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Lipolysis in Gorgonzola cheese during ripening
IN. noiry J,mxrd
5 ( 1995) I4 I I55
0 1995 Elsevier Science Lmted Printed ,n Ireland. All rights reserved 0958-6946
ELSEVIER
95’$9.50
Lipolysis in Gorgonzola Cheese during Ripening
Giovanna Contarini* lstituto Sperimentale
& Piero Maria Toppino
Lattiero-Caseario.
20075 Lodi. Milano. Italy
(Received 25 November 1992: revised version accepted 25 January 1994)
ABSTRACT
INTRODUCTION Cheese ripening is a complex process that includes the breakdown of the curd by proteolysis, lipolysis and other enzyme-catalysed reactions which cause flavour and textural changes typical of the different varieties. Enzymatic processes are responsible for the production of a considerable number of compounds which, as a result of their presence, concentration and proportions. are often characteristic of particular cheese types. As far as the evaluation of lipolysis is concerned, most investigations have dealt with the determination of free fatty acids, which are the last product of hydrolytic reactions (Downey, 1980; Woo & Lindsay, 1984; Woo ct rd., 1984a, h; Berdague et ul., 1987). In contrast, few references are available on the formation of partial glycerides during cheese ripening (De Man, 1966; Vujicic & De Man, 1966; Kalo et ul., 1988). The study of cheese flavour is rather complex because the aroma profile is derived from a large number ofcompounds having very different physico-chemical properties. *To whom correspondence
should be addressed. 141
142
G. Contarini, P. hf. Toppino
Various analytical and instrumental methods have been developed to evaluate cheese flavour; the most widely used methods are solvent extraction, distillation and headspace analysis (Dumont & Adda, 1972; Price & Manning, 1983; Bertuccioli, 1985; Badings et al., 1985; Oria et al., 1987; Barlow et al., 1989). In recent years, a new technique for dynamic extraction, thermal cold trapping, has been developed by Chrompack (Middleburg, Netherlands). This technique consists of stripping volatile substances from the cheese sample by nitrogen flow and trapping them on an adsorbent material (Tenax). A suitable thermal desorber allows the cryoconcentration on a silica trap; flash evaporation and direct injection into a capillary gas chromatograph are then performed (Badings et al., 1985). Whichever technique is used, the major components detected, for example, in blueveined cheese belong to the chemical classes of alcohols, aldehydes, methyl ketones, carboxylic acids, esters and lactones (Jackson & Hussog, 1958; Shwartz & Parks, 1963; Anderson & Day, 1966; Fan et al., 1976; Adda, 1986). Ripening of Gorgonzola cheese is due mainly to the proteolytic and lipolytic activity of selected strains of Penicillium roqueforti. Penicillium moulds are strongly hydrolytic and their action is superimposed on that of thermophilic starters (Law, 198 1; Cerning et al., 1987). By studying a cheese characterized by strong lipolysis during ripening (Gorgonzola), the purpose of this work was to evaluate the significance of the determination of both the intermediate and the final compounds of the lipolytic process using gas chromatography (GC) techniques. The investigation was extended to volatile compounds using the thermal cold trapping (TCT) system.
MATERIALS
AND METHODS
Samples Three Gorgonzola cheeses were selected at l-week intervals from the same factory. Samples of these products were taken during ripening according to the following sampling plan: pasteurized milk, milk with starters, curd, cheese collected after 3 days (immediately after sweating), 20 days (5 days after the first skewering), 30 days (8 days after the second skewering), 60 days (ready for sale), and 105 days after production (cheese over-ripened). Extraction of total and partial glycerides Five grams of cheese or curd were diluted in 20 ml of diethyl ether (purity 99.5%) and 10 ml of hexane (purity 99.5%). The mixture was shaken in a laboratory blender for 10 min, and filtered through a fluted filter paper, fast grade, containing about 20 g of anhydrous Na2S04 (Merck 6649). The filtrate was evaporated under vacuum at a low temperature. For the extraction of fat from milk and milk plus starters, 20 g of sample were submitted to the procedure reported in the FIL-IDF Standard Method 1B (1983). The following procedure was a modification of the method used by Riva et al. (1981) for the separation of lipid classes in vegetable fats. Anhydrous fat (40 mg) was dissolved in 1 ml of chloroform solution containing 1 mg of internal standard (dinonadecanoin, purity 99%, Nu Chek Prep Inc., Elysian, MN, USA); 0.4 ml of this solution was applied to a silica-gel-precoated plate (Merck 5725).
Lipoiysis
in Gorgonzola
cheese during
ripening
143
The plate, after development in hexane-diethyl ether-formic acid (70/30/0.5), was stained by spraying a 2’7’-dichlorofluorescein solution (0.15% in ethanol). Diglycerides and triglycerides bands were scraped off and extracted three times with 10 ml of chloroformdiethyl ether (l:l, v/v). Separation of monoglycerides was performed by the same method but the amount of fat was increased (80 mg) and the solvent proportions in the solvent mixture (hexane-diethyl ether-formic acid: 30/70/0.5) were modified. Monoheptadecanoin (0.25 mg/ml, purity 99%, Nu Chek Prep) in chloroform was used as an internal standard. Monoglycerides and diglycerides were converted into trimethylsilyl derivatives by addition of 200 ~1 of BFT (Supelco Inc., Bellefonte, PA, USA) and 200 ~1 of pyridine (Supelco) and diluted with 5 ml of hexane before GC analysis. Triglycerides were diluted directly with 20 ml of hexane. Analysis of triglycerides
Capillary column GC separation was performed with a Carlo Erba Mega 3500 instrument, equipped with a flame ionization detector, a cooled on-column injection port and a CP Sil 5 CB (Chrompack) fused-silica column (15 m length, 0.32 mm i.d., 0.12 pm film thickness). The oven temperature was programmed to increase at a rate of 40”C/min from 60 to 360°C. The detector temperature was 370°C. The flow rate of hydrogen carrier gas was 8 ml/min. A l-1*1sample of the solution of triglycerides was injected. Triglycerides having an even number of carbon atoms (from 28 to 54) were identified by their relative retention times. Results were expressed as percentages of the total triglycerides. This method does not allow the separation of triglycerides having the same number of carbon atoms but different degree of unsaturation or different positional isomers. Analyses of mono- and diglycerides
The same gas chromatograph, equipped with a CP Sil 8 CB (Crompack) fusedsilica capillary column (25 m length, 0.32 mm i.d., 0.12 pm film thickness), was used. Two different analyses were performed using this column to determine the concentrations of diglycerides and monoglycerides. For the analysis of diglycerides, the oven temperature was programmed from 60 to 250°C at a rate of 40”C/min, from 250 to 320°C at 8”C/min, from 320 to 345°C at 40”C/min and to hold at 345°C for 10 min. The detector temperature was 355°C; the flow rate of hydrogen carrier gas was 2 ml/min. A 1 ,LJsample of the solution of diglycerides was then injected. Four groups of diglycerides having 30-36 carbon atoms were identified, and the different positional isomers within each group were labelled with a letter according to Mariani et ul. (1989). Figure 1 shows an example of the analysis of diglycerides. The results were expressed both as total diglycerides (mg/g fat) adding up the values for all peaks, and as percentages calculating the value for each single peak with respect to total diglycerides. For the analysis of monoglycerides, the oven temperature was programmed from 60 to 250°C at a rate of 20°C min, to hold for 1 min at 250°C and then from
G. Contarini, P. hf. Toppino
I
A
II h
0
I
10
I
I
20
I
D
E
I
30
/
32
A I
34 I
30
36
I
40
TIME (MINUTES)
Fig. 1. Example of gas chromatogram of diglycerides of Gorgonzola cheese (each capital letter identities a different isomer according to Mariani et al. (1989); IS: internal standard). 250 to 320°C at 8”C/min. The detector temperature was 340°C; the flow rate of hydrogen carrier gas was 2 ml/min. A l-p1 sample of the solution of monoglycerides was injected. The monoglycerides containing fatty acids with 16 and 18 carbon atoms were detected (Fig. 2) and the different positional isomers were grouped. The results were expressed both as total monoglycerides (mg/g fat) adding up the values for all the groups and as percentages calculating the value for each single group with respect to total monoglycerides. Analysis of free fatty acids Milk plus starters, curd and cheese samples (20 g) were extracted using the procedure of Contarini et al. (1989) for the identification and quantitative evaluation of C2 to Cts fatty acids. Results were expressed both as the sum of free fatty acids (mg per 100 g sample) and as percentages summing the free fatty acids derived from bacterial fermentation (C,-Cs), from lipolysis (C&ts) or from proteolysis (C, br). Analysis of volatile compounds The sample (2 g of curd or cheese) was weighed in a 20-ml vial and dispersed in 9 ml of distilled water. Milk and milk plus starters (10 g) were weighed directly in the vial.
Lipolysis in Gorgonzola cheese during ripening
5
Fig. 2. Example
TIME (MINUTES)
._
”
of gas chromatogram of monoglycerides Internal standard.
145
1 20
._
15
of Gorgonzola
cheese.
IS:
One millilitre of internal standard (hexanal 0.0025%, w/v, in water) was added to each sample. The vial was equilibrated at 45°C in a thermostatic bath and connected to a nitrogen source (flow rate 10 ml/min) and to the Tenax trap (TA 60-80 mesh, Cat. No. 16252, Chrompack) through two separate outlets. The volatile compounds were stripped for 15 min. The Tenax trap was fitted in the thermal desorption cold trap injector (TCT, Chrompack), connected directly to the GC system. Compounds were desorbed from Tenax by heating at 250°C for 10 min and then transferred by a carrier gas stream (H,) to a fused-silica trap, cooled at -120°C by liquid nitrogen. Injection into the capillary system was performed by flash-heating (250°C for 10 min) of the cold trap on which the volatile components had been reconcentrated. A Carlo Erba HRGC 4160 gas chromatograph was equipped with a Chrompack TCT injector, a flame ionization detector and a CP Sil 5 CB (Chrompack) capillary column (50 m length, 0.32 mm i.d., 5 pm film thickness). The oven temperature was held at 50°C for 10 min, programmed to 220°C at a rate of 8”C/ min and held at 220°C for 12 min. The detector temperature was 250°C; the flow rate of hydrogen carrier gas was 1 ml/min. Twenty-two peaks were detected and numbered; some of them were identified by comparison with the retention times of chromatographic standards. Identification was confirmed by mass spectrometry (VG 70-250 mass spectrometer coupled with an HP 5890 (Hewlett Packard, Palo Alto, CA, USA) gas chromatograph equipped with the same capillary column and using the same temperature programme).
146
G. Contarini.
Determination of volatile compounds results were expressed as mg/kg sample.
P. M. Toppino
was performed
only on cheese No. I;
RESULTS AND DISCUSSION Total and partial glycerides
The determination of triglycerides was performed to establish if triglycerides having different numbers of carbon atoms were hydrolysed at the same rate by lipases throughout the ripening period. It can be noted from Table 1 that the average values of all peaks showed small variations during ripening, and that the standard deviations calculated for the three cheeses at each sampling were very low and similar to those found for the natural variability of milk fat (Zegarska & Jaworski, 1981). These results suggest that the lipases hydrolysed the triglycerides at the same rate independently of the number of carbon atoms, so that the relative composition of the triglycerides of milk did not differ significantly from those of the 105day-old cheese. It seems important to emphasize that the method used for analysis of the triglycerides, which is the method most commonly used for milk fat, does not allow the determination of triglycerides with respect to the amount of fat. A method having the performances described above is already established for vegetable oils which have, generally, a less complex triglyceride composition, but it is still under study for milk fat. The methods applied to the determination of diglycerides and monoglycerides which are, at the same time, substrates for and products of the lipolytic process, were more selective. Figures 3 and 4 show the trend of these compounds during ripening. The differences between the three cheeses were large, particularly after 20 days, but, despite the high values for sample No. 3 at 60 days (probably as a result of a greater growth of mould in the portion of the sample analysed), no specific tendency to increase or decrease in either the mono- or diglycerides was found, throughout the ripening period. Also, the values for the milk and the 105-day-old cheese were very close in all the samples. The concentration of diglycerides was always higher than the concentration of monoglycerides. These results suggested that the high rate of lipolysis did not allow the accumulation of intermediate compounds and that the hydrolysis of monoglycerides to produce free fatty acids could be the fastest step of lipolysis. The high variation is shown also by the coefficient of variation (CV) values for the individual compounds reported in Tables 2 and 3. It is worth noting that the differences observed for the concentrations of diglycerides in the three milk samples were comparable with the variability calculated by other workers (e.g. Mariani et al., 1989) for samples of butter. The increase of the variability during ripening, up to 60 days, was probably due to the different rates of development of lipase activity in the three cheeses; at 105 days the variation was less, suggesting a decrease of microbial and enzymatic activity when the cheese is over-ripened. As far as the monoglycerides are concerned, the CV values of the compounds with I6 carbon atoms were similar to those of the compounds with I8 carbon atoms.
Lipolysis
in Gnrgonzola
cheese during
TABLE1 Triglyceride Composition (wt %) of Gorgonzola Carbon
Milk
a form
Milk
+
147
ripming
Cheese Samples Cheese
Curd
starters Duys of’ ripening
28 30 32
avg SD avg SD avg
SD 34
38
avg SD avg SD avg
40
aVg
36
SD SD 42 44 46
Livg
SD avg SD avg
SD 48
avg SD
50
iivg
52
avg
54
avg
SD SD SD
0.66 0.02 1.27 0.01 2.48 0.05 5.35 0.03 IO.28 0.15 13.11 0.09 II.40 0.22 I.25 0.07 6.50 0.10 7.04 0.12 8.45 0.11 IO.68 0.17 IO.53 0.10 4.96 0.04
0.61 0.04 I.21 0.07 2.46 0.00 5.45 0.07 IO.22 0.22 13.23 0.32 II.59 0.21 7.25 0.03 6.52 0.06 7.08 0.09 8.56 0.07 IO.83 0.04 IO.32 0.10 4.66 0.17
0.64 0.03 I.22 0.01 2.44 0.04 5.45 0.05 IO.25 0.33 13.23 0.39 Il.16 0.15 7.27 0.05 6.52 0.14 7.15 0.14 8.67 0.11 IO.93 0.08 IO.32 0.27 4.15 0.10
3
20
30
60
105
0.62 0.02 1.22 0.03 2.46 0.03 5.46 0.06 IO.30 0.07 13.23 0.14 II.40 0.30 I.24 0.08 6.54 0.12 7.08 0.12 8.49 0.11 IO.79 0.12 IO.54 0.08 4.65 0.23
0.70 0.05 I.28 0.03 2.48 0.03 5.58 0.21 IO.34 0.21 13.05 0.39 II.03 0.38 7.25 0.15 6.66 0.15 7.22 0.14 8.76 0.19 10.73 0.24 IO.15 0.44 4.11 0.09
0.68 0.03 I.28 0.04 2.48 0.03 5.48 0.12 IO.31 0.33 12.98 0.19 II.01 0.26 7.19 0.11 6.52 0.00 7.14 0.09 8.58 0.26 IO.98 0.22 IO.46 0.35 4.88 0.10
0.67 0.01 I.29 0.04 2.54 0.06 5.34 0.13 IO.35 0.38 13.15 0.67 IO.97 0.48 7.24 0.08 6.63 0.18 7.33 0.36 8.75 0.35 11.05 0.48 IO.13 0.40 4.56 0.11
0.64 0.01 I.21 0.02 2.35 0.04 5.22 0.09 IO.16 0.11 12.91 0.07 IO.84 0.05 1.21 0.11 6.75 0.05 7.46 0.11 9.05 0.10 II.12 0.05 IO.44 0.17 4.52 0.19
The percentage of mono Cl6 in milk was higher than that of mono However, at the last sampling, the ratio between the two monoglycerides reversed.
Crx. was
Free fatty acids The method used to determine the free fatty acids produced by both lipolytic tation (Cz+). In Gorgonzola, butyric acid can be high redox potential of this cheese does
fatty acids permitted the measurement of processes (C-Crs) and bacterial fermenproduced only by lipolysis, because not allow the growth of Clostridia.
the
G. Contarini, P. M. Toppino
148
Sample 1
!IIzl 30
Sample 2
-
fi;
Ei
Sample 3
\” E 20
-
10
-
0 Milk W&art.
Curd
3d
20d
30d
6Od
105d
Fig. 3. Changes in the total diglycerides during ripening. 7
t
Sample 1
6 I-
Milk M+start.
m
Sample 2
El
Sample 3
Curd
36
206
306
Fig. 4. Changes in the total monoglycerides
The total value of free fatty acids increased, from cheesemaking to the end of ripening, and concentration (Fig. 5). During the early period acids produced by bacterial fermentations made
6Od
105d
during ripening.
although not at a constant rate, sample No. 3 showed the highest of ripening (O-10 days), the fatty a significant contribution to the
Lipolysis
in Gorgnnzola
149
cheese during ripcvling
TABLE2 Diglyceride
Composition
Milk
Milk + starters
Carbon a ton7.f
(wt %) of Gorgonzola Curd
3 30A
avg CV
30B
avg cv avg cv avg cv avg cv avg cv avg cv avg cv avg cv avg cv avg cv avg cv avg cv avg cv avg cv
32A 32B 32C 32D 34A 34B 34C 34D 36A 36B 36C 36D 36E
“Capital
6.91 23.13 3.83 20.03 3.49 16.37 12.99 20.81 3.46 16.26 IO.17 36.27 IO.42 21.16 8.28 13.95 9.05 14.09 9.36 30.63 3.29 3 I .40 3.93 43.75 4.29 42.53 7.69 12.57 2.83 23.90
letters identify
6.65 29.55 4.62 25.11 2.58 60.62 12.15 32.77 3.35 15.69 8.29 26.50 9.22 31-00 7.97 18.20 8.85 18.95 9.22 45.20 3.22 30.97 4.05 29.64 5.39 12.51 9.40 35.8 I 5.05 41.83 different
Cheese Samples
6.97 34.59 5.03 32.44 2.60 66.95 12.72 25.95 3.41 22.9 I 9.88 32.55 IO.85 23.96 7.98 17.16 8.92 28.79 7.80 26.4 I 3.73 15.98 4.59 25.31 5.21 6.34 7.37 13.83 2.88 21.09 isomers
211
4.19 50.36 7.27 29.68 4.01 42.23 13.87 30. I8 2.76 38.36 8.36 45.57 12.31 43.65 8.49 28.23 7.91 42.24 7.28 34.78 4.75 48.22 3.88 7.15 5.12 21.48 6.55 52.62 3.26 48.67
according
6.58 32.66 5.70 25.50 2.74 53.39 I I ,90 35.67 5.18 24.39 9.79 25.10 9.86 4.78 7.66 25.72 I I .oo 15.77 6.73 18.23 3.59 2.28 4.06 I.01 7.15 8.25 5.81 3.36 2.24 3.81
to Mariani
30
60
105
6.36 23.72 4.66 22.14 4.17 23.49 I I.34 21.53 4.53 25.29 7.78 I I.48 IO.67 15.40 7.57 18.28 9.97 I I .74 6.37 24.57 4.90 26.05 4.55 8.92 6.31 25.40 6.66 26.84 4.18 85.56
5.84 55.54 8.41 56.57 3.36 64.30 IO.42 57.20 4.16 47.16 14.34 58.85 9.79 49.63 6.54 52.89
6.60 7.55 5.66 9.86 5.05 9.87 I I .06 I I .22 3.94 IO.77 9.87 12.52 12.32 9.17 6.82 9.48 9.25 7.23 6.71 8.93 5.13 12.35 4.82 6.6X 5.47 7.41 5.18 6.58 2.12 4.00
Il.30 49.73 9.79 34.10 3.69 33.39 3.87 46.75 5.60 17.37 6.07 35.86 2.69 31.90
ct 01.(1989)
total acidity (Table 4). During this period, bacterial activity prevails over the activity of the moulds, as reported also by Ottogalli ef rrl. (1971). Isovaleric acid. which is produced by proteolytic activity, particularly from the deamination of leucine and isoleucine (Hemme et. al., 1982), was detected only after 3 days, a period which corresponds to mould growth. Free fatty acids derived from lipolysis showed a general trend to increase. A similar result was observed in blue cheese by Godinho and Fox (1981).
150
G. Contarini, P. M. Toppino
TABLE 3 Monoglyceride Composition (wt%) of Gorgonzola Cheese Samples Milk
Carbon atomf
Milk + starters
Curd
Cheese Days of ripening 3
16
avg cv
18
avg cv
56.79 13.40 43.21 17.61
45.63 14.78 54.37 12.40
69.31 12.80 30.69 28.91
46.94 47.22 53.06 41.77
20
30
60
50.72 17.12 49.28 17.62
49.17 5.61 50.83 5.43
46.60 7.62 53.40 6.65
105 32.46 20.15 67.54 9.68
“Each number represents the sum of the isomers having the same carbon atoms. Volatile compounds
Examples of the GC profiles of milk and Gorgonzola ready for sale are shown in Figs 6 and 7, respectively. The quantitative values of the principal peaks are reported in Table 5. The list of detected compounds clearly indicates that the method allowed the extraction of only the most volatile fraction of the aroma. The highest concentration of volatile compounds was found in the samples collected after the first and second skewering. Methyl ketones are produced by the /?-oxidation of free fatty acids with 4-12 carbon atoms and are characteristic of blue cheeses. The increase in 2-pentanone and 2-heptanone, which occurred after 20 days of ripening, corresponds to the 5000
Sample 1 4000
1 f 3000 A m
m
Sample 2
I_
Sample 3
0” 0 F
‘a
2000
E
1000
0 Mlk*start.
Curd
3d
204
30d
6Od
1054
Fig. 5. Changes in the total free fatty acids during ripening.
Lipolysis
in Gorgonzolu
cheese during
ripening
151
TABLE 4 Free Fatty Acid Composition Fatty
Milk
acid
starters
+
(wt%) of Gorgonzola
Curd
Cheese Samples Cheese
Days of ripening 3
cz-c3 C5 br c4-c
Ix
30
60
10.5
avg
39.56
16.26
43.92
23.82
9.35
7.05
I.82
cv
49.35
43.56
28.73
65.68
65.58
68.96
104.41
avg cv
0.00
0.00
avg
60.44 32.3 I
83.74 8.46
0.83 70.73 55.25 21.83
1.72 37.41 74.46 21.85
4.42 57.69 86.23 10.04
1.93 58.66 91.02 6.55
cv
“Designated
20
0.41 54.82 97.77 2.17
by number of carbon atoms.
development of P. roqueforti vegetative mycelium. Interestingly, the high concentration of these compounds after 30 days of ripening corresponded to the lowest concentration of C6 and Cs fatty acids at the same ageing period. As far as alcohols are concerned, 2-alkanols showed an increase after 30 days of ageing, as reported by Jackson and Hussog (1958). The high value for ethanol after 20 days of ripening may be due to the development of yeasts on the cheese.
6,
IS
:: : :
z
10
4
,_I
A
c
t
0
1
I
I
/
10
(M:NOUTESl
..-
4
I
/
30
I
40
TIME
Fig. 6. Gas chromatogram of volatile compounds of milk, extracted by the TCT technique. The identity of each numbered peak is listed in Table 5. IS: Internal standard.
G. Contarini, P. M. Toppino
152
17
!O
1
I
0
I
1
,
/
10
/
30
I
40
TIME &:“TESI
Fig. 7. Gas chromatogram of volatile compounds of 60-day-aged Gorgonzola cheese, extracted by the TCT technique. The identity of each numbered peak is listed in Table 5.
IS: Internal standard. Acetaldehyde is also produced through the metabolism of yeasts, and showed the highest value after 20 days of ripening. The proteolytic activity of P. roqueforti, according to Hemme et al. (1982), is partially responsible for the formation of the other aldehydes detected (2-methyl-propanal, butanal and 3-methyl-butanal).
CONCLUSIONS The determinations of both the total and partial glycerides and the free fatty acids, even if carried out on a small number of cheese samples, permitted a preliminary evaluation of their significance in studying cheese ripening. The triglycerides, determined on the basis of the number of carbon atoms, provided no useful information on lipolysis during ripening. Analyses of partial glycerides showed that the concentrations of diglycerides were much higher than those of monoglycerides, and neither mono- nor diglycerides accumulated in the cheese. The strong and intensive lipolytic activity during ripening was evident from the variability of the individual compounds, which was particularly high when compared with that of milk and over-ripened cheese. The free fatty acids provided more useful information on the age of the cheese, even if large variations were observed between the samples. In fact, these compounds are the substrates for the production of the carbonyl compounds which characterize blue cheeses.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
No.
Acetaldehyde Methanol Ethanol Acetone Not identified Not identified 2-Methyl-propanal Butanal 2-Butanone 2-ButanoI Ethyl acetate Acetic acid Not identified Not identified 3-Methyl-butanal Not identified 2-Pentanone 2-Pentanol Methyl butyrate 2-Methyl-butanol 3-Methyl-butanol 2-Heptanone
Compound
Volatile
0.01
0.01 0.01 0.07 0.05
0.02 0.02 0.03 0.66 0.07 7.64 0.02 0.26 0.05 0.58 0.16 0.01 -
Milk
Compounds
0.01
0.14
0.02 0.02 0.01 0.15
0.42 0.17 0.15 0.55 0.36 0.12 0.06
0.01 0.01 0.36 0.53
-
0.01 0.11
0.02
0.18
0.64
0.48 0.90 0.91 2347 0.09 1.22 0.03 0.76 0.46 -
0.02
Curd
TABLE 5 in Sample No. I (mg/kg)
Milk + Starters
Detected
0.14 3.15 0.01 0.06
0.11 0.71 0.05 I .33 0.08 0.06 6.28 0.01 0.03
1.24
0.78 0.06 0.40
1.37 0.16 0.72 0.66 0.20 0.42 0.09 0.12
2.11 0.34
6.05 0.36 73.X I 0.93 0.08 5.04 0.34 -
20
0.14 0.04 0.81 0.10
3
1.63 0.27 29.8 1 2.32 18.71 0.58 0.90 6.28 0.02 2.46
-
0.01 3.26 0.51
2.71 0,17 7.58 2.90 1.74 7.25 3.08 0.23
30
Cheese
(see Figs 6 and 7 for Chromatograms)
0.55 5.12 0.59 8.35 0.55 0,70 4.91 0.01 4.21
0.48
0.50 0.42 0.49 3.85 0.04 2.27 0.47
60
0.80 0.26 2.32 0.37 22.92 0.95 0.93 2.82 0.02 6.43
0.02 I .82 0.13
0,08 0.18 1.81 4.95 0.43 12.09 0.05 -
105
154
G. Contarini, P. M. Toppino
Study of the volatile fraction showed the presence of compounds which seem to be connected to the various steps of the technological process (sweating, and first and second skewering). This work represents a first approach to the study of cheese ripening by means of unusual techniques. The indications obtained must be confirmed by extending the research both to a larger number of samples and different kinds of cheese.
ACKNOWLEDGEMENTS The authors wish to thank the Istituto di Ricerche Farmacologiche Milan0 (Italy) for support in mass spectrometry determinations.
M. Negri
REFERENCES Adda, J. (1986). Flavour of dairy products. In Developments in Food Flavour, ed. G. G. Birch & M. G. Lindley. Elsevier Applied Science Publishers, London, pp. 151-72. Anderson, D. F. & Day, E. A. (1966). Quantitation, evaluation, and effect of certain microorganisms on flavor components of blue cheese. J. Agric. Food Chem., 14,241-5. Badings, H. T., de Jong, C., Dooper, R. P. M. & de Nijs, R. C. M. (1985). Automatic system for rapid analysis of volatile compounds in food by purge-and-cold-trapping/ capillary gas chromatography. J. High Resolution Chromatogr., 8, 755-63. Badings, H. T. & Neeter, R. (1980). Recent advances in the study of aroma compounds of milk and diary products. Neth. Milk Dairy J., 34, 9930. Barlow, I., Lloyd, G. T., Ramshaw, E. H., Miller, A. J., McCabe, G. P. & McCabe, L. (1989). Correlations and changes in flavour and chemical parameters of Cheddar cheese during maturation. Aust. J. Dairy Technol., 44, 7-18. Berdague, J. L., Jeunet, R. & Grappin, R. (1987). Affinage et qualite du Gruyere de Comte. 111. Fermentation lactique et teneur en acides gras volatils des fromages de Comte. Lait, 67, 249-63. Bertuccioli, M. (1985). I costituenti volatili de1 formaggio: stato dell’arte. In Aggiornamenti sulle metodiche analitiche per il controllo di qualita dei,fbrmaggi, Monograph 5. CNR, IPRA, Rome, pp. 125-66. Cerning, J., Gripon, J. C., Lamberet, G. & Lenoir, J. (1987). Les activites biochimiques des Penicillium utilises en fromagerie. Lait, 67, 3-39. Contarini, G., Zucchetti, S., Amelotti, G. & Toppino, P. M. (1989). Metodiche analitiche e gascromatograliche per la valutazione della frazione acidica libera nei prodotti lattierocaseari. Riv. Ital. Sostanse Grasse, 66, 561-7. De Man, J. M. (1966). Partial glycerides in the fat of Cheddar cheese. J. Dairy Sci.. 49, 343-5.
Downey, W. K. (I 980). Review of the progress of dairy science: flavour impairment from pre- and post-manufacture lipolysis in milk and dairy products. J. Dairy Res., 47, 23752.
Dumont, J. P. & Adda, J. (1972). Isolement des constituants de I’arome des fromages: comparison de methodes. Lair, 52, 31 l-23. Fan, T. Y., Hwang, D. H. 8c Kinsella, J. E. (1976). Methyl ketone formation during germination of Penicillium roquejarti. J. Agric. Food Chem.. 24,443-6. Godinho, M. & Fox, P. F. (1981). Ripening of blue cheese. Influence of salting rate on lipolysis and carbonyl formation. Milchwissenschafi, 36, 4768. Hemme, D., Bouillanne, C., Metro, F. & Desmazeaud, M. S. (1982). Microbial catabolism of amino acids during cheese ripening. Sci. Alim., 2, I 13-23.
Lipolysis
in Gorgonzolu
cheese during
ripening
155
Jackson, A. W. & Hussog, R. D. (1958). Secondary alcohols in blue cheese and their relation to methyl ketones. .I. Dairy Sci., 41,920-4. Kale, P., Elo, P. & Antila, M. (1988). Determination of mono- and diacylglycerols, free fatty acids and cholesterol in interesteritied butter fat by direct gas chromatography on a capillary column coated with immobilized phenylmethylsilicone. Mil~hcc,issmschqfi, 43,4 16-22.
Law, B. A. (1981). The formation of aroma and flavour compounds in fermented dairy products. Dairy Sci. Ahstr., 43, 143-54. Mariani, C., Contarini. G., Zucchetti, S. & Toppino, P. M. (1989). Significance of minor components in milk fat. J. High Resolution Chromatogr., 13, 356-60. Oria, R., Condon, S. & Sala, T. F. (1987). Headspace profiles sampling and recording method for cheese. Mil~h~~~issenschqft, 42, 7 13-6. Ottogalli, G., Resmini, P. P., Bianchi, B., Galli, A., Rondinini, G., Salvadori, P., Saracchi, S. & Volonterio, G. (1971). Ricerche sulla maturazione del Gorgonzola sotto it profilo chimico e microbiologico. Latte, 45, 776-89. Price, J. C. & Manning, D. J. (1983). A new technique for the headspace analysis of hard cheese. J. Dairy Res., 50, 381-5. Riva, M., Daghetta, A. & Galli, M. (1981). Analisi gascromatografica ad alta risoluzione dei mono e dei digliceridi con colonna capillare persilanizzata associata al sistema di introduzione diretta in colonna. Riv. Ital. Sostanze Grusse, 58,432-44. Shwartz. D. P. & Parks, 0. W. (1963). Quantitative analysis of methyl ketones in blue cheese fat. J. Duiry Sci., 46, 989-90. Vujicic. I. & de Man, J. M. (1966). Partial glycerides in the fat of cheese. Milchwissenschqfi,
22, 554-8.
Woo, A. H., Kollodge, S. & Lindsay, R. C. (1984~). Modified techniques for improved gas chromatographic quantification of free fatty acids in dairy foods. J. Dairy Sci.. 67, I5 17-20.
Woo. A. H., Kollodge, S. & Lindsay, R. C. (1984h). Quantification of major free fatty acids in several cheese varieties. J. Dairy Sci., 67, 874-8. Woo, A. H. & Lindsay, R. C. (1984). Concentration of major free fatty acids and flavour development in Italian cheese varieties. J. Dairy Sri., 67, 960-8. Zegarska, Z. & Jaworski, J. (1981). Effect of lactation period and seasonal feeding system on quantitative triglyceride composition of milk fat. Mi(ch~zissensLh~~t 36, 3968.
5 ( 1995) I4 I I55
0 1995 Elsevier Science Lmted Printed ,n Ireland. All rights reserved 0958-6946
ELSEVIER
95’$9.50
Lipolysis in Gorgonzola Cheese during Ripening
Giovanna Contarini* lstituto Sperimentale
& Piero Maria Toppino
Lattiero-Caseario.
20075 Lodi. Milano. Italy
(Received 25 November 1992: revised version accepted 25 January 1994)
ABSTRACT
INTRODUCTION Cheese ripening is a complex process that includes the breakdown of the curd by proteolysis, lipolysis and other enzyme-catalysed reactions which cause flavour and textural changes typical of the different varieties. Enzymatic processes are responsible for the production of a considerable number of compounds which, as a result of their presence, concentration and proportions. are often characteristic of particular cheese types. As far as the evaluation of lipolysis is concerned, most investigations have dealt with the determination of free fatty acids, which are the last product of hydrolytic reactions (Downey, 1980; Woo & Lindsay, 1984; Woo ct rd., 1984a, h; Berdague et ul., 1987). In contrast, few references are available on the formation of partial glycerides during cheese ripening (De Man, 1966; Vujicic & De Man, 1966; Kalo et ul., 1988). The study of cheese flavour is rather complex because the aroma profile is derived from a large number ofcompounds having very different physico-chemical properties. *To whom correspondence
should be addressed. 141
142
G. Contarini, P. hf. Toppino
Various analytical and instrumental methods have been developed to evaluate cheese flavour; the most widely used methods are solvent extraction, distillation and headspace analysis (Dumont & Adda, 1972; Price & Manning, 1983; Bertuccioli, 1985; Badings et al., 1985; Oria et al., 1987; Barlow et al., 1989). In recent years, a new technique for dynamic extraction, thermal cold trapping, has been developed by Chrompack (Middleburg, Netherlands). This technique consists of stripping volatile substances from the cheese sample by nitrogen flow and trapping them on an adsorbent material (Tenax). A suitable thermal desorber allows the cryoconcentration on a silica trap; flash evaporation and direct injection into a capillary gas chromatograph are then performed (Badings et al., 1985). Whichever technique is used, the major components detected, for example, in blueveined cheese belong to the chemical classes of alcohols, aldehydes, methyl ketones, carboxylic acids, esters and lactones (Jackson & Hussog, 1958; Shwartz & Parks, 1963; Anderson & Day, 1966; Fan et al., 1976; Adda, 1986). Ripening of Gorgonzola cheese is due mainly to the proteolytic and lipolytic activity of selected strains of Penicillium roqueforti. Penicillium moulds are strongly hydrolytic and their action is superimposed on that of thermophilic starters (Law, 198 1; Cerning et al., 1987). By studying a cheese characterized by strong lipolysis during ripening (Gorgonzola), the purpose of this work was to evaluate the significance of the determination of both the intermediate and the final compounds of the lipolytic process using gas chromatography (GC) techniques. The investigation was extended to volatile compounds using the thermal cold trapping (TCT) system.
MATERIALS
AND METHODS
Samples Three Gorgonzola cheeses were selected at l-week intervals from the same factory. Samples of these products were taken during ripening according to the following sampling plan: pasteurized milk, milk with starters, curd, cheese collected after 3 days (immediately after sweating), 20 days (5 days after the first skewering), 30 days (8 days after the second skewering), 60 days (ready for sale), and 105 days after production (cheese over-ripened). Extraction of total and partial glycerides Five grams of cheese or curd were diluted in 20 ml of diethyl ether (purity 99.5%) and 10 ml of hexane (purity 99.5%). The mixture was shaken in a laboratory blender for 10 min, and filtered through a fluted filter paper, fast grade, containing about 20 g of anhydrous Na2S04 (Merck 6649). The filtrate was evaporated under vacuum at a low temperature. For the extraction of fat from milk and milk plus starters, 20 g of sample were submitted to the procedure reported in the FIL-IDF Standard Method 1B (1983). The following procedure was a modification of the method used by Riva et al. (1981) for the separation of lipid classes in vegetable fats. Anhydrous fat (40 mg) was dissolved in 1 ml of chloroform solution containing 1 mg of internal standard (dinonadecanoin, purity 99%, Nu Chek Prep Inc., Elysian, MN, USA); 0.4 ml of this solution was applied to a silica-gel-precoated plate (Merck 5725).
Lipoiysis
in Gorgonzola
cheese during
ripening
143
The plate, after development in hexane-diethyl ether-formic acid (70/30/0.5), was stained by spraying a 2’7’-dichlorofluorescein solution (0.15% in ethanol). Diglycerides and triglycerides bands were scraped off and extracted three times with 10 ml of chloroformdiethyl ether (l:l, v/v). Separation of monoglycerides was performed by the same method but the amount of fat was increased (80 mg) and the solvent proportions in the solvent mixture (hexane-diethyl ether-formic acid: 30/70/0.5) were modified. Monoheptadecanoin (0.25 mg/ml, purity 99%, Nu Chek Prep) in chloroform was used as an internal standard. Monoglycerides and diglycerides were converted into trimethylsilyl derivatives by addition of 200 ~1 of BFT (Supelco Inc., Bellefonte, PA, USA) and 200 ~1 of pyridine (Supelco) and diluted with 5 ml of hexane before GC analysis. Triglycerides were diluted directly with 20 ml of hexane. Analysis of triglycerides
Capillary column GC separation was performed with a Carlo Erba Mega 3500 instrument, equipped with a flame ionization detector, a cooled on-column injection port and a CP Sil 5 CB (Chrompack) fused-silica column (15 m length, 0.32 mm i.d., 0.12 pm film thickness). The oven temperature was programmed to increase at a rate of 40”C/min from 60 to 360°C. The detector temperature was 370°C. The flow rate of hydrogen carrier gas was 8 ml/min. A l-1*1sample of the solution of triglycerides was injected. Triglycerides having an even number of carbon atoms (from 28 to 54) were identified by their relative retention times. Results were expressed as percentages of the total triglycerides. This method does not allow the separation of triglycerides having the same number of carbon atoms but different degree of unsaturation or different positional isomers. Analyses of mono- and diglycerides
The same gas chromatograph, equipped with a CP Sil 8 CB (Crompack) fusedsilica capillary column (25 m length, 0.32 mm i.d., 0.12 pm film thickness), was used. Two different analyses were performed using this column to determine the concentrations of diglycerides and monoglycerides. For the analysis of diglycerides, the oven temperature was programmed from 60 to 250°C at a rate of 40”C/min, from 250 to 320°C at 8”C/min, from 320 to 345°C at 40”C/min and to hold at 345°C for 10 min. The detector temperature was 355°C; the flow rate of hydrogen carrier gas was 2 ml/min. A 1 ,LJsample of the solution of diglycerides was then injected. Four groups of diglycerides having 30-36 carbon atoms were identified, and the different positional isomers within each group were labelled with a letter according to Mariani et ul. (1989). Figure 1 shows an example of the analysis of diglycerides. The results were expressed both as total diglycerides (mg/g fat) adding up the values for all peaks, and as percentages calculating the value for each single peak with respect to total diglycerides. For the analysis of monoglycerides, the oven temperature was programmed from 60 to 250°C at a rate of 20°C min, to hold for 1 min at 250°C and then from
G. Contarini, P. hf. Toppino
I
A
II h
0
I
10
I
I
20
I
D
E
I
30
/
32
A I
34 I
30
36
I
40
TIME (MINUTES)
Fig. 1. Example of gas chromatogram of diglycerides of Gorgonzola cheese (each capital letter identities a different isomer according to Mariani et al. (1989); IS: internal standard). 250 to 320°C at 8”C/min. The detector temperature was 340°C; the flow rate of hydrogen carrier gas was 2 ml/min. A l-p1 sample of the solution of monoglycerides was injected. The monoglycerides containing fatty acids with 16 and 18 carbon atoms were detected (Fig. 2) and the different positional isomers were grouped. The results were expressed both as total monoglycerides (mg/g fat) adding up the values for all the groups and as percentages calculating the value for each single group with respect to total monoglycerides. Analysis of free fatty acids Milk plus starters, curd and cheese samples (20 g) were extracted using the procedure of Contarini et al. (1989) for the identification and quantitative evaluation of C2 to Cts fatty acids. Results were expressed both as the sum of free fatty acids (mg per 100 g sample) and as percentages summing the free fatty acids derived from bacterial fermentation (C,-Cs), from lipolysis (C&ts) or from proteolysis (C, br). Analysis of volatile compounds The sample (2 g of curd or cheese) was weighed in a 20-ml vial and dispersed in 9 ml of distilled water. Milk and milk plus starters (10 g) were weighed directly in the vial.
Lipolysis in Gorgonzola cheese during ripening
5
Fig. 2. Example
TIME (MINUTES)
._
”
of gas chromatogram of monoglycerides Internal standard.
145
1 20
._
15
of Gorgonzola
cheese.
IS:
One millilitre of internal standard (hexanal 0.0025%, w/v, in water) was added to each sample. The vial was equilibrated at 45°C in a thermostatic bath and connected to a nitrogen source (flow rate 10 ml/min) and to the Tenax trap (TA 60-80 mesh, Cat. No. 16252, Chrompack) through two separate outlets. The volatile compounds were stripped for 15 min. The Tenax trap was fitted in the thermal desorption cold trap injector (TCT, Chrompack), connected directly to the GC system. Compounds were desorbed from Tenax by heating at 250°C for 10 min and then transferred by a carrier gas stream (H,) to a fused-silica trap, cooled at -120°C by liquid nitrogen. Injection into the capillary system was performed by flash-heating (250°C for 10 min) of the cold trap on which the volatile components had been reconcentrated. A Carlo Erba HRGC 4160 gas chromatograph was equipped with a Chrompack TCT injector, a flame ionization detector and a CP Sil 5 CB (Chrompack) capillary column (50 m length, 0.32 mm i.d., 5 pm film thickness). The oven temperature was held at 50°C for 10 min, programmed to 220°C at a rate of 8”C/ min and held at 220°C for 12 min. The detector temperature was 250°C; the flow rate of hydrogen carrier gas was 1 ml/min. Twenty-two peaks were detected and numbered; some of them were identified by comparison with the retention times of chromatographic standards. Identification was confirmed by mass spectrometry (VG 70-250 mass spectrometer coupled with an HP 5890 (Hewlett Packard, Palo Alto, CA, USA) gas chromatograph equipped with the same capillary column and using the same temperature programme).
146
G. Contarini.
Determination of volatile compounds results were expressed as mg/kg sample.
P. M. Toppino
was performed
only on cheese No. I;
RESULTS AND DISCUSSION Total and partial glycerides
The determination of triglycerides was performed to establish if triglycerides having different numbers of carbon atoms were hydrolysed at the same rate by lipases throughout the ripening period. It can be noted from Table 1 that the average values of all peaks showed small variations during ripening, and that the standard deviations calculated for the three cheeses at each sampling were very low and similar to those found for the natural variability of milk fat (Zegarska & Jaworski, 1981). These results suggest that the lipases hydrolysed the triglycerides at the same rate independently of the number of carbon atoms, so that the relative composition of the triglycerides of milk did not differ significantly from those of the 105day-old cheese. It seems important to emphasize that the method used for analysis of the triglycerides, which is the method most commonly used for milk fat, does not allow the determination of triglycerides with respect to the amount of fat. A method having the performances described above is already established for vegetable oils which have, generally, a less complex triglyceride composition, but it is still under study for milk fat. The methods applied to the determination of diglycerides and monoglycerides which are, at the same time, substrates for and products of the lipolytic process, were more selective. Figures 3 and 4 show the trend of these compounds during ripening. The differences between the three cheeses were large, particularly after 20 days, but, despite the high values for sample No. 3 at 60 days (probably as a result of a greater growth of mould in the portion of the sample analysed), no specific tendency to increase or decrease in either the mono- or diglycerides was found, throughout the ripening period. Also, the values for the milk and the 105-day-old cheese were very close in all the samples. The concentration of diglycerides was always higher than the concentration of monoglycerides. These results suggested that the high rate of lipolysis did not allow the accumulation of intermediate compounds and that the hydrolysis of monoglycerides to produce free fatty acids could be the fastest step of lipolysis. The high variation is shown also by the coefficient of variation (CV) values for the individual compounds reported in Tables 2 and 3. It is worth noting that the differences observed for the concentrations of diglycerides in the three milk samples were comparable with the variability calculated by other workers (e.g. Mariani et al., 1989) for samples of butter. The increase of the variability during ripening, up to 60 days, was probably due to the different rates of development of lipase activity in the three cheeses; at 105 days the variation was less, suggesting a decrease of microbial and enzymatic activity when the cheese is over-ripened. As far as the monoglycerides are concerned, the CV values of the compounds with I6 carbon atoms were similar to those of the compounds with I8 carbon atoms.
Lipolysis
in Gnrgonzola
cheese during
TABLE1 Triglyceride Composition (wt %) of Gorgonzola Carbon
Milk
a form
Milk
+
147
ripming
Cheese Samples Cheese
Curd
starters Duys of’ ripening
28 30 32
avg SD avg SD avg
SD 34
38
avg SD avg SD avg
40
aVg
36
SD SD 42 44 46
Livg
SD avg SD avg
SD 48
avg SD
50
iivg
52
avg
54
avg
SD SD SD
0.66 0.02 1.27 0.01 2.48 0.05 5.35 0.03 IO.28 0.15 13.11 0.09 II.40 0.22 I.25 0.07 6.50 0.10 7.04 0.12 8.45 0.11 IO.68 0.17 IO.53 0.10 4.96 0.04
0.61 0.04 I.21 0.07 2.46 0.00 5.45 0.07 IO.22 0.22 13.23 0.32 II.59 0.21 7.25 0.03 6.52 0.06 7.08 0.09 8.56 0.07 IO.83 0.04 IO.32 0.10 4.66 0.17
0.64 0.03 I.22 0.01 2.44 0.04 5.45 0.05 IO.25 0.33 13.23 0.39 Il.16 0.15 7.27 0.05 6.52 0.14 7.15 0.14 8.67 0.11 IO.93 0.08 IO.32 0.27 4.15 0.10
3
20
30
60
105
0.62 0.02 1.22 0.03 2.46 0.03 5.46 0.06 IO.30 0.07 13.23 0.14 II.40 0.30 I.24 0.08 6.54 0.12 7.08 0.12 8.49 0.11 IO.79 0.12 IO.54 0.08 4.65 0.23
0.70 0.05 I.28 0.03 2.48 0.03 5.58 0.21 IO.34 0.21 13.05 0.39 II.03 0.38 7.25 0.15 6.66 0.15 7.22 0.14 8.76 0.19 10.73 0.24 IO.15 0.44 4.11 0.09
0.68 0.03 I.28 0.04 2.48 0.03 5.48 0.12 IO.31 0.33 12.98 0.19 II.01 0.26 7.19 0.11 6.52 0.00 7.14 0.09 8.58 0.26 IO.98 0.22 IO.46 0.35 4.88 0.10
0.67 0.01 I.29 0.04 2.54 0.06 5.34 0.13 IO.35 0.38 13.15 0.67 IO.97 0.48 7.24 0.08 6.63 0.18 7.33 0.36 8.75 0.35 11.05 0.48 IO.13 0.40 4.56 0.11
0.64 0.01 I.21 0.02 2.35 0.04 5.22 0.09 IO.16 0.11 12.91 0.07 IO.84 0.05 1.21 0.11 6.75 0.05 7.46 0.11 9.05 0.10 II.12 0.05 IO.44 0.17 4.52 0.19
The percentage of mono Cl6 in milk was higher than that of mono However, at the last sampling, the ratio between the two monoglycerides reversed.
Crx. was
Free fatty acids The method used to determine the free fatty acids produced by both lipolytic tation (Cz+). In Gorgonzola, butyric acid can be high redox potential of this cheese does
fatty acids permitted the measurement of processes (C-Crs) and bacterial fermenproduced only by lipolysis, because not allow the growth of Clostridia.
the
G. Contarini, P. M. Toppino
148
Sample 1
!IIzl 30
Sample 2
-
fi;
Ei
Sample 3
\” E 20
-
10
-
0 Milk W&art.
Curd
3d
20d
30d
6Od
105d
Fig. 3. Changes in the total diglycerides during ripening. 7
t
Sample 1
6 I-
Milk M+start.
m
Sample 2
El
Sample 3
Curd
36
206
306
Fig. 4. Changes in the total monoglycerides
The total value of free fatty acids increased, from cheesemaking to the end of ripening, and concentration (Fig. 5). During the early period acids produced by bacterial fermentations made
6Od
105d
during ripening.
although not at a constant rate, sample No. 3 showed the highest of ripening (O-10 days), the fatty a significant contribution to the
Lipolysis
in Gorgnnzola
149
cheese during ripcvling
TABLE2 Diglyceride
Composition
Milk
Milk + starters
Carbon a ton7.f
(wt %) of Gorgonzola Curd
3 30A
avg CV
30B
avg cv avg cv avg cv avg cv avg cv avg cv avg cv avg cv avg cv avg cv avg cv avg cv avg cv avg cv
32A 32B 32C 32D 34A 34B 34C 34D 36A 36B 36C 36D 36E
“Capital
6.91 23.13 3.83 20.03 3.49 16.37 12.99 20.81 3.46 16.26 IO.17 36.27 IO.42 21.16 8.28 13.95 9.05 14.09 9.36 30.63 3.29 3 I .40 3.93 43.75 4.29 42.53 7.69 12.57 2.83 23.90
letters identify
6.65 29.55 4.62 25.11 2.58 60.62 12.15 32.77 3.35 15.69 8.29 26.50 9.22 31-00 7.97 18.20 8.85 18.95 9.22 45.20 3.22 30.97 4.05 29.64 5.39 12.51 9.40 35.8 I 5.05 41.83 different
Cheese Samples
6.97 34.59 5.03 32.44 2.60 66.95 12.72 25.95 3.41 22.9 I 9.88 32.55 IO.85 23.96 7.98 17.16 8.92 28.79 7.80 26.4 I 3.73 15.98 4.59 25.31 5.21 6.34 7.37 13.83 2.88 21.09 isomers
211
4.19 50.36 7.27 29.68 4.01 42.23 13.87 30. I8 2.76 38.36 8.36 45.57 12.31 43.65 8.49 28.23 7.91 42.24 7.28 34.78 4.75 48.22 3.88 7.15 5.12 21.48 6.55 52.62 3.26 48.67
according
6.58 32.66 5.70 25.50 2.74 53.39 I I ,90 35.67 5.18 24.39 9.79 25.10 9.86 4.78 7.66 25.72 I I .oo 15.77 6.73 18.23 3.59 2.28 4.06 I.01 7.15 8.25 5.81 3.36 2.24 3.81
to Mariani
30
60
105
6.36 23.72 4.66 22.14 4.17 23.49 I I.34 21.53 4.53 25.29 7.78 I I.48 IO.67 15.40 7.57 18.28 9.97 I I .74 6.37 24.57 4.90 26.05 4.55 8.92 6.31 25.40 6.66 26.84 4.18 85.56
5.84 55.54 8.41 56.57 3.36 64.30 IO.42 57.20 4.16 47.16 14.34 58.85 9.79 49.63 6.54 52.89
6.60 7.55 5.66 9.86 5.05 9.87 I I .06 I I .22 3.94 IO.77 9.87 12.52 12.32 9.17 6.82 9.48 9.25 7.23 6.71 8.93 5.13 12.35 4.82 6.6X 5.47 7.41 5.18 6.58 2.12 4.00
Il.30 49.73 9.79 34.10 3.69 33.39 3.87 46.75 5.60 17.37 6.07 35.86 2.69 31.90
ct 01.(1989)
total acidity (Table 4). During this period, bacterial activity prevails over the activity of the moulds, as reported also by Ottogalli ef rrl. (1971). Isovaleric acid. which is produced by proteolytic activity, particularly from the deamination of leucine and isoleucine (Hemme et. al., 1982), was detected only after 3 days, a period which corresponds to mould growth. Free fatty acids derived from lipolysis showed a general trend to increase. A similar result was observed in blue cheese by Godinho and Fox (1981).
150
G. Contarini, P. M. Toppino
TABLE 3 Monoglyceride Composition (wt%) of Gorgonzola Cheese Samples Milk
Carbon atomf
Milk + starters
Curd
Cheese Days of ripening 3
16
avg cv
18
avg cv
56.79 13.40 43.21 17.61
45.63 14.78 54.37 12.40
69.31 12.80 30.69 28.91
46.94 47.22 53.06 41.77
20
30
60
50.72 17.12 49.28 17.62
49.17 5.61 50.83 5.43
46.60 7.62 53.40 6.65
105 32.46 20.15 67.54 9.68
“Each number represents the sum of the isomers having the same carbon atoms. Volatile compounds
Examples of the GC profiles of milk and Gorgonzola ready for sale are shown in Figs 6 and 7, respectively. The quantitative values of the principal peaks are reported in Table 5. The list of detected compounds clearly indicates that the method allowed the extraction of only the most volatile fraction of the aroma. The highest concentration of volatile compounds was found in the samples collected after the first and second skewering. Methyl ketones are produced by the /?-oxidation of free fatty acids with 4-12 carbon atoms and are characteristic of blue cheeses. The increase in 2-pentanone and 2-heptanone, which occurred after 20 days of ripening, corresponds to the 5000
Sample 1 4000
1 f 3000 A m
m
Sample 2
I_
Sample 3
0” 0 F
‘a
2000
E
1000
0 Mlk*start.
Curd
3d
204
30d
6Od
1054
Fig. 5. Changes in the total free fatty acids during ripening.
Lipolysis
in Gorgonzolu
cheese during
ripening
151
TABLE 4 Free Fatty Acid Composition Fatty
Milk
acid
starters
+
(wt%) of Gorgonzola
Curd
Cheese Samples Cheese
Days of ripening 3
cz-c3 C5 br c4-c
Ix
30
60
10.5
avg
39.56
16.26
43.92
23.82
9.35
7.05
I.82
cv
49.35
43.56
28.73
65.68
65.58
68.96
104.41
avg cv
0.00
0.00
avg
60.44 32.3 I
83.74 8.46
0.83 70.73 55.25 21.83
1.72 37.41 74.46 21.85
4.42 57.69 86.23 10.04
1.93 58.66 91.02 6.55
cv
“Designated
20
0.41 54.82 97.77 2.17
by number of carbon atoms.
development of P. roqueforti vegetative mycelium. Interestingly, the high concentration of these compounds after 30 days of ripening corresponded to the lowest concentration of C6 and Cs fatty acids at the same ageing period. As far as alcohols are concerned, 2-alkanols showed an increase after 30 days of ageing, as reported by Jackson and Hussog (1958). The high value for ethanol after 20 days of ripening may be due to the development of yeasts on the cheese.
6,
IS
:: : :
z
10
4
,_I
A
c
t
0
1
I
I
/
10
(M:NOUTESl
..-
4
I
/
30
I
40
TIME
Fig. 6. Gas chromatogram of volatile compounds of milk, extracted by the TCT technique. The identity of each numbered peak is listed in Table 5. IS: Internal standard.
G. Contarini, P. M. Toppino
152
17
!O
1
I
0
I
1
,
/
10
/
30
I
40
TIME &:“TESI
Fig. 7. Gas chromatogram of volatile compounds of 60-day-aged Gorgonzola cheese, extracted by the TCT technique. The identity of each numbered peak is listed in Table 5.
IS: Internal standard. Acetaldehyde is also produced through the metabolism of yeasts, and showed the highest value after 20 days of ripening. The proteolytic activity of P. roqueforti, according to Hemme et al. (1982), is partially responsible for the formation of the other aldehydes detected (2-methyl-propanal, butanal and 3-methyl-butanal).
CONCLUSIONS The determinations of both the total and partial glycerides and the free fatty acids, even if carried out on a small number of cheese samples, permitted a preliminary evaluation of their significance in studying cheese ripening. The triglycerides, determined on the basis of the number of carbon atoms, provided no useful information on lipolysis during ripening. Analyses of partial glycerides showed that the concentrations of diglycerides were much higher than those of monoglycerides, and neither mono- nor diglycerides accumulated in the cheese. The strong and intensive lipolytic activity during ripening was evident from the variability of the individual compounds, which was particularly high when compared with that of milk and over-ripened cheese. The free fatty acids provided more useful information on the age of the cheese, even if large variations were observed between the samples. In fact, these compounds are the substrates for the production of the carbonyl compounds which characterize blue cheeses.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
No.
Acetaldehyde Methanol Ethanol Acetone Not identified Not identified 2-Methyl-propanal Butanal 2-Butanone 2-ButanoI Ethyl acetate Acetic acid Not identified Not identified 3-Methyl-butanal Not identified 2-Pentanone 2-Pentanol Methyl butyrate 2-Methyl-butanol 3-Methyl-butanol 2-Heptanone
Compound
Volatile
0.01
0.01 0.01 0.07 0.05
0.02 0.02 0.03 0.66 0.07 7.64 0.02 0.26 0.05 0.58 0.16 0.01 -
Milk
Compounds
0.01
0.14
0.02 0.02 0.01 0.15
0.42 0.17 0.15 0.55 0.36 0.12 0.06
0.01 0.01 0.36 0.53
-
0.01 0.11
0.02
0.18
0.64
0.48 0.90 0.91 2347 0.09 1.22 0.03 0.76 0.46 -
0.02
Curd
TABLE 5 in Sample No. I (mg/kg)
Milk + Starters
Detected
0.14 3.15 0.01 0.06
0.11 0.71 0.05 I .33 0.08 0.06 6.28 0.01 0.03
1.24
0.78 0.06 0.40
1.37 0.16 0.72 0.66 0.20 0.42 0.09 0.12
2.11 0.34
6.05 0.36 73.X I 0.93 0.08 5.04 0.34 -
20
0.14 0.04 0.81 0.10
3
1.63 0.27 29.8 1 2.32 18.71 0.58 0.90 6.28 0.02 2.46
-
0.01 3.26 0.51
2.71 0,17 7.58 2.90 1.74 7.25 3.08 0.23
30
Cheese
(see Figs 6 and 7 for Chromatograms)
0.55 5.12 0.59 8.35 0.55 0,70 4.91 0.01 4.21
0.48
0.50 0.42 0.49 3.85 0.04 2.27 0.47
60
0.80 0.26 2.32 0.37 22.92 0.95 0.93 2.82 0.02 6.43
0.02 I .82 0.13
0,08 0.18 1.81 4.95 0.43 12.09 0.05 -
105
154
G. Contarini, P. M. Toppino
Study of the volatile fraction showed the presence of compounds which seem to be connected to the various steps of the technological process (sweating, and first and second skewering). This work represents a first approach to the study of cheese ripening by means of unusual techniques. The indications obtained must be confirmed by extending the research both to a larger number of samples and different kinds of cheese.
ACKNOWLEDGEMENTS The authors wish to thank the Istituto di Ricerche Farmacologiche Milan0 (Italy) for support in mass spectrometry determinations.
M. Negri
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