Method for producing dairy products

Abstract

The present invention relates to a method for producing a heat treated milk-derived product having reduced lactose, comprising treating a milk-derived substrate with lactase and performing a heat treatment.

Description

[Technical field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0002] The present invention relates to a method for producing a heat treated milk-derived product having reduced lactose, the method comprising treating a milk-derived substrate with lactase and performing a heat treatment. [Background technology]

[0003] Most lactose-reduced or lactose-free dairy products are produced in a batch process, i.e., lactase is added to milk, which is then incubated at low temperature (usually below 10° C.) for a sufficient time to reduce the lactose content to less than 0.01% or less than 0.1% (which allows the milk to be labelled as lactose-free in most countries, for example), followed by a heat treatment such as pasteurisation, UHT treatment or ESL (extended shelf life up to 35 days) treatment. Some of the disadvantages associated with batch application of lactase include: (1) Incubation times can be up to 24 hours, and in some cases even longer, which raises concerns about the capacity and capital investment of new incubation tanks to meet the increasing demand for lactose-free dairy products. (2) Long incubation times allow psychotropic microbes the opportunity to grow and secrete enzymes, particularly proteases, some of which are heat stable and may deteriorate the quality of the final product during storage. (3) Lactose hydrolysis followed by severe heat treatment, such as by application of UHT, accelerates the Maillard reaction. When lactose is hydrolyzed to glucose and galactose, the concentration of sugars with reducing ends doubles; galactose, in particular, is much more reactive than lactose.

[0004] Recently, especially for lactose-free ESL and UHT dairy drinks, there has been a great deal of interest in advanced process engineering techniques in the form of aseptic dosing equipment, which potentially overcomes many of the concerns associated with batch processes. Such aseptic dosing equipment allows lactase to be added after ESL / UHT processing. Examples of such aseptic dosing equipment are the Tetra Pak Aldose system, the Tetra Pak Flexdose System, and the GEA Varidose system. Advantages of these systems include: (1) After the heat treatment step, the milk stream is dosed with small, precise amounts of sterile lactase, which allows lactose hydrolysis to occur during the first few days of storage. (2) The extent of Maillard reactions, browning, and advanced glycation end products (AGE) formation in lactose-free dairy beverages is reduced, especially when properly controlled storage conditions are applied. (3) Elimination of pre-incubation compared to batch processes, thereby solving capacity issues and reducing the risk of psychotropic microbial activity.

[0005] Despite the above advantages of the sterile administration system, there are several disadvantages associated with its use, including: (1) Because lactase is added after the heat treatment step, the preparation must be of the highest purity possible, since any adverse side effects in the preparation can have a significant adverse effect on the final product over its long shelf life, especially in the case of UHT products. (2) In the case of the Tetra Pak Flexdose and GEA Varidose systems, the lactase enzyme should be filled aseptically into sterile buckets and bags. (3) The requirements in (1) and (2) above mean that these sterile lactases have a higher cost per unit of activity compared to lactases used in batch processes. (4) The average capital cost of this sterile dosing system is substantial. (5) The Tetra Pak Flexdose and GEA Varidose systems incur additional running costs related to consumables, e.g., changing hoses and needles every time the sterile bucket is changed. (6) In the Tetra Pak Aldose system, the enzyme preparation is not initially sterile. It is diluted with water and then filtered in-line (at the dairy) using at least two filters to ensure the sterility of the enzyme stream before mixing with the milk stream. Problems can therefore arise as the filters introduced in-line can become clogged with poorly filterable enzyme preparations, which can cause many operational problems. Even if the filterability of the enzyme preparation works as expected, the filters still need to be changed regularly (usually daily).

[0006] A quick, smooth, easy to implement, trouble-free and cost-effective solution for the application of lactase in lactose-free UHT and ESL products that overcomes all the above limitations in both batch and aseptic dosing processes does not yet exist.

[0007] WO 2009 / 071539 (Novozymes) relates to a method for producing dairy products using an enzyme with lactase activity. Disclosed is a method for producing a low-lactose dairy product by treating a milk-derived substrate with lactose at elevated temperatures (i.e. at least 60° C., at least 62° C., at least 63° C., at least 64° C., at least 65° C., at least 67° C., at least 70° C., or at least 75° C.).

[0008] WO 2018 / 189238 (Chr. Hansen) discloses a beta-galactosidase that is said to be relatively highly active and stable over a wide range of temperatures and pH values. Disclosed is a method of producing a dairy product by treating a milk-based substrate with beta-galactosidase, which treatment, or part of the treatment, may be carried out at an elevated temperature. Three to 30 minutes after addition of beta-galactosidase, lactose concentrations may be less than 0.2% lactose.

[0009] WO 2020 / 176734 (DuPont) relates to a method for reducing the amount of lactose in a milk-derived substrate by contacting the substrate with lactase (e.g., thermostable lactase) at high temperature. Disclosed is a method for producing a lactose-free dairy product from a milk-derived substrate with an enzyme having neutral lactase activity, where more than 20% lactase activity remains in the milk-derived substrate after pasteurization at 72° C. for 15 seconds. Such pasteurization may also be referred to as high temperature short time (HTST) pasteurization.

[0010] US 2010 / 0215828 A1 relates to a method for preparing a shelf-stable low-lactose, lactose-free or carbohydrate-free dairy product, which comprises separating sugar and protein into separate fractions, heat-treating at least the protein fraction to inactivate the native plasmin enzyme system and other harmful enzymes, heat-treating the protein fraction and the sugar fraction separately (to avoid the Maillard reaction), and combining one or more fractions to obtain a dairy product with the desired composition and sweetness. Heat-treating can be performed by pasteurization, high-temperature pasteurization, using ESL treatment, or using UHT treatment. Optionally, lactose in the sugar fraction can be hydrolyzed. Hydrolysis using lactase is followed by incubation at 37°C for 4 hours, followed by heat-treating.

[0011] US Patent Application Publication No. 2013 / 0142904 (Arla) relates to a method for producing a packaged lactose-reduced dairy-based product, in which a lactose-reduced dairy-based feed is subjected to a high temperature treatment and packaged.

[0012] Deeth (2017) “Optimum Thermal Processing for Extended Shelf-Life (ESL) Milk”, Foods 6(11):102, outlines the optimal heat treatment for extended shelf-life (ESL) milk. Deeth explains that ESL milk or ultra-pasteurized milk is produced by a heat treatment that uses conditions between those used in conventional high-temperature short-time (HTST) pasteurization and those used in ultra-high-temperature (UHT) pasteurization. ESL milk should have a refrigerated shelf-life of more than 30 days. To achieve this, the heat treatment must be fairly intense. Unlike the temperature-time conditions for pasteurization, which in most countries are specified as at least 72°C for at least 15 seconds, no such conditions are generally specified for ESL processing. According to Deeth (2017), most reported commercial processing conditions for ESL milk range from 123 to 127°C for 1 to 5 seconds. In the United States regulations, the process of “ultra-pasteurization” is defined as heating milk at at least 138°C for at least 2 seconds.

[0013] The UHT treatment may be, for example, a heat treatment at 130° C. for 30 seconds, or a heat treatment at 140° C. for 3-4 seconds, or a heat treatment at 145° C. for 1 second. Summary of the Invention [Means for solving the problem]

[0014] The present invention provides a method for producing heat-treated milk-derived products (e.g., milk) having reduced lactase using this enzyme without the need for extensive pre-incubation of a milk-derived substrate with an enzyme having lactase activity and without the need for the use of a sterile dosing system for adding the enzyme after heat treatment.

[0015] The present invention relates to a method for producing a heat treated milk-derived product having reduced lactose, comprising the steps of: a) adding an enzyme having lactase activity to a milk-derived substrate containing at least 2% (w / w) lactose; b) carrying out a heat treatment of the milk-derived substrate by holding the milk-derived substrate at a holding temperature of at least 120°C for a holding time of at least 1 second after addition of the enzyme, followed by cooling to produce a heat-treated milk-derived product; and c) storing the heat-treated milk-derived product at a temperature of up to 40° C. for at least 24 hours, preferably for at least 2 days, such as at least 3 days, more preferably for at least 4 days. Including, After step c), the lactose content of the milk-derived product is at most 0.2% (w / w); A method is provided.

[0016] Preferably, after step b) but before step c), the lactose content of the milk-derived product is at least 0.5% (w / w).

[0017] Preferably, the enzyme with lactase activity is administered immediately before the heat treatment (e.g. UHT treatment). After heat treatment (e.g. UHT treatment), the enzyme has some residual activity that ensures lactose degradation to the desired low lactose level during storage at low or ambient temperature. Preferably, the enzyme has an optimum temperature of 30-60°C, more preferably 35-55°C. Without wishing to be bound by theory, enzymes with higher optimum temperatures, and perhaps active even during heat treatment, will have a somewhat rigid structure and will not have sufficient activity during storage at low or ambient temperature. Again, without wishing to be bound by theory, the enzymes used in the method of the invention, with an optimum temperature of 30-60°C, more preferably 35-55°C, may be unfolded and inactive during heat treatment, but have the ability to be refolded and reactivated thereafter, and therefore have a measurable or substantial residual activity that ensures lactose degradation during storage of the milk-derived product. It is believed that the significant residual activity of the lactase enzyme of the present invention after heat treatment, such as UHT treatment, may be due to its ability to refold and be reactivated when the temperature is reduced.

[0018] Preferably, the enzyme has a residual activity of at least 0.5%, preferably at least 1%, at least 2%, or at least 3%, more preferably at least 5%, and even more preferably at least 10%, after incubation in skim milk having a lactose content of 4.7% at 90°C for 30 seconds, at 140°C for 5 seconds, and at 70°C for 30 seconds, followed by cooling to 0-10°C, and then incubation at 23°C for 72 hours, relative to the activity of the same enzyme in skim milk without the incubation at 90°C for 30 seconds, at 140°C for 5 seconds, and at 70°C for 30 seconds, followed by cooling to 0-10°C, and then incubation at 23°C for 72 hours.

[0019] Preferably, the milk-derived substrate is not incubated with lactase prior to heat treatment, except for the time between the addition of lactase and when the milk-derived substrate reaches the holding temperature of the heat treatment (depending on the process equipment).Preferably, step b) is carried out immediately after step a), without a dedicated incubation step.

[0020] According to the method, at least three main process options can be applied:

[0021] In the first option, the milk-derived substrate may be mixed with lactase and then directly treated under UHT or ESL conditions without the need to incubate the milk-derived substrate with lactase.

[0022] In a second option, the milk-derived substrate may be directly treated under UHT or ESL conditions, in which case lactase is added to the milk-derived substrate while it is flowing through the process pipe just before the heat treatment step, optionally while the temperature of the milk-derived substrate is being increased towards the temperature of the heat treatment step. Lactase may be added just before UHT / ESL treatment at any time when the temperature of the milk-derived substrate is between 1 and 95°C, preferably between 70 and 90°C. Addition of lactase may be done by a simple dosing pump via a tube connected to the main milk flow tube. When added to the milk-derived substrate stream, the temperature is (further) increased to ESL or UHT treatment conditions. A heating medium (e.g. plate or tube heat exchanger) may be used that does not directly contact the milk-derived substrate but is separated by the contact surface of the equipment. This may be referred to as indirect heat treatment (preferably indirect UHT treatment).

[0023] In a third option, the heat treatment may be carried out by steam injection or steam infusion (preferably steam injection) using high pressure steam to heat the milk-derived substrate and the enzymes may be added together with the steam. This may be referred to as direct heat treatment (preferably direct UHT treatment). After the retention time of step b), the steam-containing milk-derived substrate may be flash cooled in vacuum to remove water equivalent to the amount of condensed steam used. In addition to heating the milk-derived substrate by steam injection or steam infusion, indirect heating may also be applied, for example using a plate or tube heat exchanger.

[0024] In either case, residual lactase activity after heat treatment ensures that lactose levels are reduced to lactose-reduced levels, preferably lactose-free levels (e.g. less than 0.1%, or less than 0.01%) during the initial stages of storage (e.g. up to 2 weeks, e.g. the first 2 or 3 days).

[0025] The methods of the present invention have many advantages over processes used today for producing ESL and UHT treated milk-derived products, such as ESL milk or UHT milk.

[0026] Compared to the batch processes applied today, the process of the present invention improves the color and quality of lactose-reduced or lactose-free dairy products due to the reduction of the Maillard reaction. The growth of psychotropic bacteria secreting enzymes such as proteases, which may survive heat treatment without pre-incubation, is reduced, and therefore dairy products may have a longer shelf life. Furthermore, the absence of pre-incubation reduces capacity costs and process time. The method of the present invention is also easy to operate, since there is no need to monitor the tank until the lactose level is below 0.1% or 0.01%.

[0027] Compared to the aseptic dosing processes applied today, the process of the present invention does not require investment in aseptic dosing equipment and does not require regular replacement of, for example, aseptic buckets. Moreover, while current aseptic lactase has a shelf life of about one year, the enzyme used in the process of the present invention can have a shelf life of more than two years. The process of the present invention is easy to operate and hassle-free, since there is no need to monitor or troubleshoot any of the aseptic dosing systems. The final quality of the milk-derived substrate is the same in terms of color and Maillard reaction.

[0028] The method of the present invention is easy to apply on an industrial level and there are no obstacles to its implementation.

[0029] The inventors have surprisingly found that lactase enzymes from the CAZy database GH2 family clade DYLGE are particularly suitable for use in the methods of the invention.Thus, in a preferred embodiment, the enzyme having lactase activity is a GH2 lactase from clade DYLGE, preferably a bacterial GH2 lactase from clade DYLGE.

[0030] In a preferred embodiment, the enzyme with lactase activity comprises in its amino acid sequence the motif WTXXDY[I / L / R]GE[P / S / A].

[0031] In a preferred embodiment, the enzyme with lactase activity comprises in its amino acid sequence the motif SR[W / Y / F]YSGSGX[Y / G]R and / or [L / V / I]X[L / V / I]PHD. [Brief description of the drawings]

[0032] [Figure 1]1 shows the dose-response curve of non-UHT treated B. bifidum lactase (SEQ ID NO: 1). B. bifidum lactase was added to skim milk at the following dosages: 2.03, 1.63, 1.30, 1.04, 0.832, 0.666, 0.532, 0.426, 0.341, 0.273, 0.218, 0.174, 0.140, 0.112, 0.0893, 0.0715 mg enzyme protein (ep) per liter skim milk and incubated at 23° C. for 3 days (without UHT treatment). The residual lactose in each sample was determined as % (g lactose per 100 ml skim milk) and a dose / response curve was generated. This curve can be used to determine the relative activity of UHT-treated lactase in % to the activity of non-UHT-treated B. bifidum lactase. The lactase to be tested is added to skim milk at a dosage of x mg lactase ep per liter skim milk, UHT treatment is carried out, followed by incubation at 23° C. for 3 days, and the residual lactose is determined in % (g lactose per 100 ml skim milk). The dose / response curve is used to determine the "corresponding dosage" y (mg lactase ep per liter skim milk) of non-UHT-treated B. bifidum lactase that will result in the same residual lactose after incubation in skim milk for 3 days at 23° C. The relative activity of UHT-treated lactase in % to the activity of non-UHT-treated B. bifidum lactase is calculated as y / x*100%. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] definition In accordance with this Detailed Description, the following definitions apply: It should be noted that the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0034] Unless otherwise defined or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0035] GH2 lactase: The term "GH2 lactase", "GH2 enzyme" or "GH2 polypeptide" in the context of the present invention refers to a lactase enzyme that is classified as a member of glycoside hydrolase family 2 in the Carbohydrate-Active EnZymes (CAZymes) database (http: / / www.cazy.org / ).

[0036] Isolated: The term "isolated" refers to a polypeptide, nucleic acid, cell, or other specified substance or component that is separated from at least one other substance or component, including but not limited to other proteins, nucleic acids, cells, etc. Thus, an isolated polypeptide, nucleic acid, cell, or other substance is in a form that is not found in nature. Isolated polypeptides include, but are not limited to, culture broths containing the polypeptide expressed and secreted in a host cell.

[0037] Lactase: The term "lactase" refers to a glycoside hydrolase capable of hydrolyzing the disaccharide lactose into the constituent galactose and glucose monomers. The group of lactases includes, but is not limited to, enzymes assigned to the subclasses EC 3.2.1.23 and EC 3.2.1.108. Enzymes assigned to other subclasses, such as EC 3.2.1.21, may also be lactases in the context of the present invention. Lactases in the context of the present invention may have activities other than lactose hydrolysis activity, such as transgalactosylation activity. In the context of the present invention, the lactose hydrolysis activity of a lactase may be referred to as its lactase activity, its beta-galactosidase activity, or its hydrolysis activity.

[0038] Lactase activity: Lactase activity can be determined, for example, using the LAU(B) assay. The activity of a particular lactase in LAU(B) can be determined by direct measurement of o-nitrophenyl (ONP) released from o-nitrophenyl β-D-galactopyranoside (ONPG) in a buffer containing 1.46 mg / ml substrate in 0.05 M MES, 1 mM MgSO4 7H2O, 450 mg / L Brij 35 at pH 6.5 and 30°C. After 600 seconds of incubation, the reaction is stopped by adding 0.2 M Na2CO3, and after 126 seconds of incubation, the released ONP is measured at 405 nm. Activity is obtained by comparison with a standard curve performed with lactases of known activity, from which the activity of unknown samples is calculated. The lactase of known activity can be, for example, Saphera® from Novozymes A / S, Denmark. Lactase activity can be determined by measuring the amount of lactose hydrolysis in milk, for example by the method described in Example 4 in the "Analysis of Residual Lactose Content" paragraph using HPAEC-PAD, where the lactose peak is related to a lactose standard of known concentration. Lactose hydrolysis can then be related to the amount of lactase added (e.g., amount per mg of enzyme protein or amount per mole of enzyme). Other methods of measuring lactase activity are also known and routinely used in the art.

[0039] Mature Polypeptide: The term "mature polypeptide" refers to the mature form of a polypeptide following N-terminal and / or C-terminal processing (eg, removal of a signal peptide).

[0040] Milk: By "milk" is meant the milky secretion obtained by milking any mammal, for example a cow, sheep, goat, buffalo, or camel.

[0041] Milk-derived products: The term "milk-derived products" refers to dairy and other products based on milk. In the present context, it is clear that the term particularly includes heat-treated milk-derived products, including pasteurized milk, but also milk that has been subjected to higher temperatures than those typically used in pasteurization (e.g. ultra-pasteurized milk, UHT (extra high temperature) milk, and ESL (extended shelf life) milk).

[0042] Purified: The term "purified" refers to a nucleic acid, polypeptide, or cell that is substantially free of other components as determined by analytical techniques known in the art (e.g., a purified polypeptide or nucleic acid may form a discrete band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, and typically is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or more pure (e.g., percent on a weight or molar basis). In a related sense, a composition is enriched with respect to a molecule if there is a substantial increase in the concentration of that molecule following application of a purification or concentration technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified substance or component that is present in a composition at a higher relative or absolute concentration as compared to the starting composition.

[0043] In one aspect, the term "purified" as used herein refers to a polypeptide or cell that is essentially free of components, particularly insoluble components, from the producing organism. In another aspect, the term "purified" refers to a polypeptide that is essentially free of insoluble components, particularly insoluble components, from the natural organism from which the polypeptide is obtained. In one aspect, the polypeptide is separated from some of the possible components of the organism from which it is recovered and the culture medium. The polypeptide may be purified (i.e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0044] Thus, a polypeptide may be purified such that only trace amounts of other proteins (particularly other polypeptides) are present. The term "purified" as used herein may refer to the removal of other components (particularly other proteins, most particularly other enzymes) present in the cell from which the polypeptide originates. A polypeptide may be "substantially pure", i.e., free of other components from the organism that produces the polypeptide (e.g., the host organism in the case of recombinantly produced polypeptides). In one embodiment, the polypeptide is at least 40% pure by weight of total polypeptide material present in a preparation. In one embodiment, the polypeptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of total polypeptide material present in a preparation. As used herein, a "substantially pure polypeptide" can refer to a polypeptide preparation that contains up to 10% by weight of other polypeptide material with which the polypeptide is naturally or recombinantly associated, preferably up to 8%, more preferably up to 6%, more preferably up to 5%, more preferably up to 4%, more preferably up to 3%, even more preferably up to 2%, most preferably up to 1%, and most preferably up to 5%.

[0045] Thus, a substantially pure polypeptide is preferably at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, and most preferably at least 99.5% pure, by weight of the total polypeptide material present in the preparation. The polypeptides of the invention are preferably in substantially pure form (i.e., the preparation is essentially free of other polypeptide material with which it is naturally or recombinantly associated). This can be accomplished, for example, by preparing the polypeptide by known recombinant or classical purification methods.

[0046] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0047] For the purposes of the present invention, sequence identity between two amino acid sequences is determined as the "longest identity" output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48; 443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified on the command line. The output of Needle labeled "longest identity" is calculated as follows: (identical residues × 100) / (length of alignment−total number of gaps in the alignment).

[0048] Variant: The term "variant" refers to a polypeptide having lactase activity and which contains an artificial mutation (i.e., a substitution, an insertion (including an extension), and / or a deletion (e.g., a truncation)) at one or more positions. A substitution refers to the replacement of an amino acid occupying a position with a different amino acid; a deletion refers to the removal of an amino acid occupying a position; and an insertion refers to the addition of 1 to 5 amino acids (e.g., 1 to 3 amino acids, specifically 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0049] Wild-type: The term "wild-type" with respect to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "naturally occurring" refers to something that is found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to something that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory, or modifications of a wild-type sequence).

[0050] Conventions for naming variants: For the purposes of the present invention, a polypeptide with a selected wild-type sequence can be used to determine the corresponding amino acid position of another lactase. The amino acid sequence of another lactase is aligned with the polypeptide with a selected wild-type sequence, and based on this alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide with a selected wild-type sequence is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48; 443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS; The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16; 276-277), version 5.0.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0051] In describing the variants of the invention, the nomenclature described below is adapted for ease of reference. The accepted IUPAC one-letter or three-letter amino acid abbreviations are employed. The amino acid numbering of the variants disclosed herein is in each case based on the numbering of the relevant wild-type sequence.

[0052] For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, a substitution of threonine with alanine at position 226 is designated "Thr226Ala" or "T226A". Multiple mutations are separated by an additional sign ("+") or simply a space, e.g., a substitution of glycine (G) with arginine (R) and a substitution of serine (S) with phenylalanine (F) at positions 205 and 411 can be represented as "Gly205Arg+Ser411Phe", "G205R+S411F", "Gly205Arg Ser411Phe", or "G205R S411F".

[0053] Detailed Description of the Invention The present invention relates to a method for producing a heat treated milk-derived product having reduced lactose, comprising the steps of: a) adding an enzyme having lactase activity to a milk-derived substrate containing at least 2% (w / w) lactose; b) carrying out a heat treatment of the milk-derived substrate by holding the milk-derived substrate at a holding temperature of at least 120°C for a holding time of at least 1 second after the addition of the enzyme, followed by cooling to produce a heat-treated milk-derived product; and c) storing the heat-treated milk-derived product at a temperature of up to 40° C. for at least 24 hours, preferably for at least 2 days, such as at least 3 days, more preferably for at least 4 days. Including, After step c), the lactose content of the milk-derived product is at most 0.2% (w / w); A method is provided.

[0054] Preferably, after step b) but before step c), the lactose content of the milk-derived product is at least 0.5% (w / w).

[0055] The milk-derived substrate preferably contains 2 to 30% lactose, preferably 2 to 17% (w / w) lactose, more preferably 4 to 5.5% (w / w) lactose.

[0056] The dairy-derived substrate can be any raw milk and / or processed dairy ingredient. Useful dairy-derived substrates include, but are not limited to, any milk or dairy-like product solution / suspension containing lactose, such as whole or low-fat milk, skim milk, buttermilk, flavored milk such as chocolate milk, reconstituted milk powder, condensed milk, a solution of dry milk, milk including skim milk powder, milk permeate, whey, whey permeate, acid whey, or cream.

[0057] The dairy-derived substrate may be milk, for example raw milk (eg raw milk that has not been pasteurized prior to step a).

[0058] In a preferred embodiment, the dairy-derived substrate is milk, including milk, condensed milk, or skim milk powder.

[0059] In a more preferred embodiment, the milk-derived substrate is milk containing 4.5 to 5% (preferably 4.5 to 5%) (w / w) lactose.

[0060] In one embodiment, the dairy-derived substrate is raw milk, preferably raw milk that has not been pasteurized prior to step a.

[0061] In one embodiment, after step a) but before step b), the milk-derived substrate is incubated for up to 4 hours, preferably for up to 60 minutes, more preferably for up to 10 minutes, even more preferably for up to 5 minutes. Such incubation may be carried out at a temperature of up to 10° C., preferably at a temperature of up to 7° C.

[0062] Preferably, however, step b) is carried out immediately after step a), without a dedicated incubation step.

[0063] Preferably, the time between addition of the enzyme and reaching the holding temperature of step b) is at most 5 minutes, more preferably at most 2 minutes, even more preferably at most 1 minute.

[0064] In a preferred embodiment, the heat treatment is carried out as an indirect heat treatment, preferably as an indirect UHT treatment. A pumping device may be installed to add the enzyme to the milk-derived substrate while it flows through a process device such as a process pipe. A heating medium (e.g. a plate heat exchanger or a tube heat exchanger) may be used that does not directly contact the milk-derived substrate but is separated by a contact surface of the device. Preferably, the enzyme is added to the milk-derived substrate just before the heat treatment step (e.g. just before reaching the holding temperature), optionally while the temperature of the milk-derived substrate is increasing towards the holding temperature of step b).

[0065] In another preferred embodiment, the heat treatment is carried out as a direct heat treatment, preferably as a direct UHT treatment. The heat treatment may be carried out by steam injection or steam infusion using high pressure steam, preferably by steam injection, to heat the milk-derived substrate. In a preferred embodiment, the enzyme is added together with the steam. Preferably, after the retention time of b), the steam-containing milk-derived substrate is flash cooled in vacuum to remove water equivalent to the amount of condensed steam used.

[0066] A combination of direct and indirect heat treatment may also be used.

[0067] The heat treatment may be an ESL treatment, an ultra-pasteurization, or a UHL treatment.

[0068] The heat treatment may be carried out at a temperature of 120 to 150°C.

[0069] In one embodiment, the heat treatment is carried out at a temperature of at least 123°C, preferably between 123 and 145°C.

[0070] In one embodiment, the heat treatment is carried out at a temperature of at least 130°C, preferably between 130 and 145°C.

[0071] In one embodiment, the heat treatment is carried out at a temperature of at least 138°C, preferably at a temperature of 138-145°C, more preferably at a temperature of 138-142°C.

[0072] The retention time in step b) may be from 1 to 30 seconds, preferably from 1 to 10 seconds, and more preferably from 1 to 5 seconds.

[0073] In one embodiment, the heat treatment is a UHT treatment, preferably carried out at a temperature of 130-145°C for a time period of 1-30 seconds, more preferably at a temperature of 138-145°C for a time period of 1-10 seconds, and even more preferably at a temperature of 138-144°C for a time period of 1-5 seconds.

[0074] In one embodiment, the heat treatment is a UHT treatment carried out at a temperature of 128-132°C for 25-35 seconds, or at a temperature of 138-140°C for 2-5 seconds, or at a temperature of 144-146°C for 0.5-2 seconds.

[0075] In one embodiment, the heat treatment is an ESL treatment or ultra pasteurization, preferably an ESL treatment or ultra pasteurization carried out at a temperature of 120-140°C for a time period of 1-5 seconds, more preferably an ESL treatment or ultra pasteurization carried out at a temperature of 120-130°C for a time period of 1-5 seconds or at a temperature of 138-140°C for a time period of 2-4 seconds.

[0076] Preferably, no enzyme with lactase activity is added to the milk-derived product after step b), e.g. after the holding time of step b). More preferably, no enzyme is added to the milk-derived product after step b), e.g. after the holding time of step b). Even more preferably, nothing is added to the milk-derived product after step b), e.g. after the holding time of step b). The reason for this is that after the heat treatment, the milk-derived product is sterile and adding anything, even something that is considered to be sterile, would be a risk of contaminating this product.

[0077] After the holding time of step b), the milk-derived product is cooled, preferably within 5 minutes, more preferably within 3 minutes, even more preferably within 2 minutes, for example within 1 minute, to a maximum of 40°C, preferably to a maximum of 35°C, more preferably to a maximum of 30°C.

[0078] Preferably, the time from addition of the enzyme until the heat treated milk-derived product has been cooled to a temperature of at most 40°C, preferably at most 35°C, more preferably at most 30°C, is at most 3.5 minutes, preferably at most 3 minutes, more preferably at most 2.5 minutes, such as at most 2 minutes or at most 1 minute.

[0079] After step b) but before step c), the dairy-derived product may be homogenized.

[0080] Alternatively, homogenization may be carried out before reaching the holding temperature of step b). In indirect UHT processing (e.g. tube or plate exchange), homogenization is preferably carried out upstream. In direct UHT processing (e.g. steam injection or steam infusion), homogenization is preferably carried out downstream.

[0081] Preferably, after step b) but before step c), the milk-derived product is aseptically packaged.

[0082] In a preferred embodiment, the milk-derived product is UHT milk. UHT milk in the context of the present invention is milk that has been subjected to a sterilization procedure intended to kill all microorganisms, including bacterial spores.

[0083] Preferably, when step b) is completed, less than 80% of the lactose is hydrolyzed and after one week, more than 90% of the lactose is hydrolyzed.More preferably, when step b) is completed, less than 60% of the lactose is hydrolyzed and after one week, more than 95% of the lactose is hydrolyzed.

[0084] Preferably, after step b), the enzyme retains at least 0.01%, more preferably at least 0.1%, more preferably at least 1%, more preferably at least 2%, more preferably at least 10%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 90% of its initial activity. The enzyme activity retained after step b) as a percentage of the enzyme activity when the enzyme was added can be determined, for example, using the "residual activity assay" of this example.

[0085] Preferably, step c) is carried out at a temperature between 2 and 40° C., preferably between 15 and 40° C., more preferably between 18 and 40° C., most preferably between 18 and 30° C. In a preferred embodiment, step c) is carried out at room temperature, which may vary during storage.

[0086] In a preferred embodiment, after step b) but before step c), the lactose content of the milk-derived product is at least 1% (w / w), such as at least 2% (w / w), at least 3% (w / w), or at least 4% (w / w).

[0087] In another preferred embodiment, after step b) but before step c), the lactose content of the milk-derived product is reduced by up to 80%, preferably reduced by up to 50%, more preferably reduced by up to 20% compared to the lactose content before step a).

[0088] In step c), the heat treated milk-derived product is stored for at least 24 hours, preferably for at least 2 days (e.g. at least 3 days), more preferably for at least 4 days (e.g. at least 7 days), even more preferably for at least 14 days (e.g. at least 21 days). It is mainly during this storage period that the lactose level is reduced to the desired level due to residual lactase activity.

[0089] In a preferred embodiment, after storage of the heat treated milk-derived product for 21 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of the milk-derived product is at most 0.2% (w / w), preferably at most 0.1%, more preferably at most 0.01%.

[0090] In another preferred embodiment, after storage of the heat-treated milk-derived product for 14 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of the milk-derived product is at most 0.2% (w / w), preferably at most 0.1%, more preferably at most 0.01%.

[0091] In another preferred embodiment, after storage of the heat-treated milk-derived product for 7 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of the milk-derived product is at most 0.2% (w / w), preferably at most 0.1%, more preferably at most 0.01%.

[0092] In another preferred embodiment, after storage of the heat-treated milk-derived product for 4 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of the milk-derived product is at most 0.2% (w / w), preferably at most 0.1%, more preferably at most 0.01%.

[0093] In another preferred embodiment, after storage of the heat-treated milk-derived product for 3 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of the milk-derived product is at most 0.2% (w / w), preferably at most 0.1%, more preferably at most 0.01%.

[0094] In another preferred embodiment, after storage of the heat-treated milk-derived product for 2 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of the milk-derived product is at most 0.2% (w / w), preferably at most 0.1%, more preferably at most 0.01%.

[0095] In another preferred embodiment, after storage of the heat-treated milk-derived product for 24 hours at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of the milk-derived product is at most 0.2% (w / w), preferably at most 0.1%, more preferably at most 0.01%.

[0096] In a preferred embodiment, after storage of the heat treated milk-derived product for 21 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C and most preferably at a temperature between 18 and 30°C, the lactose content of this milk-derived product is reduced by at least 80% or at least 85%, preferably reduced by at least 90, 91, 92, 93, 94, 95, 96 or 97%, more preferably reduced by at least 98%, even more preferably reduced by at least 99% or at least 99.5% and most preferably reduced by at least 99.8% or at least 99.9% compared to the lactose content before step a).

[0097] In another preferred embodiment, after storage of the heat treated milk-derived product for 14 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of this milk-derived product is reduced by at least 80% or at least 85%, preferably reduced by at least 90, 91, 92, 93, 94, 95, 96 or 97%, more preferably reduced by at least 98%, even more preferably reduced by at least 99% or at least 99.5% and most preferably reduced by at least 99.8% or at least 99.9% compared to the lactose content before step a).

[0098] In another preferred embodiment, after storage of the heat treated milk-derived product for 7 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, and most preferably at a temperature between 18 and 30°C, the lactose content of this milk-derived product is reduced by at least 80% or at least 85%, preferably reduced by at least 90, 91, 92, 93, 94, 95, 96 or 97%, more preferably reduced by at least 98%, even more preferably reduced by at least 99% or at least 99.5% and most preferably reduced by at least 99.8% or at least 99.9% compared to the lactose content before step a).

[0099] In another preferred embodiment, after storage of the heat treated milk-derived product for 4 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of this milk-derived product is reduced by at least 80% or at least 85%, preferably reduced by at least 90, 91, 92, 93, 94, 95, 96 or 97%, more preferably reduced by at least 98%, even more preferably reduced by at least 99% or at least 99.5% and most preferably reduced by at least 99.8% or at least 99.9% compared to the lactose content before step a).

[0100] In another preferred embodiment, after storage of the heat treated milk-derived product for 3 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, and most preferably at a temperature between 18 and 30°C, the lactose content of this milk-derived product is reduced by at least 80% or at least 85%, preferably reduced by at least 90, 91, 92, 93, 94, 95, 96 or 97%, more preferably reduced by at least 98%, even more preferably reduced by at least 99% or at least 99.5% and most preferably reduced by at least 99.8% or at least 99.9% compared to the lactose content before step a).

[0101] In another preferred embodiment, after storage of the heat treated milk-derived product for 2 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of this milk-derived product is reduced by at least 80% or at least 85%, preferably reduced by at least 90, 91, 92, 93, 94, 95, 96 or 97%, more preferably reduced by at least 98%, even more preferably reduced by at least 99% or at least 99.5% and most preferably reduced by at least 99.8% or at least 99.9% compared to the lactose content before step a).

[0102] In another preferred embodiment, after storage of the heat treated milk-derived product for 24 hours at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C and most preferably at a temperature between 18 and 30°C, the lactose content of this milk-derived product is reduced by at least 80% or at least 85%, preferably reduced by at least 90, 91, 92, 93, 94, 95, 96 or 97%, more preferably reduced by at least 98%, even more preferably reduced by at least 99% or at least 99.5% and most preferably reduced by at least 99.8% or at least 99.9% compared to the lactose content before step a).

[0103] In a preferred embodiment, after storage of the heat treated milk-derived product for 21 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of the milk-derived product is at most 1000 ppm, preferably at most 100 ppm.

[0104] In another preferred embodiment, after storage of the heat-treated milk-derived product for 14 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of the milk-derived product is at most 1000 ppm, preferably at most 100 ppm.

[0105] In another preferred embodiment, after storage of the heat-treated milk-derived product for 7 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of the milk-derived product is at most 1000 ppm, preferably at most 100 ppm.

[0106] In another preferred embodiment, after storage of the heat-treated milk-derived product for 4 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of the milk-derived product is at most 1000 ppm, preferably at most 100 ppm.

[0107] In another preferred embodiment, after storage of the heat-treated milk-derived product for 3 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of the milk-derived product is at most 1000 ppm, preferably at most 100 ppm.

[0108] In another preferred embodiment, after storage of the heat-treated milk-derived product for 2 days at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of the milk-derived product is at most 1000 ppm, preferably at most 100 ppm.

[0109] In another preferred embodiment, after storage of the heat-treated milk-derived product for 24 hours at a temperature between 2 and 40°C, preferably at a temperature between 15 and 40°C, more preferably at a temperature between 18 and 40°C, most preferably at a temperature between 18 and 30°C, the lactose content of the milk-derived product is at most 1000 ppm, preferably at most 100 ppm.

[0110] After step c), the lactose-reduced heat-treated dairy product may optionally be freeze-dried.

[0111] The enzyme having lactase activity may be added at a concentration of 100 to 50,000 LAU(B) per liter of milk-derived substrate, and preferably at a concentration of 500 to 40,000 LAU(B).

[0112] The enzyme having lactase activity may be added at a concentration of 1-150 mg of enzyme protein per liter of milk-derived substrate, preferably at a concentration of 1-100 mg of enzyme protein per liter of milk-derived substrate, and more preferably at a concentration of 2-50 or 5-50 mg of enzyme protein per liter of milk-derived substrate.

[0113] A reducing agent may be added to the milk-derived substrate prior to step b), preferably a reducing agent approved for food use, more preferably a reducing agent selected from L-cysteine, sulfite, and glutathione. The reducing agent may be added together with the enzyme, for example, the reducing agent may be part of the enzyme formulation. The reducing agent may be added, for example, to reduce or prevent oxidation of cysteines in the lactase enzyme that are not part of a disulfide bridge (sometimes referred to as "free cysteines"). Oxidation of such free cysteines may reduce the ability of the enzyme to refold after heat treatment, such as UHT treatment.

[0114] The lactase enzyme of SEQ ID NO:1 has a free cysteine ​​residue at position C372, the oxidation of which has been shown to reduce the ability of the enzyme to refold, and the corresponding cysteine ​​residue can be identified in a number of other GH2 lactase enzymes of the clade DYLGE by aligning their amino acid sequences with SEQ ID NO:1. In a preferred embodiment, the enzyme with lactase activity has an amino acid substitution of the cysteine ​​corresponding to C372 of SEQ ID NO:1, preferably to serine, alanine, or glycine.

[0115] Preferably, the enzyme having lactase activity is lactase. Preferably, this enzyme belongs to the enzyme class 3.2.1.21, 3.2.1.23 or 3.2.1.108, more preferably 3.2.1.23 or 3.2.1.108.

[0116] In a preferred embodiment, the enzyme having lactase activity is neutral lactase.

[0117] In a preferred embodiment, the enzyme having lactase activity is purified.

[0118] In a preferred embodiment, the enzyme having lactase activity is isolated.

[0119] Preferably, the enzyme having lactase activity is a bacterial lactase.

[0120] Preferably, the enzyme having lactase activity is a GH2 lactase, more preferably a GH2 lactase of the clade DYLGE. The Carbohydrate-Active enZYmes Database (CAZy) (http: / / www.cazy.org / ), online since 1998, is a specialized database that classifies carbohydrate-active enzymes (CAZymes) into families (Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B (2014) The Carbohydrate-active enzymes database (CAZy), 2013, Nucleic Acids Res 42: D490-D495; Elodie Drula, Marie-Line Garron, Suzan Dogan, Vincent Lombard, Bernard Henrissat, Nicolas Terrapon, The carbohydrate-active enzyme database: functions and literature, Nucleic Acids Research, Volume 50, Issue D1, 7 January 2022, Pages D571-D577).

[0121] The glycoside hydrolase (GH) family consists of enzymes that catalyse the hydrolysis and / or rearrangement of glycosidic bonds, which are further classified into (sub)families such as GH2, which may again be subdivided into clades based on sequence motifs. It has been found by the inventors that lactases classified in GH2 of the clade DYLGE are particularly good at retaining activity after heat treatment and are therefore particularly suitable for the method of the invention.

[0122] GH2 lactases of the DYLGE clade contain the motif WTXXDY[I / L / R]GE[P / S / A], in which the glutamic acid E is involved in galactose binding. DYLGE clade lactases may also contain additional short peptide motifs SR[W / Y / F]YSGSGX[Y / G]R and / or [L / V / I]X[L / V / I]PHD, both of which are located in the GH2 N-terminal galactose-binding domain and are important for substrate binding.

[0123] In a preferred embodiment, the enzyme with lactase activity comprises in its amino acid sequence the motif WTXXDY[I / L / R]GE[P / S / A].

[0124] In a preferred embodiment, the enzyme with lactase activity comprises in its amino acid sequence the motif SR[W / Y / F]YSGSGX[Y / G]R and / or [L / V / I]X[L / V / I]PHD.

[0125] Preferably, the enzyme having lactase activity has an optimum temperature of 30 to 60° C., preferably 35 to 55° C. This optimum temperature can be determined using Method 2 of Example 2.

[0126] Preferably, the enzyme with lactase activity has a melting temperature Tm of 50-70° C. as determined by thermal shift at pH 6. In another preferred embodiment, the enzyme with lactase activity has a melting temperature Tm of 50-70° C. as determined by thermal shift at pH 7. The melting temperature Tm may be determined using the thermal shift assay of Example 5.

[0127] Preferably, the enzyme with lactase activity has a residual activity of at least 1%, preferably at least 2%, more preferably at least 5%, even more preferably at least 10% after incubation in skim milk with a lactose content of 4.7% at 70° C. for 30 seconds and at 140° C. for 5 seconds. This residual activity may be determined as in Example 3.

[0128] In a preferred embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 50% identical to any of SEQ ID NOs: 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or the mature polypeptide of any of these, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical.

[0129] In another preferred embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 50% identical to SEQ ID NO:1, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical.

[0130] In another preferred embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 50% identical to SEQ ID NO:4, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical.

[0131] In another preferred embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 50% identical to SEQ ID NO:5, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical.

[0132] In another preferred embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 50% identical to SEQ ID NO:6, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical.

[0133] In another preferred embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 50% identical to SEQ ID NO:11, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical.

[0134] In a preferred embodiment, the enzyme having lactase activity is a polypeptide derived from any of SEQ ID NOs: 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or a mature polypeptide of any of these, having 1 to 30 modifications, such as substitutions, deletions, and / or insertions, at one or more positions, such as 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30 modifications, particularly substitutions.

[0135] In another preferred embodiment, the enzyme with lactase activity is a polypeptide derived from SEQ ID NO: 1 having 1 to 30 modifications, such as substitutions, deletions and / or insertions, at one or more positions, such as 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30 modifications, in particular substitutions.

[0136] In another preferred embodiment, the enzyme with lactase activity is a polypeptide derived from SEQ ID NO: 4, which has 1 to 30 modifications, such as substitutions, deletions and / or insertions, at one or more positions, such as 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30 modifications, in particular substitutions.

[0137] In another preferred embodiment, the enzyme with lactase activity is a polypeptide derived from SEQ ID NO: 5, which has 1 to 30 modifications, such as substitutions, deletions and / or insertions, at one or more positions, such as 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30 modifications, in particular substitutions.

[0138] In another preferred embodiment, the enzyme with lactase activity is a polypeptide derived from SEQ ID NO: 6, which has 1 to 30 modifications, such as substitutions, deletions and / or insertions, at one or more positions, such as 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30 modifications, in particular substitutions.

[0139] In another preferred embodiment, the enzyme with lactase activity is a polypeptide derived from SEQ ID NO: 11 having 1 to 30 modifications, such as substitutions, deletions and / or insertions, at one or more positions, such as 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30 modifications, in particular substitutions.

[0140] In a preferred embodiment, the enzyme with lactase activity is derived from any of SEQ ID NOs: 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 by substitution, deletion or addition of one or several amino acids.

[0141] In another preferred embodiment, the enzyme having lactase activity is derived from the mature polypeptide of any of SEQ ID NOs: 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 by substitution, deletion or addition of one or several amino acids.

[0142] In one embodiment, the number of amino acid substitutions, deletions, and / or insertions introduced into any of the polypeptides of SEQ ID NO:1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 is up to 15, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0143] The amino acid changes may be of a minor nature, that is, conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine tract, an antigenic epitope, or a binding module.

[0144] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule to identify amino acid residues critical to the activity of the molecule, and the resulting molecules are tested for lactase activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active site of an enzyme, or other biological interactions, can also be determined by physical analysis of structures determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255; 306-312; Smith et al., 1992, J. Mol. Biol. 224; 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. Identification of essential amino acids can also be inferred from alignment with related polypeptides and / or sequence homology with related polypeptides and conserved catalytic mechanisms, or within a polypeptide or protein family that typically has polypeptides / proteins derived from a common ancestor with similar three-dimensional structure, function, and significant sequence similarity. Additionally or alternatively, protein structure prediction tools can be used for protein structure modeling to identify essential amino acids and / or active sites of polypeptides. See, e.g., Jumper et al., 2021, "Highly accurate protein structure prediction with AlphaFold", Nature 596: 583-589.

[0145] Single or multiple amino acid substitutions, deletions and / or insertions may be made and tested using known mutagenesis, recombination, and / or shuffling methods followed by associated screening procedures such as those disclosed in Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86; 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that may be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30; 10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46; 145; Ner et al., 1988, DNA 7; 127).

[0146] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17;893-896). Mutagenized DNA molecules that code for active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues within a polypeptide.

[0147] The amino acid modifications can also be non-conservative modifications (e.g., non-conservative substitutions, deletions, and / or insertions, typically substitutions) that confer one or more desired properties to the enzyme. Non-limiting examples of such desirable properties include improved residual activity after exposure to high temperatures, the ability to refold after denaturation, and specific activity.

[0148] In a preferred embodiment, the enzyme having lactase activity is derived from any of SEQ ID NOs: 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, preferably any of SEQ ID NOs: 1, 4, 5, 6, or 11, by substituting 1 to 30 (preferably 1 to 20) amino acids such that the enzyme has a higher residual activity after UHT treatment compared to the enzyme from which it is derived.

[0149] The enzyme with lactase activity can be obtained from any genus of microorganism. For the purposes of the present invention, the term "obtained from", when used herein in reference to a given source, is intended to mean that the polypeptide encoded by the polynucleotide is produced by the source or is produced by the strain into which the polynucleotide of the present invention has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0150] In a preferred embodiment, the enzyme with lactase activity is obtained from the genus Bifidobacterium, preferably from Bifidobacterium bifidum, Bifidobacterium samirii, Bifidobacterium aerophilum or Bifidobacterium mongoliense, or from the genus Bacillus, preferably from Bacillus circulans or Bacillus sp. S3, from Varibaculum sp. or from the genus Urmitella, preferably from Urmitella timonensis. timonensis, or from the genus Streptococcus, preferably from Streptococcus entericus DSM 14446, or from the genus Streptomyces, preferably from Streptomyces cirratus, or from the genus Clostridium, preferably from Clostridium nexile CAG:348, or from the genus Neobacillus, preferably from Neobacillus bataviensis, Neobacillus mesonae, or Neobacillus niacini. niacini) or a variant of the enzyme obtained from any of these genera or species.

[0151] In another preferred embodiment, the enzyme with lactase activity is obtained from a Bifidobacterium, preferably from Bifidobacterium bifidum or Bifidobacterium samirii, or is a variant of an enzyme with lactase activity obtained from a Bifidobacterium, preferably from Bifidobacterium bifidum or Bifidobacterium samirii.

[0152] In another preferred embodiment, the enzyme with lactase activity is obtained from the genus Urmitella, preferably from Urmitella timonensis, or is a variant of an enzyme with lactase activity obtained from the genus Urmitella, preferably from Urmitella timonensis.

[0153] In another preferred embodiment, the enzyme with lactase activity is obtained from the genus Streptococcus, preferably from Streptococcus entericus, or is a variant of an enzyme with lactase activity obtained from the genus Streptococcus, preferably from Streptococcus entericus.

[0154] In another preferred embodiment, the enzyme with lactase activity is obtained from Varibaculum sp. or is a variant of the enzyme with lactase activity obtained from Varibaculum sp.

[0155] With respect to the aforementioned species, it will be understood that the invention encompasses both perfect and imperfect forms, as well as other taxonomic equivalents (e.g., anamorphs), regardless of the species name by which they are known. Those of skill in the art will readily recognize the identity of appropriate equivalents.

[0156] Enzymes can be identified and obtained from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.) using the above probes. Techniques for isolating microorganisms and DNA directly from natural habitats are known in the art. Polynucleotides encoding the enzymes can then be obtained by similarly screening genomic or cDNA libraries of other microorganisms, or mixed DNA samples. Probes can be used to detect the polynucleotides encoding the enzymes, and the polynucleotides can then be isolated or cloned using techniques known to those skilled in the art (see, for example, Davis et al., 2012, Basic Methods in Molecular Biology, Elsevier).

[0157] An enzyme having lactase activity may be a polypeptide derived from any of SEQ ID NOs: 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or may be a polypeptide derived from the mature polypeptide of any of these, where the N-terminus and / or C-terminus has been extended by the addition of one or more amino acids or one or more amino acids have been deleted from the N-terminus and / or C-terminus.

[0158] An enzyme having lactase activity may be C-terminally truncated compared to the wild-type enzyme from which it is derived.

[0159] In a preferred embodiment, the enzyme with lactase activity is up to about 1500 amino acids in length, such as up to about 1400 amino acids or up to about 1350 amino acids in length, such as from 850 to 1500 amino acids, preferably from 850 to 1400 amino acids, such as from 850 to 1350 or from 1100 to 1400 amino acids in length.

[0160] In another preferred embodiment the enzyme with lactase activity is C-terminally truncated and has a length of up to about 1500 amino acids, such as up to about 1400 amino acids or up to about 1350 amino acids, such as 850-1500 amino acids, preferably 850-1400 amino acids, such as 850-1350 or 1100-1400 amino acids.

[0161] In one embodiment the enzyme with lactase activity is obtained from a Bifidobacterium, preferably from Bifidobacterium bifidum, or is a variant of an enzyme with lactase activity obtained from a Bifidobacterium, preferably from Bifidobacterium bifidum, and is C-terminally truncated and has a length of 850 to 1500 amino acids, preferably 850 to 1350 amino acids, more preferably 880 to 1350, 880 to 1320, or 885 to 1310 amino acids.

[0162] In another embodiment the enzyme with lactase activity is obtained from a Bifidobacterium, preferably from Bifidobacterium bifidum, or is a variant of an enzyme with lactase activity obtained from a Bifidobacterium, preferably from Bifidobacterium bifidum, and which is C-terminally truncated and has a length of 850 to 1500 amino acids, preferably 1250 to 1500 amino acids, more preferably 1250 to 1350 or 1290 to 1350 amino acids, even more preferably 1300 to 1305 amino acids, such as 1302 or 1304 amino acids.

[0163] In one embodiment the enzyme with lactase activity is obtained from Bifidobacterium samirii or a variant of the enzyme with lactase activity obtained from Bifidobacterium samirii and is C-terminally truncated and has a length of 850 to 1500 amino acids, preferably 1250 to 1400 amino acids, more preferably 1250 to 1350 or 1280 to 1320 amino acids, for example 1290 to 1310 amino acids.

[0164] In one embodiment the enzyme with lactase activity is obtained from Streptococcus entericus DSM 14446 or is a variant of the enzyme with lactase activity obtained from Streptococcus entericus DSM 14446 and is C-terminally truncated and has a length of 850 to 1500 amino acids, preferably 1250 to 1400 amino acids, more preferably 1250 to 1350 or 1270 to 1310 amino acids, for example 1280 to 1300 amino acids.

[0165] In one embodiment, the enzyme with lactase activity is obtained from Varibaculum sp. or a variant of an enzyme with lactase activity obtained from Varibaculum sp. and is C-terminally truncated and has a length of 850 to 1500 amino acids, preferably 1250 to 1400 amino acids, more preferably 1250 to 1350 or 1290 to 1330 amino acids, for example 1300 to 1320 amino acids.

[0166] In one embodiment the enzyme with lactase activity is obtained from Urmitella timonensis or a variant of the enzyme with lactase activity obtained from Urmitella timonensis and is C-terminally truncated and has a length of 850 to 1500 amino acids, preferably 1250 to 1400 amino acids, more preferably 1250 to 1350 or 1290 to 1330 amino acids, for example 1300 to 1320 amino acids.

[0167] In one embodiment, the enzyme with lactase activity is obtained from Bacillus sp. S3 or a variant of an enzyme with lactase activity obtained from Bacillus sp. S3 and is C-terminally truncated and has a length of 850 to 1500 amino acids, preferably 1250 to 1400 amino acids, more preferably 1250 to 1350 or 1290 to 1330 amino acids, for example 1300 to 1320 amino acids.

[0168] In one embodiment the enzyme with lactase activity is obtained from Bifidobacterium aerophilum or a variant of the enzyme with lactase activity obtained from Bifidobacterium aerophilum and is C-terminally truncated and has a length of 850 to 1500 amino acids, preferably 1250 to 1400 amino acids, more preferably 1250 to 1350 or 1280 to 1320 amino acids, for example 1290 to 1310 amino acids.

[0169] In one embodiment the enzyme with lactase activity is obtained from Bifidobacterium mongoliense or a variant of the enzyme with lactase activity obtained from Bifidobacterium mongoliense and is C-terminally truncated and has a length of 850 to 1500 amino acids, preferably 1250 to 1400 amino acids, more preferably 1250 to 1350 or 1280 to 1320 amino acids, for example 1290 to 1310 amino acids.

[0170] In one embodiment the enzyme with lactase activity is obtained from Clostridium nexile CAG:348 or a variant of the enzyme with lactase activity obtained from Clostridium nexile CAG:348 and is C-terminally truncated and has a length of 850 to 1500 amino acids, preferably 1250 to 1400 amino acids, more preferably 1250 to 1350 or 1270 to 1310 amino acids, for example 1280 to 1300 amino acids.

[0171] In one embodiment the enzyme with lactase activity is obtained from Neobacillus bataviensis or a variant of the enzyme with lactase activity obtained from Neobacillus bataviensis and is C-terminally truncated and has a length of 800 to 1400 amino acids, preferably 1000 to 1200 amino acids, more preferably 1100 to 1200 or 1110 to 1150 amino acids, for example 1120 to 1140 amino acids.

[0172] In one embodiment the enzyme with lactase activity is obtained from Neobacillus mesonae or a variant of the enzyme with lactase activity obtained from Neobacillus mesonae and is C-terminally truncated and has a length of 850 to 1500 amino acids, preferably 1250 to 1400 amino acids, more preferably 1250 to 1350 or 1290 to 1330 amino acids, for example 1300 to 1320 amino acids.

[0173] In one embodiment the enzyme with lactase activity is obtained from Neobacillus niacini or a variant of the enzyme with lactase activity obtained from Neobacillus niacini and is C-terminally truncated and has a length of 850 to 1500 amino acids, preferably 1250 to 1400 amino acids, more preferably 1250 to 1350 or 1290 to 1330 amino acids, for example 1300 to 1320 amino acids.

[0174] In one embodiment the enzyme with lactase activity is obtained from Streptomyces cirratus or a variant of the enzyme with lactase activity obtained from Streptomyces cirratus and is C-terminally truncated and has a length of 1200 to 1900 amino acids, preferably 1600 to 1800 amino acids, more preferably 1650 to 1750 or 1660 to 1700 amino acids, for example 1670 to 1690 amino acids.

[0175] In a preferred embodiment, the enzyme with lactase activity has an initial lactose turnover of at least 10 / sec / enzyme molecule in milk at 5° C. This initial lactose turnover can be determined, for example, when 0-10% of lactose is hydrolyzed. Methods for determining the initial lactose turnover per enzyme molecule will be known to those skilled in the art. For example, this can be a direct measurement of lactose using HPLC or an indirect measurement using a glucose detection method.

[0176] The initial lactose turnover per enzyme molecule in milk at 5°C is 36 seconds for Lactozym® Pure. -1 and 89 seconds for Saphera®. -1 It is.

[0177] In a more preferred embodiment, the enzyme with lactase activity has an initial lactose turnover in milk at 5° C. of at least 20 / sec / enzyme molecule, preferably at least 50 / sec / enzyme molecule, more preferably at least 80 / sec / enzyme molecule.

[0178] In a preferred embodiment, the enzyme with lactase activity has an average lactose turnover from initial lactose (preferably about 4.7% lactose to 0.1% residual lactose) of at least 10 / sec / enzyme molecule in milk at 5°C.

[0179] The average lactose turnover from 4.7% initial lactose to 0.1% residual lactose was approximately 9 seconds for Lactozym® Pure. -1 In the case of Saphera, it takes about 23 seconds. -1 The lower average lactose turnover from 4.7% to 0.1% compared to the initial rate (4.7% to 4.23%) is due to a combination of several factors (e.g., higher product inhibition and lower substrate concentration closer to 0.1%).

[0180] In a preferred embodiment, the enzyme with lactase activity has an average lactose turnover from initial lactose (preferably from about 4.7% to 0.01% residual lactose) of at least 5 / sec / enzyme molecule in milk at 5°C.

[0181] The average lactose turnover from the initial lactose from 4.7% to 0.01% residual lactose was approximately 5 seconds for Lactozym® Pure. -1 In the case of Saphera, it takes about 18 seconds. -1 It is.

[0182] In a preferred embodiment, the enzyme having lactase activity has a Michaelis constant K M is at most 40 mM, preferably at most 30 mM, more preferably at most 20 mM.

[0183] In another preferred embodiment, the enzyme having lactase activity has a Michaelis constant K M is at most 40 mM, preferably at most 30 mM, more preferably at most 20 mM.

[0184] Michaelis constant K M is the substrate concentration (in this case lactose concentration) at which the reaction rate is half-maximal and is a measure of the affinity of the substrate for the enzyme. M indicates high affinity, which means that the rate is MThis means that the reaction approaches a maximum when the substrate concentration is lower than when the substrate concentration is higher.

[0185] Michaelis constant K M can be determined according to the method of Example 5 of WO 09071539 A1. In this example, K M has been determined to be 8 mM for experimental Bifidobacterium lactase and 30 mM for Lactozym (K. lactis lactase), with a K at 37°C. M has been determined to be 13 mM for experimental Bifidobacterium lactase and 30 mM for Lactozym.

[0186] Preferred Embodiments 1. A method for producing a heat-treated milk-derived product having reduced lactose, comprising: a) adding an enzyme having lactase activity to a milk-derived substrate containing at least 2% (w / w) lactose; b) carrying out a heat treatment of the milk-derived substrate by holding the milk-derived substrate at a holding temperature of at least 120°C for a holding time of at least 1 second after addition of the enzyme, followed by cooling to produce a heat-treated milk-derived product; and c) storing the heat treated milk-derived product at a temperature of up to 40° C. for at least 24 hours, preferably for at least 2 days, such as at least 3 days, more preferably for at least 4 days; Including, After step c), the lactose content of the milk-derived product is at most 0.2% (w / w); method.

[0187] 2. The method according to embodiment 1, wherein after step b) but before step c), the lactose content of the milk-derived product is at least 0.5% (w / w).

[0188] 3. The method according to embodiment 1 or 2, wherein after step a) but before step b), the milk-derived substrate is incubated for up to 4 hours, preferably for up to 60 minutes, more preferably for up to 10 minutes, even more preferably for up to 5 minutes.

[0189] 4. The method according to embodiment 3, wherein after step a) but before step b), the milk-derived substrate is incubated at a temperature of up to 10°C, preferably at a temperature of up to 7°C.

[0190] 5. The method according to embodiment 1 or 2, wherein step b) is carried out immediately after step a) without a dedicated incubation step.

[0191] 6. The method according to embodiment 1 or 2, wherein step b) is carried out immediately after step a), without a dedicated incubation step after and before step a).

[0192] 7. The method of embodiment 1 or 2, or 5 or 6, wherein a pumping device is provided for adding the enzyme to the milk-derived substrate while the milk-derived substrate is flowing through process equipment, such as process piping.

[0193] 8. The method according to any one of embodiments 1 or 2, or 5 to 7, wherein the time from addition of the enzyme to reaching the holding temperature of step b) is at most 5 minutes, preferably at most 2 minutes, more preferably at most 1 minute.

[0194] 9. The method according to any one of embodiments 1 or 2, or 5 to 8, wherein the time from addition of the enzyme until the heat treated milk-derived product has been cooled to a temperature of at most 40°C, preferably to a temperature of at most 35°C, more preferably to a temperature of at most 30°C, is at most 3.5 minutes, preferably at most 3 minutes, more preferably at most 2.5 minutes, such as at most 2 minutes or at most 1 minute.

[0195] 10. The method of any one of embodiments 5 to 9, wherein the enzyme is added to the milk-derived substrate while it is flowing through process equipment, such as process piping, immediately prior to the heat treatment step, optionally while the temperature of the milk-derived substrate is being increased towards the holding temperature of step b).

[0196] 11. A method according to any one of the preceding embodiments 1 to 10, wherein a heating medium is used that is not in direct contact with the milk-derived substrate but is separated by a contact surface of an apparatus, and preferably the heating medium is a plate heat exchanger or a tube heat exchanger.

[0197] 12. The method according to any one of the preceding embodiments, wherein the heat treatment is carried out as an indirect heat treatment, preferably as an indirect UHT treatment.

[0198] 13. The method according to any one of the preceding embodiments, wherein the heat treatment is carried out by steam injection or steam infusion using high pressure steam, preferably by steam injection, to heat the milk-derived substrate.

[0199] 14. The method according to embodiment 13, wherein step b) is carried out directly after step a) without a dedicated incubation step, and the enzyme is added together with the steam.

[0200] 15. The method according to embodiment 13 or 14, wherein after the retention time of step b), the milk-derived substrate containing the steam is flash cooled in vacuum to remove water equivalent to the amount of condensed steam used.

[0201] 16. The method according to any one of embodiments 13 to 15, wherein the heat treatment is carried out as a direct heat treatment, preferably as a direct UHT treatment.

[0202] 17. The method according to any one of embodiments 13 to 16, further comprising applying indirect heating using a plate or tube heat exchanger.

[0203] 18. The method according to any one of the preceding embodiments, wherein the milk-derived substrate comprises 2-30% (w / w) lactose, preferably 2-17% (w / w) lactose, more preferably 4-5.5% (w / w) lactose.

[0204] 19. The method of any one of the preceding embodiments, wherein the milk-derived substrate is milk containing 4-5.5% (w / w) lactose.

[0205] 20. The method according to any one of the preceding embodiments, wherein the milk-derived substrate is raw milk, preferably raw milk that has not been pasteurized prior to step a).

[0206] 21. The method of any one of the preceding embodiments, wherein the heat treatment is an ESL treatment, an ultra-pasteurization, or a UHL treatment.

[0207] 22. The method according to any one of the preceding embodiments, wherein the heat treatment is carried out at a temperature of 120 to 150° C.

[0208] 23. The method according to any one of the preceding embodiments, wherein the heat treatment is carried out at a temperature of at least 123°C, preferably at a temperature of 123 to 145°C.

[0209] 24. The method according to any one of the preceding embodiments, wherein the heat treatment is carried out at a temperature of at least 130°C, preferably at a temperature of 130-145°C.

[0210] 25. The method according to any one of the preceding embodiments 1 to 24, wherein the heat treatment is carried out at a temperature of at least 138°C, preferably at a temperature of 138 to 145°C, more preferably at a temperature of 138 to 142°C.

[0211] 26. The method according to any one of the preceding embodiments 1 to 25, wherein the holding time in step b) is from 1 to 30 seconds, preferably from 1 to 10 seconds, more preferably from 1 to 5 seconds.

[0212] 27. The method according to any one of the preceding embodiments 1 to 26, wherein the heat treatment is a UHT treatment, preferably carried out at a temperature of 130 to 145°C for a period of 1 to 30 seconds, more preferably at a temperature of 138 to 145°C for a period of 1 to 10 seconds, and even more preferably at a temperature of 138 to 144°C for a period of 1 to 5 seconds.

[0213] 28. The method according to any one of the preceding embodiments 1 to 27, wherein the heat treatment is a UHT treatment carried out at a temperature of 128-132°C for 25-35 seconds, or at a temperature of 138-140°C for 2-5 seconds, or at a temperature of 144-146°C for 1-2 seconds.

[0214] 29. The method according to any one of embodiments 1 to 22, wherein the heat treatment is ESL treatment or ultra-pasteurization, preferably ESL treatment or ultra-pasteurization carried out at a temperature of 120-140°C for a period of 1-5 seconds, more preferably ESL treatment or ultra-pasteurization carried out at a temperature of 120-130°C for a period of 1-5 seconds or at a temperature of 138-140°C for a period of 2-4 seconds.

[0215] 30. The method according to any one of the preceding embodiments, wherein after step b), preferably after the retention time of step b), no enzyme having lactase activity is added to the milk-derived product.

[0216] 31. The method according to any one of the preceding embodiments, wherein no enzyme is added to the milk-derived product after step b), preferably after the retention time of step b).

[0217] 32. The method according to any one of the preceding embodiments, wherein nothing is added to the milk-derived product after step b), preferably after the retention time of step b).

[0218] 33. The method according to any one of the preceding embodiments, wherein after the holding time of step b), the milk-derived product is cooled, preferably within 5 minutes, more preferably within 3 minutes, even more preferably within 2 minutes, for example within 1 minute, to a maximum of 40°C, preferably to a maximum of 35°C, more preferably to a maximum of 30°C.

[0219] 34. The method according to any one of the preceding embodiments, wherein after step b) but before step c), the milk-derived product is aseptically packaged.

[0220] 35. The method according to any one of the preceding embodiments, wherein after step b) but before step c), the milk-derived product is homogenized.

[0221] 36. The method of any one of the preceding embodiments, wherein the milk-derived product is UHT milk.

[0222] 37. The method according to any one of the preceding embodiments, wherein after step b) the enzyme retains at least 0.1%, preferably at least 1%, more preferably at least 2%, more preferably at least 10%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 90% of its initial activity.

[0223] 38. The method of any one of the preceding embodiments, wherein the initial activity is the activity prior to step b).

[0224] 39. The method according to any one of the preceding embodiments 1 to 38, wherein step c) is carried out at a temperature of 2 to 40°C, preferably at a temperature of 15 to 40°C, more preferably at a temperature of 18 to 40°C, and most preferably at a temperature of 18 to 30°C.

[0225] 40. The method according to any one of the preceding embodiments, wherein step c) is carried out at room temperature, which may vary during storage.

[0226] 41. The method according to any one of the preceding embodiments, wherein after step b) but before step c), the lactose content of the milk-derived product is at least 1% (w / w), such as at least 2% (w / w), at least 3% (w / w), or at least 4% (w / w).

[0227] 42. The method according to any one of the preceding embodiments, wherein after step b) but before step c), the lactose content of the milk-derived product is reduced by up to 80%, preferably by up to 50%, more preferably by up to 20% compared to the lactose content before step a).

[0228] 43. The method according to any one of the preceding embodiments, wherein in step c) the heat-treated milk-derived product is stored for at least 7 days, preferably for at least 14 days, more preferably for at least 21 days.

[0229] 44. The method according to any one of embodiments 1 to 43, wherein after storing the heat treated milk-derived product at a temperature between 2 and 40°C, preferably between 15 and 40°C, more preferably between 18 and 40°C, and most preferably between 18 and 30°C for 21 days, preferably for 14 days, more preferably for 7 days, even more preferably for 4 days, and most preferably for 3 days, the lactose content of the milk-derived product is at most 0.2% (w / w), preferably at most 0.1%, more preferably at most 0.01%.

[0230] 45. The method according to any one of the preceding embodiments, wherein after storing the heat treated milk-derived product at a temperature between 2 and 40°C, preferably between 15 and 40°C, more preferably between 18 and 40°C, and most preferably between 18 and 30°C for 21 days, preferably for 14 days, more preferably for 7 days, even more preferably for 4 days, and most preferably for 3 days, the lactose content of the milk-derived product is reduced by at least 80% or at least 85%, preferably reduced by at least 90, 91, 92, 93, 94, 95, 96, or 97%, more preferably reduced by at least 98%, even more preferably reduced by at least 99% or at least 99.5% and most preferably reduced by at least 99.8% or at least 99.9%% compared to the lactose content before step a).

[0231] 46. ​​The method according to any one of embodiments 1 to 45, wherein after storing the heat treated milk-derived product at a temperature between 2 and 40°C, preferably between 15 and 40°C, more preferably between 18 and 40°C, and most preferably between 18 and 30°C for 21 days, preferably for 14 days, more preferably for 7 days, even more preferably for 4 days, and most preferably for 3 days, the lactose content of the milk-derived product is at most 1000 ppm, preferably at most 100 ppm.

[0232] 47. The method according to any one of the preceding embodiments, wherein after step c), the lactose-reduced heat-treated dairy product is freeze-dried.

[0233] 48. The method according to any one of the preceding embodiments 1 to 47, wherein prior to step b) a reducing agent is added to the milk-derived product, preferably a reducing agent approved for food use, more preferably a reducing agent selected from L-cysteine, sulfites and glutathione.

[0234] 49. The method according to any one of the preceding embodiments, wherein the enzyme having lactase activity is added at a concentration of 100 to 50,000 LAU (B) per liter of milk-derived substrate, preferably at a concentration of 500 to 40,000 LAU (B).

[0235] 50. The method according to any one of the preceding embodiments, wherein the enzyme having lactase activity is added at a concentration of 1 to 150 mg of enzyme protein per liter of milk-derived substrate, preferably at a concentration of 1 to 100 mg of enzyme protein per liter of milk-derived substrate, more preferably at a concentration of 2 to 50 or 5 to 50 mg of enzyme protein per liter of milk-derived substrate.

[0236] 51. The method of any one of the preceding embodiments, wherein the enzyme having lactase activity is lactase.

[0237] 52. The method according to any one of the preceding embodiments, wherein the enzyme having lactase activity belongs to the enzyme class 3.2.1.21, 3.2.1.23, or 3.2.1.108, preferably 3.2.1.23 or 3.2.1.108.

[0238] 53. The method of any one of the preceding embodiments, wherein the enzyme having lactase activity is neutral lactase.

[0239] 54. The method of any one of the preceding embodiments, wherein the enzyme having lactase activity is purified.

[0240] 55. The method of any one of the preceding embodiments, wherein the enzyme having lactase activity is isolated.

[0241] 56. The method of any one of the preceding embodiments, wherein the enzyme having lactase activity is bacterial lactase.

[0242] 57. The method according to any one of the preceding embodiments, wherein the enzyme having lactase activity is a GH2 lactase, preferably a GH2 lactase of the clade DYLGE.

[0243] 58. A method according to any one of the preceding claims, wherein the enzyme having lactase activity comprises the motif WTXXDY[I / L / R]GE[P / S / A] in the amino acid sequence of the enzyme.

[0244] 59. The method according to any one of the preceding claims, wherein the enzyme having lactase activity comprises in the amino acid sequence of the enzyme the motif SR[W / Y / F]YSGSGX[Y / G]R and / or [L / V / I]X[L / V / I]PHD.

[0245] 60. The method according to any one of the preceding embodiments, wherein the enzyme having lactase activity has CBM32 or CBM71, preferably CBM32.

[0246] 61. The method according to any one of the preceding embodiments 1 to 60, wherein the enzyme having lactase activity has an optimum temperature of 30 to 60°C, preferably 35 to 55°C.

[0247] 62. The method according to any one of the preceding embodiments, wherein the enzyme having lactase activity has a melting temperature Tm of 50-70°C as determined by thermal shift at pH 6.

[0248] 63. The method according to any one of the preceding embodiments, wherein the enzyme having lactase activity has a melting temperature Tm of 50-70°C as determined by thermal shift at pH 7.

[0249] 64. The method of any one of the preceding embodiments, wherein the enzyme having lactase activity has a residual activity of at least 1%, preferably at least 2%, more preferably at least 5%, and even more preferably at least 10% after incubation at 70°C for 30 seconds and at 140°C for 5 seconds in skim milk having a lactose content of 4.7%.

[0250] 65. The method of any one of the preceding embodiments, wherein the enzyme having lactase activity has a residual activity of at least 0.1%, preferably at least 0.5%, at least 1%, or at least 2%, more preferably at least 5%, and even more preferably at least 10%, after incubation in skim milk with a lactose content of 4.7% at 90°C for 30 seconds, at 140°C for 5 seconds, and at 70°C for 30 seconds, followed by cooling to 0-10°C and subsequent incubation at 23°C for 0.5 hours, wherein the residual activity is relative to the activity of the same enzyme in skim milk without incubation at 90°C for 30 seconds, at 140°C for 5 seconds, and at 70°C for 30 seconds, followed by cooling to 0-10°C and subsequent incubation at 23°C for 0.5 hours.

[0251] 66. The method of any one of the preceding embodiments, wherein the enzyme having lactase activity has a residual activity of at least 0.5%, preferably at least 1%, at least 2%, or at least 3%, more preferably at least 5%, and even more preferably at least 10%, after incubation in skim milk with a lactose content of 4.7% at 90°C for 30 seconds, at 140°C for 5 seconds, and at 70°C for 30 seconds, followed by cooling to 0-10°C, and subsequent incubation at 23°C for 72 hours, wherein the residual activity is relative to the activity of the same enzyme in skim milk without incubation at 90°C for 30 seconds, at 140°C for 5 seconds, and at 70°C for 30 seconds, followed by cooling to 0-10°C, and subsequent incubation at 23°C for 72 hours.

[0252] 67. The method according to any one of the preceding embodiments, wherein the enzyme having lactase activity has at least 20% higher activity after incubation in skim milk with a lactose content of 4.7% at 90° C. for 30 seconds, at 140° C. for 5 seconds, and at 70° C. for 30 seconds, followed by cooling to 0-10° C., followed by incubation at 23° C. for 72 hours, compared to the activity after incubation in skim milk with a lactose content of 4.7% at 90° C. for 30 seconds, at 140° C. for 5 seconds, and at 70° C. for 30 seconds, followed by cooling to 0-10° C., followed by incubation at 23° C. for 0.5 hours.

[0253] 68. The method of any one of the preceding embodiments, wherein the enzyme having lactase activity has an amino acid sequence that is at least 50% identical, for example at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, or a mature polypeptide of any of these.

[0254] 69. The method of any one of the preceding embodiments, wherein the enzyme having lactase activity has an amino acid sequence that is at least 50% identical to any of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:11, or a mature polypeptide of any of these, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical.

[0255] 70. The enzyme having lactase activity is a) an enzyme having an amino acid sequence which is at least 50% identical to SEQ ID NO:1, e.g. at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical and which comprises at least one amino acid substitution selected from the group consisting of P65A, N275S, C372A, M386Q, R389A, G482A, T523N, P615T, T756K, A936S, T972C, I1035C, T1076C, Y1122C, A1129C, F1132C, K1168C, S1195C, and C1199S, e.g. comprising two or more of said amino acid substitutions, wherein the numbering is based on SEQ ID NO:1; b) an enzyme having an amino acid sequence which is at least 50% identical to SEQ ID NO:4, e.g. at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical, and which comprises at least one amino acid substitution selected from the group consisting of P65A, C372A, P615T, A1073C, H1122C, and C1195G, e.g. comprising two or more of said amino acid substitutions, wherein the numbering is based on SEQ ID NO:4; c) an enzyme having an amino acid sequence which is at least 50% identical to SEQ ID NO: 11, e.g. at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical, and which comprises at least one amino acid substitution selected from the group consisting of P52A, G607T, L1064C, and Y1110C, e.g. comprising two or more of said amino acid substitutions, wherein the numbering is based on SEQ ID NO: 11; and d) an enzyme having an amino acid sequence that is at least 50% identical to SEQ ID NO:5, e.g. at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical, and that contains the substitutions G386Q and / or P620T, wherein the numbering is based on SEQ ID NO:5. 70. The method according to any one of the preceding embodiments, selected from the group consisting of:

[0256] 71. The method according to any one of the preceding embodiments, wherein the enzyme having lactase activity has an amino acid substitution of cysteine ​​corresponding to C372 in SEQ ID NO:1, preferably with serine, alanine, or glycine.

[0257] 72. The enzyme having lactase activity is obtained from the genus Bifidobacterium, preferably from Bifidobacterium bifidum, Bifidobacterium samirii, Bifidobacterium aerophilum or Bifidobacterium mongoliense, or from the genus Bacillus, preferably from Bacillus circulans or Bacillus sp. S3, or from Varibaculum sp. or from the genus Urmitella, preferably from Urmitella timonensis. timonensis, or from the genus Streptococcus, preferably from Streptococcus entericus DSM 14446, or from the genus Streptomyces, preferably from Streptomyces cirratus, or from the genus Clostridium, preferably from Clostridium nexile CAG:348, or from the genus Neobacillus, preferably from Neobacillus bataviensis, Neobacillus mesonae, or Neobacillus niacini. 72. The method of any one of the preceding embodiments, wherein the enzyme is obtained from Azotobacter pneumoniae (Azotobacter niacini) or a variant of the enzyme obtained from any of these genera or species.

[0258] 73. The method according to any one of embodiments 1 to 72, wherein the enzyme having lactase activity is up to about 1500 amino acids in length, for example up to about 1400 amino acids, or up to about 1350 amino acids in length, for example 850 to 1500 amino acids, preferably 850 to 1400 amino acids in length, for example 850 to 1350 or 1100 to 1400 amino acids in length.

[0259] 74. The method of any one of the preceding embodiments, wherein the enzyme having lactase activity has an amino acid sequence that is at least 50% identical to either of SEQ ID NO:1 or 2, or to any mature polypeptide of SEQ ID NO:1 or 2, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical.

[0260] 75. The method of any one of embodiments 1 to 74, wherein the enzyme having lactase activity has an amino acid sequence that is at least 50% identical to SEQ ID NO:1, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% identical.

[0261] 76. The method according to any one of the preceding embodiments, wherein the enzyme having lactase activity is a polypeptide derived from any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or a mature polypeptide derived from any of these, having 1 to 30 modifications, such as substitutions, deletions, and / or insertions, at one or more positions, such as 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30 modifications, particularly substitutions.

[0262] 77. The method according to any one of the preceding embodiments, wherein the enzyme with lactase activity is a polypeptide derived from SEQ ID NO: 1 having 1 to 30 modifications, such as substitutions, deletions and / or insertions, at one or more positions, such as 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30 modifications, in particular substitutions.

[0263] 78. The method according to any one of the preceding embodiments, wherein the enzyme having lactase activity is a polypeptide derived from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or a mature polypeptide derived from any one of these, wherein the N-terminus and / or C-terminus has been extended by the addition of one or more amino acids or one or more amino acids have been deleted from the N-terminus and / or C-terminus.

[0264] 79. The method according to any one of the preceding embodiments, wherein the enzyme having lactase activity is C-terminally truncated compared to the wild-type enzyme from which the enzyme is derived.

[0265] 80. The method according to any one of the preceding embodiments, wherein the enzyme with lactase activity is obtained from a species of Bifidobacterium, preferably from Bifidobacterium bifidum, or from a species of Bacillus, preferably from Bacillus circulans, or a variant thereof, from a species of Bifidobacterium, preferably from Bifidobacterium bifidum, or from a species of Bacillus, preferably from Bacillus circulans, a variant of an enzyme with lactase activity.

[0266] 81. The method according to any one of the preceding embodiments, wherein the enzyme with lactase activity is obtained from a Bifidobacterium, preferably from Bifidobacterium bifidum, or is a variant, i.e. a variant of an enzyme with lactase activity obtained from a Bifidobacterium, preferably from Bifidobacterium bifidum.

[0267] 82. The method according to any one of the preceding embodiments, wherein the enzyme having lactase activity is 850 to 1500 amino acids in length, preferably 850 to 1350 amino acids in length, more preferably 880 to 1350, 880 to 1320, or 885 to 1310 amino acids in length.

[0268] 83. The method according to any one of embodiments 1 to 82, wherein the enzyme having lactase activity has a length of 850 to 1500 amino acids, preferably 1250 to 1500 amino acids, more preferably 1250 to 1350 or 1290 to 1350 amino acids, even more preferably 1300 to 1305 amino acids, for example 1302 or 1304 amino acids.

[0269] 84. The method of any one of the preceding embodiments, wherein the enzyme having lactase activity has an initial lactose turnover in milk at 5° C. of at least 10 lactose molecules / second / enzyme molecule.

[0270] 85. The method according to any one of the preceding embodiments, wherein the enzyme having lactase activity has an initial lactose turnover in milk at 5° C. of at least 20 units / sec / enzyme molecule, preferably at least 50 units / sec / enzyme molecule, more preferably at least 80 units / sec / enzyme molecule.

[0271] 86. The method of any one of the preceding embodiments, wherein the enzyme having lactase activity has an average lactose turnover from initial lactose (preferably from about 4.7% lactose to 0.1% residual lactose) of at least 10 per second per enzyme molecule in milk at 5°C.

[0272] 87. The method of any one of the preceding embodiments, wherein the enzyme having lactase activity has an average lactose turnover from initial lactose (preferably from about 4.7% to 0.01% residual lactose) of at least 5 per second per enzyme molecule in milk at 5°C.

[0273] 88. The enzyme having lactase activity has a Michaelis constant K at 5°C. M The method according to any one of the preceding embodiments, wherein the ATP is at most 40 mM, preferably at most 30 mM, more preferably at most 20 mM.

[0274] 89. The enzyme having lactase activity has a Michaelis constant K at 37°C. M The method according to any one of the preceding embodiments, wherein the ATP is at most 40 mM, preferably at most 30 mM, more preferably at most 20 mM. EXAMPLES

[0275] Method 1: LAU(B) assay The activity of a particular lactase in LAU-B / g can be determined by directly measuring the o-nitrophenyl (ONP) released from o-nitrophenyl β-D-galactopyranoside (ONPG) in a buffer containing 1.46 mg / ml substrate in 0.05 M MES, 1 mM MgSO4 7H2O, 450 mg / L Brij 35 at pH 6.5 and 30°C. After 600 seconds of incubation, the reaction is stopped by adding 0.2 M Na2CO3 and the released ONP is measured at 405 nm after 126 seconds of incubation. The activity is obtained by comparison with a standard curve performed with lactases of known activity, from which the activity of unknown samples is calculated. The lactase of known activity can be, for example, Saphera® from Novozymes A / S, Denmark.

[0276] Method 2: Assay to determine optimal temperature Assay for determining temperature profile 35°C to 75°C A temperature profile to determine the optimum temperature is prepared by placing 10 μl of diluted enzyme sample (diluted in 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl2, 1 mM MgCl2 + 0.01% triton X-100, pH 6.5) into a PCR tube, then adding 90 μl of substrate (167 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 6.5) and placing the PCR tube in a preheated PCR block with a temperature gradient of 35-75°C (using a TProfessional thermocycler, Biometra), incubating at 35-75°C (gradient) for 30 min, then placing on ice. The reaction is stopped by adding 100 μl of 0.25 M NaOH. 20 μl are transferred to a 96-well microtiter plate and 230 μl of GOD-Perid (100 mM potassium phosphate buffer, pH 7, 0.6 g / l glucose oxidase, 0.02 g / l horseradish peroxidase, 1.0 g / l ABTS) solution are added. After 30 min in the dark at room temperature, the absorbance is measured at 420 nm. The initial dilution of the enzyme should be adjusted so that the final 420 nm reading at the optimal temperature is Abs 0.5-2.5. The temperature at which the delta Abs at 420 nm is highest ("Abs 420 nm with enzyme" - "Abs 420 nm without enzyme", i.e. background) is taken as 100% and the delta Abs 420 nm reading is then related to the delta Abs 420 nm at the highest value.

[0277] Example 1 The lactase used is Saphera 2600L from Novozymes A / S (Bifidobacterium bifidum, SEQ ID NO: 1) with a published activity of 2600 LAU(B) / g.

[0278] Test 1 248.5 liters of skim milk was pumped into the incubation tank and then cooled to 5°C.

[0279] To this milk 1.5 kg Saphera (0.6% dose equivalent to 15,600 LAU(B) per litre of milk) was added and the milk was then stirred at approximately 50 rpm for 18 hours.

[0280] At the end of the incubation, the milk was treated with UHT using steam injection under the following UHT conditions: 140°C / 4 sec, and downstream two-stage homogenization (70°C 200 / 50 bar).

[0281] The milk was cooled from 140° C. to about 90° C. using a flash cooling method that instantly (in about 1 second) lowered the temperature of the milk to 90° C. The temperature was then lowered to about 60° C. over about 30 seconds (including downstream homogenization), and then lowered again to room temperature (about 20° C.) over about 30 seconds before being filled.

[0282] Samples were collected after UHT, two samples were frozen until analysis, the remaining sample was stored at room temperature for up to 3 weeks and analyzed for residual lactose.

[0283] Test 2 248.875 liters of skimmed milk was thoroughly mixed with 1.125 kg of Saphera (0.45% dosage) at low temperature (5° C.). The enzyme and milk were mixed for 45 minutes and then processed using steam injection under the following UHT conditions: UHT conditions (140° C. / 4 sec) and downstream two-stage homogenization (70° C. 200 / 50 bar).

[0284] Using flash cooling, the milk was cooled from 140°C to about 90°C in about 1 second, after which the temperature was reduced to about 60°C in about 30 seconds (including downstream homogenization), and then again to room temperature (about 20°C) in about 30 seconds before filling.

[0285] Samples were collected after UHT, two samples were frozen until analysis, the remaining sample was stored at room temperature for up to 3 weeks and analyzed for residual lactose.

[0286] Test 3 248.5 litres of skimmed milk was pumped into the UHT unit. Once the milk temperature reached 70°C, 1.5 kg of Saphera (0.6% dosage) was pumped into the hot milk stream (70°C) as the milk stream was on its way to the UHT treatment. The UHT conditions were: steam injection UHT conditions (140°C / 4 sec) and downstream two stage homogenisation (70°C 200 / 50bar).

[0287] After the enzymes were added, the milk was exposed to hot steam for a short period of time, raising the temperature of the milk instantaneously (in about 1 second) to 140°C, followed by a 4 second hold at 140°C, a 1 second (flash cooling) drop in temperature to about 90°C, a further drop in temperature to about 60°C over 30 seconds (downstream homogenization was performed during this stage), and a 30 second drop in temperature to room temperature (about 20°C) before filling.

[0288] Samples were collected after UHT, two samples were frozen until analysis, the remaining sample was stored at room temperature for up to 3 weeks and analyzed for residual lactose.

[0289] Test 4 248.5 liters of skimmed milk was thoroughly mixed with 1.5 kg of Saphera (0.6% dosage) at low temperature (5° C.). The enzyme and milk were mixed for 10 minutes and then processed using steam injection under the following UHT conditions: UHT condition (140° C. / 4 sec), and downstream two-stage homogenization (70° C. 200 / 50 bar).

[0290] Using flash cooling, the milk was cooled from 140°C to about 90°C in about 1 second, after which the temperature was reduced to about 60°C in about 30 seconds (including downstream homogenization), and then again to room temperature (about 20°C) in about 30 seconds before filling.

[0291] Samples were collected after UHT, two samples were frozen until analysis, the remaining sample was stored at room temperature for up to 3 weeks and analyzed for residual lactose.

[0292] Test 5 247 litres of skimmed milk was thoroughly mixed with 3 kg of Saphera (1.2% dosage) at low temperature (5°C). The milk was instantly processed using steam injection at the following UHT conditions: UHT conditions (140°C / 4 sec) and downstream two-stage homogenisation (70°C 200 / 50bar).

[0293] Using flash cooling, the milk was cooled from 140°C to about 90°C in about 1 second, after which the temperature was reduced to about 60°C in about 30 seconds (including downstream homogenization), and then again to room temperature (about 20°C) in about 30 seconds before filling.

[0294] Samples were collected after UHT, two samples were frozen until analysis, the remaining sample was stored at room temperature for up to 3 weeks and analyzed for residual lactose.

[0295] Test 6 249.25 litres of skimmed milk was thoroughly mixed with 0.75 kg of Saphera (0.3% dosage) at low temperature (5°C). The milk was instantly processed using steam injection at the following UHT conditions: UHT conditions (140°C / 4 sec) and downstream two-stage homogenisation (70°C 200 / 50 bar).

[0296] Using flash cooling, the milk was cooled from 140°C to about 90°C in about 1 second, after which the temperature was reduced to about 60°C in about 30 seconds (including downstream homogenization), and then again to room temperature (about 20°C) in about 30 seconds before filling.

[0297] Samples were collected after UHT, two samples were frozen until analysis, the remaining sample was stored at room temperature for up to 3 weeks and analyzed for residual lactose.

[0298] Test 7 247 liters of skim milk was pumped into the UHT unit. Once the milk temperature reached 90°C, it was incubated in a holding tube at approximately 90°C for 2 minutes. Then 3 kg of Saphera (1.2% dosage) was pumped into the hot milk stream (90°C) as it was on its way to the UHT treatment. The UHT conditions were tube exchange heating as follows: UHT conditions (140°C / 4 seconds), where a two-stage homogenization was performed upstream (70°C 200 / 50bar).

[0299] After the enzyme was added, the mixture was heated to 140°C over approximately 30 seconds, held at 140°C for 4 seconds, the temperature was reduced to approximately 45°C over approximately 40 seconds, and then the temperature was reduced to room temperature (approximately 20°C) over an additional 30 seconds before filling.

[0300] Samples were collected after UHT, two samples were frozen until analysis, the remaining sample was stored at room temperature for up to 3 weeks and analyzed for residual lactose.

[0301] Residual lactose content analysis Residual lactose analysis was performed by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

[0302] Sample preparation for HPAEC-PAD: 50ul of sample was transferred to a 5ml Eppendorf tube containing 500ul of MQ water. 10ul of Carrez I solution was added and mixed, then 10ul of Carrez II solution was added and mixed. 4.43ml of MQ water (total 5ml) was then added and mixed. Centrifugation was performed at 14200rpm for 5min. The supernatant was diluted 5 times and then 20 times for some samples. These samples were analyzed by HPAEC-PAD.

[0303] Analysis method: Equipment: Dionex IC-5000 Pump: ICS-5000 single pump model SP-5, S / N 11113787 Autosampler: Thermo Scientific AS-AP, P / N 074925, S / N 12101147 Oven ICS-5000 Detector / Chromatography Module Model DC-5, S / N: 11102695 Detector: Dionex IC-5000 ECD, P / N 072043, S / N 11113974, ECD cell P / N 072044, S / N 10198 with non-disposable gold electrodes P / N 063494, S / N 50435. Column: CarboPac PA20 Guard column, 3 x 30 mm (Dionex P / N 060144) coupled with CarboPac PA20 column, 3 x 150 mm (Dionex P / N 060142) Eluent: A: MQ water B: 200 mM NaOH C: 100 mM sodium acetate in 200 mM NaOH D: 1 mM sodium acetate in 200 mM NaOH

[0304] [Table 1]

[0305] [Table 2]

[0306] [Table 3]

[0307] Example 2 UHT Treatment 1% (w / v) Saphera 2600L (Bifidobacterium bifidum, SEQ ID NO: 1), 0.25% (w / v) Biolacta N5 (Bacillus circulans, SEQ ID NO: 2), 1% (w / v) Bonlacta (Lactobacillus delbrueckii subsp. Bulgaricus, SEQ ID NO: 3), and 1% (w / v) Lactozym Pure 6500L (Kluyveromyces lactis) were each added to skim milk with a lactose content of 4.7%. Sodium azide* was added to a final concentration of 0.025% (w / v) to avoid microbial growth, since handling / tubing etc. are not completely sterile despite the UHT step. The milk sample was applied to a laboratory scale UHT apparatus as described below. A syringe containing 10 ml of milk sample was connected to a long Teflon tube, which was immersed in three baths in sequence. The first bath, made of silicone, was at 70° C. and had 3 m of Teflon tubing, the second connected bath was also made of silicone, was at 140° C. and had 50 cm of Teflon tubing, and the last bath was a 1 m ice / water bath (0° C.) to cool the milk after the 140° C. treatment. A flow rate of 3 ml / min was applied with the syringe, ensuring that the milk sample was incubated at 70° C. for 30 seconds and at 140° C. for 5 seconds, followed by cooling in the ice / water bath for 10 seconds to cool the milk to 0-10° C. Finally, the milk was withdrawn with a 10 ml syringe after the ice / water bath, and the sample was assayed for residual activity.

[0308] *Sodium azide is not used in commercial processes where sterility is assured, but is used in this laboratory setting which may not be completely sterile and where milk is not consumed.

[0309] Residual activity assay Samples were centrifuged at 20,600g in a pre-cooled centrifuge for 45 min at 5°C and the supernatant was diluted with 20 mM sodium succinate and 0.01% triton x-100, pH 6.5 to allow a measurable absorbance reading of less than 1.5 at 405 nm. 25 μl of each sample was mixed with 175 μl of ONPG substrate (1.67 mg / ml ONPG (o-nitrophenyl β-D-galatopyranoside, approximately 5.5 mM), 0.05 M MES, 1 mM MgSO4, 150 mM KCl, 0.01% Triton X-100, pH 6.5), incubated for 2.5 h at 40°C, stopped by adding 50 μl Na2CO3 + 5 mM Na4EDTA and measured at 405 nm. Residual activity (%) was calculated using the formula = ((Abs405 加熱処理サンプル -Abs405 ブランク )*Dilution factor) / ((Abs405 非処理サンプル -Abs405 ブランク )*dilution factor)*100%.

[0310] The heat-treated sample was the enzyme mixed with skim milk and sodium azide and subjected to UHT treatment and subsequent cooling as described above. The untreated sample was the same enzyme mixed with skim milk and sodium azide but not subjected to UHT treatment. Both of these samples were diluted to give an absorbance at 405 nm in the range of 0.5-1.0. The blank was a sample without enzyme, using the same dilution factor as the enzyme-containing sample.

[0311] [Table 4]

[0312] Table 3 shows that lactases can withstand UHT treatment to different degrees, and the highest residual activity can be found in the following order (highest residual activity first): Biolacta N5, Saphera 2600L, Lactozym pure 6500L, and Bonlacta.

[0313] Biolacta® N5 (SEQ ID NO:2) and Saphera® 2600L (SEQ ID NO:1) are GH2 lactases of the clade DYLGE, while Lactozym® Pure 6500L (a yeast lactase from Kluyveromyces lactis) and Bonlacta® (SEQ ID NO:3) are GH2 lactases of the clade MGN.

[0314] Example 3: UHT Treatment A batch of PE variants of Saphera (SEQ ID NO: 1) with the amino acid substitutions shown in Table 4 was added to skim milk containing 0.025% sodium azide to a final concentration of 5% (v / v). 5% (v / v) corresponds approximately to the same amount of enzyme as 1% (w / v) in the Saphera 2600L. The milk sample was applied to a lab-scale UHT setup as described below. A syringe containing 10 ml of milk sample was connected to a long Teflon tube, which was immersed in three baths in sequence. The first bath, made of silicone, was at 70° C. and contained 3 m of Teflon tubing, the second connected bath, also made of silicone, was at 140° C. and contained 50 cm of Teflon tubing, and the last bath was a 1 m ice / water bath (0° C.) to cool the milk after the 140° C. treatment. A flow rate of 3 ml / min was applied with the syringe and milk samples were incubated steadily at 70° C. for 30 seconds, at 140° C. for 5 seconds, and then cooled in an ice / water bath for 10 seconds to cool the milk to 0-10° C. Finally, milk was withdrawn with a 10 ml syringe after the ice / water bath and samples were assayed for residual activity.

[0315] Residual activity assay The samples were centrifuged at 20,600g in a pre-cooled centrifuge for 45 min at 5°C, and the supernatants were diluted with 20 mM sodium succinate and 0.01% triton x-100, pH 6.5 to allow for a measurable absorbance reading of less than 1.5 at 405 nm. 25 μl of each sample was mixed with 175 μl of ONPG substrate (1.67 mg / ml ONPG (o-nitrophenyl β-D-galatopyranoside, approximately 5.5 mM), 0.05 M MES, 1 mM MgSO4, 150 mM KCl, 0.01% Triton X-100, pH 6.5 (preferably adjusted with NaOH)), incubated for 2.5 h at 40°C, stopped by adding 50 μl Na2CO3 + 5 mM Na4EDTA, and measured at 405 nm. The residual activity (%) was calculated using the formula: ((Abs405 加熱処理サンプル -Abs405 ブランク )*Dilution factor) / ((Abs405 非処理サンプル -Abs405 ブランク )*dilution factor)*100%.

[0316] The residual activity of each variant was then related to the residual activity of lactase of SEQ ID NO:1 using the following formula = (% residual activity of variant) / (% residual activity of lactase of SEQ ID NO:1)*100%, and these values ​​are shown in Table 4.

[0317] [Table 5]

[0318] Table 4 shows that all 10 variants V1 to V10 have improved residual activity compared to the lactase of SEQ ID NO: 1. In particular, V9 had more than 6-fold higher residual activity compared to the lactase of SEQ ID NO: 1.

[0319] Example 4: A newly identified lactase with high residual activity after UHT treatment. In this example, a number of newly identified lactases and their variants are demonstrated to be useful in the methods of the invention. The lactases are added to milk immediately prior to UHT treatment and are shown to have sufficient residual activity after UHT treatment to efficiently hydrolyze lactose in milk upon storage at room temperature for several days.

[0320] Some lactases are newly identified wild-type enzymes that have been truncated at the C-terminus to facilitate expression as secreted enzymes, while others are variants of these wild-type enzymes in which one or more amino acids have been replaced using established protein engineering (PE) techniques. The enzymes tested in this example are listed in Table 5 below, and a matrix of sequence identities between the truncated wild-type enzymes is shown in Table 6.

[0321] Some of the newly identified C-terminally truncated wild-type enzymes are further described in a co-pending application claiming priority to European Patent Application No. 22215776, filed December 22, 2022.

[0322] Expression in Bacillus subtilis The genome sequences of the bacterial donor strains (Table 5) were downloaded from EMBL-EBI (www.ebi.ac.uk / ) and analyzed for putative lactases from the CAZY database GH2 family (Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B (2014) The Carbohydrate-active enzymes database (CAZy), 2013, Nucleic Acids Res 42: D490-D495; Elodie Drula, Marie-Line Garron, Suzan Dogan, Vincent Lombard, Bernard Henrissat, Nicolas Terrapon, The carbohydrate-active enzyme database: functions and literature, Nucleic Acids Research, Volume 50, Issue D1, 7 January 2022, Pages D571-D577; http: / / www.cazy.org / ).

[0323] The genes encoding the enzymes in Table 5 were optimized for expression in Bacillus subtilis using standard methods known in the art. Alternatively, codon-optimized genes can be purchased commercially. In the case of lactase SEQ ID NO: 9 (Neobacillus bataviensis) and lactase SEQ ID NO: 12 (Streptomyces cirratus), the native DNA (for the truncated mature peptide) was used. This gene was fused with DNA encoding a Bacillus clausii secretion signal (encoding the following amino acid sequence: MKKPLGKIVASTALLISVAFSSSIASA (SEQ ID NO: 16)) that replaces the native secretion signal. In addition, this expression construct adds an amino-terminal polyhistidine tag to the mature lactase, consisting of the amino acid sequence HHHHHPR (SEQ ID NO: 17), to facilitate purification by immobilized metal affinity chromatography. The resulting gene was ordered as a fully synthetically produced DNA fragment from Twist Bioscience (San Francisco, CA, USA).

[0324] The linear integration construct was a SOE-PCR fusion product formed by fusing the gene of interest between two B. subtilis chromosomal regions with a strong promoter and a chloramphenicol resistance marker (Horton, RM, Hunt, HD, Ho, SN, Pullen, JK and Pease, LR (1989) Engineering hybrid genes without the use of restriction enzymes, gene splicing by overlap extension, Gene 77:61-68). This SOE PCR method is also described in WO2003095658.

[0325] The lactase gene was expressed under the control of a triple promoter system (as described in WO 99 / 43835) consisting of promoters from the Bacillus licheniformis α-amylase gene (amyL), the Bacillus amyloliquefaciens α-amylase gene (amyQ) and the Bacillus thuringiensis cryIIIA promoter containing stabilizing sequences.

[0326] For each of the lactase expression constructs, the SOE-PCR products were transformed into Bacillus subtilis and integrated into the chromosome by homologous recombination into the pectate lyase locus. Recombinant Bacillus subtilis clones containing the integrated expression constructs were then grown in liquid medium. The culture broth was centrifuged (20,000 × g, 20 min) and the supernatant was carefully decanted from the pellet and used for enzyme purification or the sterile filtered supernatant was used directly in the assay.

[0327] Purification of recombinant enzymes by immobilized metal affinity chromatography The pH of the clarified supernatant was adjusted to pH 8, filtered through a 0.2 μM filter and the supernatant was applied to a 5 ml HisTrap™ Excel column. Before loading, the column was equilibrated with 5 column volumes (CV) of 50 mM Tris / HCl pH 8. To remove unbound material, the column was washed with 8 CV of 50 mM Tris / HCl pH 8 and the target elution was obtained with 50 mM HEPES pH 7 + 10 mM imidazole. The eluted protein was desalted on a HiPrep™ 26 / 10 desalting column equilibrated with 3 CV of 50 mM HEPES pH 7 + 100 mM NaCl. This buffer was also used for the elution of the target, the flow rate was 10 ml / min. Relevant fractions were selected and pooled based on chromatogram and SDS-PAGE analysis.

[0328] UHT Treatment The enzymes in Table 5 were added to skim milk, UHT treatment was carried out followed by incubation at 23°C for 0.5 and 72 hours, with lactase activity and lactose measurements carried out after each of these two intervals as described below.

[0329] Depending on the enzyme, 5.5-31.6 mg enzyme protein (ep) per liter of skim milk was used. For most enzymes, 12.7 mg ep / L skim milk was used (see Table 7, column 2). As the tubes are not completely sterile despite the UHT process due to handling etc., sodium azide was added to a final concentration of 0.025% (w / v) in all milks tested to avoid microbial growth. Milk samples were applied to a laboratory-scale UHT setup as described below. A syringe containing 10 ml of milk sample was connected to a long Teflon tube (0.8 mm internal diameter) and this tube was immersed in the four baths in sequence. The first bath was silicone-lined at 90°C and had 3 m of Teflon tubing, the second connected bath was silicone-lined at 140°C and had 50 cm of Teflon tubing, the third silicone bath was at 70°C and had 3 m of Teflon tubing, and the last bath was an ice / water bath (0°C) with 1 m of Teflon tubing to cool the milk. A flow rate of 3 ml / min was applied with a syringe and the milk samples were incubated steadily at 90°C for 30 seconds, at 140°C for 5 seconds and at 70°C for 30 seconds, followed by cooling in an ice / water bath for 10 seconds to cool the milk to a temperature ranging from 0 to 10°C. Finally, the milk was collected in a tube after the ice / water bath and the samples were incubated at 23°C for 0.5 and 72 hours, respectively, and then assayed for residual activity and lactose content using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

[0330] Residual activity assay Samples were centrifuged at 20,600g in a pre-cooled centrifuge for 45 min at 5°C and the supernatant was diluted with 20 mM sodium succinate and 0.01% triton X-100, pH 6.5 to allow a measurable absorbance reading of less than 1.5 at 405 nm. 25 μl of each sample was mixed with 175 μl of ONPG substrate (1.67 mg / ml ONPG (o-nitrophenyl β-D-galatopyranoside, approximately 5.5 mM), 0.05 M MES, 1 mM MgSO4, 150 mM KCl, 0.01% Triton X-100, pH 6.5), incubated for 2.5 h at 40°C, stopped by adding 50 μl Na2CO3 + 5 mM Na4EDTA and measured at 405 nm. Residual activity (%) was calculated using the formula = ((Abs405 加熱処理サンプル -Abs405 ブランク )*Dilution factor) / ((Abs405 非処理サンプル -Abs405 ブランク )*dilution factor)*100%.

[0331] The heat treated sample was the enzyme mixed with skim milk and sodium azide, subjected to UHT treatment and subsequent cooling, and then incubated at 23°C for 0.5 and 72 hours as described above. The untreated sample was the same enzyme mixed with skim milk and sodium azide but without UHT treatment and incubation. Both of these samples were diluted to give an absorbance at 405 nm in the range of 0.5 to 1.0. The blank was a sample without enzyme, using the same dilution factor as the enzyme containing samples. The results are shown in Table 8 below.

[0332] Residual lactose content analysis Residual lactose analysis was performed by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

[0333] Sample preparation for HPAEC-PAD: The enzyme was inactivated by adding 5ul glacial acetic acid to 1ml milk sample, heated to 90°C for 5min and centrifuged at 20,000g for 10min. 50ul sample was transferred to a 5ml Eppendorf tube containing 500ul M(Milli-Q)Q water. 10ul Carrez I solution was added and mixed, then 10ul Carrez II solution was added and mixed. 4.43ml MQ water (total 5ml) was then added and mixed. Centrifugation was carried out at 20,000g for 5min. The supernatant was diluted 5 times with Milli Q water. These samples were analysed by HPAEC-PAD.

[0334] Determination of Lactose using HPAEC-PAD The analysis performed is essentially as described in Leeuwen S, Kuipers B, Dijkhuizen L, Kamerling J. Comparative structural characterization of 7 commercial galacto-oligosaccharide (GOS) products, Carbohydrate Research, 425 (2016) 48-58, with minor modifications, e.g., shorter gradients, as specified below. A Dionex ICS-6000 workstation (Dionex, Amsterdam, The Netherlands) was used, equipped with a CarboPac PA1 4 x 50 mm Guard column (Dionex, product number 043096) followed by a CarboPac PA1 4 x 250 mm (Dionex, product number 035391) and an ICS-6000 DC ECD detector (Dionex) with a complex gradient of A: Milli-Q water, B: 600 mM NaOAc in 100 mM NaOH, C: 100 mM NaOH, and D: 50 mM NaOAc. Fractionation was performed at 1.0 mL / min with 85% A, 0% B, 10% C, and 5% D, a 25 min linear gradient to 10% A, 0% B, 40% C, and 50% D, followed by a 2 min linear gradient to 0% A, 25% B, 75% C, and 0% D, immediately followed by a 5 min wash with 100% B, and a 15 min regeneration with 85% A, 0% B, 10% C, and 5% D. A lactose standard was used to determine the amount of lactose in the enzyme treated samples. The results are shown in Table 7, columns 3 and 4 below.

[0335] Dose-response curve for non-UHT treated B. bifidum lactase To be able to compare the activity of the enzyme in the UHT-treated samples with the activity of non-UHT-treated B. bifidum lactase (Enzyme #31, SEQ ID NO:1), a dose / response curve for non-UHT-treated B. bifidum lactase (Enzyme #31, SEQ ID NO:1) was generated.

[0336] The following doses of B. bifidum lactase (enzyme #31) in skim milk (+0.025% sodium azide) were prepared in duplicate: 2.03, 1.63, 1.30, 1.04, 0.832, 0.666, 0.532, 0.426, 0.341, 0.273, 0.218, 0.174, 0.140, 0.112, 0.0893, 0.0715 mg ep / liter skim milk. The milk containing the enzyme was incubated for 3 days at room temperature (23° C.). Residual lactose in each sample was determined using HPAEC-PAD as described in the paragraph "Analysis of Residual Lactose Content" above. Dose / response curves were generated by plotting the measured lactose levels against the dose of B. bifidum lactase (#31). Please refer to Figure 1.

[0337] This curve was used to determine the relative activity of each UHT lactase in % to the activity of non-UHT B. bifidum lactase (#31). The data in columns 2 and 4 of Table 7 were used. For each lactase, this curve was used to determine the "corresponding amount" (mg ep / L milk) of non-UHT B. bifidum lactase required to reach the same % lactose remaining after 72 hours of incubation at the same temperature, using the value in column 4 (% lactose remaining after 72 hours of incubation). The relative activity of UHT-treated lactase in % to the activity of non-UHT-treated B. bifidum lactase was calculated as the reciprocal of the amount of UHT-treated lactase in percentage (mg ep / L milk) (column 2 of Table 7) to the reciprocal of the "corresponding amount" (mg ep / L milk) of non-UHT-treated B. bifidum lactase (i.e. the amount required to reach the same residual lactose content after incubation at 23° C. for 72 hours), said values ​​being determined using a dose / response curve. In practice, this calculation can be simplified by dividing the "corresponding amount" (mg ep / L milk) of non-UHT-treated B. bifidum lactase by the amount of UHT-treated lactase (mg ep / L milk) found in column 2 of Table 7 and expressing the result as a percentage. The relative activities so determined for each lactase are shown in column 6 of Table 7.

[0338] This value is therefore a measure of the "enzyme activity remaining after UHT treatment" based on the lactose remaining after UHT treatment and incubation for 3 days at 23° C. A dose / response curve of non-UHT treated B. bifidum lactase (SEQ ID NO: 1) (further incubated at 23° C. for 3 days) is used to determine the "corresponding amount" of non-UHT treated B. bifidum lactase.

[0339] [Table 6]

[0340] [Table 7]

[0341] [Table 8]

[0342] [Table 9]

[0343] [Table 10]

[0344] [Table 11]

[0345] result: Rows 3 and 4 of Table 7 show the residual lactose after UHT treatment followed by incubation at 23°C for 0.5 and 72 hours, respectively. Lactose reduction occurs primarily between 0.5 hours (average 4.5 g lactose / 100 ml corresponding to 96% of the initial lactose) and 72 hours, with enzymes #1, 3, and 5 having less than 0.01% residual lactose, which is required in many countries to claim lactose-free. The enzyme dosage used (mg eq / L milk) is shown for each enzyme in column 2. As the enzyme dosage increases, the residual lactose after 72 hours decreases.

[0346] Column 5 shows the pseudo specific activity after UHT treatment (grams of lactose converted per mg of enzyme protein) calculated as (g / L lactose at 0.5 hrs - g / L lactose at 72 hrs) / mg ep / L, i.e. the value in column 3 minus the value in column 4 (but expressed in g / L instead of % lactose) divided by the value in column 2. Example using data for enzyme #10 in Table 7: (45 g / L lactose - 12.7 g / L lactose) / 5.5 mg ep / L = 5.9 g lactose / mg ep per 71.5 hrs. This pseudo specific activity is biased in that it is dose-dependent since the residual lactose is not linear with respect to the enzyme dose (see Figure 1). As a result, a relatively low enzyme dose results in a higher pseudo specific activity value compared to a higher enzyme dose.

[0347] To better compare the efficiency of the enzymes, the "relative activity of unstressed B. bifidum lactase (#31)" has been calculated as above and is shown in column 6. An example of the calculation for enzyme #10 is shown in Figure 1, where, according to Table 7, a dose of 5.5 mg ep / liter milk of enzyme #10 results in 1.27% residual lactose after 72 hours (see horizontal arrow in Figure 1). The "corresponding amount" of unstressed (non-UHT treated) B. bifidum lactase (enzyme #31) is 0.39 mg eq (vertical arrow in Figure 1). This gives a relative activity of 7.1% (0.39 / 5.5*100%), as can be shown for enzyme #10 in column 6 of Table 7. This is a very good measure of the relative activity of the enzyme after UHT treatment compared to non-UHT treated B. bifidum lactase (#31) determined as the reciprocal of the amount of ep required to obtain a certain residual lactose content after UHT treatment and incubation at 23°C for 72 hours (percentage of the reciprocal of the "corresponding amount" of ep of non-UHT treated B. bifidum lactase required to obtain the same residual lactose content after incubation at 23°C for 72 hours).

[0348] Columns 7, 8, and 9 show the calculated enzyme dosages required to reach 1.41% residual lactose (a gastro-friendly level would be a 70% reduction in lactose), 0.1% residual lactose (to claim a low lactose level in many countries), or 0.01% residual lactose (to claim a lactose-free level in many countries) after 72 hours of storage at 23° C. These figures are calculated as follows: mg ep / L of B. bifidum lactase (enzyme #31) to reach lactose levels of 1.41%, 0.1%, and 0.01% are 0.361, 0.934, and 1.56 mg ep / L milk, respectively (as determined from the dose-response curve in FIG. 1). The "% relative activity to unstressed B. bifidum lactase" value (column 6 of Table 7) was used to predict the corresponding value for each enzyme, for example, for enzyme #10, the "% relative activity to unstressed B. bifidum lactase" value is 7.1% (0.071), therefore the corresponding amounts of enzyme protein for enzyme #10 are 0.361 / 0.071, 0.934 / 0.071, and 1.56 / 0.071 mg ep / L milk, which are 5.1, 13.1, and 22.0 mg ep / L milk.

[0349] Enzyme dosages of 5-50 mg lactase ep / L milk (4.5-5.0% lactose) were typically used in industrial batch lactose reduction applications with tank incubations ranging from 8-24 hours at 4-10°C to reach lactose levels of 0.01-0.1% lactose (%=g lactose / 100ml milk). Therefore, the top candidates in Table 7 are highly suitable for lactose-free applications (0.01% lactose) and other sequences with lower relative activity may be more suitable for low-lactose applications rather than lactose-free.

[0350] Columns 2 and 3 of Table 8 show the residual activity of the enzymes at 0.5 and 72 hours, respectively, after UHT treatment. The degree of residual activity varies, and surprisingly, most enzymes have increased residual activity after 72 hours compared to 0.5 hours, an average of more than 3-fold for all of the enzymes listed.

[0351] In Example 2, the residual activity of lactase of SEQ ID NO:1 was determined to be 2.14% after incubation at 70° C. for 30 seconds and 140° C. for 5 seconds before cooling. In this example, lactase including lactase of SEQ ID NO:1 (enzyme #31) was incubated at 90° C. for 30 seconds, 140° C. for 5 seconds, and 70° C. for 30 seconds before cooling, and therefore the residual activity of lactase of SEQ ID NO:1 was lower.

[0352] The relatively poor correlation between the % remaining activity (Table 8) and the measured residual lactose levels in Table 7 is likely due to the different specific activities of each enzyme towards lactose. The ability of the enzyme to hydrolyze lactose after UHT treatment is affected by the efficiency of the enzyme to convert lactose (specific activity before UHT treatment) and the activity capacity of the enzyme after UHT treatment (% remaining activity).

[0353] Amino acid changes have improved enzyme performance, particularly the replacement of a free Cys (homologous to C372 in enzyme #31) in enzyme #1 (Bifidobacterium samirii, C372A), the introduction of disulfides in enzymes #2 and 4, and the replacement of cis-proline in enzymes #3, 6, and 7.

[0354] This example shows that all of the enzymes in Table 5 are suitable for use in the methods claimed herein.

[0355] The enzymes in Table 5 are all bacterial GH2 lactases of the clade DYLGE. They all contain the motifs WTXXDY[I / L / R]GE[P / S / A], SR[W / Y / F]YSGSGX[Y / G]R, and [L / V / I]X[L / V / I]PHD.

[0356] Notes regarding Figure 1: The first two columns of Table 9 below show the data points for FIG.

[0357] [Table 12]

[0358] Instead of manually / visually reading the curve, a fitted equation was created using Equation 1 below and the numerical values ​​for "a" and "b" in Table 10 along with the % lactase referenced in the heading. The mg ep / L calculated using the % lactose in Table 9 is shown in column 3 of Table 9. Equation 1: mg ep / L milk = a*Ln(lactose%)+b

[0359] [Table 13]

[0360] Example 5: Thermal shift assay at pH 6 and 7 Thermal shift assays (TSA) measure the melting temperature (Tm) of proteins, the temperature at which 50% is denatured. Protein denaturation is monitored by the increase in fluorescence of SYPRO Orange dye, which binds to hydrophobic residues exposed as the target protein unfolds.

[0361] Purified samples were diluted to 0.24mg / ml with MilliQ water. Thermal shift assay mix was prepared by diluting SyProOrange (Invitrogen / ThermoFisher #S6650) 200-fold with the desired pH buffer (100mM succinic acid, 100mM HEPES, 100mM glycine, 150mM KCl, 1mM CaCl2, 0.01% TritonX100, adjusted to pH 6 and 7). 10ul of diluted sample was added to 20ul of TSA mix in a 96-well (Light Cycler 480 multiwell plate, Roche #04729692001). After sealing with optical tape (Roche, #04729757001), the plate was heated from 25°C to 95°C (temperature increase: 3.2°C / min) in a LightCycler 480 II real-time PCR instrument (Roche) and fluorescence was measured continuously (excision / emission wavelengths: 465 / 510 nm). Tm is determined by plotting the first derivative of fluorescence as a function of temperature (dF / dT) and determining the temperature with maximum dF / dt.

[0362] [Table 14]

[0363] Example 6: Temperature Profile Assay for determining temperature profile 35 °C - 75 °C: A temperature profile to determine the optimum temperature is prepared by placing 10 μl of diluted enzyme sample (diluted with 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl2, 1 mM MgCl2 + 0.01% triton X-100, pH 6.5) into a PCR tube. Then, 90 μl of substrate (167 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 6.5) is added and the PCR tube is placed in a preheated PCR block with a temperature gradient of 35-75°C (using a TProfessional thermocycler, Biometra), incubated at 35-75°C (gradient) for 30 min, then placed on ice. The reaction is stopped by adding 100 μl of 0.25 M NaOH. 20 μl are transferred to a 96-well microtiter plate and 230 μl of GOD-Perid (100 mM potassium phosphate buffer, pH 7, 0.6 g / l glucose oxidase, 0.02 g / l horseradish peroxidase, 1.0 g / l ABTS) solution is added. After 30 min in the dark at room temperature, the absorbance is measured at 420 nm. The initial dilution of the enzyme should be adjusted so that the final 420 nm reading at the optimal temperature is Abs 0.5-2.5. The temperature at which the delta Abs at 420 nm is highest ("Abs 420 nm with enzyme" - "Abs 420 nm without enzyme", i.e. background) is taken as 100% (optimum temperature) and the relative activity at other temperatures based on the delta Abs 420 nm to the highest delta Abs 420 nm is used to determine the temperature profile.

[0364] The assays were performed using the lactase enzymes shown below in Table 12. The enzyme numbers (Enzyme #) are according to Table 5.

[0365] [Table 15]

[0366] The temperature profile from 35 to 75°C can be found in Table 12, showing optimum temperatures of 43.0°C, 47.5°C, 38.9°C, 43°C, and close to below 35°C for enzymes #9, 11, 30, 31, and 16, respectively. The temperatures at which 50% activity remains (after the optimum) are approximately 55°C, 55°C, 46°C, 58°C, and 52°C, respectively (evaluated from Table 12, between the two numbers marked with an asterisk *). If the unfolding of the enzyme is fully reversible, the decrease in activity after the optimum will correlate with the amount of enzyme unfolded, and the "melting point" (Tm, where half of the enzyme is unfolded) shown in Table 11 should be close to the 50% remaining activity obtained from this Table 12. However, typically the unfolding is irreversible, and there is also a temperature dependence on the rate of lactose hydrolysis, both of which affect the decrease in activity. The Tms determined in Example 5 were approximately 57°C, 55°C, 54°C, 56°C, and 63°C for enzymes #9, 11, 30, 31, and 16, respectively (see Table 11), and there was close agreement between the Tms and 50% residual activity for enzymes #9, 11, and 31, suggesting that these enzymes have a significant amount of refolding in the assay.

[0367] The small amount (2-5%) of relative activity at higher temperatures (above 70°C) appears to remain constant, likely due to lactose hydrolysis during the temperature rise from room temperature to above 70°C (heat rise times are expected to be less than 1 min).

[0368] Example 7 Reducing agents as scavengers of oxidation of free cysteine. Enzyme #31 has two free cysteines (i.e., cysteines that are not in disulfide form) (C372 and C1199) that can potentially be oxidized, thus preventing refolding. This experiment was performed to determine whether the reducing agent dithiothreitol (DTT) could be a scavenger of this Cys oxidation by reacting with oxidized compounds present in milk. UHT treatment and measurement of residual activity (RA) were performed as described in Example 4. As shown in Table 13 below, there is a positive effect of adding 0.1 mM DTT (see last column (calculated by "RA% with DTT" / "RA% without DTT")), suggesting that the removal of oxidized compounds present in milk increases the residual activity of lactase enzymes that contain one or more free cysteines. Addition of 0.1 mM DTT has a less pronounced effect on the two variants in which cysteine ​​is replaced by different amino acids (C372A and C1199S, respectively). When comparing them with the wild type (Enzyme #31), the effect of DTT addition is less. This clearly indicates that oxidation of the "free cysteine" in Enzyme #31 affects refolding. The residual activity of the wild type (Enzyme #31) at 0.1 mM DTT is almost identical to the PE variant C372A, suggesting that C372 is the most sensitive to oxidation. DTT is not suitable for food intake, but other reducing agents approved for food (e.g. L-cysteine, sulfite, or glutathione) can be added to milk or enzyme preparations.

[0369] [Table 16]

[0370] Example 8: Lactase enzyme of SEQ ID NO:1 tested in high protein chocolate milk in an industrial scale setting 44mg ep "SEQ ID NO:1" / L was placed in a high shear mixer with 4000L of high protein chocolate milk with 4.5% lactose, 8% milk protein and 1% cocoa powder. An Elecster 10800 UHT machine was used (indirect UHT treatment), the flow rate was about 10,000L / h (90% volume) and the enzymes were added 30 minutes before the start of the UHT treatment. UHT conditions were performed with protein stabilization at 80°C for 5 minutes followed by a high heating step at 140-142°C for 5 seconds (homogenization was performed upstream at 70°C, 150 / 50bar). Samples were collected after UHT and frozen, one sample was collected after 1 hour and frozen, the remaining samples were stored at ambient temperature for 1-9 days and then frozen. All samples were analyzed for residual lactose using HPAEC-PAD as described in Example 4.

[0371] [Table 17]

[0372] Table 14 shows the residual lactose after UHT treatment (data is the average of two measurements, Avg.Dev. is the "average of absolute deviation of the data points from this average"). With the dosage used, low lactose (less than 0.1% lactose) can be claimed after 9 days at ambient temperature. The more enzyme added, the faster the lactose will be reduced accordingly.

[0373] Example 9: Further PE variants of SEQ ID NO:4 In this example, additional PE variants of SEQ ID NO:4 were tested using the same conditions as in Example 4. Samples were included that were incubated at 23° C. for 24 hours after UHT treatment and then assayed for residual activity and lactose content.

[0374] [Table 18]

[0375] [Table 19]

[0376] [Table 20]

[0377] [Table 21]

[0378] Example 10: Clade and phylogenetic tree construction GH2 phylogenetic tree A phylogenetic tree of the polypeptide sequences of the present invention containing GH2 domains was constructed as defined by CAZY (Lombard, Henrissat et al, 2014. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42: D490-5, http: / / www.cazy.org / ). The phylogenetic tree was constructed from multiple alignments of mature polypeptide sequences containing at least one GH2 domain. The sequences were aligned using the MUSCLE algorithm version 3.8.31 (Edgar, 2004. Nucleic Acids Research 32(5):1792-1797) and the tree was constructed using FastTree version 2.1.8 (Price et al., 2010, PloS one 5(3):e9490) and visualized using iTOL (Letunic & Bork, 2007. Bioinformatics 23(1):127-128).

[0379] A subset of polypeptides containing a GH2 domain also contains a glycosyl hydrolase family 2 N-terminal domain, as defined by the Pfam domain ID PF02837 (The Pfam protein families database: towards a more sustainable future: RD Finn, P. Coggill, RY Eberhardt, SR Adddy, J. Mistry, AL Mitchell, SC Otter, M. Punta, M. Qureshi, A. Sangrador-Vegas, GA Salazar, J. Tate, A. Bateman, Nucleic Acids Research (2016) Database Issue 44: D279-D285). This domain is involved in binding galactose. All polypeptides of the present invention contain a GH2 domain in addition to this glycosyl hydrolase family 2 N-terminal domain. This glycosyl hydrolase family 2 N-terminal domain is referred to as the GH2N domain. As an example, in SEQ ID NO: 4 from Bifidobacterium samirii, the GH2N domain is located at positions 34-178.

[0380] Generation of clades In addition to containing a GH2 domain, the polypeptides of the invention also contain several unique short peptide motifs that are important for lactase activity, and our data indicates that these motifs are also important for the ability of the enzyme to refold after heat treatment, such as UHT treatment.

[0381] One example is WTXXDY[I / L / R]GE[P / S / A] (SEQ ID NO: 18), which is located at a position corresponding to positions 591 to 600 in Bifidobacterium samirii (SEQ ID NO: 4).

[0382] Polypeptides containing GH2 domains can be divided into different subclusters, which are defined by one or more short sequence motifs and further contain the GH2 domain.

[0383] Generation of DYLGE clades We have assigned a subcluster containing the short peptide motif WTXXDY[I / L / R]GE[P / S / A] (SEQ ID NO: 18) to the DYLGE clade. The glutamic acid E in this motif, located at position 599 of SEQ ID NO: 4, is involved in galactose binding. All polypeptide sequences containing a GH2 domain and this motif belong to the DYLGE clade. The DYLGE clade may also contain additional short peptide motifs: SR[W / Y / F]YSGSGX[Y / G]R (SEQ ID NO: 19), located at positions 164-174 of SEQ ID NO: 4, and the motif [L / V / I]X[L / V / I]PHD (SEQ ID NO: 20), corresponding to positions 62-67 of SEQ ID NO: 4. Both motifs are located in the GH2N galactose binding domain and are important for substrate binding.

[0384] Generation of MGN clades We have named another subcluster of GH2, containing the motif EYXH[A / S / D / T]MG[N / T / L] (SEQ ID NO: 21) located at positions 532-539 of SEQ ID NO: 3, as the MGN clade. The glutamic acid residue E is completely conserved within the clade and acts as a nucleophilic catalyst in the active site of the enzyme (https: / / www.uniprot.org / uniprotkb / B3GS90 / entry).

[0385] All polypeptide sequences containing the GH2 domain and this motif are considered to belong to the MGN clade.

[0386] The MGN clade may also include one or more additional motifs. The motif [I / V]RX[A / C / S]HYP[N / P / D / Q / T / S][D / H / Q / V] (SEQ ID NO: 22) is located at positions 382-390 of SEQ ID NO: 3, similarly the motif YGG[D / N]X[G / D][E / D] (SEQ ID NO: 23) corresponds to positions 577-583 of SEQ ID NO: 3, and GXXXW[D / E][W / F / Y]X[D / E / N][Q / E / H]] (SEQ ID NO: 24) corresponds to positions 558-567 of SEQ ID NO: 3. These motifs are found near the active site of SEQ ID NO: 3 and are involved in substrate binding.

[0387] Example 11 (Comparative): Non-GH2 lactases of the DYLGE clade For example, many prior art lactases with high thermostability and / or high specific activity are not GH2 lactases of the DYLGE clade, some of which are listed in Table 18.

[0388] [Table 22]

[0389] Many bacterial GH2 family lactase enzymes of clade MGN were UHT treated in the same manner as the GH2 clade DYLGE lactase in Example 4 (Tables 7-8). The data are shown in Table 19.

[0390] [Table 23]

[0391] Little change in lactose levels was observed after 72 hours, and therefore grams of lactose converted per mg of enzyme protein after UHT treatment was also very low (calculated as (g / L lactose at 0.5 hours - g / L lactose at 72 hours) / mg ep / L, e.g., for Enzyme #40, (43.75-42.27) / 15=0.098 g lactose / mg ep). Similarly, very low residual activity was observed; for all but one (Enzyme #42), the residual activity was below the level of detection.

[0392] These data show that all of the GH2 clade MGN lactase enzymes tested survived UHT treatment less than the GH2 clade DYLGE lactase enzyme tested in Example 4, even though both GH2 clade DYLGE and GH2 clade MGN belong to the GH2 family.

Claims

1. 1. A method for producing a lactose-reduced heat-treated milk-derived product, comprising: a) adding an enzyme with lactase activity to a milk-derived substrate containing at least 2% (w / w) lactose; b) heat-treating the milk-derived substrate by holding the milk-derived substrate at a holding temperature of at least 120°C for a holding time of at least 1 second after adding the enzyme, followed by cooling to produce a heat-treated milk-derived product; and c) storing the heat-treated milk-derived product at a maximum temperature of 40°C for at least 4 days; Including, after step b) but before step c), the lactose content of the milk-derived product is at least 0.5% (w / w), and after step c), the lactose content of the milk-derived product is at most 0.2% (w / w); method.

2. 2. The method of claim 1, wherein step b) is carried out immediately after step a) without a dedicated incubation step after and before step b).

3. 10. The method of claim 1, wherein a pumping system is provided for adding the enzyme to the dairy-derived substrate while the dairy-derived substrate is flowing through process equipment.

4. 2. The method of claim 1, wherein the time from adding the enzyme to reaching the holding temperature in step b) is up to 5 minutes.

5. 2. The method of claim 1, wherein the time from adding the enzyme until the heat-treated milk-derived product has cooled to a temperature of up to 40°C is up to 3.5 minutes.

6. 2. The method of claim 1, wherein the milk-derived substrate comprises 2-30% (w / w) lactose.

7. 2. The method of claim 1, wherein the milk-derived substrate is milk containing 4 to 5.5% (w / w) lactose.

8. 10. The method of claim 1, wherein the heat treatment is an ESL treatment, an ultra-pasteurization, or an UHL treatment.

9. 2. The method of claim 1, wherein the heat treatment is a UHT treatment carried out at a temperature of 128-132°C for 25-35 seconds, or at a temperature of 138-140°C for 2-5 seconds, or at a temperature of 144-146°C for 1-2 seconds.

10. 10. The method of claim 1, wherein after the holding period of step b), the dairy-derived substrate is cooled to a maximum of 40°C within 5 minutes.

11. 10. The method of claim 1, wherein after step b) but before step c), the milk-derived product is aseptically packaged.

12. 2. The method of claim 1, wherein after step b), the enzyme retains at least 0.1% of its initial activity.

13. 2. The method of claim 1, wherein the enzyme having lactase activity comprises the motif WTXXDY[I / L / R]GE[P / S / A] in the amino acid sequence of the enzyme.

14. 2. The method according to claim 1, wherein the enzyme having lactase activity has an optimum temperature of 30 to 60°C.

15. 10. The method of claim 1, wherein the enzyme having lactase activity has a residual activity of at least 0.5% after incubation in skim milk having a lactose content of 4.7% at 90°C for 30 seconds, followed by cooling to 0-10°C, and then incubation at 23°C for 72 hours, wherein the residual activity is relative to the activity of the same enzyme in skim milk without the incubation at 90°C for 30 seconds, 140°C for 5 seconds, and 70°C for 30 seconds, followed by cooling to 0-10°C, and then incubation at 23°C for 72 hours.

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