stirring, on water-bath oscillator, 30-50min is hatched in 40 DEG C of concussions.Then temperature is brought up to 52 DEG C, then add black bean powder dry weight 1.0% Alcalase(Alcalase), 5%Na
2cO
2solution adjust pH is that 7.5-8.0 continues concussion hydrolysis 1-2h.Be heated to 90 DEG C, insulation 15min.The centrifugal 15-20min of 4000-5000r/min, collects supernatant.Precipitation after centrifugal is according to initial degreasing black bean powder solid-liquid ratio 1:3-5(g/mL) amount add deionized water and fully mixing, then according to first time enzyme amount 30% add cellulase, hemicellulase, polygalacturonase and Alcalase successively, enzymatic hydrolysis condition is the same.Be hydrolyzed the enzyme centrifugal of going out.The supernatant liquor obtained mixes with first time centrifuged supernatant.
Step 4, the lactic acid solution of supernatant liquor 2mol/L adjusts supernatant liquor pH value to be 5.0, makes that protein is in iso-electric point state and coagulative precipitation is got off, the centrifugal 15min of 4000r/min, collecting precipitation; Supernatant liquor is adjusted to pH4.7 with lactic acid again, and leaves standstill the centrifugal 15min of 30min, 4000r/min, collecting precipitation; Supernatant liquor is adjusted to pH4.4 again, the centrifugal 15min of 4000r/min after standing 30min, collecting precipitation.
Mix three times centrifugal after precipitation, deionization washing and centrifugal twice, abandoning supernatant, precipitation be black soya bean albumen.After carrying out precooling treatment under 4 DEG C of low temperature, be placed in-20 DEG C of refrigerator freezings and spend the night, then freeze-drying on vacuum freeze drier, freeze temperature-25 DEG C, vacuum tightness 0.21bar, after waiting sample complete drying, the protein powder after freeze-drying is taken out, obtains black bean protein powder.87.6% is reached with the black soya bean protein extracting ratio that present method is extracted.
embodiment 2the preparation of black soya bean polypeptide powder
Water to the protein liquid concentration that the black bean protein powder getting the first step gained adds 4-10 DEG C is 3-6%, stir, adjust pH 7.5-8.5, be heated to 90-95 DEG C, 15-20min sterilising treatment makes high molecular weight protein become soft simultaneously and is easy to follow-up enzymolysis, then protein solution is cooled to 50-55 DEG C, then is hydrolyzed according to following steps:
Alcalase(Sumizyme MP) hydrolysis: the protein solution that takes a morsel adds the Sumizyme MP of black bean protein powder quality 2% times, after being uniformly mixed, add remaining protein solution, regulate temperature 40-60 DEG C, pH7.5-8.0, reaction times 30-40min, regulate temperature 95 DEG C, insulation 10min, the enzyme that goes out obtains the first hydrolyzed solution.
Flavourzyme(flavor protease) hydrolysis: the flavor protease getting black bean protein powder quality 1.5% times adds the first hydrolyzed solution, again according to black bean protein powder quality 1.5% amount, regulate temperature 50 C, reaction 20min, pH7.0-7.5, then regulate temperature 95 DEG C, insulation 10min, the enzyme that goes out obtains the second hydrolyzed solution.
Neutrase(neutral protease) hydrolysis: the neutral protease getting black bean protein powder quality 1.5% times adds in the second hydrolyzed solution, regulates temperature 50 C, pH6.5-7.5, reaction 20min.Then regulate temperature 95 DEG C, insulation 10min, makes enzyme devitalization.
After enzyme digestion reaction terminates, adjust ph to 4.5.Protein liquid good for enzyme digestion reaction is carried out pre-treatment through the microfiltration membrane through 0.2 μm, then adopts the small-sized tangential flow ultra-filtration unit of laboratory LabscaleTFF (MILLIPORE) the separation and purification black soya bean polypeptide that Pall company of the U.S. produces.Ultrafiltration operating pressure is provided by Millipore multi-speed exchanged peristaltic pump.Diaphragm material is the polyethersulfone material be attached to above polyolefine.Ultrafiltration flow velocity 15-40mL/min, ultrafiltration temperature range: 4-10 DEG C.Feed pressure 0.21MPa, top hole pressure 0.12MPa, collect phegma.
Black soya bean polypeptide liquid lactic acid is adjusted to pH4.5, be 0.15-0.3(g/L according to gac/polypeptide liquid) amount in black soya bean liquid, add the gac after cleaning, on shaking bath, 50 DEG C slowly shake 1.5h, and the centrifugal 15min of 4000r/min after standing 30min, collects supernatant.
Polypeptide solution after ultra filtration, is concentrated further by cryogenic vacuum concentrating under reduced pressure method.Hydrolyzed peptide solution Rotary Evaporators after ultrafiltration, 50 DEG C of concentrating under reduced pressure, when being 25%-50% to strength of solution, stop evaporation.
After black soya bean polypeptide solution after concentrated carries out precooling treatment under 4 DEG C of low temperature, be placed in-20 DEG C of refrigerator freezings to spend the night, then freeze-drying on vacuum freeze drier, freeze temperature-25 DEG C, vacuum tightness 0.21bar, after sample complete drying, the polypeptide powder after freeze-drying is taken out, obtains black soya bean polypeptide powder product.
performance test
1. pair gained black soya bean polypeptide powder of the present invention carries out composition detection, and the data obtained is as shown in table 1 below:
The major ingredient table of table 1 black soya bean polypeptide powder
2. the result that is separated on SephadexG-25 of black soya bean polypeptide powder
Abundant swelling good sephadex SephadexG-25 (1.2cm × 60cm) is filled post, rinses stripping equilibria with the Tris-HCl buffered soln of 20mM, pH7.0.Loading standard solution, standardized solution is: each 10mg of potassiumchromate, reduced glutathion, Sleep-promoting factor B, VB12, is dissolved in respectively in 1mL elutriant, makes standardized solution.After loading, carry out wash-out with identical buffered soln, elution speed 0.8mL/min, often 3mL collected by pipe.The efflux volume (mL) of record elutriant, take efflux volume as ordinate zou, the logarithm (lgM) of standard molecular weight is X-coordinate drawing standard curve.Standardized solution is eluted to baseline completely at ordinary times, then loading black soya bean polypeptide solution (black soya bean polypeptide dissolves with distilled water), and elution process is identical with standard.Elution curve as shown in Figure 1.
Can find out from elution curve, black soya bean peptide molecule is continuous distribution, namely there is the peptide molecule that all size does not wait in hydrolyzate.Wash-out obtains three elution peaks, according to the corresponding relation of molecular weight standard sample elution volume and standard model molecular weight, in figure, the molecular weight of first elution peak elution volume 21mL point correspondence is 9850Da, second corresponding molecular weight of elution peak elution volume 42mL point is 4326Da, the 3rd the corresponding molecular weight 875Da of elution peak wash-out 57mL point.Can find out, the little peptide of MW<1000 occupies very large proportion.From the area of elution peak, molecular weight accounts for about 60% at the black soya bean polypeptide of below 10000Da, molecular weight 1000-4000Da black soya bean polypeptide account for about 25%, more than 10000Da account for about 15%.
3. black soya bean polypeptide is to the restraining effect of two stupid generations bitter taste acyl group free radical (DPPH)
By 2mL ethanol and 2mL20mmol/LDPPH
.solution adds in same test tube, shakes up, and places 30min, measures its absorbancy with solvent as reference
a empty; During working sample rejection ability, institute's reagent of getting is the same, just add respectively 2mL sample liquid (black soya bean peptide concentration designs: 0.05,0.10,0.20,0.40,0.80,1.60 and 3.20mg/mL) replace 2mL solvent, after standing 30min.Survey as reference with same concentration sample liquid
avalue, is and treats test tube absorbance value
asample.Free radical scavenging activity (%)=(
a empty-
a sample)/
a empty× 100.As shown in Figure 2, in the dosage range of test, Vc and black soya bean polypeptide strengthen with concentration rising the scavenging(action) of DPPH result gradually, Vc has very strong scavenging(action) under lower concentration, when 0.4mg/mL, clearance rate about 94%, removes DPPH completely substantially.Black soya bean polypeptide when concentration 3.2mg/mL, clearance rate about 85%.This shows that black soya bean polypeptide has very strong scavenging(action) to DPPH and likely reaction generates more stable material, can obviously suppress DPPH oxidized, and there is significant anti-oxidant function.
4. the mensuration of black soya bean polypeptide reducing power
(blank with the replacement of lmL distilled water at lmL different concns sample, other reagent successively with under) in, add the potassium ferricyanide solution of 2.5mL1% and the phosphate buffered saline buffer of 2.5mLpH6.60.2mol/L, at 50 DEG C of insulation 20min after mixing, then the trichoroacetic acid(TCA) of 2.5mL10% is added, with the centrifugal 10min of 3000r/min after mixed, get supernatant liquor 2.5mL, add the FeCl of 2.5mL distilled water and 0.5mL0.1%
3, after mixing under 700nm wavelength colorimetric, record absorbancy
avalue.Vc contrasts as there being reducing power, and black soya bean peptide concentration designs: 0.05,0.10,0.50,1.00,5.00,20.00 and 50.00mg/mL (sample concentration refers to add the concentration of sample before reaction system, as follows).Absorbancy
abe worth relevant with the reducing power of sample,
athe reducing power being worth larger just sample is stronger.
As can be seen from Figure 3 contrast Vc and there is stronger antioxygenation.Black soya bean polypeptide is in the concentration range that experiment is chosen, and at low concentrations, reducing power is more weak, and increase with concentration, reducing power increases gradually, illustrates that the reducing power of black soya bean polypeptide has good dose-effect relationship.At black soya bean polypeptide within the scope of 8-32mg/mL, black soya bean polypeptide reducing power is stronger.
5. black soya bean polypeptide is to the restraining effect of hydroxyl radical free radical
The KH of 0.4mL50mmol/L is added successively in the test tube of clean dried
2p0
4-KOH damping fluid, certain density sample, the EDTA solution of 0.1mL1.04mmol/L, 0.1mL20mmol/LFeCl3 solution, the H202 of 0.1mL10mmol/L, the DRI (DR) (contrast does not add) of 0.1mL10mmol/L, 0.1mL20mmol/LVc, ensures that often pipe final volume is lmL, lh is incubated at 37 DEG C, take out the HCllmL adding 25%, add 1%, thiobarbituricacidα-(TBA) 1mL, on boiling water bath, boil 15min, cooling, if have muddiness to add 3mL n-butanol extraction, colorimetric under 532nm, survey absorbancy.Press formulae discovery inhibiting rate below: clearance rate (%)=(
a 0-
a 1-
a 2)/
a 0× 100, wherein
a 0: blank absorbency;
a 1: add absorbancy after inhibitor and DR;
a 2: the own absorbancy of sample (not adding DR).
Black soya bean polypeptide to the suppression situation of hydroxyl radical free radical as shown in Figure 4, in 0-50.00mg/mL concentration range, black soya bean polypeptide all shows restraining effect to a certain degree to the hydroxy radical qiao that deoxyribose-iron system produces, and along with the increase of concentration, inhibiting rate increases.Wherein under high density 15.00mg/mL-50.00mg/mL, black soya bean polypeptide restraining effect is a little more than the restraining effect of Vc.Wherein when 40.00mg/mL, the inhibiting rate of black soya bean polypeptide reaches 85%.Therefore black soya bean polypeptide has the restraining effect of stronger hydroxyl radical free radical.
Claims (7)
1. prozyme prepares a method for black soya bean antioxidation polypeptide powder, it is characterized in that, comprises the following steps:
The first step, the separation of black soya bean protein
Step 1, selects black soya bean, and removing impurity is placed in 4-10 DEG C of water and soaks 6-10h removal Testa sojae atricolor, dries 5-6h, makes its water content lower than 10%, then the peeling black soya bean after drying is ground the black bean powder that sieves to obtain at being placed in baking oven 50-60 DEG C;
Step 2, be 1:10-15g/mL mix after in 40-50 DEG C at stir with degreasing solvent according to mass volume ratio by black bean powder, incline supernatant liquor, then add sherwood oil and continue to stir, and leave standstill more than 5h, incline supernatant liquor, by air-dry for throw out degreasing black bean powder;
Step 3, extracting degreasing black bean powder adds distilled water according to 1:8-10g/mL solid-to-liquid ratio and stirs, pH to 4.5-5.5 is adjusted with lactic acid, add the cellulase of 0.5-0.9% black bean powder weight again, the hemicellulase of 0.5-0.7% and the polygalacturonase of 0.1-0.4%, shake ovum at 35-45 DEG C after stirring and educate 30-50min, be warming up to 50-55 DEG C again, add the Alcalase of 1.0% black bean powder weight again, the sodium carbonate solution of 5% adjusts pH to 7.5-8.0, continue concussion 1-2h, be heated to 85-95 DEG C, and be incubated 10-20min, the centrifugal 15-20min of 4000-5000rpm, collect supernatant liquor, precipitation after centrifugal adds deionized water according to the amount of degreasing black bean powder solid-to-liquid ratio 1:3-5g/mL, again according to first time enzyme amount 30% add cellulase successively, polygalacturonase and Alcalase, centrifugal supernatant liquor, be incorporated to first time centrifuged supernatant mixing,
Step 4, the pH to 5.0 of supernatant liquor is regulated with lactic acid, centrifugal collecting precipitation, then regulate supernatant liquor pH to 4.7 to leave standstill 30min, centrifugal collecting precipitation, last adjustment pH to 4.4, centrifugal collecting precipitation after leaving standstill, mixes three precipitations, obtains throw out with centrifugal after deionized water wash, after precooling treatment at throw out 4 DEG C, lyophilize obtains black bean protein powder;
Second step, the preparation of black soya bean polypeptide powder
The black bean protein powder getting the first step gained adds in 4-10 DEG C of water, black soya bean protein liquid mass concentration is made to be 3-6%, after stirring, adjust pH is 7.5-8.5, be heated to 90-95 DEG C, sterilizing 15-20min, be cooled to 50-55 DEG C again, successively through hydrolysis by novo, flavor protease hydrolysis and neutral proteinase hydrolysis, adjust ph to 4.5, by black soya bean protein liquid through microfiltration membrane pre-treatment, ultra-filtration and separation purifying again, regulate pH to 4.6, according to gac: black soya bean protein liquid is that the amount of 0.15-0.3g/L adds gac, 45-55 DEG C concussion 1-2h after leave standstill and centrifugal, collect supernatant liquor, being concentrated into concentration is at 4 DEG C, carry out chance deepfreeze after 25-50%, lyophilize obtains black soya bean antioxidation polypeptide powder product again.
2. prozyme according to claim 1 prepares the method for black soya bean antioxidation polypeptide powder, it is characterized in that: the first step, black bean powder at least 80 order in step 1.
3. prozyme according to claim 1 prepares the method for black soya bean antioxidation polypeptide powder, it is characterized in that: the first step, and in step 2, degreasing solvent is ethanol and petroleum ether mixtures, and wherein volume fraction of ethanol is 15%.
4. prozyme according to claim 1 prepares the method for black soya bean antioxidation polypeptide powder, it is characterized in that: the first step, and in step 3, the concentration of lactic acid is 2mol/L; The cellulase added, hemicellulase and polygalacturonase content are respectively 0.7%, 0.6% and 0.3% of black bean powder weight; Cellulase activity is greater than 100,000 u/g, and hemicellulase activity is greater than 50,000 u/g, and pectinase activity is greater than 10,000 u/g; It is 40 DEG C that concussion ovum educates temperature.
5. prozyme according to claim 1 prepares the method for black soya bean antioxidation polypeptide powder, it is characterized in that: second step, ultrafiltration flow velocity 15-40mL/min, temperature range 4-10 DEG C, and feed pressure is 0.21MPa, top hole pressure 0.12MPa.
6. prozyme according to claim 1 prepares the method for black soya bean antioxidation polypeptide powder, it is characterized in that: second step, the Sumizyme MP of hydrolysis by novo is 2% times of black bean protein powder quality, initial adjustment temperature is 40-60 DEG C, pH is 7.5-8.0, reaction times is 30-40min, regulates temperature to be 95 DEG C, insulation 10min; The flavor protease of flavor protease hydrolysis is 1.5% times of black bean protein powder quality, and initial adjustment temperature is 45-55 DEG C, pH is 7.0-7.5, reaction times 20min, regulates temperature to be 95 DEG C, insulation 10min; The neutral protease of neutral proteinase hydrolysis is 1.5% times of black bean protein powder quality, and regulate temperature to be 50 DEG C, pH is 6.5-7.5, reaction times 20min, regulates temperature to be 95 DEG C, insulation 10min.
7. prozyme according to claim 1 prepares the method for black soya bean antioxidation polypeptide powder, it is characterized in that: second step, and lyophilize is-20 DEG C of refrigerator overnight, and freeze temperature is-25 DEG C, vacuum tightness 0.21bar.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510399925.2A CN105039476A (en) | 2015-07-09 | 2015-07-09 | Method for preparing black bean antioxidant polypepetide powder through compound enzyme method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510399925.2A CN105039476A (en) | 2015-07-09 | 2015-07-09 | Method for preparing black bean antioxidant polypepetide powder through compound enzyme method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105039476A true CN105039476A (en) | 2015-11-11 |
Family
ID=54446443
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510399925.2A Pending CN105039476A (en) | 2015-07-09 | 2015-07-09 | Method for preparing black bean antioxidant polypepetide powder through compound enzyme method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105039476A (en) |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107095315A (en) * | 2017-07-06 | 2017-08-29 | 湖南楚宏生物科技有限公司 | A kind of additive for infant food |
| CN107279995A (en) * | 2017-07-06 | 2017-10-24 | 长沙湘资生物科技有限公司 | A kind of radix polygonati officinalis nutrition reconstitutes powder product |
| CN107287267A (en) * | 2016-04-11 | 2017-10-24 | 姜培 | A kind of enzymatic isolation method prepares black soya bean ace inhibitory peptide |
| CN109370902A (en) * | 2018-12-12 | 2019-02-22 | 张家口康生物科技有限公司 | A kind of continuous extraction process of oat polypeptide |
| CN110721137A (en) * | 2018-07-12 | 2020-01-24 | 盛金洲 | Antioxidant liquid and preparation method thereof |
| CN111011579A (en) * | 2019-12-27 | 2020-04-17 | 武汉中地西能科技有限公司 | Black bean selenium polypeptide and preparation method and application thereof |
| WO2020173112A1 (en) * | 2019-02-28 | 2020-09-03 | 江南大学 | Active peptide having anti-oil oxidation function, preparation method therefor and use thereof |
| CN112890206A (en) * | 2021-01-28 | 2021-06-04 | 麦和(广州)实业有限公司 | Black bean oligopeptide embedded substance and preparation method thereof |
| CN112998279A (en) * | 2021-03-15 | 2021-06-22 | 东北农业大学 | Method for preparing functional polypeptide powder by enzyme method |
| CN113197316A (en) * | 2021-05-21 | 2021-08-03 | 江南大学 | Double-functional bean-source polypeptide and preparation method thereof |
| CN114752644A (en) * | 2022-05-31 | 2022-07-15 | 北京工商大学 | Preparation method of small red bean polypeptide with antioxidation effect |
| CN117363682A (en) * | 2023-11-16 | 2024-01-09 | 山西大学 | Preparation method and application of black bean polypeptide with anti-aging activity |
| CN117384250A (en) * | 2023-07-10 | 2024-01-12 | 江苏恰瑞生物科技有限公司 | Short peptide, improved blood perfusion filler, preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1359693A (en) * | 2000-12-16 | 2002-07-24 | 武汉天天好生物制品有限公司肽类物质研究所 | Process for preparing dried soybean polypeptide powder with hypolipemic action |
| CN104126669A (en) * | 2013-05-02 | 2014-11-05 | 孙学兵 | Method for increasing product yield of bean product |
-
2015
- 2015-07-09 CN CN201510399925.2A patent/CN105039476A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1359693A (en) * | 2000-12-16 | 2002-07-24 | 武汉天天好生物制品有限公司肽类物质研究所 | Process for preparing dried soybean polypeptide powder with hypolipemic action |
| CN104126669A (en) * | 2013-05-02 | 2014-11-05 | 孙学兵 | Method for increasing product yield of bean product |
Non-Patent Citations (2)
| Title |
|---|
| 李华等: "酶法制备黑豆抗氧化肽及其分离纯化与氨基酸组成分析", 《食品科学》 * |
| 李杨等: "复合酶水酶法提取大豆蛋白的工艺优化", 《食品科学》 * |
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107287267A (en) * | 2016-04-11 | 2017-10-24 | 姜培 | A kind of enzymatic isolation method prepares black soya bean ace inhibitory peptide |
| CN107279995A (en) * | 2017-07-06 | 2017-10-24 | 长沙湘资生物科技有限公司 | A kind of radix polygonati officinalis nutrition reconstitutes powder product |
| CN107095315A (en) * | 2017-07-06 | 2017-08-29 | 湖南楚宏生物科技有限公司 | A kind of additive for infant food |
| CN110721137A (en) * | 2018-07-12 | 2020-01-24 | 盛金洲 | Antioxidant liquid and preparation method thereof |
| CN109370902A (en) * | 2018-12-12 | 2019-02-22 | 张家口康生物科技有限公司 | A kind of continuous extraction process of oat polypeptide |
| US11319569B2 (en) | 2019-02-28 | 2022-05-03 | Jiangnan University | Preparation method and application thereof of peptides with anti-lipid-oxidation capability |
| WO2020173112A1 (en) * | 2019-02-28 | 2020-09-03 | 江南大学 | Active peptide having anti-oil oxidation function, preparation method therefor and use thereof |
| CN111011579A (en) * | 2019-12-27 | 2020-04-17 | 武汉中地西能科技有限公司 | Black bean selenium polypeptide and preparation method and application thereof |
| CN112890206A (en) * | 2021-01-28 | 2021-06-04 | 麦和(广州)实业有限公司 | Black bean oligopeptide embedded substance and preparation method thereof |
| CN112998279A (en) * | 2021-03-15 | 2021-06-22 | 东北农业大学 | Method for preparing functional polypeptide powder by enzyme method |
| CN113197316A (en) * | 2021-05-21 | 2021-08-03 | 江南大学 | Double-functional bean-source polypeptide and preparation method thereof |
| WO2022242386A1 (en) * | 2021-05-21 | 2022-11-24 | 江南大学 | Bifunctional bean-derived polypeptide and preparation method therefor |
| CN114752644A (en) * | 2022-05-31 | 2022-07-15 | 北京工商大学 | Preparation method of small red bean polypeptide with antioxidation effect |
| CN114752644B (en) * | 2022-05-31 | 2024-02-13 | 北京工商大学 | Preparation method of red bean polypeptide with antioxidant effect |
| CN117384250A (en) * | 2023-07-10 | 2024-01-12 | 江苏恰瑞生物科技有限公司 | Short peptide, improved blood perfusion filler, preparation method and application thereof |
| CN117384250B (en) * | 2023-07-10 | 2024-04-19 | 江苏恰瑞生物科技有限公司 | Short peptide, improved blood perfusion filler, preparation method and application thereof |
| CN117363682A (en) * | 2023-11-16 | 2024-01-09 | 山西大学 | Preparation method and application of black bean polypeptide with anti-aging activity |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105039476A (en) | Method for preparing black bean antioxidant polypepetide powder through compound enzyme method | |
| CN102293292B (en) | Health tea with functions of yin nourishing and blood nourishing, and processing method thereof | |
| CN104342282B (en) | Method for preparing functional grease and active protein powder from abelmoschus manilhot | |
| CN101991165B (en) | Sweet potato leaf-perilla leaf composite beverage and preparation method thereof | |
| CN106699917A (en) | Ultrasonic-assisted extraction method for polysaccharide extract and pectin extract of okra | |
| CN110746515B (en) | Lycium barbarum polysaccharide, lycium barbarum red element and lycium barbarum polypeptide prepared by synchronous separation and preparation method thereof | |
| CN105475787A (en) | Dendrobium and Radix Ophiopogonis milk beverage and preparation method thereof | |
| CN103125735A (en) | Preparation method of walnut polypeptide | |
| CN101288437A (en) | Method for producing soy protein peptides and dietary fiber from defated soybean pulp | |
| CN110354141A (en) | A kind of preparation method of deer blood freeze-dried powder | |
| CN103342734A (en) | Egg white source antioxidant peptide powder and preparation method thereof | |
| CN103250865A (en) | Preparation method of peanut polypeptide | |
| CN101133837B (en) | Chickpea nutrition powder and method for preparing the same | |
| CN107385003A (en) | A kind of preparation technology of soy peptide powder | |
| CN107523602A (en) | A kind of extracting method of black-bone chicken active peptide | |
| CN106306936A (en) | Preparation method of moringa oleifera enzymolyzed drink | |
| CN106387304A (en) | Method for preparing abalone viscera phospholipid through HPEF (High Pulsed Electric Field)-coupled biological enzymolysis | |
| CN103815015B (en) | A kind of production method of medlar bud coarse cereals functional yoghourt | |
| CN107484984A (en) | A kind of stichopus japonicus oral liquid and its manufacture method rich in sea cucumber active polysaccharide | |
| CN103622879A (en) | Day cream containing peanut polypeptides and preparation method of day cream containing peanut polypeptides | |
| CN106755181A (en) | A kind of method for extracting longan seed polysaccharide | |
| CN110771902A (en) | Composite polypeptide powder and application thereof | |
| CN105029449A (en) | Healthcare food | |
| CN101810648A (en) | Pine needle product and ultrasonic preparation method thereof | |
| CN106867768A (en) | A kind of Pitaya Flower peach blossom health glutinous rice wine and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20151111 |