J Appl Phycol
https://doi.org/10.1007/s10811-017-1310-4
Influence of sea grapes (Caulerpa racemosa) supplementation
on physical, functional, and anti-oxidant properties of semi-sweet
biscuits
Anuj Kumar 1 & Elavarasan Krishnamoorthy 1 & Hanjabam Mandakini Devi 1 &
Devananda Uchoi 1 & C. S. Tejpal 2 & George Ninan 1 & A. A. Zynudheen 1
Received: 14 February 2017 / Revised and accepted: 8 October 2017
# Springer Science+Business Media B.V. 2017
Abstract Seaweeds present a promising option for inclusion
in the human diet through supplementation in various food
systems. Sea grapes (Caulerpa racemosa), a common tropical
green seaweed, has immense nutritional potential. An attempt
was made to utilize the health benefits of sea grapes in human
diet via biscuit as carrying medium. The effect of C. racemosa
incorporation on nutritional, physical, anti-oxidative and sen-
sorial characteristics of biscuits was assessed. Functional
properties of seaweed-flour mix along with the electrophoretic
pattern of dough were evaluated. Caulerpa racemosa addition
increased water and oil absorption capacity of flour mix.
Likewise, increase was noticed in solvent retention capacities
(sodium carbonate, lactic acid, and sucrose) of flour mix.
Inclusion of C. racemosa in biscuit enhanced the nutritive
potential. Protein and fiber content of biscuits increased with
rising level of C. racemosa incorporation. With increase in the
concentration of seaweed in biscuits, the phenolic content and
anti-oxidant activities (DPPH, ABTS, H2O2 radical scaveng-
ing activity and ferric reducing anti-oxidant power) increased.
The sensory rating of biscuits varied from Bneither like nor
dislike^ to Blike slightly^ on 9-point Hedonic scale. The study
demonstrated the positive nutritional effect of C. racemosa
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10811-017-1310-4) contains supplementary
material, which is available to authorized users.
* Anuj Kumar
anuj19.ak@gmail.com
1
Fish Processing Division, ICAR-Central Institute of Fisheries
Technology, Willingdon Island, Kochi, Kerala 682029, India
2
Biochemistry and Nutrition Division, ICAR-Central Institute of
Fisheries Technology, Willingdon Island, Kochi, Kerala 682029,
India
inclusion in biscuits. Further, the health benefits of different
seaweeds can be explored via incorporation in various other
food systems. This could give rise to new and significant
segment of health foods—Bcomposite seaweed food
products.^
Keywords Caulerpa racemosa . Chlorophyta . Biscuit .
Functional properties . Anti-oxidant activity . Sensory
characteristics
Introduction
Rising population, climate change, and depleting land and fresh
water sources are big challenges in way of ending hunger and
achieving food security throughout the world. To address this,
scientists and researchers across the globe are constantly explor-
ing various novel and non-conventional sources of food.
Seaweeds are varied group of predominantly marine, mul-
ticellular, photosynthetic, chlorophyll containing, eukaryotic
organisms found from the intertidal zone to about 300 m deep
(Levine 2016). Their cultivation in oceanic farms can help in
growing profuse quantities of nutrient rich foods for humans
in a sustainable manner. Unlike land, there is no need of fresh
water and chemical fertilizers in these oceanic farms (Tiwari
and Troy 2015). The role of seaweeds in human health has
been well reviewed by Brown et al. (2014) and Wells et al.
(2017). Seaweeds are good source of anti-oxidants, dietary
fiber, essential amino acids, phytochemicals, vitamins, and
minerals. Further, bioactive compound present in seaweeds
have health promoting properties, i.e., anti-diabetic activity,
anti-inflammatory, anti-oxidant, dyslipidemia, bone-health,
heart-health and mental-health benefits (Hayes 2015;
Roohinejad et al. 2017). Thus, the seaweeds have the potential
to be used as a means to counter food security concerns and
 J Appl Phycol

reduce the burden of non-communicable diseases (NCDs) The present scenario wherein functional foods are increas-
arising due to modern lifestyle. ingly becoming popular, seaweed-incorporated biscuits could
Caulerpa racemosa, commonly known as sea grapes, is a offer a palatable way of consuming the seaweed and utilizing
tropical green alga (Chlorophyta) belonging to family the health gains these provide, in the human diet. Therefore, it
Caulerpaceae. They are found on sandy rock bottoms in the was intended to incorporate C. racemosa in biscuits and ana-
upper sublittoral zone of tropical coral reefs (Horstmann lyze the compositional, textural, anti-oxidative, and sensorial
1983; Mao et al. 2011). Caulerpa racemosa is one of the most changes occurring in the biscuits.
predominant seaweeds along Indian coastline (CSIR 1985) and
has immense nutritional potential. This seaweed is consumed in
raw as well as cooked forms across the Indo-Pacific (Morris Materials and methods
et al. 2014; de Gaillande et al. 2016). In folk medicine,
C. racemosa is used for treating high blood pressure and rheu- Caulerpa racemosa (sea grapes) was harvested from Gulf of
matism (Novaczek 2001; Prud’homme van Reine and Trono Mannar, Tamil Nadu, India, and transported to the laboratory
2001). It is good source of essential minerals like calcium, iron, in cold condition within 10 h. The seaweed was cleaned and
and magnesium and natural anti-oxidants (Matanjun et al. then dried in a vacuum oven at 50 °C. The dried Caulerpa was
2008; Kumar et al. 2011; Li et al. 2012; Paul et al. 2014). The milled into powder using hammer mill, packed in low-density
bioactives present in C. racemosa have been observed to dis- polyethylene pouches, and stored in desiccator.
play anti-neoplastic, anti-bacterial, anti-viral, and anti-
proliferative activities (Barbier et al. 2001; Cavas et al. 2006;
Nagappan and Vairappan 2014; de Souza Barros et al. 2015). Preparation of biscuits
Different fractions of polysaccharides isolated from
C. racemosa have exhibited anti-tumor, anti-nociceptive, and Various ingredients required for the formulation of biscuits
anti-inflammatory properties (Ghosh et al. 2004; Ji et al. 2008; were purchased locally (Table 1). The formulation used for
Ribeiro et al. 2014). the preparation of semi-sweet biscuits was according to
Seaweeds have been utilized in various segments of food, Manley (2011). In brief, C. racemosa in defined levels was
i.e., cereals, dairy, meat/fish, and fruit/vegetable products to used to replace the refined flour (Level of substitution: A- 0%,
improve their functionality. Bakery products are a suitable car- B-1%, C-5% and D-10%). Refined flour (Elite Maida,
rier for utilization of seaweed functionality because of their Yamuna Roller Floor Mills Pvt., Ltd. Thrissur, India),
wider reach. Mamat et al. (2014) were able to incorporate up C. racemosa and sugar were blended in a mixer. Salts (sodium
to 8% red seaweed (Kappaphycus alvarezii) in bread without chloride and sodium bisulfite), leavening chemicals, and fla-
impairing the quality. Functional breads were also prepared by vor were dispersed in water. All-in-one method (AOM) was
utilizing different seaweeds, i.e., Himanthalia elongata, used for mixing the ingredients. In this method, all the ingre-
Myagropsis myagroides, and Palmaria palmata (Lee et al. dients are loaded and mixed together (single stage process).
2010; Cox and Abu-Ghannam 2013; Fitzgerald et al. 2014). The method is quick and used for manufacturing hard dough
Palmaria palmata incorporation at 4% level did not have any biscuits, i.e., semi-sweet biscuits. The aforementioned
adverse effect on the textural and sensorial attributes of the
bread. M. myagroides supplementation improved shelf life
and overall quality of the bread. Further, softer/spongier noo- Table 1 Formula for semi-sweet biscuit preparation
dles and improved quality pasta product were developed by
Ingredients (parts per 100 g of Percentage (%)
replacing a part of refined wheat flour with Monstroma and refined wheat flour)
Undaria (Prabhasankar et al. 2009; Chang et al. 2011).
Among bakery products, biscuits occupy their own niche. Refined wheat flour 100 65.2
Biscuits are favorite food widely consumed mostly due to their Sugar 25 16.3
pleasant taste, ready to eat nature, accessible cost, availability, Shortening 20 13.0
and longer shelf time (Sudha et al. 2007). Semi-sweet biscuits Skim milk powder 1.4 0.91
are healthier option when compared to cookies, due to reduced Lecithin 0.4 0.26
fat and sugar content. These are prepared from hard doughs Invert syrup 4 2.61
(developed). The critical limits of fat and sugar content in Salt 1 0.65
semi-sweet biscuit recipes are 22 and 30 units per 100 g of Sodium bicarbonate 0.6 0.39
flour. Above these limits, dough loses extensibility and acquires Ammonium bicarbonate 0.4 0.26
short dough characteristics. Short doughs are processed in dif- Sodium metabisulfite 0.05 0.03
ferent manner. Low production cost makes them favorable op- Vanilla flavor 0.05 0.03
tion for developing countries (Manley 2011).
J Appl Phycol
ingredients, including hydrogenated vegetable fat (Dalda,
Bunge India Pvt., Ltd., Mumbai, India) and lecithin, were
mixed using a mixer (A120, Hobart Corporation, UK) until
the dough temperature reached 40–42 °C. The warm dough
was sheeted (PN, La Monferrina, Alfero, Italy) quickly,
shaped using a circular mold (diameter 5.1 cm), and then
dockered manually. The biscuits were baked at 190 °C for
14–15 min, cooled, and then packed in metallized polyester
pouches.
Analyses
Functional properties of flour-seaweed mix
Water absorption capacity (WAC) was determined according
to Stojceska et al. (2008) with minor modifications. About 2 g
of C. racemosa -flour blend (seaweed powder + refined wheat
flour at desired levels) was suspended in distilled water for
30 min. The mix was vortexed intermittently and then centri-
fuged at 3500xg for 10 min at room temperature. The super-
natant was decanted and the gel obtained after decantation was
weighed. Oil absorption capacity was determined using sun-
flower oil instead of water. The solvent retention capacity was
evaluated as described by Duyvejonck et al. (2011). Caulerpa
racemosa -flour blend was weighed and vigorously mixed
with respective solvents (5% Na2CO3, 5% lactic acid, and
50% sucrose in water). Samples were kept for 30 min at room
temperature to solvate and then centrifuged at 1000xg for
15 min. Analyses were carried out in triplicates.
Electrophoresis of dough and seaweed proteins
The proteins from 1 g of dough sample were extracted in 9 mL
of 5% sodium dodecyl sulfate (SDS) solution. The electropho-
resis was performed as described by Laemmli (1970) and
protein bands were visualized by Coomassie Blue G. The
mixture was kept in the boiling water bath for 30 min. The
reaction mixture was cooled in an ice bath and mixed with
sample buffer (0.5 M Tris-HCl, pH 6.8, containing 4% SDS,
20% glycerol, and 10% β-mercapto-ethanol) at 1:1 ratio
followed by boiling for 90 s in a water bath. The samples were
cooled and centrifuged at 9000×g for 15 min. The supernatant
was collected and stored at frozen condition until the analysis.
After thawing, an aliquot of 7 μL of samples were loaded onto
polyacrylamide gels prepared with 12.5% running gel and 4%
stacking gel and subjected to electrophoresis at a constant
voltage of 90 V in a Mini Protein II unit (Bio-Rad
Laboratories, USA). After electrophoresis, the gels were
stained in Coomassie Blue G-250 (0.05% (w/v)) prepared in
8% (v/v) acetic acid and destained with 8% (v/v) acetic acid.
For seaweed protein characterization, the proteins were ex-
tracted in Tris-HCl buffer according to Wijesekara et al.
(2017). The protein bands were visualized by silver staining
using the protocol described by Schagger (2006). Wide range
of protein molecular weight markers (S8445, Sigma Chemical
Co., USA) was used to approximate the molecular weight of
the peptide units resolved.
Proximate analysis of biscuits
Proximate composition of biscuit samples was evaluated as
described in AOAC (2000). Moisture content was determined
gravimetrically by oven drying at 105 °C until constant
weight. The nitrogen content was determined by the
Kjeldahl procedure and the protein content was calculated
using the conversion factor of 6.25. Lipid content was deter-
mined gravimetrically after the extraction with petroleum
ether in a Soxhlet extractor. The ash content was determined
gravimetrically in a muffle furnace by incinerating the charred
samples at 550 °C. Carbohydrate was estimated by difference.
The analyses were carried out in duplicates.
Physical characteristics of biscuits
The color of biscuits was measured in terms of Hunter L*, a*,
and b* values using a spectro-colorimeter (Colorflex EZ 45/0,
HunterLab, USA) having illuminant D65 and visual angle of
10°. Color measurement was carried on five biscuit samples.
The thickness and diameter of biscuits were directly mea-
sured by Vernier calipers (CD-6″ CSX, Mitutoyo Corporation,
Japan). The spread ratio of biscuits was calculated as quotient
of diameter and thickness. The measurement was conducted
on five biscuit samples.
The breaking strength of biscuits (force required to break
the sample) was determined by snap test using texture analyz-
er (LRX Plus, Lloyd Materials Testing, UK). The test was
carried out using 3-point bend rig and the aluminum probe
traveled at 10 mm s−1. Fifteen biscuits were utilized for mea-
suring the break strength.
Fourier transmission-infrared (FTIR) spectroscopy of biscuits
FTIR spectroscopy was carried out by using a FTIR spectrom-
eter (Model Nicolet iS 10, Thermo Fisher Scientific, USA)
from 4000 to 600 cm−1 to measure any changes in the spectra.
A background spectrum was collected by keeping the resolu-
tion at 4 cm−1.
Polyphenol content and anti-oxidant activity
Sample extraction for evaluating the anti-oxidant activities was
carried out as described by Aprodu and Banu (2012) with minor
modifications. Extraction system used to determine the phenolic
content of C. racemosa was hydrochloric acid: methanol: water
(1:80:10). Ground biscuit samples were dispersed at 5% (w/v) in
the extraction system. The phenolic content in the sample

extracts was determined according to Kahkonen et al. (1999).
ABTS (2,2′-azino-bis (3-ethyl-benzo-thiazoline-6-sulphonic ac-
id) radical scavenging activity was measured according to
Senphan and Benjakul (2014). Ferric reducing power was de-
termined according to the method adapted from Klompong et al.
(2007). Evaluation of DPPH (2,2-diphenyl-1-picrylhydrazyl)-
and hydrogen peroxide- radical scavenging activity were carried
in hydro-methanolic (1:1) extract as described by Brand-
Williams et al. (1995) and Mukhopadhyay et al. (2016), respec-
tively. The determinations were carried out in triplicates.
Sensory evaluation
Semi-trained panel on 9-point Hedonic rating test evaluated the
sensory quality of biscuits. The semi-trained panel is generally
used for discriminative testing. Prior to the evaluation, panel
members were briefly trained about the product, its attributes,
and 9-point Hedonic rating method. The Hedonic scale is com-
monly used for measuring the acceptability/preference of the
food product. The descriptors of the sensory attributes for
Hedonic rating vary from Blike extremely^ to Bdislike
extremely,^ i.e., nine different ratings. The attributes evaluated
were: color, appearance, texture, flavor, and overall acceptability.
Statistical analysis
The biscuits preparation was carried out in single batch to
avoid batch-to-batch variation. The number of biscuits pre-
pared for each treatment varied from 75 to 80. The results
are expressed as mean ± standard deviation. One-way analysis
of variance test was performed using SPSS, and difference
between the means was tested using Tukey’s test at 95% con-
fidence level (p < 0.05).
Results
Functional properties of flour-seaweed mix
Table 2 depicts the water and oil absorption capacities (WAC
and OAC) of flour and C. racemosa -flour blend utilized for
Table 2 Functional properties of
the flour-seaweed mix Sample Water absorption
capacity
A 1.24 ± 0.01a
B 1.26 ± 0.03a
C 1.38 ± 0.01b
D 1.52 ± 0.03c
Results are expressed as mean ± standard deviation; n = 3. Values within same column having different super-
scripts are significantly different (p ≤ 0.05). Substitution level: A- 0%, B-1%, C-5%, and D-10%
J Appl Phycol
preparation of semi-sweet biscuits. Incorporation of CR in-
creased the WAC and OAC significantly (p < 0.05) and the
increase was found to be concentration dependent. WAC and
OAC of 10% seaweed-flour mix were higher by 22.5 and
27.5%, compared to control samples. Solvent retention capac-
ities (SRCs) for different samples are presented in Table 2. All
the SRCs increased significantly (p < 0.05) with the rising
level of seaweed in the mix used for biscuit preparation, ex-
cept lactic acid-SRC. The increase was 16% for sodium
carbonate-SRC and 20% for sucrose-SRC.
Protein pattern of dough
The protein pattern of dough and seaweed samples is present-
ed in Fig. 1a and b. The SDS-PAGE profile displayed intense
protein bands corresponding to peptide chains with different
molecular weights. The bands were detected in the molecular
weight range of 97, 66–97 (two bands), 55, 36–45, 29–36, and
24–29 kDa. SDS-PAGE profile of C. racemosa revealed the
presence of eight different peptide chains with the approxi-
mate molecular weight of 116, 66, 55, 45, 36, 29, 24, and
20 kDa.
Physical characteristics of biscuits
The physical characteristics viz. color, thickness, diameter,
spread ratio, and break strength are detailed in Table 3. The
biscuit images are represented in Fig. 2. The color of semi-
sweet biscuits was expressed in terms of Hunter coordinates
viz. L*, a*, and b* which deliberates the color into three
components: L*—lightness/darkness, a*—redness/green-
ness, and b*—yellowness/blueness. With increasing seaweed
concentration, Hunter L* value decreased significantly
(p < 0.05) from 66.6 to 41.1. A significant (p < 0.05) decrease
from 10.6 to 3.26 was also observed for redness/greenness
(a*) value. Likewise, yellowness/blueness component (b*)
value reduced from 32.0 to 22.9. The biscuits became darker
in color when compared to control. The diameter of biscuits
varied from 48.58–50.16 mm while the thickness varied from
3.48–5.41 mm. The variation in diameter and thickness of the
biscuits was significant (p < 0.05). Unlike the thickness which
Oil absorption Solvent retention capacity
capacity
Na2CO3 Lactic acid Sucrose
0.87 ± 0.00a 1.37 ± 0.06a 1.52 ± 0.02ab 1.98 ± 0.09a
0.88 ± 0.00b 1.43 ± 0.03a 1.50 ± 0.03ab 2.17 ± 0.20ab
0.99 ± 0.00c 1.48 ± 0.04ab 1.45 ± 0.02a 2.27 ± 0.07ab
1.11 ± 0.00d 1.59 ± 0.07b 1.56 ± 0.06b 2.38 ± 0.13b
J Appl Phycol

Fig. 1 a Electrophoretic pattern of proteins from dough samples (A- 0%, B-1%, C-5%, and D-10%). b Electrophoretic pattern of proteins from Caulerpa
racemosa

decreased significantly with increased C. racemosa incorpo-
ration, the spread ratio showed marked increase with rising
C. racemosa levels in flour mix. Spread ratio increased from
9.63 to 13.97 (approx. 45% increase). The break strength of
biscuits decreased significantly (p < 0.05) with increasing
C. racemosa level. The hardness decreased from 17.57 to
11.29 N, i.e., 35% decrease in break strength. The biscuits
with the highest inclusion level of CR (10%) exhibited the
least hardness.
was observed with increase in level of C. racemosa. Similar
trend was observed for ash content, which increased from 1.28
to 2.23%. The carbohydrate amount in the biscuit samples
decreased from 71.7 to 68.75%. A slight and non-significant
decrease was observed in the lipid content of biscuits. Ten
percent C. racemosa supplemented biscuits had higher
amount of protein and ash (mineral) content.
Anti-oxidant properties of biscuits

Proximate composition of biscuits
The basic nutritional components in semi-sweet biscuits were
quantified and presented in Table 4. An increase in the protein
content of the semi-sweet biscuit samples from 7.69 to 9.01%
The phenolic content and anti-oxidant activity of biscuits were
assessed (Table 5). Biscuits supplemented with C. racemosa
displayed significantly (p < 0.05) higher polyphenolic content
than control biscuits. The phenolic content was found to de-
crease with increasing level of CR. The level of phenolic

Table 3 Physical parameters of

Caulerpa racemosa Parameters A B C D
supplemented biscuits
Hunter color coordinates# L* 66.62 ± 2.11d 63.67 ± 1.57c 48.32 ± 1.84b 41.12 ± 0.83a
a* 10.61 ± 0.77c 5.11 ± 0.65b 3.59 ± 1.36ab 3.26 ± 0.70a
b* 32.02 ± 0.64d 27.25 ± 0.68c 25.56 ± 0.43b 22.93 ± 0.82a
Thickness# (mm) 5.10 ± 0.11d 5.41 ± 0.03c 4.16 ± 0.12b 3.48 ± 0.12a
Diameter# (mm) 49.08 ± 0.35b 50.16 ± 0.20c 49.04 ± 0.24b 48.58 ± 0.14a
Spread ratio# 9.63 ± 0.20a 9.27 ± 0.08a 11.79 ± 0.39b 13.97 ± 0.49c
Break strength$ (N) 17.54 ± 2.94c 17.57 ± 2.93c 13.45 ± 1.95b 11.29 ± 1.78a

Results are expressed as mean ± standard deviation; # n = 5, $ n = 15. Values within same rows having different
superscripts are significantly different (p ≤ 0.05). Substitution level: A- 0%, B-1%, C-5%, and D-10%
 J Appl Phycol

Fig. 2 Control and seaweed-

supplemented semi-sweet biscuits

content varied from 616.8 to 829.1 μg g−1 biscuits. Likewise,
a significant (p < 0.05) increase in the anti-oxidant activity
(measured as radical scavenging activity using DPPH,
ABTS and hydrogen peroxide radical) of biscuits fortified
with C. racemosa powder was obtained with the increase in
seaweed concentration. A similar trend was also observed for
ferric reducing power of biscuit samples.
FTIR spectroscopy
FTIR spectra of biscuits prepared without (control) and with
seaweed (10% level) are shown in Fig. 3. Compared to the
control sample, seaweed-added biscuits had distinct band pat-
tern in the wave number region of 3888–3937, 3435–3575,
1754–2926, 1479–1561, and 1328–1348 cm−1 corresponding
to CH in aromatic ring, OH group, asymmetrical stretching
vibration of methylene group and carbonyls, CN stretching
and NH bending, and functional group from sulfated polysac-
charides, respectively. The addition of seaweed helped in
enriching the biscuits with seaweed polyphenols, as evident
by spectral bands in the region of 3888–3937 cm−1. The spec-
tral region observed in the present study in the wave number
of 3435 cm−1 corresponds to amide A band indicates the par-
ticipation of NH group is in hydrogen bonding and also OH
stretching of free water molecules. The spectral region of
2926 cm−1 can be assigned to amide B band and can be related
to asymmetric stretching of CH2 groups. The band at 1463 and
1464 cm−1 in control and seaweed-added biscuits indicated
bending vibrations of CH3 and CH2 aliphatic groups. High-
intensity bands in the control samples were observed at the
wave number of 3195.8, 1510, 1233,1182,1036,827, and
560 cm−1. These bands could be assigned to NH-stretching,
amide II, C-O-C stretching, CH2 wagging, and pyranose ring
structure.
Sensorial behavior of biscuits
The sensory behavior—color, appearance, texture, flavor, and
overall acceptability—of C. racemosa-supplemented biscuits
is elucidated using a radar diagram (Fig. 4). Perusal of senso-
rial data indicated a significant (p < 0.05) decline in the sen-
sory scores of biscuits up on supplementation at higher levels.
The color score was found to decrease from 8.00 to 5.83,
texture score from 7.88 to 6.56, flavor score from 7.89 to
5.44, and overall acceptability from 7.78 to 5.64. The overall
acceptability of seaweed-supplemented biscuits lied in the
Bneither like nor dislike- like slightly^ to Blike moderately-
like very much range^.
Discussion
Water/oil absorption capacity (WAC/OAC) measures the abil-
ity of flour to bind oil/water under specified conditions
(Adebiyi et al. 2016). Flour-seaweed mix was able to absorb
and hold more water/oil than the flour alone. This could be

Table 4 Compositional analysis

of Caulerpa racemosa Parameters (%) A B C D
supplemented biscuits
Moisture 4.19 ± 0.07a 4.08 ± 0.12a 3.97 ± 0.10a 4.03 ± 0.16a
Fat 14.48 ± 1.04a 14.58 ± 0.89a 13.35 ± 0.53a 13.77 ± 0.53a
Protein 7.69 ± 0.04a 7.93 ± 0.04b 8.40 ± 0.00c 9.01 ± 0.02d
Ash 1.28 ± 0.01a 1.37 ± 0.02b 1.68 ± 0.00c 2.23 ± 0.03d
Carbohydrate* 72.37 72.04 72.59 70.95
Fiber 0.30 0.44 1.41 1.83

Results are expressed as mean ± standard deviation, n = 2. Values within same rows having different superscripts
are significantly different (p ≤ 0.05). Substitution level: A- 0%, B-1%, C-5%, and D-10%
*By difference
J Appl Phycol

Table 5 Phenolic content and

anti-oxidative parameters of Parameters A B C D
seaweed-supplemented biscuits
Total phenolic content (μg g−1) 616.8 ± 17.4a 622.1 ± 6.1a 729.2 ± 1.6b 829.0 ± 0.6c
DPPH activity (%) 84.19 ± 0.61a 84.75 ± 0.33a 87.79 ± 0.14b 88.60 ± 0.14b
ABTS activity (%) 89.34 ± 1.17a 83.99 ± 0.14b 95.06 ± 0.26c 96.61 ± 0.31c
H2O2 activity (%) 20.08 ± 1.85a 25.19 ± 0.82b 33.88 ± 0.48c 38.77 ± 0.90d
Reducing power 0.29 ± 0.01a 0.29 ± 0.00a 0.38 ± 0.02b 0.41 ± 0.01c

Results are expressed as Mean ± standard deviation, n = 3. Values within same rows having different superscripts
are significantly different (p ≤ 0.05). Substitution level: A- 0%, B-1%, C-5%, and D-10%

Fig. 3 FTIR pattern of a control and b seaweed-supplemented (10%) biscuit


Sensory attributes
Colour
9 SDS-PAGE and is depicted in Fig. 1b. As we were not able to
7
5 eight different peptide units. The relative proportion of pep-
Overall acceptability
3
1
Flavour Texture
A B C D
Fig. 4 Sensory characteristics of seaweed-supplemented biscuits (A-
0%, B-1%, C-5%, and D-10%)
due to the absorption of water/oil by the seaweed constituents
like proteins, fibers, and polysaccharides through various in-
teractions/entrapment. WAC is considered to be an important
factor which governs the end quality of baked products like
biscuits. WAC interferes with the machinability of the dough.
Further, higher WAC leads to the development of undesirable
hardening in biscuits by affecting the density and shrinkage.
WAC and OAC are related to hydrophilic and lipophilic com-
ponents present in the flour. Higher OAC signifies better fla-
vor retention ability and consequently the palatability (Kumar
et al. 2016). Solvent retention capacity (SRC) assay deter-
mines the swelling nature of flour components in the presence
of various solvents and helps in measuring the flour function-
ality. The SRC measurement evaluates the functional contri-
bution of flour components (Kweon et al. 2014) and helps in
predicting the quality of soft wheat products (Ram et al.
2005). Generally, lactic acid, sodium carbonate, and sucrose-
SRC are associated with glutenin characteristics, level of dam-
aged starch, and gliadin and pentosan characteristics, respec-
tively (Gaines 2004). The increase in SRC of flour was more
prominent at higher level of seaweed supplementation. The
constituents of seaweed imbibed the solvents and increased
the solvent retention capacity of flour-seaweed mix. Among
all the solvents, sucrose -SRC was higher compared to lactic
acid and sodium carbonate-SRC implying more absorption
effect of pentosans and gliadin in comparison to other flour
constituents. SDS-PAGE profile confirmed the presence of
high molecular weight subunits corresponding to x and y type
glutenin (HMW-GS; 67–74, 83–88 kDa), gliadin subunits
(ω5, ω1–2, α/β and γ; 49–55, 39–44, 28–35, 31–35 kDa)
and low molecular weight glutenin subunits (32–39 kDa)
(Wieser 2007). The viscoelastic and cohesive properties of
dough influence the final texture of biscuits. These properties
are affected by the presence of glutenin and gliadin proteins
and the extent of gluten development controls the hardness of
J Appl Phycol
biscuits. The protein profile of seaweed was analyzed using
detect the protein bands by Coomassie Blue staining, silver
staining was carried out. The results revealed the presence of
Appearance tides having molecular weight of 45, 36 and 24 kDa were
higher as indicated by the intensity of the peptide bands.
Mohd Rosni et al. (2015) have reported similar results on
protein pattern of C. racemosa. Further work is needed to
better understand the role of seaweed proteins.
Color is a significant parameter for food products as it
affects the consumer preference. Incorporation of additional
ingredient may alter the color of the product. The changes
observed in color were essentially due to C. racemosa supple-
mentation, which imparted its own characteristic color tone to
the biscuits. Lightness component of color declined with in-
creasing C. racemosa incorporation in biscuits. The same held
true for the decrease in redness and yellowness components.
The physical parameters like thickness, diameter etc. give the
elementary prediction of biscuit quality (Dayakar Rao et al.
2016). They not only affect the textural attributes of biscuits,
but are also important in designing and development of pack-
age. With increased C. racemosa levels, the thickness and
diameter of biscuits reduced, although a small increase in both
the parameters was observed at initial level of addition. Trend
was reflected in the spread ratio (ratio of diameter and thick-
ness) of the biscuits. Generally, higher spread ratio is preferred
for biscuits. The increase in spread ratio upon C. racemosa
addition was due to a comparatively larger decrease in the
thickness instead of the diameter. Addition of C. racemosa
hindered expansion and caused shrinkage in biscuits (biscuit
sheets were thinner). The decrease in the diameter and thick-
ness of biscuits could be attributed to the dilution effect on
gluten (Ajila et al. 2008) up on addition of seaweed which in
turn affected the formation of suitable dough. Addition of
psyllium (Raymundo et al. 2014) and bamboo shoot powder
(Choudhury et al. 2015) led to similar effect on thickness and
diameter of biscuits. Water absorption capacity of the flour
bears an inverse relationship with the spread of biscuits.
Fibers have high affinity towards water and this hampers the
development of appropriate dough required in forming oper-
ation (Villemejane et al. 2013) thereby decreasing the spread
ratio of biscuits. The texture, expressed in terms of break
strength, is an important attribute that affects the acceptability
of biscuits. Usually, the hard textured biscuits are not preferred
by the consumers. Decrease in the hardness, upon
C. racemosa incorporation, was in line with the reduction in
thickness of biscuits. Addition of buckwheat flour and green
tea powder have also been reported to reduce the hardness of
biscuits (Jan et al. 2015; Ahmad et al. 2015).
The increase in protein and mineral (ash) content of biscuits
presumably occurred due to the incorporation of C. racemosa .
Caulerpa racemosa consisted of around 30% protein and 11%
J Appl Phycol
ash (Online resource 1). The decrease observed in carbohydrate
content of C. racemosa-supplemented biscuits was due to lesser
carbohydrate content in CR than refined flour. The addition of
C. racemosa in biscuits caused a remarkable increase in the
fiber content of biscuit. CR is a reasonable source of dietary
fiber (13%) and the consumption of fiber is deemed to be help-
ful in alleviating the risk of various diseases like gastrointestinal
disorders, cancer, heart diseases, etc. In general, the addition of
C. racemosa influenced the nutritional quality of semi-sweet
biscuits positively.
Caulerpa racemosa contains various polyphenolic com-
pounds, which displays different health promoting properties.
Sea grapes have shown potential DPPH, ABTS, FRAP, and
superoxide radical scavenging activities (Chew et al. 2008;
Matanjun et al. 2008; Kumar et al. 2011; Mahendran and
Saravanan 2013). Fortification of biscuits with C. racemosa
enhanced the phenolic content of biscuits, which might have
augmented the hydrogen donating ability of the biscuits, there-
by increasing the anti-oxidant activity (reducing power and free
radical scavenging activity). The C. racemosa-supplemented
biscuits may provide various physiological benefits apart from
the basic nutrition. Therefore, the supplementation of biscuits
with C. racemosa appears to be justified. The increase in total
phenolic content of C. racemosa supplemented biscuits was
well correlated with increment in various in vitro anti-oxidant
activities assayed. Therefore, the CR can be utilized for the
development of various functional and health foods.
FTIR spectra provide useful structural insights which may
help in identifying the sampoles which are enriched with spe-
cific functional food ingredients. It is used to examine the
interaction of functional groups during processing of the food
products. Seaweed incorporation enriched the biscuits with
polyphenols as evident in the spectral bands detected in the
region of 3888–3937 cm−1. In case of seaweed enriched bis-
cuits, the same regions as in the control sample were except
3195 cm−1. However, one needs to be careful in interpreting
the FTIR spectral data of complex samples like biscuits, as
they have different ingredients with many functional groups
and the overlapping of spectra of functional groups are obvi-
ous. The results obtained for FTIR analyses indicate that the
region of 3435–3575 cm−1 could be used to identify the bis-
cuit samples enriched with seaweed. Further work needs to be
carried out in this direction.
Organoleptic evaluation is very important while develop-
ing new food product. The decrease in color score correlated
well with the decrease in Hunter L*, a*, and b* values of
biscuits. Similarly, the decrease observed in the texture score
was well associated with decreased break strength value.
Biscuits fortified with 10% C. racemosa scored least on all
organoleptic evaluation parameters. Since the decrease in or-
ganoleptic scores was inversely correlated with the
C. racemosa level in biscuits, it could be presumed that
C. racemosa imparted its sensory properties to the biscuits.
Incorporation of C. racemosa at higher levels (5 and 10%)
caused significant decrease in sensory attributes as compared
to 1% C. racemosa-supplemented biscuits. The higher con-
centrations of C. racemosa reduced the esthetic characteristics
of biscuits. But, all the biscuits were in the acceptable range,
as suggested by the sensory scores awarded by the panelists.
Degree of liking for color, flavor, and overall acceptability
scores varied from Bneither like nor dislike^ to Bliked slightly^
on 9-point Hedonic rating.
Inclusion of seaweed—Caulerpa racemosa (sea grapes)—
in biscuits appeared to be innovative and warranted approach
towards producing functional/health foods. Seaweed-added
biscuits are a unique attempt to utilize the nutritional potential
of the alga. The seaweed incorporation enhanced the basic
nutritive and anti-oxidative value of biscuits, although a de-
crease in sensorial and instrumental parameters was noticed.
Further, the sensorial attributes may be modified by the use of
legally permitted food additives, i.e., food colors, flavors, and
enzymes to enhance the quality. The present attempt could
indeed give rise to a new segment of foods—Bcomposite sea-
weed food products^—which may help in exploiting the func-
tional benefits of these abundant oceanic food resources. The
utilization of seaweed in production of composite foods may
further strengthen the nutritional and food security of growing
human population.
Acknowledgements The authors are grateful to the Director, ICAR-
CIFT for providing the necessary facilities. The work was carried out
under the Institutional Project No.1000661033 [P-96/2014(3)]. The au-
thors also wish to express their gratitude to Technical Staff of Fish
Processing Division for the support rendered during the work.
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