EA042481B1 - METHOD FOR PRODUCING A FERMENTED DAIRY PRODUCT - Google Patents

Description

Field of invention

The present invention relates to a method for producing a fermented dairy product.

Prior Art

WO 2009/071539 discloses a lactase derived from Bifidobacterium bifidum which has the ability to hydrolyze very efficiently in milk and which has activity over a wide pH range, including low pH, e.g. pH less than 5. Lactase can be used in milk production processes and fermented dairy products such as cheese, yogurt, butter, buttermilk, sour cream, etc. to reduce lactose.

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

EP-A1-2957180 in one embodiment discloses a process for making a fermented dairy product using a combination of starter cultures and conventional lactase to reduce the lactose content and the level of subsequent acidification in the fermented dairy product. EPA1-2957180, in a second embodiment, discloses a method for producing a fermented dairy product using lactose-deficient lactic acid bacteria.

Brief summary of the invention

The object of the present invention is to provide an improved process for the production of a fermented dairy product.

The object in accordance with the present invention is achieved by a method for producing a fermented dairy product, comprising the steps:

1) adding a starter culture containing at least one strain of lactic acid bacteria to the milk base,

2) fermenting milk for a period of time until the desired pH is reached,

3) where the starter culture contains at least one lactose-deficient strain that is capable of metabolizing a carbohydrate other than lactose, and

4) adding low pH stable lactase to the process at the beginning, during or at the end of the fermentation step, where the low pH stable lactase retains its activity at pH 5.0 and at 37° C. at a level of at least least 5% compared to its activity at the optimal pH for lactase.

Lactose-deficient lactic acid bacteria generally grow on a carbohydrate source other than lactose, such as sucrose, galactose, and glucose, added to milk in such an amount as to stop the fermentation process and the growth of lactic acid bacteria by eliminating the added carbohydrate source. In this way, subsequent acidification during subsequent storage is significantly reduced or even completely prevented. Lactase, which is stable at low pH, will be active during the entire course of the fermentation process and therefore ensures that most or all of the lactose present in milk is converted into glucose and galactose. Thus, it is possible to obtain a lactose-reduced fermented dairy product or a lactose-free product. It is also possible to obtain a fermented dairy product with increased natural sweetness, since glucose and galactose have a much higher sweetness than lactose.

The present invention is based on the understanding that by using a combination of low pH stable lactase and lactose-deficient lactic acid bacteria, a fermented milk product can be obtained that simultaneously has reduced lactose content, increased sweetness, and reduced subsequent acidification. Furthermore, it has been unexpectedly found that said combination results in a fermented milk product having improved texture compared to using lactose-deficient lactic acid bacteria and without lactase and compared to using low pH stable lactase and lactose-positive lactic acid bacteria.

In addition, when lactase is added prior to the fermentation step, then the method according to the invention makes it possible to reduce or even eliminate the amount of carbohydrate other than lactose added to the milk. Thus, glucose and galactose, formed due to the enzymatic action of lactase, will be available for the growth of lactose-deficient lactic acid bacteria.

Finally, when lactase is added after the fermentation step, it is then possible to take full advantage of the ability of lactose-deficient lactic acid bacteria to avoid subsequent acidification.

Around the world, a significant number of consumers are lactose intolerant or sensitive. Thus, there is currently a high demand for dairy products that include fermented dairy products that are reduced in lactose, or that are substantially lactose-free. The present invention provides a novel approach for making such a product in a simple and cost effective manner.

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Detailed description of the invention

Lactase

The lactase of the fermented dairy product according to the invention may be any lactase which retains at least 5% activity at pH 5.0 and 37° C. compared to its activity at lactase-optimal pH.

In relation to the present invention, lactase activity in LAU (lactose units) is measured as presented in the Definitions section below.

In a preferred embodiment according to the 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 optimal lactase pH.

In a preferred embodiment according to the 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 optimum for lactase pH.

In a preferred embodiment according to the 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 optimum for lactase pH.

With respect to the present invention, the optimal pH for lactase is determined by measuring lactase activity at pH using the method described in the Definitions section below and determining the pH with optimal activity.

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

With respect to the present invention, the optimum temperature for lactase is determined by measuring lactase activity at various temperatures using the method described in the Definitions section below and determining the temperature with optimum activity.

In a preferred embodiment, the lactase used in the product according to 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%, at least 70% or at least 75% of lactase activity at 37°C and pH 6.

In yet another preferred embodiment, the lactase used in the product according to 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 the lactase activity at 37°C and pH 6.

In yet another preferred embodiment, the lactase used in the product according to the present invention has an optimum pH for lactase activity at 37° C., which is greater than pH 5.5.

In yet another preferred embodiment, the lactase used in the product according to the present invention has a lactase activity at 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 lactase activity at a temperature of 38°C and pH 6.5.

In a preferred embodiment according to the present invention, the Km of lactase at 5° C. is less than 25 mM, such as 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, such as less than 20 mM or less than 15 mM. One skilled in the art knows how to determine Km for lactase activity at a particular temperature. Km can be determined in the manner described in WO 2009/071539.

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

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

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

Enzymes having lactase activity used in the method according to the present invention may be of animal, vegetable or microbial origin. Preferred lactases are obtained from microbial sources, in particular from filamentous fungi or yeasts, or from bacteria.

The enzyme may, for example, be derived from a strain of Agaricus, such as 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. fragilis, K. lactis; Mucor, e.g. M. javanicus, M. mucedo, M. subtilissimus; Neurospora, eg N. crassa; Rhizomucor, eg R. pusillus; Rhizopus, e.g. R. arrhizus, R. japonicus, R. stolonifer; Sclerotinia, e.g. S. libertiana; Torula; Torulopsis; Trichophyton, e.g. T. rubrum; Whetzelinia, e.g. W. sclerotiorum; Bacillus, eg B. coagulans, B. circulans, B. megaterium, B. novalis, B. subtilis, B. pumilus, B. stearothermophilus, B. thuringiensis; Bifidobacterium, e.g. B. longum, B. bifidum, B. animalis; Chryseobacterium; Citrobacter, such as C. freundii; Clostridium, such as C. perfringens; Diplodia, e.g. D. gossypina; Enterobacter, e.g. E. aerogenes, E. cloacae Edwardsiella, E. tarda; Erwinia, e.g. E. herbicola; Escherichia, eg E. coli; Klebsiella, e.g. K. pneumoniae; Miriococcum; myrothesium; Mucor; Neurospora, eg N. crassa; Proteus, for example P. vulgaris; Providencia, e.g. P. stuartii; Pycnoporus, e.g. Pycnoporus cinnabarinus, Pycnoporus sanguineus; Ruminococcus, eg R. torques; Salmonella, eg 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 is from a bacterium, for example 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 is from Bifidobacterium bifidum.

In a preferred embodiment, the enzyme having lactase activity used in the product according to 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 in SEQ ID NO: 1, amino acids 28-1331 in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and their fragments having lactase activity. In a more preferred embodiment, the enzyme contains 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 a sequence selected from the group consisting of amino acids 28-1931 in SEQ ID NO: 1, amino acids 28-1331 in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and their fragments having lactase activity.

Preferably the enzyme is a lactase having a sequence that 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 in SEQ ID NO: 1 or a fragment having lactase activity. Such a lactase-active fragment in SEQ ID NO: 1 may be any fragment in SEQ ID NO: 1 having lactase activity. The lactase-active fragment in SEQ ID NO: 1 may be, for example, amino acids 28-979, amino acids 28-1170, amino acids 28-1323, amino acids 28-1331, or amino acids 28-1600 in SEQ ID NO: 1.

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

In yet another embodiment, the enzyme having lactase activity used in the product according to 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 used in the product according to 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% , 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 tasks in accordance with the present invention, the degree of identity of two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch (1970) J. Mol. Biol. European Molecular Biology Open Software Suite, Rice et al (2000) Trends in Genetics 16: 276277), preferably version 3.0.0 or later. Possible parameters used are a gap insertion penalty of 10, a gap lengthening penalty of 0.5, and an EBLOSUM62 replacement matrix (EMBOSS variant of BLOSUM62). The Needle output marked as the longest identity (obtained using the non-short option) is used as a percentage identity and is calculated as follows:

(Identical residuals x 100)/(Alignment length - Total number of gaps in the alignment).

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

The lactases 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 used in the method of the present invention may be derived from any of the sources mentioned herein. The term originating means in this context that the enzyme can be isolated from an organism where it is present natively, ie. the amino acid sequence identity of the enzyme is identical to that of the native enzyme. The term originating also means that the enzymes can be produced recombinantly in the host organism, the recombinantly produced enzyme has an identity with the native enzyme or has a modified amino acid sequence, e.g. has a deletion, insertion and/or substitution of one or more amino acids, i.e. . a recombinantly produced enzyme that is a mutant and/or fragment of a native amino acid sequence. Natural variants are included in the concept of native enzyme. In addition, the term originating includes enzymes produced synthetically by, for example, peptide synthesis. The term originating also encompasses enzymes that are modified, for example, by glycosylation, phosphorylation, and the like, both in vivo and in vitro. With respect to a recombinantly produced enzyme, the term derived from refers to the identity of the enzyme, not the identity of the host organism in which it is produced recombinantly.

The lactase can be obtained from the microorganism by using any suitable method. For example, a lactase enzyme preparation can be obtained 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 using recombinant DNA techniques. Such a method typically involves growing a host cell transformed with a recombinant DNA vector comprising a DNA sequence encoding the lactase of interest and a DNA sequence operably linked to a suitable expression signal such that it is capable of expressing lactase in a culture medium under conditions that provide expression of the enzyme and isolation of the enzyme from the culture. The DNA sequence may also be included in the genome of the host cell. The DNA sequence may be genomic DNA, cDNA, or synthetic origin, or any combination thereof, and may be isolated or synthesized according to methods known in the art.

The lactases used in the process according to the present invention can be purified.

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As used herein, the term purified encompasses a lactase enzyme protein substantially free of the insoluble components of the producing organism. The term purified also encompasses a lactase enzyme protein substantially free of the insoluble components of the native organism from which it is derived. Preferably, it is also separated from some of the soluble components of the organism and the culture medium from which it is obtained. More preferably, it is separated by one or more operations: filtration, precipitation or chromatography.

Accordingly, an enzyme having lactase activity can be purified such that only minor amounts of other proteins are present. The expression other proteins refers in particular to other enzymes. As used herein, the term purified also refers to the removal of other components, in particular other proteins, and in particular other enzymes present in the cell from which the lactase originates. The lactase may be substantially pure, ie. not containing other components of 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 enzyme protein preparation, more preferably at least 50%, 60%, 70%, 80% or even at least 90% pure.

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 present in the reaction system in nature.

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

lactose-deficient lactic acid bacteria

The terms lactose metabolism deficiency and lactose deficiency are used in the context of the present invention to characterize LABs that have partially or completely lost the ability to use lactose as a source for cell growth or maintenance of cell viability. The respective LABs are capable of metabolizing one or more carbohydrates selected from sucrose, galactose and/or glucose, or another fermentable carbohydrate. Because these carbohydrates are not naturally present in milk in sufficient quantities to support fermentation with lactose-deficient mutants, it is necessary to add these carbohydrates to milk. Lactose-deficient and partially deficient LABs can be characterized as white colonies on a medium containing lactose and X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside).

In a specific embodiment according to the invention, the lactose-deficient strain is capable of metabolizing a carbohydrate other than lactose selected from the group consisting of sucrose, galactose and glucose, preferably sucrose. In a specific embodiment according to the invention, the lactose-deficient strain is capable of metabolizing galactose.

In a particular embodiment according to the invention, the lactose-deficient strain is selected from the group consisting of lactose-deficient Streptococcus thermophilus and lactose-deficient Lactobacillus delbrueckii subsp. bulgaricus.

In a particular embodiment according to the invention, the lactose-deficient strain is selected from the group consisting of:

(a) a strain of Streptococcus thermophilus, which is:

(1) a strain deposited with the German Collection of Microorganisms and Cell Cultures (DSMZDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 2014-06-12) under accession number DSM 28952;

(2) or a strain derived from DSM 28952, further characterized as having the ability to form white colonies on a medium containing lactose and X-Gal;

(b) a strain of Streptococcus thermophilus, which is:

(1) a strain deposited with the German Collection of Microorganisms and Cell Cultures (DSMZDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 2014-06-12) under accession number DSM 28953;

(2) or a strain derived from DSM 28953, further characterized as having the ability to form white colonies on a medium containing lactose and X-Gal;

(c) a strain of Lactobacillus delbrueckii subsp. bulgaricus, which is:

(1) a strain deposited with the German Collection of Microorganisms and Cell Cultures (DSMZDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 2014-06-12) under accession number DSM 28910;

(2) or a strain derived from DSM 28910, wherein the derived strain is further characterized as having the ability to form white colonies on a medium containing lactose and X-Gal.

In a specific embodiment according to the invention, the starter culture contains at least one lactose-deficient Streptococcus thermophilus and at least one lactose

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In a specific embodiment according to the invention, the starter culture comprises at least one Streptococcus thermophilus and at least one Lactobacillus delbrueckii subsp. bulgaricus, and wherein all of the strains of Streptococcus thermophilus and all of the Lactobacillus delbrueckii subsp. bulgaricus are lactose deficient.

Method steps according to the invention

In a specific embodiment according to the invention, the fermentation step is terminated in a manner selected from the group consisting of:

1) acidification of the fermented milk, leading to the fact that at least one strain of the starter culture is not able to grow,

2) cooling treatment and

3) depletion of a carbohydrate other than lactose.

In a specific embodiment according to the invention, a carbohydrate other than lactose is added to the milk base at the start of the fermentation step.

Preferably, a carbohydrate other than lactose is added to the milk base in such an amount that it is depleted and therefore results in the cessation of lactic acid bacteria growth and cessation of fermentation. Preferably, a carbohydrate other than lactose is added to the milk base in such an amount that it is depleted and therefore results in the cessation of lactic acid bacteria growth and cessation of fermentation at the selected desired pH.

The amount of carbohydrate other than lactose added to the milk base depends on a variety of parameters including the strains of lactic acid bacteria used in the starter culture, the composition of the milk base, the fermentation temperature, and the desired target pH. In addition, the amount of carbohydrate other than lactose added to the milk base may depend on the type and amount of lactase used in the process. The amount of carbohydrate other than lactose added to the milk base can be determined by experimentation and is within the knowledge of a person skilled in the art carrying out such an experiment.

In a particular embodiment according to the invention, the added carbohydrate other than lactose is selected from the group consisting of sucrose, galactose and glucose, preferably sucrose.

In a first aspect according to the invention, the low pH stable lactase is added to the milk base at the beginning of the fermentation step. In this aspect, the added lactase converts the milk base lactose into glucose and galactose, which are capable of being metabolized for the starter culture in addition to the added carbohydrate other than lactose. In this case, it is not possible to stop the fermentation by depleting the added carbohydrate other than lactose. Thus, in a specific embodiment of the first aspect according to the invention, the fermentation step is terminated in a manner selected from the group consisting of:

1) acidification of the fermented milk, resulting in at least one strain of the starter culture being unable to grow, and

2) cooling treatment.

In a specific embodiment of the first aspect according to the invention, no carbohydrate other than lactose is added during the fermentation step and at least one lactose-deficient lactic acid starter culture strain is capable of metabolizing a carbohydrate selected from the group consisting of glucose and galactose. In this embodiment, the lactose-deficient lactic acid strain of the starter culture grows exclusively on glucose and/or galactose generated by the enzymatic action of low pH stable lactase. In this embodiment, in accordance with the invention, the fermentation is stopped at the desired pH by a method selected from the group consisting of: 1) acidifying the fermented milk, resulting in at least one strain of the starter culture being unable to grow, 2) cooling treatment and 3 ) depletion of glucose and/or galactose, which are formed under the action of lactase, stable at low pH.

In a second aspect according to the invention, the low pH stable lactase is added to the fermented milk at the end of the fermentation. In this case, the lactase-containing fermented dairy product is preferably stored at a temperature of at least 2° C. for at least 1 day. In a specific embodiment of the process according to the invention, the lactase-containing fermented dairy 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 specific embodiment of the second aspect according to the invention, the fermentation step is terminated by depletion of a carbohydrate other than lactose.

In a specific embodiment according to the invention, the desired pH is from 3.2 to 4.8, more preferably from 3.6 to 4.6, more preferably from 3.8 to 4.5 and most preferably from

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4.0 to 4.4.

In a specific embodiment according to the invention, the fermentation temperature is 35 to 45°C, preferably 37 to 43°C and more preferably 40 to 43°C.

In yet another specific embodiment according to the invention, the fermentation is carried out at a temperature of 15 to 35°C, preferably 25 to 35°C and more preferably 30 to 33°C.

In a specific embodiment according to the invention, the fermented dairy product is packaged at a temperature between 15 and 45°C.

In a particular embodiment according to the invention, the pH of the fermented milk product is maintained in the range of 0.3 pH units, preferably in the range of 0.2 pH units, and most preferably in the range of 0.1 pH units, when stored after fermentation is terminated at the temperature used for fermentation over a period of 20 hours.

In a specific embodiment according to the invention, the amount of added carbohydrate other than lactose is 1 to 30 mg/g, preferably 2 to 20 mg/g and more preferably 3 to 10 mg/g of the milk base.

In a specific embodiment according to the invention, the amount of added carbohydrate other than lactose is 0.1 to 10%, preferably 0.2 to 8%, preferably 0.3 to 2%, preferably 0.4 to 1, 5% and more preferably 0.5 to 1.2%, where the % is (w/w) milk based.

The starter culture may have a strain composition of any conventional lactic acid bacteria starter culture, including a single strain culture 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 specific embodiment according to the invention, the fermented dairy product after fermentation is subjected to a temperature treatment in such a way as to reduce the level of bacteria in the starter culture to a level of no more than 1X10exp02 CFU (colony forming units) per gram of fermented dairy product. In this case, a specific embodiment is characterized in that lactase is added after heat treatment. In this case, the lactase-containing fermented dairy product is stored at a temperature of at least 2° C. for at least 1 day. In a specific embodiment of the process according to the invention, the lactase-containing fermented dairy 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.

Temperature treatment to reduce the bacteria level of the starter culture to a level of not more than 1.0X10exp02 CFU per g of fermented milk is preferably carried out by exposing the starter culture of the fermented dairy product to a temperature of from 50 to 110°C, preferably from 50 to 100°C, preferably 50 to 90°C, preferably 60 to 85°C, more preferably 65 to 82°C and most preferably 70 to 80°C. The temperature treatment is preferably carried out for a period of 5 to 180 seconds, preferably 5 to 120 seconds, more preferably 5 to 90 seconds, more preferably 5 to 60 seconds, more preferably 8 to 50 seconds, and most preferably 10 to 40 seconds. With. Preferably, the level of bacteria in the starter culture is reduced to a level of no more than 1.0 x 10exp01 CFU per g of fermented milk, more preferably 0 CFU per g. are suitable for 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.

Low pH stable lactase is added in an appropriate amount to achieve the desired degree of lactose hydrolysis under the chosen reaction conditions. In a specific embodiment according to the invention, lactase is added in an amount of 100 to 20,000 LAU per liter of milk base, preferably 100 to 10,000 LAU per liter of milk base, preferably 100 to 5,000 LAU per liter of milk base, preferably less than 3000, such as less than 1500, less than 1000, less than 750 or less than 500 LAU per liter of milk base.

In a preferred embodiment, lactase is added at a concentration of from 5 to 400 LAU per g of lactose in the milk base, preferably from 5 to 200 LAU per g of lactose in the milk base, preferably from 5 to 100 LAU per g of lactose in the milk base, 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 base.

In a preferred embodiment according to the invention, lactase is added to the milk base in an amount of 2.0 to 50 mg/ml, preferably 5 to 48 mg/ml, more preferably 10 to 46 mg/ml and most preferably 20 to 45 mg /ml

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In a preferred embodiment according to the invention, the residual lactose level at the end of fermentation is 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

In a preferred embodiment according to the invention, the milk base at the start of the fermentation step has a lactose content of 30.0 to 70 mg/ml, preferably 35 to 65 mg/ml, more preferably 40 to 60 mg/ml and most preferably 50 to 50 mg/ml. 60 mg/ml.

In a preferred embodiment according to the invention, when low pH stable lactase is added in the process at the end of the fermentation step, then the fermented dairy product to which the lactase is added has a viscosity that allows the lactase to be easily distributed in the fermented dairy product, for example, by mixing.

In a preferred embodiment of the method according to the invention, the lactase added to the process is presented in a sterile composition. In another preferred embodiment of the method according to the invention, the lactase is added in the process under aseptic conditions, for example by sterile filtration of the lactase solution.

fermented dairy product

The present invention further relates to a fermented dairy product obtainable by the method according to the invention.

In a particular embodiment, the invention relates to a fermented dairy product obtainable by a process according to the invention comprising the starter culture from step 3) of said method and the low pH stable lactase added in step 4) of said method.

In a specific embodiment according to the invention, the fermented dairy product is a product that can be obtained using a strain of lactic acid bacteria selected from the group consisting of lactose-deficient Streptococcus thermophilus and lactose-deficient Lactobacillus delbruecMi subsp. bulgaricus.

In a specific embodiment according to the invention, the fermented dairy product is selected from the group consisting of yogurt, curd cheese, curdled milk, sour cream, buttermilk, fermented whey, sour milk, sour cream, kefir, drinking yogurt and Japanese bio-yogurt. Preferably, the yogurt is selected from the group consisting of oven yogurt, stirred yogurt and drinking yogurt.

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

The fermented dairy product typically contains protein at a level of 2.0 to 3.5% by weight. The fermented dairy product may also be a low protein product having a protein level of 1.0 to 2.0% by weight. Alternatively, the fermented dairy product may be a high protein product above 3.5% by weight.

Application

The present invention further relates to use in a process for producing a fermented dairy product, comprising the steps of:

1) adding to the milk base a starter culture containing at least one strain of lactic acid bacteria, and

2) fermenting milk for a period of time until the desired pH is reached,

3) a starter culture containing at least one lactose-deficient strain that is capable of metabolizing a carbohydrate other than lactose, and

4) low pH stable lactase, added at the beginning, during or at the end of the fermentation stage, where the low pH stable lactase retains its activity at pH 5.0 and a temperature of 37°C at a level of at least 5% by compared with its activity at the optimum pH for lactase.

Definitions

The following definitions apply to the present invention:

L AU means lactose units and 1 lactase unit (1 LAU) is the amount of enzyme that releases 1 μmol of glucose per minute in M-buffer at pH 6.5 and 37° C. at a lactose concentration of 4.75% w/v. M-buffer is prepared by dissolving 3.98 g C ₆ H5Na 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 MgCM^, 3.75 g CaCl22H ₂ O and 1.4 g NaHCO ₃ in 4 liters of water, adding 12.5 ml of 4N. NaOH, adjusting to pH 6.5 using HCl and adding water to a total volume of 5 L.

- 8 042481

The LAU activity of a particular lactase can be determined by direct measurement of the glucose released from lactose under the conditions described above. One skilled in the art knows how to determine such activity. Alternatively, activity can be determined using the lactase activity assay described in Example 1 of this application. In this application, the activity is obtained by comparison with a standard curve that is built for a known activity of lactase, and from it the activity for an unknown sample is calculated. A lactase with known activity may be, for example, a lactozyme 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 that inactivates at least part of the bacteria of the starter culture. In this regard, the term inactivation means any cessation, reduction or suppression of bacterial growth, such as cell lysis.

The expression lactic acid bacteria (LAB) refers to gram-positive, microaerophilic or anaerobic bacteria that ferment sugars to produce acids, including lactic acid as the predominantly produced acid, acetic acid and propionic acid. The most industrially useful lactic acid bacteria are found in the order Lactobacillus, 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 cultures alone or in combination with other lactic acid bacteria.

Lactic acid bacteria, including bacteria of the species Lactobacillus sp. and Lactococcus sp., are commonly supplied to the dairy industry as frozen or freeze-dried cultures for growth in production starter cultures or so-called Direct Inoculation Cultures (DVS) intended for direct inoculation into a fermenter vessel or container for the production of a dairy product such as fermented milk. product or cheese. Such cultures of lactic acid bacteria are generally referred to as starter cultures or sourdoughs. 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 milk obtained by milking any mammal such as cows, sheep, goats, buffalo or camels. In a preferred embodiment, the milk is cow's milk. The term milk also includes protein/fat solutions prepared from plant materials, such as soy milk.

The term milk base can be any untreated and/or processed milk material that can be fermented in accordance with the method according to the invention. Thus, useful dairy bases include solutions/suspensions of any dairy or dairy-like products containing protein, such as whole or low fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, powdered milk, dairy but not limited to whey, whey permeate, lactose, lactose crystallized mother liquor, whey protein concentrate or cream. It is obvious that the milk base can be derived from any mammal, for example be essentially pure mammalian milk or reconstituted milk powder.

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

Used here homogenization means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is carried out before fermentation, then it can be carried out in such a way as to break the milk fat into smaller particles, such that they do not separate from the milk. The latter can be done by forcing milk at high pressure through small holes.

As used herein, pasteurization means the treatment of a milk base to reduce or eliminate the presence of living organisms such as microorganisms. Preferably, pasteurization is carried out by maintaining a specific temperature for a specific period of time. The specific temperature is usually reached by heating. The temperature and duration can be chosen to kill or inactivate certain bacteria, such as harmful bacteria. This may be followed by a rapid cooling step.

Fermentation in the methods of the present invention means the conversion of carbohydrates into alcohols or acids by the action of a microorganism. Preferably, the fermentation in the methods according to the invention comprises the conversion of lactose to lactic acid.

The fermentation methods used in the production of dairy products are well known and the person skilled in the art knows how to select the appropriate conditions.

- 9 042481 process variables such as temperature, oxygen, quantity and characteristics of the microorganism(s) and duration of the process. Obviously, the fermentation conditions are chosen to support the success of the present invention, i.e. receive a dairy product in solid (such as cheese) or liquid form (such as a fermented dairy product).

The use of terms in the singular in the context of the description of this invention (especially in the context of the following claims) should be understood as covering both the singular and the plural, unless otherwise indicated here or it is clearly not contrary to the context. The terms containing, having, including, and containing are to be considered as non-limiting terms (i.e., meaning including, but not limited to), unless otherwise indicated. The listing of ranges of values herein is intended to serve only as a shorthand way of referring individually to each individual value falling within that range, unless otherwise noted in the specification, and each individual value is included in this specification as if it were separately stated in the specification. All of the methods disclosed in the description can be performed in any suitable order, unless otherwise indicated here or it is clearly consistent with the context. The use of any or all of the examples or illustrative language (eg, such as) suggested in the specification is only intended to better illuminate the present invention and does not limit the scope of the present invention unless otherwise stated. No expression in this specification is to be understood as indicating any element not claimed to be essential to the practice of the invention.

The term fermented dairy product means a food or feed product, where the preparation of the food or feed product involves the fermentation of a milk base using lactic acid bacteria. As used herein, the term fermented dairy product includes, but is not limited to, products such as thermophilic fermented dairy products such as yogurt, mesophilic fermented dairy products such as sour cream and buttermilk, and fermented whey.

The term thermophilic as used herein refers to microorganisms that grow best at temperatures above 35°C. The thermophilic bacteria most useful for industrial applications include Streptococcus spp. and Lactobacillus spp. As used herein, the term thermophilic fermentation refers to fermentation at a temperature above about 35°C, such as from about 35 to about 45°C. The term thermophilic fermented dairy product refers to fermented dairy products obtained by the thermophilic fermentation of a thermophilic starter culture and includes fermented dairy products such as oven-baked yogurt, stirred yogurt, and drinking yogurt such as Japanese bio-yogurt.

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

In relation to the present invention, the shear stress can be measured in the following way:

Seven days after production, the fermented milk product was brought to 13°C and gently mixed by hand with a spatula (5 times) until the sample was homogeneous. The rheological properties of the sample were evaluated on a rheometer (Anton Paar Physica Rheometer with ASC, automatic sample changer, Anton Paar® GmbH, Austria) using a bob-cup. The rheometer was set to a constant temperature of 13°C during the measurement time. The settings were as follows:

Retention time (to restore to some extent the original structure) 5 min without any physical load (rocking or rotation) applied to the sample.

Rocking stage (to measure the modulus of elasticity and viscosity, G' and G respectively, then the complex modulus G* is calculated)

Constant tension=0.3%, frequency (f)=[0.5...8] Hz 6 measurement points within 60 s (one every 10 s)

Rotation stage (for measuring shear stress at 300 1/s)

Two stages have been developed:

1) Shear rate = [0.3-300] 1/s and

2) Shear rate = [275-0.3] 1/s.

- 10 042481

Each stage contained 21 measurement points for 210 s (every 10 s).

Shear stress at 300 1/s was chosen for subsequent analysis because it correlates with thickening in the mouth when swallowing a fermented dairy product.

With respect to the present invention, the hardness of a gel can be measured in the following way:

Gel hardness is measured by a back extrusion test using a texture analyzer (TA.XT Plus, Stable Micro System, Surrey, UK) equipped with a 35 mm parallel plate. The travel path was set to 15 mm and the travel speed to 2 mm/s. The test is carried out 7 days after receipt. The fermented milk product was brought to 13°C and gently stirred by hand and measured in a 250 g plastic container. mm) as degree of deformation, maximum negative force (N) as toughness.

As used herein, the term low pH stable lactase refers to a lactase that retains at least 5% activity at pH 5.0 and 37° C. compared to its activity at lactase-optimal pH.

The term activity at optimal pH means the activity of lactase at a pH value at which lactase has optimal activity.

The term carbohydrate other than lactose means any carbohydrate which is not lactose and which the lactose-deficient lactic acid bacterium used in the method according to the invention is capable of metabolizing.

The expression at the beginning of the fermentation stage means within a short time before, at the same time, or within a short time after the addition of the starter culture to the milk base. As used herein, within a short time means less than 30 minutes.

The expression during the fermentation stage means any time during the fermentation after the start and before the end of the fermentation.

The expression at the end of the fermentation stage means within a short time before, at the same time, or within a short time after the desired pH is reached. Here, the term for a short time means less than 30 minutes.

The term desired pH means the pH at which the fermentation step is completed. Depending on various process parameters, the fermentation step is terminated by a method selected from the group consisting of: 1) acidification of the fermented milk, resulting in at least one strain of the starter culture not being able to grow, 2) cooling treatment and 3) carbohydrate depletion, different from lactose.

Escrow and expert solution

The applicant requests that the deposited microorganism sample be made available only to an expert approved by the applicant.

The Streptococcus thermophilus strain was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig 2014-06-12) under accession number DSM 28952.

The Streptococcus thermophilus strain was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig 2014-06-12) under accession number DSM 28953.

The strain Lactobacillus delbrueckii subsp. bulgaricus was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig 2014-06-12) under accession number DSM 28910.

The deposits were made in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

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 assay (Werner W. et al. (1970) Z. analyt. Chem. 252: 224.).

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

- 11 042481

Solutions:

Assay buffer: 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl ₂ , 1 mM MgCl 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 (horseradish peroxidase), 0.65 g/l ABTS (2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid)).

Substrate:

160 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl ₂ , 1 mM MgCl 2 , pH 6.5.

Standard:

Lactozyme (obtained from Novozymes A/S, Denmark) with known activity in LAU/g was used as a standard diluted in assay buffer in the range of 0.09 to 0.7 LAU/r.

Samples:

Enzyme samples are diluted appropriately in assay buffer.

Way:

1. 375 µl of substrate are 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 1M NaOH

4. Transfer 20 µl to a 96-well microtiter plate and add 200 µl GOD-Perid solution. After 30 minutes at room temperature, the 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 respectively 85 or 70 ml of water with ions. This solution was poured into a flask containing a magnetic stir bar and placed in a water bath at 37°C. After 15 min, the enzyme was added. The enzymes tested were lactozyme, which is 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 coding sequence shown in SEQ ID NO: 2 and an activity of 295 LAU/g. G.

The doses for lactozyme were 4225 LAU/L milk and for Bifidobacterium lactase 2025 LAU/L milk. Milk samples were taken 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 diluted appropriately 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 lactose, glucose and galactose standards.

The results are shown below.

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 BY 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

- 12 042481

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 in both 15% and 30% whey permeate, no or very little galactooligosaccharides were produced. In addition, the levels of galactose and glucose produced are equivalent and correspond to the amount of hydrolyzed lactose. In comparison, lactozyme produces less galactose than glucose, clearly demonstrating that galactooligosaccharides are 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 hydrolyzes lactose to form glucose + galactose. Glucose is measured according to a modified version of the conventional 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 KCl, 2 mm CaCl ₂ , 1 mm MgCl ₂ and the pH was adjusted to pH 3, 4, 5, 6, 7, 8, 9 and 10 at help of NaOH.

Enzyme Sample:

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

Way:

μl of the enzyme sample diluted in enzyme dilution buffer was added to PGR (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 terminated 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 3

pH Relative activity of B. bifidum lactase (% activity at pHb) 3 0 4 4 5 75 6 100 7 85 8 39 9 10 10 4

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 the pH was adjusted to pH 6.5 with NaOH.

- 13 042481

Enzyme Sample:

An 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 KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , 0. 01% Triton X100 and pH adjusted to pH 6.5.

Way:

μl of enzyme sample diluted in enzyme dilution buffer was added to PCR tubes at room temperature;

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

The reaction was terminated 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 B. bifidum lactase activity (% activity at 38, GS) 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

Example 4

Determination of Km for lactase enzyme at 5°C.

Principle:

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

Substrate:

Various 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 the pH was adjusted to pH 6.5 with NaOH.

Enzyme Sample:

An 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 KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , 0. 01% Triton X100, pH adjusted to pH 6.5 with NaOH.

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

Way:

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

Added 90 μl of substrate (5°C) and incubated for 2 h 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 were added. After 30 minutes at com

- 14 042481 at room temperature, absorption was measured at 420 nm.

Definition of km:

We used a computer non-linear least squares fit and the Michaelis-Menten equation:

v=(Vmax*S)/(Km+S)

The determined Km for Bifidobacterium lactase and lactozyme were 8 and 30 mM, respectively.

In a similar test performed at 37° C., the determined Km for Bifidobacterium lactase and lactozyme were 13 and 30 mM, respectively.

Example 5

Making yogurt using Bifidobacterium bifidum lactase and a lactose-deficient starter culture - varying levels of sucrose and lactase.

Experiment plan

For the chosen combination of low pH stable lactase and lactose deficient starter culture, two levels of added sucrose (0.5 and 0.7%) and two levels of added lactase (800 LAU/L and 1000 LAU/L) were tested. For comparison, fermentations were carried out without the addition of lactase and with two levels of sucrose (1.0 and 1.5%). Lactase was added at the start of the fermentation along with the starter culture.

milk base

Table 5 Milk Base Composition

Quantity (g) Protein content (%) Carbohydrate (%) Fat (%) Skim milk 1000 3.5 4.8 0.1 1.5% milk 2000 3.4 4.7 1.5 Protein Powder Promilk 802FB 14 80.0 Protein Powder Milex 240 95 34.0 General content milk base 3109 4.71 4.57 1.00

starter culture

The starter culture consists of a Streptococcus thermophilus strain deposited under DSM accession number 28952, a Streptococcus thermophilus strain deposited under DSM accession number 28953, and a strain of Lactobacillus delbrueckii subsp. bulgaricus deposited under DSM accession number 28910.

Lactase:

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

Measurements:

Fat, protein and lactose levels were determined using the MilkoScan assay.

Subsequent acidification was measured over a storage period of 28 days at 5°C.

The level of sucrose, glucose, galactose, fructose and lactose in fermented milk on day 1 and day 14 after production was measured using HPLC.

Gel Hardness:

Gel hardness is measured by a back extrusion test using a texture analyzer (TA.XT Plus, Stable Micro System, Surrey, UK) equipped with a 35 mm parallel plate. The travel path was set to 15 mm and the travel speed to 2 mm/s. The test is carried out 7 days after production. The fermented dairy product was brought to 13°C, mixed gently by hand and measured in a 250 g plastic container. The maximum force (g) obtained from the distance versus force curves was used as the gel hardness parameter.

Shear stress:

Seven days after incubation, the fermented milk product was brought to 13°C and gently mixed by hand with a spatula (5 times) until the sample was homogeneous. The rheological properties of the sample were evaluated on a rheometer (Anton Paar Physica Rheometer with ASC, automatic sample changer, Anton Paar® GmbH, Austria) using a bob-cup. The rheometer was set to a constant temperature of 13°C during the measurement time.

The settings were as follows:

Retention time (to restore to some extent the original structure) 5 min without any physical load (rocking or rotation) applied to the sample.

Rocking stage (to measure the modulus of elasticity and viscosity, G' and G, respectively, then the complex modulus G* is calculated)

Constant tension = 0.3%, frequency (f) = [0.5...8] Hz 6 measurement points within 60 s (one every 10 s)

- 15 042481

Rotation stage (for measuring shear stress at 300 1/s)

Two stages have been developed:

1) Shear rate=[0.3-300] 1/s and

2) Shear rate=[275-0.3] 1/s.

Each stage contained 21 measurement points for 210 s (every 10 s).

Shear stress at 300 1/s was chosen for subsequent analysis because it correlates with thickening in the mouth when swallowing a fermented dairy product.

Way

The milk base ingredients were mixed and left to rehydrate for 2 hours at 5°C. The milk base was then pasteurized at 90° C. for 20 minutes. Fermentation was carried out at 43°C to a final pH of 4.55 with the formation of yoghurt. The yogurt was cooled in a PTU (post-treatment module) at a cooling temperature of 25° C. at 2 bar (200 kPa). Chilled yogurt was stored at 6°C.

results

Subsequent acidification

Table 6. Subsequent Acidification

Control 1 Control 2 Test sample 1 Test sample 2 Test sample 3 Test sample 4 Sucrose(%) 1 1.5 0.5 0.5 0.7 0.7 Lactase (LAU/L) 0 0 800 1000 800 1000 Enzyme time. (Hours:Min.) 7:25 7:05 7:15 7:15 7:00 7:00 final pH 4.58 4.55 4.55 4.55 4.55 4.55 pH 1 day 4.58 4.53 4.54 4.53 4.50 4.51 pH 7 days 4.54 4.49 4.50 4.50 4.47 4.49 pH 14 days 4.49 4.43 4.49 4.49 4.46 4.47 pH 21 days 4.35 4.34 4.45 4.45 4.42 4.43 pH 28 days 4.35 4.34 4.45 4.42 4.43 4.42 pH drop 0.23 0.19 0.09 0.11 0.07 0.09

For all six samples, i.e. 2 control samples and 4 samples obtained in accordance with the invention (test samples), a pH of approximately 4.55 was reached within 7 hours.

As can be seen from Table. 6, subsequent acidification over a period of 28 days was significantly reduced for samples produced in accordance with the invention compared to control samples.

Carbohydrate analysis

Table 7. Levels of residual carbohydrate 1 day after production

Control 1 Control 2 Test sample 1 Test Sample 2 Test sample 3 Test sample 4 Sucrose (%) start enzyme. 1 1.5 0.5 0.5 0.7 0.7 Lactase (LAU/L) 0 0 800 1000 800 1000 Fructose (mg/g) 1.62 1.91 0.80* 0.74* 1.27 1.18 Galactose (mg/g) <0.9 1.78* 29.01 29.87 28.78 29.29 Glucose (mg/g) 0.77* 1.73 27.60 27.84 28.08 28.19 Lactose (mg/g) 54.16 53.37 1.18 0.40 1.20 0.40 Sucrose (mg/g) <0.3 4.6 <0.3 <0.3 1.3 0.7

*value represents limit of detection and limit of quantitation.

All levels in the table. 7 are average values for two samples.

For the 4 samples tested at the end of the fermentation, the lactose level was very low and the glucose and galactose levels were very high compared to the controls, indicating a high activity of the added lactase. For testing samples using a lactase level of 1000 LAU/l, a residual lactose level of approx. 0.04% and for testing samples using a lactase level of 800 LAU/l, a residual lactose level of approx. 0.1%.

Fermented milk with low lactose and high levels of glucose and galacs

- 16 042481 tones, has a much higher level of apparent sweetness than fermented milk with high levels of lactose and low levels of glucose and galactose, as controls. The reason for this is that glucose and galactose have a much higher sweetness index than lactose.

Gel hardness

Table 8. Gel hardness

Control 1 Control 2 Test sample 1 Test sample 2 Test sample 3 Test sample 4 Sucrose (%) start enzyme. 1 1.5 0.5 0.5 0.7 0.7 Lactase (LAU/L) 0 0 800 1000 800 1000 Gel hardness (g) 45.76 47.42 48.19 54.57 50.19 46.60

All levels in the table. 8 are average values for two samples. As can be seen from Table. 8, 3 out of 4 tested samples had a much higher gel hardness than 2 control samples.

Shear stress

Table 9. Shear stress measured at 300 s ^-1

Control 1 Control 2 Test sample 1 Test sample 2 Test sample 3 Test sample 4 Sucrose (%) start enzyme. 1 1.5 0.5 0.5 0.7 0.7 Lactase (LAU/L) 0 0 800 1000 800 1000 Shear stress (Pa) 67.1 65.3 78.2 84.4 84.7 77.6

All levels in the table. 9 are average values for two samples.

As can be seen from Table. 9, the 4 test samples had a much higher shear stress measured at 300 s ^-1 than the 2 control samples. In a group of 4 test samples, the combination of 1) 0.5% added sucrose level and 1000 LAU/L lactase level and 2) 0.7% added sucrose level and 800 LAU/L lactase level had the highest shear stress.

Example 6 Yogurt production using Bifidobacterium bifidum lactase and a lactose-deficient starter culture - addition of excess sucrose levels prior to fermentation I

Experiment plan

Fermentations were carried out with a lactase-free control containing 9.5% sucrose and 2 test samples containing 800 LAU/l lactase and 6.5 and 7.0% sucrose. Lactase was added at the start of the fermentation along with the starter culture.

milk base

Table 10. Composition of the original milk base 1

Quantity (g) Protein content (%) Carbohydrate (%) Fat (%) Skim milk 984 3.5 4.8 0.1 1.5% milk 1911 3.4 4.7 1.5 Protein Powder Promilk 802FB 13.3 80.0 Protein Powder Milex 240 91.5 34.0 54.00 1.00 General content milk base 3000 4.71 4.57 1.02

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Table 11. Composition of the original milk base 2

Quantity (g) Protein content (%) Carbohydrate (%) Fat (%) Skim milk 984 3.5 4.8 0.1 1.5% milk 1911 3.4 4.7 1.5 Protein Powder Promilk 802FB 17.40 80.0 Protein Powder Milex 240 109.50 34.0 5400 1.00 sucrose 180.00 100.0 General content milk base 3202 4.70 11.75 0.96

The original milk base 2 was used to prepare the final milk bases for the control sample and two test samples by adding additional sucrose.

Starter culture:

The starter culture consists of a Streptococcus thermophilus strain deposited under DSM accession number 28952, a Streptococcus thermophilus strain deposited under DSM accession number 28953, and a strain of Lactobacillus delbrueckii subsp. bulgaricus deposited under DSM accession number 28910.

Lactase:

Bifidobacterium bifidum lactase had the encoded sequence of SEQ ID NO: 2.

Measurements:

All measurements were carried out using the same methods as in example 5.

Way

The milk base ingredients were mixed and left to rehydrate for 2 hours at 5°C. The milk base was then pasteurized at 95°C for 5 minutes. The milk base was then homogenized at 65° C. at 200 bar (20,000 kPa). Fermentation was carried out at 43°C to a final pH of 4.55 with the formation of yoghurt. The yogurt was cooled in a PTU (post-treatment module) at a cooling temperature of 25° C. at 2 bar (200 kPa). Chilled yogurt was stored at 6°C.

results

Subsequent acidification

Table 12 Post acidification

Sample 1 2 3 Lactase (LAU/l) 0 800 800 Fermentation time 6 h 15 min 6 h 15 min 6 h 15 min Final pH 4.55 4.51 4.51 pH 4 days 4.54 4.44 4.44 pH 7 days 4.51 4.42 4.42 pH 14 days 4.40 4.38 4.37 pH 21 days 4.38 4.38 4.37 pH 28 days 4.36 4.37 4.36 pH drop 0.19 0.14 0.15

Carbohydrate analysis

Table 13. Carbohydrate analysis

Sample 1 2 3 Lactase (LAU/L) 0 800 800 Sucrose(%) start enzyme. 9.5 6.5 7.0 1 day 28 day 1 day 28 day 1 day 28 day Fructose (mg/g) 2.4 3.07 1.7 2.01 1.9 2.00 Galactose (mg/g) <2.0 8.99 26.4 29.03 26.4 28.72 Glucose (mg/g) <1.0 8.84 26.2 27.52 26.2 27.22 Lactose (mg/g) 51.4 38.04 2.8 <2 2.7 <2 Sucrose (mg/g) 64.3 64.23 41.7 42.38 46.0 46.26

All levels in the table. 13 are average values for two samples.

As can be seen from Table. 13, for the 2 test samples at the end of the fermentation, the lactose level was very low and the glucose and galactose levels were very high compared to the control sample.

- 18 042481 tsami, which indicates a high activity of the added lactase.

Fermented milk having low lactose and high glucose and galactose had a much higher level of sweetness than fermented milk with high lactose and low glucose and galactose as controls. The reason for this is that glucose and galactose have a much higher sweetness than lactose.

Gel hardness and shear stress

Table 14. Gel hardness and shear stress at 300 s ^-1

Sample 1 2 3 Lactase (LAU/l) 0 800 800 Sucrose (%) start enzyme. 9.5 6.5 7.0 Gel hardness (g) 46.36 46.76 45.75 Shear stress at 1 s ¹ (Pa) 9.4 9.2 9.3 8.8 10.3 10.2 Shear stress at 30.2 s ¹ (Pa) 39.2 38.8 41.6 39.2 44.7 45.1 Shear stress at 135 s ¹ (Pa) 58.3 58.0 67.2 64.5 72.3 72.2 Shear stress at 300 s ¹ (Pa) 69.4 69.1 80.6 78.2 84.4 84.4

As can be seen from Table. 14, the shear stress at 300 s ^-1 for the two tested samples containing lactase increased significantly compared to the control samples without lactase.

Example 7 Preparation of Yogurt Using Bifidobacterium bifidum Lactase and a Lactose-Deficient Starter Culture - Adding Excess Sucrose Before Fermentation II

Experiment plan

Fermentations were carried out with two lactase-free controls containing 1.5 and 9.5% sucrose and 3 test samples containing 1000 LAU/l lactase and 0.5%, 6.5% and 7.0% sucrose. Lactase was added at the start of the fermentation along with the starter culture.

milk base

The two original milk bases according to Example 6 were used to prepare the final milk bases for controls and three test samples by adding additional sucrose.

starter culture

The starter culture consists of a Streptococcus thermophilus strain deposited under DSM accession number 28952, a Streptococcus thermophilus strain deposited under DSM accession number 28953, and a strain of Lactobacillus delbrueckii subsp. bulgaricus deposited under DSM accession number 28910.

Lactase:

Bifidobacterium bifidum lactase having the encoded sequence of SEQ ID NO: 2.

Measurements:

All measurements were carried out using the same methods as in example 5.

Way

The milk base ingredients were mixed and left to rehydrate for 2 hours at 5°C. The milk base was then pasteurized at 90° C. for 20 minutes. Fermentation was carried out at 43°C to a final pH of 4.55 with the formation of yoghurt. The yogurt was cooled in a PTU (Post-Treatment Module) at a cooling temperature of 25°C at 2 bar (200 kPa). Chilled yogurt was stored at 6°C.

results

Subsequent acidification

Table 15 Post acidification

Sample 1 2 3 4 5 Lactase (LAU/L) 0 1000 0 1000 1000 Fermentation time 6 h 50 min 6 h 55 min 6 h 35 min 6 h 10 min 5 h 40 min Final pH 4.55* 4.55* 4.55* 4.55* 4.55* pH 1 day 4.54 4.51 4.52 4.48 4.47 pH 7 days 4.45 4.45 4.40 4.34 4.35 pH 14 days 4.37 4.44 4.37 4.32 4.34 pH 21 days 4.35 4.39 4.35 4.29 4.29 pH 28 days 4.34 4.37 4.33 4.27 4.26 pH drop 0.21 0.18 0.22 0.28 0.29

*Fermentation is stopped by cooling at pH = 4.55.

As can be seen from Table. 15, post-acidification was at the same level for lactase-free controls and lactase-containing test samples.

- 19 042481

Carbohydrate analysis

Table 16. Carbohydrate analysis for 1 day

Sample 1 2 3 4 5 Lactase (LAU/L) 0 1000 0 1000 1000 Sucrose (%) start enzyme. 1.5 0.5 9.5 6.5 7.0 Fructose (mg/g) 1.96 <1 2.70 1.74 1.88 Galactose (mg/g) <2 29.19 <2 27.83 27.89 Glucose (mg/g) <1 27.37 <1 27.29 27.40 Lactose (mg/g) 56.11 <2 54.56 <2 <2 Sucrose (mg/g 5.45 <4 63.44 39.79 45.44

Table 17. Carbohydrate analysis on day 28

Sample 1 2 3 4 5 Lactase (LAU/l) 0 1000 0 1000 1000 Sucrose (%) 1.5 0.5 9.5 6.5 7.0 the beginning of the enzyme. Fructose (mg/g) 1.88 <1 4.72 2.95 3.39 Galactose (mg/g) 3.90 28.83 <2 28.79 29.15 Glucose (mg/g) 2.69 26.78 1.66 26.83 27.27 Lactose (mg/g) 50.71 <2 52.42 <2 <2 Sucrose (mg/g 4.18 <4 62.07 39.91 46.17

As can be seen from Table. 16 and 17, for the 3 test samples at the end of the fermentation, the lactose level was very low and the glucose and galactose levels were very high compared to the control samples, indicating a high activity of the added lactase.

Fermented milk having a low lactose level and a high level of glucose and galactose had a much higher level of sweetness than a fermented milk with a high level of lactose and a low level of glucose and galactose as controls. The reason for this is that glucose and galactose have a much higher sweetness than lactose.

Gel hardness and shear stress

Table 18. Gel Hardness and Shear Stress

Sample 1 2 3 4 5 Lactase (LAU/L) 0 1000 0 1000 1000 Sucrose(%) start enzyme. 1.5 0.5 9.5 6.5 7.0 Gel hardness (g) 51.13 49.41 45.04 45.94 47.89 Shear stress at 1 s ¹ (Pa) 9.4 9.3 8.4 8.5 8.1 8.5 7.7 7.8 8.3 8.5 Shear stress at 30.2 s ¹ (Pa) 39.4 38.5 38.2 37.6 34.3 34.7 37.3 37.1 38.8 38.9 Shear stress at 135 s ¹ (Pa) 61.0 59.5 65.8 65.1 56.8 56.8 72.7 72.3 74.8 74.9 Shear stress at 300 s ¹ (Pa) 72.1 70.5 78.8 77.6 69.1 69.1 86.7 86.6 89.4 89.4

As can be seen from Table. 18, the shear stress for the three tested samples containing lactase increased significantly compared to the control samples without lactase.

- 20 042481

Example 8 Production of yogurt using Bifidobacterium bifidum lactase and a lactose-deficient starter culture - addition of lactase before and after fermentation

Experiment plan

Table 19 Experiment plan

Sample Time of lactase addition Dairy the basis Sucrose (%) Lactase (LAU/L) 1 Not added 1 0.70 0 2 Together with culture 1 0.70 600 3 Together with culture 1 0.70 800 4 Together with culture 1 0.70 1000 5 Together with culture 1 0.70 1400 6 Not added 2 0.97 0 7 After fermentation 2 0.97 800 8 After fermentation 2 0.97 1600 9 After fermentation 2 0.97 2400 10 After fermentation 2 0.97 3200

The fermentation temperature was 43°C. The final pH was 4.50. The milk base ingredients were mixed and left to rehydrate for 2 hours at 6°C. The milk base was then pasteurized at 95°C for 5 minutes and homogenized at 200/50 bar (20000/5000 kPa) at 65°C. Fermentation was carried out at 43°C to a final pH of 4.50 with the formation of yoghurt. Yogurt was cooled to 6°C. Chilled yogurt was stored at 6°C.

starter culture

The starter culture consists of a Streptococcus thermophilus strain deposited under DSM accession number 28952, a Streptococcus thermophilus strain deposited under DSM accession number 28953, and a strain of Lactobacillus delbrueckii subsp. bulgaricus deposited under DSM accession number 28910.

Lactase:

Bifidobacterium bifidum lactase having the encoded sequence of SEQ ID NO: 2.

milk base

Table 20 Milk Base Composition 1

Quantity (g) Protein content (%) Carbohydrate (%) Fat (%) sucrose 22 0.0 100.0 0.0 0.5% milk 1500 3.8 4.8 0.5 1.5% milk 1500 3.6 4.7 1.5 Aria dry skimmed milk 103 34.0 54.0 1.0 General content milk base 3125 4.67 7.05 0.98

Table 21 Milk Base Composition 2

Quantity (g) Protein content (%) Carbohydrate (%) Fat (%) sucrose thirty 0.0 100.0 0.0

0.5% milk 1500 3.8 4.8 0.5 1.5% milk 1500 3.6 4.7 1.5 Aria dry skimmed milk 103 34.0 54.0 1.0 General content milk base 3133 4.66 7.30 1.00

Measurements:

All measurements were carried out using the same methods as in example 5.

- 21 042481

Results:

Subsequent acidification

Table 22

Sample Time of lactase addition milk base Lactase (LAU/L) pH day 0 pH day 7 pH day 28 pH day 42 pH drop 1 Not added 1 0 4.89* 4.89* 4.87 4.84 0.05 2 Together with culture 1 600 4.26 4.23 4.23 4.22 0.04 3 Together with culture 1 800 4.22 4.26 4.24 4.24 -0.02 4 Together with culture 1 1000 4.32 4.26 4.26 4.26 0.06 5 Together with culture 1 1400 4.36 4.30 4.28 4.27 0.09 6 Not added 2 0 4.47 4.47 4.47 4.44 0.03 7 After fermentation 800 4.43 4.40 4.42 4.38 0.05 8 After fermentation 1600 4.44 4.40 4.42 4.39 0.05 9 After fermentation 2 2400 4.41 4.43 4.40 4.40 0.01 10 After fermentation 2 3200 nd 4.43 4.41 4.40 nd

*Not enough sucrose to ferment to desired pH. Therefore, this sample cannot be used as a reference.

As can be seen from Table. 22, subsequent acidification over a period of 42 days was as low as less than 0.09 for all lactase levels tested where lactase was added at the start of fermentation, and even as low as less than 0.05 for all lactase levels tested where lactase was added at the end. fermentation. The addition of lactase at the end of the fermentation did not result in a statistically different subsequent acidification compared to controls without lactase addition. Thus, the present experiment demonstrates that the use of low pH stable lactase in combination with a lactose deficient starter culture does not result in an unacceptable increase in the low post-acidification that can be achieved with such a starter culture. The latter is true when lactase is added at the beginning and at the end of the fermentation.

- 22 042481

Gel hardness and shear stress

Table 23. Gel Hardness and Shear Stress

Sample Time of lactase addition milk base Sucrose (%) Lactase (LAU/L) Gel hardness (g) Shear stress (Pa) 1 Not added 1 0.70 0 0.187* 46.4* 2 Together with culture 1 0.70 600 0.281 63.9 3 Together with culture 1 0.70 800 0.302 65.3 4 Together with culture 1 0.70 1000 0.318 64.5 5 Together with culture 1 0.70 1400 0.307 63.7 6 Not added 2 0.97 0 0.304 53.1 7 After fermentation 2 0.97 800 0.312 51.4 8 After fermentation 2 0.97 1600 0.322 52.8 9 After fermentation 0.97 2400 0.323 52.9 10 After fermentation 0.97 3200 0.320 51.7

*Not enough sucrose to ferment to desired pH. Therefore, this sample cannot be used as a control.

As can be seen from Table. 23, high levels of gel hardness and shear stress were obtained for all lactase levels tested, where lactase was added at the start of fermentation. For lactase levels tested with lactase added at the end of fermentation, gel hardness and shear stress levels were somewhat lower as a result of lactase mixing in the yogurt, which partially destroyed the texture of the yogurt. For all lactase levels tested, where lactase was added at the end of fermentation, gel hardness was slightly higher than for the control without lactase addition. For all lactase levels tested, where lactase was added at the end of fermentation, shear stress was maintained at the same level or slightly less.

Carbohydrate analysis

Table 24. Carbohydrate analysis

Sample Lactase (LAU/L) Galactose 20 days (mg/g) Glucose 20 days (mg/g) Sucrose 20 days (mg/g) Lactose 20 days (mg/g) Lactose 24 hours (%) Lactose 20 days (%) 1 0 2.4 2.3 <1.0 50.7 ND ND 2 600 29.5 27.7 <1.0 <0.5 >0.1 0.023 3 800 30.2 28.1 <1.0 <0.5 0.028 0.016 4 1000 30.0 27.8 <1.0 <0.5 0.014 0.014 5 1400 29.5 27.4 <1.0 <0.5 0.006 0.013 6 0 4.2 4.2 <1.0 49.2 ND ND 7 800 31.2 30.7 <1.0 <0.5 >0.2 0.014 8 1600 30.7 30.2 <1.0 <0.5 >0.1 0.012 9 2400 31.2 30.6 <1.0 <0.5 0.029 0.011 10 3200 31.8 31.2 <1.0 <0.5 0.015 0.011

ND - no data

As can be seen from Table. 24, on day 20, high concentrations of galactose and glucose were formed for all lactase levels tested, where lactase was added at the beginning and at the end of fermentation. In addition, on day 20, the glucose level was less than the detected level of 0.5 mg/g, which in some countries qualifies as lactose-free. For higher lactase levels, most of the lactose removal was achieved 24 hours after the end of fermentation.

- 23 042481

Example 9 Yogurt Production Using Bifidobacterium bifidum Lactase and a Lactose-Deficient Starter Culture - Addition of Lactase Before Fermentation II

Experiment plan

Table 25 Experiment plan

Sample Sucrose(%) Lactase (LAU/l) 1 0 0 2 0 1000 3 0 1200 4 0 1600 5 0.2 0 6 0.2 1000 7 0.2 1200 8 0.2 1600 9 0.7 0 10 0.7 1000 AND 0.7 1200 12 0.7 1600

The fermentation temperature was 43°C. The final pH was 4.45. The milk base ingredients were mixed and left to rehydrate for 2 hours at 6°C. The milk base was then pasteurized at 95°C for 5 minutes and homogenized at 200/50 bar (20000/5000 kPa) at 65°C. Fermentation was carried out at 43°C to a final pH of 4.45 with the formation of yoghurt. Yogurt was cooled to 5°C. Chilled yogurt was stored at 6°C.

Starter culture:

The starter culture consists of a Streptococcus thermophilus strain deposited under DSM accession number 28952, a Streptococcus thermophilus strain deposited under DSM accession number 28953, and a strain of Lactobacillus delbrueckii subsp. bulgaricus deposited under DSM accession number 28910.

Lactase:

Bifidobacterium bifidum lactase having the encoded sequence of SEQ ID NO: 2.

milk base

Table 26 Milk Base Composition

Quantity (g) Protein content (%) Carbohydrate (%) Fat (%) 0.5% milk 1500 3.8 4.8 0.5 1.5% milk 1500 3.6 4.7 1.5 Aria dry skimmed milk 103 34.0 54.0 1.0 General content milk base 3103 4.70 6.39 0.99

Measurements:

All measurements were carried out using the same methods as in example 5.

results

Subsequent acidification

Table 27

Sample Sucrose (%) Lactase (LAU/l) pH day 0 pH day 1 pH day 14 pH day 28 pH day 42 pH drop 1 0 0 ND ND ND ND ND ND 2 0 1000 4.45 4.39 4.36 4.33 4.39 0.06 3 0 1200 4.45 4.39 4.38 4.34 4.39 0.06

- 24 042481

4 0 1600 4.46 4.39 4.38 4.34 4.39 0.07 5 0.2 0 ND ND ND ND ND ND 6 0.2 1000 4.50 4.44 4.40 4.40 4.43 0.07 7 0.2 1200 4.53 4.47 4.46 4.42 4.46 0.07 8 0.2 1600 4.53 4.49 4.46 4.44 4.47 0.06 9 0.7 0 4.76 4.73 4.69 4.68 4.70 0.06 10 0.7 1000 4.44 4.40 4.39 4.33 4.38 0.06 eleven 0.7 1200 4.46 4.40 4.39 4.33 4.39 0.07 12 0.7 1600 4.47 4.41 4.40 4.34 4.41 0.06

ND - no data

As can be seen from Table. 27, subsequent acidification over a period of 42 days was at a very low level of approx. 0.06 for all lactase and sucrose levels tested. Thus, the present experiment demonstrates that the use of low pH stable lactase in combination with a lactose deficient starter culture does not result in an unacceptable increase in the low post-acidification that can be achieved with such a starter culture.

Gel hardness and shear stress

Table 28: Gel Hardness and Shear Stress

Sample Sucrose (%) Lactase (LAU/l) Gel hardness (g) Shear stress (Pa) 1 0 0 ND ND 2 0 1000 0.386 70.3 3 0 1200 0.377 69.8 4 0 1600 0.388 69.5 5 0.20 0 ND ND 6 0.20 1000 0.380 70.7 7 0.20 1200 0.378 69.5 8 0.20 1600 0.397 70.2 9 0.70 0 ND ND 10 0.70 1000 0.362 71.2 eleven 0.70 1200 0.375 72.2 12 0.70 1600 0.362 71.9

ND - no data

As can be seen from Table. 28, high levels of gel hardness and shear stress were achieved for all lactase levels tested. Gel hardness and shear strength were of the same order of magnitude for all three lactase levels tested.

Carbohydrate analysis

Table 29. Carbohydrate analysis

Form- zetz Sucrose (%) Lactase (LAU/l) Fructose (mg/g) Galacto-za (mg/g) Glucose (mg/g) Lactose (mg/g) Sucrose (mg/g) 1 0 0 <0.5 6.3 <0.5 45.5 <2 2 0 1000 <0.5 30.7 26.0 <0.9 <2 3 0 1200 <0.5 31.5 26.6 <0.9 <2 4 0 1600 <0.5 30.9 25.8 <0.9 <2 5 0.20 0 <0.5 11.7 6.7 36.1 <2 6 0.20 1000 <0.5 30.8 27.8 <0.9 <2 7 0.20 1200 <0.5 31.6 28.3 <0.9 <2 8 0.20 1600 <0.5 31.5 28.0 <0.9 <2 9 0.70 0 1.1 9.1 9.1 43.4 <2 10 0.70 1000 1.1 31.1 29.5 <0.9 <2 eleven 0.70 1200 1.0 32.1 30.3 <0.9 1.8 12 0.70 1600 0.9 31.8 29.9 <0.9 1.9

As can be seen from Table. 29, for testing samples containing lactase, at the end of fermentation, the lactose level was very low and the glucose and galactose levels were very high compared to the control samples, indicating a high activity of the added lactase.

Fermented milk that is low in lactose and high in glucose and galactose has a much higher level of sweetness than high fermented milk.

-

Claims (15)

lactose levels and low glucose and galactose levels as controls. The reason for this is that glucose and galactose have a much higher sweetness than lactose. The sequence listing SEQ ID NO: 1 shows the sequence of the SEQ ID NO: 4 mutant. SEQ ID NO: 2 shows the sequence of the SEQ ID NO: 4 mutant. SEQ ID NO: 3 shows the sequence of Bifidobacterium bifidum lactase DSM20215. SEQ ID NO: 4 shows the sequence of lactase Bifidobacterium bifidum NCIMB41171, the nucleotide sequence of which is presented in the NCBI (National Center for Biotechnology Information) under accession number DQ448279. SEQ ID NO: 4 is discussed in the following references, in which it is referred to as 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) adding a starter culture containing at least one strain of lactic acid bacteria deficient in lactose metabolism, which is able to metabolize a carbohydrate other than lactose to a milk base, (1) a strain deposited with the German Collection of Microorganisms and Cell Cultures (DSMZDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 2014-06-12) under accession number DSM 28910; - 26 042481 (2) or a strain derived from DSM 28910, further characterized as having the ability to form white colonies on a medium containing lactose and X-Gal. (1) a strain deposited with the German Collection of Microorganisms and Cell Cultures (DSMZDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 2014-06-12) under accession number DSM 28953; (1) a strain deposited with the German Collection of Microorganisms and Cell Cultures (DSMZDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 2014-06-12) under accession number DSM 28952; 1) adding to the milk base a starter culture containing at least one strain of lactic acid bacteria deficient in lactose metabolism, which is able to metabolize a carbohydrate other than lactose, 1. A method for producing a fermented dairy product, including the steps: 2) fermentation of the milk base for a period of time until the desired pH is reached, where lactase stable at low pH is added to the process at the beginning, during or at the end of the fermentation step. (2) or a strain derived from DSM 28953, further characterized as having the ability to form white colonies on a medium containing lactose and X-Gal; (c) a strain of Lactobacillus delbrueckii subsp. bulgaricus, which is: (2) or a strain derived from DSM 28952, further characterized as having the ability to form white colonies on a medium containing lactose and X-Gal; (b) a strain of Streptococcus thermophilus, which is: 2. The method of claim 1, wherein the low pH stable lactase has an activity at 10° C. and pH 6.0 that is at least 10% of its activity at the lactase's optimum temperature. 2) fermentation of the milk base for a period of time until the desired pH is reached, 3. The method of claim 1 or 2, wherein the lactose metabolizing strain is capable of metabolizing a carbohydrate other than lactose selected from the group consisting of sucrose, galactose and glucose. 3) adding a low pH stable lactase to the process at the beginning, during or at the end of the fermentation step, where the low pH stable lactase has a lactase activity at pH 5.0 and a temperature of 37° C. that is at least 5% of its activity at the optimal pH value for this lactase. 4. The method according to any one of claims 1 to 3, wherein a carbohydrate other than lactose is added to the milk base at the beginning of the fermentation step. 5. The method of claim 4, wherein a carbohydrate other than lactose is added to the milk base in such an amount that it is depleted and therefore results in cessation of lactic acid bacteria growth and cessation of fermentation. 6. The method according to any one of claims 1 to 5, wherein the low pH stable lactase is added to the milk base at the beginning of the fermentation step. 7. The method of claim 6, wherein a carbohydrate other than lactose is not added during the fermentation step, and wherein at least one lactose-metabolizing lactose deficient starter culture strain is capable of metabolizing a carbohydrate selected from the group consisting of glucose and galactose. 8. The method according to any one of claims 1 to 5, wherein the low pH stable lactase is added to the milk base at the end of the fermentation step. 9. The method according to any one of claims 1 to 8, wherein the lactose metabolizing deficient strain is selected from the group consisting of lactose metabolizing deficient Streptococcus thermophilus and lactose metabolizing deficient Lactobacillus delbrueckii subsp. bulgaricus. 10. The method of claim 9, wherein the lactose metabolizing deficient strain is selected from the group consisting of: (a) a strain of Streptococcus thermophilus, which is: 11. The method according to claim 9 or 10, wherein the starter culture contains at least one lactose metabolizing deficient Streptococcus thermophilus and at least one lactose metabolizing deficient Lactobacillus delbrueckii subsp. bulgaricus. 12. The method according to any one of claims 9 to 11, wherein the starter culture comprises at least one Streptococcus thermophilus and at least one Lactobacillus delbrueckii subsp. bulgaricus, and wherein all strains of Streptococcus thermophilus and all strains of Lactobacillus delbrueckii subsp. bulgaricus are strains with deficiency of lactose metabolism . 13. A fermented milk product obtained by the method of claims 1 to 12, wherein the residual lactose level is less than 3 mg/ml. 14. Use of a starter culture containing at least one strain of lactic acid bacteria deficient in lactose metabolism, which is capable of metabolizing a carbohydrate other than lactose and a low pH stable lactase, wherein the low pH stable lactase has lactase activity at pH 5 0 and a temperature of 37°C, which is at least 5% of its activity at the optimal pH for this lactase, to obtain a fermented dairy product by a method including the steps: 15. Use according to claim 14, wherein the texture of the fermented milk product is improved compared to using lactose-metabolizing deficient and lactase-free lactic acid bacteria and compared to using low pH stable lactase and lactose positive lactic acid bacteria. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2