CN1274361C - Soybean peptide, its preparation and application - Google Patents
Soybean peptide, its preparation and application Download PDFInfo
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Abstract
The present invention relates to a soybean functional short peptide product with low molecular weight, the preparation of the product, and the application of the product to resisting organism fatigue and improving an organism antioxidant ability, which belongs to the technical field of biology. The soybean peptide is prepared by that low-temperature desolventized soybean pulp is used as a raw material and processed through the procedures of soybean pulp pretreatment, enzyme hydrolysis, enzyme inactivation, powder refining, powder drying, etc. Soybean peptide powder is yellowish powder, has molecular weight mainly dispersed over 200 to 900Da, has an average chain with a length of 3 to 5 amino acids, and contains various amino acids of aspartic acid, threonine, serine, glutamate, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, etc. The soybean peptide of the present invention has the advantages of simple preparation technology, high nutritive value, good physical characteristic, various physiological functions, etc., and is used as a base material used in the field of a health food, a common food additive, a medicine, cosmetics, etc.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of low-molecular-weight soybean peptide product, utilize the enzyme process low temperature desolventizing soybean meal of directly degrading to prepare the method for this soybean peptide and this soybean peptide product in resisting fatigue and improve application aspect the oxidation resistance.
Background technology
One of the most basic living matter that protein is made up of aminoacid, its fragment is called peptide.Peptide with particular physiological function is called biologically active peptide, is the important living matter of a class that plays regulating action in the vital movement.In recent years, external research worker finds that isolated multiple little peptide has the effect of the various physiologically actives of regulating human body from the enzymic hydrolysates of some food proteins.In addition, people know for a long time, and food proteins will just can be absorbed by the body through complicated digestion process.Yet, finding that medically little peptide can directly be absorbed by intestinal, and infiltration rate is also faster than the free amino acid of same composition recently, these find exploitation peptide class new function health food significant.
The mixture of the soybean peptide multiple peptide that to be soybean protein obtain through enzymatic degradation.Functional food should be the nutritious and healthy food with adequate nutrition and health care.As everyone knows, soybean protein is the very high food proteins of nutritive value, also is unique vegetable protein that can replace animal proteinum.Compare with soybean protein, soybean peptide often has better physical property and processing characteristics, and for example, the viscosity of soybean protein increases with concentration and raises rapidly, even but low-molecular-weight soybean peptide also still keeps mobile under 50% high concentration.The soybean protein aqueous solution of about 10% concentration will solidify once heating, and soybean polypeptide then can not.Aspect dissolubility, soybean protein can form precipitation in its isoelectric point, IP, but soybean polypeptide still can keep dissolved state.Moreover, soybean polypeptide also has the ability that Profilin matter forms gel.These character are very favorable factors to the development and use of soybean peptide.Except trophic function and, it has been found that in recent years soybean polypeptide also has many physiological functions, as regulating lipid metabolism, cholesterol reducing suppresses hypertension, nonallergic, raise immunity etc.
For a long time, Semen sojae atricolor is a primary raw material of producing edible oil, although sub-fraction is used for producing soybean protein isolate in the side-product bean cake of oils preparation industry, most of bean cake is used for processing feedstuff, and value is too low.Yet, concerning Semen sojae atricolor, contain whole soybean proteins in the bean cake.If can be raw material and peptide class functional food, pharmaceuticals and the other products that comprises cosmetics that directly prepares high added value with the bean cake with different physiological roles with enzyme hydrolysis.This can make the grain and oil resource obtain comprehensive utilization.
Summary of the invention
The purpose of this invention is to provide the method that a kind of raw material is easy to get, production technology simply prepares a kind of low-molecular-weight soybean peptide.
Another object of the present invention provides a kind of low-molecular-weight soybean peptide product.
A further object of the present invention provides this application of low-molecular-weight peptide product in food, medicine and cosmetic industry.
The present invention is to be raw material with Semen sojae atricolor low temperature desolventizing bean cake (rather than with soybean protein isolate), and enzyme process prepares the low-molecular-weight soybean peptide.The production technology of soybean peptide is simplified greatly, be significantly reduced to this.
The preparation method of soybean peptide of the present invention relates to steps such as soybean meal pretreatment of raw material, pickling impurity removal, enzymolysis, and pretreatment of raw material is about to the soybean meal raw material pulverizing and sieves, and purpose is to make the easy stripping of soybean protein; The purpose of pickling impurity removal is to make the sedimentary while of soybean protein, makes the water soluble ingredients of staying in the supernatant such as soluble oligosaccharide separated; In enzymolysis process, protein is degraded to peptide, enter in the aqueous solution, and the insoluble matter of cellulose family is stayed in the precipitation, promptly obtains soybean peptide stock solution after the solid-liquid separation.Adopt the ultrafilter membrane isolation technics to carry out the refining of soybean peptide at last.
It is extensive to the present invention relates to the enzyme preparation Substratspezifitaet that adopted, obtains low-molecular-weight fragments of peptides easily, and soybean peptide does not have bitterness substantially.If it is improper that enzyme preparation is selected, then soybean peptide has bitterness.
The step for preparing above-mentioned soybean peptide product is as follows:
Institute's water is deionized water in the preparation process.
1, pre-treatment of raw material
Commercially available Semen sojae atricolor low temperature desolventizing bean cake is pulverized with pulverizer, crossed the 60-80 mesh sieve;
2, acid precipitation and washing
By solid-to-liquid ratio 1: 8-1: 12 add water in bean cake powder, be warming up to 30-60 ℃, stir down and transfer pH to 3.0-6.0 with concentrated hydrochloric acid, and the precipitation soybean protein continues to stir 1-2h, and 6000-9000r/min is centrifugal, and supernatant inclines; Add water (not needing to heat up also uncomfortable pH value) by above-mentioned solid-to-liquid ratio in precipitation again, stir 15-20min and wash precipitation, 6000-9000r/min is centrifugal, collecting precipitation behind the removal supernatant; Wash primary sedimentation again by same operation, abandon supernatant, collecting precipitation;
3, enzyme hydrolysis
By solid-to-liquid ratio 1: 8-1: 12 mix the precipitation of collecting in the step 2 with water, stir, and with NaOH solution accent pH to 8.0-10.0, are warming up to 35 ℃-60 ℃, add the ratio of 40-100ml enzyme liquid, adding Ka Shi bacillus subtilis serine alkaline protease liquid in every kg bean cake
[1], the beginning hydrolysis, in the process of reaction, dropping 0.05mol/LNaOH maintenance system pH value is constant, when protein degree reaches 20-30%, stopped reaction;
4, enzyme-deactivating
Step 3 is rapidly heated to 90-100 ℃ after finishing, and keeps 10-20min to make enzyme-deactivating, and then, (in the 10-20min) reduces to room temperature fast, transfers pH to neutral, and 12,000-15,000rpm, centrifugal 20-30min, solid-liquid separation, supernatant is a soybean peptide stock solution;
5, refining
Soybean peptide stock solution is passed through by molecular weight 5000-10, the ultrafiltration membrane system of 000Da, the macromolecular gluco that removes in the soybean peptide stock solution is finished its subtractive process, and liquid is got back to step 3 continuation hydrolysis on the film;
6, concentrate
Film liquid excessively by obtaining in the step 5 is evaporated to solid content 35-40%;
7, drying
After purified soybean peptide liquid concentrated, dry powder was made in spray-dried or lyophilization, and productive rate is 20-30% (peptide dry powder/bean cake);
Soybean peptide product of the present invention has following performance:
One. biochemical characteristics
1, purified soybean peptide dry powder is a kind of yellowish powder, crude protein quality content 80%-85%, ash 5-7%, sugar, moisture content and other component contents 8-15%, wherein sugar is 4-6%, the nitrogen solubility index NSI of product is 98.0-99.9%, trichloroacetic acid soluble protein (TCA Pr.) 99.5%-99.9%;
2, purified soybean peptide is a kind of mixture of oligopeptide, contains about 10-15% free amino acid, and the molecular weight distribution of 60%-70% oligopeptide is at 200-900Da; Measuring average chain length with the TNBS method is 3-5 aminoacid;
3, soybean peptide can both be dissolved in water under all pH;
4, soybean peptide is to thermally-stabilised, and 100 ℃ can not boiled precipitation;
5, the soybean peptide product was at room temperature preserved 3 months, and peptide content does not have significant change;
6, soybean peptide ammino acid is formed: east, Tianmen propylhomoserin; Threonine; Serine; Glutamic acid; Glycine; Alanine; Cysteine; Valine; Methionine; Isoleucine; Leucine; Tyrosine; Phenylalanine; Lysine; Histidine; Arginine; Proline.
Two. biological function
1. soybean peptide antifatigue effect
(1). detection method
Detect animal: Male Kunming strain mice, 6-8 age in week, body weight 18-22g.Be divided into experimental group and matched group at random, to irritate the stomach administration.Experimental group gavages soybean peptide solution, dosage every day: experiment I organizes 100mg dry powder/kg body weight; Experiment II group, 200mg dry powder/kg body weight; Experiment III group, 400mg dry powder/kg body weight.By the every 10g body weight of mice administration 0.2ml, matched group is given the distilled water with volume behind the dry powder wiring solution-forming.
The exercise tolerance test, i.e. swimming with a load attached to the body test: 40 mices are divided into four groups, matched group and experiment I, II, III group at random.After the mice administration 25 days, be placed in the swimming groove and swim, 26 ± 0.5 ℃ of water temperatures, depth of water 28cm, mice bears a heavy burden and is 2% of body weight, and record mice free swimming is from beginning the time to death.
The blood lactic acid concn is measured: 40 mices are divided into matched group and experiment I, II, III group at random.After the administration 25 days, allow the mice 30min that in 30 ± 2 ℃ of water, swims, before the swimming when quiet and swimming back 15min, 50min respectively get tail blood once, with parazon colorimetric method for determining blood lactic acid content.
Hepatic glycogen content is measured: 40 mices are divided into matched group and experiment I, II, III group at random.Tested the 25th day, and put to death mice, take out liver immediately, with its glycogen content of anthrone colorimetric method for determining.
(2). testing result
1. mice swimming with a load attached to the body experiment
From table 1 data as seen, the animal swimming with a load attached to the body time of middle dosage and high dose group soybean peptide and matched group relatively have tangible prolongation and there were significant differences (comparing P＜0.05 with matched group).
Table 1: soybean peptide is to the influence of mice swimming time (X ± SD)
| Group | Number of animals (only) | Swimming time (min) |
| Dosage group high dose group in the matched group low dose group | 10 10 10 10 | 79.64±12.24 80.70±21.39 96.93±8.27 * 112.45±9.36 * |
*Compare P＜0.05 with matched group
2. soybean peptide is to the influence of mice blood lactic acid concn
Data from table 2 as can be seen, the mice of the middle and high dosage group of soybean peptide moved back 15 minutes and 50 minutes blood lactic acid values are starkly lower than matched group experiment the 25th day, and the tool significant difference.In this experiment, the soybean peptide experimental mice is starkly lower than matched group in back 15 minutes blood lactic acid values of motion, proof is during this, aerobic metabolism proportion in energy supply is very high, so little lactic acid that produces, the lactic acid that produces in the then zymolysis process is not easy to accumulate in muscle, thus but the generation of delay fatigue.In the experiment, the soybean peptide experimental mice stops back 50 minutes blood lactic acid value in motion and is starkly lower than matched group, shows then that in the exercise recovery phase lactic acid too much in the muscle can be eliminated rapidly, this means and can comparatively fast set up.
Table 2: soybean peptide is to the influence of mice lactic acid content (mg/100ml, X ± S)
| Group | Before the motion | Motion stops back 15 minutes | Motion stops back 50 minutes |
| Dosage group high dose group in the matched group low dose group | 28.97±2.95 27.42±2.49 27.51±2.43 28.01±2.97 | 57.48±2.98 57.24±2.37 52.38±1.56 * 48.78±1.78 ** | 49.04±1.77 48.72±2.18 44.44±2.87 ** 40.29±2.00 ** |
*P＜0.05；
**P＜0.01
3. soybean peptide is to the influence of Mouse Liver glycogen content
Data from table 3 as can be seen, mice was administration (soybean peptide) the 25th day, its hepatic glycogen content, compare with matched group, low dose group does not have significant change, and middle dosage group Mouse Liver glycogen content has increased by 22.30% (P＜0.05), and high dose group Mouse Liver glycogen content has increased by 35.98% (P＜0.01).
If body hepatic glycogen and muscle glycogen at the volley exhausts that then glyconeogenesis is obstructed, blood sugar concentration reduces, and can disturb the central nervous system and fatigue occurs.This experiment soybean peptide experimental group hepatic glycogen illustrates that than the obvious raising of matched group soybean peptide can be by increase hepatic glycogen deposit, blood sugar level when keeping motion, thus for body provides more energy, body is set up rapidly.
Table 3 soybean peptide is to the influence of Mouse Liver glycogen (X ± SD)
| Group | Hepatic glycogen content (mg/100g tissue) |
| Dosage group high dose group in the matched group low dose group | 2.78±0.30 2.81±0.26 3.48±0.21 * 3.79±0.23 ** |
*P＜0.05；
**P＜0.01
Above-mentioned experimental result shows that the prepared soybean peptide of the present invention has the function of tangible alleviating physical fatigue.In addition, soybean peptide is rich in essential amino acid, and balance is good, thereby has very high nutritive value.With the soybean peptide is base material, can be developed to health food and pharmaceuticals (as the enteral nutrition agent), has allaying tiredness rapidly, improves effects such as endurance and extra-nutrition.This series products is fit to the people of long-time study or work, the people who drives for a long time or ride and be engaged in sports or be engaged in blue-collar people edible.This product also is fit to the patient or the weak old people and the infants of digestive function of postoperative.This product has the characteristics of crowd's wide coverage.
2. soybean peptide antioxidation
(1). detection method
1. pyrogallol autoxidation
The rapid autoxidation of pyrogallol (PR) energy under alkali condition discharges O
2 -, generating colored intermediate product, available spectrophotography is measured.The starting stage of PR autoxidation process, being accumulated in behind the 30-40s of intermediate product is linear with the time, and at 320nm strong absorption is arranged, and when antioxidant exists, can remove O
2 -Thereby, stop the accumulation of intermediate product, can obtain class superoxide dismutase (SOD) activity of antioxidant in view of the above.
Reaction volume 3ml, blank pipe adds the 0.1ml double distilled water, 2.8ml 0.1mol/L Tris-HCl buffer (pH8.2), 25 ℃ of insulation 10min, the 3mmol/L pyrogallol that adds the 0.1ml preheating then, mixing picks up counting, behind the reaction 30s, do time sweep in the 320nm place, response time 4min.Blank best autoxidation rate controlled is 0.06A/min; Sample cell adds 0.1ml soybean peptide (10mg/1ml), 2.8ml 0.1mol/LTris-HCl buffer (pH8.2), and same 25 ℃ of insulation 10min add 0.1ml 3mmol/L pyrogallol.Behind the 30s, scan in the 320nm place.Calculate suppression ratio at last, be used for the activity of removing ultra-oxygen anion free radical of assess sample.
2. linoleic acid peroxidation
Chemical property is active owing to contain a plurality of pairs of keys for polyunsaturated fatty acid in the lipid, and peroxidization takes place the destruction that is vulnerable to free radical most.A monounsaturated fatty acid molecule forms conjugated double bond and malonaldehyde by radical reaction.Linoleic acid is the model compound of polyunsaturated fatty acid, also contains a plurality of pairs of keys, and linoleic peroxidating can be represented the snperoxiaized process of body lipid.
Preparation linoleic acid micelle liquid: add 0.01mol/L in light resistant container, the borate buffer of pH7.4 splashes into an amount of linoleic acid, and fully mixing to milky gets the linoleic acid micelle, and concentration is 6.4mmol/L, and 4 ℃ of stored refrigerated were used in 24 hours.
The mensuration of linoleic acid peroxidation degree: linoleic acid peroxidation is made up of three phases, i.e. induction period, extended period and amortization period.Have conjugated diene to generate at induction period, it reflects linoleic peroxidating degree, and conjugated diene has characteristic absorption at 234nm; Have the generation of linoleic acid peroxidation thing (LOOH) mid-term in the linoleic acid peroxidation reaction, and the peroxidating degree can be represented with peroxide value (POV); End-product malonaldehyde (MDA) as the linoleic acid peroxidation reaction generates at amortization period.And malonaldehyde can with thiobarbituricacid (TBA) reaction, form red product, be 1.56 * 10 at the extinction coefficient of 532nm
5Lmol
-1Cm
-1Can calculate the amount of MDA thus, and then to obtain with MDA be the lipid peroxidation value of representative.
When surveying conjugated diene, at first set up the I of system, promptly 0,01mol/L, the borate buffer of pH7.4 contains Vc 0.025m mol/L, Cu
2+10 μ mol/L, linoleic acid 64 μ mol/L.Secondly, on the basis of the I of system, be 0.02,0.04 by adding final concentration respectively, the soybean peptide of 0.08mg/ml, the glutathion of 0.04mg/ml (GSH) is set up the II of system, III, IV and V.Under 25 ℃, be blank with the linoleic acid storing solution of same concentrations, measure the OD of above five systems synchronously
234Over time, detect the ability of the inhibition linoleic acid peroxidation of soybean peptide.
When surveying the linoleic acid peroxidation thing (surveying the POV value), every 20 minutes, respectively from above-mentioned system I, III, the 20ml that takes a sample among the V, the HCl of adding 1.5ml 1N transfers to 2.0 to stop peroxidization with pH.The KI that adds 4ml 40% again after 10 minutes, uses the Na that accurately demarcates in the dark place placement
2S
2O
3Titration.When solution colour when being yellowish by xanthochromia, the starch that adds two 1% is made indicator, and titration is to colourless, record Na
2S
2O
3Amount.Parallel laboratory test three times.Because Cu
2+Also can oxidation I
-Be I
2So, at first to only containing Cu
2+Blank system carry out titration.Growing amount with LOOH in every ml reactant liquor is represented POV.
When surveying the product malonaldehyde, preparation thiobarbituricacid (TBA) solution takes by weighing 0.67g TBA earlier, uses the acetic acid of 100ml 5.6% to dissolve in 56 ℃ of water-baths, is stored in the brown bottle stand-by.Then, set up following four systems:
System I: linoleic final concentration is 64 μ mol/L, carries out autoxidation.
System II: linoleic final concentration is 64 μ mol/L, Cu
2+Final concentration be 10 μ mol/L, the concentration of Vc is 25 μ mol/L, makes it carry out peroxidating.
System III: adding final concentration in the II of system is the soybean peptide of 0.04mg/ml.
System IV: adding final concentration in the II of system is the GSH of 0.04mg/ml.
After reaction starts, the 1ml that from each system, took a sample every 20 minutes, behind the TBA of adding 0.5ml, boiling water bath 15 minutes is diluted to 3.0ml with deionized water then.With the deionized water is blank, measures OD
532Value, parallel laboratory test three times.
3. rat oxidative hemolysis of erythrocyte
The preparation of red cell suspension: Wistar rat broken end is got blood, sodium citrate with 4% is made anticoagulant, adds 2-3 normal saline doubly and suspends centrifugal (1500r/min, 5min, 4 ℃), remove supernatant and yellow thin layer, repeated washing three times, be suspended in 0.01mol/L, in the phosphate buffer of pH7.4, be made into 1% red cell suspension, cold preservation is stand-by.
Erythrocyte is from the haemolysis system:
System I:10ml red cell suspension
System II: system I+0.1mmol/L soybean peptide
System III: system I+0.1mmol/L Vc
System IV: system I+0.1mmol/L GSH
The oxidative hemolysis of erythrocyte system of hydrogen peroxide-induced:
System A:10m1 red cell suspension+100mmol/LH
2O
2
System B: system A+0.1mmol/L soybean peptide;
System C: system A+0.1mmol/LVc;
System D: system A+0.1mmol/LGSH
System A-D bathes 1h in 37 ℃ of temperature.
The mensuration of erythrocyte autoxidation haemolysis degree: when measuring content of hemoglobin, from the supernatant behind the centrifugal 5min of the I-IV 1500r/min of system, take a sample, survey OD
540When measuring the amount of oxidation product, from the supernatant behind the centrifugal 5min of the I-IV1500r/min of system, get the TBA (thiobarbituricacid) that 1.5ml adds 1ml, place boiling water bath 30min, 1500r/min centrifugal 5min in cooling back surveys OD
532OD
532The concentration linear correlation of value and colored product.
The mensuration of the erythrocyte peroxidating haemolysis degree of hydrogen peroxide-induced: comprise the mensuration of hemoglobin, method is with behind the centrifugal 5min of the A-D 1500r/min of system, gets supernatant, surveys OD
540, the content of mensuration hemoglobin; The assay method of oxidation product: from the supernatant behind the centrifugal 5min of the A-D 1500r/min of system, get 1.5ml, add the TBA solution of 1ml, boiling water bath 30min, the centrifugal 5min of cooling back 1500r/min gets supernatant and measures OD
532, the content of mensuration TBA reactant.
(2). assay
1. soybean peptide is to the autoxidizable inhibitory action of pyrogallol
Soybean peptide is calculated as follows the autoxidizable suppression ratio of adjacent triphenol:
I＝(ΔA。-ΔA)/ΔA。×100％
I represents suppression ratio; Δ A., Δ A represents not add and add pyrogallol autoxidation speed behind the antioxidant, the i.e. average rate of change of its light absorption of per minute respectively.After measured, pyrogallol autoxidation speed (Δ A.) be 0.06A/min.
Experimental result shows that along with the increase of sample concentration is linear, its suppression ratio reaches 50% soybean peptide concentration (IC to the variable concentrations soybean peptide to the autoxidizable suppression ratio of pyrogallol
50) be 4.5mg/ml.
2. soybean peptide is to the inhibitory action of linoleic acid peroxidation
The conjugated diene measurement result: with the linoleic acid peroxidation system under the experiment condition is matched group, adds the soybean peptide of variable concentrations, the variation of conjugated diene growing amount in the assaying reaction system, and the result is as shown in Figure 1.As seen from the figure, the suppression ratio when soybean peptide concentration is 20 μ g/ml has just reached 80%, shows higher antioxidant activity.But along with increasing of concentration, the increase of suppression ratio is not fairly obvious.Analyzing its reason, may be because the antioxidant activity of soybean peptide mainly shows as the chain reaction of blocking-up free radical, the peroxy radical in the Mulberry Extract reaction chain, thereby the chain reaction that can slow down free radical.And want weak than enzymes such as SOD for the ability of removing chain initiation stage free radical.Even thereby the concentration of inhibitor improves several times, the raising of suppression ratio is also little.
The linoleic measurement result of peroxidating: the LOOH that the linoleic acid peroxidation reaction generates can be I under mild acid conditions
-Be oxidized to I
2, I
2Again can with Na
2S
2O
3Carry out quantitative response, but the amount of the LOOH that generates of indirect determination reaction thus.Because Cu
2+Also can oxidation I
-So, at first to only containing Cu
2+Blank system carry out titration, to remove Cu
2+Influence to reaction.
As antioxidant, add reaction system with soybean peptide and glutathion (GSH), with the non-oxidizability of check soybean peptide and GSH, the result as shown in Figure 2.As shown in Figure 2, soybean peptide and GSH have inhibitory action to the linoleic generation of peroxidating, but the inhibitory action of soybean peptide is stronger under identical peptide concentration condition, and the suppression ratio of soybean peptide is 68.4%, and the suppression ratio of GSH is 22.4%.
The measurement result of malonaldehyde: as shown in Figure 3, compared the effect of generation that soybean peptide and GSH suppress the end-product malonaldehyde of linoleic acid peroxidation.Wherein, GSH shows stronger inhibitory action, and when concentration was 0.04mg/ml, its suppression ratio was 46.2%, and the suppression ratio of soybean peptide under identical activity is 41.7%.
3. soybean peptide is to the influence of rat oxidative hemolysis of erythrocyte
Haemolysis with the hemoglobin that do not add antioxidant is 100%, calculates the haemolysis degree of the hemoglobin that adds antioxidant.As can be seen from Table 4, when having soybean peptide to exist, the erythrocyte hemolysis degree has descended 38.6%, illustrates that soybean peptide has protective effect to erythrocytic autoxidation damage, is better than glutathion (GSH) and Vc.
Table 4: soybean peptide is to the hemolytic influence of rat erythrocyte autoxidation
| From haemolysis | Soybean peptide | GSH | Vc | |
| OD 540Haemolysis degree suppression ratio | 0.474 100％ - | 0.344 72.6％ 27.4％ | 0.389 82.1％ 17.9％ | 0.384 81.0％ 19％ |
In addition, adding external source active oxygen (H
2O
2, O
2 -And OH) act on after the erythrocyte, the system haemolysis degree that contains soybean peptide obviously descends (seeing Table 5), can infer that thus the superoxide radical that soybean peptide may make erythrocyte produce reduces.
Table 5: soybean peptide is to rat H
2O
2The influence of inductive erythrocyte hemolysis
| H 2O 2Inductive haemolysis | Soybean peptide | GSH | Vc | |
| OD 540Haemolysis degree inhibition degree | 0.786±0.05 100％ ----- | 0.333±0.04 42.4％ 57.3％ | 0.311±0.05 39.6％ 60.4％ | 0.625±0.07 79.5％ 20.5％ |
Free radical is meant from chemical constitution and contains not group, atom or the molecule of sharing electron.The oxygen-derived free radicals that exists in the human body is an active oxygen, comprises superoxide anion (O
2 *-), hydroxy radical (
*OH), hydrogen peroxide (H
2O
2), hydroperoxy (HO
2), alkoxyl (RO
*), alkane peroxy (ROO
*), hydroperoxides (ROOH) and singlet oxygen (
1O
2) etc.Free radical is the metabolite in the body normal structure, generates and be eliminated to be in dynamic equilibrium, and the radical pair body that has does not have overt toxicity, and most of radical pair body damage is serious.Just these free radicals constantly produce under physiological conditions, but also constantly by antioxidant for clearing, thereby do not show the damage of radical pair body.And under some pathologic condition, free-radical generating too much or removed slowly, it makes tissue cause damage by attacking biomacromolecule, causes body aging, even malignant disease such as initiation tumor.In order to defend the damage of free radical, can give body extra free radical scavenger, to reach the purpose of the disease that is caused owing to free agent surplus in the body.
Along with the development of free-radical theory, the mechanism of action of natural small molecule antioxidant is constantly illustrated, and particularly the mechanism of action of vitamin C (Vc) and glutathion (GSH) is understood.Above-mentioned experimental result shows that the prepared soybean peptide of the present invention also is micromolecular antioxidant.Under the molar concentration situation suitable with GSH, soybean peptide suppresses the linoleic acid peroxidation activity and is close with GSH.In first and second stage of reaction, the suppression ratio of soybean peptide is a little less than GSH, but is higher than GSH in the reaction phase III, shows that soybean peptide is more stable than sulfhydryl compound.Erythrocyte peroxidating hemolytic experiment shows that under identical molar concentration, soybean peptide suppresses erythrocyte peroxidating haemolysis damage ability, and is all strong than Vc and GSH.
The prepared Semen sojae atricolor small peptide of the present invention has the antioxidant activity of removing free radical, this physiological function can be applicable to pharmaceuticals industry and cosmetic industry, be suitable for prevention and treatment because the caused disorders such as cancers of radical damage, the destruction of organ-tissue cell and minimizing, immunologic hypofunction etc.Soybean peptide can be used for the fresh-keeping of bread and cheese, as prevents the oils oxidative rancidity.Soybean peptide can be used for producing cosmetics, as the suppurative mastitis agent, has hair care and moisture-keeping function; Facial cream and for example has the antioxidation of similar SOD, can skin care (defying age).Compare with other small molecule antioxidants, soybean peptide has more easy preparation, and is cheap, characteristics such as good stability.
Three. the soybean peptide stability test
To two batches of main functional components of soybean peptide pilot product, promptly the stability of oligopeptide is checked.Sample is preserved at ambient temperature, the content of the peptide in the every month working sample, and be three months observing time, test result sees Table 6.
Table 6: soybean peptide stability test
| Storage life (moon) | Batch I/mg/ml | Batch II/mg/ml |
| 0 1 2 3 | 30.7 29.0 31.5 30.9 | 29.3 29.3 30.9 30.1 |
By as can be seen above, soybean peptide of the present invention to have preparation technology simple, good physical behavior (low-molecular-weight, stability is high) and different physiological roles (antifatigue effect and stronger antioxidation) advantage.
Description of drawings
Fig. 1: soybean peptide concentration is to the empirical curve of linoleic acid peroxidation influence;
Fig. 2: soybean peptide and glutathion generate inhibiting empirical curve to the peroxidating linoleic acid;
Fig. 3: soybean peptide and GSH suppress the empirical curve of the end-product MDA generation of linoleic acid peroxidation.
The specific embodiment
Illustrate the preparation of soybean peptide of the present invention below:
Embodiment 1:
Get 20g soybean meal powder (protein content is 45%), add 200ml water, stir, be warming up to 45 ℃, stir down and transfer pH to 4.2 with concentrated hydrochloric acid.Stop behind the 2h stirring, centrifugal (9000rpm 30min), removes supernatant, adds 200ml water, washes precipitation, stirs 20min, and centrifugal (9000rpm 30min), removes supernatant; This step is repeated.Collecting precipitation adds 200ml water, stirs, and adds 0.5mol/LNaOH and transfers pH9.5, is warming up to 35 ℃, adds 1.1ml enzyme liquid (Ka Shi bacillus subtilis serine alkaline protease
[1], Novo Nordisk), the beginning hydrolysis.In the process of reaction, keep 50 ℃ of temperature, keep pH9.5.When degree of hydrolysis reaches 25%, stopped reaction.Reaction is rapidly heated to 95 ℃ after finishing, and keeps 10-15min to make enzyme-deactivating.Then, reduce to room temperature, transfer pH to 6.8.12,000rpm, centrifugal 30min, supernatant are rough soybean peptide liquid.It is the ultrafilter membrane of 5000Da that soybean peptide liquid is crossed by molecular weight, removes macromolecular gluco.Liquid is got back to hydrolysis once more in the enzymatic vessel on the film.Purified soybean peptide liquid is evaporated to solid content 35-40%, and spray drying obtains purified soybean peptide dry powder.Productive rate: 0.51g/g albumen,
It is thick 82.5% that soybean peptide contains albumen, ash 5.5%, sugar and other 5.0%, moisture 7.0%; Nitrogen solubility index (NSI) 99.9%, trichloroacetic acid soluble protein (TCA Pr.) 99.8%, average chain length 3.3, free amino acid 14.8%.
Embodiment 2:
Get 20g soybean meal powder (protein content is 45%), add 200ml water, stir, be warming up to 45 ℃, stir down and transfer pH to 4.2 with concentrated hydrochloric acid.Stop behind the 2h stirring, centrifugal (9000rpm 30min), removes supernatant, adds 200ml water, washes precipitation, stirs 20min, and centrifugal (9000rpm 30min), removes supernatant; This step is repeated, and collecting precipitation adds 200ml water, transfers pH8.5, is warming up to 55 ℃, adds 1.6ml enzyme liquid (Ka Shi bacillus subtilis serine alkaline protease
[1], Novo Nordisk), the beginning hydrolysis.Make pH maintain 8.5.When degree of hydrolysis reaches 30%, stopped reaction.Be rapidly heated to 95 ℃, keep 15min to make enzyme-deactivating.Then, be cooled to room temperature.Transfer pH to 7.0.12,000rpm, centrifugal 30min, supernatant are rough soybean peptide liquid.Soybean peptide liquid is crossed by molecular weight 10, the 000Da ultrafilter membrane.Cross film liquid and be evaporated to solid content 35-40%, spray drying obtains purified soybean peptide dry powder.Productive rate: 0.63g/g albumen
It is thick 82.2% that soybean peptide contains albumen, ash 6.1%, sugar and other 5.0%, moisture 6.7%; Nitrogen solubility index (NSI) 99.9%, trichloroacetic acid soluble protein (TCA Pr.) 99.6%, average chain length 4.5, free amino acid 14.5%.
Document 1:
Shui-Tein Chen，Shiah-Yun Chen，and Kung-Tsung Wang，Kinetically Controlled Peptide BondFormation in Anhydrous Alcohol Catalyzed by the Industrial Protease Alcalase，The Journal ofOrganic Chemistry，1992，57(25)：6960-6965.
Claims (7)
1, a kind of soybean peptide product, it has following feature:
(1). purified soybean peptide dry powder is a kind of yellowish powder, crude protein quality content 80%-85%, ash 5-7%, sugar, moisture content and other component contents 8-15%, wherein sugar is 4-6%, the nitrogen solubility index NSI of product is 98.0-99.9%, trichloroacetic acid soluble protein measured value 99.5%-99.9%;
(2). purified soybean peptide is a kind of mixture of oligopeptide, contains the 10-15% free amino acid, and the molecular weight distribution of 60%-70% oligopeptide is at 200-900Da; Measuring average chain length with the TNBS method is 3-5 aminoacid;
(3). soybean peptide can both be dissolved in water under all pH;
(4). soybean peptide is very stable to heat, and 100 ℃ can not boiled precipitation;
(5). soybean peptide product chambers relaxing the bowels with purgatives of warm nature is preserved 3 months peptide content no changes;
(6). soybean peptide ammino acid is formed: east, Tianmen propylhomoserin, threonine, serine, glutamic acid, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, proline;
And by the preparation of following method:
(1) pre-treatment of raw material
The Semen sojae atricolor defatted soybean meal is pulverized, and crosses the 60-80 mesh sieve;
(2) acid precipitation and washing
By solid-to-liquid ratio 1: 8-1: 12 add deionized water in bean cake powder, be warming up to 30-60 ℃, stir down and transfer pH to 3.0-6.0 with concentrated hydrochloric acid, and the precipitation soybean protein continues to stir 1-2h, and 6000-9000r/min is centrifugal, and supernatant inclines; Add deionized water by above-mentioned solid-to-liquid ratio in precipitation again, stir 15-20min and wash precipitation, 6000-9000r/min is centrifugal, and collecting precipitation behind the removal supernatant is washed primary sedimentation again by same operation, abandons supernatant, collecting precipitation;
(3) enzyme hydrolysis
By solid-to-liquid ratio 1: 8-1: 12 mix the precipitation of collecting in the step 2 with deionized water, stir, transfer pH to 8.0-10.0 with NaOH solution, be warming up to 35 ℃-60 ℃, add the ratio of 40-100ml enzyme liquid in every kg bean cake, add Ka Shi bacillus subtilis serine alkaline protease liquid, the beginning hydrolysis, in the process of reaction, dropping 0.05mol/L NaOH maintenance system pH value is constant, when protein degree reaches 20-30%, stopped reaction;
(4) enzyme-deactivating
After step 3 end, be rapidly heated, keep 10-20min to make enzyme-deactivating, then, make temperature reduce to room temperature fast, transfer pH to neutral to 90-95 ℃, 12,000-15, the centrifugal 20-30min of 000rpm, solid-liquid separation gets supernatant, is soybean peptide stock solution;
(5) refining
Soybean peptide stock solution is passed through by molecular weight 5000-10, the ultrafiltration membrane system of 000Da, the macromolecular gluco that removes in the soybean peptide stock solution is finished its subtractive process, and liquid is got back to step 3 continuation hydrolysis on the film;
(6) concentrate
Film liquid excessively by obtaining in the step 5 is evaporated to solid content 35-40%;
(7) drying
After purified soybean peptide liquid concentrated, dry powder was made in spray-dried or lyophilization.
2, the described soybean peptide product of claim 1 is used to prepare health food or the pharmaceuticals with antifatigue effect.
3, soybean peptide product as claimed in claim 2 is used to prepare health food or the pharmaceuticals with antifatigue effect, it is characterized in that: be used to prepare the enteral nutrition agent.
4, the described soybean peptide product of claim 1 is used for food fresh keeping or is used to prepare cosmetics.
5, the described soybean peptide product of claim 1 be used to prepare have the hair care and the suppurative mastitis agent of moisture-keeping function or facial cream with antioxidation.
6, the described soybean peptide product of claim 1 is used to prepare prevention and treats because the medicine of the caused disease of radical damage.
7, soybean peptide product as claimed in claim 6 is used to prepare prevention and treat because the medicine of the caused disease of radical damage is characterized in that: the medicine that is used to prepare prevention and the caused cancer of treatment radical damage, organ-tissue cytoclasis and minimizing or immunologic hypofunction.
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