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`PCT/EP2008/066624
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`METHOD FOR PRODUCING A DAIRY PRODUCT
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`SEQUENCE LISTING
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`The present invention comprises a sequence listing.
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`TECHNICAL FIELD
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`The present invention relates to a method for producing a dairy product using an enzyme
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`having lactase activity.
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`10 BACKGROUND OF THE INVENTION
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`Lactose intolerance is perhaps the best-known food sensitivity in the United States and other
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`parts of the world. It is estimated that about 70% of the world's population has a genetically
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`controlled limited ability to digest lactose. Therefore, to help dairy maldigesters keep dairy
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`foods in their diet, there is a growing demand for dairy food products that contain no or only
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`15 low levels of lactose.
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`Lactase is used commercially to break down lactose in milk to produce dairy products which
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`are suitable for people with lactose intolerance and/or have a sweeter taste. Because
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`glucose and galactose are sweeter than lactose, lactase produces a more pleasant taste.
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`at the low 20 Lactase is also used in the manufacture of ice cream. Lactose crystallises
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`temperatures of ice cream, whereas glucose and galactose stay liquid and contribute to a
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`smoother texture. Lactase is also used in the conversion of whey into syrup. Lactase is also
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`used for production of condensed milk.
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`25 Lactases have been isolated from a large variety of organisms, including microorganisms.
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`Lactase is often an intracellular component of microorganisms like Kluyveromyces and
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`Bacillus. Kluyveromyces, K. fragilis especially and K. lactis, and other fungi such as those of
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`the genera Candida, Torula and Torulopsis, are a common source of fungal lactases,
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`whereas B. coagulans and B circulans are well known sources for bacterial lactases. Several
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`such as are available derived from these organisms 30 commercial lactase preparations
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`Lactozym® (available from Novozymes, Denmark), HA-Lactase (available from Chr. Hansen,
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`Denmark) and Maxilact® (available from DSM, the Netherlands), all from K. lactis. All these
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`lactases are so called neutral lactases having a pH optimum between pH 6 and pH 8. When
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`such lactases are used in the production of, e.g., low-lactose yoghurt, the enzyme treatment
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`35 will either have to be done in a separate
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`step before fermentation or rather high enzyme
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`dosages have to be used, because their activity drop as the pH decreases during
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`fermentation. Also, these lactases are not suitable for hydrolysis of lactose in milk performed
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`at high temperature, which would in some cases be beneficial to keep the microbial count
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`low and thus ensure good milk quality.
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`Several extracellular lactases have been described having a lower pH optimum, see, e.g.,
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`US patent 5,736,374 which describes an example of such lactase, produced by Aspergillus
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`5 oryzae.
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`A lactase from Bifidobacterium bifidum h a s b e e n d e s c r i b e d h a v i n g a h i g h
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`transgalactosylating activity, both in the full-length form and especially when truncated from
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`Biotechnol., the C-terminal end (see, e.g., J0rgensen et al. (2001 ), Appl. Microbial. 57: 647-
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`10 652 or EP patent 1,283,876).
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`It is an object of the present invention to provide a method for production of dairy products,
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`e.g. fermented dairy products, such as yoghurt, having a low level of lactose by using a
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`lactase. It is also an object to provide a method for production of low-lactose beverage milk
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`by using a lactase, where the method gives rise to low 15 having an extended shelf-life
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`formation of off-flavour and/or low formation of brown colour as compared to known methods.
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`Lactase to be used according to the invention should hydrolyse lactose efficiently and
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`optimally allow for almost complete lactose hydrolysis. Especially, such lactase should have
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`a high ratio of lactase to transgalactosylase activity. For use in the production of fermented
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`20 dairy products, the lactase should be active over a broad pH range.
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`SUMMARY OF THE INVENTION
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`The present inventors have surprisingly found that a C-terminally truncated fragment of the
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`bifidum, extracellular lactase from Bifidobacterium which was originally isolated and patented
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`25 for its ability to make high amounts of galactooligosaccharides from lactose, can be used
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`very successfully for hydrolysis of lactose in milk. When tested in water + 100 g/I lactose at
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`in the 37°C, the enzyme makes galactooligosaccharides with high efficiency as described
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`prior art. However, when tested in milk, the ratio of hydrolytic to transgalactosylating activity
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`has changed markedly, resulting in efficient hydrolysis and very low production of
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`30 galactooligosaccharides.
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`Consequently, the present invention relates to a method for producing a dairy product
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`comprising
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`a)providing a milk-based substrate comprising lactose; and
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`35 b)treating said substrate with an enzyme having lactase activity and having an amino acid
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`sequence which is at least 70% identical to amino acids 28-1931 of SEQ ID NO: 1 or a
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`In a preferred aspect, the invention relates to a method for producing a dairy product
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`comprising
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`a)providing a milk-based substrate comprising lactose; and
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`b)treating said substrate with an enzyme having lactase activity and having an amino acid
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`to amino acids 28-1331 of SEQ ID NO: 2. 5 sequence which is at least 70% identical
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`Further, the inventors have surprisingly found that very low levels of lactose can be achieved
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`bifidum
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`when using the lactase from Bifidobacterium as compared to other lactases typically
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`used for treatment of milk. Another unexpected advantage of using the lactase from
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`10 Bifidobacterium bifidum
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`is that the enzyme is active at high temperatures, allowing for
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`and thereby improving the microbial count treatment of the milk at, e.g., 52°C, thus reducing
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`the quality of the milk.
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`Therefore, in another aspect, the present invention relates to a method for producing a dairy
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`15 product comprising
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`a)providing a milk-based substrate comprising lactose, and
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`b)treating said substrate with an enzyme having lactase activity,
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`where step b) takes place at a temperature of at least 50°C.
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`20 In a preferred embodiment, step b) takes place at a temperature of at least 52°C.
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`Also, the inventors have surprisingly found that the lactase from Bifidobacterium is
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`active over a broad pH range.
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`25 Therefore, in another aspect, the present invention relates to a method for producing a dairy
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`product comprising
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`a)providing a milk-based substrate comprising lactose, and
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`b)treating said substrate with an enzyme having lactase activity, where the pH optimum of
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`the lactase activity at 37°C is above pH 5, and where the lactase activity of the enzyme at pH
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`activity at pH 6 when measured at 37°C. 30 5 is at least 50% of its lactase
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`Use of a lactase enzyme being active over a broad pH spectrum is especially useful for the
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`production of fermented dairy products, where it allows for low enzyme dosage, since the
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`enzyme is still active during and after fermentation. Also, very low levels of lactose in the
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`can be reached using such enzyme. 35 fermented dairy product
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`Therefore, in a preferred aspect, the present invention relates to a method for producing a
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`low-lactose fermented dairy product comprising
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`a)providing a milk-based substrate comprising lactose,
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`b)treating said substrate with an enzyme having lactase activity, where the pH optimum of
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`the lactase activity at 37°C is above pH 5, and where the lactase activity of the enzyme at pH
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`5 is at least 50% of its lactase activity at pH 6 when measured at 37°C, and
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`with a microorganism.5 c)fermenting said substrate
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`The present inventors have also surprisingly found that in the manufacture of low-lactose
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`beverage milk having an extended shelf life, the lactose hydrolysis can preferentially be
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`carried out at high temperature, such as at a temperature of at least 60°C. Preferentially,
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`and lactase treatment. low-pasteurization simultaneous 10 such manufacture may comprise
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`Therefore, in a preferred aspect, the present invention relates to a method for producing a
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`dairy product comprising
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`a)providing a milk-based substrate comprising lactose, and
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`b)treating said substrate with an enzyme having lactase activity,
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`of 15 wherein step b) is performed for between 10 minutes and 4 hours at a temperature
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`between 62°C and 64 °C.
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`In a more preferred aspect, step b) in such method is followed by cooling to below 10°C
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`without further heat treatment. This will allow for the enzyme to be still active after the milk
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`20 has been cooled, i.e. during its storage. In another more preferred aspect, step b) in such
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`method is followed by U HT treatment.
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`Preferably, in the methods of the invention, at least 70% of the lactose in the milk-based
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`substrate is hydrolysed. More preferably, at least 80%, such as at least 85%, at least 90%, at
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`is hydrolysed. 25 least 95% or at least 98%, of the lactose in the milk-based substrate
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`In another aspect, the present invention relates to a method for producing a dairy product
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`comprising
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`a)providing a milk-based substrate comprising lactose; and
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`30 b)treating said substrate with an enzyme having lactase activity and having an amino acid
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`sequence which is at least 70% identical to SEQ ID NO: 3 or a fragment thereof.
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`In another aspect, the present invention relates to a method for producing a dairy product
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`comprising
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`lactose; andsubstrate comprising 35 a)providing a milk-based
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`b)treating said substrate with an enzyme having lactase activity and having an amino acid
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`sequence which is at least 70% identical to SEQ ID NO: 4 or a fragment thereof.
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`In another aspect, the present invention relates to a method for producing a dairy product
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`comprising
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`a)providing a milk-based substrate comprising lactose; and
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`b)treating said substrate with an enzyme having lactase activity and having an amino acid
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`thereof. to SEQ ID NO: 5 or a fragment 5 sequence which is at least 70% identical
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`In another aspect, the present invention relates to a method for producing a dairy product
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`comprising
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`a)providing a milk-based substrate comprising lactose; and
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`10 b)treating said substrate with an enzyme having lactase activity and having an amino acid
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`thereof. sequence which is at least 70% identical to SEQ ID NO: 6 or a fragment
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`In yet another aspect, the present invention relates to a method for producing a dairy product
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`comprising
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`lactose; andsubstrate comprising 15 a)providing a milk-based
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`b)treating said substrate with an enzyme having lactase activity and having an amino acid
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`sequence which is at least 70% identical to SEQ ID NO: 7 or a fragment thereof.
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`DETAILED DISCLOSURE OF THE INVENTION
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`20 Milk-based substrate
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`The term "milk", in the context of the present invention, is to be understood as the lacteal
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`secretion obtained by milking any mammal, such as cows, sheep, goats, buffaloes or
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`camels.
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`25
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`"Milk-based substrate", in the context of the present invention, may be any raw and/or
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`processed milk material. Useful milk-based substrates include, but are not limited t o
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`solutions/suspensions o f any milk or milk like products comprising lactose, such a s whole or
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`low fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, solutions of
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`30 dried milk, UHT milk, whey, whey permeate, acid whey, or cream.
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`Preferably, the milk-based substrate is milk or an aqueous solution of skim milk powder.
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`The milk-based substrate may be more concentrated than raw milk.
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`35
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`In one embodiment, the milk-based substrate has a ratio of protein to lactose of at least 0.2,
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`preferably at least 0.3, at least 0.4, at least 0.5, at least 0.6 or, most preferably, at least 0.7.
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`The milk-based substrate may be homogenized and pasteurized according to methods
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`known in the art.
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`"Homogenizing" as used herein means intensive mixing to obtain a soluble suspension or
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`5 emulsion. It may be performed so as to break up the milk fat into smaller sizes so that it no
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`longer separates from the milk. This may be accomplished by forcing the milk at high
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`pressure through small orifices.
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`"Pasteurizing" as used herein means reducing or eliminating the presence of live organisms,
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`is attained pasteurization substrate. Preferably, 10 such as microorganisms, in the milk-based
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`by maintaining a specified temperature for a specified period of time. The specified
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`temperature is usually attained by heating. The temperature and duration may be selected in
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`order to kill or inactivate certain bacteria, such as harmful bacteria, and/or to inactivate
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`enzymes in the milk. A rapid cooling step may follow.
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`Dairy product
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`A "dairy product" in the context of the present invention may be any food product wherein
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`one of the major constituents is milk-based. Preferable, the major constituent is milk-based.
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`20 More preferably, the major constituent is a milk-based substrate which has been treated with
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`an enzyme having lactase activity according to a method of the invention. In the context of
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`the present invention "one of the major constituents" means a constituent having a dry matter
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`which constitutes more than 20%, preferably more than 30% or more than 40% of the total
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`dry matter of the dairy product, whereas "the major constituent" means a constituent having a
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`25 dry matter which constitutes more than 50%, preferably more than 60% or more than 70% of
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`the total dry matter of the dairy product.
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`A dairy product according to the invention may be, e.g., skim milk, low fat milk, whole milk,
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`cream, U HT milk, milk having an extended shelf life, a fermented milk product, cheese,
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`milk drink, sour cream, whey based drink, butter milk, acidified 30 yoghurt, butter, dairy spread,
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`ice cream, condensed milk, dulce de leche or a flavoured milk drink. A dairy product may be
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`manufactured by any method known in the art.
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`A dairy product may additionally comprise non-milk components, e.g. vegetable components
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`35 such as, e.g., vegetable oil, vegetable protein, and/or vegetable carbohydrates. Dairy
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`products may also comprise further additives such as, e.g., enzymes, flavouring agents,
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`microbial cultures such as probiotic cultures, salts, sweeteners, sugars, acids, fruit, fruit
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`juices, or any other component known in the art as a component of, or additive to, a dairy
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`product.
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`In one embodiment of the invention, one or more milk components and/or milk fractions ac­
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`count for at least 50% (weight/weight), such as at least 70%, e.g. at least 80%, preferably at
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`5 least 90%, of the dairy product.
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`In one embodiment of the invention, one or more milk-based substrates having been treated
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`with an enzyme having lactase activity according to a method of the invention account for at
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`least 50% (weight/weight), such as at least 70%, e.g. at least 80%, preferably at least 90%,
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`10 of the dairy product.
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`In one embodiment of the invention, the dairy product is a dairy product which is not enriched
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`by addition of galactooligosaccharides.
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`is not dried milk-based substrate the enzyme-treated 15 In one embodiment of the invention,
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`before being used as an ingredient in the dairy product.
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`In one embodiment of the invention, the dairy product is ice cream. In the present context,
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`ice cream may be any kind of ice cream such as full fat ice cream, low fat ice cream, or ice
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`20 cream based on yoghurt or other fermented milk products. Ice cream may be manufactured
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`by any method known in the art.
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`In one embodiment of the invention, the dairy product is milk or condensed milk.
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`the dairy product is UHT milk. UHT milk in the of the invention, 25 In one preferred embodiment
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`context of the present invention is milk which has been subjected to a sterilization procedure
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`which is intended to kill all microorganisms, including the bacterial spores. UHT (ultra high
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`temperature) treatment may be, e.g., heat treatment for 30 seconds at 130°G, or heat
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`treatment for one second at 145°G.
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`In one preferred embodiment of the invention, the dairy product is ESL milk. ESL milk in the
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`context of the present invention is milk which has an extended shelf life due to microfiltration
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`and/or heat treatment and which is able to stay fresh for at least 15 days, preferably for at
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`least 20 days, on the store shelf at 2-5°C.
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`30
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`35
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`In another preferred embodiment of the invention, the dairy product is a fermented dairy
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`product, e.g., yoghurt.
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`7
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`

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`WO 2009/071539
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`Fermented dairy product
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`PCT /EP2008/066624
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`A "fermented dairy product" in the context of the present invention is to be understood as any
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`dairy product wherein any type of fermentation forms part of the production process.
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`5 Examples of fermented dairy products are products like yoghurt, buttermilk, creme fraiche,
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`quark and fromage frais. A fermented dairy product may be produced by any method known
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`in the art.
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`"Fermentation" in the method of the present invention means the conversion of
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`Preferably, or acids through the action of a microorganism. 10 carbohydrates into alcohols
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`fermentation in the method of the present invention comprises conversion of lactose to lactic
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`acid.
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`In the context of the present invention, "microorganism" may include any bacterium or fungus
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`15 being able to ferment the milk substrate.
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`The microorganisms used for most fermented milk products are selected from the group of
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`bacteria generally referred to as lactic acid bacteria. As used herein, the term "lactic acid
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`bacterium" designates a gram-positive, microaerophilic or anaerobic bacterium, which
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`lactic acid as the predominantly o f acids including 20 ferments sugars with the production
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`produced acid, acetic acid and propionic acid. The industrially most useful lactic acid bacteria
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`are found within the order "Lactobacillales" which includes
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`Lactococcus spp., Streptococcus
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`spp., Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp., Pediococcus spp.,
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`Brevibacterium spp., Enterococcus spp. and Propionibacterium spp. Additionally, lactic acid
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`i.e. bifidobacteria, bacteria, to the group of anaerobic belonging 25 producing bacteria
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`Bifidobacterium
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`spp., which are frequently used as food cultures alone or in combination with
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`lactic acid bacteria, are generally included in the group of lactic acid bacteria.
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`Lactic acid bacteria are normally supplied to the dairy industry either as frozen or freeze-
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`"Direct Vat Set" (DVS) cultures, or as so-called propagation 30 dried cultures for bulk starter
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`intended for direct inoculation into a fermentation vessel or vat for the production of a
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`fermented dairy product. Such cultures are in general referred to as "starter cultures" or
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`"starters".
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`35 Commonly used starter culture strains of lactic acid bacteria are generally divided into
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`mesophilic organisms having optimum growth temperatures at about 30°C and thermophilic
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`organisms having optimum growth temperatures in the range of about 40 to about 45°C.
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`lactis, Typical organisms belonging to the mesophilic group include Lactococcus
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`8
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`

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`WO 2009/071539
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`PCT /EP2008/066624
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`mesenteroides Leuconostoc Lactococcus lactis subsp. cremoris, subsp. cremoris,
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`Lactococcus pentosaceus, Pediococcus Pseudoleuconostoc mesenteroides subsp. cremoris,
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`Lactobacillus biovar. diacetylactis, lactis subsp. lactis casei subsp. casei and Lactobacillus
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`paracasei subsp. paracasei. Thermophilic lactic acid bacterial species include as examples
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`delbrueckii Lactobacillus Enterococcus faecium, 5 Streptococcus thermophilus, subsp. lactis,
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`Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus
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`acidophilus.
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`Bifidobacterium Also the anaerobic bacteria belonging to the genus Bifidobacterium including
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`longum are commonly used as dairy animalis and Bifidobacterium 10 bifidum, Bifidobacterium
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`starter cultures and are generally included in the group of lactic acid bacteria. Additionally,
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`species of Propionibacteria are used as dairy starter cultures, in particular in the manufacture
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`of cheese. Additionally, organisms belonging to the Brevibacterium genus are commonly
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`used as food starter cultures.
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`15
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`Another group of microbial starter cultures are fungal cultures, including yeast cultures and
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`cultures of filamentous fungi, which are particularly used in the manufacture of certain types
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`roqueforti, Penicillium of cheese and beverage. Examples of fungi include Penicillium
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`candidum, Geotrichum candidum, Torula kefir, Saccharomyces kefir and Saccharomyces
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`20 cerevisiae.
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`In one embodiment of the present invention, the microorganism used for fermentation of the
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`thermophilus milk-based substrate is Lactobacillus casei or a mixture of Streptococcus and
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`Lactobacillus delbrueckii subsp. bulgaricus.
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`25
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`Fermentation processes to be used in a method of the present invention are well known and
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`the person of skill in the art will know how to select suitable process conditions, such as
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`temperature, oxygen, amount and characteristics of microorganism/s, additives such as e.g.
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`carbohydrates, flavours, minerals, enzymes, and process time. Obviously, fermentation
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`. of the present inventionso as to support the achievement 30 conditions are selected
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`As a result of fermentation, pH of the milk-based substrate will be lowered. The pH of a
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`fermented dairy product of the invention may be, e.g., in the range 3.5-6, such as in the
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`range 3.5-5, preferably in the range 3.8-4.8.
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`35
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`In a preferred embodiment, the fermented dairy product is yoghurt.
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`9
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`

`

`WO 2009/071539
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`PCT /EP2008/066624
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`
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`Method for producing a dairy product
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`As mentioned above, the present invention in one aspect relates to a method for producing a
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`dairy product comprising:
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`lactose; andsubstrate comprising 5 a)providing a milk-based
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`b)treating said substrate with an enzyme having lactase activity and having an amino acid
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`sequence which is at least 70% identical to amino acids 28-1931 of SEQ ID NO: 1 or a
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`fragment thereof.
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`a dairy product relates to a method for producing 10 In a preferred aspect, the invention
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`comprising
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`a)providing a milk-based substrate comprising lactose; and
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`b)treating said substrate with an enzyme having lactase activity and having an amino acid
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`sequence which is at least 70% identical to amino acids 28-1331 of SEQ ID NO: 2.
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`15
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`In another aspect, the invention relates to a method for producing a dairy product comprising
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`a)providing a milk-based substrate comprising lactose; and
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`b)treating said substrate with an enzyme having lactase activity and having an amino acid
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`sequence which is at least 70% identical to SEQ ID NO: 3 or a fragment thereof.
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`20
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`In another aspect, the invention relates to a method for producing a dairy product comprising
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`a)providing a milk-based substrate comprising lactose; and
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`b)treating said substrate with an enzyme having lactase activity and having an amino acid
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`sequence which is at least 70% identical to SEQ ID NO: 4 or a fragment thereof.
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`25
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`In another aspect, the invention relates to a method for producing a dairy product comprising
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`a)providing a milk-based substrate comprising lactose; and
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`b)treating said substrate with an enzyme having lactase activity and having an amino acid
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`sequence which is at least 70% identical to SEQ ID NO: 5 or a fragment thereof.
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`30
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`In another aspect, the invention relates to a method for producing a dairy product comprising
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`a)providing a milk-based substrate comprising lactose; and
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`b)treating said substrate with an enzyme having lactase activity and having an amino acid
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`sequence which is at least 70% identical to SEQ ID NO: 6 or a fragment thereof.
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`35
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`In another aspect, the invention relates to a method for producing a dairy product comprising
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`a)providing a milk-based substrate comprising lactose; and
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`b)treating said substrate with an enzyme having lactase activity and having an amino acid
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`10
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`

`

`WO 2009/071539
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`PCT /EP2008/066624
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`sequence which is at least 70% identical to SEQ ID NO: 7 or a fragment thereof.
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`In another aspect, the invention relates to a method for producing a dairy product comprising
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`a)providing a milk-based substrate comprising lactose, and
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`5 b)treating said substrate with an enzyme having lactase activity,
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`wherein step b) takes place at a temperature of at least 50°C.
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`In yet another aspect, the invention relates to a method for producing a dairy product
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`comprising
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`10 a)providing a milk-based substrate comprising lactose,
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`b)treating said substrate with an enzyme having lactase activity, where the pH optimum of
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`the lactase activity at 37°C is above pH 5, and where the lactase activity of the enzyme at pH
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`5 is at least 50% of its lactase activity at pH 6 when measured at 37°C.
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`pH and thereby activity at different the lactase 15 The skilled person will know how to determine
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`determine the pH optimum for the enzyme. The lactase activity at different pH may be
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`determined by measuring hydrolysis of lactose at 37°C for 30 minutes, preferably in a buffer
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`comprising succinate, HEPES, CHES, KCI, CaCb and MgCb, e.g., by using a method as
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`described in the Examples of the present application. For the avoidance of doubt, HEPES is
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`20 a buffering agent, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and CHES is a
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`buffering agent, N-Cyclohexyl-2-aminoethanesulfonic acid.
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`The enzyme-treated milk-based substrate may optionally be mixed with other ingredients to
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`obtain the dairy product. In one embodiment of the invention, the enzyme-treated milk-based
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`to obtain the dairy product. 25 substrate is mixed with other ingredients
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`In one embodiment of the invention, the dairy product is milk. In another embodiment, the
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`dairy product is condensed milk. In another embodiment, the dairy product is ice cream. In
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`another embodiment, the dairy product is UHT milk. In another embodiment, the dairy
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`the dairy product is a fermented dairy 30 product is ESL milk. In yet another embodiment,
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`product, e.g., yoghurt.
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`Preferably, the dairy product is a low-lactose dairy product. "Low-lactose", in the context of
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`the present invention, means that the amount of lactose in the dairy product, such as in the
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`80%, 90%, 95%, has been reduced by at least 70%, preferably 35 fermented dairy product,
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`98%, 99% or 99,5%.
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`Method for producing a low-lactose fermented dairy product
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`11
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`

`

`WO 2009/071539
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`PCT /EP2008/066624
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`One embodiment of the present invention relates to a method for producing a low-lactose
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`fermented dairy product comprising
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`a)providing a milk-based substrate comprising lactose,
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`activity ofactivity, where the lactase with an enzyme having lactase 5 b)treating said substrate
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`the enzyme at pH 5 is at least 50% of its lactase activity at pH 6 when measured at 37°C,
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`and
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`c)fermenting said substrate with a microorganism.
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`a low-lactose relates to a method for producing 10 Another embodiment of the invention
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`fermented dairy product comprising
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`a)providing a milk-based substrate comprising lactose,
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`b)treating said substrate with an enzyme having lactase activity and having an amino acid
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`sequence which is at least 70% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
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`15 SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or a fragment of any of these,
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`and
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`c)fermenting said substrate with a microorganism.
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`Preferably, in these embodiments, step b) and step c) are performed essentially at the same
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`20 time.
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`In the context of the method of the invention, "essentially at the same time" means that
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`enzyme treatment and fermentation are not performed as separate steps, i.e., incubation of
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`the milk-based substrate with the enzyme is not performed as a separate step before
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`25 inoculation with the microorganism. Instead, the enzyme and the microorganism may be
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`added to the milk-based substrate at essentially the same time. I.e., the enzyme may be
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`added to the milk-based substrate immediately before inoculation with the microorganism.
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`"Immediately before" in this context means without a separate incubation step for the
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`enzymatic hydrolysis. Alternatively, the microorganism may be added immediately before the
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`30 enzyme, or the microorganism and the enzyme may be added at the same time. "Essentially
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`at the same time" in the context of the method of the invention may mean that the enzyme is
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`active throughout the whole of step c).
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`"Essentially at the same time" does not mean that the enzymatic hydrolysis of the lactose in
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`i.e. when the pH is completed, when the fermentation 35 the milk-based substrate is completed
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`has dropped to a level preventing further fermenting activity of the microbial starter culture.
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`In a preferred embodiment of the invention, the enzyme is still active after completion of step
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`12
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`

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`WO 2009/071539
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`PCT /EP2008/066624
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`c). In a more preferred embodiment, the enzyme has retained at least 20%, such as at least
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`30%, at least 40%, at least 50%, at least 60% or at least 70%, of its lactase activity after
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`completion of step c) as compared to its activity when added to the milk-based substrate.
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`5 After completion of step c) may mean when at least one of the below is true:
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`-temperature is lowered to below 30°C
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`-pH has reached 4.55
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`-pH does no longer decrease by more than 0.2 units per hour.
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`such as less than 70%, less than 60%, less 10 In another preferred embodiment, less than 80%,
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`than 50%, less than 40%, less than 30% or less than 20%, of the lactose has been
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`hydrolyzed after two hours of fermentation. "After two hours of fermentation" in the context of
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`the invention means that the milk-based substrate, after having been inoculated with the
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`microorganism, has been incubated for two hours at a temperature which is appropriate for
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