pathogens

Article
Estimating the Prevalence of Foodborne Pathogen Campylobacter
jejuni in Chicken and Its Control via Sorghum Extracts
Gamal M. Hamad 1 , Mariam Gerges 2 , Taha Mehany 1, * , Saleh M. Hussein 3 , Michael Eskander 4 ,
Rasha G. Tawfik 5 , Yasser El-Halmouch 6 , Alaa M. Mansour 7 , Elsayed E. Hafez 8 , Tuba Esatbeyoglu 9, *
and Eman M. Elghazaly 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
2 Department of Chemistry, Faculty of Science, Alexandria University, Alexandria 22758, Egypt
3 Department of Food Science and Technology, Faculty of Agriculture, Al-Azhar University, Assiut 71524, Egypt
4 Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Alexandria University,
Alexandria 22758, Egypt
5 Department of Microbiology, Faculty of Veterinary Medicine, Alexandria University, Alexandria 22758, Egypt
6 Department of Botany and Microbiology, Faculty of Science, Kafrelsheikh University,
Kafr Elsheikh 33516, Egypt
7 Department of Animal Hygiene and Zoonoses, Faculty of Veterinary Medicine, Alexandria University,
Alexandria 22758, Egypt
8 Department of Plant Protection and Biomolecular Diagnosis, Arid Lands Cultivation Research
Institute (ALCRI), City of Scientific Research and Technological Applications (SRTA-City),
New Borg El-Arab 21934, Egypt
9 Department of Food Development and Food Quality, Institute of Food Science and Human Nutrition,
Gottfried Wilhelm Leibniz University Hannover, Am Kleinen Felde 30, 30167 Hannover, Germany
10 Department of Microbiology, Faculty of Veterinary Medicine, Matrouh University, Mersa Matruh 51511, Egypt
* Correspondence: tmehany@srtacity.sci.eg (T.M.); esatbeyoglu@lw.uni-hannover.de (T.E.)

Abstract: Campylobacter jejuni is a Gram-negative bacterium which is considered as the most reported
Citation: Hamad, G.M.; Gerges, M.;
cause of foodborne infection, especially for poultry species. The object of this work is to evaluate
Mehany, T.; Hussein, S.M.; Eskander,
M.; Tawfik, R.G.; El-Halmouch, Y.;
the occurrence of C. jejuni in chicken meat as well its control via three types of sorghum extracts
Mansour, A.M.; Hafez, E.E.; (white sorghum (WS), yellow sorghum (YS), and red sorghum (RS)); antibacterial activity, antioxidant
Esatbeyoglu, T.; et al. Estimating the power, and cytotoxicity of sorghum extracts were also assessed. It was found that C. jejuni is very
Prevalence of Foodborne Pathogen abundant in chicken meat, especially breast and thigh. WS extract showed more effectiveness than
Campylobacter jejuni in Chicken and both yellow and red ones. Lyophilized WS extract offered high total phenolic compounds (TPCs) and
Its Control via Sorghum Extracts. total flavonoid compounds (TFCs) of 64.2 ± 0.8 mg gallic acid equivalent (GAE/g) and 33.9 ± 0.4 mg
Pathogens 2023, 12, 958. https:// catechol equivalent (CE)/g, respectively. Concerning the antibacterial and antioxidant activities,
doi.org/10.3390/pathogens12070958
WS showed high and significant antibacterial activity (p < 0.001); hence, WS displayed a minimum
Academic Editors: Csaba Varga and inhibitory concentration (MIC) of 6.25%, and revealed an inhibition zone of 7.8 ± 0.3 mm; it also
Lawrence S. Young showed an IC50 at a concentration of 34.6 µg/mL. In our study, different samples of chicken fillet
were collected and inoculated with pathogenic C. jejuni and stored at 4 ◦ C. Inoculated samples were
Received: 7 June 2023
treated with lyophilized WS extract at (2%, 4%, and 6%), the 2% treatment showed a full reduction
Revised: 2 July 2023
Accepted: 17 July 2023
in C. jejuni on the 10th day, the 4% treatment showed a full reduction in C. jejuni on the 8th day,
Published: 20 July 2023 while the 6% treatment showed a full reduction in C. jejuni on the 6th day. Additionally, 2%, 4%,
and 6% WS extracts were applied on un-inoculated grilled chicken fillet, which enhanced its sensory
attributes. In sum, WS extract is a promising natural preservative for chicken meat with accepted
sensory evaluation results thanks to its high antibacterial and antioxidant potentials.
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland. Keywords: Campylobacter jejuni; foodborne; natural antioxidant; antibacterial activity; natural preser-
This article is an open access article vatives; polyphenols; sorghum extract; chicken fillet
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Pathogens 2023, 12, 958. https://doi.org/10.3390/pathogens12070958 https://www.mdpi.com/journal/pathogens

Pathogens 2023, 12, 958 2 of 13

1. Introduction
Campylobacter jejuni is a Gram-negative, microaerophilic bacterium that is regarded
as a worldwide leading cause of foodborne illness [1]. It is responsible for most bacterial
infections associated with poultry consumption, causing symptoms ranging from mild
diarrhea to severe abdominal cramping and fever [2,3]. Campylobacter infections were
found to cause several diarrheal diseases from 2 to 7 times as regularly as infections with
Escherichia coli, Salmonella species, or Shigella species [4]. The bacterium is expected to be
responsible for over 400 million cases of gastroenteritis annually; most of these cases are in
developing countries [5].
Poultry is considered a primary host for C. jejuni, with up to 90% of chicken and turkey
flocks being colonized with the bacterium. The contamination of poultry meat during
processing is a considerable concern, as it can cause transmission of various bacteria to
humans [6,7]. The occurrence of C. jejuni in poultry is influenced by a range of diverse
factors, including the hygiene of the rearing environment, the use of antibiotics, and the
presence of other microorganisms [8–10]. The high occurrence of Campylobacter species
including C. jejuni that mainly invade poultry species has been reported to be due to
its ability to attack birds’ intestinal tracts’ epithelium and multiply inside it, owing to
their warm-blooded body nature [11,12]. Chicken ceca of age between 14 and 21 days are
colonized by C. jejuni reaching 1 × 109 CFU/g [13]. The ability of the bacterium to form
biofilms also contributes to its persistence in the poultry gut, as these structures provide a
protective environment that can resist antimicrobial treatments [14].
Recently, consumers’ demands regarding healthy and sustainable food have increased
and are currently more critical than before owing to the severe outcomes of epidemics,
climate change, and conflicts [15]. Additionally, there is a serious need for efficient interven-
tions to minimize the occurrence of pathogenic bacteria in poultry species, providing that
C. jejuni contributes most of the pathogenic contamination in poultry species that causes
dangerous public health consequences [16]. Lately, it was found that sorghum, which is a
cereal crop which belongs to the family Graminae, has been found to possess anti-bacterial
properties against a range of pathogenic bacterial species. Sorghum plants have been
identified as potential sources of natural antimicrobial compounds due to the existence of
high levels of phytochemicals, such as tannins and flavonoids [17]. Furthermore, sorghum
extracts have been shown to have a prebiotic effect, promoting the growth of beneficial gut
bacteria while inhibiting the growth of harmful pathogens [18].
In this paper, the extracts of three types of sorghum were employed as antibacterial
agents against C. jejuni, as it has been reported recently that sorghum extract can be used
as a natural alternative to antibiotics in poultry feed to control C. jejuni colonization in the
intestinal tract of broiler chickens, which could ultimately minimize the risk of human
infection [19]. Therefore, the aim of our study is the experimental application of sorghum
extract on chicken fillet inoculated experimentally with C. jejuni, and assessment of its
antibacterial activity, antioxidant activity, minimum inhibitory concentration (MIC), total
phenolic compounds (TPCs), total flavonoid compounds (TFCs), and the cytotoxicity.

2. Materials and Methods

2.1. Growth Conditions of Bacterial Strains
The C. jejuni EMCC 1835 reference strain was brought from Microbial Resource Cen-
ter (MIRCEN), Ain Shams University, Cairo, Egypt. The cell counts were accustomed to
106 CFU/mL as the infective dose is >105 CFU/g [20]. The strains were stored at a tempera-
ture of −80 ◦ C in Brain Heart Broth (Merck, 1.10493.0500, Darmstadt, Germany), including
both glycerol and lysed horse blood (LHB) (Oxoid, SR048C, Hampshire, UK), then they
were sub-cultured at a temperature of 42 ◦ C on Columbia agar base (CAB) (Oxoid, CM0331,
Hampshire, UK) under certain conditions as follow: 5% O2 , 85% N2 , and 10% CO2 .
 including both glycerol and lysed horse blood (LHB) (Oxoid, SR048C, Hampshire, UK),
then they were sub-cultured at a temperature of 42 °C on Columbia agar base (CAB) (Ox-
oid, CM0331, Hampshire, UK) under certain conditions as follow: 5% O2, 85% N2, and 10%
Pathogens 2023, 12, 958 CO2. 3 of 13

2.2. Collection of Chicken Samples and Detection of C. jejuni

2.2. Collection
A hundred of chicken
Chicken Samples and Detection
breast, thigh, of C.
liver, and jejuni samples were grouped and gath-
gizzard
ered from various
A hundred local markets
chicken located
breast, thigh, in Alexandria
liver, and gizzard Governorate,
samples were Egypt, in 2022.
grouped and Dur-
gath-
ing
eredthe collection
from variousprocess, the chicken
local markets locatedmeat that is sold
in Alexandria in pieces was
Governorate, randomly
Egypt, collected
in 2022. During
the collection
from the local process, the chicken
retail stores meat that
at refrigerated is sold in pieces
temperature was randomly
and packaged in bagscollected
made of from
pol-
the local retail
yethylene. stores at refrigerated
Immediately temperature
after collection, and packaged
samples were transferredin bags made
in a box of polyethy-
containing ice
lene.
to theImmediately
laboratory toafter collection,bacteriologically.
be examined samples were transferred in a isolated
C. jejuni was box containing ice to ac-
and applied the
laboratory
cording to be
to the examined
method bacteriologically.
illustrated by El-KhawasC. jejuni
et al.was isolated and applied according to
[21].
the method illustrated by El-Khawas et al. [21].
2.3. Sorghum Materials and Extraction
2.3. Sorghum Materials and Extraction
Three local varieties of sorghum—red, white, and yellow seeds (see Figure 1)—were
Three
obtained andlocal varietiesby
identified of the
sorghum—red, white, Research
Shandawil Sohag and yellow seeds(Sohag
Center (see Figure 1)—were
Governorate,
obtained and identified by the Shandawil Sohag Research Center
Egypt) as follows: Shandaweel 1 (white sorghum), Giza 54 (yellow sorghum), Alsabeinaa (Sohag Governorate,
Egypt)
(red as follows:
sorghum). TheShandaweel 1 (white
collected grains weresorghum),
washed and Giza 54 (yellow
dried, and 10 sorghum),
g were thenAlsabeinaa
extracted
in an ethanolic solution (70% ethanol: deionized water v/v) of up to 100 mL withextracted
(red sorghum). The collected grains were washed and dried, and 10 g were then random
in an ethanolic solution (70% ethanol: deionized water v/v) of up to 100 mL with random
shaking at 100 rpm. After incubation for 2 days, the attained extracts were centrifuged for
shaking at 100 rpm. After incubation for 2 days, the attained extracts were centrifuged for
30 min at 2.147× g, then filtered by using normal filter papers. Finally, extract lyophiliza-
30 min at 2.147× g, then filtered by using normal filter papers. Finally, extract lyophilization
tion took place at −50 °C (Telstar Model 50, Barcelona, Spain) and the resultant extracts
took place at −50 ◦ C (Telstar Model 50, Barcelona, Spain) and the resultant extracts were
were diluted in distilled H2O with specific recognized concentrations in mg/mL [22,23].
diluted in distilled H2 O with specific recognized concentrations in mg/mL [22,23].

Figure 1.
Figure Pictures of
1. Pictures of the
the investigated
investigatedsorghum
sorghumseeds
seedsin
in this
this study:
study: red,
red,yellow,
yellow, and
and white
white sorghum
sorghum
seeds (Sorghum
seeds bicolor L.).
(Sorghum bicolor L.).

2.4. Antibacterial Activity

2.4. Antibacterial Activity
2.4.1. Antibacterial Potential of Sorghum Extract
2.4.1. Antibacterial Potential of Sorghum Extract
The aptitude of sorghum extracts as a microbial agent contrary to C. jejuni EMCC
1835The aptitude
reference of sorghum
strain (preparedextracts as a microbial
in MIRCEN) agent contrary
was measured following tothe
C. jejuni EMCC
procedure of
1835 reference strain (prepared in MIRCEN) was measured following the procedure
Hamad et al. [22] and Klančnik et al. [24]. The overnight C. jejuni cultures were enriched on of
Hamad
Muelleret al. [22]
hinton and (MHM)
media Klančnik(Oxoid,
et al. [24].
UK)The
at 42overnight C. jejuni
◦ C/48 h and then cultures
spread on were
MHM enriched
plates.
on
TheMueller hinton
inhibition zonemedia
was then(MHM) (Oxoid,
recorded (mm),UK) at 42 °C/48
observing h andjejuni
the anti-C. then power
spreadofontheMHM
three
plates. The inhibition zone was then recorded (mm), observing the anti-C.
types of sorghum extracts. Additionally, a comparative study was carried out between the jejuni power of
the three types of sorghum extracts. Additionally, a comparative study was
results of the inhibition zone and those of 3 antibiotic disks which are Erythromycin (ERY), carried out
between
Gentamicinthe (GEN),
results of
and theAmoxicillin
inhibition zone
(AMX) and those of 3 antibiotic disks which are Eryth-
[25].
romycin (ERY), Gentamicin (GEN), and Amoxicillin (AMX) [25].
2.4.2. Assessment MICs of Sorghum Extract against C. jejuni
Sorghum extracts of minimum inhibitory concentrations against C. jejuni EMCC 1835
were evaluated using agar disk diffusion assay [22,25] with the following descending
concentrations of white sorghum extract: 100, 50, 25, 12.5, 6.25, 3.12 µL. C. jejuni spread on
MHM plates of grown Mueller Hinton Medium (MHM) plates and adjusted to a density of
106 CFU/mL [26] and the plates were kept at a temperature of 4 ◦ C for 30 min and then
Pathogens 2023, 12, 958 4 of 13

incubated at 42 ◦ C/24 h. A clear and sharp inhibition zone was noted in mm, taking in
consideration the anti-C. jejuni potential of the different sorghum extracts.

2.5. Phytochemical Analysis of White Sorghum Extract

2.5.1. Total Phenolic Compounds (TPCs) of White Sorghum Extract
The total phenol compounds (TPCs) of sorghum extracts was evaluated by using the
test method described by Hamad et al. [23]. Briefly, approximately 0.1 mL reconstituted
extract was incorporated into a 100 µL Folin–Ciocalteu substance, the mixture then stood
for fifteen min, and then we added 2 mL sodium carbonate (2%). After that, the mixture
was left at ambient temperature for half hour, and gallic acid as a calibration was used in
the evaluation of TPCs by using a spectrophotometer (T80, PG Instrument, Lutterworth,
UK) at 760 nm. TPC is expressed as mg of gallic acid equivalents to each gram per sample.

2.5.2. Total Flavonoid Compounds (TFCs) of White Sorghum Extract

TFCs of sorghum extracts was assessed following the method illustrated by
Hamad et al. [27]. Four milliliters of water was mixed with 1 mL of the examined sample
in a volumetric flask. After that, 0.15 mL of aluminum chloride (10% AlCl3 ) and 0.75 mL
of sodium nitrite (5% NaNO2 ) were added. Finally, and after 5 min, 500 µL of 1 M from
NaOH was added. The TFCs was measured spectrophotometrically at 510 nm.

2.5.3. Diphenyl-1-Picrylhydrazyl (DPPH) Radical Scavenging Assay

The ability of sorghum extracts to scavenge DPPH free radicals was examined [27,28]
by applying some modifications. A stock solution of each extract in methanol to 1 mg/mL
was prepared. Serialized dilutions of plant extracts were carried out by mixing about 1 mL
of every dilution with 1 mL DPPH and then measured at 517 nm spectrophotometrically.
The results were expressed as IC50 (the concentration of the extract that can suppress the
50% DPPH). Then we calculated inhibition % through this equation:

DPPH inhibition % = [(A of control − A of the sample)/A of control] × 100 (1)

where: A: Absorbance.

2.6. Safety and Cytotoxicity Assay of White Sorghum Extract

White sorghum extract was assessed for its impact on the peripheral blood mononu-
clear cell’s viability (PBMCs). Tested wells (150 µL PBMCs), control wells (150 µL PBMCs),
and blank wells (150 µL PBS) were used. Several different concentrations of white sorghum
extract were inoculated for test wells and incubated for one day, according to the approach
of Popiołkiewicz et al. [29]. Using the spectrophotometer, the absorbance was observed at
540 nm; white sorghum extract inhibition % was calculated from the following equation.
IC50 values were calculated through this portal www.aatbio.com/tools/ic50-calculator
(accessed on 22 February 2023).

White sorghum extract inhibition % = 100 − (optical density (O.D) Control − O.D Treatment/O.D Control) (2)

2.7. Experimental Application and Evaluation of the Antimicrobial Power of White Sorghum
Extract against C. jejuni Experimentally Inoculated into Chicken Fillet
2.7.1. Microbes
C. jejuni EMCC 1835 was gained from MIRCEN. Bacterial strain was set, and its
bacterial density was adjusted at a value of 1 × 107 CFU/mL [26,30].

2.7.2. Refrigerated Storage Study of Chicken Breast Fillets

Raw boneless chicken breast fillets were sliced with a sterile knife into 5 cm × 5 cm
pieces. Before doing the experiment, chicken samples were sterilized according to
Hamad et al. [22] and Morsy et al. [31]. Chicken samples were grouped into several
treatments (6 treatments): T1, chicken samples without applying any handlings (negative
Pathogens 2023, 12, 958 5 of 13

control (1)); T2, chicken samples supplemented with white sorghum extract (2%) (negative
control (2)); T3, chicken samples with 1 × 107 CFU/mL C. jejuni (positive control); T4,
chicken samples with C. jejuni and white sorghum extract (2%); T5, chicken samples with
C. jejuni and white sorghum extract (4%); T6, chicken samples with C. jejuni and white
sorghum extract (6%).
Samples were kept for a time of 15 min at room temperature, and further were cooled
◦
at 4 C and observed for C. jejuni occurrence every two days bacteriologically until complete
reduction of the bacterial count. Chicken samples were assessed bacteriologically at various
storage periods (i.e., 0, 2, 4, 6, 8, and 10 days) for determination of C. jejuni amount in the
products [32].

2.7.3. Sensory Evaluation of the Acceptability of Chicken Fillet Fortified with White
Sorghum Extract
To determine the acceptability of white sorghum extract as a food additive, organolep-
tic attributes were evaluated in chicken samples enriched with the sorghum extracts. The
experiment was carried out on four groups, the first group is chicken fillet without any
treatments (control); the other three groups are chicken samples with different concentra-
tions of white sorghum extract of 2%, 4%, and 6%. Ten experienced panelists analyzed the
samples. Panelists checked the degree of acceptability of chicken for the sensorial scores:
texture, appearance, taste, odor, color, and overall acceptability, with a scale ranging from 1
to 10 (10 points/each item), where 10 is more accepted [22,33].

2.8. Statistical Analysis

R software, version 4.2.0 (Lucent Technologies, New Providence, NJ, United States),
was employed for data analyses. One-way analysis of variance (ANOVA) of the means of
three reads (mean ± SE) using Tukey’s test at p < 0.01 or p < 0.05 and regression analysis
was used for continuous independent variables.

3. Results and Discussion

3.1. Occurrence of C. jejuni in Chicken Meat
Chicken meat makes up a high percentage of our diet, which surely leads to human
infection. Unfortunately, the processing step is the main reason various poisoning bacteria
contaminate chicken food products [22]. The occurrence of C. jejuni in chicken fillets can
vary between countries and regions and has been the subject of numerous studies. C. jejuni
affects human health negatively as it causes mild to severe symptoms such as diarrhea,
fever, nausea, and vomiting. Symptoms typically develop 2–5 days after exposure to the
bacteria and can last for up to 10 days [34].
In our study, the collected chicken meat samples showed a high presence of C. jejuni
especially in thigh and breast meat with percentages of 88% and 80%, respectively, as
illustrated in Table 1. These findings are in line with results shown by Walker et al. [35]
who isolated E. coli and C. jejuni from chicken, pork offal, lamb, and beef collected from
retail food outlets and showed a very high incidence of Campylobacter in chicken meat.

Table 1. Incidence of C. jejuni in chicken collected from different local markets (n = 100).

Samples Positive Samples

Types of Chicken Fillet No. of Samples No. %
Breast 25 20 80
Thigh 25 22 88
Liver 25 15 60
Gizzard 25 18 72
Total 100 75 75
 highlights the importance of implementing proper hygiene and sanitation practices to
prevent contamination and the spread of foodborne illness. Additionally, consumers
should be aware of the risks associated with improperly handled and cooked chicken meat
and should follow proper food safety guidelines to minimize the risk of foodborne illness.
Pathogens 2023, 12, 958 6 of 13
Table 1. Incidence of C. jejuni in chicken collected from different local markets (n = 100).

Samples Positive Samples

Types of Chicken
The chicken meat samplesNo. of in this study were collected
Samples No. from suppliers%with poor
Fillet and low sanitation levels for chicken cutting tools to observe the prevalence
hygiene levels
of C. jejuni. In addition, the high
Breast 25incidence of C. jejuni
20found in chicken samples
80 may
be causedThigh
by contamination from25microbes anywhere along 22 the supply chain.88Therefore,
this highlights
Liver the importance of25 implementing proper15 hygiene and sanitation60 practices
to prevent contamination
Gizzard and the
25spread of foodborne illness.
18 Additionally, consumers
72
should beTotal
aware of the risks associated
100 with improperly 75 handled and cooked chicken
75 meat
and should follow proper food safety guidelines to minimize the risk of foodborne illness.
3.2.Antibacterial
3.2. AntibacterialActivity
Activity of
ofLyophilized
LyophilizedSorghum
SorghumExtracts
Extracts
Three types
Three types ofof sorghum
sorghumextracts
extractswere
wereprepared
preparedandand
the the
antibacterial effecteffect
antibacterial on C.on
je-
juni was evaluated using agar disk diffusion assay as well as three types of antibiotics:
C. jejuni was evaluated using agar disk diffusion assay as well as three types of antibiotics:
Gentamicin,Erythromycin,
Gentamicin, Erythromycin,and andAmoxicillin.
Amoxicillin.Then,
Then,results
resultswere
wereobtained;
obtained;they
theyare
areshown
shown
inFigure
in Figure22and
andtabulated
tabulatedin inTable
Table2.2.

Figure2.2.Antibacterial
Figure Antibacterial activity
activity of
of red
red (R.
(R. Sorghum),
Sorghum), white
white (W.
(W. Sorghum),
Sorghum), and
and yellow
yellow sorghum
sorghum (Y.
(Y.
Sorghum) extracts against C. jejuni using agar disk diffusion assay vs. Erythromycin, Gentamicin,
Sorghum) extracts against C. jejuni using agar disk diffusion assay vs. Erythromycin, Gentamicin,
and Amoxicillin antibiotics. Inhibition zones are indicated in mm. (A) Antibacterial impact of three
and Amoxicillin antibiotics. Inhibition zones are indicated in mm. (A) Antibacterial impact of three
sorghum extracts; (B) Antibacterial power of 3 antibiotics compared to sorghum extracts.
sorghum extracts; (B) Antibacterial power of 3 antibiotics compared to sorghum extracts.
Results showed that red sorghum extract has a negative effect on C. jejuni, while the
Table 2. Antibacterial activity of lyophilized sorghum extracts against C. jejuni using agar disk
other sorghum extracts showed significant effects, especially white sorghum which exhib-
diffusion assay.
ited an inhibitory zone of 39.1 ± 0.2 mm, even higher than the three antibiotic types (p <
0.001). WhiteSorghumand yellow sorghum showed extremely high effectsZone
Inhibition compared
Diameter with
Concentration
Piskernik et al. [36], who used rosemary extract. A recent study by Chen (mm)
Extracts/Antibiotics et al. [37] reported
thatWhite
polyphenol
sorghumextract
extractsof sweet sorghum displayed antibacterial activity
100 mg/mL 39.1 ±against
0.2 a Staphy-
lococcus
Yellowaureus,
sorghum Escherichia
extracts coli, Listeria spp., and Salmonella spp. Another
100 mg/mL ± 0.2 c by Garzón
30.1report
et al.Red
[38]sorghum
confirmed extracts 100 mg/mL
that a higher antimicrobial potential of sorghum spent NDgrain could be
Gentamicin (GEN) 30 mg/mL 35.3 ± 0.1 b
Erythromycin (ERY) 100 mg/mL 29.1 ± 0.1 cd
Amoxicillin (AMX) 30 mg/mL 26.4 ± 0.1 e
ND, not detected; Data represented are the means of triplicates ± standard error of means; a, b, c, d, e Mean values
with different superscript letters in the same column are significantly different (p < 0.001); Depending on the in vitro
antibacterial evaluation results, extracts were chosen for further application in chicken fillet against C. jejuni.
 an important natural source of bioactive peptides with antimicrobial activity against Ba-
cillus cereus growth.

Table 2. Antibacterial activity of lyophilized sorghum extracts against C. jejuni using agar disk dif-
Pathogens 2023, 12, 958 7 of 13
fusion assay.

Inhibition Zone Diameter

Sorghum Extracts/Antibiotics Concentration
(mm)
Results showed that red sorghum extract has a negative effect ona C. jejuni, while
White sorghum extracts 100 mg/mL 39.1 ± 0.2
the other sorghum extracts showed significant effects, especially whitec sorghum which
Yellow sorghum extracts 100 mg/mL 30.1 ± 0.2
exhibited an inhibitory zone of 39.1 ± 0.2 mm, even higher than the three antibiotic
Red sorghum extracts 100 mg/mL ND
types (p < 0.001). White and yellow sorghum showed extremely high effects compared
Gentamicin (GEN) 30 mg/mL 35.3 ± 0.1 b
with Piskernik et al. [36], who used rosemary extract. A recent study by Chen et al. [37]
Erythromycin (ERY) 100 mg/mL 29.1 ± 0.1 cd
reported that polyphenol
Amoxicillin (AMX)
extract of sweet sorghum
30 mg/mL
displayed antibacterial
26.4 ± 0.1 e
activity against
Staphylococcus
ND, not detected;aureus, Escherichia
Data represented are coli, Listeria
the means spp., and
of triplicates Salmonella
± standard errorspp. Another
of means; a, b, c, d,report
e by
Garzón et al.with
Mean values [38]different
confirmed that aletters
superscript higher antimicrobial
in the same column potential of sorghum
are significantly different spent
(p < grain
could be an important
0.001); Depending on the in natural source evaluation
vitro antibacterial of bioactive peptides
results, withchosen
extracts were antimicrobial
for further activity
application
against in chicken
Bacillus against C. jejuni.
filletgrowth.
cereus

3.3. Minimal
3.3. Minimal Inhibitory
InhibitoryConcentration
Concentration(MIC) of Sorghum
(MIC) Plant Plant
of Sorghum ExtractExtract
In our
In our study,
study,the
theMIC
MIC ofof
lyophilized
lyophilized white sorghum
white extract
sorghum againstagainst
extract C. jejuniC.
injejuni
vitro in vitro
and the antimicrobial potential at several ratios of white sorghum extracts were examined;
and the antimicrobial potential at several ratios of white sorghum extracts were examined;
the results are shown in Figure 3 and tabulated in Table 3. The results confirmed that the
the results are shown in Figure 3 and tabulated in Table 3. The results confirmed that the
MIC value of white sorghum extract was 6.25% with an inhibition zone of 7.8 ± 0.3 mm.
MIC value of white sorghum extract was 6.25% with an inhibition zone of 7.8 ± 0.3 mm. Ad-
Additionally, the concentration of 3.12% showed negative results and the anti-C. jejuni
ditionally, the concentration
bacterial activity of 3.12%onshowed
increased gradually negative
increasing results
the extract and the anti-C.
concentration jejuni bacterial
percentage
activity increased
(p < 0.001). gradually on increasing the extract concentration percentage (p < 0.001).

Figure 3. Minimum inhibitory concentrations (MICs) of the white sorghum extracts against C. jejuni
Figure 3. Minimum inhibitory concentrations (MICs) of the white sorghum extracts against
(mm).
C. jejuni (mm).

Table 3. Minimum inhibitory concentrations (MICs) of the white sorghum extracts against
C. jejuni (mm).

White Sorghum Extracts (mg/mL)

Conc. (%) Inhibition zone (mm)
100 38.7 ± 0.6 a
50 24.8 ± 0.8 b
Strains/Extract 25 16.1 ± 0.7 c
12.5 11.8 ± 0.8 d
6.25 7.8 ± 0.3 e
3.12 ND
MIC, minimum inhibitory concentration; ND, not detected; Data represented are the means of triplicates ± standard
error of means; a, b, c, d, e Mean values with different superscript letters in the same column are significantly
different (p < 0.001).
Pathogens 2023, 12, 958 8 of 13

3.4. Total Phenolic Compounds (TPCs) and Total Flavonoid Compounds (TFCs) of Lyophilized
Sorghum Extract
Phenolic compounds and flavonoids are important antioxidant and antibacterial
agents in plants that play a significant role in preventing many diseases such as cancer and
promote human health and immunology [39].
According to the results in Table 4, TPC and TFC of white sorghum extract were
64.2 ± 0.8 mg GAE/g and 33.9 ± 0.4 mg CE/g, respectively, so it has the highest antioxi-
dant activity. It showed significant results, followed by yellow sorghum extract then red
sorghum extract. White sorghum extract showed the best results in treating C. jejuni in
chicken fillets.

Table 4. Total phenolic (mg GAE/g) and flavonoid contents (mg CE/g) of sorghum extracts.

Total Phenolic Content Total Flavonoid Content

Extracts
(mg GAE/g) (mg CE/g)
White sorghum extract 64.2 ± 0.8 a 33.9 ± 0.4 a
Yellow sorghum extract 31.6 ± 1.4 b 21.9 ± 0.4 b
Red sorghum extract 15.5 ± 0.5 c 7.20 ± 0.3 c
Data represented are the means of triplicates ± standard error of means; a, b, c Mean values with different
superscript letters in the same column are significantly different (p < 0.001).

TPC and TFC values may differ according to temperature, seasonal exchange, pH,
polarity of used solvents in extraction process, light incidence, water nutrient composition,
and salinity [22,40,41].

3.5. Antioxidant Potential and DPPH Radical Scavenging Ability

The antioxidant power can be estimated accurately via assessment of the DPPH radical
scavenging ability. The antioxidant capacity of lyophilized sorghum extract was estimated
according to the DPPH radical scavenging capacity and compared with a standard which
is ascorbic acid. Results are presented in Table 5; it was observed that the IC50 values
of the lyophilized extracts were 34.62 µg/mL for white sorghum, 51.5 µg/mL for yellow
sorghum, and 65.8 µg/mL for red sorghum, while the IC50 value of ascorbic acid was
20.1 µg/mL. The highest free radical scavenging capacity of the extracts was 99.2% at a
ratio of 100 µg/mL for white sorghum, 97.6% at a concentration of 100 µg/mL for yellow
sorghum, and 89.2% at a concentration of 100 µg/mL for red sorghum. These findings
contradict the findings of Pontieri et al. [42], who found that red sorghum extract has higher
antioxidant activity than white sorghum.
Some sorghum species contain several types of phytochemicals that have the ability
to neutralize free radicals. It has been confirmed that the high antioxidant activity of
sorghum extract gives it a potential health effect as protection against cardiovascular
disease, obesity, dyslipidemia, oxidative stress, and diabetes [42,43], as well as antimicrobial,
anti-inflammatory, and anticancer activity [42,44].

3.6. Safety Assay and Cytotoxicity of White Sorghum Extract

The PBMC cytotoxicity method involves using a number of cells obtained from various
individuals to assess the in vitro cytotoxicity of potential drugs in a high-throughput
manner. Moreover, this assay can offer valuable insights into the response of immune
cells from different donors to the compounds being developed. By utilizing this approach,
researchers can obtain a more comprehensive understanding of the efficacy and safety
of potential drugs [22]. That is why both the safety and the cytotoxicity of lyophilized
white sorghum extract were assessed due to the high concern regarding evaluation of the
safety of new antimicrobials applied on food. The estimated cytotoxic effects of white
sorghum extract on peripheral blood mononuclear cell (PBMC) viability and IC50 are
presented in Table 6 and exhibited a positive correlation with the concentration of sorghum
extract. The concentration ranged from 19.5 µg/mL to 10,000 µg/mL. It was proven
Pathogens 2023, 12, 958 9 of 13

that the lyophilized extract is toxic to PBMCs at several concentrations as the minimum
concentration showed 16% inhibition and 84% viability, while the maximum concentration
showed 100% inhibition and zero viability. Furthermore, white sorghum showed a high
IC50 at 482.4 µg/mL, which allowed its usage as a safe and promising food additive in
chicken meat.

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

White Yellow Red

Ascorbic Acid
Conc. Sorghum Extract Sorghum Extract Sorghum Extract
(µg/mL)
Inhibition IC50 Inhibition IC50 Inhibition IC50 Inhibition IC50
(%) (µg/mL) (%) (µg/mL) (%) (µg/mL) (%) (µg/mL)
10 33.2 (0.0) a 25.6 (0.0) b 11.3 (0.0) c 5.2 (0.0) d
20 49.8 (0.0) a 34.6 (0.0) b 19.2 (0.0) c 12.5 (0.0) d
30 78.4 (0.0) a 43.3 (0.0) b 27.3(0.0) c 18.6(0.0) d
40 84.5 (0.0) a 51.4 (0.0) b 36.3 (0.0) c 28.2 (0.0) d
50 90.7 (0.0) a 20.1 63.6 (0.0) b 34.6 48.5 (0.0) c 51.5 35.3 (0.0) d 65.8
60 93.3 (0.0) a 75.2 (0.0) b 56.9 (0.0) c 45.6 (0.0) d

a b c
70 95.3 (0.0) 92.5 (0.0) 71.2 (0.0) 55.5 (0.0) d
80 97.4 (0.0) a 95.2 (0.0) b 86.4 (0.0) c 69.2 (0.0) d
90 98.4 (0.0) a 97.3 (0.0) ab 95.7 (0.0) c 83.4 (0.0) d
100 99.2 (0.0) a 98.8 (0.0) ab 97.6 (0.0) c 89.2 (0.0) d
Data represented are the means of triplicates ± standard error of means; a, b, c, d Mean values with different
superscript letters in the same row are significantly different (p < 0.001).

Table 6. The safety and cytotoxicity assessment of white sorghum extract on the viability of
PBMC cells.

Concentration (µg/mL) Inhibition % Viability %

10,000 100 0
5000 100 0
2500 97 3
1250 88 12
625 69 31
312 53 47
156 43 57
78 32 68
39 28 72
19.5 16 84
IC50 = 482.4

3.7. Preparation of Chicken Fillets and Their Acceptability after the Fortification with Lyophilized
White Sorghum Extract
Gaining insight into how consumers perceive the safety and quality of chicken meat
can be a valuable resource for public health educators, as it is the most widely consumed
meat [45]. To ensure the safety of chicken meat upon applying the white sorghum extract,
experimentally inoculated chicken fillets with C. jejuni were prepared and treated with
different concentrations of white sorghum extract to determine the antibacterial effect. In
Table 7, the results are presented; they reveal that white sorghum extract has an anti-C.
jejuni effect on chicken fillet meat stored at 4 ◦ C. The 6% treatment showed a high chicken
fillet treatment and a complete reduction of C. jejuni on the 6th day, while the 2% treatment
showed a low chicken fillet treatment and a complete reduction of C. jejuni on the 10th day.
 Pathogens 2023, 12, 958 10 of 13

Table 7. Antibacterial impact of several ratios from white sorghum extract against C. jejuni experi-
mentally inoculated into chicken fillet stored at 4 ◦ C (mean ± SE).

Storage Negative Negative Positive Treatment Treatment Treatment

(Days) Control (1) Control (2) Control (2%) (4%) (6%)
0 0.00 0.00 1 × 107 1 × 107 (0.0) 1 × 107 (0.1) 1 × 107 (0.1)
2nd 0.00 0.00 1 × 107 1.81 × 106 (0.0) 6.21 × 105 (0.0) 4.1 × 104 (0.1)
4th 0.00 0.00 1 × 107 7.21 × 105 (0.0) 2.51 × 104 (0.0) 2.1 × 102 (0.0)
6th 0.00 0.00 1 × 107 3.21 × 104 (0.0) 1.4 × 102 (0.0) 0.00 (0.0)
8th 0.00 0.00 1 × 107 1.8 × 106 (0.0) 0.00 (0.0) 0.00 (0.0)
10th 0.00 0.00 1 × 107 0.00 (0.0) 0.00 (0.0) 0.00 (0.0)
C. jejuni counts are in (Log10 CFU/g); Data represented are the means of triplicates ± standard error of means;
All data have a total significant difference of p < 0.001.

In addition to the above study, sensory attributions of grilled un-inoculated chicken

fillet were assessed upon applying white sorghum extract. Results are tabulated in Figure 4
and showed that white sorghum of 2%, 4%, and 6% improved taste, color, appearance,
odor, and texture of the grilled chicken fillet. In addition to being a good preservative for
chicken fillet due to its high antibacterial activity as shown before, it was reported that
sorghum could increase the nutritional value of food due to its enrichment with proteins,
vitamins including riboflavin, vitamin B6, thiamin, and several minerals including sodium,
potassium, zinc, and iron [46]. It is also reported that sorghum flour has a water-holding
athogens 2023, 12, x FOR PEER REVIEW 11 of 13
capacity and has the ability to lower meat fat content so it could improve the texture of
chicken fillet meat and increase its juiciness [47].

Color
Odor
Taste
10 Texture
Appearance
Overall

8
Sensory score

0
Control T 2% T 4% T 6%
Sample

4. Grilled un-inoculated
Figure un-inoculated
Figure 4. Grilled chicken
chicken fillet’s fillet’s acceptability
acceptability supplemented supplemented with white
with white sorghum ex- sorghum
tract basedextract based on organoleptic
on organoleptic characteristics.
characteristics. Control:
Control: chicken chicken
fillet withoutfillet
anywithout any treatment;
treatment; T 2%: T 2%:
chicken with white
chicken sorghum
with extract 2%;
white sorghum T 4%:
extract 2%; chicken with with
T 4%: chicken whitewhite
sorghum
sorghumextract 4%;4%;
extract T 6%:
T 6%: chicken
chicken with white
with whitesorghum
sorghum extract 6%.
extract 6%.

4. Conclusions
In conclusion, C. jejuni is very abundant in chicken meat, especially breast and thigh.
WS extract showed more effectiveness than both yellow and red ones. WS extract showed
a high antibacterial effect against C. jejuni due to its large TPC and TFC content, in addi-
tion to its high antioxidant activity that could scavenge free radicals. Inoculated samples
were treated with lyophilized WS extract at several ratios; the 2% treatment showed a full
Pathogens 2023, 12, 958 11 of 13

4. Conclusions
In conclusion, C. jejuni is very abundant in chicken meat, especially breast and thigh.
WS extract showed more effectiveness than both yellow and red ones. WS extract showed
a high antibacterial effect against C. jejuni due to its large TPC and TFC content, in addition
to its high antioxidant activity that could scavenge free radicals. Inoculated samples were
treated with lyophilized WS extract at several ratios; the 2% treatment showed a full
reduction of C. jejuni on the 10th day, the 4% treatment showed a full reduction of C. jejuni
on the 8th day, and the 6% treatment showed a full reduction of C. jejuni on the 6th day.
Furthermore, WS extract showed a high acceptance upon application to chicken fillet as
it does not affect negatively its sensory attributes. The current results confirmed that WS
can be used as a promising food preservative because it is safe and is not toxic for humans;
moreover, it increases the nutritional value of food and improves both the texture and the
juiciness of meat.

Author Contributions: Conceptualization, G.M.H.; methodology, G.M.H.; software, T.M., G.M.H.,

M.G., S.M.H., M.E., R.G.T., Y.E.-H., A.M.M., E.E.H. and E.M.E.; validation, T.M., G.M.H., T.E. and
M.G.; formal analysis, T.M., G.M.H., M.G., T.E., S.M.H., M.E., R.G.T., Y.H, A.M.M., E.E.H. and E.M.E.;
investigation, T.M., G.M.H., T.E. and M.G.; resources, G.M.H., M.G.; data curation, T.M., G.M.H. and
M.G.; writing—original draft preparation, G.M.H., M.G. and T.M.; writing—review and editing, T.M.,
G.M.H., T.E. and M.G.; visualization, G.M.H., M.G., T.M., T.E., S.M.H., M.E., R.G.T., Y.E.-H., A.M.M.,
E.E.H. and E.M.E.; supervision, G.M.H.; project administration, G.M.H., M.G., T.M., T.E. and E.E.H.;
All authors have read and agreed to the published version of the manuscript.
Funding: This study received no funding. The open access publication of this article was supported
by the Open Access Fund of Leibniz Universität Hannover.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available upon request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.

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