Field of the invention

The present invention relates to a water buffalo lipase, a process for the isolation of the lipase from the water buffalo tongue root and its use in the production of cheese and enzyme modified cheese.

Background of the invention

Lipases are enzymes that catalyze the hydrolysis of ester bonds in lipid substrates, leading to the release of fatty acids. Lipases are used in the dairy applications for flavor generation, most importantly in cheese. Traditionally, ruminant lipase preparations are used derived caprine, ovine and bovine sources. They are derived from pregastric tissues from these ruminants and these lipase preparations are also referred to as pregastric esterases.

There are various commercial products in the market, such as the Capalase™ K (from caprine (kid)), Capalase™ L (from ovine (lamb)), Italase™ C (from bovine (calf)), Capalase™ KL (a blend from kid and lamb, KSHRASE ^® K (a kosher lipase from caprine (kid)), KSHRASE ^® L (a kosher lipase from ovine (lamb) and KSHRASE ^® C (a kosher lipase from bovine (calf)), all available from DSM Food Specialties, The Netherlands. These lipase products are used in the preparation of a variety of Italian, Spanish, Greek and other traditional cheeses. In particular these lipases produce a vibrant, "goaty", sharp "piccante" flavor profile in Italian-type cheeses such as Provolone or Asiago. The development of a specific flavor profile in these types of cheeses during ripening is largely due to the action of lipases on milk fat. Lipases catalyze hydrolysis of milk fat with generation of free fatty acids. Said fatty acids may have short chains (C4-C6 fatty acids, such as containing 4 or 6 carbon atoms, i.e. butyric, caproic acid) and medium to long chain (C12-C18:3 fatty acids). Subsequently free fatty acids can take part in chemical reactions, e.g. the formation of flavor compounds such as acetoacetate, betaketo acids, methyl ketons, esters and lactones.

Conversion of fatty acids in flavor components can be catalyzed by the enzymes originating from the microbial population in cheese.

The inventors have now identified a new lipase from water buffalo which, in comparison to the known ruminant lipase preparations, gives unique taste and flavor profiles to various types of cheese.

Detailed description of the invention

In a first aspect, the invention provides a pregastric lipase, or gastric lipase, from water buffalo (or abbreviated as WBL). Preferably the present lipase from water buffalo is an isolated pregastric lipase from water buffalo. The water buffalo is also known as Bubalus bubalis. A lipase is a hydrolase (Class EC 3), acting on ester bonds (Class EC 3.1 ), in particular carboxylic esters (Class EC 3.1 .1 ). The water buffalo pregastric lipase has triacylglycerol lipolytic activity (EC Class 3.1 .1 .3). The pregastric lipase of the invention is preferably a lipase derived or isolated from pharyngeal tissue (tongue root or gullet) of the water buffalo. Preferably the present pregastric lipase has a molecular weight within the range of 35 to 60 kDa, preferably within the range of 40 to 50 kDa such as 43 to 47 kDa, more preferably a molecular weight of 45 kDa or around 45 kDa. Preferably the present pregastric lipase comprises an amino acid sequence according to SEQ ID NO 1 , or an amino acid sequence having more than 99% sequence identity with SEQ ID no 1 , or more than 99.5% sequence identity. Preferably, the sequence identity is calculated over the entire amino acid length of 397 amino acids. More preferably, the present pregastric lipase comprises an amino acid sequence according to SEQ ID NO 1 having at most 5, 4, 3, 2 or 1 different amino acids.

Within the present context, sequence identity is defined as the number of corresponding positions in an alignment showing an identical amino acid 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 the computer program NEEDLE by using the NOBRIEF option and is labelled in the output of the program as "longest-identity". For purposes of the invention the level of identity (homology) between two sequences (amino acid or nucleotide) is calculated according to the definition of "longest-identity" as can be carried out by using NEEDLE (NEEDLE program from the EMBOSS package - version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice,P. LongdenJ. and BleasbyA Trends in Genetics 16, (6) pp276— 277.

The inventors of the present invention surprisingly found that despite the fact that the pregastric lipase derived from water buffalo corresponds for at most 99% with pregastric lipase from bovine (cattle) calf, the water buffalo lipase provides another advantageous fatty acid and taste profile than bovine (cattle) calf derived pregastric lipase. SEQ ID NO 2 provides the corresponding pregastric lipase from Bos Taurus, showing at most 99% sequence similarity with the water buffalo pregastric lipase according to SEQ ID NO 1 .

The invention provides also compositions comprising the present water buffalo pregastric lipase. Advantageously, the present compositions are suitable for the hydrolysis of milk fat with generation of free fatty acids. In other words, the present composition is suitable for the ripening or production of dairy products, such as for example enzyme modified cheeses. The composition may comprise more than 0.1 % (w/w), more than 0.5% (w/w) or more than 1 % (w/w) of the present pregastric lipase. Preferably, the composition comprises 0.1 % to 10% (w/w) or 0.5% to 5% (w/w) pregastric lipase. More preferably the pregastric lipase is present in the present composition in an amount of 1 % to 2% (w/w) of the composition. Such a composition may be a liquid composition, such as an aqueous solution or may be a solid composition such as a spray dried composition, a vacuum dried composition, a lyophilized composition and the like. The composition comprising the water buffalo pregastric lipase may comprise additional components, such as salt, enzyme stabilizers, filling agents and the like. Preferably the present composition is derived from pharyngeal tissue of water buffalo. More preferably the present composition is an extract 1 , extract 2 as described below, or combination thereof, which is obtainable by the present method for the isolation of lipase. The advantage of the isolated water buffalo pregastric lipase in comparison with other pregastric lipases as described herein before, is its unique properties when it is used in the manufacturing of various types of cheese. The water buffalo pregastric lipase gives unique taste and flavor profiles to various types of cheese as will be shown below.

In a second aspect, the invention provides a process for the isolation of the water buffalo pregastric lipase of the first aspect of the invention or the composition comprising the water buffalo pregastric lipase. Preferably the process comprises (a) providing tongue roots originating from one or more water buffalos; (b) chipping and/or grinding obtained tongue roots in a buffer to give a suspension of ground tongue roots and hold for preferably at least 2 hours; (c) increasing the pH of the buffer with tongue roots, and (d) extracting the lipase from ground tongue roots for a time sufficient for the lipase to be extracted from the ground tongue roots to provide a suspension; and/or separating the obtained suspension to give an extract 1 and extracted ground tongue roots.

Alternatively, the present process comprises the steps of:

a. Isolating tongue roots from one or more water buffalos;

b. Chipping and/or grinding tongue roots obtained in step (a) in a buffer with a pH preferably below 5.5 at preferably below 10°C to give a suspension of ground tongue roots and hold for preferably at least 2 hours;

c. Increasing the pH of the buffer in step (b) to preferably a pH in the range 9.0 to 10.0 at preferably below 10.0°C and extracting the lipase from the ground tongue roots for a time sufficient for the lipase to be extracted, preferably at least 3 hours, from the ground tongue roots; and/or

d. Separating the suspension obtained in step (c) to give extract 1 and extracted ground tongue roots preferably by decanting.

Step a) - The isolation of the tongue roots from water buffalos may be done in a slaughter house. The tongue roots may be used fresh in the process of the second aspect of the invention or the tongue roots may be frozen, preferably at a temperature equal to or below -12°C, preferable below -15°C, more preferably around -20°C. In case the isolation of the lipase from the tongue roots takes place at another location as the slaughtering, the tongue roots may be transported, preferably as frozen tongue roots. Frozen tongue roots may be stored from several days up to one or more years. In this way, a batch of tongue roots of sufficient size may be built up and collected which then may be subjected to the lipase isolation process.

Step b) - The fresh or frozen tongue roots obtained in step a) may be subjected to chipping and/or grinding in a buffer with a pH preferably below 5.5, preferably in the range 5.0 to 5.5, more preferably in the range 5.2 to 5.5 and/or at a temperature preferably below 10°C but above the freezing point, to give a suspension of ground tongue roots. The ratio of the [volume of the buffer] / [weight of the tongue roots] is preferably in the range of 20 to 80, more preferably in the range of 30 to 70, preferably around 60. For instance, 20 liters of buffer may be used for each 1 kg of tongue roots, or 80 liters of buffer may be used for each 1 kg of tongue roots. The suspension of the ground tongue roots may be hold under these conditions for a period of time, preferably for at least 2 hours.

Step c) - In order to extract the lipase from the tongue roots, the pH of the suspension obtained after step b) is increased, preferably to a pH in the range of 7.5 to 1 1 , preferably 8 to 10.5, more preferably 9 to 10 and/or at a temperature preferably below 10.0°C but above the freezing point and extracting the lipase from the ground tongue roots for a time sufficient for the lipase to be extracted from the ground tongue roots. The extraction time is preferably at least 3 hours.

Step d) - In a final step, the suspension obtained in step (c) is separated to give extract an 1 and extracted ground tongue roots. Any suitable separation method can be used such as decanting, centrifugation, filtration and the like. Most preferred is decanting since this is a simple and cheap method. The extracted ground tongue roots obtained after the separation step may be subjected to another extraction according to step (c) and (d) to give an extract 2.

Extract 1 or extract 2 or the combined extracts 1 and 2 may be used as such in the process for manufacturing of cheese as described below. Extract 1 or extract 2 or the combined extracts 1 and 2 may also be passed or separated through a filter, preferably a 100 μ filter, more preferably a 100 μ filter followed by a 5 μ filter and most preferably a 100 μ filter followed by a 5 μ filter, followed by 1 μ filter. The extracts which may or may not been filtered as described above, may also be concentrated by methods known in the art, e.g. by ultrafiltration or nanofiltration. Ultrafiltration is a preferred method. The ultrafiltration unit may be equipped with a filter having preferably a 10 kDa cut off. The filter may be charged or uncharged. The lipase is collected in the retentate (the concentrated retentate). The concentrating factor will depend on the lipase concentration in the extracts and the desired lipase concentration in the final lipase composition. For instance, the extracts may be concentrated between 20-fold and 80-fold. The lipase in the liquid extracts or concentrated extracts may be stabilized by methods known in the art. Extract 1 or extract 2 or the combined extracts 1 and 2 or the concentrated retentate may also be dried by methods known in the art, such as spray drying, vacuum drying or lyophilization. Preferably vacuum drying is used, preferably at a temperature below 1 10°C at a pressure below 30 mm Hg. The conditions during filtration and/or concentrating and/or drying are preferably a temperature below 10°C and at a pH in the range 8.5-10.0.

In one embodiment, salt, preferably kitchen salt or sodium chloride, is added to the concentrated retentate or to the present composition comprising the water buffalo pregastric lipase. A suitable salt concentration is in the range of 8 wt% to 16 wt%, preferably 10 wt% to 14%, more preferably 1 1 wt% to 13 wt%, most preferably around or at 12 wt%. In another embodiment, the concentrated retentate or composition is mixed with whey powder and salt before vacuum drying, suitable concentration are 5 wt% whey powder and 5 wt% salt.

In a third aspect, the invention provides a process for the manufacturing or production of a dairy product preferably the manufacture of cheese, a cheese-like product, EMC (Enzyme Modified Cheese), or EMDI (Enzyme Modified Dairy Ingredients) or in the manufacture of free fatty acid mixtures obtainable by the lipolysis of butter fat or cream, the process comprising the use of the isolated water buffalo pregastric lipase of the first aspect of the invention or the compositions comprising the isolated water buffalo pregastric lipase. Examples of EMDI (Enzyme Modified Dairy Ingredient) are lipolyzed cream, lipolyzed butter, lipolyzed butter oil and enzyme modified milk. Preferably, the present process comprises the addition of the present water buffalo pregastric lipase milk, preferably to cow milk.

In a preferred embodiment, the present invention relates to the production of a dairy product comprising a ratio of C ₄ - C10 fatty acids present in the dairy product to Ci2 - Cis fatty acids present in the dairy product of less than 100:1 , preferably less than 60:1 .

In a preferred embodiment, the process of the present invention concerns the manufacturing or production of cheese for example the manufacture of Provolone, Asiago, Romano, Parmesan, Feta, Cotija, Kassari or Kasar, Kashkaval, Caciocavallo and Tulum cheese. Preferably Provolone and/or Asiago. The manufacturing process for the production of cheese in general and Provolone, Asiago, Romano, Parmesan, Feta, Cotija, Kassari or Kasar, Kashkaval, Caciocavallo and Tulum cheese in particular is well known in the art. The process of the third aspect of the invention differs from the processes known in the art in that the isolated water buffalo pregastric lipase of the present invention is used instead of the pregastric lipases that are known in the art, such as pregastric lipase from cattle. The advantage of using the isolated water buffalo pregastric lipase in comparison with other pregastric lipases as described hereinbefore, is its unique properties when it is used in the manufacturing of cheese such as the types of cheese listed above. The water buffalo pregastric lipase gives unique taste and flavor profiles to various types of cheese as is demonstrated in the Examples. In the cheese manufacturing process, the water buffalo pregastric lipase may be added to the milk prior to adding a coagulant for clotting the milk (or on cheese curd after whey draining before press or hot water stretch or before further any cheese specific processes).

In a preferred embodiment, the process of the present invention for manufacturing of cheese comprises the use of the present lipase comprising an amino acid sequence according to SEQ ID NO 1 or sequences having more than 99% sequence identity with SEQ ID NO 1 . Alternatively, the present process comprises the addition to milk of a water buffalo lipase comprising an amino acid sequence according to SEQ ID NO 1 or sequences having more than 99% sequence identity with SEQ ID NO 1 , in combination with a cattle lipase comprising an amino acid sequence according to SEQ ID NO 2 or sequences having more than 99% sequence identity with SEQ ID NO 2.

In a fourth aspect, the invention provides the use of the present water buffalo pregastric lipase for the production of cheese, a cheese-like product, EMC (Enzyme Modified Cheese), or EMDI (Enzyme Modified Dairy Ingredients) or in the manufacture of free fatty acid mixtures obtainable by the lipolysis of butter fat or cream. Preferably, the present invention relates to use of the present lipase comprising an amino acid sequence according to SEQ ID NO 1 or sequences having more than 99% sequence identity with SEQ ID NO 1 .

In a fifth aspect, the invention provides a cheese, a cheese-like product, EMC (Enzyme Modified Cheese), or EMDI (Enzyme Modified Dairy Ingredients), which is preferably obtainable by the process of the present invention, and whereby the cheese, a cheese-like product, EMC (Enzyme Modified Cheese), or EMDI (Enzyme Modified Dairy Ingredients) have a unique taste and flavor profile compared to the cheese, a cheese-like product, EMC (Enzyme Modified Cheese), or EMDI (Enzyme Modified Dairy Ingredients) made with the other pregastric lipases known in the art. Preferred cheeses provided by the present invention are Provolone, Asiago, Romano, Parmesan, Feta, Cotija, Kassari or Kasar, Kashkaval, Caciocavallo and Tulum cheese.

In a sixth aspect, the invention provides a dairy product, preferably a dairy product obtainable by the process of the present the invention. Preferably the dairy product is cheese, a cheese-like product, enzyme modified cheese (EMC) or an EMDI (Enzyme Modified Dairy Ingredient). More preferably the cheese is Provolone, Asiago, Romano, Parmesan, Feta, Cotija, Kassari or Kasar, Kashkaval, Caciocavallo or Tulum cheese. In a preferred embodiment, the dairy product, which is preferably obtainable by the process of the second aspect of the invention, is comprising the lipase or the composition comprising the lipase as defined in the first aspect of the invention. Preferably the dairy product comprising the lipase or the composition comprising the lipase as defined in the first aspect of the invention is cheese, a cheese-like product, enzyme modified cheese (EMC) or an EMDI (Enzyme Modified Dairy Ingredient). More preferably the cheese comprising the lipase or the composition comprising the lipase as defined in the first aspect of the invention is Provolone, Asiago, Romano, Parmesan, Feta, Cotija, Kassari or Kasar, Kashkaval, Caciocavallo or Tulum cheese.

In a preferred embodiment, the present dairy product, or cheese, comprises comprising a lipase having an amino acid sequence according to SEQ ID NO. 1 or sequences having more than 99% sequence identity to SEQ ID NO. 1 .

In a preferred embodiment, the present dairy product, or cheese, is characterized in that the ratio of the sum of C ₄ to Cio fatty acids to the sum of C12 to C18 fatty acids present in the dairy product is less than 100:1 or 100, preferably less than 90:1 , or 90, more preferably less than 80:1 or 70:1 (or 80 or 70), most preferably less than 60:1 (or 60) or less than 50:1 (or 50) or even less than 40:1 (or 40). A dairy product having such a spread in fatty acids provides a more balanced flavour profile in comparison with dairy products having a higher ratio of C ₄ to C10 : C12 to C18 fatty acids. Preferably the present dairy product comprises the present ratio of the sum of C ₄ to C10 fatty acids to the sum of C12 to C18 fatty acids present in the dairy product for a time period, or aging period, of at least 1 month after inoculation, more preferably at least 3 months, most preferably for at least 6 months.

In a preferred embodiment, the present dairy product is a provolone cheese having a ratio of the sum of C ₄ to C10 fatty acids to the sum of C12 to C18 fatty acids present in the provolone cheese of less than 100:1 , preferably less than 50:1 , more preferably even less than 40:1 , preferably for a time period of at least 1 or at least 6 months.

In another preferred embodiment, the present dairy product is an asiago cheese having a ratio of the sum of C ₄ to C10 fatty acids to the sum of C12 to C18 fatty acids present in the provolone cheese of less than 100:1 , preferably less than 80:1 , more preferably even less than 60:1 , preferably for a time period of at least 6 months.

Within the context of the present invention, the term sum of C ₄ to Cio fatty acids to the sum of C12 to C18 fatty acids means the sum of the amount of C ₄ Ce, Cs, C10 fatty acids present in the product divided by the sum the amounts of C12, On, C16, C18 fatty acids present in the product.

SEQUENCE LISTING

<1 10> DSM IP Assets B.V.

<120> WATER BUFFALO LIPASE

<130> 29942-WO-PCT

<160> 2

<170> BiSSAP 1 .2

<210> 1

<21 1 > 397

<212> PRT

<213> Bubalus bubalis

<400> 1

Met Trp Trp Leu Leu Val Thr Met Cys Phe lie His Met Ser Gly Asn

Ala Phe Cys Phe Leu Gly Lys lie Ala Lys Asn Pro Glu Ala Ser Met

Asn Val Ser Gin Met lie Ser Tyr Trp Gly Tyr Pro Ser Glu Met His

Lys Val lie Thr Ala Asp Gly Tyr lie Leu Gin Val Tyr Arg lie Pro

His Gly Lys Asn Asp Ala Asn His Leu Gly Gin Arg Pro Val Val Phe

Leu Gin His Gly Leu Leu Gly Ser Ala Thr Asn Trp lie Ser Asn Leu

Pro Lys Asn Ser Leu Gly Phe Leu Leu Ala Asp Ala Gly Tyr Asp Val

Trp Leu Gly Asn Ser Arg Gly Asn Thr Trp Ala Gin Glu His Leu Tyr

Tyr Ser Pro Asp Ser Pro Glu Phe Trp Ala Phe Ser Phe Asp Glu Met

Ala Glu Tyr Asp Leu Pro Ser Thr lie Asp Phe lie Leu Arg Arg Thr

Gly Gin Lys Lys Leu His Tyr Val Gly His Ser Gin Gly Thr Thr lie Gly Phe lie Ala Phe Ser Thr Asn Pro Thr Leu Ala Glu Lys lie Lys Val Phe Tyr Ala Leu Ala Pro Val Ala Thr Val Lys Tyr Thr Lys Ser Leu Phe Asn Lys Leu Ala Leu lie Pro His Phe Leu Phe Lys lie lie Phe Gly Asp Lys Met Phe Tyr Pro His Thr Phe Leu Glu Gin Phe Leu Gly Val Glu Val Cys Ser Arg Glu Thr Leu Asp Val Leu Cys Lys Asn Ala Leu Phe Ala lie Thr Gly Val Asp Asn Lys Asn Phe Asn Met Ser Arg Leu Asp Val Tyr lie Ala His Asn Pro Ala Gly Thr Ser Val Gin Asn Thr Leu His Trp Arg Gin Ala Val Lys Ser Gly Lys Phe Gin Ala Phe Asp Trp Gly Ala Pro Tyr Gin Asn Leu Met His Tyr His Gin Pro Thr Pro Pro lie Tyr Asn Leu Thr Ala Met Asn Val Pro lie Ala Val Trp Ser Ala Asp Asn Asp Leu Leu Ala Asp Pro Gin Asp Val Asp Phe Leu Leu Ser Lys Leu Ser Asn Leu lie Tyr His Lys Gin lie Pro Asn Tyr Asn His Leu Asp Phe lie Trp Ala Met Asp Ala Pro Gin Glu Val Tyr Asn Glu lie Val Ser Leu Met Ala Glu Asp Lys Lys

<210> 2

<21 1 > 397

<212> PRT

<213> Bos taurus

<400> 2

Met Trp Trp Leu Leu Val Thr Val Cys Phe lie His Met Ser Gly Asn Ala Phe Cys Phe Leu Gly Lys lie Ala Lys Asn Pro Glu Ala Ser Met Asn Val Ser Gin Met lie Ser Tyr Trp Gly Tyr Pro Ser Glu Met His Lys Val lie Thr Ala Asp Gly Tyr lie Leu Gin Val Tyr Arg lie Pro His Gly Lys Asn Asn Ala Asn His Leu Gly Gin Arg Pro Val Val Phe Leu Gin His Gly Leu Leu Gly Ser Ala Thr Asn Trp lie Ser Asn Leu Pro Lys Asn Ser Leu Gly Phe Leu Leu Ala Asp Ala Gly Tyr Asp Val Trp Leu Gly Asn Ser Arg Gly Asn Thr Trp Ala Gin Glu His Leu Tyr Tyr Ser Pro Asp Ser Pro Glu Phe Trp Ala Phe Ser Phe Asp Glu Met Ala Glu Tyr Asp Leu Pro Ser Thr lie Asp Phe lie Leu Arg Arg Thr Gly Gin Lys Lys Leu His Tyr Val Gly His Ser Gin Gly Thr Thr lie Gly Phe lie Ala Phe Ser Thr Ser Pro Thr Leu Ala Glu Lys lie Lys Val Phe Tyr Ala Leu Ala Pro Val Ala Thr Val Lys Tyr Thr Lys Ser

Leu Phe Asn Lys Leu Ala Leu lie Pro His Phe Leu Phe Lys lie lie

Phe Gly Asp Lys Met Phe Tyr Pro His Thr Phe Leu Glu Gin Phe Leu

Gly Val Glu Met Cys Ser Arg Glu Thr Leu Asp Val Leu Cys Lys Asn

Ala Leu Phe Ala lie Thr Gly Val Asp Asn Lys Asn Phe Asn Met Ser

Arg Leu Asp Val Tyr lie Ala His Asn Pro Ala Gly Thr Ser Val Gin

Asn Thr Leu His Trp Arg Gin Ala Val Lys Ser Gly Lys Phe Gin Ala

Phe Asp Trp Gly Ala Pro Tyr Gin Asn Leu Met His Tyr His Gin Pro

Thr Pro Pro lie Tyr Asn Leu Thr Ala Met Asn Val Pro lie Ala Val

Trp Ser Ala Asp Asn Asp Leu Leu Ala Asp Pro Gin Asp Val Asp Phe

Leu Leu Ser Lys Leu Ser Asn Leu lie Tyr His Lys Glu lie Pro Asn

Tyr Asn His Leu Asp Phe lie Trp Ala Met Asp Ala Pro Gin Glu Val

Tyr Asn Glu lie Val Ser Leu Met Ala Glu Asp Lys Lys

MATERIALS AND METHODS

Lipase measurement and unit definition

Lipase activity is measured through a potentiometric titration at a constant pH. Butyric acid is released by the action of the lipase enzyme on the tributyrin substrate. The butyric acid is titrated with sodium hydroxide (NaOH) to maintain the pH at a constant value of 6.2 at 42.0°C. The activity of a lipase sample is directly proportional to the volume of NaOH required to maintain the constant pH during a given time period.

One DSM lipase unit is defined as the amount of lipase activity which liberates 5.0 micromoles of butyric acid per minute under the conditions of this assay. The activity of lipase samples is calculated by the following formula:

RxNxDFxlO ³

• R : the titrant delivery rate in ml/min

• N: Normality of titrant

• DF: Dilution factor • W : the weight in grams of final diluted sample used in assay

All chemicals used were analytical grade. The method described in FCC III (Food Chemical Codex; General Test and Apparatus, p. 493) was followed with the following exception: 0.80 gram of sodium caseinate and 1 ml of hydoxylated lecithin (10% solution) were used in substrate preparation.

Fatty acid profile determination

HS-SPME GC-MS fatty acid profile was carried out by injecting on an Agilent 6890/5973 GCMS with a ZB-FFAP (30m x 0.25mm ID x 0.25μηη) column. Sample introduction was accomplished using a CTC Analytics CombiPal Autosampler. For fatty acid analysis 3 gram of grated cheese was added to 20 ml vials in triplicate along with 3ppm heptadecanoic acid as internal standard. Vials were equilibrated for 10 min at 1 10°C with 4 second pulsed 250 rpm agitation. A single 30 μιτι PDMS 1 cm fiber was used for all analysis. The SPME fiber was exposed to the samples for 40 min at depth 3.1 cm. The fiber was retracted and injected at 5.0 cm in the GC inlet for 10 minutes. The oven program was 100°C for 2 minutes followed by a 10°C/min ramp to 245°C with a hold of 13.5 minutes. The injector was kept 250°C and set to splitless mode. A constant flow rate of 1 ml/min was maintained throughout.

FFA results were presented as μιτιοΙ of each free fatty acid per kg cheese (e.g. Table 3 and 10). In order to understand specificity of lipases, mean relative concentration of each FFA was divided by total FFA amount and stated as % μηηοΙ/total (Table 4 and 1 1 ).

Beside % μιτιοΙ FFA generation, the ratio (Rspec) of sum of C4-C10 to sum of C12-C18:3 could also be used for comparing specifity of each lipase

(Table 5 and 12). The Rspec is calculated as follows:

∑C4 - C10

R ^{spec ~} ∑C12 - C18: 3

Descriptive Sensory Analysis

Descriptive analysis of flavor used a 15 point universal intensity scale with the SpectrumTM method (Meilgaard, M., Civille, G., Carr, B. 1999. Descriptive analysis techniques in M. Meilgaard, G. Civille, and B. Carr, Sensory evaluation techniques (pp. 173-183). Boca Raton: CRC Press; Drake, M.A. and Civille, G.V. 2003. Flavor Lexicons. Compr. Rev. Food Sci. 2(1 ):33-40) and a previously established cheese flavor sensory language (Drake, M.A., Mclngvale, S.C., Cadwallader, K.R., and Civille, G.V. 2001 . Development of a descriptive sensory language for Cheddar cheese. J Food Sci. 66:1422-1427; Drake, M.A., Keziah, M.D., Gerard, P.D., Delahunty, CM. Sheehan, C, Turnbull, R.P. and Dodds, T.M., 2005. Comparison of cross-cultural differences between lexicons for descriptive analysis of Cheddar cheese flavor in Ireland, New Zealand, and the United States. International Dairy Journal. 15:473-483; Carunchia Whetstine and others 2003; Liggett, R., Drake, M.A., Delwiche, J. 2008. Impact of flavor attributes on consumer liking of Swiss cheese. J. Dairy Sci. 91 :466-476). Paper ballots were used.

A descriptive sensory panel (n=10, 9 females, 1 male, ages 22 - 46 y) with more than 200 h experience with the descriptive analysis of Cheddar cheese flavor (and more than 75 h experience each with Swiss and pasta filata cheeses) evaluated the cheeses. Consistent with SpectrumTM descriptive analysis training, panelists were presented with reference solutions of sweet, sour, salty, and bitter tastes to learn to consistently use the universal intensity scale (Meilgaard et al. 1999; Drake and Civille 2003). Following consistent use of the Spectrum TM scale with basic tastes, panelists learned to identify and scale flavor descriptors using the same intensity scale through presentation and discussion of flavor definitions, references and a wide array of cheeses. Discussion and evaluation of a wide array of cheeses (Provolones and other cheeses) was also conducted during training to enable panelists to consistently differentiate and replicate samples. Analysis of data collected from training sessions confirmed that panel results were consistent and that terms were not redundant, consistent with previous use of the developed language (Drake et al. 2001 ; Drake et al. 2005). Each panelist evaluated cheese from each treatment replication in duplicate.

Statistical analysis of data was done by a general linear model analysis of variance with Fisher's least significant difference (LSD) as a post hoc test (SAS version 9.1 , Cary, NC) as well as ANOVA with Fisher's (LSD) and modeled using Principle Component analysis (PCA) (XLSTAT version 201 1 .2.01 , New York, NY).

EXAMPLES

Example 1

Isolation of Water Buffalo Lipase (WBL) from water buffalo pharyngeal tissue (tongue root or gullet)

Frozen water buffalo tongue roots were ground in industrial scale meat grinder (chipper/grinder) in a buffer at <10°C. The ratio of gullet weigth to buffer volume was 1 :20 to 1 :60. Buffer pH was adjusted to <5.5 and held for minimum 2 hours with constant mixing. After 2 hours, buffer pH was adjusted to 9.0-10.0 and held for another 3 hours under constant agitation. When pH of buffer shifted out of 9.0-10.0, it was adjusted back into the range.

Agitation was stopped and tissues and fat layer were allowed to settle at bottom of tank and top. Extract containing lipase was decanted (extract 1 ). The decanted amount of fresh buffer at pH 9.0-10.0 was added back into the left over tissue and mixed for 5 hours. Agitation was stopped and allowed tissues and fat seperation (extract 2) for 3 hours. Both extracts were combined.

Combined extract was passed thorugh serious of filter starting from 100 μ, 5 μ and 1 μ respectively. After filtration, the extract was concentrated by passing through ultrafiltration with 10kDa cutoff. The final concentrated extract was blended with NaCI to arrive at 12g salt /100 ml for further processing. Frozen concentrate was thawed and 5%(w/w) whey powder and NaCI were added. The final blend was dried in a vacuum oven at <1 10°C and <30 Hg pressure.

Example 2

Production of Provolone cheese using buffalo lipase

Provolone cheese was manufactured in line with defined standard of identity for Provolone cheese in USA (21 CFR133.181 ). Based on 21 CFR133.181 , the minimum milk fat content is 45 percent by weight of the solids, and the maximum moisture content is 45 percent by weight. Milk was standardized by cream removal to 1 .8% milk fat level to meet Provolone cheese standard of identity. The standardized milk was pasteurized at 73°C for 19 sec. The milk was then split equally into 6 vats. Each vat randomly selected for addition of animal and microbial lipases and control (Table 1 ) .

The milk in the vats was then adjusted to a setting temperature of 34.5°C. The Ultra-Gro™ Direct™ Tempo 303 starter cultures and lipase enzymes were inoculated at a rate of 0.101 g/liter and a rate of 2.64 LU/liter cheese milk respectively. After 1 hour of ripening, Imperial CHEES-ZYME ^® was added at a rate of 78.5 IMCU/liter cheese milk. After coagulant addition the gel was allowed to form for a 10 minute time period. The gel is allowed to become very firm to incorporate more serum into the casein matrix in 20-25 minutes. This firm gel was cut with 6.35 mm knives. After 7 minutes of healing and 8 minutes of agitation, the curds and whey were cooked slowly over a 30 minute period, raising the temperature from 34.5°C to 41 °C. At the end of cooking the whey was completely separated at a curd pH of 5.85-6.0. The curd was pushed back to form a big slab before the draining process. When draining was complete, the curd slab was cut into 6 equal pieces and stacked two high immediately. The vat temperature was kept warm 41 °C. When pH of curd reached 5.20, slabs were milled to 2.54 cm x 2.54 cm x 10.16 cm size. Salt was added in two parts 5 minutes apart. The salted curd was taken into the cooker/stretcher machine for hot water kneading process.

Candida cylindracea and

6 KSHRASE ^® MBL 2.64

Rhizopus oryzae

The water temperature in the cooker/stretcher was kept at 62.7°C. The curd was held in the cooker/stretcher for about 10 minutes. The curd temperature after the cooker stretcher was 55.5°C. The provolone cheese placed in molding blocks immediately after the cooker/stretcher and placed into 5°C water for half an hour. Then the cheese was removed from molds and placed into 95% saturated brine solution at 7°C for 2 hours. The cheeses were vacuum packaged and ripened at 7°C until they were analyzed. Table 2. Chemical composition of Provolone cheese

The chemical composition and sensory properties of cheese were determined at 2 weeks and 1 month of ripening respectively. Table 2 shows chemical composition of cheese.

C18:1 0.18AB 0.18AB 0.1 1 BC 0.28A 0.21AB

C18:2 <0.0001 <0.0001 <0.0001 0.1 1A <0.0001

C18:3 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 total 606.27 574.79 392.75 431 .34 7.79

Table 3. Mean relative concentration of free fatty acid composition of Provolone cheese (μιτιοΙ/kg)

^* Values corrected from background free fatty acid concentrations in non-lipase added control Provolone Cheese

^** A-C Means that do not share common letters are significantly different. Significance lettering applies to values within row and not in column.

The Provolone cheese samples were analyzed for free fatty acid compositions (FFA) at 1 month (see Materials and Methods) and followed by decriptive sensory analysis. The FFA results are presented as μιτιοΙ of each free fatty acid per kg cheese (Table 3) and as % μηηοΙ/total (Table 4). The Rspec is summarized in Tabel 5.

Table 4. Mean relative concentration of free fatty acid composition of Provolone cheese (% μηιοΙ/total)

able 5. Specific ratio Rspec of lipases

Provolone cheeses were cut into 2 cm cubes for descriptive sensory analysis. The cheeses were placed into 58 mL souffle cups with three-digit codes. The cheeses were tempered at 10°C for one hour and were served at this temperature with spring water and unsalted crackers for palate cleansing. Sensory results are shown in Table 6.

Table 6. Descriptive flavor profiles of cheeses by trained panel

Sweet 2.3 2.0 2.0 2.4 2.3 2.5 0.3

Bitter 0.5 1 .2 1 .0 ND 0.6 ND 0.4

Umami 3.0 3.1 2.9 2.9 3.0 3.1 0.3

Prickle ND 1 .0 1 .2 ND ND ND 0.4

Soapy ND 0.5 1 .7 ND 0.9 ND 0.4

Cheeses were scored on a 0 to 15-point universal intensity scale (Spectrum method, Meilgaard et al., 1999). Most cheese flavors fall between 0 and 5 (Drake, M.A. 2004. Defining dairy flavors. J. Dairy Sci. 87:777-784.).

ND: Not detected

LSD: Least significant difference. Attribute means that differ by more than the LSD are different (p<0.05).

As can be seen from FFA analysis (Tables 3-5) and the sensory assesments (Table 6), WBL gives a different FFA profile and sensory profile compared to the other animal and microbial lipases (KSHRASE ^® MBL). The FFA results indicate that the total FFA generated by WBL was comparable to other animal lipases (Table 3). However, the total amount of FFA of from C4:C10 was the lowest among animal lipases (Table 5). WBL also generated statistically significant higher amount of C10 and longer chain FFA than other animal lipases (Table 5). WBL had the lowest Rspec value among animal lipases which means that WBL was the least specific for release of C4:C10 among other animal lipases. Even though the sensory panel results (Table 6) indicated that the FFA attributes were not statistically significant different from each other, WBL scored higher than ITALASE ^® C and KSHRASE ^® MBL.

In addition to the data in Table 6, the panellist observed a distinct FFA flavor for WBL compared to the other animal lipases, presumably as a result of the unique combination of generation of short, mid and long chain FFA. The kid lipase (CAPALASE™ K or KSHRASE ^® K) produces a peppery, lingering piccante flavor and has a sharp punch at first and then dissipates. Calf lipase (Italase™ C or KSHRASE ^® C) generates a delicate buttery, sweet, and mild piccante flavor. The lamb lipase (Capalase™ L or KSHRASE ^® L) produces a sharp, lingering peccorino flavor. The WBL flavor was perceived by the panel in between kid and calf and microbial. It tasted at the back of your tounge and had lingering flavor. All these flavor effects fall under the FFA attributes. So even though WBL has a numerical score of FFA 3.0 (Table 6) and is not statistically different than i.e Capalase L (3.6), it was established by the panel that WBL tasted differently than the other animal lipases.

Example 3

Production ofAsiago cheese using Water Buffalo Lipase

Asiago cheese was manufactured in line with defined standard of identity for Asiago medium cheese in USA (21 CFR133.103). Based on 21 CFR133.103, the minimum milk fat content is 45 percent by weight of the solids, and the maximum moisture content is 35 percent by weight and it is cured for not less than 6 months. The milk was pasteurized at 73°C for 19 sec. The milk was then split equally into 6 vats. Each vat randomly selected for addition of animal and microbial lipases and control (Table 7) .

Table 7. Addition of li ases into cheese milk

The milk in the vats was then adjusted to a setting temperature of 33°C. The Ultra-Gro™ Direct™ TD-17 and the Ultra-Gro™ Direct™ TD-MN starter cultures and lipase enzymes were inoculated at a rate of 0.158 g/liter, 0.233 g/liter and a rate of 2,33 LU/liter cheese milk respectively. After 1 hour of ripening, Imperial CHEES-ZYME® was added at a rate of 71 .43 IMCU/liter cheese milk. After coagulant addition the gel was allowed to form for a 10 minute time period. The gel is allowed to become very firm to incorporate more serum into the casein matrix in 20-25 minutes. This firm gel was cut with 6.35 mm knives. After 7 minutes of healing and 8 minutes of agitation, the curds and whey were cooked slowly over a 30 minute period, raising the temperature from 33°C to 46°C. At the end of cooking the whey was completely separated at a curd pH of 6.2. After completion of draining whey, salt was added in two parts 5 minutes apart. The salted curd was put into forms and pressed under 40 psi pressure for 30 minutes. Then cheeses were turned and pressed again for 90 min under 40 psi pressure. The cheeses were removed from forms and placed into 95% saturated brine solution at 7°C for 3 days. The cheeses were cured in drying room at 22°C for 3 weeks untill they reached desired moisture content. After curing cheeses were vacuum packaged and ripened at 7°C until they were analyzed. The chemical composition of cheese were determined at 1 month of ripening. Table 8 shows chemical composition of cheese.

Table 8. Chemical composition of Asiago cheese

Asiago cheese samples were analyzed for free fatty acid compositions (FFA) at 1 month and after 6 months, followed by decriptive sensory analysis - see Materials and Methods. The FFA results are presented as μιτιοΙ of each free fatty acid per kg cheese (Table 9 & 13) and as % μηηοΙ/total (Table 10 & 14). The Rspec is summarized in Tabel 1 1 & 15.

Table 9. Mean relative concentration of free fatty acid composition of Asiago cheese (μιτιοΙ/kg) after 1 month

*Values corrected from background free fatty acid concentrations in non-lipase added control Asiago

**A-C Means that do not share common letters are significantly different. Significance lettering applies to values rows and not in columns.

Table 10. Mean relative concentration of free fatty acid composition

Asiago cheese (% μιηοΙ/total) after 1 month

able 11. Specific ratio R _sp ec of lipases after 1 month

Table 12. Descriptive flavor profiles of cheeses by trained panel after 1 month aging

Cheeses were scored on a 0 to 15-point universal intensity scale (Spectrum method, Meilgaard et al., 1999). Most cheese flavors fall between 0 and 5 (Drake, 2004).

ND: Not detected; NA: Not applicable;

LSD: Least significant difference.

Attribute means that differ by more than the LSD are different (p<0.05).

As can be seen from the FFA analysis (Tables 9-1 1 ) and sensory assesments (Table 12), WBL gives a different FFA profile and sensory profile compared to the other animal and microbial lipases. The FFA results indicate that the total FFA generated by WBL was comparable to other animal lipases and higher than Piccantase ^® A (Table 9). WBL had the lowest Rspec value among animal lipases which means that WBL was the least specific for relase of C4:C10 among other animal lipases (Table 1 1 ). All these results are similar to those obtained with the Provolone cheese in Example 2.

Also the flavour attributes as summarized in table 12 are comparable with those obtained for the Provolone cheese in Table 6, including the additional flavour effects described for the various animal lipases in the last paragraph of Example 2.

Table 13. Mean relative concentration of free fatty acid composition of Asiago cheese (μιτιοΙ/kg) after 6 months

Table 14. Mean relative concentration of free fatty acid composition of Asiago cheese (% μιτιοΙ/total) 6 months

Table 15. Specific ratio R _sp ec of lipases after 6 months aging

Sample Rs ec Sum (C4:C10) Sum(C12:C18:3)

Piccantase ^® A 1 1.3 91.92% 8.08%

CAPALASE™ L 9998 99.98% 0.01 %

CAPALASE™ K 1427 99.93% 0.07%

ITALASE® C 139.8 99.29% 0.71 %

WBL 63.1 98.44% 1 .56%

Table 16. Descriptive flavor profiles of cheeses by trained panel after 6

As can be seen from the FFA analysis (Tables 13-15) and sensory assesments (Table 16), WBL gives a different FFA profile and sensory profile compared to the other animal and microbial lipases. The FFA results indicate that WBL had the lowest Rspec value among animal lipases which means that WBL was the least specific for relase of C4:C10 among other animal lipases (Table 1 1 ). Thus, the use of buffalo lipase for the aging of asiago cheeses provides a FFA profile which is more balanced between short, mid and longer chain FFA's. Further, it is remarkable that the perception of prickle as perceived by the panel was significantly higher after 1 or 6 months, providing a advantageous feeling of a trigeminal stimulation and a reminiscent of hot pepper burn. Prickle is one of the sensory attributes that is known to be caused by lipase. However, is was not expected that, although the water buffalo lipase originates from a relative of bovine calf, the water buffalo lipase provides a different FFA profile and sensory profile.

Example 4 LC-MS analysis of lipase preparations

Sample preparation

Approximately 50 mg of both ITALASE ^® C and the WBL as provided by example 1 were dissolved in 1 ml MilliQ water. The samples were incubated at 4°C for 10 minutes before brief centrifugation at 14.000 rpm to remove insoluble material. The protein in 20 μΙ of the supernatant was precipitated with TCA by 1 :1 dilution with 20% TCA and incubation at 4°C for 30 minutes, before centrifugation for 10 minutes at 14.000 rpm, 4°C. The supernatant was removed and the pellet was washed with 200 μΙ acetone at -20°C. Samples were again centrifuged for 10 minutes at 14.000 rpm, 4°C, the supernatants were removed. Protein pellets were dissolved in 50 μΙ 50 mM NaOH and diluted with 450 μΙ 100 mM NH4HCO3.

To each pellet 5 μΙ 500 mM tris(2-carboxyethyl)phosphine (TCEP)was added followed by 30 min incubation at room temperature. 5 μΙ 550 mM lodo acetamine (IAA) was added followed by 30 min incubation at room temperature in the dark. 2 μΙ 500 mM dithiotreitol was added followed by 10 min incubation at room temperature. 20 μΙ 250 μg ml trypsin (Gold, Promega) was added followed by overnight incubation at 37°C. Finally, 2.5 μΙ 250 μg ml trypsin was added followed by 3h incubation at 37°C

LC-MS/MS analysis

The digested samples were analyzed on an Accela - LTQ-Velos (Thermo Fisher) UHPLC-MS/MS. Samples (25ul) were injected on a Waters Acquity UPLC BEH130 C18 1 .7μηη 2.1 x 100mm Column, the column oven was set to 50 °C. Mobile phase A was: Formic Acid 0,1 % in Water (UHPLC-MS Grade, Biosolve) and mobile phase B was Formic Acid 0,1 % in Acetonitrile (UHPLC-MS Grade, Biosolve). A gradient of 70 minutes from 5-30% B was ran, followed by column washing at 80% B and column re-equilibration at 5% B. The total runtime was 90 minutes. The MS detector settings were: Nth order Double Play: MS 300- 2000 m/z in enhanced mode and MS/MS on top 10 peaks from MS scan Dynamic exclusion was on with repeat count 2, rejection time 10 seconds. Charge states 1 + and 4+ and up were rejected.

Extracts from the NBCI database of the protein sequences in taxonomies Bos taurus and Bubalus bubalis were prepared. The Italase C data was searched against the Bos taurus database and the buffalo sample was searched against the Bubalus bubalis database. The results were combined in Scaffold and the relative protein quantities were calculated based on spectral counts.

From this LC-MS analysis it becomes apparent that there are many different proteins present in both ITALASE ^® C and WBL. With the LC-MS method we could identify approximately 100 proteins in both preparations, of which many proteins are shared. In both preparations only approximately 1 .5% of the total number of identified peptides could be linked to the pregastric triacylglycerol lipase, which is thought to be responsible for the major lipase activity in these preparations. The size of the WBL was 45 kDa. Further, the WBL preparation comprises 0.4% of a liver carboxylesterase like protein having a molecular weight of 58 kDa.

When the bovine pregastric triacylglycerol lipase protein sequence (NP_776528.1 ) is directly compared with the buffalo pregastric triacylglycerol lipase protein sequence (XP_006058496) using a BLASTp comparison it becomes clear that both proteins are identical is size (397 aa) and 99% identical in amino acid sequence, see SEQ ID NO 1 and 2.

Table 16. Sequences

SEQ ID NO 1 Pregastric lipase from WBL (Bubalus bubalis)

SEQ ID NO 2 Pregastric lipase from calf (Bos taurus)