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Biogenic amine content during the manufacture of dry-cured lacón, a Spanish traditional meat product: Effect of some additives
MEAT SCIENCE Meat Science 77 (2007) 287–293 www.elsevier.com/locate/meatsci
Short Communication
Biogenic amine content during the manufacture of dry-cured laco´n, a Spanish traditional meat product: Effect of some additives Jose´ M. Lorenzo, Sidonia Martı´nez, Inmaculada Franco, Javier Carballo
*
A´rea de Tecnologı´a de los Alimentos, Facultad de Ciencias de Orense, Universidad de Vigo, 32004 Orense, Spain Received 14 December 2006; received in revised form 14 March 2007; accepted 22 March 2007
Abstract The content of nine biogenic amines (agmatine, tryptamine, 2-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine) was determined throughout the manufacture of dry-cured laco´n, a traditional dry-salted and ripened meat product made in the north-west of Spain from the fore leg of the pig following a similar process to that of dry-cured ham. The effect of the use of additives (glucose, sodium nitrite, sodium nitrate, sodium ascorbate and sodium citrate) on the biogenic amine content during manufacture was also studied. Tryptamine and spermine were the main biogenic amines in fresh meat, while tryptamine and cadaverine were the most abundant at the end of the manufacturing process. During ripening the total amine content increased significantly (P < 0.05) in the batches made both without and with additives. The use of additives significantly (P < 0.05) increased the total amine content and the content of tryptamine, tyramine and histamine. The total biogenic amine content at the end of the manufacturing process was low as expected for a product in which there is little active microbial metabolism during manufacture. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Dry-cured laco´n; Biogenic amines; Additives; Ripening; Cured meat products
1. Introduction The biogenic amines are basic nitrogen compounds usually formed by the decarboxylation of their precursor amino acids (Hala´sz, Bara´th, Simon-Sarkadi, & Holzapfel, 1994; Janz, Scheiber, & Beutling, 1983; Silla Santos, 1996). The formation of biogenic amines in the foods has importance not only for its unfavourable effect on the flavour, but also from a sanitary point of view. Biogenic amines affect blood pressure and an excessive quantity in the foods can trigger in sensitive people migraines, gastric and intestinal problems and allergic responses (Smith, 1980; Stratton, Hutkins, & Taylor, 1991; Taylor, 1985). These substances are especially dangerous in people treated with inhibitors of the monoaminooxidase enzyme (Stratton et al., 1991). *
Corresponding author. Tel.: +34 988 387052; fax: +34 988 387001. E-mail address: [email protected] (J. Carballo).
0309-1740/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2007.03.020
During the ripening of meat products, the proteins undergo degradation; firstly, large peptides are generated that, later, are degraded into smaller peptides and free amino acids. The free amino acids can in turn be converted to such compounds as ammonia, a-ketoacids, methylketones and amines. In meat products, the formation of biogenic amines is above all related to the activity of the microorganisms present in the meat (Paulsen & Bauer, 1997; Shalaby, 1996; Ten Brink, Damink, Joosten, & Huis, 1990). In the case of cured products, high quantities of certain biogenic amines can be observed as a consequence of the use of poor quality raw materials, microbial contamination and inadequate conditions during processing and storage (Bover-Cid, Migue´lezArrizado, Latorre-Moratalla, & Vidal-Carou, 2006; Ten Brink et al., 1990). High temperatures, high pH values and low salt contents can favour the accumulation of free amino acids and, therefore, stimulate the formation of biogenic amines (Joosten, 1987), but during the drying-maturation
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stage, the factors that affect enzymatic decarboxylation could be more important than the availability of precursors (Edwards & Sandine, 1981; Joosten & Van Boeckel, 1988). Dry-cured ‘‘laco´n’’ is a traditional cured meat product made in the north-west of Spain from the fore leg of the pig cut at the shoulder blade-humerus joint, following manufacturing processes very similar to those used in the production of dry-cured ham. In the Galicia region it is recognised as a Geographically Protected Identity (G.P.I.) (Official Journal of the European Communities, 2001). Studies on biogenic amine contents in fermented sausages and on the factors that affect their formation are abundant in the literature. However information on this subject in drycured meat products made from entire pieces are limited. Only in ham have amine contents been determined during the manufacturing process (Alfaia et al., 2004; Co´rdoba et al., 1994) and in the final product (Herna´ndez-Jover, Izquierdo-Pulido, Veciana-Nogue´s, Marine´-Font, & VidalCarou, 1997a; Ruiz-Capillas & Jime´nez-Colmenero, 2004). The aim of this work, that forms part of a wider study dedicated to improvement of the quality of dry-cured laco´n, is to determine the content of biogenic amines during manufacture and ripening. This product is traditionally manufactured using only coarse salt, without any additives. However, nowadays some common additives are used in to improve the appearance and the quality of the final product (development of the typical colour of the cured meat and inhibition of mould growth on the surface). Thus the effect of these additives on biogenic amine generation throughout the manufacturing process was also studied. 2. Materials and methods 2.1. Samples Six batches of dry-cured laco´n were manufactured in three different factories, each one consisted of nine laco´n pieces. Fresh pieces weighing around 4 kg were used. In three batches (one per factory), raw pieces were salted with coarse salt, forming piles alternating between meat and salt; the pieces remained in the pile for four days (a day per kg of weight), the temperature of the salting room was between 2 and 5 °C and the relative humidity between 80% and 90%. After the salting stage, the pieces were taken from the pile, brushed, washed, and transferred to a postsalting room where they stayed for 14 days at a 2–5 °C and 85–90% relative humidity. After the post-salting stage the pieces were transferred to a room at 12 °C and 74–78% relative humidity where drying-ripening took place for 84 days. In the other three batches (one per factory), before the salting process, each piece was rubbed with glucose (2 g/kg), sodium nitrite (E250) (0.125 g/kg), sodium nitrate (E251) (0.175 g/kg), sodium ascorbate (E301) (0.5 g/kg), and sodium citrate (E331) (0.1 g/kg). In these batches, the salting, post-salting and drying-ripening stages were carried out as in the batches manufactured without additives.
In each batch, samples were taken from the fresh pieces, after the end of the salting stage, after 7 and 14 days of post-salting, and after 7, 14, 28, 56, and 84 days of drying-ripening. Each sample consisted of one whole laco´n piece. Samples were transported to the laboratory under refrigerated conditions (below 4 °C) and analysed on arrival. Once in the laboratory, the entire pieces were skinned and boned, and finally minced in a high-capacity mincer. 2.2. Biogenic amine analysis The extraction of the biogenic amines was carried out following the method described by Eerola, Hinnkanen, Lindfors, and Hirvi (1993). The separation, identification and quantification of the biogenic amines were carried out by HPLC techniques following the procedure described by Eerola et al. (1993), using a Spectra System chromatograph (Thermo Finnigan, San Jose´, CA, USA) equipped with a SCM 1000 degasser, a P4000 pump, an AS 3000 automatic injector and a Photodiode Array UV6000LP detector. The separation of the different biogenic amines was carried out in a reversed phase C18 mod. Kromasil 100 column (25 cm, 4 mm ID) (Teknokroma S. Coop. C. Ltda., San Cugat del Valle´s, Barcelona, Spain). The temperature of the column was 40 ± 1 °C and the wavelength of the detector 254 nm. The chromatographic conditions used are described in Table 1; an ammonium acetate 0.1 M solution was used as eluant A and acetonitrile as eluant B. A standard containing appropriate amounts of agmatine, tryptamine, 2-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, spermine and 1,7-diaminoheptane (as internal standard) was used to quantify the biogenic amines present in the samples. All the samples and standards were injected at least in duplicate. Repeatability tests were performed by injecting a standard and sample consecutively six times in a day. Reproducibility tests were also carried out by injecting the standard and the sample twice a day for three days under the same experimental conditions. Significant differences (P < 0.05) were not found between the results obtained in these tests. Each biogenic amine was expressed as mg/kg (ppm). 2.3. Chemical analysis Moisture, NaCl, nitrate and total carbohydrate contents, and pH and aw values were determined using the Table 1 Chromatographic conditions used in the determination of the biogenic amines Time (min)
Flow (mL/min)
% Eluant A
% Eluant B
0 19.00 20.00 30.00
1.00 1.00 1.00 1.00
50 10 50 50
50 90 50 50
129.03 ± 12.06b* 113.77 ± 14.23b 114.24 ± 12.35b 87.22 ± 10.92a Total
ND = Not detected. a–d Values in the same row (corresponding to the same biogenic amine) not followed by a common letter differ significantly (P < 0.05). * Values which were significantly different (P < 0.05) when compared the batches made without additives with those made with additives.
118.62 ± 16.80b* 130.42 ± 8.05b 137.78 ± 18.74b*
3.00 ± 5.20 59.29 ± 5.36b 14.09 ± 6.68c ND 11.87 ± 3.19a 4.38 ± 0.51a 3.80 ± 0.98abc 8.06 ± 1.16a 25.92 ± 2.88ab
2.75 ± 4.76 62.83 ± 2.42b* 22.08 ± 2.42b 1.21 ± 2.10ab 6.64 ± 2.72a 4.62 ± 0.48a 1.62 ± 1.84bc 8.27 ± 1.27a 27.74 ± 3.49a ND 53.64 ± 8.09bc 10.92 ± 6.11ac ND 4.34 ± 2.36a 4.07 ± 0.04a 4.75 ± 1.67ac 7.45 ± 0.23a 29.07 ± 1.37a ND 25.03 ± 11.57ad 3.64 ± 1.05a 3.88 ± 2.00ac 6.90 ± 3.21a 4.44 ± 0.66a 3.87 ± 1.21abc 7.14 ± 1.21a 32.30 ± 3.02a Agmatine Tryptamine 2-Phenylethylamine Putrescine Cadaverine Histamine Tyramine Spermidine Spermine
ND 62.14 ± 1.74b* 22.57 ± 1.77bc 0.70 ± 1.21b 9.50 ± 8.84a 4.11 ± 0.13a 1.32 ± 1.78bc 8.38 ± 0.70a 27.89 ± 7.32a
ND 55.66 ± 3.35bc* 20.32 ± 6.23c ND 6.60 ± 4.28a 4.45 ± 0.57a 1.22 ± 1.07b 7.43 ± 0.76a 26.36 ± 2.14ab
a
14
122.06 ± 6.71b*
9.95 ± 10.66 44.14 ± 4.74cd 4.33 ± 2.41a 0.69 ± 1.19b 12.89 ± 4.04a 4.19 ± 0.11a* 4.25 ± 1.25c 8.96 ± 0.62a 24.37 ± 2.45ab 6.79 ± 6.65 49.57 ± 13.50bcd 6.41 ± 4.66a 0.46 ± 0.79b 13.21 ± 15.50a* 4.25 ± 0.39a* 2.75 ± 1.82abc* 9.29 ± 0.74a 25.88 ± 3.66ab
56
a
28
a
7 7
14
Drying-ripening (days) Post-salting (days)
Tables 2 and 3 show the content of the biogenic amines during the manufacture of dry-cured laco´n made without and with additives, respectively. The content of each biogenic amine showed great variability at each sampling time which agrees with data reported by other authors in ham (Alfaia et al., 2004; Co´rdoba et al., 1994). Spermine and the tryptamine were the main biogenic amines in the fresh laco´n pieces. The spermine values in the fresh pieces agree with those reported by other authors for fresh meat (Cantoni, 1995; Herna´ndez-Jover, IzquierdoPulido, Veciana-Nogue´s, & Vidal-Carou, 1996a; Herna´ndez-Jover, Izquierdo-Pulido, Veciana-Nogue´s, & VidalCarou, 1996b; Herna´ndez-Jover et al., 1997a; Herna´ndezJover, Izquierdo-Pulido, Veciana-Nogue´s, Marine´-Font, & Vidal-Carou, 1997b; Maijala & Eerola, 1993; Maijala, Eerola, Aho, & Rin, 1993; Rogowski & Do¨hla, 1984), while the tryptamine values were higher than reported by other authors, some did not detect it (Cantoni, 1995; Co´rdoba et al., 1994; Herna´ndez-Jover et al., 1996a, 1996b, 1997a, 1997b). Quantitatively, the third biogenic amine in the fresh pieces was spermidine, which showed an average value of 7.00 mg/kg. This value is similar than that observed by Herna´ndez-Jover et al. (1996b) and slightly higher than those reported by other authors (Cantoni, 1995; Herna´ndez-Jover et al., 1996a, 1997a, 1997b; Maijala & Eerola, 1993; Maijala et al., 1993; Rogowski & Do¨hla, 1984) in fresh meat. The quantities of 2-phenylethylamine, putrescine, cadaverine, histamine and tyramine varied from 2.89 mg/kg to 6.90 mg/kg in the fresh pieces. These values are within the range found for fresh meat by Herna´ndez-Jover et al. (1996b), although in many other works these biogenic amines were not detected (Cantoni, 1995; Herna´ndez-Jover et al., 1997a, 1997b). The agmatine content in the fresh pieces was very low, not being detected in most of the pieces. This agrees with
After salting
3. Results and discussion
Fresh piece
In order to study significant differences between the different sampling points during the ripening process in the batches of the same manufacture, and between the two systems of manufacture at each sampling point, an analysis of variance (ANOVA) was performed, with a confidence interval of 95% (P < 0.05). Means were compared by the least squares difference (LSD) test, using the computer programme Statistica 5.1 for Windows (Statsoft Inc., 1996, Tulsa, OK, USA).
Table 2 Changes in biogenic amine content (expressed as mg/kg) during the manufacturing process of dry-cured laco´n made without additives (average values ± SD of three batches)
a
2.4. Statistical analysis
289
136.61 ± 19.18b*
84
methods cited by Lorenzo, Prieto, Carballo, and Franco (2003). All chemical determinations were made in duplicate on each sample.
7.70 ± 6.91a 36.92 ± 9.80d* 6.59 ± 3.81a 6.34 ± 1.98c 39.15 ± 3.44b 4.01 ± 0.06a* 2.14 ± 1.69abc* 7.20 ± 4.04a 18.71 ± 11.30b
J.M. Lorenzo et al. / Meat Science 77 (2007) 287–293
161.72 ± 12.06c 127.01 ± 14.67b 98.18 ± 12.35a 92.79 ± 14.06a Total
ND = Not detected. a–d Values in the same row (corresponding to the same biogenic amine) not followed by a common letter differ significantly (P < 0.05).
151.38 ± 24.37bc 113.97 ± 14.60a 100.58 ± 3.26a 92.31 ± 24.50a
3.06 ± 2.76 49.68 ± 3.10cd 14.68 ± 10.09bcd ND 5.20 ± 3.19a 4.16 ± 0.10a 3.50 ± 3.55abcd 7.83 ± 0.66abc 25.86 ± 3.97a ND 33.80 ± 2.71ab 16.71 ± 2.84bd ND 10.21 ± 7.93a 5.11 ± 0.90b 2.22 ± 2.15abd 7.07 ± 0.79abc 25.46 ± 3.76a ND 28.01 ± 8.56b 20.09 ± 13.39b ND 3.90 ± 2.02a 4.35 ± 0.49ab 3.41 ± 0.96abcd 7.09 ± 1.74abc 25.46 ± 4.50a ND 27.25 ± 8.88b 15.31 ± 9.12bcd 1.14 ± 1.98b 8.92 ± 10.40a 4.19 ± 0.12a 1.38 ± 0.88b 7.22 ± 0.48abc 22.39 ± 2.28a 1.36 ± 2.36 42.44 ± 4.32ac 10.86 ± 10.41abcd ND 5.34 ± 4.47a 4.22 ± 0.25ab 2.89 ± 1.08abd 6.47 ± 1.17a 24.58 ± 2.47a
3.11 ± 2.70 33.12 ± 8.81ab 3.95 ± 2.43acd 6.15 ± 2.85a 2.89 ± 1.57a 4.06 ± 0.08a 4.78 ± 2.25acd 6.88 ± 0.83ac 27.92 ± 5.87a Agmatine Tryptamine 2-Phenylethylamine Putrescine Cadaverine Histamine Tyramine Spermidine Spermine
87.81 ± 9.19a
7.78 ± 4.88 49.43 ± 15.69cd 10.94 ± 5.55abcd 1.03 ± 1.78b 30.98 ± 25.00b 5.32 ± 1.32b 6.11 ± 2.54cd 9.75 ± 2.53bc 30.03 ± 8.18a
56
a
28
a
14
14 7
a
a
Drying-ripening (days)
7
Post-salting (days) After salting Fresh piece
Table 3 Changes in biogenic amine content (expressed as mg/kg) during the manufacturing process of dry-cured laco´n made with additives (average values ± SD of three batches)
a
7.65 ± 7.97 50.90 ± 13.62cd 5.58 ± 3.83cd 3.64 ± 4.89ab 15.41 ± 11.37a 5.40 ± 0.72b 3.74 ± 1.40abcd 8.81 ± 0.80abc 25.87 ± 4.12a
9.74 ± 4.34a 57.50 ± 10.81d 7.57 ± 2.25d 6.67 ± 2.35a 35.55 ± 5.88b 5.94 ± 0.74b 5.12 ± 1.44d 9.46 ± 3.35c 24.15 ± 8.77a
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84
290
previous studies (Cantoni, 1995; Herna´ndez-Jover et al., 1996a, 1996b, 1997b; Maijala et al., 1993). At the end of the manufacturing process, the main biogenic amines were tryptamine and cadaverine which reached average values of 36.92 mg/kg and 39.15 mg/kg in the batches made without additives and of 57.50 mg/ kg and 35.55 mg/kg in the batches made with additives. The tryptamine content evolved differently in the batches made without and with additives. In the batches made without additives its content increased significantly (P < 0.05) after the salting process and then stayed around 60 mg/kg during the post-salting stage and the first two weeks of drying-ripening, then decreasing so that at the end of the process values that did not differ significantly from those in the fresh pieces. In the batches made with additives, the tryptamine content increased in significantly (P < 0.05) during the drying-ripening stage, reaching at the end of the manufacturing process values that were significantly higher than those observed in the batches made without additives. According to the available literature, tryptamine has not been quantified in cured hams. The high contents of tryptamine at the end of the process for laco´n could be related to the high tryptophan contents, the precursor amino acid for this amine, whose levels increased during maturation of this product by a factor of 6.50 with regard to the initial content (Lorenzo, 2006). Cadaverine was the only amine that during manufacture showed a significant increase (P < 0.05) in the two types (without and with additives). The presence of high cadaverine concentrations has been associated with poor quality meat used in the manufacture of the meat products and, mainly, with high levels of microbial contamination (Bover-Cid, Izquierdo-Pulido, & Vidal-Carou, 2001; Suzzi & Gardini, 2003). Among the microbial groups producing biogenic amines, Enterobacteriaceae seem to have an important role in cadaverine production, as high Enterobacteriaceae counts are directly related to high cadaverine concentrations (Bover-Cid, Izquierdo-Pulido, & Vidal-Carou, 2000; Suzzi & Gardini, 2003). During manufacture of the laco´n batches studied, Enterobacteriaceae have been only detected on the surface of the fresh pieces, none being found after salting (Lorenzo, Garcı´a Fonta´n, Franco, & Carballo, 2007), but it is at this stage (fresh piece) when they can liberate the enzymes responsible for the later formation of the biogenic amines. Some authors (Alfaia et al., 2004; Ruiz-Capillas & Jime´nez-Colmenero, 2004) have also cited cadaverine as one of the most abundant biogenic amines in hams at the end of the manufacturing process. The final average values of cadaverine in the laco´n were higher than those reported by other authors in ham (Alfaia et al., 2004; Co´rdoba et al., 1994; Ruiz-Capillas & Jime´nez-Colmenero, 2004), but much lower than found in other meat products such as sausages. The spermine and the spermidine appear naturally in fresh meat (Herna´ndez-Jover et al., 1997a) and they are
54.98 ± 2.86e 13.14 ± 5.58b 6.30 ± 0.23abc 0.885 ± 0.032d 70.78 ± 8.67c 0.102 ± 0.078e
*
a–f
C
D
B
A
Total solids (Expressed as g/100 g). Expressed as g/100 g of total solids. Expressed as ppm. Total carbohydrates (expressed as g of glucose/100 g of total solids). Values in the same row (corresponding to the same physico-chemical parameter) not followed by a common letter differ significantly (P < 0.05). Values which were significantly different (P < 0.05) when compared the batches made without additives with those made with additives.
50.83 ± 3.50de 12.44 ± 5.63b 6.38 ± 0.12c 0.910 ± 0.034cd 68.82 ± 8.48c 0.119 ± 0.092de 48.00 ± 3.23cd 12.62 ± 4.87b 6.26 ± 0.07bc 0.928 ± 0.037bcd 76.27 ± 8.83bc 0.164 ± 0.069de 44.06 ± 4.74bc 13.35 ± 3.50b 6.13 ± 0.27bc 0.940 ± 0.022bc 80.20 ± 12.58bc 0.218 ± 0.074bd 42.18 ± 5.21bc 13.26 ± 2.49 b 6.20 ± 0.19 bc 0.945 ± 0.023ef 88.04 ± 1.36b 0.320 ± 0.099c 38.73 ± 2.25b 11.34 ± 2.84b 6.23 ± 0.31bc 0.953 ± 0.024abc 83.33 ± 12.02b 0.280 ± 0.111bc 40.21 ± 2.61 b 8.33 ± 1.45b 6.02 ± 0.10bc 0.966 ± 0.011ab 50.39 ± 5.56a 0.200 ± 0.121ade With additives 28.72 ± 4.25a TSA NaClB 2.34 ± 0.31a pH 6.58 ± 0.10a aw 0.996 ± 0.001a NitrateC 47.25 ± 7.83a TCD 0.095 ± 0.038ade
40.45 ± 3.53b 11.06 ± 1.59b 6.32 ± 0.21abc 0.956 ± 0.016abc 85.69 ± 5.31b 0.201 ± 0.086bde
57.98 ± 2.81f 13.06 ± 4.90b 6.40 ± 0.22a 0.876 ± 0.075d 39.02 ± 6.04d* 0.028 ± 0.004a* 51.09 ± 2.97e 13.88 ± 4.19b 6.25 ± 0.09a 0.900 ± 0.059cd 31.96 ± 4.45cd* 0.036 ± 0.004a* 46.03 ± 2.92de 14.26 ± 3.79b 6.25 ± 0.07a 0.930 ± 0.025bc 24.12 ± 3.53bc* 0.052 ± 0.010a* 43.90 ± 4.40cd 12.76 ± 3.38b 6.34 ± 0.24a 0.944 ± 0.016bc 33.53 ± 3.11abcd* 0.065 ± 0.018a* 39.43 ± 2.08bc 14.33 ± 1.02b 6.16 ± 0.16a 0.947 ± 0.014bc 37.06 ± 4.24acd* 0.100 ± 0.014a* 40.08 ± 3.44bcd 13.34 ± 2.00b 6.24 ± 0.21a 0.951 ± 0.015ab 32.75 ± 4.75abcd* 0.120 ± 0.013a* 36.95 ± 5.16b 13.14 ± 1.70b 6.22 ± 0.06a 0.962 ± 0.003ab 35.88 ± 10.26abcd* 0.119 ± 0.013a 36.54 ± 5.72b 9.27 ± 1.82b 6.17 ± 0.22a 0.968 ± 0.003ab 44.51 ± 8.91ad 0.120 ± 0.018a
84 56 28 14 7
291
Without additives 29.66 ± 6.23a TSA NaClB 2.49 ± 0.32a pH 6.36 ± 0.27a aw 0.997 ± 0.003a NitrateC 37.45 ± 7.19acd TCD 0.107 ± 0.008a
Drying-ripening (days)
7
14 Post-salting (days) After salting Fresh piece
always present in the lean and fat used in the manufacture of the products. Their levels are not influenced by the curing process or by the microflora present (Komprda et al., 2004) and their content usually remains constant during the maturation processes. Spermine levels can even drop in raw-cured meat products compared to the raw materials from which they come, since some microorganisms can use this polyamine as a nitrogen source (Herna´ndez-Jover et al., 1997a; Tabor & Tabor, 1985). During the manufacture process of laco´n, the spermidine content remained practically constant, while the spermine level experienced a significant drop (P < 0.05) in the batches manufactured without additives, as also observed by Co´rdoba et al. (1994) in Iberian ham. Alfaia et al. (2004) observed a significant drop (P < 0.05) of both polyamines during the ripening of Portuguese ham. The spermine and spermidine contents at the end of the manufacturing process of laco´n were of the same order as observed by other authors in ham (Alfaia et al., 2004; Herna´ndez-Jover et al., 1997a; Ruiz-Capillas & Jime´nezColmenero, 2004). Histamine did not undergo significant variations during the manufacture of the laco´n batches made without additives. However, it increased significantly (P < 0.05) during the drying-ripening stage in the batches manufactured with additives, its content in the end product being significantly higher (P < 0.05) than observed in the pieces made without additives. The histamine values at the end of the manufacturing process were, nevertheless, very low and are in the range reported for raw-cured hams by Herna´ndez-Jover et al. (1997a), and by Alfaia et al. (2004) in Portuguese hams with a five months maturation period. Henry Chin and Koehler (1986) reported that at NaCl concentrations of 3.5–5.5%, histamine production could be inhibited. Some authors (Buncic et al., 1993; Maijala et al., 1993) have also related high histamine concentrations with an inadequate drop of pH in the first days of ripening. The pH values changed little during the manufacture of laco´n (Table 4). However, this product during the manufacturing process had NaCl values higher than 5.5% (Table 4), for which histamine production could be inhibited. Agmatine was generated during the drying-ripening stage in both types (without and with additives). The values reached in the final product (around 8 mg/kg) were higher than reported in ham (Ruiz-Capillas & Jime´nez-Colmenero, 2004). 2-Phenylethylamine is only sporadically found in rawcured meat products (Herna´ndez-Jover et al., 1997a; Koehler & Eitenmiller, 1978; Pechanek, Pfannhauser, & Woiduch, 1983). It is generally formed when there is a high tyramine concentration, and its presence can be related with a nonspecific activity of tyrosine decarboxylase (Joosten, 1987). This amine has not been reported in ham. In the present study, its content increased significantly (P < 0.05) during the salting and post-salting stages, and later dropped during the drying-ripening stage, so that at the end of the process similar values to those in the fresh pieces were observed.
Table 4 Values of some physico-chemical parameters during the manufacture process of dry-cured laco´n made without and with additives (average values ± SD of three batches in each manufacture type)
J.M. Lorenzo et al. / Meat Science 77 (2007) 287–293
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J.M. Lorenzo et al. / Meat Science 77 (2007) 287–293
Putrescine and tyramine did not modify their contents in a statistically significant way during the manufacture of the laco´n. At the end of the process, the content of tyramine was significantly higher (P < 0.05) in the batches made with additives than in those made without additives. The contents observed for putrescine and tyramine at the end of the process in both laco´n types are within the values reported in the literature for other raw-cured meat products (Alfaia et al., 2004; Cantoni, 1995; Herna´ndez-Jover et al., 1997a; Ruiz-Capillas & Jime´nez-Colmenero, 2004). The content of putrescine was, nevertheless, much lower than reported by Co´rdoba et al. (1994) in Iberian ham. During the manufacture of dry-curd laco´n, a significant increase (P < 0.05) was observed in the total biogenic amine content, in the batches made both without and with additives. In the batches made without additives the increase took place mainly during the salting process and in the first week of the post-salting stage, while in the batches manufactured with additives amine production occurred in the first four weeks of the drying-ripening stage. The increase in the total biogenic amine content during ripening, also found for other meat products, has been attributed to lactic acid bacteria (Roig-Sague´s, Herna´ndez-Herrero, Lo´pez-Sabater, Rodrı´guez-Jerez, & Mora Ventura, 1999) or to the residual activity of decarboxylases from Enterobacteriaceae (Bover-Cid et al., 2000). In our study it was observed that the total biogenic amine content at the end of manufacture was significantly (P < 0.05) higher in the batches manufactured with additives than in those made without additives. In a microbiological study of the laco´n batches used in this work (Lorenzo et al., 2007) it was observed that the counts of total lactic acid bacteria and of lactobacilli were higher in the batches made with additives, possibly due to the presence, among the additives, of glucose that could stimulate the growth of the lactic acid bacteria. Higher counts of lactic acid bacteria in the batches made with additives could explain the higher contents of total biogenic amines and of some individual amines such as tryptamine, histamine and tyramine at the end of manufacture in these batches. As expected in a product in which there is no true lactic fermentation, the total biogenic amine content at the end of manufacture of dry-cured laco´n was low. Acknowledgments The authors gratefully acknowledge the financial assistance of the Xunta de Galicia (The Regional Government) (Projects 38301B98 and PGIDT01PXI38301PR). Jose´ M. Lorenzo was supported by a Pre-doctoral fellowship from the Xunta de Galicia. References Alfaia, C. M., Castro, M. F., Reis, V. A., Prates, J. M., de Almeida, I. T., Correia, A. D., et al. (2004). Changes in the profile of free amino acids and biogenic amines during the extended short ripening of Portuguese
dry-cured ham. Food Science and Technology International, 10, 297–304. Bover-Cid, S., Izquierdo-Pulido, M., & Vidal-Carou, M. C. (2000). Influence of hygienic quality of raw material on biogenic amine production during ripening and storage of dry fermented sausages. Journal of Food Protection, 63, 1544–1550. Bover-Cid, S., Izquierdo-Pulido, M., & Vidal-Carou, M. C. (2001). Effect of the interaction between a low tyramine producing Lactobacillus and proteolytic staphylococci on biogenic amine production during ripening and storage of dry sausage. International Journal of Food Microbiology, 65, 113–125. Bover-Cid, S., Migue´lez-Arrizado, M., Latorre-Moratalla, L. L., & VidalCarou, M. C. (2006). Freezing of meat raw materials affects tyramine and diamine accumulation in spontaneously fermented sausages. Meat Science, 72, 62–68. Buncic, S., Paunovic, L., Teodorovic, V., Radisic, D., Vojinovic, G., Smiljanic, D., et al. (1993). Effects of gluconodeltalactone and Lactobacillus plantarum on the production of histamine and tyramine in fermented sausages. International Journal of Food Microbiology, 17, 303–309. Cantoni, C. (1995). Amine biogene nei prodotti carnei nazionali. Industrie Alimentari, 34, 9–12. Co´rdoba, J. J., Antequera Rojas, T., Garcı´a Gonza´lez, C., Ventanas Barroso, J., Lo´pez-Bote, C., & Asensio, M. A. (1994). Evolution of free amino acids and amines during ripening of Iberian cured ham. Journal of Agricultural and Food Chemistry, 42, 2296–2301. Edwards, S. T., & Sandine, W. E. (1981). Public health significance of amine in cheese. Journal of Dairy Science, 64, 2431–2438. Eerola, S., Hinnkanen, R., Lindfors, E., & Hirvi, T. K. (1993). Liquid chromatographic determination of biogenic amines in dry sausages. Journal of AOAC International, 76, 575–577. Hala´sz, A., Bara´th, A., Simon-Sarkadi, L., & Holzapfel, W. (1994). Biogenic amines and their production by microorganisms in food. Trends in Food Science and Technology, 5, 42–49. Henry Chin, K. D., & Koehler, P. E. (1986). Effects of salt concentration and incubation temperature on formation of histamine, phenylethylamine, tryptamine and tyramine during miso fermentation. Journal of Food Protection, 49, 423–427. Herna´ndez-Jover, T., Izquierdo-Pulido, M., Veciana-Nogue´s, M. T., & Vidal-Carou, M. C. (1996a). Biogenic amine sources in cooked cured shoulder pork. Journal of Agricultural and Food Chemistry, 44, 3097–3101. Herna´ndez-Jover, T., Izquierdo-Pulido, M., Veciana-Nogue´s, M. T., & Vidal-Carou, M. C. (1996b). Ion-pair high-performance liquid chromatographic determination of biogenic amines in meat and meat products. Journal of Agricultural and Food Chemistry, 44, 2710–2715. Herna´ndez-Jover, T., Izquierdo-Pulido, M., Veciana-Nogue´s, M. T., Marine´-Font, A., & Vidal-Carou, M. C. (1997a). Biogenic amine and polyamine contents in meat and meat products. Journal of Agricultural and Food Chemistry, 45, 2098–2102. Herna´ndez-Jover, T., Izquierdo-Pulido, M., Veciana-Nogue´s, M. T., Marine´-Font, A., & Vidal-Carou, M. C. (1997b). Effect of starter cultures on biogenic amine formation during fermented sausage production. Journal of Food Protection, 60, 825–830. Janz, V. I., Scheiber, G., & Beutling, D. (1983). Die lebensmittelhygienische bedeutung von histamin und tyramin. Mh Vet-med, 38, 701–709. Joosten, H. M. L. J. (1987). Conditions allowing the formation of biogenic amines in cheese: III. Factors influencing the amounts formed. Netherlands Milk and Dairy Journal, 41, 329–357. Joosten, H. M. L. J., & Van Boeckel, M. A. J. S. (1988). Conditions allowing the formation of biogenic amines in cheese: IV. A study of the kinetics of histamine formation in an infected Gouda cheese. Netherlands Milk and Dairy Journal, 42, 3–24. Koehler, P. E., & Eitenmiller, R. R. (1978). High-performance liquid chromatographic analysis of tyramine, phenylethylamine and tryptamine in sausage, cheese and chocolate. Journal of Food Science, 43, 344–346.
J.M. Lorenzo et al. / Meat Science 77 (2007) 287–293 Komprda, T., Smela, D., Pechova, P., Kalhotka, L., Stencl, J., & Klejdus, B. (2004). Effect of starter culture, spice mix and storage time and temperature on biogenic amine content of dry fermented sausages. Meat Science, 67, 607–616. Lorenzo, J. M. (2006). Study of the biochemical and microbiological changes which occur during the manufacture process of dry-cured laco´n. Effect of the use of some additives. Ph. Doctoral European Thesis, University of Vigo, Spain. Lorenzo, J. M., Garcı´a Fonta´n, M. C., Franco, I., & Carballo, J. (2007). Microbiological succession throughout the manufacture process of dry-cured laco´n. Effect of the use of additives. Fleischwirtschaft International, 2, 88–92. Lorenzo, J. M., Prieto, B., Carballo, J., & Franco, I. (2003). Compositional and degradative changes during the manufacture of dry-cured ‘‘laco´n’’. Journal of the Science of Food and Agriculture, 83, 593–601. Maijala, R., & Eerola, S. (1993). Contaminant lactic acid bacteria of dry sausages produce histamine and tyramine. Meat Science, 35, 387–395. Maijala, R., Eerola, S., Aho, S., & Rin, J. (1993). The effect of GDLinduced pH decrease on the formation of biogenic amines in meat. Journal of Food Protection, 56, 125–129. Official Journal of the European Communities (2001). Commission Regulation (EC) No 898/2001, of 7 May 2001. L 126, Vol. 44, 8 May 2001 (p. 18). Paulsen, P., & Bauer, F. (1997). Biogenic amines in fermented sausage: II. Formation of biogenic amines in raw fermented sausage. Fleischwirtschaft, 77, 362–364. Pechanek, U., Pfannhauser, U., & Woiduch, H. (1983). Utersuchung u¨ber den Gehalt biogener Amine in vier Gruppen von Lebensmitteln des o¨sterreichischen Markets. Zeitschrift fu¨r Lebensmittel Untersuchung und Forschung, 176, 335–340.
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Rogowski, B., & Do¨hla, I. (1984). Bestimmung und gehalt biogener amine in fleisch und fleischwaren. Lebensmittelchemie und Gerichtl Chemie, 38, 1–30. Roig-Sague´s, A. X., Herna´ndez-Herrero, M., Lo´pez-Sabater, E. I., Rodrı´guez-Jerez, J. J., & Mora Ventura, M. T. (1999). Microbiological events during the elaboration of ‘‘fuet’’, a Spanish ripened sausage. Relationship between the development of histidine and tyrosine decarboxilase containing bacteria and pH and water activity. European Food Research and Technology, 209, 108–112. Ruiz-Capillas, C., & Jime´nez-Colmenero, F. (2004). Biogenic amine content in Spanish retail market meat products treated with protective atmosphere and high pressure. European Food Research and Technology, 218, 237–241. Shalaby, A. R. (1996). Significance of biogenic amines to food safety and human health. Food Research International, 29, 675–690. Silla Santos, M. H. (1996). Biogenic amines: Their importance in foods. International Journal of Food Microbiology, 29, 213–231. Smith, T. A. (1980). Amines in food. Food Chemistry, 6, 169–200. Stratton, J. E., Hutkins, R. W., & Taylor, S. L. (1991). Biogenic amines in cheese and other fermented foods: A review. Journal of Food Protection, 54, 460–470. Suzzi, G., & Gardini, F. (2003). Biogenic amines in dry fermented sausages: A review. International Journal of Food Microbiology, 88, 41–54. Tabor, C. W., & Tabor, H. (1985). Polyamines in microorganisms. Microbiological Reviews, 49, 81–99. Taylor, S. L. (1985). Histamine poisoning associated with fish, cheese, and other foods. World Health Organization. WPH/FOS, 85, 1–47. Ten Brink, B., Damink, C., Joosten, H. M. L. J., & Huis, J. H. J. (1990). Occurrence and formation of biologically active amines in foods. International Journal of Food Microbiology, 11, 73–84.
Short Communication
Biogenic amine content during the manufacture of dry-cured laco´n, a Spanish traditional meat product: Effect of some additives Jose´ M. Lorenzo, Sidonia Martı´nez, Inmaculada Franco, Javier Carballo
*
A´rea de Tecnologı´a de los Alimentos, Facultad de Ciencias de Orense, Universidad de Vigo, 32004 Orense, Spain Received 14 December 2006; received in revised form 14 March 2007; accepted 22 March 2007
Abstract The content of nine biogenic amines (agmatine, tryptamine, 2-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine) was determined throughout the manufacture of dry-cured laco´n, a traditional dry-salted and ripened meat product made in the north-west of Spain from the fore leg of the pig following a similar process to that of dry-cured ham. The effect of the use of additives (glucose, sodium nitrite, sodium nitrate, sodium ascorbate and sodium citrate) on the biogenic amine content during manufacture was also studied. Tryptamine and spermine were the main biogenic amines in fresh meat, while tryptamine and cadaverine were the most abundant at the end of the manufacturing process. During ripening the total amine content increased significantly (P < 0.05) in the batches made both without and with additives. The use of additives significantly (P < 0.05) increased the total amine content and the content of tryptamine, tyramine and histamine. The total biogenic amine content at the end of the manufacturing process was low as expected for a product in which there is little active microbial metabolism during manufacture. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Dry-cured laco´n; Biogenic amines; Additives; Ripening; Cured meat products
1. Introduction The biogenic amines are basic nitrogen compounds usually formed by the decarboxylation of their precursor amino acids (Hala´sz, Bara´th, Simon-Sarkadi, & Holzapfel, 1994; Janz, Scheiber, & Beutling, 1983; Silla Santos, 1996). The formation of biogenic amines in the foods has importance not only for its unfavourable effect on the flavour, but also from a sanitary point of view. Biogenic amines affect blood pressure and an excessive quantity in the foods can trigger in sensitive people migraines, gastric and intestinal problems and allergic responses (Smith, 1980; Stratton, Hutkins, & Taylor, 1991; Taylor, 1985). These substances are especially dangerous in people treated with inhibitors of the monoaminooxidase enzyme (Stratton et al., 1991). *
Corresponding author. Tel.: +34 988 387052; fax: +34 988 387001. E-mail address: [email protected] (J. Carballo).
0309-1740/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2007.03.020
During the ripening of meat products, the proteins undergo degradation; firstly, large peptides are generated that, later, are degraded into smaller peptides and free amino acids. The free amino acids can in turn be converted to such compounds as ammonia, a-ketoacids, methylketones and amines. In meat products, the formation of biogenic amines is above all related to the activity of the microorganisms present in the meat (Paulsen & Bauer, 1997; Shalaby, 1996; Ten Brink, Damink, Joosten, & Huis, 1990). In the case of cured products, high quantities of certain biogenic amines can be observed as a consequence of the use of poor quality raw materials, microbial contamination and inadequate conditions during processing and storage (Bover-Cid, Migue´lezArrizado, Latorre-Moratalla, & Vidal-Carou, 2006; Ten Brink et al., 1990). High temperatures, high pH values and low salt contents can favour the accumulation of free amino acids and, therefore, stimulate the formation of biogenic amines (Joosten, 1987), but during the drying-maturation
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stage, the factors that affect enzymatic decarboxylation could be more important than the availability of precursors (Edwards & Sandine, 1981; Joosten & Van Boeckel, 1988). Dry-cured ‘‘laco´n’’ is a traditional cured meat product made in the north-west of Spain from the fore leg of the pig cut at the shoulder blade-humerus joint, following manufacturing processes very similar to those used in the production of dry-cured ham. In the Galicia region it is recognised as a Geographically Protected Identity (G.P.I.) (Official Journal of the European Communities, 2001). Studies on biogenic amine contents in fermented sausages and on the factors that affect their formation are abundant in the literature. However information on this subject in drycured meat products made from entire pieces are limited. Only in ham have amine contents been determined during the manufacturing process (Alfaia et al., 2004; Co´rdoba et al., 1994) and in the final product (Herna´ndez-Jover, Izquierdo-Pulido, Veciana-Nogue´s, Marine´-Font, & VidalCarou, 1997a; Ruiz-Capillas & Jime´nez-Colmenero, 2004). The aim of this work, that forms part of a wider study dedicated to improvement of the quality of dry-cured laco´n, is to determine the content of biogenic amines during manufacture and ripening. This product is traditionally manufactured using only coarse salt, without any additives. However, nowadays some common additives are used in to improve the appearance and the quality of the final product (development of the typical colour of the cured meat and inhibition of mould growth on the surface). Thus the effect of these additives on biogenic amine generation throughout the manufacturing process was also studied. 2. Materials and methods 2.1. Samples Six batches of dry-cured laco´n were manufactured in three different factories, each one consisted of nine laco´n pieces. Fresh pieces weighing around 4 kg were used. In three batches (one per factory), raw pieces were salted with coarse salt, forming piles alternating between meat and salt; the pieces remained in the pile for four days (a day per kg of weight), the temperature of the salting room was between 2 and 5 °C and the relative humidity between 80% and 90%. After the salting stage, the pieces were taken from the pile, brushed, washed, and transferred to a postsalting room where they stayed for 14 days at a 2–5 °C and 85–90% relative humidity. After the post-salting stage the pieces were transferred to a room at 12 °C and 74–78% relative humidity where drying-ripening took place for 84 days. In the other three batches (one per factory), before the salting process, each piece was rubbed with glucose (2 g/kg), sodium nitrite (E250) (0.125 g/kg), sodium nitrate (E251) (0.175 g/kg), sodium ascorbate (E301) (0.5 g/kg), and sodium citrate (E331) (0.1 g/kg). In these batches, the salting, post-salting and drying-ripening stages were carried out as in the batches manufactured without additives.
In each batch, samples were taken from the fresh pieces, after the end of the salting stage, after 7 and 14 days of post-salting, and after 7, 14, 28, 56, and 84 days of drying-ripening. Each sample consisted of one whole laco´n piece. Samples were transported to the laboratory under refrigerated conditions (below 4 °C) and analysed on arrival. Once in the laboratory, the entire pieces were skinned and boned, and finally minced in a high-capacity mincer. 2.2. Biogenic amine analysis The extraction of the biogenic amines was carried out following the method described by Eerola, Hinnkanen, Lindfors, and Hirvi (1993). The separation, identification and quantification of the biogenic amines were carried out by HPLC techniques following the procedure described by Eerola et al. (1993), using a Spectra System chromatograph (Thermo Finnigan, San Jose´, CA, USA) equipped with a SCM 1000 degasser, a P4000 pump, an AS 3000 automatic injector and a Photodiode Array UV6000LP detector. The separation of the different biogenic amines was carried out in a reversed phase C18 mod. Kromasil 100 column (25 cm, 4 mm ID) (Teknokroma S. Coop. C. Ltda., San Cugat del Valle´s, Barcelona, Spain). The temperature of the column was 40 ± 1 °C and the wavelength of the detector 254 nm. The chromatographic conditions used are described in Table 1; an ammonium acetate 0.1 M solution was used as eluant A and acetonitrile as eluant B. A standard containing appropriate amounts of agmatine, tryptamine, 2-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, spermine and 1,7-diaminoheptane (as internal standard) was used to quantify the biogenic amines present in the samples. All the samples and standards were injected at least in duplicate. Repeatability tests were performed by injecting a standard and sample consecutively six times in a day. Reproducibility tests were also carried out by injecting the standard and the sample twice a day for three days under the same experimental conditions. Significant differences (P < 0.05) were not found between the results obtained in these tests. Each biogenic amine was expressed as mg/kg (ppm). 2.3. Chemical analysis Moisture, NaCl, nitrate and total carbohydrate contents, and pH and aw values were determined using the Table 1 Chromatographic conditions used in the determination of the biogenic amines Time (min)
Flow (mL/min)
% Eluant A
% Eluant B
0 19.00 20.00 30.00
1.00 1.00 1.00 1.00
50 10 50 50
50 90 50 50
129.03 ± 12.06b* 113.77 ± 14.23b 114.24 ± 12.35b 87.22 ± 10.92a Total
ND = Not detected. a–d Values in the same row (corresponding to the same biogenic amine) not followed by a common letter differ significantly (P < 0.05). * Values which were significantly different (P < 0.05) when compared the batches made without additives with those made with additives.
118.62 ± 16.80b* 130.42 ± 8.05b 137.78 ± 18.74b*
3.00 ± 5.20 59.29 ± 5.36b 14.09 ± 6.68c ND 11.87 ± 3.19a 4.38 ± 0.51a 3.80 ± 0.98abc 8.06 ± 1.16a 25.92 ± 2.88ab
2.75 ± 4.76 62.83 ± 2.42b* 22.08 ± 2.42b 1.21 ± 2.10ab 6.64 ± 2.72a 4.62 ± 0.48a 1.62 ± 1.84bc 8.27 ± 1.27a 27.74 ± 3.49a ND 53.64 ± 8.09bc 10.92 ± 6.11ac ND 4.34 ± 2.36a 4.07 ± 0.04a 4.75 ± 1.67ac 7.45 ± 0.23a 29.07 ± 1.37a ND 25.03 ± 11.57ad 3.64 ± 1.05a 3.88 ± 2.00ac 6.90 ± 3.21a 4.44 ± 0.66a 3.87 ± 1.21abc 7.14 ± 1.21a 32.30 ± 3.02a Agmatine Tryptamine 2-Phenylethylamine Putrescine Cadaverine Histamine Tyramine Spermidine Spermine
ND 62.14 ± 1.74b* 22.57 ± 1.77bc 0.70 ± 1.21b 9.50 ± 8.84a 4.11 ± 0.13a 1.32 ± 1.78bc 8.38 ± 0.70a 27.89 ± 7.32a
ND 55.66 ± 3.35bc* 20.32 ± 6.23c ND 6.60 ± 4.28a 4.45 ± 0.57a 1.22 ± 1.07b 7.43 ± 0.76a 26.36 ± 2.14ab
a
14
122.06 ± 6.71b*
9.95 ± 10.66 44.14 ± 4.74cd 4.33 ± 2.41a 0.69 ± 1.19b 12.89 ± 4.04a 4.19 ± 0.11a* 4.25 ± 1.25c 8.96 ± 0.62a 24.37 ± 2.45ab 6.79 ± 6.65 49.57 ± 13.50bcd 6.41 ± 4.66a 0.46 ± 0.79b 13.21 ± 15.50a* 4.25 ± 0.39a* 2.75 ± 1.82abc* 9.29 ± 0.74a 25.88 ± 3.66ab
56
a
28
a
7 7
14
Drying-ripening (days) Post-salting (days)
Tables 2 and 3 show the content of the biogenic amines during the manufacture of dry-cured laco´n made without and with additives, respectively. The content of each biogenic amine showed great variability at each sampling time which agrees with data reported by other authors in ham (Alfaia et al., 2004; Co´rdoba et al., 1994). Spermine and the tryptamine were the main biogenic amines in the fresh laco´n pieces. The spermine values in the fresh pieces agree with those reported by other authors for fresh meat (Cantoni, 1995; Herna´ndez-Jover, IzquierdoPulido, Veciana-Nogue´s, & Vidal-Carou, 1996a; Herna´ndez-Jover, Izquierdo-Pulido, Veciana-Nogue´s, & VidalCarou, 1996b; Herna´ndez-Jover et al., 1997a; Herna´ndezJover, Izquierdo-Pulido, Veciana-Nogue´s, Marine´-Font, & Vidal-Carou, 1997b; Maijala & Eerola, 1993; Maijala, Eerola, Aho, & Rin, 1993; Rogowski & Do¨hla, 1984), while the tryptamine values were higher than reported by other authors, some did not detect it (Cantoni, 1995; Co´rdoba et al., 1994; Herna´ndez-Jover et al., 1996a, 1996b, 1997a, 1997b). Quantitatively, the third biogenic amine in the fresh pieces was spermidine, which showed an average value of 7.00 mg/kg. This value is similar than that observed by Herna´ndez-Jover et al. (1996b) and slightly higher than those reported by other authors (Cantoni, 1995; Herna´ndez-Jover et al., 1996a, 1997a, 1997b; Maijala & Eerola, 1993; Maijala et al., 1993; Rogowski & Do¨hla, 1984) in fresh meat. The quantities of 2-phenylethylamine, putrescine, cadaverine, histamine and tyramine varied from 2.89 mg/kg to 6.90 mg/kg in the fresh pieces. These values are within the range found for fresh meat by Herna´ndez-Jover et al. (1996b), although in many other works these biogenic amines were not detected (Cantoni, 1995; Herna´ndez-Jover et al., 1997a, 1997b). The agmatine content in the fresh pieces was very low, not being detected in most of the pieces. This agrees with
After salting
3. Results and discussion
Fresh piece
In order to study significant differences between the different sampling points during the ripening process in the batches of the same manufacture, and between the two systems of manufacture at each sampling point, an analysis of variance (ANOVA) was performed, with a confidence interval of 95% (P < 0.05). Means were compared by the least squares difference (LSD) test, using the computer programme Statistica 5.1 for Windows (Statsoft Inc., 1996, Tulsa, OK, USA).
Table 2 Changes in biogenic amine content (expressed as mg/kg) during the manufacturing process of dry-cured laco´n made without additives (average values ± SD of three batches)
a
2.4. Statistical analysis
289
136.61 ± 19.18b*
84
methods cited by Lorenzo, Prieto, Carballo, and Franco (2003). All chemical determinations were made in duplicate on each sample.
7.70 ± 6.91a 36.92 ± 9.80d* 6.59 ± 3.81a 6.34 ± 1.98c 39.15 ± 3.44b 4.01 ± 0.06a* 2.14 ± 1.69abc* 7.20 ± 4.04a 18.71 ± 11.30b
J.M. Lorenzo et al. / Meat Science 77 (2007) 287–293
161.72 ± 12.06c 127.01 ± 14.67b 98.18 ± 12.35a 92.79 ± 14.06a Total
ND = Not detected. a–d Values in the same row (corresponding to the same biogenic amine) not followed by a common letter differ significantly (P < 0.05).
151.38 ± 24.37bc 113.97 ± 14.60a 100.58 ± 3.26a 92.31 ± 24.50a
3.06 ± 2.76 49.68 ± 3.10cd 14.68 ± 10.09bcd ND 5.20 ± 3.19a 4.16 ± 0.10a 3.50 ± 3.55abcd 7.83 ± 0.66abc 25.86 ± 3.97a ND 33.80 ± 2.71ab 16.71 ± 2.84bd ND 10.21 ± 7.93a 5.11 ± 0.90b 2.22 ± 2.15abd 7.07 ± 0.79abc 25.46 ± 3.76a ND 28.01 ± 8.56b 20.09 ± 13.39b ND 3.90 ± 2.02a 4.35 ± 0.49ab 3.41 ± 0.96abcd 7.09 ± 1.74abc 25.46 ± 4.50a ND 27.25 ± 8.88b 15.31 ± 9.12bcd 1.14 ± 1.98b 8.92 ± 10.40a 4.19 ± 0.12a 1.38 ± 0.88b 7.22 ± 0.48abc 22.39 ± 2.28a 1.36 ± 2.36 42.44 ± 4.32ac 10.86 ± 10.41abcd ND 5.34 ± 4.47a 4.22 ± 0.25ab 2.89 ± 1.08abd 6.47 ± 1.17a 24.58 ± 2.47a
3.11 ± 2.70 33.12 ± 8.81ab 3.95 ± 2.43acd 6.15 ± 2.85a 2.89 ± 1.57a 4.06 ± 0.08a 4.78 ± 2.25acd 6.88 ± 0.83ac 27.92 ± 5.87a Agmatine Tryptamine 2-Phenylethylamine Putrescine Cadaverine Histamine Tyramine Spermidine Spermine
87.81 ± 9.19a
7.78 ± 4.88 49.43 ± 15.69cd 10.94 ± 5.55abcd 1.03 ± 1.78b 30.98 ± 25.00b 5.32 ± 1.32b 6.11 ± 2.54cd 9.75 ± 2.53bc 30.03 ± 8.18a
56
a
28
a
14
14 7
a
a
Drying-ripening (days)
7
Post-salting (days) After salting Fresh piece
Table 3 Changes in biogenic amine content (expressed as mg/kg) during the manufacturing process of dry-cured laco´n made with additives (average values ± SD of three batches)
a
7.65 ± 7.97 50.90 ± 13.62cd 5.58 ± 3.83cd 3.64 ± 4.89ab 15.41 ± 11.37a 5.40 ± 0.72b 3.74 ± 1.40abcd 8.81 ± 0.80abc 25.87 ± 4.12a
9.74 ± 4.34a 57.50 ± 10.81d 7.57 ± 2.25d 6.67 ± 2.35a 35.55 ± 5.88b 5.94 ± 0.74b 5.12 ± 1.44d 9.46 ± 3.35c 24.15 ± 8.77a
J.M. Lorenzo et al. / Meat Science 77 (2007) 287–293
84
290
previous studies (Cantoni, 1995; Herna´ndez-Jover et al., 1996a, 1996b, 1997b; Maijala et al., 1993). At the end of the manufacturing process, the main biogenic amines were tryptamine and cadaverine which reached average values of 36.92 mg/kg and 39.15 mg/kg in the batches made without additives and of 57.50 mg/ kg and 35.55 mg/kg in the batches made with additives. The tryptamine content evolved differently in the batches made without and with additives. In the batches made without additives its content increased significantly (P < 0.05) after the salting process and then stayed around 60 mg/kg during the post-salting stage and the first two weeks of drying-ripening, then decreasing so that at the end of the process values that did not differ significantly from those in the fresh pieces. In the batches made with additives, the tryptamine content increased in significantly (P < 0.05) during the drying-ripening stage, reaching at the end of the manufacturing process values that were significantly higher than those observed in the batches made without additives. According to the available literature, tryptamine has not been quantified in cured hams. The high contents of tryptamine at the end of the process for laco´n could be related to the high tryptophan contents, the precursor amino acid for this amine, whose levels increased during maturation of this product by a factor of 6.50 with regard to the initial content (Lorenzo, 2006). Cadaverine was the only amine that during manufacture showed a significant increase (P < 0.05) in the two types (without and with additives). The presence of high cadaverine concentrations has been associated with poor quality meat used in the manufacture of the meat products and, mainly, with high levels of microbial contamination (Bover-Cid, Izquierdo-Pulido, & Vidal-Carou, 2001; Suzzi & Gardini, 2003). Among the microbial groups producing biogenic amines, Enterobacteriaceae seem to have an important role in cadaverine production, as high Enterobacteriaceae counts are directly related to high cadaverine concentrations (Bover-Cid, Izquierdo-Pulido, & Vidal-Carou, 2000; Suzzi & Gardini, 2003). During manufacture of the laco´n batches studied, Enterobacteriaceae have been only detected on the surface of the fresh pieces, none being found after salting (Lorenzo, Garcı´a Fonta´n, Franco, & Carballo, 2007), but it is at this stage (fresh piece) when they can liberate the enzymes responsible for the later formation of the biogenic amines. Some authors (Alfaia et al., 2004; Ruiz-Capillas & Jime´nez-Colmenero, 2004) have also cited cadaverine as one of the most abundant biogenic amines in hams at the end of the manufacturing process. The final average values of cadaverine in the laco´n were higher than those reported by other authors in ham (Alfaia et al., 2004; Co´rdoba et al., 1994; Ruiz-Capillas & Jime´nez-Colmenero, 2004), but much lower than found in other meat products such as sausages. The spermine and the spermidine appear naturally in fresh meat (Herna´ndez-Jover et al., 1997a) and they are
54.98 ± 2.86e 13.14 ± 5.58b 6.30 ± 0.23abc 0.885 ± 0.032d 70.78 ± 8.67c 0.102 ± 0.078e
*
a–f
C
D
B
A
Total solids (Expressed as g/100 g). Expressed as g/100 g of total solids. Expressed as ppm. Total carbohydrates (expressed as g of glucose/100 g of total solids). Values in the same row (corresponding to the same physico-chemical parameter) not followed by a common letter differ significantly (P < 0.05). Values which were significantly different (P < 0.05) when compared the batches made without additives with those made with additives.
50.83 ± 3.50de 12.44 ± 5.63b 6.38 ± 0.12c 0.910 ± 0.034cd 68.82 ± 8.48c 0.119 ± 0.092de 48.00 ± 3.23cd 12.62 ± 4.87b 6.26 ± 0.07bc 0.928 ± 0.037bcd 76.27 ± 8.83bc 0.164 ± 0.069de 44.06 ± 4.74bc 13.35 ± 3.50b 6.13 ± 0.27bc 0.940 ± 0.022bc 80.20 ± 12.58bc 0.218 ± 0.074bd 42.18 ± 5.21bc 13.26 ± 2.49 b 6.20 ± 0.19 bc 0.945 ± 0.023ef 88.04 ± 1.36b 0.320 ± 0.099c 38.73 ± 2.25b 11.34 ± 2.84b 6.23 ± 0.31bc 0.953 ± 0.024abc 83.33 ± 12.02b 0.280 ± 0.111bc 40.21 ± 2.61 b 8.33 ± 1.45b 6.02 ± 0.10bc 0.966 ± 0.011ab 50.39 ± 5.56a 0.200 ± 0.121ade With additives 28.72 ± 4.25a TSA NaClB 2.34 ± 0.31a pH 6.58 ± 0.10a aw 0.996 ± 0.001a NitrateC 47.25 ± 7.83a TCD 0.095 ± 0.038ade
40.45 ± 3.53b 11.06 ± 1.59b 6.32 ± 0.21abc 0.956 ± 0.016abc 85.69 ± 5.31b 0.201 ± 0.086bde
57.98 ± 2.81f 13.06 ± 4.90b 6.40 ± 0.22a 0.876 ± 0.075d 39.02 ± 6.04d* 0.028 ± 0.004a* 51.09 ± 2.97e 13.88 ± 4.19b 6.25 ± 0.09a 0.900 ± 0.059cd 31.96 ± 4.45cd* 0.036 ± 0.004a* 46.03 ± 2.92de 14.26 ± 3.79b 6.25 ± 0.07a 0.930 ± 0.025bc 24.12 ± 3.53bc* 0.052 ± 0.010a* 43.90 ± 4.40cd 12.76 ± 3.38b 6.34 ± 0.24a 0.944 ± 0.016bc 33.53 ± 3.11abcd* 0.065 ± 0.018a* 39.43 ± 2.08bc 14.33 ± 1.02b 6.16 ± 0.16a 0.947 ± 0.014bc 37.06 ± 4.24acd* 0.100 ± 0.014a* 40.08 ± 3.44bcd 13.34 ± 2.00b 6.24 ± 0.21a 0.951 ± 0.015ab 32.75 ± 4.75abcd* 0.120 ± 0.013a* 36.95 ± 5.16b 13.14 ± 1.70b 6.22 ± 0.06a 0.962 ± 0.003ab 35.88 ± 10.26abcd* 0.119 ± 0.013a 36.54 ± 5.72b 9.27 ± 1.82b 6.17 ± 0.22a 0.968 ± 0.003ab 44.51 ± 8.91ad 0.120 ± 0.018a
84 56 28 14 7
291
Without additives 29.66 ± 6.23a TSA NaClB 2.49 ± 0.32a pH 6.36 ± 0.27a aw 0.997 ± 0.003a NitrateC 37.45 ± 7.19acd TCD 0.107 ± 0.008a
Drying-ripening (days)
7
14 Post-salting (days) After salting Fresh piece
always present in the lean and fat used in the manufacture of the products. Their levels are not influenced by the curing process or by the microflora present (Komprda et al., 2004) and their content usually remains constant during the maturation processes. Spermine levels can even drop in raw-cured meat products compared to the raw materials from which they come, since some microorganisms can use this polyamine as a nitrogen source (Herna´ndez-Jover et al., 1997a; Tabor & Tabor, 1985). During the manufacture process of laco´n, the spermidine content remained practically constant, while the spermine level experienced a significant drop (P < 0.05) in the batches manufactured without additives, as also observed by Co´rdoba et al. (1994) in Iberian ham. Alfaia et al. (2004) observed a significant drop (P < 0.05) of both polyamines during the ripening of Portuguese ham. The spermine and spermidine contents at the end of the manufacturing process of laco´n were of the same order as observed by other authors in ham (Alfaia et al., 2004; Herna´ndez-Jover et al., 1997a; Ruiz-Capillas & Jime´nezColmenero, 2004). Histamine did not undergo significant variations during the manufacture of the laco´n batches made without additives. However, it increased significantly (P < 0.05) during the drying-ripening stage in the batches manufactured with additives, its content in the end product being significantly higher (P < 0.05) than observed in the pieces made without additives. The histamine values at the end of the manufacturing process were, nevertheless, very low and are in the range reported for raw-cured hams by Herna´ndez-Jover et al. (1997a), and by Alfaia et al. (2004) in Portuguese hams with a five months maturation period. Henry Chin and Koehler (1986) reported that at NaCl concentrations of 3.5–5.5%, histamine production could be inhibited. Some authors (Buncic et al., 1993; Maijala et al., 1993) have also related high histamine concentrations with an inadequate drop of pH in the first days of ripening. The pH values changed little during the manufacture of laco´n (Table 4). However, this product during the manufacturing process had NaCl values higher than 5.5% (Table 4), for which histamine production could be inhibited. Agmatine was generated during the drying-ripening stage in both types (without and with additives). The values reached in the final product (around 8 mg/kg) were higher than reported in ham (Ruiz-Capillas & Jime´nez-Colmenero, 2004). 2-Phenylethylamine is only sporadically found in rawcured meat products (Herna´ndez-Jover et al., 1997a; Koehler & Eitenmiller, 1978; Pechanek, Pfannhauser, & Woiduch, 1983). It is generally formed when there is a high tyramine concentration, and its presence can be related with a nonspecific activity of tyrosine decarboxylase (Joosten, 1987). This amine has not been reported in ham. In the present study, its content increased significantly (P < 0.05) during the salting and post-salting stages, and later dropped during the drying-ripening stage, so that at the end of the process similar values to those in the fresh pieces were observed.
Table 4 Values of some physico-chemical parameters during the manufacture process of dry-cured laco´n made without and with additives (average values ± SD of three batches in each manufacture type)
J.M. Lorenzo et al. / Meat Science 77 (2007) 287–293
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J.M. Lorenzo et al. / Meat Science 77 (2007) 287–293
Putrescine and tyramine did not modify their contents in a statistically significant way during the manufacture of the laco´n. At the end of the process, the content of tyramine was significantly higher (P < 0.05) in the batches made with additives than in those made without additives. The contents observed for putrescine and tyramine at the end of the process in both laco´n types are within the values reported in the literature for other raw-cured meat products (Alfaia et al., 2004; Cantoni, 1995; Herna´ndez-Jover et al., 1997a; Ruiz-Capillas & Jime´nez-Colmenero, 2004). The content of putrescine was, nevertheless, much lower than reported by Co´rdoba et al. (1994) in Iberian ham. During the manufacture of dry-curd laco´n, a significant increase (P < 0.05) was observed in the total biogenic amine content, in the batches made both without and with additives. In the batches made without additives the increase took place mainly during the salting process and in the first week of the post-salting stage, while in the batches manufactured with additives amine production occurred in the first four weeks of the drying-ripening stage. The increase in the total biogenic amine content during ripening, also found for other meat products, has been attributed to lactic acid bacteria (Roig-Sague´s, Herna´ndez-Herrero, Lo´pez-Sabater, Rodrı´guez-Jerez, & Mora Ventura, 1999) or to the residual activity of decarboxylases from Enterobacteriaceae (Bover-Cid et al., 2000). In our study it was observed that the total biogenic amine content at the end of manufacture was significantly (P < 0.05) higher in the batches manufactured with additives than in those made without additives. In a microbiological study of the laco´n batches used in this work (Lorenzo et al., 2007) it was observed that the counts of total lactic acid bacteria and of lactobacilli were higher in the batches made with additives, possibly due to the presence, among the additives, of glucose that could stimulate the growth of the lactic acid bacteria. Higher counts of lactic acid bacteria in the batches made with additives could explain the higher contents of total biogenic amines and of some individual amines such as tryptamine, histamine and tyramine at the end of manufacture in these batches. As expected in a product in which there is no true lactic fermentation, the total biogenic amine content at the end of manufacture of dry-cured laco´n was low. Acknowledgments The authors gratefully acknowledge the financial assistance of the Xunta de Galicia (The Regional Government) (Projects 38301B98 and PGIDT01PXI38301PR). Jose´ M. Lorenzo was supported by a Pre-doctoral fellowship from the Xunta de Galicia. References Alfaia, C. M., Castro, M. F., Reis, V. A., Prates, J. M., de Almeida, I. T., Correia, A. D., et al. (2004). Changes in the profile of free amino acids and biogenic amines during the extended short ripening of Portuguese
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