Biogenic Amine Formation in "Bez Sucuk," a Type of Turkish Traditional Fermented Sausage Produced with Different Meat: Fat Ratios.

This study aims to evaluate biogenic amine levels of bez sucuks (BS) produced with different meat:fat ratios. For this, three BS groups were manufactured with meat:fat ratios of 90:10 (BS10), 80:20 (BS20), and 70:30 (BS30). The pH and water activity values and biogenic amine amounts of sucuk samples were determined during processing and storage periods and the pH values of the initial mixtures of BS samples were in the range 5.51-5.74, decreasing to 4.72-4.94 by the 14th day. The water activity values of BS samples showed significant decreases as a result of the drying stage and reached to range 0.913-0.935 on the 14th day of processing (p<0.05). Although BS10 had the highest tyramine (434.12 mg/kg), histamine (5.69 mg/kg), cadaverine (12.48 mg/kg), putrescine (17.83 mg/kg), 2-phenylethylamine (15.43 mg/kg), and tryptamine (122.41 mg/kg) levels at the end of processing stage (p<0.05), spermine and spermidine levels did not differ between the BS samples due to their utilization of different meat:fat ratios (p>0.05). Similarly, the tryptamine (205.11 mg/kg), putrescine (43.57 mg/kg), and tyramine (766.23 mg/kg) levels of BS10 were higher than BS20 and BS30 samples at the end of storage (p<0.05). The results showed that BS10 with the highest meat ratio had the highest tryptamine, putrescine, and tyramine levels at the end of the processing and storage period.

Introduction

Biogenic amines (BA) are nitrogenous compounds that can be classified as aliphatic, aromatic, or heterocyclic amines according to their chemical structures (Bozkurt and Erkmen, 2007; Gençcelep et al., 2007; Papavergou et al., 2012; Suzzi and Gardini, 2003; Tosukhowong et al., 2011). In general, BAs are naturally found in various foods such as cheeses, fermented fish, meat products, and wine due to the amino acid decarboxylase activity of Pseudomonas, Enterobacteriaceae, and Lactobacilli (Baka et al., 2011; Suzzi and Gardini, 2003). The most important BAs in fermented foods are histamine, putrescine, cadaverine, tyramine, tryptamine, 2-phenylethylamine, spermine, and spermidine (Bover-Cid et al., 2001a; Suzzi and Gardini, 2003; Tosukhowong et al., 2011). Histamine, putrescine, tyramine, and cadaverine are the most prevalent BAs in fermented sausages; moreover, high levels of these BAs can be found in fermented sausages produced from raw materials (beef, pork, turkey meat, etc.) that have high protein content (Latorre-Moratalla et al., 2010; Papavergou, 2011; Suzzi and Gardini, 2003).

Bez sucuk (BS), which is a type of Turkish fermented beef sausage, differs from other Turkish-type fermented sausages due to the use of cloth casings sewn to size of 7 × 25 cm from uncolored cloth with 42 threads per cm2. Although these products are mainly produced by butchers and small-scale facilities that utilize traditional technologies without adding starter cultures, a few manufacturers use curing agents such as sodium nitrite. In addition, the formulation (meat:fat ratio and seasonings) and process conditions (temperature, humidity, and ripening period) of the BS show differences between manufacturers (Çiçek and Köse, 2016). However, BS generally has three production stages: mixing the sucuk batter, filling the cloth casings, and ripening for 10-14 days (Çiçek and Köse, 2016; Çiçek and Polat, 2016). During the ripening stage for fermented sausages such as BS, lipids, proteins, and carbohydrates undergo various chemical and microbiological degradation reactions (Ensoy et al., 2010; Karslıoğlu et al., 2014; Papavergou, 2011). Although the proteolysis reactions during the ripening stage resulted in increased free amino acids, which are the precursors of BAs formation (Baka et al., 2011; Bover-Cid et al., 1999a; Karslıoğlu et al., 2014; Papavergou, 2011), the microbial counts of raw materials, process conditions, utilizing starter cultures, additives (antimicrobial agents), and the pH drop during fermentation stage affects the BA formation in fermented sausages (Bover-Cid et al., 2001a; Bozkurt and Erkmen, 2007; Suzzi and Gardini, 2003). Furthermore, the extended proteolytic reactions during ripening and storage periods with both a low pH value and high counts of bacteria having decarboxylase activity may enhance the accumulation of BAs in fermented sausages (Bover-Cid et al., 1999b; Papavergou, 2011; Papavergou et al., 2012; Suzzi and Gardini, 2003); moreover, BAs may also result from the decarboxylation activity of spoiling bacteria during the storage period (Bover-Cid et al., 2001b; Bozkurt and Erkmen, 2007; Suzzi and Gardini, 2003).

Numerous studies have been conducted to determine the BA levels of fermented meat products, particularly fermented sausages. The BA amounts of these products are also important from the perspective of human health. Many researchers have reported that the consumption of foods with high levels of BAs may cause various types of foodborne diseases such as hypotension, migraine, nausea, diarrhea, abdominal cramps, etc. (Bover-Cid et al., 1999a; Casquete et al., 2011; Gençcelep et al., 2007; Komprda et al., 2001; Latorre-Moratalla et al., 2008; Papavergou, 2011; Papavergou et al., 2012; Tosukhowong et al., 2011). Additionally, cadaverine and histamine levels have been proposed as chemical indicators of meat spoilage and the hygienic conditions of the manufacturing practices of fermented sausages (Bozkurt and Erkmen, 2007; Komprda et al., 2001; Tosukhowong et al., 2011); meanwhile, there is only a legal upper limit for histamine: 100 mg per kg of food (Bozkurt and Erkmen, 2002; Latorre-Moratalla et al., 2008).

The BA levels of Turkish fermented sausages that have natural or collagen casings have been investigated. Although the formulation and processing conditions of BS show differences between manufacturers and casing sewn from uncolored cloth, no study has assessed the BA levels of BS, particularly in those manufactured with different meat:fat ratios. According to the TS 1070 Turkish Sucuk (fermented sausage) Standard (Anonymous, 2002), the maximum fat contents of first, second, and third quality sucuks are limited to 35%, 40%, and 40%, respectively. In addition, according to the Turkish Food Codex-Meat Products Communique (Anonymous, 2012) the maximum fat content of fermented sausages is limited to 40%. After producing BS in accordance with both food legislation and sucuk standards, the current study aimed to determine the effects of different meat:fat ratios and the process stages/storage periods on the formation of BAs in BS.

Materials and Methods

Sausage production

In this study, experimental BS production was repeated three times, and three groups of BS named BS10, BS20, and BS30 were manufactured with respective meat:fat ratios of 90:10, 80:20, and 70:30. Cubed beef meat was subdivided into three groups and combined with salt (1.6%), garlic (2%), cumin (0.8%), hot red pepper (0.8%), red pepper (0.6%), black pepper (0.4%), sucrose (0.5%), and allspice (0.2%) using a Mainca 400 mixer (Germany). Following the mixing stage, all sucuk mixtures were stored at 4℃ for 12 hours (initial mixture). Sheep tail fat was added to all sucuk batters to the meat:fat ratios mentioned above and ground twice using a grinding machine (Turkey). After grinding, cloth casings were filled with sucuk mixtures using a filling machine from Mainca EM-20 (Germany). Sucuk batons were hung on stainless steel and ripened for 14 days. The ripening steps were as follows: (step 1) at 22℃, 90% relative humidity for 48 h; (step 2) at 22℃, 85% relative humidity for 48 h; (step 3) at 20℃, 80% relative humidity for 72 h; (step 4) at 18℃, 75% relative humidity for 72 h; (step 5) at 18℃, 60% relative humidity for 96 h. During the ripening stage, all sucuk samples were pressed for three times and following the ripening stage, all sucuk samples were vacuum packed using a vacuum machine from La Minerva Pack 10B (Italy), and then stored at 4℃ for six months.

Three randomly selected sucuk samples were removed during processing (initial mixture, 2nd day, 4th day, 10th day, and 14th day) and storage periods (day 0, 30th day, 60th day, 90th day, 120th day, and 180th day) for further analysis.

Methods

pH and water activity values (aw)

The pH of the sample was determined using an Orion 420A pH-meter that was calibrated using standard buffer solutions at pH 4 and 7. The water activity values of samples were measured using AquaLab Series 3 TE model (USA) at each sampling stage (Çiçek et al., 2015).

Biogenic amine levels

The biogenic amine amounts of samples were analyzed using the method of Bozkurt and Erkmen (2002). The HPLC system, which consisted of a PerkinElmer Series 200 model UV/VIS Detector (USA), PerkinElmer Series 200 model pump (USA) and PerkinElmer Series 200 model Peltier column oven (USA) was used. The column was Waters Spherisorb® 10.0 μm, ODS2 (4.6 × 200 mm) (USA). Acetonitrile (solvent A) and 0.1 M ammonium acetate (solvent B) were the mobile phases (flow rate: 1 mL/min). The standard solution of the dansylated derivatives of tryptamine, 2-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine (SIGMA) was used and all standards were diluted to 1 mL using 0.4 perchloric acid to give concentrations in the range 0.5-10 mg/mL.

Statistical analysis

The data were statistically analyzed using the SPSS 20.0 statistical package (International Business Machines Corporation [IBM] Armonk, USA). Mean values for different meat:fat ratios and manufacturing/storage periods were compared using analysis of variance (ANOVA) with the Duncan multiple post hoc comparison test to evaluate statistical significance between the means (p<0.05). Comparison of means for BS groups and sampling stages was done for every tested variable. Standard deviation (SD) values are presented alongside the means in tables.

Results and Discussions

pH and water activity (aw) values

The results showed that the pH values of BS decreased during the ripening stages (p<0.05) and reached the lowest values on day 4 due to the acidification activity of lactic acid bacteria (p<0.05). The occurrence of proteolytic activity during the ripening stage resulted in the formation of ammonia and amine, which slightly increased the pH values of BS10, BS20, and BS30 to 4.94, 4.78, and 4.72, respectively. Although all sucuk formulations were prepared with the same sucrose amount of 0.5%, the highest pH value was measured in BS10 on day 14 (p<0.05) (Table 1). This difference could be the result of proteolysis reactions in BS10 having the highest meat ratio among the three. It has been stated that sugar addition with certain concentrations to the sausage mixture can improve the rapid acidification, which limits the decarboxylase activity of bacteria (Gonzalez et al., 2003; Latorre-Moratalla et al., 2010). Similar rapid pH decreases in all BS groups were also achieved with sucrose addition to BS formulations. However, Latorre-Moratalla et al. (2010) stated that sugar addition did not result in rapid pH decreases in fermented sausages and higher amounts of sugar promoted the decarboxylase activity of aminogenic microorganisms. During the storage periods, the pH values of BS groups showed slight increases and decreases, and measured 5.21, 4.91, and 4.69 on day 180 for BS10, BS20, and BS30, respectively (Table 1). It was seen that BS10 had the highest pH value among the three (p<0.05).

Table 1.

pH and water activity (aw) values of bez sucuks during processing stages and storage periods1)

	BS102)		BS20		BS30
	pH	aw	pH	aw	pH	aw
Process stages (day)
Initial mixture	5.51 ± 0.21Aa	0.978 ± 0.007Aa	5.61 ± 0.21Aa	0.976 ± 0.005Aa	5.74 ± 0.26Aa	0.973 ± 0.005Aa
2	4.89 ± 0.20Bb	0.972 ± 0.004BAa	5.06 ± 0.11ABb	0.974 ± 0.005Aa	5.16 ± 0.19Ab	0.971 ± 0.005Aa
4	4.67 ± 0.14Ac	0.968 ± 0.003Bb	4.74 ± 0.10Ad	0.971 ± 0.00Aa	4.76 ± 0.13Ac	0.967 ± 0.002Ba
10	5.04 ± 0.16Ab	0.964 ± 0.005Ac	4.90 ± 0.07Bc	0.949 ± 0.006Ab	4.84 ± 0.08Bc	0.949 ± 0.005Ab
14	4.94 ± 0.13Ab	0.913 ± 0.017Bd	4.78 ± 0.06Bd	0.922 ± 0.015ABc	4.72 ± 0.07Bc	0.935 ± 0.016Ac
Storage periods (day)
0	4.94 ± 0.13Ab	0.913 ± 0.017Ba	4.78 ± 0.06Bb	0.920 ± 0.015ABab	4.72 ± 0.07Bbc	0.935 ± 0.016Aa
30	5.26 ± 0.24Aa	0.897 ± 0.012Bb	5.04 ± 0.59Ba	0.908 ± 0.015ABcd	4.98 ± 0.18Ba	0.920 ± 0.009Ab
60	5.10 ± 0.05Aab	0.907 ± 0.006Cab	4.83 ± 0.23Bb	0.916 ± 0.008Babc	4.65 ± 0.25Bc	0.927 ± 0.008Aab
90	5.23 ± 0.04Aa	0.904 ± 0.004Cab	5.04 ± 0.22Ba	0.923 ± 0.005Ba	4.85 ± 0.16Cab	0.935 ± 0.006Aa
120	5.23 ± 0.32Aa	0.896 ± 0.009Cb	5.01 ± 0.27ABa	0.909 ± 0.010Bcd	4.80 ± 0.18Bbc	0.926 ± 0.011Aab
150	5.12 ± 0.17Aa	0.900 ± 0.008Bb	4.87 ± 0.10Bab	0.901 ± 0.008Bd	4.67 ± 0.03Cc	0.918 ± 0.010Ab
180	5.21 ± 0.07Aa	0.905 ± 0.004Bab	4.91 ± 0.11Bab	0.911 ± 0.007Bbcd	4.69 ± 0.13Cbc	0.923 ± 0.005Ab

1)Data ± standard deviation (n=6).

2)BS10: bez sucuk having meat:fat ratio of 90:10; BS20: bez sucuk having meat:fat ratio of 80:20; BS30: bez sucuk having meat:fat ratio of 70:30.

a-dMeans in a column of each group not having a common superscript letter are different (p<0.05).

A-CMeans in a row of same processing stage not having a common superscript letter are different (p<0.05).

The initial aw values of the sucuk mixtures were 0.973-0.978 and reached 0.913-0.935 due to the increasing acidity and water release at the end of the ripening stage (p<0.05) (Table 1). It was observed that BS10 had the lowest aw value on the 14th day (p<0.05). Although all sucuk samples were vacuum packaged, the aw values of BS showed differences between the storage periods due to sampling (p<0.05) and measured 0.905, 0.911, and 0.923 on day 180 for BS10, BS20, and BS30, respectively (p<0.05) (Table 1).

The levels of BAs measured in BS groups during the processing stages and storage periods are given in Tables 2 and 3, respectively. The spermine levels of the initial mixtures of BS groups were in the range 282.77-343.73 mg/kg (p<0.05) (Table 2). Many researchers noted that spermine and spermidine could be present at high levels in fresh meat used for fermented sausage manufacturing (Papavergou, 2011; Suzzi and Gardini, 2003). Papavergou et al. (2012) also stated that continued intake of foods with high amounts of spermine and spermidine may cause the facilitation of a food allergy. Spermine levels in BS groups showed slight decreases and increases during processing stages, and spermine levels were measured as 343.34, 306.95, and 321.63 mg/kg on day 14 for BS10, BS20, and BS30, respectively (Table 2). Bover-Cid et al. (1999b) also reported similar fluctuations in spermine levels during the ripening of dry fermented sausages. Contrary to our results, Kurt and Zorba (2009) stated that increasing the ripening period of sucuks resulted in higher spermine accumulation. In agreement with the results of current study, Roseiro et al. (2010) stated that spermine levels were higher than spermidine levels. The spermidine levels of BS groups increased during processing stages and reached 202.70, 141.79, and 188.42 mg/kg for BS10, BS20, and BS30, respectively (p<0.05) (Table 2). Kurt and Zorba (2009) also noted similar increases during processing stages. Utilizing different meat:fat ratios did not affect spermine and spermidine levels among BS groups on day 14 (p>0.05). Although the spermidine levels of all BS samples showed significant decreases and increases during storage periods (p<0.05), the changes in spermine levels for BS10 and BS20 were found to be significant (p<0.05) (Table 3). However, Bozkurt and Erkmen (2002) reported significant decreases in spermine concentrations for Turkish sucuks during storage.

Table 2.

Biogenic amine levels of bez sucuks during processing stages (mg/kg)1)

Group	Process stage (day)	Tryptamine	2-Phenyl ethylamine	Putrescine	Cadaverine	Histamine	Tyramine	Spermidine	Spermine
BS102)	Initial mixture	23.18 ± 6.74Ab	5.10 ± 2.33Aab	0.50 ± 0.44Ac	0.67 ± 0.13Ab	0.48 ± 0.20Ad	2.05 ± 1.28Ad	13.29 ± 2.65Ac	343.73 ± 14.49Aa
	2	41.67 ± 0.73Ab	ND3)	0.56 ± 0.46Ac	2.20 ± 1.23Ab	0.62 ± 0.12Ad	95.55 ± 18.90Bcd	62.74 ± 6.11Abc	339.40 ± 7.48Aa
	4	40.26 ± 16.51Ab	7.74 ± 5.58Aab	1.87 ± 0.51Ac	3.82 ± 0.78ABb	2.29 ± 0.18Ac	166.28 ± 37.93Abc	125.62 ± 28.00Ab	306.95 ± 43.42Aab
	10	59.75 ± 34.57Ab	14.78 ± 7.64Aa	9.05 ± 4.66Ab	13.13 ± 2.25Aa	3.60 ± 0.01Ab	247.36 ± 100.43ABb	213.23 ± 61.57Aa	245.38 ± 93.87Ab
	14	122.41 ± 23.08Aa	15.43 ± 12.87Aa	17.83 ± 2.40Aa	12.48 ± 4.72Aa	5.69 ± 0.26Aa	434.12 ± 83.20Aa	202.70 ± 77.38Aa	343.34 ± 48.83Aa
BS20	Initial mixture	16.78 ± 3.19Ab	2.28 ± 1.11Bb	0.22 ± 0.03Ab	0.78 ± 0.10Ac	0.39 ± 0.13Ab	0.95 ± 0.46Ac	8.66 ± 3.22Bb	298.10 ± 11.11Ba
	2	34.42 ± 14.15Ab	ND	0.45 ± 0.22Ab	4.00 ± 2.55Ab	0.38 ± 0.11Bb	124.13 ± 22.39Ab	146.70 ± 96.45Aa	305.51 ± 79.76ABa
	4	41.83 ± 10.21Ab	0.27 ± 0.67Bc	1.74 ± 1.27Ab	4.36 ± 1.16Ab	0.80 ± 0.20Bb	144.97 ± 27.15Ab	118.66 ± 5.84ABa	295.76 ± 32.81Aa
	10	74.98 ± 14.24Aa	18.66 ± 3.53Aa	9.06 ± 3.43Aa	8.80 ± 1.51Ba	2.16 ± 0.81Ba	311.48 ± 33.88Aa	158.44 ± 76.99ABa	321.42 ± 8.25Aa
	14	73.32 ± 32.05Ba	ND	10.34 ± 4.20Ba	8.54 ± 1.56Ba	2.70 ± 0.04Ba	303.65 ± 82.53Ba	141.79 ± 44.75Aa	306.95 ± 41.37Aa
BS30	Initial mixture	14.52 ± 0.45Abc	1.13 ± 0.67Bb	0.23 ± 0.03Ac	0.90 ± 0.38Ac	0.59 ± 0.52Aa	1.92 ± 0.30Ae	6.31 ± 2.11Bc	282.77 ± 31.70Bab
	2	ND	ND	0.23 ± 0.08Ac	1.70 ± 1.06Abc	0.31 ± 0.02Ba	66.93 ± 7.74Cd	61.82 ± 15.28Abc	232.76 ± 107.60Bb
	4	31.30 ± 2.22Ab	ND	1.14 ± 1.43Ac	2.76 ± 1.27Bb	0.38 ± 0.27Ba	103.99 ± 12.73Bc	96.06 ± 13.14Bb	294.62 ± 22.18Aab
	10	60.33 ± 17.29Aa	47.24 ± 50.67Aa	5.71 ± 2.57Ab	5.72 ± 1.77Ca	0.73 ± 0.38Ca	222.12 ± 22.20Bb	90.66 ± 14.66Bb	297.73 ± 52.07Aab
	14	73.77 ± 14.94Ba	3.68 ± 1.93Bb	9.07 ± 2.23Ba	6.44 ± 1.94Ba	0.84 ± 0.73Ca	293.99 ± 21.47Ba	188.42 ± 89.63Aa	321.63 ± 24.77Aa

1)Data ± standard deviation (n=6).

2)BS10: bez sucuk having meat:fat ratio of 90:10; BS20: bez sucuk having meat:fat ratio of 80:20; BS30: bez sucuk having meat:fat ratio of 70:30.

3)ND: Not detected.

a-eMeans in a column of each group not having a common superscript letter are different (p<0.05).

A-CMeans in a column of same processing stage not having a common superscript letter are different (p<0.05).

Table 3.

Biogenic amine levels of bez sucuks during storage periods (mg/kg)1)

Group	Storage periods (day)	Tryptamine	2-Phenyl ethylamine	Putrescine	Cadaverine	Histamine	Tyramine	Spermidine	Spermine
BS102)	0	122.41 ± 23.08Abc	15.43 ± 12.87Abc	17.83 ± 2.40Ad	12.48 ± 4.72Aa	5.69 ± 0.26Aa	434.12 ± 83.20Ab	202.70 ± 77.38Aab	343.34 ± 48.83Aa
	30	104.19 ± 26.31Ac	0.80 ± 0.95Ac	13.38 ± 3.80Ad	10.95 ± 0.72Aa	2.44 ± 2.10Aa	417.02 ± 88.72ABb	131.42 ± 47.25ABb	317.42 ± 49.43Aab
	60	150.18 ± 40.74Aabc	18.99 ± 12.70Abc	26.89 ± 4.79Abc	18.79 ± 11.13Aa	6.30 ± 5.68Aa	554.34 ± 113.07Aab	235.86 ± 71.61Aa	319.35 ± 28.74Aab
	90	111.59 ± 82.96Ac	12.27 ± 7.72ABbc	20.07 ± 12.25Acd	11.71 ± 8.87Aa	2.80 ± 3.47Aa	425.27 ± 266.83Ab	128.25 ± 113.63Ab	224.35 ± 113.17Ab
	120	157.27 ± 47.94Aabc	21.92 ± 18.95Abc	31.68 ± 8.00Ab	18.29 ± 13.20Aa	3.58 ± 4.02Aa	631.57 ± 178.73Aab	223.29 ± 65.68Aa	327.81 ± 38.84Aa
	150	184.97 ± 54.01Aab	64.45 ± 19.81Aa	35.63 ± 6.06Aab	23.42 ± 18.29Aa	3.48 ± 3.74Aa	681.73 ± 163.02Aa	256.16 ± 45.76Aa	276.03 ± 49.99Aab
	180	205.11 ± 25.64Aa	29.85 ± 23.27Ab	43.57 ± 4.96Aa	27.66 ± 24.15Aa	5.53 ± 6.42Aa	766.23 ± 171.00Aa	297.31 ± 37.02Aa	358.58 ± 120.47Aa
BS20	0	73.32 ± 32.05Bb	ND3)	10.34 ± 4.20Bc	8.54 ± 1.56Ba	2.70 ± 0.04Ba	303.65 ± 82.53Bb	141.79 ± 44.75Ab	306.95 ± 41.37Aab
	30	120.91 ± 39.02Aab	11.62 ± 10.89Aabc	19.00 ± 7.10Abc	14.76 ± 8.94Aa	3.77 ± 3.73Aa	481.84 ± 123.88Aab	214.63 ± 76.77Aab	299.83 ± 32.77Aab
	60	153.29 ± 40.08Aa	4.48 ± 2.00Bc	23.55 ± 3.42ABab	12.02 ± 2.44ABa	1.82 ± 1.88Ba	498.21 ± 83.05Aab	189.05 ± 34.96Aab	331.18 ± 12.57Aa
	90	112.71 ± 72.65Aab	24.34 ± 19.93Aa	23.89 ± 13.32Aab	17.39 ± 15.42Aa	2.65 ± 3.46Aa	441.25 ± 268.81Aab	138.13 ± 80.66Ab	242.47 ± 97.03Ab
	120	123.79 ± 34.35ABab	9.52 ± 9.31ABbc	25.84 ± 6.84Aab	13.70 ± 5.44Aa	3.48 ± 4.30Aa	457.19 ± 122.22ABab	209.28 ± 45.81Aab	299.85 ± 63.16Aab
	150	150.68 ± 18.57ABa	16.48 ± 12.93Babc	32.30 ± 4.43ABa	16.13 ± 7.47Aa	3.54 ± 4.38Aa	551.88 ± 93.70ABa	239.53 ± 44.02Aa	312.29 ± 43.04Aab
	180	146.10 ± 54.38Ba	18.20 ± 7.19Aab	30.10 ± 8.17Ba	17.89 ± 8.27Aa	3.04 ± 3.14Aa	539.60 ± 167.06Ba	249.52 ± 84.71Aa	246.31 ± 23.19Bb
BS30	0	73.77 ± 14.94Bb	3.68 ± 1.93ABb	9.07 ± 2.23Bc	6.44 ± 1.94Bb	0.84 ± 0.73Cb	293.99 ± 21.47Ba	188.42 ± 89.63Aab	321.63 ± 24.77Aa
	30	83.51 ± 30.49Aab	ND	11.50 ± 4.48Abc	6.80 ± 2.15Aab	0.75 ± 0.58Ab	200.77 ± 64.68Ba	104.88 ± 5.59Bb	255.65 ± 49.90Aa
	60	104.65 ± 27.79Aab	0.97 ± 2.38Bb	18.47 ± 5.92Babc	8.14 ± 0.72Bab	0.77 ± 0.48Bb	374.14 ± 70.54Ba	192.17 ± 70.41Aab	298.49 ± 55.83Aa
	90	90.72 ± 57.35Aab	ND	18.68 ± 10.92Aabc	6.75 ± 3.54Aab	1.39 ± 1.62Aab	339.60 ± 180.84Ab	121.52 ± 76.13Ab	258.44 ± 110.53Aa
	120	99.77 ± 45.19Bab	0.14 ± 0.35Bb	21.12 ± 9.55Aab	8.39 ± 4.60Aab	1.89 ± 2.30Aab	356.66 ± 150.06Ba	136.16 ± 46.49Bb	249.66 ± 85.30Aa
	150	109.46 ± 42.32Bab	34.87 ± 34.01ABa	24.93 ± 10.64Ba	9.87 ± 5.38Aab	1.57 ± 0.98Aab	397.09 ± 155.19Ba	234.41 ± 104.52Aa	273.73 ± 85.85Aa
	180	132.04 ± 14.08Ba	10.91 ± 4.18Ab	28.81 ± 5.25Ba	11.48 ± 4.16Aa	9.32 ± 15.32Aa	453.48 ± 62.92Ba	278.66 ± 61.90Aa	284.58 ± 46.43ABa

1)Data ± standard deviation (n=6).

2)BS10: bez sucuk having meat:fat ratio of 90:10; BS20: bez sucuk having meat:fat ratio of 80:20; BS30: bez sucuk having meat:fat ratio of 70:30.

3)ND: Not detected.

a-eMeans in a column of each group not having a common superscript letter are different (p<0.05).

A-CMeans in a column of same processing stage not having a common superscript letter are different (p<0.05).

The cadaverine and putrescine levels of initial mixtures were in the ranges of 0.67-0.90 mg/kg and 0.22-0.50 mg/kg, respectively (Table 2). The accumulation of cadaverine and putrescine significantly increased during processing stages and reached 6.44-12.48 mg/kg and 9.06-17.83 mg/kg, respectively (p<0.05). Similar increases in putrescine and cadaverine levels in Turkish sucuks were also noted by Gençcelep et al. (2007). The presence of especially high levels of cadaverine and putrescine in BAs has been attributed to meat spoilage (Baka et al., 2011; Suzzi and Gardini, 2003). Thus, the formation of cadaverine is mainly related to the decarboxylase activity of Enterobacteriaceae and Pseudomonas (Baka et al., 2011; Latorre-Moratalla et al., 2010). The low cadaverine levels of BS groups could be explained by the contribution of good hygienic conditions when handling raw materials and the manufacturing conditions. Latorre-Moratalla et al. (2010) noted that high cadaverine levels of spontaneously fermented Portuguese chouriços were due to their high counts of Enterobacteriaceae. The cadaverine levels of BS samples showed increases during storage periods, and respectively measured 27.66, 17.89, and 11.48 mg/kg for BS10, BS20, and BS30 on day 180 (Table 3). It was seen that utilizing different meat:fat ratios did not affect the cadaverine levels of BS samples at the end of storage (p>0.05). The significant increment in putrescine levels of all BS groups was observed, especially after day 10 of the ripening stage (p<0.05). Bover-Cid et al. (1999a) also stated similar putrescine production that increased after the first week due to a pH drop. In our study, it was seen that no significant putrescine formation was observed during the first two days of the ripening stage (p>005) and, although the putrescine accumulation was significant on day 10, the pH value of the BS groups dropped dramatically after day 4 of ripening. Additionally, it was determined that BS10, with the highest meat ratio and pH value, had the highest amounts of cadaverine and putrescine on day 14 (p<0.05). The results indicated that the putrescine levels of the BS groups increased during storage and BS10, with the highest meat ratio, had the highest putrescine level (43.57 mg/kg) on day 180 (p<0.05) (Table 3). Baka et al. (2011) and Papavergou (2011) stated that putrescine was the major amine in fermented sausages. The results obtained from Turkish fermented sausages showed that utilizing only nitrite/nitrate or starter cultures limited putrescine formation (Bozkurt and Erkmen, 2007; Gençcelep et al., 2007).

The histamine levels of the BS10 and BS20 groups showed significant increases during the processing stages (p<0.05) and were respectively measured as 5.59, 2.70, and 0.84 mg/kg for BS10, BS20, and BS30 (Table 2), on day 14. This could be the result of pH decreases in the first days of ripening as the pH values of all BS groups significantly decreased after the 2nd day of the ripening stages and reached the range of 4.89-5.16 (p<0.05). Many researchers also noted that an inadequate decrease of pH at the beginning of the ripening stage with extended ripening times could increase the accumulation of histamine in fermented sausages (Papavergou, 2011; Suzzi and Gardini, 2003). Bozkurt and Erkmen (2007) also stated that the ripening period had an increasing effect on the histamine levels of Turkish sucuks, but, in contrast, the researchers also noted decreases in the histamine levels after the 5th day of ripening. Furthermore, an increasing meat ratio resulted in higher histamine levels. Thus, BS10, having the highest meat ratio, had the highest histamine level (p<0.05) (Table 2); on the other hand, the histamine levels of the BS samples were under the values causing health concerns (100 mg/kg). Although the histamine levels of BS20 and BS30 showed significant decreases and increases during storage periods (p<0.05), the differences between the BS groups were not significant on day 180 (p>0.05) (Table 3).

The tyramine levels of all initial mixtures were between 0.95 and 2.05 mg/kg (p>0.05). The accumulation of tyramine showed increases during the processing stages, and the tyramine levels of BS10, BS20, and BS30 were respectively measured as 434.12, 303.65, and 293.99 mg/kg (p<0.05), on day 14 (Table 2). In all the BS groups, the major tyramine formation rate occurred during the first two days due to the pH dropping. Bover-Cid et al. (2001a) also stated that gradual biogenic amine accumulation occurred from the first week of the ripening stage, coinciding with the pH decrease, especially in noninoculated fermented sausages. Similarly, Bozkurt and Erkmen (2007) determined significant increases in the tyramine levels of Turkish sucuks during the first five days of ripening and then decreases during the ripening stage. Contrary to these findings, Komprda et al. (2001) reported a substantial increment in tyramine content after the second week of ripening. Furthermore, tyramine was the main amine in all the BS groups with the exception of the naturally occurring polyamines spermine and spermidine at the end of the processing stages. Similarly, many researchers also stated that tyramine was the major amine in fermented sausages and/or sucuks (De Mey et al., 2014; Gençcelep et al., 2007; Papavergou et al., 2012). It was also determined that utilizing different meat ratios affected the tyramine levels of the BS samples. Thus, BS10, with the highest meat ratio, had the highest tyramine level on the 14th day (p<0.05) (Table 2). The tyramine levels of BS showed significant increases during the storage periods (except BS30), and the tyramine levels of BS10, BS20, and BS30 were respectively measured as 766.23, 539.60, and 453.48 mg/kg on day 180 (Table 3). The maximum allowable limit for tyramine in foods is 100-800 mg/kg (Bozkurt and Erkmen, 2002), and it was seen that the tyramine levels of all BS groups were in this range both at the end of the processing stages and storage periods.

Although tryptamine was not detected in BS30 on the 2nd day of processing, the tryptamine levels of all initial mixtures increased during manufacturing and reached the range of 73.32-122.41 mg/kg at the end of the processing stages (Table 2). It was seen that BS10, having the highest meat ratio and pH on day 14, had the highest tryptamine level (p<0.05) (Table 2) and a significant increment in the tryptamine value of BS10 occurred after the 14th day of ripening, while the tryptamine levels of BS20 and BS30 increased after the 10th day of ripening (p<0.05). Tasic et al. (2012) stated that the tryptamine levels of Petrovska klobasa - a traditional fermented sausage - ranged between 14.7 and 75.1 mg/kg at the end of the drying stage. Papavergou (2011) indicated that the tryptamine levels of Greek fermented sausages were between 0 and 60.53 mg/kg. In another study, Ikonic et al. (2013) reported higher tryptamine levels (24.3-150 mg/kg) for Petrovska klobasa. The tryptamine levels of all the BS groups showed decreases and increases during storage and reached the range of 132.04-205.11 on day 180 (p<0.05) (Table 3). The highest tryptamine level was measured in BS10, having the highest meat ratio and pH value, on day 180 (p<0.05).

In the initial mixtures of all groups, the 2-phenylethylamine levels were in the range of 1.13-5.10 mg/kg (p<0.05). On the 2nd day of processing, no 2-phenylethylamine was determined in any groups. Additionally, 2-phenylethylamine was not detected in BS30 on the 4th day (Table 2). Roseiro et al. (2010) and Bozkurt and Erkmen (2007) noted that 2-phenylethylamine was not detected in any ripening stages of traditional fermented sausages. In the present study, on the 14th day, the 2-phenylethylamine levels of BS10 and BS30 increased to the values of 15.43 and 3.68 mg/kg, respectively (Table 2). Similar findings were also reported by Bover-Cid et al. (2001b). Furthermore, Bover-Cid et al. (2001a) detected 2-phenylethylamine only in spontaneously fermented sausages; thus, BS is a type of spontaneously fermented sausage., the 2-phenylethylamine amounts of all BS groups varied during the storage periods, and the 2-phenylethylamine levels of BS10, BS20, and BS30 were respectively measured as 29.85, 18.20, and 10.91 mg/kg (p>0.05), on day 180 (Table 3).

Conclusions

Although the histamine levels of all the BS groups were lower than 100 mg/kg, the findings of the current study showed that increasing meat ratio resulted in higher biogenic amine levels; thus, BS10, with the highest meat ratio, had the highest amounts of tryptamine, putrescine, cadaverine, and tyramine on the 14th day (ripening) and of tryptamine, putrescine, and tyramine at the end of the storage period. The effect of the ripening period was also found to be significant in tryptamine, putrescine, cadaverine, tyramine, and spermine formation in all the BS groups. The study results indicated that the meat:fat ratio of 70:30 could be suitable for BS production to decrease the BA levels of BS.