Seaweed Extracts as Potential Functional Ingredients in Yogurt
A.M. OSullivan, M.N. OGrady, Y.C. OCallaghan, T. Smyth, N.M.
OBrien, J.P. Kerry
PII:
DOI:
Reference:

S1466-8564(16)30174-6
doi: 10.1016/j.ifset.2016.07.031
INNFOO 1597

To appear in:

Innovative Food Science and Emerging Technologies

Received date:
Revised date:
Accepted date:

23 August 2015
26 July 2016
30 July 2016

Please cite this article as: OSullivan, A.M., OGrady, M.N., OCallaghan, Y.C.,
Smyth, T., OBrien, N.M. & Kerry, J.P., Seaweed Extracts as Potential Functional
Ingredients in Yogurt, Innovative Food Science and Emerging Technologies (2016), doi:
10.1016/j.ifset.2016.07.031

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Seaweed Extracts as Potential Functional Ingredients in Yogurt

A.M. OSullivan1, M.N. OGrady1, Y.C. OCallaghan1, T. Smyth2, N.M. OBrien1, and

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J.P. Kerry1*

School of Food and Nutritional Sciences, College of Science, Engineering and Food

Science, University College Cork, Ireland.

Department of Food BioSciences, Teagasc Food Research Centre, Ashtown, Dublin 15,

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Ireland.

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*Corresponding author: Professor Joseph Kerry, School of Food and Nutritional Sciences,
University College, Cork, Ireland. Phone +353 21 490 3798, Fax: +353 21 4270001,
email: Joe.Kerry@ucc.ie

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ABSTRACT
Yoghurt was manufactured containing extracts (0.25% and 0.5% (w/w)) prepared from

Ascophyllum nodosum (100% H2O (AN100), 80% ethanol : 20% H2O (AN80e)) and Fucus

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vesiculosus (60% ethanol : 40% H2O (FV60e)). Yogurt composition, shelf-life parameters,

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stability and bioactivity of seaweed extracts in yogurt was examined over 28 days.
Yellowness b* was higher (P < 0.05) in yogurts containing FV60e and AN80e. Yogurts
containing AN80e (0.5%) and FV60e (0.5%) had lower (P < 0.05) levels of lipid oxidation.

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The pH, microbiology and whey separation in yogurt were unaffected by seaweed extract

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addition. Yogurt modulus was higher in control yogurts. Control and AN100 (0.25% and
0.5%) yogurts were most accepted by sensory panellists. Antioxidant activity (DPPH) of

seaweed extracts in yogurt was stable as a function of storage time. Yogurt and digestates

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did not affect the antioxidant status (CAT, SOD and GSH assays) or protect against

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H2O2-induced DNA damage in Caco-2 cells.

INDUSTRIAL RELEVANCE

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The research work and results presented in this manuscript are of high industrial
importance.

Yogurt and related products are some of the most commonly manufactured and consumed
food products worldwide. In recent years yogurt and other dairy products have been used
as carriers for functional bioactive food ingredients, or nutraceuticals. Seaweeds contain
a range of bioactive compounds with reported health benefits and represent a potentially
exploitable source of functional ingredients for the dairy industry.

This manuscript

demonstrates that extracts from Ascophyllum nodosum can be incorporated successfully

into a fermented dairy product such as yoghurt.

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Joe P. Kerry, Ph.D.

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Keywords: Seaweed extracts; Yogurt; antioxidants; cell-culture.

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Chemical compounds studied in this article:

Ethanol (PubChem CID: 702); DPPH, Free radical (PubChem CID: 2735032); 2-

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Thiobarbituric acid (PubChem CID: 2723628); Hydrogen peroxide (PubChem CID: 784).

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1. INTRODUCTION
Yogurt and related products are some of the worlds most commonly manufactured and

consumed food products. From a nutritional perspective, yogurt is widely perceived as a

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healthy food as it contains protein, riboflavin, vitamins B6 and B12, and calcium.

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Additionally, in recent years yogurt and other dairy products have been used as carriers
for functional food ingredients, or nutraceuticals. Nutraceuticals are defined as food
components which demonstrate physiological benefits or reduced risk of chronic disease

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beyond their basic nutritional function (Shah, 2001).

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Bifidobacterium and Lactobacillus-enriched probiotic yogurt is among the most common

type of functional food products marketed worldwide. Yogurt is also used commercially

as a carrier for gut-friendly prebiotics (Thomas & Greer, 2010; Mishra Pandey & Mishra,

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2015) and cholesterol lowering phytosterols (Moreau, 2004). Laboratory scale studies
have investigated yogurt as a possible carrier for other functional food ingredients such as

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omega-3 fatty acids, vitamins and minerals (Hekmat & McMahon, 1997; Achanta,
Aryana, & Boeneke, 2007; Sabeena Farvin, Baron, Nielsen, & Jacobsen, 2010).

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Antioxidants such as polyphenols and carotenoids are popular functional ingredients

which exhibit a range of health benefits including anti-cancer, eye protective, heart
protective and anti-diabetic properties (Pandey & Rizvi, 2009). Studies have investigated
yogurt as a potential carrier for antioxidant compounds with the aim of increasing the
health benefits and quality attributes of the resulting product. Cossu, Juliano, Pisu, and
Alamanni (2009) produced yogurts enriched with crude extracts from artichoke (Cynara
scolymus L.), strawberry-tree fruit (Arbutus unedo L.) and cherry (Prunus avium L.) and
found that the fortified yogurts exhibited significantly higher antioxidant activity (total
phenol content (TPC), FRAP and DPPH scavenging activities) compared to the nonenriched yogurt.

Similarly, green and Pu-erh tea infusions (5-15%) increased the

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antioxidant capacity (FRAP and DPPH scavenging activities) of yogurt (NajgebauerLejko, Sady, Grega, & Walczycka, 2011).

Brown seaweeds such as Ascophyllum nodosum and Fucus vesiculosus contain a range of

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proteins, fats, carbohydrates, minerals, vitamins and bioactive compounds (Miyashita,

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Mikami, & Hosokawa, 2013). Polyphenolic compounds previously quantified in brown

seaweed include caffeic acid, chlorogenic acid, coumaric acid and catechins (Keyrouz et
al., 2011). Phlorotannins, polyphenolics unique to marine algae, are found at the highest

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levels (2 10%) in brown algal species (Ragan & Glombitza, 1986).

Due to the

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complexity and wide diversity of compounds present in seaweeds, characterisation and

quantification of target compounds is difficult. In vitro antioxidant assays (e.g. total

phenol content (TPC), FRAP and DPPH radical scavenging activities) are frequently used

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to assess the antioxidant activity and potency of seaweed extracts (Zaragoz et al., 2008).
Polyphenol rich seaweed extracts are commonly prepared using polar solvents such as

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water and/or alcohol and represent a concentrated form of bioactive compounds (e.g.
antioxidants) present in seaweed.

Such extracts are a potentially unique source of

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functional food ingredients. To date, the scientific literature contains limited data on the
toxicological evaluation of seaweed extracts. However, Zaragoz et al. (2008) reported
non-toxic effects of Fucus vesiculosus extracts in vivo.
A number of seaweed and seaweed-derived extracts have demonstrated superior
antioxidant activity compared to terrestrial plants (Budhiyanti, Raharjo, Marseno, &
Lelana, 2011). OSullivan et al. (2011) demonstrated that crude water-methanol prepared
extracts from a range of brown seaweeds exhibited in vitro antioxidant activity and DNA
protective effects against H2O2-induced DNA damage in Caco-2 cells. Similar findings
were reported for water and aqueous ethanol extracts of Ascophyllum nodosum and Fucus
vesiculosus (OSullivan et al., 2013). In the scientific literature, studies investigating the

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addition of seaweed extracts to dairy products are limited. A recent study by our research
group examined the potential of seaweed extracts as functional antioxidant ingredients in

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product such as yoghurt is unkown and merits investigation.

milk (OSullivan et al., 2014). The behaviour of such extracts in a fermented dairy

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The objective of this study was to manufacture yogurt containing three seaweed extracts
(Ascophyllum nodosum: 100% H2O (AN100), 80% ethanol : 20% H2O (AN80e) and Fucus
vesiculosus: 60% ethanol : 40% H2O (FV60e)). The effect of seaweed extracts (0.25 and

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0.5%) (w/w) on the quality and shelf-life (colour, lipid oxidation, pH, microbiology,

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whey separation, rheology, and sensory analysis) of yogurts was investigated over a 28
day storage period. The stability of seaweed extracts in yogurt was assessed using the
Seaweed extract enriched yogurts were

DPPH radical scavenging activity assay.

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subjected to an in-vitro digestion procedure to assess the antioxidant capacity of the

yogurts before and after digestion.

Antioxidant activity of the yogurt and yogurt

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digestates was determined using in-vitro antioxidant (DPPH radical scavenging activity
and ferrous-chelating activity (FICA)) and cellular antioxidant (catalase (CAT),

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superoxide dismutases (SOD) and glutathione (GSH)) assays. The ability of yogurt and
yogurt digestates to protect against oxidant-induced DNA damage in human
adenocarcinoma Caco-2 cells was also investigated.

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2. MATERIALS AND METHODS
2.1 Materials

Cell culture materials were as described in

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obtained from CHR Hansen (Cork).

Fresh whole milk was obtained from a local retail outlet. Yogurt culture (Yo-flex) was

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OSullivan et al. (2013). Agar was purchased from Oxoid Ltd., Basingstoke, Hampshire,
England.

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2.2 Addition of seaweed extracts to milk and yogurt manufacture

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Ascophyllum nodosum was collected from New Quay, Co. Clare, Ireland and Fucus
vesiculosus was harvested from Spiddal, Co. Galway, Ireland and transported to the

laboratory in the National University of Ireland, Galway (NUIG) for taxonomic

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identification, washing and storage at -20C. Seaweed samples were supplied to the
Teagasc Food Research Centre (Ashtown, Dublin 15, Ireland) for seaweed extract

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manufacture. Seaweeds were freeze-dried at -20C for 72 hrs, vacuum-packed and stored
at -80C prior to extraction.

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Seaweed extracts (Ascophyllum nodosum: 100% H2O (AN100), 80% ethanol : 20% H2O
(AN80e) and Fucus vesiculosus: 60% ethanol : 40% H2O (FV60e)) were manufactured and
characterised (total phenol content (TPC) - FV60e > AN80e > AN100 (P < 0.05); DPPH
radical scavenging (AN100 = AN80e = FV60e) and ferrous-ion chelating activity (FICA AN100 = AN80e > FV60e (P < 0.05) assays) as described in OSullivan et al. (2014).
Extracts (AN100, AN80e and FV60e) were added to milk (1000 ml) at concentrations of
0.25% and 0.5% (w/w) and mixed for 5 hrs at 4C with a magnetic stirrer to aid
dissolution. Seaweed extract-containing milk was heated in a waterbath until 93C was
reached and then held at this temperature for 15 minutes and subsequently cooled to
43C. Yogurt culture (Yo-flex, CHR Hansen) was added at a concentration of 0.1% (v/v)

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and milk samples were further incubated at 43C until a pH of 4.5 was reached. Yogurt
samples were subsequently stirred and ~ 95g portions were packaged aseptically in 100

ml sterile containers (Sarstedt Ltd., Co. Wexford, Ireland) and stored for 28 days at 4C.

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Quality and shelf-life measurements (colour, lipid oxidation, pH, microbiology, whey

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separation, rheology, and sensory analysis) were recorded at 7 day intervals up to 28 days
of storage.

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2.3 Compositional analysis

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The moisture and fat content of yoghurt were measured using the SMART Trac rapid
moisture/fat analyser (CEM Corporation, NC, USA).

Protein nitrogen (x 6.38) was

determined by the Kjeldahl method of the Association of Official Analytical Chemists

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(AOAC, 1995). The ash content was determined using a muffle furnace (AOAC, 1995).
The carbohydrate content was calculated by difference.

The composition of

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commercially available natural yoghurt was also analysed for comparative purposes.

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Compositional analysis results were expressed as percentage values, %.

2.4 Surface colour measurement of yogurt

The surface colour of yogurt samples was measured by Minolta colorimetry as described
in OSullivan et al. (2014).

2.5 Measurement of lipid oxidation in yogurt

Lipid oxidation in yogurt was measured following the procedure of Fenaille, Mottier,
Turesky, Ali, and Guy (2001). Results were expressed as 2-thiobarbituric acid-reactive
substances (TBARS) in mg malondialdehyde (MDA)/kg yogurt.

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2.6 pH and microbiology of yogurt
Yoghurt pH was measured using a pH meter (Seven Easy portable, Mettler-Toledo

GmbH, Switzerland) by directly inserting the pH probe into the yogurt samples.

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Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus counts were

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determined in yoghurt using the pour plate technique with M17 agar (supplemented with
sterile lactose solution (10% w/v)) (Oxoid Ltd.) and MRSA (de Man, Rogosa and Sharpe
agar), respectively. The MRSA plates were placed in heat-sealed bags in the presence of

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Anaerocult A (Merck Millipore, Germany). The MRSA and M17 plates were incubated

2.7 Whey separation in yoghurt

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at 37C for 4 days. Results were expressed as log10CFU (colony forming units)/g yogurt.

Whey separation in yogurt was assessed according to the method of Keogh and Kennedy

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(1998) with modifactions. The % whey separation was measured as follows:

Whey separation, %, = ((Original weight of yogurt - weight of yogurt after whey

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removal) / original weight of yogurt) 100.

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2.8 Rheology of yoghurt

The rheological assessment of the yogurt samples was carried out using a rheometer
(Brookfield RS, Lab Unlimited, Dublin, Ireland) with a V-40/20 vane spindle attachment.
Yogurt samples were removed from refrigerated storage immediately prior to rheology
measurements. The spindle was directly inserted into each yogurt sample, a shear rate of
0.05 s-1 was applied to each sample for 2 min and the shear modulus (G) (tendency to
deform when acted on by opposing forces) was measured.

2.9 Sensory analysis of yogurt

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Sensory analysis of yogurt was performed as described in OSullivan et al. (2014).
Sensory analysis descriptors were colour, texture, odour, flavour, off-flavour, thickness

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and overall acceptability.

2.10.1 Stability of seaweed extracts in yogurt

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2.10 Stability and bioactivity of seaweed extract enriched yogurt

The stability of seaweed extracts in yogurt samples over the storage period was

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determined using the DPPH radical scavenging activity as described in OSullivan et al.

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(2014). The % DPPH radical scavenging activity (stability) of yogurts was measured at 7
day intervals up to 28 days of storage.

2.10.2 In-vitro digestion of yogurt

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Yogurt samples (containing 0.5% AN100, AN80e and FV60e) (1 g) were dissolved in 10 ml
HBSS and shaken vigorously. In-vitro digestion of yogurt samples was carried out

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following the method of Daly, Jiwan, OBrien, and Aherne (2010).

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2.10.3 Effect of digestion on the antioxidant activity of seaweed enriched yogurts

Yogurt and subsequent digestions were diluted in water or methanol (1:10) for % FICA
and % DPPH radical scavenging activity, respectively as described by OSullivan et al.
(2014).

2.10.4 Cellular antioxidant activity of yogurt and yogurt digestates

Human colon adenocarcinoma Caco-2 cells were maintained as described by OSullivan
et al. (2011). Caco-2 cells were supplemented with increasing concentrations (0-15
mg/ml) of undigested and digested yogurt samples for 24 hrs.

The cytotoxicity of

seaweed extracts enriched yogurt and yogurt was assessed in Caco-2 cells using the MTT

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assay (MTT I proliferation kit, Roche Diagnostics, UK). Non toxic concentrations for
digested and undigested yogurt samples were determined to be at concentrations of 10

mg/ml media (0.05 mg seaweed extract) and 0.66 mg/ml media (0.0033 mg seaweed

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extract), respectively. For the determination of cellular enzymatic activity, Caco-2 cells

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were supplemented with seaweed extract enriched yogurt and yogurt digestates for 24 hrs.
Following incubation, catalase (CAT) and superoxide dismutases (SOD) activities, and
glutathione (GSH) levels were determined. The Comet assay was used to access the

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potential DNA protective effects of seaweed enriched yogurt and yogurt digestates in

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Caco-2 cells following 30 min incubation with 50 M H2O2.

2.11 Statistical analysis

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Statistical analysis for surface colour, lipid oxidation, pH, microbiology, whey separation,
and rheology measurements was by repeated measures ANOVA followed by Dunnett's

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test or Tukeys test (Prism 4.0, GraphPad Inc, San Diego, CA, USA). Each experiment
was carried out four times and results are presented as mean values the standard error of

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the mean (SEM). The level of statistical significance was P < 0.05.
ANOVA-Partial Least Squares Regression (APLSR) was used to process the mean data
accumulated from the 26 test subjects during sensory analysis and shelf-life evaluation
using instrumental methods. The X-matrix was designated as 0/1 for treatment and days
with the Y-matrix designated as sensory and instrumental variables. The optimal number
of components in the ASLSR models presented was determined to be 6 principal
components. PC1 versus PC2 is presented as the other PCs did not yield additional
information. In these models, assessor and session level effects were removed using level
correction.

The validated explained variance for the model constructed for yogurt

samples was 69% and the calibrated variance was 73.19%.

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To derive significance

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indications for the relationships determined in the quantitative APLSR, regression
coefficients were analyzed by jack-knifing which is based on cross-validation and

stability plots (Martens & Martens, 1999, 2001). All analyses were performed using

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Unscrambler Software, version 9.8 (CAMO ASA, Trondheim, Norway).

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3. RESULTS AND DISCUSSION
3.1 Compositional analysis of seaweed extract enriched yogurt

The composition of yoghurts containing seaweed extracts were similar to the control

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yoghurt and not affected by seaweed extract type or concentration (Table 1). Similar

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protein and ash concentrations in yogurt were reported by Sabeena Farvin et al. (2010).
Yoghurt containing seaweed extracts were comparable to the commercial natural yoghurt
with respect to fat and ash contents.

The commercial yoghurt contained higher

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ingredient in the yoghurt formulation.

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carbohydrate and protein levels presumably due to the addition of milk protein as an

3.2 Surface colour of yogurt

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The addition of seaweed extracts did not significantly affect the surface lightness L*
values of yogurt (Table 2). Yogurts containing aqueous extracts from A. nodosum (AN100

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(0.25%) and AN100 (0.5%)) had significantly (P < 0.05) lower -a* values compared to
other yogurts. Yellowness b* values were significantly (P < 0.05) higher in yogurts

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containing the extracts AN80e (0.5%), FV60e (0.25%) and FV60e (0.5%). Similar results
were reported in a previous study (OSullivan et al., 2014) where milk samples containing
AN80e (0.25%), AN80e (0.5%), FV60e (0.25%), and FV60e (0.5%) had higher b*
yellowness values. The L*, -a* and b* values of the seaweed extract enriched
yogurts did not change significantly over the 28 day storage period.

3.3 Lipid oxidation in yogurt

Lipid oxidation increased in each of the yogurt samples (Table 3) and was significantly
positively correlated (P < 0.001) with days 14, 21 and 28 of storage (Table 5). Yogurts
containing AN80e (0.5%) and FV60e (0.5%) exhibited significantly (P < 0.05) lower levels

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of lipid oxidation compared to the control and other yogurts, which may be attributed to
various antioxidant compounds present in the extracts. A previous study (OSullivan et

al., 2014) found that FV60e (0.25%) significantly reduced the level of lipid oxidation in

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milk.

3.4 pH, microbiology, whey separation and rheology of yogurt

On each analysis day, the pH and growth of S. thermophilus and L. bulgaricus in yoghurts

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were unaffected (P > 0.05) by seaweed extract type or concentration (Table 4). The pH

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of yoghurts was significantly negative correlated (P < 0.001) with days 14, 21 and 28 of
storage (Table 5) reflecting minor decreases in pH as a function of storage time. Whey
separation was similar for all yoghurt samples on each analysis day and overall rates

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ranged from ~ 15 21% (Table 4). Similarly, Brignac and Aryana (2012) reported that
the addition of antioxidant compounds (vitamin C, vitamin E and -carotene) did not

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influence whey separation in yoghurt. In general, the modulus of yoghurts varied as a

function of storage time (Table 4). On day 28 of storage, all yoghuts, with the exception

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of AN100 (0.5%) and AN80e (0.25%), had significantly lower modulus values compared to
the control, indicating that seaweed extracts influenced the rheology of yoghurt at the end
of the storage period. The modulus of yogurts was lower than that reported by Lee and
Lucey (2004) which may be attributed to lower starter culture inoculation rates (0.1%)
used in the present study.

3.5 Sensory evaluation of yogurt

Sensory evaluation, instrumental and chemical analysis data is presented in Table 5. In
general, positive and negative correlations indicate like and dislike of selected attributes,
respectively. The control yogurt (P < 0.001) and yogurts containing AN100 (0.25%) (P <

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0.001), AN100 (0.5%) (P < 0.05) and FV60e (E) (0.5%) (P < 0.001) were significantly
positively correlated with the colour descriptor. Yogurt containing AN80e (0.5%) and

FV60e (0.5%) exhibited a negative correlation to colour (Table 5) probably due the higher

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instrumental b* yellowness values (Table 2) observed in these yogurts.

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The control yogurt (P < 0.001) and AN100 (0.25%) (P < 0.01) and AN100 (0.5%) (P <
0.001) containing yogurts were positively correlated to texture. Yogurts containing AN80e
(0.25%) (P < 0.01) and AN80e (0.5%) (P < 0.001) were found to be negatively correlated

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to odour. The flavour of the control yogurt (P < 0.001) and yogurts containing AN100

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(0.25%) (P < 0.05) and AN100 (0.5%) (P < 0.01) were the most preferred by sensory
panellists. Yogurts containing AN80e (0.25%), AN80e (0.5%) and FV60e (0.5%) (P < 0.01)

were negatively correlated to flavour and it is postulated that the undesirable colour of

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these yogurts may have adversely biased the flavour-liking scores of sensory panellists.
In traditional foods like yogurt unexpected colours can negatively affect flavour

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perception (Sanz et al., 2008). The yogurt containing AN80e (0.5%) (P < 0.001) exhibited
a positive correlation to off-flavour however the other yogurts were not affected by the

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addition of seaweed extracts. The control yogurt (P < 0.001) and yogurts containing
AN100 (0.25%) (P < 0.01) and AN100 (0.5%) (P < 0.01) were positively correlated to
overall acceptability (Table 5). A previous study (OSullivan et al., 2014) found that the
control milk and AN100 (0.5%) containing milk were positively correlated to overall
acceptability. Yogurts containing AN80e (P < 0.01) and FV60e (P < 0.01) were negatively
correlated to overall acceptability indicating reduced acceptability by sensory panellists.
Overall the data suggested that colour, flavour and texture were the three most important
parameters governing the overall acceptability of yogurt containing seaweed extracts.
The sensory properties of yogurt as a function of time were also examined (Table 5).
Odour, flavour, and overall acceptability were positively correlated to day 1, 7 and 14 and

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negatively correlated to day 21 and 28. These results indicated that sensory attributes of
yogurt decreased after day 14 of storage.

Negative sensory associations with some of the seaweed extract enriched yogurts

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examined in the present study could potentially be addressed using food colorants,

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flavourings or by micro-encapsulation of the extracts in order to mask undesirable colour

and flavours associated with seaweed extracts (Petrotos et al., 2012).

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3.6 Stability of seaweed extracts in yogurt

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The DPPH radical scavenging activity of all yogurts was similar over the 28 day storage
period indicating stability of the seaweed extracts in yogurt (Figure 1). All seaweed

extract-enriched yogurt samples had significantly (P < 0.05) higher DPPH radical

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scavenging activities compared to the corresponding controls. Yogurts containing FV60e

(0.25%) and FV60e (0.5%) exhibited the highest DPPH radical scavenging activity similar

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to that of Trolox (0.04 M) standard. These findings indicated that the seaweed extracts
are stable antioxidant ingredients within a fermented dairy product such as yogurt.

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Similarly, OSullivan et al. (2014) reported that seaweed extracts were stable in milk as a
function of storage time.

3.7 In-vitro antioxidant activity of yogurts and digestates

The antioxidant potential of the seaweed enriched yogurts was determined before and
after an in-vitro digestion procedure by measuring the DPPH radical scavenging activity
and the ferrous iron chelating ability (FICA). The yogurt containing FV60e (0.5%) was
found to have a significantly (P < 0.05) higher radical scavenging activity than the control
before digestion. In addition, DPPH radical scavenging activity only occurred in FV60e
(0.5%) digestate following the in-vitro digestion procedure (Figure 2). DPPH scavenging

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activity has also been shown to decrease following in-vitro digestion in wild-grown caper
(Capparis spinosa L.) and sea fennel (Crithmum maritimum L.) (Siracusa et al., 2011).

Antioxidant compounds such as polyphenols may be degraded during digestion due to

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alterations in the pH and enzymatic hydrolysis (Nagah & Seal, 2005). A previous study

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(OSullivan et al., 2014) found contrasting results where the DPPH-scavenging activity of
seaweed-enriched milk samples was stable during digestion. This indicated that seaweed
extracts prepared from Ascophyllum nodosum were less stable in fermented milk than in

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non-fermented milk products.

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Prior to digestion, the yogurt containing FV60e exhibited the highest FICA (P < 0.05)
while the other yogurt samples had similar FICA. The FICA of all yogurt samples

increased following digestion and there was no significant difference between the

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different digestates (Figure 3). The increased FICA exhibited in the yogurt digestates
may be attributed to the presence of milk peptides and iron-chelating components such as

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polyphenols from seaweed extracts released during the in-vitro digestion procedure
(Hurrell, Reddy, Juillerat, & Cook, 2006).

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The addition of seaweed-enriched yogurt or digestates did not significantly alter the
antioxidant status (CAT, SOD and GSH) (data not shown) or protect against H2O2
induced DNA damage (Figure 4) in Caco-2 cells. It has been reported previously that
crude extracts and isolated compounds from seaweed enhanced antioxidant enzyme
activity and protected against oxidant induced DNA damage in cells (Kang et al., 2005;
OSullivan et al., 2011) however, at comparable seaweed extract concentrations, the
seaweed supplemented yogurts did not demonstrate antioxidant activity in Caco-2 cells.
Previous studies have found that, in the presence of milk proteins, the DPPH scavenging
and antioxidant activity of a number of polyphenol compounds was reduced due to the
formation of polyphenol-milk protein complexes (Xiao et al., 2011; Lorenz et al., 2007).

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Furthermore, heat denatured proteins, such as those found in yogurt, are more likely to
form polyphenol-milk protein complexes due to the increased exposure of polyphenol

binding sites (Siebert, Troukhanova, & Lynn, 1996). This may explain the lack of

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cellular antioxidant activity by the undigested yogurt samples. The lack of cellular

of compounds present in the seaweed extracts.

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4. CONCLUSIONS

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antioxidant activity in digested yogurt samples may be due to pH-mediated modification

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Seaweed extract enriched yogurts were manufactured containing seaweed extracts which
altered selected characteristics of the resulting yogurts. In particular, yogurts containing

FV60e (0.25%), FV60e (0.5%) and AN80e (0.5%) had higher yellowness b* values than

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other yogurt samples. From a quality perspective, yogurts containing AN80e (0.5%) and
FV60e (0.5%) exhibited lower levels of lipid oxidation.

Parameters such as pH,

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microbiology and whey separation were not affected by the addition of seaweed extracts
to yogurt suggesting that seaweed extracts may be added to yogurt without negatively

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affecting shelf-life characteristics. Sensory analysis indicated that colour, flavour and
texture were the three most important parameters governing the overall acceptability of
seaweed extract-enriched yogurt. Yogurts containing AN100 (0.25%) and AN100 (0.5%)
were the most acceptable yogurts from a sensory perspective. Results from the in-vitro
antioxidant assays indicated that the ferrous-ion chelating activity of yogurt was stable
after digestion, however the DPPH radical scavenging activity was not stable.

In

addition, the seaweed extract-enriched yogurts did not exhibit cellular antioxidant activity
indicating reduced biological activity of extracts when added to yogurt. Further research
is needed to evaluate the potential of additional seaweed derived ingredients as functional
components in fermented dairy products.

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ACKNOWLEDGEMENT
This project (Grant-Aid Agreement No. MFFRI/07/01) is carried out under the Sea

Change Strategy with the support of the Marine Institute and the Department of

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Agriculture, Food and the Marine (NutraMara programme), funded under the National

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Development Plan 2007-2013.

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Zaragoz, M. C., Lpez, D., Siz, M. P., Poquet, M., Prez, J., Puig-Parellada, P.,
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in vivo of two Fucus vesiculosus extracts.

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Chemistry, 56, 7773-7780.

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FIGURE LEGENDS

denotes significantly higher (P <

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to 28 days at 4C. Mean values standard error bars.

Figure 1. The DPPH-scavenging activity (%) (stability) of yogurt samples stored for up

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0.05) DPPH radical scavenging activity, on each measurement day, between seaweed
extract enriched yoghurt samples and the corresponding controls.

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Figure 2. The DPPH-scavenging activity (%) of yogurt samples before and after an in*

denotes significantly

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vitro digestion procedure. Mean values standard error bars.

higher (P < 0.05) DPPH radical scavenging activity in yoghurt samples.

denotes

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corresponding digestate.

significantly lower (P < 0.05) DPPH radical scavenging activity between yoghurt and the

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Figure 3. The ferrous-ion chelating activity (FICA) (%) of yogurt samples before and
after an in-vitro digestion procedure. Mean values standard error bars.
+

denotes

denotes significantly higher

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significantly higher (P < 0.05) FICA in yoghurt samples.

(P < 0.05) FICA between yoghurt digestates and the corresponding yoghurt samples.

Figure 4. DNA damage in Caco-2 cells following pre-treatment with or without seaweed
enriched yogurt or yogurt digestates for 24 hrs and exposed to 50 M H2O2. Mean values
standard error bars. No significant differences between the H2O2 challenge, yoghurt
and yoghurt digestate samples (P > 0.05).

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Fat

Protein

Ash

Carbohydrate

Control

88.00.2

2.70.2

3.00.1

0.60.1

5.70.1

AN100 (0.25%)

87.80.1

2.60.1

3.00.1

0.60.1

AN100 (0.5%)

87.70.1

2.80.2

3.10.2

IP

5.90.2

0.70.1

5.70.5

AN80e (0.25%)

88.10.3

2.80.2

2.80.1

0.70.1

5.60.4

AN80e (0.5%)

87.70.1

2.70.2

3.10.2

0.70.1

5.80.3

FV60e (0.25%)

88.20.3

2.40.1

3.00.1

0.70.1

5.80.5

FV60e (0.5%)

87.90.3

2.50.1

0.70.1

5.70.4

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Moisture

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Composition, %

3.10.2

AC

CE
P

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Commercial
84.90.1
2.40.2
4.80.2
0.70.1
7.20.4
yogurt
Table 1. Compositional analysis of yogurt containing seaweed extracts.

Mean values SEM.

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Table 2.

Effect of seaweed extract addition on the surface lightness (L* value),

greenness

89.01.5

92.51.5

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91.41.6

AN100 (0.25%)b

84.40.8

84.90.5

85.61.9

87.41.3

89.00.3

AN100 (0.5%)c

83.52.6

84.72.7

84.61.6

87.41.4

85.30.1

AN80e (0.25%)d

87.72.6

85.02.4

84.72.4

84.92.0

83.50.1

AN80e (0.5%)e

81.53.1

83.71.6

84.31.6

84.91.4

85.90.2

FV60e (0.25%)f

85.74.5

81.32.4

82.81.5

82.01.1

82.70.1

FV60e (0.5%)g

79.35.7

80.11.5

77.51.5

79.01.1

76.31.8

Controlh

-4.00.1i,j

-4.00.2i,j

-3.80.1i,j

-4.00.2i,j

-3.90.1i,j

AN100 (0.25%)i

-2.10.4h,k-n

-2.30.4h,k-n

-2.20.3h,k-n

-2.30.3h,k-n

-1.80.1h,k-n

AN100 (0.5%)j

-1.70.3h,k-n

-1.70.4h,k-n

-1.30.4h,k-n

-1.70.4h,k-n

-1.00.1h,k-n

AN80e (0.25%)k

-3.80.1i,j

-3.70.3i,j

-3.80.2i,j

-3.80. i,j

-3.40.i,j

AN80e (0.5%)l

-4.00.2i,j

-4.10.2i,j

-4.10.1i,j

-4.20.3i,j

-3.70.1i,j

FV60e (0.25%)m

-4.60.3i,j

-4.30.3i,j

-4.10.1i,j

-4.20.2i,j

-3.80.2i,j

FV60e (0.5%)n

-4.60.5i,j

-4.30.3i,j

-4.30.1i,j

-4.20.3i,j

-3.70.2i,j

Controlo

13.31.9s-u

12.51.2s-u

11.20.5s-u

12.91.2s-u

10.90.1s-u

AN100 (0.25%)p

14.91.2s-u

15.30.7s-u

13.30.1s-u

14.50.5s-u

13.80.4s-u

AN100 (0.5%)q

15.30.4s-u

15.90.7s-u

16.10.7s-u

16.60.4s-u

15.50.5s-u

AN80e (0.25%)r

15.40.3s-u

15.91.1s-u

16.11.4s-u

16.01.0s-u

17.50.2s-u

AN80e (0.5%)s

18.70.7o-r,t-u

19.60.8o-r,t-u

18.90.7o-r,t-u

19.30.5o-r,t-u

18.70.5o-r,t-u

FV60e (0.25%)t

26.11.3o-s,u

25.71.2o-s,u

25.60.5o-s,u

25.81.2o-s,u

23.50.5o-s,u

FV60e (0.5%)u

30.81.3o-t

29.61.1o-t

29.90.4o-t

29.71.3o-t

26.90.3o-t

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88.62.1

AC

Yellowness
b*

28

82.32.5

CE
P

Greenness
-a*

21

Controla

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Lightness
L*

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Storage time
at 4C, days
14

(-a*) and yellowness (b value) of yogurt stored for up to 28 days at 4C.

(a-g) Mean ( SEM) L* lightness values, data without superscripts indicates no significant differences (P
> 0.05). (h-n) Mean ( SEM) -a* greenness values, where h, i, j, k, l, m and n denote significant differences (P <
0.05) from the control, AN100 (0.25 and 0.5%), AN80e (0.25 and 0.5%) and FV60e (0.25 and 0.5%),
respectively. (o-u) Mean ( SEM) b* yellowness values where o, p, q, r, s, t and u denote significant differences
(P < 0.05) from the control, AN100 (0.25 and 0.5%), AN80e (0.25 and 0.5%) and FV60e (0.25 and 0.5%),
respectively.

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Table 3. Lipid oxidation in seaweed-enriched yogurt stored for up to 28 days at 4C.

7

14

21

28

Controla

0.120.06

0.160.03

0.210.02

0.230.02

0.220.01e,g

AN100 (0.25%)b

0.090.05

0.130.03

0.140.02

0.160.01

0.200.01e,g

AN100 (0.5%)c

0.100.05

0.170.02

0.150.03

0.180.02

0.180.03e,g

AN80e (0.25%)d

0.090.05

0.140.04

0.120.03

0.170.02

0.180.01e,g

AN80e (0.5%)e

0.090.04

0.110.04

0.110.03

0.120.03

0.16 0.01a-f

FV60e (0.25%)f

0.100.05

0.120.03

0.100.04

0.140.02

0.190.02e,g

FV60e (0.5%)g

0.110.06

0.100.03

0.090.03

0.140.02

0.15 0.01a-d,f

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Storage time at 4C, days

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CE
P

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(a-g) Mean ( SEM) values without superscripts indicates no significant differences (P > 0.05), a, b, c, d, e, f and g
denote significant differences (P < 0.05) from the control, AN100 (0.25 and 0.5%), AN80e (0.25 and 0.5%)
and FV60e (0.25 and 0.5%), respectively.

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Table 4. Effect of seaweed extract addition on the pH, microbiology, whey separation
Yoghurt parameters
Rheology,

separation,

Shear Modulus,

6.5 7.5

14.7 16.4

55.0 98.5

6.5 7.2

17.4 21.3

43.5 50.5

7.1 7.5

14.9 21.4

54.5 69.5

6.5 7.5

16.4 21.2

45.5 61.5

bulgaricus,

log10CFU/g

log10CFU/g

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thermophilus,

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pH

Whey

Streptococcus Lactobacillus

4.06 3.92

9.0 9.7

AN100 (0.25%)

4.00 3.88

8.7 9.7

AN100 (0.5%)

4.05 3.92

8.7 9.9

AN80e (0.25%)

4.03 3.93

8.7 9.9

AN80e (0.5%)

4.04 3.92

8.4 9.4

6.5 7.9

16.5 19.1

39.5 51.5

FV60e (0.25%)

4.06 3.95

8.5 9.5

6.5 7.5

15.4 17.3

40.0 60.5

4.09 3.98
and rheology of yoghurt.

8.5 9.5

6.9 7.5

16.1 19.5

33.5 56.5

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FV60e (0.5%)

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Control

AC

CE
P

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Data presented as overall ranges (minimum and maximum) of all values measured at 7 day intervals over
28 days of storage.

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Table 5. P-values of regression coefficients as derived by jack-knife uncertainty testing.

Significance of regression coefficients; ns, not significant, *95% significance, P < 0.05, *99% significance,
P < 0.01, ***99.9% significance, P < 0.001.

AN100
(0.5
%)

AN80e
(0.25
%)

AN80e
(0.5
%)

0.001

0.001

0.05*

***

***

0.506

0.01*

0.001

0.05*

Flavour

Thickne
ss

14

21

28

0.001

0.83n

0.57

0.01*

***

0.001

5ns

0.01*

0.55n

***

0.001

0.212

0.22

0.174

0.103

0.247

***

0.05*

ns

6ns

ns

0.396

0.693

ns

ns

ns

ns

0.054

0.16n

0.229

0.001

0.05

0.001

***

ns

0.01*

0.001

ns

0.107

***

***

0.001

0.001

***

***

***

0.01*

0.21n

0.01*

0.01

0.05*

0.001

0.01*

**

0.05*

0.001

0.001

0.05*

AC

flavour

0.001

***

Off-

***

CE
P

Odour

***

0.001

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Texture

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ns

FV60e
(0.5
%)

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Sensory
Attribu
te
Colour

FV60e
(0.25
%)

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Cont
rol

Stor
age
time
at
4C,
days
AN100
(0.25
%)

Yog
urt

ns

***

0.05*

***

0.21n

0.001

0.12n

0.001

0.375

***

0.73n

0.001

0.05

0.01*

***

ns

***

0.001

0.001

0.084

***

***

ns

0.001

0.77n

0.001

0.95n

0.001

0.23

0.001

***

***

0.18n

0.83n

0.41n

***

ns

***

0.01*

0.001

***

Overall

0.001

0.01*

0.01*

0.32n

0.001

0.01

0.01*

accepta

***

0.061

0.01*

0.01*

***

**

0.001

0.001

ns

***

***

0.05*

0.05*

0.056

bility
Instru
mental
and
chemic
al
analysis
L*

0.001

0.001

0.2ns

34

0.23

0.964

ACCEPTED MANUSCRIPT
***

0.001

0.001

0.13n

***

***

ns

ns

0.001

0.001

0.24n

0.67

0.63n

0.01*

0.001

***

***

***

0.001

0.001

0.001

0.05*

ns

***

***

***

***

0.79

0.18n

0.068

ns

ns

0.83n

0.001

0.001

0.34n

0.001

0.001

0.001

0.001

***

***

***

***

***

***

Lipid

0.001

0.59n

0.001

0.77n

oxidatio

***

***

0.001

0.48n

0.68

0.001

0.001

0.001

0.063

0.001

0.001

ns

***

***

***

***

ns

***

***

0.001

0.001

0.001

0.001

0.39n

0.001

0.144

0.92n

***

***

***

***

0.14

0.001

0.001

0.001

***

ns

ns

***

***

***

Whey

separati

0.001

0.01*

0.001

0.05*

on

***

***

0.001

0.001

0.43n

0.001

0.001

0.22n

0.47n

***

***

***

***

0.4ns

0.001

0.001

0.54n

0.001

***

***

***

0.19n

0.001

***

***

0.05

0.86n

0.001

0.001

7ns

***

***

0.068

ns

0.01

0.555

0.01*

0.368

**

ns

MA

0.001

ns

0.001

0.001

0.62n

***

***

0.19

0.76n

0.001

0.01*

ns

***

AC

CE
P

DPPH

0.96n

Rheolog

NU

TE

IP

pH

ns

0.001

SC
R

b*

0.15n

-a*

***

35

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HIGHLIGHTS

Seaweed extracts from Ascophyllum nodosum and Fucus vesiculosus were added to

SC
R

IP

yoghurt.

Extracts influenced quality parameters (colour and lipid oxidation) in yoghurt.

NU

Colour, flavour and texture were important for sensorial acceptance of yoghurt.

MA

Extract-enriched yogurt or digestates did not alter cellular antioxidant status.

AC

CE
P

TE

Extract-enriched yogurt or digestates did not protect against DNA damage.

36