Food Research International, Vol. 31, No. 67, pp.

441447, 1998 # 1999 Canadian Institute of Food Science and Technology Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain PII: S0963-9969(99)00011-3 0963-9969/99/$ - see front matter

Ion-exchange HPLC analysis of a broad spectrum of organic acids from matured Cheddar cheese and assessment of extraction methods
J. F. R. Lues,a* W. C. Bothab & E. J. Smita
a

Department of Environmental Sciences, Technikon Free State, Private Bag X20539, Bloemfontein, 9300, Free State Province, South Africa b Department of Food Science, University of the Orange Free State, PO Box 339, Bloemfontein, 9300, Free State Province, South Africa An ion-exclusion HPLC technique was used to separate a total of 16 organic acids from matured Cheddar cheese. Three extraction methods, reported in the literature for the purpose of extracting organic acids from cheese, were evaluated by determining the percentage recovery of the organic acids from the selected cheese. All three extraction methods evaluated yielded high recovery rates for all compounds analyzed. The method using 0.009 N H2SO4 gave best recoveries for 11 of the 16 detected compounds while the barium hydroxide/zinc sulphate extraction method yielded best recoveries for oxalic and malic acids. The bueracetonitrile extraction method showed the highest recovery percentages for propionic, isovaleric and n-valeric acids. In addition, the method using 0.009 N H2SO4 exhibited improved retention of minor peaks. With the exception of oxalic acid, organic acid concentrations determined for the mature Cheddar cheese corresponded well with values in the literature. On the basis of amount of analytes separated, resolution and ease of analysis, the 0.009 N H2SO4 extraction method and proposed ion-exchange HPLC protocol was well suited for the quantication of a broad spectrum of major and minor organic acids from Cheddar cheese. However, rapid sample pre-treatment techniques to eliminate contaminating biochemical species, require investigation. # 1999 Canadian Institute of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved Keywords: organic acids, Cheddar cheese, HPLC.

INTRODUCTION Understanding the role of organic acids in cheese maturation has been impeded by the diculty of fully separating and identifying them. Gomis (1992) remarked that, although separation of the major organic acids could be regarded as a completely solved problem, this is not the case with the minor and trace organic acids, calling for derivatisation and preconcentration techniques. According to Khalid and Marth (1990), the contribution of glycolysis and lipolysis in cheese is more dicult to dene for some varieties than even proteolysis. Good results using HPLC for the analysis of organic acids, sugars and related compounds from

*To whom correspondence should be addressed. Fax: +2751-507-3911; e-mail: rlues@mail.tofs.ac.za

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dairy products including cheese, have been reported by Marsili et al. (1981), Pirisino (1983), Morawski (1984), Panari (1986), Bevilacqua and Califano (1989), McGregor and White (1990), Bouzas et al. (1991), Lombardi et al. (1994) and Gonzalez de Llano et al. (1996). Extraction of organic acids from cheese is an equally crucial step in the analytical process, as failure to exclude undesirable compounds can lead to artefacts and interference with results. According to Gomis (1992) organic acids, due to their high water solubility, could generally be extracted from solid and semi-solid samples by cutting up and grinding an adequate portion, followed by blending in water, acidied water, 80% ethanol or 80% acetonitrile. The acids should consequently be completely extracted by stirring the slurry for up to 2 h (Gomis, 1992). Marsili et al. (1981) utilised acetonitrile (20 ml) and distilled water (5 ml) for extracting the organic acids from 5 g of cheese. After

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J. F. R. Lues et al. rm as an extra-matured speciality-line cheese. The sample was collected by means of a type C (short) stainless steel trier, according to International Dairy Federation (IDF) International Standards for sampling of milk and milk products (IDF, 50B: International Dairy Federation, 1985). Samples were kept below 10 C during transportation to the laboratory in order to limit excessive microbial proliferation at elevated temperatures. Extraction methods Three extraction methods were used. The rst was the method by Marsili (1985), with minor changes. Cheese (10 g) was added to 30 ml 0.30 N barium hydroxide per 30 ml 0.28 N zinc sulphate and macerated for 1 min at high speed with a Polytron blender. The slurry was then centrifuged at 7000 g for 5 min whereafter three layers formed (upper layer of fat, middle aqueous layer and a bottom layer of protein). The aqueous phase was collected by micropipette and ltered (Gelman 0.2 mm syringe membrane lter). The fat and protein layers were washed with distilled water and the aqueous phases combined and diluted to a total of 100 ml. Fifty microlitres of the aqueous phase were used for HPLC analysis. The second extraction method employed was similar to the one used by Bevilacqua and Califano (1992). Here 7 g of cheese were added to 50 ml of buer-acetonitrile mobile phase (0.5% w/v (NH4)2HPO40.4% w/v acetonitrile made to pH 2.24 with H3PO4). The mixture was homogenised for 1 h (magnetic stirrer) and centrifuged for 5 min at 7000 g. The supernatant was ltered through a 0.45 mm membrane lter (Gelman) whereafter 10 ml was injected for analysis. The third extraction method was described by Bouzas et al. (1993). Cheddar cheese was ground and 5 g added to 25 ml of 0.009 N H2SO4, followed by stirring of the mixture for 1 h on a magnetic stirrer. After centrifugation (5000 g for 10 min) the supernatant was ltered through a 0.2 mm membrane lter (Gelman). Ten ml was injected for analysis. All reagents used were analytical grade obtained from Merck (SA). Technique standardisation, recovery and statistical design Five HPLC separations were performed per extraction whereas the extraction procedures were performed in triplicate. Bevilacqua and Califano (1989) stated that variation in the biochemical composition of Cheddar cheese could occur, not only amongst batches of cheese, but also within a single cheese block. This biochemical variation, together with variation that might occur during extraction, could inuence the standard deviations of the various analytes. Thus, a single sample of the test block was taken to best eliminate non-analytical variables. The measure of repeatability of the extraction technique and separation methods were determined by

shaking for 1 min, the mixture was centrifuged (7000 g for 5 min) and ltered through a 0.2 mm teon membrane lter (Marsili et al., 1981). A few years later, the same author used 30 ml of 0.30 N barium hydroxide and 30 ml of 0.28 N zinc sulphate as solution in which 10 g of cheese was macerated for 1 min at high speed (Marsili, 1985). Upon centrifugation three layers formedan upper layer of fat, a middle aqueous layer and a bottom layer of casein. The aqueous layer was ltered through Whatman No. 40 lter paper and used for HPLC analyses of organic acids (Marsili, 1985). The above-mentioned barium hydroxide/zinc-sulphate method was also used by McGregor and White (1990) for HPLC analysis of organic acids in low-fat Cheddar cheese. Panari (1986) used 80 ml distilled water with 20 g of cheese to extract organic acids from cheese, followed by blending for 3 min and centrifugation. The aqueous organic acid extract was consequently applied to an anionic exchange column (Amberlite CG 400) and eluted with 0.1 N H2SO4. Extraction of the organic acids with the mobile phase (0.5% w/v (NH4)2HPO40.4% w/v acetonitrile made to pH 2.24 with H3PO4) used as extraction agent was reported by Bevilacqua and Califano (1989). Cheese (7 g) was added to 50 ml of the aforementioned solution, homogenized for 1 h, centrifuged and ltered through a 0.45 mm membrane lter. The above authors repeated the method in a more recent study on organic acid changes in Port Salut Argentino cheese (Bevilacqua and Califano, 1992). A commonly used method for the extraction of organic acids from cheese for the purpose of HPLC analysis is the method which employs, with minor dierences, 0.00490.009 N of H2SO4 as the extraction agent (Bouzas et al., 1991, 1993; St-Gelais et al., 1991; Lombardi et al., 1994; Gonzalez de Llano et al., 1996). A more-or-less 1:5 w/v mixture (7 g in 50 ml or 5 g in 25 ml) of cheese and extraction liquid is used after which centrifugation (5000 g for 10 min or 7000 g for 5 min) is done followed by ltration of the aqueous phase through a 0.2 mm or 0.45 mm lter. The aim of this study was to ascertain the applicability of the ion-exchange HPLC procedure for quantication of organic acids in matured Cheddar cheese in a single chromatographic run and to establish the optimum extraction procedure for isolating a broad spectrum of major and minor organic acids from matured Cheddar cheese for the purpose of ion-exchange HPLC analysis. MATERIALS AND METHODS Sampling technique A 10-month old matured Cheddar cheese of a selected manufacturer was obtained from the processing plant. The matured cheese was a product developed by the

Ion-exchange HPLC analysis of organic acids from Cheddar cheese calculating the standard deviations (sn1) of the concentrations and retention times of analytes between consecutive HPLC runs. Percentage recovery was determined by adding known concentrations (1000 mg kg1) of selected organic acid standards to the cheese samples during extraction. The concentration of these selected organic acids added to the cheese was then determined by running a separation as for the sample analysis and calculating the percentage recovery. The dierences between the extraction procedures and recoveries of consecutive HPLC runs were tested with the student-t test (signicance level: p 0.05). Reagents and standards for HPLC The organic acids determined were those that are commonly associated with microbiological metabolism, inherently occurring in milk and cheese or added during processing. These included acetic; citric; butyric; fumaric; formic; hippuric; iso-valeric; lactic; malic; orotic; oxalic; propionic; pyruvic; uric and n-valeric acids, including diacetyl (Marsili et al., 1981; Bevilacqua and Califano, 1989; Bouzas et al., 1991, 1993; Bevilacqua and Califano, 1992). Apart from butyric acid, the volatile fatty acids isovaleric and n-valeric, were included in the list of analytes, as they have been reported to, together with diacetyl, contribute signicantly to the avour of cheese (Ohren and Tuckey, 1969; Keen and Walker, 1974; Adda et al., 1982; Axelsson, 1993). External standards [conc. 1000 mg kg1 in HPLC grade water (Merck)] of the mentioned organic acids were injected individually to establish respective retention times and recovery. Phosphoric and sulphuric acids as well as the organic acid standards were all obtained from Merck (Midrand, SA). Separation protocol The HPLC system (Spectra Physics) was equipped with a solvent degasser (SCM 400), quaternary gradient solvent pump (P4000), multiple autosampler (AS1000) tted with a 50 ml loop, spectral array UV detector (UV3000) and system controller with OS/2 based PC 1000 and Spectacle software. A semi-preparative (30 cm7.8 mm) Supelcogel C-610H ion-exchange chromatography column developed for separation of organic acids, carbohydrates, sugar alcohols and fermentation products was used. The support for this column is sulfonated divinyl benzene-styrene copolymer with a 9 mm particle size. Operating conditions At ambient temperature an isocratic ow rate of 1 ml min1 was maintained with a 0.1% phosphoric acid mobile phase. Detection was at 210 to 290 nm scanning with 5 nm intervals. Column pressure varied between

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5170 and 5520 kPa throughout the separations. Injection volume was 40 ml and the run time was 40 min per injection. RESULTS AND DISCUSSION Extraction methods and separation Figure 1 shows typical HPLC chromatograms of organic acids extracted from Cheddar cheese by means of the various extraction methods. Although the chromatograms appeared to be relatively similar, minor differences could be observed with regard to the number of minor and the intensity of major peaks. Resolution of minor peaks appeared more dened with the 0.009 N H2SO4 extraction [Fig. 1(C)]. Apart from these dierences, HPLC chromatograms were relatively similar amongst the various extraction methods. The overall chromatogram trend obtained with the 0.009 N H2SO4 extract [Fig. 1(C)] was comparable to the chromatograms obtained with the extraction methods using 0.5% w/v (NH4)2HPO40.4% w/v acetonitrile made to pH 2.24 with H3PO4 as extraction agent [Fig. 1(B)] and 30 ml of 0.30 N barium hydroxide30 ml of 0.28 N zinc sulphate [Fig. 1(A)], respectively. From the results it could be concluded that the 0.009 N H2SO4 extraction method (Bouzas et al., 1991) was marginally more suitable for extracting the minor and major organic acids for subsequent ion-exchange separation. An initial large peak (2- to 4-min elution time) could be observed with all three extraction procedures. This peak interfered to a large extent with oxalic acid and could be ascribed to compounds (other than organic acids) retained during extraction. Bevilaqua and Califano (1989) also observed this peak [labelled `u' (unidentied)] with a reversed phase separation at 3 min retention time. It was notable that oxalic acid also had higher recovery values compared to the other compounds. A chromatogram of the organic acid composition of matured Cheddar cheese extracted with the method of Bouzas et al. (1993) is shown in Fig. 2. When the Y-axis (mV signal) of a section of the chromatogram was amplied with the Spectacle software [Fig. 2(B)] it was obvious that fusion of the peaks occurred early in the chromatogram. Analysis of the actual extract was hindered by numerous contaminants that seemingly were not removed during the extraction process [Fig. 2.(A and B]. This can be attributed to early eluting biochemical species not removed during extraction. These compounds interfered with the accuracy of the early eluting organic acids (oxalic acid in particular) and could have an additive eect on the observed concentrations of these compounds. The percentages of recovery for individual compounds extracted with the mentioned extraction procedures are depicted in Table 1. Eleven of the compounds

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J. F. R. Lues et al.

Fig. 1. Ion-exchange HPLC chromatograms of organic acids from matured Cheddar cheese extracted with (A) barium-hydroxide/ zinc-sulphate, (B) buer-acetonitrile and (C) H2SO4.

Fig. 2. Separation of matured Cheddar cheese by ion-exclusion HPLC, with (A) the normal Y-axis calibration and (B) an amplied Y-axis of a complex section of the chromatogram.

out of the total of 16 extracted with 0.009 N H2SO4, yielded the highest recovery of all three extraction methods. The barium hydroxide/zinc sulphate (Marsili, 1985) and buer acetonitrile (Bevilacqua and Califano,

1992) extraction methods had, however, higher recovery rates for a number of individual compounds. Oxalic acid and malic acid were recovered best with the barium hydroxide/zinc sulphate extraction method whereas

Ion-exchange HPLC analysis of organic acids from Cheddar cheese propionic, isovaleric and n-valeric acids showed the highest recoveries with the buer-acetonitrile extraction method. Gonzalez de Llano et al. (1996) reported that extraction assays using acetonitrile proved less ecient, whereas recovery values ranging from 96 to 103% were obtained with an extraction method using 4.5 mmol H2SO4. Oxalic acid had a relatively high recovery rate (97.8103.6%) with all three of the selected extraction methods (Table 1), in comparison with the other organic acids evaluated. This high percentage obtained for oxalic acid could be attributed to non-organic acid compounds that elute together with oxalic acid in an additive manner, and supports the previous observation of the large peak areas found for this compound. Similarly, recovery rates for formic, butyric, isovaleric, nvaleric and hippuric acids were lower than for the other compounds. This was demonstrated by an average recovery rate for these analytes of 86.5% compared to average recovery rates of 91.5% for the other acids. The recovery rates of butyric, isovaleric, n-valeric and hippuric acids could be the result of relatively longer retention times during which these compounds are subjected to mobile-stationary phase interactions. Bevilacqua and Califano (1989) reported recovery rates ranging from 85.3% (butyric acid) to 93.3% (propionic acid) with the buer-acetonitrile extraction mixture, whereas Marsili (1985) reported recoveries for all
Table 1. Percentage recovery (%) for individual organic acids extracted with three dierent extraction procedures determined by ion-exclusion HPLC* Compound 0.009 N H2SO4a 97.8 95.2 92.1 94.6 87.9 93.8 88.5 92.1 90.7 93.3 92.3 92.9 91.3 86.8 85.1 88.9 3.34 Barium hydroxide/zinc sulphateb 103.6 82.7 86.4 91.9 89.9 93.5 85.2 90.8 90.3 87.6 91.8 90 83.1 85.5 84.8 86 5.12 Bueracetonitrilec 99.3 87.9 89.7 89 88.1 91.5 88.3 88.6 90.2 92.7 90.8 94.3 86.4 87 87.6 85.9 3.4

445

Oxalic Citric Orotic Pyruvic Malic Lactic Formic Diacetyl Acetic acid Fumaric Uric Propionic Butyric Isovaleric n-Valeric Hippuric sn1

*Chromatographic conditions: 1 ml min1 ow rate; 0.1% phosphoric acid mobile phase; Supelcogel C-610H column; detector scanning from 210 to 290 nm with 5 nm intervals. Recoveries were the average of three extractions and ve HPLC separations per sample. a Method used by Bouzas et al. (1993). b Method used by Marsili (1985). c Method used by Bevilaqua and Califano (1992).

organic acids of over 90%, except for butyric acid, which was 83% with the barium hydroxide/zinc sulphate extraction method. The high recovery rates achieved with the extraction method using H2SO4 correspond with St-Gelais et al. (1991) who reported recovery rates of more than 92% for organic acids in cheese. Although overall sample recovery was high, Bouzas et al. (1991) found relatively low recovery rates for citric acid (70%) and hippuric acid (80%) with 0.009 N H2SO4 as the extraction agent followed by separation by ion-exchange HPLC [Aminex HPX-87 (H+) column]. Taking into account that the extraction agent is often related to the mobile phase used for separation, the extraction methods other than the 0.009 N H2SO4 recommended in this study could be more suitable for use with columns and chromatographic parameters other than the ion-exchange methodology proposed in this study, as in the case of reverse-phase HPLC. The types of organic acids to be separated could also play a role in the selection of an extraction method, as individual organic acids reacted dierently with dierent extraction agents (Table 1). For example, n-valeric acid showed the highest recovery rate with the buer-acetonitrile extraction procedure, whereas oxalic acid was best recovered with the barium hydroxide/zinc sulphate method. The vastly dierent biochemical characteristics (including polarity and degree of saturation) of the compounds analysed could be a reason for the variation in degree of recovery. The methodology of the 0.009 N H2SO4 extraction was simple to perform and did not require expensive equipment. Although the extraction period (1 hour) was long and more time consuming than vigorous homogenisation, it did not inuence the experimental procedure, as the run-time for the HPLC separation was set at 40 minutes per injection, allowing ample time for extraction when triplicate injections were performed. With large numbers of extractions, the 1-hour extraction period might pose a problem and the zinc-sulphate/ barium-hydroxide method involving a 1-min, high-speed maceration period, could be the method of choice. For analyses of the predominant organic acids in Cheddar cheese, any of the evaluated extraction methods would suce. However, for the purpose of analysing the selected major and minor organic acids from Cheddar cheese with the separation parameters referred to in this study, the method used by Bouzas et al. (1991) involving 0.009 N H2SO4 as extraction agent, is recommended as the appropriate method. It must be pointed out that, although this extraction method was proposed to have the advantage of being eective, simple and rapid being a single extraction (Bouzas et al., 1991), the authors are of the opinion that the extraction procedure needs optimising. The extraction method used to isolate the organic acids from the cheese must facilitate elimination of all substances that might contaminate the organic acid extract, particularly early in the separation

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J. F. R. Lues et al.
Hippuric n-Valeric Isovaleric Butyric Propionic Uric Fumaric Acetic Diacetyl Formic

Table 2. Concentrations (mg kg1) and statistical evaluation of organic acids and related compounds in matured cheddar cheese separated by ion-exclusion HPLC*

(less than 4 min). Although overall sample recovery was good, further investigation is necessary to combine the evaluated extraction procedures with solidsolid or solidliquid sample pre-treatment techniques without sacricing time and eort. Organic acid quantication Concentrations for the compounds analysed in matured Cheddar cheese are shown in Table 2. Surprisingly high concentrations of oxalic acid (1165.4 mg kg1) and nvaleric acids (876.5 mg kg1) were obtained, whereas higher concentrations of pyruvic acid were expected in comparison to amounts reported for these compounds in the literature. Orotic acid, pyruvic acid, diacetyl, formic acid, fumaric acid and uric acid were constantly present in very small concentrations. Hippuric acid was not detected in any of the HPLC separations. The data presented in Table 2 correspond with those of Bouzas et al. (1991), who reported the following organic acid values for commercial Cheddar cheese: 2.2 mg g1 (citric acid), 0.02 mg g1 (orotic acid), 0.074 mg g1 (pyruvic acid), 26.5 mg g1 (lactic acid), 0.6 mg g1 (formic acid), 0.3 mg g1 (acetic acid), 0.98 mg g1 (propionic acid), 0.2 mg g1 (butyric acid and 0.006 mg g1 (hippuric acid). However, Bouzas et al. (1991) did not report values for uric acid in commercial Cheddar cheese. In a study on time and temperature inuence on chemical ageing indicators for a commercial Cheddar cheese, Bouzas et al. (1993) indicated that after a 99-day storage period, citric acid was detected at 0.79 mmoles 1001 g cheese, acetic acid at 1.12 mmoles 1001 g, propionic acid at 2.35 mmoles 1001 g, butyric acid at 0.40 mmoles 1001 g and isovaleric acid at 1.44 mmoles 1001 g. Concentrations of 4.9 mg g1 (orotic acid), 25 mg g1 (citric acid), 26 mg g1 (pyruvic acid), 5140 mg g1 (lactic acid), 16.7 mg g1 (uric acid), 420 mg g1 (formic acid), 600 mg g1 (acetic acid) and <2 (hippuric acid) were reported by Marsili et al. (1981) for sharp Cheddar cheese, while propionic acid was not detected. In a study conducted on the chemical changes during ageing in Cheddar cheese utilising HPLC and GC techniques, Marsili (1985) reported values for pyruvic acid (66 mg kg1), lactic acid (17 000 mg kg1), acetic acid (665 mg kg1) and propionic acid (874 mg kg1) in 10month old Cheddar cheese matured at 6 C. According to St-Gelais et al. (1991), the organic acid concentrations in Cheddar cheese-like products amount to 1.595% (lactic acid), 0.025% (acetic acid), 0.075% (propionic acid) and 0.262% (butyric acid) for control Cheddar cheese matured at 10 C. Values for full-fat Cheddar cheese were reported by McGregor and White (1990) at 265 mg kg1 (citric acid), 12.5 mg kg1 (orotic acid), 23 mg kg1 (pyruvic acid), 840 mg kg1 (propionic acid), 80 mg kg1 (acetic acid) and 8300 mg kg1 (lactic acid). From these literature it is clear that the concentrations reported by authors for the various

+ *Extraction method using 0.009 N H2SO4 (Bouzas et al., 1993). " , Mathematical average of 15 repetitions. b sn1 Standard deviation (n=15). c +, Detected in negligible quantities. d nd, Not detected.
a

Malic Pyruvic Orotic Citric Oxalic

" a sn1b Min Max

1165.4 89.2 991.5 1335.7

1089 77.7 957 1220.4

+c

327.3 21.5 94 381

23 413.9 1175.7 20 463.3 26 641

Lactic

121.5 9.6 104.2 139.2

512.9 463 401.7 629.3

285.9 35.4 199.5 354.6

158.1 34 101.1 234.8

876.5 135.3 694.6 1029.8

ndd

Ion-exchange HPLC analysis of organic acids from Cheddar cheese organic acids vary considerably, however, levels of the major acids correspond well with values found in this study. In conclusion the ion-exclusion high-performance liquid chromatography technique and H2SO4 extraction method evaluated was best suited for the separation and quantication of a broad spectrum of major and minor organic acids and related compounds from matured Cheddar cheese. Reports on the role of organic acids in Cheddar cheese maturation, quality and microbiology have included organic acid and related compounds limited to the major compounds expected in Cheddar cheese, amounting to 10 compounds or less. With the methods proposed in this study, a total of 16 compounds can be isolated and analysed in a single chromatographic run. By using the HPLC separation and extraction techniques described as templates, the inuence of a wide spectrum of organic acids and related compounds on the maturation, biochemistry, avour and microbiology of Cheddar and possibly other cheeses could be assessed.

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cators for a commercial Cheddar cheese. Journal of Food Science 58, 13071312. Gomis, D. B. (1992) HPLC analysis of organic acids. In Food Analysis by HPLC, ed. L. M. L. Nollet, pp. 371385. Marcel Dekker, New York. Gonzalez de Llano, D., Rodriguez, A. and Cuesta, P. (1996) Eect of lactic starter cultures on the organic acid composition of milk and cheese during ripeninganalysis by HPLC. Journal of Applied Bacteriology 80, 570576. International Dairy Federation (1985) International IDF Standard: milk and milk productsmethods of sampling, 50B:1985. IDF General Secretariat. Keen, A. R. and Walker, N. J. (1974) The determination of acetic, propionic and butyric acids in cheese. Journal of Dairy Research 41, 397404. Khalid, N. M. and Marth, E. H. (1990) Lactobacillitheir enzymes and role in ripening and spoilage of cheese: a review. Journal of Dairy Science 73, 26692684. Lombardi, A. M., Bevilacqua, A. E. and Califano, A. N. (1994) Variation in organic acids content during ripening of Reggianito cheese in air-tight sealed bags. Food Chemistry 51, 221226. Marsili, R. T. (1985) Monitoring chemical changes in Cheddar cheese during aging by high performance liquid chromatography and gas chromatography techniques. Journal of Dairy Science 68, 31553161. Marsili, R. T., Ostapenko, H., Simmons, R. E. and Green, D. E. (1981) High performance liquid chromatographic determination of organic acids in dairy products. Journal of Food Science 46, 5257. McGregor, J. U. and White, C. H. (1990) Eect of enzyme treatment and ultraltration on the quality of lowfat Cheddar cheese. Journal of Dairy Science 73, 571578. Morawski, J. (1984) Analysis of dairy products by HPLC. Food Technology 38, 7078. Ohren, J. A. and Tuckey, L. T. (1969) Relation of avour development in Cheddar cheese to chemical changes in the fat of the cheese. Journal of Dairy Science 52, 598607. Panari, G. (1986) HPLC of organic acids: an approach to cheese analysis. Milchwissenschaft 41, 214216. Pirisino, J. F. (1983) High performance liquid chromatographic determination of lactose, glucose and galactose in lactose-reduced milk. Journal of Food Science 48, 742745. St-Gelais, D., Doyon, G., Rolland, J. R. and Goulet, J. (1991) Sugar and organic acid concentrations during ripening of Cheddar cheese-like products. Milchwissenschaft 46, 288 291.

(Received 17 March 1998; accepted 29 December 1998)