Streptococcus thermophilus strains with a zero-galactose phenotype

Abstract

The present disclosure relates to a method for generating a lactic acid bacteria strain having an active Lac-PTS system and an inactive LacSZ system; wherein said strain does not excrete galactose or has a reduced excretion of galactose as compared to the mother strain from which it is derived. Streptococcus thermophilus strains, compositions comprising the strains, method for manufacturing food products, food products, and use of said strains and compositions are disclosed.

Description

[0001] STREPTOCOCCUS THERMOPHILUS STRAINS WITH A ZERO-GALACTOSE PHENOTYPE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to lactic acid bacteria strains of the species Streptococcus thermophilus excreting no, very low, or insignificant amounts of galactose, i.e. having a zero-galactose phenotype.

[0004] BACKGROUND ART

[0005] Lactic acid bacteria (LAB) are used in the food industry. In the dairy industry LAB of the species Streptococcus thermophilus (ST) is one of the most widely used bacteria and is comprised in starter cultures to bring about acidification of milk (by fermentation). Selection and combination of specific lactic acid bacteria for fermentation are very important as the texture, organoleptic and other characteristics of the fermented product are highly impacted. The catabolism of lactose leads to the excretion of galactose by Streptococcus thermophilus strains.

[0006] A relatively high concentration of galactose can lead to "browning" during heating of cheeses as it is often described when e.g. paste filata type cheese such as mozzarella is produced by S. thermophilus for e.g. pizza production. The browning phenomenon is believed to be due to the Maillard reaction where galactose as reducing sugar is reacting with amino acids / peptides.

[0007] Mukherjee et al. (1994, J Dairy Sci 77:2839-2849) describes the use of so-called galactose non-releasing Streptococcus as starter cultures in the manufacture of low browning mozzarella cheese.

[0008] Hassan et al. (2001, International Journal of Food Microbiology 64: 199-203) describes that the ability of some strains of S. thermophilus to use galactose in capsule production could reduce browning of mozzarella cheese during baking by removing a source of reducing sugar.

[0009] Further to the issue of browning, excess amounts of free galactose may lead to post-acidification and imbalance in the flora of other fermented food products.

[0010] For individuals with galactosemia, a genetic metabolic disorder that affects their ability to metabolize galactose properly, ingestion of galactose present in food products is harmful for their health. Therefore, development of food products with low level of galactose are desirable. SUMMARY OF THE INVENTION

[0011] In a first aspect the disclosure relates to a method for generating a lactic acid bacteria strain comprising the following steps: (a) Providing a galactose excreting mother strain of the species Streptococcus thermophilus having an active lactose permease (LacSZ) system and an active phosphoenolpyruvate-dependent phosphotransferase (Lac-PTS) system; (b) Treating the mother strain whereby the LacSZ system becomes inactive; (c) Growing the treated strain under conditions enabling the differentiation between cells with an active LacSZ system from cells with an inactive LacSZ system; and (d) Screening for and isolating a strain having an active Lac-PTS system and an inactive LacSZ system; wherein said strain does not excrete galactose or has a reduced excretion of galactose as compared to the mother strain from which it is derived.

[0012] In a second aspect the disclosure relates to a lactic acid bacteria strain derived from a galactose excreting mother strain of the species Streptococcus thermophilus, wherein the strain has (a) an active phosphoenolpyruvate-dependent phosphotransferase (Lac-PTS) system; (b) an inactive lactose permease (LacSZ) system; and (c) does not excrete galactose or has a reduced excretion of galactose as compared to the mother strain.

[0013] In a third aspect the disclosure relates to a composition comprising, either as a mixture or as a kit-of-parts, one or more strains according to the present disclosure.

[0014] In a fourth aspect the disclosure relates to a method for manufacturing a food product comprising the steps: (a) providing the strain according to the present disclosure, or the composition according to the present disclosure; (b) inoculating a starting material with said strain or composition; and (c) allowing fermentation of the inoculated starting material until a target pH has been reached.

[0015] In a fifth aspect the disclosure relates to a food product comprising the strain according to the present disclosure or the composition according to the present disclosure.

[0016] In a sixth aspect the disclosure relates to use of the strain according to the present disclosure or the composition according to the present disclosure for reducing the amount of galactose in food products; and / or for reducing the browning of cheese. BRIEF DESCRIPTION OF SEQUENCES

[0017] SEQ ID No: l - DSM 34887 LacZ gene sequence.

[0018] SEQ ID No:2 - DSM 34887 LacZ protein sequence.

[0019] SEQ ID No:3 - DSM 34889 LacZ gene sequence.

[0020] SEQ ID No:4 - DSM 34889 LacZ protein sequence.

[0021] SEQ ID No:5 - DSM 34888 LacZ gene sequence.

[0022] SEQ ID No:6 - DSM 34888 LacZ protein sequence.

[0023] SEQ ID No:7 - DSM 34890 LacZ gene sequence.

[0024] SEQ ID No:8 - DSM 34890 LacZ protein sequence.

[0025] SEQ ID No:9 - DSM 34891 LacZ gene sequence.

[0026] SEQ ID No: 10 - DSM 34891 LacZ protein sequence

[0027] SEQ ID No:ll - DSM 34892 LacZ gene sequence.

[0028] SEQ ID No:12 - DSM 34892 LacZ protein sequence

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030] Lactic acid bacteria (LAB) of the species Streptococcus thermophilus (ST or S. thermophilus') have evolved into a species highly adapted to the dairy environment. The primary use of ST in the dairy industry is acidification of milk that leads to modification of the physico-chemical properties of milk. Milk acidification is a result from the production of lactic acid by fermentation of lactose, the major carbohydrate in milk.

[0031] Different systems dependent of the lactic acid bacteria have been described whereby lactose is internalized and metabolized. The lactose permease (LacSZ) system is responsible for lactose uptake in ST. The transport mechanism is an antiporter driven by the exchange of one molecule of galactose with one molecule of lactose. Lactose can also be imported by the lactose / proton cotransport reaction provided sufficient proton potential is present.

[0032] Lactose is a disaccharide composed of a / p-D-glucose and p-D-galactose and is the main carbon source in mammalian milk. Catabolism of lactose results in production of lactate and other end products. The lac operon comprising LacS and LacZ the coding genes of lactose permease and p-galactosidase respectively controls the transport and hydrolysis of lactose. Lactose catabolism in Streptococcus thermophilus starts with lactose being transported over the cell membrane into the cytoplasm by the lactose permease. Once inside the cell the disaccharide lactose is hydrolyzed into its monosaccharide's glucose and galactose by the enzyme 0- galactosidase (EC 3.2.1.23).

[0033] Glucose is being metabolized by the glycolytic pathway where it is phosphorylated by glucokinase to form glucose-6-phosphate, which is then converted to pyruvate by the Embden-Meyerhof-Parnas (EMP) pathway, and pyruvate is converted to lactic acid mainly by lactate dehydrogenase.

[0034] Galactose serves to synthesize polysaccharides, teichoic acids and nucleotide sugars. Galactose may enter glycolysis through phosphoglucomutase (PgmA; EC 5.4.2.2) and Leloir pathway. However, despite the presence of a complete Leloir pathway operon (GalKTEM) and PgmA, they are not active in most ST strains which therefore are unable to metabolize galactose. The galactose produced is thus excreted by the action of the lactose permease. It has been described that modified ST strains can become galactose positive, Gal(+), i.e. gain the ability to metabolize galactose by the Leloir pathway. However, galactose is only partially metabolized, and the majority is excreted.

[0035] Another system for taking up lactose is the phosphoenolpyruvate-dependent phosphotransferase (Lac-PTS) system which is found in other bacteria such as e.g. Lactococcus lactis. Here lactose is taken up, phosphorylated to lactose-6-phosphate with the concomitant hydrolysis of phosphoenolpyruvate into pyruvate. The phosphorylated lactose is subsequently hydrolyzed by a phospho-0-galactosidase, resulting in galactose-6-phosphate and glucose. The glucose enters glycolysis through phosphorylation by glucokinase. The galactose-6-phosphate enters the tagatose pathway, resulting in the formation of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, which enters glycolysis. Thus, the galactose is not excreted, but metabolized via the tagatose pathway. The Lac-PTS system is normally not present in S. thermophilus strains.

[0036] Method for generating a lactic acid bacteria strain.

[0037] A method for generating a lactic acid bacteria of the species Streptococcus thermophilus excreting no, very low, or insignificant amount of galactose, i.e. a zero-galactose phenotype has been developed and is described in detail herein. In a first aspect the present disclosure relates to a method for generating a lactic acid bacteria strain comprising the following steps:

[0038] (a) Providing a galactose excreting mother strain of the species Streptococcus thermophilus having an active lactose permease (LacSZ) system and an active phosphoenolpyruvate-dependent phosphotransferase (Lac-PTS) system;

[0039] (b) Treating the mother strain whereby the LacSZ system becomes inactive;

[0040] (c) Growing the treated strain under conditions enabling the differentiation between cells with an active LacSZ system from cells with an inactive LacSZ system; and

[0041] (d) Screening for and isolating a strain having an active Lac-PTS system and an inactive LacSZ system; wherein said strain does not excrete galactose or has a reduced excretion of galactose as compared to the mother strain from which it is derived.

[0042] As described supra ST strains does not have an active Lac-PTS system, and to obtain a mother strain comprising both an active LacSZ system as well as an active Lac-PTS system the latter may be introduced into a ST strain either on a plasmid or by means of genetic engineering. Accordingly, a mother strain with an active LacSZ system and an active Lac-PTS system may be derived from a strain with an active LacSZ system by transferring the genes encoding the Lac-PTS system into the strain.

[0043] The term "Lactose Permease System" or "LacSZ system" entail both the transporter protein encoded by the LacS gene and the 0-galactosidase protein encoded by the LacZ gene. By the genes "LacS" and "LacZ" are meant both the sequences encoding the protein as well at the sequences encoding regulatory elements for expressing the genes, such as e.g. the promotor etc.

[0044] To inactivate the LacSZ system the mother strain may be treated with e.g. UV mutagenesis or another method of mutagenization.

[0045] To differentiate between cells with an active lacSZ system from those with an inactive LacSZ system the cell was streaked on X-gal (5-Brom-4-chlor-3-indoxyl- p-D-galactopyranosid) containing agar plates. The observation of blue colonies indicates an active LacSZ system whereas observation of white colonies indicates an inactive LacSZ system.

[0046] The activity of the Lac-PTS was shown by a milk acidification experiment. If a strain has an inactive lacZ gene, shown by the appearance of white colonies on LM17 agar (M17 containing 1% lactose) containing X-Gal, and can ferment milk which is containing lactose as main fermentable carbohydrate, then acidification can only occur when an active Lac-PTS system is present. The same would be true if the lacS gene is inactive and lactose cannot be transported into the cell.

[0047] The galactose excretion and phenotype were confirmed by carbohydrate analysis of the fermented milk.

[0048] In one embodiment the present disclosure relates to the method, wherein the active Lac-PTS system is (a) present and active in the mother strain; (b) present and has been activated in the mother strain; or (c) has been transferred to and is active in the mother strain which were previously lacking an active Lac-PTS system.

[0049] In one embodiment the present disclosure relates to the method, wherein the active Lac-PTS system has been transferred into the mother strain by means of a plasmid or genetic engineering.

[0050] The excretion of galactose by the strain is dependent on both the lactose permease as well as the p-galactosidase. Any changes in the LacSZ nucleotide sequences or encoded amino acid sequences leading to a impaired or inactive protein will in strains with lactose uptake through the LacSZ system have a reduced excretion of galactose.

[0051] In one embodiment the present disclosure relates to the method, wherein the LacSZ system is inactivated due to a change in one or more of the LacS and LacZ nucleotide sequences and / or amino acid sequences. In one embodiment the present disclosure relates to the method, wherein the lactose permease system is inactivated due to a change in the LacS nucleotide sequence or encoded amino acid sequence for lactose permease. In one embodiment the present disclosure relates to the method, wherein the LacSZ system is inactivated due to a change in the LacZ gene encoding p-galactosidase. The change may be an insertion, a mutation, or a deletion.

[0052] In one embodiment the present disclosure relates to the method, wherein the LacSZ system is inactivated due to a mutation in the LacZ gene encoding p-galactosidase. The sequence of the lacZ gene of the mother strain is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical with SEQ ID No: l or SEQ ID No;5.

[0053] In one embodiment the present disclosure relates to the method, wherein the mutation is at a position corresponding to the nucleotide at position 1143 in SEQ ID NO:3; position 1667 in SEQ ID NO:7; position 2144 in SEQ ID NO:9; or position 2144 in SEQ ID NO: 11. In one embodiment the present disclosure relates to the method, wherein the mutation at the position corresponding to the nucleotide at position 1143 in SEQ ID NO:3 is A; position 1667 in SEQ ID NO:7 is T; position 2144 in SEQ ID NO:9 is A; or position 2144 in SEQ ID NO: 11 is A.

[0054] In one embodiment the present disclosure relates to the method, wherein the mutation in the LacZ gene leads to a mutated 0-galactosidase protein comprising an amino acid substitution at a position corresponding to position 556 in SEQ ID NO:8.

[0055] In one embodiment the present disclosure relates to the method, wherein the amino acid substitution at the position corresponding to position 556 in SEQ ID NO:8 is V.

[0056] Two ST strains DSM 34887 and DSM 34888 were shown to express both an active LacSZ system and an active Lac-PTS system. The strains were identified by a BLAST search using the Lac-PTS gene sequence from Lactococcus lactis towards the genome sequenced S. thermophilus strains from our culture collection using the CLC software (CLC Genomics Workbench, version 10.1.1 (CLC bio, Qiagen Bioinformatics)). These strains were used as mother strains from which new strains were derived wherein the LacSZ system was inactivated leaving the Lac-PTS system responsible for uptake of lactose. The galactose produced in these strains was not excreted due to the inactivated LacSZ system and was metabolized via the tagatose pathway.

[0057] In one embodiment the present disclosure relates to the method, wherein the mother strain is selected from DSM 34887; DSM 34888; or mutants or variants thereof.

[0058] In the present context, the term "mutant" or "mutant strain" should be understood as a strain derived, or a strain which can be derived, from a strain of the present disclosure (or the mother strain) by means of e.g. genetic engineering, radiation and / or chemical treatment. It is preferred that the mutant is a functionally equivalent mutant, e.g. a mutant that has substantially the same, or improved, properties (e.g. regarding texture, shear stress, viscosity, gel firmness, mouth coating, flavor, post acidification, acidification speed, and / or phage robustness) as the strain from which it is derived. Such a mutant is a part of the present invention. Especially, the term "mutant” refers to a strain obtained by subjecting a strain of the invention to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light, or to a spontaneously occurring mutant. A mutant may have been subjected to several mutagenization treatments (a single treatment should be understood one mutagenization step followed by a screening / selection step), but it is presently preferred that no more than 20, or no more than 10, or no more than 5, treatments (or screening / selection steps) are carried out. In a presently preferred mutant, less than 5%, less than 1%, less than 0.5%, or even less than 0.1% of the nucleotides in the bacterial genome have been shifted with another nucleotide, or deleted, compared to the mother strain. As will be clear to the skilled person mutants of the present invention can also be mother strains.

[0059] In the present context, the term "variant" or "variant strain" should be understood as a strain which is functionally equivalent to a strain of the invention, e.g. having substantially the same, or improved, properties or characteristics e.g. texture, acidification speed, viscosity, gel firmness, mouth coating, flavor, post acidification and / or phage robustness). Such variants, which may be identified using appropriate screening techniques, are a part of the present invention.

[0060] Streptococcus thermophilus strains.

[0061] In general, ST strains express and use the LacSZ system for lactose uptake and metabolism and which due to the lactose permease antiporter excrete galactose. Only rarely ST strains have an active Lac-PTS system. Two new ST strains DSM 34887 and DSM 34888 having both an active LacSZ system and an active Lac-PTS system were identified by screening our strain bank. In one embodiment the present disclosure relates to a strain having both an active LacSZ system and an active Lac-PTS system. In one embodiment the present disclosure relates to the strain, wherein said strain is DSM 34887 or DSM 34888. These strains are suitable starting points for generating the mutant strains of the present disclosure. In one embodiment the present relates to a mother strain selected from any of DSM 34887 or DSM 34888. In one embodiment of the present disclosure relates to use of DSM 34887 or DSM 34888 as mother strains to generate mutant strains having an inactive LacSZ system and an active Lac-PTS system.

[0062] The ST strains of the present disclosure are characterized by their ability of excreting no, very low, or insignificant amounts of galactose, i.e. having a zero- galactose phenotype. As compared to their mother strain they have a reduced excretion of galactose.

[0063] In a second aspect the present disclosure relates to a lactic acid bacteria strain derived from a galactose excreting mother strain of the species Streptococcus thermophilus, wherein the strain has (a) an active phosphoenolpyruvate-dependent phosphotransferase (Lac-PTS) system; (b) an inactive lactose permease (LacSZ) system; and (c) does not excrete galactose or has a reduced excretion of galactose as compared to the mother strain.

[0064] The ST strains may be generated by the method described supra. It is to be understood that relevant features described in the other sections supra and infra of the present disclosure are also applicable in this section. This principle applies to all sections, and thus headings are only meant as a guidance and not as a limitation. In one embodiment the present disclosure relates to the strain, wherein the strain is generated by the method according to the present disclosure.

[0065] The amount of galactose excreted by the ST strains of the present disclosure are very low and is reduced as compared to the amount of galactose excreted by the mother strain. In some instances, it was found that the amount of galactose excreted by the ST strains of the present disclosure is at the level of what is found in B-Milk. This indicates that no galactose has been excreted. In one embodiment the present disclosure relates to the strain, wherein the strain excretes less than 2.00; 1.75; 1.50; 1.25; 1.00; 0.75; 0.50; or less than 0.25g / L galactose.

[0066] In one embodiment the present disclosure relates to the strain, wherein the strain is selected from the group consisting of DSM 34889; DSM 34890; DSM 34891; and DSM 34892.

[0067] The sequences of the lacZ gene encoding the 0-galactosidase protein of the S. thermophilus strains described herein are listed below in SEQ ID NOs 1 to 12. The changes in the gene sequences are a mutation leading to introduction of a new amino acid and an inactive protein or a stop codon in the gene leading to a truncated and inactive protein.

[0068] SEQ ID No:l DSM 34887 lacZ sequence (1..3081 bp) :

[0069] ATGAACATGA CTGAAAAAAT TCAAACTTAT TTAAACGATC CAAAGATTGT TAGCGTTAAT ACTGTTGATG CTCACTCAGA TCATAAGTAT TTTGAATCTC TTGAAGAATT TTCTGAAGGG GAGATGAAGT TAAGACAATC TCTTAATGGA AAATGGAAAA TTCACTATGC TCAGAATACA AATCAGGTTT TAAAAGACTT TTATAAAACA GAATTTGATG AAACTGATTT GAATTTCATC AATGTACCAG GTCATTTAGA GCTTCAAGGT TTTGGTTCTC CACAATATGT GAATACCCAA TATCCTTGGG ATGGTAAAGA ATTCCTTCGT CCACCTCAAG TTCCTCAAGA ATCAAATGCT GTTGCATCAT ACGTTAAACA TTTTACCTTG AATGATGCAT TAAAAGATAA AAAAGTATTT ATCTCATTCC AAGGGGTTGC TACTTCCATC TTTGTATGGG TCAATGGTAA CTTTGTAGGC TACAGTGAAG ATTCATTTAC ACCTAGTGAA TTTGAAATTA GTGATTACCT TGTTGAAGGT GATAACAAGT TGGCGGTAGC TGTTTATCGT TACTCTACAG CAAGCTGGTT GGAAGACCAA GACTTCTGGA GACTTTACGG TATTTTTAGA GATGTTTACT TGTATGCTAT TCCAAAAGTT CACGTTCAAG ATCTCTTTGT TAAGGGAGAT TATGATTACC AAACAAAAGC AGGTCAATTA GATATTGATT TGAAGACTGT TGGTGATTAT GAAGACAAGA AGATTAAATA TGTTCTTTCA GATTATGAAG GCATCGTTAC AGAAGGTGAT GCATCTGTTA ATGGTGACGG TGAACTATCT GTAAGTCTTG AAAATCTTAA AATCAAACCT TGGAGTGCTG AAAGTCCTAA ACTTTACGAT TTGATCCTTC ATGTTTTGGA TGATGACCAA GTTGTTGAAG TCGTTCCAGT TAAAGTTGGA TTTAGACGCT TTGAAATTAA AGATAAACTT ATGCTTTTGA ATGGTAAGAG AATTGTCTTT AAAGGGGTTA ACAGACACGA ATTTAACGCT AGAACAGGAC GTTGTATCAC TGAAGAAGAT ATGCTTTGGG ATATCAAAGT GATGAAACAA CATAACATCA ATGCTGTTCG TACTTCACAC TATCCTAACC AAACACGTTG GTATGAATTG TGTGATGAAT ATGGACTTTA TGTTATCGAT GAAGCCAACC TTGAAACACA CGGTACATGG CAAAAACTTG GTCTATGCGA ACCTTCATGG AATATCCCAG CTAGTGAACC AGAATGGTTG CCTGCTTGTT TGGATCGTGC CAATAACATG TTCCAACGCG ATAAGAACCA TGCTAGTGTT ATCATTTGGT CTTGTGGTAA TGAATCATAT GCTGGTAAAG ATATTGCTGA CATGGCTGAT TACTTCCGTA GTGTTGACAA TACTCGTCCA GTTCACTATG AAGGTGTTAC ATGGTGTCGT GAATTTGATT ACATTACAGA CATCGAAAGT CGTATGTATG CGAAACCAGC TGATATCGAA GAATACCTCA CAACTGGTAA ACTAGTTGAT CTTTCAAGCG TTAGTGATAA ACACTTTGCT TCAGGTACCC TAACTAACAA TCCTCAAAAA CCTTATATTT CATGTGAATA CATGCACATG ATGGGTAACT CTGGTGGTGG ATTGGAACTC TACACTGACT TAGAGAAATA TCCAGAATAC CAAGGTGGAT TTATTTGGGA CTTCATTGAC CAAGCTATTT ACAAACCACT TCCAAATGGT AGCGAATTTC TATCATATGG TGGTGACTGG CATGATAGAC CTTCTGACTA CGAATTTTGT GGAAATGGTA TTGTCTTCGC AGATCGTACC CTAACTCCAA AACTTCAAAC AGTTAAACAT CTTTACTCTA ATATTAAGAT TGCTGTTGAT GAAAAATCAG TAACTATCAA GAATGATAAT GTCTTTGAAG ATCTTTCTGC TTATACTTTC CTAGCTAGAG TTTACGAAGA TGGTAGAAAA GTTAGCGAAA GTGAATATCA CTTTGATGTT AAACCAGGTG AAGAAGCAAC ATTCCCAGTA GAATTCGCTG TTGGAGCTTC AAATGCTGAA CGAATTTATG AAGTTGCTTG TGTTCAGAAT GAAGCAACTG AATGGGCTCC AAAAGGTCAT GAAATTGTTC GTGGTCAATA TGTTGCTGAA AAGATTAGCA CCGAAACACC AGTTAAAGCA CCTTTGAATG TTGTTGAAGG CGACTTCAAC ATCGGTATTC AAGGACAAAA CTTCTCAATC TTGCTTTCAC GTGCACAAAA TACTTTAGTA TCTGCTAAGT ATAATGGTGT TGAATTCATT GAGAAAGGTC CTAAACTTAG CTTCACTCGT GCTTACACTG ACAACGATCG TGGTGCTGGA TATCCATTCG AAATGGCAGG CTGGAAGGTT GCTGGAAACT ATAGTAAAGT TACAGATACT CAAATTCAAA TCGAAGACGA CTCTGTTAAA GTGACTTATG TTCATGAATT GCCAGGCTTG TCTGATGTCG AAGTTAAGGT AACTTATCAA GT T GAG TACA AGGGTCGAAT CTTTGTTACT GCAAACTATG ATGGTAAAGC AGGTTTGCCA AACTTCCCTG AATTTGGTCT AGAATTTGCT ATCGGTTCAC AATTTACAAA CCTTAGCTAT TATGGATACG GTGCAGAAGA AAGCTACCGT GATAAACTTC CTGGTGCCTA TCTTGGTCGA TATGAAACAT CTGTTGAAAA GACATTTGCT CCATATCTAA TGCCACAAGA ATCTGGTAAT CACTATGGTA CTCGTGAATT CACAGTATCT GATGATAACC ATAATGGTGT TAAATTCACC GCACTTAATA AAGCATTCGA ATTCAGTGCT TTGCGTAACA GTACTGAACA AATTGAAAAT GCTCGTCACC AATATGAGTT GCAAGAATCT GATGCTACAT GGATTAAAGT TCTTGCTGCT CAAATGGGTG TAGGTGGTGA CGACTCATGG GGTGCTCCAG TTCATGACGA ATTCTTGCTT AGCTCAGCAG ATAGCTATCA ATTAAGCTTC ATGATTGAAC CACTAAATTA G

[0070] SEQ ID No:2 DSM 34887 lacZ sequence (1..1026 aa): MNMTEKIQTY LNDPKIVSVN TVDAHSDHKY FESLEEFSEG EMKLRQSLNG KWKIHYAQNT NQVLKDFYKT EFDETDLNFI NVPGHLELQG FGSPQYVNTQ YPWDGKEFLR PPQVPQESNA VASYVKHFTL NDALKDKKVF I SFQGVATS I FVWVNGNFVG YSEDSFTPSE FEISDYLVEG DNKLAVAVYR YSTASWLEDQ DFWRLYGI FR DVYLYAI PKV HVQDLFVKGD YDYQTKAGQL DIDLKTVGDY EDKKIKYVLS DYEGIVTEGD ASVNGDGELS VSLENLKIKP WSAESPKLYD LILHVLDDDQ WEWPVKVG FRRFEIKDKL MLLNGKRIVF KGVNRHEFNA RTGRCITEED MLWDIKVMKQ HNINAVRTSH YPNQTRWYEL CDEYGLYVID EANLETHGTW QKLGLCEPSW NI PASEPEWL PACLDRANNM FQRDKNHASV I IWSCGNESY AGKDIADMAD YFRSVDNTRP VHYEGVTWCR EFDYITDIES RMYAKPAD IE EYLTTGKLVD LSSVSDKHFA SGTLTNNPQK PYI SCEYMHM MGNSGGGLEL YTDLEKYPEY QGGFIWDFID QAIYKPLPNG SEFLSYGGDW HDRPSDYEFC GNGIVFADRT LTPKLQTVKH LYSNIKIAVD EKSVTIKNDN VFEDLSAYTF LARVYEDGRK VSESEYHFDV KPGEEATFPV EFAVGASNAE RIYEVACVQN EATEWAPKGH EIVRGQYVAE KI STETPVKA PLNWEGDFN IGIQGQNFSI LLSRAQNTLV SAKYNGVEFI EKGPKLSFTR AYTDNDRGAG YPFEMAGWKV AGNYSKVTDT QIQIEDDSVK VTYVHELPGL SDVEVKVTYQ VDYKGRI FVT ANYDGKAGLP NFPEFGLEFA IGSQFTNLSY YGYGAEESYR DKLPGAYLGR YETSVEKTFA PYLMPQESGN HYGTREFTVS DDNHNGLKFT ALNKAFEFSA LRNSTEQIEN ARHQYELQES DATWIKVLAA QMGVGGDDSW GAPVHDEFLL SSADSYQLSF MIEPLN*

[0071] SEQ ID No:3 DSM 34889 LacZ T1143A mutant sequence (1..1143 bp) :

[0072] ATGAACATGA CTGAAAAAAT TCAAACTTAT TTAAACGATC CAAAGATTGT TAGCGTTAAT ACTGTTGATG CTCACTCAGA TCATAAGTAT TTTGAATCTC TTGAAGAATT TTCTGAAGGG GAGATGAAGT TAAGACAATC TCTTAATGGA AAATGGAAAA TTCACTATGC TCAGAATACA AATCAGGTTT TAAAAGACTT TTATAAAACA GAATTTGATG AAACTGATTT GAATTTCATC AATGTACCAG GTCATTTAGA GCTTCAAGGT TTTGGTTCTC CACAATATGT GAATACCCAA TATCCTTGGG ATGGTAAAGA ATTCCTTCGT CCACCTCAAG TTCCTCAAGA ATCAAATGCT GTTGCATCAT ACGTTAAACA TTTTACCTTG AATGATGCAT TAAAAGATAA AAAAGTATTT ATCTCATTCC AAGGGGTTGC TACTTCCATC TTTGTATGGG TCAATGGTAA CTTTGTAGGC TACAGTGAAG ATTCATTTAC ACCTAGTGAA TTTGAAATTA GTGATTACCT TGTTGAAGGT GATAACAAGT TGGCGGTAGC TGTTTATCGT TACTCTACAG CAAGCTGGTT GGAAGACCAA GACTTCTGGA GACTTTACGG TATTTTTAGA GATGTTTACT TGTATGCTAT TCCAAAAGTT CACGTTCAAG ATCTCTTTGT TAAGGGAGAT TATGATTACC AAACAAAAGC AGGTCAATTA GATATTGATT TGAAGACTGT TGGTGATTAT GAAGACAAGA AGATTAAATA TGTTCTTTCA GATTATGAAG GCATCGTTAC AGAAGGTGAT GCATCTGTTA ATGGTGACGG TGAACTATCT GTAAGTCTTG AAAATCTTAA AATCAAACCT TGGAGTGCTG AAAGTCCTAA ACTTTACGAT TTGATCCTTC ATGTTTTGGA TGATGACCAA GTTGTTGAAG TCGTTCCAGT TAAAGTTGGA TTTAGACGCT TTGAAATTAA AGATAAACTT ATGCTTTTGA ATGGTAAGAG AATTGTCTTT AAAGGGGTTA ACAGACACGA ATTTAACGCT AGAACAGGAC GTTGTATCAC TGAAGAAGAT ATGCTTTGGG ATATCAAAGT GATGAAACAA CATAACATCA ATGCTGTTCG TACTTCACAC TAA

[0073] SEQ ID No:4 DSM 34889 lacZ truncated mutant sequence (1..381 aa) : MNMTEKIQTY LNDPKIVSVN TVDAHSDHKY FESLEEFSEG EMKLRQSLNG KWKIHYAQNT NQVLKDFYKT EFDETDLNFI NVPGHLELQG FGSPQYVNTQ YPWDGKEFLR PPQVPQESNA VASYVKHFTL NDALKDKKVF ISFQGVATSI FVWVNGNFVG YSEDSFTPSE FEISDYLVEG DNKLAVAVYR YSTASWLEDQ DFWRLYGIFR DVYLYAIPKV HVQDLFVKGD YDYQTKAGQL DIDLKTVGDY EDKKIKYVLS DYEGIVTEGD ASVNGDGELS VSLENLKIKP WSAESPKLYD LILHVLDDDQ WEWPVKVG FRRFEIKDKL MLLNGKRIVF KGVNRHEFNA RTGRCITEED MLWDIKVMKQ HNINAVRTSH*

[0074] SEQ ID No:5 DSM 34888 lacZ sequence (1..3081 bp) :

[0075] ATGAACATGA CTGAAAAAAT TCAAACTTAT TTAAACGATC CAAAGATTGT TAGCGTTAAT ACTGTTGATG CTCACTCAGA TCATAAGTAT TTTGAATCTC TTGAAGAATT TTCTGAAGGG GAGATGAAGT TAAGACAATC TCTTAATGGA AAATGGAAAA TTCACTATGC TCAGAATACA AATCAGGTTT TAAAAGACTT TTATAAAACA GAATTTGATG AAACTGATTT GAATTTCATC AATGTACCAG GTCATTTAGA GCTTCAAGGT TTTGGTTCTC CACAATATGT GAATACCCAA TATCCTTGGG ATGGTAAAGA ATTCCTTCGT CCACCTCAAG TTCCTCAAGA ATCAAATGCT GTTGCATCAT ACGTTAAACA TTTTACCTTG AATGATGCAT TAAAAGATAA AAAAGTATTT ATCTCATTCC AAGGGGTTGC TACTTCCATC TTTGTATGGG TCAATGGTAA CTTTGTAGGC TACAGTGAAG ATTCATTTAC ACCTAGTGAA TTTGAAATTA GTGATTACCT TGTTGAAGGT GATAACAAGT TGGCGGTAGC TGTTTATCGT TACTCTACAG CAAGCTGGTT GGAAGACCAA GACTTCTGGA GACTTTACGG TATTTTTAGA GATGTTTACT TGTATGCTAT TCCAAAAGTT CACGTTCAAG ATCTCTTTGT TAAGGGAGAT TATGATTACC AAACAAAATC AGGTCAATTA GATATTGATT TGAAGACTGT TGGTGATTAT GAAGACAAGA AGATTAAATA TGTTCTTTCA GATTATGAAG GCATCATTAC AGAAGGTGAT GCATCTGTTA ATGGTGACGG TGAACTATCT GTAAGTCTTG AAAATCTTAA AATCAAACCT TGGAGTGCTG AAAGTCCTAA ACTTTACGAT TTGATCCTTC ATGTTTTGGA TGATGACCAA GTTGTTGAAG TCGTTCCAGT TAAAGTTGGA TTTAGACGCT TTGAAATTAA AGATAAACTT ATGCTTTTGA ATGGTAAGAG AATTGTCTTT AAAGGGGTTA ACAGACACGA ATTTAACGCT AGAACAGGAC GTTGTATCAC TGAAGAAGAT ATGCTTTGGG ATATCAAAGT GATGAAACAA CATAACATCA ATGCTGTTCG TACTTCACAC TATCCTAACC AAACACGTTG GTATGAATTG TGTGATGAAT ATGGACTTTA TGTTATCGAT GAAGCCAACC TTGAAACACA CGGTACATGG CAAAAACTTG GTCTATGCGA ACCTTCATGG AATATCCCAG CTAGTGAACC AGAATGGTTG CCTGCTTGTT TGGATCGTGC CAATAACATG TTCCAACGCG ATAAGAACCA TGCTAGTGTT ATCATTTGGT CTTGTGGTAA TGAATCATAT GCTGGTAAAG ATATTGCTGA CATGGCTGAT TACTTCCGTA GTGTTGACAA TACTCGTCCA GTTCACTATG AAGGTGTTAC ATGGTGTCGT GAATTTGATT ACATTACAGA CATCGAAAGT CGTATGTATG CGAAACCAGC TGATATTGAA GAATACCTCA CAACTGGTAA ACTAGTTGAT CTTTCAAGCG TTAGTGATAA ACACTTTGCT TCAGGTAACC TAACTAACAA TCCTCAAAAA CCTTATATTT CATGTGAATA CATGCACATG ATGGGTAACT CTGGTGGTGG ATTGGAACTC TACACTGACT TAGAGAAATA TCCAGAATAC CAAGGTGGAT TTATTTGGGA CTTCATTGAC CAAGCTATTT ACAAAACACT TCCAAATGGT AGCGAATTCC TATCATATGG TGGTGACTGG CATGATAGAC CTTCTGACTA CGAATTTTGT GGAAATGGTA TTGTCTTTGC AGATCGTACC CTAACTCCAA AACTTCAAAC AGTTAAACAT CTTTACTCTA ATATTAAGAT TGCTGTTGAT GAAAAATCAG TAACTATCAA GAATGATAAT CTCTTCGAAG ATCTTTCTGC TTATACTTTC CTAGCTAGAG TTTACGAAGA TGGTAGAAAA GTTAGCGAAA GTGAATATCA CTTTGATGTT AAACCAGGTG AAGAAGCAAC ATTCCCAGTT AACTTTGTAG TCGAGGCTTC AAATTCTGAA CAAATTTACG AAGTTGCTTG TGTTCTGAGG GAAGCAACTA AATGGGCTCC TAAAGGTCAT GAAATTGTTC GTGGTCAATA TGTTGCTGAA AAGATTAGCA CCGAAACACC AGTTAAAGCA CCTTTGAATG TTGTTGAAGG CGACTTCAAC ATCGGTATTC AAGGACAAAA CTTCTCAATC TTGCTTTCAC GTGCACAAAA TACTTTAGTA TCTGCTAAGT ATAATGGTGT TGAATTCATT GAGAAAGGTC CTAAACTTAG CTTCACTCGT GCTTACACTG ACAACGATCG TGGTGCTGGA TATCCATTCG AAATGGCAGG CTGGAAGGTT GCTGGAAACT ATAGTAAAGT TACAGATACT CAAATTCAAA TCGAAGACGA CTCTGTTAAA GTGACTTATG TTCATGAATT GCCAGGCTTG TCTGATGTCG AAGTTAAGGT AACTTATCAA GT T GAG TACA AGGGTCGAAT CTTTGTTACT GCAAACTATG ATGGTAAAGC AGGTTTGCCA AACTTCCCTG AATTTGGTCT AGAATTTGCT ATCGGTTCAC AATTTACAAA CCTTAGCTAT TATGGATACG GTGCAGAAGA AAGCTACCGT GATAAACTTC CTGGTGCCTA TCTTGGTCGA TATGAAACAT CTGTTGAAAA GACATTTGCT CCATATCTAA TGCCACAAGA ATCTGGTAAT CACTATGGTA CTCGTGAATT CACAGTATCT GATGATAACC ATAATGGTGT TAAATTCACC GCACTTAATA AAGCATTCGA ATTCAGTGCT TTGCGTAACA GTACTGAACA AATTGAAAAT GCTCGTCACC AATATGAGTT GCAAGAATCT GATGCTACAT GGATTAAAGT TCTTGCTGCT CAAATGGGTG TAGGTGGTGA CGACTCATGG GGTGCTCCAG TTCATGACGA ATTCTTGCTT AGCTCAGCAG ATAGCTATCA ATTAAGCTTC ATGATTGAAC CACTAAATTA G

[0076] SEQ ID No:6 DSM 34888 lacZ sequence (1..1026 aa) :

[0077] MNMTEKIQTY LNDPKIVSVN TVDAHSDHKY FESLEEFSEG EMKLRQSLNG KWKIHYAQNT NQVLKDFYKT EFDETDLNFI NVPGHLELQG FGSPQYVNTQ YPWDGKEFLR PPQVPQESNA VASYVKHFTL NDALKDKKVF I SFQGVATS I FVWVNGNFVG YSEDSFTPSE FEISDYLVEG DNKLAVAVYR YSTASWLEDQ DFWRLYGI FR DVYLYAI PKV HVQDLFVKGD YDYQTKSGQL DIDLKTVGDY EDKKIKYVLS DYEGI ITEGD ASVNGDGELS VSLENLKIKP WSAESPKLYD LILHVLDDDQ WEWPVKVG FRRFEIKDKL MLLNGKRIVF KGVNRHEFNA RTGRCITEED MLWDIKVMKQ HNINAVRTSH YPNQTRWYEL CDEYGLYVID EANLETHGTW QKLGLCEPSW NI PASEPEWL PACLDRANNM FQRDKNHASV I IWSCGNESY AGKDIADMAD YFRSVDNTRP VHYEGVTWCR EFDYITDIES RMYAKPAD IE EYLTTGKLVD LSSVSDKHFA SGNLTNNPQK PYI SCEYMHM MGNSGGGLEL YTDLEKYPEY QGGFIWDFID QAIYKTLPNG SEFLSYGGDW HDRPSDYEFC GNGIVFADRT LTPKLQTVKH LYSNIKIAVD EKSVTIKNDN LFEDLSAYTF LARVYEDGRK VSESEYHFDV KPGEEATFPV NFWEASNSE QIYEVACVLR EATKWAPKGH EIVRGQYVAE KI STETPVKA PLNWEGDFN IGIQGQNFSI LLSRAQNTLV SAKYNGVEFI EKGPKLSFTR AYTDNDRGAG YPFEMAGWKV AGNYSKVTDT QIQIEDDSVK VTYVHELPGL SDVEVKVTYQ VDYKGRI FVT ANYDGKAGLP NFPEFGLEFA IGSQFTNLSY YGYGAEESYR DKLPGAYLGR YETSVEKTFA PYLMPQESGN HYGTREFTVS DDNHNGLKFT ALNKAFEFSA LRNSTEQIEN ARHQYELQES DATWIKVLAA QMGVGGDDSW GAPVHDEFLL SSADSYQLSF MIEPLN*

[0078] SEQ ID No:7 DSM 34890 lacZ C1667T mutant sequence (1..3081 bp) :

[0079] ATGAACATGA CTGAAAAAAT TCAAACTTAT TTAAACGATC CAAAGATTGT TAGCGTTAAT ACTGTTGATG CTCACTCAGA TCATAAGTAT TTTGAATCTC TTGAAGAATT TTCTGAAGGG GAGATGAAGT TAAGACAATC TCTTAATGGA AAATGGAAAA TTCACTATGC TCAGAATACA AATCAGGTTT TAAAAGACTT TTATAAAACA GAATTTGATG AAACTGATTT GAATTTCATC AATGTACCAG GT CAT TT AGA GCTTCAAGGT TTTGGTTCTC CACAATATGT GAATACCCAA TATCCTTGGG ATGGTAAAGA ATTCCTTCGT CCACCTCAAG TTCCTCAAGA ATCAAATGCT GTTGCATCAT ACGTTAAACA TTTTACCTTG AATGATGCAT TAAAAGATAA AAAAGTATTT ATCTCATTCC AAGGGGTTGC TACTTCCATC TTTGTATGGG TCAATGGTAA CTTTGTAGGC TACAGTGAAG ATTCATTTAC ACCTAGTGAA TTTGAAATTA GTGATTACCT TGTTGAAGGT GATAACAAGT TGGCGGTAGC TGTTTATCGT TACTCTACAG CAAGCTGGTT GGAAGACCAA GACTTCTGGA GACTTTACGG TATTTTTAGA GATGTTTACT TGTATGCTAT TCCAAAAGTT CACGTTCAAG ATCTCTTTGT TAAGGGAGAT TATGATTACC AAACAAAATC AGGTCAATTA GATATTGATT TGAAGACTGT TGGTGATTAT GAAGACAAGA AGATTAAATA TGTTCTTTCA GATTATGAAG GCATCATTAC AGAAGGTGAT GCATCTGTTA ATGGTGACGG TGAACTATCT GTAAGTCTTG AAAATCTTAA AATCAAACCT TGGAGTGCTG AAAGTCCTAA ACTTTACGAT TTGATCCTTC ATGTTTTGGA TGATGACCAA GTTGTTGAAG TCGTTCCAGT TAAAGTTGGA TTTAGACGCT TTGAAATTAA AGATAAACTT ATGCTTTTGA ATGGTAAGAG AATTGTCTTT AAAGGGGTTA ACAGACACGA ATTTAACGCT AGAACAGGAC GTTGTATCAC TGAAGAAGAT ATGCTTTGGG ATATCAAAGT GATGAAACAA CATAACATCA ATGCTGTTCG TACTTCACAC TATCCTAACC AAACACGTTG GTATGAATTG TGTGATGAAT ATGGACTTTA TGTTATCGAT GAAGCCAACC TTGAAACACA CGGTACATGG CAAAAACTTG GTCTATGCGA ACCTTCATGG AATATCCCAG CTAGTGAACC AGAATGGTTG CCTGCTTGTT TGGATCGTGC CAATAACATG TTCCAACGCG ATAAGAACCA TGCTAGTGTT ATCATTTGGT CTTGTGGTAA TGAATCATAT GCTGGTAAAG ATATTGCTGA CATGGCTGAT TACTTCCGTA GTGTTGACAA TACTCGTCCA GTTCACTATG AAGGTGTTAC ATGGTGTCGT GAATTTGATT ACATTACAGA CATCGAAAGT CGTATGTATG CGAAACCAGC TGATATTGAA GAATACCTCA CAACTGGTAA ACTAGTTGAT CTTTCAAGCG TTAGTGATAA ACACTTTGCT TCAGGTAACC TAACTAACAA TCCTCAAAAA CCTTATATTT CATGTGAATA CATGCACATG ATGGGTAACT CTGGTGTTGG ATTGGAACTC TACACTGACT TAGAGAAATA TCCAGAATAC CAAGGTGGAT TTATTTGGGA CTTCATTGAC CAAGCTATTT ACAAAACACT TCCAAATGGT AGCGAATTCC TATCATATGG TGGTGACTGG CATGATAGAC CTTCTGACTA CGAATTTTGT GGAAATGGTA TTGTCTTTGC AGATCGTACC CTAACTCCAA AACTTCAAAC AGTTAAACAT CTTTACTCTA ATATTAAGAT TGCTGTTGAT GAAAAATCAG TAACTATCAA GAATGATAAT CTCTTCGAAG ATCTTTCTGC TTATACTTTC CTAGCTAGAG TTTACGAAGA TGGTAGAAAA GTTAGCGAAA GTGAATATCA CTTTGATGTT AAACCAGGTG AAGAAGCAAC ATTCCCAGTT AACTTTGTAG TCGAGGCTTC AAATTCTGAA CAAATTTACG AAGTTGCTTG TGTTCTGAGG GAAGCAACTA AATGGGCTCC TAAAGGTCAT GAAATTGTTC GTGGTCAATA TGTTGCTGAA AAGATTAGCA CCGAAACACC AGTTAAAGCA CCTTTGAATG TTGTTGAAGG CGACTTCAAC ATCGGTATTC AAGGACAAAA CTTCTCAATC TTGCTTTCAC GTGCACAAAA TACTTTAGTA TCTGCTAAGT ATAATGGTGT TGAATTCATT GAGAAAGGTC CTAAACTTAG CTTCACTCGT GCTTACACTG ACAACGATCG TGGTGCTGGA TATCCATTCG AAATGGCAGG CTGGAAGGTT GCTGGAAACT ATAGTAAAGT TACAGATACT CAAATTCAAA TCGAAGACGA CTCTGTTAAA GTGACTTATG TTCATGAATT GCCAGGCTTG TCTGATGTCG AAGTTAAGGT AACTTATCAA GT T GAG TACA AGGGTCGAAT CTTTGTTACT GCAAACTATG ATGGTAAAGC AGGTTTGCCA AACTTCCCTG AATTTGGTCT AGAATTTGCT ATCGGTTCAC AATTTACAAA CCTTAGCTAT TATGGATACG GTGCAGAAGA AAGCTACCGT GATAAACTTC CTGGTGCCTA TCTTGGTCGA TATGAAACAT CTGTTGAAAA GACATTTGCT CCATATCTAA TGCCACAAGA ATCTGGTAAT CACTATGGTA CTCGTGAATT CACAGTATCT GATGATAACC ATAATGGTGT TAAATTCACC GCACTTAATA AAGCATTCGA ATTCAGTGCT TTGCGTAACA GTACTGAACA

[0080] AATTGAAAAT GCTCGTCACC AATATGAGTT GCAAGAATCT GATGCTACAT

[0081] GGATTAAAGT TCTTGCTGCT CAAATGGGTG TAGGTGGTGA CGACTCATGG GGTGCTCCAG TTCATGACGA ATTCTTGCTT AGCTCAGCAG ATAGCTATCA ATTAAGCTTC ATGATTGAAC CACTAAATTA G

[0082] SEQ ID No:8 DSM 34890 lacZ G556V mutant sequence (1..1026 aa) :

[0083] MNMTEKIQTY LNDPKIVSVN TVDAHSDHKY FESLEEFSEG EMKLRQSLNG KWKIHYAQNT NQVLKDFYKT EFDETDLNFI NVPGHLELQG FGSPQYVNTQ YPWDGKEFLR PPQVPQESNA VASYVKHFTL NDALKDKKVF I SFQGVATS I FVWVNGNFVG YSEDSFTPSE FEISDYLVEG DNKLAVAVYR YSTASWLEDQ DFWRLYGI FR DVYLYAI PKV HVQDLFVKGD YDYQTKSGQL DIDLKTVGDY EDKKIKYVLS DYEGI ITEGD ASVNGDGELS VSLENLKIKP WSAESPKLYD LILHVLDDDQ WEWPVKVG FRRFEIKDKL MLLNGKRIVF KGVNRHEFNA RTGRCITEED MLWDIKVMKQ HNINAVRTSH YPNQTRWYEL CDEYGLYVID EANLETHGTW QKLGLCEPSW NI PASEPEWL PACLDRANNM FQRDKNHASV I IWSCGNESY AGKDIADMAD YFRSVDNTRP VHYEGVTWCR EFDYITDIES RMYAKPAD IE EYLTTGKLVD LSSVSDKHFA SGNLTNNPQK PYI SCEYMHM MGNSGVGLEL YTDLEKYPEY QGGFIWDFID QAIYKTLPNG SEFLSYGGDW HDRPSDYEFC GNGIVFADRT LTPKLQTVKH LYSNIKIAVD EKSVTIKNDN LFEDLSAYTF LARVYEDGRK VSESEYHFDV KPGEEATFPV NFWEASNSE QIYEVACVLR EATKWAPKGH EIVRGQYVAE KI STETPVKA PLNWEGDFN IGIQGQNFSI LLSRAQNTLV SAKYNGVEFI EKGPKLSFTR AYTDNDRGAG YPFEMAGWKV AGNYSKVTDT QIQIEDDSVK VTYVHELPGL SDVEVKVTYQ VDYKGRI FVT ANYDGKAGLP NFPEFGLEFA IGSQFTNLSY YGYGAEESYR DKLPGAYLGR YETSVEKTFA PYLMPQESGN HYGTREFTVS DDNHNGLKFT ALNKAFEFSA LRNSTEQIEN ARHQYELQES DATWIKVLAA QMGVGGDDSW GAPVHDEFLL SSADSYQLSF MIEPLN*

[0084] SEQ ID No:9 DSM 34891 lacZ G2144A mutant sequence (1..2145 bp):

[0085] ATGAACATGA CTGAAAAAAT TCAAACTTAT TTAAACGATC CAAAGATTGT TAGCGTTAAT ACTGTTGATG CTCACTCAGA TCATAAGTAT TTTGAATCTC TTGAAGAATT TTCTGAAGGG GAGATGAAGT TAAGACAATC TCTTAATGGA AAATGGAAAA TTCACTATGC TCAGAATACA AATCAGGTTT TAAAAGACTT TTATAAAACA GAATTTGATG AAACTGATTT GAATTTCATC AATGTACCAG GTCATTTAGA GCTTCAAGGT TTTGGTTCTC CACAATATGT GAATACCCAA TATCCTTGGG ATGGTAAAGA ATTCCTTCGT CCACCTCAAG TTCCTCAAGA ATCAAATGCT GTTGCATCAT ACGTTAAACA TTTTACCTTG AATGATGCAT TAAAAGATAA AAAAGTATTT ATCTCATTCC AAGGGGTTGC TACTTCCATC TTTGTATGGG TCAATGGTAA CTTTGTAGGC TACAGTGAAG ATTCATTTAC ACCTAGTGAA TTTGAAATTA GTGATTACCT TGTTGAAGGT GATAACAAGT TGGCGGTAGC TGTTTATCGT TACTCTACAG CAAGCTGGTT GGAAGACCAA GACTTCTGGA GACTTTACGG TATTTTTAGA GATGTTTACT TGTATGCTAT TCCAAAAGTT CACGTTCAAG ATCTCTTTGT TAAGGGAGAT TATGATTACC AAACAAAATC AGGTCAATTA GATATTGATT TGAAGACTGT TGGTGATTAT GAAGACAAGA AGATTAAATA TGTTCTTTCA GATTATGAAG GCATCATTAC AGAAGGTGAT GCATCTGTTA ATGGTGACGG TGAACTATCT GTAAGTCTTG AAAATCTTAA AATCAAACCT TGGAGTGCTG AAAGTCCTAA ACTTTACGAT TTGATCCTTC ATGTTTTGGA TGATGACCAA GTTGTTGAAG TCGTTCCAGT TAAAGTTGGA TTTAGACGCT TTGAAATTAA AGATAAACTT ATGCTTTTGA ATGGTAAGAG AATTGTCTTT AAAGGGGTTA ACAGACACGA ATTTAACGCT AGAACAGGAC GTTGTATCAC TGAAGAAGAT ATGCTTTGGG ATATCAAAGT GATGAAACAA CATAACATCA ATGCTGTTCG TACTTCACAC TATCCTAACC AAACACGTTG GTATGAATTG TGTGATGAAT ATGGACTTTA TGTTATCGAT GAAGCCAACC TTGAAACACA CGGTACATGG CAAAAACTTG GTCTATGCGA ACCTTCATGG AATATCCCAG CTAGTGAACC AGAATGGTTG CCTGCTTGTT TGGATCGTGC CAATAACATG TTCCAACGCG ATAAGAACCA TGCTAGTGTT ATCATTTGGT CTTGTGGTAA TGAATCATAT GCTGGTAAAG ATATTGCTGA CATGGCTGAT TACTTCCGTA GTGTTGACAA TACTCGTCCA GTTCACTATG AAGGTGTTAC ATGGTGTCGT GAATTTGATT ACATTACAGA CATCGAAAGT CGTATGTATG CGAAACCAGC TGATATTGAA GAATACCTCA CAACTGGTAA ACTAGTTGAT CTTTCAAGCG TTAGTGATAA ACACTTTGCT TCAGGTAACC TAACTAACAA TCCTCAAAAA CCTTATATTT CATGTGAATA CATGCACATG ATGGGTAACT CTGGTGGTGG ATTGGAACTC TACACTGACT TAGAGAAATA TCCAGAATAC CAAGGTGGAT TTATTTGGGA CTTCATTGAC CAAGCTATTT ACAAAACACT TCCAAATGGT AGCGAATTCC TATCATATGG TGGTGACTGG CATGATAGAC CTTCTGACTA CGAATTTTGT GGAAATGGTA TTGTCTTTGC AGATCGTACC CTAACTCCAA AACTTCAAAC AGTTAAACAT CTTTACTCTA ATATTAAGAT TGCTGTTGAT GAAAAATCAG TAACTATCAA GAATGATAAT CTCTTCGAAG ATCTTTCTGC TTATACTTTC CTAGCTAGAG TTTACGAAGA TGGTAGAAAA GTTAGCGAAA GTGAATATCA CTTTGATGTT AAACCAGGTG AAGAAGCAAC ATTCCCAGTT AACTTTGTAG TCGAGGCTTC AAATTCTGAA CAAATTTACG AAGTTGCTTG TGTTCTGAGG GAAGCAACTA AATAG

[0086] SEQ ID No:10 DSM 34891 lacZ truncated mutant sequence (1..714 aa) :

[0087] MNMTEKIQTY LNDPKIVSVN TVDAHSDHKY FESLEEFSEG EMKLRQSLNG KWKIHYAQNT NQVLKDFYKT EFDETDLNFI NVPGHLELQG FGSPQYVNTQ YPWDGKEFLR PPQVPQESNA VASYVKHFTL NDALKDKKVF I SFQGVATS I FVWVNGNFVG YSEDSFTPSE FEISDYLVEG DNKLAVAVYR YSTASWLEDQ DFWRLYGI FR DVYLYAI PKV HVQDLFVKGD YDYQTKSGQL DIDLKTVGDY EDKKIKYVLS DYEGI ITEGD ASVNGDGELS VSLENLKIKP WSAESPKLYD LILHVLDDDQ WEWPVKVG FRRFEIKDKL MLLNGKRIVF KGVNRHEFNA RTGRCITEED MLWDIKVMKQ HNINAVRTSH YPNQTRWYEL CDEYGLYVID EANLETHGTW QKLGLCEPSW NI PASEPEWL PACLDRANNM FQRDKNHASV I IWSCGNESY AGKDIADMAD YFRSVDNTRP VHYEGVTWCR EFDYITDIES RMYAKPAD IE EYLTTGKLVD LSSVSDKHFA SGNLTNNPQK PYI SCEYMHM MGNSGGGLEL YTDLEKYPEY QGGFIWDFID QAIYKTLPNG SEFLSYGGDW HDRPSDYEFC GNGIVFADRT LTPKLQTVKH LYSNIKIAVD EKSVTIKNDN LFEDLSAYTF LARVYEDGRK VSESEYHFDV KPGEEATFPV NFWEASNSE QIYEVACVLR EATK*

[0088] SEQ ID No:ll DSM 34892 lacZ G2144A mutant sequence (1..2145 bp) :

[0089] ATGAACATGA CTGAAAAAAT TCAAACTTAT TTAAACGATC CAAAGATTGT TAGCGTTAAT ACTGTTGATG CTCACTCAGA TCATAAGTAT TTTGAATCTC TTGAAGAATT TTCTGAAGGG GAGATGAAGT TAAGACAATC TCTTAATGGA AAATGGAAAA TTCACTATGC TCAGAATACA AATCAGGTTT TAAAAGACTT TTATAAAACA GAATTTGATG AAACTGATTT GAATTTCATC AATGTACCAG GTCATTTAGA GCTTCAAGGT TTTGGTTCTC CACAATATGT GAATACCCAA TATCCTTGGG ATGGTAAAGA ATTCCTTCGT CCACCTCAAG TTCCTCAAGA ATCAAATGCT GTTGCATCAT ACGTTAAACA TTTTACCTTG AATGATGCAT TAAAAGATAA AAAAGTATTT ATCTCATTCC AAGGGGTTGC TACTTCCATC TTTGTATGGG TCAATGGTAA CTTTGTAGGC TACAGTGAAG ATTCATTTAC ACCTAGTGAA TTTGAAATTA GTGATTACCT TGTTGAAGGT GATAACAAGT TGGCGGTAGC TGTTTATCGT TACTCTACAG CAAGCTGGTT GGAAGACCAA GACTTCTGGA GACTTTACGG TATTTTTAGA GATGTTTACT TGTATGCTAT TCCAAAAGTT CACGTTCAAG ATCTCTTTGT TAAGGGAGAT TATGATTACC AAACAAAATC AGGTCAATTA GATATTGATT TGAAGACTGT TGGTGATTAT GAAGACAAGA AGATTAAATA TGTTCTTTCA GATTATGAAG GCATCATTAC AGAAGGTGAT GCATCTGTTA ATGGTGACGG TGAACTATCT GTAAGTCTTG AAAATCTTAA AATCAAACCT TGGAGTGCTG AAAGTCCTAA ACTTTACGAT TTGATCCTTC ATGTTTTGGA TGATGACCAA GTTGTTGAAG TCGTTCCAGT TAAAGTTGGA TTTAGACGCT TTGAAATTAA AGATAAACTT ATGCTTTTGA ATGGTAAGAG AATTGTCTTT AAAGGGGTTA ACAGACACGA ATTTAACGCT AGAACAGGAC GTTGTATCAC TGAAGAAGAT ATGCTTTGGG ATATCAAAGT GATGAAACAA CATAACATCA ATGCTGTTCG TACTTCACAC TATCCTAACC AAACACGTTG GTATGAATTG TGTGATGAAT ATGGACTTTA TGTTATCGAT GAAGCCAACC TTGAAACACA CGGTACATGG CAAAAACTTG GTCTATGCGA ACCTTCATGG AATATCCCAG CTAGTGAACC AGAATGGTTG CCTGCTTGTT TGGATCGTGC CAATAACATG TTCCAACGCG ATAAGAACCA TGCTAGTGTT ATCATTTGGT CTTGTGGTAA TGAATCATAT GCTGGTAAAG ATATTGCTGA CATGGCTGAT TACTTCCGTA GTGTTGACAA TACTCGTCCA GTTCACTATG AAGGTGTTAC ATGGTGTCGT GAATTTGATT ACATTACAGA CATCGAAAGT CGTATGTATG CGAAACCAGC TGATATTGAA GAATACCTCA CAACTGGTAA ACTAGTTGAT CTTTCAAGCG TTAGTGATAA ACACTTTGCT TCAGGTAACC TAACTAACAA TCCTCAAAAA CCTTATATTT CATGTGAATA CATGCACATG ATGGGTAACT CTGGTGGTGG ATTGGAACTC TACACTGACT TAGAGAAATA TCCAGAATAC CAAGGTGGAT TTATTTGGGA CTTCATTGAC CAAGCTATTT ACAAAACACT TCCAAATGGT AGCGAATTCC TATCATATGG TGGTGACTGG CATGATAGAC CTTCTGACTA CGAATTTTGT GGAAATGGTA TTGTCTTTGC AGATCGTACC CTAACTCCAA AACTTCAAAC AGTTAAACAT CTTTACTCTA ATATTAAGAT TGCTGTTGAT GAAAAATCAG TAACTATCAA GAATGATAAT CTCTTCGAAG ATCTTTCTGC TTATACTTTC CTAGCTAGAG TTTACGAAGA TGGTAGAAAA GTTAGCGAAA GTGAATATCA CTTTGATGTT AAACCAGGTG AAGAAGCAAC ATTCCCAGTT AACTTTGTAG TCGAGGCTTC AAATTCTGAA CAAATTTACG AAGTTGCTTG TGTTCTGAGG GAAGCAACTA AATAG

[0090] SEQ ID No:12 DSM 34892 lacZ truncated mutant sequence (1..714 aa) :

[0091] MNMTEKIQTY LNDPKIVSVN TVDAHSDHKY FESLEEFSEG EMKLRQSLNG KWKIHYAQNT NQVLKDFYKT EFDETDLNFI NVPGHLELQG FGSPQYVNTQ YPWDGKEFLR PPQVPQESNA VASYVKHFTL NDALKDKKVF ISFQGVATSI FVWVNGNFVG YSEDSFTPSE FEISDYLVEG DNKLAVAVYR YSTASWLEDQ DFWRLYGIFR DVYLYAIPKV HVQDLFVKGD YDYQTKSGQL DIDLKTVGDY EDKKIKYVLS DYEGI ITEGD ASVNGDGELS VSLENLKIKP WSAESPKLYD LILHVLDDDQ WEWPVKVG FRRFEIKDKL MLLNGKRIVF KGVNRHEFNA RTGRCITEED MLWDIKVMKQ HNINAVRTSH YPNQTRWYEL CDEYGLYVID EANLETHGTW QKLGLCEPSW NIPASEPEWL PACLDRANNM FQRDKNHASV IIWSCGNESY AGKDIADMAD YFRSVDNTRP VHYEGVTWCR EFDYITDIES RMYAKPAD IE EYLTTGKLVD LSSVSDKHFA SGNLTNNPQK PYISCEYMHM MGNSGGGLEL YTDLEKYPEY QGGFIWDFID QAIYKTLPNG SEFLSYGGDW HDRPSDYEFC GNGIVFADRT LTPKLQTVKH LYSNIKIAVD EKSVTIKNDN LFEDLSAYTF LARVYEDGRK VSESEYHFDV KPGEEATFPV NFWEASNSE QIYEVACVLR EATK*

[0092] Composition.

[0093] Streptococcus thermophilus is one of the lactic acid bacteria most often used for producing fermented products and often alongside with one or more strains of same and / or different genus. In a third aspect the present disclosure relates to a composition comprising, either as a mixture or as a kit-of-parts, one or more strains according to the present disclosure.

[0094] The S. thermophilus as described herein is useful as a starter culture in the production of food products. Starter cultures comprising one or more lactic acid bacteria (LAB) are added to acidify the milk base.

[0095] The term "Starter" or "Starter culture" as used herein means a culture or composition comprising one or more lactic acid bacteria strains able to acidify a milk base according to general practice in the cheese making industry. One embodiment of the present disclosure relates to a starter culture comprising one or more LAB strains of the species Streptococcus thermophilus (ST), one embodiment of the present disclosure relates to a strain of the species Streptococcus thermophilus (ST). One embodiment of the present disclosure relates to a protease positive ST strain. As it is normal in the production of lactic acid bacterial fermentation processes to apply mixed cultures of lactic acid bacteria, the composition will in certain embodiments comprise a multiplicity of strains either belonging to the same species or belonging to different species. One embodiment of the present disclosure relates to the starter further comprising one or more strains of the genus Lactobacillus or Lactococcus. One embodiment of the present disclosure relates to a starter culture, wherein the one or more strains of the genus Lactobacillus is selected from L. delbrueckii ssp. bulgaricus, L. delbrueckii ssp. lactis, L. fermentum and L. helveticus. One embodiment of the present disclosure relates to a starter culture, wherein the one or more strains of the genus Lactococcus is selected from Lactococcus lactis.

[0096] The composition of the present invention may be provided in several forms. It may be a powder, pellets or tablets. It may be a frozen form, dried form, freeze dried form, or liquid form. Thus, a starter culture composition comprises the bacteria in a concentrated form including frozen, dried or freeze-dried concentrates typically having a concentration of viable cells, which is in the range of 104to 1014cfu (colony forming units) per gram of the composition including at least 104cfu per gram of the composition, such as at least 105cfu / g, e. g. at least 105cfu / g, such as at least 107cfu / g, e.g. at least 108cfu / g, such as at least 109cfu / g, e.g. at least IO10cfu / g, such as at least 1011cfu / g. In one embodiment the present disclosure relates to the composition, wherein the composition is a starter culture or comprised in a starter culture, and is in frozen, freeze-dried, or liquid form. In one embodiment the present disclosure relates to the composition, wherein the concentration of viable cells of each of the one or more strains in the composition is in the range of 104to 1014cfu per gram of the composition; 105to 1014; 105to 1014; or 107to 1014cfu per gram of the composition.

[0097] The composition may as further components contain cryoprotectants and / or conventional additives including nutrients such as yeast extracts, sugars and / or vitamins. The composition of the present invention may additionally comprise cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, flavorants or mixtures thereof. The composition preferably comprises one or more of cryoprotectants, lyoprotectants, antioxidants and / or nutrients, more preferably cryoprotectants, lyoprotectants and / or antioxidants and most preferably cryoprotectants or lyoprotectants, or both. Use of protectants such as cryoprotectants and lyoprotectantare known to a skilled person in the art. Suitable cryoprotectants or lyoprotectants include mono-, di-, tri-and polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (acacia) and the like), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol and the like), amino acids (such as proline, glutamic acid), complex substances (such as skim milk, peptones, gelatin, yeast extract) and inorganic compounds (such as sodium tripolyphosphate).

[0098] In one embodiment, the composition according to the present invention may comprise one or more cryoprotective agent(s) 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 a derivative of any such compounds. Suitable antioxidants include ascorbic acid, citric acid and salts thereof, gallates, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (such as vitamin B-family, vitamin C). The composition may optionally comprise further substances including fillers (such as lactose, maltodextrin) and / or flavorants.

[0099] In one embodiment of the invention the cryoprotective agent is an agent or mixture of agents, which in addition to its cryoprotectivity has a booster effect. The expression "booster effect" is used to describe the situation wherein the cryoprotective agent confers an increased metabolic activity (booster effect) on to the thawed or reconstituted culture when it is inoculated into the medium to be fermented or converted. Viability and metabolic activity are not synonymous concepts. Commercial frozen or freeze-dried cultures may retain their viability, although they may have lost a significant portion of their metabolic activity e.g. cultures may lose their acid-producing (acidification) activity when kept stored even for shorter periods of time. Thus, viability and booster effect has to be evaluated by different assays. Whereas viability is assessed by viability assays such as the determination of colony forming units, booster effect is assessed by quantifying the relevant metabolic activity of the thawed or reconstituted culture relative to the viability of the culture. The term "metabolic activity" refers to the oxygen removal activity of the cultures, its acid-producing activity, i. e. the production of e. g. lactic acid, acetic acid, formic acid and / or propionic acid, or its metabolite producing activity such as the production of aroma compounds such as acetaldehyde, (a- acetolactate, acetoin, diacetyl and 2,3-butylene glycol (butanediol)).

[0100] In one embodiment the composition of the invention contains or comprises from 0.2% to 20% of the cryoprotective agent or mixture of agents measured as % w / w of the material. It is, however, preferable to add the cryoprotective agent or mixture of agents at an amount which is in the range from 0.2% to 15%, from 0.2% to 10%, from 0.5% to 7%, and from 1% to 6% by weight, including within the range from 2% to 5% of the cryoprotective agent or mixture of agents measured as % w / w of the frozen material by weight. In a preferred embodiment the culture comprises approximately 3% of the cryoprotective agent or mixture of agents measured as %w / w of the material by weight. The amount of approximately 3% of the cryoprotective agent corresponds to concentrations in the 100 mM range. It should be recognized that for each aspect of embodiment of the invention the ranges may be increments of the described ranges.

[0101] In the present context the term "from x% to y%" means to include the end-points, thus equal to the term "from and including x% to and including y%"

[0102] In a further aspect, the composition of the present invention contains or comprises an ammonium salt (e.g. an ammonium salt of organic acid (such as ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid) as a booster (e.g. growth booster or acidification booster) for bacterial cells, such as cells belonging to the species S. thermophilus, e.g. (substantial) urease negative bacterial cells. The term "ammonium salt", "ammonium formate", etc., should be understood as a source of the salt or a combination of the ions. The term "source" of e.g. "ammonium formate" or "ammonium salt" refers to a compound or mix of compounds that when added to a culture of cells, provides ammonium formate or an ammonium salt. In some embodiments, the source of ammonium releases ammonium into a growth medium, while in other embodiments, the ammonium source is metabolized to produce ammonium. In some preferred embodiments, the ammonium source is exogenous. In some particularly preferred embodiments, ammonium is not provided by the dairy base. It should of course be understood that ammonia may be added instead of ammonium salt. Thus, the term ammonium salt comprises ammonia (NH3), NH4OH, NH4+, and the like.

[0103] In one embodiment the composition of the invention may comprise thickener and / or stabilizer, such as pectin (e.g. HM pectin, LM pectin), gelatin, CMC, Soya Bean Fiber / Soya Bean Polymer, starch, modified starch, carrageenan, alginate, and guar gum.

[0104] Method for manufacturing a food product.

[0105] The present disclosure relates to a method for manufacturing a food product by using the strain or composition of the present disclosure. The description of the strain and composition provided in other parts of the disclosure {supra and infra) also applies to the present part of the disclosure.

[0106] Starting material suitable for use in the method may be any any raw and / or processed material or base fermentable by the strain and composition. Any starting material comprising lactose such as milk, or a mixture comprising milk may be suitable. Thus, useful milk bases include, but are not limited to, solutions / suspensions of any milk or milk like products comprising protein, such as whole or low-fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, dried milk, whey, whey permeate, lactose, mother liquid from crystallization of lactose, whey protein concentrate, or cream. Obviously, the milk base may originate from any mammal, e.g. being substantially pure mammalian milk, or reconstituted milk powder or the milk base may originate partly from a plant material. Preferably, at least part of the protein in the milk base is (i) proteins naturally occurring in mammalian milk, such as casein or whey protein or (ii) proteins naturally occurring in plant milk. However, part of the protein may be proteins which are not naturally occurring in milk. The terms "Milk" or "Milk base" as used herein means a composition comprising lacteal secretion obtained from any mammal, such as an animal of a species belonging to the subfamily Bovinae (which includes the domestic cow (Bos taurus) and buffalo); an animal of a species belonging to the subfamily Caprinae (which includes goat and sheep); or an animal of the species Camelidae (which includes camels). Optionally the milk or milk base is acidified, e.g. by addition of an acid (such as citric, acetic or lactic acid) or by addition of an acid producing microorganism. The milk or milk base may be raw or processed, e.g. by filtering, sterilizing, pasteurizing, homogenizing, fractionating (e.g. reducing the fat content of the milk) etc., or it may be reconstituted dried milk. An important example of milk or milk base according to the present disclosure is pasteurized cow's milk. It is understood that the milk may be acidified, mixed or processed before, during and / or after the adding of bacterial cultures. The milk or milk base may further comprise protein, calcium or other additives added.

[0107] Non-dairy starting material with addition of milk sugars or other sugars that may be metabolized by the lactic acid bacteria. Examples of suitable sugars include but are not limited to lactose, glucose, galactose, sucrose, fructose. Suitable non-dairy starting materials include but are not limited to materials originating from plants, meat, fruit.

[0108] In a fourth aspect the present disclosure relates to a method for manufacturing a food product comprising the steps: (a) providing the strain of the present disclosure, or the composition of the present disclosure; (b) inoculating a starting material with said strain or composition; and (c) allowing fermentation of the inoculated starting material until a target pH has been reached. In one embodiment the present disclosure relates to the method according to claim 18, wherein the starting material is comprising lactose, preferably mammalian milk.

[0109] The fermented products, which are obtained by the method, include as typical examples dairy products such as fermented milk, yogurt, cheese including fresh cheese products, soft cheese products, Cheddar, mozzarella or buttermilk. In a preferred embodiment, the dairy product is soft cheese, Cheddar cheese, pasta filata cheese or mozzarella cheese - more preferably, the dairy product is pasta filata cheese, Cheddar cheese or mozzarella cheese - most preferably the dairy product is mozzarella cheese or Cheddar cheese (preferably used for making pizza). In one embodiment the present disclosure relates to the method of the present disclosure, wherein the food product is a dairy product. In one embodiment the present disclosure relates to the method of the present disclosure, wherein the food product is Fermented milk; Yogurt including stirred type, set type, and drinkable yogurt; Buttermilk; Cheese including fresh cheese, soft cheese, pasta filata cheese, Bacterial ripened cheese such as Cheddar, continental, edam / gouda, or parmesan.

[0110] In a fifth aspect the present disclosure relates to a food product comprising the strain of the present disclosure or the composition of the present disclosure. In one embodiment the present disclosure relates to a food product, wherein the food product is made by the method of the present disclosure. In one embodiment the present disclosure relates to a food product of the present disclosure, wherein said food product has a concentration of galactose below 0.25; 0.20; 0.15; 0.10; or 0.05 g / L of food product.

[0111] Use.

[0112] In a sixth aspect the present disclosure relates to use of the strain or the composition for reducing the amount of galactose in food products and / or for reducing the browning of cheese.

[0113] In one embodiment the present disclosure relates to use of the strain, or the composition or kit-of-parts for producing a pasta filata type cheese product. In one embodiment the present disclosure relates to a pasta filata type cheese product made by the method or by the use. The cheese product may be fresh cheese such as Mozzarella, Halloumi, Burrata, Stracciatelle, Scamorza, and Pizza cheese; low moisture cheese such as low-moisture mozzarella; aged cheese such as Provolone, Ragusano and Caciocavallo, and / or smoked cheese such as Provolone or Scamorza affumicata. There are many different types of pasta filata cheeses each characterized by the specific method used in the region of origin. One embodiment of the present disclosure relates to a product, wherein the cheese is Mozzarella, Burrata, Stracciatella, Scamorza, Caciocavallo, Ragusano, Kashkaval, Provolone or Pizza cheese. In one embodiment the present disclosure relates to a product, wherein the product is a low-moisture cheese. Low-moisture is defined as a moisture content measured in w / w% below 52%, below 51%, below 50%, or in the range of 43- 51%, 44-50%, 45-49%, 46-48%, or about 45%, about 46%, about 47%, about 48%, or about 49%.

[0114] DEPOSITS AND EXPERT SOLUTION

[0115] The applicant requests that a sample of the deposited microorganisms stated in Table 1 may only be made available to an expert, subject to available provisions governed by Industrial Property Offices of States Party to the Budapest Treaty, until the date on which the patent is granted.

[0116] Table 1 : Deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany.

[0117] EXAMPLES

[0118] Material & Methods

[0119] B-Milk was made from skim milk powder to a level of dry matter of 9.5% (w / v) reconstituted in distilled water and pasteurized at 99°C for 30 min, followed by cooling to 30°C.

[0120] The following strain were used in the examples: DSM 32826 described in W02020 / 254604.

[0121] Example 1 - Identification of Streptococcus thermophilus strains expressing both an active LacSZ system and an active Lactose-PTS system.

[0122] We identified two new and previously undescribed Streptococcus thermophilus strains DSM 34887, and DSM 34888 which expressed in addition to the normal active LacSZ system also an active Lactose-PTS system. The strains were identified by a BLAST search using the Lac-PTS gene sequence from Lactococcus lactis towards the genome sequenced S. thermophilus strains from our culture collection using the CLC software (CLC Genomics Workbench, version 10.1.1 (CLC bio, Qiagen Bioinformatics)). The effect of the presence of an active Lactose-PTS in Streptococcus thermophilus strains on the carbohydrate profile were studied. The strains DSM 34887 and DSM 34888 with an active LacSZ system and an active Lac-PTS system were compared to the strain DSM 32826 having an active LacSZ system and no Lac-PTS system. The strains were inoculated in an amount of 1% in B-milk at 37°C and acidified until a target pH of 4.5 was reached. Samples were analyzed for the presence of various metabolites and uninoculated B-milk was included as control.

[0123] Table 2a : Concentration of metabolites in B-Milk (g / L) after fermentation with a mother strain.

[0124] Table 2b : Concentration of metabolites in B-Milk (g / L) after fermentation with a mother strain.

[0125] Example 2 - Isolation of LacSZ-neqative and lactose-PTS positive mutants.

[0126] The mother strains identified in Example 1 were used to generate mutant strains wherein the LacSZ system was inactivated leaving the Lactose-PTS system for fermentation of lactose. The galactose produced in the mutant strains was not excreted due to the inactivated LacSZ system and was metabolized via the tagatose pathway. Mutants expressing an inactive LacSZ system and an active Lactose-PTS system were generated using a method comprising the following steps: Providing cells of a mother strain: UV-mutagenizing the cells; Plating and growing the cells on X-Gal plates; Screening the plates for white colonies.

[0127] The presence of active lacZ enzyme was indicated by blue colonies, whereas inactive LacZ enzyme was indicated by white colonies on X-Gal. The mutants could still grow on lactose with reduced activity due to the lac-PTS activity. The activity of the Lactose-PTS was shown by a milk acidification experiment. If a strain has an inactive lacZ gene, shown by the appearance of white colonies on LM17 agar (M17 agar containing 1% lactose) containing X-Gal, and can ferment milk which is containing lactose as main fermentable carbohydrate, then acidification can only occur when an active Lac-PTS system is present.

[0128] The following mutants deficient in lacZ activity were isolated DSM 34889, DSM 34890, DSM 34891, and DSM 34892. The mutants were all able to acidify B-milk to a pH at the same level as their mother strain although not as fast.

[0129] Example 3 - Carbohydrate analysis

[0130] Carbohydrate analysis of the mutant grown in B-milk to pH 4.55 showed that the mutants did not excrete any galactose (see tables below). The minor amount of measured galactose is lower than the galactose concentration of the inoculated B- milk, thus no galactose is excreted by the cells. It was shown that it is possible to develop an S. thermophilus strain which is using the lac-PTS system for lactose assimilation and the tagatose system to metabolize the galactose and excretion no significant amount of galactose. The indicated values in the tables are the average from two measurements.

[0131] Table 3a : Concentration of metabolites in B-Milk (g / L) after fermentation with DSM 34887 and the mutant.

[0132] Table 3b: Concentration of metabolites in B-M ilk (g / L) after fermentation with DSM 34888 and the mutants.

[0133]

[0134] Example 4 - Genome analysis.

[0135] The genomes were sequenced at Novonesis A / S. Total DNA was purified and used to prepare a 250-bp paired-end library for genome sequencing using Illumina MiSeq system. The sequence reads were subjected to quality trimming (Phred score < 25) and assembled into contigs using the de novo assembly algorithm in CLC Genomics Workbench, version 23.0.1 (CLC bio, Qiagen Bioinformatics). The resulting genome assembly was filtered by removing contigs with coverage of <15X and / or <20% of the median coverage of the assembly. The consensus sequences of the remaining contigs were exported in FASTA format, which is referred to as the draft genome sequence, and used in the subsequent sequence analysis.

[0136] The genes encoding the Lactose permease (LacSZ) system were analyzed by using the same software CLC. The results from the mutation analysis are listed in the table below. Table 4: Mutations in the sequences.

Claims

CLAIMS1. A method for generating a lactic acid bacteria strain comprising the following steps: a) Providing a galactose excreting mother strain of the species Streptococcus thermophilus having an active lactose permease (LacSZ) system and an active phosphoenolpyruvate-dependent phosphotransferase (Lac-PTS) system; b) Treating the mother strain whereby the LacSZ system becomes inactive; c) Growing the treated strain under conditions enabling the differentiation between cells with an active LacSZ system from cells with an inactive LacSZ system; and d) Screening for and isolating a strain having an active Lac-PTS system and an inactive LacSZ system; wherein said strain does not excrete galactose or has a reduced excretion of galactose as compared to the mother strain from which it is derived.

2. The method according to claim 1, wherein the active Lac-PTS system is (a) present and active in the mother strain; (b) present and has been activated in the mother strain; or (c) has been transferred to and is active in the mother strain which were previously lacking an active Lac-PTS system.

3. The method according to claim 2, wherein the active Lac-PTS system has been transferred into the mother strain by means of a plasmid or genetic engineering.

4. The method according to any one of claims 1-3, wherein the LacSZ system is inactivated due to a change in one or more of the LacS and LacZ nucleotide sequences and / or amino acid sequences.

5. The method according to any one of claims 1-4, wherein the LacSZ system is inactivated due to a mutation in the LacZ gene encoding 0-galactosidase.

6. The method according to the preceding claim, wherein the mutation is at a position corresponding to the nucleotide at position 1143 in SEQ ID NO:3; position 1667 in SEQ ID NO:7; position 2144 in SEQ ID NO:9; or position 2144 in SEQ ID NO: 11.

7. The method according to any one of claims 5-6, wherein the mutation at the position corresponding to the nucleotide at position 1143 in SEQ ID NO:3 is A; position 1667 in SEQ ID NO:7 is T; position 2144 in SEQ ID NO:9 is A; or position 2144 in SEQ ID NO: 11 is A.

8. The method according to any one of claims 5-7, wherein the mutation in the LacZ gene leads to a mutated 0-galactosidase protein comprising an amino acid substitution at a position corresponding to position 556 in SEQ ID NO:8.

9. The method according to the preceding claim, wherein the amino acid substitution at the position corresponding to position 556 in SEQ ID NO:8 is V.

10. The method according to any one of claims 1-9, wherein the mother strain is selected from DSM 34887; DSM 34888; or mutants or variants thereof.

11. A lactic acid bacteria strain selected from DSM 34887; or DSM 34888.

12. A lactic acid bacteria strain derived from a galactose excreting mother strain of the species Streptococcus thermophilus, wherein the strain has (a) an active phosphoenolpyruvate-dependent phosphotransferase (Lac-PTS) system; (b) an inactive lactose permease (LacSZ) system; and (c) does not excrete galactose or has a reduced excretion of galactose as compared to the mother strain.

13. The strain according to claim 12, wherein the strain is generated by the method according to any one of claims 1-10.

14. The strain according to any one of claims 12-13, wherein the strain excretes less than 2.00; 1.75; 1.50; 1.25; 1.00; 0.75; 0.50; or less than 0.25g / L galactose.

15. The strain according to any one of claims 12-14, wherein the strain is selected from the group consisting of DSM 34889; DSM 34890; DSM 34891; and DSM 34892.

16. A composition comprising, either as a mixture or as a kit-of-parts, one or more strains according to any one of claims 12-14.

17. The composition according to claim 15, wherein the composition is a starter culture or comprised in a starter culture, and is in frozen, freeze-dried, or liquid form.

18. The composition according to any one of claims 15-16, wherein the concentration of viable cells of each of the one or more strains in the composition is in the range of 104to 1014cfu per gram of the composition; 105to 1014; 105to 1014; or 107to 1014cfu per gram of the composition.

19. A method for manufacturing a food product comprising the steps: a) providing the strain according to any one of claims 11-14, or the composition according to any one of claims 15-17; b) inoculating a starting material with said strain or composition; and c) allowing fermentation of the inoculated starting material until a target pH has been reached.

20. The method according to claim 18, wherein the starting material is comprising lactose, preferably mammalian milk.

21. The method according to any one of claims 18-19, wherein the food product is a dairy product.

22. The method according to any one of claims 18-19, wherein the food product is Fermented milk; Yogurt including stirred type, set type, and drinkable yogurt; Buttermilk; Cheese including fresh cheese, soft cheese, pasta filata cheese, Bacterial ripened cheese such as Cheddar, continental, edam / gouda, or parmesan.

23. A food product comprising the strain according to any one of claims 11-14 or the composition according to any one of claims 15-17.

24. The food product according to the preceding claim, wherein the food product is made by the method according to any one of claims 18-21.

25. The food product according to any one of claims 22-23, wherein said food product has a concentration of galactose below 0.25; 0.20; 0.15; 0.10; or 0.05 g / L of food product.

26. Use of the strain according to any one of claims 11-14 or the composition according to any one of claims 15-17 for reducing the amount of galactose in food products and / or for reducing the browning of cheese.

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