Application of aspergillus niger fructosyl transferase

Abstract

The present invention relates to a method of converting sucrose contained in a liquid food product to at least one fructooligosaccharide (FOS), the method comprising contacting the liquid food product with an Aspergillus niger fructosyl transferase under conditions allowing enzymatic conversion of sucrose to at least one FOS by a fructosyl transferase, wherein the contacting is carried out at least until the sucrose concentration in the liquid food product is at most 15 g / L; and compositions, uses and products related thereto.

Description

Technical Field

[0001] The present invention relates to a method for converting sucrose contained in a liquid food product into at least one fructo-oligosaccharide (FOS), the method comprising contacting the liquid food product with an Aspergillus niger fructosyltransferase under conditions allowing enzymatic conversion of sucrose by the fructosyltransferase into at least one FOS, wherein the contacting is carried out at least until the sucrose concentration in the liquid food product is at most 15 g / L; and compositions, uses and products related thereto. Background Art

[0002] Many food products produced from fruits or beans contain high concentrations of sucrose. For many applications, it may be necessary to reduce the sucrose concentration in such food products. For this purpose, invertase has been used for a long time, and it hydrolyzes sucrose into the monomeric sugar units of sucrose: glucose and fructose. A member of the invertase family is the Aspergillus niger invertase, which is described in Yuan et al. (2006), Microbiology 152:3061 and Boddy et al. (1993), Curr Genet 24:60.

[0003] However, it is generally considered desirable to reduce the amount of monosaccharides and disaccharides in food products, particularly from a health perspective, for example to reduce the glycemic index of monosaccharides and disaccharides. One option for achieving this goal is to use fructosyltransferases, enzymes that transfer the fructose units of sucrose to sucrose or other acceptor sugars, thereby generating oligofructose containing several fructose moieties. Corresponding enzymes and methods have been described, for example, in EP 3231293 A1, EP 2294093 A1, EP 1951885 A1 and WO2008 / 102336 A2.

[0004] It is worth noting that fructosyltransferases generally also have invertase activity, especially at low acceptor concentrations; that is, for most fructosyltransferases, at relatively high sucrose concentrations, the hydrolysis of sucrose already prevails over the transfer of fructose units. For example, in EP 1951885 A1, using Aspergillus japonicus fructosyltransferase, oligofructose production was essentially stopped at sucrose concentrations below about 5% (i.e., 50 g / L). Summary of the invention

[0005] In view of the above, there is a need for improved tools and methods for reducing the sucrose content in food products. The technical problem of the present invention can be seen as providing tools and methods to meet the above needs. The technical problem is solved by the claims and the embodiments described below.

[0006] Accordingly, the present invention relates to a method for converting sucrose contained in a liquid food product into at least one fructo-oligosaccharide (FOS), said method comprising contacting said food product with an Aspergillus niger fructosyltransferase under conditions allowing enzymatic conversion of sucrose by said fructosyltransferase into at least one FOS.

[0007] In general, the terms used herein are given their common and customary meanings to those of ordinary skill in the art and are not limited to special or customary meanings unless otherwise specified. As described below, the terms "having", "including" or "comprising" or any grammatical variants thereof are used in a non-exclusive manner. Therefore, these terms can refer to the situation where there are no other features in the entity described herein in addition to the features introduced by these terms, or the situation where there are one or more other features. For example, the expressions "A has B", "A includes B" and "A includes B" can refer to the situation where there are no other elements in A except B (i.e., A is composed of B alone and exclusively), or the situation where there are one or more other elements (e.g., element C, element C and D or even other elements) in addition to B in entity A. In addition, as understood by those skilled in the art, the expressions "comprising a" and "comprising an" preferably refer to "comprising one or more", i.e., equivalent to "comprising at least one". Thus, unless otherwise stated, a statement relating to one of a plurality of items preferably relates to at least one such item, more preferably a plurality of such items; thus, for example, identifying "a cell" relates to identifying at least one cell, preferably a plurality of cells. At the same time, reference to "at least one oligofructose" relates to at least one type of oligofructose, as described below.

[0008] In addition, as described below, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in combination with optional features without limiting further possibilities. Therefore, the features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. Those skilled in the art will recognize that the present invention can be implemented by using alternative features. Similarly, the features introduced by "in an embodiment" or similar expressions are intended to be optional features, without any limitation on other embodiments of the invention, without any limitation on the scope of the invention, and without any limitation on the possibility of combining the features introduced in this way with other optional or non-optional features of the invention.

[0009] The methods specified below are preferably in vitro methods. In principle, the method steps can be performed in any arbitrary order that the skilled person considers suitable, but are preferably performed in the specified order; likewise, one or more (preferably all) of the steps can be assisted or performed by automated equipment. Furthermore, the methods may include steps other than those explicitly mentioned above. Furthermore, the methods can be performed at any scale that the skilled person considers suitable, such as laboratory scale, pilot plant scale or industrial scale.

[0010] As used herein, the term "standard conditions" (if not otherwise specified) refers to IUPAC standard ambient temperature and pressure (SATP) conditions, i.e., preferably a temperature of 25°C and an absolute pressure of 100 kPa; also preferably, standard conditions include a pH value of 7. In addition, if not otherwise indicated, the term "about" refers to an indicated value with a technical accuracy generally accepted in the relevant field, preferably to an indicated value of ±20%, more preferably ±10%, and most preferably ±5%. In addition, the term "substantially" means that there is no deviation that has an impact on the indicated result or use, i.e., the potential deviation will not cause the indicated result to deviate by more than ±20%, more preferably ±10%, and most preferably ±5%. Therefore, "substantially consisting of..." means including the specified ingredients, but excluding other ingredients, except for materials present as impurities, unavoidable materials present due to the process used to provide the ingredients, and ingredients added for purposes other than achieving the technical effects of the present invention. For example, a composition defined using the phrase "substantially consisting of..." includes any known acceptable additives, excipients, diluents, carriers, etc. Preferably, a composition consisting essentially of a group of ingredients will contain less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, most preferably less than 0.1% by weight of the non-specified ingredient or ingredients.

[0011] The degree of identity (e.g., expressed as "% identity") between two biological sequences (preferably DNA, RNA or amino acid sequences) can be determined by algorithms well known in the art. Preferably, the degree of identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the sequence fragments in the comparison window may include additions or deletions (e.g., gaps or overhangs) compared to the sequences compared to which the optimal alignment is obtained. The percentage is calculated by determining the number of positions where the same residues occur in the two sequences (preferably over the entire length of the polynucleotide or polypeptide) to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the percentage of sequence identity. The optimal alignment of sequences for comparison can be performed by searching by the local homology algorithm of Smith and Waterman (1981), the homology alignment algorithm of Needleman and Wunsch (1970), the similarity method of Pearson and Lipman (1988), computerized implementations of these algorithms (e.g., BLAST, GAP, BESTFIT, PASTA or TFASTA), or visual inspection. If two sequences have been determined to be used for comparison, preferably GAP and BESTFIT are used to determine their optimal comparison, thereby determining the degree of identity. Preferably, the default value of 5.00 for the gap weight and the default value of 0.30 for the gap weight length are used. More preferably, the comparison is performed using the basic local alignment search tool (BLAST) implementation and default parameter values. In the context of biological sequences referred to herein, the term "substantially identical" represents at least 80%, preferably at least 90%, more preferably at least 98%, and most preferably at least 99% identity value%. It is understood that the term "substantially identical" includes 100% identical. The foregoing is applicable to the term "basic complementarity" after necessary modifications.

[0012] The term "fragment" of a biomacromolecule (preferably a polynucleotide or polypeptide) is used in a broad sense herein and relates to any sub-portion (preferably a subdomain) of each biomacromolecule comprising the sequence, structure and / or function indicated. Thus, the term includes sub-portions generated by actual fragmentation of the biomacromolecule, but also sub-portions derived from the corresponding biomacromolecule in an abstract manner (e.g., on a computer). Thus, a fragment of a fructosyltransferase may in particular be a sub-portion of a fructosyltransferase as described below, which still has the activity described. Unless otherwise specifically stated herein, the specified compounds, in particular polynucleotides and polypeptides, may be included in a larger structure, for example, may be covalently or non-covalently linked to other sequences, carrier molecules, retardants and other excipients. In particular, the specified polypeptide may be included in a fusion polypeptide comprising other peptides, which may be used, for example, as tags, linkers for purification and / or detection, or to extend the in vivo half-life of the compound. However, preferably, the polypeptide consists of a specifically specified sequence.

[0013] As used herein, the term "polypeptide" refers to a molecule consisting of a plurality of (usually at least 20) amino acids covalently linked by peptide bonds. Molecules consisting of less than 20 amino acids covalently linked by peptide bonds are generally considered to be "peptides". Preferably, the polypeptide comprises 50 to 1000, more preferably 75 to 1000, more preferably 100 to 500, and most preferably 110 to 400 amino acids. Preferably, the polypeptide may be contained in a fusion polypeptide and / or a polypeptide complex.

[0014] The term "sucrose" is understood by the skilled person to relate to β-D-fructofuranosyl-α-D-glucopyranoside (No. 57-50-1), which is the main component of eg beet sugar and cane sugar.

[0015] The term "oligosaccharide" is understood by the skilled person. Preferably, an oligosaccharide is an oligomer comprising at most 10, preferably at most 6, even more preferably at most 5, still more preferably at most 4 monosaccharide units. Also preferably, the oligosaccharide comprises at least 3 monosaccharide units, i.e. is a trisaccharide, or comprises at least 4 monosaccharide units. Thus, preferably, the oligosaccharide comprises 3 to 10, preferably 3 to 5 monosaccharide units.

[0016] As used herein, the term "oligofructose", which may also be referred to herein as "FOS" or "oligofructan", relates to an oligosaccharide as defined above, comprising at least 2 fructose monomers and optionally at least 1 glucose monomer. Preferably, FOS comprises one D-glucose molecule in terminal position and from 2 to 4 D-fructose units, wherein the linkages between the fructose residues are preferably β-(2-1) glycosidic linkages. Thus, preferably, FOS is produced by transferring one or more fructose units to sucrose. Preferably, the oligofructose comprises, preferably, kestose, preferably 1-kestose (CAS No. 470-69-9), kestotetraose (preferably PubChem CID 91851626) and / or kestopentaose (preferably PubChem CID 1337599).

[0017] The term "liquid food product" is understood by a person skilled in the art. Preferably, the term "liquid" product as used herein comprises all products which are not solid under standard conditions. Thus, a liquid product is a product which, within a time frame of up to 1 hour, assumes substantially the shape of the container in which it is contained. Thus, the term liquid food product also comprises semi-solid products, such as puree. Thus, as used herein, the term "liquid food product" may also be replaced by the term "liquid or semi-solid food product". Preferably, the viscosity of the liquid food product, preferably before being treated by the method of the present invention, is from 1 mPa*s to 10000 mPa*s, more preferably from 10 mPa*s to 6000 mPa*s, wherein the viscosity is preferably a dynamic viscosity, preferably measured as described in the examples below.

[0018] As used herein, the term "food" relates to a composition of matter that is safe for human consumption; preferably, suitable for human consumption as determined in accordance with applicable EU and / or US regulations (preferably as of July 2022). Preferably, the term food product also includes a composition of matter that is expected to be safe for animal consumption and / or a composition of matter that is safe for animal consumption, which may also be referred to as "feed". Therefore, as used herein, the term food product is used in a broad sense and may preferably be replaced by the term "food or feed product". It is understood by those skilled in the art that in the context of this specification, it may not always be possible to strictly separate food products and feed products, because, for example, the liquid part of the product can be used for human consumption, while the solid component of the same product obtained, for example, by filtration or precipitation can be used as feed. Preferably, the liquid food product for contact is a food product that has not been enzymatically pretreated or has been pretreated only with cellulase and / or amylase, i.e., preferably, it has not been pretreated with one or more enzymes other than cellulase and / or amylase. More preferably, the liquid food product is a food product that has not been enzymatically pretreated; therefore, the fructosyltransferase is preferably the first enzyme that the liquid food product contacts; more preferably, the fructosyltransferase and optionally the pectinase are the first enzymes that the liquid food product contacts, and even more preferably, the fructosyltransferase and optionally the pectinase are the only enzymes that the liquid food product contacts. For the avoidance of doubt, it is pointed out that the term "food product" includes a finished product, such as a product that can be sold and / or eaten, and any intermediate product produced by the method described herein, which can be processed by further methods and / or can be used to produce at least one other intermediate product or finished product. In view of the description elsewhere herein, the liquid food product used in the contacting step is preferably an early crude product in the production process, and can therefore be, for example, a crude juice or paste, which is obtained by crushing and / or pressing, optionally pre-treated by heat treatment and / or centrifugation, fruits and / or vegetables. Preferably, the liquid food product used in the contacting step is a crude product from an early stage in the production process and may thus be a crude juice or paste obtained, for example, by crushing and / or pressing fruits and / or vegetables without further pre-treatment.

[0019] Prior to said contacting, the liquid food product comprises sucrose, preferably in a concentration of at least 1 g / L, more preferably at least 2 g / L, even more preferably at least 5 g / L. Thus, the liquid food product is preferably a plant-based food product, preferably a vegetable juice, vegetable puree, fruit juice or fruit puree. Preferably, the food product is a crude fruit juice or crude vegetable juice or a crude fruit puree or a crude vegetable puree. The fruit juice or vegetable juice may be a clear or turbid fruit juice or vegetable juice; in addition, the fruit juice or vegetable juice may be a freshly prepared fruit juice or vegetable juice, a concentrated fruit juice or vegetable juice, may be prepared from concentrated fruit juice or vegetable juice, or may be prepared from non-concentrated fruit juice or vegetable juice. Preferably, the liquid food product is or comprises a juice or puree made from apples, oranges, watermelons, grapefruits, plums, apricots, peaches, tangerines, pineapples, lychees, mangoes, honeydew melons, bananas, vegetables, onions, black roots, celery, carrots, horseradish, red beets and / or chestnuts. The liquid food product can comprise the component except the component mentioned above, particularly can comprise the sugar (for example sucrose), water, pH adjusting agent and / or other additives that those skilled in the art think suitable of adding.In addition, the liquid food product can be the mixture of at least two above-mentioned fruit juices and / or fruit puree, and the mixture can also comprise other compounds as mentioned above.However, the liquid food product should be a liquid food product in any case, and should comprise the sucrose of at least 1g / L before the contact.Preferably, the liquid food product is orange juice, pineapple juice, apple juice, apple paste, banana mud or apricot mud.

[0020] Preferably, before the contact, the liquid food product comprises a sucrose concentration of less than 50 g / L, preferably less than 25 g / L, more preferably less than 20 g / L, still more preferably less than 15 g / L, even more preferably less than 10 g / L, most preferably less than 5 g / L. Therefore, preferably, before the contact, the liquid food product is a low-sucrose food product. Also preferably, after the contact, the residual sucrose concentration in the liquid food product is at most 50 g / L, preferably at most 25 g / L, more preferably at most 15 g / L, still more preferably at most 10 g / L, even more preferably at most 5 g / L. Therefore, also preferably, after the contact, the liquid food product is a low-sucrose food product. Those skilled in the art will appreciate that the sucrose concentration prior to contact is preferably higher than the target sucrose concentration after contact; thus, in the case where contact is performed to a sucrose concentration of up to 15 g / L, the sucrose concentration prior to contact is preferably at least 16 g / L, preferably at least 17 g / L, more preferably at least 20 g / L, still more preferably at least 25 g / L, and most preferably at least 50 g / L. The above applies mutatis mutandis to other target concentrations.

[0021] The term "contacting" is understood by the skilled person. Preferably, the term relates to bringing the specified compound into physical contact with the liquid food product, and thereby, for example, allowing the liquid food product and the compound to interact. Thus, contacting preferably comprises preparing a mixture of the liquid food product and the fructosyltransferase. Preferably, the contacting is carried out under conditions that allow the enzymatic conversion of sucrose to at least one FOS by the fructosyltransferase. The conditions comprise, preferably, mixing the fructosyltransferase into the liquid food product, i.e. preferably using a water activity substantially the same as that already present in the liquid food product. The conditions preferably also comprise a time range suitable for the fructosyltransferase to be active; as will be understood by the skilled person, the time range may depend in particular on the amount of enzyme activity contacted; a typical time range may be from 10 minutes to 24 hours, preferably 15 minutes to 12 hours, more preferably 20 minutes to 6 hours, most preferably 25 minutes to 180 minutes. The conditions preferably also include a temperature suitable for the fructosyltransferase to be active; as will be understood by those skilled in the art, the temperature may depend in particular on the amount of enzyme activity contacted, and also on the sensitivity of the liquid food product to high temperatures and / or temperature variations. Typical temperature ranges may be, for example, from 0°C to 75°C, preferably from 4°C to 60°C, more preferably from 15°C to 55°C, most preferably from 18°C ​​to 55°C. Preferably, the conditions include contacting at a temperature of 20°C ± 10°C or 50°C ± 10°C. It will be understood by those skilled in the art that the liquid food product may contain one or more enzymes already present in the fruit or vegetable used to prepare the liquid food product; as referred to herein, no matter which enzyme activity is involved, such inherently present enzymes are not referred to by the term "contact". Therefore, as used herein, the term "contact" only relates to contacting with enzyme activity added exogenously to the liquid food, preferably by adding a composition of substances that are not liquid foods as described above.

[0022] The skilled person understands that the term "fructosyltransferase" relates to an enzyme which, under appropriate conditions, produces FOS by transferring a fructose unit to an appropriate acceptor. The fructosyltransferase preferably has a sucrose: sucrose fructosyltransferase activity (EC 2.4.1.99); preferably, the fructosyltransferase has the above activity at low sucrose concentrations, preferably a sucrose concentration of at most 15 g / L, preferably at most 10 g / L, more preferably at most 5 g / L. The fructosyltransferase may have one or more additional activities, in particular an invertase activity (sucrose hydrolase activity, EC 3.2.1.26). Preferably, however, the fructosyltransferase activity is higher than the invertase activity, in particular at the above sucrose concentrations. Thus, preferably, in a solution comprising sucrose at a concentration of at most 5 g / L, at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95% of the fructose contained in the sucrose is converted into FOS by the fructosyltransferase. Thus, at least the above-mentioned fructose portion contained in sucrose as fructose units is converted into at least one FOS, and the term "generating" fructose refers to the generation of fructose monomers, which may, however, be enzyme-bound intermediates of fructose, which are then transferred to a suitable acceptor molecule, such as sucrose. Therefore, the "generating" fructose referred to herein does not necessarily include the production of free fructose.

[0023] The fructosyltransferase is an Aspergillus niger fructosyltransferase. As described above, fructosyltransferases from Aspergillus strains, such as A. niger, are known in the art. Preferably, the fructosyltransferase comprises the amino acid sequence of the mature form of Genbank accession number AHC54391.1 and / or the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, still more preferably at least 98%, most preferably at least 99% identity thereto. More preferably, the fructosyltransferase comprises, preferably consists of, the amino acid sequence of the mature form of Genbank accession number AHC54391.1 and / or the amino acid sequence of SEQ ID NO: 1, which can be produced, for example, by expressing a gene encoding a sequence comprising the amino acid sequence of SEQ ID NO: 2 in a suitable host cell, such as a Trichoderma spec. cell. Preferably, the fructosyltransferase is produced by Trichoderma cells, more preferably purified from Trichoderma cultures, most preferably purified from Trichoderma culture supernatants, preferably as described in Bergès et al. (1993), Curr Genet 24:53 or in WO 2018 / 009806 A1. The fructosyltransferase can be obtained as a crude extract or a partially enriched extract from Aspergillus niger or Trichoderma cells, or can be recombinantly expressed, for example in bacterial cells. However, the fructosyltransferase can also be purified, for example by filtration, chromatography, precipitation and / or any other purification step deemed suitable by the skilled person, to a purity of, for example, at least 70%, preferably 80%, more preferably 90%.

[0024] The method for converting sucrose comprises the step of contacting the liquid food product with a fructosyltransferase under conditions allowing enzymatic conversion of sucrose into at least one FOS by the fructosyltransferase. Preferably, the contacting is carried out at least until the sucrose concentration in the liquid food product is at most 15 g / L, preferably at most 10 g / L, more preferably at most 5 g / L; i.e. the contacting is preferably continued until the above-mentioned maximum sucrose concentration is reached or the above-mentioned maximum sucrose concentration falls below the said maximum sucrose concentration. Preferably, at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95% of the fructose in the sucrose contained in the liquid food product before the contacting is converted into at least one FOS during the contacting. Thus, in view of the above, at least the above-mentioned fraction of the total fructose units released from the sucrose is contained in at least one FOS. Thus, preferably less than 5 g / L, more preferably less than 2 g / L of fructose is produced by the conversion. More preferably, during the contacting, the fructose concentration in the liquid food product does not increase significantly.

[0025] Preferably, the method described herein is an early step in the production of a liquid food product. Thus, the Aspergillus niger fructosyltransferase can in principle be added to the food product before or during one or more initial crushing and / or pressing steps. Preferably, the Aspergillus niger fructosyltransferase is added directly after the above-mentioned one or more initial crushing and / or pressing steps, and / or the contacting step is carried out directly after the addition. Optionally, the crude preparation obtained in the one or more initial crushing and / or pressing steps can be further pretreated by centrifugation. More preferably, no other enzyme pretreatment step is carried out before the contacting step with the Aspergillus niger fructosyltransferase; therefore, the above-mentioned contacting step is preferably the first enzyme treatment step, or is included in the first enzyme treatment step of the liquid food product. Also preferably, the contacting step referred to herein is a step before the optional evaporation step, for example in the manufacture of concentrated juice, preferably a step immediately before the optional evaporation step, or a step before the optional ultrafiltration step, for example in the production of apple juice, preferably a step immediately before the optional ultrafiltration step. Therefore, the contacting step may preferably be performed between the centrifugation step and the evaporation step of crude juice in a cloudy juice production process, between the crushing step and the pressing step and / or between the first pressing step and the second pressing step in apple paste processing, or between the pressing step and the ultrafiltration step in apple juice production.

[0026] Optionally, the method for converting sucrose further comprises contacting the food product with at least one pectinase. The term "pectinase" is known to the skilled person in the art to relate to a group of hydrolases that degrade pectin, preferably having endopolygalacturonase activity (EC 3.2.1.15), exopolygalacturonase (EC 3.2.1.67), arabinan endo-1,5-alpha-L-arabinanase activity (EC 3.2.1.99), arabinofuranosidase activity (EC 3.2.1.55), rhamnogalacturonan hydrolase (EC 3.2.1.171), pectin methylesterase activity (EC 3.1.1.11), pectin acetylesterase (EC 3.1.1.6), glucan 1,4-alpha-glucosidase activity (EC 3.2.1.3), pectate lyase (EC 3.2.1.7), pyruvate lyase (EC 3.2.1.9), pyruvate lyase (EC 3.2.1.10), pyruvate lyase (EC 3.2.1.112), pyruvate lyase (EC 3.2.1.13), pyruvate lyase (EC 3.2.1.14), pyruvate lyase (EC 3.2.1.15), pyruvate lyase (EC 3.2.1.16), pyruvate lyase (EC 3.2.1.173), pyruvate lyase (EC 3.2.1.18), pyruvate lyase (EC 3.2.1.19). 4.2.2.2) and / or pectin lyase activity (EC 4.2.2.10). The contacting with at least one pectinase may be carried out before, simultaneously and / or after the contacting with the fructosyltransferase, preferably simultaneously with the contacting with the fructosyltransferase. Preferably, the pectinase is an Aspergillus niger pectinase.Suitable pectinases and mixtures thereof are commercially available, for example ROHAPECT® PTE 100 (AB Enzymes), ROHAPECT® MA plusT (AB Enzymes), ROHAPECT® UF (AB Enzymes), Pectinex® Smash XXL (Novozymes), ROHAPECT® MA Plus HC (AB Enzymes), ROHAPECT® MA Plus (AB Enzymes), Pectinex® Yield Mash Extra (Novozymes), Pectinex® Yield Mash Plus (Novozymes), Pectinex® XXL (Novozymes), Pectinex® Ultra Clear (Novozymes), Pectinex® UF (Novozymes), Rapidase® Smart (DSM), Rapidase® Smart Clear (DSM), Rapidase® Power (DSM), Rapidase® Smart Plus (DSM), GAMYLOZYM® AFL (AB Enzymes), GAMMADEX® CAL (AB Enzymes), Hazyme® DCL (DSM), Amylase AG XXL (Novozymes) or Amylase AG300 L (Novozymes).Preferably, ROHAPECT® PTE 100 and / or Pectinex® Smash XXL are used in orange juice processing; ROHAPECT® MA plus T, ROHAPECT® MA Plus HC, ROHAPECT® MA Plus, Pectinex® Yield Mash Extra, Pectinex® Yield Mash Plus, Rapidase® Smart and / or Rapidase® Smart Plus are used in apple mash processing; GAMYLOZYM® AFL, ROHAPECT® UF; Pectinex® XXL; Pectinex® UF, Pectinex® Ultra Clear, Rapidase® Smart Clear, Rapidase® Power, GAMMADEX® CAL, Hazyme® DCL, Amylase AG XXL and / or Amylase AG 300 L are used in apple juice processing; ROHAPECT® B1L, Pectinex® Ultra Tropical, Pectinex® Ultra SP-L, Rapidase® Pineapple and / or Pectinex® Power for pineapple juice; ROHAPECT® PTE 100 and / or Pectinex® Smash XXL for banana or apricot puree; all in combination with the specified fructosyltransferase.

[0027] Optionally, the method for converting sucrose further comprises contacting the liquid food product with at least one cellulase and / or amylase as described above, particularly in the pretreatment of the liquid food product. Preferably, however, the method does not comprise an enzyme treatment step before and / or after the contacting. Preferably, the method for converting sucrose further comprises reducing the turbidity and / or viscosity of the liquid food product, particularly in the case where the method further comprises contacting the food product with at least one pectinase.

[0028] Optionally, the method comprises a further step of inactivating the fructosyltransferase, for example by heat inactivation. Preferably, in this case, the liquid food product is heated to a temperature of at least 85°C, more preferably at least 90°C, even more preferably at least 95°C, for at least 5 minutes, more preferably at least 10 minutes, most preferably at least 15 minutes. Preferably, the heat inactivation is carried out at a temperature of from 75°C to 100°C, more preferably at a temperature of from 80°C to 97°C, most preferably at a temperature of from 85°C to 95°C, for one of the above time ranges. Also optionally, the liquid food is heat treated prior to said contacting, preferably as described above.

[0029] After said contacting step, the liquid food product preferably has at least one altered property; the skilled person understands that the term "altered" means a comparison with an appropriate control, preferably an untreated control or a vehicle control, the vehicle control being a control contacted with a vehicle for providing the corresponding one or more enzymes, but not including said one or more enzymes. Preferably, after said contacting, preferably as described above, the liquid food product has a reduced sucrose concentration, an increased FOS content. Also preferably, the liquid food product has a reduced turbidity after said contacting, preferably compared to an untreated control and / or a vehicle control. Methods for determining turbidity are known to the skilled person; preferably, turbidity is determined as light scattering turbidity units (NTU) according to standard methods. Preferably, before the turbidity measurement, the liquid food product is centrifuged, more preferably at from 1000g to 10000g, preferably at 8000g, for from 1 to 30 minutes, preferably from 2 to 15 minutes, more preferably about 2 minutes. Also preferably, said liquid food product has a reduced viscosity, preferably a reduced dynamic viscosity, after said contacting, preferably compared to an untreated control and / or a vehicle control. Methods for determining viscosity in liquid food products are known in the art. Preferably, said dynamic viscosity is determined by rolling ball viscometry, preferably under standard conditions, preferably at a temperature of 20°C.

[0030] Advantageously, it was discovered in the work underlying the present invention that the Aspergillus niger fructosyltransferase (also referred to as "Aspergillus niger invertase") has a high fructosyltransferase activity even at low sucrose concentrations; thus, the enzyme can be used to convert sucrose into FOS with essentially no production of free fructose; furthermore, the enzyme can be used to convert sucrose into FOS even at low sucrose concentrations, for example in low-sucrose foods and / or in situations where it is desired to convert the sucrose as completely as possible.

[0031] The above definitions apply mutatis mutandis to the following. The other definitions and explanations given further below also apply mutatis mutandis to all embodiments described in this specification.

[0032] The present invention also relates to a method for reducing the turbidity and / or viscosity of a liquid food product, the method comprising contacting the liquid food product with (i) a fructosyltransferase and (ii) a pectinase under conditions allowing the fructosyltransferase and the pectinase to have enzymatic activity, wherein the contacting with (i) and (ii) is preferably simultaneous.

[0033] The method for reducing turbidity and / or viscosity may include steps in addition to the steps explicitly mentioned above. For example, further steps may involve, for example, one or more heat treatments as described above, removal of solid components from the liquid food product, etc. In addition, one or more of the steps may be assisted or performed by automated equipment. Preferably, in addition to reducing the turbidity and / or viscosity of the liquid food product, the method also converts sucrose into at least one FOS, as described above. The method may include further steps of enzymatic, chemical and / or physical treatment; however, preferably, the method does not include an enzymatic treatment step prior to the contacting.

[0034] The present invention also relates to a composition comprising (i) an Aspergillus niger fructosyltransferase and (ii) a pectinase, wherein the Aspergillus niger fructosyltransferase preferably comprises the amino acid sequence shown in SEQ ID NO: 1 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, still more preferably at least 98%, and most preferably at least 99% identity thereto.

[0035] As referred to herein, the term "composition" relates to any composition of matter comprising the indicated components. Preferably, the composition is a solid composition, such as a lyophilisate or a granulate, which may be formulated, for example, with sodium chloride and / or maltodextrin. More preferably, the composition is a liquid composition, such as a stock solution, preferably ready for incorporation into a liquid food product. In addition to the components mentioned herein, the composition may also comprise other components, such as at least one solvent, in particular water; at least one stabilizer, in particular glycerol or sorbitol; at least one salt, such as a sodium, potassium, calcium and / or magnesium salt; at least one buffer, such as a phosphate or citrate buffer; at least one preservative, such as a benzoate or a sorbate; and / or one or more other additives deemed suitable by the skilled person. The composition may also comprise a filler, a moisture regulator and / or an antioxidant, in particular in the case where the composition is a solid composition. The skilled person understands that the other components of the composition are preferably selected so that they do not interfere with the shelf life and / or activity of the fructosyltransferase and pectinase. Furthermore, the other components of the composition are preferably selected to be compatible with the use of the composition in a food product.

[0036] The present invention also relates to a kit comprising (i) an Aspergillus niger fructosyltransferase and (ii) a pectinase, wherein the Aspergillus niger fructosyltransferase preferably comprises the amino acid sequence shown in SEQ ID NO: 1 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, still more preferably at least 98%, and most preferably at least 99% identity thereto.

[0037] The term "kit" as used herein refers to a collection of the aforementioned compounds, tools or reagents, which are preferably packaged together, more preferably in a common housing. The housing may be any kind of container and / or packaging deemed suitable by a person skilled in the art. The components of the kit may consist of separate containers (i.e., as a set of separate parts) or be provided in individual vials. In addition, it should be understood that the kit may preferably be used to implement the methods mentioned elsewhere herein. Preferably, it is envisioned that all components are provided in a ready-to-use manner for implementing the methods mentioned above. In addition, the kit preferably comprises instructions for performing the methods. These instructions may be provided by a user manual in paper or electronic form. In addition, the manual may include instructions for administration and / or dosage instructions for performing the above methods using the kit of the present invention. Preferably, the kit comprises a diluent and / or a tool for administration. Preferably, the fructosyltransferase and / or pectinase is contained in a preparation containing other components, and the preparation is preferably used for the preparation of the composition as described herein.

[0038] The present invention also relates to the use of Aspergillus niger fructosyltransferase in liquid food products for converting sucrose into at least one fructo-oligosaccharide (FOS); to the use of Aspergillus niger fructosyltransferase in liquid food products for producing at least one FOS; to the use of Aspergillus niger fructosyltransferase in liquid food products for reducing the sucrose concentration to a maximum of 50 g / L, preferably a maximum of 25 g / L, more preferably a maximum of 15 g / L, still more preferably a maximum of 10 g / L, even more preferably a maximum of 5 g / l; and to the use of Aspergillus niger fructosyltransferase and pectinase for reducing the turbidity and / or viscosity of liquid food products.

[0039] The above-mentioned uses preferably comprise one or more of the above-mentioned features of the method of the present invention. Preferably, the liquid food product is a low-sucrose food product. Preferably, the liquid food product is a low-sucrose food product before being used for one of the above-mentioned uses, preferably comprising a concentration of sucrose less than 50 g / L, preferably less than 25 g / L, more preferably less than 20 g / L, still more preferably less than 15 g / L, even more preferably less than 10 g / L, most preferably less than 5 g / L; more preferably, the liquid food product is a low-sucrose food product after being used for one of the above-mentioned uses.

[0040] The invention also relates to a food product obtained or obtainable by a process comprising the steps of the process of the invention.

[0041] In view of the above, it will be appreciated by those skilled in the art that the food product may be a low sucrose food, may have reduced turbidity and / or may have reduced viscosity compared to similar food products not produced according to the present invention. The food product comprises a fructosyltransferase and optionally a pectinase, both as described above. The above enzymes may still have the activity described herein above; more preferably, the enzymes no longer have the above activity; therefore, one or more of the enzymes may have been inactivated before or during the production of the food product, for example by heat inactivation and / or may have been at least partially removed, for example using bentonite, activated carbon and / or ultrafiltration. Methods for identifying whether a food product contains one or more of the above enzymes are available, such as enzymatic tests for active enzymes, or, for example, immunological or mass spectrometric methods for inactive enzymes.

[0042] The food product may be a solid food comprising at least one liquid food of the present invention as at least one component. Preferably, the food product is a liquid food obtained or obtainable by a method comprising the steps of the method as described above as described above. Preferably, the food product is orange juice, pineapple juice, apple juice, apple puree, banana puree or apricot puree.

[0043] The present invention also relates to a liquid food product comprising an Aspergillus niger fructosyltransferase and comprising sucrose in a concentration of less than 50 g / L, preferably less than 25 g / L, more preferably less than 20 g / L, still more preferably less than 15 g / L, even more preferably less than 10 g / L, most preferably less than 5 g / L.

[0044] The above description of the food product obtained or obtainable by the method of the present invention applies mutatis mutandis to a liquid food comprising an Aspergillus niger fructosyltransferase. Preferably, the liquid food product is a fruit juice or fruit puree, preferably orange juice, pineapple juice, apple juice, apple puree, banana puree or apricot puree. In view of the above description, the skilled person understands that the Aspergillus niger fructosyltransferase is preferably an added Aspergillus niger fructosyltransferase, i.e. it is not inherently present in the liquid food.

[0045] In view of the above, the following implementation plans are specifically envisaged:

[0046] Embodiment 1: A method for converting sucrose contained in a liquid food product into at least one fructo-oligosaccharide (FOS), the method comprising

[0047] The liquid food product is contacted with an Aspergillus niger fructosyltransferase under conditions that allow enzymatic conversion of sucrose to at least one FOS by the fructosyltransferase.

[0048] Embodiment 2: The method of embodiment 1, wherein the fructosyltransferase has fructosyltransferase activity at a sucrose concentration of at most 10 g / L, preferably at most 5 g / L.

[0049] Embodiment 3: The method of Embodiment 1 or 2, wherein the liquid food product is a fruit juice, a fruit puree, a vegetable juice or a vegetable puree.

[0050] Embodiment 4: The method of any one of Embodiments 1 to 3, wherein the liquid food product is orange juice, pineapple juice, apple juice, apple puree, banana puree, or apricot puree.

[0051] Embodiment 5: The method of any one of Embodiments 1 to 4, wherein prior to said contacting, said liquid food product is a low sucrose food product.

[0052] Embodiment 6: The method of any one of Embodiments 1 to 5, wherein prior to said contacting, said liquid food product comprises sucrose in a concentration of at most 50 g / L, preferably at most 25 g / L, more preferably at most 20 g / L, still more preferably at most 15 g / L, even more preferably at most 10 g / L, most preferably at most 5 g / L.

[0053] Embodiment 7: The method of any one of Embodiments 1 to 6, wherein the liquid food product is a food product that has not been enzymatically pretreated, or, optionally, has been enzymatically pretreated only with cellulase and / or amylase.

[0054] Embodiment 8: The method of any one of embodiments 1 to 7, wherein at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95% of the fructose in the sucrose contained in the liquid food product before said contacting is converted into at least one FOS during said contacting.

[0055] Embodiment 9: The method of any one of Embodiments 1 to 8, wherein contacting is performed at least until the sucrose concentration in the liquid food product is at most 15 g / L, preferably at most 10 g / L, more preferably at most 5 g / L; and / or wherein after contacting, the residual sucrose concentration in the liquid food product is at most 15 g / L, preferably at most 10 g / L, more preferably at most 5 g / L.

[0056] Embodiment 10: The method of any one of Embodiments 1 to 9, wherein less than 5 g / L, preferably less than 2 g / L, of fructose is produced by the conversion.

[0057] Embodiment 11: The method of any one of Embodiments 1 to 10, wherein during said contacting, the fructose concentration in said liquid food product does not significantly increase.

[0058] Embodiment 12: The method of any one of Embodiments 1 to 11, wherein the method further comprises contacting the food product with at least one pectinase.

[0059] Embodiment 13: The method of any one of Embodiments 1 to 12, wherein said food product has reduced turbidity after said contacting, preferably compared to an untreated control and / or a vehicle control.

[0060] Embodiment 14: The method of any one of Embodiments 1 to 13, wherein said liquid food product has a reduced viscosity after said contacting, preferably compared to an untreated control and / or a vehicle control.

[0061] Embodiment 15: The method of any one of Embodiments 1 to 14, wherein the method comprises contacting the food product with the fructosyltransferase and the pectinase simultaneously.

[0062] Embodiment 16: The method of any one of Embodiments 1 to 15, wherein the liquid food product is a crude fruit juice or a crude fruit puree.

[0063] Embodiment 17: The method of any one of Embodiments 1 to 16, wherein the method does not comprise an enzyme treatment step before and / or after the contacting.

[0064] Embodiment 18: A method of reducing the turbidity and / or viscosity of a liquid food product, the method comprising contacting the liquid food product with (i) a fructosyltransferase and (ii) a pectinase in a manner permitting enzymatic activity of the fructosyltransferase and the pectinase.

[0065] Embodiment 19: The method of Embodiment 18, wherein said contacting with said fructosyltransferase and said pectinase is simultaneous contacting.

[0066] Embodiment 20: The method of Embodiment 18 or 19, wherein the method does not include an enzyme treatment step prior to the contacting.

[0067] Embodiment 21: The method of any one of Embodiments 18 to 20, further comprising the features of any one of Embodiments 1 to 17.

[0068] Embodiment 22: The method of any one of Embodiments 1 to 21, wherein the fructosyltransferase comprises the amino acid sequence shown in SEQ ID NO: 1 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, still more preferably at least 98%, and most preferably at least 99% identity thereto.

[0069] Embodiment 23: A composition comprising (i) a fructosyltransferase and (ii) a pectinase, wherein the fructosyltransferase comprises the amino acid sequence shown in SEQ ID NO: 1 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, still more preferably at least 98%, and most preferably at least 99% identity thereto.

[0070] Embodiment 24: The composition of Embodiment 23, wherein the composition is a liquid composition.

[0071] Embodiment 25: The composition of Embodiment 23 or 24, further comprising at least one other compound selected from the list consisting of solvents, stabilizers, salts, buffers and preservatives.

[0072] Embodiment 26: A kit comprising (i) a fructosyltransferase and (ii) a pectinase, wherein the fructosyltransferase comprises the amino acid sequence shown in SEQ ID NO: 1 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, still more preferably at least 98%, and most preferably at least 99% identity thereto.

[0073] Embodiment 27: The kit of embodiment 26, wherein the components of the kit are contained in a housing, preferably in a common housing.

[0074] Embodiment 28: The method of any one of embodiments 12 to 22, the composition of any one of embodiments 23 to 25, or the kit of embodiments 26 or 27, wherein the pectinase comprises endopolygalacturonase activity (EC 3.2.1.15), arabinan endo-1,5-α-L-arabinase activity (EC 3.2.1.99), arabinofuranosidase activity (EC 3.2.1.55), pectin methylesterase activity (EC 3.1.1.11), glucan 1,4-α-glucosidase activity (EC 3.2.1.3) and / or pectin lyase activity (EC 4.2.2.10).

[0075] Embodiment 29: Use of an Aspergillus niger fructosyltransferase for converting sucrose into at least one fructo-oligosaccharide (FOS) in a liquid food product.

[0076] Embodiment 30: Use of an Aspergillus niger fructosyltransferase for producing at least one FOS in a liquid food product.

[0077] Embodiment 31: Use of an Aspergillus niger fructosyltransferase for reducing the sucrose concentration in a liquid food product to at most 50 g / L, preferably at most 25 g / L, more preferably at most 15 g / L, still more preferably at most 10 g / L, even more preferably at most 5 g / l.

[0078] Embodiment 32: Use of Aspergillus niger fructosyltransferase and pectinase for reducing the turbidity and / or viscosity of a liquid food product.

[0079] Embodiment 33: The use of any one of Embodiments 29 to 32, wherein the Aspergillus niger fructosyltransferase comprises the amino acid sequence shown in SEQ ID NO: 1, or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, still more preferably at least 98%, and most preferably at least 99% identity thereto.

[0080] Embodiment 34: The use of any one of Embodiments 29 to 33, wherein the liquid food product is a low sucrose food product, preferably the liquid food product is a low sucrose food product after the sucrose is converted into FOS, and the low sucrose food product preferably contains sucrose in a concentration of less than 50 g / L, preferably less than 25 g / L, more preferably less than 20 g / L, still more preferably less than 15 g / L, even more preferably less than 10 g / L, most preferably less than 5 g / L.

[0081] Embodiment 35: Food product obtained or obtainable by a process comprising the steps of the process of any one of embodiments 1 to 22 or 28.

[0082] Embodiment 36: The food product of Embodiment 35, wherein the food product is a liquid food product.

[0083] Embodiment 37: The food product of Embodiment 35 or 36, which is orange juice, apple juice, pineapple juice, apple puree, banana puree or apricot puree.

[0084] Embodiment 38: A liquid food product comprising Aspergillus niger fructosyltransferase and comprising sucrose, wherein the sucrose concentration is less than 50 g / L, preferably less than 25 g / L, more preferably less than 20 g / L, still more preferably less than 15 g / L, even more preferably less than 10 g / L, and most preferably less than 5 g / L.

[0085] Embodiment 39: The liquid food product of Embodiment 38, wherein the liquid food product is a fruit juice or a fruit puree, preferably orange juice, apple juice, pineapple juice, apple puree, banana puree or apricot puree.

[0086] Embodiment 40: The liquid food product of Embodiment 38 or 39, wherein the Aspergillus niger fructosyltransferase is an added Aspergillus niger fructosyltransferase.

[0087] All references cited in this specification are incorporated by reference into their entire disclosures as well as the disclosures specifically mentioned in this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 : Results of orange juice treatment; (A) and (B) are the changes in sugar concentration over time under the specified treatments; (C) is the changes in turbidity over time under the specified treatments; x-axis: time (minutes), FOS-E: FOSEnzyme preparation.

[0089] Figure 2 : Results of apple paste treatment; (A) Sugar concentration after 60 min at 20°C; (B) Dynamic viscosity, unit [mPa*s], FOS-E: FOSEnzyme preparation.

[0090] Figure 3 : Results of apple juice treatment; sugar concentration after 90 minutes at 50°C, FOS-E: FOS Enzyme formulation.

[0091] Figure 4 : Results of pineapple juice treatment; Changes in sugar concentration over time under the indicated treatments; x-axis: time (minutes), FOS-E: FOSEnzyme formulation.

[0092] Figure 5 : Results of banana puree treatment; (A) and (B) are the changes in sugar concentration over time under the specified treatments; x-axis: time (minutes), FOS-E: FOSEnzyme preparation.

[0093] Figure 6 : Results of apricot paste treatment; (A) and (B) are the changes in sugar concentration over time under the specified treatments; x-axis: time (minutes), FOS-E: FOSEnzyme preparation.

[0094] Figure 7 : HPAEC-PAD chromatograms showing the signal intensity as a function of elution time for extracts that were untreated (blank) or treated (20 ppm PTE100 / 1000 ppm FOSEnzyme product) as described in Example 5.

[0095] Figure 8 : The amount of sugar and sc-FOS in orange juice depends on the dosage of FOSEnzyme formulations; (A) with addition of 5 ppm of Rohapect PTE 100, (B) without addition of enzyme.

[0096] Fig. 9 : The amount of sugar and sc-FOS in apple juice depends on the dosage of FOSEnzyme formulation. In addition, 50 ppm of Rohapect MA PLUS T was added.

[0097] Fig.10 : The amount of sugar and sc-FOS in banana puree depends on the dosage of FOSEnzyme formulation. In addition, 5 ppm of Rohapect PTE 100 was added.

[0098] Fig.11 : The amount of sugar and sc-FOS in carrot juice depends on the dosage of FOSEnzyme formulation. In addition, 5 ppm of Rohapect PTE 100 was added. DETAILED DESCRIPTION

[0099] The following examples are only used to illustrate the present invention. In any case, they should not be construed as limiting the scope of the present invention.

[0100] Example 1: Orange Juice

[0101] Substrate: Orange juice, directly pressed juice from organic farming REWE Bio, pH value: 3.35

[0102] Steps: Pour 50 ml of orange juice into a sealable conical bottle.

[0103] Enzyme dosage: ROHAPECT® PTE 100 5 ppm (Aspergillus niger pectin lyase, EC 4.2.2.10, batch number R212243ST, specific activity 100 PTF / mg); all specific activities were provided by the manufacturer.

[0104] FOSEnzyme product (Aspergillus niger fructosyltransferase (FOS-E), lot number R220627ST), 100, 500 and 1000 ppm; the control formulation lacking Aspergillus niger fructosyltransferase also lacked fructosyltransferase activity)

[0105] Reaction temperature: 50℃

[0106] Reaction time: 120 minutes

[0107] Sampling after 30-60-120 minutes

[0108] The sample was briefly exposed to 90°C for 10 minutes.

[0109] Analysis: Freeze inactivated samples for (S / G / F measurement) kit testing

[0110] Turbidity [NTU]: 1 ml centrifuged at 13,000 rpm for 2 minutes

[0111] The results are as follows Figure 1 As shown; co-treatment with fructosyltransferase (FOSEnzyme product) and pectinase (PTE100) resulted in a decrease in sucrose concentration and an increase in glucose concentration ( Figure 1 A), and the turbidity decreased ( Figure 1 C). The effect on sugar concentration is mainly mediated by fructosyltransferase ( Figure 1 B).

[0112] Example 2: Apple paste

[0113] Substrate: Jonagold, pH 3.49

[0114] Steps: Put the apples into a meat grinder and crush them. Take 500g of paste and put it into a 1000ml beaker.

[0115] Enzyme dosage: ROHAPECT® MA plus T 100 ppm (pectinase mixture containing endopolygalacturonase (3.2.1.15, specific activity 1100 PGX / g) and pectin methylesterase (3.1.1.11, specific activity 3150 PE / g), batch number R206179ST)

[0116] FOSEnzyme products 100 and 1000 ppm (R220627ST)

[0117] Reaction temperature: 20℃

[0118] Reaction time: 60 minutes

[0119] After the reaction, the apple paste was squeezed with a small Hafico squeezer at 0 bar for 2 minutes, 50 bar for 2 minutes, 100 bar for 2 minutes, 150 bar for 2 minutes, and 200 bar for 2 minutes.

[0120] Analysis: Freeze inactivated samples for (S / G / F measurement) kit testing

[0121] Turbidity [NTU]: 1 ml centrifuged at 13,000 rpm for 2 minutes

[0122] The results are as follows Figure 2 and Table 1; co-treatment with fructosyltransferase (FOSEnzyme product) and pectinase (MA plus T) resulted in a decrease in sucrose concentration to less than 5 g / L, while an increase in glucose concentration ( Figure 2 A), and the dynamic viscosity decreases ( Figure 2 B). The co-treatment had no negative effect on the juice yield (Table 1), which is one of the most important parameters in apple juice production. Surprisingly, an increase in the juice turbidity (NTU) of the juice squeezed from the treated apple mash was observed (Table 1), which is desirable in the manufacture of cloudy apple juice. At the same time, the viscosity of the juice obtained by mash co-treatment was significantly reduced compared to treatment with ROHAPECT MA plus alone. This is also a surprising effect, which, according to Darcy's law, will lead to better filtration of the juice in the next processing step and to an improved process, which includes better filtration in subsequent production steps in industrial juice manufacturing plants.

[0123] Example 3: Apple Juice

[0124] Substrate: Juice from Jonagold, pH 3.55

[0125] Procedure: See apple paste treatment (Example 2), with modified enzyme dosage

[0126] Enzyme dosage: EL-2022 / 000031 30 ppm (pectinase mixture containing endopolygalacturonase (EC 3.2.1.15, specific activity 1250 PGX / g, pectin methylesterase (EC 3.1.1.11, specific activity 1550 PE / g), pectin lyase (EC 4.2.2.10, specific activity 30 PTF / mg), arabinan endo-1,5-α-L-arabinase (EC 3.2.1.99, specific activity 100 ARAG / g) and glucan 1,4-α-glucosidase (150 GAU / g).

[0127] FOSEnzyme products 100 and 500 ppm (lot number R220627ST)

[0128] GAMYLOZYM AFL 15 ppm

[0129] Reaction temperature: 50°C; reaction time: 120 minutes.

[0130] After the reaction, heat at 90°C for 10 minutes and then cool to 50°C

[0131] Amicon TCF 10-UF system is available. Membrane: SARTORIUS 11107 nitrocellulose filter 0.2 μm.

[0132] Filtration parameters: air pressure is 0.5 bar. Time unit is weight method.

[0133] After the juice reaction time is over, the juice is directly transferred to the UF system and started. Temperature: 55°C in the UF system. After the reaction is over, the sample is heated (90°C for 10 minutes) and then cooled to 50°C

[0134] Analysis: Freeze inactivated samples for (S / G / F measurement) kit testing until use

[0135] Turbidity [NTU]: 1 ml centrifuged at 13,000 rpm for 2 minutes

[0136] The results are as follows Figure 3 and Table 8; co-treatment with fructosyltransferase (FOSEnzyme product) and pectinase (EL-2022 / 000031) resulted in a decrease in sucrose concentration to less than 5 g / L, while glucose concentration increased ( Figure 3 The co-treatment also unexpectedly resulted in a significant improvement or increase in the flux rate during filtration, which means a significant improvement in filtration performance in industrial manufacturing. Flux is described as the volume of juice filtered per square meter of membrane in 1 hour (Table 8).

[0137] Example 4: Pineapple Juice

[0138] Substrate: pineapple, pH 3.4

[0139] Procedure: The pineapple was ground in a meat grinder; the paste was heated to 90°C in a microwave oven (hot crushing) and then directly transferred to a finisher (Alexander Werk).

[0140] Juice enzyme dosage ROHAPECT® B1L 100 ppm (pectinase mixture containing pectin methylesterase (EC3.1.1.11, specific activity 450 PE / g) and endopolygalacturonase (EC 3.2.1.15, specific activity 400 PGX / g), as well as small amounts of mannanase (EC 3.2.1.78) and cellulase (3.2.1.4) activities);

[0141] FOSEnzyme products 100, 1000 and 1000ppm

[0142] Reaction temperature: 50℃

[0143] Reaction time: 120 minutes

[0144] Sampling after 30-60-120 minutes

[0145] The sample was briefly exposed to 90°C for 10 minutes.

[0146] Analysis: Freeze inactivated samples for (S / G / F measurement) kit testing

[0147] Turbidity [NTU]: 1 ml centrifuged at 13,000 rpm for 2 minutes

[0148] The results are as follows Figure 4 As shown; co-treatment with fructosyltransferase (FOSEnzyme product) and pectinase (B1L) resulted in a decrease in sucrose concentration and a concomitant increase in glucose concentration.

[0149] Example 5: Banana Mash

[0150] Substrate: Banana, pH 4.75

[0151] Steps: Bananas are ground in a meat grinder

[0152] The paste was heated to 90 °C in a microwave oven (hot crushing) and then directly transferred to a finisher (Alexander Werk). Ascorbic acid was added to the puree at 250 mg / kg and the puree was frozen in 500 g units until use.

[0153] Enzyme dosage ROHAPECT® PTE 100 20 ppm, batch number R212243ST, which includes pectin lyase (EC 4.2.2.10, specific activity 100 PTF / mg)

[0154] FOSEnzyme products 100 and 1000 ppm, Catalog No. R220627ST

[0155] Reaction temperature: 50℃

[0156] Reaction time: 30 minutes / 60 minutes

[0157] After the reaction, the paste was inactivated at 90 °C

[0158] Analysis: Freeze inactivated samples for (S / G / F measurement) kit testing

[0159] Turbidity [NTU]: 1 ml centrifuged at 13,000 rpm for 2 minutes, the viscosity of the mud and supernatant

[0160] Blank samples (60 min) and enzyme-treated samples (60 min; 20 ppm ROHAPECT® PTE 100 / 1000 ppm FOSEnzyme products) were analyzed by HPAEC-PAD (High Performance Anion Exchange Chromatography / Pulsed Amperometric Detection) using a Dionex CarboPac PA1 column (Thermo Scientific). Standard substances (glucose, fructose, sucrose, nitrosacetose, nitrosacetose, nitrosacetose, nitrosacetose) were used for peak identification.

[0161] The results are as follows Figure 5 and Figure 7 As shown in Tables 2 to 4. Figure 7 As shown, transferase treatment with fructosyltransferase results in the production of glucose, kestose, kestetose, and kestose, with a concomitant reduction in sucrose.

[0162] Example 6: Apricot puree

[0163] Substrate: Apricot, pH 3.55

[0164] Procedure: mince apricots in a meat grinder; heat the paste in a microwave to 90°C (hot crushing) and transfer the paste directly to a finishing machine (Alexander Werk); freeze the puree in 500g units until use

[0165] Enzyme dosage ROHAPECT® PTE 100 20 ppm

[0166] FOSEnzyme products 100 and 1000ppm

[0167] Reaction temperature: 50°C Reaction time: 30 minutes / 60 minutes

[0168] After the reaction time, the paste was inactivated at 90 °C

[0169] analyze:

[0170] Freeze-inactivated samples for (S / G / F measurement) kit testing

[0171] Turbidity [NTU]: 1 ml centrifuged at 13,000 rpm for 2 minutes, the viscosity of the mud and supernatant

[0172] Example 7: Quantification of FOS production

[0173] General approach:

[0174] Conical flasks containing the corresponding substrate solutions (orange juice, apple juice, banana puree or carrot juice) as indicated below were placed in a preheated water bath at 50°C. The substrate solutions were stirred at 50 rpm using a magnetic stirrer. After temperature equilibrium, enzymes were added at the doses indicated in the figure. Samples were taken after an incubation time of 60 minutes and heated at 90°C for 10 minutes to inactivate the enzymes.

[0175] The amounts of sucrose (S), glucose (G), fructose (F), kenetose (GF2), kenetetrose (GF3), and kenetopentaose (GF4) in the samples were analyzed by HPAEC (high performance anion exchange chromatography) and PAD (pulsed amperometric detection).

[0176] Example 7.1: Orange juice

[0177] As shown in Example 1, orange juice was basically obtained; the pH value was 3.6. Figure 8 A) or without adding ( Figure 8 B) Incubation with 5 ppm of Rohapect PTE 100; the results are shown in Figure 8 shown.

[0178] Example 7.2: Apple juice

[0179] Apple juice was made from crushed apples (apple paste) treated with enzymes at room temperature (about 22°C) before pressing. After an incubation time of 60 minutes, the apple paste was pressed to obtain the apple juice used in the examples; the pH value was 3.75. During the incubation, 50 ppm of Rohapect MA PLUS T was additionally added; the results are shown in Table 1. Fig. 9 shown.

[0180] Example 7.3: Banana mash

[0181] As shown in Example 5, banana puree was obtained; the pH value was 4.75. 5 ppm of Rohapect PTE 100 was added during incubation; the results were as follows Fig.10 shown.

[0182] Example 7.4: Carrot juice

[0183] Direct pressed carrot juice from organic farming REWE Bio; pH 5. 5 ppm of Rohapect PTE 100 was added during incubation; results Fig.11 shown.

[0184] Table 1: Apple paste treatment results

[0185]

[0186] Table 2: Banana puree results, combined treatments.

[0187]

[0188] Table 3: Banana puree results, single treatment.

[0189]

[0190] Table 4: Banana mash results, tasting.

[0191]

[0192] Table 5: Apricot puree results, combined treatments.

[0193]

[0194] Table 6: Apricot puree results, single treatment.

[0195]

[0196] Table 7: Apricot puree results, tasting.

[0197]

[0198] Table 8: Flux rates after enzyme treatment

[0199]

[0200] References:

[0201] Bergès et al. (1993), Curr Genet 24:53

[0202] Boddy et al. (1993), Curr Genet 24:60

[0203] EP 1 951 885 A1

[0204] EP 2 294 093 A1

[0205] EP 3 231 293 A1

[0206] WO 2008 / 102336 A2

[0207] WO 2018 / 009806 A1

[0208] Yuan et al. (2006), Microbiology 152:3061

Claims

1. A method for converting sucrose contained in a liquid food product into at least one fructo-oligosaccharide (FOS), the method comprising contacting the liquid food product with an Aspergillus niger fructosyltransferase under conditions allowing enzymatic conversion of sucrose by the Aspergillus niger fructosyltransferase into at least one FOS, wherein the contacting is carried out at least until the sucrose concentration in the liquid food product is at most 15 g / L.

2. The method according to claim 1, wherein after contacting, the residual sucrose concentration in the liquid food product is at most 10 g / L, preferably at most 5 g / L.

3. The method according to claim 1 or 2, wherein: At least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95% of the fructose in the sucrose contained in the liquid food product before said contacting is converted into at least one FOS during said contacting.

4. The method according to any one of claims 1 to 3, wherein less than 5 g / L, preferably less than 2 g / L fructose is produced by said conversion, preferably wherein the concentration of fructose in said liquid food product does not significantly increase during said contacting.

5. The method according to any one of claims 1 to 4, wherein the liquid food product is a food product which has not been enzymatically pretreated or has been pretreated only with cellulase and / or amylase.

6. The method according to any one of claims 1 to 5, wherein before said contacting, said liquid food product is a low sucrose food product comprising sucrose in a concentration of less than 50 g / L, preferably less than 25 g / L, more preferably less than 20 g / L.

7. The method according to any one of claims 1 to 6, wherein the method further comprises contacting the food product with at least one pectinase.

8. The method according to any one of claims 1 to 7, wherein said food product has reduced turbidity and / or viscosity after said contacting, preferably compared to an untreated control and / or a vehicle control.

9. A composition, preferably a liquid composition, comprising (i) an Aspergillus niger fructosyltransferase and (ii) a pectinase.

10. Use of an Aspergillus niger fructosyltransferase for converting sucrose to at least one fructo-oligosaccharide (FOS) in a liquid food product, wherein after said conversion the residual sucrose concentration in said liquid food product is at most 15 g / L.

11. Use of an Aspergillus niger fructosyltransferase for reducing the sucrose concentration in a liquid food product to at most 15 g / L, still more preferably at most 10 g / L, even more preferably at most 5 g / l.

12. The subject matter of any one of claims 1 to 11, wherein the Aspergillus niger fructosyltransferase comprises the amino acid sequence shown in SEQ ID NO: 1 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, still more preferably at least 98%, most preferably at least 99% identity thereto.

13. Liquid food product obtained or obtainable by a process comprising the steps of the process as claimed in any one of claims 1 to 8.

14. A liquid food product comprising an Aspergillus niger fructosyltransferase and comprising sucrose in a concentration of up to 15 g / L.

15. Subject matter according to any one of claims 1 to 14, wherein the liquid food product is a fruit juice, a fruit puree, a vegetable juice or a vegetable puree, preferably orange juice, apple juice, pineapple juice, apple juice, apple puree, banana puree or apricot puree.

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