JP2005517418A - Cheese manufacturing method - Google Patents

Abstract

The present invention relates to a process for producing cheese from enzyme-treated cheese milk and the resulting use of the resulting cheese as a component of a food product. More particularly, the present invention relates to a process for producing cheese from cheese milk treated with phospholipase A and lysophospholipase.

Description

  The present invention relates to method B for the production of cheese from enzyme-treated cheese milk and the use of the resulting resulting cheese as a component of a food product.

  In cheese products, the state of the fat phase is important for the nature of the cheese. The fat phase is particularly important for the stabilization of cheese during manufacture and ripening, but it can also be used, eaten as it is, or of the final cheese for use in instant foods such as pizza, toast, or burgers. Also important for.

  In addition, the oiling-off properties of cheese products are an important property parameter. Oiling off is the tendency to produce free oil upon storage and melting. Excess oiling-off is the defect most often associated with heated products where cheese is used, such as pizza and related foods (see, eg, Kindstedt JS; Rippe JK 1990, J. Dairy Sci. 73: 867-873) ). As consumers' interest in dietary fat levels increases, it becomes increasingly important to control / remove this deficiency. Free oil / fat in the product is recognized as having a high fat content and is generally undesirable.

There is a need for improved methods for cheese manufacture, particularly processes for improving the stability of fat in cheese.
WO 00/54601 describes a method for improving the properties of cheese, in particular the stability of the fatty phase, comprising the steps of a) treating cheese milk with phospholipase and (b) producing cheese from said cheese milk. Disclosure. The phospholipase is selected from the group consisting of phospholipase A ₁ , phospholipase A ₂ , phospholipase B, and combinations thereof, added in an amount sufficient to reduce the oiling-off effect and / or increase the cheese yield in cheese Is done.

The present invention adds a combination of phospholipase A and lysophospholipase to cheese milk in an effective amount to reduce the oiling-off effect in cheese and / or increase cheese yield, and produce cheese from the cheese milk The present invention relates to a method for producing cheese comprising.
The present invention further comprises a phospholipase selected from the group consisting of phospholipase A ₁ and phospholipase A ₂ and combinations thereof, and lysophospholipase to reduce the oiling-off effect in cheese and / or increase cheese yield. Relates to a method for producing cheese, comprising adding to cheese milk and producing cheese from said cheese milk.

  Here is provided a process for improving the properties of cheese, in particular the fat stability of cheese and cheese milk is improved by the present invention. Without being limited to any theory of operation, it is believed that the addition of phospholipase A and lysophospholipase to cheese milk during cheese manufacture promotes stability during heat treatment of cheese made from said processed cheese milk. It has been. The process of the present invention also provides a method for increasing the yield in cheese manufacture.

  The invention further relates to the combined use of phospholipase A and lysophospholipase in the manufacture of cheese products, wherein the phospholipase A and lysophospholipase treatment is carried out on cheese milk during the cheese manufacture. The present invention further relates to cheeses that can be obtained, in particular cheeses obtained by any of the processes described in the present invention.

The present invention also provides a purified phospholipase selected from the group consisting of phospholipase A ₁ and phospholipase A ₂ and combinations thereof, and lysophospholipase to reduce the oiling-off effect in cheese and / or increase cheese yield To a cheese milk, and to making cheese from the cheese milk.

In order to reduce the oiling-off effect in cheese and / or increase cheese yield, a cheese comprising adding a combination of phospholipase A and lysophospholipase to cheese milk and producing cheese from said cheese milk Method for manufacturing. Phospholipase A is preferably one selected from phospholipase A _1, phospholipase A _2, as well as the group consisting of these combinations products.
Phospholipase A and lysophospholipase are added to cheese milk during the cheese making process. As used herein, the term “cheese milk” means a milk-based composition from which cheese is prepared.

  Phospholipase A and lysophospholipase will be added at any suitable stage during the cheese making process. In a preferred embodiment of the present invention, phospholipase A and lysophospholipase are used during normalization of the fat and / or protein content of cheese milk, more preferably the cheese milk fills the cheese vat while the starter culture is cheese. As soon as it is added to the milk and cheese rennet is added to the cheese milk.

  Phospholipase A and lysophospholipase will be added simultaneously or sequentially during the cheese making process. In one embodiment, phospholipase A and lysophospholipase will be added to cheese milk at different stages in the process. For example, phospholipase A is added before lysophospholipase, or lysophospholipase is added before phospholipase A. In a preferred embodiment, phospholipase A and lysophospholipase are added simultaneously during the cheese making process.

Cheese milk and cheese production :
In the context of the present invention, the term “cheese” relates to any kind of cheese, and for example natural cheese, cheese analogues and processed cheese. Cheese is obtained by any suitable process known in the industry, for example by enzymatic flocculation of cheese milk using rennet or by acidic flocculation of cheese milk using food grade acids or acids produced by lactic acid bacteria growth Will. In one embodiment, the cheese produced by the process of the invention is rennet curd cheese. Rennet is commercially available. Examples include Naturen ™ (animal rennet), Chymax ™ (fermented chymosin), and Microlant ™ (fermented microbial flocculant), all available from the Danish company Chr. Hansen A / S It is. Cheese milk will be subjected to normal cheese making methods.

  Process cheese is preferably made from cheese analogs prepared from natural cheese or cheese and emulsified using, for example, emulsified salts (eg, phosphate and citrate). The process will further include the addition of spices / condiments. The term “cheese analogue” relates to a cheese-like product comprising a fat (eg milk fat (eg cream) as part of the composition, and further comprising a non-milk component, eg vegetable oil, as part of the composition. One example of a cheese analog is a cheese base.

  The cheeses produced by the method of the present invention are all kinds of cheeses, for example, Campesino, Chester, Danbo, Drabant, Herregard, Manchego, Probolone. (Provolone), Saint Paulin, soft cheese, Svecia, Taleggio, white cheese containing rennet curd cheese made by rennet agglomeration of cheese curd; aged cheese such as Cheddar, Colby, Edam, Muenster, Gryere, Emmenthal, Camembert, Parmesan and Romano; fresh cheeses such as Mozzarella and Feta; acids Agglomerated cheese such as cream cheese Neuchâtel (Neufchatel), quark (Quarg), cottage (Cottage) cheese and Queso Blanco (Queso Blanco); as well as pasta Firata (pasta filata) cheeses. One embodiment relates to the production of pizza cheese by the process of the present invention.

  In cheese production, agglomeration of casein in milk is preferably carried out by one of two methods, so-called rennet curd and acid curd cheese. These two curds in cheese making make up the two main groups of cheese molds. Fresh acid curd cheese relates to a variety of cheeses made by acidification or agglomeration of milk, cream or whey via a combination of acid and heat, and these cheeses once finished without aging Can be consumed immediately. Fresh acid curd cheese is generally different from rennet curd cheese species (eg Camembert, Cheddar, Emmenthal), where agglomeration is usually induced by the action of rennet at pH 6.4-6.6. That is, agglomeration usually occurs near the isoelectric point of casein, ie, at a higher pH when, for example, pH 4.6 or higher, eg, pH 6.0 and 80 ° C. for Ricotta. In a preferred embodiment of the invention, the cheese belongs to the type of rennet curd cheese.

  Mozzarella is a member of the so-called pasta filata, or stretched curd cheese. These cheeses are usually characterized by the characteristic plasticization and kneading process of fresh curd in hot water, giving the product cheese its characteristic fibrous structure and melting and stretching properties (eg “Mozzarella and Pizza cheese ”, Paul S. Kindstedt, Cheese: See Chemistry, physics and microbiology, Volume 2: Major Cheese groups, second edition, pages 337-341, Chapman & Hall). As used herein, pizza cheese includes cheese suitable for pizza and is usually pasta filata / stretched curd cheese. In one embodiment, the process of the present invention further includes a heat / stretching process when it comes to the manufacture of pasta filata cheese, such as Mozzarella.

In further embodiments of the invention, the cheese milk is, in whole or in part, a dry milk fraction, such as, for example, whole milk powder, skim milk powder, casein, casein salt, whole milk protein or buttermilk powder, or any of these Prepared from these combinations.
In a preferred embodiment, the cheese milk to which phospholipase A and lysophospholipase are to be added comprises or consists of cream. In a further embodiment, the cheese milk to which phospholipase A and lysophospholipase are to be added comprises or consists of butter. In a still further embodiment, the cheese milk to which phospholipase A and lysophospholipase are to be added comprises or consists of buttermilk.

  Milk from different animal species will be used in the manufacture of cheese. Thus, “milk” may be a milk secretion obtained, for example, by milking cows, sheep, goats, buffalos or salmon.

  Milk for cheese making will be normalized to the desired composition by removing all or part of the raw milk composition and / or adding additional amounts of such composition. This would be done by separating the raw milk into cream and skim milk when the raw milk arrives at the dairy factory. Thus, cheese milk will be prepared as is normally done by fractionating raw milk and recombining the fractions to obtain the desired final composition. Separation would be done by continuous centrifugation at a skim milk fraction with a very low fat content (ie, for example, less than 0.5%) and a cream at, for example, greater than 35% fat. Cheese milk will be prepared by mixing cream and skim milk.

  The cheese milk to which phospholipase A and lysophospholipase are to be added contains a phospholipid such as lysitin. The cheese milk will have any total fat content that has been found suitable for the cheese to be produced by the process of the present invention. For example about 25% fat (dry matter), for example in the range of 10-50% fat, for example about 0.06% is phospholipid, for example 0.02-5% of total fat content ( w / w) is a phospholipid.

Usual measures will be taken to keep the bacterial count in cheese milk low. Since heat-denatured proteins in cheese milk adversely affect milk aggregation and delay cheese ripening, sterilization of skim milk is usually not preferred. The bacterial count of the skim milk fraction will therefore be lowered by other techniques such as microfiltration and centrifugal sterilization. The cream will preferably be sterilized to reduce the number of bacteria in the product. In other preferred embodiments, the cheese milk is raw, non-sterilized milk.
In an embodiment of the invention, the cheese milk will be subjected to a homogenization process prior to cheese manufacture, for example during the manufacture of Danish Blue cheese.

Enzymatic treatment The enzymatic treatment in the method of the present invention will be carried out by dispersing phospholipase A and lysophospholipase in cheese milk so that the enzymatic reaction takes place at an appropriate temperature and at an appropriate holding time. Treatment with phospholipase A and lysophospholipase will be carried out under conditions selected to suit the enzyme selected according to principles well known in the art.
The enzyme treatment will be carried out at any suitable pH, for example within a range of 2-10, such as a pH of 4-9 or 5-7. It would be preferable to work phospholipase A and lysophospholipase at the natural pH of the cheese milk that occurs during the cheese making process.

The method will be performed such that phospholipase A and lysophospholipase act at an aggregation temperature, eg 25-45 ° C. (eg at least 5 minutes, eg at least 10 minutes or at least 30 minutes, eg 5-60 minutes). .
Optionally, phospholipase A and lysophospholipase are allowed to act on cheese milk followed by removal, reduction, inactivation, or any combination thereof of phospholipase A and / or lysophospholipase enzyme protein.

Phospholipase A and lysophospholipase are added in appropriate amounts to produce cheese with desirable properties. Preferably, phospholipase A and lysophospholipase are added in an effective amount to reduce the oiling-off effect in cheese and / or increase cheese yield. The appropriate amount of phospholipase A is usually in the range of 0.003 to 0.3 mg of enzyme protein per gram of milk fat, preferably 0.01 to 0.3 mg of enzyme protein per gram of milk fat, more preferably 0.03 mg of enzyme protein per gram of milk fat. .
The dosage of lysophospholipase is usually in the range of 0.005-0.5 mg enzyme protein per gram milk fat, preferably 0.01-0.5 mg enzyme protein per gram milk fat, more preferably 0.05 mg enzyme protein per gram milk fat. .

Enzyme phospholipids such as lecithin or phosphatidylcholine used in the method of the present invention are esterified with two fatty acids at the outer position (sn-1) and the middle position (sn-2) and esterified with phosphoric acid at the third position The phosphoric acid will then be esterified to an amino alcohol. Phospholipases are enzymes involved in phospholipid hydrolysis. One aliphatic acyl group to form a lysophospholipid the (s husband, in sn-1 and sn-2 position) comprises hydrolyzing phospholipase A ₁ and A _2, several kinds of phospholipase activity can be distinguished. Lysophospholipase hydrolyzes the aliphatic acyl group remaining in the lysophospholipid.

Enzymes used in the methods of the present invention include phospholipase A and lysophospholipase. The term phospholipase A is phospholipase A ₁ , phospholipase A ₂ , and a combination of phospholipase A ₁ and phospholipase A ₂ , such as a combination of an enzyme having phospholipase A ₁ activity and an enzyme having phospholipase A ₂ activity, or phospholipase A ₁ and It comprises a single enzyme having both activities of phospholipase a ₂ activity.

Phospholipase A ₁ is defined as EC 3.1.1.32 according to the standard enzyme EC taxonomy.
Official name: Phospholipase A ₁ .
Catalyzed reaction: phosphatidylcholine + H ₂ O ⇔
2-acylglycerophosphocholine + aliphatic anion.
Comment: Has a broader specificity than EC 3.1.1.4.

Phospholipase A ₂ according to standard enzyme EC Classification is defined as EC 3.1.1.4.
Official name: phospholipase A _2.
Alternative name: phosphatidylcholine 2-acyl hydrolase. Lecithinase a;
Phosphatidase; or phosphatidlipase.
Catalyzed reaction: phosphatidylcholine + H ₂ O ⇔
1-acylglycerophosphocholine + aliphatic anion.
Comments: Acts on phosphatidylethanolamine, choline plasmalogen, and phosphatides to remove fatty acids added in the 2-position.

Lysophospholipase is defined as EC 3.1.1.5 according to the standard enzyme EC taxonomy.
Official name: Lysophospholipase.
Alternative names: lecithinase b; lysolecithinase; phospholipase B; or PLB.
Catalyzed reaction: 2-lysophosphatidylcholine + H ₂ O ⇔
Glycerophosphocholine + aliphatic anion.

  Phospholipase A activity may be similarly provided by enzymes having other activities, such as lipases having phospholipase A activity. Phospholipase A activity may be, for example, from a lipase with phospholipase A side activity. In other embodiments of the invention, the phospholipase A enzyme activity is provided by an enzyme that has essentially only phospholipase A activity, at which time the phospholipase A enzyme activity is not a side activity.

  Phospholipase A may be of any origin. For example, from animal sources (eg, mammals), eg from the pancreas (eg cow or porcine pancreas), or from snake venom or bee venom. Alternatively, phospholipase A will be of microbial origin. For example, from filamentous fungi, yeasts or bacteria, such as, for example, Aspergillus genus or species, such as A. niger, Dictyostelium, such as D. discoideum; Mucor such as M.javanicus, M.mucedo, M.subtillissimus; Neurospora such as N.crassa; Rhizomucor ( Rhizomucor) eg R. pusillus;

  Rhizopus such as R. arrhizus, R. japonicus, R.stolonifer; Sclerotinia such as S. libertiana; tricton Trichophyton rubrum; Whetzelinia eg W. scierotiorum; Bacillus eg B. megaterium, B. subtilis; Citrobacter Citrobacter), for example, C. freundii; Enterobacter, for example, Enterobacter erogenes (E.aerogenes), Enterobacter cloacae (E. cloacae),

  Edwardsiella, Edwardsila Talba (E.tarda); Erwinia (eg W.herbicola); Escherichia (eg Escherichia) (E.coli); Klebsiella (eg Klebsiella) (K. pneumoniae; Proteus such as Proteus bulgaris; P. vulgaris; Providencia such as Providencia p. stuartii; Salmonella such as Salmonella S. typhimurium; Serratia, eg Serratia liquefasciens, Serratia marcescens; Shigella, eg Shigella flexneri; Streptomyces, eg Streptomyces Bio Will Yersinia (Yersinia) e.g. Yersinia enterocolitica (Y.enterocolltica); Seoruba (S.violeceoruber).

  Thus, phospholipase A can be obtained from species of filamentous fungi, such as Pyrenomycetes, for example from the genus Fusarium, such as F.culmorum, F.heterosporum, Fusarium solani ( F. solani) or Fusarium F. oxysporum strain. Spholipase A may also be from a filamentous strain in the genus Aspergillus. For example, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus niger or Aspergillus oryzae to Aspergillus oryzae.

  A preferred phospholipase A is a Fusarium strain, in particular F. oxysporum, for example the DSM 2672 strain as described in WO 98/26057, in particular claim 36 and SEQ ID NO: WO 98/26057. : Derived from those described in 2. In a further embodiment, the phospholipase A is a phospholipase as disclosed in WO 00/32758 (Novozymes A / S, Denmark).

Lysophospholipase The term “lysophospholipase” as used herein in the context of the enzyme of the present invention is intended to include enzymes having lysophospholipase activity.
Lysophospholipase activity may be similarly provided by enzymes having other activities, such as lipases having lysophospholipase activity. The lysophospholipase activity may be, for example, from a lipase with lysophospholipase side activity. In other embodiments of the invention, lysophospholipase enzyme activity is provided by an enzyme having essentially only lysophospholipase activity. At this time, lysophospholipase enzyme activity is not a side activity. In one embodiment of the invention, the lysophospholipase is not a lipase having lysophospholipase side activity as defined in WO 98/26057.

  The lysophospholipase may be of any origin. For example, from an animal source (eg, mammal), eg, liver (eg, rat liver). Alternatively, lysophospholipase can be obtained from microbial sources, such as filamentous fungi, yeasts or bacteria, for example Aspergillus genus or species such as Aspergillus fetidus, A. fumigatus, Aspergillus nidulans. (A.nidulens), Aspergillus niger, Aspergillus oryzae; Botrytis such as Botrytis cinerea (B.cinerea); Candida such as C. albicans ); Cryptococcus such as C. neoformans, Escherichia such as E. coli,

  Fusarium such as Fusarium spororotrichioides, F. venenatum, F. verticillioides; Hyphozyma; Kluyveromyces such as Kluyveromyces Lactis; K. lactis; Magnaporte, eg M. grisea; Metalrhizium, eg Metalhizium anisopliae; Mycosphaerella, eg Mycosphaerella, Graminicola (M Neurospora, for example Neurospora, N. crassa; Penicillium, for example Penicillium, P.notatum; Saccharomyces, for example S. cerevisi ae);

  Schizosaccharomyces such as S. pombe; Torulaspora such as T. delbrueckii; Vibrio such as V. cholerae. Preferred lysophospholipases are, for example, those contained in the Escherichia coli clone DSM 13003 or DSM 13004, or strains of Aspergillus, in particular lysophospholipase LLPL from Aspergillus niger. -1 or LLPL-2 or contained in DSM 13082 or DSM 13083 of the Escherichia coli clone described for example in WO 01/27251, in particular of WO 01/27251 Derived from lysophospholipase LLPL-1 or LLPL-2 from A. oryzae as set forth in claim 1 and SEQ ID NO: 2, 4, 6 or 8.

Enzymes with both phospholipase A and lysophospholipase activity In a preferred embodiment of the invention, the phospholipase A and lysophospholipase activities are provided by a single enzyme having both phospholipase A and lysophospholipase activities. For example, in one embodiment, a single enzyme has both phospholipase A ₁ activity and lysophospholipase activity, and in another embodiment, a single enzyme has both phospholipase A ₂ activity and lysophospholipase activity. In a further embodiment, the single enzyme has phospholipase A ₁ activity, phospholipase A ₂ activity and lysophospholipase activity.

The present invention thus also adds an enzyme having both phospholipase A activity and lysophospholipase activity to cheese milk in order to reduce the oiling-off effect in cheese and / or increase cheese yield, and It relates to a process for producing cheese comprising producing cheese from cheese milk.
Enzymes having both phospholipase A and lysophospholipase activities are known to those skilled in the art. Enzymes having both phospholipase A and lysophospholipase activities are described, for example, by Saito et al., Methods in Enzymology (1991) 197,446-456 and Lee et al., J. Biol. Chem. (1994) 269, 19725-19730.

Enzyme Sources and Formulations The phospholipase A and / or lysophospholipase used in the process of the present invention could be derived or obtained from any of the sources mentioned herein. The term “derived” in this context means that the enzyme would have been isolated from an organism that is naturally occurring, ie, the identity of the amino acid sequence of the enzyme is identical to the natural enzyme. The term “derived” also means that the enzyme would have been produced recombinantly in the host organism. The recombinantly produced enzyme has exactly the same identity as the natural enzyme or has a modified amino acid sequence. For example, having one or more amino acids that have been deleted, inserted and / or substituted. That is, a recombinantly produced enzyme that is a mutant and / or fragment of the natural amino acid sequence. Natural mutants are included within the meaning of natural enzymes.

  Furthermore, the term “derived” is meant to include enzymes produced synthetically, eg, by glycosylation. The term “derived” also encompasses enzymes that have been modified in vivo or in vitro, for example, by glycosylation, phosphorylation, and the like. Furthermore, the term “available” in this context means that the enzyme has the same amino acid sequence as the natural enzyme. The term includes an enzyme isolated from a naturally occurring organism, or an enzyme recombinantly expressed in the same or another organism, or an enzyme produced synthetically, eg, by peptide synthesis. . With respect to recombinantly produced enzymes, the terms “available” and “derived” relate to the identity of the enzyme and to the identity of the host organism in which the enzyme is produced recombinantly. is not.

  Thus, phospholipase A and / or lysophospholipase will be obtained from a microorganism by use of any suitable technique. For example, the phospholipase A and / or lysophospholipase enzyme preparation may continue to isolate the phospholipase A and / or lysophospholipase preparation from the appropriate fermentation broth and the resulting fermentation broth or microorganism by methods known in the art. Will be obtained. Phospholipase A and / or lysophospholipase may also be obtained by use of recombinant DNA technology.

  Usually such methods are operatively linked to the DNA sequence encoding the phospholipase A or lysophospholipase in question, and the appropriate expression signal, so that the conditions allow the enzyme to be expressed and recover the enzyme from the culture. A culture of host cells transformed with a recombinant DNA vector containing a DNA sequence allowing expression of phospholipase A or lysophospholipase in the culture medium. The DNA sequence will also be integrated into the genome of the host cell. The DNA sequence will be genomic, cDNA or synthetic origin or combinations thereof, and will be isolated or synthesized according to methods known in the art.

Suitable phospholipases are commercially available. As a typical example of an enzyme for practical use, phospholipase A ₂ derived from the pancreas, such as Lecitase ™ (Novozymes A / S) is preferably used. A suitable lysophospholipase is Aspergillus niger lysophospholipase LLPL-2, which can be produced recombinantly in A. niger as described for example in WO 01/27251.

  In the process of the present invention, phospholipase A and / or lysophospholipase will be purified. As used herein, the term “purified” includes phospholipase A or lysophospholipase enzyme protein that does not contain components from the organism from which the enzyme is derived. The term “purified” also includes phospholipase A or lysophospholipase enzyme protein which does not contain components derived from the natural organism from which the enzyme is obtained. This is also referred to as “essentially pure” phospholipase A or lysophospholipase, which occurs naturally and undergoes genetic modifications, such as deletion or substitution or insertion of one or more amino acid residues. Of particular relevance to non-phospholipases and lysophospholipases.

  Thus, phospholipase A and / or lysophospholipase will be purified. That is, the presence of other proteins is very small. The expression “other proteins” particularly relates to other enzymes. The term “purified” as used herein relates to the removal of other components, especially other proteins and most particularly phospholipase A or other enzymes present in cells of lysophospholipase origin. Phospholipase A or lysophospholipase will be "essentially pure", i.e. an enzyme producing organism, i.e. other components derived from a host organism for eg recombinantly produced phospholipase A or lysophospholipase Will not include. Preferably the enzyme is at least 75% (w / w) pure, more preferably at least 80%, 85%, 90%, or even at least 95% pure. In an even more preferred embodiment, the phospholipase A or lysophospholipase is an enzyme protein preparation that is at least 98% pure. In other embodiments, phospholipase A and / or lysophospholipase are not naturally present in milk.

The terms “phospholipase” and “lysophospholipase” include any auxiliary compounds that may be necessary for the catalytic activity of the enzyme, such as a suitable receptor or cofactor. These may or may not exist naturally in the reaction system.
Phospholipase A and lysophospholipase may be present in any form suitable for the use in question, for example, in dry powder or granular form, powder-free granular form, liquid, stabilized liquid, or protected enzyme form. The grains will be produced, for example, as disclosed in US Pat. No. 4,106,991 or US Pat. No. 4,661,452, and will optionally be coated by methods known in the art. Liquid enzyme preparations may be stabilized, for example, by adding stabilizers such as sugars, sugar alcohols or other polyols, lactic acid or other organic acids according to established methods. The protected enzyme will be prepared according to the method disclosed in EP 238,216.

The lecithin content of cheese by the process of the present invention, as described herein, is at least 5%, such as at least 10%, as compared to without the phospholipase A and lysophospholipase enzymatic treatments, although in a similar cheese manufacturing process. %, At least 20%, at least 30%, at least 50%, eg, within the range of 5-95%.
In milk, lecithin usually constitutes more than 95% of milk phospholipids, whereas lysolecithin is about 1% of phospholipids. Phospholipids usually represent only less than 1% of total milk lipids, but are mainly present in the milk fat globule membrane and play a particularly important role. The lecithin content of available cheeses will be lower than 90%, such as lower than 80%, such as lower than 60% or 50%, for example, by the process of the present invention. The lecithin concentration will be measured by any method known by those skilled in the art, such as HPLC.

The invention further relates to the use of cheese produced by the process of the invention in pizza, instant food, processed cheese or as an additive to other food products. Thus, cheese made according to the process of the present invention will be used in further processed food products such as processed cheese, pizza, burger, toast, sauce, dressing, cheese flour or cheese flavor.
In a further embodiment, the process of the present invention further comprises subjecting the cheese to a heat treatment, such as in the range of 150-350 ° C.
The invention also relates to cheeses that can be obtained, in particular obtained by the process of the invention.

The invention is further illustrated in the following examples, which are in no way limited to the scope of protection.
Example 1. Cheese prepared by adding phospholipase A and lysophospholipase
Enzyme preparation
A: 0.03 mg of enzyme protein phospholipase A per gram of fat (Lecitase (trademark) 10 L manufactured by Novozymes A / S, Denmark).
B: 0.05 mg lysophospholipase per gram of milk fat (Aspergillus niger lysophospholipase LLPL-2 produced recombinantly in Aspergillus niger as described in WO 01/27251 ).
C: A combination of A and B above.

Cheese-Making Full Fat Mozzarella Cheese is made using pasteurized non-homogenized cream (North Carolina State University Dairy Factory) and pasteurized non-homogenized skim milk (North Carolina State University Dairy Factory) to 3.5% fat Standardized.
A starter culture was prepared by adding 0.1 g Rhodia LH100 and 0.3 g Rhodia TA061 starter culture to 50 mL skim milk and equilibrating to 35 ° C. with slow continuous stirring. Standardized cheese milk was equilibrated to 35 ° C. and divided into 3 batches. 1.4% starter culture was added to each batch, while the above enzyme preparations A, B and C were added to each batch with slow agitation.

  When the pH reached 6.45-6.50, 0.01% rennet (fermented chymosin Chymax from Chr. Hansens Lab., Denmark) was added to each batch with vigorous stirring for 3 minutes. The stirrer was removed and the milk was agglomerated. A downward cut was made in the card with a knife or spatula to determine the appropriate cutting time. The spatula was then inserted into the card below the notch and perpendicular to the direction of the notch, and used to lift the card upward. When the card was lifted and separated, and the sharp angle was maintained on the upper surface of the cut end, the card was judged ready for cutting. Cutting was performed using a 1/2 inch knife, and the card was allowed to stand for 5 minutes after cutting.

With slow and occasional stirring, the temperature was raised to 41 ° C. and maintained until the pH was 5.65-5.70. The whey was then drained and the card pieces were poured into a stainless steel bowl. The bowl was floated in a 41 ° C. water bath to maintain the temperature of the curd and excess whey was drained periodically, leaving only enough to cover the curd to maintain heat.
When the pH reached 5.25 to 5.3, all whey was drained and the card was immersed in deionized water at 57 ° C for 5 minutes. The card was stretched by hand for 1 minute and returned to 57 ° C water if necessary to maintain the card temperature. The stretched curd was soaked in room temperature tap water for 10-15 minutes for cooling, then dampened and dried and refrigerated.

Oiling-off measurement cheese was ground in a blender for 20 seconds to homogenize the sample. Approximately 3.0 g was placed in a metal ring (diameter 2.2 cm), molded, placed in the center of Whatman # 4 filter paper, and melted in an oven set at 90 ° C. for 5 minutes. Each material was tested three times. Oiling-off was measured by image analysis as the difference between the area of the ring of oil developed during melting around the cheese and the area of the cheese circle. The oiling-off calculation is shown below:

The results (protein, moisture, fat and oiling off) are shown in Table 1 below.

  From the results in Table 1, it can be seen that the addition of phospholipase A has a higher effect on reducing cheese oiling-off than the addition of lysophospholipase. When both enzymes are added, however, the oiling off is even lower, showing a synergistic effect of the two enzymes.

Example 2 Production of long chain fatty acids in milk treated with phospholipase A and lysophospholipase
The substrate preparation and enzyme-treated cream were normalized to a fat content of 25% using skim milk. Milk samples were prepared as shown in Table 3, Phospholipase A (Lecitase ™ 10 L from Novozymes A / S, Denmark), Lysophospholipase (A.niger as described in WO 01/27251). Treated with recombinantly expressed Aspergillus niger lysophospholipase LLPL-2) and a combination of phospholipase A and lysophospholipase. Enzymes were prediluted with water if necessary and added to the cream at the appropriate concentration. Samples were incubated at 35 ° C. for 1.5 hours without shaking. The reaction was stopped by adding an organic solvent for lipid extraction.

Lipid extraction Total milk lipids were extracted by mixing each sample with 1 mL water followed by 9 mL chloroform / methanol (2: 1). The sample was mixed vigorously for 1 minute and then centrifuged at 3000 rpm for 5 minutes. 6 mL of the lower CHCl ₃ phase was removed and concentrated under vacuum. 2 mL of CHCl ₃ was added to the sample. Fatty acids were further purified by solid phase extraction (SPE). Each CHCl ₃ extract was applied to an aminopropyl SPE column under vacuum. The column was washed with 4 mL CHCl ₃ / isopropanol (2: 1) to remove neutral lipids. The fatty acid was eluted with 4 mL of diethyl ether acidified with glacial acetic acid (2% v / v). The ether extract was concentrated on a rotary evaporator, and 1.0 mL of methanol was added for HPLC analysis.

HPLC method
Reverse phase chromatography was performed using a Phenomenex (Torrance, CA) Luna C8 (150 x 4.6 mm, 5μ, 100 A) column and water containing acetonitrile and 0.1% trifluorinated acetic acid as the mobile phase. See Table 2 for the gradient elution scheme. The elution fatty acid was detected by evaporative light scattering. HPLC fatty acid standards (linolenic acid, palmitic acid, oleic acid, and stearic acid) were selected based on the incidence of total milk lipids. Stock solutions were routinely prepared in a concentration range of 2-10 mg / mL in 100% methanol. HPLC calibrators were prepared from fatty acid stock solutions by diluting in 100% methanol at appropriate concentrations.

  Table 3 shows the amount of free long chain fatty acids produced after the cream samples were treated with phospholipase A and / or lysophospholipase. 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 significantly increase the amount of free long chain fatty acids produced. When lysophospholipase is added in combination with phospholipase A, the amount of fatty acid produced increases significantly.

Claims (17)

To reduce the oiling-off effect in cheese and / or increase the cheese yield, phospholipase selected from the group consisting of phospholipase A ₁ and phospholipase A ₂ and combinations thereof, and lysophospholipase are added to cheese milk. And the cheese manufacturing method characterized by manufacturing cheese from this cheese milk.   The method according to claim 1, wherein the amount of the phospholipase A is in the range of 0.003 to 0.3 mg of enzyme protein per gram of milk fat.   The method according to claim 1, wherein the amount of the phospholipase A is in the range of 0.01 to 0.3 mg of enzyme protein per gram of milk fat.   The method according to claim 1, wherein the amount of phospholipase A is 0.03 mg of enzyme protein per gram of milk fat.   The method according to any one of claims 1 to 4, wherein the phospholipase A and / or lysophospholipase is added to cheese milk during normalization of fat and / or protein content of cheese milk.   The method according to any one of claims 1 to 4, wherein the phospholipase A and / or lysophospholipase is added simultaneously with the starter culture.   The method according to any one of claims 1 to 4, wherein the phospholipase A and / or lysophospholipase is added simultaneously with cheese rennet.   The method according to any one of claims 1 to 7, further comprising a step of removing or reducing the content of the enzyme protein of the phospholipase A and / or lysophospholipase after the enzyme acts in cheese milk. The method according to item.   The method further comprises inactivating the phospholipase A and / or lysophospholipase after the enzyme acts in cheese milk. The method according to claim 1, characterized in that:   The method according to any one of claims 1 to 9, wherein the cheese is selected from the group consisting of rennet curd cheese produced by rennet aggregation of cheese curd, aged cheese, fresh cheese, and acid agglomerated cheese. .   The cheese is Campesino, Chester, Danbo, Drabant, Herregard, Manchego, Provolone, Saint Paulin, soft cheese, Svecia (Svecia), Taleggio, White Cheese, Cheddar, Colby, Edam, Muenster, Gryere, Emmenthal, Camembert, Parmesan ( Claims 1 to 5 selected from the group consisting of Parmesan, Romano, Mozzarella, Feta; cream cheese, Neufchatel, Quarg, and Queso Blanco 10. The method according to any one of items 9.   The method according to any one of claims 1 to 10, wherein the cheese milk is subjected to a homogenization step before the production of cheese.   13. The method of claim 12, wherein the cheese is Danish Blue cheese.   14. The method according to any one of claims 1 to 13, wherein the activities of phospholipase A and lysophospholipase are provided by a single enzyme having both phospholipase A activity and lysophospholipase activity.   15. The method according to any one of claims 1 to 14, further comprising the step of processing the cheese into a food product.   16. The method of claim 15, wherein the food product is selected from the group consisting of pizza, instant food, toast, burger, lasagna, dressing, sauce, cheese flour, cheese flavor, and processed cheese.   A cheese prepared by the method of any one of claims 1-14.