As cig kofte is prepared without applying any heat treatment or using any preservative, its microbiological quality depends on personal hygiene, production method, hygienic quality of ingredients used in particular of minced meat and spices³. Several studies have been conducted on the microbiological quality of cig kofte. These studies have demonstrated that this food product can be contaminated with fecal indicator bacteria and pathogenic bacteria, including coliforms, Salmonella spp., L. monocytogenes, S. aureus, E. coli, Y. entereocolitica and B. cereus^4-6. Listed among the major food pathogens, which cause significant public health problems, Salmonella spp. are reported to have a prevalence ranging between 0-14% in cig köfte^4,7,8.

Previous research has shown that cig kofte is a high-risk food product in terms of public health and food safety. Literature reports on how to increase the microbiological safety of cig kofte are few. The scarcity of scientific information requires the use of antimicrobial food additives apart from the application of good hygiene practices. The natural composition and low toxicity of these natural antimicrobial substances have enabled them to find common use in the food industry. Of these substances, chitosan is a natural polymer obtained by the deacetylation of chitin found in the exoskeleton of crustaceans, including crabs and shrimps⁹. In the past few years, chitosan has been widely investigated by the food industry, and is categorized as “generally recognized as safe (GRAS)” by the Food and Drug Administration¹⁰. It is reported that chitosan can prevent the growth of Gram negative bacteria, including E. coli, Pseudomonas aeruginosa, Shigella dysenteria, Vibrio spp. and Salmonella spp., and that the minimum inhibitory concentration of this substance for these bacteria ranges from 100 ppm to 10.000 ppm¹¹. Essential oils are another group of natural compounds with antimicrobial property. Owing to their taste, aroma and broad bioactivity profile, they constitute a significant alternative for use in cig köfte^12-14. Several essential oils, including those of cloves, thyme and rosemary, are classified as GRAS by the FDA¹⁰. Previous research has shown that, apart from their antimicrobial property, essential oils also exhibit antibacterial and antifungal affects both in vitro and in food¹². The inhibitory effect of these essential oils on saprophytic and pathogenic Gram-negative and Gram-positive bacteria suggests that they may play an important role in enhancing the safety of risk food products¹⁵.

The present study was carried out to investigate the effects of the use of chitosan and thyme, clove and rosemary essential oils, alone or in combination, on the inactivation of Salmonella in cig kofte.

Prerapation of Inoculums: The strains used to inoculate cig kofte were three Salmonella typhimurium strains (ATCC 14028, NCTC 12416, RSKK 95091). The strains were stored at −80 °C before use. After thawing, each strain was cultured in 10 mL of tryptic soy broth (Merck, Darmstadt, Germany) and incubated at 37 ºC for 18 h. Then, the cultures were centrifuged at 4.192 × g for 10 min at 5 °C (NF800R, Nüve, Turkey), and the pellets were washed with 0.1% sterile peptone water (LAB M, England) before re-centrifuging to remove organic residues. The pellets of each strain were re-suspended in an aliquot of peptone water. These suspensions were then combined in a single beaker and completed to 10 mL with 0.1% sterile peptone water. The final level of the pathogen was approximately 10⁸ CFU/mL in the inoculum.

Salmonella Inoculation and the Addition of Essential Oils and Chitosan: For each trial, freshly prepared 1500 g of cig kofte was obtained from a local restaurant. In the present study, all ingredients used by the restaurant were presented in Table 1 as g/ kg cig kofte. Each trial was repeated three times. A cig kofte sample of 100 g was taken and analyzed for pH, water activity, salt level, and Salmonella spp. presence. The remaining 1400 g was inoculated with Salmonella mixture and mixed by hand. Subsequently, the cig kofte sample was divided into 8 portions/groups, each weighing 175 g. The control group did not include any essential oil or chitosan, and Groups I, II III, IV, V, VI and VII were added 250 mg/kg of thyme essential oil, 250 mg/kg of clove essential oil, 250 of rosemary essential oil, 500 mg/kg of chitosan, 500 mg/kg of chitosan + 250 mg/kg of thyme essential oil, 500 mg/kg of chitosan + 250 mg/kg of clove essential oil, and 500 mg/kg of chitosan + 250 mg/kg of rosemary essential oil, respectively. In all of the experimental groups, including the control group, the cig kofte portions were stored in styrofoam containers covered with stretch wrap at 4±1 °C in a cooled incubator (ES 120, Nüve, Turkey) for 24 hours. At 0, 3, 6, 12 and 24 hours of storage, microbiological and chemical analyses (pH and water activity – aw value) were performed.

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Table 1: Proximate composition of the ingredients used in producing cig kofte (g /kg)

Enumeration of Salmonella: In each group, at 0, 3, 6, 12 and 24 hours of storage, a 25-g portion of the sample was taken under aseptic conditions, added 225 ml of sterile peptone water, and homogenized for 2 minutes (Bagmixer 400W, Interscience, France). 1/10 serial dilutions were prepared up to 1/108, and each dilution was spread in volumes of 0.1 mL onto duplicate XLD agar plates using the spread plate method, and incubated at 35 °C for 24 hours. Three colonies randomly selected from the counted plates were confirmed as Salmonella colonies, based on the demonstration of the “O” antigen with a polyvalent Salmonella antiserum¹³.

Chemical Analysis: Chemical analyses were conducted in duplicate to determine the following: salt levels determined by Mohr method^16, pH was measured using a pH meter (selecta 2001 Spain) and water activity (aw) determined using a water activity meter (Testo 650 , USA).

Experimental Design and the Statistical Analysis of Data: The trials were designed as three repetitions performed at an interval of 15 days. The data converted to log10 CFU/g was subjected to variance analysis for fixed effects and interactions between variables according to the 3x8x5 factorial design as “replication x treatment groups x sampling time to be taken at a time from each test group”. In accordance with the General Linear Model (GLM) procedures, the least square means were detected by Fisher's Least Significant Difference (LSD) test, and the level of statistical significance was set at 5%. The data was analyzed using the Statistical Analysis System software¹⁷.

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Table 2: Inactivation of Salmonella in cig kofte samples, treated with alone or combination of some essential oils and chitosan, stored at 4 °C for 24 hours (log10 CFU/g).

Water activitiy (a_w) and pH values of treatment groups and control were evaluated during storage and are shown in Table 3. No significant difference was determined between the groups, indicating that the addition of the essential oils and the chitosan did not change the pH and aw in the cig kofte (P>0.05).

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Table 3: Mean values and standard errors (SEs) of pH and aw in cig kofte samples treated with alone or combination of some essential oils and chitosan stored at 4 °C for 24 hours

The results of the present study demonstrated that, the addition of 500 mg/kg of chitosan to cig kofte (Group IV) stored at 4 °C, yielded a reduction of 0.88 log CFU/g in Salmonella numbers when compared to the control group (P<0.05). The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of chitosan for Salmonella bacteria under in vitro conditions have been reported as 288 ppm and 300 ppm, respectively (20). Furthermore, in nutrient broth, chitosan concentrations of 1.0%, 1.5%, 2.0%, and 2.5% have been reported to produce a reduction greater than 3 log in the Salmonella population, during an 8-day period at a pH value of 5.5 and a temperature of 30 °C²¹. While Simpson et al.²² reported that the addition of chitosan at a concentration level of 0.01% to shrimps was sufficient to inhibit the growth of Salmonella Typhimurium, Chhabra et al.²³ reported that the addition of chitosan at concentrations of 0.5%, 1.0%, and 2.0% to raw oysters had no effect on the inactivation of Salmonella Typhimurium throughout a 12-day storage period at 4 °C. The differences between these two studies, apart from several possible variables (storage period, matrices used in modelling, etc.), are attributed to differences in the concentration, deacetylation degree and molecular weight of the chitosan used^24,25. Some research has shown that chitosan of medium molecular weight exhibits strong antibacterial effect on S. enteritidis at low temperatures^9,25. Marques et al.²⁶ reported the MIC value of chitosan against S. enterica at 10 °C as 0.05% and indicated that chitosan had no inhibitory effect on this pathogen at 20 °C. The use of chitosan with a medium molecular weight and the storage temperature being adjusted to 4 °C in the present study, also suggest that these factors may be influential on the bioactivity of chitosan.

In the present study, the greatest reduction in the numbers of Salmonella by the end of the storage period (1.42 log CFU/g) was treated in Group V, which was administered with chitosan plus thyme essential oil (P<0.05, Table 2). This suggests that thyme essential oil and chitosan may exhibit a synergistic effect when applied in combination. This is explained by the breakdown of the outer protective membrane of the Salmonella bacteria by both thyme essential oil and chitosan, and the inhibition of bacterial growth as a result of the interference with mRNA and protein synthesis by chitosan²⁷. Similarly, some other studies have shown that, in terms of the extension of the shelf life of food products and the inactivation of pathogens in food products, the combined use of chitosan and essential oils enhances antimicrobial effect^28,29. Georgantelis et al.³⁰ reported that chitosan and rosemary essential oil showed a synergistic effect in increasing the microbial quality of pork sausages, while Kanatt et al.³¹ reported that the addition of 0.1% of mint oil and chitosan mixture to lamb meat enabled a reduction of 1 log in the number of Gram-negative bacteria, including E. coli, P. fluorescens, and Salmonella Typhimurium, and up to 2-3 log in Gram-positive bacteria, including S. aureus and B. cereus.

In the present study, the pH value was determined to range between 5.52 and 5.59 in the control group and the groups applied essential oils alone, and between 5.27 and 5.40 in the groups applied chitosan alone and chitosan in combination with essential oils (P>0.05, Table 3). Furthermore, the average NaCl (salt) level of the cig kofte samples was ascertained to be 1.87±0.43%. The pH values of the groups, which contained chitosan, being lower than that of the control group and the groups that were applied essential oils was attributed to the use of a chitosan solution prepared in acetic acid. The results of the present study contradict with those reported by Georgantelis et al.^30, indicating a higher pH value in Greek-style sausages added chitosan and rosemary extract, and comply with those reported by Giatrakou et al.²⁸ indicating a lower pH value in chicken-pepper kebab added chitosan and thyme essential oil. Generally, low pH values are associated with an increased antimicrobial activity of essential oils and chitosan^11,18. However, in the present study, of the groups, which had a low pH value and were applied chitosan, the group that was treated wiht chitosan in combination with thyme essential oil displayed the greatest reduction in Salmonella bacteria (P< 0.05).

The essential oil and the chitosan concentrations used in the present study were subjected to a sensory evaluation prior to their use (data not shown). Based on the panel results, the flavor, odor and taste of the samples, which contained 500, 750 and 1000 mg/kg of thyme, clove, rosemary essential oils and 750 and 1000 mg/kg of chitosan were disliked, while the samples contained 250 mg/kg of these oils and 500 mg/kg of chitosan were considered to be acceptable by the panelists.

The present study demonstrated that the combined use of chitosan and thyme essential oil in cig kofte samples, presenting with a high risk of cross contamination and a risk of production under unhygienic conditions, and consumed without any prior heat treatment, produced a synergistic effect against Salmonella without any adverse effect on the organoleptic properties of the product.

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