Method for reducing lactose at high temperatures

Abstract

To provide a method for reducing the amount of lactose in a milk-based substrate containing lactose.SOLUTION: Provided is a method comprising contacting a substrate with an enzyme having neutral lactase activity at a temperature of more than about 50°C, wherein the enzyme has a specific sequence, and wherein the lactase reduces the amount of lactose in the substrate by at least about 70%, and wherein the enzyme having neutral lactase activity is an enzyme capable of hydrolyzing the disaccharide lactose into its galactose and glucose monomer components, and wherein the enzyme has an optimum pH of about pH 6.0 to about pH 8.0.SELECTED DRAWING: Figure 8

Description

Technical Field

[0001] The present application relates to a method for reducing the amount of lactose in a milk-based substrate containing lactose by contacting the substrate with an enzyme having a neutral lactase activity, more particularly at an elevated temperature. The present specification also refers to milk-based substrates and dairy products such as low-lactose or lactose-free obtainable or obtainable by the methods disclosed herein. The present specification also refers to enzyme preparations containing an enzyme having a neutral lactase activity as disclosed herein, nucleic acid molecules encoding the enzyme, vectors containing the nucleic acid molecules, cells capable of expressing the enzyme, and their use for preparing milk-based substrates and dairy products. The present specification also relates to a concentrated and purified lactase effective for lactose hydrolysis at 50 to 65 °C that can be used for the preparation of low-lactose or lactose-free dairy products.

Background Art

[0002] People with lactose intolerance have difficulty digesting dairy products with high lactose concentrations. Approximately 70% of the world's population is estimated to have a low ability to digest lactose. Therefore, the demand for dairy products that contain no lactose or only a small amount of lactose is increasing. The commercial use of lactase is to break down lactose in dairy products. A common manufacturing method for lactose-reduced sterilized milk involves adding lactase enzyme to milk and then incubating it at a temperature near 6°C for a long period (10 - 48 hours, often 24 hours). Lactase has been isolated from a wide variety of organisms, including microorganisms such as the genus Kluyveromyces, Bacillus, and Bifidobacterium. The genus Kluyveromyces, especially K. fragilis and K. lactis, and other fungi, such as the genus Candida, Torula, and Torulopsis, are common sources of fungal lactase, while B. coagulans and B. circulans are well-known sources of bacterial lactase. Several commercially available lactase preparations derived from these organisms, such as Lactozym® (available from Novozymes, Denmark), HA-Lactase (available from Chr. Hansen, Denmark), and Maxilact® (available from DSM, the Netherlands), are available, and all of them are derived from K. lactis. In addition, a few Bifidobacterium bifidum lactases, such as Saphera (available from Novozymes, Denmark) and NOLA Fit (available from Chr. Hansen, Denmark), are commercially available. All of these lactases are so-called neutral lactases with an optimal pH of pH 6 - 8 and an optimal temperature of around 37°C.These lactases are not suitable for the hydrolysis of lactose in milk substrates carried out at high temperatures, which, in some cases, are beneficial for maintaining low microbial counts and thus ensuring good milk quality.

[0003] Commercially available lactase preparations having an optimal temperature above 50 °C are very few and they are GOS-producing lactases and / or acidic lactases such as the commercially available Bacillus lactase from Daiwa Kasei (Japan), the Bifidumbacterium biftdum lactase from DuPont, FoodPro GOS, and NutribioGOS of acidic lactase derived from Aspergillus oryzae, which reduce the production efficiency of low-lactose or lactose-free neutral dairy products.

[0004] In U.S. Patent Nos. 4,374,861 and 4,333,954 regarding ice cream, lactose is hydrolyzed at a low temperature of 30 to 50 °C. These hydrolyses at low temperatures carry a high risk of microbial counts.

[0005] In the Czech Patent No. 201100672 regarding condensed milk, lactose is hydrolyzed at a low temperature of 2 to 50 °C. These hydrolyses at low temperatures are not efficient. Furthermore, these hydrolyses at low temperatures carry a high risk of microbial counts.

[0006] The present invention provides a neutral lactase that efficiently hydrolyzes lactose at 50 to 65 °C for producing dairy products.

Summary of the Invention

Means for Solving the Problems

[0007] Briefly described, the present specification generally discloses methods for reducing the amount of lactose in dairy products containing a milk-based substrate and lactose, as well as milk-based substrates and dairy products produced by those methods.

[0008] The present specification discloses, in part, enzyme preparations containing enzymes with neutral lactase activity, as well as vectors and nucleic acid molecules encoding the enzymes, and cells capable of expressing the enzymes.

[0009] Advantageously, the methods described herein produce milk-based substrates and / or dairy products that are lactose-free or have a low lactose concentration. Advantageously, the method results in milk-based substrates and / or dairy products with low off-odor levels and / or few texture defects.

[0010] Further advantages of the teachings herein will become apparent to those skilled in the art upon reading this specification.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Mode for Carrying Out the Invention

[0012] This detailed description is intended to inform other persons skilled in the art of the applicant's invention, its principles, and its practical applications, so that other persons skilled in the art can apply and adapt the invention to its many forms and optimize it for the requirements of specific applications. This detailed description and its specific examples show specific embodiments, but are for illustrative purposes only. Therefore, this specification is not limited to the described embodiments and may be variously modified. This specification discloses a method for reducing the amount of lactose in a milk-based substrate containing lactose, which includes contacting the substrate with an enzyme having neutral lactase activity at a temperature above about 50°C, and the lactase reduces the amount of lactose in the substrate by at least about 70%.

[0013] This specification also discloses a milk-based substrate with a reduced lactose content obtainable or obtained by the above (or other places in this specification) method.

[0014] This specification further discloses a method for manufacturing a dairy product, which includes contacting the dairy product with an enzyme having neutral lactase activity at a temperature above about 50°C, and the lactase reduces the amount of lactose in the dairy product by at least about 70%.

[0015] This specification also discloses a dairy product obtainable or obtained by the above (or other places in this specification) method.

[0016] This specification further discloses, in part, an enzyme preparation containing an enzyme having neutral lactase activity, and the enzyme can reduce the amount of lactose in the substrate by at least 70% at a temperature of 50°C or higher.

[0017] This specification also discloses, in part, an enzyme preparation containing an enzyme having neutral lactase activity, the enzyme can reduce the amount of lactose in the substrate by at least 70% at a temperature of 50°C or higher, and the enzyme is not the enzyme of SEQ ID NO: 1.

[0018] This specification further discloses, in part, a nucleic acid molecule encoding the enzyme having neutral lactase activity described in this specification or a lactase-active fragment thereof.

[0019] This specification also, in part, discloses an expression vector that can contain the nucleic acid molecules described above (or elsewhere in this specification) or can express an enzyme having the neutral lactase activity described in this specification or a lactase activity fragment thereof.

[0020] This specification also, in part, discloses a cell that can express an enzyme having the neutral lactase activity described in this specification or a lactase activity fragment thereof.

[0021] This specification further, in part, discloses a method for expressing an enzyme, the method including providing the above-described (or described elsewhere in this specification) cell, expressing the enzyme from the cell, and optionally purifying the enzyme.

[0022] This specification also, in part, discloses an enzyme having the neutral lactase activity described in this specification and having a half-life in milk of more than 4 hours at 55°C or more than 1 hour at 58°C.

[0023] This specification further, in part, discloses the use of the enzyme preparation described in this specification for preparing dairy products.

[0024] This specification further, in part, discloses a bacterial expression host that can express an enzyme having the neutral lactase activity described in this specification, wherein the host cell contains a genetic modification that reduces or eliminates lipase activity, preferably lipase A activity.

[0025] This specification further, in part, discloses an enzyme having the neutral lactase activity produced by the bacterial expression host described in this specification.

[0026] This specification further, in part, discloses an enzyme having the neutral lactase activity produced by the bacterial expression host described in this specification and being an enzyme preparation as defined in this specification.

[0027] This specification also, in part, discloses dairy products comprising the bacterial expression hosts described herein.

[0028] This specification further, in part, discloses the use of the bacterial expression hosts described herein for manufacturing dairy products.

[0029] This specification further, in part, discloses a method for manufacturing a low-lactose or lactose-free milkshake, ice cream, reduced milk product, dessert, pudding, condensed milk, sweetened condensed milk, ryazhenka, dulce de leche, or milk-based powder, the method comprising contacting a milk-based substrate with lactase at 50 to 65 °C.

[0030] This specification also, in part, discloses a method for manufacturing a milk-based powder or whey powder with reduced lactose content, wherein an enzyme having neutral lactase activity is added for hydrolysis during an evaporation / concentration process.

[0031] This specification further, in part, discloses a method for manufacturing a lactose-free dairy product from a milk-based substrate comprising an enzyme having neutral lactase activity, wherein more than 20% of the activity remains in the milk-based substrate after pasteurization at 72 °C for 15 seconds.

[0032] This specification further, in part, discloses a method for manufacturing a fermented dairy product, comprising adding lactase, for example, during cooling to the fermentation temperature after pasteurization and homogenization.

[0033] This specification further, in part, discloses a method for manufacturing a fermented dairy product, comprising adding lactase only, for example, during cooling to about 50 to 65 °C after pasteurization and homogenization. Thereafter, further cooling may be performed, and a starter culture may be added when the temperature reaches about 45 °C.

[0034] This specification further discloses, in part, a method for in situ GOS production in a milk-based substrate comprising at least 4.7% (w / w) lactose, the method comprising contacting the substrate with a lactase having a neutral lactase activity, wherein more than 30% of the lactose is converted to TGOS.

[0035] This specification further discloses, in part, a method for in situ GOS production, wherein the milk-based substrate contains 6-40% (w / w) lactose and more than 40% of the lactose is converted to TGOS.

[0036] This specification further discloses, in part, a method for reducing the amount of sugar in a milk substrate, the method comprising contacting the substrate with an enzyme having a neutral lactase activity such that lactose is converted to GOS fiber (DP3+).

[0037] This specification further discloses, in part, a method for producing a lactose-free dairy product from a milk-based substrate, the method comprising: supplying a milk-based substrate; adding an enzyme having a neutral lactase activity to the milk-based substrate; pasteurizing the milk-based substrate, wherein the enzyme retains a significant amount of activity after the pasteurization step; and storing the resulting milk-based substrate for a time sufficient to produce a lactose-free dairy product.

[0038] Enzyme having a neutral lactase activity The terms "enzyme having a neutral lactase activity" and "lactase" may be used interchangeably herein.

[0039] Enzymes with neutral lactase activity are enzymes that have the ability to hydrolyze the disaccharide lactose into its constituent galactose and glucose monomers. Furthermore, enzymes with neutral lactase activity have an optimal pH of about 6.0 to about 8.0. Neutral lactase preparations typically originate from the cytoplasm of microorganisms. Their production involves isolating lactase after (large-scale) fermentation of the microorganism. The latter requires disrupting the cell wall to release the enzyme from the cytoplasm. Several techniques can be used to obtain cell lysates, including permeabilization of the cell wall by organic solvents such as octanol, sonication, or French press. In addition to lactase, other enzymes, such as proteases, are released simultaneously from the cytoplasm.

[0040] Neutral lactase activity can be determined as neutral lactase units (NLU) using o-nitrophenyl-β-D-galactopyranoside (ONPG) as a substrate according to the procedure described in FCC (fourth ed, July 1996, p801 - 802: Lactase (neutral) β-galactosidase activity). 1 NLU / g can be defined as the amount of enzyme that liberates 1.30 mM of o-nitrophenol per minute under assay conditions.

[0041] The term "enzyme with neutral lactase activity" includes auxiliary compounds (which may or may not be naturally present in the reaction system), such as appropriate acceptors or cofactors, that may be required for the catalytic activity of the enzyme.

[0042] In one aspect, the enzyme with neutral lactase activity has an optimal pH of about 6.0 to about 8.0.

[0043] In one aspect, the enzyme with neutral lactase activity can hydrolyze lactose at a temperature of about 50°C to about 65°C.

[0044] In one aspect, the enzyme with neutral lactase activity is derived from Lactobacillus species.

[0045] In one aspect, the enzyme having neutral lactase activity is derived from Lactobacillus delbrueckii bulgaricus.

[0046] In one aspect, the enzyme having neutral lactase activity has at least about 60% identity to SEQ ID NO: 1. In one aspect, the enzyme having neutral lactase activity has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. In one aspect, the enzyme having neutral lactase activity is the enzyme of SEQ ID NO: 1, or a lactase-active fragment thereof, a homolog thereof, or a variant thereof.

[0047] "Homolog" means an entity having a certain percentage of identity, i.e., "homology", with the subject amino acid sequence. In one aspect, the subject amino acid sequence is SEQ ID NO: 1.

[0048] "Homologous sequences" include polynucleotides or polypeptides having a specific percentage, e.g., 80%, 85%, 90%, 95% or 99% sequence identity with another sequence. The identity rate means that when aligned, the percentage of bases or amino acid residues is the same when comparing two sequences. An amino acid sequence is not the same if an amino acid is substituted, deleted or added compared to the subject sequence. The sequence identity rate is generally measured compared to the mature sequence of the subject protein, i.e., after removal of the signal sequence, for example. Generally, a homolog will contain the same active site residues as the subject amino acid sequence. A homolog may retain the enzyme activity of the wild-type or reference enzyme, e.g., the enzyme activity of neutral lactase.

[0049] As used herein, "reference enzyme", "reference sequence", "reference polypeptide" mean any of the variant polypeptides, for example, the enzyme and polypeptide based on SEQ ID NO: 1. "Reference nucleic acid" means a nucleic acid sequence encoding a reference polypeptide. As used herein, the terms "reference sequence" and "subject sequence" are used interchangeably.

[0050] As used herein, "experimental DuPont lactase" and "LBul" mean a thermostable β-galactosidase derived from Lactobacillus delbrueckii bulgaricus having the amino acid sequence shown in SEQ ID NO: 1.

[0051] As used herein, "query sequence" means a foreign sequence that is aligned with a reference sequence to determine whether it is included within the scope of the present invention. Thus, such a query sequence may be, for example, a prior art sequence or a third party sequence.

[0052] As used herein, the term "sequence" may refer to a polypeptide sequence or a nucleic acid sequence depending on the context.

[0053] As used herein, the terms "polypeptide sequence" and "amino acid sequence" are used interchangeably.

[0054] "One variety" or "multiple varieties" refers to a polypeptide or a nucleic acid. The term "variety" can be used interchangeably with the term "variant". A variety includes insertions, substitutions, transversions, cleavages and / or inversions at one or more locations in each of the amino acid sequence or nucleotide sequence. The phrases "variety polypeptide", "polypeptide variety", "polypeptide", "variety" and "variety enzyme" mean a polypeptide / protein / enzyme having or containing a selected amino acid sequence such as SEQ ID NO: 1, or having an amino acid sequence modified as compared to a selected amino acid sequence such as SEQ ID NO: 1. A variety may retain the enzyme activity of a wild-type or reference enzyme, such as the enzyme activity of neutral lactase.

[0055] As used herein, the term "fragment" is defined herein as a polypeptide in which one or more (several) amino acids are deleted from the amino terminus and / or carboxyl terminus, and at this time the fragment has activity.

[0056] In one aspect, the term "fragment" is defined herein as a polypeptide in which one or more (several) amino acids are deleted from the amino terminus and / or carboxyl terminus of the polypeptide of SEQ ID NO: 1, and at this time the fragment has neutral lactase activity.

[0057] The lactase activity fragment of SEQ ID NO: 1 can be any fragment of SEQ ID NO: 1 having neutral lactase activity. For example, the lactase activity fragment of SEQ ID NO: 1 can be the amino acids at positions 1 to 618, 1 to 713, or 1 to 1002 of SEQ ID NO: 1.

[0058] Degree of sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".

[0059] In one aspect, the degree of sequence identity between a query sequence and a reference sequence is determined by: 1) aligning the two sequences using any suitable alignment program with a default scoring matrix and default gap penalty; 2) identifying the number of exact matches (where an exact match is when the alignment program identifies the same amino acid or nucleotide in the same position within the two aligned sequences); and 3) dividing the number of exact matches by the length of the reference sequence.

[0060] In one aspect, the degree of sequence identity between a query sequence and a reference sequence is determined by: 1) aligning the two sequences using any suitable alignment program with a default scoring matrix and default gap penalty; 2) identifying the number of exact matches (where an exact match is when the alignment program identifies the same amino acid or nucleotide in the same position within the two aligned sequences); and 3) dividing the number of exact matches by the length of the longest of the two sequences.

[0061] In another aspect, the degree of sequence identity between a query sequence and a reference sequence is determined by: 1) aligning the two sequences using any suitable alignment program with a default scoring matrix and default gap penalty; 2) identifying the number of exact matches (where an exact match is when the alignment program identifies the same amino acid or nucleotide in the same position within the two aligned sequences); and 3) dividing the number of exact matches by the "alignment length" (where the alignment length is the length of the entire alignment including sequence gaps and overhangs).

[0062] The comparison of sequence identity can be carried out visually or, more generally, by utilizing readily available sequence comparison programs. These commercially available computer programs use complex comparison algorithms that align two or more sequences and best reflect the evolutionary events that could have caused differences between the two or more sequences. For this reason, these algorithms operate with a scoring system that gives an alignment of identical or similar amino acids and penalizes gaps, gap extensions, and alignments of dissimilar amino acids. The scoring system of the comparison algorithm includes specifying a penalty score for each time a gap is inserted (gap penalty score), specifying a penalty score for each time an existing gap extends with an increment position (extension penalty score), specifying a high score when identical amino acids are aligned, and specifying a variable score when non-identical amino acids are aligned is included.

[0063] Most alignment programs allow the gap penalty to be changed. However, when using such software for sequence comparison, it is preferable to use the default values.

[0064] The score given for the alignment of non-identical amino acids is specified according to a scoring matrix, also called a substitution matrix. The scores provided in such a substitution matrix reflect the fact that during evolution the likelihood of one amino acid being substituted by another varies and depends on the physical / chemical properties of the amino acid being substituted. For example, the likelihood of one polar amino acid being substituted by another polar amino acid is higher compared to being substituted by a hydrophobic amino acid. For this reason, the scoring matrix will assign the highest score to identical amino acids, a lower score to non-identical but similar amino acids, and an even lower score to non-identical and dissimilar amino acids. The most frequently used scoring matrices are the PAM matrix (Dayhoff et al. (1978), Jones et al. (1992)), the BLOSUM matrix (Henikoff and Henikoff (1992)), and the Gonnet matrix (Gonnet et al. (1992)).

[0065] Suitable computer programs for performing such alignments include, but are not limited to, Vector NTI (Invitrogen Corp.) and the ClustalV, ClustalW and ClustalW2 programs (Higgins DG & Sharp PM (1988), Higgins et al. (1992), Thompson et al. (1994), Larkin et al. (2007)). A variety of excellent alignment tools are available from the ExPASy Proteomics server at www.expasy.org. Another example of software capable of performing sequence alignment is BLAST (Basic Local Alignment Search Tool), which is currently available from the web page of the National Center for Biotechnology Information, found at www.ncbi.nlm.nih.gov / , and which was first described in Altschul et al. (1990) J. Mol. Biol. 215; 403-410 as BLAST (Basic Local Alignment Search Tool).

[0066] In a preferred embodiment of the present invention, the alignment program implements a global alignment program that optimizes the alignment over the entire length of the sequences. In another preferred embodiment, the global alignment program is based on the Needleman-Wunsch algorithm (Needleman, Saul B.; and Wunsch, Christian D. (1970), “A general method applicable to the search for similarities in the amino acid sequence of two proteins”, Journal of Molecular Biology 48(3):443-53). Examples of current programs that perform global alignment using the Needleman-Wunsch algorithm are the EMBOSS Needle and EMBOSS Stretcher programs, both of which are available from www.ebi.ac.uk / Tools / psa / .

[0067] EMBOSS Needle performs an optimal global sequence alignment using the Needleman-Wunsch alignment algorithm to find the optimal alignment (including gaps) of the entire lengths of two sequences.

[0068] EMBOSS Stretcher uses a modification of the Needleman-Wunsch algorithm to globally align larger sequences.

[0069] In one embodiment, the sequences are aligned by a global alignment program and the sequence identity is calculated by dividing the number of exact matches identified by the program by the “alignment length” (where the alignment length is the length of the entire alignment including gaps and overhang portions of the sequences).

[0070] In yet another aspect, the global alignment program uses the Needleman-Wunsch algorithm and calculates sequence identity by dividing the number of exact matches identified by the program by the "alignment length" (where the alignment length is the length of the entire alignment including gaps and overhangs in the sequences).

[0071] In yet another embodiment, the global alignment program is selected from the group consisting of EMBOSS Needle and EMBOSS Stretcher, and calculates sequence identity by dividing the number of exact matches identified by the program by the "alignment length" (where the alignment length is the length of the entire alignment including gaps and overhangs in the sequences).

[0072] When software generates an alignment, it is possible to calculate the similarity rate (%) and the sequence identity rate (%). The software typically performs this as part of sequence comparison and generates numerical results.

[0073] In one aspect, it is preferable to use ClustalW software for performing sequence alignment. Preferably, the alignment using ClustalW is performed using the following parameters for pairwise alignment:

[0074] Substitution matrix:

[0075] [Table 1]

[0076] Gap open penalty:

[0077] [Table 2]

[0078] Gap extension penalty:

[0079]

Table 3

[0080] Gap end penalty:

[0081]

Table 4

[0082] ClustalW2 is available, for example, on the Internet under the EMBL-EBI web page www.ebi.ac.uk of the European Bioinformatics Institute under tools-sequence analysis-ClustalW2. Currently, the exact address of the ClustalW2 tool is www.ebi.ac.uk / Tools / clustalw2.

[0083] In another aspect, it is preferable to use the program Align X in Vector NTI (Invitrogen) for performing sequence alignment. In one aspect, Exp10 is made available using the following default settings: Gap opening penalty: 10 Gap extension penalty: 0.05 Gap separation penalty range: 8

[0084] In another aspect, the alignment of one amino acid sequence with another amino acid sequence, or to another amino acid sequence, is determined by the use of a score matrix: blosum62mt2 and VectorNTI pairwise alignment settings. Set K-tuple 1 Number of best diagonals 5 Window size 5 Gap penalty 3 Gap opening penalty 10 Gap extension penalty 0.1

[0085] In one aspect, the percentage of identity between one amino acid sequence and another amino acid sequence, or to another amino acid sequence, is determined by use of Blast with a word size of 3 and BLOSUM 62 as the substitution matrix.

[0086] Purified enzyme In one aspect, an enzyme having neutral lactase activity is purified.

[0087] In one aspect, the purified enzyme is the enzyme of SEQ ID NO: 1.

[0088] In one aspect, the term "purified", as used herein, refers to an enzyme having neutral lactase activity that is essentially free of insoluble components from the producing organism. In other aspects, the term "purified" also refers to an enzyme having neutral lactase activity that is substantially free of insoluble components from the natural organism from which it is obtained. In one aspect, the enzyme having neutral lactase activity is also separated from some of the soluble components of the organism and culture medium from which it is derived. The enzyme having neutral lactase activity can be purified (i.e., separated) by one or more of the following unit operations: filtration, precipitation, or chromatography.

[0089] Thus, an enzyme having neutral lactase activity can be purified such that very small amounts of other proteins are present. The expression "other proteins" relates in particular to other enzymes. As used herein, the term "purified" also refers to the removal of other components, in particular other proteins, and most particularly other enzymes present in cells of lactase origin. Lactase can be "substantially pure", i.e., it does not contain other components from the organism in which it is produced, i.e., for example, the host organism for recombinantly produced lactase. In one embodiment, lactase is present in an enzyme preparation that is at least 40% (w / w) pure. In one embodiment, lactase is present in an enzyme preparation that is at least 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w) or 90% (w / w) pure. As used herein, a "substantially pure enzyme" contains at most 10% by weight, preferably at most 8% by weight, more preferably at most 6% by weight, more preferably at most 5% by weight, more preferably at most 4% by weight, more preferably at most 3% by weight, even more preferably at most 2% by weight, most preferably at most 1% by weight, and even more preferably at most 0.5% by weight of other polypeptide substances that are naturally or recombinantly associated therewith.

[0090] Accordingly, a substantially pure enzyme is preferably at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably 99.5% pure, and even most preferably 100% pure, relative to the weight of all polypeptide substances present in the preparation. The enzyme of the present invention is preferably in a substantially pure form (i.e., the preparation is substantially free of other polypeptide substances that are naturally or recombinantly associated therewith). This can be achieved, for example, by preparing the polypeptide by well-known recombinant or traditional purification methods.

[0091] Thus, as used herein, the term "substantially free of lipase" means a preparation containing lipase at most 10% by weight, preferably at most 8% by weight, more preferably at most 6% by weight, more preferably at most 5% by weight, more preferably at most 4% by weight, at most 3% by weight, even more preferably at most 2% by weight, most preferably at most 1% by weight, and even more preferably at most 0.5% by weight. As used herein, the term "substantially free of ~" can thus be considered synonymous with the terms "isolated enzyme" and "enzyme in isolated form". The term "isolated" means that the polypeptide contains at least substantially no other component with which its sequence is naturally associated and is naturally found. In one aspect, an "isolated polypeptide" as used herein refers to a polypeptide that is at least 30% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure, and at least 95% pure as determined by SDS-PAGE.

[0092] Thus, as used herein, the term "substantially free of p-nitrobenzyl esterase" means a preparation containing p-nitrobenzyl esterase at most 10% by weight, preferably at most 8% by weight, more preferably at most 6% by weight, more preferably at most 5% by weight, more preferably at most 4% by weight, at most 3% by weight, even more preferably at most 2% by weight, most preferably at most 1% by weight, and even more preferably at most 0.5% by weight. As used herein, the term "substantially free of ~" can thus be considered synonymous with the terms "isolated enzyme" and "enzyme in isolated form".

[0093] Accordingly, as used herein, the term "substantially free of cellulase" means a preparation containing cellulase at most 10% by weight, preferably at most 8% by weight, more preferably at most 6% by weight, more preferably at most 5% by weight, more preferably at most 4% by weight, at most 3% by weight, even more preferably at most 2% by weight, most preferably at most 1% by weight, and still more preferably at most 0.5% by weight. As used herein, the term "substantially free of ~" can thus be considered synonymous with "isolated polypeptide" and "polypeptide in isolated form".

[0094] As used herein, the term "substantially free of mannanase" means a preparation containing mannanase at most 10% by weight, preferably at most 8% by weight, more preferably at most 6% by weight, more preferably at most 5% by weight, more preferably at most 4% by weight, at most 3% by weight, even more preferably at most 2% by weight, most preferably at most 1% by weight, and still more preferably at most 0.5% by weight. As used herein, the term "substantially free of ~" can thus be considered synonymous with "isolated polypeptide" and "polypeptide in isolated form".

[0095] As used herein, the term "substantially free of pectinase" means a preparation containing pectinase at most 10% by weight, preferably at most 8% by weight, more preferably at most 6% by weight, more preferably at most 5% by weight, more preferably at most 4% by weight, at most 3% by weight, even more preferably at most 2% by weight, most preferably at most 1% by weight, and still more preferably at most 0.5% by weight. As used herein, the term "substantially free of ~" can thus be considered synonymous with "isolated polypeptide" and "polypeptide in isolated form".

[0096] As used herein, the term "substantially free of amylase" means a preparation containing amylase at most 10% by weight, preferably at most 8% by weight, more preferably at most 6% by weight, more preferably at most 5% by weight, more preferably at most 4% by weight, at most 3% by weight, even more preferably at most 2% by weight, most preferably at most 1% by weight, and even more preferably at most 0.5% by weight. As used herein, the term "substantially free of ~" can thus be considered synonymous with "isolated polypeptide" and "polypeptide in isolated form".

[0097] In another aspect, the present disclosure is directed to a purified enzyme having neutral lactase activity produced by the methods described herein.

[0098] In other aspects, the present disclosure relates to purified enzymes that are free of lipase co-activity, phospholipase co-activity, cellulase co-activity, pectinase co-activity, amylase co-activity, protease co-activity, and / or mannanase co-activity. In certain other aspects, the present disclosure relates to dairy products containing a purified enzyme produced by the methods of the present disclosure.

[0099] As defined herein, an "endogenous gene" refers to a gene that is present at its natural location in the genome of an organism.

[0100] As defined herein, a "heterologous" gene, "non-endogenous" gene, or "foreign" gene refers to a gene (or ORF) that is not normally found in a host organism but is introduced into the host organism by genetic engineering. As used herein, the term "heterologous" gene includes a natural gene (or ORF) inserted into a non-natural organism and / or a chimeric gene inserted into a natural or non-natural organism.

[0101] As used herein, the term "foreign polynucleotide" or "heterologous" "Polynucleotide" (and its variations) is defined as (A) a polynucleotide that is not native to the host cell, (B) a polynucleotide that is native to the host cell but has been modified by the use of genetic elements (such as heterologous promoters, 5'UTR, 3'UTR, etc.) that do not naturally associate with the polynucleotide isolated from the host cell, or (C) the use of native elements that have been engineered to function in a manner not normally occurring in the host cell.

[0102] As defined herein, a "heterologous" nucleic acid construct or "heterologous" nucleic acid sequence has a sequence portion that is not native to the cell in which it is expressed.

[0103] As used herein, the term "transformed cell" includes bacterial cells (e.g., Bacillus cells) that have been transformed by the use of recombinant DNA technology. Transformation generally results from the introduction of one or more nucleotide sequences (e.g., polynucleotides) into the cell. The introduced nucleotide sequence can also be a heterologous nucleotide sequence (i.e., a nucleic acid sequence that is not endogenous to the cell).

[0104] As used herein, the term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule (e.g., a polynucleotide molecule) that is isolated from a naturally occurring gene, or otherwise modified or synthesized in a way that does not naturally exist to contain fragments of nucleic acids. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences necessary for the expression of the coding sequence.

[0105] As used herein, the term "control sequence" is defined to include all components that are necessary or advantageous for the expression of a polynucleotide encoding a polypeptide of the invention. Each control sequence may be native or foreign to the polynucleotide encoding the polypeptide, or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, as well as transcription and translation stop signals. The control sequences may include a linker to introduce specific restriction sites to facilitate the ligation of the control sequences to the coding region of the nucleotide sequence encoding the polypeptide.

[0106] As defined herein, "heterologous control sequence" refers to a gene expression control sequence (e.g., a promoter or enhancer) that does not function to regulate (control) the expression of a gene of interest in nature. Generally, heterologous nucleic acid sequences are not endogenous (native) to the cell or genome in which they are present, but are added to the cell by infection, transfection, transformation, microinjection, electroporation, etc. A "heterologous" nucleic acid construct may contain a combination of control sequences / DNA coding sequences that are the same as, or different from, the combination found in the native host cell.

[0107] As used herein, the term "promoter" is defined as a DNA sequence that binds to RNA polymerase and directs that polymerase to the correct downstream transcription start site of a polynucleotide encoding a polypeptide. RNA polymerase effectively catalyzes the assembly of messenger RNA complementary to the appropriate DNA strand of the coding region. The term "promoter" is also understood to include the 5' non-coding region (between the promoter and translation start) for post-transcriptional translation into mRNA, cis-acting transcriptional control elements such as enhancers, and / or other nucleotide sequences that can interact with transcription factors. A promoter can be a wild-type, variant, hybrid, or consensus promoter.

[0108] As used herein, the term "promoter region" is defined as a nucleotide sequence that contains one or more (several) promoter sequences (e.g., dual promoter, triple promoter, etc.).

[0109] As used herein, the term "operably linked" means a configuration in which a control sequence (e.g., a promoter sequence) is placed at an appropriate position relative to the coding sequence of a polynucleotide sequence so as to direct the expression of the coding sequence of a polypeptide. Coding sequence: As used herein, the term "coding sequence" means a nucleotide sequence that directly specifies the amino acid sequence of its protein product. The boundaries of a coding sequence are generally determined by an open reading frame, which usually begins with an ATG start codon, or alternative start codons such as GTG and TTG, and ends with a stop codon such as TAA, TAG, and TGA. A coding sequence can be a DNA sequence, cDNA sequence, synthetic sequence, or recombinant nucleotide sequence.

[0110] As used herein, "expression" includes the processes involved in the production of a polypeptide of interest (POI), examples of which include, but are not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0111] As used herein, "expression vector" and "expression construct" are used interchangeably and refer to a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide of interest and is operably linked to additional nucleotides that provide for its expression.

[0112] In the context of the present invention, "one of the main components" means a component having a dry matter that constitutes more than 20%, preferably more than 30% or more than 40% of the total dry matter of the dairy product, while "main component" means a component having a dry matter that constitutes more than 50%, preferably more than 60% or more than 70% of the total dry matter of the dairy product.

[0113] Enzyme properties In one aspect, the enzyme having neutral lactase activity is concentrated. In one aspect, the enzyme is concentrated 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 100, or 1000 times compared to the enzyme before concentration. In one aspect, lactase can be concentrated to about 10 - 1100 mg / ml.

[0114] In one aspect, the activity of the enzyme having neutral lactase activity at 60 °C is at least 50% of the activity at 50 °C.

[0115] In one aspect, the enzyme has a half-life in milk of more than about 4 hours at 55 °C or more than about 1 hour at 58 °C.

[0116] In one aspect, the enzyme having neutral lactase activity has an optimum temperature of about 60 °C.

[0117] In one aspect, the enzyme having neutral lactase activity has an optimum pH of about 5.5 - about 8.0. In one aspect, the enzyme having neutral lactase activity has an optimum pH of about 5.7 - about 7.6.

[0118] In one aspect, the lactase activity of the enzyme having neutral lactase activity at pH 6.0 is at least 50% of the lactase activity at pH 6.5 when measured at 37 °C.

[0119] In one aspect, the galactooligosaccharide is a DP3+ galactooligosaccharide. Thus, in one aspect, the enzyme having lactase activity converts about 35% or less of the lactose in the substrate to DP3+ galactooligosaccharide. Preferably, the enzyme having lactase activity converts about 30% or less of the lactose in the substrate to DP3+ galactooligosaccharide, and more preferably, the enzyme having lactase activity converts about 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% or less of the lactose in the substrate to DP3+ galactooligosaccharide.

[0120] In one aspect, the enzyme having the neutral lactase activity has an activity ratio of lactase:transgalactosylase greater than 1:1. In one aspect, the contact is carried out under conditions where the lactase activity of the enzyme is higher than the transgalactosylation activity, for example, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher. The ratio of lactase activity to transgalactosylase activity can be determined, for example, by HPLC analysis. Lactose can be converted to an equal amount of free glucose and free galactose.

[0121] "Transgalactosylase" particularly means an enzyme that can transfer galactose to the hydroxyl group of D-galactose or D-glucose, thereby producing galactooligosaccharide. In one aspect, transgalactosylase is identified by an enzymatic reaction in lactose where the amount of galactose produced in a given time is less than the amount of glucose produced.

[0122] In the context of the present invention, the term "transgalactosylation activity" means the transfer of a galactose moiety to a molecule other than water. This activity can be measured by directly quantifying [glucose]-[galactose] produced at any point during the reaction, or by directly quantifying GOS produced at any point during the reaction. This measurement can be carried out by several methods such as the HPLC method shown in the examples. When comparing the measured values of transgalactosylation activity, for example, it has been carried out at a given initial lactose concentration such as 3, 4, 5, 6, 7, 8, 9 or 10% (w / w).

[0123] In one aspect, the enzyme having lactase activity can have a transgalactosylation activity rate of less than 120%.

[0124] Transgalactosylation activity rate = (Abs420 +セロビオース / Abs420 -セロビオース ) × 100% (wherein Abs420 +セロビオース is the absorbance read at 420 nm using the method described below including cellobiose during the reaction, and Abs420 -セロビオース is the absorbance read at 420 nm using the method described below except that cellobiose is not included during the reaction. The above equation applies only to dilutions with an absorbance of 0.5 to 1.0.

[0125] Measurement of β-galactosidase activity Enzyme activity can be measured using the commercially available substrate 2-nitrophenyl-β-D-galactopyranoside (ONPG) (Sigma N1127). Without ONPG acceptor 100 mM KP04 (pH 6.0) 12.3 mM ONPG ONPG supplemented with acceptor 100 mM KP04 pH 6.0 20 mM cellobiose 12.3 mM ONPG Stop solution 10% Na 2 CO 3

[0126] A 10 μL dilution series of the purified enzyme can be added into the wells of a microtiter plate containing 90 μL of ONPG buffer with or without an acceptor. The samples are mixed and incubated at 37 °C for 10 minutes, and subsequently 100 μL of stop solution can be added to each well to terminate the reaction. The absorbance measurements can be recorded at 420 nm using a Molecular Device SpectraMax plate reader controlled by the Softmax software package.

[0127] The transgalactosylation activity rate can be calculated as follows: For the dilution with an absorbance between 0.5 and 1.0, transgalactosylation activity rate = (Abs420 +セロビオース / Abs420 -セロビオース ) × 100.

[0128] In one aspect, the activity is measured after a reaction for 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, or 180 minutes. Thus, in one aspect, by way of example, the relative transgalactosylation activity is measured 15 minutes after the addition of the enzyme, for example 30 minutes after the addition of the enzyme, for example 60 minutes after the addition of the enzyme, for example 90 minutes after the addition of the enzyme, for example 120 minutes after the addition of the enzyme, or for example 180 minutes after the addition of the enzyme.

[0129] In the context of the present invention, the term "ratio of transgalactosylation activity: lactase activity" means ([glucose] - [galactose] / [galactose]).

[0130] In the context of the present invention, the term [glucose] means the glucose concentration (% by weight) measured by HPLC method.

[0131] In the context of the present invention, the term [galactose] means the galactose concentration (% by weight) measured by HPLC method.

[0132] In the context of the present invention, the term "lactose being transgalactosylated" means that a galactose molecule is covalently bound to any free hydroxyl group of a lactose molecule, or is covalently bound to a lactose molecule generated by internal transgalactosylation, such as forming allolactose.

[0133] In one aspect, an enzyme having neutral lactase activity when hydrolyzing lactose in a milk-based substrate or dairy product has an activity ratio of lactase to transgalactosylase greater than 1:1, such as greater than 2:1, or greater than 3:1. In another aspect, the enzymatic treatment is carried out under conditions where the lactase activity of the enzyme is higher than the transgalactosylase activity, such as at least 2-fold higher, or at least 3-fold higher. The activity ratio of lactase to transgalactosylase in a milk-based substrate can be determined, for example, by HPLC analysis.

[0134] In one aspect, the enzyme having the neutral lactase activity has a lactase activity rate of more than 100%. In one aspect, the enzyme having the neutral lactase activity has a lactase activity rate of more than 120%, such as more than 150%, more than 175%, or more than 200%.

[0135] The lactases referred to herein can be extracellular. They have a signal sequence at the N-terminus, and the signal sequence is cleaved and removed during the secretion process.

[0136] The lactase referred to in this specification may be derived from any of the sources referred to in this specification. The term "derived from" in this context means that the enzyme is isolated from a naturally occurring organism, i.e., the amino acid sequence of the enzyme is identical to that of the natural enzyme. The term "derived from" also means that the enzyme can be recombinantly produced in a host organism, and the recombinantly produced enzyme is either identical to the natural enzyme or has an amino acid sequence that has been modified, e.g., one or more amino acids have been deleted, inserted and / or substituted, i.e., it is a variant and / or fragment of the natural amino acid sequence. The meaning of natural enzyme includes natural variants. Further, the term "derived from" includes enzymes that are synthetically produced, for example, by peptide synthesis. The term "derived from" also includes enzymes that have been modified, e.g., by glycosylation, phosphorylation, etc., whether in vivo or in vitro. With respect to a recombinantly produced enzyme, the term "derived from" refers to the identity of the enzyme and not to the identity of the host organism in which the enzyme is recombinantly produced.

[0137] Lactase can be obtained from microorganisms by any suitable method. For example, a lactase enzyme preparation can be obtained by fermentation of a suitable microorganism and subsequent isolation of the lactase preparation from the resulting fermentation broth or microorganism by methods known in the art. Lactase can also be obtained by the use of recombinant DNA technology. Such methods typically involve culturing a host cell transformed with a recombinant DNA vector containing a DNA sequence encoding the lactase in question, the DNA sequence being operably linked to appropriate expression signals so that it can express lactase in the culture medium under conditions that permit expression of the enzyme, and recovering the enzyme from the culture. The DNA sequence may also be integrated into the genome of the host cell. The DNA sequence may be of genomic, cDNA or synthetic origin, or a combination thereof, and may be isolated or synthesized according to methods known in the art.

[0138] The quality of lactase can be determined by the ratio of secondary activity to lactase activity. Proteases are known to cause undesirable side effects such as milk clotting or the formation of an unpleasant odor in milk. The formation of off-odors is particularly critical in products that can be stored long-term and are stored at room temperature. One such product is UHT milk, and the formation of off-odors is a known problem in lactose-hydrolyzed UHT milk. UHT milk is very sensitive to off-odor formation, and if a lactase preparation does not produce off-odors in UHT milk, it will generally not produce off-odors in other applications either. The compounds involved in off-odor formation in milk, particularly UHT milk, are related to both proteolysis and the Maillard reaction (Valero et al (2001) Food Chem. 72, 51-58). Any protease present as a secondary activity in a lactase preparation has the potential to enhance off-odor formation, and while it is unclear what level of protease is required, even very low proteolytic activity can be important over several months of storage time. UHT milk is very sensitive to off-odor formation, and if a lactase preparation does not produce off-odors in UHT milk other than the described off-odors (such as those described in Valero et al (2001) Food Chem. 72, 51-58), it will generally not produce off-odors in other applications either. Therefore, UHT application is a good method for evaluating the quality of lactase preparations with respect to the potential for off-odors. Since proteases were thought to be at least partially involved in off-odor formation, intensive efforts have been directed towards reducing the protease level of lactase products.

[0139] In one aspect, the enzyme having neutral lactase activity is in the form of an enzyme preparation. Enzyme preparation

[0140] In one aspect, the enzyme having neutral lactase activity is in the form of an enzyme preparation with reduced secondary activity levels. A "preparation" is any suitable composition containing the enzyme.

[0141] The enzyme preparation can reduce the lactose amount in the substrate by at least about 70% at a temperature of 55°C or higher, or at other temperatures as described herein.

[0142] In one aspect, the enzyme having neutral lactase activity is in the form of an enzyme preparation in which the level of one or more of the following secondary activities is reduced: lipase activity, protease activity, amylase activity, mannanase activity, pectinase activity, cellulase activity, and / or p-nitrobenzyl esterase activity.

[0143] In one aspect, the enzyme having neutral lactase activity is in the form of an enzyme preparation in which the lipase secondary activity level is reduced.

[0144] In one aspect, the enzyme having neutral lactase activity is in the form of an enzyme preparation in which the protease, amylase, mannanase, pectinase, cellulase, and / or p-nitrobenzyl esterase secondary activity levels are reduced.

[0145] In one aspect, the enzyme preparation has a reduced level of secondary activity. In one aspect, the enzyme preparation has a reduced level of one or more of the following secondary activities: lipase activity, protease activity, amylase activity, mannanase activity, pectinase activity, cellulase activity, and / or p-nitrobenzyl esterase activity.

[0146] In one aspect, the enzyme preparation has a reduced level of lipase secondary activity.

[0147] In one aspect, the enzyme preparation has a reduced level of protease, amylase, mannanase, pectinase, cellulase, and / or p-nitrobenzyl esterase secondary activity.

[0148] As used herein, the term "reduction in level" associated with the phrases "reduction in lipase secondary activity level" and "reduction in protease, amylase, mannanase, pectinase, cellulase, and / or p-nitrobenzyl esterase secondary activity level" may refer to a reduction in level compared to an enzyme preparation containing an enzyme having a neutral lactase activity produced from different host cells. This reduction in level may refer to a reduction in level compared to an enzyme preparation produced from an unmodified host cell, i.e., a host cell that has not undergone any of the modifications described herein. As shown in this example, sensory analysis by a trained panel concluded that milk produced using an enzyme from a modified host with lower secondary activity was preferred. In one aspect, the enzyme preparation has good properties from the perspective of reducing off-odor formation. Also, as described in the examples, the enzyme preparation according to the present invention can improve the texture in, for example, condensed milk.

[0149] While not wishing to be bound by theory, the secondary activities of lactase (e.g., lipase activity, protease activity, amylase activity, mannanase activity, pectinase activity, cellulase activity, and / or p-nitrobenzyl esterase activity) affect the flavor of milk-based substrates or dairy products, for example, reducing the taste of dairy products. A reduction in the secondary activity of lactase then, for example, reduces the off-odor level of milk-based substrates or dairy products.

[0150] Enzyme preparations can be advantageously used in dairy products to hydrolyze lactose without forming off-odor compounds. Lactase can be either intracellularly produced lactase or extracellularly produced lactase. In a preferred embodiment, lactase is intracellularly produced lactase. Extracellular lactases are also described. Since they contain a peptide sequence called a leader sequence, they are generally recognized as extracellular enzymes. This leader sequence is recognized in some way by the cells producing the enzyme as a signal that the enzyme should be excreted extracellularly. During secretion, the leader sequence is usually removed. Extracellular lactases are described in various species (e.g., Aspergillus oryzae). Crude preparations of extracellular lactases are characterized by the absence of intracellular enzymes and the presence of typical extracellular enzymes such as proteases. The types of extracellular enzymes found vary by organism and are typical for that organism. Low levels of intracellular enzymes may be found in such extracellular enzyme preparations due to cell lysis during fermentation or processing. Thus, lactase enzymes can be classified as extracellular or intracellular based on a comparison of their amino acid sequences with those of other known lactases. In principle, intracellular lactases can have a leader sequence. This causes the lactase to be excreted from the cell into the medium. Crude preparations of such enzymes are characterized by lactases that are classified as intracellular based on their amino acid sequences, in the presence of typical extracellular enzymes and the absence or low levels of typical intracellular enzymes.

[0151] The enzyme or enzyme preparation can be in any form suitable for the use in question, for example, in the form of a dried powder or granule, non-dusting granule, liquid, stabilized liquid, or protected enzyme.

[0152] An enzyme or enzyme preparation is added in an appropriate amount to achieve the desired degree of lactose hydrolysis under selected reaction conditions. The enzyme can be added at a concentration of about 0.1 to 10 g / L of the milk-based substrate, for example, 0.2 to 2 g / L of the milk-based substrate. In one aspect, the enzyme or enzyme preparation can be added in an amount of about 20 to 300 NLU / g of lactose, for example, about 24 to 240 NLU / g of lactose, per liter of the milk-based substrate.

[0153] As used herein, the terms "low lactose" and "lactose-free" refer to a decrease in the amount of lactose in a milk-based substrate or dairy product after contacting the milk-based substrate or dairy product with lactase.

[0154] In one aspect, the amount of lactose is reduced by at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0155] In one aspect, the lactose is reduced to less than 100 ppm or less than 1000 ppm.

[0156] In certain aspects, the lactose is reduced to less than 100 ppm within 180 minutes at a temperature above 50 °C. In certain aspects, the lactose is reduced to less than 100 ppm within 120 minutes at a temperature above 50 °C.

[0157] Milk-based substrate As used herein, the term "milk" refers to milk secretions obtained by milking any mammal, such as cows, sheep, goats, buffalo, or camels.

[0158] As used herein, the term "milk-based substrate" can be raw milk and / or processed milk substances. In one aspect, the milk-based substrate is a solution / suspension of any milk or milk-like product containing lactose, such as whole milk or low-fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, a solution of milk powder, UHT milk, whey, whey permeate, acid whey, or cream.

[0159] In one aspect, the milk-based substrate is an aqueous solution of milk or skim milk powder.

[0160] The milk-based substrate may be more concentrated than raw milk.

[0161] In one aspect, the milk-based substrate is raw milk that has not been sterilized before contacting with the enzyme.

[0162] In one aspect, the milk-based substrate contains about 3 to 60% lactose. In one aspect, the milk-based substrate contains about 3 to 30% lactose. In one aspect, the milk-based substrate contains about 3 to 16% lactose.

[0163] In one aspect, the milk-based substrate contains about 3 to about 60% lactose before contacting with an enzyme having neutral lactase activity. In one aspect, the milk-based substrate contains about 3 to about 30% lactose before contacting with an enzyme having neutral lactase activity. In one aspect, the milk-based substrate contains about 3 to about 16% lactose before contacting with an enzyme having neutral lactase activity.

[0164] In one aspect, the milk-based substrate has a protein-to-lactose ratio of at least 0.2, preferably at least 0.3, at least 0.4, at least 0.5, at least 0.6, or most preferably at least 0.7.

[0165] The milk-based substrate can be homogenized and sterilized by methods known in the art.

[0166] As used herein, the term "homogenize" means to mix intensively to obtain a soluble suspension or emulsion. It can be carried out such that the milk fat is broken down to a size so small that it no longer separates from the milk. This can be achieved by passing the milk through a small orifice under high pressure.

[0167] As used herein, the term "sterilize" means reducing or eliminating the presence of living organisms such as microorganisms in a milk-based substrate. In one aspect, sterilization is achieved by maintaining a specific temperature over a specific period of time. The specific temperature is usually achieved by heating. The temperature and duration can be selected to kill or inactivate specific bacteria such as harmful bacteria and / or inactivate enzymes in the milk. A rapid cooling step can then follow.

[0168] In one aspect, a milk-based substrate with a reduced lactose content as described herein is provided. A milk-based substrate with a reduced lactose content can be produced by the methods described herein.

[0169] Dairy product The present invention encompasses dairy products obtainable or obtained by any of the methods described herein.

[0170] As used herein, the term "dairy product" can be any food product in which one of the main components is milk-based. In one aspect, the main component is milk-based. In one aspect, the main component is a milk-based substrate that has been treated with an enzyme having neutral lactase activity by the method of the present invention. As used herein, the term "one of the main components" means a component having a dry matter content exceeding 20%, 30% or 40% of the total dry matter of the dairy product. As used herein, the term "main component" means a component having a dry matter content exceeding 50%, 60% or 70% of the total dry matter of the dairy product.

[0171] As described herein, the present invention enables the production of dairy products with a reduced lactose content. In one aspect, the lactose amount is reduced by at least about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, preferably at least about 97%. In one aspect, the lactose amount in a milk-based substrate is reduced by at least about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, preferably at least about 97%. In one aspect, the lactose amount in a dairy product is reduced by at least about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, preferably at least about 97% compared to the milk-based substrate.

[0172] In one aspect, the dairy product produced by the method described herein contains less than 100 ppm of lactose. In one aspect, the dairy product produced by the method described herein contains less than 1000 ppm of lactose.

[0173] In one aspect, lactose is reduced to less than 100 ppm at a temperature above 50°C within 180 minutes, preferably within about 170, 160, 150, 140, 130, 120, 110, 100 or 90 minutes. In one aspect, about 35 to about 100 mg of enzyme / l of milk can be used. In one aspect, about 0.75 to about 2.2 mg of enzyme / g of lactose can be used.

[0174] The dairy products described herein can be, for example, skim milk, low-fat milk, whole milk, cream, UHT milk, extended shelf-life milk, fermented dairy products, cheese, yogurt, butter, daily spread, buttermilk, acidified milk beverages, sour cream, whey-based beverages, condensed milk, dulce de leche, flavored milk beverages, sweetened condensed milk, powdered milk, reconstituted dairy products, ice cream, ryazhenka, pudding, desserts, or milkshakes. The dairy products can be produced by any method known in the art.

[0175] In one aspect, the dairy product is selected from the group consisting of skim milk, low-fat milk, whole milk, cream, UHT milk, extended shelf-life milk, fermented dairy products, cheese, yogurt, butter, daily spread, buttermilk, acidified milk beverages, sour cream, whey-based beverages, condensed milk, dulce de leche, flavored milk beverages, sweetened condensed milk, powdered milk, reconstituted dairy products, ice cream, ryazhenka, pudding, desserts, and milkshakes. In one aspect, the dairy product is condensed milk, ice cream, or a milkshake. In one aspect, the dairy product is powdered milk or whey powder.

[0176] "Fermented dairy products" should be understood to be any dairy product in which any type of fermentation forms part of the manufacturing process. Examples of fermented dairy products are products such as yogurt, buttermilk, quark, and fromage frais. Another example of a fermented dairy product is cheese. Fermented dairy products can be manufactured by any method known in the art.

[0177] The term "fermentation" means the conversion of carbohydrates into alcohol or acids by the action of microorganisms such as starter cultures. In one aspect, fermentation includes the conversion of lactose to lactic acid.

[0178] In the context of the present invention, "microorganisms" can include any bacterium or fungus capable of fermenting a milk substrate.

[0179] Dairy products can further include non-milk components, such as plant-based components, such as vegetable oils, plant proteins, and / or plant carbohydrates. Dairy products can also further include additives, such as enzymes, flavorings, microbial cultures such as probiotic cultures, salts, sweeteners, sugars, acids, fruits, fruit juices, or any other component known in the art as a component or additive of dairy products.

[0180] Method for reducing lactose In one aspect, the method includes contacting a milk-based substrate with an enzyme having neutral lactase activity at a temperature of about 50°C to about 90°C, preferably about 50°C to about 65°C, more preferably about 55°C to about 60°C.

[0181] In one aspect, the method includes contacting a milk-based substrate with an enzyme having neutral lactase activity as described herein at a temperature selected from about 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 90°C.

[0182] In one aspect, the method includes contacting a milk-based substrate with an enzyme having neutral lactase activity at a temperature of about 55°C or 58°C.

[0183] In one aspect, the temperature is about 55°C. For example, when the dairy product is condensed milk, the temperature can be about 55°C. In one aspect, the temperature can be about 63°C for, for example, about 30 minutes.

[0184] In one aspect, the contacting is carried out for several minutes or several hours, for example about 1 to 60 minutes or about 1 to 5 hours. In one aspect, the contacting is carried out for about 10 minutes to about 4 hours. In one aspect, the contacting is carried out for about 10 minutes to about 3 hours. In one aspect, the contacting is carried out for about 10 minutes to about 2 hours. In one aspect, the contacting is carried out for about 10 minutes to about 1 hour.

[0185] In one aspect, the enzyme having neutral lactase activity is added to the milk-based substrate at a concentration of about 20 to about 300 NLU / g lactose, for example about 24 to about 240 NLU / g lactose, more preferably about 60 to about 70 NLU / g lactose.

[0186] The present invention encompasses a method for producing a low-lactose or lactose-free milkshake, ice cream, reduced milk product, dessert, pudding, condensed milk, sweetened condensed milk, ryazhenka, dulce de leche, or milk-based powder, which includes contacting a milk-based substrate with lactase at a temperature of about 50 to about 65 °C.

[0187] In one aspect, the method includes a sterilization step. As used herein, "sterilizing" means reducing or eliminating the presence of living organisms such as microorganisms in the milk-based substrate. Preferably, sterilization is achieved by maintaining a specific temperature over a specific time. The specific temperature is usually achieved by heating. The temperature and duration can be selected to kill or inactivate specific bacteria such as harmful bacteria and / or inactivate enzymes in the milk. A rapid cooling step can then follow.

[0188] In one aspect, the method includes adding the enzyme having neutral lactase activity after sterilization and / or homogenization of the milk-based substrate. The contacting can occur during cooling to the fermentation temperature. In one aspect, the method includes adding the enzyme having neutral lactase activity before or during sterilization and / or homogenization. In one aspect, sterilization can be performed at about 72 °C. In one aspect, sterilization can be performed for 15 to 30 seconds, such as 10 to 20 seconds, preferably about 15 seconds. In one aspect, sterilization can be performed for about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 seconds.

[0189] In one aspect, about 10, 15, 20, 25, 30, or 35%, preferably about 20 - 30%, more preferably about 20% of lactase activity can remain in the milk-based substrate after sterilization.

[0190] In one aspect, the milk-based substrate is not sterilized. In one aspect, the substrate is raw milk that has not been sterilized prior to contact with the enzyme described herein.

[0191] As used herein, "homogenize" means to mix intensively to obtain a soluble suspension or emulsion. It can be carried out such that the milk fat is broken down to a size where it no longer separates from the milk. This can be achieved by passing the milk under high pressure through a small orifice.

[0192] In one aspect, the contact can be during an evaporation process. In one aspect, the contact can be during a concentration process. In one aspect, the contact is carried out during sterilization.

[0193] host cell The terms "host", "host cell" and "cell" may be used interchangeably herein.

[0194] In one aspect, the host cell is a bacterial expression host.

[0195] In one aspect, the bacterial expression host can express an enzyme having the neutral lactase activity described herein.

[0196] In one aspect, the host comprises a modification of a gene that reduces or eliminates lipase activity. In one aspect, a bacterial expression host that can express an enzyme having neutral lactase activity comprises a genetic modification that reduces or eliminates lipase A activity.

[0197] In one aspect, the genetic mutation comprises a deletion, disruption, or down-regulation of the gene encoding lipase. In one aspect, the modification of the gene comprises a deletion, disruption, or down-regulation of the gene encoding the lipase A polypeptide as described, for example, in SEQ ID NO: 2.

[0198] In one aspect, the host comprises a genetic modification that reduces or eliminates one or more enzyme activities selected from the following: protease, amylase, mannanase, pectinase, cellulase, and p-nitrobenzyl esterase activity.

[0199] In one aspect, the host comprises a genetic modification that reduces or eliminates lipase, preferably lipase A, protease, amylase, mannanase, pectinase, cellulase, and p-nitrobenzyl esterase activity.

[0200] In one aspect, the host comprises a modification of a gene that reduces or eliminates cellulase activity.

[0201] In one aspect, the bacterial expression host is a cell of a Bacillus species.

[0202] In one aspect, the bacterial expression host is selected from the group consisting of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. sonorensis, B. halodurans, B. pumilus, B. lautus, B. pabuli, B. cereus, B. agaradhaerens, B. akibai, B. clarkii, B. pseudofirmus, B. lehensis, B. megaterium, B. coagulans, B. circulans, B. gibsonii, and B. thuringiensis.

[0203] In one aspect, the bacterial expression host is Bacillus subtilis.

[0204] In one aspect, the bacterial expression host contains the nucleic acid molecule described herein. In one aspect, the host expresses an enzyme having the neutral lactase activity described herein.

[0205] Modification, downregulation, or inactivation of a nucleic acid molecule encoding an enzyme having neutral lactase activity can be obtained by RNA interference (RNAi) technology (FEMS Microb. Lett. 237:317 - 324, 2004). More specifically, gene expression in filamentous fungal cells can be reduced or abolished by cloning the same sense and antisense portions of the nucleotide sequence whose expression is to be affected, with a nucleotide spacer in between, in tandem, inserting them into an expression vector, and introducing this expression vector into cells in which double-stranded RNA (dsRNA) can be transcribed and then processed into shorter siRNAs that can hybridize to the target mRNA. After dsRNA is transcribed, target degradation of the affected mRNA can occur by formation of small (21 - 23 nucleotide) siRNA fragments. The disappearance of a specific mRNA can be of various degrees. The RNA interference technology described in WO 2005 / 05672 pamphlet and WO 2005 / 026356 pamphlet can be used to modify, downregulate, or inactivate a nucleic acid molecule encoding an enzyme having neutral lactase activity.

[0206] One aspect is directed to a genetically modified Bacillus host cell capable of expressing / producing one or more lactases of the present disclosure. For example, WO 2016 / 071504 pamphlet discloses recombinant Bacillus subtilis host cells transformed with polynucleotide constructs (e.g., expression constructs) encoding various β-galactosidases. Thus, Bacillus host cells are well known in the art as expression hosts and are particularly suitable host cells for the expression / production of the lactases of the present disclosure.

[0207] In certain embodiments, the present disclosure thus relates to a method of modifying a gene in a Bacillus cell, the modification comprising (a) introducing, substituting, or removing one or more nucleotides in the gene (or its ORF), or introducing, substituting, or removing one or more nucleotides in a regulatory element required for transcription or translation of the gene or its ORF, (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) downregulation of the gene, (f) site-directed mutagenesis, and / or (g) random mutagenesis.

[0208] Thus, the modified Bacillus cells of the present disclosure are constructed by decreasing or eliminating the expression of a gene encoding any of the secondary activities described herein using methods well known in the art, such as insertion, disruption, exchange, deletion, truncation, substitution, frameshift mutation, etc. The gene portion to be modified or inactivated can be, for example, a coding region or a regulatory / control element required to express the coding region.

[0209] Examples of such regulatory or control sequences can be a promoter sequence or a functional portion thereof (i.e., a portion that sufficiently affects the expression of a nucleic acid sequence). Other control sequences for modification include, but are not limited to, leader sequences, propeptide sequences, signal sequences, transcription terminators, transcription activators, and the like.

[0210] In certain other embodiments, the modified Bacillus cells are constructed by gene deletions that abolish or reduce gene expression. Gene deletion techniques allow for the partial or complete removal of genes, thereby abolishing their expression or expressing non-functional (or reduced activity) protein products. In such methods, gene deletions can be achieved by homologous recombination using plasmids constructed such that the 5' and 3' regions flanking the gene are included adjacent to each other. The adjacent 5' and 3' regions can be introduced into Bacillus cells, for example, in a temperature-sensitive plasmid such as pE194 that associates with a second selectable marker at a permissive temperature that allows the plasmid to be established intracellularly. The cells are then shifted to a non-permissive temperature to select for cells that have the plasmid integrated into the chromosome at one of the homologous flanking regions. Selection for plasmid integration is achieved by selection for the second selectable marker. After integration, the recombination event at the second homologous flanking region is facilitated by shifting the cells to the permissive temperature for several generations without selection. The cells are plated to obtain single colonies, and these colonies are tested for the loss of both selectable markers (see, for example, Perego, 1993). Thus, one of ordinary skill in the art can readily identify (e.g., by reference to the p-nitrobenzyl esterase gene (nucleic acid) sequence and its encoded protein sequence) nucleotide regions in the coding and / or non-coding sequences of genes suitable for complete or partial deletions.

[0211] In other aspects, the modified Bacillus cells of the present disclosure are constructed by introducing, substituting, or removing one or more nucleotides within a gene or regulatory element required for transcription or translation of that gene. For example, nucleotides can be inserted or removed to introduce a stop codon, remove a start codon, or cause a frameshift in the open reading frame. Such modifications can be achieved by site-directed mutagenesis or PCR-generated mutagenesis according to methods known in the art (see, for example, Botstein and Shortle, 1985; Lo et al, 1985; Higuchi et al, 1988; Shimada, 1996; Ho et al, 1989; Horton et al, 1989, and Sarkar and Sommer, 1990). Thus, in certain embodiments, the >-nitrobenzyl esterase gene of the present disclosure is inactivated by complete or partial deletion.

[0212] In another aspect, the modified Bacillus cells are constructed by the process of gene conversion (see, for example, Iglesias and Trautner, 1983). For example, in the gene conversion method, the nucleic acid sequence corresponding to a gene is mutated in vitro to generate a defective nucleic acid sequence, and then this nucleic acid sequence is transformed into a parental Bacillus cell to generate a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous gene. The defective gene or gene fragment may also desirably encode a marker that can be used for selection of transformants containing the defective gene. For example, the defective gene can be introduced by a non-replicating plasmid or a temperature-sensitive plasmid associated with a selectable marker. Selection for plasmid integration is achieved by selecting for that marker under conditions that do not permit plasmid replication. Selection for the second recombination event that results in gene replacement is achieved by examining colonies for loss of the selectable marker and acquisition of the mutant gene (Perego, 1993). Alternatively, the defective nucleic acid sequence may contain insertions, substitutions or deletions of one or more nucleotides of the gene, as described below.

[0213] In other embodiments, the modified Bacillus cell is constructed by established antisense techniques that use nucleotide sequences complementary to the nucleic acid sequence of the gene (Parish and Stoker, 1997). More specifically, the expression of a gene by a Bacillus cell can be decreased (downregulated) or abolished by introducing a nucleotide sequence complementary to the nucleic acid sequence of this gene, which can be transcribed intracellularly and hybridize to the mRNA produced intracellularly. Under conditions where the complementary antisense nucleotide sequence can hybridize to the mRNA, the amount of protein translated from the gene is decreased or eliminated. Such antisense methods include, but are not limited to, RNA interference (RNAi), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, etc., all of which are well known to those skilled in the art.

[0214] In other embodiments, the modified Bacillus cells are created / constructed by CRISPR-Cas9 editing. For example, a gene encoding any of the para-activities referred to herein can be disrupted (or deleted or downregulated) by a nucleic acid-guided endonuclease that binds to a guide RNA (e.g., Cas9) and Cpf1 or guide DNA (e.g., NgAgo) that recruits an endonuclease to a target sequence on the DNA to find their target DNA. Here, the endonuclease can generate single-stranded or double-stranded breaks in the DNA. This targeted DNA cleavage serves as a substrate for DNA repair and can recombine with an editing template provided to disrupt or delete the gene. For example, a gene encoding a nucleic acid-guided endonuclease (for this purpose, Cas9 from S. pyogenes), or a codon-optimized gene encoding Cas9 nuclease, is operably linked to a promoter active in Bacillus cells and a terminator active in Bacillus cells, thereby generating a Bacillus Cas9 expression cassette. Similarly, one or more target sites specific to the gene are readily identified by those skilled in the art. For example, to construct a DNA construct encoding a gRNA directed to a target site within a gene of interest, the variable targeting (VT) domain has nucleotides that are fused to DNA encoding the Cas9 endonuclease recognition domain (CER) for Cas9 from S. pyogenes and will include the nucleotides of the target site that are 5' of the (PAM) protospacer adjacent motif (TGG). The combination of the DNA encoding the VT domain and the DNA encoding the CER domain thereby generates the DNA encoding the gRNA. Thus, a Bacillus expression cassette for the gRNA is made by operably linking the DNA encoding the gRNA to a promoter active in Bacillus cells and a terminator active in Bacillus cells.

[0215] In still other embodiments, the modified Bacillus cells are constructed by random mutagenesis or site-directed mutagenesis using methods well known in the art, such as, but not limited to, chemical mutagenesis (see, e.g., Hopwood, 1970) and transposition (see, e.g., Youngman et al., 1983). Gene modification can be carried out by mutagenizing the parental cells and screening for mutant cells in which gene expression is reduced or absent. Mutagenesis can be either site-directed or random, and can be carried out, for example, by using a suitable physical or chemical mutagen, by using a suitable oligonucleotide, or by subjecting a DNA sequence to PCR-generated mutagenesis. Further, mutagenesis can be carried out by arbitrarily combining these mutagenesis methods.

[0216] Examples of suitable physical or chemical mutagens for the purposes of the present invention include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, ethyl methanesulfonate (EMS), sodium bisulfite, formic acid, and nucleotide analogs. When using such agents, mutagenesis is generally carried out by incubating the parental cells to be mutagenized in the presence of a suitable mutagen under suitable conditions, and selecting mutant cells that show a decrease in gene expression or that do not show gene expression.

[0217] In other aspects, the modified Bacillus cells contain disruption of an endogenous gene, and the polynucleotide disruption cassette contains a marker gene.

[0218] WO 2003 / 083125 discloses methods for modifying Bacillus cells, such as the generation of Bacillus deletion strains and DNA constructs using PCR fusion to bypass E. coli.

[0219] International Publication No. WO 2002 / 14490 pamphlet discloses a method for modifying Bacillus cells including: (1) construction and transformation of an integration plasmid (pComK); (2) random mutagenesis of a coding sequence, a signal sequence and a propeptide sequence; (3) homologous recombination; (4) increasing transformation efficiency by adding non-homologous flanks to the transforming DNA; (5) optimizing double crossover integration; (6) site-directed mutagenesis; and (7) markerless deletion.

[0220] Those skilled in the art are well aware of suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., E. coli and Bacillus species) (e.g., Ferrari et al, 1989; Saunders et al, 1984; Hoch et al, 1967; Mann et al, 1986; Holubova, 1985; Chang et al, 1979; Vorobjeva et al, 1980; Smith et al, 1986; Fisher et.al, 1981; and McDonald, 1984). In fact, methods such as transformation including protoplast transformation and conjugation, transduction, and protoplast fusion are known and suitable for use in the present disclosure. The transformation method is particularly preferred for introducing the DNA constructs of the present disclosure into host cells.

[0221] In addition to the commonly used methods, in some embodiments, the host cell is directly transformed (i.e., no intermediate cells are used to amplify the DNA construct prior to introduction into the host cell or for other processing). The introduction of the DNA construct into the host cell includes those physical and chemical methods known in the art for introducing DNA into the host cell without inserting it into a plasmid or vector. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, liposome methods, and the like. In a further embodiment, the DNA construct is co-transformed with the plasmid without being inserted into the plasmid. In yet another embodiment, the selectable marker is deleted or substantially excised from the modified Bacillus strain by methods known in the art (e.g., Stahl et al, 1984 and Palmeros et al, 2000). In some embodiments, the degradation of the vector derived from the host chromosome leaves the flanking regions within the chromosome while removing the native chromosomal region.

[0222] In one embodiment, a method for expressing an enzyme described herein is provided, which includes providing a host cell described herein, expressing the enzyme from the cell, and optionally purifying and / or concentrating the enzyme. In one embodiment, an enzyme produced by such a method is provided. Also provided is a dairy product comprising the expression host described herein or the enzyme described herein produced in the expression host.

[0223] Nucleic Acids and Vectors In one embodiment, the present invention relates to an isolated enzyme (polypeptide) having the above-described neutral lactase activity, which polypeptide hybridizes under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions to i) a nucleic acid sequence encoding the mature polypeptide of SEQ ID NO: 1, ii) the cDNA sequence of i), or iii) a polynucleotide encoding a polynucleotide that hybridizes to the complementary strand of i) or ii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York). A subsequence of the nucleic acid sequence contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Furthermore, the subsequence may encode a polypeptide fragment having neutral lactase activity.

[0224] Using the nucleotide sequences or subsequences thereof described herein, and the amino acid sequence of SEQ ID NO: 1 or fragments thereof, nucleic acid probes can be designed to identify and clone DNA encoding polypeptides having neutral lactase activity from strains of various genera or species according to methods well known to those skilled in the art. In particular, such probes can be used for hybridization according to standard Southern blotting methods for the purpose of identifying and isolating the corresponding gene in the genome or cDNA of the genus or species of interest. Such probes may be considerably shorter than the entire array, but must be at least 14 nucleotides in length, preferably at least 25 nucleotides, more preferably at least 35 nucleotides, and most preferably at least 70 nucleotides. However, the nucleic acid probe is preferably at least 100 nucleotides in length. For example, the nucleic acid probe can be at least 200 nucleotides in length, preferably at least 300 nucleotides, more preferably at least 400 nucleotides, or most preferably at least 500 nucleotides. Even longer probes can be used, for example, nucleic acid probes that are at least 600 nucleotides in length, preferably at least 700 nucleotides, more preferably at least 800 nucleotides, or most preferably at least 900 nucleotides. Both DNA probes and RNA probes can be used. These probes are usually labeled (e.g., 32 P, 3 H, 35 S, biotin, or avidin). Such probes are encompassed by the present invention.

[0225] Therefore, genomic DNA libraries prepared from such other organisms can be hybridized with the above-described probes and screened for DNA encoding a polypeptide having lactase activity. Genomic or other DNA from such other organisms can be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the library or the separated DNA can be transferred and immobilized on nitrocellulose or other suitable carrier materials. Carrier materials are used in Southern blots to identify clones or DNA homologous to the nucleic acid sequences or subsequences described herein.

[0226] For the purposes of the present invention, hybridization indicates that a nucleotide sequence hybridizes to a corresponding labeled nucleic acid probe under ultra-low to ultra-high stringency conditions to the nucleotide sequences described herein, their complementary strands, or subsequences thereof. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using X-ray film.

[0227] In another preferred embodiment, the nucleic acid probe is the mature polypeptide coding region of the nucleic acid sequence.

[0228] For probes at least 100 nucleotides in length, ultra-low to ultra-high stringency conditions are defined as prehybridization and hybridization at 42 °C in 5× SSPE, 0.3% SDS, 200 μg / mL sheared and denatured salmon sperm DNA, and 25% formamide for ultra-low and low stringency, 35% formamide for medium and medium-high stringency, or 50% formamide for high and ultra-high stringency, optimally following a standard Southern blotting procedure for 12 - 24 hours.

[0229] For long probes at least 100 nucleotides in length, the carrier material is finally washed three times for 15 minutes each with 2× SSC, 0.2% SDS, preferably at least 45 °C (ultra-low stringency), more preferably at least 50 °C (low stringency), more preferably 55 °C (medium stringency), more preferably at least 60 °C (medium-high stringency), even more preferably at least 65 °C (high stringency), and most preferably at least 70 °C (ultra-high stringency).

[0230] In certain embodiments, washing is performed using 0.2× SSC, 0.2% SDS, preferably at least 45°C (ultra-low stringency), more preferably at least 50°C (low stringency), more preferably 55°C (medium stringency), more preferably at least 60°C (medium-high stringency), even more preferably at least 65°C (high stringency), and most preferably at least 70°C (ultra-high stringency). In another certain embodiment, washing is performed using 0.1× SSC, 0.2% SDS, preferably at least 45°C (ultra-low stringency), more preferably at least 50°C (low stringency), more preferably at least 55°C (medium stringency), more preferably at least 60°C (medium-high stringency), even more preferably at least 65°C (high stringency), and most preferably at least 70°C (ultra-high stringency).

[0231] For short probes that are about 15 nucleotides to about 70 nucleotides in length, the stringency conditions are calculated using the calculations by Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris HCl (pH 7.6), 6 mM EDTA, 0.5% NP-40, 1× Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM monobasic sodium phosphate, 0.1 mM ATP, and 0.2 mg / mL yeast RNA according to standard Southern blotting procedures m and are defined as prehybridization, hybridization, and post-hybridization washes at about 5°C to about 10°C below T

[0232] For short probes that are about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6× SCC + 0.1% SDS for 15 minutes and m washed twice for 15 minutes each using 6× SSC at 5°C to 10°C below T

[0233] Under salt-containing hybridization conditions, effective T m controls the degree of identity required between the probe and the filter-bound DNA for successful hybridization. Effective T m can be determined using the following formula to determine the degree of identity required for two DNAs to hybridize under various stringency conditions.

[0234] Effective T m = 81.5 + 16.6(log M[Na + ) + 0.41(G + C%) - 0.72(formamide%)

[0235] Under medium stringency, formamide is 35% and the Na + concentration for 5× SSPE is 0.75 M.

[0236] Another related relationship is that 1% mismatch of two DNAs decreases T m by 1.4 °C. To determine the degree of identity required for two DNAs to hybridize under medium stringency conditions of 42 °C, the following formula is used.

[0237] Homology (%) = 100 - [(effective T m - hybridization temperature) / 1.4]

[0238] (See www.ndsu.nodak.edu / instruct / mcclean / plsc731 / dna / dna6.htm)

[0239] The polynucleotide diversity includes polynucleotides having a sequence identity of a specific percentage, such as 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% with the nucleic acid encoding SEQ ID NO: 1. In one aspect, a nucleic acid capable of encoding the enzyme (polypeptide) disclosed herein is provided. In another aspect, the nucleic acid has a nucleic acid sequence that is at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% identical to the nucleic acid capable of encoding the enzyme (polypeptide) disclosed herein.

[0240] In one aspect, a plasmid containing the nucleic acid described herein is provided.

[0241] In one aspect, an expression vector containing the nucleic acid described herein or capable of expressing the polypeptide described herein is provided.

[0242] Nucleic acids complementary to the nucleic acids encoding any of the polypeptide diversities defined herein and described herein are provided. Further, nucleic acids capable of hybridizing to this complement are provided. In another aspect, the sequences for use in the methods and compositions described herein are synthetic sequences. It includes, but is not limited to, sequences created using optimal codons for expression in host organisms such as yeast.

[0243] The polypeptide diversities provided herein can be produced synthetically or by recombinant expression in host cells according to procedures well known in the art. In one aspect, the polypeptides disclosed herein are recombinant polypeptides. The expressed polypeptide diversities defined herein are optionally isolated prior to use.

[0244] In another aspect, the polypeptide variants defined herein are purified after expression. Methods for genetic modification and recombinant production of polypeptide variants are described, for example, in U.S. Patent Nos. 7,371,552, 7,166,453, 6,890,572, and 6,667,065; and U.S. Patent Application Publication Nos. 2007 / 0141693, 2007 / 0072270, 2007 / 0020731, 2007 / 0020727, 2006 / 0073583, 2006 / 0019347, 2006 / 0018997, 2006 / 0008890, 2006 / 0008888, and 2005 / 0137111. The polynucleotide sequences encoding the polypeptides, primers, vectors, selection methods, host cells, purification and refolding of the expressed polypeptide variants, and the buffers, pH ranges, Ca 2+ concentrations, substrate concentrations, and enzyme concentrations useful for enzyme assays, including the characterization of the polypeptide variants defined herein, are incorporated herein by reference in their relevant teachings.

[0245] Nucleic acid sequences encoding the protein of SEQ ID NO: 1, or nucleic acid sequences having at least about 66%, 68%, 70%, 72%, 74%, 78%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleic acid encoding the enzyme of SEQ ID NO: 1 are provided. In one aspect, the nucleic acid sequence has at least about 60%, 66%, 68%, 70%, 72%, 74%, 78%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid encoding the enzyme of SEQ ID NO: 1.

[0246] In one aspect, the present invention relates to a vector comprising a polynucleotide. In one aspect, a bacterial cell contains the vector. In some aspects, a DNA construct comprising a nucleic acid encoding a polypeptide is introduced into a host cell in an expression vector that includes regulatory sequences operably linked to the encoding sequence. The vector can be any vector that can be integrated into the fungal host cell genome and replicated when introduced into the host cell. The FGSC strain catalog of the University of Missouri lists suitable vectors. Further examples of suitable expression vectors and / or integrative vectors are presented in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd rd, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001); Bennett et al., More Gene Manipulations in Fungi, Academic Press, San Diego (1991), pp. 396-428; and U.S. Patent No. 5,874,276. Exemplary vectors include pFB6, pBR322, PUC18, pUC100 and pENTR / D, pDON TM 201, pDONR TM 221, pENTR TM , pGEM®3Z and pGEM®4Z. Examples for use in bacterial cells include pBR322 and pUC19, which permit replication in E. coli, and pE194, which permits replication, for example, in Bacillus.

[0247] In some embodiments, the nucleic acid encoding the manifold is operably linked to a suitable promoter that enables transcription in the host cell. The promoter may be derived from a gene encoding a protein that is homologous or heterologous to the host cell. Suitable non-limiting examples of promoters include the cbh1, cbh2, egl1, and egl2 promoters. In one embodiment, the promoter is a promoter that is native to the host cell. For example, when P. saccharophila is the host, the promoter is the native P. saccharophila promoter. An "inducible promoter" is a promoter that is active under environmental or developmental regulation. In another embodiment, the promoter is a promoter that is heterologous to the host cell.

[0248] In some embodiments, the coding sequence is operably linked to a DNA sequence encoding a signal sequence. In another embodiment, a representative signal peptide can be the native signal sequence of the Bacillus subtilis aprE precursor. In other embodiments, the DNA encoding the signal sequence is replaced with a nucleotide sequence encoding a signal sequence from another extracellular Bacillus subtilis precursor. In one embodiment, the polynucleotide encoding the signal sequence is immediately upstream and in-frame with the polynucleotide encoding the polypeptide. The signal sequence can be selected from the same species as the host cell.

[0249] In a further embodiment, the signal sequence and promoter sequence, including the DNA construct or vector to be introduced into the fungal host cell, are derived from the same source. In some embodiments, the expression vector also includes a termination sequence. In one embodiment, the termination sequence and the promoter sequence are derived from the same source. In another embodiment, the termination sequence is homologous to the host cell.

[0250] In some embodiments, the expression vector includes a selectable marker. Examples of suitable selectable markers include markers that confer resistance to antimicrobial agents such as hygromycin or phleomycin. Nutritional selectable markers are also suitable, including amdS, argB, and pyr4. In one embodiment, the selectable marker is the amdS gene that encodes the enzyme acetamidase, which enables transformed cells to grow using acetamide as a nitrogen source. The use of the A. nidulans amdS gene as a selectable marker is described in Kelley et al., EMBO J. 4:475-479 (1985) and Penttila et al., Gene 61:155-164 (1987).

[0251] A suitable expression vector containing a DNA construct comprising a polynucleotide encoding a variant may be any vector that can self-replicate within a given host organism or can be integrated into the host's DNA. In some embodiments, the expression vector is a plasmid. In some embodiments, two types of expression vectors are contemplated for obtaining gene expression. The first expression vector contains DNA sequences in which all of the promoter, coding region, and terminator therein are derived from the gene to be expressed. In some embodiments, gene truncation is obtained by deleting unwanted DNA sequences in order to leave the domain to be expressed under the control of its own transcriptional and translational regulatory sequences. The second type of expression vector is pre-assembled and contains sequences required for high-level transcription and a selectable marker. In some embodiments, the coding region for a gene or a portion thereof is inserted into this versatile expression vector such that it is under the transcriptional control of the expression construct promoter and terminator sequences. In some embodiments, the gene or a portion thereof is inserted downstream of the strong cbhl promoter.

[0252] In yet another aspect, a method of expressing a polypeptide described herein includes obtaining a cell or host cell described herein, expressing the polypeptide from the cell or host cell, and optionally purifying the polypeptide.

[0253] The characteristics of expression mean the change in the expression level of the variant when the variant is produced in a particular host cell. Expression generally relates to the amount of active variant that can be recovered from the fermentation broth using standard techniques known in the art over a given period of time. Expression can also relate to the amount or proportion of the variant produced within or secreted by the host cell. Expression can also relate to the rate of translation of the mRNA encoding the variant polypeptide.

[0254] Methods for introducing a DNA construct or vector into a host cell include techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection-mediated and DEAE-dextran-mediated transfection), incubation with calcium phosphate DNA precipitates, high-velocity irradiation with DNA-coated microprojectiles, and protoplast fusion. General transformation techniques are known in the art. See, for example, Ausubel et al. (1987) (supra), chapter 9; Sambrook et al. (2001) (supra); and Campbell et al., Curr. Genet. 16:53-56 (1989). Expression of heterologous proteins in Trichoderma is described, for example, in U.S. Patent No. 6,022,725, U.S. Patent No. 6,268,328, Harkki et al., Enzyme Microb. Technol. 13:227-233 (1991), Harkki et al., Bio Technol. 7:596-603 (1989), European Patent No. 244,234, and European Patent No. 215,594. In one aspect, genetically stable transformants are constructed using a vector system in which a nucleic acid encoding a variant is stably integrated into the host cell chromosome. Transformants are then purified by known techniques.

[0255] In one non-limiting example, stable transformants containing the amdS marker are distinguished from unstable transformants by their faster growth rate and the formation of circular colonies with a smooth rather than a jagged outline on solid culture media containing acetamide. Further, in some examples, additional tests for stability are performed by growing the transformants on solid non-selective media, such as media lacking acetamide, harvesting spores from this culture media, and subsequently determining the proportion of these spores that germinate and grow on selective media containing acetamide. Other methods known in the art can be used to select transformants.

[0256] To evaluate the expression of the variant in the host cell, the assay can measure the expressed protein, the corresponding mRNA, or the β-lactamase activity. For example, suitable assays include Northern blotting and Southern blotting using appropriately labeled hybridization probes, RT-PCR (reverse transcription polymerase chain reaction), and in situ hybridization. Suitable assays also include measuring the activity in the sample. Suitable assays for the activity of the variant include, but are not limited to, ONPG-based assays or determining glucose in the reaction mixture as described, for example, in the methods and examples herein.

[0257] Generally, the variants produced in cell culture are secreted into the culture medium and can be purified or isolated, for example, by removing undesirable components from the cell culture medium. In some examples, the variants can be recovered from cell lysates. In such examples, the enzyme is purified from the produced cells using techniques routinely used by those skilled in the art. Examples include, but are not limited to, affinity chromatography, ion exchange chromatography methods including high resolution ion exchange, hydrophobic interaction chromatography, two-phase partitioning, ethanol precipitation, reverse phase HPLC, silica or cation exchange resin chromatography such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using Sephadex G-75. Depending on the intended use, the polypeptides disclosed herein may be prepared, for example, in lyophilized form or in solution. In one aspect, the polypeptides disclosed herein are in lyophilized form. In another aspect, the polypeptides disclosed herein are in solution. Polypeptide compositions can be prepared according to methods known in the art and they may be in the form of liquid compositions or dry compositions. For example, the polypeptide compositions may be in granular form or in microgranular form. The polypeptides included in this composition can be stabilized according to methods known in the art.

[0258] Preferred examples of the use of the enzyme or enzyme preparation of the present invention are shown below.

[0259] In one aspect, the amount of lactose in a milk-based substrate, such as fresh low-fat milk or skim milk, which typically has from about 4.7 to about 5.0% lactose, or condensed milk or concentrated milk which has from about 6.0 to about 65% lactose, can be reduced by adding a lactase capable of converting lactose to TGOS, i.e., a lactase having transgalactosylation activity. Here, "lactose" is defined as lactose (DP2), as well as monosaccharides (DPI) obtained by hydrolysis of lactose, including glucose and galactose, and other DP2 molecules such as allolactose and DP2 GOS. DP3+ fiber is not a sugar. Thus, treating a milk-based substrate containing a given amount of lactose with a lactase having transgalactosylase activity can reduce the sugar, and lactose is converted to GOS fiber (DP3+).

[0260] According to this aspect of the present disclosure, the milk-based substrate preferably contains 6-9% lactose, 9-20% lactose, 20-40% lactose, or 40-65% lactose. The lactose in the milk-based substrate preferably decreases by more than 20%, more than 25%, more than 30%, and more than 40%.

[0261] In one aspect, provided herein is a method for producing a food product by treating a substrate containing lactose with the enzyme described herein.

[0262] In one aspect, provided herein is a method for producing a dairy product by treating a milk-based substrate containing lactose with the enzyme described herein.

[0263] An enzyme preparation, such as in the form of a food ingredient prepared according to the present disclosure, may be in the form of a solution or in the form of a solid, depending on the mode of use and / or application and / or administration. The solid form can be as a dry enzyme powder or as a granular enzyme.

[0264] Examples of dry enzyme preparations include spray-dried products, mixer granulated products, layered products (e.g., fluidized bed granules), extruded granules or pelletized granules, spheronized products, and freeze-dried products.

[0265] Enzyme preparations, such as the form of food ingredients prepared according to the present disclosure, may be in the form of a solution or in the form of a solid, depending on the use and / or mode of application and / or mode of administration. The solid form can be as a dry enzyme powder or as a granular enzyme.

[0266] In one aspect, there is provided a composition comprising the host cell or enzyme described herein, preferably a food composition, more preferably a dairy product.

[0267] Furthermore, herein, based on the total amount of the enzyme in a composition having at least 70%, such as 72%, 74%, 74%, 78%, 80%, 82%, 84%, 86%, 88%, 90% sequence identity with SEQ ID NO: 1, there is disclosed a composition or preparation comprising at least 5 w / w%, such as about 10 w / w%, 15 w / w%, 20 w / w%, 25 w / w%, 30 w / w%, 35 w / w%, 40 w / w%, 45 w / w%, 50 w / w% of one or more enzymes disclosed herein. This can be evaluated by using the following techniques known to those skilled in the art. The sample to be evaluated is subjected to SDS-PAGE and visualized using a dye suitable for protein quantification, such as the Bio-Rad Criterion system. The gel is then scanned using a suitable densitometer scanner, such as the Bio-Rad Criterion system, to ensure that the resulting image is within the dynamic range. Bands corresponding to any variant / fragment are quantified, and the percentage of the polypeptide is calculated as follows: percentage of the target polypeptide = target polypeptide / (total of all polypeptides showing transgalactosylation activity) × 100. The total number of polypeptide variants / fragments in the composition can be determined by detecting the fragments by Western blotting using polyclonal antibodies by methods known to those skilled in the art.

[0268] In one aspect, the present invention provides an enzyme preparation comprising an enzyme according to the present invention, an enzyme carrier and optionally a stabilizer and / or a preservative.

[0269] In yet another aspect of the present invention, the enzyme carrier is selected from the group consisting of glycerol or water.

[0270] In yet another aspect, the preparation / composition contains a stabilizer. In one aspect, the stabilizer is selected from the group consisting of inorganic salts, polyols, sugars, and combinations thereof. In one aspect, the stabilizer is an inorganic salt such as potassium chloride. In another aspect, the polyol is glycerol, propylene glycol, or sorbitol. In yet another aspect, the sugar is any one of several sweet sugars such as low molecular weight carbohydrates, particularly glucose, galactose, fructose, and sucrose.

[0271] In yet another aspect, the preparation contains a preservative. In one aspect, the preservative is methylparaben, propylparaben, benzoate, sorbate, or other food preservatives, or mixtures thereof.

[0272] The method of the present invention can be carried out using an immobilized enzyme, such as immobilized lactase or other galactooligosaccharide-producing enzymes. The enzyme can be immobilized on any organic or inorganic support. Exemplary inorganic supports include alumina, celite, Dowex-1-chloride, glass beads, and silica gel. Exemplary organic supports include DEAE-cellulose, alginate hydrogel or alginate beads, or equivalents. In various aspects of the present invention, the immobilization of lactase can be optimized by physical adsorption onto an inorganic support. The enzyme used in the practice of the present invention can be immobilized in various media such as water, Tris HCl buffer, and phosphate buffer solution. The enzyme can be immobilized on any type of substrate, for example, on a filter, fiber, column, bead, colloid, gel, hydrogel, mesh, etc.

[0273] In one aspect, a method for producing a dairy product is provided by treating a milk-based substrate containing lactose with the enzymes described herein. In one aspect, a method is provided in which the treatment with the enzymes described herein is carried out at a temperature optimal for the activity of the enzymes, for example at a high temperature described herein.

[0274] In yet another aspect, the polypeptide is added to the milk-based substrate at a concentration of 0.01 to 1000 ppm. In yet another aspect, the polypeptide is added to the milk substrate at a concentration of 0.1 to 100 ppm. In yet another aspect, the polypeptide is added to the milk-based substrate at a concentration of 1 to 60 ppm, such as 1 to 55, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10 or 1 to 5 ppm, preferably 1 to 10 ppm.

[0275] In one aspect, a method is provided that further includes fermenting a substrate such as a dairy product with a microorganism. In yet another aspect, the dairy product is yogurt. In yet another aspect, the treatment with the enzyme and the microorganism is carried out substantially simultaneously. In one aspect, the polypeptide and the microorganism are added to the milk-based substrate essentially simultaneously.

[0276] In one aspect, a dairy product containing the cells or enzymes described herein is provided. In one aspect, the enzyme as defined herein is added at a concentration of 0.01 to 1000 ppm. In one aspect, a dairy product containing the inactivated enzyme as defined herein is provided. In one aspect, a dairy product containing the inactivated enzyme as defined herein at a concentration of 0.01 to 1000 ppm is provided. In one aspect, a dairy product containing the cells as defined herein is provided.

[0277] The dairy products described in this specification can be skim milk, low-fat milk, whole milk, cream, UHT milk, extended shelf-life milk, fermented dairy products, cheese, yogurt, butter, daily spread, buttermilk, acidified milk beverages, sour cream, whey-based beverages, condensed milk, dulce de leche, flavored milk beverages, sweetened condensed milk, powdered milk, reconstituted dairy products, ice cream, ryazhenka, pudding, desserts, and milkshakes. The dairy products can be manufactured by any method known in the art.

[0278] The dairy products can further include non-milk components, such as vegetable components, such as vegetable oils, vegetable proteins, and / or vegetable carbohydrates. The dairy products can also further include additives, such as enzymes, flavoring agents, microbial cultures such as probiotic cultures, salts, sweeteners, sugars, acids, fruits, fruit juices, or any other components known in the art as components or additives of dairy products.

[0279] In one aspect of the present invention, one or more milk components and / or milk fractions account for at least 50 wt / wt%, such as at least 70 wt / wt%, such as at least 80 wt / wt%, preferably at least 90 wt / wt% of the dairy product.

[0280] In one aspect of the present invention, one or more milk-based substrates treated with the enzymes defined herein account for at least 50 wt / wt%, such as at least 70 wt / wt%, such as at least 80 wt / wt%, preferably at least 90 wt / wt% of the dairy product.

[0281] In one aspect of the present invention, the enzyme-treated milk-based substrate is not dried before being used as one of the components in the dairy product.

[0282] In one aspect of the present invention, the dairy product is ice cream. In the context of the present invention, the ice cream can be any type of ice cream, such as high-fat ice cream, low-fat ice cream, or ice cream based on yogurt or other fermented dairy products. The ice cream can be produced by any method known in the art.

[0283] In one aspect of the present invention, the dairy product is milk or condensed milk.

[0284] In one aspect of the present invention, the dairy product is UHT milk. In the context of the present invention, UHT milk is milk that has undergone a sterilization procedure intended to kill any microorganisms, including bacterial spores. The UHT (ultra-high temperature) treatment can be, for example, a heat treatment at 130°C for 30 seconds or at 145°C for 1 second.

[0285] In one aspect of the present invention, the dairy product is ESL milk. In the context of the present invention, ESL milk is milk whose shelf life has been extended by microfiltration and / or heat treatment and that can maintain its freshness for at least 15 days, preferably at least 20 days, on the product shelf at 2 - 5°C.

[0286] In another preferred aspect of the present invention, the dairy product is a fermented dairy product, such as yogurt.

[0287] The microorganisms used for most fermented dairy products are generally selected from the group of bacteria commonly referred to as lactic acid bacteria. As used herein, the term "lactic acid bacteria" designates gram-positive, microaerophilic, or anaerobic bacteria that ferment sugars to produce acids such as lactic acid, acetic acid, and propionic acid, primarily as the main acids produced. The most industrially useful lactic acid bacteria are found among the "Lactobacillales" order, including species of the genus Lactococcus, Streptococcus, Lactobacillus, Leuconostoc, Pseudoleuconostoc, Pediococcus, Brevibacterium, Enterococcus, and Propionibacterium. Furthermore, anaerobic bacteria of the Bifidobacterium genus, i.e., lactic acid-producing bacteria belonging to the group of Bifidobacterium species, which are often used as food cultures either alone or in combination with lactic acid bacteria, are generally included in the group of lactic acid bacteria. Lactic acid bacteria are usually supplied to the dairy industry as frozen or freeze-dried cultures for bulk starter propagation or as so-called "direct vat set" (DVS) cultures intended for direct inoculation into fermentation vessels or vats for the production of fermented dairy products. Such cultures are generally referred to as "starter cultures" or "starters".

[0288] The strains of starter cultures of lactic acid bacteria commonly used are generally divided into mesophilic organisms with an optimal growth temperature of about 30°C and thermophilic organisms with an optimal growth temperature in the range of about 40 - 45°C. Representative organisms belonging to the mesophilic group include Lactococcus lactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Pseudoleuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactobacillus casei subsp. casei, and Lactobacillus paracasei subsp. paracasei. Examples of thermophilic lactic acid bacteria species include Streptococcus thermophilus, Enterococcus faecium, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus acidophilus.In addition, anaerobic bacteria belonging to the genus Bifidobacterium, such as Bifidobacterium bifidum, Bifidobacterium animalis, and Bifidobacterium longum, are also commonly used as dairy starter cultures and are generally included in the group of lactic acid bacteria. Furthermore, species of Propionibacterium are used as dairy starter cultures, particularly in the production of cheese. Additionally, organisms belonging to the genus Brevibacterium are commonly used as food starter cultures.

[0289] Another group of microbial starter cultures is fungal cultures, including yeast cultures and filamentous fungal cultures that are used particularly in the production of specific types of cheese and beverages. Examples of fungi include Penicillium roqueforti, Penicillium candidum, Geotrichum candidum, Torula kefir, Saccharomyces kefir, and Saccharomyces cerevisiae.

[0290] In one aspect of the present invention, the microorganism used for the fermentation of a milk-based substrate is Lactobacillus casei, or a mixture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

[0291] The fermentation process used in the method of the present invention is well known, and a person skilled in the art will know how to select suitable process conditions such as temperature, oxygen, the amount and characteristics of microorganisms, additives such as carbohydrates, flavorings, minerals, enzymes, etc., and the treatment time. Obviously, the fermentation conditions are selected so as to assist in the achievement of the present invention. As a result of fermentation, the pH of the milk-based substrate will decrease. The pH of the fermented dairy product of the present invention can be, for example, in the range of 3.5 to 6, for example in the range of 3.5 to 5, preferably in the range of 3.8 to 4.8.

[0292] In one aspect, provided is the use of the cells disclosed herein for manufacturing a product selected from the group consisting of yogurt, cheese, fermented dairy products, nutritional supplements, and probiotic edible products.

[0293] In one aspect, the enzyme described herein can be used in a method for preparing and manufacturing a cheese product. The cheese product can be selected from the group consisting of, for example, cream cheese, cottage cheese, and processed cheese.

[0294] The treatment of milk products with enzymes that convert lactose into monosaccharides has several advantages. First, the product can be consumed by people with lactose intolerance who would otherwise exhibit symptoms such as flatulence and diarrhea. Second, dairy products treated with lactase will be sweeter than similar untreated products because glucose and galactose are perceived as sweeter than lactose. This effect is particularly interesting for applications such as yogurt and ice cream, where a strong sweetness in the final product is desirable and a reduction in the net carbohydrates in the consumed product is possible. Third, in the manufacture of ice cream, a phenomenon called sanding is often observed, in which lactose molecules crystallize due to the relatively low solubility of lactose. When lactose is converted to monosaccharides, the mouthfeel of the ice cream is much improved compared to the untreated product. The sandy feeling due to lactose crystallization can be eliminated, and the raw material cost can be reduced by replacing skim milk powder with whey powder. The main effect of the enzyme treatment was an increase in sweetness.

[0295] In one aspect, the enzymes disclosed herein can be used together with other enzymes, such as proteases like chymosin or renin, lipases like phospholipase, amylase, transferase, and lactase. In one aspect, the enzymes disclosed herein can be used together with transgalactosylase. This can be particularly useful when it is desirable to reduce the residual lactose, especially at low lactose levels, after treatment with transgalactosylated polypeptides. An enzyme having lactase activity is any glycoside hydrolase having the ability to hydrolyze the disaccharide lactose into its constituent galactose and glucose monomers. The group of lactases includes, but is not limited to, enzymes designated in subclass EC 3.2.1.108. Enzymes designated in other subclasses, such as EC 3.2.1.23, can also be lactases. Lactases can have activities other than lactose hydrolysis activity, such as transgalactosylation activity. In the context of the present invention, the lactose hydrolysis activity of lactase can be referred to as its lactase activity or β-galactosidase activity. Enzymes having lactase activity can be of animal origin, plant origin, or microbial origin. The enzyme can be obtained from microbial origin, particularly from filamentous fungi or fungi, or from bacteria. The enzyme can be, for example, from the genus Agaricus, such as A. bisporus; the genus Ascovaginospora; the genus Aspergillus, such as A. niger, A. awamori, A. foetidus, A. japonicus, A. oryzae; the genus Candida; the genus Chaetomium; the genus Chaetotomastia; the genus Dictyostelium, such as D. discoideum; the genus Kluveromyces, such as K. fragilis, K. lactis; the genus Mucor, such as M. javanicus, M. mucedo(M.Mucedo), M. subtilissimus; Neurospora, for example, N. crassa; Rhizomucor, for example, R. pusillus; Rhizopus, for example, R. arrhizus, R. japonicus, R. stolonifer; Sclerotinia, for example, S. libertiana; Torula; Torulopsis; Trichophyton, for example, T. rubrum; Whetzelinia, for example, W. sclerotiorum; Bacillus, for example, B. coagulans, B. circulans, B. megaterium, B. novalis, B. subtilis, B. pumilus, B. stearothermophilus, B. thuringiensis; Bifidobacterium, for example, B. longum, B. bifidum, B. B. animalis; Chryseobacterium; Citrobacter, e.g., C. freundii; Clostridium, e.g., C. perfringens; Diplodia, e.g., D. gossypina; Enterobacter, e.g., E. aerogenes, E. cloacae; Edwardsiella, E. tarda; Erwinia, e.g., E. herbicola; Escherichia, e.g., E. coli; Klebsiella, e.g., K. pneumoniae; Miriococcum; Myrothesium; Mucor; Neurospora, e.g., N. crassa; Proteus, e.g., P. vulgaris; Providencia, e.g., P. stuartii; Pycnoporus, e.g., Pycnoporus cinnabarinus, Pycnoporus sanguineus; Ruminococcus, e.g., R. torques; Salmonella, e.g., S. typhimurium; Serratia, e.g., S. liquefasciens, S. marcescens; Shigella, e.g., S. flexneri; Streptomyces, e.g., S. antibioticus, S. castaneoglobisporus; S.castaneoglobisporus), S. violeceoruber; Trametes; Trichoderma, e.g., T. reesei, T. viride; Yersinia, e.g., Y. enterocolitica strains. In one embodiment, lactase is an intracellular component of microorganisms such as Kluyveromyces and Bacillus. Kluyveromyces, particularly K. fragilis and K. lactis, and other fungi such as Candida, Torula, and Torulopsis are common sources of fungal lactases, while B. coagulans and B. circulans are well-known sources of bacterial lactases. Some commercially available lactase preparations derived from these organisms, such as Lactozym.RTM. (available from Novozymes, Denmark), HA-Lactase (available from Chr. Hansen, Denmark), and Maxilact.RTM. (available from DSM, the Netherlands) (all derived from K. lactis), are available. These lactases are all so-called neutral lactases with an optimal pH of pH 6 to pH 8.

[0296] In one embodiment of the present invention, a method for producing a lactose-free dairy product from a milk-based substrate is provided, comprising the following steps: a.) providing a milk-based substrate; b.) adding an enzyme having neutral lactase activity to the milk-based substrate; c.) sterilizing the milk-based substrate (wherein the lactase having neutral lactase activity retains a significant amount of activity after the sterilization step); and d.) storing the milk-based substrate for a time sufficient to provide a lactose-free dairy product.

[0297] Preferably, the milk-based substrate has at least 4.7% (w / w) lactose. Preferably, the sterilization step is carried out at 65-75°C. More preferably, the sterilization step is carried out at 70-73°C. Most preferably, the sterilization step is carried out at 72.8°C.

[0298] Preferably, the storage step is 3-10 days. More preferably, the storage step is 6-9 days. Most preferably, the storage step is 8 days.

[0299] Preferably, the enzyme having neutral lactase activity is derived from the genus Lactobacillus. More preferably, the enzyme having neutral lactase activity is derived from Lactobacillus delbrueckii bulgaricus. Even more preferably, the enzyme has at least about 60% identity with SEQ ID NO: 1. Even more preferably, the enzyme has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. In an even more preferred embodiment, the enzyme is an enzyme of SEQ ID NO: 1 or a lactase-active fragment thereof. In other preferred embodiments, the enzyme having neutral lactase activity is purified. Even more preferably, the enzyme having neutral lactase activity is concentrated. Even more preferably, the enzyme having neutral lactase activity is in the form of an enzyme preparation with reduced lipase secondary activity levels. In the most preferred embodiment, the enzyme having neutral lactase activity is in the form of an enzyme preparation with reduced protease, amylase, mannanase, pectinase, cellulase, and / or p-nitrobenzyl esterase secondary activity levels. [Examples]

[0300] The following examples are merely illustrative and in no way limit the present specification.

Examples

[0301] Enzyme Experimental DuPont lactase: a thermostable β-galactosidase derived from Lactobacillus delbrueckii bulgaricus having the amino acid sequence shown in SEQ ID NO: 1. As an example of a hydrolytic lactase derived from Kluyveromyces lactis, GODO-YNL2 (available from DuPont, Denmark) was used. Another example of a hydrolytic lactase derived from Bifidobacterium bifidum is commercially available as Saphera (available from Novozymes, Denmark) or NOLA Fit (available from Chr. Hansen, Denmark). As examples of transgalactosylating lactases, FoodPro GOS of Bifidobacterium bifidum lactase (available from DuPont, Denmark) and Nutribio GOS L of an acidic lactase derived from Aspergillus oryzae (available from DuPont, Denmark) were utilized.

Example

[0302] HPLC Lactose Assay The following method describes the quantification procedure for lactose (and potentially allolactose) in milk samples and other matrices. The samples are derivatized and analyzed by HPLC with UV and FLD detection.

[0303] Chemicals: dimethyl sulfoxide, DMSO (CAS: 67-68-5 Sigma A8418); phosphoric acid, H 3 P0 4 , (CAS: 7664-38-2, Sigma P5811); acetic acid, 99% or more (CAS: 64-19-7, Sigma A6283); sodium phosphate, NaH 2 PO 4Above 99.0% (CAS: 7558-79-4, Sigma S7907); 4-aminobenzoic acid, above 99% (CAS: 150-13-0, Sigma A9878); 2-methylpyridine borane complex solution, 95% (CAS: 3999-38-0, Sigma 654213); tetrabutylammonium bisulfate, above 99.0% (CAS: 32503-27-8, Sigma 86868); lactose (CAS: 10039-26-6, Sigma)

[0304] The derivatization solvent was DMSO / acetic acid (70 / 30 v / v %), and the derivatization reagents 4-aminobenzoic acid and 2-methylpyridine borane complex in the solvent were freshly prepared. The sample solvent was 10 mM Na 2 HPO 4 (H 3 PO 4 (adjusted to pH 2.5 with 85%)).

[0305] An Agilent 1100 modular HPLC system equipped with an on-line vacuum degassing device, pump, autosampler, column temperature control, diode array detector and fluorescence detector was used. Agilent Technologies OpenLAB CDS software was used for quantification. Column: Knauer Prontosil RP-C18 SH (150×4.6 mm, 3 µm) from Mikrolab (KN 15EF180PSG / 15VH185PSJ). Tetrabutylammonium hydrogen sulfate was used as the ion pair reagent in the eluent system. The injection volume was 20 µL, the column temperature was 20 °C, isocratic: MP A flow rate: 0.8 mL / min, A: 20 mM tetrabutylammonium bisulfate (pH 2.0) for the washing program, B% acetonitrile-containing 10 mM sodium phosphate buffer, washed with 50 / 50 v / v% acetonitrile / water between each injection column, pressure: 250 bar, analysis time 100 min, fluorescence Ex: 313 nm and Em: 358 nm, diode array detector absorbance 303 nm. The lactose used as the calibration standard was prepared in ddH 2 O in the range of 5 - 500 mg / L.

[0306] Sample preparation: L - arabinose (75 mg / L) prepared in 10 mM NaH 2 PO 4 (pH 2.5) was used as the internal standard for all samples. For lactose - free milk and cream, and standards, 200 mL of the sample / standard was transferred to 1.5 mL Eppendorf tubes, 400 mL of the sample solvent was added, and mixed. This mixture was centrifuged at maximum speed for 10 minutes (Labnet centrifuge), and 200 mL of the supernatant was transferred to 2 mL Eppendorf tubes. For lactose - free yogurt, Skyr and Quarg, the following preparation was used: Approximately 1 g of the fermented sample was weighed into 1.5 mL Eppendorf tubes. The sample was centrifuged at maximum speed for 10 minutes (Labnet centrifuge). 300 μL of the sample was transferred to 1.5 mL Eppendorf tubes, 600 μL of the sample solvent was added, and mixed. This solution was centrifuged at maximum speed for 10 minutes, and 200 μL of the supernatant was transferred to 1.5 mL Eppendorf tubes. Subsequently, the samples were derivatized: 200 μL of the derivatization reagent was added to 2 mL Eppendorf tubes containing 200 mL of the sample. The tubes were set in a Thermomixer at 60 °C and reacted at 750 rpm for 30 minutes. After 30 minutes, the tubes containing the reaction mixture were removed from the Thermomixer, and 25 μL of the reaction mixture was mixed with 225 μL of the mobile phase in a microtiter plate. This solution was filtered by centrifuging in a microtiter plate equipped with a filter. The plate was finally sealed, and then HPLC analysis was performed. The lactose concentration in dairy product samples (including milk) was calculated by calibration standards and internal controls.

Example

[0307] Determination of neutral lactase activity

[0308] The lactase activity is determined in units of NLU / g using the following method. It is applicable to the determination of lactase activity of 2000 - 5000 neutral lactase units (NLU) / g in enzyme preparations derived from Kluyveromyces lactis and Saccharomyces species. The principle of this assay method is that lactase hydrolyzes o-nitrophenyl-β-D-galactopyranoside (ONPG) into o-nitrophenol (ONP) and galactose at 30 °C and pH 6.5. The reaction is stopped by adding sodium carbonate, and the released ONP is measured at 420 nm with a spectrophotometer or a colorimeter. 1 NLU / g is defined as the amount of enzyme that releases 1.30 mM of o-nitrophenol per minute under the assay conditions.

[0309] The following reagents were prepared: 0.1 M magnesium solution. (By quantitatively transferring 24.65 g of MgSO 4 ·7H 2 O (Sigma-aldrich) into a 1 L volumetric flask). 5 mM EDTA solution (1.86 g of Na 2 EDTA (C 10 H 14 N 2 Na 2 O 8 。2×H 2 O) was quantitatively transferred into a 1 L volumetric flask, dissolved in a small amount of distilled water, and then diluted to volume). 0.1 M PEM buffer: Phosphate buffer. - pH 6.5. 8.8 g of KH 2 PO 4 and 8 g of K 2 HPO 4 .3H 2 O was quantitatively transferred into a 1 L volumetric flask and dissolved in about 900 mL of H 2 O. 10 mL of the Mg solution and 10 mL of the EDTA solution were added. Diluted with distilled water and mixed. If necessary, adjust the pH with aqueous KOH solution (56 g / L) or 10% H 3 PO 4 solution before use. Na 2 CO 3 Stop solution - 50 g of Na 2 CO3 and 37.2 g of Na 2 Dissolve EDTA in 1000 ml of distilled water. Dissolve 250 mg of ONPG substrate (Mw: 301.55 g / mol, Sigma-aldrich #N1127) up to 100 ml in a volumetric flask containing buffer. ONP standard solutions - 0.02, 0.04, 0.06, 0.08, 0.10, 0.12 and 0.14 μM ONP / mL are prepared as follows; Transfer 139 mg of ONP (2-o-nitrophenol, Sigma-aldrich #33444-25G purity >99%, LC grade) to a 1 L volumetric flask and dissolve in 10 mL of 95% ethanol. H 2 Dilute to volume with H 2 O and mix. From this solution, pipette 2, 4, 6, 8, 10, 12 and 14 mL aliquots into separate 100 mL volumetric flasks. Add 25 mL of Na 3 solution to each flask and dilute to volume with buffer.

[0310] Assay procedure: Weigh out the lactase enzyme in at least 1 g aliquots twice, dissolve in buffer to contain 0.027 - 0.095 NLU in 1 mL of the final dilution. Record the total dilution volume. Equilibrate the ONPG substrate at 30 °C for (at least) 10 minutes. Pipette two 1 mL aliquots from each final dilution (sample and blank sample) into separate 15 x 150 mm test tubes. Place the test tubes and blank buffer (not the blank sample) in a 30 °C water bath and equilibrate exactly for 5 minutes starting at zero time. Add 5 ml of equilibrated ONPG substrate at 1 minute intervals (or 15 second intervals) (and mix by shaking and start the stopwatch when adding the substrate). After 10 minutes of incubation, stop the reaction and mix in the same order at 1 minute (or 15 second) intervals by adding 2 mL of Na 2 CO 3 solution (or 1 mL if scaling down the volume). Blank sample: 2 mL of Na 2 CO 3Add the solution (or 1 mL if scaling down the volume) to the blank sample. Mix by shaking and add 5 mL of ONPG substrate. Within 30 minutes, measure the OD420 of the sample, blank sample, blank buffer, and ONP standard, using distilled water as the blank, for example, in a cuvette. For each standard solution, plot the OD420 against the μM of ONP. A straight line passing through the origin must be obtained. Divide the absorbance of each standard solution by the μM of ONP to determine the absorbance rate a for each concentration and the average calculated value. A value of 4.65 + / - 0.05 will be obtained. If this value is not obtained, repeat the analysis using freshly prepared reagents.

[0311] Calculate the neutral lactase activity in the test sample as follows:

Number

Example

[0312] - Method for protein quantification

[0313] Protein quantification by Stain Free Imager Criterion Protein was quantified by densitometry using SDS-PAGE gels and a Gel Doc™ EZ imaging system. Reagents used in this analysis: Concentrating (2×) Laemmli Sample Buffer (Bio-Rad, catalog number 161-0737); 26-well XT4-12% Bis-Tris Gel (Bio-Rad, catalog number 345-0125); Protein Marker “Precision Plus Protein Standards” (Bio-Rad, catalog number 161-0363); Protein standard BSA (Thermo Scientific, catalog number 23208) and SimplyBlue Safestain (Invitrogen, catalog number LC 6060). The analysis was performed as follows: In a 96-well PCR plate, 50 μL of diluted enzyme sample was mixed with 50 μL of sample buffer containing 2.7 mg of DTT. The plate was sealed with a Microseal “B” Film from Bio-Rad and placed in a PCR machine and heated to 70°C for 10 minutes. Then, the chamber was filled with running buffer and the gel cassette was set. Next, 10 μL of each sample and standard (0.125 - 1.00 mg / ml BSA) were placed on the gel and 5 μL of marker was added. Then, electrophoresis was performed at 200 V for 45 minutes. Following electrophoresis, the gel was rinsed three times with water for 5 minutes each, then stained overnight in Safestain and finally destained with water. Then, the gel was transferred to the Imager. Image Lab software was used to calculate the intensity of each band. A calibration curve was generated using BSA (Thermo Scientific, catalog number 23208). The amount of target protein was determined by band intensity and the calibration curve. Enzyme samples for subsequent examples were prepared using the protein quantification method.

Example

[0314] Generation of a lactase-expressing Bacillus host cell genetically modified to lack lipA of secreted lipase

[0315] The Bacillus subtilis lipA gene encoding secreted lipase was deleted in Bacillus host cells free of p-nitrobenzyl esterase, cellulase, pectinase, amylase, protease, and mannanase activities using CRISPR technology as described in WO 2018 / 187524 pamphlet. Plasmid pRF827 containing the Cas9 Y155H variant expression cassette was constructed as described in WO / US18 / 026170 pamphlet.

[0316] Construction of the pCAS-lipA plasmid (Figure 1): Plasmid pRF827 was amplified and the serA target site in pRF827 was replaced with the lipA target site. The new lipA target site was amplified using primers CB1287 (SEQ ID NO: 3) and CB1284 (SEQ ID NO: 4).

Chemical formula

[0317] Also, a second DNA fragment was prepared using pRF827 and the aprE promoter was amplified with the new lipA target site.

Chemical formula

[0318] The two fragments were assembled in vitro and subsequently transformed into E. coli TOP10 competent cells. This plasmid contains the lipA target site (SEQ ID NO: 7, 5’-TTATGGTTCACGGTATTGGA-3’), and using this, lipA was then deleted.

[0319] Construction of the pRS426-lipA deletion plasmid (Figure 2): A lipA deletion construct with homologous regions in the genome of Bacillus subtilis (B. subtilis) was assembled using yeast assembly. Four fragments were amplified by PCR and assembled. The first fragment contains half of the pRS426 vector backbone (Genetics. 1989 May;122(l):19-27). This fragment was amplified using primers DH 18-327F and DH 18-273R.

Chem.

[0320] The second DNA fragment contains half of the pRS426 vector backbone (Genetics. 1989 May;122(l):19-27). This fragment was amplified using primers DH 18-272F and DH 18-325R.

Chem.

[0321] The third fragment was amplified using B. subtilis genomic DNA and contains the genetic region upstream of lipA including the imrB, imrA, and ansZ genes for integration into the genome, with an overhang in the downstream region of lipA to enable assembly.

Chem.

[0322] The fourth fragment was amplified from B. subtilis genomic DNA and contains the yczC, yccF, and natK genes in the downstream region of lipA, with an overhang in the upstream region to enable assembly.

Chem.

[0323] These four fragments were assembled and transformed into the research yeast strain ATCC#76625 = Saccharomyces cerevisiae yph499 using the Zymo Research Frozen-EZ Yeast Transformation II Kit. A linear deletion construct consisting of the upstream lipA homologous region and the downstream lipA homologous region was amplified from the assembled yeast plasmid using the oligonucleotides DH18-343F and DH18-346R. This fragment was used as a repair template when deleting lipA.

Chem.

[0324] Transformation and isolation of lipA deletion strains The linear deletion construct and the pCAS-lipA CRISPR plasmid were co-transformed into B. subtilis cells. The transformation reaction was plated onto 5 ppm (parts per million) kanamycin containing 1.6% skim milk. The transformants were then streaked onto 5 ppm kanamycin plates and colony PCR was performed to check for lipA deletion (the primers used are as follows).

Chem.

[0325] Transformation of the β-galactosidase expression cassette A cassette for the expression of β-galactosidase (SEQ ID NO: 1) from Lactobacillus delbrueckii subsp. bulgaricus was transformed into the lipA deletion strain and plated on X-gal plates. The transformants were re-isolated on X-gal plates for better visualization of clones expressing lactase.

[0326] Nucleotide sequence of lipA (SEQ ID NO: 20) [B. Subtilis 168]

Chem.

[0327] Amino acid sequence of Lip A (SEQ ID NO: 2) [B. Subtilis 168]

Chem.

Example

[0328] Expression and purification of β-galactosidase derived from Lactobacillus delbrueckii bulgaricus The expression of β-galactosidase derived from Lactobacillus delbrueckii bulgaricus in Bacillus subtilis is achieved under aerobic fermentation conditions. Generally, the fermentation is carried out in a culture medium at pH 6 - 8 and 37 °C containing carbon and nitrogen sources, as well as inorganic salts, in a standard bioreactor in the industry. Then, β-galactosidase is recovered from the fermentation broth using conventional procedures, such as, but not limited to, centrifugation, microfiltration, or an aggregation method combined with rotary vacuum drum or filter press filtration. The supernatant or filtrate is recovered and ultrafiltered through a 10 kDa PES membrane (Koch) until the desired concentration for purification is reached. Purification such as chromatography (i.e., European Patent No. 2280065), protein crystallization (i.e., International Publication No. 8908703 pamphlet), or precipitation (not limited to these) can be used to achieve the desired level of purity. Precipitating agents or crystallizing agents for optimal insolubilization include, but are not limited to, metal halides such as sodium chloride, magnesium chloride, potassium chloride, sodium sulfate, etc. in the range of 0.1% (w / w) - 5% (w / w) and combinations thereof. The target chromatographic fractions can be pooled and further concentrated to enable a final formulation containing 40 - 60% glycerol at pH 6 - 8. Additionally, the precipitated or crystallized β-galactosidase can be pelleted at 7000 - 10,000 rpm, where the pellet can be resuspended in water and repelleted to optimal purity. The pellet is resuspended in the aforementioned formulation and filtered through rotary drum filtration or filter press to remove any insoluble fine particles formed upstream in the process.

Example

[0329] Optimal temperature of lactase The optimal temperatures of lactase GODO-YNL, experimental Dupont lactase SEQ ID NO: 1, and Saphera lactase were evaluated at 5°C to 70°C according to the modified FCC IV method shown in Example 3. The results are shown in Figure 3 and are all presented as relative lactase activity % relative to the enzyme activity quantified at 30°C. Comparing the temperature profiles, it is clearly seen that experimental Dupont lactase SEQ ID NO: 1 has a significant optimal temperature near 60°C (300%), while GODO YNL2 and Saphera have an optimal temperature near 45°C (100 - 150%). Experimental Dupont lactase SEQ ID NO: 1 is considered to be able to be used at higher temperatures in milk-based applications compared to existing hydrolytic lactases.

Example

[0330] Stability of Lactase in Milk Hydrolytic lactase samples (experimental Dupont lactase SEQ ID NO: 1 and Nola Fit) were diluted in Arla Mini-milk samples (Arla Foods, Denmark, 0.5% fat and 4.8% lactose), aliquoted into 1 mL samples, and then incubated at 55°C and 58°C for up to 6 hours. One sample was stored at 5°C for reference. Samples removed from the incubation were rapidly cooled on ice and then refrigerated until the residual activity was quantified.

[0331] The residual lactase activity was quantified by the following procedure. 20 μL of each milk sample was diluted in 480 μL of 0.1 M MES buffer (pH 6.4) (Mw: 195.2 g / mol, Sigma-aldrich #M8250-250G). After preheating the sample to 37 °C for 2 minutes, 750 μL of 3.7 mg / ml ONPG substrate (ONPG, Mw: 301.55 g / mol, Sigma-aldrich #N1127) was added. After 10 minutes of enzyme treatment, the reaction was stopped with 750 μL of 10% sodium carbonate (Sigma-aldrich) stop solution. The sample was further diluted to 150 μL in 850 μL of stop buffer, and then Abs420 was read with a spectrophotometer. The percentage of residual activity was calculated as Abs420 (sample incubated at a temperature of 50 °C or higher) / Abs420 (reference sample stored at 5 °C). The results are shown in FIGS. 4A and 4B. It is clearly recognized that the experimental Dupont lactase SEQ ID NO: 1 retains a significantly higher specific activity compared to Nola Fit at both 55 °C and 58 °C, and can enable sufficient lactose hydrolysis in milk-based products.

Example

[0332] Use of a thermostable lactase for the production of low-lactose or lactose-free dairy products at high temperatures 50 mL of an Arla Mini-milk sample (Arla Foods, Denmark, 0.5% fat and 4.8% lactose) was temperature-adjusted to 60 °C in a water bath. To enable lactose reduction at high temperature (60 °C), various lactases were added to the milk sample. The protein concentration in the lactase sample was measured according to the method of Example 4, and the dosages shown in Table 1 were applied.

[0333]

Table 5

[0334] At time zero, various lactases were added to milk and mixed. The lmL samples were extracted at various time points in the range of 10 to 240 minutes after enzyme inactivation. Inactivation was carried out at 98 °C for 10 minutes immediately after being placed in a thermomixer. The inactivated samples were stored at -20 °C until quantification. Lactose in all samples was quantified by the HPLC method described in Example 2. The results are shown in Figure 5A.

[0335] It is clear that the high thermal stability of the experimental Dupont lactase of SEQ ID NO: 1 enabled hydrolysis at 60 °C, efficiently producing lactose-free (100 ppm, 0.01 w / v%) or low-lactose (1000 ppm, 0.1 w / v%) milk samples. The remaining set of lactases tested showed insufficient lactose hydrolysis in milk, probably due to rapid inactivation at high temperature.

[0336] Also, for the lactase of SEQ ID NO: 1, tests were carried out under the same conditions except for different temperatures. The results are shown in Figure 5B. It is clear that the lactase can significantly reduce the amount of lactose in different temperature ranges.

Example

[0337] Use of thermostable lactase for lactose hydrolysis in condensed milk A reduced milk sample containing 12% (w / v) lactose was prepared by mixing low-fat milk (Arla Foods, Denmark, 0.1% fat) (83.5%) with skim milk powder (Arla Foods, Denmark, 1.25% fat, Batch 3150035537) (16.5%). After adjusting the temperature of this milk sample to 55 °C, the experimental Dupont lactase of SEQ ID NO: 1 was added at a dose less than that shown in Table 2. Exactly 1 hour later, the milk was rapidly heated at 97 °C for 10 minutes to inactivate the lactase. Residual lactose was quantified by the HPLC method described in Example 2. The results are shown in Table 2. The experiment clearly shows that the experimental Dupont lactase of SEQ ID NO: 1 has the ability to reduce lactose at high temperature in a milk-based solution.

[0338]

Table 6

Examples

[0339] Use of a thermostable lactase for the production of reduced-sugar condensed milk Procedure; Samples 2 - 8 were prepared according to the components (w / w%) in Table 3. Skim milk powder (Arla Foods, Denmark, 1.25% fat, batch 3150035537) (22.9 w / w%) and demineralized water (24.4 w / w%) were mixed with thorough stirring at 55 °C and hydrated for about 15 minutes to completely solubilize. Experimental Dupont lactase: J15005 lot: 4863191499 corresponding to lactase SEQ ID NO: 1 (having a lactase activity of 16976 SDLU / g) was added to samples 2 - 6 at various concentrations (0.005, 0.01, 0.34, 0.50 and 0.34 w / w%), and the mixture was further incubated at 55 °C for 60 minutes. Butter oil AMF (butter oil, Corman, Belgium number A00015111) was melted and then added to the mixture at 75 °C (65 °C for test 6 only) together with sucrose (granulated sugar, 550, Nordic Sugar, Denmark) and Recodan (RECODAN® RS VEG, Dupont Nutrition Bioscience, Denmark). Then all samples were homogenized at 75 °C (65 °C for test 6 only) at 80 bar and then further sterilized at 90 °C for 15 seconds. Then the sweetened condensed milk was filled into aluminum cans and sealed. Lactose powder (Variolac® 992 BG100, Arla Foods, Denmark) was ground as finely as possible to ensure only small crystals were formed, and added only to sample 7 after sterilization, incubated at 32 °C for 60 - 90 minutes and then filled. The lactose in Samples 2 to 6 was quantified by the HPLC method described in Example 2. The results are shown in Table 4. The lactose concentration clearly decreases according to the added amount of the thermostable lactase enzyme. A photograph of the reduced-sugar condensed milk is shown in Fig. 6. The photograph clearly shows that lactose crystals were formed in Sample 8 in which neither the experimental Dupont lactase SEQ ID NO: 1 nor the micronized lactose (microcrystals) was used (which can be applied to avoid larger crystal formation). Samples 2 and 7 had a slightly sandy mouthfeel, while all of Samples 3 to 6 containing the experimental Dupont lactase SEQ ID NO: 1 had a smooth mouthfeel and no crystal formation. The data clearly show that the use of the thermostable experimental Dupont lactase SEQ ID NO: 1 at high temperatures can surprisingly prevent lactose crystal formation and reduce the sandy mouthfeel in the production of reduced-sugar condensed milk.

[0340]

Table 7

[0341]

Table 8

[0342] Cooked sweetened condensed milk procedure: Aluminum cans containing the samples prepared above were heat-treated in an autoclave at 120°C for 10 minutes to induce the Maillard reaction (cooked to obtain the Maillard reaction). Thereafter, the samples were stored at room temperature for 6 weeks. A photograph of the cooked sweetened condensed milk is shown in Fig. 7.

[0343] This photograph clearly shows that, surprisingly, the same level of desired browning can be achieved with a dose of only 0.05% (w / w) of the experimental Dupont lactase SEQ ID NO: 1 that prevented crystal formation, as compared to a control without microcrystalline lactose or added lactase. For all doses of the experimental Dupont lactase (Samples 3 to 6) above 0.05%, an increase in browning was observed as a response to the increased Maillard reaction.

Example

[0344] Use of thermostable lactase for the production of low-lactose-containing milkshakes The use of thermostable lactase for the production of low-lactose milkshakes was tested in the following examples. All liquid components according to Table 5 were mixed at 55 °C: cream (Arla Foods, Denmark, 38% fat), skim milk (Arla Foods, Denmark, 0.1% fat, 4.8% lactose), and lactase enzyme (experimental Dupont lactase enzyme SEQ ID NO: 1, Nola Fit or no enzyme). The following dry components were incorporated: non-fat dry milk (Arla Foods, Denmark, 1.25% fat, batch 3150035537), sucrose (granulated sugar, 550, Nordic Sugar, Denmark), Litesse® Two (Dupont, Denmark), Cremodan MSA (Dupont, Denmark), followed by addition of vanilla essence and colorant (annatto seed extract, Danisco, Denmark, P.no. 1211663). All components were mixed thoroughly and held at 56 °C for 70 minutes. The temperature was raised to 70 °C and three samples (1, 2, and 3) were homogenized at 70 °C / pressure according to fat content, followed by pasteurization at 84 °C for 30 seconds. Thereafter, the samples were cooled to 5 °C, filled into 2 × 180 ml TPS + 2 × 155 ml pots, and the remainder was placed in metal containers for further heat treatment. The metal containers were heated to 95 °C for 10 minutes to ensure proper enzyme inactivation. Lactose was quantified according to Example 2, and samples 1, 2, and 3 contained 4.50% (w / v), 0.17% (w / v), and 0.30% (w / v) lactose, respectively. This clearly shows that the use of the experimental Dupont lactase enzyme SEQ ID NO: 1 is effective for low-lactose milkshakes at high temperatures.

[0345]

Table 9

Example

[0346] Use of a thermostable lactase for the production of low-lactose ice cream The use of a thermostable lactase for the production of low-lactose ice cream was tested in the following examples. All liquid components according to Table 6 were mixed at 55 °C: cream (Arla Foods, Denmark, 38% fat), skim milk (Arla Foods, Denmark, 0.1% fat, 4.8% lactose), glucose syrup (32DE 95% TS), and lactase enzyme (experimental Dupont lactase enzyme SEQ ID NO: 1, Nola Fit or no enzyme). The following dry components were incorporated: non-fat dry milk (Arla Foods, Denmark, 1.25% fat, batch 3150035537), sucrose (granulated sugar, 550, Nordic Sugar, Denmark), Litesse® Two (Dupont, Denmark), Cremodan MSA (Dupont, Denmark), Cremodan® SE 30 (Dupont, Denmark), followed by addition of flavor (vanilla essence) and colorant (annatto seed extract, Danisco, Denmark, P.no.1211663). All components were thoroughly mixed and held at 56 °C for 70 minutes. The temperature was raised to 70 °C and three samples (1, 2 and 3) were homogenized at 70 °C / pressure according to fat percentage, followed by pasteurization at 84 °C for 30 seconds. Thereafter, the samples were cooled to 5 °C, filled into 2 x 180 ml TPS + 2 x 155 ml pots and the remainder was placed in metal containers for further heat treatment. The metal containers were heated to 95 °C for 10 minutes to ensure proper inactivation of the enzyme and subsequently frozen. Lactose was quantified according to Example 2 and samples 1, 2 and 3 contained 4.90% (w / v), 0.20% (w / v) and 0.74% (w / v) lactose, respectively. This clearly shows that the use of the experimental Dupont lactase enzyme SEQ ID NO: 1 is effective for low-lactose ice cream at high temperatures.

[0347]

Table 10

Example

[0348] Sensory evaluation Production of UHT milk for sensory evaluation Add different enzymes to the milk and leave it in an ice water bath at 5°C for 24 hours while circulating it. UHT-PHE direct injection: -HI: 70°C -H2: 90°C -Injection: 142°C, 3 seconds -Vacuum: 87°C -Downstream homogenization (150 / 30) -C2: 45°C -C3: 20°C -Fill into sterilized 1L bottles -Store at room temperature for 10 weeks

[0349] Sensory method To explain the influence on perceptible product characteristics, descriptive sensory analysis is selected. The basis of descriptive analysis is ISO 13299 "Sensory analysis - Methodology General guidance for establishing a sensory profile". In descriptive sensory analysis, the intensity of each descriptor is evaluated on a line scale with two anchor points indicating low intensity and high intensity respectively. The low and high anchor points are taught to the panel in a training / calibration session. All samples were evaluated three times each.

[0350] The sensory panel consists of 8 members, all of whom have passed a basic sensory screening test before being accepted onto the panel. Before participating in the descriptive analysis of this assay, panelists receive training on product characteristic recognition and intensity scaling. Figure 8 shows taste characteristics with significant differences. This clearly shows how lactase containing LipA causes off-flavors such as sour, stale, and bitter in milk, while samples without LipA, whether they are reference milk or milk with lactase without LipA added, do not show the sweet, cooked, and caramel-like taste characteristics typically seen with the addition of lactase in UHT milk.

[0351] Lipase / FFA Test Reagent Preparation R1: Prepare R1 by mixing one vial of Color A and Solvent A. After preparing R1, store it at 2 - 10 °C and use it within 1 month. R2: Prepare R2 by mixing one vial of Color B and Solvent B. After preparing R2, store it at 2 - 10 °C and use it within 1 month.

[0352] Procedure Add 200 μL of the enzyme sample to 800 μL of reference milk containing 3.5% fat. Then store the sample at 5 °C for 20 hours. Add 10 μl of reference milk and the enzyme-added milk sample to 297 μL of R1 and incubate in a thermomixer at 37 °C. After 10 minutes, add 150 mL of R2 dropwise to each tube and continue incubation at 37 °C for 8 minutes. Then centrifuge the sample and leave the milk haze in the upper fraction. After carefully aliquoting 200 μL from the bottom fraction, read the OD546 in a microtiter plate. The results showed an OD546 of 0.5673 for lactase containing LipA, 0.2213 for reference milk, and 0.3048 for lactase without LipA.

Example

[0353] Addition of lactase after sterilization and homogenization Pre-pasteurized (72°C, 15 seconds) bulk mixed skim milk (0.1% fat) (Arla Foods, Denmark) stored at 4 - 6°C was standardized to the desired protein (4 w / w%) and fat (1 w / w%) content by the addition of skim milk powder (33% protein, 1.2% fat, 54% carbohydrates) from BBA Lactalis (Laval, Mayenne, France) and cream (38% fat) from Arla Foods (Denmark). The standardized milk was then pasteurized and homogenized in a standard plate heat exchanger. Homogenization was carried out at 65°C and 200 bar, and pasteurization was carried out at 95°C for 6 minutes. Subsequently, the milk was cooled to the fermentation temperature (45°C). After reaching the fermentation temperature, the milk was inoculated with a thermophilic starter culture, such as YO-MIX 224 / 485, at an inoculation rate of 20 DCU / 100 L. Lactase was added directly to the culture. Alternatively, lactase can be added at a temperature below 60°C after pasteurization and homogenization (i.e., while cooling to the fermentation temperature). After the addition of the enzyme, heated (95°C, 20 minutes) and non-heated samples (2 mL each) were taken at various time points during fermentation. The non-heated samples were used to determine the residual lactase activity. The residual lactase activity was quantified by the following procedure: 20 μL of each milk sample was diluted in 480 μL of 0.1 M MES buffer (pH 6.4) (Mw: 195.2 g / mol, Sigma-aldrich #M8250-250G). After preheating the sample to 37°C for 2 minutes, 750 μL of 3.7 mg / ml ONPG substrate (ONPG, Mw: 301.55 g / mol, Sigma-aldrich #N1127) was added. After 10 minutes of enzyme treatment, the reaction was stopped with 750 μL of 10% sodium carbonate (Sigma-aldrich) stop solution. The sample was further diluted 150 μL in 850 μL of stop buffer and then Abs420 was read on a spectrophotometer. The residual activity % was calculated as Abs420 (sample incubated at a temperature of 50°C or higher) / Abs420 (reference sample stored at 5°C). The heated samples were used for subsequent measurement of residual lactose according to Example 2. Fermentation was followed using a CINAC multi-channel pH system (Ysebaert, Frepillon, France), and the pH progression was monitored every 5 minutes.Fermentation was carried out until pH 4.60, and the product was cooled to 24 °C with a yogurt plate heat exchanger (SPX Flow Technology, Sussex, UK) and a YTRON-ZP shear pump system (YTRON Process Technology, Bad Endorf, Germany). The resulting stirred yogurt was stored at 4 - 6 °C.

[0354] The terms "comprise", "comprises", and "comprising" are to be construed inclusively rather than exclusively. This construction is intended to be the same as the construction given these words under United States patent law at the time of this application.

[0355] The singular forms "a" and "an" are intended to include plural referents unless the context indicates otherwise. Thus, for example, reference to the presence of "an excipient" does not exclude the presence of a plurality of excipients unless the context indicates otherwise.

Example

[0356] HPLC method for quantification of GOS fiber Lactobacillus delbrueckii bulgaricus lactase is capable of GOS formation (DP3+) in skim milk containing 5.2% initial lactose (References 1, 2), and the conversion of 15% lactose to GOS with a degree of polymerization of 3 or more (DP3+) has already been described. Tien-Thanh Nguyen et al. (Reference 3) also described the testing of Lactobacillus delbrueckii bulgaricus lactase at higher substrate concentrations, which can convert up to 50% of lactose to total GOS (including DP2 GOS such as allolactose) under buffered conditions containing 20.5% initial lactose. However, it was not expected that similar conversion yields could be achieved in milk substrates. Todor Vasiljevic and Paul Jelen 2003 (Reference 4) showed that the yield using Lactobacillus delbrueckii bulgaricus lactase was approximately 40% lower in a milk substrate of 5% lactose compared to using 5% lactose dissolved in buffer as the substrate. When tested at lactose concentrations of 10 - 15%, the same trend was observed that the conversion of lactose in the milk substrate to TGOS was lower compared to buffered conditions.

[0357] We hereby show that Lactobacillus delbrueckii bulgaricus lactase is commercially suitable for in situ GOS production when enzymatizing milk substrates. Surprisingly, we have found that it is possible to obtain a sugar reduction of more than 25% in milk-based substrates with 9 - 30% (w / w) lactose. It would not be possible to reduce the sugar by 25% when using a normal lactase such as GODO YNL2.

[0358] Standard lactose (HPLC grade, Sigma Aldrich) was prepared with redistilled water (ddH 2 O) and filtered through a 0.2 μm syringe filter. A dilution series in the range of 500 - 10,000 ppm of lactose standard was prepared.

[0359] The same sample preparation for the milk-based solution was applied, but the milk sample was diluted to approximately 5% milk carbohydrates and then 200 mg of the sample (by weight) was thoroughly mixed with 800 mL of H 2 O. 50 mL of Carrez reagent A (Carrez Clarification Kit, 1.10537.001, Merck) and 50 mL of Carrez B were added to 1000 mL of the diluted sample, and each was mixed after addition to induce precipitation of proteins and lipids. The sample mixture was incubated at room temperature for 15 minutes, and 50 mL of 10 mM NaOH, 1 mM EDTA was added to the sample. The sample was centrifuged at 10,000 rpm for 4 minutes, and 300 μL of the clarified supernatant was transferred to an MTP filter plate through a 0.20 μm 96-well plate filter (centrifuged at 3000 rpm for 15 minutes) and then analyzed (Corning filter plate, PVDF hydrophilic membrane, NY, USA). All samples were analyzed twice in a 96-well MTP plate sealed with tape.

[0360] Measurement device Quantification of galactooligosaccharides (GOS), lactose, glucose, and galactose was performed by HPLC. Analysis of the samples was carried out on a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific) equipped with a DGP-3600SD Dual-Gradient analysis pump, a WPS-3000TSL thermostat autosampler, a TCC-3000SD thermostat column oven, and an RI-101 refractive index detector (Shodex, JM Science). Chromeleon data system software (Version 6.80, DU10A Build 2826, 171948) was used for data acquisition and analysis.

[0361] Chromatographic conditions An analytical guard column (Carbo-Ag + neutral, AJ0-4491, Phenomenex, The Netherlands) equipped RSO oligosaccharide column, Ag + Using 4% crosslinking (Phenomenex, The Netherlands), the sample was analyzed by HPLC. This column was eluted with redistilled water (filtered through a 0.45 μm regenerated cellulose membrane and purged with helium gas) at a flow rate of 0.3 ml / min.

[0362] During the analysis with a total run time of 45 minutes, an isocratic flow rate of 0.3 ml / min was maintained and the injection volume was set to 10 mL. The sample was held at 30 °C in the compartment of the thermostatic autosampler to ensure complete solubilization of all components. The eluate was monitored with a refractive index detector (RI-101, Shodex, JM Science), and quantification was performed by the peak area relative to the peak area of the lactose standard substance described above. Peaks with DP3 or higher (DP3+) were quantified as galacto-oligosaccharides (DP3, DP4, DP5, etc.). The assumption that all DP3+ galacto-oligosaccharide components show the same response was confirmed by mass balance. Lactose containing other DP2 components was quantified in the same manner as DP2, glucose, and galactose.

Example

[0363] Total GOS Total GOS (TGOS) is the sum of transgalactosylated molecules. This value is calculated based on multiplying the amount of galactose bound in TGOS molecules by a factor of 1.4. Here, the factor of 1.4 represents the galactose-to-glucose ratio. The galactose bound to TGOS molecules is calculated by subtracting the free galactose and the galactose bound to lactose from the total galactose (which is the sum of free galactose, galactose bound to TGOS, and galactose bound to lactose). This calculation method is in line with the AOAC 2001.02 method.

[0364] Based on the values obtained from Example 2 and Example 16, TGOS is calculated as follows: TGOS = (Total carb. (w / w%) / 1.9 - Galactose (w / w%) - Lactose (w / w%) / 1.9) × 1.4 Total carb. (w / w%) is the sum of DP3+ (w / w%), DP2 (w / w%), glucose (w / w%), and galactose (w / w%) Total carb. (w / w%) / 1.9 represents the total galactose in the sample, Galactose (w / w%) is the free galactose in the sample Lactose (w / w%) / 1.9 is the galactose bound in lactose molecules

Example

[0365] Conversion of lactose to GOS in a lactose-buffered substrate 12%, 20% and 30% lactose solutions were prepared by dissolving anhydrous lactose (Sigmaaldrich, #17814) in 100 ml of 0.1 M MES buffer (pH 6.4) (Mw: 195.2 g / mol, Sigmaaldrich #M8250-250G). The solution temperature was raised to 40 °C for the addition of GODO YNL2 or 55 °C for the addition of LBul. Once the appropriate temperature was reached, the enzyme was added at time 0 according to Table 7. After 20, 40, 60 and 80 minutes, 5 ml samples were taken for inactivation. Inactivation was carried out at 95 °C for 10 minutes. Quantification of DP3+, DP2, glucose, and galactose was performed according to Example 16, and lactose was quantified according to Example 2.

[0366]

Table 11

[0367] All data are shown in Table 8 for LBul and Table 9 for GODO YNL2, together with the calculated values of total carbohydrates (carb.), % decrease in sugars, TGOS (according to Example 17), and % lactose converted to TGOS.

[0368]

Table 12

[0369]

Table 13

[0370] Conclusion Unlike GODO YNL2 lactase used to produce lactose-free products, it is clear that LBul produces significant levels of GOS fiber (DP3+), thereby providing a sugar reduction of more than 30% in 12 - 30% lactose buffered substrates.

Example

[0371] Conversion of Lactose to GOS in a Milk-based Substrate According to Table 10, substrates of Arla Mini-milk (Arla Foods, Denmark, 0.5% fat and 4.8% lactose), or skim milk powder (Arla Foods, Denmark, 1.25% fat, batch 3150035537) were prepared in water (100 ml). The temperature was maintained at 50 °C for Mini-milk, 55 °C for 12 - 40% milk powder, and 58 °C for 60% milk powder. LBul was added at time 0 according to Table 10, and samples were taken at various time points for heat inactivation. Inactivation was carried out at 95 °C for 10 minutes. Quantification of DP3+, DP2, glucose, and galactose was performed according to Example 16, and lactose was quantified according to Example 2.

[0372] [Table 14]

[0373] All data are shown in Table 11 together with the calculated values of total carbohydrate (carb.), % decrease in sugar, TGOS (according to Example 17), and % lactose converted to TGOS.

[0374] [Table 15]

[0375] [Table 16]

[0376] Conclusion In contrast to current literature, the inventors have found that LBul not only converts 15% of lactose (starting at 5.2%) into GOS fiber (DP3+), but also exceeds 18% in normal milk with 4.7% lactose. This corresponds to a reduction of sugars by more than 18% in normal Arla Mini - milk. Similarly, the inventors have found that more than 20% of lactose is converted into DP3+ (reduction of sugars by more than 20%) in a concentrated milk substrate with 6% lactose, more than 25% of lactose is converted into DP3+ (reduction of sugars by more than 25%) in a concentrated milk substrate with 9 - 12% lactose, and more than 30% of lactose is converted into DP3+ (reduction of sugars by more than 30%) in a concentrated milk substrate with 20 - 40% lactose. Also, in contrast to current literature, the inventors have found that LBul not only converts 20% of lactose (starting at 5.2%) into TGOS, but also exceeds 30% in normal milk with 4.7% lactose. Similarly, the inventors have found that more than 35% of lactose is converted into TGOS in a concentrated milk substrate with 6% lactose, more than 40% of lactose is converted into TGOS in a concentrated milk substrate with 9% lactose, more than 45% of lactose is converted into TGOS in a concentrated milk substrate with 12% lactose, and more than 50% of lactose is converted into TGOS in a substrate with 20 - 40% lactose.

Example

[0377] Conversion of lactose to GOS in a buffered substrate with 55% lactose Two solutions of 56.2% (w / w) lactose in 0.1 M MES (pH 6.0) (for LBul) or 50 mM sodium acetate (pH 4.8) (for NutriBio GOS) were prepared in a volume of 200 ml. These solutions were heated above 90 °C in a heating block while mixing at 400 rpm. The solutions were then cooled to 59 °C on the table for about 30 minutes. Subsequently, 80 U / ml of NutriBio GOS was added to the lactose solution at pH 4.8, and 0.488 ml of LBul was added to the lactose solution at pH 6. Then the solutions were placed in another heating block at 58 °C. The reaction was carried out at 58 °C and 400 rpm for up to 3 hours. After 0.5, 1, 1.5, 2, and 3 hours, 1 ml samples were taken from each solution and inactivated by heating at 98 °C for 10 minutes. Quantification of DP3+, DP2, glucose, and galactose was performed according to Example 16, and lactose was quantified according to Example 2.

[0378] All data are shown in Table 12 together with the calculated values of total carbohydrate (carb.), % decrease in sugar, TGOS (according to Example 17), and % lactose converted to TGOS.

[0379]

Table 17

[0380] Conclusion The inventors have found that LBul can produce GOS fibers in higher yields than NutriBio GOS used in today's GOS production (resulting in a greater decrease in sugar) and can achieve a significantly higher TGOS yield.

Example

[0381] Reduction or elimination of the holding time for producing low-lactose or lactose-free high-temperature short-time sterilized (HTST) milk Belfonte skim milk was treated with experimental Dupont lactase enzyme at 4°C for 6 hours before the pasteurization step or at 4°C for 5 minutes per liter of milk before the pasteurization step. The milk was then subjected to an HTST pasteurization process including preheating at 60°C, pasteurization at 72.8°C or 73.3°C for 30 seconds, and homogenization at 1300 psi. The milk was cooled immediately after the pasteurization step and stored at 4°C for 3 weeks. The sensory evaluation was completed at 3 weeks. Current HTST low-lactose or lactose-free conditions usually require 18 - 24 hours of lactose hydrolysis before pasteurization since the enzyme is inactivated as it passes through the pasteurizer.

[0382] 1 mL milk samples were taken at the following multiple time points throughout the study and transferred to separate 1.5 mL Eppendorf tubes for lactose quantification according to Example 2: initial milk sample before enzyme treatment, 30 minutes or 2 hours after pasteurization, 24 - 26 hours after pasteurization, 42 - 43 hours after pasteurization, and 8 - 9 days after pasteurization. Immediately after the milk samples were taken, they were placed in a 95°C heat block for 10 minutes to inactivate the lactase enzyme. The milk samples were then cooled to room temperature and prepared as follows: 200 mL of the heat-treated milk sample was transferred to a new 1.5 mL Eppendorf tube, 800 mL of deionized water was added, and vortexed. 50 mL of Carrez reagent I and 50 mL of Carrez reagent II were added to each tube and vortexed. The solution was left at room temperature for 30 minutes and then centrifuged at 3000 rpm for 5 minutes. 10 mL of the supernatant was added to 990 mL of deionized water in separate 1.5 mL Eppendorf tubes, vortexed, filtered through a 0.45 micron filter, and placed in vials for lactose analysis by ion chromatography. The lactose concentration was calculated using calibration standards and internal controls.

[0383] Table 13 shows the enzyme dosage, incubation time before sterilization, and sterilization temperature in the experiments conducted, together with the milk sampling time point and lactose results for each sample. Lactose levels decreased after sterilization under all test conditions, indicating the viability of the enzyme over the sterilization process and post-sterilization shelf life. By 8 - 9 days after treatment, the lactose level was less than 0.1 w / v%. No off-odors were detected in any of the samples over a 3-week shelf life. This example illustrates the use of experimental Dupont lactase in high-temperature short-time (HTST) milk for the production of lactose-free or low-lactose milk. The viability of the enzyme over the entire sterilization process allows for a significant reduction or elimination of the holding time before sterilization, if possible.

[0384]

Table 18

[0385] References 1: European Patent No. 303201 2: Fischer, C. and Kleinschmidt, T. (2018), Synthesis of Galactooligosaccharides in Milk and Whey: A Review. Comprehensive Reviews in Food Science and Food Safety, 17: 678 - 697. 3: Tien-Thanh Nguyen, Hoang Anh Nguyen, Sheryl Lozel Arreola, Georg Mlynek, Kristina Djinovic-Carugo, Geir Mathiesen, Thu-Ha Nguyen, and Dietmar Haltrich. Homodimeric β-Galactosidase from Lactobacillus delbmeckii subsp. bulgaricus DSM 20081: Expression in Lactobacillus plantarum and Biodiemical Characterization. Journal of Agricultural and Food Chemistry 2012 60 (7), 1713-1721. DOI: 10.1021 / jf203909e4. Todor Vasiljevic and Paul Jelen (2003). Oligosaccharide production and proteolysis during lactose hydrolysis using crude cellular extracts from lactic acid bacteria. Lait, 83 6 (2003) 453-467.

[0386] All references cited herein are hereby incorporated by reference into this specification.

Claims

1. 1. A method for reducing the amount of lactose in a milk-based substrate containing lactose, comprising contacting said substrate with an enzyme having neutral lactase activity at a temperature above 50°C, said enzyme having at least 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:1, said lactase reduces the amount of lactose in said substrate by at least 70%, and said enzyme with neutral lactase activity is an enzyme having the ability to hydrolyze the disaccharide lactose into its component galactose and glucose monomers, and said enzyme has a pH optimum of between pH 6.0 and pH 8.

0.

2. 2. The method of claim 1, wherein the enzyme is the enzyme of SEQ ID NO: 1, or a lactase active fragment thereof.

3. 3. The method of claim 1 or 2, wherein the enzyme having neutral lactase activity is purified.

4. The method according to any one of claims 1 to 3, wherein the enzyme having neutral lactase activity is concentrated.

5. The method according to any one of claims 1 to 4, wherein the enzyme with neutral lactase activity is in the form of an enzyme preparation having a reduced level of lipase side activity.

6. 6. The method of claim 5, wherein the enzyme with neutral lactase activity is in the form of an enzyme preparation having reduced levels of protease, amylase, mannanase, pectinase, cellulase and / or p-nitrobenzyl esterase side activities.

7. 7. The method of claim 5 or 6, wherein the preparation is substantially free of lipase, protease, amylase, mannanase, pectinase, cellulase, and / or p-nitrobenzyl esterase side activity.

8. The method according to any one of claims 1 to 7, wherein the temperature is greater than 50°C to 65°C.

9. 9. The method of claim 1, wherein the temperature is selected from 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 90°C.

10. The method according to any one of claims 1 to 9, wherein the temperature is 55°C or 58°C.

11. The method according to any one of claims 1 to 10, wherein the activity of the enzyme at 60°C is at least 50% of its activity at 50°C.

12. The method according to any one of claims 1 to 11, wherein the enzyme has a temperature optimum of 60°C.

13. The composition according to any one of claims 1 to 12, wherein the enzyme has a pH optimum of 5.5 to 8.

0.

14. 14. The method according to any one of claims 1 to 13, wherein the lactase activity of the enzyme at pH 6.0 is at least 50% of its lactase activity at pH 6.5, measured at 37°C.

15. The method of any one of claims 1 to 14, wherein the contacting is performed for 10 minutes to 4 hours.

16. The milk-based substrate is selected from the group consisting of whole or low fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, a solution of milk powder, UHT milk, whey, whey permeate, acid whey, or cream. The method according to any one of claims 1 to 15.

17. 17. The method of any one of claims 1 to 16, wherein the milk-based substrate is raw milk that has not been pasteurized prior to contact with the enzyme.

18. The method according to any one of claims 1 to 17, wherein the substrate comprises 3 to 30% lactose.

19. 19. The method of any one of claims 1 to 18, wherein the amount of lactose is reduced by at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

20. 20. The method of any one of claims 1 to 19, wherein the lactose is reduced to less than 100 ppm or less than 1000 ppm.

21. 21. The method of claim 20, wherein the lactose is reduced to less than 100 ppm within 180 minutes at temperatures above 50°C.

22. 22. The method of any one of claims 1 to 21, wherein the enzyme is added to the milk-based substrate at a concentration of 24 to 240 NLU per gram of lactose.

23. 23. The method of any one of claims 1 to 22, wherein the enzyme has a lactase:transgalactosylase activity ratio of 1:1 or a transgalactosylation activity rate of less than 120%.

24. The method according to any one of claims 1 to 23, wherein the enzyme has a lactase activity of 100%.

25. A method according to any one of claims 1 to 24 for producing a dairy product.

26. 26. The method of claim 25, wherein the dairy product is selected from skim milk, low fat milk, whole milk, cream, UHT milk, extended shelf life milk, cultured dairy products, cheese, yogurt, butter, dairy spreads, buttermilk, acidified milk beverages, sour cream, whey-based beverages, condensed milk, dulce de leche, flavored milk beverages, sweetened condensed milk, milk powder, reconstituted dairy products, ice cream, ryazhenka, puddings, desserts, and milkshakes.

27. 27. The method of claim 26, wherein the dairy product is condensed milk, ice cream, or a milkshake.

28. 27. The method of claim 26, further comprising a sterilization step.

29. 29. The method of any one of claims 25 to 28, wherein the dairy product is condensed milk and the temperature is 55°C.

30. 1. An enzyme preparation comprising an enzyme having neutral lactase activity, said enzyme being capable of reducing the amount of lactose in a substrate by at least 70% at a temperature of 50°C or greater, said enzyme having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, said enzyme having neutral lactase activity being an enzyme having the ability to hydrolyze the disaccharide lactose into its component galactose and glucose monomers, said enzyme having an optimum pH between pH 6.0 and pH 8.

0.

31. 31. The enzyme preparation of claim 30, wherein the enzyme is not the enzyme of SEQ ID NO:

1.

32. 32. The enzyme preparation of claim 30 or 31, wherein the enzyme having neutral lactase activity is purified.

33. 33. The enzyme preparation according to any one of claims 30 to 32, wherein the enzyme having neutral lactase activity is concentrated.

34. 34. The enzyme preparation according to any one of claims 30 to 33, wherein the enzyme having neutral lactase activity has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:

1.

35. An enzyme preparation according to any one of claims 30 to 34, having a reduced level of lipase side activity.

36. 36. The enzyme preparation of any one of claims 30 to 35, having reduced levels of protease, amylase, mannanase, pectinase, cellulase and / or p-nitrobenzyl esterase side activities.

37. 37. The enzyme preparation of claim 35 or 36, wherein the preparation is substantially free of lipase, protease, amylase, mannanase, pectinase, cellulase, and / or p-nitrobenzyl esterase side activity.

38. 38. An enzyme preparation according to any one of claims 30 to 37, wherein the activity of the enzyme having neutral lactase activity at 60°C is at least 50% of its activity at 50°C.

39. 39. An enzyme preparation according to any one of claims 30 to 38, wherein the enzyme having neutral lactase activity has a temperature optimum of 60°C.

40. 40. The enzyme preparation according to any one of claims 30 to 39, wherein the enzyme having neutral lactase activity has a pH optimum of 5.5 to 8.

0.

41. 41. An enzyme preparation according to any one of claims 30 to 40, wherein the activity of the enzyme with neutral lactase activity at pH 6.0 is at least 50% of the lactase activity at pH 6.5 when measured at 37°C.

42. A nucleic acid molecule encoding an enzyme having neutral lactase activity or a lactase active fragment thereof as defined in any one of claims 30 to 41.

43. 43. An expression vector comprising a nucleic acid molecule according to claim 42 or capable of expressing an enzyme with neutral lactase activity as defined in any one of claims 30 to 41, or a lactase active fragment thereof.

44. A cell capable of expressing an enzyme with neutral lactase activity as defined in any one of claims 30 to 41 or a lactase active fragment thereof.

45. 45. A method of expressing an enzyme, comprising providing a cell according to claim 44 and expressing said enzyme from said cell.

46. 42. An enzyme with neutral lactase activity as defined in any one of claims 30 to 41 having a half-life in milk of more than 4 hours at 55°C or more than 1 hour at 58°C.

47. Use of an enzyme according to any one of claims 30 to 41 for preparing a dairy product.

48. 48. The use according to claim 47, wherein the dairy product is selected from skim milk, low fat milk, whole milk, cream, UHT milk, extended shelf life milk, cultured milk products, cheese, yoghurt, butter, dairy spreads, buttermilk, acidified milk drinks, sour cream, whey based drinks, condensed milk, dulce de leche, flavoured milk drinks, sweetened condensed milk, milk powder, reconstituted dairy products, ice cream, ice cream, ryazhenka, puddings, desserts and milkshakes.

49. 42. A bacterial expression host capable of expressing an enzyme having neutral lactase activity as defined in any one of claims 30 to 41 or a lactase active fragment thereof, wherein the host cell comprises a genetic modification which reduces or eliminates lipase activity.

50. 50. The bacterial expression host of claim 49, wherein the genetic modification comprises a deletion, disruption, or downregulation of the gene encoding the Lipase A polypeptide set forth in SEQ ID NO:

2.

51. 42. A bacterial expression host capable of expressing an enzyme having neutral lactase activity as defined in any one of claims 30 to 41 or a lactase active fragment thereof, said host cell comprising a genetic modification that reduces or eliminates one or more enzymatic activities selected from protease activity, amylase activity, mannanase activity, pectinase activity, cellulase activity and p-nitrobenzyl esterase activity.

52. 52. The bacterial expression host of claim 50 or 51, wherein the host cell comprises genetic modifications that reduce or eliminate lipase activity, protease activity, amylase activity, mannanase activity, pectinase activity, cellulase activity, and p-nitrobenzyl esterase activity.

53. 53. The bacterial expression host of any one of claims 49 to 52, wherein the bacterium is a Bacillus species.

54. B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. sonorensis, B. halodurans, B. pumilus, B. lautus, B.

54. The bacterial expression host of claim 53, selected from the group consisting of B. pabuli, B. cereus, B. agaradhaerens, B. akibai, B. clarkii, B. pseudofirmus, B. lehensis, B. megaterium, B. coagulans, B. circulans, B. gibsonii, and B. thuringiensis.

55. 55. The bacterial expression host of claim 54, wherein the bacterium is Bacillus subtilis.

56. A bacterial expression host according to any one of claims 49 to 55, wherein the bacterium comprises a nucleic acid molecule according to claim 42 or an expression vector according to claim 43.

57. 57. The bacterial expression host of any one of claims 49 to 56, wherein the host expresses an enzyme having neutral lactase activity.

58. 1. A method for producing a low-lactose or lactose-free milkshake, ice cream, reconstituted milk product, dessert, pudding, condensed milk, sweetened condensed milk, llagenca, dulce de leche, or milk-based powder, comprising contacting a milk-based substrate with an enzyme having neutral lactase activity at 50-65°C, said enzyme having at least 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:1, said enzyme having neutral lactase activity being an enzyme having the ability to hydrolyze the disaccharide lactose into its component galactose and glucose monomers, said enzyme having a pH optimum of between pH 6.0 and pH 8.

0.

59. The method of claim 11, wherein the temperature is between 55°C and 60°C.

60. 22. The method of claim 21, wherein the substrate comprises 3% to 16% lactose.

61. 61. The method of claim 60, wherein the substrate comprises 4% to 5% lactose.

62. 23. The method of claim 22, wherein the amount of lactose is reduced by at least 97%.

63. 25. The method of claim 24, wherein the lactose is reduced to less than 100 ppm within 120 minutes at temperatures above 50°C.

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