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Food Hydrocolloids 63 (2017) 50e58

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Food Hydrocolloids

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Rheological properties of agar and carrageenan from Ghanaian redseaweeds

Nanna Rhein-Knudsen a, Marcel Tutor Ale a, Fatemeh Ajalloueian b, Liyun Yu c,Anne S. Meyer a, *

a Center for Bioprocess Engineering, Dept. of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads 229, 2800 Lyngby,Denmarkb Research Group for Nano-Bio Science, National Food Institute, Technical University of Denmark, Søltofts Plads 227, 2800 Lyngby, Denmarkc Danish Polymer Center, Dept. of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads 229, 2800 Lyngby, Denmark

a r t i c l e i n f o

Article history:Received 28 March 2016Received in revised form11 August 2016Accepted 13 August 2016Available online 15 August 2016

Keywords:Red seaweedsHydrocolloidsGelling temperatureRheology

* Corresponding author.E-mail addresses: nark@kt.dtu.dk (N. Rhein-Knuds

faaj@food.dtu.dk (F. Ajalloueian), lyyu@kt.dtu.dk (L. Yu

http://dx.doi.org/10.1016/j.foodhyd.2016.08.0230268-005X/© 2016 Elsevier Ltd. All rights reserved.

a b s t r a c t

Red seaweeds contain unique galactose-rich hydrocolloids, carrageenans and agar, which find use asgelling agents in high value applications. This study examined the chemical and rheological properties ofhydrocolloids from selected wild red seaweed species collected in Ghana: Hypnea musciformis andCryptonemia crenulata, expected to hold carrageenan, contained 21e26% by weight of galactose. Acommercial Kappaphycus alvarezii carrageenan sample had 30% galactose residues by weight. Hydro-puntia dentata, expected to contain agar, contained 15% by weight of galactose-monomers. Fouriertransform infrared spectroscopy (FTIR) analysis on the hydrocolloids extracted from H. musciformis (andK. alvarezii) indicated k-carrageenan, C. crenulata hydrocolloids were mainly i-carrageenan, and theH. dentata hydrocolloids were agar. Gelling temperatures ranged from 32 to 36 �C for the k-carrageenanhydrocolloid samples. The i-carrageenan and agar samples had gelling temperatures of 70e74 �C and 38e52 �C, respectively. Gel strengths, G’ at 25 �C, of carrageenan samples extracted via alkali-treatmentwere 4000e6500 Pa. The agar gel strength was 287 Pa. The rheological properties of theH. musciformis k-carrageenans were comparable with k-carrageenan from K. alvarezii, whereas theH. dentata agar properties were different from those of a commercial agar sample. This work shows thatcertain red seaweed species in Ghana contain hydrocolloids with desirable properties for high valueapplications.

© 2016 Elsevier Ltd. All rights reserved.

1. Introduction

Seaweed hydrocolloids currently have a global value ofapproximately US$ 1.1 billion with products from the Asia-Pacificregion dominating the market (Bixler & Porse, 2011; Hurtado,Neish, & Critchley, 2015; Rhein-Knudsen, Ale, & Meyer, 2015).Red seaweed species contain particularly valuable hydrocolloidssuch as agar and carrageenan that are used in food, pharmaceutical,and biotechnological applications due to their unique physico-chemical properties and gelling characteristics (Mchugh, 2003).Extraction of such high value hydrocolloids from native redseaweed species in Ghana could enable a new type of green growth

en), mta@kt.dtu.dk (M.T. Ale),), am@kt.dtu.dk (A.S. Meyer).

in the West African coastal region.Carrageenans are principally made up of repeating di-

saccharides of D-galactopyranose units bound together with alter-nating a-1,3 and b-1,4 linkages. Some of the galactose moieties arepresent in a 3,6-anhydro-a-D-galactopyranose form and a signifi-cant portion of the galactose-moieties may also be sulfated at C-2,C-4 or at C-6 (De Ruiter & Rudolph, 1997). The presence of 3,6-anhydro-galactopyranose and the amount and position of the sul-fate substitutions form the basis for categorizing carrageenans intothree chemically distinct types of structures, that also designate themain commercially used types, namely k-, i-, and l-carrageenan. Ingeneral, k-carrageenan has one sulfate ester per galactose dimer,whereas i-and l-carrageenan have two and three sulfates perdimer, respectively (De Ruiter & Rudolph, 1997). Agar is chemicallysimilar to carrageenan, and is made up of galacto-pyranose dimers,i.e. alternating galactose and 3,6-anhydro-a-galactopyranose unitsconnected by alternating a-1,3 and b-1,4 linkages, but with the

N. Rhein-Knudsen et al. / Food Hydrocolloids 63 (2017) 50e58 51

important difference that in agar the 3,6-anhydro-a-galactopyr-anose units are in the L-configuration and not in the D-configura-tion as is the case for carrageenans (Usov, 2011).

Both carrageenans and agar form gels in aqueous environmentsvia helix formation and aggregation of the polysaccharide chainsthrough inter-molecular hydrogen bonds (Morris, 1986; Schafer &Stevens, 1995) - for carrageenans the gelation is supported by thepresence of cations that induce the formation of a stable three-dimensional gel-network (usually with potassium for k-carra-geenan and with calcium for i-carrageenan) (Montero & Pe, 2002;Rhein-Knudsen et al., 2015). However, being biologically synthe-sized in nature, natural carrageenans and agar are inherently het-erogeneous. Extracted k- and i-carrageenans may thus containtraces of their biosynthetic precursors, i.e. m- and n-carrageenan,respectively, whereas agar may hold porphyran structures, i.e. theprecursor for agar, having a-L-galactose-6-sulfate instead of 3,6-anhydro-a-L-galactopyranose, along with other hybrid structures(Rhein-Knudsen et al., 2015). The level of these different precursorsand the extent of structural differences vary in different redseaweed species, and this variation obviously affects the rheologicalproperties of the hydrocolloids.

Tanzania has long been a producer of carrageenan from seaweedfarming (Hurtado et al., 2015), but neither seaweed collection norfarming are currently practiced in Ghana, even though the 540 kmlong Atlantic coastline in the south is a habitat for differentseaweed species with potential for local hydrocolloid production.Some of the wild red seaweed species such as Hypnea spp., Cryp-tonemia crenulata and Hydropuntia spp. found along the coast ofGhana are known, however, to contain hydrocolloids, notablycarrageenan and agar (Mtolera & Buriyo, 2004; Pereira-Pacheco,Robledo, Rodríguez-Carvajal, & Freile-Pelegrín, 2007; Saito & deOliveira, 1990). Cultivation of e.g. Hypnea musciformis for extractionof k-carrageenan has been studied in India and Brazil, but has notreached a commercial stage (Berchez, Pereira, & Kamiya, 1993;Ganesan, Thiruppathi, & Jha, 2006). Only few studies have beenconducted on Ghanaian seaweeds, and these have mainly focusedon elemental analysis and assessments of iodine levels (e.g. Serfor-Armah, Nyarko, Osae, Carboo, & Seku, 1999; Serfor-Armah et al.,2000). In 1975, John and Asare (John & Asare, 1975) published apreliminary assessment study of the yields and properties of hy-drocolloids extracted from certain Ghanaian red seaweeds, but toour knowledge, no recent data are available on the rheologicalcharacteristics or hydrocolloid levels in wild red seaweed speciesfrom Ghana. The hypothesis of the present work was that redseaweed species native to Ghana hold galactose-rich hydrocolloidsof the carrageenan or agar type, and that the rheological propertiesof hydrocolloids extracted from these seaweed species could be onpar with commercially used carrageenans and agar. The presentstudy was conducted to assess the carbohydrate composition oflocal red seaweed species found along the coast in Ghana, and tocharacterize the rheological properties of the hydrocolloidsextracted as a base for considering local carrageenan and agarproduction from red seaweed resources in the region.

2. Materials and methods

2.1. Chemicals

All chemicals were purchased from Sigma-Aldrich Chemical Co.(St. Louis, MO, USA) unless stated otherwise. Guluronic acid waspurchased from Chemos GmbH (Regenstauf, Germany).

2.2. Seaweed sampling and sample preparation

Wild red seaweed samples were collected in the coastal areas of

Ghana, except the Kappaphycus alvarezii (Table 1). All samples ob-tained from Ghana were immediately frozen in aliquout portionsat �20 �C after collection. Before use, the seaweed samples weregently thawn, and then rinsed to remove epiphytes, entangledmaterials, and sand. The washed seaweed samples were thenfreeze-dried and milled, then passed through a 1 mm mesh sieve(MF 10 basic Microfine grinder drive, IKA) to obtain uniform par-ticle sizes. The milled seaweed samples were stored in sealedplastic bags at �20 �C. Cultured Kappaphycus alvareziiwas receivedin dried form from Vietnam (Nhatrang Institute of TechnologyResearch and Application) and used as a benchmark for carra-geenan extraction and rheological properties of carrageenan.

2.3. Composition analysis

The amount of dry matter and ash in the seaweed samples weredetermined according to the National Renewable Energy Labora-tory (NREL) procedure and the weight of biomass used in the ex-periments was mathematically corrected for the amount ofmoisture present in the samples (Sluiter et al., 2004, 2008). Forcarbohydrate composition analysis, the collected seaweed sampleswere hydrolyzed according to a modification of the NREL two-stepsulfuric acid hydrolysis procedure, as described by Manns et al.(Manns, Deutschle, Saake, & Meyer, 2014). In brief, 100 mg drymatter seaweed material per mL was mixed with 72% H2SO4

(weight/volume, w/v) and left to react at 30 �C for 1 h. The reactionmixture was then diluted to 4% w/v H2SO4 and hydrolyzed in anautoclave at 120 �C for 40 min (Manns et al., 2014). The acid hy-drolysate and seaweed residuals were then separated by vacuumfiltration and the supernatants filtered through a 0.22 mm nylonsyringe tip filter (Frisenette Aps, Knepel, DK) and diluted in 500mMNaOH prior to injection for high performance anion exchangechromatography (HPAEC) analysis. HPAEC separation of theseaweed polysaccharides was performed using a HPAEC-PAD,ICS3000 system (Dionex Corp. Sunnyvale, CA) equipped with aCarboPac™ PA20 column by a method principally as described byArnous and Meyer (2008). L-fucose, L-arabinose, L-rhamnose, D-galactose, D-glucose, D-xylose, D-mannose, D-galacturonic acid, D-guluronic acid, and D-glucuronic acid were used as mono-saccharide standards for quantification; quantification was doneusing Chromeleon software (Dionex Corp. Sunnyvale, CA). Recoveryvalues for the monosaccharides were estimated from parallel runsof monosaccharide standards (Arnous & Meyer, 2008).

2.4. Hydrocolloid extraction and characterization

2.4.1. Carrageenan extractionWater extraction of carrageenanwas performed using 1.5 g (dry

matter) samples from H. musciformis, C. crenulata and K. alvareziithat were hydrated overnight in 30 mL milli-Q water before directextraction at 99 �C for 1.5 h. The pH following overnight soakingwas between 7.5 and 8.5 and based on previous results, this pH washigh enough to prevent depolymerization of the seaweed galactans(Capron, Yvon, & Muller, 1996; John & Asare, 1975). Alkali-treatment was carried out using a slightly modified procedure ofthemethod described by Istinii, Ohno, and Kusunose (1994). Briefly,60 mL 6% w/v KOH was added to 1.5 g of the seaweed samples andreaction was carried out at 80 �C for 3 h. KOH was removed bywashing the seaweed samples and soaking in water overnight.Carrageenan extraction was then performed on the alkali-treated,washed, seaweed samples (1.5 g dry matter) in 30 mL milli-Q wa-ter at 99 �C for 1.5 h. The extracts were pressure filtered (filterpaper, PP filter cloth, Sigma-Aldrich) after being mixed with dia-tomaceous earth (Celite, Sigma-Aldrich), precipitated in 80% iso-propanol, filtrated, and recovered by freeze-drying. Yields were

Table 1Overview of seaweed samples, expected hydrocolloids, and origin of the red seaweed samples used in the present study.

Red seaweed samples Expected hydrocolloid type Origin

Hypnea musciformis(I) Carrageenan Old Ningo, Greater Accra, Ghana/Jan. 2015Hypnea musciformis(II) Carrageenan Prampram, Greater Accra, Ghana/Aug. 2015Cryptonemia crenulata Carrageenan Prampram, Greater Accra, Ghana/ Jan. 2015Kappaphycus alvarezii Carrageenan (benchmark) Nha Trang, Vietnam/2015Hydropuntia dentata Agar Prampram, Greater Accra, Ghana/Jan. 2015

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determined by weighing and confirmed by HPAEC analysis of HClhydrolyzed carrageenans (0.5% byweight of carrageenan in 1MHCl,105 �C, 3 h) (Istinii et al., 1994). Standard grade carrageenans ob-tained from Sigma-Aldrich Chemical Co. (k-carrageenan: 22048,Lot# BCBK1080V, i-carrageenan: C1138, Lot# SLBJ7874V) (St. Louis,MO, USA) were used for comparison. 3,6-anhydro-galactose con-tents of the hydrocolloids were determined, where practicallypossible, following the reducing acid hydrolysis proceduredescribed by Jol, Neiss, Penninkhof, Rudolph, and De Ruiter (1999).(the third addition of the reducing agent methylmorpholine-borane complex in the procedure was left out to improve thesubsequent analytical HPAEC quantitation, however). Quantifica-tion was done using HPAEC-PAD with 3,6-anhydro-galactose as astandard (purity � 95% as informed by the manufacturer) (DextraLaboratories Ltd., UK).

2.4.2. Agar extractionDirect water extraction of agar and extraction of agar after

alkali-treatment were performed according to the methodsdescribed by Freile-Pelegrín and Murano (2005). For the waterextraction, 1.5 g (dry matter) of Hydropuntia dentata was hydratedin 30 mL milli-Q water overnight. The pH of the suspensions wasadjusted to 6e6.5 and extraction was performed at 99 �C for 1.5 h.The procedure was selected from the literature where it is alsostated that agar is stable at pH 6 when extracting at boiling point(without pressure) (Armisen & Galatas, 1987; Freile-Pelegrín &Murano, 2005; Marinho-Soriano & Bourret, 2003). For extractionof agar after alkali-treatment, the seaweeds were soaked in 30 mL5% w/v NaOH overnight and modification of the seaweed carbo-hydrates was done at 90 �C for 3 h. The alkali-treated biomasseswere then washed and soaked in water overnight to removeremaining alkali and extraction was performed in 45 mL milli-Qwater, pH 6e6.5 at 99 �C for 1.5 h. The extracts were filtered asdescribed above for carrageenans. Agar was recovered by freeze-drying. Yields were determined by weighing and confirmed byHPAEC analysis of HCl hydrolyzed agar (0.5% agar in 1M HCl, 105 �C,3 h) (Freile-Pelegrín & Murano, 2005). Standard grade agar ob-tained from Sigma-Aldrich Chemical Co. (A1296, Lot# BCBQ5740V)(St. Louis, MO, USA) was used for comparison. 3,6-anhydro-galac-tose contents were determined as described in section 2.4.1.

2.4.3. Sulfate content analysisThe inorganic sulfate content was determined by turbidity

analysis as described by Jackson and McCandless (1978) after hy-drolysis of the hydrocolloids with 1M HCl at 105 �C for 3 h (Jackson& McCandless, 1978).

2.4.4. Fourier transform infrared spectroscopyAttenuated total reflectance Fourier transform infrared spec-

troscopy (ATR-FTIR) was carried out on a Nicolet iS50 FTIR spec-trometer (Thermo Fisher Scientific Inc., USA) with ATR module. Thespectra were recorded in the range of 4000e400 cm�1 by acquiring32 scans with 4 cm�1 resolution.

2.4.5. Oscillatory rheologySamples for oscillatory rheology analysis were prepared by a

modified procedure from Thrimawithana, Young, Dunstan, andAlany (2010). In brief, the carrageenans were prepared by dissolv-ing at 80 �C for the k-carrageenans and at 95 �C for the i-carra-geenans. Solutions were diluted with 4% KCl to obtain finalconcentrations of 1.5% w/v carrageenan and 1% of KCl. After addi-tion of KCl, the solutions were heated for an additional time of20 min. Agar at 1.5% w/v was dissolved in milli Q water at 95 �C andanalyzed without further addition of ions (Thrimawithana et al.,2010). The volume used for each analysis was 3 mL. Rheologicalproperties of the hydrocolloids were assessed by small angleoscillatory rheological measurements on a HAAKE MARS rotationalrheometer (Thermo Scientific Inc., Germany) equipped with aserrated parallel-plate (Reologica Instruments AB) with a diameterof 60 mm and a gap of 1.0 mm. Temperature sweep tests wereconducted at 0.1 Hz to evaluate the viscoelastic properties duringgelation by in situ cooling (80/95e20 �C) and heating (20e80/95 �C)of the hydrocolloid mixture at a rate of 1 �C/min. To avoid dehy-dration during experiment, the plate was covered with silicone oil(Hall Miba A/S, Kgs. Lyngby, Denmark). The storage modulus (G0),the loss modulus (G00), and the thermal hysteresis behavior of thegels were determined as a function of temperature.

2.5. Statistics

Carbohydrate composition analyses, hydrocolloid extractions,and sulfate content determinations were performed in triplicatesand the data are presented as mean ± standard deviations (SD).Analyses of variances (ANOVA) were used to determine significancedifferences in yields and compositions with the Tukey-Kramer testfrom pooled standard deviations (JMP 11 Statistical Software, SAS).Values of P < 0.05 were considered statistically significant.

3. Results and discussion

3.1. Seaweed composition

All the seaweed samples were found to contain significantamounts of galactose, namely from 21 to 26% by weight (w/w) ofthe dry matter for the Hypnea musciformis and Cryptonemia cren-ulata samples, and 15% w/w for the Hydropuntia dentata sample(Table 2), which supports the expectation that the seaweed speciescould contain carrageenan and agar, respectively (Table 1). For thecompositional analysis of the whole seaweed samples the quanti-fied galactose was assumed to represent both the galactose moi-eties and the 3,6-anhydro-galactose, though it is known that the3,6-anhydro bridges are acid labile (Jol et al., 1999). The mono-saccharide composition showed that the C. crenulata andH. musciformis samples had almost as high galactose levels(constituting 21e26% by weight of the seaweed dry matter) as thatfound in the carrageenan benchmark red seaweed sample fromK. alvarezii (30% % by weight of the seaweed dry matter) (Table 2) ethe latter was used as benchmark since K. alvarezii is currently themost significant commercial k-carrageenan source (Hurtado et al.,

Table 2Monomeric carbohydrate yields [mean ± SD]1 determined by HPAEC-PAD analysis after two-step sulfuric acid hydrolysis and ash content determined gravimetrically afterigniting at 550 �C. Different roman superscript letters column-wise indicate significant differences (P < 0.05) by one-way ANOVA.

Seaweed samples Monosaccharides and ash levels

Galactose[% dry material]

Glucose[% dry material]

Others2

[% dry material]Ash content[% dry material]

Hypnea musciformis(I) 26a,b±0.9 11d ± 0.7 2c±0.1 22d ± 0.8Hypnea musciformis(II) 21b ± 0.9 13c±0.6 3c±0.5 30b ± 0.2Cryptonemia crenulata 25a,b±1.7 18a±0.9 4c±0.6 19e±0.6Kappaphycus alvarezii 30a±4.5 11d ± 0.3 14a±4.8 23c±0.1Hydropuntia dentata 15c±0.8 15b ± 0.7 9b ± 0.8 36a±0.2

1 All monosaccharide values are given on per weight basis as dehydrated monomers.2 Others: mannose, rhamnose, arabinose, xylose, galacturonic acid, guluronic acid, and glucuronic acid.

N. Rhein-Knudsen et al. / Food Hydrocolloids 63 (2017) 50e58 53

2015). Comparison of the polysaccharide contents in the Ghanaianseaweeds and K. alvarezii was done even though the post-harvesthandling differed. The ash contents and glucose levels variedslightly in the two H. musciformis samples, which were collectedfrom two different sites along the Ghanaian coast (Table 1), but thegalactose levels were similar (Table 2).

Several studies involve estimation of carbohydrate content inK. alvarezii and Meinita et al. (2012) estimated the carbohydratecontent to range from 35 to 78% by weight in different K. alvareziitissues collected from various places in Indonesia (Meinita et al.,2012). By extraction of crude carbohydrates, Arunkumar,Palanivelu, and Darsis (2014) found a carbohydrate content of40% for aH. musciformis sample collected in India (Arunkumar et al.,2014). When adding the monosaccharide levels the carbohydratelevels found in the H. musciformis samples collected in Ghana of37e39% w/w (Table 2) were thus in complete accordance with thetotal level of crude carbohydrates found in the Indian sample byArunkumar et al. (2014). Glucosewas found in levels of 11e18%w/w(Table 2). Since red seaweeds are generally believed to contain lessthan 10% w/w cellulose, some of the glucose may derive from flo-ridean starch, the storage carbohydrate of red algae that is built of1,4-linked a-D-glucopyranose chains with branches at position 6(Usov, 2011). Other minor monosaccharide constituents wereidentified in the red seaweed hydrolysates, namely: mannose,rhamnose, arabinose, xylose, galacturonic acid, guluronic acid andglucuronic acid. These minor saccharides have also been identifiedin red seaweeds earlier (Usov, 2011). For the C. crenulata, minormonosaccharides were mainly rhamnose, while the other seaweedsamples containedmainly xylose and some traces of uronic acids asminor monosaccharides. These findings are in agreement withprevious findings reporting the presence of rhamnose in the redalgae Rhodella maculata (Fareed & Percival, 1977), while xylosesubstitutions on agar and carrageenans from red seaweed specieshave been described by Araki, Arai, and Hirase (1967) and Estevez,Ciancia, and Cerezo (2000) respectively. The ash contents in sea-weeds are generally higher than those of terrestrial plants, andranged from 19 to 36% w/w (Table 2), which is in agreement withprevious findings for these seaweed species (Arunkumar et al.,2014).

The observed differences in the composition of these redseaweed species may be attributed to the differences in the algalsource and the growth environment (Table 1). It is well known thatwild seaweeds show significant variation in nutrient contents atdifferent environmental conditions such as water temperature,water salinity, nutrients, and light (Marinho-Soriano, Fonseca,Carneiro, & Moreira, 2006).

Once the exact impact of the different factors is understood, itmay be possible to optimize the monosaccharide composition andhydrocolloid levels.

3.2. Hydrocolloid yield and characteristics

3.2.1. Hydrocolloid yieldTo validate whether the galactose units found in the red sea-

weeds, section 3.1, were actually constituents of hydrocolloidpolysaccharides, the seaweeds were subjected to direct water-extraction and alkali-treatment methods as described in Section2.4.

Evidently, most of the extracted material contained galactoseand sulfate, which are the main components of agar and carra-geenan. Extraction yields for the putative carrageenans rangedfrom 19 to 27% by weight while yields were slightly lower for theextracted agar hydrocolloids, but generally with no profound dif-ferences in yields between water- and alkali-treatment (Table 3).The lower (agar) hydrocolloid extraction yield for H. dentata is inagreement with the lower level of galactose-moieties found inH. dentata than in the other seaweed samples expected to containcarrageenan (Table 2). The carrageenan and agar yields fromH. musciformis and H. dentata were in accordance with the resultspublished by John and Asare (1975) showing GhanaianH. musciformis carrageenan extraction yields of 25e45% by weightand Ghanaian H. dentata (referred to as Gracilaria dentata in theirstudy) agar extraction yields of approximately 10e30% dry weight,depending on season of harvest (John& Asare, 1975). A comparisonof the total content of galactose-monomers found in the seaweedsamples (Table 2) and the hydrocolloid extraction yields and thehydrocolloids compositions (Table 3) showed that 48e89% byweight of the galactose residues present in the different seaweedswere extracted as hydrocolloids. Generally, the alkali-treatmentwas most effective for obtaining more galactose-rich hydrocol-loids, resulting in high extraction of galactose in the hydrocolloids,e.g. reaching a yield of 89% of the galactose-moieties present inH. musciformis(II) in the extracted hydrocolloid (calculated from theData in Tables 2 and 3). In comparison, 81% of the galactose-moieties present in the H. musciformis (II) were released as hy-drocolloid by water-extraction. Although we did not observe asignificant difference in hydrocolloid extraction yield betweendirect water-extraction and alkali-treated the modification of theseaweed hydrocolloids was successfully accomplished by thealkali-treatment, since the water-extracted hydrocolloid containedsignificantly higher amounts of sulfate compared to the onesextracted via alkali-treatment (Table 3). The analysis of the sulfatecontent supported the expectation that the hydrocolloid poly-saccharides from the two Ghanaian Hypnea musciformis samplescould be k-carrageenans. The water-extracted hydrocolloids fromC. crenulata had sulfate contents that were comparable with thestandard grade i-carrageenan, whereas the sulfate level in thealkali-extracted hydrocolloids was more at the level of sulfate in k-carrageenan (Table 3). Literature regarding C. crenulata is limitedbut has been described as producing both i-carrageenan (Saito& de

Table 3Overview of seaweed type (hydrocolloid source), hydrocolloid extraction method (direct water-extraction or after alkali treatment), hydrocolloid and monomer1 yields, andsulfate levels [data given as means ± SD]. Different roman superscript letters indicate significant differences (P < 0.05) column-wise for carrageenans and agar yields,monosaccharides, and sulfate content by one-way ANOVA.

Hydrocolloidsource

Hydrocolloidextraction method

Hydrocolloid extraction yield[% dry material]

Hydrocolloid composition

Galactose (3,6-anhydrogalactose3)[% hydrocolloid]

Glucose[% hydrocolloid]

Others4

[% hydrocolloid]Sulfate content[% hydrocolloid]

H. musciformis(I) Direct water-extraction 24a±1.7 72b,c±1.0 (24) 4d,e±0.8 1d ± 0.01 20c,d±1.2Alkali-treated 26a±1.6 84a±0.9 (48) 2f±0.3 2b ± 0.2 16d ± 0.4

H. musciformis (II) Direct water-extraction 24a±1.9 71b,c±1.5 (n.a.) 8b,c±0.7 1.45c,d 21c,d±2.4Alkali-treated 27a±4.5 70c±1.5 (n.a.) 4d,e,f±0.6 1c,d±0.5 16d ± 1.2

C. crenulata Direct water-extraction 21a±1.9 61d ± 2.7 (n.a.) 4d,e±0.6 1d ± 0.1 32b ± 4.3Alkali-treated 19a±0.1.8 68c±1.6 (n.a.) 3e,f±0.2 3b ± 0.2 25c±2.5

K. alvarezii Direct water-extraction 21a±2.8 68c,d±5.3 (23) 3e,f±0.3 2b,c±0.1 20c,d±0.7Alkali-treated 23a±5.9 77b ± 2.4 (52) 6c,d±0.5 2b,c±0.1 16d ± 0.6

k-carrageenan2 47e±1.5 (17) 30a±1.5 n.d 22c,d±0.6i-carrageenan2 40e±2.0 (8) 9b ± 0.6 4a± <0.1 39a±3.8H. dentata Direct water-extraction 15b ± 1.7 79b ± 2.5 (34) 1b ± 0.1 6a±0.4 8a±0.2

Alkali-treated 13b ± 0.02 79b ± 2.6 (46) 2a ±0.6 6a±0.2 5b ± 0.2Agar2 89a±1.1 (48) 2a,b±0.3 3b ± 0.6 7b ± 0.6

1 All monosaccharides values are given as dehydrated monomers,2 purchased from Sigma-Aldrich,3 data in parenthesis are amounts of 3,6 anhydrogalactose out of total galactose quantified by reducing TFA hydrolysis, n.a. not analyzed (i.e. not done),4Fucose, xylose, mannose; n.d: not detected.

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Oliveira, 1990) and a hybrid galactan composed of l-carrageenanand agar (Zibetti, Noseda, Cerezo, & Duarte, 2005). The 3,6-anhydro-galactose contents were quantified by HPAEC-PAD anal-ysis following reductive hydrolysis (Table 3). The results supportedthat the alkali-treatment was effective as evident from the lower3,6-anhydro-galactose content in the water-extracted samples(Table 3). The standard grade i-carrageenan was shown to containonly 8% 3,6-anhydro-galactose, while the k-carrageenan containedhigher amounts (Table 3). The commercial i-carrageenan wascommercial grade Type II: predominantly i-carrageenan and maycontain higher amounts of its precursor n-carrageenan that pro-duce flexible gels thus has low content of 3,6-anhydro-galactose.

Small amounts of glucose, fucose and xylosewere also identifiedin the extracted hydrocolloids (Table 3). Several types of mono-saccharide substitutions on the galactose-rich hydrocolloids,including xylose substitutions on the galactose-moieties in agar,have been described in the literature, e.g. by Araki et al. (1967).Estevez et al. (2000) have similarly described xylose-substitutionson carrageenan derived from the red seaweed Kappaphycus alvar-ezii (Estevez et al., 2000). Surprisingly high amounts of glucosewere found in the standard grade k-carrageenan sample (Table 3).With reductive acid hydrolysis the level of glucose remained thesame (data not shown), which verified that the detection of glucosewas not a result of decomposition of anhydro-galactose moieties.Unfortunately, the manufacturing method for this commercial k-carrageenan sample was not specified by the manufacturer. Thepresence of glucose could be a result of insufficient separation ofthe carrageenan during extraction.

3.2.2. Fourier transform infrared (FTIR) spectroscopyThe FTIR spectra of carrageenan products from the

H. musciformis, K. alvarezii, and C. crenulata seaweed samples ob-tained from two different extraction procedures, namely water-and extractions and alkali-treatments, were compared with thoseof commercial k-carrageenan and i-carrageenan (Fig. 1A). Thespectra of the carrageenans (Fig.1A) showed themain features of k-and i-carrageenan, since notably the moderately strong intensity atapproximately 845 cm�1 is assigned to CeOeSO4 on C4 of D-galactose-4-sulfate (Pereira, Amado, Critchley, van de Velde, &Ribeiro-Claro, 2009; Pereira, Sousa, Coelho, Amado, & Ribeiro-Claro, 2003). The weak intensity band at approximately 867 cm�1

(Fig. 1A) also indicates CeOeSO4, but on C6 of galactose. This sulfatesubstitution is present on m and n-carrageenans, the precursors fork- and i-carrageenan, respectively, as well as on l-carrageenan. Aswe see a strong intensity band at 845 cm�1 from D-galactose-4-sulfate, which is not part of l-carrageenan, the weak band at867 cm�1 is appointed to m and n-carrageenan (Pereira et al., 2009).The findings are in accord with the chemical composition data(Table 3) showing that although sulfate contents were decreased inthe alkali-extracted samples, the results indicated that only partialremoval of sulfate substitutions from the galactose-moieties in thecarrageenan took place during the alkali-treatment. The presenceof a strong band at approximately 930 cm�1 indicated the presenceof 3,6-anhydro-D-galactose and was thus assigned as CeO bonds(Matsuhiro, 1996). In Fig. 1Ahej, an absorption band around805 cm�1 was also visible, indicating the presence of a sulfate esterat C2 of the anhydro-D-galactose, i.e. CeOeSO4 on C2 of the 3,6-anhydro-D-galactose-unit, which is a profound band characteristicfor i-carrageenan and q-carrageenan (Matsuhiro, 1996; Pereiraet al., 2009). No clear bands were observed between 820 cm�1

and 830 cm�1 to verify the presence of l-carrageenan as obtainedfrom the CeOeSO4 on C2 and C6 on galactose (Pereira et al., 2009).These findings suggest that the main type of hydrocolloid presentin the Ghanaian H. musciformis was indeed k-carrageenan (and asexpected, the K. alvarezii hydrocolloid was also confirmed to be k-carrageenan), whereas C. crenulata is expected to contain i-carra-geenan, based on the presence of a band at strong intensity band at845 cm�1, from CeOeSO3 on C4 of galactose, and a weaker band at805 cm�1, assigned to the CeOeSO3 of C2 on 3,6-anhydro-galactose.

The glucose, especially the high amount of glucose detected inthe commercial k-carrageenan, having characteristic bands be-tween 990 and 1150 cm�1, was not clearly revealed in the FTIRspectrum. As the samples for FTIR analysis were not weighed foraccurate estimation of contents, it was difficult to make furtherconclusions about this issue; this conclusion is in accord with aprevious FTIR report (Adina, Florinela, Adbelmoumen, & Carmen,2010).

Although the FTIR spectra of the agar samples were generallysignificantly different from the FTIR spectra of the carrageenansamples, some similarities between the two sets of spectra wereevident (Fig. 1B). For example, as in the carrageenan hydrocolloidspectra, the presence of a strong band in the region around

Fig. 1. A) FTIR analysis of carrageenans from a) Sigma-Aldrich (k-carrageenan), b)H. musciformis(I) (water-extracted), c) H. musciformis (II) (water extracted), d)H. musciformis(II) (alkali-treated), e) H. musciformis(I) (alkali-treated), f) K. alvarezii(alkali-treated), g) K. alvarezii (water-treatedh) C. crenulata (water-extracted), i)C. crenulata (alkali-treatedand j) Sigma-Aldrich (i-carrageenan. B) FTIR analysis of agarfrom a) Sigma-Aldrich, b) H. dentata (water-extracted), c) H. dentata (alkali-treated).

N. Rhein-Knudsen et al. / Food Hydrocolloids 63 (2017) 50e58 55

930 cm�1 for the agar samples (Fig. 1B), could be attributed to theCeO bonds of 3,6-anhydro-a-L-galactopyranose. The characteristicbroad band of a sulfate ester between 1210 and 1260 cm�1 wasmuch stronger in carrageenan than in agar (Fig. 1A and B) indi-cating that the amount of sulfates was higher in the extractedcarrageenan hydrocolloids than in the agar, in accord with thecompositional data (Table 3). The bands at 740 and 770 cm�1 in thespectra of the commercial agar and the water-extracted H. dentatahydrocolloids (Fig. 1B) were assigned to the skeletal bending of thegalactose ring and especially in the anomeric region(700e950 cm�1), where agar and carrageenan have previouslybeen reported to show several similar bands (Pereira, Gheda, &Ribeiro-claro, 2013).

The spectral feature of agar at approximately 890 cm�1 (Fig. 1B)is interpreted to be mainly associated with the CeH bending at theanomeric carbon in b-galactose residues (Matsuhiro, 1996). Thespectra of the agar hydrocolloids extracted fromH. dentata revealedthat effective modification had occurred during the alkali

treatment, as the band at 867 cm�1, indicating CeOeSO4, in thewater-extracted sample (Fig. 1Beb), was not present in the equiv-alent alkali-extracted sample (Fig. 1Bec) (Guerrero, Etxabide,Leceta, Pe~nalba, & De La Caba, 2014). These findings confirmedthat the alkali-treatment used modified the sulfates in the hydro-colloids, in agreement with the sulfate content analysis, thatshowed that the sulfate content was consistently lower in thealkali-extracted than in the water-extracted samples (Table 3).

3.2.3. Rheological propertiesOscillatory rheological measurements were performed to assess

the gelling characteristics of the extracted hydrocolloids from theGhanaian seaweed samples. 1.5% carrageenans with 1% KCl addedwere used for the carrageenan gel analysis, while the gellingproperties of the agar samples were determined using 1.5% agardissolved in milli-Q water. The storage modulus (G0) and the lossmodulus (G00) were measured over temperature ranges from80 �Ce20 �C for k-carrageenan and 95 �Ce20 �C for i-carrageenanand agar. (Rheological analysis from80 �Ce20 �Cwas performed fori-carrageenan and agar as well, and the same rheological patternwas observed (Results not shown). The temperature was increasedas no cross-over of G0 and G00 was observed). To evaluate thereversible gelling properties, the parameters were determined byheating back to 80 �C and 95 �C, respectively. For all the testedhydrocolloids, G0 increased as a result of gel formation as thetemperature decreased (Fig. 2, and Supplementary materialFigures S1-S3). The gel strengths were estimated at 25 �C as theaverage of the G’ values measured at the three temperatures closestto 25 �C and are summarized in Table 4. Clear differences in the gelstrengths were evident between the hydrocolloids obtained byalkali-treatment and those obtained by water extraction (Table 4).These differences are most likely attributable to the formation ofanhydro-galactose by the alkali-treatment for extraction, ascorroborated by the sulfate content differences described in section3.2. The alkali-treated carrageenans are expected to have higher gelstrengths after removal of sulfate in the b-1,4-linked galactose unitsforming higher amounts of the gel-inducing 3,6-anhydro-galactoseunits. Statistical analysis revealed that the water-extracted carra-geenans from the Ghanaian seaweeds had similar gel strengths tothe k-carrageenan extracted by from the Vietnamese K. alvareziisample (Table 4). No significant difference in gel strength wasobserved between the alkali-extracted carrageenans from theH. musciformis samples collected at different sites along the coastand at different times, indicating that both samples contain hy-drocolloids with remarkable gelling potential and that the collec-tion site and time had no influence in this particular study.

The extracted k-carrageenans from the seaweed samplesshowed significantly higher gel strengths at 25 �C than the com-mercial k-carrageenan obtained from Sigma-Aldrich (Fig. 2A andTable 4). It is difficult to make any conclusive statements whencomparing the k-carrageenan in the present study to the standardgrade k-carrageenan, as the origin and extraction technique of thestandard grade compound is undisclosed. Nevertheless, it isevident from the composition analysis that the lower gel strengthobtained from the commercial k-carrageenan is likely related to thehigher glucose to galactose ratio as the sulfate levels were the same(Table 3). In addition, several other factors have an impact on thegelling properties, such as molecular weight and ionic content, andfurther analyses are thus required for a full understanding on theorigin of the differences in the rheological behaviors of the hy-drocolloid gels (Chen, Liao, & Dunstan, 2002). As was the case forthe k-carrageenans (Fig. 2A), the storage modulus (G0) values forthe i-carrageenan from C. crenulata also increased upon cooling(Fig. 2B). The increase in G’ for i-carrageenan occurred earlier thanfor k-carrageenan, indicating that sol-gel transition occurred at

Fig. 2. Storage modulus, G’ [Pa], measured from 80 �C to 20 �C at a rate of 1 �C/min forA) 1.5% k-carrageenan with 1% added KCl, and the storage modulus, G’ [Pa], measuredfrom 95 �C to 20 �C at a rate of 1 �C/min for B) 1.5% i-carrageenan with 1% added KCland C) 1.5% agar in milli-Q water.

Table 4Overview of the parameters determined by oscillatory rheology for 1.5% carrageenanwiththe averages of the three values determined closest to 25 �C ± SD; different roman superssamples assessed separately from agar samples).

Hydrocolloid source Extraction method Gel strength at z25

H. musciformis(I) Direct water-extraction 3126d ± 209.7Alkali-treated 6409b ± 147.0

H. musciformis(II) Direct water-extraction 3075d ± 218.4Alkali-treated 6538a,b±79.8

C. crenulata Direct water-extraction 1590e±38.00Alkali-treated 4035c±135.3

K. alvarezii Direct water-extraction 3126d ± 209.8Alkali-treated 6905a±241.7

k-carrageenan1 1263e±11.0i-carrageenan1 790f±10.3H. dentata Direct water-extraction 10b ± 1.1

Alkali-treated 287a±13.7Agar1 238a±71.2

1 Purchased from Sigma-Aldrich,2 Average values of the three measurements closest to 25 �C, - could not be determined

N. Rhein-Knudsen et al. / Food Hydrocolloids 63 (2017) 50e5856

higher temperatures in the i-carrageenan hydrocolloids than in thek-carrageenan. The gel strengths for the i-carrageenans were esti-mated as described above and the data are summarized in Table 4.The lower gel strengths observed for the i-carrageenan samples arein accord with theory, and the characteristics of k-carrageenan toproduce stronger and more rigid gels than i-carrageenan is alsowell established (Rhein-Knudsen et al., 2015).

The estimated gel strengths were based on gel formation bypotassium ions. Calcium ions are far more effective at increasingthe gel strength for i-carrageenan than potassium, which is best foroptimization of gel strength in k-carrageenan samples(Thrimawithana et al., 2010). We observed a significant differencein gel strength between the water e and alkali-extracted i-carra-geenan samples, corroborating that an effective alkali-modificationprocedure was accomplished (Table 3). For the i-carrageenansamples, a change in slope occurred close to 30e40 �C which wasevident only in a non-logarithmic plot (Fig. 2B). This change inslope may be attributable to the co-existence of k-carrageenan inthe sample as occurrence of hybrid carrageenans has beendescribed previously (Blanco-Pascual, Alem�an, G�omez-Guill�en, &Montero, 2014; van de Velde, Peppelman, Rollema, & Hans,2001). The observed type of gelling behavior is believed to be aresult of a two-step gelation process (Parker, Brigand, Miniou, &Trespoey, 1993). The presence of k-carrageenan should ideally in-crease the gel strength, since k-carrageenan makes stronger gelsthan i-carrageenan, but the abrupt drop in gel strength appears tocontradict this expectation. Nevertheless, the sudden drop in slope(Fig. 2B) was not evident when examining the logarithmic plot fori-carrageenan more closely (Figure S2). Serrated plates were usedto avoid risk of sliding of the samples. Thus the observed drop in gelstrength may be due to minor syneresis, which may influence themeasurements due to the formation of a solvent layer (Chen et al.,2002; Parker et al., 1993; Richardson & Goycoolea, 1994).

The water-extracted agar from H. dentata had gel strength at25 �C of around 10 Pa, much lower than the one obtained from thestandard grade agar (Fig. 2C). The alkali-extracted agar fromH. dentata on the other hand, showed a gel strength which wasstatistically similar to the commercial sample (Table 4). The gelstrength of commercial agar kept increasing when further cooled,while the one extracted from H. dentata seemingly reached itsmaximum around 20e25 �C (Fig. 2C). The gel strength measuredfor the agar samples were generally much lower than those for k-

1% added KCl and 1.5% agar in milli-Q water. Gel strength at 25 �C was determined ascript letters indicate significantly different values (p < 0.05) by ANOVA (carrageenan

�C [Pa]2 (G0 at z25 �C) Tgel [�C] (G' > G00) Tmelt [�C] (G' > G00)

33 5636 6033 5636 6074 e

71 e

32 5335 5541 5770 8038 7452 e

48 e

.

N. Rhein-Knudsen et al. / Food Hydrocolloids 63 (2017) 50e58 57

and i-carrageenan (Fig. 2). The low gel strength of the putative agarhydrocolloids, is likely due to the agar beingmeasured in absence ofionic interaction (i.e. no ions added for gelation), whereas the gelstrengths for the carrageenans were measured according to thestandard procedure in the presence of potassium ions to promotethe gel formation.

The gelling temperatures Tgel and melting temperatures Tmeltwere estimated for all the hydrocolloid samples and were definedas the point in which the storage modulus G0 exceeded the value ofthe loss modulus G’’ (G’ > G00), during cooling and re-heatingrespectively (Table 4) (Hossain, Miyanaga, Maeda, & Nemoto,2001). Comparing the water-extracted with the alkali-extractedhydrocolloids, we observed that both the gelling and meltingtemperatures were slightly lower for the water-extracted hydro-colloids. The alkali- and water-extracted hydrocolloid fromC. crenulata, assumed to contain mainly i-carrageenan poly-saccharides, had higher gelling temperature compared to that of k-carrageenan, which was in agreement to standard grade sample i-carrageenan. This result highly indicated that the gelling charac-teristics of C. crenulata carrageenan were similar to those of iotacarrageenan as exemplified by the benchmark sample. The deter-mined gelling and melting temperatures for H. dentata andH. musciformis was similar to those reported earlier by John andAsare (John & Asare, 1975). The gelling temperature is dependenton the concentration of the negatively charged sulfate groups, asthey inhibit gel formation due to repulsion of the carrageenanchains. The potassium ions promotes the gel formation as theystabilize the “junction zones” between the hydrocolloid helices bybinding to the sulfate groups without hindering cross-linking of thetwo helices. The higher sulfate content and the absence of counterions in the water-extracted hydrocolloids will hence decrease thegelling temperature due to the repulsion effects (Rhein-Knudsenet al., 2015). The gelling temperature for the commercial k-carra-geenan was different from the carrageenans extracted from theGhanaian seaweed used in the present study. As shown in Table 3,the k-carrageenan sample had significantly higher amounts ofsulfate than the other k-carrageenan samples. In addition it alsocontained high amounts of glucose that promotes the increase ofthe gelling temperature. The glucose thus appears to have a highereffect than the charged provided by the sulfate esters.

The gels obtained from seaweed sources are thermo-reversibleand the melting temperatures and corresponding G0 values wereestimated as described above for the gelling temperatures. Theprofiles of sulfate dependences of Tmelt were similar to thosedetermined of Tgel (Table 4). The melting temperatures for thecarrageenan extracted from C. crenulata and the agar derived fromH. dentata could not be determined, as the cross-over between G0

and G00 was not observed within the chosen temperature range(Supplementary material Figures S2, S3).

Carrageenans and agar are commercially extracted by either hotwater- or alkali-treatment techniques. Hot water extraction is usedfor the extraction of native agar and carrageenan, whereas thealkali-treatment is a combined chemical modification and extrac-tion method. The alkali removes the sulfate esters from the pre-cursors, i.e. from porphyran, m-carrageenan, and n-carrageenan,and moreover causes formation of the 3,6-anhydro-bridge on thegalactopyranose moieties, which in turn promotes gel formation asthe de-sulfatation and 3,6-anhydro-bridge formation causeconformational transitions of the polysaccharides (Genicot-Joncouret al., 2009). Hence, both the chemistry and the rheological prop-erties of the red seaweed hydrocolloids are influenced by severalfactors, notably the algal source, life-stage, growth environment,and the final rheological properties are moreover affected by thehydrocolloid extraction method (Rhein-Knudsen et al., 2015).

4. Conclusion

Selected wild red seaweed samples collected along the coastalareas in Ghana were assessed for their potential as new source forhydrocolloid production. The assessments were based on compar-ison of monosaccharides composition, yields and characteristics ofthe extracted hydrocolloids with carrageenan from Kappaphycusalvarezii, the most important source of commercial carrageenan(Hurtado et al., 2015) Rheological characteristics and gellingproperties were also compared with benchmark samples ofstandard-grade carrageenan and agar. Two-step sulfuric acid hy-drolysis and HPAEC-PAD analysis showed that the Ghanaian redseaweeds contained high and similar amounts of galactose, themain component of carrageenan and agar. The extraction tech-nology adopted in this study i.e. water e and alkali-treatment re-quires overnight treatment, high temperature and long extractionhours which can be optimized using milder extraction technologywhich is crucial to preserving the integrity of the hydrocolloids.Nevertheless the yield for carrageenan (21e27%) was very satis-factory similar to the carrageenan yield of K. alvarezii. Moreover, thecomposition analysis of the extracted hydrocolloids indicated suc-cessful modification during alkali-treatment, which was supportedby FTIR analysis and oscillatory rheological measurements. Esti-mation of hydrocolloids rheological properties showed only minordifferences between the hydrocolloids extracted from the Ghanaianred seaweed (H. musciformis) and from K. alvarezii from Vietnam. Acomparison with commercial hydrocolloids revealed that the car-rageenans from the Ghanaian H. musciformis produced gels ofhigher strength due to the absence of glucose and the formation ofanhydro-bridge in the 4-linked galactose unit resulting in lowersulfate content. The C. crenulata collected in Ghana was found tocontain a hybrid carrageenan having the characteristics similar tocommercial i-carrageenan but with k-carrageenan co-existing inthe i-carrageenan matrix. The modification during extraction wasable to promote gelation, resulting in a gel strength exceeding theone for the commercial i-carrageenan. The same trend wasobserved from agar extracted from H. dentata, which showed poorgelling abilities when extracted with water, but was improvedduring alkali-treatment. Analysis of rheological parameters indi-cated that the alkali-treated agar from H. dentata did not producegood gel as the commercially used agar sample, if used at lowertemperatures. Based on the presented analysis of the seaweedhydrocolloids and its characteristics, it is evident that the Ghanaianseaweed samples have potential to be used for extraction of func-tional hydrocolloids. In particular, the carrageenans from theGhanaian H. musciformis showed pronounced similarities with thecarrageenan derived from K. alvarezii and even surpassed the gel-ling abilities of the standard grade k-carrageenan. In addition, theresults clearly showed that the seaweed hydrocolloids compositionand rheological parameters could be enhanced by modificationduring extraction, which is worth noting when considering appli-cation for these hydrocolloid polysaccharides.

Conflict of interest

All authors declare no conflict of interest.

Acknowledgements

This work was funded via the Seaweed Biorefinery ResearchProject in Ghana (SeaBioGha) supported by Denmark's develop-ment cooperation (Grant DANIDA-14-01DTU), Ministry of ForeignAffairs of Denmark. We will also like to thank the Water ResearchInstitute, Council for Scientific Research, Accra, Ghana for theirassistance in collecting the seaweed samples.

N. Rhein-Knudsen et al. / Food Hydrocolloids 63 (2017) 50e5858

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.foodhyd.2016.08.023.

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