CN101608206B - Method for preparing soy protein immune-active peptides and product thereof - Google Patents
Method for preparing soy protein immune-active peptides and product thereof Download PDFInfo
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Abstract
The invention discloses a method for preparing soy protein immune-active peptides and a product thereof. The method for preparing the soy protein immune-active peptide comprises the following steps of: carrying out hydrolysis reaction on soy protein isolate with proteolytic enzyme; ultrafiltering the hydrolysate of the soy protein isolate with an ultrafiltration membrane; and collecting ultrafiltrate so as to obtain the soy protein immune-active peptides. The prepared soy protein immune-active peptides occupy 70.95 percent of molecular weight of 3400Da, the protein content is 89.2 percent, and the nitrogen content is 14.272 percent. The prepared soy protein immune-active peptides with different dosages are given to mice through mouths for 30 days to carry out three immunity function experiments, experiment results show that the prepared soy protein immune-active peptides can increase half hemolysis values of the mice and enhance carbon expurgation function; in addition, the mice have no delayed type transformation reaction so as to show that the soy protein immune-active peptides have the functions of enhancing humor immunity function and devouring mononuclear-macrophage phagotrophy.
Description
Technical field
The present invention relates to a kind of preparation method of soybean active peptide, relate in particular to a kind of preparation method of the active peptide of soybean protein with immunologic function and the product for preparing by this method, belong to the soyabean processing field.
Background technology
China is large agricultural country and is the native place of soybean that the output of soybean occupies the 4th in the world, and the processing and utilization of soybean lags far behind external advanced country.To realize that as the soybean of farm crop high added value just must improve processing technology, converted products class.In recent years, the soybean intensive processing product of China has had certain development, has all demonstrated fully the progress of processing technology from the soya-bean milk powder to soybean protein isolate to the exploitation of soybean physiological function composition.But owing to management and many-sided reason, the soybean protein isolate enterprise of China is all depressed, the Sunlover 10 enterprise of domestic nearly various schools of thinkers, wherein most of the stopping production or half stopping production.With regard to product, soybean protein isolate itself is exactly a soybean intensive processing product that added value is high, owing to its various functional performance is widely used in every field; And why depressed the protein isolates production of China is; Be that kind is single because product performance are single, quality is unstable; Cause its application to be very limited, this is a unsalable major cause.Thereby each functional performance of exploitation soybean, improve the quality of products, increase range of product, and synergistic application research will be the another ground zero of China's Sunlover 10 exploitation.
As everyone knows; The bio-modification technology is a first-elected safe and reliable new and high technology of 21st century; Enzyme-modified wherein also is the first-selection of foodstuffs industry, utilizes the bio-modification technology that Sunlover 10 is carried out modification promptly through the protease hydrolysis Sunlover 10, changes its functional performance; Be a kind of manufacturing LV, polymolecularity; A kind of effective ways of performance product special characteristics, and action condition is gentle, specificity is strong, and this processing comprises the change of Sunlover 10 functional property and increases solubleness, stops thermo-coagulating, increases lather volume.Hydrolysis control appropriateness not only can reach ideal effect but also do not destroy the requirement of special purpose to function.Because therefore the security and the suitability of bio-modification have good application prospects, at present domestic this research still is in the starting stage, the proteic product of various functional soys also is imported product on the market.
Research and development at the U.S., Japanese bio-modification Sunlover 10 have got into the industrialization stage; Main gordian technique is the selection of proteolytic enzyme and the control of product bitter taste; Carried out from simple composite hydrolysis thing to single component functional hydrolysate Study on Separation, and exploitation multi-series product.
The processing industry developmental level of external soybean is higher than China, is representative western countries with the U.S., Japan.No matter all very advanced in fundamental research or production application, grouping of the world economy soybean industry forms already, and is solid financial strength, and product diversification is widely used, and output is big.Soybean food production has realized mass-producing and modernization, the healthy soybean composition has been carried out deep exploitation, comprehensive exploitation.Soy material sales volumes such as U.S. soybean protein, soybean fibers and soybean isoflavones increased to 6.529 hundred million dollars in 2005.At present; Technological trend is to adopt biotechnologies such as modern food stripping technique, microcapsule agglomeration technique, enzymatic reaction; Wherein enzymatic reaction technology can keep the soybean nutritional composition in the course of processing, do not produce other chemical residues, can improve the function value that flavour of food products improves food; Widen the scope that the soybean intensive processing utilizes greatly, improved the comprehensive exploitation ability.
Soybean peptides is a kind of than Sunlover 10 more high-quality, novel Sunlover 10 enzyme-modified product.Soybean peptides has and is prone to digest and assimilate; Can supply with human body energy rapidly, no protein denaturation, free from beany flavor; Liquid viscosity is little characteristic such as does not solidify with being heated, especially have the serum cholesterol of reduction, hypotensive, increase immunity and promote special physiological properties such as metabolism of fat.Soybean peptides all has DEVELOPMENT PROSPECT and huge market potential in fields such as food, medicine, daily-use chemical industry.
Up to the present, the research of immune-active peptides still is in experimental level, does not also have comparatively effectively preparation method of a cover.Bioactive peptide is broadly divided into four types by the acquisition methods difference: (one) separation and purification natural radioactivity peptide; (2) chemical synthesis; (3) recombinant DNA method; (4) protein digestion method.Diverse ways is fit to different purpose, depends on length, quantity, purposes and the preparation cost of purpose peptide.Every kind of method also has its relative merits.Natural radioactivity peptide source is few, and extraction cost is high, can not use by large-scale development, and the remaining organic solvent of biologically active peptides that extracts in addition can bring toxicity problem.Chemical synthesis is widely used in the pharmaceutical peptide of producing synthetic high nutritive value, moderate-length, but its cost is high, and residual compounds such as sub product is harmful, and this method is only used in experimental size.Recombinant DNA technology is applicable to macromole peptide and proteinic production, also is widely used in laboratory and the industrial production now, develops prematurity still but compare with other method.The Production by Enzymes functional peptides has many good qualities, and as working condition is gentle, Product Safety is high and can locate the specific peptide of production, cost is low, has become the topmost working method of biologically active peptides.
Summary of the invention
The purpose of this invention is to provide a kind of preparation method of soy protein immune-active peptides and by the resulting soy protein immune-active peptides product of this method.
The objective of the invention is to realize through following technical scheme:
A kind of preparation method of soy protein immune-active peptides may further comprise the steps: with soybean protein isolate with the proteolytic enzyme reaction that is hydrolyzed; Resulting soybean separation proteolysis liquid is carried out ultrafiltration with ultra-filtration membrane; Collect ultrafiltrated, promptly get.
In order to reach better preparation effect, described proteolytic enzyme can be proteolytic enzyme such as papoid, compound protease, Sumizyme MP or neutral protease; Be preferably compound protease or Sumizyme MP;
Wherein, when adopting compound protease to be hydrolyzed, best hydrolysising condition is: temperature of reaction is 45 ℃, concentration of substrate 5% (w/w), and enzyme dosage 10000IU/g, reaction times 6hr, pH value in reaction is 7; When adopting Sumizyme MP to be hydrolyzed, best hydrolysising condition is: 50 ℃ of temperature of reaction, concentration of substrate are 5% (w/w), and enzyme dosage is 12000IU/g, react 8 hours, and pH value in reaction is 9; The employing papoid is hydrolyzed, and the optimum hydrolysis combination condition is: concentration of substrate 5%, 50 ℃ of temperature of reaction, reaction times 6hr, enzyme dosage 7000IU/g, reaction pH6; The employing neutral protease is hydrolyzed, and optimum hydrolysising condition is: 45 ℃ of temperature of reaction, concentration of substrate are 5%, and enzyme dosage is 7000IU/g, react 6 hours, under pH6.5, carry out.
Described ultra-filtration membrane preferably molecular weight is the ultra-filtration membrane of 3000Da;
Ultrafiltration is preferably carried out in described ultrafiltration under the pressure of 15psi to 40psi, more preferably under the pressure of 30psi, carry out ultrafiltration; In order to reach better ultrafiltration effect; The inventor optimizes the other factors that influences ultrafiltration and screens; The result finds; The weight percent concentration of soybean hydrolyzed solution has remarkably influenced for the ultrafiltration effect, and the effect of ultrafiltration is better when the weight percent concentration of soybean hydrolyzed solution is 3%-6%, and the effect of ultrafiltration is for best when its weight percent concentration is 5%.In addition, the pH value of soybean protein hydrolysis liquid also has certain influence for the ultrafiltration effect, and the ultrafiltration effect is better when the pH of soybean protein hydrolysis liquid value is 5-8, and when the pH value was 7, the effect of ultrafiltration was best.
The inventive method is the method that enzyme process and embrane method combine, and promptly behind the enzymatic hydrolysis of soybean protein isolates, carries out separation and concentration to needed functional peptide fragment targetedly through membrane ultrafiltration again, and the bioactive peptide product function that obtains is stronger, and purity is higher.The prepared soy protein immune-active peptides that obtains of the inventive method is 70.95% in the shared ratio of the molecular weight of 3400Da; Protein contnt: 89.2%; Nitrogen content is: 14.272%.
Oral administration was given the prepared soy protein immune-active peptides of mouse various dose the present invention 30 days, had carried out the test (the mouse carbon that promptly reflects the delayed allergy (the sufficient sole of the foot thickens method) of cellular immune function, the mensuration that reflects the half hemolysis value (HC50) of humoral immune function, reflection monokaryon-macrophage phagocytic function is cleaned up the detection of experiment) of 3 para-immunity functions.Experimental result shows that the prepared soy protein immune-active peptides of the inventive method can strengthen half hemolysis value and the carbon of mouse and clean up function, but does not see the delayed allergy of increase mouse.Point out soy protein immune-active peptides of the present invention to have the humoral immune function of enhancing and monokaryon-macrophage phagocytic function.
Description of drawings
Fig. 1 hydrolysate of soybean protein process flow sheet.
Fig. 2 papain hydrolysis soybean protein isolate K value is to the result that influences of hydrolysis degree.
Fig. 3 conjugated protein enzyme hydrolysis of soybean protein isolates K value is to the result that influences of hydrolysis degree.
Fig. 4 hydrolysis by novo soybean protein isolate K value is to the result that influences of hydrolysis degree.
Fig. 5 neutral proteinase hydrolysis soybean protein isolate K value is to the result that influences of hydrolysis degree
Fig. 6 different pressures permeation flux changes.
The different pH value of Fig. 7 permeation flux changes.
Fig. 8 different material concentration permeation flux changes.
Fig. 9 polyoxyethylene glycol is working curve under corresponding chromatographic condition.
The MWD of the immune peptide that Figure 10 the inventive method is prepared.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
The optimum process condition research experiment of experimental example 1 enzymatic hydrolysis Sunlover 10
1. experiment material and method
1.1 experiment material and plant and instrument
1.1.1 experiment material
Soybean protein isolate: Ha Gaoke soybean Food Co., Ltd
Papoid: the Wuxi is outstanding can biological ltd of section
Sumizyme MP: NoVo company
Compound protease: NoVo company
Formaldehyde: chemical plant in the new year, analytical pure Harbin City
NaOH: analytical pure Qiqihar chemical general factory
Neutral protease: the Wuxi is outstanding can biological ltd of section
1.1.2 plant and instrument
Electric-heated thermostatic water bath: WSZ-133-75 Shantou City, Guangdong Province broadcasting instrument plant
Acidometer: PHS-3C Shanghai thunder magnetic instrument plant
Magnetic stirring apparatus: 79-1 Jiangsu Medical Instruments factory
Electronic balance: AE200 Sweden Tecator company
1.2 experimental technique
1.2.1 raw materials quality analysis
1) determination of moisture: GB 5497-95
2) protein content determination: GB 5511-95
1.2.2 enzyme activity determination
Protease activity amylograph ZB X 66030-87
1.2.3 the mensuration of hydrolysate degree of hydrolysis
FTM: water intaking is separated protein liquid 5.00mL and in small beaker, is added people 60mL and remove CO
2: zero(ppm) water and magnetic agitation, drip to pH=8.2 with accurate pH meter its pH value of indication and with 0.1moL/LNaOH, add the formaldehyde solution 20mL that the people becomes reconciled in then, record with its pH value droplet to 9.2 o'clock consume the mL number of 0.1mol/LNaOH; Do blank test; A proteinic NH in the Hydrolyzed protein liquid then: content is:
M is the volumetric molar concentration of used NaOH solution in the formula, and Vl, V.: be the sample titration, consume the volume number (mL) of NaOH during blank test.
1.2.5 screening of Sunlover 10 enzyme class and optimum hydrolysising condition research
1.2.5.1 papain hydrolysis Sunlover 10 optimum hydrolysising condition is selected
According to data record and simulation test, consider influencing each other of factors such as enzyme concentration, temperature of reaction, time, pH value in reaction, concentration of substrate, to intend and adopt orthogonal experiment to confirm optimum hydrolysising condition, this experiment is through five factors, four horizontal L
16(4
5) orthogonal test accomplishes, scheme is seen table 1.
The test of table 1 papain hydrolysis Sunlover 10
1.2.5.2 conjugated protein soy proteins top condition is selected
This experimental basis simulation test is considered from factors such as temperature of reaction, concentration of substrate, enzyme dosage, reaction times, pH, design L
16Orthogonal test is accomplished, and sees table 2.
The test of table 2 conjugated protein soy proteins top condition
1.2.5.3 hydrolysis by novo Sunlover 10 top condition is selected
L is adopted in this experiment
16Orthogonal test is accomplished, and scheme is seen table 3.
The test of table 3 hydrolysis by novo Sunlover 10 top condition
1.2.5.4 neutral proteinase hydrolysis Sunlover 10 top condition is selected
This experiment is intended and is adopted the L16 orthogonal design, sees table 4.
The test of table 4 neutral proteinase hydrolysis Sunlover 10 top condition
2. experimental result and analysis
2.1 raw materials quality analysis
Index through measuring the raw soybeans protein isolates is: protein content: 87.23%; Moisture content: 4.27%.
The content of material protein is the foundation that enzyme addition and degree of hydrolysis calculate in the experiment; The Sunlover 10 protein contnt of finding out employing from data is higher; It is minimum that the hydrolysate that obtains contains the ratio of fat, carbohydrate; So also ignored the influence of these components to hydrolysis effect under study for action, it is desirable, direct promptly adopting soybean protein isolate to carry out this research.
2.2 enzymatic hydrolysis of soybean albumen best-of-breed technology research
2.2.1 the papoid optimum hydrolysising condition is selected
Enzyme activity through measuring papoid is 13300IU/g.
Table 5 papoid orthogonal experiments
Find out that from the range analysis of table 5 orthogonal experiments each factor of papain hydrolysis Sunlover 10 is followed successively by enzyme dosage＞reaction times＞concentration of substrate＞pH＞temperature of reaction the influence of hydrolysis degree.The excellent A1B4C2D4E5 that is combined as of reaction conditions in theory.
The result that influences to the papain hydrolysis degree finds out from each level of factor K value: concentration of substrate is the line of delimitation when influence of hydrolysis degree is concentration 8%, and concentration is less than 8%, and the K value raises very fast; Rise to 27.28 by 23.55, and concentration is greater than 8%, it is little that the K value changes; Basically be in about 20, it is low more promptly to draw concentration of substrate, and hydrolysis effect is good more; But consider the suitability and the operability of production, the needs of the energy, pricing are not considered below 5%; This test design is a minimum with 5% concentration of substrate, and the feasible state of manufacturing enterprise that coincide basically suits so concentration of substrate elects 5% as.
Temperature of reaction is less to the influence of hydrolysis degree, and influence not quite tends towards stability basically in the time of 40-50 ℃; But when temperature rose to 50 ℃-70 ℃, the K value slightly rose, but changed little; In the single factor experiment,, will make enzyme deactivation to a certain extent when temperature is higher than 70 ℃; Based on this, the design will on be defined as 70 ℃.Can find out that from the R value extreme difference is very little, so this factor is little to the hydrolysis degree influence, each level is more or less the same, so temperature of reaction is elected 60 ℃ as.
Reaction times is bigger to the influence of hydrolysis degree, and the reaction times is suitable for reaction during from 4-6 hour, prolongs with the reaction times, and the DH value changes greatly; And from 6-8-12 hour, with the reaction times prolongation, though the DH value increases; But more slow, because soy bean proteinous soln is nutritious substratum, reaction at a certain temperature also just is being suitable for microbial growth; Be prone to cause microbiological contamination, so consider production application, the reaction times is difficult for long; Surpass 12hr and be unfavorable for producing, in conjunction with test-results, the reaction times is elected 6hr as.
Enzyme dosage is the main factor that influences hydrolysis degree, and enzyme dosage increases with enzyme dosage from 3000IU/g to 7000IU/g; Hydrolysis degree changes greatly; And enzyme dosage is greater than 7000IU/g, and it is not obvious that hydrolysis is carried out, and promptly influence is not very big to hydrolysis degree; Consider from the angle of economy again, so the best enzyme dosage that adopts is decided to be 7000IU/g.
PH is little to the influence of hydrolysis degree, and is promptly little to hydrolysis degree influence between the pH6-8, to inhibited reaction then more than the pH9, so pH elects 6 as.
The optimum hydrolysis combination condition that draws the papain hydrolysis Sunlover 10 from above analysis is A1B4C2D4E5, and promptly concentration of substrate 5%, 50 ℃ of temperature of reaction, reaction times 6hr, enzyme dosage 7000IU/g, reaction pH6.Under the optimum hydrolysising condition, the degree of hydrolysis of Sunlover 10 can reach 8.99%.
2.2.2 the compound protease optimum hydrolysising condition is selected
Through measuring the compound protease enzyme activity is 12437.6IU/g.
Table 6 conjugated protein soy proteins orthogonal experiments
Find out from the range analysis of table 6 orthogonal experiments; Each factor of conjugated protein enzyme hydrolysis of soybean protein isolates is followed successively by the influence of hydrolysis degree: in reaction pH＞concentration of substrate＞temperature of reaction＞enzyme dosage＞reaction times, the reaction conditions best of breed is A2B1C4D4C3.
As can be seen from Figure 3: temperature of reaction influences between 35-45 ℃ hydrolysis degree, and is on the rise with temperature rising K value, and temperature is greater than 45 ℃, raises with temperature, and K value is extremely acute to descend, and reaction is had considerable influence, so temperature is selected in 45 ℃ and suits.Concentration of substrate is bigger to the hydrolysis degree influence, and with the rising of concentration, the K value descends obviously; Mainly be because with the increasing of soybean protein isolate concentration, the chance minimizing that substrate contacts with the enzyme molecule, and with the increase of concentration of substrate; The viscosity that solution is relative is also increasing; Hindered the motion of enzyme molecule, and wrapped up, reduced the chance that the enzyme molecule plays a role by substrate.Consider that the practicality concentration of production is minimum and select 5% for use, therefore the righttest hydrolysis substrate concentration confirms as 5%.Enzyme dosage is to the influence of hydrolysis degree, and in the range of choice of enzyme dosage, its consumption is little to whole process influence; This scope selected is on the single factor experiment basis and consider to use and economically reduce rolling up of development cost and set, but can find out from the variation of K value, along with the increase of enzyme dosage; Reaction K value is in rising trend, and promptly addition is big more, and hydrolysis effect is good more; Take all factors into consideration this research, and suitable according to experimental result enzyme dosage 10000IU/g.The influence of reaction times to hydrolysis degree changed not quite from 4-10 hour, peaked reaction in 6 hours, so the reaction times is selected in 6hr.Reaction pH has the greatest impact to whole hydrolysis reaction, and promptly hydrolysis effect changes greatly between the pH5-8, increases by 5 to 7 with pH; Hydrolysis degree becomes positive correlation; And when pH continued to raise by 7, then level of response descended, and this mainly is because proteolytic enzyme has the suitability of reaction; In certain pH medium, just give full play to its vigor, reach the optimum hydrolysis effect.So ph optimum confirms as 7.
The top condition optimum combination that is drawn conjugated protein enzyme hydrolysis of soybean protein isolates by above analysis is A2B1C4D2E3; Be that temperature of reaction is 45 ℃, concentration of substrate 5%, enzyme dosage 10000IU/g; Reaction times 6hr; Reaction pH is 7, and under optimum hydrolysising condition, the degree of hydrolysis that can obtain soybean separation proteolysis liquid is 19.48%.
2.2.3 the Sumizyme MP optimum hydrolysising condition is selected
Enzyme activity through measuring Sumizyme MP is 13535.9IU/ml.
Table 7 hydrolysis by novo soybean protein isolate orthogonal experiments
Range analysis by table 7 hydrolysis by novo soybean protein isolate orthogonal experiments finds out, the secondary factors that influences hydrolysis degree be temperature of reaction＞concentration of substrate＞reaction times＞reaction pH＞enzyme dosage.
The variation of K value can be found out from reaction, and temperature of reaction is maximum to the influence degree of hydrolysis reaction, and degree of hydrolysis is the highest in the time of 50 ℃, and K value is maximum, promptly 50 ℃ become activation basic protein enzyme activity should temperature promptly adopt 50 ℃ to suit.
Reaction density is to the influence of hydrolysis degree: in the elevation process of concentration by 5%-10%, with the increase of concentration of substrate, hydrolysis degree weakens gradually; Excessive mainly due to concentration; Protein molecule capsule parcel enzyme molecule makes it motion and slows down, and only has an effect with the protein molecule of periphery; And reduced and other protein molecular bonded chance, so concentration of substrate confirms as 5%.Enzyme dosage is little to the hydrolysis degree influence in this experiment, and theoretical enzyme dosage is to influence the maximum factor of hydrolysis degree, but through simulation test; Enzyme dosage is added to 28000IU/g when following, and degree of hydrolysis is 25.13%, the optimum hydrolysis degree only high 3.51% of this experiment; But caused the huge waste of cost virtually, thus this research when taking all factors into consideration experiment effect and production cost, select for use in this horizontal extent, from these four levels; The enzyme dosage is when 8000IU/g, and the K value changes milder, and ascensional range is little; When 16000IU/g, the K value is high slightly, and it is suitable therefore to be suitable for 12000IU/g.The influence of reaction times to hydrolysis reaction shows as: the reaction times is a weight break point that influences hydrolysis degree when being 8 hours, is lower than 8 hours, prolongs in time; Hydrolysis degree is strengthened, and surpasses 8 hours, has but suppressed reaction; This mainly is because the peptide of hydrolysis has the possibility of local reversible reaction again; Polymerization again, thus the reduction of degree of hydrolysis shown, be to suit in 8 hours so select the reaction times for use.PH is not very big to reaction influence comparatively speaking, but hydrolysis effect is best can find out pH9 the time, and promptly pH9 is the ph optimum that the alkaline protease activity center plays a role, and also meets its action rule.Therefore pH elect as 9 suitable.
The optimum hydrolysising condition that is drawn the hydrolysis by novo soybean protein isolate by above-mentioned analysis is A2B1C3D3E4; Be that reaction conditions is: 50 ℃ of temperature of reaction, concentration of substrate are 5%, and enzyme dosage is 12000IU/g; Reacted 8 hours; Under pH9, carry out, with this understanding, obtaining the soybean separation proteolysis degree is 21.62%.
2.2.4 neutral proteinase hydrolysis soybean protein isolate optimum hydrolysising condition is selected
Enzyme activity through measuring neutral protease is 15443.8IU/g.
Table 8 neutral proteinase hydrolysis soybean protein isolate orthogonal experiments
Range analysis by table 8 neutral proteinase hydrolysis soybean protein isolate orthogonal experiments finds out that each factor is followed successively by concentration of substrate＞temperature of reaction＞pH＞reaction times＞enzyme dosage to the influence of hydrolysis degree.
Find out from K value audio-visual picture (Fig. 5): temperature of reaction is to the influence of hydrolysis degree: in rising trend when being increased to 45 ℃ with temperature by 35 ℃; And continue to raise on a declining curve by 45 ℃; Be that temperature surpasses 45 ℃; With the neutral protein enzymatic inactivation, the vigor reduction causes to some extent, so select for use 45 ℃ to suit.Concentration of substrate has the greatest impact to hydrolysis degree in this research, changes from the K value and sees, with the rising of concentration of substrate; The K value descends obviously, i.e. the increase of concentration has hindered the enzyme molecular motion; Fully contact with protein molecule and to cause hydrolysis degree to descend, so concentration of substrate is selected in 5%.Enzyme dosage is little to the hydrolysis degree influence in this test, and promptly each level difference is not very remarkable, and it is selected mainly is from economic consideration; In addition through simulated experiment, use 30000IU/g when above when the enzyme dosage, react degree of hydrolysis and reach 9.47%; After tend to balance; Select degree of hydrolysis approaching basically with the level of factor of test, see that from each level enzyme dosage 7000IU/g is suitable.
3. experiment conclusion
3.1 the papoid optimum hydrolysising condition is: concentration of substrate 5%, 50 ℃ of temperature of reaction, reaction times 6hr, enzyme dosage 7000IU/g, pH value in reaction 6.Optimum hydrolysis Du Keda 8.03%.
3.2 the optimum combination of conjugated protein enzyme hydrolysis of soybean protein isolates top condition is: temperature of reaction is 45 ℃, concentration of substrate 5%, and enzyme dosage 10000IU/g, reaction times 6hr, pH value in reaction is 7, the optimum hydrolysis degree is 19.48%.
3.3 the optimum hydrolysising condition of hydrolysis by novo soybean protein isolate is: reaction conditions is: 50 ℃ of temperature of reaction, concentration of substrate are 5%, and enzyme dosage is 12000IU/g, react 8 hours, under pH9, carry out, and the optimum hydrolysis degree is 21.62%.
3.4 the optimum hydrolysising condition of neutral proteinase hydrolysis soybean protein isolate is: reaction conditions is: 45 ℃ of temperature of reaction, concentration of substrate are 5%, and enzyme dosage is 7000IU/g, react 6 hours, under pH6.5, carry out, and the optimum hydrolysis degree is 9.47%.
Experimental example 2 Sunlover 10 immune peptide method for separating and concentrating and functional performance experiment thereof
1. materials and methods
1.1 main experiment material and equipment
(1) main experiment material
Soybean protein isolate Hagaoke Soybean Food Co., Ltd.
The Sumizyme MP Wuxi is outstanding can biological ltd of section
The compound protease Wuxi is outstanding can biological ltd of section
Sheep red blood cell (SRBC) (SRBC) straits, Harbin City trade Co., Ltd
Complement (GPS) straits, Harbin City trade Co., Ltd
The present of disease prevention and control center, Dou Shi reagent Heilongjiang Province
Chemical plant in the west, india ink Beijing
Vernier callipers (precision 0.01mm) Harbin Measuring & Cutting Tools Factory
(2) laboratory animal
The Kunming mouse that provides by Beijing Vital River Experimental Animals Technology Co., Ltd..Select 60 of Healthy female mouse, body weight 18～24g is divided into 5 groups at random, 12 every group, as immune one group, carries out delayed allergy experiment and half hemolysis value (HC50) determination experiment.Select 60 of Healthy female mouse, body weight 18～24g is divided into 5 groups at random, 12 every group, as immune two groups, carries out carbon and cleans up experiment and dirty animal body ratio measurement.
1.1.2 key instrument and reagent
Acidometer pHS-25 type Shanghai great achievement instrument plant
Electronic analytical balance Mei Lete-Tuo benefit instrument (Shanghai) Co., Ltd.
Whizzer Beijing Medical Centrifugal Machine Factory
Accurate electric blender Jiangsu Province Jintan City's high honour instrument Manufacturing Co., Ltd
Electric-heated thermostatic water bath Yuyao City east electric instrument factory
Albumen automatic analyzer Sweden FOX company
722-type spectrophotometer Shanghai Precision Scientific Apparatus Co., Ltd
Labscale TFF System ultrafiltration system Millipore
Poly (ether sulfone) film (3000D) U.S. is that company quite
UV-1901 ultraviolet-visible pectrophotometer Beijing General Corporation
East, clean bench Harbin joins
The great rich bio tech ltd of timing register
1.2 main experimental methods
1.2.1 the selection of immunologic function peptase research
Compare soybean protein isolate Sumizyme MP and the compound protease enzymolysis solution disconnected distribution situation of peptide behind the 3000Da membrane sepn.
1.2.2 the research of immunologic function peptide ultrafiltration optimal conditions
This experiment selects for use the 3000Da film to carry out the research of ultrafiltration optimal conditions.
(1) ultrafiltration pressure is to the influence of ultrafiltration
At room temperature, transferring pH is 7, investigates permeation flux different ultrafiltration pressures (15psi, 20psi, 30psi, 40psi) under, through protein contnt.
(2) the pH value is to the influence of ultrafiltration
Select the righttest ultrafiltration pressure according to test (1), investigate permeation flux under the different pH values (5,6,7,8), through protein contnt.
(3) material concentration is to the influence of ultrafiltration
Select the righttest ultrafiltration pressure according to test (1), select optimum pH, investigate the following permeation flux of different material concentration (3%, 4%, 5%, 6%), through protein contnt according to test (2).
(4) the righttest ultrafiltration condition confirms
Ultrafiltration hydrolysate of soybean protein liquid, key are to improve proteic transmitance.According to above single factor experiment analytical results, this Project design the orthogonal experiment of three factors, three levels, respectively with film see through protein contnt as investigating index, confirm the optimum process scheme of ultrafiltration.The material elements level is seen table 9 in the orthogonal experiment.
Table 9 orthogonal experiment level of factor table
1.2.3 the production of immunologic function peptide product preparation
Operational path:
The 5% albumen emulsion → enzymolysis → enzyme postcooling that goes out (is index determining with the DH value) → separation → protein enzymatic hydrolyzate → membrane sepn (3000D) → spraying drying → immunologic function peptide powder
The 5% albumen emulsion → enzymolysis → enzyme postcooling that goes out (is index determining with the DH value) → separation → protein enzymatic hydrolyzate → spraying drying → thick peptide powder (without ultrafiltration)
1.2.4 thick peptide powder and the immunocompetent research of immunologic function peptide powder
Through consulting pertinent literature, soybean protein hydrolysis peptide (through the 3000D ultrafiltration) is established three dose groups, is respectively 0.85g/kg body weight (low high dosage), 1.7g/kg body weight (middle dosage) and 5.1g/kg body weight (high dosage).Establish negative control group (irritating the stomach cleaning water) and the thick peptide powder of soy bean protein hydrolysate (without ultrafiltration) group simultaneously, (5.1g/kg body weight).Each treated animal is irritated the stomach volume by 2% of body weight and is irritated stomach, and the continuous irrigation stomach is surveyed each item immune indexes after 30 days.Immunocompetence detects test and cleans up experiment by the mouse carbon of the mensuration of the half hemolysis value of the delayed allergy (the sufficient sole of the foot thickens method) of reacting cells immunity, reaction humoral immunization and reaction non-specific immunity and form.
Delayed allergy (the sufficient sole of the foot thickens method): with every mouse of an immune treated animal through abdominal injection 2% (V/V prepares with saline water) hematocrit SRBC (2000rpm, 10 minutes) 0.2ml; After the sensitization 4 days; Measurement of left metapedes sole of the foot portion thickness, same position is measured three times, averages.The subcutaneous injection 20% in the measuring point (V/V prepares with saline water) hematocrit SRBC 20 μ l in back 24 hours measurement of left metapedes sole of the foot portion thickness of injection, represent the degree of DTH with the difference (swelling degree of the paw) of sufficient sole of the foot thickness before and after attacking then.
The mensuration of half hemolysis value: at the 2nd day of above-mentioned delayed allergy end, extract eyeball and get blood in centrifuge tube, placed about 1 hour, solidification blood and tube wall are peeled off, serum is fully separated out, centrifugal 10 minutes of 2000rpm collects serum., get 1ml and put in vitro 250 times of serum dilutions with the SA damping fluid, add 10% (V/V is with the preparation of SA damping fluid) hematocrit SRBC 0.5ml successively, complement 1ml (diluting by 1: 10) with the SA damping fluid.Other establishes the not control tube of increase serum (replacing with the SA damping fluid).Put in 37 ℃ of waters bath with thermostatic control insulation after 30 minutes, the ice bath termination reaction.Centrifugal 10 minutes of 2000rpm; Get supernatant 1ml, add Dou Shi reagent 3ml, get 10% simultaneously (V/V is with the preparation of SA damping fluid) hematocrit SRBC 0.25ml, add Dou Shi reagent to 4ml in another test tube; Abundant mixing; Place after 10 minutes, sentence control tube in 540nm and make blank, measure respectively and respectively manage OD value.The amount of hemolysin is calculated as follows with half hemolysis value (HC50) expression.
OD value * extension rate during sample HC50=sample OD value/SRBC HD50
Mouse carbon is cleaned up experiment: mouse is pressed the india ink of 1: 3.5 times of dilution of 0.1ml/10g body weight tail vein injection, treats that prepared Chinese ink injects timing immediately.
Injected behind the prepared Chinese ink 2,10 minutes, and got blood 20 μ l from the angular vein clump respectively, and it is added in the 2mL Na2CO3 solution.The mouse back cervical vertebra dislocation of weighing is put to death, get liver, spleen and thymus gland and remove most manadesma, blot the organ surface blood stains, weigh with filter paper.
At 600nm wavelength photometry density value (OD), make blank with 722 spectrophotometers with Na2CO3 solution.Be calculated as follows phagocytic index α:
K＝(lgOD1-lgOD2)/(t2-t1)
By formula calculate spleen/body weight ratio and thymus gland/body weight ratio.
Internal organs/body weight ratio=internal organs weight/body weight * 100.
Data processing and result judge: adopt variance analysis method, carry out homogeneity test of variance earlier by the program of variance analysis.Neat like variance, calculate the F value.F value＜F0.05, conclusion does not have significance for each group mean differences; F value>=F0.05, P≤0.05 is added up with the comparative approach in twos of mean between a plurality of experimental group and control group again.The data of abnormal or heterogeneity of variance are carried out the conversion of suitable variable, after waiting to satisfy normal state or homoscedasticity and requiring, add up with the data after the conversion.
1.2.5 the immunologic function peptide molecular weight distributes and the mensuration of nitrogen content
MWD testing conditions: Agilent1100 high performance liquid chromatograph, (state Agilent company) pump: Agilent G1311A (quaternary pump); Differential detector: Agilent G1315A/B (RID detector); Thermostat container: Agilent G1316A (thermostat container); Chromatic spectrum sample feeder: Agilent G1313A (automatic sampler); Chromatographic column: Agilent79911GF-083, the series connection of 79911GF-084 twin columns; 35 ℃ of column temperatures; Chromatographic working station: Angilent LC Chemstation; Moving phase: water; Flow velocity: 1.0ml/min; Sample size: 20 μ l.
Protein and nitrogen analysis: GB 5511-95
2. experimental result
2.1 the selection of immunologic function peptase research
The different 3000D membrane sepn with Sumizyme MP of table 10 compound protease relatively
Enzyme class＞3000D peptide content＜3000D peptide content (%)
(％)
Compound protease 64.1% 35.9%
Sumizyme MP 67.9% 32.1%
The separatory membrane that 3000D is chosen in this experiment carries out the separation and purification of immune peptide; Relatively compound protease and Sumizyme MP can be known through the distribution situation of peptide content behind the 3000D membrane sepn: the peptide content compound protease enzyme liquid below two kinds of enzymolysis solution 3000D is higher than the Sumizyme MP enzymolysis solution; And the enzymolysis time of separating most of compound protease is to be lower than 8 hours of Sumizyme MP in 6 hours, thus we to select prozyme for use be the lytic enzyme of immunologic function peptide.
2.2 the research of immunologic function peptide ultrafiltration optimal conditions
(1) ultrafiltration pressure is to the influence of ultrafiltration
Ultrafiltration is the membrane sepn process of pressure-driven type, and pressure reduction is the impellent of ultra-filtration process, and ultrafiltration material osmosis flux is exerted a decisive influence.
Pressure has decisive influence as the unique impellent in the ultra-filtration process to ultrafiltration material filterability.Macromolecular substance in the feed liquid can gather on the film surface, when this gathering can form gel coat when making film surface solute reach finite concentration, and the pressure when emergent pressure is firm formation gel coat.During actually operating, pressure should be chosen near the emergent pressure, has so both guaranteed certain filtrate flux; The energy consumption of having avoided meaningless pressure boost and having caused increases, and has prevented again under the excess pressure that macromole is clamp-oned pore membrane; And it is serious that pore membrane is stopped up, and avoided the infringement to film.
Can find out by Fig. 6; Rising pressure has remarkably influenced to the initial flux of seeing through, and the high more initial permeation flux of pressure is big more, but along with the prolongation of time; Proteins throughput presents remarkable decline under 15psi, 20psi, 30psi, the 40psi; 40psi particularly, this mainly is because the rising of pressure aggravates, causes permeation flux to descend rapidly along with the prolongation of time makes concentration polarization pollute with film.Therefore, can not raise pressure simply and should select a pressure that helps the mild infiltration of protein.The pressure of finding out 20psi to 30psi thus helps proteinic infiltration.
Table 11 different pressures sees through the white content of liquid eggs
Can find out by table 11, raise along with the rising protein transmitance of pressure, but the protein transmitance when proteinic transmitance is not apparently higher than 30psi under the 40psi; Major cause is that the ultrafiltration initial stage is excessive owing to pressure, and protein has seen through film, but the ultrafiltration later stage; Excessive pressure has aggravated the process of concentration polarization and film pollution; Make protein be trapped within on " film ", saying so accurately has been trapped within on the protein adsorption layer on the face, makes protein and film to separate.Therefore comprehensive permeation flux, protein transmitance, it is comparatively suitable to select to carry out ultrafiltration under the 30psi.
(2) the pH value is to the influence of ultrafiltration
Table 12 different PH albumen is held back and protein content
Shown in Fig. 7, table 12, protein is when neutral pH 7, and proteinic permeation flux is best; And the protein transmitance is also the highest because at this moment when the higher iso-electric point of pH value, protein institute electrically charged with the film surface charge same sex; Because the same sex is repelled each other; Protein granule is difficult for assembling, and therefore is difficult for forming the protein adsorption layer, and rejection is also bigger.But when the pH value was excessive, permeation flux, protein transmitance reduced on the contrary.The pH value also receives the restriction of mould material and material properties to the influence of ultrafiltration.Therefore, it is vital in the protein ultra-filtration process, selecting suitable pH value.
Comprehensive permeation flux, two factor analyses of protein transmitance, pH7 is optimum ultrafiltration pH condition.
(4) material concentration is to the influence of ultrafiltration
Table 13 different material concentration sees through protein content content
Can be found out that by Fig. 8 the feed liquid membrane permeation flux that material concentration is low is higher than the high feed liquid of material concentration, this is because in the ultra-filtration process; The increase of main body strength of solution causes the rising of protein concn, and theoretical according to concentration polarization, the decline of flux is proportional to proteinic logarithm concentration; After material concentration surpasses a certain scope; This linear relationship just no longer keeps, and this is because the increase of protein concn can cause the increase of viscosity of sludge, reduces protein molecule spread coefficient in solution; The consequent is increasing the weight of of concentration polarization phenomenon; Interaction between the protein molecule is simultaneously strengthened, thereby the resistance increase, and rejection increases and the reduction of albumen transmitance.Can find out rising from table 13 along with material concentration; The total protein transmitance of feed liquid raises; The highest during material concentration 6%; But it takes all factors into consideration the changing conditions of infiltration energy and two factors of protein transmitance only a little more than 5%, and the material concentration that this research is selected is chosen 5% and is suitable material concentration.
(5) the righttest ultrafiltration condition confirms
Table 14 Orthogonal experiment results extreme difference table
The variance analysis of table 15 protein content experimental result
| Source of error | S (sum of squares) | F (degree of freedom) | V (all sides) | F | Significance |
| Factor A B C error e summation ∑ | 238.46 9.9566 29.27 4.34 282.03 | 2 2 2 2 8 | 119.23 4.978 14.636 2.168 | 54.99 2.2957 6.75 | ** ?* |
F
0.05(2，6)＝5.14
F
0.01(2，6)＝10.92
See that from the protein content experimental result influence factor primary and secondary is: pressure＞material concentration＞pH value, wherein pressure is the most remarkable, and material concentration is remarkable for generally, and the pH value is not remarkable.The top condition of ultrafiltration is: pressure: 30psi, and pH value 7, material concentration is 5%.
2.3 immunologic function peptide molecular weight distribution situation and nitrogen analysis
1. standard curve making:
Prepare serial narrow distribution polyoxyethylene glycol standard specimen, according to the solution of molecular weight size preparation respective concentration.Molecular weight is respectively 106,194,620,400,1010,4020,1900,6450,11840,22450.. under corresponding chromatographic condition, obtain Z 150PH standard specimen HPGPC correction work curve.
(Fig. 9) can know from the typical curve analysis, and As time goes on promptly along with the increase of mobile phase volume, the descending order of molecular weight goes out the peak.
The MWD of 2 immune peptides:
See Figure 10.Can know that by the spectrum analysis of sample Mn, Mw, Mz are respectively 774.32,945.48,136.72, polydispersity coefficient D=Mw/Mn=1.22, the range of molecular weight distributions of sample is narrow.And the MWD large percentage about 13min, contrasting further analysis with typical curve can know, mainly being distributed in below the 5000Da of sample, the shared ratio of the molecular weight of 3400Da is 70.95%.
3 nitrogen content results:
Protein contnt through measuring the immune protein peptide is: 89.2%, and nitrogen content is: 14.272%
2.4 the research of soybean peptides immunologic function
2.4.1 the soy bean protein hydrolysate peptide is to the influence of mouse body weight
Mouse give the soy bean protein hydrolysate peptide after 30 days the influence to the mouse body weight see table 16 and 17 respectively.
Table 16 soy bean protein hydrolysate peptide is to the influence of immune one group of mouse body weight
Table 17 soy bean protein hydrolysate peptide is to the influence of immune two groups of mouse body weight
There are table 16 and table 17 visible; Compare with negative control group; The initial body weight of each immunization experiment treated animal and former powder treated animal and whole opisthosoma weight average there was no significant difference (P＞0.05) explain that soy bean protein hydrolysate peptide under this experiment condition (through the 3000D ultrafiltration) and the thick peptide powder of soy bean protein hydrolysate peptide (without ultrafiltration) do not have influence to weight of mice.
2.4.2 the soy bean protein hydrolysate peptide is to the influence of mouse internal organs/body weight ratio
Mouse gave the soy bean protein hydrolysate peptide after 30 days, and table 18 is seen in the influence of internal organs/body weight ratio.
Table 18 soy bean protein hydrolysate peptide is to the influence of mouse internal organs/body weight ratio
Visible by table 18; Per os gives soybean protein hydrolysis peptide (through the 3000D ultrafiltration) and the former powder (without ultrafiltration) 30 days of mouse various dose; And compare between control group; The spleen of each dose groups and former powder treated animal/body weight ratio and thymus gland/body weight ratio there are no significant difference (P＞0.05), promptly internal organs/body weight the ratio to mouse does not have influence.
2.4.3 the soy bean protein hydrolysate peptide is to the influence of mouse delayed allergy (DTH)
Mouse gives the soy bean protein hydrolysate peptide and after 30 days table 19 is seen in the influence of mouse delayed allergy (DTH).
Table 19 soy bean protein hydrolysate peptide is to the influence of mouse delayed allergy (DTH)
Visible by table 4; Per os gives soy bean protein hydrolysate peptide (through the 3000D ultrafiltration) and the thick peptide powder (without ultrafiltration) 30 days of mouse various dose; Compare with negative control group; Initial sufficient sole of the foot thickness there was no significant difference (P＞0.05), each dose groups of toes thickness difference and former powder treated animal there was no significant difference (P＞0.05), promptly soy bean protein hydrolysate peptide (through the 3000D ultrafiltration) can not strengthen the delayed allergy of mouse.
2.4.4 the soy bean protein hydrolysate peptide is to the influence of mouse half hemolysis value (HC50)
Mouse gives the soy bean protein hydrolysate peptide and after 30 days table 20 is seen in the influence of mouse half hemolysis value (HC50).
Table 20 soy bean protein hydrolysate peptide is to the influence of mouse half hemolysis value (HC50)
Annotate:
*With negative control group significant difference P＜0.05 is arranged relatively;
*With negative control group utmost point significant difference P＜0.01 is arranged relatively
Visible by table 20; Per os gives the soy bean protein hydrolysate peptide 30 days of mouse various dose; Compare with negative control group; There were significant differences (P＜0.05) for middle dose groups half hemolysis value, and high dose group and thick peptide powder group half hemolysis value significant difference (P＜0.01) show that the soy bean protein hydrolysate Toplink improves the mouse half hemolysis value.
2.4.5 the soy bean protein hydrolysate peptide is cleaned up the influence of function to mouse carbon
Give the soy bean protein hydrolysate peptide and after 30 days table 21 is seen in the influence that mouse carbon is cleaned up ability.
Table 21 soy bean protein hydrolysate peptide is cleaned up the influence of function to mouse carbon
Annotate:
*With negative control group significant difference P＜0.05 is arranged relatively
Visible by table 21; The soy bean protein hydrolysate peptide that per os gives the mouse various dose and thick peptide powder 30 days; Compare with negative control group; In dose groups and high dose group phagocytic index significant difference (P＞0.05) is arranged, promptly the soy bean protein hydrolysate peptide can increase mouse carbon and cleans up function, former powder group does not see that increase mouse carbon cleans up function.
3 experiment conclusion
3.1 selecting prozyme for use is that enzyme is used in the production of immune peptide; Best ultrafiltration condition is: pH=7, pressure are 0.3psi, and material concentration is 5%; The soybean immunologic function peptide that under best ultrafiltration condition, obtains is 70.95% in the shared ratio of the molecular weight of 3400Da; Protein contnt: 89.2%; Nitrogen content is: 14.272%.
3.2 oral administration is given the soy bean protein hydrolysate peptide 30 days of mouse various dose; Carried out the test of 3 para-immunity functions, reflected that promptly the mouse carbon of the delayed allergy (the sufficient sole of the foot thickens method) of cellular immune function, the mensuration that reflects the half hemolysis value (HC50) of humoral immune function, reflection monokaryon-macrophage phagocytic function is cleaned up the detection of experiment.The result shows that half hemolysis value and carbon that the prepared soy bean protein hydrolysate peptide of preparation method of the present invention can strengthen mouse cleans up function, but does not see and increase the mouse delayed allergy.Explain that soy bean protein hydrolysate peptide of the present invention has the humoral immune function of enhancing and monokaryon-macrophage phagocytic function.Stoste does not see that increasing mouse carbon cleans up function, and the half hemolysis value effect that strengthens mouse is not as the soy bean protein hydrolysate peptide of dosage.
Claims (1)
1. the preparation method of a soy protein immune-active peptides; May further comprise the steps: with the proteolytic enzyme reaction that is hydrolyzed, described proteolytic enzyme is selected from compound protease with soybean protein isolate, and the reaction conditions that is hydrolyzed is: temperature of reaction is 45 ℃; Concentration of substrate is 5%; Enzyme dosage 10000IU/g, in 6 hours reaction times, pH value in reaction is 7; With resulting weight percent concentration is that 5% soybean separation proteolysis liquid uses molecular weight to carry out ultrafiltration as the ultra-filtration membrane of 3000Da, and the pH value of said soybean separation proteolysis liquid is 7, and described ultrafiltration is under the pressure of 30psi, to carry out ultrafiltration; Collect ultrafiltrated, promptly get the bioactive peptide molecular weight and be 70.95% soy protein immune-active peptides in the shared ratio of 3400Da.
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