Graphical Abstract
Food Hydrocolloids 2010,-,---Microalgae biomass interaction in biopolymer gelled systems
A.P. Batistaa,b,*, M.C. Nunesa, A. Raymundoa, L. Gouveiab, I. Sousac,
F. Cordob?sd, A. Guerrerod, J.M. Francoe
aN?cleo de Investiga??o de Eng.a Alimentar e Biotecnologia, Instituto Piaget, ISEIT de Almada,
Quinta da Arreinelade Cima, 2800-305 Almada, Portugal
bLaborat?rio Nacional de Energia e Geologia (LNEG), Unidade de Bioenergia, Estrada do Pa?o do Lumiar,
1649-038 Lisboa, Portugal
cInstituto Superior de Agronomia, DAIAT, Universidade T?cnica de Lisboa, Tapada da Ajuda,
1349-017 Lisboa, Portugal
dUniversidad de Sevilla, Facultad de Qu?mica, Dpto. Ingenieria Qu?mica, R. Prof. Garcia Gonzalez,
41012 Sevilla, Spain
eUniversidad de Huelva, Faculdad de Ci?ncias Experimentales, Dpto. Ingenier?a Qu?mica, Campus del Carmen,
21071Huelva, Spain
Contents lists available at ScienceDirect
Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd
Food Hydrocolloids xxx (2010) 1
FOOHYD1645_grabs ? 12 October 2010 ? 1/1
Pleasecitethisarticleinpressas:Batista,A.P.,etal.,Microalgaebiomassinteractioninbiopolymergelledsystems,Food Hydrocolloids(2010),
doi:10.1016/j.foodhyd.2010.09.018
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Microalgae biomass interaction in biopolymer gelled systems
A.P. Batistaa,b,*, M.C. Nunesa, A. Raymundoa, L. Gouveiab, I. Sousac, F. Cordob?sd,
A. Guerrerod, J.M. Francoe
aN?cleo de Investiga??o de Eng.a Alimentar e Biotecnologia, Instituto Piaget, ISEIT de Almada, Quinta da Arreinela de Cima, 2800-305 Almada, Portugal
bLaborat?rio Nacional de Energia e Geologia (LNEG), Unidade de Bioenergia, Estrada do Pa?o do Lumiar, 1649-038 Lisboa, Portugal
cInstituto Superior de Agronomia, DAIAT, Universidade T?cnica de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal
dUniversidad de Sevilla, Facultad de Qu?mica, Dpto. Ingenieria Qu?mica, R. Prof. Garcia Gonzalez, 41012 Sevilla, Spain
eUniversidad de Huelva, Faculdad de Ci?ncias Experimentales, Dpto. Ingenier?a Qu?mica, Campus del Carmen, 21071 Huelva, Spain
a r t i c l e i n f o
Article history:
Received 1 April 2010
Accepted 24 September 2010
Keywords:
Microalgal biomass
Spirulina
Haematococcus
Pea protein
kappa-Carrageenan
Starch
Gels
a b s t r a c t
Microalgae are an enormous biological resource, representing one of the most promising sources for the
development of new food products and applications. Pea protein/k-carrageenan/starch gels, interesting
vegetarian alternatives to dairy desserts, served as model systems to study the addition of microalgal
biomass, its effect, and subsequent rheological behaviour. Spirulina and Haematococcus gels presented
a markedly different rheological behaviour compared to the control mixed biopolymer gelled system.
The present goal is toclarify howthese microalgae affect the gelation and interact with each biopolymer
present in the complex mixed gel system. Hence, the aim of the present work is to study the effect of
Spirulina and Haematococcus microalgal biomass addition on the rheological behaviourof pea protein, k-
carrageenan and starch simple gels, as well as in pea protein/k-carrageenan and pea protein/starch
systems. The gelation process was monitored in-situ through dynamic oscillatory measurements
(temperature,timeandfrequencysweeptests)fora24hmaturationperiod,andrheologicalresultswere
supported with uniFB02uorescence optical microscopy observations. The addition of Spirulina and Haemato-
coccus to biopolymer gelled systems induced signiuniFB01cant changes in the gels? rheological behaviour and
microstructure.Ingeneral,itwasobservedthatthegellingmechanismisruledbythebiopolymers,while
microalgae seem to be embedded in the gel network acting as active particle uniFB01llers. The addition of
Haematococcus resulted in more structured gels in comparison to the control and Spirulina systems. In
the case of k-carrageenan gels, both microalgae induced a large increase in the rheological parameters,
which should be related to the high ionic content of microalgal biomass. Spirulina addition on starch
systems promoted a decrease in the gels? rheological parameters. This should be related to the starch
gelatinization process, probably by competing for water binding zones during the granules? hydration
process.
C211 2010 Published by Elsevier Ltd.
1. Introduction
Microalgae are an enormous biological resource, representing
one of the most promising sources for the development of new
products and applications. They can be used to enhance the
nutritionalvalueoffoodandanimalfeed,duetotheirwellbalanced
chemical composition.
Spirulina (Arthrospira) is a microscopic uniFB01lamentous prokaryotic
alga (cyanobacteria), which has been used in the human diet forat
least 700 years. Its origins date back to the Native Americans (lake
Texcoco,Mexico)andAfricans(lakeChade),whereitwasusedasan
alternative source of protein (protein content>50%). Nowadays, it
is broadlyused as a dietary food supplementworldwide, due to its
balanced composition in terms of essential amino acids, essential
fatty acids (e.g. g-linolenic acid), phycobiliproteins, vitamins (e.g.
B12) and minerals. Spirulina has between 15 and 25% of total
carbohydrates, with glycogen being the main storage sugar. Pepti-
doglycan, a polymer made of sugars (b-1,4-N-acetylglucosamine
and N-acetylmuramic acid) and amino acids, forms a mesh-like
layer outside the plasma membrane of the cyanobacteria, thus
forming cell walls similar to Gram positive bacterias (Belay, 2008).
Haematococcus pluvialis is a unicellular eukaryotic green alga
and the organism known to accumulate the highest level of
* Corresponding author at: N?cleo de Investiga??o de Eng.a Alimentar e Bio-
tecnologia, Instituto Piaget, ISEIT de Almada, Quinta da Arreinela de Cima, 2800-
305 Almada, Portugal. Tel.: ?351 21 294 62 50; fax: ?351 21 294 62 51.
E-mail address: pbatista@almada.ipiaget.org (A.P. Batista).
Contents lists available at ScienceDirect
Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd
0268-005X/$ e see front matter C211 2010 Published by Elsevier Ltd.
doi:10.1016/j.foodhyd.2010.09.018
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astaxanthin in nature (1.5e3.0%dw) (Todd-Lorenz & Cysewski,
2000). When green vegetative cells come across stress conditions
(e.g.nitrogendeuniFB01ciency,highlightintensity,saltaddition)thealga
rapidly differentiates into encysted cells which accumulate astax-
anthininfatglobules(usuallyesteriuniFB01edwitholeicacid)outsidethe
chloroplast. This carotenogenesis process induces large changes in
the microalgal cell composition, namely lowering the protein and
increasing the lipid contents. The encysted cell walls are mainly
composed of carbohydrates (hexoses), cellulose and proteins, and
the main storage sugar within the cell is starch. It has been sug-
gested that the accumulated astaxanthin might function as
aprotectiveagentagainstoxidativestressdamage(Kobayashietal.,
1997). This carotenoid pigment has been primarily used for uniFB01sh
(e.g. salmon, trout) and shrimp pigmentation in aquaculture.
Currently, the primary Haematococcus production target is focused
in its potential nutraceutical use.
Freeze-dried biomass of various microalgae has been success-
fully used as a colouring agent and as a source of u3 poly-
unsaturated fatty acids in model food products developed by our
research group, such as oil-in-water emulsions (Gouveia, Batista,
Raymundo, Sousa, & Empis, 2006; Raymundo, Gouveia, Batista,
Empis, & Sousa, 2005), vegetable puddings (Batista, Gouveia,
Nunes, Franco, & Raymundo, 2008; Gouveia, Batista, Raymundo,
& Bandarra, 2008), biscuits (Gouveia, Batista, Miranda, Empis, &
Raymundo, 2007; Gouveia, Coutinho, et al., 2008) and pasta
(Fradique et al., 2010). It has been observed that besides colouring
and nutritional purposes, the addition of microalgal biomass to
food systems imparts signiuniFB01cant changes in its microstructure and
rheological properties.
Most food products have a complex composition, including at
least three biopolymer types whose interaction determines the
products? structural and mechanical properties (Tolstoguzov,
2003). The impact of microalgae addition in different food
matrixes reuniFB02ects their interactions with other food components
such as biopolymers (e.g. proteins and polysaccharides).
Recently, pea protein/k-carrageenan/starch gel systems have
been extensively studied as an interesting alternative to dairy
desserts(Nunes,Raymundo,&Sousa,2006a,2006b).Recentresearch
(Batista,Gouveia,etal.,2008),hasshownthatthesebiopolymergels
served as model systems to study the effect ofuniFB01ve different micro-
algae on the rheological behaviour of these gels. Spirulina and Hae-
matococcus gels presented very different rheological properties
comparedtootheralgae(ChlorellavulgarisandDiacronemavlkianum)
and the control gel. Haematococcus promoted a structural rein-
forcement expressed by improved rheological properties, while the
addition of Spirulina promoted a reduction in the rheological
parameters of the gels. To continue with this research, this study?s
goal is to clarify how these two microalgae inuniFB02uence and interact
witheachbiopolymerpresentinthecomplexmixedgelsystemepea
protein, k-carrageenan and starch in aqueous media through
dynamicrheologyandmicroscopymeasurements.
2. Materials and methods
2.1. Microalgae production
Spirulina maxima (Sp) and Haematococcus pluvialis (Hp) micro-
algae were produced at the LNEG e Bioenergy Unit (Lisbon). The
microalgae were cultivated in appropriate growth media (Vonshak,
1986), in airlift bioreactors with bubbling air, under low light
conditions (150mEmC02sC01), and optimal growth temperatures (Sp:
34C14C; Hp: 25C14C). Spirulina was harvested during the stationary
phase,whileHaematococcuswasuniFB01rstsubmittedtoacarotenogenesis
process by nitrogen starvation, NaCl addition, and high luminosity
enhancedbyculturedilution(Gouveia&Empis,2003),beforebeing
harvested. Microalgal biomass was harvested by simply stopping
agitation,concentratedbycentrifugationandfreezedrying.
The gross chemical composition (%dw) of the microalgal
biomass is (Batista, Gouveia, et al., 2008):
- Sp: 44.9% protein; 3.6% fat; 16.6% carbohydrates; 30.9% ashes
(2.1% K; 0.8% Ca; 0.3% Mg; 7.0% Na);
- Hp: 10.2% protein; 40.7% fat; 33.6% carbohydrates; 8.9% ashes
(0.9% K; 0.2% Ca; 0.2% Mg; 5.5% Na).
2.2. Gel preparation
Pea protein isolate (Pisane F9C210, Cosucra, Belgium), k-carra-
geenan(SatiagelAMP45C210,Degussa,France)andnativemaizestarch
(Vitena AC210, Copam, Portugal) biopolymers were used to study
microalgae interactions in gelled systems. All these ingredients
were kindly provided by the respective manufacturers.
Biopolymer model systems were prepared at a concentration
near to the critical gelling concentration for each biopolymer: pea
protein 12%, k-carrageenan 0.75%, starch 5.0% (all percentages are
w/w).Peaprotein(4%)/k-carrageenan(0.15%)andpeaprotein(6%)/
starch (3.75%) binary systems were also studied.
Microalgal biomass was added to these systems in the same
proportion for each biopolymer as in a mixed geluniFB01nal formulation
proposedandoptimizedinpreviousstudies(Batista,Gouveia,etal.,
2008; Nunes et al., 2006a, 2006b): 4% pea protein isolate, 0.15% k-
carrageenan, 2.5% maize starch, 0.75% microalgal biomass. Accord-
ingly,thecompositionsofthesystemspresentlystudiedwere:
- pea protein 12%?microalga 2.25% (pH: C 6.5eSp 8.3eHp 6.6);
- k-carrageenan 0.75%?microalga 3.75% (pH: 8.6e9.0e6.6);
- starch 5%?microalga 1.5% (pH: 5.7e8.8e6.1);
- pea protein 4%?k-carrageenan 0.15%?microalga 0.75% (pH:
6.9e6.8e6.7);
- pea protein 6%?starch 3.75%?microalga 1.125% (pH:
6.7e8.3e6.7).
The ingredients were dispersed in demineralised water by
mechanical agitation (300rpm, 1h) at room temperature. No
adjustmentsweremadetothenaturalpHofthesystems(indicated
aboveforcontroleSpeHp) and no salts wereadded tothe systems,
inorderfortheioniccontentofthedispersionstoonlybeduetothe
salts present in the biopolymers and microalgae.
2.3. Rheological measurements
Thegelationprocesswasmonitoredin-situinacontrolledstress
rheometer (RS-300, Haake, Germany) coupled to an UTCePeltier
system, using cone-plate geometry (C35/2C14), through dynamic
small amplitude oscillatory shear measurements (SAOS). The
samples were heated from 20C14C to 90C14C, maintained at this
temperature for 5min, and then cooled down to 5C14C, at 1C14C/min
(u?6.28rad/s), according to previously optimized gel setting
conditions for these systems (Batista, Nunes, et al., 2008; Batista
et al., 2009). Subsequently, time sweep tests were conducted at
5C14Cduring24h(u?6.28rad/s)followedbyfrequencysweeptests
(u?0.01e111.7rad/s). All tests were carried out at stress values
within the linear viscoelastic region. The samples were covered
with parafuniFB01n oil to prevent moisture loss.
2.4. Fluorescence microscopy
The gels were analysed by uniFB02uorescence microscopy using an
Olympus BX61 optical microscope in epiuniFB02uorescence mode with
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4C2and 10C2(UPlanApo)objectives lenses. A Mercuryarclamp was
used as an excitation source and DAPI, TRITC, FITC uniFB02uorescence
uniFB01lter sets were used to select different excitation and emission
wavelengths.
After thermal treatment, samples were poured between
a microscope slide and a coverslip, sealed with varnish to prevent
dehydration,andkeptinarefrigeratorat5e7C14Cfora24hperiodto
allow gel maturation.
Spirulina, Haematococcus and pea protein presented auto-
uniFB02uorescence.Starchwasnon-covalentlystainedwithRhodamineB
(Sigma), in the absence of pea protein. Rhodamine was added
during the mechanical dispersion of the biopolymers at a 0.002%
concentration level.
3. Results and discussion
3.1. Microalgaeepea protein systems
Pea protein dispersions at 12% appear to be near the limiting-
concentration for gelling to occur (Batista, Portugal, Sousa, Crespo,
& Raymundo, 2005; Nunes, 2005). In some experiments (repli-
cates), the gel formed was sometimes weaker and at other times
stronger suggesting that at this concentration level the gels were
Fig.1. EvolutionoftheviscoelasticfunctionsG0 (closedsymbol)andG00 (opensymbol),
of 12% pea protein dispersions along thermal treatment (T e line).
Fig. 2. Fluorescence microscopy images of 12% pea protein gel systems (aeb) with 2.25% Spirulina (ced) and Haematococcus (e) microalgal biomass addition (DAPI uniFB01lter).
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not formed in a systematic manner. This has also been reported by
Batistaetal.(2005)whenpeaproteindispersionsat12.5%gelledin
therheometerdevicebut notinthe refrigerator.Fig.1presents the
evolution of the viscoelastic moduli along thermal treatment for
differentreplicasof12%peaproteindispersionsamples.Adecrease
inG0 andG00,whenheatingthesamplesfrom20C14Cupto90C14C,was
followed by an increase when cooling to 5C14C. This is the general
behaviour of gels from globular protein systems, as reported by
several authors (e.g. Van Vliet, Martin, & Bos, 2002). However, it is
clear that different sol-gel transition (G0>G00) temperatures were
found for replicates of the same material system. In one replicate,
the transitionwas identiuniFB01ed at 73C14C, whereas in another replicate
the transition was detected at only 7.3C14C, resulting in a much
weaker gel. This can be conuniFB01rmed by the microscope photographs
in Fig. 2, where one image (Fig. 2a) shows a continuous dense
network of protein aggregates (bright areas) and the other
a discontinuous and disrupted structure (Fig. 2b).
When Spirulina was added to the above system, a slight rein-
forcementofthepeaproteingelstructurewasobserved(Fig.2ced),
i.e. the brighter density of the red areas on the picture, especially
when comparing Fig. 2bwith Fig. 2d. This reinforcement of the gel
structure is conuniFB01rmed by the rheological results shown in Fig. 3b,
where G0 and G00 values are slightly higher for the pea protein/
Spirulina binary gel. In general, the microalgae appear to be
embedded (red colored) within the protein network and much
denser in the case of Haematococcus (Fig. 2e).
Adding Haematococcus causes a much more pronounced rein-
forcement of gel strength, as can be observed from the rheological
data from Fig. 3. In general, gel maturation kinetic curves are
characterized by a marked increase in G0 in the uniFB01rst few hours,
followed by a slower evolution of this modulus along time. This
behaviour is typical of biopolymer gelation processes (Clark,
Kavanagh, & Ross-Murphy, 2001), where there is a continuous
reorganizationofthegelnetworkduetotheformationandrupture
of entanglements between the polymeric chains of the macro-
molecules. At the end of the 24h maturation period the pea pro-
teineHaematococcus system presented a G0 value around 2000Pa,
oneorderofmagnitudehigherthanthepeaproteinsimplegeland
thepeaproteineSpirulinagelsystem(Fig.3a).Theseresultsarewell
supportedbythemechanicalspectra(Fig.3b),whereitisobserved
that, in all cases, G0 is higher than G00 although a stronger gel is
obtained by adding Haematococcus. However, the SAOS functions
frequency dependence is not modiuniFB01ed by microalgae addition,
indicating that pea protein is the dominant biopolymer in the gel
network.
The impact of microalgal biomass addition can be related to
modiuniFB01cationsinthepHandsaltcontentofthegels,byaffectingpea
protein electrostatic interactions during the aggregation mecha-
nism. These should be more relevant in the case of Spirulina
biomass which presents higher ionic content and a higher pH.
However, pH values are always above pea protein isoelectric point
(4.5), not affecting the global charge of this biopolymer which
Fig. 3. (a) Temperature and maturation kinetics curves (a) and mechanical spectra (b)
at 5C14C, of 12% pea protein gel systems (-) with 2.25% Spirulina (C) and Haemato-
coccus (:) microalgal biomass addition. G0 (closed symbol), G00 (open symbol).
Fig. 4. Maturation kinetics curves (a) and mechanical spectra (b) at 5C14C, of 0.75% k-
carrageenan gel systems (-) with 3.75% Spirulina (C) and Haematococcus (:)
microalgal biomass addition. G0 (closed symbol), G00 (open symbol).
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remains negative. In the case of Haematococcus, the structural
reinforcement action could also be related to its high fat content
(41%)(Batista,Gouveia,etal.,2008).TheinuniFB02uenceoffatcontenton
gelling behaviour has been studied in milk gelled systems, such as
acidmilk gels (Houz?, Cases,Colas, &Cayot,2005; Lucey,Munro, &
Singh, 1998) and dairy custard model systems (V?lez-Ruiz,
Gonz?lez-Tom?s, & Costell, 2005) from which it was concluded
that using full fat milk rather than skimmed milk resulted in
strongergels.ThemodiuniFB01cationontherheologicalpropertiesoffat-
containing gels is usuallyattributed to fat droplets acting as active
uniFB01ller particles embedded in the protein matrix.
3.2. Microalgaeek-carrageenan systems
k-Carrageenan forms a very weak gel at 0.75% which should be
near its critical gelling concentration (Nickerson & Paulson, 2004;
Nunes, 2005) in the absence of added ions. The solegel transition
ofk-carrageenandispersionsischaracterizedbyasharpincreasein
G0 upon cooling, and in parallel with a marked decrease in phase
angle (d) (Nunes, 2005). For the k-carrageenan 0.75% system, this
transition occurred in the 5e7C14C temperature range. However,
when adding Spirulina and Haematococcus the gelation tempera-
tureincreased to 30C14C and 25C14C, respectively(results notshown).
This means that gel formation occurs earlier during the cooling
process, making it possible to obtain k-carrageenan gels at room
temperature through the addition of microalgae biomass. In all
cases, the resulting microalgaeek-carrageenan maturated gels
presented impressively enhanced rheological properties, with G0
values two orders of magnitude higher than the simple k-carra-
geenan gel (Fig. 4a). However, Haematococcus gel maturation
curves are not typical, showing a decrease in G0, probably because
the gel was too hard and brittle, disturbing the rheological data
measurement. The mechanical spectra of these systems is pre-
sented in Fig. 4b, conuniFB01rming the strong nature of the gel imparted
by both microalgae.
k-Carrageenan gels are formed through intermolecular associ-
ation (junction zones) of double helices into stable structured
aggregates (Morris, Rees, & Robinson, 1980). The formation and
Fig. 5. Fluorescencemicroscopyimagesof0.75% k-carrageenangelsystems with3.75%Spirulina (aeb)andHaematococcus(ced) microalgalbiomassaddition(DAPI&TRITCuniFB01lters).
Fig. 6. EvolutionofG0 andG00 alongthermaltreatmentand24hmaturationat5C14C(a);
and mechanical spectra (b), of 5% starch gel systems (-) with 1.5% Spirulina (C) and
Haematococcus(:)microalgalbiomassaddition.G0 (closedsymbol),G00 (opensymbol).
T (line).
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aggregation of these double helices is induced by cooling and is
highly dependent on the presence of electrolytes even at very low
concentrations (Morris et al.,1980; Rochas & Landry,1987). Potas-
sium cations (K?) have a speciuniFB01c afuniFB01nity to k-carrageenan and are
usually the most effective in inducing gelliuniFB01cation, followed by
divalentcationssuchasCa2?andthenbyothermonovalentcations
such as Na? (Chen, Liao, & Dunstan, 2002).
Microalgal biomass contains signiuniFB01cant amounts of minerals
(see Section 2.1), derived from its intrinsic chemical composition
and from culture media residues. Systems with 0.75% k-carra-
geenan and 3.75% microalgal biomass include 0.08% K? (0.02M),
0.03% Ca2? (0.01M) and 0.26% Na? (0.1M) for Spirulina and 0.03%
K? (0.01M), 0.01% Ca2? (0.002M) and 0.21% Na? (0.1M) for Hae-
matococcus. This can explain the contribution of microalgal addi-
tion tothe hugeincrease in k-carrageenangelsrheological moduli.
This is in agreement with previous research conducted by other
authors, such as Chen et al. (2002) where the formation of very
weakk-carrageenangelswasobservedforconcentrationsbetween
0.7%and1.4%intheabsenceofsalts.Aftertheadditionoflowlevels
of potassium and calcium, the gels rheological moduli (G0, G00)
increased considerably, indicating a higher degree of gel structure.
k-Carrageenan/microalgae gel structures can be observed in
Fig. 5. k-Carrageenan was not detected in the uniFB02uorescence micro-
scope since there is no uniFB02uorescence marker available for
non-covalent staining. It is however possible to observe the k-car-
rageenanemicroalga systems, since Hp and Sp present auto-
uniFB02uorescence enabling k-carrageenan to be observed by contrast
(darkareas).TheimagesinFig.5aandcwereobtainedwithaDAPI-
UVuniFB01lter,andtheonesinFig.5banddwithaTRITCuniFB01lter.Thelatter
enables the collection of light emitted by the sample at higher
wavelengths, enhancing the contrast when microalgae are present
in the gel system.
3.3. Microalgaeestarch systems
A marked increase in G0 was observed by heating 5% starch
dispersions to 66.6C14C, corresponding to starch gelatinization
(Fig. 6a). This phenomenon is related to the swelling of the starch
Fig. 7. Fluorescence microscopy images of 5% starch gel systems (a) with 1.5% Spirulina (b) and Haematococcus (c) microalgal biomass addition (DAPI uniFB01lter).
Fig. 8. Maturation kinetics curves (a) and mechanical spectra (b) at 5C14C, of 4% pea
proteine0.15% k-carrageenan gel systems (-) with 0.75% Spirulina (C) and Haema-
tococcus (:) microalgal biomass addition. G0 (closed symbol), G00 (open symbol).
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granules, and subsequent amylose solubilization, which causes an
increase in the system?s viscosity (Morris, 1990). The addition of
HaematococcusandSpirulinatothissystemcausesaslightincrease
in the gelatinization temperature to 70.2C14C and 73.8C14C, respec-
tively. This indicates that microalgal biomass interferes with the
starch gelatinization process perhaps by competing for water
binding sites during the granules? hydration process.
In the case of Haematococcusestarch systems, this setback was
overcome in the cooling ramp, resulting in a strongly maturated
gel,withG0andG00valueshigherthanthesimplestarchgel(Fig.6a).
This can be related to a concentration effect of starch as a result of
exclusion from the microalgae domain. It is also expected that
Haematococcus high fat content (41%) can play a signiuniFB01cant role in
the gels? rheological properties, as discussed previously for micro-
algaeepea protein systems (Section 3.1).
Alternatively, Spirulinaestarch systems, presented much lower
G0 values than the control sample. This is in accordance with
previous studies which reported the formation of weaker gels
when Spirulina was added to pea protein/k-carrageenan/starch
mixed gels, particularly for faster gel setting conditions (Batista,
Gouveia, et al., 2008; Batista, Nunes, et al., 2008). Microscope
images (Fig. 7) show large particles of microalgae that could
imprintdiscontinuitiesinthestarchnetwork,especiallyinthecase
ofSpirulina,leadingtoamorefragilegelstructure.Unliketheother
microalgae studied by the authors (Batista, Gouveia, et al., 2008)
Spirulina is a cyanobacteria, and therefore its prokaryotic cells lack
a rigid cell wall which could lead to higher water absorption rates
by its cellular components (mainly proteinaceous), destabilizing
the starch gelation mechanism.
3.4. Microalgaeepea proteinek-carrageenan systems
Peaproteinek-carrageenanbinarysystemsformwellstructured
gels, at low concentrations for both biopolymers, suggesting
a synergistic effect. The rheological behaviour of the mixed gel is
similar to the simple k-carrageenan gel, namely the gelation
temperature at 7.3C14C, the shape of the maturation curves and
mechanical spectra (Fig. 8). This suggests that k-carrageenan
Fig. 9. Fluorescence microscopy images of 4% pea proteine0.15% k-carrageenan gel systems (a) with 0.75% Spirulina (b) and Haematococcus (c) microalgal biomass addition (DAPI
uniFB01lter).
Fig. 10. Evolution of G0 and G00 along thermal treatment and 24h maturation at 5C14C
(a); and mechanical spectra (b), of 6% pea proteine3.75% gel systems (-) with 1.125%
Spirulina (C)and Haematococcus (:) microalgal biomass addition. G0 (closed symbol),
G00 (open symbol), T (line).
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doi:10.1016/j.foodhyd.2010.09.018
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constitutes the continuous phase of the mixed system, which is in
accordance with the uniFB01ndings from Nunes (2005) regarding pea
protein and k-carrageenan as being phase separated, forming two
independent networks dispersed in each other.
Rheologicalresults(Fig.8)forpeaprotein/k-carrageenansystems
aresurprising,sincetheadditionofmicroalgaetothisbinarysystem
results inweaker gels, as opposed to their behaviour when applied
separatelyreinforcingeachbiopolymergelindividually.Microscope
images(Fig.9)suggestthatphasesegregationbecomesmoreevident
upon microalgae addition, negatively affecting the biopolymers?
interaction. Therefore, proteinepolysaccharide thermodynamical
incompatibility seems to be affected by the presence of microalgal
biomass, which may be related to the volume exclusion effect as
a consequence of macromolecular competition for space in the
solution (Tolstoguzov, 2003). This could have a negative impact on
the gel structure considering the low biopolymer levels (4% pea
protein,0.15%k-carrageenan,0.75%microalga)usedinthisstudy.
3.5. Microalgaeepea proteinestarch systems
Pea proteinestarch binary systems also formed well structured
gels, at low concentrations for both biopolymers, suggesting
asynergeticeffect.Whenheatingthesesystemsfrom20C14Cto90C14C,
a marked increase in G0 is observed at 73.8e75.6C14C (Fig.10a). This
temperature corresponds to the starch gelatinization temperature,
which is higher compared to the simple 5% starch gel (66.6C14C), as
reportedinSection3.3.TheeffectofpeaproteinorSpirulina(Section
3.3)additiontostarchgelsischaracterizedbysimilargelatinization
temperatures. This suggests that hydration of starch granules? is
impaired by pea protein and Spirulina to the same extent, demon-
strating that Spirulina?s protein fraction should be competing with
starchgranulesforhydrationasinSpirulinaestarchgellingsystems.
Pea protein/starch systems presented stronger gels in the
presence of both microalgae. Haematococcusepea proteinestarch
systems presented starch gelatinization at 73.8C14C (no effect from
Hp addition) and resulted in a stronger gel with increasing
viscoelastic functions (even after 24h maturation) (Fig. 10). The
additionofSpirulinaretardedstarchgelatinizationto81e83C14C,and
the gel seems fully maturated before the 24h period, presenting
higher G0 values when compared to the control gel.
FromSection3.3,adestabilizingeffectwasnotedafterSpirulina
addition to the starch gel. However, the pea proteinestarch
synergistic effect in microalgaeepea proteinestarch systems seem
to be stronger than the Spirulinaestarch destabilizing mechanism,
resulting in gels with higher viscoelastic properties (Fig.10).
Microscope images (Fig.11) present densely packed continuous
structures, which are in accordance with the rheological results.
From these results it seems clear that proteinepolysaccharide
interaction plays a dominant role on the development of the gel
microstructure, and will determine the extent and direction of the
microalgae addition effect.
4. Conclusions
The addition of Spirulina and Haematococcus to biopolymer
gelled systems, induced signiuniFB01cant changes in the gels? rheological
behaviour and microstructure. Haematococcus induced a general
structuralreinforcementactionwhichmayberelatedtoitshighfat
content (41%) (Batista, Gouveia, et al., 2008), considering that fat
dropletscanactasactiveuniFB01llerparticlesembeddedinthegelmatrix
asobservedformilkgelledsystems.Spirulinaadditionalsoresulted
in stronger gel systems (but always lower than Haematococcus)
exceptin the case of starch, whereit seems to negativelyaffect the
gelatinization process. This could be related to competition for
water binding sites by Spirulina protein molecules, hindering the
hydration of starch granules.
For all the systems studied, it was observed that protein and
polysaccharide biopolymers e alone or in binary combinations e
are responsible for the formation of the gel structure and resulting
rheological behaviour. In most cases, microalgae seem to be
embedded in the gel?s network, causing denser microstructures
with improved rheological parameters. However, the fact that
Fig. 11. Fluorescence microscopy images of 6% pea proteine3.75% starch gel systems (a) with 1.125% Spirulina (b) and Haematococcus (c) microalgal biomass addition (DAPI uniFB01lter).
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doi:10.1016/j.foodhyd.2010.09.018
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microalgalbiomasscontainssigniuniFB01cantamountsofionsmeansthat
this should also have a signiuniFB01cant impact on the biopolymers?
interaction associated with the gelling mechanism, particularly in
the case of k-carrageenan gels.
Fluorescence optical microscopy proved to be a simple and
effective technique to observe these materials? microstructures,
with microalgae being easily detected due to their natural
pigments? autouniFB02uorescence.Themicroscopyresultscorrelatedwell
and supported the rheological uniFB01ndings.
Acknowledgements
This work is part of a research project ?Pigments, antioxidants
and PUFA?s in microalgae based food products e functional implica-
tions? (PTDC/AGR-ALI/65926/2006) sponsored by Funda??o para
a Ci?ncia e a Tecnologia (FCT). A.P. Batista acknowledges the PhD
research grant from FCT (SFRH/21388/BD/2005), and Patricia Fra-
dinho for Technical Support.
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