Critical Reviews in Food Science and Nutrition

ISSN: 1040-8398 (Print) 1549-7852 (Online) Journal homepage: https://www.tandfonline.com/loi/bfsn20

Applications of Raman spectroscopic techniques

for quality and safety evaluation of milk: A review
of recent developments

Huirong He, Da-Wen Sun, Hongbin Pu, Lijun Chen & Li Lin

To cite this article: Huirong He, Da-Wen Sun, Hongbin Pu, Lijun Chen & Li Lin (2019) Applications
of Raman spectroscopic techniques for quality and safety evaluation of milk: A review of
recent developments, Critical Reviews in Food Science and Nutrition, 59:5, 770-793, DOI:
10.1080/10408398.2018.1528436

To link to this article: https://doi.org/10.1080/10408398.2018.1528436

Published online: 07 Jan 2019.

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CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION
2019, VOL. 59, NO. 5, 770–793
https://doi.org/10.1080/10408398.2018.1528436

REVIEW

Applications of Raman spectroscopic techniques for quality and safety

evaluation of milk: A review of recent developments
Huirong Hea,b,c, Da-Wen Suna,b,c,d, Hongbin Pua,b,c, Lijun Chene, and Li Line
a
School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China; bAcademy of Contemporary
Food Engineering, South China University of Technology, Guangzhou Higher Education Mega Centre, Guangzhou 510006, China;
c
Engineering and Technological Research Centre of Guangdong Province on Intelligent Sensing and Process Control of Cold Chain Foods,
Guangzhou Higher Education Mega Centre, Guangzhou 510006, China; dFood Refrigeration and Computerized Food Technology (FRCFT),
Agriculture and Food Science Centre, University College Dublin, National University of Ireland, Dublin 4, Ireland; eBeijing Sanyuan Foods Co.,
Ltd, Beijing, China

ABSTRACT KEYWORDS
Milk is a complete nutrient source for humans. The quality and safety of milk are critical for both Surface-enhanced Raman
producers and consumers, thereby the dairy industry requires rapid and nondestructive methods spectroscopy (SERS);
to ensure milk quality and safety. However, conventional methods are time-consuming and labori- Fourier-transform (FT)
Raman spectroscopy; micro-
ous, and require complicated preparation procedures. Therefore, the exploration of new milk ana- Raman spectroscopy; milk
lytical methods is essential. This current review introduces the principles of Raman spectroscopy compositions; microorgan-
and presents recent advances since 2012 of Raman spectroscopic techniques mainly involving isms; antibiotic residues;
surface-enhanced Raman spectroscopy (SERS), fourier-transform (FT) Raman spectroscopy, near- adulterants
infrared (NIR) Raman spectroscopy, and micro-Raman spectroscopy for milk analysis including milk
compositions, microorganisms and antibiotic residues in milk, as well as milk adulterants.
Additionally, some challenges and future outlooks are proposed. The current review shows that
Raman spectroscopic techniques have the promising potential for providing rapid and nondestruc-
tive detection of milk parameters. However, the application of Raman spectroscopy on milk ana-
lysis is not common yet since some limitations of Raman spectroscopy need to be overcome
before making it a routine tool for the dairy industry.

1. Introduction nitrogen-rich chemicals (e.g., melamine, dicyandiamide,

Milk as a complex natural food matrix, is one of the most
important dietary products, which contains nearly all the
nutrients necessary to sustain life (Finley 1995), including
nearly 90% water, different proportions of lipid, protein and
carbohydrates, and small amounts of minerals and other
micronutrients (Varnam & Sutherland 1994). The health
and economic benefits of milk, and the improvement of
human living standards lead to rapid increase in global milk
consumption, especially in developing countries (Jaiswal
et al. 2015). With the increasing demand for high quality
life, the quality and safety of milk are gaining more and
more attention for consumers and producers.
The composition of milk is an important indicator to
evaluate the quality of dairy products. For the dairy indus-
try, contents of fat, protein and carbohydrate in milk must
be labeled on commercial products, in addition, special milk
products such as low-lactose dairy goods should offer the
information on lactose contents. In order to meet these milk
indicators and gain more economic benefits, some unscru-
pulous producers add extraneous water, nondairy proteins,
melamine, urea, animal fat in milk. High-protein materials
(e.g., whey powder, soy proteins, egg white protein) and
urea, ammonium sulfate) are common adulterants for
increasing protein contents in milk products because they
can significantly raise the nitrogen concentration (Qin et al.
2017). However, the excessive addition of these nitrogenous
compounds can damage the kidneys, causing illness or even
deaths for consumers. In addition, milk produced by cows
with mastitis has a lot of harmful microorganisms (e.g.,
E. coli, L. monocytogenes, S. aureus), which poses a threat to
human health as pathogenic bacteria can transmit from milk
to humans (Dong et al. 2011). Therefore, it is necessary to
detect these harmful microorganisms in milk effectively and
accurately. In addition, antibiotics such as penicillin G,
ampicillin and tetracyclines are used for treating cattle with
mastitis, strains of antibiotic resistant bacteria may thus
vastly generate as a result of overusing these antibiotics
(Durso & Cook 2014; Singer & Williams-Nguyen 2014).
Recently, the frequent reports of food safety incidents have
drawn widespread public attention to the quality and safety
of food products. Therefore the food industry not only needs
processing techniques such as drying (Qu et al. 2017; Ma et
al. 2017; Ma, Qu & Sun 2017; Lui, Pu & Sun 2017; Pu & Sun
2015), cooling (Zhu et al. 2018; Zhou et al. 2017; Feng et al.

CONTACT Da-Wen Sun dawen.sun@ucd.ie; www.ucd.ie/refrig; www.ucd.ie/sun School of Food Science and Engineering, South China University of
Technology, Guangzhou 510641, China; Lijun Chen Beijing Sanyuan Foods Co., Ltd, Yinghai, Daxing District, Beijing 100085, China.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/bfsn.
ß 2019 Taylor & Francis Group, LLC

2014; Kiani, Sun & Zhang 2012) and freezing (Xie et al. 2015;
Xie et al. 2016; Qu et al. 2017; Cheng et al. 2018; Zhu et al.
2018; Zhu et al. 2018; Cheng et al. 2018; Li, Zhu & Sun 2018;
Luo et al. 2018) to maintain food quality and safety attributes,
but also requires methods to evaluate and control these attrib-
utes. For the dairy industry, the sources of raw milk, process-
ing methods as well as ways of transportation and storage
have a significant impact on the quality of dairy products, so
that each step in production should be carefully monitored
and evaluated. Consequently, the demand for milk quality and
safety evaluation methods has been dramatically increased.
Up to now, traditional methods such as chromatographic


techniques (Alvarez et al. 2013; Manzi & Pizzoferrato 2013),
mass spectrometry (Soukoulis et al. 2012), and enzyme bio-
sensors (Alexandre et al. 2018) are available for food quality
and safety evaluation, however these methods are destructive
and require complicated pretreatments and lengthy sample
analysis procedures and hyperspectral imaging (Liu et al.
2018; Ma, Pu and Sun 2018; Pan et al. 2018; Cheng et al.
2018; Xu, Esquerre, Sun 2018; Xu, Gowen and Sun 2018;
Liu, Pu and Sun 2017; Cheng et al. 2017; Cheng and Sun
2017; Ma, Sun and Pu 2017; Cheng, Nicolai and Sun 2017;
Xu, Riccioli, Sun 2016; Cheng, Sun and Cheng 2016; Cheng,
Sun, Pu and Liu 2016; Dai et al. 2016; Cheng et al. 2016; Pu
et al. 2016; Pu et al. 2015) techniques have been developed
for effective and efficient food quality and safety evaluation.
In particular, IR spectroscopy (Boubellouta & Dufour
2012; Jha et al. 2015; Shao & He 2009; Wu et al. 2012) has
been widely used in dairy products analysis due to its high
sensitivity, and simple and rapid detection process, but it
has limitation in detecting liquid milk due to water interfer-
ence (Li-Guo et al. 2014). Therefore Raman spectroscopy
has been proposed to avoid the problem of water interfer-
ence. Raman spectroscopy can provide accurate and reliable
information about the tested molecules in different sample
states including liquid milk and powdered milk. Therefore,
Raman spectroscopy has been gradually applied in milk
enterprises for rapid, sensitive, nondestructive and conveni-
ent detection (Vinogradova et al. 2014). The current review
presents recent advances of Raman spectroscopic techniques
for milk quality and safety evaluation since 2012.
2. Principles of raman spectroscopy
Raman spectroscopy was first documented by Raman and
Krishnan (1928), which is based on an inelastic scattering
effect (Raman & Krishnan 1928). When a sample is irradi-
ated by a high-intensity monochromatic light, most of the
scattered photons have no energy exchange with the sample
molecules, and these scattered photons just change the direc-
tion of propagation with their frequency being the same as
the excitation photons, thus such elastic scattering is called
Rayleigh scattering. On the other hand, a few of the scattered
photons (10
1010
6) can have energy exchange with the
sample molecules, and the direction and frequency of the
scattered photons can also change, thus such inelastic scatter-
ing is called Raman scattering (Fig. 1). When the scattered
photons lose or gain energy, they will shift to longer or
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 771
shorter wavelengths, consequently, Stokes lines or anti-Stokes
lines appear in the spectra (Lord 1990). The frequency differ-
ence between the Stokes scattered light (or anti- Stokes scat-
tered light) and the incident light is called Raman shift (Dv
in cm
1). The intensity of Stokes scattering is usually much
stronger than anti-stokes scattering, and Stokes scattering is
used for the application of Raman spectroscopy in food ana-
lysis (Morris 2006).
There are different forms of vibration in sample molecules,
however only a very few of the vibrational forms can give
Raman signals. As the polarizability of the molecule changes
during molecular vibration, Raman signals can therefore be
observed due to this change (Dijkstra et al. 2005). Stronger
Raman signals of functional groups, e.g., C–X (X ¼ F, Cl, Br
or I), C–NO2, C–S, S–S, C¼C, C¼N, can appear in spectrum
due to the stronger polarizability changes of these molecule
(Dijkstra et al. 2005). As shown in Fig. 2, the strongest
Raman peak of melamine, urea, penicillin G, ampicillin and
lactose are assigned to the in-plane ring breathing II mode,
the symmetric N-C-N stretching, b-lactam ring, phenyl ring
and C-O-C stretching vibration, respectively.
The frequency shifts of scattered light can be exhibited as
spectral bands in Raman spectrum. Different spectral bands
represent different chemical bonds or/and functional groups
of samples. On one hand, Raman spectroscopy can deter-
mine the fingerprint of specific molecule presented in sam-
ple, thereby realizing structural analysis and qualitative
analysis. On the other hand, Raman spectroscopy can be
successfully applied for quantitative determination as the
band intensity is linearly proportional to the concentration
of the tested molecule (Yang & Ying 2011). As shown in
Fig. 2, the Raman band intensity of analyte-spiked milk is
proportional to its concentration.
As a mature spectral analysis technology, Raman spec-
troscopy has been developed into different analysis technolo-
gies including surface-enhanced Raman spectroscopy
(SERS), Fourier-transform (FT) Raman spectroscopy, micro-
Raman spectroscopy, near infrared (NIR) Raman spectros-
copy, and spatially offset Raman spectroscopy (SORS),
which have been gradually employed for milk analysis.
Table 1 compares different applications of Raman spectros-
copy in milk detection.
3. Applications of raman spectroscopy in milk
Sample preparation is a necessary step in Raman analysis in
order to obtain reliable detection results, as milk is a com-
plex food matrix. As shown in Table 1, for sample prepar-
ation, actual milk samples are normally used for
determining intrinsic analytes such as milk components
(e.g., protein, fats and lactose), while pretreated samples by
spiking the analyte in milk are needed for evaluating con-
tamination analytes such as microorganisms, antibiotics and
additives. In the latter case, reliable results are unlikely to be
obtained as the spiked milk samples could not substitute
real contaminated milk samples. In addition, Raman results
are easily interfered by other components in milk, therefore
sample pretreatments including fat extraction, protein
772 H. HE ET AL.
Figure 1. Energy level diagram of Rayleigh and Raman scattering (Boyaci et al. 2015).
precipitation and removal of other big molecules are
often required.
Raman spectral pretreatment is another essential step to
obtain effective results in milk analysis. Reportedly, robust
summation, upper-bound spectrum (UBS), smoothing/filter-
ing techniques and nearest neighbor comparison are used
for eliminating cosmic spikes (Zhang & Henson 2007). As
shown in Table 2, the baseline correction algorithms (poly-
nomial fitting, wavelet transformation, airPLS) can be
applied for suppressing fluorescence background, while
Savitzky-Golay filter, Fourier transform, and wavelet shrink-
age are commonly used to improve signal-to-noise.
Moreover, normalization algorithms (standard normal vari-
ate, multiplicative scatter correction, peak normalization) are
employed for evaluating real samples. More approaches and
their applicability in Raman analysis are detailed in several
literatures (Lohumi et al. 2017; Luca, Dholakia, and Mazilu
2015; Schulze et al. 2005).
3.1. Detection of milk compositions
The composition of milk is very important because it greatly
determines the quality, nutritional value and economic value
of dairy products. Therefore, a rapid, nondestructive, and
sensitive method to determine the main nutritional parame-
ters including fats and fatty acids (FAs), proteins and lactose
in milk is quite necessary for the purpose of providing reli-
able nutritional value information to consumers.
3.1.1. Fats and FAs
Milk fat is an important component in milk, which contains
various FAs. Most FAs provide benefits to human health,
such as short chain FAs, C18:2 cis-9, trans11, A-linolenic
acid and oleic acid (Shingfield et al. 2008). However, some
harmful FAs exist in milk such as medium chain saturated
FAs, trans 9-C18:1 and trans10-C18:1, pose a potential
threat to human health (Bauchart et al. 2007). In some
European countries, the composition of milk fat is a stand-
ard indicator for milk prices (Coppa et al. 2014). Therefore,
the accurate determination of fat content and FAs types in
milk products is drastically needed for the dairy industry,
and Raman spectroscopy is proved to be an effective tool.
Excitation wavelength is inversely proportional to Raman
signal. Shorter laser excitation wavelengths can generate
stronger Raman signals than longer laser excitation wave-
lengths. However, Raman signal is easily interfered by fluor-
escence at shorter excitation wavelengths. Therefore,
selection of the excitation wavelength is important for
 CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 773

Figure 2. Representative SERS spectra of (A - D) melamine spiked milk samples (Li et al. 2016; Lin et al. 2014; Rajapandiyan, Tang, and Yang 2015; Yang et al.
2017), (E) urea spiked milk sample (Khan et al. 2015), (F) PENG spiked milk sample (Chen et al. 2017), (G) ampicillin spiked water and milk (Andreou et al. 2015),
and (H) lactose spiked milk sample (Li et al. 2015).

Raman analytical results. For milk, dispersive Raman spec-
troscopy and Fourier transform (FT) Raman spectroscopy
have been commonly used for fats detection
Dispersive Raman spectroscopy is the most common and
basic technique, which is very suitable for aqueous-phase
samples (Yang & Ying 2011). El-Abassy et al. (2011) selected
a 514.5 nm emission light to detect fat content in liquid
milk within three different sample pretreatment methods.
The partial least squares (PLS) model for determining milk
fat content had a high correlation coefficient (R2) when milk
sample in an unclose Al dish or dried milk droplets on glass
dish covered with Al foil, while a poor model with low R2
Table 1. Applications of Raman spectroscopy for detecting different attributes of milk quality and safety. 774

Substrates or Detection of
Targets Specific targets chemometric methods peaks (cm
1) Results Samples preparation References
Compositions Fat PLS 800–3050 R2¼0.99 RMSE ¼ 0.15 Real milk samples El-Abassy et al. (2011)
Conjugated linoleic MLR, PLS 1652, 1438, 3006 R ¼ 0.972,0.975 (MLR) Real milk samples (anhydrous milk-fat Meurens et al. (2005)
acid (CLA) R ¼ 0.954, samples: cream separate, churn, melt,
0.951 (PLS) centrifugate)
H. HE ET AL.

Total trans FAs PLS 900–1798 RMSE ¼ 0.48 Real milk samples (fatty acid samples: Zhao et al. (2015)
melt, centrifugate, separate
and methylate)
FAs PLS 1265, 1650, 2850, 3005 R2 ¼ 0.99, RMSE ¼ 0.03 Real milk samples El-Abassy et al. (2012)
Fats — 1443, 1658, 860, 2850 Just qualita- Real milk samples (fat extraction) Yao et al. (2016)
tive analysis
Total proteins Ag NPs 871 LOD ¼ 1.5 ug/mL Real milk samples (diluted 30-fold Huang et al. (2016b)
in water)
Lactose Crystal violet as an 1085 1173 LOD ¼ 0.019 mol/L Real milk samples Li et al. (2015)
internal standard
2
Lactose Crystal violet as 1087, 1003, 1173 I1087/I1003: R ¼ 0.9992, Spiked milk samples Vaskova and
internal standard I1173/ Buckova (2016)
I1003: R2 ¼ 0.9847
Proteins, fats PCA 1005, 1745 23.34–25.02% w/w, Real milk powder samples Mcgoverin
0.26–29.68% w/w et al. (2010)
Proteins, total Least 600–1800 RMSE ¼ 0.49, 0.38, Real milk samples (homogenized) Silveira et al. (2016)
fat, lactose squares modeling 0.61, 0.27 mg/ml
Fat, proteins, carbohy- PLS Single reflection 100–3700 RMSE¼ 5.3–5.8%, Real milk samples (evaporated Mazurek et al. (2015)
drates, dry matter ATR dia- 5.6–6.1%, or diluted)
mond accessory 3.5–4.8%, 3.4–4.8%.
Microorganisms S. aureus PLS, KPLS 1110, 610–630 Just qualita- Spiked milk samples (bacteria inoculated Nicolaou, Xu, and
tive analysis milk, incubated at 37  C for 24 h and Goodacre (2011)
preserved at -80 0C)
Brucella spp Support 750–3000 Accuracies¼ 94% Spiked milk samples (bacteria inoculated Meisel et al. (2012)
Vector Machine milk, incubated at room temperature
for 24 h, inactivated via a formalde-
hyde treatment for 1 h, then shaken,
centrifuged, washed and resuspended
in 0.9% sodium chloride solution and
air dried)
L. monocytogenes PCA, HCA, DA, SIMCA 724, 280, 1003, 1453, 1667 CA ¼ 96.67 Spiked milk samples (bacteria inoculated Wang et al. (2015)
milk, incubated at 22  C for 24h, then
centrifuged, top layer removed, PBS
added and resuspended in deion-
ized water)
SE1045 4-MPBA functionalized 740 LOD ¼ 102 cfu/ml Spiked milk samples (bacteria inoculated Wang et al. (2017)
Ag dendrites milk, incubated at room temperature
for 1 h, then centrifuged, washed and
resuspended in NH4HCO3 solution)
E. coli S. aureus Fe3O4@Au@PEI 729, 733 <103 cfu/ml Spiked milk samples (bacteria inoculated Wang et al. (2016)
microspheres milk and substrates added, incubated
at room temperature for 10 min, then
washed and resuspended in water)
E. coli S. aurues Electrospun polymer 720–730 EF ¼ 106 Bacteria spiked in milk samples Szymborski
mat covered with a et al. (2014)
gold layer
Antibiotics Tetracycline Ni/Au core-shell MPs 1320 1595 LOD ¼ 0.1 ug/L Spiked milk samples Li et al. (2011)
Tetracycline Au/PATP/SiO2 1043 LOD ¼ 0.001 ng/mL Spiked milk samples Li et al. (2017)
 Tetracycline Cyclodextrin capped 1317, 1322 LOD ¼ 0.1 ppm Spiked milk samples Dhakal et al. (2018)
Ag NPs
Neomycin Lateral flow immuno- 1078 LOD ¼ 0.216 pg/mL Spiked milk samples (neomycin added, Shi et al. (2018)
assay strip centrifuged for 10 min to discard
some fat or precipitates, then diluted)
Dicyandiamide Ag NPs 866 LOD ¼ 0.1 mg/mL Spiked milk samples Lin et al. (2015)
Penicillin G Ag NPs 1000 LOD ¼ 2.54  10
9 Spiked milk samples (stored in dark glass Chen et al. (2017)
mol/L bottles at 4  C for 7 days)
Ampicillin Microfluidic system, 1115 LOD >10 ppb Spiked milk samples (lipids removed) Andreou et al. (2015)
Ag NPs
Ampicillin PCA 1064 LOD <0.5 ug/ml Spiked milk samples (raw milk samples: Acar-Soykut,
centrifuged at 4  C for 5 min to Tayyarcan, and
remove milk fat, lactic acid added to Boyaci (2017)
collapse casein micelles, filtered and
ampicillin added)
Adulterants Melamine Ag colloid on Al tape 701 LOD ¼ 10
2 ppb Spiked milk samples Lin et al. (2014)
Melamine CD-Ag NPs 704 LOD ¼ 3.0 ug/ L Spiked milk samples (added with aceto- Ma et al. (2013)
nitrile, ultrasonically shaken, centri-
fuged, supernatant collected
and diluted)
Melamine Paper-based Ag 681 LOD ¼ 0.27 mg/L Spiked milk samples Han et al. (2017)
NPs arrays
Melamine Ag SHIN monolayer 686 LOD ¼ 0.03 ppm Spiked milk samples (HCl added, centri- Yang et al. (2017)
fuged and filtered)
Melamine Au@SiO2 NPs 676 LOD ¼ 1 mg/ L Spiked milk samples (shaken and centri- Li et al. (2016)
fuged for removing big molecule)
Melamine PMMA rod deco- 700 LOD ¼ 2.5 ppb Spiked milk samples (placed in 4  C for Rajapandiyan, Tang,
rated AgNPs at least 24 h and diluted 75 times) and Yang (2015)
Urea PLS 1003 LOD ¼ 100 mg/dL Spiked milk samples Khan et al. (2015)
Whey ANN 1430 1540 — Spiked milk samples (homogenized) Alves et al. (2015)
Starch PLS-DA 477 LOD ¼ 0.34% (w/w) Spiked milk samples Oliveira et al. (2016)
Ammonium sulfate, Image classifica- 1009 R2¼0.994, 0995, 0.994, Spiked milk power samples (used a poly- Qin et al. (2014)
urea, dicyandia- tion algorithm 0.996. 0.1%-5.0% propylene centrifuge tubes and a vor-
mide, melamine tex mixer, respectively)
Melamine, urea Thresholding method 1009 673 R2¼0.997, 0.997 Spiked milk power samples (used a Qin et al. (2017)
0.1%-5.0% bench-top acoustic mixer, a mechan-
ical resonator and a polystyrene mix-
ing vessel, respectively)
Thiocyanate Ag NPs 445 704 LOD ¼ 0.49 mg/L, Spiked milk samples (acetic acid added Yang et al. (2014)
ion, melamine 0.0030 mg/kg and centrifuged for removing protein)
Melamine, dicyandia- PLS-DA 683, 926, 1008, 976 LOD ¼ 162 ppm, Spiked milk samples Nieuwoudt
mide, urea, ammo- 302 ppm, 230 ppm, et al. (2016c)
nium sul- 199 ppm, 1.36%
fate, sucrose (w/w)
Note: ANN ¼ artificial neural network, ATR ¼ attenuated total reflection, Ag NPs ¼ Ag nanoparticles, CA ¼ classification accuracy, DA ¼ discriminant analysis, EF ¼ enhancement factor, HCA ¼ hierarchical cluster analysis,
KPLS ¼ kernel partial least squares, LOD ¼ limit of detection, MLR ¼ multiple linear regression, PCA ¼ principal component analysis, PLS ¼ partial least squares, PLS-DA ¼ partial least discriminant analysis,
PMMA ¼ polymethyl methacrylate, R2 ¼ correlation coefficient, RMSE ¼ root mean square error, SIMCA ¼ soft independent modeling of class analogies, 4-MPBA ¼ 4-mercaptophenylbornic acid.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION
775
Table 2. Applications of different Raman spectroscopic techniques in milk detection. 776

Raman Laser Laser Exposure Spectral Spectra

techniques Raman instruments Detection targets wavelength power (mW) time (s) range (cm
1) resolution Spectral preprocessing References
Surface Portable Raman Total proteins 785 nm 100 150 600–1700 4 cm
1 — Huang
enhanced spectrometer et al.
Raman (2016b)
DXR Raman spec- Salmonella 780 nm 5 1 500–2000 — — Wang
H. HE ET AL.

tro-microscope et al. (2017)

Portable Raman E.coli, S. aurues 785 nm 25 20 600–1800 — — Wang
spectrometer et al. (2016)
Confocal Raman Ampicillin 633 nm 3.8 1 400–2000 — Peak normalization Andreou
spectrometer et al. (2015)

1
Raman spectrometer Penicillin G 633 nm 17 10 300–1050 4 cm — Chen
et al. (2017)
Portable compact laser Dicyandiamide 785 nm 150 5 68–3200 3 cm
1 Boxcar averaging Lin et al. (2015)
Raman spectrometer

1
Confocal microprobe Neomycin 632.8 nm 4 5 1000–1800 1 cm — Shi et al. (2018)
Raman system
Confocal laser micro- Tetracycline 785 nm — 20 1100–1700 — Polynomial fitting Li et al. (2011)
Raman spectrometer and smoothing

1
Raman spectrometer Tetracycline 785 nm — — 111–2563 14 cm Upper-bound spectrum Dhakal
et al. (2018)
Micro-Raman Tetracycline 785 nm — — 150–2000 — — Li et al. (2017)
spectroscopy
Portable miniature Raman Thiocyanate 785 nm 150 10 250–1000 — — Yang
spectrometer ion, melamine et al. (2014)
Portable compact laser Melamine 785 nm 150 5 68–3200 3 cm
1 Boxcar averaging Lin et al. (2014)
Raman spectrometer

1
Portable miniature Raman Melamine 785 nm 150 10 250–800 3 cm — Ma et al. (2013)
spectrometer
iHR 320 Raman system Melamine 632.8 nm 35 5 450–1200 0.06 nm — Rajapandiyan,
Tang, and
Yang (2015)
Confocal Raman Melamine 632.8 nm 20 10 350–1800 — — Li et al. (2016)
spectrometer
Confocal Raman Melamine 638 nm 20 1 400–1000 — — Yang
spectrometer et al. (2017)
Portable Raman Melamine 785 nm 20 5 500–1600 — — Han
spectrometer et al. (2017)
NIR-Raman ID dispersive Raman Trans FAs 785 nm — 5 200–2000 — Wavelet transformation, Zhao
spectrometer spine interpolation et al. (2015)
Raman setup Phospholipids 785 nm 200 15 600–1800 10 cm
1 Savitzky-Golay smoothing, Ullah
third-order polyno- et al. (2017)
mial fitting

1
NIR Raman spectrometer Multiple 830 nm 300 2 200–1800 2 cm Fifth-order polynomial Silveira et
contaminants curve fitting al. (2016)
Raman spectrometer Urea 785 nm 80 1 750–1800 — Second-order Savitzky- Khan
Golay smoothing, et al. (2015)
RIA, SNV
Raman spectrometer S. aurues 785 nm 2 30 400–2000 — SNV Nicolaou, Xu,
and
Goodacre
(2011)
FT-Raman FT Raman spectrometer Lactose 1064 nm 100 — 400–3500 4 cm
1 Savitzky-Golay smoothing, Stephani
polynomial fitting et al. (2017)
and MSC
 FT Raman spectrometer Lactose 1064 nm 100 — 50–3500 4 cm
1 Weighted least- Torres
squares baseline et al. (2017)
FT Raman spectrometer Maltodextrin 1064 nm 100 — 400–3500 4 cm
1 Savitzky-Golay smoothing, Rodrigues
second-order polyno- J
unior
mial fitting et al. (2016)
FT Raman spectrometer Multiple 1064 nm 400 — 100–3700 8, 16, 32 cm
1 Fast fourier transform, Mazurek
contaminants mean and variance et al. (2015)
normalization (MNV)
FT Raman spectrometer proteins, fat 1064 nm 220 — 250–3500 4 cm
1 SNV Mcgoverin
et al. (2010)
FT Raman spectrometer Starch 1064 nm 150 — 50–3500 4 cm
1 Savitzky-Golay smoothing, Oliveira
second-order polyno- et al. (2016)
mial fitting
Confocal Fats and FAs 532 nm 5 10 400–3200 — Polynomial fitting Yao
Raman microscopy et al. (2016)
Confocal Whey 514.5 nm — 1200–1600 0.5675 cm
1 ANN Alves
Raman microscopy et al. (2015)
Confocal inViaTM basis L. monocytogenes 785 nm 300 10 400–1800 — Savitzky-Golay smoothing, Wang
Raman Raman microscope polynomial fitting, first et al. (2015)
derivative
transformation
Raman microscopy system Fats and FAs 514.5 nm 20 — 800–1800, — Eighth-order polyno- El-Abassy
2500–3100 mial fitting et al. (2011)
Raman microscopy system Phage 785 nm 100 20 200–2000 2 cm
1 — Acar-Soykut,
and antibiotic Tayyarcan,
and
Boyaci
(2017)
inViaTM basis Lactose 785 nm 20/300 5 100–3200 2 cm
1 — Vaskova and
Raman microscope Buckova
(2016)
Raman chem- Raman imaging Multiple 785 nm 170 — 102–2538 3.7 cm
1 Savitzky-Golay smoothing, Qin, Chao, and
ical imaging spectrometer contaminants polynomial curve fit- Kim (2013);
ting, airPLS Qin et al.
(2014); Qin
et al. (2017)
High-performance port- Lactose 785 nm 100 20 800–1300 — — Li et al. (2015)
able Raman
spectrometer
Others Portable miniature Raman Multiple 785 nm 200 2 500–3000 — Second-order polyno- Nieuwoudt
spectrometer contaminants mial fitting et al. (2016c)
Note: ANN ¼ artificial neural network, airPLS ¼ adaptive iteratively reweighted penalized least squares, DXR ¼ deep X-ray, FT ¼ fourier transform, MSC ¼ multiplicative scatter correction, NIR ¼ near infrared,
RIA ¼ range-independent background subtraction algorithm, SNV ¼ standard normal variate.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION
777
778 H. HE ET AL.
was obtained for liquid milk in quartz cuvettes due to a tur-
bid and inhomogeneous milk layer formed on the inner
wall, thereby poor Raman signals were obtained. Based on
the optimal sample pretreatment methods, the strong
Raman signal of fat content in real milk sample could be
obtained by selecting this short excitation wavelength. El-
Abassy et al. (2012) also proved the feasibility of visible
Raman spectroscopy with a 514.5 nm laser for analyzing the
unsaturation degree of milk fat in liquid milk. The reso-
lution of Raman spectra of the extracted milk fat was higher
than no extracted milk fat, and an obvious difference of
four distinct Raman peaks at 1650, 1265, 3005 and
2850 cm
1 was applied for establishing PLSR models. In the
case of excluding outliers, the R2 and RMSE of this model-
ing method were 0.93 and 0.64, 0.95 and 0.20, respectively
for liquid milk and extracted fat. In a separate study, Yao
et al. (2016) used the excitation wavelength of 532 nm to
rapidly determine the fat globules (MFGs) components from
different types of liquid milk, e.g., human, bovine and cap-
rine milk. In their study, the extraction of total lipids from
milk was needed before milk fat analysis. The differences in
fat components of human, caprine and bovine MFGs were
shown according to the sizes of these MFGs. Through the
intensity of bands in Raman spectra, results showed that the
fat unsaturation level (representative band at 1658 and
1643 cm
1) and carotenoids (1010, 1160 and 1530 cm
1) in
human milk lipids were more than that in bovine and cap-
rine milk, while the phospholipids (860 cm
1) and choles-
terol (2850 cm
1) were lower. The results indicated that
human milk was more appropriate for infant according to
the digestibility and absorbing ability of different lipid com-
positions. Subsequently, Zhao et al. (2015) compared near
infrared (NIR), Fourier-transform mid-infrared (FT-MIR),
dispersive Raman spectrometer equipped with a 785 laser,
coupled with multivariate analysis techniques for predicting
total trans FAs (TT) and classifying naturally-existing (NT)
and industrially-producing trans FAs (IT ¼ TT-NT) in butter
(n ¼ 60), dairy spreads (n ¼ 54) and Cheddar cheese
(n ¼ 44). The PLSR models established using NIR and FT-
MIR spectra showed relatively good results than those using
Raman spectra for predicting TT and NT in butter due to
their low signal-to-noise ratio. However, the models estab-
lished by all the spectroscopy methods could not predict
total fatty acids in dairy spreads and cheese due to strong
signals of protein in them. On the other hand, Raman spec-
troscopy with a diode laser at 830 nm was employed for the
successful quantitative prediction of fat in liquid milk with-
out pretreatment (Silveira et al. 2016). A good model was
developed to measure total milk fat, and their results
showed a strong correlation for total fat (R2 ¼ 0.93) with the
standard errors of prediction of 0.61. More recently, Ullah
et al. (2017) used Raman spectroscopy with a 785 nm diode
emitting laser light to discriminate infant gender on the
basis of milk fat content with PCA models. PCA models
were developed by 50 buffalo whole milk samples (20 sam-
ples with male infant and 30 with female infant) and showed
that the signal intensity of fat from female buffalo was rela-
tively higher than that from male buffalo. As mentioned
above, specific sample preprocessing was required for reduc-
ing fluorescence interference when a shorter wavelength
laser (514.5 and 532 nm) was used. However, with 785 or
830 nm laser, a better Raman signal without complicated
sample preprocessing could be obtained as it could help
eliminating the fluorescence interference. However, no lit-
erature about dispersive Raman spectroscopy with 1064 nm
laser was found in milk analysis, although 1064 nm has low
fluorescence and also low signal strength compared to
785 nm or shorter lasers.
FT-Raman spectroscopy normally uses an Nd:YAG laser
(1064 nm) with NIR interferometer, which can greatly
reduce laser-reduced fluorescence and possesses high spec-
tral resolution (Yang & Ying 2011). However, FT-Raman
spectroscopy is vulnerable to water due to the use of infra-
red wavelength. As reported by Mazurek et al. (2015), the
spectra from liquid milk using FT-Raman spectroscopy was
dominated by water scattering. Therefore, FT-Raman spec-
troscopy is commonly applied for powdered milk or anhyd-
rous samples. Meurens et al. (2005) used FT-Raman
spectroscopy for measuring conjugated linoleic acid (CLA)
content in 50 anhydrous milk-fat samples. Their results
showed that three presentative Raman peaks in spectra at
1652 cm
1, 1438 cm
1 and 3006 cm
1 were associated with
the cis, trans conjugated C¼C in rumenic and trans-10, cis-
12-obtadecadienoic acids, which could accurately determine
the CLA concentration. Bernuy et al. (2008) also proved the
potential capacity of FT-Raman spectroscopy in the detec-
tion of milk total CLA in 57 anhydrous milk fat samples
(two-thirds for calibration and one third for validation set).
Moros, Garrigues, and Guardia (2007) chose FT-Raman
spectroscopy to quantify fat content in milk powder. In their
work, Raman spectra showed no fluorescence interference,
and no preprocessing step was needed when collecting the
spectra. A partial least squares regression (PLSR) modeling
with an 8% mean relative prediction error (MRPE) was
achieved, which met the statutory requirement established
by the US Food and Drug Administration. In order to
obtain a better signal, effects of spectra acquisition tempera-
ture conditions were investigated by Stefanov et al. (2010)
for predicting odd-chain and branched-chain FAs in milk
using a FT-Raman spectroscopy. In their study, a stronger
Raman band was acquired when the sample was pretreated
with
80  C than pretreated at room temperature, and pre-
diction models formed by these spectra could effectively
(R2 > 0.65) determine individual FAs and grouped FAs.
Another work was carried out by Stefanov et al. (2011) to
prove the potential ability of FT-Raman spectroscopy com-
bined with PLSR for the determination of individual or
grouped trans-monounsaturated FAs (trans-MUFA) and
CLA in milk at two temperature conditions. Their results
showed that Raman spectra at
80  C revealed well-defined
Raman scattering bands than Raman spectra at room tem-
perature. This method could be used to accurately quantify
individual CLAs and trans-MUFAs even though the concen-
trations of these components were less than 1.0 g/100 g.
Furthermore, Mcgoverin et al. (2010) also verified the feasi-
bility of FT-Raman spectroscopy for quantifying the fat
 CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 779

Figure 3. Representative results of Raman spectroscopy combined with (A) PLS (Nicolaou, Xu, and Goodacre 2011), (B) PCA (Wang et al. 2015), (C) PCA (Wang et al.
2016), and (D) PCA (Wang et al. 2017) for bacterial analysis.
780 H. HE ET AL.

Figure 4. Representative SERS measurements based on (A) droplet-based method (Rajapandiyan, Tang, and Yang 2015), (B) pen-written SERS arrays (Han et al.
2017), (C) 4-MPBA functionalized Ag dendrites (Wang et al. 2017), (D) CD-AgNPs (Ma et al. 2013), (E) two configurations and sample droplets on different substrates
(Lin et al. 2014), (F) SERS- Lateral flow immunoassay strip (Shi et al. 2018), (G) two-step pretreatment process (Chen et al. 2017), (H) the SERS platform directly from
the solution (Szymborski et al. 2014), (I) the microfluidic junction (Andreou et al. 2015), (J) Au HSs and two Au@SiO2 NPs in the atmosphere or Cu substrate
(Li et al. 2011, 2016).

content in skim and whole milk powders. In their work,
quantification models were established to quantify a fat
concentration range of 26.26–29.68% w/w for the whole
milk powder, and low root mean square error (RMSE) of
0.20%-0.29% were obtained after four different sample
presentations. The results also indicated the orientation
of sample did not influence the calibrations by using
FT-Raman technique.
3.1.2. Milk proteins
Protein is one of the three major nutrients in milk, which
has extensive functional and biological activities (Pihlanto
2006). In recent year, some spectroscopy techniques have
been attracting increasing attention in the detection of milk
proteins. However, there are very few applications of Raman
spectroscopy in milk protein detection as the Raman signals
of most proteins in milk are extremely similar and relatively
weak, and the fluorescence interference from other milk
compositions such as fats and carbohydrates, can affect the
Raman signal of protein in milk (Han, Zhao, & Ozaki 2009;
He et al. 2011). Therefore, it is difficult to detect milk pro-
tein directly without specific extraction of protein before
detection. A number of studies published show that SERS
could quantify milk proteins associated with special groups
because these special groups can produce a stronger
enhanced Raman signal (Becker et al. 2009; Kang et al.
2013; Wood et al. 2012).
Casein is the main protein in milk, which is often used
as a reference for evaluating milk protein (He et al. 2013).
Therefore, Huang et al. (2016b) employed a phosphomolyb-
dic acid (PMA)-mediated SERS method for quantitative ana-
lysis of milk protein with a limit of detection (LOD) of
1.5 lg/mL. In their study, a significant SERS peak at
871 cm
1 for PMA was found, and PMA could be used as a
SERS reporter for determining the total protein in liquid
milk due to its rapid and irreversible redox reaction with
proteins when pre-mixture of PMA on proteins. In addition,
a good linear relationship (R2 ¼ 0.987) between various
casein concentrations and the intensity of PMA was
obtained. The result indicated this PMA-mediated SERS
method had the capability for simple, rapid and quantitative
analysis milk total protein.
3.1.3. Lactose
Lactose is a disaccharide molecule composed of glucose and
galactose, which is the main carbohydrate in milk, account-
ing for 4–5% in bovine milk (Hadizadeh, Hassanpour, and
Mohajeri, 2013). However, there are about 75% of adults in
the world have lactose intolerance, and excess intestinal gas,
nausea, cramps and diarrhea are common symptoms of lac-
tose intolerance (Wilt et al. 2010). Therefore, milk and dairy
products with low lactose content, such as cheese, yoghurt
and lactose-free milk powder, are thus vastly required for
these specific populations. Consequently, sensitive and rapid
methods for real-time analysis of the lactose content in milk
or dairy products have been investigated (Safina, Ludwig, &
Gorton 2010), and Raman spectroscopy has unique
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 781
advantages over traditional methods in milk lactose
determination.
As Raman spectroscopy is applied for milk lactose ana-
lysis, both apparatus and tested samples can reduce the
intensity of Raman signals, which in turn negatively affect
lactose analysis results. Bell and Sirimuthu (2008) demon-
strated the internal standard was the most common method
to eliminate these negative effects. To date, carbon tetra-
chloride (459 cm
1) is often regarded as an internal standard
of non-aqueous solutions, while in aqueous solutions, nitrate
ion (1050 cm
1) and perchlorate ion (930 cm
1) are usually
used. Other innovative internal standards have also been
proposed in recent years. Li et al. (2015) used crystal violet
as an internal standard and employed Raman spectroscopy
with a 785 nm laser light to quantitative detect lactose in
milk. In his study, the problem of small normal Raman
cross section and weak absorption were settled when sample
and standard solution were placed on the silicon wafer,
making Raman analysis of the aqueous lactose easier. Two
characteristic peaks at 1085 cm
1and 1173 cm
1 were identi-
fied to represent lactose and crystal violet, respectively, and
no interference and fluorescence background were observed.
The result presented a good linear relationship between lac-
tose and peak intensity with R2 of 0.9992 and the LOD of
milk lactose was 0.019 mol/L. More recently, Vaskova and
Buckova (2016) compared phenylalanine and crystal violet
for normalizing Raman determination of milk lactose. In
their study, significant differences of three kinds of saccha-
rides (lactose, glucose and galactose) in Raman spectra were
observed for distinguishing the milk lactose. C-O-H bending
mode at 1087 cm
1 was used for lactose measurement, while
the crystal violet peak at 1173 cm
1 and phenylalanine
(which is phenylalanine ring breathing band) peak at
1003 cm
1 (Mcgoverin et al. 2010) were selected for internal
standard measurements. The results showed that normaliza-
tion using phenylalanine achieved better accuracy than using
crystal violet and its procedure was easier without adding
other substances.
Meanwhile, a few studies have been reported on combin-
ing Raman spectroscopy with chemometrics for identifying
dairy products by observing lactose states, therefore optimiz-
ing dairy production. FT-Raman spectroscopy in combin-
ation with partial least discriminant analysis (PLS-DA) was
used by Rodrigues J
unior et al. (2016) for evaluating and
classifying different milk powder samples according to the
lactose state and the addition of maltodextrin (2.5  50% w/w)
in a total of 45 milk powder samples. In their study, the
band at 1086 cm
1, which was attributed to C-O-C stretch-
ing vibration of lactose, only appeared in whole milk
powder. Two new bands at 525 and 424 cm
1 appeared in
low-lactose milk. Besides, the bands at 940, 856 and
481 cm
1 only appeared in maltodextrin-spiked milk, and
the intensity of peaks increased as maltodextrin concentra-
tion was increased. PLS-DA models were established for
discriminating different milk samples by significant differen-
ces in FT-Raman spectra. In addition, Stephani et al. (2017)
demonstrated the capacity of Raman spectroscopy combined
with chemometrics to identify the quality of dairy powders
782 H. HE ET AL.
by observing the changes in lactose structure of whey pro-
teins concentrate (WPC) during production and storage. In
their study, significant variations near 2900 cm
1 and
800  1200 cm
1 in Raman spectra were observed after the
samples were stored at room temperature and humidity for
three weeks, however, there existed no changes when the
samples were stored in a desiccator for one year, which was
owing to the phase change of amorphous lactose into crys-
talline lactose, thereby increasing the level of modification of
WPC. In addition, process parameters of WPC production
could be optimized by monitoring the phase change of lac-
tose. More recently, Torres et al. (2017) successfully eval-
uated the effects of five different levels of enzymatic lactose
hydrolysis during the production and storage of milk pow-
der by employing Raman spectroscopy combined with che-
mometrics. Their results showed that the adhesion to the
inner wall of the drying chamber was increased by increas-
ing the rate of lactose hydrolysis, which was attributed to
higher levels of particle agglomeration. Besides, more brown
powder was observed with the increase in the degree of lac-
tose hydrolysis, thereby the rehydration ability was reduced
and modification of microstructure was generated. From the
above studies, it has been demonstrated that Raman technol-
ogy could realize rapid analysis of lactose in milk.
3.1.4. Multiple compositions
In addition, Raman spectroscopy allows simultaneous and
automatic determination of fats, proteins, and lactose in
milk without pretreatment (Bernuy et al. 2008; Silveira et al.
2016; Stefanov et al. 2011). However, there are some defi-
ciencies in the simultaneous detection of multiple composi-
tions in milk.
Mcgoverin et al. (2010) using FT-Raman spectroscopy to
simultaneous evaluate both proteins and fat in 80 whole or
80 skim milk powder samples. The residual predictive devi-
ation values of fat and proteins in whole milk powder and
skim milk powder were 4.41, 3.06, 4.76, respectively, these
fat prediction results were corresponded to those reported
by Moros, Garrigues, and Guardia (2007), while protein was
poorly predicted. Additionally, the capacity of Raman spec-
troscopy for the identification crystalline constituents of
whole and skim powder samples was proved, however, it
was not effective in the quantification of moisture in the
presence of other constituents. Later, Mazurek et al. (2015)
compared the feasibility of IR single reflection attenuated
total reflection (ATR) spectroscopy and FT-Raman spectros-
copy in combination with PLS models for simultaneous
quantitative measurements of various macronutrients in 75
commercial bovine milk samples. In their study, the RSEP
errors for the determination of fat, protein, carbohydrates
and dry matter by Raman spectroscopy were in the range of
3.4  5.6%. A better prediction was obtained from the PLS
model established by IR spectra registered using single
reflection ATR diamond accessory, while no evident differ-
ence was observed in the accuracy of the models based on
Raman spectra registered with different resolution. Besides,
low signal to noise ratio (S/N) of Raman spectra had a nega-
tive effect on the quantification of milk fat especially for
strongly defatted milk samples. Most recently, Czaja et al.
(2018) used FT-Raman spectroscopy coupled with PLS
analysis to provide quantitative analysis of fat, protein and
lactose in commercial yoghurt samples.
Although simultaneous detection of multiple composi-
tions in milk could be realized as FT-Raman can improve
signal-to-noise ratio through multiple acquisition of the sig-
nals and greatly reduce fluorescence interference by the
usage of near-infrared excitation light source. However,
studies reported that results were not satisfactory, largely
due to relatively weak Raman signals of these milk composi-
tions when using 1064 nm excitation.
3.2. Detection of microorganisms
Foodborne diseases are the most prominent health problems
in the world, which seriously threaten human health. The
presence of harmful microorganisms in milk can affect milk
quality and safety. Mastitis is the most common disease of
dairy cows (Halasa et al. 2007) and milk produced by cows
affected with mastitis contains many harmful microorgan-
isms, such as S. aureus, L. monocytogenes, Salmonella,
E. coli, etc. In addition, bacteria including lactobacillus and
bifidobacterium also play a significant role in the fermenta-
tion process of yogurt and cheese. Therefore, accurate and
rapid detection of microorganisms in milk is required.
Raman spectroscopic techniques including resonance
Raman, surface enhance Raman and confocal micro-Raman
have been widely used to determine the presence of micro-
organisms. Raman signals of microorganisms are attributed
to the composition of bacterial cell membrane or cell wall
(e.g., proteins, phospholipids, polysaccharides). However,
spectral features of different biochemical components in the
cell walls of different bacteria are very similar, therefore,
chemometric methods such as principal component analysis
(PCA), hierarchical cluster analysis (HCA), discriminant
analysis (DA), and soft independent modeling of class analo-
gies (SIMCA) are drastically required. These chemometrics
are used to establish qualitative or quantitative analysis
models for the amplification of minor Raman spectral differ-
ences, thereby interpreting the minor spectral features of
tested molecules and providing great potential for bacterial
enumeration and discrimination (Lu et al. 2011). Hence, an
approach of selecting an appropriate Raman spectroscopic
technique in conjunction with different chemometrics is
effective for the identification and discrimination of micro-
organisms in milk.
3.2.1. Qualitative analysis
Ultraviolet resonance Raman spectroscopy is the earliest
Raman technique for biological analyses, as the fluorescence
interference from biological samples can be avoided effect-
ively. Chromophoric species (e.g., aromatic amino acids,
nucleic acids, lipids) can be sensitively determined with this
technique (Jarvis & Goodacre 2004), allowing for rapid ana-
lysis of bacteria (L
opez-D
ıez & Goodacre 2004). However,
slight differences in biochemical composition of different

bacteria cannot be recognized due to its poor selectivity,
resulting in difficulty in identifying bacteria types.
Therefore, micro-Raman spectroscopy combined with che-
mometrics has been proposed for analysis and distinction of
bacteria in milk. S. aureus is one of the main pathogenic
microorganisms in dairy products (Asao et al. 2003; Balaban
& Rasooly 2000; Buyser et al. 2001), mainly found in milk
originated from cows with mastitis (Gilmour & Harvey
1990). A related study was carried out by Nicolaou, Xu, and
Goodacre (2011), who used micro-Raman spectroscopy in
combination with multivariate analysis technologies for the
rapid detection and separation of S. aureus and Lactococcus
lactis ssp. cremoris in milk, as well as for studying the
growth interaction between S. aureus and LAB in milk. In
their study, traditional plating method was used to obtain
the data of viable bacteria counts. Principal component ana-
lysis (PCA) and discriminant function analysis (DFA) were
used for cluster analysis, and canonical correlation analysis
(CCA), partial least-squares (PLS), and kernel partial least
squares (KPLS) multivariate statistical techniques were
employed for supervised analysis. Their study showed that
Raman spectroscopy coupled with chemometrics could pro-
vide a satisfactory prediction, and S. aureus in milk had an
antagonistic effect on LAB while no antagonism existed on
S. aureus when growth with LAB (Fig. 3A). Subsequently,
Meisel et al. (2012) showed that confocal micro-Raman
spectroscopy coupled with multivariate analysis could be
directly used to identify Brucella spp. in milk on the basis of
single cell level. In another study, six Listeria species includ-
ing L. monocytogenes, in both liquid media and milk were
distinguished by Wang et al. (2015) by confocal micro-
Raman spectroscopy coupled with chemometric analysis.
L. monocytogenes pose a serious threat to human health as
they may cause a disease known as listeriosis in susceptible
populations (Drevets & Bronze 2008). In their study,
unsupervised chemometric models including PCA cluster
models established from Raman spectra of bacteria samples,
which were successfully employed to distinguish gram-posi-
tive and gram-negative bacterial strains by PC 1 and PC 2
scores. Furthermore, six Listeria species were also separated
from other bacteria (Fig. 3B). Supervised chemometrics
including two DA and SIMCA were utilized to validate the
performance and reliability of these unsupervised chemo-
metric models. Satisfied average classification accuracies in
both media and milk were acquired by employing Raman
spectroscopy coupled with two DA and SIMCA models.
3.2.2. Quantitative analysis
The above studies are about dealing with qualitative analysis
of the bacteria in milk by micro-Raman techniques. In
recent years, SERS in combination with chemometrics has
attracted much attention in quality and safety evaluation for
the food industry (Wang et al. 2018; Yaseen et al. 2018;
Jiang et al. 2018; Pan et al. 2018; Yaseen, Pu, and Sun 2018;
Pu, Xiao, and Sun 2017; Pan et al. 2017; Pan, Pu, and Sun
2017; Yaseen, Sun, and Cheng 2017; Li et al. 2017). It can
also be used in quantitative detection of bacteria in milk as
SERS can greatly amplify the intensity of the Raman signals
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 783
when sample molecules are adsorbed onto a roughened
metal surface (Chen et al. 2015; Chisanga et al. 2017; Jarvis
& Goodacre 2008; Jarvis, Brooker, and Goodacre 2006;
Zhou et al. 2014).
The employment of nanostructured metallic substrates
can strengthen the intrinsic weak Raman signals and can
significantly limit the fluorescence interference. In recent
years, a number of scholars have proposed a series of SERS
substrates for quantitative analysis of microorganisms in
milk. Szymborski et al. (2014) (Fig. 4H) proposed a new
SERS substrates for concentrating and fixing E. coli and S.
aureus on the SERS platform, as well as enhancing the weak
Raman bands of bacteria. This new SERS platform consisted
of an electrospun polymer mat covered with a 90 nm gold
layer by physical vapor deposition (PVD) method. The elec-
trospun polymer mat could simultaneously deposit and fix
bacteria directly from the solution onto the surface of SERS
platform via vacuum-pumping method, thereby preventing
the contamination caused by the transfer process of bacteria
as well as enhancing bacteria Raman signals. The result
revealed that the novel SERS substrate had a good stability
and the bacteria signals were significantly enhanced with an
enhancement factors of 106. Additionally, an promising
Raman signal enhancement approach of polyethylenimine
(PEI)-modified Au-coated magnetic microspheres
(Fe3O4@Au@PEI) coupled with Au@Ag nanoparticles (NPs)
was developed by Wang et al. (2016). In their work, the bac-
teria with negative charges could be rapidly and effectively
absorbed on the surface of the positively charged
Fe3O4@Au@PEI microspheres owing to a strong electrostatic
interaction, thus enhancing the bacteria SERS signal.
Besides, the combination of Fe3O4@Au@PEI microspheres
and Au@Ag NPs was proved to dramatically strengthen the
Raman signals of bacteria in milk samples. Their result veri-
fied that the promising approach had the capture-enrich-
ment-enhancement (CEE) three-step effect and its
determination of E. coli and S. aureus in milk with a low
LOD of 103 cfu/mL. In addition, based on PCA models of
two bacterial spectra, E. coli and S. aureus in milk could be
clearly separated (Fig. 3C).
More recently, another novel Raman enhancement
method of functionalized 4-mercaptophenylbornic acid
(4-MPBA) coupled with Ag dendrites SERS mapping
method was invented by Wang et al. (2017) (Fig. 4C) for
detecting and distinguishing different bacteria including
Salmonella presented in milk. By using 4-MPBA functional-
ized Ag dendrites, the capture efficiency for Salmonella
enterica subsp enterica BAA1045 (SE1045) was better than
the undecorated Ag dendrites, besides, four strains were also
successfully differentiated by the PCA models established
from the bacterial cells spectra (Fig. 3D). The LODs for
SE1045 in 50 mM NH4HCO3, 1% casein and skimmed milk
was 103 cfu/mL, 102 cfu/mL and 102 cfu/mL respectively,
and these results confirmed that this enhancement method
was reliable for detecting milk microorganisms. However,
their results also demonstrated that milk compositions
covering nonspecifically on the Ag dendrites would greatly
reduce signals, therefore, further study should focus on
784 H. HE ET AL.
developing effective washing solutions for eliminating the
problem of interfering matrix compositions.
3.3. Detection of antibiotic residues
Antibiotics is an effective veterinary drug common used in
dairy farming to treat diseases such as mastitis. However,
over-dosage uses of antibiotics can lead to excessive anti-
biotic residues in milk, which has potential harmful effects
to human body (Ortelli et al. 2009). In addition, excessive
antibiotic resides in milk can inhibit the normal fermenta-
tion of fermented dairy products such as yogurt, which
greatly reduces the quality and production of fermented
dairy products, thus causing huge economic losses to
milk producers.
Over past few years, the accuracy and sensitivity of anti-
biotic residue detection methods in dairy products have
been greatly improved, and complicated testing processes
have also been avoided. Although the Raman signals of
antibiotics are quiet weak, SERS as a highly sensitive, no-
destructive and rapid analytical technique can effective amp-
lify the Raman signals, thereby determining the antibiotic
residues in milk accurately. In relevant studies, the specific
modification of the rough metal surface of the SERS sub-
strates can significantly improve the adsorption capacity of
the tested molecules, thereby greatly enhancing the Raman
signals of the detection molecules. The metal colloids includ-
ing Ag and Au colloids is the most common SERS substrate
as it only requires an easy and simple sample preparation.
3.3.1. Au colloids as SERS-based substrates
Tetracycline (TC) as a kind of polyketone antibiotic is com-
monly used to prevent or cure bacterial infectious diseases
such as mastitis in cows. However, excessive uses of TC in
animal feeding can induce the production of drug resistant
strains, thus posing an extremely threat to human health,
such as liver and kidney disease as well as gastrointestinal
disease (Kaufmann et al. 2010). For this problem, Li et al.
(2011) fabricated a new Au HSs-based substrate to detect
TC in milk rapidly and accurately. In their study, the syn-
thesis of Au HSs consisted of two procedures (Fig. 4J), the
first step was the preparation of Ni/Au core-shell MPs by a
redox-transmetalation reaction, and the second procedure
was the acidification process by etching the Ni core of Ni/
Au core-shell MPs in HCl solution. Their results presented a
particularly strong Raman signal of R6G (as low as
10
15 mol/L), in addition, a good linear relationship between
the concentration of TC aqueous solution and the intensity
of Raman peaks at 1595 cm
1and 1320 cm
1 was obtained,
and the LOD of TC mixed in aqueous solution was 0.1 mg/L.
Their work indicated that the Au HSs substrate could sig-
nificantly strengthen Raman signals of TC as the specific
structure of Au HSs could promote the absorption ability of
TC on Au HSs. Recently, a novel magnetic nanospheres
(MNs)-targeting aptasensor (cDNA- Au/PATP/SiO2) was
proposed for sensitive quantifying TC in milk by Li et al.
(2017). In their study, the aptasensor was synthesized by
binding the positively charged cDNA- Au/PATP/SiO2 to the
surface of the negatively charged MNs and the signal mole-
cules PATP in cDNA- Au/PATP/SiO2 offered specific and
efficient detection of TC in spiked milk.
Neomycin (NEO) is an aminoglycoside antibiotics, which
is known as broad-spectrum antibiotic as it can simultan-
eously prevent the invasion of multiple bacteria, such as
S. aureus and E. coli (Lechevalier 1975). While excessive
aminoglycoside antibiotic residues can negatively affect the
final quality of fermented milk products (Suhren & Walte
2000), real-time, effective and rapid methods for supervising
NEO residue in milk are thus urgently needed. An novel
SERS-lateral flow immunoassay (LFI) strip was invented for
the measurement of NEO residue in milk (Shi et al. 2018)
(Fig. 4F). In this study, NEO residue determination was
based on a competitive interaction with immunoprobe
between frees NEO and artificial antibody (NEO-OVA). The
immunoprobe was established by Au NPs combined with
anti-neomycin monoclonal antibody (NEO-mAb) and 4-
aminothiophenol (PATP, a Raman probe molecule), and the
SERS-LFI strip was assembled with a conjugate pad, sample
pad and absorbent pad. A low LOD of NEO (0.216 pg/mL)
was acquired by the SERS-LFI strip prepared under optimal
conditions, in addition, the recovery of the NEO ranged
from 89.7%105.6% with relative standard deviations (RSD)
in range of 2.4%5.3% and the cross-reactivity (CR) was as
low as 0.01%. These results indicated the high sensitivity,
specificity and reproducibility of the SERS-LFI strip for
NEO residue detection in milk.
3.3.2. Ag colloid as SERS-based substrates
Dicyandiamide (DCD) is a recognized nitrogen-rich com-
pound commonly applied to improve the quality and yield
of herbage, which can enter milk through the food chain.
Effective monitoring and strictly controlling DCD in milk
products are hence necessary as excessive DCD residues in
milk poses serious toxic-threat to humans. Recently, Lin
et al. (2015) used Ag NPs as SERS substrate to determine
DCD residue in milk. In their study, discrete Fourier trans-
form (DFT) calculation was employed to analyze Raman
spectra collected from samples, in addition, the effect of
three aggregating agents (Na2SO4, NaNO3 and NaCl) and
two conditions (acid or alkaline condition) for Ag NPs sub-
strate preparation was studied. Results showed that Na2SO4
coupled with NaOH could significantly enhance the Raman
signals with an enhancement factor of 2.88  105, and a
good linear relationship (R2 ¼ 0.99869) and a lower LOD
were acquired when detecting DCD in an aqueous solution.
For DCD detection in milk, a good linear relationship
(R2 ¼ 0.99747) and a lower LOD (1  10
4 g/mL) were also
acquired. The results revealed that Ag colloid substrate
could rapidly and accurately determine DCD in milk.
Penicillin G is a kind of typical b-lactam compound
(Kukusamude et al. 2010), which is widespread applied for
the inhibition of bacterial infectious disease such as cattle
mastitis, thereby leading to antibiotic residues presented in
milk (Anna et al. 2016; Lirio et al. 2016). In 1999, the
European Union (EU) has regulated that residues of penicillin

G in milk must be less than 4 lg/kg (or 1.2  10
8 mol/L)
(Thavarungkul et al. 2007). Recently, Chen et al. (2017)
(Fig. 4G) proved that SERS technique combined with a two-
step sample preprocessing had a capacity to achieve the
trace detection of penicillin G in milk samples. In their
study, the two-step sample preprocessing containing twice
ultrasound, centrifuge and extract supernatant, could suc-
cessfully remove the interference of other components in
milk, which was the key to the determination of penicillin G
residues in milk. Under the optimal conditions, the intensity
of the tested Raman peak at 1000 cm
1 was linearly propor-
tional to the concentration of penicillin G with a correlation
coefficient (R2) of 0.9902 in the concentration range from
1.0  10
8 mol/L to 1.0  10
3 mol/L, and the LOD of peni-
cillin G residue (0.85 lg/kg) was far less than the maximum
residue limits of EU (4 lg/kg). Their results indicated that
this detection method could provide reliable and accurate
information on penicillin G residues in milk, thereby ena-
bling its practical application in milk antibiotic analysis.
Ampicillin is a broad-spectrum b-lactamide antibiotic,
which is widely used in veterinary clinic. In dairy farming,
incorrect use of ampicillin can result in residual drugs in
milk, causing allergic reactions. SERS have been successfully
used in the detection of ampicillin (Clarke et al. 2005). A
microfluidics-based SERS methodology was proposed by
Andreou et al. (2015) (Fig. 4I) to detect ampicillin residue
in milk. Under the microfluidic system, the aggregation of
the Ag NPs was optimized and the analyte was successfully
absorbed to the Ag NPs substrate, thereby the Raman signal
of ampicillin was enhanced. Their study showed that this
microfluidic device could offer rapid and automatic deter-
mination of ampicillin in milk, however, sample pretreat-
ment procedure including the extraction of fat from milk
samples was required. Recently, Dhakal et al. (2018) used
cyclodextrin capped Ag NPs as substrate for on-site screen-
ing of TC in whole milk. Correlation coefficient of 0.92 for
water-tetracycline samples and 0.88 for milk-tetracycline
samples were obtained with the peaks at 1317 cm
1 and
1322 cm
1. There is still much work needed in order to real-
ize practical applications of this technology in milk analysis.
3.3.3. Other raman spectroscopic techniques
As mentioned above, SERS-based technique has been gener-
ally used for qualitative or quantitative determination of
antibiotics in milk as SERS can greatly enhance the Raman
signals of antibiotics and realize detection of antibiotics in
milk at trace levels. However, only a few studies use other
Raman spectroscopy techniques to detect antibiotics in milk.
A novel method of Raman spectroscopy combined with
PCA analysis was applied by Acar-Soykut, Tayyarcan, and
Boyaci (2017) for the identification of antibiotic and phage
in raw milk samples. In their study, the Raman spectra
derived from the samples were applied to establish a PCA
model. Under the PCA model, phage and antibiotic were
clearly distinguished. The results showed that the LOD of
phage in milk was less than 107 pfu/mL and the LOD of
antibiotic in milk was below 0.5 lg/mL, which could ensure
normal fermentation of milk products.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 785
3.4. Detection of milk adulteration
With the rapid development of the global dairy industry,
competition between enterprises increases, leading to pos-
sible adulteration in milk. Therefore, reliable and accurate
determination of milk ingredients is of great significance to
ensure milk quality, prevent artificial adulteration and guar-
antee human health.
3.4.1. Detection of melamine in milk by SERS
In 2008, melamine adulteration incidents had a negative
impact on society, which prompted consumers to pay more
attention to milk safety, thus enabling enterprises to develop
a series of fast and effective melamine determination meth-
ods. However, the LOD of some methods based on Raman
spectroscopy was in the range of hundreds of milligrams per
liter (Nieuwoudt et al. 2016a; b), which was unable to meet
the specified standard. Therefore, the SERS-based substrate
for the signal enhancement has raised much interest in
recent years.
To date, multiple substrates such as Au/Ag colloid
(Mecker et al. 2012), AgNP-modified single Ag nanowire
(Chen & Liu 2012), Au nanofinger (Kim et al. 2012) and
cyclodextrin-decorated Ag NPs (Ma et al. 2013) (Fig. 4D),
have been developed for melamine determination in milk.
However, the poor stability of Ag/Au-based substrates was
observed due to the Ag/Au oxidation, and their SERS
enhancement ability was hence significantly reduced, which
greatly prevented their practical application in SERS analysis.
For this purpose, Li et al. (2016) (Fig. 4J) developed an
Au@SiO2 nanoparticles substrate for the quantitative detec-
tion of melamine in milk. In their study, the purpose of
coating the silica shells onto Ag NPs was to prevent the oxi-
dation. The results showed that aggregated Au@SiO2 NPs
on Cu substrate could significantly strengthen the Raman
signals of melamine in milk, which was owing to a stronger
electric field produced in the gap (“hot spots”) between NPs
and Cu substrate. A good linear relationship (R2 ¼ 0.99) in
the concentration range of 0.5  5 mg/L and the LOD lower
than 1 mg/L were observed. In addition, the sample pretreat-
ment only contained hydrochloric acid treatment and twice
centrifugation, and the stability and SERS activity of this
substrate were higher than that of bare Ag NPs. As previous
results showed that the enhancement effect of Ag-based sub-
strates was more obvious than that of Au-based substrates,
Yang et al. (2017) proposed a SERS substrate of Ag NPs
coated with a 1  2 nm SiO2 shell (Ag SHIN monolayer
film) to quantitatively analyze melamine in milk samples. In
their study, the thin SiO2 shell could effectively protect the
Ag NPs from oxidation, thereby making this Ag-based sub-
strate more stable. In addition, the adsorption ability of the
tested molecule was greatly promoted by the SiO2 shell with
pinhole. The results showed that Raman intensity linearly
increased with the increase of melamine concentrations
(0.001  5 ppm), and the LOD of melamine in milk was
0.03 ppm, illustrating that this SERS technique based on Ag-
based substrates could offer accurate and reliable evaluation
of melamine in milk.
786 H. HE ET AL.
In addition to the effect of metal oxidation, the SERS
enhancement ability of metal colloid is also easily influenced
by other factors including synthetic conditions, the structure
and size of metal colloid, as well as testing conditions. The
common use of metal colloids for SERS detection requires a
lengthy time for the mixture droplets (sample and metal col-
loid) to dry completely. During the drying period, the phe-
nomenon of “coffee ring” may occur, resulting in an uneven
Raman response and an unreliable Raman signal (Xu et al.
2014). On the other hand, a droplet configuration from a
hydrophobic surface was developed by Avci and Culha
(2013), which offered reliable Raman signals, but the drying
process was still unavoidable and thus the time-consuming
problem was still unsolved. Therefore, Lin et al. (2014)
(Fig. 4E) developed a novel SERS analysis device for directly
testing the sample in droplet state without the time-
consuming drying process. Their study showed that the
Al-tape was more suitable to be applied as a testing platform
than quartz and silicon, which was due to the strongest
reflectivity and hydrophobicity of the Al-tape. By using
Al-tape and a particular order of agent addition, a good linear
relationship with R2 of 0.9963 was obtained at concentrations
ranging of 0.05  10 ppm. Their results indicated that this
novel SERS analysis device could be used to detect melamine
of milk rapidly and sensitively without any drying procedure.
Rajapandiyan and Yang (2012) proposed another new
droplet-based method for SERS detection of melamine in
milk. In their method, a cylindrical Ag-based substrate used
for loading droplet sample was prepared by decorating the
AgNPs into a polymethyl methacrylate (PMMA) rod via sil-
ver mirror reaction, which not only protected the Ag NPs
from oxidation but also offered direct detection of sample
droplet in solution. Besides, a certain level of sample dilu-
tion could greatly suppress the matrix interference. The
results showed that the cylindrical substrate combined with
the sample dilution method was the key to provide an
accurate and reliable melamine detection. Furthermore,
Rajapandiyan, Tang, and Yang (2015) optimized the cylin-
drical Ag-based substrate for supporting more Ag NPs
(Fig. 4A). In their study, the polymethyl methacrylate
(PMMA) rod was coated with polyvinylidene difluoride
(PVDF) in order to make the inner wall of the PMMA rod
much rougher, and more Ag NPs could hence be loaded.
The matrix interference was effectively removed by an opti-
mal sample dilution factor of 75. The result showed that the
enhancement factor of this optimum substrate was up to
107. Compared with undecorated PMMA rod, this substrate
for melamine detection could obtain a lower LOD of 2 ppm.
Generally speaking, for the purpose of practical applica-
tions of SERS in milk routine analysis, the SERS substrates
should be low-cost, easy to fabricate and with high stability
and reproducibility. For this purpose, Han et al. (2017)
(Fig. 4B) developed an attractive SERS-based substrate for
the trace detection of melamine in milk. The pen-written
SERS arrays used for sample detection was fabricated by
writing Ag NPs ink on the surface of hydrophobic paper
through pen-written method. In their study, the SERS activ-
ity of Ag NPs ink was still well even after preservation for
12 weeks due to the inhibition of oxidation. In addition, the
Raman signal of melamine in milk was greatly enhanced by
using the arrays. Compared with HPLC, this disposable
SERS arrays could provide a lower LOD of 0.27 mg/L, con-
firming that this low-cost substrate had great application
prospects in milk evaluation.
3.4.2. Detection of other single adulterant in milk
In addition to melamine, other potential adulterants such as
urea, whey, starch and other cheap substances are also artifi-
cially added to milk by unethical milk producers for increas-
ing or replacing the compositions in milk, thereby reducing
production cost. Compared with melamine adulteration in
milk, the application of Raman spectroscopy in the detection
of milk adulterated with urea, whey and starch is relatively
rare. Nevertheless, the combination of Raman spectroscopy
and chemometric methods has been proved to be an effect-
ive analytical tool for quantitative detection of these adulter-
ants presented in milk.
Urea is a natural ingredient in milk, and generally speak-
ing, the concentration of urea in milk should not exceed the
recommended limit of 70 mg/dL, otherwise human health
can be seriously threatened (Trivedi et al. 2009). Therefore,
the quantitative determination of urea in milk is crucial for
controlling milk quality and ensuring consumers health.
Khan et al. (2015) successfully proved the feasibility of NIR-
Raman spectroscopy with a 785-nm diode laser combined
with PLS analysis for accurate determining the urea content
in milk. The results showed that the LOD of urea in milk
was lower than the recommended limit value, and the accuracy
of detecting urea in milk was higher than 90% (>50 mg/dL).
In addition, the Raman signal of urea was affected by
the matrix effects in their study. Hence further study should
focus on the inhibition of matrix effects for the purpose of
promoting Raman spectroscopy for practical applications in
milk analysis.
Whey is a by-product in the dairy industry with a low
commercial value, which is attractive for milk producers to
increase protein content without affecting milk quality.
Although whey adulteration in milk has not been found to
have negative effects on human health, whey adulteration
violates the consumer protection law. Most works reported
in identifying whey in milk products are on the determin-
ation of glycomscropeotide (GMP), and applications of
Raman spectroscopy in the area are limited. de Almeida
et al. (2012) showed that the combination of FT-Raman
spectroscopy with chemometrics had a great potential for
the determination of whey adulteration in milk powder.
Alves et al. (2015) coupled Raman microscopy with artificial
neural network (ANN) and PLS algorithm to predict whey
adulteration in milk, a good prediction model was estab-
lished by ANN technique with R2 of 0. 9999. As no compli-
cated sample pretreatment and spectral processing were
needed in their work, the approach was feasible for milk
adulteration analysis. However, SERS as a promising tech-
nique has not been applied in this area yet, thus attempts
should be made to investigate the possibility of using SERS
technology for detecting whey adulteration in milk.

Starch as a food additive is widely used in dairy products
due to its specific physical properties, low cost and wide
sources available. However, the amount of adding starch
should be clearly labeled in commercial dairy products. In
order to obtain reliable information of the starch addition in
milk products, Oliveira et al. (2016) developed a method of
using FT-Raman spectroscopy together with PLS-DA to
determine starch addition in 11 cheese samples (1 sample
without adulterant, 5 samples with starch 2560, and 5 sam-
ples with starch 4051). In their work, the PLS-DA model
was used to identify whether milk samples contain starch or
not. Besides, a good PLS model was established for quantita-
tive determining the starch content in milk samples, with a
LOD of 0.34% (w/w). The results indicated that the two che-
mometric models were useful for evaluating the quality of
different types of cheese.
From the literature discussed above, it was found that
Raman spectroscopy, especially SERS technology, has not yet
been widely used in the detection of these single adulterants
in milk. Therefore, there is still much work to be done to
make Raman technology for regular detection of these single
adulterants in milk.
3.4.3 Simultaneous detection of multiple adulterants
In recent years, there are a number of studies for simultan-
eous detection of multiple adulterants in milk by Raman
spectroscopy have been published. Yang et al. (2014)
employed SERS technique to examine thiocyanate ion and
melamine presented in milk and milk powder simultan-
eously. In their study, Ag NPs colloid was used as SERS sub-
strate, and two significant Raman peaks at 445 cm
1 and
704 cm
1 were identified for SCN- and melamine determin-
ation, respectively. Under optimal detection conditions, the
intensity of SCN- peak and melamine peak were linearly
proportional to their concentrations, with R2 of 0.998 and
0.999 in the concentration ranges of 2.00  191.00 mg/L and
0.01  4.80 mg/L, respectively. In addition, chemical imaging
technologies have been effectively used for the detection of
multiple adulterants in milk powder. The same depth
(3 mm) of penetration of laser through milk powder for
adulterant detection was determined by using NIR hyper-
spectral imaging (Huang et al. 2016a) and Raman imaging
(Dhakal et al. 2016; Qin et al. 2015). Compared with the
result of NIR hyperspectral imaging (Huang et al. 2016a),
Raman spectral imaging could further realize adulterants
quantification (Dhakal et al. 2016) and detect multiple adul-
terants in milk powder. Qin, Chao, and Kim (2013) used a
point-scan 785 nm Raman image combined with self-model-
ing mixture analysis (SMA) for clearly classifying four adul-
terants (ammonium sulfate, dicyandiamide, melamine, and
urea) in milk powder based on spectral information diver-
gence (SID) values. However, the SMA algorithm was slow
and only suitable for offline data analysis. Therefore, a faster
multispectral detection algorithm was proposed for on-line
discrimination of these four adulterants in milk powder
based on the point-scan Raman imaging coupled with poly-
nomial curve-fitting method (Qin et al. 2014). A thin sample
layer prepared by dividing each sample into more parts is
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 787
ideal for detection, while long sampling time of the point-
scan system prevents fast inspection to milk powder. Line-
scan Raman image system can scan large-area sample more
efficiently and greatly reduce sampling time. Herein, Qin
et al. (2017) developed a line-scan 785 nm Raman image for
succesful and efficient identifying and quantifying both
melamine and urea mixed in milk powder based on airPLS
correction. Moreover, a novel Raman detection system
equipped with an immersion fiber optic probe was devel-
oped for simultaneously measuring melamine, dicyandia-
mide, ammonium sulfate and sucrose in milk (Nieuwoudt
et al. 2016c). In their study, PLS model and PLS-DA model
were established for predicting multiple adulterants in milk.
The results showed good linear responses of these adulter-
ants except urea. In addition, better accuracy of the predic-
tion of these adulterants were acquired. These results
indicated a good prospect of this detection system for simul-
taneous screening of multiple adulterants in milk.
Raman spectroscopy has not yet widely used in detecting
milk adulteration. As indicated in Table 1, most of studies
are about SERS for determination of melamine in milk due
to its ability of providing more satisfactory detection limit.
In addition, research on the application of Raman techni-
ques to detect other adulterants in milk is very limited and
results of these studies are not satisfactory. Raman spectros-
copy as a promising technique, especially SERS, should be
more widely investigated for the determination of adulter-
ants in milk.
4. Limitations and future perspectives
The application prospect of Raman spectroscopy in quality
and safety evaluation of milk is considerable, owing to its
nondestructive ability to offer information about concentra-
tion and structure of the molecules by molecular vibration
modes, thus realizing qualitative and quantitative determin-
ation of samples. Raman spectroscopy is also suitable for
detecting samples from different states including liquid milk
and powdered milk. However, most applications of Raman
spectroscopy are still at experimental stage, and some exist-
ing theoretical problems and technical obstacles need to be
solved and overcome before using the technique to replace
the traditional detection methods for routine evaluation.
The cost of the Raman system should be reduced. As an
emerging detection technology, the cost of Raman spectros-
copy is very expensive. High up-front investment of the
Raman system such as laser light source, data acquisition
system, detection system, signal processing and control sys-
tem, may be unaffordable for small and medium-scale milk
producers, thereby hindering the widespread usage of this
technique in routine detection for the dairy industry. With
the development of laser, detector and computer techniques,
although micro Raman spectrometers with better perform-
ance are now available, novel portable Raman systems with
low-cost and high-performance still should be developed for
on-site determination of milk products.
Raman signals of the analyte should be enhanced. Raman
spectroscopy is a molecular structure characterization
788 H. HE ET AL.
technique based on Raman scattering effect. However, the
intensity of normal Raman scattering is very weak. SERS as
an emerging detection technology has been widely applied
for enhancing the intensity of Raman scattering and hence
improving the sensitivity of Raman spectroscopy. As indi-
cated in Table 2, applications of SERS technology in milk
analysis are more common than other Raman techniques.
The preparation of SERS active substrates is the basis for
the development of SERS technology in milk analysis. To
date, the developed SERS active substrates are mainly
derived from metal nanoparticle colloids due to their simple
and easy preparation. However, Au/Ag based substrates
exposing to air is particularly prone to oxidation, which
greatly reduces the SERS enhancement effect. Therefore, Ag
SHIN monolayer (Yang et al. 2017), cylindrical supporter
(Rajapandiyan, Tang, & Yang 2015) and cyclodextrin-deco-
rated Ag NPs (Ma et al. 2013) have been developed to pre-
vent the oxidation of active metals. In order to obtain a
better SERS enhancement effect, Ni/Au core-shell MPs
(Li et al. 2011), 4-MPBA functionalized Ag dendrites (Wang
et al. 2017), metal colloids on Cu substrate and aluminum
tape (Li et al. 2016; Lin et al. 2014) have also been proposed
for trace detection of molecules in milk, and their results
indicated these modified substrates could present better
enhancement effects than bare metal colloids substrates.
However, preparation of the above SERS substrates is not a
simple job although good enhancement effects can be
achieved. Therefore, the development of a SERS substrate
with the characteristics of easy preparation, high stability,
good uniformity and reproducibility is an important task to
enhance Raman signals and increase detectability. In add-
ition, the intensity of Raman signal is affected by laser wave-
length, laser power and expose time. However, information
on how to select these parameters is limited, therefore, more
studies should be carried out to accumulate analytical data
as shown in Table 2 and build databases for future
milk analysis.
Efficiency and accuracy of detection should be improved.
The efficiency and accuracy of dairy products analysis are
mainly affected by the occurrence of fluorescence and the
matrix effect from natural complex samples. Fluorescence is
another scattering light besides Raman scattering light, and
its intensity is much stronger than Raman scattering light.
Therefore it is necessary to take appropriate measures to
effectively restrict the fluorescence interference. To date, the
common fluorescent elimination methods include physical
or chemical processing methods (fluorescence quenching
method, light bleaching method and SERS method, etc.),
computer algorithm processing methods, and eliminating
fluorescence by structural modification of Raman systems.
Although various fluorescent removal methods have been
developed, their fluorescent removal effects may change with
samples used, therefore further studies are required to find
efficient and reliable fluorescence elimination methods. On
the other hand, the complicated compositions in milk can
produce the matrix effects, which have adverse effects on
the determination of milk sample, leading to inaccurate
detection results. In order to obtain reliable results, some
simple sample preparations (extraction, concentration, etc.)
may be required before Raman measurements. Moreover,
selecting appropriate Raman instruments and analysis condi-
tions is of great benefit to obtain reliable detection results.
In addition, as shown in Table 1, Raman spectroscopies
coupled with multiple novel data processing techniques such
as chemometric methods and artificial neural networks
(Alves et al. 2015) is of great benefits to milk analysis, and
Raman Spectroscopy in conjunction with microfluid system
has also been successfully applied in this field (Andreou
et al. 2015). Therefore, the combination of Raman techni-
ques with other technologies such as NIR, FTIR, or chemo-
metric methods such as PLS and PCA is promising for
improving the accuracy and sensitivity of the analyses.
5. Conclusions
Ensuring the quality and safety of dairy products is critical
for both producers and consumers. Raman spectroscopy as a
promising detection method that can replace traditional
detection methods due to its many advantages such as non-
destructive detection, no sample preparation, and rapid
measurement. In this review, the principles and types of
Raman spectroscopic techniques are introduced, recent
developments on reliable applications of this technique in
milk evaluation including milk compositions, microorgan-
isms and antibiotics in milk, and milk adulteration are pre-
sented, and some limitations and future perspectives for
Raman spectroscopy are also discussed. For making Raman
spectroscopy as a routine tool for daily evaluation of milk
products, future studies should focus on reducing the cost
of the Raman systems, enhancing the signals by developing
novel SERS substrates, improving detection efficiency and
combing Raman spectroscopies with other techniques or
chemometric methods to overcome these limitations.
Acknowledgments
The authors are grateful to the Collaborative Innovation Major Special
Projects of Guangzhou City (201604020007) for its support. This
research was also supported by the Guangdong Provincial Science and
Technology Plan Projects (2015A020209016, 2016A040403040), the
Fundamental Research Funds for the Central Universities (2017MS067,
2017MS075), the International and Hong Kong – Macau - Taiwan
Collaborative Innovation Platform of Guangdong Province on
Intelligent Food Quality Control and Process Technology & Equipment
(2015KGJHZ001), the Guangdong Provincial R & D Centre for the
Modern Agricultural Industry on Non-destructive Detection and
Intensive Processing of Agricultural Products, the Common Technical
Innovation Team of Guangdong Province on Preservation and
Logistics of Agricultural Products (2016LM2154) and the Innovation
Centre of Guangdong Province for Modern Agricultural Science and
Technology on Intelligent Sensing and Precision Control of
Agricultural Product Qualities.
Nomenclature
ANN Artificial neural network
CCA Canonical correlation analysis
DA Discriminant analysis
DFT Discrete Fourier transform
FAs Fatty acids

FT Fourier transform
FTIR Fourier transform infrared
HCA Hierarchical cluster analysis
KPLS Kernel partial least squares
LOD Limit of detection
NIR Near infrared
PCA Principal component analysis
PLS Partial least squares
PLS-DA Partial least discriminant analysis
PLSR Partial least squares regression
PVD Physical vapor deposition
SERS Surface-enhanced Raman spectroscopy
SIMCA Soft independent modeling of class analogies
RMSE Root mean square error
CLA Conjugated linoleic acid
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