Mango Peels as an Industrial

foods
Article
Mango Peels as an Industrial By-Product: A Sustainable
Source of Compounds with Antioxidant, Enzymatic, and
Antimicrobial Activity
Nika Kučuk 1 , Mateja Primožič 1 , Petra Kotnik 1,2 , Željko Knez 1,2 and Maja Leitgeb 1,2, *
1 Faculty of Chemistry and Chemical Engineering, University of Maribor, Smetanova ulica 17,
2000 Maribor, Slovenia; nika.kucuk@um.si (N.K.); mateja.primozic@um.si (M.P.); petra.kotnik@um.si (P.K.);
zeljko.knez@um.si (Ž.K.)
2 Faculty of Medicine, University of Maribor, Taborska ulica 8, 2000 Maribor, Slovenia
* Correspondence: maja.leitgeb@um.si; Tel.: +386-2-2294-462
Abstract: Plant waste materials are important sources of bioactive compounds with remarkable
health-promoting benefits. In particular, industrial by-products such as mango peels are sustainable
sources of bioactive substances, with antioxidant, enzymatic, and antimicrobial activity. Appropriate
processing is essential to obtain highly bioactive compounds for further use in generating value-
added products for the food industry. The objective of the study was to investigate and compare the
biological activity of compounds from fresh and dried mango peels obtained by different conven-
tional methods and unconventional extraction methods using supercritical fluids (SFE). The highest
total phenolic content (25.0 mg GAE/g DW) and the total content of eight phenolic compounds
(829.92 µg/g DW) determined by LC-MS/MS were detected in dried mango peel extract obtained by
the Soxhlet process (SE). SFE gave the highest content of proanthocyanidins (0.4 mg PAC/g DW). The
ethanolic ultrasonic process (UAE) provided the highest antioxidant activity of the product (82.4%)
using DPPH radical scavenging activity and total protein content (2.95 mg protein/g DW). Overall,
Citation: Kučuk, N.; Primožič, M.; the dried mango peels were richer in bioactive compounds (caffeic acid, chlorogenic acid, gallic
Kotnik, P.; Knez, Ž.; Leitgeb, M. acid, catechin, and hesperidin/neohesperidin), indicating successful preservation during air drying.
Mango Peels as an Industrial
Furthermore, outstanding polyphenol oxidase, superoxide dismutase (SOD), and lipase activities
By-Product: A Sustainable Source of
were detected in mango peel extracts. This is the first study in which remarkable antibacterial
Compounds with Antioxidant,
activities against the growth of Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa)
Enzymatic, and Antimicrobial
and Gram-positive bacteria (Bacillus cereus and Staphylococcus aureus) were evaluated by determining
Activity. Foods 2024, 13, 553. https://
doi.org/10.3390/foods13040553
the microbial growth inhibition rate after 12 and 24 h incubation periods for mango peel extracts
obtained by different methods. Ethanolic SE and UAE extracts from dried mango peels resulted in
Academic Editors: Carmen
the lowest minimum inhibitory concentrations (MIC90 ) for all bacterial species tested. Mango peels
Soto-Maldonado, Andrés Moure,
are remarkable waste products that could contribute to the sustainable development of exceptional
Cristian Ramírez and Ma
products with high-added value for various applications, especially as dietary supplements.
Salomé Mariotti
Received: 16 January 2024 Keywords: Mangifera indica; peels; bioactive substances; LC-MS/MS; proteins; enzymes;
Revised: 1 February 2024 antibacterial activity
Accepted: 7 February 2024
Published: 11 February 2024
1. Introduction
Copyright: © 2024 by the authors.
Fruit peels account for about 15–60% of waste products in fruit processing and are
Licensee MDPI, Basel, Switzerland. usually discarded [1–3]. A total of around 89 million tons of food waste is produced in the
This article is an open access article European Union [4,5]. Due to their high biodegradability and fermentability, fruit peels
distributed under the terms and contribute to water and soil pollution, eutrophication, global warming, the greenhouse
conditions of the Creative Commons effect, etc. However, similar to the pulp, the waste parts of the fruit produced during fruit
Attribution (CC BY) license (https:// processing are a rich source of phytonutrients and valuable biologically active compounds.
creativecommons.org/licenses/by/ By applying appropriate extraction or isolation processes (low operating temperature, no
4.0/). use of toxic solvents) for bioactive ingredients from the waste parts of fruits, especially
Foods 2024, 13, 553. https://doi.org/10.3390/foods13040553 https://www.mdpi.com/journal/foods

Foods 2024, 13, 553 2 of 31
from peels, it is possible to obtain products with exceptional added value, which can be
used mainly in the food, cosmetic, and pharmaceutical industries [6,7].
Mango (Mangifera indica L.) belongs to the Anacardiaceae family. It is one of the
most important and popular tropical fruits for its attractive color, excellent taste and
aroma, and high nutritional, phytochemical, and medicinal value [8]. The edible part is
largely industrially processed into various pulps or juices, with the inedible byproducts
discarded. However, mango by-products, especially peels, represent an important source
of biologically active compounds that could be used for various applications. Mango
by-products account for 35–55% of the total mass. Moreover, their further use could reduce
the amount of waste and negative impact on the environment [9,10]. Therefore, a new
approach is urgently needed to reduce their accumulation and the resulting pollution and
generate value-added products.
Mango peels contain even more specific bioactive compounds than the edible part of
the fruit. They are a rich source of various polyphenolic compounds (PCs), dietary fiber,
pectin, carotenoids, vitamins (ascorbic acid, tocopherol), minerals (potassium, magnesium,
sodium, calcium, copper, iron, manganese, zinc, chromium, phosphorus, chlorine), and
enzymes. The main PCs in mango peels are gallic acid, ellagic acid, caffeic acid, ferulic
acid, rutin, catechin, quercetin, and kaempferol [11,12]. It is essential to highlight the
presence of mangiferin, which belongs to the xanthone class, and is the main PCs of mango.
Mangiferin has antioxidant, antidiabetic, anticancer, antimicrobial, and anti-inflammatory
effects [13,14]. However, the peels have been found to contain a higher concentration
of mangiferin than the pulp and seeds [15,16]. In addition, some enzymes have been
shown to be present in active form in mango peels, such as superoxide dismutase (SOD),
catalase, peroxidase, protease, and amylase [17]. Different studies also confirmed the good
antimicrobial potential of samples obtained from different parts of the mango plant [18,19].
Ethanolic extracts from the peels of different varieties of mango show an excellent inhibitory
effect on the growth of various types of Gram-negative bacteria, such as Escherichia coli,
Salmonella typhimurium, and Vibrio parahaemolyticus, and Gram-positive bacteria, such as
Staphylococcus aureus and Bacillus cereus [20]. In addition, acetonic, ethanolic, and aqueous
extracts obtained by the conventional maceration method exhibit efficient antimicrobial
activities against various foodborne pathogenic microorganisms [21].
It is important to emphasize that the isolation of bioactive compounds is mainly
influenced by the extraction method used, the operating conditions, and the extraction
solvent, as different polarities of the solvents directly affect the extraction efficiency [22].
However, the quality and final composition of the extract obtained are mainly influenced
by the method of cultivation and location of the plant, weather conditions, plant species,
stage of development, the process and temperature of drying and storage, and the final
water content of the raw material [23]. Moreover, the antimicrobial activity of the extracted
samples is related to the content and synergistic effects of the bioactive substances in the
extracts obtained [24].
Some sensitive but important bioactive substances may be degraded during drying
and storage. On the other hand, harmful microorganisms may develop if the fresh peels
are not processed immediately and when unsuitable drying conditions are used [25,26].
Therefore, the present study focused on the determination of the bioactive compound
content in fresh and dried mango peels obtained by different extraction methods. Dried
mango peels were obtained by air-drying at room temperature, as this is considered one
of the simplest, least demanding, and most economical methods [27]. On the other hand,
excessively high temperatures during oven-drying can degrade bioactive compounds [28].
While oven-drying is energy inefficient, it is a cost-effective method [29]. Sun-drying is
also a cost-effective method, but it is more susceptible to microbial contamination due
to unhygienic conditions. It can also have a negative effect on the plant material by
reducing the nutrient content and causing color changes due to direct exposure to UV
radiation [30,31]. Although freeze-drying is one of the most suitable methods to dry plant
Foods 2024, 13, 553 3 of 31
material and successfully preserve valuable substances, it requires very high investment
and operating costs, which is very unfavorable [32,33].
Furthermore, the study compares conventional methods (Soxhlet method (SE),
ultrasound-assisted method (UAE), homogenization-assisted method (HAE)) and an uncon-
ventional technique (supercritical fluid technique (SFE)) to obtain the bioactive compounds
from mango peels. The main objective of the study was to investigate the content of
bioactive compounds in terms of total phenols (TPC), proanthocyanidins (PACs), certain
important PCs, and antioxidant activities. In addition, the total protein (TP) content and
activities of specific enzymes in mango peel extracts were determined for the first time.
Furthermore, one of the main objectives was also to validate the antimicrobial activity
against various Gram-negative and Gram-positive bacterial strains and fungi. To the best of
our knowledge, this is the first study to quantitatively evaluate the growth inhibitory prop-
erties of mango peel extracts against bacteria, determined as microbial growth inhibition
rates (MGIRs).
This study makes a significant contribution to raising awareness of the increasing
use of underutilized mango by-products, which, due to their high content of important
bioactive compounds, contribute to the production of high-value-added products that can
be used in various industries (food, cosmetics, biomedicine, pharmaceuticals), focusing on
the use of mango peels dried by a low-cost air-drying process.
2. Materials and Methods

2.1. Chemicals and Reagents
The following chemicals were used in the study: malt extract, potato dextrose broth,
Triton X-100, and tryptic soy broth, obtained from Fluka, Buchs, Switzerland. Mueller–
Hinton broth (MHB) and potato dextrose agar (PDA) were purchased from Biolife, Mi-
lano, Italy. In addition, 4-Aminoantipyrine (4-APP), Coomassie Blue G-250, ethanol
(EtOH, ≥99.5%), hydrochloric acid (HCl, 37.0%), meat extract, meat peptone, phospho-
ric acid (85.0%), potassium dihydrogen phosphate (KH2 PO4 ), sodium chloride (NaCl),
sodium dihydrogen phosphate monohydrate (NaH2 PO4 ·H2 O), and sodium hydrogen
phosphate (Na2 HPO4 ) were obtained from Merck, Darmstadt, Germany. Calcium chlo-
ride (CaCl2 ), D-(+)-glucose anhydrous, and ferrous sulfate heptahydrate (Fe(SO4 )·7H2 O)
were purchased from Kemika, Zagreb, Croatia. Carbobenzoxy-L-Glutaminylglycine (CBZ-
Gln-Gly) was obtained from Zedira GmbH, Darmstadt, Germany. Acetic acid (glacial,
≥99.7%), agar, 2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic) acid, bovine serum albu-
min (BSA), caffeic acid (99.0%), casein, (+)-catechin (≥98.0%), chlorogenic acid (≥99.0%),
L-3,4-dihydroxyphenylalanine (L-DOPA, ≥98.0%), 3,5-dinitrosalicylic acid (DNS), 2,2-
diphenyl-1-picrylhydrazyl (DPPH, ≥97.0%), ellagic acid (≥95.0%), ethylenediaminete-
traacetic acid (EDTA, 98.5–101.5%), Folin–Ciocalteu’s phenol reagent, gallic acid (GA,
≥97.5%), glucose assay, hesperidin/neohesperidin (≥97.0%), hydrogen peroxide (H2 O2 ),
hydroxylamine hydrochloride (HONH2 ·HCl), L-glutamic acid (99.0%), L-glutathione re-
duced (≥98.0%), maltose, mangiferin (≥98.0%), methanol (MeOH, ≥99.9%), yeast extract,
peptone from soybean, p-nitrophenyl butyrate (p-NPB, ≥98.0%), potassium sodium tartrate
tetrahydrate (KNaC4 H4 O6 ·4H2 O, 99.0%), pyrogallol (≥99.0%), Sigmacell cellulose, sodium
acetate (CH3 COONa, ≥99.0%), sodium carbonate (Na2 CO3 , ≥99.5%), sodium hydroxide
(NaOH, ≥95.0%), starch, trichloroacetic acid (TCA, ≥99.0%), rutin (≥97.0%), and Trizma
Base (NH2 C(CH2 OH)3 , ≥99.7%) were purchased from Sigma-Aldrich, St. Louis, MO, USA.
Carbon dioxide (CO2 , purity 2.5) was obtained from Messer, Ruše, Slovenia. Ciprofloxacin
(400 mg/200 mL) was obtained from University Medical Centre Maribor.
2.2. Microorganisms
Selected bacteria (B. cereus (DSM 345), E. coli (DSM 498), Pseudomonas aeruginosa (DSM
1128), S. aureus (DSM 346)) and fungi (black mold Aspergillus brasiliensis (DSM 1988),
yeast Candida albicans (DSM 1386)) were purchased from DSMZ-German Collection of
Microorganisms and Cell Cultures GmbH from Berlin, Germany.
Foods 2024, 13, 553 4 of 31
2.3. Preparation of Plant Material

Washed, fully ripe, Keitt-type mango fruits (country of origin: Puerto Rico) were
peeled with a fruit peeler. Some of the obtained peels were air-dried without pretreatment
at room temperature and protected from direct sunlight to avoid possible degradation
of bioactive compounds. Before the extraction process, the completely dried peels were
ground to a uniform size. The mean particle size was determined by sieve analysis and
amounted to 1.0 mm. The dried peels were then stored at room temperature until further
use. The remainder of the fresh peels was used immediately to avoid possible microbial
contamination and putrefaction processes. The fresh peels were cut into smaller pieces and
then subjected to an extraction process.
2.4. Moisture Content

The moisture content in fresh and dried mango peels was analyzed with the Mettler
Toledo HX204 Halogen Moisture Analyzer. The moisture content was determined according
to the thermogravimetric principle based on the weight loss of the dried analysis material
by heating to 115 ◦ C.
2.5. Thermogravimetric Analysis/Differential Scanning Calorimetry

The mango peels (fresh and dried) were subjected to thermal stability testing, including
thermogravimetric analysis/differential scanning calorimetry (TGA/DSC). Both analyses
were performed simultaneously on a TGA/DSC instrument (TGA/DSC1, Mettler Toledo
AG (MTANA), Zurich, Switzerland) in a nitrogen atmosphere. Samples were weighed
into aluminum pans and analyzed at a temperature range from 25 to 600 ◦ C and a rate of
10 ◦ C/min.
2.6. Extraction Procedures

Three different conventional extraction methods, namely SE, UAE, and HAE, were
used to extract bioactive compounds from fresh and dried mango peels. Two polar ex-
traction solvents, H2 O and EtOH, were used. In addition, unconventional SFE using
supercritical carbon dioxide (SC CO2 ) and EtOH as a co-solvent was also performed. For
this method, the use of dry raw material is mandatory. Therefore, only the extraction of
dried mango peels was performed. All extraction solvents used are considered GRAS
(Generally Recognized as Safe).
2.6.1. Soxhlet Extraction

SE was performed with both fresh and dried mango peels. Peels were transferred
into a cellulose thimble and placed in a Soxhlet extraction chamber. The extraction solvent
EtOH was added to a round-bottom flask connected to a Soxhlet apparatus with a reflux
condenser. The solvent was then heated to reflux. The extraction was carried out until the
condensate became colorless (3 refluxes). The ratio of solvent (EtOH) to fresh and dried
mango peels was 5:1 and 10:1, respectively.
2.6.2. Ultrasound-Assisted Extraction

UAE was performed on fresh and dried mango peels using EtOH and H2 O as ex-
traction solvents, respectively. Fresh or dried mango peels were mixed with an extraction
solvent and sonicated at 20 ◦ C and 40 kHz in an ultrasonic bath. Subsequently, filtering
was performed through a filter flask and a Buechner funnel to remove solid, insoluble
particles. The ratio of solvent (EtOH and H2 O) to fresh and dried mango peels was 5:1 and
10:1, respectively.
2.6.3. Homogenization-Assisted Extraction

HAE, using H2 O as an extraction solvent, was performed with fresh and dried mango
peels. The homogenization process was carried out for 30 min. Then, the supernatant was
Foods 2024, 13, 553 5 of 31
centrifuged to achieve complete separation of the supernatant from the insoluble material.
The ratio of solvent (H2 O) to fresh and dried mango peels was 5:1 and 10:1, respectively.
2.6.4. Supercritical Fluid Extraction

SFE was performed in a semi-continuous apparatus using SC CO2 to obtain bioactive
compounds from dried mango peels. Dried peels were placed in the autoclave and pre-
heated to an operating temperature of 40 ◦ C in a water bath. The CO2 was compressed
to an operating pressure of 200 bar and continuously introduced into the autoclave at a
2–3 mL/min flow rate. At the same time, EtOH was added as a co-solvent to achieve better
extraction of the polar bioactive compounds. The ratio of the final consumption of the
solvent (EtOH and CO2 ) to the dried mango peels was 5:1 and 30:1, respectively.
After each extraction procedure, the extraction solvent (EtOH or H2 O) was evaporated
using a rotary evaporator (Büchi® Rotavapor R-144, Flawil, Switzerland). The extracts were
stored in a freezer at −20 ◦ C until further analysis.
2.6.5. Extraction Yield Determination

Each individual extraction was performed three times. The extraction yield was
calculated as the ratio between the weight of the extract obtained and the initial weight of
the material used. The results are given as mean values.
2.7. Quantitative Determination of Total Phenols and Proanthocyanidins

2.7.1. Determination of Total Phenolic Content (TPC)
The TPC content in the extracts obtained was determined by the Folin–Ciocalteu
method. The analysis procedure for TPC is described in detail by Škerget et al. [34]. The
results are given as mean values and are expressed as mg of GA equivalents (GAE) per
gram of fresh weight (FW) for fresh peels or dry weight (DW) for dried peels.
2.7.2. Determination of Total Proanthocyanidin Content (PAC)

Condensed tannins or PAC were determined by a colorimetric method using HCl
and n-butanol, in which acid-catalyzed oxidative depolymerization of PAC leads to the
formation of the corresponding anthocyanidin compounds [34]. The results are reported as
mean values expressed as mg of PAC per g of FW or DW.
2.8. Determination of Antioxidant Activity

The antioxidant activity of the extracts was determined using the stable free radical 2,2-
diphenyl-1-picrylhydrazyl (DPPH) [35]. Antioxidant activity was expressed as a percentage
of inhibition regarding the reference solution.
2.9. Liquid Chromatography with Tandem Mass Spectrometry Analysis

Liquid chromatography with mass spectrometry (LC-MS/MS) was used for the iden-
tification and quantitative determination of selected phenolic compounds in mango peel
extracts, using Agilent 1200 HPLC together with Agilent 6464 QQQ with JetStream technol-
ogy, as described in detail by Hrnčič et al. [36].
2.10. Determination of Total Protein (TP) Content

The Bradford method [37] was used to determine TP content using bovine serum
albumin (BSA) as the standard. All analyses were performed in three replicates and
expressed as mean values in mg of protein per g of FW or DW.
2.11. Determination of Enzyme Activities

The enzyme activity of α-amylase [38], cellulase [39], glucoamylase [40], laccase [41],
lipase [38], peroxidase [35], polyphenol oxidase (PPO) [42], protease [39], SOD [43], and
transglutaminase (TGM) [44] were examined with specific spectrophotometric enzymatic as-
says for each selected enzyme using a UV spectrophotometer (Varian—CARY® 50 UV–VIS
Foods 2024, 13, 553 6 of 31
Spectrophotometer, Varian Inc., Middelburg, The Netherlands). Each assay was performed
in three replicates. The results are reported as mean values expressed in units (U) per g
of protein.
2.12. Determination of Antimicrobial Activity

For the determination of the inhibitory properties of the extracts obtained on the
growth of selected microbial cells (Gram-negative bacteria (E. coli and P. aeruginosa), Gram-
positive bacteria (B. cereus and S. aureus), and fungi (black mold A. brasiliensis and yeast
C. albicans)), the qualitative disk diffusion method and the quantitative microbroth dilution
method were used.
2.12.1. Qualitative Disk Diffusion Method

The antimicrobial properties of the mango peel extracts were verified by the qualitative
Kirby–Bauer disk diffusion method, which was previously described in detail in the study
by Kupnik et al. [45], using 6 mm sterile cellulose discs. Ciprofloxacin was used as a
positive control. All tests were performed with three replicates, and the results are given as
mean values.
2.12.2. Quantitative Microbroth Dilution Method

The microbroth dilution method was used, which is best suited for the in vitro deter-
mination of the susceptibility or resistance of microorganisms [45]. Therefore, the minimum
inhibitory concentrations (MICs) and the MGIRs were determined. The method was per-
formed only for those microorganisms that were found to be susceptible when exposed to
the mango samples, as determined by the disk diffusion method. All experiments were
performed in three replicates and are expressed as mean values.
2.13. Statistical Analysis

All statistical data analyses were performed using IBM® SPSS® Statistics. Statistical
data analysis was performed to study the differences between extraction methods. The
normality of the distribution of data was tested using Shapiro–Wilk’s test. The homogeneity
of variances was determined using Levene’s test. Differences between extractions were
determined with one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test
(for normally distributed data) and with the nonparametric Kruskal–Wallis H test, followed
by pairwise comparison using the Dunn–Bonferroni post hoc method (for abnormally
distributed data).
3. Results and Discussion

The present study focused on the influence of various parameters (drying method, ex-
traction method) on obtaining highly active bioactive compounds with good antimicrobial
and antioxidant activity.
3.1. Thermal Stability of Mango Peels

For comparison, the moisture content in fresh and dried mango peels was first ana-
lyzed. Fresh mango peels contained 77.74% moisture, while completely air-dried peels
stored at room temperature in an airtight container still contained 11.06% moisture.
The differences in thermal behavior of fresh and dried mango peels were further
investigated using TGA/DSC analysis. Figure 1a shows the thermal degradation of fresh
and dried mango peels as determined by thermogravimetric analysis. Due to the high
moisture content, fresh mango peels showed a drastic weight loss in the temperature range
of 39–202 ◦ C. On the other hand, the weight of dried mango peels gradually decreased
throughout the entire temperature range, decreasing by 73.3%. The total weight loss of
fresh peels was 92.0%. The two curves are generally comparable after excessive evaporation
of moisture in the fresh peels, indicating a similar composition. The residue represents the
ash fraction, which contains minerals that cannot be degraded [46]. The residue accounted
and dried mango peels as determined by thermogravimetric analysis. Due to the high
moisture content, fresh mango peels showed a drastic weight loss in the temperature
range of 39–202 °C. On the other hand, the weight of dried mango peels gradually de-
creased throughout the entire temperature range, decreasing by 73.3%. The total weight
Foods 2024, 13, 553
loss of fresh peels was 92.0%. The two curves are generally comparable after excessive 7 of 31
evaporation of moisture in the fresh peels, indicating a similar composition. The residue
represents the ash fraction, which contains minerals that cannot be degraded [46]. The
for 8.0% accounted
residue of the fresh
forpeels
8.0%and 26.7%
of the freshofpeels
the dried peels,ofbased
and 26.7% on the
the dried total
peels, massonofthe
based the
studied peel samples.
total mass of the studied peel samples.
Figure 1. TGA (a) and DSC (b) of fresh and air-dried mango peels.
Figure 1. TGA (a) and DSC (b) of fresh and air-dried mango peels.
Figure 1b
Figure 1b shows
shows the the DSC
DSC thermogram
thermogram for for fresh
fresh and
and dried
dried mango
mango peels.peels.An Aninitial
initial
melting peak at 99 °C
◦ for the dried peels and at 133 °C
◦ for the fresh
melting peak at 99 C for the dried peels and at 133 C for the fresh mango peels indicates mango peels indicates
theevaporation
the evaporationof ofHH2O O and the beginning of the degradation of the carbohydrates, indi-
2 and the beginning of the degradation of the carbohydrates, indicat-
cating the melting point
ing the melting point (endothermic (endothermic peak).
peak). For For example,
example, the
the meltingpoint
melting pointofofthe
thedifferent
differ-
sugars is 126.5 C for fructose, 159.6 C for glucose, and 190.4 C for sucrose [47],which
ent sugars is 126.5
◦ °C for fructose, 159.6◦ °C for glucose, and 190.4 °C
◦ for sucrose [47], which
arepresent
are presentininmangomangofruitfruit[48].
[48].AAslight
slightshift
shiftininthe
themelting
meltingpeak
peak inin fresh
fresh peels
peels is due
is due to to
the
the different
different binding
binding of H2of OH 2O and sugars compared to the dried peels. Moreover, the two
and sugars compared to the dried peels. Moreover, the two curves
curves
of fresh of
andfresh
driedand dried peels
mango mango dopeels do notfurther.
not differ differ further. The second
The second peak (198–254
peak (198–254 ◦ C) and°C)the
and the
third peakthird peak (304–368
(304–368 ◦ C), shown°C),asshown as exothermic
exothermic peaks, are peaks,
dueare duedegradation
to the to the degradation
of long-
of long-chain,
chain, high-molecular-weight
high-molecular-weight polymerspolymers [46] orsalts,
[46] or various various salts, for example,
for example, sulfur-
sulfur-containing
containing
salts, such as salts, such hydrogen
sodium as sodiumsulfite.
hydrogen sulfite.
Sodium Sodium sulfite,
hydrogen hydrogen whichsulfite, which de-at
decomposes
composes
315 at 315 °C,as
◦ C, is classified is classified as a food
a food additive additive
[49]. Therefore,[49]. Therefore, it can betoapplied
it can be applied to theof
the surface
surface
mango peelsof mango
during peels during post-harvest
post-harvest treatmenttreatment
to preserve to preserve their freshness
their freshness and prevent and pre-
them
vent rotting
from them from ro ing too
too quickly andquickly and experiencing
experiencing discoloration discoloration
during longduring long transpor-
transportation periods.
tation periods.
Since the thermal stability of fresh and dried peels was comparable, we performed
Since the thermal
further extractions stability of
of differently freshpeels
dried and todried
comparepeels bioactive
was comparable,
compound we content
performed and
further extractions
biological activity. of differently dried peels to compare bioactive compound content and
biological activity. when visually assessing the color of the plant material used (fresh or
Furthermore,
dried peels), it was when
Furthermore, found visually assessing
that the dried peelsthe
had color of thecolor
a darker plantduematerial
to the used
lower(fresh or
moisture
dried peels), it was found that the dried peels had a darker color due
content. Consequently, the extracts obtained also had a slightly darker color (Figure 2). to the lower moisture
content. Consequently,
Therefore, the color of the the extracts
materialobtained
may also alsobe had a slightly
related darker in
to a change color
the(Figure
content2).of
bioactive compounds.
3.2. Extraction Yield Determination

Different extraction methods were performed using fresh or dried mango peels as
raw material to compare the effects of the extraction procedure (conventional and un-
conventional) and the extraction solvent (CO2 + EtOH, EtOH, H2 O) used on the profile
of bioactive compounds and biological activity. Figure 3 presents the efficiency of each
extraction performed, expressed as extraction yield (%).
Foods 2024, 13, 553 8 of 31
Foods 2024, 13, 553 Therefore, the color of the material may also be related to a change in the content of bio-
8 of 31
active compounds.
Figure 2. Plant material used (fresh and dried mango peels) and HAE (H2O) extracts obtained from
fresh and dried mango peels.

Different extraction methods were performed using fresh or dried mango peels as
raw material to compare the effects of the extraction procedure (conventional and uncon-
ventional) and the extraction solvent (CO2 + EtOH, EtOH, H2O) used on the profile of bi-
Figure2.2.Plant
Plantmaterial
materialused
used(fresh
(freshand
anddried
driedmango
mangopeels)
peels)and
andHAE
HAE(H
(H2O)
O)extracts
extractsobtained
obtainedfrom
from
oactive
Figure compounds and biological activity. Figure 3 presents the efficiency
2 of each extrac-
fresh and dried mango peels.
tion and
fresh performed, expressed
dried mango peels. as extraction yield (%).

Different extraction methods were performed using fresh or dried mango peels as
raw material to compare the effects of the extraction procedure (conventional and uncon-
ventional) and the extraction solvent (CO2 + EtOH, EtOH, H2O) used on the profile of bi-
oactive compounds and biological activity. Figure 3 presents the efficiency of each extrac-
tion performed, expressed as extraction yield (%).
Figure 3.
Figure 3. Extraction
Extractionyields
yieldsobtained
obtainedbybydifferent extraction
different procedures
extraction (SFE—supercritical
procedures fluidfluid
(SFE—supercritical ex-
traction; SE—Soxhlet extraction; UAE—ultrasound-assisted extraction; HAE—homogenization-as-
extraction; SE—Soxhlet extraction; UAE—ultrasound-assisted extraction; HAE—homogenization-
sisted extraction) from fresh or dried mango peels with different solvents (CO2 + EtOH, EtOH, H2O).
assisted extraction) from fresh or dried mango peels with different solvents (CO2 + EtOH, EtOH,
Different le ers indicate significant difference (p < 0.05).
H2 O). Different letters indicate significant difference (p < 0.05).
The highest extraction yield was obtained with conventional extraction techniques
The highest extraction yield was obtained with conventional extraction techniques
(SE (EtOH) from dried peels (35.52%) and HAE (H2O) from dried peels (33.76%)). The
(SE (EtOH) from dried peels (35.52%) and HAE (H2 O) from dried peels (33.76%)). The
Figure 3. Extraction
explanation lies in yields obtained by
the prolonged different
action extraction
of the procedures
solvent fluid ex-
(EtOH) on the mango
traction;
peels SE—Soxhlet
using SE. Duringextraction;
HAE,UAE—ultrasound-assisted extraction;
efficient cell wall disruption wasHAE—homogenization-as-
achieved, causing the
sistedbioactive
polar extraction)compounds
from fresh or dried
of themango peelstowith
cell wall different
dissolve in solvents (CO2 + EtOH,
H2 O. However, theEtOH, H2O).
operating
Different le ers indicate significant difference (p < 0.05). ◦
temperature in SE was above the boiling temperature of EtOH (>78 C), which could have
a negative effect on the extraction yield of sensitive bioactive compounds. In the other
The highest
extraction extraction
techniques yieldthe
performed, was obtainedtemperature
operating with conventional
was lowerextraction techniques
(40 ◦ C in the case
(SE
of (EtOH)
SCF, and 22from dried
◦ C for HAE peels
and (35.52%)
UAE). and HAE (H2O) from dried peels (33.76%)). The
On the other hand, when comparing UAE with the different extraction solvents used,
higher extraction yields were obtained with H2 O as the extraction solvent than with EtOH,
which can be attributed to the greater polarity of H2 O. In general, a higher amount of
ature in SE was above the boiling temperature of EtOH (>78 °C), which could have a neg-
ative effect on the extraction yield of sensitive bioactive compounds. In the other extrac-
tion techniques performed, the operating temperature was lower (40 °C in the case of SCF,
and 22 °C for HAE and UAE).
Foods 2024, 13, 553 On the other hand, when comparing UAE with the different extraction solvents used, 9 of 31
higher extraction yields were obtained with H2O as the extraction solvent than with EtOH,
which can be a ributed to the greater polarity of H2O. In general, a higher amount of
extract was
extract was obtained
obtained fromfrom thethe dried
dried peels.
peels. This
This isis due
due to
to the
the large
large amount
amount ofof moisture
moisture in in
fresh peels. Dorta et al. [50] also obtained higher extraction yields when
fresh peels. Dorta et al. [50] also obtained higher extraction yields when fresh peels were fresh peels were
extracted by
extracted by microwave-assisted
microwave-assisted extraction
extraction (MAE)
(MAE) compared
compared to to dried
dried peels
peels by
by different
different
drying methods. The extraction yields of air-dried mango peels determined
drying methods. The extraction yields of air-dried mango peels determined in a study by in a study by
Souza et al. [51] are in line with our
Souza et al. [51] are in line with our results. results.
Regarding SFE,
Regarding SFE, since
since CO CO22 is
is aa nonpolar
nonpolar solvent,
solvent, aa polar
polar co-solvent
co-solvent such
such asas EtOH
EtOH
should be used to allow the extraction of important polar bioactive
should be used to allow the extraction of important polar bioactive compounds. There- compounds. Therefore,
the efficiency
fore, of SFE
the efficiency was was
of SFE the lowest
the lowestcompared
compared to conventional
to conventional methods.
methods. Sánchez-Ca-
Sánchez-
margo et al. [52] optimized the SFE operating conditions for mango peel
Camargo et al. [52] optimized the SFE operating conditions for mango peel extraction, with extraction, with
extraction yields ranging from 1.60 to 6.25%. The highest yields were
extraction yields ranging from 1.60 to 6.25%. The highest yields were obtained at operatingobtained at operat-
ing conditions
conditions of 300ofbar
300andbar60and 60 °C
◦ C and andEtOH
using usingas EtOH as a co-solvent.
a co-solvent. This confirms
This confirms the
the presence
presence of polar secondary metabolites
of polar secondary metabolites in the plant material. in the plant material.
A comprehensive
A comprehensive comparison
comparisonwith withthetheresults
resultsofof
other studies
other is difficult
studies because
is difficult the
because
operating conditions of the extractions performed and the extraction
the operating conditions of the extractions performed and the extraction solvent used solvent used are dif-
ferent.
are This significantly
different. affectsaffects
This significantly both the
bothextraction yield yield
the extraction and the
and phytochemical
the phytochemical and
nutraceutical
and profile
nutraceutical of theofextracts.
profile Also, usually,
the extracts. the origin,
Also, usually, maturity,
the origin, and growth
maturity, condi-
and growth
tions of theof
conditions fruit
the are
fruitnot
arethe
notsame.
the same.
3.3. Content
3.3. Content of
of Secondary
Secondary Metabolites
Metabolites in
in Mango
Mango Peel
Peel Extracts
Extracts and
and Their
Their Antioxidant
Antioxidant Activity
Activity
AA quantitative investigation of the content of TPC and PAC of the extracts obtained
quantitative investigation of the content of TPC and PAC of the extracts obtained
from
from fresh
fresh and
and dried
dried mango
mango waste
waste was
was performed.
performed. The
The results
results are
are shown
shown in
in Figure
Figure 4a.
4a.
Figure 4.
Figure 4. Content
Content of
of total
total phenols
phenols (TPC)
(TPC) and
and proanthocyanidins
proanthocyanidins (PAC)
(PAC) (a)
(a) and
and antioxidant
antioxidant activity
activity
(b) of mango peel extracts from fresh and dried peels obtained by different extraction procedures
(b) of mango peel extracts from fresh and dried peels obtained by different extraction procedures
(SFE—supercritical fluid extraction; SE—Soxhlet extraction; UAE—ultrasound-assisted extraction;
(SFE—supercritical fluid extraction; SE—Soxhlet extraction; UAE—ultrasound-assisted extraction;
HAE—homogenization-assisted extraction) with different solvents (CO2 + EtOH, EtOH, H2O). Dif-
HAE—homogenization-assisted extraction)(p
ferent le ers indicate significant difference with different solvents (CO2 + EtOH, EtOH, H2 O). Dif-
< 0.05).
ferent letters indicate significant difference (p < 0.05).
The highest TPC content was achieved in samples obtained by SE (EtOH) (25.0 mg
GAE/g DW), as seen in Figure 4a. Since the lowest extraction yield was obtained in the
SFE, consequently, the TPC content was also the lowest (1.7 mg GAE/g DW). The extracts
obtained from dried mango waste generally had higher TPC content than those obtained
from fresh peels using the same extraction procedure. Furthermore, this indicates that
the TPC content is maintained regardless of the air drying of the mango peels. Ethanolic
extracts, also when SFE (CO2 + EtOH) was used, resulted in higher TPC content compared
to aqueous extracts.
The content of extracted TPC during UAE is strongly influenced by operating con-
ditions such as the solid–liquid ratio, temperature, amplitude, and time [53–56]. Borrás-
Foods 2024, 13, 553 10 of 31
Enríquez et al. [57] obtained an even higher TPC content (18.14 mg GAE/g DW) using
an aqueous solution of EtOH (50%, v/v), as in our study, where separate solvents (EtOH
and H2 O) were used. The use of EtOH as an extraction solvent proved to be more efficient
than the use of MeOH in the extraction of TPC [55]. Moreover, using SFE under the same
operating conditions as in the present study, only without a co-solvent, a 2.4-fold lower
TPC value was obtained compared to our results. This indicates that EtOH as a co-solvent
significantly affects the recovery of bioactive compounds from mango by-products. Overall,
the TPC values showed a tendency to decrease with increasing operating temperature and
increase with increasing operating pressure [51,58].
Furthermore, the PAC content was determined in the extracts obtained from mango
waste peels. PACs are colorless flavonoids that have beneficial effects on human health due
to their antioxidant, anticancer, antimicrobial, anti-inflammatory, antidiabetic, neuroprotec-
tive, and hypolipidemic properties [59,60].
The results for the PAC content between ethanolic and aqueous extracts varied between
0.2 and 0.4 mg PAC/g DW and between 0.1 and 0.2 mg PAC/g FW. The highest amount of
PACs was determined for the SFE extract (0.4 mg PAC/g DW), followed by the SE (EtOH)
extract with 0.3 mg PAC/g DW. Thus, the operating conditions used for SFE extraction
(temperature, time, pressure, addition of co-solvent) were beneficial.
According to the reviewed literature, different methods can be used to determine the
PAC concentration in extracts. However, very few studies report the analysis of the content
of PACs in mango peel extracts. The presence of PACs in mango peels is mainly influenced
by the geographical area of the species and the extraction process [61]. The content of
PACs, expressed as leucoanthocyanidin equivalents (LE), ranged from 1.2 to 5.8 mg LE/g
DW, which was mainly influenced by the solvent used, while temperature had a negligible
effect [62]. The highest PAC values were obtained with the mango peel extract using MeOH,
EtOH:H2 O, or Ace:H2 O as a solvent. In another study by Dorta et al. [63], MAE yielded
0.0018–0.0048 mg LE/g DW of mango peels. The PAC content in mango peels increases
with increasing fruit ripeness in different varieties. However, mango peels contain higher
PAC concentrations compared to pulp, as confirmed by different studies [61,64].
The antioxidant activity of the mango peel extracts was determined using the DPPH
method. This method is commonly used to evaluate the ability of substances that act as
free radical scavengers. These substances are antioxidants that can donate a proton in the
presence of the DPPH free radical, which is then converted into a reduced form [65]. The
results were expressed as a percentage of inhibition of free radical DDPH and are presented
in Figure 4b.
Furthermore, due to the different extraction methods and solvents used, different pro-
files of the extracted bioactive compounds were obtained. The extracts in which EtOH was
used as an extraction solvent (UAE and SE) or as a co-solvent (SFE) exhibited significantly
higher DPPH radical scavenging activity. This indicates that EtOH has an important influ-
ence on the extraction of bioactive compounds with antioxidant properties. The highest
antioxidant activity was determined in UAE (EtOH) extract from dried peels, with 82.4%
inhibition, which is 3.6 times higher than in HAE (H2 O) extract from fresh peels (22.7%),
where the highest % inhibition was achieved among aqueous extracts. On the other hand,
no significant differences in the antioxidant potential of fresh and dried raw peels processed
by the same method and under the same conditions were detected.
The antioxidant activity of extracts from dried mango peels obtained by UAE reached
87% DPPH inhibition using 50% (v/v) EtOH [57], which is comparable with our results
obtained by UAE with 100% EtOH. Moreover, Kaur et al. [56] demonstrated the influence
of operating conditions (liquid/solid ratio, temperature, amplitude, time) in the UAE on
the determined antioxidant activity of mango peel extracts.
Souza et al. [51] achieved only 25% DPPH inhibition of mango peel extract obtained
by SFE without a co-solvent. The present study achieved a 1.7-fold higher inhibition
percentage, indicating that the co-solvent EtOH significantly improves the recovery of
Foods 2024, 13, 553 11 of 31
polar bioactive compounds with antioxidant potential when similar operation conditions
are used.
3.4. Content of Important Phenolic Compounds in Mango Peel Extracts

Fruits are an essential source of various PCs with health-promoting properties. There-
fore, a quantitative study was performed for eight important PCs, including xanthone
(mangiferin), four phenolic acids (caffeic acid, chlorogenic acid, ellagic acid, gallic acid),
and three flavonoids (catechin, hesperidin/neohesperidin, rutin) in the obtained mango
peel extracts. The results are presented in Table 1.
Numerous studies in the reviewed literature have already shown that all parts of
the mango tree, including peels, are a rich source of important bioactive compounds [66].
However, to the best of our knowledge, no study has addressed the effects of the drying
process of the starting material on the retention of important bioactive compounds and
their contribution to the antimicrobial activity of mango peel extracts obtained by different
conventional and unconventional methods.
The results revealed that the obtained mango peel extracts varied in the composition
of the analyzed bioactive compounds, considering the extraction procedure, the extraction
solvent, and the raw material used (fresh or dried peels).
Mangiferin is one of the most important PCs of the mango tree having health-promoting
properties [67]. Most of the analyzed extracts were found to contain mangiferin, with the
highest content determined in the aqueous extracts of dried mango peels. In contrast,
mangiferin was either not detected in the aqueous extracts of fresh mango peels (UAE
extract) or only found in small amounts (HAE extract). The opposite results were obtained
with ethanolic extracts, where a higher content of mangiferin was successfully extracted
from fresh mango peels compared to dried peels. It can be concluded that air-drying has
no significant effect on the possible degradation of mangiferin. Kaur et al. [68] reported
that shade-drying at room temperature resulted in higher mangiferin content in the plant
material than oven- or sun-drying. On the other hand, the final content of mangiferin is
mainly influenced by the choice of extraction method [69] and extraction solvent [70]. In
the reviewed literature, the mangiferin content in extracts obtained by different methods
and operating conditions ranged from 0.331 to 150 µg/mg DW [57,67,71–73]. Furthermore,
Marcillo-Parra et al. [74] found that the mangiferin content in mango peel extracts also
varied widely depending on the mango variety, ranging from 57.2 to 3140 µg/g DW.
Mangiferin is only slightly soluble in EtOH and H2 O [75] but has a very low solubility in
SC CO2 [76], which is consistent with our results, as the lowest mangiferin concentration
was obtained with SFE (0.15 µg/g DW).
Foods 2024, 13, 553 12 of 31
Table 1. The presence of important phenolic compounds (PCs) in mango peel extracts from fresh and dried peels obtained by different extraction procedures
with different solvents (CO2 + EtOH, EtOH, H2 O). SFE—supercritical fluid extraction; SE—Soxhlet extraction; UAE—ultrasound-assisted extraction; HAE—
homogenization-assisted extraction. Different letters in the same row indicate significant difference (p < 0.05).
Content (µg PC/g DW) Content (µg PC/g FW)

Phenolic Compound SFE SE UAE UAE HAE SE UAE UAE HAE
(CO2 + EtOH) (EtOH) (EtOH) (H2 O) (H2 O) (EtOH) (EtOH) (H2 O) (H2 O)
Xanthones
Mangiferin 0.15 ± 0.01 a 1.73 ± 0.03 c 0.24 ± 0.01 a 39.74 ± 0.54 d 60.43 ± 0.72 b 3.86 ± 0.06 c 24.87 ± 0.41 d - 1.36 ± 0.02 c
Phenolic acids
Caffeic acid 2.80 ± 0.12 c 6.60 ± 0.24 a 2.86 ± 0.01 c 5.11 ± 0.12 a 1.81 ± 0.03 c 0.56 ± 0.01 d 0.25 ± 0.01 d - 0.08 ± 0.00 b
2.54 ± 0.06 a 26.54 ± 0.17 c 4.69 ± 0.04 a b 3.06 ± 0.02 a 3.04 ± 0.04 a
Chlorogenic acid 10.82 ± 0.09 - - -
Ellagic acid 37.00 ± 0.17 a 310.00 ± 1.32 c 96.94 ± 0.65 d 97.23 ± 1.28 d 50.37 ± 0.64 e 531.98 ± 4.09 b 268.89 ± 1.41c 539.42 ± 3.50 b 324.49 ± 3.02 c
Gallic acid 58.24 ± 1.23 c 373.53 ± 0.42 a 71.31 ± 0.53 d 405.72 ± 0.75 a 370.12 ± 2.72 a 92.75 ± 0.51 d 37.59 ± 0.55 c 8.03 ± 0.12 b 10.68 ± 0.04 b
Flavonoids
Catechin 15.35 ± 0.73 c 109.39 ± 0.6 a 67.59 ± 0.27 d 100.13 ± 0.44 a 46.59 ± 0.16 d 31.91 ± 0.12 d 1.95 ± 0.02 e - 0.38 ± 0.01 b
0.23 ± 0.01 a b 0.59 ± 0.01 c 0.88 ± 0.02 c b d b 0.62 ± 0.02 c 0.69 ± 0.02 c
Hesperidin/Neohesperidin 2.12 ± 0.04 2.38 ± 0.03 1.45 ± 0.02 2.67 ± 0.01
Rutin - - - 0.80 ± 0.03 - - - - -
Total content of analyzed
101.08 ± 1.58 716.67 ± 2.14 175.80 ± 1.23 518.89 ± 2.24 422.30 ± 3.38 628.35 ± 4.63 309.77 ± 2.00 547.45 ± 3.62 335.24 ± 3.07
phenolic acids
15.57 ± 0.74 111.51 ± 0.40 68.18 ± 0.28 101.81 ± 0.49 48.98 ± 0.19 33.37 ± 0.15 4.62 ± 0.03 0.62 ± 0.02 1.07 ± 0.03
flavonoids
116.80 ± 2.32 829.92 ± 2.57 244.22 ± 1.52 660.43 ± 3.24 531.71 ± 4.29 665.58 ± 4.84 339.26 ± 2.44 548.07 ± 3.36 337.67 ± 3.12
phenolic compounds
Foods 2024, 13, 553 13 of 31
Regarding the content of phenolic acids in the extracts of mango waste peels, ellagic
acid and gallic acid are the most abundant. The highest composition of ellagic acid was
found in UAE (H2 O) extract (539.42 µg/g FW) and SE (EtOH) extract (531.98 µg/g FW),
while the lowest composition was present in SFE (CO2 + EtOH) extract (37.50 µg/g DW),
as ellagic acid is poorly soluble in SC CO2 [77]. Furthermore, all extracts obtained from
fresh mango peels had higher ellagic acid content than those obtained from dried peels by
the same extraction method and under the same conditions. This indicates that the drying
process may have a destructive effect on ellagic acid and its composition [78]. Ellagic acid
content has already been determined in mango peel extracts, where its content ranged from
15.995 to 298.69 µg/g DW [73,79,80]. On the contrary, the highest content of gallic acid was
determined in UAE (H2 O) extract (405.72 µg/g DW), while the lowest content was found in
UAE (H2 O) extract (8.03 µg/g DW). Gallic acid is soluble in EtOH and H2 O [81], whereas
in SFE, the addition of a polar co-solvent (EtOH) is needed due to its low solubility in SC
CO2 [82]. Extracts from dried peels were found to have a higher gallic acid composition
than their fresh counterparts. Drying methods generally do not significantly affect gallic
acid and its content in dried samples [83]. Higher gallic acid contents in extracts from dried
peels indicate the high moisture content of fresh mango peels used for extraction. In the
reviewed literature, different compositions of gallic acid in mango peel extracts have been
reported, ranging from 23 to 23,816 µg/g DW [80,84–87] and 14.5 to 791.5 µg/g FW [88]. A
similar phenomenon was observed in the composition of caffeic acid and chlorogenic acid.
The content of the aforementioned phenolic acids was higher in extracts from dried peels
than in extracts from fresh peels, comparing the same extraction procedure. This indicates
that air-drying has no significant effect on caffeic and chlorogenic acids [89,90]. The highest
content of chlorogenic acid was determined in SE (EtOH) extract (26.54 µg/g DW), whereas
no chlorogenic acid or a very low amount was present in H2 O extracts. Furthermore, the
extract with the highest caffeic acid composition was the SE (EtOH) extract (6.60 µg/g DW),
while no caffeic acid was detected in the UAE (H2 O) extract, obtained from fresh peels.
This indicates higher chlorogenic and caffeic acid solubility in EtOH than in H2 O [91,92].
Compared with the reviewed literature, the content of chlorogenic acid in various mango
peel extracts was found to be 760–2280 µg/g DW [93] and 44.05–271.9 µg/g FW [88], while
the content of caffeic acid was 16.6–67.3 µg/g DW [93] and 33.03–144.3 µg/g FW [88].
Among the flavonoids, catechin was the most abundant in mango peel extracts. The
highest content was observed in the SE (EtOH) extract (109.39 µg/g DW), while the lowest
catechin concentration was found in the UAE (H2 O) extract, with only 0.38 µg/g FW, since
catechin is soluble in H2 O and organic solvents, including EtOH [94]. However, in SFE, the
solubility of catechin is mainly influenced by the operating conditions and the addition of
a co-solvent, such as EtOH [95]. Other studies have also confirmed the presence of catechin
in various mango peels, ranging from 45.73 to 115.5 µg/g DW [55,73]. A similar pattern
was observed for hesperidin/neohesperidin content, except for the UAE (EtOH) extract,
where more hesperidin/neohesperidin was extracted from fresh mango peels. Overall,
hesperidin/neohesperidin was found in lower amounts in all extracts. The highest level
was observed in the UAE (EtOH) extract (2.67 µg/g FW). Due to its polarity, hesperidin
is very poorly soluble in SC CO2 [96]. According to the reviewed literature, to the best
of our knowledge, the content of hesperidin/neohesperidin has not been determined or
detected in mango peels. However, in a study by Abdel-Aty et al. [97], hesperidin was
found to be the major PC in mango seed kernel extract (55.6% of total compounds). Rutin
was only detected in the UAE (H2 O) extract at a concentration of 0.80 µg/g DW. Other
studies have already established that the rutin content in mango peel extracts ranges from
37.15 to 390 µg/g DW, which is mainly influenced by the mango variety [55,80].
The total content of the eight phenolic compounds analyzed was determined in the
SE (EtOH) extract (829.92 µg/g DW). Overall, extracts from dried mango peels contained
higher total levels of analyzed PCs than extracts from fresh peels obtained by the same
extraction procedure and under the same conditions. This is an important fact, because
most of the PCs analyzed were retained during the air-drying process.
Foods 2024, 13, 553 14 of 31
Foods 2024, 13, 553

extraction procedure and under the same conditions. This is an important fact, because
14 of 31
most of the PCs analyzed were retained during the air-drying process.
3.5.
3.5. Total
TotalProtein
ProteinContent
Contentand
andActivities
ActivitiesofofCertain
CertainEnzymes
EnzymesininMango
MangoPeel
PeelExtracts
Extracts
The
The results of the determination of total proteins in extracts from fresh
results of the determination of total proteins in extracts from fresh and
and dried
dried
mango peels are presented in Figure
mango peels are presented in Figure 5. 5.
Figure 5.
Figure 5. Total
Total protein
protein (TP)
(TP) content
contentininmango
mangopeel
peelextracts
extractsfrom
fromfresh
freshand
anddried peels
dried obtained
peels obtainedby
different
by extraction
different procedures
procedures fluid fluid
(SFE—supercritical extraction; SE—Soxhlet
extraction; extraction;
SE—Soxhlet UAE—
extraction;
ultrasound-assisted extraction; HAE—homogenization-assisted extraction) with different solvents
UAE—ultrasound-assisted extraction; HAE—homogenization-assisted extraction) with different
(CO2 + EtOH, EtOH, H2O). TP is expressed as mg protein per g of DW for dried peels or FW for
solvents (CO2 + EtOH, EtOH, H2 O). TP is expressed as mg protein per g of DW for dried peels or FW
fresh peels. Different le ers indicate significant difference (p < 0.05).
for fresh peels. Different letters indicate significant difference (p < 0.05).
The highest TP content was observed in the extract obtained from dried peels using
The highest TP content was observed in the extract obtained from dried peels using
UAE (EtOH) (2.95 mg protein/g DW), as presented in Figure 5. The explanation lies in the
UAE (EtOH) (2.95 mg protein/g DW), as presented in Figure 5. The explanation lies in
effective mechanical disintegration of the cell wall of mango peels, causing the release of
the effective mechanical disintegration of the cell wall of mango peels, causing the release
intracellular proteins. Consequently, since fresh mango peels had a high moisture content
of intracellular proteins. Consequently, since fresh mango peels had a high moisture
(77.74%), a higher protein concentration was observed in extracts from dried peels, indi-
content (77.74%), a higher protein concentration was observed in extracts from dried peels,
cating successful
indicating successful protein
proteinretention
retentionduring
duringthetheair-drying
air-dryingprocess.
process. The
The lowest TP content
lowest TP content
was detected in the UAE (H 2O) extract, with only 0.21 mg protein/g FW, and the SE (EtOH)
was detected in the UAE (H2 O) extract, with only 0.21 mg protein/g FW, and the SE (EtOH)
extract, with
extract, with 0.27
0.27mg mgprotein/g
protein/g FW.FW.
The TP content of
The TP content of fresh fresh mango
mango peels
peels was
was 1.9
1.9 to
to 2.3%,
2.3%, based
based on
on proximate
proximate composition
composition
analysis [17,98]. Using the Bradford method, the TP content of fresh mango peels peels
analysis [17,98]. Using the Bradford method, the TP content of fresh mango pro-
processed
cessed with acid-washed sand and sodium phosphate buffer
with acid-washed sand and sodium phosphate buffer using a mortar and pestle was using a mortar and pestle
was estimated
estimated to be to be 4.0–12.7
4.0–12.7 mg/gmg/g DW. Umbreen
DW. Umbreen et al.determined
et al. [99] [99] determined a TP content
a TP content of 6.55%of
6.55% in hot air-dried mango peel powder at 50–60
◦ °C. Hot-air-dried
in hot air-dried mango peel powder at 50–60 C. Hot-air-dried raw and ripe mango peel raw and ripe mango
peel powder
powder yieldedyielded 0.160.18
0.16 and andmg 0.18protein/g
mg protein/g
DW, DW, respectively
respectively [100].[100].
However, a comparison with the total protein
However, a comparison with the total protein concentration ofconcentration of extracts
extracts from
from mango
mango
peels could not be performed because, to the best of our knowledge, no study has been been
peels could not be performed because, to the best of our knowledge, no study has pub-
published
lished usingusing the same
the same extraction
extraction procedures
procedures as in
as in the the present
present studySE,
study (SFE, (SFE,
UAE,SE,HAE).
UAE,
HAE).Additionally, the activities of certain enzymes in mango peel extracts have been
Additionally,
determined the activities
using specific enzymaticof certain
assays,enzymes
as mango inpeels
mango peel
have extracts
been havetobeen
reported de-
contain
termined using specific enzymatic assays, as mango peels have been
various types of enzymes [101]. However, only a few studies are available regarding the reported to contain
various types
enzymatic of enzymes
activities of mango [101]. However,
peel extracts. only a fewobjective
Our main studies are
wasavailable
to extractregarding
and identifythe
enzymatic activities of mango peel extracts. Our main objective
valuable enzymes from mango peels, which could make an important contribution as was to extract and identify
valuable
an enzymes
exceptional fromofmango
source enzymes peels,
for which
furthercould make anin
applications important contribution
various areas as an
of medicine,
exceptionaland
cosmetics, source of enzymes
nutrition. for further enzyme
The determined applications in various
activities, definedareas of medicine,
as U/g protein, cos-
are
metics,inand
shown Tablenutrition.
2. The determined enzyme activities, defined as U/g protein, are
shown in Table 2.
Foods 2024, 13, 553 15 of 31
Table 2. Activities of certain enzymes in mango peel extracts from fresh and dried peels obtained by different extraction procedures with different solvents
(CO2 + EtOH, EtOH, H2 O), expressed as U per g of protein obtained in each extract. SFE—supercritical fluid extraction; SE—Soxhlet extraction; UAE—ultrasound-
assisted extraction; HAE—homogenization-assisted extraction. Different letters in the same column indicate significant difference (p < 0.05).
Activity (U/g protein ± SD)

Sample α-Amylase Cellulase Glucoamylase Laccase Lipase Peroxidase PPO Protease SOD TGM
Dried mango peels
SFE (CO2 + EtOH) 272.78 ± 7.91 d 37.93 ± 2.69 a 303.04 ± 15.12 a 154.23 ± 9.65 b 2155.15 ± 34.84 d 0.17 ± 0.00 a 329,109.98 ± 370.77 d 5.43 ± 0.08 a 12,400.67 ± 185.61 d 1.05 ± 0.00 a
SE (EtOH) 66.92 ± 4.85 c 72.63 ± 4.23 a 206.00 ± 16.3 a 36.08 ± 1.74 a 5502.79 ± 102.31 b 0.01 ± 0.00 a 99,992.16 ± 143.32 c 6.19 ± 0.31 a 9368.46 ± 42.63 b 3.88 ± 0.02 d
UAE (EtOH) 429.08 ± 12.88 e 124.15 ± 9.63d 260.77 ± 6.33 a 173.23 ± 5.32 b 1505.90 ± 34.65 a 0.19 ± 0.01 a 14,036.01 ± 125.32 a 3.08 ± 0.51b 106,502.73 ± 421.05 a 1.85 ± 0.01 a
UAE (H2 O) 110.36 ± 5.64 d 132.86 ± 9.61d 232.99 ± 8.51 a 35.05 ± 0.95 a 2885.41 ± 38.21 d 0.06 ± 0.00 a 122,938.17 ± 531.61 c 2.42 ± 0.09 b 17,302.85 ± 105.92 d 1.27 ± 0.04 a
HAE (H2 O) 9.94 ± 0.52 a 122.51 ± 10.32 d 592.60 ± 22.16 b 30.78 ± 0.75 a 18,643.62 ± 86.96 c - 407,622.96 ± 540.49 b - 47,844.09 ± 103.61 c 9.76 ± 0.02 b
Fresh mango peels
SE (EtOH) 1372.05 ± 53.65 b 22.97 ± 3.51 a 1375.13 ± 81.19 c 41.41 ± 2.36 a 3551.50 ± 58.45 e 0.01 ± 0.00 a 106,299.10 ± 654.61 c 8.05 ± 0.51 c 31,349.82 ± 205.15 c 10.06 ± 0.08 b
UAE (EtOH) 226.39 ± 9.71 d 38.98 ± 2.15 a 584.46 ± 27.77 b 124.74 ± 5.54 b 1160.53 ± 44.99 a 0.14 ± 0.01 a 27,350.05 ± 259.28 a 2.56 ± 0.25 b 95,360.96 ± 329.58 a 1.86 ± 0.03 a
b b c a b d b b
UAE (H2 O) 414.98 ± 25.91 e 238.14 ± 12.19 606.13 ± 32.84 228.88 ± 8.13 1847.06 ± 21.62 0.91 ± 0.05 342,415.39 ± 456.32 1.69 ± 0.23 7445.10 ± 89.51 2.99 ± 0.03 d
HAE (H2 O) 230.26 ± 21.71 d 460.42 ± 18.21 c 665.78 ± 25.48 b 53.24 ± 0.58 a 5189.92 ± 44.21 b - 430,837.99 ± 653.94 b - 30,210.78 ± 213.62 c 120.31 ± 0.21 c
Foods 2024, 13, 553 16 of 31
Antioxidant enzymes successfully protect fruits from oxidative stress and reduce
the content of reactive oxygen compounds (ROS), as they are involved in the catalytic
conversion of ROS and their by-products into stable, non-toxic molecules [102,103]. In
the present study, the activities of peroxidase, PPO, and SOD were determined among
the antioxidant enzymes. The activity of peroxidase was the lowest compared to the
activities of all the enzymes studied. However, in the reviewed literature, peroxidase
activity was significantly higher [17,98], though different substrates and methods for
activity determination were used. On the contrary, the activity of PPO, responsible for
enzymatic browning and melanogenesis [104], was very high in all mango peel extracts.
HAE was the most outstanding technique to obtain PPO in a highly active form. The cell
wall was successfully disrupted, but the mechanical force did not affect the enzyme. The
PPO activities ranged from 14,036.01 to 430,837.99 U/g protein, which is higher than in a
study by Tokas et al. [17] (25,800–75,500 U/g protein), where the mango peel extracts were
obtained by the maceration of fresh peels. The obtained extracts from mango peels are
rich in SOD, since the SOD activity was remarkable in the UAE (EtOH) extracts obtained
from fresh and dried peels. The enzyme SOD acts as an excellent therapeutic agent against
diseases caused by ROS and provides essential antioxidant protection against oxidative
stress [105]. The results of SOD activities in the present study are comparable to the SOD
activities in extracts from fresh mango peels obtained using Tris-HCl buffer as the extraction
medium [17]. It was found that the activity of SOD was successfully maintained during
drying, as the activities of extracts obtained from dried peels by the same extraction were
higher than those of fresh peels. In contrast, opposite results were obtained for PPO activity,
where the drying process partially affected the decrease in activity.
Furthermore, activities of various digestive enzymes were found in mango peel ex-
tracts. α-Amylase and glucoamylase are starch-degrading enzymes and are among the
most frequently used biocatalysts in the food industry [106]. Both enzymes were present in
their active forms in all extracts. For both enzymes, air-drying generally resulted in a loss
of their activities. Further, proteases are protein-degrading enzymes. Protease activity was
detected in most extracts obtained from mango peels using different extraction methods,
which coincides with the results in the literature [17,98]. Cellulase activity was generally
higher in all aqueous extracts, especially those from fresh peels. Regarding ethanolic
extracts, cellulase activities were higher in extracts obtained from dried peels, with the
highest activity obtained by the UAE (EtOH) extract from dried peels (124.15 U/g protein).
Cellulase plays an important role in the degradation of insoluble cellulose to soluble sugars.
It shows great potential for various industries, including agriculture, brewing, textiles,
pulp and paper, food, and biofuel production [107]. Most digestive enzyme activities
decreased with the drying process of mango peels. However, lipase seems to be resistant to
drying, as the activities were higher in the extracts obtained from dried peels regardless
of the extraction process and solvent used. These enzymes are also important in various
industries, including food, biofuels, detergents, and animal feed [108].
All extracts from mango peels also yielded laccase activity, ranging from 30.78 U/g
protein (HAE (H2 O) extract from dried peels) to 228.88 U/g protein (UAE (H2 O) extract
from fresh peels). Laccases are multifunctional biocatalysts that play an important role
in various applications, including the environmental field, the cosmetics industry, food
processing, and the textile industry [109]. TGM was also found in its active form in all
extracts, although the activity in the HAE (H2 O) extract from fresh mango peels was
significantly higher (120.31 U/g protein) than in other extracts. This can be attributed to
the extraction process used (HAE), as the cell wall was successfully disrupted, and thus,
the extraction of the TGM in its active form was achieved. In general, TGM activity was
lower in extracts from dried mango waste. TGMs are also multifunctional enzymes [110].
From a health perspective, TGMs can reduce allergies, control energy intake from food,
and act as mediators in wound healing [111].
The ripening of the mango fruit can lead to a progressive decrease in the activity of
antioxidant enzymes, while the activity of digestive enzymes increases [17]. In addition, the
Foods 2024, 13, 553 17 of 31
extraction procedure, solvent, and operating conditions also significantly impact enzyme
activities in the final product [98].
3.6. Antimicrobial Activity of Mango Peel Extracts

Infectious diseases represent a growing global problem, making searching for new
potential antimicrobial agents increasingly important. Due to the improper and excessive
use of synthetic antimicrobials, various pathogenic microorganisms that cause serious
diseases have developed resistance [112,113]. For example, E. coli can cause different ill-
nesses, including diarrhea, urinary tract infections, respiratory illness, and bloodstream
infections [114]. P. aeruginosa is associated with causing severe infections and the spread
of antimicrobial resistance in vivo [115]. B. cereus is known as a foodborne pathogen that
can cause two types of gastrointestinal disease: emetic (vomiting) syndrome and diarrheal
syndrome [116]. S. aureus is responsible for various skin infections up to severe invasive in-
fections of the lungs or heart [117]. A. brasiliensis is mostly responsible for allergic reactions
and lung infections [118], while C. albicans is the main cause of candidiasis and primary
fungal infections [119]. Therefore, natural materials and extracts are receiving increasing
attention, as they represent a new source of important biologically active compounds
with potential antimicrobial properties [120]. For example, extracts from fruit wastes that
contain high levels of bioactive compounds with antimicrobial properties, such as mango
peels, could be used as effective, alternative, and safe natural remedies [121]. Thus, a
comprehensive and comparative antimicrobial study of the different extracts of fresh and
dried mango peels was conducted.
3.6.1. Qualitative Antimicrobial Determination of Mango Peel Extracts

The antimicrobial properties of mango peel extracts were preliminarily evaluated
using the qualitative disk diffusion method. The susceptibility of Gram-negative (E. coli,
P. aeruginosa) and Gram-positive bacterial species (S. aureus, B. cereus) and fungi (yeast
C. albicans, black mold A. brasiliensis) to the addition of mango peel extracts was studied.
Table 3 shows the measured diameters of the inhibition zone (in mm) of the individual
microorganism tested after exposure to the mango peel extracts and ciprofloxacin as a
positive control.
B. cereus appeared to be the most susceptible to all mango peel extracts among the
tested bacterial species. The largest inhibition zone was measured during exposure of
B. cereus to the EtOH extract obtained by UAE (22 mm) and SF (19 mm) from fresh peels,
followed by extracts from dried peels. B. cereus, on the other hand, was the most resistant
to the UAE (H2 O) extract from dried peels. The second most sensitive bacterial strain to the
mango peel extracts tested was E. coli. Exposure of E. coli to the UAE (EtOH) extract from
dried and fresh peels resulted in the largest zone of inhibition of all the samples tested.
The HAE (H2 O) extract showed the lowest inhibitory effect against E. coli. Similar growth
inhibition was obtained regarding the antimicrobial efficacy of mango peel extracts on
P. aeruginosa and S. aureus growth. Both bacteria were the least susceptible to the addition of
the HAE (H2 O) extract from fresh and dried peels. Table 3 also shows that ciprofloxacin was
effective against all bacterial species tested. The most sensitive strains were P. aeruginosa
and B. cereus, as the highest zone of inhibition was determined.
All the extracts obtained from mango peels successfully inhibited the growth of the
tested bacterial species, which is an important observation for the possibility of further
use as successful natural antibacterial agents. Generally, higher inhibition zones were
achieved with extracts obtained by extraction procedures using EtOH as an extraction
solvent or co-solvent. The most outstanding sample with the highest microbial sensitivity
when exposed to all four bacterial species was the UAE (EtOH) extract from fresh peels.
The extraction solvents were almost completely removed before analysis. Therefore, their
influence on the antimicrobial potential of the extracts is negligible. In addition, using the
same extraction method, slightly higher growth inhibition was obtained using extracts
from fresh mango peels than dried peels.
Foods 2024, 13, 553 18 of 31
Table 3. Antimicrobial activity of mango peel extracts from fresh and dried peels obtained by different
extraction procedures with different solvents (CO2 + EtOH, EtOH, H2 O) and ciprofloxacin as a posi-
tive control, expressed as a zone of inhibition (mm). SFE—supercritical fluid extraction; SE—Soxhlet
extraction; UAE—ultrasound-assisted extraction; HAE—homogenization-assisted extraction.
Zone of Inhibition (mm ± SD)

Sample Gram-Positive Bacteria Gram-Negative Bacteria
E. coli P. aeruginosa B. cereus S. aureus
Dried mango peel
SFE (CO2 + EtOH) 17 ± 1 10 ± 2 14 ± 1 10 ± 2
SE (EtOH) 18 ± 1 14 ± 1 18 ± 0 15 ± 1
UAE (EtOH) 19 ± 1 14 ± 2 16 ± 2 14 ± 1
UAE (H2 O) 14 ± 2 7±1 10 ± 1 9±1
HAE (H2 O) 12 ± 1 9±1 12 ± 1 7±1
Fresh mango peel
SE (EtOH) 18 ± 2 16 ± 2 19 ± 1 16 ± 1
UAE (EtOH) 21 ± 1 16 ± 1 22 ± 2 15 ± 2
UAE (H2 O) 17 ± 2 10 ± 1 15 ± 1 11 ± 1
HAE (H2 O) 10 ± 1 8±1 13 ± 1 7±0
Ciprofloxacin 43 ± 1 55 ± 0 52 ± 1 41 ± 1
The results are in agreement with the reviewed literature, which also indicates good
antibacterial activity of various extracts from mango peels, with the inhibitory potential
of EtOH extracts being higher than those of H2 O extracts. Methanolic mango peel extract
successfully inhibited the growth of S. aureus, Staphylococcus epidermidis, P. aeruginosa,
E. coli, and Salmonella sp. [8,122]. The results of the antibacterial activity of ripe and unripe
methanolic mango peel extracts show a larger inhibition zone against all tested bacterial
species by ripe mango peel extracts [123]. Also, ethanolic mango peel extract showed
inhibition properties against E. coli, S. aureus, B. cereus, P. aeruginosa, S. typhimurium, and
V. parahaemolyticus [20,124].
The remarkable antibacterial potential of mango peel extract obtained by various
methods is consistent with the fact that various phytochemicals and bioactive compounds
are responsible for the disruption of the cell membrane of microbial cells, in addition
to their ability to scavenge free radicals, activate endothelial cell migration, and inhibit
inflammatory and pain pathways [8]. In contrast, no inhibitory properties of mango peel
extracts were found against the tested fungal species (yeast C. albicans and black mold
A. brasiliensis).
3.6.2. Quantitative Antimicrobial Determination of Mango Peel Extracts

Considering the excellent inhibitory effect on the tested bacterial strains (E. coli,
P. aeruginosa, S. aureus, and B. cereus), the antimicrobial activity was additionally deter-
mined after exposure to seven different concentrations of the extracts (0.05–5.0 mg/mL)
using a more accurate quantitative microbroth dilution method. MGIR values were deter-
mined after 12 and 24 h exposure periods of the bacterial strain to different concentrations
of the tested mango peel extracts. In addition, MIC90 values were determined based on the
MGIR value when at least 90% MGIR was achieved.
Figure 6 shows the results of the antimicrobial study on the growth of Gram-negative
bacterium E. coli as performed by the microbroth dilution method.
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31
Figure 6. Microbial growth inhibition rate (%) and MIC90 values (mg/mL) for different concentra-
Figure 6. Microbial growth inhibition rate (%) and MIC90 values (mg/mL) for different concentrations
tions of mango peel extracts from dried (a) and fresh peels (b) obtained by different extraction pro-
of mango peel extracts from dried (a) and fresh peels (b) obtained by different extraction procedures
cedures (SFE—supercritical fluid extraction; SE—Soxhlet extraction; UAE—ultrasound-assisted ex-
(SFE—supercritical fluid extraction; SE—Soxhlet
traction; HAE—homogenization-assisted extraction;
extraction) UAE—ultrasound-assisted
with different extraction;
solvents (CO2 + EtOH, EtOH,
H2O) against Gram-negative bacterium E. coli.
HAE—homogenization-assisted extraction) with different solvents (CO 2 + EtOH, EtOH, H 2 O) against
Gram-negative bacterium E. coli.
All mango peel extracts, with the exception of the HAE (H2O) extract from fresh
peels,All mango
were verypeel extracts,inwith
successful the exception
inhibiting of the
the growth ofHAE (H2 O) extract
Gram-negative from fresh
bacterium peels,
E. coli at
were very successful in inhibiting the growth of Gram-negative bacterium E. coli at the
the highest concentration tested (5 mg/mL) after 12 h and 24 h of incubation. In general,
highest concentration tested (5 mg/mL) after 12 h and 24 h of incubation. In general, E. coli
E. coli was more susceptible to ethanolic than aqueous extracts. A slightly higher suscep-
was more susceptible to ethanolic than aqueous extracts. A slightly higher susceptibility of
tibility of E. coli to aqueous extracts from dried peels was observed, indicating successful
E. coli to aqueous extracts from dried peels was observed, indicating successful retention
retention of bioactive compounds regardless of the drying process. With respect to the
of bioactive compounds regardless of the drying process. With respect to the HAE (H2 O)
HAE (H2O) extract from fresh mango peels, the MIC90 value of the tested concentrations
extract from fresh mango peels, the MIC90 value of the tested concentrations could not be
could not be determined. The analyses would have to be performed at higher extract con-
determined. The analyses would have to be performed at higher extract concentrations to
centrations to obtain more reliable results. Nevertheless, a relatively high MIGR (76.26%)
obtain more reliable results. Nevertheless, a relatively high MIGR (76.26%) was achieved at
was achieved at the highest applied extract concentration after 24 h of incubation.
the highest applied extract concentration after 24 h of incubation.
Figure 7 shows the results of the antimicrobial study on the growth of the second
Figure 7 shows the results of the antimicrobial study on the growth of the second Gram-
Gram-negative bacterial strain, P. aeruginosa, as performed by the microbroth dilution
negative bacterial strain, P. aeruginosa, as performed by the microbroth dilution method.
method.
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31
HAE—homogenization-assisted extraction) with
H2O) against Gram-negative bacterium P. aeruginosa.different solvents (CO 2 + EtOH, EtOH, H 2 O) against
Gram-negative bacterium P. aeruginosa.
P. aeruginosa proved to be slightly more resistant to mango peel extracts than E. coli.
P. aeruginosa proved to be slightly more resistant to mango peel extracts than E. coli.
Overall, P. aeruginosa, like E. coli, was generally more sensitive to ethanolic than aqueous
Overall, P. aeruginosa, like E. coli, was generally more sensitive to ethanolic than aqueous
extracts. Among the aqueous extracts, only the UAE (H2O) extract from dried mango peels
extracts. Among the aqueous extracts, only the UAE (H2 O) extract from dried mango peels
successfully inhibited the growth of the above bacterium, with an effective inhibition of
successfully inhibited the growth of the above bacterium, with an effective inhibition of
more than 90% at 5.0 mg/mL. Exceptional results were obtained at the lowest concentra-
more than 90% at 5.0 mg/mL. Exceptional results were obtained at the lowest concentration
tion (0.05 mg/mL) of the extracts used, with MGIR values above 35% for seven extracts
(0.05 mg/mL) of the extracts used, with MGIR values above 35% for seven extracts and
and above 50% for four of the nine tested extracts, with the highest percentage for the SFE
above 50% for four of the nine tested extracts, with the highest percentage for the SFE
(CO2 + EtOH) extract (63.11%). This is a remarkable result, because P. aeruginosa has be-
(CO2 + EtOH) extract (63.11%). This is a remarkable result, because P. aeruginosa has become
come highly resistant to antibiotics. Therefore, new approaches to the treatment of infec-
highly resistant to antibiotics. Therefore, new approaches to the treatment of infections
tions are needed. In this regard, mango peel extracts, especially the SFE (CO2 + EtOH)
are needed. In this regard, mango peel extracts, especially the SFE (CO2 + EtOH) extract,
extract,make
would wouldanmake an important
important contribution
contribution even at
even at lower lower concentrations.
concentrations.
Figure 8 shows the results of the antimicrobial study on
Figure 8 shows the results of the antimicrobial study on the
the growth
growth of
of the
the Gram-
Gram-
positive bacterium B. cereus performed by the microbroth dilution method.
positive bacterium B. cereus performed by the microbroth dilution method.
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H2O) against Gram-positive bacterium
HAE—homogenization-assisted B. cereus.
extraction) with different solvents (CO2 + EtOH, EtOH, H2 O) against
Gram-positive bacterium B. cereus.
Excellent results in terms of inhibitory potential were obtained when Gram-positive
Excellent
bacterium results
B. cereus was inexposed
terms oftoinhibitory potential
mango peel were
extracts. obtained
Complete when Gram-positive
inhibition was achieved
bacterium B. cereus
at the highest concentration tested for all extracts, except the HAE (H2O)was
was exposed to mango peel extracts. Complete inhibition achieved
extract from
at the highest concentration tested for all extracts, except the HAE (H2 O) extract from
dried peels. Ethanolic extracts, especially extracts obtained by UAE from fresh and dried
dried peels. Ethanolic extracts, especially extracts obtained by UAE from fresh and dried
mango peels and SE from dried peels, are promising for achieving exceptional growth
mango peels and SE from dried peels, are promising for achieving exceptional growth
inhibition of B. cereus. Even at a lower added concentration of extracts (0.25 mg/mL), the
inhibition of B. cereus. Even at a lower added concentration of extracts (0.25 mg/mL), the
bacterium was very susceptible to the mentioned extracts, as the MGIR was still above
bacterium was very susceptible to the mentioned extracts, as the MGIR was still above 90%.
90%. After 24 h exposure to mango peel extracts, B. cereus proved to be highly or com-
After 24 h exposure to mango peel extracts, B. cereus proved to be highly or completely
pletely resistant at lower concentrations of the extracts tested (0.10 and 0.05 mg/mL). Only
resistant at lower concentrations of the extracts tested (0.10 and 0.05 mg/mL). Only the
the SFE (CO2 + EtOH) extract still strongly inhibited the growth of the bacterium consid-
SFE (CO2 + EtOH) extract still strongly inhibited the growth of the bacterium considered,
ered, with 60.75% MGIR at 0.10 mg/mL. B. cereus mainly causes intestinal infections but
with 60.75% MGIR at 0.10 mg/mL. B. cereus mainly causes intestinal infections but can
can also cause various eye infections, wounds, skin infections, and respiratory infections.
also cause various eye infections, wounds, skin infections, and respiratory infections.
Therefore, mango peel extracts, especially the SFE (CO2 + EtOH) extract, would contribute
Therefore, mango peel extracts, especially the SFE (CO2 + EtOH) extract, would contribute
significantly to
significantly to maintaining
maintainingeffective
effectiveantimicrobial
antimicrobialactivity
activityagainst
against
B.B. cereus
cereus growth
growth even
even at
at lower
lower concentrations.
concentrations.
Figure 99 shows
Figure shows thethe results
results of
of the
the antimicrobial
antimicrobial study
study on
on the
the growth
growth ofof the
the second
second
Gram-positive bacterium,
Gram-positive bacterium, S.S. aureus,
aureus, performed
performed byby the
the microbroth
microbroth dilution
dilution method.
method.
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Figure 9. Microbial growth inhibition rate (%) and MIC90 value (mg/mL) for different concentrations
Figure 9. Microbial growth inhibition rate (%) and MIC90 value (mg/mL) for different concentrations
of mango peel extracts from dried (a) and fresh peels (b) obtained by different extraction procedures
(SFE—supercritical fluid extraction; SE—Soxhlet extraction; UAE—ultrasound-assisted extraction;
(SFE—supercritical fluid extraction;
HAE—homogenization-assisted SE—Soxhlet
extraction) withextraction;
different UAE—ultrasound-assisted extraction;
solvents (CO2 + EtOH, EtOH, H2O)
against Gram-positive bacteriumextraction)
HAE—homogenization-assisted S. aureus. with different solvents (CO2 + EtOH, EtOH, H2 O) against
Gram-positive bacterium S. aureus.
The second Gram-positive bacterium tested was S. aureus, which was very sensitive
to allThe second
mango peelGram-positive
extracts afterbacterium
12 h of incubation. S. aureus, which
tested wasHowever, after 24was very sensitive
h exposure, to
the in-
all mango peel extracts after 12 h of incubation. However, after 24 h exposure, the inhibitory
hibitory potential of the HAE (H2O) extract and UAE (H2O) extract from fresh peels de-
potential of the HAE (H2 O) extract and UAE (H2 O) extract from fresh peels decreased.
creased. Moreover, complete resistance of S. aureus was observed at lower added concen-
Moreover, complete resistance of S. aureus was observed at lower added concentrations of
trations of the extracts. In contrast, their counterparts, the extracts obtained from dried
the extracts. In contrast, their counterparts, the extracts obtained from dried peels, showed
peels, showed excellent inhibition even after 24 h, although similar complete resistance
excellent inhibition even after 24 h, although similar complete resistance was obtained at
was obtained at the lowest concentrations. On the other hand, ethanolic extracts were
the lowest concentrations. On the other hand, ethanolic extracts were found to possess
found to possess remarkable antimicrobial properties, with an MGIR above 90% in the
remarkable antimicrobial properties, with an MGIR above 90% in the case of exposure of
case of exposure of S. aureus to the UAE (EtOH) extract from dried peels and the SE (EtOH)
S. aureus to the UAE (EtOH) extract from dried peels and the SE (EtOH) extract from fresh
extract from fresh peels after 24 h of incubation, resulting in 0.5 mg/mL as MIC90. In the
peels after 24 h of incubation, resulting in 0.5 mg/mL as MIC90 . In the case of the UAE
case of the UAE (EtOH) extract from fresh peels and the SFE (CO2 + EtOH) extract, a lower
(EtOH) extract from fresh peels and the SFE (CO2 + EtOH) extract, a lower MIC90 was
MIC90 was obtained after 12 h than after 24 h. This indicates that S. aureus can adapt to the
obtained after 12 h than after 24 h. This indicates that S. aureus can adapt to the bioactive
bioactive compounds
compounds with antimicrobial
with antimicrobial potential inpotential in the
the extracts extracts
during duringexposure,
prolonged prolongedtoexpo-
such
sure, to such an extent that the MGIR at 1.0 mg/mL decreased
an extent that the MGIR at 1.0 mg/mL decreased from 94.61 to 41.04% when from 94.61 to 41.04% when
exposed to
exposed
the SFE (COto the+ SFE
EtOH)(COextract.
2 + EtOH) extract. The antimicrobial properties of plant materials
The antimicrobial properties of plant materials are mainly
2
are mainlyto
attributed a the
ributed to the
content of content
variousof various bioactive
bioactive compounds compounds
[125–127].[125–127]. The mech-
The mechanism of
anism of
action of PCs
action of PCsto
is related is bacterial
related to bacterial damage,
membrane membrane damage,ofinhibition
inhibition virulence of virulence
factors such
factors such as enzymes and toxins, and suppression of bacterial biofilm formation [128].
Foods 2024, 13, 553 23 of 31
as enzymes and toxins, and suppression of bacterial biofilm formation [128]. All phenolic
acids investigated in the present study, caffeic acid [129,130], chlorogenic acid [131,132],
ellagic acid [133,134], and gallic acid [135,136], were found to have significant antimicro-
bial activity. In addition, flavonoids have multiple bacteria cellular targets due to their
chemical structure. Antibacterial activity is related to their interaction with cell mem-
branes [125]. Flavonoids such as catechin [137,138], hesperidin/neohesperidin [139,140],
and rutin [141,142] have antimicrobial potential, according to studies by other authors.
Furthermore, mangiferin, an important PC found in all parts of the mango plant, exhibits
significant antibacterial activity against both Gram-negative and Gram-positive bacterial
strains [19,143].
Thus, we can confirm that the presence of specific PCs (xanthones, phenolic acids, and
flavonoids) detected in the extracts obtained contributes significantly to the antimicrobial
properties of the samples studied. The SE (EtOH) extracts contained all the PCs tested,
with the exception of rutin. Since the content of chlorogenic acid, ellagic acid, caffeic
acid, and catechin was very high, it can be confirmed that they synergistically affected the
inhibition of the tested bacterial species. The UAE (EtOH) extract of dried peels yielded
lower amounts of the tested PCs. However, the synergistic effect of the different PCs was
remarkable, as the tested bacterial species were most susceptible to the presence of the UAE
(EtOH) extract. On the other hand, the aqueous extracts were rich in certain PCs studied,
such as gallic acid, ellagic acid, and catechin. However, chlorogenic acid was only detected
in the UAE (H2 O) extract from the dried peels. Due to the absence of chlorogenic acid
in the aqueous extracts, the synergistic effect was not the same as in the case of the UAE
(H2 O) extract from dried peels and ethanolic extracts. Therefore, a lower inhibitory effect
on bacterial growth of the aqueous extracts was achieved. In the SFE (CO2 + EtOH) extract,
all investigated PCs were present except rutin. Since CO2 is a non-polar solvent, moderate
amounts of PCs were detected, which were successfully extracted by the addition of the
polar co-solvent EtOH. Nevertheless, good antimicrobial efficacy was observed. In this
case, the antimicrobial properties can most likely be attributed to the synergistic effect of
different polar PCs and non-polar compounds.
Although antimicrobial properties are attributed to the whole mango tree due to
its content of numerous important bioactive compounds, there are only a small number
of studies covering the quantitative antimicrobial activity of mango peel extracts. Zafra
Ciprián et al. [144] determined much higher MIC values for the ethanolic extract and the
hydroethanolic extract of mango peel, namely over 100 mg/mL for E. coli and 25 mg/mL
and 12.5 mg/mL, respectively, for S. aureus. On the other hand, an MIC of the UAE (EtOH)
extract of 0.25 mg/mL was found for E. coli [145]. Riberio et al. [146] determined an
MIC value of 2.50 mg/mL for E. coli and 0.30 mg/mL for S. aureus for methanolic mango
peel extracts, whereas no inhibitory effect was found for aqueous extracts. Vega-Vega
et al. [147] achieved complete inhibition of E. coli and S. aureus with various extracts of
mango peels (methanolic polar, methanolic nonpolar, ethanolic polar, and ethanolic non-
polar) at a concentration of 25 mg/mL. In a study by Espinosa-Espinosa et al. [8], the
MIC values for methanolic mango peel extract were 4.0 mg/mL for E. coli, S. aureus, and
P. aeruginosa. Rakholiya et al. [148] determined MIC values of 1.25 mg/mL for B. cereus,
E. coli, P. aeruginosa, and C. albicans with aqueous mango peel extract. Furthermore, mango
peel extracts also show improvement in the antimicrobial activity of antibiotics. The addi-
tion of ethanolic mango peel extract to three different antibiotics (norfloxacin, erythromycin,
and tetracycline) showed a synergistic interaction that resulted in a reduction in S. aureus
antibiotic resistance [149].
According to the reviewed literature, the quantitative determination of the microbial
growth inhibition rate of mango peels has not yet been performed, especially concerning
the comparison of extracts from fresh and dried peels and the preservation of antimicrobial
potential during the drying of raw peels.
Foods 2024, 13, 553 24 of 31
3.7. The Most Promising Mango Peel Extracts for Various Applications
The study revealed that most of the bioactive compounds studied were successfully
preserved, indicating that the air-drying method is a successful and economical method
for the pretreatment of mango waste before the extraction process. Overall, extraction
methods using EtOH as a solvent (SE, UAE) or a co-solvent (SEF) (Table 4) were found to
be more effective compared with methods using H2 O (UAE, HAE). In addition, extracts
from dried peels generally exhibited a higher profile of bioactive compounds than extracts
from fresh mango peels due to the high moisture content in fresh peels (77.74%); the SE
(EtOH) extract resulted in the highest TPC content and total content of analyzed PCs.
The SFE (CO2 + EtOH) extract was the most abundant in PAC content. Furthermore, the
UAE (EtOH) extract exhibited the highest antioxidant activity and TP content. Among the
analyzed enzymes, antioxidant enzymes SOD and PPO activities stood out. Furthermore,
the extracts were also found to contain lipase in a highly active form.
Table 4. The properties and content of the three most promising mango peel extracts suitable for
various applications.
SFE (CO2 + EtOH) SE (EtOH) UAE (EtOH)

Total phenolic content (mg GAE/g DW) 1.7 ± 0.0 25.0 ± 0.4 7.9 ± 0.2
Proanthocyanidin content (mg PAC/g DW) 0.4 ± 0.1 0.3 ± 0.1 0.2 ± 0.0
Phenolic compounds present in high concentrations Chlorogenic acid, ellagic acid, gallic acid, catechin
Antioxidant activity (% inhibition) 41.4 ± 1.3 59.9 ± 1.8 82.4 ± 1.8
E. coli 2.5 1.0 1.0
Antibacterial Gram-negative bacteria
P. aeruginosa 2.5 2.5 2.5
potential (MIC90
after 24 h) B. cereus 0.5 0.25 0.25
Gram-positive bacteria
S. aureus 2.5 1.0 0.5
Total protein content (mg protein/g DW) 0.41 ± 0.04 2.68 ± 0.06 2.95 ± 0.10
Enzymes present in their highly active form α-Amylase, cellulase, glucoamylase, laccase, lipase, PPO, SOD
Regarding antimicrobial potential, mango peel extracts, especially ethanolic extracts,

represent a potential natural antimicrobial agent as an alternative to conventional antimi-
crobial agents, as successful growth inhibition was observed in all bacterial species tested.
The most susceptible bacterial strain tested was the Gram-positive bacterium B. cereus,
while the most resistant was the Gram-negative bacterium P. aeruginosa.
Besides UAE (EtOH) and SE (EtOH) extracts, the SFE (CO2 + EtOH) extract was a
promising extract despite the slightly lower inhibition rates for bacterial growth. SFE is
considered a green, sustainable, and environmentally friendly process, in which only small
amounts of EtOH can be used as a co-solvent. The use of an unconventional extraction
technique is becoming increasingly important, as concerns about the impact of organic
solvents on the environment and on extracts have increased in recent years.
The UAE (EtOH), SE (EtOH), and SFE (CO2 + EtOH) extracts are important for provid-
ing antibacterial and antioxidant activity and, in addition, represent a good source of certain
enzymes in their highly active form, for potential use in various areas of bioengineering.
4. Conclusions
With increasing health and healthy diet awareness, more fruits are being processed,
resulting in the generation of enormous amounts of agro-industrial waste from fruit by-
products, which has a negative impact on the environment. As they have proven to be a rich
source of important bioactive substances with health-promoting benefits, mango peels have
been analyzed as a promising source of various bioactive compounds with antioxidant,
enzymatic, and antimicrobial activities. Furthermore, a comparison was conducted between
Foods 2024, 13, 553 25 of 31
extracts from fresh and dried peels to show the effects of an undemanding and inexpensive
air-drying method on the maintenance of biological activity. Various conventional (SE,
UAE, HAE) and unconventional (SFE) methods were used for the extraction of mango peels.
Differences in the extraction yields obtained, bioactive compound content, and biological
activity were found due to the different methods, extraction solvents (EtOH, H2 O, CO2
with EtOH as co-solvent), operating conditions, and starting material (fresh or dried peels).
Of all the extracts tested, the UAE (EtOH), SE (EtOH), and SFE (CO2 + EtOH) extracts
from dried mango peels generally stood out as a good source of bioactive compounds
in high concentrations. They, therefore, showed good antibacterial, antioxidant, and
enzymatic activity.
To the best of our knowledge, there is no comprehensive and detailed study in the
literature comparing different extracts of fresh and dried mango peels and their biological
activity as obtained by conventional and unconventional methods. Therefore, the present
study makes an important contribution to this field. Mango peels thus represent remarkable
waste products of the fruit industry that could contribute to the sustainable development
of exceptional high-value-added products for various industries, such as pharmaceuticals,
cosmetics, and food. For example, bioactive compounds from mango peels are beneficial
for the stabilization of cosmetic products due to their antimicrobial and antioxidant activity.
They can also provide a soothing effect on the skin. In this case, however, further studies
need to be carried out, particularly with regard to the cytotoxicity of the mango peel extracts
obtained. Additionally, mango peel extracts can also be used in food packaging to prevent
microbial contamination and in the manufacture of food supplements.
Author Contributions: Conceptualization, M.L. and M.P.; methodology, M.L., M.P., P.K. and N.K.;
formal analysis, N.K. and P.K.; data curation, N.K.; writing—original draft preparation, N.K.;
writing—review, M.L. and M.P.; writing—editing, N.K.; visualization, N.K.; supervision, M.L. and
M.P.; funding acquisition, M.L. and Ž.K. All authors have read and agreed to the published version
of the manuscript.
Funding: This research was supported by the Slovenian Research Agency (ARIS) within the frame
program P2-0046 (Separation Processes and Production Design), project No. J2-3037 (Bionanotech-
nology as a tool for stabilization and applications of bioactive substances from natural sources),
project No. L2-4430 (Production, Isolation and Formulation of Health Beneficial Substances from
Helichrysum italicum for Applications in Cosmetic Industry), and young researcher ARIS fellowship
contract number No. 1514/FKKT-2023.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Acknowledgments: The authors acknowledge the use of research equipment for the production
of biological substances and their detection, procured within the project “Upgrading national re-
search infrastructures—RIUM”, which was co-financed by the Republic of Slovenia, the Ministry of
Higher Education, Science and Innovation, and the European Union from the European Regional
Development Fund, and the system of downstream processes for performance of obtaining biological
substances (Package 21, ARIS).
Conflicts of Interest: The authors declare no conflicts of interest.
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Foods 2024, 13, 553. https://doi.org/10.3390/foods13040553 https://www.mdpi.com/journal/foods

2. Materials and Methods

2.3. Preparation of Plant Material

2.4. Moisture Content

2.5. Thermogravimetric Analysis/Differential Scanning Calorimetry

2.6. Extraction Procedures

2.6.1. Soxhlet Extraction

2.6.2. Ultrasound-Assisted Extraction

2.6.3. Homogenization-Assisted Extraction

2.6.4. Supercritical Fluid Extraction

2.6.5. Extraction Yield Determination

2.7. Quantitative Determination of Total Phenols and Proanthocyanidins

2.7.2. Determination of Total Proanthocyanidin Content (PAC)

2.8. Determination of Antioxidant Activity

2.9. Liquid Chromatography with Tandem Mass Spectrometry Analysis

2.10. Determination of Total Protein (TP) Content

2.11. Determination of Enzyme Activities

2.12. Determination of Antimicrobial Activity

2.12.1. Qualitative Disk Diffusion Method

2.12.2. Quantitative Microbroth Dilution Method

2.13. Statistical Analysis

3. Results and Discussion

3.1. Thermal Stability of Mango Peels

3.2. Extraction Yield Determination

3.2. Extraction Yield Determination

3.2. Extraction Yield Determination

3.4. Content of Important Phenolic Compounds in Mango Peel Extracts

Content (µg PC/g DW) Content (µg PC/g FW)

Foods 2024, 13, 553

Activity (U/g protein ± SD)

3.6. Antimicrobial Activity of Mango Peel Extracts

3.6.1. Qualitative Antimicrobial Determination of Mango Peel Extracts

Zone of Inhibition (mm ± SD)

3.6.2. Quantitative Antimicrobial Determination of Mango Peel Extracts

SFE (CO2 + EtOH) SE (EtOH) UAE (EtOH)

Regarding antimicrobial potential, mango peel extracts, especially ethanolic extracts,

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