R. POSER2
F. SCHWÄGELE2
1Institute for Microbiology and Toxicology
2Institute for Chemistry and Physics,
Federal Centre for Meat Research,
Kulmbach, Germany
LR 1H NMR was used to investigate microbial growth in eggs in relation to egg age and hen age. Standardized eggs from caged 'Lohmann Brown' hens were inoculated with a defined pool of bacteria and stored under defined conditions. Characteristic changes in the NMR relaxation times indicated bacterial growth to levels > 107 - 108 c.f.u./g of egg content. These changes frequently correlated with optical-olfactorial deviations of the egg contents. The resistance of the eggs against microbial growth decreased with increasing storage time before inoculation. Eggs from younger hens showed a higher resistance than the eggs from older hens. The percentage of 'NMR¬positive eggs' increased with decreasing Haugh Units.
Introduction
LR 1H NMR may be used for the analysis of large food stuff samples (Capozzi et al. 1999) and has a potential for application in the quality control of shell eggs (Schwägele et al. 2001). It also allows the detection of microbially spoiled eggs (Kröckel et al. 2001) and thus offers a possibility for studying microbial growth in intact eggs without the need for destroying the eggs. The present study was performed to identify possible correlations between egg quality parameters and microbial growth in egg.
Material and methods
Standardized eggs from caged hens (Lohmann Brown) at 26, 31, 38 and 46 weeks of age were obtained one day after laying and stored at 20°C and 60 % r.h. for 1, 8, 15 and 22 days before and up to 9 days after inoculation with a pool of washed bacterial cells consisting of Serratia marcescens BAFF-SM19, Salmonella (6-strain pool of S. Enteritidis, one strain each of S. Senftenberg and S. Typhimurium) Pseudomonas aeruginosa BAFF-P15 and Staphylococcus aureus DSM1104.
Bacteria were suspended in 0.1 ml saline and injected into the albumen next to the shell at concentrations of about 103 (Salmonella), 102 (Pseudomonas, Staphylococcus) and 10 (Serratia) per egg. At each egg age as indicated above ten eggs were inoculated. The transversal relaxation times T2(1) and T2(2) of eggs were determined using a specially constructed 'minispec mq 10' (Bruker). Eggs were judged 'NMR-positive' if their relaxation times deviated significantly from the negative controls by day 9 after inoculation. At this time positive eggs were broken and subjected to visual-olfactorial evaluation and bacterial count determinations. Haugh Units were determined from uninoculated eggs.
Results and discussion
During storage of shell eggs at 20°C the NMR relaxation times T2(1) and T2(2) show characteristic changes (Schwägele et al., 2001). The T2(2) values decrease from 400¬500 ms exponentially within the first 7 days after laying to 330-430 ms. Then, a slower linear decrease occurs, e.g. from 430 ms on day 7 to 360 ms on day 34 at a rate of about 2.6 ms per day. The average rate of decrease from day 1 to day 7 is about 3-4 times higher. Microorganisms influence this development if they reach sufficiently high levels within the egg to induce chemical changes which usually are accompanied by optical-olfactorial changes such as deviations in odour, colour and integrity of the egg content (Kröckel et al., 2001). In these cases the T2(2) values start to increase during storage and reach values which are 130-200 ms higher than without microorganisms. While the initial T2(2) values may differ between individual eggs by up to 100 ms the initial T2(1) values are in a rather narrow range of 23-26 ms. During storage there is almost no change. From day 1 after laying to day 2-3 there is a decrease by 1-2 ms. Within the following 15 days the values increase at maximum by 3 ms and remain at this level for the rest of the storage time. Microbially induced chemical changes within the egg are recognised by an increase of T2(1) at a significantly higher rate than in the absence of microorganisms and T2(1) values may reach 40-50 ms within 13-25 days.
While in response to microbial activities the T2(1) values increase continuously the T2(2) values reach a maximum and then decrease again. Therefore, T2(1) is an important control when longer measuring intervals (e.g. once a week) are used. For a given breed subjected to constant husbandry conditions and a defined feeding regime egg quality is affected by egg age and storage conditions. Aging effects, including the breakdown of hurdles important for the egg's defence against microbial growth, are expected to proceed more rapidly at 20°C than e.g. at 5°C. The resistance of the eggs against microbial growth decreased with increasing storage time before inoculation (Fig. 1). In general, the percentage of NMR-positive eggs 9 days after inoculation was lower than the one of growth-positive eggs as evaluated by bacterial count determinations of the egg contents (Figure 1). Eggs were judged 'growth positive' when the bacterial counts per gram egg content were at least 100 times higher than the inoculum. Increasing NMR values were only observed when bacterial counts were at least 107-108 c.f.u./g and when these bacteria through their metabolic activity induced changes in the chemical composition of the egg.
In all cases microbially induced changes of the egg contents were due to Serratia and Salmonella. Upon breaking of the eggs these changes frequently correlated with optical-olfactorial deviations. When Salmonella spp. were dominant turbid albumen and a fishy smell were the characteristic deviations. The Serratia strain used for inoculation caused red discolourations and a fruity smell. Eggs from younger hens (age 26 - 38 weeks) showed a higher resistance than the eggs from older hens (age 46 weeks) (Figure 2). For very fresh eggs (day 1) hen age did not influence egg defence until living week 38. At present, it is unknown which egg components relevant as barriers for microbial growth are influenced by hen age.
Haugh units is a common measure related to albumen viscosity which both are decreasing with egg age (Haugh, 1937; Heath, 1977). The percentage of NMR-positive eggs increased with decreasing Haugh units (HU) (Figure 3). In summary, LR 1H NMR spectroscopy is an interesting technique for studying microbiologically induced changes in hen eggs. According to our results egg defence against microbial growth is primarily dependent on the age of the eggs. However, they also indicate that younger layers produce eggs which are microbiologically more stabile than eggs from older layers.
References
Capozzi, F., Cremonini, M.A., Luchinat, C., Placucci, G., Vignali, C. 1999. A low frequency 1H-NMR external unit for the analysis of large food stuff samples. Journal of Magnetic Resonance. 138: 277 – 280.
Haugh, R.R. 1937. The Haugh unit for measuring egg quality. US Poultry Magazine.
43: 552 – 555.
Heath, J.L. 1977. Chemical related osmotic changes in egg albumen during storage. Poultry Science. 56: 822 – 828.
Kröckel, L., Poser, R., Schwägele, F. 2001. Mikrobiell induzierte Änderungen der Relaxationszeiten bei intakten Eiern: Niederauflösende Kernspinresonanz-messungen (NMR). Fleischwirtschaft 81 (11), 113-116.
Schwägele, F., Poser, R., Kröckel, L. 2001. Anwendung der niederauflösenden NMR-Spektroskopie bei intakten Eiern: Messung qualitätsbestimmender physikalischer Merkmale. Fleischwirtschaft 81 (10), 103-106.
Acknowledgements
This work was supported by the EU project EggDefence QLK5-CT-2001-01606.
From Proceedings of the "XVI European Symposium on the Quality of Poultry Meat" and the "X European Symposium on the Quality of Eggs and Egg Products", Saint-Brieuc Ploufragan, France
