Nitrosamine formation in a semi-dry fermented sausage: Effects of nitrite, ascorbate and starter culture and role of cooking

Meat Science 159 (2020) 107917

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Nitrosamine formation in a semi-dry fermented sausage: Effects of nitrite, ascorbate and starter culture and role of cooking Selen Sallana, Güzin Kabana, Şeyma Şişik Oğraşa, Murat Çelikb, Mükerrem Kayaa, a b

T

⁎

Atatürk University, Faculty of Agriculture, Department of Food Engineering, 25240 Erzurum, Turkey Ataturk University, Faculty of Science, Department of Chemistry, 25420 Erzurum, Turkey

A R T I C LE I N FO

A B S T R A C T

Keywords: Heat-treated sucuk Nitrosamine NDMA NPIP Residual nitrite GC/MS

In this study, effects of ingoing nitrite level (0, 50, 100 and 150 mg/kg), use of sodium ascorbate, addition of starter culture (Lactobacillus plantarum GM77 + Staphylococcus xylosus GM92) and cooking level (control, medium, medium well, well done and very well done) on nitrosamine formation in heat-treated sucuk, a type of semi-dry fermented sausage, were investigated. The use of ascorbate had no significant effect on NDMA (NNitrosodimethylamine) and NPIP (N-Nitrosopiperidine) contents in the presence of starter culture. A higher NPYR (N- Nitrosopyrrolidine) content was detected in the group with starter culture at 150 mg/kg nitrite level in comparison to the group without starter culture. Cooking level affected all identified nitrosamines very significantly. Ingoing nitrite level × cooking level interaction was only effective on NPIP and advanced cooking levels (well done and very well done) at higher ingoing nitrite levels (100 and 150 mg/kg) resulted in significant increases in NPIP content.

1. Introduction Nitrate and nitrite, which are used as curing agents in meat products, are important additives for their antimicrobial and antioxidant properties as well as for their contributions to color, flavor and aroma. However, nitrate and nitrite play an important role in the formation of N-nitrosamines (Drabik-Markiewicz et al., 2010; Herrmann, Granby, & Duedahl-Olesen, 2015). International Agency for Research on Cancer classifies N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) as probably carcinogens and N-nitrosodibutylamine (NDBA), N-nitrosopiperidine (NPIP) and N-nitrosopyrrolidine (NPYR) as possible carcinogens (IARC, 1987). Nitrosamines are generally formed by the reaction between a nitrosation agent and a secondary amine (De Mey, De Maere, Paelinck, & Fraeye, 2017). The formation of nitrosamines is a complex process. In meat products, the formation of these compounds depends on many factors such as ingoing nitrite level, processing steps (fermentation, drying, heat treatment etc.), decarboxylase activity of microorganisms, residual nitrite, water activity and pH (Herrmann, Granby, and Duedahl-Olesen, 2015; Wang et al., 2015; De Mey et al., 2017). It has also been reported that cooking temperature / time, cooking method, nitrosation catalysts / inhibitors and storage conditions affect nitrosamine formation (Gloria, Barbour, & Scanlan, 1997; Sen, Donaldson, Charbonneau, & Miles, 1974; Yurchenko & Mölder, 2007). ⁎

In the case of fermented sausage, many compounds that take part in nitrosamine formation are formed as a result of protein degradation which occurs during fermentation and drying/ripening. Among protein degradation products, secondary amines such as dimethylamine are direct precursors of nitrosamines (De Mey et al., 2017). Furthermore, acidification during fermentation may stimulate the formation of nitrosamines (De Mey et al., 2017; Honikel, 2008). On the other hand, it is reported that biogenic amines formed during fermentation and drying/ripening may be good sources for nitrosatable amines (DrabikMarkiewicz et al., 2011). However, growth of decarboxylase positive microorganisms can be prevented through a rapid drop of pH when proper starter cultures are used in fermentation (Suzzi & Gardini, 2003). Moreover, De Mey, De Klerck, et al. (2014) suggested that there is no correlation between the presence of N-nitrosamines and biogenic amines content in dry-fermented sausage. Ascorbate, reported as one of the effective inhibitors in nitrosamine formation (Rywotycki, 2002), is a common agent that plays a part in nitrite reduction and formation of NO (Hammes, 2012). However, prooxidant effect of ascorbate has also been reported (Berardo et al., 2016). On the other hand, Sallan, Kaban, and Kaya (2019) recently reported that the effect of ascorbate use on nitrosamine formation in dry-fermented sausage varies according to the type of nitrosamine. There are many studies on the occurrence and formation of nitrosamines in processed meat products including fermented sausages

Corresponding author. E-mail address: [email protected] (M. Kaya).

https://doi.org/10.1016/j.meatsci.2019.107917 Received 18 January 2019; Received in revised form 16 August 2019; Accepted 16 August 2019 Available online 19 August 2019 0309-1740/ © 2019 Elsevier Ltd. All rights reserved.

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undergone cooking treatment were accepted as control group. Each surface of sausages was cooked at the same time and, accordingly, a total of 1 min was set for medium, 3 min for medium well cooked, 5 min for well done cooked and 7 min for very well done cooked.

such as salami, Rohwurst, cervelat, pepperoni and chorizo (Ellen, Eqmond, & Sahertian, 1986; Gavinelli, Fanelli, Bonfanti, Davoli, & Airoldi, 1988; Mavelle, Bouchikhi, & Bebry, 1991; Yurchenko & Mölder, 2007; De Mey, De Klerck, et al., 2014; Herrmann, Granby, and DuedahlOlesen, 2015; Herrmann, Duedahl-Olesen, & Granby, 2015). Nitrosamines in sucuk, a kind of dry fermented sausage, are investigated in Turkey, as well (Özdemir, Batı, & Gökalp, 1984; Ozel, Gogus, Yagcı, Hamilton, & Lewis, 2010; Sallan et al., 2019). However, there are no studies on nitrosamines in heat-treated sucuk (raw fermented cooked and dried) to the best of our knowledge. The production includes three processing steps; a short- fermentation, heat treatment and drying. The product is an industrial product which is produced using starter culture. Heat-treated sucuk is generally consumed after cooking. Production of heat-treated sucuk has been increasing significantly in recent years. Heat treatment application after fermentation stage and short production process, low production cost and safety perception of the product are important determinants in this increase (Kaban, 2013). The aim of this study was to investigate effects of ingoing nitrite level (0, 50, 100 or 150 ppm), use of sodium ascorbate and addition of starter culture on nitrosamine formation in heat-treated sucuk. The effects of five different cooking levels (control-final product, medium (1 min), medium well (3 min), well done (5 min) and very well done (7 min)) on nitrosamine formation were also determined in the study.

2.4. Determination of pH, aw and residual nitrite In the final product, pH and aw values were determined using a pH meter (ATI Orion 420, USA) and an aw sprint device (Novasina, Model TH-500, Switzerland), respectively. Residual nitrite value was determined by the method suggested by Tauchmann (1987) and residual nitrite amount was given as mg/kg NaNO2.

2.5. Determination of N-nitrosamines To determine nitrosamines, the method described by Wang et al. (2015) was used. Briefly, 10 g homogenized sample was added to 0.1 M NaOH and then sonicated. After additon of methanol, the homogenate was centrifuged at 11.963 g at 4 °C and filtered with a glass microfiber filter (70 mm diameter, Whatman, UK). 5 mL of 20% NaCl and 15 mL of filtrate were loaded to the ChemElut column (Agilent ChemElut, 20 mL, Unbuffered, USA). After 20 min, the column was eluted with 50 mL dichloromethane. The eluent was concentrated to 1 mL with the Kuderna Danish apparatus. The concentrate was evaporated under a stream nitrogen using a N-EVAP-111 evaporator (Organomation, Berlin, MA, USA) at 40 °C. GC/MS (Agilent 6890 N / Agilent 5973, USA) was used for the detection of nitrosamines. Helium as carrier gas and DB-5MS (30 m × 0.25 mm × 0.25 μm, Agilent) as column were used in the system, and it was operated in SIM mode. N-Nitrosodipropylamine-d14 (N525482, TRC, Canada) was used as an internal standard. The oven was programmed as follows: 50 °C held for 2 min, ramp to 100 °C at 3 °C/min, held for 5 min, and then ramped to 250 °C at 20 °C/min. Nitrosamine standard (EPA 521 Nitrosamine Mix, Supelco, Bellefonte PA USA) was used for identification. N-Nitrosodimethylamine (NDMA, R2 = 0.9998, LOD = 0.82 μg/kg, LOQ = 2.48 μg/kg), NNitrosopyrrolidine (NPYR, R2 = 0.9998, LOD = 0.78 μg/kg, LOQ = 2.36 μg/kg), N-Nitrosopiperidine (NPIP, R2 = 0.9998, LOD = 0.64 μg/kg, LOQ = 1.30 μg/kg) and N-Nitrosodibutylamine (NDBA, R2 = 0.9996, LOD = 1.30 μg/kg, LOQ = 3.96 μg/kg) could be separated and determined. The results were given as μg/kg.

2. Materials and methods 2.1. Material Beef meat and fat were obtained from Erzurum Meat Processing Plant of General Directory of Meat and Milk Board. Raw meat was trimmed of visible fat and connective tissue. Lean meat and beef fat were separately cut in small pieces. These small pieces were mixed by hand and divided into sixteen parts. Then, these parts were vacuumpackaged and stored at −20 °C until production. All treatments, 5 kg each (4 kg lean meat+1 kg beef meat fat), were repeated three times at three different dates. Thus, a total of 48 batters were prepared. 2.2. Heat-treated sucuk production In heat-treated sucuk production, 20 g salt, 10 g garlic, 4 g sucrose, 7 g red pepper, 5 g black pepper, 9 g cumin, 2.5 g allspice were used per kg of beef meat and beef fat (80:20). Sucuk batters were prepared using different levels of nitrite (0, 50, 100 or 150 mg/kg) and sodium ascorbate (0 or 500 mg/kg). Lactobacillus plantarum GM77 (107 cfu/ g) + Staphylococcus xylosus GM92 (106 cfu/g) strains isolated and identified from sucuk were used as starter culture (Kaban & Kaya, 2009). The batters without starter culture were evaluated as control group. Batters were prepared in a laboratory-type cutter (MADO Typ MTK 662, Dornhan, Germany). Batters then stuffed in collagen casings (38 mm, Naturin Darm, Germany) using a laboratory-type filling machine (MADO Typ MTK 591, Dornhan, Germany). The sausages were fermented for 2 days at 22 ± 1 °C and relative humidity of 90 ± 2% in an automatic climate unit (Reich, Thermoprozestechnik GmbH, Schechingen, Germany). After fermentation stage, a gradual heat treatment process was applied in a steam cooking chamber (Mauting, VKM Kompakt-P, Mikulovska, Czech Republic) to achieve an internal temperature of 68 °C. After heat treatment, the sausages were dried under controlled conditions in the automatic climate unit (Reich, Thermoprozestechnik GmbH, Schechingen, Germany) for 3 days at 16 °C.

2.6. Statistical analysis The experimental design was 2 × 2 × 4 factorial in a completely randomized design with the addition of starter culture (without and with starter culture), the use of sodium ascorbate (without and with ascorbate) and ingoing nitrite level (0, 50, 100 and 150 mg/kg) as factors for pH, aw and residual nitrite. In addition, cooking level (control-final product, medium, medium well, well done and very well done) was also considered in nitrosamine analysis as a factor (2 × 2 × 4 × 5 factorial design). The addition of starter culture, the use of ascorbate and ingoing nitrite level as well as cooking level (only nitrosamine analysis) were treated as independent variables. Data were evaluated by analysis of variance (ANOVA) using a general linear model (GLM) considering the factors and their interactions as fixed effects and the replicates as a random effect. Where factors and interactions were significant, differences between means were determined using the Duncan's multiple range tests at the P < .05 level. For each batter of sausage, all experiments were carried out in triplicate. All statistical analyses were performed using the SPSS version 24 statistical program (SPSS Inc., Chicago, IL, USA).

2.3. Cooking procedure Before nitrosamine analysis, samples from each group were sliced at a thickness of 0.5 cm and cooked at five different cooking levels (control-final product, medium, medium well, well done and very well done) at 180 °C on a hot plate. Final product samples that were not 2

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Table 1 Overall effects of starter culture, sodium ascorbate and ingoing nitrite on the pH, aw and residual nitrite values of heat-treated sucuk.

Starter culture (SC) Without SC 24 With SC 24 Significance

pH

5.2 5.0

SEM

Mean

SEM

Mean

SEM

5.13a 4.78b **

0.01

0.933a 0.926b **

0.001

6.98 7.78 NS

0.29

0.01

0.931 0.928 NS

0.001

9.28a 5.49b **

0.29

0.01

0.928 0.930 0.927 0.933 NS

0.001

4.68c 7.30b 7.83b 9.74a **

0.40

Ingoing nitrite (mg/kg) (IN) 0 12 4.91c 50 12 4.95bc 100 12 5.01a 150 12 4.96b Significance **

aA

Residual nitrite (mg/kg)

Mean

Sodium ascorbate (SA) Without SA 24 4.91b With SA 24 5.01a Significance **

SC x SA SC x IN SA x IN SCxSAxIN

aw

pH

N

5.4 aB

bA

4.8

bA

4.6 4.4

** ** * *

NS NS NS NS

without with Starter culture

5.4

without starter culture

with starter culture

(b)

aA aA

abA

5.2 bA

pH

Variance source

(a)

without sodium ascorbate with sodium ascorbate

NS NS NS NS

5 aB bB

4.8

abB

bcB

4.6

a–c: Any two means in the same column having the same letters in the same section are not significantly different (P > .05). ** P < .01, * P < .05, NS: not significant; SEM: standard error of the mean.

4.4 0

50

100

150

Ingoing nitrite level (mg/kg)

3. Results and discussion Fig. 1. (a) a-b: Different small letters indicate significant differences between samples without or with starter culture groups for ascorbate. A-B: Different capital indicate significant differences between without or with ascorbate groups for starter culture. (b) a-c: Different small letters indicate significant differences between samples with different nitrite levels for starter culture; A-B: Different capital indicate significant differences between samples without or with starter culture in different nitrite level.

3.1. pH, aw and residual nitrite Starter culture, sodium ascorbate and ingoing nitrite level had a significant effect on pH value of heat-treated sucuk (P < .01) (Table 1). Use of starter culture significantly reduced pH value. In contrast, the group with sodium ascorbate showed a higher average pH than that of the group without ascorbate (Table 1). However, this effect was not observed in the presence of starter culture (Fig. 1a). Ingoing nitrite level caused a slight difference in pH value (Table 1). In the presence of starter culture, the effect of nitrite level on pH value was also rather limited. When starter culture was not used (spontaneous fermentation), the lowest pH value was determined in the group produced without nitrite. However, no significant difference was observed between samples without nitrite and with 50 mg/kg nitrite (Fig. 1b). Starter culture affected significantly water activity (P < .01). A lower average aw value was determined in the presence of starter culture compared to the group without starter culture. This result is due to the fact that starter cultures accelerate drying by lowering pH (Lücke, 1985). Residual nitrite was significantly affected by sodium ascorbate and ingoing nitrite level (P < .01). Residual nitrite amount increased depending on the level of ingoing nitrite. It is thought that the residual nitrite content determined in the groups produced without nitrite is due to the spices used in the production. Nitrate from spices can be converted to nitrite during production (Honikel, 2008). The use of the starter culture had no significant effect on the mean residual nitrite (P > .05). The mean residual nitrite of the group with sodium ascorbate was significantly lower (P < .05) than the control group (Table 1). This result can be due to the fact that nitrite degradation enhances in the presence of sodium ascorbate (Honikel, 2008; De Mey, De Maere, et al., 2014).

NPYR (N-Nitrosopyrrolidine), NPIP (N-Nitrosopiperidine) and NDBA (N-Nitrosodibutylamine) contents of heat-treated sucuk were given in Table 2. There was no significant difference in NDMA content in sausages produced using starter culture compared to the group without starter culture (P > .05). In contrast, the addition of sodium ascorbate caused a slight increase in the mean content of NDMA (Table 2). However, the use of ascorbate in the presence of starter culture did not have a significant effect on NDMA level. In contrast, use of ascorbate in spontaneous fermentation resulted in an increase in NDMA level (Fig. 2a). On the other hand, it was indicated that ascorbic acid competes for nitrosating species by reducing nitrous acid, which resulted from protonation of the nitrite, to yield nitrogen oxide (De Mey et al., 2017). However, the chemistry of ascorbate, which is an important ingredient in fermented meat products, is quite complex (Berardo et al., 2016). It was reported that the addition of ascorbic acid might slow down the formation of some nitrosamines, but it might accelerate the formation of others by transnitrosation (Chang, Harrington, Rothstein, Swern, & Vohra, 1979). The highest mean NDMA content was detected in the ingoing nitrite level of 150 mg/kg. However, the use of 50 mg/kg nitrite did not cause a significant increase in NDMA content (Table 2). Similarly, it has been demonstrated in another study that high nitrite concentrations and high processing temperatures lead to a significant increase in NDMA content (Drabik-Markiewicz et al., 2011). Herrmann, Granby, and DuedahlOlesen (2015) also reported that NDMA content was relatively unaffected by the increase in nitrite level in both dried sausages and driedfried sausages. In contrast, Yurchenko and Mölder (2007) reported direct relation between nitrosamine content and nitrite concentration in fried and raw mutton samples. On the other hand, as shown in Fig. 2b, in case of addition of 150 mg/kg nitrite, the group with ascorbate

3.2. N-Nitrosamines The overall effects of starter culture, sodium ascorbate, ingoing nitrite level and cooking level on NDMA (N-Nitrosodimethylamine), 3

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Table 2 Overall effects of starter culture, sodium ascorbate, ingoing nitrite and cooking level on the nitrosamine contents of heat-treated sucuk. Variance source

N

NDMA (μg/kg)

NPYR (μg/kg) SEM

Mean

SEM

Mean

SEM

Mean

SEM

0.13

83.44b 93.91a **

2.76

0.31 0.37 NS

0.05

0.13

77.06b 100.30a **

2.76

0.35 0.33 NS

0.05

5.20 5.07 5.07 5.18 NS

0.18

68.48d 79.46c 92.32b 114.46 **

3.90

0.24b 0.31b 0.22b 0.58 a **

0.06

2.76d 4.45c 5.95b 5.65b 6.83a **

0.20

4.36

0.06d 0.17cd 0.34bc 0.50 ab 0.63a **

0.07

120 120

0.29 0.23 NS

0.04

4.63b 5.63a **

Sodium ascorbate (SA) Without SA With SA Significance

120 120

0.15b 0.38a **

0.04

5.40 4.85 **

Ingoing nitrite (mg/kg) (IN) 0 60 50 60 100 60 150 60 Significance

0.11c 0.21bc 0.26b 0.47a **

0.05

Cooking level (CL) Control Medium Medium well Well done Very well done Significance

0.05c 0.16bc 0.27 b 0.31b 0.53a **

0.06

SC x SA SC x IN SC x CL SA x IN SA x CL IN x CL SCxSAxIN SCxSAxCL SCxINxCL SAxINxCL SCxSAxINxCL

NDBA (μg/kg)

Mean Starter culture (SC) Without SC With SC Significance

48 48 48 48 48

NPIP (μg/kg)

* NS NS ** NS NS NS NS NS NS NS

a b

NS * ** * NS NS NS NS NS NS NS

a

1.54e 17.21d 55.29c 118.23b 251.12a ** ** NS ** NS ** ** NS ** NS NS NS

NS NS NS NS NS NS NS NS NS NS NS

a–d: Any two means in the same column having the same letters in the same section are not significantly different (P > .05), ** P < .01, * P < .05, NS: not significant; SEM: standard error of the mean.

contents in salami samples obtained from Denmark and Belgium markets as 4 μg/kg and 7.2 μg/kg, respectively, and added that NDMA content in processed meat products changes according to the type of the product and/or the heat treatment applied. The mean NPYR content of heat-treated sucuk increased with use of starter culture (Table 2). Acidity is one of the most important factors in the formation of nitrosamine and pH must be low enough to generate NO+ (Honikel, 2008). In this study, the average pH value was found to be 5.13 in the group without starter culture and 4.78 in the group with starter culture (Table 1). It is thought that the drop of pH promotes the formation of nitrosamines in sausage with starter culture. However, as Fig. 3a shows, starter culture statistically increased NPYR only at 150 mg/kg nitrite level. In other words, the combination of starter culture (low pH value) and high nitrite (150 mg/kg) level is effective in NPYR formation. Addition of sodium ascorbate reduced the formation of NPYR in heat-treated sausages by showing inhibitory effect. The mean NPYR value, determined as 5.40 μg/kg in the group without ascorbate, decreased to 4.85 μg/kg when ascorbate was used (Table 2). However, sodium ascorbate was more effective as an inhibitor at 50 mg/kg nitrite level (Fig. 3b). Li, Shao, Zhu, Zhou, and Xu (2013) reported that NPYR level increased during ripening, nevertheless, plant polyphenols and ascorbic acid reduced significantly nitrosamine level. On the other hand, Yurchenko and Mölder (2007) detected that NPYR content increased as the nitrite level increased in mutton samples prepared by adding 0, 50, 100, 150 or 200 mg/kg of nitrite. In contrast, Herrmann, Granby, and Duedahl-Olesen (2015) reported that the increase of nitrite levels in sausage relatively unaffected the NPYR content. The results of

showed a higher average NDMA content than the group without ascorbate. In addition, nitrite level did not have a significant effect on NDMA when ascorbate is not used (Fig. 2b). The mean NDMA amount in heat-treated sucuk samples that are not subjected to additional cooking process was determined as 0.05 μg/kg (Table 2). In contrast, a higher NDMA (0.78 μg/kg) content was determined in sucuk which is a dry fermented sausage (Ozel et al., 2010). This is probably due to formation of more NDMA precursors as a result of longer ripening of sucuk than heat-treated sucuk. The NDMA level increased with increasing cooking level, however, 1 min of cooking (medium) did not cause a significant increase in NDMA content in heat-treated sucuk (Table 2). Moreover, no statistically significant differences were observed between medium, medium well and well done cooking levels (Table 2). The highest average NDMA content was determined in samples that were cooked very well done. There is limited knowledge on the effects of cooking methods on the formation of nitrosamines in dry or semi-dry fermented sausages. Li, Wang, Xu, and Zhou (2012) reported that while frying (deep-frying or pan-frying) promotes NDMA formation in dry cured raw sausage, boiling and microwave applications do not have any effects on nitrosamine formation. On the other hand, in a study on sucuk (raw fermented and dried sausage), it was found that cooking level (180 °C, on hot plate) had a significant effect on NDMA (Sallan et al., 2019). Drabik-Markiewicz et al. (2009) reported NDMA content was affected by nitrite and temperature (> 120 °C). NDMA is the most frequently detected nitrosamine in cured meat products, including fermented sausages (Herrmann, Duedahl-Olesen, and Granby, 2015). Herrmann, Duedahl-Olesen, and Granby (2015) determined the highest NDMA

4

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(a)

1.0

(a)

with starter culture

8

with ascorbate

0.8

aA

0.6

NPYR (µg/kg)

NDMA(µg/kg)

without ascorbate

without starter culture

aA aA

0.4

aA aB

0.2 0.0 without

with

6

aA

aA

aA

aA aA

aA

aB

4 2 0

Starter culture

0

50

100

150

Ingoing nitrite level (mg/kg)

8

0.8 0.6 0.4

bA

bA bA

0.2

aB

without ascorbate

(b)

with ascorbate

aA

NPYR (µg/kg)

NDMA (µg/kg)

(b)

without ascorbate with ascorbate

1.0

aA

aB

aA

6

aA

aA aA

aA

aA

aA

aB

4 2

aA

0

0.0 0

0

50 100 150 Ingoing nitrite level(mg/kg)

50

100

150

Ingoing nitrite level (mg/kg)

Fig. 2. (a) a-b: Different small letters indicate significant differences between samples without or with starter culture groups for ascorbate. A-B: Different capital indicate significant differences between without or with ascorbate groups for starter culture. (b) a-b: Different small letters indicate significant differences between samples with different nitrite levels for ascorbate; A-B: Different capital indicate significant differences between samples without or with ascorbate in different nitrite level.)

without starter culture

with starter culture

8

aA

NPYR(µg/kg)

bA

present study also showed that ingoing nitrite level does not has an effect on NPYR content (Table 2). NPYR, like NDMA, is a commonly identified nitrosamine in cured meat products (Herrmann, DuedahlOlesen, and Granby, 2015). Likewise, Drabik-Markiewicz et al. (2009) stated that NPYR formation was not related to nitrite. It was also reported that the formation of NPYR was affected by proline and temperature (≥200 °C) (Drabik-Markiewicz et al., 2009). In another study, it was also indicated that proline and pyrrolidine promote the formation of NPYR and pyrrolidine is more effective in NPYR formation than nitrite concentration (Drabik-Markiewicz et al., 2010). Cooking level factor resulted in an increase in NPYR content and the highest average content was determined in the samples cooked very well done (Table 2). No increases in parallel to increases in cooking level were observed. Similarly, Sallan et al. (2019) also reported that cooking is an important factor of NPYR formation in sucuk, however, increasing the cooking levels do not cause a statistically significant change in NPYR content. In the same fashion, Yurchenko and Mölder (2007) reported that there is no NPYR increase observed in mutton samples in parallel to increase in temperature. On the other hand, the group without starter culture showed the highest value at the 7 min of cooking (very well done), however, the difference between the samples with or without starter culture had no a significant effect in this cooking level (Fig. 3c). The use of starter culture or sodium ascorbate increased the mean NPIP content of heat-treated sucuk (Table 2). However, no significant effect of ascorbate was observed in the group containing starter culture, whereas use of ascorbate increased the NPIP content in the group without starter culture (Fig. 4a). A similar finding was also obtained for NDMA (Fig. 2a). The samples containing sodium ascorbate had higher

6

(c) aA

abA

bB

cA

bcB

cA dA

4 dB

2 0 control

medium

medium well

Cooking level

well done

very well done

Fig. 3. (a) a-b: Different small letters indicate significant differences between samples with different nitrite levels for starter culture; A-B: Different capital indicate significant differences between samples without or with starter culture in different nitrite level. (b) a-b: Different small letters indicate significant differences between samples with different nitrite levels for ascorbate; A-B: Different capital indicate significant differences between samples without or with ascorbate in different nitrite level. (c) a-b: Different small letters indicate significant differences between samples with different cooking levels for starter culture; A-B: Different capital indicate significant differences between samples without or with starter culture in different cooking level.

NPIP contents than the samples without ascorbate at all cooking levels except control. According to this, ascorbate use does not have an important effect on NPIP formation in control group (final product, without additional heat treatment). Moreover, NPIP content was increased in parallel to increases in cooking level in both groups with and without ascorbate. A striking finding is that NPIP content did not show a significant increase under medium application in comparison with the control (Fig. 4b). De Mey, De Maere, et al. (2014) reported that inhibitor role of ascorbate was observed in the beginning of the production. On the other hand, as can be seen from Fig. 4c, use of starter culture did not have a significant effect on NPIP in the control samples. NPIP content increased as cooking level increased and higher NPIP 5

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without ascorbate

aA

aA

NPIP (µg/kg)

contents were observed under medium, medium well and well done applications. There was no significant difference between groups with and without starter culture under very well done application (Fig. 4c). According to these results, cooking level comes forward as an important factor rather than starter culture and ascorbate use. Among all identified nitrosamines, NPIP is the one which is most affected by both ingoing nitrite level and cooking level. Average NPIP level increased as nitrite level and cooking level increase (Table 2). However, as it can be seen from the cooking level × ingoing nitrite level interaction, the effect of nitrite level was quite related with cooking level. In control (final product) groups, 0 mg/kg nitrite level showed lower NPIP content than only 100 and 150 mg/kg nitrite levels (Fig. 4d). Ingoing nitrite level (0, 50, 100 and 150 mg/kg) did not cause a significant change in NPIP content under medium application. In other words, 1 min of cooking does not have an effect on NPIP even at 150 mg/kg nitrite level. However, NPIP level increased at 100 and 150 mg/kg nitrite levels from medium well level through very well done application. 150 mg/kg nitrite level produced higher NPIP values in both well done and very well done cooked samples. This result indicates that NPIP increases more in case of intensive cooking. On the other hand, NPIP content increased as cooking level increased at 0 and 50 mg/kg nitrite levels but no statistically significant differences were observed between medium and medium well levels at both nitrite levels (Fig. 4d). Similar to the findings of this study, Drabik-Markiewicz et al. (2011) also pointed out that application of high heat treatement and high nitrite concentration have a significant effect on the increase of NPIP content, yet they particularly emphasized that heat treatement has the largest effect in this increase. In the same fashion, Herrmann, Duedahl-Olesen, and Granby (2015) also showed that there is a clear positive effect of heat treatment on the level of NPIP. Black pepper included in the formulation of fermented sausage may be an important source of NPIP (Yurchenko & Mölder, 2007; Herrmann, Granby, and Duedahl-Olesen, 2015; Sallan et al., 2019). However, it was reported in another study on sucuk that high black pepper level (15 g/kg) causes a drop in NPIP due to antioxidant compounds (Sallan et al., 2019). The ingoing nitrite and cooking factors showed significant effect (P < .01) on NDBA, whereas the use of starter culture or sodium ascorbate had no significant effect on the NDBA (P > .05). While this nitrosamine does not show a significant increase after 1 min of cooking, extension of cooking time caused an increase in NDBA content (Table 2). None of the interactions of the main variation sources showed a significant effect on the NDBA content of samples (P > .05) (Table 2). NDBA is reported a nitrosamine which detected at very low levels in meat products or none (Gloria et al., 1997; Yurchenko & Mölder, 2007; Ozel et al., 2010; Herrmann, Granby, and DuedahlOlesen, 2015). Nevertheless, NDBA contamination can happen via the migration of precursors from the packaging material (Fiddler, Pensabene, Gates, & Adam, 1998; Pensabene, Fiddler, & Gates, 1995; Sen, Baddoo, & Seaman, 1987).

(a)

with ascorbate

aA

120 100 aB

80 60 40 20 0

without

with Starter culture

without ascorbate

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300

with ascorbate aA

250

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100 cB cA

50

dB dA

dA dA

0 control

medium medium well done very well well done

Cooking level

NPIP (µg/kg)

without starter culture

270 240 210 180 150 120 90 60 30 0

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(c)

aA aA

bB cB dA dA

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bA

cA

dB dA

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0mg/kg

350 300 250 200 150 100 50 0

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(d)

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aA aB aBC aC bB bB cC cBC

bB

bA

cAB cA

dB dAB dA dA cdA cdA dA dA

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medium

medium well

Cooking level

well done

very well done

4. Conclusion

Fig. 4. a-b: Different small letters indicate significant differences between samples without or with starter culture groups for ascorbate. A-B: Different capital indicate significant differences between without or with ascorbate groups for starter culture. (b) a-c: Different small letters indicate significant differences between samples with different cooking levels for ascorbate; A-B: Different capital indicate significant differences between samples without or with ascorbate in different cooking level. (c) a-c: Different small letters indicate significant differences between samples with different cooking levels for starter culture; A-B: Different capital indicate significant differences between samples without or with starter culture in different cooking level. (d) a-c: Different small letters indicate significant differences between samples with different cooking levels for different nitrite level; A-C: Different capital indicate significant differences between samples with different nitrite levels for different cooking level. 80). Kulmbac)

The total volatile nitrosamine level in heat-treated sucuk (< 5 μg/ kg) is lower than the declared value of total volatile nitrosamines for cured meat products in the United States (10 μg/kg) (Crews, 2010), if an additional heat treatment is not applied. Application of heat treatment to this product at higher temperatures (at 180 °C on hot plate) causes an increase in total nitrosamine level depending on the intensity of heat treatment. Cooking level and ingoing nitrite level interaction is only significant for NPIP and significant increases were observed in NPIP level at advanced cooking levels in the presence of high amounts of nitrite. An interesting result here is that ingoing nitrite level does not cause an increase in NPIP content at medium cooking level. While ingoing nitrite level does not have an effect on NPYR, starter culture (low pH value) and high nitrite (150 mg/kg) level combination has an effect 6

Meat Science 159 (2020) 107917

S. Sallan, et al.

on the formation of NPYR. Moreover, unlike NPIP, no increases in NPYR and NDMA contents are observed in parallel to advancing cooking levels. The effect of ascorbate on nitrosamines differs as to type of nitrosamine. The use of ascorbate in spontaneous fermentation results in an increase in both NDMA and NPIP contents.

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