Method for producing GOS comprising yogurt

Abstract

The invention relates to a method for producing a yogurt comprising galacto-oligosaccharide (GOS), wherein the method comprises the steps of (a) providing a milk base comprising lactose (b1) treating the milk base with Sporobolomyces singularis beta-hexosyltransferase or a variant thereof to generate GOS followed by fermenting the treated milk base with a lactic acid bacterial strain until a pH below is reached, or (b2) treating and fermenting the milk base at the same time by treating the milk base with Sporobolomyces singularis beta-hexosyltransferase or a variant thereof to generate GOS and by fermenting the milk base with a lactic acid bacterial strain until a pH below 5 is reached such as to produce a yogurt comprising GOS, and wherein said method does not comprise a step for inactivating the Sporobolomyces singularis beta-hexosyltransferase or a variant thereof.

Description

[0001] DSM IP Assets B.V. 2024P00107WO

[0002] METHOD FOR PRODUCING GOS COMPRISING YOGURT

[0003] Field of the invention

[0004]

[0001] The invention relates to a method for producing yogurt comprising galactooligosaccharides (GOS). The invention further relates to a GOS comprising yogurt and to the use of beta-hexosyltransferase or a variant thereof for obtaining storage stable GOS in yogurt.

[0005] Background of the invention

[0006]

[0002] Galacto-oligosaccharides (GOS) are known to enhance the growth of Bifidobacteria. This effect is likely achieved through the unique ability of Bifidobacteria to exploit GOS as a carbon source. Dietary supplements with galacto-oligosaccharides are furthermore thought to have a number of long-term disease protecting effects. There is therefore a great interest in developing cheap and efficient methods for producing GOS for improving dietary supplements and dairy products.

[0007]

[0003] GOS can be produced from lactose using the enzyme p-galactosidase which hydrolyses lactose to the monosaccharides D-glucose and D-galactose. However, at high lactose concentrations some p-galactosidases are able to transfer galactose to the hydroxyl groups of D-galactose or D-glucose in a process called transgalactosylation whereby galacto-oligosaccharides (GOS) are produced.

[0008]

[0004] Enzymes capable of transgalactosylation have been isolated from a wide range of microorganisms, including bacteria, fungi and yeasts (Torres et al. (2010) Comprehensive Reviews in Food Science and Food Safety, 9: 438-454). GOS yield from lactose can vary depending on the origin of the enzyme, lactose concentration, pH, enzyme dosage, time and temperature of the incubation, and is therefore difficult to compare. It is known that several basidiomycetous yeasts have potent transgalactosylation activities, and GOS production by Sporobolomyces singularis (formerly called Bullera singularis and recently renamed to Hamamotoa singularis), Cryptococcus laurentii, Sterigmatomyces eleviae, Rhodotorula minuta, and Sirobasidium magnum has been reported. A major difficulty is producing these hexosyltransferases in sufficient amounts, making the commercial production of GOS with these enzymes inhibitory expensive. An obstacle in producing these enzymes is their cellular attachment and poor secretion, necessitating cell-wall extraction and extensive purification of the enzyme with poor yield (e.g. Ishikawa et al. (2005) Journal of Bioscience and Bioengineering, 4:331-339).

[0009]

[0005] Examples of commercial GOS producing enzymes are Nurica (IFF) and Saphera Fiber (Novonesis). However, both enzymes show some disadvantages. W02020 / 117548, WO2015 / 086746 and WO2022189568 describe that stable GOS content can only be obtained if the transgalactosylating enzyme is inactivated. DSM IP Assets B.V. 2024P00107WO

[0010]

[0006] Industry is calling for improved transgalactosylating enzymes which can for example be used in the production of fermented milk products without the need for an enzyme inactivation step. Alternatively, industry is looking for transgalactosylating enzymes which result in increased GOS concentration at the end of yogurt fermentation. Yet another desire of industry is to obtain GOS comprising products, such as yoghurt, which have reduced sugar content (DP1 and / or DP2).

[0011] Summary of the invention

[0012]

[0007] The examples herein show that beta-hexosyltransferase (BHT) from Sporobolomyces singularis as well as variants thereof can be used in a fermented milk product (FMP) without a need for enzyme (heat) inactivation before storage. Surprisingly, the amount of GOS is stable during storage.

[0013]

[0008] Accordingly, in a first aspect, the present invention provides a method for producing a yogurt comprising galacto-oligosaccharide (GOS), wherein the method comprises the steps of (a) providing a milk base comprising lactose

[0014] (b1) treating the milk base with Sporobolomyces singularis beta-hexosyltransferase or a variant thereof to generate GOS followed by fermenting the treated milk base with a lactic acid bacterial strain until a pH below 5 is reached, or

[0015] (b2) treating and fermenting the milk base at the same time by treating the milk base with Sporobolomyces singularis beta-hexosyltransferase or a variant thereof to generate GOS and by fermenting the milk base with a lactic acid bacterial strain until a pH below 5 is reached such as to produce a yogurt comprising GOS,

[0016] and wherein said method does not comprise a step for inactivating the Sporobolomyces singularis beta-hexosyltransferase or a variant thereof.

[0017] Preferably, the claimed method does not comprise a step of irreversible inactivating the Sporobolomyces singularis beta-hexosyltransferase or a variant thereof. An example of irreversible inactivating would be subjecting the Sporobolomyces singularis beta-hexosyltransferase or a variant thereof to a step of heat inactivating such as a heat inactivating step at a temperature of at least 50 degrees Celsius, such as 60, 65, 70, 75, 80, 85, 90, 95 or 100 degrees Celsius.

[0018]

[0009] In a second aspect, the invention provides a yogurt obtainable by a method according to the first aspect or any of its dependent embodiments.

[0019]

[0010] In a third aspect, the invention provides use of Sporobolomyces singularis beta-hexosyltransferase or a variant thereof for obtaining storage stable GOS in yogurt without an enzyme inactivation step.

[0020]

[0011] The above aspects according to the invention advantageously allow for a GOS comprising yogurt production process comprising less steps, i.e. a production process which can omit the inactivation of the used GOS producing enzyme without significant reduction of the GOS amount DSM IP Assets B.V. 2024P00107WO

[0021] upon storage of the GOS comprising yogurt. Preferably, the GOS amount is stable for at least 24 hours after yogurt production, more preferably, the GOS amount is stable for at least 2 days such as for at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 days after yogurt production.

[0022] Brief description of the drawings

[0023]

[0012] The invention is illustrated by the following figures:

[0024]

[0013] Figure 1: GOS-fiber (g / kg) in yoghurt with added BHT (3.4 mg / L) during making and storage.

[0025]

[0014] Figure 2: GOS-fiber (g / L) in milk with added BHT or control enzymes during incubation.

[0026]

[0015] Figure 3: GOS-fiber (g / kg) in yoghurt with added BHT or competitor enzymes during making and storage.

[0027]

[0016] Figure 4: Relative pH profile of the different BHT variants.

[0028] Brief description of the sequence listing

[0029]

[0017] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. An overview of the sequences is provided by Table 1 below.

[0030] Table 1 : Overview of sequence listing:

[0031] SEQ ID No: Enzyme / gene Name Origin Type (herein also

[0032] referred to as)

[0033] SEQ ID NO: 1 Full length mature BHT Sporobolomyces singularis Protein (BHT_01)

[0034] SEQ ID NO: 2 Truncated mature BHT Synthetic construct Protein (BHT_02)

[0035] SEQ ID NO: 3 Variant of truncated mature Synthetic construct Protein (BHT_03) BHT; S295L mutant of SEQ

[0036] ID NO:2

[0037] SEQ ID NO: 4 Variant of truncated mature Synthetic construct Protein (BHT_04) BHT; R321 H mutant of

[0038] SEQ ID NO:2

[0039] SEQ ID NO: 5 Variant of truncated mature Synthetic construct Protein (BHT_05) BHT; S295L + R321 H

[0040] mutant of SEQ ID NO:2

[0041] SEQ ID NO: 6 Signal sequence Alpha-mating factor signal protein

[0042] sequence DSM IP Assets B.V. 2024P00107WO

[0043] SEQ ID NO: 7 Coding sequence of SEQ Synthetic construct DNA

[0044] ID NO:2

[0045] SEQ ID NO: 8 Coding sequence of SEQ Synthetic construct DNA

[0046] ID NO:3

[0047] SEQ ID NO: 9 Coding sequence of SEQ Synthetic construct DNA

[0048] ID NO:4

[0049] SEQ ID NO: 10 Coding sequence of SEQ Synthetic construct DNA

[0050] ID NO:5

[0051] Detailed description of the invention

[0052] Definitions

[0053]

[0018] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0054]

[0019] 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.

[0055]

[0020] 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.

[0056]

[0021] Unless explicitly indicated otherwise, the various embodiments of the invention described herein can be cross-combined.

[0057]

[0022] The term "milk" is intended to encompass milks from mammals, milks from microbial sources and / or mixtures thereof. Preferably, the milk is from a mammal source. Mammal sources of milk include, but are not limited to cow milk, sheep milk, goat milk, buffalo milk, camel milk, llama milk, horse milk or reindeer milk. In an embodiment, the milk is from a mammal selected from the group consisting of cow, sheep, goat, buffalo, camel, llama, horse and deer, and combinations thereof. Microbial sources of milk, supplemented with lactose, include milk and milk proteins produced by recombinant micro-organisms in a laboratory (also referred to as “lab-grown milk”) or bioreactor. Preferably the milk is a non-recombinant, naturally occurring and / or naturally produced, milk. Cows are most preferred as a source for milk. Bovine milk is most preferred. In addition, the term "milk" refers to not only whole milk, but also skim milk or any liquid component derived thereof or reconstituted milk.

[0058]

[0023] The term "milk-base" (also referred to as milk-based substrate) refers to a base composition, comprising or consisting of milk or milk ingredients, or derived from milk or milk ingredients. The milk-base can be used as a raw material for the dairy product. The milk-base may for example comprise or consist of skimmed or non-skimmed milk, or reconstituted milk. Optionally DSM IP Assets B.V. 2024P00107WO

[0059] the milk-base may be concentrated, or in the form of powder, or may be reconstituted from such. By reconstituted milk is herein understood liquid milk obtained by adding liquid, such as water, to a skim milk powder, skim milk concentrate, whole milk powder or whole milk concentrate. Furthermore, the milk-base may or may not have been subjected to a thermal processing operation which is at least as efficient as pasteurization. Preferably the milk-base is from a bovine source. The milk-based substrate may have been subjected to treatments known in the art such as - for example- homogenization, pasteurization, sterilization or an extended shelf life (ESL) treatment or an ultra-heat treatment (UHT).

[0060] The milk-based substrate may be skim milk, full fat milk, semi-skimmed milk, condensed milk, reconstituted (skim) milk, butter milk, milk protein concentrate (MPC), or whey such as sweet whey or acid whey, whey protein concentrate (WPC), whey protein isolate (WPI) or whey permeate. Preferably, the milk-based substrate may be skim milk, full fat milk, semi-skimmed milk, condensed milk, reconstituted (skim) milk, butter milk or milk protein concentrate (MPC).

[0061] The herein used milk-based substrate comprises lactose and can thus also be described as “a lactose comprising milk-based substrate”. In general, cow’s milk comprises approximately 5% lactose. Preferably, the milk-based substrate only comprises the lactose which is naturally present in the milk-based substrate. Preferably, the milk-based substrate does not comprise added lactose. Preferably, the milk-based substrate comprises lactose in the range of 4 to 10% (w / v) lactose, preferably 4 to 8% (w / v) and even more preferably 4 to 6% (w / v) or 4.5 to 5.5% (w / v) lactose.

[0062]

[0024] Any references to “% (w / V)” herein, such as for example references to 12% w / v reconstituted skim milk (RSM), refer to weight in grams present per volume of 100 ml solution, for example 12% w / v RSM corresponds to 12 grams of skim milk powder dissolved per 100 ml water.

[0063]

[0025] Beta-galactosidases are enzymes which hydrolyse terminal non-reducing beta-D-galactose residues in beta-D-galactosides, for example lactose is hydrolysed to galactose and glucose. These enzymes belong to the enzyme class EC 3.2.1.23. Besides hydrolysing, this enzyme class is also able to transfer galactose to other sugars and thereby produce galactooligosaccharides (GOS). The different enzymes of class EC 3.2.1 .23 have various preferences for hydrolytic (beta-galactosidase) activity and transgalactosylase activity and the preference can be expressed for example by the ratio of transgalactosylating activity to hydrolysing activity.

[0064]

[0026] The enzyme having transgalactosylase activity preferably belongs to subclass EC 3.2.1.23. As used herein, the terms “enzyme having transgalactosylase activity”, “enzyme having transgalactosylating activity”, “transgalactosylating enzyme” and “transgalactosylase” are used interchangeably herein and all refer to an enzyme capable of transferring galactose from lactose to a hydroxyl group of, for example, lactose, D-galactose (gal) or D-glucose (glu) whereby galactooligosaccharides are produced.

[0065]

[0027] As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a polypeptide as described herein. A gene may DSM IP Assets B.V. 2024P00107WO

[0066] include coding sequences, non-coding sequences, introns and regulatory sequences. That is to say, a “gene”, as used herein, may refer to an isolated nucleic acid molecule as defined herein. Accordingly, the term “gene”, in the context of the present application, does not refer only to naturally-occurring sequences.

[0067]

[0028] As used herein, the terms “polynucleotide” “nucleic acid sequence” or “nucleic acid molecule” are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.

[0068]

[0029] As used herein, the terms "peptide" and "oligopeptide" are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by peptidyl linkages. The word "polypeptide" is used herein for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus. The one-letter code of amino acids used herein is commonly known in the art and can be found in Berg, Tymoczko and Stryer, Biochemistry, 6thedition, chapter 2, W.H Freeman and Company, New York, 2007.

[0069]

[0030] The terms “homology” and “percent identity” are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid for optimal alignment with a second amino or nucleic acid sequence). The amino acid or nucleotide residues at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (i.e. overlapping positions) x 100). Preferably, the two sequences are the same length.

[0070]

[0031] A sequence comparison may be carried out over the entire lengths of the two sequences being compared or over fragment of the two sequences. Suitably the comparison can be carried out over the full length of the two sequences being compared. However, sequence identity may be carried out over a region of, for example, twenty, fifty, one hundred or more contiguous amino acid residues.

[0071]

[0032] The skilled person will be aware of the fact that several different computer programs are available to determine the homology between two sequences. For instance, a comparison of DSM IP Assets B.V. 2024P00107WO

[0072] sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. 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.

[0073]

[0033] The protein sequences or nucleic acid 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 BLASTN and BLASTP programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 — 10. BLAST protein searches can be performed with the BLASTP program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. 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., BLASTP and BLASTN) can be used. See the homepage of the National Center for Biotechnology Information at http: / / www.ncbi.nlm.nih.gov / .

[0074]

[0034] The term "yoghurt" or “yogurt” (the terms are used interchangeably herein) refers to products comprising or obtained by means of lactic acid bacteria that include at least Streptococcus salivarius thermophilus and Lactobacillus delbruekii subsp. bulgaricus, but may also, optionally, include further microorganisms such as Lactobacillus delbruekii subsp. lactis, Bifidobacterium animalis subsp. lactis, Lactococcus lactis, Lactobacillus acidophilus and Lactobacillus casei, or any microorganism derived therefrom. Such lactic acid strains other than Streptococcus salivarius thermophilus and Lactobacillus delbruekii subsp. bulgaricus, can give the finished product various properties, such as the property of promoting the equilibrium of the gut microbiota. As used herein, the term "yoghurt" encompasses set yoghurt, stirred yoghurt, drinking yoghurt, heat treated yoghurt and yoghurt-like products. More preferably, the term "yoghurt" encompasses, but is not limited to, yoghurt as defined according to French and European regulations, e.g. coagulated dairy products obtained by lactic acid fermentation by means of specific thermophilic lactic acid bacteria only (i.e. Lactobacillus delbruekii subsp. bulgaricus and Streptococcus salivarius thermophilus) which are cultured simultaneously and are found to be live in the final product in an amount of at least 10 million CFU (colony-forming unit) / g. Preferably, the yoghurt is not heat-treated after fermentation. Yoghurts may optionally contain added dairy raw materials (e.g. cream) or other ingredients such as sugar or sweetening agents, one or more flavouring(s), fruit, cereals, or nutritional substances, DSM IP Assets B.V. 2024P00107WO

[0075] especially vitamins, minerals and fibers. Such yoghurt advantageously meets the specifications for fermented milks and yoghurts of the AFNOR NF 04-600 standard and / or the codex StanA-lla-1975 standard. In order to satisfy the AFNOR NF 04-600 standard, the product must not have been heated after fermentation and the dairy raw materials must represent a minimum of 70% (m / m) of the finished product.

[0076]

[0035] The term "starter" or "starter culture" as used herein refers to a culture of one or more foodgrade micro-organisms, more preferably a culture comprising lactic acid bacteria, which are responsible for the acidification of the milk base. Starter cultures may be fresh (liquid), frozen or freeze-dried. Freeze dried cultures need to be regenerated before use. For the production of a yoghurt, the starter can for example be added in an amount from 0.01 to 3% by weight of the total amount of milk base. For the production of cheese, lower dosages can be used such as from 0.006% by weight of the total amount of milk base.

[0077]

[0036] As used herein, the term "lactic acid bacteria", "LAB", "lactic acid bacterial strains" and "lactic bacteria" are used interchangeably and refer to food-grade bacteria producing lactic acid as the major metabolic end-product of carbohydrate fermentation. These bacteria are related by their common metabolic and physiological characteristics and are usually Gram positive, low-GC, acid tolerant, non- sporulating, non-respiring, rod-shaped bacilli or cocci. During the fermentation stage, the consumption of lactose by these bacteria causes the formation of lactic acid, reducing the pH and leading to the formation of a protein coagulum. These bacteria are thus responsible for the acidification of milk and for the texture of the dairy product. As used herein, the term "lactic acid bacteria" or "lactic bacteria" encompasses, but is not limited to, bacteria belonging to the genus of Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., Lactococcus spp., such as Lactobacillus delbruekii subsp. bulgaricus, Streptococcus salivarius thermophilus, Lactobacillus lactis, Bifidobacterium animalis, Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus acidophilus and Bifidobacterium breve.

[0078] Galacto-oligosaccharides

[0079]

[0037] A GOS can be a disaccharide (Degree of Polymerization (DP) 2, i.e. DP2) (except lactose which is not GOS), a trisaccharide (DP3), tetrasaccharide (DP4), pentasaccharide (DP5) or longer oligosaccharides.

[0080]

[0038] An example of a disaccharide is galactosyl-galactose (gal-gal) or allolactose (galactosylbeta 1-6-glucose). An example of a trisaccharide (DP3) is galactosyl-lactose (gal-gal-glu). An example of a tetrasaccharide (DP4) is galactosyl-galactosyl-lactose (gal-gal-gal-glu) An example of a pentasaccharide (DP5) is galactosyl-galactosyl-galactosyl-lactose (gal-gal-gal-gal-glu). An example of an even longer oligosaccharide is (galactose)n-lactose with n>3.

[0081]

[0039] The term “DPx+ GOS” means the sum of GOS molecules having DPx or higher DP. For example the term “DP3+ GOS” means the sum of GOS molecules having DP3, DP4, DP5 etc. The DSM IP Assets B.V. 2024P00107WO

[0082] term GOS as used herein refers to the combination of DP2+ GOS. DP2+ GOS excludes lactose (which is by definition not GOS).

[0083]

[0040] The level of GOS molecules can be determined by using different analysis techniques. For example, one can determine the amount of lactose, glucose and galactose in the lactose comprising milk-based substrate and after incubation with the enzymes, determine the amount of lactose, glucose and galactose using an enzymatic analysis, NMR or HPAEC-PAD. Quantification of mono-, disaccharides can also be performed by using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis.

[0084]

[0041] In yet another alternative method, GOS production can be established by reaction of an enzyme on lactose in which the amount of galactose generated is less than the amount of glucose generated at a given time.

[0085]

[0042] The amount of GOS can then be determined by subtracting the sum of the amount of lactose, glucose and galactose (as present in the product) from the sum of the starting amount of lactose, glucose and galactose (as present in the substrate). The difference is then the amount of GOS. In a formula: [GOS]product = ([lac]+[glu]+[gal])SUbstrate - ([lac]+[glu]+[gla])prOduct

[0086]

[0043] Determining the level of GOS with this formula can be performed in respect of a method of the invention, because the skilled person has the opportunity to determine the level of lactose in the milk-based substrate.

[0087] Sporobolomyces sinqularis beta-hexosyltransferase (BHT) or a variant thereof

[0088]

[0044] In a method according to the invention a milk base comprising lactose is treated with a Sporobolomyces singularis beta-hexosyltransferase (BHT) or a variant thereof.

[0089]

[0045] The term “Sporobolomyces singularis beta-hexosyltransferase (BHT)” refers to an enzyme having transgalactosylase activity and preferably belongs to subclass EC 3.2.1.23. As used herein, the terms “enzyme having transgalactosylase activity”, “enzyme having transgalactosylating activity”, “transgalactosylating enzyme” and “transgalactosylase” are used interchangeably herein and all refer to an enzyme capable of transferring galactose from lactose to a hydroxyl group of, for example, lactose, D-galactose (gal) or D-glucose (glu) whereby galacto-oligosaccharides are produced.

[0090]

[0046] Preferably, the Sporobolomyces singularis beta-hexosyltransferase (BHT) has an amino acid with equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2.

[0091]

[0047] The Sporobolomyces singularis beta-hexosyltransferase (BHT) enzyme may be produced in sufficient amounts using for example K. lactis or A. niger as a production host.

[0092]

[0048] Preferably, the Sporobolomyces singularis beta-hexosyltransferase (BHT) variant has an amino acid sequence with equal to or more than 80%, more preferably equal to or more than 85%, DSM IP Assets B.V. 2024P00107WO

[0093] more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2.

[0094]

[0049] More preferably, the variant used in a method according to the invention, has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position 295 or 321 , wherein said position is defined with reference to SEQ ID NO: 2.

[0095]

[0050] SEQ ID NO: 2 is a truncated version of SEQ ID NO:1 (mature sequence of the wildtype of an enzyme having transgalactosylase activity originally found in Sporobolomyces singulars') by deleting a potential cell-binding domain / anchor at the start of the BHT sequence. Hence, the variant used in the herein claimed method is a variant polypeptide of the polypeptide having the amino acid sequence of SEQ ID NO:2. The variant described herein is a beta-hexosyltransferase and is also referred to as BHT.

[0096]

[0051] Even more preferably, the variant used in a method according to the invention has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position 295, wherein said position is defined with reference to SEQ ID NO: 2. Alternatively, the variant used in a method according to the invention has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position 321 , wherein said position is defined with reference to SEQ ID NO: 2.

[0097]

[0052] Preferably, the substitution at position 295, is S295L, wherein said position is defined with reference to SEQ ID NO: 2. Alternatively, the substitution at position 321 is R321H, wherein said position is defined with reference to SEQ ID NO: 2.

[0098]

[0053] In one of its embodiments, the variant used in a method according to the invention has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least two substitutions of the amino acid residues corresponding to the amino acids in position 295 or 321 , wherein said positions are defined with reference to SEQ ID NO: 2. Preferably, the at least two substitutions are S295L and R321H, wherein said positions are defined with reference to SEQ ID NO: 2

[0099]

[0054] Preferably the variant used in a method according to the invention has an amino acid sequence with equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2. Hence, the variant used in a method according to the invention - has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position 295, wherein said position is defined with reference to SEQ ID NO: 2, wherein the polypeptide has an amino acid sequence with equal to or more than 80%, more preferably equal DSM IP Assets B.V. 2024P00107WO

[0100] to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2

[0101] - has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position 321 , wherein said position is defined with reference to SEQ ID NO: 2, wherein the polypeptide has an amino acid sequence with equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2

[0102] - has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position 295, wherein said position is defined with reference to SEQ ID NO: 2 and wherein the substitution is S295L, wherein the polypeptide has an amino acid sequence with equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2 - has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position R321 H, wherein said position is defined with reference to SEQ ID NO: 2 and wherein the substitution is R321H, wherein the polypeptide has an amino acid sequence with equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2 - has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least two substitutions of the amino acid residues corresponding to the amino acids in position 295 and 321 , wherein said positions are defined with reference to SEQ ID NO: 2, wherein the polypeptide has an amino acid sequence with equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2 or

[0103] - has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least two substitutions of the amino acid residues corresponding to the amino acids in position 295 and 321 , wherein said positions are defined with reference to SEQ ID NO: 2 and wherein the substitutions are S295L and R321H, wherein the polypeptide has an amino acid sequence with equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2.

[0104]

[0055] Preferably, the Sporobolomyces singularis beta-hexosyltransferase (BHT) or a variant thereof used in a method of the invention has beta-hexosyltransferase activity, i.e. is capable of transferring galactose from lactose to a hydroxyl group of, for example, lactose, D-galactose (gal) or D-glucose (glu) whereby galacto-oligosaccharides are produced or in short the claimed isolated DSM IP Assets B.V. 2024P00107WO

[0105] and / or recombinant polypeptide uses lactose as a substrate to produce galacto-oligosaccharides (GOS).

[0106]

[0056] Most preferably the polypeptide is:

[0107] - a polypeptide that has an amino acid sequence of SEQ ID NO: 3, SEQ ID NO:4 or SEQ ID NO:5, or

[0108] - has an amino acid sequence with more than 80% sequence identity with SEQ ID NO: 3 and wherein said polypeptide comprises substitution S295L; or

[0109] - has an amino acid sequence with more than 80% sequence identity with SEQ ID NO: 4 and wherein said polypeptide comprises substitution R321 H; or

[0110] - has an amino acid sequence with more than 80% sequence identity with SEQ ID NO: 5 and wherein said polypeptide comprises substitutions S295L and R321H.

[0111]

[0057] The Sporobolomyces singularis beta-hexosyltransferase (BHT) or a variant thereof used in a method of the invention can be an isolated naturally occurring polypeptide or a non-naturally occurring polypeptide. By a non-naturally occurring polypeptide is herein understood a polypeptide that is not naturally produced by any organism. Preferably the polypeptide is a non-naturally occurring polypeptide.

[0112]

[0058] By an "isolated" polypeptide or protein is herein understood a polypeptide or protein removed from its native environment. For example, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention as are recombinant polypeptides which have been substantially purified by any suitable technique. A polypeptide variant according to the invention can be recovered and purified from recombinant cell cultures by methods known in the art.

[0113]

[0059] The Sporobolomyces singularis beta-hexosyltransferase (BHT) or a variant thereof is allowed sufficient time to produce a certain level of GOS molecules. The exact time will depend on the specific enzyme used, the amount of enzyme used but also on the used temperature and the lactose concentration in the milk-based substrate. The skilled person is capable to identify an enzyme dosage, incubation temperature and incubation time to allow the production of GOS in the milk-based substrate. Preferably, the amount of BHT enzyme or a variant thereof which is added to the milk base is 1.0 to 10 mg BHT protein I L milk base, more preferably 2.0 to 7.0 mg BHT protein I L milk base.

[0114]

[0060] The GOS is produced in situ which means that the GOS is produced in the milk-based substrate from the lactose present in said milk-based substrate. Preferably, a method, use or product according to the invention does not comprises added GOS (i.e. non in situ produced GOS).

[0115] Lactic acid bacterial strains

[0116]

[0061] The method as claimed herein describes 2 ways how a yogurt comprising GOS (alternatively: a GOS comprising yogurt) can be prepared. The first one is to first treat a milk base DSM IP Assets B.V. 2024P00107WO

[0117] with Sporobolomyces singular BHT or a variant thereof such as to generate GOS and subsequently ferment the obtained GOS comprising milk base with a lactic acid bacterial strain. This first option is a so-called two-step method. The second option is a so-called one-step method wherein the production of GOS and the fermentation step are performed at the same time.

[0118]

[0062] More preferably (independent of whether a one or a two-step method is used) the milk base is fermented in the presence of two or more lactic acid bacterial strains.

[0119]

[0063] Preferably the lactic acid bacterial strain(s) is / are selected from the group consisting of Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., Lactococcus spp. Leuconostoc spp., Pediococcus spp. and Propionobacterium spp.

[0120]

[0064] More preferably, the lactic acid bacterial strain(s) is / are selected from the group consisting of Lactobacillus delbruekii subsp. bulgaricus, Streptococcus (salivarius) thermophilus, Lactobacillus lactis, Bifidobacterium animalis, Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus acidophilus Bifidobacterium breve and / or combinations thereof. Most preferably the lactic acid bacterial strains comprise or consist of Lactobacillus delbruekii subsp. bulgaricus and Streptococcus thermophilus.

[0121]

[0065] Preferably the milk base is fermented in the presence of a starter culture comprising at least:

[0122] - a Streptococcus thermophilus strain; and

[0123] - a Lactobacillus delbreuckii subsp. bulgaricus stain.

[0124]

[0066] The starter culture can comprise lactic acid bacterial strains consisting of only a Streptococcus thermophilus strain and a Lactobacillus delbreuckii subsp. bulgaricus stain. That is, suitably the milk base can be fermented in the presence of a Streptococcus thermophilus strain and a Lactobacillus delbreuckii subsp. bulgaricus stain as the sole lactic acid bacterial strains. However, more preferably the starter culture comprises lactic acid bacterial strains that include a Streptococcus thermophilus strain and a Lactobacillus delbreuckii subsp. bulgaricus stain and in addition one or more further lactic acid bacterial strain(s). That is, suitably the milk base can be fermented in the presence of a Streptococcus thermophilus strain and a Lactobacillus delbreuckii subsp. bulgaricus stain and one or more further lactic acid bacterial strain(s).

[0125]

[0067] For example, the milk base can be fermented in the presence of a strain of Streptococcus thermophilus, a strain of Lactobacillus delbreuckii spp. bulgaricus and in addition a strain of Lactobacillus acidophilus and / or a strain of Lactococcus lactis spp. lactis.

[0126]

[0068] The total amount of lactic acid bacterial stains in the milk base preferably lies in the range from equal to or more than 0.001 % (w / w), more preferably equal to or more than 0.005 % (w / w), even more preferably equal to or more than 0.01 % w / w, still more preferably equal to or more than 0.05 % (w / w) and most preferably equal to or more than 0.1 % (w / w) to equal to or less than 10.0 % (w / w), more preferably equal to or less than 5.0% w / w, even more preferably equal to or less than 3.0 % (w / w), still more preferably equal to or less than 1.0 % (w / w) and most preferably equal DSM IP Assets B.V. 2024P00107WO

[0127] to or less than 0.5 % (w / w), based on the total weight of the milk base (i.e. based on the total weight of the milk base, including any supplemented sucrose, glucose, fructose and optionally any invertase contained therein).

[0128] Starter culture

[0129]

[0069] The one or more lactic acid bacterial strains (and optionally the Sporobolomyces singularis beta-hexosyltransferase or a variant thereof) can be added to the milk base each separately or together. More preferably the lactic acid bacterial strains are combined in one starter culture. This starter culture can advantageously be contacted with the milk base. This advantageously allows for simultaneous contacting of the one or more lactic acid bacterial strains with the milk base.

[0130]

[0070] The present invention therefore also describes a starter culture comprising:

[0131] - a lactic acid bacterial strain; and

[0132] - a Sporobolomyces singularis beta-hexosyltransferase or a variant thereof.

[0133]

[0071] More preferably such a starter culture comprises:

[0134] - one or more lactic acid bacterial strains, preferably including a Streptococcus thermophilus strain and a Lactobacillus delbreuckii subsp. bulgaricus stain; and

[0135] - a Sporobolomyces singularis beta-hexosyltransferase or a variant thereof

[0136]

[0072] Preferably the starter culture is a frozen starter culture comprising the Sporobolomyces singularis beta-hexosyltransferase or a variant thereof in the form of separate frozen enzyme pellets.

[0137]

[0073] Suitably the frozen Sporobolomyces singularis beta-hexosyltransferase or a variant thereof and the frozen lactic acid bacterial strains can even be present in the same frozen pellet. Such frozen pellets can for example be prepared by mixing the lactic acid bacterial strains and Sporobolomyces singularis beta-hexosyltransferase or a variant thereof and subsequently producing frozen pellets therefrom. A frozen pellet comprising both the frozen Sporobolomyces singularis beta-hexosyltransferase or a variant thereof and lactic acid bacterial strains can be advantageously to allow for addition of both to a milk base in a constant molar or weight ratio.

[0138]

[0074] The invention therefore also describes a kit of parts comprising:

[0139] - frozen pellets comprising a lactic acid bacterial strain,

[0140] - frozen pellets comprising a Sporobolomyces singularis beta-hexosyltransferase or a variant thereof.

[0141] The kit of parts can conveniently be provided in a package, for example with a package size in the range from equal to or more than 1 grams to equal to or less than 1000 kilograms, more preferably in the range from equal to or more than 1 kilograms to equal to or less than 100 kilograms.

[0142]

[0075] Preferably the above starter culture, respectively the above kit of parts, comprises a total weight of one or more lactic acid bacterial strains in the range from equal to or more than 20.0 % by weight (w / w), more preferably equal to or more than 40.0 % (w / w), even more preferably equal DSM IP Assets B.V. 2024P00107WO

[0143] to or more than 50.0 % (w / w) and still more preferably equal to or more than 70.0 % (w / w) or even equal to or more than 90.0 % (w / w) or equal to or more than 95.0 % (w / w) to equal to or less than 99.9 % (w / w), more preferably equal to or less than 99 % (w / w) and possibly equal to or less than 95% (w / w) or even equal to or less than 90% (w / w), based on the total weight of the starter culture, respectively kit of parts.

[0144]

[0076] Preferably the starter culture comprises a total weight of Sporobolomyces singularis beta-hexosyltransferase or a variant thereof in the range from equal to or more than 0.01 % by weight (w / w), more preferably equal to or more than 0.05 % (w / w), even more preferably equal to or more than 0.1 % (w / w) and still more preferably equal to or more than 0.5 % (w / w) or even equal to or more than 1.0 % (w / w) or equal to or more than 2.0 % (w / w) to equal to or less than 30 % (w / w), more preferably equal to or less than 20 % (w / w) and possibly equal to or less than 10% (w / w) or even equal to or less than 8% (w / w), based on the total weight of the starter culture.

[0145]

[0077] The remainder of the starter culture can comprise one or more other compounds or materials, such as for example fillers, excipients or protectants, such as cryoprotectants and / or lyo protectants. These compounds or materials can be added to ensure or increase the stability of the lactic acid bacterial strain(s) or the enzyme(s), for example during long term storage or that are added to improve disability or flowability. Cryoprotectants and / or lyoprotectants can be used to protect the lactic acid bacteria and / or the Sporobolomyces singularis beta-hexosyltransferase or a variant thereof from damage during freezing and thawing, respectively during freeze-drying. Such a cryoprotectant, respectively lyoprotectant, may be any additive as long as it protects the lactic acid bacterial cells or the enzyme from damage during freezing and thawing, respectively freeze-drying.

[0146]

[0078] Suitable excipients and / or protectants include proteins, carbohydrates including monosaccharides (e.g. galactose, glucose, fructose, D-mannose, sorbose), disaccharides (e.g. lactose, trehalose, sucrose), polysaccharides (e.g. raffinose, starch, gums, celluloses, maltodextrin, cyclodextrin, dextran), polyalcohols (e.g. glycerol, sorbitol, mannitol), polyethers (e.g. polypropylene glycol, polyethylene glycol, polybutylene glycol), antioxidants (e.g. natural antioxidants such as ascorbic acid, beta-carotene, vitamin E, glutathione, chemical antioxidants), oils (e.g. rapeseed oil, sunflower oil, olive oil), surfactants (e.g. Tween®20, Tween®80, fatty acids), peptones (e.g. soy peptones, wheat peptone, whey peptone), tryptones, vitamins, minerals (e.g. iron, manganese, zinc), hydrolysates (e.g. protein hydrolysates such as whey powder, malt extract, soy), amino acids (e.g. monosodium glutamate, glycine, alanine, arginine, histidine), nucleobases (e.g. cytosine, guanine, adenine, thymine, uracil, xanthine, hypoxanthine, inosine), yeast extracts (e.g. yeast extracts of Saccharomyces spp., Kluyveromyces spp., or Torula spp.), beef extract, growth factors, and lipids and combinations of all of these.

[0147]

[0079] Preferably, the starter culture has a content of viable lactic acid bacterial cells of at least 1x107colony forming units (cfu) per gram (g) starter culture, more preferably at least 1x108cfu / g, DSM IP Assets B.V. 2024P00107WO

[0148] more preferably at least 1x109cfu / g, even more preferably at least 1x1010cfu / g, still more preferably at least 1x1011cfu / g, yet even more preferably at least 1x1012cfu / g and most preferably at least 1x1013cfu / g starter culture. The advantage of such high concentrations of lactic acid bacteria in the starter culture is that small amounts of starter culture are sufficient for the inoculation of large amounts of milk base.

[0149]

[0080] Preferably the weight ratio of Sporobolomyces singularis beta-hexosyltransferase or a variant thereof to lactic acid bacteria, in the starter culture and / or during fermentation, lies in the range from equal to or more than 0.001 :1 , preferably equal to or more than 0.01 :1 to equal to or less than 1 :1 , more preferably equal to or less than 0.1 :1.

[0150] Fermenting

[0151]

[0081] The milk base is fermented in the presence of a lactic acid bacterial strain and a Sporobolomyces singularis beta-hexosyltransferase or a variant thereof. During this fermentation, the milk base is acidified.

[0152]

[0082] That is, the invention also provides the use of a combination of a lactic acid bacterial strain and a Sporobolomyces singularis beta-hexosyltransferase or a variant thereof for the purpose of acidification of a milk base comprising lactose. Thus the invention provides the use of a combination of a lactic acid bacterial strain and a Sporobolomyces singularis beta-hexosyltransferase or a variant thereof for the production of a fermented milk product.

[0153]

[0083] More preferably the invention provides the use of a starter culture, comprising a combination of a lactic acid bacterial strain and a Sporobolomyces singularis beta-hexosyltransferase or a variant thereof for the purpose of acidification and / or fermentation of a milk base comprising lactose. That is, preferably the invention provides the use of a starter culture, comprising a combination of a lactic acid bacterial strain and a Sporobolomyces singularis beta-hexosyltransferase or a variant thereof for the production of a fermented milk product.

[0154]

[0084] The process conditions during such acidification, respectively fermentation, can be varied widely.

[0155]

[0085] As described above, the composition of the milk base can be adjusted to arrange for the desired concentrations of lactose in the milk base. The milk base may further be adjusted to arrange for the desired amounts of fat and / or proteins. If so desired, stabilizers and / or other additives may be added.

[0156]

[0086] The milk base is preferably heated before fermentation thereof and before adding the Sporobolomyces singularis beta-hexosyltransferase or a variant thereof. More preferably the milk base is heated at a temperature equal to or more than 80 °C, more preferably a temperature equal to or more than 85°C, for a period of preferably equal to or more than 20 minutes, more preferably equal to or more than 30 minutes. In the alternative or in addition, the milk base may be heated at a temperature of equal to or more than 95°C, preferably for a period of equal to or more than 10 DSM IP Assets B.V. 2024P00107WO

[0157] minutes. The heat treatments advantageously allow for the elimination of pathogens. In addition, the heat treatments can help to create a better environment for the lactic acid bacterial cells to grow.

[0158]

[0087] Optionally the milk base can be homogenized (e.g. stirred or mixed) before fermentation. Without wishing to be bound by any kind of theory, such homogenization may allow for an improved consistency of the fermented milk product.

[0159]

[0088] After heating and before inoculation of the milk base with the lactic acid bacterial strain(s), the milk base is preferably cooled to the desired fermentation temperature. More preferably the temperature of the milk base is adjusted to a fermentation temperature in the range from equal to or more than 18°C, preferably equal to or more than 22°C to equal to or less than 45°C, more preferably equal to or less than 42°C.

[0160]

[0089] Fermentation of the milk base can suitably be carried out in a so-called fermentation vat or fermentation tank.

[0161]

[0090] The milk base can be inoculated with the starter culture in any manner known by the person skilled in the art. For example, the starter culture can be dosed batchwise, semi-batchwise or continuously, including for example by inline dosing.

[0162]

[0091] Although the temperature can be adjusted during fermentation, the temperature during fermentation is preferably kept constant. Preferably a constant fermentation temperature is chosen in the range from equal to or more than 18°C, preferably equal to or more than 22°C to equal to or less than 45°C, more preferably equal to or less than 42°C. During the fermentation the pH decreases. Preferably the fermentation is continued until a certain desired pH, preferably a pH in the range from equal to or more than pH 4.0 to equal to or less than pH 4.8, is reached. More preferably the fermentation is at least continued for a certain period of time until a pH of for example pH 4.8, pH 4.7, pH 4.6, pH 4.5, pH 4.4, pH 4.3, pH 4.2, pH 4.1 or pH 4.0 is reached. The time period until the desired pH is reached is herein also referred to as "acidification time". Preferably the time to reach a pH of fer example pH 4.6 is equal to or less than 10 hours, more preferably equal to or less than 8 hours, even more preferably equal to or less than 7 hours and most preferably equal to or less than 6 hours.

[0163]

[0092] In the process according to the invention, the time period during which the milk base is fermented (the "fermentation time") can therefore advantageously be equal to or less than 22 hours, more preferably equal to or less than 20 hours, still more preferably equal to or less than 18 hours, even more preferably equal to or less than 16 hours, still even more preferably equal to or less than 14 hours or even equal to or less than 12 hours. More preferably the milk base is fermented during a time period that is equal to or less than 10 hours, still more preferably equal to or less than 8 hours, even more preferably equal to or less than 7 hours and most preferably equal to or less than 6 hours. Hence advantageously the time period for the fermentation of the milk base in the process according to the invention can lie in the range from equal to more than 3 hours, more preferably DSM IP Assets B.V. 2024P00107WO

[0164] equal to or more than 4 hours, still more preferably equal to or more than 5 hours, to equal to or less than 12 hours, more preferably equal to or less than 10 hours, even more preferably equal to or less than 8 hour, still more preferably equal to or less than 7 hours and most preferably equal to or less than 6 hours.

[0165]

[0093] When the desired pH is reached, the fermentation can be stopped in any manner known to the person skilled in the art. Preferably the fermentation is stopped by cooling the fermented milk product, for example by reducing the temperature to a temperature equal to or less than 10°C, more preferably equal to or less than 8°C, and most preferably equal to or less than 7°C. The fermented milk product can suitably be removed from the fermentation vat or fermentation tank.

[0166]

[0094] Optionally the fermented milk product can be stirred and / or fruit and / or flavors can be added to the fermented milk product. Subsequently the fermented milk product can be packaged as desired.

[0167] No enzyme inactivation step needed

[0168]

[0095] The examples shows that beta-hexosyltransferase (BHT) from Sporobolomyces singularis as well as variants thereof can be used in a fermented milk product (FMP) without a need for inactivation before storage. Surprisingly, the amount of GOS is stable during storage. This is in sharp contrast with commercial products such as Nurica (IFF) and Saphera Fiber (Novonesis) which both show reduction in the amount of GOS when the enzyme is not inactivated before storage of the FMP product.

[0169]

[0096] Hence, a method of the invention does not need a heat treatment step to inactivate the GOS producing enzyme.

[0170]

[0097] More preferably, a method as claimed herein does not include a heat treatment step, such as a heat treatment of from about 70 degrees centigrade to 95 degrees centigrade and for between about 5 minutes to 30 minutes. Alternatively, a method as claimed herein does not include a heat treatment of from about 95 degrees centigrade for 5 to 30 minutes. Alternatively, a method as claimed herein does not include a heat treatment of from about 135 degrees centigrade to about 150 degrees centigrade for about 2 seconds to about 15 seconds.

[0171]

[0098] The term “enzyme inactivation” refers to irreversible enzyme inactivation, i.e. a method as claimed herein does not need a step of irreversible enzyme inactivation.

[0172] Storage

[0173]

[0099] As described above when the desired pH is reached, the fermentation can be stopped in any manner known to the person skilled in the art. The fermented milk product is typically stored until sold and / or consumed. Storage for example involves putting the fermented milk product at a temperature equal to or less than 10°C, more preferably equal to or less than 8°C, and most preferably equal to or less than 7°C. More preferably, storage involves putting the fermented milk DSM IP Assets B.V. 2024P00107WO

[0174] product at a temperature equal to or less than 10°C, more preferably equal to or less than 8°C, and most preferably equal to or less than 7°C, wherein said storage time is at least 1 day, preferably at least 2 days, more preferably at least 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0175]

[0100] Preferably, a method as claimed herein, further comprises a step of storing the yogurt comprising GOS for at least 7 days at 3 to 10 degrees Celsius. More preferably, storing the GOS comprising yogurt for at least 7 days at 3 to 8°C or 3 to 7°C.

[0176] Storage stability

[0177]

[0101] The phrase “storage stable GOS” refers to the GOS or GOS fiber in the fermented milk product or more preferably in the yogurt, having a variance of less than about 10 percent when stored for 40 days at 3 to 10 degrees Celsius.

[0178] A composition comprising a Sporobolomyces singularis beta-hexosyltransferase or a variant thereof

[0179]

[0102] The Sporobolomyces singularis beta-hexosyltransferase or a variant thereof can be added as such or as an enzyme composition to the milk base.

[0180]

[0103] Such a composition comprises a Sporobolomyces singularis beta-hexosyltransferase or a variant thereof and at least one component selected from milk powder, gluten, granulated fat, an additional enzyme, an amino acid, a salt, an oxidant, a pH buffer, a reducing agent, an emulsifier, sodium stearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters of fatty acids and diacetyl tartaric acid esters of mono- and diglycerides, a gum, a flavour, an acid, a starch, a modified starch, a humectant, a polyol and a preservative.

[0181]

[0104] Such a composition may be a solid (for example a powder or a granulate) or fluid (i.e. liquid) or frozen composition. If liquid, the composition is preferably a solution, suspension or emulsion.

[0182]

[0105] A composition may comprise one or more compounds selected from the group consisting of: milk powder, gluten, granulated fat, an additional enzyme, an amino acid, a salt (like sodium or potassium chloride), an oxidant, a pH buffer, a reducing agent, an emulsifier, sodium stearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters of fatty acids and diacetyl tartaric acid esters of mono- and diglycerides, a gum, a flavour, an acid, a starch, a modified starch, a humectant, a polyol (like glycerol) and a preservative. The term composition includes a pre-mix.

[0183]

[0106] A composition may comprise one or more further enzyme(s) such as an amylase such as an alpha-amylase, for example a fungal alpha-amylase, a beta-amylase; a glucanotransferase; a peptidase for example an exopeptidase or endopeptidase; a transglutaminase; a protein glutaminase; a cellulase; a hemicellulase, in particular a pentosanase such as xylanase; protease; a protein disulfide isomerase, e.g., a protein disulfide isomerase as disclosed in WO 95 / 00636; a glycosyltransferase; a peroxidase; a laccase; an oxidase, such as an hexose oxidase, a glucose oxidase, aldose oxidase, pyranose oxidase; a lipoxygenase; L-amino acid oxidase; a DSM IP Assets B.V. 2024P00107WO

[0184] glucoamylase, a lactase, a catalase ora phytase and / or an asparaginase. Preferably, a preferred further enzyme is lactase, glucose oxidase or catalase.

[0185]

[0107] A suitable commercial example of an enzyme having hydrolytic activity on lactose (i.e. a lactase) is Maxilact (dsm-firmenich), Lactozyme (Novozymes / Novonesis), Saphera (Novozymes 1 Novonesis), Nolafit (Chr. Hansen I Novonesis), Ha-lactase (Chr. Hansen I Novonesis), Godo YNL- 2 (Dupont) and some other enzymes described as lactases. Other suitable lactases have been described in the scientific literature and / or in patent publications.

[0186] Yogurt

[0187]

[0108] In a further aspect, the invention provides a yogurt obtainable by any of the methods as described above. Such a yogurt comprises an enzymatically active Sporobolomyces singularis hexosyltransferase or a variant thereof. The details provided above, for example in respect of the Sporobolomyces singularis hexosyltransferase or a variant thereof, equally apply to this part of the invention.

[0188] Use according to the invention

[0189]

[0109] In yet a further aspect, the invention provides use of Sporobolomyces singularis hexosyltransferase or a variant thereof for obtaining storage stable GOS in yogurt without an enzyme inactivation step. The details provided above, for example in respect of the Sporobolomyces singularis hexosyltransferase or a variant thereof, equally apply to this part of the invention.

[0190]

[0110] Preferable, the term storage stable refers to less than 10 % reduction of GOS when the produced fermented milk product such as yogurt is stored for 40 days at 3 to 10 degrees Celsius.

[0191]

[0111] The present invention is further illustrated by the following, non-limiting, Examples. DSM IP Assets B.V. 2024P00107WO

[0192] Examples

[0193] Methods and materials

[0194] Medium composition

[0195]

[0112] YEP2D medium: 10 g / l yeast extract, 20 g / l bacto-peptone, 40 g / l glucose. pH was set to pH 6.7 with 4N NaOH. Medium was autoclaved for 30 minutes at 110°C.

[0196]

[0113] YEP2D / MES medium: 10 g / l yeast extract, 20 g / l bacto-peptone, 40 g / l glucose, 20 g / l MES. pH was set to pH 6.7 with 4N NaOH. Medium was autoclaved for 30 minutes at 110°C.

[0197]

[0114] YEP2D plates contain YEP2D medium with 1.8-2% agar. Medium was autoclaved for 30 minutes at 110°C and poured in petridishes.

[0198] Strains

[0199]

[0115] K. lactis strain GG799: The Kluyveromyces lactis strain is used as a wild-type strain. The strain is obtained from New England Biolabs, Ipswich, Massachusetts, USA.

[0200] Molecular biology techniques

[0201]

[0116] Molecular biology techniques known to the skilled person were used (see: Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor, NY, 2001). Examples of the general design of expression vectors for gene over expression, transformation, use of markers and selective media can be found in W02007060247, WO2010102982 and US4943529 and references herein.

[0202] Activity determination BHT

[0203]

[0117] The activity of BHT was determined using an assay which is essentially similar to the assay described (Workman and Day (1982) Appl. Environm. Microbiol. 44(6) 1289-1295). Assay conditions were slightly adapted and performed at 37 °C in sodium acetate buffer pH 4.40 at a p-nitrophenyl-p-D-glucopyranoside (PNPG) concentration of 4.7mM. After termination of the reaction the pNP released was measured at 405 nm, and the enzyme activity was expressed as nanomole of p-nitrophenol released per second (BHTU).

[0204] Protein quantification for BHT, Nurica and Saphera Fiber

[0205]

[0118] Enzyme protein concentration was determined by qSDS-PAGE relative to a BSA standard. SDS-PAGE gels were stained with SYPRO™ Ruby protein gel stain and scanned with a GelDoc Go imaging system (Bio-Rad).

[0206] GOS analysis DSM IP Assets B.V. 2024P00107WO

[0207]

[0119] For GOS analysis the following HPLC method was used:

[0208]

[0209] GOS oligosaccharides of different degrees of polymerisation (DP) were quantified relative to a standard such as maltose or lactose. The DP of GOS oligosaccharide peaks was determined using commercial maltose oligosaccharide standards. For GOS-fiber quantification, the GOS species of DP3 and higher were summed up. The values as shown in the Examples were obtained by using lactose as a standard.

[0210] Lactose analysis

[0211] Lactose was quantified based on lactose standard using the following HPLC method:

[0212]

[0213] Sample preparation was performed by dispersing approximately 1 g sample in 25 mL Milli-Q purified water, vigorously mixing for 30 min on a magnetic stirrer and centrifuging for 10 min at 20,000xg. Approximately 0.5-0.6 mL of the supernatant was transferred into an ultrafiltration tube, avoiding the upper fat layer, and ultrafiltrated through a 10 kDa filter at 14,000 x g, for at least 15 min. The clear filtrate was diluted with Milli-Q water to a lactose concentration of approximately 1 mg / L.

[0214] Example 1 : DNA constructs and transformation

[0215]

[0120] Synthetic DNA constructs were designed to start with a Xhol restriction site, encoding amino acids L and E, followed in frame with DNA encoding a kex-protease cleavage site with amino acids K and R, followed by in frame genes encoding variants of BHT and ending with a Pad restriction site just after the stop codon. As an example, a DNA fragment encoding the truncated DSM IP Assets B.V. 2024P00107WO

[0216] BHT sequence is listed as SEQ ID NO: 7 Codon usage was adapted according to the method described in patent application US090286280. All variants were designed in a similar fashion and cloned as Xhol Pad fragments in vector pKLACI (New England Biolabs, Ipswich, Massachusetts, USA).

[0217]

[0121] The resulting open reading frames start with the leader sequence of the K. lactis Mating Factor alpha and progresses over the kex processing site to the BHT variants.

[0218]

[0122] Following the design of synthetic DNA constructs were designed. Amino acid changes were introduced resulting in 189 variants.

[0219]

[0123] Transformation and strain selection were performed by electroporation and selection on acetamide containing plates essentially as described in W02007060247. The plasmids were linearized by digestion with Sacll and transformed to Kluyveromyces lactis strain GG799 by electroporation. Of each construct six transformants were tested for BHT production using shake flask fermentations, and the best producing transformant was selected for further analysis.

[0220]

[0124] Starting from 189 variants, we selected 7 variants with superior trans-galactosylation activity with lactose as substrate. These samples were tested in milk and yoghurt application after which some variants were selected for further analysis.

[0221]

[0125] A selection of the new variants and their mutations is depicted in Table 1. Position of the change is indicated in comparison with the truncated mature BHT sequence (SEQ ID NO: 2).

[0222] Example 2: Cultivation, purification and concentration

[0223]

[0126] The Kluyveromyces lactis strains harbouring a variant BHT gene were placed on YEP2D agar plate and grown for 48 hours at 30°C. A pre-culture in 20 ml of YEP2D medium in 100 ml Erlenmeyer flasks was inoculated with the yeast cells taken from the plates. The cultures were grown for 24 hours in an incubator shaker at 30°C and 250 rpm. The amount of pre-culture for inoculation of new 500 ml Erlenmeyer flasks with 100 ml YEP2D / MES medium was calculated to give a GD600=0.01. These main cultures were grown for 65 hours in an incubator shaker at 30°C and 250 rpm.

[0224]

[0127] Broth from each flask was centrifuged and the supernatant containing the target enzymatic activity was clarified using a dead-end filter. The clear liquid, containing the enzyme, was subsequently concentrated using a 10kDa MWCO PES spiral wound ultra-filtration system. The concentrate was then filtered again and stored frozen until use.

[0225] Example 3: Use of BHT-02, -03 and -04 in FMP

[0226]

[0128] Retail pasteurized semi-skimmed cow milk was fortified with 2.32% skim milk powder, pasteurized for 30 min at 85°C and stored refrigerated over-night. Prior to yoghurt preparation, the milk was equilibrated to 42°C for 30 min. Then yoghurt fermentation was performed in duplicates for each enzyme variant at 42°C. 200 mL milk was inoculated with 2U / WOOL of YS-141 (dsm- DSM IP Assets B.V. 2024P00107WO

[0227] firmenich; blend consisting of Streptococcus thermophilus, Lactobacillus delbrueckii bulgaricus) and BHT-02, -03 or -04 enzymes were dosed at 3.4 mg BHT protein / L milk. The inoculated milk was divided in two parts:100 mL was used to monitor milk acidification at 42°C until the end of yoghurt fermentation at pH 4.6 was reached; the remaining 100 mL were aliquoted at ~10mL into Falcon tubes for yoghurt preparation at small scale at 42°C. These aliquots were withdrawn during the yoghurt fermentation after 1 , 2, 3 hr and at the end of fermentation (when pH 4.6 was reached in the control tube - EoF). After reaching pH 4.6 all remaining yoghurt aliquots were stored refrigerated for the period of 1 to 40 days, to determine GOS stability. Upon withdrawal of a sample at given time points (1 hr, 2hr, 3 hr, EoF, 1 day, 7 days, 14 days, 28 days, 40 days) enzymes were immediately inactivated by heating at 90°C for 10 min and stored frozen until analysis for galactose and glucose (DP1), lactose and other disaccharides (DP2) and GOS fiber (DP3 and higher). Control yoghurt preparation without enzymes addition was included in duplicates.

[0228]

[0129] Table 2 shows GOS fiber (sum of all oligosaccharides with a degree of polymerization (DP) of 3 and higher) and sugar (DP 1 and 2) concentrations analyzed at the end of yoghurt fermentation (when pH 4.6 reached at 42°C; EoF) and after 40 days shelf-life storage at 4°C forthetop 2 variants (BHT_03 and BHT_04) and wild-type BHT (BHT_02) applied at the concentration 3.4 mg / L milk in comparison with the plain yoghurt using only YS-141 :

[0229] Table 2: Determination of sugar and GOS in yoghurt at the EoF and after 40 days storage

[0230]

[0231] *DP2 is lactose plus other disaccharides

[0232] The GOS fiber concentration reached 11.0-15.3 g / kg at the end of fermentation depending on the BHT variant applied. GOS fiber content was further monitored during yoghurt shelf-life of 40 days stored refrigerated. GOS fiber remained stable, in contrast to the faith of GOS produced with enzymes known in the art, during storage in yoghurt (see Example 5). DSM IP Assets B.V. 2024P00107WO

[0233] Both variants BHT-03 and BHT-04 showed superior performance in comparison with wild-type BHT-02 with improved GOS fibers concentration at the end of fermentation, which remained stable during shelf-life of 40 days.

[0234] Control yoghurt preparation without enzymes addition showed no GOS formation. The sugar content (all DP1 + DP2) of the yoghurt produced with these enzymes is reduced 30-40% in comparison with a control yoghurt recipe without enzymes, both at the end of fermentation and after 40 days storage (see Table 2).

[0235] GOS-fiber formation during fermentation and stability during storage of the yoghurt is also depicted in Figure 1. Further increasing the BHT concentration at the start of the fermentation led to only a slight increase in GOS-fiber content at EoF for all BHT samples (data not shown), but both BHT-03 and BHT-04 were always improved in GOS-fiber content compared to BHT-02. A similar improvement of GOS fiber production at EoF with variants BHT-03 and BHT-04 compared to BHT-02 was also seen when the enzymes were dosed at 2.0, 4.7 or 6.7 mg protein / L milk.

[0236] Example 4: Use of BHT-02, -03 and -04 in milk

[0237]

[0130] GOS production in semi skimmed pasteurized milk (containing 4.8 g lactose, 3.5 g proteins and 1.5 g fat per 100 mL) by BHT variants, Nurica (IFF) and Saphera Fiber (Novozymes) was tested by dosing at 104 mg enzyme protein / L milk (for Nurica this corresponds to 3.2 g / L and for Saphera Fiber with 3.4 g / L of volume dosing). Before the start of the incubation the milk was equilibrated at 8°C. The hydrolysis was performed in 250 ml Schott bottles in an incubator (T= 8°C) at continuous gentle stirring using magnetic stirring plates. A control sample (without addition of enzyme) was included. All BHT, Nurica and Saphera Fiber samples were first diluted to an equal enzyme protein concentration and an equal volume was introduced in each test to prevent volumetric differences between the samples. During the incubation, samples of ~5 ml were taken at different timepoints and heat-treated for 10 minutes at 95°C in order to inactivate the enzyme, cooled on ice and stored at -18°C until analysis.

[0238] Table 3 shows GOS fiber (DP3 and higher) concentrations analyzed after milk incubation for 24 hr for all the samples. At 24 hr incubation both control samples Nurica and Saphera Fiber reach their optimal GOS fiber production at this dosage, so this is a good time point to compare BHT samples with these controls. For the BHT samples, the GOS fiber concentration reached 15-18.3 g / L after 24hrs, wherein both BHT-03 and BHT-04 showed optimal GOS fiber concentration already in the period shorter than 24 hrs (see Figure 2). Also in milk the total sugar reduction (reduction in DP1+DP2) was 30-40%, as was also seen in yoghurt (see Example 3).

[0239] Table 3: Determination of sugar and GOS in milk after 24 hr incubation DSM IP Assets B.V. 2024P00107WO

[0240]

[0241] < <

[0242] *DP2 is lactose plus other disaccharides

[0243] BHT has higher specificity towards GOS formation than both Nurica and Saphera Fiber, yielding higher amounts of total GOS fiber (DP3-6) within 24 hours but lower amounts of monosaccharides (Table 4 - DP1). Hence, BHT has less hydrolase activity and more transferase activity in milk compared to these commercial references. None of the enzymes reached lactose-free concentration (0.1% or 0.01%).

[0244] When the milk incubation was extended to 48 hr, a clear difference between the BHT samples and the controls Nurica and Saphera Fiber was seen. While the GOS fiber content in all BHT samples seems to be stable during this extended incubation period, GOS fiber in milk made with both control enzymes seems to be degraded. Having a higher GOS fiber stability in milk may give flexibility in the production conditions at an industrial scale.

[0245] Example 5: Comparisons of GOS production by BHT with commercial references, in FMP

[0131] FMP preparation: Semi-skimmed milk (Plus) was fortified with 2.32 % skimmilk powder and after pasteurization under regular conditions (30 min, 85°C) stored refrigerated over-night. Prior to the yoghurt preparation, milk was equilibrated to 42°C for 30 min. CINAC fermentation (pH profile) was performed in duplicates for each enzyme dosage of Nurica, Saphera Fiber and BHT. Milk was inoculated with 2U / WOOL of YS-141 , a blend consisting of Streptococcus thermophilus and Lactobacillus delbrueckii bulgaricus.

[0246] Table 4 shows the accumulation of GOS fiber at the end of the fermentation (EoF) using different dosages of BHT-02, BHT-03 and control enzymes. When all enzymes were added at the start of the fermentation in similar dosage (e.g. 4.7 mg / L; for Nurica this corresponds to 0.54 g / L and for Saphera Fiber with 0.58 g / L of volume dosing), the BHT enzymes clearly were superior to the control enzymes and accumulated much more GOS fiber. W.r.t. the accumulation of GOS fiber, a dosage of 17.5 mg / L of Nurica is comparable to 6.7 mg / L BHT-02 or 4.7 mg / L BHT-03, and a dosage of 17.5 mg / L of Saphera Fiber is comparable to less than 3.4 mg / L BHT-02 or 2.5 mg / L BHT-03. This experiment and Example 3 show that the BHT enzymes can be used at a much lower dosage than prior art enzymes in the FMP application. Both control enzymes Nurica and DSM IP Assets B.V. 2024P00107WO

[0247] Saphera Fiber accumulate much more DP1 , again indicating that these control enzymes are much more hydrolytic then BHT.

[0248] Table 4: Determination of sugar and GOS in yoghurt

[0249]

[0250] DP2 is lactose plus other disaccharides

[0251] The experiment above indicates that for a fair comparison of the GOS production during fermentation, the control enzymes have to be dosed at much higher amount than the BHT enzymes. Therefore, we repeated this experiment and dosed BHT-02 at 6.7 mg / L, BHT-03 at 4.7 mg / L and Nurica and Saphera Fiber at 17.5 mg / L. Samples were taken during fermentation (1 , 2, 3, 5 hours and EoF) and storage of the yoghurt (1 , 7, 14, 21 , 28 and 40 days) as described in Example 3. Results of this experiment are shown in Figure 3. Due to the difference in enzyme dosage, GOS-fiber accumulation during the yoghurt making was similar in the BHT variants and Nurica, and all yoghurts accumulated ~16 g / kg GOS-fiber. As was already expected from Table 4, Saphera Fiber was clearly less effective in GOS formation.

[0252] During yoghurt storage it became clear that the GOS-fiber made with BHT variants was clearly more stable than that of the controls Nurica and Saphera Fiber. Hence, prior art enzymes need inactivation either before or after yoghurt making to prevent GOS fiber degradation during shelflife, as was also indicated previously (W02020 / 117548, WO2015 / 086746 and WO2022189568). Surprisingly, GOS-fiber in yoghurt produced with BHT is much more stable during shelf-life without enzyme inactivation. Clearly this extra process step is not required when BHT is used for the yoghurt making and storage.

[0253] Example 6: pH activity profile of BHT variants

[0254]

[0132] The activity of the different BHT variants at different pH was measured using the assay with p-nitrophenyl-p-D-glucopyranoside as substrate as described above. For setting the pH buffer DSM IP Assets B.V. 2024P00107WO

[0255] A (50 mM citric acid) was mixed with buffer B (100 mM disodium phosphate) to obtain the right pH. Relative activity of each BHT variant compared to the optimum activity at pH5.0 is depicted in Figure 4. The activity profile of BHT-03 was shifted to a slightly less acidic optimum compared to BHT-02.

[0256] Example 7: Use of BHT-03 in FMP with varying milk base and culture blends

[0257]

[0133] GOS - containing yoghurts were prepared as in example 3 with or without starch addition or using fat-free milk base without addition of milk powder. Milk base was inoculated with 2U / WOOL milk of commercial dsm-firmenich cultures blends FVV-121 , FVV-221 Pioneer-01 , YS-051 , YS-241 consisting of Streptococcus thermophilus and Lactobacillus delbrueckii bulgaricus at 42°C with BHT-03 enzyme at dosage range 2.0 - 4.7 mg / L milk, resulting in similar results as in example 3.

Claims

DSM IP Assets B.V. 2024P00107WOClaims1. A method for producing a yogurt comprising galacto-oligosaccharide (GOS), wherein the method comprises the steps of(a) providing a milk base comprising lactose(b1) treating the milk base with Sporobolomyces singularis beta-hexosyltransferase or a variant thereof to generate GOS followed by fermenting the treated milk base with a lactic acid bacterial strain until a pH below 5 is reached, or(b2) treating and fermenting the milk base at the same time by treating the milk base with Sporobolomyces singularis beta-hexosyltransferase or a variant thereof to generate GOS and by fermenting the milk base with a lactic acid bacterial strain until a pH below 5 is reached such as to produce a yogurt comprising GOS,and wherein said method does not comprise a step for inactivating the Sporobolomyces singularis beta-hexosyltransferase or a variant thereof.

2. The method according to claim 1 , wherein the Sporobolomyces singularis beta-hexosyltransferase or a variant thereof has an amino acid sequence with equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2.

3. The method according to claim 1 or 2, wherein the variant has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position 295 or 321 , wherein said position is defined with reference to SEQ ID NO: 2.

4. The method according to any of the previous claims, wherein the variant comprises at least the mutation S295L or R321H, wherein said positions are defined with reference to SEQ ID NO: 2.

5. The method according to any of the previous claims, wherein the variant comprises at least 2 substitutions, preferably S295L and R321H, wherein said positions are defined with reference to SEQ ID NO: 2.

6. The method according to any of the previous claims, wherein the variant is a polypeptide that has an amino acid sequence of SEQ ID NO: 3, SEQ ID NO:4 or SEQ ID NO:5.29DSM IP Assets B.V. 2024P00107WO7. The method according to any of the previous claims, wherein said method further comprises a step of storing the yogurt comprising GOS for at least 7 days at 3 to 10 degrees Celsius.

8. The method according to any of the previous claims, wherein the yogurt is a set, stirred or drinking yogurt.

9. A yogurt obtainable by a method according to any one of claims 1 to 8.

10. Use of Sporobolomyces singularis beta-hexosyltransferase or a variant thereof for obtaining storage stable GOS in yogurt without an enzyme inactivation step.

11. Use according to claim 10, wherein storage stable is less than a 10% reduction when stored for 40 days at 3 to 10 degrees Celsius.30

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