G.-J.E. Nychas Agricultural University of Athens, Department of Food Science and Technology, Laboratory of Microbiology and Biotechnology of Foods, Iera Odos 75, Athens 11855, Greece (e-mail: email@example.com).
Aims: This study aimed to determine the combined effect of packaging (air, modified atmosphere) with or without the addition of essential oil not only on the selection of microbial association of meat but also to determine any significant difference in microbial metabolites produced from the prevailing bacteria.
Methods and Results: Samples of minced meat were mixed with different concentration of oregano essential oil (0, 0·05, 0·5 and 1% v/w) and packed under aerobic or with modified atmosphere (Mixed Gas Modified Atmosphere – MGMA, 40% CO2/30% N2/30% O2; or CO2 Modified Atmosphere – COMA, 100% CO2) and stored at 5°C. In all packaging conditions, only concentrations of 0·5% and 1% oregano oil were effective.
Inhibition was evident in the order air < MGMA < COMA. Oregano essential oil delayed glucose and lactate consumption aerobically as well as under MGMA. pH changes were also evident. Furthermore, proteolysis was significantly inhibited in aerobically stored samples, and so was the production of acetate under MAP. Similar results were obtained for the other organic acids eluted from HPLC column.
Conclusions: Oregano essential oil delayed microbial growth and suppressed the final counts of the spoilage micro-organisms. It also caused a pronounced alteration in the physico-chemical properties of the minced meat.
Significance and Impact of the Study: Microbial analysis alone as spoilage index may misrepresent the effect of a hurdle such as essential oils on spoilage.
Modified atmosphere packaging (MAP) has gained considerable popularity as a modern method for food preservation. The combination of carbon dioxide, nitrogen and oxygen in MAP packs is able to suppress the aerobic spoilage flora of perishable foods, such as meat, fish and related products, and to sustain their visual appearance (Davies 1995). Spoilage is commonly detected by sensory or/and microbiological analysis (Dainty 1996). However, the former often requires trained panelists to minimize subjectivity, whereas the latter is laborious, time consuming and requires extensive knowledge of the specific spoilage organisms. An alternative method to the above-mentioned analyses involves the measurement of chemical changes associated with the growth of specific spoilage organisms in meat and meat products (Jay 1986; Dainty 1996; Nychas et al. 1998). Among these changes, glucose, gluconic acid, D- and L-lactic acid, acetic acid and ethanol have been proposed as potential indicators of spoilage (Kakouri and Nychas 1994; Boers et al. 1994; Seymour et al. 1994; Dainty 1996; Lambropoulou et al. 1996). To date, most efforts to determine spoilage by chemical/biochemical means have questionable results under practical application, probably due to the fact that such measurements are likely to be influenced by the packaging method (e.g. vacuum pack/MAP), or the use of preservatives, including essential oils, since the latter act as additional hurdles on the microbial association (Davies 1995; Tassou et al. 1996; Tsigarida et al. 2000). Essential oils are regarded as ‘natural’ alternatives of chemical preservatives and their use in foods meets the demands of consumers for mildly processed or natural products (Nychas 1995). However, their practical application is limited because of flavour considerations, as well as because their effectiveness is moderated due to interaction with food ingredients and structure (Juven et al. 1994; Skandamis and Nychas 2000; Skandamis et al. 2000). Indeed, to compromise between effective doses of such flavouring agents and sensory acceptability is a difficult task. The antimicrobial properties of essential oils are well documented in liquid media or in model structured system (Paster et al. 1990, 1995; Skandamis et al. 2000; Tassou et al. 2000) but there is a lack of detailed knowledge on the effectiveness of combinations of various hurdles, including the popular MAP technology, with these novel antimicrobial substances. For instance, studies of the effect of oregano and rosemary essential oils on spoilage flora and pathogenic micro-organisms in meat and fish, stored aerobically, revealed that bacterial counts were significantly suppressed in both (Tassou et al. 1996; Tsigarida et al. 2000). Although the suppression of bacterial counts is currently used as the measure of effectiveness of essential oils, the possible impact on the physico-chemical attributes of spoilage is poorly elucidated.
Considering the above, the aim of the present study is to investigate: (a) the potential of MGMA and COMA to extend the shelf life of minced meat at 5°C, with or without the addition of various concentrations of oregano essential oil on the microbial association; and (b) the profile of potential spoilage indicators of minced meat stored aerobically and under the above-mentioned storage conditions.
MATERIALS AND METHODS
Preparation of samples
Minced beef of normal pH (pH 5·4–5·6) was transported to the laboratory within 30 min of purchase and held at about 1°C for 1–2 h. Then, each batch (12 kg) was divided into four portions of 3 kg. Each portion was further mixed with the appropriate volume of oregano essential oil (final concentrations 0, 0·05, 0·5 & 1% v/w) and samples of 40 g were placed into 144 plastic containers. The containers were assigned to one of three packaging treatments, namely air, MGMA and COMA. Samples stored aerobically were simply enclosed into permeable polyethylene bags. Samples stored under modified atmospheres were packed individually into plastic pouches 300 mm wide × 200 mm long, with oxygen permeability of 1·7 cm3 m–2 24 h–1 at 23°C and 75% RH, which were evacuated and flushed three times before being filled (3 l). After filling, the pouches were double heat-sealed. The whole packaging was carried out using a HencoVac Machine. All packs were stored at 5°C. A limited number of samples were freeze-stored to serve as controls during sensory evaluation of colour and odour.
Extraction of essential oil
Five hundred (500) g of dried oregano (Origanum vulgare) was purchased from a local retail spice market and placed in a 2-l flask and 1 l of distilled water was added. A continuous steam distillation extraction was performed for approximately 3 h and the oil was collected and stored at 4°C (Skandamis et al. 2000; Tsigarida et al. 2000).
Samples (25 g) of minced meat were aseptically weighed, added to sterile quarter-strength Ringer’s solution (90 ml), and homogenized in a stomacher (Laboratory Blender 400, Seward Medical, London) for 60 s at room temperature. Decimal dilutions in quarter-strength Ringer’s solution were prepared and duplicate 1 ml or 0·1 ml samples of appropriate dilutions were poured or spread on the following media: Plate Count Agar (PCA; Merck, 1·05463, Darmstadt, Germany) for total viable count (TVC), incubated at 25°C for 72 h; STAA medium supplemented with streptomycin sulphate, thallous acetate and cycloheximide (actidione) for Brochothrix thermosphacta– this medium was made from basic ingredients in the laboratory, and incubated at 25°C for 72 h; MRS (Merck, 1·10660, Darmstadt, Germany) for lactic acid bacteria, overlaid with the same medium and incubated at 25°C for 96 h under anaerobic conditions; cetrimide–fucidin–cephaloridine (CFC) agar (Oxoid, CM559 supplemented with selective supplement SR 103E, Basingstoke, UK) for Pseudomonas spp., incubated at 25°C for 48 h; Rose Bengal Chloramphenicol Agar (Lab M, 36, supplemented with chloramphenicol supplement, X009, Bury, UK) for yeasts, incubated at 25°C for 5 d; Violet Red Bile Dextrose Agar (Merck, 1·10275, Darmstadt, Germany) for Enterobacteriaceae, incubated at 37°C for 24 h.
The pH value was recorded by a pH meter (Metrohm 691 pH meter), the glass electrode being immersed in the homogenate of minced meat after the end of microbiological analysis.
The concentration of glucose in minced meat during storage was assayed as follows: minced meat (25 g) was reduced to a fine suspension with 100 ml cold water (3–5°C) in an Omni mixer (Waring, New Hartford, UK). The suspension was agitated (orbital shaker, 100 rev min–1, 45 min) at 3°C, centrifuged (4000 × g, 5 min, 3°C), filtered (Millipore 0·22 μm) and the clear filtrate used to determine glucose using the GOD-PERID kit (Boehringer, Mannheim GmBH). The above filtrate was also used to determine α-amino groups by using the spectrophotometric assay described by Church et al. (1983).
HPLC analysis of organic acids
The profile of the organic acids (treated with Trifluoroacetic acid) of minced meat were analysed by high performance liquid chromatography (Spectra Physics P2000 two pump system with UV/VIS detector using Low Inertia Scanning Technology – similar to Photodiode array – and software, San Jose CA, USA) using a Rheodyne 7125 injector and a 300 × 7·8 mm Aminex HPX-87H 5 μm column (Bio-Rad Laboratories, Richmont, CA) as described by Koutsoumanis and Nychas (1999). The compounds were separated isocratically with 0·009 mol l–1 H2SO4 in distilled water (flow rate 0·7 ml min–1). Peak width was 12, peak threshold 600 and 0·034 AUFS. The whole spectra (190–330 nm) of the chromatograms were analysed. The solvents were HPLC grade and for the identification of peaks, solutions of reference substances (citric, lactic, acetic, tartaric, malic, succinic, formic and propionic) were analysed using the same program and their retention times (RT) and spectra were compared. The contribution of each identified compound was expressed as the percentage (%) of its peak area to the total area of all peaks eluted in each chromatograph. The precision of the results was always better than ± 5%.
In order to monitor microbiological and chemical changes during chilled storage of minced meat under different packaging conditions and supplemented with oregano essential oil, a two-way analysis of variance experiment was designed. Samples with three gaseous atmospheres (air, MGMA, COMA), four concentrations of oregano essential oil (0, 0·05, 0·5 and 1% v/w) were stored at 5°C. The selected concentrations of oregano essential oil were organoleptically acceptable (and almost undetectable) after cooking of raw minced beef (180°C/20). The above procedure was performed twice and duplicate samples for each treatment were taken. The growth data from plate counts were transformed to log10 values. The Baranyi model (Baranyi et al. 1993) was fitted to the logarithm of the viable cell concentration. For curve fitting, the in-house program DMFit (Institute of Food Research, Reading, UK) was used, which was kindly provided by Dr J. Baranyi.
Sensory evaluation of minced-meat samples was performed during storage according to Gill and Jeremiah (1991) by a four member sensory panel composed of staff from the laboratory. The same trained persons were used in each evaluation, and all were blinded to which product was being tested. The sensory evaluation was carried out in artificial light and the temperature of packed product approximated the ambient temperature. Special attention was given to the colour and the presence of exudate in the pack prior to opening and the assessment of abnormal odours during the opening of the pack (Kotzekidou and Bloukas 1996). Each attribute was scored on a 3-point hedonic scale where: 1=acceptable; 2=marginal; and 3=unacceptable. Assessment was designed to identify spoilage conditions exclusively. Odours typical of raw minced meat as exemplified by special samples from frozen storage that were thawed prior to each sensory evaluation, were regarded as acceptable. Distinct putrid, sweet, sour or cheesy odours were regarded as indicative of spoilage and therefore unacceptable. Bright colours typical of fresh oxygenated meat were considered acceptable. A persistent dull appearance, or unusual colour or appearance were considered unacceptable. The time in days before the taste panel considered the quality to be at the limit of acceptability (score=2–1) was defined as the organoleptical shelf-life of samples, under the specific packaging conditions and oregano essential oil concentrations. The shelf-life limit was defined as the point when 50% of the panelists rejected the sample.
The initial microflora of normal minced meat comprised, in decreasing order of magnitude, of Pseudomonas spp., B. thermosphacta, yeasts, lactic acid bacteria and Enterobacteriaceae. The contribution of these groups to the final flora depended on the packaging system used and on levels of oregano essential oil (Table 1). It needs to be stressed that the rate of growth, lag phase and final counts were affected by the packaging conditions, as well as by the addition of oregano essential oil (Table 1).
Table 1. The effect of packaging* in air, 40% CO2/30% O2/30% N2 and 100% CO2 modified atmosphere (MAP) and the addition of oregano† essential oil on the final population, lag period and maximum specific growth rate of spoilage microorganisms of minced-meat stored at 5°C
During the aerobic storage of minced meat, the TVC reached the highest levels within 6 d, with Pseudomonas spp. being the dominant micro-organism, followed by B. thermosphacta (Table 1) and then lactic acid bacteria. Packaging under MGMA delayed and suppressed growth of pseudomonads, whereas it enhanced the increase of B. thermosphacta and lactic acid bacteria (Table 1). Lactic acid bacteria, which started from lower levels increased more rapidly, with a higher growth rate than that found for pseudomonads or B. thermosphacta, and attained the same levels as the other two groups by the end of storage. In contrast, under COMA, lactic acid bacteria predominated after the 8th day of storage, having the highest growth rate among the members of the association (Table 1). By comparison with aerobic storage, maximum TVC were suppressed by 2 log cfu g–1 and 3 log cfu g–1 in MGMA and COMA, respectively (Table 1). Accordingly, extension of shelf life, defined as the time (d) required for 100-fold increase, was 5 and 12 d. As far as yeasts and Enterobacteriaceae are concerned, the former managed to grow faster than lactic acid bacteria and Enterobacteriaceae in air, whereas under MAP, yeasts counts remained almost constant or indicated the slowest growth rate (Table 1). Enterobacteriaceae were capable of reaching the same numbers as B. thermosphacta and lactic acid bacteria mainly at the end of storage under both MAP (Table 1).
The addition of oregano essential oil influenced the microbial association of minced meat stored under MAP, but no pronounced inhibition was evident in aerobically developed microbiota (Table 1). Furthermore, the inhibitory effect of the oil was proportional to its concentration in samples, with higher levels (0·5 and 1% v/w) having a more pronounced effect. The statistically (P < 0·05) significant changes in microbial attributes (compared to the control) due to oregano essential oil (Table 1) can be summarized in three primary effects: (i) reduction of initial microbial load (0·3–0·9 log10 cfu g–1 of minced meat), immediately after mixing with essential oil; (ii) reduction of growth rates and maximum population density of minced-meat spoilage organisms; and (iii) in some cases (Enterobacteriaceae and lactic acid bacteria under MAP) increase in lag phases. In general, inhibition occurred selectively towards B. thermosphacta, lactic acid bacteria and Enterobacteriaceae under MGMA, whereas in COMA, lactic acid bacteria was the only target group for oregano essential oil, since no other member of the initial microbial association of minced meat managed to grow in this atmosphere, regardless of the presence of essential oil (Table 1). Pseudomonads and yeasts were not significantly affected at all three packaging atmospheres.
The scores for colour and odour of minced meat stored at 5°C, are shown in Table 2. It was evident that colour remained acceptable for longer period in samples stored under MGMA in comparison with samples stored in air and COMA. The best score as far as the odour is concerned was evident with samples stored under COMA, followed by samples in MGMA. The addition of 1% of oregano essential oil did affect positively the odour and the colour of the minced meat. In particular the meaty odour of minced meat supplemented with oregano essential oil, remained 7 d longer during storage under MGMA, in comparison to samples without oregano oil. Furthermore, colour remained stable even in samples flushed with COMA (Table 2). In addition to the positive effects on colour and odour of minced meat, oregano essential oil also delayed the appearance of exudates in aerobically and under MGMA stored packs. It needs to be noted that oregano flavour was not strong enough to hamper the sensory evaluation of samples by panelists.
Table 2. Sensory evaluation of minced meat in air or packaged under MAP, with or without oregano essential oil at 5°C
The chemical attributes of minced meat stored aerobically or in modified atmospheres are presented in Figs 12–3. Glucose and lactate were utilized sequentially under aerobic conditions as well as under MGMA (Table 3; Fig. 1). At the same time there was a significant rise in pH at both cases, with the highest pH values obtained aerobically (Fig. 2). Increase in α-amino groups aerobically (Fig. 3) was concomitant with rise in pH (Fig. 2), whereas under MAP α-amino groups remained almost constant throughout the storage period with slight fluctuations in MGMA packs (Fig. 3). Storage under COMA resulted in the most pronounce delay in glucose utilization. During the first three days of storage under this atmosphere, lactate was slightly reduced and then it was raised up to the initial levels (Table 3). pH changed inversely to lactate (Table 3; Fig. 2). At all three packaging conditions, acetate increased towards the end of storage (Table 3). The highest levels occurred in samples stored under MGMA, whereas the lowest levels were observed in aerobic storage (Table 3). Formate showed a marked increase in air, and a sequential rise (initial) and decrease (late) under MGMA. In CO2 saturated atmosphere, formate declined progressively from the beginning and fell below the detection level by the end of storage (Table 3). In addition, three unknown peaks (RT=10·9, Acid A; RT=14·2, Acid B; RT=15·2, Acid C) were eluted (Table 3). Peak B and C indicated a fluctuation during storage at any atmosphere, whereas peak A was not detected in aerobic storage (Table 3).
Table 3. Changes in the areas under, lactic, formic and acetic acid peaks and unknown peaks with RT of 10·93 (A), 14·22 (B) and 15·23 (C) min during storage of minced meat under different packaging atmospheres, without or with 1% (v/w) oregano essential oil at 5°C
Oregano essential oil also influenced the physico-chemical changes of minced meat. The less pronounce effects were evident in the presence of 0·05 and 0·5% oregano essential oil (results not shown). On the contrary, increasing the concentration of oregano essential oil to 0·5 and 1% not only decreased the rate of glucose utilization under MGMA and COMA (Fig. 1), but also suppressed the production of acetate (Table 3) and the increase in α-amino groups (Fig. 3). In MGMA atmosphere, lactate indicated a pronounce increase rather than the recorded reduction in the control (Table 3). Formate reached higher maximum and lower minimum levels compared to the control (Table 3). Under COMA, addition of 0·5 and 1% oregano essential oil resulted in greater initial reduction in lactate and the accumulated levels towards the end of storage were higher than those in untreated samples (Table 3). Marked differences were also evident in the profile of formate and pH, as well as in the profile of the unknown peaks (Table 3; Fig. 2).
The present communication suggested quantitative changes in the profile of organic acids α-amino acids and pH of minced meat stored under MAP (Table 3; Figs 12–3) with or without the addition of oregano essential oil in comparison with samples stored under aerobic conditions. For instance, under COMA, higher quantities of lactate were accumulated in the presence of 0·5 and 1% oregano essential oil, compared to the control. In contrast, growth of lactic acid bacteria was significantly inhibited by the addition of oregano essential oil (Table 1). The above observation, in relation to suppression of acetate accumulation (Table 3), can be associated with either the influence on the metabolism of lactic acid bacteria (Axelsson 1998), or a selection of homo-fermentative lactic acid bacteria due to the essential oil. A shift in the microbial metabolism of lactic acid bacteria in meat stored under vp/MAP conditions has also been reported in the literature (Borch and Agerhem 1992). Indeed, similar metabolic conversion was induced by oxygen limitation (MAP) and glucose starvation in continuous fermentation, in a model system, of meat and dairy products (Borch et al. 1991; Borch and Agerhem 1992; Marshall 1992; Kakouri and Nychas 1994; Tsigarida and Nychas 2001). This metabolic shift may be influenced by the addition of oregano essential oil. However, the contribution of mixed acid fermentation of Enterobacteriaceae should also be taken into account (Gottschalk 1979), since their growth was rather unexpectedly higher in meat samples with the presence of 0·5 and 1% oregano essential oil, compared to samples stored under 100% CO2 without oregano essential oil (Table 1). It has previously been reported that the metabolism of glucose, lactate and amino acids by various bacteria, such as Staphylococcus aureus and Salmonella typhimurium, is influenced (e.g. rate and type of end-product formation) by the presence of essential oil in nutrient broth and in model systems (Skandamis et al. 2000; Tassou et al. 2000).
With respect to general principles of meat spoilage, MAP contributes to the extension of shelf life of meat: (i) by reducing the growth rate of specific spoilage bacteria (Table 1); (ii) by delaying the deterioration of meat colour and retaining the fresh meaty odour (Table 2); and (iii) by decreasing the rate of consumption of glucose and lactate, the limitation of which also affected the metabolic products produced by the microbial association of meat (iv) by producing relatively inoffensive compounds compared to typical spoilage odours produced by pseudomonads (Tables 2, 3) (Nychas et al. 1998). This observation was accentuated with the presence of oregano essential oil.
It is notable that the effect of oregano essential oil, as a potential ‘hurdle’, was more pronounced on chemical changes than on microbial numbers of minced meat during storage. Since the efficacy of ‘hurdles’ in foods is based entirely on inhibition of microbiological counts, such observations on physicochemical changes as that of the present study could be hardly comprehended.
These chemical changes may also account for the failure or for the benefit of potential microbial substrates and/or products that are considered to be useful for assessing meat quality (Jay 1986; Dainty 1996; Nychas et al. 1998).
As an overall conclusion, oregano essential oil was found to affect the contribution of spoilage micro-organisms to the microbial association as well as to the physico-chemical changes of the minced meat. However, differences in numbers of bacterial populations did not necessarily reflect changes in glucose, pH and organic acid profiles since these may be unpredictably related with the effect of additional hurdles, such as oregano essential oil on each specific member of meat microbial flora. Consequently, microbial analysis alone as a spoilage index may misrepresent the effect of a hurdle such as an essential oils on spoilage. For these reasons, further research on the benefits of essential oils in food preservation is needed through the scope of microbiological and physico-chemical aspects of preservation.
This research is funded by EU (DGXII), project FAIR-ct-1066. One of us (P.S.) would like to thank the Greek Scholarship Foundation for the financial support of his Ph.D. Thesis.