HK1128202B - Process flavours with low acrylamide - Google Patents

Description

Processing flavoring agent with low acrylamide

Technical Field

The present invention relates to yeast extracts, autolysed yeasts, protein hydrolysates and methods for their production, and their use in food or feed or food or feed ingredients. The invention also relates to a process flavour (process flavour), a process for its production and its use in food or feed or food or feed ingredients.

Background

Processed foods and instant meals are increasingly used in our society. Typically, several types of flavoring (flavouring), such as hydrolysed vegetable proteins, yeast extracts, cheese, spices (balls) and the like, are added to these types of food products during or after processing to make the food products more palatable. It is known that the flavour of food products is caused by a complex combination of different reaction pathways that occur during cooking. A problem that may be encountered in the production of processed foods or instant meals is that the heating step in the production process may not be long enough to develop a satisfactory aroma. However, flavor defects that may be encountered in such food products can be filled by adding a processing flavoring agent (also referred to as a "reaction flavoring agent") to the processed food product. Process flavours are typically added to the processed food after the main processing steps are completed.

The term "process flavour" is used throughout the present specification for compositions having a unique flavour, for example meat flavours, which are obtainable by the following process: the mixture of ingredients comprising at least the compound containing nitrogen in amino form, preferably at least the reducing carbohydrate, is heated at a pH, temperature, pressure and reaction time sufficient to develop the fragrance. The ingredient mixtures used in the process for producing the flavoring agents may also contain one or more lipids, sulfur-containing compounds, carbonyl-containing compounds, and the like.

Processing flavours is obtained by a complex combination of reaction pathways that occur between the ingredients during the heating step. For an overview of several reaction pathways involved in the production of process Flavours, see for example "Savory flavors", 1995, t.w.nagoda within ana, Esteekay associates inc., Wisconsin, USA, page 103-.

In the process flavours produced according to the invention, the "nitrogen compound containing the amino form" can be obtained from a source selected from yeast extract, autolysed yeast, protein hydrolysate or a mixture of one or more of these ingredients, optionally in combination with one or more supplemental amino acids.

Acrylamide has long been produced commercially for a variety of technical applications, which is believed to be potentially carcinogenic to animals and humans. In 1991, the Scientific Committee on Food was studied for the contact of acrylamide with Food materials and evaluated to conclude that acrylamide is a genotoxic carcinogen.

Recently, the presence of acrylamide in various foods and baked foods (tare et al chem. res. toxicol.13, 517-. Further studies revealed that considerable amounts of acrylamide can be detected in a variety of baked, fried and roasted food products, indicating that the presence of acrylamide in the food product is a result of the baking process.

The presence of acrylamide in process flavours (which are subsequently used in many types of food) is highly undesirable. Surprisingly, it has now been found that unfortunately, process flavours can contain considerable amounts of acrylamide (which can be as high as, for example, 10000 ppb). Acrylamide in process flavours may even be generated at temperatures below 120 ℃, where acrylamide formation is not at all expected. The problem of the presence of acrylamide in process flavours is not known at present.

Yeast extracts, autolysed yeast and protein hydrolysates can advantageously be used as amino acid sources for the production of process flavours.

The applicant has now surprisingly found that high levels of acrylamide may be produced during the production of process flavours using conventional yeast extracts, conventional autolysed yeast and or conventional protein hydrolysates as the amino acid source.

The invention therefore also relates to novel yeast extracts, novel autolysed yeasts and novel protein hydrolysates suitable for the production of process flavours with low acrylamide. Furthermore, the present invention relates to novel process flavours with low acrylamide.

Detailed Description

The present invention relates in a first aspect to a yeast extract having no more than 1mg/g free asparagine on dry matter, preferably no more than 0.2mg/g, more preferably no more than 0.1 mg/g. The amount of free asparagine in the yeast extract according to the first aspect of the invention may be as low as-0 mg/g.

Yeast extract is defined as a composition comprising water soluble components extracted from yeast cells. Typically, yeast extracts comprise amino acids, proteins, peptides, vitamins, carbohydrates and salts (e.g. phosphates). The yeast extract may also comprise 5' -ribonucleotides. Yeast extracts can be divided, for example, into autolytic and hydrolytic yeast extracts.

Autolytic yeast extracts are concentrates of soluble material obtainable from yeast after cell disruption and digestion (lysis) of polymeric yeast material. Active yeast enzymes released in the medium after cell disruption act to lyse. These types of yeast extracts are rich in amino acids and usually do not contain 5 '-ribonucleotides because the natural RNA is broken down or modified during the autolytic process into a form that cannot be degraded into 5' -ribonucleotides. They are used in the food industry as basic taste providers. The amino acids present in the yeast extract add a bouillon (bouillon) -type bouillon (brothy) taste to the food product.

Hydrolytic yeast extracts are concentrates of soluble materials which can be obtained from yeast after cell disruption, digestion (lysis) and addition of extraneous enzymes (such as proteases and/or peptidases and especially nucleases, such as 5 '-phosphodiesterase and optionally 5' -adenylate deaminase) to the yeast suspension during lysis. The yeast enzymes themselves are usually inactivated prior to lysis. During this process, 5 ' -ribonucleotides of guanine (5 ' -guanine monophosphate; 5 ' -GMP), uracil (5 ' -uracil monophosphate; 5 ' -UMP), cytosine (5 ' -cytosine monophosphate; 5 ' -CMP) and adenine (5 ' -adenine monophosphate; 5 ' -AMP) may be formed. When 5 '-adenylate deaminase is added to the mixture, 5' -AMP is converted to 5 '-inosine monophosphate (5' -IMP). The hydrolysed yeast extract obtained by this process is therefore rich in 5 ' -ribonucleotides, in particular rich in 5 ' -GMP and 5 ' -IMP. Typically, yeast extracts are also rich in monosodium glutamate (MSG). 5 '-IMP, 5' -GMP and MSG are known to have flavour enhancing properties. They can enhance a palatable and pleasant taste in certain types of food. This phenomenon is described as "mouthfeel" or umami (umami).

In one embodiment of the first aspect of the invention, the yeast extract may be an autolysed yeast extract or a hydrolysed yeast extract or a mixture thereof. The yeast extract may comprise 5' -ribonucleotides. The term "5 ' -ribonucleotides" means herein a mixture of 5 ' -GMP, 5 ' -CMP, 5 ' -UMP, and also 5 ' AMP and/or 5 ' IMP, wherein the 5 ' IMP in the mixture is obtained by partial or complete conversion of 5 ' AMP to 5 ' IMP.

In a second aspect, the present invention relates to an autolysed yeast having a free asparagine of not more than 1mg/g, preferably not more than 0.2mg/g, more preferably not more than 0.1mg/g on a dry matter basis. The amount of free asparagine in the autolysed yeast extract may be as low as-0 mg/g.

Autolytic yeast is a precursor of autolytic yeast extracts. It comprises a concentrate of soluble material obtainable from yeast after cell disruption and digestion (lysis) of polymeric yeast material, wherein active yeast enzymes released in the culture medium after cell disruption act to lyse, and insoluble matter formed during lysis, which is mainly due to degraded yeast cell wall fraction.

In a third aspect, the present invention relates to a protein hydrolysate having no more than 1mg/g free asparagine, preferably no more than 0.2mg/g, more preferably no more than 0.1mg/g on a dry matter basis.

Protein hydrolysates are acid-or enzymatically-treated protein substrates that contain a mixture of amino acids and peptides in varying proportions, which are determined by the degree of hydrolysis and/or the type of enzyme used. Typical protein substrates for the preparation of protein hydrolysates are plant proteins, such as wheat gluten, corn gluten, soy protein, canola protein, pea protein, alfalfa protein, sunflower protein, broad bean (fabaceous) protein, cotton or sesame seed protein, corn gluten, barley protein, sorghum protein, potato protein, rice protein, coffee protein. Other possible protein substrates are animal proteins such as milk proteins (e.g., casein, whey protein), egg proteins, fish proteins, meat proteins including gelatin, collagen, blood proteins (e.g., hemoglobin), hair, feathers, and fish meat.

The yeast extract, autolysed yeast or protein hydrolysate may be in any form, for example dissolved in a liquid or dried. Typically, the yeast extract, autolysed yeast or protein hydrolysate will be in dry form, e.g. in powder or granular form.

In a fourth aspect, the present invention relates to a process for producing a yeast extract of the first aspect, an autolysed yeast of the second aspect or a protein hydrolysate of the third aspect, said process comprising treating a starting yeast extract, a starting autolysed yeast or a starting protein hydrolysate, which all contain free asparagine, with an enzyme, with a physical method, with a chemical method or with a combination thereof, said enzyme, physical method, chemical method or combination thereof being capable of reducing the amount of free asparagine in the final product and preferably to obtain in the final product no more than 1mg/g, more preferably no more than 0.2mg/g, most preferably no more than 0.1mg/g of free asparagine on a dry matter basis.

The starting yeast extract, the starting autolysed yeast or the starting protein hydrolysate, all containing free asparagine, are also referred to as "starting products" throughout the present specification. The starting products to be used in the process of the invention may be commercially available products, may be the end products of the preparation process or may be intermediate products obtained in steps of the preparation process for the end products. Thus, in one embodiment of the invention, the treatment with enzymes, with physical means, with chemical means or with a combination thereof is carried out on a free asparagine-containing intermediate obtained in a step of a preparation process for the preparation of a yeast extract or of an autolysed yeast from a yeast cell or in a step of a preparation process for the preparation of a protein hydrolysate from a protein substrate.

The starting product to be used in the process of the invention may be any starting product containing free asparagine as described above. The starting product may be in any form, for example dissolved in a liquid or dried. Typically, the starting product will be in dry form, e.g., powder or granular form.

In one embodiment of the method according to the fourth aspect of the invention, the treatment with an enzyme, physical means, chemical means or a combination thereof capable of reducing the amount of free asparagine is performed on the starting product, preferably after suspending and/or dissolving it in a suitable solvent, wherein the starting product is a commercially available or end product of the manufacturing process yeast extract, autolysed yeast or protein hydrolysate. In general, the solvents may be those suitable for allowing the enzyme to react with free asparagine or suitable for use in physical or chemical processes. Generally, the solvent may be a water-based solvent, more preferably, the solvent is water.

In the context of the present invention, an "enzyme, physical process, chemical process or combination thereof capable of reducing the amount of free asparagine" is any enzyme or mixture of enzymes, any physical process or any chemical process or combination thereof capable of removing all or part of the free asparagine and/or capable of degrading asparagine to a form which does not result in the formation of acrylamide when heated in the presence of a reducing sugar.

Physical methods that can be used in the treatment to reduce the amount of free asparagine are methods that remove some or all of the free asparagine from the starting product. Such methods may include, for example, the use of separation techniques, such as chromatographic techniques. Chemical methods are methods that modify and/or degrade free asparagine to a form that does not result in the formation of acrylamide. Such methods may include, for example, using chemical reactions such as oxidation, reduction, deamination.

In a preferred embodiment of the method of the fourth aspect of the invention, the treatment is carried out with an enzyme capable of reducing the amount of free asparagine in the final product under conditions of pH, temperature and reaction time sufficient for the enzyme to react with free asparagine.

Preferably, the treatment with the enzyme is with an enzyme capable of modifying the free asparagine side chain, more preferably with an enzyme capable of hydrolyzing the amide group of the free asparagine side chain, even more preferably with asparaginase (EC3.5.1.1).

The enzyme or enzyme mixture used in a preferred embodiment of the method of the third aspect of the invention may be obtained from a variety of sources, for example, from plants, animals and microorganisms (e.g., bacteria, fungi or yeast). Examples of suitable microorganisms are species of Escherichia, Erwinia, Streptomyces, Pseudomonas, Aspergillus and Bacillus. An example of a suitable Escherichia strain is Escherichia coli. An example of a suitable Erwinia strain is Erwinia chrysanthemi. Examples of suitable Streptomyces strains are Streptomyces lividans or Streptomyces murinus. Examples of suitable Aspergillus strains are Aspergillus oryzae, Aspergillus nidulans or Aspergillus niger. Examples of suitable Bacillus strains are Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coemulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis or Bacillus thuringiensis. Suitable methods for producing asparaginase from strains of Bacillus, Streptomyces, Escherichia or Pseudomonas are described in WO 03/083043. Preferably, the asparaginase is obtained from Aspergillus niger or Bacillus subtilis, more preferably from Aspergillus niger. Suitable asparaginases from Aspergillus niger are described in WO 2004/030468.

The enzyme capable of reducing the amount of free asparagine used in a preferred embodiment of the method of the fourth aspect of the invention will be used under conditions of pH, temperature and reaction time sufficient to allow the enzyme to react with the free asparagine.

It is known that pH and temperature will affect the activity of the enzyme. Depending on the type of enzyme used, the skilled person will be able to determine the optimal pH and temperature conditions for the enzymatic reaction. Furthermore, such conditions may be obtained via textbooks and/or provided by the supplier of the enzyme. The reaction time sufficient for the enzyme to react with free asparagine will depend on: the amount and type of enzyme used, the amount of conversion of asparagine desired in the final product, and the like. One skilled in the art will be able to determine the optimal reaction time.

The amount of enzyme added to the starting product that will reduce the amount of free asparagine will depend, among other things, on the type of enzyme used and the activity of the enzyme. One skilled in the art can determine the appropriate amount to add.

In a preferred embodiment of the method of the fourth aspect of the invention, the treatment with an enzyme, physical means, chemical means or a combination thereof capable of reducing the amount of free asparagine, preferably the treatment with an enzyme capable of reducing the amount of free asparagine, is performed on an intermediate product containing free asparagine obtained in a step of a process for the production of a yeast extract or an autolysed yeast from a yeast cell.

In this case, the process for producing yeast extract or autolysed yeast from yeast cells may start with yeast cells, e.g. with an aqueous suspension of yeast cells, e.g. a fermentation broth of yeast cells. Any type of yeast cell can be used in the methods of the invention. In particular, yeast strains belonging to the genera Saccharomyces, Kluyveromyces or Candida may be suitably used. Yeast strains belonging to the genus Saccharomyces, for example Saccharomyces cerevisiae, are preferred.

Fermentation processes suitable for producing yeast cell suspensions are known in the art. In some cases, the fermentation broth may be concentrated, for example by centrifugation or filtration, prior to the process for producing yeast extract or autolysed yeast from yeast cells. For example, cream yeast (baker's yeast concentrated to 15-27% w/w/dry matter content) may be used.

In a preferred embodiment of the method of the fourth aspect of the invention, the treatment with an enzyme, a physical method, a chemical method or a combination thereof capable of reducing the amount of free asparagine is performed on an intermediate product containing free asparagine obtained in a step of a method for producing a yeast extract or an autolysed yeast from yeast cells, preferably obtained during or more preferably after a treatment of the yeast cells to release and optionally at least partially degrade the cell contents. The cell wall is thereby (partially) damaged and/or ruptured, resulting in the release of the cell contents and, optionally, the at least partial degradation of the cell contents (e.g. proteins and/or RNA and/or polysaccharides).

In order to release the cell contents from the cells and optionally at least partially degrade the cell contents, the cells may be treated chemically, mechanically, enzymatically or by a combination of two or more of these methods using methods known to those skilled in the art. Mechanical methods include high pressure homogenization techniques. For this purpose, a high-pressure homogenizer may be used. Other homogenization techniques may involve: mixed with particles, such as sand and/or glass beads, or using a milling device (e.g., bead milling). Chemical treatments include the use of salts, bases, and/or one or more surfactants or detergents. In some cases, chemical treatments are less preferred because they may lead to partial degradation of the RNA, especially when bases are used, followed by the formation of 2 '-ribonucleotides and 3' -ribonucleotides.

Preferably, the cells are treated enzymatically, optionally after chemical and/or mechanical treatment. The enzymatic treatment may be carried out by: the yeast cells are acted upon with the yeast enzymes themselves and/or with added exogenous enzymes. The enzymatic treatment may be carried out at a pH between 4 and 10 and/or at a temperature between 40 ℃ and 70 ℃, depending on the type of enzyme used. Generally, the enzymatic treatment may be carried out for a time between 1 and 24. Enzymatic treatment may not only release the cell contents by (partially) damaging and/or rupturing the cell wall, which may also lead to degradation of the cell contents (e.g. proteins, RNA and polysaccharides), depending on the enzyme used.

One or more exogenous enzymes may be added to the yeast cells to effect the enzymatic treatment. Preferably, a protease, more preferably, an endoprotease, may be used as the foreign enzyme. Alternatively, the one or more exogenous enzymes are added after the native yeast enzyme has been inactivated. The person skilled in the art knows how to inactivate the yeast enzymes themselves. The deactivation can be effected, for example, by pH treatment or thermal shock, the latter method being preferred. The thermal shock may suitably be carried out by subjecting the yeast cells to a treatment at a temperature of 80-97 ℃ for 5 to 10 minutes. When the process of the invention is used to produce autolysed yeast extracts or autolysed yeast, the yeast enzymes themselves are generally not inactivated.

Optionally, one or more of, e.g., 5 ' -phosphodiesterase (5 ' -Fdase) and optionally deaminase for converting RNA to 5 ' -ribonucleotides may also be added together with or subsequent to the treatment with the above-mentioned enzymes. Preferably, 5 '-Fdase is used to convert RNA into 5' -ribonucleotides. The 5' -Fdase can be obtained from a microbial or plant source (e.g., malt root extract). An example of a commercially available microorganism, 5' -Fdase, is the enzyme RP-1 produced by Amano (Japan). Alternatively, 5 ' -AMP is converted to 5 ' -IMP by a deaminase, e.g., 5 ' -adenylate deaminase. An example of a commercially available Deaminase is deamidase 500 produced by Amano (japan).

As mentioned above, in a preferred embodiment of the method of the fourth aspect of the invention, the treatment is performed on an intermediate product containing free asparagine obtained in a step of the method for producing a yeast extract or an autolysed yeast from a yeast cell. Preferably, the treatment is performed during, more preferably after, the treatment of the yeast cells to release and optionally at least partially degrade the cell contents. However, if the treatment is performed to convert RNA to 5' -ribonucleotides, the treatment with an enzyme capable of reducing the amount of free asparagine can be performed before or after the RNA conversion. The treatment with an enzyme capable of reducing the amount of free asparagine can be performed before or after inactivation of the enzymes and/or chemicals used to release and optionally at least partially degrade the cell contents from the yeast cell. Inactivation of the enzyme may be carried out as described above. Treatment with an enzyme capable of reducing the amount of free asparagine may require adjustment of the pH of the mixture in which the reaction takes place, depending on the pH at which the previous step is carried out and the optimum pH of the enzyme used. After the treatment, the enzyme capable of reducing the amount of free asparagine is preferably inactivated using one of the methods described above.

When the yeast extract is to be treated with the method of the fourth aspect, the insoluble fraction obtained after (partial) damaging and/or rupturing of the yeast cell wall and optionally degrading the yeast cell contents may be removed after all enzymatic treatments have been completed, i.e. after degradation of the RNA to 5' -ribonucleotides or treatment with an enzyme capable of hydrolysing the amide group of the side chain of free asparagine, depending on which step was performed last. The insoluble fraction can be separated from the supernatant by any conventional solid-liquid separation method, such as centrifugation or filtration. When the autolysed yeast is to be produced by the method of the third aspect, the step of removing the insoluble fraction may be omitted.

The liquid fraction obtained after removal of the solid fraction may be concentrated or dried. The liquid fraction may be concentrated to produce the yeast extract in liquid form (typically having a dry matter content of about 40-65% w/w), or it may be further concentrated to produce the yeast extract in paste form (typically having a dry matter content of about 70-80% w/w). The yeast extract may be dried, e.g. to a dry powder having a dry matter content of about 95% w/w or more. In the case of production of autolysed yeast, the suspension comprising the liquid fraction and the insoluble fraction may be concentrated or dried in a similar manner to that described for the yeast extract.

In another embodiment of the invention, the starting product for use in the process of the fourth aspect of the invention may be an intermediate product containing free asparagine obtained in a step of the process for producing a protein hydrolysate from a protein substrate (preferably during, more preferably after, a treatment of the protein substrate to hydrolyse it to a mixture of peptides and amino acids).

In this embodiment, the process for producing a protein hydrolysate from a protein substrate may start with the protein substrate, e.g. with a suspension of the protein substrate in a suitable solvent (e.g. water). Any of the protein substrates mentioned aboveEither substance or a combination thereof may be used. Preferably, the protein substrate is treated to hydrolyze it to a mixture of peptides and amino acids. The treatment may be carried out by chemical or enzymatic means. Chemical treatment is typically carried out using hydrochloric acid according to methods known in the art (e.g. "Savory flavors", 1995, t.w. nagodawithin, esseekay Associates inc., Wisconsin, USA, page 233-. Preferably, the treatment is carried out by enzymatic means. Preferably, the enzymatic treatment is performed using a mixture of endo-and exo-proteases. Suitable endoproteases may be derived from animal, plant or microbial material. They include recombinant enzymes, for example, enzymes obtained by genetic engineering techniques. Suitable endoproteases may be trypsin (EC3.4.21.4), chymotrypsin (EC3.4.21.1), subtilisin (EC3.4.21.14) and papain (EC3.4.22.2). Suitable exoproteases may include carboxypeptidases (ec3.4.16 and EC3.4.17) and/or aminopeptidases (ec 3.4.11). Exoproteases may be derived from animal, plant or microbial material. They may be recombinant enzymes. An example of a suitable carboxypeptidase can be, for example, carboxypeptidase B (EC 3.4.17.2). Examples of suitable aminopeptidases may be, for example, Peptidase R from Amano-JapanOr Corolase LAP from ABEnzymes (UK). In addition, complex enzyme preparations comprising endoprotease, carboxypeptidase and aminopeptidase can be used. An example of such a formulation is Flavourzyme(NOVO, Denmark) or Sumizyme FP(Shin Nihon, Japan).

Depending on the type of enzyme used, a suitable pH and temperature may be used. Suitable pH may vary between about pH3 to 9. Suitable temperatures may vary from about 5 to 75 ℃. The mixture of exoprotease and endoprotease and the proteinaceous substrate may be incubated together or may be incubated with the exoprotease after the proteinaceous substrate is incubated with the endoprotease (optionally after inactivation of the endoprotease).

The treatment with an enzyme capable of reducing the amount of free asparagine may be performed during the enzymatic treatment with an endoprotease and/or an exoprotease. Depending on the pH optimum of the enzyme, the treatment with an enzyme capable of reducing the amount of free asparagine can be carried out during the enzymatic treatment with an endoprotease or during the enzymatic treatment with an exoprotease. Alternatively, the treatment with an enzyme capable of reducing the amount of free asparagine may be performed after the treatment with an endoprotease and an exoprotease.

Once the desired protein hydrolysate has been obtained, one or more of the enzymes used in the process may be inactivated. The inactivation may be performed under conditions similar to those under which the enzyme is inactivated in the method of the third aspect. The skilled person will be able to select the best conditions for inactivating the enzyme.

The protein hydrolysate obtained by the fourth aspect may be worked up according to methods known to the person skilled in the art. For example, the aqueous suspension comprising the protein hydrolysate may be, for example, centrifuged and/or ultrafiltered, and then concentrated, for example, by evaporation and optionally dried in any convenient manner (e.g., spray drying, freeze drying, fluid bed processing, or a combination of these methods).

In a fifth aspect, the present invention provides the use of a yeast extract according to the first aspect of the invention or a yeast extract obtainable by a method according to the fourth aspect of the invention in a food, feed or food or feed ingredient or a process for the preparation thereof.

In a sixth aspect, the present invention provides the use of an autolysed yeast according to the second aspect of the invention or obtainable by a method according to the fourth aspect of the invention in a food, feed or food or feed ingredient or a method of preparation thereof.

In a seventh aspect, the present invention provides the use of a protein hydrolysate according to the third aspect of the invention or a protein hydrolysate obtainable by a method according to the fourth aspect of the invention in a food, feed or food or feed ingredient or a process for the preparation thereof. Preferably, the protein hydrolysate is used in the preparation of a process flavour.

In a preferred embodiment of the fifth, sixth or seventh aspect of the invention, the yeast extract, autolysed yeast or protein hydrolysate is used in the production of a process flavour.

Surprisingly, when the yeast extract, autolysed yeast or protein hydrolysate according to the invention is used in the production of a process flavour, a process flavour with a low acrylamide content is obtained, which is considerably lower than the amount present in process flavours obtained using conventional yeast extracts, autolysed yeast or protein hydrolysates.

In an eighth aspect, the present invention relates to a process flavour having acrylamide not higher than 800ppb, preferably not higher than 600ppb, more preferably not higher than 400ppb, most preferably not higher than 200ppb on a dry matter basis. The definition of process flavours is herein shown in the "background of the invention". The process flavouring may be obtained from an amino acid source selected from yeast extract, autolysed yeast or protein hydrolysate or a combination of one or more of these ingredients, optionally in combination with one or more supplemental amino acids. Advantageously, the process flavours of the present invention contain low levels of the toxic component acrylamide. Preferably, in the process flavour of the present invention, acrylamide is as low as 50ppb, more preferably as low as 20ppb, even more preferably as low as 10ppb, most preferably almost absent. This feature makes the process flavoring agent of the present invention particularly useful for flavoring food products or food ingredients.

In a ninth aspect, the present invention relates to a method of producing the processed seasoning of the eighth aspect of the present invention. The method comprises the following steps: heating a mixture comprising at least an amino acid source selected from the group consisting of yeast extract, autolysed yeast, protein hydrolysate, or a mixture thereof, all containing no more than 1mg/g, preferably no more than 0.2mg/g, more preferably no more than 0.1mg/g of free asparagine, and preferably a reducing carbohydrate, at a pH, temperature, pressure, and reaction time sufficient to develop a flavour.

Quite surprisingly, when the above amino acid source is used in the process of this aspect, the amount of acrylamide in the final product is reduced considerably compared to the amount present in process flavours using conventional amino acid sources. The latter is quite evident when the process flavour is produced by an extruder, as shown in the examples.

The amount of acrylamide in the final product is not more than 800ppb, preferably not more than 600ppb, more preferably not more than 400ppb, most preferably not more than 200ppb, based on dry matter.

In one embodiment, the method according to the ninth aspect of the invention starts with an initial mixture comprising an amino acid source selected from a yeast extract, an autolysed yeast, a protein hydrolysate containing no more than 1mg/g, preferably no more than 0.2mg/g, more preferably no more than 0.1mg/g of free asparagine, or a mixture thereof, optionally in combination with one or more complementary amino acids.

In another embodiment, the method according to the ninth aspect of the invention may also be started with an initial mixture comprising an amino acid source selected from yeast extract, autolysed yeast, protein hydrolysate or mixtures thereof, said source containing more than 1mg/g of free asparagine. In this embodiment, the mixture may, for example, comprise a commercially available yeast extract, autolysed yeast or protein hydrolysate. Prior to subjecting the mixture to conditions suitable for producing a process flavoring as described below, the mixture is first treated with an enzyme, physical method, chemical method, or combination thereof as described above that reduces the amount of free asparagine to a level of less than 1 mg/g. The step of reducing the amount of free asparagine can partially overlap with the step of producing the processed flavoring agent.

Preferably, the starting mixture described in the two above embodiments further comprises at least a reducing carbohydrate. The reducing carbohydrate may be a monosaccharide, disaccharide, polysaccharide or a mixture of one or more of these components. Preferably, the reducing carbohydrate is selected from the group of monosaccharides, preferably from the group of C5-monosaccharides or C6-monosaccharides, more preferably from the group of L-ribose or D-ribose, dextrose (D-glucose), L-arabinose, L-rhamnose, L-fructose. The present invention does not exclude the possibility of combining more than one reducing carbohydrate. In this latter case, the hydrolysate obtained from the chemical or enzymatic degradation of the polysaccharide can be used as a source of reducing carbohydrates, e.g., maltodextrin. Preferably, the reducing carbohydrate is present in the mixture in an amount between 0 and 25% w/w, more preferably 1 to 25% w/w, even more preferably 2 to 25% w/w based on dry matter of the mixture. When a polysaccharide, such as maltodextrin, is present in the mixture, the reduced carbohydrate may vary between 5 and 50% w/w, preferably 10 to 40% w/w, more preferably 15 to 40% w/w, based on dry matter of the mixture.

As indicated in the background of the invention, when other ingredients are present in the starting mixture in addition to the amino acid source, the amount of amino acid source may be between 30 and 98% w/w based on the total mixture dry matter.

The ingredient mix used to produce the process flavoring may also contain one or more lipids (e.g., oils or fats), sulfur-containing compounds, carbonyl-containing compounds, and the like.

When oils or fats are present in the mixture, they may be present in an amount of 0 to 5% w/w based on the total mixture dry matter.

Preferably, the reaction mixture comprises a solvent, preferably water, and typically the content of dry matter in the reaction mixture may be between 60 and 98% w/w, more preferably between 75 and 95% w/w, based on the total mixture including water.

The ingredients may be mixed according to any method known in the art, depending also on the amount of water present in the mixture. The ingredients may be added to the mixture simultaneously or subsequently. Mechanical stirrers may also be used.

In the method of this aspect, a mixture comprising at least the above-indicated amino acid source or mixture thereof (optionally in combination with one or more amino acids and preferably also at least a reducing carbohydrate) is heated under conditions of pH, temperature, pressure and reaction time sufficient to develop a fragrance.

To obtain good flavour, the pH of the mixture in the process of the invention may generally be at least 2. Preferably, the pH is between 4 and 8. More preferably between 5 and 7, even more preferably between 5.5 and 6. The pH can be adjusted using edible acids or bases well known to those skilled in the art.

Generally, to produce the process flavour, the reaction mixture may be heated to a temperature between 70 and 200 ℃, preferably 70 to 190 ℃, also depending on the reaction time. Longer reaction times generally require lower reaction temperatures, while shorter reaction times generally require higher reaction temperatures. The reaction time may vary between seconds (e.g., 2 seconds) to 6 hours, preferably between 10 seconds and 4 hours, more preferably between 20 seconds and 2 hours. To avoid loss of solvent during heating, the reaction mixture may be maintained under reflux conditions.

Depending on the type of flavour that should be obtained and the system used to produce the processed flavour, the process of the ninth aspect may vary from reduced pressure (e.g. down to 50mbar, e.g. in a vacuum oven) to pressures above atmospheric pressure (e.g. up to 2-5 bar).

Alternatively, when the water content in the processed flavouring after the heating step is 4% w/w or more, the method of the ninth aspect comprises the steps of: drying the processed flavoring agent. Methods known in the art, for example, oven drying, belt drying, spray drying, can be used in this regard.

Depending on the type of scent one wishes to develop, different combinations of mixture dry matter content and/or temperature of the heating step and/or duration of the heating step and/or pressure used during the heating step may be used.

For example, to produce a processed flavour with a roasted taste, a mixture comprising 80% w/w dry matter based on the total mixture including water may typically be heated at a temperature above 100℃ (e.g. 110C and 120℃) under reduced pressure (e.g. 50-400mbar) for 4-6 hours.

For example, to produce a processed flavour with a cooked taste, a mixture comprising 40 to 60% w/w dry matter, based on the total mixture including water, may be heated at a temperature of about 100 ℃ for about 1-2 hours.

In a preferred embodiment of the process of the ninth aspect of the invention, the ingredients of the mixture comprising at least the source of amino acids as indicated above and preferably also at least the reducing carbohydrate are introduced into an extruder, the mixture is kneaded and heated under conditions of pH, temperature and reaction time sufficient to develop the flavour, and the reaction mixture is subsequently extruded from the extruder.

The extruder may be any extruder suitable for producing process flavours, for example a twin screw extruder (twin extruder). Extruders, such as twin-screw extruders, are known to those skilled in the art. The ingredients may be introduced into the extruder by the same or separate feeders. When an extruder is used, the mixture is preferably kneaded and heated at a temperature of 110 to 190 ℃, preferably 130 to 165 ℃. Preferably, a pressure of 1 to 3bar is used. The reaction time is preferably 2 seconds to 30 minutes, more preferably 10 seconds to 5 minutes. When an extruder is used, the amount of dry material in the reaction mixture is preferably: at least 90% w/w based on the total weight of the mixture including water.

The reaction product may exit the extruder at an extruder-external pressure varying from a reduced pressure (e.g. 5mbar) to atmospheric pressure (e.g. about 1 bar). The extruded product may be further cooled and/or dried using a cooling belt or any suitable method.

The method of the ninth aspect may further comprise, if desired: treatment is performed to further reduce the amount of acrylamide in the final processed flavor. The treatment may be any treatment suitable for reducing the amount of acrylamide in the product and applicable during the production of process flavours.

In one embodiment, the pH, temperature, pressure and reaction time in the process of the ninth aspect are adjusted to reduce the amount of acrylamide in the process. For example, the processed flavor is dried under reduced pressure. The pressure can generally be between 20 and 400 mbar.

In another embodiment, the final product is treated with an enzyme that modifies or degrades acrylamide. The treatment is carried out under conditions of pH, temperature and reaction time sufficient to react the enzyme with acrylamide. Preferably, amidases may be used. Examples of enzymes that can be used and of conditions under which enzymes can be used are given in international patent application No. PCT/EP2005/055242, filed 10/13/2005.

The process flavouring according to the eighth aspect of the invention or obtainable by the process according to the ninth aspect of the invention is very suitable for use as a flavouring in food or feed or food or feed ingredients because of its very low acrylamide level.

Examples

Materials and methods

Acrylamide measurement

Sample pretreatment

600mg of the dried and homogenized sample was extracted using 5ml of milliQ water. Adding 1 μ g internal standard to the extract¹³C₃Acrylamide (in solution, CIL). After centrifugation (6000rpm) for 10 minutes, 3ml of the upper layer was placed on an Extreluut-3BT column (Merck). Acrylamide was eluted from the column using 15ml of ethyl acetate. The ethyl acetate was evaporated under a gentle stream of nitrogen to about 0.5 ml.

Chromatographic conditions

The ethyl acetate solution was analyzed using gas chromatography. The separation was achieved using a CP-Wax-57(Varian) column (length 25m, internal diameter 0.32mm, membrane 1.2 μm) with helium as carrier gas at a constant flow of 5.4 ml/min. Mu.l of split-less injection was used. The furnace temperature was held at 50 ℃ for 1 minute, after which the temperature was raised to 220 ℃ at 30 ℃/minute. After a constant temperature of 220 ℃ for 12 minutes, the furnace was cooled and stabilized before the next injection.

Detection was performed using on-line chemical ionization mass spectrometry in positive ion mode using methane as the ionized gas. Monitoring the characteristic ions m/z 72 (acrylamide) and m/z 75: (¹³C₃Acrylamide) was used for quantification.

Apparatus for use

GC： HP6890(Hewlet Packard)

MSD (quality selection detector): HP5973(Hewlet Packard)

Method for measuring free amino acids

The amount of free amino acids (e.g., in yeast extract) is measured using the following method. An accurately weighed sample of yeast extract material was dissolved in dilute acid and the precipitate was removed by centrifugation in an Eppendorf centrifuge. Amino Acid Analysis of the clear supernatant was performed according to the PicoTag method as indicated in the operating manual of the Amino Acid Analysis System of Waters (Milford MA, USA). To this end, a suitable sample is taken from the liquid, diluted acid is added, and homogenization is carried out. A new sample was taken from the latter solution, dried and derivatized with phenylisothiocyanate. The presence of various derivatized amino acids was quantified using an HPLC method and summed to calculate the total level of free amino acids in the weighed sample.

Example 1 production of autolysed yeast extracts with Low free asparagine

Definition of enzyme units: 1. mu. mol NH released from L-asparagine per minute at pH5.5 and 37 deg.C₃。

200 liters of 20% autolysed yeast extract (Gistex) were preparedLS, DSM food specialties-the netherlands). The solution pH was adjusted to 5.1 using 4N KOH. To the yeast extract solution was added 20ml asparaginase solution (which contained 4602 enzyme units/ml) and the mixture was incubated for 4 hours at 51 ℃. Once the reaction is terminated, the enzyme is inactivated by heat treatment. After adjusting the pH to pH6.5, the resulting solution was spray dried. The final product contained less than 3.5% w/w water. The amino acid composition of the mixture was measured at the beginning and after the enzymatic treatment as described above. The results are shown in Table 1.

The asparaginase used was Aspergillus niger asparaginase as described in WO 2004/030468.

The results in table 1 clearly show that the yeast extract after asparaginase treatment is free of asparagine. In addition, the amount of aspartic acid is increased, which is similar to the amount of asparagine reduction.

Table 1: effect of asparaginase treatment on autolytic Yeast extracts

^*Mg per gram of yeast extract dry matter

Example 2 preparation of process flavours Using ovens of different temperatures during the heating step

Formulation 2a

81.3 grams of GistexLS powder (DSM Food Specialties-Holland)

16.0 grams of MaxhromePlus HS powder (DSM Food Specialties-Holland)

24.3 grams of dextrose monohydrate

Formulation 2b

81.3 g of the yeast extract, powder obtained in example 1

16.0 grams of MaxhromePlus HS powder (DSM Food Specialties-Holland)

24.3 grams of dextrose monohydrate

Gistex LS powder is an autolysed yeast extract comprising less than 1% w/w sodium chloride, 62% w/w protein and 40-50% w/w free amino acids relative to the total protein, all weight percentages being based on dry matter.

Maxarome Plus HS powder is a hydrolysed yeast extract comprising about 40% w/w sodium chloride on a dry matter basis. Furthermore, it comprises 3% w/w 5 '-GMP (measured as disodium salt heptahydrate) and 3% w/w 5' -IMP (measured as disodium salt heptahydrate), 5% w/w glutamic acid (measured as free acid), 72% w/w protein and approximately 20% w/w free amino acids of the total protein, all weight percentages being based on yeast extract dry matter without sodium chloride.

In both cases, the powder was stirred with a spoon. To the powder mixture was added dropwise 3.4g sunflower oil with stirring.

The powder mixture was divided into aluminum pans.

The powder was heated in a furnace at different temperatures for a fixed time. The obtained powder has a roasted beef flavor.

The powder was analyzed for the presence of acrylamide. The results are shown in Table 2.

TABLE 2

Conditions of the experiment	Acrylamide (mu g/kg)
		Powder mixture, formulation 2a, 165 ℃, 40 minutes	935
Powder mixture, formulation 2a, 180 ℃, 40 minutes	1497
		Powder mixture, formulation 2b, 165 ℃, 40 minutes	255
Powder mixture, formulation 2b, 180 ℃, 40 minutes	296

Example 3 preparation of a processed flavoring agent Using a batch kneader

A30 gram sample of formulation 3, 3a (see Table 3 for composition) was transferred to a 50cc batch kneader preheated to the specified temperature and mixed at the same temperature for 180 seconds.

Temperature for formulations 3, 3 a: 145 and 150 ℃.

TABLE 3

The obtained processed seasoning has roasted chicken flavor.

The resulting processed flavor was analyzed for acrylamide content.

The results are shown in Table 4.

TABLE 4

Formulation of	Temperature (. degree.C.)	Acrylamide (ppb)
			3	145	5928
3a	145	143
			3	150	6227
3a	150	124

Example 4 preparation of a processing flavor Using an extruder

The process is described as follows:

the mixture of sugar and yeast extract was added to a twin-screw extruder equipped with a feed unit and an injector for water and a feed unit and an injector for oil, using separate feeders. Two different formulations were used (formulations 4 and 4a, see table 4). 8 kg of each formulation were processed in an extruder at 165 ℃ for 1 hour.

The product was extruded from the extruder at room temperature and atmospheric pressure, cooled and dried on a cooling belt equipped with a pressure roller, ground and sampled.

TABLE 4

The obtained product has deep-baked beef flavor. The samples were analyzed for acrylamide.

As a result:

and (4) formula: 4568 ppb.

Formulation 4 a: 400 ppb.

Example 5 production of Low free asparagine Casein hydrolysate

Definition of enzyme units: 1 μmol NH Release from L-asparagine per minute at pH5.5 and 37 deg.C₃。

1 liter of a 10% aqueous casein hydrolysate solution was prepared. The solution pH was adjusted to 5.1. 62ml of asparaginase (containing 14472 enzyme units/mg) was added to the casein solution and the mixture was incubated at 51 ℃ for 2 hours. Once the reaction is terminated, the enzyme is inactivated by heat treatment. The resulting solution was freeze-dried. The amino acid composition of the mixture was measured at the start of and after the enzyme treatment as described above. The results are shown in Table 6.

Table 6: effect of asparaginase treatment on Casein hydrolysate

^*Mg per gram of dry matter of casein hydrolysate

The asparaginase used was Aspergillus niger asparaginase as described in WO 2004/030468.

Example 6 production of Process flavours based on Casein hydrolysate

A mixture of 4.7g glucose, 0.4g glycine, 1.6g maltodextrin and 13.4g casein hydrolysate (prepared as described in example 5 without asparagine) was prepared. As a reference, a similar mixture was prepared using casein hydrolysate which was not treated with asparaginase instead of the casein hydrolysate of example 5.

Both mixtures were oven treated at 155 ℃ for 45 minutes. Finally, the acrylamide content was measured using the method described in the materials and methods section.

The results of the acrylamide analysis are listed in table 7.

TABLE 7

Casein hydrolysate	Acrylamide (ppb)
		Asparaginase-treated casein hydrolysate	614
Untreated cheeseProtein hydrolysate	2801

Example 7 production of autolysed Yeast with Low free asparagine

In the presence of 2 g of the endoprotease Alcalase(Novozymes-Denmark) 2 liters of Saccharomyces cerevisiae cream yeast (18.5% dry solids) were autolysed at pH5.1 and 51 ℃ for 24 hours. The reaction mixture is then subjected to a heat treatment to inactivate all enzyme activities.

1 liter of the reaction mixture was incubated for a further 2 hours at pH5.1 and 51 ℃ in the presence of 53mg of asparaginase (with 14772 units/mg). Subsequently, the enzyme is inactivated by heat treatment. The resulting solution was spray dried. The reference sample (not treated with asparaginase) was also spray dried. The amino acid composition of the dried material (with or without asparaginase treatment) was measured. The results are shown in Table 8.

Table 8: effect of asparaginase treatment on autolysed Yeast

^*Mg per g of autolysed yeast dry matter

The asparaginase used was Aspergillus niger asparaginase as described in WO 2004/030468.

Example 8 is based onProduction of process flavours from autolysed yeast

A mixture of 4.7g glucose, 0.4g glycine, 1.6g maltodextrin and 13.4g autolysed yeast (prepared as described in example 7, without asparagine) was prepared. As a reference, autolysed yeast without asparaginase treatment was used to prepare a similar mixture.

Both mixtures were oven treated at 155 ℃ for 45 minutes. Finally, the acrylamide content was measured using the method described in the materials and methods section.

The results of the acrylamide analysis are shown in Table 9.

TABLE 9

Autolytic yeast	Acrylamide (ppb)
		Asparaginase-treated autolysed yeast	710
Untreated autolysed yeast	2551

Example 9 production of Yeast extracts with Low free asparagine

In the presence of 2 g of the endoprotease Alcalase(Novozymes-Denmark) 2 liters of Saccharomyces cerevisiae cream yeast (18.2% dry solids) were autolysed at pH5.1 and 51 ℃ for 17.5 hours. The reaction mixture was incubated for a further 2 hours at pH5.1 and 51 ℃ in the presence of 613mg of asparaginase (with 1802 units/mg). Cell walls were removed by centrifugation and the supernatant was treated at 95 ℃ for 5 minutes to inactivate all enzyme activity present. Subsequently, the supernatant was concentrated and spray-dried.

The asparagine concentration in the reaction mixture before and after the asparaginase treatment and in the final extract powder was measured. The results are shown in Table 10.

Table 10: results of asparagine assay

Sample (I)	Asparagine concentration (mg/g dry matter)
		Before asparaginase treatment	3.29
After asparaginase treatment	＜0.01
		Yeast extract powder	＜0.03

The asparaginase used was Aspergillus niger asparaginase as described in WO 2004/030468.

Claims (10)

1. A method of producing a processed seasoning having an amount of acrylamide of not more than 800ppb on a dry matter basis, comprising:

introducing into the extruder a mixture comprising an amino acid source having an amount of free asparagine of not more than 1mg/g on a dry matter basis,

kneading and heating the mixture under conditions of pH, temperature, pressure and reaction time sufficient to develop a fragrance,

the resulting processed flavor is then extruded from the extruder,

wherein the amino acid source is selected from yeast extract, autolysed yeast, protein hydrolysate or mixtures thereof.

2. The method of claim 1, the method comprising: treating a mixture comprising an amino acid source selected from yeast extract, autolysed yeast, protein hydrolysate or mixtures thereof containing more than 1mg/g of free asparagine with an enzyme, physical means, chemical means or a combination thereof capable of reducing the amount of free asparagine to a level below 1 mg/g.

3. The method of claim 1 or 2, wherein the mixture further comprises a reducing carbohydrate.

4. The method according to any one of claims 1 to 2, wherein the amino acid source is a yeast extract.

5. The method according to any one of claims 1 to 2, wherein the amino acid source is autolysed yeast.

6. The process according to any one of claims 1 to 2, wherein the amino acid source is a protein hydrolysate.

7. The method of any one of claims 1-2, wherein the conditions of pH, temperature, pressure, and reaction time are adjusted to reduce the amount of acrylamide in the process flavor.

8. The method of claim 7, wherein the process flavor is dried under reduced pressure.

9. The method of claim 8, wherein the processed flavor is further treated with an enzyme that modifies or degrades acrylamide.

10. The method of claim 9, wherein the enzyme capable of modifying or degrading acrylamide is an amidase.