EP3435771A1 - Composition enzymatique et préparation d'un produit laitier ayant des propriétés améliorées - Google Patents

Composition enzymatique et préparation d'un produit laitier ayant des propriétés améliorées

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Publication number
EP3435771A1
EP3435771A1 EP17714718.8A EP17714718A EP3435771A1 EP 3435771 A1 EP3435771 A1 EP 3435771A1 EP 17714718 A EP17714718 A EP 17714718A EP 3435771 A1 EP3435771 A1 EP 3435771A1
Authority
EP
European Patent Office
Prior art keywords
seq
activity
sequence
enzyme composition
lactase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP17714718.8A
Other languages
German (de)
English (en)
Inventor
Petrus Jacobus Theodorus Dekker
Cornelis Marinus Muijlwijk
Marten Aalt PAASMAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
DSM IP Assets BV
Original Assignee
DSM IP Assets BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by DSM IP Assets BV filed Critical DSM IP Assets BV
Publication of EP3435771A1 publication Critical patent/EP3435771A1/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G9/00—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
    • A23G9/32—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds
    • A23G9/36—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
    • A23C9/1206—Lactose hydrolysing enzymes, e.g. lactase, beta-galactosidase
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A23C9/137—Thickening substances
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A23C9/154—Milk preparations; Milk powder or milk powder preparations containing additives containing thickening substances, eggs or cereal preparations; Milk gels
    • A23C9/1544—Non-acidified gels, e.g. custards, creams, desserts, puddings, shakes or foams, containing eggs or thickening or gelling agents other than sugar; Milk products containing natural or microbial polysaccharides, e.g. cellulose or cellulose derivatives; Milk products containing nutrient fibres
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G9/00—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
    • A23G9/32—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds
    • A23G9/40—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds characterised by the dairy products used
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01108—Lactase (3.2.1.108)

Definitions

  • the present invention relates to a dairy product obtainable by a process in which a milk-based substrate comprising lactose is treated with an enzyme composition comprising lactase activity.
  • the invention also relates to the dairy product itself, to use of an enzyme composition comprising lactase activity in the production of a dairy product and to the enzyme composition itself.
  • Lactose intolerance is perhaps the best-known food sensitivity 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. Accordingly, 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 crystallizes 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 and for the production of condensed milk.
  • high temperatures are used. These high temperatures may lead to higher activity of contaminating side activities present in a lactase composition such that additional low temperature steps need to be incorporated into the process for making the dairy product.
  • the use of a lactase composition which has reduced protease activity means that such a composition can be used at higher temperatures without the protease destabilising processing the dairy product.
  • the invention relates to a dairy product obtainable by a process which comprises:
  • lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2, a truncated version either thereof or a sequence having at least about 60% sequence identity to any thereto, and
  • the enzyme composition has reduced protease activity.
  • the invention further relates to a dairy product obtainable by a process which comprises:
  • lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or a sequence having at least about 60% sequence identity to any thereto, and
  • the enzyme composition has reduced protease activity.
  • the invention also relates to a dairy product obtainable by a process which comprises:
  • lactase activity is provided by a polypeptide having lactase activity which is derived from Bifidobacterium bifidum, and
  • the invention also provides:
  • a method for the production of a dairy product which method comprises:
  • lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2, a truncated version either thereof or a sequence having at least about 60% sequence identity to any thereto, and
  • the enzyme composition has reduced protease activity
  • a method for the production of a dairy product which method comprises:
  • lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or a sequence having at least about 60% sequence identity to any thereto, and
  • the enzyme composition has reduced protease activity
  • a method for the production of a dairy product which method comprises:
  • lactase activity is provided by a polypeptide having lactase activity which is derived from Bifidobacterium bifidum, and
  • the enzyme composition has reduced protease activity
  • dairy product obtainable by a process which comprises:
  • the enzyme composition has reduced protease activity
  • dairy product obtainable by a process which comprises:
  • lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or a sequence having at least about 60% sequence identity to any thereto, and
  • the enzyme composition has reduced protease activity
  • dairy product obtainable by a process which comprises:
  • lactase activity is provided by a polypeptide having lactase activity which is derived from Bifidobacterium bifidum, and
  • the enzyme composition has reduced protease activity
  • an enzyme composition comprising lactase activity, wherein at least a part of the lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ I D NO: 1 or SEQ ID NO: 2, a truncated version either thereof or a sequence having at least about 60% sequence identity to any thereto, which has reduced protease activity;
  • an enzyme composition comprising lactase activity, wherein at least a part of the lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or a sequence having at least about 60% sequence identity to any thereto, which has reduced protease activity and
  • an enzyme composition comprising lactase activity, wherein at least a part of the lactase activity is provided by a polypeptide having lactase activity which is derived from Bifidobacterium bifidum which has reduced protease activity.
  • SEQ ID NOs: 1 and 2 set out the polypeptide sequences of lactase enzymes from Bifidobacterium bifidum.
  • SEQ ID NO: 1 may also be defined with reference to SEQ ID NO: 2 in WO01/90317.
  • SEQ ID NO: 2 may also be defined with reference to the Bbg3 sequence described in Goulas T.K., Goulas A.K., Tzortzis G., Gibson G.R., Appl. Microbiol. Biotechnol. 76(6), 1365 and 1372 (2007).
  • SEQ ID NO: 3 a C-terminal truncation variant, may also be defined with reference to SEQ ID NO: 2 as described in WO2009/071539.
  • SEQ ID NO: 4 another C-terminal truncation variant, may also be defined with reference to SEQ ID NO: 2 as described in WO2014/184189.
  • the treatment of the substrate may involve any process wherein a substrate is contacted with the enzyme preparation.
  • the treatment may involve any process wherein the substrate is incubated in the presence of the enzyme preparation.
  • the process described herein is a high temperature process.
  • a high temperature process herein may be a process which is carried out at at least about 45°C, for example at at least about 45°C or at a higher temperature such as at least 50°C, at least 55°C, at least 60°C or at least above 61 , 62, 63, 64 or 65°C.
  • the enzyme preparation may be added to the substrate in any suitable manner.
  • the process may be any process wherein a product is produced, for instance a nutritive product, preferably a dairy product.
  • Milk-based substrate in this 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 milk, low fat milk, skim milk, buttermilk, reconstituted milk from milk powder, condensed milk, solutions of dried milk, UHT milk or cream.
  • a dairy product encompasses any composition that contains milk protein, for instance casein and/or whey protein.
  • milk protein for instance casein and/or whey protein.
  • milk-derived products e.g. yoghurt, for example stirred, set or greek
  • condensed milk e.g. yoghurt, for example stirred, set or greek
  • condensed milk e.g. yoghurt, for example stirred, set or greek
  • condensed milk e.g. yoghurt, for example stirred, set or greek
  • condensed milk evaporated milk
  • dry milk frozen milk
  • ice cream ice cream
  • whey e.g. whey
  • cheese for example fresh or hard cheese.
  • the dairy product may also be a milk powder and/or a hydrolysate.
  • a dairy product may be plain or flavoured.
  • dairy products which are to be made using high temperature conditions, for example a starch containing product such as a custard. Yet another preferred dairy product is ice cream. Preferred dairy products are custard and ice cream.
  • the milk protein may be derived from a mammal or a plant sources or mixtures thereof.
  • the milk protein is from a mammal source.
  • Mammals sources of milk protein include, but are not limited to cow, sheep, goat, buffalo, camel, llama, mare and deer.
  • the milk protein is from a mammal selected from the group consisting of cow, sheep, goat, buffalo, camel, llama, mare and deer, and combinations thereof.
  • Plant sources of milk protein include, but are not limited to, milk extracted from soy bean, pea, peanut, barley, rice, oat, quinoa, almond, cashew, coconut, hazelnut, hemp, sesame seed and sunflower seed. Soy bean milk protein is preferred.
  • milk refers to not only whole milk, but also skim milk or any liquid component derived thereof.
  • the invention also relates to the use of the enzyme preparation according to the invention to improve processability, especially in higher temperature process and/or prevent or reduce the development of off-flavor and/or to improve texture.
  • an enzyme preparation has a reduced activity of a given enzyme if the activity of the given enzyme in the preparation has been reduced as compared with an identical preparation in which no steps are taken to reduce activity of the enzyme.
  • an enzyme preparation having lactase activity and having reduced activity of a given enzyme, such as protease activity may be one which is subject to a biochemical step intended to reduce protease activity (as compared to process in which such a step is not used).
  • An enzyme preparation with reduced protease activity may also thus encompass an enzyme preparation obtained by purifying a crude enzyme preparation which lactase activity, wherein at least some of the protease activity is separated from the lactase.
  • a host microorganism used to express a lactase may be modified so that the activity of one or more given enzymes, such as one or more proteases (expressed by the host), is reduced partly or completely, for example by modification or part or complete deletion of a nucleic acid sequence encoding a protease or by modification or part or complete deletion of a nucleic acid sequence encoding a polypeptide involved in the regulation of a protease. That is to say, a genetic approach may be used to produce an enzyme composition comprising lactase activity having reduced activity of a given enzyme, such as protease activity.
  • the invention provides a process in which a modified host is used to express a lactase having reduced protease activity and then that lactase composition is used as described herein.
  • the activity of a given enzyme, such as protease activity, in an enzyme composition comprising lactase activity may be reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more.
  • the percentage decrease represents the reduction as compared with an identical composition where no steps are taken to reduce protease activity.
  • one or more genetic modifications may be made to reduce protease activity by the desired amount (as compared to a non-modified host).
  • An enzyme composition of the invention may be substantially free from protease activity. This typically implies a reduction in activity of at least about 50%, but more typically of at least about 80% or higher, such as a reduction of at least about 95% or a greater reduction.
  • a purification step that has the effect that the activity of protease relative to the activity of lactase is reduced may be used.
  • the purifying results in a reduction of protease activity of at least 50%, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99%.
  • this is understood to mean that preferably
  • ur activity of lactase in purified enzyme preparation (unit/ml)
  • protease, crude protease activity in crude enzyme preparation (unit/ml)
  • aiactase.cmde activity of lactase in crude enzyme preparation (unit/ml)
  • the purification can be effected in any suitable manner.
  • the purifying is by chromatography.
  • Processes for purifying enzyme preparations using chromatography are known per se. Selecting the most appropriate chromatographic separation methods depend on molecular characteristics of both the relevant enzyme and of the relevant arylsulfatase activity present. Relevant molecular characteristics are the isoelectric point, hydrophobicity, molecular surface charge distribution, molecular weight of the relevant enzyme and the side activity as well as several other protein chemical properties. A practical background on the use of these characteristics in selecting the appropriate chromatographic separation process, can be found in a.o. the Protein Purification Handbook (issued by Amersham Pharmacia Biotech, nowadays GE Healthcare Bio-Sciences, Diegem, Belgium). Suitable chromatographic separation methods comprise ion exchange chromatography, affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography and others. For the present invention ion exchange chromatography or hydrophobic interaction chromatography are preferred.
  • An enzyme preparation of the invention may encompass an enzyme preparation wherein the ratio of the protease activity divided by the activity of the lactase is below a specified value. Preferred ratio's may vary depending on the enzyme and application used.
  • protease activity is meant the activity able to carry out proteolysis.
  • An enzyme composition of the invention having lactase activity and having reduced protease activity may also have one or more of reduced arylsulfatase activity, reduced invertase activity, reduced lipase activity and/or reduced amylase activity.
  • An enzyme composition of the invention may have reduced activity of all of these enzymatic activities.
  • An enzyme composition of the invention may be substantially free from all of these enzyme activities.
  • the enzyme composition is substantially free from protease activity and amylase activity.
  • arylsulfatase activity is meant the sulphuric ester hydrolase activity able to cleave a phenol sulfate into the phenol and sulfate moiety as described for EC 3.1 .6.1.
  • invertase activity is meant the activity that catalyzes the hydrolysis (breakdown) of sucrose as described in EC 3.2.1 .26.
  • lipase activity is meant the activity that catalyzes the hydrolysis of lipids.
  • amylase activity is meant the activity that catalyzes the hydrolysis of starch into sugars.
  • protease activity is meant the activity that catalyzes the hydrolysis of proteins.
  • At least a part of the lactase activity in the enzyme composition may be provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ I D NO: 1 or SEQ ID NO: 2, a truncated version either thereof or a sequence having at least about 60% sequence identity to any thereto.
  • Reference to a truncated version refers to a C-terminal truncated version.
  • At least a part of the lactase activity in the enzyme composition may be provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ I D NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or a sequence having at least about 60% sequence identity to any thereto.
  • lactase activity is provided by a polypeptide having lactase activity which is derived from Bifidobacterium bifidum.
  • the term “derived from” also includes the terms “originated from,” “obtained from,” “obtainable from,” “isolated from,” and “created from,” and generally indicates that one specified material find its origin in another specified material or has features that can be described with reference to the another specified material.
  • a substance e.g., a nucleic acid molecule or polypeptide "derived from" a microorganism preferably means that the substance is native to that microorganism or is a variant based on a substance native to the microorganism (for example a truncated version of a polypeptide derived from the microorganism and/or a version which contains one or more substitutions as compared to a polypeptide derived from the microorganism).
  • polypeptide set out in SEQ ID NO: 1 or SEQ ID NO: 2 of the invention is the full-length wild-type sequence of a lactase from Bifidobacterium bifidum.
  • a polypeptide having lactase activity suitable for use in the invention may have comprise an amino acid sequence having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about, 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.
  • SEQ ID NO: 1 and 2 both comprise a signal sequence which is positioned at amino acids 1 to 32 for both of them.
  • a polypeptide having lactase activity for use in the invention preferably comprises:
  • amino acid sequence having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about, 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to amino acids 33 to 1752 of SEQ ID NO: 1 .
  • amino acid sequence having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about, 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to amino acids 33 to 1935 of SEQ ID NO: 2.
  • a truncated version of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2, or more preferably a truncated version of amino acids 33 to 1752 of SEQ ID NO: 1 or a truncated version of amino acids 33 to 1935 of SEQ ID NO:2, may be used or a polypeptide comprising an amino acid sequence having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about, 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to such a truncated version of SEQ ID NO: 1 OR SEQ ID NO: 2 or more preferred to a truncated version of amino acids 33 to 1752 of SEQ ID NO:
  • polypeptide having lactase activity suitable for use in the invention is the Bbg3 polypeptide described in Goulas T.K., Goulas A.K., Tzortzis G., Gibson G.R., Appl. Microbiol. Biotechnol. 76(6), 1365 and 1372 (2007).
  • Suitable truncated versions of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2 include SEQ ID NO: 2 in WO2009/071539 (herein SEQ ID NO: 3) and SEQ ID NOs: 1 or 2 in WO2014/184189 (SEQ ID NO: 2 of WO2014/184189 is SEQ ID NO: 4 in this patent application).
  • the invention provides a dairy product or a method according to the invention in which SEQ ID NO: 3 or 4 is used or a polypeptide comprising an amino acid sequence having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about, 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.
  • SEQ ID NO: 3 and 4 both comprise a signal sequence which is positioned at amino acids 1 to 27 for both of them.
  • a polypeptide having lactase activity for use in the invention preferably comprises:
  • amino acid sequence having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about, 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to amino acids 28 to 1341 of SEQ I D NO: 3, more preferably amino acids 28-1331 of SEQ ID NO: 3 (as amino acids 1332-1341 comprise a purification tag which is preferably removed)
  • amino acid sequence having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about, 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to amino acids 28 to 1331 of SEQ ID NO: 4.
  • the invention concerns a dairy product obtainable by a process which comprises:
  • lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in SEQ ID NO: 1 , 2, 3 or 4 or a sequence having at least 60% sequence identity to SEQ ID NO: 1 , 2, 3 or 4 wherein the enzyme composition has reduced protease activity.
  • At least part of the lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in amino acids 33 to 1752 of SEQ I D NO: 1 , amino acids 33 to 1935 of SEQ ID NO: 2, amino acids 28 to 1331 of SEQ ID NO:3 or amino acids 28 to 1331 of SEQ ID NO: 4 or a sequence having at least 60% sequence identity to any thereto.
  • the invention also concerns a method for the production of a dairy product, which method comprises:
  • At least part of the lactase activity is provided by a polypeptide having an amino acid sequence which comprises the sequence set out in amino acids 33 to 1752 of SEQ ID NO: 1 , amino acids 33 to 1935 of SEQ I D NO: 2, amino acids 28 to 1331 of SEQ ID NO:3 or amino acids 28 to 1331 of SEQ ID NO: 4 or a sequence having at least 60% sequence identity to any thereto.
  • sequence homology or “sequence identity” or “homology” or “identity” are used interchangeably herein.
  • sequences are aligned for optimal comparison purposes.
  • gaps may be introduced in any of the two sequences that are compared.
  • alignment can be carried out over the full length of the sequences being compared.
  • the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/based or amino acids.
  • sequence identity is the percentage of identical matches between the two sequences over the reported aligned region.
  • a comparison of sequences and determination of percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm.
  • the skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the identity between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1 -44 Addison Wesley).
  • the percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol.
  • the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment.
  • the identity defined as herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as "longest-identity".
  • nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403—10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • Example 1 Preparation of a lactase composition comprising substantially no protease activity and its use in the preparation of an ice-cream
  • a lactase composition is prepared by expressing lactases in Bacillus subtilis.
  • Lad 6 is described in WO2009/071538 (SEQ ID NO: 2; herein SEQ ID NO: 3) and lad 9 (herein SEQ ID NO: 2) is described as Bbg3 in Goulas T.K., Goulas A.K., Tzortzis G., Gibson G.R., Appl. Microbiol. Biotechnol. 76(6), 1365 and 1372 (2007).
  • a lactase having SEQ ID NO: 3 or 4, or more preferably amino acids 28- 1331 of SEQ ID NO: 3 or 4, is used.
  • Standard genetic techniques such as overexpression of enzymes in the host cells, genetic modification of host cells, or hybridisation techniques, are known methods in the art, such as described in Sambrook and Russel (2001 ) "Molecular Cloning: A Laboratory Manual (3 rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987). Methods for transformation, genetic modification etc of fungal host cells are known from e.g.
  • Enzymes were isolated from the fermentation broth of Bacillus subtilis strains and purified using standard centrifugation and filtration techniques.
  • compositions are prepared so that they comprise substantially no protease activity. This can be carried out using a biochemical method to reduce the amount of protease in the composition. Alternatively, it can be carried out by modifying the host organism in which the lactase is prepared such that the amount of protease produced by the host organism is reduced.
  • compositions are then used in the manufacture of an ice-cream. This comprises the steps of: • Reception raw materials (liquid and dry)
  • the raw materials can be split into 2 groups liquid and dry raw materials.
  • the liquid raw materials are usually: condensed milk; cream; corn sugar; cane sugar and water.
  • the dry ingredients are usually flavors; fruits; nuts; stabilizers; milk powder; whey powder; emulsifiers; egg-powder; and sugar.
  • the dry ingredients are blended with the liquid ingredients prior to pasteurization of the total ice-cream mix together with the enzyme composition comprising lactase.
  • the dry ingredients are given enough time to completely hydrate in order to prevent lumps formation and ensure proper solubilization at the moment of pasteurization.
  • the lactase composition may be added with the wet ingredients.
  • the temperature of hydration of the dry ingredients is typically above 60°C and no hydrolysis of lactose-containing ingredients is carried out prior to blending with the other ice-cream mix ingredients.
  • the ice-cream is evaluated by a sensory panel for taste and texture

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Abstract

La présente invention concerne un produit laitier pouvant être obtenu par un procédé consistant à : a) fournir un substrat à base de lait comprenant du lactose ; et b) traiter ledit substrat avec une composition enzymatique comprenant une activité lactase, au moins une partie de l'activité lactase étant fournie par un polypeptide ayant une séquence d'acides aminés qui comprend la séquence définie dans SEQ ID NO: 1 ou SEQ ID NO: 2, une version tronquée de l'une ou de l'autre de celles-ci ou une séquence ayant au moins environ 60 % d'identité de séquence par rapport à l'une de celles-ci, et la composition enzymatique ayant une activité de protéase réduite.
EP17714718.8A 2016-03-31 2017-03-30 Composition enzymatique et préparation d'un produit laitier ayant des propriétés améliorées Pending EP3435771A1 (fr)

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WO2022268875A1 (fr) 2021-06-23 2022-12-29 Firmenich Sa Compositions modifiant l'arôme contenant de la lactase

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WO1990014423A1 (fr) 1989-05-18 1990-11-29 The Infergene Company Transformation de microorganismes
ATE196505T1 (de) 1989-07-07 2000-10-15 Unilever Nv Verfahren zur herstellung eines proteins mittels eines durch mehrfachkopie-integrierung eines expressionsvektors tranformierten pilzes
EP0635574B1 (fr) 1993-07-23 2003-04-23 Dsm N.V. Souches récombinantes dépourvues de marqueurs de sélection: procédé pour leur obtention et utilisation de ces souches
DK0979294T3 (en) 1997-04-11 2015-08-31 Dsm Ip Assets Bv Gene conversion AS A TOOL FOR CONSTRUCTION OF RECOMBINANT INDUSTRIAL filamentous fungi
US6265186B1 (en) 1997-04-11 2001-07-24 Dsm N.V. Yeast cells comprising at least two copies of a desired gene integrated into the chromosomal genome at more than one non-ribosomal RNA encoding domain, particularly with Kluyveromyces
DE69938129D1 (de) 1998-05-19 2008-03-27 Dsm Ip Assets Bv Verbesserte in vivo-produktion von cephalosporinen
JP2002533092A (ja) 1998-12-22 2002-10-08 デーエスエム・ナムローゼ・フェンノートシャップ セファロスポリンの改良されたｉｎｖｉｖｏ産生
KR100878968B1 (ko) 2000-05-26 2009-01-19 아를라 푸즈 에이엠비에이 비피도박테륨에서 단리한 베타-갈락토시다제
AU2013200779C1 (en) * 2005-11-28 2022-11-10 Dsm Ip Assets B.V. Enzyme preparations yielding a clean taste
ES2591359T3 (es) 2007-12-03 2016-11-28 Novozymes A/S Método para producir un producto lácteo de bajo contenido en lactosa
WO2009071538A2 (fr) 2007-12-04 2009-06-11 Shell Internationale Research Maatschappij B.V. Procédé et appareil permettant de refroidir et/ou de liquéfier un flux d'hydrocarbure
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JP6341911B2 (ja) * 2013-05-13 2018-06-13 合同酒精株式会社 ラクターゼ含有組成物の製造法
US9840723B2 (en) 2013-05-14 2017-12-12 Novozymes A/S Process for simultaneous saccharfication and fermentation of whey permeate

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