Manufacturing method of allolactose

Abstract

The present invention relates to a method for obtaining a composition comprising allolactose by applying one or more glucose-deficient lactic acid bacteria strains. The present invention also relates to the use of said strains for preparing a food product comprising allolactose and for increasing the allolactose content in a food product. The present invention also relates to a food product comprising allolactose and one or more glucose-deficient lactic acid bacteria strains.

Description

[Technical field]

[0001] The present invention relates to a method for producing allolactose. [Background technology]

[0002] Functional and health foods, and their effects on the gut microbiota, are attracting increasing attention from consumers worldwide.

[0003] Functional foods can be defined as dietary products that, in addition to providing nutrients and energy, beneficially modulate one or more target functions in the body by enhancing specific physiological responses and / or reducing the risk of disease ( Nicoletti, 2012 ).

[0004] Remarkable properties of the gut microbiota include its functionality and resilience. Several strategies have been proposed to modulate the composition and / or function of the gut microbiota. One such strategy is the application of functional foods, such as probiotics and other live microorganisms, prebiotics, and synbiotics, to modulate the composition and / or function of the gut microbiota. The current scientific definition of prebiotics is "substrates that are selectively utilized by the host microorganisms to confer health benefits." Thus, the concept contains three important parts: substance, physiological beneficial effect, and mechanism.

[0005] Galactooligosaccharides (GOS) non-digestible fibers with prebiotic activity. They are indigestible oligosaccharides, composed of 2-20 molecules of galactose and one molecule of glucose. GOS are recognized as important prebiotics that stimulate the growth of Lactobacillus and Bifidobacteria in the intestine. Thus, they beneficially affect the host by selectively stimulating the growth and / or activity of many gastrointestinal microorganisms (probiotics) that confer health benefits. GOS have been shown to help regulate the colonic microflora to a healthy balance. This typically involves an increase in Bifidobacteria and Lactobacillus and a decrease in less desirable microorganisms.

[0006] Allolactose is a disaccharide similar to lactose and is considered a common GOS. It is composed of the monosaccharides D-galactose and D-glucose linked through a β1-6 glycosidic bond instead of the β1-4 bond of lactose. Allolactose may result from the transglycosylation of lactose by β-galactosidase.

[0007] Given the high demand for functional and healthy foods by global consumers, there is growing commercial interest in the food industry to produce GOS such as allolactose as functional ingredients in a wide range of products, especially raw dairy products. While the production of allolactose itself is important to the industry, there is an even greater demand, for example in raw dairy products where allolactose is not actively added to the product but is in fact produced directly within the product. Healthy foods include foods such as raw dairy products containing GOS, as well as low-calorie and sweetened foods.

[0008] For example, sugar in fermented foods such as raw dairy products is increasingly being replaced by sweeteners such as aspartame, acesulfame K, sucralose and saccharin, which provide sweetness with a lower calorie intake. In recent years, as consumers have become more aware of the drawbacks of artificial sweeteners, there has been an increasing demand for fermented dairy products that contain only natural sweeteners or preferably no added sweeteners. A particular challenge lies in the development of food products such as raw dairy products that have a high natural (internal) sweetness. Summary of the Invention [Problem to be solved by the invention]

[0009] Based on the above, there is a need to provide a method by which fermented foods, such as fresh dairy products, can be obtained that simultaneously contain (i) allolactose and (ii) increased natural sweetness, and in which the allolactose content and increased natural sweetness are primarily produced directly in the product during fermentation. [Means for solving the problem]

[0010] The above-mentioned objects are achieved by the present invention, inter alia, by providing a method for obtaining a composition comprising allolactose by using at least one or more glucose-deficient lactic acid bacteria strains. The present invention also provides a method for obtaining a composition, such as a fermented food product, in particular a raw dairy product, comprising allolactose by using said strains. The present invention also relates to a composition comprising allolactose and said strains, as well as a method for increasing the content of allolactose in a composition using said strains.

[0011] Thus, according to one aspect, the present invention relates to a method for producing a composition comprising allolactose, said method comprising the steps of: a) inoculating a substrate containing lactose with one or more glucose-deficient lactic acid bacteria strains, wherein the glucose deficiency of said strains is caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein; and b) fermenting the inoculated substrate to obtain a composition comprising allolactose.

[0012] According to a further aspect, the present invention relates to a composition comprising allolactose obtainable by the process of the present invention.

[0013] A further aspect of the invention relates to a composition comprising at least 0.04% w / w allolactose, said composition further comprising one or more glucose deficient lactic acid bacteria strains, wherein the glucose deficiency of said strains is caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein.

[0014] Another aspect of the present invention relates to the use of one or more glucose-deficient lactic acid bacteria strains for preparing a composition comprising allolactose, wherein the glucose deficiency of said strains is caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein.

[0015] A further aspect of the present invention relates to the use of one or more glucose deficient lactic acid bacteria strains to increase the allolactose content in a composition, wherein the glucose deficiency of said strains is caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Before outlining the present invention in more detail, a set of terms and conventions will first be defined.

[0017] The term "genus" refers to a genus as defined at the website www.ncbi.nlm.nih.gov / taxonomy. As used herein, a bacterial "strain" refers to a bacterium that does not genetically change as it grows or multiplies. It includes many identical bacteria.

[0018] In the present context, the term "mutant" or "mutant strain" is to be understood as a strain derived or derivable from the strain of the invention (or the mother strain), for example by genetic engineering, radiation and / or chemical treatment. The mutant is preferably a functionally equivalent mutant, for example a mutant having substantially the same or improved properties (for example in terms of texture, shear stress, viscosity, gel consistency, mouth coating, flavor, post-acidification, acidification rate and / or phage robustness) as the strain from which it is derived. Such mutants are part of the present invention. In particular, the term "mutant" refers to a strain obtained by subjecting the strain of the invention to any conventionally used mutagenic treatment, including treatment with chemical mutagens such as ethane methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light, or a spontaneous mutant. The mutant may have undergone multiple mutagenesis treatments (a single treatment should be understood as one mutagenesis step followed by a screening / selection step), but it is currently preferred to carry out no more than 20, or no more than 10, or no more than 5 treatments (or screening / selection steps). In currently preferred mutants, less than 5%, or no more than 1%, or even no more than 0.1% of the nucleotides in the bacterial genome have been shifted to another nucleotide or deleted compared to the mother strain. As will be apparent to one skilled in the art, the mutant of the present invention may also be the mother strain.

[0019] As used herein, the term "mutant" or "variant" should be understood as a strain that is functionally equivalent to a strain of the invention, e.g., a strain that has substantially the same or improved properties or characteristics (e.g., texture, acidification rate, viscosity, gel hardness, mouth coating, flavor, post-acidification and / or phage robustness). Such mutants can be identified using appropriate screening techniques and are part of the present invention.

[0020] For the purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453), as implemented in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. (2000) Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. Optional parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "Longest Identity" (obtained using the -no Brief option) is used as the percent identity, calculated as follows: (identical residues x 100) / (length of alignment - total number of gaps in the alignment).

[0021] In the present specification and claims, conventional single-letter and three-letter codes for amino acid residues are used. For ease of reference, the amino acid changes in the mutants and variants of the present invention are described using the following nomenclature: amino acid residue of the parent enzyme:position:substituted amino acid residue. According to this nomenclature, for example, the substitution of an alanine residue for a glycine residue at position 20 is indicated as Ala20Gly or A20G. The deletion of an alanine at the same position is indicated as Ala20Gly or A20G. * or A20 *Insertions of additional amino acid residues (such as glycine) are indicated as Ala20AlaGly or A20AG. Deletions of consecutive amino acid residues (e.g., between alanine at position 20 and glycine at position 21) are indicated as DELTA(Ala20-Gly21) or DELTA(A20-G21). When a parent enzyme sequence contains a deletion (e.g., an alanine at deleted position 20) compared to the enzyme sequence used to number insertions at such positions, * 20Ala or * A20A is designated as A20G+E21S or A20G / E21S. Multiple mutations are separated by a plus sign or a slash. For example, two mutations at positions 20 and 21 substituting alanine and glutamic acid for glycine and serine, respectively, are designated as A20G+E21S or A20G / E21S. When an amino acid residue at a particular position is substituted with more than one alternative amino acid residue, these residues are separated by a comma or a slash. For example, the substitution of alanine at position 30 with glycine or glutamic acid is designated as A20G,E or A20G / E, or A20G,A20E. When a position suitable for modification is identified herein without suggesting any particular modification, it should be understood that the amino acid residue present at that position can be substituted with any amino acid residue. Thus, for example, where modification of an alanine at position 20 is mentioned but not specified, it should be understood that the alanine may be deleted or substituted with another amino acid residue (i.e., any one of R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V).

[0022] In the context of the present invention, a mutation in a gene (genetic mutation) should be understood as a change in the nucleotide sequence of the genome of an organism that results in a change in the phenotype of the organism, where the change can be a deletion of a nucleotide, a replacement of a nucleotide by another nucleotide, an insertion of a nucleotide, or a frameshift. In the context of the present invention, a deletion should be understood as a genetic mutation that results in the removal of one or more nucleotides of the nucleotide sequence of the genome of an organism; an insertion should be understood as the addition of one or more nucleotides to a nucleotide sequence; a substitution (or point mutation) should be understood as a genetic mutation in which a nucleotide of a nucleotide sequence is replaced by another nucleotide; a frameshift should be understood as a genetic mutation caused by the insertion or deletion of a number of nucleotides not divisible by 3 in a nucleotide sequence, resulting in a change in the reading frame and a translation that is completely different from the original reading frame; the introduction of a stop codon should be understood as a point mutation in the DNA sequence that results in a premature stop codon; an inhibition of substrate binding of an encoded protein should be understood as any mutation in the nucleotide sequence that results in a change in the protein sequence that prevents the binding of a substrate to the catalytic site of the protein. Furthermore, a knockout mutant should be understood as a genetic mutation that results in the removal or deletion of a gene, such as an entire gene or an entire open reading frame, from the genome of an organism.

[0023] In the present specification and claims, the conventional single-letter code for nucleotides is used following similar principles as described for the amino acid nomenclature above.

[0024] Algorithms for aligning sequences and determining the degree of sequence identity between them are well known in the art. For the purposes of the present invention, the process for aligning nucleotide sequences can be carried out using blastn, provided by the National Center for Biotechnology Information (NCBI) at https: / / blast.ncbi.nlm.nih.gov, applying standard parameters.

[0025] As used herein, "mutant bacteria" or "mutant strain" refers to naturally occurring or induced mutant bacteria that contain one or more mutations in their genome (DNA) that are not present in the wild-type DNA. An "induced mutant" is a bacterium in which a mutation has been induced by artificial treatment, such as treatment with chemical mutagens, ultraviolet light or gamma radiation. In contrast, a "spontaneous mutant" or "spontaneous mutant" has not been induced by humans. As used herein, mutant bacteria are non-GMO (non-genetically modified organisms), i.e., not modified by recombinant DNA technology.

[0026] Allolactose is to be understood as a disaccharide consisting of the monosaccharides D-galactose and D-glucose linked via a β1-6 glycosidic bond.

[0027] The amounts of fructose, galactose, glucose, sucrose, lactose and allolactose are determined as disclosed in Example 1.

[0028] For strains of the genus Lactobacillus, the term "CFU" refers to colony forming units as determined by growth (formation of colonies) on MRS agar plates incubated at 37° C. for 3 days under anaerobic conditions. The composition of MRS agar medium is as follows (g / l): Bacto Proteose Peptone No.3:10.0 Bacto Beef Extract: 10.0 Bacto Yeast Extract: 5.0 Glucose: 20.0 Sorbitan monooleate complex: 1.0 Ammonium citrate: 2.0 Sodium acetate: 5.0 Magnesium sulfate: 0.1 Manganese sulfate: 0.05 Dipotassium phosphate: 2.0 Bacto agar: 15.0 Milli-Q water: 1000ml. The pH is adjusted to 5.4 or 6.5: for L. rhamnosus, L. casei and L. paracasei, the pH is adjusted to 6.5. For all other Lactobacillus species, the pH is adjusted to 5.4. In particular, for L. delbrueckii subsp. bulgaricus, L. acidophilus and L. helveticus, the pH is adjusted to 5.4. For L. rhamnosus, L. casei and L. paracasei, the pH is adjusted to 6.5.

[0029] With respect to S. thermophilus, the term "CFU" refers to colony forming units as determined by growth (formation of colonies) on M17 agar plates incubated aerobic at 37° C. for 3 days. The composition of M17 agar (g / l) is as follows: Tryptone: 2.5g Meat digesta: 2.5g Soybean meal and papaya digest: 5.0g Yeast extract: 2.5g Meat extract: 5.0g Lactose: 5.0g Sodium glycerophosphate: 19.0g Magnesium sulfate, 7H2O: 0.25 g Ascorbic acid: 0.5g Agar: 15.0g Milli-Q water: 1000 ml. The pH is adjusted to a final pH of 7.1 ± 0.2 (25°C).

[0030] The term "lactic acid bacteria" as used herein refers to gram-positive, microaerophilic or anaerobic bacteria that ferment sugars producing acids, with lactic acid, acetic acid and propionic acid being the main acids produced. The most industrially useful lactic acid bacteria are found within the order "Lactobacillales", which includes the genera Lactococcus, Streptococcus, Lactobacillus, Leuconostoc, Pediococcus, Brevibacterium, Enterococcus and Propionibacterium. Lactic acid bacteria, including bacteria of the Lactobacillus and Streptococcus thermophilus species, are usually supplied to the dairy industry as frozen or freeze-dried cultures for bulk starter growth or as so-called "direct vat set" (DVS) cultures intended for direct inoculation of fermentation vessels or vats for the production of dairy products, such as fermented dairy products. Such cultures are commonly called "starter cultures" or "starters".

[0031] The inventors of the present invention have surprisingly discovered a method for obtaining a composition comprising allolactose by applying one or more glucose-deficient lactic acid bacteria strains, caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein, which was able to obtain allolactose.

[0032] Thus, according to one aspect of the present invention, there is provided a method for producing a composition comprising allolactose, said method comprising the steps of: a) inoculating a substrate containing lactose with one or more glucose-deficient lactic acid bacteria strains, wherein the glucose deficiency of said strains is caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein; and b) fermenting the inoculated substrate to obtain a composition comprising allolactose.

[0033] The term "2-deoxyglucose resistance" herein with respect to L. bulgaricus is defined by the ability of a particular mutant bacterial strain to grow into colonies when streaked onto a plate of M17 medium containing 20 mM 2-deoxyglucose after 20 hours of incubation at 40°C. The presence of 2-deoxyglucose in the medium prevents the growth of non-mutant strains, while the growth of mutant strains is unaffected or not significantly affected. Thus, for selection purposes, 2-deoxyglucose can be applied in the selection process.

[0034] The term "galactose positive strain", "gal positive strain" or "gal+ strain" in this context is defined as a strain that can metabolize / grow on / use galactose for growth. Galactose can be obtained by hydrolysis of lactose or by transport of galactose into the cell.

[0035] The term "galactose-fermenting strain" or "gal-fermenting strain" in this context refers to a strain having at least 10 4 It is defined as a strain that, when inoculated at a cell / ml, reduces the pH by a value of at least 1.0 after 16 h of incubation at 37°C in M17 supplemented with 2% galactose as the only carbohydrate.

[0036] Galactokinase, encoded by the galK gene, is an enzyme in the Leloir pathway of galactose metabolism that converts α-galactose to galactose-1-phosphate.

[0037] The term "mutations that inactivate a glucokinase protein" as used herein refers to mutations that result in an "inactivated glucokinase protein", i.e., a glucokinase protein that is unable to perform its normal function when present in a cell, and mutations that prevent the formation of the glucokinase protein or cause the degradation of the glucokinase protein. In particular, an inactivated glucokinase protein is a protein that is unable to promote the phosphorylation of glucose to glucose-6-phosphate or promotes the phosphorylation of glucose to glucose-6-phosphate at a significantly reduced rate compared to a functional glucokinase protein. A gene encoding such an inactivated glucokinase protein contains a mutation in the open reading frame (ORF) of the gene compared to a gene encoding a functional glucokinase protein, where the mutation includes, but is not limited to, a deletion, a frameshift mutation, the introduction of a stop codon, or a mutation that results in an amino acid substitution that alters the functional properties of the protein, or a promoter mutation that reduces or eliminates the transcription or translation of the gene.

[0038] As used herein, the term "functional glucokinase protein" refers to a glucokinase protein that, when present in a cell, promotes the phosphorylation of glucose to glucose-6-phosphate.

[0039] In preferred embodiments, the mutation reduces the activity of the glucokinase protein (rate of phosphorylation of glucose to glucose-6-phosphate) by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% when compared to a strain that does not have a mutation in the DNA sequence of the glcK gene.

[0040] Thus, if the mutation inactivates the encoded glucokinase protein, the activity of the protein is reduced by 100% compared to a S. thermophilus strain that does not have the mutation in the DNA sequence of the glcK gene, whereas if the mutation adversely affects expression of the gene, the activity of the glucokinase protein is reduced by at least at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to a S. thermophilus strain that does not have the mutation in the DNA sequence of the glcK gene.

[0041] Glucokinase activity can be measured by the glucokinase enzyme assay as described by Pool et al. (2006) Metabolic Engineering 8; 456-464.

[0042] The method may preferably further comprise the steps of: c) concentrating the composition containing allolactose to obtain a composition having an increased amount of allolactose compared to the composition before concentration.

[0043] The concentration in step c) can be carried out using filtration, including but not limited to diafiltration, membrane filtration, centrifugation, sedimentation, evaporation and / or chromatography, but any method applicable in the art can be used for this purpose.

[0044] Depending on the use of the allolactose, it is contemplated that the method further comprises the steps of: d) purifying the composition containing allolactose to obtain a composition having increased purity of allolactose compared to the composition before purification.

[0045] Purification can be carried out using any method known in the art, including, but not limited to, diafiltration, membrane filtration, centrifugation, precipitation, evaporation and / or chromatography.

[0046] The allolactose-containing composition obtainable according to the invention may be a fermented food product, more particularly a fermented product such as a fermented dairy product.

[0047] According to one embodiment, the one or more glucose deficient lactic acid bacteria strains are galactose positive.

[0048] According to one embodiment, the one or more glucose deficient lactic acid bacteria strains have a mutation in the galK gene, which encodes the galactokinase protein.

[0049] According to one embodiment, the glucose deficient lactic acid bacteria strain or strains are galactose fermenting.

[0050] It may be preferred that one or more of the glucose-deficient lactic acid bacteria strains carry a mutation that reduces or inactivates the transport of glucose into the cell. According to a preferred embodiment, the lactic acid bacteria is Streptococcus thermophilus (S. thermophilus) carrying a mutation that reduces or inactivates the transport of glucose into the cell.

[0051] As used herein, the term "mutations that reduce glucose transport into the cell" refers to mutations in genes encoding proteins involved in glucose transport that result in the accumulation of glucose in the cellular environment. Glucose levels in the culture medium of S. thermophilus or L. bulgaricus strains can be easily measured by methods known to those skilled in the art.

[0052] As used herein, the term "mutations that inactivate a glucose transporter" refers to mutations that result in an "inactivated glucose transporter," i.e., a glucose transporter protein that is unable to perform its normal function when present in a cell, and mutations that prevent the formation of or cause degradation of the glucose transporter protein.

[0053] The glucose-deficient lactic acid bacteria strain(s) may also harbor a mutation in a gene encoding a component of a glucose transporter, which mutation reduces or inactivates the glucose transporter or adversely affects expression of the gene.

[0054] As used herein, the term "functional glucose transporter protein" refers to a glucose transporter protein that, when present in a cell, facilitates the transport of glucose across the cell membrane.

[0055] The term "glucose deficient" is used in the context of the present invention to characterize lactic acid bacteria (LAB) that have partially or completely lost the ability to use glucose as a source for cell growth or for maintaining cell viability. The respective deficiency in glucose metabolism can be caused, for example, by a mutation in a gene that inhibits or inactivates the expression or activity of the glucokinase protein and / or a glucose transporter protein involved in glucose uptake.

[0056] LAB with defective glucose metabolism may increase the glucose concentration in the medium when grown with lactose as a carbohydrate source. The increase in glucose is caused by glucose secretion of the glucose-deficient LAB. The increase in glucose concentration in the medium can be measured by HPLC analysis using, for example, a Dionex CarboPac PA 20 3* 150mm column (Thermo Fisher Scientific, product number 060142).

[0057] The term "glucose fermenting" is used in the context of the present invention to characterize LAB that partially or completely maintain the ability to use glucose as a source for cell growth or maintenance of cell viability.

[0058] Thus, one or more glucose-deficient lactic acid bacteria strains, such as S. thermophilus strains and / or some Lactobacillus strains, can be both galactose-fermenting and glucose-fermenting. If a strain is able to ferment both galactose and glucose, it may be preferred that this strain carries a mutation that reduces or inactivates the transport of glucose into the cells. When grown on a substrate containing lactose, these strains do not transport glucose from the milk source into the cells and metabolize it, or at least to a lesser extent - whereas they transport lactose into the cells, metabolize it to glucose and galactose, and further metabolize the galactose and excrete glucose into the environment, further increasing the glucose concentration in the medium and thereby the "intrinsic sweetness" of the product.

[0059] Likewise, it may be preferred that one or more of the glucose deficient lactic acid bacteria strains have a mutation in the DNA sequence encoding the glucose / mannose phosphotransferase system.

[0060] According to more specific embodiments, one or more glucose-deficient lactic acid bacteria strains, such as, but not limited to, S. thermophilus strains, have a mutation in the DNA sequence of the manM gene encoding the PTS mannose / glucose / fructose subunit IIC (also called IICMan protein of the glucose / mannose phosphotransferase system, and therefore the two are used interchangeably herein), where said mutation inactivates the IICMan protein or negatively affects the expression of the gene. According to preferred embodiments, the mutation reduces the transport of glucose into the cell by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a cell without such a mutation.

[0061] Glucose transport into cells can be measured by the glucose uptake assay described by Cochu et al. (2003) Appl Environ Microbiol 69(9); 5423-5432.

[0062] According to a further embodiment, the one or more glucose deficient lactic acid bacteria strains, such as, but not limited to, S. thermophilus strains, are 6 ~10 7 When inoculated into a milk substrate at a concentration of CFU / ml and grown for 18 hours at 43° C., it is capable of producing allolactose in an amount of at least 0.04% w / w of milk in the milk substrate, said milk substrate comprising 4% protein, 1.5% fat, and 0.1% added sucrose. The milk substrate is prepared as disclosed in Example 1.

[0063] In a further embodiment, one or more glucose deficient lactic acid bacteria strains, such as, but not limited to, a Thermophilus strain, are cultured together with at least one further lactic acid bacteria strain (mixed culture) in a concentration of 1000 mM. 6 ~10 7 When inoculated into a milk substrate at a concentration of CFU / ml and grown at 43° C. until a pH of 4.55 was reached, it was capable of producing allolactose in the milk substrate in an amount of at least 0.04% w / w of milk, where the milk substrate contained 4% protein and 1.5% fat. The milk source was prepared as disclosed in Example 2.

[0064] According to one embodiment, the one or more glucose deficient strains are selected from the group consisting of Streptococcus and Lactobacillus.

[0065] According to another embodiment, the streptococcus is Streptococcus thermophilus.

[0066] According to another embodiment, the Lactobacillus genus is selected from the group consisting of Lactobacillus delbrueckii subsp. bulgaricus, Lacticaseibacillus rhamnosus, and Lactobacillus helvticus, Lacticaseibacillus paracasei subsp. Paracasei.

[0067] According to a preferred embodiment, the Streptococcus is Streptococcus thermophilus and the Lactobacillus is Lactobacillus delbrueckii subsp. bulgaricus.

[0068] In a preferred embodiment, the glucose deficient S. thermophilus is selected from the group consisting of DSM 25850, DSM 26722, DSM 28889, DSM 33719, DSM 32227 and DSM 33762 and mutant strains derived therefrom, wherein said mutant strain was obtained by using one of the deposited strains as starting material and said mutant strain retains or even improves the lactose fermentation and / or glucose secretion properties of the deposited strain.

[0069] Glucose-deficient Lactobacillus species, such as Lactobacillus delbrueckii subsp. bulgaricus, can produce 10% glucose in 9.5% B milk. 6 ~10 7 When inoculated at a concentration of CFU / ml and grown at 40°C for at least 20 h, it is expected to produce allolactose in 9.5% B milk in an amount of at least 0.04% w / w of B milk.

[0070] Glucose-deficient Lactobacillus species, such as Lactobacillus delbrueckii subsp. bulgaricus, can produce 10% glucose in 9.5% B milk. 6 ~10 7 When inoculated with at least one further strain of lactic acid bacteria (mixed culture) at a concentration of CFU / ml and grown at 40°C for at least 20 hours, it is expected to produce allolactose in 9.5% B milk in an amount of at least 0.04% w / w of B milk.

[0071] In the method according to the invention it may be envisaged that one or more glucose deficient S. thermophilus and / or at least one glucose deficient L. bulgaricus strains are inoculated in step a) together with at least one non-glucose deficient S. thermophilus strain or at least one non-glucose deficient L. bulgaricus strain, wherein the glucose deficiency of said strains is caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein.

[0072] A combination of S. thermophilus and L. bulgaricus is often used to obtain the best combination of acidity, taste and texture for compositions such as dairy products such as yogurt.

[0073] It is conceivable that the substrate in step a) of the method of the invention is inoculated with: i.DSM 25850, DSM 26722, DSM 28889 and DSM 28910DSM 28910, DSM 32227 and DSM 33762; ii. DSM 25850, DSM 26722, DSM 28889, DSM 28910, and DSM 33720; or iii. DSM 28910, DSM 32227, and DSM 33719.

[0074] It is also contemplated that the compositions of the invention include: i. DSM 25850, DSM 26722, DSM 28889 and DSM 28910; ii. DSM 28910, DSM 32227 and DSM 33762; iii. DSM 25850, DSM 26722, DSM 28889, DSM 28910 and DSM 33720; or iv. DSM 28910, DSM 32227 and DSM 33719.

[0075] The probiotic bacterial strains can be added to the process of the invention either before or after fermentation. If added before fermentation, the probiotic bacterial strains also function as fermenters.

[0076] The term "probiotic bacteria" refers to live bacteria that are administered in appropriate amounts to a consumer with the aim of achieving a health-promoting effect on the consumer. Probiotic bacteria are capable of surviving the conditions of the gastrointestinal tract after ingestion and colonize the intestine of the consumer.

[0077] It is understood that the taxonomy of the genus Lactobacillus was updated in 2020. The new taxonomy is disclosed in Zheng et al. 2020 and will be consistently described herein unless otherwise indicated. For purposes of the present invention, the following table provides a list of the new and old names of some Lactobacillus species relevant to the present invention.

[0078] [Table 1]

[0079] According to a particular embodiment of the invention, the probiotic strain according to the invention is selected from the group consisting of Lactobacillus species, such as Lactobacillus acidophilus, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lacticaseibacillus casei, Lactobacillus delbrueckii, Lactobacillus lactis, Lactiplantibacillus plantarum, Limosilactobacillus reuteri and Lactobacillus johnsonii, Bifidobacteria, such as Bifidobacterium longum. longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis, Bifidobacterium dentium, Bifidobacterium catenulatum, Bifidobacterium angulatum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum and Bifidobacterium infantis.

[0080] According to a particular embodiment of the invention, the probiotic lactic acid bacteria strain is selected from the group consisting of: Lactobacillus acidophilus, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus lactis, Lactiplantibacillus plantarum, Limosilactobacillus reuteri and Lactobacillus johnsonii.

[0081] According to a particular embodiment of the invention, the probiotic strain is Lactobacillus acidophilus deposited under DSM 13241 (LA-5®).

[0082] According to a particular embodiment of the invention, the probiotic Lactobacillus strain is selected from the group consisting of Lactocaceibacillus rhamnosus and Lactocaceibacillus paracasei strains. According to a particular embodiment of the invention, the probiotic strain is Lactocaceibacillus rhamnosus strain LGG® deposited under ATCC 53103. According to a particular embodiment of the invention, the probiotic strain is Lactocaceibacillus paracasei strain CRL 431 deposited under ATCC 55544.

[0083] In a particular embodiment of the invention, the probiotic Bifidobacterium strain is selected from the group consisting of Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis, Bifidobacterium dentium, Bifidobacterium catenulatum, Bifidobacterium angulatum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum and Bifidobacterium infantis. According to a particular embodiment of the invention, the probiotic Bifidobacterium probiotic strain is Bifidobacterium animalis subsp. lactis BB-12®, deposited under DSM 15954.

[0084] Depending on the nature of the allolactose-containing composition, it may be preferred that the lactose-containing substrate is an animal and / or plant-derived substrate, or it may be preferred that the lactose-containing substrate is a dairy substrate, such as a milk substrate.

[0085] The term "milk" should be understood as the lacteal secretion obtained by milking any mammal, such as a cow, sheep, goat, buffalo or camel. According to a preferred embodiment, the milk is cow's milk. The term milk also includes protein / fat solutions made partly or entirely from plant material.

[0086] Dairy ingredients can be partially replaced with plant materials, for example using plant-based milks derived from legumes (such as soy), nuts (such as coconut) and cereals (such as oats).

[0087] The term "legume" refers to any plant belonging to the family Fabaceae. Fabaceae is a large and economically important family of flowering plants commonly known as the legume family, pea family, bean family, or pulse family. Many different types of pulses are available. Legumes usually have a pod or husk that opens along two sutures when the seeds are ripe.

[0088] The term "nut" as used herein may refer to true nuts from trees or shrubs, or edible nuts which may be drupe nuts or nut-like seeds. In botanical terms, nuts are dry, single-seeded fruits that are nondehiscent (i.e., do not split along a distinct seam at maturity). Culinary nuts are nuts that do not qualify botanically as nuts but have a similar appearance and culinary role. Many edible nuts are the seeds of drupes, referred to herein as drupe nuts. Drupes are nondehiscent fruits in which the outer fleshy portion surrounds a hardened endocarp of a single shell (pit or stone) that contains the seed. Drupes are the seeds of stone fruits.

[0089] The term "cereal" refers to both true cereals and pseudocereals. True cereals refer to the seeds of plants that belong to the grass family. Pseudocereals are the seeds of plants that do not belong to the grass family, but are used in much the same way as cereals.

[0090] When the substrate is entirely plant-based, lactose is added to obtain a lactose-containing substrate, which may be obtained from a dairy side stream.

[0091] The term "dairy substrate" may be any raw and / or processed milk material that can be subjected to fermentation according to the method of the present invention. Useful dairy substrates thus include, but are not limited to: solutions / suspensions of milk or milk-like products containing proteins, such as whole or low-fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, milk powder, whey, whey permeate, lactose, mother liquor from lactose crystallization, whey protein concentrate, or cream. Obviously, the dairy substrate may be derived from any mammal, e.g. substantially pure mammalian milk, or reconstituted milk powder, or the dairy substrate may be derived from a plant material. Preferably, at least a portion of the proteins in the dairy substrate are (i) proteins naturally occurring in mammalian milk, such as casein or whey protein, or (ii) proteins naturally occurring in plant milk. However, some of the proteins may be proteins that do not naturally occur in cow's milk.

[0092] Prior to fermentation, the dairy substrate may be homogenized and pasteurized according to methods known in the art.

[0093] "Homogenization" as used herein means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed before fermentation, it can be performed to break down the milk fat into smaller sizes so that it does not separate from the milk. This can be accomplished by forcing the milk through small orifices at high pressure.

[0094] "Pasteurization" as used herein means the treatment of a dairy substrate to reduce or eliminate the presence of live microorganisms, such as bacteria. Preferably, pasteurization is achieved by maintaining a specific temperature for a specific time. The specified temperature is usually achieved by heating. The temperature and duration may be selected to kill or inactivate specific bacteria, such as harmful bacteria. A rapid cooling step may follow.

[0095] The fermentation processes used for the production of fermented milk products are well known and the skilled person will know how to select the appropriate process conditions such as temperature, oxygen, amount and characteristics of microorganisms, and process time. Obviously, the fermentation conditions are selected to support the achievement of the invention, i.e. to obtain a fermented product such as a dairy product or dairy analogue product (fermented dairy product) in solid or liquid form.

[0096] The lactose-containing composition may be a fermented product, such as a fermented milk product selected from the group consisting of yoghurt (set, stirred or drinkable), buttermilk, sour milk, sour cream, kefir, quark, tvarog and cheese. According to one embodiment, the cheese may be selected from the group consisting of fresh cheese, cream cheese or pasta filata.

[0097] Depending on the product and intended consumer, the composition comprising allolactose may further comprise ingredients selected from the group consisting of fruit concentrates, syrups, probiotic bacterial strains or cultures, colorants, thickeners, flavors, preservatives and mixtures thereof, and probiotic bacterial strains or cultures.

[0098] Similarly, an enzyme may be added to the lactose-containing substrate before, during and / or after fermentation, the enzyme being selected from the group consisting of enzymes capable of cross-linking proteins, transglutaminase, aspartic protease, lactase, chymosin, rennet and mixtures thereof.

[0099] The composition containing allolactose, for example, the fermented product containing allolactose, may be in any form, such as a stirred type, a solidified type, or a drinkable type.

[0100] According to one aspect, the present invention relates to a composition comprising allolactose obtainable by the method according to the present invention.

[0101] According to one aspect, the present invention relates to a composition comprising at least 0.04% w / w, at least 0.06% w / w, at least 0.08% w / w, at least 0.10% w / w, at least 0.15% w / w, at least 0.20% w / w, at least 0.25% w / w, at least 0.30% w / w, at least 0.35% w / w, at least 0.40% w / w, at least 0.45% w / w, at least 0.50% w / w, at least 0.55% w / w, at least 0.60% w / w, at least 0.65% w / w, at least 0.70% w / w, at least 0.75% w / w, at least 0.80% w / w, at least 0.85% w / w, at least 0.90% w / w, at least 0.95% w / w, at least 1% or 0.04% w / w to 1% w / w, 0.06% w / w to 0.95% w / w, 0.08% w / w to 0.90% w / w, 0.10% w / w to 0.85% w / w, 0.15% w / w to 0.80% w / w, 0.20% w / w to 0.75% w / w, 0.25% w / w to 0.70% w / w, 0.30% w / w to 0.65% w / w, 0.35% w / w to 0.60% w / w, 0.40% w / w to 0.55% w / w, or 0.45% w / w to 0.50% w / w of allolactose, and said composition further comprises one or more glucose deficient lactic acid bacteria strains, Here, the glucose deficiency of the strain is caused by a mutation in the DNA sequence of the glcK gene, which codes for the glucokinase protein.

[0102] The composition of the present invention can be provided in several forms. It can be a powder, a pellet or a tablet. It can be in a frozen form, a dry form, a lyophilized form or a liquid form. Thus, according to one embodiment, the composition is in a frozen, dry, lyophilized or liquid form.

[0103] The composition of the present invention may further comprise a cryoprotectant, a cryoprotectant, an antioxidant, a nutrient, a filler, a flavoring, or a mixture thereof.The composition preferably comprises one or more cryoprotectants, cryoprotectants, antioxidants, and / or nutrients, more preferably a cryoprotectant, a cryoprotectant, and / or antioxidants, most preferably a cryoprotectant or a cryoprotectant, or both.The use of protective agents such as cryoprotectants and cryoprotectants is known to those skilled in the art. Suitable cryoprotectants or cryoprotectants include monosaccharides, disaccharides, trisaccharides, polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch, gum arabic (acacia), etc.), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol, etc.), amino acids (such as proline, glutamic acid), complex substances (such as skim milk, peptone, gelatin, yeast extract, etc.), and inorganic compounds (such as sodium tripolyphosphate).

[0104] According to one embodiment, the composition according to the invention may contain one or more cryoprotectants selected from the group consisting of inosine-5'-monophosphate (IMP), adenosine-5'-monophosphate (AMP), guanosine-5'-monophosphate (GMP), uranosine-5'-monophosphate (UMP), cytidine-5'-monophosphate (CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, hypoxanthine, orotidine, thymidine, inosine, and derivatives of any such compound. Suitable antioxidants include ascorbic acid, citric acid and its salts, gallates, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (vitamin B family, vitamin C, etc.). The composition may optionally contain further substances including bulking agents (such as lactose, maltodextrin, etc.) and / or flavourings.

[0105] According to one embodiment of the invention, the cryoprotectant is a drug or mixture of drugs that has a booster effect in addition to its cryoprotective properties.

[0106] The expression "booster effect" is used to describe the situation where a cryoprotectant confers an increased metabolic activity (booster effect) to a thawed or reconstituted culture when inoculated into a medium to be fermented or converted. Viability and metabolic activity are not synonymous concepts. Commercially available frozen or freeze-dried cultures may have lost a significant part of their metabolic activity but may retain viability, for example cultures may lose acidogenic (acidifying) activity if stored even for a short period of time. Thus, viability and booster effect need to be evaluated with different assays. Viability is evaluated by viability assays such as the measurement of colony forming units, while booster effect is evaluated by quantifying the relevant metabolic activity of a thawed or reconstituted culture compared to the viability of the culture. The term "metabolic activity" refers to the oxygen scavenging activity of a culture, its acidogenic activity, i.e. the production of lactic acid, acetic acid, formic acid and / or propionic acid, or its metabolite-producing activity, for example the production of aroma compounds such as acetaldehyde (α-acetolactate, acetoin, diacetyl and 2,3-butylene glycol (butanediol)).

[0107] According to one embodiment, the composition of the invention contains or comprises 0.2% to 20% of a cryoprotectant or mixture of cryoprotectants, measured as % w / w of the material. However, it is preferred to add the cryoprotectant or mixture of cryoprotectants in amounts ranging from 0.2% to 15%, 0.2% to 10%, 0.5% to 7%, and 1% to 6% by weight, including within the range of 2% to 5% by weight of the cryoprotectant or mixture of cryoprotectants, measured as % w / w of the weight of the frozen material. According to a preferred embodiment, the culture comprises about 3% by weight of a cryoprotectant or mixture of cryoprotectants, measured as % w / w of the weight of the material. An amount of about 3% of a cryoprotectant corresponds to a concentration in the 100 mM range. It should be appreciated that for each aspect of the embodiment of the invention, the ranges may be increments of the stated ranges.

[0108] In this context, the term "x% to y%" is meant to be inclusive of the endpoints and is thus equivalent to the term "x% (inclusive) to y% (inclusive)."

[0109] According to a further aspect, the composition of the invention comprises or consists of: an ammonium salt (e.g. an ammonium salt of an organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid) as a booster (e.g. a growth booster or an acidification booster) of bacterial cells, such as cells belonging to the species S. thermophilus, e.g. a (substantially) urease negative bacterial cell. The terms "ammonium salt", "ammonium formate" and the like should be understood as a source of salt or a combination of ions. For example, the term "source" of "ammonium formate" or "ammonium salt" refers to a compound or a mixture of compounds that provides ammonium formate or ammonium salt when added to a cell culture. In some embodiments, the ammonium source releases ammonium into the growth medium, while in other embodiments, the ammonium source is metabolized to produce ammonium. According to some preferred embodiments, the ammonium source is exogenous. According to some particularly preferred embodiments, the ammonium is not provided by the dairy substrate. Of course, it should be understood that ammonia may be added instead of an ammonium salt. Thus, the term ammonium salt includes ammonia (NH3), NH4OH, NH4 + etc.

[0110] According to one embodiment, the compositions of the present invention may include thickeners and / or stabilizers such as pectin (e.g., HM pectin, LM pectin), gelatin, CMC, soy fiber / soy polymers, starch, modified starch, carrageenan, alginates, and guar gum.

[0111] According to one embodiment, where the microorganisms produce polysaccharides (such as EPS) that cause a thick / smooth texture in the acidified dairy product, the acidified dairy product is produced substantially free or completely free of added thickeners and / or stabilizers, such as pectins (e.g. HM pectin, LM pectin), gelatin, CMC, soy fiber / soy polymers, starch, modified starch, carrageenan, alginates, guar gum. By substantially free, it should be understood that the product comprises 0%-20% (w / w) (e.g. 0%-10%, 0%-5%, or 0%-2%, or 0%-1%) of thickeners and / or stabilizers.

[0112] Of course, all embodiments disclosed with respect to the method of the present invention are equally applicable to the composition aspects and embodiments of the present invention.

[0113] Another aspect of the present invention relates to the use of one or more glucose deficient strains for producing a composition comprising allolactose, wherein the glucose deficiency of said strains is caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein.

[0114] According to a further aspect, the present invention relates to the use of one or more glucose deficient strains for increasing the allolactose content in a composition, wherein the glucose deficiency of said strains is caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein.

[0115] It goes without saying that all embodiments disclosed with respect to the inventive method and the inventive composition are equally applicable to the various application aspects and embodiments of the invention.

[0116] The use of the terms "a", "an", "the" and similar referents in the context of the present description (particularly in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each individual value falling within the range, unless otherwise indicated herein, and each individual value is incorporated into the specification as if it were individually set forth herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any examples or exemplary language (e.g., "such as") provided herein is merely intended to facilitate a better understanding of the present invention, and does not impose limitations on the scope of the present invention, unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0117] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0118] Preferences, options, and embodiments of a given aspect, feature, or parameter of the present invention should be considered as disclosed in combination with all preferences, options, and embodiments of all other aspects, features, and parameters of the present invention, unless the context indicates otherwise. This is particularly true for the description of the fat-encapsulated microbial culture and all its features, which may be part of the final composition or product readily obtained by the methods described herein. The embodiments and features of the present invention are also outlined in the following paragraphs.

[0119] item A1. A method for producing a composition containing allolactose, comprising the steps of: a) inoculating a substrate containing lactose with one or more glucose-deficient lactic acid bacteria strains, wherein the glucose deficiency of said strains is caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein; and b) fermenting the inoculated substrate to obtain a composition comprising allolactose.

[0120] A2.The following process: The method according to item A1, further comprising the step of: c) concentrating the composition containing allolactose to obtain a composition having an increased amount of allolactose compared to the composition before concentration.

[0121] A3. The following process: The method according to item A1 or A2, further comprising the step of: d) purifying the composition containing allolactose to obtain a composition having an increased purity of allolactose compared to the composition before purification.

[0122] A4. The method according to any one of items 1 to 3, wherein the composition comprising allolactose is a food product, more specifically a fermented milk product.

[0123] A5. The method according to any one of items 1 to 4, wherein step c) is carried out using filtration, including but not limited to diafiltration, membrane filtration, centrifugation, sedimentation, evaporation and / or chromatography.

[0124] A6. The method of any one of items 1 to 5, wherein the one or more strains are galactose positive.

[0125] A7. The method according to any one of items 1 to 6, wherein the one or more strains have a mutation in the galK gene encoding the galactokinase protein.

[0126] A8. The method according to any one of items 1 to 7, wherein the one or more strains are galactose fermenting.

[0127] A9. The method according to any one of items 1 to 8, wherein the one or more strains have a mutation in a gene that reduces or inactivates glucose transport into and / or out of the cell.

[0128] A10. The method according to any one of items 1 to 9, wherein the gene encodes a component of a glucose transport system.

[0129] A11. The method according to any one of items 1 to 10, wherein the glucose transporter system is a glucose / mannose phosphotransferase system.

[0130] A12. The one or more strains are 6 ~10 7 12. The method of any one of items 1 to 11, wherein the strain produces allolactose in an amount of at least 0.04% w / w in a milk substrate when inoculated into the milk substrate at a concentration of CFU / ml and grown at 43° C. for 18 hours, wherein the milk substrate comprises 4% protein, 1.5% fat, and 0.1% added sucrose.

[0131] A13. The method according to any one of items 1 to 12, wherein the one or more glucose deficient strains are selected from the group consisting of Streptococcus and Lactobacillus.

[0132] A14. The method according to any one of items 1 to 13, wherein the streptococcus is Streptococcus thermophilus and the lactobacillus is Lactobacillus delbrueckii subsp. bulgaricus.

[0133] A15. The method according to any one of items 1 to 14, wherein the Streptococcus thermophilus strain is selected from the group consisting of DSM 25850, DSM 26722, DSM 28889, DSM 33719, DSM 32227 and DSM 33762 and mutants derived therefrom, wherein the mutant strain is obtained by using one of the deposited strains as starting material, and wherein the mutant retains or further improves the lactose fermentation and / or glucose secretion properties of the deposited strain.

[0134] A16. The method according to any one of items 1 to 15, wherein one or more glucose-deficient S. thermophilus strains and / or at least one glucose-deficient L. bulgaricus strain are inoculated with at least one non-glucose-deficient S. thermophilus strain or at least one non-glucose-deficient L. bulgaricus strain in step a), and the glucose deficiency of said strains is caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein.

[0135] A17. The substrate of step a) of item 1 is added with: i.DSM 25850, DSM 26722, DSM 28889 and DSM 28910DSM 28910, DSM 32227 and DSM 33762; ii. DSM 25850, DSM 26722, DSM 28889, DSM 28910, and DSM 33720; or iii. DSM 28910, DSM 32227, and DSM 33719; 17. The method according to any one of items 1 to 16, wherein the mouse is inoculated with

[0136] A18. The method according to any one of items 1 to 17, wherein the amount of allolactose in the composition is at least 0.04% w / w.

[0137] A19. The method according to any one of items 1 to 18, wherein the lactose-containing substrate is an animal- and / or plant-derived substrate.

[0138] A20. The method according to any one of items 1 to 19, wherein the lactose-containing substrate is a dairy substrate.

[0139] A21. The method according to any one of items 1 to 20, wherein the fermented dairy product is selected from the group consisting of yogurt (set, stirred or drinking), buttermilk, sour milk, sour cream, kefir, quark, tvorog and cheese.

[0140] A22. The method according to any one of items 1 to 21, wherein the cheese is selected from the group consisting of fresh cheese, cream cheese, or pasta filata.

[0141] A23. The method according to any one of items 1 to 22, wherein the allolactose-containing product, such as a fermented raw milk product, further comprises an ingredient selected from the group consisting of fruit concentrates, syrups, probiotic bacterial strains or cultures, colourants, thickeners, flavours, preservatives and mixtures thereof.

[0142] A24. The method according to any one of items 1 to 23, wherein an enzyme is added to the lactose-containing substrate before, during and / or after fermentation, said enzyme being selected from the group consisting of enzymes capable of cross-linking proteins, transglutaminases, aspartic proteases, chymosin, rennet, and mixtures thereof.

[0143] A25. The method according to any one of items 1 to 24, wherein the fermented product is in the form of a stirred product, a stationary product, or a drinkable product.

[0144] B1. A composition comprising allolactose obtained by the method according to any one of items 1 to 25.

[0145] C1. A composition comprising at least 0.04% w / w allolactose and further comprising one or more glucose deficient lactic acid bacteria strains, wherein the glucose deficiency of said strains is caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein.

[0146] D1. Use of one or more glucose-deficient strains caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein to produce a composition comprising allolactose.

[0147] E1. Use of one or more glucose-deficient strains caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein to increase the allolactose content in a composition.

[0148] Deposits and expert solutions Applicant requests that samples of the deposited microorganisms set forth in the table below be made available to expert personnel only until the date a patent is granted.

[0149] [Table 2] EXAMPLES

[0150] Example 1 - Production of allolactose by a single strain . All cultures tested were provided as frozen pellets by Chr. Hansen.

[0151] Milk supplemented with 4% protein, 1.5% fat, and 0.1% sucrose was prepared by mixing skim milk powder, semi-skim milk powder, and sucrose until fully hydrated. The milk was pasteurized and poured into 200 mL bottles.

[0152] Prior to inoculation, the cultures were thawed and prediluted in milk as recommended by Chr. Hansen. The cultures were inoculated at an inoculation rate of 0.01% (CFU 10 6 ~ 10 7 The inoculated milk was added to milk at 10 mL (equivalent to 100 mL of ethanol). The inoculated milk was incubated at 43 °C for 18 h, cooled to 6 °C, and sampled for chemical analysis. Fermented milk samples for carbohydrate analysis were prepared by weighing 1 g of sample into a 10 mL centrifuge tube, followed by the addition of 2 mL of ice-cold 96% ethanol. Samples were mixed on a vortex and stored at -50 °C until analysis.

[0153] Concentrations of fructose, galactose, glucose, sucrose, lactose and allolactose were measured by high performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD). Samples were quenched with 96% EtOH. Analytes (e.g. carbohydrates) were extracted from the samples and proteins were deproteinized. Samples were further diluted to fit the dynamic range of quantification. Fucose was added as an internal standard. Diluted samples were analyzed on a Dionex ICS-5000, 6000 or Integrion system (Thermo Fischer Scientific, Waltham (MA), USA) using an analytical anion exchange column and a pulsed amperometric detector (PAD). An eight-point calibration curve was used for quantification. Concentrations were calculated based on the chromatographic peak heights after normalization to the internal standard (fucose).

[0154] [Table 3]

[0155] The results clearly show that measurable levels of allolactose are produced when the glucose deficient strain is present in the culture, but no allolactose is produced when the glucose deficient strain is not present in the culture.

[0156] Example 2 - Allolactose produced by cultures of the present invention [Table 4]

[0157] The cultures were inoculated into milk bases prepared from semi-skimmed milk, skimmed milk powder, and sucrose blended to contain 4.0% protein, 1.5% fat, and various levels of sucrose according to the recommended usage and inoculation rate (0.2 U / L) (see Table 2). The inoculated milk samples were incubated at 43°C until a pH of 4.55 was reached, then cooled to 6°C and sampled for chemical analysis. Samples were prepared as disclosed in Example 1 and the concentrations of glucose, galactose, fructose, sucrose, and lactose were measured.

[0158] [Table 5]

[0159] The results clearly show a higher presence of allolactose in samples produced with cultures containing the glucose deficient strain compared to the control culture premium 1.0 that did not contain the glucose deficient strain.

[0160] Example 3 - Lactic acidification and carbohydrate analysis of milk fermented with different strain combinations and ratios . [Table 6]

[0161] Two different milk bases were produced before fermentation. Skim milk powder and sucrose were added to milk, followed by hydration for 2 h without stirring, homogenization and pasteurization at >80 °C for 1 min. Milk base 1 contained 0.1% sucrose, 4% protein (adjusted with skim milk powder) and 1.5% fat (adjusted with 1.5% fat milk and 9% cream). Milk base 2 contained 5% sucrose, 4% protein (adjusted with skim milk powder) and 1.5% fat (adjusted with 1.5% fat milk and 9% cream).

[0162] The milk base was transferred to 200 mL feeding bottles. The bottles were inoculated with 1:100 dilutions prepared from frozen pellets dissolved in B milk (F-DVS). Separate dilutions were prepared for each strain. To create blends, several strains were inoculated in one CINAC bottle in different ratios and volumes. The final inoculum rate of the blends corresponded to 1E+6–1E+8 CFU / mL.

[0163] All strains were inoculated from the first dilution made from F-DVS, except for DSM 33762, which was used in culture 2. To prepare the DSM 33762 inoculum, 1 mL of M17 BK medium containing 4% sucrose and excess casein peptone was inoculated with a scrap of DSM 33762 and incubated aerobically at 43°C for at least 18 h.

[0164] The inoculated bottle was placed in a water bath and heated to 43° C. The pH was monitored at 43° C. according to conventional techniques using CINAC hardware and software until the mixture reached a final pH of 4.55.

[0165] Fermented milk samples were prepared for allolactose analysis by weighing 1 g±0.5 g into a 10 mL centrifuge tube. 2 mL of ice-cold 96% ethanol was added, the sample mixed vigorously and placed at -50°C until analysis. Allolactose concentration was analyzed according to the method disclosed in Example 1.

[0166] [Table 7]

[0167] The tests were carried out in a milk base 1. Culture 3 and Culture 4 results are obtained from different blends, i.e. the eight Culture 3 blends each contain the same strains but in different amounts and ratios.

[0168] Fermentation times and allolactose concentrations vary among cultures. Allolactose is produced by the cultures of the present invention, Culture 1, Culture 3, and Culture 4, but no allolactose was detected when using yogurt cultures Premium 1.0 and YF-L904, both sold by Chr. Hansen A / S. Different ratios of strains within the blend variants may result in variations in fermentation times, allolactose levels, etc. Thus, culture and blend composition can be adjusted according to the specific needs required for the final fermented product.

[0169] [Table 8]

[0170] The table shows the results obtained with culture 1 and culture 2 (blends 1–6), which are six blend variations containing the same strains in different amounts and ratios. It can be seen that the fermentation time and concentration of allolactose depend on the culture and the milk base.

[0171] Example 4 - Different ratios of strains in the culture alter the properties of the fermented milk product Fermented milk was produced and analyzed as described in Example 3.

[0172] [Table 9]

[0173] [Table 10]

[0174] [Table 11]

[0175] Different strain ratios of culture 4 variants (S03b, S03, and P07) resulted in different fermentation times and concentrations of allolactose. In milk base 1, culture 4 variants produced higher allolactose levels compared to culture 1. However, this was reversed in milk base 2, where culture 4 variants produced higher allolactose levels compared to culture 1. The level of allolactose produced was dependent on the culture composition and milk base.

Claims

1. A method for producing a composition comprising allolactose, comprising the steps of: a) inoculating a substrate containing lactose with one or more glucose deficient lactic acid bacteria strains, wherein the glucose deficiency of said strains is caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein; and b) fermenting the inoculated substrate to obtain a composition comprising allolactose.

2. The following steps: The method of claim 1, further comprising the step of: c) concentrating the composition containing allolactose to obtain a composition having an increased amount of allolactose compared to the composition before concentration.

3. The method of claim 1 , wherein the one or more strains are galactose positive.

4. The method of claim 1 , wherein the one or more strains have a mutation in the galK gene, which encodes a galactokinase protein.

5. The method of claim 1 , wherein the one or more strains are galactose fermenting.

6. 2. The method of claim 1, wherein the one or more strains have a mutation in a gene that reduces or inactivates glucose transport into and / or out of the cell.

7. The method of claim 6 , wherein the gene encodes a component of a glucose transport system.

8. 2. The method of claim 1, wherein the one or more glucose deficient strains are selected from the group consisting of Streptococcus and Lactobacillus.

9. 2. The method of claim 1, wherein the glucose-deficient strain is Streptococcus thermophilus and is selected from the group consisting of the deposited strains DSM 25850, DSM 26722, DSM 28889, DSM 33719, DSM 32227 and DSM 33762 and mutants derived therefrom, said mutant strain being obtained by using one of the deposited strains as starting material, and said mutant retains or even improves the lactose fermentation and / or glucose secretion properties of the deposited strain.

10. The substrate of step a) of claim 1 comprises: i. DSM 25850, DSM 26722, DSM 28889 and DSM 28910 DSM 28910, DSM 32227 and DSM 33762; ii. DSM 25850, DSM 26722, DSM 28889, DSM 28910, and DSM 33720; or iii. DSM 28910, DSM 32227, and DSM 33719; The method of claim 1, wherein the

11. 2. The method of claim 1, wherein the amount of allolactose in the composition is at least 0.04% w / w.

12. A composition comprising allolactose obtainable by the method of claim 1.

13. A composition comprising at least 0.04% w / w allolactose, further comprising one or more glucose-deficient lactic acid bacteria strains, wherein the glucose deficiency of said strains is caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein.

14. The use of one or more glucose-deficient strains caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein to produce a composition comprising allolactose.

15. The use of one or more glucose-deficient strains caused by a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein to increase the allolactose content in a composition.