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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Effect of chitosan on spoilage bacteria, Escherichia coli and Lissteria monocytogenes in cured chicken meat
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Seyed Shahram Shekarforoush ∗ , Sara Basiri, Hadi Ebrahimnejad, Saeid Hosseinzadeh Department of Food Hygiene and Public Health, School of Veterinary Medicine, Shiraz University, Shiraz 71441-69155, Iran
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Article history: Received 21 January 2015 Received in revised form 23 February 2015 Accepted 23 February 2015 Available online xxx
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Keywords: Listeria monocytogenes E. coli O157:H7 Oregano Chitosan Chicken meat
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1. Introduction
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The effects of essential oil (EO) of oregano and chitosan on the microbial quality and growth inhibition of Listeria monocytogenes and Escherichia coli O157:H7 in the Iranian traditional ready-to-barbecue chicken was evaluated. Thus, three groups of samples were prepared. One of them was considered to evaluate for aerobic plate count (APC), lactic acid bacteria (LAB), psychrophilic and enterobacteriacae counts and the other two groups were inoculated with E. coli O157:H7 and L. monocytogenes 4b to investigate the effect of oregano EO and chitosan on pathogenic bacteria. All groups were stored at 3, 8 and 20 ◦ C. Oregano showed antibacterial effects against APC, LAB, psychrophilics, enterobacteriacae and E. coli O157:H7, whereas, such an effect was not observed against L. monocytogenes. Chitosan individually did not show an inhibitory effect on the spoilage-inducing bacteria and E. coli, but was effective against L. monocytogenes. Using chitosan and oregano EO in combination can reduce the number of spoilage and safety indicators and also the two food-borne pathogens in ready-to-barbecue chicken meat. © 2015 Published by Elsevier B.V.
Food decomposition is a metabolic process which may cause some undesirable organoleptic changes in food, even though decomposed food is not a cause of food borne illness, the changes in its appearance, taste or consistency are matters that affect the retail value of the food. Various microorganisms are responsible for the decomposition of food products which may induce food poisoning as well [1]. L. monocytogenes and E. coli O157:H7 are considered as two major food-borne pathogens. Although Listeria can spread via direct contact or congenital routes, the use of food is nonetheless considered to be the primary route of infection. This microorganism can survive at 2–4 ◦ C and also in frozen foods [2]. Due to thehigh mortality rates for cases of severe food poisoning and its psychrophilic characterization [3], Listeria is considered as an important foodborne pathogen during the last decade [4]. As a case to verify this, the Canadian listeriosis outbreak was a widespread outburst of the food infection in which twenty people died out of 56 total confirmed cases [5]. E. coli O157: H7 is also discussed as an important fecal bacterium which may spread through direct contact and consumption of contaminated food and can survive at 15% concentration of acetic,
∗ Corresponding author. Tel.: +98 71 32286950; fax: +98 71 32286940. E-mail address: [email protected] (S.S. Shekarforoush).
citric and lactic acids [1]. Consumption of undercooked meat [6] and bovine raw milk [7] are the main sources of several outbreaks. E. coli O157:H7 can lead to hemorrhagic colitis, hemolytic uremic syndrome, and thrombocytopenic purpura, and severe cases, death [8]. Enterohaemorrhagic E. coli O157:H7 was first implicated in human disease in the early 1980s, with the ruminants cited as the primary reservoirs. Preliminary studies indicated cattle to be the sole source of E. coli O157:H7 outbreaks in humans, however, further, epidemiological studies have demonstrated that E. coli O157:H7 was widespread in other food sources and that a number of transmission routes existed. Chemical preservatives are currently replaced by natural ones in food stuffs due to the various side effects on consumers [9,10]. Efficacy of essential oil (EO) of oregano (Origanum vulgare) has been previously addressed. Elgayyar et al. (2001) reported the antimicrobial effects of the EO against L. monocytogenes and Staphylococcus aureus [9]. Antonio et al. (2008) showed strong inhibitory effects of three substances, garlic, oregano and chitosan, against Salmonella enterica, at low temperatures [11]. For instance, as a result of delay in the growth of microorganisms and physiochemical changes of meat samples, the extension of shelf life due to the synergistic effects of oregano (EO) and the modified atmosphere changes, was formerly demonstrated [12]. Chitosan, a natural and biodegradable biopolymer was originated from crab shells, is currently approved by the United States Food and Drug Administration (FDA). Different kinds of chitosan
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are available commercially giving a chance to use them as a popular natural preservative [13]. The antimicrobial activity of chitosan increases at pH less than six due to the positive charge of its glucosamine monomers [14]. Compared to the frozen ready-to-barbecue chicken meat, fresh meat has greater retail value. In these circumstances, using natural antimicrobial components such as chitosan and EO can potentially reduce the risk factors associated with fresh poultry meat. In recent years, many researchers are focused on the organoleptic changes coming simultaneously by using natural preservatives [15,16]. The objective of the present study was to further shed light on the antibacterial effects of chitosan and the oregano EO against the main food spoilage inducer microorganisms with a special spotlight on two main food-borne pathogens in the Iranian readyto-barbecue chicken meat.
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2. Materials and Methods
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2.1. Bacterial strains and preparation
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A proven strain of L. monocytogenes 4b obtained from Razi Institute, Hesarak-Karaj, Iran; and E. coli O157:H7 (ATCC, 43895) was provided by Department of Microbiology, School of Veterinary Medicine, Shiraz University.
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2.2. Production of nalidixic acid-resistant strain of E. coli O157:H7
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The nalidixic acid-resistant E. coli O157:H7 was prepared, as previously described and employed to easy differentiation from background bacteria [17]. Briefly, the wild-type strain of E. coli O157:H7 was inoculated into 100 ml Trypticase soy broth (TSB, Merck, Germany) and incubated at 37 ◦ C for 24 h. A total of 100 ml TSB containing 200 g/ml nalidixic acid (1451000, Sigma-Aldrich, St. Louis, USA) were added to the incubated culture, which was subsequently incubated at 37 ◦ C for a further 24 h. After incubation, 0.1 ml of the culture was spread on the surface of a Trypticase soy agar plate (TSA, Merck, Germany) containing 100 g/ml nalidixic acid and incubated at 37 ◦ C for 24 h. A single colony was selected and re-streaked onto another TSA plate in order to confirm its resistance to nalidixic acid. 2.3. Essential oil of oregano
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The pure EO of oregano (density: 0.960 at 20 ◦ C; tested by gas chromatography–mass spectrometry [GC–MS]; origin: Bulgaria; steam distillation extraction) was obtained from Kobashi Company (Ide, Devon-shire, UK).
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The low molecular weight chitosan (product number: 448869) was obtained from Sigma-Aldrich Compan, St. Louis, USA.
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2.5. Preparation of chicken meat
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Iranian ready-to-barbecue chicken meat cubes were prepared according to the local chicken meat processing industries. Briefly, salt (4.7 g), red pepper (1.4 g), 1% acetic acid (50 ml), chopped onion (47 g), saffron (0.1 g) and sunflower oil (20 ml) were added to 1000 g chicken meat. According to the recipe, 4 kg of breast meat was cut into 1 cm3 and mixed with the ingredients excluding acetic acid and vegetable oil. The mixture was then divided into four portions and treated as follows: Group 1 (control): 50 ml acetic acid 1% and 20 ml sunflower oil. Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2015.02.033
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Group 2 (chitosan): 50 ml chitosan solution 2% in acetic acid 1% and 20 ml sunflower oil. Group 3 (oregano): 50 ml acetic acid 1% and 20 ml oregano EO 15% in sunflower oil. Group 4 (chitosan + oregano): 50 ml chitosan solution 2% in acetic acid 1% and 20 ml oregano EO 15% in sunflower oil. 2.6. Effects of chitosan and EO of oregano on microbial quality of ready-to-barbecue chicken Each pack of the sample was divided into 10 g portions in a stomacher bag and stored at 3 ± 1, 8 ± 1 and 20 ± 1 ◦ C for further use. Aerobic plate count (APC), psychrophilic count, lactic acid bacterial (LAB) and enterobacteriacae counts were performed at different storage conditions. The content of the bags was then diluted in 90 ml of 0.1% peptone water and homogenized for 2 min using the stomacher (Seward, BA6021, UK). Homogenates were then ten-fold serially diluted in the peptone water. Plate count agar (Merck, Germany) was used to count APC and psychrophilic bacteria. Man Rogosa Sharpe Agar (MRSA; Merck, Germany) and Violet Red Bile Glucose Agar (VBRGA; Merck, Germany) were employed to count LAB and enterobacteriacae, respectively. To count APC and psychrophilic bacteria, plates were respectively incubated at 37 ◦ C for 48 h and 10 ◦ C for 7 days. MRS agar was incubated at 25 ◦ C for 3 days and VRBGA was also incubated at 37 ◦ C for 24 h. All colonies were finally, enumerated and the results were represented as CFU/g. For each condition, three independent samples were analyzed in duplicate. 2.7. Effects of chitosan and EO of oregano on the survival of E.coli O157:H7 and L. monocytogenes in ready-to-barbecue chicken To establish a correlation between the colony forming unit (CFU)/ml and absorbance of the dilutions of the resistant strain of E. coli O157: H7 and L. monocytogenes at 600 nm, a standard curve were prepared. The bacteria were grown in TSA at 37 ◦ C for 24 h. A single colony from the TSA plates were inoculated into 10 ml of TSB and were incubated overnight at 37 ◦ C. The bacterial suspension were pelleted three times by centrifugation at 3000 × g for 20 min, and washed with sterile normal saline. Final cell pellets were resuspended in 10 ml of sterile normal saline. Appropriate serial dilutions were made and their absorbances were measured at 600 nm. To determine the log10 CFU/ml corresponding to the absorbance of that same dilution, viable cell counts were performed in duplicate by plating serial dilutions onto TSA and incubating at 37 ◦ C for 24 h. Prior to inoculating the bacterial culture into the samples, the absorbance of inocula were matched to the standard curve and if necessary, the density of the bacterial culture were adjusted by adding sterile normal saline. One ml of 1 × 107 CFU/ml of each bacterial suspension was added to 1 kg of each experimental group, which were subsequently massaged manually for 2 min. The samples were finally divided into 10 g portions and kept at 3 ± 1, 8 ± 1 and 20 ± 1 ◦ C, for further use. To determine the bacterial counts in different storing conditions, the contents of each 10 g sample was homogenized using stomacher for 2 min and ten-fold serially diluted in the 0.1% buffered peptone water. 0.1 ml of each dilution was spread on Palcam agar (Merck, Germany) containing 5 mg/L acriflavin and 40 mg/L nalidixic acid and McConkey agar media (Merck, Germany) containing 40 mg/L nalidixic acid that were used to count of L. monocytogenes and E. coli, respectively. The plates were finally incubated at 37 ◦ C for 48 h (to grow L. monocytogenes) and at 37 ◦ C for 24 h (to grow E. coli O157: H7). The plates counted accordingly [18].
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Fig. 1. Bacterial growth in ready-to-barbecue chicken affected by oregano EO and chitosan stored at 3 ◦ C. A. Aerobic plate count, B. psychrophilic count, C. lactic acid bacteria, D. enterobacteriacae. Treatment groups: control (), 1 mg/g chitosan (), 3 l/g oregano EO (), 1 mg/g chitosan + 3 l/g oregano EO (䊉). Different letters means the significant differences within each time point (P < 0.05).
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For each condition, three independent samples were analyzed in duplicate.
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3. Results
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3.1. Samples stored at 3 ◦ C for 192 h
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3.2. Samples stored at 8 ◦ C for 72 h
The number of CFU was initially converted into log10 and analyzed using ANOVA and the general linear model procedure using SPSS, version 16 (SPSS, Chicago, Ill.). The Scheffe post hoc test was used to determine if any significant differences existed among logs CFU/g of bacteria.
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APC was increased to 0.34 and 1.0 logs in the oregano and control groups, respectively (P < 0.05). The number of APC was not affected by chitosan (P > 0.05). Number of APC in oregano + chitosan treatment group decrease from 4.46 to 4.35 CFU/g during the storage period which was significantly different from the other treatments (P < 0.05) (Fig. 1A). Growth of psychrophilic bacteria and enterobacteriacae were not affected by EO of oregano and chitosan (P > 0.05) (Figs. 1B and D). The population of LAB was increased to 5.44 and 5.49 logs CFU/g after 192 h in the control and chitosan groups, respectively (P > 0.05). They were 4.54 and 3.92 logs CFU/g for oregano and Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2015.02.033
chitosan + oregano groups, which were significantly lower than control and chitosan groups (P < 0.05) (Fig. 1C).
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During the storage period, growth of APC, psychrophilic bacteria and enterobacteriacae were not inhibited by oregano EO, chitosan and their combinations (P > 0.05) (Fig. 3A, 3B and 3D). Oregano EO and chitosan were not influenced the growing of LAB, whilst et
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Following 72 h incubation, APC increased to 4.83, 4.86, 4.45 and 4.09 logs CFU/g, in the control, chitosan, oregano and chitosan + oregano groups, respectively. At the end of the storage period, the number of APC in the oregano + chitosan group was significantly lower than other treatments (P < 0.05) (Fig. 2A). Chitosan had no significant effect on psychrophils at 8 ◦ C for 72 h (P > 0.05). However, in the oregano group, number of psychrophils increased by 0.68 logs after 72 h incubation, which was significantly lower than the control and chitosan groups (P < 0.05) (Fig. 2B). Oregano EO and chitosan have not shown any inhibitory effects on the LAB (P > 0.05), whereas, the combinations showed considerable effects on the growing rates (P < 0.05) (Fig. 2C). Following 72 h incubation, numbers of enterobacteriacae were reduced by up to 0.44 and 0.46 logs in the oregano and chitosan + oregano groups, respectively. The inhibitory effect was only induced by oregano EO (P < 0.05) (Fig. 2D).
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Fig. 2. Bacterial growth in ready-to-barbecue chicken affected by oregano EO and chitosan stored at 8 ◦ C. A. Aerobic plate count, B. psychrophilic count, C. lactic acid bacteria, D. enterobacteriacae. Treatment groups: control (), 1 mg/g chitosan (), 3 l/g oregano EO (), 1 mg/g chitosan + 3 l/g oregano EO (䊉). Different letters means the significant differences within each time point (P < 0.05).
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their combinations showed a significant inhibitory effect (P < 0.05) (Fig. 3C).
was observed for just 24 h of storage (P < 0.05), even though no synergistic effects were evidenced (P > 0.05) (Table 2).
3.4. Effects of chitosan and EO of oregano on survival of E.coli O157:H7 in ready-to-barbecue chicken
4. Discussion
Following 144 h storage at 3 ◦ C, Log CFU/g of E. coli was decreased from 4.55 to 4.09, 3.98, 3.47 and 3.34 in the control, chitosan, oregano and chitosan + oregano groups, respectively. In contrast to chitosan, the survival of bacterium was inhibited by oregano EO (P < 0.05). Furthermore, the inhibitory effect of oregano was not enhanced by chitosan (P > 0.05). The same results were also demonstrated in the samples stored at 8 ◦ C and 20 ◦ C (Table 1). 3.5. Effects of chitosan and EO of oregano on the survival of L. monocytogenes in ready-to-barbecue chicken The treated samples kept at 3 ◦ C for 144 h revealed that using oregano and chitosan separately or in conjunction, did not show any inhibitory effects (P > 0.05) (Table 2). No inhibitory effects were shown, when the samples incubated with oregano at 8 ◦ C for 72 h (P > 0.05). Surprisingly, using chitosan separately was effective against L. monocytogenes (P < 0.05) but its effect was not increased when it used synergistically with oregano (P > 0.05). Moreover, when the samples were stored at 20 ◦ C, chitosan was effective until 48 h of storage. In this case the number of bacteria increased to 8.09 log CFU/g, while, it was 8.54 log CFU/g in the control group (P < 0.05). The inhibitory effect of oregano EO Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2015.02.033
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Oregano, as a natural food additive with proven antimicrobial effects, has been demonstrated to inhibit the growth of spoilage inducer and food-borne pathogenic bacteria [19,20]. The antimicrobial activity of oregano has been attributed mainly to the presence of volatile compounds found in its EO, especially carvacrol and thymol [21]. In the current study, we have demonstrated the antibacterial effects of oregano EO against APC and LAB (at 3 ◦ C), psychrophilic bacteria and enterobacteriacae (at 8 ◦ C), and E. coli (at 3, 8 and 20 ◦ C). Even though, the antimicrobial effects of oregano against L. monocytogenes have already shown, in vitro [22], no considerable effects were proven in barbecued chicken. It has been verified that the presence of some nutrients such as carbohydrates, proteins and salts and pH may influence the efficacy of EOs in foods [23–26]. The physical structure [27], lower water activity [28], adipose components [29] of food stuff may reduce the antimicrobial effects of EOs. This phenomenon might be due to the absorption of some antimicrobial components by proteins or adipose tissues [26]. In the present study, we have shown the antimicrobial effects of oregano against spoilage-inducing bacteria in the samples kept at 3 and 8 ◦ C. In contrast, no antimicrobial effects were revealed at 20 ◦ C. These findings may suggest that increasing, storing temperature
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Fig. 3. Bacterial growth in ready-to-barbecue chicken affected by oregano EO and chitosan stored at 20 ◦ C. A. Aerobic plate count, B. psychrophilic count, C. lactic acid bacteria, D. enterobacteriacae. Treatment groups: control (), 1 mg/g chitosan (), 3 l/g oregano EO (), 1 mg/g chitosan + 3 l/g oregano EO (䊉). Different letters means the significant differences within each time point (P < 0.05).
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induces bacterial growth and dramatically decreases the shelf life of foods. Although using chitosan and oregano showed substantial effects on the growth of decomposer microorganisms, chitosan individually as a single dose was not effective. Additionally, chitosan was not effective against enterobacteriacae count and E. coli O157: H7 for all storage temperatures and in the samples inoculated with L. monocytogenes; chitosan was only effective at 3 and 8 ◦ C, in the absence of oregano EO. Tsai et al.
(2006) demonstrated that chitosan is more effective at lower temperatures [30]. In the food covered by chitosan film, an overwhelming efficacy against L. monocytogenes and Staphylococcus aureus were observed [31]. The antimicrobial characteristics of chitosan were also confirmed against L. monocytogenes in fried cattle meats when the load of the bacteria was less than 6.5 log10 CFU/g, moreover, chitosan coats on the meats, reduced the number of colonies to 2–3 log10 CFU/g [32].
Table 1 Survival and growth of E. coli O157:H7 in ready-to-barbecue chicken affected by oregano essential oil and chitosan stored at different temperature. Temp. ◦ C
Treatments
Mean (±SD) population recovered (Log CFU/g)a 0h
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Con Chi Org Chi + Org con Chi Org Chi + Org con Chi Org Chi + Org
4.55 4.55 4.55 4.55 4.41 4.41 4.41 4.41 4.06 4.06 4.06 4.06
24 h
48 h
4.29 4.40 4.16 3.89 7.11 7.04 7.14 6.97
4.21 4.13 3.90 3.75 4.80 4.79 4.48 4.37 8.83 8.84 8.53 8.37
± 0.05a ± 0.05a ± 0.05a ± 0.05a ± 0.05a ± 0.05a ± 0.05a ± 0.05a ± 0.06a ± 0.06a ± 0.06a ± 0.06a
± ± ± ± ± ± ± ±
0.13a 0.04a 0.02b 0.07b 0.02a 0.03a 0.02a 0.09a
72 h ± ± ± ± ± ± ± ± ± ± ± ±
0.02a 0.14a 0.12b 0.09b 0.11a 0.11a 0.10b 0.03b 0.17a 0.16a 0.06b 0.21b
96 h 3.89 4.08 3.66 3.62
4.90 4.93 4.13 4.23 8.88 8.95 8.57 8.53
± ± ± ± ± ± ± ±
144 h ± ± ± ±
0.08a 0.21a 0.07b 0.11b
4.09 3.98 3.47 3.34
± ± ± ±
0.24a 0.04a 0.02b 0.08b
0.17a 0.02a 0.04b 0.16b 0.15a 0.24a 0.08b 0.15b
n = 3 at each time point. The different letter means the significant differences within each temperature, time point (P < 0.05). Con = control; Chi = chitosan (1 mg/g); Org = oregano essential oil (3 l/g); Chi + Org = Chitosan (1 mg/g) + oregano essential oil (3 l/g).
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Table 2 Survival and growth of Listeria monocytogenes in ready-to-barbecue chicken affected by oregano essential oil and chitosan stored at different temperature. Temp. ◦ C
Treatments
Mean (±SD) population recovered (Log CFU/g)a 0h
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8
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Con Chi Org Chi + Org Con Chi Org Chi + Org Con Chi Org Chi + Org
4.69 4.69 4.69 4.69 4.76 4.76 4.76 4.76 4.89 4.89 4.89 4.89
24 h ± ± ± ± ± ± ± ± ± ± ± ±
0.15a 0.15a 0.15a 0.15a 0.04a 0.04a 0.04a 0.04a 0.06a 0.06a 0.06a 0.06a
48 h 3.81 3.63 3.61 3.75 4.80 4.38 4.71 4.46 8.51 8.33 8.56 8.17
4.72 ± 0.08 a 4.23 ± 0.05 b 4.62 ± 0.10 a 4.26 ± 0.19 b 6.10 ± 0.08 a 5.98 ± 0.18 a 5.82 ± 0.13 a 6.04 ± 0.11 a
72 h ± ± ± ± ± ± ± ± ± ± ± ±
0.19a 0.16a 0.12a 0.29a 0.08a 0.03b 0.06a 0.11b 0.01a 0.06a 0.05a 0.01b
96 h 3.61 3.55 3.62 3.36
4.73 4.28 4.75 4.29 8.56 8.52 8.66 8.68
± ± ± ± ± ± ± ±
144 h ± ± ± ±
0.17a 0.09a 0.05a 0.43a
3.40 3.46 3.32 3.25
± ± ± ±
0.47a 0.11a 0.31a 0.14a
0.12a 0.29b 0.03a 0.17b 0.14a 0.01a 0.06a 0.03a
n = 3 at each time point. The different letter means the significant differences within each temperature, time point (P < 0.05). Con = control; Chi = chitosan (1 mg/g); Org = oregano essential oil (3 l/g); Chi + Org = chitosan (1 mg/g) + oregano essential oil (3 l/g).
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Zheng and Zhu (2003) found the effectiveness of chitosan (with the molecular weight of less than 300 KDa) against Staphylococcus aureus [33]. Bacterial strain, incubation temperature and food substrate are the main variables greatly determine the antimicrobial activity of chitosan [34]. Previous works on chitosan revealed that gram negative bacteria seemed to be more resistance against chitosan probably due to their outer membrane [13,35]. In the case of using chitosan as a protective coverage layer in cheese, 100 and 77% effectiveness was shown against Gram positive bacteria and Pseudomonas aeruginosa, respectively [31]. The antimicrobial films using incorporating acetic or propionic acid into a chitosan matrix were shown to be effective against surface spoilage bacteria, such as enterobacteriacae, and Serratia [36]. In the in vitro inoculation of shrimp, more than 90% reduction of Vibrio parahaemolyticus was achieved by chitosan. Chaiyakosa et al. (2007) suggested that chitosan might be a suitable replacement for chlorine, which is extensively used in the frozen shrimp industries and causes severe respiratory tract damage [37]. Chitosan exists in two water soluble/insoluble forms. It has been suggested that the water soluble form is more effective which might be due to the production of a non-permeable coat around the bacterial cells [38]. In the current study, we have demonstrated the synergistic antibacterial effects of chitosan and EO of oregano against spoilage inducers in chicken meat stored at 3 and 8 ◦ C. Burt et al. (2005) showed that stabilizers significantly improved the effectiveness of carvacrol against E. coli O157:H7 in broth and they concluded that stabilizers delay the separation of the hydrophobic substrate from the aqueous phase of the medium [19]. Due to the stabilizing and emulsifying nature of chitosan [39], increases in the efficacy of EOs can be evident when used synergistically. From a practical point of view, the results showed an important antimicrobial effect of chitosan and oregano on microbial quality, E. coli O157:H7 and L. monocytogenes 4b on ready-to-barbecue poultry meat. The combined use of chitosan and EO of oregano can also reduce the number of spoilage and safety indicators and also the two food-borne pathogenic bacteria in ready-to-barbecue chicken meat. From a practical point of view, the results showed that the combined use of chitosan and EO of oregano can reduce the number of spoilage and safety indicator bacteria and also E. coli O157:H7 and L. monocytogenes 4b in ready-to-barbecue chicken meat.
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This research was financially supported by “Natural Antimicrobials Centre of Excellence (NACE)” which is gratefully Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2015.02.033
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acknowledged. We would like to thank Miss M. Aghazi and Mr. G. Niknia for their technical assistance and Miss Z. Shekarforoush for revising the manuscript.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Effect of chitosan on spoilage bacteria, Escherichia coli and Lissteria monocytogenes in cured chicken meat
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Seyed Shahram Shekarforoush ∗ , Sara Basiri, Hadi Ebrahimnejad, Saeid Hosseinzadeh Department of Food Hygiene and Public Health, School of Veterinary Medicine, Shiraz University, Shiraz 71441-69155, Iran
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Article history: Received 21 January 2015 Received in revised form 23 February 2015 Accepted 23 February 2015 Available online xxx
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Keywords: Listeria monocytogenes E. coli O157:H7 Oregano Chitosan Chicken meat
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1. Introduction
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The effects of essential oil (EO) of oregano and chitosan on the microbial quality and growth inhibition of Listeria monocytogenes and Escherichia coli O157:H7 in the Iranian traditional ready-to-barbecue chicken was evaluated. Thus, three groups of samples were prepared. One of them was considered to evaluate for aerobic plate count (APC), lactic acid bacteria (LAB), psychrophilic and enterobacteriacae counts and the other two groups were inoculated with E. coli O157:H7 and L. monocytogenes 4b to investigate the effect of oregano EO and chitosan on pathogenic bacteria. All groups were stored at 3, 8 and 20 ◦ C. Oregano showed antibacterial effects against APC, LAB, psychrophilics, enterobacteriacae and E. coli O157:H7, whereas, such an effect was not observed against L. monocytogenes. Chitosan individually did not show an inhibitory effect on the spoilage-inducing bacteria and E. coli, but was effective against L. monocytogenes. Using chitosan and oregano EO in combination can reduce the number of spoilage and safety indicators and also the two food-borne pathogens in ready-to-barbecue chicken meat. © 2015 Published by Elsevier B.V.
Food decomposition is a metabolic process which may cause some undesirable organoleptic changes in food, even though decomposed food is not a cause of food borne illness, the changes in its appearance, taste or consistency are matters that affect the retail value of the food. Various microorganisms are responsible for the decomposition of food products which may induce food poisoning as well [1]. L. monocytogenes and E. coli O157:H7 are considered as two major food-borne pathogens. Although Listeria can spread via direct contact or congenital routes, the use of food is nonetheless considered to be the primary route of infection. This microorganism can survive at 2–4 ◦ C and also in frozen foods [2]. Due to thehigh mortality rates for cases of severe food poisoning and its psychrophilic characterization [3], Listeria is considered as an important foodborne pathogen during the last decade [4]. As a case to verify this, the Canadian listeriosis outbreak was a widespread outburst of the food infection in which twenty people died out of 56 total confirmed cases [5]. E. coli O157: H7 is also discussed as an important fecal bacterium which may spread through direct contact and consumption of contaminated food and can survive at 15% concentration of acetic,
∗ Corresponding author. Tel.: +98 71 32286950; fax: +98 71 32286940. E-mail address: [email protected] (S.S. Shekarforoush).
citric and lactic acids [1]. Consumption of undercooked meat [6] and bovine raw milk [7] are the main sources of several outbreaks. E. coli O157:H7 can lead to hemorrhagic colitis, hemolytic uremic syndrome, and thrombocytopenic purpura, and severe cases, death [8]. Enterohaemorrhagic E. coli O157:H7 was first implicated in human disease in the early 1980s, with the ruminants cited as the primary reservoirs. Preliminary studies indicated cattle to be the sole source of E. coli O157:H7 outbreaks in humans, however, further, epidemiological studies have demonstrated that E. coli O157:H7 was widespread in other food sources and that a number of transmission routes existed. Chemical preservatives are currently replaced by natural ones in food stuffs due to the various side effects on consumers [9,10]. Efficacy of essential oil (EO) of oregano (Origanum vulgare) has been previously addressed. Elgayyar et al. (2001) reported the antimicrobial effects of the EO against L. monocytogenes and Staphylococcus aureus [9]. Antonio et al. (2008) showed strong inhibitory effects of three substances, garlic, oregano and chitosan, against Salmonella enterica, at low temperatures [11]. For instance, as a result of delay in the growth of microorganisms and physiochemical changes of meat samples, the extension of shelf life due to the synergistic effects of oregano (EO) and the modified atmosphere changes, was formerly demonstrated [12]. Chitosan, a natural and biodegradable biopolymer was originated from crab shells, is currently approved by the United States Food and Drug Administration (FDA). Different kinds of chitosan
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are available commercially giving a chance to use them as a popular natural preservative [13]. The antimicrobial activity of chitosan increases at pH less than six due to the positive charge of its glucosamine monomers [14]. Compared to the frozen ready-to-barbecue chicken meat, fresh meat has greater retail value. In these circumstances, using natural antimicrobial components such as chitosan and EO can potentially reduce the risk factors associated with fresh poultry meat. In recent years, many researchers are focused on the organoleptic changes coming simultaneously by using natural preservatives [15,16]. The objective of the present study was to further shed light on the antibacterial effects of chitosan and the oregano EO against the main food spoilage inducer microorganisms with a special spotlight on two main food-borne pathogens in the Iranian readyto-barbecue chicken meat.
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A proven strain of L. monocytogenes 4b obtained from Razi Institute, Hesarak-Karaj, Iran; and E. coli O157:H7 (ATCC, 43895) was provided by Department of Microbiology, School of Veterinary Medicine, Shiraz University.
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The nalidixic acid-resistant E. coli O157:H7 was prepared, as previously described and employed to easy differentiation from background bacteria [17]. Briefly, the wild-type strain of E. coli O157:H7 was inoculated into 100 ml Trypticase soy broth (TSB, Merck, Germany) and incubated at 37 ◦ C for 24 h. A total of 100 ml TSB containing 200 g/ml nalidixic acid (1451000, Sigma-Aldrich, St. Louis, USA) were added to the incubated culture, which was subsequently incubated at 37 ◦ C for a further 24 h. After incubation, 0.1 ml of the culture was spread on the surface of a Trypticase soy agar plate (TSA, Merck, Germany) containing 100 g/ml nalidixic acid and incubated at 37 ◦ C for 24 h. A single colony was selected and re-streaked onto another TSA plate in order to confirm its resistance to nalidixic acid. 2.3. Essential oil of oregano
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The pure EO of oregano (density: 0.960 at 20 ◦ C; tested by gas chromatography–mass spectrometry [GC–MS]; origin: Bulgaria; steam distillation extraction) was obtained from Kobashi Company (Ide, Devon-shire, UK).
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The low molecular weight chitosan (product number: 448869) was obtained from Sigma-Aldrich Compan, St. Louis, USA.
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Iranian ready-to-barbecue chicken meat cubes were prepared according to the local chicken meat processing industries. Briefly, salt (4.7 g), red pepper (1.4 g), 1% acetic acid (50 ml), chopped onion (47 g), saffron (0.1 g) and sunflower oil (20 ml) were added to 1000 g chicken meat. According to the recipe, 4 kg of breast meat was cut into 1 cm3 and mixed with the ingredients excluding acetic acid and vegetable oil. The mixture was then divided into four portions and treated as follows: Group 1 (control): 50 ml acetic acid 1% and 20 ml sunflower oil. Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2015.02.033
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Group 2 (chitosan): 50 ml chitosan solution 2% in acetic acid 1% and 20 ml sunflower oil. Group 3 (oregano): 50 ml acetic acid 1% and 20 ml oregano EO 15% in sunflower oil. Group 4 (chitosan + oregano): 50 ml chitosan solution 2% in acetic acid 1% and 20 ml oregano EO 15% in sunflower oil. 2.6. Effects of chitosan and EO of oregano on microbial quality of ready-to-barbecue chicken Each pack of the sample was divided into 10 g portions in a stomacher bag and stored at 3 ± 1, 8 ± 1 and 20 ± 1 ◦ C for further use. Aerobic plate count (APC), psychrophilic count, lactic acid bacterial (LAB) and enterobacteriacae counts were performed at different storage conditions. The content of the bags was then diluted in 90 ml of 0.1% peptone water and homogenized for 2 min using the stomacher (Seward, BA6021, UK). Homogenates were then ten-fold serially diluted in the peptone water. Plate count agar (Merck, Germany) was used to count APC and psychrophilic bacteria. Man Rogosa Sharpe Agar (MRSA; Merck, Germany) and Violet Red Bile Glucose Agar (VBRGA; Merck, Germany) were employed to count LAB and enterobacteriacae, respectively. To count APC and psychrophilic bacteria, plates were respectively incubated at 37 ◦ C for 48 h and 10 ◦ C for 7 days. MRS agar was incubated at 25 ◦ C for 3 days and VRBGA was also incubated at 37 ◦ C for 24 h. All colonies were finally, enumerated and the results were represented as CFU/g. For each condition, three independent samples were analyzed in duplicate. 2.7. Effects of chitosan and EO of oregano on the survival of E.coli O157:H7 and L. monocytogenes in ready-to-barbecue chicken To establish a correlation between the colony forming unit (CFU)/ml and absorbance of the dilutions of the resistant strain of E. coli O157: H7 and L. monocytogenes at 600 nm, a standard curve were prepared. The bacteria were grown in TSA at 37 ◦ C for 24 h. A single colony from the TSA plates were inoculated into 10 ml of TSB and were incubated overnight at 37 ◦ C. The bacterial suspension were pelleted three times by centrifugation at 3000 × g for 20 min, and washed with sterile normal saline. Final cell pellets were resuspended in 10 ml of sterile normal saline. Appropriate serial dilutions were made and their absorbances were measured at 600 nm. To determine the log10 CFU/ml corresponding to the absorbance of that same dilution, viable cell counts were performed in duplicate by plating serial dilutions onto TSA and incubating at 37 ◦ C for 24 h. Prior to inoculating the bacterial culture into the samples, the absorbance of inocula were matched to the standard curve and if necessary, the density of the bacterial culture were adjusted by adding sterile normal saline. One ml of 1 × 107 CFU/ml of each bacterial suspension was added to 1 kg of each experimental group, which were subsequently massaged manually for 2 min. The samples were finally divided into 10 g portions and kept at 3 ± 1, 8 ± 1 and 20 ± 1 ◦ C, for further use. To determine the bacterial counts in different storing conditions, the contents of each 10 g sample was homogenized using stomacher for 2 min and ten-fold serially diluted in the 0.1% buffered peptone water. 0.1 ml of each dilution was spread on Palcam agar (Merck, Germany) containing 5 mg/L acriflavin and 40 mg/L nalidixic acid and McConkey agar media (Merck, Germany) containing 40 mg/L nalidixic acid that were used to count of L. monocytogenes and E. coli, respectively. The plates were finally incubated at 37 ◦ C for 48 h (to grow L. monocytogenes) and at 37 ◦ C for 24 h (to grow E. coli O157: H7). The plates counted accordingly [18].
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Fig. 1. Bacterial growth in ready-to-barbecue chicken affected by oregano EO and chitosan stored at 3 ◦ C. A. Aerobic plate count, B. psychrophilic count, C. lactic acid bacteria, D. enterobacteriacae. Treatment groups: control (), 1 mg/g chitosan (), 3 l/g oregano EO (), 1 mg/g chitosan + 3 l/g oregano EO (䊉). Different letters means the significant differences within each time point (P < 0.05).
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APC was increased to 0.34 and 1.0 logs in the oregano and control groups, respectively (P < 0.05). The number of APC was not affected by chitosan (P > 0.05). Number of APC in oregano + chitosan treatment group decrease from 4.46 to 4.35 CFU/g during the storage period which was significantly different from the other treatments (P < 0.05) (Fig. 1A). Growth of psychrophilic bacteria and enterobacteriacae were not affected by EO of oregano and chitosan (P > 0.05) (Figs. 1B and D). The population of LAB was increased to 5.44 and 5.49 logs CFU/g after 192 h in the control and chitosan groups, respectively (P > 0.05). They were 4.54 and 3.92 logs CFU/g for oregano and Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2015.02.033
chitosan + oregano groups, which were significantly lower than control and chitosan groups (P < 0.05) (Fig. 1C).
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During the storage period, growth of APC, psychrophilic bacteria and enterobacteriacae were not inhibited by oregano EO, chitosan and their combinations (P > 0.05) (Fig. 3A, 3B and 3D). Oregano EO and chitosan were not influenced the growing of LAB, whilst et
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Following 72 h incubation, APC increased to 4.83, 4.86, 4.45 and 4.09 logs CFU/g, in the control, chitosan, oregano and chitosan + oregano groups, respectively. At the end of the storage period, the number of APC in the oregano + chitosan group was significantly lower than other treatments (P < 0.05) (Fig. 2A). Chitosan had no significant effect on psychrophils at 8 ◦ C for 72 h (P > 0.05). However, in the oregano group, number of psychrophils increased by 0.68 logs after 72 h incubation, which was significantly lower than the control and chitosan groups (P < 0.05) (Fig. 2B). Oregano EO and chitosan have not shown any inhibitory effects on the LAB (P > 0.05), whereas, the combinations showed considerable effects on the growing rates (P < 0.05) (Fig. 2C). Following 72 h incubation, numbers of enterobacteriacae were reduced by up to 0.44 and 0.46 logs in the oregano and chitosan + oregano groups, respectively. The inhibitory effect was only induced by oregano EO (P < 0.05) (Fig. 2D).
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Fig. 2. Bacterial growth in ready-to-barbecue chicken affected by oregano EO and chitosan stored at 8 ◦ C. A. Aerobic plate count, B. psychrophilic count, C. lactic acid bacteria, D. enterobacteriacae. Treatment groups: control (), 1 mg/g chitosan (), 3 l/g oregano EO (), 1 mg/g chitosan + 3 l/g oregano EO (䊉). Different letters means the significant differences within each time point (P < 0.05).
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their combinations showed a significant inhibitory effect (P < 0.05) (Fig. 3C).
was observed for just 24 h of storage (P < 0.05), even though no synergistic effects were evidenced (P > 0.05) (Table 2).
3.4. Effects of chitosan and EO of oregano on survival of E.coli O157:H7 in ready-to-barbecue chicken
4. Discussion
Following 144 h storage at 3 ◦ C, Log CFU/g of E. coli was decreased from 4.55 to 4.09, 3.98, 3.47 and 3.34 in the control, chitosan, oregano and chitosan + oregano groups, respectively. In contrast to chitosan, the survival of bacterium was inhibited by oregano EO (P < 0.05). Furthermore, the inhibitory effect of oregano was not enhanced by chitosan (P > 0.05). The same results were also demonstrated in the samples stored at 8 ◦ C and 20 ◦ C (Table 1). 3.5. Effects of chitosan and EO of oregano on the survival of L. monocytogenes in ready-to-barbecue chicken The treated samples kept at 3 ◦ C for 144 h revealed that using oregano and chitosan separately or in conjunction, did not show any inhibitory effects (P > 0.05) (Table 2). No inhibitory effects were shown, when the samples incubated with oregano at 8 ◦ C for 72 h (P > 0.05). Surprisingly, using chitosan separately was effective against L. monocytogenes (P < 0.05) but its effect was not increased when it used synergistically with oregano (P > 0.05). Moreover, when the samples were stored at 20 ◦ C, chitosan was effective until 48 h of storage. In this case the number of bacteria increased to 8.09 log CFU/g, while, it was 8.54 log CFU/g in the control group (P < 0.05). The inhibitory effect of oregano EO Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2015.02.033
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Oregano, as a natural food additive with proven antimicrobial effects, has been demonstrated to inhibit the growth of spoilage inducer and food-borne pathogenic bacteria [19,20]. The antimicrobial activity of oregano has been attributed mainly to the presence of volatile compounds found in its EO, especially carvacrol and thymol [21]. In the current study, we have demonstrated the antibacterial effects of oregano EO against APC and LAB (at 3 ◦ C), psychrophilic bacteria and enterobacteriacae (at 8 ◦ C), and E. coli (at 3, 8 and 20 ◦ C). Even though, the antimicrobial effects of oregano against L. monocytogenes have already shown, in vitro [22], no considerable effects were proven in barbecued chicken. It has been verified that the presence of some nutrients such as carbohydrates, proteins and salts and pH may influence the efficacy of EOs in foods [23–26]. The physical structure [27], lower water activity [28], adipose components [29] of food stuff may reduce the antimicrobial effects of EOs. This phenomenon might be due to the absorption of some antimicrobial components by proteins or adipose tissues [26]. In the present study, we have shown the antimicrobial effects of oregano against spoilage-inducing bacteria in the samples kept at 3 and 8 ◦ C. In contrast, no antimicrobial effects were revealed at 20 ◦ C. These findings may suggest that increasing, storing temperature
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Fig. 3. Bacterial growth in ready-to-barbecue chicken affected by oregano EO and chitosan stored at 20 ◦ C. A. Aerobic plate count, B. psychrophilic count, C. lactic acid bacteria, D. enterobacteriacae. Treatment groups: control (), 1 mg/g chitosan (), 3 l/g oregano EO (), 1 mg/g chitosan + 3 l/g oregano EO (䊉). Different letters means the significant differences within each time point (P < 0.05).
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induces bacterial growth and dramatically decreases the shelf life of foods. Although using chitosan and oregano showed substantial effects on the growth of decomposer microorganisms, chitosan individually as a single dose was not effective. Additionally, chitosan was not effective against enterobacteriacae count and E. coli O157: H7 for all storage temperatures and in the samples inoculated with L. monocytogenes; chitosan was only effective at 3 and 8 ◦ C, in the absence of oregano EO. Tsai et al.
(2006) demonstrated that chitosan is more effective at lower temperatures [30]. In the food covered by chitosan film, an overwhelming efficacy against L. monocytogenes and Staphylococcus aureus were observed [31]. The antimicrobial characteristics of chitosan were also confirmed against L. monocytogenes in fried cattle meats when the load of the bacteria was less than 6.5 log10 CFU/g, moreover, chitosan coats on the meats, reduced the number of colonies to 2–3 log10 CFU/g [32].
Table 1 Survival and growth of E. coli O157:H7 in ready-to-barbecue chicken affected by oregano essential oil and chitosan stored at different temperature. Temp. ◦ C
Treatments
Mean (±SD) population recovered (Log CFU/g)a 0h
3
8
20
Con Chi Org Chi + Org con Chi Org Chi + Org con Chi Org Chi + Org
4.55 4.55 4.55 4.55 4.41 4.41 4.41 4.41 4.06 4.06 4.06 4.06
24 h
48 h
4.29 4.40 4.16 3.89 7.11 7.04 7.14 6.97
4.21 4.13 3.90 3.75 4.80 4.79 4.48 4.37 8.83 8.84 8.53 8.37
± 0.05a ± 0.05a ± 0.05a ± 0.05a ± 0.05a ± 0.05a ± 0.05a ± 0.05a ± 0.06a ± 0.06a ± 0.06a ± 0.06a
± ± ± ± ± ± ± ±
0.13a 0.04a 0.02b 0.07b 0.02a 0.03a 0.02a 0.09a
72 h ± ± ± ± ± ± ± ± ± ± ± ±
0.02a 0.14a 0.12b 0.09b 0.11a 0.11a 0.10b 0.03b 0.17a 0.16a 0.06b 0.21b
96 h 3.89 4.08 3.66 3.62
4.90 4.93 4.13 4.23 8.88 8.95 8.57 8.53
± ± ± ± ± ± ± ±
144 h ± ± ± ±
0.08a 0.21a 0.07b 0.11b
4.09 3.98 3.47 3.34
± ± ± ±
0.24a 0.04a 0.02b 0.08b
0.17a 0.02a 0.04b 0.16b 0.15a 0.24a 0.08b 0.15b
n = 3 at each time point. The different letter means the significant differences within each temperature, time point (P < 0.05). Con = control; Chi = chitosan (1 mg/g); Org = oregano essential oil (3 l/g); Chi + Org = Chitosan (1 mg/g) + oregano essential oil (3 l/g).
Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2015.02.033
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Table 2 Survival and growth of Listeria monocytogenes in ready-to-barbecue chicken affected by oregano essential oil and chitosan stored at different temperature. Temp. ◦ C
Treatments
Mean (±SD) population recovered (Log CFU/g)a 0h
3
8
20
Con Chi Org Chi + Org Con Chi Org Chi + Org Con Chi Org Chi + Org
4.69 4.69 4.69 4.69 4.76 4.76 4.76 4.76 4.89 4.89 4.89 4.89
24 h ± ± ± ± ± ± ± ± ± ± ± ±
0.15a 0.15a 0.15a 0.15a 0.04a 0.04a 0.04a 0.04a 0.06a 0.06a 0.06a 0.06a
48 h 3.81 3.63 3.61 3.75 4.80 4.38 4.71 4.46 8.51 8.33 8.56 8.17
4.72 ± 0.08 a 4.23 ± 0.05 b 4.62 ± 0.10 a 4.26 ± 0.19 b 6.10 ± 0.08 a 5.98 ± 0.18 a 5.82 ± 0.13 a 6.04 ± 0.11 a
72 h ± ± ± ± ± ± ± ± ± ± ± ±
0.19a 0.16a 0.12a 0.29a 0.08a 0.03b 0.06a 0.11b 0.01a 0.06a 0.05a 0.01b
96 h 3.61 3.55 3.62 3.36
4.73 4.28 4.75 4.29 8.56 8.52 8.66 8.68
± ± ± ± ± ± ± ±
144 h ± ± ± ±
0.17a 0.09a 0.05a 0.43a
3.40 3.46 3.32 3.25
± ± ± ±
0.47a 0.11a 0.31a 0.14a
0.12a 0.29b 0.03a 0.17b 0.14a 0.01a 0.06a 0.03a
n = 3 at each time point. The different letter means the significant differences within each temperature, time point (P < 0.05). Con = control; Chi = chitosan (1 mg/g); Org = oregano essential oil (3 l/g); Chi + Org = chitosan (1 mg/g) + oregano essential oil (3 l/g).
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Zheng and Zhu (2003) found the effectiveness of chitosan (with the molecular weight of less than 300 KDa) against Staphylococcus aureus [33]. Bacterial strain, incubation temperature and food substrate are the main variables greatly determine the antimicrobial activity of chitosan [34]. Previous works on chitosan revealed that gram negative bacteria seemed to be more resistance against chitosan probably due to their outer membrane [13,35]. In the case of using chitosan as a protective coverage layer in cheese, 100 and 77% effectiveness was shown against Gram positive bacteria and Pseudomonas aeruginosa, respectively [31]. The antimicrobial films using incorporating acetic or propionic acid into a chitosan matrix were shown to be effective against surface spoilage bacteria, such as enterobacteriacae, and Serratia [36]. In the in vitro inoculation of shrimp, more than 90% reduction of Vibrio parahaemolyticus was achieved by chitosan. Chaiyakosa et al. (2007) suggested that chitosan might be a suitable replacement for chlorine, which is extensively used in the frozen shrimp industries and causes severe respiratory tract damage [37]. Chitosan exists in two water soluble/insoluble forms. It has been suggested that the water soluble form is more effective which might be due to the production of a non-permeable coat around the bacterial cells [38]. In the current study, we have demonstrated the synergistic antibacterial effects of chitosan and EO of oregano against spoilage inducers in chicken meat stored at 3 and 8 ◦ C. Burt et al. (2005) showed that stabilizers significantly improved the effectiveness of carvacrol against E. coli O157:H7 in broth and they concluded that stabilizers delay the separation of the hydrophobic substrate from the aqueous phase of the medium [19]. Due to the stabilizing and emulsifying nature of chitosan [39], increases in the efficacy of EOs can be evident when used synergistically. From a practical point of view, the results showed an important antimicrobial effect of chitosan and oregano on microbial quality, E. coli O157:H7 and L. monocytogenes 4b on ready-to-barbecue poultry meat. The combined use of chitosan and EO of oregano can also reduce the number of spoilage and safety indicators and also the two food-borne pathogenic bacteria in ready-to-barbecue chicken meat. From a practical point of view, the results showed that the combined use of chitosan and EO of oregano can reduce the number of spoilage and safety indicator bacteria and also E. coli O157:H7 and L. monocytogenes 4b in ready-to-barbecue chicken meat.
340
Acknowledgments
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This research was financially supported by “Natural Antimicrobials Centre of Excellence (NACE)” which is gratefully Please cite this article in press as: http://dx.doi.org/10.1016/j.ijbiomac.2015.02.033
S.S.
acknowledged. We would like to thank Miss M. Aghazi and Mr. G. Niknia for their technical assistance and Miss Z. Shekarforoush for revising the manuscript.
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