1 EFFECTS OF FEEDING ON NUTRITIONAL AND ...

1 EFFECTS OF FEEDING ON NUTRITIONAL AND AROMATIC CHARACTERISTICS OF CACIOCAVALLO PALERMITANO CHEESE A. Bonanno1*, A. Di Grigoli1, G. Tornambè1, B. Formoso1, M. L. Alicata1, G. Procida2, P. Manzi3, S. Marconi3, and L. Pizzoferrato3 1

Dipartimento S.En.Fi.Mi.Zo., sezione di Produzioni Animali, Università degli Studi di Palermo, Facoltà di Agraria, Viale delle Scienze, 90128 Palermo, Italy; 2Dipartimento di Economia e Merceologia delle Risorse Naturali e della Produzione, Università degli Studi di Trieste, and 3Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione (INRAN), Roma.

[email protected] ABSTRACT The aim of this research, carried out in the typical Cinisara cow-breeding area, was to highlight the effects on cheese quality characteristics, of a diet based exclusively on pasture exploitation and its integration with concentrates varying in their speed of ruminal degradation. In a typical, coastal farm in the province of Palermo, 24 Cinisara cows were distributed in four groups, homogeneous for lactation order, stage of lactation and milk yield, and were fed different diets: continuous natural pasture (P); continuous natural pasture with 5 kg/head/d slow degradable concentrate (60% corn, 20% tick-bean, 20% fine bran) (PSD); continuous natural pasture with 5 kg/head/d rapid degradable concentrate (60% barley, 20% chick pea, 20% fine bran) (PRD); hay of “sulla” (Hedysarium coronarium L.) ad libitum with 7 kg/head/d of farm concentrate (65% tick-bean, 35% fine bran) (H). The P, PSD and PRD cows were grazed during the day and only moved to cowsheds for milking and concentrate administration; the cows from H group were housed in a half-open cowshed. The bulk milk from each group was utilised for making Caciocavallo Palermitano cheese, this being four times during the experiment (29th April, 19th May, 2nd and 9th June). After 2 months of ripening, the cheese samples were analysed for dry matter, fat, total and soluble N, ash, cholesterol, α-tocopherol, all trans retinol, 13cis retinol and -carotene. Analysis of volatile fraction was carried out for both milk and cheese by a headspace-cold trap technique. Cheeses produced on 19th May and 2nd June were examined by triangle test. The rapid degradable concentrate reduced fat in cheese. A lower level of fat cholesterol and a higher content of α-tocopherol and 13cis retinol were observed in cheeses produced from milk obtained from grazing cows in comparison with the H cheese. Terpenes were detected in larger quantities in milk and cheese from P group in comparison with the H group. The concentrate supplement reduced the terpene content to an intermediate level. The cheese produced on 19th May from P milk differed greatly from the others in the triangle test. Cheese Art 2004 – 6th International Meeting on Mountain Cheese – Ragusa, Donnafugata Castle, June 1st-2nd, 2004

2 INTRODUCTION The Cinisara cow is an indigenous breed, reared mainly in the area around Palermo (Sicily). The breeding system is mostly extensive, and based on utilization of natural pasture, integrated with hay and concentrate only during periods of low forage availability (Di Grigoli et al., 1998). Cinisara milk is traditionally processed in order to make Caciocavallo Palermitano cheese, a typical stretched curd cheese, either consumed fresh or at various periods of ripening. As for all traditional cheeses, Caciocavallo Palermitano also has profound ties to the area of origin, particularly to the natural forage resources grazed by cows. The effects of fresh-pasture grazing on the nutritional and organoleptic characteristics of dairy products are already widely-known, these being linked to the presence of fat-soluble vitamins, ageing as natural antioxidants, and aroma compounds. The content of antioxidants, such as -tocopherol and -carotene, increases in milk and cheese produced from animals fed at pasture as compared to animals fed conserved forage (Pizzoferrato et al., 2000); moreover, their level is known to be seasonal, being higher in summer than winter (Thompson et al., 1964). These antioxidants play an important role in human health (Pizzoferrato, 1998) and prevent oxidation degradation of non-saturated fatty acids and cholesterol in cheese, improving its stability and preservation (Focant et al., 1998; Pizzoferrato and Manzi, 1999). A wide variety of volatile organic compounds is responsible for flavour in milk and cheese; the presence of a volatile fraction is strongly affected by feeding (Urbach, 1990; Moio et al., 1996). In particular, certain aroma compounds, such as terpenes, are directly transferred from plant tissues to milk fat, and can be found in cheese (Viallon et al., 1999; Fedele et al., 2000). Terpenes are plant secondary metabolites, well-known for the intensity of their smell, which contributes to cheese flavour (Duke, 1992); they could be used as markers of dairy products with regard to the feeding regimen of animals and the production period (Cornu et al., 2002; Viallon et al., 1999). In this experiment, the effects of the feeding system on chemical composition, fat-soluble vitamin content and presence of organic volatile compounds in cheese produced by Cinisara cows, were examined. In order to define the effects induced by pasture, the extensive system, based on grazing, was compared with the confined system, in which hay constituted the forage resource; additionally, the effect of concentrate supplement in pasture, and the different speeds of ruminal degradation of the concentrate, were evaluated. The effects of experimental treatments on milk yield and quality, and the body condition of cows, have already been reported in detail (Di Grigoli et al., 2000).

Cheese Art 2004 – 6th International Meeting on Mountain Cheese – Ragusa, Donnafugata Castle, June 1st-2nd, 2004

3 MATERIALS AND METHODS Experimental Site and Groups The research was carried out from 12th April to 10th June in a farm sited in a typical coastal Cinisara cattle-breeding area in the province of Palermo in Sicily (altitude 300-400 m a.s.l.; latitude 38° 01’ N - 13° 11’ E). A total of 24 Cinisara cows were distributed in four groups, homogeneous for lactation order (from 1 to 3), stage of lactation (calving period November-January) and milk yield, and were fed different diets: P: grazing on natural pasture, without supplementation; PSD: grazing on natural pasture and supplement of 5 kg/head/d slow degradable concentrate (60% corn, 20% tick-bean, 20% fine bran; CP 15.4%, ENl 1.14 Mcal/kg on DM basis); PRD: grazing on natural pasture and 5 kg/head/d rapid degradable concentrate (60% barley, 20% chick pea, 20% fine bran; CP 14.7%, ENl 1.15 Mcal/kg on DM basis); H: zero-grazing, hay of “sulla” (Hedysarium coronarium L.) ad libitum (CP 9.0%, ENl 0.58 Mcal/kg on DM basis) and 7 kg/head/d farm concentrate (65% tick-bean, 35% fine bran; CP 22.8%, ENl 1.08 Mcal/kg on DM basis). The P, PSD and PRD cows were grazed freely and continuously all day long, and were only moved twice to cowshed, for milking and feed supplementation; during the first period (12th April-19th May), cows were grazed in 50 ha pasture at 450 m a.s.l.; afterwards they were grazed in 45 ha pasture at lower altitude (180 m a.s.l.). The cows from H group were permanently housed in a half-open cowshed. Measurements During the experimental period, four series of measurements were taken (29th April, 19th May, 2nd and 9th June). After observing animals, representative samples of forage selected by cows at pasture were collected, simulating their biting off parts of plants; composition was determined in terms of the main botanic families of selected herbage. The bulk milk from each group was processed for making Caciocavallo Palermitano cheese in line with traditional method (Fulco et al., 1984). After 2 months of ripening, cheeses were weighted for cheese yield calculation. Analysis Herbage selected at pasture, concentrate and hay were analysed for dry matter (DM), crude protein (CP), ether extract, ash (AOAC, 1990), NDF (Goering and Van Soest, 1970), ADF and ADL (Van Soest and Robertson, 1980), and assessed for net energy value for lactation (NEl) (INRA, 1988). After 2 months of ripening, the cheese samples were analysed for dry matter, fat, total and soluble N and ash (G.U.R.I., 1986). Cholesterol and fat-soluble vitamins (α-tocopherol, all trans and 13cis Cheese Art 2004 – 6th International Meeting on Mountain Cheese – Ragusa, Donnafugata Castle, June 1st-2nd, 2004

4 retinol and -carotene) in cheese were extracted in line with procedure described by Panfili et al. (1994) and determined by High-Performance Liquid Chromatography (Pizzoferrato and Manzi, 1999). Analysis of volatile fraction was carried out for both milk and cheese by headspace capillary gas chromatography-mass spectrometry. The headspace sampling technique used was assessed by Barcarolo et al. (1992) and Barcarolo and Casson (1997), and based on a cold trap. Milk (40 ml) or cheese (10 g fine grinded) samples were stripped for 150 s with helium, at a rate of 10 ml/min. Volatile components were driven into a capillary tube inside a cryogenic trap maintained by liquid nitrogen to -90°C for milk and -110°C for cheese, and connected to a capillary gas chromatograph Carlo Erba GC 8000. At the end of the sampling time, desorption of volatile components took place by heating the trap to 240°C. The analytical column used was a capillary fused-silica column 50 m X 0.32 mm I.D., coated with PS 264 (Mega, Milan, Italy), 3 µm film thickness. The capillary GC system was coupled directly to a Carlo Erba MD 800 mass spectrometer. Identification of compounds was carried out by comparison with the NIST library. Cheeses produced on 19th May and 2nd June were assessed by a panel of 10 members in triangle tests, conducted in two sessions. Each panellist assessed the experimental diets in both triangular possibilities. For each set, panellists were asked to select the odd sample out. Statistical Analysis Experimental data was subjected to analysis of variance, using the GLM procedure of SAS 6.12 (1989). The statistical model included the effects of diet (4 levels: P, PSD, PRD, H) and period (2 levels: 1, including 29th April and 19th May; 2, including 2nd and 9th June). Their interaction was omitted because it was not significant. Sum of squares for diet were separated using orthogonal contrasts into single degree of freedom comparisons, as follows: P+PSD+PRD vs. H = comparison between pasture and hay; P vs. PSD+PRD = comparison between pasture and concentrate supplement; PSD vs. PRD = comparison between slow and rapid degradable concentrate. For the sensory triangle test, standard references tables (Amerine et al., 1965) were used to calculate the significance of differences.

RESULTS AND DISCUSSION The botanic composition of the herbage selected from cows at pasture (Table 1) shows how, in the first period (29th April and 19th May), the Gramineous species, being in their early stage, were consumed at a greater extent than Leguminous ones, because of both their higher availability and better palatability.


5 Table 1. Botanic (% DM) and chemical composition (% DM), and energy value (Mcal/kg DM) of herbage selected from cows at pasture. Herbage selected at pasture 29th April 19th May

2nd June

9th June

Leguminous

21.7

5.4

41.7

36.5

Gramineous

57.4

44.9

36.4

41.5

Other species

20.9

49.7

21.9

22.0

Dry matter

24.8

30.3

36.4

44.7

Crude protein

13.6

9.5

12.3

9.8

Ether extract

3.1

3.2

3.4

2.8

Ash

9.8

7.4

9.3

12.3

NDF

43.6

50.7

50.5

50.3

ADF

31.7

35.3

35.6

38.8

ADL

4.4

4.5

5.8

7.0

NEl Mcal/kg.

1.94

1.96

1.84

0.99

On 19th May, the incidence of the other botanic families increased to 50% of intake. In the second period (2nd and 9th June), Gramineous and Leguminous species were selected on an equal basis, indicating the increased preference for Leguminous families in a late phenologic stage. The chemical composition trend of selected herbage (Table 1) followed the physiological development of plants, with an increasing of dry matter, ADF and ADL, and a consequent reduction of energy value. The low protein content of selected herbage on 19th May and 9th June must be related to the lower presence of Leguminous species in the first case, and to herbage desiccation in the second case. The yield and chemical composition of cheese was affected more by period than diet (table 2), even though the cheese yield was higher for H milk and cheese fat was lower in PRD milk, in conformity with milk composition (Di Grigoli et al., 2000).

Table 2. Cheese yield and composition.


6 Significance P

PSD

PRD

H

SEM

Cheese yield kg/100 l 8.2 8.0 7.9 8.3 0,31 % Dry matter 68.1 67.5 67.1 65.2 0.83 % Fat 43.7 43.6 41.8 42.2 1.36 % Total N 7.4 7.2 7.4 7.4 0.22 % Soluble N 0.7 0.5 0.5 0.5 0.12 % Ash 8.5 8.2 8.8 8.4 0.33 mg/100 g 102.6 108.6 101.0 106.8 4.45 Cholesterol mg/g fat Cholesterol 3.5 3.7 3.6 3.9 0.09 α-tocopherol µg/100 g 1301 1314 1197 823 58.0 α-tocopherol µg/g fat 43.3 44.5 42.4 29.4 2.7 Trans retinol µg/100 g 235.3 256.8 206.2 205.9 18.2 µg/g fat Trans retinol 7.9 8.7 7.3 7.4 0.44 µg/100 g 13-cis retinol 59.9 42.6 37.0 32.7 5.3 13-cis retinol µg/g fat 2.0 1.5 1.3 1.2 0.19 µg/100 g β-carotene 104.8 139.7 103.4 110.2 14.7 µg/g fat β-carotene 3.6 4.7 3.7 4.1 0.41 * = P≤0.10; ** = P≤0.05; *** = P≤0.01; **** = P≤0.001.

Diet

** **** ***

** **

P+PSL+ Perio PRD vs. d H **** *** **

**

** **** *** **

** **** ****

**

** *

P vs. PSD+ PRD

PSD vs. PRD

* **

***

*** **

Root MSE 0.62 1.65 2.7 0.44 0.25 0.65 8.9 0.2 116.0 5.4 36.4 0.88 10.5 0.39 29.4 0.83

Cholesterol level in cheese was affected by diet as regards fat content; it was lower in cheese produced from grazing groups (P 0.05) than confined cows, in accordance with Pizzoferrato et al. (2000). The trans retinol was higher in PSD cheese and cheese fat in comparison with the PRD (P 0.05); the 13cis retinol resulted in a higher amount in P cheese than in the other diets. Among retinol cis isomers, 13 cis retinol has the largest biopotency as vitamin A (75%); it is generally absent in fresh raw milk, but can be formed by a reaction of cis-trans isomerization promoted by light, heat treatments, and fermentation processes (Panfili et al., 1998). According to these results, animal diet seems to be an isomerization determinant not reported before. A pasture-induced selection of specific natural micro-organisms in milk, acting during cheese making and possibly during ripening, might promote retinol isomerization, but further research is necessary to confirm this hypothesis. The cheese content of α-tocopherol was significantly affected by diet and period. Cheese from pasture-based diets contained higher amounts of α-tocopherol than H diet (P 0.001), as also evidenced by Pizzoferrato et al. (2000) in goat milk and cheese. Figure 1 shows how the αtocopherol level increased in passing from spring to early summer, especially in cheese from grazing cows. The β-carotene did not show any differences between the groups. Jensen et al. (1999) also found a similar concentration of β-carotene in milk produced from pasture or grass silage. Nevertheless, a tendency can be observed for a higher content of β-carotene, expressed on both cheese and fat basis, for PSD cows fed a concentrate with 60% of corn, particularly rich in βCheese Art 2004 – 6th International Meeting on Mountain Cheese – Ragusa, Donnafugata Castle, June 1st-2nd, 2004

7 carotene, as is well-known; on the other hand, the level of β-carotene is thought to be related to

µg/100 g

intake (Thompson et al., 1964; Weiss, 1998).

1800 1600 1400 1200 1000 800 600 400 200 0

P PSD PRD H

29th April 19th May 2nd June 9th June

Figure 1. Variation of α-tocopherol in cheese in relation to time.

The analysis of the volatile fraction permitted the identification of 42 compounds in milk and 61 in cheese. The compounds were grouped according to their chemical class. Most of them were ketones in milk (Table 3), and alcohols, derived from fermentation activity of natural microflora, followed by esters in cheese (Table 4). No effects of the feeding regimen emerged in volatile compounds in milk and cheese, with the exception of terpenes, which showed a tendency towards a higher content with the pasture diets in in milk and, especially, in cheese (P 0.05) in comparison with a hay-based diet. The intermediate values of terpenes in milk and cheese, due to the effects of concentrate supplemented diets, might indicate how concentrate, with an accompanying reduction in herbage intake, contributed to diluting terpene content, in accordance with Fedele et al. (2000). In relation to time (Figure 2), the terpenes in cheese showed an increasing trend in all diets, but the amount was almost always higher in cheese from pasture-fed cows. The main isolated terpenes were α-pinene e p-menth-l-ene, the incidence of which was similar among the diets, being equal to 40-50% and 20-30% in milk, and 50% and 40% in cheese, respectively; the remaining amount was made up by limonene.

Table 3. Volatile organic compounds in milk (µg/kg).


8 Significance P

PSD

PRD

H

SEM

Diet

Carboxylics acids 10.9 8.3 5.0 2.9 5.21 Alchols 11.5 2.6 5.7 4.5 3.27 Aldehydes 11.9 12.4 11.0 19.8 4.72 Ketones 127.4 129.3 105.7 95.6 20.65 Esters 31.5 33.9 44.4 41.9 10.54 Hydrocarbons 5.8 6.7 5.3 5.3 1.13 Sulphoureous 5.1 2.6 2.8 2.0 1.20 Terpenes 4.9 3.7 4.0 2.3 0.95 * = P≤0.10; ** = P≤0.05; *** = P≤0.01; **** = P≤0.001.

Period

P+PSL +PRD vs. H

P vs. PSD+ PRD

PSD vs. PRD

Root MSE 10.4 6.5 8.1 41.3 21.1 2.3 2.4 1.9

*

****

Table 4. Volatile organic compounds in cheese (µg/kg). Significance P

PSD

PRD

H

SEM

Diet

µg/kg

Carboxylics acids 447.7 323.2 639.9 326.5 225.8 Alchols 5389 3769 5415 7454 2065 Aldehydes 184.8 214.6 191.5 234.7 46.9 Ketones 467.4 520.4 570.1 570.2 153.6 Esters 817.7 781.1 539.1 852.5 203.4 Hydrocarbons 96.2 85.4 109.8 75.5 16.8 Sulphoureous 28.7 43.4 41.1 36.8 9.1 * Terpenes 35.4 21.9 27.6 11.5 6.1 * = P≤0.10; ** = P≤0.05; *** = P≤0.01; **** = P≤0.001.

P+PSL Period +PRD vs. H

P vs. PSD+ PRD

**

****

80 70 60 50 40 30 20 10 0

**

PSD vs. PRD

Root MSE 451.5 4131 93.7 307.3 406.9 33.5 18.1 12.3

P PSD PRD H

29th April

19th May

2nd June

9th June

Figure 2. Variation of terpenes in cheese in relation to time.

At the triangle tests (Table 5), the panellists recognized different organoleptic characteristics among cheeses produced on 19th May. The P and PSD cheeses seemed different from each other and from all others. The diversity of P cheese did not emerge on 2nd June, while the PSD cheese was again found to be different from PRD and H cheeses. It was not possible to justify these differences on the Cheese Art 2004 – 6th International Meeting on Mountain Cheese – Ragusa, Donnafugata Castle, June 1st-2nd, 2004

9 mere basis of identified terpenes coming from pasture plants. For 19th May cheese, the role of pasture appeared to be marked, probably because the fresh herbage selected by cows was made up of almost 50% of other species, widely-recognised for their high supply of aroma active terpenes. The differences in 2nd June cheese were not so evident, probably because the herbage at pasture was still dried up. The aroma profile of cheese derives from the combinations between compounds possessing high odour potencies. Thus, a series of factors may play a role in modulating the changes during cheese-making and ripening, including the milk composition in volatile compounds, surely linked to the feeding regimen and, especially, to the quality of grazed herbage.

Table 5. Triangle test on cheese: incidence (%) of differences found by panel of judges. Comparison Significance 19th May 2nd June 85.0 *** 10.0 P vs H 75.0 *** 15.0 P – PSD 75.0 *** 40.0 P – PRD 70.0 *** 75.0 PSD vs H 50.0 n.s. 35.0 PRD vs H 70.0 *** 65.0 PSD vs PRD * = P≤0.05; ** = P≤0.01; *** = P≤0.001; n.s. = not significant.

Significance n.s. n.s. n.s. *** n.s. **

CONCLUSIONS The experiments permitted the highlighting, in the typical cattle-breeding area of Cinisara, of certain effects of natural pasture grazing and concentrate supplementation on cheese quality. Feeding at pasture, supplemented by rapidly degradable concentrate, reduced cheese fat. Cheeses produced from the milk of grazing cows showed lower fat cholesterol levels and higher amounts of α-tocopherol and 13cis retinol. The level of terpenes was higher in cheese from pasture-fed cows, than from cows fed a hay-based diet, and increased in the passing from spring to early summer. Intermediate values of terpenes were recorded in milk and cheese from diets supplemented by concentrate. These findings confirm the possibility of using terpenes to develop a tracer method for dairy products, with regard to animal feeding, production season and geographic origins. During triangle tests, sensory differences were observed between the cheese produced in spring, from cows fed only pasture and the others, but these were not easily referable to terpene content or volatile profile drawn from analysis. REFERENCES Amerine, M.A., R.M. Pangborn, and E.B. Roessler. 1965. Principles of sensory evaluation of food. Academic Press, New York and London. Cheese Art 2004 – 6th International Meeting on Mountain Cheese – Ragusa, Donnafugata Castle, June 1st-2nd, 2004

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