Raman spectroscopy is already finding uses in the food industry, but now a new research project in Wales aims to develop an updated version of the technology so that it can be used for tasks such as assuring food authenticity.
As with many other manufacturing sectors, the food industry is beginning to replace its more traditional processing technologies with bioprocesses. The attraction is obvious: bioprocesses have great chemical specificity, desirable reaction kinetics and relatively benign reaction conditions.
However, as the ability to control a bioprocess is paramount for product yield optimisation, it is imperative that the concentration of the fermentation product is assessed accurately. The development of such monitoring methods is driven by economic and ecological needs, and by the requirements for better process documentation.
While many spectroscopic studies have concentrated on measurements of biomass and nutrient supply, comparatively few have attempted to gain quantitative information on the product, unless they are non-complex chemical processes. In addition, within microbial physiology and microbial systematics there is a continued need for the development of non-invasive methods to characterise bacteria at the biochemical level.
One of the main European centres for such development work is at the University of Wales at Aberystwyth (UWA). There the Molecular and Spectroscopic Systematics Group is focusing on research directed towards the rapid, accurate characterisation and identification of clinically, industrially and ecologically important microorganisms, and in generic approaches to measure microbial biodiversity.
These research aims are achieved via a tandem analysis using analytical instrumentation to produce rapid physiological metabolome or proteome fingerprints of the microbial cells. In associated projects, molecular methods are employed for the phylogenetic analysis of bacteria and fungi.
A potential and extremely attractive method for on-line monitoring of bioprocesses for the rapid, precise and accurate analysis of the biochemical composition of the group's fermenter broths, and the characterisation of the organisms which they contain, is based on Raman spectroscopy. This has gained favour as a novel, rapid and non-destructive tool for analysing all kinds of biotechnological processes from the products of fermentations, the identification of clinically important bacterial species, the assessment of the origin and authenticity of foodstuffs, to the discrimination of various perfumes and cosmetics. The UWA team is developing a number of different inelastic light scattering methods based on dispersive, surface enhanced (SERS), and deep UV resonance (UVRR) Raman spectroscopies.
A new role for Raman
Dispersive Raman spectroscopy is a physico-chemical method that measures the vibrations of bonds within functional groups by measuring the exchange of energy with EM radiation of a particular wavelength of light. This exchange of energy results in a measurable Raman shift in the wavelength of the incident laser light. Since different bonds scatter different wavelengths of EM radiation, these Raman spectra or fingerprints are made up of the vibrational features of all the samples components. Therefore, this method will give quantitative information about the total chemical composition of a biological sample, without its destruction (that is to say be totally non-invasive), and produce fingerprints which are reproducible and distinct for different materials.
In previous work, the Aberystwyth team has used dispersive Raman spectroscopy for non-invasive, online determination of the biotransformation by yeast of glucose to ethanol. Software was developed which automatically removed the effects of cosmic rays and other noise, normalised the spectra to an invariant peak, then removed the abaseline' arising from interference by fluorescent impurities, to obtain the atrue' Raman spectra. The multivariate calibration models so formed were sufficiently robust to be able to predict the concentration of glucose and ethanol in a completely different fermentation with a precision better than five per cent. Dispersive Raman spectroscopy, when coupled with the appropriate chemometrics, is a very useful approach to the non-invasive, on-line determination of the progress of microbial fermentations.
Nevertheless, the dispersive Raman effect is very weak since typically only one in every 108 photons exchange energy with a molecular bond vibration, the rest of the photons being Rayleigh scattered. Consequently data acquisition for spectra from samples with only a modest concentration of determinand with a suitably high signal-to-noise ratio still often takes 5-15 minutes. So the team has concentrated on enhancing the Raman signal via SERS and UVRR.
In SERS the Raman scattering can be enhanced (by some 106-108-fold) if the molecules are attached to, or microscopically close to, a suitably undulating (roughened) surface usually of the coinage metals Cu, Ag or Au. In UVRR the inelastic light scattering occurs when the sample is excited with a frequency of light that is within the molecular absorption bands of the sample, in particular double and triple bonds, and aromatic ring vibrations.
Excitation of this type is in resonance with the electronic transition and yields Raman scattering that is resonance enhanced. The enhancement factor of resonance Raman scattering compared to normal Raman scattering can be as high as 108 but is typically 103-104.
Now the potential of UVRR is to be thoroughly investigated. Dr Roy Goodacre and his colleagues in the Institute of Biological Sciences have been awarded a grant worth £306 291 by the Engineering and Physical Sciences Research Council (EPSRC) to develop and to exploit ultra-violet resonance Raman spectroscopy for the on-line, non-invasive measurement of fermentation samples of biotechnological interest, and the characterisation of the organisms they contain.
Practical applications of interest to the food industry could include proving whether or not a bottle of olive oil has been adulterated, or the exact diet of honey bees. UVRR also has wider applications, for example the rapid and accurate identification of an infecting organisms in a hospital and optimising the production of substances such as antibiotics which involve fermentation.
"UVRR is a very promising technique for studying biological materials,“ said Goodacre. p