nir meat milk

INFRARED SPECTROSCOPY: AN ANALYTICAL TOOL IN FOOD SCIENCE

S. Jhaumeer-Laulloo and P. Ramasami

University of Mauritius

ABSTRACT Infrared spectroscopy (IR) is a type of absorption spectroscopy, which is used to measure the ability of a sample to absorb different wavelengths of infrared radiation. The infrared energy band is defined for convenience as the near infrared (NIR) (14 000-4 000 cm-1), the infrared or mid infrared (MIR) (4 000-400 cm-1) and the far infrared (400-50 cm-1). The MIR range consists of fundamental molecular vibrations while the NIR is composed of overtones and combination molecular vibrations. Therefore the absorption coefficients of the NIR bands are weaker than those of MIR. The NIR spectral region carries information related to CH, OH, CO and NH functional groups and this allows quantitative measurement of chemical concentrations in a matrix. However due to the strong absorption of water in the MIR and complex spectra obtained, the use of MIR. was restricted for the analysis of food products. Both MIR and NIR have been developed into a powerful tool for food analysis with the advent of Fourier Transform and the use of powerful data analysis techniques. The field of application of IR in food industry is very wide; it covers the quantification of major constituents such as water, proteins, lipids and sugars. Additionally NIR has an advantage over MIR because NIR spectra can be obtained with no sample preparation and it can be used as a continuous and real time processing monitoring tool. Key words: infrared spectroscopy, food products, analysis INTRODUCTION In recent years, infrared spectroscopy has been developed into an important and extremely useful method of analysis. In fact in food industry it has become an indispensable analytical tool because this fast and cost effective type of spectroscopy provides qualitative and quantitative information not available from other techniques. The most appealing feature of infrared spectroscopy is that many diverse parameters can be quickly assessed by a single scan of the ingredient. This stems from industrial developments, extensive use of computers and the development of appropriate chemometrics techniques. Applications of infrared spectroscopy in the fields of chemistry, drugs, the agro-food sector, life sciences and environmental analysis have been reported. This paper presents the characteristics, advantages, limitations and potentials of MIR and NIR. Special emphasis is placed on the application of these techniques in the food industry. INFRARED SPECTROSCOPY MIR and NIR are based on absorption techniques. The absorption bands in both MIR and NIR are due to molecular vibrations. While the bands in MIR are associated with fundamentals vibrations those associated with NIR are overtones and combination molecular vibrations. The MIR spectral region (4 000-400 cm-1) gives distinctive patterns for many spectra and this permits the identification of different functional groups and compounds. The absorption bands of NIR (14 000-4 000 cm-1) radiation by organic molecules are due to overtone and combination bands primarily of OH, CH, NH and CO groups whose fundamental molecular stretching and bending absorb in the MIR region. These overtones are anharmonic making NIR spectra complex and not directly interpretable as MIR. Moreover, due to the lower transition probabilities in NIR, the absorption coefficient is lower by a factor of 10-100 for each step from the fundamental to the next overtone.

MAS 2005. Food and Agricultural Research Council, Réduit, Mauritius. 43

Infrared Spectroscopy: An Analytical Tool in Food Science. S Jhaumeer-Laulloo and P Ramasami.

Infrared spectroscopy is based on the Beer-Lambert Law, which relates the absorbance of an observed band to the path length of the sample that the infrared energy passes through to its concentration and absorption coefficient. It is expressed as:

A= εcL Where A is the absorbance and ε is the absorption coefficient, c is the concentration and L is the path length (or thickness of the sample). As absorption coefficient decreases, the path length must increase to measure the absorbance of the material. On the contrary if the absorption coefficient is large the path length must be small, otherwise the measured value for the absorbance will saturate the detector of the infrared spectrometer. The weaknesses of these absorption bands proved to be benefit as samples can be analysed directly without dilution. Instrument The field of instrument in infrared is constantly evolving. The first generation of MIR instruments used a high-resolution diffraction monochromator and this has been mainly used for qualitative analysis for the identification of chemical compound. With the advent of Fourier Transform spectroscopy, MIR has developed considerably with the use of powerful microcomputers and the advent of new techniques such as ATR (attenuated total reflectance) cells (Crocombe et al., 1987, Van de Voort and Ismail, 1991, Cadet et al., 1991). In NIR spectroscopy, the most common type of instrument used in the analysis of food is the sequential instrument, where absorbances are collected sequentially in time. However, the new trend is that analysis is moving closer to sampling point allowing real time analysis. Therefore the future generation of IR instrument is evolving into Fourier Transform spectroscopy, IR imaging spectrometry and hand held IR spectrometry. Infrared Calibration MIR spectra normally contain well-defined peaks, which are associated to different functional groups. However, very often the peaks overlap in the fingerprint region of the spectra. NIR spectra show overlapping bands, which are the result of the first and second overtones as well as of combination bands. As a result NIR spectra cannot be used to determine analytes concentrations directly because of the way in which near infrared radiation passes through and is reflected from the sample. Meaningful information can be extracted from both NIR and MIR spectra with the help of sophisticated chemometrics techniques (Bertrand et al. 1984, Cowe and McNicol (1985). Robust prediction equations are normally based on calibration data sets. The calibration procedure includes:

• Selecting the calibration set • Determine standard concentrations using classical chemistry and biochemical tests • Collecting spectral data for the samples • Developing the calibration model • Validating the calibration model using separate set of samples

Analytical characteristics The analytical characteristics of NIR and MIR display certain advantages that make these techniques attractive alternatives to classical analysis. First they are rapid, do not require chemicals and are non-destructive. The best illustration is the determination of protein content. The AOAC (Association of the Official Analytical Chemists) method for the determination of protein is the Kjeldhal method which takes 3 hours and use chemicals such as sulphuric acid, which are pollutants. The growing concern of scientists is to have environmentally safe by-products. In fact with NIR and MIR, it takes less than a minute to get the protein content. The other advantage of spectroscopic technique is that it requires no to minimal sample preparation. A similar illustration can be provided with regard to quantification of fat. The time required for a single determination using classical method (i.e. soxhlet



extraction) is 6-8 hours. In infrared spectroscopic method all these parameters can be detected simultaneously within minutes. Sample size or physical state (solid, liquid, gas) is also not a problem. The main disadvantage of spectroscopic techniques is that it is based on correlations derived from calibration set. APPLICATIONS Near Infrared spectroscopy NIR is the most commonly used spectroscopic method in the food industry for the quantification of major biochemical constituents (Cadet et al., 2000, Pasquini et al., 2003, Bakeev, 2003). Its success is primarily due to its simplicity and its rapidity. NIR spectra are normally collected either as transmittance (light passing through translucent media) or reflectance (light diffusely reflected from opaque media) mode. Norris developed the first NIR apparatus for the quantification of water in foodstuffs (Norris, 1962 and Hart, 1965). The measurement of the components such as proteins, lipids and carbohydrates was hindered by the presence of water. But with the advent of multiregression analysis to spectral data this problem was solved and it has also allowed the measurements different constituents. NIR has been used to measure moisture, sugars, protein and fat in food (Davies and Grant, 1987, Hong and Tsou, 1998 and Osborne, 1993). Cereals and Cereal Products NIR is currently being used as a quality testing of crossbred material from wheat breeding programmes. The application of NIR analysis gives the protein and moisture in the wheat, which in turn is an indication of the quality of the wheat and flour obtained. Breeders use this as an indication to verify the quality of wheat and the yield of flour to be obtained. NIR has now been used over the world for example in Canada, Australia and Europe to monitor grower’s deliveries. The use of NIR to determine the protein and the moisture content has become a common practice in flourmills and it is replacing conventional chemical test. Since spectral analysis can be done on solid grains this has greatly reduced analysis time. Many bakeries also monitor the quality of flour using NIR spectroscopy. NIR is applicable to the analysis of moisture, protein, fat, starch, sugars and fibre in intact cereal foods such as bread, cakes, mixes, breakfast cereals, pasta and snack foods (Osborne 1993). Milk and Dairy Products NIR has a key role in the analysis of dairy products (Ozaki, 2001). It offers flexibility in the analysis of protein, fat and lactose content in a wide variety of dairy products including:

• Liquid milk • Dried whole milk, skim milk and whey powders • Cream • Traditional processed cheese

Many of these products are emulsions whose classical analysis is difficult. For example blending such samples changes their physical characteristics. NIR offers the possibility of on-line analysis. Meat NIR spectroscopy is widely used in the meat industry (Cozzolino and Murray, 2002). A special interactance fibre optic probe has been designed to spear carcasses and determine the fat content. The protein, fat and moisture contents of ground meat and meat products are available for processed meat. The meat samples are minced then blended in a food processor before being packed in an open sample cell.



The spectra of meat are dominated by water bands at 1450 nm (first overtone of the OH stretching mode) and 1934 nm (the combination band of OH vibrations). However, bands at 762, 960 and 1152 nm are well defined in the second derivative of the spectrum of water for the moisture content (McClure, 2002) Fish The analysis of fish flesh by NIR spectroscopy has been reported (Gjerde and Mertens, 1987; Mathias et al., 1987), where a good correlation has been obtained between laboratory and spectral data for fat and moisture contents on the lyophilized material. Non-destructive NIR analysis (760-200 nm) of fish has been reported by Rasco et al., 1991; Lee et al., 1992; Sollid and Solberg, 1992 Wold et al., 1996 and Downey, 2003. Salmon fish is high a value food product. The salt and moisture contents are critical factors that influence and inhibit the growth of foodborne pathogens and spoilage bacteria (Euckland, 1995, Gram and Huss, 2000). Knowing the salt concentration in fish will help to control salt content of the final product. There is normally a large variation in the salt absorption among samples within the same production and also because of high value of fish material, only a rapid and non-destructive method for moisture and salt determination is useful. Huang et al. 2003 have determined the salt and moisture content in cured Atlantic salmon using short-wavelength near infrared spectroscopy (600-1100 nm). Fruits and vegetables In the past ripeness of fruits was sorted by optical spectroscope in the visible region. However appearance of fruits is not a reliable guide to sweetness. NIR spectroscopy has been used as an automatic online method for evaluating qualities of foods. A number of researchers have used NIR spectroscopy coupled with regression analysis to determine compositions of different types of fruits and vegetables. Athansia et al., 2003, used NIR to measure the moisture, sugar, acid protein and salt in a variety of tomato juice. These are important criteria in the determining the nutritious energetic value, which also influences the physical characteristics hence the quality of the food products. Moisture limits are often specified in the product regulations. But in addition to moisture, sugar, acid, protein and salt must be analysed routinely in order to achieve standardisation of the product according to the label specification. In Japan it is a common practice to use NIR in the food industry for sorting fruits such as peaches, kiwi, apples and melons (Hasegawa et al., 2000, Tsuta et al., 2002), by visualizing the sugar content based on the absorption band in the NIR wavelength region. The second derivatives absorbances at 874 and 902 nm correlate with the sugar content. Muramatsu et al., 2003, coupled NIR with neural computing self-organisation map (SOM) have developed a non-destructive quality evaluation technique for apples. Fermentation Arnold et al. 2002 have reported that NIR spectroscopy can improve fermentation processes by incorporating rapid (analysis within seconds or minutes), non-destructive, multiconstituent analyses of fermentation broth media directly into monitoring and control strategies with minimal or no sample preparation or pretreatment. In fact NIR spectroscopy (Blanco et al. 2004) has been used for analysis and control of fermentation processes in both support laboratories and directly in the manufacturing environment. During fermentation process biomass accumulates, the initially translucent media transforms opaque media that strongly scatters light. The important absorption bands in the NIR spectra of fermentation samples are at 1450 nm and 1940 nm corresponding to OH first overtone and the OH combination band of water. Because of the large absorption of water molecule it is a common practice to work with a second derivative data, where absorbance maxima are converted into minima flanked by positive lobes. In this way spectral data band-with is reduced and baselines differences are greatly reduced between spectra.



Because of the complexity of fermentation process, it remains difficult to relate spectral variations observed in the broth spectra to changes in the concentration levels of the individual broth constituents. Basic assignments can only be made if the fermentation broth is unmodified by comparing the NIR of pure component. Interfering absorption bands from other components, matrix variations from complex media and widely varying accumulation profiles affect spectroscopic measurements. Hence proper calibration set of samples must be included and the spectral data are treated using mathematical model. Mid-infrared spectroscopy Mid-infrared spectroscopy is perhaps one of the most widely used vibrational spectroscopic techniques. However its use has been restricted in the food industry because:

• Water being a major component in biological samples is also a strong infrared absorber • Sample preparation • Complex spectra • Weak penetration of incident rays

In MIR, because of the strong absorption coefficients, samples have to be diluted before making transmission measurement. Unlike NIR, MIR has been more currently used in off-line analyses. Sugars MIR spectroscopy has been used since 1950’s for the study of carbohydrates. The combination of mid-infrared spectroscopy and multivariate statistics for determining glucose, fructose, and sucrose in aqueous mixtures has been investigated (Ramasami et al., 2004). In contrast with other classical methods the different sugars present can be determined in one run. Cadet et al., 1991 has used MIR spectroscopy for the study of sucrose in raw sugarcane juice. The absorption band at 997cm-1 was used to quantify the sucrose content. This is an important criterion since the level of sucrose determines the price of sugar. Fat The rapid control of the quality of lipids is a major preoccupation in the food industry. Wheeler (1954) has intensively investigated the structural analysis through their MIR absorption spectra. MIR is now currently used for the determination of cis and trans fatty acids in oils and fats (Belton et al., 1988, Gobhurdhun et al., 2000) and has been adopted as an official routine method by the American Oil Society (AOCS) and the International Union of Pure and Applied Chemistry (IUPAC). The bands at 3 015 and 967 cm-1 are associated to the cis and trans of fatty acid (Stuart, 2004). MIR has been used to analyze the fat content of milk and cream (Tay, 2001). The attenuated total reflectance (ATR) sampling technique was found to be fast and easy to use as compared to the transmittance sampling technique. The absorption band in the region 1,730 - 1,760 cm-1 (characteristic of ester group) is used to quantify the amount of lipids. This study showed that the ATR technique is fast, easy to use and can be implemented for on line monitoring of fat composition. Protein Etzion et al. 2004 have investigated the use of ATR MIR for the determination of protein concentration in raw milk. The determination of protein concentration is based on two absorbance bands in the 1 500-1,700 cm-1 range, known as amide band I and amide bands II, and 1,060 – 1,100 cm-1 range which is associated with phosphate groups covalently bind to casein proteins. The absorption bands due to water spectra were subtracted to reduce the effect of water, which overlaps with the amide bands (1,640 cm-1).



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