CHANGES IN BIOCHEMICAL AND MICROBIOLOGICAL ...

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CHANGES IN BIOCHEMICAL AND MICROBIOLOGICAL CHARACTERISTICS OF TURKEY SUCUKS AS AFFECTED BY PROCESSING AND STARTER CULTURE UTILIZATION ˘ AN2 and ÜMRAN ENSOY1,3, NURAY KOLSARICI2, KEZBAN CANDOG 2 ˘ BETÜL KARSLIOGLU 1

Agricultural Faculty Food Engineering Department Tasliciftlik Campus, Gaziosmanpas¸a University 60250, Tokat, Turkey 2 Engineering Faculty Food Engineering Department Diskapi Campus, Ankara University 06110, Ankara, Turkey.

Accepted for Publication February 22, 2008

ABSTRACT The effects of starter culture and processing on the microbiological and biochemical characteristics of turkey sucuk were studied during production and storage. No differences (P > 0.05) in proximate composition and salt contents were found in sucuks produced either by traditional or heated methodologies. Both traditional (P < 0.05) and heat-processed (P > 0.05) sucuks that incorporated S1 had the lowest pH value, and thus the highest titratable acidity. Heating process resulted in about 1 log unit reduction in yeast counts and 1.5 log unit reduction in lactic acid bacteria, total mesophilic aerobic bacteria and micrococci–staphylococci counts of all sucuks. Within the heat-processed sucuks, heating stage did not result in a significant change in nonprotein nitrogen contents (P > 0.05). Changes in myofibrillar and sarcoplasmic protein were observed because of proteolytic changes during ripening, but, in general, no major difference was observed between sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns as a result of starter culture treatments.

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Corresponding author. TEL: 90 (356) 252 1616 (3255-ext.); FAX: 90 (356) 252 1488; EMAIL: [email protected]

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Journal of Muscle Foods 21 (2010) 142–165. © 2009, The Author(s) Journal compilation © 2009, Wiley Periodicals, Inc.

BIOCHEMICAL AND MICROBIOLOGICAL CHANGES IN TURKEY SUCUKS

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PRACTICAL APPLICATIONS Results of this study can be applied to improve some biochemical and microbiological characteristics of fermented turkey sausages. The findings showed that utilizing commercial starter culture mix, containing Lactobacillus sake, Staphylococcus carnosus and Staphylococcus xylosus II resulted in lower pH value with a higher titratable acidity than traditional style-fermented turkey sausages. These parameters are responsible for texture development and characteristic taste of fermented sausages.

INTRODUCTION Dry fermented sausage is a typical Mediterranean fermented meat product (Bolumar et al. 2006). Sucuk, Turkish dry fermented sausage, is similar to that found in many other Middle Eastern countries and in Europe, and produced in large amounts in various parts of Turkey (Çön et al. 2001, Bozkurt and Erkmen 2002, 2007; Kaban and Kaya 2006; Çolak et al. 2007). Manufacturing of sucuk varies regionally, but in general, sucuk batter consists of beef and water buffalo meat, beef fat, sheep tail fat, salt, garlic and other additives such as sugar, nitrite and/or nitrate and various spices (Aksu and Kaya 2004; Soyer et al. 2005; Bozkurt and Erkmen 2007; Çolak et al. 2007). After mixing, sausage batter is filled into natural casings, and dried under climatic conditions (Bozkurt and Erkmen 2007). The ripening and drying stages of sucuk produced traditionally are carried out under natural climatic conditions, and ripening periods change from 6 to 20 days according to temperature (Soyer et al. 2005). During the last decade, modern food facilities have modified the traditional methods. In these facilities, sucuk is produced under controlled climatic conditions, and starter culture mixes are utilized to manufacture sucuk (Bozkurt and Erkmen 2002; Soyer et al. 2005). The addition of desirable microorganisms to meat improves the quality (by inhibiting the undesirable microorganisms) and safety (by inactivation of pathogens) of the product, and standardizes the manufacturing process (Lucke 2000; Ferreira et al. 2006; Leroy et al. 2006). The homo-fermentative lactobacilli and/or pediococci, and gram-positive, catalase-positive cocci are the main starter cultures that play a significant role in fermented sausages (Drosinos et al. 2005; Leroy et al. 2006; Rantsiou and Cocolin 2006). Lactic acid bacteria are responsible for rapid acidulation of the sausage during fermentation, depending on the starter applied, carbohydrate used and sources of meat and additives (Talon et al. 2000; Tjener et al. 2004; Comi et al. 2005; Drosinos et al. 2005). Lactic acid bacteria reduce the pH of the sausage by producing lactic acid from carbohydrates with their main metabolic activity (Rantsiou

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and Cocolin 2006). They are also responsible for tangy flavor, texture development and water release of fermented sausages (Smith and Palumbo 1983; Vignolo et al. 1989; Ordonez et al. 1999; Comi et al. 2005). Staphylococci are well known for ensuring the color development by nitrate reductase activity (Talon et al. 2000; Tjener et al. 2004; Comi et al. 2005; Drosinos et al. 2005). Furthermore, nitrate reduction produces nitrite that can limit lipid oxidation (Rantsiou and Cocolin 2006). In addition to nitrate reductase activity, staphylococci are responsible for decomposition of peroxides and aroma formation because of their proteolytic and lipolytic activities (Olesen and Stahnke 2004; Comi et al. 2005; Rantsiou and Cocolin 2006). Proteolysis is considered to be one of the most important biochemical changes occuring during the ripening of fermented sausages, which involves aroma development by formation of peptides, amino acids, aldehydes, organic acids and amines (Diaz et al. 1997; Molly et al. 1997; Hughes et al. 2002; Olesen and Stahnke 2004). In recent years, the availability of turkey meat has greatly increased because of its alleged characteristics of presenting low levels of cholesterol and total lipids, and high levels of polyunsaturated fatty acids. In addition to these properties, it has a neutral taste and smooth texture (Baggio et al. 2002). Thus, the objective of this study was to determine some chemical and microbiological properties of turkey sucuks at manufacturing steps and during refrigerated storage.

MATERIALS AND METHODS Materials Freshly slaughtered turkey thigh meat was obtained from BOLCA Turkey Processing Plant of Bolu Animal Feeding Company Ltd. Co. (Bolu, Turkey) at different time periods (for two replications) and transferred to the meat technology laboratory of Food Engineering Department in Ankara University Faculty of Agriculture the day before the production, and kept at +4C. Frozen sheep tail fat was used. The spice mix used in the formulation of fermented turkey sausage batters (contaning spice mix, spice extract, dextrose, sodium ascorbate, monosodium glutamate, sodium polyphosphate) was obtained from Bayramog˘lu-PABAY Chemical Material Marketing and Distribution Ltd. Co. (Istanbul, Turkey). Beef intestines were used for stuffing the sausages. Sodium nitrite and sodium nitrate (food grade, Merck, Darmstadt, Germany) were utilized as curing agents. The freeze-dried starter cultures used for the fermentation of turkey sausage batters were Texel (Groupe Rhöne Poulenc, Saint-Romain, France)


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named as S1, containing a mixture of Lactobacillus sake, Staphylococcus carnosus and Staphylococcus xylosus II, and Biobak K (Wiberg-Salzburg) named as S2, containing L. sake, S. xylosus and Pediococcus pentosaceus. Methods Preparation of Fermented Turkey Sausages. Three batches of turkey sucuk mixes were prepared each of which contained 16.5 kg turkey thigh meat and 2 kg of sheep tail fat ground using using a meat grinder (Arı Torna Co., Istanbul, Turkey) having a plate with 12 mm orifices. The other ingredients utilized in sucuk mixtures were 800 g spice mix, 375 g NaCl, 7.5 g sodium nitrite and 1.8 g sodium nitrate (food grade, Merck). Two of the sucuk batches were inoculated with 4.6 g of freeze-dried starter cultures Texel (S1), or with 9.3 g of freeze-dried Biobak K a second starter culture (S2), and one batch was not inoculated with starter culture named as control (C). The preparation of these starter cultures was completed using the respective manufacturer’s instructions. Meat, sheep tail fat and other ingredients were mixed using a Yuneka mixer (Yuneka Metal Co., Bursa, Turkey). The sucuk mixtures were then reground through a plate with 3 mm orifices, stored overnight at 4C (initial mix) and then stuffed into natural beef intestines using a hydraulic filling machine (Yuneka Metal Co.). After stuffing, the sucuks were hung on stainless steel hangers, allowed to equilibrate at 18C for 6 h and placed in a Biogen SCK 4500 ¥ 1 model ripening chamber (Biogen Co., Ankara, Turkey). The conditions of relative humidity (RH) and temperature applied are reported in Table 1.

TABLE 1. RIPENING PHASE AND CONDITIONS OF FERMENTED TURKEY SAUSAGES PREPARED WITH OR WITHOUT STARTER ADDITION Step Fermentation I II Heating* Drying I II

Temperature (C)

Time

RH (%)

26 23 55 (internal)

36 h 12 h 5 min

95–98 80 Dry air

22 18

48 h 120 h

50–55 50–55

* For heated sucuks only.

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After fermentation, sucuk chubs from each group were randomly subdivided into two parts. One group was placed in a drying room, and dried at the conditions given in Table 1 (traditional-style processing without heat application). The other group was heat processed following fermentation in a Biogen Oven programmed to 70C until an internal temperature of approximately 55C was attained, and then kept at this temperature for 5 min. The internal temperature of the sucuk was measured using microprocessor digital thermometer with type K thermocouple. Following heating, the sucuks were cooled with tap water, surface dried, placed in a drying room and dried for 7 days using the same drying conditions indicated above. After drying, each chub was vacuum packed individually, and stored in refrigerated conditions for 4 months for futher analysis. Sampling. Two randomly selected chubs were removed at each stage of processing: (1) after the initial mix; (2) after fermentation; (3) after heating; and (4) after drying and during storage. For storage periods, the samples were taken on the first day (day 0), and days 30, 60, 90 and 120. The pH value, titratable acidity and nonprotein nitrogen (NPN) contents were evaluated using all steps mentioned. During processing, alterations of meat proteins were monitored with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the same production steps. In addition, moisture, fat, protein and ash contents of sucuks were measured at day 0. Compositional Analysis. Moisture, protein (N ¥ 6.25), fat and ash contents were determined in duplicate following AOAC (1990) methods. The pH Value and Titratable Acidity. Duplicate 10 g samples of sucuks were blended with 100 mL of distilled water for 60 s using an UltraTurrax T25 Basic Model homogenizer. The pH of the slurries was determined using an Orion 420A pH-meter (Orion Inc., Küstnacht, Switzerland). Sample slurries were then titrated with 0.1 N NaOH to an end point of pH 8.30. The mEq of NaOH (Merck) was converted to and expressed as percent lactic acid (Acton and Keller 1974). NPN Content. At each of the sampling intervals previously noted, 20 g of sucuk sample was homogenized with 200 mL of distilled water for 1 min using Ultra-Turrax T25 Basic Model homogenizer, placed on a magnetic stirrer, slowly blended at 4C for 45 min and then filtered using Whatman no. 4 filter paper. To 100 mL of filtrate, 5.0 g of sulfosalicylic acid, 0.455 g LiCl and 0.450 g LiOH were added. The 5% sulfosalicylic acid addition precipitated proteins present in the initial extraction. Following 15 min for precipitation, coagulated protein was removed by centrifugation at 6.000 ¥ g for


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20 min. Duplicate samples from each supernatant were analyzed for nitrogen (as NPN) following the AOAC (1990) Kjeldahl method. The NPN concentration was expressed as mg N/100 g dry matter (DeMasi et al. 1990). SDS-PAGE Analysis of Meat Extracts for Protein Alteration. Alteration of meat proteins in sarcoplasmic and myofibrillar protein fractions during processing was monitored with SDS-PAGE according to the method described by both Laemmli (1970) and Toldra et al. (1992). Samples of two replications from each sucuk group were analyzed. Protein Extraction. Four grams of sample was homogenized with 40 mL of 0.03 M potasium phosphate buffer, pH 7.4. After centrifugation at 10.000 ¥ g at 4C for 20 min, the supernatant was collected by filtering through glass wool. The pellet was dispersed in 40 mL of phosphate buffer, and the same procedure was repeated. Supernatants were combined as the sarcoplasmic protein fraction. The resulting pellet was dispersed in 40 mL of 8 M urea containing 1% b-mercaptoethanol and homogenized for 3 min. After centrifugation of homogenate at 4C at 10.000 ¥ g for 20 min, the supernatant was collected as the myofibrillar protein fraction (Toldra et al. 1992). Protein Assay. Protein concentrations of both sarcoplasmic and myofibrillar protein extracts were determined using a standard assay with Bradford Reagent (Sigma, St. Louis, MO) and bovine serum albumin (Sigma). Absorbance at 595 nm was used for protein concentration as described in the manufacturer’s instructions (Sigma). Because of lower protein content of sarcoplasmic proteins (0.063 mg/mL extract) extracted from heated sucuks after heating and drying, SDS-PAGE could not be conducted for these stages. SDS-PAGE Electrophoresis. SDS-PAGE electrophoresis was conducted as described by Laemmli (1970) using 4–20% gradient polyacrylamide precast gel (Bio-Rad Laboratories, Hercules, CA). Standard proteins (high and low molecular weight) of Bio-Rad were run for protin identification as described in the manufacturer’s instructions. These standard proteins were myosin (200,000 Da), b-galactosidase (116,250), phosphorylase b (97,000 Da), serum albumin (66,200 Da), ovalbumine (45,000 Da), carbonic anhydrase (31,000 Da), trypsin inhibitor (21,500 Da) and lyzozyme (14,400 Da). The gel was run on a Bio-Rad Mini Protean III elecrophoresis aparatus, with Bio-Rad Model Power Pac Basic. The gel was stained with PhastGel Blue R 350 for 45 min, and destained with the solution containing 10% methanol and 5% acetic acid until the background was clear. Gel images were captured with Bio-Rad VersaDoc 1000 Gel Documentation System with one version 4.5.2. software.

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Microbiological Analysis. Sucuk samples (10 grams) were aseptically transferred to sterile plastic bags and homogenized for 1 min at 200 rpm in a Stomacher 400 Circulator (England), with 90 mL of 0.85% NaCl (Merck). Appropriate decimal dilutions (10–8) of the samples were prepared. The following media and incubation conditions were used: (1) Baird Parker agar (Oxoid, CM961, Basingstoke, England) with 3.5% egg yolk tellurite emulsion incubated at 37C for 48 h for Micrococcus–Staphylococcus spp. Count (Johansson et al. 1994); MRS agar (Merck, 1.10660) incubated at 37C for 48 h for the lactic acid bacteria (LAB) count (Komprda et al. 2004); plate count agar (Merck, 1.05463) incubated at 28C for 72 h for total mesophilic aerobic bacteria (TMAB) count (Gelabert et al. 2003); potato dextrose agar (Merck, 1.10130) incubated at 28C for 72 h for yeast count (Coppola et al. 2000). The counts were expressed as log10 colony-forming units (cfu)/g. Statistical Analysis. All experimental data collected during the processing steps were examined statistically using analysis of variance (ANOVA), a two factorial design, and of storage periods were evaluated by applying ANOVA according to a three factorial design with repeated measurements in time. If required, a Duncan multiple comparison test was performed to investigate the differences between mean values.

RESULTS AND DISCUSSION Proximate Composition, pH and Titratable Acidity Moisture, protein, fat and ash contents of traditional style sucuks were in the range of 46.4–54.7%, 21.1–25.3%, 20.6–21.6% and 3.9–4.7%, respectively (Table 2). For heated sucuks, moisture, protein, fat and ash contents were measured between 41.1 and 48.3%, 25.2 and 27.4%, 22.3 and 25.6% and 4.3 and 4.7%, respectively (Table 2). No statistical difference (P > 0.05) in proximate composition was found in sucuks produced either by traditional or heated methods. The pH of all sucuks decreased after fermentation (P < 0.05) as a result of increasing concentration of lactic acid formed during fermentation (Table 3). The lowest pH value was observed in traditional style S1 group, which was statistically significant after drying (P < 0.05). It was stated by many investigators that the fermented sausages produced with L. sake had the lowest pH value as compared with other fermented sausages inoculated with other starter cultures (Vignolo et al. 1989; Montel et al. 1993; Hammes and Knauf 1994; Hagen et al. 1996; Sanz et al. 1997a,b). Bozkurt and Erkmen (2002) reported that the pH of the sausages fermented with starter culture mix containing


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TABLE 2. COMPOSITION OF FERMENTED TURKEY SAUSAGES (%)* Sausage group Traditional C S1 S2 Heated C S1 S2

Moisture

Protein

Fat

Ash

54.73 ⫾ 3.56 46.43 ⫾ 0.99 50.87 ⫾ 5.40

21.08 ⫾ 0.29 25.28 ⫾ 0.04 24.41 ⫾ 1.70

21.64 ⫾ 1.22 21.14 ⫾ 1.67 20.61 ⫾ 2.29

3.97 ⫾ 0.45 4.68 ⫾ 0.03 4.10 ⫾ 0.23

48.31 ⫾ 3.34 41.09 ⫾ 3.87 46.47 ⫾ 1.70

25.69 ⫾ 0.99 27.47 ⫾ 3.71 25.25 ⫾ 0.46

22.29 ⫾ 2.00 25.60 ⫾ 0.07 23.15 ⫾ 0.84

4.33 ⫾ 0.25 4.70 ⫾ 0.63 4.52 ⫾ 0.53

* Data are the mean ⫾ standard deviation (n = 4).

Pediococcus acidilactici, Lactobacillus plantarum and S. carnosus had pH value of 4.53 after fermentation, which was lower than the pH of turkey sucuks produced in the present study. This was caused by the higher pH value of turkey meat used as raw material than that of beef. There was a slight increase (P > 0.05) in the pH of traditional-style sucuks after drying, possibly because enzymatic activity resulted in ammonia and amine production. Traditional sucuks had final pH of 5.34; 5.19 and 5.34 for C, S1 and S2, respectively. This increase in pH value in fermented sausages was similarly reported by Montel et al. (1993), Garcia de Fontan et al. (2007) and Soriano et al. (2007). For heat-processed sucuks, there was no change in pH of C during processing stages after fermentation, while pHs of S1 and S2 showed increases after heating (P > 0.05) and after drying (P < 0.05). Candog˘an (2000) also reported increases in pH of fermented and heat-processsed beef sausages after drying. The storage period did have a decreasing effect (P < 0.05) on pH in both traditional and heat-processed sucuks, especially after day 60 (Table 4). But the pH values showed increases at the 120th day of storage; however, no effect of individual treatment groups was observed on pH values of sucuks. Similarly, Komprda et al. (2001) stated that although lactic acid bacterial counts of fermented sausages did not decrease, the pH was increased at day 72 of storage. Titratable acidity of initial mixes expressed as lactic acid % (0.70–0.74%) increased to 1.55–1.59% after fermentation (P < 0.05), showing a typical inverse relationship to pH decline (Table 3). Candog˘an (2000) reported similar results of titrable acidity of fermented sausages, which was between 1.36 and 1.68% after fermentation. Increases in titratable acidity values of all sucuks in processing stages after fermentation were observed for traditionally processed sucuks after drying, and for heated sucuks after drying and heating, which was not statistically significant (P > 0.05). Candog˘an (2000) stated that the titrable acidity of fermented sausages after drying stage was between 1.84 and 2.13%,

a,b

A–C

*

6.45 ⫾ 0.02Aa 5.16 ⫾ 0.01Ba 5.19 ⫾ 0.04Bb 6.45 ⫾ 0.02A 5.16 ⫾ 0.01C 5.22 ⫾ 0.03BC 5.30 ⫾ 0.05B

0.74 ⫾ 0.06B 1.58 ⫾ 0.02A 2.12 ⫾ 0.38A 0.74 ⫾ 0.06B 1.58 ⫾ 0.02A 1.55 ⫾ 0.08A 2.00 ⫾ 0.46A

6.30 ⫾ 0.07Ab 5.26 ⫾ 0.01Ba 5.34 ⫾ 0.05Ba

6.30 ⫾ 0.07A 5.26 ⫾ 0.01B 5.29 ⫾ 0.05B 5.43 ⫾ 0.10B

PH

pH

T.A.

S1

C

Sausage groups

Data are the mean ⫾ standard deviation (n = 4). Means in a column not having common supercript letter are different (P < 0.05). Means in a row not having a common supercript letter are different (P < 0.05).

Traditional method Initial mix Fermentation Drying Heated Initial mix Fermentation Heating Drying

Processing stage

0.70 ⫾ 0.04B 1.59 ⫾ 0.01A 1.62 ⫾ 0.04A 2.22 ⫾ 0.40A

0.70 ⫾ 0.04B 1.59 ⫾ 0.01A 2.19 ⫾ 0.36A

T.A.

6.45 ⫾ 0.02A 5.21 ⫾ 0.04C 5.31 ⫾ 0.06BC 5.42 ⫾ 0.06B

6.44 ⫾ 0.03Aa 5.21 ⫾ 0.04Ba 5.34 ⫾ 0.05Ba

pH

S2

0.70 ⫾ 0.00B 1.55 ⫾ 0.03A 1.54 ⫾ 0.08A 1.91 ⫾ 0.36A

0.70 ⫾ 0.00B 1.55 ⫾ 0.03A 2.14 ⫾ 0.44A

T.A.

TABLE 3. THE pH AND TITRATABLE ACIDITY (T.A., %LACTIC ACID) VALUES AT VARIOUS PROCESSING STAGES FOR FERMENTED TURKEY SAUSAGES*

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TABLE 4. THE pH AND TITRATABLE ACIDITY (T.A., %LACTIC ACID) VALUES AT STORAGE PERIODS FOR FERMENTED TURKEY SAUSAGES* Processing stage

Sausage groups C

TraditionalAb 0 days 30 days 60 days 90 days 120 days HeatedBa 0 days 30 days 60 days 90 days 120 days * A,B,a,b

S1

S2

pH

T.A.

PH

T.A.

pH

T.A.

5.34 ⫾ 0.05 5.25 ⫾ 0.04 5.13 ⫾ 0.06 5.12 ⫾ 0.23 5.33 ⫾ 0.02

2.12 ⫾ 0.38 2.64 ⫾ 0.13 3.02 ⫾ 0.33 3.19 ⫾ 0.55 2.91 ⫾ 0.24

5.19 ⫾ 0.04 5.22 ⫾ 0.02 5.07 ⫾ 0.09 5.09 ⫾ 0.24 5.31 ⫾ 0.03

2.19 ⫾ 0.36 2.77 ⫾ 0.21 2.81 ⫾ 0.25 3.33 ⫾ 0.36 2.82 ⫾ 0.37

5.34 ⫾ 0.05 5.31 ⫾ 0.05 5.12 ⫾ 0.07 5.20 ⫾ 0.26 5.42 ⫾ 0.08

2.14 ⫾ 0.44 2.61 ⫾ 0.16 2.84 ⫾ 0.23 3.28 ⫾ 0.51 2.89 ⫾ 0.46

5.43 ⫾ 0.10 5.39 ⫾ 0.02 5.35 ⫾ 0.03 5.29 ⫾ 0.26 5.52 ⫾ 0.08

2.00 ⫾ 0.46 2.37 ⫾ 0.30 2.50 ⫾ 0.21 2.72 ⫾ 0.35 2.50 ⫾ 0.24

5.30 ⫾ 0.05 5.31 ⫾ 0.01 5.18 ⫾ 0.13 5.18 ⫾ 0.15 5.39 ⫾ 0.03

2.22 ⫾ 0.40 2.63 ⫾ 0.25 2.66 ⫾ 0.18 2.98 ⫾ 0.42 2.69 ⫾ 0.28

5.42 ⫾ 0.06 5.35 ⫾ 0.02 5.30 ⫾ 0.12 5.25 ⫾ 0.19 5.48 ⫾ 0.07

1.91 ⫾ 0.36 2.31 ⫾ 0.24 2.50 ⫾ 0.28 2.65 ⫾ 0.33 2.49 ⫾ 0.20

Data are the mean ⫾ standard deviation (n = 4). Means in a column not having common supercript letters are different (P < 0.05).

which was similar to lactic acid contents of traditional-style turkey sucuks. Similar results were also reported by Acton and Dick (1976). During refrigerated storage, traditional-style sucuks had higher titratable acidity than heated sucuks (P < 0.05); whereas there was no significant difference in titratable acidity between treatment groups (C, S1, S2) over storage (P > 0.05). NPN Changes NPN contents of sucuks produced by both traditional and heated processes are given in Table 5. The NPN content of initial traditional-style sucuk mixes showed increases after fermentation from 834.2, 841.3 and 805.2 mg/ 100 g dry matter to 1,193.0, 1,345.9 and 1,287.7 mg/100 g dry matter for C (P > 0.05), S1 and S2 (P < 0.05), respectively. Wardlaw et al. (1973) reported that the NPN content of fermented sausages increased from 2.98 mg/g sample to 4.45 mg/g sample after fermentation. Beriain et al. (2000) observed an increase in NPN content of fermented sausages from 485 mg/100 g dry material to 772 mg/100 g dry matter. There were slight, but statistically insignificant, increases determined in NPN contents of traditional-style sucuks (P > 0.05). Many investigators also reported increases in NPN content of fermented sausages during drying, and

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TABLE 5. NONPROTEIN NITROGEN CONTENTS AT VARIOUS PROCESSING STAGES FOR FERMENTED TURKEY SAUSAGES (MG/100 G DM)* Processing stage

Traditional method Initial mix Fermentation Drying Heated Initial mix Fermentation Heating Drying * A,B

Sausage groups C

S1

S2

834.2 ⫾ 63.0B 1,193.0 ⫾ 179.0AB 1,573.0 ⫾ 163.0A

841.3 ⫾ 44.5B 1,345.9 ⫾ 82.6A 1,395.0 ⫾ 108.0A

805.2 ⫾ 83.2B 1,287.7 ⫾ 63.0A 1,490.1 ⫾ 60.0A

834.2 ⫾ 63.0B 1,193.0 ⫾ 179.0AB 1,382.5 ⫾ 34.2A 1,439.3 ⫾ 73.7A

841.3 ⫾ 44.5B 1,345.9 ⫾ 82.6A 1,304.4 ⫾ 42.1A 1,354.0 ⫾ 55.8A

805.2 ⫾ 83.2B 1,287.7 ⫾ 63.0A 1,375.6 ⫾ 49.4A 1,362.0 ⫾ 108.0A


associated this increase with effect of proteolysis (Wardlaw et al. 1973; Dierick et al. 1974; DeMasi et al. 1990; Verplaetse 1994; Beriain et al. 2000; Candog˘an 2000). Within heated sucuks, fermentation resulted in increasing effect (P < 0.05) on NPN contents except C; however, the increase found during heating and drying was not significant (P > 0.05). Wardlaw et al. (1973) and Candog˘an (2000) reported significant increases after the heating stage of fermented sausage processing. The NPN contents of heated sucuks at day 0 were between 1,354.0 and 1,439.3 mg/100 g dry matter, which was higher than the findings of DeMasi et al. (1990). Refrigerated storage had no significant effect (P > 0.05) on NPN content of all sucuks produced by either traditional or heated processes (Table 6). Protein degradation occurs during fermentation because of bacterial and indigenous muscle proteases already present in the meat. This protein degradation is the main biochemical change that occurs during sucuk manufacturing, and produces low-molecular weight NPN compounds such as peptides, amino acids, organic acids and amines (Verplaetse 1994; Hierro et al. 1999; Lucke 2000; Hughes et al. 2002). Some investigators have noted that protein degradation during fermentation is a result of indigenous proteases, particularly cathepsin D, already present in the meat or from bacterial proteinases present during the ripening stage of production (Demeyer et al. 1995; Molly et al. 1997; Stahnke 1999; Lucke 2000; Hughes et al. 2002; Herranz et al. 2006).


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TABLE 6. NONPROTEIN NITROGEN CONTENTS AT STORAGE PERIODS FOR FERMENTED TURKEY SAUSAGES (MG/100 G DM)* Processing stage

TraditionalA 0 days 30 days 60 days 90 days 120 days HeatedB 0 days 30 days 60 days 90 days 120 days * A.B

Sausage groups C

S1

S2

1,573.0 ⫾ 163.0 1,578.2 ⫾ 56.1 1,521.0 ⫾ 67.3 1,523.0 ⫾ 145.0 1,702.0 ⫾ 169.0

1,395.0 ⫾ 108.0 1,355.3 ⫾ 60.2 1,339.0 ⫾ 100.0 1,172.1 ⫾ 78.2 1,245.0 ⫾ 138.0

1,490.1 ⫾ 60.0 1,450.8 ⫾ 76.6 1,450.3 ⫾ 40.8 1,359.6 ⫾ 64.4 1,469.0 ⫾ 178.0

1,439.3 ⫾ 73.7 1,474.6 ⫾ 85.0 1,380.2 ⫾ 65.9 1,358.0 ⫾ 129.0 1,398.0 ⫾ 93.4

1,354.0 ⫾ 55.8 1,321.1 ⫾ 56.6 1,313.0 ⫾ 102.0 1,135.5 ⫾ 34.8 1,260.8 ⫾ 67.0

1,362.0 ⫾ 108.0 1,368.3 ⫾ 43.3 1,265.2 ⫾ 85.4 1,232.1 ⫾ 27.3 1,248.9 ⫾ 13.6


SDS-PAGE Electrophoresis The effect of processing and starter addition on the protein fraction was evaluated by SDS-PAGE. The protein profiles from hydrolysis of muscle sarcoplasmic and myofibrillar proteins of all treatments are presented in Figs. 1–4. In general, no major difference occured between the SDS-PAGE patterns because of starter culture treatments. This result can be explained by the fact that the first step in protein hydrolysis during processing is degradation of muscle proteins to polypeptides by indigenous meat enzymes. Further degradation of polypeptides to peptides with low-molecular weight and free amino acids is achieved by enzymes of microbial origin (Verplaetse 1994). However, changes in both sarcoplasmic and myofibrillar proteins at processing stages were observed. Changes in sarcoplasmic protein profiles during fermentation and drying are shown in Fig. 1. Protein band with a molecular weight of about 120 kDa disappeared after drying in all groups, and a band with an appraximate molecular weight of 75 kDa appeared in all groups. Additionally, a band with an approximate molecular weight of 105 kDa appeared in C and S1 after drying. The intensity of this band is higher than that of C. For all groups, the intensity of the band with an approximate weight of 65 kDa increased after drying. The intensity of the band with a molecular weight of about 30 kDa increased in C and S2 after fermentation and decreased after drying. However, the intensity of this band showed increase after fermentation in S1. Casaburi et al. (2007) reported similar electro-

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A

B

C

D

E

F

G

H

I

J

200 kDa 116 kDa 97 kDa 66,2 kDa 45 kDa 31 kDa

215 kDa 144 kDa

FIG. 1. SDS-PAGE GEL ELECTROPHORETIC PATTERNS OF SARCOPLASMIC PROTEINS FOR TRADITIONAL-STYLE TURKEY SUCUKS (INITIAL MIX, FERMENTATION AND DRYING AFTER FERMENTATION)

Myofibrillar Control A

B

C

D

E

F

G

200 kDa 116 kDa 97 kDa 66,2 kDa 45 kDa 31 kDa

21,5 kDa 14,4 kDa

FIG. 2. SDS-PAGE GEL ELECTROPHORETIC PATTERNS OF MYOFIBRILLAR PROTEINS FOR CONTROL GROUPS OF TURKEY SUCUKS

phoretic profile of fermented sausage produced with two different strains of proteolytic S. xylosus. The researchers explained this low proteolytic activity in uninoculated fermented sausages by the high pH value that could have effected the indigenous proteases activity. Furthermore, the pH values of uninoculated fermented sausages analyzed by Casaburi et al. (2007) were


155

Myofibrillar S2 A

B

C

D

E

F

G

200 kDa 116 kDa 97 kDa 66,2 kDa 45 kDa 31 kDa 21,5 kDa 14,4 kDa

FIG. 3. SDS-PAGE GEL ELECTROPHORETIC PATTERNS OF MYOFIBRILLAR PROTEINS FOR S2 GROUPS OF TURKEY SUCUKS

Miyofibrilar S1 A

B

C

D

E

F

G

200 kDa 116 kDa 97 kDa 66,2 kDa 45 kDa 31 kDa 21,5 kDa 14,4 kDa

FIG. 4. SDS-PAGE GEL ELECTROPHORETIC PATTERNS OF MYOFIBRILLAR PROTEINS FOR S1 GROUPS OF TURKEY SUCUKS

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higher than the pH of turkey sausages. The similarity between the electrophoretic profile of C treatment, and S1 and S2 treatments may be explained by the similar pH value of all sucuk treatments, whereas S1 had the lowest pH. Changes in myofibrillar protein profile were also observed over processing stages. It was observed that the intensity of myosin heavy chain (200 kDa) was decreased after fermentation, and the band with a molecular weight of 140 kDa appeared after fermentation in all groups probably because of degradation of myosin heavy chain. In S1, myosin heavy chain disappeared after drying in traditional-style group, and after heating and drying in heated group while the intensity of the band corresponding to myosin heavy chain decreased after drying in heated C and S2. Similar results were stated by Candog˘an (2000) and Hughes et al. (2002) within the fermented beef sausages and fermented salami. Casaburi et al. (2007) also reported that the band corresponding to myosin heavy chain decreased during ripening. Significant degradation of myosin heavy chain during ripening of naturally fermented sausages was also reported by Diaz et al. (1997). The actin band (approximately 45 kDa) also decreased in intensity during processing stages. This decrease in intensity of actin was apparent after drying in traditional-style and after heating in heated-style S1 groups (Fig. 4); however, the intensity of the corresponding band increased at the end of drying in heated S1. Casaburi et al. (2007) reported decreases in early stages of ripening and increases at the end of ripening in the intensity of actin band in starter culture-inoculated fermented sausages, and attributed this increase in intensity of corresponding band to the co-migration of peptides arrising from a myosin heavy chain degradation. For C and S2, however, the intensity of actin band decreased after drying in heated sucuks (Figs. 2 and 3). Degradation of myosin and actin during fermented sausage processing was also reported by Verplaetse et al. (1989), Garcia de Fernando and Fox (1991) and Hughes et al. (2002). Microbiological Analysis C had lower initial LAB counts (4.51 log cfu/g) than S1 (6.23 log cfu/g) and S2 (6.29 log cfu/g) (P < 0.01). After fermentation, LAB increased rapidly (P < 0.01) to about 8.03, 8.97 and 8.47 log cfu/g for C, S1 and S2 (Table 7). C had lower LAB counts than S1 (P < 0.01) and S2 (P > 0.01) over fermentation. When dried, the number of LAB in sucuks did not differ between groups, and ranged from 8.60 to 9.11 log cfu/g. Candog˘an (2000) reported that the LAB counts of fermented sausages in which L. sake was used as a starter culture increased after fermentation. The number of LAB in the present study showed similarities to the number of LAB observed by other researchers (Vignolo et al. 1989; Samelis et al. 1993; Sanz et al. 1997b; Gonzalez and Diez 2002; Sakhare and Rao 2003).

A,B

x–z

X,Y

*

4.51 ⫾ 0.44Yy 8.03 ⫾ 0.33Xy 8.60 ⫾ 0.24Xx 4.51 ⫾ 0.44By 8.03 ⫾ 0.33Ay 6.04 ⫾ 1.03AB 5.19 ⫾ 1.19AB

5.45 ⫾ 0.01Yz 8.23 ⫾ 0.15Xy 8.38 ⫾ 0.07Xy

5.45 ⫾ 0.01Bz 8.23 ⫾ 0.15Ay 6.41 ⫾ 0.99AB 6.25 ⫾ 1.00AB 6.64 ⫾ 0.13Yy 8.95 ⫾ 0.19Xx 6.54 ⫾ 1.12 7.04 ⫾ 2.12

6.64 ⫾ 0.13Yy 8.95 ⫾ 0.19Xx 8.88 ⫾ 0.05Xx

TMAB

TMAB

LAB

S1

C

Sausage groups

Data are the mean ⫾ standard deviation (n = 8). Means in a column not having common superscript letters are different (P < 0.01). Means in a row not having a common supercript letter are different (P < 0.01). Means in a column not having common supercript letter are different (P < 0.05).


Processing stage

6.23 ⫾ 0.08Yx 8.97 ⫾ 0.14Xx 6.28 ⫾ 1.18 6.34 ⫾ 2.65

6.23 ⫾ 0.08Yx 8.97 ⫾ 0.14Xx 9.11 ⫾ 0.11Xx

LAB

6.99 ⫾ 0.09Yx 8.88 ⫾ 0.05Xx 6.45 ⫾ 1.32 6.721.55

6.99 ⫾ 0.09Yx 8.88 ⫾ 0.05Xx 8.96 ⫾ 0.08Xx

TMAB

S2

6.29 ⫾ 0.05Yx 8.47 ⫾ 0.05Xxy 6.03 ⫾ 1.19 5.98 ⫾ 2.02

6.29 ⫾ 0.05Yx 8.47 ⫾ 0.05Xxy 8.63 ⫾ 0.23Xx

LAB

TABLE 7. TOTAL MESOPHILIC AEROBIC BACTERIA (TMAB) AND LACTIC ACID BACTERIA (LAB) COUNTS AFTER THE PROCESSING STAGES FOR FERMENTED TURKEY SAUSAGES (LOG CFU/G)*

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The initial counts of TMAB were 5.45, 6.64 and 6.99 log cfu/g in C, S1 and S2, respectively. The differences in initial TMAB counts of groups were significant (P < 0.01). TMAB counts of sucuks showed increases in each group after fermentation, (P < 0.01), and S1 and S2 had higher TMAB counts than C after fermentation and drying in traditional-style sucuks (P < 0.01). Similar results for TMAB counts of fermented sausages after fermentation were reported by many investigators (Holey et al. 1988; Samelis et al. 1993; Olesen and Stahnke 2000; Gelabert et al. 2003; Sakhare and Rao 2003; Visessanguan et al. 2006). TMAB and LAB counts in the heated treatments are given in Table 7. The heating stage resulted in an approximately 1.5 log unit reduction of TMAB and LAB counts (P < 0.05). Candog˘an (2000) reported similar results and stated that heating process significantly reduces the LAB and TMAB counts of fermented sausages. The counts of TMAB and LAB in sucuk produced either by traditional or heated methodologies did not change during 120 days of refrigerated storage (P > 0.05) (data not shown). Micrococci–staphylococci counts of initial mixes were between 4.58 and 6.78 log cfu/g (Table 8). The highest numbers of micrococci–staphylococci were observed in the S1 and S2 treatments because of utilization of starter culture mixes containing S. xylosus and S. carnosus (P < 0.05) (Table 8). Although micrococci–staphylococci counts in each sucuk increased at the end of the fermentation stage, this increase was only significant in C (P < 0.05). Micrococci–staphylococci counts decreased slightly to 6.81, 6.19 and 7.54 log cfu/g after the drying stage in the traditional preparation for C, S1 and S2, respectively (P > 0.05). Soyer et al. (2005) reported similar counts of micrococci–staphylococci in naturally fermented sausages at the end of fermentation and the drying process. In agreement with the results of this study, several studies have shown that micrococci–staphylococci counts in fermented sausages showed an initial increase after fermentation, then, a decrease after ripening. Johansson et al. (1994) and Visessanguan et al. (2006) also reported similar changes during processing stages in micrococci–staphylococci counts. The heating step resulted in a decrease in micrococci–staphylococci counts of sucuks, but this decrease was not statistically significant (P > 0.05) (Table 8). Although the micrococci–staphylococci counts observed in the heated sucuk were lower than those observed in the traditional preparation overall 120 days of storage (P < 0.01), the difference between micrococci– staphylococci counts of storage periods was not statistically significant (P > 0.05) (data not shown). The yeast counts of about 5.67, 5.90 and 5.87 log cfu/g were found in C, S1 and S2, respectively, then, the yeast count of the S1 and S2 treatments decreased while those observed in the C treatment increased after fermentation

X,Y,a,b

A,B

*

6.36 ⫾ 0.03Aa 7.08 ⫾ 0.32Aa 6.19 ⫾ 0.08Ab 6.36 ⫾ 0.03Aa 7.08 ⫾ 0.32Aa 5.69 ⫾ 0.74 5.42 ⫾ 1.59

5.67 ⫾ 0.30 6.98 ⫾ 0.60 5.74 ⫾ 1.45 6.07 ⫾ 0.96

4.59 ⫾ 0.32Bb 7.78 ⫾ 0.37Aa 5.90 ⫾ 0.98 5.32 ⫾ 1.65

Micrococcus– Staphylococcus spp.

5.67 ⫾ 0.30 6.98 ⫾ 0.60 7.43 ⫾ 0.07

YeastX

S1

4.58 ⫾ 0.32Bb 7.78 ⫾ 0.37Aa 6.81 ⫾ 0.61Aab


C

Sausage groups

Data are the mean ⫾ standard deviation (n = 8). Means in a column not having common supercript letters are different (P < 0.05). Means in a row not having a common supercript letter are different (P < 0.05).


Processing stage

5.90 ⫾ 0.56 4.76 ⫾ 0.57 4.03 ⫾ 0.15 4.24 ⫾ 0.77

5.90 ⫾ 0.56 4.76 ⫾ 0.57 6.14 ⫾ 0.08

YeastY

6.78 ⫾ 0.14Aa 7.87 ⫾ 0.50Aa 5.51 ⫾ 1.14 6.09 ⫾ 1.90

6.78 ⫾ 0.14Aa 7.87 ⫾ 0.50Aa 7.54 ⫾ 0.59Aa


S2

5.87 ⫾ 0.55 5.58 ⫾ 0.52 4.66 ⫾ 0.25 5.38 ⫾ 0.54

5.87 ⫾ 0.55 5.58 ⫾ 0.52 6.72 ⫾ 0.48

YeastXY

TABLE 8. MICROCOCCUS–STAPHYLOCOCCUS SPP. AND YEAST COUNTS AFTER THE PROCESSING STAGES FOR FERMENTED TURKEY SAUSAGES (LOG CFU/G)*

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(P > 0.05). S1 had the lowest yeast counts with 6.14 log cfu/g (P < 0.05). The changes in yeast counts observed in traditional process stages were significant, especially after drying stage (P < 0.05), while the heating stage resulted in about 1 log unit reduction in yeast counts within all sucuk groups (P > 0.05) (Table 8). The interaction between the sucuk treatments and processing methodologies was found to be significant, and the S1 treatment in both processing methods had the lowest yeast counts (P < 0.05). The refrigerated storage (120 days) had no effect on the yeast counts in any of the treatment groups examined (P > 0.05) (data not shown). CONCLUSIONS The findings of this study show that incorporation of commercial starter culture mixes containing L. sake, S. carnosus, S. xylosus II, L. sake, S. xylosus and P. pentosaceus had some differences in chemical and microbiological properties of turkey sucuks as compared with uninoculated control during processing stages. Turkey sucuks containing L. sake, S. carnosus and S. xylosus II had the lowest pH value after drying in traditional processing. Utilization of different starter culture mixes and heat application in turkey sucuk manufacturing resulted in differences in protein degradation during processing stages. In general, no significant changes were observed in biochemical characteristics of over-refrigerated storage for 120 days.

ACKNOWLEDGMENT This work was supported by Ankara University Scientific Research Project Funding (BAP 2003-07-11-075).

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