essential oils against yeasts and moulds causing food spoilage

Essential oils against yeasts and moulds causing food spoilage

Judit Krisch1, Rentsenkhand Tserennadmid 2, Csaba Vágvölgyi3 1Institute of Food Engineering, Faculty of Engineering, University of Szeged, Mars tér 7., H-6724 Szeged, Hungary 2Institute of Biology, Mongolian Academy of Sciences, Ulaanbaatar-51, Mongolia 3Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52., H-6726 Szeged,

Hungary

In foodstuffs with low pH, low water activity, or high carbohydrate content spoilage bacteria are, with some exception, usually not present because this environment is unfavourable for their growth. Food-spoilage yeasts and moulds, however, can grow under these circumstances and cause deterioration of various products, such as fruit and vegetable juices and purees, soft drinks, pickled vegetables, dairy products, bread, dried fruits, sausages. Heat treatment and antiseptic packaging exclude yeast and mould spoilage as long as the packaging is intact. Products that cannot be pasteurized are usually treated with weak acid preservatives: sorbic, propionic or benzoic acid or their salts. However, there is a strong consumers’ demand to avoid or diminish the use of artificial substances in their food. Chemical preservatives also present some problems: it was recently reported that benzene can be formed from benzoic acid in foods by decarboxylating action of some spoilage microorganisms. The use of plant-derived essential oils (EOs) or their components as natural preservatives can overcome these problems. Most EOs are regarded as safe (GRAS) and are accepted by consumers. EOs can be added directly to the food or can be applied in active packaging in vapour phase. Both our experiments and data from the literature showed that EOs and their components increase the lag phases and diminish the maximum cell count in the stationary phase of yeast growth. The colony forming ability of moulds was also reduced or stopped by the EOs. The strong aroma of the EOs can affect the organoleptic properties of the foods but the synergistic combinations of EOs with each other or with other hurdle techniques can reduce this effect. Essential oils represent a natural, effective, and consumer-accepted tool against food spoilage caused by yeast and moulds.

Keywords yeasts; moulds; essential oils; food spoilage; antifungal

1. Food spoilage by yeasts and moulds

Spoilage fungi, yeasts and moulds can grow on raw and processed foods where the environmental conditions for most bacteria are unfavourable (low pH, low water activity, aw). The nutrients and oxygen available in the food are the main factors determining the kind of fungal spoilage. Moulds require oxygen for their growth, but dissolved oxygen in the foodstuffs is more important here than atmospheric oxygen tension. Fermentative yeasts are able to grow without oxygen. Moulds produce a vast number of enzymes: lipases, proteases, carbohydrases for the degradation of complex molecules, and can utilize nitrogen and carbon sources in many forms from nitrates to proteins and from simple sugars to complex carbohydrates. On the contrary, many types of yeast are unable to assimilate nitrate or complex carbohydrates such as starch, and require vitamins for their growth [1]. Ethanol fermenting yeasts, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces strains, cause the deterioration of fruit juices, soft drinks, fruit purees and dairy products. The film-forming yeast Pichia anomala has been reported to cause spoilage in wines, fruit juices, soft drinks, pickled vegetables, yoghurts, and cream-filled cakes. The filamentous yeast Geotrichum candidum can be found in raw milk used for production of soft cheeses and other dairy products, and causes a bitter taste [1-3]. The yeast Endomyces fibuliger, called “chalk mold”, is an important spoiler of rye bread [4]. Mould growth on raw or processed foods leads to textural and sensorial changes: softening, off-odours and off-flavours. The most important aspect is, however, the formation of mycotoxins. Mycotoxins are secondary fungal metabolites and are toxic to humans and animals, causing severe disorders like cancer, immune suppression, or endocrine disruption. Since mycotoxins are very stable and mainly resistant against heat treatment and acidic environment, they remain in the food during processing and storage, causing a serious food safety problem [5]. The main mould spoilers in fruits and vegetables are Mucor and Rhizopus species from Zygomycetes, and Aspergillus and Penicillium species from Ascomycetes. Alternaria alternata and Botrytis cinerea are also very common causes of fungal rot in fruits. Aspergillus, Penicillium and Fusarium are the main associated fungi of wheat, rye and corn grains under field and storage conditions. P. commune and P. nalgiovense is associated with cheeses, fermented sausages and salamis. Mycotoxins found in juices made from pomaceous or stone fruits are patulin and citrinin. Patulin is a strong antibiotic but it is toxic to humans. In cereals, flours and bakery products ochratoxin A, aflatoxin and the Fusarium toxins: deoxynivalenol (DON), zearalenone and fumonisins can be found [5]. Yeast and mould spoilage results in considerable loss in food supply and enhances food safety problems.

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2. Classic methods for prevention of fungal growth in foodstuffs

The prevention of fungal growth on crops means spraying fungicides over the fields. Most of these fungicides are synthetic chemicals with a directed site-specific effect on the pests; mainly by inhibiting an important metabolic pathway. Unfortunately, fungi can develop resistance against these fungicides and non-target organisms, insects, birds, mammals are also affected. Sometimes the degradation products of these chemicals are more toxic to humans than the parent molecules. Fungicide residues in foods present a food safety risk. For fruit and vegetable juices and purees, heat treatment, pasteurization, and antiseptic packaging are used to avoid microbiological deterioration. Breads and bakery products are baked at high temperatures but some heat resistant fungal spores can survive. Fungal contamination of heat treated and packaged foods occur with airborne spores, mainly after opening the packaging. In many processed foods where heat treatment is not recommended, but also in heat-treated commodities, preservatives are added to prevent microbial growth. These preservatives are mainly weak organic acids and their salts, or sulphites. Weak acids can penetrate the cell membrane in their undissociated form. In the cell, the acids dissociate and lower the cytoplasmic pH, but glycolysis and respiratory pathways are also affected. Foods with acidic pH are successfully treated with these preservatives because most weak acids are in undissociated form below pH 5 [6]. However, a growing number of consumers refuse the use of synthetic chemicals in their daily food, and there are also other problems with preservatives. Benzoic acid can be transformed by decarboxylation into benzene, one of the most carcinogenic substances. Yeasts and moulds are able to degrade sorbic acid to 1,3-pentadiene, causing a kerosene-like off-odour [5, 6]. S. pombe may produce off-flavours when sulphite is used as preservative in wines. There is growing interest to replace synthetic pesticides and preservatives, at least partly, by natural compounds possessing the same inhibitory effect.

3. Essential oils

Essential oils (EOs) are plant-derived volatiles with a hydrophobic character. They are extracted from various plant organs; leaves, fruits, flowers, bulbs, seeds, roots, wood and bark of aromatic plants. EOs are known to possess antiviral, antibacterial, antifungal and insecticide properties [7]. They can have more than 50 components, of which 1-3 are the main components representing 85-95% of the whole volume, while the others are minor components, sometimes below 1%. The chemical character of the compounds influences their antimicrobial efficacy and the mechanism of action on the target organism. The two main groups of EOs are terpenes and terpenoids, and aromatic and aliphatic constituents [8]. EOs have several targets in the cell. Degradation of the cell wall, and weakening the membrane causing enhanced permeability, lead to the loss of intracellular components. In Candia albicans yeast cells, tea tree oil increased the plasma membrane permeability that led to the loss of chemiosmotic control [9]. Lesion formation in the membrane and considerable reduction of ergosterol content (the major sterol component in fungal cell membrane) was reported for Thymus pulegoides (thyme) EO in Candida albicans [10]. Genes involved in ergosterol biosynthesis and sterol uptake were affected by α-terpinene, a cyclic monoterpene in S. cerevisiae [11]. EOs can also react with important cell membrane proteins depleting their function [7]. The hydroxyl groups of phenolic and alcoholic EO components can form hydrogen bonds with amino acid residues in the active site of enzymes [12, 13]. Enzymes in the energy regulation can be involved: the monoterpenes -pinene and limonene inhibited the respiratory activity in intact yeast cells and also in isolated mitochondria [14]. Spore formation of Aspergillus species was reduced by lemongrass [15] and cassia, cinnamon or clove EO [16]. Spore germination of Aspergillus species, as well as B. cinerea, Cladosporium herbarum and Rhizopus stolonifer, was also inhibited by lemongrass and oregano EO [17, 18]. Hyphal morphology of Phytophtora infestans causing late blight disease of tomato was affected by thyme, lavender and rosemary oil leading to cytoplasmic coagulation, vacuolization, hyphal shrivelling and protoplast leakage [19]. It seems that the disruption of ergosterol biosynthesis in fungi, similarly to the action of azole fungicides, contributes to the antifungal activity of EOs.

4. Methods for determination of the antifungal activity of essential oils

4.1 Agar well and paper disc diffusion methods

In these methods, agar plates are overlaid with yeast cell or fungal spore suspensions (usually in the concentration of 104-106 cfu/ml). After drying, wells are cut into the agar with a sterile cork borer and filled with EOs solved in methanol, ethanol, dimethyl-sulfoxide (DMSO) or in some other solvent; or sterile paper discs containing the appropriate amount of the investigated EO are laid over the inoculated medium. After incubation, the diameter of the inhibition zones is measured. These methods are useful for screening or preliminary testing of antimicrobial effect on a large number of EOs or microorganisms. The type of solvent and the thickness of the medium can affect the diffusion rate of the EOs so that comparison of literature data is not feasible [7].

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4.2 Determination of growth parameters

For yeasts, duration of lag phase and growth rate is determined in liquid culture medium in the absence and presence of the investigated EO. Essential oils are added to the medium directly or a solvent is used. Yeast growth is monitored by absorbance measurements or by viable cell count. Absorbance change or logarithmic viable cell number is plotted against time. Growth rate (1/h) is calculated from the slope of the straight line fitted to the growth curves in the exponential phase. Lag phase is determined as the X intercept of this straight line. In the case of moulds, changes in the colony diameter during the incubation period are measured and expressed as colony growth rate (mm/day). Two different methods are used for the evaluation of the antifungal effect of EOs: poisoned food or agar dilution technique and the inverted Petri dish technique. In the first method, the EOs are mixed to the medium, while in the latter the moulds grow in the vapor phase of the EO (a paper disc soaked in the EO is placed on the centre of the lid, the Petri dish sealed with parafilm, and is incubated in inverted position) [20, 21].

4.3 Determination of growth inhibition

In these methods, the microorganisms are incubated for a given period (24-48 h for yeasts, 72-96 h or more for moulds) and the achieved amount in the presence of EO is compared to the amount in the control without EOs.

4.3.1 Broth dilution methods

In broth macro- or microdilution assays cell density is measured and the absorbance of control is taken as 100%. Sometimes viable cell count is also determined [7].

4.3.2 Dry weight determination

Moulds are cultured in liquid medium with and without EOs. After the incubation period the mycelium is filtered and dried in oven to constant weight. Inhibition is calculated from the dry weight of the mycelia, taking the control for 100% growth [22, 23].

4.3.3 Colony growth measurements

A fungal disc cut from the periphery of a 3-7 days old culture is placed on the centre of a solid medium. Poisoned food or inverted Petri dish technique is used. After incubation the colony diameters are measured, and the percentage of inhibition is calculated by the formula: (dc-dt)/dc x 100, where dc is the average diameter of the control fungal colony and dt is the average diameter of treated fungal colony [24].

4.4 Determination of minimum inhibitory concentration (MIC)

Macro- or micro-dilution assays or agar dilution techniques are used to determine the MIC value. In different publications MIC is defined in different terms. In macro-dilution assays MIC is the lowest EO concentration where no visible growth occurs. In microdilution assays (where absorbance is measured) MIC is usually defined as the lowest concentration where >90 % growth inhibition is determined. In agar dilution tests MIC is the lowest EO concentration where no colony growth is observed at the end of the incubation period [7, 24]. The nature of MIC (fungistatic or fungicidal) is determined by re-inoculation of the fungi into fresh medium.

4.5 Determination of the effect of EO combinations – the checkerboard method

The checkerboard method is performed by macro or microdilution assay. Dilutions of different EOs (usually 2 EOs are used) are combined with each other in all possible combinations. After incubation, growth or no growth is determined visually or by absorbance measurements. For an EO pair, the lowest concentration of the one component (A) which caused no growth in the presence of the other component (B) is determined. These values are divided by the MICs for component A and B, yielding FICA (=MICA+B/MICA) and FICB, respectively (FIC is the fractional inhibitory concentration). FIC index (FICI) is calculated as the sum of FICA+FICB [25]. Results are interpreted as synergy (FICI<0.5), addition (0.5≤FICI≤1), indifference (1<FICI≤4) or antagonism (FICI>4).

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5. Anti-yeast activity of essential oils

Anti-yeast effect of EOs was mainly tested by disc diffusion methods. Numerous EOs have been investigated and it is hard to establish an order based on anti-yeast activity, but oregano and thyme EOs are apparently among the best inhibitors [26-29]. These EOs contain the phenolic compounds carvacrol and thymol as main constituents with membrane disrupting ability. Other EOs, such as juniper, lemon, marjoram, clary sage, basil, ginger or lemon balm, containing non-phenolic main compounds have been also found to show high toxicity against yeasts [28-31]. According to Sachetti [28], the susceptibility of yeasts against 12 EOs in decreasing order was: S. pombe > S. cerevisiae > Yarrowia lipolytica > Rhodotorula glutinis with MIC values from 0.03 mg/ml up to 0.54 mg/ml. Similar results were found in another study where the decreasing order of sensitivity was: S. pombe > S. cerevisiae > Pichia anomala > Geotrichum candidum; with MIC values from 0.0625 μl/ml to 2.0 μl/ml [30]. MIC values for cumin, cassia, allspice and thyme against Candida species and S. cerevisiae varied between 0.04 μl/ml and 1.25 μl/ml [27]. EO components were also investigated for anti-yeast activity: the MIC for the monoterpenes α-terpinene and limonene against Kluyveromyces and Candida strains varied in a broad range from 4.9 to 312 μg/ml [32]. In general, MIC of the main components of the EOs was higher than the MIC for the parent EOs, suggesting the synergistic effect of EO components. It seems that monoterpenes (α-pinene, β-pinene, α-terpinene) play, beside phenolics, a considerable role in disturbing the membrane function in yeasts [9, 11, 32].

6. Anti-moulds activity of essential oils

Moulds can attack crops under pre- or post harvest conditions or can spoil processed foods. Since discovery of the mycotoxins and their effect on human health, intensive research has been done for preventing mould contamination and spoilage. The most frequently investigated species in the antifungal tests are strains from the Aspergillus, Penicillium and Fusarium genera. A broad spectrum of EOs and EO components has been used against moulds: basil, citrus EOs, fennel, lemongrass, oregano, rosemary, thyme etc. It is difficult to compare the antifungal activities of the different EOs because MICs are given in different units like ppm, mg/ml, %, μg/ml or μl/ml. In Table 1, MICs published in ppm units are presented. In some cases μl/ml units were converted into ppm to give a base for comparison. It can be seen that even the same EO against the same mould gave different MIC values, suggesting a need for standardization of antifungal methods. In mycotoxigenic fungi, not only growth inhibition but also the reduction of toxin production was investigated (Table 2). In many cases the EO concentration for total inhibition of toxin production was below the MIC for colony growth, suggesting that enzymes involved in the mycotoxin pathway are targets of the EOs. In the antifungal tests, not only the rate of mycelial growth reduction is determined but the inhibition of sporulation and spore germination is also investigated. The spore formation of A. flavus was totally inhibited by lemongrass EO at the concentration of 2.80 mg/ml [15]. Spore germination of different Aspergillus species was inhibited by oregano EO at 40-80 μl/ml concentration [17] and germination of B. cinerea, Cladosporium herbarum and R. stolonifer was inhibited by lemongrass at 500 ppm concentration [18]. Sometimes EOs can accelerate spore germination: lemongrass up to 100 ppm accelerated the germination of A. niger conidia. Spore germination of the citrus fruit post harvest pathogen Penicillium digitatum is stimulated by the combination of limonene, α-pinene, sabinene, β-myrcene, acetaldehyde, ethanol, ethylene and CO2, released from the wounded orange peel [5]. In many cases, EOs in volatile phase (inverted Petri dish method) are more efficient than dissolved in the solid medium. The MIC of laurel EO against Phytophtora infestans was 2.0 μg/ml air in volatile phase but 51.2 μg/ml in contact phase [19]. Environmental factors such as water activity and temperature are also important in the antifungal and anti-mycotoxigenic effect of EOs. Fumonisin B1 production by F. proliferatum, and zearalenone and DON production by F. graminearum, were more strongly inhibited by EOs at high water activity (aw = 0.995) than at aw = 0.950 [33, 34]. The role of temperature was more pronounced at higher water activity. The higher the water activity of the grain was, the better was the inhibition effect of the EOs; so they could be used as pre-harvest agents against moulds.



Table 1 Minimum inhibitory concentration (MIC) of various essential oils against moulds

Mould Essential oil MIC (ppm) References Alternaria alternate thyme 700 [35] Alternaria solani clary sage 3200 [36] Aspergillus flavus anise 500 [37] basil 800; 3000 [37, 38] cinnamon 1000 [37] lemongrass 1200 [38] oregano 20 000 – 40 000a [17]b thyme 250; 700; 1000 [35, 37, 38] A. fumigatus basil 600 [38] lemongrass 1200 [38] oregano 80 000 [17] thyme 600/700 [35, 38] A. niger oregano 40 000 [17] A. ochraceus anise 500 [37] basil 3000 [37] cinnamon 1000 [37] lemongrass 500 [18] oregano 40 000 [17] thyme 500 [37] A. parasiticus anise 500 [37] basil 3000 [37] cinnamon 1000 [37] oregano 40 000 – 80 000a [17]b thyme 500 [37] A. terreus oregano 40 000 [17] thyme 700 [37] Botrytis cinerea clary sage 1600 [36] lemongrass 500 [18] Cladosporium herbarum lemongrass 500 [18] Fusarium moniliforme anise 500 [37] basil 500/3000 [37, 38] cinnamon 1000 [37] lemongrass 500 [38] thyme 250/500 [37, 38] Fusarium oxysporum clary sage 3200 [36] thyme 700 [35] Rhizoctonia solani clary sage 800 [36]

a Different strains were used b MIC was converted from μl/ml to ppm

7. Antifungal activity of essential oils in real foods

7. 1 EOs in fruit and vegetable juices, soft drinks and purees

Lemon oil was used to extended “open” shelf life of clear and cloudy apple juice. Lag phases of the fermentative yeasts S. cerevisiae and S. pombe was significantly lengthened, especially in clear apple juice [30]. The taste of the product was evaluated as refreshing and harmonic. In citrus based non-carbonated beverages the combination of linalool and β-pinene, together with a mild (55 °C) heat treatment, led to a lower spoilage probability. The used concentrations of the EO components (40 and 60 μl/L) had no negative impact on the flavour of the beverages, and the mild thermal treatment below the usual temperature range (65-75 °C) reduced the energy costs of the product [39, 40]. In tomato paste 500 ppm thyme EO reduced the growth of A. flavus by 87%. The taste was accepted by panellists [41].

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Table 2 Inhibition of mycotoxin production by the EOs

Mould Mycotoxin Essential oil MIC Mycotoxin total inhibition

References

A. flavus aflatoxins lemongrass 1 mg/ml 0.1 mg/ml [15] A. flavus Aflatoxin B1 thyme 0.7 μl/ml 0.6 μl/ml [35] A. flavus + A. parasiticus

aflatoxins anise 500 ppm 1% [37]

cinnamon 1000 ppm 2% spearmint 3000 ppm >2% thyme 250 ppm 1% A. parasiticus Aflatoxin B1 sweet basil 5% (v/v) 5% (v/v) [22] Aflatoxin G1 sweet basil 5% (v/v) 5% (v/v) A. ochraceus ochratoxin A anise 500 ppm 1% [37] cinnamon 1000 ppm 2% spearmint 2000 ppm 2% thyme 500 ppm 1% Fusarium moniliforme

fumonisins anise 500 ppm 2% [37]

cinnamon 1000 ppm 2% spearmint 3000 ppm 2% thyme 250 ppm 2%

7.2 Essential oils as pre- and post harvest preservation agents

Soft fruits are highly perishable products with a short shelf life, even under refrigeration. Essential oils can be used to prolong the shelf life of these fruits, in vapour phase in active packaging or as coating on the surface of the fruits. Cinnamaldehyde vapour used in the packaging of apricot reduced Rhizopus fungal rot on inoculated fruits [42]. Citral at 0.5% concentration inhibited the fungal growth of Colletotrichum gloesporoides on papaya to 70% [43]. Spraying summer savory oil (6.25 μl/ml) on lemon fruit 7 days before pathogen inoculation prevented decay completely for 20 days [44]. Whole sweet basil leaves added to sorghum, groundnut, maize and melon seeds reduced aflatoxin production by 89-91% for 35 days [22]. Oregano, cinnamon, clove, lemongrass and palmarose EOs can be used as pre-harvest natural fungicides on maize grain under field conditions [33, 34].

7.3 Essential oils and bread

Sliced bread is very sensitive to mould spoilage. Mustard and lemongrass EO in modified atmosphere (MAP) active packaging reduced the growth of the inoculated bread spoilage fungi, P. communis, P. roqueforti, A. flavus and E. fibuliges on rye and wheat bread [4, 45]. It was reported that smaller, more volatile compounds, such as allyl-isothiocianate from mustard EO, were more effective in vapour phase than added to the substrate [46, 47] therefore; EOs in the packaging could be more effective than in the dough. Taste changes posed a limit to the application of EOs in bakery products.

8. Conclusions

Essential oils represent an alternative to synthetic preservatives in the food industry against spoilage yeasts and moulds. Most investigated fungi showed some (higher or lower) sensitivity to EOs or EO components. It is difficult to compare the results of antifungal tests because of the diversity of methods and units used. It seems that the main target of EOs is the cell membrane also in fungi, causing increased permeability and disruption of membrane integrity. Both monoterpenes and phenolics are involved in the action against the cell membrane and key enzymes important for energy regulation or synthetic pathways; and mycotoxin production of moulds is also affected by the EOs. Spore formation and germination is sometimes accelerated by EOS, especially in case of fungi that can attack aromatic plants or fruits. The choice of EO and its concentration in a particular food is important because a small amount can cause sensory alterations. Combinations of EOs with each other or with other preservation techniques can solve this problem. Although the literature data about the antimicrobial effect of EOs are ample there are new areas of application to be discovered.

Acknowledgment This work was supported in part by the grant NKTH TéT MN-1/2009.



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essential oils against yeasts and moulds causing food spoilage

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