Eur Food Res Technol (2006) 222: 250257 DOI 10.

1007/s00217-005-0021-2

ORIGINAL PAPER

Ana Bel n Fl rez Patricia Ruas-Madiedo e o Leocadio Alonso Baltasar Mayo

Microbial, chemical and sensorial variables of the Spanish traditional blue-veined Cabrales cheese, as affected by inoculation with commercial Penicillium roqueforti spores
Received: 28 February 2005 / Published online: 17 September 2005 C Springer-Verlag 2005

Abstract This paper reports the technological effects of inoculating Cabrales cheese (a traditional, Spanish, blueveined cheese) with Penicillium roqueforti spores. Three batches of inoculated Cabrales cheese were manufactured and a number of their microbial and biochemical variables recorded. The results were compared with those obtained for three batches of control cheese made using traditional technology (i.e., adding neither starter cultures nor fungal spores). Although mould and yeast populations grew more quickly in the inoculated cheeses, their normally dominant and representative microbial populations were not affected neither was their gross biochemical composition changed. The variations observed were thought to be caused by the uncontrolled environmental conditions of manufacture and ripening. The development of free amino acids and volatile compounds was signicantly increased at 30 days in the inoculated cheeses, although the values for both types of cheese were almost identical at 90 days. The inoculated cheeses obtained higher scores in a hedonistic sensorial evaluation. Thus, inoculation improved the standardization and quality of the cheese. Keywords Traditional cheese . Artisan cheese . Blue-veined cheese . Cabrales cheese . Penicillium roqueforti Introduction Cabrales is by far the most famous of the traditional, Spanish, blue-veined cheeses, and it has enjoyed Protected Designation of Origin (PDO) since 1981. This cheese is manufactured in the mountainous area of the Picos de Europa (Principality of Asturias, Northern Spain) from cows
A. B. Fl rez P. Ruas-Madiedo L. Alonso B. Mayo ( ) o Instituto de Productos L cteos de Asturias (CSIC), Carretera de a Inesto s/n, 33300-Villaviciosa Asturias, Spain e-mail: baltasar.mayo@ipla.csic.es Tel.: +34 985 89 21 31 Fax: +34 985 89 22 33

milk, with seasonal additions of goats and sheeps milk. The traditional manufacture of Cabrales involves curdling mixtures of evening and morning milk at 2830 C with farm-made kid rennet. Neither starter culture nor mould spores are added during the manufacturing process. The curd is then cut into hazelnut-size cubes and placed in cylindrical moulds for whey drainage without pressing. After 48 h at room temperature, during which cheeses are turned over several times, following which they are covered under coarse salt and kept at room temperature for further 1015 days. Finally, they are placed in natural caves in the production area, where ripening takes place at a nearly constant temperature (912 C) and relative humidity (90 95%) [1, 2]. Under these conditions, Penicillium roqueforti gains access to the cheese matrix and develops leading to biochemical changes during ripening. Although ripening continues to take place in natural caves, some producers have recently started to add commercial P. roqueforti spores in response to seasonal technological problems of different nature and origin, but this nally results in incomplete and heterogeneous fungal development. The aim of adding these spores is to achieve more satisfactory fungal development and thus to produce cheeses of consistent quality (including texture and avor). Moreover, microbial (and biochemical) standardization and the improvement of both hygienehealth and organoleptic standards is the only way for traditional varieties to face market demands [3]. Our group recently reported the biochemical and microbial characterization of Cabrales cheese as part of work aimed at developing specic starter cultures using native microorganism [4]. Representative strains from all microbial groups with a potential technological role were identied and studied in detail, including different strains of P. roqueforti (Fl rez and Mayo, unpublished). These strains o are thought to be adapted to the milk produced in the manufacturing area, and ensure the original sensorial properties of the mature cheese [5]. This paper reports the development of the dominant and representative microbial populations, and the changes in a number of chemical and sensorial variables in three batches

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of Penicillium-inoculated Cabrales cheese. The work was undertaken to know the microbial and biochemical effects of this inoculation and to investigate if changes in its technololgy should be introduced.The results were compared to those of three control series in which cheeses were produced in the traditional manner. Material and methods Cheese manufacture and sampling Two series of three batches of Cabrales cheese were made by two different manufacturers using the same traditional technology. These batches were produced in the month of October, February and April. The rst batch from each manufacture was exclusively made from cows milk; the others were made from a mixture of cows (75%), goats (15%) and sheeps (10%) milk. Before renneting, the cheese milk of one of the series was inoculated (approximately 1106 cfu ml1 ) with commercial P. roqueforti spores (Strain PB6; Christian Hansen, Hlstrom, Denmark). The milk, curd and 3, 7, 15, 30, 60 and 90 day-old cheeses were sampled according to standard procedures [6]. Microbial analyses Ten grams of each cheese sample were homogenized with 90 ml of a 2% (w/v) sterilized sodium citrate solution at 45 C for 1 min in a Colworth Stomacher 400 (Seward Ltd., London, UK). Ten-fold dilutions were prepared with sterile Ringers solution (VWR International, Darmstadt, Germany) and plated onto specic media (in duplicate). Filtered sterilized cycloheximide (50 g ml1 ) (Sigma, Chemical Co., St. Louis, Miss., USA) was added to these media when it was believed that the number of yeasts and moulds might interfere with bacterial enumeration. Total bacterial counts. Total bacterial counts were determined on plate count agar (PCA; VWR International) containing 10 g l1 skimmed milk powder. All plates were incubated at 30 C for 72 h. Lactococci. Lactococci were enumerated on M17 agar (Scharlau Chemie, Barcelona, Spain). The medium was supplemented with 40 g ml1 nalidixic acid for enumeration in milk and curd samples. Incubation was at 30 C for 48 h. Lactobacilli. Lactobacilli were scored on MRS agar (VWR International), pH 5.4, after 72 h of incubation at 30 C in an enriched CO2 atmosphere (Gaspak; BBL, Benton Dickinson & Co., Franklin Lakes, NJ, USA). Leuconostocs. Leuconostocs were grown on MSE agar (Biokar Diagnostics, Beauvais, France) containing 30 g ml1 vancomycin (Sigma) and enumerated after 35 days of incubation at 25 C. Yeasts and moulds. Dilutions of the milk and cheese samples were plated on the surface of yeast extract glucose chloramphenicol agar (YGC) (VWR International) and incubated for 35 days at 25 C.

Enterobacteria and coliforms. Violet red bile lactose agar (VRBLA) (VWR International) was used to enumerate enterobacteria and coliforms, using the pourplate and overlay technique. Incubations were for 2448 h at 30 C. Enterococci. Enterococci were scored after 24 h of incubation at 44 C in Slanetz and Bartley Medium (SB; VWR International). Staphylococci. Dilutions were plated on BairdParker agar (BP; VWR International) and incubated for 24 h at 37 C. Black colonies with or without egg yolk clearing were recorded. Basic biochemical analyses The total solids, fat, NaCl, pH, titratable acidity, protein and total nitrogen were determined in accordance with standard methods [6]. The water activity (Aw ) was measured in duplicate using an AquaLab apparatus (Decagon Devices Inc., Pullman, Wa., USA). Changes in free amino acid and biogenic amine concentrations. Cheese samples weighing between 0.1 and 1.0 g were dispersed in 10 ml of HCl 0.1 mol l1 plus 0.2% 3,3 thiodipropionic acid (TDPA) and 250 nmol ml1 of norvaline and norleucine (internal standards). The suspension was homogenized using an Ultraturrax apparatus at 20,000 rpm for 2 min. This mixture was then incubated in an ultrasound water bath for 30 min and centrifuged at 10,000 rpm for defatting. The supernatant was adjusted to 10 ml with HCl 0.1 mol l1 and ltered through a 0.45 m diameter membrane. The solution was deproteinized by centrifugation using an ultraltration cartridge (Amicon Biomax 5; Millipore, Millipore Corporation, Bedford, Ma., USA). Amino acids were derivatized in 20 l of deproteinized sample by reacting with dabsilo chloride (Sigma) at 70 C for 15 min in the presence of 0.15 mol l1 of NaHCO3 pH 8.6. The products were separated by HPLC in a Novapack C18 column (Waters Corporation, Milford, Mass., USA) and detected at 436 nm. Analysis of the main volatile compounds The analysis of the volatile fraction was performed by a headspace gas chromatographic mass spectrometric (GCMS) method, essentially as described by Alonso et al. [7]. To 10 g of cheese previously homogenized, 80 l of an aqueous solution of propionic acid ethyl ester (1.14 mg/ml), as internal standard, and 10 g of anhydrous sodium sulphate, to retain water, were added. Prior to be analysed in a static headspace apparatus (Model HSS 19395; Hewlett-Packard), the samples were maintained at 80 C for 60 min until the sample and gaseous phase reached the thermodynamic equilibrium. The headspace apparatus was programmed as follows: 5s pressurization, 18 equilibrium

252

and lling and 2 min for injection. Helium was employed as the carrier gas at ow 18 rate of 17.5 ml/min. A HewlettPackard GC Model 5890 coupled to a selective MS Model 5972 was employed for volatile compounds analysis. Samples were injected in the split mode (split rate of 7:1) on a capillary silica column with polyethylene glycol (HP Innovax, 60 m, 0.25 mm 18 ID, 0.25 m lm thickness, Hewlett Packard). Helium was used as the carrier gas, at a ow rate of 18, 36.5 cm/s. The column temperature program was: 33 C per min, increase at 1 C/min up to 38 C and then at 7 C/min up to 210 C, and held for 10 min. Injection was carried out at 200 C and the interface line of MS at 280 C. Electronic ionisation energy and photomultiplier voltage 18 were 70 eV and 1647 V, respectively. Sensorial analysis The quality of the cheeses was assessed at 2 and 3 months by a trained taste panel comprising an average of 15 people at the Instituto de Productos L cteos de Asturias, Villaviciosa, a Spain. Samples were held for 2 h at room temperature before presentation to the panel members. The descriptors and values scored were as dened by the Queso de Cabrales PDO Regulatory Council [8]. Statistical analysis Statistical analysis was performed using SPSS 11.0 software for Windows (SPSS Inc., Chicago, Il., USA). Bacterial counts, the data for chemical variables and sensorial values were subjected to one-way ANOVA taking into account the two categories of cheese type (control and inoculated).

Results and discussion Microbial development in control and inoculated cheeses The count differences observed between different batches of control and Penicillium-inoculated samples were moderate to large (Table 1). Such differences are a common feature of cheeses made from raw milk. In both series, the microbiological load of the starting milk was high, especially in the case of the inoculated batches. The maximum levels of predominant and indicator microbial populations were reached between day 3 and day 7, with a small lag in the control cheeses. Most bacteria (including total mesophilic bacteria, lactococci, leuconostocs and coliforms) developed faster in the Penicillium-inoculated batches, although these populations reached similar numbers at day 3 in both series. Thus, lactic acid bacteria species (especially lactococci and lactobacilli) dominated the two series (Table 1), and showed a similar pattern of growth during ripening. These results agree well with those reported by other authors [2, 9] and with our own previous results [4]. The mould and yeast populations of the inoculated and control cheeses developed quite differently. Moulds were already dominant in the curd of the inoculated batches, in which they reached counts of 5.37106 cfu g1 . Moreover, maximum numbers were reached in these cheeses at day 15; in control cheeses the maximum number of moulds was not reached until day 30. The dominant populations in all control cheese samples were formed by yeasts. However, at the end of ripening, the control cheeses had the same population of moulds and yeasts as the inoculated

Table 1 Mean Log counts (cfu g1 ) and standard deviations of the principal microbial groups throughout manufacture and ripening in the three batches of control and Penicillium-inoculated Cabrales cheeses Milk Total mesophilic bacteria Lactococci Lactobacilli Leuconostoc Moulds and yeast Coliforms Enterococci Staphylococci Control Inoculated Control Inoculated Control Inoculated Control Inoculated Control Inoculated Control Inoculated Control Inoculated Control Inoculated 6.101.30 7.761.77 5.571.36 6.510.15 4.902.57 4.590.94 2.900.12 4.820.85 2.042.28 4.521.34 2.342.14 5.290.25 3.141.63 3.901.28 3.631.61 4.360.37 Curd 6.741.17 9.151.42 6.610.89 8.030.51 5.630.37 6.491.39 3.640.36 5.580.45 3.681.37 6.732.81 4.570.34 7.290.30 4.911.33 5.440.98 5.051.16 5.560.30 Cheese of the following ripening time (days) 3d 7d 15 d 30 d 9.550.42 9.440.27 7.992.30 9.460.28 8.621.16 8.421.27 5.471.04 7.100.20 5.401.30 6.871.68 6.911.10 7.460.28 5.130.11 6.291.01 6.020.18 5.950.97 9.200.16 9.700.85 9.500.49 9.851.44 8.360.87 8.091.04 5.271.11 7.110.41 4.430.45 6.521.21 5.811.13 5.780.26 5.811.30 6.000.88 4.571.10 5.471.77 8.780.41 9.030.25 8.910.22 9.080.22 8.000.88 8.050.44 6.030.89 6.910.96 7.310.18 8.070.12 5.210.90 5.740.63 5.761.16 6.230.97 5.090.27 5.951.34 8.350.32 8.380.69 8.170.05 8.470.89 8.020.10 7.830.67 5.850.75 6.930.71 8.070.35 7.540.85 4.621.06 5.310.50 5.831.02 6.240.65 6.081.29 6.510.33 60 d 7.750.28 8.050.21 7.700.18 7.910.32 7.050.32 7.150.92 4.920.78 7.240.52 7.460.24 7.470.67 4.360.58 4.460.82 5.840.77 5.950.78 5.651.02 5.041.85 90 d 7.370.16 7.540.07 7.240.28 7.650.30 7.161.03 7.160.24 5.950.94 6.330.32 6.860.34 7.460.50 3.541.21 3.190.88 5.360.86 6.120.79 4.830.96 6.732.87

253 Table 2 Mean values and standard deviations for the gross composition of the control and Penicillium-inoculated batches of Cabrales cheese analyzed during manufacture and ripening Parameter pH Control Inoculated Acidity Control Inoculated (g 100 g1 ) TS (%, w/w) Control Inoculated Fat (%, w/v) Control Inoculated Total protein Control Inoculated (g 100 g1 ) Total nitrogen Control Inoculated (g 100 g1 ) NaCl (%, w/w) Control Inoculated Control Aw Inoculated Milk 6.650.32 6.530.06 0.180.01 0.160.00 12.710.60 12.310.93 3.700.26 3.850.15 3.480.25 3.340.09 0.540.04 0.730.31 0.170.01 0.090.01 0.9990.002 0.9990.001 Curd 6.350.48 6.270.43 0.360.11 0.260.15 23.623.46 24.163.19 10.501.50 13.500.00 8.461.54 11.460.95 1.320.24 1.790.15 0.220.07 0.170.06 0.9990.001 0.9990.001 Cheese of the ripening time (days) 3d 7d 15 d 5.260.24 5.070.11 1.190.22 1.190.30 50.33.85 49.023.18 26.004.36 27.501.80 20.662.12 18.460.26 3.230.33 2.890.04 1.221.03 0.170.06 0.9880.014 0.9950.008 5.130.28 4.970.09 1.260.26 1.330.47 55.872.84 52.880.42 28.674.16 30.672.08 23.791.88 19.440.62 3.730.30 3.040.10 0.830.42 1.441.45 0.9840.011 0.9730.012 5.430.66 5.560.28 0.830.32 0.870.10 57.272.65 57.760.83 30.501.80 35.173.75 23.311.61 19.604.30 4.311.39 3.070.67 1.570.93 1.700.52 0.9770.006 0.9590.013 30 d 5.910.63 6.010.17 0.760.28 0.820.11 59.563.12 63.781.71 33.502.50 38.335.51 23.851.08 23.520.67 3.740.17 3.680.10 2.100.87 2.271.58 0.9550.009 0.9290.017 60 d 5.930.18 6.300.27 1.130.61 1.040.06 61.362.24 64.173.13 25.501.50 36.334.16 23.091.20 24.682.60 3.610.19 3.860.40 2.570.55 1.870.40 0.9250.011 0.9040.013 90 d 5.970.23 6.320.07 1.470.34 1.160.11 60.992.64 62.814.56 32.004.24 35.750.35 24.922.83 22.441.06 3.910.45 3.510.17 2.000.56 2.400.44 0.9280.010 0.9170.014

cheeses. Furthermore, the types of yeast and fungi found in the control cheeses were more diverse (data not shown). In conclusion, it seems that the faster growth of moulds in the inoculated cheeses does not inuence the growth of the bacterial populations examined. These populations were more inuenced by their initial concentrations in the cheese milk, which are known to vary between the two manufacturing dairies [4]. Biochemical analysis and gross composition of inoculated and control cheeses Table 2 shows the mean values and standard deviations for the biochemical test results. Again, moderate to high variation was observed in several variables, especially with respect to total solids and the fat content (due to the different mixtures of milk used in manufacture). The pH of the inoculated batches declined and their acidity increased more rapidly than in the control cheeses as a consequence of their greater microbial development. In all other respects, the differences between the series were generally not statistically signicant; the differences between batches was actually greater than those between series. These variations are presumably due to uncontrolled environmental conditions (in particular, temperature and relative humidity), as has been noted in the production of other traditional, blueveined cheeses [1012]. Changes in free amino acid and biogenic amine concentrations in control and inoculated cheeses Blue cheeses undergo profound proteolysis during ripening. Consequently, the concentration of free amino acids

strongly increases as ripening progresses [1113]. Table 3 shows the mean values for the different free amino acids detected in the control and inoculated cheeses. With a few exceptions, amino acid concentrations increased over maturation, a property that allows them to be used as a ripening index [14]. Isoleucine was not detected as a free amino acid until day 7, and no serine was found until day 15. Further, in the inoculated batches, threonine was not detected at day 3. As reported elsewhere [15], the most abundant amino acids at the end of ripening of Cabrales cheese were glutamic acid, leucine, lysine, valine and proline. These amino acids have been repeatedly reported as the main types in blue-veined cheeses [10, 13, 16, 17]. In general, free amino acid levels were signicantly higher in the inoculated cheeses until day 30, at which time the differences with the control cheeses were maximum (Table 3). However, the concentrations of most amino acids were almost equal at day 60 and 90. The standard deviations of the values between samples were frequently high. This may due to sample heterogeneity, which depends on the growth of the fungi and their proteolytic activities. Free amino acids are precursors of biogenic amines; for this reason the latter follow a similar but delayed pattern of appearance to that of amino acids (Table 3). The levels of both histamine and tyramine increased over ripening. At day 60, the age at which cheeses are allowed to be marketed by the PDO Regulatory Council, the average values were 557.38 and 537.34 g g1 for histamine and 982.08 and 334.81 g g1 for tyramine in control and Penicilliuminoculated cheeses, respectively. Levels of biogenic amines similar to those found in this work have previously been reported for Cabrales cheese [18], and are in the normal range for other blue-veined cheeses [11, 13, 19]. It is assumed that these levels have no health-related signicance [20]. The concentration of did not exactly follow the

Table 3 Mean values of the free amino acids and biogenic amines (g g1 ) analyzed in control and Penicillium-inoculated batches of Cabrales cheese throughout ripening Cheese ripening time (days) 3d 7d 15 d 30 d 60 d 90 d

254

Amino acid

Glutamic acid

Leucine

Lysine

Valine

Proline

Isoleucine

Phenylalanine

Alanine

Tyrosine

Aspartic acid

Serine

Threonine

Histidine

Histamine

Tyramine

Control Inoculated Control Inoculated Control Inoculated Control Inoculated Control Inoculated Control Inoculated Control Inoculated Control Inoculated Control Inoculated Control Inoculated Control Inoculated Control Inoculated Control Inoculated Control Inoculated Control Inoculated

150.11 101.23 80.22 48.72 14.77 11.13 15.21 11.81 34.32 42.83 nd nd 43.39 27.72 28.88 22.01 235.91 201.53 17.79 25.76 nd nd 8.57 nd 3.81 4.47 1.81 16.30 5.0 nd

98.83 53.51 235.74 197.96 100.06 46.92 43.59 32.54 69.41 65.92 71.79 40.99 466.26 493.18 43.07 20.39 140.28 128.80 48.35 37.66 nd nd 44.01 42.43 14.44 3.18 21.57 39.79 12.79 2.25

218.96 376.64 244.14 250.71 104.02 252.78 99.83 94.78 87.79 58.66 31.21 48.19 315.13 172.44 92.80 104.91 61.12 90.90 63.91 76.17 5.90 36.30 34.55 32.76 20.27 51.26 30.27 20.20 48.94 6.91

157.13 1862.75 338.97 1758.15 233.69 1698.63 105.74 794.01 123.77 327.40 56.36 731.90 234.94 912.15 73.56 356.44 222.78 612.99 67.98 466.92 7.63 112.77 75.08 128.55 36.12 325.12 37.54 86.99 78.76 87.18

2870.90 5187.75 4338.70 4326.47 3006.32 2876.49 2596.47 2644.06 2152.51 1481.09 2187.40 2576.59 2309.68 2256.77 1026.94 1012.50 529.21 1491.72 964.84 1393.99 116.81 429.07 118.66 457.46 41.07 547.68 557.38 537.34 982.08 334.81

8241.61 12332.87 7187.53 7196.14 5568.15 5754.04 4325.84 4695.14 4222.42 4015.55 3618.86 4032.83 4032.83 3589.09 2611.24 2639.25 3872.44 2191.42 2445.67 3034.12 620.12 1162.92 643.34 1161.91 307.52 863.26 1079.65 732.22 933.25 926.76

p0.1; p0.05; p0.01; p0.001 nd, not detected

Table 4 Mean values and standard deviations (g 100 g1 ) of the principal volatile compounds of blue-veined cheeses in control and and Penicillium-inoculated batches of Cabrales cheese at 30, 60 and 90 days of ripening Days of ripening 30 d 60 d 90 d

Type

Compound

Methylketones

2-propanone

2-pentanone

2-heptanone

2-nonanone

Control Inoculated Control Inoculated Control Inoculated Control Inoculated

17.04.2 31.63.5 61.110.8 87.88.0 90.08.4 108.511.3 38.39.2 60.94.1

31.04.3 52.08.6 75.010.5 112.013.8 116.016.8 141.015.9 52.412.8 91.920.7

46.68.8 77.917.5 89.218.3 136.621.9 130.117.1 166.220.7 64.015.1 109.423.4

Secondary alcohols

2-propanol

2-pentanol

2-heptanol

2-nonanol

Control Inoculated Control Inoculated Control Inoculated Control Inoculated

9.62.5 18.92.7 13.73.3 25.34.0 10.72.6 19.62.1 3.10.8 3.50.7

16.34.8 39.811.3 20.76.3 47.612.1 17.26.5 37.611.6 3.70.7 5.50.6

28.510.4 55.012.4 31.76.9 65.916.8 25.36.1 49.111.4 4.81.0 7.61.4

Table 5 Mean and standard deviations of sensory attributes of control and Penicillium-inoculated Cabrales batches of two and three months old Penicillium development 3.841.21 3.131.33 4.770.86 4.290.99 4.630.68 4.720.66 4.090.95 3.880.97 4.170.90 4.151.08 4.080.96 4.451.05 Eyes Flavor Texture Mouth appeal 4.260.77 4.220.68 4.220.91 4.270.88 4.020.94 4.200.94 4.221.07 4.390.85 Savor Retro-savor Globala

Cheese sample 3.851.11 3.191.06 4.400.87 4.190.96 4.740.73 4.890.69 4.320.83 3.671.24

Time of evaluation (Months)

Cheese descriptors (value 06) Shape Rind Color

Control

2 3

4.720.75 3.971.20

3.950.88 3.791.19 4.261.28 4.581.01

3.891.00 3.841.45 4.081.31 4.400.99

101.8816.55 95.6220.73 110.0616.47 113.9214.54

Inoculated

2 3

4.940.78 4.790.80

Although descriptors have all the same nominal value (06), for the global score shape and rind are multiplied by a factor of 1, mouth appeal for a factor or 2 and the values of all other descriptors for a factor of 3

255

256

pattern of tyrosine and histidine, which were always more abundant in the inoculated cheeses. This suggests that aminodecarboxylating agents (whether of bacterial or fungal origin) might be the limiting factor for biogenic amine concentrations. Changes in the concentration of the main volatile compounds in control and inoculated cheeses Carbonyl compounds, which are strongly aromatic, make a signicant contribution to the avor of many cheese varieties and methyl ketones and secondary alcohols make a large contribution to the avor of blue cheeses. The predominant volatile compounds of blue cheeses, as in other cheese varieties, are methylketones and secondary alcohols [21, 22]. These are well known cheese components whose formation has been attributed to -oxidation of fatty acids. The concentration of these compounds in traditional Cabrales cheese has been reported elsewhere [7]. In this work, 29 compounds were detected. The mean values and standard deviations of the dominant and representative compounds in control and inoculated cheeses after 30, 60 and 90 days of ripening are presented in Table 4. Most of the ketones in Cabrales cheese studied were methylketones. It should be noted the presence of 2-propanone, 2-pentanone, 2-heptanone and 2-nonanone in all lots of cheese. Among them, 2-pentanone and 2heptanone were the most abundant ketones at all ripening times analysed. Those compounds are usually formed from acid oxidation, although other routes are also possible. The concentration of the volatile compounds increased with the ripening. Although the concentrations in control and inoculated cheeses did not reach to the same nal values (at least during the rst 90 days). The total methylketones and secondary alcohols were always higher in the inoculated cheeses. Their concentrations are directly related to mould development and proteolytic activities, which lead to the nal sensorial attributes of the cheeses [22, 23]. Values for Cabrales sensorial descriptors in control and inoculated cheeses Table 5 shows the sensorial variables dened by the Queso de Cabrales PDO Regulatory Council and the mean values for the control and inoculated cheeses. The global scores for both the control and inoculated series were higher than the mean for different manufacturers in analyses performed by the PDO Regulatory Council in 2003 [8]. In general, control batches received lower scores for all variables except for avor and texture, which were comparable in the two series. The number of descriptors with higher values in the inoculated cheeses greatly increased from day 60 to 90, indicating that the inoculation of Cabrales cheese with Penicillium spores has a positive inuence on the nal quality of the mature cheese.

Inoculation with Penicillium spores did not inuence the traditional technology used in the making of Cabrales cheese. Neither were its basic biochemical or microbiological variables affected, except for the faster growth of moulds in the inoculated series. A similar concentration of free amino acids was obtained at the end of the 90 day assay period in both the inoculated and control cheeses, although the former reached high levels earlier. This may favour a more rapid formation of avoring compounds [24, 25]. The main mouldy volatile compounds developed more quickly, and higher concentrations were always found in the inoculated cheeses. Inoculation resulted in cheeses with higher scores for most sensorial attributes. Thus, this inoculation procedure is strongly recommended for achieving standardization and higher overall quality. Several P. roqueforti strains isolated from Cabrales cheeses are currently being characterized for use as specic maturation cultures.
Acknowledgements This work was supported by a research project from the Spanish Ministry of Education and Science (PTR19950556-OP). The authors wish to thank the Queso de Cabrales PDO Regulatory Council for the nancial support received. The skilful technical assistance of M. J. Gonz lez is gratefully a acknowledged.

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