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

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, 2^nd day, 4^th day, 10^th day, and 14^th day) and storage periods (day 0, 30^th day, 60^th day, 90^th day, 120^th day, and 180^th day) for further analysis.

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).

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.

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.

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).

The initial a_w 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 a_w value on the 14^th day (p<0.05). Although all sucuk samples were vacuum packaged, the a_w 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.

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 2^nd 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 5^th 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 14^th 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 2^nd 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 14^th day of ripening, while the tryptamine levels of BS20 and BS30 increased after the 10^th 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 2^nd day of processing, no 2-phenylethylamine was determined in any groups. Additionally, 2-phenylethylamine was not detected in BS30 on the 4^th 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 14^th 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).