Method for confirming that proteolytic enzyme has been removed, and applications thereof

Abstract

The present invention addresses the problem of providing: a method for confirming that a proteolytic enzyme has been removed from a protein-containing composition; a method for producing a protein-containing composition from which a proteolytic enzyme has been removed; and a method for confirming that an autolysate of a proteolytic enzyme is not contained in a final product. This method for confirming that a proteolytic enzyme has been removed comprises: using an ultrafiltration membrane having a molecular weight cutoff of at most 10 kDa to exclusively ultrafilter a proteolytic enzyme; and measuring the proteolytic enzyme activity and / or protein content of a permeated liquid and / or a non-permeated liquid.

Description

Method for confirming removal of proteolytic enzymes and its application

[0001] The present invention relates to a method for confirming the removal of protease, a method for producing a protein-containing composition from which enzymes have been removed, and a method for confirming that the final product does not contain autolysis products of the protease.

[0002] Cow's milk proteins are often used in the manufacture of infant formula. When producing infant formula for infants at risk of developing cow's milk allergy, the milk proteins are treated with enzymes to remove allergens. Therefore, infant formula for infants at risk of developing cow's milk allergy contains partial hydrolysates of milk proteins.

[0003] In the step of purifying the partially hydrolyzed milk protein, filtration may be performed using an ultrafiltration membrane, etc. For example, Patent Document 1 discloses a method for producing infant formula, which includes, as a purifying step, recovering a filtrate using a membrane having a molecular cutoff in the range of 10 to 100 kDa or size exclusion chromatography.

[0004] International Publication No. 2019 / 141662

[0005] To obtain a partial protein hydrolysate, a protein-containing composition is generally treated with a protease by adding the protease. The protease is then inactivated by a process such as heat treatment, but for food safety reasons, it is necessary to completely remove the protease. Ultrafiltration membranes and size exclusion chromatography are used in the process of removing the protease. However, since the protein and the protease contained in the protein-containing composition are the same protein, no efficient method has been established to confirm that the protease has been removed from the protein-containing composition.

[0006] Therefore, an object of the present invention is to provide a method for confirming that proteases have been removed from a protein-containing composition, a method for producing a protein-containing composition from which proteases have been removed, and a method for confirming that the final product does not contain autolyzed products of the proteases.

[0007] Examples of specific embodiments of the present invention are given below.

[0008] [1] A method for confirming the removal of a protease, comprising ultrafiltration of the protease alone using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or less, and measuring the protease activity and / or protein content of the permeate and / or retentate. [2] The method for confirming the removal of a protease according to [1], wherein measuring the protease activity and / or protein content of the permeate and / or retentate means measuring the protease activity and protein content of the permeate and retentate. [3] The method for confirming the removal of a protease according to [1] or [2], wherein the protease is an enzyme derived from a filamentous fungus or a bacterium. [4] The method for confirming the removal of a protease according to any of [1] to [3], wherein the protease is at least one selected from the group consisting of enzymes derived from the genus Aspergillus, enzymes derived from the genus Bacillus, and enzymes derived from the genus Geobacillus. [5] A method for confirming the removal of a protease according to any one of [1] to [4], wherein the molecular weight cutoff of the ultrafiltration membrane is 5 kDa or less. [6] A method for producing a protein-containing composition from which a protease has been removed, comprising ultrafiltration of a protein-containing composition treated with a protease using an ultrafiltration membrane having a molecular weight cutoff of 10 kDa or less, wherein the ultrafiltration membrane is one that has been confirmed to remove the protease by ultrafiltration of the protease alone and then measuring the protease activity and / or protein content of the permeate and / or retentate. [7] The method for producing a protein-containing composition from which the protease has been removed according to [6], further comprising ultrafiltering the protease alone using an ultrafiltration membrane having a molecular weight cutoff of 10 kDa or less, and measuring the protease activity and / or protein content of the permeate and / or the retentate, wherein ultrafiltration of the protein-containing composition treated with the protease using an ultrafiltration membrane having a molecular weight cutoff of 10 kDa or less comprises ultrafiltration of the protein-containing composition treated with the protease using the ultrafiltration membrane that was used for measurement.[8] The method for producing a protein-containing composition from which proteases have been removed according to [6] or [7], wherein measuring the protease activity and / or protein content of the permeate and / or retentate is measuring the protease activity and protein content of the permeate and retentate. [9] The method for producing a protein-containing composition from which proteases have been removed according to any of [6] to [8], wherein the molecular weight cut-off of the ultrafiltration membrane is 5 kDa or less.

[10] The method for producing a protein-containing composition from which proteases have been removed according to any of [6] to [9], wherein the protease is an enzyme derived from a filamentous fungus or a bacteria.

[11] The method for producing a protein-containing composition from which enzymes have been removed according to any of [6] to

[10] , wherein the protease is at least one selected from the group consisting of enzymes derived from the genus Aspergillus, enzymes derived from the genus Bacillus, and enzymes derived from the genus Geobacillus.

[12] The method for producing a protease-free protein-containing composition according to any one of [6] to

[11] , wherein the protein-containing composition is a milk protein-containing composition.

[13] A method for confirming that the final product does not contain autolyzed products of the protease, comprising ultrafiltration of the protease alone using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or less, and measuring the protease activity and / or protein content of the permeate and / or retentate.

[14] The method according to

[13] , wherein measuring the protease activity and / or protein content of the permeate and / or retentate is measuring the protease activity and protein content of the permeate and retentate.

[15] The method according to

[13] or

[14] , wherein the protease is an enzyme derived from a filamentous fungus or a bacteria.

[16] The method according to any one of

[13] to

[15] , wherein the protease is at least one selected from the group consisting of enzymes derived from the genus Aspergillus, enzymes derived from the genus Bacillus, and enzymes derived from the genus Geobacillus.

[17] The method according to any one of

[13] to

[16] , wherein the molecular weight cut-off of the ultrafiltration membrane is 5 kDa or less.

[18] The method according to any one of

[13] to

[17] , further comprising treating the protein-containing composition with a protease.

[19] The method according to

[18] , wherein the protein-containing composition is a milk protein-containing composition.

[0009] According to the method of the present invention, it is possible to efficiently confirm that proteases have been removed from a protein-containing composition. Furthermore, by using the method of the present invention, it is possible to produce a protein-containing composition from which proteases have been removed, and further to confirm that the final product does not contain autolyzed products of the proteases.

[0010] The present invention will be described in detail below. The following description may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0011] (Method for Confirming Removal of Protease) A first embodiment of the present invention relates to a method for confirming removal of a protease, comprising ultrafiltration of the protease alone using an ultrafiltration membrane having a molecular weight cutoff of 10 kDa or less, and measuring the protease activity and / or protein content of the permeate and / or retentate. The method of the first embodiment is a method for confirming removal of the protease from the permeate after ultrafiltration, and more preferably a method for confirming removal of the protease from the permeate obtained by ultrafiltration of a protein-containing composition containing a protein decomposed by the protease.

[0012] More specifically, the first embodiment preferably includes the following steps: (1) ultrafiltrating a protease solution alone using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or less; and (2) measuring the protease activity and / or protein content of the permeate and / or retentate after ultrafiltration.

[0013] In this embodiment, the protease solution is ultrafiltered alone. As used herein, "ultrafiltering the protease solution alone" means that the protease solution is substantially free of proteins other than the protease during ultrafiltration. The protease solution may contain trace amounts of contaminating proteins. In this case, "trace amounts" means that the protease solution may contain 10% by mass or less (preferably 5% by mass or less, 1% by mass or less, or 0.1% by mass or less) of contaminating proteins relative to the total protein amount. The protease solution may also contain other components (e.g., additives contained in protease preparations) in addition to the protease and contaminating proteins.

[0014] The method for measuring protease activity and protein content is not particularly limited. The method for measuring protease activity is not particularly limited as long as it can detect the activity of the enzyme used. For example, protease activity can be measured by the Folin method using casein as a substrate. Methods for measuring protein content include, for example, methods for quantifying total protein amount (BCA method, Bradford method, Lowry method, methods equivalent thereto, absorbance measurement method, etc.), and methods for quantifying the amount of individual proteins or peptides (HPLC, etc.), preferably the Bradford method.

[0015] In the first embodiment, for example, it is possible to efficiently confirm that a protease used in a production process for an enzymatically treated protein-containing composition has been removed. Although the protease may undergo autolysis during the enzymatic treatment process, the method of this embodiment makes it possible to confirm whether autolysis has occurred and whether the final product contains no autolyzed products of the protease.

[0016] In the first embodiment, it is preferable to measure the protease activity and protein content of both the permeate and the retentate. That is, the step of measuring the protease activity and / or protein content of the permeate and / or the retentate is preferably a step of measuring the protease activity and protein content of the permeate and the retentate. By measuring the protease activity and protein content of both the permeate and the retentate, it is possible to efficiently confirm that the protease has been removed, including inactivated proteases. Furthermore, by measuring the protease activity and protein content of both the permeate and the retentate, it is possible to confirm whether or not the protease has self-digested. Furthermore, if self-digestion of the protease has occurred, it is possible to confirm that the autolyzed product has been removed. In this embodiment, combining the measurement of the protease activity and the protein content in this way makes it possible to efficiently confirm that the protease used has been removed, and further, it is possible to confirm whether or not autolysis has occurred and whether or not the final product contains autolyzed products of the protease.

[0017] Specifically, the protease activity of the permeate is preferably 100 U / mL or less, more preferably 50 U / mL or less, even more preferably 30 U / mL or less, even more preferably 20 U / mL or less, even more preferably 10 U / mL or less, even more preferably 5 U / mL or less, even more preferably 2 U / mL or less, and particularly preferably 1 U / mL or less. The protease activity of the permeate is most preferably 0 U / mL. The protein content of the permeate is preferably 2.0 μg / mL or less, more preferably 1.7 μg / mL or less, even more preferably 1.5 μg / mL or less, even more preferably 1.25 μg / mL or less, and particularly preferably 1.0 μg / mL or less. The protein content of the permeate is most preferably 0 μg / mL.

[0018] On the other hand, the protease activity of the retentate is preferably equal to or greater than the protease activity before ultrafiltration, and the protein content of the retentate is preferably equal to or greater than the protein content before ultrafiltration. Specifically, the enzyme residual rate of the protease is preferably 75% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 99% or more, and particularly preferably 100% or more. In this specification, the enzyme residual rate is calculated from the protease activity value before and after ultrafiltration or the total protein amount before and after ultrafiltration. In this embodiment, it is preferable that both the enzyme residual rates calculated from the protease activity value before and after ultrafiltration and the total protein amount before and after ultrafiltration are within the above ranges.

[0019] When the protease activity and protein content of the permeate and non-permeate satisfy the above conditions, it can be determined that the protease has been removed from the permeate.

[0020] The molecular weight cutoff of the ultrafiltration membrane used in the first embodiment is preferably 10 kDa or less, more preferably 9 kDa or less, 8 kDa or less, 7 kDa or less, 6 kDa or less, 5 kDa or less, 4 kDa or less, or 3 kDa or less. A smaller molecular weight cutoff is preferable so as to ensure the removal of the protease, but there are no particular limitations as long as it can reliably remove the protease used. It is preferable that the ultrafiltration membrane has a molecular weight cutoff of a certain level or more so that the protein hydrolyzate can be recovered from the permeate. The lower limit of the molecular weight cutoff of the ultrafiltration membrane is preferably 0.5 kDa or more, more preferably 1 kDa or more, 1.5 kDa or more, or 2 kDa or more.

[0021] <Enzyme> The protease used in this embodiment may be a protease or a peptidase, preferably a protease. The protease may be any enzyme with any type or origin, as long as it can be removed using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or less, preferably 5 kDa or less. In particular, the protease is preferably an enzyme derived from a filamentous fungus or a bacteria. More specifically, examples of proteolytic enzymes include proteases derived from filamentous fungi such as those of the genus Aspergillus, Mucor, Neurospora, Penicillium, Rhizomucor, Rhizopus, and Sclerotinia; proteases derived from yeasts of the genus Saccharomyces; proteases derived from bacteria such as those of the genus Bacillus and Geobacillus; and proteases derived from actinomycetes of the genus Streptomyces. These proteases may be used singly or in combination of two or more. Among these, the protease is more preferably an enzyme derived from a filamentous fungus or an enzyme derived from a gram-positive bacterium, even more preferably at least one selected from the group consisting of an enzyme derived from the genus Aspergillus, an enzyme derived from the genus Bacillus, and an enzyme derived from the genus Geobacillus, still more preferably at least one selected from the group consisting of an enzyme derived from Aspergillus oryzae, an enzyme derived from Bacillus amyloliquefaciens, and an enzyme derived from Geobacillus stearothermophilus, and particularly preferably at least one selected from an enzyme derived from Aspergillus oryzae and an enzyme derived from Bacillus amyloliquefaciens.

[0022] Proteases can be prepared from the culture medium of a microorganism from which the proteases are derived. Specific preparation methods include recovering the proteases from the culture medium or cells of the microorganism. For example, when a protease-secreting microorganism is used, the cells can be recovered from the culture medium by filtration, centrifugation, or the like, as needed, and the enzyme can then be separated and / or purified. When a non-protease-secreting microorganism is used, the cells can be recovered from the culture medium by pressure treatment, ultrasonication, or the like, as needed, and then the enzyme can be isolated and / or purified. The enzyme can be separated and / or purified by any known protein separation and / or purification method, without any particular limitation. Examples of the enzyme separation and / or purification method include centrifugation, UF concentration, salting out, and various chromatography methods using ion exchange resins. The separated and / or purified enzyme can be powdered by drying methods such as lyophilization and vacuum drying, or by using an appropriate excipient and / or drying aid in the drying method. The isolated and / or purified enzyme can also be liquefied by adding an appropriate additive and sterilizing by filtration.

[0023] Commercially available protease products can also be used, and preferred examples of commercially available products include Bacillus amyloliquefaciens-derived protease, Aspergillus oryzae-derived protease, and Geobacillus stearothermophilus-derived protease, all of which are manufactured by Amano Enzyme Inc.

[0024] The amount of protease used is not particularly limited, but may be, for example, 0.01 U or more per mL of protease preparation. From the viewpoint of preventing autolysis, the amount used per mL of protease preparation may be, for example, 0.01 U or more, 0.1 U or more, 1.0 U or more, or 10.0 U or more. The upper limit of the range of the amount used per mL of protease preparation is not particularly limited, but may be, for example, 100,000 U or less, 50,000 U or less, 10,000 U or less, 5,000 U or less, or 4,000 U or less.

[0025] Protease activity can be measured by the Folin method using casein as a substrate. Protease activity is measured by a standard enzyme reaction using casein as a substrate, and the amount of enzyme that causes an increase in the amount of Folin test solution color substance equivalent to 1 μg of tyrosine per minute is defined as 1 unit (1 U).

[0026] (Method for producing a protein-containing composition from which proteases have been removed) A second embodiment of the present invention relates to a method for producing a protein-containing composition from which proteases have been removed, comprising ultrafiltration of a protein-containing composition treated with a protease using an ultrafiltration membrane having a molecular weight cutoff of 10 kDa or less. The ultrafiltration membrane used in the second embodiment has been confirmed to remove proteases by ultrafiltration of the protease alone and then measuring the protease activity and / or protein content of the permeate and / or retentate. The ultrafiltration membrane used in the second embodiment may be the same ultrafiltration membrane that has been confirmed to remove proteases, or it may be the same type of ultrafiltration membrane (having similar ultrafiltration performance) as the ultrafiltration membrane that has been confirmed to remove proteases.

[0027] In the second embodiment, the concentration of the enzyme solution when treating the protein-containing composition with the protease is not particularly limited, and is preferably approximately the same as the enzyme concentration when removal of the protease is confirmed.

[0028] The method for producing a protein-containing composition of the second embodiment preferably further comprises ultrafiltration of the protease alone using an ultrafiltration membrane having a molecular weight cutoff of 10 kDa or less, and measuring the protease activity and / or protein content of the permeate and / or retentate. The protein-containing composition treated with the protease is then subjected to ultrafiltration using the ultrafiltration membrane having a molecular weight cutoff of 10 kDa or less, which has been used for measurement. That is, the method for producing a protein-containing composition of the second embodiment preferably comprises step 1 of ultrafiltration of the protease alone using an ultrafiltration membrane having a molecular weight cutoff of 10 kDa or less, and measuring the protease activity and / or protein content of the permeate and / or retentate, and step 2 of ultrafiltration of the protein-containing composition treated with the protease using the ultrafiltration membrane having a molecular weight cutoff of 10 kDa or less.

[0029] Step 2 may be a step performed continuously following step 1, or steps 1 and 2 may be discontinuous steps. In this specification, "discontinuous steps" includes cases where steps 1 and 2 are performed on different days or in different facilities. The ultrafiltration membrane used in step 2 may be the same ultrafiltration membrane used when the measurement was performed in step 1, or may be the same type of ultrafiltration membrane (having the same level of ultrafiltration performance) as the ultrafiltration membrane used when the measurement was performed in step 1.

[0030] In the second embodiment, it is preferable to measure the protease activity and protein content of both the permeate and the retentate. That is, the step of measuring the protease activity and / or protein content of the permeate and / or the retentate is preferably a step of measuring the protease activity and protein content of the permeate and the retentate. By measuring the protease activity and protein content of both the permeate and the retentate, it is possible to efficiently confirm that the protease has been removed, including inactivated proteases. Furthermore, by measuring the protease activity and protein content of both the permeate and the retentate, it is possible to confirm whether or not the protease has self-digested. Furthermore, if self-digestion of the protease has occurred, it is possible to confirm that the autolyzed product has been removed. In the second embodiment, combining the measurement of the protease activity and protein content in this way makes it possible to efficiently confirm that the protease used has been removed, thereby improving the production efficiency of a protein-containing composition from which the protease has been removed.

[0031] The molecular weight cutoff of the ultrafiltration membrane used in the second embodiment is preferably 10 kDa or less, more preferably 9 kDa or less, 8 kDa or less, 7 kDa or less, 6 kDa or less, 5 kDa or less, 4 kDa or less, or 3 kDa or less. A smaller molecular weight cutoff is preferable so as to ensure the removal of the protease, but there are no particular limitations as long as it can reliably remove the protease used. It is preferable that the ultrafiltration membrane has a molecular weight cutoff of a certain level or more so that the protein hydrolyzate can be recovered from the permeate. The lower limit of the molecular weight cutoff of the ultrafiltration membrane is preferably 0.5 kDa or more, more preferably 1 kDa or more, 1.5 kDa or more, or 2 kDa or more.

[0032] More specifically, the second embodiment preferably includes the following steps: (1) ultrafiltration of the protease alone using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or less, (2) measurement of the protease activity and / or protein content of the permeate and / or retentate after ultrafiltration, (3) preparation of an enzyme-treated protein-containing composition by allowing the protease to act on a protein-containing composition, (4) ultrafiltration of the enzyme-treated protein-containing composition, and (5) recovery of the permeate from the ultrafiltration. Note that a step of inactivating the protease may be included between (3) and (4).

[0033] The protease activity of the permeate in step (2) is preferably 100 U / mL or less, more preferably 50 U / mL or less, even more preferably 30 U / mL or less, even more preferably 20 U / mL or less, even more preferably 10 U / mL or less, even more preferably 5 U / mL or less, even more preferably 2 U / mL or less, and particularly preferably 1 U / mL or less. The protease activity of the permeate is most preferably 0 U / mL. The protein content of the permeate is preferably 2.0 μg / mL or less, more preferably 1.7 μg / mL or less, even more preferably 1.5 μg / mL or less, even more preferably 1.25 μg / mL or less, and particularly preferably 1.0 μg / mL or less. The protein content of the permeate is most preferably 0 μg / mL.

[0034] On the other hand, the protease activity of the retentate in step (2) is preferably equal to or greater than the protease activity before ultrafiltration, and the protein content of the retentate is preferably equal to or greater than the protein content before ultrafiltration. Specifically, the enzyme residual rate of the protease is preferably 75% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 99% or more, and particularly preferably 100% or more.

[0035] The reaction time, temperature, pH of the reaction solution, etc. for allowing the protease to act on the protein-containing composition are not particularly limited, but it is preferable to allow the protease to act under conditions that allow the protein in the protein-containing composition to be degraded to an appropriate size while making the protease less susceptible to self-digestion. The reaction temperature is, for example, 10 to 80°C, preferably 20 to 70°C, and more preferably 30 to 60°C. The pH of the reaction solution is, for example, 2 to 10, preferably 3 to 9, and more preferably 4 to 8. The reaction time is, for example, 30 seconds to 48 hours, preferably 1 minute to 24 hours, and more preferably 30 minutes to 12 hours.

[0036] The conditions for ultrafiltration are not particularly limited. The molecular weight cutoff of the ultrafiltration membrane may be selected so that the product to be recovered permeates among the proteolysis products derived from the raw material produced by the enzymatic treatment, but that the protease used does not permeate. The temperature, pH, and filtration time during ultrafiltration are also not particularly limited, but it is preferable to perform the ultrafiltration under conditions that make it difficult for the protease to self-digest.

[0037] The protein-containing composition (enzyme-removed composition) obtained in the second embodiment from which proteases have been removed contains a hydrolyzed product of the raw protein but does not contain the proteases used. Therefore, a protein-containing composition (enzyme-removed composition) with improved safety can be provided. For example, a specific example of a protein-containing composition (enzyme-removed composition) is a milk peptide-containing composition.

[0038] <Enzyme> Examples of the protease used in the second embodiment include the same protease as used in the first embodiment, and preferred forms are also the same.

[0039] <Protein-Containing Composition> The protein-containing composition used in the second embodiment is not particularly limited as long as it contains protein. The origin of the contained protein is not particularly limited, and examples thereof include animal-derived (milk, livestock meat, fish meat, etc.), plant-derived (cereals such as barley, rice, wheat, rye, oats, buckwheat, sorghum, barnyard millet, foxtail millet, teff, quinoa, and corn; pulses such as soybeans, lentils, broad beans, peas, chickpeas, mung beans, lupin beans, and kidney beans; nuts and seeds such as hemp, canary seeds, flaxseed, almonds, cashew nuts, hazelnuts, pecan nuts, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, coconuts, pili nuts, chestnuts, sesame seeds, and pine nuts), insect-derived, and microbial-derived raw materials. Among these, it is preferable that the protein-containing composition is a milk protein-containing composition.

[0040] The state of the protein-containing composition when subjected to the protease treatment is not particularly limited as long as it does not impair the effects of the present invention, but is preferably in the form of a liquid or a slurry.

[0041] (Method for Confirming Absence of Protease Autolysis Products) A third embodiment of the present invention relates to a method for confirming that a final product does not contain protease autolysis products, comprising ultrafiltration of a protease alone using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or less, and measuring the protease activity and / or protein content of the permeate and / or retentate. The method of the third embodiment is a method for confirming that the final product (permeate after ultrafiltration) does not contain protease autolysis products (protease degradation products), and more preferably, a method for confirming that the permeate obtained by ultrafiltration of a protein-containing composition containing a protein decomposed by a protease does not contain protease autolysis products (protease degradation products).

[0042] More specifically, the third embodiment preferably includes the following steps: (1) ultrafiltrating a protease solution alone using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or less; and (2) measuring the protease activity and / or protein content of the permeate and / or retentate after ultrafiltration.

[0043] The protease activity of the permeate in step (2) is preferably 100 U / mL or less, more preferably 50 U / mL or less, even more preferably 30 U / mL or less, even more preferably 20 U / mL or less, even more preferably 10 U / mL or less, even more preferably 5 U / mL or less, even more preferably 2 U / mL or less, and particularly preferably 1 U / mL or less. The protease activity of the permeate is most preferably 0 U / mL. The protein content of the permeate is preferably 2.0 μg / mL or less, more preferably 1.7 μg / mL or less, even more preferably 1.5 μg / mL or less, even more preferably 1.25 μg / mL or less, and particularly preferably 1.0 μg / mL or less. The protein content of the permeate is most preferably 0 μg / mL.

[0044] On the other hand, the protease activity of the retentate is preferably equal to or greater than the protease activity before ultrafiltration, and the protein content of the retentate is preferably equal to or greater than the protein content before ultrafiltration. Specifically, the enzyme residual rate of the protease is preferably 75% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 99% or more, and particularly preferably 100% or more.

[0045] In the third embodiment, for example, in the process for producing an enzyme-treated protein-containing composition, it is possible to efficiently confirm that autolysis products (protease degradation products) of the protease used do not remain in the final product. Although the protease may itself be autolyzed in the enzyme treatment process, the method of this embodiment can be used to confirm that the final product does not contain autolysis products of the protease.

[0046] In the third embodiment, it is preferable to measure the protease activity and protein content of both the permeate and the retentate. That is, the step of measuring the protease activity and / or protein content of the permeate and / or the retentate is preferably a step of measuring the protease activity and protein content of the permeate and the retentate. By measuring the protease activity and protein content of both the permeate and the retentate, it is possible to confirm whether or not autolysis of the protease has occurred, and further, if autolysis of the protease has occurred, it is possible to confirm that the autolysis product has been removed. In this embodiment, by combining the measurement of the protease activity and the protein content in this way, it is possible to confirm whether or not autolysis has occurred and whether or not the final product contains autolysis product of the protease.

[0047] The molecular weight cutoff of the ultrafiltration membrane used in the third embodiment is preferably 10 kDa or less, more preferably 9 kDa or less, 8 kDa or less, 7 kDa or less, 6 kDa or less, 5 kDa or less, 4 kDa or less, or 3 kDa or less. A smaller molecular weight cutoff is preferable so as to ensure the removal of the protease, but there are no particular limitations as long as it can reliably remove the protease used. It is preferable that the ultrafiltration membrane has a molecular weight cutoff of a certain level or more so that the protein hydrolyzate can be recovered from the permeate. The lower limit of the molecular weight cutoff of the ultrafiltration membrane is preferably 0.5 kDa or more, more preferably 1 kDa or more, 1.5 kDa or more, or 2 kDa or more.

[0048] The method of the third embodiment may further include a step of treating the protein-containing composition with a protease. In such a case, more specifically, the third embodiment preferably includes the following steps: (1) ultrafiltration of the protease solution alone using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or less, (2) measuring the protease activity and / or protein content of the permeate and / or retentate after ultrafiltration, (3) preparing an enzyme-treated protein-containing composition by treating the protein-containing composition with the protease, (4) ultrafiltration of the enzyme-treated protein-containing composition, and (5) recovering the permeate from the ultrafiltration. Note that a step of inactivating the protease may be included between (3) and (4).

[0049] <Enzyme> Examples of the protease used in the third embodiment include the same protease as used in the first embodiment, and preferred forms are also the same.

[0050] <Protein-Containing Composition> Examples of the protein-containing composition used in the third embodiment include the same ones as the protein-containing composition used in the second embodiment, and preferred forms are also the same.

[0051] The features of the present invention will be explained in more detail below with reference to examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[0052] <Method for Measuring Enzyme Activity (Method for Measuring Protease Activity)> Protease activity was calculated using the following method based on the 9th edition of the Japanese Standards for Food Additives. 5 mL of 0.6% (w / v) casein solution (0.05 mol / L sodium hydrogen phosphate, pH 8.0) was heated at 37°C for 10 minutes, and then 1 mL of appropriately diluted sample solution (each enzyme solution, permeate, and concentrate described below) was added and immediately shaken. This solution was then left at 37°C for 10 minutes, followed by the addition of 5 mL of trichloroacetic acid test solution (containing 1.8% (w / v) trichloroacetic acid, 1.8% (w / v) sodium acetate, and 0.33 mol / L acetic acid), shaking, and again at 37°C for 30 minutes. The first 3 mL of filtrate was discarded, and 2 mL of the next filtrate was measured. 5 mL of 0.55 mol / L sodium carbonate test solution and 1 mL of Folin's test solution (1→3) were added, shaken well, and left at 37°C for 30 minutes. The absorbance AT of this solution (enzyme reaction solution) at a wavelength of 660 nm was measured, using water as a control. Separately, 1 mL of sample solution containing protease was measured, and 5 mL of trichloroacetic acid test solution (containing 1.8% (w / v) trichloroacetic acid, 1.8% (w / v) sodium acetate, and 0.33 mol / L acetic acid) was added and shaken. Then, 5 mL of 0.6% (w / v) casein solution (0.05 mol / L sodium hydrogen phosphate, pH 8.0) was added, immediately shaken, and left at 37°C for 30 minutes. The absorbance AB of a blank solution was measured in the same manner as the enzyme reaction solution described above, except that the blank was left at 37°C for 30 minutes. One unit (1 U) of enzyme was defined as the amount of enzyme that increases the Folin test solution color product equivalent to 1 μg of tyrosine per minute. 1 mL, 2 mL, 3 mL, or 4 mL of 1 mg / mL tyrosine standard stock solution (0.2 mol / L hydrochloric acid) was measured, and 0.2 mol / L hydrochloric acid test solution was added to each solution to make a 100 mL volume. 2 mL of each solution was measured, and 5 mL of 0.55 mol / L sodium carbonate test solution and 1 mL of Folin test solution (1 → 3) were added. The solution was immediately shaken and left at 37 °C for 30 minutes. The absorbances A1, A2, A3, and A4 at a wavelength of 660 nm were measured for these solutions. Furthermore, 2 mL of 0.2 mol / L hydrochloric acid test solution was measured, and 5 mL of 0.55 mol / L sodium carbonate test solution and 1 mL of Folin test solution (1 → 3) were added. The solution obtained by immediately shaking and leaving at 37 °C for 30 minutes was used as a control (blank solution), and the same measurements were performed.A calibration curve was prepared with absorbances A1, A2, A3, and A4 on the vertical axis and the amount of tyrosine (μg) in 2 mL of each solution on the horizontal axis, and the amount of tyrosine (μg) per absorbance difference of 1 was determined. Protease activity (U / g, U / mL) = (AT - AB) x F x 1 1 / 2 x 1 / 10 x 1 / M AT: absorbance of enzyme reaction solution AB: absorbance of blank F: amount of tyrosine (μg) when the absorbance difference was 1, determined from the tyrosine calibration curve 1 1 / 2: conversion coefficient to total solution volume after reaction was stopped 1 / 10: conversion coefficient to reaction time per minute M: dilution factor of sample.

[0053] <Total Protein Quantification Method> Total protein quantification was measured by a colorimetric method based on the Bradford method. Specifically, each enzyme solution, permeate, and concentrate described below was mixed with 200 μL of staining solution (protein assay dye reagent concentrate, Bio-Rad) filtered through filter paper (Whatman #1), and after standing at room temperature for 10 minutes, the absorbance AT was measured at 595 nm. Separately, 10 μL of purified water or 10 μL of bovine serum albumin (BSA) solution prepared at 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL was mixed with 200 μL of diluted and filtered staining solution, and after standing at room temperature for 10 minutes, the absorbance AT was measured at 595 nm. A calibration curve was created from the amount of BSA (mg) and its absorbance AT. The total protein amount (mg) of each enzyme solution, permeate, and concentrate was calculated from the calibration curve.

[0054] The following proteolytic enzymes were used:

[0055] (Test Example 1) (1) Preparation of Enzyme Solution An enzyme solution was prepared by adding 2 mmol / L calcium acetate and 10 mmol / L sodium chloride solution to a Bacillus amyloliquefaciens-derived protease concentration of 0.5% (w / v). An enzyme solution was prepared by adding chilled 0.02 mol / L phosphate buffer (pH 8.0) to a Aspergillus oryzae-derived protease concentration of 0.5% (w / v). An enzyme solution was prepared by adding 2 mmol / L calcium acetate and 10 mmol / L sodium chloride solution to a Geobacillus stearothermophilus-derived protease concentration of 0.5% (w / v) or 3.7% (w / v).

[0056] (2) Molecular weight fractionation by ultrafiltration membrane 4 ml of each prepared enzyme solution was loaded onto an ultrafiltration membrane (Amicon Ultra-4 filter, manufactured by Merck) with a molecular weight cutoff of 3 kDa. Then, the centrifugal separation step was repeated under cooling at 7,500 g until the volume of the permeate and the volume of the concentrate became equal.

[0057] <Evaluation 1> (Protease activity measurement) Each 0.5% (w / v) enzyme solution, permeate, and concentrate was diluted appropriately with purified water, and the protease activity was calculated according to the activity measurement method described above. The results are shown in the table below. The enzyme residual rate was also calculated using the following formula: Enzyme residual rate (%) = "Protease activity (U / ml) of concentrate or permeate" x "Volume (ml)" / "Protease activity (U / ml) of 0.5% enzyme solution used for ultrafiltration" x "Volume (ml)" x 100

[0058]

[0059] <Evaluation 2> (Quantification of Total Protein Amount) A 0.5% (w / v) Bacillus amyloliquefaciens-derived protease solution, a 0.5% (w / v) Aspergillus oryzae-derived protease solution, a 3.7% (w / v) Geobacillus stearothermophilus-derived protease solution, the permeate, and the concentrate were appropriately diluted with purified water, and the protease activity was calculated according to the total protein quantification method described above. The results are shown in the table below. The enzyme residual rate was also calculated using the following formula: Enzyme residual rate (%) = "Total protein amount (μg / mL) of the concentrate or permeate" × "Volume (ml)" / "Total protein amount (μg / mL) of the 0.5% enzyme solution used for ultrafiltration" × "Volume (ml)" × 100

[0060]

[0061] For all proteases, the total protein amount in the permeate was below the detection limit (1.25 μg / mL), confirming that proteases can be removed using a membrane with a molecular weight cutoff of 3 kDa.

Claims

1. A method for confirming that a protease has been removed, comprising ultrafiltration of the protease alone using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or less, and measuring the protease activity and / or protein content of the permeate and / or retentate.

2. A method for confirming that proteases have been removed as described in claim 1, wherein measuring the protease activity and / or protein content of the permeate and / or retentate comprises measuring the protease activity and protein content of the permeate and retentate.

3. The method for confirming the removal of a protease according to claim 1, wherein the protease is an enzyme derived from a filamentous fungus or a bacteria.

4. The method for confirming the removal of proteases according to claim 1, wherein the proteases are at least one selected from the group consisting of enzymes derived from the genus Aspergillus, enzymes derived from the genus Bacillus, and enzymes derived from the genus Geobacillus.

5. A method for confirming the removal of protease according to claim 1, wherein the molecular weight cutoff of the ultrafiltration membrane is 5 kDa or less.

6. A method for producing a protein-containing composition from which proteases have been removed, comprising ultrafiltration of a protein-containing composition treated with a protease using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or less, wherein the ultrafiltration membrane has been confirmed to be capable of removing proteases by ultrafiltration of the protease alone and then measuring the protease activity and / or protein content of the permeate and / or retentate.

7. The method for producing a protein-containing composition from which a protease has been removed according to claim 6, further comprising ultrafiltering the protease alone using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or less and measuring the protease activity and / or protein content of the permeate and / or non-permeate, wherein ultrafiltration of the protein-containing composition treated with the protease using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or less comprises ultrafiltration of the protein-containing composition treated with the protease using the ultrafiltration membrane used for the measurement.

8. A method for producing a protein-containing composition from which proteases have been removed, as described in claim 6, wherein measuring the protease activity and / or protein content of the permeate and / or retentate comprises measuring the protease activity and protein content of the permeate and retentate.

9. A method for producing a protein-containing composition from which proteases have been removed according to claim 6, wherein the molecular weight cut-off of the ultrafiltration membrane is 5 kDa or less.

10. The method for producing a protein-containing composition from which proteases have been removed according to claim 6, wherein the proteases are derived from filamentous fungi or bacteria.

11. The method for producing an enzyme-free protein-containing composition according to claim 6, wherein the proteolytic enzyme is at least one selected from the group consisting of enzymes derived from the genus Aspergillus, enzymes derived from the genus Bacillus, and enzymes derived from the genus Geobacillus.

12. The method for producing a protein-containing composition from which proteases have been removed according to claim 6, wherein the protein-containing composition is a milk protein-containing composition.

13. A method for confirming that the final product does not contain autolysis products of the protease, comprising ultrafiltration of the protease alone using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or less, and measuring the protease activity and / or protein content of the permeate and / or retentate.

14. The method of claim 13, wherein measuring the protease activity and / or protein content of the permeate and / or retentate comprises measuring the protease activity and protein content of the permeate and retentate.

15. The method according to claim 13, wherein the proteolytic enzyme is an enzyme derived from a filamentous fungus or a bacteria.

16. The method according to claim 13, wherein the protease is at least one selected from the group consisting of enzymes derived from the genus Aspergillus, enzymes derived from the genus Bacillus, and enzymes derived from the genus Geobacillus.

17. The method according to claim 13, wherein the molecular weight cutoff of the ultrafiltration membrane is 5 kDa or less.

18. The method of claim 13, further comprising treating a protein-containing composition with said proteolytic enzyme.

19. The method of claim 18, wherein the protein-containing composition is a dairy protein-containing composition.

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