EA045126B1 - FERMENTED DAIRY PRODUCT WITH REDUCED LACTOSE CONTENT - Google Patents

Description

Field of invention

The present invention relates to a fermented milk product with reduced lactose content.

Prior Art

WO 2009/071539 discloses a lactase derived from Bifidobacterium bifidum, which is capable of very efficient hydrolysis in milk, and which is active over a wide pH range, including low pH, for example pH less than 5. This lactase can be used in milk and fermentation processes. dairy products such as cheese, yogurt, butter, buttermilk, sour cream, etc., to reduce the lactose content.

WO 2013/160413 discloses a method for producing a fermented dairy product using a combination of glucose-negative strains of lactic acid bacteria and conventional lactase to reduce the lactose content of the fermented dairy product while increasing the glucose content.

EP-A1-2957180 discloses a method for producing a fermented dairy product using a combination of starter cultures and conventional lactase to reduce the lactose content and subsequent acidification level in the fermented dairy product.

Summary of the invention

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

The object of the present invention is achieved by means of a fermented milk product which has a pH of 3.0 to 5.0 and a lactose content of at least 1.5 mg/ml, wherein said product contains lactase which remains active at a pH of 5.0 and a temperature of 37 °C at a level of at least 5% compared to its activity at the optimal pH for lactase.

The present invention has made it possible to modify a lactose-containing food product with a pH of 3.0 to 5.0 during storage at ambient temperature, i.e. without the need for refrigeration, after completion of production at the point of production, for example during transport and storage in a retail establishment, in such a way as to reduce lactose levels. The present invention is based on the recognition that for a product having a low pH and intended to be transported and stored at ambient temperatures, for example, fermented dairy products after pasteurization, it is possible to reduce the lactose content after completion of production at the point of production by adding lactase active at low pH, and after the completion of the normal food production process, and allowing lactase to convert lactose into galactose and glucose during subsequent phases of the product's life until its final consumption by the end consumer. Thus, the present invention provides the lactose conversion step in a simple and cost-effective manner off-site, and therefore, the on-site process for producing a fermented dairy product can be simplified by eliminating the lactose conversion completion step, thereby saving the life and productivity of the production equipment.

In addition, when adding lactase after heat treatment of a fermented dairy product, less lactase can be used than when the lactase is active during fermentation, since lactase activity can be maintained for a long period of time, while in the case of a production fermentation method it is desirable to this method as quickly as possible in order to reduce costs. Most of the lactase will be inactivated during heat treatment, and therefore will not be active after heat treatment. Thus, the present invention has provided the ability to reduce the amount of lactase required to achieve removal of the lactose content of a fermented milk product. Also, when lactase is added after heat treatment of a fermented dairy product, the level of Maillard reaction caused by heat treatment of the fermented dairy product is reduced, since lactose causes a Maillard reaction to a lesser extent than the carbohydrate metabolites of lactose. Finally, by adding lactase after heat treatment of a fermented dairy product, any detrimental effect of the lactic acid bacteria used in fermentation on lactase activity can be avoided.

Worldwide, a significant number of consumers are lactose intolerant or lactose sensitive. Consequently, there is currently a high demand for dairy products, including fermented dairy products, that are reduced in lactose or that are substantially lactose-free. The present invention provides a new approach for producing such a product in a simple and cost-effective manner.

Detailed Description of the Invention

Lactase.

The lactase of the fermented milk product of the invention can be any lactase that retains its activity at pH 5.0 and a temperature of 37° C. at a level of at least 5% compared to its activity at the optimal pH for lactase.

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According to the present invention, lactase activity in LAU (lactase activity units) is measured as defined in the Definitions section below.

In a preferred embodiment of the present invention, lactase retains its activity at pH 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% compared to its activity at the optimal lactase pH.

In a preferred embodiment of the present invention, lactase retains its activity at pH 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% compared to its activity at the optimal lactase pH.

In a preferred embodiment of the present invention, lactase retains its activity at pH 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% compared to its activity at the optimal lactase pH.

According to the present invention, the optimal pH of lactase is determined by measuring lactase activity at different pH using the method specified below in the Definitions section, and determining the pH with optimal activity. In particular, lactase activity at different pH values is measured in M buffer and at 37°C. Alternatively, lactase activity, reported herein as a percentage of activity at the lactase optimal pH, is considered as a percentage of lactase activity at pH 6.5.

In a preferred embodiment of the present invention, lactase retains its activity at a temperature of 10°C and pH 6.0 at a level of at least 10% compared to its activity at the optimal temperature for lactase. Preferably, the lactase retains its activity at a temperature of 10°C and pH 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 at least 60%, more preferably at least 70% and most preferably at least 80% compared to its activity at the optimal lactase temperature.

According to the present invention, the optimal temperature of lactase is determined by measuring lactase activity at different temperatures using the method specified below in the Definitions section, and determining the temperature with optimal activity. Alternatively, lactase activity reported herein as the percentage of activity at the optimal lactase temperature is considered as the percentage of lactase activity at 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 that is at least 55%, such as at least 60%, at least 65%, according to 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 that 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 an optimum pH of lactase activity at 37° C., which is higher than 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 pH 6.5 that 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 pH 6.5.

In a preferred embodiment of the present invention, the Km (Michaelis constant) of lactase at 5°C is less than 25 mM, for example less than 20 mM, less than 15 mM or less than 10 mM. In another preferred embodiment, the Km of lactase at 37°C is less than 25 mM, for example less than 20 mM or less than 15 mM. One skilled in the art will know how to determine Km for lactase activity at a specific temperature. Km can be determined by the method described in WO 2009/071539.

In another preferred embodiment, when hydrolyzing lactose in a dairy product, the enzyme has a ratio of lactase to transgalactosylase activity greater than 1:1, for example greater than 2:1, or greater than 3:1. In another preferred embodiment, the enzymatic treatment is carried out under conditions where the lactase activity of the enzyme is higher than the transgalactosylase activity, for example at least twice as high or at least three times as high.

The ratio of lactase to transgalactosylase activity in a dairy product, for example, can be determined by HPLC (high performance liquid chromatography) analysis. In another preferred embodiment, the enzymatic treatment is carried out under conditions where at least 50% (% w/w) of the hydrolyzed lactose is converted to free galactose. In another preferred embodiment, the enzymatic treatment is carried out under conditions where the hydrolyzed lactose is converted to equal amounts of free glucose and free galactose.

Lactase in the context of the present invention is a glycoside hydrolase having the ability to hydrolyze the disaccharide lactose into its constituent monomers galactose and glucose. The group of lactases to which the lactase of the invention belongs includes, but is not limited to, enzymes assigned to subclass EC 3.2.1.108. Enzymes assigned to other subclasses, such as, for example, EC 3.2.1.23, may also be lactases in the context of the present invention. Lactase in the context of the present invention may have other activities other than lactose hydrolyzing activity, such as, for example, transgalactosylation activity. In the context of the present invention, the lactose hydrolyzing activity of lactase may be referred to as its lactase activity or its beta-galactosidase activity.

Enzymes having lactase activity to be used in the method of the present invention may be of animal, plant or microbial origin. Preferred lactases are obtained from microbial sources, in particular from a filamentous fungus or yeast or bacterium.

This enzyme may, for example, come from an Agaricus strain, for example A. bisporus; Ascovaginospora; Aspergillus, for example A. niger, A. awamori, A. foetidus, A. japonicus, A. oryzae; Candida; Chaetomium; Chaetomastia; Dictyostelium, for example D. discoideum; Kluveromyces, for example K. fragilis, K. lactis; Mucor, for example M. javanicus, M. mucedo, M. subtilissimus; Neurospora, eg N. crassa; Rhizomucor, for example R. pusillus; Rhizopus, for example R. arrhizus, R. japonicus, R. stolonifer; Sclerotinia, for example S. libertiana; Torula; Torulopsis; Trichophyton, for example T. rubrum; Whetzelinia, for example W. sclerotiorum; Bacillus, for example B. coagulans, B. circulans, B. megaterium, B. novalis, B. subtilis, B. pumilus, B. stearothermophilus, B. thuringiensis; Bifidobacterium, for example B. longum, B. bifidum, B. animalis; Chryseobacterium; dtrobacter, for example C. freundii; Clostridium, for example C. perfringens; Diplodia, for example D. gossypina; Enterobacter, for example E. aerogenes, E. cloacae Edwardsiella, E. tarda; Erwinia, for example E. herbicola; Escherichia, for example E. coli; Klebsiella e.g. K. pneumoniae; Miriococcum; Myrothesium; Mucor; Neurospora, eg N. crassa; Proteus, for example P. vulgaris; Providencia, for example R. stuartii; Pycnoporus, for example Pycnoporus cinnabarinus, Pycnoporus sanguineus; Ruminococcus, for example R. torques; Salmonella, for example S. typhimurium; Serratia, for example S. liquefasciens, S. marcescens; Shigella, for example S. flexneri; Streptomyces, for example S. antibioticus, S. castaneoglobisporus, S. violeceoruber; Trametes; Trichoderma, for example T. reesei, T. viride; Yersinia, for example Y. enterocolitica.

In a preferred embodiment, the lactase is derived from a bacterium, for example from the family Bifidobacteriaceae, for example from the genus Bifidobacterium, for example from a strain of B. bifidum, B. animalis or B. longum. In a more preferred embodiment, the lactase is derived from Bifidobacterium bifidum.

In a preferred embodiment, the enzyme having lactase activity to be used in the product of the present invention contains an amino acid sequence that is at least 50% identical to a sequence selected from the group consisting of amino acids 28-1931 SEQ ID NO: 1, amino acids 28 -1331 SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and fragments thereof with lactase activity. In a more preferred embodiment, the enzyme contains an amino acid sequence that is at least 60%, for example 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 SEQ ID NO: 1, amino acids 28-1331 SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and fragments thereof with lactase activity.

A preferred enzyme is lactase having a sequence that is at least 50%, for example 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 SEQ ID NO: 1 or a fragment thereof with lactase activity. Such a fragment of SEQ ID NO: 1 with lactase activity may be any fragment of SEQ ID NO: 1 having lactase activity. The portion of SEQ ID NO: 1 with lactase activity, for example, may be amino acids 28-979, amino acids 28-1170, amino acids 28-1323, amino acids 28-1331, or amino acids 28-1600 SEQ ID NO: 1.

In a preferred embodiment, the enzyme having lactase activity to be used in the product of the present invention contains an amino acid sequence that is at least 50% identical to amino acids 28-1331 SEQ ID NO: 2. In a more preferred embodiment, the enzyme contains an amino acid sequence , which

- 3 045126 is at least 60%, for example at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to amino acids 28- 1331 SEQ ID NO: 2.

In another embodiment, the enzyme having lactase activity to be used in the product of the present invention has an amino acid sequence that is at least 50% identical to SEQ ID NO: 3. Preferably, the enzyme has an amino acid sequence that 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, the enzyme having lactase activity to be used in the product of the present invention has an amino acid sequence that is at least 50% identical to SEQ ID NO: 4. Preferably, the enzyme has an amino acid sequence that is at least 60%, for example at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to SeQ ID NO: 4.

For the purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453) as implemented in the 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 a gap open penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 substitution matrix (EMBOSS version of BLOSUM62).

The identity percentage uses the Needle output marked as the longest identity (obtained by using the -no brief option) and is calculated as follows:

(Identical residues x 100)/(Alignment length - Total number of gaps in alignment)

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

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

The lactases to be used in the method of the present invention may come from any of the sources mentioned herein. The term originating in this context means that the enzyme can be isolated from an organism where it is naturally present, i.e. the nature of the amino acid sequence of the enzyme is identical to the native enzyme. The term "derived" also means that the enzymes may have been recombinantly produced in the host, wherein the recombinantly produced enzyme is either of a nature identical to the native enzyme or has a modified amino acid sequence, such as having one or more amino acids that are deleted, inserted and/or substituted , i.e. is a recombinantly produced enzyme that is a mutant and/or fragment of the native amino acid sequence. Natural variants are included within the meaning of native enzyme. In addition, the term derived includes enzymes produced synthetically, for example through peptide synthesis. The term originating also includes enzymes that have been modified, for example by glycosylation, phosphorylation, etc., either in vivo or in vitro. In relation to a recombinantly produced enzyme, the term derived from refers to the nature of the enzyme and not the nature of the host organism in which it is recombinantly produced.

Lactase can be obtained from a microorganism using any suitable technique. For example, a lactase enzyme preparation can be produced by fermenting a suitable microorganism and then isolating the lactase preparation from the resulting fermentation broth or microorganism by methods known in the art. Lactase can also be produced through the use of recombinant DNA technologies. Such a method typically involves culturing a host cell transformed with a recombinant DNA vector containing a DNA sequence encoding the lactase in question, which DNA sequence is operably linked to a suitable expression signal such that it is capable of expressing the lactase in the culture medium under conditions allowing expression this enzyme and isolating this enzyme from the culture. This DNA sequence may also be incorporated into the genome of the host cell. This DNA sequence may be genomic, cDNA, or synthetic in origin, or any combination thereof, and may be isolated or synthesized according to methods known in the art.

The lactases to be used in the method of the present invention can be purified. The term purified as used here covers the enzyme protein

- 4 045126 lactase, substantially free of insoluble components from the producing organism. The term purified also includes lactase enzyme protein substantially free of insoluble components from the natural organism from which it is obtained. Preferably it is also separated from some of the soluble components of the body and culture medium from which it originates. More preferably, it is separated by one or more separate operations: filtration, precipitation or chromatography.

Accordingly, the enzyme having lactase activity may be purified, that is, only minor amounts of other proteins are present. The expression other proteins specifically refers to other enzymes. The term purified as used herein also refers to the removal of other components, in particular other proteins and, most specifically, other enzymes present in the cell of origin of the lactase. The lactase may be substantially pure, i.e. not containing other components from the organism in which it is produced, i.e., for example, the host organism for recombinantly produced lactase. Preferably, the lactase is at least 40% (w/w) pure enzymatic protein preparation, more preferably having at least 50%, 60%, 70%, 80%, or even at least 90% purity.

The term enzyme having lactase activity includes any auxiliary compounds that may be necessary for the catalytic activity of the enzyme, such as, for example, a suitable acceptor or cofactor, which may or may not be naturally present in the reaction system.

This enzyme may be in any form suitable for the application in question, such as, for example, in the form of a dry powder or granulate, non-dusting granule, liquid, stabilized liquid or protected enzyme.

Fermented milk product.

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

In one embodiment of the invention, the fermented milk product is a chemically acidified dairy product. Acidification can be accomplished by any acidifying agent approved for food use, 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 fermented milk product is a fermented milk product obtained by fermentation using a starter culture. The starter culture can be any conventional lactic acid bacteria starter culture, including single-strain cultures and mixtures of cultures, used to produce a particular type of fermented dairy product. Other beneficial bacteria that can 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 dairy product, after fermentation, is subjected to heat treatment in such a way as to reduce the bacterial count of the starter culture to no more than 1 x 10 ² CFU (colony forming units) per gram, and lactase is added after heat treatment.

The starter culture can be any conventional lactic acid bacteria starter culture, including single-strain cultures and mixtures of cultures, used to produce a particular type of fermented dairy product. In a preferred embodiment of the product according to the invention, the fermentation is carried out so as to obtain a pH of from 3.0 to 5.0, preferably from 3.2 to 4.8, more preferably from 3.4 to 4.6 and most preferably from 3.6 up to 4.4.

Heat treatment to reduce the bacterial count of the starter culture to no more than 1.0 x 10 ² CFU/g of fermented milk is preferably carried out by exposing the starter culture of the fermented milk product to a temperature of from 50°C to 110°C, preferably from 50°C to 100°C , preferably from 50°C to 90°C, preferably from 60°C to 85°C, more preferably from 65°C to 82°C, and most preferably from 70°C to 80°C. The heat treatment is preferably carried out for a period of from 5 seconds to 180 seconds, preferably from 5 seconds to 120 seconds, more preferably from 5 seconds to 90 seconds, more preferably from 5 seconds to 60 seconds, more preferably from 8 seconds to 50 seconds and most preferably 10 to 40 seconds. Preferably, the bacterial count of the starter culture is reduced to no more than 1.0 x 10 CFU/g of fermented milk, more preferably to 0 CFU/g.

The enzyme is added in an amount suitable to achieve the desired degree of lactose hydrolysis under the selected reaction conditions. In a specific embodiment of the invention, the dairy product contains lactase in an amount of from 100 to 20,000 LAU per liter of dairy product, preferably from 100 to 10,000 LAU per liter of dairy product, preferably from 100 to 5000 LAU per liter of dairy product, preferably less than 3000, for example less , less than 1500, less than 1000, less than 750

- 5 045126 or less than 500 LAU per liter of dairy product.

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

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

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

A fermented milk product typically contains protein in an amount ranging from 2.0% by weight to 3.5% by weight. The fermented milk product may also be a low protein product, with protein levels ranging from 1.0% by weight to 2.0% by weight. Alternatively, the lactic acid product may be a high protein product, with a protein level greater than 3.5% by weight. In a specific embodiment, the fermented milk product of the invention is a mixture of a fermented milk product and a cereal product, such as an oat product, wherein said cereal product may be a fermented cereal product, such as a fermented oat product.

In a specific embodiment of the invention, the fermented milk product contains a fermented cereal product. Said fermented cereal product can be obtained by grinding grains of the original cereal biological material to obtain cereal flour, which is then fermented. Fermentation of the cereal flour can be carried out using the same lactic acid bacteria (starter culture) used to ferment the dairy substrate, as described elsewhere in this application.

In a specific embodiment of the invention, the fermented milk product contains a fruit drink. The fruit drinks may additionally contain, for example, oats, soy, almonds, whey and/or unfermented milk, for example in the form of milk powder.

In a specific embodiment of the invention, the fermented milk product of the invention is a chemically acidified product. Acidification can be carried out using any acidifying agent suitable for addition to food products, such as lactic acid, citric acid, fruit juice, fruit pulp and fruit compound. In a specific embodiment, the fermented milk product is acidified with fruit juice.

In a specific embodiment of the invention, the fermented milk product contains a chemically acidified cereal product. The chemically acidified cereal product can be produced by grinding grains of the original cereal biological material to produce cereal flour, which is then used to obtain an aqueous suspension, and the pH of said suspension is then adjusted to the required level.

A fermented milk product containing acid whey or acid whey permeate.

In a particular aspect of the present invention, the fermented milk product contains an acid whey product selected from the group consisting of acid whey and acid whey permeate. Specifically, an acid whey product is produced by concentrating a fermented dairy product into a concentrated fraction and a separated acid whey fraction.

Acid whey and acid whey permeate are by-products of the production of a number of young cheeses, such as cottage cheese, ricotta, skyr, Greek yogurt, cottage cheese, quark and labneh. Acid whey and acid whey permeate have a pH of less than 5.1 in a 10% solution and have historically been difficult to find uses for by-products, for example due to their acidity. Acid whey is obtained by concentrating yoghurt in a separator. Acid permeate is obtained by concentrating yogurt through ultrafiltration.

This present aspect of the invention has provided the opportunity to use acid whey and acid whey permeate to produce a dairy product, such as a milk drink. This aspect is based on the recognition that acid whey and acid whey permeate are suitable for the production of the reduced lactose dairy product of the invention, since the production of this product requires enzymatic hydrolysis of lactose at low pH.

In a specific embodiment of the third aspect of the method of the invention, the starter culture-fermented dairy product is subjected to a concentration step to separate the starter culture-fermented dairy product into a concentrated fraction and a separated acid whey fraction, wherein the separated acid whey fraction but not the concentrated fraction , subjected to subsequent steps of this method.

Method for producing fermented dairy product.

In a first aspect, the present invention also relates to a method for producing fermented milk

- 6 045126 product, including the steps of providing a base fermented milk product, which has a pH from 3.0 to 5.0 and a lactose content of at least 1.5 mg/ml, adding lactase to said base fermented milk product, which retains its activity at pH 5.0 and a temperature of 37°C at a level of at least 5% compared to its activity at the optimal pH for lactase, to obtain a lactase-containing fermented milk product, and storing said lactase-containing fermented milk product at a temperature of at least 2°C for at least at least 1 day.

In a second aspect, the present invention relates to a method for producing a fermented milk product, comprising the steps of providing a base fermented milk product that has a pH of 3.0 to 5.0 and a lactose content of at least 1.5 mg/ml, subjecting said base fermented milk product to heat treatment in such a way as to reduce the number of bacteria to no more than 1x10 ² CFU/g, with the production of a heat-treated fermented milk product, adding lactase to the specified heat-treated fermented milk product, which retains its activity at pH 5.0 and a temperature of 37 ° C at a level of at least 5% compared to its activity at the optimal pH for lactase, to obtain a lactase-containing fermented milk product, and storing said lactase-containing fermented milk product at a temperature of at least 2°C for at least 1 day.

In other words, the second aspect is a specific embodiment of the first aspect, comprising the steps of subjecting the base fermented milk product to heat treatment so as to reduce the number of bacteria to no more than 1x10 ² CFU/g, to obtain a heat-treated fermented milk product, adding to said heat-treated fermented milk product a lactase product that retains its activity at pH 5.0 and a temperature of 37°C at a level of at least 5% compared to its activity at the optimal pH for lactase, to obtain a lactase-containing fermented milk product, and storing said lactase-containing fermented 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 method for producing a fermented dairy product, comprising the steps of fermenting a dairy substrate using a starter culture of lactic acid bacteria to produce a starter culture-fermented dairy product that has a pH of 3.0 to 5.0 and a lactose content of at least 1 .5 mg/ml, subjecting said starter culture-fermented dairy product to heat treatment in such a way as to reduce the number of bacteria to no more than 1x10 ² CFU/g, to obtain a heat-treated fermented milk product, adding lactase to said heat-treated fermented milk product, which retains its activity at pH 5.0 and a temperature of 37°C at a level of at least 5% compared to its activity at the optimal pH for lactase, to obtain a lactase-containing fermented dairy product, and storing said lactase-containing fermented dairy product at a temperature of at least 2°C for at least 1 day.

In other words, the third aspect is a specific embodiment of the first aspect, wherein the resulting fermented milk product is a fermented dairy product, and wherein the method includes the steps of fermenting a dairy substrate using a starter culture of lactic acid bacteria to produce a starter culture-fermented dairy product that has a pH of 3. 0 to 5.0 and a lactose content of at least 1.5 mg/ml, subjecting said starter culture-fermented milk product to heat treatment so as to reduce the bacterial count to not more than 1 x 10 ² CFU/g, to obtain a heat-treated fermented milk product product, adding to the specified heat-treated fermented dairy product lactase, which retains its activity at pH 5.0 and a temperature of 37°C at a level of at least 5% compared to its activity at the optimal pH for lactase, to obtain a lactase-containing fermented dairy product , and storing said lactase-containing fermented dairy product at a temperature of at least 2°C for at least 1 day.

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

In one embodiment of the method of the invention, the base fermented milk product or starter culture-fermented milk product may be heat treated in such a way as to reduce the bacterial count to no more than 1 x 10 ² CFU/g. Such a heat-treated acidified product is also referred to as a post-pasteurized product, thereby indicating that the product has been pasteurized after the chemically acidified dairy product or fermented dairy product has been produced. This heat-treated acidified product does not need to be stored at low temperature and is therefore suitable for transportation and storage at ambient temperature.

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Thus, in a preferred embodiment of the method according to the invention, the lactase-containing acidified or fermented milk product is maintained 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 specific embodiment of the method according to the invention, the lactase-containing acidified or fermented milk product is aged 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 days and most preferably at least 7 days.

In a specific embodiment of the invention, the lactase-containing acidified or fermented milk product has, after aging, a lactose content 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 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 can be any conventional lactic acid bacteria starter culture, including single-strain cultures and mixtures of cultures, used to produce a particular type of fermented dairy product. In a preferred embodiment of the product according to the invention, the fermentation is carried out so as to obtain a pH of from 3.0 to 5.0, preferably from 3.2 to 4.8, more preferably from 3.4 to 4.6 and most preferably from 3.8 up to 4.4.

Heat treatment to reduce the starter culture bacteria level to no more than 1.0 x 10 ² CFU/g fermented dairy product is preferably carried out by exposing the starter culture fermented dairy product to a temperature of from 50°C to 110°C, preferably from 50°C to 100 °C, preferably 50°C to 90°C, preferably 60°C to 85°C, more preferably 65°C to 82°C, and most preferably 70°C to 80°C. The heat treatment is preferably carried out for a period of 5 seconds to 180 seconds, preferably 10 seconds to 180 seconds, preferably 12 seconds to 120 seconds, more preferably 14 seconds to 90 seconds, more preferably 16 seconds to 60 seconds, more preferably from 18 seconds to 50 seconds, and most preferably from 20 to 40 seconds. Preferably, the bacterial count of the starter culture is reduced to no more than 10 CFU/gram of fermented milk, more preferably to 0 CFU/g. Fermented dairy products that have been heat treated to reduce bacterial levels to not more than 1 x 10 ² CFU/g are suitable for use with storage at ambient temperatures, such as storage at 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 method for producing a fermented dairy product, comprising the steps of fermenting a dairy substrate using a starter culture of lactic acid bacteria to produce a starter culture-fermented dairy product that has a pH of 3.0 to 5.0 and a lactose content of at least 1 .5 mg/ml, subjecting said starter culture-fermented dairy product to heat treatment so as to reduce the bacterial level to not more than 1 x 108 CFU/g to obtain a heat-treated fermented milk product, adding to said heat-treated fermented milk product lactase, which preserves its activity at pH 5.0 and a temperature of 37°C at a level of at least 5% compared to its activity at the optimal pH for lactase to obtain a lactase-containing fermented dairy product, and storing said lactase-containing fermented dairy product at a temperature of at least 2° C for at least 1 day. Preferably, the bacterial level of the starter culture is reduced to no more than 1.0 x 107 CFU/g fermented milk, more preferably to no more than 1.0 x 106 CFU/g fermented milk, and most preferably to no more than 1.0 x 105 CFU/g fermented milk. Fermented dairy products that are heat treated to reduce bacterial levels to no more than, for example, 1x108 CFU/g have an increased shelf life at low temperatures.

The enzyme is added in a suitable amount to achieve the required degree of lactose hydrolysis under the selected reaction conditions. In a specific embodiment of the invention, the dairy product contains lactase in an amount of from 100 to 20,000 LAU per liter of dairy product, preferably from 100 to 10,000 LAU per liter of dairy product, preferably from 100 to 5000 LAU per liter of dairy product, preferably less than 3000, for example less , less than 1500, less than 1000, less than 750 or less than 500 LAU per liter of dairy product.

In a preferred embodiment, lactase is added at a concentration of from 5 to 400 LAU per g of lactose in the milk product, preferably from 5 to 200 LAU per g of lactose in the milk product, preferably from 5 to 100 LAU per g of lactose in the milk product, preferably less than 50 , For example

- 8 045126 less than 40, less than 30, less than 20 or less than 10 LAU per g of lactose in the milk product.

In a preferred embodiment of the invention, the lactic acid product has a lactose content of from 2.0 mg/ml to 45 mg/ml, preferably from 5 mg/ml to 40 mg/ml, more preferably from 10 mg/ml to mg/ml and most preferably from 20 mg/ml to 37 mg/ml.

In a preferred embodiment of the invention, the fermented milk product to which the lactase is to be added has a viscosity that allows the lactase to easily spread throughout the fermented milk product, for example by mixing. In a preferred embodiment of the method according to the invention, the lactase to be added to the fermented milk product is provided in a sterile preparation. In another preferred embodiment of the method according to the invention, lactase is added to the fermented milk product under aseptic conditions, for example by sterilizing filtration of the lactase solution.

In a preferred embodiment of the method according to the invention, lactase is added to the fermented milk product by in-line dosing. According to the present invention, the term in-line dosing means dosing directly into a pipeline through which the fermented milk product flows. As used herein, the term conduit means conduit or any synonym thereof, including channel, pipe, trough, tube, line, standpipe, passage and hose. Examples of commercially available in-line dosing systems suitable for use in the present invention are the Tetra FlexDos aseptic in-line dosing system and the Tetra Aldose aseptic in-line dosing system.

In a preferred embodiment of the method of the invention, lactase is added to the fermented milk product by in-line dosing into a pipeline, and the lactase is then mixed with the yoghurt in the pipeline via a mixing device. Preferably, the mixing device is selected from the group consisting of at least one pipe bend, check valve spring, static mixer or rotor/static mixer. An example of a commercially available rotor/static mixer is the Ytron-Z homogenizer (pump-disperser).

Two-step addition of lactase.

In a specific embodiment of the third and fourth aspects of the method of the invention, lactase is added prior to heat treatment of the fermented dairy product seed culture. The previously added lactase can be any lactase, including lactase selected from the group consisting of H-lactase and lactase used in the method of the invention, i.e. lactase, which retains its activity at pH 5.0 and a temperature of 37°C at a level of at least 5% compared to its activity at the optimal pH for lactase. Na-lactase is a lactase obtained from yeasts of the genus Klyveromyces, in particular selected from the group consisting of Klyveromyces lactis and Klyveromyces fragilis.

In a specific embodiment, the previously added lactase is added at the beginning of the fermentation along with the starter culture. In this case, the previously added lactase is preferably H-lactase.

In another specific embodiment, the previously added lactase is added after fermentation has begun.

If lactase is added either at the start of fermentation or after heat treatment of the starter cultured dairy product, a portion of the lactose will be hydrolyzed during the fermentation step and the remainder of the lactose will be hydrolyzed during the storage of the heat treated fermented milk product. If lactase is added both at the beginning of fermentation and after heat treatment of the starter cultured dairy product, the total amount of lactase added can be reduced in order to reduce the lactose concentration in the fermented milk product to the required level compared to the case of adding lactase only after heat treatment a dairy product fermented with a starter culture. Thus, lactase added at the beginning of fermentation will be active at a higher pH than pH 3.0 to 5.0, for example pH 5.0 to 7.0, and most lactases have higher activity at this higher pH. Therefore, a certain amount of lactase added at the beginning of fermentation will result in more lactose hydrolysis than the same amount of lactase added after heat treatment of a dairy product fermented with a starter culture.

Application.

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

Definitions.

For the purposes of the present invention, the following definitions apply.

LAU stands for lactose units, and 1 lactase unit (1 LAU) represents the amount

- 9 045126 enzyme production that releases 1 micromol of 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 Na ₃ O ₇ 2H ₂ O, 8.31 g citric acid, 0.9 g K2SO4, 2.6 g K2HPO4, 7.35 g KH2PO4, 5.45 g KOH, 4.15 g MgCl ₂ 6H ₂ O, 3.75 g CaCl ₂ -2H ₂ O and 1.4 g NaHCO ₃ in 4 liters of water, adding 12.5 ml 4 N. NaOH, adjusting the pH to 6.5 using HCl and adding water up to a total volume of 5 liters.

The LAU activity of a particular lactase can be determined by directly measuring the glucose released from lactose under the conditions described above. One skilled in the art will know how to detect such activity. Alternatively, activity can be determined using the lactase activity assay described in Example 1 of this application. The activity is obtained by comparing a standard curve obtained with lactase of known activity, and the activity of the unknown sample is calculated from this. A lactase with known activity, for example, can be lactozym (Lactozym), purchased 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 that inactivates at least a portion of the bacteria in the starter culture. In this regard, the term inactivate means any stopping, stopping or inhibiting the growth of bacteria, such as lysing cells.

The expression lactic acid bacteria refers to Gram-positive microaerophilic or anaerobic bacteria that ferment sugars to produce acids, including lactic acid as the predominantly formed acid, acetic acid and propionic acid. The most productive 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. They are often used as food crops, alone or in combination with other lactic acid bacteria.

Lactic acid bacteria, including bacteria of the species Lactobacillus sp. and Lactococcus sp., are usually supplied to the dairy industry either as frozen or lyophilized cultures for bulk propagation of starters, or as so-called direct application cultures (DVS) intended for direct inoculation into a fermentation vessel or vat for the production of a dairy product, such as fermented dairy product or cheese. Such cultures of lactic acid bacteria are generally called starter cultures or starter cultures. Typically, a yogurt starter culture contains Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and in most countries, yogurt is legally defined as a fermented dairy product produced using a starter culture containing these two strains.

The term milk should be understood as the secretion of the mammary glands obtained by milking any animal 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 from plant matter, such as soy milk.

The term milk substrate can refer to any raw and/or processed milk substance that can be fermented according to the method of the invention. Thus, useful milk substrates include solutions/suspensions of any milk or milk-like products containing protein, such as whole or reduced fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, milk powder, whey, acid whey, whey permeate , including but not limited to acid whey permeate, whey powder, including acid whey powder, sweet whey powder, demineralized whey powder and lactose-free whey powder, lactose, lactose mother liquor, whey protein concentrate or cream. It will be appreciated that the milk substrate may be from any mammal, for example being substantially pure mammalian milk or reconstituted milk powder.

The term acid whey permeate means the whey permeate obtained by concentrating a fermented dairy product by ultrafiltration in the production of young cheese such as cottage cheese, ricotta, skyr, Greek yogurt, cottage cheese, quark curd and labneh obtained by acid coagulation by fermentation with lactic acid bacteria , and where the pH of the acid whey permeate in a 10% solution is less than 5.1.

The term acid whey means the whey fraction obtained by concentrating the fermented dairy product in a separator during the production of young cheese, such as cottage cheese, ricotta, skyr, Greek yogurt, cottage cheese, quark curd and labneh, obtained by acid coagulation by fermentation with lactic acid bacteria, and where the pH of the acid whey fraction in a 10% solution is less than 5.1.

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

The term homogenization as used here means vigorous mixing to produce a soluble suspension or emulsion. When homogenizing before

- 10 045126 fermentation can be carried out in such a way as to break down the milk fat to smaller sizes so that it no longer separates from the milk. This can be done by forcing milk under high pressure through small holes.

The term pasteurization as used herein means the treatment of a milk substrate to control or eliminate the presence of living organisms such as microorganisms. Preferably, pasteurization is achieved by maintaining a certain temperature for a certain period of time.

A certain temperature is usually achieved by heating. Temperature and duration can be selected to kill or inactivate specific bacteria, such as harmful bacteria. A rapid cooling step may then 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 involves converting lactose to lactic acid.

Fermentation methods to be used in the production of dairy products are well known, and one skilled in the art will know how to select appropriate process conditions such as temperature, oxygen content, number and characteristics of microorganism(s), and process time. Obviously, the fermentation conditions are chosen to support the achievement of the present invention, i.e. obtain a dairy product in solid (such as cheese) or liquid form (such as a fermented dairy product).

The use of singular terms in the context of the description of the present invention (especially in the context of the following claims) should be construed to cover both singular and plural terms, unless otherwise indicated or clearly inconsistent with the context. The terms containing, having, including and including should be construed as open terms (i.e., meaning including but not limited to) unless otherwise noted. The listing of ranges of values in this application is intended only to serve as a concise means of referring individually to each individual value falling within a given range unless otherwise indicated herein, and each individual value is included in this specification as if it were individually listed. All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contrary to context. The use of any and all examples or exemplary language used herein (eg, such as) is intended to better illustrate the invention only and is not intended to limit the scope of the invention unless otherwise stated. Nothing in the specification should be construed as indicating any unclaimed element essential to the practice of the present invention.

The expression fermented dairy product means a food or feed product where the production of the food or feed product involves fermentation of the milk substrate by lactic acid bacteria. The term fermented dairy product as used herein includes, but is not limited to, thermophilically fermented dairy products such as yogurt, mesophilically fermented dairy products such as sour cream and buttermilk, and fermented whey.

The term thermophile as used herein refers to microorganisms that grow best at temperatures above 35°C. The most industrially useful thermophilic bacteria include Streptococcus spp. and Lactobacillus spp. The term thermophilic fermentation here refers to fermentation at temperatures above about 35°C, such as from about 35°C to about 45°C. The term thermophilically fermented dairy product refers to fermented dairy products produced by thermophilic fermentation using a thermophilic starter culture, and includes fermented dairy products such as thermostatic yogurt, broken curd yogurt, and drinking yogurt such as Yakult.

The term mesophilic as used herein refers to microorganisms that grow best at moderate temperatures (15°C-35°C). The most industrially useful mesophilic bacteria include Lactococcus spp. and Leuconostoc spp. The term mesophilic fermentation refers here to fermentation at a temperature of from about 22°C to about 35°C. The term mesophilic fermented dairy product refers to fermented dairy products produced by mesophilic fermentation using a mesophilic starter culture and includes fermented dairy products such as buttermilk, curdled milk, fermented milk, sour cream, fermented cream, kefir and young cheese such as cottage cheese quark, cottage cheese and cream cheese.

Graphic materials

In fig. 1 shows the amounts of residual lactose in samples treated with lactase added to yoghurt after post-pasteurization for lactase amounts of 1000, 2000 and 3000 LAU/L.

In fig. Figure 2 shows the amounts of residual lactose in samples treated with lactase added to yoghurt after post-pasteurization, for lactase amounts of 200, 400, 600, 800, 1000 and 1200 LAU/L in 8.

- 11 045126

24, 48 and 76 hours.

In fig. 3 shows the data from FIG. 2 at the 48 hour point.

In fig. 4 shows the data from FIG. 2 at 76 hours.

Examples

Example 1.

Analysis of lactase activity in Eppendorf tubes at 37°C, pH 6.5.

Principle.

Lactase hydrolyzes lactose to glucose and galactose. Glucose is measured according to a modified version of the conventional glucose oxidase/peroxidase analytical method (Werner, W. et al. (1970) Z. analyt. Chem. 252: 224.).

LAU is defined as the amount of enzyme that releases 1 micromol of glucose per minute at 37°C, pH 6.5 in M-buffer (M-buffer is defined in the specification of this patent application). Alternatively, LAU activity for a particular lactase can be determined by the method described herein. The value obtained is compared with a standard curve obtained with lactase of known activity, and the activity of the unknown sample is calculated from this. A lactase with known activity, for example, can be lactozyme purchased from Novozymes A/S, Denmark.

Solutions.

Assay buffer: 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 _mM CaCl2, 1 mM _MgCl2 , 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 (horseradish peroxidase), 0.65 g/L ABTS (2,2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid).

Substrate.

160 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, ₂ mM CaCl2, 1 mM MgCl2, pH 6.5.

Standard.

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

Samples.

Enzyme samples are suitably diluted in assay buffer.

Methodology.

1. 375 µl of substrate is incubated for 5 minutes at 37°C.

2. Add 25 µl of enzyme diluted in assay buffer.

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

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

Example 2.

100 ml of 15% or 30% (w/w) whey permeate containing predominantly lactose and ions was prepared by mixing 15 or 30 g of spray-dried whey permeate powder (Variolac, Arla) in 85 or 70 ml of ion-containing water, respectively. This solution was poured into a flask containing a magnetic stir bar and placed in a water bath at 37°C. After 15 minutes, enzyme was added. The enzymes tested were Lactozyme, a commercial lactase from Novozymes A/S, Denmark, having an activity of 3060 LAU/g, and an experimental lactase from Bifidobacterium bifdum, having the encoded sequence shown in SEQ ID NO: 2 and an activity of 295 LAU/g.

The dosages were as follows: 4225 LAU/L of lactozyme milk and 2025 LAU/L of Bifidobacterium lactase milk. Milk samples were collected at regular intervals up to 5.5 hours and the enzyme was inactivated by heating to 99°C for 10 minutes in a thermomixer. Samples were suitably diluted and filtered through a 0.20 μm filter.

Lactose hydrolysis was measured using a Dionex BioLC equipped with a Dionex PA1 column and a pulsed amperometric detector (PAD). Peaks were identified and quantified by comparison with known standards of lactose, glucose and galactose.

The results are shown below.

- 12 045126

Table 1

Lactose, glucose and galactose in 15% DS whey permeate after treatment with lactozyme or Bifidobacterium lactase

Lactozyme Bifidobacterium lactase Time min Lactose mM Glucose mM Galactose mM Lactose mM Glucose mM Galactose mM 0 499 1 2 499 1 2 thirty 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 lactozyme or Bifidobacterium lactase.

Lactozyme Bifidobacterium lactase Time min Lactose mM Glucose mM Galactose mM Lactose mM Glucose mM Galactose mM 0 848 1 4 848 1 4 thirty 824 109 75 819 43 45 60 615 253 150 788 86 83 120 420 370 242 651 159 158 180 291 459 300 625 232 230 240 246 559 373 501 283 273 330 154 544 367 391 333 324 1440 54 649 545 20 727 739

Also, when tested at higher concentrations of lactose, such as in 15% or 30% whey permeate, no or very little galactooligosaccharides are produced. In addition, the levels of galactose and glucose produced are equal and correspond to the amount of hydrolyzed lactose. In comparison, lactozyme produces less galactose than glucose, clearly indicating 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, determined using lactose as substrate.

Principle.

Lactase hydrolyzes lactose to form glucose and galactose. Glucose is measured according to a modified version of the conventional glucose oxidase/peroxidase method (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 KCl, 2 mM CaCb, 1 mM MgCl ₂ , and pH adjusted to pH 3, 4, 5, 6, 7, 8, 9 and 10 s We use NaOH.

Enzyme sample.

Experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID NO: 2 was suitably diluted in 150 mM KCl, 2 mM CaCl ₂ , 1 mM MgCl 2 , 0.01% Triton X100.

Methodology.

µL of enzyme sample diluted in enzyme dilution buffer was added to PCR (polymerase chain reaction) tubes at room temperature.

µL of substrate was added at room temperature 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 μl of 0.25 M NaOH.

µL was transferred to a 96-well microtiter plate, and 230 µL of 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, absorbance was measured at 420 nm.

- 13 045126

Temperature profile.

Substrate.

167 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ and adjusted to pH 6.5 using NaOH.

Enzyme sample.

Experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID NO: 2 was suitably diluted in 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , 0, 01% Triton X100, and the pH was adjusted to pH 6.5.

Procedure: 10 µl of enzyme sample diluted in enzyme dilution buffer was added to PCR tubes at room temperature.

µl of prewarmed (Peltier thermal cycler, 30-70°C) substrate was added and incubation was carried out with a temperature gradient from 30 to 70°C over 30 min, and then placed on ice.

The reaction was stopped by adding 100 μl of 0.25 M NaOH.

µL was transferred to a 96-well microtiter plate, and 230 µL of 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, absorbance was measured at 420 nm.

Table 4

Pace. °C Relative lactase activity of B. bifidum (% of activity at 38.1 °C) 20 54 21 63 22 64 24 68 26 73 29 81 31 88 34 94 36 96 38 100 43 96 48 91 52 83 57 76 62 58 66 32 69 20 70 17

- 14 045126

Example 4.

Determination of Km for lactase enzymes at 5°C.

Principle.

Lactase hydrolyzes lactose to form glucose and galactose. Glucose is measured according to a modified version of the conventional glucose oxidase/peroxidase method (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 KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , and pH adjusted to pH 6.5 using NaOH.

Enzyme sample.

Experimental lactase from Bifidobacterium bifdum having the encoded sequence shown in SEQ ID NO: 2 was suitably diluted in 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , 0, 01% Triton X100, and the pH was adjusted to pH 6.5 with NaOH.

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

Methodology.

µL of enzyme sample (5°C) was added to a 96-well microtiter plate on ice.

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

The reaction was stopped by adding 100 μl of 0.25 M NaOH.

µL was transferred to a 96-well microtiter plate, and 230 µL of 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, absorbance was measured at 420 nm.

Determination of Km.

Computerized nonlinear least squares approximation and Michaelis-Menten equation were used:

V = (Vmax*S)/(Km+S).

It was determined that Km for Bifidobacterium lactase and lactozyme is 8 and 30 mM, respectively.

In a similar analysis carried out at 37°C, the Km for Bifidobacterium lactase and lactozyme was determined to be 13 mM and 30 mM, respectively.

Example 5.

Preparation of post-pasteurized yogurt containing lactase added after pasteurization.

Milk substrate.

Fat amount 2.8%*.

Protein amount 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%.

*Quantity in the final product, i.e. after heat treatment, adding a strain for storage at ambient temperature and storage for 150 days.

Starter culture.

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

Lactase.

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

Methodology for producing the product.

1. Dispersing dry ingredients in milk.

2. Remain at rest for 3 hours with light stirring.

3. Heating the milk until the temperature reaches 65°C.

4. Homogenization at 150 bar (15 MPa).

5. Treatment by heating to 95°C for 5 minutes.

6. Cooling to fermentation temperature 43°C.

7. Pumping milk into the fermentation tank.

8. Inoculation of YoFlex culture type YF-L904.

9. Fermentation until pH 4.30 is reached.

10. Destruction of the coagulum and mixing until a homogeneous structure is obtained.

11. Heat treatment at 75°C for 30 s.

12. Cooling to 25°C.

13. Add lactase in the amount of 1420 LAU/l of yoghurt product, and mix slowly

- 15 045126 mixing yogurt in such a way as to evenly distribute the lactase in the yogurt.

14. Filling containers with yogurt.

15. Keep the yogurt at a temperature of 20°C for a period of 7 days.

Example 6.

Subsequent addition of lactase to post-pasteurized yogurt.

The purpose of the experimental work carried out was to demonstrate that it is possible to add a suitable lactase after the last heat treatment of post-pasteurized yogurt to achieve lactose residuals of less than 0.01%.

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

Milk substrate.

Ingredients Specification Dosage Fresh milk 3.5% fat, commercial milk, Aria Foods 74.15% Water 16.6% Sucrose 7.0% Whey Protein Concentrate Nutrilac YO-7830, Aria Food Ingredients 0.60% Modified starch Clearam CJ 5025, Roquette 1.50% Pectin LM LM 106-AS YA, CP Kelco 0.12% Gellan gum Kelcogel YSS, CP Kelco 0.03% Cultures FD-DVS YF-L904 200 units/metric tons

Milkoscan analysis: fat level 2.8%, protein level 2.9%.

The following parameters were used for fermentation and processing.

Stirring temperature: 10°C.

Hydration time: 3 hours with light stirring.

Lactase.

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

Starter culture.

YoFlex® culture type YF-L904 from the YoFlex® Chr product line. Hansen, containing two species: Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus.

Method for producing yogurt.

Homogenization pressure: 150 bar (15 MPa).

Pasteurization conditions: 95°C, 5 minutes.

Fermentation temperature: 43°C.

Final pH: 4.30.

The coagulum is destroyed manually and mixed until a homogeneous structure is obtained.

Cooling to 15 °C in a plate-type heat exchanger.

Thermalization in a plate-type heat exchanger, current 414 l/h.

Homogenization pressure: 0 Pa.

Thermization conditions: 74°C, 20 s.

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

Adding lactase.

Lactase was subjected to sterilizing filtration and added to 100 ml glasses of post-pasteurized yogurt in the following dosages:

a) 0 LAU/l - control,

b) 1000 LAU/l,

c) 3000 LAU/l,

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d) 10000 LAU/l.

Samples were kept at room temperature (22°C) and samples were collected for residual lactose analysis at the following time points: 24 hours, 48 hours and 1 week.

Results.

In fig. Figure 1 shows the residual lactose of samples with added lactase. The control sample's residual lactose was measured to be 2.4% residual lactose. As will be seen from Fig. 1, lactose concentration decreased to a level of approximately 0.005% at lactase concentrations of 3000 LAU/L and 10,000 LAU/L after 24 hours of addition. Also, the lactose concentration decreased to a level of approximately 0.005% for all three lactase concentrations after 48 hours and 1 week after addition.

Example 7.

Subsequent addition of lactase to post-pasteurized yogurt.

The purpose of the experimental work carried out was to demonstrate that it is possible to add a suitable lactase after the final heat treatment of post-pasteurized yogurt to achieve residual lactose levels of less than 0.1%.

Post-pasteurized yogurt was produced with 2.87% protein and 2.89% fat. The milk substrate was fermented to pH 4.30 using YoFlex culture type YF-L904. After reaching a pH of 4.30, the yogurt was cooled to 15°C in a plate heat exchanger and kept in an insulated buffer vessel at 15°C for three hours before the final heat treatment. The final heat treatment was carried out at 74°C for 20 s in a plate type heat exchanger and the product was filled into sterile beakers at 25°C. The samples were kept at 25°C for one day before adding sterile lactase.

Milk substrate.

Ingredients Specification Dosage Fresh milk 3.5% fat, commercial milk, Aria Foods 74.15% Water 16.6% Sucrose 7.0% Whey Protein Concentrate Nutrilac YO-7830, Aria Food Ingredients 0.60% Modified starch Clearam CJ 5025, Roquette 1.50% Pectin LM LM 106-AS YA, CP Kelco 0.12% Gellan gum Kelcogel YSS, CP Kelco 0.03% Cultures FD-DVS YF-L904 200 units/metric tons

Milkoscan analysis: fat level 2.87%, protein level 2.89%.

The following parameters were used for fermentation and processing.

Stirring temperature: 10°C.

Hydration time: 3 hours with light stirring.

Lactase.

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

Starter culture.

YoFlex® culture type YF-L904 from the YoFlex® Chr product line. Hansen, containing two species: Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus.

Method for producing yogurt.

Homogenization pressure: 150 bar (15 MPa).

Pasteurization conditions: 95°C, 5 minutes.

Fermentation temperature: 43°C.

Final pH: 4.30.

The coagulum is destroyed manually and mixed until a homogeneous structure is obtained.

Cooling to 15 °C in a plate-type heat exchanger.

Thermalization in a plate-type heat exchanger, current 414 l/h.

Thermization conditions: 74°C, 20 s.

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

Adding lactase.

Lactase was subjected to sterilizing filtration and added to 100 ml post-pasteurized glasses.

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a) 0 LAU/l - control,

b) 200 LAU/l,

c) 400 LAU/l,

d) 600 LAU/l,

e) 800 LAU/l,

e) 1000 LAU/l,

g) 1200 LAU/l.

Samples were kept 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 fig. Figures 2-4 show the residual lactose of samples with added lactase. The control sample's residual lactose was measured to be 2.4% residual lactose. The horizontal lines at residual lactose levels of 0.1 and 0.5 indicate the level of lactose that qualifies as lactose-free under food regulatory laws in the EU (European Union) and China, respectively.

As will be seen from Fig. 2-4, the lactose concentration after 24 hours decreased to a level of approximately 0.355% at a lactase concentration of 400 LAU/L and to less than 0.01% at higher lactase concentrations. After 48 hours, the lactose concentration dropped to 0.015% at lactase concentrations of 400 LAU/L and to less than 0.01% at higher lactase concentrations. After 76 hours, the lactose concentration dropped to less than 0.01% at lactase concentrations of 400 LAU/L and higher lactase concentrations.

Thus, the present experiments demonstrate that it is possible to meet a target residual lactose level of 0.01% or less using a lactase dose of 600 LAU/L at 48 hours and a lactase dose of 400 LAU/L at 76 hours.

Example 8.

Subsequent addition of lactase to post-pasteurized yogurt at different pH levels (from 4.0 to 4.4).

The purpose of the experimental work was to demonstrate that the lactose removal process in post-pasteurized yoghurt (PPY) using lactase can achieve the required lactose removal for PPY with different pH ranging from 4.0 to 4.4.

Milk substrate.

Ingredients Specification Dosage with standard lactose level Dosage with high lactose levels Fresh milk 3.5% fat, commercial milk, Aria Foods 74.30% 74.0% Water 16.45% 15.95% Sucrose 7.0% 7.0% Whey Protein Concentrate Nutrilac YO-7830, Aria Food Ingredients 0.60% 0.60% Modified starch Clearam CJ 5025, Roquette 1.50% 1.5% Pectin LM LM 106-AS YA, CP Kelco 0.12% 0.12% Gellan gum Kelcogel YSS, CP Kelco 0.03% 0.03% Cultures FD-DVS YF-L904 200 units/metric tons 200 units/metric tons

Milkoscan analysis.

Milk base with standard lactose level: 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 coded sequence SEQ ID NO. 2.

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Method for producing yogurt.

Homogenization pressure: 150 bar (15 MPa) at 60°C.

Pasteurization conditions: 95 °C, 5 minutes.

Fermentation temperature: 43 °C.

Final pH for milk base with standard lactose level: 4.0; 4.1; 4.2; 4.3 and 4.4.

Final pH for high lactose milk base: 4.3.

The coagulum is destroyed manually and mixed until a homogeneous structure is obtained.

Cooling to 15 °C in a plate-type heat exchanger.

Thermalization in a plate-type heat exchanger, current 414 l/h.

Thermization conditions: 74°C, 20 s.

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

Adding lactase.

Lactase was subjected to sterilizing filtration and added to 100 ml glasses of post-pasteurized yogurt in the following dosages:

a) 0 LAU/l - control,

b) 200 LAU/l,

c) 400 LAU/l,

d) 600 LAU/l,

e) 800 LAU/l.

Samples were maintained 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 brevity, only results from 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 Final pH Lactase dosage (LAU/l) 24 hours (%) 48 hours (%) 72 hours (%) 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 BUT (not defined) BUT BUT

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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 BUT BUT BUT 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 without added lactase

Milk base pH Lactase dosage (LAU/l) 24 hours (%) Standard 4.4 0 2.6 Standard 4.3 0 2.7 Standard 4.2 0 2.6 Standard 4.1 0 2.5 Standard 4.0 0 2.5 High 4.3 0 3.2 Standard (before fermentation) 0 z, z High (before fermentation) 0 4.0

As will be seen from Tables 5 and 6, the lower the final pH level of yogurt, the higher the residual lactose content. These results also show that at the lowest final pH level, it is possible to achieve a target residual lactose level of less than 0.1% with a lactase dose of 800 LAU/L after 48 hours. In addition, at a final pH of 4.1, it is possible to achieve a residual lactose level of 0.108% with a lactase dose of 1000 LAU/L after 24 hours.

Example 9.

Subsequent addition of lactase to post-pasteurized yogurt at low levels of added lactase.

The purpose of the experimental work was to test the effectiveness of low dosages of lactase in the lactase removal process for post-pasteurized yoghurt (PPY) in order to establish the minimum dose required to obtain the required target level of residual lactose.

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Milk substrate.

Ingredients Specification Dosage Fresh milk 3.5% fat, commercial milk, Aria Foods 74.30% Water 16.45% Sucrose 7.0% Whey Protein Concentrate Nutrilac YO-7830, Aria Food Ingredients 0.60% Modified starch Clearam CJ 5025, Roquette 1.50% Pectin LM LM 106-AS YA, CP Kelco 0.12% Gellan gum Kelcogel YSS, CP Kelco 0.03% Cultures FD-DVS YF-L904 200 units/metric tons

Milkoscan analysis.

Dairy base: fat level 2.81%, protein level 3.11%.

Lactase.

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

Method for producing yogurt.

Homogenization pressure: 150 bar (15 MPa) at 60°C.

Pasteurization conditions: 95 °C, 5 minutes.

Fermentation temperature: 43 °C.

Final pH for milk base with standard lactose level: 4.15; 4.20; 4.25 and 4.30.

Final pH for high lactose milk base: 4.3.

The coagulum is destroyed manually and mixed until a homogeneous structure is obtained.

Cooling to 15 °C in a plate-type heat exchanger.

Thermalization in a plate-type heat exchanger, current 414 l/h.

Thermization conditions: 74°C, 20 s.

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

Adding lactase.

Lactase was subjected to sterilizing filtration and added to 100 ml glasses of post-pasteurized yogurt in the following dosages:

a) 0 LAU/l - control,

b) 50 LAU/l,

c) 100 LAU/l,

d) 200 LAU/l,

e) 300 LAU/l.

Samples were kept 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 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.

Milk base Final pH Lactase dosage (LAU/l) 24 hours (%) 48 hours (%) 72 hours (%) Standard 4.30 50 2.1 1.8 1.5 Standard 4.25 100 1.8 1.3 0.9 Standard 4.20 100 1.9 1.4 1.1 Standard 4.15 100 1.9 1.5 1.2 Standard 4.30 200 1.2 0.4 0.087 Standard 4.25 200 1.1 0.5 0.119 Standard 4.20 200 1.4 0.7 0.237

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Standard 4.15 200 1.4 0.7 0.317 Standard 4.30 300 0.7 0.055 0.010 Standard 4.25 300 0.8 0.102 0.017 Standard 4.20 300 0.9 0.2 0.037 Standard 4.15 300 1.0 o, s 0.081

Pasteurized yoghurt base with pH 4.3; 4.25; 4.20 and 4.15 and without added lactase had a lactose level of 2.57; 2.52; 2.47 and 2.40, respectively.

As will be seen from the table. 7, it is possible to achieve a target residual lactose level of less than 0.5% using a lactase dose of 200 LAU/L after 48 hours for samples with pH 4.3 and 4.25, and with a lactose level of less than 0.3% using a lactase dose of 300 LAU/L after 48 hours for all samples tested.

Example 10.

Production of an acid whey drink with a low lactose level by adding lactase.

The purpose of the experimental work carried out was to demonstrate that it was possible to remove lactose from acid whey permeate and to investigate the effect of different lactase dosages, reaction times and temperatures.

Milk substrate.

Ingredients Specification Dosage (g/1000 g) Acid whey Acid whey permeate (by-product from skyr production) 800 Sucrose Nordic Sugar 70 Water (ionized) 90 Pectin NM YM-115-H, CP-Kelco 4.5 Sum 979.5

Acid whey permeate (from ultrafiltration) is a by-product from the 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 coded sequence SEQ ID NO. 2. Method.

1. Heat the acidic permeate to 72°C/2 min.

2. Cool until:

a) 5°C (2 x 1000 ml),

b) 40°C (3 x 1000 ml).

3. Lactase dosing:

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 lactose analysis.

a) Before adding 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 a drink based on acid whey.

a) Adding a solution of LM pectin, flavoring agent and sucrose.

b) Adjust the pH to 3.9 with citric acid.

c) Heating to 80°C for 2 minutes.

d) Homogenization at 15 MPa at 80°C.

e) Filling in a bottle.

Results.

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Claims (5)

Table 8 Residual lactose levels (g/l) for samples tested Pace. 5°С 5°С Lactase (LAU/l) 0 2500 5000 0 hours 2.7235 1 hour 2 hours 3 hours 4 hours 5 o'clock 6 hours 7 o'clock 24 hours 0.5646 0.1018 40°С 40°С 40°С 0 500 1000 2500 2.7235 1.7793 0.5527 0.3117 0.1681 0.1376 0.4381 0.0830 0.0378 As will be seen from the table. 8, it was possible to reduce the lactose level to an amount of about 0.1 g/L for the samples tested 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 two other samples it was possible to reduce lactose levels to levels less than 0.5 g/L. List of sequences. SEQ ID NO. 1: Shows the sequence of the mutant SEQ ID NO. 4. SEQ ID NO. 2: shows the sequence of the mutant SEQ ID NO. 4. SEQ ID NO. 3: Shows the sequence of lactase from Bifidobacterium bifidum DSM20215. SEQ ID NO. 4: shows the sequence of lactase from Bifidobacterium bifidum NCIMB41171, the nucleotide sequence of which is listed in NCBI (US National Center for Biotechnology Information) with accession number DQ448279. SEQ ID NO. 4 is discussed in the following links, where it is called bbgIII: Appl Microbiol Biotechnol (2007) 76:1365–1372 T K Goulas et al. Appl Microbiol Biotechnol (2009) 82:1079–1088 T Goulas et al. Appl Microbiol Biotechnol (2009) 84:899–907 T Goulas et al. CLAIM 1. A method for producing a fermented dairy product, including the stages: a) fermentation of the milk substrate using a starter culture of lactic acid bacteria to produce a starter culture-fermented milk product that has a pH of 3.0 to 5.0 and a lactose content of at least 1.5 mg/ml, b) subjecting said starter culture-fermented dairy product to heat treatment in such a way as to reduce the number of starter culture bacteria to no more than 1x10 ² CFU/g (colony forming units), to obtain a heat-treated fermented dairy product, c) adding lactase to the specified heat-treated fermented milk product, which retains its activity at pH 5.0 and a temperature of 37°C at a level of at least 5% compared to its activity at pH 6.5, optimal for lactase, to obtain lactase-containing fermented dairy product, and d) keeping the specified lactase-containing fermented dairy product at a temperature of 2 to 25 ° C for 1 to 7 days to hydrolyze lactose. 2. The method according to claim 1, where the lactase-containing fermented dairy product is kept at a temperature of 15°C. 3. The method according to claim 1 or 2, where the lactase-containing fermented dairy product is kept for 7 days. 4. The method according to any one of claims 1 to 3, wherein the starter culture-fermented dairy product is subjected to a concentration step to separate said starter culture-fermented dairy product into a concentrated fraction and a separated acid whey fraction, wherein said separated acid whey fraction, but not a concentrated fraction, subjected to subsequent stages of this method. 5. Lactase-containing fermented dairy product obtained by the method according to claim 1, where -