J. Dairy Sci. 84:6673 American Dairy Science Association, 2001.

A Survey of Lipolytic and Glycolytic End-Products in Commercial Cheddar Enzyme-Modied Cheese

K. N. Kilcawley,* M. G. Wilkinson,* and P. F. Fox
*Cheese Department, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland. Department of Food Chemistry, University College, Cork, Ireland

ABSTRACT The concentrations of L- and D-lactic acid and free fatty acids, C4:0 to C18:3, were quantied in a range of commercial enzyme-modied Cheddar cheeses. Lactic acid in Cheddar enzyme-modied cheeses varied markedly depending on the manufacturer. Differences in the ratio of L- to D-lactic acid indicate that cheeses of different age were used in their manufacture or contained varying levels of nonstarter lactic acid bacteria. The level of lipolysis in enzyme-modied cheese was higher than in natural Cheddar cheese; butyrate was the predominant free fatty acid. The addition of exogenous acetate, lactate, and butyrate was also indicated in some enzyme-modied cheeses and may be used to confer a specic avor characteristic or reduce the pH of the product. Propionate was also found in some enzymemodied cheese products and most likely originated from Swiss-type cheese used in their manufacture. Propionate is not normally associated with natural Cheddar cheese avor; however, it may be important in the avor and aroma of Cheddar enzyme-modied cheese. Levels of lipolysis and glycolysis appear to highly controlled as interbatch variability was generally low. Overall, the production of enzyme-modied Cheddar cheese involves manipulation of the end-products of glycolysis (lactate, propionate, and acetate) and lipolysis to generate products for specic applications. (Key words: Cheddar enzyme-modied cheese, glycolysis, lipolysis)) Abbreviation key: BHA = butylated hydroxyanisole, EMC = enzyme-modied cheese, NSLAB = nonstarter lactic acid bacteria, PBP = p-bromophenacyl. INTRODUCTION Concentrated cheese avors produced enzymatically from cheese, immature cheese curd, or other dairy sub-

strates are commonly known as enzyme-modied cheese (EMC)(19). EMC are used as a cost-effective source of cheese avor in a range of processed consumer foods and are available in a range of avors, differing in purported intensity and avor attributes (19, 23, 27). Cheddar EMC is of particular importance due to its inclusion in a wide range of foods. In a previous survey (20) it was found that the manipulation of compositional and proteolytic parameters was important in the manufacture of commercial Cheddar EMC products for specic applications. However, other avor-generating pathways, such as lipolysis and glycolysis, are likely involved in the production of Cheddar EMC. Lipolysis of triglycerides in natural Cheddar cheese produces FFA, which inuence cheese avor directly by imparting specic avor notes or indirectly as precursors for other avor compounds (10). While lipolysis has been reported in the production of EMC (18, 23, 24, 28), its signicance in a range of commercially available Cheddar EMC products has not been quantied. Glyolysis in natural cheese principally concerns the metabolism of lactose to L-lactic acid and its subsequent conversion to D-lactic acid or its degradation to acetate, propionate or, in certain cases, to butyrate (12). Lactic acid and its degradation products make a major contribution to the avor of natural Cheddar cheese. However, to our knowledge the signicance of glycolytic endproducts in the manufacture of Cheddar EMC is unknown. Indeed, as nonstarter lactic acid bacteria (NSLAB convert L-lactic acid to D-lactic acid during the ripening of natural Cheddar cheese, the concentration and ratio of these compounds in Cheddar EMC may provide information on the maturity and initial NSLAB levels in the cheese substrate used in EMC manufacture. Therefore, this study was undertaken to quantify and assess the signicance of lipolytic or glycolytic endproducts in a range of commercial Cheddar EMCs. MATERIALS AND METHODS

Received May 4, 2000. Accepted August 4, 2000. Corresponding author: K. N. Kilcawley; e-mail: kkilcawley@ moorepark.teagasc.ie.

Commercial Cheddar EMC Products Fifteen commercial Cheddar EMC products were obtained as gifts from eight suppliers of EMC: Interna66

LIPOLYSIS GLYCOLYSIS ENZYME-MODIFIED CHEESE

67

tional Flavors and Fragrances (GB) Ltd, Duddery Hill, Haverhill, Suffolk, UK; Golden Vale plc, Charleville, Co. Cork, Ireland (Dairy Produce Packers, Coleraine, Northern Ireland); Chr Hansens Laboratories (Ireland) Ltd, Little Island, Co. Cork; Einar Willumsen, Brondby, Denmark; Danisco Ingredients, Brabrand, Denmark; Systems Bio-Industries (SBI), Newbury, Berks, UK: Firmenich, Harrogate, North Yorkshire, UK and Stern, Altrincham, Cheshire, UK. The same products were previously assessed for compositional and proteolytic parameters (20). To respect the condentiality of the suppliers and their products, each product was coded. Thirteen EMC were in paste form and two were powders; three of the suppliers provided more than one EMC product. To evaluate the effect of interbatch variation, a number of batches of certain EMC were analysed over a 6- to 12mo period. Natural Cheddar cheeses were purchased at a local supermarket. All products were stored at 4C before analysis. Each parameter was measured in duplicate for each product. Mean and standard deviation values for different batches of the same products are given. Determination of Lactic Acid Content The concentrations of L- and D-lactic acid were determined in Cheddar EMC and in natural Cheddar cheese with a UV test kit (catalogue number 1112821, Boehringer BCL, Blackrock, Dublin, Ireland). EMC or natural cheese was prepared for analysis (5), with the following modications: 5 g of product were weighed into a glass beaker, to which 25 ml of 0.005 M H2SO4 was added. The mixture was stirred at room temperature for 1 h, after which the cheese particles were allowed settle for 2 min. Aliquots (4 ml) were centrifuged for 5 min at 20,000 g; the fat and protein pellet were removed and the aqueous intermediate layer was ltered through a 0.22 m nylon acrodisc lter (Gelman Sciences, Ann Arbour, MI) before analysis. The increase in the concentration of NADH was measured spectrophotometrically at 340 nm in a Hitachi U-1100 UV-Vis spectrophotometer (Hitachi Ltd, Tokyo, Japan). Results for D- and L-lactic acid were expressed as mg/g dry weight of product to allow comparison between products. Determination of Acetate, Propionate, and Butyrate Content Acetate, propionate, and butyrate (C2:0, C3:0, C4:0) were recovered by steam distillation and subsequently quantied by ligand-exchange, ion-exclusion HPLC. Samples were prepared by adding 20 g of EMC or grated

natural cheese to a distillation tube, followed by the addition of 10 ml of 10% (wt/vol) H2SO4, one drop of silicone antifoam agent and 40 ml of deionized water. This mixture was then distilled into 40 ml of deionized water until a nal volume of 200 ml had been collected. Each sample was mixed and ltered through a 0.2 m nylon acrodisk syringe lter (Gelman Sciences) and stored at 4C before analysis. A sample (50 l) was injected onto the column. Individual standard curves for acetate, propionate, and butyrate were prepared from 10% (wt/vol) stock solutions made up in distilled water. Equal volumes of each stock solution were then pooled and used to prepare standard curves in the range of 0 to 0.2 g per 20 ml of distilled water. Each 20-ml batch of standard was then treated as for 20 g of product, described above. Separation was achieved using an isocratic mobile phase of 0.01NH2SO4 at a ow rate of 0.7 ml/min for 30 min at 50C on a Rezex Monosaccharide H+ column (Phenomenex, Melville House, Hurdseld Industrial Estate, Maccleseld, Cheshire, with a Micro-guard cation H+ cartridge (Bio-Rad, Marylands Avenue, Hemel Hempstead, Hertfordshire, UK). The chromatography system used was a HPLC Waters Alliance 2690 separation module coupled to a Waters 2487 dual-wavelength absorbance detector (220 nm) controlled by Waters Millenium32 software (Waters Corporation, Milford, MA). Results are expressed as mg/g dry weight of product. Determination of FFA (C4:0 to C18:3) Content Individual FFA from C4:0 to C18:3 in Cheddar EMC and natural Cheddar cheese were quantied by reversephase HPLC. This method allows the quantitative determination of the major FFA in cheese and EMC by resolution of p-bromophenacyl (PBP) ester derivatives of saturated and unsaturated FFA, C4:0 to C18:3. The method is a modication of a number of chromatographic techniques (9, 14, 26). The Waters HPLC system described above was used at a wavelength of 254 nm. The column (250 mm 4.6 mm) used was a Kingsorb C18, 5m bead size, 90 nm pore size, with associated guard column (Phenomenex). The column was eluted at 33C using the gradient outlined in Table 1. A standard curve was prepared for each of the following individual FFA, C4:0, C6:0, C8:0, C9:0, C10:0, C12:0, C14:0, C16:0, C17:0, C18:0, C18:1, C18:2, and C18:3, from stock solutions containing 0.1 M/L in a 1:1 (vol/vol) methanol:chloroform solution. Varying amounts of the stock solutions were used to prepare standard curves in the range 0.0025 to 0.03 mM. Each standard solution was made up to a nal volume of 3.9 ml with 1:1 (vol/vol) methanol:chloroform. One ml of each standard solution was
Journal of Dairy Science Vol. 84, No. 1, 2001

68

KILCAWLEY ET AL. Table 1. Composition of the mobile phase used to separate individual FFA by RP-HPLC1. Time (min) 0.0 2.0 24.0 25.0 40.0 45.0 46.0 58.0 60.0
1 2

Flow rate (mL/min) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.1

Acetonitrile (%) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

Water (%) 45.0 24.0 13.0 1.9 1.6 0.0 45.0 45.0 45.0

Methanol (%) 50.0 71.0 82.0 93.1 93.4 95.0 50.0 50.0 50.0

Curve type2 2 6 2 2 6 6 6 6

RP-HPLC = Reverse-phase HPLC. Denotes the type of gradient applied to the changing components of the mobile phase. Low values indicate a convex curve, while high values indicate a concave curve (1 to 12).

added to 0.75 ml of 0.05% butylated hydroxyanisole (BHA) in 1:1 (vol/vol) methanol:chloroform, and 100l of this solution was transferred to a glass test tube with a sealed cap. Two milliliters of a solution (2 g/L) of PBP in acetonitrile was added, followed by 80 l of 5 g/L 18crown-6-ether in acetonitrile and 0.2 g of K2CO3. These solutions must be added in this order; otherwise, extraneous peaks will appear on the chromatogram. The solution was mixed and heated to 75C in a water bath for 30 min to promote derivatization to PBP fatty acid esters, after which the tubes were removed and allowed to cool to room temperature; 40 l of 40 g/L of formic acid in acetonitrile was added. The solution was mixed and heated at 75C for 5 min, cooled to room temperature, and placed at 4C for 1 h. Each standard solution was then ltered through a 0.2-m nylon acrodisk syringe lter (Gelman Sciences), and 40 l of ltrate was applied to the column. EMC or natural Cheddar cheeses were prepared as follows: 2.5 g of product was ground with a mortar and pestle with 10 g anhydrous Na2SO4 for EMC and 5 g Na2SO4 for natural Cheddar cheese. The mixture was transferred to a glass screw-cap container, and 3 ml of diethyl ether was added, agitated, and centrifuged at 2500 g for 4 min at room temperature. The solvent layer containing the fat was decanted and stored in a second glass screw-cap container. The remaining pellet was broken up with a spatula and reextracted twice, and the solvent layers were pooled on completion. The volume of solvent collected was measured and 1.0 ml of extract was added to 0.75 ml of 0.05% BHA in 1:1, vol/vol, methanol:chloroform and treated as for the standards described above. A portion of extract (10l for EMC and 40 l for natural Cheddar cheese) was applied to the column. RESULTS AND DISCUSSION Lactic Acid The concentrations of L- or D-lactic acid and total lactic acid (mg/g dry weight of product) are shown in
Journal of Dairy Science Vol. 84, No. 1, 2001

Table 2. The concentrations of L- and D- or total lactic acid differed between EMC and were dependent mainly on the manufacturer. Three distinct groups of EMC could be differentiated on the basis of the total lactic acid content. Products 1 (A to D), 4, 5, 7A, 7B, and 8 contained ~10 to 20 mg/g of lactic acid, which in some products was similar to the levels found in the natural Cheddar cheeses in this study (17 to 22 mg/g) and in agreement with values found by others for natural Cheddar cheeses (~16 to 24 mg/g)(16). It is therefore probable that natural Cheddar cheese was used in their manufacture, and as the ratio of L- to D-lactic acid in these EMC differed, it is likely that the natural cheeses used differed in maturity or in their NSLAB levels. The second group consisted of products 2 (A to D) and 6 which contained higher concentrations of lactic acid (~35 to 80 mg/g) with approximately equal amounts of L- and D-lactic acid. As these concentrations of lactic acid are signicantly higher than those in natural Cheddar cheese, it is likely that some exogenous lactic acid was added in their manufacture. Product 3 contained the highest concentration of lactic acid (280 mg/g). This level was most likely achieved through the addition of L-lactic acid, as 92% was L-lactic acid. L-lactic acid may have been added to adjust pH or to confer a specic avor character to the nal product. Indeed, lactic acid may be added to an EMC base to accentuate avor (31). Interbatch variation in lactic acid content was low for most EMC. Acetate, Propionate, and Butyrate The levels of acetate, propionate, and butyrate in commercial Cheddar EMC and natural Cheddar cheeses are shown in Table 3. A chromatogram obtained by this method is shown in Figure 1. Overall, the concentration of these compounds varied in the EMC from different manufacturers, with higher concentrations found in EMC compared with natural Cheddar cheeses.

LIPOLYSIS GLYCOLYSIS ENZYME-MODIFIED CHEESE Table 2. Concentrations (mg/g dry wt) of total, and D- and L-lactic acid in commercial Cheddar enzyme-modied cheese or in natural Cheddar cheese. Code 1A Batch 1 3 4 Mean SD 1 3 4 Mean SD 1 2 3 Mean SD 1 2 Mean 1 2 Mean 1 2 Mean 1 2 Mean 1 2 Mean 1 2 3 Mean SD 1 2 Mean 1 2 Mean 1 1 2 Mean 1 2 3 Mean SD 1 1 1 1
D-Lactic

69

acid

L-Lactic

acid

Total lactic acid 17.12 16.78 14.8 16.2 1.3 11.21 15.29 15.26 13.9 2.3 9.26 10.31 8.83 9.5 0.8 16.93 11.33 14.13 54.54 59.40 57.0 50.83 45.63 48.23 59.21 35.70 47.5 69.67 93.84 81.8 268.84 286.19 286.56 280.5 10.1 13.22 14.88 14.0 16.54 15.23 15.9 39.54 25.39 12.68 19.0 13.32 14.12 12.95 13.5 0.6 12.81 22.17 19.1 17.84

1B

1C

1D 2A 2B 2C 2D 3

4 5 6 7A 7B1

81 NC2 NC NC
1

0.26 1.14 1.33 0.9 0.6 0.39 0.16 0.92 0.5 0.4 0.54 0.00 0.51 0.3 0.3 0.13 2.15 1.1 30.03 26.94 28.5 26.14 33.98 30.1 26.25 19.81 23.0 20.58 45.22 32.9 2.85 3.56 2.22 2.9 0.7 6.27 5.79 6.0 7.26 4.9 6.1 20.16 4.37 1.38 2.9 3.47 3.43 3.31 3.4 0.1 5.64 0.61 0.98 7.13

16.86 15.64 13.47 15.3 1.7 10.82 15.13 14.34 13.4 2.3 8.73 10.31 8.32 9.1 1.1 16.8 9.18 13.0 24.51 32.47 28.5 24.69 11.65 18.2 32.95 15.90 24.4 49.09 48.61 48.8 265.99 282.62 284.34 277.6 10.1 6.96 9.09 8.0 9.28 10.33 9.8 19.38 21.02 11.30 16.2 9.84 10.69 9.64 10.1 0.6 7.17 21.56 18.12 10.7

(11, 12, 16, 21, 29). Acetate is thought to contribute positively to the avor of Cheddar cheese; however, when present at high concentrations, it may also impart off-avors (2). The upper limit of acetate concentration in natural Cheddar cheese is about 1% (wt/wt) or 16

Table 3. Concentrations (mg/g dry wt) of acetate, propionate and butyrate in commercial Cheddar enzyme-modied cheese or in natural Cheddar cheese. Code 1A Batch 1 3 4 Mean SD 1 3 4 Mean SD 1 2 3 Mean SD 1 2 Mean 1 2 Mean 1 2 Mean 1 2 Mean 1 2 Mean 1 2 3 Mean SD 1 2 Mean 1 2 Mean 1 1 2 Mean 1 2 3 Mean SD 1 1 1 1 Acetate 1.3 3.8 0.8 2.0 1.6 1.3 1.3 1.1 0.5 0.4 373.4 412.7 287.1 357.7 64.2 0.8 0.8 0.8 15.2 8.1 11.7 12.0 11.5 11.8 22.6 26.3 24.5 37.4 29.7 33.6 3.9 4.2 3.4 2.9 0.7 5.0 5.5 5.3 2.0 1.1 1.6 12.7 4.2 1.0 2.6 3.4 4.7 4.5 4.2 0.7 2.5 0.3 1.0 0.8 Propionate 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.7 2.4 3.1 2.7 2.6 2.6 11.4 12.8 12.1 19.5 17.1 18.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 8.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Butyrate 112.7 116.8 128.1 119.2 8.0 103.6 103.0 104.2 103.6 0.6 36.2 37.8 29.2 34.4 4.6 28.8 22.6 25.7 14.9 13.0 14.0 14.7 14.2 14.5 17.0 19.7 18.4 17.3 10.1 13.7 23.9 25.9 29.6 26.5 2.9 19.7 20.3 20.0 37.0 31.9 34.5 16.8 25.7 26.4 26.1 3.7 6.5 4.5 4.9 1.4 1.6 0.6 0.6 0.5

1B

1C

1D 2A 2B 2C 2D 3

4 5 6 7A 7B1

Powdered product. 2 NC = Natural Cheddar cheese.

Acetate, propionate, and butyrate in Cheddar EMC or natural Cheddar cheese may originate through a number of pathways. Metabolism of citrate and lactose by heterofermentative NSLAB is the principal mechanism for acetate formation in natural Cheddar cheese

81 NC2 NC NC
1 2

Powdered product. NC = Natural Cheddar cheese. Journal of Dairy Science Vol. 84, No. 1, 2001

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KILCAWLEY ET AL.

Figure 1. Chromatogram of acetic, propionic and butyric acids in a Cheddar enzyme-modied cheese (EMC) and a natural Cheddar cheese.

diacetyl, lactic, acetate, and propionate known to be inhibitory to Gram-negative bacteria (1, 8). Butyrate has been identied widely in Cheddar cheese, where it is formed principally by lipolysis of milk fat (3, 4, 22, 32, 33). The concentration of butyrate found in the natural Cheddar cheeses was much lower than that found in the EMC. Wide variations in the level of butyrate were evident in the EMC, with most products (1C, 1D, 2A, 2B, 2C, 2D, 3, 4, 5, 6, and 7A) containing ~12 to 35 mg/g of butyrate. The extremely high level of butyrate in products 1A and 1B (> 100 mg/ g) was probably due to its direct addition to inuence the nal avor characteristics or indirectly by the inclusion of lipolyzed butterfat. The powdered products, 7B and 8, contained low levels of both acetate and butyrate, which is most likely due to the loss of volatile compounds on spray drying. Other less likely possibilities are the use of young Cheddar cheese curd in the formulation or a dilution effect due to the addition of materials to aid spray drying such as lactose or maltodextrins (20). Interbatch variability for acetate, propionate, and butyrate was higher than for lactic acid. FFA (C4:0 to C18:3) A reverse-phase HPLC chromatogram of individual FFA (C4:0 to C18:3) obtained from a Cheddar EMC and a natural Cheddar cheese sample is shown in Figure 2. The levels of individual and total FFA (C4:0 to C18:3) in commercial Cheddar EMC and natural Cheddar cheese are shown in Tables 4 and 5. The total FFA (C4:0 to

mg/g on a dry weight basis, assuming 38% moisture (29). The concentration of acetate found in EMC 1B, 1D, and 5 was similar to that found in the natural Cheddar cheese samples and, together with their lactic acid content, suggests that natural Cheddar cheese was probably used in their manufacture. Products 1A, 3, 4, 7A, 7B, and 8 contained slightly higher levels, which may have also come from the use of natural Cheddar cheese in their manufacture. The higher level of acetate in products 2 (A to D) and 6 may have arisen from the inclusion of Swiss-type cheese in the formulation, its exogenous addition (e.g., in the form of starter distillate) or as a result of the growth of heterofermentative organisms during processing. The extremely high level of acetate found in product 1C (350 mg/g) suggests its addition during production, possibly to impart a specic avor note and/or to reduce pH. Propionate occurs in Swiss-type cheeses as a result of the metabolism of lactic acid by Propionibacteria to propionate, acetate, and CO2 in the ratio of 2:1:1 (25, 30). In Swiss-type cheese, propionate is an important contributor to avor providing the sweet or nutty notes characteristic of these varieties (34). Propionate is not normally present in Cheddar cheese (3, 4, 17) and does not contribute to Cheddar avor. Propionate was present in EMC 2 (A to D) and 6 to impart a specic avor note, or as a preservative or both. Propionate may originate from the use of Swiss cheese in the formulation, its exogenous addition (e.g., as starter distillate), or through the growth of Propionibacteria generating
Journal of Dairy Science Vol. 84, No. 1, 2001

Figure 2. A chromatogram of the FFA (C4:0 to C18:3) in a Cheddar enzyme-modied cheese (EMC) and a natural Cheddar cheese.

LIPOLYSIS GLYCOLYSIS ENZYME-MODIFIED CHEESE Table 4. Concentrations (mg/g dry wt) of individual FFA (C4:0 to C18:3) in commercial Cheddar enzymemodied cheese or in natural Cheddar cheese. Code 1A Batch 1 3 4 Mean SD 1 3 4 Mean SD 1 2 3 Mean SD 1 2 Mean 1 2 Mean 1 2 Mean 1 2 Mean C4:0 3.49 3.49 3.82 3.60 0.2 2.53 2.39 3.71 2.88 0.7 0.54 0.63 0.60 2.88 0.7 1.25 1.15 1.20 3.81 3.67 3.74 1.80 0.86 1.33 3.03 3.76 3.39 C6:0 0.70 0.68 0.78 0.72 0.1 0.59 0.56 0.74 0.63 0.1 0.38 0.47 0.41 0.63 0.1 0.83 0.76 0.83 1.96 1.67 1.82 1.26 0.96 1.11 2.00 2.27 2.21 C8:0 0.55 0.57 0.64 0.59 0.0 0.48 0.44 0.64 0.52 0.1 0.39 0.47 0.39 0.52 0.1 0.62 0.58 0.62 0.99 0.83 0.91 0.90 0.78 0.84 1.19 1.28 1.23 C10:0 0.68 0.75 0.88 0.77 0.1 0.48 0.43 0.93 0.61 0.3 0.14 0.18 0.13 0.15 0.0 0.89 0.82 0.89 1.59 1.24 1.41 1.35 1.14 1.25 1.23 1.81 1.52 C12:0 1.13 1.19 1.36 1.23 0.1 0.92 0.85 1.38 1.05 0.3 0.57 0.69 0.61 0.62 0.1 1.26 1.24 1.26 2.45 2.11 2.28 2.30 2.00 2.15 1.82 2.45 2.13 C14:0 3.29 3.62 4.08 3.66 0.4 3.02 2.63 4.06 3.24 0.7 1.39 1.82 1.65 1.62 0.2 3.85 3.57 3.71 4.65 3.36 4.01 5.39 5.00 5.20 3.35 3.73 3.54 C16:0 8.13 8.68 9.95 8.92 0.9 7.02 6.51 9.71 7.75 1.7 3.63 4.83 4.11 4.19 0.6 9.58 9.09 9.34 6.87 6.79 6.83 13.49 11.72 12.61 5.19 6.50 5.84 C18:0 3.58 3.85 3.80 3.74 0.1 3.40 3.12 3.90 3.47 0.4 1.62 2.16 1.84 1.87 0.3 3.07 3.67 3.37 1.81 1.38 1.60 4.88 4.22 4.55 0.97 1.50 1.23 C18:1 5.41 5.58 7.07 6.02 0.9 4.67 4.41 7.32 5.47 1.6 1.74 2.58 1.73 2.02 0.5 6.04 7.61 6.82 2.99 2.49 2.74 5.78 7.91 6.85 1.57 2.40 1.99 C18:2 1.84 1.75 1.85 1.81 0.1 1.56 1.49 1.92 1.66 0.2 0.91 1.19 1.01 1.04 0.1 1.99 1.95 1.97 1.06 1.03 1.05 1.37 2.22 1.80 0.85 1.13 0.99 C18:3 0.28 0.22 0.35 0.28 0.1 0.24 0.24 0.36 0.30 0.1 0.13 0.16 0.19 0.16 0.0 0.33 0.32 0.33 0.21 0.24 0.23 0.47 0.39 0.43 0.20 0.24 0.22 Total 29.08 30.38 34.58 31.35 2.9 24.91 23.07 34.67 27.55 6.2 11.44 15.18 12.67 13.10 1.9 29.71 30.76 30.24 28.39 24.81 26.60 38.99 37.20 38.10 21.40 27.07 24.24

71

1B

1C

1D 2A 2B 2C

Table 5. Concentrations (mg/g dry wt) of individual FFA (C4:0 to C18:3) in commercial Cheddar enzymemodied cheese or in natural Cheddar. Code 2D 3A Batch 1 2 Mean 1 2 3 Mean SD 1 2 Mean 1 2 Mean Mean 1 2 Mean 1 2 3 Mean SD Mean 1 1 1 C4:0 0.83 0.87 0.85 0.44 0.53 0.47 0.48 0.0 1.32 1.37 1.35 1.24 1.40 1.32 3.36 0.97 0.95 0.96 0.05 0.07 0.15 0.09 0.1 0.12 0.16 0.23 0.13 C6:0 0.90 0.91 0.91 0.30 0.32 0.30 0.31 0.0 0.77 0.90 0.84 0.78 0.85 0.82 1.64 0.45 0.46 0.46 0.27 0.29 0.17 0.24 0.1 0.08 0.09 0.12 0.07 C8:0 0.83 0.87 0.85 0.36 0.38 0.35 0.36 0.0 0.56 0.67 0.62 0.56 0.59 0.58 0.86 0.67 0.71 0.69 0.10 0.12 0.18 0.13 0.0 0.14 0.11 0.10 0.07 C10:0 0.59 0.69 0.64 0.11 0.12 0.10 0.11 0.0 0.68 0.89 0.79 0.70 0.83 0.77 1.43 1.03 1.20 1.12 0.17 0.18 0.26 0.20 0.0 0.21 0.1 0.11 0.03 C12:0 1.14 1.52 1.33 0.56 0.61 0.54 0.57 0.0 1.04 1.30 1.17 1.11 1.20 1.16 1.33 1.25 1.62 1.44 0.10 0.14 0.49 0.24 0.2 0.59 0.36 0.39 0.15 C14:0 3.17 3.59 3.38 1.19 1.24 1.30 1.24 0.1 1.99 2.54 2.27 3.14 3.60 3.51 3.62 4.09 3.56 3.83 0.32 0.46 1.48 0.75 0.6 1.46 0.60 0.47 0.25 C16:0 8.49 9.38 8.93 2.98 3.15 3.21 3.11 0.1 6.16 8.20 7.18 8.22 9.16 8.69 6.50 9.13 10.95 10.04 0.90 1.15 3.81 1.95 1.6 3.16 0.98 0.79 0.39 C18:0 3.58 3.57 3.58 0.92 0.91 1.05 0.96 0.1 3.39 4.42 3.91 4.77 4.67 4.72 1.46 2.56 3.01 2.79 0.43 0.55 1.74 0.91 0.7 1.10 0.50 0.32 0.17 C18:1 3.89 5.62 4.75 1.75 1.48 1.69 1.64 0.1 4.99 6.08 5.54 7.59 8.50 8.05 2.85 6.36 8.25 7.31 0.18 0.35 3.00 1.18 1.6 3.62 1.79 0.93 0.42 C18:2 1.06 1.46 1.26 0.77 0.81 0.77 0.78 0.0 1.28 1.61 1.45 1.91 2.43 2.17 1.20 1.92 2.42 2.17 0.17 0.21 0.88 0.42 0.4 0.94 0.40 0.26 0.13 C18:3 0.27 0.29 0.28 0.17 0.18 0.20 0.18 0.0 0.13 0.33 0.23 0.34 0.29 0.32 0.28 0.27 0.30 0.29 0.11 0.11 0.12 0.11 0.0 0.01 0.15 0.11 0.07 Total 24.75 28.77 26.76 9.55 9.73 9.98 9.75 0.2 22.31 28.31 25.31 30.63 33.52 32.08 25.53 28.70 33.43 31.07 2.80 3.63 12.28 6.42 5.3 11.43 5.24 3.83 1.88

4 5 6 7A 7B1

81 NC2 NC NC
1 2

Powdered product. NC = Natural Cheddar cheese. Journal of Dairy Science Vol. 84, No. 1, 2001

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KILCAWLEY ET AL.

Figure 3. Individual short (C4:0 to C8:0) medium (C10:0 to C14:0), and long (C16:0 to C18:3) chain FFA as a percentage of the total FFA in commercial Cheddar enzyme-modied cheeses and in natural Cheddar cheese. NC = Natural Cheddar cheese.

C18:3) content varied between EMC from different suppliers and between EMC from the same supplier. Overall, the total level of FFA (C4:0 to C18:3) was higher in the EMC than in natural Cheddar cheese. Total FFA level (C4:0 to C18:3) was highest in product 2B (~38 mg/g) and most EMC (1A, 1B, 1D, 2A, 2C, 2D, 4, 5, 6, and 7A) contained ~24 to 32 mg/g. The lowest levels of total FFA were found in products 1C, 3, 7B, and 8, which contained on average ~10 mg/g. The total FFA values obtained for the EMC in this study were lower than those reported previously for four commercial Cheddar EMC, which had a total FFA content of 60.5, 135.5, 190, and 194 mg/g of FFA on a dry wt basis (28). The predominant FFA in most EMC were C16:0 (palmitic acid), C18:1 (oleic acid), C14:0 (myristic acid), and C18:0 (stearic acid), which represented ~30, ~19, ~13, and ~11% of the total FFA, respectively. In natural Cheddar cheese, the same FFA predominated, but not in the same order, with C18:1, representing ~27% of the total FFA, C16:0 ~20%, C14:0 ~12%, and C18:0 ~9%. The fact that these FFA predominated is not due to their preferential release from triglycerides, but rather reects their abundance in milk fat (7, 15). Individual FFA expressed as a percentage of total FFA and grouped into short (C4:0 to C8:0), medium (C10:0 to C14:0), or long chain (C16:0 to C18:3) FFA are shown in Figure 3. Overall, the FFA prole in most Cheddar EMC was similar to that for the natural Cheddar cheeses. However, there is some evidence for the selective release of short and medium chain FFA in products 1A, 1B, 2A, 2C, and 6 and long chain FFA in product 5. This would indicate the use of lipase or esterase
Journal of Dairy Science Vol. 84, No. 1, 2001

enzymes with dened chain length specicity to generate particular Cheddar EMC avor proles. Interbatch variation of FFA content was low for most products, except product 2D. In this study, the level of butyrate measured by the reverse-phase HPLC method was considerably lower than that obtained by the steam distillation method (~2 to ~38% of those obtained by the steam distillation method), with the closest agreement between the natural Cheddar cheeses. The differences in butyrate concentration between methods is most likely due to two factors; (1) short chain fatty acid esters are volatile, and some may have been lost during the extraction and derivatization step in the reverse-phase HPLC method; and (2) short chain FFA are partitioned mainly in the aqueous phase with the remaining FFA associated with the fat phase (15). The latter factor makes it difcult to completely recover all FFA in natural cheese; however, the problem is further exacerbated in EMC due to their highly emulsied state (19). The levels of FFA in natural Cheddar, as determined by reverse-phase HPLC, were comparable to other studies which used different extraction and separation methods (4, 6, 22, 32, 33). CONCLUSIONS In this study, the levels of L- and D-lactic acid and FFA were quantied in a range of commercial Cheddar EMC. It was apparent from the results that the concentration of lactic acid and acetate in the products differed between manufacturers. Much of the lactic acid and acetate in the EMC appeared to have originated from the use of Cheddar cheese of varying maturity in their manufacture, as the levels of L- and D-lactic acid and acetate were similar to that found in natural Cheddar cheese for the majority of EMC. It was also apparent that the levels of lactate were dependent mainly on manufacturing source. However, results also indicated that exogenous lactic acid and acetate were added to certain products to impart a specic avor note, to reduce pH for bacteriostatic purposes, or both. The presence of propionate in certain EMC was most likely due to the use of Swiss-type cheese in their formulation as a preservative. While propionate is not generally regarded as a major contributor to natural Cheddar cheese avor, it may have a role in the overall avor and aroma of Cheddar EMC. Higher levels of FFA in Cheddar EMC compared with natural Cheddar cheese indicate the signicance of lipolysis in their production. The use of specic lipases and esterases, which preferentially release short chain free fatty acids such as butyrate, was indicated in some EMC products. The selection of enzymes specic for

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73

the release of butyrate highlights the contribution of butyrate to Cheddar EMC avor and aroma and, indeed, this compound is known to be an important contributor to the avor of natural Cheddar cheese (10, 13). Butyrate levels were very high in a number of EMC, which suggests the possible addition of exogenous sources of butyrate. The level of volatile acids (acetate, propionate, and butyrate) was lower in the powdered EMC than in their paste counterparts, which may indicate the loss of volatile compounds during spray drying. The loss of volatile aroma compounds on spray drying is an important factor to consider in the manufacture of EMC and requires further quantication. Results indicate that levels of lipolysis and glycolysis were controlled, as interbatch variability was low for different batches of identical products. These results conrm the signicance of lipolytic and glycolytic end products in the manufacture of commercial Cheddar EMC. REFERENCES
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Journal of Dairy Science Vol. 84, No. 1, 2001