PACKAGING TECHNOLOGY AND SCIENCE Packag. Technol. Sci.

2010; 23: 1925 Published online 4 September 2009 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/pts.875

Effect of Different Modied Atmosphere Packaging on Microbial Quality, Oxidation and Colour of a Seasoned Ground Beef Product (Meatball)
By Aylin Ozturk, Neriman Yilmaz and Gurbuz Gunes*

SUMMARY The effects of different modied atmosphere packaging (MAP) on microbial quality, oxidation and colour of meatballs were investigated. Meatballs were prepared in our laboratory and packaged in vacuum or in different gases: combinations of O2 (0, 2, 21%) and CO2 (0, 33, 66, 100%) with N2 as balance gas. Total microbial count, thiobarbituric acid reactive substances and colour were determined during storage at 3C for 3 weeks. Total microbial count remained unchanged at around 6.3 LogCFU/g in vacuum and 100% CO2 packages after 7 days, and slightly increased afterwards. The microbial count was higher in other packages and increased to 7.6 LogCFU/g after 21 days storage. Oxidation was inhibited by reduced O2 and increased CO2 in the packages. Packages with low O2 maintained colour (a-values) to a greater extent than the packages with higher O2 levels. MAP containing 13%O2 with 33%CO2 inhibited microbial growth, oxidation and colour change in meatballs. Copyright 2009 John Wiley & Sons, Ltd.
Received 17 July 2008; Revised 20 July 2009; Accepted 12 August 2009 KEY WORDS: meatball; modied atmosphere packaging; oxidation; colour; total plate count

INTRODUCTION There has been a signicant increase in consumer demand for minimally processed ready-to-cook and ready-to-eat food products throughout the world. Ready-to-cook or partially cooked pre-portioned and marinated fresh beef and poultry products, hamburger patties, meatballs and kebabs are some examples of these types of products. Spices and herbs such as onion, garlic, parsley, black pepper and cumin are commonly used to give special avour to these products. Meatballs are prepared with ground beef mixed with various spices and herbs depending on cultural preferences. The product is commonly served in fast food restaurants or cooked at home by consumers. Because of the changes in consumers lifestyle and trend in food services, convenient ready-to-cook meatballs can be a valueadded product if produced with high quality and sufcient shelf life. The shelf life of ready-to-cook meatballs is limited by microbial spoilage as they can easily be contaminated by microorganisms from raw materials (ground beef and spices), equipments and employees.1 The product can also be contaminated by potential pathogens including Escherichia coli O157:H7, Salmonella and Listeria monocytogenes, creating a great risk for foodborne diseases. In fact, during the last couple of decades, there have been a number of foodborne disease outbreaks associated with the consumption of meat products.2 Lipid oxidation and colour changes also affect the quality and shelf life of meatballs. Oxidation of fats in meat products can adversely affect avour and acceptability during storage.3,4 Determination of thiobarbituric acid reactive substances (TBARS)

* Correspondence to: Gurbuz Gunes, Istanbul Technical University, Faculty of Chemical and Metallurgical Engineering, Food Engineering Department, Istanbul, Turkey. E-mail: gunesg@itu.edu.tr Copyright 2009 John Wiley & Sons, Ltd.

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value is commonly used in assessing oxidation in meat products. Maintaining the characteristic colour of meat products is also important for consumer acceptance.4 Meatballs containing several spices have a unique colour depending on the added spices and the ground beef. Modied atmosphere packaging (MAP) is commonly used to extend the shelf life of foods by controlling microbial growth and chemical degradations. Elevated CO2 and reduced O2 levels in MAP can inhibit growth of various spoilage and pathogenic microorganisms.5 MAP has been shown to extend the shelf life of various meat products.69 Reduced O2 and elevated CO2 in MAP can also control oxidative degradation and colour changes in the product. High O2 atmospheres are suggested for fresh beef to maintain bright red colour although it can cause oxidation depending on the fat level. Meatball, which is a mixture of ground beef and several spices, has a greenish-brown colour that is totally different from the bright red colour of ground beef. Therefore, it is not necessary to have high O2 in package atmosphere for colour retention because high O2 can accelerate oxidation in meatballs. MAP, along with chiller storage, without use of any chemical preservatives, can delay microbial spoilage and quality degradations due to oxidation in meatballs. Effects of O2 and CO2 in MAP on these quality attributes of meatballs need to be studied. The objective of this research was to study the effects of MAP with different O2 and CO2 concentrations on the microbial quality, oxidation and colour of meatballs during storage.

MATERIALS AND METHODS Materials Ground beef, red pepper, black pepper, cumin, salt, bread powder, garlic powder and onion powder were purchased from a local supermarket. Plate count agar (PCA) and peptone were purchased from Oxoid (Hampshire, UK); NaCl and thiobarbituric acid (TBA) were purchased from Merck (Darmstadt, Germany); butylated hydroxytoluene (BHT), 1,1,3,3-tetraethoxypropane (TEP), trichloroacetic acid (TCA) were purchased from Fluka (Buschs, Switzerland). Packaging lms [62 m polyethylene terephthalate/polyethyleneethylene vinyl alcoholpolyethylene (PET/PEEVOHPE) with an oxygen transmission rate (OTR) of 1.2 cc O2/m2.day.atm at 0C 0% relative humidity (RH), and 50 m lowdensity polyethylene (LDPE) with a OTR of 3800 cc O2/m2.day.atm at 0C 0% RH as given by the manufacturer] were provided by Korozo Packaging Company GmbH (Istanbul, Turkey). Preparation of meatballs, packaging and storage Meatballs were prepared by mixing and kneading of 82.4% ground meat, 8% bread powder, 0.1% black pepper, 2% red pepper, 3% onion, 0.5% garlic, 2% salt and 2% cumin for 30 min by hand with sanitized gloves. Then, meatball samples (18 3 g) were formed from the mix. Samples were placed in bags made of a high-barrier multilayered lm (PET/PEEVOHPE). Gas mixtures containing different levels of O2 and CO2 were prepared through a gas mixer (PBI Dansensor Map Mix 9000, Ringsted, Denmark) and fed to a packaging machine (Multivac C200, Multivac Sepp Haggenmller GmbH & Co. KG, Wolfertschwenden, Germany). The packages were ushed with the gas mixture before sealing. As control treatments the samples were vacuum packaged in the high-barrier packaging lm or packaged in air in bags made of a low-barrier LDPE lm. Packaging treatments with 11 different atmospheres were as follows: 21%O2 + 0%CO2 (air), 21%O2 + 33%CO2, 21%O2 + 66%CO2, 1%O2 + 0%CO2, 1%O2 + 33%CO2, 1%O2 + 66%CO2, 0%O2 + 0%CO2, 0%O2 + 33%CO2, 0%O2 + 66%CO2, 0%O2 + 100%CO2 and vacuum. Gas mixtures in packages were completed to %100 with N2 when needed. The packaging treatments were replicated twice. Packaged meatball samples were stored at 3 1C for 21 days. Gas analysis The O2 and CO2 concentrations in the package atmosphere were measured by a gas analyser (PBI Dansensor CheckMate) at each sampling period.
Copyright 2009 John Wiley & Sons, Ltd. Packag. Technol. Sci. 2010; 23: 1925 DOI: 10.1002/pts

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Microbial analysis Samples (18 g each) were homogenized in 162 ml of peptone water (0.1%) using a stomacher (AES Easymix, Combourg, France) at medium speed for 2 min. Appropriate dilutions were made in peptone water and pour-plated in duplicate using PCA. The plates were incubated at 35C for 48 h, and the colony forming units (CFU) were counted. TBARS analysis Lipid oxidation was determined according to the TBA (thiobarbituric acid) method.10 A 10 g sample was mixed with 34 ml TCA solution (5% in water) and 1 ml BHT solution (7.2% in ethanol), and homogenized (IKA Ultraturax T18, Staufen, Germany) for 2 min. The homogenate was ltered through a lter paper (Whatman No. 4) into a 50 ml ask and diluted to volume with the TCA solution. Next, 5 ml of this solution was mixed with 5 ml 0.02 M TBA solution in a test tube and incubated at 80C in a water bath for 20 min to develop colour. Absorbance at 532 nm was measured against a blank using a spectrophotometer (PG Instruments T80, Leicester, UK). Standard curve was obtained using TEP as the manondialdehyde (MDA) standard at different concentrations. TBARS values were expressed as mg MDA/kg sample. Colour analysis Colour (Hunter L, a, b values) of meatballs were measured at three different positions on each samples by using a chromameter (CR-400, Konica Minolta, Tokyo, Japan). The averaged values were provided by the chromameter. Statistical analysis Data from two replicated experiments were analysed by ANOVA, and Tukey multiple comparisons were made using a statistical program (MINITAB 12.2, Minitab Inc., State College, PA, USA)

RESULTS AND DISCUSSION There was a slight decrease in O2 and a slight increase in CO2 levels in the packages possibly due to microbial growth during storage, but the gas compositions remained fairly close to the target levels during storage (data not shown). Microorganisms consume O2 and produce CO2 during growth. Packaging treatment affected total microbial count signicantly (Table 1). Initial total plate count (TPC) was 6.32 0.07 LogCFU/g. This high initial level was expected from uncooked ground beef
Table 1. Changes in TPC (given in LogCFU/g) of meatballs packaged in vacuum or different gas compositions during storage at 3C. Day 7 0%O2
Vacuum 100%CO2 66%CO2 33%CO2 0%CO2 6.78 (0.35) 6.60 (0.02) 8.24 (0.13) 6.90 (0.26) 6.99 (0.40)

Day 14 21%O2
7.22 (0.09) 7.78 (0.15) 7.82 (0.55)

Day 21 21%O2
6.94 (0.20) 7.19 (0.23) 7.19 (0.48)

1%O2
7.61 (0.18) 7.16 (0.15) 8.01 (0.45)

0%O2
7.30 (0.09) 6.80 (0.04) 7.90 (0.03) 7.23 (0.20) 7.33 (0.15)

1%O2
7.36 (0.26) 6.83 (0.19) 7.55 (0.30)

0%O2
7.42 (0.08) 7.24 (0.16) 7.84 (0.14) 7.48 (0.02) 7.75 (0.03)

1%O2
7.93 (0.13) 7.68 (0.03) 7.42 (0.05)

21%O2
7.55 (0.00) 7.78 (0.11) 7.80 (0.28)

Numbers in brackets are standard deviations. The initial TPC was 6.32 0.07 LogCFU/g. Copyright 2009 John Wiley & Sons, Ltd. Packag. Technol. Sci. 2010; 23: 1925 DOI: 10.1002/pts

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with spices as observed in other studies.1113 The average microbial load in the spices used in our study was 106 CFU/g. CO2 at 33%, resulting in a signicant inhibition of microbial growth in samples during storage. On the other hand, the effect of 66% CO2 was not different from 0% CO2 level. The reason behind this was not clear. It may be related with CO2 effects on some subpopulation (such as anaerobic) of microorganisms. It appears that 100% CO2 was high enough to inhibit the growth of anaerobic microorganisms, but 66% or lower was too low to inhibit them. A more detailed study of the changes in subpopulations of microorganisms on samples as a result of CO2 levels is required. Packages with 100% CO2 and vacuum resulted in the lowest microbial counts (Table 1). CO2 at 20 40% CO2 is usually used in meat packaging as lower levels have no effect on microorganisms while higher levels may result in package collapse due to the solubility of CO2 in the product.14 O2 levels had no effect on microbial growth in the samples (P > 0.05). Overall, TPC increased from the initial 6.3 LogCFU/g to 7.6 LogCFU/g at the end of 21 days storage. The initial microbial load in the samples was quite high possibly because of a high level of contamination in the ground beef and the spices purchased from a local supermarket. The effects of high CO2 and low O2 on microbial growth were lower than expected in our study possibly because of the high initial microbial load. The TPC in all packages exceeded 7 LogCFU/g at the end of the 21 days. Microbial spoilage of meat usually occurs when TPC reaches 107 to 108 CFU/g.4,5 Shelf-life extension of fresh meats was best achieved at 100% CO2 atmosphere.8 Jimenez et al.15 found that the TPC of chicken breast decreased signicantly with increased CO2 levels in packages. TBARS values of samples in packages with high O2 levels were signicantly higher than the samples in the other packages (Figure 1). However, there was no signicant difference among TBARS values of the samples in the packages with 0% and 1% target O2 levels or vacuum (P > 0.05). High CO2 levels (66% and 100%) resulted in lower TBARS values compared with 0% and 33% CO2 levels (P < 0.05). This was more apparent after 21 days storage (Figure 1). Overall, TBARS values increased with storage time (P < 0.05). Products of lipid oxidation are associated with off-avour in products. TBARS values in packages containing 21% O2 exceeded the threshold levels of 25 mg MDA/kg above which consumers can detect the oxidation and reject the product.3,4 The slight increase in TBARS of samples in packages with low or no O2 in our study was due to the residual O2 levels measured. Residual O2 levels in the range of 02% were found to be sufcient for lipid oxidation to occur.3 Packaging treatment signicantly affected colour of meatballs. Vacuum packaging maintained the initial a-value (14.1) during storage (Figure 2). Packaging treatment with 1% or 0% O2 resulted in only a small reduction (from 14.1 to 10.0) in the a-value during 21 days storage. There was no difference between the effects of 0% and 1% O2 on a-values (P > 0.05). On the other hand, a-values decreased substantially (from 14.1 to 4) by the packaging treatment with 21% O2 at the end of 21 days storage (Figure 2). CO2 levels in packages did not affect the a-values (P > 0.05). Overall, avalues decreased with storage time (P < 0.05). The reduction in a-values indicates loss of red colour, which was mainly from the colour of ground beef and the colour of the added red pepper in meatball samples. The red colour contributed by the red pepper due to its carotenoid content may have been lost because of a high degree of oxidation in 21% O2 packages. Further investigation is needed to determine the reason behind the red colour loss in samples. Berruga et al.16 found that colour stability in lamb meat was maintained in vacuum packaged samples and packaging with 20% O2 caused a larger reduction in a-values during storage. Higher a-values were observed for deer meat stored in packages with no O2 compared with packages containing O2.9 No differences in L- and b-values of the samples due to the packaging treatment were detected (data not shown). The results indicate that packaging with 100% CO2 or vacuum maintains the quality of the product well during storage and seems to be the best treatment among the tested conditions. However, anaerobic conditions with high CO2 can stimulate the production of Clostridium botulinum toxin in the product under abused temperatures.17,18 Therefore, to eliminate this risk, it is important to have some residual O2 (13%) in packages during storage. Moreover, very high levels of CO2 can dissolve in the product and result in package collapse.7 The amount of CO2 absorbed varies from 0 to 1.8 l CO2 per kilogram of meat depending upon the applied packaging and storage conditions.19 However, we did not observe a signicant collapse in the packages with high CO2 levels. Vacuum packaging resulted in compression damage to the meatballs, negatively affecting their texture and appearance.
Copyright 2009 John Wiley & Sons, Ltd. Packag. Technol. Sci. 2010; 23: 1925 DOI: 10.1002/pts

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DAY 7
vacuum 33%CO2 100CO2 66%CO2 0%CO2

TBARS (mgMDA/kg)

4
TBARS (mgMDA/kg)

3 2 1 0
4

DAY 14

DAY 21

TBARS (mgMDA/kg)

3 2 1 0 Vacuum 100%CO2 0%O2 1%O2 21%O2

Figure 1. Changes in TBARS values of meatballs packaged in vacuum or different gas compositions during storage at 3C. Error bars represent standard deviations. The initial TBARS value was 1.03 0.10. The samples in vacuum packages lost their shape because of the compression by external atmosphere. Keeping the initial microbial contamination at low levels in meatballs is very important in quality maintenance and shelf-life extension. This can be achieved by using ground beef and other ingredients with small microbial populations. Meatballs can be preserved using a hurdle approach in which spices and herbs, refrigeration, MAP and other non-thermal treatments such as gamma irradiation can be used. Spices used in meatballs have certain antimicrobial and antioxidant properties through which they can contribute to the quality maintenance of the product.11,20,21 Gamma irradiation has been shown to decrease the level of spoilage microorganisms in meatballs effectively.11 Therefore, further research on the combination of MAP and gamma irradiation in the preservation of ready-to-cook refrigerated meatballs is suggested.
Copyright 2009 John Wiley & Sons, Ltd. Packag. Technol. Sci. 2010; 23: 1925 DOI: 10.1002/pts

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16 12

DAY 7 vacuum 33%CO2

100CO2 66%CO2

0%CO2

a-values

8 4 0
16 12

DAY 14

a-values

8 4 0

16 12 a-values 8 4 0 Vacuum 100%CO2 0%O2 1%O2 21%O2

DAY 21

Figure 2. Changes in a-values of meatballs packaged in vacuum or different gas compositions during storage at 3C. Error bars represent standard deviations. The initial a-value was 14.23 1.17.

CONCLUSIONS TPC and TBARS values of samples were high in packages containing high O2 levels. On the other hand, a-values decreased in 21% O2 concentration packages at the end of 21 days storage. High CO2 levels (66% and 100%) resulted in lower TBARS values. The results indicate that packaging with 100% CO2 atmosphere or vacuum maintains the quality of the product well during storage and is the best treatment among the tested conditions. However, the samples in vacuum packages lost their shape because of the compression by external atmosphere, which adversely affected the meatballs appearance. Inclusion of residual O2 (13%) in packages may eliminate the risk of C. botulinum growth in the product under abused temperatures during storage. Therefore, a package atmosphere containing low O2 (13) with 33% CO2 (balanced with N2) is suggested to maintain the microbial quality, colour and oxidative stability of meatballs during refrigerated storage. Further studies with sensory evaluation of uncooked and cooked samples should be carried out to investigate the effects of different packaging atmospheres on the sensory quality of the product.
Copyright 2009 John Wiley & Sons, Ltd. Packag. Technol. Sci. 2010; 23: 1925 DOI: 10.1002/pts

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ACKNOWLEDGEMENTS

This research was funded by TUBITAK (Turkey). We acknowledge Korozo Packaging Company GmbH (Istanbul, Turkey) for donating the packaging materials.

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Copyright 2009 John Wiley & Sons, Ltd.

Packag. Technol. Sci. 2010; 23: 1925 DOI: 10.1002/pts