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Influence of starter cultures and sugar concentrations on biogenic amine contents in chorizo dry sausage
FOOD MICROBIOLOGY Food Microbiology 20 (2003) 275–284
www.elsevier.nl/locate/jnlabr/yfmic
Influence of starter cultures and sugar concentrations on biogenic amine contents in chorizo dry sausage Consuelo Gonza! lez-Ferna! ndez, E.M. Santos, Isabel Jaime, Jordi Rovira* Departamento de Tecnolog!ıa de los Alimentos, Facultad de Ciencias, Universidad de Burgos, Plaza Misael Banuelos * s/n, 09001 Burgos, Spain Received 23 April 2002; received in revised form 12 November 2002; accepted 12 November 2002
Abstract The production of biogenic amines in chorizo dry sausage by using different decarboxylase negative starter cultures and different concentrations of sugar added in its manufacture has been studied. Biogenic amines were determined as dansyl derivates by highperformance liquid chromatography (HPLC). Putrescine and tyramine were the most significant biogenic amines in the final product, followed by cadaverine and tryptamine. The highest concentrations of biogenic amines were found at the end of the ripening process in the control sausage with no starter culture irrespective of the use of different sugar concentrations. However, when a starter culture and sugar concentrations equal to 0.5% or 1% were used, the presence of biogenic amines in the sausage decreased considerably in comparison with control and low sugar concentration chorizos. Among all starter cultures tested Lactobacillus sakei K29 was really efficient in reducing the amine production since this strain caused a quick pH drop during sausage fermentation. On the other hand, the production of high amounts of putrescine (223–252 mg/K) and tyramine (64–102 mg/K) was not avoided when the concentration of sugar in the sausage was only 0.1%, even in sausages with a starter culture. Therefore, the use of decarboxylase negative lactic acid bacteria as starter cultures, which produces a rapid decrease on the pH of the meat mixture, and the use of a sugar concentration (mainly glucose) in a range of 0.5–1%, are two important factors to be considered in order to reduce the levels of biogenic amines in chorizo dry sausage. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Biogenic amines; Chorizo; Fermented sausage; Starter culture
1. Introduction Biogenic amines are organic molecules with low molecular weight, biologically active and normally formed by bacterial decarboxylation of their precursor amino acids (Hala! sz et al., 1994). These molecules are generally either psychoactive or vasoactive. Psychoactive amines, such as dopamine and serotonin, are neurotransmitters in the central nervous system (Bard! ocz, 1993). Tyramine, tryptamine, b-phenylethylamine, histamine and serotonin play a vasoactive role, either directly or indirectly, on the vascular system (Bover-Cid et al., 1999). Furthermore, biogenic amines such as tyramine and also diamines such as putrescine and cadaverine have been described as precursors of carcinogenic nitrosamines (Ten Brink et al., 1990). *Corresponding author. Tel.: +34-947-258814; fax: +34-947258831. E-mail address: [email protected] (J. Rovira).
It is not unusual to find high amounts of biogenic amines in some foods, such as fermented and spoiled products. Among other products, high levels of biogenic amines have been observed in dry fermented sausages (Eerola et al., 1993; Herna! ndez-Jover et al., 1997b). The ripening of fermented sausages offers favourable conditions for biogenic amines production, due to the growth of active microbial populations, acidification and the proteolysis that increases the amounts of free amino acids available for decarboxylation (Hala! sz et al., 1994). According to some studies, the use of starter cultures was not sufficient to avoid the presence of biogenic amines in some fermented meat products (Buncic et al., 1993; Eerola et al., 1996; Paulsen and Bauer, 1997). However, other works have indicated that the use of starter cultures reduces the production of some amines, but not of others (Maijala et al., 1995; Herna! ndez-Jover et al., 1997b; Ayhan et al., 1999; Bover-Cid et al., 2000a, b). This fact could be related with the lack of effectiveness of the starter culture depending on the
0740-0020/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0740-0020(02)00157-0
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hygienic quality of the raw material used (Bover-Cid et al., 2001a). The objective of this work was to study the influence of three decarboxylase negative starter cultures and different composition and concentration of sugars, on the presence of biogenic amines in chorizo dry sausage. Chorizo is a typical Spanish dry fermented sausage characterized by its red colour due to the presence of red pepper in its composition. In industrial formulations some sugars, mainly glucose, sucrose and lactose, are added in order to improve the fermentation process by the lactic acid bacteria (LAB). In this sense, the relation between the starter culture and the different sugars and their concentration, as well as the way LAB decrease the pH in the meat stuff could play an important role in biogenic amines production in dry fermented sausages, together with other important factor: the hygienic quality of raw materials.
rinsing steps, they were kept frozen in phosphate buffer (pH 7.4), with monosodium glutamate as cryoprotector, until they were used in chorizo manufacture. The days 1 (meat mixture before stuffing step), 3 (midfermentation, 24 h), 4 (end of fermentation, 48 h), 8, 15 and 22 (end of ripening process) a LAB count in MRS agar plates (Biokar Diagnostics, Beauvais, France) was done. A number of LAB strains equal to the square root of the number of colonies obtained in these plates were chosen for further plasmid profile analysis on days 1, 4 and 22 according to the method described by Anderson and McKay (1983) and modified by Reinkemeier et al. (1996). The plasmid profile together with some phenotypic features, as colony morphology, acetoin production and cell shape, were used to monitor the presence of the different starter cultures strains throughout the ripening process of chorizo sausages.
2.2. Manufacture of chorizo sausage 2. Materials and methods 2.1. Starter cultures Three strains of LAB were used as starter cultures. Two of them belonged to the culture collection of the Food Technology Department (University of Burgos) and were isolated from traditional chorizo ! sausages from the Spanish Region, Castilla and Leon, i.e. Lactobacillus sakei K29 and Pediococcus P22 (Santos et al., 1998). The third strain was a commercial Pediococcus P208, from Rhodia Food (Dange Saint Romain, France). The morphological, biochemical and physiological characterization of the three bacteria was carried out according to Gonza! lez-Ferna! ndez et al. (1997b). The ability of the starter cultures used to produce biogenic amines ‘‘in vitro’’ was tested following the method described by Maijala and Eerola (1993). According to this method, each starter culture was incubated separately in 50 ml of MRS broth (Biokar Diagnostics, Beauvais, France), together with the different types and concentrations of sugars used in the manufacture of chorizos and 1% of different precursor amino acids: llysine hydrochloride, l-histidine, l-phenylalanine, lornithine hydrochloride, l-tryptophan and l-tyrosine all of them purchased to Sigma (San Louis, Missouri, USA). After 24 h incubation at 301C, under anaerobic conditions, a sample of 1 ml from the homogenate was used to determine biogenic amines production by highperformance liquid chromatography (HPLC), as it is reported later. Before the production of the chorizo sausage, freezedryed strains of the starter cultures were inoculated in MRS broth, and incubated at 301C for 24 h, under anaerobic conditions. After different centrifugation and
Six batches of chorizo sausage were elaborated. Their formulation differs only in the sugar concentration. Each batch was made up of four different chorizo sausages according to the type of starter culture used: control (Cn) without a starter culture, chorizo sausage made with L. sakei K29 (chorizo A), with Pediococcus P22 (chorizo B), and with Pediococcus P208 (chorizo C). The manufacture process began with the formulation of a mixture (w/w) consisting of 50% lean pork meat, 50% fat pork meat, 2.4% salt, 1.9% red pepper, 0.15% dried garlic, 0.1% phosphate, 0.1% oregano and 0.1% black pepper, 0.045% ascorbic acid, 0.015% nitrites, 0.01% nitrates. The type and concentration of sugars were chosen according to the most characteristic formulations in this kind of meat products: 0.1% glucose (batch 1), 0.5% glucose (batch 2), and 1% glucose (batch 3), 0.5% glucose and 0.5% lactose (batch 4), 0.5% glucose and 0.3% sucrose (batch 5) and 0.5% glucose, 0.5% lactose and 0.3% sucrose (batch 6). The meat was ground in a meat mincer (CATO, Sabadell, Spain) with a plate diameter of 15 mm at 01C. Then, it was mixed with the rest of the ingredients and with the starter culture dissolved in 40 ml of water for chorizos A–C to obtain a concentration of 106–107 cfu/g. All the components were mixed at vacuum for 2 min, and the sausage mixture was stuffed into 45 mm diameter collagen casings (Naturin, Weinheim, Germany). The sausages were placed in a drying chamber, and after a resting day, in which the temperature of the chamber was slowly increased till 231C, fermentation step started and it lasted 48 h at 231C and 90% of relative humidity (RH). Thereafter, sausages were held in the ripening chamber at 131C and 80–70% RH during 18 days more.
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2.3. Microbiological and physicochemical analysis For the microbiological analysis, 10 g of chorizo without casing were taken aseptically, transferred to sterile bags and homogenized for 60 s with 90 ml peptone-water (Biokar Diagnostics, Beauvais, France). Decimal dilutions of the samples were prepared using the peptone-water. LAB were analysed in MRS agar (Biokar Diagnostics, Beauvais, France) at 301C for 48 h in anaerobic conditions. Catalase-positive cocci were enumerated on MSA (Biokar Diagnostics, Beauvais, France) at 301C for 48 h, and enterobacteria were analysed in VRBGA (Biokar Diagnostics, Beauvais, France) with double layer and incubated for 24 h at 301C. Two samples per duplicate were taken from each chorizo sausage and batch the 1st, 3rd, 4th, 8th, 15th and 22nd days of the ripening process.
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with Aqua Lab CX-2 equipment (Decagon, Washington, USA) and moisture content in accordance with the ISO norm (ISO, 1973). Four measurements of each parameter were carried out the 1st, 3rd, 4th, 8th, 15th and 22nd days of the ripening process. 2.6. Statistical analysis Statistical calculations were performed using the Statgraphics Plus software for Windows. Analysis of variance (ANOVA) of analytical parameters was carried out. In order to determine whether the values were significantly different from each other, the Fisher PLSD test was used. Furthermore, the lineal correlation between biogenic amines, moisture contents, pH and aw was determined.
2.4. Determination of biogenic amines 3. Results and discussion Biogenic amines (both in vitro and in the sausages) were determined by using HPLC according to the method described by Eerola et al. (1993). In the sausages, the amines were extracted from 10 g of sample with 40 ml of acetone, after centrifugation at 12 000 g during 10 min, 40 ml of 5% trichloracetic acid solution were added to the pellet. Sample extracts (1.5 ml) were derivatized with 125 mg dansyl chloride (Sigma, San Louis, Missouri, USA) in 2 ml acetone. Liquid chromatographic separations were performed with HewlettPackard 110 liquid chromatography (Waldbronn, Germany), with a reverse phase Spherisorb ODSO column (150 4.6 mm2, 5 mm) (Symta, Madrid, Spain), a quaternary pump system, a manual injector and a diode array detector. The mobile phase was acetic acid and acetonitrile 9/1 v/v as solvent A and acetic acid, acetonitrile and methanol 2/9/9 v/v as solvent B. The flow rate was 0.7 ml/min, biogenic amines were detected at 254 nm. The limits of determination were for all biogenic amines 1 mg/kg. 1-8 diaminooctane was used as internal standard. Eight biogenic amines were analysed: tryptamine, phenyletylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine. To quantify these amines standard solutions were prepared in the same way as samples from tryptamine hydrochloride, phenyletylamine, putrescine dihydrochloride, cadaverine dihydrochloride, histamine dihydrochloride, tyramine, spermidine and spermine dihydrochloride (Sigma, San Louis, Missouri, USA). Biogenic amines only were analysed in final products. 2.5. Physical and chemical analysis pH was measured in the sausage by means of a pHmeter with a penetration probe electrode (Crison, Barcelona, Spain). Water activity (aw ) was measured
3.1. Microbiological development According to the results, none of the three strains of LAB used as a starter culture produced biogenic amines in vitro from the amino acids tested. Table 1 shows the results of the LAB counts during the ripening process, with special reference to the fermentation step. The counts of LAB in the meat mixture before stuffing are slightly lower in Control chorizo (4.4–5.2 log cfu/g) than in the rest of chorizo sausages inoculated with starter cultures (6.5–7.1 log cfu/g), which means that inoculation with the starter cultures has been done properly and that meat raw materials posses an important amount of wild LAB microbiota. During the fermentation period (days 3 and 4) the strain L. sakei K29 grew very fast in comparison with the other starter strains and the wild LAB of the Control chorizo, probably because it is a very well adapted strain to this kind of product. Pediococcus strains also grew faster than the LAB of the Control chorizo during the same period. During the rest of the ripening process all chorizo sausages kept more or less the same LAB counts (8.1–9.2 log cfu/g) that they had in day 4. From the practical point of view no significant differences were observed among all batches. In order to follow the implantation degree of the different starter cultures during the ripening process, the plasmid profile of the strains was analysed. L. sakei K29 showed two plasmids (14.29 kb and over 55 kb), Pediococcus sp. P208 showed only one plasmid over 55 kb as it was in L. sakei K29, and no plasmids appeared in Pediococcus sp. P22. Table 2 shows that only L. sakei K29 survive during all ripening process and batches. On the contrary, the implantation of Pediococcus strains was lower and particularly the strain Pediococcus P22.
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Table 1 Lactic acid bacteria counts in different batches throughout the ripening process of chorizo (log cfu/g) (n ¼ 4) Batch 1 0.1% G
Batch 2 0.5% G
Batch 3 1% G
Batch 4 0.5% G 0.5% L
Batch 5 0.5% G 0.3% S
Batch 6 0.5% G 0.5% L 0.3% S
a
a
a
a
Day 1
Control Chorizo A Chorizo B Chorizo C
5.1 6.7b 6.9b 6.8b
4.8 6.8b 6.8b 6.6b
5.0 6.8b 7.1c 6.7b
4.4 6.5b 6.9b 6.7b
5.2a 6.5b 6.5b 6.8b
4.6a 6.7b 7.0b 6.6b
Day 3
Control Chorizo A Chorizo B Chorizo C
7.5a 9.0c 8.3b 7.6a
6.1a 8.7c 8.0b 7.7b
6.8a 9.0c 8.5b 8.4b
6.5a 9.2c 8.7b 8.4b
7.0a 8.2c 7.8b 7.2b
5.9a 8.7c 7.7b 7.3b
Day 4
Control Chorizo A Chorizo B Chorizo C
8.9c 9.1c 8.3a 8.7b
8.1a 9.2c 8.6b 8.7b
8.8b 9.0c 8.7b 8.6a
8.4a 9.2c 8.7b 8.8b
8.4a 9.2c 8.7b 8.8b
7.7a 9.1c 8.1b 8.7b
Day 15
Control Chorizo A Chorizo B Chorizo C
9.1b 8.9b 8.7a 8.8ab
8.6a 9.0b 8.7a 8.7a
9.0b 8.8ab 8.8ab 8.6a
8.9b 9.3c 8.7a 8.7a
9.1b 9.0b 8.5a 8.7a
8.8ab 9.1b 8.6a 8.7a
Day 22
Control Chorizo A Chorizo B Chorizo C
9.1b 9.1b 8.6a 8.7a
8.7a 9.0b 8.9ab 8.8a
9.3c 8.9b 8.9b 8.7a
9.2b 9.0b 8.5a 8.6a
9.0b 8.7b 8.3a 8.5a
9.0b 8.9b 8.3a 8.7b
G: glucose; L: lactose; S: sucrose. Control: chorizo without starter culture; chorizo A: with L. sakei K29; chorizo B: with Pediococcus sp. P22; chorizo C: with Pediococcus sp. P208. Different letters in the same column and in each day indicate statistically significant difference (Po0:05).
Table 2 Plasmid profile analysis of the different strains used as starter culture (%) Batch 1 0.1% G
Batch 2 0.5% G
Batch 3 1% G
Batch 4 0.5% G 0.5% L
Batch 5 0.5% G 0.3% S
Batch 6 0.5% G 0.5% L 0.35 S
Day 1
Chorizo A Chorizo B Chorizo C
100 100 100
100 100 100
100 100 100
100 100 100
100 100 100
100 100 100
Day 4
Chorizo A Chorizo B Chorizo C
100 100 100
100 75 100
100 50 100
100 80 100
100 50 50
100 75 100
Day 22
Chorizo A Chorizo B Chorizo C
100 80 100
100 50 100
100 10 50
100 10 80
100 50 50
100 50 100
G: glucose; L: lactose; S: sucrose. Control: chorizo without starter culture; chorizo A: with L. Sakei K29; chorizo B: with Pediococcus sp. P22; chorizo C: with Pediococcus sp. P208. Different letters in the same column and in each day indicate statistically significant difference (Po0:05).
Dealing with the evolution of the catalase-positive cocci, the counts were similar in all batches from the beginning to the end of the ripening process, about 4– 5 log cfu/g.
The low initial values of enterobacteria (Table 3) proved the good hygienic quality of raw meat. However, during the ripening process some differences could be observed between chorizo sausages with and without a
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Table 3 Enterobacteria counts in different batches throughout the ripening process of chorizo (log cfu/g) (n ¼ 4) Batch 1 0.1% G
Batch 2 0.5% G
Batch 3 1% G
Batch 4 0.5% G 0.5% L
Batch 5 0.5% G 0.3% S
Day 1
Day 4
Day 15
Day 22
a
Control Chorizo A Chorizo B Chorizo C
2.7 2.5 2.7 2.6
Control Chorizo A Chorizo B Chorizo C
1.9 1.9 1.8 1.7
a
Control Chorizo A Chorizo B Chorizo C
1.3 — — 1.0
a
Control Chorizo A Chorizo B Chorizo C
1.2 — — —
a
a a a
a a a
a
a
2.5 2.6a 2.3a 1.7a 1.7a — — 1.3a
a
Batch 6 0.5% G 0.5% L 0.3% S
2.4 2.2 a 2.1 a 2.3a
a
3.1 2.9a 2.7a 2.3a
2.7a 3.0a 3.0a 2.8a
3.5a 2.7a 3.1a 3.1a
1.7b 1.4ab 1.0a
2.7b 0.7a 1.0a
2.3a 1.9a 2.0a 2.2a
2.8a 2.1a 2.2a 2.8a
1.0 — — —
1.0 — — —
1.7 — — —
— — — —
— — — —
1.2a — — —
1.7b 1.0a 1.3a 1.4ab 1.5a — — 1.2a
1.7 — — — 2.0a — — —
G: glucose; L: lactose; S: sucrose. Control: chorizo without starter culture; chorizo A: with L. sakei K29; chorizo B: with Pediococcus sp. P22; chorizo C: with Pediococcus sp. P208. Different letters in the same column and in each day indicate statistically significant difference (Po0:05).
starter culture and between batches. In this sense, Control chorizo had enterobacteria during almost the whole ripening time. On the contrary, in chorizo A almost all enterobacteria had disappeared by day 4 of process. Surprisingly all batches with 0.3% of sucrose showed higher counts of enterbacteria during all the ripening process. 3.2. pH, moisture content and water activity (aw ) The starter cultures and the different sugar patterns and concentrations showed differences in pH drop during the fermentation step (Table 4). In general, Control chorizo sausages showed a slower pH drop than the chorizo sausages inoculated with starter cultures in all batches till day 15th of manufacture and they never reached pH values below 5. If the different batches are compared, it is possible to observe that in all of them, except batch 1, the Control chorizo had a higher pH than the rest of chorizos. In this batch, the pH of the four chorizos decreased similarly and in all cases over 5.3 on day 4. In the other batches with higher sugar concentrations, the pH fall was usually faster in chorizos with starter culture than in control, especially in chorizo A (L. sakei K29), which always presented the lower pH (days 3 and 4) of all chorizos. This rapid acidifying capability of L. sakei K29 could be related with the good adaptation to the meat matrix of this strain. Some pH differences existed, mainly in chorizos A, between the
batches elaborated with added sucrose (pH: 5.56–5.40) and those elaborated without this sugar (pH: 5.20–5.02). This small ‘‘lag phase’’ in the decrease of pH in those batches could be due to the fact that LAB metabolized sucrose before glucose in first place. According to Liepe et al. (1990), sugar molecules diffusion inside the meat matrix is quicker for big size molecules, and these molecules are available to bacteria before the small ones. The moisture content and the water activity (aw ) decreased similarly in the chorizos irrespective of whether a starter culture was used or not, and the concentration of sugar used in their formulation (Table 5). Nevertheless, it is remarkable that in all Control chorizo sausages the final value of aw (day 22) was higher in each batch (0.886–0.902) than in chorizos with starter culture. 3.3. Biogenic amines The biogenic amines values in final products are shown in Table 6. Generally, tyramine, cadaverine and putrescine are the main amines found in meat products. In this work, tyramine and putrescine were the most abundant biogenic amines in all chorizos, as it was observed in others studies (Herna! ndez-Jover et al., 1996, 1997b). However, tyramine and cadaverine have been reported as major biogenic amines in other fermented products (Bover-Cid et al., 2000a, b, 2001a, b). In the present study, histamine was not found in any of the
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280
Table 4 Mean values of pH throughout the manufacture process of chorizo sausage (n ¼ 4) Batch 1 0.1% G
Batch 2 0.5% G
Batch 3 1% G
b
b
a
Batch 4 0.5% G 0.5% L a
Batch 5 0.5% G 0.3% S
Batch 6 0.5% G 0.5% L 0.3% S
Day 1
Control Chorizo A Chorizo B Chorizo C
5.86 5.78ab 5.70a 5.76a
5.68 5.64b 5.55a 5.65b
5.81 5.80a 5.77a 5.79a
6.04 6.01a 6.02a 6.00a
5.79b 5.76ab 5.76ab 5.70a
5.85a 5.83a 5.93b 5.90ab
Day 3
Control Chorizo A Chorizo B Chorizo C
5.90c 5.44a 5.43a 5.69b
5.68c 5.20a 5.52b 5.64c
5.70d 5.02a 5.31b 5.46c
6.07d 5.19a 5.51b 5.74c
5.80c 5.56a 5.66b 5.74c
5.73c 5.40a 5.61b 5.69bc
Day 4
Control Chorizo A Chorizo B Chorizo C
5.37ab 5.41b 5.31a 5.35ab
5.62d 4.86a 4.97b 5.05c
5.42c 4.84a 4.91b 4.94b
5.87b 5.14a 5.17a 5.19a
5.32c 4.79a 5.01b 5.05b
5.68c 4.92a 5.07b 5.14b
Day 15
Control Chorizo A Chorizo B Chorizo C
5.58c 5.55c 5.42a 5.45b
5.13d 4.80a 4.87b 4.96c
4.87d 4.69a 4.79c 4.74b
5.20b 5.07a 5.06a 5.11a
4.91b 4.80a 4.80a 4.84a
5.02b 4.87a 4.87a 4.88a
Day 22
Control Chorizo A Chorizo B Chorizo C
5.62b 5.66c 5.55a 5.60b
5.11b 4.93a 4.95a 5.06b
5.12c 4.95a 4.96ab 5.00b
5.55c 5.27a 5.26a 5.37b
5.25c 4.99a 5.33b 5.27b
5.08b 4.82a 4.87a 4.87a
G: glucose; L: lactose; S: sucrose. Control: chorizo without starter culture; chorizo A: with L. sakei K29; chorizo B: with Pediococcus sp. P22; chorizo C: with Pediococcus sp. P208. Different letters in the same column and in each day indicate statistically significant difference (Po0:05).
four chorizos irrespective of the type or concentration of sugar used. The same phenomenon has been observed with other fermented sausages (Herna! ndez-Jover et al., 1997a; Bover-Cid et al., 2000b). The fact that bacteria with the capacity to decarboxylate the histidine are uncommon in meat (Paulsen and Bauer, 1997) and the good hygienic quality of raw meat could explain the absence of this biogenic amine in these products. The values of spermine (2–10 mg/kg) and spermidine (2–5 mg/kg) were low and very similar among the four chorizos in each batch. This result could confirm that these amines are not the result of enzymatic decarboxylation by the micro-organisms, but physiological ! micro-components of the original meat (Bardocz, 1995). Finally, the aromatic mono amine phenylethylamine either does not appear, or its levels are insignificant (o2 mg/kg). The amounts of this biogenic amine found in other fermented sausages were also very low, and rarely higher than 50 mg/kg (Paulsen and Bauer, 1997). When batches are compared, the batch prepared with only 0.1% glucose showed the highest concentrations of biogenic amines in chorizo. Furthermore, it is the only batch in which the levels of tyramine and putrescine were high and very similar for the four chorizos,
irrespective of whether or not a starter culture was used. This result may be explained by the pH value, which did not decrease below 5.3 in any of the four chorizos in batch 1. So, this pH was too high to prevent the development of decarboxylase positive bacteria, which were present in the raw material. In this sense, for example Eitenmiller et al. (1978) stated that most of the wild bacteria in fermented natural sausage have a high capacity to decarboxylate tyrosine. Concerning to the effect of sugar on biogenic amine contents in fermented sausages, Bower-Cid et al. (2001b) found contents of tyramine and cadaverine significantly higher in sausages without sugar in their formulation; nevertheless, the sugar concentrations recommended to reduce biogenic amine production were slightly lower than the amounts used in this work. The relationship between pH and levels of biogenic amines in fermented sausages is confused. Some authors have reported that the main responsible factor for low levels of biogenic amines is the low pH reached during the manufacture process (Maijala and Eerola, 1993; Gonza! lez-Ferna! ndez et al., 1997a). In contrast, other researchers have found that the low pH favour biogenic amines production (Maijala et al., 1995; Roig-Sague! s
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Table 5 Mean values of physical–chemical parameters throughout the manufacture process of chorizo sausage (n ¼ 4). Batch 1 0.1% G
Moisture content Day 1 Control Chorizo A Chorizo B Chorizo C
Batch 2 0.5% G
Batch 3 1% G
Batch 4 0.5% G 0.5% L
Batch 5 0.5% G 0.3% S
Batch 6 0.5% G 0.5% L 0.3% S
60.1a 61.4a 60.4a 59.9a
63.3ab 65.8b 62.3ab 59.6a
59.8a 59.5a 59.8a 57.9a
61.6a 61.2a 61.7a 61.2a
58.8ab 57.2a 59.5ab 61.0b
62.9b 59.2a 60.0ab 60.8ab
Day 4
Control Chorizo A Chorizo B Chorizo C
58.6a 57.7a 56.3a 58.2a
58.3a 63.5b 61.7ab 62.2ab
60.4a 59.8a 58.1a 60.0a
59.2a 61.0a 60.2a 60.6a
56.0a 56.8ab 60.0b 55.7a
58.3ab 56.0a 56.3ab 58.8b
Day 22
Control Chorizo A Chorizo B Chorizo C
35.2ab 33.7a 35.7ab 37.7b
36.7b 30.5a 34.1ab 33.7 ab
32.3ab 29.7a 32.2ab 33.1b
34.7b 33.1a 33.3a 35.1b
33.4bc 30.7a 32.5ab 35.2c
35.9b 31.5a 32.4a 33.4a
aw Day 1
Day 4
Day 22
Control Chorizo A Chorizo B Chorizo C Control Chorizo A Chorizo B Chorizo C
0.972a 0.972a 0.970a 0.972a 0.965a 0.968a 0.965a 0.965a
0.972a 0.972a 0.970a 0.970a 0.969ab 0.968a 0.970ab 0.971b
0.970a 0.970a 0.970a 0.970a 0.969b 0.965a 0.966ab 0.965a
0.970a 0.970a 0.971a 0.971a 0.964a 0.965a 0.966a 0.962a
0.966a 0.967a 0.969a 0.969a 0.965a 0.965a 0.965a 0.964a
0.969a 0.968a 0.968a 0.969a 0.966a 0.964a 0.965a 0.964a
Control Chorizo A Chorizo B Chorizo C
0.902b 0.898b 0.890a 0.898b
0.898c 0.871a 0.875b 0.872a
0.886d 0.864a 0.869b 0.874c
0.893c 0.881b 0.881b 0.872a
0.901c 0.881a 0.888b 0.890b
0.897b 0.875a 0.876a 0.877a
G: glucose; L: lactose; S: sucrose. Control: chorizo without starter culture; chorizo A: with L. sakei K29; chorizo B: with Pediococcus sp. P22; chorizo C: with Pediococcus sp. P208. Different letters in the same column and in each day indicate statistically significant difference (Po0:05).
et al., 1998). These authors point out that bacteria increase the decarboxylase activity to defend themselves against acid pH. The significant correlation between putrescine and tyramine and pH (r=0.829 and 0.567, respectively) found in this study indicates that the high pH increased the biogenic amine formation. Probably, the high pH favoured the development of decarboxylase positive bacteria. Furthermore, the positive correlation between these biogenic amines and aw (r ¼ 0:841 and 0.826) and moisture content (r ¼ 0:579 and 0.541) point out that the high values of both parameters (aw and moisture content) also favoured the development of decarboxylase positive bacteria. Regarding to the differences between chorizo sausages elaborated with and without starter culture it was observed that the Control chorizo (except in batch 1 as it has been indicated before) had significantly higher values of putrescine and tyramine than the chorizos inoculated with starter cultures. Among these, the
chorizo A (L. sakei K29), had significantly lower concentrations of biogenic amines than chorizo B (Pediococcus sp. P22) and chorizo C (Pediococcus sp. P208). Again, there is a relationship between the production of biogenic amines and pH drop, since in the batches with sugar concentration above 0.5%, it was always the chorizo inoculated with the L. sakei K29 the one whose pH decreased the fastest, followed by the two pediococci, and finally, by the control. Cadaverine and putrescine production in fermented sausages is generally associated with lysine- and ornithine-decarboxylase activity, respectively, of Enterobacteriaceae (Edwards et al., 1978, Hala! sz et al., 1994, Bover-Cid et al., 2001b). According to Table 3, the levels of enterobacteria found were, in general, low in the raw meat materials, although in some batches as batch 5, the survival of these bacteria during the ripening process was surprisingly high. This could be the reason why high concentrations of putrescine were found in this batch in comparison with other batches.
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Table 6 Concentration of biogenic amines (mg/kg) in chorizo sausage at the end of ripening process (day 22) (n ¼ 4) Batch 1 0.1% G
c
Batch 2 0.5% G
b
Batch 3 1% G
d
Batch 4 0.5% G 0.5% L
Batch 5 0.5% G 0.3% S
Batch 6 0.5% G 0.5% L 0.3% S
Tryptamine
Control Chorizo A Chorizo B Chorizo C
27 5a 16b 30c
50 4a 20a 5a
17 6b 2a 8c
ND ND ND ND
37b ND 24a 35b
22b 6a ND ND
Phenylethylamine
Control Chorizo A Chorizo B Chorizo C
5b ND ND 2a
ND 2a ND 2a
ND 2b 1a 1a
ND ND ND ND
ND ND ND ND
ND ND ND ND
Putrescine
Control Chorizo A Chorizo B Chorizo C
223a 238ab 252b 231ab
107b 2a 7a 12a
78d 6a 13b 25c
193d 38c 7a 21b
200c 30a 147b 207c
98b 5a 4a ND
Cadaverine
Control Chorizo A Chorizo B Chorizo C
16d 12b 10a 15c
12b ND 4a ND
7c 5b 5b 2a
ND ND ND ND
19b ND ND 9a
2a 2a 2a 19b
Histamine
Control Chorizo A Chorizo B Chorizo C
ND ND ND ND
ND ND ND ND
ND ND ND ND
ND ND ND ND
ND ND ND ND
ND ND ND ND
Tyramine
Control Chorizo A Chorizo B Chorizo C
85b 73a 64a 102c
106c 2a 8a 20b
62c 11a 44b 65c
125b 20a 11a 12a
132d 39a 85b 117c
106c 4c 8c 36b
Spermidine
Control Chorizo A Chorizo B Chorizo C
3a 3a 4b 5c
3a 3a 3a 3a
4b 2a 2a 2a
ND 2a 2a 2a
ND ND ND ND
ND ND ND ND
Spermine
Control Chorizo A Chorizo B Chorizo C
2a 9b 11b 9b
ND ND ND ND
ND 4b 5b 2a
ND ND ND ND
ND 9a 10a ND
ND ND ND ND
ND: not detected; G: glucose; L: lactose; S: sucrose. Control: chorizo without starter culture; chorizo A: with L. sakei K29; chorizo B: with Pediococcus sp. P22; chorizo C: with Pediococcus sp. P208. Different letters in the same column and in each day indicate statistically significant difference (Po0:05).
From health point of view, one must bear in mind that the minimum amount of tyramine, which can cause an increase in the blood pressure in the arteries for patients under classical monoamine oxidase inhibitor treatment, is 6 mg (Vidal et al., 1990). Then, all chorizos with more than 120 ppm or mg/kg of this amine could lead to the described disorder when such sensitive individuals ingest 50 g of sausage. The Control chorizo (with no starter culture) had levels of this amine very close to this limit value in all batches, being even higher in batches 4 and 5. Thus, the production of putrescine in these chorizos may increase
even more the toxic effect of tyramine (Ten Brink et al., 1990). Several factors have to exert their role together to prevent production of biogenic amines in fermented dry sausages. The three main factors seem to be a suitable starter culture, a proper level of sugar and good quality raw materials. Previous works have shown a beneficial effect of starter culture to reduce amine accumulation. Some of them pointing out the importance of raw material quality to prevent high biogenic amines contents in fermented sausages (Bover-Cid et al., 2000a, b, 2001a). These authors found small differences
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between spontaneously fermented and starter inoculated sausages, when good quality meat was used, except for tyramine, and in a lesser extent putrescine and cadaverine. However, in the present work, the highquality raw materials used have not been effective to prevent the production of putrescine and tyramine in control sausage (Table 6), and low contents of these amines were obtained only when a starter culture was included in sausage formulation. On the other hand, the use of sugar in sausage formulation, as the only factor to reduce amine contents, had not shown a significant effect if fermentation was not controlled by starter cultures (Table 6). In conclusion, to avoid the presence of high concentrations of biogenic amines in chorizo, it is advisable to use a competitive starter culture such as L. sakei K29, a negative-decarboxylate strain, which may decrease the pH quickly during fermentation step and be predominant throughout the process, thus it would prevent the growth of bacteria which can produce biogenic amines. The use of glucose concentrations above 0.5% is another factor that may contribute to reduce the production of amines in chorizo, since it favours the rapid pH decrease in the sausage when a suitable starter culture is used.
Acknowledgements This work was supported by a grant (ALI-94-0956! Interministerial de Ciencia CO2-01) from the Comision ! y Tecnologıa of the Spanish Government.
References Anderson, D.G., McKay, L.L., 1983. Simple and rapid method for isolating large plasmid DNA from lactic streptococc. Appl. Environ. Microbiol. 46, 549–552. . Ayhan, K., Kolsarici, N., Ozcan, G.A., 1999. The effects of a starter culture on the formation of biogenic amines in Turkish soudjoucks. Meat Sci. 53, 183–188. ! Bardocz, S., 1993. The role of dietary polyamines. Eur. J. Clin. Nutr. 47, 683–690. ! Bardocz, S., 1995. Polyamines in food and their consequences for food quality and human health. Trends Food Sci. Technol. 6, 341–346. Bover-Cid, S., Schoppen, S., Izquierdo-Pulido, M., Vidal-Carou, M.C., 1999. Relationship between biogenic amines contents and the size of dry fermented sausages. Meat Sci. 51, 305–311. Bover-Cid, S., Hugas, M., Izquierdo-Pulido, M., Vidal-Carou, M.C., 2000a. Reduction of biogenic amine formation using a negative amino acid-decarboxylase starter culture for fermentation of ‘‘Fuet’’ sausages. J. Food Protect. 63, 237–243. Bover-Cid, S., Izquierdo-Pulido, M., Vidal-Carou, M.C., 2000b. Mixed starter cultures to control biogenic amine production in dry fermented sausages. J. Food Protect. 63, 1556–1562. Bover-Cid, S., Izquierdo-Pulido, M., Vidal-Carou, M.C., 2001a. Effectiveness of a Lactobacillus sakei starter culture in the reduction of biogenic amine accumulation as a function of the raw material quality. J. Food Protect. 64, 367–373.
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Bover-Cid, S., Izquierdo-Pulido, M., Vidal-Carou, M.C., 2001b. Changes in biogenic amine and polyamine contents in slightly fermented sausages manufactured with and without sugar. Meat Sci. 57, 215–221. Buncic, S., Paunovic, L., Teodorovic, V., Radisic, D., Vojinovic, G., Smiljanic, D., Baltic, M., 1993. Effects of gluconodeltalactone and Lactobacillus plantarum on the production of histamine and tyramine in fermented sausages. Int. J. Food Microbiol. 17, 303–309. Edwards, R.A., Dainty, R.H., Hibbard, C.M., Remantanis, S.V., 1978. Amines in fresh beef of normal pH and the role of bacteria in changes in concentration observed during storage in vacuum packs at chill temperature. J. Appl. Bacteriol. 63, 427–434. Eerola, S., Hinkkanen, R., Lindfors, E., Hirvi, T., 1993. Liquid chromatographic determination of biogenic amines in dry sausages. Int. J. AOAC 76, 575–577. Eerola, H.S., Maijala, R., Roig-Sagu!es, A.X., Salminen, M., Hirvi, T.K., 1996. Biogenic amines in dry sausages as affected by starter culture and contaminant amine-positive Lactobacillus. J. Food Sci. 61, 1243–1246. Eitenmiller, R.R., Koehler, P.E., Reagan, P.E., 1978. Tyramine in fermented sausages: factor-affecting formation of tyrosine and tyrosine decarboxylase. J. Food Sci. 43, 689–693. Gonz!alez-Fern!andez, C., Jaime, I., Rovira, J., Stevens, G., Raemaekers, M., Demeyer, D., 1997a. The importance of starter strains on the production of biogenic amines in a ‘‘chorizo’’ dry sausage. In: Proceedings of Lactic 97, Caen, France. Gonz!alez-Fern!andez, C., Santos, E.M., Jaime, I., Rovira, J., 1997b. ! de cultivos iniciadores en la elaboracion ! de chorizo y su Utilizacion influencia en las propiedades sensoriales. Food Sci. Technol. Int. 3, 31–42. Hal!asz, A., Bar!ath, A., Simon-Sarkadi, L., Holzapfel, W., 1994. Biogenic amines and their production by micro-organisms in food. Trends Food Sci. Technol. 5, 42–49. Hern!andez-Jover, T., Izquierdo-Pulido, M., Veciana-Nogu!es, M.T., Vidal-Carou, M.C., 1996. Ion-pair high-performance liquid chromatographic determination of biogenic amines in meat and meat products. J. Agric. Food Chem. 44, 2710–2715. Hern!andez-Jover, T., Izquierdo-Pulido, M., Veciana-Nogu!es, M.T., Marin!e-Font, A., Vidal-Carou, M.C., 1997a. Effect of starter cultures on biogenic amine formation during fermented sausage production. J. Food Protect. 60, 825–830. Hern!andez-Jover, T., Izquierdo-Pulido, M., Veciana-Nogu!es, M.T., Marin!e-Font, A., Vidal-Carou, M.C., 1997b. Biogenic amine and polyamine contents in meat and meat products. J. Agric. Food Chem. 45, 2098–2102. ISO, 1973. Determination of moisture content, ISO 1442-1973. In: International Standards Meat and Meat Products, International Organization for Standardization, Geneve. Liepe, H.U., Pfeil, E., Porobic, R., 1990. Influence of sugars and bacteria on dry sausage souring. Fleischwirtschaft 70, 189–192. Maijala, R., Eerola, S., 1993. Contaminant lactic acid bacteria of dry sausages produce histamine and tyramine. Meat Sci. 35, 387–395. Maijala, R., Nurmi, E., Fischer, A., 1995. Influence of processing temperature on the formation of biogenic amines in dry sausages. Meat Sci. 39, 9–22. Paulsen, P., Bauer, F., 1997. Biogenic amines in fermented sausages, II: factors influencing the formation of biogenic amines in raw fermented sausage. Fleischwirtschaft 77, 362–364. . Reinkemeier, M., Rocken, W., Leitzmann, C., 1996. A rapid mechanical lysing procedure for routine analysis of plasmids from lactobacilli isolated from sourdoughs. Int. J. Food Microbiol. 29, 93–104. Roig Sagu!es, A.X., Hern!andez Herrero, M.M., Rodr!ıguez Jerez, J.J., Quinto Fern!andez, E.J., Mora Ventura, M.T., 1998. Aminas
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www.elsevier.nl/locate/jnlabr/yfmic
Influence of starter cultures and sugar concentrations on biogenic amine contents in chorizo dry sausage Consuelo Gonza! lez-Ferna! ndez, E.M. Santos, Isabel Jaime, Jordi Rovira* Departamento de Tecnolog!ıa de los Alimentos, Facultad de Ciencias, Universidad de Burgos, Plaza Misael Banuelos * s/n, 09001 Burgos, Spain Received 23 April 2002; received in revised form 12 November 2002; accepted 12 November 2002
Abstract The production of biogenic amines in chorizo dry sausage by using different decarboxylase negative starter cultures and different concentrations of sugar added in its manufacture has been studied. Biogenic amines were determined as dansyl derivates by highperformance liquid chromatography (HPLC). Putrescine and tyramine were the most significant biogenic amines in the final product, followed by cadaverine and tryptamine. The highest concentrations of biogenic amines were found at the end of the ripening process in the control sausage with no starter culture irrespective of the use of different sugar concentrations. However, when a starter culture and sugar concentrations equal to 0.5% or 1% were used, the presence of biogenic amines in the sausage decreased considerably in comparison with control and low sugar concentration chorizos. Among all starter cultures tested Lactobacillus sakei K29 was really efficient in reducing the amine production since this strain caused a quick pH drop during sausage fermentation. On the other hand, the production of high amounts of putrescine (223–252 mg/K) and tyramine (64–102 mg/K) was not avoided when the concentration of sugar in the sausage was only 0.1%, even in sausages with a starter culture. Therefore, the use of decarboxylase negative lactic acid bacteria as starter cultures, which produces a rapid decrease on the pH of the meat mixture, and the use of a sugar concentration (mainly glucose) in a range of 0.5–1%, are two important factors to be considered in order to reduce the levels of biogenic amines in chorizo dry sausage. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Biogenic amines; Chorizo; Fermented sausage; Starter culture
1. Introduction Biogenic amines are organic molecules with low molecular weight, biologically active and normally formed by bacterial decarboxylation of their precursor amino acids (Hala! sz et al., 1994). These molecules are generally either psychoactive or vasoactive. Psychoactive amines, such as dopamine and serotonin, are neurotransmitters in the central nervous system (Bard! ocz, 1993). Tyramine, tryptamine, b-phenylethylamine, histamine and serotonin play a vasoactive role, either directly or indirectly, on the vascular system (Bover-Cid et al., 1999). Furthermore, biogenic amines such as tyramine and also diamines such as putrescine and cadaverine have been described as precursors of carcinogenic nitrosamines (Ten Brink et al., 1990). *Corresponding author. Tel.: +34-947-258814; fax: +34-947258831. E-mail address: [email protected] (J. Rovira).
It is not unusual to find high amounts of biogenic amines in some foods, such as fermented and spoiled products. Among other products, high levels of biogenic amines have been observed in dry fermented sausages (Eerola et al., 1993; Herna! ndez-Jover et al., 1997b). The ripening of fermented sausages offers favourable conditions for biogenic amines production, due to the growth of active microbial populations, acidification and the proteolysis that increases the amounts of free amino acids available for decarboxylation (Hala! sz et al., 1994). According to some studies, the use of starter cultures was not sufficient to avoid the presence of biogenic amines in some fermented meat products (Buncic et al., 1993; Eerola et al., 1996; Paulsen and Bauer, 1997). However, other works have indicated that the use of starter cultures reduces the production of some amines, but not of others (Maijala et al., 1995; Herna! ndez-Jover et al., 1997b; Ayhan et al., 1999; Bover-Cid et al., 2000a, b). This fact could be related with the lack of effectiveness of the starter culture depending on the
0740-0020/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0740-0020(02)00157-0
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hygienic quality of the raw material used (Bover-Cid et al., 2001a). The objective of this work was to study the influence of three decarboxylase negative starter cultures and different composition and concentration of sugars, on the presence of biogenic amines in chorizo dry sausage. Chorizo is a typical Spanish dry fermented sausage characterized by its red colour due to the presence of red pepper in its composition. In industrial formulations some sugars, mainly glucose, sucrose and lactose, are added in order to improve the fermentation process by the lactic acid bacteria (LAB). In this sense, the relation between the starter culture and the different sugars and their concentration, as well as the way LAB decrease the pH in the meat stuff could play an important role in biogenic amines production in dry fermented sausages, together with other important factor: the hygienic quality of raw materials.
rinsing steps, they were kept frozen in phosphate buffer (pH 7.4), with monosodium glutamate as cryoprotector, until they were used in chorizo manufacture. The days 1 (meat mixture before stuffing step), 3 (midfermentation, 24 h), 4 (end of fermentation, 48 h), 8, 15 and 22 (end of ripening process) a LAB count in MRS agar plates (Biokar Diagnostics, Beauvais, France) was done. A number of LAB strains equal to the square root of the number of colonies obtained in these plates were chosen for further plasmid profile analysis on days 1, 4 and 22 according to the method described by Anderson and McKay (1983) and modified by Reinkemeier et al. (1996). The plasmid profile together with some phenotypic features, as colony morphology, acetoin production and cell shape, were used to monitor the presence of the different starter cultures strains throughout the ripening process of chorizo sausages.
2.2. Manufacture of chorizo sausage 2. Materials and methods 2.1. Starter cultures Three strains of LAB were used as starter cultures. Two of them belonged to the culture collection of the Food Technology Department (University of Burgos) and were isolated from traditional chorizo ! sausages from the Spanish Region, Castilla and Leon, i.e. Lactobacillus sakei K29 and Pediococcus P22 (Santos et al., 1998). The third strain was a commercial Pediococcus P208, from Rhodia Food (Dange Saint Romain, France). The morphological, biochemical and physiological characterization of the three bacteria was carried out according to Gonza! lez-Ferna! ndez et al. (1997b). The ability of the starter cultures used to produce biogenic amines ‘‘in vitro’’ was tested following the method described by Maijala and Eerola (1993). According to this method, each starter culture was incubated separately in 50 ml of MRS broth (Biokar Diagnostics, Beauvais, France), together with the different types and concentrations of sugars used in the manufacture of chorizos and 1% of different precursor amino acids: llysine hydrochloride, l-histidine, l-phenylalanine, lornithine hydrochloride, l-tryptophan and l-tyrosine all of them purchased to Sigma (San Louis, Missouri, USA). After 24 h incubation at 301C, under anaerobic conditions, a sample of 1 ml from the homogenate was used to determine biogenic amines production by highperformance liquid chromatography (HPLC), as it is reported later. Before the production of the chorizo sausage, freezedryed strains of the starter cultures were inoculated in MRS broth, and incubated at 301C for 24 h, under anaerobic conditions. After different centrifugation and
Six batches of chorizo sausage were elaborated. Their formulation differs only in the sugar concentration. Each batch was made up of four different chorizo sausages according to the type of starter culture used: control (Cn) without a starter culture, chorizo sausage made with L. sakei K29 (chorizo A), with Pediococcus P22 (chorizo B), and with Pediococcus P208 (chorizo C). The manufacture process began with the formulation of a mixture (w/w) consisting of 50% lean pork meat, 50% fat pork meat, 2.4% salt, 1.9% red pepper, 0.15% dried garlic, 0.1% phosphate, 0.1% oregano and 0.1% black pepper, 0.045% ascorbic acid, 0.015% nitrites, 0.01% nitrates. The type and concentration of sugars were chosen according to the most characteristic formulations in this kind of meat products: 0.1% glucose (batch 1), 0.5% glucose (batch 2), and 1% glucose (batch 3), 0.5% glucose and 0.5% lactose (batch 4), 0.5% glucose and 0.3% sucrose (batch 5) and 0.5% glucose, 0.5% lactose and 0.3% sucrose (batch 6). The meat was ground in a meat mincer (CATO, Sabadell, Spain) with a plate diameter of 15 mm at 01C. Then, it was mixed with the rest of the ingredients and with the starter culture dissolved in 40 ml of water for chorizos A–C to obtain a concentration of 106–107 cfu/g. All the components were mixed at vacuum for 2 min, and the sausage mixture was stuffed into 45 mm diameter collagen casings (Naturin, Weinheim, Germany). The sausages were placed in a drying chamber, and after a resting day, in which the temperature of the chamber was slowly increased till 231C, fermentation step started and it lasted 48 h at 231C and 90% of relative humidity (RH). Thereafter, sausages were held in the ripening chamber at 131C and 80–70% RH during 18 days more.
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2.3. Microbiological and physicochemical analysis For the microbiological analysis, 10 g of chorizo without casing were taken aseptically, transferred to sterile bags and homogenized for 60 s with 90 ml peptone-water (Biokar Diagnostics, Beauvais, France). Decimal dilutions of the samples were prepared using the peptone-water. LAB were analysed in MRS agar (Biokar Diagnostics, Beauvais, France) at 301C for 48 h in anaerobic conditions. Catalase-positive cocci were enumerated on MSA (Biokar Diagnostics, Beauvais, France) at 301C for 48 h, and enterobacteria were analysed in VRBGA (Biokar Diagnostics, Beauvais, France) with double layer and incubated for 24 h at 301C. Two samples per duplicate were taken from each chorizo sausage and batch the 1st, 3rd, 4th, 8th, 15th and 22nd days of the ripening process.
277
with Aqua Lab CX-2 equipment (Decagon, Washington, USA) and moisture content in accordance with the ISO norm (ISO, 1973). Four measurements of each parameter were carried out the 1st, 3rd, 4th, 8th, 15th and 22nd days of the ripening process. 2.6. Statistical analysis Statistical calculations were performed using the Statgraphics Plus software for Windows. Analysis of variance (ANOVA) of analytical parameters was carried out. In order to determine whether the values were significantly different from each other, the Fisher PLSD test was used. Furthermore, the lineal correlation between biogenic amines, moisture contents, pH and aw was determined.
2.4. Determination of biogenic amines 3. Results and discussion Biogenic amines (both in vitro and in the sausages) were determined by using HPLC according to the method described by Eerola et al. (1993). In the sausages, the amines were extracted from 10 g of sample with 40 ml of acetone, after centrifugation at 12 000 g during 10 min, 40 ml of 5% trichloracetic acid solution were added to the pellet. Sample extracts (1.5 ml) were derivatized with 125 mg dansyl chloride (Sigma, San Louis, Missouri, USA) in 2 ml acetone. Liquid chromatographic separations were performed with HewlettPackard 110 liquid chromatography (Waldbronn, Germany), with a reverse phase Spherisorb ODSO column (150 4.6 mm2, 5 mm) (Symta, Madrid, Spain), a quaternary pump system, a manual injector and a diode array detector. The mobile phase was acetic acid and acetonitrile 9/1 v/v as solvent A and acetic acid, acetonitrile and methanol 2/9/9 v/v as solvent B. The flow rate was 0.7 ml/min, biogenic amines were detected at 254 nm. The limits of determination were for all biogenic amines 1 mg/kg. 1-8 diaminooctane was used as internal standard. Eight biogenic amines were analysed: tryptamine, phenyletylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine. To quantify these amines standard solutions were prepared in the same way as samples from tryptamine hydrochloride, phenyletylamine, putrescine dihydrochloride, cadaverine dihydrochloride, histamine dihydrochloride, tyramine, spermidine and spermine dihydrochloride (Sigma, San Louis, Missouri, USA). Biogenic amines only were analysed in final products. 2.5. Physical and chemical analysis pH was measured in the sausage by means of a pHmeter with a penetration probe electrode (Crison, Barcelona, Spain). Water activity (aw ) was measured
3.1. Microbiological development According to the results, none of the three strains of LAB used as a starter culture produced biogenic amines in vitro from the amino acids tested. Table 1 shows the results of the LAB counts during the ripening process, with special reference to the fermentation step. The counts of LAB in the meat mixture before stuffing are slightly lower in Control chorizo (4.4–5.2 log cfu/g) than in the rest of chorizo sausages inoculated with starter cultures (6.5–7.1 log cfu/g), which means that inoculation with the starter cultures has been done properly and that meat raw materials posses an important amount of wild LAB microbiota. During the fermentation period (days 3 and 4) the strain L. sakei K29 grew very fast in comparison with the other starter strains and the wild LAB of the Control chorizo, probably because it is a very well adapted strain to this kind of product. Pediococcus strains also grew faster than the LAB of the Control chorizo during the same period. During the rest of the ripening process all chorizo sausages kept more or less the same LAB counts (8.1–9.2 log cfu/g) that they had in day 4. From the practical point of view no significant differences were observed among all batches. In order to follow the implantation degree of the different starter cultures during the ripening process, the plasmid profile of the strains was analysed. L. sakei K29 showed two plasmids (14.29 kb and over 55 kb), Pediococcus sp. P208 showed only one plasmid over 55 kb as it was in L. sakei K29, and no plasmids appeared in Pediococcus sp. P22. Table 2 shows that only L. sakei K29 survive during all ripening process and batches. On the contrary, the implantation of Pediococcus strains was lower and particularly the strain Pediococcus P22.
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Table 1 Lactic acid bacteria counts in different batches throughout the ripening process of chorizo (log cfu/g) (n ¼ 4) Batch 1 0.1% G
Batch 2 0.5% G
Batch 3 1% G
Batch 4 0.5% G 0.5% L
Batch 5 0.5% G 0.3% S
Batch 6 0.5% G 0.5% L 0.3% S
a
a
a
a
Day 1
Control Chorizo A Chorizo B Chorizo C
5.1 6.7b 6.9b 6.8b
4.8 6.8b 6.8b 6.6b
5.0 6.8b 7.1c 6.7b
4.4 6.5b 6.9b 6.7b
5.2a 6.5b 6.5b 6.8b
4.6a 6.7b 7.0b 6.6b
Day 3
Control Chorizo A Chorizo B Chorizo C
7.5a 9.0c 8.3b 7.6a
6.1a 8.7c 8.0b 7.7b
6.8a 9.0c 8.5b 8.4b
6.5a 9.2c 8.7b 8.4b
7.0a 8.2c 7.8b 7.2b
5.9a 8.7c 7.7b 7.3b
Day 4
Control Chorizo A Chorizo B Chorizo C
8.9c 9.1c 8.3a 8.7b
8.1a 9.2c 8.6b 8.7b
8.8b 9.0c 8.7b 8.6a
8.4a 9.2c 8.7b 8.8b
8.4a 9.2c 8.7b 8.8b
7.7a 9.1c 8.1b 8.7b
Day 15
Control Chorizo A Chorizo B Chorizo C
9.1b 8.9b 8.7a 8.8ab
8.6a 9.0b 8.7a 8.7a
9.0b 8.8ab 8.8ab 8.6a
8.9b 9.3c 8.7a 8.7a
9.1b 9.0b 8.5a 8.7a
8.8ab 9.1b 8.6a 8.7a
Day 22
Control Chorizo A Chorizo B Chorizo C
9.1b 9.1b 8.6a 8.7a
8.7a 9.0b 8.9ab 8.8a
9.3c 8.9b 8.9b 8.7a
9.2b 9.0b 8.5a 8.6a
9.0b 8.7b 8.3a 8.5a
9.0b 8.9b 8.3a 8.7b
G: glucose; L: lactose; S: sucrose. Control: chorizo without starter culture; chorizo A: with L. sakei K29; chorizo B: with Pediococcus sp. P22; chorizo C: with Pediococcus sp. P208. Different letters in the same column and in each day indicate statistically significant difference (Po0:05).
Table 2 Plasmid profile analysis of the different strains used as starter culture (%) Batch 1 0.1% G
Batch 2 0.5% G
Batch 3 1% G
Batch 4 0.5% G 0.5% L
Batch 5 0.5% G 0.3% S
Batch 6 0.5% G 0.5% L 0.35 S
Day 1
Chorizo A Chorizo B Chorizo C
100 100 100
100 100 100
100 100 100
100 100 100
100 100 100
100 100 100
Day 4
Chorizo A Chorizo B Chorizo C
100 100 100
100 75 100
100 50 100
100 80 100
100 50 50
100 75 100
Day 22
Chorizo A Chorizo B Chorizo C
100 80 100
100 50 100
100 10 50
100 10 80
100 50 50
100 50 100
G: glucose; L: lactose; S: sucrose. Control: chorizo without starter culture; chorizo A: with L. Sakei K29; chorizo B: with Pediococcus sp. P22; chorizo C: with Pediococcus sp. P208. Different letters in the same column and in each day indicate statistically significant difference (Po0:05).
Dealing with the evolution of the catalase-positive cocci, the counts were similar in all batches from the beginning to the end of the ripening process, about 4– 5 log cfu/g.
The low initial values of enterobacteria (Table 3) proved the good hygienic quality of raw meat. However, during the ripening process some differences could be observed between chorizo sausages with and without a
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Table 3 Enterobacteria counts in different batches throughout the ripening process of chorizo (log cfu/g) (n ¼ 4) Batch 1 0.1% G
Batch 2 0.5% G
Batch 3 1% G
Batch 4 0.5% G 0.5% L
Batch 5 0.5% G 0.3% S
Day 1
Day 4
Day 15
Day 22
a
Control Chorizo A Chorizo B Chorizo C
2.7 2.5 2.7 2.6
Control Chorizo A Chorizo B Chorizo C
1.9 1.9 1.8 1.7
a
Control Chorizo A Chorizo B Chorizo C
1.3 — — 1.0
a
Control Chorizo A Chorizo B Chorizo C
1.2 — — —
a
a a a
a a a
a
a
2.5 2.6a 2.3a 1.7a 1.7a — — 1.3a
a
Batch 6 0.5% G 0.5% L 0.3% S
2.4 2.2 a 2.1 a 2.3a
a
3.1 2.9a 2.7a 2.3a
2.7a 3.0a 3.0a 2.8a
3.5a 2.7a 3.1a 3.1a
1.7b 1.4ab 1.0a
2.7b 0.7a 1.0a
2.3a 1.9a 2.0a 2.2a
2.8a 2.1a 2.2a 2.8a
1.0 — — —
1.0 — — —
1.7 — — —
— — — —
— — — —
1.2a — — —
1.7b 1.0a 1.3a 1.4ab 1.5a — — 1.2a
1.7 — — — 2.0a — — —
G: glucose; L: lactose; S: sucrose. Control: chorizo without starter culture; chorizo A: with L. sakei K29; chorizo B: with Pediococcus sp. P22; chorizo C: with Pediococcus sp. P208. Different letters in the same column and in each day indicate statistically significant difference (Po0:05).
starter culture and between batches. In this sense, Control chorizo had enterobacteria during almost the whole ripening time. On the contrary, in chorizo A almost all enterobacteria had disappeared by day 4 of process. Surprisingly all batches with 0.3% of sucrose showed higher counts of enterbacteria during all the ripening process. 3.2. pH, moisture content and water activity (aw ) The starter cultures and the different sugar patterns and concentrations showed differences in pH drop during the fermentation step (Table 4). In general, Control chorizo sausages showed a slower pH drop than the chorizo sausages inoculated with starter cultures in all batches till day 15th of manufacture and they never reached pH values below 5. If the different batches are compared, it is possible to observe that in all of them, except batch 1, the Control chorizo had a higher pH than the rest of chorizos. In this batch, the pH of the four chorizos decreased similarly and in all cases over 5.3 on day 4. In the other batches with higher sugar concentrations, the pH fall was usually faster in chorizos with starter culture than in control, especially in chorizo A (L. sakei K29), which always presented the lower pH (days 3 and 4) of all chorizos. This rapid acidifying capability of L. sakei K29 could be related with the good adaptation to the meat matrix of this strain. Some pH differences existed, mainly in chorizos A, between the
batches elaborated with added sucrose (pH: 5.56–5.40) and those elaborated without this sugar (pH: 5.20–5.02). This small ‘‘lag phase’’ in the decrease of pH in those batches could be due to the fact that LAB metabolized sucrose before glucose in first place. According to Liepe et al. (1990), sugar molecules diffusion inside the meat matrix is quicker for big size molecules, and these molecules are available to bacteria before the small ones. The moisture content and the water activity (aw ) decreased similarly in the chorizos irrespective of whether a starter culture was used or not, and the concentration of sugar used in their formulation (Table 5). Nevertheless, it is remarkable that in all Control chorizo sausages the final value of aw (day 22) was higher in each batch (0.886–0.902) than in chorizos with starter culture. 3.3. Biogenic amines The biogenic amines values in final products are shown in Table 6. Generally, tyramine, cadaverine and putrescine are the main amines found in meat products. In this work, tyramine and putrescine were the most abundant biogenic amines in all chorizos, as it was observed in others studies (Herna! ndez-Jover et al., 1996, 1997b). However, tyramine and cadaverine have been reported as major biogenic amines in other fermented products (Bover-Cid et al., 2000a, b, 2001a, b). In the present study, histamine was not found in any of the
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280
Table 4 Mean values of pH throughout the manufacture process of chorizo sausage (n ¼ 4) Batch 1 0.1% G
Batch 2 0.5% G
Batch 3 1% G
b
b
a
Batch 4 0.5% G 0.5% L a
Batch 5 0.5% G 0.3% S
Batch 6 0.5% G 0.5% L 0.3% S
Day 1
Control Chorizo A Chorizo B Chorizo C
5.86 5.78ab 5.70a 5.76a
5.68 5.64b 5.55a 5.65b
5.81 5.80a 5.77a 5.79a
6.04 6.01a 6.02a 6.00a
5.79b 5.76ab 5.76ab 5.70a
5.85a 5.83a 5.93b 5.90ab
Day 3
Control Chorizo A Chorizo B Chorizo C
5.90c 5.44a 5.43a 5.69b
5.68c 5.20a 5.52b 5.64c
5.70d 5.02a 5.31b 5.46c
6.07d 5.19a 5.51b 5.74c
5.80c 5.56a 5.66b 5.74c
5.73c 5.40a 5.61b 5.69bc
Day 4
Control Chorizo A Chorizo B Chorizo C
5.37ab 5.41b 5.31a 5.35ab
5.62d 4.86a 4.97b 5.05c
5.42c 4.84a 4.91b 4.94b
5.87b 5.14a 5.17a 5.19a
5.32c 4.79a 5.01b 5.05b
5.68c 4.92a 5.07b 5.14b
Day 15
Control Chorizo A Chorizo B Chorizo C
5.58c 5.55c 5.42a 5.45b
5.13d 4.80a 4.87b 4.96c
4.87d 4.69a 4.79c 4.74b
5.20b 5.07a 5.06a 5.11a
4.91b 4.80a 4.80a 4.84a
5.02b 4.87a 4.87a 4.88a
Day 22
Control Chorizo A Chorizo B Chorizo C
5.62b 5.66c 5.55a 5.60b
5.11b 4.93a 4.95a 5.06b
5.12c 4.95a 4.96ab 5.00b
5.55c 5.27a 5.26a 5.37b
5.25c 4.99a 5.33b 5.27b
5.08b 4.82a 4.87a 4.87a
G: glucose; L: lactose; S: sucrose. Control: chorizo without starter culture; chorizo A: with L. sakei K29; chorizo B: with Pediococcus sp. P22; chorizo C: with Pediococcus sp. P208. Different letters in the same column and in each day indicate statistically significant difference (Po0:05).
four chorizos irrespective of the type or concentration of sugar used. The same phenomenon has been observed with other fermented sausages (Herna! ndez-Jover et al., 1997a; Bover-Cid et al., 2000b). The fact that bacteria with the capacity to decarboxylate the histidine are uncommon in meat (Paulsen and Bauer, 1997) and the good hygienic quality of raw meat could explain the absence of this biogenic amine in these products. The values of spermine (2–10 mg/kg) and spermidine (2–5 mg/kg) were low and very similar among the four chorizos in each batch. This result could confirm that these amines are not the result of enzymatic decarboxylation by the micro-organisms, but physiological ! micro-components of the original meat (Bardocz, 1995). Finally, the aromatic mono amine phenylethylamine either does not appear, or its levels are insignificant (o2 mg/kg). The amounts of this biogenic amine found in other fermented sausages were also very low, and rarely higher than 50 mg/kg (Paulsen and Bauer, 1997). When batches are compared, the batch prepared with only 0.1% glucose showed the highest concentrations of biogenic amines in chorizo. Furthermore, it is the only batch in which the levels of tyramine and putrescine were high and very similar for the four chorizos,
irrespective of whether or not a starter culture was used. This result may be explained by the pH value, which did not decrease below 5.3 in any of the four chorizos in batch 1. So, this pH was too high to prevent the development of decarboxylase positive bacteria, which were present in the raw material. In this sense, for example Eitenmiller et al. (1978) stated that most of the wild bacteria in fermented natural sausage have a high capacity to decarboxylate tyrosine. Concerning to the effect of sugar on biogenic amine contents in fermented sausages, Bower-Cid et al. (2001b) found contents of tyramine and cadaverine significantly higher in sausages without sugar in their formulation; nevertheless, the sugar concentrations recommended to reduce biogenic amine production were slightly lower than the amounts used in this work. The relationship between pH and levels of biogenic amines in fermented sausages is confused. Some authors have reported that the main responsible factor for low levels of biogenic amines is the low pH reached during the manufacture process (Maijala and Eerola, 1993; Gonza! lez-Ferna! ndez et al., 1997a). In contrast, other researchers have found that the low pH favour biogenic amines production (Maijala et al., 1995; Roig-Sague! s
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281
Table 5 Mean values of physical–chemical parameters throughout the manufacture process of chorizo sausage (n ¼ 4). Batch 1 0.1% G
Moisture content Day 1 Control Chorizo A Chorizo B Chorizo C
Batch 2 0.5% G
Batch 3 1% G
Batch 4 0.5% G 0.5% L
Batch 5 0.5% G 0.3% S
Batch 6 0.5% G 0.5% L 0.3% S
60.1a 61.4a 60.4a 59.9a
63.3ab 65.8b 62.3ab 59.6a
59.8a 59.5a 59.8a 57.9a
61.6a 61.2a 61.7a 61.2a
58.8ab 57.2a 59.5ab 61.0b
62.9b 59.2a 60.0ab 60.8ab
Day 4
Control Chorizo A Chorizo B Chorizo C
58.6a 57.7a 56.3a 58.2a
58.3a 63.5b 61.7ab 62.2ab
60.4a 59.8a 58.1a 60.0a
59.2a 61.0a 60.2a 60.6a
56.0a 56.8ab 60.0b 55.7a
58.3ab 56.0a 56.3ab 58.8b
Day 22
Control Chorizo A Chorizo B Chorizo C
35.2ab 33.7a 35.7ab 37.7b
36.7b 30.5a 34.1ab 33.7 ab
32.3ab 29.7a 32.2ab 33.1b
34.7b 33.1a 33.3a 35.1b
33.4bc 30.7a 32.5ab 35.2c
35.9b 31.5a 32.4a 33.4a
aw Day 1
Day 4
Day 22
Control Chorizo A Chorizo B Chorizo C Control Chorizo A Chorizo B Chorizo C
0.972a 0.972a 0.970a 0.972a 0.965a 0.968a 0.965a 0.965a
0.972a 0.972a 0.970a 0.970a 0.969ab 0.968a 0.970ab 0.971b
0.970a 0.970a 0.970a 0.970a 0.969b 0.965a 0.966ab 0.965a
0.970a 0.970a 0.971a 0.971a 0.964a 0.965a 0.966a 0.962a
0.966a 0.967a 0.969a 0.969a 0.965a 0.965a 0.965a 0.964a
0.969a 0.968a 0.968a 0.969a 0.966a 0.964a 0.965a 0.964a
Control Chorizo A Chorizo B Chorizo C
0.902b 0.898b 0.890a 0.898b
0.898c 0.871a 0.875b 0.872a
0.886d 0.864a 0.869b 0.874c
0.893c 0.881b 0.881b 0.872a
0.901c 0.881a 0.888b 0.890b
0.897b 0.875a 0.876a 0.877a
G: glucose; L: lactose; S: sucrose. Control: chorizo without starter culture; chorizo A: with L. sakei K29; chorizo B: with Pediococcus sp. P22; chorizo C: with Pediococcus sp. P208. Different letters in the same column and in each day indicate statistically significant difference (Po0:05).
et al., 1998). These authors point out that bacteria increase the decarboxylase activity to defend themselves against acid pH. The significant correlation between putrescine and tyramine and pH (r=0.829 and 0.567, respectively) found in this study indicates that the high pH increased the biogenic amine formation. Probably, the high pH favoured the development of decarboxylase positive bacteria. Furthermore, the positive correlation between these biogenic amines and aw (r ¼ 0:841 and 0.826) and moisture content (r ¼ 0:579 and 0.541) point out that the high values of both parameters (aw and moisture content) also favoured the development of decarboxylase positive bacteria. Regarding to the differences between chorizo sausages elaborated with and without starter culture it was observed that the Control chorizo (except in batch 1 as it has been indicated before) had significantly higher values of putrescine and tyramine than the chorizos inoculated with starter cultures. Among these, the
chorizo A (L. sakei K29), had significantly lower concentrations of biogenic amines than chorizo B (Pediococcus sp. P22) and chorizo C (Pediococcus sp. P208). Again, there is a relationship between the production of biogenic amines and pH drop, since in the batches with sugar concentration above 0.5%, it was always the chorizo inoculated with the L. sakei K29 the one whose pH decreased the fastest, followed by the two pediococci, and finally, by the control. Cadaverine and putrescine production in fermented sausages is generally associated with lysine- and ornithine-decarboxylase activity, respectively, of Enterobacteriaceae (Edwards et al., 1978, Hala! sz et al., 1994, Bover-Cid et al., 2001b). According to Table 3, the levels of enterobacteria found were, in general, low in the raw meat materials, although in some batches as batch 5, the survival of these bacteria during the ripening process was surprisingly high. This could be the reason why high concentrations of putrescine were found in this batch in comparison with other batches.
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Table 6 Concentration of biogenic amines (mg/kg) in chorizo sausage at the end of ripening process (day 22) (n ¼ 4) Batch 1 0.1% G
c
Batch 2 0.5% G
b
Batch 3 1% G
d
Batch 4 0.5% G 0.5% L
Batch 5 0.5% G 0.3% S
Batch 6 0.5% G 0.5% L 0.3% S
Tryptamine
Control Chorizo A Chorizo B Chorizo C
27 5a 16b 30c
50 4a 20a 5a
17 6b 2a 8c
ND ND ND ND
37b ND 24a 35b
22b 6a ND ND
Phenylethylamine
Control Chorizo A Chorizo B Chorizo C
5b ND ND 2a
ND 2a ND 2a
ND 2b 1a 1a
ND ND ND ND
ND ND ND ND
ND ND ND ND
Putrescine
Control Chorizo A Chorizo B Chorizo C
223a 238ab 252b 231ab
107b 2a 7a 12a
78d 6a 13b 25c
193d 38c 7a 21b
200c 30a 147b 207c
98b 5a 4a ND
Cadaverine
Control Chorizo A Chorizo B Chorizo C
16d 12b 10a 15c
12b ND 4a ND
7c 5b 5b 2a
ND ND ND ND
19b ND ND 9a
2a 2a 2a 19b
Histamine
Control Chorizo A Chorizo B Chorizo C
ND ND ND ND
ND ND ND ND
ND ND ND ND
ND ND ND ND
ND ND ND ND
ND ND ND ND
Tyramine
Control Chorizo A Chorizo B Chorizo C
85b 73a 64a 102c
106c 2a 8a 20b
62c 11a 44b 65c
125b 20a 11a 12a
132d 39a 85b 117c
106c 4c 8c 36b
Spermidine
Control Chorizo A Chorizo B Chorizo C
3a 3a 4b 5c
3a 3a 3a 3a
4b 2a 2a 2a
ND 2a 2a 2a
ND ND ND ND
ND ND ND ND
Spermine
Control Chorizo A Chorizo B Chorizo C
2a 9b 11b 9b
ND ND ND ND
ND 4b 5b 2a
ND ND ND ND
ND 9a 10a ND
ND ND ND ND
ND: not detected; G: glucose; L: lactose; S: sucrose. Control: chorizo without starter culture; chorizo A: with L. sakei K29; chorizo B: with Pediococcus sp. P22; chorizo C: with Pediococcus sp. P208. Different letters in the same column and in each day indicate statistically significant difference (Po0:05).
From health point of view, one must bear in mind that the minimum amount of tyramine, which can cause an increase in the blood pressure in the arteries for patients under classical monoamine oxidase inhibitor treatment, is 6 mg (Vidal et al., 1990). Then, all chorizos with more than 120 ppm or mg/kg of this amine could lead to the described disorder when such sensitive individuals ingest 50 g of sausage. The Control chorizo (with no starter culture) had levels of this amine very close to this limit value in all batches, being even higher in batches 4 and 5. Thus, the production of putrescine in these chorizos may increase
even more the toxic effect of tyramine (Ten Brink et al., 1990). Several factors have to exert their role together to prevent production of biogenic amines in fermented dry sausages. The three main factors seem to be a suitable starter culture, a proper level of sugar and good quality raw materials. Previous works have shown a beneficial effect of starter culture to reduce amine accumulation. Some of them pointing out the importance of raw material quality to prevent high biogenic amines contents in fermented sausages (Bover-Cid et al., 2000a, b, 2001a). These authors found small differences
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between spontaneously fermented and starter inoculated sausages, when good quality meat was used, except for tyramine, and in a lesser extent putrescine and cadaverine. However, in the present work, the highquality raw materials used have not been effective to prevent the production of putrescine and tyramine in control sausage (Table 6), and low contents of these amines were obtained only when a starter culture was included in sausage formulation. On the other hand, the use of sugar in sausage formulation, as the only factor to reduce amine contents, had not shown a significant effect if fermentation was not controlled by starter cultures (Table 6). In conclusion, to avoid the presence of high concentrations of biogenic amines in chorizo, it is advisable to use a competitive starter culture such as L. sakei K29, a negative-decarboxylate strain, which may decrease the pH quickly during fermentation step and be predominant throughout the process, thus it would prevent the growth of bacteria which can produce biogenic amines. The use of glucose concentrations above 0.5% is another factor that may contribute to reduce the production of amines in chorizo, since it favours the rapid pH decrease in the sausage when a suitable starter culture is used.
Acknowledgements This work was supported by a grant (ALI-94-0956! Interministerial de Ciencia CO2-01) from the Comision ! y Tecnologıa of the Spanish Government.
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