EP1478237A1 - Procede de production de fromage - Google Patents

Procede de production de fromage

Info

Publication number
EP1478237A1
EP1478237A1 EP03702377A EP03702377A EP1478237A1 EP 1478237 A1 EP1478237 A1 EP 1478237A1 EP 03702377 A EP03702377 A EP 03702377A EP 03702377 A EP03702377 A EP 03702377A EP 1478237 A1 EP1478237 A1 EP 1478237A1
Authority
EP
European Patent Office
Prior art keywords
cheese
phospholipase
lysophospholipase
milk
enzyme
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.)
Withdrawn
Application number
EP03702377A
Other languages
German (de)
English (en)
Inventor
Per Munk Nielsen
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.)
Novozymes AS
Original Assignee
Novozymes AS
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 Novozymes AS filed Critical Novozymes AS
Publication of EP1478237A1 publication Critical patent/EP1478237A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01004—Phospholipase A2 (3.1.1.4)
    • 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
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
    • A23C19/0328—Enzymes other than milk clotting enzymes, e.g. lipase, 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
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/04—Making cheese curd characterised by the use of specific enzymes of vegetable or animal origin
    • A23C19/043—Enzymes other than proteolytic enzymes or milk clotting enzymes, e.g. lipase, lysosyme
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01005—Lysophospholipase (3.1.1.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01032—Phospholipase A1 (3.1.1.32)

Definitions

  • the present invention relates to a process for producing cheese from enzyme-treated cheese milk, and the use of the resulting produced cheese as ingredient in food products.
  • the state of the fat phase is important to the properties of the cheese.
  • the fat phase is particularly important for the stabilisation of the cheese during production and ripening, but also for the final cheese to be used, eaten as such, or used in prepared ready-to-eat dishes e.g. pizza, toast or burgers.
  • Oiling-off properties of cheese products are important quality parameters. Oiling-off is the tendency to form free oil upon storage and melting. Excessive oiling-off is a defect most often related to heated products wherein cheese is used, e.g. pizza and related foods (cf. e.g. Kindstedt J.S; Rippe J.K. 1990, J Dairy Sci. 73: 867-873. It becomes more and more important to control/eliminate this defect, as the consumer concern about dietary fat levels increases. Free oil/fat in a product is perceived as a high fat content, and is generally undesirable.
  • WO 00/54601 discloses a method for improving the properties of cheese, particularly the stability of the fat phase, comprising the steps of a) treating the cheese milk with a phospholipase and b) producing cheese from the cheese milk.
  • the phospholipase is selected from the group consisting of phospholipase A ⁇ , phospholipase A 2 , phospholipase B and combinations thereof and added in an amount sufficient to decrease the oiling-off effect in cheese and/or to increase cheese yield.
  • the present invention relates to a process for producing cheese comprising adding to the cheese milk a combination of phospholipase A and a lysophospholipase; in an amount effective to decrease the oiling-off effect in cheese and/or to increase cheese yield, and producing cheese from the cheese milk.
  • the invention further relates to a process for producing cheese comprising adding to the cheese milk a phospholipase selected from the group consisting of phospholipase A t and phospholipase A 2 and combinations thereof; and a lysophospholipase; to decrease the oiling-off effect in cheese and/or to increase cheese yield; and producing cheese from the cheese milk.
  • a phospholipase selected from the group consisting of phospholipase A t and phospholipase A 2 and combinations thereof; and a lysophospholipase
  • the invention further relates to the combined use of phospholipase A and lysophospholipase in the manufacturing of cheese products, wherein the phospholipase A and lysophospholipase treatment is conducted on the cheese milk during the production of the cheese.
  • the invention also relates to cheeses obtainable, in particular obtained, by any of the processes described herein.
  • the invention also relates to a process for producing cheese comprising adding to the cheese milk a purified phospholipase selected from the group consisting of phospholipase A and phospholipase A 2 and combinations thereof; and a lysophospholipase; to decrease the oiling-off effect in cheese and/or to increase cheese yield; and producing cheese from the cheese milk.
  • a purified phospholipase selected from the group consisting of phospholipase A and phospholipase A 2 and combinations thereof; and a lysophospholipase
  • a process for producing cheese comprising adding to the cheese milk a combination of a phospholipase A and a lysophospholipase to decrease the oiling-off effect in cheese and/or to increase cheese yield, and producing cheese from the cheese milk.
  • the phospholipase A is preferably selected from the group consisting of phospholipase A ⁇ phospholipase A 2 and combinations thereof.
  • the phospholipase A and lysophospholipase are added to the cheese milk during cheese making process.
  • cheese milk is the milk-based composition from which the cheese is prepared.
  • the phospholipase A and lysophospholipase may be added at any appropriate stage during the cheese making process.
  • the phospholipase A and lysophospholipase are added during standardization of the fat and/or protein content of the cheese milk, more preferably, at the same time as the cheese milk is filled into the cheese vat, at the same time as the starter culture is added to the cheese milk, and at the same time as cheese rennet is added to the cheese milk.
  • the phospholipase A and the lysophospholipase may be added simultaneously or sequentially during the cheese making process.
  • the phospholipase A and lysophospholipase are added to the cheese milk at different stages in the process, such as, for example, the phospholipase A is added before the lysophospholipase or the lysophospholipase is added before the phospholipase A.
  • the phospholipase A and lysophospholipase are added simultaneously in the cheese making process.
  • the term "cheese” refers to any kind of cheese and such as, e.g., natural cheese, cheese analogues and processed cheese.
  • the cheese may be obtained by any suitable process known in the art, such as, e.g., by enzymatic coagulation of the cheese milk with rennet, or by acidic coagulation of the cheese milk with food grade acid or acid produced by lactic acid bacteria growth.
  • the cheese manufactured by the process of the invention is rennet-curd cheese. Rennet is commercially available, e.g. as Bachen ® (animal rennet), Chy-max ® (fermentation produced chymosin), Microlant ® (Microbial coagulant produced by fermentation), all from Chr. Hansen A/S, Denmark).
  • the cheese milk may be subjected to a conventional cheese-making process.
  • Processed cheese is preferably manufactured from natural cheese or cheese analogues by cooking and emulsifying the cheese, such as, with emulsifying salts (e.g. phosphates and citrate).
  • the process may further include the addition of spices/condiments.
  • the term "cheese analogues" refers to cheese-like products which contain fat (such as, e.g., milk fat (e.g., cream) as a part of the composition, and, in which further contain, as part of the composition, a non-milk constituents, such as, e.g., vegetable oil.
  • An example of a cheese analogue is cheese base.
  • the cheeses produced by the process of the present invention comprise all varieties of cheese, such as, e.g. Campesino, Chester, Danbo, Drabant, Herregard, Manchego, Provolone, Saint Paulin, Soft cheese, Svecia, Taleggio, White cheese, including rennet-curd cheese produced by rennet-coagulation of the cheese curd; ripened cheeses such as Cheddar, Colby, Edam, Muenster, Gryere, Emmenthal, Camembert, Parmesan and Romano; fresh cheeses such as Mozzarella and Feta; acid coagulated cheeses such as cream cheese, Neufchatel, Quarg, Cottage Cheese and Queso Blanco; and pasta filata cheese.
  • One embodiment relates to the production of pizza cheese by the process of the invention.
  • the coagulation of the casein in milk is preferably performed in one of two ways: the so-called rennet-curd and acid-curd cheese.
  • rennet-curd In cheese production these two types of curds makes up two major groups of cheese types.
  • Fresh acid-curd cheeses refer to those varieties of cheese produced by the coagulation of milk, cream or whey via acidification or a combination of acid and heat, and which are ready for consumption once the manufacturing without ripening are completed.
  • Fresh acid-curd cheeses generally differ from rennet-curd cheese varieties (e.g.
  • the cheese belongs to the class of rennet curd cheeses.
  • Mozzarella is a member of the so-called pasta filata, or stretched curd, cheeses which are normally distinguished by a unique plasticizing and kneading treatment of the fresh curd in hot water, which imparts the finished cheese its characteristic fibrous structure and melting and stretching properties, cf. e.g. "Mozzarella and Pizza cheese" by Paul S. Kindstedt,
  • Pizza cheese as used herein includes cheeses suitable for pizzas and they are usually pasta filata/stretched curd cheeses.
  • the process of the invention further comprises a heat/stretching treatment as for pasta filata cheeses, such as for the manufacturing of Mozzarella.
  • the cheese milk is prepared, totally or in part, from dried milk fractions, such as, e.g., whole milk powder, skim milk powder, casein, caseinate, total milk protein or buttermilk powder, or any combination thereof.
  • the cheese milk, to which phospholipase A and lysophospholipase are to be added comprises or consists of cream.
  • the cheese milk, to which phospholipase A and lysophospholipase are to be added comprises or consists of butter.
  • the cheese milk, to which phospholipase A and lysophospholipase are to be added comprises or consists of0 buttermilk.
  • milk from different species of animals may be used in the production of cheese.
  • milk may be the lacteal secretion obtained by milking, e.g., cows, sheep, goats, buffaloes or camels. 5
  • the milk for production of cheese may be standardised to the desired composition by removal of all or a portion of any of the raw milk components and/or by adding thereto additional amounts of such components. This may be done by separation of the raw milk into cream and skim milk at arrival to the dairy.
  • the cheese milk may be prepared as done0 conventionally by fractioning the raw milk and recombining the fractions so as to obtain the desired final composition of the cheese milk.
  • the separation may be made in continuous centrifuges leading to a skim milk fraction with very low fat content (i.e. e.g. ⁇ 0.5%) and cream with e.g. > 35% fat.
  • the cheese milk may be prepared by mixing cream and skim milk.
  • the cheese milk to which phospholipase A and lysophospholipase are to be added, comprises phospholipids, such as e.g. lecithin.
  • the cheese milk may have any total fat content which is found suitable for the cheese to be produced by the process of the invention, such as, e.g., about 25% fat (of dry matter), such as e.g. in the range 10-50% fat, of which, e.g., about 0.06% is phospholipids, such as e.g. 0.02-5% (w/w) of the total fat content is phospholipids.
  • the cheese milk is pasteurise the skim milk because heat denatured proteins in the cheese milk have a negative influence on the coagulation of the milk, and retard the ripening of the cheese.
  • the bacterial count of the skim milk fraction may thus be lowered by other technologies, such as, for example, by microfiltration or bactofugation.
  • the cream is preferably pasteurised to lower the bacterial count in the product.
  • the cheese milk is raw, unpasteurised milk.
  • the cheese milk may be subjected to a homogenization process before the production of cheese, such as e.g. in the production of Danish Blue Cheese.
  • the enzymatic treatment in the process of the invention may be conducted by dispersing the phospholipase A and lysophospholipase into cheese milk, and allowing the enzyme reaction to take place at an appropriate holding-time at an appropriate temperature.
  • the treatment with phospholipase A and lysophospholipase may be carried out at conditions chosen to suit the selected enzymes according to principles well known in the art.
  • the enzymatic treatment may be conducted at any suitable pH, such as e.g., in the range 2- 10, such as, at a pH of 4-9 or 5-7. It may be preferred to let the phospholipase A and lysophospholipase act at the natural pH of the cheese milk as it develops during the cheese making process.
  • the process may be conducted so that the phospholipase A and lysophospholipase are allowed to react at coagulation temperature, such as, 25-45°C (e.g., for at least 5 minutes, such as, e.g., for at least 10 minutes or at least 30 minutes, e.g., for 5-60 minutes).
  • coagulation temperature such as, 25-45°C
  • the phospholipase A and the lysophospholipase have been allowed to act on the cheese milk
  • the phospholipase A and/or lysophospholipase enzyme protein is removed, reduced, inactivated or any combination thereof.
  • the phospholipase A and the lysophospholipase are added in suitable amounts to produce the cheese having the desired properties.
  • the phospholipase A and lysophospholipase are added in amounts effective to decrease the oiling-off effect in cheese and/or to increase cheese yield.
  • a suitable dosage of phospholipase A will usually be in the range 0.003-0.3 mg enzyme protein per g milk fat, preferably 0.01-0.3 mg enzyme protein per g milk fat, more preferably, 0.03 mg enzyme protein per g milk fat.
  • Dosage of lysophospholipase will usually be in the range 0.005-0.5 mg enzyme protein per g milk fat, preferably 0.01-0.5 mg enzyme protein per g milk fat, more preferably 0.05 mg enzyme protein per g milk fat.
  • Enzymes to be used in the process of the invention are enzymes to be used in the process of the invention:
  • Phospholipids such as lecithin or phosphatidylcholine, consist of glycerol esterified with two fatty acids in an outer (sn-1) and the middle (sn-2) positions and esterified with phosphoric acid in the third position; the phosphoric acid, in turn, may be esterified to an amino-alcohol.
  • Phospholipases are enzymes which participate in the hydrolysis of phospholipids. Several types of phospholipase activity can be distinguished, including phospholipases Ai and A 2 which hydrolyze one fatty acyl group (in the sn-1 and sn-2 position, respectively) to form lysophospholipid. Lysophospholipase hydrolyzes the remaining fatty acyl group in lysophospholipid.
  • the enzymes used in the process of the present invention comprise a phospholipase A and a lysophospholipase.
  • phospholipase A includes phospholipase Ai, phospholipase A 2 and the combination of phospholipase A ⁇ and phospholipase A 2 , such as e.g. the combination of an enzyme with phospholipase A-i activity and an enzyme with phospholipase A 2 activity, or a single enzyme with both phospholipase Ai and phospholipase A 2 activity.
  • Phospholipase Ai is defined according to standard enzyme EC-classification as EC 3.1.1.32. Official Name: Phospholipase A ⁇ . Reaction catalyzed: phosphatidylcholine + H(2)O ⁇ > 2-acylglycerophosphocholine + a fatty acid anion
  • Phospholipase A? is defined according to standard enzyme EC-classification as EC 3.1.1.4
  • phosphatidylcholine 2-acylhydrolase lecithinase a; phosphatidase; or phosphatidolipase.
  • Lysophospholipase is defined according to standard enzyme EC-classification as EC 3.1.1.5.
  • the phospholipase A activity may be provided by enzymes having other activities as well, such as e.g. a lipase with phospholipase A activity.
  • the phospholipase A activity may e.g. be from a lipase with phospholipase A side activity.
  • the phospholipase A enzyme activity is provided by an enzyme having essentially only phospholipase A activity and wherein the phospholipase A enzyme activity is not a side activity.
  • the phospholipase A may be of any origin, e.g. of animal origin (such as, e.g. mammalian), e.g. from pancreas (e.g. bovine or porcine pancreas), or snake venom or bee venom.
  • animal origin such as, e.g. mammalian
  • pancreas e.g. bovine or porcine pancreas
  • snake venom or bee venom e.
  • the phospholipase A may be of microbial origin, e.g. from filamentous fungi, yeast or bacteria, such as the genus or species Aspergillus, e.g. A. niger, Dictyostelium, e.g.
  • D. discoideum D. discoideum; Mucor, e.g. M. javanicus, M. mucedo, M. subtilissimus; Neurospora, e.g. N. crassa; Rhizomucor, e.g. R. pusillus; Rhizopus, e.g. R. arrhizus, R. japonicus, R. stolonifer, Sclerotinia, e.g. S. Iibertiana; Trichophyton, e.g. T. rubrum; Whetzelinia, e.g. W. sclerotiorum; Bacillus, e.g. ⁇ . megaterium, B.
  • subtilis Citrobacter, e.g. C. freundii; Enterobacter, e.g. E aerogenes, E. cloacae Edwardsiella, E. tarda; Erwinia, e.g. E. herbicola; Escherichia, e.g. E. coli; Klebsiella, e.g. K. pneumoniae; Proteus, e.g. P. vulgaris; Providencia, e.g. P. stuartii; Salmonella, e.g. S. typhimurium; Serratia, e.g. S. liquefasciens, S. marcescens; Shigella, e.g. S.
  • the phospholipase A may be fungal, e.g. from the class Pyrenomycetes, such as the genus Fusarium, such as a strain of F. culmorum, F. heterosporum, F. solani, or a strain of F. oxysporum.
  • the phospholipase A may also be from a filamentous fungus strain within the genus Aspergillus, such as a strain of Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus niger or Aspergillus oryzae.
  • a preferred phospholipase A is derived from a strain of Fusarium, particularly F. oxysporum, e.g. from strain DSM 2672 as described in WO 98/26057, especially described in claim 36 and SEQ ID NO. 2 of WO 98/26057.
  • the phospholipase A is a phospholipase as disclosed in WO 00/32758 (Novozymes A/S, Denmark).
  • lysophospholipase used herein in connection with an enzyme of the invention is intended to cover an enzyme with lysophospholipase activity.
  • the lysophospholipase activity may be provided by enzymes having other activities as well, such as e.g. a lipase with lysophospholipase activity.
  • the lysophospholipase activity may e.g. be from a lipase with lysophospholipase side activity.
  • the lysophospholipase enzyme activity is provided by an enzyme having essentially only lysophospholipase activity and wherein the lysophospholipase enzyme activity is not a side activity.
  • the lysophospholipase is not lipases having lysophospholipase side activity as defined in WO 98/26057.
  • the lysophospholipase may be of any origin, e.g. of animal origin (such as, e.g. mammalian), e.g. from liver (e.g. rat liver).
  • the lysophospholipase may be of microbial origin, e.g. from filamentous fungi, yeasts or bacteria, such as the genus or species Aspergillus, e.g. A. foetidus, A. fumigatus, A. nidulans, A. niger, A. oryzae; Botrytis, e.g. S. cinerea; Candida, e.g. C. albicans; Cryptococcus, e.g. C.
  • neoformans Escherichia, e.g. E. coli, Fusarium, e.g. F. sporotrichioides, F. venenatum, F. verticillioides; Hyphozyma; Kluyveromyces, e.g. K. lactis; Magnaporte, e.g. M. grisea; Metarhizium, e.g. M. anisopliae; Mycosphaerella, e.g. M. graminicola; Neurospora, e.g. N. crassa; Penicillium, e.g. P. notatum; Saccharomyces, e.g. S.
  • a preferred lysophospholipase is derived from a strain of Aspergillus, particularly lysophospholipase LLPL-1 or LLPL-2 from A. niger, e.g. as contained in the Escherichia coli clones DSM 13003 or DSM 13004, or lysophospholipase LLPL-1 or LLPL-2 from A. oryzae, e.g. as contained in the E. coli clones DSM 13082 or DSM 13083 as described in WO 01/27251 , especially described in claim 1 and SEQ ID NOs. 2, 4, 6 or 8 of WO 01/27251.
  • Enzymes with both phospholipase A and lysophospholipase activity are provided by a single enzyme having both phospholipase A activity and lysophospholipase activity.
  • a single enzyme has both phospholipase Ai activity and lysophospholipase activity
  • a single enzyme has both phospholipase A 2 activity and lysophospholipase activity
  • a single enzyme has both phospholipase A T activity, phospholipase A 2 activity, and lysophospholipase activity.
  • the invention thus also relates to a process for producing cheese comprising adding to the cheese milk an enzyme with both phospholipase A activity and lysophospholipase activity to decrease the oiling-off effect in cheese and/or to increase cheese yield; and producing cheese from the cheese milk.
  • Enzymes with both phospholipase A and lysophospholipase activity are known to the person skilled in the art. Enzymes with both phospholipase A and lysophospholipase activity are e.g. described by Saito et al., Methods in Enzymology (1991) 197, 446-456, and by Lee et al., J. Biol. Chem. (1994) 269, 19725-19730.
  • the phospholipase A and/or lysophospholipase used in the process of the invention may be derived or obtainable from any of the sources mentioned herein.
  • the term "derived” means in this context that the enzyme may have been isolated from an organism where it is present natively, i.e. the identity of the amino acid sequence of the enzyme are identical to a native enzyme.
  • the term "derived” also means that the enzymes may have been produced recombinantly in a host organism, the recombinant produced enzyme having either an identity identical to a native enzyme or having a modified amino acid sequence, e.g. having one or more amino acids which are deleted, inserted and/or substituted, i.e.
  • a recombinantly produced enzyme which is a mutant and/or a fragment of a native amino acid sequence.
  • a native enzyme are included natural variants.
  • the term “derived” includes enzymes produced synthetically by e.g. peptide synthesis.
  • the term “derived” also encompasses enzymes which have been modified e.g. by glycosylation, phosphorylation etc., whether in vivo or in vitro.
  • the term “obtainable” in this context means that the enzyme has an amino acid sequence identical to a native enzyme.
  • the term encompasses an enzyme that has been isolated from an organism where it is present natively, or one in which it has been expressed recombinantly in the same type of organism or another, or enzymes produced synthetically by e.g. peptide synthesis.
  • the terms “obtainable” and “derived” refer to the identity of the enzyme and not the identity of the host organism in which it is produced recombinantly.
  • the phospholipase A and/or lysophospholipase may be obtained from a microorganism by use of any suitable technique.
  • a phospholipase A and/or lysophospholipase enzyme preparation may be obtained by fermentation of a suitable microorganism and subsequent isolation of a phospholipase A and/or lysophospholipase preparation from the resulting fermented broth or microorganism by methods known in the art.
  • the phospholipase A and/or lysophospholipase may also be obtained by use of recombinant DNA techniques.
  • Such method normally comprises cultivation of a host cell transformed with a recombinant DNA vector comprising a DNA sequence encoding the phospholipase A or lysophospholipase in question and the DNA sequence being operationally linked with an appropriate expression signal so that it is capable of expressing the phospholipase A or lysophospholipase in a culture medium under conditions permitting the expression of the enzyme and recovering the enzyme from the culture.
  • the DNA sequence may also be incorporated into the genome of the host cell.
  • the DNA sequence may be of genomic, cDNA or synthetic origin or any combinations of these, and may be isolated or synthesized in accordance with methods known in the art.
  • Suitable phospholipases are available commercially. As typical examples of the enzymes for practical use, pancreas-derived phospholipase A 2 such as Lecitase® (manufactured by Novozymes A/S) is preferably used.
  • a suitable lysophospholipase is e.g. Aspergillus niger lysophospholipase LLPL-2 that can be produced recombinantly in A. niger as described in WO 01/27251. In the process of the invention the phospholipase A and/or lysophospholipase may be purified.
  • purified covers phospholipase A or lysophospholipase enzyme protein free from components from the organism from which it is derived.
  • purified also covers phospholipase A or lysophospholipase enzyme protein free from components from the native organism from which it is obtained, this is also termed “essentially pure” phospholipase A or lysophospholipase and may be particularly relevant for phospholipases and lysophospholipases which are naturally occurring and which have not been modified genetically, such as by deletion, substitution or insertion of one or more amino acid residues.
  • the phospholipase A and/or lysophospholipase may be purified, viz. only minor amounts of other proteins being present.
  • the expression "other proteins” relate in particular to other enzymes.
  • the term “purified” as used herein also refers to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the phospholipase A or lysophospholipase.
  • the phospholipase A or lysophospholipase may be "substantially pure", i.e.
  • the enzymes are at least 75% (w/w) pure, more preferably at least 80%, 85%, 90% or even at least 95% pure.
  • the phospholipase A or lysophospholipase is an at least 98% pure enzyme protein preparation.
  • the phospholipase A and/or lysophospholipase is not naturally present in milk.
  • phospholipase and "lysophospholipase” include whatever auxiliary compounds that may be necessary for the catalytic activity of the enzyme, such as, e.g. an appropriate acceptor or cofactor, which may or may not be naturally present in the reaction system.
  • the phospholipase A and lysophospholipase may be in any form suited for the use in question, such as e.g. in the form of a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a protected enzyme.
  • Granulates may be produced, e.g. as disclosed in US 4,106,991 and US 4,661,452, and may optionally be coated by methods known in the art.
  • Liquid enzyme preparations may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, lactic acid or another organic acid according to established methods.
  • Protected enzymes may be prepared according to the method disclosed in EP 238,216.
  • the lecithin content of the cheese may be reduced by at least 5%, such as at least 10%, at least 20%, at least 30%, at least 50%, such as in the range of 5-95% compared to a similar cheese making process but without the enzymatic treatment of a phospholipase A and a lysophospholipase, as described herein.
  • the lecithin constitutes normally more than 95% of the phospholipids in milk whereas the lysolecithin is approximately 1 % of the phospholipids.
  • the phospholipids represent normally less than 1% of the total lipids in cow milk, they play a particularly important role, being present mainly in the milk fat globule membrane.
  • the lecithin content in the obtainable cheese may be less than 90%, such as e.g. less than 80%, e.g. less than 60% or less than 50% of the total content of phospholipid in the cheese.
  • the lecithin content may be measured by any method known by the skilled person, e.g. by HPLC.
  • the present invention further relates to use of the cheese produced by the process, of the invention in pizza, ready-to-eat dishes, processed cheese or as an ingredient in other food products. Accordingly, the cheese produced according to the process of the invention may be used in further processed food products like processed cheese, pizza, burgers, toast, sauces, dressings, cheese powder or cheese flavours.
  • the process of the invention further comprises the step of subjecting the cheese to a heating treatment, such as, e.g., in the range 150-350°C.
  • the invention also relates to a cheese obtainable, in particular obtained, by the process of the invention.
  • Pasteurized, non-homogenized cream (North Carolina State University Dairy Plant) was used to standardize pasteurized, non-homogenized skim milk (North Carolina State University Dairy Plant) to 3.5% fat for production of full fat Mozzarella cheese.
  • Starter culture was prepared by adding 0.1 g Rhodia LH100 and 0.3 g Rhodia TA061 starter cultures to 50 ml of the skim milk and equilibrating to 35°C with gentle, continuous stirring. Standardised cheese milk was equilibrated to 35°C and divided into 3 batches. 1.4 %, starter culture was added to each batch, and at the same time the enzyme preparations A, B and C above were added to the respective batches under gentle stirring.
  • the temperature was increased to 41 °C under gentle and intermittently stirring and kept until pH reached 5.65 - 5.70. Then the whey was drained off and curd particles poured into stainless steel bowls. The bowls were floated in 41 °C water bath to maintain curd temperature and excess whey was drained off periodically, leaving only enough to cover curds for maintenance of heat.
  • curd pH reached 5.25 - 5.3
  • all whey was drained off and the curd flooded with deionised water at 57°C for 5 min.
  • the curd was stretched by hand for 1 min and returned to
  • Oiling-Off Total Area - Area of Cheese x 100
  • Cream was standardized to a fat content of 25% using skim milk. Milk samples were treated with phospholipase A (Lecitase ® 10 L manufactured by Novozymes A/S, Denmark), Lysophospholipase (Aspergillus niger lysophospholipase LLPL2 expressed recombinantly in A. niger as described in WO 01/27251) and combinations of phospholipase A and lysophospholipase as indicated in table 3. The enzymes were pre-diluted in water, if
  • Fatty Acids were eluted with 4 ml of diethylether acidified with glacial acetic acid (2% v/v). Ether extracts were dried down in a rotary evaporator and reconstituted in 1.0 ml of methanol for HPLC analysis.
  • HPLC Method Reverse phase chromatography was conducted using a Luna C8 (150 x 4.6 30 mm, 5 ⁇ , 100 A) column from Phenomenex (Torrance, CA USA), and a mobile phase of acetonitrile and water containing 0.1 % trifluoroacetic acid. See Table 2 for the gradient elution scheme. Detection of eluted Fatty Acids was done by evaporative light scattering. HPLC Fatty Acid standards (linoleic, palmitic, oleic, and stearic acids) were chosen based on their prevalence in total lipids of bovine milk. Stock solutions were routinely prepared in 35 100% methanol in the concentration range of 2-10 mg/ml. HPLC calibrators were prepared from Fatty Acid stock solutions by dilution to the appropriate concentration in 100% methanol.
  • the amount of generated free long chain fatty acids after treatment of the cream samples with phospholipase A and/or lysophospholipase is shown in table 3. It can be seen that increasing the amount of phospholipase A from 0.04 mg/(g fat) to 0.18 mg/(g fat) does not increase the amount of generated free fatty acids considerably. When lysophospholipase is added in combination with the phospholipase A, however, the amount of generated fatty acid is considerably increased.

Abstract

L'invention concerne un procédé de production de fromage à partir de lait de fromagerie traité aux enzymes. L'invention concerne également l'utilisation du fromage ainsi produit comme ingrédient de produits alimentaires. L'invention concerne plus particulièrement un procédé de production de fromage à partir de lait de fromagerie traité à la phospholipase A et à la lysophospholipase.
EP03702377A 2002-02-20 2003-02-20 Procede de production de fromage Withdrawn EP1478237A1 (fr)

Applications Claiming Priority (3)

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DK200200262 2002-02-20
DKPA200200262 2002-02-20
PCT/DK2003/000113 WO2003070013A1 (fr) 2002-02-20 2003-02-20 Procede de production de fromage

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AU2003205559A1 (en) 2003-09-09
BR0307350A (pt) 2004-12-14

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