Control of Penicillium sp . on the Surface of Italian Salami Using Essential Oils

Condiments have always been used in the food industry to preserve food. Consumer concerns over excessive use of synthetic additives has motivated the industry to seek for new alternatives to reduce condiment use. In this context, the use of natural products in food preservation is a promising alternative to ensure food quality, extend shelf life and meet the needs and expectations of consumers (1). Among industrialised foods, Italian salami has a signifi cant share of the meat market (2); however, the characteristic presence of mould is a natural consequence of the technological process of salami manufacturing (3).


Introduction
Condiments have always been used in the food industry to preserve food.Consumer concerns over excessive use of synthetic additives has motivated the industry to seek for new alternatives to reduce condiment use.In this context, the use of natural products in food preservation is a promising alternative to ensure food quality, extend shelf life and meet the needs and expectations of consumers (1).Among industrialised foods, Italian salami has a signifi cant share of the meat market (2); however, the characteristic presence of mould is a natural consequence of the technological process of salami manufacturing (3).
The growth of fi lamentous fungi on the surface of salami during ripening is considered a quality factor that should complement the biochemical changes involved in the maturation of the product.Among these biochemical changes are the typical fl avour produced by the oxidation of lactate, amino acid degradation and proteolysis, lipolysis, oxygen consumption, protection against light and colonization by undesirable mould (4,5).
However, many of these fungi can cause changes in colour and fl avour and also represent a public health problem because of toxin production (6).Contaminating fungi are green, brown or black, colours that are unacceptable to most consumers, and may also have a negative impact on fl avour or delay curing time (7).Green moulds are usually Penicillium and Aspergillus, and brown or black moulds are Cladosporium, Alternaria and Aspergillus (6).Natamycin (pimaricin) is used in the food industry for prevention against harmful fungi and can be sprayed on the surface of cheese and meat products (3).However, this treatment adds a signifi cant cost and, at high doses, can cause nausea, vomiting and diarrhoea (8).
Essential oils derived from secondary plant metabolism are composed of a mixture of compounds such as monoterpenes, sesquiterpenes, phenyl propanoids, alcohols, aldehydes, esters, ketones, phenols and oxides.These metabolites confer organoleptic characteristics, have therapeutic and pharmacological activity and are also studied for their antimicrobial, antifungal, cytotoxic, anti-infl ammatory and insecticide potential (9).
Studies on the antifungal activity of essential oils in food are scarce in the literature; however, this issue has aroused enormous interest industrially and commercially.Given the importance of studies related to the manufacture of safe and healthy food products and the emphasis on the use of natural preservatives and additives, this study aims to evaluate the in vitro antifungal activity of the oils of rosemary (Rosmarinus offi cinalis), salvia (Salvia sclarea), oregano (Origanum vulgare) and clove (Eugenia caryophyllata).The oils with the highest antifungal activity in vitro were tested as curative treatments for controlling harmful fungi on the surface of Italian salamis in an industrial plant.Subsequently, the salamis treated with different volume fractions of the oils were subjected to a sensory analysis of fl avour.

Collection and isolation of a fi lamentous fungus, an industrial contaminant
A contaminating fungus was collected in the curing room of an Italian salami industrial plant and immediately inoculated on potato dextrose agar (PDA) plates and incubated in a bacteriological oven (model TE-393/2, Tecnal Piracicaba-SP, Brazil) at 30 °C for 72 h.Aft erwards, the fungus was transferred to the PDA with chloramphenicol to isolate the contaminant (a green mould, most likely Penicillium verrucosum) from the inoculated fungus (a white mould, P. nalgiovense), based on its macroscopic characteristics, aft er incubation at 30 °C for seven days (10).The procedure was repeated until the contaminant was isolated.

Characterization and identifi cation of the fungus
Fungal identifi cation was based on classical taxonomy (6) via morphological examination (macroscopic and microscopic).For microscopic identifi cation, we used a microcultivation technique (11).The fungus was inoculated on a slice of agar laid on a sterile glass slide and covered with a sterile coverslip.The slide was then placed in a Petri dish and incubated for 5 days at 25 °C.The coverslip containing adhered hyphae was withdrawn and stained with Cott on Blue dye.The same procedure was adopted to examine spores and hyphae bound to the slide.For a morphological description of Penicillium, we used the criteria adopted by Pitt (12), Frisvald and Filtenborg (13) and Samson and Pitt (14).A molecular analysis for their identifi cation was not performed because the scope of the study was to establish the eff ects of the essential oil on fungi during the industrial production of salami, regardless of contamination.
The column temperature was programmed to 50 °C for 3 min, increased by 5 °C/min to 130 °C and then by 15 °C/min to 210 °C, and held for 5 min.Helium was used as the carrier gas, and the detector and injector temperatures were 250 °C.A volume of 0.5 mL was injected into the GC-MS system.This device operated at a fl ow rate of 1 mL/min with an electron impact of 70 eV in split (split ratio 1:3) mode.Compounds were identifi ed by comparing the mass spectra with those in the Wiley library (15) using the soft ware provided with the equipment and by comparison with the GC retention times of standard compounds.
The inhibition zone of mycelial growth was evaluated on agar plates according to Silva and Bastos (16).A preinoculum (10 6 spores/mL) was added to the PDA medium before solidifi cation at a ratio of 1:10 (10 5 spores/ mL) and then poured into sterile Petri dishes.Aft er solidifi cation, four cavities were made with sterile glass tubes (diameter 6 mm).
In each cavity, 50 μL of diluted oil were added, 50 μL of Tween 80 (1 %) were used as a negative control, and 50 μL of the antifungal ketoconazole were used as a standard positive control.The system was then incubated for 72 h at 30 °C.At the end of the incubation period, the diameter of each inhibition zone was measured in millimetres.Six replicates of each experiment were conducted.

Preparation of Italian salami
T he salamis were prepared according to the standard formulation of the food industry, which is registered at the Ministry of Agriculture, Livestock and Food Supply of Brazil.The normal procedure in the industrial manufacturing plant was followed until the curing phase.Once the salamis entered the curing room, they were monitored daily.Fift een days aft er the placement in the curing room, the appearance of contaminating fungi on salami that had been spray-coated with test oil was observed.

Application of oils on the tips of salami
The essential oils that showed the best results in the in vitro tests were sprayed on the tips of salami previously inoculated with Penicillium nalgiovense that showed visual contamination with the green fungus in the industrial plant.The following oils and volume fractions were utilized (10 replicates for each treatment were performed): clove oil at 125 and 250 μL/mL, oregano oil at 250 and 500 μL/mL and a mixture (1:1 by volume) of the two oils at 100 μL/mL.Other 20 ends of salami were left untreated as a negative control for visual and fl avour analyses.The dilutions were prepared with distilled water and 1 % Tween 80.The diff erent volume fractions were applied by spraying (100±10) μL/cm 2 , without overlapping the spray.
For the visual analysis, dilutions were applied to the tips of the salami (5 cm), where fungal growth generally occurs.For the sensory analysis of fl avour, the oil was applied to the entire salami.Aft er the oil was applied, the salamis were stored for 15 days in the curing room and then collected for the visual and fl avour analyses.

Sensory characteristics
Forty untrained panellists of both genders, aged between 22 and 50, participated in the sensory analyses.They were all consumers of Italian salami and familiar with it.

Flavour analysis
The fl avour analysis was carried out to determine if there was a noticeable diff erence between the control sample (without oil) and the samples treated with the essential oils as described above.A paired comparison test was employed.

Visual analysis
Visual analysis was performed by comparing the samples to the control (17).The presence of fungi in the clip (tip) region of Italian salami treated with 125 and 250 μL/mL of clove oil, 250 and 500 μL/mL of oregano oil or 100 μL/mL of the mixture (1:1 by volume) of the two oils was monitored.The tests were performed individually under white light by comparing the clip region of the control with clip regions of fi ve samples, each coded with a random three-digit number (a sample of each dilution is described in the section Application of oils on the tips of salami).
The panellists were asked to carefully observe the clip region of the coded samples, comparing them with the untreated standard (control), and to evaluate the degree of diff erence between the standard and the coded samples using a mixed, structured 8-point scale, ranging from no growth (grade 0) to fungal growth equivalent to that on the control sample (grade 8).

Statistical analysis
The in vitro evaluation of the antifungal eff ects of essential oils was performed in six replicates, and the aver-age values were obtained by analysis of variance (ANO-VA) and compared with Tukey's test (p<0.05)using the SPSS Statistics soft ware package (IBM Corporation, Armonk, NY, USA).
The results of the visual sensory analysis of treated samples were compared with standard sample (control) to test the diff erences (Dunnett 's test with 95 % confi dence intervals).The results of the fl avour analysis were evaluated with a paired comparison test that analysed the signifi cance of the number of hits (other than the standard) in relation to the number of errors (17).

Isolation and identifi cation of contaminating fungi
The white mould isolated in the curing room of the industrial plant was identifi ed as Penicillium sp., which is most likely Penicillium nalgiovense because this fungus had been deliberately inoculated into the product at the plant where the collections were performed.
The green mould was identifi ed as Penicillium sp., most likely Penicillium verrucosum, but it was not molecularly identifi ed.According to Andersen (18) and Bremmelgaard (19), these fungi can produce black or brown spots that are not acceptable to consumers, have a negative impact on aroma and fl avour and are associated with the production of penicillin, which can cause allergies when consumed in large quantities.

Chemical composition of the essential oils
The essential oil of clove contained the volatile compound eugenol as the major component (89.58 %).This result was similar to the results obtained by Silvestri et al. (20), who reported that eugenol represented 90.3 % of the volatile compounds.Arenas et al. (21) analysed clove essential oil and also found eugenol as the principal component but at a lower percentage (60.5 %).
The phenolic monoterpenoid carvacrol, which has known antimicrobial activity, was the major component found in oregano essential oil (60.71 %).Silva et al. (22) evaluated the essential oil of oregano of fi ve diff erent commercial brands, and all of the chromatograms showed one major peak with a retention time similar to a carvacrol standard; the peaks were identifi ed as carvacrol, which was present in percentages ranging between 61.7 and 93.4 % of the total volatile compounds.Busatt a et al. (23) found only 11.67 % carvacrol in oregano essential oil.The major compounds found in the essential oils of clove and sage were bornyl acetate (39.64 %) and linalool (39.26 %), respectively.
In the 1:1 combination of essential oils, the major component was eugenol (56.42 %), while the second highest component was carvacrol (15.39 %).

Antifungal activity of essential oils
The results of the antifungal activity of the essential oils tested on the isolated green mould (Penicillium sp.) are shown in Table 1.Rosemary oil had an inhibitory effect only at 1000 μL/mL, producing an inhibition zone of 9.6 mm.
Hillen et al. (24) studied the eff ect of essential oils extracted from diff erent species, including Rosmarinus offi cinalis (rosemary), on the mycelial growth of fungi (Alternaria carthami, Alternaria sp. and Rhizoctonia solani).Candle and rosemary oils were more eff ective when aliquots larger than 200 μL were added.
Pereira et al. (25) also evaluated the in vitro effi cacy of diff erent essential oils, including rosemary (Rosmarinus offi cinalis L.), on the mycelial growth of the fungi Aspergillus niger, Aspergillus ochraceus, Aspergillus fl avus and Fusarium sp.They concluded that the rate of inhibition of the mycelial growth of the fungus A. ochraceus by rosemary oil was proportional to the volume fraction tested.Mycelial growth of the fungi Fusarium sp. and A. fl avus was aff ected at volume fractions of 1500 and 2000 μL/mL, respectively.However, the mycelial growth of the fungus A. niger was not aff ected at any volume fractions tested.Si milar results were obtained in this study -rosemary oil showed an inhibition zone only at a volume fraction of 1000 μL/mL. 1 indicate that the sage oil produced an inhibitory eff ect at volume fractions of 500, 750 and 1000 μL/mL, with inhibition zones of 10.5, 11.2 and 12.2 mm in diameter, respectively.Pozzatt i et al. (26) investigated the antifungal activity of the essential oil of sage and failed to demonstrate any antifungal activity of this oil against various strains of Candida spp.

The results in Table
The best results were obtained with oregano oil at higher volume fractions (from 500 to 1000 μL/mL), followed by clove oil.These oils were superior to rosemary and sage oils at all volume fractions, producing zones of inhibition even at the lowest volume fractions test ed, thus demonstrating their potential as fungicides (Table 1).A gradual increase in the diameter of the inhibition zones with increasing volume fraction was observed, up to 750 μL/ mL of oregano and up to 500 μL/mL of clove oil.
Omidbeygi et al. (27) tested the antifungal activity of the essential oils of thyme and clove on the development of Aspergillus.The essential oils were added at volume fractions of 0, 50, 200, 350 and 500 ppm.The results showed that both essential oils inhibited the growth of the microorganism.
Pereira et al. (25) evaluated the in vitro eff ects of condiment essential oils, including oregano (Origanum vulgare L.), on the mycelial growth of the fungi Aspergillus niger, Aspergillus ochraceus, Aspergillus fl avus and Fusarium sp.They reported that all fungi were signifi cantly inhibited by 500 mg/mL of oregano oil with the exception of A. niger, which showed a decrease only at a mass concentration of 1000 mg/mL, demonstrating the antifungal power of oregano.
Several studies have shown the capacity of essential oils to inhibit fungal growth; however, the mechanisms by which the oils prevent the growth of microorganisms have rarely been explained.Some authors claim that the antifungal action of the oils is linked to disturbances of the cytoplasmic membrane, changes that cause alterations in membrane proteins, inhibition of the proton motive force, electron fl ow, active transport and coagulation of cellular contents.Others explain further that the antifungal activity is related to the hydrophobic characteristics of the oil, which helps to break down the cell membrane and mitochondrial lipids, causing changes in the structure and increasing permeability, which in turn causes the leakage of cellular components such as ions (28)(29)(30)(31).

Analyses of Italian salami
Sensory analysis of fl avour Although essential oils have been extensively studied, their use in foods as antimicrobial substances has been quite limited due to changes in fl avour because the doses eff ective against microorganisms can also change the acceptability of the product.Consequently, a growing demand is present to determine the volume fractions of essential oils that achieve a balance between antimicrobial effi cacy and sensory acceptability (32).In this regard, a sensory analysis of fl avour of Italian salami with the applied diff erent volume fractions of the essential oils of clove and oregano was performed to determine how much oil could be used in the product without producing a signifi cant change of fl avour.The results of the fl avour analysis are shown in Table 2.
The fl avour analysis showed that the samples treated with 125 μL/mL of clove oil or a mixture of clove oil and oregano (1:1) at a dose of 100 μL/mL showed no significant diff erences in fl avour when compared to the control sample (p>0.05); that is, the panellists did not notice any fl avour diff erences between the treated samples and the control (without added oil), indicating that the oil could be used at that volume fraction without changing the standard product fl avour.A volume fraction of 250 μL/ mL changed the fl avour of the product, so it was found unacceptable.

Visual analysis of fungal growth on salami
Fig. 1 shows the tip regions of the analysed salami samples.The visual analysis indicated that, compared to the control, there were signifi cant diff erences among the samples in fungal growth in the region of the salami where the oil was applied.A large reduction in fungal growth was observed in the oil-treated samples with scores lower than 2 (Table 3).
It was observed that 125 μL/mL of clove oil or a mixture of 100 μL/mL of clove oil and oregano oil (1:1) had the best overall eff ect on the appearance and fl avour, and can be recommended as a curative treatment to control the growth of undesirable fungi on the surface of salami during the curing process without changing the original fl avour characteristics of the product.These results further indicate that the major compound present in the essential oil of oregano, carvacrol, is primarily responsible for the high antifungal activity, although a synergistic effect of eugenol could be present in the treatment that used the mixture of clove oil and oregano oil.

Conclusions
The results of the in vitro analyses indicated that rosemary and sage oil did not have a pronounced antifungal eff ect but that clove and oregano oil were inhibitory even at lower volume fractions.The fl avour evaluation showed that 250 μL/mL of clove oil or 500 and 250 μL/mL of oregano oil caused noticeable fl avour diff erences compared to the untreated salami.However, this result does not indicate that the fl avour caused by the oil was unacceptable but only that a noticeable diff erence was present.However, samples treated with 125 μL/mL of clove oil or 100 μL/ mL of the mixture of clove and oregano oil (1:1) showed no signifi cant fl avour changes when compared to the untreated sample.Visual analysis showed that the sprayed oil inhibited the growth of the fungi and that the inhibition of the fungal growth was visually perceptible when samples with added oil and untreated samples were compared.Mean values followed by the same lett er do not diff er signifi cantly from the control sample (standard) with 95 % confi dence (Dun nett ´s test).Score range: 0=without growth, 2=large reduction of the standard, 4=moderate reduction of the standard, 6=small reduction of the standard, 8=equal to standard.N(panellists)=40 ISSN 1330-9862 scientifi c note doi: 10.17113/ft b.53.03.15.3877

Table 1 .
Mean diameter of inhibition zones of the fungus Penicillium sp. when using diff erent volume fractions of essential oils (17) a signifi cant diff erence (p<0.05),minimum of correct answers of the panellists is 26(17).N(panellists)=40

Table 3 .
Visual analysis scores of fungal growth on salami treated with diff erent volume fractions of essential oils compared to control sample (without essential oil)