WO2017167847A1

WO2017167847A1 - Production of milk with low lactose content

Info

Publication number: WO2017167847A1
Application number: PCT/EP2017/057488
Authority: WO
Inventors: Petrus Jacobus Theodorus Dekker; Cornelis Marinus Muijlwijk; Marten Aalt PAASMAN
Original assignee: Dsm Ip Assets B.V.
Priority date: 2016-03-31
Filing date: 2017-03-30
Publication date: 2017-10-05
Also published as: EP3435770A1

Abstract

The invention provides a milk obtainable by a process which comprises: a) providing a milk comprising lactose; and b) treating said milk in a low temperature batch process or an aseptic process with an enzyme composition comprising lactase activity, wherein at least a part of the lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ I D NO: 1 OR SEQ ID NO: 2, a truncated version of either thereof or a sequence having at least about 60% sequence identity to any thereto.

Description

PRODUCTION OF MILK WITH LOW LACTOSE CONTENT

Field of the invention

The invention relates to a milk obtainable by a process in which a milk comprising lactose is treated with an enzyme composition comprising lactase activity. The invention also relates to the process itself and to use of an enzyme composition comprising lactase activity in the production of a milk with a low lactose content.

Background to the invention

Lactose intolerance is perhaps the best-known food sensitivity in the United States and other parts of the world. It is estimated that about 70% of the world's population has a genetically controlled limited ability to digest lactose. Accordingly, there is a growing demand for dairy food products that contain no or only low levels of lactose.

Lactase is used commercially to break down lactose in milk to produce dairy products which are suitable for people with lactose intolerance and/or have a sweeter taste. Because glucose and galactose are sweeter than lactose, lactase produces a more pleasant taste. Lactase is also used in the manufacture of ice cream. Lactose crystallizes at the low temperatures of ice cream, whereas glucose and galactose stay liquid and contribute to a smoother texture. Lactase is also used in the conversion of whey into syrup and for the production of condensed milk.

It is desirable to provide new dairy products having low-amounts of lactose and methods for their production.

Summary of the invention

The present invention relates to a milk, to a process for production of such a milk and to the use of a lactase composition in a process for the production of a milk having low lactose. The milk may have a low level of lactose (in view of the use of the lactase, i.e. low lactose lactase-treated milk). According to the invention, there is thus provided a milk comprising about 50 mg/L or less lactose obtainable by a process which comprises:

a) providing a milk comprising lactose; and

b) treating said milk in a low temperature batch process with an enzyme composition comprising lactase activity,

wherein at least a part of the lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2, a truncated version of either thereof or a sequence having at least about 60% sequence identity to any thereto.

The invention also provides:

a milk comprising about 50 mg/L or less lactose obtainable by a process which comprises:

a) providing a milk comprising lactose; and

wherein at least a part of the lactase activity is provided by a polypeptide having lactase activity which is derived from Bifidobacterium bifidum; a process for the production of a milk comprising about 50 mg/L or less lactose, which process comprises:

a) providing a milk comprising lactose; and

wherein at least a part of the lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2, a truncated version of either thereof or a sequence having at least about 60% sequence identity to any thereto; a process for the production of a milk comprising about 50 mg/L or less lactose, which process comprises:

a) providing a milk comprising lactose; and b) treating said milk in a low temperature batch process with an enzyme composition comprising lactase activity,

wherein at least a part of the lactase activity is provided by a polypeptide having lactase activity which is derived from Bifidobacterium bifidum a milk comprising about 50 mg/L or less lactose obtainable by a process which comprises:

a) providing a milk comprising lactose; and

b) treating said milk in an aseptic process with an enzyme composition comprising lactase activity,

wherein at least a part of the lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2, a truncated version of either thereof or a sequence having at least about 60% sequence identity to any thereto; a milk comprising about 50 mg/L or less lactose obtainable by a process which comprises:

a) providing a milk comprising lactose; and

wherein at least a part of the lactase activity is provided by a polypeptide having lactase activity which is derived from Bifidobacterium bifidum; a process for the production of a milk comprising about 50 mg/L or less lactose which comprises:

a) providing a milk comprising lactose; and

wherein at least a part of the lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2, a truncated version of either thereof or a sequence having at least about 60% sequence identity to any thereto; a process for the production of a milk comprising about 50 mg/L or less lactose which comprises:

a) providing a milk comprising lactose; and

wherein at least a part of the lactase activity is provided by a polypeptide having lactase activity which is derived from Bifidobacterium bifidum; use of an enzyme having lactase activity in the production of milk with a low lactose content, which milk is obtainable by a process which comprises:

a) providing a milk comprising lactose; and

wherein at least a part of the lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2, a truncated version of either thereof or a sequence having at least about 60% sequence identity to any thereto; use of an enzyme having lactase activity in the production of milk with a low lactose content, which milk is obtainable by a process which comprises:

a) providing a milk comprising lactose; and

wherein at least a part of the lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2, a truncated version of either thereof or a sequence having at least about 60% sequence identity to any thereto; and use of an enzyme having lactase activity in the production of milk with a low lactose content, which milk is obtainable by a process which comprises:

a) providing a milk comprising lactose; and

wherein at least a part of the lactase activity is provided by a polypeptide having lactase activity which is derived from Bifidobacterium bifidum.

Description of the sequence listing

SEQ ID NOs: 1 and 2 set out the polypeptide sequences of lactase enzymes from Bifidobacterium bifidum. SEQ ID NO: 1 may also be defined with reference to SEQ ID NO: 2 in WO01/90317. SEQ ID NO: 2 may also be defined with reference to the Bbg3 sequence described in Goulas T.K., Goulas A.K., Tzortzis G., Gibson G.R., Appl. Microbiol. Biotechnol. 76(6), 1365 and 1372 (2007).

SEQ ID NO: 3, a C-terminal truncation variant, may also be defined with reference to SEQ ID NO: 2 as described in WO2009/071539.

SEQ ID NO: 4, another C-terminal truncation variant, may also be defined with reference to SEQ ID NO: 2 as described in WO2014/184189.

Detailed description of the invention

Throughout the present specification and the accompanying claims, the words "comprise", "include" and "having" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element. The invention provides a milk comprising about 50 mg/L or less lactose. The inventors of the present patent application were surprised that a (lactase-treated) milk with such low levels of lactose could be produced/obtained, the invention thus provides a low lactose, lactase-treated milk.

The invention concerns a milk comprising about 50 mg/L or less lactose obtainable by a process which comprises:

a) providing a milk comprising lactose; and

b) treating said milk in a low temperature batch process with an enzyme composition comprising lactase activity.

The invention also concerns a milk comprising about 50 mg/L or less lactose obtainable by a process which comprises:

a) providing a milk comprising lactose; and

b) treating said milk in an aseptic process with an enzyme composition comprising lactase activity.

Further provided is a process for the production of a milk comprising about 50 mg/L or less lactose, which process comprises:

a) providing a milk comprising lactose; and

In addition is provided a process for the production of a milk comprising about 50 mg/L or less lactose which comprises:

a) providing a milk comprising lactose; and

At least a part of the lactase activity in the enzyme composition may be provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2, a truncated version of either thereof or a sequence having at least about 60% sequence identity to any thereto. Reference to a truncated version refers to a C-terminal truncated version.

Alternatively, at least a part of the lactase activity in the enzyme composition may be provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 or SEQ I D NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or a sequence having at least about 60% sequence identity to any thereto In addition or alternatively, at least a part of the lactase activity is provided by a polypeptide having lactase activity which is derived from Bifidobacterium bifidum. The term "derived from" also includes the terms "originated from," "obtained from," "obtainable from," "isolated from," and "created from," and generally indicates that one specified material find its origin in another specified material or has features that can be described with reference to the another specified material. As used herein, a substance (e.g., a nucleic acid molecule or polypeptide) "derived from" a microorganism preferably means that the substance is native to that microorganism or is a variant based on a substance native to the microorganism (for example a truncated version of a polypeptide derived from the microorganism and/or a version which contains one or more substitutions as compared to a polypeptide derived from the microorganism).

The polypeptide set out in SEQ ID NO: 1 or SEQ ID NO: 2 of the invention is a full-length wild-type sequence of a lactase from Bifidobacterium bifidum.

A polypeptide having lactase activity suitable for use in the invention may comprise an amino acid sequence having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about, 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

SEQ ID NO: 1 and 2 both comprise a signal sequence which is positioned at amino acids 1 to 32 for both of them. As the signal sequence will most likely be cleaved off during production in a host cell, a polypeptide having lactase activity for use in the invention preferably comprises:

an amino acid sequence having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about, 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to amino acids 33 to 1752 of SEQ ID NO: 1 .

an amino acid sequence having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about, 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to amino acids 33 to 1935 of SEQ ID NO: 2.

A truncated version of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2, or more preferably a truncated version of amino acids 33 to 1752 of SEQ ID NO: 1 or a truncated version of amino acids 33 to 1935 of SEQ ID NO:2, may be used or a polypeptide comprising an amino acid sequence having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about, 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to such a truncated version of SEQ ID NO: 1 or SEQ ID NO: 2 or more preferred to a truncated version of amino acids 33 to 1752 of SEQ ID NO: 1 or amino acids 33 to 1935 of SEQ ID NO:2.

An example of such a polypeptide, i.e. a polypeptide having lactase activity suitable for use in the invention, is the Bbg3 polypeptide described in Goulas T.K., Goulas A.K., Tzortzis G., Gibson G.R., Appl. Microbiol. Biotechnol. 76(6), 1365 and 1372 (2007).

Suitable truncated versions of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2 include SEQ ID NO: 2 in WO2009/071539 (herein SEQ ID NO: 3) and SEQ ID NOs: 1 or 2 in WO2014/184189 (SEQ ID NO: 2 of WO2014/184189 is SEQ ID NO: 4 in this patent application).

In a preferred embodiment, the invention provides a milk or a process according to the invention in which SEQ ID NO: 3 or 4 is used or a polypeptide comprising an amino acid sequence having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about, 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. SEQ ID NO: 3 and 4 both comprise a signal sequence which is positioned at amino acids 1 to 27 for both of them. As the signal sequence will most likely be cleaved off during production in a host cell, a polypeptide having lactase activity for use in the invention preferably comprises:

an amino acid sequence having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about, 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to amino acids 28 to 1341 of SEQ I D NO: 3, more preferably amino acids 28-1331 of SEQ ID NO: 3 (as amino acids 1332-1341 comprise a purification tag which is preferably removed)

an amino acid sequence having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about, 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to amino acids 28 to 1331 of SEQ ID NO: 4.

Preferably, the invention concerns a milk comprising about 50 mg/L or less lactose obtainable by a process which comprises:

a) providing a milk comprising lactose; and

a) providing a milk comprising lactose; and b) treating said milk in a low temperature batch process with an enzyme composition comprising lactase activity.

a) providing a milk comprising lactose; and

wherein at least part of the lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 , 2, 3 or 4 or a sequence having at least 60% sequence identity to SEQ ID NO: 1 , 2, 3 or 4.

More preferably, at least part of the lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in amino acids 33 to 1752 of SEQ ID NO: 1 , amino acids 33 to 1935 of SEQ I D NO: 2, amino acids 28 to 1331 of SEQ ID NO:3 or amino acids 28 to 1331 of SEQ ID NO: 4 or a sequence having at least 60% sequence identity to any thereto.

A low temperature batch process is one which is typically carried out a temperature of from about 5°C to about 10°C, for example at about 6°C to about 9°C.

In a batch process, the enzyme composition comprising lactase activity may be used so that from about 2000 to about 5000 NLU/L lactase activity is provided. More preferably, the skilled person determines the suitable amount of enzyme based on the specific enzyme characteristics. The skilled person is very well capable of determining the suitable amount of enzyme needed to achieve the desired degree of lactose hydrolysis. The enzyme activity may also be expressed in a different unit, for example in LAU.

The used incubation time depends on the amount of NLU/L milk and is typically in the range of 20 to 48 hours.

Preferably, the low temperature batch process is performed at from about 5°C to about 10°C and incubation is performed from about 20 to about 48 hours.

An aseptic process is one which is typically carried out at ambient temperature, for example a temperature of at least about 20°C, for example at least about 25°C.

In an aseptic process, the enzyme composition comprising lactase activity may be used so that from about 200 to about 500 NLU/L lactase activity is provided.

An aseptic process may be the FlexDos™ or Aldose systems. An aseptic process is a process in which milk is first pasteurized or sterilized and cooled to ambient temperature, at which time sterile filtered lactase is added and the product sterile packed. The lactase is allowed to react with lactose in the milk over several days. An example of a resulting milk is low lactose UHT milk.

The invention provides a milk obtainable by the process of the invention. The term "milk" is intended to encompass milks from mammals and plant sources or mixtures thereof. Preferably, the milk is from a mammal source. Mammals sources of milk include, but are not limited to cow, sheep, goat, buffalo, camel, llama, mare and deer. In an embodiment, the milk is from a mammal selected from the group consisting of cow, sheep, goat, buffalo, camel, llama, mare and deer, and combinations thereof. Plant sources of milk include, but are not limited to, milk extracted from soy bean, pea, peanut, barley, rice, oat, quinoa, almond, cashew, coconut, hazelnut, hemp, sesame seed and sunflower seed. Soy bean milk is preferred. In addition, the term "milk" refers to not only whole milk, but also skim milk or any liquid component derived thereof.

As described herein, the present invention relates to low lactose milk. The term low lactose milk refers to a milk which comprises about 50mg/L or less lactose, about 40mg/L or less lactose, about 30mg/L or less lactose, about 20mg/L or less lactose or about 10mg/L or less lactose. Such concentrations may be reached in about 48 hours or less, about 32 hours or less or about 24 hours or less (when a milk is treated as described herein).

The milk could be a pasteurized milk, a UHT-milk or milk reconstituted from all or part of its original constituents with or without intermediate processing steps such as protein hydrolysis.

Treatment with an enzyme composition comprising lactase activity may be done prior to and after the heat-treatment of the milk. The lactase treatment may be done by adding the enzyme to the milk.

The invention also provides:

use of an enzyme having lactase activity in the production of milk with a low lactose content, which milk is obtainable by a process which comprises:

a) providing a milk comprising lactose; and

b) treating said milk in a low temperature batch process with an enzyme composition comprising lactase activity; and use of an enzyme having lactase activity in the production of milk with a low lactose content, which milk is obtainable by a process which comprises:

a) providing a milk comprising lactose; and

The enzyme composition comprising lactase activity may be as described herein.

The term "milk with a low lactose content" refers to a milk which comprises about 50mg/L or less lactose, about 40mg/L or less lactose, about 30mg/L or less lactose, about 20mg/L or less lactose or about 10mg/L or less lactose.

The conditions cited above for the low temperature batch process and the aseptic process (such as for example temperature and amount of lactase) are applicable to the use claims as well.

Moreover, also the description of the suitable lactases are applicable to the use claims as well.

The terms "sequence homology" or "sequence identity" or "homology" or "identity" are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percentage of sequence homology or sequence identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/based or amino acids. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region.

A comparison of sequences and determination of percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the identity between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1 -44 Addison Wesley). The percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. LongdenJ. and BleasbyA Trends in Genetics 16, (6) pp276— 277, http://emboss.bioinformatics.nl/). For protein sequences EBLOSUM62 is used for the substitution matrix. For nucleotide sequence, EDNAFULL is used. The optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

After alignment by the program NEEDLE as described above the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity defined as herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as "longest-identity".

The nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403—10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules as described herein. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules as described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See the homepage of the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.

A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

The present invention is further illustrated by the following Examples:

EXAMPLES

Lactase enzymes were expressed in Bacillus subtilis for comparison with Maxilact LGI (DSM Food Specialties, Delft, NL). Lad 6 is described in WO2009/071538 (SEQ ID NO: 2; herein SEQ ID NO: 3) and Iac19 (herein SEQ ID NO: 2) is described as Bbg3 in Goulas T.K., Goulas A.K., Tzortzis G., Gibson G.R., Appl. Microbiol. Biotechnol. 76(6), 1365 and 1372 (2007).

Standard genetic techniques, such as overexpression of enzymes in the host cells, genetic modification of host cells, or hybridisation techniques, are known methods in the art, such as described in Sambrook and Russel (2001 ) "Molecular Cloning: A Laboratory Manual (3^rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987). Methods for transformation, genetic modification etc of fungal host cells are known from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO 00/37671 , WO90/14423, EP-A- 0481008, EP-A-0635 574 and US 6,265,186.

Enzymes were isolated from the fermentation broth of Bacillus subtilis strains and purified using standard centrifugation and filtration techniques.

Example 1 : application of the lactase enzymes in the milk pre-sterilization process (6°C)

Substrate: "Halfvolle melk from Jumbo supermarket". For each enzyme 2 vials each containing 18.4 ml of semi-skimmed pasteurized and homogenized milk was equilibrated at 6°C before start of the experiment. Lactase was dosed based on the amount of specific protein at 20 mg/l milk in duplicate and incubation is continued at 6°C.

After exactly 0.5, 2, 6, 24 and 48 h incubation, vials were gently mixed and 1 .8 mL sample was taken from each vial into an Eppendorf cup (all "incubations vials" were kept in the cold room during the complete experiment). This cup was placed in a thermomixer (10 minutes 90°C). Subsequently the cup was cooled down to room temperature and exactly 1 mL was pipetted in MTP-plate and stored frozen until further analysis (NMR for lactose, glucose and galactose analysis). After 48 hours extra samples were taken to be analyzed using HPEAC-PAD to determine low lactose content. Control sample without enzyme addition was included as well.

In Table 1 the dosage of the various lactases in the application experiments is presented.

Table 1 : dosage of the various lactases in the application experiments

The different Bifidobacterium lactases were tested for activity in pasteurized milk by incubation at 6°C and taking samples at various time points, as described above. This method represents the batch process (also called pre-sterilization process).

Dosage of all lactases was based on the protein giving however the most fair comparison between the Bifidobacterium lactases and Maxilact. Since low lactose concentrations are difficult to measure accurately using NMR analysis, we decided to analyze the last time points (48 hr) of these samples using HPAEC-PAD. A detailed description of this method can be found in: Determination fo lactose in lactose-free milk products by high performance anion-exchange

chromatography with pulsed amperometric detection, Pranathi Perati, Brian de Borba and Jeffrey Rohrer, Thermo Fisher Scientific, Sunnyvale, Ca, USA, Application Note 248, pages 1 -8, 2015). With this method very low amounts of lactose can be detected. Table 2 shows a summary of the average amount of lactose in these samples after 48 hr.

These experiments show that there is a clear difference between the milk hydrolyzed with Maxilact and the milk hydrolyzed with the Bifidobacterium lactases.

Table 2: Amount of lactose in milk after 48 hr at 6°C incubated with different lactases

^* calculated based on a generally accepted density of 1 .03

Example 2: Application of the enzymes in the milk post-sterilization process (25°C)

Substrate: "Houdbare Halfvolle melk from Jumbo supermarket" = UHT milk

For each enzyme 2 vials each containing 18.4 ml of semi-skimmed UHT milk was equilibrated at 25°C before start of the experiment. Azide was added to the milk to prevent infection during incubation. Lactases were dosed based on the amount of specific protein at 2 mg/l milk in duplicate and incubation was continued at 25°C. Exactly after 0.5, 2, 6, 24 and 48 h vials were gently mixed and 1.8 mL sample is taken from each vial into an Eppendorf cup. This cup was placed in a thermomixer (10 minutes 90°C). Subsequently the cup was cooled down to room temperature and exactly 1 mL was pipetted in MTP-plate and stored frozen until further analysis (NMR for lactose, glucose and galactose analysis). After 48 hours extra samples were taken to be analyzed using HPEAC-PAD to determine low lactose content. Control sample without enzyme addition was included as well.

In Table 3 the dosage of the various lactases in the application experiments is presented.

Table 3: dosage of the various lactases in the application experiments

The different Bifidobacterium lactases were dosed in UHT milk and incubated at 25°C, representing the aseptic process (also called post-sterilization process). Dosage of all lactases was based on the protein giving however the most fair comparison between the Bifidobacterium lactases and Maxilact.

Since low lactose concentrations are difficult to measure accurately using NMR analysis, we decided to analyze the last time points (48 hr) of these samples using HPAEC-PAD. With this method very low amounts of lactose can be detected. Table 4 shows a summary of the average amount of lactose in these samples after 48 hr.

These experiments show that there is a clear difference between the milk hydrolyzed with Maxilact and the milk hydrolyzed with the Bifidobacterium lactases. Table 4: Amount of lactose in milk after 48 hr at 25°C incubated with different lactases

^* calculated based on a generally accepted density of 1.03

Claims

1 . A milk comprising about 50 mg/L or less lactose obtainable by a process which comprises:

a) providing a milk comprising lactose; and

2. A milk comprising about 50 mg/L or less lactose obtainable by a process which comprises:

a) providing a milk comprising lactose; and

wherein at least a part of the lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or a sequence having at least about 60% sequence identity to any thereto.

3. A milk comprising about 50 mg/L or less lactose obtainable by a process which comprises:

a) providing a milk comprising lactose; and

4. A milk according to claim 1 or 2 or 3, wherein the process is carried out at a temperature of from about 5°C to about 10°C.

5. A milk according to any one of claims 1 to 4, wherein the process is carried out for a time of 20 to 48 hours.

6. A process for the production of a milk comprising about 50 mg/L or less lactose, which process comprises:

a) providing a milk comprising lactose; and

7. A process for the production of a milk comprising about 50 mg/L or less lactose, which process comprises:

a) providing a milk comprising lactose; and

8. A process for the production of a milk comprising about 50 mg/L or less lactose, which process comprises:

a) providing a milk comprising lactose; and

9. A process according to any one of claims 6 to 8, wherein the process is carried out at a temperature of from about 5°C to about 10°C.

10. A process according to any one of claims 6 to 9, wherein the process is carried out for a time of 20 to 48 hours.

1 1 . A milk comprising about 50 mg/L or less lactose obtainable by a process which comprises:

a) providing a milk comprising lactose; and

12. A milk comprising about 50 mg/L or less lactose obtainable by a process which comprises:

a) providing a milk comprising lactose; and

wherein at least a part of the lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO:4 or a sequence having at least about 60% sequence identity to any thereto.

13. A milk comprising about 50 mg/L or less lactose obtainable by a process which comprises:

a) providing a milk comprising lactose; and

14. A milk according to claim 1 1 or 13, wherein the process is carried out at a temperature of at least about 20°C.

15. A process for the production of a milk comprising about 50 mg/L or less lactose which comprises:

a) providing a milk comprising lactose; and

16. A process for the production of a milk comprising about 50 mg/L or less lactose which comprises:

a) providing a milk comprising lactose; and

17. A process for the production of a milk comprising about 50 mg/L or less lactose which comprises:

a) providing a milk comprising lactose; and

18. A process according to any one of claims 15 to 17, wherein the process is carried out at a temperature of at least about 20°C.

19. Use of an enzyme having lactase activity in the production of milk with a low lactose content, which milk is obtainable by a process which comprises:

a) providing a milk comprising lactose; and

20. Use of an enzyme having lactase activity in the production of milk with a low lactose content, which milk is obtainable by a process which comprises:

a) providing a milk comprising lactose; and

21 . Use of an enzyme having lactase activity in the production of milk with a low lactose content, which milk is obtainable by a process which comprises:

a) providing a milk comprising lactose; and

wherein at least a part of the lactase activity is provided by a polypeptide having lactase activity which is derived from Bifidobacterium bifidum

22. Use of an enzyme having lactase activity in the production of milk with a low lactose content, which milk is obtainable by a process which comprises:

a) providing a milk comprising lactose; and b) treating said milk in an aseptic process with an enzyme composition comprising lactase activity,

23. Use of an enzyme having lactase activity in the production of milk with a low lactose content, which milk is obtainable by a process which comprises:

a) providing a milk comprising lactose; and

24. Use of an enzyme having lactase activity in the production of milk with a low lactose content, which milk is obtainable by a process which comprises:

a) providing a milk comprising lactose; and