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Contribution to the Evaluation of Physicochemical Properties, Total Phenolic

Content, Antioxidant Potential, and Antimicrobial Activity of Vinegar
Commercialized in Morocco

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 molecules
Article
Contribution to the Evaluation of Physicochemical Properties,
Total Phenolic Content, Antioxidant Potential, and
Antimicrobial Activity of Vinegar Commercialized in Morocco
Mohammed Kara 1, * , Amine Assouguem 2 , Mohamed El Fadili 3 , Safaâ Benmessaoud 1 ,
Samar Zuhair Alshawwa 4 , Omkulthom Al Kamaly 4, * , Hamza Saghrouchni 5 , Abdou Rachid Zerhouni 1
and Jamila Bahhou 1

1 Laboratory of Biotechnology, Conservation and Valorisation of Natural Resources (LBCVNR), Faculty of

Sciences Dhar El Mehraz, Sidi Mohamed Ben Abdallah University, Fez 30000, Morocco;
[email protected] (S.B.); [email protected] (A.R.Z.);
[email protected] (J.B.)
2 Laboratory of Functional Ecology and Environment, Faculty of Sciences and Technology, Sidi Mohamed Ben
Abdellah University, Fez 30000, Morocco; [email protected]
3 Laboratory of Engineering Materials Modeling and Environmental, Faculty of Sciences Dhar El Mahraz, Sidi
Mohamed Ben Abdellah University, Fez 30000, Morocco; [email protected]
4 Department of Pharmaceutical Sciences, College of Pharmacy, Princess Nourah bint Abdulrahman University,



P.O. Box 84428, Riyadh 11671, Saudi Arabia; [email protected]

 5 Department of Biotechnology, Institute of Natural and Applied Sciences, Çukurova University,
Citation: Kara, M.; Assouguem, A.; Adana 01250, Turkey; [email protected]
Fadili, M.E.; Benmessaoud, S.; * Correspondence: [email protected] (M.K.); [email protected] (O.A.K.)
Alshawwa, S.Z.; Kamaly, O.A.;
Saghrouchni, H.; Zerhouni, A.R.; Abstract: Vinegar is a natural product widely used in food and traditional medicine thanks to
Bahhou, J. Contribution to the its physicochemical properties and its richness in bioactive molecules. However, its direct use by
Evaluation of Physicochemical consumers can have complications and undesirable effects. Therefore, this study contributes to
Properties, Total Phenolic Content,
investigating the physicochemical and biological properties of eleven vinegars marketed in Mo-
Antioxidant Potential, and
rocco. Determination of pH, acetic acid, conductivity, total soluble solids and alcohol content in
Antimicrobial Activity of Vinegar
vinegar was carried out. The polyphenols (TP), flavonoids (TF), and condensed tannins (CT) con-
Commercialized in Morocco.
tent was determined, and their antioxidant activities were evaluated using 2,2-diphenyl-1-picryl
Molecules 2022, 27, 770.
https://doi.org/10.3390/
Hydrazyl (DPPH), Ferric Reducing Antioxidant Power (FRAP) and Phosphomolybdenum Reduction
molecules27030770 Assay (TAC). Then, the antimicrobial activity was studied against four pathogenic bacteria and
two fungal strains, using the disk diffusion and the microdilution method. This study showed a
Academic Editors: Alice Martins and
wide range of acetic acid values from 0.65 ± 0.29 to 5.15 ± 0.20%. The high value of TP, TF, and
Joaquina Pinheiro
CT in our samples V10, V9, and V4 was 655.00 ± 22.2 µgGAE/mL, 244.53 ± 11.32 µgQE/mL and
Received: 21 December 2021 84.63 ± 1.00 µgTAE/mL, respectively. The tested strains showed variable sensitivities to the different
Accepted: 21 January 2022 samples with inhibition zones ranging from 6.33 ± 2.08 to 34.33 ± 0.58 mm. The lowest minimum
Published: 25 January 2022 inhibition concentrations were recorded against Staphylococcus aureus ATCC29213 ranging from
Publisher’s Note: MDPI stays neutral 1.95 to 7.81 µL/mL. While Aspergillus niger ATCC16404 showed resistance against all of the analyzed
with regard to jurisdictional claims in samples. In general, vinegar commercialized in Morocco presents a variable range of products with
published maps and institutional affil- variable properties. Indeed, must take into account this diversity when using it. A future study
iations. is needed to identify the phytochemical composition that will further the comprehension of this
variability and contribute to its valorization.

Keywords: vinegar; polyphenols; fermented fruits; antioxidant activity; antimicrobial activity;

Copyright: © 2022 by the authors.
bioactive molecules
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
1. Introduction
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
Around the world, the use of vinegar is becoming increasingly important. It is used
4.0/). in various fields of application such as the nutritional, medicinal, and pharmaceutical

Molecules 2022, 27, 770. https://doi.org/10.3390/molecules27030770 https://www.mdpi.com/journal/molecules

Molecules 2022, 27, 770 2 of 13

fields. Its richness in biomolecules allows it to be a product of preference. Vinegar is a

condiment that can be found throughout the market [1–4]. It can be made from various
fruits containing carbohydrates and is obtained by alcoholic fermentation and oxidation,
leading to the formation of acetic acid. This production is performed with traditional
or industrial fermentation processes [5]. In the traditional method, the raw material is
fermented with spontaneous microorganisms, taking weeks or months, while the industrial
method is carried out to produce a fast fermentation using a liquid of fruits submerging
the bacterial starter culture [3,6]. The final product of vinegars produced by different
methods differs in its profile. In other words, there are various factors influencing the
physicochemical and phytochemical parameters of the final product of vinegar, such as
the raw material, production methods, temperature, pH and microorganisms involved in
the fermentation process [7–9]. In fact, this variability influences its pharmaceutical and
medicinal effects [3].
Several research studies have reported that the effect of vinegar on the human body
is related to its phytochemical composition and its concentration [4], which provides ex-
cellent biological activity. According to Ozturk et al. [6], almost all of the traditional and
industrial vinegar samples showed antibacterial activity at varying levels. The vinegar
was shown to be effective as an antimicrobial agent against several foodborne agents and
pathogenic strains such as Klebsiella pneumoniae, which causes community-onset infections
and Escherichia coli, Bacillus cereus, Salmonella typhi, Pseudomonas aeruginosa and Staphylococ-
cus aureus responsible for diarrhea [10–14]. The antifungal activity of vinegar was tested
against various fungal strains of Aspergillus, Fusarium, and Candida [15,16]. Indeed, the
acetic acid content in vinegar confers it an activity which can be used for the treatment
of various infections even at low concentrations [3,17]. In addition, vinegar was used
recently in certain dietary behaviors to prevent non-severe and severe acute respiratory
syndrome (SARS) caused by a coronavirus (SARS-CoV) or as an effective disinfecting agent
to stop its transmission. Several studies reported that acetic acid content was responsible
for this antivirus effect by its application alone or combined (at 0.34% concentration) with
hydroxychloroquine [18–20]. The polyphenol content in vinegar is known for its consid-
erable biological effect on the human body as an antioxidant agent involved in several
biological processes [4,21]. The major phenolic compounds that can be found in vinegar
are phenolic acids, flavonoids, tannins, anthocyanins, and stilbenoids [22–24]. In addition,
vinegar is rich in mineral and volatile compounds [6]. These properties of vinegar mean it
experiences remarkably widespread consumption in the whole world. This leads us to pay
particular attention to the quality of the vinegar produced and to question the products
available on the market. Therefore, the present study aimed to investigate the variation
of physicochemical parameters in different kinds of vinegar commercialized in Morocco.
Meanwhile, we characterized the total phenolic content, total flavonoid, and condensed
tannins and their antioxidant and antimicrobial activities. This study is considered the
first to be conducted in Morocco and would be helpful to characterize different kinds of
Moroccan vinegar in order of its valorization in the future.
2. Results and Discussion
2.1. Physicochemical Analysis
The results of the physicochemical analysis of vinegar commercialized in Morocco
are shown in Table 1. The pH measurement showed variable values ranging between
2.37 ± 0.09 and 4.47 ± 0.08 for V4 and V8, respectively. In general, pH values of tradi-
tionally produced vinegar were higher than the industrially produced ones, and many
other samples. These results are in line with previous studies established by [25–27]. The
TSS values were between 1.03 ± 0.18 ◦ Brix and 8.67 ± 0.12 ◦ Brix. The V9 and V8 values
represent higher conductivity values in the order of 6.19 ± 0.29 and 5.67 ± 0.50 µS/cm,
respectively. Compared to the other research, these values are similar to those reported
by [14,28–30]. The acetic acid contents in vinegar V2 (5.15%), V9 (4.98%), V1 and V5 (3.75%)
are higher than those of the other samples. The alcohol content in the vinegar samples of
Molecules 2022, 27, 770 3 of 13

this research ranged from 0.03 ± 0.02 to 1.00 ± 0.00% for V2 and V3, respectively. Except
for V2, these values were not in conformity with the standard which must be less than 0.1%
for residual alcohol content and more than 5% for the degree of acidity [31].
2.2. Determination of Total Phenolic, Total Flavonoids, and Condensed Tannins Content
Polyphenols and flavonoids are the main bioactive compounds in vinegar that can
be obtained from various raw materials, which are responsible for several positive ef-
fects on health [32,33]. Table 1 summarizes the results of the phenolic compounds in
the different types of vinegar studied. It was demonstrated that V10, V7, V9 are very
rich in total polyphenols (655.00 ± 22.2, 577.89 ± 13.47 and 521.22 ± 12.73 µg GAE/mL,
respectively) compared to the other samples. The lowest value was 6.22 µg GAE/mL,
which was recorded in V6. The flavonoid content range between 18.67 ± 4.56 and
244.53 ± 11.32 µg QE/mL in all samples except V6 and V11, which do not show any traces
of flavonoids. The highest concentration of Flavones and Flavonols recorded in our vinegar
samples was 225.20 ± 17.6 µg QE/mL for V9, followed in descending order by V7 with
114.72 ± 11.16 µg QE/mL, while the lowest values were 2.33 ± 0.58 µg QE/mL and
3.67 ± 3.50 µg QE/mL for V2 and V3, respectively. These results are in line with pre-
vious research that reported that TPC and TFC in apple vinegar are lower than in most
fruit vinegar [6,12,14,34]. Sengun et al. reported that the TPC in grape and apple vine-
gar was 1025 ± 2.83 and 988 ± 2.83 mg GAE/L, and the TFC was 221.81 ± 3.43 and
174.79 ± 3.40 mg Catechin/L, respectively [14]. Yun et al. found that TPC and TFC con-
tained in peach were 437.6 ± 29.8 mg/L and 30.3 ± 2.1 mg/L, respectively [35]. Moreover,
the total phenolic content (TPC) and total flavonoid content (TFC) of traditional vinegar are
significantly higher than those of industrial vinegars [6,12]. In the case of condensed tannins,
the concentration varies in all the studied samples between 84.63 ± 1.00 µg TAE/mL and
0.69 ± 0.53 µg TAE/mL. V6 record the lowest concentration, while V4 has a very high
concentration compared to the other samples. According to the scientific literature, pro-
cyanidins and condensed tannins are the major substances of most apple varieties [36].
In general, the phytochemical compound in vinegar varies widely depending on the raw
material used in its production [6] and on the strain of yeast and acetic acid bacteria
involved [9].
2.3. In Vitro Antioxidant Activity
The antioxidant activity of the different types of vinegar studied in this research
was evaluated using three complementary tests: DPPH, FRAP, and TAC assay. The re-
sults are shown in Figure 1. It is shown in these results (Figure 1A) that V10, V3, V2,
V9, and V8 have a lower DPPH IC50 value (6.441 µg/mL, 6.581 µg/mL, 10.508 µg/mL,
12.852 µg/mL, and 13.465 µg/mL, respectively), while 50% inhibition for V6 and V11 was
not attained. According to the results found by [35] on various fruit vinegars, the peach vine-
gar and the grape vinegar record a higher value of antioxidant activity using DPPH assay
(94.7 ± 0.7% and 82.4 ± 1.5%, respectively) in comparison with apple vinegar which was
between 61.2 ± 0.4% and 72.2 ± 1.4% [35]. In addition, Ozturk et al. reported several
ranges of DPPH values, which ranged from 4.93% to 89.53% for grape vinegar, while they
ranged from 0.53% to 65.12% for apple vinegar [6]. In addition, the antioxidant prop-
erty of fruit vinegar, including traditional balsamic vinegar and peach vinegar, was over
90% [35]. The antioxidant power based on the FRAP assay indicated significant outcomes
between different samples (Figure 1B). The IC50 values of V8, V7, and V2 were much lower
(8.369 µg/mL and 19.072 µg/mL, respectively) than V1, V4, V5, V9, and V10 while the
highest IC50% value was recorded in the V10 sample (461.536 µg/mL). According to [12],
the FRAP values of apple vinegar were lower than those of grape vinegar. As shown in
Figure 1C, among the tested vinegar samples, V1 and V8 were found to exhibit the highest
total antioxidant capacities with the values of 134.068 and 119.903 µg AAE/mL, respectively.
While the lowest values were recorded in V11 (31.814 µg AAE/mL). In a related study, [35]
recorded that, in peach vinegar, the IC50 was (13.1 ± 0.6 µg/mL). Several studies have
 µg/mL and 19.072 µg/mL, respectively) than V1, V4, V5, V9, and V10 while the highest
IC50% value was recorded in the V10 sample (461.536 µg/mL). According to [12], the
FRAP values of apple vinegar were lower than those of grape vinegar. As shown in Figure
1C, among the tested vinegar samples, V1 and V8 were found to exhibit the highest total
Molecules 2022, 27, 770 antioxidant capacities with the values of 134.068 and 119.903 µg AAE/mL, respectively. 4 of 13

While the lowest values were recorded in V11 (31.814 µg AAE/mL). In a related study,
[35] recorded that, in peach vinegar, the IC50 was (13.1 ± 0.6 µg/mL). Several studies have
reported that
reported thatthe
thedifference in in
difference antioxidant capacity
antioxidant is due
capacity is to
duediffering their phytochemical
to differing their phytochemical
profiles and initial raw materials [14].
profiles and initial raw materials [14].

Molecules 2022, 27, x FOR PEER REVIEW 5 of 13

Figure1.
Figure Antioxidant activity
1.Antioxidant activityofofdifferent vinegar
different samples
vinegar tested
samples by 2,2-diphenyl-1-picryl
tested hydroxyl
by 2,2-diphenyl-1-picryl hydroxyl
radical scavenging assay (A), ferric reducing antioxidant power assay (B), and phosphomolybdenum
radical scavenging assay (A), ferric reducing antioxidant power assay (B), and phosphomolyb-
reduction
denum assay (C).
reduction ND (C).
assay = 50% NDinhibition not determined.
= 50% inhibition Values that Values
not determined. do not share thenot
that do same letter
share the same
are significantly
letter different.
are significantly different.

2.4. Antimicrobial Analysis

2.4.1. Disk Diffusion Assay
Molecules 2022, 27, 770 5 of 13

2.4. Antimicrobial Analysis

2.4.1. Disk Diffusion Assay
The antimicrobial activities of the vinegar samples were tested against bacterial and
fungal strains using the disc diffusion method, and the outcomes are shown in Table 2.
The sensitivity of the bacteria to the various vinegar samples was highly variable. The
highest value of inhibition was recorded against K. pneumonia (ESBL-KP) in the range of
8.67 ± 1.15 mm and 32.67 ± 2.52 mm for V11 and V5, respectively. The V11 and V10
revealed a higher antibacterial activity (25.67 ± 0.58 and 24.67 ± 0.58 mm) against the
E. coli ATCC strain followed by V9, V3, and V2 with DIZ of 18.67 ± 2.31, 18.33 ± 1.53 and
18.00 ± 0.00 mm, respectively., while the lowest activity was 7.33 ± 0.58 and
6.67 ± 0.58 mm for V4 and V6, respectively. Except for sample V11, we can note that
all samples have antibacterial activity against E. coli CIP ranging between the lowest value
of 6.33 ± 2.06 mm and the highest of 25.33 ± 0.58 mm for V6 and V10, respectively. An-
tibacterial activity against S. aureus was shown to be the lowest in this study with DIZs
ranging from 9.00 ± 1.73 mm to 16.33 ± 1.15 mm for V4 and V9, respectively. Concerning
A. niger strains, we can clearly note its resistance to all the vinegar samples used in this study,
while the antimicrobial activity against C. albicans yeast ranged between 8.00 ± 0.00 and
34.33 ± 0.58 mm for V7 and V1, respectively. Generally, the peach, grape, and apple vinegar
purchased from cooperatives and ACV obtained from the local market had a stronger
antibacterial activity. Several studies showed that apple vinegar had low antimicrobial
activity against almost all strains of microorganisms [16,37,38]. The antimicrobial activity
of each vinegar sample is strongly correlated with their phytochemical compounds [4,39].
Indeed, the qualitative and quantitative difference of organic acids, polyphenols and pri-
mary metabolites contained in each kind of vinegar are the main factors responsible for
the variation of the antimicrobial activity [40,41]. The presence of bioactive compounds,
such as Gallic acid, Epicatechin-3-gallate, Caffeic acid, Catechins, amino acids and acetic
acid, in the vinegar can inhibit the bacteria strains at low concentration such as S. aureus,
S. mutans, E. coli O157:H7 and P. aeruginosa [40,41]. The previous literature showed that
polyphenols contained in the apple vinegar is generally lower than that found in the other
fruits’ vinegar [42,43].

2.4.2. Determination of the Minimum Inhibitory Concentration (MIC)

The results of the antimicrobial activity of the samples obtained through the microdi-
lution method were presented in Table 2. The Minimum Inhibitory Concentration (MIC)
recorded in this study was in the range of 1.95 µL/mL and 500 µL/mL. The MIC values
recorded against E. coli ATCC were 1.95 µL/mL for (V6 and V9), 3.9 µL/mL for V8, and
7.81 µL/mL for the other samples. The lowest antimicrobial activity recorded against
E. coli CIP was 62.5 µL/mL by V7 sample and the highest was 1.95 µL/mL. The antimicro-
bial activity of all samples against S. aureus ranged between 1.95 µL/mL and 7.81 µL/mL.
Generally, the lowest antimicrobial activity in this study was recorded against the yeast
C. albicans which falls within the range of 31.25 µL/mL and 500 µL/mL for V6 and V5,
respectively, and the highest activity was against S. aureus with MIC of 1.95 µL/mL. Ac-
cording to Yagnik et al., the MIC for C. Albicans was required at 1/2 ACV and for S. aureus
was 1/25 dilution ACV was required, whereas for E. coli cultures the value was 1/50 ACV
dilution [16]. As reported by Sengun et al., apple vinegar had lower antimicrobial activity
(MIC=12.5–25%, v/v) than that recorded in grape vinegar (MIC=3.12–6.25%, v/v) against
all studied bacterial strains. In general, apple vinegar had higher inhibitory concentrations
than grape vinegar [14].
Molecules 2022, 27, 770 6 of 13

Table 1. Physicochemical properties and bioactive compounds of different vinegar samples.

Flavones et
Alcohol Conductivity TPC
Samples ID Acetic Acid (%) pH TSS (◦ Brix) TFC µgQE/mL Flavonols CTC µgTAE/mL
Content (%) (µS/cm) µgGAE/mL
µgQE/mL
V1 3.75 b ± 0.14 3.33 b ± 0.14 5.30 de ± 0.14 0.50 bc ± 0.10 3.78 b ± 0.25 278.40 e ± 21.4 131.09 c ± 4.06 67.18 d ± 8.84 44.12 d ± 0.74
V2 5.15 a ± 0.20 2.60 de ± 0.10 4.96 e ± 0.10 0.03 d ± 0.02 3.41 bc ± 0.16 480.67 c ± 16.91 21.56d de ± 5.77 2.33 e ± 0.58 53.91 b ± 1.36
V3 2.82 c ± 0.11 2.70 dce ± 0.11 5.23 e ± 0.11 1.00 a ± 0.00 2.82 cd ± 0.07 34.56 gh ± 5.85 18.67 de ± 4.56 3.67 e ± 3.50 27.21 e ± 1.73
V4 1.90 d ± 0.09 2.37 e ± 0.09 5.47 de ± 0.09 0.93 a ± 0.09 2.92 cd ± 0.45 299.00 e ± 5.00 43.349 d ± 1.550 9.810 e ± 4.72 84.63 a ± 1.00
V5 3.75 b ± 0.15 2.77 cd ± 0.15 7.87 b ± 0.15 0.50 bc ± 0.15 3.05 bcd ± 0.08 117.33 f ± 8.33 37.49 d ± 12.81 15.25 e ± 4.09 55.68 b ± 0.34
V6 1.02 e ± 0.18 2.63 de ± 0.18 1.03 f ± 0.18 0.50 bc ± 0.18 ND 6.22 h ± 4.81 ND ND 0.69 f ± 0.53
V7 1.05 e ± 0.03 3.07 bc ± 0.03 6.03d ± 0.03 0.10 d ± 0.03 2.47 d ± 0.21 577.89 b ± 13.47 131.79 c ± 4.01 114.72 b ± 11.16 46.72 cd ± 2.37
V8 2.15 d ± 0.08 4.47 a ± 0.08 7.23 bc ± 0.08 0.90 a ± 0.08 5.67 a ± 0.50 395.10 d ± 29.6 194.37 b ± 16.78 89.81 c ± 2.65 45.72 cd ± 1.36
V9 4.96 a ± 0.50 2.70 cde ± 0.20 7.07 c ± 0.20 0.50 bc ± 0.20 6.19 a ± 0.29 521.22 c ± 12.73 244.53 a ± 11.32 225.20 a ± 17.6 82.18 c ± 1.49
V10 1.80 d ± 1.00 2.57 de ± 0.30 8.67 a ± 0.12 0.73 ab ± 0.58 2.58 d ± 0.32 655.00 a ± 22.2 105.07 b ± 21.33 47.81 d ± 3.31 48.80 c ± 1.20
V11 0.65 e ± 0.29 2.80 cd ± 0.09 1.17 f ± 0.20 0.27 cd ± 0.29 ND 82.00 fg ± 34.67 ND ND 2.85 f ± 0.86
Values in the same column with different superscripts are significantly different (p < 0.05). ND: not determined.

Table 2. Antimicrobial activity of the different vinegars against various pathogens.

E. coli ATCC E. coli CIP S. aureus ATCC K. pneumonia ATCC C. albicans ATCC A. niger ATCC
Samples ID
DIZ MIC DIZ MIC DIZ MIC DIZ MIC DIZ MIC DIZ MIC
bc b bcd cd a
34.33 ± 0.58
V1 17.67 ± 2.52 7.81 17.67 ± 0.58 15.62 11.67 ± 2.89 1.95 20.67 ± 4.04 3.91 62.5 Rs Rs
V2 18.00 b ± 0.00 7.81 12.67 bc ± 2.52 3.91 14.33 abc ± 0.58 1.95 29.67 ab ± 0.58 3.91 26.67 bc ± 2.89 125 Rs Rs
V3 18.33 b ± 1.53 7.81 17.67 b ± 0.58 7.81 16.00 ab ± 1.73 1.95 24.67 bc ± 1.53 31.25 26.67 bc ± 2.89 250 Rs Rs
V4 7.33 f ± 0.58 7.81 7.00 cd ± 0.00 15.62 9.00 d ± 1.73 3.91 26.00 bc ± 1.00 15.63 23.33 c ± 2.89 62.5 Rs Rs
V5 13.00 de ± 1.00 7.81 16.67 b ± 1.53 3.91 14.00 abc ± 1.00 1.95 32.67 a ± 2.52 31.25 29.67 ab ± 0.58 500 Rs Rs
V6 6.67 f ± 0.58 1.95 6.33 d ± 2.08 15.62 10.00 cd ± 0.00 3.91 17.67 d ± 2.52 15.63 23.33 c ± 2.89 31.25 Rs Rs
V7 8.67 ef ± 2.31 7.81 7.00 cd ± 0.00 62.5 Rs Rs 10.33 e ± 0.58 250 8.00 e ± 0.00 250 Rs Rs
V8 13.33 cd ± 2.89 3.9 12.67 bc ± 6.43 15.62 12.67 abcd ± 2.31 7.81 31.67 a ± 1.53 7.81 15.67 d ± 1.15 62.5 Rs Rs
V9 18.67 b ± 1.15 1.95 15.00 b ± 0.00 1.95 16.33 a ± 1.15 1.95 29.00 ab ± 1.00 3.91 30.00 ab ± 0.00 62.5 Rs Rs
V10 24.67 a ± 0.58 3.9 25.33 a ± 0.58 3.9 14.00 abc ± 1.00 1.95 22.67 cd ± 0.58 3.91 Rs Rs Rs Rs
V11 25.67 a ± 0.58 3.9 Rs Rs 11.00 cd ± 1.00 1.95 8.67 e ± 1.15 31.25 Rs Rs Rs Rs
Voriconazole * - - - - - - - - - - 12 0.5
Fluconazole * - - - - - - - - 21 0.4 - -
Ampicilline * Rs Rs Rs Rs Rs Rs Rs Rs - - - -
Streptomycine * Rs 0.25 Rs 0.5 9 Rs Rs 0.003 - - - -
Values in the same column with different superscripts letters are significantly different (p < 0.05). DIZ: diameter of inhibition zone (mm); MIC: minimum inhibitory concentration
(µL/mL); * MIC expressed in mg/mL; Rs: resistant.
 cipal components represent 73.1% of the variation in the data. The first principal compo-
nent shows a strong positive correlation with acetic acid, Brix, conductivity, and DIZ of
K. pneumonia. The second component shows a positive correlation with DIZ of S. aureus,
and a negative association with MIC of E. coli CIP and MIC of K. pneumonia. While the
third component is correlated positively with flavonoids and flavonols/flavanone, and
Molecules 2022, 27, 770 7 of 13
negatively with MIC of E. coli ATCC and C. albicans. The fourth one shows a negative
correlation with polyphenols, and positively correlated with pH and MIC of S. aureus. In
general, the variables are distributed on the different sides of the axis. However, the pro-
2.4.3. Principal Component Analysis of Various Studied Parameters
jection of the scoring diagram and the contribution diagram visually shows a positive
Results of
contribution of physicochemical
PCA obtained in properties
Figure 2 show thatfirst
on the the main
eigenvalues
axis in of the first
positive four prin-
correlation
cipal components represent 73.1% of the variation in the data. The first principal compo-
with V1, V2, V5, V8, and V9, while V6 and V11 were correlated negatively. The second
nent shows a strong positive correlation with acetic acid, Brix, conductivity, and DIZ of
component measures some of the antimicrobial activity of V3, V7, and V10.
K. pneumonia. The second component shows a positive correlation with DIZ of S. aureus,
We could conclude, based on these data, that the vinegar produced by the coopera-
and a negative association with MIC of E. coli CIP and MIC of K. pneumonia. While the
tives and those obtained from peach, quince, and grapes have important physicochemical
third component is correlated positively with flavonoids and flavonols/flavanone, and
and biological properties. The samples obtained from the herbalists show variable char-
negatively with MIC of E. coli ATCC and C. albicans. The fourth one shows a negative
acteristics. In general, the vinegar marketed in Morocco presents a variable range of prod-
correlation with polyphenols, and positively correlated with pH and MIC of S. aureus.
ucts with variable properties. According to several studies, the therapeutic effects of vin-
In general, the variables are distributed on the different sides of the axis. However, the
egar are highly due to its bioactive compounds content [44–49]. Apple vinegar is highly
projection of the scoring diagram and the contribution diagram visually shows a positive
rich in organic acids, phenolic acids, tannins, flavonoids, and carotenoids, which confer it
contribution of physicochemical properties on the first main axis in positive correlation
with a high level of antioxidants and antibacterial properties. For a reasonable application
with V1, V2, V5, V8, and V9, while V6 and V11 were correlated negatively. The second
of vinegar, its
component use by consumers
measures some of themust take into account
antimicrobial this
activity of V3,diversity
V7, andof product charac-
V10.
teristics.

Figure 2. Principal component analysis (PCA) of the various commercialized vinegar samples using
the assessed parameters.

We could conclude, based on these data, that the vinegar produced by the cooperatives
and those obtained from peach, quince, and grapes have important physicochemical and bi-
ological properties. The samples obtained from the herbalists show variable characteristics.
In general, the vinegar marketed in Morocco presents a variable range of products with vari-
able properties. According to several studies, the therapeutic effects of vinegar are highly
due to its bioactive compounds content [44–49]. Apple vinegar is highly rich in organic
acids, phenolic acids, tannins, flavonoids, and carotenoids, which confer it with a high level
of antioxidants and antibacterial properties. For a reasonable application of vinegar, its use
by consumers must take into account this diversity of product characteristics.

3. Materials and Methods

3.1. Raw Material
Eleven commercial kinds of vinegar were used in this study. Ten of these were
traditionally prepared from apple fruits (V1, V3, V4, V5, V6, V10, and V11), quince (V7),
peach (V8), and grape (V9), and one sample was industrially prepared from apple cider
(V2). The V2 and V9 samples were purchased from the local supermarket of Fez, Morocco,
while V3 and V5 were obtained from vinegar-producing cooperatives located in Imouzzer
Kander, Morocco, and V10 from cooperative located in Sefrou, Morocco. The other samples
(V1, V4, V6, V7, V8, and V11) were purchased from Fez herbalists. All samples were
centrifuged to reduce the turbidity and were stored at 4 ◦ C for later use.
Molecules 2022, 27, 770 8 of 13

3.2. Physicochemical Analysis

The percentage of acetic acid in the samples was calculated using NaOH (0.1 mol/L) [50].
The pH value was determined using a previously calibrated pH Meter SELECTA pH-2005
(SOMESTIM, Rabat, Morocco). Conductivity (µS/cm) was measured by direct reading
using a previously calibrated conductivity meter SELECTA CD 2005 (Lab Associates,
Oudenbosch, Netherlands). A handheld refractometer with Automatic Temperature Com-
pensation (ATC) Model ATAGO Pocket Refractometer (ATOGO, Fujian, China) was used
to measure the total soluble solids (TSS) (◦ Brix) and alcohol content (%) of the samples [51].

3.3. Determination of Total Phenolic Content (TPC)

The TPC of the samples was determined using the method described by [52,53]. Briefly,
0.1 mL of Folin–Ciocalteu reagent (25%) is mixed with 0.1 mL of vinegar. The mixture is
stirred vigorously and 2 mL of 2% sodium carbonate was added. After 30 min of incubation
at room temperature, spectrophotometry measurement (SOMESTIM, Rabat, Morocco) was
applied to the set at 760 nm. The results are expressed as µg GAE/mL of vinegar sample
using standard concentration curve y = 0.0006x + 0.0936, R2 = 0.94.

3.4. Determination of Total Flavonoids Content (TFC)

Total flavonoids content was determined by the methods previously described by [54].
In a test tube, 1 mL of vinegar and 1 mL of AlCl3 (2%) methanol solution were mixed.
The absorbance of the set was measured by a UV-visible spectrophotometer UV-1600PC
(VWR, Fontenay-sous-Bois, France) at 430 nm after 15 min of incubation. Ethanol and AlCl3
solution served as a control. All experiments were conducted in triplicate. A quercetin
calibration curve (y = 0.0149x + 0.0528, R2 = 0.99) was used to determine flavonoid concen-
tration as micrograms of Quercetin Equivalent per mL of vinegar (µg QE/mL).

3.5. Determination of Flavones/Flavonols Content

Flavone and Flavonol contents of the samples were determined by mixing 0.5 mL
of vinegar with 1.5 mL of ethanol, 0.1 mL of AlCl3 (10%) methanol solution, 0.1 mL of
(CH3 COO, Na), and 2.8 mL of distilled water in a test tube. The set was incubated for
30 min at room temperature. Absorbance of the set was measured at 415 nm using a UV-
visible spectrophotometer UV-1600PC (VWR, Fontenay-sous-Bois, France). Our samples’
concentration of flavones and flavonols was calculated using the calibration curve obtained
using Quercetin as the standard (y = 0.0019x + 0.0447, R2 = 0.98). All operations were
repeated three times [55].

3.6. Determination of Condensed Tannins Content (CTC)

Calorimetric testing as described by [56] was used to determine the tannin content of
the samples. Briefly, 100 µL of the sample was mixed with 500 µL of Folin–Ciocalteu and
1 mL of sodium carbonate (7.5%). The absorbance was measured at 760 nm after 30 min
of incubation. The values of condensed tannins of vinegar were expressed as micrograms
of Tannic Acid Equivalent per mL of vinegar (µg TAE/mL of vinegar) using standard
concentration curve y = 0.0051x + 0.0289, R2 = 0.99.

3.7. The 2,2-Diphenyl-1-picryl Hydrazyl Radical Scavenging Activity of Vinegar

The antioxidant activity of the samples was determined using DPPH assay as described
by [57]. This method involves mixing 100 µL of each methanol solution of the tested vinegar
samples with 750 µL DPPH in methanol (0.004%). Its optical density is then determined at
517 nm after incubation at laboratory temperature for 30 min. Methanol serves as a blank
control. The following equation was used to determine the percentage inhibition of DPPH:

PI (%) = (1 − (Ac /As ))∗100 (1)

Molecules 2022, 27, 770 9 of 13

where Ac = the absorbance of the control sample and As = the absorbance of the tested
sample. The half-maximal inhibitory concentration (IC50) was determined graphically.

3.8. Ferric Reducing Antioxidant Power of Vinegar

This test was performed using the method described by [58]; briefly, 500 µL of phos-
phate buffer (0.2 M; pH = 6.6) and 500 µL of potassium ferricyanide (1%) were added
to 100 µL of the sample at different concentrations prepared in methanol. After 20 min
incubation at 50 ◦ C in a water bath, 500 µL of aqueous TCA (10%) solution, 100 µL FeCl3
(0.1%), and 0.5 mL of distilled water were added to the reaction medium. The absorbance
of the resulting preparation was determined by the colorimetric method using UV-visible-
spectrophotometer UV-1600PC (VWR, France) at 700 nm. The tubes containing all the
reagents except samples were used as a blank test. Determination of IC50 reflects the
concentration of antioxidants required to obtain an absorbance of 0.5 nm. Higher ab-
sorbance indicates the higher reducing power of the sample. Assays were carried out in
triplicate [58].

3.9. Phosphomolybdenum Reduction Assay of Vinegar

Determination of the total antioxidant capacity (TAC) of the samples was conducted
by mixing 25 µL of the sample with 1 mL of liquid reactive solution (0.6 M sulfuric acid,
28 mM sodium phosphate, and four mM ammonium molybdate). Then, the mixture was
incubated for 90 min in a water bath at 95 ◦ C. The absorbance of the incubated mixture was
determined using a spectrophotometer UV-1600PC (VWR, Fontenay-sous-Bois, France) at
695 nm absorbance. The antioxidant capacity was expressed as micrograms of ascorbic acid
equivalent per mL of vinegar (µg AAE/mL of the sample). The equation of the standard
concentration curve was y = 10.761x + 0.248, R2 = 0.98. Methanol was used instead of the
sample as a negative control [59].

3.10. Antimicrobial Analysis

3.10.1. Microbial Strains and Inoculums Standardization
Six microbial strains were used in this study. Four bacterial strains: Escherichia coli
ATCC 25922, Escherichia coli CIP 53126, Klebsiella pneumonia (ESBL-KP) were Gram-negative,
and the one Gram-positive strain was Staphylococcus aureus ATCC 29213. Candida albicans
ATCC 10231 and Aspergillus niger ATCC 16404 were used as fungal strains. All micro-
bial strains were provided from the Microbiology Laboratory, Faculty of Medicine and
Pharmacy of Fez, Morocco. The cultures were stored on Muller–Hinton agar at 4 ◦ C.
The different microbial strains were standardized and inoculated following the method
described in [60] for bacteria and [61] for fungal strains.

3.10.2. Disk Diffusion Assay

Disk diffusion assay (DD), based on the Kirby–Bauer method of [62] was used to
determine the antimicrobial activity, with slight modification. The standardized suspension
(1–5 × 108 CFU/mL) of the previously prepared isolates were inoculated onto Mueller–
Hinton agar (MHA) for the bacteria strain. Whatman paper discs (6 mm) impregnated with
20 µL of the vinegar samples were gently placed onto the surface of the pre-inoculated
agar. The plates were left to dry for 10 min, after which they were incubated for 24 h at
37 ◦ C [40]. After incubation, the diameters of the inhibition zones were measured in mm.
Fluconazole, Ampicilline, Streptomycine and voriconazole were used as positive controls.

3.10.3. Determination of the Minimum Inhibitory Concentration

The minimum inhibitory concentration (MIC) of the samples was determined using
microdilution assays according to the standards of the NCCLS [63], with slight modification.
Under sterile conditions, ten concentrations ranging from 500 to 3.91 µL/mL of each vinegar
sample were prepared by successive two-fold dilutions in distilled water. Then, 50 µL of
the culture medium MH broth was deposited into each well of the microplate, while the
Molecules 2022, 27, 770 10 of 13

first and the last wells were devoted to a negative control containing 100 µL of the vinegar
and positive growth control, respectively. Microdilutions were made by transferring 50 µL
by a factor of 12 into each well. The microplate was then inoculated with 50 µL of the
microbial suspension. The inoculated microplate was incubated for 24 h at 37 ◦ C for
bacteria, and 25 ◦ C for fungal strains. The colorimetric method based on the reagents
of 2,3,5-triphenyltetrazolium chloride (TTC) was used to read the results. After 2 h of
incubation, the MIC was determined as the lowest concentration that does not produce a
pinkish coloration where there is growth due to the activity of the dehydrogenases [40].

3.11. Statistical Analysis

The experiments were conducted in triplicate. Multiple comparison and separation of
the mean values was performed by a post hoc Tukey test at p < 0.05. Principal component
analyses (PCA) were accomplished using Minitab19.1 software to classify each kind of
vinegar’s in relationship with the parameters analyzed.

4. Conclusions
This study contributes to evaluating the physicochemical, biochemical properties, an-
tioxidant potential, and antimicrobial activity of different kinds of vinegar commercialized
in Morocco. The results showed a large diversity of vinegar products intended for direct
use by the consumer. The high values of phytochemical were 655.00 ± 22.2 µg GAE/mL
for TPC, 244.53 ± 11.32 µg QE/mL for TFC, and 84.63 ± 1.00 µg TAE/mL for CTC in V10,
V9, and V4, respectively. The strains tested showed variable sensitivities to the different
samples studied with inhibition zones ranging from 6.33 ± 2.08 mm to 34.33 ± 0.58 mm.
The lowest minimum inhibition concentrations (MIC) were recorded against Staphylococcus
aureus ATCC 29213 ranging from 1.95 to 7.81 µL/mL of vinegar, while the filamentous
fungi strain studied showed resistance against all of the analyzed samples. Therefore, the
application of vinegar must take into account its phytochemical characteristics. A future
study is needed to identify the phytochemical composition that will better elucidate this
variability and contribute to its valorization.

Author Contributions: Conceptualization, M.K. and A.A.; methodology, M.K., M.E.F. and O.A.K.;
data curation, A.A. and S.B.; writing—original draft preparation, M.K., H.S., and S.Z.A.; writing—
review and editing, J.B., and A.R.Z.; supervision, J.B. All authors have read and agreed to the
published version of the manuscript.
Funding: The paper is funded by Princess Nourah bint Abdulrahman University Researchers Sup-
porting Project number (PNURSP2022R165), Princess Nourah bint Abdulrahman University, Riyadh,
Saudi Arabia.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors extend their appreciation to Princess Nourah bint Abdulrahman
University Researchers Supporting Project number (PNURSP2022R165), Princess Nourah bint Abdul-
rahman University, Riyadh, Saudi Arabia.
Conflicts of Interest: Data are available upon request.
Sample Availability: Samples are available from the authors upon reasonable request.

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