Applications of Raman Spectroscopy in Agricultural Products and Food Analysis: A Review

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Applications of Raman Spectroscopy in Agricultural Products and

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Article in Applied Spectroscopy Reviews · October 2011

DOI: 10.1080/05704928.2011.593216
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Applied Spectroscopy Reviews, 46:539–560, 2011
Copyright © Taylor & Francis Group, LLC
ISSN: 0570-4928 print / 1520-569X online
DOI: 10.1080/05704928.2011.593216
Applications of Raman Spectroscopy in Agricultural

Products and Food Analysis: A Review
DANTING YANG1 AND YIBIN YING2

1
Zhejiang University, College of Biosystems Engineering and Food Science,
Hangzhou, China
2
Zhejiang University, Faculty of Agriculture, Life and Environment Sciences,
Hangzhou, China
Downloaded by [Zhejiang University] at 02:35 08 October 2011
Abstract: Raman spectroscopy has been gaining popularity as an analytical tool due
to advances in development of Raman spectrometry and the power of personal comput-
ers. Due to to its narrow and highly resolved bands, Raman spectroscopy allows for
nondestructive extraction of chemical and physical information about samples and aids
in rapid on-line analysis without any special sample preparation. In this review, Raman
spectroscopic techniques such as dispersive Raman spectroscopy, Fourier transform Ra-
man spectroscopy, surface-enhanced Raman spectroscopy, and spatially offset Raman
spectroscopy are briefly introduced. In addition, applications of Raman spectroscopy
are explored, within various fields of agricultural products and food, including fruits
and vegetables, crops, meat and dairy products, oil, as well as beverages. In addition,
some discussion on the importance of Raman spectroscopy as fundamental and applied
research of agricultural products and food is provided.
Keywords: Raman spectroscopy, agricultural products, food, quality and safety control
Introduction
Agricultural products and food are essential to life and are also important to the world
economy. With the increasing demand for a high quality life, quality and safety control
are gaining the attention of researchers. Several techniques have been employed for the
detection and identification of the agricultural products and food. Traditional methods such
as gas chromatography (GC), high-performance liquid chromatography (HPLC), and gas
chromatography–mass spectrometry (GC-MS) are all powerful for the study of component
qualification and composition determination (1, 2), but they are time consuming, solvent
wasting, and require the instruments to come in contact with hazardous samples. Near-
infrared spectroscopy (NIR) is another method to monitor and assess the composition and
quality of products in the food industry (3, 4), but it shows low spectral resolution for samples
in aqueous solutions because of the very strong infrared absorption of water. Fluorescence
spectroscopy is a very sensitive tool to provide information about molecules and their
environment in food samples; however, it is limited to samples that have the fluorescence
effect (5, 6). In contrast, Raman spectroscopy, an inelastic effect discovered in 1928 by
Address correspondence to Yibin Ying, Zhejiang University, Faculty of Agriculture, Life and
Environment Sciences, 388 Yuhangtang Road, Hangzhou, China. E-mail: yingyb@zju.edu.cn
539
540 D. Yang and Y. Ying
Figure 1. Simple diagram for Raman Effect.
C. V. Raman, overcomes most of the above drawbacks and presents distinct advantages
(7). It can be used for both solid samples and aqueous solutions, offering information
on the structural changes in agricultural products, classification of different species, or
determination of the internal component content in samples (8, 9).
Theoretical Basis of Raman Spectroscopy

When a sample is irradiated by a laser beam, a tiny portion of photons with a known
frequency and polarization are scattered from the sample. Figure 1 shows a schematic
of the Raman effect. In this process, an inelastic collision between the incident photon
and the molecule of the sample occurs. As a result, the vibrational or rotational energy of
the molecule is changed, and the scattered radiation is shifted to a different wavelength.
The frequency difference between scattered radiation and incident radiation is called a
Raman shift. If the molecule gains energy, scattered photons are shifted to longer wave-
lengths, giving rise to Stokes lines in the Raman spectrum; otherwise, they are shifted to
shorter wavelengths, giving rise to anti-Stokes lines in the Raman spectrum (10). Figure
2 shows an energy level diagram for Raman scattering. The frequency shifts of scattered
light can be analyzed and presented as spectra. Spectral bands represent vibrational char-
acteristics for chemical bonds and functional groups that constitute the components in
the examined samples. The spectra can provide a fingerprint of a specific substance, fa-
voring the analysis of this compound presented in several kinds of samples, which offers
the basis for structural analysis and qualitative analysis. Raman spectroscopy can also be
used to perform quantitative determination, because the intensity of an analyte band is
linearly proportional to the analyte concentration, which can be presented as the following
Figure 2. Energy level diagram for Raman scattering.

Raman Spectroscopy and Food Analysis 541
equation.
Iv = I0 Kv C (11)
where Iv is the measured Raman intensity; I 0 is the excitation intensity; Kv is the constant;
and C is the analyte concentration.
Raman spectroscopy has considerable advantages that are significant in food analysis,
such as high specificity, good compatibility with aqueous systems, no special sample
preparation, and short timescale.
1. High specificity: Raman bands have a good signal-to-noise ratio (SNR) and are
nonoverlapping, which allows Raman spectroscopy to be used for fingerprinting of
samples to conduct the real analysis.
2. Good compatibility with aqueous systems: Because the Raman spectra of water are
weak and unobtrusive, it is a good tool for application in aqueous solutions.

3. No special sample preparation: Raman spectra require no special sample prepara-
tion and no contact with the sample because a Raman spectrometer involves only
illuminating a sample with a laser and collecting the scattered photons.
4. Short timescale: Raman spectroscopy can complete the analysis in a few seconds,
making it able to monitor industry processes in real time.
Techniques
The main Raman techniques commonly applied in agricultural products and food analy-
sis include dispersive Raman spectroscopy, Fourier transform (FT) Raman spectroscopy,
surface-enhanced Raman spectroscopy (SERS), and spatially offset Raman spectroscopy
(SORS). A common Raman spectrometer consists of four parts: a laser light source, a
sampling system, a detection system, and a computer to collect and store the data. A brief
outline of these techniques with their advantages is given below and is also shown in
Table 1.
Table 1
Advantages of Raman techniques
Techniques Advantages
Dispersive Raman Is suitable for aqueous samples, samples with elevated
spectroscopy temperature, black samples
Suppresses fluorescence at 780 or 830 nm
Fourier transform Raman Reduces fluorescence
spectroscopy Eases operation with FTIR spectrometer
Possesses high spectral resolution
Surface-enhanced Raman Possesses high molecule specificity, sensitivity, and
spectroscopy resolution
Spatially offset Raman Has more effective illumination
spectroscopy Suppresses fluorescence
Facilitates analysis of a variety of samples
Figure 3. Schematic diagram of dispersive Raman spectrometer.
Dispersive Raman Spectroscopy

Dispersive Raman spectroscopy is the most common and basic technique, with intense
monochromatic radiation, which is usually provided by continuous-wave gas lasers such as
argon (488.0 and 514.5 nm), krypton (647.1 nm) ions, or diodes (780 or 830 nm) combined
with a charge-coupled device (CCD) array detector with a single-grating spectrograph.
Figure 3 shows a schematic of a dispersive Raman spectrometer. Dispersive Raman spec-
troscopy has some instinct advantages: (1) It is very suitable for aqueous-phase samples;
(2) it can be used for samples with elevated temperatures, even above 1000◦ C; (3) it allows
black samples to be analyzed; and (4) with a 780- or 830-nm laser, it can help suppress the
fluorescence of samples.
Fourier Transform Raman Spectroscopy

Fourier transform Raman spectroscopy systems have been available since 1987. Com-
mercial systems use an Nd:YAG laser (1,064 nm) with an NIR interferometer coupled to
either a liquid nitrogen–cooled Ge or an InGaAs detector. Figure 4 shows a schematic of
an FT-Raman spectrometer. FT-Raman has three main advantages over dispersive Raman
systems: (1) reducing the laser-induced fluorescence that a number of samples exhibit; (2)
easing the operation as with a Fourier transform infrared (FTIR) spectrometer; and (3)
showing a high spectral resolution with good wavelength accuracy.
Surface-Enhanced Raman Spectroscopy

Surface-enhanced Raman spectroscopy, which has advantages such as inherent molecule
specificity, higher resolution, and relatively larger sensitivity over the normal spontaneous
Raman spectroscopy (12), is regarded as a promising quantitative analysis tool. With the
help of two main classes of enhancing media—solid substrates and colloidal nanoparticles
(13)—the total enhancement of the observed Raman signal can be as high as 1014 times
that of the unenhanced signal. The effect is attributed to two mechanisms: a chemical
mechanism and electromagnetic mechanism. The chemical mechanism is often attributed
Figure 4. Schematic diagram of FT-Raman spectrometer.
to a charge transfer intermediate state, which takes place at the strong electron coupling
between the analyte and the metal surface. The electromagnetic mechanism, with a larger
number of scattered photons arising from the oscillations of conduction band electrons at
a metal surface, may play a greater part than chemical enhancement (14).
Spatially Offset Raman Spectroscopy

Spatially offset Raman spectroscopy is based on earlier investigations into the Raman
photon migration process. Unlike conventional Raman spectroscopy, shown in Figure 5A,
Raman spectra for SORS are collected from spatial regions offset from the point of illu-
mination on the sample surface. Due to wider lateral diffusion of photons emerging from
greater depths, SORS contains different signals from sample layers at different depths (15).
With a larger spatial offset, a greater suppression of surface layer signal will be achieved
in SORS. Standard SORS (Figure 5B) does not automatically permit the simultaneous
interrogation of diffusely scattering and transparent samples. Eliasson et al. (16) demon-
strated a variant of SORS (Figure 5C) to adapt SORS for a wide variety of packaging and
samples analysis. In adapted SORS, the container acts not only as a container but also as
a source of diffuse radiation illuminating a large area of liquid, which generates Raman
photons emanating in all directions. This mechanism facilitates the effective illumination
of the container wall and the liquid content directly under the Raman collection area, which
Figure 5. Schematic diagram of conventional Raman and SORS approach.

ensures that the Raman and fluorescence contributions from the container wall will not
overwhelm the weak Raman signal of the liquid.
Applications
The characteristics of the Raman spectroscopic technique, such as inherent rapid analysis,
minimal sample preparation, and compatibility with aqueous solutions, make it a versatile
tool for at-line or on-line analysis in different fields, including agricultural and food (17–19),
pharmaceutical and biomedical (20–23), material science (24–26), biochemical (27–29),
geological (30–32), and environmental (33, 34). Considering that agricultural products and
food are an important factor in people’s daily lives, current applications are reviewed in this
article that illustrate the exploitation of Raman spectroscopy in agricultural products and
food, including structural and qualitative and quantitative analysis of fruits and vegetables,
crops, meat and dairy products, oils, and beverages, most of which were published between
2001 and 2011.
Fruits and Vegetables

Both for optimal economic value and health benefits, it is important to maintain high inter-
nal and external quality of fruits and vegetables using rapid, low-cost analysis in modern
agricultural and food industries. Many researchers have endeavored to apply Raman spec-
troscopic techniques in structural analysis, safety control, classification, and quantification
of fruits and vegetables.
Structural Analysis. FT-Raman spectroscopy was used by Veraverbeke et al. (35) to evalu-
ate the natural, intact wax layers on the surface of whole fruits, but no stable interferogram
was obtained due to the high fluorescence of apple tissues. López-Casado et al. (36) obtained
success in biomechanical behavior analysis of isolated tomato (Solanum lycopersicum L.)
fruit cuticles using Raman spectroscopy. Synytsya et al. (37) illustrated that FT-Raman
spectroscopy is a valuable tool in structural analysis of commercial citrus and sugar beet
pectin and indicated that the combination of FT-Raman and FTIR spectroscopic methods
provided more complete characterization of pectin samples than either method alone. Nu-
mata and Tanaka (38) found that the concentrations of quercetin determined by Raman
spectroscopy were in good agreement with HPLC and ultraviolet-visible (UV-Vis) spec-
troscopies with relative errors of about 1%. Da Silva et al. (39) provided a spectroscopic
description of Rosa aff. rubiginosa flesh and seeds with Raman spectroscopy to clarify the
important chemical aspects of this fruit, which helped to produce a spectroscopic protocol
for characterization of products made from these fruits. Confocal Raman spectroscopy with
excitation at 785 nm and micrometer-level spatial resolution over the conventional Raman
spectroscopy was successfully used to examine the distribution of components within food
microstructures. In research by Pudney et al. (40), three different varieties of carotenoids
found in tomatoes, lycopene, β-carotene, and lutein were examined and the physical state
of the carotenoids was identified by this method, which provided essential knowledge that
Raman spectroscopy could be fruitfully applied in nutrient bioavailability trials.
Safety Control. Raman spectroscopic techniques can be used for safety control of fruits
and vegetables. Yang and Irudayaraj (41) employed an FT-Raman approach to detect
and classify foodborne microorganisms on the whole apple surface for the first time. The
successful discrimination of five different strains of Escherichia coli and the 100% accuracy
of differentiating pathogens from nonpathogens demonstrated that FT-Raman spectroscopy
is an excellent tool for examination of microorganism contamination on food surfaces and
classification of microbial cultures. Bonora et al. (42) used FT-Raman spectroscopy for
identification of microbial disease. The structural characteristics of ‘Hayward’ kiwifruits
from elephantiasis-affected plants were studied by means of an FT-Raman technique. From
the pulp spectra of the elephantiasis fruits, they determineed that the increased peaks at
1530 and 1661 cm−1 unequivocally related to the infection may be used as an early marker
of evidencing fruits from diseased plants. Pesticides are a key component in protecting
fruits and vegetables, but excessive concentrations pose a threat to human health. However,
rapid chemical analysis of pesticide residues is unavailable. Zhang et al. (43) measured
Raman spectra from clean fruits and pesticides and found that the FT-Raman technique
could be used to measure the effective components in fruit and vegetables as well as
the pesticides left on the surface. Based on the extreme sensitivity and ability to identify
molecular structure of pesticides, an SERS sampling device was developed by Shende et al.
(44) to detect pesticides on fruit surfaces and in juices extracted from fruit. They achieved a
series of organophosphorus pesticides in solution phase. Fonofos was successfully detected
at 1% on an apple and other kinds of pesticides were measured at 100 to 10 ppm by SERS-
active capillaries. When an improvement of new alkoxide precursor in metal-doped sol-gel
sampling devices with a more open structure with both polar and nonpolar characteristics
was used, the estimated limit of detection was elevated to 10 ppb (45).
Classification. A very small portion of bad fruits can ruin the whole batch. Discriminating
good from bad ones, better from common ones is the key to maintaining good quality of fruit.
Combined with chemometric methods, FT-Raman spectroscopy is capable of extracting
chemical information from complex matrices. Classification of olives is an important first
step in improving production of extra virgin olive oil, but the currently employed method
is still far less suitable for on-line process control. Muik et al. (46) combined FT-Raman
spectroscopy and partial least squares (PLS) to classify olives based on the fatty acids (FFA)
content in olives and achieved a correctness of 80%. In a later study (47), FT-Raman and
pattern recognition were employed to discriminate olives of different qualities, including
sound olives, olives with frostbite, olives collected from the ground, fermented olives, and
olive samples with diseases. The best results were provided by soft independent modeling
of class analogy (SIMCA) with prediction abilities of 95% for sound, 93% for frostbite,
96% for ground, 92% for fermented olives, and 100% for damaged olives. The research
above emphasized that Raman spectroscopy worked directly on the olives and therefore
enabled a screening of olives according to their quality, which could help optimize the
production of high-quality virgin olive oil. Changes that occurred during the development
and maturation of olive fruits, such as the increased content in carotenoids and phenolic
compounds during olive growing and their decrease during the ripening phase, could be
successfully monitored by Raman spectroscopy (48), providing a rapid tool to classify
olives depending on their maturation stage.
Quantification. Raman spectroscopy is usually used to determine carotenoids in fruits and

vegetables, because carotenoids are known to contribute to the beneficial effects of fruits
and vegetables. In a study by Darvin et al. (49), resonance Raman spectroscopy was used
for the determination of the carotenoid antioxidant substances in fruits and vegetables.
Schulz et al. (50) constructed an NIR-FT-Raman spectroscopy method for measurement
of lycopene and employed the Raman mapping technique to obtain deeper knowledge
of the individual carotenoid distribution. Different 7-, 8-, and 9-double-bond conjugated
carotenoids could be analyzed independently in the same sample, which demonstrated that
Raman spectroscopy was helpful for providing information on carotenoids in plant material
and related food products. Baranska et al. (51) compared the determination of lycopene
and α-carotene content in tomato fruits and related products by means of FT-Raman and
NIR spectroscopy. Raman spectra of tomato products showed characteristic key bands
of the investigated carotenoids, which provided a basis for quantitative measurements
of lycopene and β-carotene. The prediction quality of FT-Raman (R2 = 0.91 and 0.89,
standard error of cross-validation [SECV] = 74.34 and 0.34 for lycopene and α-carotene,
respectively) was better than that of NIR (R2 = 0.85 and 0.80, SECV = 91.19 and 0.41
for lycopene and α-carotene, respectively). Similar research was carried out by Bicanic
et al. (52), who assessed the trans-lycopene content of fresh tomato homogenates using
micro-Raman spectroscopy. The most intense characteristic Raman band appeared at 1520
cm−1, but the concentration of trans-lycopene in homogenate pointed to a poor correlation
(R2 = 0.52), which was most likely due to the fact that Raman measurements could not be
performed after one week during a storage time of 1–20◦ C. Resonant Raman spectroscopy
(RRS) could enlarge the signal of samples to improve the quantitative analysis. Bicanic
et al. employed RRS to estimate the concentration of beta carotene in mango homogenates
and obtained a good correlation with R2 = 0.9618 between the intensity of Raman signals
and the concentration (53). Except for carotenoid, a feasibility study performed to predict
the individual piperine and essential oil content in various ground pepper samples by NIR-
FT-Raman spectrometry showed that a high correlation between Raman spectroscopic and
chromatographic data was obtained with R2 = 0.82–0.84 for piperine and R2 > 0.95 for
essential oil (54).
Crops
For structural and qualitative analysis of crops, Raman spectroscopy shows great potential
to provide information about biochemical molecules within intact cells, tissues, and even
plants without extraction or any other use of labeling agents. The application of Raman
spectroscopy has spread in several kinds of crops, such as cacao, tobacco, cotton, rice,
wheat, and so on.
Structural Analysis. Cacao, also called kakao, is the starting material used in chocolate pro-
duction and is a popular medicine as well, used as an antiseptic, diuretic, and parasiticide.
The analytical determination of the chemical components in cacao is of great importance
industrially, especially for quality control. An investigation by Celedon and Aguilera (55)
showed the capability of Raman spectroscopy in analyzing the fat content of cacao but could
not identify the vibrational bands that are characteristic of the main components responsible
for the spectral signatures. Edwards et al. (56) employed FT-Raman spectroscopy in the
identification of a biomarker band for cacao (Sterculiaceae theobroma cacao L.) seeds and
their extracts. The spectral bands at 1744 and 1659 cm−1, (C O) and (C C) stretching
modes representative of fatty material and mainly oleic acid, were found to be the key
marker of this chemical species. Adebajo et al. (57) discussed whether FT-Raman spec-
troscopy could indicate successful acetylation of raw cotton as shown by highly sensitive
FTIR. The degree of acetylation calculated from Raman data was found to increase linearly
with that calculated from the FTIR technique, which indicated that FT-Raman spectroscopy
could also determine the acetylation level of natural cellulose fibers. Kizil and Irudayaraj
(58) applied molecular fingerprints obtained from spectral analysis and spectral variations
in the C-H stretch and O-H stretch as well as bending regions of FT-Raman spectra to
discriminate gels based on the extent of irradiation. A complete discrimination of irradiated
starches with hybrid PLS and canonical variate analysis (CVA) models was achieved. Elle-
pola et al. (59) used FT-Raman to study the conformation of rice globulin, the second major
storage protein fraction in rice, under various buffer environment and heat treatments and
revealed that hydrophobic interactions and disulfide bonds played a major role in stabilizing
the conformation and thermal aggregation of rice globulin. Siu-Mei and Ching-Yung (60)
used a common buckwheat sample and found, from changes in the Raman characteristics,
that when protein suffered extreme pHs and increased heating time at 100◦ C it showed
unfolding and denaturation. Barron et al. (61) showed that analysis of Raman spectra of
arabino-xylo-oligosaccharides allowed identification of specific spectral features of disub-
stitution and revealed the structural heterogeneity of wheat arabinoxylans. Schulte et al.
(62) reported in situ characterization of tree pollen molecular composition based on Raman
spectroscopy and successfully classified different species of pollen using the fingerprint-
like chemical information. In a further study, the resonant Raman spectra obtained from
carotenoid species upon high-performance thin-layer chromatography (HPTLC) separation
could provide evidence that in situ Raman difference spectra represented an average of the
overall carotenoid constitution in pollen (63). Almeida et al. (64) explored the applica-
tion of FT-Raman spectroscopy in classification of pure corn, cassava starch samples, and
mixtures of both starches, as well as in quantification of the amylose content in corn and
cassava starch samples. The principal component analysis (PCA) method with FT-Raman
was used, which could successfully differentiate the botanical type (corn and cassava).
Prediction errors (lower than 6%) similar to those of the standard method showed that
FT-Raman combined with PLS could be used for rapid determination of apparent amylose
in starch samples.
Classification. Raman spectroscopy was applied to discriminate between transgenic and

normal crops in crop breeding. Stewart et al. (65) reported evidence of differences between
the transgenic tobacco (Nicotiana tabacum L.) and the wild type. For transgenic tobacco,
when cinnamaldehyde was incorporated into the lignin, the expression of cinnamyl alcohol
dehydrogenase was greatly depleted. In addition, the possibility of Raman technique to dis-
tinguish seedlings of the classic rapeseed line ‘Drakkar’ from the new genetically modified
line ‘t-mix’ derived from ‘Drakkar’ was investigated. Analysis by this method showed that
95.1% of the seedlings were classified correctly. The iodine values and the lipid content
as well as composition of seedlings were calculated, with errors between 1.0 and 12.6%,
which correlated very well with traditional methods such as GC-MS (66). Research has
also been conducted on classification of geographic originals of crops and different species
of crops. Ootake and Kokot (67) reported the initial steps for FT-Raman spectroscopy for
good discrimination of glutinous and nonglutinous rice. Kim et al. (68) applied a wide area
illumination scheme (WAI) to collect Raman spectra for differentiation of rice of two geo-
graphical origins: Chinese rice and Korean rice. The research exhibited that a WAI scheme
could efficiently produce Raman spectra with a more reliable sample representation, as
well as better reproducibility. It indicated that a Raman technique employing the WAI
scheme presented good potential for analysis of diverse agricultural samples consisting
of solid granules. Visible micro-Raman spectroscopy combined with PCA was applied by
El-Abassy et al. (69) for fast discrimination between two coffee species: Arabica and Ro-
busta beans. A clear separation between Arabica and Robusta with 93% of the total spectral
variation based on chlorogenic acid (CGA) and 85% based on lipid contents was achieved.
Results obtained for internal quality detection were satisfactory. Eidler-Lozykowska et al.
(70) employed mapping accessory and point acquisition modes to accelerate Raman
measurements of essential oil content and composition of caraway (Carum carvi L.) sin-
gle fruit. A high congruence between data from Raman measurements and reference GC
indicated that an FT-Raman technique is a reliable tool for selection of fruit with desired
essential oil composition without fruit destruction.
Meat and Dairy Products

Dairy Products. A fast, nondestructive, and accurate method to determine the main nutri-
tional parameters in dairy products is of great importance to establish nutritional values
and provide consumer information. Following the first determination of fat in milk powder
with Raman spectroscopy by Fehrmann et al. (71), Moros et al. (72) used an FT-Raman
spectroscopy method in conjunction with hierarchical cluster analysis (HCA) and PLS for
quantification of nutritional parameters in powdered milk and infant food formulas, such
as energetic value and total carbohydrates, protein, and fat. Good quality coefficient (QC)
values of 3, 3, 4, and 9 were obtained. An initial data pretreatment in the quantitative anal-
ysis of Raman spectroscopy was indispensable to obtain reasonable results. McGoverin
et al. (73) focused on the quantification of protein and fat in skim and whole milk powders.
Sample temperature was shown to alter the predicted fat and protein content within milk
powder, but the accuracy for protein was lower, which may be due to the increased protein
variance. Meurens et al. (74) made the first successful trial of conjugated linoleic acids
(CLA) determination in cow’s milk by FT-Raman spectroscopy. Three specific signals at
1652, 1438, and 3006 cm−1 were found to be well suited for accurate and reliable mea-
surement of CLA concentration in milk. Bernuy et al. (75) compared the performance of
UV and FT-Raman spectroscopic techniques in the determination of CLA in milk. The best
calibration for FT-Raman was obtained by a PLS model of seven factors with a standard
error of prediction (SEP) of 0.246, indicating that FT-Raman spectroscopy may be used as a
reference spectroscopic method for CLA determination in the future. El-Abassy et al. (76)
employed UV-Vis Raman spectroscopy in combination with PLS to directly analyze milk
fat in liquid milk in an open dish, in quartz cuvettes, and dried milk droplets on glass plates
covered with Al foil. The application in liquid milk in quartz cuvettes was not very useful
due to the formation of an undefined milk layer on the inner surface of the cuvette. The
other two models obtained low root mean square errors and high correlation coefficients.
They also pointed out the effect of varying temperature on Raman spectroscopy would be
investigated in their further study. Because fatty acid composition and lipid content varied
according to the size of the globules, confocal Raman microscopy was used by Gallier
et al. (77) to determine the chemical fingerprint of individual bovine milk fat globules
of different sizes and from different breeds. The results suggested that small fat globules
delivered information about the lipids of the milk fat globule membrane and the milk fat
globules showed different properties according to their sizes.
In the dairy products industry, some adulterated milk consists of whey which could
compromise the nutritional values. Discrimination of adulterated and unadulterated milk
powder samples as well as identification of the types of milk powder (whole, low-fat,
skimmed, or modified) was explored by FT-Raman spectroscopy (78). A 100% classification
of the adulterated samples was obtained with a PLS-DA model, and the separation of milk
powder samples according to types was performed with a PCA model. Recently, some high-
protein milk products have contained large doses of melamine, which is harmful to health
and can result in urinary calculi, acute renal failure, and even infant death (79). Traditionally,
melamine analysis has been carried out by GC, LC, and so on, which are time consuming
and also chemical reagent consuming. Raman spectroscopy provides a very rapid screening
test for melamine-adulterated dried milk in both the food chemistry and forensic toxicology
(80). Okazaki et al. (81) used a Raman spectroscopic method based on an intense band at
676 cm−1 to detect melamine in dried milk with a detection limit of about 1% (w/w), which
is a new method for melamine determination. Qin et al. (82) reported that micro-Raman
spectroscopy with a spatial resolution as high as 0.1 mm could visualize the quantity and
spatial distribution of melamine particles in mixtures with concentrations (w/w) ranging
from 0.2 to 10.0%. In order to make Raman spectroscopy suitable for on-line analysis,
Cheng et al. (83) constructed a portable compact Raman spectrometric system to determine
melamine adulteration in milk powder. Two characteristic vibrational modes at 673 and
982 cm−1 were identified with good reproducibility. When the first mode was employed, a
detection limit (DL) of 0.13% was obtained. SERS was reported to have a great potential
for detecting samples with a limit of detection (LOD) down to parts per billion (ppb) or even
a single-molecule level. He et al. (84) achieved a limit of detection of obtained LODs of
33 ppb in milk aqueous solution with gold nano-substrates. Lee et al. (85), 100 ppm by
the gold nanosubstrate, and a better 200 ppb by the roughened gold substrate in powdered
milk, respectively.
Meat. Raman spectroscopy has considerable potential for determination of meat and fish
quality parameters, including protein, fat, and so on. Raman spectroscopy with a 780-nm
laser is suitable for investigating the effect of external condition changes on meat and fish
structure, because meat or fish exhibits little fluorescence with a 780-nm laser. Beattie
et al. (86) created a Raman spectroscopic method for predicting consumer-perceived beef
quality, including texture, tenderness, juiciness, and overall acceptability. The success of
Raman spectroscopy for the texture and tenderness quality attributes, the predominant
factors in consumer-perceived beef quality was proven. Furthermore, the effects of aging
and cooking on the Raman spectra of porcine longissimus dorsi were investigated (87).
Good correlations and standard errors of prediction in unhomogenized and untreated raw
samples and cooked pork were obtained, with R2 = 0.77, Root-mean square error of
prediction (RMSEP) = 12% for shear force and R2 = 0.71, RMSEP = 10% for cooking
loss. The first trial of Raman spectroscopy in predicting FFA composition of unextracted
adipose tissue of pork, beef, lamb, and chicken was done by Beattie’s team (88). The
predicted accuracy of bulk properties and detailed FFA was at least as good as that of
other spectroscopic methods such as FTIR and NIR. Based on the study by Beattie et al.
above all (86–88), it can be concluded that although the accuracy of Raman spectroscopy
does not approach that of conventional GC methods, it is still suitable for on-line analysis,
because the lower accuracy obtained can be compensated for by the ability to make rapid,
noncontact measurements with no additional sample preparation. Olsen et al. (89) achieved
good correlations for polyunsaturated FFA and monounsaturated FFA in pork adipose tissue
with R2 = 0.98, Root-mean square error of cross validation (RMSECV) = 1.0% and R2
= 0.96, RMSECV = 1.0%, respectively. The success may be ascribed to the use of a ball
probe with a sapphire spherical lens, which helped improve sampling precision. The results
strongly indicated that Raman spectroscopy with a ball probe could be used to measure the
degree of unsaturation in a single pork carcass on-line. Marquardt and Wold (90) conducted
an exploratory study for quantitative measurements of carotenoid, collagen, and fat in fish
muscle. The Raman spectra extracted by PCA from the samples contained detailed spectral
and relative concentration information on all the studied constituents. Afseth et al. (91)
provided three Raman measurements on intact salmon muscles, ground salmon samples,
as well as oil extracts. Higher prediction errors were obtained from intact salmon muscle
than the other samples, which may be explained by sampling uncertainties in the relation
between Raman measurements and reference analysis. But all of the partial least squares
regression (PLSR) models that were based on sound regression coefficients suggested that
the information regarding FFA unsaturation was readily available from Raman spectra
even in systems with high contents of protein and water. He et al. (92) reported the lowest
detectable concentration for dye molecules of 0.2 ppb in select seafoods using an SERS
technique.
Thawornchinsombut et al. (93) elucidated the structural changes of alkali-treated pro-
tein isolates (AKPIs) in rockfish during frozen storage using a Raman spectrometer. A
greater reduction in protein quality was observed when AKPI were kept frozen at pH 5.5
than at pH 7.0. Because lipid oxidation can affect the solubility and extractability of fish
myofibrillar proteins, Sarkardei and Howell (94) observed the effect of freeze drying and
storage on Atlantic mackerel (Scomber scombrus) and horse mackerel (Trachurus trachu-
rus) using FT-Raman spectroscopy. The spectra showed that freeze drying affected the
structure and composition of fish lipids, with particularly prominent changes during the
long-term storage of freeze-dried fish at 22◦ C. Herrero et al. (95) showed the fact that
Raman bands were more prone to changes due to different storage temperatures. The cal-
ibration regression coefficients (R2) and the prediction of PLSR models ranged from 0.99
to 0.97 and 0.95 to 0.89 in the 600–1800 cm−1, respectively. The results indicated the
potential of Raman spectroscopy for establishing the time–temperature history of frozen
fish muscle. Then a Raman spectroscopic evaluation of meat batter structural changes
induced by thermal treatment and salt addition was made (96). A significant correlation
(p < 0.05) between textural properties of meat batter and the structural changes in meat
proteins and water binding revealed by Raman analysis was found. The research opened
new possibilities that the development of meat products could be improved when thermal
treatment and/or salt addition was used. The effect of plasma powder addition, with or with-
out NaCl to meat emulsion, was also studied (97). A good correlation (p < 0.05) between
meat protein structural changes and rheological properties of meat systems was found. The
effect of pressure on the structure of pork was studied by Wackerbarth et al. (98) with RRS
combined with a 413-nm excitation. It was found that a shift of the electronic transitions of
the heme did not only cause a color change but also initiated unwanted oxidative side reac-
tions involving further components of meat. Soo-Yeun and Li-Chan (99) investigated the
influence of ingredients such as glucosamine, sucrose, ascorbic acid, and/or polyethylene
glycol on the release of beefy aroma components of simulated beef flavor in the presence
of soy protein isolate. The research paved the way for further study to elucidate strategies
maximizing the perception of a beefy aroma in soy products. Fu et al. (100) applied Raman
spectroscopy to investigate the effect of formaldehyde (FA) on the structural changes in
squid (Loligo japonica) collagen. When collagen was treated with 2 mmol L−1 FA, the
ratio of I940 /I1005 and tyrosine doublet ratio were increased, suggesting that the α helix,
β sheet type, and random coil structures were changed by FA. Sánchez-González et al.
(101) indicated that the combination of isotopic Hydrogen/Deuterium (H/D) exchange and
Raman spectroscopy assisted by monitoring of rheological characteristics could reveal the
protein and water structural changes in fish surimi during gelation.
Oil
Adulteration. In the olive oil industry, there is a tendency toward mislabeling or adulterating
extra-virgin olive oil with refined olive oil or cheaper and similar oils. The identification
of different oils and the detection of oil adulteration are of great importance from both
market and health perspectives. Many efforts (102–105) have been made to classify oils
in different categories and analyze adulterated oils. Lopez-Diez et al. (106) successfully
discriminated the adulteration of virgin olive oils with hazelnut oils and quantified the levels
of hazelnut oils in the range of commercial interest from 0 to 20%. Zou et al. (107) used a
portable Raman spectroscope with intensity ratios of the vibration bands for discrimination
between the various grades of olive oils and seed oils. Genuine olive oils can be reliably
distinguished from the olive oils containing 5% (volume percentage) or more of other
edible oils, such as soybean oil, rapeseed oil, sunflower seed oil, or corn oil. Due to the high
sensitivity of micro-Raman spectroscopy, El-Abassy et al. (108) investigated the potential of
visible Raman spectroscopy for classification of different vegetable oils and quantification
of adulteration of virgin olive oils. Ninety-six percent of the spectral variation of different
vegetable oils as well as sunflower oil and extra-virgin olive oil was characterized by PCA.
A quantitative LOD down to 500 ppm (0.05%) with a low error (RMSECV = 0.036) and
high correlation coefficient (R2 = 0.971) was achieved. In addition, the essential oil was
used to provide information for different plant spices discrimination (109), fast essential
oil classification, and selection of requested individual essential oil types.
Quantification. Generally, Raman spectroscopy has seldom been used to identify the unsat-
uration of FFA in quantitative analysis. In recent years, with the introduction of chemometric
methods, such applications have been found in literature. Barthus et al. (110) applied NIR-
FT Raman spectroscopy for determination of the total degree of unsaturation (iodine values
ranging from 17 to 130) in vegetable oils. A correlation coefficient of 0.996 indicated good
agreement with the data obtained from the official titrimetric methodology. A group of
acyclic, unsaturated sulfur-containing components present in garlic oil was quantitatively
analyzed with FT-Raman spectroscopy by Kimbaris et al. (111). Band intensities at 1636
and 1606 cm−1 were used from untreated garlic oil spectra to determine the percentage
content in acyclic, unsaturated sulfur-containing compounds. Ghesti et al. (112) employed
Raman spectroscopy to monitor the trans-esterification of soybean oil. The best results
were achieved by Raman/PLS models with R2 of 0.9985 for calibration and 0.9977 for
validation. Quantitative evaluation of a total conjugated linoleic acid was seen in the re-
search of Bernuy et al. (113). The results demonstrated that the PLS calibration model
containing sufficient information of I2 -photoisomerized soybean oil was developed using
the (C C) region (1642–1680 cm−1). The model was reliable for measurement of total
CLA without the interference of other compounds. El-Abassy et al. (114) introduced a
method for determining the FFA content of extra virgin olive oil with multivariate analysis.
The spectral window (945–1600 cm−1) including the carotenoid bands was the significant
fingerprint region for the prediction of FFA with a high R2 (0.963167) and small root mean
square error (RMSE) values (RMSEC = 0.01193, RMSEV = 0.034114).
Beverages
Raman spectroscopy can be applied in quality and security control of beverages, including
soft drinks, alcoholic beverages, and other liquid mixtures for their important role in
people’s daily lives.
Quality Control. Studies on the structure and properties of alcoholic solutions are of im-
portance and interest from both basic scientific and practical points of view (115). Most of
the research was conducted by Nose’s group and was related to the role of the properties
of water in the quality of alcoholic beverages, such as flavor and salty components. Nose
et al. (116–119) devoted their efforts to clarifying what affected hydrogen-bonding struc-
ture of water–ethanol in alcoholic beverages. From the Raman spectra of Japanese sake and
ethanol–water mixtures, they found that the strength of the hydrogen bond of water–ethanol
was correlated with that total concentration of organic acids, amino acids, (poly) phenols,
and conjugate base anions of weak acids. However, the concentration of glucose or sac-
charides should not have a strengthening effect of the hydrogen bond of water-ethanol but
should moderate the sour and astringent taste the beverage. Zhu et al. (120) presented a
collection of Raman spectra of 18 kinds of amino acids (l-alanine, l-arginine, l-aspartic
acid, cystine, l-glutamic acid, l-glycine, l-histidine, l-isoluecine, l-leucine, l-lysine, l-
phenylalanine, l-methionone, l-proline, l-serine, l-threonine, l-tryptophan, l-tyrosine,
l-valine) and their aqueous solutions, which could serve as references for the interpretation
of Raman spectra of proteins and biological materials.
Frausto-Reyes et al. (121) elaborated a PCA method based on the OH region profile
of Raman spectra to qualitatively study the ethanol content in tequila samples. Three
PCs, which represented 99% of the total variance of the data set, were used for sample
classification. The results indicated that the use of ethanol and distilled water as standards
is important. Silveira et al. (122) combined dispersive Raman spectroscopy with PLSR
to quantify the amount of sucrose in four commercial lemon-flavored soft drinks. The
predicted sugar content ranged from 8.1 to 10.9 g mL−1 and a prediction error ranging
from 1.1 to 5.5% proved that Raman spectroscopy is an effective method of quantifying
sucrose content. Similar research was carried out for the qualification of glucose content in
sports drinks (123). Although the model, interval partial least square regression (IPLS), was
different, good agreement with the values of a biochemical assay was obtained. Eliasson
et al. (124) reported a new concept, a variant of SORS, in which any interfering Raman and
fluorescence contributions from the bottle could be efficiently suppressed. The method can
be used to detect cocaine concealed inside transparent glass bottles containing alcoholic
beverages with an LOD of 0.04 mol L−1 in 1 s acquisition time.
Different types of Raman spectrometers have been used for determining alcoholic
content. Sanford and Mantooth (125) described a modular Raman spectrometer combining
the peak area method to determine ethanol content. The LOD could reach 1%. Mendes
et al. (126) compared the ability of FT-NIR and FT-Raman in determination of alco-
hol content of alcoholic beverage. The FT-Raman model using PLS presented an ac-
curacy equivalent to the reference method (ASTM D4052) as evaluated using t-test,
slightly better than NIR, and 1% better than GC. The results also indicated that the
external standard (hexachloro-1,3-butadiene) could acquire better precision and accu-
racy of the calibration models than the internal standard. Nordon et al. (127) also
compared the suitability of NIR and Raman spectrometry for detection of percentage
ethanol content. The precision of the two methods was found to be comparable (aver-
age relative standard deviation [RSD] of 0.4 and 0.5% for NIR and Raman spectrome-
try, respectively). Though the accuracy of NIR was slightly better than Raman, Raman
spectrometry provided a more direct measurement and provided an easier calibration
model than NIR. Meneghini et al. (128) developed a liquid-core air-clad microstruc-
tured fiber for determination of ethanol concentrations in aqueous solutions using Ra-
man spectroscopy. The concentration values agreed with the expected values within an
estimated uncertainty of ±0.1 wt% concentrations. Nah et al. (129) proposed a novel
and reliable quantitative Raman measurement scheme (WAI) for the analysis of ethanol
and isopropanol solutions. The use of Teflon tubing as the intensity correction standard and
sample cell could accomplish quantitative analysis of diverse liquid samples with better
reproducibility and prediction performance than conventional Raman analysis.
Safety Control. Raman spectroscopic techniques are not only applied in quality control
but also in safety control of beverages, in particular for the detection of adulterants and
microorganisms. When Paradkar and Irudayaraj (130) detected adulterants such as cane
and beet invert in honey by FT-Raman with linear discriminant analysis (LDA) and CVA,
they found that FT-Raman was efficient in predicting beet and cane invert adulterants
with R2 > 0.91 in all types of honey considered. Schmilovitch et al. (131) obtained a
clear distinction between samples containing bacteria and clean samples using a Raman
spectrometer. The Raman method could detect the Erwinia carotovora pv. carotovora
(ECC) and Clavibacter michiganense (CBM) with a concentration of 10 to 100 cells mL−1
in mixed bacteria suspensions. Mizrach et al. (132) achieved the best detection (100%) at
the predicted concentration of 10 colony-forming units (CFU) mL−1 of yeast in apple juice
using Raman spectroscopy. Camerlingo et al. (133) reported the possibility of using Raman
spectroscopy for on-line monitoring of the production of clarified fruit juice, apple juice,
and apricot juice. Data analysis enabled the clear identification of pectin, which played
a strategic role in the production of clarified juice. Aspartame is commercial sweetener

widely used in the beverage. Mazurek and Szostak (134) applied Raman spectroscopy in
commercial preparations containing between 17% and 36% of aspartame by weight. The
results agreed perfectly with the results of the UV-Vis reference analysis, with recoveries
in the 98.7–100.8%, 98.6–101.1%, and 97.8–102.2% ranges for PLS, Principal component
regression (PCR), and counter-propagation artifical neural networks (CP-ANN) models,
respectively (134). Naja et al. (135, 136) found that the SERS technique was capable of
detection 103 cell mL−1 E. coli in milk and apple juice without any preenrichment.
Conclusion
In recent years, advances in Raman spectroscopy instrumentation have allowed the de-
velopment of this spectroscopic technique, providing new perspectives and paving ways
for applications of Raman spectroscopy in agricultural products and food analysis. Raman
spectroscopy is a powerful tool to assess internal quality and safety, due to many advan-
tages such as nondestructive detection, no sample preparation, and fast measurement. Due
to its narrow and sharply resolved bands, Raman spectroscopy shows great potential in
qualitative, structural, and quantitative analysis. However, there is still a long way to go.
Such features as fluorescence from the sample, absorbance of the reaction mixture, as well
as analysis conditions including laser power, scan times, and orientation of the sample can
jeopardize accurate quantification. The introduction of internal or external standards and
the development of new techniques such as SERS, micro-Raman spectroscopy, or SORS
techniques are promising for improvement of the precision and sensitivity of analysis. The
combination of Raman spectroscopy with other techniques such as NIR, FTIR, or chemo-
metric methods such as PLS and PCA is promising for overcoming these defects. The
results are encouraging and we expect that Raman spectroscopy will replace traditional
methods and play an important role in on-line quality and safety control of agricultural
products and food in the future.
Acknowledgments
We are grateful for the financial support provided by the National Natural Science Foun-
dation of China (No. 30825027).
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Applications of Raman Spectroscopy in Agricultural Products and

Article in Applied Spectroscopy Reviews · October 2011

Danting Yang Yibin Ying

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Applications of Raman Spectroscopy in Agricultural

DANTING YANG1 AND YIBIN YING2

Figure 1. Simple diagram for Raman Effect.

Theoretical Basis of Raman Spectroscopy

Figure 2. Energy level diagram for Raman scattering.

weak and unobtrusive, it is a good tool for application in aqueous solutions.

Figure 3. Schematic diagram of dispersive Raman spectrometer.

Dispersive Raman Spectroscopy

Fourier Transform Raman Spectroscopy

Surface-Enhanced Raman Spectroscopy

Figure 4. Schematic diagram of FT-Raman spectrometer.

Spatially Offset Raman Spectroscopy

Figure 5. Schematic diagram of conventional Raman and SORS approach.

2001 and 2011.

Fruits and Vegetables

Quantification. Raman spectroscopy is usually used to determine carotenoids in fruits and

Classification. Raman spectroscopy was applied to discriminate between transgenic and

Meat and Dairy Products

a strategic role in the production of clarified juice. Aspartame is commercial sweetener

fluorescence excitation-emission matrices in combination with PARAFAC, as fingerprints of

Spectrochim. Acta A, 60: 2231–2234.

and pattern recognition techniques. J. Agr. Food Chem., 52: 6055–6060.

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