Abstract: This study was aimed to determine microbiological changes in various periods (raw material, after mixing, after stuffing, after heat application and in 10 days of storage) of Turkish semi-dry fermented sausage produced in a meat products plant located in Konya, Turkey. Coliforms, Enterobacteriaceae, yeasts-moulds counts of sausages stuffed into both natural and collagen casing decreased (p<0.001) after heat application and Staphylococcus-Micrococcus at the end of storage (p<0.01, p<0.05). On the other hand, significant increase (p<0.01, p<0.05) in lactic acid bacteria counts at the end of storage were found. In order to prolong the shelf life and to improve the microbiological quality of semi-dry fermented sausage, lower initial microbial levels of meat and other ingredients, effective heat treatment during heat application, careful handling of sausage and maintenance of appropriate chill temperature during storage are necessary.

INTRODUCTION

Sausage is very popular and highly relished meat product all over the world. Recently, consumer’s awareness has increased for microbiological quality of sausages. Thus, an understanding of microbial characteristics of sausage is vital. Microbial ecology of sausages is closely related to meat and other ingredients as well as environment, equipment, handling practices, processing, packaging and storage temperature (Sachindra et al., 2005). Microflora of sausage is composed of technological microorganisms (Lactic acid bacteria and Gram-positive cocci) that are useful for fermentation and flavour, spoilage microorganisms that can cause negative changes in appearance, odour, flavour and consistency of the final product due to their metabolic activity and may also include some pathogenic microorganisms (Lebert et al., 2007). In recent years, food operators have been urged to develop food hygiene procedures based on the principles of HACCP and good manufacturing practices, requiring that the safety of final products be demonstrated prior to marketing (Barbuti and Parolari, 2002).

Fermented sausages are divided into 2 subgroups. That is, they are named semidry or quickly fermented and dry or slowly fermented sausages (Savic, 1985). Both semidry and dry fermented sausages are produced in Turkey. Turkish dry fermented sausage has traditionally been produced without the addition of starter cultures. Both ripening and drying are carried out under natural climatic conditions during September and December (Soyer et al., 2005). Due to the long processing time and dependence on natural climatic conditions, dry fermented sausage production has been replaced by rapid ripening methods which mainly rely on the use of controlled drying chambers and starter cultures and heat application process to guarantee the safety and quality of the final product (Sanz et al., 1998).

During the last decade, modern plants in Turkey started to produce semi-dry fermented sausages with the addition of starter cultures and heat application. However, the characteristics of these sausages produced with the addition of starter cultures and heat application are quite different from the naturally fermented ones with respect to taste and flavour. Currently, there is a much higher market share for the sausages produced with starter cultures and heat application than the naturally fermented sausages (Soyer et al., 2005).

Microbiological control of sausage at either production or storage stages for preparation of high quality product is the major concern. The safety of sausage is generally achieved by controlling or preventing growth of pathogen and spoilage microorganism during the process and reducing contamination to the lowest possible level. There are a great number of studies (Bozkurt and Erkmen, 2002; Aksu and Kaya, 2004; Kaban and Kaya, 2006; Colak et al., 2007) on dry fermented sausages in Turkey. On the other hand, information in the literature related to semi-dry fermented sausage very limited (Vural, 2003; Siriken et al., 2006a, b) and these studies have been generally carried out in final product and during storage. Thus, the aim of this study, was to investigate microbiological changes in some periods (raw material, after mixing, after stuffing, after heat application and in ten days of storage) of Turkish semi-dry fermented sausage produced in a meat products plant located in Konya, Turkey.

MATERIALS AND METHODS

Samples collection: Samples were taken four times in different periods of sausage production from a private meat products factory in Konya/Turkey. A flow diagram of Turkish semi-dry fermented sausage production is given in detail in Fig. 1. Samples were collected from raw material (meat), after grinding of meat, after mixing of meat with other ingredients, after stuffing and after heat application during production stages and in 10 days of storage. All samples were stored at 4°C and processed within 12 h of collection.

Microbiological analysis: Except Ringer’s tablet, which was purchased from Merck (Darmstadt, Germany), all other microbiological media were obtained from Oxoid (Basingstoke, UK). For microbiological analysis, 25 g of samples were diluted in 225 mL of l/4-strength Ringer’s solution and homogenised in a Colworth Stomacher Lab-Blender 400 (Seward Medical, London, UK) for at least 2 min. The homogenate was decimally diluted in the same solution and each dilution was plated in duplicate on the specific media required for the different microbial groups to be examined (Messer et al., 1992).

Fig. 1:	Flow diagram of Turkish semi-dry fermented sausage production

The pour plate technique (1 mL) was used to determine Total Aerobic Mesophilic Bacteria (TAMB), yeasts and moulds and Staphylococcus-Micrococcus or the pour plate and overlay technique was utilized for determining coliforms, Enterobacteriaceae and Lactic Acid Bacteria (LAB) (Swanson et al., 1992). Total aerobic mesophilic bacteria were enumerated on standard plate count agar after incubation at 30°C for 72 h. Yeasts and moulds were counted on potato dextrose agar acidified with 10% tartaric acid and incubated at 25°C for 5 days. Staphylococcus-Micrococcus were enumerated on mannitol salt agar at 37°C for 48 h. Coliforms were determined on violet red bile lactose agar after incubation at 37°C for 24 h. Enterobacteriaceae was enumerated on violet red bile glucose agar after incubation at 37°C for 24 h. LAB were determined on De Man, Rogosa, Sharpe agar after incubation at 30°C for 72 h. Plates with 30-300 colonies were counted and the results were expressed as logarithm of colony forming units (cfu) per gram (log₁₀ cfu g^-1).

Measuring of pH: The pH values of samples were measured by pH meter (InoLab pH 720 model; WTW GmbH, Hamburg, Germany) equipped with a combined electrode.

Statistical analysis: A one way analysis of variance was performed on data obtained at different stages of production after a log transformation for bacterial counts, using the SPSS/PC version 10.00 (SPSS Inc, Chicago, IL, USA). Differences between the groups and the processing stages were identified using Duncan’s multiple range test.

RESULTS AND DISCUSSION

Microorganisms gain access into sausage from meat, spices and other ingredients, intestine, environment, equipment and handlers during processing stages and this affect microbiological quality of final product. Lower initial microbial load of sausage mix and maintenance of adequate temperature during storage would improve the microbiological quality and enhance the shelf life of sausage (Sachindra et al., 2005; Siriken et al., 2006b).

The changes in the counts of microbial groups enumerated during processing stages and storage of Turkish semi-dry fermented sausages stuffed into natural and collagen casing are presented in Table 1 and 2. Processing stages and storage significantly affected coliforms, Enterobacteriaceae, Staphylococcus-Micrococcus and yeasts-moulds counts of sausages, which had lower counts in sausages stuffed into both natural and collagen casing after heat application for coliforms (p<0.001), Enterobacteriaceae (p<0.001) and yeasts-moulds (p<0.001) and at the end of storage for Staphylococcus-Micrococcus (p<0.01, p<0.05). On the other hand, LAB were significantly increased in sausages stuffed into both natural and collagen casing at the end of storage (p<0.01, p<0.05).

Table 1:	Changes in microbiological profile during processing stages of Turkish semi-dry fermented sausage stuffed into natural casing (log10 cfu g-1)1

Table 2:	Changes in microbiological profile during processing stages of Turkish semi-dry fermented sausage stuffed into collagen casing (log10 cfu g-1)1
1Mean values and standard error of 4 samples; a-cMeans within the same line with different superscript letters are different (p<0.05) according to Duncan’s multiple range test; nd: not detected

TAMB count of raw material (meat), which was detected 6.26 log₁₀ cfu g^-1, was within the microbiological standards of raw red meat according to the Turkish Food Codex (2006). This initial TAMB count increased up to 7.16 log₁₀ cfu g^-1 in the sausage stuffed into natural casing and 7.20 log₁₀ cfu g^-1 in the sausage stuffed into collagen casing at the end of storage. However, the TAMB counts in both groups did not change significantly (p>0.05) during production stages and storage. TAMB counts of final product were lower than the findings of Siriken et al. (2006b), who found 73% (73/100) of the samples contained TAMB between the levels of 10⁷-10¹⁰ cfu g^-1.

The initial count of coliforms in raw material was 4.54 log₁₀ cfu g^-1 and count increased at level 4.88 log₁₀ cfu g^-1 in sausage stuffed into natural casing and reached at level 4.92 log₁₀ cfu g^-1 in sausage stuffed into collagen casing after stuffing stage. But, the increase of coliforms count did not show any significance (p>0.05) during mixing and stuffing stages. After heat application, significant decrease was observed (p<0.001). This result demonstrates that heat application process was effective in reducing the coliforms counts. While coliforms were not detected in s a usage stuffed into natural casing at the end of storage (p<0.001), its count decreased to 0.75 log₁₀ cfu g^-1 in sausage stuffed into collagen casing (p<0.001). This observation confirms the strong competitive effect of LAB on the rest of the endogenous flora as is observed in sausage (Drosinos et al., 2005) and in other fermentations (Spyropoulou et al., 2001). Result obtained in this study was in agreement with those of Lücke (2000) and Chevallier et al. (2006), who stated that coliforms count declined very quickly and was totally inhibited within 7 days in more acidified sausages. On the other hand, Siriken et al. (2006b) have reported that contamination of semi-dry fermented sausages with coliforms and Enterobacteriaceae in high levels. This may be due to recontamination during handling of sausage.

The initial count of Enterobacteriaceae in raw material was 4.45 log₁₀ cfu g^-1. Similar changes like coliforms were observed in the counts of Enterobacteriaceae in sausages stuffed into both natural casing and collagen casing (p<0.001).

The LAB may have an important contribution to the final flavour of the products due to their fermentation of carbohydrates (Lizaso et al., 1999). The initial count of LAB was low and other microorganisms such as Staphylococcus-Micrococcus had higher initial counts. LAB counts increased during processing stages and reached levels up to 7.88 and 7.77 log₁₀ cfu g^-1 of sausages stuffed into both natural and collagen casing at the end of storage, respectively (p<0.01, p<0.05). The count of LAB exceeded 7 log₁₀ cfu g^-1 and constituted the major microflora at the end of storage in sausages stuffed into both natural and collagen casing. Because of the good adaptation of LAB to the meat environment, the presence of NaCl and nitrite and their faster growth rates, which were displayed during fermentation and ripening of sausages (Drosinos et al., 2005; Chevallier et al., 2006), they became the dominant microflora at the end of storage.

Micrococcus and Staphylococcus are beneficial for their ability to reduce nitrates, hydrolyze lipids and destroy peroxides (Samelis et al., 1994; Fista et al., 2004). The initial count of Staphylococcus-Micrococcus (5.20 log₁₀ cfu g^-1) was increased at level 5.91 log₁₀ cfu g^-1 in sausage stuffed into natural casing and reached at level 5.69 log₁₀ cfu g^-1 in sausage stuffed into collagen casing after stuffing stage. Thereafter, the counts decreased after heat application and storage stages in sausages stuffed into both natural and collagen casing. Staphylococcus-Micrococcus counts at the end of storage significantly lower than the other stages (p<0.01, p<0.05). Several authors have reported that the acidification and anaerobic condition inhibited the growth of Staphylococcus-Micrococcus during ripening of fermented sausage (Aksu and Kaya, 2004; Hu et al., 2008). Fista et al. (2004) stated that low temperatures of storage combined with high numbers of LAB and pH values below 5 cause growth of Micrococcus and Staphylococcus to cease. Our results were lower than those determined by Siriken et al. (2006b), who stated that 72% of the samples contained Staphylococcus-Micrococcus at the level ≥10⁴ cfu g^-1.

Yeasts and moulds count level of raw material was within the microbiological standards of raw red meat according to the Turkish Food Codex (2006). The initial count of yeasts and moulds in raw material was 3.76 log₁₀ cfu g^-1 and this count reached at level 4.59 log₁₀ cfu g^-1 after mixing of meat with other ingredients. Yeasts and moulds count reached 5.24 in sausage stuffed into natural casing and decreased 4.37 log₁₀ cfu g^-1 in sausage stuffed into collagen casing after stuffing stage. After heat application, yeasts and moulds were not detected in the samples (p<0.001). Similar results, were observed by Sachindra et al. (2005). They stated that cooking process was effective in reducing the yeasts and moulds counts substantially in sausage. Conversely, the counts of yeasts and moulds recorded in this study at the end of storage are not in agreement with the finding of Siriken et al. (2006b). They reported that 17% of the semi-dry fermented sausage samples were found highly contaminated with yeasts and moulds at the level of 10⁴ cfu g^-1 and 34% of the contaminated samples were considered as non-consumable products with relation to Turkish Food Codex (2000).

Fig. 2:	Changes in pH values during production stages of Turkish semi-dry fermented sausage stuffed into natural and collagen casing

According to Turkish Food Codex (2000), maximum pH value of sausage applied heat treatment should be 5.8. The changes in the pH values during processing stages and storage of Turkish semi-dry fermented sausage stuffed into natural and collagen casing are presented in Fig. 2. The initial pH value of raw material was 5.76. This value increased in grinding stage and decreased after mixing stage. After stuffing and heat application stages, pH values of sausages stuffed into both natural and collagen casing were again increased. During storage, pH values significantly decreased to 5.11 and 5.36 in both groups, respectively (p<0.001, p<0.05). The drop of pH because of the production of lactic acid by the increasing population of LAB at the end of storage, antagonism by other metabolic products produced by LAB and depletion of nutrients may have prevented the increase. In this respect, pH values was correlated to LAB counts at the end of storage and significant correlation (r = -627, p<0.01 for sausage stuffed into natural casing; r = -598, p<0.01 for sausage stuffed into collagen casing) was found between LAB and pH. Many researchers (Muguerza et al., 2002; Kayaardi and Gok, 2003; Vural, 2003) stated organic acids, mainly lactic acid are formed in fermented sausages as a result of carbohydrate breakdown during fermentation giving rise to the reduction in pH. The pH values obtained in this study at the end of storage were lower than those reported in semi-dry fermented sausages by Siriken et al. (2006a, b).

CONCLUSION

Hygienic quality of raw material has an important effect on final microbial load of sausage. Heat application is also the main stage for the elimination of non-desired microorganisms during the production of semi-dry fermented sausage. In order to prolong the shelf life and to improve the microbiological quality of semi-dry fermented sausage, lower initial microbial levels of meat and other ingredients, effective heat treatment during heat application, careful handling of sausage and maintenance of appropriate chill temperature during storage are necessary. In addition, the use of HACCP based control programs improves the quality and safety of the sausage during processing stages because hygienic status of the processing environment and equipment plays an essential role in the microbial stability and safety of the final products.