High-pressure processing (HPP) of food products is often accompanied by colour changes. Although these discolorations do not affect the actual safety of the product, they may give the end-consumer the false impression of spoiled or decomposing food. The mechanisms behind these colour changes are highly complex and are therefore difficult to analyse.
In this article, the basic principles of Raman Resonance Spectroscopy (RSS) are presented. The technique is a promising analytical tool for the investigation and monitoring of HPP-induced colour alterations. RSS may also allow the determination of optimal HPP conditions so that discolouration may be minimised.
High-pressure processing of foods
In many ways high-pressure processing (HPP) of foodstuffs satisfies customer demands for fresh products by enabling a prolonged shelf-life without the use of artificial preservatives. In HPP, foods are exposed to high hydrostatic pressure in the range of 400-600 MPa at moderate temperatures; in the process, pathogenic microorganisms are inactivated thus preventing microbial spoilage and decay. Unlike thermal preservation methods, HPP treatment causes fewer changes in flavours or in the levels of vitamins. However, HPP has some major drawbacks when applied to meat, fish, fruits and vegetables. In these products, many of the chromophoric components such as globins or carotenoids are pressure-sensitive and are therefore affected by the high pressures used in HPP. Although the safety and storability of the foods are actually improved, the resulting overall colour changes of the products can be perceived by the customers as a hint of diminished quality and freshness. The precise mechanisms by which the various chromophoric components give rise to the colour changes in the product undergoing HPP are as yet poorly understood and are the subject of active investigation.
Raman resonance spectroscopy
One analytical method suitable for such a purpose is Raman Resonance Spectroscopy (RRS). This techniques makes use of the phenomenon that occurs when light of a certain wavelength is scattered by a polyatomic molecule. When the light interacts with the scattering molecule, each photon in the illuminating radiation either gains or loses energy leading to a shift Δv of its frequency, usually expressed in terms of wavenumbers (cm-1). This effect is known as Raman scattering [Figure 1a]. If the frequency of the incoming light matches that of an electronic transition in the irradiated molecule, an enhancement effect of the shift can be observed. This is the resonance effect of Raman scattering [Figure 1b ]. Under resonance conditions, chromophoric moeities such as haems, chlorophylls and carotenoids can be selectively observed, even if they are not fully purified or embedded in a bioorganic matrix, which is an obvious advantage for in situ measurements. Furthermore, Raman spectroscopy is largely unaffected by water background signals because, unlike infrared (IR) spectroscopy, the Raman bands derive from nonpolar groups. This makes this RRS a useful complement to infrared spectroscopy.
One of the major limitations of RRS is however the fact that the probability that the molecular conditions needed for Raman scattering are fulfilled is very low: only a very few combinations of interacting molecules and the frequency of the irradiation give rise to Raman resonance. Signal strength may therefore be rather weak with consequent difficulties in detection [1, 2].
RRS has already been used to analyse the colour changes caused by HPP in pork, poultry and smoked salmon [3]. Chromophoric pigments in the raw material all showed a pressure-dependent loss of redness, the so-called “whitening effect”, which seems to be dependent on the pressure threshold and not on the time of exposure. The colour of smoked salmon is more pressure-stable than that of pork and chicken. This is likely because of the different biochemical composition of the chromophores.
Meat
Meat discolouration is related to globin denaturation and/or haem release or displacement. In smoked salmon, the main colour pigment is the carotenoid molecule astaxanthin. Using RRS, it was shown that oxidised myglobin (oxyMy) in pressurised pork undergoes a conformational transition to methylated myglobin (metMy) and to probably other denatured ferric myoglobin species. This was illustrated by a shift of the Raman bands in the region of 1300 – 1700 cm-1, where the oxidation marker v4 can be found. This marker is a recognised parameter for the oxidation state of haem iron. Methylated myoglobins and other derivatives of myoglobin are undesired side products of the HPP process not only because of the brownish colour they impart to the product but also because of their potential to induce a further oxidative degeneration of other meat components, in particular, lipids. Non-oxidised myglobin (deoxyMy) species do not carry that risk of undesirable meat modification. The RRS experiments described above showed the potential of the analytical technique to limit whitening effects in the pressurisation of pork by optimising the oxMy/deoxyMy ratio in the meat prior to high pressure treatment [4].
Salmon
A different mechanism for colour change has been proposed for pressurised smoked salmon. With RRS measurements at an excitation wavelength of 514 nm, which is the wavelength corresponding to electronic transitions in astaxanthin molecules, no shift in the Raman bands could be detected. When the excitation wavelength was changed to 413 nm, however, some substantial pressure-induced changes were observed. It should also be noted that, in the pressurised samples, there is an increase in the signal at 1358 cm-1 and a decrease in the signal at 1370 cm-1. These signals cannot be attributed to astaxanthin but are related to the oxidation of haem iron in globins, as described above in the experiments on pork.
Thus, a mechanism was put forward involving a yet unknown reduction reaction of metmyglobin/ metha emoglobin, that would lead to an oxidative attack of astaxanthin, which finally results in the observed whitening of the carotenoid. The experiments performed on smoked salmon are also remarkable in another way, namely the fact that all the RRS measurements were obtained from samples of fish in vacuum-packs. The packing foil did not interfere with the measurements, highlighting a major advantage of RRS, namely the possibility of applying the technique in situ [5].
Further applications of RRS Fruit and vegetables
RRS also may develop into an interesting tool for the monitoring of HPP of fruits and vegetables [6]. Changing life-styles and greater health-consciousness are giving rise to an increasing consumer demand for a large range of natural, storable products or those that have undergone only minimal processing. Fruits and vegetables are perceived as healthy food sources, rich in vitamins and with a low fat and cholesterol content. Though the important vitamins and flavour components remain largely unaffected at high pressures, in some cases colour changes can be observed. The chromophoric pigments in fruits and vegetables mainly consist of chlorophylls, carotenoids and anthocynins and, while most of these chromophores are relatively stable under HPP conditions at moderate temperatures, colour changes can occur in products pressurised at elevated temperatures. This is often attributed to residual enzymatic activity that leads to browning reactions, e.g. by polyphenoloxidases. The detection and quantification of such colour alterations can be carried out in a way similar to that used in the quality controls of meat products described, since the chromophores in fruits and vegetables display distinctive Raman scattering.
Flavour
RRS might also find a niche role in the detection and assignation of flavour components. Although most flavour-associated substances are relatively small molecules that are usually considered as being unaffected by high pressures, nevertheless they can undergo chemical or enzymatic modification or degradation. In a method analoguous to the measurements of chromophoric pigments in fruits and vegetables, the formation and loss of flavour components that have Raman scattering properties can be monitored by RRS.
Conclusion
It has been established that HPP is a technique with a promising potential in the field of food preservation. The technique prolongs the shelf-life by inactivation and/or destruction of pathogenic microorganisms in a purely physical fashion, i.e. without the use of artificial and potentially risky preservatives. Nutritional value, health benefits and flavour are not negatively affected by pressurisation, so making the method particularly attractive for the preservation of raw foodstuffs, which are often not amenable to other preservation methods such as heat treatment or pasteurisation. HHP is however limited by the fact that high pressures tend to alter certain sensory properties of foods such as texture and colour, which are important criteria for end-consumers. Since there are many individual, poorly understood, molecular mechanisms behind such sensory changes, powerful analytical methods are needed to understand them so that optimised HPP conditions can be established. RRS is a promising tool for the investigation, monitoring and documentation of colour changes induced by high pressure treatment. RSS is a rapid method which does not require labour-intensive sample preparation steps, thus facilitating in situ controls and measurements.
References
1. Li-Chan E.C.Y. The applications of Raman spectroscopy in food science.Trends in Food Science & Technology 1996; 71: 361.
2. Robert B. Resonance Raman spectroscopy. Photosynth Res 2009; 101: 147.
3. Tintchev F et al. Molecular effects of high-pressure processing on food studied by resonance Raman. Ann NY Acad Sc 2010; 1189: 34.
4. Wackerbarth H et al. Structural changes of myoglobin in pressure-treated pork meat probed by resonance Raman spectroscopy. Food Chemistry 2009; 115: 1194.
5. Tintchev F et al. Redox processes in pressurised smoked salmon studied by resonance Raman spectroscopy. Food Chemistry 2009; 112: 482.
6. Oey I, Effect of high pressure processing on colour, texture and flavour of fruit and vegetable-based food products: a review. Trends in Food Science & Technology 2008; 19: 320.
The authors
Martin Linde, Ph.D., Christian Hertel, Ph.D., and Volker Heinz, Ph.D.
German Institute of Food Technologies (DIL)
Prof.-von-Klitzing-Str. 7
D - 49610 Quakenbrück
Germany
