BRPI0620656A2 - low acrylamide process flavorings - Google Patents

Abstract

PROCESS FLAVORIZERS WITH LOW ACRYLAMIDE CONTENT. This is a new yeast extract, an autolysed yeast and protein hydrolyzate with an amount of free asparagine that is not greater than 1 mg / g. This yeast extract, autolyzed yeast or protein hydrolyzate can be used advantageously in the production of a process flavoring with an amount of acrylamide that is not greater than 800 ppb. This process flavoring agent is particularly suitable for use in food or food seasoning.

Description

LOW ACRYLAMIDE PROCESS FLAVORIZERS

Field of the Invention

The present invention relates to a yeast extract, an autolysed yeast, a protein hydrolyzate, processes for producing them and their use in food or feed or in food or feed ingredients. The present invention also relates to a process flavoring, a process for producing it and its use in food or feed or in food or feed ingredients.

Background of the Invention

The use of processed foods and ready meals in our society is increasing day by day. Often, various flavors such as hydrolyzed vegetable protein, yeast extracts, cheese, seasonings, etc., are added to such foods during or after processing to make the food more palatable. The taste of food is known to be the result of a complex combination of different reaction paths that occur during cooking. One problem that can be encountered in producing processed food or ready meals is that the heating step in producing them may not be long enough to develop a satisfactory taste. However, the flavor gap that can be found in processed foods can be filled by adding process flavorings (also called "reaction flavorings") to such foods. Process flavorings are usually added to the processed food after the main processing steps have been completed. The term "process flavoring" is used throughout this report for a composition that has a distinct flavor, for example a meat flavor, that can be obtained by heating an ingredient mixture comprising at least one nitrogen-containing compound. as an amino group and preferably at least one reducing carbohydrate under conditions of pH, temperature, pressure and reaction time sufficient for a flavor to develop. The mixture of ingredients used in the production of process flavorings may further comprise one or more lipids, sulfur containing compounds, carbonyl containing compounds, etc.

Process flavorings are obtained through a complex combination of reaction paths that occur between the ingredients during the heating step. An overview of the various reaction pathways involved in the production of process flavorings is given, for example, in "Savory Flavors" 1995 by T. W.

Nagodawithana, Esteekay Associates Inc., Wisconsin, USA, pages 103-163.

In process flavorings that are produced according to this invention, "at least one nitrogen-containing compound in the form of an amino group" may be obtained from a source of amino acids selected from a yeast extract, a autolysed yeast, a protein hydrolyzate or a mixture of one or more of these ingredients, optionally in combination with one or more complementary amino acids.

Acrylamide, which has long been commercially produced for a number of technical applications, is considered to be probably carcinogenic to animals and humans. In 1991, the Scientific Committee on Food investigated monomeric acrylamide in food contact materials and, in its assessment, concluded that acrylamide is a genotoxic carcinogen.

Recently, the occurrence of acrylamide in a range of food and baked foods has been published (Tareke et al., Chem. Res. Toxicol. 13, 517-522 (2000)) and this has resulted in worldwide concern. Further research has revealed that considerable amounts of acrylamide can be detected in a range of common baked, baked and fried foods and it has been shown that the occurrence of acrylamide in food was the result of the roasting process.

The presence of acrylamide in process flavorings, which are subsequently used in various types of foods, is highly undesirable. Unexpectedly, the applicant has found that, unfortunately, process flavors can comprise a considerable amount of acrylamide (contents that can be as high as, for example, 10,000 ppb). Acrylamide in process flavorings can be produced even at temperatures below 120 ° C, when acrylamide formation would not be expected. The problem of the presence of acrylamide in process flavorings was not known at this time.

Yeast extracts, autolysed yeast and protein hydrolysates may advantageously be used as a source of amino acids for the production of process flavorings.

Surprisingly, we have found that when regular yeast extracts, regular autolyzed yeast or regular protein hydrolyzate yeast are used as a source of amino acids in the production of process flavorings, a high level of acrylamide can be produced.

Thus, the present invention also relates to novel yeast extracts, novel autolysed yeast and novel protein hydrolysates suitable for the production of low acrylamide process flavorings. Furthermore, the present invention relates to new low acrylamide process flavorings.

Detailed Description of the Invention

The present invention relates, in a first aspect, to a yeast extract with an amount of free dry matter-based asparagine, which is not greater than 1 mg / g, preferably not greater than 0, 2 mg / g, more preferably not greater than 0.1 mg / g. The amount of free asparagine in the yeast extract according to a first aspect may be as low as ~ 0 mg / g.

Yeast extract is defined as a composition comprising water-soluble components extracted from yeast cells. In general, yeast extracts comprise amino acids, proteins, peptides, vitamins, carbohydrates and phosphate-like salts. Yeast extracts may also comprise 5'-ribonucleotides. Yeast extracts can be divided, for example, into autolytic and hydrolytic yeast extracts.

Autolytic yeast extracts are concentrated from soluble materials that can be obtained from yeast after cell disruption and digestion (lysis) of the polymeric yeast material. Active yeast enzymes released in the medium after cell disruption contribute to lysis. These types of yeast extract are rich in amino acids and generally do not comprise 5'-ribonucleotides because during the autolytic process, native RNA is decomposed or modified in a form that is not degradable to 5'-ribonucleotides. They are used in the food industry as basic flavor providers. The amino acids present in yeast extract add a meat-like broth flavor.

Hydrolytic yeast extracts are concentrated from soluble materials and can be obtained from yeast after cell disruption, digestion (lysis) and addition of exogenous enzymes such as proteases and / or peptidases and especially nucleases such as 5'-phosphodiesterase and, optionally 5'-adenyl deaminase to the yeast suspension during lysis. Native yeast enzymes are generally deactivated prior to lysis. During this process guanine 5'-ribonucleotides (5'-guanine monophosphate, 5'-GMP), uracil (5'-uracil monophosphate, 5'-UMP), cytosine (5-cytosine monophosphate, 5'- CMP) and adenine (5'-adenine mono phosphate, 5'-AMP) can be formed. When adenyl deaminase is added to the mixture, 5'-AMP is transformed into 5'-inosine mono phosphate (5'-IMP). The hydrolytic yeast extracts obtained by this method are therefore rich in 5'-ribonucleotides, especially rich in 5'-GMP and 5'-IMP. Often, yeast extracts are also rich in monosodium glutamate (MSG). 5'-IMP, 5'-GMP and MSG are known for their taste enhancing properties. They are capable of enhancing the tasty and delicious taste in certain types of foods. This phenomenon is described as a "sensory impression left by food in the mouth" or umami (fifth basic taste).

In one embodiment of the first aspect of the invention, the yeast extract may be an autolytic or hydrolytic yeast extract or a mixture thereof. The yeast extract may comprise 5'-ribonucleotides. By the term "5" -ribonucleotides "is meant a mixture of 5'-GMP, 5" - CMP, 5 "-UMP and additionally 5'-AMP and / or 5'-IMP where 5 "- IMP in the mixture is obtained by partial or complete conversion of 5'-AMP to 5'-IMP.

In a second aspect, the invention relates to an autolysed yeast with an amount of free asparagine, based on dry matter, which is not greater than 1 mg / g, preferably not greater than 0.2 mg / g. more preferably not greater than 0.1 mg / g. The amount of free asparagine in autolysed yeast can be as low as ~ 0 mg / g.

Autolysed yeast is a precursor to an autolysed yeast extract. It comprises concentrates of soluble materials, which can be obtained from yeast after cell disruption and digestion (lysis) of polymeric yeast material (where active yeast enzymes released into the medium after cell disruption contribute to lysis) and the insolubles formed during lysis, mainly due to the degraded yeast cell wall fraction. In a third aspect, the invention relates to a protein hydrolyzate with an amount of free asparagine, based on the dry matter of the product, which is not greater than 1 mg / g, preferably not greater than 0.2. mg / g, more preferably not greater than 0.1 mg / g.

Protein hydrolysates are enzymatically treated acid or protein substrates containing mixtures of amino acids and peptides in varying proportions that are determined by the degree of hydrolysis and / or the type of enzymes used. Typical protein substrates for the preparation of protein hydrolysates are vegetable proteins such as wheat gluten, corn gluten, soy protein, rapeseed protein, pea protein, alfalfa protein, sunflower protein, fabanaceous bean protein, cottonseed or sesame seed protein, corn protein, barley protein, sorghum protein, potato protein, rice protein, coffee protein. Other possible protein substrates are animal proteins such as milk protein (eg casein, whey protein), egg white, fish protein, meat protein including gelatin, collagen, blood protein (eg hemoglobin ), hair, feathers and fish meat.

Yeast extract, autolysed yeast or protein hydrolyzate may be in any form, for example, dissolved in a liquid or in dry form. Generally, the yeast extract, autolysed yeast or protein hydrolyzate will be in a dry form, for example in powdered or granular form.

In a fourth aspect, the invention relates to a method for producing a yeast extract of the first aspect, an autolysed yeast of the second aspect, or a protein hydrolyzate of the third aspect, the method comprising treating a yeast extract. starting yeast, an autolysed starting yeast or a starting protein hydrolyzate, all containing free asparagine, an enzyme, a physical method, a chemical method or a combination thereof, capable of reducing the amount of free asparagine preferably in the final product, and preferably to obtain an amount of free asparagine in the final product that is not greater than 1 mg / g, more preferably not greater than 0.2 mg / g, more preferably, not more than 0,1 mg / g based on dry matter.

The composition of starting yeast extract, starting autolysed yeast or starting protein hydrolyzate, all containing free asparagine, is also referred to as a "starting product" throughout this report. The starting product to be used in the method of the invention may be a commercially available product, may be an end product of a preparation process or may be an intermediate product obtained in one step of a preparation process for an end product. Thus, in one embodiment of the invention, treatment with an enzyme, a physical method, a chemical method or a combination thereof is carried out in a free asparagine-containing intermediate obtained in a process step of preparation for extract of yeast or autolysed yeast from yeast cells or to a protein hydrolyzate from a protein substrate. The starting product to be used in the method of the invention may be any free asparagine containing starting product as mentioned above. The starting product may be in any form, for example, dissolved in a liquid or dried. Generally, the starting product will be in a dry form, for example in a powdered or granular form.

In one embodiment of the method according to the fourth aspect of the invention, 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 a product. preferably after its suspension and / or solubilization in a suitable solvent, wherein the starting product is a commercially available yeast extract, autolysed yeast or protein hydrolyzate or as the end product of a preparation process. . Generally, the solvent may be such that it is suitable for allowing the enzyme to react with free asparagine or is suitable for use in the physical or chemical method. Generally, the solvent may be a water based solvent, more preferably, the solvent is water.

In the context of the present invention, "an enzyme, a physical method, a chemical method or a combination thereof capable of reducing the amount of free asparagine" is any enzyme or mixture of enzymes, any tax method or any chemical method or combination thereof. capable of removing part or all of free asparagine and / or degrading free asparagine in a way that cannot lead to the formation of acrylamide when heated in the presence of a reducing sugar.

Physical methods that can be used in treatment to reduce the amount of free asparagine are methods that remove part or all of the free asparagine from the starting product. Such methods may comprise, for example, the use of separation techniques as chromatographic techniques. Chemical methods are methods that modify and / or degrade free asparagine in a way that cannot lead to acrylamide formation. Such methods may comprise, for example, the use of chemical reactions such as oxidation, reduction, removal of an amino group.

In a preferred embodiment of the fourth aspect method 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 enzyme to react with free asparagine. .

Preferably, treatment with the enzyme is performed using an enzyme capable of modifying the free asparagine side chain, more preferably with an enzyme capable of hydrolyzing the amide group in the free asparagine side chain, even more preferably with the enzyme. asparaginase enzyme (EC 3.5.1.1).

The enzyme or enzyme mixture used in the preferred embodiment of the method of the third aspect of the invention may be added as an enzyme preparation. Asparaginase can be obtained from a variety of sources, such as from plants, animals and microorganisms such as a bacterium, fungus or yeast. Examples of suitable microorganisms are Escherichia, Erwinia, Streptomyces, Pseudomonas, Aspergillus and Bacillus. An example of a suitable Escherichia strain is Eseherichia eoli. An example of a suitable Erwinia strain is Erwinia ehrysanthemi. 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 amyloliquefaeiens, Bacillus brevis, Bacillus eireulans, Bacillus eagulans, Bacillus lautus, Bacillus lieheniformis, Bacillus megateruim, Bacillus stearothemophilus or Bacillus thuringi, Bacillus subtilis. An example of suitable methods for obtaining asparaginase from strains of Bacillus, Streptomyees, Eseheriehia or Pseudomonas is described in WO 03/083043. preferably asparaginase is derived from Aspergillus niger or Bacillus subtilis, more preferably Aspergillus niger. A suitable asparaginase of Aspergillus niger is described in WO 2004/030468.

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

You know that pH and temperature influence enzyme activity. Depending on the type of enzyme used, those skilled in the art will be able to determine the optimal pH and temperature conditions under which the enzyme can react. In addition, such conditions are available via books and / or are provided by enzyme providers. The sufficient reaction time for the enzyme to react with free asparagine will depend, among other things, on the amount and type of enzyme used and the amount of asparagine conversion desired in the final product. Those skilled in the art will be able to determine the optimal reaction time.

The amount of enzyme added to the starting product capable of reducing the amount of free asparagine will depend, among other things, on the type of enzyme used and the activity of the enzyme. The appropriate amount to be added may be determined by those skilled in the art.

In a preferred embodiment of the method of the fourth aspect of the invention, treatment with an enzyme, a physical method, a chemical method or a combination thereof, capable of reducing the amount of free asparagine, preferably an enzyme capable of In order to reduce the amount of free asparagine, it is carried out in an intermediate product containing free asparagine contained in one step of a method for producing yeast extract or an autolysed yeast from yeast cells.

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

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

In a preferred embodiment of the fourth aspect method of the invention, treatment with an enzyme, a physical method, a chemical method or a combination thereof, capable of reducing the amount of free asparagine, is carried out in an intermediate product containing free asparagine obtained in one step of a method for producing a yeast extract or autolysed yeast from yeast cells, preferably during treatment, more preferably after treatment, of yeast cells in order to release and optionally at least partially degrading the contents of the cell. Thus, cell walls are (partially) damaged and / or ruptured resulting in the release of cell contents and optionally cell contents such as proteins and / or RNA and / or polysaccharides are at least partially degraded.

In order to release cell contents and optionally at least partially degrade cell contents, 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. in technique. Mechanical treatments include homogenization techniques. For this purpose it is possible to use high pressure homogenizers. Other homogenization techniques may involve mixing with particles, for example sand and / or glass beads or using milling apparatus (for example, a bead crusher). Chemical treatments include the use of salts, alkali and / or one or more surfactants or detergents. Chemical treatments may in some cases be less preferred because they may lead to partial RNA degradation, especially when using alkali, with the consequent formation of 2'-ribonucleotides and 3'-ribonucleotides.

Preferably, the cells are enzymatically treated, optionally after chemical and / or mechanical treatment. Enzymatic treatment can be performed by subjecting the yeast cells to the action of native yeast enzymes and / or added exogenous enzymes. Enzymatic treatment may be carried out at a pH between 4 and 10 and / or at a temperature between 40 ° C and 70 ° C, depending on the type of enzyme (s) used. Generally, enzymatic treatment can be performed for a period of time between 1 and 24 hours. Enzymatic treatment can not only release cell contents by (partially) damaging and / or disrupting cell walls, but, depending on the enzymes involved, can also contribute to the degradation of cell contents such as proteins, RNA and polysaccharides.

One or more exogenous enzymes may be added to yeast cells to perform enzyme treatment. Preferably, a protease, more preferably an endoprotease, may be used as an exogenous enzyme. Optionally, one or more exogenous enzymes are added after the native yeast enzymes have been deactivated. Those skilled in the art know how to inactivate native yeast enzymes. Deactivation may be effected, for example, by a pH treatment or heat shock, the latter being the preferred method. A heat shock may suitably be accomplished by treating the yeast cells at a temperature of 80 to 97 ° C for 5 to 10 minutes. When an autolytic yeast extract or autolysed yeast has to be produced with the method of the invention, native yeast enzymes are generally not deactivated.

Optionally, one or more enzymes used to transform RNA into 5'-ribonucleotides, such as 5'-phosphodiesterase (5'-Fdase) and optionally deaminase, may also be added together with or subsequent to treatment with the enzymes mentioned above. 5'-Fdase is preferably used to convert RNA to 5-ribonucleotides. 5'-Fdase may be obtained from a microbial or plant source (for example, a malt root extract). An example of commercially available microbial 5'-Fdase is Enzyme RP-1, produced by Amano (Japan). Optionally, 5'-AMP is converted to 5'-IMP by deaminase, for example adenyl deaminase. An example of a commercially available deaminase is Deaminase 500, produced by Amano (Japan).

As mentioned above, in a preferred embodiment of the fourth aspect method of the invention, the treatment is performed on a free asparagine-containing intermediate product obtained in one step of a method for producing a yeast extract or an autolysed yeast from yeast cells. Preferably, the treatment is performed during treatment, more preferably after treatment, of the yeast cells to release and optionally at least partially degrade the cell contents. However, if a treatment is performed to transform RNA into 5'-ribonucleotides, enzyme treatment capable of reducing the amount of free asparagine can be performed before or after RNA transformation. Enzyme treatment capable of reducing the amount of free asparagine may be performed before or after deactivation of the enzyme (s) and / or chemical (s) used to release and optionally degrade at least partially the cell content from the yeast cells. Deactivation of enzymes can be performed according to the methods mentioned above. Treatment with the enzyme capable of reducing the amount of free asparagine may require adjusting the pH of the mixture in which the reaction will take place, depending on the pH at which the previous steps were conducted and the optimal pH of the enzyme used. After treatment, the enzyme capable of reducing the amount of free asparagine is preferably deactivated using one of the methods mentioned above. When a yeast extract is to be produced by the fourth aspect method, the insoluble fraction obtained after (partially) damaging and / or breaking the yeast cell walls and optionally degrading the yeast cell, the contents may be removed after all. enzymatic treatments have been completed, ie either after degradation of RNA into 5'-ribonucleotides or enzyme treatment capable of hydrolyzing the amide group in the free asparagine side chain, depending on which step will be performed last. The insoluble fraction may be separated from the supernatant by any solid-liquid communication separation method, such as centrifugation or filtration. Where an autolysed yeast is to be produced by the third aspect method, removal of the insoluble fraction may be dispensed with.

The liquid fraction obtained after removal of the solid fraction may be concentrated or dried. The liquid fraction may be concentrated to provide a liquid yeast extract (generally with a dry matter content of approximately 40-65% w / w) or additionally concentrated to provide a yeast extract in a liquid form. pulp (generally with a dry matter content of approximately 70-80% w / w). The yeast extract may be dried to, for example, a dry powder with a dry matter content of approximately 95% w / w or more. In the case of autolysed yeast production, the suspension comprising the liquid fraction and the insoluble fraction may be concentrated or dried according to methods similar to those described for yeast extracts.

In another embodiment of the invention, the starting product to be used in the method of the fourth aspect of the invention may be a free asparagine-containing intermediate obtained in one step of a method for the production of a protein hydrolyzate from a preferably during treatment, more preferably after treatment of the protein substrate to hydrolyze it to a mixture of peptides and amino acids.

In this embodiment, the method for producing protein hydrolyzate from a protein substrate may start with a protein substrate, for example by suspending it in an appropriate solvent, preferably in water. Any of the above mentioned protein substrates 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 performed chemically or enzymatically. Chemical treatment can generally be performed using hydrochloric acid according to methods known in the art, for example, as described in Savory Flavors, 1995, by TW Nagodawithana, Esteekay Associates Inc., Wisconsin, USA, pages 233-237. . Preferably, the treatment is performed enzymatically. Preferably, a mixture of endo proteases and exothermic proteases is used to perform the enzymatic treatment. Suitable endoproteases may originate from animal, plant or microbial material. They include recombinant enzymes, for example enzymes obtained by genetic engineering techniques. Examples of suitable endoproteases may be, among others, trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), subtilisin (EC 3.4.21.14) and papain (EC

3.4.22.2). Suitable exoproteases may include carboxypeptidase (EC 3.4.16 and EC 3.4.17) and / or aminopeptidases (EC 3.4.11). Exoproteases may originate from animal, plant or microbial material. They may be recombinant enzymes. An example of suitable carboxypeptidases may be, for example, carboxypeptidase B (EC 3.4.17.2). An example of a suitable aminopeptidase may be, for example, Amano-Japan Peptidase R® or ABEnzymes Corolase LAP® (United Kingdom). In addition, complex enzyme preparations comprising both endoproteases, carboxypeptidases and aminopeptidases may be used. Examples of such preparations are Flavourzyme® (NOVO, Denmark) or Sumizyme FP® (Shin Nihon, Japan).

Depending on the types of enzymes used, a suitable pH and temperature may be used. The suitable pH may range from about 3 to 9. A suitable temperature may range from about 5 to 75 ° C. The protein substrate and a mixture of exoprotease and endoprotease may be incubated together or the exoprotease may be incubated after incubation of the protein substrate with endoprotease, optionally after endoprotease deactivation.

Enzyme treatment capable of reducing the amount of free asparagine may occur during enzyme treatment with endoprotease and / or exoprotease. Depending on the optimal pH of the enzymes, enzyme treatment capable of reducing the amount of free asparagine may be performed during endoprotease treatment or during exoprotease treatment. Optionally, enzyme treatment capable of reducing the amount of free asparagine may occur following treatment with endoprotease or exoprotease.

Once the desired protein hydrolyzate has been obtained, the enzyme or enzymes used in the method can be deactivated. Deactivation may occur under conditions similar to those used for deactivation of enzymes in the third aspect method. A person skilled in the art will be able to select the best conditions for deactivating enzymes.

The protein hydrolyzate obtainable by the fourth aspect method may be worked up according to methods known to those skilled in the art. For example, the aqueous suspension comprising the protein hydrolyzate may be, for example, centrifuged and / or ultrafiltrated, then concentrated for example by evaporation and optionally dried in any convenient manner such as jet drying, freeze drying. , fluidized bed treatment or a combination of these methods.

In a fifth aspect, the invention provides for 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 food, feed or food ingredients. or feeding or in the preparation thereof.

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

In a seventh aspect, the invention relates to the use of a protein hydrolyzate according to the third aspect or a protein hydrolyzate obtainable by a method of the fourth aspect in food, feed or in food or feed ingredients or in the preparation. of the same. Preferably the protein hydrolyzate is used in the preparation of process flavorings.

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

Surprisingly, when a yeast extract, autolysed or hydrolyzed protein yeast according to the invention is used in the production of a process flavoring, a process flavoring is obtained with a low amount of acrylamide, considerably less than the amount. present in a process flavoring obtained using a

regular yeast, autolysed or hydrolyzed protein yeast.

In an eighth aspect, the invention relates to a process flavorant having a dry matter-based amount of acrylamide of the product, which is not higher than 800 ppb, preferably not greater than 600 ppb, more preferably. , not greater than 400 ppb, more preferably not greater than 200 ppb. Process flavoring is defined herein as indicated in

"Foundations of the Invention". The process flavorant is obtainable from a source of amino acids selected from a yeast extract, an autolysed yeast, a protein hydrolyzate or a mixture of one or more of these ingredients, optionally in combination with one or more supplemental amino acids. Advantageously, the process flavorant of the invention comprises a low amount of the toxic component acrylamide. Preferably the acrylamide is as low as 50 ppb, more preferably as low as 20 ppb, even more preferably as low as 10 ppb and even more preferably almost absent in the process flavoring of the invention. This feature makes the process flavoring of the invention particularly suitable for use as a flavoring of food products or food ingredients.

In a ninth aspect, the invention relates to a method for producing a process flavoring of the eighth aspect of the invention. The method comprises the step of subjecting a mixture containing at least one source of amino acids selected from a yeast extract, an autolysed yeast, a protein hydrolyzate, all containing an amount of free asparagine not greater than 1 mg / g, preferably no greater than 0.2 mg / g, more preferably no greater than 0.1 mg / g or a mixture, and preferably at least one reducing carbohydrate, heat under conditions of pH, temperature, pressure and sufficient reaction time to develop a flavor.

Most surprisingly, when the above mentioned amino acid source is used in the process of this aspect, the amount of acrylamide in the final product is considerably reduced compared to the amount present in process flavorings using regular amino acid sources. The latter is particularly evident when the process flavorant is produced by means of an extruder as shown in the examples.

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

In one embodiment, the method according to the ninth aspect of the invention begins with an initial mixture comprising a source of amino acids selected from a yeast extract, an autolysed yeast, a protein hydrolyzate, containing an amount of free asparagine. which is not greater than 1 mg / g, preferably not greater than 0.2 mg / g, more preferably not greater than 0.1 mg / g or a mixture thereof, optionally in combination with one or more. supplemental amino acids.

In another embodiment, the method according to the ninth aspect of the invention may also start with an initial mixture comprising a source of amino acids selected from a yeast extract, an autolysed yeast, a protein hydrolyzate or a mixture of the same. containing an amount of free asparagine that is greater than 1 mg / g,. In this embodiment, the mixture may comprise, for example, commercially available yeast extract, autolysed or hydrolyzed protein. Prior to subjecting the mixture to conditions suitable for the generation of a process flavorant as described below, the mixture is first subjected to treatment with an enzyme, a physical method, a chemical method or a combination thereof capable of reduce the amount of free asparagine as described above to decrease the amount of asparagine to a level of less than 1 mg / g. The step of reducing the amount of free asparagine may partially overlap with the step of generating process flavorings.

Preferably, the starter mixture as described in the two embodiments above further comprises at least one reducing carbohydrate. The reducing carbohydrate may be a monosaccharide, a disaccharide, a polysaccharide or a mixture of one or more of these ingredients. Preferably, the reducing carbohydrate is selected from the group of monosaccharides, preferably C5- or C6 monosaccharides, more preferably selected from the group of: L- or D-ribose, D-xylose, 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 the latter case, hydrolysates obtained from chemical or enzymatic degradation of polysaccharides may also be used as a source of reducing carbohydrates such as maltodextrin. Preferably, the reducing carbohydrate is present in the mixture in an amount between 0 and 25% w / w, more preferably between 1 and 25% w / w, even more preferably between 2 and 25% w / w, based on dry matter of the mixture. When polysaccharides, such as maltodextrin, are present in the mixture, the amount of reducing carbohydrate may range from 5 to 50% w / w, preferably from 10 to 40% w / w, more preferably from 15 to 40% w / w. p, based on the dry matter of the mixture.

The amount of the amino acid source as specified in the background of the invention may be from 30 to 98% w / w based on the dry matter of the total mixture when, in addition to the amino acid source, other ingredients are present in the initial mixture.

The mixture of ingredients used in the production of process flavorings may further comprise one or more lipids such as oils or fats, sulfur containing compounds, carbonyl containing compounds, etc.

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

Preferably, the reaction mixture comprises a solvent, preferably water, generally the dry matter content in the reaction mixture may be from 60 to 98% w / w, more preferably from 75 to 95% w / w based in 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 simultaneously or subsequently to the mixture. Mechanical mixers may also be used.

In the method of this aspect, the mixture comprising at least one source of amino acids, as specified above, or a mixture thereof, optionally in combination with one or more amino acids and preferably at least one reducing carbohydrate, is heated under heating conditions. pH, temperature, pressure and reaction time sufficient to develop a flavor.

To obtain a good taste profile, the pH of the mixture in the method 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 and 7. , 5 and 6. The pH may be adjusted using food acceptable acids or bases within the knowledge of those skilled in the art.

Generally, in order to produce the process flavorant, the reaction mixture may be heated to a temperature between 70 and 200 ° C, preferably between 70 and 190 ° C, also depending on the reaction time. Longer reaction times usually require a lower reaction temperature while shorter reaction times usually require a longer reaction time. The reaction time may range from a few seconds (e.g. 2 seconds) to 6 hours, preferably from 10 seconds to 4 hours, more preferably from 20 seconds to 2 hours. To avoid loss of solvent during heating, the reaction mixture may be kept under reflux conditions.

Depending on the type of flavor to be obtained and the system used to produce the process flavoring, the ninth aspect method may be performed at a pressure ranging from reduced pressure (e.g., as low as 5 kPa, for example in a vacuum oven) to a pressure above atmospheric pressure, for example as high as 200-500 kPa. Optionally, when the amount of water in the process flavoring after the heating step is 4% w / w or more, the ninth aspect method comprises a step in which the process flavoring is dried. Methods known in the art, such as oven drying, belt drying, spray drying, can be used.

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

In order to produce, for example, toast-flavored process flavorings, generally a mixture comprising approximately 80% w / w dry matter, based on the total mixture, including water, may be heated above 100 ° C, for example. at a temperature of 110-120 ° C for a period of 4 to 6 hours at a reduced pressure, for example at 5 to 40 kPa.

In order to produce, for example, a process flavoring with a baked flavor, generally a mixture comprising a dry matter content based on the total mixture, including water, of 40 to 60% w / w may be heated to a temperature. approximately 100 ° C for a period of approximately 1 to 2 hours.

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

The extruder may be any type of extruder suitable for the production of process flavorings, such as a twin extruder. Extruders, for example twin extruders, are known to those skilled in the art. Ingredients may be introduced into the extruder through the same feeder or through separate feeders. When an extruder is used, preferably the mixture is kneaded and heated to a temperature of 110 to 190 ° C, preferably 130 to 165 ° C. Preferably, a pressure of 100 to 300 kPa is used. The reaction time is preferably from 2 seconds to 30 minutes, more preferably from 10 seconds to 5 minutes. When an extruder is used, the amount of dry matter 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 leave the extruder at a pressure outside the extruder ranging from reduced pressure (eg 0.5 kPa) to atmospheric pressure (eg approximately 100 kPa). The extruded product may be further cooled and / or dried using a cooling belt or any suitable method thereof.

If necessary, the ninth aspect method may further comprise a treatment aimed at further reducing the amount of acrylamide in the final process flavoring. The treatment may be any suitable treatment to reduce the amount of acrylamide in the product that can be applied in the production of process flavorings.

In one embodiment, the pH, temperature, pressure, and reaction time conditions in the ninth aspect method are adjusted to reduce the amount of acrylamide in the process. For example, the process flavoring is dried under reduced pressure. The pressure may generally be from 2 to 40 kPa.

In another embodiment, the final product is treated with an enzyme capable of modifying or degrading acrylamide. The treatment is carried out under conditions of sufficient pH, temperature and reaction time for the enzyme to react with the acrylamide.

Preferably, an amidase enzyme may be used. Examples of enzymes that could be used and the conditions under which the enzymes could be used are given in international patent application, filed 13-10-2005, application number PCT / EP2005 / 055242.

The process flavorant according to the eighth aspect of the invention, or obtainable with the method according to the ninth aspect of the invention, is very suitable, thanks to the very low level of acrylamide, to be used as a flavoring in food or food. or in food or feed ingredients.

Examples Materials and Methods Acrylamide Measurement Sample Pretreatment

Dry, homogenized 600 mg sample is extracted using 5 ml of milliQ water. 1 μg of standard internal 13C3 acrylamide in solution (CIL) is added to the extract. After 10 minutes of centrifugation (6000 rpm), 3 ml of the top layer is placed on an Extreluut-3BT column (Merck). Using 15 ml of ethylacetate, acrylamide is eluted from the column. The ethyl acetate is evaporated under a gentle stream of nitrogen to approximately 0.5 ml.

Chromatographic Conditions

The ethyl acetate solution is analyzed using gas chromatography. Separation is achieved using a CP-Wax 57 (Varian) column (length 25 m, inner diameter 0.32mm, 1.2 μιτι film) and helium as the carrier gas with a constant flow rate of 5.4 ml / min. Injection without separation of 3 μΐ is performed. The oven temperature is kept at 50 ° C for 1 minute, after which the temperature is increased by 30 ° C / minute to 220 ° C. After 12 minutes of constant temperature of 220 ° C, the oven is cooled and stabilized before the next injection.

Detection is performed using online ionization mass spectrometry in positive ion mode, using methane as the ionization gas. The characteristic ions m / z 72 (acrylamide) and m / z 75 (13C3 acrylamide) are monitored for quantification.

Used Equipment

GC: HP6890 (Hewlet Packard)

MSD: HP5973 (Packard Hewlet)

Method for Measuring Free Amino Acids

The following method was used to measure the amount of free amino acids (eg in yeast extract). A precisely weighed sample of yeast extract material was dissolved in dilute acid and the precipitates were removed by centrifugation in an Eppendorf centrifuge. Amino acid analysis was performed on the transparent supernatant according to the PicoTag method as specified in the Amino Acid Analysis System of Waters Operator's Manual (Milford MA, USA).

For this purpose, a suitable sample was obtained from the liquid, added to the diluted acid and homogenized. From this latter solution, a new sample was taken, dried and derived using phenylisothiocyanate. The various derived amino acids present were quantified using HPLC methods and summed to calculate the total level of free amino acids in the weighed sample.

Example 1 Production of a Free Low Asparagine Autolytic Yeast Extract

Enzyme unit definition: 1 μmole and NH3 released from L-asparagine per minute at pH 5.5 and 37 ° C.

200 l of a 20% solution of an autolytic yeast extract (Gistex® LS, DSM Food Specialties - Netherlands) was made in water. The pH of this solution was adjusted to 5.1 using 4 N KOH. 20 ml of asparaginase solution (the solution contained 4602 enzyme units / ml) was added to the yeast extract solution and the mixture was incubated for 4 hours. 51 ° C. Once the reaction was completed, the enzyme was deactivated by heat treatment. The resulting solution was spray dried, after pH was adjusted to pH 6.5. The final product contained less than 3.5% w / w water. The amino acid composition of the mixture was measured at the start end after enzyme treatment as indicated above. Results are listed in Table 1.

The asparaginase used was Aspergillus niger asparaginase, described in WO2004 / 030468.

The results in Table 1 clearly demonstrate that yeast extract after Asparaginase treatment is free of asparagine. In addition, the amount of aspartic acid increased with an amount similar to that of asparagine reduction.

Table 1: Effect of Asparaginase Treatment on an Autolytic Yeast Extract

<table> table see original document page 33 </column> </row> <table> <table> table see original document page 34 </column> </row> <table>

* mg per gram of yeast extract dry matter

Example 2

Preparation of Process Flavorers Using an Oven of Different Temperatures During the Heating Step

Formulation 2a

- 81.3 g Gistex® LS powder (DSM Food Specialties

- Netherlands)

16.0 g Maxarorae® Plus HS Powder (DSM Food Specialties - Netherlands)

24.3 g dextrose monohydrate

Formulation 2b

81.3 g yeast extract obtained in Example 1, powder

16.0 g Maxarome® Plus HS Powder (DSM Food Specialties - Netherlands)

24.3 g dextrose monohydrate

Gistex® Powder is an autolytic yeast extract comprising less than 1% w / w sodium chloride, a protein amount of 62% w / w and a free amino acid amount of 40 to 50% w / w of the total protein. , all weight percentages based on dry matter.

Maxarome Plus HS Powder is a hydrolytic yeast extract comprising -4% w / w sodium chloride based on dry matter. Furthermore, it comprises approximately 3% w / w each of 5'-GMP and 5'-IMP (measured as dihydrate sodium heptahydrate), 5% w / w glutamic acid (measured as free acid), 72% w / w protein and an amount of free amino acids that is -20% w / w of total protein, all weight percentages based on dry matter of sodium chloride free yeast extract.

In both cases, the powders were shaken with a spoon. In droplet form, 3.4 grams of sunflower oil was added under stirring to the powder mixture.

The powder mixtures were divided into aluminum trays.

The powders were heated in the oven for a fixed period of time at different temperatures. The powders obtained had a taste of grilled beef.

The powders were analyzed for the presence of acrylamide. Results are reported in Table 2.

Table 2

<table> table see original document page 35 </column> </row> <table>

Example 3

Preparation of Process Flavorers Using a Batch Kneader

30 grams of samples from formulations 3, 3a (see Table 3 for their compositions) were transferred to a 50 cc batch kneader, which was preheated to a specific temperature and mixed for 180 seconds at the same temperatures.

Temperature used for formulation 3, 3a: 14 5 and 150 ° C

Table 3

<table> table see original document page 36 </column> </row> <table>

The process flavorings obtained had a grilled chicken flavor.

The resulting process flavorings were analyzed for acrylamide content.

Results are reported in Table 4.

Table 4

<table> table see original document page 36 </column> </row> <table>

Example 4

Process Flavoring Preparation Using an Extruder Process Description

To a twin screw extruder, equipped with a dosing unit and water injector and a dosing unit and oil injector, a mixture of sugars and yeast extract was added using separate feeders. Two different formulations (formulation 4 and 4a, see Table 4) were used. 8 pounds of each formulation was processed in the extruder for one hour at 165 ° C.

The product was extruded from the extruder in an atmospheric pressure environment and was cooled and dried on a pressure roller cooling belt, ground and the sample was made.

Table 4

<table> table see original document page 37 </column> </row> <table>

The products obtained had a taste of dark grilled beef. Samples were analyzed for acrylamide.

Results:

Formulation 4: 4568 ppb

Formulation 4a: 400 ppb

Example 5

Production of Caselna Hydrolyzate, low in free asparagine

Enzyme unit definition: 1 μιηοΐβ released NH3 L-asparagine per minute at pH 5.5 and 37 ° C.

1 l of a 10% solution of casein hydrolyzate in water was made. The pH of this solution was adjusted to 5.1. 62 mg of asparaginase (having 14772 units / mg) was added to the casein solution and the mixture was incubated for 2 hours at 51 ° C. Once the reaction was completed, the enzyme was deactivated by heat treatment. The resulting solution was freeze dried. The amino acid composition of the mixture was measured at baseline and after enzyme treatment as indicated above. Results are listed in Table 6.

Table 6:

<table> table see original document page 38 </column> </row> <table> <table> table see original document page 39 </column> </row> <table>

* mg per gram of casein hydrolyzate dry matter

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

Example 6

Production of a Case Flavorizer Based on Casein Hydrolyzate

A mixture of 4.7 g glucose, 0.4 g glycine, 1.6 g maltodextrin and 13.4 g casein hydrolyzate was prepared as per Example 5 (asparagine free). As a reference, a similar mixture was prepared using asparaginase untreated casein hydrolyzate instead of the casein hydrolyzate of example 5.

Both mixtures were oven-baked for 45 minutes at 155 ° C. Finally, the acrylamide content was measured using the method described in the Materials and Methods section.

The result of the acrylamide analysis is listed in Table 7.

Table 7

<table> table see original document page 39 </column> </row> <table> <table> table see original document page 40 </column> </row> <table>

Example 7

Production of an autolysed yeast low in free asparagine

2 l of 18.5% Saccharomyces cerevisae cream yeast were autolyzed from 51.5 ° C dry solids for 24 hours at pH 5.1 in the presence of 2 grams of Alcalase® endoprotease (Novozymes - Denmark). Thereafter, the reaction mixture was heat treated to disable all enzyme activity.

1 l of the reaction mixture is further incubated for 2 hours at pH 5.1 and 51 ° C in the presence of 53 mg Asparaginase having 14772 units / mg). Thereafter, the enzyme was deactivated by heat treatment. The resulting solution was spray dried. A reference sample (not treated with asparaginase) was also spray dried. The amino acid composition of the dried materials (with and without asparaginase treatment) was measured. Results are listed in Table 8.

Table 8: Effect of Asparaginase Treatment on an Autolysed Yeast

<table> table see original document page 40 </column> </row> <table> <table> table see original document page 41 </column> </row> <table>

* mg per gram of autolysed yeast dry matter

The asparaginase used was Aspergillus niger asparaginase, described in W02004 / 030468.

Example 8

Production of a Process Flavorizer based on autolysed yeast

A mixture consisting of 4.7 g glucose, 0.4 g glycine, 1.6 g maltodextrin and 13.4 g autolysed yeast was prepared as prepared in Example 7 (asparagine free). As a reference, a similar mixture was prepared using untreated autolysed yeast.

Both mixtures were oven-baked for 45 minutes at 155 ° C. Finally, the acrylamide content was measured using the method described in the Materials and Methods section. The result of the acrylamide analysis is listed in Table 9.

Table 9

<table> table see original document page 42 </column> </row> <table>

Example 9

Yeast Extract Production, Low in Free Asparagine 2 1 of Yeast Cream

Saccharomyces cerevisae of 18.2 dried solids at 51 ° C for 17.5 hours at pH 5.1 in the presence of 2 grams of Alcalase® endoprotease (Novozymes - Denmark). The reaction mixture was further incubated for 2 hours at pH 5.1 and 51 ° C in the presence of 613 mg Asparaginase (having 180 units / mg). The cell walls were removed by centrifugation and the supernatant was heat treated for 5 minutes at 95 ° to disable all enzyme activity present. Then the supernatant was concentrated and spray dried.

Asparagine concentrations were measured in the reaction mixture before and after asparaginase treatment and in the final extract powder. Results are listed in Table 10.

Table 10: Results of Asparagine Analysis

<table> table see original document page 42 </column> </row> <table> <table> table see original document page 43 </column> </row> <table>

The asparaginase used was Aspergillus niger asparaginase, described as W02004 / 030468.

Claims (17)

Yeast extract characterized by the fact that it has a free dry matter-based amount of asparagine, which is not greater than 1 mg / g, preferably not greater than 0.2 mg / g, more preferably , is not greater than 0.1 mg / g. 2. Autolysed yeast characterized by the fact that it has a free dry matter-based amount of asparagine which is not greater than 1 mg / g, preferably not greater than 0.2 mg / g, more preferably is not greater than 0.1 mg / g. 3. Protein hydrolyzate characterized by the fact that it has a free dry matter-based amount of asparagine which is not greater than 1 mg / g, preferably not greater than 0.2 mg / g, more preferably , is not greater than 0.1 mg / g. Method for producing the yeast extract of claim 1, the autolysed yeast of claim 2, or the protein hydrolyzate of claim 3, comprising treating a starting yeast extract of an autolysed yeast of or an asparagine-free starting protein hydrolyzate with an enzyme, a physical method, a chemical method or a combination thereof, capable of reducing the amount of free asparagine in the yeast extract, autolysed yeast or protein hydrolyzate to obtain an amount of free asparagine in the yeast extract, autolysed yeast or protein hydrolyzate of not more than 1 mg / g, more preferably not greater than 0,2 mg / g more preferably not greater than 0.1 mg / g based on dry matter. Method according to claim 4, characterized in that 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 the enzyme. free asparagine. Method according to claim 5, characterized in that the enzyme treatment is performed using an enzyme capable of modifying the free asparagine side chain, preferably with an enzyme capable of hydrolyzing the amide group in the side chain. free asparagine, more preferably with the enzyme asparaginase (EC 3.5.1.1). Method according to any one of claims 4, 5 or 6, characterized in that the starting yeast extract, the starting autolysed yeast or the starting protein hydrolyzate is an intermediate product obtained in one step. a method for producing a yeast extract or autolysed yeast from yeast cells, preferably during treatment, more preferably after treatment, of yeast cells to release and optionally at least partially degrade the cell content or intermediate obtained in one step of a method for the production of a protein hydrolyzate from a protein substrate, preferably during treatment, more preferably after treatment of the protein substrate so as to hydrolyze it into a mixture of peptides and amino acids. Use of the yeast extract of claim 1 or a yeast extract obtainable by a method of any one of claims 4, 5, 6 or 7, characterized in that it is in food, feed or food or feed ingredient or in the preparation thereof, preferably in the preparation of a process flavorant. Use of the autolysed yeast of claim 2 or an autolysed yeast obtainable by a method of any one of claims 4, 5, 6 or 7, characterized in that it is in food, feed or food or feed ingredients or in the preparation preferably in the preparation of a process flavorant. Use of the protein hydrolyzate of claim 3 or a protein hydrolyzate obtainable by a method of any one of claims 4, 5, 6 or -7, characterized in that it is in food, feed or in food ingredients or feed or preparation thereof, preferably in the preparation of a process flavorant. 11. Flavoring process, characterized in that it has an amount of acrylamide, based on the dry matter of the product, which is not greater than 800 ppb, preferably not greater than 600 ppb, more preferably not greater than 400 ppb, more preferably not greater than 200 ppb. A method for producing the process flavoring agent of claim 11, which comprises subjecting a mixture containing a source of amino acids selected from the yeast extract of claim 1 to the autolysed yeast of claim 2 to the protein hydrolyzate of claim 12. 3 or a mixture thereof, and preferably at least one reducing carbohydrate, to heat under conditions of pH, temperature, pressure and reaction time sufficient to develop a taste. A method for producing the process flavorant of claim 11, which comprises subjecting a mixture containing a source of amino acids selected from a yeast extract, an autolysed yeast, a protein hydrolyzate or a mixture thereof. contains an amount of free asparagine that is greater than 1 mg / g, by treatment with an enzyme, a physical method, a chemical method or a combination thereof, capable of reducing the amount of free asparagine, preferably to a level of less than 1 mg / g and warming under conditions of pH, temperature, pressure and reaction time sufficient to develop a taste. Method according to any one of claims 12 or 13, characterized in that the mixture ingredients are introduced into an extruder, the mixture is kneaded and heated under conditions of pH, temperature, pressure and reaction time sufficient to develop a flavor and the resulting process flavor is subsequently extruded from the extruder. A method according to any one of claims 12, 13 or 14, characterized in that the conditions of pH, temperature, pressure and / or reaction time are adjusted to reduce the amount of acrylamide in the reaction flavorant of preferably, wherein the process flavoring is dried under reduced pressure. Method according to any one of claims 12, 13, 14 or 15, characterized in that the reaction flavorant is further treated with an enzyme capable of modifying or degrading acrylamide, preferably with an amidase. Use of the process flavoring agent of claim 11, or obtainable by a process of any one of claims 12, 13, 14, 15 or 16, characterized in that it is in providing flavor to food or feed or food or feed ingredients. .