Improved methods for gos stabilization in yogurt

Abstract

A method for preparing a dairy product having a stable content of GOS fiber, using a neutral lactase that will be inactive at pH below 4.7, and to a fermented GOS enriched milk-based product prepared by the method.

Description

[0001] TITLE IMPROVED METHODS FOR GOS STABILIZATION IN YOGURT CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 513,275, filed July 12, 2023, which is incorporated herein by reference in its entirety. TECHNICAL FIELD The present invention relates to a method for preparing a dairy product having a stable content of GOS fiber, using a neutral lactase that will be inactive at pH below 4.7, and to a fermented GOS enriched milk-based product prepared by the method. REFERENCE TO A SEQUENCE LISTING The contents of the electronic submission of the text file Sequence Listing, named “NB42220USPSP_SequenceListing.xml” was created on July 12, 2023, and is 3 KB in size, which is hereby incorporated by reference in its entirety. BACKGROUND Galactooligosaccharides (GOS) are carbohydrates which are nondigestable in humans and animals comprising two or more galactose molecules, typically up to nine, linked by glycosidic bonds. GOS may also include one or more glucose molecules. One of the beneficial effects of GOS is its ability of acting as prebiotic compounds by selectively stimulating the proliferation of beneficial colonic microorganisms such as bacteria to give physiological benefits to the consumer. The established health effects have resulted in a growing interest in GOS as food ingredients for various types of food. The enzyme β-galactosidase (EC 3.2.1.23) usually hydrolyses lactose to the monosaccharides D-glucose and D-galactose. In the normal enzyme reaction of β- galactosidases, the enzyme hydrolyses lactose and transiently binds the galactose monosaccharide in a galactose-enzyme complex that transfers galactose to the hydroxyl group of water, resulting in the liberation of D-galactose and D-glucose. However, at high lactose concentrations some β-galactosidases are able to transfer galactose to the hydroxyl groups of D- galactose or D-glucose in a process called transgalactosylation whereby galacto-oligosaccharides are produced. At high lactose concentrations some β -galactosidases are able to transfer galactose to the hydroxyl groups of lactose or higher order oligosaccharides. Enzymes and methods for creating high levels of GOS have been developed. See, e.g., Polypeptides Having Transgalactosylating Activity, WO 2013 / 182686. The challenges in production of fresh fermented products having stable GOS is however clear. WO 2015 / 86746 describes, how the content of GOS produced in situ in a milk-based dairy application is not stable over time and that it is highly dependent on the very low amounts of residual beta- galactosidase present in the milk-based product (either milk or fermented product). The residual beta-galactosidase present in the milk-based product after the high temperature pasteurization step (95 °C, 5min), that normally is applied, in for example yogurt production, can be a challenge for the GOS stability in a fresh fermented product. This is in spite of the decrease in pH during fermentation that further retards the beta-galactosidase activity but current GOS yielding beta-galactosidases are not inactivated by this decrease. Currently, the commercially available beta-galactosidases producing relevant yields of GOS are therefore mainly used in the milk-base prior to fermentation, which is an extra step in the process and require a refrigerated holding tank. Extra pasteurization time of the milk can be required to ensure full inactivation of the beta-galactosidase before yogurt production. Alternatively, it is described in WO 2022 / 189568 an example of using the commercially available beta-galactosidases producing relevant yields of GOS in co-fermentation, meaning addition of the beta-galactosidase simultaneous with the addition of the yoghurt culture. However, as the beta-galactosidases are not inactivated by the decrease in pH during fermentation it is required to apply an extra pasteurization step after fermentation in order to obtain stable GOS. This type of extra pasteurization at low pH of 4.3 after fermentation is though only routinely performed for production of ambient stable long shelf life yogurts. Hence, there is a continuing need for methods of stabilizing GOS in fresh fermented and yogurt applications. SUMMARY OF THE INVENTION In accordance with an aspect of the present invention, it has surprisingly been found that a fresh fermented product with stable GOS can be produced when adding a pH sensitive, neutral beta-galactosidase that is inactivated at pH below 4.6, simultaneous with the addition of the yoghurt culture, and without using any additional pasteurization steps. The method according to the present invention is much simpler and can be applied for all fresh fermented products. In accordance with an aspect of the present invention, a method is presented of producing a yoghurt having a stable composition of DP3+ GOS having the steps of: a. providing a milk- based substrate having lactose; b. treating the milk-based substrate with a pH sensitive, neutral beta-galactosidase enzyme to generate DP3+ GOS and c. acidifying the milk-based substrate to a pH 5.0 or below to inactivate the beta-galactosidase to provide the yoghurt having the stable composition of DP3+ GOS. Optionally, the pH is below 4.9, 4.8 or 4.7. Optionally, the pH is at 4.6. Optionally, the milk-based substrate has a lactose concentration of between 1-60% (w / w); 2–50 % (w / w), 3-40 % (w / w); or 4-30 % (w / w); or 4-15 % (w / w). Optionally, the beta-galactosidase has less than 10, 5, 4, 3, 2, 1, or 0.1 % of its maximal activity at a pH 5.0 or under. Optionally, the beta-galactosidase has less than 10, 5, 4, 3, 2, 1, or 0.1 % of its maximal activity at pH 4.6. Optionally, the pH sensitive, neutral beta-galactosidase is derived from Lactobacillus delbrueckii bulgaricus. Optionally, the pH sensitive beta-galactosidase is a polypeptide having at least 70% sequence identity to SEQ ID NO:1 or a beta-galactosidase active fragment thereof. Optionally, the polypeptide has at least 80% sequence identity to SEQ ID NO:1 or a beta- galactosidase active fragment thereof. Optionally, the polypeptide has at least 90% sequence identity to SEQ ID NO:1 or a beta-galactosidase active fragment thereof. Optionally, the polypeptide has at least 95% sequence identity to SEQ ID NO:1 or a beta-galactosidase active fragment thereof. Optionally, the polypeptide has at least 98% sequence identity to SEQ ID NO:1 or a beta-galactosidase active fragment thereof. Optionally, the polypeptide has at least 99% sequence identity to SEQ ID NO:1 or a beta-galactosidase active fragment thereof. Optionally, the polypeptide is a sequence according to SEQ ID NO:1 or a beta-galactosidase active fragment thereof. Optionally, the polypeptide is a sequence according to SEQ ID NO:1. Optionally, the step of acidifying comprises adding a culture and fermenting. Optionally, the yoghurt is an acidified milk product, a set yoghurt, a standing yoghurt, a stirred yoghurt, or a drinking yoghurt. Optionally, the composition of DP3+ GOS in the yoghurt decreases by less than 5, 4, 3, 2, 1, or 0.1% after 5, 10, 15, 21, 28 or 56 days of storage after step c. Optionally, the beta-galactosidase produces at least 1.5, 2, 3, 4, or 5 g of DP3+ GOS per 100 Calories of yoghurt. Optionally, the step of acidifying comprises adding a chemical acidifying agent. Optionally, the acidifying agents is acetic acid, citric acid, lactic acid, malic acid, succinic acid, tartaric acid or glucono-delta-lactone. BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES SEQ ID NO: 1 – depicts the amino acid sequence of the mature form of B-galactosidase from Lactobacillus delbrueckii bulgaricus. DESCRIPTION OF FIGURES FIG. 1. Depicts % Galactose relative to total carbohydrate of trial 1-7 measured in samples extracted over 56 days of shelf life. DETAILED DESCRIPTION Definitions To employ the claim “source of fiber” on a food product, various regulatory authorities require that the product contains at least 3 g of fiber per 100 g or at least 1.5 g of fiber per 100kcal. The claim “high in fiber” may only be made where the product contains at least 6 g of fiber per 100 g or at least 3 g of fiber per 100 kcal. It is estimated that >1% GOS would be required for a full fat yoghurt to be a “source of fiber” or at least 1.25% DP3+ to be “high in fiber” in a low fat fresh fermented product. Relevant levels of DP3+ is therefore defined as >= 1.0% (w / w) DP3+. A “beta-galactosidase” is glycoside hydrolase that catalyzes the hydrolysis of beta- galactosides including lactose, into monosaccharides. A b-galactosidase is also sometimes called a “lactase.” Under some conditions, a beta-galactosidase catalyzes the formation of GOS. The term “galactooligosaccharides” also referred to herein as “GOS” refers to nondigestable oligosaccharides composed of from 2 to 20 molecules of predominantly galactose. GOS is typically formed by beta-galactosidase enzymes, also called lactases by degrading lactose in e.g. milk and / or milk-based products. The term “GOS fiber” herein refers to nondigestible galactooligosaccharides with a degree of polymerization of 3 or more (DP3+). The term “amino acid sequence” is synonymous with the terms “polypeptide,” “protein,” and “peptide,” and are used interchangeably. Where such amino acid sequences exhibit activity, they may be referred to as an “enzyme.” The conventional one-letter or three- letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino-to-carboxy terminal orientation (i.e., N→C). The term “nucleic acid” encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded. The terms “nucleic acid” and “polynucleotide” are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5′-to-3′ orientation. A “vector” refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like. An “expression vector” refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation. In addition to the specific amino acid sequences and polynucleotides mentioned herein, the present invention encompasses variants, homologues, derivatives and fragments thereof. The term "variant" is used to mean a nucleotide sequence or amino acid sequence which differs from a wild-type sequence. For example, a variant may include substitutions, insertions, deletions, truncations, transversions and / or inversions at one or more position(s) relative to a wild-type sequence. Variants can be made using methods known in the art for example site scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis and directed-evolution as well as using recombinant methods well known in the art. Polynucleotide sequences encoding variant amino acid sequences may readily be synthesized using methods known in the art. In some aspects, the variant is a naturally occurring nucleotide sequence or amino acid sequence which differs from a wild-type sequence. For example, the variant may be a natural genetic variant. In some aspects, the variant is an engineered variant. For example, the variant may be engineered by recombinant methods. The protein sequences of the instant invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine. Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other as set forth in Table 1.

[0002] Table 1 ALIPHATIC Non-polar G A P I L V e presen nven on a so encompasses omo ogous su s u on (su stitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine. Replacements may also be made by synthetic amino acids (e.g. unnatural amino acids) include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br- phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, ß-alanine*, L-a-amino butyric acid*, L-g- amino butyric acid*, L-a-amino isobutyric acid*, L-e-amino caproic acid#, 7-amino heptanoic acid*, L-methionine sulfone#*, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L- hydroxyproline#, L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl- Phe*, pentamethyl-Phe*, L-Phe (4-amino)#, L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionic acid#and L-Phe (4- benzyl)*. The notation * has been utilized for the purpose of the discussion above (relating to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative whereas # has been utilized to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics. Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or b-alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, “the peptoid form” is used to refer to variant amino acid residues wherein the a-carbon substituent group is on the residue’s nitrogen atom rather than the a-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134. The nucleotide sequences for use in the present invention may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and / or the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences of the present invention. The present invention also encompasses the use of nucleotide sequences that are complementary to the sequences presented herein. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. In addition, other homologues may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridizing to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries or genomic DNA libraries made from other animal species and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention. Variants and strain / species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants / homologues. Sequence alignments can be performed using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used. The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences. Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterized sequences. This may be useful where for example silent codon sequence changes are required to optimize codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides. The present invention employs, unless otherwise indicated, conventional techniques of biochemistry, molecular biology, microbiology and recombinant DNA, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N. Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IrI Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference. As used herein, “percent (%) sequence identity” means that a particular sequence has at least a certain percentage of amino acid residues identical to those in a specified reference sequence, when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the CLUSTAL W algorithm are: Gap opening penalty: 10.0 Gap extension penalty: 0.05 Protein weight matrix:BLOSUM series DNA weight matrix: IUB Delay divergent sequences %: 40 Gap separation distance: 8 DNA transitions weight: 0.50 List hydrophilic residues: GPSNDQEKR Use negative matrix: OFF Toggle Residue specific penalties: ON Toggle hydrophilic penalties: ON Toggle end gap separation penalty: OFF Deletions are counted as non-identical residues, compared to a reference sequence. Deletions occurring at either terminus are included. For example, a variant with five amino acid deletions of the C-terminus of the mature 617 residue polypeptide would have a percent sequence identity of 99% (612 / 617 identical residues × 100, rounded to the nearest whole number) relative to the mature polypeptide. Such a variant would be encompassed by a variant having “at least 99% sequence identity” to a mature polypeptide. In accordance with the instant invention, proteins, including enzymes, of the present invention exist in multiple forms. Proteins of the instant invention may be clipped or trimmed (i.e., removing amino acids) from the N-terminus and / or the C-terminus, resulting in a shorter protein. Proteins of the instant invention can also have internal deletions. Shorter proteins as described herein can have higher activity or lower activity than longer counterparts. Without being bound by theory, as used herein the term “pre-pro-protein” is a protein, including an enzyme, which has an N-terminal signal peptide that targets the protein for secretion. A pre-pro- protein is sometimes referred to herein as “full length” or “full length protein”. The N-terminal signal peptide is cleaved off in the endoplasmic reticulum to yield a “pro-protein”. A pro- protein, as used herein, is shorter in length than the full length protein (it is missing the signal peptide) but longer than the mature protein. In general, a pro-protein is inactive or less active than the mature protein. A pro-protein can be activated or converted to a more active mature form by post-translational modification such as N- or C- terminal clipping. A pro-protein which is an enzyme may be called a “proenzyme” or a “zymogen.” The clipped active protein (derived from the pro-protein) is also referred to herein as the mature protein. It is to be noted that the above terms are used for convenience and are not meant to override or determine the activities of a protein of the instant invention. It is also to be noted that any particular protein of the instant invention can have more than one variant described by the same term. All references cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings of all references herein specifically referred to are incorporated by reference. Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention. Other definitions are set forth below. Production of enzymes The enzymes of the present invention can be produced in host cells, for example, by secretion or intracellular expression. A cultured cell material (e.g., a whole-cell broth) having an enzyme can be obtained following secretion of the enzyme into the cell medium. Optionally, the enzyme can be isolated from the host cells, or even isolated from the cell broth, depending on the desired purity of the final enzyme. Suitable host cells include bacterial, fungal (including yeast and filamentous fungi), and plant cells (including algae). Particularly useful host cells include Aspergillus niger, Aspergillus oryzae or Trichoderma reesei. Other host cells include bacterial cells, e.g., Bacillus subtilis or B. licheniformis, as well as Streptomyces, E. coli. Vectors A DNA construct comprising a nucleic acid encoding an enzyme can be constructed to be expressed in a host cell. Because of the well-known degeneracy in the genetic code, variant polynucleotides that encode an identical amino acid sequence can be designed and made with routine skill. It is also well-known in the art to optimize codon use for a particular host cell. Nucleic acids encoding enzymes of the present invention can be incorporated into a vector. Vectors can be transferred to a host cell using well-known transformation techniques, such as those disclosed below. The vector may be any vector that can be transformed into and replicated within a host cell. For example, a vector comprising a nucleic acid encoding an enzyme can be transformed and replicated in a bacterial host cell as a means of propagating and amplifying the vector. The vector also may be transformed into an expression host, so that the encoding nucleic acids can be expressed as a functional enzyme. Host cells that serve as expression hosts can include filamentous fungi, for example. The Fungal Genetics Stock Center (FGSC) Catalogue of Strains lists suitable vectors for expression in fungal host cells. See FGSC, Catalogue of Strains, University of Missouri, at www.fgsc.net (last modified January 17, 2007). A representative vector is pJG153, a promoterless Cre expression vector that can be replicated in a bacterial host. See Harrison et al. (June 2011) Applied Environ. Microbiol. 77: 3916-22. pJG153can be modified with routine skill to comprise and express a nucleic acid encoding an enzyme. A nucleic acid encoding an enzyme can be operably linked to a suitable promoter, which allows transcription in the host cell. The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Exemplary promoters for directing the transcription of the DNA sequence encoding an enzyme, especially in a bacterial host, are the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA or celA promoters, the promoters of the Bacillus licheniformis α-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens α-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, A. niger acid stable α-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, or A. nidulans acetamidase. When a gene encoding an enzyme is expressed in a bacterial species such as E. coli, a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter. Examples of suitable promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters. cbh1 is an endogenous, inducible promoter from Trichoderma reesei. See Liu et al. (2008) “Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbh1) promoter optimization,” Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158-65. The coding sequence can be operably linked to a signal sequence. The DNA encoding the signal sequence may be the DNA sequence naturally associated with the enzyme gene to be expressed or from a different Genus or species. A signal sequence and a promoter sequence comprising a DNA construct or vector can be introduced into a fungal host cell and can be derived from the same source. For example, the signal sequence is the cbh1 signal sequence that is operably linked to a cbh1 promoter. An expression vector may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding a variant enzyme. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter. The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1, and pIJ702. The vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance such as, e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and xxsC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, such as known in the art. See e.g., International PCT Application WO 91 / 17243. Intracellular expression may be advantageous in some respects, e.g., when using certain bacteria or fungi as host cells to produce large amounts of enzyme for subsequent enrichment or purification. Extracellular secretion of enzyme into the culture medium can also be used to make a cultured cell material comprising the isolated enzyme. The expression vector typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes. The expression vector normally comprises control nucleotide sequences such as a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activator genes. Additionally, the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the enzyme to a host cell organelle such as a peroxisome, or to a particular host cell compartment. Such a targeting sequence includes but is not limited to the sequence, SKL. For expression under the direction of control sequences, the nucleic acid sequence of the enzyme is operably linked to the control sequences in proper manner with respect to expression. The procedures used to ligate the DNA construct encoding an enzyme, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (see, e.g., Sambrook et al., MOLECULARCLONING: A LABORATORYMANUAL, 2nded., Cold Spring Harbor, 1989, and 3rded., 2001). Transformation and Culture of Host Cells An isolated cell, either comprising a DNA construct or an expression vector, is advantageously used as a host cell in the recombinant production of an enzyme according to the instant invention. The cell may be transformed with the DNA construct encoding the enzyme, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells. Examples of suitable bacterial host organisms are Gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis; Streptomyces species such as Streptomyces murinus; lactic acid bacterial species including Lactococcus sp. such as Lactococcus lactis; Lactobacillus sp. including Lactobacillus reuteri; Leuconostoc sp.; Pediococcus sp.; and Streptococcus sp. Alternatively, strains of a Gram negative bacterial species belonging to Enterobacteriaceae including E. coli, or to Pseudomonadaceae can be selected as the host organism. A suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as but not limited to yeast species such as Pichia sp., Hansenula sp., or Kluyveromyces, Yarrowinia, Schizosaccharomyces species or a species of Saccharomyces, including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyces such as, for example, S. pombe species. A strain of the methylotrophic yeast species, Pichia pastoris, can be used as the host organism. Alternatively, the host organism can be a Hansenula species. Suitable host organisms among filamentous fungi include species of Aspergillus, e.g., Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or Aspergillus nidulans. Alternatively, strains of a Fusarium species, e.g., Fusarium oxysporum or of a Rhizomucor species such as Rhizomucor miehei can be used as the host organism. Other suitable strains include Thermomyces and Mucor species. In addition, Trichoderma sp. can be used as a host. A suitable procedure for transformation of Aspergillus host cells includes, for example, that described in EP 238023. An enzyme expressed by a fungal host cell can be glycosylated, i.e., will comprise a glycosyl moiety. The glycosylation pattern can be the same or different as present in the wild-type enzyme. The type and / or degree of glycosylation may impart changes in enzymatic and / or biochemical properties. It may be advantageous to delete genes from expression hosts, where the gene deficiency can be cured by the transformed expression vector. Known methods may be used to obtain a fungal host cell having one or more inactivated genes. Gene inactivation may be accomplished by complete or partial deletion, by insertional inactivation or by any other means that renders a gene nonfunctional for its intended purpose, such that the gene is prevented from expression of a functional protein. Any gene from a Trichoderma sp. or other filamentous fungal host that has been cloned can be deleted, for example, cbh1, cbh2, egl1, and egl2 genes. Gene deletion may be accomplished by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art. Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, e.g., lipofection mediated and DEAE-Dextrin mediated transfection; incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion. General transformation techniques are known in the art. See, e.g., Sambrook et al. (2001), supra. The expression of heterologous protein in Trichoderma is described, for example, in U.S. Patent No. 6,022,725. Reference is also made to Cao et al. (2000) Science 9:991-1001 for transformation of Aspergillus strains. Genetically stable transformants can be constructed with vector systems whereby the nucleic acid encoding an enzyme is stably integrated into a host cell chromosome. Transformants are then selected and purified by known techniques. The preparation of Trichoderma sp. for transformation, for example, may involve the preparation of protoplasts from fungal mycelia. See Campbell et al. (1989) Curr. Genet. 16: 53- 56. The mycelia can be obtained from germinated vegetative spores. The mycelia are treated with an enzyme that digests the cell wall, resulting in protoplasts. The protoplasts are protected by the presence of an osmotic stabilizer in the suspending medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate, and the like. Usually, the concentration of these stabilizers varies between 0.8 M and 1.2 M, e.g., a 1.2 M solution of sorbitol can be used in the suspension medium. Uptake of DNA into the host Trichoderma sp. strain depends upon the calcium ion concentration. Generally, between about 10-50^mM CaCl2is used in an uptake solution. Additional suitable compounds include a buffering system, such as TE buffer (10^mM Tris, pH^7.4; 1^mM EDTA) or 10^mM MOPS, pH 6.0 and polyethylene glycol. The polyethylene glycol is believed to fuse the cell membranes, thus permitting the contents of the medium to be delivered into the cytoplasm of the Trichoderma sp. strain. This fusion frequently leaves multiple copies of the plasmid DNA integrated into the host chromosome. Usually, transformation of Trichoderma sp. uses protoplasts or cells that have been subjected to a permeability treatment, typically at a density of 105to 107 / mL, particularly 2x106 / mL. A volume of 100 μL of these protoplasts or cells in an appropriate solution (e.g., 1.2^M sorbitol and 50^mM CaCl2) may be mixed with the desired DNA. Generally, a high concentration of PEG is added to the uptake solution. From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension; however, it is useful to add about 0.25 volumes to the protoplast suspension. Additives, such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like, may also be added to the uptake solution to facilitate transformation. Similar procedures are available for other fungal host cells. See, e.g., U.S. Patent No. 6,022,725. As used herein, Protein Identification (“JGI PID”) numbers for native Trichoderma genes reference Version 2 of the Trichoderma reesei QM6a genome sequence assembly generated by the Department of Energy Joint Genome Institute (JGI). (The Genome Portal of the Department of Energy Joint Genome Institute, Grigoriev et al., Nucleic Acids Res 2012 Jan;40(Database issue):D26-32. doi: 10.1093 / nar / gkr947). The JGI assembled Scaffold sequences and annotated genes have also been deposited in GeneBank (The National Center for Biotechnology) under the nucleotide accession numbers GL985056.1 through GL985132.1. Expression A method of producing an enzyme of the instant invention may comprise cultivating a host cell as described above under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and / or culture medium. The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of an enzyme. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection). An enzyme secreted from the host cells can be used in a whole broth preparation. In the present methods, the preparation of a spent whole fermentation broth of a recombinant microorganism can be achieved using any cultivation method known in the art resulting in the expression of an enzyme. Fermentation may, therefore, be understood as comprising shake flask cultivation, small- or large-scale fermentation (including continuous, batch, fed-batch, or solid- state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the enzyme to be expressed or isolated. The term “spent whole fermentation broth” is defined herein as unfractionated contents of fermentation material that includes culture medium, extracellular proteins (e.g., enzymes), and cellular biomass. It is understood that the term “spent whole fermentation broth” also encompasses cellular biomass that has been lysed or permeabilized using methods well known in the art. An enzyme secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like. The polynucleotide encoding an enzyme in a vector can be operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators. The control sequences may in particular comprise promoters. Host cells may be cultured under suitable conditions that allow expression of an enzyme. Expression of the enzymes may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG or Sophorose. Polypeptides can also be produced recombinantly in an in vitro cell-free system, such as the TNT™ (Promega) rabbit reticulocyte system. An expression host also can be cultured in the appropriate medium for the host, under aerobic conditions. Shaking or a combination of agitation and aeration can be provided, with production occurring at the appropriate temperature for that host, e.g., from about 25°C to about 75°C (e.g., 30°C to 45°C), depending on the needs of the host and production of the desired enzyme. Culturing can occur from about 12 to about 100 hours or greater (and any hour value there between, e.g., from 24 to 72 hours). Typically, the culture broth is at a pH of about 4.0 to about 8.0, again depending on the culture conditions needed for the host relative to production of an enzyme. Methods for Enriching and Purifying enzymes Fermentation, separation, and concentration techniques are well known in the art and conventional methods can be used in order to prepare an enzyme polypeptide-containing solution. After fermentation, a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques in order to obtain an enzyme solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultra- filtration, extraction, or chromatography, or the like, are generally used. It is desirable to concentrate an enzyme polypeptide-containing solution in order to optimize recovery. Use of unconcentrated solutions requires increased incubation time in order to collect the enriched or purified enzyme precipitate. The enzyme containing solution is concentrated using conventional concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme containing solution may be achieved by any of the techniques discussed herein. Exemplary methods of enrichment and purification include but are not limited to rotary vacuum filtration and / or ultrafiltration. The enzyme solution is concentrated into a concentrated enzyme solution until the enzyme activity of the concentrated enzyme polypeptide-containing solution is at a desired level. Concentration may be performed using, e.g., a precipitation agent, such as a metal halide precipitation agent. Metal halide precipitation agents include but are not limited to alkali metal chlorides, alkali metal bromides and blends of two or more of these metal halides. Exemplary metal halides include sodium chloride, potassium chloride, sodium bromide, potassium bromide and blends of two or more of these metal halides. The metal halide precipitation agent, sodium chloride, can also be used as a preservative. The metal halide precipitation agent is used in an amount effective to precipitate an enzyme. The selection of at least an effective amount and an optimum amount of metal halide effective to cause precipitation of the enzyme, as well as the conditions of the precipitation for maximum recovery including incubation time, pH, temperature and concentration of enzyme, will be readily apparent to one of ordinary skill in the art, after routine testing. Generally, at least about 5% w / v (weight / volume) to about 25% w / v of metal halide is added to the concentrated enzyme solution, and usually at least 8% w / v. Generally, no more than about 25% w / v of metal halide is added to the concentrated enzyme solution and usually no more than about 20% w / v. The optimal concentration of the metal halide precipitation agent will depend, among others, on the nature of the specific enzyme polypeptide and on its concentration in the concentrated enzyme solution. Another alternative way to precipitate the enzyme is to use organic compounds. Exemplary organic compound precipitating agents include: 4-hydroxybenzoic acid, alkali metal salts of 4-hydroxybenzoic acid, alkyl esters of 4-hydroxybenzoic acid, and blends of two or more of these organic compounds. The addition of the organic compound precipitation agents can take place prior to, simultaneously with or subsequent to the addition of the metal halide precipitation agent, and the addition of both precipitation agents, organic compound and metal halide, may be carried out sequentially or simultaneously. Generally, the organic precipitation agents are selected from the group consisting of alkali metal salts of 4-hydroxybenzoic acid, such as sodium or potassium salts, and linear or branched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 12 carbon atoms, and blends of two or more of these organic compounds. The organic compound precipitation agents can be, for example, linear or branched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 10 carbon atoms, and blends of two or more of these organic compounds. Exemplary organic compounds are linear alkyl esters of 4- hydroxybenzoic acid, wherein the alkyl group contains from 1 to 6 carbon atoms, and blends of two or more of these organic compounds. Methyl esters of 4-hydroxybenzoic acid, propyl esters of 4-hydroxybenzoic acid, butyl ester of 4-hydroxybenzoic acid, ethyl ester of 4-hydroxybenzoic acid and blends of two or more of these organic compounds can also be used. Additional organic compounds also include but are not limited to 4-hydroxybenzoic acid methyl ester (named methyl PARABEN), 4-hydroxybenzoic acid propyl ester (named propyl PARABEN), which also are both preservative agents. For further descriptions, see, e.g., U.S. Patent No. 5,281,526. Addition of the organic compound precipitation agent provides the advantage of high flexibility of the precipitation conditions with respect to pH, temperature, enzyme concentration, precipitation agent concentration, and time of incubation. The organic compound precipitation agent is used in an amount effective to improve precipitation of the enzyme by means of the metal halide precipitation agent. The selection of at least an effective amount and an optimum amount of organic compound precipitation agent, as well as the conditions of the precipitation for maximum recovery including incubation time, pH, temperature and concentration of enzyme, will be readily apparent to one of ordinary skill in the art, in light of the present disclosure, after routine testing. Generally, at least about 0.01% w / v of organic compound precipitation agent is added to the concentrated enzyme solution and usually at least about 0.02% w / v. Generally, no more than about 0.3% w / v of organic compound precipitation agent is added to the concentrated enzyme solution and usually no more than about 0.2% w / v. The concentrated polypeptide solution, containing the metal halide precipitation agent, and the organic compound precipitation agent, can be adjusted to a pH, which will, of necessity, depend on the enzyme to be enriched or purified. Generally, the pH is adjusted at a level near the isoelectric point of the enzyme. The pH can be adjusted at a pH in a range from about 2.5 pH units below the isoelectric point (pI) up to about 2.5 pH units above the isoelectric point. The incubation time necessary to obtain an enriched or purified enzyme precipitate depends on the nature of the specific enzyme, the concentration of enzyme, and the specific precipitation agent(s) and its (their) concentration. Generally, the time effective to precipitate the enzyme is between about 1 to about 30 hours; usually it does not exceed about 25 hours. In the presence of the organic compound precipitation agent, the time of incubation can still be reduced to less about 10 hours and in most cases even about 6 hours. Generally, the temperature during incubation is between about 4°C and about 50°C. Usually, the method is carried out at a temperature between about 10°C and about 45°C (e.g., between about 20°C and about 40°C). The optimal temperature for inducing precipitation varies according to the solution conditions and the enzyme or precipitation agent(s) used. The overall recovery of enriched or purified enzyme precipitate, and the efficiency with which the process is conducted, is improved by agitating the solution comprising the enzyme, the added metal halide and the added organic compound. The agitation step is done both during addition of the metal halide and the organic compound, and during the subsequent incubation period. Suitable agitation methods include mechanical stirring or shaking, vigorous aeration, or any similar technique. After the incubation period, the enriched or purified enzyme is then separated from the dissociated pigment and other impurities and collected by conventional separation techniques, such as filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, press filtration, cross membrane microfiltration, cross flow membrane microfiltration, or the like. Further enrichment or purification of the enzyme precipitate can be obtained by washing the precipitate with water. For example, the enriched or purified enzyme precipitate is washed with water containing the metal halide precipitation agent, or with water containing the metal halide and the organic compound precipitation agents. During fermentation, an enzyme polypeptide accumulates in the culture broth. For the isolation, enrichment, or purification of the desired enzyme, the culture broth is centrifuged or filtered to eliminate cells, and the resulting cell-free liquid is used for enzyme enrichment or purification. In one embodiment, the cell-free broth is subjected to salting out using ammonium sulfate at about 70% saturation; the 70% saturation-precipitation fraction is then dissolved in a buffer and applied to a column such as a Sephadex G-100 column and eluted to recover the enzyme-active fraction. For further enrichment or purification, a conventional procedure such as ion exchange chromatography may be used. Enriched or purified enzymes can be made into a final product that is either liquid (solution, slurry) or solid (granular, powder). Description of the Preferred Embodiments In accordance with an aspect of the present invention, a method is presented of producing a yoghurt having a stable composition of DP3+ GOS having the steps of: a. providing a milk- based substrate having lactose; b. treating the milk-based substrate with a pH sensitive, neutral beta-galactosidase enzyme to generate DP3+ GOS and c. acidifying the milk-based substrate to a pH 5.0 or below to inactivate the beta-galactosidase to provide the yoghurt having the stable composition of DP3+ GOS. Preferably, the pH is below 4.9, 4.8 or 4.7. More preferably, the pH is at 4.6. Preferably, the milk-based substrate has a lactose concentration of between 1-60% (w / w); 2–50 % (w / w), 3-40 % (w / w); or 4-30 % (w / w); or 4-15 % (w / w). Preferably, the beta-galactosidase has less than 10, 5, 4, 3, 2, 1, or 0.1 % of its maximal activity at a pH 5.0 or under. More preferably, the beta-galactosidase has less than 10, 5, 4, 3, 2, 1, or 0.1 % of its maximal activity at pH 4.6. Preferably, the pH sensitive, neutral beta-galactosidase is derived from Lactobacillus delbrueckii bulgaricus. More preferably, the pH sensitive beta-galactosidase is a polypeptide having at least 70% sequence identity to SEQ ID NO:1 or a beta-galactosidase active fragment thereof. Yet more preferably, the polypeptide has at least 80% sequence identity to SEQ ID NO:1 or a beta-galactosidase active fragment thereof. In still more preferred embodiments, the polypeptide has at least 90% sequence identity to SEQ ID NO:1 or a beta-galactosidase active fragment thereof. In still more preferred embodiments, the polypeptide has at least 95% sequence identity to SEQ ID NO:1 or a beta-galactosidase active fragment thereof. Still more preferably, the polypeptide has at least 98% sequence identity to SEQ ID NO:1 or a beta-galactosidase active fragment thereof. Yet more preferably, the polypeptide has at least 99% sequence identity to SEQ ID NO:1 or a beta-galactosidase active fragment thereof. In yet more preferred embodiments, the polypeptide is a sequence according to SEQ ID NO:1 or a beta-galactosidase active fragment thereof. In the most preferred embodiments, the polypeptide is a sequence according to SEQ ID NO:1. Preferably, the step of acidifying comprises adding a culture and fermenting. Preferably, the yoghurt is an acidified milk product, a set yoghurt, a standing yoghurt, a stirred yoghurt, or a drinking yoghurt. Preferably, the composition of DP3+ GOS in the yoghurt decreases by less than 5, 4, 3, 2, 1, or 0.1% after 5, 10, 15, 21, 28 or 56 days of storage after step c. Preferably, the beta-galactosidase produces at least 1.5, 2, 3, 4, or 5 g of DP3+ GOS per 100 Calories of yoghurt. Preferably, the step of acidifying comprises adding a chemical acidifying agent. Preferably, the acidifying agents is acetic acid, citric acid, lactic acid, malic acid, succinic acid, tartaric acid or glucono-delta-lactone. EXAMPLES Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, certain changes and modifications can be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety for all purposes to the same extent as if each reference was individually incorporated by reference. To the extent the content of any citation, including website or accession number may change with time, the version in effect at the filing date of this application is meant. Unless otherwise apparent from the context any step, element, aspect, feature of embodiment can be used in combination with any other. Method for quantification of GOS (DP3 and above) Quantification of galacto-oligosaccharides by HPLC The standard lactose (HPLC analytical grade, Sigma Aldrich) was prepared in double distilled water (ddH2O) and filtered through 0.2µm syringe filters. A dilution series ranging from 500 to 10000 ppm of the lactose standard was created. Similar sample preparation of milk-base and yogurt samples was applied, however utilizing a dilution factor of 5x and 10x respectively. A milk-base sample was diluted 5x: 200mg sample (weight noted) in 800µL H2O, mixed thoroughly and inactivated 20 minutes in boiling water and following cooled. 50µL Carrez reagent A (Carrez Clarification Kit, 1.10537.001, Sigma Aldrich) and 50 µL Carrez B was added to 1000µL diluted sample to induce protein and lipid precipitation. The sample mixture was incubated 15 minutes at room temperature and 50µL 10mM NaOH, 1 mM EDTA was added to the sample. The sample was centrifuged at 10.000 rpm for 4 minutes and 300µL clarified supernatant was transferred to an MTP filter plate, through 0.20 µm 96 well plate filters (centrifuged 3000 rpm in 15 minutes) before analysis (Corning filter plate, PVDF hydrophile membrane, NY, USA). All samples were analyzed duplicate and in 96 well MTP plates sealed with tape. Instrumentation Quantification of galacto-oligosaccharides (GOS), lactose, glucose and galactose were performed by HPLC. Analysis of samples was carried out on a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific) equipped with a DGP-3600SD Dual-Gradient analytical pump, WPS-3000TSL thermostated autosampler, TCC-3000SD thermostated column oven, and a RI-101 refractive index detector (Shodex, JM Science). Chromeleon datasystem software (Version 6.80, DU10A Build 2826, 171948) was used for data acquisition and analysis. Chromatographic conditions The samples were analyzed by HPLC using an RSO oligosaccharide column, Ag+4% crosslinked (Phenomenex, The Netherlands) equipped with an analytical guard column (Carbo- Ag+neutral, AJ0-4491, Phenomenex, The Netherlands) operated at 70°C. The column was eluted with double distilled water at a flow rate of 0.3 ml / min. Isocratic flow of 0.3 ml / min was maintained throughout analysis with a total run time of 45 minutes and injection volume was set to 20 µL. Samples were held at 30°C in the thermostated autosampler compartment to ensure solubilization of all components. The eluent was monitored by means of a refractive index detector (RI-101, Shodex, JM Science) and quantification was made by the peak area relative to the peak area of lactose as described above. Peaks with a degree of three or higher (DP3+) were quantified as galactooligosaccharides (DP3, DP4, DP5 and so forth). The assumption of the same response for all DP3+ galacto-oligosaccharides components was confirmed with mass balances. Lactose including other GOS DP2 components was quantified as DP2, glucose, galactose and sucrose in a similar manner. Example 1. Enzyme residual activity Experimental Dupont lactase: A thermostable β-galactosidase from Lactobacillus delbrueckii bulgaricus having the amino acid sequence shown in SEQ ID NO:1. As an example of a hydrolyzing lactase from Kluyveromyces lactis, GODO-YNL2 (available from DuPont, Denmark) was used. Another example of a transgalactosylating lactase from Bifidobacterium bifidum is commercially available as Saphera fiber (available from Novozymes, Denmark). As another example of a transgalactosylating lactase Nurica, a Bifidumbacterium bifidum lactase (available from DuPont, Denmark) were utilized. A 10% (w / w) skimmed milk solution was prepared and aliquoted in 100ml portions. pH was adjusted with 20% lactic acid to either 4.0 - 4.6 - 5.0 - 5.5 - 6.0 or kept at pH 6.6. All samples were preheated to 43°C and then added either 0.8 g / L LBul, 0.7 g / L KLac or 2.1 g / L Nurica. The samples were further incubated for 5 hours at 43°C, before transfer to ice. A reference sample without enzyme was also incubated for each pH. All samples were centrifuged 300 rpm for 10 min and supernatant was collected. The supernatant was diluted either 10 (LBul added samples), 3 (KLac added samples) or 2 times (Nurica added samples) in deionized water. 500µL of the diluted supernatants were preheated 2 min to 30°C before addition of 750µL of 3.7mg / ml ONPG substrate in 100mM MES Buffer pH 6.4. After 10 min incubation, the reaction was stopped with 750 µL sodium carbonate stop buffer (50 g of Na2CO3 and 37.2 g Na2EDTA in 1000 ml of distilled water). OD420 was read. Residual activity was calculated at % relative to the sample with highest OD420 response. The results showed (see Table 2) that both LBul and KLac had less than 10% activity remaining after incubation at pH 5 and would both be inactive after incubation at pH 4.6. Table 2. Enzyme Activity at Various pHs pH LBul KLac Nurica Example 2. Yoghurt production Pre-pasteurized (72^°C for 15 s) bulk blended skimmed milk (0.1^% fat) (Arla Foods, Denmark) stored at 4-6 °C was standardized to 7.5 % w / w Lactose content by addition of Variolac® 992 BG100 (from Arla Foods Denmark) either with or without 5% sucrose (Granulated Sugar 500, Nordic Sugar A / S, Denmark). The standardized milk was then pasteurized and homogenized in a standard plate heat exchange pasteurizer. Homogenization was performed at 65 °C at 200 bar and pasteurization at 95^°C for 6 minutes, and then milk was cooled to 43^°C. Following, the milk-base was fermented to a yoghurt. Various amounts of either KLac, LBul, Nurica or Saphera fiber lactase was added according to table 3 together with starter culture YO-MIX 410 or YO-MIX Prime 900 (20DCU / 100L). Fermentation was carried out until a pH of 4.6. All samples were then cooled to 5°C. Samples were extracted and frozen at various timepoints over shelf life until they were analyzed for DP3+ GOS and other carbohydrates according to the method. Table 3 Trial nr LBul (g / L) Lactose (%) Sucrose (%) Culture Results from analysis of day 0 samples are shown in table 4 in which calories are calculated from the measured carbohydrates, 3.94% protein and 0.1% fat. It was clear that relevant levels of > 1% DP3+ GOS had been produced in all trials except for trial 5. Hence, in trial 5 the DP3+ pr 100 calories were less than 1.5g and therefore these were not relevant levels. Table 4. Carbohydrates and calories in the yoghurts at day 0. DP3+ Total Calories g DP3+ % b h d 100 Samples extracted over shelf life was also analyzed according to the method. The results of DP3+ GOS stability is shown in table 5 and the galactose levels are shown in figure 1. It was found that the DP3+ GOS levels were stable in trial 1-5 which is supported by the fact that there was no significant increase in galactose over shelf life for these trials either. This support the fact that both LBul and KLac are inactivated by pH decrease during fermentation. In contrast, it was observed that the DP3+ GOS levels were not stable in trial 6 and 7 and that an increase in galactose was observed. This clearly show that the Saphera fiber and Nurica are not inactivated by pH during fermentation and will therefore degrade the formed DP3+ GOS and release galactose over shelf life. It was therefore only possible to reach relevant levels of DP3+ GOS as well as stable GOS if using a GOS producing neutral beta-galactosidase that is inactivated during fermentation at pH below 4.7. Table 5. % DP3+ relative to total carbohydrate of trial 1-7 measured in samples extracted over 56 days of shelf life. % DP3+ relative to total carbohydrate Da 0 Da 1 Da 14 Da 28 Da 42 Da 56 1 3 0 4 8 1 1

Claims

CLAIMS What is claimed is:

1. A method of producing a yoghurt having a stable composition of DP3+ GOS comprising the steps of: a. providing a milk-based substrate comprising lactose; b. treating the milk-based substrate with a pH sensitive, neutral beta- galactosidase enzyme to generate DP3+ GOS. c. acidifying the milk-based substrate to a pH 5.0 or below to inactivate said beta-galactosidase to provide the yoghurt having the stable composition of DP3+ GOS.

2. The method of claim 1 wherein the pH is below 4.9, 4.8 or 4.

7.

3. The method of claim 2 wherein the pH is at 4.

6.

4. The method of any of the preceding claims wherein the milk-based substrate has a lactose concentration of between 1-60% (w / w); 2–50 % (w / w), 3-40 % (w / w); or 4-30 % (w / w); or 4-15 % (w / w).

5. The method of any of the preceding claims wherein the beta-galactosidase has less than 10, 5, 4, 3, 2, 1, or 0.1 % of its maximal activity at a pH 5.0 or under.

6. The method of any of claim 5 wherein the beta-galactosidase has less than 10, 5, 4, 3, 2, 1, or 0.1 % of its maximal activity at pH 4.

6.

7. The method of any of the preceding claims wherein the pH sensitive, neutral beta- galactosidase is derived from Lactobacillus delbrueckii bulgaricus.

8. The method of any of the preceding claims wherein the pH sensitive beta-galactosidase comprises a polypeptide having at least 70% sequence identity to SEQ ID NO:1 or a beta- galactosidase active fragment thereof. 9 The method of claim 8 wherein the polypeptide has at least 80% sequence identity to SEQ ID NO:1 or a beta-galactosidase active fragment thereof.

10. The method of claim 9 wherein the polypeptide has at least 90% sequence identity to SEQ ID NO:1 or a beta-galactosidase active fragment thereof.

11. The method of claim 10 wherein the polypeptide has at least 95% sequence identity to SEQ ID NO:1 or a beta-galactosidase active fragment thereof.

12. The method of claim 11 wherein the polypeptide has at least 98% sequence identity to SEQ ID NO:1 or a beta-galactosidase active fragment thereof.

13. The method of claim 12 wherein the polypeptide has at least 99% sequence identity to SEQ ID NO:1 or a beta-galactosidase active fragment thereof.

14. The method of claim 13 wherein the polypeptide comprises a sequence according to SEQ ID NO:1 or a beta-galactosidase active fragment thereof.

15. The method of claim 14 wherein the polypeptide comprises a sequence according to SEQ ID NO:

1.

16. The method of any of the preceding claims wherein the step of acidifying comprises adding a culture and fermenting.

17. The method of any of the preceding claims wherein the yoghurt is an acidified milk product, a set yoghurt, a standing yoghurt, a stirred yoghurt, or a drinking yoghurt.

18. The method of any of the preceding claims, wherein the composition of DP3+ GOS in the yoghurt decreases by less than 5, 4, 3, 2, 1, or 0.1% after 5, 10, 15, 21, 28 or 56 days of storage after step c.

19. The method of any of the preceding claims wherein the beta-galactosidase produces at least 1.5, 2, 3, 4, or 5 g of DP3+ GOS per 100 Calories of yoghurt.

20. The method of claim 1, wherein the step of acidifying comprises adding a chemical acidifying agent.

21. The method of claim 20 wherein the acidifying agents is acetic acid, citric acid, lactic acid, malic acid, succinic acid, tartaric acid or glucono-delta-lactone.

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