CN105166321A - High immune activity soybean peptide preparation method obtained through combination of microcalorimetric method and immune activity evaluation experiment - Google Patents
High immune activity soybean peptide preparation method obtained through combination of microcalorimetric method and immune activity evaluation experiment Download PDFInfo
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Abstract
The invention discloses a high immune activity soybean peptide preparation method obtained through combination of a microcalorimetric method and an immune activity evaluation experiment and belongs to the technical field of soybean deep processing. According to the method, the microcalorimetric method is adopted, and the optimum protease for preparing the high immune activity soybean peptide is obtained through Michalis constant comparison of an enzymatic hydrolysis to serve as the alkaline protease; the high immune activity soybean peptide preparation method is obtained in combination with the product immunity activity evaluation experiment. By means of the high immune activity soybean peptide preparation method, the immunity activity of a product can be effectively improved, it is proved by the immune activity evaluation experiment that the immunity activity of the product can be improved remarkably, the phagocytic rate of phagocytes can reach 57.23%, the IgG content can reach 119.53 mg/100 ml, and the NK cell activity can reach 26.46%. The high immune activity soybean peptide preparation method is free of pollution, low in cost, simple in process, high in product immunity activity, good in water solubility, safe and free of toxic and side effect, has high popularization value, can generate high economic value and social benefits and is quite wide in prospect.
Description
Technical field
The invention belongs to soybean deep processing technology field, especially a kind of preparation method of microcalorimetric method binding immunoassay active appraisal experiment determination high immunological activity Soybean Peptide.
Background technology
The phagocytic activity that immune-active peptides can not only stimulate the propagation of body Autologous lymphocyte, promote the release of cell factor, strengthens macrophage in body, and the immunological rejection of body can not be caused.Compared with the macromolecular drug for improving body immunity, immune-active peptides has obvious advantage, it can give full play to the initiative of body self immune system, promote the raising of body autoimmunity ability, immune-active peptides is as small molecule material in recent years, the unique advantage low with its molecular weight, activity is strong, consumption is few, causes the attention of people more and more.
Large quantity research proves to have immune active ingredient in the Soybean Peptide that soybean protein is formed through protease hydrolytic, but these Soybean Peptide product immune active ingredient content are not very high.The method Chinese scholars being prepared by protease hydrolytic to Soybean Peptide has carried out large quantifier elimination, be mostly the character by measuring product, determine the condition such as optimal reaction temperature, acidity, test method is more complicated, randomness is comparatively large, can not the optimised process of accurate quantitative analysis determination protease hydrolytic reaction.Cause protease hydrolytic technology controlling and process to be short of theoretical direction, it is difficult to control, and product yield is low.Microcalorimetric method has highly sensitive, simple operation and other advantages.The present invention is intended to utilize microcalorimetric method directly to measure the thermal change of trace in enzyme-catalyzed reaction by highly sensitive micro-calorimeter, obtain protease to the thermal power-time graph of soybean protein catalytic reaction, quantitatively determine protease hydrolytic soybean protein isolate optimal reaction temperature, pH.The present invention utilizes microcalorimetric method binding immunoassay active appraisal experiment quantitatively can determine the preparation method of high immunological activity Soybean Peptide more accurately.
Summary of the invention
The object of the invention is to, overcome the deficiencies in the prior art, a kind of method preparing high immunological activity Soybean Peptide is provided.A kind of new research method microcalorimetric method of the present invention, produce the suitableeest protease of Soybean Peptide to hydrolysate of soybean protein, optimum condition carries out determining quantifier elimination, binding immunoassay active appraisal experiment determines a kind of preparation method of new high immunological activity Soybean Peptide.
For solving the problems of the technologies described above, technical scheme of the present invention is: a kind of preparation method of microcalorimetric method binding immunoassay active appraisal experiment determination high immunological activity Soybean Peptide, and step is as follows;
1. obtain solution:
Solution is 1.: accurately take soybean protein isolate sample, with the cushioning liquid of different pH value (pH3.3-pH10.0) respectively compound concentration be 5-10gL
-1soybean protein isolate solution.
Solution is 2.: compound concentration is 0.1-0.3gL
-1neutral proteinase (enzyme activity 5.3 × 10
4u/g) solution, trypsase (enzyme activity 5.1 × 10
4u/g) solution, bromelain (enzyme activity 6.0 × 10
4u/g) solution, alkali protease (enzyme activity 5.7 × 10
4u/g) solution.
2.90 DEG C of soybean protein isolate solution carry out water bath processing 10min;
3. measure thermal power time graph: get solution 1. 2ml put into stainless steel ampoule.On the connecting rod of titration micro-calorimeter around thin plastic pipe, Guan Zhongsheng protein enzyme solution 2. 1ml, first preheating, after temperature (306.15K-336.15K) is constant, stirring system is laid, rotating speed is 60-120rpm, with peristaltic pump, 2. protein enzyme solution is injected ampoule, then carries out the mensuration of thermal power-time graph;
4. by thermal power-time graph, michaelis constant Km and maximal rate Vmax when parsing neutral proteinase, trypsase, papain, each temperature of alkali protease (306.15K-336.15K), pH value (pH6.13-pH9.22) according to thermokinetic theory and Reduced extent method, and the relational expression set up between michaelis constant and temperature, pH value, thus obtain neutral proteinase optimum temperature 319.19K ﹑ optimal pH 7.13; Trypsase optimum temperature 323.27K ﹑ optimal pH 7.77; Bromelain optimum temperature 314.63K ﹑ optimal pH 6.99; Alkali protease optimum temperature 329.5K ﹑ optimal pH 8.25.
5., by thermal power-time graph, try to achieve the michaelis constant Km=24.02gL of neutral proteinase at its most thermophilic Du ﹑ optimal pH
-1, trypsase is at the michaelis constant Km=42.41gL of its most thermophilic Du ﹑ optimal pH
-1, bromelain is at the michaelis constant Km=50.31gL of its most thermophilic Du ﹑ optimal pH
-1, alkali protease is at the michaelis constant Km=16.01gL of its most thermophilic Du ﹑ optimal pH
-1, thus can know that the affinity of basic protein enzyme-to-substrate soybean protein isolate is the strongest, binding immunoassay active appraisal experiment determines that alkali protease is the suitableeest protease of catalyzing hydrolysis soybean protein isolate further.
6. the Soybean Peptide product obtained under different tests condition (at the bottom of concentration of substrate, enzyme ratio, pH, temperature, enzyme combination) carries out immunocompetence evaluation experimental, by result of the test determination high immunological activity Soybean Peptide preparation method: for the temperature control enzyme reactor tank with agitator put into by soybean protein, add water and size mixing, slurry concentration is about 5-10%; After sizing mixing, the 1-3% heavy by substrate protein adds alkali protease, and limit is enzyme-added, limit is stirred, add alkali at any time, keep pH value ≈ 8.25, mixing speed=60-120r/min, hydrolysis temperature remains on about 329.5K, enzymolysis, after 3 hours, adds the neutral proteinase with alkali protease 1:3, at 319.19K, pH7.13 to be hydrolyzed after 1h through separation and Extraction → remove peculiar smell → remove inorganic salts → sterilising and enzyme inactivating → concentrated, spraying dry, obtains high immunological activity Soybean Peptide.
The invention has the beneficial effects as follows: the microcalorimetric method that the present invention uses has highly sensitive, the advantages such as simple to operate, experimental result is accurate; The invention provides a kind of method preparing high immunological activity Soybean Peptide, effectively can improve the immunocompetence of product, prove through immunocompetence evaluation experimental, the present invention more can significantly improve the immunocompetence of product, phagocyte phagocytic rate can reach 57.23%, IgG content can reach 119.53mg/100ml, and NK cytoactive can reach 26.46%.The present invention is pollution-free, and cost is low, and technique is simple, and product immunocompetence is high, good water solubility, safe, nontoxic, have no side effect, have higher promotional value, can produce higher economic worth and social benefit, its prospect is boundless.
Accompanying drawing explanation
Fig. 1 at pH=6.50, the thermal power-time graph of neutral protein enzymic catalytic reaction during different temperatures
Fig. 2 at T=318.15, the thermal power-time graph of neutral protein enzymic catalytic reaction during different acidity
Detailed description of the invention:
The present invention will be described below to enumerate specific embodiment.It is to be noted that embodiment is only for the invention will be further described, do not represent invention protection domain, other people point out the nonessential amendment and adjustment made according to the present invention, still belong to scope.
Embodiment 1
1. the preparation of solution:
Solution is 1.: accurately take soybean protein isolate sample, with pH value be 6.13,6.60,7.10,7.61,8.03 cushioning liquid respectively compound concentration be 10gL
-1soybean protein isolate solution.
Solution is 2.: compound concentration is 0.2gL
-1neutral protein enzyme solutions.
2.90 DEG C of soybean protein isolate solution carry out water bath processing 10min;
3. experiment is carried out in 4ml stainless steel ampulla (titration micro-calorimeter).Get soybean protein isolate solution 2ml and put into stainless steel ampoule.On the connecting rod of titration micro-calorimeter around thin plastic pipe, Guan Zhongsheng alkaline protease solution 1ml, first preheating, as temperature (306.15K; 311.15K; 316.15K; 321.15K; 326.15K) constant after, stirring system is laid, rotating speed is 120rpm, with peristaltic pump, neutral protein enzyme solutions is injected ampoule, then the mensuration of thermal power-time graph is carried out, recorder starts record, thinks that experiment terminates, stir and stop when recording pen draws the straight line parallel with baseline.Every individual system repeats measuring three times, averages.
Experiment records at pH=6.50, and during different temperatures, the thermal power-time graph of neutral protein enzymic catalytic reaction is as Fig. 1, obtains different temperatures (306.15K by Fig. 1; 311.15K; 316.15K; 321.15K; The michaelis constant (Km) of neutral protein enzymic catalytic reaction and maximum rate (Vmax) 326.15K):
T=306.15K, K
m=36.12 and V
max=0.1207; T=311.15K, K
m=31.93 and V
max=0.1351; T=316.15K, K
m=29.64 and V
max=0.1410; T=321.15K, K
m=30.59 and V
max=0.1371; T=326.15K, K
m=32.33 and V
max=0.1238
According to the data of Km and Vmax, with computer fitting, obtain Km-T curvilinear equation, namely
K
m=4398-27.45T+0.043T
2, R=0.9930, asking extreme value to obtain neutral protein enzymatic soybean separation proteolysis optimum temperature is 319.19K.
Experiment records at T=318.15, during different acidity, the thermal power-time graph of neutral protein enzymic catalytic reaction is as Fig. 2, T=318.15K can be obtained by Fig. 2, the michaelis constant (Km) of enzymic catalytic reaction and maximum rate (Vmax) time different pH (6.13,6.60,7.10,7.61,8.03): PH=6.13, K
m=29.52 and V
max=0.1166; PH=6.60, K
m=27.71 and V
max=0.1328; PH=7.10K
m=26.64 and V
max=0.1415; PH=7.61, K
m=27.23 and V
max=0.1353; PH=8.03, K
m=29.16 and V
max=0.1236, with computer fitting, can obtain K
m-pH curvilinear equation, i.e. K
m=175.7-41.80pH+2.932pH
2, R=0.9935, the optimal pH asking extreme value to obtain neutral protein enzymatic hydrolysis soybean protein isolate is 7.13.
Embodiment 2
1. size mixing
Protein isolate is put into the temperature control enzyme reactor tank with agitator, add water and size mixing, slurry concentration about 10%;
2. add enzyme reaction
After sizing mixing, by substrate protein heavy 1% add alkaline microbial protease, limit is enzyme-added, and limit is stirred, and adds alkali at any time, keep pH value ≈ 8.25, mixing speed=60r/min, hydrolysis temperature remains on about 329.5K, and enzymolysis adds the neutral proteinase with alkali protease 1:3 after 3 hours, 1h is hydrolyzed at 319.19K, pH7.13.
3. separation and Extraction
Go out the mixed liquor after enzymolysis ferment treatment, pH4.5, and make non-enzymolysis protein precipitating, mixed liquor pumps in seperator, carries out Separation of Solid and Liquid, and effluent is all acid-soluble Soybean Peptide.Solid formation is water insoluble ingredients, adds alkali neutralization, can be used as bean dregs dry feed after oven dry;
4. remove peculiar smell
Soybean Peptide is taste compound in low or soybean at enzymolysis degree---when isoflavones, saponin(e etc. are without separation and Extraction process, often there is unacceptable peculiar smell, for removing peculiar smell, Soybean Peptide liquid can be processed by activated-charcoal column, Soybean Peptide liquid and active carbon ratio be about 1:10 (namely by time Soybean Peptide flow quantity be 1/10 of activated carbon grain volume), cross column temperature ≈ 40 DEG C, after charcoal treatment, the Soybean Peptide liquid of transparent color and luster, substantially tasteless can be obtained;
5. remove inorganic salts
Inorganic salts are removed through anion-cation exchange resin.
6. sterilising and enzyme inactivating
Through de-taste, except the Soybean Peptide liquid after Ficus caricaL, high temperature sterilization goes out enzyme.
7. concentrated, spraying dry
Through the Soybean Peptide liquid of sterilising and enzyme inactivating, pump into the economic benefits and social benefits enrichment facility that steam pressure is 180KPa, vacuum is 90Kpa, temperature is 55 DEG C, concentrated concentration reaches ≈ 40%, in pump-in pressure formula spray drying tower, inlet temperature is 170 DEG C, outlet temperature 80 DEG C, products obtained therefrom is " high immunological activity Soybean Peptide ".
Embodiment 3
Immunocompetence evaluation experimental
Animal: mouse, healthy Kunming kind, weight (25 scholar 2) g, be SPF level (no-special pathogen), by Tai'an, Tai Bang company provides.
Tested products: embodiment 2 product " high immunological activity Soybean Peptide "
Animal grouping and administration:
Prerun 3d, raises and train observation to mouse between preliminary trial period, observes its adaptive condition to new environment, trains its fixed point to search for food; Quantity delivered every day of feeding is raised rate table according to throwing and is determined, and is adjusted accordingly according to the situation of ingesting.Throw something and feed and follow fixed point, regularly, quantitatively, determine the principle of matter.Eliminate physique and locomitivity and on average have the mouse of notable difference.After prerun 3d, mouse is divided into 4 groups at random, often organizes 40: control group, high immunological activity Soybean Peptide basic, normal, high dosage group 7d empty stomach in the morning amount of weighing starts experiment, control group gavage 300mgkg
-1d
-1physiological saline, basic, normal, high dosage group gavage 100,200,300mgkg respectively
-1d
-1high immunological activity Soybean Peptide, continuous gavage 30d.Observe the situations such as the drinking-water of mouse, spirit, hair and stool and urine every day.
1. high immunological activity Soybean Peptide is on the impact of mouse immune organ weight and index thereof
Mouse is normally raised, on time administration.When experiment is to 30d, every dosage group and control group get mouse 10.After administration terminates, weigh, spleen is got in execution and thymus gland is weighed.Computation immunity shoot formation.Immune Organs Index represents with the ratio of immune organ weight and body weight.Experimental result is in table 1.
Table 1 high immunological activity Soybean Peptide is on the impact of mouse immune organ weight index
Table1effectsofpeptideontheweightofimmuneorgansinmice
Note:
represent p<0.05,
represent p<0.01, compare with control group
By data analysis in table 1, the each dosage group of high immunological activity Soybean Peptide all can improve the immunocompetence of body by promotion spleen and thymus development, high dose group conspicuousness can improve the Thymus and spleen index of normal mouse, to the fundamental immunity level effect of being significantly improved of animal.
2. high immunological activity Soybean Peptide is on the impact of mice serum immunoglobulin (Ig) (IgG) content
After raising 30d, often organize taking-up 10, eyeball gets blood, and separation obtains serum.Get mice serum 20 μ L, add buffer solution 1mL and mix, then get the blood sample 20 μ L serum of dilution, separately add buffer solution 1mL and mix, then illustrate by the concrete operations of IgG radioimmunological kit and measure.Measurement result is in table 2.
Table 2 high immunological activity Soybean Peptide is on the impact of normal mouse IgG content
Table2effectsofpeptideonIgGcontentinnormalmice
Analytical table 2 data are known, and each dosage group of product all can improve IgG content in mice serum, and the raising effect of high dose is the most obvious.Illustrate that high immunological activity Soybean Peptide can largely improve humoral immunity of organism ability.
3. high immunological activity Soybean Peptide is on the impact of NK cells in mice activity
Before experiment, target cell is carried out Secondary Culture by 24h.Washing 3 times with front with Hank ' S liquid, is 4 × 10 with RPMl-l640 complete culture solution adjustment cell concentration
5individual/mL.
Prepare splenocyte suspension.Each treated animal is aseptic gets spleen, is placed in and fills appropriate aseptic Hank ' S liquid plate, ground by spleen gently, make individual cells suspension with tweezers.Filter through 200 eye mesh screens, wash 2 times with Hank ' S liquid, each centrifugal 10min (1000r/min).Abandon supernatant cytoplasm is upspring, after sterilized water for injection splitting erythrocyte, add 1% glacial acetic acid diluting cells suspension.The concentration of adjustment splenocyte suspension is 2 × 10
7after, add in 96 orifice plates and cultivate.Each animal is divided into: reacting hole (splenocyte suspension and each 100 μ L of YAC-1 cell suspension, effect target ratio is 50: 1); Spontaneous release hole (YAC-1 cell suspension and each 100 μ L of nutrient solution); Maximum release aperture (YAC-1 cell suspension and each 100 μ L of 1%NP40).Everyly above all make 3 parallel pipes.96 orifice plates at 37 DEG C, 5%CO
2cultivate 4h in incubator, after adding LDH matrix liquid and 1moL/LHCl, merge the solution of each parallel hole, measure OD value (OD) at ELIASA 490nm place.Calculate NK cytoactive.
The results are shown in Table 3.
Table 3 high immunological activity Soybean Peptide is on the impact of normal mouse boosting cell NK activity
Table3effectsofpeptidesonNKactivityofspleencellsinmice
Note:
represent p<0.05,
represent p<0.01, compare with control group
Table 3 shows that product each dosage group NK cells in mice activity all has enhancing.Middle and high dosage group all has conspicuousness.
4. high immunological activity Soybean Peptide is on the impact of macrophage phagocytic function
After raising 30d, often organize taking-up 10, the chicken erythrocyte suspension of every mouse lumbar injection lmL20%; Put to death mouse after 30min, faced upward position and be fixed on mouse plate, open abdomen, through Intraperitoneal injection physiological saline 2mL.Rotate mouse plate lmin, then, sucking-off abdominal cavity washing lotion lmL, average mark drips on 2 slides, 37 DEG C of incubation 30min; Use physiological saline rinsing afterwards, dry, fix with the acetone methanol solution of 1:1,4%Giemsa-phosphate buffer dyeing 3min, then dry with distilled water rinsing.Under oil mirror, counting 100 macrophages, calculate phagocytic rate and phagocytic index.
The results are shown in Table 4.
Form 4 high immunological activity Soybean Peptide is on the impact of macrophage phagocytosis of mice
Table4effectsofpeptidesonphagocytosisofmacrophageinmice
Note:
represent p<0.05,
represent p<0.01, compare with control group
From data in table 4, each dosage group Turnover of Mouse Peritoneal Macrophages phagocytic rate is with phagocytic index compared with control group, and general performance ascendant trend, wherein middle and high dosage group has extremely significant difference, significantly can promote the phagocytic function of Turnover of Mouse Peritoneal Macrophages.
Claims (4)
1. a preparation method for microcalorimetric method binding immunoassay active appraisal experiment determination high immunological activity Soybean Peptide, is characterized in that: the suitableeest protease, optimal pH, the optimum temperature that adopt microcalorimetric method determination hydrolyzed soy protein isolate; The preparation method of binding immunoassay active appraisal experiment determination high immunological activity Soybean Peptide.
2. the preparation method of a kind of microcalorimetric method binding immunoassay active appraisal experiment determination high immunological activity Soybean Peptide as claimed in claim 1, it is characterized in that, microcalorimetric method is utilized to measure the thermal power-time graph of neutral proteinase, trypsase, bromelain, alkali protease hydrolyzed soy protein isolate when different temperatures (306.15K-336.15K) respectively, calculate the michaelis constant of each enzyme digestion reaction and maximum reaction rate respectively, quantitatively obtain the optimum temperature of above-mentioned protease hydrolytic soybean protein isolate.
3. the preparation method of a kind of microcalorimetric method binding immunoassay active appraisal experiment determination high immunological activity Soybean Peptide as claimed in claim 1, it is characterized in that, microcalorimetric method is also utilized to measure the thermal power-time graph of neutral proteinase, trypsase, papain, alkali protease hydrolyzed soy protein isolate when different pH (pH6.13-pH9.22), calculate the michaelis constant of each enzyme digestion reaction and maximum reaction rate respectively, quantitatively obtain the optimal pH of above-mentioned protease hydrolytic soybean protein isolate.
4. the preparation method of a kind of microcalorimetric method binding immunoassay active appraisal experiment determination high immunological activity Soybean Peptide as claimed in claim 1, it is characterized in that, the suitableeest described protease is alkali protease; Its hydrolyzed soy protein isolate optimal pH is 8.25; Optimum temperature is 329.5K; The preparation method of described high immunological activity Soybean Peptide is enzyme reaction → separation and Extraction of sizing mixing → add → remove peculiar smell → remove inorganic salts → sterilising and enzyme inactivating → concentrated, spraying dry → high anti-fatigue activity Soybean Peptide product.Wherein add at the bottom of enzyme reaction substrate concentration 5%-10%, pH value 8.25, enzyme than 1-3%, hydrolysis temperature 329.5K, the neutral proteinase with alkali protease 1:3 is added after hydrolysis by novo soybean protein 3h, be hydrolyzed 1h at 319.19K, pH7.13, obtain high immunological activity Soybean Peptide.
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