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Nutrition & Food ScienceVol. 38 No. 5, 2008pp. 492-501# Emerald Group Publishing Limited0034-6659DOI 10.1108/00346650810907010

Spirulina maxima andDiacronema vlkianum

microalgae in vegetable gelleddessertsLuısa Gouveia

Instituto Nacional de Engenharia e Tecnologia Industrial – INETI-DER –Unidade Biomassa, Estrada do Paco do Lumiar, Lisboa, Portugal

Ana Paula Batista and Anabela RaymundoInstituto Piaget, Centro de Investigacao em Eng Alimentar e Biotecnologia,ISEIT de Almada, Quinta da Arreinela de Cima, Almada, Portugal, and

Narcisa BandarraDepartamento de Inovacao tecnologica e Valorizacao dos Produtos da Pesca,Instituto de Investigacao das Pescas e do Mar – IPIMAR, Lisboa, Portugal

Abstract

Purpose – The purpose of this paper is to develop microalgae vegetable-based gelled desserts(similar to ‘‘dairy desserts’’) prepared with pea protein isolate, Spirulina maxima and Diacronemavlkianum biomass, rich in essential fatty acids (omega-3 polyunsaturated fatty acid’s (PUFA)). Theeffect of microalgae concentration and gelling temperature on the colour, texture and fatty acid profileof the gels was investigated.Design/methodology/approach – Pea protein isolate (4 per cent), �-carrageenan (0.15 per cent)and starch (2.5 per cent) were used to produce a gelled dessert that was further enriched withmicroalgal biomass (0.1-1.0 per cent). The gels were prepared using different temperatures, rangingfrom 75 to 90 �C. Colour, total pigments (carotenoids), texture and fatty acid profile of the gels wereevaluated.Findings – The addition of Diacronema and Spirulina microalgal biomass to vegetable gelleddeserts, results in a novel alternative food product, with PUFA’s (e.g. EPA, DHA and GLA), withfavourable texture characteristics, particularly for Diacronema, due to the structural reinforcement,representing a new food market niche.Practical implications – Nowadays there is an increasing consumer demand for more natural foodproducts, presenting health benefits. Microalgae are able to enhance the nutritional content ofconventional food and hence to positively affect humans health due to their original chemicalcomposition, namely as a source of long chain omega-3 PUFA.Originality/value – As far as the authors are aware the use of these microalgae in gelled desserts, inassociation with vegetable proteins, is an innovative food application.

Keywords Micro-organisms, Food products

Paper type Research paper

IntroductionRecent trends to efficiently develop food products with higher quality and morehealthful, leads to the use of vegetable proteins as an alternative to animal ones. The

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This work is part of a research project ‘‘Pigments, antioxidants and PUFA’s in microalgae-basedfood products – functional implications’’ (PTDC/AGR-ALI/65926/2006) sponsored by thePortuguese Foundation for the Science and Technology (‘‘Fundacao para a Ciencia e aTecnologia’’ – FCT). A.P. Batista acknowledges the PhD research grant from FCT (SFRH/21388/BD/2005). The authors thank Ricardo Antunes and Daniel Oliveira for technical support.

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current trend is to use natural ingredients with functional properties, such asmicroalgae rich in natural colourings, antioxidants and omega-3 polyunsaturated fattyacids (PUFAs).

Vegetable proteins, namely pea protein isolate, present good functional properties,including gelling, emulsifying, fat-absorbing and water-binding properties, and havealready been successfully tested by the authors in oil-in-water food emulsions(Raymundo et al., 2002) and gelled desserts (Nunes et al., 2003, 2006a, b). These proteinspresent some additional advantages such as no cholesterol, positive agriculturalimpact and expectable superior microbiological quality.

Microalgae biomass have also been studied by the authors as a source of naturalcolourings and fatty acids, in a wide range of food products, such as oil-in-wateremulsion (Raymundo et al., 2005; Gouveia et al., 2006), biscuits (Gouveia et al., 2007,2008a) and food gels (Batista et al., 2007) with success. Natural antioxidants, such ascarotenoids, are now thought to possibly prevent the incidence of cancers,cardiovascular and age-related diseases, by protecting cells against oxidative damage(Pszczola, 1998; Mazza, 1998; Downham and Collins, 2000).

Omega-3 PUFA positively affect human health, especially in the prevention ofcardiovascular diseases and cancer, reduction of blood cholesterol (Bønaa et al., 1990),atherosclerosis (Thies et al., 2003), diabetes, as well as improvement of properdevelopment and function of the brain and retina (Connor, 2000; Crawford, 2000).Usually, these compounds are provided by fish and fish oils, but global fish stocks aredeclining due to general over fishing and overly efficient fishing methods, and the oilsderived from fish are sometimes contaminated with a range of pollutants, heavy metalsand toxins. Alternative sources of PUFA’s are clearly desirable, and some microalgae,which synthesize these fatty acids, are particularly attractive.

Without radical changing of eating habits, an alternative of increase PUFA’s dailyconsumption is the use of traditional foods, largely consumed, with the incorporation ofmicroalgae biomass, as vehicles to high impact functional ingredients like PUFA’s,carotenoids, antioxidants, fibers and oligoelements.

Microalgae cultures can extend into areas and climates unsuitable for agriculturalactivities (including deserts and seashore areas) showing no season dependence andmost of all they can contribute to reduce the greenhouse effect by CO2 biofixation andrecycling of fixed C in their cells, contributing to avoid climate alterations.

MethodologyMicroalgaThe microalga Diacronema vlkianum (Di) used in this study was obtained from MaryPark collection and maintained in IPIMAR since 1973. The microalga was cultivated inWallertein’s associated with Miquel’s medium enriched seawater in the ratio 3:1.Spirulina maxima (LB 2342) (Sp) was cultivated in INETI in the appropriategrowth medium (Vonshak, 1986). Both microalgae were cultivated in plastic airliftbioreactors with bubbling air, at low light conditions (150�E m�2 s�1), 24 h light, and25 �C temperature. Biomass was recovered in the stationary growth phase withoutflocculation by simply stopping agitation, concentrating by centrifugation and freezedrying.

Gelled dessertsThe gels were prepared using a formulation previously optimised by Nunes et al.(2006a, b): 4 per cent (w/w) pea protein isolate (Pisane H9w, Cosucra, Belgium), 0.15

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per cent (w/w) �-carrageenan (Satiagel AMP45w, Degussa, France), 2.5 per cent (w/w)native maize starch (Vitena Aw, Copam, Portugal) and 15 per cent sugar. All theseingredients were kindly provided by the respective manufacturers.

Microalgal biomass was added at 0.1, 0.25, 0.5 and 0.75 per cent (w/w) concentrationlevels. A control sample without algal addition was also prepared.

The ingredients were dispersed in deionised water, under magnetic stirring(300 rpm, 1 h) at room temperature. The gels were prepared using differenttemperatures, ranging from 75 to 90 �C, for 5 min. The samples were placed incylindrical glass containers (33 mm diameter and 33 mm height) and allowed to set at5 �C in a refrigerator, being colour, carotenoids, texture and fatty acids measured after24 h maturation.

Gels analysisColour. The gels colour was measured instrumentally with a Minolta CR-300 (Japan)tristimulus colorimeter which was calibrated using a white standard porcelain plate(Y¼ 92.7, x¼ 0.3161, y¼ 0.3322).

Food gels are translucent materials that are seen partially by reflected light andpartially by transmitted light that passes through them. These samples require specialhandling since any variations in their thickness ( path length) or background will affecttheir colour (Good, 2003). To overcome this, a fixed amount of sample (10 mm height)was poured into a measuring cup, which was then surrounded with a black paper strip,against a white backing. The measurements were conducted at 20 �C under the samelight conditions, and replicated six times (50 mm2 measuring area per measurement).

The results were expressed in accordance to the CIELAB uniform colour systemwith reference to standard illuminant D65 (average daylight conditions) and a visualangle of 2� (C.I.E., 1978). The parameters determined were L* (lightness), a*(greenness/redness) and b* (blueness/yellowness). The total colour difference betweensamples with different gelling temperatures and different microalgal biomassconcentrations were calculated according to the following equation:

�E ¼ ½ð�L�Þ2 þ ð�a�Þ2 þ ð�b�Þ2�1=2

Total carotenoids. Total pigment content (dry weight basis) of the gels was determinedspectrophotometrically after extraction with acetone and expressed on a dry basisusing extinction coefficients (E1%

1 cm¼ 2,150) at their absorption maximum in acetone(Gouveia et al., 1996). Microalgae biomass total pigment content was also determined,being 2.5 per cent for Di and 0.9 per cent for Sp.

Texture. The gels texture was measured objectively using a texturometerTA.XTplus (Stable MicroSystems, UK) in penetration mode with a cylinder probe of10 mm diameter, plunged 7 mm at 0.5 mm/s, with 5 s waiting time between penetrationcycles. Firmness was considered as the height of the force peak during the firstcompression cycle of the force vs time curve, and was the parameter with betterdiscriminating ability to compare this type of gels (Nunes et al., 2003). Measurementswere done in triplicate.

Fatty acid profile. Fatty acid methyl esters were prepared according to Lepage andRoy (1986) modified by Cohen et al. (1988). The fatty acid methyl esters preparationwas carried out using 300 mg lyophilized sample (Sp, Di and gels) and 5 ml of theacetylchloride:methanol mixture (5 per cent v/v). The esterification was done at 80 �Cduring 1 h. After cooling, 1 ml of water and 2 ml of n-heptane were added to the

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mixture, which was stirred and centrifuged at 2,000 g for 5 min. The organic phasewas collected, filtered and dried with anhydrous sodium sulphate. The solvent wasremoved under nitrogen and the methyl esters solubilised with n-heptane to aconcentration of 100 mg/ml. Fatty acid methyl esters were separated and quantifiedusing a CP-3800 GC (Varian, USA) equipped with 30 m DB-WAX ( J&W, Agilent)capillary column (0.25 mm of internal diameter and 0.25�m of film thickness). Injector(split 1:100) and detector (flame ionization) temperatures were kept constant at 250 �C.The oven temperature program started at 180 �C for 5 min, increased at 4 �C/min until220 �C, and kept constant at this temperature for 25 min. The velocity of the carrier gas,He, was kept constant at 1 ml/min.

Statistical. Data are presented as mean� standard deviation and subjected toANOVA – Post Hoc Comparisons – Scheffe test, at 0.05 probability level, using StatSoftSTATISTICA program, version 6.0.

ResultsColour and pigmentsDiacronema showed a higher colouring capacity than Spirulina, presenting highernegative a* (green hue) and positive b* (yellow hue) values for gels with 0.75 per centalgae prepared at 90 �C (Figure 1). The Sp gels presented an unattractive tonalitywhich is reflected by a* and b* values close to zero.

When changing the microalgal biomass concentration from 0.10 to 0.75 per cent thecolour variations (�E*) observed are relatively small, being practically undetectablebetween higher concentrations (Table I). For both microalgae, the total colour differencebetween samples with different gelling temperatures was not detectable by visualobservation (�E* < 5) (Castellar et al., 2006) (Table I). These results are supported bythe gels total pigments content, which remained relatively constant for the differentgelling temperatures used (Figure 2). Considering the amount of pigments frommicroalgae (0.9 per cent – Sp and 2.5 per cent – Di) that were added to the gels, thepigment losses were less than 20 per cent, as it can be seen for Sp gels in Figure 2.

Figure 1.Location of the vegetable

gelled puddings with 0.75per cent (w/w) microalgae

biomass in the two-dimensional colour space,

for 90 �C gellingtemperature

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TextureThe addition of Diacronema resulted in a structural reinforcement of the gels whichpresented higher firmness values, while Sp gels were less firm than the control. Bothmicroalgae, there were no significant differences on the gels firmness with increasingbiomass concentration (Figure 3).

Di gels presented a tendency of increasing firmness with processing temperature, as itwas observed for the control gel (0 per cent alga). However, Sp gels showed a differentbehaviour with a steep decrease on firmness for gelling temperatures above 85 �C (Figure 4).

Fatty acids profileBoth microalgae present a high percentage of PUFAs ranging between 36.7 per cent in Spand 44.8 per cent in Di (Table II). Spirulina showed the presence of linoleic (LA, 18:2!6)(~18 per cent) and �-linolenic acid (GLA, 18:3!6) (17 per cent) as the main PUFAs followedby palmitic (16:0) (~40 per cent) and palmitoleic acid (16:1!7) (~7 per cent) (Table II).Diacronema microalga is rich in eicosapentaenoic (EPA, 20:5!3) (~21 per cent) anddocosahexaenoic (DHA, 22:6!3) which is present in Diacronema gels (3 and 4 per cent, 0.7and 0.9 per cent for EPA and DHA, at 75 and 90 �C, respectively) in spite of control gel(without alga addition) in which linoleic (~47 per cent), oleic (~19 per cent) and palmitic(~14 per cent) are the main fatty acids, similar with Spirulina gels composition.

DiscussionThe addition of Spirulina and Diacronema microalgal biomass in gelled food systems,similar to ‘‘ready-to-eat desserts’’ resulted in products with poor sensory properties

Figure 2.Temperature effect ontotal pigments content ofgelled desserts with 0.75per cent (w/w) Spirulinabiomass. Bold linerepresents the amount ofpigments added to the gel,considering the microalgapigment content(0.9 per cent w/w)

Table I.Total colour difference(�E *) between sampleswith different microalgalbiomass concentrationand different gellingtemperatures ofvegetable gelledpuddings with Spirulina(Sp) and Diacronema(Di) microalgæ biomass

�E *Temperature (�C) Concentration (%) Sp gels Di gels

90 0.10 – –0.25 12.7 6.20.50 5.8 5.20.75 5.7 3.8

75 0.75 – –80 0.8 3.585 1.1 5.190 2.5 2.2

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(particularly colour) in relation to other microalgae (e.g. Chlorella vulgaris andHaematococcus pluvialis) and other food products (e.g. emulsions and biscuits)(Gouveia et al., 2008b).

The gels colour and pigment content presented a good thermal stability whenincreasing the gelling temperature from 75 to 90 �C, revealing an efficient pigmentprotection inside the Sp and Di cells.

For both algae, no significant differences (p < 0.05) were observed on the gels colourand texture properties, in the range of concentrations used (0.10-0.75 per cent). Then, itis possible to reduce the amount of microalgal biomass used in order to achieve adesired tonality or texture.

Figure 4.Effect of gelling

temperature on the gelsfirmness (0.75 per cent

w/w microalgal biomass)

Figure 3.Effect of microalgal

biomass concentration onthe gels firmness (gelling

temperature – 90 �C)

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Di gels attained higher firmness values than the control, as it was also observed for C.vulgaris microalgal biomass addition in other colloidal systems – oil-in-wateremulsions (Raymundo et al., 2005). Di gels firmness increased linearly withtemperature, showing a similar behaviour to the control, typical of biopolymer systems(Clark et al., 2001). Thermal gelation of biopolymers is a process that requiresdenaturation and subsequent interaction of the exposed residues to form aggregates(Clark et al., 2001). High temperatures favours biopolymer interactions by inducingprotein unfolding, �-carrageenan maximal hydration and total starch gelatinization, asdemonstrated by Nunes et al. (2006a, b).

Contrarily, Sp gels presented much lower firmness values than the control, which isparticularly evident for higher temperatures. This destabilization process could beassociated to a competitive interaction, or thermodynamic incompatibility, betweenthe microalgal protein (44.9 per cent of the dry biomass) and pea protein isolate.In fact, proteins isolated from Spirulina have been reported (Chronakis, 2001) asbeing quite intricate biomaterials, likely to be protein and/or protein-pigment(phycocyanin) complexes rather than individual protein molecules. Therefore,Spirulina denaturation and gel formation is a complex phenomenon per se, which canprobably interfere with the gelling process of the biopolymers present in the mixedgel system.

The microalgae present an interesting fatty acid profile, particularly Di with EPAand DHA. The high percentage of EPA (21.4 per cent) in this microalga was alsoreferred by Donato et al. (2003) during all the life cycle, recording the same value in the

Table II.Fatty acid composition(percentage of total fattyacids) of the microalgaeSpirulina (Sp) andDiacronema (Di), gelsprepared with thesealgae (at 75 and 90 �C)and control gel (withoutalga addition)

Sp Di Control gel Sp gel 75 �C Sp gel 90 �C Di gel 75 �C Di gel 90 �C

SFA14:0 – miristic 0.2 13.0 0.2 – 0.2 2.7 3.216:0 – palmitic 39.7 8.8 14.3 15.8 15.8 13.3 12.218:0 – stearic 1.2 – 3.1 3.5 3.5 2.8 2.3P

SFA 42.7 23.8 17.7 19.7 19.2 19.4 18.2MUFA16:1!7 – palmitoleic 7.0 16.1 – 0.5 0.5 3.2 3.818:1!9 – oleic 4.3 – 18.7 19.7 19.2 16.7 15.6P

MUFA 15.0 24.0 19.1 20.8 20.6 20.7 20.3

PUFA-!318:3!3 – �-linolenic 1.5 – 5.8 6.9 6.7 5.8 6.218:4!3 – 7.4 – – – 1.2 1.520:5!3 – EPA – 21.3 – – – 3.1 4.122:6!3 – DHA – 5.5 – – – 0.7 0.9EPA+DHA – 26.8 – – – 3.9 5.0P

PUFA-!3 1.5 35.9 6.6 6.9 6.7 11.1 12.6

PUFA-!618:2!6 – linoleic 17.9 0.3 47.1 50.4 49.1 41.9 43.018:3!6 – �-linolenic 16.8 0.7 – 1.3 1.3 0.2 0.222:5!6 – 6.5 – – – 0.9 1.1P

PUFA-!6 35.2 8.9 47.1 51.8 50.4 42.1 43.3PPUFA total 36.7 44.8 53.7 58.7 57.1 53.2 55.9

Notes: SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturatedfatty acid

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stationary phase. Nevertheless, a high level of linoleic acid was registered in the finalproduct gels, even in the samples prepared with Di, where linoleic acid contribution isvery low (0.3 per cent). This result is probably due to the addition of residual lipidsfrom maize starch and pea protein isolate used as ingredients in the preparationstep, because linoleic acid is the main fatty acid present in maize and pea oil (He et al.,2007; Chavan et al., 1999; Yoshida et al., 2007). The range of temperatures used inthe preparation of the gels seems do not affect GLA, EPA and DHA percentage,suggesting that these microalgae cells could resist to thermal treatments, as wasreferred by Gouveia et al. (2008a) in a previous work with Isochrysis galbana submittedto a temperature of 121 �C in a biscuit matrix. However, this type of food productspresents very low lipid content (0.4 per cent) contributing to a limited level of thesecomponents.

ConclusionDiacronema vlkianum and S. maxima microalgae are a rich source of carotenoidpigments and omega-3 fatty acids (EPA 20:5!3 and GLA 18:3!3, respectively). Inaddition, when Di and Sp were included in a protein-polysaccharide gelled system itwas observed a significant thermal resistance of these biomolecules. A similar effectwas previous observed for microalgae incorporation in biscuits systems. Theresistance of the microalgae bioactive molecules to different heat transfer processes,through ‘‘dry’’ and ‘‘wet’’ food matrices, evidences the potential of microalgae as foodingredients and/or nutraceutical delivery systems.

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Corresponding authorLuısa Gouveia can be contacted at: luisa.gouveia@ineti.pt

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