Analytical Strategies For Characterizati

Trends in Analytical Chemistry, Vol. xxx, No.
x, 2012 Trends
Analytical strategies for

characterization and validation
of functional dairy foods
Dina Rodrigues, Teresa A.P. Rocha-Santos, Ana C. Freitas,
Ana M.P. Gomes, Armando C. Duarte
Functional foods (FFs) are food products to be consumed as part of a balanced diet. They provide physiological benefits or reduce
the risk of chronic disease beyond basic nutritional functions. Functional foods containing probiotics and/or prebiotics have
gained much interest in recent years due to their health-promoting capacity.
The main objective of this review is to discuss the analytical strategies that have been used to validate FFs associated with dairy
products containing probiotics and/or prebiotics. In these products, the biochemical events, carried out by enzymes of different
sources (milk, bacteria, rennet) leading to the transformation of milk to diverse products (e.g., yoghurt and cheese), are glycolysis,
proteolysis and lipolysis.
We present the analytical methodologies used to study the microbial probiotic flora and to evaluate the biochemical trans-
formations, the associated functionality in terms of intestinal microbiome and the safety of such FFs. We address the analytical
figures of merit. We cover the advantages and the disadvantages of such analytical methodologies and comment on future
applications and potential research interest within this field.
ª 2012 Elsevier Ltd. All rights reserved.
Keywords: Analytical methodology; Dairy; Enzyme; Functional food; Functionality; Glycolysis; Lipolysis; Prebiotic; Probiotic; Proteolysis
Dina Rodrigues*,
1. Introduction beverages account for an important frac-
Teresa A.P. Rocha-Santos, tion of this sector [4].
Ana C. Freitas, Nutrient supply through foods has an Probiotics and prebiotics may be found
Armando C. Duarte important role in the health of each in some FFs, namely dairy products. Pro-
CESAM & Department of
person; consciousness of this factor is biotics are live microorganisms that confer
Chemistry, University of Aveiro,
3810-193 Aveiro, Portugal
increasing and consumersÕ awareness is health benefits to the host when adminis-
noticeable through the increased demand tered in adequate amounts [5,6] (e.g.,
Teresa A.P. Rocha-Santos, for functional foods (FFs) [1]. Although modulation of the immune system,
Ana C. Freitas there is no consensus about the exact reduction of lactose intolerance, reduction
ISEIT/Viseu, Instituto Piaget,
definition of FFs, it is accepted that these of cholesterol levels, antimicrobial activity,
Estrada do Alto do Gaio,
Galifonge, 3515-776 Lordosa,
foods should come from natural sources, and anti-diarrheal, anti-mutagenic and
Viseu, Portugal and their nutritional characteristics anti-carcinogenic properties) [6,7]. Addi-
should also be able to provide physiologi- tionally, from a safety point of view, pro-
Ana M.P. Gomes cal benefits by improving health and well- biotic strains cannot be associated with
CBQF/Escola Superior de
being [2] or to reduce the risks of chronic any type of disease or transmission of
Biotecnologia, Catholic
University, Rua Dr. António
diseases (e.g., cardiovascular diseases, antibiotic-resistant genes [6].
Bernardino de Almeida, overweight or obesity, dyslipidemia, In order to promote health benefit to the
P-4200-072 Porto, Portugal hypertension, osteoporosis and diabetes) host, probiotic viable cells should be
[1,2]. According to Jankovic et al. [3], the technologically resistant to survive the
main role of FFs is to be able to maintain different processing steps [6,8] and storage
host health or to help recovery upon conditions, survive the passage through
temporary illness. the gastrointestinal tract and adhere to
In the worldwide FF market, dairy intestinal cells where they will be poten-
*
Corresponding author. products are key products and, among tially beneficial [9]. Recommended mini-
E-mail: dinarodrigues@ua.pt those dairy-based products, functional mum doses of probiotic viable cells to
0165-9936/$ - see front matter ª 2012 Elsevier Ltd. All rights reserved. doi:http://dx.doi.org/10.1016/j.trac.2012.08.009 1
Please cite this article in press as: D. Rodrigues et al., Trends Anal. Chem. (2012), http://dx.doi.org/10.1016/j.trac.2012.08.009
Trends Trends in Analytical Chemistry, Vol. xxx, No. x, 2012
ensure health benefits to the host must be in the range conjugated linoleic acid)] that are important for digestive,
7–9 log cfu per gram or mL [5] of a product, which must gastrointestinal and cardiovascular functions, gastroin-
be consumed on a regular basis. The majority of probi- testinal microflora, hemodynamics and immunoregula-
otic strains used in functional dairy foods (FDFs) belong tion. There may be a direct probiotic effect (interaction
to the Lactobacillus and Bifidobacterium genera [9], but with ingested beneficial microorganisms) or an indirect
other species (e.g., Saccharomyces cerevisiae (boulardii), biogenic effect (action of generated microbial metabolites,
Bacillus subtilis, Escherichia coli) are also being marketed including vitamins, proteins, peptides, oligosaccharides,
with similar purposes [6]. An overview of probiotic dairy and organic acids) [2]. These products are encouraged
products considered as good vehicles for delivering pro- and this overview concerns an investigation on the cur-
biotic bacteria to the gastrointestinal tract (e.g., yoghurts rent analytical techniques that are being used to char-
and cheese) is given in the review by Granato et al. [10]; acterize and to validate FDFs (e.g., cheeses, yoghurts, and
beverages are also included. Certain factors (e.g., fat fermented milks) where probiotics or lactic-acid bacteria
content, concentration and type of protein, non-protein (LAB) play a vital role.
nitrogen, sugars and pH of food) may affect growth and Assessment of probiotic bacteria or LAB viability and/
viability of probiotic bacteria. Cheese characteristics or survivability must be assured throughout processing
(e.g., higher pH, lower titratable acidity, higher buffering phases and storage until consumption. Therefore, ana-
capacity, greater fat content, higher nutrient availabil- lytical strategies to monitor the viable microflora and
ity, lower oxygen content and a denser matrix) are their metabolic action require discussion. Additionally, in
favorable factors for the survivability of probiotic bacte- most fermented dairy products, the biochemical events,
ria [11,12]. performed by enzymes of different sources (milk, bacteria,
Prebiotics are non-digestible oligosaccharides resistant rennet) leading to the transformation of milk to diverse
to hydrolysis and absorption in the digestive system [5], products (e.g., yoghurt and cheese) are glycolysis, pro-
reaching the colon almost intact where they promote the teolysis, and lipolysis. The analytical methodologies used
proliferation and activity of desirable bacteria in the gut to evaluate these biochemical transformations have
[13] because they are selectively fermented especially by evolved. Nowadays, foodomics, a new discipline that
probiotic bacteria. The prebiotics most commonly used studies food and nutrition via application of advanced
are oligosaccharides whose degree of polymerization ‘‘omics’’ techniques, is changing the analytical
varies between 2 and 20 monomers [e.g., fructo- approaches and strategies in food-science research;
oligosaccharides (FOSs), inulin, galacto-oligosaccharides classical methods are being confronted with advanced
(GOSs) and xylo-oligosaccharides (XOSs)]. Their benefits omics strategies, which include analytical techniques
have been related to increase of calcium absorption, [e.g., mass spectrometry (MS), and nuclear magnetic
improvement of bone-mineral content and bone-mineral resonance (NMR) spectrometry] [15,16]. Foodomics
density, blood-glucose formation, reduction of choles- covers many fields of research including genomics,
terol and serum lipids levels, and obesity control [1]. transcriptomics, proteomics and/or food metabolomics
Prebiotic compounds are important for probiotic micro- studies for analysis of biomarkers, and compound profil-
organisms, since they favor their growth and survival in ing for food quality/authenticity purposes and research
foods and the gastrointestinal tract; food products con- in food bioactivity and its effect on human health
taining both probiotics and prebiotics are designated as through nutrigenomics and/or nutrigenetics approaches,
synbiotic [14]. among others.
Different analytical strategies have been applied to This review mainly focuses on and discusses the
study probiotic microflora and prebiotic compounds, and major analytical approaches, confronting classical with
to characterize biochemical transformations in FDFs and advanced analytical techniques that have been applied
the associated functionality and safety, including legal to study FDFs containing probiotics and/or prebiotics.
requirements to uphold associated claims.
The main goal of this review is to discuss the advan- 2.1. Viability and/or survivability of probiotic bacteria
tages and the disadvantages of such analytical method- The presence of viable and metabolic active cells of
ologies, and to comment on future applications and their probiotic bacteria in dairy products is essential in order
potential research interest. to assure the beneficial health benefits in the host.
According to Gomes et al. [17], the physiological func-
tionality of fermented dairy foods is inherent to the
2. Analytical strategies intrinsic biological activity of the microorganisms.
However, viability of probiotic organisms can be affected
In milk and fermented dairy products, there is a plethora by several factors resulting from processing or even
of beneficial compounds [e.g., functional proteins, bio- storage conditions (e.g., temperature, pH, oxygen, and
active peptides, vitamins, antioxidants, oligosaccharides, antimicrobial substances) and by their resistance
organic acids, and polyunsaturated fatty acids (e.g., throughout the gastrointestinal tract. Generally, the
2 http://www.elsevier.com/locate/trac
Trends in Analytical Chemistry, Vol. xxx, No. x, 2012 Trends
incorporation of probiotic organisms in diverse food permeability or enzyme activities [20]. Łaniewska-
vehicles requires: Trokenheim et al. [21] compared fluorescent microscopy
(1) a selection of probiotic strains that should rely on (direct epifluorescent filter using carboxyfluorescein
their technological performance (e.g., genetic stabil- diacetate stain) with a plating method where MRS agar
ity, growth rate, and acidification rate) besides the was incubated anaerobically at optimal temperature for
safety requirements [17]; and, 48 h for several species of Lactobacillus. Statistically
(2) product and process adaptations in order to significant higher values of cell counts were obtained by
promote higher survival enhancing the probiotic direct fluorescence than by plate counts in MRS agar; the
functionality [3]. magnitude of the differences were strain dependent and
Detection, enumeration and identification of these higher for strains of L. acidophilus (P<0.05).
probiotic strains in dairy products are indeed tools of An alternative method is Fourier transform infrared
utmost importance to assure the consumer about the (FT-IR) spectroscopy that has been applied for detection,
veracity of claims placed on product labels concerning discrimination, identification and classification of LAB
both bacterial species and associated concentration and and probiotics and also provides information about cell
viability. Undoubtedly, more care and attention is metabolism [22] from cultures and foods. FT-IR is able to
deemed necessary for accurate identification as well as discriminate viable, injured and dead bacteria and is
safety and functionality monitoring of probiotic agents used in the analysis of structural components of bacteria
[18,19]. [23]. FT-IR is a method based on the measurement of the
Currently, there are several methods available for modes of vibration of a molecule that is excited by
detection, enumeration and identification of microor- radiation in the infrared region producing a spectrum
ganisms that are also applied to probiotic microorgan- which represents a ‘‘fingerprint’’ characteristic of any
isms. We discuss these methods in the following chemical/biochemical substance. The fingerprint results
sub-sections divided between non-molecular and from the stretching, contracting and bending vibrations
molecular approaches. of molecular bonds or functional groups present in pro-
teins, nucleic acids, lipids, sugars, and lipopolysaccha-
2.1.1. Non-molecular methods. The non-molecular rides, because there is a correlation between IR-band
approach to detect, to enumerate and to identify probiotic position and chemical structures in the molecule. Since
strains in dairy products includes those methods consid- the molecular composition varies from species to species,
ered conventional, based on the growth of the microor- each bacterium will give rise to a unique, characteristic
ganism in culture media, and those not requiring this FT-IR spectrum [23]. Several sampling techniques and
step (Fig. 1). The basis of the conventional methods relies spectra recording have been used for microbial charac-
on standard procedures encompassing isolation, count- terization; some of the more common are transmittance,
ing and identification of the probiotic strains at the genus diffuse reflectance, attenuated total reflectance and
and species levels based on cell ability to reproduce and to microspectroscopy. Since FT-IR spectroscopy generates
form colonies on selective/differential agar media plates large amounts of data, their interpretation requires
[17]. The monitoring of viability and survival of probiotic appropriate multivariate statistical methods – supervised
bacteria through differential counting is important to and unsupervised methods [e.g., principal component
assess product quality and to estimate the storage time, analysis (PCA) and hierarchical cluster analysis (HCA)].
but we do not discuss it, since this review is more focused FT-IR is a relatively fast, simple, sensitive technique,
on the most advanced and latest analytical strategies. requiring very little sample, and the biological cell
Alternative methodologies have been applied to remains intact during analysis. It is possible to make
quantify probiotic bacteria in a more accurate way able qualitative and quantitative analysis and the sample can
to reduce the underestimation of viable bacteria obtained be in the form of liquid, gas, powder, solid, or film.
by plate-count methods (Fig. 1). Fluorescence methods However, variations in the spectra can be caused by
have been applied in bacterial viability studies [20]; environmental conditions around the FT-IR instrument,
these are able to detect and to differentiate between so, for the same sample, background scans and multiple
viable, injured, stressed and dead bacterial cells through scans are required. If the sample is complex, then it can
fluorescent dyes, which, in turn, can be detected by produce overlapping spectra, and so previous separation
fluorescence microscopy, fluorometry, flow cytometry, or or purification steps may be needed or may require
fluorescence in situ hybridization (FISH). Different standardization, rigorous data collection, and expertise
fluorescent dyes are used as indicators of dead or alive in the chemometric analyses of spectra [23].
cells. In order to assess intact and metabolically active Vodnar et al. [22] used FT-IR spectroscopy to finger-
cells but not culturable, a nucleic acid dye, TOTO-1, can be print L. plantarum, L. casei, B. infantis and B. breve during
used since it will stain only membrane damaged cells [17]. fermentation by identifying specific markers located at
Fluorescent physiological indicators are also available, several wavelengths, especially at 2845 cm 1 and
permitting detection of differences in membrane 2929 cm 1.
http://www.elsevier.com/locate/trac 3
Figure 1. Non-molecular versus molecular-based approaches for microbial analysis of probiotic bacteria in functional dairy foods targeting assessment of microbial diversity, identification and
quantification.
2.1.2. Molecular methods. With the use of molecular salts). Commercially-available DNA-extraction kits are
methods, the ability to detect and to identify food mi- able to yield pure DNA [37]. The selection of DNA or
crobes, including probiotic bacteria, has made tremen- RNA extraction relies on the choice to obtain informa-
dous advances in recent years, especially after the tion of entire microbial population not differentiating
introduction of PCR in the 1980s. Several detection between alive or dead cells, which is achieved by DNA
techniques have been developed based on PCR, namely extraction whereas RNA or DNA staining provides
denaturing gradient gel electrophoresis (DGGE), real- information about live cells.
time PCR (qPCR), terminal restriction fragment length Following DNA/RNA extraction, the amplification of
polymorphism (T-RFLP), random amplified polymorphic encoding genes by PCR is a step that directs the outcome
DNA (RAPD) [17,24]. Identification of probiotic species, of results. Universal primers (e.g., 16S and 23S rRNA),
including their differentiation in different strains by intergenic spacers, functional gene primers or specific
culture-dependent or culture-independent techniques, primers should be carefully selected to study microbial
the study of microbial community composition, and diversity, functional diversity and/or search of targeted
assessment of its different populations and interactions in species. According to Justé et al. [25], microbial popu-
food products are some of the main achievements of lations can be characterized considering three types of
molecular techniques [17,25]. Procedures involving outcome information: i) diversity; ii) identity; and, iii)
culture-independent techniques, which are essentially quantity.
based on the analysis of amplified nucleic acids by PCR Different protocols involving molecular methods have
present in the sample, overcome the major drawback of been described in the literature, some of them indicated
the culture-dependent techniques that are unable to in Fig. 1. Table 2 summarizes some of the studies
detect non-culturable cells [25,26]. The possibility to involving molecular-based protocols that have been
differentiate or to find new strains of probiotic bacteria, recently applied to probiotic bacteria, especially in FDFs.
namely of lactobacilli or bifidobacteria, is of the utmost As safety and functionality of probiotics are strain
importance, since it is known that probiotic capacities/ dependent, the results published by Coereut et al. [27]
functionality are strain-dependent, and therefore the demonstrated the need to control probiotic bacteria
reliable identification of these strains and of their sur- present in commercial FFs at not only the level of species
vival in a food product are indispensable for quality and their viable cells but also the strain level (Table 2) to
control of FFs [17]. Nowadays, the availability of a large ensure product quality and protect consumer. This
number of bifidobacteria and lactobacilli genome concern was also the main driving force in Collado et al.
sequences allows further insights into the evolutionary studies [18], where different molecular techniques were
development of these genera using a so-called tested to evaluate the presence of B. animalis subsp. lactis
phylogenomics approach, permitting more insights into in commercial Spanish fermented milks. Multiplex PCR
gene functions [24]. A detailed list of food microbes and RAPD were revealed as the most suitable, rapid and
including probiotic bacteria (e.g., B. animalis subsp. precise for identification until strain level, being able to
lactis, B. longum; L. acidophilus, L. brevis, and L. casei) be used routinely in quality control procedures as
with complete or genome sequencing in progress was confirmed by follow-up study [18].
published by OÕFlaherty and Klaenhammer [24]. Garcı́a-Cayuela et al. [32] used a procedure based on
In recent years, several review papers were published the combined use of propidium monoazide (PMA) with
describing the application of molecular techniques to quantitative qPCR for the specific detection and enu-
analyze microbial composition in food products, including meration of viable bacteria of four species of LAB and of
functional dairy products [17,26–29]; specifically, RAPD, B. lactis mixed culture in fermented milk products.
PFGE, DGGE and qPCR reveal great potential to detect, to According to Justé et al. [25], the use of molecular
enumerate and to identify probiotic species at strain level techniques does not have to eliminate the classical
[30–33], which is of utmost important for a relationship approaches to study microorganisms; instead, the com-
of trust between the food industry, the consumer and the plementarities between both approaches will enable
regulators. Fig. 1 displays the major steps involved in better understanding of the cell itself and overcome
culture-dependent or culture-independent techniques, technological limitations of non-molecular methods
whereas Table 1 briefly sets out the main genomics-based (e.g., those involving the growth of cells in agar media).
methods that can be used to analyze microorganisms,
namely probiotic species/strains in FDFs. 2.2. Metabolic assessment
The extraction of DNA or RNA is an important step, Nowadays, the analytical strategies to characterize and
especially in assessment based on independent culture, to control the quality and the safety of food products are
because a representative DNA/RNA extraction of total tools that have been implemented in routine laboratory
microbial population is needed with high concentration procedures and food research. These analytical strategies
and purity [26]. This step may suffer interferences from comprise a tool for food-product differentiation,
the matrix composition of dairy food (i.e. fats, proteins, validation and authentication, since all food constituents
6
Trends
Table 1. An overview of the main molecular techniques used by dependent or independent culture approach to study microbial communities in dairy food* for which an example of specific
application is given
http://www.elsevier.com/locate/trac
Method Brief description of technique Major achievement Advantages/Limitations Sample Ref.

Amplified Digestion of amplified ribosomal. Microbial identification and A: Relatively simple PCR-based Probiotic [19]
ribosomal DNA DNA followed by gel electrophoresis microbial communities and fingerprinting technique. fermented milks
restriction being all fragments detected. dynamics analysis. L: Due to a complexity of the profiles,
analysis multiple restriction enzymes need to be
(ARDRA) used to attain higher resolution; Limited
staining sensitivity of gels; Best results in
low diversity communities.
Random Use of short and low stringency Identification of LAB including A: Reliable method to distinguish species Probiotic [31]
amplified hybridization to randomly amplify. probiotic bacteria. in dairy products and to monitor changes fermented milks
polymorphic DNA fragments originating a in the LAB community during and cheeses
DNA (RAPD) fingerprinting pattern. fermentation.
L: Careful controlled conditions are
needed to assure reproducibility.
Denaturing and Electrophoretic separation of small Microbial diversity analysis and A: Affordability and outcome data Probiotic [30]
temperature PCR-amplified DNA fragments on microbial communities and relative easy to interpret. fermented milks
gradient gel acrylamide gel by denaturing dynamics analysis. L: PCR small fragments can difficult
electrophoresis chemical gradient (DGGE) using urea reliable identification; Possible co-
(DGGE/TGGE) or formamide or by temperature migration of different fragments with
gradient (TGGE) with a constant identical electrophoretic mobility; Lack
denaturants concentration. of reproducibility that can be enhanced
by use of internal standard; Low
sensitivity due to gel staining that can be
improved by use of fluorescent labeled
primers.
Single-strand Electrophoretic separation of PCR- Microbial diversity analysis. A: Similar limitations and advantages as No studies [34]
Trends in Analytical Chemistry, Vol. xxx, No. x, 2012

conformation amplified products based on DGGE/TGGE but with more PCR found in
polymorphism conformational differences of folded amplification since no clamped primers functional dairy
(SSCP) single-stranded products obtained by are required. food;
denaturation on non-denaturating L: Formation of several stable Livarot cheese
acrylamide gel being able to separate conformations out of one single stranded
fragments with similar molecular DNA fragment resulting in possible
weight. multiple bands.
Terminal Marker genes are amplified with Comparative microbial A: Precise length assignment with single- No studies [35]
restriction fluorescent labeled primer followed community analysis. base pair resolution by use of internal size found in
fragment length by restriction digestion and standard which by comparison to functional dairy
polymorphism separation and detection. Since only sequence database of Ribosomal food;
(T-RFLP) labeled terminal restriction fragments Database Project allows predictions of Yoghurt and
are detected on electropherogram, the microorganisms present in the hard cheeses
their length heterogeneity is an sample.
indication of a complex microbial L: Multiple restriction enzymes are
community. needed to improve specificity and
reliability; Primers and labeling dyes
should be selected carefully.
may be meticulously identified and qualified, and verifi-

[36]
[32]
[31]
The brief descriptions of each technique, their major achievements, and their advantages (A) and limitations (L) were based on reviews published between 2008 and 2011 [17,25,26,28].
cation of compliance with nutritional labeling of FFs,
validating their claims regarding composition in benefi-
cial compounds.
Reviews on analytical methods for evaluation of food
fermented milks
fermented milks
functional dairy
identity, in general [39], and for dairy products, in
and cheeses
Camembert
No studies
particular [40], show physicochemical analysis, liquid

Probiotic
Probiotic
found in
cheese
chromatography (LC) and gas chromatography (GC)

food;
techniques with different detectors as some of the tradi-

tional approaches used for the determination of the
quality and/or authenticity of dairy products based on
chemical characteristics. While measurement of the
L: Single organisms can origin more than
accurate template quantification; Enables

should be selected to improve sensitivity
fragments than the flanking 16S and 23S
L: Limited targets because of the limited

online detection of PCR product; Target
A: Accurate quantification of DNA and
A: PFGE profile of whole genome with

A: The intergenic region displays more
traditional nutrients (i.e. proteins, fat, carbohydrates,

one signal; suitable primers for ARISA
DNA can be quantified by calibration
number of available fluorescent dyes.
differentiate at subspecies and strain

length and nucleotide heterogeneity
and minerals) relies on basic chemical techniques, the

concentration of template DNA).
identification of more specific components (e.g., individ-

L: Time-consuming technique.
curve (threshold cycles versus
discriminatory power; able to
ual phenols, bioactive peptides, and polyunsaturated

fatty acids) demands complex processes involving
extraction, detection and quantification.
and reproducibility.
Due to the complexity of food matrices, the use of

ribosomal genes.
advanced analytical techniques able to generate a

chemical fingerprint of a product is required, especially
for FDFs in order to identify and to quantify the beneficial
level.
compounds resulting from biochemical reactions,

assessing their bioactivity and bioaccessibility/bioavail-
ability throughout the food-product life-cycle. MS, NMR
spectroscopy, capillary electrophoresis (CE) and high-
Microbial diversity analysis with
Microbial diversity analysis and
performance LC (HPLC), coupled with several detectors,

are some of the advanced techniques to study food
Able to detect and quantify
subspecies and strain level.

identification of microbial
specific target organisms.
matrices [15,41]. These techniques are also used in

identification ability at
combination, designated ‘‘hyphenated’’ techniques, to

Studies of microbial
analyze multiple components (i.e. HPLC-NMR, HPLC-

MS, GC-MS, and CE-MS) in food analysis [15,41].
communities.
The analytical technique to be employed depends on

species.
the target compounds and the matrix, where physico-

chemical properties (e.g., polarity, size, and volatility)
will be determinant for the choice of:
(1) sample-preparation procedures;
(2) separation mechanism and technique (GC, HPLC,
sizes can be explored for subtyping of
Amplification of the targeted DNA in
probes; Able to use specific-species

PCR amplification of total bacterial
bacterial strains; Use of fluorescent
primers of gene or microorganism.

real-time monitored by fluorescent
and CE); and,

ribosomal genes; Different spacers
community DNA of the intergenic
fragments obtained by restriction

region between the 16S and 23S
digestion resulting in fingerprint
(3) type of detector [ultraviolet spectroscopy (UV),

Separation of the large DNA
primers with laser detection.
refractive-index detector (RID), fluorescence spec-

troscopy detector (FLD), flame ionization detector
(FID), and MS] [41].
Fig. 2 gives an overview of the most commonly used
analytical techniques and the alternative advanced
technologies to analyze parameters able to reflect gly-
profiles.
colysis, proteolysis and lipolysis in traditional dairy foods

and/or in FDFs, which we discuss in the following sub-
sections.
2.2.1. Glycolysis. In the majority of dairy products (e.g.,

Pulse Field Gel
spacer analysis
Electrophoresis
Real time PCR
fermented milks or cheeses), glycolysis is mainly char-

Automated
acterized by degradation of lactose. Depending on LAB or

intergenic
ribosomal
(ARISA)
probiotic bacteria and their fermentation pathways,

(qPCR)
(PFGE)
lactose is degraded, originating small organic acids (e.g.,

*
lactic acid, propionic acid, citric acid, and acetic acid).
8
Trends
Table 2. A short description of protocols based on molecular methods for detection, enumeration and identification of potential probiotic lactobacilli and bifidobacteria bacteria in dairy foods
and their main results in studies published between 2004 and 2010
Study Procedure description Main results Ref.

Determination of 1. DNA extraction in culture dependent technique (growth in MRS broth) – Lactobacilli genus and species (L. casei, L. zeae, L. rhamnosus, L. del- [31]
lactobacilli counts by phenol-chloroform method; brueckii subsp. bulgaricus) were identified;
and species 2. Genus specific PCR, followed by multiplex PCR to group lactobacilli and – The auditors found that many products were incorrectly labeled both
probiotic fermented species-specific PCR to identify lactobacilli species; in terms of probiotic species and their viable counts in the products.
milks and cheeses 3. RAPD to differentiate potential replicates;
4. Isolates typed by PFGE.
Evaluation of 1. DNA extraction in culture dependent technique (growth in BFM agar) by – Bifidobacterium genus was identified by both Genus specific PCR [18]
B. animalis subsp. phenol-chloroform method; and FISH;
lactis in commercial 2. Genus specific PCR and FISH using 16S rRNA probe specific of bifido- – Multiplex PCR was able to identify B. animalis subsp. lactis in all
fermented milk bacteria followed by ARDRA-PCR using Lm26 and Lm3 primers to fermented samples. A rapid, easy and low cost technique useful
identify all known strains of Bifidobacterium and distinguish them from for rapid monitoring of this strain in dairy products;
other bacteria genera; – According to ARDRA results, all the isolated strains belong to B.
3. Multiplex PCR by application of oligonucleotide pair Bflact2-Bflact5 for animalis subsp. lactis;
fragment amplification of DNA from B. animalis subsp. lactis; – RAPD-PCR results demonstrated that the strains were not all the
4. RAPD-PCR analysis in genomic DNA with Bif primer and fragments same although very similar among them;
were separated by NuSieve agarose gel electrophoresis; – AFLP results were similar as those obtained by RAPD-PCR.
5. AFLP analysis in genomic DNA digested with restriction enzyme HindIII
and restriction fragments were ligated with ADH1 and ADH2 adapters;
amplified fragments were separated by agarose gel electrophoresis;
6. ARDRA, RAPS and amplified ribosomal length polymorphism (AFLP)
stained gels were processed by software package.
Enumeration and 1. DNA extraction from fermented milk (culture independent technique); – The species-specific PCR primers were suitable for identification [30]
identification of 2. Species-specific primers PCR for Streptococcus thermophillus, L. del- each specie by culture independent analysis in the fermented milk

mixed cultures in brueckii subsp. bulgaricus, L. acidophilus, L. paracasei subsp. paracasei using the PCR-DGGE methods;
fermented milks and B. lactis; – The combination of species-specific PCR and DGGE analysis dem-
3. DGGE was performed with a DCode system. onstrate great potential for identification purposes and for evaluation
of the accuracy in the species-labeling in commercial fermented milk
by an independent culture technique.
Enumeration and 1. PMA treatment of cultures of pure strains (culture dependent technique) – Loss of viability of the species through 28 days of storage at 4C was [32]
identification of in order to be distinguishable viable cells from irreversibly damaged able to be quantified by PMA-quantitative qPCR;
mixed cultures in cells; – Enumeration of viable bacteria was able in 3 h;
fermented milks 2. DNA extraction by DNA kit; – The use of species-specific primers in the PMA-quantitative qPCR
3. Quantitative qPCR using species-specific primers PCR for Streptococcus assay allowed the identification and enumeration of the viable
thermophillus, L. delbrueckii subsp. bulgaricus, L. acidophilus, L. paracasei bacteria in complex mixture present in fermented milk products.
subsp. paracasei and B. lactis; the quantification by RTi-PCR was per-
formed by a multicolor qPCR detection system cycler.
Other sugars, also present in milk, are used as fermen-

[38]
[33]
tation substrates, namely galactose, glucose and sucrose.
The classical approach used to study glycolysis and
fermentative pathways involves HPLC coupled to detec-
– By PFGE differentiation of B. animalis subsp. lactis isolates enabled
– Bifidobacteria counts were determined by quantitative qPCR allow-

– All the isolates were identified by PCR as B. animalis subsp. lactis;
– Biochemical fermentation tests revealed that all strains were charac-
B. longum subsp. longum was able to confirm these species in the
– Effective method to evaluate the accuracy in the species-labeling in

– PCR assay with primers specific to B. animalis subsp. animalis and
tors (e.g., UV/VIS, RID or FID) to monitor organic acids,
ing the enumeration of both viable/culturable and unculturable;

sugars (Table 3), diacetyl, acetoin and the first inter-
mediates of amino-acid degradation. Rodrigues et al.
commercial yoghurts by an independent culture technique.

[42] monitored glycolysis in probiotic and synbiotic
dairy matrices inoculated with different probiotic bacte-
ria throughout 7 days, analyzing the variation of
organic acids (i.e. lactic, acetic, formic and citric acids)
terized by a unique fermentation pattern.
by HPLC-UV and sugars (i.e. lactose, fructose and

glucose) by HPLC-RID. The limit of detection (LOD) for
HPLC/UV obtained by Rodrigues et al. [42] in the
assessment of glycolysis in probiotic and synbiotic cur-
dled milk is reported in Table 4. This was the only figure
of merit (FOM) found among examples listed in Table 3
to distinguish strains;
with HPLC-UV and HPLC-RID; this may be explained by

yogurt samples;
the fact that the authors [42] used a classical approach

where techniques have been previously validated, so
FOMs will naturally be scarcer. Nevertheless, it is
important to remember that FOMs are not only used for
validation of a new methodology (sensitivity, selectivity,
LOD which are related to the methods and analyte) but
should also be used for validation of the final results
(traceability, uncertainty, and representativity), since,
3. Genomic bacterial DNA from each pure strain culture was digested with
2. PCR using primer sets specific for the Bifidobacterium genus, B. animalis
5. Carbohydrate fermentation patterns of the Bifidobacterium strains were

restriction enzymes; macrorestriction fragments were separated in 1%
3. Bifidobacterium spp. determined by PCR were subjected to quantitative

1. DNA extraction in culture independent technique with 0.1 mL yoghurt;
being quantifiable, they may indicate the quality of the

2. PCR assay with primers specific to bifidobacteria genus and species;
process [51]. Despite the several advantages pointed out

for HPLC-UV, including being considered a straightfor-
1. DNA extraction in culture dependent technique using DNA kit;
ward, robust and reproducible technique, other ap-

proaches (e.g., CE) are indicated as also suitable for
4. PFGE patters were analyzed by TotalLab 1v1 software;
analysis of small organic compounds variations {e.g.,

lactic-acid enantiomers were determined in milk and
qPCR using the SYBR Green PCR Master kit.
yoghurt samples by CE with contactless conductivity

subsp. lactis and B. animalis subsp. animalis;
detection [52]}.
Certain oligosaccharides (i.e. GOS, FOS and XOS) and
determined using the API 50 CHL.
inulin are considered prebiotic agents due to their

capacity to stimulate microbial growth and metabolism of
pulsed field certified agarose;
several lactobacilli and bifidobacteria. They have been

studied by diverse analytical approaches, ranging from
classical to advanced. Ignatova et al. [53], in their study
on the effect of oligosaccharides on the growth of
L. delbrueckii subsp. bulgaricus, monitored the variation of
fermentation metabolites (e.g., D -lactic acid, acetic acid
and ethanol) enzymatically through commercially-
available kits, whereas the sugars and the oligosaccha-
rides in the fermentation broth were analyzed by
HPLC-RID.
High-performance anion-exchange chromatography
coupled with pulsed amperometric detection (HPAEC-
animalis subsp. lactis
Differentiation and
identification of B.
from bio-yoghurts
PAD) comprised an alternative tool to evaluate FOS and

Enumeration and
Bifidobacteria in
identification of
inulin, namely the changes in their chain-length distri-

bution by bifidobacteria, as demonstrated by Corradini
et al. [54], who reported that HPAEC-PAD provided
yogurt
important information about the potential use of the

oligosaccharides and inulin as prebiotic agents. A review
Figure 2. A general overview of the most used analytical techniques as well as the alternative advanced technologies to analyze parameters able
to evaluate glycolysis, proteolysis and lipolysis in functional dairy foods.
of the principles and applications of HPAEC-PAD to DIGE was able to differentiate 28 protein spots, which
evaluate carbohydrates of food interest was recently were submitted to matrix-assisted laser desorption
published by Corradini et al. [55]. ionization – time-of-flight (MALDI-TOF)-MS analysis,
Alternative approaches to characterize molecular which identified 25 proteins; carbohydrate-metabolizing
weight and structure of potential prebiotic oligosaccha- proteins, including XOS-metabolizing proteins and en-
rides have been performed using electrospray ionization zymes of the bifid shunt, accounted for the majority of
(ESI) MS and 1H-NMR [56]. Bengal gram-husk and the proteins identified. This analytical approach was able
wheat-bran oligosaccharides obtained by enzymatic to show that B. animalis subsp. lactis BB-12 possessed
hydrolysis and separated by gel permeation chromato- metabolic pathways to utilize XOS as a carbon source
graphy were collected in fractions, which were submitted and also to enable the authors to propose a trans-
to 1H-NMR. Proton-NMR spectra of oligosaccharides criptomics/proteomics-based model for XOS catabolism.
indicated chemical shifts in the region of d Metabolic profiling is a powerful tool to probe benefi-
3.00–5.00, characteristic of a-linked arabinofurose and cial probiotic mechanisms in both the food and the
b-linked xylopyranoside residues. Further analysis by human host, as we discuss below.
ESI-MS was able to identify tri, penta, hexa and tetra
saccharides [56]. 2.2.2. Proteolysis. The degradation of milk proteins,
In order to gain knowledge about metabolism of XOS designated proteolysis, is an important biochemical
by B. animalis subsp. lactis BB-12, Gilad et al. [57] event that determines textural and flavor characteristics
combined the proteomic and transcriptomic approaches, of dairy products. In cheeses, this event starts with milk
involving DNA microarrays, qPCR and two-dimensional coagulation and continues during ripening [58],
difference gel electrophoresis (2D-DIGE) analysis of strain whereas, in fermented milks, this event is not as evident.
samples grown in cultures with XOS or glucose. The It is known that hydrolysis of milk proteins can release
concentration of XOS with different degrees of polymer- bioactive peptides with specific biological activities (e.g.,
ization measured by HPAEC-PAD decreased during antihypertensive, antimicrobial, opioid, antioxidant,
growth of B. animalis subsp. lactis BB-12. Microarray immunomodulant, antithrombotic or mineral binding)
analysis showed that 9–10 genes encoded proteins in- [59,60] that can be found in fermented milks, yoghurt,
volved in XOS catabolism, including degrading and cheese and in dairy by-products (e.g., whey) [61].
metabolizing enzymes, transport enzymes and a regula- Proteolysis is catalyzed by enzymes derived from the
tory enzyme. Comparative proteome analysis using 2D- residual coagulant (chymosin or pepsin) if cheese is
Table 3. Glycolysis, proteolysis and lipolysis assessment on different functional dairy products
BE* Sample Composition Methodology/analyzed parameters Results Ref.

Glycolysis Sweet whey cheese stored 21 – Whey with 10% ovine milk precipitated at Analysis of organic acids (lactic acid, acetic Statistically significant increase of lactic [12]
d at 7C 95C acid) by HPLC-UV; acid over storage time (P < 0.05) but no
– L. paracasei L26 Analysis of sugars (lactose, sucrose, fructose, statistical differences between sweet whey
– Sweet whey cheeses with sugar, aloe vera, glucose) by HPLC-RID. cheese types.
chocolate fiber and powder, strawberry Concomitant degradation of lactose over
jam time
Probiotic and synbiotic – CowÕs milk curdled with animal rennet Analysis of organic acids (lactic acid, acetic Lactic acid increase correlated with lactose [42]
curdled milk matrices – L. casei-01; L. acidophilus La5; B. lactis acid, formic acid, citric acid) by HPLC-UV; consumption in both probiotic and
ripened over 60 d at 12C B94 Analysis of sugars (lactose, sucrose, fructose) synbiotic matrices.
– 50:50 mix FOS:Inulin by HPLC-RID. Higher increase of acetic acid in probiotic
matrix with B. lactis B94.
Fos:Inulin did not affect overall post-
acidification
Probiotic Pecorino cheeses – Ovine milk curdled with lamb rennet Analysis of organic acids (lactic acid, acetic Variation of lactic acid content over 30 d of [43]
ripened over 30 d – Starter cultures: S. thermophilus/ L. del- acid, citric acid) by HPLC-UV diode-array ripening.
brueckii spp. bulgaricus with or without detector. Increase of acetic acid by 30 d of ripening in
probiotic bacteria (L. acidophilus, B. lactis, Pecorino cheese containing bifidobacteria
B. longum) species
Probiotic yoghurt with – CowÕs milk and skim milk fermented at Analysis of organic acids (lactic acid, acetic Probiotic metabolism enhanced by higher [44]
glucose oxidase stored for 30 45C until pH = 4.6 € 0.5 acid) by HPLC-UV/Vis; amounts of glucose oxidase during
d at 3–5C – Starter cultures: S. thermophilus/ L. del- Analysis of diacetyl and acetaldehyde by GC- fermentation.
brueckii spp. bulgaricus MS. Higher amounts of diacetyl and
– B. longum BL05, L. acidophilus La14 acetaldehyde in probiotic yoghurt with
glucose oxidase.
Proteolysis Traditional and probiotic – Ovine milk fermented at 42C until Nitrogen fractions+: WSN and N-TCA quanti- Most peptides derived from b-casein. [45]
yoghurt stored for 26 d at pH = 4.7 fied by Kjeldahl method; Similar peptide profiles but quantitative differ-
4C – Starter cultures: S. thermophilus/ L. del- Soluble peptide profiles by RPLC-UV/Vis; ences over storage time
brueckii spp. bulgaricus Isolated peptides by RPLC were characterized
– Probiotic adjunct cultures: L. paracasei by pulsed liquid-phase protein-peptide sequencer
subsp. paracasei DC412
Cremoso soft cheese with – CowÕs milk curdled with chymosin Nitrogen fractions: WSN, N-TCA and N-PTA No significant differences for all nitrogen frac- [46]
potential probiotic – Starter cultures: S. thermophilus quantified by Kjeldahl method; tions between cheeses with or without
lactobacilli ripened over 60 – Adjunct cultures with probiotic potential: Protein degradation in insoluble fraction at lactobacilli.
d at 50±0.5C L. casei I90, L. plantarum I91, L. rhamno- pH = 4.6 by UREA-PAGE; Higher as1-casein breakdown but not affected
sus I73/I77 Soluble peptide profiles by RPLC-UV/Vis; by the addition of lactobacilli.
FAA by HPLC-UV/Vis L. rhamnosus had the strongest influence in
peptidolysis and increase of FAA
concentration.
Trends
11
Table 3. Continued
12
Trends
BE* Sample Composition Methodology/analyzed parameters Results Ref.

Semi-hard cheese (Pategrás – CowÕs milk curdled with chymosin Nitrogen fractions: WSN, N-TCA and N-PTA L. acidophilus had a significant influence [47]
Argentino cheese) with – Starter cultures: S thermophilus quantified by Kjeldahl method; on increase of small nitrogen compounds,
probiotic strains ripened over – Adjunct cultures: L. paracasei subsp. para- Protein degradation in insoluble fraction at FAA and peptide profiles.
60 d at 12C. casei, L. acidophilus and B. lactis used as pH = 4.6 by UREA-PAGE; Synergistic effects observed for the three-
single strain or three-strain mixed culture Soluble peptide profiles by RPLC-UV/Vis; strain mixed culture.
FAA by HPLC-UV/Vis.
Probiotic Pecorino cheeses – Ovine milk curdled with lamb rennet Nitrogen fractions: WSN quantified by Kjel- Probiotic cheeses displayed higher [43]
ripened over 30 d – Starter cultures: S. thermophilus/ L. del- dahl method; percentages of as1-I-casein fraction up to
brueckii spp. bulgaricus with or without Protein degradation in insoluble fraction at 15 d.
probiotic bacteria (L. acidophilus, B. lactis, pH = 4.6 by UREA-PAGE; Higher accumulation of FFAA in cheeses
B. longum) Soluble peptide profiles by RPLC-UV/Vis; with L. acidophilus and with a mix of
Total and individual AA determined in WSN bifidobacteria
by EZ:fast physiological AA CG/FID.
Lipolysis Probiotic Pecorino cheeses – Ovine milk curdled with lamb rennet Extracted underivatized FFA determined by Total FFA was higher in ovine milk cream [48]
ripened over 60 d – Starter cultures: S. thermophilus/ L. del- CGC-FID; containing probiotic strains.
brueckii spp. bulgaricus with or without Extracted and methylated CLAÕs# determined Increase of cheese FFA over the first 30 d of
probiotic bacteria (L. acidophilus, B. lactis, by HPLC with diode array detector ripening period.
B. longum) Cheese with L. acidophilus with higher
amounts of CLA1 and CLA2.
Probiotic Pecorino cheeses – Ovine milk curdled with lamb rennet Extracted and derivatized FFA were deter- Higher content of FFA, CLA1, CLA2 and [43]
ripened over 30 d – Starter cultures: S. thermophilus/ L. del- mined by CGC-FID. CLA3 in probiotic cheeses especially
brueckii spp. bulgaricus with or without containing L. acidophilus.
probiotic bacteria (L. acidophilus, B. lactis,
B. longum)
Probiotic and synbiotic – CowÕs milk curdled by animal rennet Extracted and methylated FFA and CLAs Increases of total FFA and total CLA over [14]

cheeses ripened over 60 d at – L. casei-01; B. lactis B94 determined by GC-MS. ripening period in probiotic and synbiotic
12C – FOS and 50:50 mix FOS:Inulin cheeses.
Synbiotic cheeses containing B. lactis B94
and 50:50 FOS/Inulin with higher content
of CLA especially of CLA1 and CLA3.
Probiotic fermented milk – Fermentation at 42C until pH = 4.7 of Extracted and methylated FFA and CLAs Fermented organic milks: Lower levels of [49]
conventional and organic cowÕs milk determined by GC-FID. saturated FA, higher levels of
– B. animalis subsp. lactis BB12, BL04, monounsaturated FA and of CLA.
B94, HN019
*
Biochemical event
+
WSN – Water-soluble nitrogen at pH = 4.6; N-TCA – Nitrogen soluble in 12% trichloroacetic acid; N-PTA – Nitrogen soluble in 2.5% phosphotungstic acid.
#
CLAs – CLA1: cis-9, trans-11 octadecadienoic acid; CLA2: trans-9, trans-11 octadecadienoic acid; CLA3: trans-10, cis-12 octadecadienoic acid.
Table 4. Analytical parameters of methodologies for evaluation of glycolysis and lipolysis in functional dairy foods (FDFs)
BE* Method Sample Compounds Linear range LOD# Ref.

Glycolysis HPLC-UV Probiotic and synbiotic Acetic acid – 0.19 mg/g curdled milk [42]
HPLC-RID curdled milk Formic acid 0.20 mg/g curdled milk
Fructose 0.049 mg/g curdled milk
Lipolysis GC-MS Probiotic curdled milk Linoleic acid (C18:2) methyl ester 5–130 lg 1.92 lg [8]
Oleic acid (C18:1) methyl ester 5–130 lg 2.57 lg
CLA1 methyl ester 5–130 lg 1.72 lg
GC-MS Probiotic and synbiotic CLA1 – 0.048 lg/g cheese [14]
cheeses CLA3 0.059 lg/g cheese
CLA4 0.050 lg/g cheese
a-linolenic acid (C18:3) 0.077 lg/g cheese
c-linolenic acid (C18:3) 0.066 lg/g cheese
GC-FID Probiotic cheese CLA3 – 0.01% (of total) cheese [50]
(Ras cheese) Pentadecylic acid (C15:1) 0.01% (of total) cheese
c-linolenic acid (C18:3) 0.01% (of total) cheese
Tricosylic acid (C23:0) 0.01% (of total) cheese
Nervonic acid (C24:1) 0.01% (of total) cheese
Docosahexaenoic acid (C22:6) 0.01% (of total) cheese
GC-OF Probiotic curdled milk Linoleic acid (C18:2) methyl ester 5–130 lg 1.94 lg [8]
Oleic acid (C18:1) methyl ester 5–130 lg 2.56 lg
CLA1 methyl ester 5–130 lg 1.92 lg
*
Biochemical event.
#
Limit of detection; CLAs – CLA1: cis-9, trans-11-C18:2; CLA2: trans-9, trans-11-C18:2; CLA3: trans-10, cis-12-C18:2; CLA4: trans-9, trans-12-
C18:2.
considered, milk (plasmin) and the starter/non-starter (3) peptide profiles by reversed-phase LC (RPLC)-
bacterial/probiotic microflora [58,62], so a general UV–Vis and FAAs by HPLC-UV/Vis [46,63].
pattern of milk proteolysis is recognized – initial degra- This classical approach takes time, consumes reagents
dation of as1-casein by bacteria and/or rennet enzymes and is very laborious, but it is still in use in proteolytic
or by acids – whereas as2-casein, j-casein and b-casein studies of FDFs – some examples are described in Table 3.
are degraded by plasmin-originating polypeptides/pep- According to De Simone et al. [61], the use of classical
tides. If production or storage conditions favor further analytical methodologies to characterize milk-derived
enzymatic activities, the residual rennet and/or bacterial peptides is very challenging, due to the complexity of the
endo-cellular or exo-cellular proteinases and peptidases milk-protein fraction and the high relative abundance of
will continuously generate smaller peptides and free peptides.
amino acids (FAAs), some of which may be precursors of Alternative approaches to avoid methods that are
the flavor compounds (e.g., alcohols, aldehydes, ketones time-consuming, expensive and involving extensive
and acids) [62]. chemical use have been studied, namely the use of FT-IR
In order to assure product quality, characterization to monitor amino acids, organic acids and ripening
and control of the proteolysis level is important, since changes in cheese [64] and of NIR spectroscopy for
peptide and protein content can affect both functional amino acids during cheese ripening [62] and peptides in
and biological properties of functional and/or traditional cheeses with different ripening times [58]. No studies
dairy products. In general, the classical approaches to using this approach were found for FDFs, namely
assess and to quantify proteolysis in dairy products, probiotic or synbiotic cheeses. Subramanian et al. [64]
namely in cheeses, have mainly been based on: observed that FT-IR spectroscopy has the potential to
(1) fractionation procedures in order to obtain nitrogen monitor 20 amino acids and three organic acids simul-
fractions soluble in different extractants with differ- taneously, besides age characterization during cheese
ent contents in polypeptides, peptides of different ripening, in 20 min while requiring less than 1 mL of
sizes and/or FAAs, which are quantified by the Kjel- solvent per sample.
dahl method, giving information about proteolysis NMR is also considered a valuable tool to study struc-
indexes [12,42,63]; tural and compositional aspects of food chemistry and
(2) analysis of casein (as1-, as2-, b- or j-) degradation food analysis. 1H nucleus is the most exploited, but 13C
by urea-polyacrylamide gel electrophoresis (PAGE), and 31P have also been applied in food matrices. Metabolic
whereby use of casein standards and image treat- profiling of fermented foods and potential probiotic or
ment of the gel quantification of casein degradation synbiotic cheeses by 1H-NMR have demonstrated the
is performed [46]; potential of this technique to study metabolic processes in
fermented foods [65] and probiotic and/or synbiotic dairy

matrices [63]. In the study by Choi et al. [65], the major De Simone et al. [61] used this combined approach for
peaks of 1H-NMR spectra assigned to amino acids (iso- whey-peptide extracts from Mozzarela di Bufala
leucine/leucine), organic acids (lactate, acetic acid, citric Campana cheese; peptide fractions obtained from ultra-
acid), choline and sugars (fructose, glucose, sucrose) filtration with a 3-kDa cut-off membrane were charac-
analyzed by PCA were able to discriminate the different terized by MALDI-TOF-MS analysis, whereas peptide
samples with different periods of fermentation. extracts fractionated by RP-HPLC were characterized by
Although the term proteome refers to the proteins ESI-MS (off-line or on-line with RP-HPLC for fraction-
expressed by a genome at a particular point in time, and ation). They identified two peptides derived from b-casein
proteomic tools have been shown as important to screen (b-CN f57-68 and f60-68), precursors of agonist opioid
for proteins expressed by microorganisms in several fer- b-casomorphin 7 and b-casomorphin 5, which appear to
mented foods, these have also been applied to milk- inhibit cell proliferation of particular interest to target
protein analysis to study proteolysis [66]. According to some cancers.
several authors [66–68], proteomic approaches by the To complement proteomics studies focused on identi-
use of high-resolution two dimensional gel electropho- fying proteins and peptides expressed by microorgan-
resis (2D-GE), mono- or multi-dimensional LC coupled isms, quantitative proteomic analysis based on MS has
with MS, MALDI-TOF-MS and ESI-MS can provide been developed – a topic also developed in Gagnaire et al.
information on: [67]. Indeed, MS-based approaches involving isotopic
(1) proteins, including minor proteins in complex mix- labeling for protein or peptide quantification purposes
tures; have been proposed as a way to overcome difficulties
(2) peptides produced in food matrices, some of them normally encountered when trying to quantify proteins
exhibiting functional or bioactive properties, and in gels [72].
the proteases responsible for their release in situ; Jardin et al. [72] applied quantitative proteomic
(3) reference proteomic maps to detect strain-strain analysis to bacterial proteins in Swiss-type cheese
variation, including that of probiotic lactobacilli extracts with different ripening times based on the
[69] and bifidobacteria [70], elucidating the mech- analysis of standardized protein cheese samples by:
anisms of in vitro and in vivo adaptation to environ- (1) 2D-GE for qualitative analysis;
mental stresses or predominant metabolic pathways (2) nano-LC coupled on-line with ESI-QTOF-MS analy-
that are active. sis of samples submitted to trypsinolysis and labeled
According to Mamone et al. [68], MS is the core tech- with iTRAQ tag, one per ripening time. 30 proteins
nology to characterize food proteins and peptides, being of bacterial (carbon-metabolism enzymes, stress
able to perform qualitative and quantitative analysis, proteins) and milk origin could be identified and
helping to increase knowledge about their nature, struc- quantified and, depending on the protein, a
ture, functional properties and impact on human health. 2.5–20 times increase in quantity was registered
The peptidome can be defined as the whole peptide within 7–69 d of ripening.
pool present in food products or raw materials or ob- According to Jardin et al. [72], this dynamic approach
tained during food processing or storage, namely those can contribute to a better insight into in situ starter
produced by proteolysis [67]. Gagnaire et al. [67] sug- metabolism.
gest that food peptidomics can provide information The use of the proteomic and peptidomic approach in
about product authenticity, origin, functional and/or the near future will certainly permit more insight into
biological activities, allergenicity and sensory properties. FDFs, namely with respect to bioactive peptides produced
Panchaud et al. [71] describe how to analyze food bio- by probiotic bacteria.
actives and their health effect, and discuss the techno-
logical challenges related to small, medium and large 2.2.3. Lipolysis. Lipolysis in dairy foods is also a
bioactive peptides based on MS techniques. Integrated biochemical event resulting from enzymes (esterases/
analytical strategies for the characterization of bioactive lipases) originating from the milk, coagulation agents
peptides from food (milk, yoghurt, cheese) are described and microbial flora (starters/non-starters/probiotic bac-
by Mamone et al. [68], including a food bioactive pep- teria) that cleave the ester linkage between free fatty
tide-analysis workflow for in vitro or in vivo analysis, acids (FFAs) and glycerol in the triacylglycerides. From
where the main steps involve: their activity, FFAs are released, and, in turn, comprise
(1) protein/peptide separation by techniques such as precursors of certain compounds (e.g., methyl ketones,
2D-GE, RPLC, or CE; esters, secondary alcohols, lactones and aldehydes),
(2) analysis of protein and peptide fractions by MS; and, which may influence the flavor or the aroma of dairy
(3) structural characterization by ESI-MS, MALDI-TOF- products. Analysis of the lipidic composition and the FFA
MS, and the potential of in silico analysis for predict- profile in milk and dairy products has therefore been a
ing possible bioactive sequences. topic of interest for decades, especially analysis of FFAs of
potential interest for human health produced by probi- molecular structures of purified lipids whereas 31P-NMR
otic bacteria, e.g.: is more suitable for analysis of phospholipid mixtures.
(1) mono and polyunsaturated FFAs; Microbial production of CLNA by c-linolenic acid with
(2) conjugated linoleic acids (CLAs), to which antiadip- L. plantarum AKU 1009a, a potential probiotic bacte-
ogenic, anticarcinogenic, antiatherogenic, antidia- rium, was studied by Kishino et al. [73], who found that
betogenic and anti-inflammatory properties have this strain is able to produce a mix of two CLNAs. These
been attributed and found in FDFs [14,42,50]; isomers (cis-6, cis-9, trans-11 and cis-6, trans-9, trans-11
(3) other conjugated FFAs {e.g., conjugated c-linolenic octadecatrienoic acid) were purified by HPLC and
acid (CLNA), and conjugated stearidonic acid pro- confirmed by MS and NMR analysis.
duced by probiotic bacteria, which are currently
becoming of interest because of their biological prop-
erties (e.g., anti-inflammatory, immunomodulatory, 3. Advanced omics approaches and microbiome
anti-obese and anti-carcinogenic effects) [73,74]}.
The classical approach to evaluate lipolysis through Increasing awareness of the intestinal microbiota (also
study of the main changes in FFA and CLA profiles in designated as intestinal microbiome) has promoted
functional dairy products or produced by probiotic considerable efforts to define their complex role better in
bacteria (bifidobacteria) is mainly based on certain host physiology and to discover the underlying mecha-
methodologies (e.g., GC-FID, GC-MS or Ag+-HPLC-UV) nisms [78]. Recall that the gastrointestinal (GI)
[14,48,63,75]. Table 3 displays studies of lipolysis microbiome comprises about 1014 bacteria that are
assessment in FDFs containing probiotics and/or mainly located in the large intestine. These have been
prebiotics. associated with multiple functions, including energy
In Carrasco-Pancorbo et al. [76], there can be found a homeostasis, bioavailability of nutrients, prevention of
description of lipid-extraction procedures from different mucosal infections, maintenance of an intact intestinal
samples and discussion about: barrier, and regulation of the mucosal immune system
(1) chromatography procedures [e.g., GC-FID, high- by acting as an important source of stimulators [78,79].
performance thin-layer chromatography (HPTLC) Any interruption of these functions may be closely
commonly coupled with densitometric quantifica- related to alterations or maladaptations of the GI
tion, HPLC and Ag+-HPLC with various detection microbiome and can consequently impair many
methods (UV, RID, electrochemical detection homeostatic and physiological signals, resulting in a
(ECD)] and the multi-dimensional chromatographic number of disease states, including allergy, inflamma-
systems (GC-GC, LC-LC, LC-GC and two-dimensional tory bowel disease (IBD) and obesity [80].
supercritical fluid chromatography); Diet and food-associated bacteria (that play a role in
(2) electrophoretic methods based on CE, where some food-fermentation processes, as previously described)
of the most used for lipid determination include may interact with the gut microbiome and impact on
capillary-zone electrophoresis (CZE) or capillary host response [24], although the associated molecular
electrochromatography (CEC). mechanisms are still unknown. In order to understand
A review of analysis of bioactive fatty acids (CLAs, these effects better, and to contribute to the validation of
c-linoleic acid, stearidonic acid) based on recent FDF functionality, for example, there is a need to char-
advances in GC and HPLC techniques, including sample- acterize not only the phylogenetic profile of human
preparation procedures, was published by Ruis- microbial communities but also the functional capacity
Rodrigues et al. [77]. of their members. Approaches toward this goal have
An alternative methodology based on an optical fiber included direct bacterial culture, 16S rRNA sequencing,
(OF) to screen the effect of probiotic bacteria on CLA in shotgun metagenomic sequencing, PCR probing for
curdled milk was proposed by Silva et al. [8]. The OF- specific genes, and chemical profiling of microbial
based methodology was validated by comparison with metabolites [80]. Results have shed light on develop-
GC-MS and showed comparable linearity, accuracy and mental changes in the composition of the GI microbiota
LODs, which were 1.92–2.56 mg for CLA methyl ester during infancy and childhood [81], the influence of diet
and oleic acid methyl ester, respectively (Table 4). It is on composition of the intestinal microbiota [82] and
important to highlight that, for this study, since the alterations in the bacterial phylotypes and altered
validation of a new method was the issue, other FOMs microbial profiles in humans with IBD [80].
(e.g., sensitivity, uncertainty and analytical error) were Omics technology combined with potent software
determined, besides LOD and linear range. systems, through high-throughput sequencing of 16S
More recently, NMR has also become a universal rRNA, may constitute a powerful tool to distinguish
method for lipid analysis, providing information about between species and to assess bacterial diversity in dif-
structures, and qualitative and quantitative analysis ferent communities and environmental niches within a
[76]; 1H- and 13C-NMR are indicated for elucidation of short period of time, an advantage as far as analytical
strategies are concerned [24]. For example, the 16S (e.g., 16S rRNA gene analysis) is unable to differentiate
rRNA PhyloChip is a high-density micro-array based on bacterial isolates. 92% specificity was obtained by
this technology, which has the ability to sample and to MALDI-TOF-MS in comparison to molecular analysis
detect species that represent as little as 0.01% of the (sequencing of tuf and 16S rDNA genes) in probiotic food
bacterial community, allowing high-resolution profiling and yoghurts [86].
of approximately 8500 bacterial taxa (defined as groups The large MetaHit consortium of European and Chi-
of organisms that share at least 97% 16S rRNA se- nese researchers developed a new approach, termed
quence identity) in a single experiment. It has been quantitative metagenomics, in order to characterize gut
successfully used to demonstrate the positive effect of microbial communities. A central element was the use of
L. casei subsp. rhamnosus GG (LGG) supplementation on the Illumina sequencing platform to create a reference
the infant gut microbiome [83]. The positive effect of catalogue composed of 3.3 million, non-redundant
LGG on children with high risk of atopic disease was due intestinal microbial genes [89], on which a high number
to not only its presence in high numbers but also the of short sequences generated from total stool DNA of an
impact of these high numbers on the modulation of individual are mapped, which enables determination of
the bacterial community structure. Confirmation of the the presence and the abundance of each catalogue gene
variation in relative abundance observed by the micro- harbored by that individual. Use of this approach has led
array in infant-stool samples was confirmed by inde- to detection of three robust gut enterotypes, character-
pendent quantitative qPCR analysis (n = 11) – an ized by different bacterial communities, to which
important step, if one considers that the 16S rRNA gene humans belong, spanned across geography, age, gender,
has a confined representativeness of the microbial gen- and disease [90]. These enterotypes will allow stratifi-
ome (represents only a small region), so inference of cation of individuals and assessment of the microbial
phylogenies from one gene alone has greater misclassi- communities associated with health and disease; several
fication possibilities. Regression analysis demonstrated a authors are of the opinion that these stratifications may
correlation between the two independent molecular be helpful in predicting individual responses to thera-
methods (r = 0.63; P<0.05), upholding the versatile peutics [90] and eventually to FFs containing probiotics
application capacity of this array. Such an approach has and/or prebiotics.
also enabled identification of new candidates as benefi- As understanding of microbial variation and corre-
cial microbes that, after further study and analysis, may sponding genetic parameters deepens, the information
be potential probiotic bacteria. For example, the probiotic generated may be applied to restructure the gut micro-
Faecalibacterium prausnitzii, which was identified by high- bial communities and their associated functions, thus
throughput sequencing of the 16S rRNA gene of the contributing to improvement of human health; specific
mucosa-associated microbiota of CrohnÕs patients was probiotic strains may provide missing microbial compo-
shown to prompt strong anti-inflammatory responses in nents with proved beneficial functions for the human
in vitro co-culture models and to protect against host, whereas prebiotics may increase the proliferation of
trinitrobenzene sulphonic acid-induced colitis in mice; probiotics or other beneficial bacteria, in order to maxi-
such results may ultimately lead to the use of this species mize sustainable alterations in the human microbiome
in healthy humans or IBD patients [84]. [79]. In this context, natural probiotic strains may be
Further limitations to the 16S rRNA sequencing ap- useful in FFs.
proach (e.g., nucleotide composition bias within the 16S In terms of food science, microarrays have contributed
rRNA gene which can lead to incorrect phylogenetic significantly to knowledge of food microbes [e.g., trans-
characterizations) may be overcome by metagenomics criptomics of food microbes during growth in stressful
using shotgun sequencing [24,85]. This technology se- environments and various foods (e.g., milk, cheese, and
quences the total community DNA directly from a yoghurt)] [24]. To study the impact of three lactic-acid
sample, providing information about both the phyloge- bacteria strains, with proven clinical functions, gene-
netic profile and the functional genes. Undoubtedly, 16S expression studies from the duodenum of humans were
rRNA sequencing is much cheaper if only community performed and results demonstrated that administration
profiling is required (yet limitations should be main- of L. plantarum can stimulate immunomodulatory re-
tained present), however metagenomic profiles are sponses [91].
essential for understanding the functions encoded in Metagenomics is without doubt a powerful analytical
those genomes. strategy even though it does require very complex
The use of MALDI-TOF-MS Biotype (MTB) has also computation, data-storage and handling procedures,
been used to detect, to identify and to characterize food and more sophisticated algorithms to help improve se-
products, including probiotic ones [86] and human quence assemblies in complex microbial communities
microbiota [87]. According to Emami et al. [88], MTB (e.g., the gut). An important aspect to consider in omics
supported by reliable, accessible bacterial databases be- technology is to link specific functional capacities to
comes a useful method when the molecular analysis specific microrganisms, so that vector recommendation
(food, supplement) or strain selection can be better tar- Independently of their degree of complexity, the overall
geted. Although metagenomics provides information objective is to enable characterization of food-matrix
about functional gene content in the gut, it is not known composition, to validate functionality associated with
whether the genes are actively expressed and have any bioactive compounds present, to increase knowledge on
functional role in a given sample. In order to obtain added value of food matrix and to identify all potential
information about active functions, it is necessary to applications.
look at the expression profile (metatranscriptomics) or In FDF, probiotics and prebiotics are among the most
the protein products (metaproteomics). These two tech- common ingredients employed. Their technological role
nologies are still technically demanding and have only and specific contributions in the FF need to be assessed
recently begun to be applied for the study of microbial by many the analytical strategies available to charac-
communities in the gut [92]. Metabonomics is also an terize:
important approach to couple to metagenomics if the (1) species/strains of the microflora, their viability/sur-
functional capacity of the gut is to be assessed. Metab- vivability and their metabolic action;
onomics can be described as the computational analysis (2) the biochemical events – glycolysis, proteolysis and
of spectral metabolic data (obtained by MS and NMR lipolysis.
spectroscopy platforms) in order to determine the time- Among these methods, there are those that have
specific metabolic changes in a complex system, thus undoubtedly accelerated knowledge and understanding
providing an important overview of the metabolic state of the complexities of food matrices, in particular, dairy
of the host [80]. matrices, and have simultaneously contributed to the
The many advances in omics strategies are promising validation of aspects important for human nutrition and
for the validation of FDFs discussed herein, including health. Despite its recent character, omics technology
biomarker discovery and health-promoting strategies. has greatly contributed to this endeavor, at both bacte-
Support for this statement is provided by recent omics rial and molecular levels. For example, by probing the
studies that have demonstrated the relevance of diet on proteome, peptidome and lipidome in food, specific
modulating gut microbiota. For example, the charac- markers can be identified that substantiate the specific
terization of fecal samples of 98 individuals revealed role of the probiotic or the prebiotic – namely by pro-
clustering of fecal communities into enterotypes strongly duction of specific bioactive peptides or polyunsaturated
correlated with long-term diets, with Bacteroides associ- acids (e.g., CLA or CLNA). The use of the proteomic and
ated with diets high in protein and animal fat versus peptidomic approach in the near future will certainly
Prevotella associated with carbohydrate-rich diets [93]. give more insight into FDFs, namely with respect to
In another perspective, Gordon and colleagues showed bioactive peptides produced by probiotic bacteria.
in a microbial profiling study that the ratio of Firmicutes/ Independently of the parameter being analyzed, ana-
Bacteroides is higher in obese than in lean individuals; lytical strategies need to be sound in order to guarantee
this trend was associated with a higher capacity for effectiveness, sensitivity, reproducibility and traceability.
polysaccharide fermentation [94]. FOMs are parameters that may indicate the extent of the
This new omics approach has opened many avenues quality of these strategies, and their assessment is re-
of understanding, and, as more studies are published, the quired to ensure the quality of results, even more so
symbiosis between gut microbiome and host will become during characterization and validation of FDFs. The
clearer. The impact of diet on the currently known availability of FOMs concerning the final results obtained
microbial communities needs to be further explored and by quality-control and quality-assurance procedures
properly designed FFs may find in these strategies should be strongly recommended as far as FDFs are
important answers to uphold their beneficial role. These concerned, since the FOM is a measure that may guar-
initiatives will continue to benefit the study of food mi- antee concordance between product composition and
crobes, among which probiotics are naturally included. functionality and wording on the associated label.
As the analytical approaches are further improved and
applied to food microorganisms and food matrices, not
4. Conclusions and future trends only will quality and nutritional value be assured but
also we expect that a better understanding of their
Nutrition plays an important role in promoting health beneficial aspects on gut microbiota and human health
and well-being. FDFs may help in this role, yet their will be achieved. That will naturally impact on the food
functionality needs to be characterized and maintained. industry, the scientific community and above all the
Several technologies are available toward this goal and consumer.
go from conventional physicochemical and chromato- Metabolic profiling is recognized as a powerful tool to
graphic/spectroscopic techniques to more advanced probe probiotic beneficial mechanisms further in both
new-generation methodologies, which rely highly on food and human host. The new omics approach has
molecular methods allied to MS and NMR analysis. enabled new knowledge on gut microbiome, and, as
more studies are published, the symbiosis between gut [24] S. OÕFlaherty, T.R. Klaenhammer, Annu. Rev. Food Sci. Technol.
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Trends in Analytical Chemistry, Vol. xxx, No.

Analytical strategies for

Method Brief description of technique Major achievement Advantages/Limitations Sample Ref.

Trends in Analytical Chemistry, Vol. xxx, No. x, 2012

may be meticulously identified and qualified, and verifi-

identity, in general [39], and for dairy products, in

particular [40], show physicochemical analysis, liquid

chromatography (LC) and gas chromatography (GC)

techniques with different detectors as some of the tradi-

accurate template quantification; Enables

L: Limited targets because of the limited

A: PFGE profile of whole genome with

traditional nutrients (i.e. proteins, fat, carbohydrates,

DNA can be quantified by calibration

number of available fluorescent dyes.

differentiate at subspecies and strain

and minerals) relies on basic chemical techniques, the

identification of more specific components (e.g., individ-

discriminatory power; able to

ual phenols, bioactive peptides, and polyunsaturated

Due to the complexity of food matrices, the use of

advanced analytical techniques able to generate a

compounds resulting from biochemical reactions,

performance LC (HPLC), coupled with several detectors,

subspecies and strain level.

specific target organisms.

matrices [15,41]. These techniques are also used in

combination, designated ‘‘hyphenated’’ techniques, to

analyze multiple components (i.e. HPLC-NMR, HPLC-

The analytical technique to be employed depends on

the target compounds and the matrix, where physico-

Amplification of the targeted DNA in

probes; Able to use specific-species

bacterial strains; Use of fluorescent

primers of gene or microorganism.

and CE); and,

fragments obtained by restriction

digestion resulting in fingerprint

(3) type of detector [ultraviolet spectroscopy (UV),

refractive-index detector (RID), fluorescence spec-

colysis, proteolysis and lipolysis in traditional dairy foods

2.2.1. Glycolysis. In the majority of dairy products (e.g.,

fermented milks or cheeses), glycolysis is mainly char-

acterized by degradation of lactose. Depending on LAB or

probiotic bacteria and their fermentation pathways,

lactose is degraded, originating small organic acids (e.g.,

lactic acid, propionic acid, citric acid, and acetic acid).

Study Procedure description Main results Ref.

Trends in Analytical Chemistry, Vol. xxx, No. x, 2012

Other sugars, also present in milk, are used as fermen-

– Bifidobacteria counts were determined by quantitative qPCR allow-

– Biochemical fermentation tests revealed that all strains were charac-

B. longum subsp. longum was able to confirm these species in the

– Effective method to evaluate the accuracy in the species-labeling in

ing the enumeration of both viable/culturable and unculturable;

commercial yoghurts by an independent culture technique.

by HPLC-UV and sugars (i.e. lactose, fructose and

with HPLC-UV and HPLC-RID; this may be explained by

the fact that the authors [42] used a classical approach

5. Carbohydrate fermentation patterns of the Bifidobacterium strains were

3. Bifidobacterium spp. determined by PCR were subjected to quantitative

being quantifiable, they may indicate the quality of the

process [51]. Despite the several advantages pointed out