Electrical impedance to easily discover undeclared freeze-thaw cycles in slaughtered bovine meat

From a structural point of view, the trimmed meat from bovine muscle is an anisotropic tissue, which is characterized by a composite network of muscle fiber bundles containing aligned myofibers surrounded by a fine endomysial envelope of connective tissue. Several structural levels that characterize the muscle's morphology have highly contrasted electrical and dielectric properties. In fact, the meat's electrical properties result in part from non-frequency-dependent materials, which define the meat's resistance, i.e. an array of highly elongated structures with high longitudinal conductance due to both intra- and extra-cellular presence of ions, which are surrounded by connective sheaths with a very low conductance. Electrical characteristics of meat are also due to frequency-dependent materials, which define its capacitive reactance due to the cell membrane property of maintaining the separation of the negative from positive electrical charges across it, as in an electrical capacitor. Both these electrical specificities give rise to an electric anisotropy of the muscle, which is closely dependent on its histological characteristics.

In slaughtered meat, it has been found that during the post-rigor period (which begins 2 – 3 days after slaughter) the behavior of its electrical impedance (Z_m) reflects major changes occurring in the meat's structure [1], and these modifications are related to the disruption of the myofibrillar organization of the cytoskeleton and of cell membranes, due to protease activity [2]. In fact, during this time-dependent proteolysis, i.e. the meat's maturation, degradation occurs in proteins with structural tears and myofibril fragmentation, together with degradation in cytoskeleton architecture [3]. These structural modifications of trimmed muscle give rise to a progressive loss of structural anisotropy from which a reduction of Z_m also occurs [4,5].

The optimization of the slaughtered meat maturation period, paying close attention that the maturation process takes place properly, is one of the main goals of the meat industry, particularly of the beef sector [6]. In fact, this production sector has to contend with the broad-ranging variability of the raw material and with the low process control on the marketed end product, especially concerning the freeze-thaw cycles that the meat often has to suffer with a worsening of its quality. In order to guarantee good quality of meat products the industry is looking for instrumental systems to assess and certify the product quality of this food supply chain.

In their paper, Banach et al. [7] showed that only 72 hours post-slaughter, a sample of bovine meat presented an impedance module (149 Ω), which practically coincided with the module of its resistive component (145 Ω) when a current with a frequency of 10 kHz was injected into that meat sample. From these latter data it can be argued that, when an electrical current with a high frequency circulates in the meat, the capacitive membrane-dependent component of the Z_m could be totally nullified and, at the same time, the impedance module reflects only its resistive component which could consent the expression of quality attributes of meat in the form of numerical values [7].

Starting from all the above considerations, the aim of this study was that of developing and testing a portable and cheaper device able to safely and easily assess the Z_m values of slaughtered beef muscles by injecting an electrical current with a high frequency, the preset value of which surely short-circuits the capacitive reactance of the cell membranes and thereby reduces the Z_m to just the intra- and extra-cellular resistive components [8].

The present work is available on preprints.org (https://www.preprints.org/manuscript/201905.0136/v1).

Table 2 shows that 7 days after the animals’ slaughter the Z_mNF samples of LDm which aged 4 days at 5°C in the chilled room after the Z_mB measurements, reduced its value slowly (about −12%), even though significantly, with respect to that of the Z_mB. On the contrary, the Z_mF samples of LDm which stayed 4 days after the Z_mB measurements at −18°C before thawing, showed a larger and significant reduction (about −39%) with respect to that of the Z_mB.

To highlight the different behavior of Z_m when LDm were submitted or not to a freezing treatment, Figure 5 shows a graphic representation of a typical behavior of the impedance in the longissimus dorsi muscle samples concerning one of the tested animals. Due to the asymmetric distribution of the data, the visualization of the assessed values of the specific impedance was made by means of box and whiskers plots [26,27] in which we have also included the spread of data. Figure 5 shows a practically complete overlap between the third quartile (Q3) of the Z_mB box and the first quartile (Q1) of the Z_mNF box, thus their respective median lines are very close (Z_mB = 6.6 Ω/cm and Z_mNF = 5.8 Ω/cm) with no statistically significant difference between them.

The same Figure 5 clearly shows that the box of Z_mB data is placed totally above the box of Z_mF data, and the median value of this latter group is lower than half that of the base group of data. For this reason, statistical comparison between these two groups of data showed high significant difference. When comparing both the Z_mNF and the Z_mF box, it appears that the Z_mNF box is also placed above the Z_mF one, and the median of non-frozen group data is 1.9 times higher than that of the frozen one. Also, this difference between Z_m values turned out to be highly significant.

Fig.5

Results arising from these experimental measures of the meat's electrical impedance reasonably consent us to consider 65 kHz of frequency for the applied signal for Z_m monitoring, i.e. that released from the Z_Meat, as a reliable frequency for giving information concerning the ageing-dependent maturation of bovine meat.

In fact, it must be taken into account that ageing-dependent maturation of meat is due to a progressive disruption of the composite network of myofibres with their surrounding envelopes of connective tissue. This leads to an irreversible morpho-functional alteration of the cytoskeleton and cell membranes of myocytes [2] with a progressive loss of the histological anisotrophy of the muscle tissue. This loss of the morphological anisotropy induces a parallel reduction in the muscle's electrical anisotropy [28] which is revealed by a progressive falling of its electrical impedance [21] which coincides with its real, i.e. resistive, component.

Considering that in its hardware, the Z_Meat implements a current generator which delivers a current flow into muscle tissue with a frequency of 65 kHz, or a frequency which is higher than 10 kHz, which has been found to nullify the muscle capacitive reactance [7], then it is reasonable to assume that our Z_Meat measures the resistive components as described in the muscle's electrical analog model [9], i.e. this Z_m monitor could give information on the ageing-dependent maturation of meat in the form of numerical values.

Paying attention to the results from these experiments, data indicate that, at the 7^th day from slaughter, a portion of the beef longissimus dorsi muscle frozen at −18° C in the previous 4 days, when thawed, showed a Z_m value which was almost half that of the value assessed in the 2^nd day post-slaughter, before its freezing. These results were in good agreement with those previously found by Banach et al. [7] that, in a bovine meat sample which was frozen for three months at −24° C, when thawed, showed a Z_m which was 3.6-fold lower than that measured just after slaughter.

That described above, from both ours and Banach et al.'s experiments, is supported by robust histological evidence that highlights the quick and dramatic damage done to the sophisticated fibrillar organization of muscles by the very rapid formation of ice crystals when the meat has been frozen.

In fact, in the mile-stone paper by Rahelić and Puać [29] it was clearly demonstrated that when samples of beef longissimus dorsi muscle were frozen at temperatures between −10 °C and −22 °C ice crystals formed both between muscle cells and inside these cells. In fact, these authors showed that histological changes in muscles frozen at −10 °C presented in cross-section groups of several tens of fibers. These groups of fibers were separated by large intercellular spaces due to ice crystal formation. In these groups, muscle fibers were attached to one another and individual groups were linked by one or two rows of fibers. Otherwise, when the muscle samples were frozen at −22 °C the fiber groups were smaller with respect to those frozen at −10 °C and were separated by smaller interspaces. However, there are frequent gaps in the middle of fibers due to ice crystals formed inside the cells. This study concluded that damage is most severe in the muscles frozen at −22 °C. To justify this claim they proposed that opposing pressures through cell membranes are created from outside the cell by large ice crystals formed intercellularly and from inside the cell by pressures created by the formation of ice crystals here too. As a result of such opposing pressures, the tearing of the fibers will be greater.

More recently, Egelandsdal et al. [30] utilized the method of the Cryo-SEM (cryo-scanning electron microscopy) in samples of pork loin muscle frozen for 3 days at −25°C. After 14 hours of thawing the meat samples up to 3°C, the Cryo-SEM images at 500 × magnification identified large cavities (more than 1 μm) in the muscle samples indicating ice-crystal formation due to freeze damage [31]. In these muscle samples the regular arrangement of fibers interspersed with perimysial connective tissue was essentially lost, and typical anatomic structures of meat became non-identifiable since extensive cavity formation was observed in the frozen meat.

Even though in the present experiment no histological examination of the meat samples was carried out either at the beginning or at the end of the two experimental treatments, on the basis of the previous experimental results as reported above, it is reasonable to assume that in our bovine longissimus dorsi muscle samples that were frozen for 4 days at −18 °C, the histological structure underwent dramatic changes due to formation of ice crystals both outside and inside the muscle fibers.

These occurrences are even more supported by a recent histological study of samples of both longissimus lomborum and semitendinosus beef muscles, which were frozen at −20 °C for 3 days after slaughter [32]. Observing muscle micro slices under the light microscope the authors of these experiments found the presence of ice crystal formation in more elongated shapes and widely distributed patterns. Moreover, in the same experiments, muscle samples frozen at −20 °C for 3 weeks and after thawing, showed more gaps between muscle fibers compared to the ones that underwent maturation at 2 °C for 2 weeks. This occurrence could further justify the disruption of the histological anisotropy, as described by Rahelić and Puać [30], which could happen when muscle samples are frozen at low temperatures around −18 °C, i.e. what could happen to our left side longissimus dorsi muscles of beef samples.

This evidence would lead us to expect a remarkably better electrolytic conductance in frozen meat, in comparison to the non-frozen one, which is linked with the rapid and profound change in its anisotropic structure, due to the formation of ice crystals, i.e. an alteration of insulating properties of cellular membranes allowing a great mobility of ions could happen [11,33]. The degree of this change depends on the rate of the freezing process and the amount of water inside the structure of muscle tissue [34]. In the present experiment we also found that, as expected, after 7 days post-slaughter the Z_m of the sample muscles which had been frozen in the previous 4 days and then thawed were almost a half that of the only-aged one. This latter result reinforces the validity of this 65 kHz Z_Meat monitor to also discriminate between fresh and frozen-thawed beef meat.

These experimental results reasonably demonstrate that our Z_Meat prototype applied to Z_m monitoring is a reliable device to assess resistive components of the electrical impedance in trimmed longissimus dorsi muscle of slaughtered bovine since impedance values assessed by it behaved in agreement with those of previous experiments concerning the same matter.

The use of a custom designed device with a dedicated control software allowed the development of a low cost, compact, portable tool able to automatically and wirelessly perform all the measurements of the resistive components of the electrical impedance in the trimmed meat in real-time. So, this device might meet the needs of the bovine meat industry especially when it is necessary to rapidly check, in the place of production or storage, if such meat is fresh or previously frozen.

A possible criticism for this manuscript could be drawn due to the lack of histological evaluations of our meat samples both at the beginning and the end of the two experimental treatments. However, many previous experiments [29,30,31,32] have already investigated in great depth into the ultra-structural changes that animal elongated muscles undergo when frozen at temperatures close to that used in this experiment, and the results of these previous experiments can reasonably be used as a reference of the possible histological damage that also occurred in our samples of bovine longissimus dorsi muscles. Nevertheless, we are looking for a skilled histological laboratory which could be capable of providing information concerning the ultrastructural changes of beef muscle samples to be compared with our Z_Meat data.

Another possible criticism could be drawn due to the relatively small number of animals studied. However, it is quite possible that calves coming from the same breeding and of the same age and weight would also present very similar values of morphological and functional parameters due to the high affinity in genetic architecture of livestock breeds [20]. Nevertheless, the data reported in this paper are based on a pilot study to be followed as soon as possible by a confirmatory study based on a larger sample of tested animals.