CN107400690B - Rice protein preparation method - Google Patents
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- CN107400690B CN107400690B CN201610335290.4A CN201610335290A CN107400690B CN 107400690 B CN107400690 B CN 107400690B CN 201610335290 A CN201610335290 A CN 201610335290A CN 107400690 B CN107400690 B CN 107400690B
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- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 79
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- 238000003257 protein preparation method Methods 0.000 title description 2
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- 108010064851 Plant Proteins Proteins 0.000 description 3
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- 108090001060 Lipase Proteins 0.000 description 2
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- 235000021312 gluten Nutrition 0.000 description 2
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
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- 229910052799 carbon Inorganic materials 0.000 description 1
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
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- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
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- VUZPPFZMUPKLLV-UHFFFAOYSA-N methane;hydrate Chemical compound C.O VUZPPFZMUPKLLV-UHFFFAOYSA-N 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 239000004223 monosodium glutamate Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 108090000021 oryzin Proteins 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
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- 229940116540 protein supplement Drugs 0.000 description 1
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- 210000001938 protoplast Anatomy 0.000 description 1
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- 238000007670 refining Methods 0.000 description 1
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- 235000019991 rice wine Nutrition 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明涉及一种大米蛋白的制备方法，包括将大米蛋白粉与水，以木瓜蛋白酶及中性或碱蛋白酶的组合酶解处理，采用本发明的制备方法可改善大米蛋白的溶解度，从而获得高蛋白质收率的大米蛋白产品。The invention relates to a preparation method of rice protein, which comprises enzymolysis treatment of rice protein powder and water with a combination of papain and neutral or alkaline protease. The preparation method of the invention can improve the solubility of rice protein, thereby obtaining high Rice protein products for protein yield.
Description
Technical Field
The invention relates to a preparation method of rice protein.
Background
The rice protein is a high-quality plant protein, contains rich essential amino acids, has an amino acid composition mode similar to the recommended mode of World Health Organization (WHO) and Food and Agriculture Organization (FAO) of the United Nations, and is easy to digest and absorb by human bodies. In addition, the rice protein has the characteristic of hypoallergenic property, and is very suitable to be used as a protein supplement for special people, especially patients. However, due to the particularity of the structure of the major component gluten of the rice protein, and a large number of sulfydryl and disulfide bonds exist among molecules and in molecules, the rice protein is difficult to dissolve in water under a neutral condition, and the application of the rice protein in the food and health care product industry is influenced, so that the key for solving the problem is to improve the solubility of the rice protein.
The method for improving the solubility of the protein comprises a physical method, a chemical method and a biological method, wherein the enzymatic modification has the characteristics of strong specificity, mild conditions, no toxic or side effect and the like, and is widely applied to the modification research of the solubility of various plant proteins. Generally, protease such as soybean protein, peanut protein, rice bran protein and the like is selected, so that the peptide chain of protein molecules can be hydrolyzed and broken, the solubility of the protein is improved, and the effect of promoting the solubility of the insoluble plant protein powder is achieved.
Chinese patent CN1417344C, published in 2003, discloses a new process for extracting rice protein from rice grains, which is characterized in that the ratio of the byproduct rice grains of a monosodium glutamate factory to complex enzyme is 1000: 1.5-4.5 by weight; wherein the complex enzyme consists of 0.05 to 0.15 percent of amylase, 0.025 to 0.075 percent of lipase, 0.025 to 0.075 percent of cellulase and 0.05 to 0.15 percent of protease; the process comprises the following steps: adding rice wine into a container, regulating the pH value to 3 by using hydrochloric acid, regulating the temperature to 35-40 ℃, adding cellulase according to the proportion, reacting for 30 minutes, adding 30% NaOH, regulating the pH value to 5.0-5.5, adding lipase, reacting for 30 minutes, adding NaOH to regulate the pH value to be neutral 7.0, adding amylase and protease, reacting for 20 minutes, heating to 60-70 ℃, reacting for 30 minutes, filtering, adding dilute alkali solution with the pH value of 9.5 into filter residues, dissolving, controlling the temperature to be below 60 ℃ to obtain protein solution, regulating the pH value to 5.5 by using acid, and precipitating the protein to obtain the concentrated rice protein with the content of 75%.
Another chinese patent CN1257984C, published in 2006, discloses, for example, a method for preparing rice protein peptides, which is characterized in that rice dregs and water are mixed at a weight ratio of 1: 4-6, the temperature is 55-60 ℃, the temperature is kept for 30 minutes, then, according to the activity of an enzyme preparation of 50,000U/mg, alpha-amylase and neutral protease are added in an amount of 0.05-0.1% of the weight of the rice dregs, respectively, wherein the weight ratio of the alpha-amylase to the neutral protease is 1: 1, the mixture is stirred at a ph of 6.5-7.5 and a temperature of 50-60 ℃ for 5-7 hours, the reaction product is filtered, and the obtained liquid is spray-dried to obtain a white powder product, wherein the content of the rice protein peptides is about 80%.
At present, a better preparation method of rice protein still needs to be continuously developed to solve the problem of difficult dissolution of rice protein and improve the protein yield.
Disclosure of Invention
In order to solve the problem that the rice protein is difficult to dissolve and improve the protein yield, the invention provides a preparation method of the rice protein, the invention utilizes the compound enzyme composed of papain and neutral or alkaline protease to carry out enzymolysis on the rice protein powder, the solubility of the rice protein can be obviously improved, the solubility of the rice protein is improved by more than 60 percent, and the protein yield of the obtained rice protein product is more than 90 percent.
In one aspect, the present invention provides a method for preparing rice protein by improving the solubility of rice protein powder to increase the protein yield, which is characterized in that the rice protein powder is mixed with water in a proper proportion and treated by an enzyme combination, wherein the enzyme combination is a complex enzyme consisting of papain and neutral protease or a complex enzyme consisting of papain and neutral protease, and the weight ratio of the papain to the neutral or alkaline protease is 1: 1-3.
In a specific embodiment of the invention, the weight ratio of the papain to the neutral protease or the papain to the alkaline protease in the complex enzyme is 1: 2.
according to the invention, the enzyme combination further comprises a flavour enzyme to improve bitterness.
In one embodiment of the invention, the flavourzyme is a combination of Aspergillus oryzae (Aspergillus oryzae) and aminopeptidases (aminopeptidases).
In one embodiment of the present invention, the method of the present invention is characterized by comprising the steps of: the rice protein powder is prepared by adding water according to a proper proportion, uniformly stirring, adjusting the pH value of the obtained solution to be neutral to alkaline, adding the enzyme combination for enzymolysis, continuously stirring for a period of time, heating to inactivate enzyme, filtering, concentrating and drying.
In another aspect, the present invention provides a rice protein product made by the method of the present invention, wherein the rice protein product has a rice protein solubility of greater than 60% and a rice protein yield of greater than 90%.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a sample" includes a plurality of such samples and equivalents thereof known to those skilled in the art.
As used herein, the term "Papain" (Papain), or simply Papain, is a cysteine protease present in the fruit of papaya. Papain plays an important role in tenderizing meat, is endoprotease and has protease and esterase activities.
As used herein, the term "Neutral Protease" refers to a class of proteases having an optimum pH of 6.0-7.5, a molecular weight of 35000-40000 and an isoelectric point of 8-9. The endoprotease is prepared by deep fermentation culture of the bred bacillus subtilis and refining by an advanced extraction process, and can decompose macromolecular protein into products such as polypeptide, amino acid and the like to form unique hydrolysis flavor. The neutral protease is a protease preparation which is originally discovered and widely applied to industrial production, and can be used in the baking food industry, the synthesis of aspartame, the bitter taste removal of protein hydrolysate and the like.
The term "Alkaline Protease" as used herein refers to a proteolytic enzyme obtained by submerged fermentation, extraction and purification of a Bacillus subtilis strain cultivated by bacterial protoplast mutagenesis, and belongs to the serine fragile exo-high Alkaline Protease which hydrolyzes a peptide chain of a protein molecule under Alkaline conditions to form a polypeptide or an amino acid.
The term "Aspergillus oryzae" as used herein is a flavourzyme which comprises both endoprotease and exopeptidase activities and is used to remove the bitter-peptide chain of low-hydrolysis products and degrade it completely into amino acids.
The term "aminopeptidases" as used herein is an exopeptidase which selectively cleaves amino acid residues from the amino terminus of proteins or peptide chains, leaving the amino acids free one by one from the N-terminal order of the polypeptide chain.
The rice protein is insoluble in water under neutral conditions due to the fact that a large number of sulfydryl and disulfide bonds exist between molecules and in molecules of the rice protein which is the main component of the gluten. According to the invention, the rice protein anti-degradation enzyme composition is characterized in that an enzyme composition comprising a compound enzyme consisting of papain and neutral protease or a compound enzyme consisting of the papain and the neutral protease is used, so that the problem of difficult dissolution of the rice protein is solved, and the protein yield is improved.
The present invention provides a method for producing rice protein using a specific combination of enzymes to improve the solubility of rice protein and to increase the yield of protein. The method comprises the steps of mixing rice protein powder and water in a proper proportion, and treating the mixture by using an enzyme combination, wherein the enzyme combination is a compound enzyme consisting of papain and neutral protease or a compound enzyme consisting of the papain and alkaline protease. However, the enzyme treatment often has a bitter taste, and therefore, the addition of flavor enzymes improves the bitter taste.
In the invention, the ratio of the papain to the neutral or alkaline protease is preferably 1: 1-3, preferably 1: 2. flavor enzymes may be added as necessary to improve bitterness.
According to the present invention, the comparison between table 2 and table 3 in the following examples shows that the protein yield of the compound enzyme of papain and neutral protease or the compound enzyme of papain and neutral protease of the present invention is as high as 90% or more, which is much higher than that of the compound enzyme alone, wherein the protein yield of the compound enzyme of papain, neutral protease, alkaline protease, aspergillus oryzae or aminopeptidase alone is less than 75%. While Aspergillus oryzae has a bitter taste-improving effect, it was unexpectedly found that Aspergillus oryzae in combination with aminopeptidase has the best bitter taste-improving effect.
The rice protein powder obtained by the method of the present invention can be obtained by any conventional method including, but not limited to, physical or chemical methods. For example, the enzymatic hydrolysis of the method of the present invention can be carried out by pulverizing rice, purifying, drying, and the like, which are generally used for protein extraction, to obtain a powder. According to one embodiment of the invention, the rice is crushed and primarily treated to obtain rice protein powder, and water is added according to a proper proportion and uniformly stirred, for example, the weight ratio of the water to the material is 1: 3-10, preferably 1:4, and adjusting the resulting solution to neutral or slightly basic, e.g., about ph 8.0. The enzymatic hydrolysis is generally carried out at a relatively high temperature, for example, about 50-60 deg.C, preferably about 55 deg.C, with continuous stirring, heating to a high temperature, for example, about 85 deg.C, inactivating the enzyme, filtering, concentrating, and drying to obtain rice protein product. The resulting product can be filtered, concentrated, dried, and further purified by conventional methods.
The following examples are provided to illustrate the present invention and are not to be construed as limiting the scope of the invention in any way.
Examples
Example 1
Grinding rice for primary treatment to obtain rice protein powder, adding water according to a proper proportion, and uniformly stirring, wherein the weight ratio of the materials to the water is 1: 3-10, adjusting the pH of the resulting solution to slightly alkaline, e.g., about pH8.0, adding various enzyme combinations as shown in Table 1 below, performing enzymolysis at a higher temperature, e.g., about 50-60 deg.C, continuously stirring, heating to about 85 deg.C to inactivate enzyme, filtering, concentrating, drying to obtain rice protein product, and testing.
TABLE 1 enzyme combinations for the respective experimental groups
| Group of | Contents of enzyme combination |
| Experimental group 1 | Composite protease A (papain + alkaline protease) + flavor enzyme A (aspergillus oryzae) |
| Experimental group 2 | Compound protease A (papain + alkaline protease) + flavor enzyme B (Aspergillus oryzae + aminopeptidase) |
| Experimental group 3 | Compound protease B (papain + neutral protease) + flavor enzyme A (Aspergillus oryzae) |
| Experimental group 4 | Compound protease B (papain + neutral protease) + flavor enzyme B (Aspergillus oryzae + aminopeptidase) |
| Experimental group 5 | Blank space |
Example 2
The rice protein product obtained in example 1 was examined by the following test methods.
1. Solubility test method
Performing hydrolysis test on a certain amount of raw materials X according to a test process, performing centrifugal separation after hydrolysis, collecting supernatant, and drying to obtain dried weight Y with solubility of
2. Amino nitrogen determination experimental method
The amphoteric effect of amino acid is utilized, formaldehyde is added to fix the alkalinity of amino acid, carboxyl is displayed with acidity, and the quantification is carried out after titration by sodium hydroxide standard solution. Firstly, precisely sucking 1ml of sample to be detected, adding 79ml of distilled water, starting a magnetic stirrer, titrating to pH8.2 by using a sodium hydroxide standard solution with the concentration of 0.05mol/l, precisely adding 10ml of formaldehyde, uniformly mixing, continuously titrating to pH9.2 by using a sodium hydroxide standard solution with the concentration of 0.05mol/l, and recording the volume of the consumed sodium hydroxide standard solution after adding the formaldehyde. Then, 80ml of distilled water was taken, 10ml of formaldehyde was added precisely, and the solution was titrated to pH9.2 with a sodium hydroxide standard solution having a concentration of 0.05mol/l, and the volume of the sodium hydroxide standard solution consumed was recorded as an experimental blank. Calculated according to the following formula:
v1: determining the volume (ml) of the sodium hydroxide standard solution consumed by the solution to be determined after adding formaldehyde
V2: actual blank volume (ml) of sodium hydroxide standard solution consumed after addition of Formaldehyde
C: concentration (mol/l) of sodium hydroxide Standard solution
W: weighing the sample
0.0141: the mass of amino nitrogen expressed in grams, corresponding to 1ml of standard sodium hydroxide solution
3. Protein quantitative test (Kjeldahl method)
Heating and digesting the sample together with concentrated sulfuric acid and a catalyst to hydrolyze protein, wherein carbon and hydrogen are oxidized into carbon dioxide and water to escape, organic nitrogen in the sample is converted into ammonia and sulfuric acid to combine into ammonium sulfate, then adding alkali for distillation to evaporate ammonia, absorbing with boric acid and then titrating with a standard hydrochloric acid solution, and calculating the content of the protein according to the consumption volume of the hydrochloric acid. Firstly, accurately weighing 0.2-2 g (liquid 1-2ml) of sample, carefully transferring into a dry and clean digestion bottle, then adding 0.5 g of copper sulfate, 9.5 g of potassium sulfate and 20ml of concentrated sulfuric acid, shaking up lightly, and then placing on an electric furnace to heat with small fire. After the contents in the bottle are completely carbonized and the foam stops generating, the firepower is increased, the liquid in the bottle is kept slightly boiling until the liquid turns blue-green and transparent, and then the liquid is continuously heated and slightly boiled for 1-2 hours (the time is determined by the protein content). Then, after the solution is cooled, 200ml of pure water is added, the solution is moved into a distillation flask, 150ml of 40% NaOH solution is added, the mouth of the distillation flask is tightly plugged, the lower end of a condenser tube is inserted below the liquid level of an absorption flask, 50ml of 4% boric acid solution and 2-3 drops of mixed indicator are contained in the absorption flask, and heating and distillation are carried out until ammonia is completely evaporated (the distillate is more than 250 ml). Titration to gray with 0.1mol/L HCl standard solution was performed while blanking. Calculated according to the following formula:
v1 volume (ml) of hydrochloric acid standard solution consumed by sample
V2 volume (ml) of actual blank hydrochloric acid standard solution
W: weighing the sample
0.014: the mass of amino nitrogen in grams equivalent to 1ml of hydrochloric acid standard
C: concentration of hydrochloric acid Standard solution (mol/l)
F: factor of nitrogen conversion to protein, F6.25
The test results are summarized in table 2, the protein yield of each experimental group is up to more than 90%, and the solubility is obviously improved to more than 60%. Particularly, the test group 4 (compound protease B + flavor enzyme B) is preferred, the protein hydrolysis degree is more than 98%, the solubility is more than 70%, and no bitter taste exists.
TABLE 2 examination results of the respective experimental groups
| Experimental group | AN％(g/100mg) | Protein yield% | Solubility in water | Flavor (I) and flavor (II) |
| Experimental group 1 | 0.3397 | 96.5％ | 68.9％ | Light rice flavor and obvious bitter taste |
| Experimental group 2 | 0.3739 | 97.5％ | 72.0％ | Light rice flavor and bitter taste |
| Experimental group 3 | 0.3654 | 96.2％ | 63.8％ | Light rice flavor and slight bitter taste |
| Experimental group 4 | 0.3839 | 98.6％ | 73.5％ | Light rice fragrance and slight sweet taste |
| Experimental group 5 | 0.0093 | 0.4％ | 6.1％ | Is tasteless |
The same method steps are adopted, single enzyme preparation is used for enzymolysis, the results are shown in table 3 after inspection, the protein yield of each group is more than 40-75%, and obviously, the enzyme combination obviously improves the solubility of the rice protein powder and improves the solubility of the rice protein to more than 60%. Particularly, the test group 4 (compound protease B + flavor enzyme B) is preferred, the protein hydrolysis degree is more than 98%, the solubility is more than 70%, and no bitter taste exists.
TABLE 3 test results for the individual enzyme preparation groups
| Group number | Using enzyme preparations | AN％(g/100mg) | Protein yield% | Flavor (I) and flavor (II) |
| 1 | Papain | 0.2450 | 69.7％ | Light rice flavor and bitter taste |
| 2 | Alkaline enzyme protease | 0.2733 | 73.5％ | Light rice flavor and heavy bitter taste |
| 3 | Neutral protease | 0.2061 | 63.2％ | Light rice flavor and slight bitter taste |
| 4 | Aspergillus oryzae | 0.1468 | 43.5％ | Light rice flavor and no bitter taste |
In conclusion, the compound enzyme combining papain and neutral or alkaline protease can improve the solubility of the rice protein and obviously improve the protein yield of the rice protein (both are as high as more than 90 percent). Especially preferred are papain and neutral protease, and flavourzyme B), and the protein hydrolysis degree is more than 98%, the solubility is more than 70%, and the product has no bitter taste. The same method steps are adopted, single enzyme preparation is used for enzymolysis, the results are shown in table 3 after inspection, the protein yield of each group is more than 40-75%, and obviously, the enzyme combination can obviously improve the solubility of the rice protein powder and improve the solubility of the rice protein to be more than 60%. Particularly, the test group 4 (compound protease B + flavor enzyme B) is preferred, the protein hydrolysis degree is more than 98%, the solubility is more than 70%, and no bitter taste exists.
The above embodiments are merely exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
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| CN102524736A (en) * | 2011-12-27 | 2012-07-04 | 广州合诚实业有限公司 | Method for preparing functional flavor-developing base material from rice protein |
| CN103789383A (en) * | 2014-03-11 | 2014-05-14 | 江苏联海生物科技有限公司 | Rice peptide production method |
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| CN102524736A (en) * | 2011-12-27 | 2012-07-04 | 广州合诚实业有限公司 | Method for preparing functional flavor-developing base material from rice protein |
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