Kitosan ve Bazı Esansiyel Yağların (Kekik, Karanfil ve Biberiye) Tek Başlarına ve Kombine Halde Çiğ Köftede Salmonella Typhimurium Üzerine Etkileri

Bu araştırma, kitosan ve bazı esansiyal yağların çiğ köftede Salmonella Typhimurium bakterisinin inaktivasyonu üzerine etkilerinin belirlenmesi amacıyla gerçekleştirildi. Bu amaçla Salmonella Typhimurium ile kontamine edilen çiğ köfte; C (kontrol), I. grup (250 mg/kg kekik), II. grup (250 mg/kg karanfil), III. grup (250 mg/kg biberiye), IV. grup (500 mg/kg kitosan), V. grup (500 mg/kg kitosan + 250 mg/kg kekik), VI. grup (500 mg/kg kitosan + 250 mg/kg karanfil) ve VII. grup (500 mg/kg kitosan + 250 mg/kg biberiye) olmak üzere 8 gruba ayrıldı. 4±1 °C’de 24 saat muhafaza edilen gruplar, muhafazanın 0., 3., 6., 12. ve 24. saatlerinde Salmonella Typhimurium, pH ve su aktivitesi (a_w) yönünden analiz edildi. Muhafaza sonunda (24. saat) en fazla patojen azalması V. grupta (1.60 log₁₀ CFU/g) tespit edilirken I., II., III., IV., VI. ve VII. gruplarda ise sırasıyla 0.57, 0.55, 0.54, 0.88, 0.58 ve 0.68 log₁₀ CFU/g olarak tespit edildi.

This study was undertaken to determine the effects of chitosan and some essential oils on the inactivation of Salmonella Typhimurium bacteria in “cig kofte” (raw meatball). For this purpose, cig kofte was inoculated with Salmonella Typhimurium and divided into 8 portions, such that they were treated differently in 8 groups, including Groups C (control), I (250 mg/kg thyme essential oil), II (250 mg/kg clove essential oil), III (250 mg/kg rosemary essential oil), IV (500 mg/kg chitosan), V (500 mg/kg chitosan + 250 mg/kg thyme essential oil), VI (500 mg/kg chitosan + 250 mg/kg clove essential oil) and VII (500 mg/kg chitosan + 250 mg/kg rosemary essential oil). The eight cig kofte portions were stored at 4±1 °C for 24 hours, and analyses for the numbers of Salmonella Typhimurium, pH value, and water activity (a_w) were performed at 0, 3, 6, 12 and 24 hours of storage. At the end of the storage period (at the 24^th hour), the highest level of pathogen reduction was determined in group V (1.60 log₁₀ CFU/g), while the level of pathogen reduction in groups I, II, III, IV, VI and VII were determined as 0.57, 0.55, 0.54, 0.88, 0.58 and 0.68 log₁₀ CFU/g respectively.

Giriş

“Cig kofte (raw meatball)” is a popular traditional appetizer in Turkey and some Middle East countries. This dish is prepared by kneading finely ground lean beef, cracked wheat, chopped onions, garlic, spices (red pepper, black pepper, cumin and pimento, etc.) salt and tomato paste. The amount and variety of additives used in the preparation of this dish varies, as no standard exists for cig kofte. Cig kofte is usually consumed shortly after preparation, but can be stored in the refrigerator for up to 24 hours[1]. According to Turkish Food Codex of 2013, the use of ground meat and all food additives except citric acid is prohibited in cig köfte[2]. However, some particular restaurants and some local sale points or homemade cig kofte can still be made with raw ground meat.

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 spices[3]. 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 shrimps[9]. 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 Administration[10]. 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 ppm[11]. 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 FDA[10]. Previous research has shown that, apart from their antimicrobial property, essential oils also exhibit antibacterial and antifungal affects both in vitro and in food[12]. 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 products[15].

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.

Gereç ve Yöntem

Chitosan and Essential Oils: Chitosan (Sigma C3646, ≥75 % deacetylated) was purchased from Sigma Company (St. Louis, MO, USA). Stock solution of chitosan (1%) was prepared in 1% acetic acid (food grade). Essential oils were purchased from the Kalsec Company (Kalamazoo, Mich.) which supplies food grade oils. Undiluted 100% oil of clove, and oil of thyme, and oil of rosemary, were used.

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.

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 antiserum[13].

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 software[17].

Bulgular

Changes of the Salmonella numbers in cig kofte samples during storage treated with some essential oils and chitosan are shown in Table 2. Initial number of the Salmonella was determined to be between 7.50 and 7.60 log CFU/g in control and treatment groups. The pathogen number in the treatment groups were decreased during the storage, whereas the control group had a slight increase. However, no significant difference was observed between group I, II, III, VI, VII and the control group during the storage (P>0.05). A significant difference was observed between group IV, V and control group from 12^th hours to the end of storage period (P<0.05). At the end of the storage period (at the 24^th hour), highest level of the pathogen reduction was determined in group V (1.60 log₁₀ CFU/g), but the other treatment groups were below the 1 log of reduction. A significant difference was found only within the group V at the end of storage (P<0.05) except in the other treatment groups (P>0.05).

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).

Tartışma

In recent years, the demand for ready-to-eat products, including meat and meat products, has increased and is on the rise. Of such products, cig kofte has a particular significance due to its risk of being contaminated with pathogens, including Listeria monocytogenes, Salmonella spp., and E. coli 0157:H7, and thus because of the risk it poses for food safety and public health. In this context, cig kofte belongs to the group of ready-to-eat food products with the highest risk, as it is consumed without any prior heat treatment. The very few studies conducted on the viability of Salmonella bacteria in cig kofte have demonstrated that these bacteria may remain viable within the consumption period (up to 24 hours) of the product[13,14]. Uzunlu and Yıldırım[6] determined that in cig kofte samples contaminated with Salmonella enteritidis and stored at 4 °C for 24 hours, the Salmonella levels at 0 and 24 hours of storage were 3.57 log CFU/g and 3.54 log CFU/g, respectively. Similarly, in the present study, the Salmonella typhimurium level determined as 7.60 log CFU/g in the control group at 0 hours was ascertained to have increased to 7.68 log CFU/g by the end of the 24^th hour of storage (Table 2). These results clearly suggest that the ingredients of cig kofte have bacteriostatic effect on Salmonella bacteria. On the other hand, these results also show that the spices added to cig kofte did not suffice in eliminating Salmonella typhimurium from the product. However, essential oils (i.e. thyme essential oil, clove essential oil, rosemary essential oil, sage essential oil) obtained from aromatic plants (i.e. spices) and compounds found in aromatic plants (i.e. thymol, eugenol, carvacrol) display strong antibacterial effect against foodborne pathogens[12]. In the present study, when added to cig kofte samples at a concentration level of 250 mg/kg, thyme, clove and rosemary essential oils (in Groups I, II and III) did not produce any significant inhibitory effect on Salmonella typhimurium throughout the storage period, yet it was shown that the addition of these essential oils to cig kofte reduced the bacterial growth rate (P>0.05). In previous laboratory studies carried out on the antibacterial effect of spice extracts, although these extracts were determined to significantly reduce pathogen levels even when applied at concentrations below 0.1%, it has been underlined that, to ensure such an effect in food, depending on the particular essential oil or compound used, much higher concentrations are required to be applied[18]. In a study in which eugenol, linalool, carvone, cineole and limonene oil were each added separately to cig kofte samples at a concentration level of 1.8%, Çalıcıoğlu and Dikici[13] determined the reduction in the number of Salmonella bacteria, enabled by these compounds, at 3 hours as 6.5, 2.54, 1.81, 0.92 and 0.25 log CFU/g, respectively. Both these results and the observation of the addition of eugenol at concentrations of 0.5%, 1% and 1.5% resulting in a reduction of approximately 1, 2 and 3 log CFU/g; respectively, are in support of the need for higher concentrations in food. In general, literature reports indicate that Gram-negative bacteria are more resistant to essential oils than Gram-positive bacteria[12]. While the resistance of Gram-negative bacteria has been explained by these bacteria possessing an outer membrane composed of lipopolysaccharides and proteins, some other research has shown that Gram-positive bacteria may also be resistant to the effects of essential oils[14,19].

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 °C[21]. 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 chitosan[27]. 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.

References

1. Durmaz H, Sagun E, Sancak H, Sagdic O. The fate of two Listeria monocytogenes serotypes in “cig kofte” at different storage temperatures. Meat Sci 2007; 76, 123-127.
2. Tukish Food Codex. Revised Turkish Food Codex Regulation on Food Additives Official Gazette no. 28693 dated 06/30/2013.
3. Erol I, Kuplulu O, Karagoz S. Microbiological quality of some spices consumed in Ankara. Ankara Univ Vet Fak 1999; 46: 115–126.
4. Cetin O, Bingol EB, Akaya H. The microbiological, serological and parasitological quality of cig kofte (raw meatball) and its lettuce marketed in Istanbul. Pol J Environ Stud 2008; 17: 701-706.
5. Küplülü Ö, Sarımehmetoğlu B, Oral N. The microbiological quality of cig kofte sold in Ankara. Turk J Vet Anim Sci 2003; 27: 325-329.
6. Uzunlu S, Yıldırım I. Microbiolgical quality of raw meat ball and investigation of its microbial variation at the different storage time and temperature. Gıda 2003; 28: 553-558.
7. Cetinkaya F, Mus TE, Cibik R, Levent B, Gulesen R. Assessment of microbiological quality of cig kofte (raw consumed spiced meatball): Prevalence and antimicrobial susceptibility of Salmonella. Food Control 2012; 26: 15-18.
8. Tuncel G, Tiryaki G. Terms of food safety assessment of raw meatballs. Dünya Gıda 2001; 12: 55-61.
9. Roller S, Covill N. The antimicrobial properties of chitosan in mayonnaise and mayonnaise-based shrimp salads. J Food Prot 2000; 63: 202-209.
10. US FDA (US Food and Drug Administration) “GRAS notifications (2005)”. http://www.fda.gov/30 March 2014.
11. Helander IM, Nurmiaho-Lassila E-L, Ahvenainen R, Rhoades J, Roller S. Chitosan disrupts the barrier properties of the outer membrane of Gram-negative bacteria. Int J Food Microbiol 2001; 71: 235-244.
12. Burt S. Essential oils: their antibacterial properties and potential applications in foods–a review. Int J Food Microbiol 2004; 94: 223-253.
13. Çalıcıoğlu M, Dikici A. Effect of essential oils on inactivation of Salmonella in cig kofte. Fırat Üniversitesi Sağlık Bilimleri Dergisi 2008; 22: 283-288.
14. Dikici A, Ilhak OI, Çalicioğlu M. Effects of essential oil compounds on survival of Listeria monocytogenes and Escherichia coli O157:H7 in cig kofte. Turk J Vet Anim Sci 2013; 37: 177-182.
15. Holley RA, Patel D. Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiol 2005; 22: 273-292.
16. Official Methods of Analysis of the Association of Official Analytical Chemists. 15th edition, Association Official Analytical Chemists (AOAC) (1990)
17. SAS. Statistical Analyses System Inst. Inc. Cary. 6.1. Version, North Caroline, USA (1999)
18. Gutierrez J, Barry-Ryan C, Bourke P. The antimicrobial efficacy of plant essential oil combinations and interactions with food ingredients. Int J Food Microbiol 2008; 124: 91-97.
19. Oussalah M, Caillet S, Saucier L, Lacroix M. Inhibitory effects of selected plant essential oils on the growth of four pathogenic bacteria: E. coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus and Listeria monocytogenes. Food Control 2007; 18: 414-420.
20. Islam, MM, Masum SM, Mahbub KR. In vitro antibacterial activity of shrimp chitosan against Salmonela paratyphi and Staphylococcus aureus. Journal of the Bangladesh Chemical Society 2011; 24: 185-190.
21. Wang GH. Inhibition and inactivation of five species of foodborne pathogens by chitosan. J Food Prot 1992; 55: 916-919.
22. Simpson BK, Gagne N, Ashie INA, Noroozi E. Utilization of chitosan for preservation of raw shrimp (Pandalus borealis). Food Biotechnol 1997; 11: 25-44.
23. Chhabra P, Huang YW, Frank JF, Chmielewski R, Gates K. Fate of Staphylococcus aureus, Salmonella enterica serovar typhimurium, and Vibrio vulnificus in raw oysters treated with chitosan. J Food Prot 2006; 69: 1600-1604.
24. Kong M, Chen XG, Xing K, Park HJ. Antimicrobial properties of chitosan and mode of action: A state of the art review. Int J Food Microbiol 2010; 144: 51-63.
25. No HK, Kim SH, Lee SH, Park NY, Prinyawiwatkul W. Stability and antibacterial activity of chitosan solutions affected by storage temperature and time. Carbohydr Polym 2006; 65: 174-178.
26. Marques A, Encarnaçao E, Pedro S, Leonor Nunes M. In vitro anti-microbial activity of garlic, oregano and chitosan against Salmonella enterica. World J Microbiol Biotechnol 2008; 24: 2357-2360.
27. Zivanovic S, Basurto CC, Chi S, Davidson PM, Weiss J. Molecular weight of chitosan influences antimicrobial activity in oil-in-water emulsions. J Food Prot 2004; 67: 952-959.
28. Giatrakou V, Kykkidou S, Papavergou A, Kontominas MG, Savvaidis IN. Potential of regano essential oil to extend the shelf-life of fresh swordfish. A comparative study with ice storage. Journal of Food Sci 2008; 73: 167-173.
29. Ojagh SM, Rezaei M, Razavi SH, Hosseini SMH. Effect of chitosan coatings enriched with cinnamon oil on the quality of refrigerated rainbow trout. Food Chem 2010; 120: 193-198.
30. Georgantelis D, Ambrosiadis I, Katikou P, Blekas G, Georgakis SA, Effect of rosemary extract, chitosan and α-tocopherol on microbiological parameters and lipid oxidation of fresh pork sausages stored at 4 °C. Meat Sci 2007; 76: 172-181.
31. Kanatt SR, Chander R, Sharma A. Chitosan and mint mixture: A new preservative for meat and meat products. Food Chem 2008; 107: 845-852.