Effects of Autochthonous Yeast Cultures on Some Quality Characteristics of Traditional Turkish Fermented Sausage “Sucuk”

Fresh lean of beef from round region, tallow fat, spices (red pepper, black pepper, cumin, and pimento) and other ingredients (salt, fresh garlic, sugar, and sodium nitrite) were supplied from local suppliers in Kayseri, Turkey.

In order to produce about 1 kg of sucuk, main ingredients [(ground lean beef (800 g) and ground tallow fat (200 g)] and additives [salt (25 g), minced garlic (10 g), cumin (9 g), red pepper (7 g), black pepper (5 g), pimento (2.5 g), sugar (4 g) and sodium nitrite (NaNO₂ 150 mg)] were prepared and incorporated together. First, all the ingredients were homogenized by kneading in a kitchen bowl approximately 10 min and the mass was divided into 8 batches as following; S1: Control (without starter culture), S2: LAB (Lactobacillus curvatus, L. plantarum and L. sakei), S3: LAB+C. zeylanoides, S4: LAB+D. hansenii, S5: LAB+C. zeylanoides+D. hansenii, S6: C. zeylanoides, S7: D. hansenii and S8: C. zeylanoides+D. hansenii. The LAB and yeast cultures were added at levels of approximately 10⁸ and 10⁶ CFU/g, respectively. The mixtures were filled into standard artificial collagen casings (diameter 30–32 mm) by using a filling machine (Tefal Le Hachoir 1500, France). Production of sucuk was carried out at room temperature (~22°C). The ripening of sucuks was carried out according to the following program: First 3 days at 24±1°C and 90±2% relative humidity (RH), then following 4 days at 22±1°C and 85±2% RH and finally for 5 days at 18±1°C and 80±2% RH in fermentation cabinets (Nüve, TK 252, Ankara, Turkey). The sucuk samples were analyzed at 1^st, 6^th, and 12^th days of ripening. All the batches were produced as triplicate.

The 25 g of the sample was weighed into the sterile stomacher bag and homogenized with 225 mL of sterile Maximum Recovery Diluent solution (MRD, Merck, Darmstadt, Germany) for 1.5 min using a homogenizer (Stomacher, IUL, Barcelona, Spain). Total mesophilic aerobic bacteria (TMAB) counts of the samples were determined after 48 h incubation at 30°C on Plate Count Agar (PCA, Merck). LAB counts of the samples were determined in pour plates of De Man Rogosa and Sharpe Agar (MRS, Merck), after incubation for 48 h at 37°C in anaerobic conditions. The number of Micrococcaceae on Mannitol Salt Phenol-Red agar (MSA, Merck) was determined after incubation for 48 h at 37°C. The Enterobacteriaceae counts were determined after incubation for 48 h at 35°C, in anaerobic conditions on Violet Red Bile Glucose Agar (VRBG, Merck). Yeast and mold counts were determined for 5 days at 25°C on Dichloran Rose Bengal Chloramphenicol Agar (DRBC, Merck). The microbiological analyses were carried out as triplicate.

The moisture contents of the sucuk samples were determined by using oven air drying method in a drying oven (Nüve FN 120, Ankara, Turkey) (AOAC, 2000). Water activities (a_w) of the samples were determined using a_w meter (Aqua Lab 2.0, Pullman, WA, USA). To determine the pH values of the sucuk samples, 10 g of the sucuk sample was homogenized with 100 mL of distilled water using Ultraturrax (IKA T18 Basic, Staufen, Germany) and the values were measured by using pH meter (WTW, Inolab 720, Weilheim, Germany). Weight loss of the sucuk samples was determined by recording the weights of the samples at the first day and 12^th day of ripening. It was calculated using the following equation:

Residual nitrite level was determined based on the method described by Taucmann (1987). They were calculated the residual nitrite level using a calibration curve and expressed as ppm of sodium nitrite (NaNO₂). The physicochemical analyses of the sucuk samples were carried out as triplicate.

Color properties of the sucuk samples were determined using an automatic colorimeter (Konica Minolta, model CM-5, Mississauga, ON, Canada) at an observer angle of 10° with the illuminant D₆₅ and specular-component-excluded mode, according to The Commission Internationale de l’Eclairage (CIE)-Lab color scales. Artificial collagen casing was removed to measure color of the exterior part of the samples, and the cross-section of the sliced sucuk samples was used to measure the internal color. The color results were expressed as L*, a*, and b*. L* values measure the level of brightness (0–100), a* redness (+=red and −=green), and b* yellowness (+=yellow and −=blue). Color parameter values of the sucuk samples were measured in ten replicates.

Volatile compounds profile of the sucuk samples produced with different yeast and/or LAB starter cultures was determined using a Gas Chromatographic-Mass Spectrometry (GC–MS) (Agilent 7890A GC system, Agilent, Santa Clara, CA, USA) equipped with a mass selective detector (Agilent Technologies, Agilent) and HP5-MS capillary column (60 m×0.250 mm i.d.; film thickness 0.25 μm) (Agilent). The 5 g of the sucuk sample was weighed into GC-MS vial (Agilent). and the vial was sealed with PTFE-faced silicone septum (Supelco, Bellefonte, PA, USA). Then vial was kept in a termoblock (IKA, RCT basic, Germany) at 40°C for 1 h. Then SPME fiber (75 μm, 23 ga, carboxen/polydimethylsiloxane (CAR/PDMS)) (Supelco) was subjected to the headspace while maintaining the sample at 40°C for 1 h. The volatile compounds adsorbed by the fibers were desorbed from the injection port for 20 min at 50°C and injected to GC–MS in the splitless mode. The compounds were identified by comparison with spectra from the libraries of Flavor 2, Nist05 and Wiley7n. GC–MS conditions were adjusted according to Kaban and Kaya (2009). The volatile compound analyses were run as triplicate.

To determine textural properties of the sucuk samples, TPA test was conducted using a texture analyzer (TA.XT Plus Texture Analyzer, Texture Technologies, Scarsdale, NY/Stable Micro System, Surrey, UK). The sucuk samples ripened were cut into small pieces having a 20 mm diameter and 20±0.5 mm thickness. Firstly, the parameters of measurement were set to be following: pre test speed 2 mm/s, test speed 1 mm/s, post test speed 1 mm/s and compression (strain) level 25%. For the determination of TPA parameters, a spherical probe (SMS/1S) and 30 kg load cell were used. Hardness (g), adhesiveness (g s), springiness (mm), cohesiveness, gumminess (g), chewiness (g mm) and resilience values of the sucuk samples were measured by calculation using TPA curves (Bozkurt and Bayram, 2006). Texture parameters of the sucuk samples were measured in ten replicates.

All the data were means of triplicate data with their standard deviations. Analysis of data was performed by using one-way ANOVA and/or two-way ANOVA. Duncan’s Multiple Range Test was also applied to determine significant differences between means at the p<0.05 significance level using SAS 8.0 statistical software (SAS, 2000).

Some physicochemical properties of the sucuk samples prepared with C. zeylanoides (CZ), D. hansenii (DH) and LAB cultures are given in Table 1. Moisture contents of the sucuk samples varied from 54% to 57% in the first day of the ripening. Then, it decreased during ripening as expected depending on the dehydration of sucuks. Moisture contents of the sucuk samples (S2-S4) with LAB cultures were lower than that of the samples with CZ and DH at end of the ripening, and these results were also insignificant (p>0.05). Water activity (a_w) levels of the sucuk samples correlated with their moisture contents. Again, a_w values of the samples containing LAB were lower when compared to other samples at the 6^th and 12^th days. The pH levels of the samples may be the reason for low moisture and a_w values of the samples containing LAB. Low pH causes to decrease in water-holding capacity of the meat proteins. This promotes the drying process of sucuk or fermented sausages (Lücke, 1998; Ordóñez et al., 1999; Toldrá et al., 2001). Additionally, higher pH value of meat is considered as a problem in sucuk or fermented sausage production. When a meat having high pH is used in sucuk or fermented sausage production, sufficient drying cannot be achieved due to high water retention capacity (Toldrá, 2007). Therefore, pH values of meats which will be used in sucuk or fermented sausage should be in between 5.4 and 5.8 (Öztan, 2005). The pH values of the sucuks were approximately 5.8 at the first day of the ripening, and the pH values of sucuks produced with LAB cultures were lower than that of the control and samples inoculated yeast culture at the end of the ripening. However, pH results were not (p>0.05) different at first and 12^th days, but they were significantly different (p<0.05) at 6^th days of ripening. Andrade et al. (2010) investigated effects of three D. hansenii strains on microbiological, physiochemical properties and volatile compounds of salchichón, a dry fermented sausage. They found a_w and pH values to be high in the samples with D. hansenii as compared to the control sample at the end of ripening (54 days). In the same study, however, a_w and pH values of fermented sausage changed depending on the D. hansenii strains. Kaban and Kaya (2009) reported that pH and a_w values of sucuk samples produced with L. plantarum and Staphylococcus xylosus were lower than that of the control sucuks. LAB are the bacteria group that mainly responsible for pH decrease in fermented sausage and sucuks, and it affects moisture and a_w values of fermented meat products during the ripening. In another study, dry matter, a_w and pH values of dry fermented sausage produced with different yeast strains (C. famata, Yarrowia lipolytica, D. hansenii and Trichosporon mucoides) were 66%–68%, 0.81%–0.82% and 4.6%–4.7%, respectively, after 21 days of ripening (Selgas et al., 2003). These results are not in accordance with our findings probably due to fermentation time and conditions, process applied and materials used such as starter culture, meat, spices and other additives. The Turkish Food Codex (2000) states that ripened sucuk which is high quality should have pH between 5.2 and 5.4. Again pH levels of the sucuk samples produced in the current study are not in line with Turkish Food Codex.

Weight loss values of the sucuk samples are seen in Fig. 1. The highest weight loss values were determined in the sucuks produced with LAB cultures, and the differences were significant (p<0.05). However, use of the yeast cultures decreased weight loss of the sucuks. Weight loss is an important parameter for the economical reasons for the meat processors, it means saving money for the manufacturers.

Fig. 1. Weight loss (%) of the ripened sucuk samples. S1, Control; S2, LAB; S3, LAB+C. zeylanoides; S4, LAB+D. hansenii; S5, LAB+C. zeylanoides+D. hansenii; S6, C. zeylanoides; S7, D. hansenii; S8, C. zeylanoides+D. hansenii. ^a–e Different lowercase indicate the statistical difference (p<0.05) for weight loss of sucuk samples. LAB, lactic acid bacteria.

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Residual nitrite values of the sucuk samples are shown in Table 1. At the first day of ripening, nitrite values of the sucuks varied from 53.3 to 80.9 ppm, and it was the lowest in the sucuks produced with LAB cultures. The nitrite level significantly (p<0.05) decreased with the ripening time. Again, the lowest residual nitrite values were in the sucuks produced with LAB culture and ranged between 8.1–9.8 ppm. Our residual nitrite results were in agreement with the results of Gençcelep et al. (2007) who reported that residual nitrite values of sucuks with starter culture were lower than those of the control group (without starter culture) sucuks. Nitrite is used to improve color and oxidative properties of the product, and it also inhibits Clostridium botulinum as well (Oh et al., 2004). However, it is at higher levels in foods a potential carcinogen and toxic agent for human (Cammack et al., 1999). According to the Turkish Food Codex (2000), residual nitrite levels of the fermented Turkish sucuks must be lower than 150 mg/kg. It has been reported that nitrite levels can be reduced by LAB having a nitrite reductase enzyme system (Fournaud and Mocquot, 1966). The nitrite values of the cured meat products decrease spontaneously during the storage period (Oh et al., 2004). Several studies have shown that certain LAB strains are able to degrade nitrite in the fermented meat products (Dodds and Collins-Thompson, 1984; Oh et al., 2004; Yan et al., 2008).

Microbiological properties of the sucuk samples are given in Table 2. TMAB counts of the samples were approximately 9.0 Log CFU/g at the beginning of ripening. Then, it increased at 6^th days of the ripening and decreased at the end of ripening. TMAB and LAB counts of samples were 9.85–10.21 Log CFU/g and 9.89–10.17 Log CFU/g, respectively and the differences were not significant (p>0.05) in the 12^th days of ripening. However, Micrococcaceae population of the sucuk samples was different (p<0.05) at the 6^th days and 12^th days of ripening. Enterobacteriaceae numbers decreased during the ripening (Table 2). Yeast and mold counts of the sucuk samples ranged from 4.12 to 6.10 Log CFU/g at the beginning of the ripening but they were closer to each other at the 6^th days of ripening. Additionally, use of CZ and DH in the sucuk production affected (p<0.05) the yeast and mold counts as expected and Micrococcaceae and Enterobacteriaceae counts as well, while the TMAB and LAB counts of sucuk samples were not affected (p>0.05) from the presence of these yeasts at the end of ripening. It was reported that LAB, Micrococcaceae and yeast counts of fermented sausage produced with different D. hansenii strains were approximately 10⁷, 10³, and 10⁴–10⁵ CFU/g at the end of ripening, respectively (Andrade et al., 2010). In another study, Bolumar et al. (2006) used D. hansenii and L. sakei to improve sensory properties of fermented dry sausage, and yeast and LAB counts of ripened fermented sausage were determined as approximately 3.5 and 8.5 Log CFU/g, respectively. At the same time, yeast count of fermented sausage produced with only D. hansenii increased at the end of ripening while the number of LAB did not change considerably during ripening. Durá et al. (2004) reported that yeast number decreased during ripening of fermented sausage produced with two different D. hansenii strains while the yeast count decreased in the control sample during ripening. Additionally, LAB counts of the samples were approximately 7 Log CFU/g at the beginning of ripening and reached to approximately 9.5 Log CFU/g within the ripening. These results were in accordance with our findings related to yeast and LAB counts (Table 2). Kaban and Kaya (2009) reported that Enterobacteriaceae was not detected in the control and the sucuk samples produced with starter culture (L. plantarum and S. xylosus) at the 7^th and 3^rd days of ripening, respectively. In this study, Enterobacteriaceae was present in the all sucuk samples after ripening, but their counts were low. It is known that Enterobacteriaceae is sensitive against low acidity and water activity (Kaban and Kaya, 2009). In this study, the pH and a_w values of the sucuks were higher than pH and a_w results of the sucuk samples produced by Kaban and Kaya (2009). Microbiological characteristics of fermented meat products are generally influenced from natural meat microbiota and starter cultures used.

Color properties of the sucuk samples were determined in exterior and interior sections as shown in Table 3. The L* values of sucuks were found in the range 43.26–43.80 and 29.99–32.95 in the interior and exterior section, respectively. Use of LAB, CZ and DH starter cultures did not affect (p>0.05) the L* values of the sucuks in the neither interior nor exterior section. The a* values of sucuks were higher (p>0.05) in interior section. The highest a* value was in the S8 (CZ+DH), while the lowest value was determined in S2 sample (only LAB). However, the a* value of sucuks was lower (p>0.05) in the exterior part. Again, the highest and lowest a* values were in the S8 (CZ+DH) and S2 (only LAB) in the exterior part of sucuks, respectively (Table 3). The a* value is one of the most important color parameters for the quality of sucuk. The a* value is generally low at the beginning of ripening of dry-cured fermented meat products. However, it increases during the ripening due to the formation of nitrosomyoglobin which is associated with the red color of fermented meat products and moisture loss in the fermented meat products (Pérez-Alvarez et al., 1999). The reason of low a* values may be acidity in the sucuks produced with LAB cultures. Because, lactic acid produced by LAB might denature (partly or totally) the myoglobin during ripening (Pérez-Alvarez et al., 1999). The b* values of sucuk samples were variable (p<0.05) in both interior and exterior section. As L* and a* values of sucuks, the b* values were high in the interior section of sucuk samples.

Textural properties including hardness, adhesiveness, springiness, cohesiveness, gumminess, chewiness and resilience parameters were determined in the ripened sucuk samples (at the 12^th days of ripening), and these results are shown in Fig. 2. Hardness values of the sucuks with LAB cultures were higher than those of the sucuks without LAB cultures and control group. In general, use of LAB and yeast cultures affected (p<0.05) the hardness of the sucuk samples. The highest hardness value was found in the sucuk sample produced by only LAB cultures (S2) while the lowest hardness was determined for the sucuk with DH culture. Again, in general, the presence of CZ and DH cultures decreased hardness values of sucuk samples. This result might be due to low pH value and higher moisture loss of the sucuk samples. Adhesiveness values of sucuk were different (p<0.05) from each other, and it was higher in sucuk samples with yeast cultures compared to the sample produced with only LAB culture (S2). Adhesiveness values were –27.46 (g.sn) and –20.42 (g.sn) for S2 and S3 sucuks, respectively, and it was –9.99 (g.sn) in control sample (S1). However, it ranged from –4.60 to –13.58 (g.sn) and was higher for the sucuk sample produced without LAB culture. Springiness and cohesiveness values of the sucuk produced using yeast culture were lower than those of the sample with LAB culture. Use of only yeast culture on sucuk significantly (p<0.05) decreased both springiness and cohesiveness values (Fig. 2). Especially, it was lower in sucuks only with DH (S7) or CZ+DH (S8) samples, and it was 0.57 and 0.59 for in S7, respectively. However, springiness and cohesiveness values were higher in S4 and S5 samples (yeast+LAB cultures) than that of the S7 and S8 sucuks (yeast cultures). Although use of LAB cultures increased gumminess values of sucuk, use of only yeast cultures in formulation decreased that parameter. Gumminess values of the samples ranged from 316.7 to 701.7. The lowest gumminess values were determined as 316.7 and 360.4 in S7 and S8 samples, respectively. Chewiness values were similar to gumminess and ranged between 181.6 and 469.5 (Fig. 2). Again, both parameters were lower in sucuk samples (S5–S8) produced with only used yeast cultures. Both gumminess and chewiness values of the sucuk samples were significantly (p<0.05) different. Resilience values of the sucuk samples varied from 0.18 to 0.20, and it was lower in the samples with CZ and DH.

Fig. 2. Textural properties of the sucuks produced with yeast cultures. S1, Control; S2, LAB; S3, LAB+C. zeylanoides; S4, LAB+D. hansenii; S5, LAB+C. zeylanoides+D. hansenii; S6, C. zeylanoides; S7, D. hansenii; S8, C. zeylanoides+D. hansenii. ^a–f Different lowercase for each parameters indicate the statistical difference (p<0.05) for textural properties of sucuk samples. LAB, lactic acid bacteria.

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Limited numbers of studies are available related to effects of yeast starter cultures (D. hansenii) on textural properties of fermented sausages, and there is no report for the sucuk in the literature. In a study performed on Portuguese traditional sausage, use of Lactobacillus spp., Micrococcaceae and yeasts in production of fermented sausage slightly improved the cohesiveness properties of sausage, and other textural properties including hardness, adhesiveness, springiness, gumminess, chewiness and resilience were not significantly affected (Elias et al., 2014). In another study, D. hansenii affected the hardness and chewiness values, while no effect was observed on springiness and cohesiveness properties of sausage (Corral et al., 2014). In the current study, use of yeast culture significantly (p<0.05) affected the textural properties of sucuk (Fig. 2).

In general, microbial growth decreases pH level of sausages during the ripening. It leads to drying of sausage, and the denaturation and gelation properties of meat proteins. This affects hardness values of sausage (Bozkurt and Bayram 2006; Wu et al., 2010). Lower moisture levels in sausage also decrease the adhesiveness values, improving cutting ability of the product (Bozkurt and Bayram, 2006).

Volatile composition of the sucuk samples is given in Table 4. In this study, total 85 volatile compounds were identified in the sucuk samples, and volatile compounds were in the following groups: 5 aldehydes, 2 alkanes, 2 alkines, 7 acids, 8 alcohols, 5 esters, 19 sulphur compounds, 31 terpenes, 3 aromatic hydrocarbons and 3 other components. Cuminaldehyde, di-2-propenyl disulfide and p-cymene were the major volatile aldehydes, sulphur compounds and terpenes, respectively. Again, eugenol, carvacrol and naphthalene were the aromatic hydrocarbons observed in the sucuk samples.

Cuminaldehyde level in the volatile compounds decreased gradually during the ripening, and it was lower in the sucuk samples produced with LAB cultures at the end of ripening. However, the p-cymene, γ-terpinene, β-caryophyllene and limonene levels were higher in the samples produced with LAB cultures at the end of the ripening. Safranal was one of the major terpenes and was higher level in the control group and the sample produced with only yeast cultures. Propionic (propanoic) acid was detected in the sucuk samples produced only with yeast cultures at the end of ripening, and its level was 0.31% and 0.32% in S6 and S8, respectively. These samples were prepared with CZ yeast culture. Butyric acid was not detected at 6^th and 12^th days of ripening while it was observed at the beginning of the ripening. However, stearic acid, palmitic acid and 9-octadecenoic acid (oleic) which are fatty acids were determined in sucuk samples produced only with yeast cultures at the end of ripening. It was speculated that the degradation of free fatty acids to volatile compounds may have caused to these results (Flores and Olivares, 2014). Also the yeast species having lipolytic activity are able to hydrolyze fatty acids during the ripening, because CZ and DH cultures used in this study have lipolytic activity too (Ozturk and Sagdic, 2014).

The flavor properties of fermented sausages are associated with breakdown of carbohydrates, lipids and proteins by enzymes which are microbial origin and/or endogenous meat enzymes (Ansorena et al., 2001; Flores and Olivares, 2014; Kaban and Kaya 2009). Volatile compounds of fermented sausages can be also affected from meat origin, type of starter culture, process conditions and the spices used in production (Kaban and Kaya 2009; Leroy et al., 2006; Toldrá et al., 2001). Terpenes and sulphur compounds are important volatile compound groups in ripened meat products. Terpenes generally originate from spices used in production of fermented sausages as well as arise from the meat which based on animal nutrition with some terpenes (Ansorena et al., 2001). Again, cuminaldehyde, limonene, carvacrol and safranal can be resulted from cumin used in production of sucuk (Ağaoğlu et al., 2007, Li and Jiang 2004). While benzaldehyde, benzeneacetaldehyde and phenethyl alcohol compounds determined in the samples are the yields of degradation of amino acids (bacterial metabolism), 1-hexanol, propanoic acid and hexanoic acid comprise by lipid autooxidation in fermented sausages (Corral et al., 2013; Olivares et al., 2011). Kaban and Kaya (2009) found that major volatile compounds were 2-methyl-3-phenyl propanal, o-cymene, γ-terpinene and di-2-propenyl disulfide in sucuks produced with L. plantarum and S. xylosus. In another study, major volatile compounds of traditional sucuk samples were terpenes (o-cymene, γ-terpinene), acids (acetic acid), aldehydes (propanal,2-methyl-3-phenyl) and sulphur compounds (1-Propene,3,3’-thiobis) (Kaban, 2010). Küçüktaş (2012) isolated 38 volatile compounds from sucuk samples produced with yeast cultures (Y. lipolytica and D. hansenii) during ripening and major volatile compounds of sucuk were terpenes and sulphur compounds at the 9^th day of ripening. However, some volatile compounds such as acetic acid, 2-methylpentanoic acid, 2-hydroxypropanoic acid, hexanoic acid ethanol, 1-pentanol, 1-hexanol and benzyl alcohol were reported as yields of lipid autooxidation, carbohydrate fermentation and amino acid degradation in that study. When considering this volatile composition of fermented sausages, some differences are available between the findings of the current study and literature. Analysis of volatile compounds of fermented sausage and other similar fermented meat products could be affected from several other factors such as solid phase microextraction (SPME), column and method conditions as well as meat origin, type of starter culture, process conditions and the spices used.