Free fatty acid profiles of Reggianito Argentino cheese produced with different starters

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International Dairy Journal 15 (2005) 1150–1155 www.elsevier.com/locate/idairyj

Free fatty acid profiles of Reggianito Argentino cheese produced with different starters M.C. Perotti,1, S.M. Bernal, C.A. Meinardi, C.A. Zalazar2 Programa de Lactologı´a Industrial, Facultad de Ingenierı´a Quı´mica, Universidad Nacional del Litoral, Santiago del Estero 2829, S3000AOM Santa Fe, Argentina Received 15 September 2003; accepted 12 November 2004

Abstract Reggianito Argentino cheeses were made at pilot plant scale using a natural whey starter (control) and different combinations of strains of selected Lactobacillus helveticus (experimental cheeses). Free fatty acids (FFAs) were extracted from cheese samples at different ripening times, and FFAs from C6:0 to C18:2 were analysed by gas chromatography. The characteristics of the chromatographic profiles were studied by principal components and linear discriminant analysis were performed. Levels of individual FFAs from caproic (C6:0) to linoleic (C18:2) acids increased significantly ðpo0:05Þ during ripening in control and experimental cheeses. Palmitic (C16:0) and oleic (C18:1) acids were the most abundant FFAs throughout ripening in all cheeses. No significant differences were found between FFA profiles of control and experimental cheeses. The possibility of using selected strains of Lactobacillus to replace traditional ‘‘natural whey starter’’ in the production of the cheese is discussed. r 2005 Elsevier Ltd. All rights reserved. Keywords: Reggianito Argentino; Starters; Lipolysis

1. Introduction During cheese ripening numerous chemical and biochemical reactions occur involving protein, fat and lactose, producing important changes in texture and sensory characteristics. For certain types of cheese, fat is as great importance to flavour through the generation of free fatty acids (FFAs) and some compounds derived from their transformation (Poveda, Pe´rez–Coello, & Cabezas, 1999; McSweeney & Sousa, 2000). The following lipolytic agents are important to cheese ripening: certain starters microorganisms, lipases from milk clotting enzymes preparations such as pregastric Corresponding author. Tel.: +54 342 4530 302; fax: +54 342 4571 162. E-mail address: cperotti@fiqus.unl.edu.ar (M.C. Perotti). 1 Postdoctoral fellowship of Consejo Nacional de Investigaciones Cientı´ ficas y Tecnolo´gicas. 2 Researcher of Consejo Nacional de Investigaciones Cientı´ ficas y Tecnolo´gicas.

0958-6946/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.11.005

lipase of lamb rennet paste, lipases from moulds included as secondary starters, the natural lipases from milk for raw milk cheeses, and other exogenous lipases. In cheeses where moulds are encouraged to grow during ripening such as Camembert, Cabrales, Roquefort and Gorgonzola, lipolysis is very important in the formation of flavour. In such cases, the agents responsible for the hydrolysis and the products formed have been well studied and characterized (Gonzalez de Llano, Ramos, Polo, Sanz, & Martinez-Castro, 1990; McSweeney & Sousa, 2000). On the other hand, in the Italian cheeses Grana Padano and Pamigiano Reggiano, and in Reggianito Argentino, limited hydrolysis of fat occurs during the long ripening period. This lipolysis, however, makes an important contribution to the formation of cheese flavour. The agents responsible for lipolysis in these cheeses are bacteria from natural whey starters, non-starter lactic acid bacteria (NSLAB) and the indigenous milk lipoprotein lipase in cheeses produced from raw milk (Battistotti & Corradini, 1993).

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In Argentina, natural whey starters are widely used in the production of Reggianito Argentino cheese (Hynes, Bergamini, Sua´rez, & Zalazar, 2003). These starters, composed mainly of Lactobacillus helveticus and L. delbrueckii subsp. lactis, have some disadvantages such as variations in their microbiological composition and the presence of undesirable contaminants, mainly moulds and yeasts (Reinheimer, Quiberoni, Tailliez, Binetti, & Sua´rez, 1996; Reinheimer, Sua´rez, Bailo, & Zalazar, 1995). In order to avoid the disadvantages of natural whey cultures, but preserving the typical technology, the addition of single and mixed cultures of selected strains of L. helveticus grown in whey, has been proved in our laboratory (Candioti et al., 2002; Perotti, Bernal, Meinardi, Candioti, & Zalazar, 2004). Few studies on FFA profiles and their evolution during ripening are available for hard cheeses in general and for Reggianito Argentino in particular (Lindsay, 1983; Woo & Lindsay, 1984). No results have been presented regarding the influence on the profile when natural whey starters are replaced by selected strains in the production. In the present work, we studied the evolution of FFAs during the ripening of Reggianito cheeses manufactured using natural whey starter cultures or with single and mixed cultures of selected strains of L. helveticus and to detect possible differences produced in these profiles during ripening when selected starters replace the natural whey starter.

2. Materials and methods 2.1. Cheese making Control and experimental Reggianito Argentino cheeses were produced at our pilot plant according to a standard process (Hynes et al., 2003). Raw bulk milk (170 L), pH 6:60  0:05; acidity 18  1 D (1 D ¼ 100 mg lactic acid L1) was supplied by a nearby dairy plant on each cheese making day and divided into two vats. One cheese of about 7 kg was obtained from each vat. The milk was standardized to 2.50% fat, and batchpasteurized at 65 1C for 20 min. After cooling to 33 1C, CaCl2 (Merck, Darmstadt, Germany) was added to a final concentration of 0.02% (w/v). A volume of starter was added sufficiently to increase the milk acidity by 4 D. The procedure of measuring the acidity of the milk instead of the starter culture volume is traditional for hard cheeses like Reggianito, and it provides a rough uniformity in the number of viable cells per millilitre of milk. The inoculum volume was 4% (v/v) of the milk, for a whey culture having a pH between 6.3 and 6.4. After 10 min of mechanical stirring (100 rpm), adult bovine coagulant (230 IMCU mL1, 0.29 mL L1of

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milk) (Naturen, Chr. Hansen, Quilmes, Buenos Aires) was added. After 18–20 min, the curd was cut to the required grain size (approximately half the size of a rice grain) and the mixture of the curd particles and whey was gently stirred and heated at 0.5 1C min1, until it reached 44 1C to reduce moisture in the curd grains. The mixture was then more rapidly (1 1C min1) heated to 51 1C. When the temperature reached 51 1C the stirring was stopped. The curds were separated from the whey, put into moulds and the remaining whey was discarded. The curd was pressed for 24 h after which it was brined in 20% (w/v) brine, pH 5.4, at 12 1C for 6 days. During this period, the cheese was inverted every other day. Ripening was carried out at 12 1C and 80% relative humidity for 6 months. Samples were taken from curd just after manufacture and from cheeses according to the IDF standard method (International Dairy Federation, 1995) at 90 and 180 days of ripening, and were stored at –18 1C until analysis. 2.2. Starter cultures Natural whey starter cultures from our region (Santa Fe, Argentina) have roughly constant microbiological composition: 66% L. helveticus strains and 33% L. delbrueckii subsp. lactis strains. No strains of Streptococcus thermophilus were found (Reinheimer et al., 1996). These natural whey starters were used in control cheeses. Experimental cheeses were made using three strains of L. helveticus (Lh 133, Lh 138 and Lh 209) single or in different combinations, which were isolated from natural whey starters as described before (Reinheimer et al., 1995,1996; Quiberoni, Tailliez, Que´nee, Sua´rez, & Reinheimer, 1998). Samples of natural whey starter were taken from a nearby Reggianito Argentino dairy plant after the cooking step, and immediately brought to our laboratory under refrigerated conditions in a container with ice. Once in the laboratory, the pH of the whey was adjusted to 6.3 with 40% (w/v) NaOH (Merck, Darmstadt, Germany) under controlled microbiological conditions, and the whey was divided into two fractions of 1.8 L. One of the fractions was incubated for 24 h at 45 1C in order to obtain the natural whey starter for manufacture of the control cheese. The remaining fraction was heated for 5 min at 85 1C to destroy all vegetative cells. After cooling to 45 1C, it was inoculated (2% v/v) with active cultures of L. helveticus 133, 138 and 209 alone or in the following combinations: Lh133+Lh138, Lh133+Lh 209, Lh138+Lh 209 and Lh133+Lh138+Lh209. The single strains were cultured at 45 1C for 24 h in sterilized (110 1C, 10 min), reconstituted (10% w/v) low-heat skim milk powder. When combinations of strains were used, the mixture was prepared just before adding to the vat.

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2.3. Experimental design Eight Reggianito Argentino cheeses were manufactured. Control cheeses with natural whey starter (1), three types of cheese made with single strain Lh 133 (2), Lh 138 (3) and Lh 209 (4), three types of cheese made with two strains Lh133+Lh138 (5), Lh133+Lh209 (6) and Lh138+Lh209 (7) and a cheese made with three strains Lh133+Lh138+Lh209 (8). For each cheese, three replications were made, which gave a total of 24 cheeses made in 12 cheese making days (two cheeses per day). The distribution of control and experimental cheeses on making days was randomized.

equipped with a fused silica capillary column PE-Wax (polyethylene glycol, 30 m  0.25 mm  0.25 mm), was used. The oven temperature was programmed as follows: 50 1C (4 min), 10 1C min1, 150 1C (3 min), 10 1C min1, 230 1C (5 min). The injector was thermostatized at 220 1C 1 at a split mode (split ratio 52 ). The FID was maintained at a constant temperature of 275 1C and nitrogen was employed as carrier gas at a rate of 3.3 mL min1. The sampling of cheeses made with eight different starters at three times of ripening and with three replications for each cheese, gave 72 samples. From the chromatographic analysis, 648 concentrations in mg kg1 of the nine fatty acids were obtained.

2.4. Cheese composition and microbiology

2.6. Principal component analysis

Dry matter and total protein were determined in cheeses at 180 days of ripening, according to IDF standard methods (International Dairy Federation, 1982, 1993), and fat content by the APHA method (Bradley et al., 1993). The lactic acid bacteria was determined during ripening and at the end of the ripening according to Candioti et al., 2002.

Multivariate statistical analysis of the experimental data was performed by principal component analysis (PCA) and linear discriminant analysis (DA) (Statistix 7, Analytical Software, Tallahassee, FL, USA and Statgraphics plus 3.1, Manugistics, Inc., Rockville, MD, USA) to study and emphasize the differences in FFA profiles between the starters investigated and also the variations produced by the time of ripening. The influence of lipolysis in the cheese on the amount of different FFA was observed by considering the PCA loadings. The PCA scores were employed as predictor variables for the linear discriminant analysis with the aim of verifying the influence of ripening time and starter type on the profiles of lipolysis (Gardiner, 1997; Hair, Anderson, Tatham, & Black, 1999).

2.5. Free fatty acid analysis Individual FFA from C6:0 to C18:2 were extracted and quantified by GC in samples of control and experimental cheeses (24 cheeses analyzed at three times of ripening). The extraction was performed on 5 g of curd or cheese samples acidified to a pH lower than 2 with 50% (v/v) sulfuric acid. In order to dehydrate samples, anhydrous sodium sulfate (20 g) was added. A solid–liquid extractor (Twyselmann) was employed with 100 mL of n-hexane at 60 1C for 2 h (Mehlenbacher, 1970). Enantic (C7:0) and margaric (C17:0) acid added to the extraction mixture were used as internal standards. At the end of this extraction, the FFA in the fatty phase were converted to sodium salts by titration with 0.1 M NaOH. The sodium salts of the FFA were then separated from the fatty phase by successive washing with water. The salt solution was then evaporated to dryness as described by Contarini, Zucchetti, Amelotti, & Toppino (1989). The salts were esterified with 8 mL of ethanol–sulphuric acid (5% v/v) in cap tubes at 7072 1C for 1 h. After cooling the tubes, 6 mL of water and 1 mL of n-hexane were added with vigorous stirring for 1 min. The upper phase containing the ethyl esters was analysed by triplicate in a gas chromatograph, injecting 1 mL with a Hamilton syringe. The FFAs C6:0 to C18:2 and the internal standards were quantified by means of calibration curves prepared using a mixture of FFA standards (1 mg mL1 in isopropanol for each acid). A Perkin Elmer model 9000 gas chromatograph with a Turbocrom v4.0 software fitted with a split-splitless injector and a flame ionization detector (FID) and

3. Results and discussion 3.1. Cheese composition and microbiology Gross composition of all cheeses at 180 days of ripening were within the ranges established by Mercosur Regulations for Reggianito cheese (Mercosur Res. GMC, 1997). No significant differences ðp40:05Þ in dry matter, fat matter and total protein were found among cheeses made with the different starters. Mean values were 62:61  1:50% for dry matter, 24:00  1:37% for fat and 28:95  0:59% for total protein. As was reported by previous workers (Candioti et al., 2002; Perotti et al., 2004), the lactic acid bacteria population in curd before moulding was about 107 CFU g1 for control and experimental cheeses. After 24 h all cheeses achieved a maximum of about 109 CFU g1. During ripening, lactic flora decreased by 2 or 3 log orders, reaching values near 107 CFU g1 at the end of the ripening. For cheese made with strain Lh 209 alone or in different combinations somewhat lower counts were observed, but differences were very small as was shown by Candioti et al. (2002).

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3.2. Free fatty acid profiles

3.3. Multivariate data analysis The results on FFA content of 72 samples were used in the construction of the matrix for PCA analysis. A

Fig. 1. Changes in the chromatographic FFA profile of Reggianito Argentino cheese during ripening. Cheese made with strain Lactobacillus helveticus 138 grown in whey. Peaks: 1, caproic; 2, enantic (internal standard); 3, caprilic; 4, capric; 5, lauric; 6, myristic; 7, palmitic; 8, margaric (internal standard); 9, stearic; 10, oleic; 11, linoleic.

1600

mg kg-1 of cheeses

In Fig. 1, examples of typical chromatographic profiles of FFAs for curd and cheese 3 at 0, 90 and 180 days or ripening, are presented. Fig. 2 shows the FFA profile for Reggianito Argentino cheese made with natural whey starter (mean of three replicate cheeses) at 180 days of ripening. For comparison, the figure includes also a profile for Parmigiano Reggiano cheese (mean for 60 samples) (Caboni, Zanoni, & Lercker, 1988). From these profiles, it can be seen that the distribution of FFAs in cheeses is not different from the distribution of fatty acids in milk-fat. As a consequence, a non-selective fat hydrolysis occurs during cheese ripening. However, quantitatively speaking, a more intense lipolysis is produced in Italian cheese, which has higher values (Parmigiano Reggiano approximately three times higher than Reggianito) for all the FFAs determined. The fat hydrolysis in Reggianito cheese expressed as percentage of FFAs in relation to total cheese fat, reached a value lower than 1%. A value of 1.7% has been reported for Parmigiano Reggiano. The raw milk used in the production of Parmigiano Reggiano and the longer ripening period of this cheese, could be the reasons of these differences. Myristic, palmitic, stearic and oleic acids were the main FFAs for both Reggianito and Parmigiano Reggiano cheeses. During the 180 days of ripening, concentration of all the FFAs in Reggianito increased from curd to ripened cheese (180 days) (data not shown).

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1400

Reggianito Argentino

1200

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1000 800 600 400 200 0 caproic caprilic capric

lauric myristic palmitic stearic

C18 insat.

Free fatty acids

Fig. 2. Comparison between free fatty acid profiles of Reggianito Argentino and Parmigiano Reggiano cheeses. Reggianito Argentino data from this paper and Parmigiano Reggiano data from Caboni et al. (1988).

high positive correlation among all variables was found ðpo0:05Þ and consequently, the application of principal component analysis was justified (Hair et al., 1999). Variables were standardised to zero mean. This was equivalent to making the principal component analysis using the variance–covariance matrix (Everitt & Dunn, 1997; Pripp, Stepaniak, & Sørhaug, 2000). The first and second principal components were selected (PC1 and PC2) from the PCA according to the Cattell criterion (Everitt & Dunn, 1997; Hair et al., 1999). These two components accumulated 97.8% of the total variability from the original data matrix (PC1 94.7%, and PC2 3.1%). In Fig. 3, loadings for PC1 and PC2 are represented in a two-dimensional space. In relation to PC1 loading, three categories of positive values were observed. In a decreasing order, the first level was formed by oleic and palmitic acid, the second level by myristic and stearic acid and the third by the remaining acids. This order is a representation of the importance of each FFA to the total profile during ripening, as it was already seen in the FFA profile of Fig. 1. The fact that all loadings of PC1 were positive means that the concentrations of the nine fatty acids increased during ripening for control and experimental cheeses. In relation to PC2 loadings, it can be observed that oleic, myristic and palmitic acid had the higher loadings, loadings for the first acids being positive and for the second and third negative. The remaining fatty acids had low loadings. The different signs observed in PC2 loadings are probably associated with different lipolytic actions among the starters used. In particular, myristic acid showed different evolutions for different starters during ripening (data not presented). Representation in two dimensions for PC1 and PC2 scores is another aspect of the PCA which allows the

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-1.0 PC1 loading caproic

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C1 H1 E1

C2 H2 E2

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C4 H4 E4

C5 H5 E5

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C8 H8 E8

Fig. 3. Bidimensional representation of PC1 and PC2 loadings of PCA for the free fatty acid; profiles of cheeses made with natural whey starter and different combinations of selected strains of Lactobacillus helveticus at 0, 90 and 180 days of ripening.

Fig. 4. C1–C8 curds, H1–H8 cheeses at 90 days (half ripening time), E1–E8 cheeses 1 at 180 days (end of ripening). Numbers 1–8 refer to cheeses produced with natural whey starter and different combinations of selected strains of L. helveticus (3 replications for each cheese). Circled: samples containing Lh 138.

visual comparison of the samples for establishing possible associations among them (Fig. 4). The origin of coordinates represents a mean lipolysis profile. Visual observation of the dispersion among samples permitted some conclusions. Curds were grouped in the negative zone of PC1, with low values, either positive or negative for PC2. Samples of 90-dayold cheese were grouped in a zone near the coordinates origin and samples of 180-day were grouped in the positive quadrant of PC1 with profiles higher than the mean. From this analysis, an increase in levels of all FFA with time in the sense of positive PC1 is clear, an observation already stated from the evolution of FFA profiles during ripening (see Fig. 1). In order to validate the previous conclusion based on the visual observation of scores, Linear Discriminant Analysis (DA) was applied using PC1 and PC2 scores as predictors. Time of ripening and type of starter were used as classification factors. A significantly discriminative function ðpo0:05Þ was found with respect to the time factor. This function allowed the correct classification in the 3 pre-defined groups (0, 90 and 180 days of ripening). The situation was not the same for the starter type factor since we found no significant discriminate function to make a distinction among the 8 predetermined levels (natural whey starter and starters composed of L. helveticus strains) when a PCA analysis was made for all the samples. However, from Fig. 4 it is possible to estimate visually some differences among the scores. Cheeses with 180 days of ripening and produced with strain Lh 138 alone or in different associations with other strains (E3, E5, E7 and

E8, circled in Fig. 4), were located far from the origin and they showed high values of PC1 and PC2 as a consequence of the importance of lipolytic profiles. This situation could be interpreted as a higher lipolytic activity of L. helveticus 138 in relation to strains Lh 133 and Lh 209.

4. Conclusions The FFA profiles for Reggianito Argentino cheese made with natural whey starter at 180 days of ripening were presented. Only limited information is available about these profiles and therefore these results will be useful to understand better the ripening process of this typical Argentinean hard cheese. From comparison between the FFA profiles for Reggianito Argentino and Parmigiano Reggiano it was concluded that the lipolysis in the Italian cheese is more important possibly as a consequence of the raw milk used for its manufacture and its long ripening period. However, the proportion of FFAs in the profile was similar for both cheeses and levels of all FFAs increased with ripening time. PCA and DA did not show important differences between control cheeses made with natural whey starter and experimental cheeses made with selected bacteria, from the point of view of lipolysis. Taking into account that Reggianito Argentino is a cheese with a ripening period considerably shorter than that for Parmigiano Reggiano, and with less intense flavour, these results together with results in relation to proteolysis reported by other workers (Hynes et al.,

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2003), would allow the use of selected Lactobacillus helveticus strains isolated from natural whey starter in place of natural whey starter without significant changes in the physicochemical and sensory properties of cheeses. These selected strains developed in whey maintain the advantages of natural whey cultures and allow a greater control of the cheese-making and ripening processes.

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