I 1111111111111111 1111111111 1111111111 111111111111111 111111111111111 IIII IIII
`US010306902B2
`
`c12) United States Patent
`Hendriksen et al.
`
`(IO) Patent No.: US 10,306,902 B2
`(45) Date of Patent:
`*Jun. 4, 2019
`
`(54) METHOD FOR PRODUCING A DAIRY
`PRODUCT
`
`(71) Applicant: Novozymes A/S, Bagsvaaerd (DK)
`
`(72)
`
`Inventors: Hanne Vang Hendriksen, Holte (DK);
`Steffen Ernst, Broenshoej (DK);
`Reinhard Wilting, Farum (DK); Jeppe
`Wegener Tams, Gentofte (DK); Mette
`Oerhrstroem Runge, Snekkersten
`(DK); Helle Skov Guldager, Vedbaek
`(DK)
`
`CN
`CN
`EP
`EP
`EP
`EP
`WO
`WO
`WO
`WO
`WO
`WO
`
`FOREIGN PATENT DOCUMENTS
`
`1807609 A
`1810161 A
`0026672 Al
`0119329 A2
`0323201 A2
`1283876 Bl
`01/90317 A2
`02/081673 Al
`2003/094623 Al
`2007/021204 Al
`2009/071539 Al
`2013/182686 Al
`
`7/2006
`8/2006
`8/1981
`9/1984
`7 /1989
`1/2007
`11/2001
`10/2002
`11/2003
`2/2007
`6/2009
`12/2013
`
`(73) Assignee: Novozymes A/S, Bagsavaerd (DK)
`
`OTHER PUBLICATIONS
`
`( *) Notice:
`
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by O days.
`
`This patent is subject to a terminal dis(cid:173)
`claimer.
`
`(21) Appl. No.: 16/021,048
`
`(22) Filed:
`
`Jun. 28, 2018
`
`(65)
`
`Prior Publication Data
`
`US 2018/0295846 Al
`
`Oct. 18, 2018
`
`Related U.S. Application Data
`
`(63) Continuation of application No. 15/433,642, filed on
`Feb. 15, 2017, now Pat. No. 10,058,107, which is a
`continuation of application No. 12/744,508, filed as
`application No. PCT/EP2008/066624 on Dec. 2,
`2008, now abandoned.
`(60) Provisional application No. 61/055,164, filed on May
`22, 2008, provisional application No. 60/992,783,
`filed on Dec. 6, 2007.
`
`(30)
`
`Foreign Application Priority Data
`
`Dec. 3, 2007
`May 21, 2008
`
`(EP) ..................................... 07122110
`(EP) ..................................... 08156674
`
`(51)
`
`Int. Cl.
`C12N 9/38
`A23C 9112
`A23C 9/123
`(52) U.S. Cl.
`CPC ............ A23C 9/1206 (2013.01); A23C 9/123
`(2013.01)
`
`(2006.01)
`(2006.01)
`(2006.01)
`
`( 58) Field of Classification Search
`CPC .............................................. C12Y 302/01023
`USPC ............................................... 435/207, 252.3
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5,736,374 A
`7,081,355 B2 *
`
`4/1998 Berka et al.
`7/2006 Jorgensen
`
`2008/0286412 Al
`2017/0156357 Al*
`
`11/2008 Dekker et al.
`6/2017 Hendriksen.
`
`Cl2Y 302/01023
`426/582
`
`A23C 9/1206
`
`Anonymous, Asia Pacific Food Industry, Special Supplement, Ingre(cid:173)
`dients & Additives, "Lactase: An Optimum Enzyme for Low
`Lactose Dairy Products", pp. 40-43 (Jun. 2001).
`Anonymous, Food Ingredients South America Innovation Awards
`(2017).
`Anonymous, Food Ingredients Innovation Awards, "Novozymes is
`Finalist of the Fi Innovation Awards 2017 with Saphera" (2017).
`Anonymous, Sequence Alignment between Amino Acid Sequences
`Disclosed in EP 2234501 and WO 01/90317 (date unknown).
`Arla Foods Ingredients Group, brochure on "WheyPermeate Taste
`and Texture Enhancement at a Cost Benefit" (date unknown).
`Broome et al., Australian Journal of Dairy Technology, pp. 35-37
`(Mar. 1983).
`CHR. Hansen, Ha-lactase(TM) application sheet (2010).
`Cruz et al., Revista de Microbiologia, vol. 30, pp. 265-271 (1999).
`De Vrese et al., Am. J. Clin. Nutr., vol. 73(suppl.), pp. 421-429
`(2001).
`Goulas et al., Appl. Microbiol. Biotechnol., DOI 10.1007/s00253-
`007-l 099-1 (2007).
`Goulas et al., UniProt Accession No. A4K5H9 (2007).
`Hussein et al., Journal of Food Protection, vol. 52, No. 1, pp. 30-34
`(1988).
`Ismail, Journal of the Society of Dairy Technology, vol. 36, No. 2,
`pp. 52-55 (1983).
`Jorgensen et al., Appl. Microbiol. Biotechnol., vol. 57, pp. 647-652
`(2001).
`Jurado et al., Enzyme and Microbial Technology, vol. 34, pp. 33-40
`(2004).
`Kang et al., Enzyme and Microbial Technology, vol. 37, pp.
`655-662 (2005).
`Kenward, "Novozymes: SAPHERA Enables New Innovation in
`Lactose-Free, Fermented Dairy Products" (2016).
`Ma et al., Science and Technology of Food Industry, DOI:10.13386/
`j .issnl 002-0306.2002.01.021 (2002).
`Madsen et al., UniProt Accession No. Q9F4D5 ((2003).
`Mahoney et al., Journal of Food Science, vol. 43, pp. 584-591
`(1978).
`Martinez-Villaluenga et al., Food Chemistry, vol. 107, pp. 258-264
`(2008).
`Moller et al., Applied and Environmental Microbiology, vol. 67, No.
`5, pp. 2276-2283 (2001).
`
`(Continued)
`
`Primary Examiner - Tekchand Saidha
`(74) Attorney, Agent, or Firm - Elias Lambiris
`
`ABSTRACT
`(57)
`The present invention relates to a method for producing a
`dairy product using an enzyme having lactase activity.
`
`19 Claims, No Drawings
`Specification includes a Sequence Listing.
`
`

`

`US 10,306,902 B2
`Page 2
`
`(56)
`
`References Cited
`
`OTHER PUBLICATIONS
`
`Myers et al., UniProt Accession No. QOTR23 (2006).
`Passerat et al., Nutrition Research, vol. 15, No. 9, pp. 12871295
`(1995).
`Rao, Food Science and Technology International, vol. 3, pp. 87-92
`(1997).
`Rubio-Texeira., Biotechnology Advances, vol. 24, pp. 212-225
`(2006).
`Shimizu et al., UniProt Accession No. Q8XKX6 (2002).
`Sudarsanam et al., UniProt Accession No. A5KPK7 (2007).
`Tamm et al., Scandinavian Journal of Gastroenterology, "Manage(cid:173)
`ment of Lactose Intolerance", vol. 29, Suppl. 202, pp. 55-63 (1994).
`Anonymous, Alignment of BIF 1331 with QH-tag, BIF 1331 without
`QH-tag, and SEQ ID No. 2 of EP 2234501 (date unknown).
`Anonymous, Alignment ofBIF1331 without HQ (date unknown).
`Anonymous, Alignment of SEQ ID No. 2 of EP 2 234 501 and B.
`bifidum ATCC 29521 (UniProtKB Cccession No. U2C0I1) (date
`unknown).
`Bornhorst et al., Methods Enzymol., vol. 326, pp. 245-254 (2000).
`Dechter et al., Food Biotechnology, vol. 12, pp. 73-89 (1998).
`Dumortier et al., Biotechnol. Appl. Biochem., vol. 19, pp. 341-354
`(1994).
`Novozymes, Application Sheet-Lactose reduction (2009).
`
`* cited by examiner
`
`

`

`US 10,306,902 B2
`
`1
`METHOD FOR PRODUCING A DAIRY
`PRODUCT
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`2
`are not suitable for hydrolysis of lactose in milk performed
`at high temperature, which would in some cases be benefi(cid:173)
`cial to keep the microbial count low and thus ensure good
`milk quality.
`Several extracellular lactases have been described having
`a lower pH optimum, see, e.g., U.S. Pat. No. 5,736,374
`which describes an example of such lactase, produced by
`Aspergillus oryzae.
`A
`lactase from Bifidobacterium bifidum has been
`10 described having a high transgalactosylating activity, both in
`the full-length form and especially when truncated from the
`C-terminal end (see, e.g., Jorgensen et al., 2001, Appl.
`Microbial. Biotechnol. 57: 647-652 or EP Patent No. 1,283,
`876).
`It is an object of the present invention to provide a method
`for production of dairy products, e.g., fermented dairy
`products, such as yoghurt, having a low level of lactose by
`using a lactase. It is also an object to provide a method for
`production oflow-lactose beverage milk having an extended
`20 shelf-life by using a lactase, where the method gives rise to
`low formation of off-flavour and/or low formation of brown
`color as compared to known methods. Lactase to be used
`according to the invention should hydrolyze lactose effi(cid:173)
`ciently and optimally allow for almost complete lactose
`25 hydrolysis. Especially, such lactase should have a high ratio
`of lactase to transgalactosylase activity. For use in the
`production of fermented dairy products, the lactase should
`be active over a broad pH range.
`
`This application is a continuation of U.S. application Ser.
`No. 15/433,642 filed Feb. 15, 2017, now U.S. Pat. No.
`10,058,107, which is a continuation of U.S. application Ser.
`No. 12/744,508 filed May 25, 2010, now abandoned, which
`is a 35 U.S.C. 371 national application of PCT/EP2008/
`66624 filed Dec. 2, 2008, which claims priority or the benefit
`under 35 U.S.C. 119 of European application nos.
`07122110.5 and 08156674.7 filed Dec. 3, 2007 and May 21,
`2008, respectively, and U.S. provisional application Nos. 15
`61/055,164 and 60/992,783 filed May 22, 2008 and Dec. 6,
`2007, respectively. The content of each application is fully
`incorporated herein by reference.
`
`SEQUENCE LISTING
`
`The present invention comprises a sequence listing, which
`is incorporated herein by reference.
`
`TECHNICAL FIELD
`
`The present invention relates to a method for producing a
`dairy product using an enzyme having lactase activity.
`
`BACKGROUND OF THE INVENTION
`
`30
`
`SUMMARY OF THE INVENTION
`
`Lactose intolerance is perhaps the best-known food sen(cid:173)
`sitivity in the United States and other parts of the world. It
`is estimated that about 70% of the world's population has a
`genetically controlled limited ability to digest lactose.
`Therefore, to help dairy maldigesters keep dairy foods in
`their diet, there is a growing demand for dairy food products
`that contain no or only low levels of lactose.
`Lactase is used commercially to break down lactose in
`milk to produce dairy products which are suitable for people
`with lactose intolerance and/or have a sweeter taste. Because
`glucose and galactose are sweeter than lactose, lactase
`produces a more pleasant taste. Lactase is also used in the
`manufacture of ice cream. Lactose crystallises at the low
`temperatures of ice cream, whereas glucose and galactose
`stay liquid and contribute to a smoother texture. Lactase is
`also used in the conversion of whey into syrup. Lactase is
`also used for production of condensed milk.
`Lactases have been isolated from a large variety of
`organisms, including microorganisms. Lactase is often an 50
`intracellular component of microorganisms like Kluyvero(cid:173)
`myces and Bacillus. Kluyveromyces, especially K. fragilis
`and K. lactis, and other fungi such as those of the genera
`Candida, Torula and Torulopsis, are a common source of
`fungal lactases, whereas B. coagulans and B circulans are 55
`well known sources for bacterial lactases. Several commer-
`cial lactase preparations derived from these organisms are
`available such as Lactozym® (available from Novozymes,
`Denmark), HA-Lactase (available from Chr. Hansen, Den(cid:173)
`mark) and Maxilact® (available from DSM, the Nether(cid:173)
`lands), all from K. lactis. All these lactases are so called
`neutral lactases having a pH optimum between pH 6 and pH
`8. When such lactases are used in the production of, e.g.,
`low-lactose yoghurt, the enzyme treatment will have to be
`done in a separate step before fermentation or rather high
`enzyme dosages have to be used, because their activity drops
`as the pH decreases during fermentation. Also, these lactases
`
`The present inventors have surprisingly found that a
`C-terminally truncated fragment of the extracellular lactase
`from Bifidobacterium bifidum, which was originally isolated
`35 and patented for its ability to make high amounts of galac(cid:173)
`tooligosaccharides from lactose, can be used very success(cid:173)
`fully for hydrolysis of lactose in milk. When tested in
`water+l00 g/1 lactose at 37° C., the enzyme makes galac(cid:173)
`tooligosaccharides with high efficiency as described in the
`40 prior art. However, when tested in milk, the ratio of hydro(cid:173)
`lytic to transgalactosylating activity has changed markedly,
`resulting in efficient hydrolysis and very low production of
`galactooligosaccharides.
`Consequently, the present invention relates to a method
`45 for producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose;
`and
`b) treating said substrate with an enzyme having lactase
`activity and having an amino acid sequence which is at least
`70% identical to amino acids 28-1931 of SEQ ID NO: 1 or
`a fragment thereof.
`In a preferred aspect, the invention relates to a method for
`producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose;
`and
`b) treating said substrate with an enzyme having lactase
`activity and having an amino acid sequence which is at least
`70% identical to amino acids 28-1331 of SEQ ID NO: 2.
`Further, the inventors have surprisingly found that very
`60 low levels oflactose can be achieved when using the lactase
`from Bifidobacterium bifidum as compared to other lactases
`typically used for treatment of milk. Another unexpected
`advantage of using the lactase from Bifidobacterium bifidum
`is that the enzyme is active at high temperatures, allowing
`65 for treatment of the milk at, e.g., 52° C., thus reducing the
`microbial count and thereby improving the quality of the
`milk.
`
`

`

`US 10,306,902 B2
`
`15
`
`4
`b) treating said substrate with an enzyme having lactase
`activity and having an amino acid sequence which is at least
`70% identical to SEQ ID NO: 3 or a fragment thereof.
`In another aspect, the present invention relates to a
`method for producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose;
`and
`b) treating said substrate with an enzyme having lactase
`activity and having an amino acid sequence which is at least
`70% identical to SEQ ID NO: 4 or a fragment thereof.
`In another aspect, the present invention relates to a
`method for producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose;
`and
`b) treating said substrate with an enzyme having lactase
`activity and having an amino acid sequence which is at least
`70% identical to SEQ ID NO: 5 or a fragment thereof.
`In another aspect, the present invention relates to a
`method for producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose;
`and
`b) treating said substrate with an enzyme having lactase
`activity and having an amino acid sequence which is at least
`70% identical to SEQ ID NO: 6 or a fragment thereof.
`In yet another aspect, the present invention relates to a
`method for producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose;
`and
`b) treating said substrate with an enzyme having lactase
`activity and having an amino acid sequence which is at least
`70% identical to SEQ ID NO: 7 or a fragment thereof.
`
`DETAILED DISCLOSURE OF THE INVENTION
`
`3
`Therefore, in another aspect, the present invention relates
`to a method for producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose,
`and
`b) treating said substrate with an enzyme having lactase 5
`activity,
`where step b) takes place at a temperature of at least 50° C.
`In a preferred embodiment, step b) takes place at a
`temperature of at least 52° C.
`Also, the inventors have surprisingly found that the 10
`lactase from Bifidobacterium bifidum is active over a broad
`pH range.
`Therefore, in another aspect, the present invention relates
`to a method for producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose,
`and
`b) treating said substrate with an enzyme having lactase
`activity, where the pH optimum of the lactase activity at 37°
`C. is above pH 5, and where the lactase activity of the 20
`enzyme at pH 5 is at least 50% of its lactase activity at pH
`6 when measured at 37° C.
`Use of a lactase enzyme being active over a broad pH
`spectrum is especially useful for the production of fermented
`dairy products, where it allows for low enzyme dosage, 25
`since the enzyme is still active during and after fermentation.
`Also, very low levels of lactose in the fermented dairy
`product can be reached using such enzyme.
`Therefore, in a preferred aspect, the present invention
`relates to a method for producing a low-lactose fermented 30
`dairy product comprising
`a) providing a milk-based substrate comprising lactose,
`b) treating said substrate with an enzyme having lactase
`activity, where the pH optimum of the lactase activity at 37°
`C. is above pH 5, and where the lactase activity of the
`enzyme at pH 5 is at least 50% of its lactase activity at pH
`6 when measured at 37° C., and
`c) fermenting said substrate with a microorganism.
`The present inventors have also surprisingly found that in
`the manufacture of low-lactose beverage milk having an 40
`extended shelf life, the lactose hydrolysis can preferentially
`be carried out at high temperature, such as at a temperature
`of at least 60° C. Preferentially, such manufacture may
`comprise simultaneous low-pasteurization and lactase treat(cid:173)
`ment. Therefore, in a preferred aspect, the present invention 45
`relates to a method for producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose,
`and
`b) treating said substrate with an enzyme having lactase
`activity,
`wherein step b) is performed for between 10 minutes and 4
`hours at a temperature of between 62° C. and 64 ° C.
`In a more preferred aspect, step b) in such method is
`followed by cooling to below 10° C. without further heat
`treatment. This will allow for the enzyme to be still active 55
`after the milk has been cooled, i.e., during its storage. In
`another more preferred aspect, step b) in such method is
`followed by UHT treatment.
`Preferably, in the methods of the invention, at least 70%
`of the lactose in the milk-based substrate is hydrolyzed. 60
`More preferably, at least 80%, such as at least 85%, at least
`90%, at least 95% or at least 98%, of the lactose in the
`milk-based substrate is hydrolyzed.
`In another aspect, the present invention relates to a
`method for producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose;
`and
`
`50
`
`35 Milk-Based Substrate
`The term "milk", in the context of the present invention,
`is to be understood as the lacteal secretion obtained by
`milking any mammal, such as cows, sheep, goats, buffaloes
`or camels.
`"Milk-based substrate", in the context of the present
`invention, may be any raw and/or processed milk material.
`Useful milk-based substrates include, but are not limited to
`solutions/suspensions of any milk or milk like products
`comprising lactose, such as whole or low fat milk, skim
`milk, buttermilk, reconstituted milk powder, condensed
`milk, solutions of dried milk, UHT milk, whey, whey
`permeate, acid whey, or cream.
`Preferably, the milk-based substrate is milk or an aqueous
`solution of skim milk powder.
`The milk-based substrate may be more concentrated than
`raw milk.
`In one embodiment, the milk-based substrate has a ratio
`of protein to lactose of at least 0.2, preferably at least 0.3, at
`least 0.4, at least 0.5, at least 0.6 or, most preferably, at least
`0.7.
`The milk-based substrate may be homogenized and pas(cid:173)
`teurized according to methods known in the art.
`"Homogenizing" as used herein means intensive mixing
`to obtain a soluble suspension or emulsion. It may be
`performed so as to break up the milk fat into smaller sizes
`so that it no longer separates from the milk. This may be
`accomplished by forcing the milk at high pressure through
`small orifices.
`"Pasteurizing" as used herein means reducing or elimi-
`65 nating the presence of live organisms, such as microorgan(cid:173)
`isms, in the milk-based substrate. Preferably, pasteurization
`is attained by maintaining a specified temperature for a
`
`

`

`US 10,306,902 B2
`
`5
`specified period of time. The specified temperature is usually
`attained by heating. The temperature and duration may be
`selected in order to kill or inactivate certain bacteria, such as
`harmful bacteria, and/or to inactivate enzymes in the milk.
`A rapid cooling step may follow.
`Dairy Product
`A "dairy product" in the context of the present invention
`may be any food product wherein one of the major con(cid:173)
`stituents is milk-based. Preferable, the major constituent is
`milk-based. More preferably, the major constituent is a 10
`milk-based substrate which has been treated with an enzyme
`having lactase activity according to a method of the inven(cid:173)
`tion. In the context of the present invention "one of the major
`constituents" means a constituent having a dry matter which
`constitutes more than 20%, preferably more than 30% or 15
`more than 40% of the total dry matter of the dairy product,
`whereas "the major constituent" means a constituent having
`a dry matter which constitutes more than 50%, preferably
`more than 60% or more than 70% of the total dry matter of
`the dairy product.
`A dairy product according to the invention may be, e.g.,
`skim milk, low fat milk, whole milk, cream, UHT milk, milk
`having an extended shelf life, a fermented milk product,
`cheese, yoghurt, butter, dairy spread, butter milk, acidified
`milk drink, sour cream, whey based drink, ice cream, 25
`condensed milk, duke de leche or a flavoured milk drink. A
`dairy product may be manufactured by any method known
`in the art.
`A dairy product may additionally comprise non-milk
`components, e.g., vegetable components such as, e.g., veg(cid:173)
`etable oil, vegetable protein, and/or vegetable carbohy(cid:173)
`drates. Dairy products may also comprise further additives
`such as, e.g., enzymes, flavouring agents, microbial cultures
`such as probiotic cultures, salts, sweeteners, sugars, acids,
`fruit, fruit juices, or any other component known in the art 35
`as a component of, or additive to, a dairy product.
`In one embodiment of the invention, one or more milk
`components and/or milk fractions ac-count for at least 50%
`(weight/weight), such as at least 70%, e.g., at least 80%,
`preferably at least 90%, of the dairy product.
`In one embodiment of the invention, one or more milk(cid:173)
`based substrates having been treated with an enzyme having
`lactase activity according to a method of the invention
`account for at least 50% (weight/weight), such as at least
`70%, e.g., at least 80%, preferably at least 90%, of the dairy 45
`product.
`In one embodiment of the invention, the dairy product is
`a dairy product which is not enriched by addition of galac(cid:173)
`tooligosaccharides.
`In one embodiment of the invention, the enzyme-treated 50
`milk-based substrate is not dried before being used as an
`ingredient in the dairy product.
`In one embodiment of the invention, the dairy product is
`ice cream. In the present context, ice cream may be any kind
`of ice cream such as full fat ice cream, low fat ice cream, or 55
`ice cream based on yoghurt or other fermented milk prod(cid:173)
`ucts. Ice cream may be manufactured by any method known
`in the art.
`In one embodiment of the invention, the dairy product is
`milk or condensed milk.
`In one preferred embodiment of the invention, the dairy
`product is UHT milk. UHT milk in the context of the present
`invention is milk which has been subjected to a sterilization
`procedure which is intended to kill all microorganisms,
`including the bacterial spores. UHT (ultra high temperature) 65
`treatment may be, e.g., heat treatment for 30 seconds at 130°
`C., or heat treatment for one second at 145° C.
`
`6
`In one preferred embodiment of the invention, the dairy
`product is ESL milk. ESL milk in the context of the present
`invention is milk which has an extended shelf life due to
`microfiltration and/or heat treatment and which is able to
`5 stay fresh for at least 15 days, preferably for at least 20 days,
`on the store shelf at 2-5° C.
`In another preferred embodiment of the invention, the
`dairy product is a fermented dairy product, e.g., yoghurt.
`Fermented Dairy Product
`A "fermented dairy product" in the context of the present
`invention is to be understood as any dairy product wherein
`any type of fermentation forms part of the production
`process. Examples of fermented dairy products are products
`like yoghurt, buttermilk, creme fraiche, quark and fromage
`frais. A fermented dairy product may be produced by any
`method known in the art.
`"Fermentation" in the method of the present invention
`means the conversion of carbohydrates into alcohols or acids
`through the action of a microorganism. Preferably, fermen-
`20 tation in the method of the present invention comprises
`conversion of lactose to lactic acid.
`In the context of the present invention, "microorganism"
`may include any bacterium or fungus being able to ferment
`the milk substrate.
`The microorganisms used for most fermented milk prod-
`ucts are selected from the group of bacteria generally
`referred to as lactic acid bacteria. As used herein, the term
`"lactic acid bacterium" designates a gram-pos1t1ve,
`microaerophilic or anaerobic bacterium, which ferments
`30 sugars with the production of acids including lactic acid as
`the predominantly produced acid, acetic acid and propionic
`acid. The industrially most useful lactic acid bacteria are
`found within the order "Lactobacillales" which includes
`Lactococcus spp., Streptococcus spp., Lactobacillus spp.,
`Leuconostoc spp., Pseudoleuconostoc spp., Pediococcus
`spp., Brevibacterium spp., Enterococcus spp. and Propioni-
`bacterium spp. Additionally, lactic acid producing bacteria
`belonging to the group of anaerobic bacteria, bifidobacteria,
`i.e., Bifidobacterium spp., which are frequently used as food
`40 cultures alone or in combination with lactic acid bacteria, are
`generally included in the group of lactic acid bacteria.
`Lactic acid bacteria are normally supplied to the dairy
`industry either as frozen or freeze-dried cultures for bulk
`starter propagation or as so-called "Direct Vat Set" (DYS)
`cultures, intended for direct inoculation into a fermentation
`vessel or vat for the production of a fermented dairy product.
`Such cultures are in general referred to as "starter cultures"
`or "starters".
`Commonly used starter culture strains of lactic acid
`bacteria are generally divided into mesophilic organisms
`having optimum growth temperatures at about 30° C. and
`thermophilic organisms having optimum growth tempera(cid:173)
`tures in the range of about 40 to about 45° C. Typical
`organisms belonging to the mesophilic group include Lac-
`tococcus lactis, Lactococcus lactis subsp. cremoris, Leu(cid:173)
`conostoc mesenteroides subsp. cremoris, Pseudoleuconos-
`subsp.
`toc mesenteroides
`cremoris,
`Pediococcus
`lactis subsp.
`lactis biovar.
`pentosaceus, Lactococcus
`diacetylactis, Lactobacillus casei subsp. casei and Lactoba-
`60 cillus paracasei subsp. paracasei. Thermophilic lactic acid
`bacterial species include as examples Streptococcus ther(cid:173)
`mophilus, Enterococcus faecium, Lactobacillus delbrueckii
`subsp. lactis, Lactobacillus helveticus, Lactobacillus del-
`brueckii subsp. bulgaricus and Lactobacillus acidophilus.
`Also the anaerobic bacteria belonging to the genus Bifi(cid:173)
`dobacterium including Bifidobacterium bifidum, Bifidobac(cid:173)
`terium animalis and Bifidobacterium longum are commonly
`
`

`

`US 10,306,902 B2
`
`7
`used as dairy starter cultures and are generally included in
`the group of lactic acid bacteria. Additionally, species of
`Propionibacteria are used as dairy starter cultures, in par(cid:173)
`ticular, in the manufacture of cheese. Additionally, organ(cid:173)
`isms belonging to the Brevibacterium genus are commonly
`used as food starter cultures.
`Another group of microbial starter cultures are fungal
`cultures, including yeast cultures and cultures of filamentous
`fungi, which are particularly used in the manufacture of
`certain types of cheese and beverage. Examples of fungi 10
`include Penicillium roqueforti, Penicillium candidum,
`Geotrichum candidum, Torula kefir, Saccharomyces kefir
`and Saccharomyces cerevisiae.
`In one embodiment of the present invention, the micro- 15
`organism used for fermentation of the milk-based substrate
`is Lactobacillus casei or a mixture of Streptococcus ther(cid:173)
`mophilus and Lactobacillus delbrueckii subsp. bulgaricus.
`Fermentation processes to be used in a method of the
`present invention are well known and the person of skill in 20
`the art will know how to select suitable process conditions,
`such as temperature, oxygen, amount and characteristics of
`microorganism/s, additives such as, e.g., carbohydrates,
`flavours, minerals, enzymes, and process time. Obviously,
`fermentation conditions are selected so as to support the
`achievement of the present invention.
`As a result of fermentation, pH of the milk-based sub(cid:173)
`strate will be lowered. The pH of a fermented dairy product
`of the invention may be, e.g., in the range 3.5-6, such as in
`the range 3.5-5, preferably in the range 3.8-4.8.
`In a preferred embodiment, the fermented dairy product is
`yoghurt.
`Method for Producing a Dairy Product
`As mentioned above, the present invention in one aspect
`relates to a method for producing a dairy product compris- 35
`ing:
`a) providing a milk-based substrate comprising lactose;
`and
`b) treating said substrate with an enzyme having lactase
`activity and having an amino acid sequence which is at least
`70% identical to amino acids 28-1931 of SEQ ID NO: 1 or
`a fragment thereof.
`In a preferred aspect, the invention relates to a method for
`producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose;
`and
`b) treating said substrate with an enzyme having lactase
`activity and having an amino acid sequence which is at least
`70% identical to amino acids 28-1331 of SEQ ID NO: 2.
`In another aspect, the invention relates to a method for
`producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose;
`and
`b) treating said substrate with an enzyme having lactase
`activity and having an amino acid sequence which is at least
`70% identical to SEQ ID NO: 3 or a fragment thereof.
`In another aspect, the invention relates to a method for
`producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose;
`and
`b) treating said substrate with an enzyme having lactase
`activity and having an amino acid sequence which is at least
`70% identical to SEQ ID NO: 4 or a fragment thereof.
`In another aspect, the invention relates to a method for
`producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose;
`and
`
`8
`b) treating said substrate with an enzyme having lactase
`activity and having an amino acid sequence which is at least
`70% identical to SEQ ID NO: 5 or a fragment thereof.
`In another aspect, the invention relates to a method for
`5 producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose;
`and
`b) treating said substrate with an enzyme having lactase
`activity and having an amino acid sequence which is at least
`70% identical to SEQ ID NO: 6 or a fragment thereof.
`In another aspect, the invention relates to a method for
`producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose;
`and
`b) treating said substrate with an enzyme having lactase
`activity and having an amino acid sequence which is at least
`70% identical to SEQ ID NO: 7 or a fragment thereof.
`In another aspect, the invention relates to a method for
`producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose,
`and
`b) treating said substrate with an enzyme having lactase
`activity,
`25 wherein step b) takes place at a temperature of at least 50°
`C.
`In yet another aspect, the invention relates to a method for
`producing a dairy product comprising
`a) providing a milk-based substrate comprising lactose,
`b) treating said substrate with an enzyme having lactase
`activity, where the pH optimum of the lactase activity at 37°
`C. is above pH 5, and where the lactase activity of the
`enzyme at pH 5 is at least 50% of its lactase activity at pH
`6 when measured at 37° C.
`The skilled person will know how to determine the lactase
`activity at different pH and thereby determine the pH opti(cid:173)
`mum for the enzyme. The lactase activity at different pH
`may be determined by measuring hydrolysis of lactose at
`37° C. for 30 minutes, preferably in a buffer comprising
`40 succinate, HEPES, CHES, KC!, CaCl 2 and MgCl2 , e.g., by
`using a method as described in the Examples of the present
`application. For the avoidance of doubt, HEPES is a buff(cid:173)
`ering agent, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
`acid, and CHES is a buffering agent, N-Cyclohexyl-2-
`45 aminoethanesulfonic acid.
`The enzyme-treated milk-based substrate may optionally
`be mixed with other ingredients to obtain the dairy product.
`In one embodiment of the invention, the enzyme-treated
`milk-based substrate is mixed with other ingredients to
`50 obtain the dairy product.
`In one embodiment of the invention, the dairy product is
`milk. In another embodiment, the dairy product is condensed
`milk. In another embodiment, the dairy product is ice cream.
`In another embodiment, the da