End of Project ReportEnzyme-modified cheeses (EMCs) are defined as concentrated cheese
flavours produced enzymatically from cheeses of various ages and are principally used as an ingredient in processed foods, where they provide a cost-effective
alternative to natural cheese. They can be used as the sole source of cheese flavour to
intensify an existing cheese taste, or to impart a specific cheese character to a more bland
product. Their main applications are in processed cheese, analogue cheese, cheese
spreads, snack foods, soups, sauces, biscuits, dips and pet foods. Their main advantages
over other cheese flavour ingredients are: low production costs, consistency, high flavour
intensity, diverse flavour range, extended shelf- life, low storage costs and increased
functionality.
EMCs are generated utilising the same flavour pathways that occur in natural cheese
ripening i.e. proteolysis, lipolysis and glycolysis. They are not as easy to differentiate as
natural cheeses, as they are characterised by flavour and aroma alone as texture is not a
factor in EMC production. The relationship of the flavour of EMCs to the flavour of the
corresponding natural cheese remains unclear. This is especially true for Cheddar EMC
which is commercially available in a range of  Cheddar flavours . Despite the fact that a
wide range of commercial EMCs are available, there is very little detailed information
available regarding their properties or the specific production processes used.
The main objective of this research was to build a knowledge base on EMC products and
to utilise this to develop a biotechnological process for the production of improved enzyme
modified cheeses for use as flavour ingredients. The strategy was to establish quantitative
relationships between the compositional, proteolytic and lipolytic parameters and the
sensory characteristics of EMCs.  This data would then be used to develop a predictive
model for flavour development in EMC production and the subsequent generation of an
optimised EMC process enabling the generation of a range of cheese flavours from single
or multiple substrates.Department of Agriculture, Food and the Marin

Kilcawley, Kieran

Mulholland, E.

Wilkinson, M.G.

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DAIRY PRODUCTS RESEARCH CENTREMOOREPARKEND OF PROJECT REPORT 1999:    DPRC No. 10Enzyme Modified Cheese FlavourIngredientsDr. M. Wilkinson and Mr. K. KilcawleyThese studies provided an in-depth understanding of the keyprocess parameters and methods of flavour production usedin the manufacture of enzyme modified cheese (EMC)products, thereby facilitating the successful production ofthese cheese flavour ingredients.The Science of Farming and Foodprincipally used as an ingredient in processed foods, where they provide a cost-effectivealternative to natural cheese.  They can be used as the sole source of cheese flavour tointensify an existing cheese taste, or to impart a specific cheese character to a more blandproduct.  Their main applications are in processed cheese, analogue cheese, cheesespreads, snack foods, soups, sauces, biscuits, dips and pet foods.  Their main advantagesover other cheese flavour ingredients are: low production costs, consistency, high flavourintensity, diverse flavour range, extended shelf- life, low storage costs and increasedfunctionality.EMCs are generated utilising the same flavour pathways that occur in natural cheeseripening i.e. proteolysis, lipolysis and glycolysis.  They are not as easy to differentiate asnatural cheeses, as they are characterised by flavour and aroma alone as texture is not afactor in EMC production.  The relationship of the flavour of EMCs to the flavour of thecorresponding natural cheese remains unclear.  This is especially true for Cheddar EMCwhich is commercially available in a range of Cheddar flavours.  Despite the fact that awide range of commercial EMCs are available, there is very little detailed informationavailable regarding their properties or the specific production processes used.The main objective of this research was to build a knowledge base on EMC products andto utilise this to develop a biotechnological process for the production of improved enzymemodified cheeses for use as flavour ingredients. The strategy was to establish quantitativerelationships between the compositional, proteolytic and lipolytic parameters and thesensory characteristics of EMCs.  This data would then be used to develop a predictivemodel for flavour development in EMC production and the subsequent generation of anoptimised EMC process enabling the generation of a range of cheese flavours from singleor multiple substrates.Main Conclusions and AchievementsHigh levels of proteolysis and lipolysis are an integral feature of Cheddar EMC products.However, most commercial products tested were not perceived as having a Cheddar cheeseflavour profile.  Manipulation of proteolytic, lipolytic and compositional parameters, as wellas the addition of flavour potentiators, organic acids and bulking agents were identified asbeing used to produce Cheddar EMC products of different characteristics.A number of commercial enzyme preparations were identified for their flavour generatingpotential in EMC systems.  Commercial proteinase, peptidase and lipase preparations frommicrobial and animal sources were found to contain large differences in active enzymes andprotein content.A laboratory scale EMC system was developed and successfully used to assess the de-bittering potential of a combination of purified peptidase enzymes (aminopeptidases).A prototype pilot plant EMC process has been developed and used to generate Cheddarflavoured EMC products.32This report is based on two complementary projects:* Development of flavour ingredients for processed consumer foods through the use of enzymetechnology (Armis No. 4338)* Process optimisation for the generation of a range of hydrolysed dairy fractions for use in theflavouring of processed consumer foods (Armis No. 4540)(Enzyme Modified Cheese Flavour Ingredients)Project Team:M. Wilkinson (Leader)K. KilcawleyE. MulhollandThe Dairy Products Research CentreMoorepark, Fermoy, Co. Cork.Teagasc acknowledges with gratitude grant aid under the Food Sub-Programme (Sub-Measure 3 (ii) - Institutional R&D) of the OperationalProgramme for Industrial Development.  The Programme is managed by theDepartment of Agriculture and Food supported by EU and national funds.ISBN:  1 901138  62 3DPRC No. 10© Teagasc, September 2000Teagasc 19 Sandymount Avenue EUROPEAN UNIONBallsbridge Dublin 4 European RegionalDevelopment FundSummary andConclusionsEnzyme-modified cheeses (EMCs) are defined as concentrated cheeseflavours produced enzymatically from cheeses of various ages and areThe Science of Farming and FoodDatabase of Cheddar EMC productsInitially, a wide range of EMC flavours were investigated, such as Blue, Swiss, Gouda,Mozzarella and Parmesan.  However, because of its commercial importance in Ireland itwas decided to focus on Cheddar EMC.  Cheddar EMC was also of interest because it isavailable as a range of purported Cheddar flavours and intensities from differentmanufactures.Fifteen commercial Cheddar EMC products were obtained from eight suppliers in paste andpowdered formats.  The compositional parameters analysed were moisture, protein, fat,NaCl, pH, ash, phosphate and calcium.  Proteolysis was measured by urea-PAGE, thepercentage of total N soluble in water at pH 4.6 (pH 4.6 WSN) and 5% phosphotungsticacid (PTA-N), the molecular mass distribution of soluble peptides in the pH 4.6 watersoluble extract (pH 4.6 WSE), the free amino acid content (FAA) of the pH 4.6 WSE, andpeptide profiles by Reverse Phase -HPLC of pH 4.6 WSE.Results shown in Table 1 are the averages of the compositional parameters analysed forpaste Cheddar EMC products and for typical natural Cheddar cheeses.  Table 2 shows theaverage proteolytic data from these same products.  The compositional and proteolyticindices varied widely, with most products having high levels of proteolysis.  It was evidentfrom these results that manipulation of both compositional and proteolytic parameters is afeature of Cheddar EMCs. The use of emulsifying salts, flavour potentiators, bulking agents,cheeses of varying fat and/or the addition of exogenous protein or fat was also indicated inthese commercial products.  This work is presented in full in Kilcawley et al. (2000).Table 1: Compositional data for commercial Cheddar EMC pastes and typical natural Cheddar cheeseMoisture Protein           Fat NaCl pH(%) (mg/g*)        (mg/g*) (mg/g*)n          Av       sd     Av       sd     Av       sd    Av      Sd    Av     sdCheddar EMC 13        54.4     7.9    35.1    7.8    52.2   10.9    7.4     4.6     4.8     0.7Cheddar cheese 4       36.5     1.0    40.6    1.1    54.0    2.3    2.8    0.2     5.0    0.1* dry wt basisTable 2: Proteolytic data for commercial Cheddar EMC pastes and typical natural Cheddar cheesesWSN PTA FAA (%) (%) (mg/g*)n     Mean        sd       Mean       sd Mean       sdCheddar EMC                         13    49.7 18.5 26.7 21.9 60.7 40.7Cheddar cheese                       4 22.2 6.9 5.6 2.8 22.3 13.9* dry wt basisWSN = pH 4.6 water soluble nitrogen. PTA = 5% Phosphotungstic acid soluble nitrogen.FAA = Free amino acid.5Research and ResultsBasis of EMC productionThe basis of EMC production is the utilisation of specific enzymes, under optimumconditions, to produce intense cheese flavours rapidly from dairy substrates (usually cheesecurd).  The technology was developed in the late 1960s where fresh curd and a NaClsolution were mixed to make an emulsion of approximately 40% solids.  Enzymes andpreservatives were added and the mixture was incubated under controlled conditions, afterwhich characteristic cheese flavours developed.  This work demonstrated the potential ofgenerating a range of intense cheese flavours in a short time from cheese substrates bymodification of process parameters.Many commercial procedures are based on similar principles and in general, productioninvolves incubating mature or immature cheese with specific exogenous enzymes and/ormicro-organisms, terminating the process by heat treatment, and standardising the finalproduct to a desired flavour intensity and composition.  EMCs are generally producedeither by a one step process (Fig 1 - Page 10), in which hydrolysis of fat and protein occursimultaneously, or by a component approach (Fig 2 - Page 11), where several differentflavour components are created separately and then blended together.  The componentapproach allows greater flexibility, facilitating greater diversity of products.The actual processing conditions used depend upon many factors and directly relate to theenzymes being used.  In general, EMCs are produced between 12 to 72h in a temperaturerange of 30 - 45°C, and between  pH of 5 - 7 (Kilcawley et al., 1998).  Once a product hasbeen produced it may be formulated to a desired composition depending upon its finalapplication.  The pH may be altered to improve storage stability, or flavour, while bulkingagents or carriers may be added to increase yield, obtain a desired flavour intensity or tofacilitate spray drying.Experimental ApproachThe major objective of this research was to develop a biotechnological process for theproduction of EMCs for use as flavour ingredients.The steps to achieving this objective involved:- Compilation of a database of the compositional, proteolytic, lipolytic indices and sensoryevaluation of a range of commercial EMC products- Compilation of a database of the enzyme activities (proteinase, peptidase, lipase andesterases) present in commercial enzyme preparations used in the manufacture of EMCs - Development of a laboratory-scale EMC process to investigate the flavour generatingand/or de-bittering potential of various enzyme combinations- Development of a prototype pilot biotechnological process for the manufacture of EMC4API-ZYM test method.  Each preparation was also analysed for specific proteinase activityat pH 5.5 and pH 7.0 at 37°C using azocasein.  Proteinases of interest were further evaluated in a sodium caseinate system, monitoringhydrolysis by urea-PAGE and RP-HPLC and free amino acid analysis and assessed forspecific peptidase, lipase and esterase content at pH 7.0 at 37°C.  Peptidase preparationswere assessed for a number of specific peptidase activities; at both pH 7.0 and pH 5.5 at37°C (general amino peptidase activity; leucine aminopeptidase, Post-proline dipeptidylaminopeptidase; PepX, proline imino-peptidase; PepI, aminopeptidase A; PepA,aminopeptidase P; PepP, aminopeptidase M; PepM, endoproteinase O; PepO,carboxypeptidase and endopeptidase activity).Lipase preparations were assessed for both esterase and lipase activity at pH 7.0 at 37°C.Brief summary of evaluationsProteinasesThe protein content of the proteinase preparations was found to vary as was their specificproteinase activities. All preparations were found to contain other contaminating sideactivities.  The preparations derived from Aspergillus and Rhizomucor species containedmore peptidase side activities than the preparations derived from Bacillus species.  Onlytwo products contained lipase activity, but most contained esterase activity.Bacillus species had higher proteinase activity at pH7.0 and Aspergillus and Rhizomucorspecies had higher proteinase activity at pH5.5.PeptidasesThe protein content and proteinase activities of these preparations were also found to vary.All preparations contained varying amounts of different specific peptidase activities, withhigher activities at pH 7.0 than at pH 5.5. Most preperations contained contaminatingenzyme side activities.In general those derived from Aspergillus had the highest levels of generalaminopeptidase activity, with those derived from Lactococcus lactis having the highestlevels of PepX activity.LipasesThe protein content of the lipase preparations was found to vary.  Little or no peptidaseactivity was determined in these preparations.  Large differences were determined in theesterase and lipase activities of each preparation.Novel purified enzymesThe de-bittering potential of a combination of purified aminopeptidases was evaluated ina laboratory-scale EMC system.  A curd slurry substrate was inoculated with a high dosage76The free fatty acid (FFA) and lactic acid contents of these same Cheddar EMC productswere also quantified.  The lactic acid content was determined using a UV test kit method.Short chain volatile FFA (C2:0, C3:0, C4:0) were extracted by steam distillation andquantified using ion - partition HPLC,  medium and long chain FFAs (C6:0, C8:0, C10:0,C12:0, C14:0, C16:0, C18:0, C18:1, C18:2, C18:3) were isolated, esterified and quantified by RP-HPLC.  Results for FFA and lactic acid contents of the paste Cheddar EMCs and naturalcommercial Cheddar cheeses are summarised in Table 3.  These results confirm that levelsof lipolysis were higher in commercial Cheddar EMCs than in natural Cheddar cheese.Levels of lipolysis varied widely between individual EMCs indicating that manipulation oflipolysis is used to generate a range of Cheddar EMC products (in addition to manipulationof composition and proteolysis).  Addition of exogenous acetic acid, butyric acid and lacticacid was evident in certain EMCs and indicates their possible uses as flavour potentiatorsand/or to reduce pH.  (Kilcawley et al. [In Press]).Table 3: Levels of different acids in commercial Cheddar EMC pastes and typical natural CheddarcheeseAcetic Acid    Propionic Acid    Butyric Acid    Lactic Acid   Total FFA(mg/g*) (mg/g*) (mg/g*) (mg/g*)     (C6:0-C18:3)*(mg/g*)n Av      Sd          Av      Sd         Av      Sd        Av      Sd    Av      SdCheddar EMC 13  36.1   97.1        3.4     5.9       36.0    34.4     50.6   72.5  24.4    9.5Cheddar cheese 4    0.7     0.4         nd                  0.6      0.1     19.7     2.2    3.5    1.7*dry wt basisThe sensory characteristics of EMCs were determined by quantitative descriptive analysisby a trained sensory panel.  Due to the high flavour intensity, the commercial EMCs wereassessed in a bland cheese slurry at 10% (w/w) of its original concentration.  The sensoryprofiles of the Cheddar EMCs were found to be significantly different to the sensorycharacteristic used to describe natural Cheddar cheese.  Positive correlation was foundbetween flavour intensity and certain compositional parameters.  This study is described indetail in Hulin-Bertaud et al. (In Press).Database on commercial and novel enzymesFifty commercially available enzyme preparations were obtained from 10 different enzymesuppliers.  These preparations consisted of proteinases, peptidases, lipases or mixturesthereof.  All of the proteinases were derived from microbial sources (Bacillus, Aspergillus orRhizomucor spp.), as were the peptidases (Aspergillus, Rhizomucor or Lactococcus lactis).The lipases were derived from both animal (calf, kid and lamb) and microbial sources(Aspergillus, Rhizopus, Mucor, Candida or Penicillium spp.).All enzyme preparations were analysed for protein content, semi-quantitative tests for thepresence of lipase, proteinase, peptidase, phosphatase and carbohydrase activity, using theFig 2: EMC Process - Component Approach for the Production of Enzyme Modified Cheeses9of the commercial proteinase, Neutrase 0.5L (Novo Nordisk A/S) derived from Bacillussubtilis which had previously been shown to accelerate Cheddar cheese flavourdevelopment, but accumulate bitterness.  Neutrase 0.5L was used in this study to generatean excessively bitter curd slurry which was then treated with a combination of three purifiedpeptidases isolated from Lactococcus lactis subsp. cremoris AM2; aminopeptidase KpNA-H(lysyl-nitranilide hydrolyase), PepX and PepP.  KpNA-H cleaves all amino acids from the N-terminal of peptides up to the imido bond (the peptide bond preceding the proline residue).The proline present in the second position was then removed by the action of PepX, whichalso enables the general aminopeptidase to continue to hydrolyse the peptide.  However ifthe imido bond is followed by consecutive proline residues (e.g. in positions 2 and 3) PepXcannot remove the Pro-X dipeptide due to the presence of this second proline.  PepP canhowever, remove the amino acid preceding proline (position 1) which then enables PepXto remove the Pro-Pro dipeptide, thus allowing the general aminopeptidase to continue tohydrolyse the peptide and reduce bitterness.  Addition of these purified peptidases wereshown to significantly reduce bitterness (~ 40%) in the Neutrase-treated cheese slurry. Thiswork has demonstrated the potential of utilising purified peptidases to alleviate the problemof bitterness in specific applications, such as in EMC manufacture.Development of a prototype EMC processPreliminary work to develop a prototype pilot plant EMC process has utilised theknowledge gained of commercial Cheddar EMCs, commercial enzymes and the novellaboratory scale de-bittering process.  Preliminary trials of this process have enabled thesuccessful production of Cheddar and Blue EMC flavours at laboratory scale and CheddarEMC flavour at pilot scale.Fig 1: EMC Process - One Step Approach for the Production of Enzyme Modified Cheeses 811SubstrateCheese curdMixProteases /PeptidasesProcessParameters(Temperature,time and pH)Water,EmulsifiersSlurry,PasteuriseIncubate and MixStop,Pasteurise andFormulateSubstrateButter fatMixLipases/EsterasesProcess Parameters(Temperature, timeand pH)Water,EmulsifiersSlurry,PasteuriseIncubate and MixStop,Pasteurise andFormulateBlend10SubstrateCheese curd, butter fatMixEnzyme(s),Flavour Potentiators(optional)Process Parameters(Temperature, timeand pH)Water, EmulsifiersSlurry, Pasteurise PasteuriseIncubate and MixStop reaction.Pasteurise andFormulateDAIRY PRODUCTS RESEARCH CENTREMoorepark, Fermoy, Co. Cork, IrelandTel: +353 (0) 25 42222 - Fax: +353 (0) 42340E-mail: reception@moorepark.teagasc.ieDr. M. Wilkinson Mr. K. KilcawleyThe Science of Farming and FoodPublicationsKilcawley, K.N., Wilkinson, M.G. and Fox, P.F. (1998).  Enzyme-modifiedcheese. International Dairy Journal 8, 1-10.Kilcawley, K.N., Wilkinson, M.G. and Fox, P.F. (2000).  A survey of thecompositional and proteolytic indices of commercial enzyme-modifiedCheddar cheese. International Dairy Journal 10, 181-190.Hulin-Bertaud, S., Kilcawley, K.N., Wilkinson, M.G. and Delahunty, C.M.(In Press).  Determination of the sensory and compositional relationshipsbetween commercial enzyme modified Cheddar cheeses and naturalCheddar cheeses. Journal of Food Science.Kilcawley, K.N., Wilkinson, M.G. and Fox, P.F. (In Press).  A survey of thelipolytic and glycolytic end products in commercial Cheddar enzyme-modified cheese. Journal of Dairy Science.For further information, please contact Dr. Martin Wilkinson or Mr. KieranKilcawley

Enzyme Modified Cheese Flavour Ingredients

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