marine drugs
Article
Fillet Fish Fortified with Algal Extracts of Codium tomentosum
and Actinotrichia fragilis, as a Potential Antibacterial and
Antioxidant Food Supplement
Mohamed S. M. Abd El Hafez 1,2, * , Sarah H. Rashedy 1 , Neveen M. Abdelmotilib 3 ,
Hala E. Abou El-Hassayeb 1 , João Cotas 4 and Leonel Pereira 4, *
1
2
3
4
*
Citation: Hafez, M.S.M.A.E.;
Rashedy, S.H.; Abdelmotilib, N.M.;
El-Hassayeb, H.E.A.; Cotas, J.;
Pereira, L. Fillet Fish Fortified with
Algal Extracts of Codium tomentosum
and Actinotrichia fragilis, as a Potential
Antibacterial and Antioxidant Food
Supplement. Mar. Drugs 2022, 20, 785.
https://doi.org/10.3390/
md20120785
Academic Editor: You-Jin Jeon
Received: 17 November 2022
Accepted: 16 December 2022
National Institute of Oceanography and Fisheries, NIOF, Cairo 11516, Egypt
Center of Excellence for Drug Preclinical Studies (CE-DPS), Pharmaceutical and Fermentation Industries
Development Center (PFIDC), City of Scientific Research and Technological Applications (SRTA-City),
New Borg El-Arab City 21934, Egypt
Department of Food Technology, Arid Lands Cultivation Research Institute (ALCRI), City of Scientific
Research and Technological Applications (SRTA-CITY), New Borg El-Arab City 21934, Egypt
MARE—Marine and Environmental Sciences Centre, Department of Life Sciences, University of Coimbra,
Calçada Martim de Freitas, 3000-456 Coimbra, Portugal
Correspondence: mohamedsaid80@yahoo.com (M.S.M.A.E.H.); leonel.pereira@uc.pt (L.P.)
Abstract: With respect to the potential natural resources in the marine environment, marine macroalgae or seaweeds are recognized to have health impacts. Two marine algae that are found in the
Red Sea, Codium tomentosum (Green algae) and Actinotrichia fragilis (Red algae), were collected.
Antibacterial and antioxidant activities of aqueous extracts of these algae were evaluated in vitro.
Polyphenols from the extracts were determined using HPLC. Fillet fish was fortified with these algal
extracts in an attempt to improve its nutritional value, and sensory evaluation was performed. The
antibacterial effect of C. tomentosum extract was found to be superior to that of A. fragilis extract.
Total phenolic contents of C. tomentosum and A. fragilis aqueous extract were 32.28 ± 1.63 mg/g
and 19.96 ± 1.28 mg/g, respectively, while total flavonoid contents were 4.54 ± 1.48 mg/g and
3.86 ± 1.02 mg/g, respectively. Extract of C. tomentosum demonstrates the highest antioxidant activity, with an IC50 value of 75.32 ± 0.07 µg/mL. The IC50 of L-ascorbic acid as a positive control
was 22.71 ± 0.03 µg/mL. The IC50 values for inhibiting proliferation on normal PBMC cells were
33.7 ± 1.02 µg/mL and 51.0 ± 1.14 µg/mL for C. tomentosum and A. fragilis, respectively. The results
indicated that both algal aqueous extracts were safe, with low toxicity to normal cells. Interestingly,
fillet fish fortified with C. tomentosum extract demonstrated the greatest overall acceptance score.
These findings highlight the potential of these seaweed species for cultivation as a sustainable and
safe source of therapeutic compounds for treating human and fish diseases, as well as effective food
supplements and preservatives instead of chemical ones after performing in vivo assays.
Published: 17 December 2022
Publisher’s Note: MDPI stays neutral
Keywords: marine natural products; seaweed; HPLC; DPPH; cytotoxicity
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Copyright: © 2022 by the authors.
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Attribution (CC BY) license (https://
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4.0/).
1. Introduction
Marine natural products are extremely useful substances for use in food additives and
drug discovery as a result of their broad variety of bioactivities [1–3]. Marine organisms use
powerful secondary metabolites to prevent the growth of invading neighbors and attract
food. Such survival conditions stimulate the production of an extremely abundant variety
of biologically active substances [4–7]. Secondary metabolites isolated from different alga
are playing an important role as lead components, natural medicine or nutraceuticals in
drug discovery research and pharmaceutical industries and natural food preservatives [8,9].
Recently, due to the resistance of different pathogenic bacteria and pests to antibiotics and
insecticidal agents, finding new active components against these health and environmental
Mar. Drugs 2022, 20, 785. https://doi.org/10.3390/md20120785
https://www.mdpi.com/journal/marinedrugs
Mar. Drugs 2022, 20, 785
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problems is one of the major areas of medical and agricultural research. The peculiarity
of the marine environment and the high biological activity of marine natural products
make algal metabolites a fascinating source for finding new antimicrobial and insecticidal
compound [10–12].
Seaweeds are large and diverse groups of plants that are rich in active metabolites
and a source of novel ingredients for functional foods. Nutritional studies on seaweeds
indicate that brown, green and red seaweeds possess good nutritional quality and could
be used as an alternative source of dietary fiber, protein and minerals. Seaweeds are also
considered as a source of bioactive compounds as they are able to produce a great variety
of secondary metabolites, characterized by a wide range of biological activities such as
antimicrobial, anti-inflammatory, antiviral as well as antitumor activities [13–15]. Moreover,
many studies show that some algae extracts display substantial antioxidant activities.
Antioxidant substances in seaweeds contribute to the endogenous defense mechanism
against external stressful conditions. Antioxidant properties of some red, brown and green
algae extracts have shown that they vary in proportion to the content of antioxidative
compounds [16,17].
Algae are a unique raw material for the production of a number of substances with
a wide range of useful properties. Their composition is characterized by the specific content of minerals, pigments, lipids, polyphenols, proteins, amino acids, cellulose, polysaccharides, etc. One of the most significant groups of compounds that determines the
biomedical importance of marine algae is polyphenols. The largest proportion of phenolic
compounds in green and red algae is represented by bromophenols, phenolic acids and
flavonoids [18]. Seaweed phenolic compounds are metabolites that are scientifically defined as molecules with hydroxylated aromatic rings [19–21]. These phytochemicals have
a diverse chemical structure, ranging from simple moieties to large molecular polymers.
The shikimate or acetate pathway is the main metabolic pathway which generates these
phytochemicals [22–24]. Bromophenols, flavonoids, phenolic acids, phenolic terpenoids
and mycosporine-like amino acids account for the majority of phenolic chemicals found in
green and red seaweeds [25–28]. These compounds are classified as secondary metabolites
because they are protective agents that are produced in response to various stimuli and
serve as seaweed defense mechanisms against herbivory and UV exposure [29].
The majority of phenolic compounds have anti-diabetic, anti-inflammatory, antimicrobial, antiviral, anti-allergic, antioxidant, antiphotoaging, antipruritic, hepatoprotective,
hypotension, neuroprotective and anticancer properties [19,28,30–41]. Given their many
bioactivities, seaweeds are ideal candidates for the creation of goods or components utilized in commercial applications such as medicines, cosmetics, functional foods and even
bioactive food packaging films to preserve food quality [38,40,42–44]. Because of their
structural similarities and proclivity to react with other chemicals, phenolic compounds
are extremely difficult to extract quantitatively on an industrial scale [19]. Thus, seaweed
extracts can be a solution to reduce costs and attain economic feasibility at a large scale.
Therefore, the aim of the present study was to determine the polyphenol content
and antioxidant and antibacterial activities of Codium tomentosum (Chlorophyta) and
Actinotrichia fragilis (Rhodophyta) from the Red Sea (Egypt). Furthermore, we fortified fillet
fish with these algal extracts in an attempt to improve nutritional values and food safety
while maintaining a pleasant taste.
2. Results and Discussion
In the present study, the aqueous extracts of C. tomentosum and A. fragilis were investigated for their antioxidant and antimicrobial activity, and their polyphenol contents
were also investigated to provide clarification of the chemical constituents. Macroalgae
are ecologically and biologically important natural sources. They are an important basis
for therapeutically useful substances. Due to their biological and chemical variations, the
marine environment may be a source of novel types of antimicrobial agents and biologically
active compounds.
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2.1. Antibacterial Activity and MIC Determination
Antibacterial activities expressed as inhibition zone diameters of algal aqueous extracts against the tested bacterial strains are shown in Table 1. The antibacterial effects
of C. tomentosum aqueous extract were found to be superior to those of A. fragilis extract
in the experiment.
Table 1. Antibacterial potential of algal extracts.
Inhibition Zone Diameter (mm) 1
Strains
Gram-positive bacteria
Staphylococcus aureus (ATCC25923)
Streptococcus pyogenes (EMCC1772)
Gram-negative bacteria
Escherichia coli (ATCC25922)
Klebsiella pneumonia (ATCC700603)
Codium tomentosum
(Green Algae)
Actinotrichia fragilis
(Red Algae)
22 ± 0.04 a
20 ± 0.01 a
18 ± 0.05 b
16 ± 0.01 b
14 ± 0.08 a
18 ± 0.02 a
12 ± 0.05 b
14 ± 0.07 b
1
All results are expressed as the means ± standard deviation; n = 3. Diameter includes 5 mm well diameter.
Different letters (a and b ) indicate significant differences at p ≤ 0.05.
The minimum inhibitory concentration (MIC) is an essential factor that assesses
microorganism resistance and sensitivity to specific substances. The MIC of C. tomentosum
and A. fragilis against the bacterial strains is shown in Table 2.
Table 2. Minimum inhibitory concentration (MIC) of algal extracts against pathogenic bacteria
indicated as a zone of inhibition for each concentration.
Strains
Gram-positive bacteria
Staphylococcus aureus (ATCC25923)
Streptococcus pyogenes (EMCC1772)
Gram-negative bacteria
Escherichia coli (ATCC25922)
Klebsiella pneumonia (ATCC700603)
MIC (mg/mL) 1
Codium tomentosum
Actinotrichia fragilis
0.4 ± 0.05 b
0.4 ± 0.04 b
0.8 ± 0.03 a
0.8 ± 0.02 a
0.8 ± 0.01 b
0.6 ± 0.04 b
1.2 ± 0.03 a
1.2 ± 0.01 a
1
Diameter includes 5 mm well diameter. MIC—minimum inhibition concentration (mg/mL). Different letters
(a and b ) indicate significant differences at p ≤ 0.05.
Seaweeds are potential renewable resources of bioactive compounds with diverse
beneficial effects. Several studies reported that bioactive secondary metabolites isolated
from various seaweed exhibit potential to be used as antimicrobial molecules. Nevertheless, species belonging to the genus Codium have been the least investigated among all
members of Chlorophyta for their biological activities and their possible use in food and
biomedical applications. Aqueous extracts of C. tomentosum and A. fragilis were subjected
to antibacterial assay against a wide array of bacterial pathogens. The selected bacteria are
among the most common causes of foodborne and infectious diseases. They showed an
extended spectrum of inhibitory activity against all the bacterial pathogens [45].
In addition, there are reported studies that describe the antibacterial capability
(derived from secondary and primary metabolites) of seaweeds against medically important pathogenic bacteria. As previously shown in many studies, marine macroalgae
have antimicrobial components that inhibit the growth of some bacteria. It has also been
reported that the efficacy of macroalgae extracts against microorganisms is mostly influenced by factors such as location and seasonality. Another study of macroalgae showed
a high percentage of species with antimicrobial activity, 73% in the case of Chlorophyta
(green algae), 69% in Rhodophyta (red algae) and 53% in Phaeophyceae (brown algae) [46].
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Rajauria et al. used aqueous phenolic extract to demonstrate that Himanthalia elongata have
antimicrobial activity against Listeria monocytogenes ATCC 19115, Salmonella abony NCTC
6017, Enterococcus faecalis ATCC 7080 and Pseudomonas aeruginosa ATCC 27853 [47].
The findings of the present investigation are analogous to those observed by
Elkhateeb et al. [48], who reported that the crude extract of C. tomentosum showed strong
antimicrobial activity against two Gram-positive bacteria, Staphylococcus aureus ATCC 6538
and Bacillus subtilis ATCC 6633, as well as two Gram-negative bacteria, Escherichia coli
ATCC 19404 and Vibrio alginolyticus MK 170250.
However, in other studies such as that of Koz et al. [46], antibacterial activity of Codium
fragile extract against E. aerogenes, E. coli and B. subtilis was observed. Antibacterial activity of C. intricatum extract in opposition to S. aureus and MRSA is similar to that reported
from methanol extracts of Codium species (C. tomentosum, C. tomentosum, C. dichotomum and
C. fragile), where high inhibitory activity was noted [45]. Pasdaran et al. [10] reported the
antibacterial activity of A. fragilis volatile oil against Pseudomonas aeruginosa, E. coli and Staphylococcus aureus. Salem et al. [49] reported the antibacterial activity of A. fragilis extracts against
E. coli, S. aureus, E. feacalis, Salmonella sp., B. cereus and P. aeruginosa. Alghazeer et al. [50]
reported that extracts from C. tomentosum possess in vitro antibacterial activity against eight
bacterial strains, namely S. aureus, B. subtilis, Bacillus spp., S. epidermidis, S. typhi, E. coli,
P. aeruginosa and klebsiella spp., similar to the findings of this study.
The sensitivity of a specific kind of bacteria to the activity of bioactive substances
found in the algal extracts is attributed to the difference in structure and composition of the
cell walls. Gram-positive bacteria are marked by dense peptidoglycan in the outer layer
of the cell wall, while Gram-negative bacteria have a composite, multilayered cell wall
structure that makes the entry of bioactive compounds more difficult [51].
2.2. Antioxidant Activity
Phenolic compounds are key antioxidant and antibacterial agents with numerous
benefits for disease prevention and human health. Flavonoids are natural substances with
a polyphenolic structure; as a result, they have antibacterial and antioxidant activity and
can help prevent illnesses including Alzheimer’s disease, cancer and atherosclerosis [52].
Total phenolic and flavonoid contents of Codium tomentosum and Actinotrichia fragilis are
shown in Table 3.
Table 3. Total phenolic and flavonoid contents of Codium tomentosum and Actinotrichia fragilis.
1
Test 1
Codium tomentosum
Actinotrichia fragilis
Total phenolic content
(mg/g of extract)
Total flavonoid content
(mg/g of extract)
32.28 ± 1.63 a
19.96 ± 1.28 b
4.54 ± 1.48 c
3.86 ± 1.02 d
Means in the same column followed by different lowercase letters are significantly different (p < 0.05).
Total phenolic contents of C. tomentosum and A. fragilis aqueous extract were
32.28 ± 1.63 mg/g and 19.96 ± 1.28 mg/g of extract, respectively, while TFCs were
4.54 ± 1.48 mg/g and 3.86 ± 1.02 mg/g of extract, respectively. TFC’s significance comes
mostly from its redox characteristics that might account for its antibacterial and antioxidant
activities against a variety of microorganisms and free radicals. Furthermore, the action
of TFC as an antioxidant agent is closely related to the hydroxyl groups in its structures,
which are responsible for scavenging lipid peroxy-radicals, singlet oxygen, superoxide
anion and free radical stabilization [53–55].
The DPPH is a significant metric for determining an extract’s antioxidant activity.
The IC50 is the extract concentration needed to scavenge 50% of the DPPH radicals. The
IC50 values of C. tomentosum and A. fragilis aqueous extracts are presented in Table 4. The
better the antioxidant properties, the smaller the IC50 value. According to the results,
C. tomentosum aqueous extract demonstrates the highest antioxidant activity, with an
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IC50 value of 75.32 ± 0.07 µg/mL. The IC50 of L-ascorbic acid as a positive control was
22.71 ± 0.03 µg/mL.
Table 4. Antioxidant activity of Codium tomentosum and Actinotrichia fragilis as measured by DPPH assay.
Extracts
DPPH (IC50 ) µg/mL
Ascorbic acid
Codium tomentosum
Actinotrichia fragilis
22.71 ± 0.03 c
75.32 ± 0.07 b
94.43 ± 0.02 a
IC50 (µg/mL): Inhibitory concentrations at which 50% of DPPH radicals are scavenged. Means in the same column
followed by different lowercase letters are significantly different (p < 0.05).
Basically, antioxidants delay oxidation and reduce oxidative damage, which is a significant causative factor in the development of many chronic diseases. Oxidative stress is
an important factor in the pathogenesis of various diseases such as atherosclerosis, cancer
and aging. Naturally generated reactive oxygen species can attack cell components and
then exert several types of biological damage and oxidative stress [56–58]. Antioxidants
protect against these reactions which occur in vital systems and increase shelf-life when
added to lipids and lipid-containing foods [46,52]. Although synthetic antioxidants such
as butylatedhydroxyanisole, butylatedhydroxytoluene and propyl gallate have been used
for many years, they have started to be restricted in recent years because of their carcinogenicity. Thus, there is a gradual increase in the investigations to identify new natural
antioxidants [46,52].
Seaweeds are considered to be important sources of antioxidants. The results from
antioxidant activity screening in the extracts suggested that algae extracts have radical
scavenging inhibitor activity. The findings of the present investigation are similar to
those observed by Alghazeer et al. [16], who reported that C. tomentosum extracts possess
antioxidant and antiproliferative activities which might be helpful in preventing or slowing
the progress of various oxidative stress-related disorders. The findings of the present
investigation are similar to those observed by Baskaran et al. [59], who reported that
methanol crude extracts of A. fragilis contain different potential antioxidant compounds
able to scavenge different types of free radicals. Acanthophora specifera flavonoid separation
reveals a combination of chlorogenic acid (69.64%), caffeic acid (12.86%), vitexin-rahmnose
(12.35%), quercetin (1.41%) and catechol (0.59%) [60]. The antioxidant activity of the
flavonoid-enriched extract has been proven to be very high [61]
Phenolic compounds have exceptional antioxidant properties due to their capacity to
function as chelating agents with reactive oxygen species, avoiding oxidative stress and
cell damage [29,62]. As a result, scavenging of oxidants is critical for disease prevention,
and phenolic compounds found in seaweeds are particularly helpful as a natural supply
of antioxidant agents [52]. The study of Agregán et al. [63] demonstrated that aqueous
polyphenolic extracts of Ascophyllum nodosum, Bifurcaria bifurcata and Fucus vesiculosus have
antioxidant activity, which stabilize the canola oil oxidation level.
Nonetheless, synthetic ingredient limits in the food business may represent a tipping
point for the use of seaweed compounds as safe replacements [64], since they also exhibit
anti-microbial activity against key food spoilage and food pathogenic microorganisms [65].
Seaweed phenolic antioxidant extracts have been used to improve oxidative stability and
to preserve or boost the intrinsic quality and nutritional content of foods [66,67]. The
antioxidant potential is useful in the food industry not only for nutraceutical compounds
on functional food products, where they are indisputably valuable for health improvement
(as food supplements), but also to extend the shelf-life period when used in processed food
(functional foods) [68,69]. Furthermore, the antibacterial activity of seaweed phenolics
suggests that they can be valuable in the food industry [70].
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2.3. Cytotoxicity Assay
The influence of various concentrations of Codium tomentosum and Actinotrichia fragilis
aqueous extracts on PBMC viability are given in Tables 5 and 6. The IC50 value for inhibiting
proliferation on normal PBMC cells was 33.7 ± 1.02 µg/mL and 51.0 ± 1.14 µg/mL for
C. tomentosum and Actinotrichia fragilis, respectively. The results indicated that both algal
aqueous extracts were safe with low toxicity to normal cells and could be used as novel
and effective food preservatives instead of chemical ones after performing in vivo assays,
acting also as nutraceuticals [52].
Table 5. The influence of various concentrations of Codium tomentosum aqueous extract on
PBMC viability.
Concentration (µg/mL)
Inhibition %
250
125
62.5
31.2
15.6
7.8
IC50
69
69
64
30
29
27
= 33.7 ± 1.02 µg/mL
Viability %
31
31
36
70
71
73
Table 6. The influence of various concentrations of Actinotrichia fragilis aqueous extract on
PBMC viability.
Concentration (µg/mL)
Inhibition %
250
125
62.5
31.2
15.6
7.8
IC50
73
60
47
39
37
11
= 51.0 ± 1.14 µg/mL
Viability %
27
40
53
61
63
89
The current study’s findings were consistent with those published by
Alghazeer et al. [16], who reported that seaweeds rich in bioactive compounds may be
used in anticancer drug research programs. However, further investigations are essential
to reveal the molecular mechanisms of the anticancer activities of these algae.
2.4. HPLC Analysis
To identify bioactive compounds in C. tomentosum and A. fragilis aqueous extracts,
high-performance liquid chromatography (HPLC) was used. The HPLC chromatogram of
standards and phenolic compounds of C. tomentosum and A. fragilis aqueous extracts are
shown in Table 7 and Figure 1.
The HPLC analysis results confirmed the presence of several polyphenolic compounds
when compared to the HPLC standard chromatogram. These findings agreed with those
of Tanna et al. [36], who revealed phenolic, flavonoid and amino acid compositions in
some species of seaweeds that are promising as functional food ingredients or dietary
supplements for daily intake.
HPLC analysis results of C. tomentosum and A. fragilis extracts confirmed the in vitro
antioxidant and antimicrobial studies, which demonstrated their biochemical components.
These active components are also considered the main reason for free radical scavenging
activities of our target seaweeds, demonstrating the nutraceutical potential of these extracts and also highlighting them as a raw source of interesting natural compounds with
pharmacological potential [52].
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μ
Table 7. Phenolic compounds of algal extracts identified by HPLC.
Phenolic Compounds
Gallic acid
Chlorogenic acid
Catechin
Methyl gallate
Caffeic acid
Syringic acid
Pyro catechol
Rutin
Ellagic acid
Coumaric acid
Vanillin
Ferulic acid
Naringenin
Daidzein
Quercetin
Cinnamic acid
Apigenin
Kaempferol
Hesperetin
ND: Not detected.
Figure 1. Cont.
Codium tomentosum
Actinotrichia fragilis
Conc. (µg/g)
174.32
ND
29.92
12.91
70.21
50.03
ND
ND
232.69
ND
ND
26.13
61.09
791.39
48.03
35.51
224.88
77.28
75.90
303.68
29.50
87.10
0.00
12.79
0.00
0.00
22.73
190.62
0.00
3.36
15.99
23.77
10.47
65.78
6.10
30.28
0.00
0.00
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Figure 1. HPLC chromatograms of (a) Codium tomentosum extract, (b) Actinotrichia
Figure 1. HPLC chromatograms of (a) Codium tomentosum extract, (b) Actinotrichia fragilis extract and
(c) standards.
2.5. Sensory Evaluation of Fillet Fish Fortified with Algal Extracts
Regarding the bioactivity and biochemical potential of these two extracts, it is important to understand if they can be used as food additives and applied in the food
industry [52,71]. Table 8 shows the sensory evaluation scores of fillet fish fortified with
algal extracts. According to the findings, fillet fish fortified with algal extracts has been
accepted as having high nutritional value for human diet. Interestingly, fillet fish fortified
with C. tomentosum extract demonstrated the greatest overall acceptance score, as well as
the highest antibacterial and antioxidant activity.
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Table 8. Sensory evaluation of fillet fish fortified with algal extracts.
T1 (Control)
T2 (1% of C. tomentosum
extract)
T3 (1% A. fragilis extract)
T4 (0.5% of C.
tomentosum and 0.5% of
A. fragilis extracts)
Appearance
Consistency
Tenderness
Flavor (Odor and
Taste)
Overall
Acceptance
4.60 ± 1.06 a
5.07 ± 1.03 a
3.53 ± 1.69 b
4.13 ± 1.64 a
4.53 ± 1.18 b
5.80 ± 1.15 a
5.67 ± 1.18 a
5.13 ± 1.41 a
5.60 ± 1.63 a
5.93 ± 1.10 a
4.73 ± 1.16 a
4.87 ± 1.19 a
4.80 ± 1.61 ab
4.67 ± 1.11 a
4.80 ± 0.77 b
5.13 ± 1.41 a
5.67 ± 1.05 a
5.40 ± 1.59 a
5.33 ± 1.39 a
5.47 ± 1.45 a
Means in the same column followed by different lowercase letters are significantly different (p < 0.05).
Water extracts were used because they are safer solvents than other organic solvents.
Water extracts contain a variety of constituents, including polysaccharides and polyphenols,
but we focused on polyphenols in this study because they are powerful antioxidants and
antibacterial agents, as reported previously by several studies. This important observation
will be made in subsequent work.
In this preliminary work, we treated the fillet fish with aqueous extracts mixed with
spices immediately before frying in oil. Only sensory evaluation was performed on the fillet
fish fortified with algal extracts in an attempt to improve its nutritional value in comparison
to our standard fillet fish diets.
Our future studies will focus on fortifying fillet fish or yogurt with natural seaweed
extracts as natural preservatives instead of chemical ones. pH values, titratable acidities
(TAs), total soluble solids, water holding capacity (WHC), thiobarbituric acid reactive
substances (TBARS), lipid oxidation, protein oxidation (modified DNPH carbonyl assay)
and microbiological properties will be evaluated over 30 days of storage.
The constraints on synthetic components in the food business may be a tipping moment for the use of seaweed compounds as safe replacements [64], as they also exhibit
antimicrobial properties against major food spoilage and pathogenic microbes [65]. Seaweed phenolic antioxidant extracts have been used to improve oxidative stability and to
preserve or boost the intrinsic quality and nutritional content of foods [66,67].
The antioxidant potential is beneficial in the food business not only as nutraceutical
compounds in functional food items, where they are undeniably valuable for health improvement (as food supplements), but also to extend the shelf-life period when included
in processed foods (functional foods) [68,69]. Furthermore, the antibacterial properties of
seaweed phenolics suggest that they may be valuable in the food business [70].
Furthermore, bromophenols enriched extract from Ulva lactuca and Pterocladiella capillacea were investigated as “marine flavour” agents in farmed fish and other aquatic organisms, because farming final products can differ in flavor from wild catches, and this can be
incorporated as a feed ingredient or a seaweed bromophenol-enriched sauce [72].
3. Materials and Methods
3.1. Algae Collection
Two marine algae species, Codium tomentosum Stackhouse, a green marine macroalgae
(phylum Chlorophyta, class Ulvophyceae), and Actinotrichia fragilis Forsskal, Borgesen, a
red marine macroalgae (phylum Rhodophyta, class Florideophyceae), were harvested in
summer 2021 from the tidal zone of the Red Sea shore at Hurghada, Egypt, between latitude
27◦ 28.15′ N and longitude 33◦ 77.13′ E (Figure 2). The seaweed species were identified
under a stereo microscope according to their morphological characteristics with taxonomic
references following the descriptions of Aleem [73]. The two macroalgal species were
collected randomly from the site in the summer season. Because of the large amount of
collected algae used in the extraction, no reproductive stage was recorded.
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Figure 2. The collected marine algae: (a) Codium tomentosum (green algae) and (b) Actinotrichia fragilis
(red algae).
3.2. Preparation of Algal Extracts
About 0.5 kg of each alga was washed with water, shade-dried and powdered in a mixer
grinder. Hot water (80 ◦ C) was used to extract the compounds from the samples (1:2, w/v) for
one hour before separating the filtrate and residue. The extraction was repeated three times
under similar conditions. Last, freeze-drying the filtrate yielded powdered algae aqueous
extracts. The extracts were then kept at −20 ◦ C for subsequent−study.
3.3. Antibacterial Activity and MIC Determination
Agar well diffusion tests described by Abd El Hafez et al. [5] and Kadaikunnan et al. [74]
were applied to detect the action of the extracts against the reference pathogenic strains—
Gram-negative bacteria Klebsiella pneumonia (ATCC700603) and E. coli (ATCC25922), and
Gram-positive bacteria Staphylococcus aureus (ATCC6538) and Streptococcus pyogenes
(EMCC1772)—obtained from City of Scientific Research and Technological Applications,
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by using 100 µL of the inoculums (1 × 108 CFU/mL). Using descending concentrations,
the minimum inhibitory concentration (MIC) of algal extracts toward the reference strains
was established. In sterile saline, growing culture suspensions of the reference pathogenic
strains were prepared and adapted to a concentration of 106 cells/mL. The reference
pathogenic strain suspension was inoculated onto a plate of nutrient agar (Oxoid, U.K.)
and left to dry for 15 min at room temperature. The algal extracts were serially diluted, and
100 µL of each dilution was independently applied to each well. The plates were incubated
for 24 h at 37 ◦ C before the MIC values were recorded. For each culture algal extract, the
test was performed in triplicate.
3.4. Determination of Total Phenolic Contents
The total phenolic content was determined using the Folin–Ciocalteu reagent
following [75] using gallic acid as a reference. Each sample was evaluated in three replicates.
A 0.1 mL aliquot of Folin–Ciocalteu reagent was added to 0.1 mL of reconstituted extract.
Then, 2.0 mL saturated sodium carbonate (7%) was added to the mixture after 15 min. For
30 min, the mixture was left at room temperature. TPC was determined using a spectrophotometer at 760 nm with gallic acid as a reference. The linear regression equation generated
from the standard gallic acid calibration curve was used to calculate TPC as milligrams of
gallic acid equivalent per gram sample. Each sample was evaluated in triplicate.
3.5. Determination of Total Flavonoid Contents
The total flavonoid contents were determined using a colorimetric method described
by Sakanaka et al. [76]. First, 1.0 mL of the sample and 4.0 mL of water were mixed in a
flask. Then, 0.75 mL of 5% sodium nitrite and 0.150 mL of 10% aluminum chloride were
added to the mixture. After 5 min at room temperature, 0.5 mL of 1 M sodium hydroxide
was added. A UV/VIS spectrophotometer was used to measure the absorbance at 510 nm.
The results were expressed as milligram catechol equivalent per gram sample from the
standard catechol calibration curve.
3.6. Antioxidant Potentials and DPPH Radical Scavenging Activity
The extracts’ free radical scavenging activity was determined using the DPPH method
described by Catarino et al. [77]. Ascorbic acid was used as a standard with different ranges
(10–100 µg/mL) to generate the standard curve. The absorbance was measured at 517 nm
using a spectrophotometer. The activity of DPPH radical scavenging was determined as mg
ascorbic acid equivalent (AAE)/g dry sample. The percentage of DPPH radical scavenging
activity was calculated using the following Equation (1):
DPPH radical scavenging activity (% inhibition) =
Abscontrol − Abssample
× 100
Abscontrol
(1)
3.7. HPLC Analysis
The phenolic profile of algal aqueous extracts was screened using an HPLC (Agilent
1260 series). An Eclipse C18 column with (4.6 mm × 250 mm i.d., 5 µm) was used and
its temperature was kept constant at 40 ◦ C. The mobile phase was water (A) and 0.05%
trifluoroacetic acid in acetonitrile (B) and the volume of injection was 5 µL. The flow rate
was 0.9 mL/min and the multi-wavelength detector was monitored at 280 nm.
3.8. Cytotoxicity Assay
In a 96-well tissue culture plate, the IC50 value of algal extracts on peripheral blood
mononuclear cells (PBMCs) was defined using the neutral red cytotoxicity assay [78,79].
Extract concentrations of 250, 125, 62.5, 31.2, 15.6 and 7.8 µg/mL were obtained using
double-fold serial dilutions. The cells in the wells were individually treated with vari-
Mar. Drugs 2022, 20, 785
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ous concentrations of extract, 150 µL in every well except for the blank. The following
Formula (2) was utilized to determine the percentage of cytotoxicity (inhibition):
% Inhibition =
O.D Control − O.D Treatment
O.D Control
(2)
3.9. Sensory Evaluation of Fillet Fish Fortified with Algal Extracts
A total of 1 kg of fillet fish was treated by adding 1% of powdered algae aqueous
extracts (C. tomentosum and A. fragilis) and then fried in oil. The samples were divided into
four treatments: Treatment 1 (T1) as control (without extracts), Treatment 2 (T2) with 1% of
C. tomentosum extract, Treatment 3 (T3) with 1% of A. fragilis extract and Treatment 4 (T4)
as a mix with 0.5% of C. tomentosum and 0.5% of A. fragilis extracts.
The sensory evaluation panel included 15 members from the Food Technology Department and other departments, Arid Lands Cultivation Research Institute (ACRI), City
of Scientific Research and Technological Applications (SRTA-City). The panel members
were asked to observe some items (appearance, consistency, tenderness, flavor and overall
eating quality) and give scores from 1 to 7 (excellent 7; very good 6; good 5; medium 4;
fair 3; poor 2; very poor 1).
3.10. Statistical Analysis
All data collected were analyzed using one-way ANOVA, Fisher’s grouping test
Minitab® (Version 16) software, n = 3, and p < 0.05 revealed a significant difference. Significant means were compared by Duncan’s post hoc multiple comparison test.
4. Conclusions
It is well established that seaweeds contain various phytochemicals with diverse
biological activities, are considered safe for human consumption and have significant health
benefits. The current study provides approximate data on the phytochemical constituents
of marine seaweeds Codium tomentosum and Actinotrichia fragilis aqueous extracts for use
as food supplements fortified in fillet fish. Bioactive compounds found in seaweeds are
awaiting a significant breakthrough to be used as natural antibacterial and antioxidant
supplements in various food products. Moreover, these seaweed extracts have antibacterial
activity against bacterial infections, lending credence to their traditional use and implying
a future role for these seaweeds in combating microbial populations. The nutritional and
physiological benefits of seaweeds suggest that they may be introduced to our regular diets
as natural preservatives to promote health and immune system function, as well as enhance
new pharmaceutical and food applications.
These results demonstrate that C. tomentosum aqueous extract can be further exploited.
There is a need to advance our understanding of this interesting extract’s impact after fortified
fillet consumption to understand if the bioactivity is maintained, improved or lowered.
Author Contributions: Conceptualization, M.S.M.A.E.H., S.H.R., N.M.A. and H.E.A.E.-H.; methodology, M.S.M.A.E.H., S.H.R., N.M.A. and H.E.A.E.-H.; validation, J.C. and L.P.; formal analysis,
M.S.M.A.E.H., S.H.R., N.M.A., H.E.A.E.-H. and J.C.; investigation, M.S.M.A.E.H., S.H.R., N.M.A. and
H.E.A.E.-H.; writing—original draft preparation, M.S.M.A.E.H., S.H.R., N.M.A. and H.E.A.E.-H.;
writing—review and editing, J.C. and L.P.; visualization, J.C. and L.P.; supervision, S.H.R. and L.P.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are available from authors.
Mar. Drugs 2022, 20, 785
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Acknowledgments: João Cotas and Leonel Pereira are grateful for the support of the FCT—Foundation
for Science and Technology, I.P., within the scope of the project LA/P/0069/2020 granted to the
Associate Laboratory ARNET, UIDB/04292/2020 granted to MARE—Marine and Environmental
Sciences Centre.
Conflicts of Interest: The authors declare no conflict of interest.
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