Patent Description:
In order to grow on milk, lactose hydrolysis is a good way for lactic acid bacteria to obtain glucose and galactose as carbon source. Lactase (beta-galactosidase; EC <NUM>. <NUM>) is the enzyme that performs the hydrolysis step of the milk sugar lactose into monosaccharides. The commercial use of lactase is to break down lactose in dairy products. Lactose intolerant people have difficulties to digest dairy products with high lactose levels. It is estimated that about <NUM>% of the world's population has a limited ability to digest lactose. Accordingly, there is a growing demand for dairy food products that contain no or only low levels of lactose.

Lactases have been isolated from a large variety of organisms, including microorganisms like Kluyveromyces and Bacillus. Kluyveromyces, especially K. fragilis and K. lactis, and other fungi such as those of the genera Candida, Torula and Torulopsis, are a common source of fungal lactases, whereas B. coagulans and B. circulans are well known sources for bacterial lactases. Several commercial lactase preparations derived from these organisms are available such as Lactozym® (available from Novozymes, Denmark), HA-Lactase (available from Chr. Hansen, Denmark) and Maxilact® (available from DSM, the Netherlands), all from K. All these lactases are so-called neutral lactases having a pH optimum between pH <NUM> and pH <NUM>, as well as a temperature optimum around <NUM>. When such lactases are used in the production of, e.g. low-lactose yoghurt, the enzyme treatment will either have to be done in a separate step before fermentation or rather high enzyme dosages have to be used because their activity will drop as the pH decreases during fermentation.

A typical process for production of pasteurized milk with reduced lactose comprises addition of the lactase enzyme to the milk followed by prolonged incubation (<NUM>-<NUM>, often <NUM>) at temperatures around <NUM>. Because the Ha-Lactase and NOLA® Fit activity is in the range of <NUM>-<NUM>µmol per min per mg of enzyme, enzyme doses in the range of <NUM>-<NUM>/L and <NUM>-<NUM>/L respectively for pasteurized milk are required to achieve the desired residual lactose level. The Ha-Lactase and NOLA® Fit enzymes have temperature optimum around <NUM>. Longer incubation of milk at <NUM> can result in microbial growth.

Also, these lactases are not suitable for hydrolysis of lactose in milk performed at high or low temperatures, which would in some cases be beneficial in order to keep the microbial count low and thus ensure high milk quality. Furthermore, the known lactases would not be suitable for use in a desired process for the production of ultra-heat treated (UHT) milk, wherein enzymes were added prior to the UHT treatment.

<CIT> relates to compositions comprising lactase activity obtained from sonication of microbial cells of bacteria or yeast. <CIT> and <CIT> relate to cold-active beta-galactosidases. <CIT> relates to beta-galactosidase with high transgalactosylating activity, whereas <CIT> relates to beta-galactosidase with lower transgalactosylating activity. <CIT> relates to a beta-galactosidase for production of lactose depleted milk products which is inactivated at a pasteurization temperature.

It is an object of embodiments of the invention to provide methods using a beta-galactosidase that enable the production of improved lactose-free or low-lactose products at low temperatures.

It is a further object of embodiments of the invention to provide methods using a beta-galactosidase with properties that improve the lowering of lactose in a product, such as lactose-free or low-lactose products.

The present inventor(s) have identified beta-galactosidases with properties not previously described that enable the production of improved lactose-free or low-lactose products as well as enabling improved production methods for such lactose-free or low-lactose products. In particular these beta-galactosidases have been shown to be very stable with relatively high activity at a very broad range of both temperatures as well as pH values. They are also useable at specific temperatures, such as at high temperatures and pH values not normally seen with these enzymes. First of all, this enables to the use of beta-galactosidases at specific pH values and temperatures that were not known to be possible. It also enables the use of the same specific enzyme in several different applications, which is highly requested in the industry.

In a first aspect the present invention provides methods for producing a dairy product comprising:.

In a related embodiment the present invention provides methods for reducing the lactose content in a milk-based substrate comprising:.

The methods of the present invention are advantageous as they only require a low concentration of the peptide exhibiting beta-galactosidase activity and still significantly reduce the lactose concentration. In a preferred alternative, the peptide exhibiting beta-galactosidase activity is added in a concentration of <NUM> to <NUM>/L, in a concentration of <NUM> to <NUM>/L or in a concentration of <NUM> to <NUM>/L.

The milk-based substrate can be any substrate containing milk. In one aspect the above methods use a milk-based substrate which is:.

In a particularly preferred embodiment, the above methods use cow milk comprising lactose in a concentration of about <NUM> to <NUM>/L or a heat treated, pasteurized, raw and/or filtered form thereof as the milk-based substrate.

The above methods provide for a significant reduction of the concentration of lactose in a short period of time. In certain embodiments, the concentration is reduced to a value of less than <NUM>/l lactose after incubation for at least <NUM> hours, at least <NUM> hours, at least <NUM> hours or at least <NUM> hours.

One of the advantages of the methods of the present invention resides in reduction of the concentration of lactose at low temperatures.

The methods provide a significant reduction of the concentration of lactose and preferably the incubation in step (b) reduces the lactose concentration in the mixture to less than <NUM>/L, to less than <NUM>/L, or to less than <NUM>/L.

Specific the peptide exhibiting beta-galactosidase activity to be used in the methods of the invention are not only highly active at low temperatures, but also at high temperatures. In one aspect the invention thus provides method as described above, wherein the mixture comprising the milk-based substrate and the peptide exhibiting beta-galactosidase activity is heated to a temperature of at least <NUM> for at least four seconds before or after incubating the mixture at a temperature from <NUM>-<NUM>. In particular, the method may comprise a heating step including heating to a temperature of <NUM> for about <NUM> seconds before or after incubating the mixture at low temperatures in step (b).

In one alternative, the methods of the present invention are used for producing a dairy product. These methods may further comprise a step of fermenting the milk-based substrate with lactic acid bacteria. The fermentation step is carried out before or after the incubation with a peptide exhibiting beta-galactosidase activity.

The methods are particularly suitable for producing dairy products, such as a fermented milk product, cheese, yoghurt, butter, dairy spread, butter milk, acidified milk drink, sour cream, whey based drink, ice cream, condensed milk, dulce de leche or a flavored milk drink.

In a further embodiment the present invention relates to the use of a peptide exhibiting beta-galactosidase activity for producing a dairy product with reduced lactose content at a temperature from <NUM>-<NUM> for a period of time sufficient to reduce the lactose concentration in the mixture to less than <NUM>/L, wherein the peptide exhibiting beta-galactosidase activity is a peptide having the amino acid sequence represented by SEQ ID NO: <NUM>.

The present inventors have found that certain peptides exhibiting beta-galactosidase enzyme activity are surprisingly stabile at many different physical conditions giving a relatively high activity outside of the ranges normally seen to be optimal for this class of enzymes.

Accordingly, these by the present inventors identified enzymes have a relatively high activity around <NUM> or <NUM> and may thus be used for lactose hydrolysis in the production of e.g. fresh milk. The novel enzymes are thus particularly suitable for reducing the lactose content of milk-based products, such as dairy products, at low temperatures.

A further advantage of these novel improved peptides exhibiting beta-galactosidase enzyme activity is that they have a relatively low degree of galactose inhibition. The lower galactose inhibition of these novel enzymes is highly relevant for applications wherein very low lactose concentrations are desired.

In terms of applicability for fermented products it is highly advantageous that the enzymes as described herein have a high beta-galactosidase enzymatic activity at a relatively broad temperature range of between <NUM> and <NUM>, such as around <NUM>, where fermentation would normally be optimal, but also that this activity of the beta-galactosidase enzyme is present at low pH, such as down to <NUM>, or down to <NUM>, or down to <NUM>, or even down to pH <NUM>.

In summary, it has been found by the present inventors that some peptides exhibiting beta-galactosidase enzyme activity is active over wide range of temperature, active over wide range of pH, has a general high hydrolytic activity without side activities, that these peptides have no or little galactose inhibition, such as less than <NUM>%, and that they are stable over long-term storage.

The beta-galactosidase activity may be determined by measuring the amount of released glucose after incubation with lactose at set conditions. Released glucose can be detected by a coloring reaction.

The term "milk", as used herein and in the context of the present invention, is to be understood as the lacteal secretion obtained by milking any mammal, such as cow, sheep, goats, buffalo or camel.

The term "composition containing lactose" as used herein refers to any composition, such as any liquid that contain lactose in significant measurable degree, such as a lactose content higher than <NUM>% (<NUM>/<NUM>). Encompassed within this term are milk and milk-based substrates.

The term "milk-based substrate", in the context of the present invention, may be any raw and/or processed milk material. Useful milk-based substrates include, but are not limited to solutions/suspensions of any milk or milk like products comprising lactose, such as whole or low fat milk, skim milk, buttermilk, low-lactose milk, reconstituted milk powder, condensed milk, solutions of dried milk, UHT milk, whey, whey permeate, acid whey, cream, fermented milk products, such as yoghurt, cheese, dietary supplement and probiotic dietary products. Typically the term milk-based substrate refers to a raw or processed milk material that is processed further in order to produce a dairy product.

The term "pasteurization" as used herein refers to the process of reducing or eliminating the presence of live organisms, such as microorganisms in a milk-based substrate. Preferably, pasteurization is attained by maintaining a specified temperature for a specified period of time. The specified temperature is usually attained by heating. The temperature and duration may be selected in order to kill or inactivate certain bacteria, such as harmful bacteria, and/or to inactivate enzymes in the milk. A rapid cooling step may follow.

The term "dairy product" as used herein may be any food product wherein one of the major constituents is milk-based. Usually the major constituent is milk-based and in some embodiments, the major constituent is a milk-based substrate which has been treated with an enzyme having beta-galactosidase activity according to a method of the present invention.

A dairy product according to the invention may be, e.g., skim milk, low fat milk, whole milk, cream, UHT milk, milk having an extended shelf life, a fermented milk product, cheese, yoghurt, butter, dairy spread, butter milk, acidified milk drink, sour cream, whey based drink, ice cream, condensed milk, dulce de leche or a flavored milk drink.

A dairy product may additionally comprise non-milk components, e.g. vegetable components such as, e.g., vegetable oil, vegetable protein, and/or vegetable carbohydrates. Dairy products may also comprise further additives such as, e.g., enzymes, flavoring agents, microbial cultures such as probiotic cultures, salts, sweeteners, sugars, acids, fruit, fruit prep, fruit juices, or any other component known in the art as a component of, or additive to, a dairy product.

The terms "fermented dairy product" or "fermented milk product" as used herein is to be understood as any dairy product wherein any type of fermentation forms part of the production process. Examples of fermented dairy products are products like yoghurt, buttermilk, creme fraiche, quark and fromage frais. A fermented dairy product may be produced by or include steps of any method known in the art.

The term "fermentation" as used herein refers to the conversion of carbohydrates into alcohols or acids through the action of a microorganism. In some embodiments fermentation according to the present invention comprises the conversion of lactose to lactic acid. In the context of the present invention, "microorganism" may include any bacterium or fungus being able to ferment the milk substrate.

The term "increased beta-galactosidase enzyme activity" as used herein refers to a relatively higher specific activity of a beta-galactosidase enzyme in comparison to a reference sequence.

The term "peptide exhibiting beta-galactosidase enzyme activity" as used herein refers to any peptide, which has enzymatic activity to catalyze the hydrolysis of the disaccharide lactose into its component monosaccharides glucose and galactose. This peptide may also be referred to as a lactase or simply a beta-galactosidase (EC: <NUM>.

In a preferred embodiment the beta-galactosidase activity is determined by incubating <NUM>µl of a solution comprising a known amount of a purified lactase enzyme with a solution comprising <NUM> of lactose at pH <NUM> and <NUM> for <NUM>, terminating the lactase reaction by increasing the temperature to <NUM> for <NUM>. The amount of glucose formed was determined by incubating the reaction product at <NUM> for <NUM> with a <NUM>µL solution of glucose oxidase (<NUM>/L), <NUM>,<NUM>'-azino-bis(<NUM>-ethylbenzothiazoline-<NUM>-sulfonic acid diammonium salt) (<NUM>/L ABTS) and horseradish peroxidase (<NUM>/L) and determining the absorbance at <NUM> using a FLUOphotometer. The absorbance is correlated to the concentration of glucose formed per minute and the maximum value determined (in µmol of glucose formed/min) is determined as the Unit of Lactase Activity <NUM> (also designated herein UAL-<NUM>). The Specific Activity of Lactase (also herein designated SUAL-<NUM>) at pH <NUM> at <NUM> is defined as µmol of glucose formed/min/mg of enzyme and is determined by dividing UAL-<NUM> by the lactase protein concentration in mg. Full details of a preferred alternative of carrying out this assay are illustrated in Example <NUM>.

While characterizing beta-galactosidase activity by reference to values of the unit µmol of glucose formed/min/mg of enzyme represents the standard approach for the determination of the activity, other units may equally be used to characterize the activity of the lactase enzymes using the above test. Accordingly, some of the examples characterize the lactase enzyme activity by reference to µM of glucose formed per second per µM of enzyme.

In alternative embodiments the assay can be carried out using a different temperature or different pH values for the lactase incubation.

The terms "peptide" and "oligopeptide" as used in the context of this present application are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by peptidyl linkages. The word "polypeptide" is used herein for chains containing more than ten amino acid residues. All peptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus. "Proteins" as used herein refers to peptide sequences as they are produced by some host organism and may include posttranslational modification, such as added glycans.

The terms "amino acid" or "amino acid sequence," as used herein, refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. In this context, "fragment" refer to fragments of a peptide exhibiting beta-galactosidase enzyme activity, which retain some enzymatic activity. Where "amino acid sequence" is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited peptide molecule.

Typically, the specific beta-galactosidase enzyme activity will be measured and indicated as µmol of glucose formed/min/mg of enzyme used. This specific value however will vary depending on conditions applied, such as temperature, and pH. Accordingly, values for beta-galactosidase enzyme activity may also be referred to as relative to a reference known enzyme, such as the beta-galactosidase enzyme defined by SEQ ID NO:<NUM> OR SEQ ID NO:<NUM>.

Unless otherwise stated the term "Sequence identity" for amino acids as used herein refers to the sequence identity calculated as (nref - ndif)·<NUM>/nref, wherein ndif is the total number of non-identical residues in the two sequences when aligned and wherein nref is the number of residues in one of the sequences.

In some embodiments the sequence identity is determined by conventional methods, e.g., <NPL>, by the search for similarity method of <NPL>, using the CLUSTAL W algorithm of <NPL>, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group). The BLAST algorithm (<NPL>) for which software may be obtained through the National Center for Biotechnology Information www. gov/) may also be used. When using any of the aforementioned algorithms, the default parameters for "Window" length, gap penalty, etc., are used.

A peptide with a specific amino acid sequence as described herein may vary from a reference peptide sequence by any of amino acid substitutions, additions/insertions, or deletions.

All embodiments according to the present invention refer to the use of a peptide with the amino acid sequence represented by SEQ ID NO: <NUM>.

The term "host cell", as used herein, includes any cell type which is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide encoding the peptide of the present invention. A host cell may be the cell type, where a specific enzyme is derived from or it may be an alternative cell type susceptible to the production of a specific enzyme. The term includes both wild type and attenuated strains.

Suitable host cell may be any bacteria including lactic acid within the order "Lactobacillales" which includes Lactococcus spp. , Streptococcus spp. , Lactobacillus spp. , Leuconostoc spp. , Pseudoleuconostoc spp. , Pediococcus spp. , Brevibacterium spp. , Enterococcus spp. and Propionibacterium spp. Also included are lactic acid producing bacteria belonging to the group of anaerobic bacteria, bifidobacteria, i.e. Bifidobacterium spp. , which are frequently used as food cultures alone or in combination with lactic acid bacteria. Also included within this definition are 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 and thermophilic lactic acid bacterial species include as examples Streptococcus thermophilus, Enterococcus faecium, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus. Other specific bacteria within this definition includes bacteria of the family Bifidobacteriaceae, such as from the genus Bifidobacterium, such as from a strain of bifidobacterium animalis or bifidobacterium longum, bifidobacterium adolescentis, bifidobacterium bifodum, bifidobacterium breve, bifidobacterium catenulatum, bifidobacterium infantus or from the genus Lactobacillus, such as L. amylovorus, L. delbrueckii subsp. Lactis, and L. helveticus.

Also included within this definition of host cells include strain of Agaricus, e.g. A. bisporus; Ascovaginospora; Aspergillus, e.g. A. awamori, A. foetidus, A. japonicus, A. oryzae; Candida; Chaetomium; Chaetotomastia; Dictyostelium, e.g. D. discoideum; Kluveromyces, e.g. K. fragilis, K. lactis; Mucor, e.g. M. javanicus, M. subtilissimus; Neurospora, e.g. N. crassa; Rhizomucor, e.g. R. pusillus; Rhizopus, e.g. R. arrhizus, R. japonicus, R. stolonifer; Sclerotinia, e.g. S. libertiana; Torula; Torulopsis; Trichophyton, e.g. T. rubrum; Whetzelinia, e.g. W. sclerotiorum; Bacillus, e.g. B. coagulans, B. circulans, B. megaterium, B. novalis, B. subtilis, B. pumilus, B. stearothermophilus, B. thuringiensis; Bifidobacterium, e.g. B. bifidum, 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. violeceoruber; Trametes; Trichoderma, e.g. T. viride; Yersinia, e.g. Y. enterocolitica.

To produce lactose free milk pasteurized milk (<<NUM>% residual lactose level) at cold temperatures (<NUM>-<NUM>) in <NUM> hr, the recommended dose of the Ha-Lactase and NOLA® are <NUM>-<NUM>/L (<NUM> NLU/L) and <NUM>-<NUM>/L respectively (<NUM> BLU/L), respectively. The enzymes of the present invention provided very low residual lactose concentrations at low temperatures (<<NUM>% to <NUM>%). The specific activity measurements shows that the novel enzymes have <NUM>-<NUM> higher activity than Ha-Lactase and NOLA® Fit, therefore they will require lesser time to produce the lactose free milk.

The Examples below show that the novel lactases are faster than Ha-Lactase and NOLA® Fit and results in lactose free pasteurized milk in significantly shorter time. These new enzymes can reduce the overall process time. Additionally, with novel enzymes it is possible to further reduce the enzyme dose between <NUM>-<NUM>% to produce lactose free/reduced pasteurized milk.

Techniques for restriction enzyme digestions, ligation, transformation and other standard molecular biology manipulations were based on methods described in the literature (<NPL>;<NPL>; <NPL>); or as suggested by the manufacturer. The PCR was carried out in a DNA thermal cycler obtained from (Bio-Rad, USA). DNA sequencing was performed by LGC, Berlin, Germany. Proteins were analyzed by polyacrylamide gel electrophoresis (PAGE) under the denaturation conditions using sodium dodecyl sulphate on gels containing <NUM>% SDS (Mini-PROTEAN® TGX stain-free™ gel, Biorad, USA). Protein concentrations were determined using BCA method by following the protocol supplied with the kit.

Escherichia coli strain TOP10 (Invitrogen) was used for the cloning and isolation of plasmids. The beta-galactosidase deficient E. coli strain BW25113 (Δ(araD-araB)<NUM>, ΔlacZ4787(::rrnB-<NUM>), λ-, rph-<NUM>, Δ(rhaD-rhaB)<NUM>, hsdR514) (<NPL>) was used in combination with the pBAD/His vector (obtained from Invitrogen™ Life Technologies Corporation Europe BV) for recombinant protein production.

2xPY medium containing (<NUM>/L BD BBL™ Phyton TM Peptone, <NUM>/L Yeast Extract, <NUM>/L NaCl) was used for the recombinant protein production. The growth medium was supplemented with ampicillin (<NUM>µg/ml) to maintain the plasmid. Protein production was initiated by adding <NUM>% of arabinose in to the culture medium.

The genomic DNA of the lactic acid bacteria or bifidobacteria was extracted using commercial genomic extraction kit by following the supplied protocol (DNeasy, Qaigen, Germany). The lactase gene was amplified by PCR using two synthetic primers, using the purified genomic DNA source as biomass, and the PCR reagents were supplied in the Phusion U Hot start DNA polymerase (Thermo Scientific, USA) kit. The lactase gene was cloned into the start codon of the expression vector pBAD/His using the USER cloning method (<NPL>), resulting in the expression construct. With the USER cloning method long, complementary overhangs in both PCR product and destination vector were generated. These overhangs can anneal to each other to form a stable hybridization product which was used to transform into E. coli without ligation. For the generation of overhangs in the PCR product, a single deoxyuradine residue is included in the upstream region of each primer to amplify target DNA. The lactase gene was amplified using the forward primer (<NUM>'-ATTAACCAUGCGACGCAACTTCGAATGGCC-<NUM>') and reverse primer (ATCTTCTCUTTACCGCCTTACCACGAGCACG) containing a uridine at 9th position (as shown in bold), followed by the lactase gene sequence. In parallel, the vector DNA was PCR amplified using the forward (<NUM>'-AGAGAAGAUTTTCAGCCTGATACAGATTAAATC-<NUM>') and reverse primer (<NUM>'-ATGGTTAAUTCCTCCTGTTAGCCCAAAAAACGG-<NUM>') pair containing single deoxyuracil residue at 9th positions (as highlighted in bold) followed by vector DNA sequence. The PCR products were purified using the commercial PCR purification kit (Qiagen, Denmark). The purified PCR products (lactase gene and the vector DNA) were mixed in equimolar amount and incubated with a commercial USER enzyme mix (New England Biolabs, USA) by following the supplied protocol. These enzymes remove the uracil residue and also the short fragment upstream of the uridine, thereby creating complementary overhang in the PCR products. These complementary overhangs anneal with each other resulting in the pBAD-lactase expression vector. Aliquots of the ligation mixture were transformed into chemically competent E. coli TOP <NUM> cells. Transformants were selected at <NUM> on LB-Amp plates (LB; Luria-Bertani, Amp; <NUM>µg/ml ampicillin). The following day, colony PCR was carried out using a small biomass from the overnight grown transformant using the vector primers (primer <NUM>; <NUM>'-CGGCGTCACACTTTGCTATGCC-<NUM>' and primer <NUM>; <NUM>'-CCGCGCTACTGCCGCCAGGC-<NUM>'). The positive clones from the colony PCR were cultured in <NUM> LB-Amp medium and plasmid DNA was isolated from the cells. The cloned lactase gene was sequenced to verify that no additional mutations had been introduced during the amplification of the gene. The plasmid DNA was transformed in to the expression host E. coli strain BW25113.

The lactase enzyme was produced in E. coli BW25113 using the pBAD expression system. Freshly transformed E. coli BW25113 cells carrying the plasmid DNA were collected from a Lb-Amp plate using a sterile loop and used to inoculate <NUM> of Lb-Amp medium. The overnight grown culture (<NUM>µL) was used to inoculate <NUM> 2x PY medium (containing <NUM>µg/mL ampicillin) in a <NUM> flask in a shaker (Innova® <NUM>). The culture was grown at <NUM> at <NUM> rpm until the OD600 reached between <NUM>-<NUM>. The lactase expression was initiated by adding <NUM>% arabinose into the culture medium and the cells were cultured for additional <NUM>-<NUM> hours at <NUM> at <NUM> rpm. Cells were harvested by centrifugation (<NUM> rpm, <NUM> at <NUM>) and were stored at -<NUM> until further use.

Cells from <NUM> culture was thawed on ice and the cells were lysed using <NUM> mixture of lysis buffer (BugBuster® (Novagen) containing <NUM>/mL Lysozyme (Sigma Aldrich), <NUM> unit Benzonase (Sigma Aldrich), and 1X Complete Protease inhibitor cocktail (EDTA-free, Roche)) by incubating the cells at room temperature for <NUM>. After <NUM>, the cell debris was removed by centrifugation at <NUM> rpm for <NUM> at <NUM>. The obtained supernatant was filtered through <NUM> pore diameter filter. A gravity flow Ni-Sepharose (GE Healthcare) column was prepared with <NUM> slurry by washing out the ethanol and water. The column was then equilibrated with washing buffer (<NUM> of NaH<NUM>PO<NUM>, pH <NUM> containing <NUM> of NaCl and <NUM> of Imidazole). The cell-free extract was applied to the column and the non-bound proteins were eluted from the column. The column was washed with <NUM> of washing buffer and the retained proteins were eluted with <NUM> of elution buffer (<NUM> of NaH<NUM>PO<NUM>, pH <NUM> containing <NUM> of NaCl and <NUM> of imidazole). The collected fractions were analyzed by SDS-PAGE on gels containing <NUM>% acrylamide and those contained the purified lactase enzymes combined together. The buffer was exchanged against the storage buffer (<NUM> KH<NUM>PO<NUM> buffer pH <NUM> containing <NUM> NaCl, <NUM> MgCl<NUM>), using a prepacked PD-<NUM> desalting G-<NUM> gel filtration column (GE Healthcare). The purified enzymes were stored at <NUM> until further use.

Cells from <NUM> culture was thawed on ice and the cells were lysed using <NUM> mixture of lysis buffer (BugBuster® (Novagen) containing <NUM>/ml lysozyme, <NUM> unit Benzonase (Sigma Aldrich), and 1x Complete Protease inhibitor cocktail (EDTA-free, Roche)) by incubating the cells at room temperature (<NUM>) for <NUM>. After <NUM>, the cell debris was removed by centrifugation at <NUM> rpm for <NUM> at <NUM>. The obtained supernatant was filtered through <NUM> pore diameter filter. The clear cell-free extract was concentrated by filtering through a <NUM> Dalton filter (Vivaspin <NUM>, GE Healthcare) by following the supplied protocol. A gravity flow Sephadex G50 superfine (Pharmacia Chemicals, Sweden) column was prepared with <NUM> of column material (prepared by boiling in <NUM> water for <NUM> hour, cooled to room temperature). A column was prepared by applying <NUM> of the cooled slurry on a <NUM> filtration column. The column was washed with MilliQ water and equilibrated with wash buffer B (<NUM> of NaH<NUM>PO<NUM> buffer, pH <NUM>). <NUM>µL of the concentrated supernatant was applied on the column and allowed the supernatant to enter in the column bed. The wash buffer (<NUM> of NaH<NUM>PO<NUM> buffer, pH <NUM>) was applied on top of the column and the eluent fractions were collected individually. The collected fractions were analyzed on SDS-PAGE gel (containing <NUM>% acrylamide). The protein fractions were combined together and buffer was exchanged against the storage buffer (<NUM> KH<NUM>PO<NUM> buffer pH <NUM> containing <NUM> NaCl, <NUM> MgCl<NUM>) with the desalting column as described in earlier section. The purified enzymes were stored at <NUM> until further use.

The concentration of purified lactases was determined using Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Germany) by following the protocol supplied with the kit.

To measure the beta-galactosidase activity, the purified lactases were diluted to 40x in buffer A (<NUM> NaH<NUM>PO<NUM> buffer pH <NUM> containing <NUM> of MgSO<NUM>). In a separate reaction, the diluted enzyme was incubated with lactose solution prepared in buffer B (<NUM> of lactose prepared in <NUM> sodium-citrate buffer of pH <NUM>, containing <NUM> of MgSO<NUM>). The reaction mixture was prepared by mixing <NUM>µL of diluted enzyme and <NUM>µL of lactose solution in a PCR tube. The reaction mixture was incubated in a DNA thermal cycler with the following incubation parameters (reaction time; <NUM> at <NUM>, enzyme inactivation; <NUM> at <NUM>, cooling; <NUM>). The reaction mixtures were stored at -<NUM> until further use. To determine the amount of glucose formed during the reaction, <NUM>µL of the reaction mixture was transferred to one well of standard microtiter plate (Thermo Fischer Scientific, Denmark) containing <NUM>µL of buffer C (<NUM> of NaH<NUM>PO<NUM> buffer, pH <NUM>, containing glucose oxidase; <NUM>/L (Sigma Aldrich), <NUM>,<NUM>'-azino-bis(<NUM>-ethylbenzothiazoline-<NUM>-sulfonic acid diammonium salt); ABTS: <NUM>/L (Sigma Aldrich), horseradish peroxidase; <NUM>/L (Sigma Adrich)) and incubated at <NUM> for <NUM>. After <NUM>, the absorbance was determined at <NUM> using FLUOStar Omega UV-plate reader (BMG Labtech, Germany). The absorbance values between <NUM> and <NUM> were used for calculations, if the A610 nm value ><NUM>, the reaction mixture was diluted up to 10x with buffer A. With each purified enzyme, the reactions were carried out in triplicate and the mean value of the triplicate measurement was used for calculation. The protein purification performed with the E. coli cells transformed with the empty pBAD/His was used for normalization. Using a known concentration of glucose (<NUM>-<NUM>), a standard curve was drawn and the slope of the curve was used to calculate the glucose formed during the reaction. The maximum absorbance value for each lactase was used to determine µmol of glucose formed per min (for example by correlating the absorbance value to the glucose concentration formed using a standard or calibration curve) and is also designated Unit of Lactase Activity <NUM> (or UAL-<NUM>) at pH <NUM> at <NUM>. The Specific Activity (designated as SUAL-<NUM>) at pH <NUM> at <NUM> is defined as µmol of glucose formed per min per mg of enzyme (µmol of glucose/min/mg of enzyme) and is determined by dividing UAL-<NUM> by the protein concentration in mg. The specific activity of SEQ ID NO: <NUM> and SEQ ID NO: <NUM> were determined under essentially the same conditions. The high specific activity at pH <NUM> is highly desired for robustness for the enzyme in fresh and fermented milk applications. The detailed results of the specific activity of enzymes at pH <NUM> at <NUM> are described in <FIG>. Additionally the activity was described as µM of glucose formed per second per µM of enzyme added. The results are shown in <FIG>.

The specific activity of the enzymes was determined at pH <NUM> and at <NUM> and used to calculate the approximate time required for hydrolysis of lactose using a fixed enzyme dose based activity units at pH <NUM> at <NUM> and <NUM> lactose as substrate (SUAL-<NUM>). The results in terms of time calculated for lactose hydrolysis are shown in Table <NUM>:.

The purified lactases were diluted to 40x in buffer A (<NUM> NaH<NUM>PO<NUM> buffer pH <NUM> containing <NUM> of MgSO<NUM>). In separate reactions, the diluted enzymes were incubated with buffer D (<NUM> of lactose and <NUM> of galactose prepared in <NUM> sodium-citrate buffer of pH <NUM>, containing <NUM> of MgSO<NUM>). The reaction mixture consists of <NUM>µL of the diluted enzyme and <NUM>µL of buffer D in a PCR tube. The reaction mixture was incubated in thermal cycler with the following incubation parameters (reaction time: <NUM> at <NUM>, enzyme inactivation: <NUM> at <NUM>, cooling: <NUM>). The reaction mixtures were stored at -<NUM> until further use. To determine the amount of glucose formed during the reaction, <NUM>µL of the reaction mixture was transferred to one well of standard microtiter plate (Thermo Fischer Scientific, Denmark) containing <NUM>µL of buffer C (<NUM> of NaH<NUM>PO<NUM> buffer, pH <NUM>, containing glucose oxidase; <NUM>/L (Sigma Aldrich), <NUM>,<NUM>'-azino-bis(<NUM>-ethylbenzothiazoline-<NUM>-sulfonic acid diammonium salt); ABTS: <NUM>/L (Sigma Aldrich), horseradish peroxidase; <NUM>/L (Sigma Adrich)) and incubated at <NUM> for <NUM>. After <NUM>, the absorbance was determined at <NUM> using FLUOStar Omega UV-plate reader (BMG Labtech, Germany). The absorbance values between <NUM> and <NUM> were used for calculations, if the A610 nm value ><NUM>, the reaction mixture was diluted up to 10x with buffer A. With each purified enzyme, the reactions were carried out in triplicate and the mean value of the triplicate measurement was used for calculation. The protein purification performed with the E. coli cells transformed with the empty pBAD/His was used for normalization. Using a known concentration of glucose (<NUM>-<NUM>), a standard curve was drawn and the slope of the curve was used to calculate the absorbance corresponding to <NUM> of glucose formed during the reaction. The maximum absorbance value for each lactase was used to determine µM of glucose formed per sec, described as <NUM> Unit of Activity with Galactose at pH <NUM> at <NUM> (UAG). The specific activity at pH <NUM> at <NUM> in presence of galactose is defined as µM of glucose formed per second per µM of enzyme (µM of glucose/sec/µM of enzyme) and determined by dividing UAG by the protein concentration in µM, described as SUAG.

The percentage inhibition of enzymes with galactose is calculated by using the formula <MAT>.

Where A is specific activity in of enzymes with lactose at pH <NUM> at <NUM> (SUAL) as described in the example <NUM>, and B stand for the specific activity of enzymes in presence of galactose at pH <NUM> at <NUM> (SUAG) as described in the example <NUM>. The detail results of the % galactose inhibition are described the <FIG> and <FIG>. The lower galactose inhibition is highly relevant for the applications where very low lactose concentration is desired.

Additionally the activity was described as µmole of glucose formed per minute per milligram of enzyme added. The results are shown in <FIG>.

Note: relatively high standard deviations in galactose inhibition measurement are due to trace amounts of glucose impurities in purchased galactose.

The purified lactases were diluted up to 40x in buffer A (<NUM> NaH<NUM>PO<NUM> buffer pH <NUM> containing <NUM> of MgSO<NUM>). In a separate reaction, the diluted enzyme was incubated with lactose solution prepared in buffer B (<NUM> of lactose prepared in <NUM> sodium-citrate buffer of pH <NUM>, containing <NUM> of MgSO<NUM>). The reaction mixture was prepared by mixing <NUM>µL of diluted purified enzyme and <NUM>µL of lactose solution in a PCR tube. The reaction mixture was incubated in a DNA thermal cycler using the following incubating parameters (reaction time; <NUM> at <NUM>, enzyme inactivation; <NUM> at <NUM>, storage; <NUM>). The reaction mixtures were stored at -<NUM> freezer until further use. The amount of glucose formed during the reaction was determined by following the protocol described in example <NUM>. The maximum absorbance value for each lactase was used to determine µM of glucose formed per sec, described as <NUM> Unit of Activity with Lactose at pH <NUM> at <NUM> (UAL-<NUM>). The specific activity at pH <NUM> at <NUM> is defined as µM of glucose formed per second per µM of enzyme (µM of glucose/sec/µM of enzyme), and is determined by dividing UAL-<NUM> by the protein concentration in µM, described as SUAL-<NUM>. The high specific activity at pH <NUM> at <NUM> is highly desired for the lactose hydrolysis for fresh/pasteurized milk applications. The detail results of the specific activity of enzymes at pH <NUM> at <NUM> are described in the <FIG>.

The purified lactases were diluted to 40x in buffer A (<NUM> NaH<NUM>PO<NUM> buffer pH <NUM> containing <NUM> of MgSO<NUM>). In a separate reaction, the diluted enzyme was incubated with lactose solution prepared in buffer B (<NUM> of lactose prepared in <NUM> sodium-citrate buffer of pH <NUM>, containing <NUM> of MgSO<NUM>). The reaction mixture was prepared by mixing <NUM>µL of diluted purified enzyme and <NUM>µL of lactose solution in a PCR tube. The reaction mixture was incubated in a DNA thermal cycler using the following incubating parameters (reaction time; <NUM> at <NUM>, enzyme inactivation; <NUM> at <NUM>, storage; <NUM>). The reaction mixtures were stored at -<NUM> freezer until further use. The amount of the glucose formed during the reaction was determined by following the protocol described in example <NUM>. The maximum absorbance value for each lactase was used to determine µM of glucose formed per sec, described as <NUM> Unit of Activity with Lactose at pH <NUM> at <NUM> (UAL-<NUM>). The specific activity at pH <NUM> at <NUM> is defined as µM of glucose formed per second per µM of enzyme (µM of glucose/sec/µM of enzyme), and is determined by dividing UAL-<NUM> by the protein concentration in µM, described as SUAL-<NUM>. The high specific activity at pH <NUM> at <NUM> is highly desired for the lactose hydrolysis for the fermented milk applications. The detail results of the specific activity of enzymes at pH <NUM> at <NUM> are described in <FIG>.

The purified lactases were diluted up to 40x in buffer A (<NUM> NaH<NUM>PO<NUM> buffer pH <NUM> containing <NUM> of MgSO<NUM>). In a separate reaction, the diluted enzyme was incubated with lactose solution prepared in buffer E (<NUM> of lactose prepared in <NUM> sodium-citrate buffer of pH <NUM>, containing <NUM> of MgSO<NUM>). The reaction mixture was prepared by mixing <NUM>µL of diluted purified enzyme and <NUM>µL of lactose solution in a PCR tube. The substrate solution was prepared in a buffer of pH <NUM> and enzyme solution had a pH of <NUM>. To initiate the reaction, <NUM>µL of enzyme was added to <NUM>µL of substrate solution. This mixing of these two buffers eventually increases the reaction pH from <NUM> to <NUM>.

The reaction mixture was incubated in a DNA thermal cycler using the following incubating parameters (reaction time; <NUM> at <NUM>, enzyme inactivation; <NUM> at <NUM>, storage; <NUM>). The reaction mixtures were stored at -<NUM> freezer until further use. To determine the amount of glucose formed during the reaction, <NUM>µL of the reaction mixture was transferred to one well of standard microtiter plate containing <NUM>µL of buffer C and incubated at <NUM> for <NUM>. After <NUM>, the absorbance was determined at <NUM> using FLUOStar Omega UV-plate reader (BMG Labtech, Germany). The absorbance value between <NUM> and <NUM> were used for calculations, if the A610 nm value ><NUM>, the reaction mixture was diluted up to 5x with buffer A. With each purified enzyme, the reactions were carried out in triplicate and the mean value of the triplicate measurement was used for calculations. The maximum absorbance value for each lactase was used to determine µM of glucose formed per sec, described as <NUM> Unit of Activity with Lactose at pH <NUM> at <NUM> (UAL-<NUM>). The specific activity at pH <NUM> at <NUM> is defined as µM of glucose formed per second per µM of enzyme (µM glucose/sec/µM of enzyme), and is determined by dividing UAL-<NUM> by the protein concentration in µM, described as SUAL-<NUM>. The high specific activity at pH <NUM> at <NUM> is relevant for the lactose hydrolysis in the fermented milk applications. The detail results of the specific activity of enzymes at pH <NUM> at <NUM> are described in the <FIG>.

The reaction mixture was incubated in a DNA thermal cycler using the following incubating parameters (reaction time; <NUM> at <NUM>, enzyme inactivation; <NUM> at <NUM>, storage; <NUM>). The reaction mixtures were stored at -<NUM> until further use. The amount of glucose formed during the reaction was determined by following the protocol as described in the example <NUM>. The maximum absorbance value for each lactase was used to determine µM of glucose formed per sec, described as <NUM> Unit of Activity with Lactose at pH <NUM> at <NUM> (UAL-<NUM>). The specific activity at pH <NUM> at <NUM> is defined as µM of glucose formed per second per µM of enzyme (µM of glucose/sec/µM of enzyme), and is determined by dividing UAL-<NUM> by the protein concentration in µM, described as SUAL-<NUM>. The high specific activity at pH <NUM> at <NUM> is relevant for the lactose hydrolysis in the fermented milk applications and sweet whey lactose hydrolysis. The detail results of the specific activity of enzymes at pH <NUM> at <NUM> are described in the <FIG>.

The reaction mixture was incubated in a DNA thermal cycler using the following incubating parameters (reaction time; <NUM> at <NUM>, enzyme inactivation; <NUM> at <NUM>, storage; <NUM>). The reaction mixtures were stored at -<NUM> until further use. The amount of glucose formed during the reaction was determined by following the protocol described in the example <NUM>. The maximum absorbance value for each lactase was used to determine µM of glucose formed per sec, described as <NUM> Unit of Activity with Lactose at pH <NUM> at <NUM> (UAL-<NUM>). The specific activity at pH <NUM> at <NUM> is defined as µM of glucose formed per second per µM of enzyme (µM of glucose/sec/µM of enzyme), and is determined by dividing UAL-<NUM> by the protein concentration in µM, described as SUAL-<NUM>. The high specific activity at pH <NUM> at <NUM> is relevant for the lactose hydrolysis in the fermented milk applications and sweet whey lactose hydrolysis. The detail results of the specific activity of enzymes at pH <NUM> at <NUM> are described in the <FIG>.

The purified lactases were diluted up to 40x in buffer A (<NUM> NaH<NUM>PO<NUM> buffer pH <NUM> containing <NUM> of MgSO<NUM>). In a separate reaction, the diluted enzyme was incubated with lactose solution prepared in buffer F (<NUM> of lactose prepared in <NUM> sodium-citrate buffer of pH <NUM>, containing <NUM> of MgSO<NUM>). The reaction mixture was prepared by mixing <NUM>µL of diluted purified enzyme and <NUM>µL of lactose solution in a PCR tube. The substrate solution was prepared in a buffer of pH <NUM> and enzyme solution had a pH of <NUM>. To initiate the reaction, <NUM>µL of enzyme was added to <NUM>µL of substrate solution. This mixing of these two buffers eventually increases the reaction pH from <NUM> to <NUM>.

The reaction mixture was incubated in a DNA thermal cycler using the following incubating parameters (reaction time; <NUM> at <NUM>, enzyme inactivation; <NUM> at <NUM>, storage; <NUM>). To determine the amount of glucose formed during the reaction, <NUM>µL of the reaction mixture was transferred to one well of standard microtiter plate containing <NUM>µL of buffer C (as described in example <NUM>) and incubated at <NUM> for <NUM>. After <NUM>, the absorbance was determined at <NUM> using FLUOStar Omega UV-plate reader. The absorbance value between <NUM> and <NUM> were used for calculations, if the A610 nm value ><NUM>, the reaction mixture was diluted up to 5x with buffer A. With each purified enzyme, the reactions were carried out in triplicate and the mean value of the triplicate measurement was used for calculation. The maximum absorbance value for each lactase was used to determine µM of glucose formed per sec, described as <NUM> Unit of Activity with Lactose at pH <NUM> at <NUM> (UAL-<NUM>). The specific activity at pH <NUM> at <NUM> is defined as µM of glucose formed per second per µM of enzyme (µM of glucose/sec/µM of enzyme), and is determined by dividing UAL-<NUM> by the protein concentration in µM, described as SUAL-<NUM>. The high specific activity at pH <NUM> at <NUM> is relevant for the lactose hydrolysis in the fermented milk applications. The detail results of the specific activity of enzymes at pH <NUM> at <NUM> are described in the <FIG>.

The reaction mixture was incubated in a DNA thermal cycler using the following incubating parameters (reaction time; <NUM> at <NUM>, enzyme inactivation; <NUM> at <NUM>, storage; <NUM>). The reaction mixtures were stored at -<NUM> until further use. The amount of glucose formed during the reaction was determined by following the protocol described in the example <NUM>. The maximum absorbance value for each lactase was used to determine µM of glucose formed per sec, described as <NUM> Unit of Activity with Lactose at pH <NUM> at <NUM> (UAL-<NUM>). The specific activity at pH <NUM> at <NUM> is defined as µM of glucose formed per second per µM of enzyme (µM of glucose/sec/µM of enzyme), and is determined by dividing UAL-<NUM> by the protein concentration in µM, described as SUAL-<NUM>. The high specific activity at pH <NUM> at <NUM> is relevant for the lactose hydrolysis in the fermented milk applications and acidic whey lactose hydrolysis. The detail results of the specific activity of enzymes at pH <NUM> at <NUM> are described in the <FIG>. Additionally the activity was described as µmole of glucose formed per minute per milligram of enzyme added. The results are shown in <FIG>.

The purified lactases were diluted up to 40x in buffer A (<NUM> NaH<NUM>PO<NUM> buffer pH <NUM> containing <NUM> of MgSO<NUM>). In a separate reaction, the diluted enzyme was incubated with lactose solution prepared in buffer F (<NUM> of lactose prepared in <NUM> sodium-citrate buffer of pH <NUM>, containing <NUM> of MgSO<NUM>). The reaction mixture was prepared by mixing <NUM>µL of diluted purified enzyme and <NUM>µL of lactose solution in a PCR tube. The substrate solution was prepared in a buffer of pH <NUM> and enzyme solution had a pH of <NUM>. To initiate the reaction, <NUM>µL of enzyme was added to <NUM>µL of substrate solution. This mixing of these two buffers eventually increases the reaction pH from <NUM> to <NUM>. The reaction mixture was incubated in a DNA thermal cycler using the following incubating parameters (reaction time; <NUM> at <NUM>, enzyme inactivation; <NUM> at <NUM>, storage; <NUM>). The reaction mixtures were stored at -<NUM> until further use. The amount of glucose formed during the reaction was determined by following the protocol described in the example <NUM>. The maximum absorbance value for each lactase was used to determine µM of glucose formed per sec, described as <NUM> Unit of Activity with Lactose at pH <NUM> at <NUM> (UAL-<NUM>). The specific activity at pH <NUM> at <NUM> is defined as µM of glucose formed per second per µM of enzyme (µM of glucose/sec/µM of enzyme), and is determined by dividing UAL-<NUM> by the protein concentration in µM, described as SUAL-<NUM>. The high specific activity at pH <NUM> at <NUM> is relevant for the lactose hydrolysis in the fermented milk applications and acidic whey lactose hydrolysis. The detail results of the specific activity of enzymes at pH <NUM> at <NUM> are described in the <FIG>.

The commercially available NOLA® Fit enzyme (Chr-Hansen, Denmark) was diluted in a range from <NUM> BLU/mL to <NUM> BLU/mL in buffer G (<NUM> NaH<NUM>PO<NUM> buffer pH <NUM> containing <NUM> of MgSO<NUM>, <NUM>% Brij, Sigma Aldrich). The diluted enzyme was incubated with lactose solution prepared in buffer H (<NUM> of lactose prepared in <NUM> sodium-citrate buffer of pH <NUM>, containing <NUM> of MgSO<NUM>). The reaction mixture was prepared by mixing <NUM>µL of diluted purified enzyme and <NUM>µL of lactose solution in a PCR tube. The reaction mixture was incubated in a DNA thermal cycler using the following incubating parameters (reaction time; <NUM> at <NUM>, enzyme inactivation; <NUM> at <NUM>, storage; <NUM>). The amount of glucose conversion was determined by transferring <NUM>µL of the reaction mixture in a single well of standard microtiter plate containing <NUM>µL of buffer C and incubated at <NUM> for <NUM>. After <NUM>, the absorbance was determined at <NUM> using FLUOStar Omega UV-plate reader (BMG Labtech, Germany). The measured absorbance values were used to draw a standard curve against BLU/mL. The maximum slope of the curve was used to determine the activity of new enzymes in BLU/mL.

The purified lactases were diluted up to 50x in buffer A (<NUM> NaH<NUM>PO<NUM> buffer pH <NUM> containing <NUM> of MgSO<NUM>). In a separate reaction, the diluted enzyme was incubated with lactose solution prepared in buffer H (<NUM> of lactose prepared in <NUM> sodium-citrate buffer of pH <NUM>, containing <NUM> of MgSO<NUM>). The reaction mixture was prepared by mixing <NUM>µL of diluted purified enzyme and <NUM>µL of lactose solution in a PCR tube. The reaction mixture was incubated in a DNA thermal cycler using the following incubating parameters (reaction time; <NUM> at <NUM>, enzyme inactivation; <NUM> at <NUM>, storage; <NUM>). After the reaction, <NUM>µL of the reaction mixture was transferred to one well of standard microtiter plate containing <NUM>µL of buffer C (as described in example <NUM>) and incubated at <NUM> for <NUM>. After <NUM>, the absorbance was determined at <NUM> using FLUOStar Omega UV-plate reader. The absorbance value between <NUM> and <NUM> were used for calculations, if the A610 nm value ><NUM>, the reaction mixture was diluted up to 5x with buffer A. The maximum absorbance values were used to calculate the enzyme activity in BLU/mL, using standard curve described in example <NUM>.

<NUM> of commercial pasteurized milk (<NUM> % Fat pasteurized milk, Arla Food) was mixed with <NUM>-<NUM>µL of enzyme (equivalent to <NUM> BLU/mL) as determined in the example <NUM>, in <NUM> glass tube. The samples were incubated under constant conditions for <NUM> hours at <NUM>. After the incubation, the reaction was stopped by heat inactivation at <NUM> for <NUM>, followed by storage at -<NUM> until further use. The amount of remaining lactose in the milk was analyzed using an HPLC assay. Samples for analysis were treated with <NUM> protein precipitation solution (<NUM> PCA and <NUM> Na-EDTA) and <NUM> of MQW prior to centrifugation at <NUM> rpm for <NUM> at <NUM>. An aliquot of the supernatant was diluted a total of <NUM>-fold using a Janus dilution robot (PerkinElmer, Waltham, MA, USA). The diluted samples were analyzed on a Dionex ICS-<NUM> system (Thermo Fischer Scientific, Waltham (MA), USA) using <NUM> x <NUM> CarboPac SA20 analytical column (Thermo Fischer Scientific, Waltham, MA, USA) and a pulsed amperometric detector. The detector was set to a simple three-step potential waveform, selective for detection of carbohydrates. The eluent was set to <NUM> KOH and was continuously regenerated through a trap column (CR-TC, Thermo Fischer Scientific, Waltham (MA), USA). The flow rate of the eluent was <NUM>/min and the analysis time was <NUM> per injection. The lactose in each sample was quantified using a three-point external calibration curve prepared by adding known amounts of lactose monohydrate (Sigma-Aldrich, St. Louis, MO, USA) to MQW. Concentrations were calculated based on the chromatographic peak heights. The measured percentage residual lactose in fresh milk is shown in <FIG>.

<NUM> of UHT milk (<NUM> % Fat UHT milk, Arla Food) was mixed with <NUM>-<NUM>µL of enzyme (equivalent to <NUM> BLU/mL) as determined in example <NUM>, in <NUM> glass tube. The samples were incubated under constant conditions for <NUM> hours at <NUM>. After the incubation, the reaction was stopped by heat inactivation at <NUM> for <NUM>, followed by storage at -<NUM> until further use. The amount of residual lactose in UHT milk was analyzed using HPLC by following the protocol as described in example <NUM>. The percentage of residual lactose in fresh milk after hydrolysis is listed in the <FIG>.

The purified enzyme was diluted to <NUM> BLU/mL in buffer A (<NUM> NaH<NUM>PO<NUM> buffer pH <NUM> containing <NUM> of MgSO<NUM>). In a separate reaction, <NUM>µL of the diluted enzyme was incubated in a DNA thermal cycler with lactose solution (<NUM> lactose prepared in <NUM> sodium-citrate buffer of pH <NUM>, containing <NUM> of MgSO<NUM>). The reaction mixture was prepared by mixing <NUM>µL of enzyme and <NUM>µL of lactose solution in a PCR tube. The reaction mixture was incubated in a DNA thermal cycler using the following incubating parameters (reaction time; <NUM> at <NUM>, enzyme inactivation; <NUM> at <NUM>, storage; <NUM>). After the reaction, <NUM>µL of the reaction mixture was transferred to one well of standard microtiter plate containing <NUM>µL of buffer C (as described in example <NUM>) and incubated at <NUM> for <NUM>. After <NUM>, the absorbance was determined at <NUM> using FLUOStar Omega UV-plate reader. The absorbance value between <NUM> and <NUM> were used for calculations, if the A610 nm value ><NUM>, the reaction mixture was diluted up to 5x with buffer A. The measured absorbance was called Abs37°C, and considered as reference value for calculations.

To measure the impact of heat treatment on enzyme activity, in a separate reaction, <NUM>µL of the diluted enzyme (<NUM> BLU/mL) was incubated in a DNA thermal cycler using the following incubating parameter (at <NUM> for <NUM> sec or <NUM> for <NUM> sec or <NUM> for <NUM> sec or <NUM> for <NUM> sec or <NUM> for <NUM> sec or <NUM> for <NUM> sec or <NUM> for <NUM> sec or <NUM> for <NUM> sec, followed by storage at <NUM>°°C). The activity of the heat treated enzyme was determined by incubation with the lactose solution (<NUM> lactose prepared in <NUM> sodium-citrate buffer of pH <NUM>, containing <NUM> of MgSO<NUM>), as described above. The measured absorbance at different temperature (for example at <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>) was called as Abs72°C, Abs74°C, Abs76°C, Abs78°C, Abs80°C, Abs85°C, Abs90°C, Abs95°C.

The percentage residual activity at high temperature was determined using the formula, <MAT>.

The percentages residual activities of different enzymes at different temperature are described in <FIG>.

The effect of heat treatment on the enzyme performance in pasteurized milk was determined by incubating a fixed amount of enzyme in the milk followed by a heat treatment. In separate reactions, <NUM>µL of the pasteurized milk was mixed with <NUM> BLU/mL of purified enzyme (as determined in example <NUM>), in a PCR tube. The milk sample was incubated at <NUM> for <NUM> or <NUM> for <NUM> sec or <NUM> for <NUM> sec and <NUM> for <NUM> sec, followed by incubation at <NUM> for <NUM>. After <NUM> at <NUM>, the reaction was stopped by heating the reaction at <NUM> for <NUM>, followed by storage at -<NUM>. The residual lactose was measured by using the LactoSens® assay kit (Chr. Hansen, Denmark), by following the supplied protocol. The measured residual lactose was determined in g/L was determined at different temperature. The detection limit of the LactoSens® kit is between <NUM>/L to <NUM>/L. The results are described in the table <NUM>:.

<NUM> of commercial pasteurized milk (<NUM>% fat milk containing <NUM>% lactose, Arla Foods, Denmark) was mixed with <NUM>/mL of enzyme, in a <NUM> Eppendorf tube. The enzyme was mixed in the milk with gentle vortex or pipetting. <NUM>µL of the milk, containing the enzyme, was transferred to a PCR tube. For each enzyme the reaction was performed in 2x50 µL reaction volume. The reaction mixture was incubated in a DNA thermal cycler with the following incubation parameters (reaction temperatures and time; <NUM> for <NUM> or <NUM> for <NUM> or <NUM> for <NUM>, enzyme inactivation temperature and time; <NUM> for <NUM>, storage temperature: <NUM>). During the enzyme addition, pipetting and mixing the milk samples were kept on ice-water mixture to minimize the effect of temperature on enzyme performance. After the reaction, the milk samples were either used directly for the residual lactose measurement or stored at -<NUM> until further use. The residual lactose in the milk was analyzed using LactoSens® assay kit (Chr. Hansen, Denmark) by following the protocol supplied with the kit. The measured percentage residual lactose in the pasteurized milk is shown in <FIG>.

To test the lactose hydrolysis potential of these novel lactases, we incubated <NUM>,<NUM> enzyme per milliliter of the pasteurized milk and incubated at <NUM>, <NUM> and <NUM> for <NUM>. After <NUM> incubation, the enzymes were inactivated by heating at <NUM>. The residual lactose was determined using LactoSens® assay kit (Chr. Hansen, Denmark). At their optimal temperature (<NUM>), both the Ha-Lactase and NOLA® fit showed a high residual lactose (><NUM>% of residual lactose), suggesting that enzymes have lower activity and are not producing lactose free pasteurized milk in the given time frame. Moreover, a similar level of residual lactose was measured at <NUM> and <NUM>. On the contrary, the G33, G44, G95 andG158 enzymes showed <<NUM>% residual lactose at <NUM>, <FIG>. Because of their high activity at elevated temperatures (<NUM> or <NUM>), the novel enzymes showed <<NUM>% residual lactose after <NUM> incubation. This shows that by using the current enzyme dose it is possible to produce essentially lactose free pasteurized and filtered milk in less than <NUM>. Filtered milk is more like raw milk than like pasteurized milk. The lactose hydrolysis at elevated temperature (<NUM>-<NUM>) in short time reduces the chance of microbial growth without affecting the milk quality.

<NUM> of commercial pasteurized milk (<NUM>% fat milk containing <NUM>% lactose, Arla Foods, Denmark) was mixed with <NUM>/mL of enzyme, in a <NUM> Eppendorf tube. The enzyme was mixed in the milk with gentle vortex or pipetting. <NUM>µL of the milk, containing the enzyme, was transferred to a PCR tube. For each enzyme the reaction was performed in 2x50 µL reaction volume. The reaction mixture was incubated in a DNA thermal cycler with the following incubation parameters (reaction temperatures and time; <NUM> for <NUM> or <NUM> for <NUM>, enzyme inactivation temperature and time; <NUM> for <NUM>, storage temperature: <NUM>). During the enzyme addition, pipetting and mixing the milk samples were kept on ice-water mixture to minimize the effect of temperature and time. After the reaction, the milk samples either used directly for the residual lactose measurement or stored at -<NUM> until further use. The residual lactose in the milk was analyzed using LactoSens® assay kit (Chr. Hansen, Denmark), as described in the example <NUM>. The measured percentage residual lactose in the pasteurized milk is shown in <FIG>.

<NUM> of commercial pasteurized milk (<NUM>% fat milk containing <NUM>% lactose, Arla Foods, Denmark) was mixed with either different enzyme doses (<NUM>/mL or <NUM>/mL), in <NUM> Eppendorf tube. The enzyme was mixed in the milk with gentle vortex or pipetting. <NUM>µL of the milk, containing the enzyme, was transferred to a PCR tube. For each enzyme the reaction was performed in 2x50 µL reaction volume. The reaction mixture was incubated in a DNA thermal cycler with the following incubation parameters (reaction temperatures and time; <NUM> for <NUM>, enzyme inactivation temperature and time; <NUM> for <NUM>, storage temperature: <NUM>). After the reaction, the samples either used directly for the residual lactose measurement or stored at -<NUM> until further use. The residual lactose in the milk was analyzed by following the same protocol as described in example <NUM>. The measured percentage residual lactose in the pasteurized milk is shown in <FIG>.

<NUM> of commercial pasteurized milk (<NUM>% fat milk containing <NUM>% lactose, Arla Foods, Denmark) was mixed with different enzyme dose (<NUM> or <NUM>/mL), in <NUM> Eppendorf tube. The enzyme was mixed in the milk with gentle vortex or pipetting. <NUM>µL of the milk, containing the enzyme, was transferred to a PCR tube. For each enzyme the reaction was performed in 2x50 µL reaction volume. The reaction mixture was incubated in a DNA thermal cycler with the following incubation parameters (reaction temperatures and time; <NUM> for <NUM> or <NUM> for <NUM>, enzyme inactivation temperature and time; <NUM> for <NUM>, storage temperature: <NUM>). During the enzyme addition, pipetting and mixing the milk samples were kept on ice-water mixture to minimize the effect of temperature and time. After the reaction, the samples either used directly used the residual lactose measurement or stored at -<NUM> until further use. The residual lactose was determined using the protocol described in example <NUM>. The measured percentage residual lactose in the pasteurized milk is shown in <FIG>.

<NUM> of commercial micro-filtered semi skimmed milk (<NUM>% fat milk containing <NUM>% lactose, Marguerite, France) was mixed with <NUM>/mL of enzyme, in <NUM> Eppendorf tube. The enzyme was mixed in the milk with gentle vortex or pipetting. <NUM>µL of the milk, containing the enzyme, was transferred to a PCR tube. For each enzyme the reaction was performed in 2x50 µL reaction volume. The reaction mixture was incubated in a DNA thermal cycler with the following incubation parameters (reaction temperatures and time; <NUM> for <NUM>, enzyme inactivation temperature and time; <NUM> for <NUM>, storage temperature: <NUM>). During the enzyme addition, pipetting and mixing the milk samples were kept on ice-water mixture to minimize the effect of temperature and time. After the reaction, the samples either used directly for the residual lactose measurement or stored at -<NUM> until further use. The amount of remaining lactose in the milk was analyzed using LactoSens® assay kit (Chr. Hansen, Denmark) by following the protocol supplied with the kit. The measured percentage residual lactose in the filtered milk is shown in <FIG>.

This shows that by using the current enzyme dose it is possible to produce lactose free filtered milk (filtered milk is more like raw milk than pasteurized) in less than <NUM>. The lactose hydrolysis at elevated temperature (<NUM>-<NUM>) in short time reduces the chance of microbial growth without affecting the milk quality.

<NUM> of commercial micro-filtered semi skimmed milk (<NUM>% fat milk containing <NUM>% lactose, Marguerite, France) was mixed with different enzyme doses (<NUM>/mL, <NUM> or <NUM>/mL, <NUM>), in <NUM> Eppendorf tube. The enzyme was mixed in the milk with gentle vortex or pipetting. <NUM>µL of the milk, containing the enzyme, was transferred to a PCR tube. For each enzyme the reaction was performed in 2x50 µL reaction volume. The reaction mixture was incubated in a DNA thermal cycler with the following incubation parameters (reaction temperatures and time; <NUM> for <NUM>, enzyme inactivation temperature and time; <NUM> for <NUM>, storage temperature: <NUM>). During the enzyme addition, pipetting and mixing the milk samples were kept on ice-water mixture to minimize the effect of temperature and time. After the reaction, the samples were either used directly for the residual lactose measurement or stored at -<NUM> until further use. The residual lactose in the milk was analyzed by following the protocol described in example <NUM>. The measured percentage residual lactose in the filtered milk is shown in <FIG>.

<NUM> of commercial micro-filtered semi skimmed milk (<NUM>% fat milk containing <NUM>% lactose, Marguerite, France) was mixed with <NUM>/mL (<NUM>) of enzyme, in <NUM> Eppendorf tube. The enzyme was mixed in the milk with gentle vortex or pipetting. <NUM>µL of the milk, containing the enzyme, was transferred to a PCR tube. For each enzyme the reaction was performed in 2x50 µL reaction volume. The reaction mixture was incubated in a DNA thermal cycler with the following incubation parameters (reaction temperatures and time; <NUM> for <NUM> or <NUM> for <NUM> or <NUM> for <NUM>, enzyme inactivation temperature and time; <NUM> for <NUM>, storage temperature: <NUM>). During the enzyme addition, pipetting and mixing the milk samples were kept on ice-water mixture to minimize the effect of temperature and time. After the reaction, the samples either used directly for residual lactose measurement or stored at -<NUM> until further use. The amount of remaining lactose in the milk was analyzed using LactoSens® assay kit (Chr. Hansen, Denmark) by following the protocol supplied with the kit. The measured percentage residual lactose in the filtered milk is shown in <FIG>. This shows that by using the current enzyme dose it is possible to produce lactose free pasteurized and filtered milk (filtered milk is more like raw milk than pasteurized) in less than <NUM>-<NUM>. The lactose hydrolysis at elevated temperature (<NUM>-<NUM>) in short time reduces the chance of microbial growth without affecting the milk quality.

To analyze the kinetics of lactose hydrolysis by the novel enzymes in pasteurized milk, <NUM> enzyme was added per milliliter of commercial pasteurized milk (<NUM>% fat milk containing <NUM>% lactose, Arla Foods, Denmark). The enzyme was mixed well by gentle vortex and transferred into PCR tube, 10x100 µL of each. The reaction mixtures were incubated at <NUM>, and after a fixed interval the samples was withdrawn. The reaction was stopped by heating at <NUM> for <NUM> in PCR machine. The samples were cooled to room temperature and the residual lactose was measured using LactoSens® assay kit (Chr. Hansen, Denmark). The measured value of residual lactose was plotted against reaction time.

At <NUM>-<NUM> the known commercial products, NOLA® Fit (G600) and Ha-Lactase™ (G500) require between <NUM>-<NUM> hr and <NUM>-<NUM> hr to reduce the concentration of residual lactose in cow milk to less than <<NUM>% and <<NUM>%, respectively (as shown in <FIG> and <FIG>).

The lactases of the present invention are significantly more active under these conditions. For example, the G95 (the most active enzyme) reaches a residual concentration of lactose of <<NUM>% level (<NUM> hr). The G158 and G33 are able to reduce the residual concentration of lactose to a level of <<NUM>% in between <NUM>-<NUM> hr and a level of <<NUM>% lactose in <NUM>-<NUM> hr. After <NUM> hr incubation, several of the lactases showed lower residual lactose than control enzymes (shown in <FIG> and <FIG>). These results show that the novel lactases are faster than Ha-Lactase™ and NOLA® Fit and result in lactose free pasteurized milk in significantly shorter time. These new enzymes can reduce the overall process time by <NUM>%. Additionally, the novel enzymes provide the possibility to reduce the enzyme dose further between <NUM>-<NUM>% to produce lactose free/reduced pasteurized milk (shown in <FIG>).

These results thus show that the novel lactases can produce lactose free pasteurized milk in significantly shorter time (<NUM>-<NUM> hr) with <NUM>/L enzyme dose. Moreover, it is possible to lower the enzyme dose by <NUM>-<NUM>%, depending on the required lactose level.

To compare enzyme activity in different milk types, pasteurized and filtered milk was incubated using lactase enzyme in a concentration of <NUM>/L. The samples were mixed and stored at <NUM> for <NUM> hr.

Claim 1:
A method for producing a dairy product comprising:
(a) mixing a milk-based substrate comprising lactose in a concentration of at least <NUM>/L and a peptide exhibiting beta-galactosidase activity in a concentration of <NUM> to <NUM>/L;
(b) incubating the mixture at a temperature from <NUM>-<NUM> for a period of time sufficient to reduce the lactose concentration in the mixture to less than <NUM>/L,
wherein the peptide exhibiting beta-galactosidase activity is:
a peptide having the amino acid sequence represented by SEQ ID NO: <NUM>.