End of Project ReportNatural cheese flavour ingredients, in the form of enzyme modified
cheeses (EMCs), are widely used in the convenience food industry
and can provide high volume added opportunities for the cheese
industry.
Many EMCs are produced using commercial enzyme preparations
and previous studies have indicated that they contain side activities in
addition to their stated main activity (see DPRC Report No.10).
Therefore, it is critical that the exact enzyme complement of these
preparations are known before they can be used to produce EMC of
specific requirements on a consistent basis.
The scientific basis of rapid enzyme mediated flavour formation in the
production of EMCs is not fully understood. Consequently this
knowledge gap is a major obstacle in the development of high value
cheese flavour ingredients.
Hence, a major objective of this project was to deepen the scientific
understanding of flavour formation with a view to the production of
natural enzyme-mediated dairy flavour ingredients with commercial
potential.
The ultimate aim was to develop the technology to produce
customised high value dairy flavour ingredients in an optimised
process.Dairy LevyDepartment of Agriculture, Food and the Marin

Beresford, Tom

Kilcawley, Kieran

Lee, B.

Wilkinson, M.G.

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A model system was developed and successfully used to generate a range of Cheddar-type enzyme-modified cheese (EMC) flavours from base dairy ingredients usingselected commercial enzymes and lactic acid bacteria. This process can be used at laboratory scale to screen enzymes and lactic acid bacteriafor their flavour generating capacity, or scaled up to produce EMC products.  Theprocess is easily modified to create a myriad of natural cheese-type flavours.DAIRY PRODUCT RESEARCH CENTREMOOREPARKEND OF PROJECT REPORT 2001  DPRC No. 45Model System for the Production ofEnzyme Modified Cheese (EMC) FlavoursMr. Kieran Kilcawley and Dr. Tom Beresford Summary and ConclusionsNatural cheese flavour ingredients, in the form of enzyme modifiedcheeses (EMCs), are widely used in the convenience food industryand can provide high volume added opportunities for the cheeseindustry.Many EMCs are produced using commercial enzyme preparationsand previous studies have indicated that they contain side activities inaddition to their stated main activity (see DPRC Report No.10).Therefore, it is critical that the exact enzyme complement of thesepreparations are known before they can be used to produce EMC ofspecific requirements on a consistent basis.The scientific basis of rapid enzyme mediated flavour formation in theproduction of EMCs is not fully understood.  Consequently thisknowledge gap is a major obstacle in the development of high valuecheese flavour ingredients. Hence, a major objective of this project was to deepen the scientificunderstanding of flavour formation with a view to the production ofnatural enzyme-mediated dairy flavour ingredients with commercialpotential.The ultimate aim was to develop the technology to producecustomised high value dairy flavour ingredients in an optimisedprocess.Optimising Flavour Formation in Enzyme Mediated DairyFlavour Ingredient Systems(Model System for the Production of Enzyme Modified Cheese [EMC]Flavours)Armis No. 4771Project Team:Mr. K.N. Kilcawley and Dr. T. Beresford (Leaders)Prof. B. Lee*Dr. M.G. Wilkinson***   McGill University, Department of Food Science & Agriculture Chemistry,   Montreal / Agriculture & Agri-Food Canada, Food R&D Centre, St-Hyacinthe, Quebec, Canada** Department of Life Sciences, University of LimerickThe Dairy Products Research CentreMoorepark, Fermoy, Co. Cork.This project was primarily funded from the Dairy Levy.Teagasc also acknowledges with gratitude grant aid under the Food Sub-programme (Sub-Measure 3 (ii) - Institutional R&D) of the Operational Programmefor Industrial Development.  The Programme is managed by the Department ofAgriculture and Food supported by EU and national funds.ISBN 1 8417 258 7DPRC No.45'  Teagasc, April 2002Teagasc 19 Sandymount Avenue                                                     EUROPEAN UNIONBallsbridge Dublin 4                                              European Regional Development Fund32Development of an EMC Model SystemIn this study the main component of the substrate used for EMCproduction consisted of a high moisture, high pH, rennet cheese curd,produced from full fat milk without starter culture.  This rennet curdwas blended with anhydrous butterfat, de-ionised water, salt andemulsifying salts in a Stephan cooker until homogeneous andsubsequently heat-treated at 80…C for 20 min.  Rennet curd waschosen as it is cheap to produce and any flavour subsequentlydeveloped in the production processes could be directly attributed toadded enzymes rather than indigenous enzymes in the cheese curd.Anhydrous butterfat was added to provide more substrate for lipolysisas previous studies have shown that the level of lipolysis in EMCs islinked to flavour intensity.In the process, enzymes and/or lactic acid bacteria are added to thesubstrate in a reaction vessel with constant agitation under controlledtemperatures for a fixed period, after which the product is batch heat-treated to at least 80…C for 20 min.  This system can be used atlaboratory scale (1 - 2 kg) or production scale (1000 kg).  The choiceof processing conditions depends upon the enzymes or startercultures added, and the flavour required.A temperature of 45…C and a processing time of 24 h period wasused.Investigating the Flavour Potential of Combinations ofCommercial Enzymes in a Model SystemThe model system developed was used to investigate the potential ofa selected number of commercial enzyme preparations to produceMain Conclusions and Achievements* A model system was developed to evaluate the potential ofcommercial enzymes and lactic acid bacteria to produce customisedCheddar flavour EMC products in an optimised environment.The importance of selected hydrolysis, compositional and productionparameters in the production of EMC were identified.* The model EMC system can be used to produce a range ofdiffering cheese flavoured EMC products from base dairy ingredientscomparable to commercial products utilising combinations ofcommercial enzymes and/or lactic acid bacteria.* Manipulation of compositional, proteolytic and lipolyticparameters, and the inclusion of glutamic acid can be used to developdistinctly different products for specific applications.Research and ResultsCommercial Enzyme StudyA comprehensive study on the enzyme activities of a range of foodgrade commercial enzymes was completed and reported in DPRCReport No.10.  The study highlighted the necessity for more detailedinformation on the specificity of the main enzyme component and forinformation relating to side enzyme activities from suppliers.54Table 1: Description and dose rates of commercial proteolytic enzymes used in theproduction of preliminary EMC products in Step 1.Proteinase Preparation Peptidase Preparation(Conc. w/w)  + (Conc. w/w)EMC 1 Glutaminase F* (0.026% w/w)            +   Debritase DBP20** (0.3% w/w)EMC 2 Protease A Amano 6* (0.005% w/w)   +   Debritase DBP20** (0.3% w/w)EMC 3 Bioprotease A Conc*** (0.50% w/w)   +   Debritase DBP20** (0.3% w/w)EMC 4 Neutrase 0.5L**** (0.01%w/w)           +   Debritase DBP20** (0.3% w/w)* Amano Enzyme Europe Ltd, Chipping Norton, Oxfordshire OX7 5SR, UK** Rhodia-Foods UK, Stockport, Cheshire SK6 1PQ, UK*** Quest International, Carrigaline, Co. Cork, Ireland**** Novozymes A/S, Bagsvaerd, DenmarkTable 2: Primary and secondary proteolytic levels in EMC products 1 - 4 and the commercialtarget EMC.Product WSN (% TN) PTA-N (% TN) Total FAA (ug/g)Av sd Av sd Av sdEMC 1 58.90      0.13 12.20 0.30 10165 85 EMC 2 53.10      0.19 11.30 0.12 10908 1067EMC 3 54.80 5.08 7.30        0.00 9449 2723EMC 4 55.40 0.40 6.80        0.19 7548 263Commercial 52.07 1.90 21.39 1.40 23496 308EMC WSN =   pH 4.6 water soluble nitrogenPTA-N    =   5% Phosphotungstic acid soluble nitrogenFAA =   Free amino acids7good Cheddar-type EMC flavour in a two step process: Step 1consisting of hydrolysis using only proteolytic enzymes, followed byStep 2 which involves treatment with lipase preparations.  The products produced were compared to a commercial EMCproduct previously shown to have good Chedder-type flavours by atrained descriptive sensory panel. All products were assessed forcompositional, proteolytic, lipolytic and sensory properties; trialswere carried out in triplicate.Four EMC products (EMC 1 - 4) were produced using only proteolyticenzymes, and in the second step one of these products wassubsequently used as the substrate to produce four EMC finalproducts (EMC 5 - 8) using lipase preparations.  These final productswere compared to the target commercial EMC product.Step 1: Preliminary proteolysed EMC products (EMC 1 - 4)Four commercial proteinase preparations were used which werederived from Bacillus subtilis, Aspergillus oryzae or Aspergillus niger.These enzymes were selected on the basis of their differing activitytowards sodium caseinate.  One of the preparations, Glutaminase Fcontained glutaminase activity, which produces free glutamic acid, anatural flavour enhancer, known to be useful in EMC production.A peptidase preparation derived from  Aspergillus oryzae andLactococcus lactis was added to each EMC product (1 - 4) to aidflavour development and control bitterness. A description of each enzyme preparation and the dose rates used aregiven in Table 1.6most likely due to a number of factors including higher levels ofprotein in the commercial product, the use of higher levels ofpeptidase in its manufacture, the use of pre-hydrolysed substrate(mature cheese) or from high levels of free glutamic acid which couldhave been added exogenously.Statistical analysis of the sensory results by ranked preference foundno significant difference between EMCs 1 - 4; however as EMC 4 hadthe highest preferred score, it was thus used as a source of substratefor the production of the final EMC products in Step 2 of the process.Step 2: Final lipolysed EMC products (EMC 5 - 8)Lipase preparations were selected based on their specificity and werederived from Penicillium roqueforti, Mucor miehei, Candida rugosaor pancreatic tissue.  A description of each enzyme preparation andthe dose rates used are given in Table 3.The gross compositions of the resulting products were similar.Levels of proteolysis only increased in EMC 6, which was probablydue to the presence of a trypsin-like side proteinase activity,previously found in the lipase preparation used in its production(Table 4).  Fatty acid profiles of EMCs 5 - 8 and the commercialproduct are shown in Fig. 2.  The amount of free fatty acids in theseproducts appear quite similar despite the fact that lipases of differingspecificity were used in their production.This appears to indicate that lipase specificity is not an issue insystems where very high levels of lipolysis are attained.9Only slight differences were found in terms of composition betweenEMCs 1 - 4, each having similar levels of primary and secondaryproteolysis as measured by WSN (%TN) and PTA - N (%TN)respectively (Table 2).  However, differences in free amino acidcontents (Table 2 and Fig. 1) were apparent between these productsand can be directly attributed to differences in the specificity of theproteinases used in their preparation.  EMC 1 had the highest level offree glutamic acid, confirming the presence of glutaminase activity inthe enzyme preparation used in its production.  High levels of certainfree amino acids (leucine, lysine, valine, phenylalanine and histidine)in EMCs 1 - 4 were due to the action of the peptidase preparation,which had been previously shown to have high levels of generalaminopeptidase activity.Levels of primary proteolysis in EMCs 1 - 4 were similar to thecommercial product, but levels of secondary proteolysis wereconsiderably lower.  This difference in secondary proteolysis was8Fig. 1: Free Amino Acid Profiles of EMCs 1 - 4 and the commercial product.Statistical analysis of the sensory results by ranked preference foundno significant difference between preference of these products and thecommercial Cheddar-type EMC.This result appears to indicate that the overall sensory perception ofthese products was quite similar indicating that they are comparableto the commercial product.Detailed descriptive sensory analysis found that EMCs 5, 7 and 8were the most similar and this could be attributed to the fact that theircompositional, proteolytic and lipolytic parameters were also themost similar.  EMC 6 was distinguished from all other products andwas most likely due to its different levels of proteolysis and lipolysis.The commercial product was distinguished from EMCs 5, 7 and 8which appeared to be due to differences in composition andsecondary proteolysis and levels of free glutamic acid as their lipolyticprofiles were quite similar.11Table 3: Description and dose rates of commercial lipolytic enzymes used in the productionof final EMC products in Step 2.EMC 5 Lipomod 338* (0.50% w/w)EMC 6 Lipomod 299* (0.90% w/w)EMC 7 Palatase 20,000L** (5.50% w/w)EMC 8 Lipase AY30 Amano***(0.50% w/w)* Biocatalysts Ltd, Main Avenue, Pontypridd CF37 5UT, UK** Novozymes A/S, Bagsvaerd, Denmark*** Amano Enzyme Europe Ltd, Chipping Norton, Oxfordshire OX7 5SR, UKTable 4: Proteolytic and lipolytic indices of EMCs.Product WSN PTA-N Total FAA   Acid Degree     Total FFA(% TN) (% TN) (ug/g) Value (ug/g)Av sd Av        sd        Av       sd Av         sd           Av        sdEMC 5 50.40    1.44 9.90     0.60    6968    383      20.7      0.7       63290    4416EMC 6 78.30    3.60     36.10   10.76    9756    578      15.3      0.4       26993    4979EMC 7     51.20    1.47     10.33     0.15    7292     218      22.6       2.1       66445     4002EMC 8     50.33    0.47      9.33     0.31   7816     359      22.8     0.5       65843     7171Commercial EMC 52.07    1.90    21.39    1.40    23496    308      20.4     0.1   57978    5749WSN =   pH 4.6 water soluble nitrogenPTA-N   =   5% Phosphotungstic acid soluble nitrogenFAA =   Free amino acidsFFA =   Free fatty acids (C4:0 - C18:3)10Fig. 2: Free Fatty Acid Profiles of EMCs 5 - 8 and the commercial product.enzyme preparations Savourase RST100 (Rhodia-Foods UK) andLipomod 338 (Biocatalysts, UK) for 6 h at 45…C and at 0.42% w/vand 0.11% w/v, respectively.  After heat treatment at 80…C for 30 min,one batch was inoculated with viable (1010 cfu/ml) cells ofLb. helveticus, while a second was inoculated with an equivalentamount of heat-shocked cells (optimum 69…C for 25 s) and incubatedfor a further 24 h at 37…C.  The reaction was terminated by heattreatment at 80…C for 30 min.  This experiment was carried out intriplicate and samples were taken for analysis at 0, 8, 16 and 24 h.All samples had essentially similar compositions, except for a morepronounced reduction in pH in the product produced using viablecells.  In general, proteolysis increased in both products, with aslightly higher level in the heat-shocked cells, presumably due to earlyrelease of intracellular enzymes.  Levels of volatile short chain freefatty acids (C4:0 and C6:0) remained constant in the viable cellproduct, but increased slightly in the heat-shocked product.  Somedifferences in the volatile flavour profiles as measured by GC/MSwere also noted between both products.  Sensory evaluation on the basis of ranked preference showed anincrease in acceptability for heat-shocked product over the viable cellproduct.  A decrease in sensory acceptability appeared to correspondwith decreasing in pH, which in turn was linked to an increase aceticacid, which became more pronounced in the viable cell product.The results show that it is possible to produce acceptable intenseCheddar-type EMC flavours using starter cultures, specificallyLb. helveticus (DPC 4571) in combination with commercial enzymesutilising the model system.  This mechanism could be furtherexploited to produce a range of intensely cheesey-flavoured EMC.13This work has shown that a model EMC system can be used toproduce different Cheddar-type EMCs comparable to commercialproducts from base dairy substrates.  Differences in aroma and flavouridentified between these products appears to be related to differencesin defined compositional, proteolytic, lipolytic parameters and levelsof free glutamic acid.Assessing the Potential to Develop EMC UtilisingCommercial Enzymes and Lactic Acid Bacteria in a ModelSystemCommercial EMCs have some limitations which preclude their use insome processed consumer foods.These limitations generally relate to inconsistent flavour developmentand an inability to mimic natural cheese flavours.  To overcome theseproblems it was decided to investigate the potential of utilising lacticacid starter bacteria cultures used in natural cheese production forEMC production, since Lactobacilli are a good source of importantflavour producing enzymes.In this study a strain of Lactobacillus helveticus (DPC 4571) waschosen as previous work has shown that it is autolytic and acceleratednatural Cheddar cheese ripening.  The objective of this study was toinvestigate the potential of viable and heat-shocked cells (heatshocking causes lysis and inactivates the cells lactose fermentingability without affecting residual enzyme activity) of Lactobacillushelveticus DPC 4571 in the production of Cheddar-flavoured EMC.Like the previous study a two step approach was taken.  Using themodel system the substrate was hydrolysed by two commercial1215Publications  Wilkinson, M.G., Kilcawley, K.N. and Mulholland, E. (2000).  Enzymemodified cheese flavour ingredients.  Teagasc End of Project Report(1999),  DPRC No.10, Armis Nos. 4338 and 4540.Lee, B., Kilcawley, K.N., Hannon, J.A., Wilkinson, M.G andBeresford, T.P. (2001).  Influence of viable and heat-shockedLactobacillus helveticus DPC 4571 cells on the flavour of enzyme-modified cheese. EuroLab Conference, University College Cork. 3rd- 6th July 2001.Kilcawley, K.N. (2001).  The enzyme effect. Dairy IndustriesInternational. 66 (7) 26-29.Kilcawley, K.N., Wilkinson, M.G. and Fox, P. F.  (2002). Properties ofcommercial proteinase preparations.  Food Biotechnology. 16 (1)29-55.Kilcawley, K.N., Wilkinson, M.G. and Fox, P. F.  Determination of keyenzyme activities in commercial peptidase and lipase preparationsfrom microbial and animal sources.  Enzyme and MicrobialTechnology. (In press).For further information, please contact:Mr. Kieran Kilcawley or Dr. Tom BeresfordCover Picture:Commercial scale LIMITECH in MTL, Moorepark, used in the production of EMCs.14Mr. Kieran Kilcawley Dr. Tom BeresfordDAIRY PRODUCTS RESEARCH CENTREMoorepark, Fermoy, Co. Cork, IrelandTel: +353 (0) 25 42222 - Fax: +353 (0) 25 42340E-Mail: reception@moorepark.teagasc.ie

Model System for the Production of Enzyme Modified Cheese (EMC) Flavours.

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