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WO2025178073A1 - Procédé pour confirmer qu'une enzyme protéolytique a été éliminée, et ses applications - Google Patents

Procédé pour confirmer qu'une enzyme protéolytique a été éliminée, et ses applications

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Publication number
WO2025178073A1
WO2025178073A1 PCT/JP2025/005769 JP2025005769W WO2025178073A1 WO 2025178073 A1 WO2025178073 A1 WO 2025178073A1 JP 2025005769 W JP2025005769 W JP 2025005769W WO 2025178073 A1 WO2025178073 A1 WO 2025178073A1
Authority
WO
WIPO (PCT)
Prior art keywords
protease
protein
permeate
containing composition
less
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/JP2025/005769
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English (en)
Japanese (ja)
Inventor
翠 小川
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Amano Enzyme Inc
Original Assignee
Amano Enzyme Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amano Enzyme Inc filed Critical Amano Enzyme Inc
Publication of WO2025178073A1 publication Critical patent/WO2025178073A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/20—Accessories; Auxiliary operations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase

Definitions

  • Cow's milk proteins are often used in the production of infant formula.
  • the milk proteins are enzymatically treated to remove allergens.
  • infant formula for infants at risk of developing cow's milk allergy contains partial hydrolysates of milk proteins.
  • Patent Document 1 discloses a method for producing infant formula, which includes a purification step in which the filtrate is recovered using a membrane with a molecular cutoff in the range of 10 to 100 kDa or size exclusion chromatography.
  • a protein-containing composition When obtaining a partial protein hydrolysate, a protein-containing composition is generally treated with an enzyme by adding a protease. The protease is then inactivated through 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, because the protein and 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.
  • the present invention therefore aims 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.
  • 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, A manufacturing method in which the ultrafiltration membrane is one in which the removal of protease has been confirmed by ultrafiltration of the protease alone and then measuring the protease activity and/or protein content of the permeate and/or non-permeate.
  • the method of the present invention makes it 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.
  • a first embodiment of the present invention relates to a method for confirming the removal of a protease, comprising ultrafiltrating 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.
  • the method of the first embodiment is a method for confirming the removal of the protease from the permeate after ultrafiltration, and more preferably, a method for confirming the removal of the protease from the permeate obtained by ultrafiltration of a protein-containing composition containing a protein decomposed by the protease.
  • the first embodiment preferably includes the following steps. (1) ultrafiltration of 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.
  • Methods for measuring protease activity and protein content are not particularly limited. There are no particular limitations on the method for measuring protease activity, as long as the activity of the enzyme used can be detected. 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, equivalent methods, absorbance measurement method, etc.), and methods for quantifying the amount of individual proteins or peptides (HPLC, etc.), with the Bradford method being preferred.
  • the first embodiment for example, it is possible to efficiently confirm that a protease used in the manufacturing process of an enzyme-treated protein-containing composition has been removed.
  • Protease may undergo autolysis during the enzyme treatment process, but by using the method of this embodiment, it is possible to confirm whether autolysis has occurred and whether the final product contains any autolyzed products of the protease.
  • 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 ⁇ g/mL or less, more preferably 1.7 ⁇ g/mL or less, even more preferably 1.5 ⁇ g/mL or less, even more preferably 1.25 ⁇ g/mL or less, and particularly preferably 1.0 ⁇ g/mL or less.
  • the protein content of the permeate is most preferably 0 ⁇ g/mL.
  • the protease activity of the retentate is preferably equal to or greater than the protease activity before ultrafiltration
  • the protein content of the retentate is preferably equal to or greater than the protein content before ultrafiltration.
  • the enzyme residual rate of the protease is preferably 75% or greater, more preferably 80% or greater, even more preferably 85% or greater, even more preferably 90% or greater, even more preferably 95% or greater, even more preferably 99% or greater, and particularly preferably 100% or greater.
  • the enzyme residual rate is calculated from the protease activity values 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 values before and after ultrafiltration and the total protein amount before and after ultrafiltration are within the above ranges.
  • the molecular weight cutoff of the ultrafiltration membrane used in the first embodiment is preferably 10 kDa or less, and 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 to ensure reliable removal of the protease, but there are no particular restrictions as long as it can reliably remove the protease used.
  • the ultrafiltration membrane have a molecular weight cutoff above a certain level so that protein hydrolysates 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, and more preferably 1 kDa or more, 1.5 kDa or more, or 2 kDa or more.
  • the protease used in this embodiment may be a protease or a peptidase, preferably a protease.
  • the protease may be of 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.
  • the protease is preferably an enzyme derived from a filamentous fungus or a bacterium.
  • 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.
  • 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.
  • Protease enzymes can be prepared from the culture medium of the microorganism from which the protease is derived. Specific preparation methods include recovering the protease from the culture medium or cells of the microorganism. For example, when using a protease-secreting microorganism, the cells can be recovered from the culture medium by filtration, centrifugation, or other methods as needed, and the enzyme can then be separated and/or purified. When using a non-protease-secreting microorganism, the cells can be recovered from the culture medium by pressure treatment, ultrasonic treatment, or other methods as needed, disrupting the cells to expose the enzyme, and the enzyme can then be separated and/or purified.
  • Enzyme separation and/or purification methods can be any known protein separation and/or purification method, without any particular limitations. Examples 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 freeze-drying and vacuum drying. Furthermore, powderization can also be achieved by using appropriate excipients and/or drying aids in the drying methods. Additionally, the isolated and/or purified enzyme can be liquefied by adding appropriate additives and sterilizing by filtration.
  • 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 manufactured by Amano Enzyme Inc.
  • the amount of protease used is not particularly limited, but examples of the amount used per 1 mL of protease preparation include 0.01 U or more. From the perspective of preventing autolysis, examples of the amount used per 1 mL of protease preparation include 0.01 U or more, 0.1 U or more, 1.0 U or more, and 10.0 U or more.
  • the upper limit of the range of the amount used per 1 mL of protease preparation is not particularly limited, but examples include 100,000 U or less, 50,000 U or less, 10,000 U or less, 5,000 U or less, and 4,000 U or less.
  • Protease activity can be measured by the Folin method using casein as a substrate. Protease activity is measured by performing an enzyme reaction using casein as a substrate in the usual manner, and the amount of enzyme that causes an increase in the amount of Folin test solution colored substance equivalent to 1 ⁇ g of tyrosine per minute is defined as 1 unit (1 U).
  • 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 followed by measurement of 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 confirmed to remove proteases, or it may be the same type of ultrafiltration membrane (having similar ultrafiltration performance) as the ultrafiltration membrane confirmed to remove proteases.
  • the concentration of the enzyme solution when treating the protein-containing composition with the protease is not particularly limited.
  • the concentration is approximately the same as the enzyme concentration when removal of the protease is confirmed.
  • the method for producing a protein-containing composition of the second embodiment preferably further comprises 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 retentate.
  • the protein-containing composition treated with the protease is then ultrafiltered using the ultrafiltration membrane with a molecular weight cutoff of 10 kDa or less, using the same ultrafiltration membrane used for measurement.
  • the method for producing a protein-containing composition of the second embodiment preferably comprises Step 1 of 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 retentate, and Step 2 of ultrafiltering the protein-containing composition treated with the protease using the same ultrafiltration membrane used for measurement.
  • Step 2 may be a step that is carried out continuously following step 1, but steps 1 and 2 may be discontinuous steps.
  • discontinuous steps includes cases where steps 1 and 2 are carried out on different days or in different facilities.
  • the ultrafiltration membrane used in step 2 may be the same ultrafiltration membrane that was used when the measurement was made in step 1, or it may be the same type of ultrafiltration membrane (having the same level of ultrafiltration performance) as the ultrafiltration membrane used when the measurement was made in step 1.
  • 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.
  • the molecular weight cutoff of the ultrafiltration membrane used in the second embodiment is preferably 10 kDa or less, and 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 to ensure reliable removal of the protease, but there are no particular restrictions as long as it can reliably remove the protease used.
  • the ultrafiltration membrane have a molecular weight cutoff above a certain level so that protein hydrolysates 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, and more preferably 1 kDa or more, 1.5 kDa or more, or 2 kDa or more.
  • the second embodiment preferably includes the following steps. (1) A step of ultrafiltrating the protease alone using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or less; (2) A step of measuring the protease activity and/or protein content of the permeate and/or non-permeate after ultrafiltration; (3) A step of preparing an enzyme-treated protein-containing composition by treating a protein-containing composition with a protease; (4) A step of ultrafiltrating the enzyme-treated protein-containing composition; and (5) A step of recovering the permeate from the ultrafiltration. Note that a step of inactivating the protease may be included between (3) and (4).
  • 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 ⁇ g/mL or less, more preferably 1.7 ⁇ g/mL or less, even more preferably 1.5 ⁇ g/mL or less, even more preferably 1.25 ⁇ g/mL or less, and particularly preferably 1.0 ⁇ g/mL or less.
  • the protein content of the permeate is most preferably 0 ⁇ g/mL.
  • the protease activity of the retentate in step (2) is preferably equal to or greater than the protease activity before ultrafiltration
  • the protein content of the retentate is preferably equal to or greater than the protein content before ultrafiltration.
  • 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.
  • 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 degrade the proteins in the protein-containing composition to an appropriate size while making self-digestion difficult.
  • 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.
  • the conditions for ultrafiltration are not particularly limited.
  • the molecular weight cutoff of the ultrafiltration membrane should be the molecular weight cutoff through which the desired product, among the proteolysis products derived from the raw material produced by enzymatic treatment, passes, but which the protease used does not.
  • the temperature, pH, and filtration time during ultrafiltration are also not particularly limited, but it is preferable to carry out the ultrafiltration under conditions that make it difficult for the protease to self-digest.
  • the protein-containing composition (enzyme-removed composition) obtained in the second embodiment from which the protease has been removed contains a hydrolyzed product of the raw material protein but does not contain the protease used. This makes it possible to provide a protein-containing composition (enzyme-removed composition) with improved safety.
  • a specific example of a protein-containing composition (enzyme-removed composition) is a milk peptide-containing composition.
  • Examples of the protease used in the second embodiment include the same protease as used in the first embodiment, and the preferred forms are also the same.
  • 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, meat, fish, 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 materials.
  • the state of the protein-containing composition when subjected to protease treatment is not particularly limited, as long as it does not impair the effects of the present invention.
  • it is in liquid or slurry form.
  • the method of the third embodiment is a method for confirming that the final product (permeate after ultrafiltration) does not contain autolyzed products of the protease (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 the protease does not contain autolyzed products of the protease (protease degradation products).
  • the third embodiment preferably includes the following steps. (1) ultrafiltration of 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.
  • 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 ⁇ g/mL or less, more preferably 1.7 ⁇ g/mL or less, even more preferably 1.5 ⁇ g/mL or less, even more preferably 1.25 ⁇ g/mL or less, and particularly preferably 1.0 ⁇ g/mL or less.
  • the protein content of the permeate is most preferably 0 ⁇ g/mL.
  • the protease activity of the retentate is preferably equal to or greater than the protease activity before ultrafiltration
  • the protein content of the retentate is preferably equal to or greater than the protein content before ultrafiltration.
  • 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.
  • Examples of the protease used in the third embodiment include the same protease as used in the first embodiment, and the preferred forms are also the same.
  • the absorbance AT of this solution was measured at a wavelength of 660 nm, using water as a control.
  • 1 mL of a sample solution containing protease was measured, 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 the mixture was shaken.
  • 5 mL of 0.6% (w/v) casein solution (0.05 mol/L sodium hydrogen phosphate, pH 8.0) was then added, the mixture was immediately shaken, and the mixture was allowed to stand at 37°C for 30 minutes.
  • the vertical axis represents absorbances A1, A2, A3 and A4, and the horizontal axis represents the amount of tyrosine ( ⁇ g) in 2 mL of each solution, to prepare a calibration curve, and the amount of tyrosine ( ⁇ g) per absorbance difference of 1 was calculated.
  • Protease activity (U/g, U/mL) (AT-AB) x F x 11/2 x 1/10 x 1/M
  • AT absorbance of enzyme reaction solution
  • AB absorbance of blank
  • F amount of tyrosine ( ⁇ g) when the absorbance difference is 1 obtained from the tyrosine calibration curve
  • 11/2 Conversion factor for total liquid volume after reaction has stopped 1/10: Conversion factor for reaction time per minute
  • M Dilution factor of sample
  • Total protein quantification method The total protein content was measured by colorimetry based on the Bradford method. Specifically, each enzyme solution, permeate, and concentrate described below was mixed with 200 ⁇ L of a staining solution (protein assay dye reagent concentrate, Bio-Rad) filtered through a Whatman #1 filter paper. After allowing to stand at room temperature for 10 minutes, the absorbance (AT) was measured at 595 nm.
  • a staining solution protein assay dye reagent concentrate, Bio-Rad
  • proteolytic enzymes were used:
  • An enzyme solution was prepared by adding 2 mmol/L calcium acetate and 10 mmol/L sodium chloride solution so that the concentration of Geobacillus stearothermophilus-derived protease was 0.5% (w/v) or 3.7% (w/v).
  • ⁇ Rating 2> Quantification of total protein amount
  • the 0.5% (w/v) Bacillus amyloliquefaciens-derived protease solution, 0.5% (w/v) Aspergillus oryzae-derived protease solution, 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 calculated using the following formula.

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Abstract

La présente invention a pour but de procurer : un procédé permettant de confirmer qu'une enzyme protéolytique a été éliminée d'une composition contenant des protéines ; un procédé permettant de produire une composition contenant des protéines dont une enzyme protéolytique a été éliminée ; et un procédé permettant de confirmer qu'un autolysat d'une enzyme protéolytique n'est pas contenu dans un produit final. Ce procédé pour confirmer qu'une enzyme protéolytique a été éliminée consiste à utiliser une membrane d'ultrafiltration présentant un seuil de poids moléculaire d'au plus 10 kDa pour ultrafiltrer exclusivement une enzyme protéolytique, et à mesurer l'activité de l'enzyme protéolytique et/ou la teneur en protéines d'un liquide perméabilisé et/ou d'un liquide non perméabilisé.
PCT/JP2025/005769 2024-02-22 2025-02-20 Procédé pour confirmer qu'une enzyme protéolytique a été éliminée, et ses applications Pending WO2025178073A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10511556A (ja) * 1995-08-22 1998-11-10 イスチチュート デイ リセルシュ デイ バイオロジア モレコラレ ピー．アンジェレッティ ソシエタ ペル アチオニ Ｈｃｖ ｎｓ３プロテアーゼのタンパク質分解活性を有するポリぺプチドを製造し、精製し、分析する方法
JP2003339326A (ja) * 2002-05-27 2003-12-02 Morinaga Milk Ind Co Ltd 乳清蛋白質加水分解物及びその製造方法
JP2015065921A (ja) * 2013-09-30 2015-04-13 味の素株式会社 調味料の製造方法
JP2023013986A (ja) * 2021-07-16 2023-01-26 財團法人工業技術研究院 生物的毛髪形状改変組成物およびキット、ならびに毛髪形状を変えるための方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
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