Lactase enzymes and methods of producing milk-based products

Abstract

The present invention relates to a novel enzyme having lactase activity, a variant of said enzyme, a lactose-reduced milk-based product comprising lactase, and a method of producing a lactose-reduced heat-treated milk-based product using said lactase.

Description

[0001] References to sequence listings

[0002] This application contains a sequence listing in computer readable form, which is incorporated herein by reference. Technical Field

[0003] The present invention relates to novel enzymes having lactase activity, variants of said enzymes, methods for producing heat-treated milk-based products with reduced lactose using lactase, and reduced lactose milk-based products comprising lactase. Background Art

[0004] Most lactose-reduced or lactose-free milk-based products are produced in a batch process, whereby 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% (in most countries this allows, for example, milk to be labeled as lactose-free), followed by a heat treatment such as pasteurization, UHT, or ESL (extending shelf life to up to 35 days). Some disadvantages associated with the batch application of lactase include:

[0005] (1) Incubation times can be up to 24 h, sometimes even longer, which has raised concerns about capacity and capital investment in new incubation tanks to respond to the growing demand for lactose-free dairy products.

[0006] (2) Prolonged incubation time gives psychrophilic microorganisms the opportunity to grow and secrete their enzymes (especially proteases), some of which are thermostable and may reduce the quality of the final product during the shelf life.

[0007] (3) Severe heat treatment after lactose hydrolysis (such as in UHT applications) will cause the Maillard reaction to proceed at an accelerated rate. When lactose is hydrolyzed to glucose and galactose, the concentration of sugars with reducing ends doubles, and galactose, in particular, is much more reactive than lactose.

[0008] Recently and in particular in the case of lactose-free ESL and UHT milk beverages, advanced process engineering technology in the form of aseptic dosing equipment has attracted considerable attention and has made it possible to overcome many of the problems associated with batch processes. Such aseptic dosing equipment makes it possible to add lactase after ESL / UHT treatment. An example of such an aseptic dosing equipment is the Tetra Aldose system, Tetra Flexdose system and GEA Varidose system. The advantages of these systems are:

[0009] (1) After the heat treatment step, a relatively small amount of sterile lactase is dosed with high precision into the milk stream, allowing lactose hydrolysis to occur during the first few days of storage.

[0010] (2) Reduce the extent of Maillard reactions, browning, and advanced glycation end-product (AGE) formation in lactose-free milk beverages, especially when appropriately controlled storage conditions are applied.

[0011] (3) Compared with the batch method, the preincubation step is omitted, thus solving the capacity problem and reducing the risk of psychrophilic microbial activity.

[0012] Despite the advantages of aseptic dosing systems, there are a number of disadvantages associated with their use, such as:

[0013] (1) Since lactase is added after the heat treatment step, its formulation should have the highest possible purity. This is because any harmful side activities in its formulation may have a very negative impact on the final product during its long shelf life, especially in the case of UHT products.

[0014] (2) In Tetra In the case of the Flexdose and GEA Varidose systems, lactase should be aseptically filled in sterile drums and bags.

[0015] (3) The requirements of points (1) and (2) above mean that these sterile lactases have a higher cost per unit activity than lactases used in batch processes.

[0016] (4) The average capital cost of these aseptic dosing systems is quite high.

[0017] (5) Using Tetra When using the Flexdose and GEA Varidose systems, additional running costs are incurred related to consumables. Examples of this are the need to replace the tubing and needle every time the sterile container is changed.

[0018] (6) In Tetra In the Aldose system, the enzyme formulation is not initially sterile. The enzyme formulation is diluted with water and then filtered inline (in the milk product) using at least two filters to ensure the sterility of the enzyme stream before mixing it with the milk stream. This can be problematic because the inline filters can become clogged by the difficult-to-filter enzyme formulation, which can cause difficulties during operation. Even if the filterability of the enzyme formulation works as expected, the filters still need to be replaced regularly (usually daily).

[0019] There is currently no fast, smooth, easy to implement, trouble-free, cost-effective solution for using lactase in lactose-free UHT and ESL products that overcomes all the above limitations of batch and aseptic dosing methods.

[0020] WO 2009 / 071539 (Novozymes) relates to a method for producing a dairy product using an enzyme having lactase activity. It discloses a method for producing a low-lactose dairy product by treating a milk-based substrate with lactase at a high temperature (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).

[0021] WO 2018 / 189238 (Chr. Hansen) discloses β-galactosidases that are said to be stable over a wide range of temperatures and pH values and have relatively high activity. A method for producing a dairy product by treating a milk-based substrate with β-galactosidase is disclosed, wherein the treatment, or a portion thereof, can be performed at an elevated temperature. A lactose concentration of less than 0.2% lactose can be achieved within 3-30 minutes after addition of the β-galactosidase.

[0022] WO 2020 / 176734 (DuPont) relates to a method for reducing the amount of lactose in a milk-based substrate by contacting the substrate with a lactase, such as a thermostable lactase, at high temperature. Disclosed is a method for producing a lactose-free dairy product from a milk-based substrate using an enzyme having neutral lactase activity, wherein more than 20% of the lactase activity remains in the milk-based substrate after pasteurization at 72°C for 15 seconds. Such pasteurization is sometimes also referred to as high-temperature, short-time (HTST) pasteurization.

[0023] Deeth (2017) "Optimum Thermal Processing for Extended Shelf-Life (ESL) Milk", Foods 6(11):102 reviews the optimal thermal processing of extended shelf-life (ESL) milk. Deeth explains that ESL or ultra-pasteurized milk is produced by thermal processing using conditions that are between those used for conventional high temperature, short time (HTST) pasteurization and those used for ultra-high temperature (UHT) sterilization. The refrigerated shelf life of ESL milk should exceed 30 days. To achieve this, the thermal treatment must be quite intense. Unlike the temperature-time conditions for pasteurization (which in most countries are specified as at least 72°C for at least 15 seconds), ESL processing generally does not have such specified conditions. According to Deeth (2017), the reported commercial processing conditions for ESL milk are mainly in the range of 123°C-127°C for 1-5 seconds. US regulations define the method of "ultra-pasteurization" as heating milk to a temperature of at least 138°C for at least 2 seconds.

[0024] European Patent Application No. 21216998.1 discloses a method for producing a heat-treated dairy-based product (e.g., milk) with reduced lactose using an enzyme having lactase activity without the need for extensive pre-incubation of the dairy-based substrate with the enzyme and without the need for adding the enzyme using a sterile dosing system after heat treatment. In this method, after heat treatment (such as UHT treatment), the enzyme has some residual activity, which ensures that lactose is degraded to the desired low lactose level during storage at low or ambient temperatures.

[0025] The UHT treatment may be, for example, heat treatment at 130° C. for 30 seconds, heat treatment at 140° C. for 3-4 seconds, or heat treatment at 145° C. for 1 second.

[0026] There remains a need for improved methods for producing heat-treated milk-based products with reduced lactose, such as UHT or ESL milk, and for lactases suitable for use in such methods. Summary of the Invention

[0027] The present invention provides novel enzymes having lactase activity and variants thereof, as well as milk-based products comprising such enzymes, wherein the lactase has sufficient residual enzyme activity after heat treatment (such as UHT or ESL treatment) for the production of milk-based products with reduced lactose. This avoids the need for extensive pre-incubation of the milk-based substrate with the enzyme and the need to add the enzyme using a sterile dosing system after heat treatment. The present invention further provides methods for producing milk-based products using the lactase.

[0028] Accordingly, one aspect of the invention relates to a milk-based product comprising an enzyme having lactase activity, wherein the enzyme having lactase activity comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13.

[0029] Another aspect of the invention relates to lactase itself and variants thereof.

[0030] Another aspect of the present invention relates to a method for producing a heat-treated dairy-based product with reduced lactose, the method comprising:

[0031] a) adding an enzyme having lactase activity to a milk-based substrate comprising at least 2% lactose (w / w), wherein the enzyme having lactase activity comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13,

[0032] b) after adding the enzyme, heat treating the dairy-based substrate by holding the dairy-based substrate at a holding temperature of at least 120° C. for a holding time of at least 1 second, followed by cooling to produce a heat-treated dairy-based product, and

[0033] c) storing the heat-treated dairy-based product at a temperature of at most 40° C. for at least about 24 hours, preferably at least 2 days, such as at least 3 days, preferably at least 4 days,

[0034] wherein after step c), the lactose content of the milk-based product is at most 0.2% (w / w).

[0035] Preferably, the lactose content of the milk-based product after step b) but before step c) is at least 0.5% (w / w), preferably at least 1% (w / w).

[0036] Another aspect of the present invention relates to a method for producing a reduced lactose milk product, the method comprising adding an enzyme having lactase activity to a milk-based substrate comprising at least 2% lactose (w / w), and subjecting the milk-based substrate comprising the enzyme to a heat treatment at a temperature of at least 120°C for a holding time of at least 1 second, wherein the enzyme having lactase activity comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13.

[0037] A still further aspect of the invention relates to the use of an enzyme having lactase activity in a method for producing a heat-treated milk-based product with reduced lactose, wherein a milk-based substrate comprising at least 2% lactose (w / w) to which an enzyme has been added is heat-treated at a temperature of at least 120°C for a holding time of at least 1 second, wherein the enzyme having lactase activity comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13.

[0038] Preferably, the enzyme with lactase activity is added immediately prior to heat treatment (such as UHT treatment). After heat treatment (such as UHT treatment), the enzyme has some residual activity, which ensures that lactose is degraded to the desired low lactose level during storage at low or ambient temperatures. Preferably, the enzyme has an optimum temperature of 30°C-60°C, more preferably 35°C-55°C. Without wishing to be bound by theory, it is believed that enzymes with higher optimum temperatures and possibly even active during heat treatment will have a somewhat rigid structure and will not have sufficient activity during storage at low or ambient temperatures. Likewise, without wishing to be bound by theory, it is believed that enzymes with an optimum temperature of 30°C-60°C, more preferably 35°C-55°C used in the method of the present invention may be unfolded and inactive during heat treatment, but then have the ability to refold and reactivate, and therefore have a measurable or even substantial residual activity, ensuring lactose degradation during storage of the dairy-based product. It is expected that the high residual activity of the lactase of the present invention after heat treatment (such as UHT treatment) may be due to the ability to refold and reactivate once the temperature is lowered.

[0039] Preferably, the enzyme 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.

[0040] More 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%, even more preferably at least 10% in skim milk with a lactose content of 4.7% after 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 subsequently incubating 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 subsequently incubating at 23°C for 72 hours.

[0041] Preferably, the milk-based substrate is not incubated with the lactase prior to heat treatment, except for the time required from the addition of the lactase to the milk-based substrate to the time the holding temperature for heat treatment is reached (depending on the process equipment). Preferably, step b) is performed immediately after step a), without requiring a dedicated incubation step between steps a) and b).

[0042] At least three main process options are available under this approach:

[0043] In the first option, the milk-based substrate can be mixed with the lactase and then processed directly under UHT or ESL conditions without incubating the milk-based substrate with the lactase.

[0044] In a second option, the milk-based substrate can be treated directly under UHT or ESL conditions, wherein lactase is added to the milk-based substrate as it flows through the process piping, optionally as the temperature of the milk-based substrate increases towards the temperature of the heat treatment step, and then immediately subjected to the heat treatment step. Lactase can be added at any point in time just before UHT / ESL treatment when the temperature of the milk-based substrate is between 1°C and 95°C, preferably between 70°C and 90°C. The addition of lactase can be carried out via a simple dosing pump through a tube connected to the main milk flow pipe. Once added to the flowing milk-based stream, the temperature is (further) raised to ESL or UHT treatment conditions. A heating medium can be used that is not in direct contact with the milk-based substrate but is separated by equipment contact surfaces, such as a plate heat exchanger or a tubular heat exchanger. This can be referred to as indirect heat treatment, preferably indirect UHT treatment.

[0045] In a third option, the dairy-based substrate is heated using high-pressure steam by steam injection or steam injection, preferably steam injection, and the enzyme can be added together with the steam. This can be referred to as direct heat treatment, preferably direct UHT treatment. After the hold time in step b), the steam-containing dairy-based substrate can be rapidly cooled in a vacuum to remove water equivalent to the amount of condensed steam used. In addition to heating the dairy-based substrate by steam injection or steam injection, indirect heating can also be applied, for example using a plate or tube heat exchanger.

[0046] In any case, the residual activity of the lactase after heat treatment ensures that the lactose level will be reduced to a lactose-reduced level, preferably a lactose-free level (e.g., less than 0.1% or less than 0.01%) during the initial period of storage (e.g., up to 2 weeks, e.g., during the first 2 or 3 days).

[0047] The process of the present invention has many advantages over today's processes for producing ESL and UHT treated milk based products such as ESL or UHT milk.

[0048] Compared to currently used batch processes, the method of the present invention provides improved color and quality of lactose-reduced or lactose-free dairy-based products due to the reduction of the Maillard reaction. Without pre-incubation, the growth of psychrophilic bacteria that secrete enzymes (including proteases) that may survive heat treatment is reduced, and thus the dairy-based product can have a longer shelf life. In addition, without pre-incubation, production capacity costs and process time are reduced. The method of the present invention is also easier to operate because there is no need to monitor the tank until the lactose level is below 0.1% or 0.01%.

[0049] Compared to currently used sterile dosing methods, the method using the lactase of the present invention does not require investment in sterile dosing equipment and, for example, regular replacement of sterile barrels. Furthermore, while currently available sterile lactase has a shelf life of approximately one year, the lactase used in the method of the present invention can have a shelf life of two years or longer. The method of the present invention is easy to operate and troubleshoots, as no sterile dosing system needs to be monitored or troubleshooted. The final quality of the dairy-based product, in terms of color and Maillard reaction, is the same.

[0050] The method using the lactase of the present invention is easily applicable on an industrial scale and has no implementation barriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Shown is an example of a dose-response curve depicting the residual lactose content in milk as a function of lactase concentration.

[0052] Sequence Overview

[0053] SEQ ID NO: 1 is a C-terminally truncated lactase from Bifidobacterium samirii.

[0054] SEQ ID NO: 2 is a C-terminally truncated lactase from Streptococcus entericus DSM 14446.

[0055] SEQ ID NO: 3 is a C-terminally truncated lactase from Varibaculum sp.

[0056] SEQ ID NO: 4 is a C-terminally truncated lactase from Urmitella timonensis.

[0057] SEQ ID NO: 5 is a C-terminally truncated lactase from Bifidobacterium bifidum.

[0058] SEQ ID NO: 6 is a C-terminally truncated lactase from Bacillus sp. S3.

[0059] SEQ ID NO: 7 is a C-terminally truncated lactase from Bifidobacterium aerophilum.

[0060] SEQ ID NO: 8 is a C-terminally truncated lactase from Bifidobacterium mongoliense.

[0061] SEQ ID NO: 9 is a C-terminally truncated lactase from Clostridium nexile CAG: 348.

[0062] SEQ ID NO: 10 is a C-terminally truncated lactase from Neobacillus bataviensis.

[0063] SEQ ID NO: 11 is a C-terminally truncated lactase from Neobacillus mesonae.

[0064] SEQ ID NO: 12 is a C-terminally truncated lactase from Neobacillus niacin.

[0065] SEQ ID NO: 13 is a C-terminally truncated lactase from Streptomyces cirratus.

[0066] SEQ ID NO: 14 is the secretion signal from Bacillus clausii.

[0067] SEQ ID NO: 15 is a polyhistidine tag.

[0068] definition

[0069] In light of this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0070] Unless defined otherwise or clearly indicated by the 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.

[0071] Isolated: The term "isolated" means a polypeptide, nucleic acid, cell, or other specified material or component that has been separated from at least one other material or component (including, but not limited to, other proteins, nucleic acids, cells, etc.). Thus, an isolated polypeptide, nucleic acid, cell, or other material is in a form not found in nature. An isolated polypeptide includes, but is not limited to, a culture medium containing a secreted polypeptide expressed in a host cell.

[0072] Lactase: The term "lactase" means a glycoside hydrolase that has the ability to hydrolyze the disaccharide lactose into its component galactose and glucose monomers. The group of lactases includes, but is not limited to, enzymes designated as subclass EC 3.2.1.108. Enzymes designated as other subclasses (such as, for example, EC 3.2.1.23 or 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 other activities in addition to lactose hydrolysis activity, such as, for example, 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 β-galactosidase activity, or its hydrolytic activity.

[0073] Lactase activity: Lactase activity can be determined using, for example, the LAU(B) assay. The activity of a particular lactase in LAU(B) units 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 MgSO47H2O, 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 can be obtained by comparing a standard curve run with a lactase of known activity to the activity of the unknown sample calculated therefrom. The lactase of known activity can be, for example, obtained from Novozymes A / S, Denmark. Lactase activity can also be determined by measuring the amount of lactose hydrolyzed in milk, for example using HPAEC-PAD, as described in Example 1 in the section "Analysis of residual lactose content," where the lactose peak is correlated with a lactose standard of known concentration. Lactose hydrolysis can then be correlated with the amount of lactase added (e.g., per mg of enzyme protein or per mole of enzyme). Other methods for measuring lactase activity are known in the art and are routinely used in the art.

[0074] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its mature form following N-terminal and / or C-terminal processing (eg, removal of a signal peptide).

[0075] Milk: The term "milk" means the milky secretion obtained by milking any mammal such as cows, sheep, goats, buffaloes or camels.

[0076] Dairy-based products: The term "dairy-based products" refers to dairy products and other products based on milk. In the context of the present invention, it will be apparent that the term particularly includes heat-treated dairy-based products, including not only pasteurized milk, but also milk subjected to temperatures higher than those typically used for pasteurization, such as ultra-pasteurized milk, UHT (Ultra High Temperature) milk and ESL (Extended Shelf Life) milk.

[0077] Purified: The term "purified" means a nucleic acid, polypeptide, or cell that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid can form discrete bands in an electrophoretic gel, a chromatography eluate, and / or a culture medium subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, typically 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., percentages by weight or molar). In a related sense, a composition is enriched for a molecule when the concentration of the molecule is substantially increased following application of a purification or enrichment technique. The term "enriched" refers to the presence of a compound, polypeptide, cell, nucleic acid, amino acid or other designated material or component in a composition at a relative or absolute concentration greater than that of the starting composition.

[0078] In one aspect, the term "purified" as used herein refers to a polypeptide or cell that is substantially free of components (especially insoluble components) from the producing organism. In other aspects, the term "purified" refers to a polypeptide that is substantially free of insoluble components (especially insoluble components) from the native organism from which it was obtained. In one aspect, the polypeptide is separated from some soluble components of the organism and culture medium from which it was recovered. The polypeptide can be purified (i.e., isolated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0079] Accordingly, the polypeptide can be purified so that only small amounts of other proteins, particularly other polypeptides, are present. As used herein, the term "purified" can refer to the removal of other components present in the cell from which the polypeptide originates, particularly other proteins and most particularly other enzymes. A polypeptide can be "substantially pure," i.e., free of other components from the organism from which it is produced (e.g., a host organism for recombinant production of the polypeptide). In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" can refer to a polypeptide preparation containing at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide materials with which the polypeptide is natively or recombinantly associated.

[0080] Thus, it is preferred that a substantially pure polypeptide is 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, based on the weight of the total polypeptide material present in the preparation. The polypeptides of the present invention are preferably in substantially pure form (i.e., the preparation is substantially free of other polypeptide material with which it is naturally or recombinantly associated). This can be achieved, for example, by preparing the polypeptide using well-known recombinant methods or using classical purification methods.

[0081] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0082] For the purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of "longest identity" using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (preferably version 6.6.0 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. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of the "longest identity" of the Needle tag is calculated as follows:

[0083] (Identical residues x 100) / (Alignment length - Total number of gaps in the alignment)

[0084] Variant: The term "variant" refers to a polypeptide having lactase activity that comprises an artificial mutation (i.e., substitution, insertion (including extension) and / or deletion (e.g., truncation)) at one or more positions. Substitution means replacing the amino acid occupying a certain position with a different amino acid; deletion means removing the amino acid occupying a certain position; and insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, particularly 1 amino acid) adjacent to and immediately following the amino acid occupying a certain position.

[0085] Wild-type: When referring to an amino acid sequence or a nucleic acid sequence, the term "wild-type" means that the amino acid sequence or nucleic acid sequence is a native or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance (e.g., a protein, amino acid or nucleic acid sequence) found in nature. In contrast, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in the laboratory, or modifications of a wild-type sequence). However, as explained below, the enzyme having lactase activity of the present invention is preferably C-terminally truncated compared to the full-length wild-type enzyme from which it is obtained, i.e., truncated compared to the full-length amino acid sequence encoded by the complete genomic DNA sequence.

[0086] Variant naming conventions

[0087] For the purposes of the present invention, a polypeptide having a selected wild-type sequence can be used to determine the corresponding amino acid position in another lactase. The amino acid sequence of the other lactase is aligned with the polypeptide having the selected wild-type sequence, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide having the selected wild-type sequence is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (preferably version 5.0.0 or later). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0088] When describing the variants of the present invention, the nomenclature described below has been adjusted for ease of reference. The generally accepted IUPAC single-letter amino acid abbreviations are used. The amino acid numbering of the variants disclosed herein is in each case based on the numbering of the relevant wild-type sequence.

[0089] For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 by alanine is represented as "T226A." Multiple mutations are separated by a plus sign ("+"), a comma, or simply by a space. For example, the substitution of glycine (G) at position 205 and serine (S) at position 411 by arginine (R) and phenylalanine (F), respectively, can be represented as "G205R+S411F" or "G205R S411F." DETAILED DESCRIPTION

[0090] As described above, the present invention provides a milk-based product comprising an enzyme having lactase activity, wherein the enzyme having lactase activity comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13. Preferably, the enzyme has at least 70%, at least 75% or at least 80% sequence identity to any of said sequences.

[0091] The present invention also provides an enzyme having lactase activity, wherein the enzyme having lactase activity comprises an amino acid sequence having at least 60%, such as at least 70%, at least 75% or at least 80% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13.

[0092] Preferably, the dairy based product is a product that has been heat treated and has a lactose content of at most 0.2% (w / w).The lactose content may for example be at most 0.1% or at most 0.01% (w / w).

[0093] Preferably, the milk-based product is UHT milk, ESL milk or ultra-pasteurized milk.

[0094] The enzyme having lactase activity may, for example, comprise a nucleic acid sequence corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13, or a nucleic acid sequence corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13. The mature polypeptide of any one of NO:13 has an amino acid sequence of at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.

[0095] In a preferred embodiment, the enzyme having lactase activity comprises an amino acid sequence having at least 70% sequence identity, preferably at least 75% sequence identity, such as at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or to the mature polypeptide of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0096] The enzyme having lactase activity can be, for example, a variant of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13, the variant comprising one or more alterations, such as substitutions, deletions and / or insertions, typically substitutions. In a preferred embodiment, the enzyme is a variant of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. In some more preferred embodiments, the enzyme is a variant of SEQ ID NO: 1.

[0097] In one embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 1.

[0098] In one embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 2.

[0099] In one embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 3.

[0100] In one embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO:4.

[0101] In one embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO:6.

[0102] In one embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO:7.

[0103] In one embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 8.

[0104] In one embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO:9.

[0105] In one embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 10.

[0106] In one embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 11.

[0107] In one embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 12.

[0108] In one embodiment, the enzyme having lactase activity has an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 13.

[0109] In some preferred embodiments, the enzyme having lactase activity is based on any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13, or SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13. The polypeptide of any one of the mature polypeptides in NO:13, this polypeptide has 1-30 change in one or more positions, for example replaces, disappearance and / or insertion, for example 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 changes, particularly replaces.In the context of the present invention, "based on " means that enzyme can be derived from any wild-type sequence of listing with one or more changes, and / or can be an enzyme (in the preferred restriction of sequence length disclosed elsewhere herein) with an amino acid sequence longer than one of the wild-type sequences listed, for example, extended by the C-terminus.

[0110] In one embodiment, the enzyme having lactase activity is a polypeptide derived from or based on SEQ ID NO: 1 or derived from the mature polypeptide of SEQ ID NO: 1, which polypeptide has 1-30 alterations, e.g. substitutions, deletions and / or insertions, typically substitutions, e.g. 1-20 alterations, such as 1-15 alterations, at one or more positions.

[0111] In a preferred embodiment, the enzyme having lactase activity is a variant of SEQ ID NO: 1 having one or more substitutions selected from the group consisting of P65A, C372A, P615T, A1073C, H1122C, and C1195G, such as two, three, four or more of said substitutions. Some non-limiting examples of such substitution combinations in SEQ ID NO: 1 are A1073C + H1122C, C372A + A1073C + H1122C, C372A + C1195G, A1073C + H1122C + C1195G, and C372A + A1073C + H1122C + C1195G. The variant of this embodiment can have an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 1.

[0112] In one embodiment, the enzyme having lactase activity is a polypeptide derived from or based on SEQ ID NO: 2 or derived from the mature polypeptide of SEQ ID NO: 2, which polypeptide has 1-30 alterations, e.g., substitutions, deletions and / or insertions, typically substitutions, e.g., 1-20 alterations, such as 1-15 alterations, at one or more positions.

[0113] In a preferred embodiment, the enzyme having lactase activity is a variant of SEQ ID NO: 2 having one or more substitutions selected from the group consisting of P52A, G607T, L1064C and Y1110C, for example two or more of said substitutions, such as L1064C+Y1110C. The variant of this embodiment may have an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 2.

[0114] In one embodiment, the enzyme having lactase activity is a polypeptide derived from or based on SEQ ID NO: 3 or derived from the mature polypeptide of SEQ ID NO: 3, which polypeptide has 1-30 alterations, e.g., substitutions, deletions and / or insertions, typically substitutions, e.g., 1-20 alterations, such as 1-15 alterations, at one or more positions.

[0115] In one embodiment, the enzyme having lactase activity is a polypeptide derived from or based on SEQ ID NO: 4 or derived from the mature polypeptide of SEQ ID NO: 4, which polypeptide has 1-30 alterations, e.g., substitutions, deletions and / or insertions, typically substitutions, e.g., 1-20 alterations, such as 1-15 alterations, at one or more positions.

[0116] In a preferred embodiment, the enzyme having lactase activity is a variant of SEQ ID NO: 4 having the substitutions G386Q and / or P620T. The variant of this embodiment may have an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 4.

[0117] In one embodiment, the enzyme having lactase activity is a polypeptide derived from or based on SEQ ID NO: 6 or derived from the mature polypeptide of SEQ ID NO: 6, which polypeptide has 1-30 alterations, e.g., substitutions, deletions and / or insertions, typically substitutions, e.g., 1-20 alterations, such as 1-15 alterations, at one or more positions.

[0118] In one embodiment, the enzyme having lactase activity is a polypeptide derived from or based on SEQ ID NO: 7 or derived from the mature polypeptide of SEQ ID NO: 7, which polypeptide has 1-30 alterations, e.g., substitutions, deletions and / or insertions, typically substitutions, e.g., 1-20 alterations, such as 1-15 alterations, at one or more positions.

[0119] In one embodiment the enzyme having lactase activity is a polypeptide derived from or based on SEQ ID NO: 8 or derived from the mature polypeptide of SEQ ID NO: 8, which polypeptide has 1-30 alterations, e.g. substitutions, deletions and / or insertions, typically substitutions, e.g. 1-20 alterations, such as 1-15 alterations, at one or more positions.

[0120] In one embodiment, the enzyme having lactase activity is a polypeptide derived from or based on SEQ ID NO: 9 or derived from the mature polypeptide of SEQ ID NO: 9, which polypeptide has 1-30 alterations, e.g., substitutions, deletions and / or insertions, typically substitutions, e.g., 1-20 alterations, such as 1-15 alterations, at one or more positions.

[0121] In one embodiment, the enzyme having lactase activity is a polypeptide derived from or based on SEQ ID NO: 10 or derived from the mature polypeptide of SEQ ID NO: 10, which polypeptide has 1-30 alterations, e.g., substitutions, deletions and / or insertions, typically substitutions, e.g., 1-20 alterations, such as 1-15 alterations, at one or more positions.

[0122] In one embodiment, the enzyme having lactase activity is a polypeptide derived from or based on SEQ ID NO: 11 or derived from the mature polypeptide of SEQ ID NO: 11, which polypeptide has 1-30 alterations, e.g., substitutions, deletions and / or insertions, typically substitutions, e.g., 1-20 alterations, such as 1-15 alterations, at one or more positions.

[0123] In one embodiment, the enzyme having lactase activity is a polypeptide derived from or based on SEQ ID NO: 12 or derived from the mature polypeptide of SEQ ID NO: 12, which polypeptide has 1-30 alterations, e.g., substitutions, deletions and / or insertions, typically substitutions, e.g., 1-20 alterations, such as 1-15 alterations, at one or more positions.

[0124] In one embodiment, the enzyme having lactase activity is a polypeptide derived from or based on SEQ ID NO: 13 or derived from the mature polypeptide of SEQ ID NO: 13, which polypeptide has 1-30 alterations, e.g., substitutions, deletions and / or insertions, typically substitutions, e.g., 1-20 alterations, such as 1-15 alterations, at one or more positions.

[0125] In one embodiment, the number of amino acid substitutions, deletions and / or insertions introduced into any one of the polypeptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, 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 or SEQ ID NO: 13 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0126] The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions of typically 1-30 amino acids; small amino-terminal 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 tag, antigenic epitope, or binding module.

[0127] 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, and the resulting molecules are tested for lactase activity to identify amino acid residues that are critical for the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. Active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutating putative contact site amino acids. See, for example, 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. The identity of essential amino acids can also be inferred from alignments with related polypeptides and / or from sequence homology and conserved catalytic mechanisms with related polypeptides or polypeptides / proteins within a family of polypeptides or proteins derived from a common ancestor (typically having 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 a polypeptide. See, e.g., Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold,” Nature 596:583-589.

[0128] Single or multiple amino acid substitutions, deletions and / or insertions can be made and tested using known mutagenesis, recombination and / or shuffling methods, followed by relevant screening procedures, such as those disclosed by 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 can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; US 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0129] 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 encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0130] Amino acid changes can also be non-conservative changes, such as non-conservative substitutions, deletions and / or insertions, typically substitutions, which provide one or more desired characteristics for the enzyme. Non-limiting examples of such desired characteristics include improved performance, increased residual activity after exposure to elevated temperatures, increased refolding capacity after denaturation, increased specific activity, and reduced oxidative sensitivity.

[0131] In a preferred embodiment, the enzyme having lactase activity is derived from or based on any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, 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 or SEQ ID NO: 13 by substitution of 1-30, preferably 1-20 amino acids, such that the enzyme has higher residual activity after UHT treatment compared to the wild-type enzyme from which it is derived.

[0132] Preferably, the enzyme having lactase activity is a bacterial enzyme, i.e., an enzyme obtained from a bacterial source or a variant of an enzyme obtained from a bacterial source.

[0133] In a preferred embodiment, the enzyme having lactase activity is obtained from the genus Bifidobacterium, preferably Bifidobacterium samirii, Bifidobacterium aerophilum or Bifidobacterium mongoliense, or from the genus Bacillus, preferably Bacillus species S3, obtained from Campylobacter species, obtained from Urmitella, preferably Urmitellatimonensis, obtained from the genus Streptococcus, preferably Streptococcus intestinalis DSM 14446 or Streptomyces cirratus, obtained from the genus Clostridium, preferably Clostridium nexile CAG:348, or from the genus Neobacillus, preferably Neobacillus bataviensis, Neobacillus mesonae or Neobacillus niacini, or is a variant of an enzyme obtained from the genera or species listed above.

[0134] The above probes can be used to identify and obtain the enzyme from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples directly obtained from natural materials (e.g., soil, compost, water, etc.). Techniques for directly isolating microorganisms and DNA from natural habitats are well known in the art. Then, the polynucleotide encoding the enzyme can be obtained by similarly screening the genomic DNA or cDNA library of another microorganism or a mixed DNA sample. Once the polynucleotide encoding the enzyme has been detected with the probe, the polynucleotide can be isolated or cloned by using techniques known to those of ordinary skill in the art (see, for example, Davis et al., 2012, Basic Methods in Molecular Biology, Elsevier).

[0135] The enzyme having lactase activity may be a polypeptide derived from any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, 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 or SEQ ID NO: 13, or derived from the mature polypeptide of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, 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 or SEQ ID NO: 13, wherein the N-terminus and / or C-terminus has been extended by the addition of one or more amino acids, or wherein one or more amino acids have been deleted from the N-terminus and / or C-terminus.

[0136] The enzyme having lactase activity is preferably C-terminally truncated compared to the full-length wild-type enzyme from which it is obtained, ie the enzyme is encoded by a genomic DNA sequence.

[0137] Thus, the enzyme having lactase activity of the present invention may be derived from any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, 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 or SEQ ID NO: 13 or derived from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, 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 or SEQ ID NO: 12. NO:12, wherein the polypeptide 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, for example 850-1500 amino acids, preferably 850-1400 amino acids, such as 850-1350 amino acids; or derived from SEQ ID NO:13, wherein the polypeptide has a length of up to about 1750 amino acids.

[0138] In a preferred embodiment, the enzyme having lactase activity is obtained from Bifidobacterium, preferably Bifidobacterium samieri, Bifidobacterium aerophilum or Bifidobacterium mongolicum, or from Bacillus, preferably Bacillus species S3, from Curvularia species, from Urmitella, preferably Urmitella timonensis, from Streptococcus, preferably Streptococcus enterica DSM 14446, obtained from Clostridium, preferably Clostridium knottys CAG:348, or from Bacillus novogenes, preferably Bacillus novogenes batavia, Bacillus novogenes herbes or Bacillus novogenes nicotinoviceps, or is a variant of an enzyme having lactase activity obtained from any of these genera or species, and which has been 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, for example, 850-1500 amino acids, preferably 850-1400 amino acids, such as 850-1350 amino acids, more preferably 880-1350, 880-1330, 880-1320 or 885-1310 amino acids.

[0139] In the case of the lactase obtained from Streptomyces tenuissima, it may have a length of up to about 1750 amino acids, such as up to about 1700 amino acids, for example up to about 1680 amino acids.

[0140] In one embodiment, the enzyme having lactase activity is obtained from Bifidobacterium samiyi, or is a variant of an enzyme having lactase activity obtained from Bifidobacterium samiyi, and has a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1280-1320 amino acids, such as 1290-1310 amino acids. The enzyme can, for example, be derived from the polypeptide of SEQ ID NO: 1 and have a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1280-1320 amino acids, such as 1290-1310 amino acids.

[0141] In one embodiment, the enzyme having lactase activity is obtained from Streptococcus enterica DSM 14446, or is a variant of an enzyme having lactase activity obtained from Streptococcus enterica DSM 14446, and has a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1270-1310 amino acids, such as 1280-1300 amino acids. The enzyme can, for example, be derived from the polypeptide of SEQ ID NO: 2, and have a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1270-1310 amino acids, such as 1280-1300 amino acids.

[0142] In one embodiment, the enzyme having lactase activity is obtained from a species of C. minor, or is a variant of an enzyme having lactase activity obtained from a species of C. minor, and has a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1290-1330 amino acids, such as 1300- 1320 amino acids. The enzyme can, for example, be derived from the polypeptide of SEQ ID NO: 3, and have a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1290-1330 amino acids, such as 1300-1320 amino acids.

[0143] In one embodiment, the enzyme having lactase activity is obtained from Urmitella timonensis, or is a variant of an enzyme having lactase activity obtained from Urmitella timonensis, and has a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1290-1330 amino acids, such as 1300- 1320 amino acids. The enzyme can, for example, be derived from the polypeptide of SEQ ID NO: 4, and have a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1290-1330 amino acids, such as 1300-1320 amino acids.

[0144] In one embodiment, the enzyme having lactase activity is obtained from Bacillus sp. S3, or is a variant of an enzyme having lactase activity obtained from Bacillus sp. S3, and has a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1290-1330 amino acids, such as 1300- 1320 amino acids. The enzyme can, for example, be derived from the polypeptide of SEQ ID NO: 6, and have a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1290-1330 amino acids, such as 1300-1320 amino acids.

[0145] In one embodiment, the enzyme having lactase activity is obtained from Bifidobacterium aerobicum, or is a variant of an enzyme having lactase activity obtained from Bifidobacterium aerobicum, and has a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1280-1320 amino acids, such as 1290-1310 amino acids. The enzyme can, for example, be derived from the polypeptide of SEQ ID NO: 7, and have a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1280-1320 amino acids, such as 1290-1310 amino acids.

[0146] In one embodiment, the enzyme having lactase activity is obtained from Bifidobacterium mongolicum, or is a variant of an enzyme having lactase activity obtained from Bifidobacterium mongolicum, and has a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1280-1320 amino acids, such as 1290-1310 amino acids. The enzyme can, for example, be derived from the polypeptide of SEQ ID NO: 8, and have a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1280-1320 amino acids, such as 1290-1310 amino acids.

[0147] In one embodiment, the enzyme having lactase activity is obtained from Clostridium agglutinans CAG:348, or is a variant of an enzyme having lactase activity obtained from Clostridium agglutinans CAG:348, and has a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1270-1310 amino acids, such as 1280-1300 amino acids. The enzyme can, for example, be derived from the polypeptide of SEQ ID NO:9 and have a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1270-1310 amino acids, such as 1280-1300 amino acids.

[0148] In one embodiment, the enzyme having lactase activity is obtained from Bacillus novogenes batavia, or is a variant of an enzyme having lactase activity obtained from Bacillus novogenes batavia, and has a length of 800-1400 amino acids, preferably 1000-1200 amino acids, more preferably 1100-1200 or 1110-1150 amino acids, such as 1120-1140 amino acids. The enzyme can, for example, be derived from the polypeptide of SEQ ID NO: 10, and have a length of 800-1400 amino acids, preferably 1000-1200 amino acids, more preferably 1100-1200 or 1110-1150 amino acids, such as 1120-1140 amino acids.

[0149] In one embodiment, the enzyme having lactase activity is obtained from Bacillus aurantii, or is a variant of an enzyme having lactase activity obtained from Bacillus aurantii, and has a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1290-1330 amino acids, such as 1300-1320 amino acids. The enzyme can, for example, be derived from the polypeptide of SEQ ID NO: 11 and have a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1290-1330 amino acids, such as 1300-1320 amino acids.

[0150] In one embodiment, the enzyme having lactase activity is obtained from Bacillus nicotinonovae, or is a variant of an enzyme having lactase activity obtained from Bacillus nicotinonovae, and has a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1290-1330 amino acids, such as 1300-1320 amino acids. The enzyme can, for example, be derived from the polypeptide of SEQ ID NO: 12, and have a length of 850-1500 amino acids, preferably 1250-1400 amino acids, more preferably 1250-1350 or 1290-1330 amino acids, such as 1300-1320 amino acids.

[0151] In one embodiment, the enzyme having lactase activity is obtained from Streptomyces circinelloides, or is a variant of an enzyme having lactase activity obtained from Streptomyces circinelloides, and has a length of 1200-1900 amino acids, preferably 1600-1800 amino acids, more preferably 1650-1750 or 1660-1700 amino acids, such as 1670-1690 amino acids. The enzyme can, for example, be derived from the polypeptide of SEQ ID NO: 13 and have a length of 1200-1900 amino acids, preferably 1600-1800 amino acids, more preferably 1650-1750 or 1660-1700 amino acids, such as 1670-1690 amino acids.

[0152] The present invention further provides a method for producing a heat-treated milk-based product with reduced lactose, the method comprising:

[0153] a) adding an enzyme having lactase activity to a milk-based substrate comprising at least 2% lactose (w / w), wherein the enzyme having lactase activity comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13,

[0154] b) after adding the enzyme, heat treating the dairy-based substrate by holding the dairy-based substrate at a holding temperature of at least 120° C. for a holding time of at least 1 second, followed by cooling to produce a heat-treated dairy-based product, and

[0155] c) storing the heat-treated dairy-based product at a temperature of at most 40° C. for at least about 24 hours, preferably at least 2 days, such as at least 3 days, preferably at least 4 days,

[0156] wherein after step c), the lactose content of the milk-based product is at most 0.2% (w / w).

[0157] Preferably, the lactose content of the milk-based product after step b) but before step c) is at least 0.5% (w / w), preferably at least 1% (w / w).

[0158] The enzyme having lactase activity used in the method can, for example, comprise the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13, or the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13. The mature polypeptide of any one of NO:13 has an amino acid sequence of at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.

[0159] In a preferred embodiment, the enzyme having lactase activity used in the method comprises an amino acid sequence having at least 70% sequence identity, preferably at least 75% sequence identity, such as at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or to the mature polypeptide of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0160] It will be apparent that the lactase used in the method of the invention may be selected from any of the lactases or variants described above and in more detail elsewhere herein.

[0161] The milk-based substrate preferably comprises 2%-30%, preferably 2%-17% (w / w), more preferably 4%-5.5% (w / w) lactose.

[0162] The dairy substrate may be any raw dairy material and / or processed dairy material. Useful dairy substrates include, but are not limited to, solutions / suspensions of any milk or dairy-like product containing lactose, such as whole or low-fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, solutions of dried milk, milk containing skim milk powder, milk permeate, whey, whey permeate, acid whey, cream, or flavored milk (such as chocolate milk).

[0163] The milk-based substrate may be milk, such as raw milk, for example raw milk that has not been pasteurized prior to step a).

[0164] In a preferred embodiment, the dairy-based substrate is milk, condensed milk or milk containing skimmed milk powder.

[0165] In a more preferred embodiment, the milk-based substrate is milk comprising 4%-5.5%, preferably 4.5%-5% lactose (w / w).

[0166] In one embodiment, the milk-based substrate is raw milk, preferably raw milk that has not been pasteurized prior to step a).

[0167] In one embodiment, after step a) but before step b), the milk-based substrate is incubated for at most 4 hours, preferably at most 60 minutes, more preferably at most 10 minutes, even more preferably at most 5 minutes.Such incubation may be carried out at a temperature of at most 10°C, preferably at most 7°C.

[0168] However, preferably, step b) is performed immediately after step a) without requiring a dedicated incubation step between step a) and step b).

[0169] Preferably, the time from addition of the enzyme until the holding temperature of step b) is reached is at most 5 minutes, more preferably at most 2 minutes, even more preferably at most 1 minute.

[0170] In a preferred embodiment, the heat treatment is performed as an indirect heat treatment, preferably an indirect UHT treatment. Pumping equipment may be installed to add the enzyme to the milk-based substrate as it flows through process equipment, such as process piping. A heating medium that is not in direct contact with the milk-based substrate but is separated from it by equipment contact surfaces, such as a plate heat exchanger or a tubular heat exchanger, may be used. Preferably, the enzyme is added to the milk-based substrate immediately before the heat treatment step (such as immediately before reaching the holding temperature), optionally as the temperature of the milk-based substrate increases towards the holding temperature of step b).

[0171] In another preferred embodiment, the heat treatment is performed as a direct heat treatment, preferably a direct UHT treatment. The heat treatment can be performed using high-pressure steam by steam injection or steam injection, preferably steam injection, to heat the dairy-based substrate. In a preferred embodiment, the enzyme is added together with the steam. Preferably, after the holding time in step b), the dairy-based substrate containing the steam is rapidly cooled in a vacuum to remove water in an amount equivalent to the condensed steam used.

[0172] Combinations of direct and indirect heat treatments may also be used.

[0173] The heat treatment may be ESL treatment, ultra pasteurization or UHT treatment.

[0174] The heat treatment may be performed at a temperature of 120°C to 150°C.

[0175] In one embodiment, the heat treatment is performed at a temperature of at least 123°C, preferably 123°C-145°C.

[0176] In one embodiment, the heat treatment is performed at a temperature of at least 130°C, preferably 130°C-145°C.

[0177] In one embodiment, the heat treatment is performed at a temperature of at least 138°C, preferably 138°C-145°C, more preferably 138°C-142°C.

[0178] The holding time of step b) may be 1-30 seconds, preferably 1-10 seconds, more preferably 1-5 seconds.

[0179] In one embodiment, the heat treatment is UHT treatment, preferably at a temperature of 130-145°C for a time of 1-30 seconds, more preferably at a temperature of 138-145°C for a time of 1-10 seconds, even more preferably at a temperature of 138-144°C for a time of 1-5 seconds.

[0180] In one embodiment, the heat treatment is UHT treatment at a temperature of 128-132° C. for 25-35 seconds, at a temperature of 138-140° C. for 2-5 seconds, or at a temperature of 144-146° C. for 1-2 seconds.

[0181] In one embodiment, the heat treatment is ESL treatment or ultra-pasteurization, preferably ESL treatment or ultra-pasteurization is performed at a temperature of 120°C-140°C for 1-5 seconds, more preferably at a temperature of 120°C-130°C for 1-5 seconds or at a temperature of 138°C-140°C for 2-4 seconds.

[0182] Preferably, no enzyme with lactase activity is added to the dairy product after step b), such as after the holding time of step b). More preferably, no enzyme is added to the dairy product after step b), such as after the holding time of step b). Even more preferably, nothing is added to the dairy product after step b), such as after the holding time of step b). This is because, after the heat treatment, the dairy product is sterile and adding anything, even something that is considered sterile, would risk contaminating the product.

[0183] After the holding time of step b), the dairy based product is preferably cooled to at most 40°C, preferably at most 35°C, more preferably at most 30°C within 5 minutes, more preferably within 3 minutes, even more preferably within 2 minutes, such as within 1 minute.

[0184] Preferably, the time from adding the enzyme until cooling the heat-treated dairy-based product 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.

[0185] After step b) but before step c), the dairy-based product may be homogenized.

[0186] Alternatively, homogenization can be performed before reaching the holding temperature of step b). In indirect UHT treatment (e.g. tube exchange or plate exchange), homogenization is preferably performed upstream. In direct UHT treatment (e.g. steam injection or steam jet), homogenization is preferably performed downstream.

[0187] Preferably, after step b) but before step c), the dairy-based product is aseptically packaged.

[0188] In a preferred embodiment, the milk-based product is UHT milk.In the context of the present invention, UHT milk is milk that has been subjected to a sterilization procedure aimed at killing all microorganisms, including bacterial spores.

[0189] Preferably, when step b) is completed, less than 80% of the lactose has been hydrolyzed, and after one week, more than 90% of the lactose has been hydrolyzed. More preferably, when step b) is completed, less than 60% of the lactose has been hydrolyzed, and after one week, more than 95% of the lactose has been hydrolyzed.

[0190] Preferably, after step b), the enzyme retains at least 0.1%, more preferably at least 0.5%, 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%, most preferably at least 90% of its initial activity. The enzyme activity remaining after step b) (as a percentage of the enzyme activity at the time of enzyme addition) can be determined, for example, by using the "residual activity assay" in the examples.

[0191] Preferably, step c) is performed at a temperature of 2-40° C., preferably 15-40° C., more preferably 18-40° C., most preferably 18-30° C. In a preferred embodiment, step c) is performed at room temperature, which may vary during the storage period.

[0192] In preferred embodiments, the lactose content in the dairy-based product after step b) but before step c) is at least 1% (w / w), such as at least 2% (w / w), at least 3% (w / w) or at least 4% (w / w).

[0193] In another preferred embodiment, after step b) but before step c), the lactose content in the dairy based product has been reduced by at most 80%, preferably at most 50%, more preferably at most 20% compared to the lactose content before step a).

[0194] In step c), the heat-treated dairy-based product is stored for at least about 24 hours, preferably at least 2 days, such as at least 3 days, preferably at least 4 days, preferably at least 7 days, more preferably at least 14 days, such as at least 21 days. It is primarily during this storage period that the lactose level is reduced to the desired level by the residual activity of the lactase.

[0195] In a preferred embodiment, the lactose content in the dairy based product is at most 0.2% (w / w), preferably at most 0.1%, more preferably at most 0.01% after storage of the heat treated dairy based product at a temperature of 2°C-40°C, preferably 15°C-40°C, more preferably 18°C-40°C, most preferably 18°C-30°C for 21 days.

[0196] In another preferred embodiment, the lactose content in the dairy based product is at most 0.2% (w / w), preferably at most 0.1%, more preferably at most 0.01% after storage of the heat treated dairy based product at a temperature of 2°C-40°C, preferably 15°C-40°C, more preferably 18°C-40°C, most preferably 18°C-30°C for 14 days.

[0197] In another preferred embodiment, the lactose content in the dairy based product is at most 0.2% (w / w), preferably at most 0.1%, more preferably at most 0.01% after storage of the heat treated dairy based product at a temperature of 2°C-40°C, preferably 15°C-40°C, more preferably 18°C-40°C, most preferably 18°C-30°C for 7 days.

[0198] In another preferred embodiment, the lactose content in the dairy based product is at most 0.2% (w / w), preferably at most 0.1%, more preferably at most 0.01% after storage of the heat treated dairy based product at a temperature of 2°C-40°C, preferably 15°C-40°C, more preferably 18°C-40°C, most preferably 18°C-30°C for 4 days.

[0199] In another preferred embodiment, the lactose content in the dairy based product is at most 0.2% (w / w), preferably at most 0.1%, more preferably at most 0.01% after storage of the heat treated dairy based product at a temperature of 2°C-40°C, preferably 15°C-40°C, more preferably 18°C-40°C, most preferably 18°C-30°C for 3 days.

[0200] In a preferred embodiment, the lactose content in the dairy based product has been reduced by at least 80% or at least 85%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97%, more preferably at least 98%, even more preferably at least 99% or at least 99.5%, and most preferably at least 99.8% or at least 99.9% compared to the lactose content before step a) after storage of the heat treated dairy based product at a temperature of 2°C-40°C, preferably 15°C-40°C, more preferably 18°C-40°C, most preferably 18°C-30°C for 21 days.

[0201] In another preferred embodiment, after storage of the heat-treated dairy-based product at a temperature of 2°C-40°C, preferably 15°C-40°C, more preferably 18°C-40°C, most preferably 18°C-30°C for 14 days, the lactose content in the dairy-based product has been reduced by at least 80% or at least 85%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97%, more preferably at least 98%, even more preferably at least 99% or at least 99.5%, and most preferably at least 99.8% or at least 99.9% compared to the lactose content before step a).

[0202] In another preferred embodiment, after storage of the heat-treated dairy-based product at a temperature of 2°C-40°C, preferably 15°C-40°C, more preferably 18°C-40°C, most preferably 18°C-30°C for 7 days, the lactose content in the dairy-based product has been reduced by at least 80% or at least 85%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97%, more preferably at least 98%, even more preferably at least 99% or at least 99.5%, and most preferably at least 99.8% or at least 99.9% compared to the lactose content before step a).

[0203] In another preferred embodiment, after storing the heat-treated dairy-based product at a temperature of 2°C-40°C, preferably 15°C-40°C, more preferably 18°C-40°C, most preferably 18°C-30°C for 4 days, the lactose content in the dairy-based product has been reduced by at least 80% or at least 85%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97%, more preferably at least 98%, even more preferably at least 99% or at least 99.5%, and most preferably at least 99.8% or at least 99.9% compared to the lactose content before step a).

[0204] In another preferred embodiment, after storing the heat-treated dairy-based product at a temperature of 2°C-40°C, preferably 15°C-40°C, more preferably 18°C-40°C, most preferably 18°C-30°C for 3 days, the lactose content in the dairy-based product has been reduced by at least 80% or at least 85%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97%, more preferably at least 98%, even more preferably at least 99% or at least 99.5%, and most preferably at least 99.8% or at least 99.9% compared to the lactose content before step a).

[0205] In a preferred embodiment, the lactose content in the dairy based product is at most 1000 ppm, preferably at most 100 ppm after storage of the heat treated dairy based product at a temperature of 2°C-40°C, preferably 15°C-40°C, more preferably 18°C-40°C, most preferably 18°C-30°C for 21 days.

[0206] In another preferred embodiment, the lactose content in the dairy based product is at most 1000 ppm, preferably at most 100 ppm after storage of the heat treated dairy based product at a temperature of 2°C-40°C, preferably 15°C-40°C, more preferably 18°C-40°C, most preferably 18°C-30°C for 14 days.

[0207] In another preferred embodiment, the lactose content in the dairy based product is at most 1000 ppm, preferably at most 100 ppm after storage of the heat treated dairy based product at a temperature of 2°C-40°C, preferably 15°C-40°C, more preferably 18°C-40°C, most preferably 18°C-30°C for 7 days.

[0208] In another preferred embodiment, the lactose content in the dairy based product is at most 1000 ppm, preferably at most 100 ppm after storing the heat treated dairy based product at a temperature of 2°C-40°C, preferably 15°C-40°C, more preferably 18°C-40°C, most preferably 18°C-30°C for 4 days.

[0209] In another preferred embodiment, the lactose content in the dairy based product is at most 1000 ppm, preferably at most 100 ppm after storing the heat treated dairy based product at a temperature of 2°C-40°C, preferably 15°C-40°C, more preferably 18°C-40°C, most preferably 18°C-30°C for 3 days.

[0210] After step c), the heat-treated lactose-reduced milk product may, if desired, be freeze-dried.

[0211] The enzyme having lactase activity may be added at a concentration of 100-50,000, preferably 500-40,000 LAU(B) per liter of milk-based substrate.

[0212] The enzyme having lactase activity may be added in a concentration of 1-150 mg enzyme protein per liter of milk-based substrate, preferably 1-100, more preferably 2-50 or 5-50 mg enzyme protein per liter of milk-based substrate.

[0213] Prior to step b), a reducing agent, preferably a food-approved reducing agent, more preferably a reducing agent selected from L-cysteine, sulfite, and glutathione, may be added to the milk-based substrate. 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 to reduce or prevent oxidation of cysteines that do not form disulfide bridges (sometimes referred to as "free cysteines") in, for example, lactase. Oxidation of such free cysteines may reduce the ability of the enzyme to refold after heat treatment (e.g., UHT treatment).

[0214] The lactase of SEQ ID NO: 5 has a free cysteine residue at position C372, oxidation of which has been shown to reduce the ability of the enzyme to refold, and corresponding cysteine residues can be identified in many other lactases by aligning their amino acid sequences with SEQ ID NO: 5. In a preferred embodiment, the enzyme having lactase activity has an amino acid substitution of the cysteine corresponding to C372 of SEQ ID NO: 5, preferably serine, alanine, or glycine.

[0215] Preferably, the enzyme having lactase activity is lactase.Preferably, the enzyme belongs to 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.

[0216] In a preferred embodiment, the enzyme having lactase activity is neutral lactase.

[0217] In preferred embodiments, the enzyme having lactase activity is purified.

[0218] In preferred embodiments, the enzyme having lactase activity is isolated.

[0219] Preferably, the enzyme having lactase activity has an optimum temperature of 30° C. to 60° C., preferably 35° C. to 55° C. The optimum temperature can be determined using Method 2 in the Examples.

[0220] Preferably, the enzyme having lactase activity has a melting temperature, Tm, of 50° C. to 70° C. as determined by thermal shift at pH 6. In another preferred embodiment, the enzyme having lactase activity has a melting temperature, Tm, of 50° C. to 70° C. as determined by thermal shift at pH 7. The melting temperature, Tm, can be determined using the thermal shift assay in Example 2.

[0221] Preferably, the enzyme having 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. The residual activity can be determined as described in the examples.

[0222] In a preferred embodiment, an enzyme having lactase activity has an initial lactose conversion of at least 10 times per enzyme molecule per second in milk at 5°C. For example, the initial lactose conversion can be determined when 0-10% of the lactose has been hydrolyzed. A skilled artisan will know how to determine the initial lactose conversion per enzyme molecule. For example, this can be a direct measurement of lactose using HPLC or an indirect measurement using a glucose detection method.

[0223] Initial lactose conversion per enzyme molecule in milk at 5°C Pure is about 36 seconds -1 ,for About 89 seconds -1 , Both enzymes were obtained from Novozymes, Denmark.

[0224] In a more preferred embodiment, the enzyme having lactase activity has an initial lactose conversion per enzyme molecule per second in milk at 5°C of at least 20, preferably at least 50, more preferably at least 80.

[0225] In a preferred embodiment, the enzyme having lactase activity has an average lactose conversion from initial lactose (preferably about 4.7% lactose) to 0.1% residual lactose in milk at 5°C of at least 10 per enzyme molecule per second.

[0226] The average lactose conversion from 4.7% initial lactose to 0.1% residual lactose was Pure is about 9 seconds -1 ,for About 23 seconds -1 The lower average lactose conversion from 4.7% to 0.1% compared to the initial rate (4.7% to 4.23%) is due to a combination of several factors, such as higher product inhibition and lower substrate concentration close to 0.1%.

[0227] In a preferred embodiment, the enzyme having lactase activity has an average lactose conversion from initial lactose (preferably about 4.7%) to 0.01% residual lactose in milk at 5°C of at least 5 per enzyme molecule per second.

[0228] The average lactose conversion from 4.7% initial lactose to 0.01% residual lactose was Pure is about 5 seconds -1 ,for About 18 seconds -1 .

[0229] In a preferred embodiment, the enzyme having lactase activity has a Michaelis constant K of at most 40 mM, preferably at most 30 mM, more preferably at most 20 mM at 5°C. M .

[0230] In another preferred embodiment, the enzyme having lactase activity has a Michaelis constant K of at most 40 mM, preferably at most 30 mM, more preferably at most 20 mM at 37°C. M .

[0231] Michaelis constant K Mis the substrate concentration, in this case lactose, at which the reaction rate is half-maximum and is a measure of the affinity of the substrate for the enzyme. M indicates high affinity, which means that with a larger K M In contrast, at lower substrate concentrations the rate will be close to its maximum.

[0232] Michaelis constant K M It can be determined according to the method of Example 5 of WO 09071539 A1. In this example, K at 5°C M The KM at 37°C was determined to be 13 mM for the experimental Bifidobacterium lactase and 30 mM for Lactozym (K. lactis lactase), and 13 mM for the experimental Bifidobacterium lactase and 30 mM for Lactozym.

[0233] Preferred embodiment

[0234] 1. A dairy-based product comprising an enzyme having lactase activity, wherein the enzyme having lactase activity comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13.

[0235] 2. The dairy product of embodiment 1 , wherein the enzyme having lactase activity comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12 or SEQ ID NO: 13.

[0236] 3. The milk-based product of embodiment 2, wherein the enzyme having lactase activity comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0237] 4. The milk-based product of embodiment 3, wherein the enzyme having lactase activity is selected from the group consisting of:

[0238] a) a variant of SEQ ID NO: 1 comprising at least one substitution selected from the group consisting of P65A, C372A, P615T, A1073C, H1122C and C1195G, such as two, three, four or more of said substitutions, wherein the numbering is based on SEQ ID NO: 1;

[0239] b) a variant of SEQ ID NO: 2 comprising at least one substitution selected from the group consisting of P52A, G607T, L1064C and Y1110C, such as two or more of said substitutions, wherein the numbering is based on SEQ ID NO: 2; and

[0240] c) a variant of SEQ ID NO: 4, the variant comprising the substitutions G386Q and / or P620T, wherein the numbering is based on SEQ ID NO: 4.

[0241] 5. The milk-based product of any one of embodiments 1-4, wherein the enzyme having lactase activity has a length of at most about 1500 amino acids, such as at most about 1400 amino acids or at most about 1350 amino acids, for example 850-1500 amino acids, preferably 850-1400 amino acids, such as 850-1350 amino acids.

[0242] 6. The dairy-based product of any one of embodiments 1-5, wherein the product has been heat treated and has a lactose content of at most 0.2% (w / w).

[0243] 7. The milk-based product of any one of embodiments 1-6, wherein the product is UHT milk, ESL milk or ultra-pasteurized milk.

[0244] 8. An enzyme having lactase activity, wherein the enzyme is selected from the group consisting of:

[0245] a) a polypeptide having an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 1, wherein the polypeptide 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;

[0246] b) a polypeptide having an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 2, wherein the polypeptide 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;

[0247] c) a polypeptide having an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 3, wherein the polypeptide 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;

[0248] d) a polypeptide having an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 4, wherein the polypeptide 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;

[0249] e) a polypeptide having an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 6, wherein the polypeptide 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;

[0250] f) a polypeptide having an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 7, wherein the polypeptide 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;

[0251] g) a polypeptide having an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 8, wherein the polypeptide 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;

[0252] h) a polypeptide having an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 9, wherein the polypeptide 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;

[0253] i) a polypeptide having an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 10, wherein the polypeptide 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;

[0254] j) a polypeptide having an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 11, wherein the polypeptide 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;

[0255] k) a polypeptide having an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 12, wherein the polypeptide 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; and

[0256] l) a polypeptide having an amino acid sequence that is at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 13, wherein the polypeptide has a length of up to about 1750 amino acids, such as up to about 1700 amino acids.

[0257] 9. The enzyme of embodiment 8, wherein the enzyme has lactase activity and is selected from the group consisting of:

[0258] a) a variant of SEQ ID NO: 1 comprising at least one substitution selected from the group consisting of P65A, C372A, P615T, A1073C, H1122C and C1195G, such as two, three, four or more of said substitutions, wherein the numbering is based on SEQ ID NO: 1;

[0259] b) a variant of SEQ ID NO: 2 comprising at least one substitution selected from the group consisting of P52A, G607T, L1064C and Y1110C, such as two or more of said substitutions, wherein the numbering is based on SEQ ID NO: 2; and

[0260] c) a variant of SEQ ID NO: 4, the variant comprising the substitutions G386Q and / or P620T, wherein the numbering is based on SEQ ID NO: 4.

[0261] 10. A method for producing a heat-treated dairy-based product with reduced lactose, the method comprising:

[0262] a) adding an enzyme having lactase activity to a milk-based substrate comprising at least 2% lactose (w / w), wherein the enzyme having lactase activity comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13,

[0263] b) after adding the enzyme, heat treating the dairy-based substrate by holding the dairy-based substrate at a holding temperature of at least 120° C. for a holding time of at least 1 second, followed by cooling to produce a heat-treated dairy-based product, and

[0264] c) storing the heat-treated dairy-based product at a temperature of at most 40° C. for at least about 24 hours, preferably at least 2 days, such as at least 3 days, preferably at least 4 days,

[0265] wherein after step c) the lactose content in the dairy based product is at most 0.2% (w / w), and preferably wherein after step b) but before step c) the lactose content in the dairy based product is at least 0.5% (w / w), preferably at least 1% (w / w).

[0266] 11. The method of embodiment 10, wherein the enzyme having lactase activity comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0267] 12. The method of embodiment 11, wherein the enzyme having lactase activity is selected from the group consisting of:

[0268] a) a variant of SEQ ID NO: 1 comprising at least one substitution selected from the group consisting of P65A, C372A, P615T, A1073C, H1122C and C1195G, such as two, three, four or more of said substitutions, wherein the numbering is based on SEQ ID NO: 1;

[0269] b) a variant of SEQ ID NO: 2 comprising at least one substitution selected from the group consisting of P52A, G607T, L1064C and Y1110C, such as two or more of said substitutions, wherein the numbering is based on SEQ ID NO: 2; and

[0270] c) a variant of SEQ ID NO: 4, the variant comprising the substitutions G386Q and / or P620T, wherein the numbering is based on SEQ ID NO: 4.

[0271] 13. The method of any one of embodiments 10-12, wherein step b) is performed immediately after step a) without a dedicated incubation step between step a) and step b).

[0272] 14. The method of any one of embodiments 10-13, wherein pumping equipment is installed to add the enzyme to the dairy-based substrate as it flows through process equipment such as process piping.

[0273] 15. The method of any one of embodiments 10-14, wherein the time from adding the enzyme until the holding temperature of step b) is at most 5 minutes, preferably at most 2 minutes, more preferably at most 1 minute.

[0274] 16. The method of any one of embodiments 10-15, wherein the time from adding the enzyme until cooling the heat-treated dairy-based product 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.

[0275] 17. The method of any one of embodiments 10-16, wherein the milk-based substrate comprises 2%-30%, preferably 2%-17%, more preferably 4%-5.5% lactose (w / w).

[0276] 18. The method of any one of embodiments 10-17, wherein the milk-based substrate is milk comprising 4%-5.5% lactose (w / w).

[0277] 19. The method of any one of embodiments 10-18, wherein the heat treatment is ESL treatment, ultrapasteurization or UHT treatment, preferably UHT treatment.

[0278] 20. The method of any one of embodiments 10-19, wherein the heat treatment is UHT treatment at a temperature of 128-132°C for 25-35 seconds, at a temperature of 138-140°C for 2-5 seconds, or at a temperature of 144-146°C for 1-2 seconds.

[0279] 21. The method of any one of embodiments 10-20, wherein after the holding time of step b), the milk-based substrate is cooled to at most 40°C, preferably at most 35°C, more preferably at most 30°C, preferably within 5 minutes, more preferably within 3 minutes, even more preferably within 2 minutes, such as within 1 minute.

[0280] 22. The method of any one of embodiments 10-21, wherein after step b) but before step c), the dairy-based product is aseptically packaged.

[0281] 23. The method of any one of embodiments 10-22, wherein after step b), the enzyme retains at least 0.1%, preferably at least 0.5%, 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%, most preferably at least 90% of its initial activity.

[0282] 24. The method of any one of embodiments 10-23, wherein the lactose content in the heat-treated dairy-based product is at most 0.2% (w / w), preferably at most 0.1%, more preferably at most 0.01% after storage of the heat-treated dairy-based product at a temperature of 2°C-40°C, preferably 15°C-40°C, more preferably 18°C-40°C, most preferably 18°C-30°C for 21 days, preferably 14 days, more preferably 7 days, even more preferably 4 days, most preferably 3 days.

[0283] 25. The method of any one of embodiments 10-24, wherein the enzyme having lactase activity has a temperature optimum of 30-60°C, preferably 35-55°C.

[0284] 26. The method of any one of embodiments 10-25, wherein the enzyme having 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.

[0285] 27. The method of any one of embodiments 10-26, 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%, even more preferably at least 10% in skim milk with a lactose content of 4.7% after 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 incubating at 23°C for 0.5 hour, 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 then incubating at 23°C for 0.5 hour.

[0286] 28. The method of any one of embodiments 10-27, wherein 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%, even more preferably at least 10% in skim milk with a lactose content of 4.7% after 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 incubating 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 then incubating at 23°C for 72 hours.

[0287] 29. The method of any one of embodiments 10-28, wherein the enzyme having lactase activity is added at a concentration of 1-150 mg enzyme protein / liter of milk-based substrate, preferably 1-100, more preferably 2-50 or 5-50 mg enzyme protein / liter of milk-based substrate.

[0288] 30. The dairy-based product of any one of embodiments 1-7 or the method of any one of embodiments 10-29, wherein the enzyme is as defined in embodiment 8.

[0289] 31. A method for producing a reduced lactose milk product, the method comprising adding an enzyme having lactase activity to a milk-based substrate comprising at least 2% lactose (w / w), and subjecting the milk-based substrate comprising the enzyme to a heat treatment at a temperature of at least 120°C for a holding time of at least 1 second, wherein the enzyme having lactase activity comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13.

[0290] 32. Use of an enzyme with lactase activity in a method for producing a heat-treated milk-based product with reduced lactose, wherein a milk-based substrate comprising at least 2% lactose (w / w) to which an enzyme has been added is heat-treated at a temperature of at least 120°C for a holding time of at least 1 second, wherein the enzyme with lactase activity comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13.

[0291] The present invention is further described in the following non-limiting examples.

[0292] Examples

[0293] Determination method

[0294] Method 1:

[0295] LAU(B) determination

[0296] The activity of a specific 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 0.05 M MES, 1 mM MgSO4 7H2O, 450 mg / L Brij TM35 in 1.46 mg / ml substrate. After incubation for 600 seconds, the reaction is stopped by adding 0.2 M Na2CO3, and the released ONP is measured at 405 nm after incubation for 126 seconds. The activity can be obtained by comparing a standard curve run with a lactase of known activity with the activity of the unknown sample calculated therefrom. The lactase of known activity can be, for example, obtained from Novozymes AB, Denmark.

[0297] Method 2:

[0298] Determination of the optimum temperature

[0299] The temperature curve for determining the optimal temperature was prepared by adding 10 μl of diluted enzyme sample (50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl , 1 mM MgCl + 0.01% Triton X-100, pH 6.5) to a PCR tube. 90 μl of substrate (167 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl , 1 mM MgCl , pH 6.5) was then added and the tube was placed in a preheated PCR block with a temperature gradient of 35° C. to 75° C. (using a T Professional thermal cycler, Biometra) and incubated at 35° C. to 75° C. (gradient) for 30 min, then placed on ice. The reaction was terminated by adding 100 μl of 0.25 M NaOH. Transfer 20 μl to a 96-well microtiter plate and add 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. After 30 minutes in the dark at room temperature, measure the absorbance at 420 nm. The initial enzyme dilution should be adjusted so that the final absorbance reading at 420 nm at the optimal temperature is between 0.5–2.5.

[0300] The temperature with the highest ΔAbs at 420 nm (“Abs 420 nm with enzyme” minus “Abs 420 nm without enzyme”, i.e., background) was set to 100% (optimum temperature), and the temperature profile was determined using the relative activities at other temperatures based on ΔAbs 420 nm relative to the ΔAbs 420 nm with the highest value.

[0301] Example 1

[0302] Testing new lactase enzymes and their variants

[0303] In this example, it was demonstrated that a number of newly identified lactases and variants thereof can be used in the method of the present invention for producing a heat-treated milk product (such as UHT milk). The lactases were added to the milk before UHT treatment and have been shown to have sufficient residual activity after UHT treatment to effectively hydrolyze lactose in the milk during storage at room temperature for several days.

[0304] Some lactases are newly identified wild-type enzymes that have been C-terminally truncated to facilitate expression as secreted enzymes, while other lactases are variants of these wild-type enzymes in which one or more amino acids have been substituted using established protein engineering techniques. The enzymes tested in this example are listed in Table 1 below.

[0305] Expression in Bacillus subtilis

[0306] The genes encoding the enzymes were optimized for expression in Bacillus subtilis using standard methods known in the art, with the exception of the lactases of SEQ ID NO: 10 (Bacillus novobatii) and SEQ ID NO: 13 (Streptomyces volvaceus), in which native DNA (of the truncated mature peptide) was used. The genes were fused with DNA encoding the secretion signal of Bacillus clausii (encoding the following amino acid sequence: MKKPLGKIVASTALLISVAFSSSIASA (SEQ ID NO: 14)) in place of the native secretion signal. In addition, the expression construct resulted in the addition of an amino-terminal polyhistidine tag consisting of the amino acid sequence HHHHHHPR (SEQ ID NO: 15) to the mature lactase to facilitate easy purification by immobilized metal affinity chromatography. The resulting genes were ordered as fully synthetically produced DNA fragments from Twist Bioscience (San Francisco, CA, USA).

[0307] The linear integration construct is a SOE-PCR fusion product (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) consisting of the gene of interest fused to a strong promoter and a chloramphenicol resistance marker between two Bacillus subtilis chromosomal regions. The SOE PCR method is also described in patent application WO 2003 / 095658.

[0308] The lactase gene was expressed under the control of a triple promoter system (as described in WO 99 / 43835) consisting of the promoter of the Bacillus licheniformis α-amylase gene (amyL) containing a stabilizing sequence, the promoter of the Bacillus amyloliquefaciens α-amylase gene (amyQ) and the Bacillus thuringiensis cryIIIA promoter.

[0309] For each lactase expression construct, the SOE-PCR product is transformed into the subtilis and integrated into the pectin lyase locus on the chromosome by homologous recombination. The recombinant subtilis clone that will comprise this integrated expression construct is grown in liquid culture medium subsequently. The nutrient solution is centrifugal (20,000xg, 20min) and the supernatant is carefully separated with the sediment decantation, and is used for the purification of enzyme, or alternatively the aseptically filtered supernatant is directly used for measuring.

[0310] Purification of recombinant enzymes by immobilized metal affinity chromatography

[0311] The pH of the clear supernatant was adjusted to pH 8, filtered through a 0.2 μM filter, and the supernatant was applied to a 5 ml HisTrap TM Excel column. Before loading, the column had been equilibrated in 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 elution of the target was achieved with 50 mM HEPES pH 7 + 10 mM imidazole. The eluted protein was washed with a HiPrep TM Desalting was performed on a 26 / 10 desalting column, which was equilibrated with 3 CV of 50 mM HEPES (pH 7) + 100 mM NaCl. This buffer was also used for elution of the target at a flow rate of 10 ml / min. Relevant fractions were selected and combined based on chromatogram and SDS-PAGE analysis.

[0312] UHT treatment

[0313] The enzymes of Table 1 were added to skim milk and subjected to UHT treatment, followed by incubation at 23°C for 0.5 hours and 72 hours, and lactase activity and lactose were measured after each of these two intervals as described below.

[0314] Depending on the enzyme, between 5.5 mg and 31.6 mg of enzyme protein (ep) / liter of skim milk was used. For most enzymes, 12.7 mg ep / L skim milk was used (see Table 2 below, column 2). Sodium azide* was added to a final concentration of 0.025% (w / v) in all milks tested to avoid microbial growth because, despite the UHT step, the tubes were not completely sterile due to handling, etc. These milk samples were 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 (0.8 mm internal diameter) that was immersed in four baths in sequence. The first bath was in silicone at 90°C with 3 m of Teflon tubing, the second was in silicone at 140°C with 50 cm of Teflon tubing, the third silicone bath was at 70°C with 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 to the syringe, ensuring that the milk sample was incubated at 90° C. for 30 seconds, at 140° C. for 5 seconds, and at 70° C. for 30 seconds, and then cooled in an ice / water bath for 10 seconds, thereby cooling the milk to a temperature in the range of 0° C. 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 h and 72 h, and then the residual activity and lactose content were determined using high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD).

[0315] *Sodium azide should not be used in commercial processes where sterility is otherwise ensured, but may be used in laboratory settings where complete sterility may not be achieved and the milk is not intended for drinking.

[0316] Residual activity determination

[0317] The samples were centrifuged at 20,600 g for 45 min at 5 ° C in a pre-cooled centrifuge and the supernatant was diluted with 20 mM sodium succinate and 0.01% Triton X-100 (pH 6.5) to obtain an 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-galactopyranoside, about 5.5 mM), 0.05 M MES, 1 mM MgSO 4 , 150 mM KCl, 0.01% Triton X-100, pH 6.5 (preferably adjusted with NaOH)) and incubated at 40 ° C for 2.5 hours and stopped by adding 50 μl Na 2 CO 3 + 5 mM Na 4 EDTA and measured at 405 nm. The residual activity (in %) was calculated using the following formula = ((Abs405 热处理的样品 -Abs405 空白 )*dilution factor) / ((Abs405 未处理的样品-Abs405 空白 )*dilution factor)*100%.

[0318] The heat-treated samples contained enzyme mixed with skim milk and sodium azide and were subjected to UHT treatment and subsequent cooling as described above, followed by incubation at 23°C for 0.5h and 72h. The untreated samples were the same enzyme mixed with skim milk and sodium azide, but without UHT treatment and incubation. Both samples were diluted to obtain an absorption within the range of 0.5-1.0 at 405nm. The blanks were samples without enzyme, using the same dilution factor as the enzyme-containing samples. The results are shown in Tables 2 and 3 below.

[0319] Analysis of residual lactose content

[0320] Analysis of residual lactose was performed by high performance anion exchange chromatography equipped with pulsed amperometric detection (HPAEC-PAD).

[0321] Sample preparation for HPAEC-PAD

[0322] The enzyme was inactivated by the following method: 5 μl glacial acetic acid was added to 1 ml milk sample, after which the sample was heated to 90°C for 5 min and centrifuged at 14,200 rpm for 10 min. 50 μl samples were transferred to 5 ml Eppendorf tubes containing 500 μl MQ (Milli-Q) water. 10 μl Carrez I solution was added and mixed, followed by 10 μl Carrez II solution and mixed. 4.43 ml MQ water (total volume 5 ml) was then added and mixed. Centrifuged at 14,200 rpm for 5 min. The supernatant was diluted 5 times with Milli-Q water. These samples were analyzed on HPAEC-PAD.

[0323] Lactose determination using HPAEC-PAD

[0324] The analysis was performed 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, but with slight modifications, such as using a shorter gradient as specified below. TMICS-6000 workstation (Dionex, Amsterdam, The Netherlands) equipped with CarboPac TM A PA1 4x50 mm Guard column (Dionex, product number 043096) was used, followed by a CarboPac PA1 4x 250 mm (Dionex, product number 035391) and an ICS-6000DC ECD detector (Dionex) using the following complex gradient: A: Milli-Q water, B: 600 mM NaOAc in 100 mM NaOH, C: 100 mM NaOH, and D: 50 mM NaOAc. Fractionation was performed by a linear gradient at 1.0 mL / min with 85% A, 0% B, 10% C, and 5% D over 25 min 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, followed by a 5 min wash with 100% B and a 15 min reset with 85% A, 0% B, 10% C, and 5% D. Lactose standards were used to determine the amount of lactose in the enzyme-treated samples. The results are shown in columns 3 and 4 of Table 2 below.

[0325] Dose-response curve of lactase from Bifidobacterium bifidum without UHT treatment

[0326] In order to be able to compare the activity of the enzyme in the UHT treated samples with the activity of the un-UHT treated B. bifidum lactase (enzyme number 15, SEQ ID NO: 5), a dose / response curve of the un-UHT treated B. bifidum lactase (enzyme number 15, SEQ ID NO: 5) was prepared.

[0327] The following doses of Bifidobacterium bifidum wild-type lactase (enzyme number 15) 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 and the milk with enzyme was incubated at room temperature (23°C) for 3 days. The residual lactose in each sample was determined using HPAEC-PAD as described above ("Analysis of residual lactose content"). A dose / response curve was generated by plotting the measured lactose levels against the dose of Bifidobacterium bifidum lactase (enzyme number 15). See Figure 1 .

[0328] The data in columns 2 and 4 of Table 2 were used together with the curve to determine the relative activity of each UHT-treated lactase as a percentage of the activity of the un-UHT-treated Bifidobacterium bifidum lactase (enzyme number 15). Specifically, for each lactase, the value in column 4 (residual lactose (%) after 72 hours) was used to determine the corresponding amount of un-UHT-treated Bifidobacterium bifidum reference lactase (mgep / L milk) required to achieve the same residual lactose (%) after incubation at the same time and temperature ("corresponding amount"). This allowed the relative activity of the UHT-treated lactase to be calculated as a percentage of the activity of the un-UHT-treated Bifidobacterium bifidum reference lactase.

[0329] The relative activity can be calculated as the reciprocal of the amount of UHT-treated lactase (mg ep / L milk) (column 2 of Table 2) as a percentage of the reciprocal of the "corresponding amount" (mg ep / L milk) of the non-UHT-treated Bifidobacterium bifidum lactase. In practice, the calculation can be simplified by dividing the "corresponding amount" (mg ep / L milk) of the non-UHT-treated Bifidobacterium bifidum lactase by the amount of UHT-treated lactase (mg ep / L milk) found in column 2 of Table 2 and expressing the result as a percentage. This measure of relative activity is shown in column 6 of Table 2, and an example of the calculation is provided in the "Results" section below.

[0330] This value is therefore a measure of the "survival of enzyme activity after UHT treatment" based on the lactose remaining after UHT treatment and 3 days incubation at 23° C. A dose / response curve for an un-UHT treated Bifidobacterium bifidum lactase (SEQ ID NO: 5), which had also been incubated at 23° C. for 3 days, was used to determine the "corresponding dose" of the un-UHT treated Bifidobacterium bifidum lactase.

[0331] The lactases tested included both wild-type enzymes and engineered variants containing one or more substitutions to provide improved performance compared to one of the wild-type enzymes. These substitutions included replacing a free Cys (homologous to C372 in SEQ ID NO: 5 / enzyme number 15), such as in enzyme number 2 (Bifidobacterium samieri lactase with substitution C372A), introducing a disulfide, such as in enzyme numbers 3 and 8, and replacing cis-proline, such as in enzyme numbers 4, 5, and 13. An overview of the lactases (including SEQ ID NOs. of the wild-type sequences) is provided in Table 1 below.

[0332] Table 1: Overview of lactase

[0333]

[0334]

[0335]

[0336] result:

[0337] The results of the above measurements are shown in Tables 2 and 3. Columns 3 and 4 of Table 2 show the residual lactose at 0.5 hours and 72 hours after UHT treatment, respectively. The amount of lactose decreased from 0.5 hours (average 4.5 g lactose / 100 ml, corresponding to 96% of the initial lactose) to even lower levels after 72 hours, with enzyme numbers 1, 3, and 5 showing residual lactose below 0.01%, a requirement in many countries for milk products to be "lactose-free." For each enzyme, the enzyme dosage used (mg ep / L milk) is shown in column 2. If the enzyme dosage is increased, the residual lactose after 72 hours will be even lower.

[0338] Column 5 shows the pseudo-specific activity after UHT treatment (grams of lactose converted / mg enzyme protein), calculated as ("g lactose / L at 0.5 h" minus "g lactose / L at 72 h") / "mg ep / L", i.e. the value in column 3 minus the value in column 4 (although expressed in g / L rather than % lactose) divided by the value in column 2. As an example, using the data for enzyme number 12 in Table 2:

[0339] (45 g / L lactose - 12.7 g / L lactose) / 5.5 mg ep / L = 5.9 g lactose / mg ep (at 71.5 hours).

[0340] Since the residual lactose is not linear with respect to enzyme dosage (see Figure 1 ), so this pseudo-specific activity is biased in terms of dose dependence. Therefore, a relatively low enzyme dose gives a higher pseudo-specific activity value than a higher enzyme dose.

[0341] In order to better compare the efficiency of the enzymes, the "relative activity of unstressed B. bifidum lactase (enzyme number 15)" was calculated as described above and is shown in column 6 of Table 2. Figure 1 Example of calculation for enzyme No. 12. Using a dose of 5.5 mg ep of enzyme No. 12 per liter of milk (Table 2, column 2), 1.27% residual lactose was produced after 72 hours (Table 2, column 4). Figure 1 As shown by the horizontal arrow in FIG, which corresponds to 0.39 mg ep of unstressed Bifidobacterium bifidum lactase (enzyme number 15), as Figure 1 As shown by the vertical arrow in FIG, in this case, 0.39 mg ep / L milk is the “corresponding amount”.

[0342] Using the calculation method described above ("corresponding amount" of non-UHT-treated B. bifidum lactase (mg ep / L milk) divided by the amount of UHT-treated lactase (mg ep / L milk)), we obtain (0.39 / 5.5 x 100%), i.e. 7.1%, which is the value shown for enzyme No. 12 in column 6 of Table 2. This is a very good measure of the relative activity of the enzyme after UHT treatment compared to the non-UHT-treated B. bifidum lactase (No. 15).

[0343] Columns 7, 8, and 9 show the calculated enzyme dosage required to reach 1.41% residual lactose (a gastro-friendly level with a 70% reduction in lactose), 0.1% residual lactose (a low lactose level required in many countries), or 0.01% residual lactose (a lactose-free level required in many countries) after 72 hours of storage at 23°C. These numbers are calculated as follows:

[0344] The amount of B. bifidum lactase (enzyme number 15) required to reach lactose levels of 1.41%, 0.1% and 0.01% was 0.361, 0.934 and 1.56 mg ep / L milk, respectively (according to Figure 1 (determined by the dose-response curves in Table 2). These values were used to predict the corresponding amount of a given enzyme required to achieve the same lactose level by dividing them by the "Relative activity (%) of unstressed B. bifidum lactase" value (column 6 of Table 2). For example, for enzyme number 12, the "Relative activity (%) of unstressed B. bifidum lactase" was 7.1% (0.071), so the calculated (extrapolated) amount of enzyme number 12 protein required to achieve the three lactose levels was 0.361 / 0.071, 0.934 / 0.071, and 1.56 / 0.071 mg ep / L milk, which is 5.1, 13.1, and 22.0 mg ep / L milk, respectively, as shown in columns 7, 8, and 9 of Table 2.

[0345] An enzyme dosage of 5-50 mg lactase ep / L milk (typically containing 4.5%-5.0% lactose) is typically used in industrial batch lactose reduction applications, with tank incubation times ranging from 8-24 hours at 4°C-10°C to achieve lactose levels of 0.01%-0.1% lactose (% = g lactose / 100 ml milk). Therefore, the first few candidates in Table 2 are highly relevant to lactose-free applications (0.01% lactose), while other sequences with lower relative activities may be more relevant to low-lactose applications rather than "lactose-free" applications.

[0346] The residual activities of the enzymes at 0.5 h and 72 h after UHT treatment are shown in columns 2 and 3 of Table 3, respectively. It can be seen that the enzymes exhibit varying degrees of residual activity, and surprisingly, the residual activity of most enzymes increases after 72 h compared to 0.5 h, with the average increase for the listed enzymes exceeding 3-fold.

[0347] The relatively low correlation between the residual activity % (Table 3) and the residual lactose levels measured in Table 2 is believed to be due to the different specific activities of each enzyme. The ability of an enzyme to hydrolyze lactose after UHT treatment is affected by the efficiency with which the enzyme converts lactose (specific activity before UHT treatment) and the enzyme activity after UHT treatment (residual activity %).

[0348] Table 2: Residual lactose and relative lactase activity after UHT treatment

[0349]

[0350]

[0351] Table 3: Residual lactase activity after UHT treatment

[0352]

[0353]

[0354]

[0355] Example 2

[0356] Determine Tm using thermal shift assay at pH 6 and 7

[0357] Thermal shift assay (TSA) measures the melting temperature (Tm) of proteins, which is the temperature at which 50% denaturation occurs. Protein denaturation was performed by SYPRO TM The protein is monitored by the increase in fluorescence of the Orange dye, which binds to hydrophobic residues exposed upon unfolding of the target protein.

[0358] The purified sample was diluted to 0.24 mg / ml in Milli-Q water. TM The thermal shift assay mixture was prepared by diluting Orange (Invitrogen / ThermoFisher No. S6650) 200-fold into the desired pH buffer (100 mM succinate, 100 mM HEPES, 100 mM glycine, 150 mM KCl, 1 mM CaCl2, 0.01% Triton-X 100, adjusted to pH 6 or 7). 10 μl of the diluted sample was added to the 96-well plate. 480 multiwell plates, Roche No. 04729692001). After sealing with optical tape (Roche, No. 04729757001), the plates were placed in The cells were heated from 25°C to 95°C (temperature ramp: 3.2°C / min) in a 480II real-time PCR machine (Roche) and fluorescence was measured continuously (excitation / emission wavelength: 465 / 510 nm). Tm was determined by plotting the first derivative of fluorescence as a function of temperature (dF / dT) and determining the temperature at which dF / dt was maximum.

[0359] The determined Tm values are shown in Table 4.

[0360] Table 4: Melting temperatures Tm (°C) determined by TSA at pH 6 and 7

[0361]

[0362]

[0363] Example 3

[0364] Determination of the optimal temperature of lactase

[0365] The temperature profiles of five lactases at 35°C-75°C were determined using Method 2 above.

[0366] The results can be seen in Table 5 below, which shows the relative activity (percentage of maximum activity) of the five enzymes at different temperatures. The table shows that the optimum temperatures for enzymes No. 1, No. 12, No. 7, No. 15, and No. 11 were approximately 43.0°C, 47.5°C, 38.9°C, 43°C, and <35°C (indicated by "100"), respectively. The table also shows the temperatures (after the optimum temperature) at which 50% activity was maintained, which were approximately 55°C, 55°C, 46°C, 58°C, and 52°C, respectively (assessed from Table 5, between two numbers with an asterisk (*)).

[0367] The temperature profile is typically related to the temperature dependence of lactose hydrolysis and the rate of enzyme unfolding. If the unfolding of the enzyme is completely reversible, the decrease in activity after the optimum temperature should be related to the amount of unfolded enzyme, and the "melting temperature" (Tm, the temperature at which half of the enzyme is unfolded) should be close to the value of 50% residual activity determined from Table 5. However, typically, unfolding is irreversible, and the rate of lactose hydrolysis is also temperature-dependent, both of which contribute to the decrease in activity.

[0368] Since the Tm values for enzymes 1, 12, 7, 15, and 11 have been determined to be approximately 57°C, 55°C, 54°C, 56°C, and 63°C, respectively (see Table 4 in Example 2), there is a close match between the Tm (Table 4) and 50% residual activity (Table 5) for enzymes No. 1, 12, and 15. This indicates that these enzymes have a significant amount of refolding in the assay.

[0369] It is believed that the small amount of relative activity (2%-5%) that appears fairly constant at higher temperatures (>70°C) is due to the ramp time from room temperature to >70°C (estimated ramp time is less than 1 min).

[0370] Table 5: Relative activity of lactase at different temperatures

[0371]

Claims

1. A dairy-based product comprising an enzyme having lactase activity, wherein the enzyme having lactase activity comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO:

13.

2. The dairy-based product of claim 1 , wherein the enzyme having lactase activity comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12 or SEQ ID NO:

13.

3. The dairy-based product of claim 2, wherein the enzyme having lactase activity comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

4.

4. The dairy-based product of claim 3, wherein the enzyme having lactase activity is selected from the group consisting of: a) a variant of SEQ ID NO: 1, said variant comprising at least one substitution selected from the group consisting of P65A, C372A, P615T, A1073C, H1122C and C1195G, such as two, three, four or more of said substitutions, wherein the numbering is based on SEQ ID NO: 1; b) a variant of SEQ ID NO: 2, comprising at least one substitution selected from the group consisting of: P52A, G607T, L1064C, and Y1110C, such as two or more of said substitutions, wherein the numbering is based on SEQ ID NO: 2; and c) a variant of SEQ ID NO: 4, said variant comprising the substitutions G386Q and / or P620T, wherein the numbering is based on SEQ ID NO:

4.

5. The milk-based product of any one of claims 1 to 4, wherein the enzyme having lactase activity has a length of at most about 1500 amino acids, such as at most about 1400 amino acids or at most about 1350 amino acids, for example 850-1500 amino acids, preferably 850-1400 amino acids, such as 850-1350 amino acids.

6. A dairy based product according to any one of claims 1 to 5, wherein the product has been heat treated and has a lactose content of at most 0.2% (w / w).

7. The milk-based product according to any one of claims 1 to 6, wherein the product is UHT milk, ESL milk or ultra-pasteurized milk.

8. An enzyme having lactase activity, wherein the enzyme is selected from the group consisting of: a) a polypeptide having an amino acid sequence that is at least 75%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 1, wherein the polypeptide 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; b) a polypeptide having an amino acid sequence that is at least 75%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 2, wherein the polypeptide 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; c) a polypeptide having an amino acid sequence that is at least 75%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 3, wherein the polypeptide 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; d) a polypeptide having an amino acid sequence that is at least 75%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 4, wherein the polypeptide 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; e) a polypeptide having an amino acid sequence that is at least 75%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 6, wherein the polypeptide 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; f) a polypeptide having an amino acid sequence that is at least 75%, such as at least 80%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 7, wherein the polypeptide 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; g) a polypeptide having an amino acid sequence that is at least 75%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 8, wherein the polypeptide 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; h) a polypeptide having an amino acid sequence that is at least 75%, such as at least 80%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 9, wherein the polypeptide 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; i) a polypeptide having an amino acid sequence that is at least 75%, such as at least 80%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 10, wherein the polypeptide 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; j) a polypeptide having an amino acid sequence that is at least 75%, such as at least 80%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 11, wherein the polypeptide 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; k) a polypeptide having an amino acid sequence that is at least 75%, such as at least 80%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 12, wherein the polypeptide 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; and l) a polypeptide having an amino acid sequence that is at least 75%, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 13, wherein the polypeptide has a length of up to about 1750 amino acids, such as up to about 1700 amino acids.

9. The enzyme of claim 8, wherein the enzyme has lactase activity and is selected from the group consisting of: a) a variant of SEQ ID NO: 1, said variant comprising at least one substitution selected from the group consisting of P65A, C372A, P615T, A1073C, H1122C and C1195G, such as two, three, four or more of said substitutions, wherein the numbering is based on SEQ ID NO: 1; b) a variant of SEQ ID NO: 2, comprising at least one substitution selected from the group consisting of: P52A, G607T, L1064C, and Y1110C, such as two or more of said substitutions, wherein the numbering is based on SEQ ID NO: 2; and c) a variant of SEQ ID NO: 4, said variant comprising the substitutions G386Q and / or P620T, wherein the numbering is based on SEQ ID NO:

4.

10. A method for producing a heat-treated dairy-based product with reduced lactose, the method comprising: a) adding an enzyme having lactase activity to a milk-based substrate comprising at least 2% lactose (w / w), wherein the enzyme having lactase activity comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13, b) after adding the enzyme, heat treating the dairy-based substrate by holding the dairy-based substrate at a holding temperature of at least 120° C. for a holding time of at least 1 second, followed by cooling to produce a heat-treated dairy-based product, and c) storing the heat-treated dairy-based product at a temperature of at most 40° C. for at least about 24 hours, preferably at least 2 days, such as at least 3 days, preferably at least 4 days, wherein the lactose content in the dairy based product after step b) but before step c) is at least 0.5% (w / w), preferably at least 1% (w / w), and wherein after step c) the lactose content in the dairy based product is at most 0.2% (w / w), and preferably wherein after step b) but before step c) the lactose content in the dairy based product is at least 0.5% (w / w), preferably at least 1% (w / w).

11. The method of claim 10, wherein the enzyme having lactase activity comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

12. The method of claim 11, wherein the enzyme having lactase activity is selected from the group consisting of: a) a variant of SEQ ID NO: 1, said variant comprising at least one substitution selected from the group consisting of P65A, C372A, P615T, A1073C, H1122C and C1195G, such as two, three, four or more of said substitutions, wherein the numbering is based on SEQ ID NO: 1; b) a variant of SEQ ID NO: 2, comprising at least one substitution selected from the group consisting of: P52A, G607T, L1064C, and Y1110C, such as two or more of said substitutions, wherein the numbering is based on SEQ ID NO: 2; and c) a variant of SEQ ID NO: 4, said variant comprising the substitutions G386Q and / or P620T, wherein the numbering is based on SEQ ID NO:

4.

13. The method of any one of claims 10 to 12, wherein step b) is performed immediately after step a) without a dedicated incubation step between step a) and step b).

14. A method for producing a reduced lactose milk product, the method comprising adding an enzyme having lactase activity to a milk-based substrate comprising at least 2% lactose (w / w), and subjecting the milk-based substrate comprising the enzyme to a heat treatment at a temperature of at least 120°C for a holding time of at least 1 second, wherein the enzyme having lactase activity comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO:

13.

15. Use of an enzyme having lactase activity in a method for producing a heat-treated milk-based product with reduced lactose, wherein a milk-based substrate comprising at least 2% lactose (w / w) to which an enzyme has been added is heat-treated at a temperature of at least 120°C for a holding time of at least 1 second, wherein the enzyme having lactase activity comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, 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 or SEQ ID NO: 13.

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