FERMENTED MILK PRODUCT WITH A REDUCED CONTENT OF LACTOSE

FIELD OF THE INVENTION The present invention relates to an acidified milk product with a reduced content of lactose.

BACKGROUND OF THE INVENTION WO 2009/071539 discloses a lactase originating from Bifidobacterium bifidum, which is capable of very efficient hydrolysis in milk, and which is active over a broad pH range, including low pH, e.g. a pH below 5. The lactase may be used in processes for producing milk and fermented milk products, such as cheese, yogurt, butter, butter milk, sour cream etc., for reducing the content of lactose.

WO 2013/160413 discloses a method of producing a fermented milk product using a combination of glucose-negative lactic acid bacteria strains and a conventional lactase with an object of reducing the content of lactose in the fermented milk product while increasing the content of glucose.

EP-A1-2 957 180 discloses a method of producing a fermented milk product using a combination of a starter cultures and a conventional lactase with an object of reducing content of lactose and the level of post-acidification in the fermented milk product.

SUMMARY OF THE INVENTION

The object of the present invention is to provide an improved acidified milk product with reduced lactose content.

The object of the present invention is obtained by an acidified milk product, which has a pH of between 3.0 and 5.0 and a content of lactose of at least 1.5 mg/ml, wherein the product contains a lactase, which retains its activity at a pH of 5.0 and a temperature of 37°C at a level of at least 5% as compared to its activity at the optimum pH of the lactase. The present invention has provided a possibility of modifying a lactose-containing food product with a pH of between 3.0 and 5.0 in a storage phase at ambient storage temperature, i.e. without the need for refrigeration, after completion of the production at a production site, e.g. during transport and storage at the retailer, so as to reduce the level of lactose. The present invention is based on the recognition that for a product of low pH and intended for transport and storage at ambient temperature, e.g. post-pasteurized fermented milk products, it is possible to carry out a reduction of the lactose content after completion of the production at a production site by means of adding a low pH-active lactase after the usual food production process has been completed and allowing the lactase to convert lactose to galactose and glucose during the subsequent life phases of the product up until its final consumption by the end consumer. Thus, the invention allows for obtaining the step of the lactose conversion in a simple and cost-effective manner outside the production site, and hence the process for producing the fermented milk product at the production site may be simplified by exclusion of the step of completing the conversion of lactose thereby saving time and production equipment capacity.

Furthermore, when the lactase is added after heat treatment of the fermented milk product, it is possible to use a lower amount of lactase than when the lactase is active during the fermentation, because the lactase may be allowed to be active for a prolonged period of time, whereas in a fermentation process in a production it is desired to conduct the process as quickly as possible in order to reduce costs. Most of the lactase will be inactivated during heat treatment and will hence not be active after heat treatment. Thus, the present invention has provided a possibility of reducing the amount of lactase needed for obtaining a removal of the lactose content of a fermented milk product. Also, when the lactase is added after heat treatment of the fermented milk product, the level of Maillard reaction caused by the heat treatment of the fermented milk product is reduced, because lactose give rise to less Maillard reaction than the carbohydrate metabolites of lactose. Finally, when the lactase is added after heat treatment of the fermented milk product, any adverse effect of the lactic acid bacteria used in the fermentation on the activity of the lactase can be avoided.

Worldwide, a significant numbers of consumers are intolerant or sensitive to lactose. Therefore, there is presently a high demand for dairy products, including fermented milk products, with a reduced content of lactose or which is substantially free of lactose. The present invention has provided a new approach for producing such product in a simple and cost-efficient manner. DETAILED DISCLOSURE OF THE INVENTION Lactase

The lactase of the fermented milk product of the invention may be any lactase, which retains its activity at a pH of 5.0 and a temperature of 37°C at a level of at least 5% as compared to its activity at the optimum pH of the lactase.

In relation to the present invention the activity in LAU of the lactase is measured as specified in the "Definitions" section below.

In a preferred embodiment of the invention, the lactase retains its activity at a pH of 5.0 and a temperature of 37°C at a level of at least 10 %, preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, and most preferably at least 80%, as compared to its activity at the optimum pH of the lactase.

In a preferred embodiment of the invention, the lactase retains its activity at a pH of 4.0 and a temperature of 37°C at a level of at least 5%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, and most preferably at least 80%, as compared to its activity at the optimum pH of the lactase.

In a preferred embodiment of the invention, the lactase retains its activity at a pH of 3.0 and a temperature of 37°C at a level of at least 5%, preferably at least 10%, more preferably at least 20 %, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, and most preferably at least 80%, as compared to its activity at the optimum pH of the lactase. In connection with the present invention the optimum pH of the lactase is determined by measuring the lactase activity at different pH using the method indicated in the "Definitions" section below and determining the pH with optimum activity. In particular, the lactase activity at different pH is measured in M-buffer and at 37°C. Alternatively, the lactase activity indicated in the present application as a percentage of the activity at the optimum pH of the lactase is instead to be considered to be a percentage of the lactase activity at pH 6.5.

In a preferred embodiment of the invention, the lactase retains its activity at a temperature of 10°C and a pH of 6.0 at a level of at least 10% as compared to its activity at the optimum temperature of the lactase. Preferably, the lactase retains its activity at a temperature of 10°C and a pH of 6.0 at a level of at least 20%, more preferably at least 30%, more preferably at least 40 %, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, and most preferably at least 80%, as compared to its activity at the optimum temperature of the lactase. In connection with the present invention the optimum temperature of the lactase is determined by measuring the lactase activity at different temperatures using the method indicated in the "Definitions" section below and determining the temperature with optimum activity. Alternatively, the lactase activity indicated in the present application as a percentage of the activity at the optimum temperature of the lactase is instead to be considered to be a percentage of the lactase activity at a

temperature of 37°C.

In a preferred embodiment, the lactase to be used in the product of the present invention has a lactase activity at 37°C and pH 5 which is at least 55%, such as at least 60%, at least 65%, at least 70% or at least 75%, of its lactase activity at 37°C and pH 6.

In another preferred embodiment, the lactase to be used in the product of the present invention has a lactase activity at 37°C and pH 4.5 which is at least 10%, such as at least 20%, at least 30%, at least 35% or at least 40%, of its lactase activity at 37°C and pH 6.

In another preferred embodiment, the lactase to be used in the product of the present invention has a pH optimum of the lactase activity at 37°C which is above pH 5.5.

In another preferred embodiment, the lactase to be used in the product of the present invention has a lactase activity at a temperature of 52°C and a pH of 6.5 which is at least 50%, such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%, of its lactase activity at a temperature of 38°C and a pH of 6.5.

In a preferred embodiment of the present invention, Km (Michaelis constant) of the lactase at 5°C is below 25 mM, such as below 20 mM, below 15 mM or below 10 mM. In another preferred embodiment, Km of the lactase at 37°C is below 25 mM, such as below 20 mM or below 15 mM. The skilled person will know how to determine Km for the lactase activity at a specific temperature. Km may be determined by the method described in WO2009/071539.

In another preferred embodiment, the enzyme when hydrolyzing the lactose in the milk product has a ratio of lactase to transgalactosylase activity of more than 1 : 1, such as more than 2 : 1 or more than 3 : 1. In another preferred embodiment, the enzyme treatment is performed under conditions where the lactase activity of the enzyme is higher than the transgalactosylase activity, such as at least two times higher or at least three times higher. The ratio of lactase to transgalactosylase activity in the milk product may, e.g., be determined by HPLC analysis. In another preferred embodiment, the enzyme treatment is performed under conditions where at least 50% (w/w%) of the hydrolyzed lactose is converted into free galactose. In another preferred embodiment, the enzyme treatment is performed under conditions where the hydrolyzed lactose is converted into equal amounts of free glucose and free galactose.

A lactase in the context of the present invention is a glycoside hydrolase having the ability to hydrolyze the disaccharide lactose into constituent galactose and glucose monomers. The group of lactases, to which the lactase of the invention belongs, comprises but is not limited to enzymes assigned to subclass EC 3.2.1.108. Enzymes assigned to other subclasses, such as, e.g ., EC 3.2.1.23, may also be lactases in the context of the present invention. A lactase in the context of the invention may have other activities than the lactose hydrolyzing activity, such as for example a transgalactosylating activity. In the context of the invention, the lactose hydrolyzing activity of the lactase may be referred to as its lactase activity or its beta- galactosidase activity. Enzymes having lactase activity to be used in a method of the present invention may be of animal, of plant or of microbial origin. Preferred lactases are obtained from microbial sources, in particular from a filamentous fungus or yeast, or from a bacterium.

The enzyme may, e.g., be derived from a strain of Agaricus, e.g. A. bisporus;

Ascovaginospora; Aspergillus, e.g. A. niger, A. awamori, A. foetidus, A. japonicus, A. oryzae; Candida; Chaetomium; Chaetotomastia; Dictyostelium, e.g. D. discoideum; Kluveromyces, e.g. K. frag His, K. lactis; Mucor, e.g. M. javanicus, M. mucedo, M. subtilissimus; Neurospora, e.g. N. crassa; Rhizomucor, e.g. R. pusillus; Rhizopus, e.g. R. arrhizus, R. japonicus, R. stolon ifer; 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. longum, B. bifid um, 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. reesei, T. viride; Yersinia, e.g. Y. enterocolitica.

In a preferred embodiment, the lactase originates from a bacterium, e.g. from the family Bifidobacteriaceae, such as from the genus Bifidobacterium, such as from a strain of B. bifidum, B. animalis or B. longum. In a more preferred embodiment, the lactase originates from Bifidobacterium bifidum.

In a preferred embodiment, an enzyme having lactase activity to be used in the product of the present invention comprises an amino acid sequence which is at least 50% identical to a sequence selected from the group consisting of amino acids 28- 1931 of SEQ ID NO: 1, amino acids 28-1331 of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and lactase active fragments thereof. In a more preferred embodiment, the enzyme comprises an amino acid sequence which is at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to a sequence selected from the group consisting of amino acids 28-1931 of SEQ ID NO: 1, amino acids 28-1331 of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO : 4 and lactase active fragments thereof.

A preferred enzyme is a lactase having a sequence which is at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to amino acids 28-1931 of SEQ ID NO: 1 or to a lactase active fragment thereof. Such lactase active fragment of SEQ ID NO: 1 may be any fragment of SEQ ID NO: 1 having lactase activity. A lactase active fragment of SEQ ID NO: 1 may be, e.g., amino acids 28-979, amino acids 28-1170, amino acids 28-1323, amino acids 28-1331, or amino acids 28-1600 of SEQ ID NO: 1.

In a preferred embodiment, an enzyme having lactase activity to be used in the product of the present invention comprises an amino acid sequence which is at least 50% identical to amino acids 28-1331 of SEQ ID NO: 2. In a more preferred embodiment, the enzyme comprises an amino acid sequence which is at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to amino acids 28-1331 of SEQ ID NO: 2.

In another embodiment, an enzyme having lactase activity to be used in product of the present invention has an amino acid sequence which is at least 50% identical to SEQ ID NO: 3. Preferably, the enzyme has an amino acid sequence which is at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to SEQ ID NO: 3.

In another embodiment, an enzyme having lactase activity to be used in the product of the present invention has an amino acid sequence which is at least 50% identical to SEQ ID NO: 4. Preferably, the enzyme has an amino acid sequence which is at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to SEQ ID NO: 4.

For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. (2000) Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labelled "longest identity" (obtained using the -no brief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

A particular commercial lactase suitable for use in the present invention is Lactase F "Amano" 100SD available from Amano Enzyme Europe.

Lactases to be used in a method of the present invention may be extracellular. They may have a signal sequence at their N-terminus, which is cleaved off during secretion.

Lactases to be used in a method of the present invention may be derived from any of the sources mentioned herein. The term "derived" means in this context that the enzyme may have been isolated from an organism where it is present natively, i.e. the identity of the amino acid sequence of the enzyme are identical to a native enzyme. The term "derived" also means that the enzymes may have been produced recombinantly in a host organism, the recombinantly produced enzyme having either an identity identical to a native enzyme or having a modified amino acid sequence, e.g. having one or more amino acids which are deleted, inserted and/or substituted, i.e. a recombinantly produced enzyme which is a mutant and/or a fragment of a native amino acid sequence. Within the meaning of a native enzyme are included natural variants. Furthermore, the term "derived" includes enzymes produced synthetically by, e.g., peptide synthesis. The term "derived" also encompasses enzymes which have been modified e.g. by glycosylation, phosphorylation etc., whether in vivo or in vitro. With respect to recombinantly produced enzyme the term "derived from" refers to the identity of the enzyme and not the identity of the host organism in which it is produced recombinantly.

The lactase may be obtained from a microorganism by use of any suitable technique. For instance, a lactase enzyme preparation may be obtained by fermentation of a suitable microorganism and subsequent isolation of a lactase preparation from the resulting fermented broth or microorganism by methods known in the art. The lactase may also be obtained by use of recombinant DNA techniques. Such method normally comprises cultivation of a host cell transformed with a recombinant DNA vector comprising a DNA sequence encoding the lactase in question and the DNA sequence being operationally linked with an appropriate expression signal such that it is capable of expressing the lactase in a culture medium under conditions permitting the expression of the enzyme and recovering the enzyme from the culture. The DNA sequence may also be incorporated into the genome of the host cell. The DNA sequence may be of genomic, cDNA or synthetic origin or any combinations of these, and may be isolated or synthesized in accordance with methods known in the art.

Lactases to be used in a method of the present invention may be purified. The term "purified" as used herein covers lactase enzyme protein essentially free from insoluble components from the production organism. The term "purified" also covers lactase enzyme protein essentially free from insoluble components from the native organism from which it is obtained . Preferably, it is also separated from some of the soluble components of the organism and culture medium from which it is derived. More preferably, it is separated by one or more of the unit operations: filtration, precipitation, or chromatography. Accordingly, the enzyme having lactase activity may be purified, viz. only minor amounts of other proteins being present. The expression "other proteins" relate in particular to other enzymes. The term "purified" as used herein also refers to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the lactase. The lactase may be "substantially pure", i.e. free from other components from the organism in which it is produced, i.e., e.g., a host organism for recombinantly produced lactase. Preferably, the lactase is an at least 40% (w/w) pure enzyme protein preparation, more preferably at least 50%, 60%, 70%, 80% or even at least 90% pure. The term enzyme having lactase activity includes whatever auxiliary compounds that may be necessary for the enzyme's catalytic activity, such as, e.g., an appropriate acceptor or cofactor, which may or may not be naturally present in the reaction system. The enzyme may be in any form suited for the use in question, such as, e.g., in the form of a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a protected enzyme. Acidified milk product

In a particular embodiment of the invention, the acidified product of the invention has a pH of between 3.2 and 4.8, more preferably between 3.4 and 4.6 and most preferably between 3.6 and 4.4.

In one embodiment of the invention, the acidified milk product is a chemically acidified milk product. The acidification may be carried out by means of any acidifying agent approved for food products, such as lactic acid, citric acid, malic acid, tartaric acid, phosphoric acid, fumaric acid, fruit juice, fruit pulp and fruit compound.

In another embodiment of the invention, the acidified milk product is a fermented milk product produced by fermentation using a starter culture. The starter culture may be any conventional starter culture of lactic acid bacteria, including single strain culture and culture blends, used for producing a specific type of fermented milk product. Other useful bacteria, which may be added to the product in addition to the starter culture, include the probiotic bacteria Bifidobacterium spp.

In a preferred embodiment of the invention, the fermented milk product after fermentation has been subjected to a heat treatment so as to reduce the level of bacteria of the starter culture to no more than lX10exp02 CFU per g, and wherein the lactase has been added after heat treatment.

The starter culture may be any conventional starter culture of lactic acid bacteria, including single strain culture and culture blends, used for producing a specific type of fermented milk product. In a preferred embodiment of the product of the invention, the fermentation is carried out so as to obtain a pH of between 3.0 and 5.0, preferably between 3.2 and 4.8, more preferably between 3.4 and 4.6 and most preferably between 3.6 and 4.4.

The heat treatment so as to reduce the level of bacteria of the starter culture to no more than 1.0X10exp02 CFU per g fermented milk is preferably carried out by subjecting the starter culture fermented milk product to a temperature of between 50°C and 110°C, preferably between 50°C and 100°C, preferably between 50°C and 90°C, preferably between 60°C and 85°C, more preferably between 65°C and 82°C, and most preferably between 70°C and 80°C. The heat treatment is preferably carried out for a period of between 5 seconds and 180 seconds, preferably between 5 seconds and 120 seconds, more preferably between 5 seconds and 90 seconds, more preferably between 5 seconds and 60 seconds, more preferably between 8 seconds and 50 seconds and most preferably between 10 and 40 seconds.

Preferably, the level of bacteria of the starter culture is reduced to no more than l .OXlOexpOl CFU per g fermented milk, more preferably 0 CFU per g.

The enzyme is added in a suitable amount to achieve the desired degree of lactose hydrolysis under the chosen reaction conditions. In a particular embodiment of the invention, the milk product contains lactase in an amount of between 100 and 20000 LAU per liter milk product, preferably between 100 and 10000 LAU per liter milk product, preferably between 100 and 5000 LAU per liter milk product, preferably less than 3000, such as less than 1500, less than 1000, less than 750 or less than 500, LAU per liter milk product.

In a preferred embodiment, the lactase is added at a concentration of between 5 and 400 LAU per g lactose in the milk product, preferably between 5 and 200 LAU per g lactose in the milk product, preferably between 5 and 100 LAU per g lactose in the milk product, preferably less than 50, such as less than 40, less than 30, less than 20 or less than 10, LAU per g lactose in the milk product.

In a preferred embodiment of the invention, the acidified milk product has a content of lactose of between 2.0 mg/ml and 50 mg/ml, preferably between 5 mg/ml and 48 mg/ml, more preferably between 10 mg/ml and 46 mg/ml, and most preferably between 20 mg/ml and 45 mg/ml.

In a preferred embodiment of the invention, the acidified milk product contains a further food product selected from the group consisting of fruit beverage, fermented cereal products, chemically acidified cereal products, soy milk products and any mixture thereof.

The acidified milk product typically contains protein in a level of between 2.0% by weight to 3.5% by weight. The acidified milk product may also be a low protein product with a protein level of between 1.0% by weight and 2.0% by weight.

Alternatively, the acidified milk product may be a high protein product with a protein level of above 3.5% by weight. In a particular embodiment of the acidified milk product of the invention the product is a mixture of an acidified milk product and a cereal product, e.g. an oat product, wherein the cereal product may be a fermented cereal product, e.g. a fermented oat product. In a particular embodiment of the invention, the acidified milk product contains a fermented cereal product. The fermented cereal product may be prepared by milling the grains of a cereal biological source material to produce a cereal flour, which is then subjected to fermentation. The fermentation of the cereal flour may be carried out using the same lactic acid bacteria (starter culture) as used for fermentation of a milk substrate as described elsewhere in this application.

In a particular embodiment of the invention, the acidified milk product contains a fruit beverage. The fruit beverages may further contain e.g. oat, soy, almond, whey and/or non-fermented milk, e.g. in the form of milk powder.

In a particular embodiment of the invention the acidified milk product of the invention is a chemically acidified product. The acidification may be carried out using any acidifying agent suitable for adding to food products, such as lactic acid, citric acid, fruit juice, fruit pulp and fruit compound. In a particular embodiment, the acidified milk product is acidified with fruit juice.

In a particular embodiment of the invention, the acidified milk product contains a chemically acidified cereal product. The chemically acidified cereal product may be prepared by milling the grains of a cereal biological source material to produce a cereal flour, which is then used to produce an aqueous suspension, and the pH of the said suspension is then adjusted to a desired level.

Acidified milk product containing acid whey or acid whey permeate In a particular aspect of the invention, the acidified milk product contains an acid whey product selected from the group consisting of acid whey and acid whey permeate. In particular, the acid whey product is obtained from concentration of a fermented milk product to divide it into a concentrated fraction and a separated acid whey fraction.

Acid whey and acid whey permeate are by-products from the production of a number of fresh cheeses, such as cottage cheese, ricotta, Skyr, Greek Yogurt, Tvoroq, quark and Labneh. Acid whey and acid whey permeate have a pH of less than 5.1 in a 10% solution, and historically it has been difficult to find a use for the by-products, e.g. due to its acidity. Acid whey is obtained by concentration of yogurt in a separator. Acid permeate is obtained by concentration of yogurt in

ultrafiltration.

This present aspect of the invention has provided a possibility of using acid whey and acid whey permeate to produce a milk product, e.g. a milk beverage. The aspect is based on the recognition that acid whey and acid whey permeate are suitable for producing the milk product of the invention with a reduced content of lactose, since the production of the product requires enzymatic hydrolysis of lactose at a low pH .

In a particular embodiment of the third aspect of the process of the invention, the starter culture fermented milk product is subjected to a concentration step to divide the starter culture fermented milk product into a concentrated fraction and a separated acid whey fraction, wherein the separated acid whey fraction and not the concentrated fraction is subjected to the subsequent steps of the process.

Process of producing a fermented milk product

In a first aspect the present invention further relates to a process for producing an acidified milk product comprising the steps of providing a basic acidified milk product, which has a pH of between 3.0 and 5.0 and a content of lactose of at least 1.5 mg/ml, adding to the basic acidified milk product a lactase, which retains its activity at a pH of 5.0 and a temperature of 37°C at a level of at least 5% as compared to its activity at the optimum pH of the lactase to obtain a lactase- containing acidified milk product, and storing the lactase-containing acidified milk product at a temperature of at least 2°C for at least 1 day. In a second aspect the present invention relates to a process for producing an acidified milk product comprising the steps of providing a basic acidified milk product, which has a pH of between 3.0 and 5.0 and a content of lactose of at least 1.5 mg/ml, subjecting the basic acidified milk product to a heat treatment so as to reduce the level of bacteria to no more than lX10exp02 CFU per g to obtain a heat treated acidified milk product, adding to the heat treated acidified milk product a lactase, which retains its activity at a pH of 5.0 and a temperature of 37°C at a level of at least 5% as compared to its activity at the optimum pH of the lactase to obtain a lactase-containing acidified milk product, and storing the lactase-containing acidified milk product at a temperature of at least 2°C for at least 1 day.

In an alternative wording the second aspect is a particular embodiment of the first aspect comprising the steps of subjecting the basic acidified milk product to a heat treatment so as to reduce the level of bacteria to no more than lX10exp02 CFU per g to obtain a heat treated acidified milk product, adding to the heat treated acidified milk product a lactase, which retains its activity at a pH of 5.0 and a temperature of 37°C at a level of at least 5% as compared to its activity at the optimum pH of the lactase to obtain a lactase-containing acidified milk product, and storing the lactase- containing acidified milk product at a temperature of at least 2°C for at least 1 day.

In a third aspect the present invention relates to a process for producing a fermented milk product comprising the steps of fermentation of a milk substrate using a starter culture of lactic acid bacteria to obtain a starter culture fermented milk product, which has a pH of between 3.0 and 5.0 and a content of lactose of at least 1.5 mg/ml, subjecting the starter culture fermented milk product to a heat treatment so as to reduce the level of bacteria to no more than lX10exp02 CFU per g to obtain a heat treated fermented milk product, adding to the heat treated fermented milk product a lactase, which retains its activity at a pH of 5.0 and a temperature of 37°C at a level of at least 5% as compared to its activity at the optimum pH of the lactase to obtain a lactase-containing fermented milk product, and storing the lactase-containing fermented milk product at a temperature of at least 2°C for at least 1 day.

In an alternative wording the third aspect is a particular embodiment of the first aspect, wherein the acidified milk product produced is a fermented milk product, and wherein the process comprises the steps of fermentation of a milk substrate using a starter culture of lactic acid bacteria to obtain a starter culture fermented milk product, which has a pH of between 3.0 and 5.0 and a content of lactose of at least 1.5 mg/ml, subjecting the starter culture fermented milk product to a heat treatment so as to reduce the level of bacteria to no more than lX10exp02 CFU per g to obtain a heat treated fermented milk product, adding to the heat treated fermented milk product a lactase, which retains its activity at a pH of 5.0 and a temperature of 37°C at a level of at least 5% as compared to its activity at the optimum pH of the lactase to obtain a lactase-containing fermented milk product, and storing the lactase-containing fermented milk product at a temperature of at least 2°C for at least 1 day.

In one embodiment of the process of the invention, the acidified milk product is a chemically acidified milk product. In another embodiment of the invention, the acidified milk product is a fermented milk product produced by fermentation using a starter culture.

In one embodiment of the process of the invention, the basic acidified milk product or the a starter culture fermented milk product may be subjected to a heat treatment so as to reduce the level of bacteria to no more than lX10exp02 CFU per g. Such a heat treated acidified product is also referred to as a post-pasteurized product, by which it is indicated that the product has been subjected to

pasteurization after production of the chemically acidified milk product or fermented milk product. Such a heat treated acidified product need not be kept at refrigerated temperature, and hence it is suitable for transport and storage at ambient temperature.

Thus, in a preferred embodiment of the process of the invention, the lactase- containing acidified or fermented milk product is stored at a temperature of at least 5°C, preferably at least 10°C, more preferably at least 15°C, and most preferably at least 20°C.

In a particular embodiment of the process of the invention, the lactase-containing acidified or fermented milk product is stored for at least two days, preferably at least 3 days, more preferably at least 4 days, more preferably at least 5 days, more preferably at least 6 days, and most preferably at least 7 days.

In a particular embodiment of the invention, the lactase-containing acidified or fermented milk product after storage has a content of lactose of less than 40 mg/ml, preferably less than 35 mg/ml, more preferably less than 30 mg/ml, more preferably less than 25 mg/ml, more preferably less than 20 mg/ml, more preferably less than 15 mg/ml, more preferably less than 10 mg/ml, more preferably less than 5 mg/ml, more preferably less than 3 mg/ml, and most preferably less than 1.5 mg/ml. The starter culture may be any conventional starter culture of lactic acid bacteria, including single strain culture and culture blends, used for producing a specific type of fermented milk product. In a preferred embodiment of the product of the invention, the fermentation is carried out so as to obtain a pH of between 3.0 and 5.0, preferably between 3.2 and 4.8, more preferably between 3.4 and 4.6 and most preferably between 3.8 and 4.4. The heat treatment so as to reduce the level of bacteria of the starter culture to no more than 1.0X10exp02 CFU per g fermented milk is preferably carried out by subjecting the starter culture fermented milk product to a temperature of between 50°C and 110°C, preferably between 50°C and 100°C, preferably between 50°C and 90°C, preferably between 60°C and 85°C, more preferably between 65°C and 82°C, and most preferably between 70°C and 80°C. The heat treatment is preferably carried out for a period of between 5 seconds and 180 seconds, preferably between 10 seconds and 180 seconds, preferably between 12 seconds and 120 seconds, more preferably between 14 seconds and 90 seconds, more preferably between 16 seconds and 60 seconds, more preferably between 18 seconds and 50 seconds and most preferably between 20 and 40 seconds. Preferably, the level of bacteria of the starter culture is reduced to no more than l.OXlOexpOl CFU per g fermented milk, more preferably 0 CFU per g. Fermented milk products subjected to a heat treatment so as to reduce the level of bacteria to no more than lX10exp02 CFU per g are suitable for use storage at ambient temperature, such as storage at a temperature of at least 5°C, preferably at least 10°C, more preferably at least 15°C, and most preferably at least 20°C.

In a fourth aspect the present invention relates to a process for producing a fermented milk product comprising the steps of fermentation of a milk substrate using a starter culture of lactic acid bacteria to obtain a starter culture fermented milk product, which has a pH of between 3.0 and 5.0 and a content of lactose of at least 1.5 mg/ml, subjecting the starter culture fermented milk product to a heat treatment so as to reduce the level of bacteria to no more than lX10exp08 CFU per g to obtain a heat treated fermented milk product, adding to the heat treated fermented milk product a lactase, which retains its activity at a pH of 5.0 and a temperature of 37°C at a level of at least 5% as compared to its activity at the optimum pH of the lactase to obtain a lactase-containing fermented milk product, and storing the lactase-containing fermented milk product at a temperature of at least 2°C for at least 1 day. Preferably, the level of bacteria of the starter culture is reduced to no more than 1.0X10exp07 CFU per g fermented milk, more preferably no more than 1.0X10exp06 CFU per g fermented milk, and most preferably no more than 1.0X10exp05 CFU per g fermented milk. Fermented milk products subjected to a heat treatment so as to reduce the level of bacteria to no more than e.g.

lX10exp08 CFU per g have an extended shelf life at refrigerated temperature.

The enzyme is added in a suitable amount to achieve the desired degree of lactose hydrolysis under the chosen reaction conditions. In a particular embodiment of the invention, the milk product contains lactase in an amount of between 100 and 20000 LAU per liter milk product, preferably between 100 and 10000 LAU per liter milk product, preferably between 100 and 5000 LAU per liter milk product, preferably less than 3000, such as less than 1500, less than 1000, less than 750 or less than 500, LAU per liter milk product.

In a preferred embodiment, the lactase is added at a concentration of between 5 and 400 LAU per g lactose in the milk product, preferably between 5 and 200 LAU per g lactose in the milk product, preferably between 5 and 100 LAU per g lactose in the milk product, preferably less than 50, such as less than 40, less than 30, less than 20 or less than 10, LAU per g lactose in the milk product.

In a preferred embodiment of the invention, the acidified milk product has a content of lactose of between 2.0 mg/ml and 45 mg/ml, preferably between 5 mg/ml and 40 mg/ml, more preferably between 10 mg/ml and 37 mg/ml, and most preferably between 20 mg/ml and 37 mg/ml.

In a preferred embodiment of the invention, the acidified milk product, to which lactase is to be added, has a viscosity, which allows easy distribution of the lactase in acidified milk product, e.g. by mixing. In a preferred embodiment of the process of the invention the lactase to be added to the fermented milk product is provided in a sterile formulation. In another preferred embodiment of the process of the invention the lactase is added to the fermented milk product under aseptic conditions, e.g. by sterile filtration of a solution of the lactase.

In a preferred embodiment of the process of the invention, the lactase is added to the acidified milk product by in-line dosing. In connection with the present invention, the term "in-line dosing" means dosing directly into a pipe through which the acidified milk product flows. Here, the term "pipe" means pipe or any synonym thereof, incl. channel, conduit, duct, leader, line, penstock, through and tube.

Examples of commercial in-line dosing systems suitable for use in the present invention are Tetra FlexDos ^® Aseptic in-line dosing and Tetra Aldose ^® Aseptic in-line dosing.

In a preferred embodiment of the process of the invention, the lactase is added to the acidified milk product by in-line dosing into a pipe, and subsequently the lactase is mixed into the yogurt in the pipe by a mixing device. Preferably, the mixing device is selected from the group consisting of at least one bend of the pipe, a back pressure spring, a static mixer or a rotor / static mixer. A commercial example of a rotor / static mixer is Ytron-Z homogenizer (shear pump). Two-step addition of lactase

In a particular embodiment of the third and fourth aspect of the process of the invention, a lactase is added upstream of the heat treatment of the starter culture fermented milk product. The upstream-added lactase may be any lactase, including a lactase selected from the group consisting of a Ha-lactase and the lactase used in the process of the invention, i.e. a lactase, which retains its activity at a pH of 5.0 and a temperature of 37°C at a level of at least 5% as compared to its activity at the optimum pH of the lactase. A Ha-lactase is a lactase obtained from yeast from the genus Klyveromyces, in particular selected from the group consisting of

Klyveromyces lactis and Klyveromyces fragilis.

In a particular embodiment the upstream-added lactase is added at the start of the fermentation together with the starter culture. In this case the upstream-added lactase is preferably a Ha-lactase.

In another particular embodiment the upstream-added lactase is added after the start of the fermentation.

When adding lactase both at the start of the fermentation and after heat treatment of the starter culture fermented milk product, part of the lactose will be hydrolyzed during the fermentation step and the remaining part of the lactose will be

hydrolyzed during storage of the heat-treated fermented milk product. When adding lactase both at the start of the fermentation and after heat treatment of the starter culture fermented milk product, it is possible to reduce to total amount of lactase added in order to reduce the concentration of lactose in the fermented milk product to a desired level as compared to adding lactase only after heat treatment of the starter culture fermented milk product. Thus, the lactase added at the start of the fermentation will be active at a higher pH than a pH of between 3.0 and 5.0, e.g. at a pH of between 5.0 and 7.0, and most lactases have a higher activity at such a higher pH. Therefore, a given amount of lactase added at the start of the

fermentation will result in a higher hydrolysis of lactose than the same amount of lactase added after heat treatment of the starter culture fermented milk product.

Use

The present invention further relates to use of a lactase, which retains its activity at a pH of 5.0 and a temperature of 37°C at a level of at least 5% as compared to its activity at the optimum pH of the lactase, for adding to an acidified milk product, which has a pH of between 3.0 and 5.0 and a content of lactose of at least 1.5 mg/ml, in order to reduce the said lactose during storage. Definitions

In connection with the present invention the following definitions apply:

"LAU" means "Lactose Units" and 1 lactase unit (1 LAU) is the amount of enzyme which releases 1 micromole glucose per minute in M-buffer at pH 6.5 and 37°C with a lactose concentration of 4.75% w/v. M-buffer is prepared by dissolving 3.98 g C ₆ H ₅ Na307-2H ₂ 0, 8.31 g citric acid, 0.9 g K ₂ S0 ₄ , 2.6 g K ₂ HP0 ₄ , 7.35 g KH ₂ P0 ₄ , 5.45 g KOH, 4.15 g MgCI ₂ -6H ₂ 0, 3.75 g CaCI ₂ -2H ₂ 0 and 1.4 g NaHC0 ₃ in 4 liter water, adding 12.5 ml 4N NaOH, adjusting to pH 6.5 using HCI, and adding water up to a total volume of 5 liter.

The activity in LAU of a specific lactase may be determined by direct measurement of glucose released from lactose under the conditions described above. The skilled person will know how to determine such activity. Alternatively, the activity may be determined by using the lactase activity assay described in Example 1 of the present application. Here, the activity is obtained by comparing to a standard curve run with a lactase of known activity, and the activity of the unknown sample calculated from this. The lactase of known activity may, e.g., be Lactozym obtained from Novozymes A/S, Denmark.

The expression "heat treatment" means any treatment using any temperature, for any period of time and by any means or equipment, which inactivates at least a portion of the bacteria of the starter culture. In this connection the term "inactivate" means any stop, reduction or inhibition of growth of the bacteria, e.g. cell lysing.

The expression "lactic acid bacteria" designates a gram-positive, microaerophilic or anaerobic bacteria, which ferment sugars with the production of acids including lactic acid as the predominantly produced acid, acetic acid and propionic acid. The industrially most useful lactic acid bacteria are found 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. These are frequently used as food cultures alone or in combination with other lactic acid bacteria.

Lactic acid bacteria, including bacteria of the species Lactobacillus sp. and

Lactococcus sp., are normally supplied to the dairy industry either as frozen or freeze-dried cultures for bulk starter propagation or as so-called "Direct Vat Set" (DVS) cultures, intended for direct inoculation into a fermentation vessel or vat for the production of a dairy product, such as a fermented milk product or a cheese. Such lactic acid bacterial cultures are in general referred to as "starter cultures" or "starters". Typically, a starter culture for yogurt comprises Streptococcus

thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and in most countries a yogurt is by legislation defined as a fermented milk product produced using a starter culture comprising the two said strains.

The term "milk" is to be understood as the lacteal secretion obtained by milking of any mammal, such as cows, sheep, goats, buffaloes or camels. In a preferred embodiment, the milk is cow's milk. The term milk also includes protein/fat solutions made of plant materials, e.g. soy milk.

The term "milk substrate" may be any raw and/or processed milk material that can be subjected to fermentation according to the method of the invention. Thus, useful milk substrates include, but are not limited to, solutions/-suspensions of any milk or milk like products comprising protein, such as whole or low fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, dried milk, whey, acid whey, whey permeate, including acid whey permeate, whey powder, including acid whey powder, sweet whey powder, demineralized whey powder and delactosed whey powder, lactose, mother liquid from crystallization of lactose, whey protein concentrate, or cream. Obviously, the milk substrate may originate from any mammal, e.g. being substantially pure mammalian milk, or reconstituted milk powder.

The term "acid whey permeate" means whey permeate obtained from concentration of a fermented milk product by ultrafiltration in production of fresh cheese, such as cottage cheese, ricotta, Skyr, Greek Yogurt, Tvoroq, quark and Labneh,

manufactured by acid coagulation by lactic acid bacterium fermentation, and wherein the pH of the acid whey permeate in a 10% solution is lower than 5.1. The term "acid whey" means whey fraction obtained from concentration of a fermented milk product in a separator in production of fresh cheese, such as cottage cheese, ricotta, Skyr, Greek Yogurt, Tvoroq, quark and Labneh, manufactured by acid coagulation by lactic acid bacterium fermentation, and wherein the pH of the acid whey fraction in a 10% solution is lower than 5.1.

Prior to fermentation, the milk substrate may be homogenized and pasteurized according to methods known in the art.

"Homogenizing" as used herein means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed prior to fermentation, it may be performed so as to break up the milk fat into smaller sizes so that it no longer separates from the milk. This may be accomplished by forcing the milk at high pressure through small orifices. "Pasteurizing" as used herein means treatment of the milk substrate to reduce or eliminate the presence of live organisms, such as microorganisms. 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. A rapid cooling step may follow.

"Fermentation" in the methods of the present invention means the conversion of carbohydrates into alcohols or acids through the action of a microorganism.

Preferably, fermentation in the methods of the invention comprises conversion of lactose to lactic acid.

Fermentation processes to be used in production of dairy products are well known and the person of skill in the art will know how to select suitable process conditions, such as temperature, oxygen, amount and characteristics of microorganism(s) and process time. Obviously, fermentation conditions are selected so as to support the achievement of the present invention, i.e. to obtain a dairy product in solid (such as a cheese) or liquid form (such as a fermented milk product).

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having",

"including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly

contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The expression "fermented milk product" means a food or feed product wherein the preparation of the food or feed product involves fermentation of a milk substrate with a lactic acid bacteria. "Fermented milk product" as used herein includes but is not limited to products such as thermophilic fermented milk products, e.g. yoghurt, mesophilic fermented milk products, e.g. sour cream and buttermilk, as well as fermented whey.

The term "thermophile" herein refers to microorganisms that thrive best at temperatures above 35°C. The industrially most useful thermophilic bacteria include Streptococcus spp. and Lactobacillus spp. The term "thermophilic fermentation" herein refers to fermentation at a temperature above about 35°C, such as between about 35°C to about 45°C. The term "thermophilic fermented milk product" refers to fermented milk products prepared by thermophilic fermentation of a thermophilic starter culture and include such fermented milk products as set-yoghurt, stirred- yoghurt and drinking yoghurt, e.g. Yakult.

The term "mesophile" herein refers to microorganisms that thrive best at moderate temperatures (15°C-35°C). The industrially most useful mesophilic bacteria include Lactococcus spp. and Leuconostoc spp. The term "mesophilic fermentation" herein refers to fermentation at a temperature between about 22°C and about 35°C. The term "mesophilic fermented milk product" refers to fermented milk products prepared by mesophilic fermentation of a mesophilic starter culture and include such fermented milk products as buttermilk, sour milk, cultured milk, smetana, sour cream, Kefir and fresh cheese, such as quark, tvarog and cream cheese.

FIGURES shows the levels of residual lactose in samples treated with lactase added to a yogurt downstream of post pasteurization for lactase levels of 1000, 2000 and 3000 LAU/L.

shows the levels of residual lactose in samples treated with lactase added to a yogurt downstream of post pasteurization for lactase levels of 200, 400, 600, 800, 1000 and 1200 LAU/L at 8, 24, 48 and 76 hours, shows the data of Fig. 2 at 48 hours only,

shows the data of Fig. 2 at 76 hours only.

EXAMPLES Example 1

Lactase activity-assay in Eppendorf tubes at 37°C, pH 6.5

Principle:

Lactase hydrolyses lactose into glucose and galactose. Glucose is measured after a modified version of the common glucose oxidase / peroxidase assay (Werner, W. et al. (1970) Z. analyt. Chem. 252: 224.).

LAU is defined as the amount of enzyme liberating 1 micromole of glucose per min at 37°C, pH 6.5 in M-buffer (M-buffer is defined in the description of the present patent application). Alternatively, the activity in LAU for a specific lactase may be determined by the method described here. The value obtained is compared to a standard curve run with a lactase of known activity, and the activity of the unknown sample calculated from this. The lactase of known activity may, e.g., be Lactozym obtained from Novozymes A/S, Denmark. Solutions:

Assay buffer: 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCI, 2 mM CaCI ₂ , 1 mM MgCI ₂ , 0.01% Triton X100, pH 6.5

GOD-Perid solution : 65 mM sodium phosphate, pH 7, 0.4 g/l Glucose oxidase, 0.013 g/l HRP (Horse Radish Peroxidase), 0.65 g/l ABTS (2,2'-azino-bis(3- ethylbenzthiazoline-6-sulphonic acid)).

Substrate:

160 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCI, 2 mM CaCI ₂ , 1 mom MgCI ₂ , pH 6.5.

Standard :

Lactozym (available from Novozymes A/S, Denmark) with a known activity in LAU/g is used as standard, diluted in assay buffer in the range from 0.09 - 0.7 LAU/g.

Samples:

Enzyme samples are diluted appropriately in assay buffer. Procedure:

1. 375 μΙ substrate is incubated 5 minutes at 37°C.

2. 25 μΙ enzyme diluted in assay buffer is added.

3. The reaction is stopped after 30 minutes by adding 60 μΙ 1 M NaOH

4. 20 μΙ is transferred to a 96 well microtiter plate and 200 μΙ GOD-Perid solution is added. After 30 minutes at room temperature, the absorbance is measured at 420 nm.

Example 2

100 ml 15 or 30%(w/w) whey permeate containing primarily lactose and ions was made by mixing 15 or 30 g spray-dried whey permeate powder (Variolac, Aria) in 85 or 70 ml ionic water respectively. The solution was poured in a flask containing a magnetic stirring bar and placed in a water bath at 37°C. After 15 min, enzyme was added. Enzymes tested were Lactozym, a commercially available lactase from Novozymes A/S, Denmark, having an activity of 3060 LAU/g, and an experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID NO: 2 and an activity of 295 LAU/g. Dosages were 4225 LAU/I milk of Lactozym and 2025 LAU/I milk of the Bifidobacterium lactase. Milk samples were taken at regular intervals up till 5.5 hrs. and the enzyme was inactivated by heating to 99°C for 10 min in a thermomixer. Samples were diluted appropriately and filtered through a 0.20 μιτι filter. Lactose hydrolysis was measured using a Dionex BioLC equipped with a Dionex PAl column and a Pulsed Amperiometrisk Detektor (PAD). Peaks were identified and quantified by comparing with known standards of lactose, glucose and galactose.

Results are given below.

Table 1 : Lactose, glucose and galactose in 15% DS whey permeate after treatment with Lactozym or Bifidobacterium lactase.

Lactozym Bifidobacterium lactase

Time Lactose Glucose Galactose Lactose Glucose Galactose min mM mM mM mM mM mM

0 499 1 2 499 1 2

30 312 135 106 410 61 63

60 211 224 155 349 119 122

120 110 295 221 220 199 202

180 66 324 249 149 281 290

240 50 346 279 84 336 348

330 37 372 312 31 350 368 Table 2 : Lactose, glucose and galactose in 30% DS whey permeate after treatment with Lactozym or Bifidobacterium lactase.

Also when tested at higher lactose concentrations as in 15% or 30% whey permeate no or very little galactooligosaccharides are produced. Again, the produced galactose and glucose levels are equal and match the amount of lactose hydrolyzed. For comparison, Lactozym produces less galactose than glucose, clearly showing that galactooligosaccharides have been produced. Example 3

pH profile (at 37°C) and temperature profile (at pH 6.5) of experimental lactase from Bifidobacterium bifidum using lactose as substrate.

Principle:

Lactase hydrolyses lactose and glucose + galactose is formed. Glucose is measured after a modified version of the common glucose oxidase / peroxidase assay (Werner, W. et al. (1970) Z. analyt. Chem. 252: 224.)

pH profile Substrate:

167 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCI, 2 mM CaCI ₂ , 1 mM MgCI ₂ and pH adjusted to pH 3, 4, 5, 6, 7, 8, 9 and 10 with NaOH. Enzyme Sample:

Experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID NO: 2 was diluted appropriately in 150 mM KCI, 2 mM CaCI ₂ , 1 mM MgCI ₂ , 0.01% Triton X100.

Procedure:

• 10 μΙ enzyme sample diluted in enzyme dilution buffer was added to PCR tubes at room temp.

• 90 μΙ substrate was added at room temp, and quickly placed in a Peltier Thermal Cycler (PCT-200, MJ research) at 37°C and incubated for 30 min and then placed on ice.

• The reaction was stopped by adding 100 μΙ 0.25 M NaOH.

• 20 μΙ was transferred to a 96 well microtitre plate and 230 μΙ 65 mM sodium phosphate, pH 7, 0.4 g/l Glucose oxidase, 0.013 g/l HRP, 0.65 g/l ABTS solution was added. After 30 minutes at room temperature, the absorbance was measured at 420 nm.

Table 3 :

Temperature profile Substrate:

167 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCI, 2 mM CaCI ₂ , 1 mM MgCI ₂ and pH adjusted to pH 6.5 with NaOH. Enzyme Sample:

Experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID NO: 2 was diluted appropriately in 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCI, 2 mM CaCI ₂ , 1 mM MgCI ₂ , 0.01% Triton X100 and pH adjusted to pH 6.5.

Procedure:

• 10 μΙ enzyme sample diluted in enzyme dilution buffer was added to PCR tubes at room temp.

· 90 μΙ preheated (in a Peltier Thermal Cycler 30-70°C) substrate was added and incubation was performed with a temp, gradient from 30-70°C for 30 min. and then placed on ice.

• The reaction was stopped by adding 100 μΙ 0.25 M NaOH.

• 20 μΙ was transferred to a 96 well microtitre plate and 230 μΙ 65 mM sodium phosphate, pH 7, 0.4 g/l Glucose oxidase, 0.013 g/l HRP, 0.65 g/l ABTS solution was added. After 30 minutes at room temperature, the absorbance was measured at 420 nm.

Table 4:

Example 4

Determination of Km for lactase enzymes at 5°C

Principle:

Lactase hydrolyses lactose and glucose + galactose is formed. Glucose is measured after a modified version of the common glucose oxidase / peroxidase assay (Werner, W. et al. (1970) Z. analyt. Chem. 252: 224.)

Substrate:

Different lactose concentrations ranging from Km/5 to 10*Km, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCI, 2 mM CaCI ₂ , 1 mM MgCI ₂ and pH adjusted to pH 6.5 with NaOH. Enzyme Sample:

Experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID NO: 2 was diluted appropriately in 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCI, 2 mM CaCI ₂ , 1 mM MgCI ₂ , 0.01% Triton X100, pH adjusted to pH 6.5 with NaOH.

12 g/l Lactozym (commercially available lactase from Novozymes A/S, Denmark) was diluted 6000 times in the same buffer.

Procedure:

- 10 μΙ enzyme sample (5°C) was added to a 96 well microtitre plate on ice.

- 90 μΙ substrate (5°C) was added and incubated for 2 hours at 5°C.

- The reaction was stopped by adding 100 μΙ 0.25 M NaOH.

- 20 μΙ was transferred to a 96 well microtitre plate and 230 μΙ 65 mM sodium phosphate, pH 7, 0.4 g/l Glucose oxidase, 0.013 g/l HRP, 0.65 g/l ABTS solution was added. After 30 minutes at room temperature, the absorbance was measured at 420 nm.

Km determination :

Computerized nonlinear least-squares fitting and the Michaelis-Menten equation : v = (Vmax*S)/(Km+S)

was used. Km for the Bifidobacterium lactase and Lactozym were determined to be 8 mM and 30 mM, respectively.

In a similar test performed at 37°C, Km for the Bifidobacterium lactase and Lactozym were determined to be 13 mM and 30 mM, respectively. Example 5

Production of post-pasteurized yogurt containing lactase added after pasteurization

Milk substrate

Fat level 2.8%*

Protein level 2.8%*

Lactose 3.0%

Sucrose 5.0% (added) Modified Starch E1442 Cargill type 75720 1.50%

Pectin type LMA CP Kelco type LM 106 AS-YA 0.25%

Gellan Gum type Kelcogel YSS 0.05%

* Level in final product, i.e. after heat treatment, addition of the ambient storage strain and storage for 150 days.

Starter culture

YoFlex ^® starter culture type FD-DVS YF-L904 containing the two strains Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus.

Lactase

Lactase from Bifidobacterium bifidum having the encoded sequence of SEQ ID NO. 2 and an activity of 295 LAU/g.

Procedure for producing product

1. Dispersing the dry ingredients into the milk

2. Resting for 3 hours with gentle stirring

3. Heating the milk until a temperature of 65°C is reached

4. Homogenization at 150 Bar

5. Heat treatment to 95°C for 5 min.

6. Cooling to fermentation temperature 43°C

7. Pump the milk into fermentation vat

8. Inoculation of YoFlex Culture type YF-L904.

9. Fermentation until pH reaches 4.30.

10. Break the curd and stir until smooth structure is obtained

11. Heat treatment at 75°C for 30 sec.

12. Cooling to 25°C

13. Adding the lactase at a level of 1420 LAU/L yogurt product and gently mixing the yogurt so as to distribute the lactase evenly in the yogurt.

14. Filling the yogurt into containers.

15. Storing the yogurt at a temperature of 20°C for a period of 7 days. Example 6

Downstream addition of lactase to post pasteurized yogurt

The purpose of the experimental work carried out was to show that it is possible to add a suitable lactase after final heat treatment of post pasteurized yogurt to reach residual lactose levels of <0.01%.

Post pasteurized yogurt with a composition of 2.9% protein and 2.8% fat was produced. The milk substrate was fermented to pH of 4.30 with YoFlex Culture type YF-L904. After reaching a pH of 4.30 the yogurt was cooled down to 15°C in a plate heat exchanger and kept in insulated buffer tank at 15°C for three hours before performing the final heat treatment. The final heat treatment was done at 74°C, 20s. in a plate heat exchanger, and the product was filled at 25°C into sterile beakers. The samples were stored at ambient temperature (22°C) for one day prior to addition of sterile lactase.

Milk substrate

Milkoscan analysis: Fat level 2.8% Protein level 2.9%

The following parameters were used for fermentation and processing :

Mixing temperature: 10°C

Hydration time: 3 hours with gentle stirring

Lactase

Lactase from Bifidobacterium bifidum having the encoded sequence of SEQ ID NO. 2. Starter culture

YoFlex® Culture type YF-L904 from Chr. Hansen YoFlex® product range containing the two species Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus.

Process for producing yogurt

Homogenization pressure: 150 bar

Pasteurization condition : 95°C, 5 minutes

Fermentation temperature: 43°C

End pH : 4.30

Break the curd manually and stir until smooth structure is obtained.

Cooling to 15°C in Plate heat exchanger

Thermisation in plate heat exchanger, flow 414L/h

Homogenization pressure: 0 bar

Termisation condition : 74°C, 20s

Filling into sterile 100 ml cups, filling temperature 25°C. Lactase addition

The lactase was sterile filtered and added to 100 ml beakers of post pasteurized yogurt with the following dosages:

a) 0 LAU/L - Ref

b) 1000 LAU/L

c) 3000 LAU/L

d) 10000 LAU/L

The samples were stored at room temperature (22°C) and samples were taken for residual lactose analysis at the following time points; 24 hours, 48 hours and 1 week.

Results

In Figure 1 the residual lactose of the samples with lactase addition is shown. The residual lactose of the reference sample was measured to be 2.4% residual lactose. As will appear from Figure 1 the lactose concentration decreased to a level of about 0.005% for lactase concentrations 3000 LAU/L and 10000 LAU/L after 24 hours from addition. Also, the lactose concentration decreased to a level of about 0.005% for all three lactase concentrations after 48 hours and after 1 week from addition. Example 7

Downstream addition of lactase to post pasteurized yogurt The purpose of the experimental work carried out was to show that it is possible to add a suitable lactase after final heat treatment of post pasteurized yogurt to reach residual lactose levels of <0.1%.

Post pasteurized yogurt with a composition of 2.87% protein and 2.89% fat was produced. The milk substrate was fermented to pH of 4.30 with YoFlex Culture type YF-L904. After reaching a pH of 4.30 the yogurt was cooled down to 15°C in a plate heat exchanger and kept in insulated buffer tank at 15°C for three hours before performing the final heat treatment. The final heat treatment was done at 74°C, 20s. in a plate heat exchanger, and the product was filled at 25°C into sterile beakers. The samples were stored at 25°C for one day prior to addition of sterile lactase.

Milk substrate

Milkoscan analysis: Fat level 2.87% Protein level 2.89%

The following parameters were used for fermentation and processing :

Mixing temperature: 10°C

Hydration time: 3 hours with gentle stirring Lactase

Lactase from Bifidobacterium bifidum having the encoded sequence of SEQ ID NO. 2.

Starter culture

YoFlex® Culture type YF-L904 from Chr. Hansen YoFlex® product range containing the two species Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus.

Process for producing yogurt

Homogenization pressure: 150 bar

Pasteurization condition : 95°C, 5 minutes

Fermentation temperature: 43°C

End pH : 4.30

Break the curd manually and stir until smooth structure is obtained.

Cooling to 15°C in Plate heat exchanger

Thermization in plate heat exchanger, flow 414L/h

Thermization condition : 74°C, 20s

Filling into sterile 100 ml cups, filling temperature 25°C.

Lactase addition

The lactase was sterile filtered and added to 100 ml beakers of post pasteurized yogurt with the following dosages:

a) 0 LAU/L - Ref

b) 200 LAU/L

c) 400 LAU/L

d) 600 LAU/L

e) 800 LAU/L

f) 1000 LAU/L

g) 1200 LAU/L

The samples were stored at 25°C and samples were taken for residual lactose analysis at the following time points; 8 hours, 24 hours, 48 hours and 76 hours. Results

In Figures 2-4 the residual lactose of the samples with lactase addition is shown. The residual lactose of the reference sample was measured to be 2.4% residual lactose. The horizontal lines at a Residual Lactose level of 0.1 and 0.5 signifies the level of lactose, which according to food regulatory legislation qualifies as lactose-free in the EU and China, respectively.

As will appear from Figure 2-4 the lactose concentration at 24 hours decreased to a level of about 0.355% for a lactase concentration of 400 LAU/L and to a level of below 0.01% for higher concentrations of lactase. At 48 hours, the lactose concentration had dropped to a level of 0.015% for a lactase concentration of 400 LAU/L and to a level of below 0.01% for higher concentrations of lactase. At 76 hours, the lactose concentration had dropped to a level of below 0.01% for a lactase concentration of 400 LAU/L and higher concentrations of lactase.

Thus, the present experiments show that it is possible to meet a target of residual lactose of 0.01% or below with a lactase dose of 600 LAU/I at 48 hours and a lactase dose of 400 LAU/I at 76 hours.

Example 8

Downstream addition of lactase to post pasteurized yogurt at different pH levels (from 4.0 to 4.4) The purpose of experimental work carried out was to show that in a process for removing lactose in Post Pasteurized Yogurt (PPY) using lactase it is possible to obtain a desired lactose removal for PPY with different pH in the range of 4.0 to 4.4.

Milk substrate

Milkoscan analysis:

Standard Lactose milk base: Fat level 2.88%, Protein level 2.91%

High Lactose milk base: Fat level 2.91%, Protein level 2.94%

Lactase

Lactase from Bifidobacterium bifidum having the encoded sequence of SEQ ID NO. 2.

Process for producing yogurt

Homogenization pressure: 150 bar at 60 °C

Pasteurization condition : 95°C, 5 minutes

Fermentation temperature: 43°C

End pH for standard lactose milk base: 4.0, 4.1, 4.2, 4.3 and 4.4

End pH for high lactose milk base: 4.3

Break the curd manually and stir until smooth structure is obtained.

Cooling to 15°C in Plate heat exchanger

Thermisation in plate heat exchanger, flow 414L/h

Termisation condition : 74°C, 20s

Filling into sterile 100 ml cups, filling temperature 25°C. Lactase addition

The lactase was sterile filtered and added to 100 ml beakers of post pasteurized yogurt with the following dosages:

a) 0 LAU/L - Ref

b) 200 LAU/L

c) 400 LAU/L

d) 600 LAU/L

e) 800 LAU/L

The samples were stored at 15°C, 20°C, 25°C and 30°C and samples were taken for residual lactose analysis at the following time points; 24 hours, 48 hours and 72 hours.

Results

For reasons of brevity only results for samples stored at 25°C are shown.

Corresponding results were obtained for samples stored at 15°C, 20°C and 30°C.

Table 5: Residual lactose levels for samples stored at 25°C.

Milk Base End pH Dosage of 24 Hours 48 hours 72 Hours

Lactase (%) (%) (%)

(LAU/L)

Standard 4.4 200 0.918 0.220 0.034

Standard 4.4 400 0.106 0.004 0.003

Standard 4.4 600 0.077 0.003 0.003

Standard 4.4 800 0.009 <0.002 0.002

Standard 4.4 1000 0.004 0.002 0.002

Standard 4.3 200 1.322 0.567 0.223

Standard 4.3 400 0.566 0.043 0.008

Standard 4.3 600 0.063 0.005 0.004

Standard 4.3 800 0.015 0.003 0.003

Standard 4.3 1000 0.007 <0.002 0.002

Standard 4.2 200 1.422 0.0803 0.356

Standard 4.2 400 0.671 0.071 0.013

Standard 4.2 600 0.177 0.008 0.004

Standard 4.2 800 0.128 0.005 0.003

Standard 4.2 1000 ND ND ND

Standard 4.1 200 1.719 1.177 0.808

Standard 4.1 400 0.923 0.266 0.051

Standard 4.1 600 0.572 0.066 0.019

Standard 4.1 800 0.245 0.010 0.005

Standard 4.1 1000 0.108 0.005 0.003

Standard 4.0 200 1.725 1.196 0.933

Standard 4.0 400 1.236 0.589 0.265

Standard 4.0 600 0.773 0.187 0.039

Standard 4.0 800 0.502 0.049 0.010

Standard 4.0 1000 ND ND ND

High 4.3 200 2.011 1.139 0.584

High 4.3 400 0.876 0.121 0.018

High 4.3 600 0.250 0.009 0.005

High 4.3 800 0.053 0.006 0.003

High 4.3 1000 0.017 0.004 0.003 Table 6: Residual lactose level with no addition of lactase.

As will appear from Table 5 and 6, the lower the end pH of the yogurt, the higher the residual lactose content is. The results further show that at the lowest end pH it is possible to reach a target of residual lactose of below 0.1% with a lactase dose of 800 LAU/L at 48 hours. Furthermore, at an end pH of 4.1 it is possible to reach a residual lactose of 0.108% with a lactase dose of 1000 LAU/L at 24 hours. Example 9

Downstream addition of lactase to post pasteurized yogurt at low levels of added lactase

The purpose of experimental work carried out was to test the effectiveness of low dosages of lactase in a process for removing lactose in Post Pasteurized Yogurt (PPY) using lactase in order to establish the minimum dose required to obtain a desired target of residual lactose. Milk substrate

Milkoscan analysis:

Milk base: Fat level 2.81%, Protein level 3.11%

Lactase

Lactase from Bifidobacterium bifidum having the encoded sequence of SEQ ID NO. 2.

Process for producing yogurt

Homogenization pressure: 150 bar at 60°C

Pasteurization condition : 95°C, 5 minutes

Fermentation temperature: 43°C

End pH for standard lactose milk base: 4.15, 4.20, 4.25 and 4.30

End pH for high lactose milk base: 4.3

Break the curd manually and stir until smooth structure is obtained.

Cooling to 15°C in Plate heat exchanger

Thermisation in plate heat exchanger, flow 414L/h

Termisation condition : 74°C, 20s

Filling into sterile 100 ml cups, filling temperature 25°C.

Lactase addition

The lactase was sterile filtered and added to 100 ml beakers of post pasteurized yogurt with the following dosages:

a) 0 LAU/L - Ref b) 50 LAU/L

c) 100 LAU/L

d) 200 LAU/L

e) 300 LAU/L

The samples were stored at 25°C and samples were taken for residual lactose analysis at the following time points; 24 hours, 48 hours and 72 hours.

Results

For reasons of brevity only results for samples stored at 25°C are shown.

Corresponding results were obtained for samples stored at 15°C, 20°C and

Table 7: Residual lactose levels for samples

The pasteurized yogurt base with a pH of 4.3, 4.25, 4.20 and 4.15 and with no added lactase had a lactose level of 2.57, 2.52, 2.47 and 2.40, respectively.

As will appear from Table 7, it is possible to reach a target of residual lactose of below 0.5% with a lactase dose of 200 LAU/L at 48 hours for samples with a pH of 4.3 and 4.25 and with a lactase level of below 0.3 with a lactase dose of 300 LAU/L at 48 hours for all samples tested. Example 10

Production of acid whey beverage with low level of lactose by addition of lactase

The objective of the experimental work carried out was to show that it is possible to remove lactose from an acid whey permeate and investigate the effect of various lactase dosages, reaction times and temperatures.

Milk substrate

Acid whey permeate (from ultrafiltration) is a side-product from production of Skyr.

The acid whey permeate had the following composition :

Protein 0.2%

Sugars 3.7%

Fat 0.01%

Ash 0.8%

Moisture 94.7%

pH 4.27

Lactase

Lactase from Bifidobacterium bifidum having the encoded sequence of SEQ ID NO. 2.

Process

1. Heat acid permeate to 72°C, / 2 min.

2. Cooling to

a. 5°C (2 x 1000 ml)

b. 40°C (3 x 1000 ml)

3. Dosing of lactase:

a. 5°C: 2500 and 5000 LAU/L (2 x 1000 ml) b. 40°C: 500, 1000 and 2500 LAU/L (3 x 1000 ml)

4. Samples for analyzing lactose:

a. Before addition of lactase

b. At 40 °C and 2500 LAU/L: every 1 hour from 3 to 7 hours and 24 hours

c. Other samples: 24 hours.

5. Processing of beverage based on acid whey

a. Addition of HM pectin solution, flavor and sucrose.

b. Adjust pH to 3.9 by citric acid

c. Heat to 80°C 2 min.

d. Homogenization at 150 bar at 80°C

e. Filling into bottles.

Results

Table 8: Residual lactose levels (g/L) for test sampl

As will appear from Table 8, it was possible to reduce the level of lactose to levels around O. lg/L for test samples at a temperature of 40°C and for lactase concentrations of 1000 and 2500 LAU/L as well as at a temperature of 5°C for a lactase concentration of 5000 LAU/L. For the two other samples it was possible to reduce the level of lactose to levels below 0.5g/L. Sequence listing

SEQ ID NO. : 1 shows the sequence of a mutant of SEQ ID NO. 4.

SEQ ID NO. : 2 shows the sequence of a mutant of SEQ ID NO. 4.

SEQ ID NO. : 3 shows the sequence of a lactase from Bifidobacterium bifidum

DSM20215.

SEQ ID NO. : 4 shows the sequence of a lactase from Bifidobacterium bifidum

NCIMB41171, the nucleotide sequence of which is listed in NCBI with the accession number DQ448279.

SEQ ID NO: 4 is discussed in the following references, wherein it is referred to as bbglll :

AppI Microbiol Biotechnol (2007) 76 : 1365-1372, T K Goulas et al.

AppI Microbiol Biotechnol (2009) 82 : 1079-1088, T Goulas et al.

AppI Microbiol Biotechnol (2009) 84: 899-907, T Goulas et al.