Professional Documents
Culture Documents
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:
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
This is a PDF le of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its nal form. Please note that during the production process
errors may be discovered which could aect the content, and all legal disclaimers that
apply to the journal pertain.
ACCEPTED MANUSCRIPT
Seaweed Extracts as Potential Functional Ingredients in Yogurt
A.M. OSullivan1, M.N. OGrady1, Y.C. OCallaghan1, T. Smyth2, N.M. OBrien1, and
SC
R
IP
J.P. Kerry1*
School of Food and Nutritional Sciences, College of Science, Engineering and Food
Department of Food BioSciences, Teagasc Food Research Centre, Ashtown, Dublin 15,
NU
AC
CE
P
TE
MA
Ireland.
_______________________________
*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
ACCEPTED MANUSCRIPT
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
IP
vesiculosus (60% ethanol : 40% H2O (FV60e)). Yogurt composition, shelf-life parameters,
SC
R
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.
NU
The pH, microbiology and whey separation in yogurt were unaffected by seaweed extract
MA
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
TE
did not affect the antioxidant status (CAT, SOD and GSH assays) or protect against
CE
P
AC
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
ACCEPTED MANUSCRIPT
Joe P. Kerry, Ph.D.
IP
SC
R
Ethanol (PubChem CID: 702); DPPH, Free radical (PubChem CID: 2735032); 2-
AC
CE
P
TE
MA
NU
Thiobarbituric acid (PubChem CID: 2723628); Hydrogen peroxide (PubChem CID: 784).
ACCEPTED MANUSCRIPT
1. INTRODUCTION
Yogurt and related products are some of the worlds most commonly manufactured and
IP
healthy food as it contains protein, riboflavin, vitamins B6 and B12, and calcium.
SC
R
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
NU
MA
as a carrier for gut-friendly prebiotics (Thomas & Greer, 2010; Mishra Pandey & Mishra,
TE
2015) and cholesterol lowering phytosterols (Moreau, 2004). Laboratory scale studies
have investigated yogurt as a possible carrier for other functional food ingredients such as
CE
P
omega-3 fatty acids, vitamins and minerals (Hekmat & McMahon, 1997; Achanta,
Aryana, & Boeneke, 2007; Sabeena Farvin, Baron, Nielsen, & Jacobsen, 2010).
AC
ACCEPTED MANUSCRIPT
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
IP
SC
R
NU
Due to the
MA
phenol content (TPC), FRAP and DPPH radical scavenging activities) are frequently used
TE
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
CE
P
water and/or alcohol and represent a concentrated form of bioactive compounds (e.g.
antioxidants) present in seaweed.
AC
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
ACCEPTED MANUSCRIPT
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
IP
milk (OSullivan et al., 2014). The behaviour of such extracts in a fermented dairy
SC
R
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
NU
0.5%) (w/w) on the quality and shelf-life (colour, lipid oxidation, pH, microbiology,
MA
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
TE
CE
P
digestates was determined using in-vitro antioxidant (DPPH radical scavenging activity
and ferrous-chelating activity (FICA)) and cellular antioxidant (catalase (CAT),
AC
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.
ACCEPTED MANUSCRIPT
2. MATERIALS AND METHODS
2.1 Materials
IP
Fresh whole milk was obtained from a local retail outlet. Yogurt culture (Yo-flex) was
SC
R
OSullivan et al. (2013). Agar was purchased from Oxoid Ltd., Basingstoke, Hampshire,
England.
NU
MA
Ascophyllum nodosum was collected from New Quay, Co. Clare, Ireland and Fucus
vesiculosus was harvested from Spiddal, Co. Galway, Ireland and transported to the
TE
identification, washing and storage at -20C. Seaweed samples were supplied to the
Teagasc Food Research Centre (Ashtown, Dublin 15, Ireland) for seaweed extract
CE
P
manufacture. Seaweeds were freeze-dried at -20C for 72 hrs, vacuum-packed and stored
at -80C prior to extraction.
AC
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)
ACCEPTED MANUSCRIPT
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.
IP
Quality and shelf-life measurements (colour, lipid oxidation, pH, microbiology, whey
SC
R
separation, rheology, and sensory analysis) were recorded at 7 day intervals up to 28 days
of storage.
NU
MA
The moisture and fat content of yoghurt were measured using the SMART Trac rapid
moisture/fat analyser (CEM Corporation, NC, USA).
TE
(AOAC, 1995). The ash content was determined using a muffle furnace (AOAC, 1995).
The carbohydrate content was calculated by difference.
The composition of
CE
P
commercially available natural yoghurt was also analysed for comparative purposes.
AC
ACCEPTED MANUSCRIPT
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.
IP
SC
R
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
NU
Anaerocult A (Merck Millipore, Germany). The MRSA and M17 plates were incubated
MA
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
TE
CE
P
AC
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.
ACCEPTED MANUSCRIPT
Sensory analysis of yogurt was performed as described in OSullivan et al. (2014).
Sensory analysis descriptors were colour, texture, odour, flavour, off-flavour, thickness
IP
SC
R
The stability of seaweed extracts in yogurt samples over the storage period was
NU
determined using the DPPH radical scavenging activity as described in OSullivan et al.
MA
(2014). The % DPPH radical scavenging activity (stability) of yogurts was measured at 7
day intervals up to 28 days of storage.
TE
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
CE
P
AC
The cytotoxicity of
seaweed extracts enriched yogurt and yogurt was assessed in Caco-2 cells using the MTT
10
ACCEPTED MANUSCRIPT
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
IP
extract), respectively. For the determination of cellular enzymatic activity, Caco-2 cells
SC
R
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
NU
potential DNA protective effects of seaweed enriched yogurt and yogurt digestates in
MA
TE
Statistical analysis for surface colour, lipid oxidation, pH, microbiology, whey separation,
and rheology measurements was by repeated measures ANOVA followed by Dunnett's
CE
P
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
AC
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
11
To derive significance
ACCEPTED MANUSCRIPT
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
AC
CE
P
TE
MA
NU
SC
R
IP
12
ACCEPTED MANUSCRIPT
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
IP
yoghurt and not affected by seaweed extract type or concentration (Table 1). Similar
SC
R
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.
MA
NU
carbohydrate and protein levels presumably due to the addition of milk protein as an
TE
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
CE
P
(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
AC
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.
13
ACCEPTED MANUSCRIPT
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
SC
R
IP
milk.
NU
were unaffected (P > 0.05) by seaweed extract type or concentration (Table 4). The pH
MA
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
TE
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
CE
P
AC
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.
14
ACCEPTED MANUSCRIPT
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
IP
SC
R
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
NU
to odour. The flavour of the control yogurt (P < 0.001) and yogurts containing AN100
MA
(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
TE
these yogurts may have adversely biased the flavour-liking scores of sensory panellists.
In traditional foods like yogurt unexpected colours can negatively affect flavour
CE
P
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
AC
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
15
ACCEPTED MANUSCRIPT
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
IP
examined in the present study could potentially be addressed using food colorants,
SC
R
NU
MA
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
TE
CE
P
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.
AC
Similarly, OSullivan et al. (2014) reported that seaweed extracts were stable in milk as a
function of storage time.
16
ACCEPTED MANUSCRIPT
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).
IP
alterations in the pH and enzymatic hydrolysis (Nagah & Seal, 2005). A previous study
SC
R
(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
NU
MA
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
TE
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
CE
P
polyphenols from seaweed extracts released during the in-vitro digestion procedure
(Hurrell, Reddy, Juillerat, & Cook, 2006).
AC
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).
17
ACCEPTED MANUSCRIPT
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
IP
cellular antioxidant activity by the undigested yogurt samples. The lack of cellular
NU
4. CONCLUSIONS
SC
R
MA
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
TE
other yogurt samples. From a quality perspective, yogurts containing AN80e (0.5%) and
FV60e (0.5%) exhibited lower levels of lipid oxidation.
CE
P
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
AC
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.
18
ACCEPTED MANUSCRIPT
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
IP
Agriculture, Food and the Marine (NutraMara programme), funded under the National
AC
CE
P
TE
MA
NU
SC
R
19
ACCEPTED MANUSCRIPT
REFERENCES
Achanta, K., Aryana, K. J., & Boeneke, C. A. (2007). Fat free plain set yogurts fortified
Association of Official
IP
AOAC (1995).
SC
R
NU
Budhiyanti, S. A., Raharjo, S., Marseno D. W., & Lelana, I. Y. B. (2011). Free radical
MA
298.
Effects of
TE
CE
P
AC
Daly, T., Jiwan, M. A., OBrien, N. M., & Aherne, A. S. (2010). Carotenoid content of
commonly consumed herbs and assessment of their bioaccessibility using an in-vitro
digestion model. Plant Foods for Human Nutrition, 65, 164169.
Fenaille, F., Mottier, P., Turesky, R. J., Ali, S., & Guy, P. A. (2001). Comparison of
analytical techniques to quantify malondialdehyde in milk powders.
Journal of
20
ACCEPTED MANUSCRIPT
Hurrell, R. F., Reddy, M. B., Juillerat, M., & Cook, J. D. (2006). Meat protein fractions
enhance nonheme iron absorption in humans. The Journal of Nutrition, 136, 2808
2812.
IP
Kang, K. A., Lee, K. H., Chae, S., Koh, Y. S., Yoo, B. S., Kim, J. H., Ham, Y. M., Baik,
SC
R
J. S., Lee, N. H., & Hyun, J. W. (2005). Triphlorethol-A from Ecklonia cava protects
V79-4 lung broblast against hydrogen peroxide induced cell damage. Free Radical
Research, 39, 883-892.
NU
Keogh, M., & OKennedy, B. T. (1998). Rheology of stirred yogurt as affected by added
MA
milk fat, protein and hydrocolloids. Journal of Food Science, 63, 108112.
Keyrouz, R., Abasq, M. L., Le Bourvellec, C., Blanc, N., Audibert, L., ArGall, E., &
Total phenolic contents, radical scavenging and cyclic
Hauchard, D. (2011).
TE
3164.
CE
P
of inoculation rate and incubation temperature. Journal of Dairy Science, 87, 3153
AC
Lorenz, M., Jochmann, N., von Krosigk, A., Martus, P., Baumann, G., Stangl, K., &
Stangl, V. (2007).
21
ACCEPTED MANUSCRIPT
using response surface methodology. LWT-Food Science and Technology, 62, 458467.
Chemical and nutritional
IP
SC
R
1507-1517.
NU
New York:
MA
Nagah, A. M., & Seal, C. J. (2005). In vitro procedure to predict apparent antioxidant
release from wholegrain foods measured using three different analytical methods.
TE
Najgebauer-Lejko, D., Sady, M., Grega, T., & Walczycka, M. (2011). The impact of tea
supplementation on microflora, pH and antioxidant capacity of yogurt. International
CE
P
OSullivan, A. M., OCallaghan, Y. C., OGrady, M. N., Hayes, M., Kerry, J. P. &
AC
22
ACCEPTED MANUSCRIPT
activities of seaweed extracts prepared from five brown seaweeds harvested in spring
from the west coast of Ireland. Food Chemistry, 126, 10641070.
Pandey, K. B., & Rizvi, S. I. (2009). Plant polyphenols as dietary antioxidants in human
IP
SC
R
Petrotos, K. B., Karkanta, F. K., Gkoutsidis, P. E., Giavasis, I., Papatheodorou, K. N., &
Ntontos, A. C. (2012). Production of novel bioactive yogurt enriched with olive fruit
polyphenols. World Academy of Science, Engineering and Technology, 64, 867872.
NU
MA
Round & D. J. Chapman (Eds.), Progress in Phycological Research (4 ed., pp. 129 -
Sabeena Farvin, K. H., Baron, C. P., Nielsen, N. S., & Jacobsen, C. (2010). Antioxidant
TE
activity of yogurt peptides: Part 1-in vitro assays and evaluation in -3 enriched milk.
Food Chemistry, 123, 10811089.
CE
P
Sanz, T., Salvador, A., Jimenez, A., & Fiszman, S. M. (2008). Yogurt enrichment with
functional asparagus fibre. Effect of fibre extraction method on rheological properties,
European Food Research and Technology, 227,
AC
Shah, N. P. (2001). Functional foods from probiotics and prebiotics. Food Technology,
55, 4653.
Siebert, K. J., Troukhanova, N. V., & Lynn P. Y. (1996). Nature of polyphenol-protein
interactions. Journal of Agricultural and Food Chemistry, 44, 8085.
Siracusa, L., Kulisic-Bilusic, T., Politeo, O., Krause, I., Dejanovic, B., & Ruberto, G.
(2011). Phenolic composition and antioxidant activity of aqueous infusions from
Capparis spinosa L. and Crithmum maritimum L. before and after submission to a two-
23
ACCEPTED MANUSCRIPT
step in vitro digestion model.
1245312459.
Probiotics and prebiotics in pediatrics.
IP
SC
R
Xiao, J., Mao, F., Yang, F., Zhao, Y., Zhang, C., & Yamamoto, K. (2011). Interaction of
dietary polyphenols with bovine milk proteins: Molecular structureaffinity
relationship and influencing bioactivity aspects.
NU
MA
Zaragoz, M. C., Lpez, D., Siz, M. P., Poquet, M., Prez, J., Puig-Parellada, P.,
Mrmol, F., Simonetti, P., Gardana, C., Lerat, Y., Burtin, P., Inisan, C., Rousseau, I.,
Besnard, M., & Mitjavila, M. T. (2008). Toxicity and antioxidant activity in vitro and
TE
AC
CE
P
24
ACCEPTED MANUSCRIPT
FIGURE LEGENDS
IP
Figure 1. The DPPH-scavenging activity (%) (stability) of yogurt samples stored for up
SC
R
0.05) DPPH radical scavenging activity, on each measurement day, between seaweed
extract enriched yoghurt samples and the corresponding controls.
NU
Figure 2. The DPPH-scavenging activity (%) of yogurt samples before and after an in*
denotes significantly
MA
denotes
TE
corresponding digestate.
significantly lower (P < 0.05) DPPH radical scavenging activity between yoghurt and the
CE
P
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
AC
(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).
25
AC
CE
P
TE
MA
NU
SC
R
IP
ACCEPTED MANUSCRIPT
26
AC
CE
P
TE
MA
NU
SC
R
IP
ACCEPTED MANUSCRIPT
27
AC
CE
P
TE
MA
NU
SC
R
IP
ACCEPTED MANUSCRIPT
28
AC
CE
P
TE
MA
NU
SC
R
IP
ACCEPTED MANUSCRIPT
29
ACCEPTED MANUSCRIPT
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
SC
R
Moisture
NU
Composition, %
3.10.2
AC
CE
P
TE
MA
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.
30
ACCEPTED MANUSCRIPT
Table 2.
greenness
89.01.5
92.51.5
IP
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
MA
SC
R
88.62.1
AC
Yellowness
b*
28
82.32.5
CE
P
Greenness
-a*
21
Controla
TE
Lightness
L*
NU
Storage time
at 4C, days
14
31
ACCEPTED MANUSCRIPT
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
NU
IP
SC
R
AC
CE
P
TE
MA
(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.
32
ACCEPTED MANUSCRIPT
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
IP
thermophilus,
SC
R
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
MA
FV60e (0.5%)
NU
Control
AC
CE
P
TE
Data presented as overall ranges (minimum and maximum) of all values measured at 7 day intervals over
28 days of storage.
33
ACCEPTED MANUSCRIPT
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
TE
Texture
SC
R
IP
ns
FV60e
(0.5
%)
NU
Sensory
Attribu
te
Colour
FV60e
(0.25
%)
MA
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
ACCEPTED MANUSCRIPT
HIGHLIGHTS
Seaweed extracts from Ascophyllum nodosum and Fucus vesiculosus were added to
SC
R
IP
yoghurt.
NU
Colour, flavour and texture were important for sensorial acceptance of yoghurt.
MA
AC
CE
P
TE
36