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Valorizing date seeds through ultrasonication to enhance quality attributes
of dough and biscuit: Part 2 – Study on bioactive properties, sensory
acceptance, in vitro gastrointestinal digestion and shelf life of biscuits
Meththa Ranasinghe
a
, Mariam Alghaithi
a
, Constantinos Stathopoulos
b,c
, Balan Sundarakani
d
,
Sajid Maqsood
a,*
a
Department of Food Science, College of Agriculture and Veterinary Medicine, United Arab Emirates University, Al-Ain 15551, United Arab Emirates
b
Food Futures Institute, Murdoch University, Australia
c
Faculty of Health, University of Canberra, Australia
d
Faculty of Business, University of Wollongong in Dubai, 20183, United Arab Emirates
ARTICLE INFO
Keywords:
Sustainable food processing
Date seeds
Baked products
Bioaccessibility
Ultrasonication
Phenolic retention
ABSTRACT
Aligning with sustainable food system development, in this study, date seeds derived compounds were utilized as
functional ingredient to formulate value-added biscuits. Ultrasound-assisted extraction (UAE) was employed as a
non-thermal method to extract polyphenolic compounds from small, medium and large particles of defatted date
seed powder (DDSP). The remaining ber-rich fraction (residue) was further utilized. Water content in biscuit
formulation was replaced by the extract, and the ber-rich fraction was substituted at three substitution levels;
2.5 %, 5 % and 7.5 %. Effects of baking on bioactive properties of dough, nutrient composition, sensory analysis,
bioaccessibility of polyphenols, and shelf-life of biscuits were analyzed. Total phenolic content (TPC) increased in
dough and biscuit with incorporated ber-rich fraction. TPC of dough decreased with increasing particle size of
ber-rich fraction while biscuits exhibited an opposite trend. Similar tendency was observed with antioxidant
activity of dough and biscuit. TPC was higher in biscuits than dough, with the highest values of 0.46 mg gallic
acid equivalents (GAE)/g and 2.26 mg GAE/g in dough and biscuit, respectively. Fiber and moisture contents in
biscuits increased while protein content decreased with fortication. Consumers showed moderate acceptance of
fortied biscuits with overall acceptability comparable with the control biscuits. Bioaccessibility index of
polyphenols upon gastrointestinal digestion was high in biscuits with 5 % and 7.5 % substitution of small and
medium sized particles of ber-rich fraction. Phenolic retention increased with ber fortication and at the end
of 6 months the lowest thiobarbituric acid reactive substances (TBARS) value of 18.23 nmol malondialdehyde
(MDA)/g sample, was observed in 7.5 % large particle substituted biscuit. Thus, utilizing date seeds in the form
of green extracted polyphenols and ber-rich fraction, as functional and bioactive ingredients highlight sus-
tainable processing and utilization of date-fruit processing by-products which is in line with the circular economy
approach.
1. Introduction
In recent years, there has been a growing interest in enhancing the
nutritional prole of baked goods to meet consumer demands for
healthier food options. Biscuits, a popular bakery item consumed
worldwide, are often lacking essential micronutrients and functional
components [1]. In response to the growing consumer demand for
functional foods with added health benets, researchers have turned to
novel approach to enrich traditional bakery products [2,3]. Date seeds,
on the other hand, often discarded as waste in date processing, are rich
in valuable components such as ber, antioxidants and other bioactive
compounds that can enhance the nutritional prole of food products [4].
Integrating the nutritional prole of date seeds with a prevalent and
easily accessible food product represents a promising approach for
addressing consumer health concerns associated with maintaining
nutritious dietary habits. Prior studies have focused on either direct
incorporation of date seed powder (DSP) or the incorporation of the
extract of DSP into baked goods including cookies, breads etc [2,3,5–7].
* Corresponding author.
E-mail address: sajid.m@uaeu.ac.ae (S. Maqsood).
Contents lists available at ScienceDirect
Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultson
https://doi.org/10.1016/j.ultsonch.2024.107160
Received 23 September 2024; Received in revised form 18 October 2024; Accepted 13 November 2024
Ultrasonics Sonochemistry 112 (2025) 107160
Available online 14 November 2024
1350-4177/© 2024 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC license (
http://creativecommons.org/licenses/by-
nc/4.0/ ).
Green and non-thermal processing technologies are gaining impor-
tance in the food industry. Ultrasound-assisted extraction (UAE) is a
cutting-edge technique that has gained the attention of the food industry
for its capability to improve the extraction of bioactive compounds from
plant materials [8–10]. This process enhances the extraction yield and
reduces the extraction time compared to traditional extraction methods.
The accessibility to phenolic compounds in plant tissues can increase
with ultrasound waves due to the phenomenon known as sonication
[9,11]. Ultrasound waves create cavitation bubbles in a liquid medium,
leading to the formation of microjets and shockwaves [11]. These
physical effects can disrupt cell walls and membranes, enhancing the
release of phenolic compounds from plant materials [8,9]. The increased
surface area and improved mass transfer resulting from ultrasound
treatment [9,11] can improve the extraction efciency of phenolic
compounds, as well as making the remaining compounds within the
extraction residue more accessible. Besides, the properties of DSP may
alter when subjected to ultrasonication. For example, following UAE,
the particle size, color, and bulk density of DSP were reduced, while its
water holding capacity increased. Therefore, the inclusion of aqueous-
phase ultrasound-assisted extracts of defatted date seed powder
(DDSP), along with the ber-rich fraction, in biscuit formulations is
anticipated to be more efcient than direct incorporation of DSP.
Additionally, ultrasound-assisted extraction can help preserve the
quality of the extracted compounds by operating at lower temperatures
and minimizing degradation. The UAE of polyphenols utilizing aqueous
binary mixtures of organic solvents resulted in higher extraction yields
compared to aqueous phase extraction [12]. However, disposal of
organic solvents has negative impacts on the environment. Therefore, it
is worthwhile to explore ways to improve extraction efciency using
water-based extractions that are environmentally friendly. In addition,
the water-extracts can be directly added to food products without the
need for additional purication steps, making the process more
convenient.
However, there is still insufcient research focusing on the efcacy
of incorporating aqueous polyphenolic extracts and bre-rich fraction
obtained through UAE of DSP into baked product recipes, to enhance the
nutritional quality as well bioactive properties beyond direct incorpo-
ration. Through systematic formulation studies and sensory evaluations,
researchers can optimize the incorporation of ultrasound-assisted ex-
tracts and the ber-rich fraction of DDSP to achieve a balance between
health benets, sensory appeal, and overall product quality. Therefore,
the effect of particle size and the substitution level of DDSP ber-rich
fraction on the bioactive properties and the sensory attributes, have
been investigated. Overall, this study seeks to explore the untapped
potential of date seeds as a functional food ingredient in bakery appli-
cations, highlighting its nutritional benets, sustainability implications,
and sensory considerations. By addressing these aspects comprehen-
sively, researchers aim to contribute valuable insights to the eld of food
science and innovation, ultimately offering consumers a delicious and
nutritious snack option that aligns with current trends in health and
sustainability.
2. Materials and methods
2.1. Materials
Date seeds (from the Khalas variety) were sourced from the Al Foah
Date company in Al Ain, United Arab Emirates. The ingredients for the
biscuit formulations were procured from local markets in Al Ain. Re-
agents and chemicals were obtained from Sigma–Aldrich Chemical Co.
(St. Louis, MO) and Fisher Scientic (Nepean, ON), all of which were of
analytical grade.
2.2. UAE and the preparation of ber-rich fraction
Fine DSP was obtained using Retsch ZM 200 Ultra Centrifugal Mill
(Retsch GmbH, Germany). Then DSP was sieved through mesh lters of
125 µm, 300 µm, and 500 µm, to obtain three different particle size
categories, i.e., <125 µm [small (S)], 125 µm–300 µm [medium (M)],
and 300 µm–500 µm [large (L)]. DSP was defatted with n-hexane for 2 h
and dried at 70 ◦C. UAE was conducted according to the method fol-
lowed by Ranasinghe et al. [10]. Briey, 90 % amplitude, 8 min time and
a solid to solvent ratio of 1:25 (w/v) was used for the extraction using an
ultrasonic probe system (model VCX750, Sonics & Materials, INC.,
USA). After UAE, the solution was centrifuged at 10,528×g for 15 min
and ltered to obtain the extract, and the remaining insoluble residue
(ber-rich fraction) was separated which was oven dried at 70 ◦C.
2.3. Dough and biscuit preparation
Basic ingredients used for the dough preparation are indicated in
Table 1. For the composite samples wheat our was replaced with ber-
rich fractions at levels of 2.5 %, 5 %, and 7.5 %, and water was
substituted with the corresponding aqueous-based phenolic extracts
(derived from small, medium, and large particles of DDSP). Total
phenolic content (TPC) of the extracts obtained from small, medium and
large particles were 0.70 mg gallic acid equivalents (GAE)/ml water,
0.60 mg GAE/ml water and 0.31 mg GAE/ml water, respectively. The
control dough (CD) and biscuit (CB) did not contain any extract or ber-
rich fraction. Three additional biscuit samples were prepared, each
incorporating DDSP extracts from three different particle sizes in place
of water, with no substitution of ber-rich fraction. Half of the dough
was used for the analysis while the other half was baked. To prepare
biscuits, the dough was rolled out to a uniform thickness of 3 mm and cut
into circular shapes with 4.5 cm diameter. The dough samples were
baked at 180 ◦C for 12–14 min. After cooling to room temperature, the
biscuits were stored in airtight sealed packaging bags for future use. C
denotes the control, S, M and L denote small, medium and large sized
particles, SE, ME, LE denote extracts obtained from small, medium and
large sized particles and 2.5, 5 and 7.5 denote fortication level of ber-
rich residue. The acronyms used for the samples are detailed in Table 2.
2.4. Bioactive properties of dough and biscuit
TPC, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ac-
tivity and ferric reducing power (FRAP) assay was conducted according
to Ranasinghe et al. [10]. Detailed procedures are presented in the
Supplementary material (S1).
2.4.1. Analysis of TPC
Methanolic extract of the sample was mixed with Folin-Ciocalteu
reagent and Na
2
CO
3
followed by an incubation of 1 h at 37 ◦C. TPC
was expressed as mg of gallic acid equivalents (GAE) per g of sample.
2.4.2. DPPH radical scavenging activity
Methanolic extracts of samples were mixed with 15 mM DPPH so-
lution followed by an incubation of 30 min at 37 ◦C. Scavenging activity
was presented in mmol trolox equivalents (TE) per g of sample.
Table 1
Ingredients used in biscuit formulation.
Ingredient Amount (g)
Wheat our 80
Icing sugar 38
Butter 13
Oil 13
NaHCO
3
1.2
NaCl 1
Milk powder 4
Water/extract 19
M. Ranasinghe et al.
Ultrasonics Sonochemistry 112 (2025) 107160
2
2.4.3. Ferric reducing antioxidant power (FRAP) assay
Sample extract was mixed with FRAP solution and incubated for 30
min at 37 ◦C. The concentration was expressed as mmol trolox equiva-
lents (TE) per g of sample.
2.5. Nutrient composition
Protein, fat, moisture and ash contents of selected biscuit samples
(2.5SB, 5SB, 7.5SB) were conducted according to the AOAC methods
[13]. Kjeldahl method was used to analyze the crude protein content by
Kjeldahl apparatus (Buchi Auto Kjeldal unit K-370, Switzerland). Crude
fat was determined using Soxtec Auto fat extraction system (FOSS
Analytical, Denmark). Moisture content was determined by oven drying
method. Ash content was analyzed by direct analysis. Fiber content was
determined in terms of neutral detergent ber (NDF) using the
Ankom2000I ber analyzer (ANKOM Technology, Macedon, NY, USA).
2.6. Cytotoxicity assay
For the cytotoxicity assay, the method followed by Elfahri et al. [15]
was used with slight modications. Briey, human colon adenocarci-
noma cell line Caco-2 was utilized to assess the cytotoxicity of the
extract with the highest concentration of TPC. The cells were cultured in
Dulbecco’s Modied Eagle medium (Gibco, Invitrogen, Carlsbad, CA)
supplemented with 10 % heat-inactivated bovine serum and 1 % peni-
cillin and streptomycin in a humidied incubator at 37 ◦C with 5 % CO
2
.
After cell counting using a hemocytometer, they were seeded at a con-
centration of 1x103 cells overnight in a 96-well plate. Various concen-
trations of the extract [2.5 % to 40 % (v/v)] were added to each well and
incubated under the same conditions for 72 h. Subsequently, 20
μ
L of
prewarmed cell cytotoxicity assay solutions (Abcam ab112118) were
introduced, followed by a 5 h incubation period. Absorbance readings
were taken at 570 and 605 nm, and the percentage of cell death was
calculated using the equation:
CellDeath(%) = [100 − (Rs −R0/Rc −R0)] × 100
where R indicates the absorbance ratio of the optical density at 570/
optical density at 605, Rs indicates the absorbance ratio in the presence
of extract, Rc indicates the absorbance ratio in the absence of the extract,
and Ro indicates the averaged background absorbance ratio.
2.7. Sensory analysis of biscuit
Selected biscuit samples (CB, EMB, 2.5MB, 5MB and 7.5MB) were
subjected to a sensory evaluation along with the control biscuit using 95
untrained consumer panellists. Sensory properties; colour, odor, texture,
taste, mouthfeel and overall acceptability were rated using a 9-point
hedonic scale ranging from 9 (like extremely) to 1 (dislike extremely).
All evaluators signed an Informed Consent to be voluntarily included in
the study, which was approved by the UAEU Social Sciences Ethics
Committee (application No: ERSC_2022_1730).
2.8. In-vitro simulated gastrointestinal digestion (SGID) of biscuits
enriched with polyphenolic extract and ber-rich fraction of DDSP
In order to simulate the oral, gastric and intestinal digestion of
polyphenols, SGID was carried out according to the INFOGEST protocol
[16] with slight modications. Briey, the simulated salivary uid
(SSF), simulated gastric uid (SGF) and simulated intestinal uid (SIF)
were prepared (refer Supplementary material S2). First, the sample was
dissolved in simulated salivary uid (SSF) in 1:1 ratio followed by the
addition of
α
-amylase (75 U/ml) at pH 7. Then the Falcon tubes con-
taining the samples were incubated at 37 ◦C for 2 min with shaking to
complete the simulated oral phase digestion. For gastric digestion, the
resultant oral mixture was mixed with SGF (with 0.15 mM of CaCl
2
, pH-
2) in 1:1 ratio. Then porcine pepsin (2000 U/ml), lipase (60 U/ml) and
0.0672 ml of deionized water were added and mixed. Tubes containing
the gastric phase were incubated for 2 h (at 37 ◦C) while shaking to
complete the simulated gastric phase digestion. For the intestinal
digestion, SIF (with 0.6 mM of CaCl
2
, pH-7) and gastric mixture was
mixed in 1:1 ratio and 10 mM of bile, 2000 U/ml lipase and pancreatin
(trypsin activity 100 U/ml) solutions were added. To complete the in-
testinal digestion tubes were incubated for another 2 h (at 37 ◦C) with
shaking. Gastric phase digestion was stopped by adjusting the pH to 8
with NaOH and in intestinal phase cooling shock (instant cooling in ice
and frozen at −20 ◦C) was used to stop the reaction. Before the analysis,
the tubes from each phase were centrifuged (10,956 xg at 4 ◦C) for 20
min and the TPC of the supernatant was determined.
2.8.1. Bioaccessibility index
Bioaccessibility index of the phenolic components after the digestion
was calculated according to Tomsone et al. [17] using the equation:
Bioaccessibility index =TPCAD
TPCBD
where TPC
AD
represents the total phenolic content after digestion and
TPC
BD
represents the total phenolic content of the biscuit before
digestion.
2.9. Shelf-life studies of biscuit
The shelf-life of selected biscuits was assessed using microbiological
stability and lipid oxidation . The methods outlined by Alkaabi et al.
[18] using potato dextrose agar (PDA) for yeast and mold count, and
Table 2
Acronyms used for different samples and their explanation.
Dough Sample Acronym Biscuit Sample Acronym
2.5 % substitution of
residue +extract of small
particles
2.5SD 2.5 % substitution of
residue +extract of small
particles
2.5SB
2.5 % substitution of
residue +extract of
medium particles
2.5MD 2.5 % substitution of
residue +extract of
medium particles
2.5MB
2.5 % substitution of
residue +extract of large
particles
2.5LD 2.5 % substitution of
residue +extract of large
particles
2.5LB
5 % substitution of residue
+extract of small
particles
5SD 5 % substitution of residue
+extract of small particles
5SB
5 % substitution of residue
+extract of medium
particles
5MD 5 % substitution of residue
+extract of medium
particles
5MB
5 % substitution of residue
+extract of large
particles
5LD 5 % substitution of residue
+extract of large particles
5LB
7.5 % substitution of
residue +extract of small
particles
7.5SD 7.5 % substitution of
residue +extract of small
particles
7.5SB
7.5 % substitution +
extract of medium
particles
7.5MD 7.5 % substitution +
extract of medium particles
7.5MB
7.5 % substitution of
residue +extract of large
particles
7.5LD 7.5 % substitution of
residue +extract of large
particles
7.5LB
0 % substitution of residue
+extract of small
particles
ESD 0 % substitution of residue
+extract of small particles
ESB
0 % substitution of residue
+extract of medium
particles
EMD 0 % substitution of residue
+extract of medium
particles
EMB
0 % substitution of residue
+extract of large
particles
ELD 0 % substitution of residue
+extract of large particles
ELB
0 % substitution of residue
+water (control)
CD 0 % substitution of residue
+water (control)
CB
M. Ranasinghe et al.
Ultrasonics Sonochemistry 112 (2025) 107160
3
plate count agar (PCA) for total bacterial count were used to determine
microbiological stability. Detailed procedure is described in the Sup-
plementary material (S3). The thiobarbituric acid reactive substances
(TBARS) method was employed to assess lipid oxidation following the
method by Maqsood and Benjakul [19] with slight modications. The
standard curve was constructed using 1,1,3,3-tetramethoxypropane
(malondialdehyde). Thiobarbituric acid (0.375 %), trichloroacetic acid
(15 %) and 0.25 N HCl was used to prepare the TBA solution, which was
then mixed with 0.5 g of the sample. Mixture was kept in boiling water
for 10 min followed by cooling in running water for 5 min. The mixture
was centrifuged at 10,956 xg for 5 min at 25 ◦C and the absorbance of
the supernatant was taken at 532 nm. TBARS results were presented in
nmol malondialdehyde (MDA)/g of sample.
2.10. Phenolic retention during storage
The total phenolic content (TPC) of biscuits were assessed every 2
months over a 6-month storage period using the method outlined by
Ranasinghe et al. [10]. Refer to Supplementary material (S1) for the
detailed procedure of the analysis of TPC.
2.11. Statistical analysis
Statistical analyses were carried out using Minitab 21 software.
Formulations of the dough and biscuit were carried out in three batches.
All the analyses were carried out in triplicates. Analysis of the results
were conducted using the analysis of variances (ANOVA) and compar-
isons were made using Tukey’s multiple comparison test to determine
the signicance (P <0.05).
3. Results and discussion
3.1. Effect of baking on the bioactive properties of dough and biscuit
fortied with the polyphenolic extract and ber-rich fraction obtained
through UAE
In order to study the changes in TPC and antioxidant activities of
dough with baking, the TPC and antioxidant activities in terms of DPPH
and FRAP, were determined in both dough and biscuit samples
(Table 3).
3.1.1. Total phenolic content (TPC)
TPC of the dough samples increased signicantly with the fortica-
tion of polyphenols and ber-rich fraction obtained through UAE of
DDSP except the sample ELD. The extract from small and medium sized
particles contains more polyphenols due to increased surface area,
compared to large particles. Hence higher phenolic concentration in the
extracts obtained from small and medium particles could attribute to the
signicantly higher (P <0.05) TPC in dough even with 0 % of ber-rich
fraction incorporation, whereas the extract obtained from large particles
did not contribute signicantly to the increase of TPC. Additionally, the
TPC of dough samples increased with increasing substitution level with a
signicant difference (P <0.05) between 2.5 % and 7.5 % ber-rich
fraction level from all three particle sizes. Samples with polyphenolic
extract and 0 % ber-rich residue of DDSP, did not show a signicant
difference (P >0.05) in TPC compared to the control whereas ber-rich
fraction incorporation resulted in a signicant increase (P <0.05) in
TPC. Besides, TPC of biscuits with 5 % and 7.5 % substitution level of
ber-rich fraction were signicantly higher (P <0.05) compared to 2.5
% in all three particle sizes with the highest TPC in the biscuits fortied
with 7.5 % ber-rich fraction from large particle size date seeds.
Despite the absence of added phenolic extract in the control dough,
there was still some TPC detected. This could be due to the Folin-
Ciocalteu reagent’s ability to react with compounds capable of
reducing it [20]. Alternatively, it might be attributed to the presence of
phenolic acids, such as ferulic acid, in the endosperm and pericarp of the
wheat kernel [21,22]. In all the formulations TPC of the biscuit was
higher than the corresponding dough. Maillard reaction products can
contribute in increasing the TPC of baked products [23]. The results are
in accordance with Vitali et al. [24] in which the TPC was analyzed in
both dough and biscuit after the incorporation of soy our, amaranth
our, carob our, apple ber and oat ber. Similarly, cookies with
incorporated buckwheat, oats and spices showed increased TPC
compared to the dough [25]. Some studies claim that there can be a
reduction in TPC while baking due to degradation of polyphenols under
thermal conditions [21,26]. According to Abdel-Aal and Rabalski [27],
the process of baking can elevate the levels of free phenolic compounds
in bakery products such as mufns, bread and cakes while reducing the
levels of bound phenolic compounds. This is attributed to the conversion
of bound phenolic compounds into free phenolic compounds during the
baking process. However, the factors such as the structure of the
phenolic components, the ratio between free and bound phenolics, our
type, product type and processing conditions play a vital role in deciding
Table 3
Changes in TPC and antioxidant activity in dough and biscuit with the fortication of the polyphenolic extract and the ber-rich fraction obtained from UAE, and the
changes during baking.
Particle size of residue Residue % Acronym TPC (mg GAE/ g sample) DPPH (mmol TE/ g sample) FRAP (µmol TE/ g sample)
Dough Biscuit Dough Biscuit Dough Biscuit
−0 C 0.30 ±0.01
eA
1.26 ±0.04
hB
0.28 ±0.02
eP
0.70 ±0.01
fQ
1.35 ±0.01
ghR
2.32 ±0.11
gS
Small 0 ES 0.36 ±0.01
dA
1.34 ±0.1
ghB
0.30 ±0.01
deP
0.75 ±0.04
defQ
1.39 ±0.02
fgR
2.22 ±0.05
gS
2.5 2.5S 0.41 ±0.02
bA
1.58 ±0.03
efB
0.35 ±0.01
bcdP
0.73 ±0.03
defQ
1.46 ±0.02
efR
2.31 ±0.03
gS
5 5S 0.45 ±0.01
abA
1.77 ±0.07
cdB
0.39 ±0.02
bcP
0.80 ±0.00
dQ
1.68 ±0.03
cR
2.86 ±0.04
deS
7.5 7.5S 0.46 ±0.01
aA
1.90 ±0.06
bcB
0.46 ±0.01
aP
0.93 ±0.02
bcQ
2.07 ±0.02
aR
3.23 ±0.09
bcS
Medium 0 EM 0.35 ±0.01
dA
1.40 ±0.11
fghB
0.29 ±0.01
deP
0.71 ±0.02
efQ
1.32 ±0.04
ghR
2.17 ±0.03
gS
2.5 2.5 M 0.36 ±0.04
cdA
1.59 ±0.04
deB
0.32 ±0.01
cdeP
0.78 ±0.01
deQ
1.43 ±0.02
efR
2.30 ±0.19
gS
5 5 M 0.42 ±0.01
abA
1.83 ±0.05
cB
0.34 ±0.01
bcdP
0.89 ±0.03
cQ
1.51 ±0.02
deR
2.66 ±0.09
efS
7.5 7.5 M 0.44 ±0.02
abA
1.92 ±0.07
bcB
0.40 ±0.04
abP
1.06 ±0.01
aQ
1.84 ±0.06
bR
3.03 ±0.10
cdS
Large 0 EL 0.32 ±0.01
deA
1.31 ±0.03
hB
0.29 ±0.02
deP
0.75 ±0.03
defQ
1.29 ±0.02
hR
2.40 ±0.05
fgS
2.5 2.5L 0.36 ±0.02
dA
1.50 ±0.04
efgB
0.32 ±0.01
cdeP
0.89 ±0.06
cQ
1.40 ±0.03
fgR
2.45 ±0.19
fgS
5 5L 0.41 ±0.01
bcA
2.06 ±0.04
bB
0.33 ±0.01
cdeP
0.94 ±0.01
bcQ
1.45 ±0.02
efR
3.44 ±0.10
bS
7.5 7.5L 0.42 ±0.02
abA
2.26 ±0.08
aB
0.38 ±0.05
bcP
0.98 ±0.00
bQ
1.59 ±0.02
dR
4.06 ±0.07
aS
1
Means ±SD are presented. Different lowercase superscript letters in a column and uppercase superscript letters in a row (TPC, DPPH and FRAP values separate)
denote signicant differences, P < 0.05.
M. Ranasinghe et al.
Ultrasonics Sonochemistry 112 (2025) 107160
4
the fate of phenols during baking [21,24,26–28]. Usually, phenolic acids
are more stable in cookies and biscuits compared to bread with baking
[27]. Within 2.5 % substitution level of dough samples with small par-
ticles showed a signicant higher (P <0.05) TPC compared to large
particles. The decrease in TPC with increase in the particle size was not
signicant (P >0.05) at 5 % and 7.5 % substitution levels of dough. In
contrast, TPC of biscuits increased signicantly (P <0.05) with
increasing particle size in all three substitution levels with the highest
TPC of 2.26 mg GAE/g sample, in the biscuit fortied with 7.5 % ber-
rich fraction of large particle size. When the particle size of the ber-rich
fraction increases, the surface area decreases, leading to less extraction
efciency of phenolic components in dough with large particles.
Simultaneously, the decomposition of polyphenols in large particles
during the baking process will be less due to the reduced surface area,
resulting in a higher TPC in biscuits with large particles compared to
small particles.
Although the TPC of dough samples fortied with the extract and 0 %
ber-rich fraction was signicantly higher compared to the control
dough before baking, the difference became non-signicant after
baking. In contrast, samples fortied with ber-rich fraction showed
signicantly higher TPC even after baking. This observation suggests
that phenolic retention increased with the incorporation of ber-rich
fraction during the baking process. The non-extractable phenolics that
remain in the extraction residue are signicantly higher than the water-
extractable phenolics in DDSP, which might have been released during
the baking process [27]. As a result, biscuits with incorporated ber-rich
fraction showed a signicant increase (P <0.05) in TPC compared to
biscuits incorporated only with the extract. The aforementioned obser-
vation indicates that ber-rich fraction can act as a protector for poly-
phenols during baking. Fiber can inuence the extractability of
phenolics in a positive manner which was evident by several baked food
such as apple ber fortied snack bars and bread formulations with
added ber [29,30]. The reason could be the structural conformation of
ber which contribute to the retention of phenolic components, pre-
venting their decomposition at elevated temperatures. There is a pos-
sibility of decomposition of thermolabile polyphenols under heating
conditions above 60 ◦C [31]. Hydroxyl groups present in phenolic
compounds can interact with amino acids in wheat as well as carboxylic
groups present in ber components ensuring the stability during baking
[30]. Increase in the extractable phenolics during baking was evident
with the biscuits fortied with apple and oat ber [24] which is com-
parable with the results of the current study.
The direct incorporation of DSP led to a TPC ranging from 290.20 µg
GAE/g sample −1020.70 µg GAE/g sample, with substitution levels
increasing from 2.5 % to 7.5 %, respectively [6]. Similarly, 10 % and 30
% incorporation of DDSP resulted in TPC of 510.50 mg/kg and 999.4
mg/kg, respectively [32]. The ndings of the current study suggest that
fortifying biscuits with polyphenolic extract and the ber-rich fraction
obtained through UAE leads to higher levels of polyphenolic compounds
compared to direct incorporation. The disruption of cell walls during
sonication can enhance access to phenolic compounds [9,11], and
phenolic components not extracted with water may remain within the
extraction residue, such as bound phenolics to ber [24,27]. These
retained phenolic components in the extraction residue could be
released during baking and might have contributed to the increased TPC
observed in this study. The physical changes occurring in plant cells
during sonication facilitate greater mass transfer during UAE [33].
Furthermore, these alterations may enhance the accessibility of phenolic
components that are bound to the ber within the sample matrix.
3.1.2. Antioxidant properties
Control dough and biscuit samples also exhibited antioxidant prop-
erties which showed an increase upon baking (Table 3). Biscuits and
other wheat-based food contain a signicant amount of bioactive com-
ponents mainly phenolic acids which could be in the form of free or
bound [27,34,35]. The increase in antioxidant activity observed during
baking could be attributed to the higher extractable (free) phenolic
content resulting from the impact of elevated temperatures [24].
Furthermore, this increase may also be linked to the generation of spe-
cic Maillard reaction products like melanoidins, which possess potent
antioxidant properties which are formed through a condensation reac-
tion between amino acids from proteins and reducing sugars under high
temperature conditions during the baking process [36]. Additionally,
oxidation products of polyphenols have the potential to act as antioxi-
dants [21].
DPPH radical scavenging activity and Ferric reducing antioxidant
power (FRAP) of the dough samples fortied with polyphenolic extract
and 0 % ber-rich fraction were not signicantly different (P >0.05)
compared to the control. A similar trend was observed in the corre-
sponding biscuit samples. This could be attributed to the existence of
phenolic acids in wheat components of the control samples, which
contribute to the antioxidant activity, thereby resulting in minimal
variation in antioxidant activity compared to extract incorporated
samples. It is noteworthy that polyphenols can interact with Maillard
reaction products and can contribute to various mechanisms such as
scavenging of radicals, substitution and nucleophilic addition, thus can
enhance the antioxidant activity [36]. The effect of such mechanism will
depend on the structure of polyphenol due to the complexity of their
structural arrangement with the presence of multiple interconnected
components and hence the interactions formed with the free radicals
[36,37].
In both dough and biscuit samples, DPPH radical scavenging activity
and FRAP value increased with increasing substitution level of ber-rich
fraction with a signicant difference (P <0.05) between 2.5 % and 7.5
% substitution levels, in all three particle sizes. Antioxidant activity of
dough samples decreased with increasing particle size of the ber-rich
fraction. The reason might be the low surface area of large particles
results in lower extraction of bioactive components. However, in forti-
ed biscuits, DPPH scavenging activity increased with increasing par-
ticle size at 2.5 % and 5 % substitution levels of the ber-rich fraction,
but at 7.5 % substitution level the highest antioxidant activity was
achieved with medium sized particles with a signicantly high (P <
0.05) value of 1.06 mmol trolox equivalent (TE)/g sample. Yet, the
lowest FRAP reducing power was observed in the biscuits with medium
sized particles in all three substitution levels of ber-rich fraction. The
highest FRAP antioxidant activity of 4.06 µmol TE/g sample, was ach-
ieved in the biscuit sample fortied with 7.5 % of large sized ber-rich
fraction particles. Overall, in contrast to dough samples, antioxidant
activity of biscuits were higher in large particles compared to small
particles of the ber-rich fraction. Throughout baking process under
high temperature conditions, the breakdown of polyphenols is more
pronounced in small particles because of their larger surface area, as
opposed to large particles. This ultimately results in lower antioxidant
activity in the small particle-ber-rich fraction fortied biscuits. How-
ever, a positive correlation was observed between TPC and antioxidant
activity of fortied dough and biscuit samples. Since polyphenols are
known as the bearers of antioxidant activity, observed increase of
antioxidant activity in fortied samples could be explained by higher
TPC in comparison to the control samples. Such observations were
evident with cookies incorporated with DSP and DDSP [6,32]. However,
direct inclusion of DSP led to an antioxidant activity of 48.20–120.65 µg
TE/g of cookies [6] while with DDSP incorporation the antioxidant ca-
pacity was <50 mmol TE/kg even with 30 % substitution [32]. Present
study’s ndings suggest that fortication of biscuits with ultrasound
assisted polyphenolic extract and ber-rich fraction contributes to
increasing the antioxidant activity of biscuits compared to direct
incorporation or using defatted date seeds alone. As explained in Section
3.1.1, increased accessibility to phenolic components due to ultrasonic
waves could be a reason for the above observation. Polyphenols can
interact with ber [37] and hence can retain in the extraction residue
during the UAE process, which will contribute to the antioxidant prop-
erties of the dough and biscuit fortied with ber-rich fraction. Dietary
M. Ranasinghe et al.
Ultrasonics Sonochemistry 112 (2025) 107160
5
bers also can act as good antioxidants due to their structural arrange-
ment with hydroxyl groups and hydrophobic ends [37–39]. Dietary ber
present in most of the plant materials contain potentially bioactive
compounds, especially phenolic compounds, which are embedded in
them [39]. Different structural formations due to the interactions be-
tween polyphenols and dietary ber, will enhance the antioxidant
properties of food products including bakery products [35,37–39].
Phenolic compounds are mostly residing in the insoluble dietary ber
fraction in the form of esteried phenolic acids [34]. The interactions
formed between dietary ber and polyphenols will breakdown under
high thermal conditions such as baking, further enhancing the antioxi-
dant potential of biscuits compared to dough. Dietary ber, through its
interactions with phenolic compounds as well as with Maillard reaction
products [27,34,38] has the ability to show enhanced antioxidant ac-
tivity which was reected by the results of the current study. The ber
components linked to polyphenols have shown remarkable antioxidant
properties in various fruit and vegetable by-products, such as seeds,
pomace, peels, including date seed, orange seed, lemon seed, mango
peel, grape pomace, guava pulp, pineapple shell and so on [10,38–41].
In summary, the phenolic content and, consequently, the antioxidant
capacity of biscuits exhibited a signicant increase with the fortication
with polyphenolic extract and ber-rich fraction, with the enhancement
being more pronounced as particle size and ber-rich fraction percent-
age increased. In addition to fortication, the baking process also played
a role in boosting the antioxidant potential.
3.2. Nutrient composition of biscuit
Biscuits with medium-sized particles of ber-rich fraction were
chosen for nutrient content analysis, based on sensory analysis results
and tabulated in Table 4. Biscuits fortied with the polyphenolic extract
(EMB) without the ber-rich fraction, showed no signicant difference
(P >0.05) in composition except for moisture content (6.49 % vs control
5.87 %). Polyphenols can modify the binding afnity of gluten, thereby
inuencing its interactions with water [42]. The moisture content of
biscuits has increased signicantly (P <0.05) with the fortication of
ber-rich fraction at all three substitution levels. The high water-
absorbing capacity of ber facilitates water absorption during dough
formation and enhances water retention within the matrix during the
baking process. Comparable results were observed with cookies
substituted with 10 % DDSP [32], breads (5 % and 10 %) and cookies
(7.5 %) substituted with DSP [14,43] while pita bread substituted with
DSP did not show a signicant difference in moisture content between
fortied samples and the control [44]. The acceptable moisture content
of biscuit is <14 % [45] and hence the date seed polyphenols and ber
enriched biscuits possess an acceptable moisture level.
The ash and fat contents of biscuits fortied with the ber-rich
fraction did not differ signicantly from the control or among each
other. Since the DSP was defatted prior to use and the quantity of fat
added in biscuit formulations remains consistent, the fat content in the
end products is likely to be equivalent. Najjar et al.[14] showed a similar
observation in ash content of the cookies fortied with DSP with 2.5 %,
5 % and 7.5 % substitution. In contrast to the ndings of the current
study, bread [43] and biscuits [46] with added DSP resulted in increased
ash content, which could be attributed to differences in formulations
compared to the current study. Protein content did not signicantly
differ between 2.5 % and 5 % substitution levels, but a signicant
decrease (P <0.05) was observed at 7.5 % incorporation of the ber-rich
fraction compared to the control. Substituting the ber-rich fraction for
wheat resulted in decreased protein content, with the most notable
reduction at the highest substitution level. Similarly, biscuits incorpo-
rated with 10 %, 20 % and 30 % of DSP [46], and bread incorporated
with 5 % and 10 % of DSP [43] exhibited signicant decrease in protein
content [43]. Yet the bread with incorporated DSP [44,47] and cookies
with incorporated DDSP [32] exhibited no signicant difference (P >
0.05), possibly due to variations in ingredients like eggs.
Fiber content in terms of neutral detergent ber (NDF) increased
with the fortication of the extraction ber-rich residue obtained
through UAE of DDSP, which was signicant at 5 % and 7.5 % substi-
tution levels of the residue. As DSP is a rich source of functional dietary
ber [48] the nutritional value of biscuits can be enhanced by the
fortication of DSP. Comparable results were obtained with breads
substituted with 1 %–10 % of DSP [43], biscuits with 10 %–30 % DSP
[46] and pita bread with 5 %–20 % DSP [47]. However, the ber content
ranged between 1 % and 9 % in the above-mentioned studies while in
the current study the ber content ranged between 10.17 %–15.11 %
even at the lower substitution levels. Cookies with incorporated DDSP
showed 6–14 % ber content when increasing the DDSP level from 10 to
30 % [32], yet the achieved ber content was observed with higher
substitution levels compared to the present study. Phenolic compounds
are able to interact with ber components (as explained in detail in
Section 3.1). Through aqueous phase extraction of DDSP using UAE,
water-soluble polyphenols were mostly extracted, and these poly-
phenolic extracts were incorporated into biscuit formulations. This
process enables various interactions between polyphenols and ber,
encompassing hydrophilic, hydrophobic, and covalent interactions [37].
Besides, the elevated thermal conditions utilized in bakery goods pro-
mote the increase of dietary ber levels. The gelatinization and retro-
gradation of starch, along with the formation of Maillard reaction
products, facilitate the cross-linking of ber components [34]. Addi-
tionally, the interactions established between ber and phenolic com-
pounds might undergo oxidation, further enhancing the cross-linking of
dietary ber. Consequently, the total dietary ber content of the food
product can be heightened [34], which is reected by the NDF content of
the present study. Overall, fortication of biscuits with polyphenolic
extract and ber-rich fraction has the potential to improve the nutri-
tional value of biscuits while maintaining the acceptable levels of
moisture which is a critical factor related to the quality of bakery
products.
3.3. Cytotoxicity assay
Cytotoxicity assays are used to determine the ability of a compound
to cause cell death which in turn helps predict its toxicity in humans or
animals [49]. The highest concentration of TPC which was utilized in
biscuit formulations was used for the cytotoxicity assay with a TPC of
0.70 mg GAE/ml water. The effect of polyphenolic extract on cell
viability was examined using Caco-2 cell lines. The selection of the
above-mentioned cell lines was made because they have been widely
utilized in research on intestinal absorption and cytotoxicity [50–52].
Cytotoxicity against Caco-2 cell lines was assessed and there was no
indication of increased cytotoxicity effects. The highest cell death per-
centage was 11.91 % (Fig. 1). If cells demonstrate a cell death per-
centage of <30 % with a viability exceeding 70 %, upon exposure to a
substance, it can be considered non-cytotoxic [53]. Therefore, the
Table 4
Changes in protein, fat, moisture, ash and NDF contents of biscuits fortied with
the phenolic extract and ber-rich fraction of DDSP obtained by UAE.
Sample Protein
(%)
NDF (%) Moisture
(%)
Ash (%) Fat (%)
CB 9.35
±0.42
a
8.79±0.54
c
5.87±1.17
b
1.60
±0.09
a
16.02
±0.17
a
EMB 8.74
±0.56
ab
9.03
±0.38
bc
6.49±0.20
a
1.56
±0.20
a
15.62
±0.58
a
2.5MB 8.25
±0.15
ab
10.17
±0.18
bc
7.01±0.43
a
1.43
±0.14
a
15.60
±0.10
a
5MB 8.24
±0.01
ab
10.60
±0.18
b
6.80±0.52
a
1.59
±0.20
a
15.72
±0.98
a
7.5MB 8.00
±0.02
b
15.11
±0.53
a
7.88±0.02
a
1.68
±0.13
a
15.71
±1.74
a
1
Means ±SD are presented. Different lowercase superscript letters in a column
denote signicant differences, P < 0.05.
M. Ranasinghe et al.
Ultrasonics Sonochemistry 112 (2025) 107160
6
aqueous phenolic extract obtained through UAE of DDSP in the current
study can be considered as non-cytotoxic even up to higher percentages
like 40 %. However, further studies using different cell lines and animal
trails are worth, to have a deep understanding on cytotoxic activity of
the extract. Additionally, prior research has concluded that date seed
extracts are deemed safe for use, showing non-signicant cytotoxicity at
clinically acceptable doses of less than 1 mg/ml [54].
3.4. Sensory analysis of biscuits
The samples including 2.5MB, 5MB, 7.5MB, 7.5SB and 7.5LB were
selected to conduct the sensory analysis. Sensory scores (using 9-point
hedonic scale) of the biscuit samples fortied with the polyphenolic
extract and the ber-rich fraction, compared to the control, are pre-
sented in Table 5. In terms of odour, taste, mouthfeel and overall
acceptability biscuits fortied with the polyphenolic extract and ber-
rich fraction, did not show a signicant difference compared to the
control. Consumer preference in terms of colour has decreased with
increasing the level of substitution of ber-rich fraction. In a previous
study, the cookies incorporated with DSP did not show such trend in
acceptance in terms of colour, as those cookies earned high scores even
for darker ones compared to the control [14] but the cookies with
incorporated DDSP showed a decreased score for appearance when the
substitution level increases [32]. Increasing the amount of plant mate-
rials with high ber contents in formulations, results in darker colour of
biscuits, affecting the consumer preference which was evident by bis-
cuits supplemented with banana peel, rice bran, wheat bran, Murraya
koenigii leaves and grape pomace [55–58]. Additionally, biscuits with
7.5 % small particles (7.5SB) of the ber-rich fraction scored signi-
cantly lower scores compared to medium (7.5MB) and large particles
(7.5LB). Small particles have the potential to disperse more evenly
within the food matrix, occupying a larger proportion of the volume
compared to large particles. This can have a greater impact on the
overall color of the biscuit. The darker color of the biscuits due to small
particle dispersion may have a negative effect on consumer acceptance.
Consumer acceptability with regard to texture was not signicantly
different in fortied samples except 7.5LB with 7.5 % large particles of
the ber-rich fraction. The inclusion of high levels of ber may lead to a
hard texture, while larger particles of external material could impact the
smoothness of the biscuit, resulting in a rough, granular surface that may
reduce consumer acceptance [58]. Similarly, DDSP incorporated cookies
showed reduced scores for texture as a result of the grainy mouthfeel
associated with higher levels of DDSP addition [32]. Comparable results
were obtained with biscuits with incorporated grape pomace and wheat
bran [56,58]. In order to obtain consumer acceptance, it is important to
maintain the particle size and substitution level of the ber-rich fraction.
The ndings of the current study suggest that 5 % substitution level of
medium particles can be selected as the most accepted fortied sample
with minimal difference compared to control biscuit. However, the
scores of overall acceptability according to the hedonic scale ranged
between 5.26 and 5.97 indicating that the fortied biscuits have
acceptance by consumers which was comparable with the control
biscuits.
3.5. In-vitro digestibility of biscuit
Phenolic compounds are widely recognized as one of the main con-
tributors to health-related benets by plant-derived foods and their by-
products. In order to demonstrate their biological effects, phenolic
compounds need to be released from the food matrix during digestion in
a form that can be absorbed (or a bioaccessible form) and eventually
absorbed and transported to the bloodstream making them bioavailable
[59]. The bioaccessibility and bioavailability of phenolic compounds
may be hindered by structural arrangements and interactions within the
food matrix, which can be addressed through food processing tech-
niques. The baking process has evidently altered the structural ar-
rangements of compounds to enhance their bioaccessibility and
bioavailability, simultaneously boosting the phenolic content within the
food matrix [27,34,60,61].
In order to assess the potential of date seed polyphenol and ber-rich
fraction fortied biscuits as a viable source of bioaccessible polyphenolic
compounds, the TPC of the extracts obtained post exposure to a simu-
lated in-vitro digestion model was evaluated (Table 6). In all the samples
the TPC has increased in the order of oral <gastric <intestinal. There
was no signicant difference in TPC of the fortied biscuits with control
in oral phase. A brief contact time of the food with the amylase enzyme
could limit polyphenol release, apart from the inadequate digestion with
minimal pH changes and enzyme activity in the oral phase [61–63],
resulting a the lowest TPC compared to gastrointestinal phase with no
signicant difference among samples. TPC of gastric digesta ranged
between 0.82 and 1.36 mg GAE/g sample which was signicantly higher
(P <0.05) compared to oral phase. The release of phenolic compounds
from the matrices is signicantly inuenced by the gastric conditions
(enzyme and acidic pH). This effect is primarily attributed to the acidic
pH and enzymatic activity, which facilitate the release of certain
phenolic compounds bound to other constituents such as proteins, iron
etc., into the gastric juice [64]. The highest TPC was observed in the
intestinal phase ranged from 0.81–1.44 mg GAE/ g sample, suggesting a
high phenolic release and hence bioaccessibility. During the intestinal
phase, changes in chemical structures can occur due to the pH condi-
tions (pH 7) and effects of pancreatin-bile salts can come into action,
leading to the formation of new compounds with altered bioactivity and
Fig. 1. Cell death % in Caco-2 cell line with different percentages of extract
obtained through ultrasound-assisted extraction of defatted DSP. Different
lowercase letters on bars denote signicant differences, P <0.05.
Table 5
Sensory scores of the biscuits as affected by the fortication of phenolic extract and ber-rich fraction of DDSP obtained by UAE.
Sample Color Odor Texture Taste Mouthfeel Overall Acceptability
CB 7.14±2.09
a
6.52±1.98
a
5.61±2.34
a
5.80±2.05
a
5.60±2.25
a
5.77±2.00
a
2.5MB 6.39±1.86
ab
6.07±1.81
a
5.30±2.28
ab
6.07±2.02
a
5.66±2.08
a
5.86±2.03
a
5MB 6.19±1.79
b
6.04±1.75
a
5.38±2.03
ab
6.39±1.75
a
5.90±1.97
a
5.97±1.82
a
7.5SB 5.18±2.12
c
5.83±1.74
a
4.93±2.17
ab
5.68±1.95
a
5.18±2.03
a
5.26±2.03
a
7.5MB 6.11±1.68
b
6.20±1.62
a
5.17±2.01
ab
5.96±1.94
a
5.83±2.06
a
5.73±1.78
a
7.5LB 6.28±1.84
b
6.12±1.70
a
4.48±2.37
b
5.63±2.13
a
5.10±2.21
a
5.53±1.90
a
1
Means ±SD are presented. Different lowercase superscript letters in a column denote signicant differences, P < 0.05.
M. Ranasinghe et al.
Ultrasonics Sonochemistry 112 (2025) 107160
7
bioaccessibility. Besides, the polyphenols which are not absorbed in
gastric phase, reach the intestine where the intestinal microbiota can
convert them into low molecular weight, bioactive forms, facilitating
their absorption [65]. Additionally, the increased absorption during the
intestinal phase might be attributed to the extended digestion period in
intestinal conditions, enhancing the extraction of phenolics from the
matrix in to intestinal digesta [61].
Biscuits fortied only with polyphenolic extract (ESB, EMB and ELB)
exhibited non-signicant difference in TPC compared to control in both
gastric and intestinal digesta. The ndings of the current study suggest
that the polyphenols directly added in the form of extract might be less
stable in gastrointestinal conditions compared to the phenolic com-
pounds which are embedded within ber-rich matrix. TPC of the digesta
obtained from the biscuits with 7.5 % substitution was signicantly
higher compared to 2.5 % both in gastric and intestinal phases indi-
cating that release of phenolics has increased with increasing substitu-
tion level of the ber-rich fraction. As the ber content in biscuits
increases, the bound phenolic components within the ber will also
increase. Due to the pH and enzymatic conditions within gastrointestinal
phase, phenolic compounds associated with ber components can be
released further enhancing the TPC [34,39]. The phenolic compounds
linked with dietary ber could have signicant impacts on intestinal
health [39]. The ndings of the present study reveal that the bio-
accessibility of polyphenols in biscuits fortied with polyphenolic
extract and ber-rich fraction increases as the fortication level in-
creases (Fig. 2). Increased bioaccessibility values indicate the liberation
of metabolized compounds from intricate phenolic structures [66]. In
contrast, [67] has observed that polyphenols linked to ber exhibit poor
solubility and limited extractability in gastrointestinal uids. The in-
teractions within polyphenol-ber complexes are inuenced by their
structural organization, and the release of phenolics is closely tied to the
arrangement of these complex molecules. Furthermore, the release of
phenolics is subject to variation based on their interactions with other
molecules such as starch and protein, which is contingent upon the type
of food. There was a signicant increase in bioaccessibility at 5 % and
7.5 % fortication levels compared to 2.5 %. The incorporation of UAE
in the present study might have facilitated the partial breakdown of the
ber matrix during extraction, potentially enhancing the efcient
release of bound phenolics during SGID. Consequently, fortication with
the ber-rich fraction resulted in a higher bioaccessibility of phenolics
compared to fortication with the phenolic extract. The increase in
bioaccessibility in metabolism, indicate that the cookie matrix may have
a protective impact on polyphenols. This protection allows these com-
pounds to be trapped within the matrix during oral and gastric phases,
leading to enhanced release during the intestinal digestion [61,62].
Biscuits containing a 5 % substitution of ber-rich fraction exhibited the
highest TPC in gastric digesta compared to those with 2.5 % and 7.5 %
substitutions across all three particle sizes. However, biscuits fortied
Table 6
TPC of biscuits after the simulated in vitro gastrointestinal digestion.
Particle size of
residue
Residue
%
Sample TPC (mg GAE/ g sample)
Oral Gastric Intestine
−0 CB 0.54 ±
0.01
aB
0.86 ±
0.03
ghA
0.83 ±
0.01
eA
Small 0 ESB 0.51 ±
0.01
aB
0.82 ±
0.02
hA
0.84 ±
0.01
deA
2.5 2.5SB 0.51 ±
0.01
aB
0.90 ±
0.04
efghA
0.94 ±
0.01
cA
5 5SB 0.50 ±
0.01
aC
1.03 ±
0.04
cdB
1.39 ±
0.06
bA
7.5 7.5SB 0.51 ±
0.1
aC
1.16 ±
0.03
bB
1.35 ±
0.01
abA
Medium 0 EMB 0.51 ±
0.01
aC
0.91 ±
0.04
efgA
0.81 ±
0.04
eB
2.5 2.5MB 0.50 ±
0.01
aB
0.84 ±
0.01
ghA
0.81 ±
0.04
eA
5 5MB 0.52 ±
0.01
aC
1.30 ±
0.03
aB
1.37 ±
0.04
abA
7.5 7.5MB 0.52 ±
0.01
aB
1.36 ±
0.04
aA
1.41 ±
0.05
abA
Large 0 ELB 0.53 ±
0.01
aC
0.89 ±
0.02
fghA
0.84 ±
0.02
deB
2.5 2.5LB 0.51 ±
0.03
aB
0.95 ±
0.03
defA
0.93 ±
0.03
cdA
5 5LB 0.52 ±
0.01
aC
0.98 ±
0.03
cdeB
1.37 ±
0.03
abA
7.5 7.5LB 0.51 ±
0.01
aC
1.06 ±
0.01
cB
1.44 ±
0.03
aA
1
Means ±SD are presented. Different lowercase superscript letters in a column
and uppercase superscript letters in a row denote signicant differences, P <
0.05.
Fig. 2. Bioaccessibility indexes based on TPC after in vitro gastrointestinal digestion of the control biscuit and the biscuits fortied with the polyphenolic extract and
the ber rich fraction of DDSP obtained after UAE extraction. Different lowercase letters on bars denote signicant differences (P <0.05).
M. Ranasinghe et al.
Ultrasonics Sonochemistry 112 (2025) 107160
8
with small, medium and large particle sizes at the same substitution
level of ber-rich fraction did not show signicant differences in the TPC
of the intestinal digesta, indicating that there is no clear trend in particle
size of the ber-rich fraction in the release during gastrointestinal
digestion. Regardless of the particle size the extreme gastrointestinal
conditions in the simulated system, are strong enough to dissociate and
release the polyphenolic compounds that are associated with ber
components. It is noteworthy that association and dissociation reactions
between polyphenols and ber are dependent on the conditions
including pH and nature of the medium of gastrointestinal system, apart
from their structural arrangements [39]. Biscuits fortied with quinoa,
buckwheat, amaranth, and horseradish, showed increased TPC after
simulated in vitro digestion with increased bioaccessibility [17,60,68].
Comparable results were observed in bread enriched with buckwheat
and rice bran, showing an increase in the bioaccessibility of polyphenols
as the substitution levels increased [69,70]. The enhanced antioxidant
activity of the intestinal digesta from bread samples enriched with
wholegrain and multigrain our was attributed to interactions between
ber and phenolic compounds, and their reactions during digestion,
facilitating the release of TPC [34], and these observations support the
ndings of the present study. Besides, biscuits enriched with ber
extracted from artichoke by-products, appeared to be high in bioavail-
able phenolics in human digestion models [71]. Overall, the fortication
of polyphenolic extract and ber-rich fraction obtained through UAE of
DDSP in the biscuits resulted in enhanced bioaccessibility of poly-
phenols in simulated in vitro gastrointestinal digestion, with the
enhancement being more pronounced as the substitution level of the
ber-rich fraction increased.
3.6. Phenolic retention in biscuits during storage
TPC of the biscuits has decreased signicantly by the end of 6 months
compared to the initial concentration (Fig. 3). At the end of 2nd month,
TPC of the biscuits fortied with the polyphenolic extract, and 0 % and
2.5 % of the ber-rich fraction, did not show a signicant difference with
the control while biscuits with 5 % and 7.5 % ber-rich fraction
exhibited signicantly higher TPC. By the end of 4 months and 6 months
the above trend was the same except the samples 5SB and 7.5SB where
the reductions of TPC were from 1.25 to 1.14 mg GAE/g sample and
from 1.21 to 1.13 mg GAE/g sample, respectively. The differences in
TPC among the samples stored for 0, 2 and 4 months were not signicant
in the control biscuit and the fortied samples (ESB, EMB, ELB, 2.5SB
and 2.5MB). In contrast the biscuits fortied with 5 % and 7.5 % ber-
rich fraction exhibited a drastic and signicant decrease in TPC at each
storage time. No consistent trend was observed in the retention of
phenolic compounds during storage among the samples with varying
particle sizes of the ber-rich fraction. Reduction of TPC in biscuits
during storage could be attributed to the decomposition of phenolic
compounds due to chemical and environmental factors such as oxidation
and temperature conditions [72,73]. As suggested by Spigno et al. [74]
throughout storage, numerous radical products are generated through
several reactions, including lipid oxidation. These products can poten-
tially interact with polyphenols, diminishing their availability and their
detection by Folin-Ciocalteu assay. Additionally, the Maillard reaction
might progress during storage, resulting in the formation of Maillard
reaction products that could potentially interact with polyphenols
making them unavailable [75]. Moreover, polyphenols can scavenge
free radicals generated during storage from lipid oxidation, leading to a
decrease in TPC [76]. When the polyphenol content in a product is
increased, there will be a greater availability of phenols for scavenging,
resulting in a higher quantity of phenolic components remaining in the
food matrix during storage. Besides, the structural changes induced by
ultrasonication might enhance the interactions among phenolic com-
ponents, thereby increasing the retention of polyphenols within the food
matrix. According to the ndings of the current study, biscuits fortied
with ber-rich fraction had higher TPC deterioration rate compared to
Fig. 3. Changes in TPC values of the biscuit during storage. 0: 0 months, 2: after 2 months, 4: after 4 months, 6: after 6 months. Different lowercase letters on bars
denote signicant differences (P <0.05) among samples at the same time point and uppercase letters denote signicant differences (P <0.05) among storage times
within the same sample.
M. Ranasinghe et al.
Ultrasonics Sonochemistry 112 (2025) 107160
9
the control and 0 % ber-rich fraction incorporated samples.
Decrease of TPC at the end of 6-month storage ranged between 26.7
% to 44.3 % in the biscuits fortied with ber-rich fraction while the
reduction was only 19.37 % in the control. ESB, EMB and ELB showed
TPC reductions of 16.11 %, 21.02 % and 19.13 %, respectively. As the
initial TPC was higher in fortied biscuits the decrease of polyphenols
could be more prominently visible compared to the control which was
having lower TPC than the fortied biscuits. A higher initial TPC leads to
a greater reduction in polyphenol content during storage [75]. Though
the deterioration rate is high, the TPC at the end of 6 months, was still
signicantly higher in ber enriched samples compared to the control,
suggesting that TPC retention has increased with DSP ber fortication.
The retention of polyphenols is inuenced by key factors such as oxygen,
pH, temperature, light, moisture, enzymes, and metal ions [73,76].
Various interactions between ber components and polyphenols [37,39]
will facilitate the stability of polyphenols from such factors.
Increased retention of polyphenols in biscuits during storage, were
evident with several studies conducted on biscuits fortied with poly-
phenols and ber rich powders [71,72,77]. Comparable results were
observed with the biscuits fortied with artichoke ber, exhibiting
signicantly higher TPC compared to control biscuits, after a storage
period of seven months with the polyphenol reduction of 28 % [71].
Similarly, blueberry, poppy seed and grape seed incorporated biscuits
showed a decrease in TPC by 19.76 % after a ve-month storage period
[72]. The differences in TPC reduction percentages of the current study
compared to the previous research could be attributed to the differences
in storage time, formulation, environmental conditions and type of
fortication ingredients [72]. In general, the results of the present study
indicate that fortifying biscuits with polyphenolic extract and ber-rich
fraction obtained through UAE of DDSP, leads to enhanced retention of
phenolic compounds.
3.7. Storage stability of biscuits
The storage stability of biscuits fortied with polyphenolic extract
and ber-rich fraction obtained through UAE was assessed based on
microbial stability and lipid oxidation. Bakery products with low
moisture and high fat content, such as biscuits, are more susceptible to
lipid oxidation than microbial growth during extended storage periods.
This susceptibility can result in a decline in quality over time. Over the
storage period of 6 months, the fortied biscuits were microbiologically
safe as evident by the results of bacterial counts obtained using PCA and
PDA. Please refer to Supplementary material (Fig. S3) for the images.
Lipid oxidation is the primary reason for rancidity, which diminishes the
quality of high-fat foods like bakery products, leading to undesirable
avors and odors. Biscuits, being high in fat content, are particularly
prone to oxidation. The progress of lipid oxidation in biscuits can be
assessed by the levels of secondary lipid oxidation products, such as
aldehydes, which are volatile compounds [77]. TBARS values of all the
biscuits has increased signicantly (P <0.05) over the storage period of
6 months (Fig. 4). The initial fat content added to the biscuit formulation
was consistent, and the DSP used was defatted beforehand. As a result,
all the biscuits exhibited similar initial TBARS values, with no statisti-
cally signicant difference (P >0.05). After 2 months, biscuits fortied
with large particles of ber-rich fractions at three incorporation levels
showed signicantly lower (P <0.05) TBARS values compared to the
control (no extract added) and biscuits with 0 % ber-rich incorpora-
tion. By the end of 4 months, 2.5LB, 5MB, 5LB, and all 7.5 % ber-
fortied samples exhibited signicantly lower TBARS values than the
control (P <0.05). A similar trend was observed after 6 months, except
for 2.5LB, 5SB and 5MB, which were not signicantly different (P >
0.05) compared to the control. The overall results of the study suggest
that fortifying biscuits with ber-rich fraction enhances their oxidative
stability, with signicant decrease in TBARS (P <0.05) at high incor-
poration levels and large particle sizes. The inclusion of natural in-
gredients with antioxidant properties helps prevent nutrient losses by
slowing down or inhibiting oxidation reactions. For instance, the free
radical scavenging property of polyphenols plays a signicant role in
inhibiting the oxidation of biscuits and cookies [78–81]. The non-
signicant difference in the control (no extract added) and 0 % ber
incorporated biscuits could be attributed to the lower concentrations
being extracted while UAE due to the retention of polyphenols in the
ber matrix in a bound form, thus, exhibiting a minimal variation in TPC
compared to the control sample. Additionally, the process conditions,
formulations and environmental conditions can affect the outcomes of
the study.
As demonstrated by the results of the current study, fortication of
biscuits with polyphenolic extract and ber-rich fraction of DDSP was
more efcient towards retardation of lipid oxidation. The entrapment
and interactions of polyphenols, specially bound polyphenols in ber,
result in more polyphenol concentration within samples (as explained in
Section 3.1). Therefore, polyphenolic content in the fortied biscuits
Fig. 4. Changes in TBARS values of the biscuit during storage. 0: 0 months, 2: after 2 months, 4: after 4 months, 6: after 6 months. Different lowercase letters on bars
denote signicant differences (P <0.05) among samples at the same time point and uppercase letters denote signicant differences (P <0.05) among storage times
within the same sample.
M. Ranasinghe et al.
Ultrasonics Sonochemistry 112 (2025) 107160
10
increase with increasing the incorporation level of ber-rich fraction.
Additionally, ber compounds baring hydrophilic and hydrophobic
ends, can also act as good antioxidants [37–39] contributing in sup-
pressing the free radical formed, hence retarding lipoid oxidation.
Physical changes that occur during ultrasonication may enhance the
accessibility of phenolic components within the ber matrix [33],
leading to improved bioactive properties of the nal product when
compared to the direct incorporation of DSP [6]. Large particles with
low surface area facilitate the retention of polyphenols during baking
and storage, further contributing to the increase of phenolic content.
Comparable results were observed by Asadi et al. [82] with cookies
fortied with sapota ber and beet root leaf powder, showing increased
shelf-life in terms of lipid oxidation with 7 % sapota ber supplemented
cookies. After a 4-month storage period, among the fortied samples,
ber-rich fraction incorporated samples exhibited TBARS values ranging
from 14.84–17.57 nmol MDA/g sample, whereas 0 % ber-enriched
samples values were between 16.21 and 16.93 nmol MDA/g sample.
The highest value of 17.48 nmol MDA/g sample, was observed in the
control sample after 4 months. At the end of the storage period of 6
months, biscuit samples exhibited high TBARS values above the
accepted level of 20 nmol MDA/g sample [83,84], except 5LB and 7.5LB.
Therefore, only the fortied biscuits containing the ber-rich fraction of
large particles at 5 % and 7.5 % substitution, were stable in terms of
rancidity by the end of 6 months. Numerous studies have demonstrated
signicant oxidative degradation in biscuits during a 6-month storage
period [80,85], while other studies have shown oxidative stability in
biscuits even after 12 months of storage [86,87] after the fortication of
plant materials with antioxidant properties. It is important to note that
factors such as processing conditions, ingredients, storage and pack-
aging conditions greatly impact the stability of bakery products,
including biscuits and cookies [88,89]. Overall, the fortication of
polyphenol extract and ber-rich fraction from DDSP to biscuits signif-
icantly reduced lipid oxidation, noticeable at higher fortication levels
with large particles of the ber-rich fraction.
4. Conclusion
The fortication of polyphenolic extract and ber-rich fraction ob-
tained through UAE of DDSP signicantly enhanced the antioxidant
properties of both the dough and biscuit, with a corresponding increase
in antioxidant activity observed post-baking. The nutritional value of
biscuits was improved through fortication with the polyphenolic
extract and ber-rich fraction obtained from DDSP, without negatively
impacting consumer acceptance. Fortied biscuits showed retarded lipid
oxidation during storage with enhanced phenolic retention as well as
increased bioaccessibility of polyphenols upon simulated in vitro diges-
tion. The impact of fortifying biscuit with ber-rich fraction of DDSP
obtained after UAE, on its properties and sensory acceptance, is signif-
icantly inuenced by both the particle size and the substitution level.
Based on the current data, it can be inferred that fortifying with aqueous
phase polyphenolic extract and ber-rich fraction from date seeds ob-
tained through UAE would lead to nutritionally enhanced biscuits that
meet consumer preferences. Most importantly, this research focuses on a
green approach without using organic solvents for extraction. Addi-
tional research, including clinical studies, would help validate the pos-
itive effects of fortication on specic health promoting properties. It is
worth trying different bakery products to understand various impacts on
those products in order to expand the utilization of DSP in bakery
industry.
CRediT authorship contribution statement
Meththa Ranasinghe: Writing – original draft, Software, Method-
ology, Investigation, Formal analysis, Data curation. Mariam Alghai-
thi: Formal analysis. Constantinos Stathopoulos: Writing – review &
editing, Supervision, Methodology, Conceptualization. Balan
Sundarakani: Project administration, Funding acquisition. Sajid
Maqsood: Writing – review & editing, Supervision, Resources, Project
administration, Methodology, Funding acquisition, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Acknowledgement
Authors would like to acknowledge the funding received by the
United Arab Emirates Ministry of Education; Project ‘READY’ [grant
number G00003563]. Authors extend their thank to Dr. Jaleel Kizhak-
kayil, Department of Nutrition and Health, College of Medicine and
Health Sciences, United Arab Emirates University, for conducting the
cytotoxicity assay.
Ethical statement
All procedures in the sensory analysis were performed in compliance
with relevant laws and guidelines and have been approved by the UAEU
Social Sciences Ethics Committee (application No: ERSC_2022_1730) on
25/10/2022.
The privacy rights of human subjects have been observed and
informed consent was obtained for experimentation.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ultsonch.2024.107160.
References
[1] M. Goubgou, L.T. Songr´
e-Ouattara, F. Bationo, H. Lingani-Sawadogo, Y. Traor´
e,
A. Savadogo, Biscuits: a systematic review and meta-analysis of improving the
nutritional quality and health benets, Food Prod. Process. Nutr. 3 (2021) 26.
[2] V. Melini, F. Melini, F. Luziatelli, M. Ruzzi, Functional ingredients from agri-food
waste: effect of inclusion thereof on phenolic compound content and
bioaccessibility in bakery products, Antioxidants 9 (2020) 1216.
[3] M. Ranasinghe, I. Manikas, S. Maqsood, C. Stathopoulos, Date components as
promising plant-based materials to be incorporated into baked goods—a review,
Sustainability 14 (2022) 605.
[4] S. Maqsood, O. Adiamo, M. Ahmad, P. Mudgil, Bioactive compounds from date
fruit and seed as potential nutraceutical and functional food ingredients, Food
Chem. 308 (2020) 125522, https://doi.org/10.1016/j.foodchem.2019.125522.
[5] S. Basha, H. Ahfaiter, A.M. Zeitoun, A.E. Abdalla, Physicochemical properties and
nutritional value of egyptian date seeds and its applications in some bakery
products, 23 (2018) 260–279.
[6] Z. Najjar, J. Kizhakkayil, H. Shakoor, C. Platat, C. Stathopoulos, M. Ranasinghe,
Antioxidant potential of cookies formulated with date seed powder, Foods 11
(2022) 448.
[7] M. Ranasinghe, N. Sivapragasam, H. Mostafa, J.O. Airouyuwa, I. Manikas,
B. Sundarakani, S. Maqsood, C. Stathopoulos, Valorizing date seeds in biscuits: a
novel approach to incorporate bioactive components extracted from date seeds
using microwave-assisted extraction, Resour. Environ. Sustain. 15 (2024) 100147.
[8] I. Usman, M. Hussain, A. Imran, M. Afzaal, F. Saeed, M. Javed, A. Afzal, I. Ashfaq,
E. Al Jbawi, S.A. Saewan, Traditional and innovative approaches for the extraction
of bioactive compounds, Int. J. Food Prop. 25 (2022) 1215–1233.
[9] L. Shen, S. Pang, M. Zhong, Y. Sun, A. Qayum, Y. Liu, A. Rashid, B. Xu, Q. Liang,
H. Ma, A comprehensive review of ultrasonic assisted extraction (UAE) for
bioactive components: principles, advantages, equipment, and combined
technologies, Ultrason. Sonochem. 101 (2023) 106646.
[10] M. Ranasinghe, H. Mostafa, N. Sivapragasam, C. Stathopoulos, I. Manikas,
S. Maqsood, Sustainable approach for defatted date seed valorization through
ultrasonication-based green extraction: a prospective approach for nutraceutical
applications, Sustain. Chem. Pharm. 35 (2023) 101138.
[11] K. Kerboua, D. Mazouz, I. Hasaounia, O. Hamdaoui, Mechanical technologies:
ultrasound and cavitation in food processing, in: Nonthermal Process. Agri-Food-
Bio Sci. Sustain. Futur. Goals, Springer, 2022, pp. 189–221.
[12] E. Herrera-Pool, A.L. Ramos-Díaz, M.A. Lizardi-Jim´
enez, S. Pech-Cohuo, T. Ayora-
Talavera, J.C. Cuevas-Bernardino, U. García-Cruz, N. Pacheco, Effect of solvent
polarity on the Ultrasound Assisted extraction and antioxidant activity of phenolic
M. Ranasinghe et al.
Ultrasonics Sonochemistry 112 (2025) 107160
11
compounds from habanero pepper leaves (Capsicum chinense) and its
identication by UPLC-PDA-ESI-MS/MS, Ultrason. Sonochem. 76 (2021) 105658.
[13] AOAC, Ofcial methods of analysis of the Association of Ofcial Analytical
Chemists., 17th ed., Assoc. Off. Anal. Chem., Washington, DC, USA, 2005.
[14] Z. Najjar, M. Alkaabi, K. Alketbi, C. Stathopoulos, M. Ranasinghe, Physical
chemical and textural characteristics and sensory evaluation of cookies formulated
with date seed powder, Foods 11 (2022) 1–13, https://doi.org/10.3390/
foods11030305.
[15] K.R. Elfahri, T. Vasiljevic, T. Yeager, O.N. Donkor, Anti-colon cancer and
antioxidant activities of bovine skim milk fermented by selected Lactobacillus
helveticus strains, J. Dairy Sci. 99 (2016) 31–40.
[16] A. Brodkorb, L. Egger, M. Alminger, P. Alvito, R. Assunç˜
ao, S. Ballance, T. Bohn,
C. Bourlieu-Lacanal, R. Boutrou, F. Carri`
ere, A. Clemente, M. Corredig, D. Dupont,
C. Dufour, C. Edwards, M. Golding, S. Karakaya, B. Kirkhus, S. Le Feunteun,
U. Lesmes, A. Macierzanka, A.R. Mackie, C. Martins, S. Marze, D.J. McClements,
O. M´
enard, M. Minekus, R. Portmann, C.N. Santos, I. Souchon, R.P. Singh, G.
E. Vegarud, M.S.J. Wickham, W. Weitschies, I. Recio, INFOGEST static in vitro
simulation of gastrointestinal food digestion, Nat. Protoc. 14 (2019) 991–1014,
https://doi.org/10.1038/s41596-018-0119-1.
[17] L. Tomsone, R. Galoburda, Z. Kruma, K. Majore, Physicochemical properties of
biscuits enriched with horseradish (Armoracia rusticana L.) products and
bioaccessibility of phenolics after simulated human digestion, Polish J. Food Nutr.
Sci. 70 (2020) 419–428, https://doi.org/10.31883/pjfns/130256.
[18] S. Alkaabi, B. Sobti, P. Mudgil, F. Hasan, A. Ali, A. Nazir, Lemongrass essential oil
and aloe vera gel based antimicrobial coatings for date fruits, Appl. Food Res. 2
(2022) 100127, https://doi.org/10.1016/j.afres.2022.100127.
[19] S. Maqsood, S. Benjakul, Effect of bleeding on lipid oxidation and quality changes
of Asian seabass (Lates calcarifer) muscle during iced storage, Food Chem. 124
(2011) 459–467, https://doi.org/10.1016/j.foodchem.2010.06.055.
[20] J.C. S´
anchez-Rangel, J. Benavides, J.B. Heredia, L. Cisneros-Zevallos, D.A. Jacobo-
Vel´
azquez, The Folin–Ciocalteu assay revisited: improvement of its specicity for
total phenolic content determination, Anal. Methods 5 (2013) 5990–5999.
[21] M. Paciulli, M. Grimaldi, M. Rinaldi, A. Cavazza, F. Flamminii, C. Di Mattia,
M. Gennari, E. Chiavaro, Microencapsulated olive leaf extract enhances
physicochemical stability of biscuits, Futur. Foods 7 (2023) 100209, https://doi.
org/10.1016/j.fufo.2022.100209.
[22] L. Wang, Y. Yao, Z. He, D. Wang, A. Liu, Y. Zhang, Determination of phenolic acid
concentrations in wheat ours produced at different extraction rates, J. Cereal Sci.
57 (2013) 67–72.
[23] P. Gelinas, C.M. McKinnon, Effect of wheat variety, farming site, and bread-baking
on total phenolics, 41 (2006) 329–332.
[24] D. Vitali, I.V. Dragojevi´
c, B. ˇ
Sebeˇ
ci´
c, Effects of incorporation of integral raw
materials and dietary bre on the selected nutritional and functional properties of
biscuits, Food Chem. 114 (2009) 1462–1469.
[25] M. Starowicz, S. Arpaci, J. Topolska, M. Wronkowska, Phytochemicals and
antioxidant activity in oat-buckwheat, Molecules 226 (2021) 1–12.
[26] C.M. Ajila, K. Leelavathi, U.J.S.P. Rao, Improvement of dietary ber content and
antioxidant properties in soft dough biscuits with the incorporation of mango peel
powder, J. Cereal Sci. 48 (2008) 319–326.
[27] E.-S.-M. Abdel-Aal, I. Rabalski, Effect of baking on free and bound phenolic acids in
wholegrain bakery products, J. Cereal Sci. 57 (2013) 312–318.
[28] J. Xu, W. Wang, Y. Li, Dough properties, bread quality, and associated interactions
with added phenolic compounds: a review, J. Funct. Foods 52 (2019) 629–639.
[29] A.S. Sivam, D. Sun-Waterhouse, S.Y. Quek, C.O. Perera, Properties of bread dough
with added ber polysaccharides and phenolic antioxidants: a review, J. Food Sci.
75 (2010), https://doi.org/10.1111/j.1750-3841.2010.01815.x.
[30] D. Sun-Waterhouse, A. Teoh, C. Massarotto, R. Wibisono, S. Wadhwa, Comparative
analysis of fruit-based functional snack bars, Food Chem. 119 (2010) 1369–1379,
https://doi.org/10.1016/j.foodchem.2009.09.016.
[31] T. Mihic, D. Rainkie, K.J. Wilby, S.A. Pawluk, The therapeutic effects of camel
milk: a systematic review of animal and human trials, J. Evid.-Based Complement.
Altern. Med. 21 (2016) NP110–NP126, https://doi.org/10.1177/
2156587216658846.
[32] A. Mrabet, A. Hamdi, R. Rodríguez-Arcos, R. Guill´
en-Bejarano, A. Jim´
enez-Araujo,
Date seed by-products as source of bioactive ingredient for healthy cookies, Food
Biosci. 61 (2024) 104543, https://doi.org/10.1016/j.fbio.2024.104543.
[33] L. Cao, Y. Park, S. Lee, D.O. Kim, Extraction, identication, and health benets of
anthocyanins in blackcurrants (Ribes nigrum l.), Appl. Sci. 11 (2021) 1–14,
https://doi.org/10.3390/app11041863.
[34] V. Benítez, R.M. Esteban, E. Moniz, N. Casado, Y. Aguilera, E. Moll´
a, Breads
fortied with wholegrain cereals and seeds as source of antioxidant dietary bre
and other bioactive compounds, J. Cereal Sci. 82 (2018) 113–120, https://doi.org/
10.1016/j.jcs.2018.06.001.
[35] B. ˇ
Sebeˇ
ci´
c, I. Vedrina-Dragojevi´
c, D. Vitali, M. Heˇ
cimovi´
c, I. Dragiˇ
cevi´
c, Raw
materials in bre enriched biscuits production as source of total phenols, Agric.
Conspec. Sci. 72 (2007) 265–270.
[36] Z. Han, M. Zhu, X. Wan, X. Zhai, C.-T. Ho, L. Zhang, Food polyphenols and Maillard
reaction: regulation effect and chemical mechanism, Crit. Rev. Food Sci. Nutr. 15
(2022) 4904–4920.
[37] L. Jakobek, P. Mati´
c, Non-covalent dietary ber – polyphenol interactions and their
inuence on polyphenol bioaccessibility, Trends Food Sci. Technol. 83 (2019)
235–247, https://doi.org/10.1016/j.tifs.2018.11.024.
[38] V. Eskicioglu, S. Kamiloglu, D. Nilufer-Erdil, Antioxidant dietary bres: potential
functional food ingredients from plant processing by-products, Czech J. Food Sci.
33 (2015) 487–499.
[39] A.E. Quir´
os-Sauceda, H. Palafox-Carlos, S.G. S´
ayago-Ayerdi, J.F. Ayala-Zavala, L.
A. Bello-Perez, E. ´
Alvarez-Parrilla, L.A. De La Rosa, A.F. Gonz´
alez-C´
ordova, G.
A. Gonz´
alez-Aguilar, Dietary ber and phenolic compounds as functional
ingredients: Interaction and possible effect after ingestion, Food Funct. 5 (2014)
1063–1072, https://doi.org/10.1039/c4fo00073k.
[40] M.I. Gabriela, G. Girish, Fruit Processing By-Products: A Rich Source for Bioactive
Compounds and Value Added Products, Food Process. By-Products Their Util, John
Wiley Sons, Hoboken, NJ, USA, 2017, pp. 11–26.
[41] S. Hussain, I. J˜
oudu, R. Bhat, Dietary ber from underutilized plant resources—a
positive approach for valorization of fruit and vegetable wastes, Sustainability 12
(2020) 5401.
[42] A.L. Girard, J.M. Awika, Effects of edible plant polyphenols on gluten protein
functionality and potential applications of polyphenol–gluten interactions, Compr.
Rev. Food Sci. Food Saf. 19 (2020) 2164–2199, https://doi.org/10.1111/1541-
4337.12572.
[43] E. Jahan, A.H. Nupur, S. Majumder, P.C. Das, L. Aunsary, M.G. Aziz, M.A. Islam, M.
A.R. Mazumder, Physico-chemical, textural and sensory properties of breads
enriched with date seed powder, Food Humanit. 1 (2023) 165–173.
[44] C. Platat, H.M. Habib, I.B. Hashim, H. Kamal, F. AlMaqbali, U. Souka, W.
H. Ibrahim, Production of functional pita bread using date seed powder, J. Food
Sci. Technol. 52 (2015) 6375–6384.
[45] A.P. Rebellato, B.C. Pacheco, J.P. Prado, J.A. Lima Pallone, Iron in fortied
biscuits: a simple method for its quantication, bioaccessibility study and
physicochemical quality, Food Res. Int. 77 (2015) 385–391, https://doi.org/
10.1016/j.foodres.2015.09.028.
[46] A. Abd El-Salam, H. Hafez, A. Khorshid, Utilization of germinated date seeds as a
source of functional ingredients in biscuits, Food Technol. Res. J. 3 (2024)
126–137, https://doi.org/10.21608/ftrj.2024.351550.
[47] C. Platat, H.M. Habib, W.H. Ibrahim, I.B. Hashim, A.K. Eldin, Date seed powder-
containing bread exhibits higher levels of avonoids and antioxidant capacity
compared to regular and whole wheat bread, in: Expermantal Biol. 2013 FASEB,
Wiley Online Library, 2013, pp. 371–376.
[48] A. Mrabet, H. Hammadi, G. Rodríguez-Guti´
errez, A. Jim´
enez-Araujo, M. Sindic,
Date palm fruits as a potential source of functional dietary ber: a review, Food Sci.
Technol. Res. 25 (2019) 1–10.
[49] G.G. Ouedraogo, S. Moukha, T.A. Mobio, M. Ouedraogo, P.I. Guissou, E.E. Creppy,
Cytotoxicity assessment of aqueous extract from root barks of calotropis procera
(Ait.) R. Br. in human intestinal Caco-2 and mouse neuroblastoma neuro-2a cell
lines, J. Appl Pharm. Sci. 4 (2014) 1–7, https://doi.org/10.7324/
JAPS.2014.40401.
[50] Y. Sambuy, I. De Angelis, G. Ranaldi, M.L. Scarino, A. Stammati, F. Zucco, The
Caco-2 cell line as a model of the intestinal barrier: Inuence of cell and culture-
related factors on Caco-2 cell functional characteristics, Cell Biol. Toxicol. 21
(2005) 1–26, https://doi.org/10.1007/s10565-005-0085-6.
[51] A.G.F. Boim, J. Wragg, S.G. Canniatti-Brazaca, L.R.F. Alleoni, Human intestinal
Caco-2 cell line in vitro assay to evaluate the absorption of Cd, Cu, Mn and Zn from
urban environmental matrices, Environ. Geochem. Health 42 (2020) 601–615.
[52] M. Kus, I. Ibragimow, H. Piotrowska-Kempisty, Caco-2 cell line standardization
with pharmaceutical requirements and in vitro model suitability for permeability
assays, Pharmaceutics 15 (2023) 2523, https://doi.org/10.3390/
pharmaceutics15112523.
[53] T. Ferreira, S.M. Gomes, L. Santos, Elevating cereal-based nutrition: Moringa
oleifera supplemented bread and biscuits, Antioxidants 12 (2023) 2069, https://
doi.org/10.3390/antiox12122069.
[54] S. Hilary, J. Kizhakkayil, U. Souka, F. Al-Meqbaali, W. Ibrahim, C. Platat, In-vitro
investigation of polyphenol-rich date (Phoenix dactylifera L.) seed extract
bioactivity, Front. Nutr. 8 (2021) 1–15, https://doi.org/10.3389/
fnut.2021.667514.
[55] C.R. Drisya, B.G. Swetha, V. Velu, D. Indrani, R.P. Singh, Effect of dried Murraya
koenigii leaves on nutritional, textural and organoleptic characeteristics of cookies,
J. Food Sci. Technol. 52 (2015) 500–506.
[56] R. Ellouze-Ghorbel, A. Kamoun, M. Neifar, S. Belguith, M.A. Ayadi, A. Kamoun,
S. Ellouze-Chaabouni, Development of ber-enriched biscuits formula by a mixture
design, J. Texture Stud. 41 (2010) 472–491, https://doi.org/10.1111/j.1745-
4603.2010.00237.x.
[57] W.S. Ayoub, I. Zahoor, A.H. Dar, N. Anjum, R. Pandiselvam, S. Farooq, A.V. Rusu,
J.M. Rocha, M. Trif, G. Jeevarathinam, Effect of incorporation of wheat bran, rice
bran and banana peel powder on the mesostructure and physicochemical
characteristics of biscuits, Front. Nutr. 9 (2022) 1016717.
[58] W. Lou, H. Zhou, B. Li, G. Nataliya, Rheological, pasting and sensory properties of
biscuits supplemented with grape pomace powder, Food Sci. Technol. 42 (2021)
e78421.
[59] A. Ribas-Agustí, O. Martín-Belloso, R. Soliva-Fortuny, P. Elez-Martínez, Food
processing strategies to enhance phenolic compounds bioaccessibility and
bioavailability in plant-based foods, Crit. Rev. Food Sci. Nutr. 58 (2018)
2531–2548.
[60] M. Wronkowska, W. Wiczkowski, J. Topolska, D. Szawara-Nowak, M.K. Piskuła,
H. Zieli´
nski, Identication and bioaccessibility of maillard reaction products and
phenolic compounds in buckwheat biscuits formulated from our fermented by
Rhizopus oligosporus 2710, Molecules 28 (2023) 2746.
[61] D. Pinto, M.M. Moreira, J. ˇ
Svarc-Gaji´
c, A. Vallverdú-Queralt, T. Brezo-Borjan,
C. Delerue-Matos, F. Rodrigues, In-vitro gastrointestinal digestion of functional
cookies enriched with chestnut shells extract: Effects on phenolic composition,
bioaccessibility, bioactivity, and
α
-amylase inhibition, Food Biosci. 53 (2023)
102766, https://doi.org/10.1016/j.fbio.2023.102766.
M. Ranasinghe et al.
Ultrasonics Sonochemistry 112 (2025) 107160
12
[62] A.L. Mas, F.I. Brigante, E. Salvucci, P. Ribotta, M.L. Martinez, D.A. Wunderlin, M.
V. Baroni, Novel cookie formulation with defatted sesame our: Evaluation of its
technological and sensory properties. Changes in phenolic prole, antioxidant
activity, and gut microbiota after simulated gastrointestinal digestion, Food Chem.
389 (2022) 133122.
[63] R. Lucas-Gonzalez, S. Navarro-Coves, J.A. P´
erez-´
Alvarez, J. Fern´
andez-L´
opez, L.
A. Mu˜
noz, M. Viuda-Martos, Assessment of polyphenolic prole stability and
changes in the antioxidant potential of maqui berry (Aristotelia chilensis (Molina)
Stuntz) during in vitro gastrointestinal digestion, Ind. Crops Prod. 94 (2016)
774–782.
[64] H. Kamal, M. Hamdi, P. Mudgil, M. Aldhaheri, M.A. Baig, H.M. Hassan, A.
S. Alamri, C.M. Galanakis, S. Maqsood, Nutraceutical and bioactive potential of
high-quality date fruit varieties (Phoenix dactylifera L.) as a function of in-vitro
simulated gastrointestinal digestion, J. Pharm. Biomed. Anal. 223 (2023) 115113,
https://doi.org/10.1016/j.jpba.2022.115113.
[65] J.C. Espín, A. Gonz´
alez-Sarrías, F.A. Tom´
as-Barber´
an, The gut microbiota: a key
factor in the therapeutic effects of (poly) phenols, Biochem. Pharmacol. 139 (2017)
82–93.
[66] N.P. Nørskov, M.S. Hedemann, P.K. Theil, I.S. Fomsgaard, B.B. Laursen, K.E.
B. Knudsen, Phenolic acids from wheat show different absorption proles in
plasma: a model experiment with catheterized pigs, J. Agric. Food Chem. 61
(2013) 8842–8850.
[67] R. Lucas-Gonz´
alez, M. Viuda-Martos, J.A. P´
erez-Alvarez, J. Fern´
andez-L´
opez, In
vitro digestion models suitable for foods: opportunities for new elds of application
and challenges, Food Res. Int. 107 (2018) 423–436.
[68] A. Hidalgo, A. Ferraretto, I. De Noni, M. Bottani, S. Cattaneo, S. Galli,
A. Brandolini, Bioactive compounds and antioxidant properties of pseudocereals-
enriched water biscuits and their in vitro digestates, Food Chem. 240 (2018)
799–807, https://doi.org/10.1016/j.foodchem.2017.08.014.
[69] M. Irakli, D. Katsantonis, F. Kleisiaris, Evaluation of quality attributes,
nutraceutical components and antioxidant potential of wheat bread substituted
with rice bran, J. Cereal Sci. 65 (2015) 74–80.
[70] D. Szawara-Nowak, N. Bączek, H. Zieli´
nski, Antioxidant capacity and
bioaccessibility of buckwheat-enhanced wheat bread phenolics, J. Food Sci.
Technol. 53 (2016) 621–630.
[71] F.J.S. Jos´
e, M. Collado-Fern´
andez, P.P. ´
Alvarez-Castellanos, Variation, during shelf
life, of functional properties of biscuits enriched with bers extracted from
artichoke (Cynara scolymus L.), Nutrients 15 (2023) 1–19, https://doi.org/
10.3390/nu15153329.
[72] Z. Aksoylu, ¨
O. Ça˘
gindi, E. K¨
ose, Effects of blueberry, grape seed powder and poppy
seed incorporation on physicochemical and sensory properties of biscuit, J. Food
Qual. 38 (2015) 164–174, https://doi.org/10.1111/jfq.12133.
[73] A. Tseng, Y. Zhao, Wine grape pomace as antioxidant dietary bre for enhancing
nutritional value and improving storability of yogurt and salad dressing, Food
Chem. 138 (2013) 356–365, https://doi.org/10.1016/j.foodchem.2012.09.148.
[74] G. Spigno, F. Donsì, D. Amendola, M. Sessa, G. Ferrari, D.M. De Faveri,
Nanoencapsulation systems to improve solubility and antioxidant efciency of a
grape marc extract into hazelnut paste, J. Food Eng. 114 (2013) 207–214, https://
doi.org/10.1016/j.jfoodeng.2012.08.014.
[75] H. Zieli´
nski, M.D. Del Castillo, M. Przygodzka, Z. Ciesarova, K. Kukurova,
D. Zieli´
nska, Changes in chemical composition and antioxidative properties of rye
ginger cakes during their shelf-life, Food Chem. 135 (2012) 2965–2973, https://
doi.org/10.1016/j.foodchem.2012.07.009.
[76] X. Sui, Anthocyanins as functional ingredients in biscuits: their stability,
antioxidant capacity, and preventive effect on retarding lipid oxidation, 2017.
https://doi.org/10.1007/978-981-10-2612-6_8.
[77] M. Sakaˇ
c, M. Pestori´
c, A. Mandi´
c, A. Miˇ
san, N. Nedeljkovi´
c, D. Jambrec,
P. Jovanov, V. Lazi´
c, L. Pezo, I. Sedej, Shelf-life prediction of gluten-free rice-
buckwheat cookies, J. Cereal Sci. 69 (2016) 336–343, https://doi.org/10.1016/j.
jcs.2016.04.008.
[78] T. Ismail, S. Akhtar, M. Riaz, A. Ismail, Effect of pomegranate peel supplementation
on nutritional, organoleptic and stability properties of cookies, Int. J. Food Sci.
Nutr. 65 (2014) 661–666.
[79] L. Leliana, W. Setyaningsih, M. Palma, U.S. Supriyadi, Incorporation of young
coconut (Cocos nucifera) mesocarp increases the antioxidant activity, phenolic
compounds and oxidative stability of cookies, Trends Sci. 21 (2024) 1–11, https://
doi.org/10.48048/tis.2024.7199.
[80] M. Yaqoob, P. Aggarwal, N. Rasool, W.N. Baba, P. Ahluwalia, R. Abdelrahman,
Enhanced functional properties and shelf stability of cookies by fortication of
kinnow derived phytochemicals and residues, J. Food Meas. Charact. 15 (2021)
2369–2376, https://doi.org/10.1007/s11694-021-00827-8.
[81] M. Kozłowska, A. ˙
Zbikowska, E. Gruczy´
nska, K. ˙
Zontała, A. P´
ołtorak, Effects of
spice extracts on lipid fraction oxidative stability of cookies investigated by DSC,
J. Therm. Anal. Calorim. 118 (2014) 1697–1705, https://doi.org/10.1007/
s10973-014-4058-y.
[82] S.Z. Asadi, M.A. Khan, S. Zaidi, A study on the shelf life of cookies incorporated
with sapota and beetroot leaf powders, J. Food Sci. Technol. 59 (2022) 3848–3856.
[83] A. Miˇ
san, N. Mimica-Duki´
c, M. Sakaˇ
c, A. Mandi´
c, I. Sedej, O. ˇ
Simurina, V. Tumbas,
Antioxidant activity of medicinal plant extracts in cookies, J. Food Sci. 76 (2011)
1239–1244, https://doi.org/10.1111/j.1750-3841.2011.02400.x.
[84] K. Guti´
errez-Luna, I. Astiasaran, D. Ansorena, Fat reduced cookies using an olive
oil-alginate gelled emulsion: sensory properties, storage stability and in vitro
digestion, Food Res. Int. 167 (2023) 1–8, https://doi.org/10.1016/j.
foodres.2023.112714.
[85] F. Caponio, C. Summo, V.M. Paradiso, A. Pasqualone, T. Gomes, Evolution of the
oxidative and hydrolytic degradation of biscuits’ fatty fraction during storage,
J. Sci. Food Agric. 89 (2009) 1392–1396.
[86] M.I. Bhanger, S. Iqbal, F. Anwar, M. Imran, M. Akhtar, M. Zia-Ul-Haq, Antioxidant
potential of rice bran extracts and its effects on stabilisation of cookies under
ambient storage, Int. J. Food Sci. Technol. 43 (2008) 779–786, https://doi.org/
10.1111/j.1365-2621.2007.01515.x.
[87] O. Daglioglu, M. Tasan, U. Gecgel, F. Daglioglu, Changes in oxidative stability of
selected bakery products during shelf life, Food Sci. Technol. Res. 10 (2007)
464–468.
[88] S. Romani, S. Tappi, F. Balestra, M.T. Rodriguez Estrada, V. Siracusa, P. Rocculi,
M. Dalla Rosa, Effect of different new packaging materials on biscuit quality during
accelerated storage, J. Sci. Food Agric. 95 (2015) 1736–1746.
[89] E. Gebreselassie, H. Clifford, in: Oxidative Stability and Shelf Life of Crackers,
Cookies, and Biscuits, Elsevier Inc., 2016, https://doi.org/10.1016/B978-1-63067-
056-6.00012-4.
M. Ranasinghe et al.
Ultrasonics Sonochemistry 112 (2025) 107160
13
