Free fatty acids and other volatile compounds for the characterisation of “Vastedda della valle del Belìce” cheese
Acidos grasos libres y otros constituyentes volátiles para la caracterización de queso “Vastedda della vella del Belìce”

Abstract

The analysis of the volatile constituents of “Vastedda della valle del Belìce”, a typical Sicilian pasta filata cheese, was performed using solid phase microextraction and high-resolution gas chromatography/mass spectrometry. The research aimed to verify if the volatile fraction, determinant for cheese flavor, differs among producers and/or production seasons. The samples were provided by four producers from the area of the Protected Designation of Origin (PDO) production, during two different seasons of production. A total of 42 volatile components were identified: the main components were found to be butanoic, hexanoic, octanoic, and decanoic acid. Free fatty acids were quantified using the standard addition method, their concentrations were in the range 1273.7–1918.0 mg/kg. The analysis of variance and a multivariate approach showed that producer and season factors significantly influenced the content of almost all the identified volatile components; the artisanal cheese-making, the utilization of raw milk, and the environmental differences that characterized the various farmers are strong causes of cheeses variability.

El análisis de los constituyentes volátiles de ‘Vastedda della valle del Belìce’, un queso de pasta hilada (pasta filata) típica siciliana, fue llevado a cabo usando Microextracción en Fase Sólida y Cromatografía de Gases/Espectrometría de Masa de Alta Resolución. El objetivo de la investigación fue verificar si la fracción volátil, determinante para el sabor del queso, difiere entre productores y/o temporadas de producción. Las muestras fueron proporcionadas por cuatro productores del área de la Denominación de Origen Protegida (DOP) durante dos temporadas de producción diferentes. Se identificaron un total de cuarenta y dos componentes volátiles: los principales componentes encontrados fueron ácido butanoico, hexanoico, octanoico y decanoico. Los ácidos grasos libres fueron cuantificados usando el método de adición estándar, su concentración se encontró en el intervalo de 1273, 7–1918,0 mg/kg. El análisis de varianza y un a aproximación multivariada mostraron que productor y temporada influyeron significativamente en el contenido de casi todos los componentes volátiles identificados; la elaboración artesanal de queso, la utilización de leche cruda y las diferencias ambientales que caracterizaban a los diferentes productores son la causa de la variabilidad de los quesos.

Palabras clave:

• queso Vastedda della valle del Belìce
• volátiles
• ácidos grasos libres
• resultados cuantitativos
• SPME
• GC-MS

Keywords:

• Vastedda della valle del Belìce cheese
• volatiles
• free fatty acids
• quantitative results
• SPME
• GC-MS

Introduction

“Vastedda della valle del Belìce” is a semi-hard fresh Sicilian cheese from the Belice valley among the provinces of Palermo, Agrigento, and Trapani. It is a pasta filata cheese traditionally made with raw ewes’ milk without the addition of any starter cultures and using rennet paste to coagulate the milk. “Vastedda della valle del Belìce” cheese is today manufactured in 18 small dairies mainly near the sheep shelter; it is a small round cheese without rind, characterized by the unusual shape of a bun, weighing about 500–700 g. It is traditionally consumed within few days of production mainly in the nearly area but, nowadays, thanks to the growing interest towards traditional foods, it is marketed and appreciated also outside of Sicily (Mucchetti, Bonvini, Remagni, & Ghiglietti, 2008). “Vastedda della valle del Belìce” has been officially classified as a “historical dairy product” by Regione Sicilia; since it has already obtained the National Transitory Protection (Official Journal of the Italian Republic, 14 December 2007, No. 290) and protected designation of origin (PDO) status is approaching. The typical characteristic of the milk, the special geoclimatic conditions of this region, the natural pastures and the traditional technology used for the production may explain its unique sensorial characteristics. The knowledge about “Vastedda della valle del Belìce” cheese-making technology and composition were restricted to few researches (Conte & Barreca, 1996; Reale et al., 2007); no studies on the volatile fraction composition have been reported so far.

Flavor is one of the most important quality criteria both for fresh and aged cheeses; the consumer's acceptance of cheese mainly depends on its sensory qualities and, in this context, the flavor is decisive. During cheese processing and ripening, the enzymatic activity on milk protein, fat, lactose and citrate lead to the formation of a wide range of volatile compounds, of different polarity and reactivity, responsible for cheese flavor (Engels, Dekker, de Jong, Neeter, & Visserr, 1997; Nijsse, Visscher, Maarse, Willemsens, & Boelens, 1996). Consequently, volatiles are useful to define and improve cheese quality, to detect off flavors, to evaluate changes during the ripening process, and to choose the right ripening time (Mulet, Eschiriche, Rossello, & Tarrazò, 1999); they are also important for characterizing the cheese linking it with its typical sensorial features and its original environment, especially in case of PDO cheeses, where quality continues to be a challenge for cheese markers.

In the context of researches which aim to protect the quality of Sicilian cheeses (Condurso, Verzera, Romeo, Ziino, & Conte, 2008; Condurso, Ziino, Palmegiano, Romeo, & Verzera, 2006; Verzera, Ziino, Condurso, Romeo, & Zappalà, 2004; Ziino, Condurso, Romeo, Giuffrida, & Verzera, 2005), free fatty acids and other volatile components of “Vastedda della valle del Belìce” cheese have been studied. Particularly, this study intends to verify if the volatile constituents differ among producers and/or production seasons as it was already observed for chemical and microbiological characteristics (Conte & Barreca, 1996; Mucchetti et al., 2008).

Material and methods

Origin of the cheeses

Forty-eight “Vastedda della valle del Belìce” samples were provided by four different producers from the area of PDO production. For each producer 12 cheeses, within seven days from the cheese-making process, were collected during two different production seasons, exactly six samples in winter and six samples in spring. The cheeses were transferred from the farmhouse to the laboratory in a refrigerated box at 4–6 °C; from each cheese sample, slices were cut from the inner part of the mould and then vacuum-wrapped and stored at −20 °C until sampling. One day before the analysis, the samples were defrosted at 4 °C and the sample slices were finely grated and homogenized.

SPME procedures

The volatile components were extracted by the static headspace solid-phase microextraction method (HS-SPME). SPME was performed with a commercially available fibre housed in its manual holder (Supelco, Bellefonte, PA). All extractions were carried out using a DVB/CAR/PDMS (divinylbenzene/carboxen/polydimethylsiloxane) fibre, 50/30 μm film thickness (Supelco). Each sample (6 g) was dissolved in 12 mL water in a 40 mL vial. Extraction was performed in the headspace vial, keeping the vial at 60 °C. The sample was equilibrated for 30 min; the extraction time was 40 min; during extraction the sample was stirred continuously with a magnetic stir-bar on a stir-plate revolving at 750 rpm. The fibre was carefully placed in the same location for each exposure to the headspace to achieve maximum repeatability. After sampling, the SPME fibre was introduced into the GC–MS injector. The fibre was kept in the splitless injector, maintained at 260 °C, for 3 min for thermal desorption of the analytes.

GC–MS analysis

A Varian 3800 gas chromatograph, directly interfaced with a Varian 2000 ion-trap mass spectrometer (Varian, Milan, Italy), was used to analyze the cheese headspace components. Injector temperature, 260 °C; injection mode, splitless; capillary column, CP-Wax 52 CB (Chrompack Italy, Milan, Italy), 60 m, 0.25 mm i.d., 0.25 μm film thickness; GC oven temperature, 45 °C held for 5 min, then increased to 80 °C at 10 °C /min and to 240 °C at 2 °C /min; carrier gas, helium at a constant pressure of 68.95 kPa. Transfer line temperature, 250 °C; ionization technique, electronic impact (EI) at 70 eV; acquisition range, 30–200 m/z; scan rate, 1 μs. The components were identified by comparing their experimental spectra with those of NIST ′98 (NIST/EPA/NIH Mass Spectra Library, version 1.7, USA), by use of linear retention indices, and by injection of standards where possible; the linear retention indices (LRI) were calculated according to the Van der Dool and Kratz equation (Van den Dool and Kratz, 1963).

Quantitative analysis

Short-chain fatty acids from C₂ to C₁₂ were quantified in all the cheese samples. Each peak quantified was required to have a minimum signal-to-noise ratio (S/N) of 5. Quantitative results were obtained by using the method of standard additions. Linear saturated FFA from C₂ to C₁₂ was purchased from Sigma-Aldrich (Milan, Italy) of the highest purity available. Stock solutions of individual free fatty acid standards were prepared by dissolving the appropriate amount of each compound in alcohol to obtain a final concentration of 150 mg/mL for butanoic and hexanoic acids, 120 mg/mL for octanoic acid, 60 mg/mL for decanoic acid, and 10 mg/mL for each of even number carbon fatty acids, acetic acid and dodecanoic acid. Two mixed stock solutions, namely A and B, were prepared from stock solutions of individual standards by mixing and diluting with alcohol; solution A contained butanoic acid (50 mg/mL), hexanoic acid (50 mg/mL), octanoic acid (20 mg/mL), and decanoic acid (10 mg/mL), whereas solution B contained acetic acid (4 mg/ml), pentanoic acid (1.5 mg/ml), heptanoic acid (1.5 mg/ml), nonanoic acid (1.5 mg/ml), dodecanoic acid (1.5 mg/ml). Stock solutions were stored at under −30 °C. Calibration standards containing the fatty acid standards in a ratio 0.2–1 times those of the corresponding analytes were prepared by appropriate dilution of the mixed stock solutions with alcohols. Furthermore, three different concentrations of free fatty acids were added to multiple aliquots of each cheese sample. The sample alone was also analyzed. Quantification was based on a calibration curve generated by plotting detector response versus the amount spiked of each standard. The samples were extracted and analyzed by SPME–GC–MS as previously described. Each sample measurement was repeated three times.

Statistical analysis

Statistical analysis was carried out using univariate and multivariate approaches. A two factor ANOVA model was utilized to evaluate the effects of producer and production season on the cheese volatile compounds. Moreover, a canonical discriminant analysis was used to evaluate the differences between producers. The squared Mahalanobis distances between class means were computed too. Statistical analyses were carried out using the GLM and the CANDISC procedures of the SAS software package (V 9.1, SAS Inst., Cary, NC).

Results

Forty-four volatile components were identified in the cheese samples analyzed, namely fatty acids, ethyl esters, ketones, aldehydes and alcohols, aromatic compounds, hydrocarbons, monoterpenes and lactones (Table 1).

Table 1. Least square means of the abundance^a of volatile components detected in “Vastedda della valle del Belìce” cheese samples with different producers and production season.

Tabla 1. Mínimos cuadrados promedio de la abundancia^a de la constituyentes volátiles por los distintos productores y temporadas productivas.

	Dairies				Season
Compounds	1	2	3	4	Winter	Spring
Alcohols
3-methyl-1-butanol	95 A^b	80 A	57 B	53 B	143 A	−B
1-pentanol	266 A	239 A	−C	173 B	240 A	99 B
1-hexanol	90 AB	59 B	117 A	96 AB	162 A	20 B
1-heptanol	109 A	590 B	−B	64 B	292 A	90 B
2-heptanol	−B	97 A	22 B	−B	−B	60 A
2-ethyl-1-hexanol	28 B	12 C	−D	66 A	32 a	21 b
1-nonanol	43 A	25 B	−C	42 A	55 A	−B
phenylethanol	23 B	16 B	48 A	10 B	49 A	−B
phenol	12 C	21 C	47 B	130 A	69 A	36 B
Aldehydes
nonanal	95 A	−B	−B	55 A	75 A	−B
limonene	311 BC	1204 A	637 B	147 C	207 B	942 A
o-cymene	56 A	82 A	7 B	21 B	−B	84 A
β-caryophyllene	−B	51 A	57 A	50 A	8 B	71 A
Esters
ethyl octanoate	102 B	128 B	448 A	131 B	235 A	169 B
methyl decanoate	−B	63 A	−B	59 A	61 A	−B
ethyl decanoate	22 C	98 A	65 B	55 B	89 A	31 B
Free fatty acids
acetic acid	739 A	558 B	731 A	587 B	539 B	768 A
butanoic acid	3827 B	7673 A	7046 A	6311 A	4991 B	7438 A
pentanoic acid	80 A	67 A	72 A	43 B	71 a	60 b
hexanoic acid	11541 A	10962 A	19047 A	6322 B	10939 B	12996 A
heptanoic acid	85 A	−B	111 A	25 B	69	41
octanoic acid	4592 B	4199 B	5391 A	4301 B	4288 B	4954 A
nonanoic acid	99 B	−C	127 A	37 C	70	61
decanoic acid	1329 B	1450 B	2597 A	2799 A	1634 B	2454 A
benzoic acid	126 B	156 B	358 A	44 C	292 A	51 B
dodecanoic acid	59 B	17 C	132 A	B	99 A	31 B
Hydrocarbons
toluene	108 A	101 A	−B	97 A	130 A	23 B
heptadecane	−B	−B	40 A	16 B	28 A	−B
octadecane	−BA	13 B	62 A	11 B	43 A	−B
Ketones
acetone	30 B	−C	−C	75 A	53 A	−B
2-pentanone	258 A	286 A	84 B	0 C	245 A	69 B
2-heptanone	157 A	143 A	21 B	159 A	157 A	83 B
3-hydroxy-2-butanone	40 A	42 A	−B	44 A	63 A	−B
2-nonanone	170 A	61 B	159 A	162 A	180 A	96 B
2-undecanone	−B	76 A	88 A	64 A	114 A	−B
2-tridecanone	−B	12 B	20 A	5 BC	19 A	−B
Monoterpenes and sesquiterpenes
α-pinene	41 B	33 B	340 A	−B	4 B	204 A
β-pinene	47 B	79 B	−C	295 A	−B	211 A
limonene	311 C	1204 A	637 B	147 C	207 B	942 A
o-cymene	56 A	82 A	7 B	21 B	−B	84 A
caryophyllene	−B	51 A	57 A	50 A	8 B	71 A
Lactones
δ-octalactone	20 B	9 B	35 A	11 B	38 A	−B
δ-decalactone	39 B	33 B	103 A	50 B	112 A	−B
δ-dodecalactone	−C	−C	49 A	26 B	37 A	−B
Notes: Means followed by different capital letters in the same row and within each factor indicate significant differences at p ≤ 0.01; different small letters at p ≤ 0.05.
^aAbundance expressed as peak area, arbitrary scale.
Notas: Medias con letras mayúsculas distintas en la misma fila son diferentes (p ≤ 0,01); letras minúsculas (p ≤ 0,05). ^aAbundancia expresada en términos de área bajo pico, escala arbitraria.

The main components of the volatile fraction of “Vastedda della valle del Belìce” were free fatty acids, above all butanoic, hexanoic, octanoic and decanoic acid; the total concentration of free fatty acids was in the range 1273.7–1918.0 mg/kg (Table 2).

Table 2. Least square means of the amount (mg/kg) of free fatty acids in “Vastedda della valle del Belìce” cheese samples with different producers and production season.

Tabla 2. Mínimos cuadrados promedio de la abundancia de la cantidad de ácidos grasos por los distintos productores y temporadas productivas.

	Dairies				Season
	1	2	3	4	Winter	Spring
Free fatty acids
acetic acid	59.6 A	48.2 B	59.1 A	50.0 B	47.0 B	61.4 A
butanoic acid	253.6 A	495.2 A	455.8 A	409.7 A	326.7 B	480.4 A
pentanoic acid	20.0 A	17.4 A	17.7 A	15.9 B	17.6 a	17.8 b
hexanoic acid	477.5 B	455.0 B	769.5 A	274.5 C	454.1 B	534.1 A
heptanoic acid	11.9 A	−B	20.1 A	8.1 B	10.9	9.2
octanoic acid	301.7 B	277.0 B	351.9 A	283.4 B	282.6 B	324.4 A
nonanoic acid	12.8 B	−C	21.1 A	15.5 B	14.3 a	10.4 b
decanoic acid	96.6 B	104.3 B	176.3 A	189.0 A	115.8 AB	167.3 A
dodecanoic acid	16.9 B	14.2 CB	21.4 A	16.4 B	19.4 A	15.1 B
all	1273.7 C	1436.2 B	1918.0 A	1287.4 C	1308.4 B	1649.3 A
Notes: Means followed by different capital letters in the same row and within each factor indicate significant differences at p ≤ 0.01; different small letters at p ≤ 0.05.
Notas: Medias con letras mayúsculas distintas en la misma fila son diferentes (p ≤ 0,01); letras minúsculas (p ≤ 0,05). ^aAbundancia expresada en términos de área bajo pico, escala arbitraria.

Free fatty acids in cheese originate from lipolysis of milk fat; in our samples lipolysis is mainly due to the pregastric esterase of the lamb rennet paste used to coagulate the milk and its activity is responsible for the high amount of short-chain free fatty acids imparting a desirable piquant flavor to the cheese described as “natural lamb rennet flavour” (Virto et al., 2003). The free fatty acid profile in Vastedda cheese samples resulted similar to that reported for other Italian cheeses manufactured with lamb rennet paste both from ewes’ (Di Cagno et al., 2003) and from cows' milk (Ziino et al., 2005), such as Pecorino Romano, Fiore sardo, and Provola dei Nebrodi. Similar free fatty acid profile in ewes' and cows' milk cheeses manufactured with lamb rennet paste should be expected because, despite the different concentration of esterified forms of octanoic and decanoic acids in bovine and ovine milk fat, it was demonstrated (Ha & Lindsay, 1993) that the amount of free octanoic and decanoic acids released by pregastric lipases activity from bovine and ovine milk fat did not show any significant difference. Moreover, the total amount of free fatty acids of Vastedda della valle del Belìce cheese was in agreement to that reported for fresh Provola dei Nebrodi, another Sicilian pasta filata cheese consumed both as fresh (2–4 weeks ripened) and aged product (3–4 months) (Verzera et al., 2004).

The volatile free fatty acids are important components of cheese flavor not only when they give the main note to the cheese flavor but also because they act as precursors of other important aroma components, such as methylketones, alcohols, aldehydes, and esters. In the volatile fraction of Vastedda, these classes of substances were less represented if compared with that of free fatty acids. It was expected since Vastedda is a fresh cheese and concentrations of aldehydes, alcohols, esters, etc. increased significantly with ripening time, as previously demonstrated in other raw ewes' milk cheese (Fernández-García, Gaya, Medina, & Nuñez, 2004). Among the determined esters, ethyl octanoate showed the highest area value. Esters are mainly produced by enzymatic or chemical reaction of fatty acids with primary alcohols (Engels, Dekker, de Jong, Neeter, & Visserr, 1997) and the alcohol concentration is usually the limiting factor in ester production; esters are abundant for example, in soft and mold-ripened cheeses (Molimard & Spinnler, 1996). Nonanal was the only aldehyde identified. Aliphatic aldehydes appear at low concentrations in the volatile fraction of most cheeses; however, they have been found as major components in Parmigiano cheese (Engels et al., 1997) and in cheeses made using strains of Lactococcus lactis as starter cultures (Ayad, Verheul, Wouters, & Smit, 2000).

Even if in a low amount, 2-methyl ketones from C₂ to C₁₃ were found, with 2-pentanone, 2-heptanone and 2-nonanone as the main components. 2-Methyl ketones are the result of enzymatic oxidation of free fatty acids to β-ketoacids and their consequent decarboxylation to alkan-2-ones with the loss of one carbon atom (McSweeney & Sousa, 2000). High quantities of these compounds are usually present in mold-ripened cheeses and also in most hard cheese such as Parmigiano and Cheddar (Barbieri et al., 1994; Bellesia et al., 2003; Engels et al., 1997; Liebich, Douglas, Bayer, & Zlatkis, 1970). Primary and secondary alcohols, which contribute with fruity and nutty notes to the flavor of cheese (Gallois & Langlois, 1990), have been identified; 1-pentanol, 1-hexanol and 1-heptanol were more abundant than other alcohols. 3-Methyl-l-butanol has been also identified; it is produced by the reduction of the corresponding aldehyde which is derived from the amino acid leucine (Larrayoz, Addis, Gauch, & Bosset, 2001). 3-Methyl-l-butanol is responsible for the pleasant aroma of fresh cheese and, in general, it is abundant in rennet curd cheese during early ripening (Moio, Dekimpe, Etievant, & Addeo, 1993). A small amount of phenol and 2-ethylphenol have been determined; phenol and ethyl substituted phenols are important components of the volatile fraction of ewes' cheese because they significantly contribute to sheep-like flavor notes (Ha & Lindsay, 1991). δ-Octalactone, δ-decalactone, and δ-dodecalactone were found to be present in small amounts in the volatile fraction of Vastedda. Lactones contribute to cheese flavor giving fruity and sweet creamy fermented notes (Dufosse, Latrasse, & Spinnler, 1994) and are present in higher amounts in cheese obtained from pasteurized milk. The terpenes and hydrocarbons identified are probably because of the sheep feeding (Dumont & Adda, 1979). Some authors demonstrated that benzyl compounds, including toluene, come from the degradation of carotene in milk (Johnson, Nursten, & Self, 1969), whereas terpenes come from the pasture diet of the animals (Mariaca et al., 1997). Particularly, monoterpenes such as limonene (citrus, light, weak), 1,8-cineole (fresh, eucalyptus), pinenes (light, pine), etc. are well known components of plant essential oils, especially of citrus species which are widely diffused in Sicily (Dugo, Cotroneo, Verzera, & Bonaccorsi, 2002).

The analysis of variance showed that producer and season factors significantly influenced the content of almost all the identified volatile components. As regards the samples from different producers, the artisanal cheese-making, the utilization of raw milk, and the environmental differences that characterized the various farmers are causes of the cheeses variability. The differences between producers were amplified by those among the production seasons. For example, the amount of free fatty acids and terpenes that resulted were significantly higher in spring samples whereas the opposite happened for the other classes of substances. The content of terpenes could be correlated to the fresh pasture typical of the spring season; as regards the other classes of components, the observed behavior would indicated a greater rate of lypolisis in winter samples. In fact, in winter, the lower environmental temperatures determined a higher time for curd acidification that could be one of the main factors responsible for the observed differences.

The multivariate approach confirmed the differences observed between dairies; the Canonical discriminant correlation showed that the Mahalanobis distances between the class means of different producers were statistically significant for both hydrocarbons and terpenes and free fatty acids (Figures 1 and 2).

Figure 1. Plot of sample distribution of canonical variables using hydrocarbon and terpene amount. Samples made by producer 1 (1), 2 (2), 3 (3), 4 (4).

Figura 1. Gráfico de la distribución de las variables canónicas en relación con la cantidad de hidrocarburo y terpeno. Muestras elaboradas por los productores 1 (1), 2 (2), 3 (3), 4 (4).

Figure 2. Plot of sample distribution of canonical variables using free fatty acid amount. Samples made by producer 1 (1), 2 (2), 3 (3), 4 (4).

Figura 2. Gráfico de la distribución de las variables canónicas en relación con la cantidad de ácidos grasos libres. Muestras elaboradas por los productores 1 (1), 2 (2), 3 (3), 4 (4).

Conclusion

The study of the volatile fraction of “Vastedda della valle del Belìce” cheese by using the SPME–GC–MS method described, can produce useful information for improving the cheese quality. A large number of components belonging to different classes of substances were identified and, using the method of standard additions, quantitative results were obtained on the main components, the free fatty acids. Statistical treatment of the quantitative results enables the variation of the volatile composition between the different producers and during the production seasons. The strong variability among producers, probably related to the artisanal cheese making procedures, agrees with the results previously reported by Mucchetti et al. (2008). With the exact application of the Disciplinary of the Production, linked to the PDO acknowledgment, a major uniformity of the “Vastedda della valle del Belìce” cheese is expected.

Acknowledgments

This research was partially supported financially by the “Consorzio per la tutela del formaggio Vastedda della valle del Belìce”.