LWT - Food Science and Technology 153 (2022) 112540

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LWT
journal homepage: www.elsevier.com/locate/lwt

The effects of nitrite, sodium ascorbate and starter culture on volatile

compounds of a semi-dry fermented sausage
Selen Sallan a, *, Güzin Kaban b, Mükerrem Kaya b
a
Bandırma Onyedi Eylul University, Bandırma Vocational School, Department of Food Processing, 10200, Bandırma, Balıkesir, Turkey
b
Department of Food Engineering, Faculty of Agriculture, Atatürk University, 25240, Erzurum, Turkey

A R T I C L E I N F O A B S T R A C T

Keywords: This study investigated the effects of nitrite level (0, 50, 100 or 150 mg/kg), sodium ascorbate (with or without)
Semi-dry fermented sausage and starter culture (Lactobacillus plantarum GM77 + Staphlyococcus xylosus GM92 or without starter culture) on
Ascorbate the volatile compounds of heat-treated sucuk, a type of semi-dry fermented sausage. The volatile compounds of
Starter culture
each final product were analyzed by gas chromatography/mass spectrometry (GC/MS) with solid-phase
Nitrite
Volatile compounds
microextraction (SPME). Aliphatic and aromatic hydrocarbons, alcohols, aldehydes, esters and terpenes were
not affected by sodium ascorbate. 3-hydroxy 2-butanone was significantly affected by the nitrite level. Starter
culture addition reduced the hexanal level. The starter culture also resulted in a decrease in diallyl disulfide in
heat-treated sucuk. The use of sodium ascorbate lowered the 2- pentyl-furan, butanoic acid and propanoic acid
level. According to PCA results, the nitrite level of 100 mg/kg was the most associated with volatile compounds
than with other treatments.

1. Introduction
The flavor of fermented sausages is significantly influenced by
physical, chemical and microbiological processes which are influenced
by external factors such as initial fermentation temperature and internal
factors such as pH, nitrate/nitrite and salt content (Tjener & Stahnke,
2007). Lactic acid bacteria and Gram (+) catalase positive cocci are
technologically important microorganisms for fermented sausages. The
acidification formed by lactic acid bacteria plays an important role for
flavor, color, and texture development as well as for the control of un­
desired microbiota, including foodborne pathogens, during fermenta­
tion. Gram (+) catalase (+) cocci are also involved in the color
formation and delay lipid oxidation and flavor formation with their
nitrate reductase, catalase, lipolytic and proteolytic activities (Ordonez,
Hierro, Bruna, & de la Hoz, 1999; Fonseca, Cachaldora, Gomez, Franco,
& Carballo, 2013).
Nitrite is preferred as a curing agent in rapid fermented sausages due
to rapid drop in pH whereas nitrate is commonly used in slow fermented
sausage (Lücke, 1998). The use of nitrite in fermented sausages has been
restricted by various regulations up to 150 mg/kg (Anonymous, 2011).
However, it is reported that 40–50 mg/kg of ingoing nitrite will ensure
typical quality characteristics (color, flavor, antioxidant properties) in
meat products (Sebranek & Bacus, 2007). The addition of nitrite to
fermented sausages contributes to development of cured color, retards
lipid oxidation and inhibits growth of pathogens and spoilage micro­
organisms (Huang et al., 2020; Sebranek & Bacus, 2007). Some chemical
and biochemical reactions, occurring between the degradation products
of nitrite and protein, fat, carbohydrate degradation products, play an
important role in the development of the typical flavor in cured meat
products. Volatile compounds have significant role in typical flavor of
cured meat products. These compounds arise from carbohydrate
fermentation, lipolysis, proteolysis, lipid oxidation and amino acid
catabolism (Bolumar et al., 2006; Ordonez et al., 1999). Volatile com­
pounds, originated from amino acid degradation and carbohydrate
fermentation, has been found to be more abundant in dry fermented
sausages cured with nitrate (Marco, Navarro, & Flores, 2006). Similarly,
Olesen, Sthanke, and Talon (2004) asserted that the addition of nitrate
to dry fermented sausage increased the level of volatile compounds
compared to nitrite. Moreover, Stahnke (1995a) reported that dry fer­
mented sausage cured with nitrite had higher amounts of volatile acids,
while it had lower levels of ethyl esters, certain short chain aldehydes
and lipid oxidation products, compared to sausages cured with nitrate.
In the other hand, it was reported that the effects of curing agents on
aroma depend on balance between aldehydes formed during oxidation

* Corresponding author. Bandırma Onyedi Eylul University, Bandırma Vocational School, Department of Food Processing, 10200, Bandırma/Balıkesir, Turkey.
E-mail address: ssallan@bandirma.edu.tr (S. Sallan).

https://doi.org/10.1016/j.lwt.2021.112540
Received 30 April 2021; Received in revised form 22 September 2021; Accepted 26 September 2021
Available online 27 September 2021
0023-6438/© 2021 Elsevier Ltd. All rights reserved.
S. Sallan et al.
reactions and key sulfur odor compounds (Flores, 2018; Flores & Toldra,
2021).
Ascorbate, another ingredient added to fermented sausages, is a
reducing agent that accelerates the curing process (Flores & Toldra,
2021). It is also effective in scavenging reactive oxygen species and
radicals, and thus preventing lipid oxidation in fermented sausages
(Berardo et al., 2016). Moreover, it has been found that addition of
ascorbate to fermented sausage reduced the level of volatile compounds
arising from lipid oxidation (Olesen et al., 2004).
Fermentation is a key stage in the production of fermented sausages.
In this stage, besides the starter culture, hurdle effects such as salt and
nitrite are of great importance for ensuring product safety (Lücke,
1998). However, it is very difficult to achieve the desired sensory
characteristics in rapid fermented sausages such as heat-treated sucuk.
Heat-treated sucuk is made from beef and beef fat by using processes
that include short-fermentation, heat treatment and drying. Rapid
fermentation is usually used for this type of sausage, and therefore,
starter culture is commonly used in the manufacture. After fermenta­
tion, heat treatment is carried out until the internal temperature reaches
60–68 ◦ C, depending on the combination of temperature and time
(Armutçu, Hazar, Yılmaz Oral, Kaban, & Kaya, 2020). In this product,
the pH value should be 5.6 or less, and the moisture/protein ratio should
be below 3.6/1 (Anonymous, 2019). According to these parameters,
heat-treated sucuk (raw fermented cooked and dried) can be included
into the group of semi-dry fermented sausages (Sallan, Kaban, Şişik
Oğraş, Çelik, & Kaya, 2020). The rapid pH drop in the fermentation of
this product and the subsequent heat treatment are of great importance
in preventing growth of undesirable microorganisms. However, a suf­
ficient flavor cannot be formed in this product (Kaban & Bayrak, 2015).
Yılmaz Oral and Kaban (2021) investigated the effect of autochthonous
Lactobacillus sakei S15 alone or together autochthonous Staphylococcus
xylosus GM92 as starter culture for heat treated sucuk production on the
volatile compounds and technological properties. However, there is no
study about the effects of different levels of nitrite in the presence of
sodium ascorbate on the volatile compounds of heat-treated sucuk with
or without starter culture. In addition, there is also no study investi­
gating the use of L. plantarum as a starter culture in this product with
coagulase negative staphylococci. Therefore, the aim of the study was to
investigate the effects of autochthonous strains (Lactobacillus plantarum
GM77 + Staphlyococcus xylosus GM92), use of ascorbate (0 or 500
mg/kg) and different nitrite levels (0, 50, 100 or 150 mg/kg) on volatile
compounds of heat treated sucuk.
2. Material and method
2.1. Material
Beef and beef meat fat were used as raw materials in the study. After
trimming the visible fat and connective tissue, large pieces of meat from
beef carcasses were cut into small pieces. Then, they were vacuum
packed in Polyamide/Polyethylene (PA/PE) bags and stored at − 20 ◦ C.
Beef meat fat was also chopped into small pieces, then packed in PA/PE
bags by applying a vacuum and stored at − 20 ◦ C. For each treatment, 4
kg of beef +1 kg of beef meat fat were used, and the preparation was
repeated three times. Thus, a total of 48 batters were prepared.
Autochthonous Lactobacillus plantarum GM77 and Staphylococcus xylosus
GM92 isolated from sucuk (Kaban & Kaya, 2009) were used as a starter
culture.
2.2. Heat-treated sucuk production
To produce heat-treated sucuk, per kg of beef and fat, 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. Sixteen different sucuk batters were prepared ac­
cording to the experimental design (ingoing nitrite level: 0, 50, 100 or
150 mg/kg; use of sodium ascorbate: 0 or 500 mg/kg and starter culture:
2
LWT 153 (2022) 112540
control or Lactobacillus plantarum GM77 (107 cfu/gr) + Staphylococcus
xylosus GM92 (106 cfu/gr)). Batters were prepared in laboratory type
cutter (MADO Typ MTK 662, Dornhan/Schwarzwald) and stuffed in
collagen casings (38 mm, Naturin Darm, Germany) by using a
laboratory-type filling machine (MADO Typ MTK 591, Dornhan/
Schwarzwald). The fermentation process was applied to the sausages for
2 days at 22 ± 1 ◦ C at 90 ± 2% relative humidity in the climate chamber
(Reich, Thermoprozestechnik GmbH, Schechingen, Germany), where
temperature, relative humidity and air flow can be controlled auto­
matically. Following the fermentation, samples were subjected to a heat
treatment process, starting from 40 ◦ C in the cooking chamber (Mauting,
VKM Kompakt-P, Mikulovska, Czech Republic) to reach an internal
temperature of 68 ◦ C. After the heat treatment, the heat-treated sausages
were dried in the climate chamber (Reich, Thermoprozestechnik GmbH,
Schechingen, Germany) for 3 days at 16 ◦ C.
2.3. Method
2.3.1. Analysis of volatile compound
Five g each of the homogenized samples were weighed into 40 ml
vials (Supelco, Bellefonte PA, USA) and stored at − 20 ◦ C. After the vials
were kept in a thermal block (Supelco, Bellefonte PA, USA) for 1 h at
30 ◦ C for the extraction of headspace volatile compounds, a 75 μm
Carboxen/Polydimethylsiloxane (CAR/PDMS fiber, Supelco, 75 μm,
USA) was placed in vials for adsorption of the compounds and kept for 2
h. The compounds adsorbed by the fibre were desorbed from the in­
jection port of the gas chromatography (GC, Agilent Technologies
6890N) for 6 min at 250 ◦ C. The compounds were identified by mass
spectrometry (MS, Agilent Technologies 5973). Helium was used as
carrier gas in the system and DB-624 (60 m × 0.250 mm x 1.40 μm,
Agilent Tech, USA) was used as column. The oven temperature of the gas
chromatography was initially set at 40 ◦ C for 5 min, and then gradually
increased to 110 ◦ C with 3 ◦ C/min, 150 ◦ C with 4 ◦ C/min and 210 ◦ C
with 10 ◦ C/min, then kept at 210 ◦ C for 12 min. The injector port was in
the splitless mode. The GC/MS interface was maintained at 280 ◦ C. The
mass spectrometer was operated in the electron impact mode with the
electron energy set at 70 eV. The identification was carried out by
comparing the retention times and mass spectra of volatiles to those of
authentic compounds generated in the laboratory, by mass spectra ob­
tained from NIST, WILEY and FLAVOR libraries, and by determining
kovats’ retention indexes (Supelco 44585-U, Bellefonte PA USA) and
comparing them with those reported in the literature (Kaban, 2009).
2.3.2. Statistical analysis
In the study, addition of starter culture (without or with starter
culture), the use of sodium ascorbate (without or with sodium ascor­
bate) and ingoing nitrite level (0, 50, 100 or 150 mg/kg) were evaluated
as factors. For each treatment, three batters were prepared. The exper­
iments were performed according to the completely randomized design
with three replicates. The obtained analysis results were analyzed with
one-way ANOVA using the general linear model. The factors were
evaluated as fixed effects and replicates as random effect. The means of
significant sources of variation were compared using Duncan’s multiple
comparison test at the P < 0.05 level. All statistical analyzes were car­
ried out using the SPSS version 24 statistical program. (SPSS Inc., Chi­
cago, IL, USA). Principal component analysis (PCA) was performed with
the aid of The Unscrambler software (CAMO software version 10.1).
3. Results and discussion
Cured flavor is seen as an important quality feature of sausage
products as it influences the consumer’s purchase decision (Sebranek &
Bacus, 2007). A great number of compounds contribute to the devel­
opment of aroma and flavor in fermented sausages (Lücke, 1998). In our
study, seventy-four volatile compounds were identified in all the
heat-treated sucuk samples including 12 sulfur compounds, 7 aliphatic
S. Sallan et al. LWT 153 (2022) 112540

hydrocarbons, 5 esters, 6 aromatic hydrocarbons, 4 acids, 6 alcohols, 6
aldehydes, 1 furan, 5 ketones, 22 terpenes (Table 1, Table 2, Table 3).
3.1. Sulfur compounds
Due to their low aroma and taste threshold values, sulfur compounds
can affect the sensory properties of the product even at low concentra­
tions (McGorrin, 2011). In this study, 12 sulfur compounds were
detected (Table 1). Sulfur compounds can stem from garlic used in the
production or can be formed as a result of amino acid catabolism
(Ordonez et al., 1999). Garlic is comprised of sulfur-containing char­
acter impact compounds. The aromatic effect of garlic is based on diallyl
disulfide and diallyl thiosulfinate (McGorrin, 2011). Diallyl disulfide is
widely detected volatile sulfur compounds in heat-treated sucuk. Be­
sides, 3,3′ -thiobis-1-propene and allyl methyl sulfide are also identified
in heat-treated sucuk (Kaban & Bayrak, 2015; Yılmaz Oral & Kaban,
2021). Diallyl disulfide, methyl-2-propenyl-disulfide, 3,3′ -thio­
bis-1-propene, allyl methyl sulfide were all reduced by the addition of
starter culture in heat-treated sucuk (P < 0.05) (Table 1). However, it
has been reported that autochthonous strains (L. sakei and/or S. xylosus)

Table 1
Overall effect of starter culture, sodium ascorbate, nitrite level on the sulfur compounds, aliphatic hydrocarbons and esters of heat-treated sucuk (means ± SD) (AU x
106).
Compounds RI KI Starter Culture (SC) Sodium Ascorbate (Asc) Nitrite (mg/kg)

Without With SC P Without With Asc P Nitrite_0 Nitrite_50 Nitrite_100 Nitrite_150 P

SC Asc

Sulfur Compounds
Methanethiol c <500 2.44 ± 1.23 ± 0.001 1.80 ± 1.88 ± ns 1.84 ± 1.84 ± 1.97 ± 1.72 ± ns
2.06a 1.18b 1.73a 1.85a 1.67a 1.74a 1.97a 1.83a
1-Propanethiol c 538 0.98 ± 0.68 ± 0.046 0.61 ± 1.06 ± 0.003 1.28 ± 0.69 ± 0.71 ± 0.65 ± 0.010
0.89a 0.71b 0.65b 0.91a 0.87a 0.71b 0.81b 0.75b
2-propene-1- b 574 189.29 ± 164.16 ns 184.25 ± 169.22 ns 176.17 ± 189.13 ± 181.29 ± 160.33 ± ns
thiol 79.11a ± 82.71a 84.06a ± 78.98a 80.76a 96.91a 77.7a 70.78a
Allyl methyl b 730 26.56 ± 16.33 ± <0.001 23.38 ± 19.52 ± ns 26.38 ± 20.33 ± 20.07 ± 19.02 ± ns
sulfide 13.63a 8.29b 13.40a 10.99a 15.66a 11.42a 10.99a 9.92a
1-(metilthio)-1- c 832 1.13 ± 0.73 ± 0.004 0.97 ± 0.89 ± ns 0.82 ± 0.77 ± 1.17 ± 0.97 ± ns
propene 0.79a 0.51b 0.63a 0.76a 0.55a 0.74a 0.74a 0.69a
3,3′ -tiobis- 1- b 888 28.24 ± 18.95 ± 0.006 28.09 ± 19.10 ± 0.007 29.52 ± 20.45 ± 21.09 ± 22.53 ± ns
propene 19.03a 12.67b 18.74a 13.20b 17.58a 14.09a 15.88a 18.62a
Methyl c 958 31.53 ± 16.57 ± <0.001 27.26 ± 20.84 ± 0.043 31.36 ± 22.61 ± 22.28 ± 19.95 ± ns
2-propenyl- 19.42a 10.05b 18.81a 14.74b 19.67a 17.15a 16.22a 13.68a
disulfide
3-methylthio- c 973 0.34 ± 0.22 ± ns 0.24 ± 0.33 ± ns 0.49 ± 0.32 ± 0.19 ± 0.13 ± 0.005
propanal 0.49a 0.24a 0.48a 0.27a 0.51a 0.46 ab 0.27b 0.11b
Methyl-trans- b 977 1.83 ± 1.24 ± 0.001 1.27 ± 1.80 ± 0.003 1.17 ± 1.73 ± 1.75 ± 1.49 ± ns
propenyl- 1.00a 0.73b 0.75a 1.00b 0.80a 1.00a 0.98a 0.81a
disulfide
Diallyl disulfide a 1138 47.56 ± 32.47 ± <0.001 44.97 ± 35.06 ± 0.005 48.39 ± 36.32 ± 36.72 ± 38.63 ± 0.048
20.66a 14.30b 20.77a 16.31b 20.31a 16.78b 19.29b 18.97b
di-2-propenyl- b 1171 7.83 ± 6.31 ± 0.022 7.00 ± 7.14 ± ns 6.20 ± 7.51 ± 7.26 ± 7.32 ± ns
disulfide 3.55a 2.55b 3.39a 2.97a 2.90a 3.49a 3.23a 3.05a
2,5-dimethyl- c >1500 2.06 ± 2.69 ± ns 2.42 ± 2.34 ± ns 2.17 ± 2.82 ± 2.06 ± 2.45 ± ns
benzo[b] 2.00a 1.77a 1.79a 2.03a 1.96a 1.97a 1.77a 1.93a
thiophene
Aliphatic hydrocarbons
Hexane a 600 3.31 ± 2.13 ± 0.004 2.64 ± 2.80 ± ns 2.49 ± 2.96 ± 2.86 ± 2.57 ± ns
2.44a 1.09b 1.74a 2.21a 1.62a 2.15a 2.24a 1.92a
Heptane a 700 0.74 ± 0.31 ± ns 0.65 ± 0.39 ± ns 1.14 ± 0.35 ± 0.31 ± 0.30 ± 0.014
1.45a 0.47a 1.45a 0.54a 1.98a 0.44b 0.41b 0.42b
Octane a 800 0.76 ± 0.34 ± 0.014 0.71 ± 0.39 ± ns 0.79 ± 0.63 ± 0.45 ± 0.35 ± ns
1.11a 0.35b 1.08a 0.47a 1.14a 0.95a 0.67a 0.44a
Undecane a 1100 1.84 ± 0.98 ± 0.007 1.57 ± 1.26 ± ns 1.50 ± 1.33 ± 1.46 ± 1.35 ± ns
1.91a 0.64b 1.79a 1.09a 1.75a 1.54a 1.18a 1.49a
Dodecane a 1200 1.81 ± 0.91 ± 0.006 1.58 ± 1.14 ± ns 1.46 ± 1.36 ± 1.43 ± 1.19 ± ns
1.91a 0.79b 1.81a 1.15a 1.43a 1.67a 1.56a 1.48a
Tridecane a 1300 1.59 ± 1.14 ± ns 1.60 ± 1.12 ± ns 1.43 ± 1.50 ± 1.36 ± 1.14 ± ns
1.18a 1.23a 1.29a 1.12a 1.10a 1.39a 1.34a 1.06a
Tetradecane a 1400 1.70 ± 0.73 ± 0.006 1.46 ± 0.98 ± ns 1.12 ± 1.45 ± 1.25 ± 1.04 ± ns
1.86a 1.27b 1.96a 1.27a 1.51a 1.98a 1.64a 1.55a
Esters
Ethyl acetate a 639 1.48 ± 1.08 ± ns 1.17 ± 1.39 ± ns 1.68 ± 1.31 ± 1.25 ± 0.89 ± ns
1.52a 1.28a 1.27a 1.55a 1.69a 1.50a 1.41a 0.88a
Butyl b 824 0.65 ± 0.59 ± ns 0.69 ± 0.56 ± ns 0.81 ± 0.71 ± 0.66 ± 0.32 ± ns
propanoate 0.83a 0.85a 0.92a 0.75a 0.86a 0.73a 1.10a 0.50a
Propyl b 1151 2.65 ± 1.97 ± ns 2.14 ± 2.49 ± ns 2.51 ± 2.49 ± 2.53 ± 1.71 ± ns
hexanoate 2.05a 1.53a 1.56a 2.07a 1.91a 1.84a 2.26a 1.12a
Hexyl butanoate b 1215 3.53 ± 2.76 ± ns 3.07 ± 3.22 ± ns 3.22 ± 3.69 ± 3.11 ± 2.56 ± ns
2.43a 1.49a 1.96a 2.14a 2.38a 2.32a 1.66a 1.66a
2-hydroxy- c 1269 0.98 ± 0.51 ± 0.001 0.82 ± 0.66 ± ns 0.76 ± 0.90 ± 0.72 ± 0.59 ± ns
methyl- 0.87a 0.37b 0.78a 0.60a 0.74a 0.79a 0.70a 0.56a
benzoate

P: statistical significance; ns: not significant P > 0.05. Results are expressed in Arbitrary Area Units ( × 106); RI: Reliability of identification; a: mass spectrum and
retention time identical with an authentic sample; b: mass spectrum and Kovats index from literature in accordance; c: tentative identification by mass spectrum; KI:
Kovats index calculated for DB-624 capillary column (60 m × 0.25 mm × 1.4 μm) installed on a gas chromatograph equipped with a mass selective detector.

3
S. Sallan et al. LWT 153 (2022) 112540

Table 2
Overall effect of starter culture, sodium ascorbate, nitrite level on the aromatic hydrocarbons, acids, alcohols, aldehydes and furans of heat-treated sucuk (means ± SD)
(AU x 106).
Compounds RI KI Starter Culture (SC) Sodium Ascorbate (Asc) Nitrite (mg/kg)

Without With SC P Without With Asc P Nitrite_0 Nitrite_50 Nitrite_100 Nitrite_150 P

SC Asc

Aromatic hydrocarbons
1-methyl-1H- c 786 2.72 ± 2.71 ± ns 2.96 ± 2.48 ± ns 3.72 ± 2.97 ± 2.24 ± 1.93 ± 0.004
pyrole 2.26a 1.43a 2.22a 1.45a 2.49a 1.91 ab 1.33b 1.01b
Toluen a 796 3.80 ± 2.37 ± 0.001 3.18 ± 2.99 ± ns 3.39 ± 3.33 ± 3.01 ± 2.63 ± ns
2.39a 1.36b 2.22a 1.92a 2.10a 2.31a 2.05a 1.81a
p-xylene a 898 1.53 ± 0.66 ± 0.001 1.31 ± 0.89 ± ns 1.04 ± 1.28 ± 1.09 ± 0.98 ± ns
1.61a 0.56b 1.59a 0.82a 0.95a 1.59a 1.21a 1.33a
Styrene b 935 1.04 ± 0.75 ± 0.011 0.95 ± 0.85 ± ns 0.97 ± 0.88 ± 0.85 ± 0.89 ± ns
0.61a 0.37b 0.63a 0.39a 0.49a 0.46a 0.68a 0.45a
1-methoxy-4-(1- b 1251 2.12 ± 1.66 ± ns 1.96 ± 1.82 ± ns 1.96 ± 2.03 ± 1.65 ± 1.92 ± ns
propenyl)- 1.38a 0.96a 1.38a 1.02a 1.25a 1.29a 1.30a 0.99a
benzene
1,2-dimethoxy-4- c 1434 6.81 ± 7.19 ± ns 7.16 ± 6.85 ± ns 6.72 ± 7.09 ± 6.51 ± 7.67 ± ns
(2-propenyl)- 2.45a 2.40a 2.59a 2.25a 2.52a 2.43a 1.96a 2.68a
benzene
Acids
Acetic acid a 710 8.93 ± 20.26 ± <0.001 15.44 ± 13.76 ± ns 17.14 ± 13.24 ± 13.31 ± 14.71 ± ns
6.79a 9.64b 9.38a 10.73a 10.42a 9.26a 9.49a 11.09a
Butanoic acid a 882 0.95 ± 0.95 ± ns 1.17 ± 0.73 ± 0.010 1.21 ± 0.94 ± 0.86 ± 0.78 ± ns
0.99a 0.64a 1.00a 0.54b 1.09a 0.86a 0.68a 0.58a
Propanoic acid c 890 3.25 ± 3.61 ± ns 4.09 ± 2.77 ± 0.002 4.50 ± 3.98 ± 2.87 ± 2.37 ± 0.002
2.38a 2.15a 2.55a 1.73b 2.39a 2.58 ab 1.91bc 1.46c
3-Hexanoic acid c 910 0.17 ± 0.24 ± ns 0.18 ± 0.22 ± ns 0.27 ± 0.14 ± 0.18 ± 0.22 ± ns
0.15a 0.24a 0.18a 0.23a 0.26a 0.15a 0.19a 0.19a
Alcohols
Ethanol a 512 20.81 ± 18.12 ± ns 19.62 ± 19.32 ± ns 21.45 ± 21.69 ± 16.46 ± 18.26 ± ns
9.91a 7.07a 10.15a 7.01a 8.03a 11.87a 6.53a 6.53a
1-propen-2-ol b 527 1.00 ± 0.98 ± ns 0.86 ± 1.13 ± ns 1.35 ± 1.02 ± 0.95 ± 0.65 ± 0.041
0.93a 0.85a 0.97a 0.78a 0.83a 0.76 ab 1.18 ab 0.59b
1-hexanol a 1070 1.42 ± 0.92 ± 0.042 1.36 ± 0.98 ± ns 1.38 ± 1.24 ± 1.09 ± 0.97 ± ns
1.42a 0.75b 1.39a 0.84a 1.24a 1.46a 1.00a 0.87a
2-ethyl-1-hexanol b 1084 1.07 ± 0.53 ± 0.004 0.89 ± 0.71 ± ns 0.68 ± 0.96 ± 0.84 ± 0.72 ± ns
1.11a 0.43b 1.06a 0.66a 0.72a 1.06a 1.08a 0.62a
α-2-propenly- c 1391 6.79 ± 6.56 ± ns 6.41 ± 6.94 ± ns 6.15 ± 6.13 ± 6.68 ± 7.72 ± ns
benzemethanol 2.42a 2.30a 2.38a 2.31a 2.31a 1.78a 2.22a 2.76a
α–(1-methyethyl)- c 1398 18.92 ± 25.88 ± <0.001 21.95 ± 22.85 ± ns 21.99 ± 22.90 ± 21.42 ± 23.28 ± ns
benzemethanol 8.19a 9.33b 9.01a 9.86a 9.49a 10.80a 9.46a 8.18a
Aldehydes
Acetaldehyde b <500 41.52 ± 39.64 ± ns 41.23 ± 39.93 ± ns 42.48 ± 39.02 ± 40.16 ± 40.65 ± ns
8.76a 9.56a 9.34a 9.05a 8.33a 10.03a 9.37a 9.12a
Pentanal a 742 1.13 ± 0.65 ± 0.003 1.02 ± 0.76 ± ns 1.35 ± 0.73 ± 0.68 ± 0.79 ± 0.011
1.14a 0.37b 1.14a 0.46a 1.53a 0.52b 0.37b 0.38b
Hexanal a 837 2.86 ± 1.84 ± 0.016 2.38 ± 2.33 ± ns 2.91 ± 2.42 ± 1.87 ± 2.23 ± ns
2.42a 1.33b 1.99a 2.06a 2.31a 2.39a 1.39a 1.78a
Heptanal a 955 1.69 ± 0.99 ± 0.001 1.48 ± 1.22 ± ns 1.43 ± 1.34 ± 1.32 ± 1.29 ± ns
1.25a 0.61b 1.21a 0.82a 1.16a 1.03a 1.09a 0.92a
Octanal a 1051 2.02 ± 1.30 ± 0.045 1.72 ± 1.59 ± ns 1.72 ± 1.82 ± 1.76 ± 1.32 ± ns
1.99a 1.29b 1.91a 1.49a 0.93a 1.63a 1.65a 1.65a
2-methyl-3- b 1344 222.89 ± 191.46 0.004 200.51 ± 213.84 ns 213.35 ± 201.11 ± 202.97 ± 211.27 ± ns
phenyl-propanal 56.73a ± 59.42a ± 75.85a 47.81a 46.2a 51.84a
51.25b 52.23a
Furan
2-pentyl-furan b 1021 0.62 ± 0.44 ± ns 0.68 ± 0.39 ± 0.032 0.49 ± 0.69 ± 0.49 ± 0.45 ± ns
0.84a 0.34a 0.82a 0.36b 0.44a 0.85a 0.68a 0.54a

P: statistical significance; ns: not significant P > 0.05. Results are expressed in Arbitrary Area Units ( × 106); RI: Reliability of identification; a: mass spectrum and
retention time identical with an authentic sample; b: mass spectrum and Kovats index from literature in accordance; c: tentative identification by mass spectrum; KI:
Kovats index calculated for DB-624 capillary column (60 m × 0.25 mm × 1.4 μm) installed on a gas chromatograph equipped with a mass selective detector.

used as starter culture had no significant effects on methanethiol,
1-methylthio-1-propene (Z), 3,3′ -thiobis-1-propene, 2,4-dimethyl-thio­
phene, methyl 2-propenyl disulfide and diallyl disulfide compounds
during processing of heat-treated sucuk (Yılmaz Oral & Kaban, 2021).
Similarly, in another study conducted on a Spanish type of traditional
dry fermented sausage, it was reported that allyl methyl sulfide, diallyl
sulfide and diallyl disulfide were derived from garlic, but the using
starter culture (L. sakei + S. equorum, L. sakei + S. epidermis or L. sakei+
S. saprophyticus) had no effect on these compounds (Fonseca et al.,
2013).
The addition of sodium ascorbate reduced the amounts of diallyl
disulfide, methyl-2-propenyl-disulfide and 3,3′ -thiobis-1-propene, while
increasing the amounts of methyl-trans-propenyl-disulfide and 1-pro­
panethiol significantly (P < 0.05) (Table 1). In the other hand, 2-pro­
pene-1-thiol, detected in the highest amounts among sulfur
compounds, was not influenced by the addition of sodium ascorbate as
well as the use of starter culture or different nitrite levels (Table 1). The
use of nitrite decreased the abundance of diallyl disulfide (P < 0.05)
(Table 1). In studies examining the effects of reducing nitrate levels on
the aroma profile of dry fermented sausages produced using commercial

4
S. Sallan et al. LWT 153 (2022) 112540

Table 3
Overall effect of starter culture, sodium ascorbate, nitrite level on the ketones and terpens of heat-treated sucuk (means ± SD) (AU x 106).
Compounds RI KI Starter Culture (SC) Sodium Ascorbate (Asc) Nitrite (mg/kg)

Without With SC P Without With P Nitrite_0 Nitrite_50 Nitrite_100 Nitrite_150 P

SC Asc Asc

Ketones
2-pentanone c 728 0.51 ± 0.27 ± 0.043 0.51 ± 0.27 ± 0.042 0.64 ± 0.44 ± 0.29 ± 0.19 ± 0.042
0.76a 0.29b 0.77a 0.27b 0.95a 0.51 ab 0.32b 0.24b
3-hydroxy-2- b 779 4.81 ± 6.80 ± 0.016 6.46 ± 5.15 ± ns 7.88 ± 5.68 ± 5.24 ± 4.42 ± 0.023
butanone 3.52b 4.35a 4.55a 3.44a 4.85a 4.13 ab 3.57b 2.86b
2-heptanone b 946 0.67 ± 0.76 ± ns 0.62 ± 0.81 ± ns 0.71 ± 0.69 ± 0.74 ± 0.71 ± ns
0.59a 0.59a 0.57a 0.61a 0.71a 0.61a 0.60a 0.47a
6-methyl-5- c 1050 1.06 ± 0.68 ± 0.015 0.99 ± 0.74 ± ns 0.88 ± 0.95 ± 0.85 ± 0.78 ± ns
hepten-2-one 0.91a 0.43b 0.88a 0.52a 0.73a 0.82a 0.75a 0.66a
1-(4- c 1272 1.91 ± 1.59 ± ns 1.75 ± 1.76 ± ns 1.60 ± 1.82 ± 1.79 ± 1.80 ± ns
methylphenyl)- 1.19a 1.20a 1.23a 1.19a 1.22a 1.16a 1.22a 1.26a
ethanone
Terpens
α-Thujen b 944 4.40 ± 3.50 ± 0.015 4.16 ± 3.74 ± ns 4.15 ± 3.89 ± 3.98 ± 3.77 ± ns
1.98a 1.40b 1.99a 1.49a 1.61a 1.78a 1.93a 1.82a
1R-α-pinene c 996 19.27 ± 16.41 ± ns 18.78 ± 16.90 ± ns 18.93 ± 18.08 ± 17.33 ± 17.03 ± ns
8.08a 6.82a 8.48a 6.50a 7.83a 7.94a 7.35a 7.52a
β- myrcene b 998 0.45 ± 0.32 ± 0.030 0.42 ± 0.34 ± ns 0.41 ± 0.34 ± 0.42 ± 0.35 ± ns
0.32a 0.25b 0.33a 0.24a 0.34a 0.23a 0.34a 0.25a
α-phellandrene c 1047 31.01 ± 27.26 ± ns 30.71 ± 27.56 ± ns 31.76 ± 29.85 ± 28.43 ± 26.49 ± ns
16.39a 15.96a 16.07a 16.33a 16.61a 15.69a 17.44a 15.54a
D-Limonene a 1054 28.28 ± 22.98 ± ns 26.46 ± 24.81 ± ns 26.02 ± 25.26 ± 26.59 ± 24.66 ± ns
15.16a 10.73a 14.83a 11.76a 12.47a 12.95a 14.42a 14.17a
β- phellandrene c 1060 6.11 ± 4.45 ± 0.027 5.88 ± 4.67 ± ns 5.55 ± 5.32 ± 5.06 ± 5.18 ± ns
3.99a 2.73b 4.02a 2.82a 3.64a 3.44a 3.43a 3.71a
β-Ocimene c 1065 6.67 ± 6.95 ± ns 6.85 ± 6.76 ± ns 7.09 ± 6.84 ± 6.69 ± 6.61 ± ns
2.49a 2.63a 2.41a 2.71a 2.70a 2.80a 2.63a 2.16a
4-Carene c 1135 1.58 ± 1.56 ± ns 1.57 ± 1.57 ± ns 1.76 ± 1.59 ± 1.64 ± 1.29 ± ns
1.33a 0.94a 1.26a 1.03a 1.26a 1.03a 1.16a 1.13a
Terpinolene c 1139 6.26 ± 5.50 ± ns 5.89 ± 5.86 ± ns 6.07 ± 6.42 ± 5.32 ± 5.71 ± ns
3.44a 2.37a 2.70a 3.23a 2.53a 3.88a 2.91a 2.37a
Linalool a 1161 22.71 ± 20.97 ± ns 22.47 ± 21.21 ± ns 21.94 ± 22.17 ± 21.89 ± 21.33 ± ns
8.03a 8.42a 8.32a 8.17a 7.47a 8.81a 9.06a 7.97a
Ocimene c 1197 2.97 ± 1.44 ± 0.013 2.04 ± 1.69 ± ns 1.81 ± 2.02 ± 1.83 ± 1.80 ± ns
1.98a 0.98b 1.83a 1.38a 1.59a 1.73a 1.65a 1.60a
Camphor c 1233 1.43 ± 0.88 ± 0.002 1.28 ± 1.03 ± ns 1.17 ± 1.22 ± 1.19 ± 1.05 ± ns
1.04a 0.35b 0.95a 0.65a 0.84a 0.84a 0.96a 0.64a
Terpinen-4-ol b 1235 2.92 ± 3.06 ± ns 3.01 ± 2.96 ± ns 3.14 ± 2.95 ± 2.95 ± 2.90 ± ns
0.83a 0.82a 0.78a 0.87a 0.89a 1.00a 0.74a 0.62a
α- terpineol c 1258 1.75 ± 3.07 ± <0.001 2.47 ± 2.35 ± ns 2.81 ± 2.24 ± 2.19 ± 2.39 ± ns
1.56b 1.21a 1.76a 1.31a 1.39a 1.51a 1.38a 1.84a
Camphene c 1362 1.92 ± 2.91 ± 0.001 2.63 ± 2.20 ± ns 2.41 ± 2.28 ± 2.31 ± 2.65 ± ns
1.13b 1.61a 1.37a 1.55a 1.42a 1.34a 1.58a 1.59a
Carvacrol c 1412 1.84 ± 2.08 ± ns 2.08 ± 1.84 ± ns 1.81 ± 2.04 ± 1.96 ± 2.02 ± ns
0.99a 0.84a 1.14a 0.62a 0.83a 0.76a 1.27a 0.77a
Copaene c 1433 6.46 ± 7.04 ± ns 6.93 ± 6.58 ± ns 6.63 ± 7.03 ± 6.13 ± 7.22 ± ns
1.99a 2.47a 2.54a 1.94a 2.16a 2.43a 1.84a 2.50a
β-elemene c 1438 1.52 ± 1.46 ± ns 1.59 ± 1.39 ± ns 1.85 ± 1.50 ± 1.19 ± 1.40 ± ns
1.22a 0.85a 0.68a 1.23a 1.68a 0.80a 0.48a 0.35a
Eugenol b 1450 5.12 ± 5.33 ± ns 5.18 ± 5.27 ± ns 4.99 ± 5.32 ± 5.04 ± 5.54 ± ns
1.32a 1.42a 1.51a 1.23a 1.39a 1.37a 1.19a 1.51a
Cis-caryophyllene c 1470 5.35 ± 4.79 ± ns 5.39 ± 4.75 ± ns 4.59 ± 5.19 ± 4.77 ± 5.71 ± ns
2.42a 2.41a 2.85a 1.86a 2.43a 2.46a 2.04a 2.69a
Caryophyllene c 1490 26.94 ± 29.37 ± ns 29.05 ± 27.25 ± ns 28.66 ± 29.60 ± 25.51 ± 28.83 ± ns
5.34a 7.79a 6.72a 6.73a 5.85a 8.03a 6.37a 6.23a
α-caryophyllene c 1504 1.68 ± 1.77 ± ns 1.80 ± 1.65 ± ns 1.65 ± 1.86 ± 1.58 ± 1.81 ± ns
0.54a 0.49a 0.57a 0.45a 0.56a 0.54a 0.49a 0.47a

P: statistical significance; ns: not significant P > 0.05. Results are expressed in Arbitrary Area Units ( × 106); RI: Reliability of identification; a: mass spectrum and
retention time identical with an authentic sample; b: mass spectrum and Kovats index from literature in accordance; c: tentative identification by mass spectrum; KI:
Kovats index calculated for DB-624 capillary column (60 m × 0.25 mm × 1.4 μm) installed on a gas chromatograph equipped with a mass selective detector.

starter culture containing L. sakei, S. xylosus and S. carnosus, there was
no significant difference associated with added nitrate level in the
presence of sodium ascorbate in terms of dimethyl disulfide (Perea-Sanz,
Montero, Belloch, & Flores, 2018; Perea-Sanz, Montero, Belloch, &
Flores, 2019). In another study, similiar results were also found for dry
fermented sausages cured with nitrate and nitrite (Perea-Sanz,
Lopez-Diez, Belloch, & Flores, 2020).
3.2. Aliphatic hydrocarbons
Hexane, heptane, octane, undecane, dodecane, tridecane and tetra­
decane were determined in the heat-treated sucuk samples (Table 1).
Due to their high odor thresholds, these compounds do not contribute
significantly to the odor profile (Kaban & Kaya, 2009). The use of starter
culture had no significant effect on heptane and tridecane compounds
(P > 0.05), however other compounds decreased significantly in the
presence of starter culture (P < 0.05). In another study examining the

5
S. Sallan et al.
effects of different starter cultures on heat-treated sucuk, it was reported
that seven aliphatic hydrocarbons were determined and the use of the
starter culture was only effective on undecane (Yılmaz Oral & Kaban,
2021). It was believed that these differences might be related due to the
differences of strains used as starter culture in the heat-treated sucuk.
Among aliphatic hydrocarbons identified, only heptane was signifi­
cantly influenced from different nitrite levels (P < 0.05), however, no
significant differences between 50, 100 or 150 mg/kg nitrite levels were
observed (P > 0.05). The use of sodium ascorbate did not have a sig­
nificant effect on all aliphatic hydrocarbons compounds (P > 0.05)
(Table 1).
3.3. Esters
Esters are generally formed by the esterification of carboxylic acids
and alcohols in meat products. In addition, lactic acid bacteria and
staphylococci also show esterification activity (Gutsche, Thao Tran, &
Vogel, 2012). The use of sodium ascorbate and different nitrite levels did
not cause a significant difference in groups in terms of esters (P > 0.05).
In contrary, Sidira, Kandylis, Kanellaki, and Kourkoutas (2016) reported
that reduction of curing salts led to increased levels of esters in
dry-fermented sausages. Ethyl acetate, butyl propanoate, propyl hex­
anoate and hexyl butanoate previously determined in heat-treated sucuk
(Kaban & Bayrak, 2015; Armutçu et al., 2020; Yılmaz Oral & Kaban,
2021) were not affected by the use of starter culture (P > 0.05). Among
esters, ethyl butyrate, ethyl isovalerate and ethyl hexanoate are reported
to be the most important aroma contributors in the sausages (Wen, Sun,
Li, Chen, & Kong, 2021). 2-hydroxy methyl benzoate was significantly
influenced by the use of starter culture (P < 0.05) (Table 1). In another
study of heat-treated sucuk, it was found that the use of starter culture
had a statistical effect on butyl propanoate (Table 1) (Yılmaz Oral &
Kaban, 2021). Stahnke (1994), Stahnke (1995a; 1995b) and Sun, Zhao,
Zhao, Zhao, and Yang (2010) determined several ethyl esters in sau­
sages. In a study conducted for the impact of Micrococcaceae on the
aromatic profiles in a dry sausage model, ethyl 2- or 3- methylbutanoate
were found only in samples with S. carnosus (Montel, Mason, & Talon,
1996). Esters are not always present in meat products due to the poor
esterification activity of microbiota (Sidira, Kandylis, Kanellaki, &
Kourkoutas, 2015; Xiao, Liu, Chen, Xie, & Li, 2020) and its generation
depend on mainly high amounts of acids and alcohols (Montel, Reitz,
Talon, Berdague, & Rousset-Akrim, 1996). Being essential for proper
sausage aroma, esters are effective compounds in regards of giving fruity
note to the aroma and masking the rancid odors (Gomez & Lorenzo,
2013; Stahnke, 1994).
3.4. Aromatic hydrocarbons
The amounts of toluene, ρ-xylene and styrene in heat-treated sucuk
samples decreased when starter culture used (P < 0.05) (Table 2) and
these compounds also detected in previous studies Kaban & Bayrak,
2015; Yılmaz Oral & Kaban, 2021). Aromatic hydrocarbons, owing to
their abundance, may play an important role in the flavor of meat
products (Gomez & Lorenzo, 2013). Aromatic hydrocarbons, such as
o-xylene and p-xylene, are described to add different characteristics to
aroma like sweet and fruity respectively (Flores, Grimm, Toldra, &
Spanier, 1997). Sodium ascorbate had no significant effect on aromatic
hydrocarbons (P > 0.05). On the other hand, abundance of 1-methyl-1­
H-pyrrole significantly reduced in 100 mg/kg or 150 mg/kg nitrite level
(P < 0.05) (Table 2).
3.5. Acids
Acetic acid, butanoic acid, propanoic acid and 3-hexenoic acid were
detected in the heat-treated sucuk samples (Table 2). In heat-treated
sucuk, acetic acid and butanoic acid were also determined by Armutçu
et al. (2020). However, acids in fermented sausages are not major
6
LWT 153 (2022) 112540
contributors to the flavor profile because of their high thresholds (Wen
et al., 2021). The autochthonous strains (L. plantarum GM77 and
S. xylosus GM92) increased the acetic acid level of the heat-treated sucuk
(P < 0.05) (Table 2). Acetic acid can be formed by lactic acid bacteria
and Staphylococcus as well as by oxidation of fatty acids and by alanine
catabolism (Montel, Mason, & Talon, 1998). The use of sodium ascor­
bate decreased the amounts of butanoic acid and propanoic acid (P <
0.05). In contrast, Stahnke (1999) found that acetic acid and butanoic
acid were not significantly influenced by the ascorbate in model system
added S. xylosus (Table 2). In our study, ingoing nitrite level had a sig­
nificant effect on propanoic acid and the lowest level of propanoic acid
was determined in the group with 150 mg/kg nitrite. Similarly, Sidira
et al. (2016) also reported that reduction of curing salts content resulted
in significant increase of organic acids during ripening of dry-fermented
sausage.
3.6. Alcohols
Alcohols are produced in a number of ways, such as amino acid
metabolism, lipid oxidation, and methyl ketone reduction (Sidira et al.,
2015). In the present study, ethanol had the greatest abundance among
alcohols in heat-treated sucuk, as did other studies (Kaban & Bayrak,
2015; Armutçu et al., 2020; Yılmaz Oral & Kaban, 2021), but use of
starter culture, sodium ascorbate or nitrite level had no significant effect
on this compound (P > 0.05). 1-hexanol, 2-ethyl-1-hexanol and
α-(1-methylethyl)-benzethanol were significantly affected by the use of
starter culture (P < 0.05) (Table 2). While 1-hexanol and 2-ethyl-1-hex­
anol levels decreased with starter culture, α-(1-methyl­
ethyl)-benzethanol compound increased. 1-propen-2-ol was influenced
by the nitrite level and its abundance decreased at 150 mg/kg nitrite
level (P < 0.05). In addition, the study showed that the addition of so­
dium ascorbate had no significant effect on the abundance of alcohols (P
> 0.05) (Table 2), even though ascorbate is a reducing agent. In a study
carried out by Stahnke (1999), it was reported that the addition of
ascorbate has a significant influence on ethanol.
3.7. Aldehydes
Six aldehydes were identified, namely acetaldehyde, pentanal,
hexanal, heptanal, octanal, 2-methyl-3-phenyl-propanal. All com­
pounds, except acetaldehyde, showed statistically significant decreases
with the use of starter culture (P < 0.05) (Table 2). Among aldehydes, 2-
methyl-3-phenyl-propanal had significant share in heat-treated sucuk
and was in agreement with other studies (Kaban & Bayrak, 2015;
Armutçu et al., 2020; Yılmaz Oral & Kaban, 2021). The abundance of
2-methyl-3-phenyl-propanal lowered in the presence of starter culture
(P < 0.05) while the other factors had no significant effect on this
compound (P > 0.05) (Table 2). In the study conducted by Yılmaz Oral
and Kaban (2021), eight aldehydes were determined and it was reported
that two of these aldehydes (trans-2-heptenal and decanal) were affected
by the use of starter culture. In our study, addition of nitrite to sucuk
batter lowered the abundance of pentanal (P < 0.05), but there was no
significant change between ingoing nitrite levels for this compound (P >
0.05) (Table 2). Stahnke (1995a) found no significant effect of nitrite on
pentanal and hexanal levels, but interaction of fermentation tempera­
ture and potassium nitrate (KNO3) was significantly effective for these
compounds in their study. In the present study, sodium ascorbate was
found to have no significant effect on aldehydes (P > 0.05) (Table 2).
Similarly, Stahnke (1999) emphasized that ascorbate showed no sig­
nificant effect on aldehydes including acetaldehyde in the samples with
S. xylosus or S. carnosus. Aldehydes are compounds being effective on
aroma with low threshold values and are formed as a result of lipid
oxidation and amino acid catabolism (Ordonez et al., 1999). Most of
aldehydes, such as hexanal, heptanal, octanal, pentanal, nonanal,
2-nonenal, 2-octenal, 2-decenal, 2-dodecenal, are associated with
certain off-flavours. Hexanal has been described as an indicator of lipid
S. Sallan et al. LWT 153 (2022) 112540

oxidation because of its abundance and shows a characteristic odor of Other factors such as sodium ascorbate and nitrite level did not affect
green grass (Sidira et al., 2015). terpenes abundance in this study (P > 0.05) (Table 3).

3.8. Furans 3.11. Principal component analysis (PCA)

Only 2-pentyl-furan was determined as the furan compound in the
sausages (Table 2). This compound had also been identified in another
studies on heat-treated sucuk. It was also reported that this compound
was generated by heat treatment (Kaban & Bayrak, 2015; Yılmaz Oral &
Kaban, 2021). In addition, Yin et al. (2021) reported that 2-pentyl-furan
plays an important role in the overall flavour in all sausages due to its
low aroma thereshold value. In our study, starter culture showed no
significant effect on 2-pentyl-furan (P > 0.05). Sodium ascorbate
decreased the amount 2-pentyl-furan (P < 0.05) (Table 2). Forss (1973)
stated that 2-pentylfuran could be derived from oxidation of linoleic
acid, and, this results can be explained with antioxidant effect of
ascorbate. However, it was reported that sodium ascorbate had limited
effect on many compounds in fermented sausage. In addition, it was
expressed that main effect of sodium ascorbate was related with
decreased the level of volatile compounds, originating from lipid
oxidation (Olesen et al., 2004).
3.9. Ketones
Five ketones were determined in the heat-treated sucuk samples and
the use of starter culture significantly increased the level of 3-hydroxy 2-
butanone (acetoin) among these compounds (P < 0.05). 3-Hydroxy-2-
butanone was previously observed in heat-treated sucuk (Kaban &
Bayrak, 2015; Armutçu et al., 2020) and in cooked meat, the presence of
this ketone creates a buttery flavor (Ordonez et al., 1999). 3-hydroxy
2-butanone was significantly affected by the nitrite level and differed
from the other groups with a nitrite level of 150 mg/kg (P < 0.05)
(Table 3). In another study, it was determined that 3-hydroxy 2-buta­
none decreased by S. xylosus in the model system with nitrate (Stahnke,
1999).
3.10. Terpenes
PCA was applied to assess the relationships between factors (starter
culture, ascorbate and nitrite level) and chemical groups of volatile
compounds (Fig. 1). The eigenvalues, variances and their cumulative
proportions are shown in Table 4, coefficients of first five principal
components (PC) are given in Table 5. The first five principal compo­
nents with eigenvalues greater than 1 were analyzed. The first PC is
enough to explain 89% of the variation. The PC2 accounted 9% of the
total variance (Table 4). In other words, the first two principal compo­
nents explained 98% of the total variance. The treatment without starter
culture was placed on the positive side of PC1, while the treatment with
starter culture was on the negative side of PC1 (Fig. 1). The same dis­
tribution was observed between treatments with and without ascorbate.
In addition, nitrite_0 (without nitrite addition) and nitrite_50 (50 mg/kg
nitrite addition) placed on positive side in PC1, nitrite_100 (100 mg/kg
nitrite addition) and nitrite_150 (150 mg/kg nitrite addition) were in
negative side for PC1 (Fig. 1). Moreover, all chemical groups except
alcohols and acids were placed on positive side in PC1 (Table 4 and
Fig. 1). The treatment of without starter culture, without ascorbate,
nitrite_0 and nitrite_50 showed a positive correlation with sulfur com­
pounds, aliphatic hydrocarbons, esters, aromatic hydrocarbons, alde­
hydes, furan, ketones and terpenes in PC1, in contrast the treatments of
starter culture, ascorbate, nitrite_100 and nitrite_150 were correlated
with acids and alcohols in PC1.
The biplot obtained from PCA indicated that the chemical groups of
volatile compounds were usually collected at the center point, and ni­
trite_100 was more related with chemical groups than other factors
Table 4
Eigenvalue scores, variances and their cumulative proportions of first five
components.
Principal component Eigenvalue Explained variance
For PC Cumulative For PC Cumulative

PC1 847.20 847.20 89.10 89.10

Terpenes has an important share in volatile profile of heat-treated
PC2 84.47 931.67 8.90 98.00
sucuk (Kaban & Bayrak, 2015). Terpenes can be obtained from spices, PC3 12.47 944.14 1.30 99.30
although some of them may be found in meat due to their presence in PC4 5.17 949.31 0.50 99.80
animal feed (Sidira et al., 2015). However, the use of starter culture was PC5 1.52 950.83 0.20 100.00
only effective on 7 compounds out of 22 terpenes identified (P < 0.05).

Fig. 1. Principal component analysis biplot of the relationships between factors (starter culture, ascorbate and nitrite level) and chemical groups of vola­
tile compounds.

7
S. Sallan et al. LWT 153 (2022) 112540

Table 5 Anonymous. (2019). Turkish Food codex notification of meat, prepared meat mixtures and
Eigenvectors of the variance–covariance matrix used for PCA. meat products. No: 2018/52. Ankara: Republic of Turkey Ministry of Agriculture and
Forestry.
Variable PC1 PC2 PC3 PC4 PC5 Armutçu, Ü., Hazar, F. Y., Yılmaz Oral, Z. F., Kaban, G., & Kaya, M. (2020). Effects of
different internal temperature applications on quality properties of heat-treated
Sulfur compounds 0.942 0.224 0.125 − 0.147 − 0.073 sucuk during production. Journal of Food Processing and Presevation, 1–8. https://doi.
Aliphatic hydrocarbons 0.054 − 0.004 0.078 0.083 − 0.008 org/10.1111/jfpp.14455
Esters 0.028 0.007 0.048 − 0.048 − 0.594 Berardo, A., De Maere, H., Stravropoulou, D. A., Rysman, T., Leroy, F., & De Smet, S.
Aromatic hydrocarbons 0.035 0.013 − 0.108 0.150 − 0.103 (2016). Effect of sodium ascorbate and sodium nitrite on protein and lipid oxidation
Acids − 0.050 0.224 − 0.653 − 0.626 0.170 in dry fermented sausages. Meat Science, 121, 359–364. https://doi.org/10.1016/j.
Alcohols − 0.013 0.040 − 0.473 0.319 − 0.640 meatsci.2016.07.003
Aldehydes 0.256 − 0.927 − 0.256 − 0.065 0.055 Bolumar, T., Sanz, Y., Flores, M., Aristoy, M.-C., Toldra, F., & Flores, J. (2006). Sensory
Furan 0.003 0.007 0.004 0.026 − 0.000 improvement of dry-fermented sausages by the addition of cell-free extracts from
Ketones 0.014 0.071 − 0.188 − 0.240 − 0.178 Debaryomyces hansenii and Lactobacillus sakei. Meat Science, 72, 457–466. https://doi.
Terpenes 0.196 0.184 − 0.463 0.625 0.397 org/10.1016/j.meatsci.2005.08.010
Flores, M. (2018). Understanding the implications of current health trends on the aroma
of wet and dry cured meat products. Meat Science, 144, 53–61. https://doi.org/
10.1016/j.meatsci.2018.04.016
(Fig. 1). In PC1 explaining 89% of the variance, starter culture, ascor­ Flores, M., Grimm, C. C., Toldra, F., & Spanier, A. M. (1997). Correlations of sensory and
bate, nitrite_100 and nitrite_150 were placed on the negative side, while volatile compounds of Spanish “Serrano” dry-cured ham as a function of two
without starter culture, without ascorbate, nitrite_0 and nitrite_50 were processing times. Journal of Agricultural and Food Chemistry, 45, 2178–2186. https://
doi.org/10.1021/jf960862c
placed on the positive side in PC1. The factors affecting chemical groups Flores, M., & Toldra, F. (2021). Chemistry, safety, and regulatory considerations in the
of volatile compounds of heat-treated sucuk can be separated by PCA use of nitrite and nitrate from natural origin in meat products - invited review. Meat
and as can be seen in Table 5, sulfur compounds, aldehydes and terpenes Science, 171, 108272. https://doi.org/10.1016/j.meatsci.2020.108272
Fonseca, S., Cachaldora, A., Gomez, M., Franco, I., & Carballo, J. (2013). Effect of
have a great impact on PC1. different autochthonous starter cultures on the volatile compounds profile and
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CRediT authorship contribution statement Montel, M. C., Mason, F., & Talon, R. (1998). Bacterial role in flavour development. Meat
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Montel, M. C., Reitz, J., Talon, R., Berdague, J.-L., & Rousset-Akrim, S. (1996).
Selen Sallan: Methodology, Investigation, Writing – original draft, Biochemical activities of Micrococcaceae and their effects on the aromatic profiles
Formal analysis. Güzin Kaban: Methodology, Validation, Writing – re­ and odours of a dry sausage model. Food Microbiology, 13, 489–499. https://doi.org/
10.1006/fmic.1996.0056
view & editing. Mükerrem Kaya: Supervision, Project administration,
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Writing – review & editing. the amount of flavour compounds produced from leucine by Staphylococcus xylosus
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Declaration of competing interest Ordonez, J. A., Hierro, E. M., Bruna, J. M., & de la Hoz, L. (1999). Changes in the
components of dry-fermented sausages during ripening. Critical Reviews in Food
The authors have declared no confict of interest. Science and Nutrition, 39(4), 329–367. https://doi.org/10.1080/
10408699991279204
Perea-Sanz, L., Lopez-Diez, J. J., Belloch, C., & Flores, M. (2020). Counteracting the
Acknowledgement effect of reducing nitrate/nitrite levels on dry fermented sausage aroma by
Debaryomyces hansenii inoculation. Meat Science, 164, 108103. https://doi.org/
10.1016/j.meatsci.2020.108103
This work was supported by the Scientific and Technological Perea-Sanz, L., Montero, R., Belloch, C., & Flores, M. (2018). Nitrate reduction in the
Research Council of Turkey (TÜBİTAK) (Project number: 215 O 277) fermentation process of salt reduced dry sausages: Impact on microbial and
and was presented at the 3rd International Congress on Food Technol­ physicochemical parameters and aroma profile. International Journal of Food
Microbiology, 282, 84–91. https://doi.org/10.1016/j.ijfoodmicro.2018.06.004
ogy (10–12 October 2018), Nevşehir, Turkey. Perea-Sanz, L., Montero, R., Belloch, C., & Flores, M. (2019). Microbial changes and
aroma profile of nitrate reduced dry sausages during vacuum storage. Meat Science,
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