WO2025141023A1

WO2025141023A1 - Methods for the production of protein hydrolysates with low levels of nitrosamines

Info

Publication number: WO2025141023A1
Application number: PCT/EP2024/088310
Authority: WO
Inventors: Christian MARKIEFKA; David Melchior; Sybille HIETSCH; Selina NEUHAEUSLER; Gisbert Schaefer
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 2023-12-29
Filing date: 2024-12-23
Publication date: 2025-07-03
Anticipated expiration: 2026-06-29

Abstract

The invention relates to methods for the preparation of protein hydrolysates having low concentrations of undesired nitrosamines as well as the thus obtained protein hydrolysates, compositions containing them and uses thereof. The methods comprise the steps of (a) contacting at least one protein with at least one oxidant, preferably a peroxide, at a concentration of 10 to <500 ppm relative to the weight of the at least one protein, at a pH in the range of 2.0 to <7.0; and (b) subjecting the at least one protein of step (a) to a hydrolysis reaction to obtain a protein hydrolysate.

Description

METHODS FOR THE PRODUCTION OF PROTEIN HYDROLYSATES WITH LOW LEVELS OF NITROSAMINES

Field of the invention

The invention relates to the field of cosmetics and concerns methods for the preparation of protein hydrolysates having low concentrations of undesired nitrosamines as well as the thus obtained protein hydrolysates, compositions containing them and uses thereof.

Background of the invention

Proteins, and especially keratin, are important ingredients for the cosmetics industry. Because of their film-forming properties, proteins may form a protective layer on hair and skin and thereby care for them. Proteins have many other advantages and are therefore widely used in cosmetic compositions for skin and hair; with keratin being predominantly used in haircare.

Because of their poor solubility, however, proteins are not usually used in their natural form in cosmetic formulations but rather as what are referred to as “hydrolysates”. In hydrolysis, the peptide bridges within a protein are cleaved by the action of strong acids, bases or catalysts and protein hydrolysates are obtained, that is to say mixtures of smaller fragments such as peptides and in some cases even amino acids. Depending on the type of preparation method, the protein hydrolysates contain other protein fragments. Since chemical hydrolysis using acids or bases occurs in a non-specific manner and also commonly leads to products with poorer qualities, especially in terms of color, odor and stability, to date enzymatic hydrolysis of proteins is the predominant method, particularly for cosmetic applications. In contrast to chemical hydrolysis, enzymatic hydrolysis occurs under moderate reaction conditions in terms of pH, temperature and pressure. Furthermore, the enzymes used are known for their specificity; i.e., in contrast to chemical hydrolysis the composition of the resultant protein hydrolysates is more uniform and thus leads to consistent product compositions.

Protein hydrolysates, such as the economically important keratin hydrolysates, are often prepared from natural sources of raw materials such as wool, hair, feathers, hoof or horn by chemical and/or enzymatic hydrolysis. Enzymes widely used in such applications include proteinases, in particular alkaline proteinases, such as those from Bacillus strains, such as Bacillus licheniformis, Bacillus alcaophilus, Bacillus subtilis, Bacillus mesentericus and Bacillus firmus as well engineered and/or optimized variants thereof.

However, all existing methods for the production of such protein hydrolysates suffer from the drawback that during the processing of the proteins undesired side products are formed. In particular nitrosamines formed are undesired in any product that is applied to or consumed by humans and animals, as these are known to be highly cancerogenic and genotoxic. It is thus generally desirable to minimize the concentrations of nitrosamines in any protein hydrolysate produced. Summary of the invention

The inventors have surprisingly found that many commercially available protein hydrolysates are contaminated with detectable amounts of nitrosamines and have developed an improved method for the production of protein hydrolysates which address this issue.

Specifically, the inventors have found that pretreatment of the protein before it undergoes the hydrolysis reaction with an oxidizing agent, such as peroxy compounds, leads to an oxidation of nitrite that is frequently found in such protein preparations to nitrate, which in turn minimizes the formation of nitrosamines (which require nitrite). It has further been found that such pretreatment prevents the accumulation of nitrite and as a result nitrosamines over the following processing steps of the protein. Still further, it has been found that such pretreatment can advantageously also inactivate enzymatic contaminants in the protein.

In a first aspect, the present invention is thus directed to a method for the production of a protein hydrolysate, the method comprising (a) contacting at least one protein with at least one oxidant, preferably a peroxide, at a concentration of 10 to <2000 ppm, preferably 10 to <1000 or 10 to <500 ppm, relative to the weight of the at least one protein, at a pH in the range of 2.0 to <7.0; and (b) subjecting the at least one protein of step (a) to a hydrolysis reaction to obtain a protein hydrolysate.

In various embodiments of these methods, the at least one protein (that is to undergo hydrolysis) is provided in form of a solution or suspension, typically an aqueous solution or suspension. The protein may have undergone some earlier processing steps or may be a protein raw material.

In various embodiments, the contacting step (a) is carried out at a temperature of up to 85°C, preferably up to 80 °C, more preferably 20 to 80°C.

In various other embodiments, the contacting step (a) is carried out at a temperature of up to 70°C, for example 5 to 65°C, such as 20 to 60°C.

The at least one oxidant may be at least one peroxide, such as an inorganic peroxide. Suitable compounds include, without limitation, hydrogen peroxide and peroxy acids and salts thereof, such as percarbonates and perborates. Preferred are hydrogen peroxide and hydrogen peroxide-generating precursor compounds.

The oxidant is typically used in concentrations of 10 to less than 2000 ppm, for example 20 to less than 1600, or 20 to less than 1500 or 30 to less than 1000 ppm, relative to the total weight of the protein. These amounts may also relate to hydrogen peroxide generated (in situ) by a chemical reaction of a precursor compound. In various embodiments, the concentration is 20 to 1500 ppm, or 30 to 1000 ppm, or 40 to 900 ppm. In various other embodiments, the oxidant is used in concentrations of 10 to less than 500 ppm, relative to the total weight of the protein. In various embodiments, the concentration is 10 to 300 ppm, preferably 20 to 250 ppm, more preferably 30 to 200 ppm.

In various embodiments, the pH in step (a) is in the range of from 3.0 to 6.5, 3.5 to 6.0, 4.0 to <7.0, 4.0 to 6.5, or 4.5 to 5.5. It is generally preferred that the pH is in the acidic range, i.e. less than 7 but at least 2, at least 2.5 or at least 3.0. The pH may be adjusted such that it is above or below the isoelectric point of the at least one protein.

The at least one protein may be a protein fraction with an isoelectric point (IEP) in the range of 4.0 to 5.5, preferably 4.1 to 5.4, more preferably 4.8 to 5.4.

In various embodiments, the pH is adjusted by addition of an acid, preferably an organic acid, such as citric acid or acetic acid, or an inorganic acid, such as hydrochloric acid or sulfuric acid. In various embodiments, the acid does not contain nitrogen atoms, e.g. is not nitric acid, nitrous acid or the like.

In various embodiments, the contacting step (a) reduces the nitrite in the at least one protein to less than 100, preferably less than 50 ppb. Accordingly, the protein obtained after step (a) does, in various embodiments, contain less than 100 ppb, preferably less than 50 ppb nitrite.

In various embodiments, after step (a) and prior to step (b), the at least one protein or the reaction mixture obtained in step (a) is adjusted to a pH of >7, preferably 7.5 to 10, and contacted with a reducing agent. In such embodiments, the reducing agent is used in an amount sufficient to reduce the complete oxidant remaining after step (a) and establishing reducing conditions. In various embodiments, the reducing agent is a sulfite (SO₃ ² ) or a bisulfite (HSO₃ ) or a metabisulfite (S₂O₅ ² ). The counterion may be any ion, but can be an alkali metal ion, such as sodium or potassium. The reducing agent is typically used in a concentration of 10 ppm to 2000 ppm relative to the total weight of reaction mixture or at least one protein.

In various embodiments, the hydrolysis reaction is an enzymatic reaction. In such an enzymatic reaction, the at least one protein is typically contacted with at least one protein-hydrolyzing enzyme under conditions that allow the hydrolysis reaction to occur. Alternatively, the at least one protein may also be subjected to highly acidic or highly alkaline conditions and temperatures that allow the hydrolysis to occur, also the enzymatic reaction is generally preferred. In some embodiments, both types of reaction may be combined in that first a chemical hydrolysis and then an enzymatic hydrolysis are carried out.

In the enzymatic hydrolysis reactions, the enzyme used is preferably a protease, preferably an endopeptidase. These enzymes may be of bacterial or fungal origin, optionally engineered to improve their properties. Suitable proteases are well known in the art and used widely for a variety of purposes, including laundry and home care applications in which they are used to remove proteinaceous stains. Such proteases are often alkaline proteases, i.e. they work best under alkaline conditions, and may be derived from Bacillus strains, such as Bacillus licheniformis, Bacillus alcaophilus, Bacillus subtilis, Bacillus mesentericus and Bacillus firmus. While the native enzymes can be used, to date a variety of optimized enzymes that have been engineered to improve catalytic activity and stability are available. One enzyme type that may be used for this purpose includes, without limitation, subtilisins, one prominent member being Alcalase®.

The enzymatic hydrolysis is preferably carried out under conditions that allow the enzymatic reaction to occur and are typically optimized for enzymatic activity. In various embodiments, the hydrolysis is thus carried out at a temperature of up to 60°C, preferably in the range of 20 to 50°C.

In some embodiments, the hydrolysis reaction includes contacting the at least one protein successively with two or more protein-hydrolyzing enzyme under conditions that allow the hydrolysis reaction to occur. For example, the first enzyme may be an endopeptidase and the second or subsequent enzyme used is an exopeptidase.

In various embodiments of the methods, after the hydrolysis step (b) the concentration of nitrosamines in the protein hydrolysate obtained is <200 ppb, preferably <100 ppb, more preferably <50 ppb (preferably relative to the weight of the protein hydrolysate, i.e. the entirety of protein fragments obtained). In various embodiments, the protein hydrolysate obtained after step (b) does also contain less than 1 mg/kg, preferably less than 0.1 mg/L or less than 0.1 mg/kg nitrite.

In the methods of the invention, the hydrolysis step may be followed by additional processing steps, if desired. Therefore, in some embodiments, after step (b) the protein hydrolysate is desalted, preferably by addition of calcium ions. The desalting may include precipitation (typically of the salts) and separation of the precipitate from the protein hydrolysate-containing supernatant.

In various embodiments, the hydrolysis step (b) or any of the individual hydrolysis steps (b), if more than one are carried out, is followed by another step (c) of contacting the protein hydrolysate with at least one oxidant, preferably a peroxide and/or preferably at a concentration of 10 to <2000 ppm, especially 10 to <1000 ppm, for example 10 to <500 ppm, relative to the weight of the protein hydrolysate, at a pH in the range of 2.0 to <7.0. This may be done before an optional desalting step or subsequently to it.

After a second oxidation step (c), if carried out, the protein hydrolysate obtained may be again adjusted to a pH >7, for example 7.5 to 10.0, and contacted with a reducing agent to reduce any remaining oxidant and establish reducing conditions. This can (again) be followed by a desalting step, as described above.

Once the desired protein hydrolysate is obtained, it may be further concentrated. Said concentration may be by evaporation of water, typically at elevated temperature.

In another aspect, the present invention relates to a protein hydrolysate obtained or obtainable by the methods described herein. These hydrolysates are characterized by a reduced level of nitrosamines relative to protein hydrolysates obtained by other methods. In various embodiments, the protein hydrolysates are not subjected to any other processing steps than those described herein. The concentration of nitrosamines in said protein hydrolysate may be <200 ppb, preferably <100 ppb, more preferably <50 ppb, even more preferably <25ppb, most preferably <10 ppb or <5 ppb. The concentration of nitrite in said protein hydrolysate may be less than 1 mg/kg, preferably less than 0.1 mg/L or less than 0.1 mg/kg.

In still another aspect, the present invention is directed to a composition comprising the protein hydrolysate of the invention or obtained or obtainable according to the methods of the invention. The composition may be a cosmetic, pharmaceutical, nutraceutical, food, beverage or animal feed composition.

In still another aspect, the invention is also directed to the use of an oxidant, preferably a peroxide, at a concentration of less than 2000 ppm, preferably 40 to 1000 ppm or less than 500 ppm, for treatment of a protein prior and/or subsequent to, preferably at least prior to, a hydrolysis reaction to reduce the concentration of nitrite and/or nitrosamines in the protein hydrolysate obtained.

Detailed description

Before describing preferred embodiments of the present invention in detail, definitions important for understanding the present invention are given.

As used in this specification and in the appended claims, the singular forms of "a" and "an" also include the respective plurals unless the context clearly dictates otherwise.

“At least one” as used herein, means one or more, i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more of the referenced species. Similarly, “one or more”, as used herein, relates to at least one and comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more. In connection with a given species, the term does not relate to the total number of molecules, but rather to the type of species. “At least one oxidant”, for example, thus means that one type of oxidant or two or more different types of oxidants may be present. In connection with amounts, the term relates to the total amount of the referenced species.

In the context of the present invention, the terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±10%, preferably ±5%.

It is to be understood that the term "comprising" is not limiting. Forthe purposes of the present invention, the term "consisting of is considered to be a preferred embodiment of the term "comprising of. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group, which preferably consists of these embodiments only. Furthermore, the terms "first", "second", "third" or "(i)", "(ii)", "(iii)", "(iv)" etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

When referring to compositions and the weight percent of the therein comprised ingredients it is to be understood that according to the present invention the overall amount of ingredients does not exceed 100% (± 1 % due to rounding).

If numerical values are given herein with no or a certain number of digits, it is understood that these values comprise all values with more digits that are rounded according to standard procedures to the given value. The value 2.5 thus includes the value 2.50 as well as any value in the range of 2.45 to 2.54.

Any amounts given herein relate to the total weight or mass, unless explicitly indicated to the contrary. The amounts “ppm” and “ppb” relate to parts per million and parts per billion, respectively, typically in terms of mass/weight. A value of <50 ppm thus means that the respective component is present in amounts of less than 50 parts per million in weight, i.e. less than 0.0001 % by weight. Similarly, ppb thus relates to 0.0000001 % by weight.

Numeric ranges specified in the format “from x to y” include the specified values. If multiple preferred numeric ranges are specified in this format, it is understood that all ranges created by combining the different endpoints are also included.

“Hydrogen peroxide-generating precursor”, as used herein, relates to compounds that release hydrogen peroxide by degrading or being degraded, such as sodium percarbonate. This may occur “in situ”, i.e. within the reaction mixture and as part of the methods.

The term “protein”, as used herein, relates to a polymer of amino acids that are linked by peptide bonds. Proteins are naturally occurring polymers and typically comprised of the natural 20 proteinogenic amino acids (all in L-configuration). “Hydrolysis” refers to breaking the peptide bonds between the amino acid units by using water as the nucleophile, thus releasing either individual amino acids or shorter (poly)peptide fragments of variable length. The term “endopeptidase”, as used herein, relates to protein cleaving enzymes that cleave within the chain and release two fragments of more than 1 amino acid in length. In contrast, the term “exopeptidase” relates to enzymes that cleave off single terminal amino acids from either the N- and/or C-terminal end of the protein. The enzymes used for such a hydrolysis reaction may have varying specificity for the cleavage site. It is however generally preferred to use enzymes with broad substrate specificities to allow hydrolysis of a plurality of different proteins into a plurality of smaller fragments. “Protein hydrolysate”, as used herein, relates to the product of a protein hydrolysis reaction. The hydrolysate comprises a mixture of protein fragments that have been generated by hydrolysis of the longer protein and can comprise free amino acids as well as peptide-bound amino acids with different molecular weights and compositions. In the context of the present invention, the term “protein fragments” is defined as a mixture of fragments obtained from the degradation of the original protein, for example keratin or any other protein. Such mixtures comprise free amino acids and peptide-bound amino acids. In the context of the present invention, the amino group of the amino acids may be present free, protonated or derivatized and the carboxyl group of the amino acid may be present free, deprotonated or derivatized in the protein hydrolysate. In various embodiments, proportions of at least 30% of the protein fragments obtained may have a molecular weight of less than 1000 Da and optionally more than 100 Da. In the context of the present invention, the molecular weight of proteins and protein fragments may be defined in Daltons (Da) and determined by liquid chromatography. In order to determine molecular weight distribution of protein fragments, the liquid chromatography apparatus Agilent 1260 Infinity with binary pump and degasser can be used in combination with PSS WinGPC UniChrom. PSS WinGPC UniChrom is a macromolecular chromatography data system with manufacturer-independent real-time data acquisition for comprehensive analysis of macromolecules, and is particularly suitable for the analysis of polymers, biopolymers and proteins. The chromatographic column used may be a specific chromatographic column (Superdex Peptide 10/300 GL from GE Healthcare Life Science, pore width 100 A, 5 pm particle size) for high-resolution separation of proteins and peptides. This column is particularly well-suited to determine biomolecules with molecular weights between 100 and 7000. Dilute hydrochloric acid (0.05 M) with a flow rate of 0.5 ml/min can be used as eluent. Detection can be carried out with a diode array detector (DAD) at 214 nm.

It is to be understood that this invention is not limited to the particular methodology, protocols, reagents etc. described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention that will be limited only by the appended claims.

In a first aspect, the present invention is thus directed to a method for the production of a protein hydrolysate, the method comprising (a) contacting at least one protein with at least one oxidant, preferably a peroxide, at a concentration of 10 to <2000 ppm or 10 to <500 ppm relative to the weight of the at least one protein, at a pH in the range of 2.0 to <7.0; and (b) subjecting the at least one protein of step (a) to a hydrolysis reaction to obtain a protein hydrolysate.

In various embodiments of these methods, the at least one protein (that is to undergo hydrolysis) is provided in form of a solution or suspension, typically an aqueous solution or suspension. The protein may have undergone some earlier processing steps or may be a protein raw material. The at least one protein may include two or more different proteins or may be a mixture of different proteins. The term is also intended to cover a mixture of a protein and various fragments thereof. Keratinous waste products such as hair or wool from animals, especially sheep's wool, may be used as starting material. Additionally or alternatively, plant proteins may be used, such as wheat, almond, soy or rice protein. Soy in the form of soy flour, soy flakes, soy milk, soy milk powder, soy protein powder and/or soy protein concentrate, especially soy protein powder, can be used as starting material. Rice in the form of rice flour, rice protein powder and/or rice protein concentrate, especially rice protein powder, can be used as starting material. The same applies to wheat and almond, which may be used in form of flour, flakes, powder or protein concentrate. It is understood that these proteins are exemplary only and a plethora of other proteins, either from animal, plant or microbial sources or even recombinant sources may be used.

The oxidative pretreatment step (a) differs from other pretreating steps that have been described in the art, such as WO 2018/050483 A1 using, for example, 5g of a 30% solution of hydrogen peroxide per 10 g of protein, in that it is carried out with a significantly lower concentration of oxidant, since it has been found that higher concentrations of oxidant rather have a negative effect in that the species that cause the formation of nitrosamines, in particular nitrite, are produced. It also differs from known methods that use the oxidant to chemically hydrolyze a protein. For such methods again much higher concentrations of the oxidant than those disclosed herein are required. It is thus important to keep the concentration of the oxidant within the given limits to ensure that nitrite present in the protein (composition) is oxidized to nitrate but that no extensive oxidation of other moieties occurs.

Accordingly, the concentration of the oxidant is kept below 2000 ppm, preferably below 1900 ppm, below 1800 ppm, below 17000 ppm, below 1600 ppm, below 1500 ppm, below 1400 ppm, below 1300 ppm, below 1200 ppm, below 1100 ppm, below 1000 ppm, below 900 ppm, or below 800 ppm. In some embodiments, it may be even below 700 ppm, below 600 ppm, below 500 ppm, below 450 ppm, below 400 ppm, below 350 ppm, below 300 ppm, below 250 ppm or below 200 ppm. The minimum concentration may be 10 ppm, for example 15 ppm, 20 ppm, 25 ppm or 30 ppm, such as 35 ppm, 40 ppm, 45 ppm, 50 ppm, 55 ppm, 60 ppm, 65 ppm, 70 ppm, 75 ppm, 80 ppm, 85 ppm, 90 ppm, 95 ppm or 100 ppm. Preferred ranges are thus 10 to 1900 ppm, 20 to 1600, 20 to 1500 ppm, 30 to 1200 ppm, 40 to 1000 ppm, and 50 to 800 ppm. In some embodiments, the range may be 10 to 300 ppm, 15 to 250 ppm, 20 to 250 ppm, 30 to 250 ppm, or 30 to 200 ppm. The concentration of the oxidant given relates to the total mass of the at least one protein used. Alternatively, it may also relate to the protein solution or suspension used in step (a). The concentration of the oxidant is typically low enough to prevent substantial hydrolysis of the protein by the oxidant, i.e. afterthe treatment with the oxidant typically less than 10 wt.-% of the total protein are hydrolyzed. The amount of oxidant used may depend on the type of protein used and may be adapted accordingly within the given ranges. In various embodiments, the concentration of oxidant may be 100 to 1000 ppm, for example 300 to 800 ppm, in particular for wheat, rice or soy protein. In various embodiments, the concentration of oxidant may be 500 to 1600 ppm, for example 900 to 1600 ppm, in particular for almond or sheep wool protein.

Another important parameter is the pH. It has been found by the inventors that it is highly important that the pH during said oxidative pretreatment is kept in the acidic range, i.e. below 7. Therefore, the pH is preferably <7, such as 6.9 or less, 6.8 or less, 6.7 or less, 6.6 or less, 6.5 or less, 6.4 or less, 6.3 or less, 6.2 or less, 6.1 or less, 6.0 or less, 5.9 or less, 5.8 or less, 5.7 or less, 5.6 or less or 5.5 or less. The pH minimum is typically about 2.0, for example at least 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8., 2.9, or 3.0. In various embodiments, the pH range is thus within the range of 2.0 to 6.5, 3.0 to 6.5, 3.5 to 6.0, 4.0 to <7.0, 4.0 to 6.5, 4.0 to 6.0, 4.0 to 5.5, or 4.5 to 5.5.

The pH may be adjusted within the given range such that it is above or below the isoelectric point of the at least one protein. This may help to ensure that the protein is at least partially denatured or unfolded. The at least one protein may be a protein fraction with an isoelectric point (IEP) in the range of 4.0 to 5.5, for example 4.1 to 5.4, or 4.2 to 5.3, or 4.3 to 5.2, or 4.5 to 5.5, or 4.6 to 5.5, or 4.7 to 5.5, or 4.8 to 5.4.

The pH may be adjusted by any known pH adjustment agent, such as an acid or base. In various embodiments, this is done by addition of an acid, preferably an organic acid, such as citric acid or acetic acid, or an inorganic acid, such as hydrochloric acid or sulfuric acid. In various embodiments, the acid does not contain nitrogen atoms, e.g. is not nitric acid, nitrous acid or the like. It may further be preferred that the anion can be later removed in a desalting step, for example by precipitation with alkaline earth metal ions.

The pH is typically measured in form of an aqueous solution or suspension, for example as used in step (a). If the at least one protein is not provided in form of such a solution or suspension, it may be brought into such an aqueous solution/suspension and the pH then measured. In various embodiments, the pH may be measured as a 20%, 10%, 5% or 1 % solution/suspension of the at least one protein in deionized water. It is generally measured under standard conditions, i.e. at 20°C and 1013 mbar.

Step (a), i.e. the oxidative pretreatment, is typically carried out at a temperature of up to 85°, for example up to 82°C or up to 80°C. In some embodiments, the temperature may be up to 70°C, for example up to 65, up to 60, up to 55, or up to 50°C. The lower limit may be 5, 10, 15 or 20°C. Preferred ranges are 5 to 85°C, preferably 20 to 80°C. Other ranges are 5 to 65°C and 20 to 60°C.

The pretreatment with the oxidant may be carried out for minutes to several hours, such as at least 10 minutes, at least 30 minutes or at least 1 h. The treatment may be longer, for example at least 2, at least 4, at least 6 or at least 8 hours.

The at least one oxidant can be selected from any suitable oxidant. It is an important that it is capable of oxidizing essentially all nitrite ions present in the at least one protein (composition) to nitrate, i.e. of causing the nitrification reaction. Such oxidants may be selected from the group of peroxides, such as an inorganic peroxide. Suitable compounds include, without limitation, hydrogen peroxide and peroxy acids and salts thereof. Preferred are hydrogen peroxide and hydrogen peroxide-generating precursor compounds, such as percarbonates and perborates. For the latter, it is known that once they come into contact with water, such as in the protein solution, they degrade and form hydrogen peroxide. It may however be preferred to use hydrogen peroxide as such, for example in form of an aqueous solution, such as a 30%, 35%, 40%, 45%, 50%, 55% or 60% (v/v) aqueous solution. The preferred amounts for the oxidant given above also imply to the peroxy compounds and in particular hydrogen peroxide. In case hydrogen peroxide-generating compounds are used, the concentrations given may alternatively also relate to the amount of hydrogen peroxide produced.

It is particularly preferred that in step (a) the nitrite concentration in the at least one protein (composition/formulation) is reduced as much as possible. This means that it is preferred that the nitrite content of the protein composition after step (a) is less than 100, less than 90, less than 80, less than 70, less than 60, or less than 50 ppb. It may be even lower than that, such as less than 40, less than 30, or less than 20 ppb. Accordingly, the protein obtained after step (a) does, in various embodiments, contain less than the above given amounts of nitrite. As described above, the level of nitrite is typically reduced by its oxidation to nitrate. The nitrate content is less critical, as nitrosamines are not as readily formed as from nitrite.

Once the oxidation pretreatment of step (a) has been completed, the at least one protein (composition/formulation) obtained in step (a) can be pH adjusted to a pH of >7, for example 7.1 or more, 7.2 or more, 7.3 or more, 7.4, or more, or 7.5 or more, for example to 7.5 to 12.0, or 7.5 to 11 .0, or 7.5 to 10.0, or 7.5 to 9.5, or 7.5 to 9.0, or 8.0 to 9.0, for example to about 7.5, 8.0, 8.5 or 9.0. This may be achieved by adding a base, for example an alkaline metal hydroxide, such as sodium or potassium hydroxide or an aqueous solution thereof. The at least one protein (composition/formulation) may after step (a) alternatively or additionally be contacted with a reducing agent. The reducing agent may be used in an amount sufficient to reduce the complete amount of oxidant remaining after step (a), preferably in a surplus, i.e. in excess beyond the amount necessary to achieve said reduction of the oxidant. It is important in this step to establish reducing conditions before the hydrolysis reaction is carried out. Preferred reducing agents used for this purpose are sulfites (SO₃ ² ), bisulfite (HSO₃ ), and metabisulfite (S₂O₅ ² ) as well as combinations thereof. The counterion may be any ion, but can be an alkali metal ion, such as sodium or potassium. The reducing agents is typically used in a concentration of 10 ppm to 2000 ppm relative to the total weight of the reaction mixture (of step (a)) or at least one protein. The hydrolysis reaction may be started directly after the pH has been adjusted and/or the reducing agent added.

In various embodiments, the hydrolysis is performed by using an enzyme. In the enzymatic hydrolysis reactions, the enzyme used is preferably a protease, preferably an endopeptidase. These enzymes may be of bacterial or fungal origin, optionally engineered to improve their properties. Suitable proteases are well known in the art and used widely for a variety of purposes, including laundry and home care applications in which they are used to remove proteinaceous stains. A class of enzymes widely used and particularly suitable for this purpose are the subtilisins. The endopeptidases are preferably selected from those derived from Bacillus strains, especially Bacillus licheniformis, Bacillus alcaophilus, Bacillus subtilis, Bacillus mesentericus or Bacillus firmus. While the native enzymes can be used, to date a variety of optimized enzymes that have been engineered to improve catalytic activity and/or stability are available. One enzyme type that may be used for this purpose includes, without limitation, subtilisins. Those endopeptidases from Bacillus licheniformis that are commercially available, for example as Alcalase® from Novozymes, are especially suitable.

The enzymatic hydrolysis can be carried at an alkaline pH in the range from 8 to 9. This is particularly suitable as most of the enzymes suitable for the hydrolysis are alkaline proteases, i.e. enzymes that have a pH optimum at slightly alkaline pH values between 7.5 and 10.0, such as 8.0 to 9.0.

In such an enzymatic hydrolysis reaction, the at least one protein obtained after the pretreatment in step (a) is contacted with at least one protein-hydrolyzing enzyme under conditions that allow the hydrolysis reaction to occur and are preferably optimized for enzymatic activity. In various embodiments, the hydrolysis is thus carried out at a temperature of up to 60°C, preferably in the range of 20 to 50°C. The temperature and time may be adapted based on the enzyme used and desired degree of hydrolysis.

In some embodiments, the hydrolysis reaction may include contacting the at least one protein, preferably obtained in step (a), simultaneously or successively with two or more protein-hydrolyzing enzyme under conditions that allow the hydrolysis reaction to occur. Two enzymes may for example be combined to allow the hydrolysis of various different proteins that may be present. If two enzymes are used successively, the first enzyme may be an endopeptidase and the second or subsequent enzyme used may be an exopeptidase. Examples for such reactions are described in international patent publications WO 2018/050483 A1 and WO 2018/068947 A2.

It has been found that by performing the methods as described herein, the concentration of nitrosamines in the protein hydrolysate obtained after step (b) may be reduced to <200 ppb, preferably 150 ppb or less, more preferably 100 ppb or less, even more preferably 50 ppb or less, most preferably less than 50 ppb, or less than 30 ppb or less than 10 ppb, with the amounts preferably being relative to the weight of the protein hydrolysate, i.e. the entirety of protein fragments obtained, or the dry weight of the protein hydrolysate. Similarly, the concentration of nitrite in the protein hydrolysate obtained after step (b) may be reduced to <0.1 mg/L or <0.1 mg/kg relative to the weight of the protein hydrolysate, i.e. the entirety of protein fragments obtained, or the dry weight of the protein hydrolysate.

If the concentration of nitrite and/or nitrosamines is still undesirable high, any of the individual hydrolysis steps (b), if more than one are carried out, can be followed by another step (c) of contacting the protein hydrolysate with at least one oxidant, preferably a peroxide and/or preferably at a concentration of 10 to <2000 ppm or 10 to <500 ppm relative to the weight of the protein hydrolysate, at a pH in the range of 2.0 to <7.0. This means that step (a) is essentially repeated for the protein hydrolysate. All the above preferred embodiments for step (a) similarly apply to this step (c), in particular as regards oxidant concentration, pH value , temperatures and reaction times. Once this step has been completed, the protein hydrolysate may be again adjusted to a pH >7, for example 7.5 to 10.0, and contacted with a reducing agent to reduce any remaining oxidant and establish reducing conditions. Here again the above-described preferred embodiments and conditions for said adjustment step apply. In the methods of the invention, each of the above-described steps, such as the oxidative pretreatment step, the hydrolysis step and/or any subsequent oxidation or adjustment step may be followed by one or more additional processing steps, if desired. In various embodiments, after step (b) the protein hydrolysate is therefore subjected to a desalting step, preferably by precipitation with a precipitating agent, for example containing alkaline earth metal ions. The precipitate, for example water-insoluble salts, may then be separated from the protein hydrolysate-containing supernatant, for example by filtration or centrifugation or any other suitable method. Such desalting steps are preferably carried out after the hydrolysis step, and if carried out, any oxidation and reduction steps carried out after the hydrolysis has occurred.

The protein hydrolysate is typically obtained in form of an aqueous solution or suspension and may be further concentrated to remove excess solvent, such as water. Said concentration may be done by any conventional means known to those skilled in the art, including distillation, such by evaporation of water, for example at elevated temperature and/or reduced pressure. Typical dry mater contents are in the range of 10 to 50 wt%, for example 15 to 30 wt%. It is also possible to provide the protein hydrolysates in dry, for example powder, form.

It may be preferred that the protein hydrolysate is not subject to any other processing steps than those described herein. Accordingly, the methods of the invention may, in various embodiments, consist of steps (a) and (b) and, optionally, any one or more of the additional steps described herein but no further steps not described herein, in particular no further purification steps that aim to remove nitrosamines.

In another aspect, the present invention relates to a protein hydrolysate obtained or obtainable by the methods described herein. These hydrolysates are characterized by a reduced level of nitrosamines relative to protein hydrolysates obtained by other methods. In various embodiments, the protein hydrolysates are not subjected to any other processing steps than those described herein. The concentration of nitrosamines in said protein hydrolysate may be <200 ppb, preferably <150 ppb, more preferably <100 ppb, even more preferably <50 ppb, still more preferably <25 ppb, most preferably <10 ppb or <5 ppb. The concentration of nitrite in said protein hydrolysate may be < 1 mg/kg, <0.1 mg/L or <0.1 mg/kg.

In still another aspect, the present invention is directed to a composition comprising the protein hydrolysate of the invention or obtained or obtainable according to the methods of the invention. The composition may be a cosmetic, pharmaceutical, nutraceutical, food, beverage or animal feed composition. The protein hydrolysate concentration said compositions may be in the range of from 0.001 to 5.0, for example 0.001 to 1 .0, such as 0.002 to 0.5 wt% based on dry protein hydrolysate weight and total weight of the composition.

Protein hydrolysates are widely used in cosmetic compositions, in particular hair care compositions. In these compositions and any other compositions disclosed herein, the protein hydrolysates may be comprised in combination with any one or more of additional components, such as other actives, auxiliaries, solvents, stabilizers and the like. Typical components that may additionally be included in these compositions comprise, without limitation, surfactants, emulsifiers, co-surfactants, (cationic) polymers, oil carriers, opacifiers, pearlescent waxes, thickeners, rheology modifiers, stabilizers, waxes, lipids, phospholipids, biogenic actives, vitamins, antioxidants, film forming agents, hydrotropes, solvents, conservatives, biocides, bittering agents, silicones, swelling agents, anti-dandruff agents, UV filters, perfumes and fragrances, colorants and the like.

In still another aspect, the invention is also directed to the use of an oxidant, preferably a peroxide, at a concentration of less than 500 ppm for treatment of at least one protein prior or subsequent to a hydrolysis reaction to reduce the concentration of nitrite and/or nitrosamines in the protein hydrolysate obtained.

All embodiments disclosed herein in relation to the methods of the invention are similarly applicable to the protein hydrolysate as such, the compositions and uses of the invention and vice versa.

Examples

Example 1

1500 kg of wheat gluten (Roquette) were dispersed in 9500 kg of water at 80°C and heated to 85°C for 30 minutes. It was then cooled to 70°C. The pH of the obtained dispersion was 5.6 and a nitrite content of 1 ppm was measured (MQuant nitrite test 0.6-24 ppm Merck KGaA).

2.0 kg of 50% hydrogen peroxide solution was added (667 ppm of peroxide relative to the wheat gluten used) as follows: 1 .5 kg hydrogen peroxide 50% solution (Brenntag GmbH) was added to the dispersion, and the pH was lowered to 4.0 by adding 35 kg anhydrous citric acid (Jungbunzlauer). The mixture was stirred for 30 minutes. During stirring two further partial doses of 0.25 kg hydrogen peroxide 50% each were added. After 30 minutes no nitrite was detectable.

After peroxide treatment the pH was adjusted to 8.5 to 9.0 and 1 kg sodium disulfite (Staub & Co. Silbermann) was added to establish reductive conditions. Sulfite content was maintained throughout further processing at 10-40 ppm (MQuant sulfite test 10-400 ppm Merck KGaA).

The protein dispersion was then subjected to enzymatic hydrolysis followed by purification/filtration and concentration according to established routine protocols.

The resulting protein hydrolysate had a dry matter concentration of 40% and a total N-nitroso content measured by ATNC method (apparent total nitroso content) of < 45 ppb.

Example 2 1500 kg of wheat gluten (supplied by Roquette) were dispersed in 9500 kg of water at 80°C and heated to 85°C for 20 minutes and then cooled to 70°C. The pH of the dispersion was 5.5 and a nitrite content of 1 ppm was measured (MQuant nitrite test 0.6-24 ppm Merck KGaA). The pH was reduced to 4.1 by adding 28 liters of 32% hydrochloric acid (TIB Chemicals AG).

1 .75 kg of 50% hydrogen peroxide solution was added (583 ppm peroxide based on the wheat gluten used) as follows: 1.5 kg of hydrogen peroxide 50 % solution (Brenntag GmbH) was added, and the mixture stirred for 30 minutes. During stirring a further partial dose of 0.25 kg of hydrogen peroxide 50 % was added. After 30 minutes no nitrite was detectable.

After this treatment the pH was adjusted to 8.5 to 9.0 and 1 kg sodium disulfite (Staub & Co. Silbermann) was added to establish reductive conditions. Sulfite content was maintained throughout further processing at 10-40 ppm (MQuant sulfite test 10-400 ppm Merck KGaA).

The protein dispersion was then subjected to a enzymatic hydrolysis followed by purification/filtration and concentration according to established routine protocols.

Example 3

The following results were obtained with different protein sources under otherwise analogue conditions:

Limit of quantification

Total N-nitroso content is expressed as N-nitroso diethanolamine (NDELA)

Comparative Example 1

1500 kg of wheat gluten (supplied by Roquette) were dispersed in 9500 kg of water at 75°C and stirred at 75°C for 15 minutes and then cooled to 59°C. The pH was adjusted to 8.5 to 9.0 and 1 kg sodium disulfite (Staub&Co. Silbermann) was added to establish reductive conditions. Sulfite content was maintained throughout further processing at 10-40 ppm (MQuant sulfite test 10-400 ppm Merck KGaA). The protein dispersion was then subjected to an enzymatic hydrolysis followed by purification/filtration and concentration according to established routine protocols. The resulting protein hydrolysate had a dry matter concentration of 40% and a total N-nitroso content measured by ATNC method (apparent total nitroso content) of 130 ppb. With the exception of the treatment with hydrogen peroxide, all other steps were identical to those carried out in Examples 1-3.

Claims

1. Method for the production of a protein hydrolysate, the method comprising

(a) contacting at least one protein with at least one oxidant, preferably a peroxide, at a concentration of 10 to <2000 ppm relative to the weight of the at least one protein, at a pH in the range of 2.0 to <7.0; and

(b) subjecting the at least one protein of step (a) to a hydrolysis reaction to obtain a protein hydrolysate.

2. The method of claim 1 , wherein in step (a) said at least one protein is provided in form of a solution or suspension, preferably an aqueous solution or suspension.

3. The method of claim 1 or 2, wherein the contacting step (a) is carried out at a temperature of up to 85°C, preferably up to 80°C, more preferably 20 to 80°C.

4. The method of any one of the preceding claims, wherein

(1) the at least one peroxide is an inorganic peroxide, preferably selected from hydrogen peroxide and peroxy acids, more preferably from hydrogen peroxide and hydrogen peroxide-generating precursor compounds; and/or

(2) the concentration of the at least one oxidant is 20 to 1500 ppm, or 30 to 1000 ppm, or 40 to 900 ppm.

5. The method of any one of the preceding claims, wherein the pH is in the range of from 3 to 6.5, 3.5 to 6.0, 4.0 to <7.0, 4.0 to 6.5, or 4.5 to 5.5.

6. The method of any one of the preceding claims, wherein the pH is adjusted by addition of an acid, preferably an organic acid, preferably citric acid or acetic acid, hydrochloric acid, or sulfuric acid.

7. The method of any one of the preceding claims, wherein after step (a) and prior to step (b), the at least one protein is adjusted to a pH of >7, preferably 7.5 to 10, and contacted with a reducing agent, wherein the reducing agent is preferably used in an amount sufficient to reduce the complete oxidant remaining after step (a) and establishing reducing conditions.

8. The method of claim 7, wherein

(1) the reducing agent is a sulfite or bisulfite, preferably bisulfite.

(2) the reducing agent is used in a concentration of 10 ppm to 2000 ppm.

9. The method of any one of the preceding claims, wherein the hydrolysis reaction is an enzymatic reaction and includes contacting the at least one protein with at least one protein-hydrolyzing enzyme under conditions that allow the hydrolysis reaction to occur.

10. The method of claim 9, wherein the hydrolysis is carried out at a temperature of up to 60°C, preferably in the range of 20 to 50°C.

11 . The method of any one of the preceding claims, wherein after step (b) the protein hydrolysate is desalted, preferably by addition of calcium ions, wherein the desalting preferably includes precipitation and separation of the precipitate from the protein hydrolysate-containing supernatant.

12. The method of any one of the preceding claims, wherein the hydrolysis step (b) or any of the individual hydrolysis steps (b) is followed by another step (c) of contacting the protein hydrolysate with at least one oxidant, preferably a peroxide and/or preferably at a concentration of 10 to <2000 ppm relative to the weight of the protein hydrolysate, at a pH in the range of 2.0 to <7.0, wherein after the second oxidation step (c) the protein hydrolysate is preferably adjusted to a pH >7, for example 7.5 to 10.0, and contacted with a reducing agent to reduce any remaining oxidant and establish reducing conditions.

13. Protein hydrolysate obtainable by the method of any one of claims 1 to 12, wherein the concentration of nitrosamines in said protein hydrolysate is <200 ppb, preferably <100 ppb, more preferably <50 ppb and/or the concentration of nitrite in said protein hydrolysate is <1 mg/kg, preferably <0.1 mg/L or <0.1 mg/kg.

14. Composition comprising the protein hydrolysate of claim 13, wherein the composition preferably is a cosmetic, pharmaceutical, nutraceutical, food, beverage, or animal feed composition.

15. Use of an oxidant, preferably a peroxide, at a concentration of less than 2000 ppm, preferably 40 to 1600 or 40 to 1000 ppm, for treatment of a protein prior and/or subsequent to, preferably at least prior to, a hydrolysis reaction to reduce the concentration of nitrite and/or nitrosamines in the protein hydrolysate obtained.