CN1256032C - Soybean protein enzymolysis method capable of reducing colour change - Google Patents
Soybean protein enzymolysis method capable of reducing colour change Download PDFInfo
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- CN1256032C CN1256032C CNB031341888A CN03134188A CN1256032C CN 1256032 C CN1256032 C CN 1256032C CN B031341888 A CNB031341888 A CN B031341888A CN 03134188 A CN03134188 A CN 03134188A CN 1256032 C CN1256032 C CN 1256032C
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- soybean protein
- enzyme
- protease
- enzymolysis
- alkali
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- 239000003513 alkali Substances 0.000 claims abstract description 30
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 29
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- 235000013305 food Nutrition 0.000 description 5
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 2
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Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention belongs to the technical field of enzyme catalysis, which relates to a method for preparing oligopeptide mixtures containing less than 10 amino acids by enzyme hydrolysis soybean proteins capable of reducing color change. The present invention is characterized in that inhibitors which can prevent reducing sugar from generating Maillard reactions with amino acids and amino groups of proteins are simultaneously added in a hydrolyzing tank when the proteins are hydrolyzed under the condition that pH values gradually change without alkali. The present invention has the advantage that desalting steps and decoloring steps are saved in the downstream treatment of hydrolysate of the soybean proteins.
Description
Technical field
The invention belongs to the enzyme technology field, provide a kind of under the pH value gradual change condition of exogenously added alkali not the enzymatic hydrolysis of soybean protein Preparation suppress the method that the enzymolysis liquid look becomes simultaneously less than 10 amino acid whose oligo peptides.
Background technology
Studies show that in recent years, Soyprotein peptide also have multiple physiological hygiene function except that having the nutritive value the same with soybean protein.Therefore, the research soybean protein method that is converted into Soyprotein peptide is subjected to people's attention.
Obtain Soyprotein peptide with enzymatic isolation method and have condition and relax, environmental friendliness and do not destroy benefit such as nutrition is the direction of soybean protein hydrolysis technological development.Disclosed the concrete grammar of enzymatic hydrolysis of soybean albumen among patent CN1151848A, CN1168770A, CN1323535A and the CN1344138, wherein all require in enzymolysis process, constantly in enzymolysis liquid, to add strong base solution, keep the enzymolysis activity of alkali protease by keeping enzymolysis liquid pH value, and then obtain satisfied raw material (substrate) degradation rate and oligopeptides content.The problem of these methods is that the aqueous slkali of keeping the pH value can produce tangible alkaline peculiar smell and introduce salt impurity in zymolyte, therefore must adopt complicated desalination to remove miscellaneous operation in downstream.For this reason, we are in the Chinese patent of declaring early stage (03110987.X), proposed a kind ofly to utilize alkali protease and acid protease or/and the coordinated enzymatic hydrolysis effect of neutral proteinase the method for enzymatic hydrolysis of soybean protein Preparation oligopeptides under the pH value gradual change condition of exogenously added alkali not.But the weak point of this method is, hydrolysate of soybean protein is converted into and often is attended by coloring matter in the process of oligopeptides and generates, and causes zymolyte to present dark brown and even pitchy, has limited it in Application in Food.
In fact, in fermentation industry, the look change problem of zymotic fluid has certain generality.Following patent documentation and open source literature provide some discoloration methods: open source literature amino acid and living resources, reported ispol that the pig Hydrolysis of dried blood powder is made condition and effect among 18,1 (1996) 18-19 with the active carbon method decolouring; Food Science has been reported the fish albumen hydrolysis solution that the fish proteolysis the is made condition and the effect of activated carbon decolorizing among 19,7 (1998) 17-20; Food industry science and technology has been reported among 20,3 (1999) 27-28 fresh pig watery blood has been separated condition and the effect that the edible protein zymolyte that makes is used activated carbon decolorizing; Grain and grease have been reported the Soyprotein peptide hydrolysate that soybean protein hydrolysis the is made condition and the effect of activated carbon decolorizing among 3 (2001) 5-7.Disclosed the method for the soy sauce that makes with dregs of beans, wheat bran and wheat being used charcoal treatment among the patent documentation CN 1291449A; Disclosed the method that ion exchange resin is used for the syrup adsorption bleaching among the patent documentation CN 1336441A; Disclosed the protein hydrolyzate H that hydrolysis makes to the degreasing dried silkworm chrysalis meal among the patent documentation CN 1397192A
2O
2The method of oxidative decoloration.The discoloration method that above patent documentation and open source literature disclosed all belongs to the post processing discoloration method, needs independently unit operations of at least one special equipment and on implementing process.Moreover, the decoloring ability of above the whole bag of tricks is limited.Wherein, also can reduce the productive rate of purpose product with various absorption method decolourings.
Obviously, suppressing coloring matter and generate in the protein digestion process, is the optimum method that solves protein digestion thing color problem.But, do not see open so far as yet or patent documentation relates to carry out the details that pigment suppresses in the protein digestion process.
Summary of the invention
The purpose of this invention is to provide a kind of method that the enzymolysis liquid look becomes that suppresses when the enzymatic hydrolysis of soybean protein Preparation is less than 10 amino acid whose oligo peptides under the pH value gradual change condition of exogenously added alkali not.
Find after deliberation, under the pH value gradual change condition of exogenously added alkali not, when soybean protein at alkali protease and acid protease or/and under the synergy of neutral proteinase during enzymolysis, it is main relevant with Mei Lade side reaction (Maillard reaction) that the look of enzymolysis liquid becomes.Maillard reaction that is carbonyl ammonia react are meant that amino in amino acid and the protein and reduced sugar or reduced sugar decompose the reaction that takes place between the carbonyl in the thing.This reaction is final to be generated the macromolecular substances melanoidin of brown even black or claims to intend melanocyte.In hydrolysate of soybean protein, the reduced sugar and the reduced sugar decomposition thing that participate in Maillard reaction come from the compound sugar that contains in the soybean protein.Therefore, the carbonyl that the present invention utilizes a small amount of look that joins in advance in the enzymatic vessel to become inhibitor (oxidant or reducing agent) discharges soybean protein in enzymolysis process compound sugar decomposes or is converted into hydroxyl, reach the inhibition Maillard reaction, and then suppress the purpose that the enzymolysis liquid look becomes.
Technical scheme of the present invention and implementation step are:
In the common fermentation jar that has mechanical stirring device, add an amount of running water, and require to wherein adding a certain amount of soybean protein according to concentration of substrate.Start mechanical stirring arm, make it to stir slurries, or/and water vapour directly contacts the reacting slurry temperature is mentioned assigned temperature by water leg simultaneously with suitable speed.With pH meter and the pH meter determination of electrode enzyme digestion reaction slurries acid-base value before that is fixed in the fermentor.Then, the protease and the look that add metering in fermentation tank become inhibitor (oxidant or reducing agent), the beginning enzyme digestion reaction.At last, with the variation of pH meter monitoring enzymolysis liquid acid-base value, and by different raw material degradation rate, the protein degrees constantly of sampling (enzymolysis liquid that stirs) analysis.Enzyme digestion reaction boils deactivation in 5-10 minute with enzymolysis liquid at 95 ℃ after finishing.
Be fit to alkali protease of the present invention as: alkaline mold protease (derives from aspergillus oryzae, use pH value 6.5-8.5, serviceability temperature 45-60 ℃), alkaline bacterial protease (derives from bacillus licheniformis, use pH value 6.5-8.5, serviceability temperature 55-70 ℃), the Alcalase alkaline bacterial protease (derives from hay bacillus, use pH value 6.5-8.5, serviceability temperature 55-70 ℃).
Be fit to acid protease of the present invention as acid mold protease (derive from aspergillus niger, use pH value 2-5.0, serviceability temperature 45-55 ℃), pepsin (deriving from the animal stomach, use pH value 1.0-3.0, serviceability temperature 30-40 ℃).
Be fit to neutral proteinase of the present invention as: neutral mold protease (derives from aspergillus oryzae, use pH value 5.0-7.5, serviceability temperature 50-65 ℃), neutral bacterialprotease (derive from bacillus subtilis, use pH value 4.5-7.0, serviceability temperature 45-60 ℃), Protamex
TMBacterialprotease (belong to bacillus protease complex, use pH value 5.5-7.5, serviceability temperature 55-60 ℃).
In the present invention, alkali protease will be with acid protease or/and neutral proteinase be used.If allow the serviceability temperature scope close, then acid protease is or/and neutral proteinase can add enzymatic vessel simultaneously with alkali protease.If acid protease is or/and the permission serviceability temperature of neutral proteinase is lower than the serviceability temperature of alkali protease, (the pH value that is enzymolysis liquid stops after the decline) adds acid protease again or/and neutral proteinase after then should disappearing substantially at the enzymolysis activity of alkali protease in fermentation tank.And hydrolysis temperature adjusted to acid protease or/and the suitable serviceability temperature of neutral proteinase.
Or/and neutral proteinase is used in the scheme, alkali protease can be a kind of enzyme, also can be compound alkali protease at above said alkali protease and acid protease.Equally, acid protease also can be that combination of acidic protease is or/and compound neutral proteinase or/and neutral proteinase can be a kind of enzyme.
In order to give full play to alkali protease and acid protease or/and the coordinated enzymatic hydrolysis effect of neutral proteinase, alkali protease, acid protease are or/and the injected volume of neutral proteinase (is expressed as enzyme solutions and substrate ratio in the present invention, or the enzyme solid accounts for the percentage of substrate weight) can determine by the Utility Engineers's customary way in this field according to its vigor difference.The selection of hydrolysis temperature depends primarily on the serviceability temperature of enzyme.In the mode that alkali protease and acid protease or/and neutral proteinase add simultaneously, it is fixed that hydrolysis temperature should come according to the minimum enzyme of serviceability temperature.In the mode that alkali protease and acid protease or/and neutral proteinase add step by step, hydrolysis temperature can be adjusted according to the permission serviceability temperature of employed enzyme of different enzymolysis stage.According to the present invention, the range of choice of enzyme digestion reaction temperature is 30-70 ℃.
Be fit to soybean protein raw material of the present invention (substrate) as soybean protein isolate, FSPC, defatted soy flour, big dregs of beans and soya-bean cake etc.Wherein, preferred soybean protein isolate and FSPC.According to the present invention, the range of choice of concentration of substrate is the 20-100 grams per liter.When concentration of substrate was too high, the enzymolysis activity of enzyme was low, was difficult to obtain satisfied degradation of substrates rate.On the other hand, concentration of substrate is too low, though the enzymolysis activity height of enzyme can increase in the downstream processes difficulty of evaporating, concentrating enzymolysis liquid.When under agitation adding an amount of soybean protein isolate or defatted soy flour in the running water and be heated to aforesaid hydrolysis temperature scope, the initial pH value of soy protein slurry can reach 7-7.5, can satisfy the requirement of alkali protease to acid-base value.
Soybean protein suitable time scope of enzymolysis under above-described condition is 2-24 hour.In enzymolysis process, degradation of substrates rate and protein degree increase with the prolongation of enzymolysis time, but the color of zymolyte also changes dark with the prolongation of enzymolysis time.Enzymolysis time is unfavorable for obtaining satisfied degradation of substrates rate and protein degree very much in short-term.On the contrary, if enzymolysis time is long, then not only be unprofitable to suppress the look change of enzymolysis liquid, and enzymolysis liquid is rotten under microbial action easily.
About utilizing alkali protease and acid protease under the pH value gradual change condition of exogenously added alkali not or/and the coordinated enzymatic hydrolysis effect of neutral proteinase obtains the implementation method of oligopeptides zymolyte and effect from soybean protein has done the patent of invention early stage at us and set forth (Chinese invention patent application number 03110987.X).Therefore, key of the present invention is that look becomes selection of inhibitors and use.The particularly important is, look becomes inhibitor should belong to the medicament that allows use in the food processing, and its consumption should not influence the activity of the enzyme that uses, and more can not be higher than limit the quantity of (the considering by dried zymolyte) of alimentary codex.
The look that the present invention adopts becomes inhibitor and belongs to Oxidizing and Reducing Agents.Wherein oxidant refers to H
2O
2, reducing agent refers to Na
2SO
3, Na
2S
2O
5And NaHSO
3Above medicament uses in food and has obtained China's " food additives use sanitary standard " and (GB2760-1996) permit: as Na
2S
2O
5(sodium pyrosulfite), Na
2SO
3(sodium sulfite) and NaHSO
3(sodium hydrogensulfite) can be used for preserved fruit, biscuit, glucose, sugar, rock sugar, maltose, candy, liquid glucose, bamboo shoots, mushroom, canned mushroom, liquid and solid products such as preserved fruit, adding limits the quantity of is not higher than 6/1000ths, generally in thousand/nought point four below five.And hydrogen peroxide (H
2O
2) to be used for raw milk fresh-keeping, adds that to limit the quantity of be 2.0 milliliters/liter of 30% hydrogen peroxide.
Owing to will not do desalting processing according to enzymolysis liquid, therefore look be become inhibitor Na with gained of the present invention
2SO
3, Na
2S
2O
5And NaHSO
3Consumption be limited to thousand/below 0. 5.For hydrogen peroxide, limiting consumption is below 2/1000ths, takes off because can borrow heating condition to divide voluntarily in the downstream processes of hydrogen peroxide behind enzymolysis and enzymolysis.In above-mentioned amount ranges, various looks become inhibitor does not all influence enzymatic activity.
Implementation result of the present invention is the available substrates degradation rate, and protein degree and look become inhibiting rate and weigh.
The degradation of substrates rate is defined as the percentage that is accounted for the soybean protein raw material weight that feeds intake by the soybean protein raw material weight of enzymolysis; Degree of hydrolysis is defined as the percentage that the peptide bond quantity of opening accounts for the total peptide bond quantity of protein molecule in enzymolysis process.
The assay method of degradation rate is as follows: enzymolysis liquid is centrifugal 10min under 4000r/min, supernatant liquor and solids separation, solid insoluble is dried to constant weight under 110 ℃, weighs.Be calculated as follows the degradation of substrates rate then:
Wherein, the soybean protein inventory is converted to butt.The water content of soybean protein is measured after being dried to constant weight under 110 ℃.The degradation of substrates rate is high more, the one way utilization rate height of expression raw material.
Determination of Hydrolysis Degree of Protein adopts formol titration, concrete assay method is as follows: with enzymolysis liquid centrifugal 10min under 4000r/min, get the 5ml supernatant liquor, NaOH solution with 0.1M is titrated to pH=8.2, add the formalin (used formalin is titrated to pH=8.2 with NaOH earlier) of 5ml 37%, the NaOH with 0.1M is titrated to pH=8.2 again.Record is titrated to pH-8.2 institute alkali consumption to solution after adding formaldehyde.The pH value is measured and is used accurate pH meter.Calculate the enzymolysis degree of protein then according to following formula:
Wherein, the method for the thorough hydrolysis of soybean protein be with soybean protein in 6M HCl in 105-110 ℃ of following hydrolysis 24 hours.The degree of hydrolysis height of protein, the mean molecule quantity of expression zymolyte is little.
In the present invention, look becomes the inhibition effect represents that with look change inhibiting rate look becomes inhibiting rate employing spectrophotometry.Concrete assay method is as follows: the centrifuged supernatant of getting enzymolysis liquid is with 0.22 micron filtering with microporous membrane, then with ultraviolet-visible spectrophotometer at its absorbance of wavelength 400nm place survey.The pigment inhibiting rate calculates with following formula:
In the formula: A
0Become the enzymolysis liquid absorbance of inhibitor for additive color not; A is the enzymolysis liquid absorbance after additive color becomes inhibitor.
Benefit of the present invention is, utilize alkali protease and acid protease or/and the coordinated enzymatic hydrolysis effect of neutral proteinase, under the pH value gradual change condition of exogenously added alkali not the degradation rate of soybean protein raw material is reached more than 70%, the enzymolysis degree of protein reaches more than 20%.Simultaneously, utilizing look to become inhibitor can make the look change inhibiting rate of enzymolysis liquid reach more than 50%.Utilize the present invention, can save desalination and bleaching process in the zymolyte downstream processes, help reducing the production cost of oligopeptides zymolyte.
The specific embodiment
Below be described in detail specific embodiments of the invention.
Embodiment 1 (comparative example)
8g soybean protein isolate (butt weight) and the adding of 200ml running water are had in the enzymatic vessel of water leg, under agitation the temperature in the enzymatic vessel is brought up to 60 ℃.The pH value that records feed liquid in the enzymatic vessel by pH meter is about 7.0.Then, in enzymatic vessel, add 120 μ l Alcalase alkali proteases (2.4L, sign vigor are the 2.4AU/ gram, and Denmark NOVO company produces) and 0.48 gram aspergillus niger acid protease (enzyme activity 3000u/g), under constantly stirring, begin enzymolysis.In this example, concentration of substrate is 40 gram soybean protein isolate/premium on currency, Alcalase solution is 15 μ l/ gram soybean protein isolate with the ratio of substrate, the aspergillus niger acid protease accounts for substrate 6% (is benchmark with substrate butt weight), total enzymolysis time is 6 hours, the degradation of substrates rate is about 71%, protein degree about 22%.The enzymolysis clear liquid is a dark brown.
Embodiment 2 (according to the present invention)
Repeat embodiment 1, but when in enzymatic vessel, adding Alcalase and aspergillus niger acid protease, to wherein adding the hydrogen peroxide (promptly 30% hydrogen peroxide is 4.8 milligrams) that is equivalent to soybean protein raw material dry basis 0.12 ‰.Then the degradation of substrates rate still is about 71%, and protein degree still is about 22%, but enzymolysis clear liquid color obviously shoals, and look becomes inhibiting rate and reaches 29.3%.
Embodiment 3 (according to the present invention)
Repeat embodiment 2, but the hydrogen peroxide that adds is followed successively by 0.06 ‰, 0.25 ‰, 0.50 ‰ and 1.9 ‰ of soybean protein raw material dry basis.Then under these four kinds of situations, degradation of substrates rate and protein degree still all remain on about 71% and 22%, but enzymolysis clear liquid color shoals along with the increase of hydrogen peroxide addition, and look becomes inhibiting rate and is followed successively by 25%, 32.4%, 35% and 51.8%.In being 1.9 ‰ enzymolysis liquid, the hydrogen peroxide addition do not detect remaining hydrogen peroxide with iodimetry.Illustrate that hydrogen peroxide decomposes through 95 ℃ high-temperature inactivation.
Embodiment 4 (according to the present invention)
Repeat embodiment 2, but use Na
2SO
3(sodium sulfite) replaces hydrogen peroxide to become inhibitor as look.Na
2SO
3Addition be equivalent to 0.12 ‰, 0.25 ‰, 0.48 ‰ of soybean protein raw material dry basis successively.Then under these three kinds of situations, degradation of substrates rate and protein degree all remain on about 71% and 22%, but enzymolysis clear liquid color is along with Na
2SO
3The increase of addition and shoaling, look become inhibiting rate and are followed successively by 18.4%, 22.0% and 24.2%.
Embodiment 5 (according to the present invention)
Repeat embodiment 2, but use NaHSO
3(sodium hydrogensulfite) replaces hydrogen peroxide to become inhibitor as look.NaHSO
3Addition be equivalent to 0.06 ‰, 0.12 ‰, 0.25 ‰ and 0.50 ‰ of soybean protein raw material dry basis successively.Then under these four kinds of situations, degradation of substrates rate and protein degree all remain on about 71% and 22%, but enzymolysis clear liquid color is along with NaHSO
3The increase of addition and shoaling, look become inhibiting rate and are followed successively by 19.0%, 20.6% and 22.0% and 27.1%.
Embodiment 6 (according to the present invention)
Repeat embodiment 2, but use Na
2S
2O
5(sodium pyrosulfite) replaces hydrogen peroxide to become inhibitor as look.Na
2S
2O
5Addition be equivalent to 0.06 ‰, 0.12 ‰, 0.25 ‰ and 0.48 ‰ of soybean protein raw material dry basis successively.Then under these four kinds of situations, degradation of substrates rate and protein degree all remain on about 71% and 22%, but enzymolysis clear liquid color is along with Na
2S
2O
5The increase of addition and shoaling, look become inhibiting rate and are followed successively by 15.2%, 17.9%, 21.4% and 25.8%.
Claims (2)
1. one kind can alleviate the hydrolysate of soybean protein method that look becomes, it is characterized in that, enzyme digestion reaction, carries out in the pH value gradual change environment of exogenously added alkali not or/and under the synergy of neutral proteinase at alkali protease and acid protease, also adds the reducing agent Na below 5/1000ths in the enzymatic vessel
2SO
3, Na
2S
2O
5And NaHSO
3The oxidant H that perhaps adds 30% hydrogen peroxide below 2/1000ths
2O
2, suppress the generation of coloring matter; Enzyme digestion reaction is at 30-70 ℃, and concentration of substrate 20-100 grams per liter carries out under the hydrolysis time 2-24 hour condition; Soybean protein as the enzymolysis raw material refers to FSPC, soybean protein isolate, defatted soy flour, any in big dregs of beans and the soya-bean cake.
2. a kind of hydrolysate of soybean protein method that can alleviate the look change according to claim 1, it is characterized in that, alkali protease refers to alkaline fungous enzyme, alkalescence bacterial enzyme and any compound thereof, acid protease refers to acid fungous enzyme, pepsin and any compound thereof, neutral proteinase refers to neutral fungous enzyme, neutral bacterial enzyme and any compound thereof.
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| CNB031341888A CN1256032C (en) | 2003-08-28 | 2003-08-28 | Soybean protein enzymolysis method capable of reducing colour change |
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| CN1256032C true CN1256032C (en) | 2006-05-17 |
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| CN102260725A (en) * | 2010-05-27 | 2011-11-30 | 安徽丰原生物化学股份有限公司 | Starch raw material preprocessing method |
| CN102260726B (en) * | 2010-05-27 | 2013-07-10 | 中粮生物化学（安徽）股份有限公司 | Pretreatment method of starchiness raw material |
| CN108208345A (en) * | 2016-12-15 | 2018-06-29 | 丰益(上海)生物技术研发中心有限公司 | A kind of technique that fermented bean dregs production is carried out the environment suitable for edible fat production factory |
| CN109567117A (en) * | 2018-11-20 | 2019-04-05 | 安徽强旺调味食品有限公司 | A kind of cryopreservation answers heat-staple flavouring and preparation method thereof |
| CN109651618B (en) * | 2018-12-12 | 2020-06-05 | 东北林业大学 | Soybean protein-based water-based acrylic resin and preparation method and application thereof |
| CN115777941B (en) * | 2022-12-17 | 2023-08-29 | 寿光市沃野化工有限责任公司 | Method for preparing compound amino acid by soybean enzymolysis |
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