CN101704877A - Method for preparing soybean peptide - Google Patents
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- CN101704877A CN101704877A CN200910224026A CN200910224026A CN101704877A CN 101704877 A CN101704877 A CN 101704877A CN 200910224026 A CN200910224026 A CN 200910224026A CN 200910224026 A CN200910224026 A CN 200910224026A CN 101704877 A CN101704877 A CN 101704877A
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Abstract
The invention relates to a soybean peptide product and a preparation method thereof, in particular to a method for using isolated soybean protein as raw materials and adopting alkaline protease and flavourzyme in combination of the synergetic hydrolysis technology to prepare the target soybean peptide. The method solves the problem of the bitterness of the product in the process of enzyme modification of the isolated soybean proteins and the enrichment of the soybean peptide with low molecular weight of not more than 1000.
Description
Technical field
The present invention relates to a kind of preparation method of soybean peptide, be specifically related to a kind ofly prepare the method for low-molecular-weight soybean peptide, belong to food processing field by the combinative enzyme hydrolysis method.
Background technology
Peptide is the structure that 2 to 50 amino acid connect, and it shows and is different from activity of proteins and function.Generally may be defined as " proteinic a kind of " or " proteinic short-movie ", more precisely is the nontoxic synthetics of becoming to assign to make up in the multiple amino acids part of specific vigor of human body and technical ability.Mainly use as pharmaceuticals (as therapeutical agent, clinical diagnosis etc.) and daily necessities (as functional food and makeup), the market of daily necessities aspect enlarges day by day at present.
At present, soybean peptide is emphasis, the focus that domestic and international soyabean processing, food, trophology and medical circle are extremely paid close attention to and studied.The research of developed country such as the U.S., Japan early, formed peptide produce market widely abroad at present, market potential is very big, product mainly contains two classes: a class is peptide medicine and reagent, more than 100 kind of peptide medicine listing arranged in the world, this series products purity is very high, and price is also very expensive; Another kind of is to be the low antigen protective foods of functional factor and the bread and cheese that contains peptide with the peptide.At present external peptide food has formed industry, many famous companies have carried out the peptide series product development, for example peptide foodstuff additive and the pantry etc. that adds peptide have beverage, children's lunch, the elderly's set meal, motion food, short calcium absorption food and blood pressure-reducing food etc.
China starts late to the research of peptide, but prospect is very good, because nourishing function and physiologically active that soybean peptide had, the Application Areas in functional food is more and more wider.Existing market is in the access phase, does not also have competent product on the market, and competition situation is also not obvious, and especially the peptide product below the 1000Da is few.
More to extraction production, the research of soybean peptide at present.Wherein most of raw material that is adopted is based on defatted soy flour, soybean protein isolate or defatted soybean meal, wherein defatted soy flour and low temperature soy meal crude protein content are not high, purity of protein is lower, but soybean protein isolate content is greater than 90%, and protein quality is better.The employing technology is enzymic hydrolysis or microbial fermentation, but the sanitary condition that the applied bacterial classification of microbial fermentation needs is higher, and bacterial classification to produce proteinase activity lower, therefore using commercial enzyme, to carry out enzymolysis comparatively convenient.Centrifugal infiltration reverse osmosis, spraying drying form behind the enzymolysis.Finished product mostly is compound peptide, can be used as the intermediates use that processed food adds.Wherein enzyme hydrolysis method can adopt papoid, neutral protease, Validase TSP Concentrate II and hydrolysis by novo.Yet present method exists the various severe reaction conditions of enzyme class, only pay attention to the height of degree of hydrolysis, molecular weight 1000 is on the low side with the extraction yield of interior soybean peptides, and during enzymatic hydrolysis of soybean albumen, its inner hydrophobic grouping is exposed on the outside, and product has defectives such as bitter taste.
Summary of the invention
For the bitter taste of the product that exists in the enzymatic modification that solves soybean protein isolate remove and molecular weight 1000 with the enrichment problem of interior soybean peptide, the object of the present invention is to provide a kind of new prozyme preparation method to improve the extraction yield and the content of soybean peptide.
In order to achieve the above object, present method adopts following technical scheme:
The invention provides a kind of soybean peptide and preparation method thereof, is to be raw material with the soybean protein isolate, adopts Sumizyme MP and the compound synergetic hydrolysis technology of flavor protease that soybean protein is hydrolyzed and produces the target soybean peptide.
Soybean peptide of the present invention, it contains the soybean polypeptide of molecular weight less than 1000Da.
Described soybean peptide adopts following method to prepare:
1) method that adopts heating is carried out to a certain degree pre-treatment to soybean protein isolate;
2) adding Sumizyme MP is hydrolyzed;
3) the hydrolysis by novo substrate is gone out behind the enzyme, adopt flavor protease to continue hydrolysis;
4) hydrolyzed solution that obtains is after centrifugal, and the poly (ether sulfone) film that is respectively 3000Da and 1000Da by molecular weight cut-off carries out ultrafiltration successively, promptly flows through the 3000Da film earlier, after flow through the 1000Da film, finally obtain the polypeptide solution of molecular weight less than 1000Da.
Wherein, described pre-treatment is to get soybean protein isolate to add 10-20 times of water, is made into 5%-10% solution, and 75 ℃-90 ℃ were heated 10-20 minute.Obtain by preheating temperature and the experiment of degree of hydrolysis relation, degree of hydrolysis is better than the processing of other temperature in the time of 85 ℃, because proteic disulfide linkage is constantly opened, makes the enzyme action site expose, and makes degree of hydrolysis increase.Can improve degree of hydrolysis in 15 minutes 85 ℃ of preheatings.Because temperature is too high, make the partial protein sex change more than 90 ℃ the time, the enzyme action site is destroyed on the contrary.
The hydrolysising condition of described hydrolysis by novo is pH8.5-9.5, and the weight that adds enzyme is the 3-7% of substrate weight, hydrolysis temperature 50-65 ℃, and enzymolysis time 3-6h; Be preferably pH9.0, the weight that adds enzyme is 5% of substrate weight, 55 ℃ of hydrolysis temperatures, enzymolysis time 4h.
The hydrolysising condition of described flavor protease is pH5.0-8.0, and the weight that adds enzyme is the 3-7% of substrate weight, 45 ℃-55 ℃ of hydrolysis temperatures, enzymolysis time 3-12h; Be preferably pH6.5, the weight that adds enzyme is 5% of substrate weight, 50 ℃ of hydrolysis temperatures, enzymolysis time 6h.
The described enzyme condition of going out is the enzyme 20-25min that goes out under 90-95 ℃.
Described ultrafiltration condition is under neutral pH, pressure 0.1-0.3MPa, temperature 35-45 ℃; Be preferably pressure 0.2MPa, 40 ℃ of temperature.
Preferably, can also carry out drying to the gained polypeptide solution.For example adopt spraying drying, its condition is temperature in 160-180 ℃, temperature out 80-90 ℃, and vacuum tightness value 70-80Mpa, sample introduction value 20-30Mpa mixes material component, epigranular, the finished product dispersiveness is up to more than 80%.Preferably, described condition is 175 ℃ of inlet temperatures, 82 ℃ of temperature outs, vacuum tightness value 75Mpa, sample introduction value 25Mpa.
The present invention also provides the application of described soybean peptide in preparation peptide similar drug and reagent, the application of described soybean peptide as foodstuff additive also is provided, preferably as functional factor at low antigen protective foods with contain application in the bread and cheese of peptide.
The product of soybean peptide of the present invention is the off-white powder shape.At first adopt Sumizyme MP to be hydrolyzed by the inventive method, the soluble nitrogen content that obtains is the highest, makes the soybean protein hydrolysis degree reach 24.2%; And can reach one-step hydrolysis just by present method processing, not need middle-chain that hydrolytic process is monitored; Adopt flavor protease to continue hydrolysis afterwards, reduced bitter taste effectively when improving degree of hydrolysis, make degree of hydrolysis bring up to 25.3%, bitterness value reaches 0.2-0.5; And the yield of product reaches 20.3-24.3% after the ultrafiltration.
The Study on Antioxidtive Activities in Vitro of soybean peptide product of the present invention shows that its anti-oxidant value is higher, is 9.78-10.4mg/g, is ascorbic about 1.5 times; The IC50 value that soybean peptide is removed free radical (OH) is 6.220mg/ml.
Description of drawings
Fig. 1 is preparation method's process flow sheet of soybean peptide of the present invention.
Embodiment
The invention will be further described below in conjunction with embodiment, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.
In the orthogonal experiment of Sumizyme MP enzymatic hydrolysis of soybean albumen soluble protein content of the present invention and degree of hydrolysis, adopt four factors, three levels, the result is as shown in table 1.
Table 1
The hydrolysising condition that is obtained described hydrolysis by novo by orthogonal experiment is pH8.5-9.5, and the weight that adds enzyme is the 3-7% of substrate weight, hydrolysis temperature 50-65 ℃, and enzymolysis time 3-6h; Be preferably pH9.0, the weight that adds enzyme is 5% of substrate weight, 55 ℃ of hydrolysis temperatures, enzymolysis time 4h.
Further in the orthogonal experiment of food flavor enzyme enzymatic hydrolysis of soybean albumen soluble protein content and degree of hydrolysis, adopt four factors, three levels, the result is as shown in table 2.
Table 2
The hydrolysising condition that is obtained described flavor protease by orthogonal experiment is pH5.0-8.0, and the weight that adds enzyme is the 3-7% of substrate weight, 45 ℃-55 ℃ of hydrolysis temperatures, enzymolysis time 3-12h; Be preferably pH6.5, the weight that adds enzyme is 5% of substrate weight, 50 ℃ of hydrolysis temperatures, enzymolysis time 6h.
Embodiment 1
1, gets soybean protein isolate and add 10 times of water, be made into 10% solution, be heated to 75 ℃, be incubated 10 minutes;
2, preheating is good soy bean proteinous soln is cooled to 50 ℃, regulates pH value to 8.5, and (NOVO 2.4LMG), is incubated hydrolysis 3 hours to add the Sumizyme MP that accounts for protein-based in the soy bean proteinous soln (being the per-cent of albumen dry matter content) weight 3%;
3, with soy bean proteinous soln after the hydrolysis at 90-95 ℃, went out enzyme 20-30 minute;
4, will go out that soy bean proteinous soln is cooled to 45 ℃ behind the enzyme, add flavor protease (NOVO, 500MG), hydrolysising condition is: pH5.0, hydrolysis 3 hours, the flavor protease consumption accounts for 3% of protein-based weight in the substrate;
5, centrifugal: 3000 rev/mins of revolutions;
6, ultrafiltration: the above-mentioned solution that neutralizes (for example adopting the NaOH solution of 0.1mol/L) is to neutral, and 35 ℃ of attemperation adopt ultra-filtration membrane to be respectively the poly (ether sulfone) film of 3000Da and 1000Da, under 0.1MPa pressure, flow through the 3000Da film earlier, after flow through the 1000Da film, collect peritoneal effluent;
The mensuration of degree of being hydrolyzed: formol titration is measured degree of hydrolysis
Get the 8.0mL hydrolyzed solution behind the enzyme that goes out, place the flask of 150mL, add 60mL and remove CO
2Water, regulating the pH value is 7.0.In flask, add 10.0mL neutral formalin solution, mixing.Starting magnetic stirring apparatus, is the NaOH standard solution titration most 9.20 of 0.05mol/L with concentration, and the volume of noting the NaOH solution in drop-burette this moment consumes number V
1(mL).
Simultaneously, get not protolysate solution 8.0mL of same concentrations, do empty test certainly as stated above, the volume of the NaOH standardized solution in record drop-burette this moment consumes number V
2(mL).Then the calculation formula of proteic degree of hydrolysis is
DH%=M(V
1-V
2)(1000/C
woV)(1/h
tot)×100
In the formula: M is the concentration (mol/L) of NaOH standardized solution; V
1The volume (mL) of the NaOH standardized solution of the 0.05mol/L that consumes for the titration sample diluting liquid; V
2Volume (mL) for the NaOH standardized solution of 0.05mol/L that blank assay disappears; C
WoBe the protein mass of used WPC solution dense (g/L); V is the volume number (mL) that is used for the hydrolyzed solution of formol titration; h
TotBe the mmole number of peptide bond in every gram material protein, to soybean protein isolate, this value gets 7.8.
7, sterilization: 90-95 ℃, 20-30 minute (heating in water bath);
8, spraying drying: adopt the Buchi Mini Spray DryerB-290 of company, 160 ℃ of inlet temperatures, 80 ℃ of temperature outs, vacuum tightness value 70, sample introduction value 20.
Products obtained therefrom is the off-white powder shape, and bitterness value is 0.35.The yield of product is 24.3% after the ultrafiltration, and through the degree of hydrolysis monitoring, degree of hydrolysis reaches 25.3%.
Study on Antioxidtive Activities in Vitro shows that its anti-oxidant value (AOV) is higher, is 10.4mg/g, is ascorbic 1.5 times; The IC50 value of removing free radical (OH) is 6.220mg/ml.
Embodiment 2
1, gets soybean protein isolate and add 20 times of water, be made into 5% solution, be heated to 85 ℃, be incubated 15 minutes;
2, preheating is good soy bean proteinous soln is cooled to 55 ℃, regulates pH value to 9.0, adds the Sumizyme MP that accounts for soy bean proteinous soln (protein-based) weight 5%, insulation hydrolysis 4 hours;
3, with soy bean proteinous soln after the hydrolysis at 90-95 ℃, went out enzyme 20-30 minute;
4, will go out that soy bean proteinous soln is cooled to 50 ℃ behind the enzyme, and add flavor protease, hydrolysising condition is: pH6.5, and hydrolysis 6 hours, the flavor protease consumption accounts for 5% of substrate (protein-based) weight;
5, centrifugal: 3000 rev/mins of revolutions;
6, ultrafiltration: neutralization solution is to neutral, and 40 ℃ of attemperation adopt ultra-filtration membrane to be respectively the poly (ether sulfone) film of 3000Da and 1000Da, under 0.2MPa pressure, flow through the 3000Da film earlier, after flow through the 1000Da film, collect peritoneal effluent;
The mensuration of degree of hydrolysis is with embodiment 1;
7, sterilization: 90-95 ℃, 20-30 minute; (water-bath)
8, spraying drying: adopt the Buchi Mini Spray DryerB-290 of company, 175 ℃ of inlet temperatures, 82 ℃ of temperature outs, vacuum tightness value 75, sample introduction value 25.
Products obtained therefrom is the off-white powder shape, bitterness value 0.28.The yield of product is 20.3% after the ultrafiltration, and through the degree of hydrolysis monitoring, degree of hydrolysis reaches 25.3%.
Study on Antioxidtive Activities in Vitro shows that its anti-oxidant value (AOV) is higher, is 9.20mg/g, is ascorbic 1.5 times; The IC50 value of removing free radical (OH) is 6.220mg/ml.
Embodiment 3
1, gets soybean protein isolate and add 10 times of water, be made into 10% solution, be heated to 90 ℃, be incubated 20 minutes;
2, preheating is good soy bean proteinous soln is cooled to 65 ℃, regulates pH value to 9.5, adds the Sumizyme MP that accounts for soy bean proteinous soln (protein-based) weight 7%, insulation hydrolysis 6 hours;
3, with soy bean proteinous soln after the hydrolysis at 90-95 ℃, went out enzyme 20-30 minute;
4, will go out that soy bean proteinous soln is cooled to 55 ℃ behind the enzyme, and add flavor protease, hydrolysising condition is: pH8.0, and hydrolysis 12 hours, the flavor protease consumption accounts for 4% of substrate (protein-based) weight;
5, centrifugal: 3000 rev/mins of revolutions;
6, ultrafiltration: neutralization solution is to neutral, and 45 ℃ of attemperation adopt ultra-filtration membrane to be respectively the poly (ether sulfone) film of 3000Da and 1000Da, under 0.3MPa pressure, flow through the 3000Da film earlier, after flow through the 1000Da film, collect peritoneal effluent;
The mensuration of degree of hydrolysis is with embodiment 1;
7, sterilization: 90-95 ℃, 20-30 minute; (water-bath)
8, spraying drying: adopt the Buchi Mini Spray DryerB-290 of company, 180 ℃ of inlet temperatures, 90 ℃ of temperature outs, vacuum tightness value 80, sample introduction value 30.
Products obtained therefrom is the off-white powder shape, bitterness value 0.42.The yield of product is 22.5% after the ultrafiltration, and through the degree of hydrolysis monitoring, degree of hydrolysis reaches 25.3%.
Study on Antioxidtive Activities in Vitro shows that its anti-oxidant value (AOV) is higher, is 9.78mg/g, is ascorbic 1.5 times; The IC50 value of removing free radical (OH) is 6.220mg/ml.
The above only is preferred embodiment of the present invention, only is illustrative for the purpose of the present invention, and nonrestrictive.Those skilled in the art is understood, and can carry out many changes to it in the spirit and scope that claim of the present invention limited, revise, even equivalence, but all will fall within the scope of protection of the present invention.
Claims (10)
1. a soybean peptide is characterized in that containing the soybean polypeptide of molecular weight less than 1000Da.
2. soybean peptide according to claim 1 is characterized in that its bitterness value below 0.5, preferred 0.2-0.5.
3. a method for preparing the described soybean peptide of claim 1 is characterized in that comprising the steps:
1) adopt the method for heating that soybean protein isolate is carried out pre-treatment;
2) adopt Sumizyme MP to be hydrolyzed;
3) the hydrolysis by novo substrate is gone out behind the enzyme, adopt flavor protease to continue hydrolysis;
4) hydrolyzed solution that obtains is respectively the poly (ether sulfone) film of 3000Da and 1000Da after centrifugal successively by molecular weight cut-off, promptly flow through the 3000Da film earlier, after flow through the 1000Da film, finally obtain polypeptide solution less than 1000Da.
4. preparation method according to claim 3 is characterized in that described pre-treatment is to get soybean protein isolate to add 10-20 times of water, is made into 5%-10% solution, and 75-90 ℃ was heated 10-20 minute; Preferred 85 ℃ of heating 15 minutes.
5. preparation method according to claim 3, the hydrolysising condition that it is characterized in that described hydrolysis by novo is pH8.5-9.5, the weight that adds enzyme is the 3-7% of substrate weight, hydrolysis temperature 50-65 ℃, enzymolysis time 3-6h; Be preferably pH9.0, the weight that adds enzyme is 5% of substrate weight, 55 ℃ of hydrolysis temperatures, enzymolysis time 4h.Be pH9.0, enzyme is lived, 55 ℃ of hydrolysis temperatures, enzymolysis time 4h.
6. preparation method according to claim 3, the hydrolysising condition that it is characterized in that described flavor protease is pH5.0-8.0, the weight that adds enzyme is the 3-7% of substrate weight, 45 ℃-55 ℃ of hydrolysis temperatures, enzymolysis time 3-12h; Be preferably pH6.5, the weight that adds enzyme is 5% of substrate weight, 50 ℃ of hydrolysis temperatures, enzymolysis time 6h.
7. preparation method according to claim 3 is characterized in that described ultrafiltration condition under neutral pH, pressure 0.1-0.3MPa, temperature 35-45 ℃; Be preferably pressure 0.2MPa, 40 ℃ of temperature, pH is 7.0.
8. preparation method according to claim 3 is characterized in that further the gained polypeptide solution being carried out drying.
9. preparation method according to claim 8 is characterized in that described spray-dired condition is temperature in 160-180 ℃, temperature out 80-90 ℃, and vacuum tightness value 70-80Mpa, sample introduction value 20-30Mpa; Preferably, described condition is 175 ℃ of inlet temperatures, 82 ℃ of temperature outs, vacuum tightness value 75Mpa, sample introduction value 25Mpa.
10. the application of the described soybean peptide of claim 1 in preparation peptide similar drug and reagent, or as the application of foodstuff additive; Preferably as functional factor at low antigen protective foods with contain application in the bread and cheese of peptide.
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CN102326666A (en) * | 2011-07-29 | 2012-01-25 | 天津春发生物科技集团有限公司 | Method for preparing isolated soy protein hydrolysis liquid |
CN102511648A (en) * | 2011-12-28 | 2012-06-27 | 天津滨海诺奥酶工程技术有限公司 | Method for producing soybean polypeptide powder |
CN104489848A (en) * | 2014-12-02 | 2015-04-08 | 内蒙古天奇生物科技有限公司 | Postnatal lactation promoting solid beverage and preparation method thereof |
CN105476030A (en) * | 2015-12-15 | 2016-04-13 | 北京天肽生物科技有限公司 | Multi-functional composite oligopeptide nutrition powder |
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CN102326666A (en) * | 2011-07-29 | 2012-01-25 | 天津春发生物科技集团有限公司 | Method for preparing isolated soy protein hydrolysis liquid |
CN102326666B (en) * | 2011-07-29 | 2013-07-10 | 天津春发生物科技集团有限公司 | Method for preparing isolated soy protein hydrolysis liquid |
CN102511648A (en) * | 2011-12-28 | 2012-06-27 | 天津滨海诺奥酶工程技术有限公司 | Method for producing soybean polypeptide powder |
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CN105476030A (en) * | 2015-12-15 | 2016-04-13 | 北京天肽生物科技有限公司 | Multi-functional composite oligopeptide nutrition powder |
CN105639642A (en) * | 2016-03-14 | 2016-06-08 | 福格森(武汉)生物科技股份有限公司 | Nutritious food for rehabilitation after childbirth and induced abortion |
CN106072668A (en) * | 2016-06-29 | 2016-11-09 | 傅志勇 | Health product of eliminating toxin and beautifying the skin and preparation method thereof |
CN107232391A (en) * | 2017-04-14 | 2017-10-10 | 荣海生物科技有限公司 | A kind of method that use compound protease removes Soybean Peptide bitter taste |
CN108220374A (en) * | 2018-02-08 | 2018-06-29 | 金华市艾力生物科技有限公司 | The preparation method of Soybean Peptide |
CN108949887A (en) * | 2018-09-04 | 2018-12-07 | 哈尔滨工业大学 | A kind of preparation method of the multi-functional incretin peptide of soybean |
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