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Scientific
Publications - Work Done by Microbiology Reader
Letters in Applied Microbiology, 2002, 34 (1), pp. 27-31 Antimicrobial effect of essential oils on the seafood spoilage micro-organism Photobacterium phosphoreum in liquid media and fish productsO. Mejlholm & P. Dalgaard
ABSTRACT Aims: To evaluate the antimicrobial effect of nine essential oils (EO) on P. phosphoreum and determine the effect of oregano oil on the shelf-life of modified atmosphere-packed (MAP) cod fillets. Methods and Results: The antimicrobial effect of EO was studied in a liquid medium and in product storage trials. Oils of oregano and cinnamon had strongest antimicrobial activity, followed by lemongrass, thyme, clove, bay, marjoram, sage and basil oils. Oregano oil (0·05%, v/w) reduced growth of P. phosphoreum in naturally contaminated MAP cod fillets and extended shelf-life from 11-12 d to 21-26 d at 2°C. Conclusions: Oregano oil reduced the growth of P. phosphoreum and extended the shelf-life of MAP cod fillets. Significance and Impact of the Study: Mild and natural preservation using EO can extend the shelf-life of MAP seafood through inhibiting the specific spoilage organism P. phosphoreum.
INTRODUCTION The shelf-life of fresh seafood is short and this represents a substantial practical problem for the distribution of these products. Spoilage reactions can be inhibited by traditional processing and preservation procedures but there is increasing interest in products with milder and more natural preservatives (Gould 1996). Essential oils (EO) are natural antimicrobials with potential to extend the shelf-life of seafood when used alone or in combination with other preservation techniques. However, some food components decrease the antimicrobial effect of EO and the use of EO for mild preservation of seafood remains little studied (Pirie and Clayson 1964; Sofos et al. 1998; López-Malo et al. 2000; Nychas and Tassou 2000). Photobacterium phosphoreum is the specific spoilage organism (SSO) responsible for spoilage of modified atmosphere-packaged (MAP) fillets of cod (Dalgaard et al. 1997). We have found no previous studies of the effect of EO on P. phosphoreum but other marine vibrios such as V. natrigens, V. vulnificus, and V. parahaemolyticus are sensitive to various EO (Deans and Ritchie 1987; Sofos et al. 1998). Distribution of fresh MAP seafood is increasing in many countries and it seems relevant to develop new packed products where shelf-life is extended through mild preservation with selected EO that inhibit P. phosphoreum. The objectives of this study were to evaluate the effect of commercially available EO on the growth of P. phosphoreum and on the shelf-life of fresh MAP fish fillets. The inhibitory effects of nine EO were evaluated in liquid media and on naturally contaminated cod-muscle blocks. In addition, product storage trials with MAP cod fillets were carried out to determine the effect of oregano oil on sensory, chemical and microbial changes during storage at 2°C. Finally, the antimicrobial effect of oregano oil was compared for cod and salmon, chosen to represent a lean and a fatty fish species.
MATERIALS AND METHODS Screening studies Experiments with liquid media. The effect of nine EO on growth of a mixture of five strains of P.
phosphoreum (MIX-P, Dalgaard et al. 1998) was studied in a liquid medium at
pH 6·6 (GMB, Dalgaard et al. 1998). The effects of temperature (2°C and 15°C)
and inoculum level (102 and 105 cfu ml
where
Screening experiments with naturally contaminated cod fillets. The antimicrobial effect of 0·05% (v/w) basil, lemongrass, marjoram, sage and
thyme oils, as well as 0·01% (v/w) oregano oil and 0·005% (v/w) cinnamon oil was
studied individually in MAP cod fillets. Three cod fillet pieces of ca.
100 g were stored at 0°C with and without addition of EO as described below.
They were packed with two volumes of an atmosphere comprising 60% CO2
and 40% N2 (Dalgaard et al. 1998). For all treatments P.
phosphoreum was enumerated at the time of sensory rejection of the MAP cod
fillets without EO (see Analyses below).
Product storage experiment Preparation, packaging, and storage of fish samples. Fillets of cod (Gadus morhua) from the Baltic Sea and salmon (Salmo salar) from a sea-based aquaculture facility in Norway were studied. Approx. 100 g of skin-off fillet of both fishes were stored at 2°C with and without addition of 0·05% (v/w) oregano oil and in two volumes of a modified atmosphere comprising 60% CO2 and 40% N2. Fillets were prepared and packed as previously described (Dalgaard et al. 1998). Cod and salmon fillets were randomly divided into two portions, respectively. 1·0% (w/w) sterile deionized water was added to all portions and 0·05% (v/w) oregano oil and water was added to one portion for each fish species. Fillets were manually tumbled for 1 min to ensure even distribution of the EO. All packs of fish were stored at 2°C and packs were removed for analysis at regular intervals. Analyses. Temperature of fillets was recorded by loggers (Tinytag, Gemini Data Loggers, UK) and gas composition within packs was determined using a gas analyser (PBI, Dansensor, Denmark). At each sampling time, three packs from each sub-batch were analysed using microbiological, chemical and physical methods, and two packs using sensory analyses. Samples of salmon were only analysed by microbiological analyses. Aerobic plate counts (APC) were ascertained by spread plating (15°C, 7 d) on Long and Hammer agar with 1% NaCl (LH) and P. phosphoreum was determined by a conductance method (Dalgaard et al. 1997). Trimethylamine (TMA), pH, and percentage drip loss were determined at each sampling time (Dalgaard et al. 1998). Sensory evaluation of MAP cod fillets was performed by a panel of 4-5 skilled assessors. The quality attributes for sensory evaluation of the cod samples were appearance, taste, odour and texture. Each quality attributes was described using a linear scale with low and high scores for each attribute. The assessors evaluated each sample individually and then a common evaluation was discussed and decided upon. Statistical analyses Paired comparison of samples and the t-test were used to tests treatment effects in screening studies and product-storage trials. In addition, one-way ANOVA was used to test significant effects of treatments.
RESULTS Screening studies All of the EO studied reduced the growth rate of P. phosphoreum. The
antimicrobial effect varied between oils, and concentrations from 0·005% (v/v)
to 0·1% (v/v) were required to prevent detectable growth of P. phosphoreum
(Table 1). Oregano and cinnamon oils had strongest antimicrobial activity
followed by lemongrass, thyme, clove, bay, marjoram, sage, and basil oils
(Table 1). The inoculation level of P. phosphoreum and incubation
temperature did not influence the relative effect of EO on growth of P.
phosphoreum. In fact, when
For practical application of EO in seafood, it is important to know if combinations of oils at very low concentrations, and with a mild flavour, have any antimicrobial effects. In this study, lemongrass (0·005%, v/v), oregano (0·0025%, v/v) and thyme (0·005%, v/v) oils had no inhibitory effect on P. phosphoreum when tested alone or in combination in the liquid medium. Product storage trials Oregano oil (0·05%, v/w) extended the shelf-life of naturally contaminated MAP cod fillets from 11-12 d to 21-26 d at 2°C (Table 2). Initially MAP cod fillets with 0·05% (v/w) oregano oil had a distinctive herbal flavour which decreased gradually during storage. Samples were rejected for sour and metallic off-flavours, but not for the ammonia-like off-flavour characteristic of spoiled MAP cod fillets without oregano oil (Table 2). Although TMA was produced in MAP cod fillets with oregano oil (Fig. figr rid="f1">1), none of the sensory assessors described these fillets as TMA/ammonia-like at anytime during the 26 d of storage at 2°C. Neither texture nor appearance of the MAP cod fillets was affected by the addition of oregano oil. Growth of P. phosphoreum was significantly inhibited by 0·05% (v/w)
oregano oil (P=0·035) but
FIGURES
DISCUSSION The antimicrobial effect of the nine EO on P. phosphoreum (Table 1) corresponded to effects previously observed for other marine vibrios and numerous food-related bacteria. Previous studies also found liquid media could be used to screen the effects of EO, but product storage trials are required to determine the effect of EO on spoilage micro-organisms more precisely (see, e.g. Deans and Ritchie 1987; Sofos et al. 1998). Activeantimicrobial substances in many spices and EO interact with food components (Shelef 1983) and, if used as the only preservative in foods, more than 1% (w/w) of spice can be required to extend shelf-life. These high levels often convey a very strong flavour and they are primarily useful in sauces and products that are mixed with other food ingredients (Pirie and Clayson 1964). Nevertheless, successful application of EO as antimicrobials in palatable foods includes; (i) pelargonium leave oil (0·025-0·05%) added to quiche fillings; (ii) various EO used together with high levels of NaCl (> 7%), e.g. in bean paste, fish roe or fish sauce; and (iii) vanillin (0·3%) in fruit purée with reduced pH and water activity(Kurita and Koike 1982; Lis-Balchin et al. 1998; López-Malo et al. 2000; Nychas and Tassou 2000). In this study, 0·05% (v/w) oregano oil yielded a distinctive but pleasant flavour to cod fillets and the oil significantly extended shelf-life and delayed spoilage reactions (Fig. figr rid="f1">1, Table 2). The shelf-life extension resulted from reduced growth of P. phosphoreum and the various EO that inhibit this specific spoilage organism may be used to delay spoilage in other lean MAP fishes. EO can be added to fish fillets by simple tumbling or spraying. As compared to several other mild preservation procedures like smoking, low dose irradiation, addition of protective cultures, or high pressure treatment, EO is inexpensive and uncomplicated as method of extending shelf-life of MAP fish fillets. Oregano oil and many other EO are relatively cheap and addition of 0·05% v/w costs approx. 1% of fish raw material like cod fillets. EO typically influence the sensory characteristic of seafood and they should primarily be used to develop new products and not only to extend shelf-life of existing ones. Due to a high mineral requirement of P. phosphoreum (Dalgaard et al. 1998) the combined antimicrobial effect of EO and chelating compounds is likely to be substantial. Therefore, it seems relevant in future studies to evaluate the effect of low levels of EO together with citrate or other natural chelating compounds on spoilage of mildly flavoured MAP fish fillets. The very different antimicrobial effect of oregano oil on growth of P. phosphoreum in MAP fillets of cod and salmon corresponded to results from other foods with low and high lipid content, respectively(Pirie and Clayson 1964; Shelef 1983; Sofos et al. 1998; Nychas and Tassou 2000). Most likely the active components of oregano oil, pimarily thymol and carvacrol, dissolved in the lipid phase of salmon fillets and therefore had no effect on P. phosphoreum growth in the aqueous phase. Interestingly, the antimicrobial effect of wasabi extracts was more pronounced in fatty than in lean tuna (Hasegawa et al. 1999). This allyl-isothiocyanate-containing extract or EO with active components that are more water soluble than those of oregano oils could possibly be used to inhibit P. phosphoreum and fatty fishes.
ACKNOWLEDGEMENTS We thank professor G.-J.E. Nychas from the Agricultural University of Athens in Greece for valuable comments to the manuscript.
REFERENCES 1 Dalgaard, P. & Koutsoumanis, K. (2001) Comparison of maximum specific growth rates and lag times estimated from absorbance and viable count data by different mathematical models. Journal of Microbiological Methods 43, 183-196. 2 Dalgaard, P., Mejlholm, O., Christiansen, T.J., Huss, H.H. (1997) Importance of Photobacterium phosphoreum in relation to spoilage of modified atmosphere-packed fish products. Letters in Applied Microbiology 24, 373-378. 3 Dalgaard, P., Munoz, L.G., Mejlholm, O. (1998) Specific inhibition of Photobacterium phosphoreum extends the shelf life of modified-atmosphere-packed cod fillets. Journal of Food Protection 61, 1191-1194. 4 Deans, S.G. & Ritchie, G. (1987) Antibacterial properties of plant essential oils. International Journal of Food Microbiology 5, 165-180. 5 Gould, G.W. (1996) Industry perspectives on the use of natural antimicrobials and inhibitors for food applications. Journal of Food Protection Suppl. 82-86. 6 Hasegawa, N., Matsumoto, Y., Hoshino, A., Iwashita, K. (1999) Comparison of effects of Wasabia japonica and allyl isothiocyanate on the growth of four strains of Vibrio parahaemolyticus in lean and fatty tuna meat suspensions. International Journal of Food Microbiology 49, 27-34. 7 Kurita, N. & Koike, S. (1982) Synergistic antimicrobial effect of sodium chloride and essential oil components. Agric. Biol. Chem 46, 159-165. 8 Lis-Balchin, M., Buchbauer, G., Hirtenlehner, T., Resch, M. (1998) Antimicrobial activity of Pelargonium essential oils added to a quich filling as a model food system. Letters in Applied Microbiology 27, 207-210. 9 López-Malo, A., Alzamora, S.M., Guerrero, S. (2000) Natural antimicrobials from plants. In: Minimally Processed Fruits and Vegetables. Fundamental Aspects and Applications ed. Alzamora, S.M., Tapia, M.S & López-Malo, A., pp. 237-263. Gaithersburg: Aspen Publishers, Inc. 10 Nychas, G.-J.E. & Tassou, C.C. (2000) Traditional preservatives - oils and spices. In: Encyclopedia of Food Microbiology ed. Robinson, R.K., Batt, C.A & Patel, P.D., pp. 1717-1722. San Diego: Academic Press. 11 Pirie, D.G. & Clayson, D.H.F. (1964) Some causes of unreliability of essential oils as microbial inhibitors in foods. In Microbial Inhibitors in Food. 4th International Symposium on Food Microbiology ed. Molin, N., pp. 145-150. Uppsala: Almqvist & Wiksell. 12 Shelef, L.A. (1983) Antimicrobial effects of spices. Journal of Food Safety 6, 29-44. 13 Sofos, J.N., Beuchat, L.R., Davidson, P.M., Johnson, E.A. (1998) Naturally Occurring Antimicrobials in Food. Council of Agricultural Science and Technology report no. 132. Ames, Iowa, USA.
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1)
were evaluated. The antimicrobial effect of commercially available basil, bay,
cinnamon, clove, lemongrass, marjoram, oregano, sage and thyme oils (Urtegĺrden
Ltd, Allingĺbro, Denmark) were studied (Table 1). In addition, combinations of
lemongrass (0·001 and 0·005%, v/v), oregano (0·001 and 0·0025%, v/v) and thyme
(0·001 and 0·005%, v/v) oils were evaluated by using a full factorial experiment
(23). For each treatment, growth of MIX-P was determined in duplicate
by automated absorbance measurements at 540 nm (Bioscreen C, Labsystems,
Helsinki, Finland). For each absorbance growth curve, the maximum specific
growth rate (
max ) was determined by using the Logistic model (Dalgaard and
Koutsoumanis 2001). The effect of EO on growth of P. phosphoreum was
expressed as the percentage reduction in growth rate (% RGR) calculated as:
ln10)/te,
where te is the time (h) when the experiment was stopped, Nabs
is the cell concentration (cfu ml
1).
