Effect of processing on proteolysis and biogenic amines formation in a Portuguese traditional dry-fermented ripened sausage “Chouriço Grosso de Estremoz e Borba PGI”

Meat Science 84 (2010) 172–179

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Effect of processing on proteolysis and biogenic amines formation in a Portuguese traditional dry-fermented ripened sausage ‘‘Chouriço Grosso de Estremoz e Borba PGI” L.C. Roseiro a,*, A. Gomes a, H. Gonçalves a, M. Sol a, R. Cercas b, C. Santos a a b

Instituto Nacional de Engenharia, Tecnologia e Inovação, DTIA (Edificio S), Estrada do Paço do Lumiar, 1649-038 Lisboa, Portugal Salsicharia Estremocense, Lda, Outeiro de São José, Apartado 161, 7104-909 Estremoz, Portugal

a r t i c l e

i n f o

Article history: Received 29 October 2008 Received in revised form 11 February 2009 Accepted 21 August 2009

Keywords: Dry-fermented ripened sausage Proteolysis Free amino acids Biogenic amines

a b s t r a c t The influence of alternative drying environmental conditions on the proteolysis of a traditional Portuguese fermented sausage was evaluated, in relation to different ripening periods. Traditional sausages (batch T) had lower pH than counterparts (batch M), with differences (P < 0.05) focused at the fermentation stage. A remarkable accumulation of free amino acids (FAA) was detected in both batches along the ripening process, with batch T having higher mean levels than batch M (1795.2 mg 100 g 1 DM vs. 1742 mg 100 g 1 DM in S7, respectively), but with differences being significant for long ripened products. In both batches, glutamic acid became the most concentrated FAA in end products currently consumed (101.6 mg 100 g 1 DM and 111.0 mg 100 g 1 DM – S6; 233.8 mg 100 g 1 DM and 220.9 mg 100 g 1 DM – S7 from batches T and M, respectively) followed by leucine > alanine > taurine > serine > valine and taurine > alanine > leucine > serine > valine sequences in the former product from batches T and M, respectively, and, coincidently, the same sequence for both batches in the later (serine > leucine > alanine > proline > valine). Such effect on FAA concentrations led to a distinct (P < 0.05) expression of sweet, bitter, acidic and aged sensorial attributes between batches, in S8 and S9 products. BA typical quantitative sequences varied between batches according to the ripening stage, with differences in S6 and S7 end products also reflecting the distinct microbial development rates and profiles observed. Overall, the total BA mean concentration was higher (P < 0.05) in products from batch T. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Industrial meat products processing usually leads to increased productions at lower costs, ensuring more uniform but, necessarily, different quality standards in final products when compared to those produced from traditional/artisanal conditions. In Portugal, this last sector is mostly represented by very small/micro industries, which have been progressively stressed during the last decade by new regulations and legislation concerning hygiene practices and processing environmental conditions. In order to accomplish such recommendations, new production approaches have been implemented, which could possibly change the ‘‘in house-flora” and modify the intrinsic enzymes activity, responsible for the main proteolytic/lipolytic phenomena, the basic source for taste and aroma characteristics. Since consumers are increasingly informed about the multiple components of food quality, which they pay for, these trends towards uncharacteristic traditional pro-

* Corresponding author. Tel.: +351 217127107; fax: +351 217127162. E-mail address: [email protected] (L.C. Roseiro). 0309-1740/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2009.08.044

duction could determine less trust and popularity loss, recently regained by these agents. In traditional/artisanal activity, fermented meat products are made without inoculation of commercial starter cultures and addition of sugars, with the fermentation process being spontaneously achieved in a moderate rate by the natural occurring micro flora, under room environmental conditions difficult to standardize. When the composition of the flora does not show a suitable balance among species, hetero-fermentative agents can give rise to abnormal quality characteristics. Free amino acids are often studied as quality indicators for the impact of the technology with their concentration directly affecting important quality parameters and promoting the presence of some trace compounds responsible for safety and sensorial aspects. Among these late compounds, biogenic amines (BA) are extremely relevant because they may interfere with human metabolism (e.g. vasoactive and psychoactive properties) and contribute to flavour specificity. In addition, BAs are considered potential precursors in the formation of carcinogenic N-nitroso compounds (Karovicova & Kohajdova, 2005). Because the BA formation is mainly generated by microbial decarboxylation of corresponding amino acids, the following requisites

L.C. Roseiro et al. / Meat Science 84 (2010) 172–179

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have to be observed in the products: 1-availability of free amino acids; 2-presence of decarboxylase positive microorganisms; 3conditions for bacterial growth, decarboxylase synthesis and activity (Suzi & Gardini, 2003). Free amino acids naturally occur in raw meat and are mainly produced through intrinsic and extrinsic proteolysis during such meat product processing and storage stages (Roseiro, Santos, Sol, Silva, & Fernandes, 2006). Amino acid decarboxylase activity is higher in an acidic environment (pH between 4.0 and 5.5) (Halász, Baráth, Simon-Sarkadi, & Holzapfel, 1994), with bacteria being encouraged to produce these enzymes as part of their defence mechanism or to transduce the energy used in drive energy-requiring processes (Konings et al., 1997; Maijala, Eerola, Aho, & Rin, 1993). Amines formation by bacteria is also though to be decisively influenced by temperature, with optimum values ranging between 20–37 °C. In traditional ‘‘Chouriço Grosso”, product pH and temperature both reach values varying within those ranges. The existing technical and scientific information about this type of sausage is still scarce, being important to improve the knowledge concerning the chemical and physico-chemical changes associated to proteolysis occurring through the manufacturing process and during the storage period, before consumption. This study was undertaken to determine whether sausages alternatively processed during the initial drying phase under much lower temperatures would have different acidity and free amino acids and BA profiles, when compared to those counterparts from traditional/artisanal production. Other parameters related to proteolysis development and water activity (aw) were also followed up to help on interpretation of results.

ditional smoking room until a similar final weight loss (35–40%) in both batches was attained.

2. Materials and methods

2.4. Free amino acids and biogenic amines analysis

2.1. Preparation of ‘‘Chouriço Grosso Estremoz e Borba PGI”

Free amino acids (FAA) and ammonia were determined according to the Directive 98/64/EC, using an amino acid analyser Biochrom 20 (Pharmacia Biotech). The free amino acids were extracted with diluted hydrochloric acid. Coextracted nitrogenous macromolecules were precipitated with sulfosalicyclic acid and then removed by filtration. After this procedure, pH was adjusted to 2.2 and the amino acids separated by ion exchange chromatography. Free amino acid contents were determined by reaction with ninhydrin with photometric detection at 2 wavelengths, 570 nm and 440 nm (for proline) and expressed as mg 100 g 1 DM of sample. In the text, the terms ‘‘sweet”, ‘‘bitter”, ‘‘acidic” and ‘‘aged” flavour amino acids correspond to the sum of glycine, alanine, serine, threonine, proline; histidine, arginine, methionine, valine, leucine, isoleucine, phenylalanine; glutamic acid, aspartic acid, histidine; and lysine, tyrosine, aspartic acid, respectively (Ordonez, Hierro, Bruna, & De La Hoz, 1999).

Chouriço Grosso de Estremoz e Borba PGI (Protected Geographical Indication) has been manufactured from a mixture of lean minced pork (75%) and pig fat (25%) (about 2 cm sized) obtained from carcasses of rustic Alentejano pure breed animals, reared outdoors in an extensive feeding system. Raw materials were mixed for about 5 min under vacuum and seasonings (NaCl – 1.3%; paprika paste – 4%; raw garlic paste – 2%) and iced cool tap water (7%) were added during this operation. The seasoned batter was immediately stuffed in natural pig casings (approximately 20 cm long and about 6 cm in diameter) and raw sausages were entirely processed in traditional smoking/drying room (batch T) or, alternatively, in a chilling room (batch M) (temperature/relative humidity profiles depicted in Fig. 1) during about 12 days. After a weight loss of approximately 25%, batch M products were transferred to the tra-

2.2. Samples Samples of batches T and M, picked up at distinct processing stages, included raw meat/fat mixtures just after mincing and seasoning operations (R0) and stuffed products with 2 (S1), 8 (S2), 14 (S3), 22 (S4), 30 (S5) and 40 (S6) days of drying and smoking. In relation to the ripening period occurred during the storage phase, analysis were carried out in products packaged under vacuum at room temperature (between 15 °C and 18 °C), after 3 (S7), 6 (S8) and 12 (S9) months. Values reported to different parameters resulted from the mean of three sausages, sampled randomly from the same batch. 2.3. Physico-chemical analysis To determine aw of samples was used a Rotronic Hygromer with a probe AwVC-DIO (Rotronic AG, Switzerland). Dry matter (DM) was measured by drying the samples at 103 ± 2 °C to constant weight (ISO 1442:1997). The pH was measured according to the protocol described in NP-3441 (1990), with a 654 pH meter (Metrohm Herisau, Switzerland) equipped with combined pH glass electrode (Mettler Toledo, Switzerland). Determinations of non-protein nitrogen (NPN), free amino acids nitrogen (FAAN) and total volatile basic nitrogen (TVBN) fractions were carried out according to the methods described in Roseiro et al. (2008). The nitrogen fractions contents were expressed as g 100 g 1 DM of sample.

Fig. 1. Environmental temperature and relative humidity evolution recorded during drying/smoking processing of batches T and M.

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In same row, means with different letters are significantly different. ns = not significant. SE – standard error; NPN – non-protein nitrogen; TVBN – total volatile basic nitrogen; FAAN – free amino acids nitrogen. * P < 0.05. ** P < 0.01. *** P < 0.001.

M T

0.82j 5.63ef 10.47 1.36b 0.18a 0.64b 423.6a 0.84j 5.66e 8.83 1.22c 0.12c 0.54c 220.9bc

M T

0.84i 5.77d 9.37 1.35b 0.14b 0.63b 237.3b 0.83i 5.80cd 6.00 0.93e 0.09d 0.37e 187.3cde

M T

0.86h 5.82cd 6.40 1.06d 0.12c 0.47d 194.3bcd 0.89f 5.56g 1.64 0.85ef 0.08e 0.25fg 132.2fgh

M T

0.88g 5.54g 2.14 0.81fg 0.08e 0.26fg 143.6efgh 0.90e 5.59fg 1.68 0.83efg 0.12c 0.22hij 161.1defg

M T

0.89fg 5.45h 1.68 0.79fg 0.09de 0.24ghi 167.4defg 0.92d 5.65ef 0.79 0.62hij 0.02ij 0.21ijk 118.3gh

M T

0.91e 5.44h 1.36 0.65hi 0.06f 0.25gh 150.3defgh 0.93bc 5.85c 0.61 0.63hij 0.03hi 0.18kl 62.4ij

M T

0.93cd 5.46h 1.36 0.73gh 0.03gh 0.27fg 160.6defg 0.94a 5.92b 0.78 0.63hij 0.05g 0.19jk 64.6ij

M T

0.94ab 5.56g 1.03 0.73gh 0.04gh 0.29f 100.9hi 0.95a 6.02a 0.67 0.59ij 0.03gh 0.15l 19.2j

M T

0.94ab 5.65ef 0.95 0.65hi 0.03gh 0.20ijk 55.3ij aw pH Acidity (%) NPN TVBN FAAN Ammonia

0.94ab 6.03a 0.25 0.52j 0.01j 0.15l 27.1j Batch

0.82j 5.66e 10.70 1.50a 0.18a 0.69a 178.1cdef

0.004 0.022 0.16 0.04 0.005 0.013 17.48

RT B SE S9 S8 S7

DM) during the ripening of ‘‘Paínho de Portalegre” manufactured with traditional and modified processing.

S6 S5 S4 S3 S2

Sausages produced under processing conditions used for batches T and M presented close aw values for the same processing and storage periods analysed, with differences being noticeable (P < 0.05) at processing stages S4, S5 and S6 and at the first storage period (S7) (Table 1). During the processing phase aw values were lower in batch T but, unexpectedly, the relative position of batches in S7 inverted. These variations were due to the different environmental temperature and relative humidity conditions used in the respective drying/smoking operations, which also affected the development of the natural microflora and the intrinsic proteolytic metabolism. Associated to the impact in the microbial population, pH between batches for almost all processing stages differed (P < 0.05), in a higher degree during the initial fermentation period (Table 1), with values obtained from batch T being lower. Minimal pH never fall down below the safety limit of 5.20 (5.44 and 5.56 in batches T and M, respectively), which makes both products to be classified as low acid. The slightly higher ammonia concentrations detected in products of batch T along the processing stages (P < 0.05 at S3) could contribute to the difference observed on pH evolutional behaviour between batches, being possibly responsible for the earlier inversion detected in the running decreasing trend (between day 22 and day 30 of processing) comparatively to their counterparts (3 months storage period). This increase in pH was moderate in both situations, however being slightly higher in batch T (0.38 units) than in batch M (0.24 units). Taking into consideration the accumulation of ammonia with ripening time, the decreasing in pH observed in both batches when the storage was extended up to 6 and 12 months (from 5.82 to 5.77 and to 5.63 – batch T; from 5.80 to 5.66 in batch M) was not expected. The increase in the concentration of compounds originated from lipolysis and fatty acids oxidation could contribute to this occurrence, in accordance with the results obtained for the acidity. The evolution in pH observed in our work confirms other studies regarding the complex ripening process of raw fermented dry sausages (Franco,

S1

3. Results and discussion

1

The effects of processing technology and storage periods on the variables studied were analysed by two-way ANOVA (Statistica 6.0 – StatSoft Inc., 2001). The Fisher’s least significant difference (LSD) post hoc test was used for comparison of mean values. Differences were considered significant at P < 0.05.

DM) and ammonia (expressed as mg 100 g

2.6. Statistical analysis

1

Twenty grams of each sample were mixed with 180 mL of tryptone-salt broth (Biokar) and homogenized in a rotary homogenizer for 1.5 min. Tenfold dilutions were made in the same diluent and the following analysis were carried out: total aerobic mesophilic flora on plate count agar (Biokar) incubated at 30 °C for 3 days; Enterobacteriaceae on violet red bile glucose agar (Biokar) incubated at 37 °C for 24 h; Enterococci on kanamycin aesculin azide agar (Liofilchem) incubated at 37 °C for 2 days; Lactic acid bacteria (LAB) on MRS agar (Biokar) pH 6.2 and Lactobacillus on MRS agar pH 5.7 (Biokar) incubated at 30 °C for 3 days; Total aerobic psychrotrophic flora on plate count agar (Biokar) incubated at 6.5 °C for 10 days; Micrococcaceae on mannitol salt agar (Biokar) incubated at 30 °C for 3 days; Yeasts and molds on rose Bengal chloramphenicol agar (Biokar) incubated at 25 °C for 5 days.

Table 1 Changes in aw, pH, nitrogen fractions (expressed as g 100 g

2.5. Microbiological analysis

RT  B

Biogenic amines (BA) were determined by HPLC according to Eerola, Hinkkanen, Lindfors, and Hirvi (1993) as described in Roseiro et al. (2006).

ns

L.C. Roseiro et al. / Meat Science 84 (2010) 172–179

Ripening length R0

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4.96e <1.0 1.74e 1.51f 1.26d <1.0 <1.0 3.01g <1.00 2.67e 1.57efg <1.00 <1.00 <1.00 4.96e <1.00 2.21e <1.00 1.29de <1.00 <1.00 5.06e <1.00 3.95d 3.53bcde <1.00 <1.00 <1.00 5.73d <1.00 4.47d <1.00 0.81ef <1.00 <1.00 6.06d <1.00 6.11c <1.00 4.93ab <1.00 <1.00 8.38ab <1.00 8.60a 5.57a 2.29d <1.00 4.62 8.80a 0.80de 8.71a 5.65a 5.47a <1.00 3.94 8.24b 1.00cdef 8.30ab 2.58cdef 3.52c <1.00 2.75 8.76ab 3.26ab 8.60a 1.93defg 5.43a <1.00 2.00 8.33ab 0.51e 8.17ab 3.69abcd 3.91bc <1.00 3.44 8.69ab 2.37abcd 8.54a 4.12abc 5.26a <1.00 3.55 7.28c <1.00 7.22b 4.08abc 2.13d <1.00 3.37 In same row, means with different letters are significantly different. ns = not significant. TAMF – total aerobic mesophilic flora; SE –standard error. * P < 0.05. ** P < 0.01. *** P < 0.001.

8.56ab 2.64abc 8.59a 4.08abc 5.37a <1.00 1.22 4.81e 1.48bcde 2.69e 2.48cdef <1.00 <1.00 2.84 8.32ab 3.52 a 8.25ab 5.11ab 5.98a 0.50 3.00 3.82f <1.00 2.42e <1.00 0.51ef 1.15 2.38 TAMF Enterobacteriaceae Lactic acid bacteria Micrococcaceae Enterococci Molds Yeasts

3.88f <1.00 2.06e <1.00 <1.00 1.70 3.00

7.41c 2.03abcde 7.42b 4.97ab 4.11bc 1.00 4.11

T M T

S8

M T

S7

M T

S6

M T

S5

M T

S4

M T

S3

M T

S2

M T

S1 R0

Batch

Ripening length

Table 2 Variability of microbial counts (log cfu g

1

) at different processing stages and storage periods of ‘‘Chouriço Grosso” manufactured by traditional and modified processing methods.

S9

M

SE

0.13 0.40 0.26 0.47 0.25 0.23 0.36

RT

B

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RT  B

L.C. Roseiro et al. / Meat Science 84 (2010) 172–179

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Prieto, Cruz, López, & Carballo, 2002; Lizaso, Chasco, & Beriain, 1999; Mateo, Domínguez, Aguirrezábal, & Zumalacárregui, 1996), which is influenced by several raw materials quality parameters and processing conditions used. S6 and S7 products, the most currently consumed, as well as those ripened for 12 months, did not differ in pH. Nevertheless, the distinct evolution trend referred to the initial stages of processing could be the source of important modifications on their proteolysis profiles (Tables 1 and 3), in agreement with conclusions reported by Verplaetse, Gerard, Buys, and Demeyer (1992) and Molly et al. (1997). According to these authors, higher pH values clearly depress the proteolysis phenomena, affecting the activity of muscle cathepsine D-like enzymes, considered the most predominant agents involved. In fact, those products with lower pH values presented higher peptide and amino acid contents but lower ammonia levels (Sentandreu & Toldrá, 2001; Toldrá, 2002; Toldrá, Cerveró, & Part, 1993). In this study, the environmental temperature profile used for batch T treatment speeded up the fermentation process, increasing LAB and Micrococcaceae counts up to 7.42 log cfu and 4.97 log cfu, respectively, at day 2 of the drying/smoking phase, while in products from batch M those microbial groups only reached a similar level of development (7.22 log cfu and 4.08 log cfu, respectively) at day 14 (S3) (Table 2). Also Enterobacteriaceae and Enterococcus groups differed significantly between batches T and M along the processing, with the former presenting also higher values. Nevertheless, irrespective of the batch, Enterobacteriaceae counts were generally acceptable (less than 3.52 log cfu), from a hygiene perspective. In relation to Enterococcus, differences between batches could account to a distinct impact in products. While in traditional processing (batch T) the maximum value reached 5.98 log cfu at day 8 and remained with identical counts in products stored for 90 days, in batch M that level was considerably lower (3.91 log cfu) and was detected at day 22 of processing. The development of moulds was irrelevant in both batches while yeasts attained similar maximum concentrations during the processing phase in both batches, earlier in traditional processing (4.11 log cfu – day 2) than in modified counterpart (4.62 log cfu – day 40). Both groups were, as expected, not detected in samples analysed during the storage period under vacuum. The microbial flora was substantial reduced with storage time in products from both technological methods. In general, sausages from batch T had slightly higher NPN levels than their counterparts, with the respective evolution differing in behaviour along the processing and storage phases. In fact, while in products traditionally manufactured this fraction increased progressively up to 6 months of storage and then stabilized further on, in those obtained from batch M the initial concentration detected in raw material mixture tended to remain almost unchanged up to day 22 of processing and then increased up to the last sampling analysed (1 year in storage). The effect from the processing technology was mainly seen in FAAN, with differences between batches being significant in samples S1, S2 and S3 and also along all the storage period (S7, S8 and S9). Comparing with the existing scientific information on this concern (Franco et al., 2002; León Crespo et al., 1985; Lois, Gutiérrez, Zumalacárregui, & López, 1987; Salgado, Garcia Fontán, Franco, López, & Carballo, 2005), data obtained in this study just confirms the extreme variation that exists around the world among the distinct products manufactured, regarding raw materials quality characteristics (Flores, Romero, Aristoy, Flores, & Toldrá, 1994; Rosell & Toldrá, 1998; Toldrá, Flores, Aristoy, Virgili, & Parolari, 1996) and processing conditions (Domínguez Fernández & Zumalacárregui Rodríguez, 1992–1994; Franco et al., 2002; León Crespo et al., 1985; Lois et al., 1987). However, it can also be inferred that proteolysis persists for long periods after processing, almost exclusively promoted by intrinsic enzymes (Toldrá & Etherington, 1988; Toldrá et al., 1993), since the counts obtained

176

Table 3 Changes in free amino acids content (mg 100 g Ripening time R0 Batch

S1

1

DM) at different processing stages and storage periods of ‘‘Chouriço Grosso” manufactured by traditional and modified processing methods.

S2

S3

S4

S5

S6

S7

S8

S9

SE

RT B

RT  B

M

T

M

T

M

T

M

T

M

T

M

T

M

T

M

T

M

19.3j 7.9l 32.0a 2.1fg 7.3i 0.3g 8.7f 45.8f 24.5ij 15.6de 12.7j 51.8kl 27.8g 10.0f 25.6gh 37.3h 98.9efg 454.6l

41.3hi 18.4hi 18.3cd N.D. 26.9gh 9.9efg 22.2f 55.1ef 84.4gh 24.4de 22.9ij 75.2hijk 47.3defg 22.2de 30.2fgh 68.2fg 100.8efg 722.9ijk

21.7j 10.1jl 11.7defg 1.0g 12.5hi 1.5g 18.9f 15.2h 14.0j 14.1e 15.1j 57.4jkl 30.8g 12.2ef 18.8h 37.8h 92.1gh 403.9l

53.8gh 28.2fg 4.1hi 0.3g 41.7ef 14.2efg 31.8ef 74.0bc 107.6efg 32.8de 34.2gh 93.3fghi 58.7de 26.3d 42.1def 80.8ef 113.5abcd 938.3fghi

32.8ij 14.1ijl 18.6cd 3.0fg 21.7hi 2.0g 12.4f 30.6g 30.9ij 20.9de 21.3ij 41.2l 34.7fg 11.8ef 23.9gh 46.5gh 110.0bcde 541.0kl

57.4fg 32.2f 4.6ghi 5.7efg 50.5def 18.2efg 38.8ef 78.4b 115.9defg 33.1de 45.8gh 104.8efgh 61.3d 30.5d 47.5de 89.4ef 120.3ab 1095.2fgh

27.1ij 16.1ij 26.8ab 12.2de 26.9gh 5.4fg 19.3f 50.2f 54.9hi 21.7de 23.2ij 72.2ijk 35.4efg 11.9ef 27.2gh 47.0gh 114.6abc 654.5jkl

45.0gh 32.1f 4.2i 6.1efg 52.6de 22.3ef 37.6ef 70.0bcd 120.3def 30.3de 43.2gh 98.7fghi 59.1d 30.8d 43.5de 84.6ef 102.4def 1033.0fgh

56.0fgh 23.4gh 17.0cde 10.9def 37.6fg 14.3efg 29.6ef 61.8e 91.9fg 27.1de 34.9hi 86.9ghij 50.2defg 23.9d 37.5efg 67.0fg 107.7cde 896.1hij

70.6ef 40.7e 21.2bc 2.9fg 60.5d 23.0ef 46.6cdef 92.0a 129.5de 34.6de 52.0fg 117.5def 69.0d 33.0d 52.1d 94.9e 119.5ab 1227.1f

74.9e 41.3e 4.6ghi 4.5efg 52.7de 26.0e 43.4def 78.9b 127.0de 41.5d 47.8gh 115.5defg 64.9d 28.4d 48.3de 87.7ef 121.1a 1169.6fg

52.0gh 31.1f 7.7fghi 5.5efg 53.3de 19.5efg 36.0ef 64.6cde 101.6efg 37.4de 37.8gh 80.1hijk 56.8def 29.3d 45.1de 80.5ef 75.6ij 957.6ghij

58.8fg 34.2f 11.0efgh 8.5defg 45.5ef 17.4efg 39.3ef 73.3bc 111.0efg 21.6de 40.0gh 92.7fghi 58.3de 30.0d 44.4de 79.3ef 94.7fg 992.2fghi

152.5c 61.7d 10.7efgh 26.4c 87.4c 70.4c 80.3c 63.0de 233.8c 111.8b 72.8de 141.2d 103.6c 59.0c 88.4c 146.2cd 91.7gh 1795.2d

142.0c 62.4d 14.7cdef 14.8d 91.5c 80.3c 78.2cd 50.5f 220.9c 111.6b 80.6d 131.9de 99.1c 60.6c 90.6c 141.8d 83.3hi 1742.1d

192.1b 103.0b 4.2hi 35.5b 151.2a 155.9b 134.3b 30.4g 330.7b 177.8a 129.0b 210.2b 134.9b 104.3a 150.0a 212.8b 114.1abcd 2607.9b

179.7b 90.6c 1.8i 23.9c 123.0b 142.1b 120.7b 30.8g 304.3b 74.8c 112.6c 197.2bc 121.7bc 88.2b 127.3b 190.4b 100.1efg 2250.1c

378.5a 118.1a 2.1i 72.3a 129.8b 238.1a 264.4a 24.5gh 470.6a 196.4a 172.7a 449.7a 250.4a 86.0b 139.6ab 494.9a 92.3gh 4003.9a

120.8d 59.9d 3.7hi 0.0g 94.8c 47.9d 62.7cde 51.1f 148.1d 23.8de 63.0ef 171.5c 110.8c 52.3c 82.4c 164.9c 69.3j 1505.3e

5.38 2.28 2.58 3.10 5.20 6.66 12.92 3.65 12.38 8.73 5.16 10.75 8.27 4.05 4.88 8.03 3.75 79.57

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FlavoursA Sweet Bitter Acidic Aged

134.5j 147.8j 32.7i 21.7l

199.8ij 231.4ghi 112.7gh 51.2hijl

120.6j 133.9j 25.5i 24.1l

266.1fghi 281.9fg 150.1efg 68.3ghij

126.4j 171.3ij 47.0i 37.8jl

300.9efgh 316.1ef 166.3efg 81.4fgh

186.6ij 191.3hij 76.4hi 44.7ijl

288.3fgh 314.3ef 178.3ef 77.5fghi

240.3hi 256.6fgh 129.6fgh 81.2fgh

342.9ef 371.4e 193.2e 96.5fg

327.0efg 327.9ef 194.3e 105.4f

255.9ghi 303.8efg 152.2efg 76.9fghi

266.9fghi 302.6efg 162.6efg 84.8fgh

469.1c 557.0d 365.9c 249.3d

452.8cd 560.7d 363.5c 237.1d

681.6b 860.6b 589.5b 383.5b

536.1c 743.1c 536.9b 345.6c

1107.7a 1220.9a 826.8a 688.8a

372.2de 568.9d 255.9d 168.7e

29.77 25.83 20.07 12.37

***

***

***

***

***

***

***

***

***

***

***

***

In same row, means with different letters are significantly different. SE – standard error. P P P A Bitter flavour = of leucine, valine, isoleucine, methionine and phenylalanine. Sweet flavour = of alanine, glycine, threonine, serine and proline. Acid flavour = of glutamic acid, aspartic acid and histidine. Aged P flavour = of lysine, tyrosine and aspartic acid. * P < 0.05. ** P < 0.01. *** P < 0.001.

L.C. Roseiro et al. / Meat Science 84 (2010) 172–179

T

Lysine Histidine Arginine Tyrosine Phenylalanine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Taurine Total AA

ns

*** ***

*** ***

*** ***

***

*** ***

*** ***

ns

*** ***

***

ND 0.9e 31.1i 83.9h 10.5cde 98.3hi 0.3 78.3gh 303.3hi ND ND ND ND 2.7gh 6.8i ND 105.6bcde 115.1i Tryptamine Phenilethilamine Putrescine Cadaverine Histamine Tyramine Spermidine Spermine Total BA

ND – not detected. In same row, means with different letters are significantly different. ns = not significant. SE – standard error. * P < 0.05. ** P < 0.01. *** P < 0.001.

ND 2.3e 6.4i 10.0h 5.3efgh 6.7i ND 63.1hi 93.9i

ND 0.9e 170.9hi 135.2gh 14.5cd 154.4ghi ND 94.2defg 570.1ghi

ND 2.0e 5.4e 17.0h 21.0ab 13.0i ND 81.2fgh 139.7i

ND ND 417.2fgh 289.7f 24.9a 298.4efg 1.59 100.1cde 1131.9fg

7.04 21.1de 158.2hi 19.3h 5.3efgh 166.7ghi 3.26 102.9cde 483.9hi

10.9 21.9de 803.4de 509.6e 4.8efgh 454.2de 7.9 93.3efg 1905.9e

ND ND 186.6hi 59.0h 8.6defg 156.9ghi 2.3 94.6defg 508.0hi

ND 21.1de 486.5efgh 299.8f 1.06h 292.6efg 5.9 52.7i 1159.6fg

ND ND 327.0ghi 137.6gh 15.4bc 232.8fgh 1.7 122.6ab 1837.1gh

5.6 33.2de 751.6ef 710.6cd 14.4cd 359.3ef 4.9 82.4fgh 1962.1e

3.4 14.9de 613.7efg 275.3fg 6.3efgh 616.5cd 3.7 94.5defg 1628.3ef

36.2 125.9ab 1671.9b 1807.2b 7.6efg 860.5b 43.6 113.3abcd 3946.3b

26.7 87.7c 1505.9b 668.4cd 4.2fgh 838.9b 30.9 125.9a 3288.8cd

35.4 43.3d 1111.5cd 807.5c 8.6defg 603.9cd 15.9 94.9defg 2720.0d

31.9 93.7bc 1452.3bc 566.3de 3.25gh 734.9bc 11.9 108.3abcde 3002.8cd

30.3 129.5a 2720.7a 1237.0a 9.5cdef 1289.2a 8.8 99.5cdef 5524.6a

27.9 137.4a 1756.6b 639.0de 6.3efgh 868.4b 14.2 115.8abc 3565.6bc

3.87 ns 12.06 ns 123.85 *** 52.48 *** * 2.15 62.18 ** 4.8 ns ** 6.91 210.69 ***

RT B SE M S9

T M

S8

T M

S7

T M

S6

T M

S5

T M

S4

T M

S3

T M

S2

T M

Ripening length

R0

S1

T Batch

Table 4 Biogenic amine concentrations (mg kg

1

DM) at different processing stages and storage periods of ‘‘Chouriço Grosso” manufactured by traditional and modified processing methods.

*** *

RT  B

L.C. Roseiro et al. / Meat Science 84 (2010) 172–179

177

for the different microbial groups decreased substantially, as shown in Table 2. Despite the assumption that muscle proteinases predominate in proteolysis development along the dry-fermented sausages ripening, those from bacteria also participate in the process, mainly during the fermentation stage (Hughes et al., 2002; Molly et al., 1997; Verplaetse, 1994). Raw materials mixture had free amino acids profile similar to those detected in products analysed at different processing and storage stages, having taurine as the highest concentrated FAA (98.9 mg 100 g 1 DM), followed, in a decreasing order, by alanine, serine, leucine, arginine, valine, isoleucine and glutamic acid (all having values higher than 20 mg 100 g 1 DM) and the aspartic acid in last with only 0.3 mg 100 g 1 DM (Table 3). Concerning the amino acids precursors of BA followed up in the present study, apart from arginine (32.0 mg 100 g 1 DM), they all had mean concentrations lower than 20 mg 100 g 1 DM. These results do not totally agree with those reported by Díaz, Fernández, Garcia de Fernando, De la Hoz, and Ordóñez (1993), Zapelena, Zalacain, De Peña, Astiasarán, and Bello (1997), Bolumar, Nieto, and Flores (2001) and Roseiro et al. (2008), with the variation being assigned to factors like animal breed, diet and also intrinsic quality condition (PSE/ DFD) (Flores, Moya, Aristoy, & Toldrá, 2000). Arginine and taurine were the only exceptions having concentrations in raw material mixture (32.0 and 98.9 mg 100 g 1 DM) higher than those observed in products for the processing and storage periods analysed (e.g. 7.7 mg 100 g 1 and 10.7 mg 100 g 1 DM of arginine-S6 and S7 of batch T; 94.7 mg 100 g 1 and 83.3 mg 100 g 1 DM of taurine-S6 and S7 of batch M). A remarkable accumulation of FAA was detected along the ripening process, developed during processing (454.6 mg 100 g 1 in raw materials vs 957.6 mg 100 g 1 and 992.2 mg 100 g 1 DM in S6 of batch T and M, respectively) and storage (1795.2 mg 100 g 1 and 1742.1 mg 100 g 1 DM in S7 from batch T and M, respectively), confirming a relevant muscle aminopeptidases activity (Toldrá, 1992; Toldrá, Miralles, & Flores, 1992). Aspartic acid appeared as the most representative case of increasing in the initial concentration (0.3 mg 100 g 1 DM), reaching around 72 and 65-fold in S6 and approximately 261- and 297-fold in products ripened in storage conditions for 3 months (S7), from batches T and M, respectively. Apart very few exceptions, related to arginine and taurine, the FAA mean concentrations in batch T were superior to those from batch M. Regarding arginine, the lower levels observed in samples from batch T could be associated to their dynamics in Enterobacteriaceae development, referred as having great ability in metabolise this AA into BA. The use of fermentation temperatures in batch T ranging between 23 °C and 25 °C strongly enhanced the activity of muscle cathepsines and exopeptidases (Sentandreu & Toldrá, 2001). Otherwise, the use of alternating milder aging temperatures (14–16 °C), which also characterized the processing of batch T, has also been referred to produce the development of FAA at a faster rate (Kemp, Smith, & Moody, 1968). Significant differences (P < 0.05) between batches were obtained for every FAA analysed, but they were mostly focused in the fermentation stage of processing, up to day 14, and in final products held in storage for 6 and 12 months (Table 3). The most frequently consumed end products (S6 and S7), did not differ (P > 0.05) in total FAA between batches, despite the differences (P < 0.05) obtained for taurine, serine and arginine, in agreement with the results obtained by Bolumar et al. (2001). Irrespective of the batch, the glutamic acid became the most concentrated FAA in both ripening stages (101.6 mg 100 g 1 DM and 111.0 mg 100 g 1 DM – S6; 233.8 mg 100 g 1 DM and 220.9 mg 100 g 1 DM – S7, of batches T an M, respectively), followed by leucine > alanine > taurine > serine > alanine – S6 of batch T; taurine > alanine > leucine > serine > lysine – S6 of batch M and, coincidently

178

L.C. Roseiro et al. / Meat Science 84 (2010) 172–179

in S7 of both batches, by serine > leucine > alanine > proline > valine. Among the 6 more concentrated FAA detected in S6 and S7 end products, only minor differences existed in their ranking between batches, but the relative concentration obtained in the former ripening stage increased about 100% when the storage period was extended for 3 months. Differently, in S8 and S9 products the processing technology affected significantly the total FAA (2607.9 mg 100 g 1 DM – batch T vs. 2250.1 mg 100 g 1 DM – batch M and 4003.9 mg 100 g 1 DM – batch T vs. 1505.3 mg 100 g 1 DM – batch M, respectively), reflecting the involvement of 8 and 16 FAA, respectively (Table 3). The ripening length under storage conditions also influenced the concentration of FAA involved in the expression of sweet, bitter, acidic and aged sensorial attributes. This was traduced by important rises in the values of those FAA groups, in a straight way in products from batch T but causing a significant decrease from S8 to S9 condition of batch M. Such effect on concentration was more pronounced during the first 3 months of storage, with values reaching about +80% for the groups related to sweetness and bitterness, +140% and +225% for those contributing to the acidic and aged traits in products from batch T and a little less intense in those from batch M (approximately +70%, +80%, +120% and +180%, respectively). Differences between batches only emerged after 6 months of storage, with products from batch T having significantly higher levels of FAA related to sweet, bitter and aged tastes when compared to those from batch M. This trend substantially increased in products ripened for 1 year and extended to the group responsible for the acidic sensorial characteristic as well (826.8 mg 100 g 1 DM vs. 255.9 mg 100 g 1 DM). Reflecting the effect operated in the microbial populations, the processing conditions and the ripening time also influenced significantly most of the BA analysed, individually or as result of their interaction (Table 4). However, a large variability in individual BA concentration occurred even when similar microbiological profiles existed, proving that their formation is dependent on a complex interaction of factors (Suzi & Gardini, 2003), being strain dependent rather than species dependent. Overall, the total BA mean content (including endogenous polyamines) was higher in products from batch T, with differences being particularly important (P < 0.05) for S3, S4, S7 and S9. Comparatively to the amount of putrescine + cadaverine + tyramine detected immediately after processing (1821.5 mg kg 1 DM and 1505.5 mg kg 1 DM in S6 of batches T and M, respectively), that BA concentration doubled in products ripened for additional 3 months (S7). This fact occurred when Enterobacteriaceae were already not detected, which could meant that these microorganisms are capable of releasing decarboxylases which remain active for long periods (Bover-Cid, Izquierdo-Pulido, & Vidal-Carou, 2001). The substantial increasing of BA concentration in products ripened for 6 months and 1 year could also derive from transamination of aldehydes and ketones (VidalCarou, Izquierdo-Pulido, Martin-Morro, & Mariné-Font, 1990; Halász et al., 1994). BA typical quantitative sequence varied between batches T and M according to the ripening stage of the processing and storage phases. Batch T products showed a tyramine > cadaverine > putrescine sequence at the very early fermentation stage (S1), which changed to putrescine > tyramine > cadaverine in S2 and S3 samples. Afterwards, putrescine > cadaverine > tyramine prevailed between S4 and S8, with S9 samples turning back to putrescine > tyramine > cadaverine sequence. Differently, batch M products had cadaverine > tyramine > putrescine sequence before the occurrence of the fermentation process (S1 and S2), but when in S3 sample the total microbial counts attained similar numbers to those verified in batch T, the sequence changed to tyramine > putrescine > cadaverine. However, putrescine became rapidly the most concentrated compound in S4, followed by tyramine and cadaverine, keeping their

relative positions further on. Differences in BA profiles and concentrations between batches during the processing phase reflect, basically, the distinct evolution of microbial populations. The almost immediate significant rising in LAB counts in batch T promoted, in a first instance, the tyramine as the highest concentrated amine, but the concomitant development of Enterobacteriaceae also originated the increase of putrescine and cadaverine levels, with the later showing, in most samples, the second higher concentration. If tyramine is closely related to lactic acid fermentation due to the potential of many LAB in tyrosine decarboxylation, the source of putrescine is more controversial as it may appear through the ornithine pathway (LAB) or, alternatively, from arginine decarboxylation by many Enterobacteriaceae (Miguélez-Arrizado, Bover-Cid, Latorre-Moratalla, & Vidal-Carou, 2006). Comparatively, the holding of batch M products in chilling proximate conditions after casings filling delayed significantly the development of natural flora, which determined the lower development of tyrosine and putrescine, the 2nd and 3rd most concentrated BA. However, under such environmental conditions, the multiplication of Enterobacteriaceae was also significantly delayed, which explains the highest concentration of tyramine in S3 samples. The trend observed by Bover-Cid, Miguelez-Arrizado, Latorre-Moratalla, and Vidal-Carou (2006), with cadaverine appearing in higher levels than putrescine when raw materials were not frozen before sausage manufacturing was not confirmed in this work. Histamine was present in both products for every ripening stages, but the levels in end products were low. The concentrations found in S6 products of batches T (14.4 mg kg 1 DM) and M (6.29 mg kg 1 DM) stayed almost unchanged for longer ripening periods. The capability to decarboxylate histidine does not seem to be widely distributed among meat microrganisms, but seems to be a characteristic of specific strains of some Enterobacteriaceae and LAB species (Bover-Cid and Holzapfel, 1999; Bover-Cid, Hugas, Izquierdo-Pulido, & Vidal-Carou, 2001; Suzi & Gardini, 2003). The direct relationship between histamine accumulation and the pH of fermented sausages reported by Maijala et al. (1993) was not confirmed, since batch T, despite the lower pH of their products also showed higher histamine levels than counterparts of batch M, in more than half of samples analysed during the processing phase. Nevertheless, our results are similar to those reported by other authors (González-Fernández, Santos, Jaime, & Rovira, 2003; Hernández-Jover, Izquierdo-Pulido, Veciana-Nogué, Mariné-Font, & Vidal-Carou, 1997; Parente et al., 2001). Phenylethylamine and tryptamine represented minor BA in ‘‘Chouriço Grosso de Estremoz e Borba”, they were not always present and their concentration denoted a irregular evolution along the ripening process. Concerning the physiological polyamines, the amounts of spermine were higher than those of spermidine, with this compound, unexpectedly, not being detected in raw material mixtures and during the initial stages of processing in both batches. The concentrations of spermine were, in general, expressively higher than those detected in similar fermented sausages studied before (Roseiro et al., 2006), also disagreeing with values reported by other authors for fresh pork (Hernández-Jover, Izquierdo-Pulido, Veciana-Nogué, Mariné-Font, & Vidal-Carou, 1996; Lorenzo, Martínez, Franco, & Carballo, 2007; Maijala & Eerola, 1993). It showed irregular evolution behaviour, specially during the fermentation stage of processing, possibly due to the use by some microrganisms as a nitrogen source (Hernández-Jover et al., 1997; Tabor & Tabor, 1985).

4. Conclusions The processing environmental conditions applied during the initial stages of drying/smoking operation had an influence on

L.C. Roseiro et al. / Meat Science 84 (2010) 172–179

the accumulation of free amino acids in final products, with those developing a long ripening process in storage before consumption tending to present different concentrations in compounds involved in the expression of sweet, bitter, acidic and aged sensorial attributes. Also the typical quantitative sequences of biogenic amines varied according to the processing temperature along the ripening stages, reflecting the distinct pH values and microbial development rates and profiles observed during the fermentation process. Due to the increasing concentrations of total BA observed along the processing and storage stages under both production systems, reaching alarming levels of tyramine, putrescine and cadaverine, the end products should not be extend ripened without decarboxylase negative starter cultures addition.

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