foods

Article
Utilization of Algae Extracts as Natural Antibacterial and
Antioxidants for Controlling Foodborne Bacteria in Meat Products
Gamal M. Hamad 1, *, Haneen Samy 2 , Taha Mehany 1 , Sameh A. Korma 3 , Michael Eskander 4 ,
Rasha G. Tawfik 5 , Gamal E. A. EL-Rokh 6 , Alaa M. Mansour 7 , Samaa M. Saleh 8 , Amany EL Sharkawy 9 ,
Hesham E. A. Abdelfttah 6 and Eman Khalifa 10, *

1 Food Technology Department, Arid Lands Cultivation Research Institute (ALCRI), City of Scientific Research
and Technological Applications (SRTA-City), New Borg El-Arab 21934, Egypt; tmehany@srtacity.sci.eg
2 Biotechnology and Chemistry Department, Faculty of Science, Alexandria University,
Alexandria 22758, Egypt; haneen.samy781@gmail.com
3 Department of Food Science, Faculty of Agriculture, Zagazig University, Zagazig 44519, Egypt;
sameh.hosny@zu.edu.eg
4 Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Alexandria University,
Alexandria 22758, Egypt; michaelmagdy9090@yahoo.com
5 Department of Microbiology, Faculty of Veterinary Medicine, Alexandria University, Alexandria 22758, Egypt;
rasha.gomaa@alexu.edu.eg
6 Department of Food Science and Technology, Faculty of Agriculture, Al-Azhar University,
Assiut 71524, Egypt; dr.gamalelsayed@azhar.edu.eg (G.E.A.E.-R.);
heshamabdel-mobdy4919@azhar.eud.eg (H.E.A.A.)
7 Department of Animal Hygiene and Zoonoses, Faculty of Veterinary Medicine, Alexandria University,
Alexandria 22758, Egypt; alaa.m.mansour@alexu.edu.eg
8 Department of Food Science, Faculty of Agriculture (Saba Basha), Alexandria University,
Alexandria 21531, Egypt; samaasaleh@alexu.edu.eg
9 National Institute of Oceanography and Fisheries (NIOF), Cairo 11516, Egypt; as.sharkawy@niof.sci.eg
10 Department of Microbiology, Faculty of Veterinary Medicine, Matrouh University, Matrouh 51511, Egypt
* Correspondence: ghamad@srtacity.sci.eg (G.M.H.); khalifa.eman@alexu.edu.eg (E.K.);
Tel.: +20-1024934006 (G.M.H.); +20-1002546397 (E.K.)

Citation: Hamad, G.M.; Samy, H.; Abstract: Padina pavonica, Hormophysa cuneiformis, and Corallina officinalis are three types of algae
Mehany, T.; Korma, S.A.; Eskander, that are assumed to be used as antibacterial agents. Our study’s goal was to look into algal extracts’
M.; Tawfik, R.G.; EL-Rokh, G.E.A.;
potential to be used as food preservative agents and to evaluate their ability to inhibit pathogenic
Mansour, A.M.; Saleh, S.M.; EL
bacteria in several meat products (pastirma, beef burger, luncheon, minced meat, and kofta) from the
Sharkawy, A.; et al. Utilization of
local markets in Alexandria, Egypt. By testing their antibacterial activity, results demonstrated that
Algae Extracts as Natural
Padina pavonica showed the highest antibacterial activity towards Bacillus cereus, Staphylococcus aureus,
Antibacterial and Antioxidants for
Controlling Foodborne Bacteria in
Escherichia coli, Streptococcus pyogenes, Salmonella spp., and Klebsiella pneumoniae. Padina pavonica extract
Meat Products. Foods 2023, 12, 3281. also possesses most phenolic and flavonoid content overall. It has 24 mg gallic acid equivalent/g
https://doi.org/10.3390/ and 7.04 mg catechol equivalent/g, respectively. Moreover, the algae extracts were tested for their
foods12173281 antioxidant activity, and the findings were measured using ascorbic acid as a benchmark. The IC50
of ascorbic acid was found to be 25.09 µg/mL, while Padina pavonica exhibited an IC50 value of
Received: 24 July 2023
267.49 µg/mL, Corallina officinalis 305.01 µg/mL, and Hormophysa cuneiformis 325.23 µg/mL. In this
Revised: 28 August 2023
Accepted: 29 August 2023
study, Padina pavonica extract was utilized in three different concentrations (Treatment 1 g/100 g,
Published: 1 September 2023 Treatment 2 g/100 g, and Treatment 3 g/100 g) on beef burger as a model. The results showed that
as the concentration of the extract increased, the bacterial inhibition increased over time. Bacillus
cereus was found to be the most susceptible to the extract, while Streptococcus pyogenes was the least.
In addition, Padina pavonica was confirmed to be a safe compound through cytotoxicity testing. After
Copyright: © 2023 by the authors. conducting a sensory evaluation test, it was confirmed that Padina pavonica in meat products proved
Licensee MDPI, Basel, Switzerland.
to be a satisfactory product.
This article is an open access article
distributed under the terms and
Keywords: food preservative; algae; natural products; Padina pavonica; Hormophysa cuneiformis;
conditions of the Creative Commons
Corallina officinalis; antimicrobial; antioxidant
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Foods 2023, 12, 3281. https://doi.org/10.3390/foods12173281 https://www.mdpi.com/journal/foods

Foods 2023, 12, 3281 2 of 18

1. Introduction
Nearly 1 in 10 people worldwide, or 600 million, are expected to get sick from eating
contaminated food, and 420,000 people will die as a result. This results in the loss of
33 million DALYs (disability-adjusted life years), or years of good life, foodborne illnesses
obstruct socioeconomic progress by taxing health care systems and damaging international
trade, tourism, and national economies [1]. Bacterial foodborne illnesses are a frequent
occurrence, with several types of foodborne pathogens posing a significant risk. Among
them are Bacillus cereus, Staphylococcus aureus, E. coli, Streptococcus pyogenes, Salmonella
spp., and Klebsiella pneumoniae. These bacteria can result in a variety of issues, including
the production of harmful toxins, triggering mild inflammatory responses, abdominal
cramping, and severe diarrhea. In some cases, they can even lead to extensive ulceration of
the colonic mucosa [2]. Among the various organisms collaboration with animals, some
of these pathogens may gotten to be zoonotic and cause sickness among people, posing a
risk to public health and the economy. Animal-derived nourishment items, counting milk,
meat, and eggs, are considered fundamental components of human nourishment [3].
Based on this information and projections that Egypt is expected to consume approxi-
mately 3.2 million metric tons of meat by 2026 [4], there is a potential problem with meat
and meat products being an ideal breeding ground for foodborne diseases and spoiling
microorganisms. This is a consequence of their high levels of nutritional content and
water activity, which foster growth of these pathogenic microorganisms [5]. Consumers
have recently begun thinking of meat and meat products as unhealthy foods. In order
to overcome this difficulty, re-formulation is a useful technique for creating customized
meat-based products that include substances with specific health-beneficial properties and
exclude other traits seen as damaging [6].
As a result, taking precautionary measures to prevent the spread of foodborne illness
and ensure the safety of meat products is essential. This can be accomplished through
a variety of procedures, including proper storage, handling, and the use of preservation
agents. There are two types of preservative substances: natural and synthetic. Most
countries prefer chemical substances, which can be synthetic or semi-synthetic, given their
affordable price, and antimicrobial effect, and ability to prolong food goods’ shelf lives
without compromising their flavor, color, or texture, but it has been demonstrated that
these chemical substances have negative health effects [7].
Furthermore, each chemical substance provides a specific purpose, with some serving
as antibacterial agents, others as antifungal agents, and still others as antioxidants. In
order to maintain the safety and quality of food items, the food industry significantly
relies on a range of preservatives, for example, potassium nitrate may reduce the oxygen-
carrying capacity of blood and potentially cause cancer, calcium benzoate may inhibit
digestive enzyme function [8], and sorbic acid, benzoic acid, and their salts can promote
the production of substances with mutagenic and carcinogenic effects [9].
Macroalgae, or seaweeds, are an enormous category of varied autotrophs [10]. Algae-
Base now has 172,223 species and infraspecific names, 23,355 images, 67,900 bibliographic
articles, and 540,684 distributional information [11]. Marine macroalgae create an abun-
dance of naturally occurring secondary metabolites with varied functions as a result of
constant exposure to various biotic and abiotic stimuli that promote the development of
such compounds [12]. Algae are particularly noteworthy as photosynthetic organisms that
possess exceptional nutritional value, and along with colors like carotenoids, chlorophyll,
and phycobilins, bioactive substances include polyphenols, tocopherols, vitamin C, and
amino acids that resemble mycosporine [13].
Seaweeds include a variety of active substances, such as polyphenols, alkaloids, ter-
penes, and phlorotannins, in addition to their remarkable concentration of polysaccharides,
amino acids, fatty acids, carotenoids, and phycobiliproteins and phycocolloides [14]. These
ingredients have strong antioxidant, anti-proliferative, anti-inflammatory, anti-clotting,
anti-diabetic, and anti-hepatitis effects [15]. Marine macroalgae represent a potential source
of bioactive substances for a variety of uses in the cosmetics, pharmaceutical, agro-food,
Foods 2023, 12, 3281 3 of 18

and, more recently, functional food and chemistry industries [16]. Seaweeds are abundant
along Egypt’s Mediterranean, Suez Canal, and Red Sea shores [17], but none are used for
commercial purposes, and there is little scientific research on their potential as functional
foods and sources of bioactive compounds [18].
Marine algae could be utilized as healthy food sources, nutrition boosters, and preser-
vatives [19]. Additionally, phenolic compounds, aromatic secondary metabolites of marine
algae, are important for food’s color and nutritional value [20]. According to reports,
seaweeds include significant amounts of dietary fiber (non-starch polysaccharides), essen-
tial and non-essential amino acids, minerals, polyunsaturated fatty acids, polyphenolic
compounds, vitamins, and other nutrients that are crucial for healthy development [21].
A variety of useful metabolites, including vitamins, enzymes, proteins, lipids, carotenoids,
polysaccharides, sterols, antibiotics, and a large number of fine compounds, can be found
in algae, particularly macroalgae [22]. The secondary metabolite Padina pavonica, which
was found near Abu Qir Bay in Alexandria, Egypt, is abundant in phenolics, flavonoids,
saponins, tannins, and alkaloids. As a result, they are regarded as promising natural
candidates for treatment of cancer, diabetes, inflammation, and other diseases. It is advised
to continue applied research on such extracts of the examined species for additional thera-
peutic, medicinal uses, and in vivo studies in the future in order to better understand the
bioactive chemicals responsible for the actions [23].
The importance of macroalgae as a food source and their abundance of bioactive
chemicals are well established. Given that they are palatable, secure, and affordable, these
bioactive chemicals can be added to food products for preservation [24]. Determining the
qualitative and quantitative phytochemical content of macroalgae has received significant
attention. From tropical to temperate seas, Padina pavonica is extensively distributed
because of its accessibility and ecological characteristics [25]. P. pavonica could be used
in the production of food [26], and P. pavonica (Linnaeus) brown algae with antioxidant,
antibacterial, and anticancer properties, is frequently used in soups, salads, and other
foods [27]. Hormophysa cuneiformis has been shown to have a specific phenolic content,
antioxidant properties, and antibacterial properties. As a result, it may be used as a source
of biologically active substances [28]. The marine seaweed, Corallina officinalis, extracts also
maintain strong antimicrobial and antioxidant properties [29,30].
In our study, we aimed to utilize preservative substances that not only ensure safety
but also offer multiple beneficial properties, such as antibacterial and antioxidant. We have
selected three types of algae species, namely Padina pavonica (brown algae), Hormophysa
cuneiformis (brown algae), and Corallina officinalis (red algae), to investigate their effec-
tiveness as food preservatives in various meat products, which are pastirma, beef burger,
luncheon, minced meat, and kofta.

2. Materials and Methods

2.1. Collection of Meat Products and Detection of Pathogenic Bacteria
In Alexandria Governorate, Egypt, a comprehensive study was conducted on 125 meat
samples: 25 samples from each product from 5 meat products as follows: pastirma, beef
burger, luncheon, minced meat, and kofta, which were obtained randomly from local
markets, refrigerated, cut into pieces, and packaged in polyethylene bags then promptly de-
livered in an ice box for bacteriological examination, and pathogenic bacteria isolated using
various selective media were employed to isolate the anticipated harmful microorganisms,
according to El-Khawas et al. [31].

2.2. Bacterial Strains

The study employed six strains of pathogenic bacteria, consisting of 3 Gram-positive
strains (Bacillus cereus EMCC 1006, Staphylococcus aureus EMCC 1351, and Streptococcus
pyogenes EMCC 1772) and 3 Gram-negative strains (Salmonella spp., Escherichia coli ATCC
25,922, and Klebsiella pneumoniae EMCC 1637). The strains were obtained from the Mi-
crobiological Resources Center (MIRCEN), Faculty of Agriculture, Ain Shams University,
 2.2. Bacterial Strains
The study employed six strains of pathogenic bacteria, consisting of 3 Gram-positive
strains (Bacillus cereus EMCC 1006, Staphylococcus aureus EMCC 1351, and Streptococcus
pyogenes EMCC 1772) and 3 Gram-negative strains (Salmonella spp., Escherichia coli ATCC
Foods 2023, 12, 3281 4 of 18
25,922, and Klebsiella pneumoniae EMCC 1637). The strains were obtained from the Micro-
biological Resources Center (MIRCEN), Faculty of Agriculture, Ain Shams University,
Cairo, Egypt. Prior to use, the bacterial strains were equipped and customized to a bacte-
Cairo,
rial Egypt.
density of Prior
1 × 10to use, the bacterial
7 CFU/mL, followingstrains were equipped
the method outlinedand
by customized to al.
Bahi-Eldin et a bacterial
[32].
density of 1 × 107 CFU/mL, following the method outlined by Bahi-Eldin et al. [32].
2.3. Algal Materials and Extraction
2.3. Algal Materials and Extraction
Total of 3 types of seaweed obtained at Hurghada City, Red Sea Governorate, Egypt:
Total of 3 types of seaweed obtained at Hurghada City, Red Sea Governorate, Egypt:
Padina pavonica (P.P) (brown algae), Hormophysa cuneiformis (H.C) (brown algae), Corallina
Padina pavonica (P.P) (brown algae), Hormophysa cuneiformis (H.C) (brown algae), Corallina
officinalis (C.O) (red algae), and Figure 1. The seaweed specimens were meticulously
officinalis (C.O) (red algae), and Figure 1. The seaweed specimens were meticulously
cleansed of epiphytes, then dehydrated, and transformed into a powdered form. Each
cleansed of epiphytes, then dehydrated, and transformed into a powdered form. Each type
type of powdered seaweed was transformed into a lyophilized ethanolic extract (70% eth-
of powdered seaweed was transformed into a lyophilized ethanolic extract (70% ethanol:
anol: deionized water v/v). Recognition of algae was accomplished utilizing the methods
deionized water v/v). Recognition of algae was accomplished utilizing the methods
described
describedby bySalem
Salemetetal.
al.and
andYang
Yanget
etal.
al.[33,34].
[33,34].

Imagesofofthe
Figure1.1.Images
Figure theinvestigated
investigatedseaweeds
seaweedsininthe
thepresent
presentstudy:
study:(A) Hormophysacuneiformis
(A)Hormophysa cuneiformis(H.C)
(H.C)
(brownalgae);
(brown algae);(B) Padinapavonica
(B)Padina pavonica(P.P)
(P.P) (brown officinalis (C.O) (red algae).
(brown algae), and (C) Corallina officinalis

2.4.Antibacterial
2.4. AntibacterialActivity
Activity
2.4.1. Assessment of the Antibacterial Activity of Algae Extracts by Agar Disk
2.4.1. Assessment
Diffusion Assay of the Antibacterial Activity of Algae Extracts by Agar Disk Diffusion
Assay
Agar disk diffusion assay was assessed according to Hamad et al. [35] to determine
Agar disk diffusion
the antibacterial propertiesassay was extracts
of algae assessedagainst
according to Hamad
reference etof
strains al.pathogenic
[35] to determine
bacteria
the antibacterial
purchased fromproperties
MIRCEN,of algae extracts
Faculty against reference
of Agriculture, Ain Shams strains of pathogenic
University, bacte-
Cairo, Egypt).
ria purchased from MIRCEN, Faculty of Agriculture, Ain Shams
They enriched overnight bacterial cultures on Mueller Hinton Medium (MHM) broth University, Cairo,
Egypt).
(Oxoid, They enriched
Cheshire, UK) atovernight
37 ◦ C forbacterial
48 h andcultures on Mueller
poured the cultures onHinton
MHM Medium (MHM)
plates. Once the
broth
plates were dried, they were loaded with each disc that was impregnated with 20plates.
(Oxoid, Cheshire, UK) at 37 °C for 48 h and poured the cultures on MHM µL of
Once the plates werealgal
each corresponding dried, they were
extract with loaded with each
a concentration ofdisc
100 that
mg/mLwas each
impregnated with
and the plates
20 µL incubated
were at 4 ◦ C for 30algal
of each corresponding min,extract
followedwithbyaincubation at 37of◦100
concentration C/24mg/mL each and
h. Inhibitory the
zones
plates were incubated
were measured at 4 °C forto30evaluate
in millimeters min, followed by incubationbacterial
the anti-pathogenic at 37 °C/24 h. Inhibitory
activities of the
zones
variouswere measured
algae extracts.in millimeters to evaluate the anti-pathogenic bacterial activities of
the various algae extracts.
2.4.2. Evaluation of Minimum Inhibitory Concentrations MIC of Padina pavonica Extract
2.4.2. After
Evaluation of Minimum
assessing Inhibitory
antibacterial Concentrations
activity of three types MIC of Padina
of algae pavonica
extracts, Extracton
we focused
the one that demonstrated the highest ability to combat pathogenic bacteria. We then
determined the MIC of P. pavonica algal extracts, the lowest dose of algal extract that still
prevents detectable growth is known as MIC, towards microorganisms that are harmful,
following the methodology outlined by Kadaikunnan et al. [36]. To accomplish this, we
used varying concentrations of the algal extract that were 100, 50, 25, 12.5, 6.25, and
3.12 mg/mL. Next, we prepared pathogenic bacteria suspensions from cultures that had
been grown and adjusted their density to 107 colony forming units (CFU)/mL, as per
Bahi-Eldin et al. [32].
Foods 2023, 12, 3281 5 of 18

2.5. Phytochemical Analysis of the Algae Extracts

2.5.1. Total Phenolic Content (TPC) of Algae Extracts
To evaluate TPC of algae extracts, we used Folin–Ciocalteu test, following the tech-
nique outlined by Hamad et al. [37]. We analyzed all samples in triplicate.

2.5.2. Total Flavonoid Content (TFC) of Algae Extracts

We evaluated TFC of algae extracts by a technique outlined by Hamad et al. [38].

2.5.3. Assessment of the Antioxidant Activity Diphenyl-1-Picrylhydrazyl (DPPH) Radical

Scavenging Capacity
We evaluated algae extracts’ capacity to eliminate DPPH free radicals by modifying
the method proposed by Hamad et al. and Catarino et al. [38,39]. First, we prepared a
stock solution of each extract in methanol at a concentration of 1 mg/mL. Next, we created
serial dilutions of the plant extract by combining 1 mL of each dilution with 1 mL of a
methanol solution containing DPPH at a concentration of 1 mg/mL. After incubating the
mixture in darkness for 30 min, we measured the absorbance at 517 nm. As a positive
control, we used ascorbic acid. The results were expressed as IC50 , which represents the
extract concentration required to reduce 50% of the DPPH. To calculate the IC50 , we used a
non-linear regression algorithm to plot the inhibition percentage against the concentration.
The inhibition percentage was calculated using the following formula: DPPH inhibition
percentage (%) = [(A of control − A of the sample)/A of control] × 100, where A refers
to absorbance.

2.6. Safety and Cytotoxicity Assay of Padina pavonica Algal Extract

The potential impact of P. pavonica algal extract on PBMCs’ (peripheral blood mononu-
clear cells) vitality was thoroughly examined. To evaluate cell viability, PBMCs were
cultured in Roswell Park Memorial Institute (RPMI) medium. Initially, whole blood was
diluted with phosphate-buffered saline (PBS) and formerly carefully layered over an equal
volume of Ficoll in a Falcon tube. The mixture was centrifuged for 30 min at 500 rpm to
isolate PBMCs. Then, blank wells (150 µL PBS), control wells (150 µL PBMCs), and tested
wells (150 µL PBMCs) were placed on a 96-well microtiter plate. Various concentrations
of P. pavonica algal extracts were then introduced into the tested wells, followed by an
incubation period of 24 h in line [40]. Neutral red (150 µL) was added to the wells, and
the mixture was incubated at 37 ◦ C for 2 h. The plates were cleaned with a de-staining
solution (1% acetic acid: 49% deionized water: 50% ethanol, 150 L/well) after the cells
were washed. A T80 UV/VIS spectrophotometer previously set to 540 nm was used to
measure absorbance, as mentioned by Ryan and Deci [41]. Using the following equation,
P. pavonica algal extract inhibition% was computed, and IC50 values were obtained through
an online calculator [42]. The equation for calculating P. pavonica algal extract inhibition%
is: Lyophilized HO algal extract inhibition% = 100 − (O.D Control − O.D Treatment
O.D)/O.D. Control, where O.D. stands for optical density, control refers to 150 µL PBMCs,
and treatment denotes 150 µL P. pavonica algal extract.

2.7. Shelf-Life for Padina pavonica Extract as Antibacterial on Beef Burger after Treatment
Prior to the experiment, minced meat was sterilized using ultraviolet light (UV) for
15 min on each side to control the microorganisms present, as suggested by Morsy et al. [43].
The beef burger was prepared according to the method outlined in the Egyptian standard
specification for burgers (ESS 1688/1991), as described by Kassem et al. [44]. Fresh beef was
transported to the laboratory in an ice box and minced using an electric mincer (Moulinex,
2000 Watt, France) through a 4 mm plate. A mixture of 65 g/100 g minced meat, 20 g/100 g
fat, 5 g/100 g soybean, 0.3 g/100 g black pepper, 1.8 g/100 g salt, and 10 g/100 g water
was thoroughly mixed for five min in a mixer using a spiral dough hook at medium speed
(80 rpm) and passed through a smaller hole plate to ensure homogeneity. The resulting
mix was divided into 7 portions, with one portion serving as a negative control without
Foods 2023, 12, 3281 6 of 18

any additives, six portions inoculated with 107 CFU/mL of pathogenic bacteria, including
Bacillus cereus, Staphylococcus aureus, Streptococcus pyogenes, Salmonella spp., Escherichia coli,
and Klebsiella pneumoniae, serving as positive controls, and 18 portions with the addition of
P. pavonica extract at 1, 2, and 3 g/100 g, which were also inoculated with 107 CFU/mL of
pathogenic bacteria to test their survival. The mixtures were shaped into approximately
50 g cylindrical beef burgers using a commercial forming tool with a 10 cm internal diameter
and firmly plasticized film encased to stop moisture evaporation. The burgers were kept
in foam plates at 6 ◦ C. Samples were taken at 0, 1, 2, 3, 4, 7, 10, and 15 days of storage for
examination of the pathogenic bacteria present. To isolate pathogenic bacteria, we followed
a procedure in which 25 g of meat was added to a plastic bag containing 225 mL of 1%
peptone water. After homogenizing samples for a minute by a stomacher, we incubated
them at 35 ◦ C/24 h. Following this pre-enrichment, we added 1 mL of the mixture to
9 mL of peptone broth and incubated it at 35 ◦ C/24 h. To count pathogenic bacteria, we
surface-plated the samples with the proper dilutions and duplicated them on Tryptic Soy
Blood Agar (TSBA) medium plates. We conducted three individual replicates of each
experiment to ensure accuracy as reported by Ragab et al. and Hamad et al. [45,46].

2.8. Assessment of the Acceptability of Beef Burger Fortified with the Padina pavonica
Algal Extract
At the Food Technology Department in the City of Scientific Research and Techno-
logical Applications in New Borg El Arab, Egypt, a group of 10 experienced evaluators
conducted a sensory assessment on a grilled beef burger that was fortified with an ex-
tract from P. pavonica algae. The objective was to determine the burger’s acceptability
as a potential food additive. The evaluators assessed four different groups of samples,
including a control group with no treatment and three treatment groups with increasing
levels of P. pavonica extract [Treatment (1%): beef burger treated with P. pavonica extract
1%, Treatment (2%): beef burger treated with P. pavonica extract 2% and Treatment (3%):
beef burger treated with P. pavonica extract 3%. Before evaluation, samples left at room
temperature for 10 min Evaluators assessed samples based on odor, taste, color, texture,
and overall acceptance, with each criterion being scored on a scale from 1 to 10, where
higher ratings denoted a more palatable condition. The sensory attribute data, including
standard deviations, were analyzed and recorded according to Hamad et al. [46].

2.9. Statistical Analysis

SPSS, version 23 (IBM SPSS Statistics for Windows, IBM Corp., Armonk, NY, USA)
was used to implement all calculations. For the data analyses, the means standard error (SE)
was employed. The Duncan test was employed in one-way analysis of variance (ANOVA),
and the probability was regarded as statistically significant when p < 0.05.

3. Results and Discussion

3.1. Detection of Pathogenic Bacteria in Samples
After conducting a bacteriological examination of 125 samples of different meat prod-
ucts (pastirma, beef burger, luncheon, minced meat, and kofta) collected from the local
markets in Alexandria Governorate, Egypt, it was discovered that most of the samples
were positive and contained pathogenic bacteria. Among the various samples, beef burgers
exhibited the highest percentage of positive isolates, with 92%, followed closely by kofta
at 88%, minced meat at 84%, and luncheon at 80%. In contrast, pastirma exhibited the
lowest percentage of positive isolates at 76%, as detailed in Table 1. This highlighted the
hazard of foodborne bacteria in meat products, which affect human health, and the possible
source of those bacteria may be from the infected animal or through the slaughtering or
contamination during handling. Similar results were obtained by Hassanien [47] who
isolated pathogenic bacteria from some meat products, such as kofta, beef burger, and
luncheon. Our results confirm those obtained by Bintsis [48] who said that meat-borne
diseases are either caused by a number of bacterial infections by contaminating livestock or
Foods 2023, 12, 3281 7 of 18

meat during the meat handling or processing. Our findings are also in line with Ali and
Alsayeqh [49], who declared that the influence of meat-borne bacteria on global disease
transmission and food safety had a substantial impact on public health.

Table 1. Incidence of pathogenic bacteria isolates from products collected from different local markets
(n = 125).

Positive Samples Negative Samples

Meat Products No. of Samples
No. % No. %
Pastirma 25 19 76 6 24
Beef burger 25 23 92 2 8
Luncheon 25 20 80 5 20
Minced meat 25 21 84 4 16
Kofta 25 22 88 3 12
Total 125 105 84 20 16

3.2. Antibacterial Activity of Lyophilized Algae Extract

The antibacterial activities of three investigated types of marine algae, namely Padina
pavonica, Hormophysa cuneiformis, and Corallina officinalis by agar disk diffusion against
various strains of pathogenic bacteria, Bacillus cereus, Staphylococcus aureus, Escherichia
coli, Streptococcus pyogenes, Salmonella spp., and Klebsiella pneumoniae, were evaluated, as
illustrated in Table 2 and Figure 2. The results showed that all tested algae extracts have
antibacterial effects and inhibit the growth of both tested Gram-positive and Gram-negative
strains as followed: P. pavonica had the highest antibacterial activity with inhibition zones of
38.83 ± 0.27 and 38.23 ± 0.15 mm against B. cereus and Staph. aureus, respectively, and the
antibacterial activity of H. cuneiformis was also notable with inhibition zones of 37.13 ± 0.09
and 34.97 ± 0.15 mm against K. pneumoniae and E. coli, respectively. Additionally, C. offici-
nalis displayed considerable antibacterial activity with inhibition zones of 35.07 ± 0.23 mm
against B. cereus and 33.17 ± 0.09 mm against E. coli, this may be due to marine algae
have secondary bioactive substances, such as phenolic and flavonoid compounds, which
are known to possess antibacterial properties, this agreed with Al-Saif et al. and Jimenez-
Lopez et al. [50,51], who stated that the phenolic contents responsible for the antibacterial
activities of algae extract and Jimenez-Lopez et al. and Mohy El-Din and El-Ahwany [51,52]
that mentioned the flavonoid composition of algae extracts responsible for the antimicrobial
activities. Our results provide promising alternatives of antibacterial agents as antibiotics to
overcome the resistant bacteria. This in line with Lee et al. and El Shafay et al. [53,54] who
claimed that in order to prevent the emergence of microbial resistance to antibiotics, algae
phenolic compounds, or synergistic mixtures of these compounds with other substances,
such as fatty acids, halogenated compounds, or terpenes, are a potential new approach.
For instance, the antibacterial activity of P. pavonia against K. pneumoniae in our findings
was higher, 34.97 ± 0.09 mm, than that obtained by Al-Enazi et al. [55], who reported
23.40 ± 0.58 mm, and the antibacterial activity of the algae extract against E. coli was found
to be 18.20 ± 0.63 mm, whereas it was 36.97 ± 0.09 mm against E. coli in our study. Another
study on C. officinalis from the Aegean Sea (Turkey) reported similar results of approxi-
mately 32.00 ± 1.73 mm against E. coli obtained by Taskin et al. [56], while in our study, it
was 33.17 ± 0.09 mm. Therefore, depending on our in vitro antibacterial evaluation results,
P. pavonica (brown algae) extracts was chosen for further application in meat products
against pathogenic bacteria. While Al-Enazi et al. [55] found that extract of P. pavonica sig-
nificantly inhibited tested bacteria by studying (the inhibition zone in mm with correlated
concentration of algae extract in mg/mL) as follows: The greatest activities were against
K. pneumonia (22.60 ± 2.10 mm; 03.90 mg/mL), and against Staph. aureus (21.7 ± 0.58 mm;
1.95 mg/mL), while on Bacillus, it was (21.7 ± 1.5 mm; 1.95 mg/mL), and against Strept.
Pyogenes, it was (20.7 ± 1.2 mm; 1.95 mg/mL). Osman et al. [57] declared that H. cuneiformis
extract at a concentration of 2000 µg/disc (conc. 2) was efficient against both Gram-positive
and Gram-negative bacteria, although the concentration of 200 µg/disc (conc. 1) was less
Foods 2023, 12, 3281 8 of 18

effective. Against E. coli, it was 6 ± 0.12 mm (conc. 1), 8 ± 0.6 mm (conc. 2), against Staph.
aureus gave-ve (conc. 1), 9 ± 0.12 mm (conc. 2), and against Bacillus, it was 8.5 ± 0.01 mm
(conc. 1) and 10.5 ± 0.2 mm (conc. 2). Mofeed et al. [29] also recorded the antibacterial
inhibition activity of C. officinalis on E. coli, Salmonella, and S. aureus, the results showed that
the extract of C. officinalis inhibited 100% of S. aureus cells at 100 µg/mL concentration, the
extract of C. officinalis causes the highest inhibition (80%) against Salmonella. In conclusion,
their results proved that the extract of C. officinalis possess an effective antibacterial activity
against Salmonella, S. aureus, and E. coli pathogenic bacteria. The variation in inhibition
zone size may be due to the concentration of the algae extracts used.

Table 2. Antibacterial activity of lyophilized algae extracts against pathogenic bacteria using agar
disk diffusion assay.

Extracts (Inhibition Zone Diameter (mm))

Strains Concentration Padina pavonica Corallina officinalis Hormophysa cuneiformis
(Brown Algae) (Red Algae) (Brown Algae)
Gram-positive strains
Bacillus cereus EMCC 1006 38.83 ± 0.27 c 35.07 ± 0.23 b 32.5 ± 0.29 a
Staphylococcus aureus EMCC 1351 100 mg/mL 38.23 ± 0.15 c 33.00 ± 0.17 b 30.03 ± 0.20 a
Streptococcus pyogenes EMCC 1772 36.00 ± 0.125 c 32.33 ± 0.20 a 33.10 ± 0.06 b
Gram-negative strains
Salmonella spp. 33.97 ± 0.09 c 31.23 ± 0.15 b 28.2 ± 0.12 a
Escherichia coli ATCC 25922 100 mg/mL 36.97 ± 0.09 c 33.17 ± 0.09 a 34.97 ± 0.15 b
Klebsiella pneumoniae EMCC 1637 34.97 ± 0.09 b 30.20 ± 0.12 a 37.13 ± 0.09 c
Foods 2023, 12, x FOR PEER REVIEW 9 of 18
Data represented are the means of triplicates ± standard error of means, a,b,c data in the same column followed by
different superscript letters differ significantly (p < 0.05).

Figure 2.
Figure 2. Antibacterial
Antibacterial activity
activity of
of algae
algae extracts
extracts Padina
Padina pavonica
pavonica (brown
(brown algae),
algae), Corallina
Corallina officinalis
officinalis
(red algae), and Hormophysa cuneiformis (brown algae) against pathogenic bacteria using agar disk
(red algae), and Hormophysa cuneiformis (brown algae) against pathogenic bacteria using agar disk
diffusion assay.
diffusion assay.

3.3. MICs of Lyophilized Padina pavonica Extract

Out of
Out of all
allsamples
sampleswewetested,
tested,P. P. pavonica
pavonica performed
performed bestbest against
against bacteria.
bacteria. To deter-
To determine
mine the MIC, we experimented with numerous concentrations of P. pavonica
the MIC, we experimented with numerous concentrations of P. pavonica extract. The resultsextract. The
results 3)
(Table (Table 3) indicated
indicated that
that the the for
MIC MICB.for B. cereus
cereus andand Staph.
Staph. aureus
aureus waswas 3.1mg/mL,
3.1 mg/mL, with
an inhibition zone of 5.04 ± 0.08 mm and 5.11 ± 0.07 mm, respectively, prooving that they
are the most susceptible bacteria to P. pavonica. Additionally, the MIC for Strept. pyogenes
and E. coli was 6.2 mg/mL, resulting in an inhibition zone of 6.14 ± 0.09 mm and 4.97 ± 0.09
mm, correspondingly. Finally, we found that the MIC for Salmonella spp. and K. pneu-
Foods 2023, 12, 3281 9 of 18

an inhibition zone of 5.04 ± 0.08 mm and 5.11 ± 0.07 mm, respectively, prooving that
they are the most susceptible bacteria to P. pavonica. Additionally, the MIC for Strept.
pyogenes and E. coli was 6.2 mg/mL, resulting in an inhibition zone of 6.14 ± 0.09 mm
and 4.97 ± 0.09 mm, correspondingly. Finally, we found that the MIC for Salmonella spp.
and K. pneumoniae was 12.5 mg/mL, resulting in an inhibition zone of 6.07 ± 0.07 mm
and 7.1 ± 0.09 mm, consistently, that confirmed they are the less sensitive bacteria to
P. pavonica. The obtained results also highlighted the broad spectrum antibacterial activity
of P. pavonica. A similar study carried out by Ertürk and Taş [58] showed that the MIC of
P. pavonica extract was >1.25 mg/mL to E. coli, >10 mg/mL to B. cereus, >1.25 mg/mL to
Staph. aureus, >2.5 mg/mL to Salmonella. Al-Enazi et al. [55] also found that MIC on E. coli
0.00781 mg/mL, K. pneumoniae 0.00390 mg/mL, Bacillus 0.00195 mg/mL, Staph. aureus
0.00195 mg/mL, Strept. pyogenes 0.00195 mg/mL.

Table 3. MICs (mg/mL) of P. pavonica extracts against pathogenic bacteria (Inhibition zone diameter
in mm).

Inhibition Zone Diameter (mm) ** with Regard to Each Extract Concentration * (mg/mL) MIC mg/mL
Strains
100 * 50 * 25 * 12.5 * 6.2 * 3.1 * MIC
Gram-positive strains
Bacillus cereus EMCC 1006 38.83 ± 0.27 f 24.97 ± 0.03 e 19.10 ± 0.06 d 12.03 ± 0.09 c 8.07 ± 0.04 b 5.04 ± 0.08 a 3.1
Staphylococcus aureus f e d c b a
EMCC 1351 38.23 ± 0.15 23.10 ± 0.06 17.17 ± 0.09 11.03 ± 0.03 7.24 ± 0. 14 5.11 ± 0.07 3.1
Streptococcus pyogenes e d c b a
EMCC 1772 36.00 ± 0.12 20.17 ± 0.12 14.93 ± 0.12 10.23 ± 0.12 6.14 ± 0.09 ND 6.2
Gram-negative strains
Salmonella spp. 33.97 ± 0.09 d 18.03 ± 0.09 c 11.00 ± 0.06 b 6.07 ± 0.07 a ND ND 12.5
Escherichia coli
ATCC 25922 36.97 ± 0.09 e 21.97 ± 0.20 d 16.10 ± 0.21 c 9.17 ± 0.12 b 4.97 ± 0.09 a ND 6.2
Klebsiella pneumoniae
EMCC 1637 34.97 ± 0.09 d 20.14 ± 0.09 c 15.23 ± 0.19 b 7.1 ± 0.09 a ND ND 12.5

Data represented are the means of triplicates ± standard error of means, a,b,c,d,e,f data in the same column followed
by different superscript letters differ significantly (p < 0.05), MIC; Minimum Inhibition Concentration in mg/mL;
* Concentrations of extract in mg/mL; ** Diameter include 5 mm well diameter; ND; Not detected.

3.4. Total Phenolic and Flavonoids Content of Algae Extracts

Phenolic compounds are frequently present in seaweeds, which contain a range of
organic and inorganic compounds that can be beneficial to human health [59]. Algae
generally exhibit a greater antioxidant activity because of increased levels of non-enzymatic
antioxidant components, including phenols, flavonoids, ascorbic acid, and reduced glu-
tathione [60]. The discovery of new medications and nutritious meals with antioxidant
capabilities has spurred interest in marine bio-sources as potential natural sources of bioac-
tive substances. TPC and TFC of P. pavonica, C. officinalis, and H. cuneiformis extracts are
demonstrated in Table 4 as: P. pavonica (24.13 ± 0.35 and 7.18 ± 0.08 mg GAE/g); (Gallic
Acid Equivalent per gram GAE/g); C. officinalis (20.03 ± 0.55 and 5.23 ± 0.13 mg GAE/g)
and H. cuneiformis (15.53 ± 0.29 and 1.83 ± 0.02 mg GAE/g) TPC and TFC; respectively.
This was higher than that obtained by Čagalj et al. [61] who said that the TPC of P. pavonica
ranged from 0.44 ± 0.034 to 4.32 ± 0.15 mg GAE/g and Mannino et al. [62] also determined
low TPC in P. pavonica. This may be due to environmental challenges and sea temperature
as proofed by Mancuso et al. [63] storage, drying, and extraction methods. Čagalj et al. [61]
also recorded TFC of P. pavonica was 2.25 ± 0.12 mg QE/g (quercetin equivalent per gram
QE/g) and said that despite the lack of investigations on the flavonoid content of algae, a
few reports have suggested that P. pavonica extracts are very flavonoid-rich. Our results for
TPC and TFC may be the reason for the antibacterial properties of algae extracts. This is in
accordance with with Bansemir et al. [64], who claimed that numerous bioactive substances
from marine macro-algae were shown to have antibacterial activity, including alcohols,
phenolic hydrocarbons, terpenes, acids, phenols, sulfur-containing compounds, aldehydes,
and the skeleton of naphthalene.
Foods 2023, 12, 3281 10 of 18

Table 4. Total Phenolic (mg GAE/g) and flavonoid contents (mg catechol/g) of algae extracts.

Extracts Total Phenolic Content Total Flavonoids Content

Padina pavonica 24.13 ± 0.35 c 7.18 ± 0.08 c
Corallina officinalis 20.03 ± 0.55 b 5.23 ± 0.13 b
Hormophysa cuneiformis 15.53 ± 0.29 a 1.83 ± 0.02 a
Data represented are the means of triplicates ± standard error of means. a,b,c Data in the same column followed
by different superscript letters differ significantly (p < 0.05).

3.5. Antioxidant Activity and DPPH Radical Scavenging Capacity

During the processing and storage of meat products, the quality of the meat dete-
riorates due to a process called lipid oxidation. This process results in the formation of
primary and secondary oxidation products, reduces the nutritional quality of the meat, and
changes its flavor, all of which can pose health risks and result in economic losses due to the
production of substandard products [65]. Free radicals play a role in a number of illnesses,
such as cancer, AIDS, and neurological diseases. Antioxidants’ scavenging abilities are
highly helpful in the management of certain disorders. The most popular approach for
evaluating the antioxidant activity of various plant, fungal, or algal extracts is the DPPH
assay, which is a sensitive procedure [66]. The DPPH radical has an odd electron, making
it a stable free radical that has the maximum absorbance at 517 nm. This electron will be
combined by antioxidants with a hydrogen donor to cause a change in color from purple to
yellow. The resulting discoloration is stoichiometric in terms of the quantity of electrons
consumed [67]. The ability of seaweed extracts to donate electrons and their capacity to
scavenge them are what allow the solution containing the DPPH to be bleached [68]. Our
results of algae extracts showed antioxidant activity in different concentrations (µg/mL)
and compared with standard ascorbic acid in a concentration-dependent manner, the algae
extract demonstrated DPPH radical scavenging action. Ascorbic acid’s IC50 was discov-
ered to be 25.09 µg/mL, P. pavonica was 267.49 µg/mL, C. officinalis was 305.01 µg/mL,
H. cuneiformis was 325.23 µg/mL, as shown in Table 5. This was higher than DPPH scaveng-
ing activity obtained by Al-Enazi et al. [55] who found that IC50 = 5.59 µg/mL in P. pavonica,
and the range of P. pavonica extracts’ free antioxidant activity against the DPPH radical
was 2.33 ± 1.34% to 62.88 ± 3.13% obtained by Čagalj et al. [61], while our results were
lower than those obtained by TPC extraction. Antioxidant activity performed by Pin-
teus et al. [69] found that P. pavonica has a DPPH IC50 of 338.8 g/mL (338.8 × 106 µg/mL)
and a TPC of 44.61 mg GAE/g dry extract. Osman et al. [57] found the IC50 of H. cuneiformis
to be 676.9 µg/mL. This may be due to the methods of inhibition as the measurement
conditions, the methods of extraction, or season of collection of algae have a big impact
on IC50 results. These suggestions also agree with Kandhasamy and Arunachalam [70].
Čagalj et al. [61] claimed that the extraction parameters, solid-to-solvent ratio, and solvent
selections in the extraction procedures need to be investigated and optimized in order to
identify conditions that would produce most of the targeted compounds while maintaining
their biological activity.

Table 5. Antioxidant activity and DPPH radical scavenging capacity of the algae extracts.

Extracts
Concentration Padina Corallina Hormophysa
µg/mL Ascorbic Acid
pavonica officinalis cuneiformis
Inhibition
(Brown Algae) (Red Algae) (Brown Algae)
25 49.82 ± 0.004 d 3.74 ± 0.02 c 2.06 ± 0.03 b 1.27 ± 0.01 a
50 78.43 ± 0.008 d 8.10 ± 0.06 c 6.78 ± 0.03 b 4.11 ± 0.03 a
75 84.31 ± 0.12 d 12.04 ± 0.02 c 9.19 ± 0.04 b 7.20 ± 0.03 a
100 90.72 ± 0.03 d 15.54 ± 0.04 c 11.85 ±0.03 b 10.11 ± 0.03 a
125 93.29 ± 0.01 d 22.12 ± 0.06 c 14.24 ± 0.05 b 12.60 ± 0.04 a
Foods 2023, 12, 3281 11 of 18

Table 5. Cont.

Extracts
Concentration Padina Corallina Hormophysa
µg/mL Ascorbic Acid
pavonica officinalis cuneiformis
Inhibition
(Brown Algae) (Red Algae) (Brown Algae)
150 95.29 ± 0.01 d 26.30 ± 0.01 c 20.75 ± 0.05 b 15.15 ± 0.05 a
175 97.39 ± 0.01 d 29.14 ± 0.01 c 25.66 ± 0.02 b 19.08 ± 0.04 a
200 98.37 ± 0.003 d 33.49 ± 0.07 c 28.42 ± 0.06 b 23.88 ± 0.09 a
225 99.26 ± 0.04 d 38.20 ± 0.08 c 34.18 ± 0.05 b 27.61 ± 0.04 a
250 102.20 ± 0.04 d 46.70 ± 0.07 c 39.70 ± 0.01 b 32.33 ± 0.07 a
275 104.10 ± 0.04 d 60.27 ± 0.02 c 45.10 ± 0.01 b 38.29 ± 0.02 a
300 107.30 ± 0.05 d 63.49 ± 0.01 c 60.08 ± 0.01 b 46.16 ± 0.03 a
IC50 (µ g/mL) 25.09 267.49 305.01 325.23
Data represented are the means of triplicates ± standard error of means, a,b,c,d Data in the same row between
different antioxidant activities followed by different superscript letters significantly differ (p < 0.05).

3.6. Cytotoxicity Effect of Padina Pavonica Extract

Founded on the given data, the cytotoxicity test of P. pavonica against PBMCs demon-
strates that the extract is not completely safe as concentrations increase. The extract revealed
complete inhibition of PBMCs, meaning that it caused cell death at higher concentrations
(10,000 µg/mL gave 0% viability followed by 5000 µg/mL gave 4% viability). The lower
the concentration, the safer extract will be. The IC50 value of 885.8 g/mL indicates that
a relatively high concentration of the extract is required to inhibit PBMC cells by 50%,
as shown in Table 6. However, it is significant to remember that the IC50 value does not
always indicate safety, instead representing the concentration at which the extract can cause
significant cytotoxicity to cells. As a result, based on the information provided, it would be
a bit early to determine that the extract is completely safe to use. More research is needed
to assess its safety and potential cytotoxic impacts on other cell types, as well as in vivo
studies to determine its safety in humans. Based on the results, P. pavonica extract does
not show any genotoxicity effect [27]. A similar study carried out by Kosanić et al. [71]
confirmed that despite the richness of marine macroalgae in the Adriatic Sea, very little is
known about their total phenolic content (TPC) and antioxidant characteristics. Most of
these marine macroalgae have not yet been examined for biological activities, P. pavonica,
from the Adriatic coast of Montenegro, have recently demonstrated that their acetone
extracts exhibit antioxidant, antibacterial, and cytotoxic properties. Our results are also
consistent with the finding that, after 48 h of treatment, no toxicity was seen in any of
the concentrations (0–400 g/mL) of the methanolic and aqueous extract of Padina spp. on
peripheral blood mononuclear cells (PBMCs) [72].

Table 6. Evaluation of the safety and cytotoxicity assay to Padina pavonica extracts on the viability of
PBMC cells.

Concentration (µg/mL) Inhibition % Viability %

10,000 100 0
5000 96 4
2500 81 19
1250 63 37
625 46 54
312 31 69
156 18 82
78 9 91
39 4 96
19.5 1 99
IC50 885.8
Foods 2023, 12, 3281 12 of 18

3.7. Shelf Life of Meat Fortified with Padina pavonica Extract

According to the results, the P. pavonica extract has antibacterial properties against
all the bacteria tested. By increasing concentration, the inhibition increases. Furthermore,
as storage time increased, the extract’s antibacterial effect increased. All concentrations
of the extract show an effect on all strains, besides, treatments 2 and 3 show complete
inhibition in all pathogenic strains by day 10. As shown in Table 7, the extract was most
effective against B. cereus and least effective against Strept. pyogenes. This matched with
Ozogul et al. [73] who found that extracts of P. pavonica showed potential as antioxidant
and antibacterial agents in the sea food sector, extending the shelf life of sea bass fillets by
up to 18 days in comparison to the control (12 days). They attributed that to the presence
of phenolic chemicals, which may be connected to the studied extracts’ antioxidant and
antibacterial activities. Therefore, the plant-based extracts can be used as antioxidant and
antibacterial agents in the seafood sector because they were able to increase the microbi-
ological and oxidative stability of treated sea bass fillets when compared to the control.
Roohinejad et al. [74] also found that seaweeds are a great source of beneficial active ingre-
dients with antibacterial and antioxidant properties. As previously indicated, phenolics,
carotenoids pigments, phlorotannins, and sulfated polysaccharides, to name a few, are
the primary phytochemicals responsible for these advantageous qualities. Seaweeds and
their extracts may be used in meat products to slow down oxidation reactions and micro-
biological growth, according to numerous studies. Gullón et al. [6] studied the seaweeds’
and their extracts’ function in maintaining the quality and preventing the rotting of meat
products focused on the macroalgae species from which the extracts were obtained. They
studies the concentration of the extract or seaweed used, the meat product in which the
extract or seaweed was incorporated, and obtained noticeable results. They also included
analyses of the incorporation of seaweed or seaweed extracts in meat products and their
role in oxidative deterioration.

3.8. Sensory Evaluation of Beef Burger Treated through Padina pavonica Extract
According to the data from four groups as control, beef burger fortified with P. pavonica
extract 1%, 2%, and 3%, the changes in sensory properties are minor, and the general
acceptance scores for all extract concentrations remain high. This suggests that at the tested
concentrations, adding P. pavonica extract to meat may not have a major adverse effect on the
sensory properties of the meat, but it also shows a better result when using 3% compared
with the control. As a result, our algae extract also acts as a food enhancer, as shown in
Table 8. The sensory qualities of the beef burger augmented with P. pavonica extract were
assessed, and the majority of panelists preferred the P. pavonica-fortified version over the
control in terms of texture, taste, odor, color, and overall acceptability.
The overall acceptability of the beef burger strengthened with the P. pavonica ex-
tract treatment 3% was slightly higher (score of 8.10 ± 0.10) than the control non-treated
beef burger (score of 8.05 ± 0.12), while P. pavonica extract treatment 1% and 2% (score
of 7.95 ± 0.14 and 7.95 ± 0.12, respectively) were slightly lower than both control and
treatment 3% groups, even though they were all accepted organoleptically.
Foods 2023, 12, 3281 13 of 18

Table 7. The effect of Padina pavonica extract on shelf-life as an antibacterial effect of different concentrations against pathogenic bacteria experimentally inoculated
into beef meat stored at 4 ◦ C (mean ± SE).

Strains/Conc. Inhibition (CFU/g) Storage (Days)

Extract 0 1 2 3 4 6 8 10
Negative control (1) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Bacillus cereus
Positive control 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107
Treatment 1 g/100 g 1.01 × 107 ± 0.01 Ca 1.29 × 106 ± 0.02 Da 1.10 × 105 ± 0.06 Cb 1.76 × 104 ± 0.03 Ea 1.22 × 104 ± 0.01 Db 4.09 × 103 ± 0.05 Fc 0.41 × 102 ± 0.06 Bb 0.00 ± 0.00 Aa
Treatment 2 g/100 g 1.04 × 107 ± 0.03 Ba 1.31 × 105 ± 0.01 Ca 1.05 × 104 ± 0.02 Bb 5.24 × 103 ± 0.03 Fc 2.51 × 103 ± 0.01 Ec 2.10 × 102 ± 0.01 Db 0.00 ± 0.00 Aa 0.00 ± 0.00 Aa
Treatment 3 g/100 g 1.00 × 107 ± 0.003 Da 3.28 × 104 ±0.09 Eb 0.50 × 104 ± 0.004 Ca 3.20 × 103 ± 0.01 Eb 0.40 × 102 ± 0.01 Ba 0.00 ± 0.00 Aa 0.00 ± 0.00 Aa 0.00 ± 0.00 Aa
Staphylococcus aureus
Positive control 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107
Treatment 1 g/100 g 1.00 × 107 ± 0.003 Da 1.00 × 106 ± 0.003 Da 3.10 × 105 ± 0.01 Fc 2.54 × 104 ± 0.03 Eb 0.61 × 104 ± 0.01 Ca 5.31 × 103 ± 0.05 Gc 0.31 × 104 ± 0.01 Bb 0.00 ± 0.00 Aa
Treatment 2 g/100 g 1.01 × 107 ± 0.01 Ca 3.35 × 105 ± 0.04 Eb 1.20 × 104 ± 0.05 Db 0.45 × 104 ± 0.03 Ba 4.48 × 103 ± 0.08 Gb 3.67 × 102 ± 0.07 Fb 0.00 ± 0.00 Aa 0.00 ± 0.00 Aa
Treatment 3 g/100 g 1.04 × 107 ± 0.04 Da 5.31 × 104 ± 0.07 Fc 0.70 × 104 ± 0.01 Ca 5.09 × 103 ± 0.02 Ec 0.60 × 102 ± 0.01 Ba 0.00 ± 0.00 Aa 0.00 ± 0.00 Aa 0.00 ± 0.00 Aa
Streptococcus pyogenes
Positive control 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107
Treatment 1 g/100 g 1.01 × 107 ± 0.05 Aa 7.35 × 106 ±0.03 Gb 6.43 × 105 ± 0.10 Fc 5.60 × 104 ± 0.08 Eb 3.28 × 104 ± 0.04 Bb 8.52 × 103 ± 0.05 Hc 4.14 × 103 ± 0.03 Dc 3.66 × 102 ± 0.05 Cb
Treatment 2 g/100 g 1.01 × 107 ± 0.01 Ba 5.25 × 105 ± 0.03 Fa 4.25 × 104 ± 0.04 Ea 1.22 × 104 ± 0.02 Ca 7.26 × 103 ± 0.03 Hc 6.64 × 102 ± 0.10 Gb 2.16 × 102 ± 0.04 Db 0.00 ± 0.00 Aa
Treatment 3 g/100 g 1.08 × 107 ± 0.06 Ba 8.60 × 104 ± 0.03 Gc 5.40 × 104 ± 0.01 Eb 6.39 × 104 ± 0.03 Fc 2.54 × 103 ± 0.12 Ca 4.11 × 102 ± 0.06 Da 0.00 ± 0.00 Aa 0.00 ± 0.00 Aa
Salmonella spp.
Positive control 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107
Treatment 1 g/100 g 1.07 × 107 ± 0.07 Aa 8.35 × 106 ± 0.04 Fb 7.56 × 105 ± 0.08 Eb 6.98 × 104 ± 0.07 Db 5.32 × 104 ± 0.11 Ba 9.62 × 103 ± 0.06 Gc 6.45 × 103 ± 0.11 Cc 5.37 × 102 ± 0.05 Bb
Treatment 2 g/100 g 1.07 × 107 ± 0.07 Ba 7.36 × 105 ± 0.07 Fa 6.35 × 104 ± 0.04 Ea 3.62 × 104 ± 0.06 Ca 9.07 × 103 ± 0.09 Hb 8.75 × 102 ± 0.10 Gb 4.28 × 102 ± 0.10 Db 0.00 ± 0.00 Aa
Treatment 3 g/100 g 1.01 × 107 ± 0.01 Ba 9.66 × 104 ± 0.09 Gc 8.11 × 104 ± 0.06 Ec 8.50 × 103 ± 0.03 Fc 5.32 × 103 ± 0.07 Ca 7.52 × 102 ± 0.09 Da 0.00 ± 0.00 Aa 0.00 ± 0.00 Aa
Escherichia coli
Positive control 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107
Treatment 1 g/100 g 1.01 × 107 ± 0.01 Ba 6.29 × 106 ± 0.08 Fb 5.36 × 105 ± 0.17 Ec 4.50 × 104 ± 0.03 Dc 2.64 × 104 ± 0.03 Cb 7.31 × 103 ± 0.06 Gc 0.56 × 103 ± 0.02 Ab 2.24 ± 0.09 Cb
Treatment 2 g/100 g 1.00 × 107 ± 0.003 Da 4.25 × 105 ± 0.04 Fa 3.48 × 104 ± 0.03 Ea 0.61 × 104 ± 0.02 Ba 6.30 × 103 ± 0.03 Hc 5.49 × 102 ± 0.04 Gb 0.75 × 102 ± 0.03 Cc 0.00 ± 0.00 Aa
Treatment 3 g/100 g 1.08 × 107 ± 0.06 Ca 7.50 × 104 ± 0.12 Fc 4.11 × 104 ± 0.05 Eb 3.07 × 103 ± 0.15 Db 0.62 × 103 ± 0.08 Ba 1.14 × 102 ± 0.09 Ca 0.00 ± 0.00 Aa 0.00 ± 0.00 Aa
Klebsiella pneumoniae
Positive control 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107 1 × 107
Treatment 1 g/100 g 1.05 × 107 ± 0.03 Aa 7.67 × 106 ± 0.09 Fb 7.22 × 105 ± 0.07 Eb 6.30 × 104 ± 0.15 Db 4.53 × 104 ± 0.12 Ba 9.31 × 103 ± 0.06 Gc 5.90 × 103 ± 0.05 Cc 4.61 ± 0.15 Bb
Treatment 2 g/100 g 1.00 × 107 ± 0.01 Ba 6.45 × 105 ± 0.03 Fa 5.21 × 104 ± 0.05 Ea 2.49 × 104 ± 0.19 Ca 8.24 × 103 ± 0.09 Gb 8.31 × 102 ± 0.07 Gb 3.53 × 102 ± 0.06 Db 0.00 ± 0.00 Aa
Treatment 3 g/100 g 1.00 × 107 ± 0.00 Ba 9.30 × 104 ± 0.06 Gc 7.30 × 104 ± 0.06 Eb 8.14 × 103 ± 0.03 Fc 4.57 × 103 ± 0.09 Ca 6.50 × 102 ± 0.06 Da 0.00 ± 0.00 Aa 0.00 ± 0.00 Aa
Data represented are the means of triplicates ± standard error of means. Pathogenic bacteria counts are in (Log10 CFU/g). A,B,C,D,E,F,G,H Data in the same column between the same
treatment at different storage periods followed by different superscript letters significantly differ (p < 0.05). a,b,c Data in the same column between different treatments at the same
storage periods followed by different superscript letters significantly differ (p < 0.05).
Foods 2023, 12, 3281 14 of 18

Table 8. Acceptability of un−inoculated beef burger fortified with Padina pavonica extract depending
on sensory attributes.

Sensorial Properties
Mean (SD)
Treatment/Group
Overall
Color Odor Taste Texture Appearance
Acceptability
Control 7.70 ± 0.15 b 7.90 ± 0.12 a 8.00 ± 0.11 a 7.90 ± 0.15 ab 7.85 ± 0.13 a 8.05 ± 0.12 a
Treatment 1% 7.70 ± 0.13 b 7.80 ± 0.15 a 7.90 ± 0.12 a 8.05 ± 0.14 a 7.65 ± 0.13 b 7.95 ± 0.14 a
Treatment 2% 7.80 ± 0.15 a 7.90 ± 0.15 a 7.95 ± 0.17 a 7.95 ± 0.16 a 7.85 ± 0.18 a 7.95 ± 0.12 a
Treatment 3% 7.90 ± 0.19 a 7.90 ± 0.12 a 8.05 ± 0.14 a 8.05 ± 0.14 a 7.80 ± 0.15 a 8.10 ± 0.10 a
Control: Beef burger without any treatment, Treatment (1%): Beef burger treated with P. pavonica extract 1%,
Treatment (2%): Beef burger treated with P. pavonica extract 2%, Treatment (3%): Beef burger treated with P. pavonica
extract 3%, a,b Data in the same column between different treatments followed by different superscript letters
significantly differ (p < 0.05).

When compared to beef burgers supplemented with P. pavonica extract treatments

1%, 2%, and 3%, the texture of the control non-treated beef burger (score of 7.90 ± 0.15)
received a lower score, compared to scores of 8.05 ± 0.14, 7.95 ± 0.16, and 8.05 ± 0.14,
respectively, the superior texture characteristics of the fortified samples may be attributable
to the physicosensory capabilities of P. pavonica extract, resulting in improved features
in the finished product. The color, odor, and appearance of beef burgers fortified with
P. pavonica extract treatments 1%, 2%, and 3%, were nearly similar to those of the control beef
burgers, although the score of fortified groups was still higher compared to the control. A
promising, affordable, and secure method for treating beef burgers to regulate antibacterial,
antioxidant, and ideal sensory evaluation in the finished product might be to fortify it with
P. pavonica extract. This agrees with a study by Gullón et al. [6], who found that they are a
good natural source of nutrients and biocompounds with a wide range of functions. Edible
seaweeds have been suggested to offer intriguing opportunities in the meat industry to
make functional foods.

4. Conclusions
In conclusion, our study on algae extracts of Padina pavonica, Hormophysa cuneiformis,
and Corallina officinalis has demonstrated their antibacterial and antioxidant properties.
Therefore, there is a possibility to use algae extracts as food preservatives in meat products.
All of the studied seaweed extracts showed a wide range of antibacterial activity, thus, the
current study has demonstrated that macroalgae produce antibacterial compounds. Of the
extracts tested, P. pavonica showed the most significant antibacterial and antioxidant effects
against a range of pathogenic strains. Moreover, this extract resulted to be completely
safe for human use, with satisfactory features and no negative impact on the sensory
properties of meat. Consequently, our findings sustain the prospect of P. pavonica and
other algae extracts as natural food preservatives in the meat industry that maintain potent
antimicrobial activities, making them a future promising source of new antimicrobial as
well as preservative agents. This method could provide a viable alternative to traditional
preservatives, which might be safe to both the environment and human health. Therefore,
additional studies are required to fully comprehend the potential of these algae extracts
and their usage in food production.
Foods 2023, 12, 3281 15 of 18

Author Contributions: G.M.H.: Conceptualization: methodology, software, validation, formal anal-

ysis, investigation, resources, data curation, writing—original draft preparation, writing—review
and editing, visualization, supervision, administration. H.S.: software, writing—review and editing,
visualization. T.M.: software, investigation, validation, data curation, formal analysis, visualization,
writing—review and editing. S.A.K.: software, formal analysis, visualization. M.E.: software, formal
analysis, visualization. R.G.T.: software, formal analysis, visualization. G.E.A.E.-R.: software, formal
analysis, visualization. A.M.M.: software, formal analysis, visualization. S.M.S.: software, formal
analysis, visualization, review and editing. A.E.S.: software, formal analysis, visualization, review
and editing. H.E.A.A.: software, formal analysis, visualization, review and editing. E.K.: Software,
validation, formal analysis, investigation, data curation, writing—review and editing, visualization.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Reference number FTD-003-04-2023.
Data Availability Statement: The data presented in this study are available and contained within
the article.
Conflicts of Interest: The authors declare no conflict of interest.

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