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.     

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。     

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%。     

Antimicrobial activity of cinnamon and clove oils under modified atmosphere conditions

Mixtures of cinnamon and clove oils were tested for inhibitory activity against important spoilage microorganism of intermediate moisture foods. Four fungal species (Aspergillus flavus, Penicillium roqueforti, Mucor plumbeus and Eurotium sp.), four yeasts species (Debaryomyces hansenii, Pichia membranaefaciens, Zygosaccharomyces rouxii and Candida lipolytica), and two bacteria species (Staphylococcus aureus and Pediococcus halophilus) inoculated separately on agar plates were sealed in a barrier pouch and exposed to essential oil volatiles under a modified atmosphere of low O2 (<0.05-10%) and high CO2 (20% or 40%), with the balance being N2. A. flavus and Eurotium sp. proved to be the most resistant microorganisms. Cinnamon and clove oils added between 1000 and 4000 microL at a ratio of 1:1 were tested for minimum inhibitory volume (MIV) against molds and yeasts. The gas phase above 1000 microL of the oil mixture inhibited growth of C. lipolytica and P. membranaefaciens; 2000 microL inhibited growth of A. flavus, P. roqueforti, M. plumbeus, Eurotium sp., D. hansenii, and Z. rouxii, while inhibition of A. flavus required the addition of 4000 microL. Higher ratios of cinnamon oil/clove oil were more effective for inhibiting the growth of A. flavus.     

复合精油对黄曲霉联合抑菌作用研究

采用联合抑菌评价方法,通过测定植物精油抑菌圈直径、最低抑菌浓度(Minimum Inhibition Concentration,MIC)、最低生长下降浓度(Reduction Concentration,RC)、联合抑菌指数(Fractional Inhibitory Concentration,FIC),对植物精油单独及联合使用后的抑菌效果进行评价,筛选最佳抑制黄曲霉活性作用的复合精油种类及配比。结果表明:大蒜、肉桂、丁香、薄荷精油单独或复合使用均有较强抑制黄曲霉活性的作用。大蒜与肉桂精油联合抑菌评价为相加作用。当大蒜精油与肉桂精油复合体积比为1∶3时,复合精油具有最佳抑制黄曲霉活性的效果。因此,复合精油作为有效防霉保鲜成分具有良好的研发与应用前景。     

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.     

Effects of Essential Oils from Plants on Growth of Food Spoilage Yeasts

Thirty-two essential oils from plants were screened for inhibitory effects on 13 food-spoilage and industrial yeasts. Of these, essential oils of allspice, cinnamon, clove, garlic, onion, oregano, savory, and thyme were most inhibitory. Oils were subsequently tested for their effects on biomass production and pseudomycelium formation of eight genera of yeasts. Garlic oil was a potent inhibitor of yeast growth at concentrations as low as 25 ppm. The oils of onion, oregano and thyme were also strongly inhibitory. Essential oils (100 ppm) had no effect on pseudomycelium production by Candida lipolytica. However, all eight essential oils delayed pseudomycelium formation by Hansenula anomala, whereas six oils stimulated pseudomycelium production by Lodderomyces elongisporus. Cinnamon and clove oils were clearly stimulatory to pseudomycelium production by Saccharomyces cerevisiae.     

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.     

Screening of Argentine plant extracts: impact on growth parameters and aflatoxin B1 accumulation by Aspergillus section Flavi

The effect of essential oils, ethanolic and aqueous extract of 41 vegetable species on Aspergillus section Flavi growth was evaluated. The in vitro screen was a two-stage process. A wide-spectrum initial screen which identified promising antifungal plant extracts was carried out first. After that, identified extracts were studied in more detail by in vitro assays. A total of 96 plant extracts were screened. Essential oils were found to be the most effective extract controlling aflatoxigenic strains. Clove, mountain thyme, poleo and eucalyptus essential oils were selected to study their antifungal effect. Studies on percentage of germination, germ-tube elongation rate, growth rate, and aflatoxin B1 accumulation were carried out. Clove, mountain thyme and poleo essential oils showed the most antifungal effect under all growth parameters analyzed as well as aflatoxin B1 accumulation. Our results suggest that mountain thyme and poleo, which are native vegetal species of Argentina, and clove essential oils could be used alone or in conjunction with other substances to control the presence of aflatoxigenic fungi in stored maize.     

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.     

Application of plant essential oils in prevention of fungal growth on Para rubber wood

Antifungal activities of essential oils (cinnamon, clove, eucalyptus, peppermint and lemongrass oils) against moulds (Penicillium sp., Aspergillus niger and Aspergillus versicolor) isolated from rubber wood surfaces were examined. Clove oil possessed consistent antifungal activity with the minimal inhibitory concentration (MIC) of 5 μl ml^?1 against all these fungi, while cinnamon oil had MICs from 2.5 to 10 μl ml^?1. However, only dip treatment with cinnamon oil inhibited the growth of A. niger on rubber wood for at least 25 weeks.     

Antifungal activity evaluation of Mexican oregano (Lippia berlandieri Schauer) essential oil on the growth of Aspergillus flavus by gaseous contact

The antifungal activity of Mexican oregano (Lippia berlandieri Schauer) essential oil by gaseous contact on the growth of Aspergillus flavus at selected essential oil concentrations (14.7, 29.4, 58.8, or 117.6 μl of essential oil per liter of air) and temperatures (25, 30, or 35°C) was evaluated in potato dextrose agar formulated at water activity of 0.98 and pH 4.0. Mold growth curves were adequately fitted (0.984 < R(2) < 0.999) by the modified Gompertz model. The effect of the independent variables (concentration of essential oil and temperature) on the estimated model parameters (reciprocal of growth rate [1/ν(m)] and lag time [λ]) were evaluated through polynomial equations. Both ν(m) and λ were significantly (P < 0.05) affected by the independent variables; ν(m) decreased and λ increased as essential oil concentration increased and temperature decreased, which suggests that Mexican oregano essential oil retards or inhibits mold germination stage. Further, minimum fungistatic and fungicide essential oil concentrations at 30 and 35°C were determined. Mexican oregano essential oil applied in gas phase exerts important antifungal activity on the growth of A. flavus, suggesting its potential to inhibit other food spoilage molds.     

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.     

Inhibition of heat resistant molds: Aspergillus fumigatus and Paecilomyces variotii by some plant essential oils

In this study, the inhibitory properties of some essential oils including citrus (Citrus sinensis L. Osbeck), laurel (Laurus nobilis L.), myrtle (Myrtus communis L.), oregano (Origanum vulgare L.), and savory (Satureja thymbra L.) were investigated against the heat resistant molds Aspergillus fumigatus and Paecilomyces variotii isolated from margarine in a previous study in order to assess the potential for using these essential oils as a natural food preservative. In this study, the essential oils of the plants were obtained by steam distillation using Clevenger apparatus and were tested for antifungal activities at 0.25, 0.50, and 1.00% concentrations. Inhibitory effects of the essential oils on the growth of the fungi followed the sequence: oregano=citrus>savory>laurel>myrtle. P. variotii was more resistant against the essential oils than A. fumigatus.     

Antibacterial activity of different essential oils obtained from spices widely used in Mediterranean diet

Raw and processed foods are open to contamination during their production, sale and distribution. At present, therefore, a wide variety of chemical preservatives are used throughout the food industry to prevent the growth of food spoiling bacteria. However health and economic considerations have led to a search for alternatives, such as essentials oils that can safely be used as substitutes for fungicides and bactericides to partially or completely inhibit the growth of fungi and bacteria. The aim of this work was to determine the effectiveness of the essentials oils from oregano (Origanum vulgare), thyme (Thymus vulgaris), rosemary (Rosmarinus officinalis), sage (Salvia officinalis), cumin (Cuminum cyminum) and clove (Syzygium aromaticum) on the growth of some bacteria commonly used in the food industry, Lactobacillus curvatus, Lactobacillus sakei, Staphylococcus carnosus and Staphylococcus xylosus or related to food spoilage Enterobacter gergoviae, Enterobacter amnigenus. The agar disc diffusion method was used to determine the antibacterial activities of the oils. All six essential oils analysed had an inhibitory effect on the six tested bacteria. Oregano essential oil showed the highest inhibition effect followed by cumin and clove.     

Effects of Tropical Citrus Essential Oils on Growth, Aflatoxin Production, and Ultrastructure Alterations of Aspergillus flavus and Aspergillus parasiticus

Ethyl acetate extracts and hydrodistillated essential oils from five cultivars of tropical citrus epicarps were evaluated for their inhibitory activities against Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus, and Penicillium sp. using disk diffusion and broth microdilution assays. Essential oils prepared from kaffir lime (Citrus hystrix DC) and acid lime (Citrus aurantifolia Swingle) epicarps exhibited stronger antifungal activity to all fungi than their ethyl acetate extracts with minimum inhibitory concentration and minimum fungicidal concentration values of 0.56 and 1.13 mg/ml (dry matter), respectively, against aflatoxin-producing A. flavus and A. parasiticus. The dominant components of the essential oil from kaffir lime were limonene, citronellol, linalool, o-cymene, and camphene, whereas limonene and p-cymene were major components of acid lime essential oil. Pure limonene, citronellal, and citronellol were five to six times less fungicidal than the natural essential oils, indicating the synergistic activity of many active compounds present in the oils. Kaffir and acid lime essential oils significantly reduced aflatoxin production of A. flavus and A. parasiticus, particularly lime essential oil, which completely inhibited growth and aflatoxin production of A. flavus at the concentration of 2.25 mg/ml. Target cell damage caused by acid lime essential oil was investigated under transmission electron microscopy. Destructive alterations of plasma and nucleus membrane, loss of cytoplasm, vacuole fusion, and detachment of fibrillar layer were clearly exhibited in essential-oil-treated cells.     

Inhibition of Growth and Germination of C. botulinum 33A, 40B, and 1623E by Essential Oil of Spices

Effects of clove, thyme, black pepper, pimenta, origanum, garlic, onion, and cinnamon oils on growth and germination of Clostridium botulinum types 33A, 40B, and 1623E were studied. At 200 ppm, ail oils highly inhibited growth of C. botulinum 33A, 40B, and 1623E. At 10 ppm, inhibitory activity of most oils diminished. By activity on C. botulinum growth, oils could be divided into three categories: (1) very active: cinnamon, origanum, and clove; (2) active: pimenta, and thyme; (3) least active: garlic, onion, and black pepper. Effectiveness on germination was quite different. At 150 and 200 ppm all oils totally prevented germination. At 10 ppm garlic and onion showed higher activity than the others. Spores of 33A were more sensitive than 40B and 1623E.     

Biocontrol of Salmonella Typhimurium in RTE foods with the virulent bacteriophage FO1-E2

Antimicrobial agents can be incorporated into edible films to provide microbiological stability, since films can be used as carriers of a variety of additives to extend product shelf life and reduce the risk of microbial growth on food surfaces. Addition of antimicrobial agents to edible films offers advantages such as the use of small antimicrobial concentrations and low diffusion rates. The aim of this study was to evaluate inhibition by vapor contact of Aspergillus niger and Penicillium digitatum by selected concentrations of Mexican oregano (Lippia berlandieri Schauer), cinnamon (Cinnamomum verum) or lemongrass (Cymbopogon citratus) essential oils (EOs) added to amaranth, chitosan, or starch edible films. Essential oils were characterized by gas chromatography-mass spectrometry (GC/MS) analysis. Amaranth, chitosan and starch edible films were formulated with essential oil concentrations of 0.00, 0.25, 0.50, 0.75, 1.00, 2.00, or 4.00%. Antifungal activity was evaluated by determining the mold radial growth on agar media inoculated with A. niger and P. digitatum after exposure to vapors arising from essential oils added to amaranth, chitosan or starch films using the inverted lid technique. The modified Gompertz model adequately described mold growth curves (mean coefficient of determination 0.991 ± 0.05). Chitosan films exhibited better antifungal effectiveness (inhibition of A. niger with 0.25% of Mexican oregano and cinnamon EO; inhibition of P. digitatum with 0.50% EOs) than amaranth films (2.00 and 4.00% of cinnamon and Mexican oregano EO were needed to inhibit the studied molds, respectively). For chitosan and amaranth films a significant increase (p<0.05) of lag phase was observed among film concentrations while a significant decrease (p<0.05) of maximum specific growth was determined. Chitosan edible films incorporating Mexican oregano or cinnamon essential oil could improve the quality of foods by the action of the volatile compounds on surface growth of molds.     

Effects of essential oils and oleoresins of plants on ethanol production,respiration and sporulation of yeasts

Ethanol production, respiration, and sporulation of yeasts as effected by essential oils and oleoresins of allspice, cinnamon, clove, garlic, onion, oregano, savory, and thyme were investigated. Essential oils of allspice, cinnamon, and clove had little or no effect on ethanol production by Sacchraromyces cerevisiae. Oils of onion, oregano, savory, and thyme delayed and/or reduced the production of ethanol. Overall, essential oils effectively suppressed ethanol production by Hansenula anomala. At the highest concentrations tested (500 μg/ml), only cinnamon, clove, garlic and thyme oleoresins substantially delayed and/or reduced ethanol production by S. cerevisiae. Most of the essential oils (100 μm/ml) impaired the respiratory activity of S. cerevisiae as evidenced by a reduction in CO₂ production. Thyme oleoresin was the strongest inhibitor. Allspice and garlic oils impaired sporulation by H. anomala. All oils delayed sporulation of Lodderomyces elongisporus).     

Effects of Essential Oil Vapour Treatment on the Postharvest Disease Control and Different Defence Responses in Two Mango (<Emphasis Type="Italic">Mangifera indica</Emphasis> L.) Cultivars

The purpose of this study was to investigate the inhibitory effect of essential oils (thyme, clove and cinnamon) in vapour phase against the major fungal diseases of mango in vitro and in vivo. Thyme oil vapour (5 μL/Petri plate) completely inhibited the mycelial growth of Colletotrichum gloeosporioides and Lasiodiplodia theobromae under in vitro condition. Thyme oil vapour at 66.7 μL L^?1 significantly reduced artificially inoculated C. gloeosporioides and L. theobromae in mangoes for 4 days. GC/MS analysis revealed thymol, eugenol and benzofuran, 3-methyl as the dominant compounds in thyme, clove and cinnamon oils, respectively. The activities of defence and antioxidant enzymes including peroxidase, chitinase, phenylalanine ammonia-lyase, β-1,3-glucanase, catalase and superoxide dismutase were enhanced by thyme oil (66.7 μL L^?1) treatment and also help to maintain the phenolic content. Hence, postharvest thyme oil vapour treatment may prove to be an alternative means of controlling disease in mangoes.