Article pp.221-232 du Vol.24 n°3 (2004)

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ARTICLE ORIGINAL ORIGINAL PAPER

Study of the proteolysis and cathepsin D activity of commercial dry-cured Iberian and Serrano hams

C. Mariscal, A. García Ruiz¹, A. Soriano, M. D. Cabezudo

Department of Analytical Chemistry and Food Technology. University of Castilla-La Mancha. Faculty of Chemistry. Campus Universitario s/n. 13071 Ciudad Real. Spain.

SUMMARY

Physicochemical characteristics, proteolysis, and cathepsin D activity of the Semimembranosus and Biceps femoris muscles from commercial Iberian and Serrano hams were evaluated.

Total nitrogen, sodium chloride, fat, and amino acid nitrogen contents were the only parameters of physicochemical composition and nitrogen fractions that enabled Iberian ham to be differentiated from Serrano ham in the two muscles considered.

A wide variety of polypeptides in sarcoplasmic and myofibrillar protein fractions were found in the Semimembranousus and Biceps femoris muscles from Serrano and Iberian commercial samples. The sarcoplasmic proteins present in both muscles from both ham varieties had molecular weights of 80, 50, 45 (creatine kinase), 30 (phosphoglycerate mutase), 18, 16 (myoglobin), and 9 kDa.

The common myofibrillar proteins in both muscles from both ham varieties had molecular weights of 102 (α-actinin), 90, 80, 60, 50, 35 (Tropomyosin), 30, 20 (C-troponin), 15 (Myosin light chain III), and 11 kDa .

The protein profile showed that, in general, the myofibrillar protein fraction underwent more intense proteolysis than the sarcoplasmic protein fraction, giving rise to a larger number of low-molecular-weight peptides.

Residual cathepsin D activity in the samples was very low, with no differences between either the muscle tissues or the ham varieties, indicating that the hams are relatively stable, as regards to proteolysis, once they have reached the marketplace.

Key words

commercial Iberian and Serrano ham, muscle type, proteolysis, cathepsin D activity.

1. Correspondence: [email protected]

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1 – INTRODUCTION

Dry-cured hams are typical products of Spain and are extremely popular on account of their organoleptic properties.

Production of dry-cured hind leg and fore leg hams in Spain came to 234,000 tonnes in 2002, of which exports accounted for 14,000 tonnes; exports have increased substantially in recent years (AICE, 2003). Different varieties of dry-cured hams are available, the most important being Serrano and Iberian hams, though production levels for the latter are considerably lower than the levels for the first.

The quality of dry-cured hams depends on the raw materials and the ripening conditions. Iberian cured ham, the most valuable cured meat product of Spain, is manufactured from pure-breed Iberian pigs or a cross between Iberian (75 %) and Duroc pigs. Thighs, with weights usually of 10-12 kg, are processed following a traditional method which takes at least 18 months. These characteristics differ from those of other dry cured hams (such as Spanish Serrano, Italian Parma and San Danielle, French, etc.) that are obtained from 6-10 kg hind legs of intensively fed White breed pigs, and processed for only 4-12 months.

During the dry-curing process, a progressive increase in soluble non-protein nitrogen amount and free amino acids has been reported (BELLATI et al., 1983a;

FLORES et al., 1984) indicating that muscle proteins have undergone intense proteolysis. Nitrogen components play a key role in determining texture, colour, odour, and flavour as a result of their functional properties and insolubilization due to denaturation and proteolysis. Furthermore, lysosomal proteinases such as cathepsins B, D, H, and L have been found to be still active after 8 months of dry curing (TOLDRÁ and ETHERINGTON, 1988) suggesting that dry-cured hams were still undergoing proteolytic changes. These changes will normally be more pronounced in Iberian ham than in other meat products, on account of the longer curing time and the relatively high temperature (up to 30ºC) reached in certain stages of processing.

In Spain there have been numerous studies on the physicochemical composition (ASTIASARÁN et al., 1988; CARRASCOSA and CORNEJO, 1991); microbial flora (CARRASCOSA and CORNEJO, 1991; RODRÍGUEZ et al., 1994; GARCIA-RUIZ et al., 2001); progression of proteolysis and lipolysis (FLORES et al., 1985; TOLDRÁ et al., 1992a; CÓRDOBA, 1990; ARMERO et al., 1999; SORIANO, 2001; GARCÍA-RUIZ et al., 2002; SORIANO et al., 2003a, b); sensory characteristics (FLORES et al., 1997;

VIRGILIet al., 1999), Maillard reaction (VENTANAS et al., 1992); and formation of volatile compounds (GARCÍA et al., 1991 ; LÓPEZ et al., 1992) in both Serrano and Iberian hams. However, the literature review has disclosed few studies dealing with the behaviour of these hams in the marketplace.

Therefore, the object of the present study was to compare the physicochemical composition, proteolysis, and cathepsin D activity of two different muscles in commercial Iberian and Serrano hams.

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2 – MATERIALS AND METHODS

2.1 Material

Eight samples of Iberian ham and eleven samples of Serrano hams were purchased at different commercial retail establishments in the cities of Ciudad Real and Toledo in Spain. The samples were transported to the laboratory under refrigeration and the Semimembranosus and Biceps femoris muscles were removed from all the hams. Intermuscular fat and connective tissue were trimmed away from both muscles and they were then cut into small pieces and homogenized in a household kitchen blender (Moulinex). Water activity, pH and dry matter were assayed immediately, and the remainder of the samples were then placed in hermetically sealed containers and stored at –20ºC for later analysis.

2.2 Methods of analysis

2.2.1 Physicochemical composition

Water activity (a_w) was measured using a dew-point hygrometer (Decagon Devices, model CX-2). pH was determined directly using an Ingold electrode probe connected to a Crison model 2001 pH-meter. Dry matter (DM) was measured according to ISO standard R 1442 (1997), fat as described by ISO standard R 1443 (1973), sodium chloride (NaCl) according to ISO standard R 1841 (1996) and total nitrogen (TN) according to the Kjeldahl method, with the protein nitrogen (PN) being calculated by multiplying the TN by 6.25 (MAPYA, 1994).

2.2.2 Nitrogen fractions

Water-soluble nitrogen (WSN) was determined as described by MONIN et al.

(1997), non-protein nitrogen (NPN) by precipitation of the proteins with trichloroacetic acid followed by determination according to the Kjeldahl method (MAPYA, 1994), and amino acid nitrogen (AAN) was measured according to the method of Moore and Stein (1948).

2.2.3 Extraction, dialysis, and quantification of the sarcoplasmic and myofibrillar proteins

The sarcoplasmic proteins were extracted using the method of MONIN et al.

(1997). The myofibrillar proteins were determined using the method described by Helander (1957). Protein concentration in samples was determined by the Bradford method (1976) using bovine serum albumin as standard.

The samples were dialysed using a membrane with a cut-off point of 7000 Da in 0.01 M phosphate buffer, pH 7.1, during 16-18 h at 4ºC. The dialysed fractions were kept at –20ºC until electrophoresis.

Denaturation of the protein fractions was carried out according to the method of TOLDRÁ et al. (1990).

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2.2.4 Electrophoresis

The proteins were separated by horizontal SDS-PAGE using a PhastSystem apparatus (Amersham Pharmacia Biotech).

Porosity gradient gels (0.112 M acetate, 0.112 M Tris, pH 6.4) were used for separation of the proteins: PhastGel 10-15 for determination of the sarcoplasmic proteins with molecular weights ranging from 10 to 250 kDa, and PhastGel 8-25 for separation of the myofibrillar proteins with molecular weights ranging from 6 to 300 kDa.

Solid electrode buffer strips consisted of 0.2 M tricine, 0.2 M Tris, 0.55%

SDS, pH 8.1.

Separations of both the sarcoplasmic and the myofibrillar proteins for each sample were performed at 15ºC in duplicate.

Gels were stained with a tablet of Coomassie Blue R-350 dissolved in water/

methanol/acetic acid (6:3:1). Gels were destained twice with methanol:acetic acid:water (3:1:6), 13% (v/v) glycerol was used to give the gels consistency.

Quantification of the bands was carried out by first scanning the gels using the Photophinish program (TDI, Spain) and then making densitometric readings with the 1D-Manager program (TDI, Spain).

The molecular weights of the protein bands were calculated from the R_f values by interpolation of the calibration curve, constructed using a Sigma marker (from 6.5 to 205 kDa).

2.2.5 Cathepsin D activity

The extraction method described by ETHERINGTON et al. (1990) was employed for the lysosomal proteases. Cathepsin D was assayed on bovine haemoglobin (ETHERINGTON, 1972) and the resulting trichloroacetic acid-soluble peptides were quantified by the method of LOWRY et al. (1951) using L-tyrosine as standard. The results were expressed as µg tyrosine/mL min.

2.2.6 Statistical processing

The data for the physicochemical composition, nitrogen fractions, and enzymatic activity were tested for significance using the Student-Newman-Keuls (SNK) test for multiple comparisons of means using the program SPSS 10.0.

3 – RESULTS AND DISCUSSION

3.1 Physicochemical composition

Table 1 shows the mean and the standard deviation for the a_w, pH, and physicochemical composition parameters for the Semimembranosus and Biceps femoris muscles from the Serrano and Iberian ham samples.

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Table 1

Mean and standard deviation values for the a_w, pH, and physicochemical composition parameters of the Semimembranosus (SM) and Biceps femoris (BF) muscles from

the Serrano and Iberian ham samples.

Significant differences between the two varieties of hams were found in the intramuscular fat and NaCl content in the Biceps femoris muscle, showing that Iberian hams had the highest values for the intramuscular fat and the lowest for the NaCl content, perhaps because the higher concentration of intramuscular fat hindered the diffusion of the salt during curing. The amount of protein nitrogen was higher for the Serrano hams than for the Iberian hams in the Semi- membranosus muscle.

These findings were consistent with the results reported by VENTANAS and CÓRDOBA (1992) for Iberian ham and MONIN et al., (1997) for Bayonne ham cured for 8 months.

3.2 Nitrogen fractions and total proteins contents

Table 2 sets out the mean and standard deviation values for the nitrogen fractions and total sarcoplasmic and myofibrillar proteins contents for the Semi- membranosus and Biceps femoris muscles from both ham varieties.

MUSCLE HAM

VARIETY a_w pH DM (%) Fat (%DM) NaCl (%DM) PN (%DM) SM SERRANO (n=11)

IBERIAN (n=8)

0.86±0.02 0.88±0.01

5.94±0.13 5.96±0.06

56.76±4.14 58.65±2.94

12.75±2.00 15.49±1.52

9.44±1.1

8.20±1.83 75.67±4.55^a 71.09±3.64^b BF SERRANO (n=11)

IBERIAN (n=8)

0.88±0.02 0.89±0.02

5.87±0.06 5.91±0.12

47.21±5.86

50.46±5.97 14.31±8.64^a 28.48±11.74^b

11.95±1.92^a 10.52±1.45^b

65.51±5.34 56.81±11.92 a,b: different supercripts in each column for each muscle indicate significant differences (P<0.05) n: Number of samples; a_w: Water activity; DM: Dry matter; PN: Protein nitrogen

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Table 2 Mean and standard deviation values for the nitrogen fractions and total sarcoplasmic and myofibrillar proteins of the Semimembranosus (SM) and Biceps femoris (BF) muscles from the Serrano and Iberian ham samples. MUSCLEHAM VARIETYWSN (%DM)WSN/TN (%)NPN (%DM)NPN/TN (%)AAN (%DM)Sarcoplasmic Proteins (%DM)Myofibrillar Proteins (%DM) SMSERRANO (n=11) IBERIAN (n=8)3.19±0.47 2.55±1.1226.68±4.39 23.88±8.211.62±0.86 1.73±1.0313.31±6.81 16.42±11.477.75±4.09a 5.25±1.51b3.79±0.83 3.11±1.253.11±1.18 3.10±0.95 BFSERRANO (n=11) IBERIAN (n=8)3.34±0.95 3.05±1.2631.92±9.00 32.87±9.852.23±1.64 2.04±0.7621.34±15.49 22.56±6.719.48±4.20a 5.09±1.09b4.20±0.72 3.00±1.443.38±1.27 3.52±0.56 a,b: different supercripts in each column for each muscle indicate significant differences (P<0.05) n: number of samples; WSN: water soluble nitrogen; NPN: non protein nitrogen; AAN: amino acid nitrogen; TN: total nitrogen; DM: Dry matter

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The WSN values were similar to those published by BELLATI et al. (1983a,b) for Parma hams, and by FLORES et al. (1984) and TOLDRÁ et al. (1993) for Span- ish Serrano hams.

The NPN content was higher in the Biceps femoris muscle. This difference may be ascribable to diffusion of low molecular mass nitrogen compounds from the Semimembranosus muscle to the Biceps femoris muscle, namely, along the moisture gradient. The proteolysis index (NPN/TN) for the Biceps femoris muscle samples was consistent with the values of 22% reported by Soriano (2001) for Spanish Serrano hams cured for 11 months and 24% reported by BUSCAIL- HON et al. (1994) and VIRGILI (1996) for Bayonne hams.

The AAN content in both muscles was higher in Serrano hams than in Ibe- rian hams. A possible explanation for this finding is that since Iberian hams undergo longer curing times (at least 18 months) under higher ambient temperature conditions, the amino acids generated by proteolysis may take part in such other reactions as Strecker degradation and the Maillard reaction, thus giving rise to other aroma and flavour components and lowering the free amino acid content.

Only significant differences between muscles for the AAN content were found in Serrano hams, showing the Biceps femoris muscle the highest values. This finding was in accordance with the results of MONIN et al. (1997) and SORIANO (2001).

No significant differences in total sarcoplasmic and myofibrillar protein amounts were found either between the muscles or between the two ham varieties considered.

3.3 SDS-PAGE

A wide variety of electrophoretic patterns were found in sarcoplasmic and myofibrillar proteins extracted from the Semimembranosus and Biceps femoris muscles of Serrano and Iberian hams. Variation in proteins bands was attributed to different curing process times among commercial ham samples.

The only sarcoplasmic proteins presented in both muscles in both ham varieties corresponded with the molecular weights of 80 kDa, 50 kDa, 45 kDa (creatine kinase), 30 kDa (phosphoglycerate mutase), 18 kDa, 16 kDa (myoglobin), and 9 kDa.

The sarcoplasmic proteins, present at very low relative densities in some ham samples analysed were:

• Semimembranosus muscle:

– For Serrano hams: 111, 19, and 14 kDa.

– For Iberian hams: 153, 142, 122, 116, 108, 104, 93, 89, 85, 77, 67, and 28 kDa.

• Biceps femoris muscle:

– For Serrano hams: 138, 105, 88, 72, 60, 47, 28, 14, and 10 kDa.

– For Iberian hams : 149, 131, 110, 98, 91, 85, 80, and 65 kDa.

Table 3 shows the maximum and minimum relative density values for the sarcoplasmic proteins common in both ham varieties and both muscles. Since the samples

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were commercial the amount of the polypeptides were highly variable. The bands were identified following the criteria of MCCORNICK et al. (1988) and SAVAGE et al. (1990). The 50 kDa polypeptide was the main protein present in the two muscles in both varieties of ham. The 9 kDa fragment was the smallest detected in all the samples.

Table 3

Maximun and minimun relative density values (%) for the sarcoplasmic proteins common to both Semimembranosus and Biceps femoris muscle in Serrano

and Iberian ham samples.

The myofibrillar proteins were identified according to PORZIO and PEARSON

(1977) and IVERSEN (1995). Protein profiles showed that the myofibrillar protein fraction underwent more intense proteolysis than the sarcoplasmic protein fraction, giving rise to a larger number of low-molecular-weight peptides in both muscles from both ham varieties.

The fragments corresponding with molecular weights of 102 kDa (α-actinin), 90 kDa, 80 kDa, 60 kDa, 50 kDa, 35 kDa (tropomyosin), 30 kDa, 20 kDa (troponin- C), 15 kDa (myosin light chain III), and 11 kDa were common in both muscles and in both hams varieties. Their maximum and minimum relative density values are showed in Table 4. The myofibrillar proteins found at very low density in some ham samples were:

• Semimembranosus muscle:

– For Serrano hams: 181, 170, 68, 22, 17, 14, 9, and 8 kDa.

– For Iberian hams: 136, 112, 25, and 12 kDa.

• Biceps femoris muscle:

– For Serrano hams: 177, 141, 119, 110, 100, 75, 70, 68, 55, 45, 38, 28, 9, and 8 kDa.

Electrophoretic band

Semimembranosus Biceps femoris SERRANO

(n=11)

IBERIAN (n=8)

SERRANO (n=11)

IBERIAN (n=8)

80 kDa Max

Min

30.23 32.71

31.16 32.52

29.49 17.18

15.30 33.73

50 kDa Max

Min

34.55 34.98

37.70 10.39

49.70 30.20

26.87 34.01 Creatine kinase (45 kDa) Max

Min

44.28 36.85

10.84 35.93

35.33 30.10

18.94 35.04 Phosphoglycerate

mutase (30 kDa)

Max Min

34.27 36.20

17.31 35.35

21.47 34.58

10.54 36.70

18 kDa Max

Min

14.77 32.45

34.22 32.50

11.93 32.02

34.83 31.67 Myoglobin (16 kDa) Max

Min

10.31 33.15

10.73 32.75

33.69 32.35

38.65‘

30.99

9 kDa Max

Min

39.81 35.41

38.46 36.23

11.73 34.23

15.51 34.80 n: number of samples

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– For Iberian hams: 136, 130, 122, and 97 kDa .

A large number of polypeptides in the region between 50 and 102 kDa were recorded (102, 90, 80, 60, and 50 kDa), in accordance with the results of TOLDRÁ et al. (1992) in hams cured for 8 months. YATES et al. (1983) suggested that myosin fragments could fall in the region between 50 and 100 kDa.

Table 4

Maximun and minimun relative density values (%) for the myofibrillar proteins common to both Semimembranosus and Biceps femoris muscle in Serrano

and Iberian ham samples.

3.4 Residual cathepsin D activity

The values for the residual activity of cathepsin D for both muscles and both varieties were quite similar, and were not significant different (Serrano SM:

4.81±0.72; Serrano BF: 4.06±0.87; Iberian SM: 4.89±0.45; Iberian BF:

5.87±0.53 µg tyrosine/mL min.

The results indicate that low residual cathepsin D activity continued taking place in the hams while they were for sale in retail shops, after completion of the dry-curing process. Activity levels may be higher or lower depending on the conditions of storage and may affect the final product quality.

Electrophoretic band

Semimembranosus Biceps femoris SERRANO

(n=11)

IBERIAN (n=8)

SERRANO (n=11)

IBERIAN (n=8) α-actinin (102 kDa) Max

Min

18.46 33.46

20.21 32.32

18.75 32.75

13.19 36.27

90 kDa Max

Min

14.39 32.08

15.17 33.78

18.16 33.58

10.21 34.67

80 kDa Max

Min

11.08 32.01

12.24 32.57

14.62 30.88

15.54 35.36

60 kDa Max

Min

31.65 34.96

32.4 334.10

41.82 36.28

24.04 36.24

50 kDa Max

Min

30.54 33.50

20.26 34.78

13.34 33.93

17.32 34.25 Tropomyosin (35 kDa) Max

Min

11.82 33.77

12.01 35.70

16.44 32.54

14.91 33.83

30 kDa Max

Min

10.46 33.88

11.52 34.80

9.28 35.12

10.45 35.10 C-troponin (20 kDa) Max

Min

13.41 33.75

38.71 33.48

14.50 32.72

39.91 34.70 Myosin light chain III

(15 kDa)

Max Min

12.01 32.58

38.66 33.34

39.78 35.26

39.37 35.09

11 kDa Max

Min

14.72 32.20

37.55 34.05

35.78 33.33

10.69 35.26 n: number of samples

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4 – CONCLUSIONS

Protein nitrogen, sodium chloride, fat, and amino acid nitrogen contents were the only parameters of physicochemical composition and nitrogen fractions that enabled Iberian ham to be differentiated from Serrano ham.

Qualitatively, the sarcoplasmic and myofibrillar profiles of the Semimembra- nousus and Biceps femoris muscles from the commercial Serrano and Iberian hams were quite different. The myofibrillar protein fraction underwent more intense proteolysis than the sarcoplasmic protein fraction, giving rise to a larger number of low-molecular-weight peptides.

The low residual cathepsin D activity indicated that commercial Serrano and Iberian hams are relatively stable, as regards to proteolysis, in the marketplace.

5 – ACKNOWLEDGEMENTS

The authors gratefully acknowledge the financial support for this study in the form of a research grant provided by the Government of Castilla-La Mancha Autonomus Region.

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