Fungi and aflatoxins associated with spices in the Sultanate of Oman

One hundred and five samples of seven spices (cumin, cinnamon, clove, black pepper, cardamom, ginger, and coriander) were purchased from five popular companies in the Sultanate of Oman. The spices were surveyed for the mycoflora and aflatoxins. Twenty fungal species were isolated in which Aspergillus flavus , A. niger . Penicillium , Rhizopus , and Syncephalastrum racemosum were the most dominant. When colony forming units per gm (cfu/gm) of fungi were compared, significant differences were found among spices and companies. Of the seven spices studied, clove was found to be the least contaminated, while cumin was the most contaminated. None of the 15 selected samples of the spices contaminated by A. flavus were found to contain aflatoxins. Nevertheless, nine isolates (45%) of the twenty A. flavus strains screened for aflatoxins were aflatoxigenic. The moisture content of most of the spices was below the maximum standard limit. The results showed that the spices were contaminated by some fungi that might constitute health hazards for humans. This revised version was published online in June 2006 with corrections to the Cover Date.     

Efficacy of Essential Oils on Fungi Isolated from Archaeological Objects in Saqqara Excavation,Egypt

The archeological objects constitute an important part of the worldwide cultural heritage. The impact of the fungal activity on the deterioration of cultural heritage is a global problem and their preservation over time is a challenging task. Antifungal activities of 12 essential oils (EOs) (black cumin, castor, cinnamon, clove, cumin, garlic, geranium, lavender, lemongrass, menthe, olive, and thyme) were examined against 16 fungal species isolated from three tested archaeological objects (wall painting stone, wooden statue, and pottery coffin) from Saqqara stores in Egypt. Molecular identification was carried out for the highly frequent species (Aspergillus niger, A. flavus and Rhizopus oryzae) in the three tested archaeological samples. Antifungal activity and minimal inhibitory concentration (MIC) of the tested EOs with different concentrations (0.125, 0.25, 0.5, 0.75, 1?μl/ml) were measured.

The most efficient EOs were thyme (MIC ranged from 0.25–0.75μl/ml) followed by clove (MIC ranged from 0.25–1?μl/ml) and geranium, (MIC ranged from 0.5–1?μl/ml). Thymol (37.1%) and p-Cymene (26.32%) were the active constituents of thyme, while Triacetin (69.36%) and eugenol (28.67) were the most efficient components of clove oil followed by geranium active components (à-Citronellol 20.62% and Geraniol 14.43%). Aspergillus niger was the most resistance species, while Fusarium oxysporum and Penicilium citrinium were the most susceptible ones.     

Effect of some essential oils on mycelial growth of <Emphasis Type="Italic">Penicillium digitatum</Emphasis> Sacc.

The antifungal action of four essential oils of Foeniculum vulgare (fennel), Thymus vulgaris (thyme), Eugenia caryophyllata (Clove) and Salvia officinalis (sage) was tested in vitro against Penicillium digitatum Sacc. Direct contact and vapour phase were used to test the antifungal activity of these essential oils against P. digitatum that is responsible for green mould rot of citrus fruits. The vapour phase and direct contact of clove and thyme essential oils exhibited the strongest toxicity and totally inhibited the mycelial growth of the test fungus. Thyme and clove essential oils completely inhibited P. digitatum growth either when added into the medium 600 μl l⁻¹ or by their volatiles with 24 μl per 8 cm diameter Petri dish. In in vitro mycelial growth assay showed fungistatic and fungicidal activity by clove and thyme essential oils. Sage and fennel oils did not show any inhibitory activity on this fungus. Scanning electron microscopy (SEM) was done to study the mode of action of clove oil in P. digitatum and it was observed that treatment with the oil leads to large alterations in hyphal morphology.     

Control of Fusarium verticillioides on corn with a combination of Bacillus subtilis TM3 formulation and botanical pesticides

The aim of this study was to evaluate the effectiveness of the combination of Bacillus subtilis TM3 formulation with botanical pesticides in suppressing Fusarium verticilloides infection in corn. The research was carried out at the Plant Pathology Laboratory and the Experimental Farm of Indonesian Cereals Research Institute (ICERI) from February to November 2019. The research consisted of two stages, namely an in vitro test of antagonists of botanical pesticides against F. verticilloides using 5 types of plant extracts namely betel leaf extract, turmeric, galangal, cosmos, and clove leaf. The second stage was to test the effectiveness of the combination of the formulation of B. subtilis TM3 with the best 3 types of plant extracts in vitro testing in suppressing F. verticilloides infection in plants. The results of the in vitro study showed that the plant extracts of betel leaf, clove leaf and galangal had the best inhibitory ability on the mycelia growth of F. verticilloides. Meanwhile, the field test found that the application of the B. subtilis TM3 formulation, either alone or in combination with plant extracts, was able to suppress F. verticilloides infection. The combination of B. subtilis TM3 formulation with betel leaf extract showed the best inhibition of 20% against stem rot disease and 13.33% against corn cob rot. This treatment did not affect production quantitatively, but was able to suppress the decline in seed quality due to F. verticilloides infection. Seeds grown by the Plastic Rolled Paper Test (PRPT) method were not only infected with F. verticilloides, but also infected with other seed-borne pathogens, such as Aspergillus niger and A. falvus. The presence of these two pathogens did not inhibit the growth of F. verticilloides in kernels.     

Effect of some spices as preservatives for storage of lentil (Lens esculenta L.) seeds

The high antimycotic activity of four kinds of spices,viz. Chinese cassia, cinnamon, clove and thyme, were tested as preservatives for agricultural commodities. Clove, thyme and a mixture of the four kinds of spice completely inhibited aflatoxin production on lentil seeds during eight weeks of incubation. Chinese cassia and cinnamon partially inhibited the aflatoxin production.     

Effect of Clove (Syzygium aromaticum) spice as microbial inhibitor of resistant bacteria and Organoleptic Quality of meat

For centuries, spices have been utilized as flavourants, colourants and as preservatives in food. Moreover, spices possess various antimicrobial properties with massive health benefits for the treatment and management of ailments and diseases. The present study was focused on three (3) aspects; (1) isolation and molecular identification of bacteria from the meat; (2) to determine the antimicrobial activity of the spices against the pathogens; (3) to assess the organoleptic properties of the spiced meat. A total of twelve (n = 12) spices evaluated against forty (n = 40) spoilage food-borne pathogenic bacteria (Escherichia coli and Enterococci spp.). The spice extracts were tested using disk diffusion method to determine the inhibition abilities. The results show that clove and black seed cumin extract exhibited excellent antibacterial activity against most pathogenic bacteria. Clove displayed the highest inhibition zone of 18 mm against E. coli (EcFwS1). Clove extract was the most inhibitor followed by black cumin, whereas extracts of thyme and cinnamon showed weak antibacterial activities against the tested strains. The most sensitive strain to spice extracts was Enterococcus spp. (EnFmL1) and the most resistant strain being E. coli. (EcFmS1 and EcFpL1). Untreated meat showed that E. coli and Enterococcus spp. count was 4.4 * 10⁵ ± 3.4 * 10⁵ and 2.2 * 10⁵ ± 3.6 * 10⁴ cfu/mL respectively after 7 days while the single dose of clove showed 5.4 * 10⁴ ± 4.4 * 10² cfu/mL of E. coli and 1.7 * 10⁵ ± 4.1 * 10⁴ cfu/mL of Enterococcus spp. The organoleptic characteristics such as colour, texture, odour, pH, shape of the single dose of clove on the meat was overall acceptable.     

The use of clove oil to induce anaesthesia,and its effects on blood chemistry,in Lates niloticus from Lake Victoria,Uganda

The efficacy of clove oil as an anaesthetic and its effects on blood parameters in Nile perch Lates niloticus were evaluated in 2010. Clove oil concentrations of 49.3, 73.9 and 98.5?mg l^?1 induced anaesthesia in <3?min, while the average recovery time from anaesthesia was 11?min 22 s. The optimal oil clove oil concentration was 49.3?mg l^?1, inducing anaesthesia in 4?min 33 s, with recovery in 3?min 31 s. No stress response was elicited. Clove oil at a concentration of 24.6?mg l^?1 was an effective sedative, whereas a concentration of 49.3?mg l^?1 was sufficient for measuring fish and stripping gametes. A concentration of 73.9?mg l^?1 induced anaesthesia within 4?min and fish recovered in 10?min. Therefore, clove oil was an effective anaesthetic and sedative for the handling of Nile perch within a mass range of 0.4–12?kg fish^?1.     

Enhanced diversity and aflatoxigenicity in interspecific hybrids of Aspergillus flavus and Aspergillus parasiticus

Aspergillus flavus and A. parasiticus are the two most important aflatoxin‐producing fungi responsible for the contamination of agricultural commodities worldwide. Both species are heterothallic and undergo sexual reproduction in laboratory crosses. Here we examine the possibility of interspecific matings between A. flavus and A. parasiticus. These species can be distinguished morphologically and genetically, as well as by their mycotoxin profiles. Aspergillus flavus produces both B aflatoxins and cyclopiazonic acid (CPA), B aflatoxins or CPA alone, or neither mycotoxin; Aspergillus parasiticus produces B and G aflatoxins or the aflatoxin precursor O‐methylsterigmatocystin, but not CPA. Only four of forty‐five attempted interspecific crosses between opposite mating types of A. flavus and A. parasiticus were fertile and produced viable ascospores. Single ascospore strains from each cross were shown to be recombinant hybrids using multilocus genotyping and array comparative genome hybridization. Conidia of parents and their hybrid progeny were haploid and predominantly monokaryons and dikaryons based on flow cytometry. Multilocus phylogenetic inference showed that experimental hybrid progeny were grouped with naturally occurring A. flavus L strain and A. parasiticus. Higher total aflatoxin concentrations in some F1 progeny strains compared to midpoint parent aflatoxin levels indicate synergism in aflatoxin production; moreover, three progeny strains synthesized G aflatoxins that were not produced by the parents, and there was evidence of allopolyploidization in one strain. These results suggest that hybridization is an important diversifying force resulting in the genesis of novel toxin profiles in these agriculturally important fungi.     

Antibacterial Potent of Acetylated and Non-Acetylated Clove Bud Essential Oils and Their Main Compounds

Clove bud is a medicinal plant used traditionally in Asia for the treatment of various disease. Previously, Clove oil is a potential source of an antimicrobial compounds especially vis-a-vis bacterial pathogens. However, the compound responsible for this activity remains to be investigated. Essential oil (EO) clove, acetylated essential oil clove, eugenol, and acetyleugenol were evaluate as an antibacterial potential agent against Staphyloccocus aureus (SE), Escherichia coli (EC) and Pseudomonas aeruginosa (PA). Essential oil containing eugenol was extracted from buds of Eugenia caryophyllata commonly named clove (Syzygium aromaticum (L.) (Family Myrtaceae) by a simple hydrodistillation. The analysis of the essential oils (EOs) using gas chromatography-mass spectrometry (GC-MS) shows eugenol as the major constituent with 70.14 % of the total. The Eugenol was isolated from the EO using chemical treatment. Afterwards, the EO and eugenol were converted to acetylated EO and acetyleugenol, respectively using acetic anhydride. The antibacterial result revealed that all compounds showed a strong activity against the three strains. The Staphyloccocus aureus and Pseudomonas aeruginosa were extremely sensitive against eugenol with an inhibition diameters of 25 mm. The MIC values of eugenol versus S. aureus and P. aeruginosa were 0.58 and 2.32 mg/mL, respectively, while the MIB values were 2.32 mg/mL and 9.28 mg/mL.     

Reducing saturated fatty acids in Arabidopsis seeds by expression of a Caenorhabditis elegans 16:0–specific desaturase

Plant oilseeds are a major source of nutritional oils. Their fatty acid composition, especially the proportion of saturated and unsaturated fatty acids, has important effects on human health. Because intake of saturated fats is correlated with the incidence of cardiovascular disease and diabetes, a goal of metabolic engineering is to develop oils low in saturated fatty acids. Palmitic acid (16:0) is the most abundant saturated fatty acid in the seeds of many oilseed crops and in Arabidopsis thaliana. We expressed FAT–5, a membrane‐bound desaturase cloned from Caenorhabditis elegans, in Arabidopsis using a strong seed‐specific promoter. The FAT‐5 enzyme is highly specific to 16:0 as substrate, converting it to 16:1?9; expression of fat‐5 reduced the 16:0 content of the seed by two‐thirds. Decreased 16:0 and elevated 16:1 levels were evident both in the storage and membrane lipids of seeds. Regiochemical analysis of phosphatidylcholine showed that 16:1 was distributed at both positions on the glycerolipid backbone, unlike 16:0, which is predominately found at the sn‐1 position. Seeds from a plant line homozygous for FAT–5 expression were comparable to wild type with respect to seed set and germination, while oil content and weight were somewhat reduced. These experiments demonstrate that targeted heterologous expression of a desaturase in oilseeds can reduce the level of saturated fatty acids in the oil, significantly improving its nutritional value.     

Beneficial effects of thyme oil on age-related changes in the phospholipid C20 and C22 polyunsaturated fatty acid composition of various rat tissues

The aim of this study was to determine any age-related changes in phospholipid polyunsaturated fatty acid composition, in particular C₂₀ and C₂₂ fatty acids in rat liver, brain, kidney and heart, and to assess and compare the effects of dietary supplementation (42.5 mg/kg body weight/day) of the natural antioxidant thyme oil and its major component thymol throughout the rat life span. The fatty acid composition in the various tissues from young (7 months) and aged (28 months) rats was determined and compared. Livers from aged control, thyme oil and thymol treated rats exhibited an increase in 22:6(n–3). In contrast, 22:6(n–3) content of brain, kidney and heart declined in aged rats in all three dietary groups. However, aged rats treated with thyme oil and thymol displayed significantly higher levels of 22:6(n–3) than the respective age-matched controls. Tissue compositions of 20:4(n–6) were found to be significantly lower in the liver and kidney from aged control rats but not those fed either thyme oil or thymol. In aged rats, the composition of 20:4(n–6) in all tissues was highest in rats fed either thyme oil or thymol. These results show that dietary supplementation with thyme oil tended to maintain higher PUFA levels in all tissues studied. The majority of protection provided by thyme oil was by virtue of its thymol component, which comprises 49% of the whole oil. Thymol administered alone did not provide significantly higher protection than the whole oil, suggesting that other components within thyme oil are also contributing antioxidant activity.     

Fungal flora and aflatoxin contamination of feedstuff samples in Beida Governorate,Libya

Fungi of 19 genera, 30 species, and one variety were isolated from 25 samples of sheep-, cattle- and camel feedstuffs collected from different farms in the Beida Governorate, Libya.Aspergillus, Penicillium andFusarium were the most common genera in the three substrates tested. TLC was used to establish the identity of aflatoxins in the chloroform extract of all samples and the ability to produce aflatoxins byAspergillus flavus in a synthetic liquid medium. Twenty samples out of 25 tested were naturally contaminated and 21 isolates ofA. flavus out of 30 produced at least one of the following aflatoxins: B₁; B₁, G₁; and B₁, B₂, G₁, G₂. This is the first report about the natural occurrence of aflatoxins and aflatoxin-producers of the genusAspergillus in Libya.     

Physico-Chemical Properties of Linseed Oil as Affected by Various Seed-Borne Fungi

The effect of six seed-borne fungi on linseed oil was studied. Alternaria alternate, Aspergillus flavus, Aspergillus repens, Fusarium culmorum and Torula allii reduced oil content while Cladosporum herbarum slightly increased it. All fungi increased the free fatty acid content and saponification number linseed oil. Iodine number was decreased except in Cladosporium herbarum and Fusarium culmorum. Aspergillus flavus was the most destructive parasite in altering quantity and quality of linseed oil.     

Folic acid rivals methylenetetrahydrofolate reductase (MTHFR) gene-silencing effect on MEPM cell proliferation and apoptosis

Rates of fatty acid oxidation increase rapidly in both rat heart and skeletal muscle in the early postnatal period. Therefore, we examined in heart and soleus muscle, (a) whether there were rapid changes in fatty acid transporter (FAT/CD36, FABPpm) mRNA and protein expression early in life (days 10 –36) and thereafter (days 84, 160, 365), and (b) whether the rates of fatty acid transport and the plasmalemmal content of FAT/CD36 and FABPpm were altered. Protein expression was altered rapidly from day 10–36 in both heart (FAT/CD36 only, +21%, P < 0.05)) and soleus muscle (FAT/CD36 + 100%, P < 0.05; FABPpm −20%, P < 0.05), with no further changes thereafter (P < 0.05). Rates of fatty acid transport (day 10 vs day 160) were increased in heart (+33%, P < 0.05) and muscle (+85%, P < 0.05), and were associated with concomitant increases in plasmalemmal FABPpm (+44%, P < 0.05) and FAT/CD36 (+16%, P < 0.05) in the heart, and only plasmalemmal FAT/CD36 in muscle (+90%, P < 0.05). Therefore, known changes in the rates of fatty acid oxidation in heart and muscle early in life appear to be accompanied by a concurrent upregulation in the rates of fatty acid transport and the expression of FAT/CD36 in heart and muscle, as well as an increase in plasmalemmal FAT/CD36 and FABPpm in the heart, and only plasmalemmal FAT/CD36 in soleus muscle. We speculate that the rapid upregulation of fatty acid transport rates in heart and muscle are needed to support the increased rates of fatty oxidation that have been previously observed in these tissues.     

Production of cyclopiazonic acid,aflatrem, and aflatoxin by <Emphasis Type="Italic">Aspergillus flavus</Emphasis> is regulated by <Emphasis Type="Italic">veA</Emphasis>, a gene necessary for sclerotial formation

The plant pathogenic fungus Aspergillus flavus produces several types of mycotoxins. The most well known are the carcinogenic compounds called aflatoxins. In addition, A. flavus produces cyclopiazonic acid and aflatrem mycotoxins, contributing to the toxicity of A. flavus infected crops. Cyclopiazonic acid is a specific inhibitor of calcium-dependent ATPase in the sarcoplasmic reticulum that results in altered cellular Ca⁺⁺ levels. Aflatrem is a potent tremorgenic mycotoxin known to lead to neurological disorders. Previously we showed that a gene called veA controls aflatoxin and sclerotial production in A. parasiticus. In this study in A. flavus, we show that the veA homolog in A. flavus not only is necessary for the production of aflatoxins B1 and B2 and sclerotia, but also regulates the synthesis of the mycotoxins cyclopiazonic acid and aflatrem. The A. flavus ΔveA mutant was completely blocked in the production of aflatrem and showed greater than twofold decrease in cyclopiazonic acid production. The genes involved in the synthesis of cyclopiazonic acid are unknown; however, the aflatrem gene cluster has been characterized. Northern hybridization analysis showed that veA is required for expression of the A. flavus aflatrem genes atmC, atmG, and atmM. This is the first report of a regulatory gene governing the production of cyclopiazonic acid and aflatrem mycotoxins.     

Considerations on the distribution of aflatoxigenic Aspergillus flavus in feeds

The distribution of aflatoxin producing isolates of the Aspergillus flavus group in feeds was studied. Aflatoxin production was investigated by a sequential method previously reported (fluorescence in Coconut Agar Medium, rapid extraction from a wheat medium, and total extraction from the same wheat medium). Twenty-seven of 32 samples contained A. flavus, and 21 of them had at least one aflatoxicogenic isolate of A. flavus. Of the 115 isolates analysed, 65 produced aflatoxins, mainly B aflatoxins.     

The Phylogenetics of Mycotoxin and Sclerotium Production in Aspergillus flavus and Aspergillus oryzae

Aspergillus flavus is a common filamentous fungus that produces aflatoxins and presents a major threat to agriculture and human health. Previous phylogenetic studies of A. flavus have shown that it consists of two subgroups, called groups I and II, and morphological studies indicated that it consists of two morphological groups based on sclerotium size, called “S” and “L.” The industrially important non-aflatoxin-producing fungus A. oryzae is nested within group I. Three different gene regions, including part of a gene involved in aflatoxin biosynthesis (omt12), were sequenced in 33 S and L strains of A. flavus collected from various regions around the world, along with three isolates of A. oryzae and two isolates of A. parasiticus that were used as outgroups. The production of B and G aflatoxins and cyclopiazonic acid was analyzed in the A. flavus isolates, and each isolate was identified as “S” or “L” based on sclerotium size. Phylogenetic analysis of all three genes confirmed the inference that group I and group II represent a deep divergence within A. flavus. Most group I strains produced B aflatoxins to some degree, and none produced G aflatoxins. Four of six group II strains produced both B and G aflatoxins. All group II isolates were of the “S” sclerotium phenotype, whereas group I strains consisted of both “S” and “L” isolates. Based on the omt12 gene region, phylogenetic structure in sclerotium phenotype and aflatoxin production was evident within group I. Some non-aflatoxin-producing isolates of group I had an omt12 allele that was identical to that found in isolates of A. oryzae.     

Incidence of aflatoxin producing strains and aflatoxin contamination in dry fruit slices of quinces (Cydonia oblonga Mill.) from the Indian State of Jammu and Kashmir

An investigation was undertaken to obtain data on the occurrence of aflatoxins and the aflatoxin producing potential of Aspergillus flavus strains isolated from dry fruit slices of quinces produced in jammu and Kashmir, India. A total of 147 A. flavus isolates recovered from dr fruit slices were grown in liquid rice flour medium and screened for the production of various aflatoxins by thin layer chromatography. The results showed that 23.14% of the tested isolates were aflatoxigenic, producing aflatoxins B₁and B₂ in varying amounts. Aflatoxins G₁ and G₂ were not detected. All 25 of the investigated market samples were also found to be aflatoxin B₁ positive and the level of contamination ranged from 96 to 8164 g/kg of the dry fruit which is quite high in comparison to the permissible level of 30 ppb. As per these results biochemical composition of dry fruit slices of quinces, along with climatic conditions seem to be very favourable for aflatoxin production by the toxigenic A. flavus strains. Therefore,monitoring of aflatoxins in dry fruit slices of quincesis recommended for this region.This revised version was published online in October 2005 with corrections to the Cover Date.     

Comparison of the ability of three Aspergillus strains to form aflatoxins on bakery products and on nutrient agar

The growth of Aspergillus parasiticus NRRL 2999, A. parasiticus NRRL 3000 and A. flavus NRRL 3251 on whole wheat bread and on cake (Rührkuchen) was compared and the formation of the aflatoxins B₁, B₂, G₁, G₂ and M₁ on these substrates and, for purpose of comparison, on malt extract agar was determined. On cake the moulds grew better than on bread and formed the highest yields of aflatoxins. Malt extract agar was the most unfavourable substrate for toxin production. The ratio M₁/B₁ on bread and cake was in the order of 0.1–0.4 and was higher than the data reported for grains. The highest yields of aflatoxin B₁ (1.0 g/g) were produced by A. flavus NRRL 3251 on cake.     

Novel strategies of essential oils,chitosan, and nano- chitosan for inhibition of multi-drug resistant: E. coli O157:H7 and Listeria monocytogenes

Despite the wide range of available antibiotics, food borne bacteria demonstrate a huge spectrum of resistance. The current study aims to use natural components such as essential oils (EOs), chitosan, and nano-chitosan that have very influential antibacterial properties with novel technologies like chitosan solution/film loaded with EOs against multi-drug resistant bacteria. Two strains of Escherichia coli O157:H7 and three strains of Listeria monocytogenes were used to estimate antibiotics resistance. Ten EOs and their mixture, chitosan, nano-chitosan, chitosan plus EO solutions, and biodegradable chitosan film enriched with EOs were tested as antibacterial agents against pathogenic bacterial strains. Results showed that E. coli O157:H7 51,659 and L. monocytogenes 19,116 relatively exhibited considerable resistance to more than one single antibiotic. Turmeric, cumin, pepper black, and marjoram did not show any inhibition zone against L. monocytogenes; Whereas, clove, thyme, cinnamon, and garlic EOs exhibited high antibacterial activity against L. monocytogenes with minimum inhibitory concentration (MIC) of 250–400 μl 100^?1 ml and against E. coli O157:H7 with an MIC of 350–500 μl 100^?1 ml, respectively. Among combinations, clove, and thyme EOs showed the highest antibacterial activity against E. coli O157:H7 with MIC of 170 μl 100^?1 ml, and the combination of cinnamon and clove EOs showed the strongest antibacterial activity against L. monocytogenes with an MIC of 120 μl 100^?1 ml. Both chitosan and nano-chitosan showed a promising potential as an antibacterial agent against pathogenic bacteria as their MICs were relatively lower against L. monocytogenes than for E. coli O157:H7. Chitosan combined with each of cinnamon, clove, and thyme oil have a more effective antibacterial activity against L. monocytogenes and E. coli O157:H7 than the mixture of oils alone. Furthermore, the use of either chitosan solution or biodegradable chitosan film loaded with a combination of clove and thyme EOs had the strongest antibacterial activity against L. monocytogenes and E. coli O157:H7. However, chitosan film without EOs did not exhibit an inhibition zone against the tested bacterial strains.