Impact of volatile compounds generated by essential oils on Aspergillus section Flavi growth parameters and aflatoxin accumulation

BACKGROUND: The advantage of essential oils is their bioactivity in the vapour phase, a characteristic that makes them attractive as possible fumigants for stored grain protection. In this study the antifungal and antiaflatoxigenic effects of the volatile fractions of five essential oils (EOs) were evaluated by vapour contact on Aspergillus section Flavi isolates. RESULTS: In maize meal extract agar the volatile fractions of Pimpinella anisum L. (anise), Pëumus boldus Mol. (boldus), Hedeoma multiflora Benth. (mountain thyme), Lippia turbinata var. integrifolia (Griseb.) (poleo) and Syzygium aromaticum L. (clove) were able to decrease the growth rate and lag phase of aflatoxigenic isolates. Boldus EO showed the best antifungal effect on Aspergillus section Flavi growth rate. In sterilised maize grains, boldus and poleo EOs showed antifungal effects on growth rate and aflatoxin accumulation. The volatile fraction of boldus EO completely inhibited the growth of isolates at water activity (a_w) levels of 0.955, 0.930 and 0.900, while poleo EO showed this effect only at the lower a_w levels (0.930 and 0.900). All aflatoxigenic isolates showed reduced total aflatoxin accumulation in the presence of boldus EO under all a_w conditions. CONCLUSION: These findings clearly indicate that the volatile fraction of boldus EO could be used to control aflatoxigenic fungi in stored maize. Copyright © 2009 Society of Chemical Industry     

Application of essential oils in maize grain: impact on Aspergillus section Flavi growth parameters and aflatoxin accumulation

The antifungal activity of Pimpinella anisum L. (anise), Pëumus boldus Mol (boldus), Hedeoma multiflora Benth (mountain thyme), Syzygium aromaticum L. (clove), and Lippia turbinate var. integrifolia (griseb) (poleo) essential oils (EOs) against Aspergillus section Flavi was evaluated in sterile maize grain under different water activity (a_w) condition (0.982, 0.955, and 0.90). The effect of EOs added to maize grains on growth rate, lag phase, and aflatoxin B₁ (AFB₁) accumulation of Aspergillus section Flavi were evaluated at different water activity conditions. The five EOs analyzed have been shown to influence lag phase and growth rate. Their efficacy depended mainly on the essential oil concentrations and substrate water activity conditions. All EOs showed significant impact on AFB₁ accumulation. This effect was closely dependent on the water activity, concentration, and incubation periods. Important reduction of AFB₁ accumulation was observed in the majority of EO treatments at 11 days of incubation. Boldus, poleo, and mountain thyme EO completely inhibited AFB₁ at 2000 and 3000 μg g⁻¹. Inhibition of AFB₁ accumulation was also observed when aflatoxigenic isolates grew with different concentration of EOs during 35 days.     

Influence of Some Spice Essential Oils on Aspergillus Parasiticus Growth and Production of Aflatoxins in a Synthetic Medium

Gas-liquid chromatography was used to determine the essential oil compositions of thyme, cumin, clove, caraway, rosemary, and sage. The basic components of these oils were thymol, cumin aldehyde, eugenol, carvonc, borneol and thujonc, respectively. The antifungal potential of the oils against Aspergillus parasiticus were investigated. The essential oils caused complete inhibition of both mycelial growth and aflatoxin production. The effectiveness followed the sequence: thyme > cumin > clove > caraway > rosemary > sage. The major components of the essential oils produced an inhibitory effect at minimum inhibitory concentrations equal to those obtained with the oils.     

ANTIFUNGAL ACTIVITIES OF THYME, CLOVE AND OREGANO ESSENTIAL OILS

The antifungal potential of essential oils of oregano (Origanum vulgare), thyme (Thymus vulgaris) and clove (Syzygium aromaticum) was determined. To establish this antifungal potential, two molds related to food spoilage, Aspergillus niger and Aspergillus flavus, were selected. The agar dilution method was employed for the determination of antifungal activities. The three essential oils analyzed presented inhibitory effects on both molds tested. Oregano essential oil showed the highest inhibition of mold growth, followed by clove and thyme. Aspergillus flavus was more sensitive to thyme essential oil than A. niger. Clove essential oil was a stronger inhibitor against A. niger than against A. flavus.     

The effects of citrus oils and some spices on growth and aflatoxin production by Aspergillus parasiticus NRRL 2999

The inhibitory effect of various concentrations of mint, sage, bay leaves, thyme, aniseed, red pepper and citrus peel oils on the growth of an aflatoxin-producing strain of Aspergillus parasiticus and its production of aflatoxin was studied. Amongst the herbs used, thyme was found a highly effective antifungal agent. Growth and aflatoxin formation was depressed by orange and lemon oils at a concentration of 1.6% through 10 days incubation.     

Effect of Equisetum arvense and Stevia rebaudiana extracts on growth and mycotoxin production by Aspergillus flavus and Fusarium verticillioides in maize seeds as affected by water activity

Cereals are a very important part of the human and animal diets. However, agricultural products can be contaminated by moulds and their mycotoxins. Plant extracts, particularly those of Equisetum arvense and Stevia rebaudiana have been reported previously to contain antioxidant compounds which may have antifungal properties. In this study, E. arvense and S. rebaudiana extracts were tested for their control of mycotoxigenic fungi in maize. The extracts were tested separately and as a mixture for their effect on growth of Aspergillus flavus and Fusarium verticillioides. Extracts were added to unsterilised inoculated maize at different water activity (a(w)) levels (0.85-0.95). Moulds were inoculated and incubated for 30 days. Results confirmed that the extract of E. arvense and a mixture 1:1 of Equisetum-Stevia may be effective for the inhibition of both growth of A. flavus and aflatoxin production at high water activity levels (pre-harvest conditions). In general, growth of the F. verticillioides was reduced by the use of plant extracts, especially at 0.95 a(w). However, fumonisin presence was not significantly affected. E. arvense and S. rebaudiana extracts could be developed as an alternative treatment to control aflatoxigenic mycobiota in moist maize.     

Integrated management of insect vectors of Aspergillus flavus in stored maize, using synthetic antioxidants and natural phytochemicals

The purpose of this study was to investigate the insecticidal activity of two benzoic acids 2(3)-tert-butyl-4 hydroxyanisole (BHA) and 2,6-di(tert-butyl)-p-cresol (BHT); two phenolic acids 3-phenyl-2-propenoic acid (CA) and trans-4-hydroxy-3-methoxycinnamic acid (FA) and two essential oils of Eugenia caryophyllata (clove tree) and Thymus vulgaris (thyme) against Sitophilus zeamais, Tribolium confusum and Rhyzopertha dominica, vector carriers of aflatoxigenic fungi in stored maize. The susceptibility of insects, the frequency of isolation of Aspergillus section Flavi in insects and maize, and the analysis of aflatoxin B₁ in maize were determined. BHA, BHT, BHA/BHT mixture and the natural phytochemicals AF and AF/AC mixture showed the highest insecticidal activity against S. zeamais, T. confusum and R. dominica after 120 days of incubation. The insecticidal efficacy of the volatile fraction of essential oils of clove and thyme showed less inhibition. There was no contamination of Aspergillus section Flavi in dead and live insects collected from maize treated with BHA. No aflatoxin B₁ accumulation was detected in the control and treatments. The information obtained shows that these substances have the potential to control pest insect vectors of aflatoxigenic fungi in stored maize in microcosms during 120 days.     

复配精油对粮油中霉菌防治的增效作用及机理研究

开发新型天然防霉剂控制粮食霉变是保障粮食质量安全的重要途径之一。为研究复配植物精油对粮油储藏过程中常见霉菌赭曲霉(Aspergillus ochrator)、黄曲霉(Aspergillus flavus)和黑曲霉(Aspergillus niger)的防霉效果,挑选活性较强的植物精油进行复配并对联合防霉效果进行评价。通过复配植物精油对霉菌孢子萌发、菌丝干重和细胞完整性的影响,对其防霉机理进行初步探究。结果显示,牛至精油对赭曲霉和黑曲霉的防霉效果最好,抑菌圈直径分别为（27.83±0.58 ）、（15.33±0.29）mm,肉桂醛对黄曲霉的防霉效果最好,抑菌圈直径为（18.50±0.87 ）mm;山苍子精油与牛至精油、肉桂醛与牛至精油复配体积比为2：8时对3种霉菌的防霉效果较优;通过对部分抑菌浓度指数判读,两组植物精油复配对黑曲霉和赭曲霉的防治效果为协同作用,对黄曲霉的防治效果为相加作用;山苍子精油与牛至精油按体积比2：8复配可抑制霉菌孢子萌发和菌丝生长、破坏细胞的完整性、改变孢子和菌丝结构;山苍子精油和牛至精油按体积比2：8复配施用于含黄曲霉的玉米上,可有效降低玉米中黄曲霉毒素B1和赭曲霉毒素的含量。本研究为山苍子精油与牛至精油复配作为防霉剂提供理论支持。     

Synergistic antifungal effects of thyme essential oil and Lactobacillus plantarum cell‐free supernatant against Penicillium spp. and in situ effects

This study evaluates the synergistic antifungal effects between thyme essential oils and Lactobacillus plantarum cell‐free supernatant (LCFS) against Penicillium spp. and in situ antifungal activity in rice grains. Thyme essential oil and LCFS showed remarkable antifungal activities against Penicillium spp. with the minimum inhibitory concentration (MIC) of 40 and 80 µL/mL, respectively. The analysis of fractional inhibitory concentration (FIC) index showed the antifungal synergism between thyme essential oil and LCFS against Penicillium spp. with FIC index of 0.5. This synergism also resulted in fourfold reduction in their MICs when applied in combination. The antifungal modes of action were characterized by observing the changes in cell membrane permeability and degradation of fungal cell wall. The combination of thyme essential oil and LCFS (2 × MIC of each) showed remarkable in situ antifungal effect and completely inhibit the growth of Penicillium in rice seeds. The results suggested the possible applications of the observed synergism on actual crops.

Practical applications

Essential oils are used as preservative in food industry and high concentration of essential oil is associated with negative organoleptic characteristics. This study presented a novel approach for synergistic antifungal effects by using the combination of thyme essential oil and Lactobacillus plantarum cell‐free supernatant (LCFS) against Penicillium spp. and systematic evaluation of the antifungal effect by using fractional inhibitory concentration (FIC) index method. This approach will be a role model for future research on synergism and overcome the major drawbacks of using live bacteria and the negative effects arising from antimicrobial activities of essential oils.     

Antifungal Activity of Lactic Acid Bacteria Isolated from Peanuts,Gari, and Orange Fruit Juice against Food Aflatoxigenic Molds

This study aims to evaluate the antifungal activity of lactic acid bacteria (LAB) from some Cameroonian food commodities against mycotoxigenic and spoilage molds. Following LAB isolation, the antifungal activity of the isolates was assessed. The organic acids were quantified using high-performance liquid chromatography and the ability of the LAB to reduce mold biomass and aflatoxin production was evaluated. The LAB were identified and the biopreservative potential of strain LO3 was evaluated on tomato paste. Nine percent of the strains isolated showed broad antifungal activity. The activity was due to the effect of organic acids comprising lactic, acetic, 4-hydroxy-3-phenyllactic and 3-phenyllactic acids. Lactobacillus plantarum LO3 exhibiting the highest and broadest antifungal activity was selected and showed the capacity to inhibit fungal growth and aflatoxin production in vitro. Moreover, this strain and its cell-free supernatant showed the ability to prevent aflatoxigenic mold growth in tomato paste without altering its physico-chemical and organoleptic properties.     

Antifungal activity of five different essential oils in vapour phase for the control of Colletotrichum gloeosporioides and Lasiodiplodia theobromae in vitro and on mango

Mango fruit has high commercial value; however, major postharvest losses are encountered throughout the supply chain due to postharvest diseases. These results lead to the search for natural fungicide for postharvest diseases control. The antifungal effects of five essential oils (thyme, clove, cinnamon, anise and vitex) were assessed by disc volatilisation method. Thyme oil vapours at 5 μL per Petriplate, and clove and cinnamon oil at 8 μL per Petriplate showed 100% growth inhibition of mango pathogens in vitro. GC/MS analysis of essential oil showed thymol (23.88), o‐cymol (23.88) and terpinolene (23.88) as the major constituents of thyme oil. Clove and cinnamon oils contain 3‐allyl‐2‐methoxyphenol (37.42%) and benzofuran 3‐methyl (17.97%), respectively. Thyme oil as a fumigant at 66.7 μL L^?1 showed a significant (P < 0.05) inhibition on postharvest pathogens of mango fruits stored at 25 °C for 6 days. Results of our study suggest the possibility of using thyme oil as an alternate natural fungicide to manage postharvest diseases in mango.     

Assessment of Thymus vulgaris L. essential oil as a safe botanical preservative against post harvest fungal infestation of food commodities

A total of 14 odoriferous angiospermic essential oils were tested against the toxigenic strain of Aspergillus flavus. The essential oil of Thymus vulgaris L. showed highest antifungal efficacy. The thyme oil absolutely inhibited the mycelial growth of A. flavus at 0.7 μl ml^− 1 and exhibited a broad fungitoxic spectrum against eight different food contaminating fungi viz. Fusarium oxysporum, Cladosporium herbarum, Curvularia lunata, Aspergillus terreus, Aspergillus niger, Aspergillus fumigatus, Alternaria alternata and Botryodiploidia theobromae. The oil also showed significant antiaflatoxigenic efficacy as it completely arrested the aflatoxin B₁ production at 0.6 μl ml^− 1. Thyme oil as fungitoxicant was also found superior over most of the prevalent synthetic fungicides. The LC₅₀ of thyme oil against mice was recorded as 7142.85 μl kg^− 1 body weight indicating its non-mammalian toxicity and strengthening its safe exploitation as preservative for stored food commodities. The findings recommend the thyme oil as potential botanical preservative in eco-friendly control of biodeterioration of food commodities during storage.

Industrial relevance

The thyme essential oil may be recommended for large scale application as a plant based preservative for stored food items because of its strong antifungal as well as antiaflatoxigenic efficacy. Because of broad antimicrobial spectrum, more efficacy over prevalent synthetic preservatives as well as non-mammalian toxicity, the thyme essential oil may be formulated as a safe and economical plant based preservative against post harvest fungal infestation and aflatoxin contamination of food commodities.     

鲜切西兰花贮藏期病原菌分离鉴定及植物精油对其抑制效果

以‘冬至绿’西兰花为试材,分离、纯化并鉴定鲜切西兰花贮藏期病原菌,筛选出抑制病原菌效果较好的植物精油。通过分离、纯化及致病性实验得到病原真菌菌株AB01,以病原菌内源转录间隔区序列进行进化树分析,鉴定该病原菌为链格孢菌（Alternaria alternata）。选用肉桂、茴香、牛至、丁香、百里香和香茅6 种精油对AB01进行抑菌性实验,通过体外抑菌和活体抑菌筛选出最优的精油种类及含量。结果发现,肉桂精油对AB01的抑菌效果最好,其最低抑菌浓度和最低杀菌浓度分别为0.05 μL/mL和0.15 μL/mL,其次是茴香和牛至精油。0.15 μL/mL肉桂精油用于鲜切西兰花可有效降低花球黄花率和切面褐变率。     

Antioxidant Effect of Poleo and Oregano Essential Oil on Roasted Sunflower Seeds

The objective was to evaluate the stability of sensory and chemical parameters in roasted sunflower seeds supplemented with oregano and poleo essential oils; and the consumer acceptability of this product. Four samples were prepared: plain roasted sunflower seeds (Control = RS‐C), and sunflower seeds added with oregano (RS‐O) or poleo (RS‐P) essential oils or BHT (RS‐BHT). Consumer acceptance was determined on fresh samples. The overall acceptance averages were 6.13 for RS‐C, 5.62 for RS‐P, and 5.50 for RS‐O (9‐point hedonic scale). The addition of BHT showed greater protection against the oxidation process in the roasted sunflower seeds. Oregano essential oil exhibited a greater antioxidant effect during storage than poleo essential oil. Both essential oils (oregano and poleo) provided protection to the product, inhibiting the formation of undesirable flavors (oxidized and cardboard). The antioxidant activity that presents essential oils of oregano and poleo could be used to preserve roasted sunflower seeds.     

植物复合精油对5种稻谷霉变优势菌株抑菌作用研究

为了研究天然植物精油(百里香、丁香、肉桂)对霉变稻谷的抑菌效果,以5种稻谷霉变优势菌株为受试菌,以霉菌抑菌圈直径大小和最低抑菌浓度（MIC）为指标,通过混料设计方法建立复合精油抑菌模型,结合方差分析得到抑菌效果最佳的植物精油配比。研究结果表明,单一精油抑菌活性对亮白曲霉（A. candidus）,杂色曲霉（A. versicolor）和聚多曲霉（A. sydowii）为肉桂精油>丁香精油>百里香精油;对稻黑孢霉（N. oryzae）为肉桂精油=丁香精油>百里精香油;对布罗克青霉菌（P. brocae）为丁香精油=百里香精油<肉桂精油。当肉桂精油:丁香精油:百里香精油的体积比为55.2%︰26.9%︰17.9%时,3种植物精油对5种菌株抑制效果最佳,复合抑制值大于90.9%。     

A pilot study into the chemical and sensorial effect of thyme and pennyroyal essential oil on hens eggs

Previously, it has been shown that thyme and pennyroyal essential oils have potential as acaricides against the poultry red mite Dermanyssus gallinae . The effect of these oils on the occurrence of taint in hens' eggs was investigated using in vitro immersion tests and in vivo methods where poultry huts containing laying hens were sprayed weekly with oil. Analysis of extracts from eggs by gas chromatography mass spectrometry (GCMS) showed that no detectable taint was present in hens' eggs. However, consumer sniff tests, although restricted and only preliminary in nature, showed a significant negative response to the smell of both unbroken and cracked open eggs between those taken from poultry huts treated with pennyroyal essential oil and all other eggs tested. Some essential oils, such as thyme, may be more suitable as an acaracidal product than others, such as pennyroyal, for the use within a commercial poultry system for laying hens.     

Biodegradable gelatin–chitosan films incorporated with essential oils as antimicrobial agents for fish preservation

Essential oils of clove (Syzygium aromaticum L.), fennel (Foeniculum vulgare Miller), cypress (Cupressus sempervirens L.), lavender (Lavandula angustifolia), thyme (Thymus vulgaris L.), herb-of-the-cross (Verbena officinalis L.), pine (Pinus sylvestris) and rosemary (Rosmarinus officinalis) were tested for their antimicrobial activity on 18 genera of bacteria, which included some important food pathogen and spoilage bacteria. Clove essential oil showed the highest inhibitory effect, followed by rosemary and lavender. In an attempt to evaluate the usefulness of these essential oils as food preservatives, they were also tested on an extract made of fish, where clove and thyme essential oils were the most effective. Then, gelatin–chitosan-based edible films incorporated with clove essential oil were elaborated and their antimicrobial activity tested against six selected microorganisms: Pseudomonas fluorescens, Shewanella putrefaciens, Photobacterium phosphoreum, Listeria innocua, Escherichia coli and Lactobacillus acidophilus. The clove-containing films inhibited all these microorganisms irrespectively of the film matrix or type of microorganism. In a further experiment, when the complex gelatin–chitosan film incorporating clove essential oil was applied to fish during chilled storage, the growth of microorganisms was drastically reduced in gram-negative bacteria, especially enterobacteria, while lactic acid bacteria remained practically constant for much of the storage period. The effect on the microorganisms during this period was in accordance with biochemical indexes of quality, indicating the viability of these films for fish preservation.     

植物精油对烟草甲触杀、熏蒸和驱避作用研究

为筛选有效的贮烟用植物精油杀虫物质,利用点滴法,锥形瓶(密闭熏蒸)法,室内测定了9种植物精油(留兰香油、艾叶油、青蒿油、茶树油、丁香油、冬青油、薄荷油、桉叶油、百里香油)对烟草甲的触杀,熏蒸和驱避活性.结果表明:9种植物精油中表现触杀作用较好的是:丁香油、冬青油、茶树油、艾叶油、百里香油;表现熏蒸作用较好的是:留兰香油...     

Antibacterial properties of plant essential oils

Fifty plant essential oils were examined for their antibacterial properties against 25 genera of bacteria. Four concentrations of each oil were tested using an agar diffusion technique. The ten most inhibitory oils were thyme, cinnamon, bay, clove, almond (bitter), lovage, pimento, marjoram, angelica and nutmeg. The most comprehensively inhibitory extracts were angelica (against 25 genera), bay (24), cinnamon (23), clove (23), thyme (23), almond (bitter) (22), marjoram (22), pimento (22), geranium (21) and lovage (20).     

In vitro antifungal activity of terpinen-4-ol, eugenol, carvone, 1,8-cineole (eucalyptol) and thymol against mycotoxigenic plant pathogens

The aim of this study was to examine the effect of five naturally occurring compounds from essential oils on 10 different species of mycotoxigenic fungi involved in several plant diseases. The antifungal activities of terpinen-4-ol, eugenol, carvone, 1,8-cineole (eucalyptol) and thymol were observed in?vitro on Fusarium subglutinans, Fusarium cerealis, Fusarium verticillioides, Fusarium proliferatum, Fusarium oxysporum, Fusarium sporotrichioides, Aspergillus tubingensis, Aspergillus carbonarius, Alternaria alternata and Penicillium sp. The naturally occurring compounds tested showed toxic effects on in?vitro mycelium growth of all fungal species but with different level of potency. The results are encouraging for further investigations of in planta antifungal activities of these essential oils components.