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The effects of a starter culture on the formation of biogenic amines in Turkish soudjoucks
Meat Science 53 (1999) 183±188
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The eects of a starter culture on the formation of biogenic amines in Turkish soudjoucks $
Kamuran Ayhan a,*, Nuray Kolsarici a, GuÈleren Alsancak OÈzkan b a
Department of Food Engineering, Faculty of Agriculture, University of Ankara, Ankara, Turkey b Department of Chemistry, Faculty of Science, University of Ankara, Ankara, Turkey
Received 15 February 1999; received in revised form 18 February 1999; accepted 28 February 1999
Abstract The eects of starter culture containing Lactobacillus sake, Pediococcus pentosaceus, Staphylococcus carnosus plus Staphylococcus xylosus on the formation of biogenic amines during ripening of Turkish soudjoucks were investigated. Determination of eight different biogenic amines was carried out by reverse-phase high performance liquid chromatography (HPLC) using diode array detection. It was found that aerobic plate counts (APC) decreased in the samples with added starter culture. Lactic acid bacteria counts increased in both the controls and samples with added starter during ripening. Total Enterobacteriaceae (ENT) counts decreased on the 30th day for vacuum packed and starter added samples. Yeast counts decreased only in vacuum packed and starter added samples on the 30th day. No biogenic amines were found in ground meat samples. Putrescine (PU) and tyramine (TYR) were determined in all control samples during the storage at 4 C. Starter addition inhibited formation of PU but not TYR. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Biogenic amines are the basic nitrogenous compounds occurring in food, such as ®shery products, wine, cheese, beer and other fermented foods, resulting from decarboxylation of amino acids or transamination of aldehydes and ketonesÐor formed by bacteria, including starter culture (Maijala, Eerola, Lievonen, Hill, & Hirvi, 1995; Maijala, Nurmi, & Fisher, 1995). The presence of biogenic amines in food should not exceed hazardous limits for human beings. Two biogenic amines, histamine and tyramine (TYR), are used as indicators, especially in ®sh and ®sh products (HernandezJover, Izquierdo-Pulido, Vecina-Nogues, & Vidal-Crou, 1996; Hwang, Chang, Shiau, & Cheng, 1995). Food poisoning may occur by exogenous biogenic amines when combined with factors such as monoamine oxidaseÐinhibiting drugs, alcohol, other food amines and gastro-intestinal diseases (Maijala & Eerola 1993;
$ This study was supported by Ankara University Fund (Project no. 97-11-12-02). * Corresponding author. Tel.: +90-312-317-0550 ext.1455; fax: +90-312-317-8711. E-mail address: [email protected] (K. Ayhan)
Hernandez-Jover, Izquierdo-Pulido, Vecina-Nogues, & Videl-Crou, 1997). The toxic limits for biogenic amines are that histamine levels of 8±40 mg, 40±100 mg and over 100 mg may cause slight, intermediate and intensive poisoning, respectively, and TYR contents over 100 mg may cause migraine (Maijala & Eerola 1993). Hernandez-Jover et al. (1997) reported that the toxicity level of b-phenylethylamine was 30 mg/kg in foods and suggested that putrescine (PU) and cadaverine (CAD) were histamine/ TYR potentiators. However, they did not recommend toxicity limits (Hernandez-Jover et al., 1997a). The formation of biogenic amines in fermented foods is related to the presence of micro-organisms which are able to produce decarboxylase under appropriate conditions for the synthesis of the decarboxylase enzyme and co-factors, when appropriate amino acids are present (Maijala & Eerola, 1993; Maijala, Eerola, 1995). There are several studies concerning the levels of biogenic amines in meat and meat products (Cantoni et al., 1986; Eitenmiller et al., 1978; Hernandez-Jover et al., 1997a,b; SantosBuelgo, Pena-Egido, & Rivas-Gonzalo, 1986; RoigSagues, Hernandez-Herrero, Rodriguez-Jerez, LopezSabater, & Mora-Ventura, 1997; Maijala et al., 1993, 1995a,b; Slemr et al., 1985) and the aim of this study was to determine the eects of the starter culture on the
0309-1740/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(99)00046-7
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K. Ayhan et al. / Meat Science 53 (1999) 183±188
formation of biogenic amines during the ripening of Turkish soudjoucks. 2. Materials and methods 2.1. Soudjouck production Turkish style soudjouck consisting of beef and tallow fat (rendered from the tail of mutton added at the rate of 15% and cut into small pieces) ground together and mixed with other ingredients (2.5% ascorbate, 1% hot pepper, 0.06% pepper, 1.0% cumin, 2.0% NaCl, 0.6% sucrose, 2.5% garlic, and 0.05% sodium nitrate) was held at +4 C overnight before stung. The soudjouck mixture was divided into two portions. One was fermented by the starter culture (Biobak-K, Wiberg, Germany) containing Lactobacillus sake, Pediococcus pentosaceus, Staphylococcus carnosus plus Staphylococcus xylosus and the other was not fermented. The starter culture (50 g/100 kg) was added and mixed thoroughly before stung, both mixes were stued into cows intestine casings to make weights between 350 and 400 grams. After fermentation for 3 days at 242 C and 90±95% relative humidity followed by 5 days at 222 C and 80±85% relative humidity. Each group was divided into two (for vacuum and non-vacuumed packed by using polyethylene bags which are 5 micronthick ®lm constructed of polyamide, ionomer and polyethylene combinations, having O2 permeability of 45 cm3/m2/24 h at 25 C and 0% RH, and water transmission of <3.5 g/m2/24 h at 38 C and 90% RH). All samples were stored for 30 days at +4 C (Kolsarici, Soyer, & Turhan, 1993). 2.2. Sampling Two samples were taken from each group after stuing and on the 1st, 2nd, 3rd, 6th, 8th, 15th, 30th day of storage and their microbiological and biogenic amine contents determined. 2.3. Analytical methods Ten-gram samples were cut into small pieces, placed in 90 ml distilled water and the pH measured using a pH meter (Knick, pH-mV-meter, Germany) (Lees, 1975). Five grams of sample was dried at 1052 C to determine its dry matter content (Lees, 1975). All analyses were performed in duplicate. 2.4. HPLC analyses 2.4.1. Apparatus High performance liquid chromatography (HPLC) determinations were performed with a LC 10 AD
Schimadzu LC and CTO 10 A Schimadzu column oven (37 C) and SIL-10 AXL, autoinjecter with a 50 ml loop. Detection was at 254 nm with a Schimadzu diode array detector. LC column C18-Phenomenex Luna (2504.6 mm, 5 mm). was used. 2.4.2. Reagents Amine standard solutions were prepared in 0.4 M perchloric acid to a ®nal concentration of 10 mg/50 ml for each biogenic amine. Tyramine (TYR), putrecine (PUT), cadaverine (CAD), tryptamine (TRY), phenylethylamine (PHA), spermine (SPM), spermidine (SPD) were obtained from Sigma Chemical Co. (St. Louis, MO). For analyses, 5 ml of each stock solution was diluted to 50 ml with 0.4 M perchloric acid. Ten mg dansyl chloride was made up in 1 ml acetone. All solutions were prepared daily (BuÈtikofer, Funchs, Hurni, & Bosset, 1990). 2.4.3. Mobile phase Buer (pH 8, 0.1 M tris/0.1 M acetic acid/water, 2/1/ 2); solvent A: buer (30 ml)/acetonitrile (550 ml)/water/ 420 ml); solvent B: buer (2 ml)/acetonitrile (900 ml)/ water/100 ml, were used. 2.4.4. Sample preparation and extraction Two grams of sample was mixed with 10 ml 0.4 M perchloric acid using a Vortex mixer and then 40 ml perchloric acid was added and the whole mixed in a Waring blender. Ten ml of this solution was centrifuged at 1250g for 20 min and 1 ml of the supernatant kept in ÿ200 C (Eerola, Hinkkanen, Lindfors, & Hirvi, 1993). 2.4.5. Derivatization of sample extracts and mixed standards Four-hundred ml of sodium carbonate solution was added to 400 ml of the extract, mixed with 400 ml dansyl chloride and held at 40 C in a water bath for 30 min. To remove residual dansyl chloride, 200 ml sodium glutaminate was added and the solution held at 40 C for a further 60 min. After adding 1 ml of acetonitrile the samples were centrifuged at 1250g for 20 min and the supernatant ®ltered through a 0.45 mm ®lter. Dansyl derivatives of the calibration standards were mixed with the samples as previously described (Eerola et al., 1993; BuÈtikofer et al., 1990). 2.4.6. Microbiological analyses A 25-g of sample was mixed with 225 ml sterile peptone solution in a Waring blender and homogenized for 2 min. Serial dilutions were made with the same solution. Aerobic plate counts (APC) were performed by plating appropriate dilutions on standard plate count agar for total counts (Merck), MRS agar plates for lactic acid bacteria (de Man, Rogose, Sharpe agar, Merck),
K. Ayhan et al. / Meat Science 53 (1999) 183±188
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counts increased during fermentation for both control and starter added samples. Dierences were not statistically signi®cant after the 3rd day. Initial LAB counts in the starter added samples were statistically higher than in the control samples. No signi®cant dierences were observed in total ENT counts. Yeast counts increased gradually during the ®rst 3 days for both control and starter added samples. However, decreasing numbers were observed after the 3rd day. Yeasts counts in the starter added samples were lower than in the controls (p < 0:05). pH values in 2 treatments were similar and decreased slightly on the 2nd day of fermentation. Signi®cant decreases were observed in (APC) for nonvacuum packed and starter-added samples on the 1st and 15th day of storage. Although some decreases were observed in LAB counts during storage in both nonvacuum and vacuum packed and starter-added samples, dierences were not signi®cant (p < 0:05). Total ENT
VRBG agar for Enterobacteriaceae (Merck), OGYE agar for moulds and yeasts (Oxoid), which were incubated at 28 C for 48 h, 30 C for 48 h, 37 C for 48 h and 28 C for 72 h (yeasts) and 5 days (moulds), respectively. 2.4.7. Statistical analyses Results were analysed using SPSS and multiple analyses of variance (ANOVA). 3. Results and discussion Microbial counts are presented in Tables 1 and 2. increased in both the controls and starter added samples during production. Dierences in APC for the starter added samples after the 1st day and for the control samples after the 2nd day were not signi®cant (p < 0:05). However, APC was less in the starter added samples for days 1 to 8. Lactic acid bacteria (LAB)
Table 1 Microbiological counts during production of Turkish soudjoucks (log10CFU/g)a Bacteria b
b
APC 5.6
Ground meat
ENTb 2.4
LAB 3.0
Yeasts 4.3
pH
Days
Control
Starter
Control
Starter
Control
Starter
Control
Starter
Control
Starter
0 1 2 3 6 8 LSD
5.60c 7.45b 7.50a 7.85a 7.97a 7.78a 0.49
6.00c 6.69a 6.80a 6.82a 6.87a 6.95a
4.00c 6.37b 6.46b 7.71a 7.71a 7.39a 0.45
5.00d 6.88c 7.04bc 7.48ab 7.65a 7.31ab
3.20 3.28 2.49 2.58 2.78 2.30 NS
3.20 2.50 2.30 1.84 1.85 2.25
4.30b 5.42ab 5.54a 5.09bc 4.97c 4.20d 0.42
4.30bc 4.77a 4.70ab 4.35bc 4.12c 4.11c
6.14 5.48 4.96 5.03 5.04 5.14
6.14 5.13 4.94 4.96 5.08 5.05
a b
Within one parameter, values with the same letter are not signi®cantly dierent (p<0.05). APC, aerobic plate counts; LAB, lactic acid bacteria counts; ENT, total Enterobacteriaceae counts.
Table 2 Microbiological counts during storage of Turkish soudjoucks at +4 C (log10 CFU/g)a Control b
Starter b
Bacteria
Days
NV
pH
V
Aerobic plate counts
0 15 30 0 15 30 0 15 30 0 15 30
7.78ab 7.67a 7.45a 7.39a 7.60a 7.45a 2.30a 1.75ab 1.85a 4.20a 4.48a 4.43a
5.14 4.94 5.07 ± ± ± ± ± ± ± ± ±
7.78ab 7.72a 7.40a 7.39a 7.68a 7.30a 2.30a 1.65a 1.15b 4.20a 4.42a 4.64a
Lactic acid bacteria Enterobacteriaceae Yeasts
a b
pH
NV
pH
V
pH
5.15 5.03 5.01 ± ± ± ± ± ± ± ± ±
6.95bc 6.95bc 6.57a 7.31abc 6.43b 6.75ab 2.25a 2.25a 2.05a 4.11a 4.22a 4.83a
5.03 5.06 5.30 ± ± ± ± ± ± ± ± ±
6.95bc 6.50b 6.00b 7.31abc 6.47b 5.95b 2.25a 1.65a 1.15b 4.11a 4.89a 3.08b
5.09 5.04 5.05 ± ± ± ± ± ± ± ± ±
Within one parameter, values with the same letter are not signi®cantly dierent (p<0.05). NV, non-vacuum; V, vacuum packaged (LSD=0,8601, p<0.05).
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K. Ayhan et al. / Meat Science 53 (1999) 183±188
counts started to decrease signi®cantly in vacuum packed for both the controls and starter-added samples on the 30th day signi®cantly. Yeast counts decreased only in vacuum packed and starter added samples on the 30th day (p < 0:05). Biogenic amine contents are shown in Table 3. Biogenic amines were not found in the ground meat samples. PU, TRY and PHA were found in the control samples during production. PU and TRY were found in all controls during storage at 4 C. PU was not found in samples containing starter, but TYR was. Starter addition thus decreases biogenic amine contents as reported by Hernandez et al. (1997a) and Maijala, Nurmi, 1995). All the amines were well separated on reverse-phase HPLC. The order of elution was tryptamine, b-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine (Fig. 1) and the
chromatogram for amines extracted from a soudjouck sample are shown in Fig. 2. Reproducibility in the chromatograms was assured by injecting a standard solution 4 times and determining the standard deviation of the resulting peak areas. This was always less than 1%. Daily checks of the retention times (11 injections), was less than 0.3% for all biogenic amines. Peak purity was evaluated with the diode-array detector (Moret, Bartolomeazzi, & Lercker, 1992). Although there are no legal limits for biogenic amines, the values found in this study are close to the lower recommended limits, 100±800 mg of TYR (Hernandez et al., 1997b) and 396 mg of PU (Stratton et al., 1991) per kg of food. As reported previously, it is possible to ®nd high levels of biogenic amines from high quality raw materials if incorrectly processed (Maijala, Eerola et al., 1995). According to our results, none of
Table 3 Levels of biogenic amines during Turkish soudjouck production and storage (mg/kg)a Ground meat Days 2 3 6 8 15 30 a b c
Putrecine
Tyramine
Phenylethylamine
ND
ND
ND
Control
Starter
Control
Starter
Control
Starter
NDc 1698.10 2227.16
ND ND ND
ND 2746.94 3106.14
ND ND ND
ND ND ND
ND ND ND
NVb 3966.36 3235.83 41214.5
Vb 2456.36 3406.36 22211.5
NV ND ND ND
V ND ND ND
NV 3495.45 2874.62 2535.00
V 2305.45 2895.45 2665.45
NV 2875.45 2835.00 2835.45
V 2735.00 2115.45 2057.00
NV ND ND ND
All values are the meansSD of two determinations. NV, non-vacuum; V, vacuum packaged. ND, not determined.
Fig. 1. Chromatographic separation of dansyl derivatives of biogenic amines by gradient elution.
V ND ND ND
NV ND ND ND
V ND ND ND
K. Ayhan et al. / Meat Science 53 (1999) 183±188
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Fig. 2. Chromatogram of amines extracted from a soudjouck sample (peak identi®cation as Fig. 1).
the biogenic amines were found in the ground meat, but both PU and TYR levels increased during storage in the absence of starter. The addition of starter stopped the formation of PU, but had no aect on TYR. These results suggest that dierent starter cultures and/or processing regimes could control the formation of these amines. Finally, the HPLC method presented in this work is a precise, reliable and easily used method to determine these amines.
Acknowledgements The authors would like to thank MSc. Nilufer Vural for help with the HPLC analyses.
References BuÈtikofer, U., Funchs, D., Hurni, D., & Bosset, J. O. (1990). Bestimmung biogener Amine in Kase. Mitteilungen dee Gebiete der Lebensmittelhygine, 81, 120±133. Cantoni, C., Bersani, C., Damenes, C., & Comi, G. (1994). Amines in Italian meat products. Industrie Alimentari, 33(332), 1239±1243. Eerola, S., Hinkkanen, R., Lindfors, E., & Hirvi, T. (1994). Liquid chromatographic determination of biogenic amines in dry sausages. Journal of AOAC International, 76, 575±577. Eitenmiller, R. R., Koehler, P. E., & Reagan, J. O. (1978). Tyramine in fermented sausages: Factors eecting formation of tyramine and tyrosine decarboxylase. Journal of Food Science, 43, 689±693. Hwang, D. F., Chang, S. C., Shiau, C. Y., & Cheng, C. C. (1995). Biogenic amines in the ¯esh of sail®sh (Istiophourus playpterus) responsible for scrombroid poisoning. Journal of Food Science, 60(5), 926±928.
Hernandez-Jover, T., Izquierdo-Pulido, M., Vecina-Nogues, M. T., & Vidal-Crou, M. C. (1996). Biogenic amine sources in cooked cured shoulder pork. Journal of Agricultural and Food Chemistry, 44, 3097±3101. Hernandez-Jover, T., Izquierdo-Pulido, M., Vecina-Nogues, M. T., & Vidal-Crou, M. C. (1997a). Biogenic amine and polyamine contents in meat and meat products. Journal of Agricultural and Food Chemistry, 45, 2098±2102. Hernandez-Jover, T., Izquierdo-Pulido, M., Vecina-Nogues, M. T., & Vidal-Crou, M. C. (1997b). Eect of starter cultures on biogenic amine formation during fermented sausage production. Journal of Food Protection, 60(7), 825±830. Kolsaricõ, N., Soyer, A., & Turhan, K. (1993). A study on the usability of Angora goat meat in soudjouck production. Ankara University Publications, no.1292, 16. Lees, R. (1975). In Food Analysis analytical and quality control methods for the food manufacture and buyer (3rd ed). London: Leonard Hill Books (p. 245 ). Maijala, R., & Eerola, S. (1993). Contaminant lactic acid bacteria of dry sausages produce histamine and tyramine. Meat Science, 35(3), 387±395. Maijala, R., Eerola, S., Lievonen, S., Hill, P., & Hirvi, T. (1995a). Formation of biogenic amines during ripening of dry sausages. Journal of Food Science, 60(6), 1187±1190. Maijala, R., Nurmi, F., & Fischer, A. (1995b). In¯uence of processing temperature on the formation of biogenic amines in dry sausages. Meat Science, 39(1), 19±22. Moret, S., Bortolomeazzi, R., & Lercker, G. (1992). Improvement of extraction procedure for biogenic amines in foods and high-performance liquid chromatographic determination. Journal of Chromatography, 591, 175±180. Roig-Sagues, A. X., Hernandez-Herrero, M., Rodriguez-Jerez, J. J., Lopez-Sabater, E. I., & Mora-Ventura, M. T. (1997). Occurrence of tyramine producing microorganisms in ``Salchichon'' and tyramine production in sausages inoculated with a producing strain of Lactobacillus brevis. Journal of Food Safety, 17, 13±22. Santos-Buelgo, C., Pena-Egido, M. J., & Rivas-Gonzalo, J. C. (1986). Changes in tyramine during chorizo-sausage ripening. Journal of Food Science, 51(2), 518±519.
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Slemr, J., & Beyermann, K. (1985). Concentration pro®les of diamines in fresh and aerobically stored pork and beef. Journal of Agricultural and Food Chemistry, 33(3), 336±339.
Stratton, J. E., Hutkins, R. W., & Taylor, S. L. (1991). Biogenic amines in cheese and other fermented foods: A review. Journal of Food Protection, 54(6), 460±470.
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The eects of a starter culture on the formation of biogenic amines in Turkish soudjoucks $
Kamuran Ayhan a,*, Nuray Kolsarici a, GuÈleren Alsancak OÈzkan b a
Department of Food Engineering, Faculty of Agriculture, University of Ankara, Ankara, Turkey b Department of Chemistry, Faculty of Science, University of Ankara, Ankara, Turkey
Received 15 February 1999; received in revised form 18 February 1999; accepted 28 February 1999
Abstract The eects of starter culture containing Lactobacillus sake, Pediococcus pentosaceus, Staphylococcus carnosus plus Staphylococcus xylosus on the formation of biogenic amines during ripening of Turkish soudjoucks were investigated. Determination of eight different biogenic amines was carried out by reverse-phase high performance liquid chromatography (HPLC) using diode array detection. It was found that aerobic plate counts (APC) decreased in the samples with added starter culture. Lactic acid bacteria counts increased in both the controls and samples with added starter during ripening. Total Enterobacteriaceae (ENT) counts decreased on the 30th day for vacuum packed and starter added samples. Yeast counts decreased only in vacuum packed and starter added samples on the 30th day. No biogenic amines were found in ground meat samples. Putrescine (PU) and tyramine (TYR) were determined in all control samples during the storage at 4 C. Starter addition inhibited formation of PU but not TYR. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Biogenic amines are the basic nitrogenous compounds occurring in food, such as ®shery products, wine, cheese, beer and other fermented foods, resulting from decarboxylation of amino acids or transamination of aldehydes and ketonesÐor formed by bacteria, including starter culture (Maijala, Eerola, Lievonen, Hill, & Hirvi, 1995; Maijala, Nurmi, & Fisher, 1995). The presence of biogenic amines in food should not exceed hazardous limits for human beings. Two biogenic amines, histamine and tyramine (TYR), are used as indicators, especially in ®sh and ®sh products (HernandezJover, Izquierdo-Pulido, Vecina-Nogues, & Vidal-Crou, 1996; Hwang, Chang, Shiau, & Cheng, 1995). Food poisoning may occur by exogenous biogenic amines when combined with factors such as monoamine oxidaseÐinhibiting drugs, alcohol, other food amines and gastro-intestinal diseases (Maijala & Eerola 1993;
$ This study was supported by Ankara University Fund (Project no. 97-11-12-02). * Corresponding author. Tel.: +90-312-317-0550 ext.1455; fax: +90-312-317-8711. E-mail address: [email protected] (K. Ayhan)
Hernandez-Jover, Izquierdo-Pulido, Vecina-Nogues, & Videl-Crou, 1997). The toxic limits for biogenic amines are that histamine levels of 8±40 mg, 40±100 mg and over 100 mg may cause slight, intermediate and intensive poisoning, respectively, and TYR contents over 100 mg may cause migraine (Maijala & Eerola 1993). Hernandez-Jover et al. (1997) reported that the toxicity level of b-phenylethylamine was 30 mg/kg in foods and suggested that putrescine (PU) and cadaverine (CAD) were histamine/ TYR potentiators. However, they did not recommend toxicity limits (Hernandez-Jover et al., 1997a). The formation of biogenic amines in fermented foods is related to the presence of micro-organisms which are able to produce decarboxylase under appropriate conditions for the synthesis of the decarboxylase enzyme and co-factors, when appropriate amino acids are present (Maijala & Eerola, 1993; Maijala, Eerola, 1995). There are several studies concerning the levels of biogenic amines in meat and meat products (Cantoni et al., 1986; Eitenmiller et al., 1978; Hernandez-Jover et al., 1997a,b; SantosBuelgo, Pena-Egido, & Rivas-Gonzalo, 1986; RoigSagues, Hernandez-Herrero, Rodriguez-Jerez, LopezSabater, & Mora-Ventura, 1997; Maijala et al., 1993, 1995a,b; Slemr et al., 1985) and the aim of this study was to determine the eects of the starter culture on the
0309-1740/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(99)00046-7
184
K. Ayhan et al. / Meat Science 53 (1999) 183±188
formation of biogenic amines during the ripening of Turkish soudjoucks. 2. Materials and methods 2.1. Soudjouck production Turkish style soudjouck consisting of beef and tallow fat (rendered from the tail of mutton added at the rate of 15% and cut into small pieces) ground together and mixed with other ingredients (2.5% ascorbate, 1% hot pepper, 0.06% pepper, 1.0% cumin, 2.0% NaCl, 0.6% sucrose, 2.5% garlic, and 0.05% sodium nitrate) was held at +4 C overnight before stung. The soudjouck mixture was divided into two portions. One was fermented by the starter culture (Biobak-K, Wiberg, Germany) containing Lactobacillus sake, Pediococcus pentosaceus, Staphylococcus carnosus plus Staphylococcus xylosus and the other was not fermented. The starter culture (50 g/100 kg) was added and mixed thoroughly before stung, both mixes were stued into cows intestine casings to make weights between 350 and 400 grams. After fermentation for 3 days at 242 C and 90±95% relative humidity followed by 5 days at 222 C and 80±85% relative humidity. Each group was divided into two (for vacuum and non-vacuumed packed by using polyethylene bags which are 5 micronthick ®lm constructed of polyamide, ionomer and polyethylene combinations, having O2 permeability of 45 cm3/m2/24 h at 25 C and 0% RH, and water transmission of <3.5 g/m2/24 h at 38 C and 90% RH). All samples were stored for 30 days at +4 C (Kolsarici, Soyer, & Turhan, 1993). 2.2. Sampling Two samples were taken from each group after stuing and on the 1st, 2nd, 3rd, 6th, 8th, 15th, 30th day of storage and their microbiological and biogenic amine contents determined. 2.3. Analytical methods Ten-gram samples were cut into small pieces, placed in 90 ml distilled water and the pH measured using a pH meter (Knick, pH-mV-meter, Germany) (Lees, 1975). Five grams of sample was dried at 1052 C to determine its dry matter content (Lees, 1975). All analyses were performed in duplicate. 2.4. HPLC analyses 2.4.1. Apparatus High performance liquid chromatography (HPLC) determinations were performed with a LC 10 AD
Schimadzu LC and CTO 10 A Schimadzu column oven (37 C) and SIL-10 AXL, autoinjecter with a 50 ml loop. Detection was at 254 nm with a Schimadzu diode array detector. LC column C18-Phenomenex Luna (2504.6 mm, 5 mm). was used. 2.4.2. Reagents Amine standard solutions were prepared in 0.4 M perchloric acid to a ®nal concentration of 10 mg/50 ml for each biogenic amine. Tyramine (TYR), putrecine (PUT), cadaverine (CAD), tryptamine (TRY), phenylethylamine (PHA), spermine (SPM), spermidine (SPD) were obtained from Sigma Chemical Co. (St. Louis, MO). For analyses, 5 ml of each stock solution was diluted to 50 ml with 0.4 M perchloric acid. Ten mg dansyl chloride was made up in 1 ml acetone. All solutions were prepared daily (BuÈtikofer, Funchs, Hurni, & Bosset, 1990). 2.4.3. Mobile phase Buer (pH 8, 0.1 M tris/0.1 M acetic acid/water, 2/1/ 2); solvent A: buer (30 ml)/acetonitrile (550 ml)/water/ 420 ml); solvent B: buer (2 ml)/acetonitrile (900 ml)/ water/100 ml, were used. 2.4.4. Sample preparation and extraction Two grams of sample was mixed with 10 ml 0.4 M perchloric acid using a Vortex mixer and then 40 ml perchloric acid was added and the whole mixed in a Waring blender. Ten ml of this solution was centrifuged at 1250g for 20 min and 1 ml of the supernatant kept in ÿ200 C (Eerola, Hinkkanen, Lindfors, & Hirvi, 1993). 2.4.5. Derivatization of sample extracts and mixed standards Four-hundred ml of sodium carbonate solution was added to 400 ml of the extract, mixed with 400 ml dansyl chloride and held at 40 C in a water bath for 30 min. To remove residual dansyl chloride, 200 ml sodium glutaminate was added and the solution held at 40 C for a further 60 min. After adding 1 ml of acetonitrile the samples were centrifuged at 1250g for 20 min and the supernatant ®ltered through a 0.45 mm ®lter. Dansyl derivatives of the calibration standards were mixed with the samples as previously described (Eerola et al., 1993; BuÈtikofer et al., 1990). 2.4.6. Microbiological analyses A 25-g of sample was mixed with 225 ml sterile peptone solution in a Waring blender and homogenized for 2 min. Serial dilutions were made with the same solution. Aerobic plate counts (APC) were performed by plating appropriate dilutions on standard plate count agar for total counts (Merck), MRS agar plates for lactic acid bacteria (de Man, Rogose, Sharpe agar, Merck),
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counts increased during fermentation for both control and starter added samples. Dierences were not statistically signi®cant after the 3rd day. Initial LAB counts in the starter added samples were statistically higher than in the control samples. No signi®cant dierences were observed in total ENT counts. Yeast counts increased gradually during the ®rst 3 days for both control and starter added samples. However, decreasing numbers were observed after the 3rd day. Yeasts counts in the starter added samples were lower than in the controls (p < 0:05). pH values in 2 treatments were similar and decreased slightly on the 2nd day of fermentation. Signi®cant decreases were observed in (APC) for nonvacuum packed and starter-added samples on the 1st and 15th day of storage. Although some decreases were observed in LAB counts during storage in both nonvacuum and vacuum packed and starter-added samples, dierences were not signi®cant (p < 0:05). Total ENT
VRBG agar for Enterobacteriaceae (Merck), OGYE agar for moulds and yeasts (Oxoid), which were incubated at 28 C for 48 h, 30 C for 48 h, 37 C for 48 h and 28 C for 72 h (yeasts) and 5 days (moulds), respectively. 2.4.7. Statistical analyses Results were analysed using SPSS and multiple analyses of variance (ANOVA). 3. Results and discussion Microbial counts are presented in Tables 1 and 2. increased in both the controls and starter added samples during production. Dierences in APC for the starter added samples after the 1st day and for the control samples after the 2nd day were not signi®cant (p < 0:05). However, APC was less in the starter added samples for days 1 to 8. Lactic acid bacteria (LAB)
Table 1 Microbiological counts during production of Turkish soudjoucks (log10CFU/g)a Bacteria b
b
APC 5.6
Ground meat
ENTb 2.4
LAB 3.0
Yeasts 4.3
pH
Days
Control
Starter
Control
Starter
Control
Starter
Control
Starter
Control
Starter
0 1 2 3 6 8 LSD
5.60c 7.45b 7.50a 7.85a 7.97a 7.78a 0.49
6.00c 6.69a 6.80a 6.82a 6.87a 6.95a
4.00c 6.37b 6.46b 7.71a 7.71a 7.39a 0.45
5.00d 6.88c 7.04bc 7.48ab 7.65a 7.31ab
3.20 3.28 2.49 2.58 2.78 2.30 NS
3.20 2.50 2.30 1.84 1.85 2.25
4.30b 5.42ab 5.54a 5.09bc 4.97c 4.20d 0.42
4.30bc 4.77a 4.70ab 4.35bc 4.12c 4.11c
6.14 5.48 4.96 5.03 5.04 5.14
6.14 5.13 4.94 4.96 5.08 5.05
a b
Within one parameter, values with the same letter are not signi®cantly dierent (p<0.05). APC, aerobic plate counts; LAB, lactic acid bacteria counts; ENT, total Enterobacteriaceae counts.
Table 2 Microbiological counts during storage of Turkish soudjoucks at +4 C (log10 CFU/g)a Control b
Starter b
Bacteria
Days
NV
pH
V
Aerobic plate counts
0 15 30 0 15 30 0 15 30 0 15 30
7.78ab 7.67a 7.45a 7.39a 7.60a 7.45a 2.30a 1.75ab 1.85a 4.20a 4.48a 4.43a
5.14 4.94 5.07 ± ± ± ± ± ± ± ± ±
7.78ab 7.72a 7.40a 7.39a 7.68a 7.30a 2.30a 1.65a 1.15b 4.20a 4.42a 4.64a
Lactic acid bacteria Enterobacteriaceae Yeasts
a b
pH
NV
pH
V
pH
5.15 5.03 5.01 ± ± ± ± ± ± ± ± ±
6.95bc 6.95bc 6.57a 7.31abc 6.43b 6.75ab 2.25a 2.25a 2.05a 4.11a 4.22a 4.83a
5.03 5.06 5.30 ± ± ± ± ± ± ± ± ±
6.95bc 6.50b 6.00b 7.31abc 6.47b 5.95b 2.25a 1.65a 1.15b 4.11a 4.89a 3.08b
5.09 5.04 5.05 ± ± ± ± ± ± ± ± ±
Within one parameter, values with the same letter are not signi®cantly dierent (p<0.05). NV, non-vacuum; V, vacuum packaged (LSD=0,8601, p<0.05).
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counts started to decrease signi®cantly in vacuum packed for both the controls and starter-added samples on the 30th day signi®cantly. Yeast counts decreased only in vacuum packed and starter added samples on the 30th day (p < 0:05). Biogenic amine contents are shown in Table 3. Biogenic amines were not found in the ground meat samples. PU, TRY and PHA were found in the control samples during production. PU and TRY were found in all controls during storage at 4 C. PU was not found in samples containing starter, but TYR was. Starter addition thus decreases biogenic amine contents as reported by Hernandez et al. (1997a) and Maijala, Nurmi, 1995). All the amines were well separated on reverse-phase HPLC. The order of elution was tryptamine, b-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine (Fig. 1) and the
chromatogram for amines extracted from a soudjouck sample are shown in Fig. 2. Reproducibility in the chromatograms was assured by injecting a standard solution 4 times and determining the standard deviation of the resulting peak areas. This was always less than 1%. Daily checks of the retention times (11 injections), was less than 0.3% for all biogenic amines. Peak purity was evaluated with the diode-array detector (Moret, Bartolomeazzi, & Lercker, 1992). Although there are no legal limits for biogenic amines, the values found in this study are close to the lower recommended limits, 100±800 mg of TYR (Hernandez et al., 1997b) and 396 mg of PU (Stratton et al., 1991) per kg of food. As reported previously, it is possible to ®nd high levels of biogenic amines from high quality raw materials if incorrectly processed (Maijala, Eerola et al., 1995). According to our results, none of
Table 3 Levels of biogenic amines during Turkish soudjouck production and storage (mg/kg)a Ground meat Days 2 3 6 8 15 30 a b c
Putrecine
Tyramine
Phenylethylamine
ND
ND
ND
Control
Starter
Control
Starter
Control
Starter
NDc 1698.10 2227.16
ND ND ND
ND 2746.94 3106.14
ND ND ND
ND ND ND
ND ND ND
NVb 3966.36 3235.83 41214.5
Vb 2456.36 3406.36 22211.5
NV ND ND ND
V ND ND ND
NV 3495.45 2874.62 2535.00
V 2305.45 2895.45 2665.45
NV 2875.45 2835.00 2835.45
V 2735.00 2115.45 2057.00
NV ND ND ND
All values are the meansSD of two determinations. NV, non-vacuum; V, vacuum packaged. ND, not determined.
Fig. 1. Chromatographic separation of dansyl derivatives of biogenic amines by gradient elution.
V ND ND ND
NV ND ND ND
V ND ND ND
K. Ayhan et al. / Meat Science 53 (1999) 183±188
187
Fig. 2. Chromatogram of amines extracted from a soudjouck sample (peak identi®cation as Fig. 1).
the biogenic amines were found in the ground meat, but both PU and TYR levels increased during storage in the absence of starter. The addition of starter stopped the formation of PU, but had no aect on TYR. These results suggest that dierent starter cultures and/or processing regimes could control the formation of these amines. Finally, the HPLC method presented in this work is a precise, reliable and easily used method to determine these amines.
Acknowledgements The authors would like to thank MSc. Nilufer Vural for help with the HPLC analyses.
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