foods

Article
Manufacture of a Potential Antifungal Ingredient Using Lactic
Acid Bacteria from Dry-Cured Sausages
Tiago de Melo Nazareth * , Jorge Calpe , Carlos Luz , Jordi Mañes and Giuseppe Meca

Department of Food Science and Toxicology, Faculty of Pharmacy, University of Valencia, Ave. Vicent Andrés
Estellés s/n, 46100 Burjassot, Spain
* Correspondence: [email protected]; Tel.: +34-96-354-4959

Abstract: The growing interest in functional foods has fueled the hunt for novel lactic acid bacteria
(LAB) found in natural sources such as fermented foods. Thus, the aims of this study were to isolate,
identify, characterize, and quantify LAB’s antifungal activity and formulate an ingredient for meat
product applications. The overlay method performed a logical initial screening by assessing isolated
bacteria’s antifungal activity in vitro. Next, the antifungal activity of the fermented bacteria-free
supernatants (BFS) was evaluated by agar diffusion assay against six toxigenic fungi. Subsequently,
the antifungal activity of the most antifungal BFS was quantified using the microdilution method in
96-well microplates. The meat broth that showed higher antifungal activity was selected to elaborate
on an ingredient to be applied to meat products. Finally, antifungal compounds such as organic acids,
phenolic acids, and volatile organic compounds were identified in the chosen-fermented meat broth.
The most promising biological candidates belonged to the Lactiplantibacillus plantarum and Pediococcus
pentosaceus. P. pentosaceus C15 distinguished from other bacteria by the production of antifungal
compounds such as nonanoic acid and phenyl ethyl alcohol, as well as the higher production of lactic
and acetic acid.

Keywords: Pediococcus pentosaceus; Lactiplantibacillus plantarum; antifungal activity; organic acids;

phenolic acids; volatile organic compounds

Citation: Nazareth, T.d.M.; Calpe, J.;

Luz, C.; Mañes, J.; Meca, G.
Manufacture of a Potential
1. Introduction
Antifungal Ingredient Using Lactic
Acid Bacteria from Dry-Cured Due to their high nutritional content, meat and its derivatives are an important food
Sausages. Foods 2023, 12, 1427. category in many people’s diets. Consuming processed meats such as sausages, hot dogs,
https://doi.org/10.3390/ and luncheon meats has grown mainstream [1]. Between 1998 and 2018, global meat
foods12071427 consumption rose by 58%, reaching a total of 360 million tonnes [2].
Dry-cured meat products are a type of traditional food that are manufactured and
Academic Editors: Luis M. Medina
and Fernando Pérez-Rodríguez
eaten across the globe. Their market dominance is well-known, owing to customers’
stringent expectations for high-quality and safe foodstuffs. Consumption of these fungus-
Received: 22 February 2023 infected foods increases the risk of exposure to mycotoxins; this is a worldwide public
Revised: 20 March 2023 health concern [3].
Accepted: 23 March 2023 Dry-cured meats mostly comprise muscle tissue, and their surface physical-chemical
Published: 27 March 2023
characteristics, such as low water activity (Aw ), neutrality to low pH, high salt content,
and protein content, influence the microbial flora that grows on their exterior layers [4].
Although dry-fermented sausages have low Aw and high salt content, alterations of these
Copyright: © 2023 by the authors.
properties influence the metabolism by facilitating mycotoxin biosynthesis [5].
Licensee MDPI, Basel, Switzerland. Several species are recognized for producing mycotoxins, including Aspergillus, Al-
This article is an open access article ternaria, Claviceps, Fusarium, and Penicillium [6]. In certain environmental and substrate
distributed under the terms and conditions over meat products, Aspergillus flavus, A. parasiticus, A. ochraceus, P. nordicum, P.
conditions of the Creative Commons polonicum, and P. verrucosum produce mycotoxins, posing a risk to consumers through their
Attribution (CC BY) license (https:// growth in salami, dry-cured hams, and other meat products [7,8].
creativecommons.org/licenses/by/ Penicillium nordicum is the most important ochratoxin A (OTA) producing species fre-
4.0/). quently detected in cold-chain protein foods such as dry-cured ham, salami, and salted fish.

Foods 2023, 12, 1427. https://doi.org/10.3390/foods12071427 https://www.mdpi.com/journal/foods

Foods 2023, 12, 1427 2 of 22

In particular, this fungus grows well at temperatures of about 15 ◦ C and salt concentrations
of more than 5% NaCl [9].
The consumption of cereals contaminated with mycotoxins may result in the accumu-
lation of these toxins in the organs of both animals and humans, thereby increasing the risk
of various acute and chronic diseases. This is due to the carcinogenic, mutagenic, genotoxic,
teratogenic, neurotoxic, and estrogenic effects associated with mycotoxins [10]. Mycotoxin
contamination of these products may occur at any stage along the manufacturing chain,
from animals contaminated in feed through the end product’s manufacture or storage. To
make matters worse, despite having been detected in dry-cured meats and cheese world-
wide, most nations do not regulate mycotoxins in this kind of food. Although the EU
has published a new amending regulation and concluded that additional monitoring for
OTA occurrence is required before setting maximum levels, this mycotoxin will soon be
regulated (Commission Regulation (EC) No 2022/1370).
Various physical and chemical methods have been developed to manage the growth
of fungi and their toxins. However, an effective strategy for reducing the occurrence of
mycotoxins remains elusive. Furthermore, certain molds have developed resistance to
chemical treatments and preservatives. Thus, reducing the prevalence of these molds in
food production is of utmost importance, and significant efforts are being made to develop
safe and efficient methods for this purpose. Biopreservation, which involves controlling
the growth of one organism using natural substances, has garnered considerable interest in
the past decade as a promising solution [11]. The quest to make healthier meat products
has prompted studies to minimize saturated fat, salt, and cholesterol. Better composition of
unsaturated fatty acids and integration of postbiotics, probiotics, and prebiotics were also
encouraged. These functional additives may benefit human health while improving meat
products’ nutrition [12]. Thus, contemporary customers desire high-quality, safe, minimally
processed, and chemical-free foodstuffs.
Lactic Acid Bacteria (LAB) are either naturally present in meals or introduced as pure
cultures to a variety of dietary items. LAB have a GRAS classification (generally regarded
as safe), and it is estimated that fermented foods make up 25% of the European diet and
60% of the diet in many developing nations [13]. LAB are often used as starting cultures
in the production of acidophilus milk, yogurt, buttermilk, cottage cheeses, hard cheeses,
and soft cheeses, among other dairy products [14]. The cohabitation of LAB and yeast is
also crucial for the success of other biotechnological applications, such as the production
of sourdough bread [15]. Ancient traditions of employing LAB in food and animal feed,
together with a new understanding of the favorable health benefits of probiotic LAB use,
imply that they might serve as viable alternatives to chemical preservatives.
Regarding the antifungal activity of bioprotective cultures, it is often the consequence
of the synergistic impact of many compounds since organic acids are not the only known
active molecules. Antifungal action may require other molecules, such as fatty acids [16],
reuterin [17], cyclic dipeptides, and proteinaceous substances [18], among others.
Phenolic acids are the most representative subgroup of phenolic compounds; they are
important for fermented products because of their relationship with the sensory charac-
teristics of foods. LAB have the ability to metabolize and release bound phenolic acids in
their free form, and some of them have demonstrated a broad spectrum of antimicrobial
activity, making them an important preservative to consider for foods [19]. For instance,
salicylic and gallic acid have shown antifungal potential against postharvest fungi such as
P. expansum and F. graminearum [20,21].
The use of probiotics in meat products is regarded as an attractive strategy for en-
hancing their healthfulness, as the fermentation carried out by probiotics can generate
health-improving compounds, typically through the hydrolysis of polysaccharides, pro-
teins, and fats, as well as biologically active compounds such as peptides, organic acids,
and conjugated linoleic acid [22]. In addition, fermented sausages are significant matrices
for probiotic delivery since they may be taken without heat treatment, which increases the
survival rates of bacteria and fungi.
Foods 2023, 12, 1427 3 of 22

Therefore, a continuous survey of OTA-producing strains, especially during ripening,

could support safety assurance in dry-cured meat production. Against this background,
the objective of this study was to isolate LAB with antifungal properties from traditional
dry-cured sausages and to develop an antifungal ingredient with potential application in
the manufacture of dry-cured meat products. Additionally, the antifungal properties of
the ingredient were characterized, and its responsible metabolites were identified using
various analytical techniques, including high-performance liquid chromatography with
diode array detection (HPLC-DAD), Quadrupole Time-of-Flight (QTOF) mass spectrometry,
and Headspace Solid-Phase Microextraction (HS-SPME).

2. Materials and Methods

2.1. Chemicals
Each analyte had at least 95% of purity. Sigma-Aldrich (St. Louis, MO, USA) provided
the phenolic acids 1,2–dihydroxybenzene, 3–(4–hydroxy–3–methoxyphenyl) propionic,
benzoic acid, caffeic acid, gallic acid, p–Coumaric acid, sinapic acid, syringic acid, vanillic
acid, and vanillin. Bachem (Bubendorf, Switzerland) provided the DL–3–phenyllactic acid
(PLA). MP Biomedicals (Santa Ana, CA, USA) provided the ferulic acid. The lactic acid and
acetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Solvents suitable for liquid chromatography (≥99.9% purity), including acetonitrile
(ACN), ethyl acetate, formic acid, and methanol, were provided by VWR Chemicals (Rad-
nor, PA, USA). Sigma-Aldrich supplied ammonium formate (≥99.5%), C18, magnesium
sulfate (MgSO4 ), sodium chloride (NaCl), and potato dextrose broth (PDB) (P6685). Li-
ofilchem (Roseto degli Abruzzi, Teramo, Italy) provided de Man, Rogosa Sharpe broth
(MRSb) (Oxoid CM359), and MRS agar (MRSa) (Oxoid CM361), plate count agar (PCA)
(Oxoid CM0463) and potato dextrose agar (PDA) (Oxoid CM0139). Water was deionized
with <18 MΩ/cm using a Milli-Q purification system (Millipore Corp., Bedford, MA, USA).

2.2. Sampling Isolation, Cultivation, and Preliminary Characterization of LAB Isolates

Sampling, isolation, cultivation, and preliminary characterization LABs were collected
from various Spanish homemade-fermented products obtained from local shops in the
Valencia region: “longaniza de payés”, “fuet”, “longaniza de Pascua”, and “longaniza
classic”. Samples (20 g) were added to 180 mL of sterile physiological saline solution and
were homogenized for 5 min. Simultaneously, bacteria were isolated by scraping a swab
dipped in 0.1% peptone water (Liofilchem, Roseto degli Abruzzi, Teramo, Italy; Oxoid
LP 0037) over the food surface. The swabs were then submerged in tubes containing
MRSb to recover the bacteria, and the tubes were kept anaerobic at 37 ◦ C for LAB culture.
Appropriate dilutions were plated onto MRSa and cultures anaerobically for 48 h at 37 ◦ C.
Colonies were purified on MRSa after being randomly chosen from MRSa plates containing
15 to 300 colonies [23]. Microorganisms were first evaluated microscopically for Gram
reaction and morphology as well as catalase production. Only Gram-positive and catalase-
negative isolates were investigated, and the strains were maintained at −80 ◦ C in MRSb
containing 20% glycerol.
Biochemical and physiological tests were used to describe the microorganisms. The
bacteria were evaluated for their capacity to grow at different temperatures (15, 30, and
45 ◦ C), at different pH values (3, 5, and 6), and MRSb supplemented with NaCl (3, 6.5, and
10%).

2.3. Fungal Culture and Inoculum Preparation

Fungal strains purchased from the Spanish Type Culture Collection (Valencia, Spain)
were Aspergillus parasiticus CECT 2681, Penicillium commune CECT 20767, P. griseofulvum
CECT 2605–T, and P. nordicum CECT 2320. A. flavus ITEM 8111 was acquired from the
Institute of Sciences for Food Production’s microbial culture collection (Bari, Italy). The VTT
Culture Collection provided P. verrucosum VTT D–01847 (Espoo, Finlandia). The fungal
strains were stored at −80 ◦ C in sterile glycerol (30% v/v).
Foods 2023, 12, 1427 4 of 22

The fungal strains were recovered by adding 1 mL of the glycerinated solution to 9 mL

PDB media. After incubating the infected PDB for 72 h at 25 ◦ C, aliquots were plated on
PDA Petri plates to harvest spores. The experiments were performed with these spores.

2.4. Screening of Antifungal Properties by Overlay Method

The first screening of antifungal activity was performed using bacterial isolates grown
overnight in 10 mL of MRSb at 37 ◦ C. Then, the overlay assay was used to determine the
antifungal activity of LAB isolates [24]. Bacterial suspensions (10 µL) were inoculated on
MRSa plates and incubated at 37 ◦ C for 48 h. Afterward, the agar plates were filled with
10 mL of soft (7% agar) PDA containing 106 conidia/mL and were incubated at 30 ◦ C.
The inhibition growth zone was determined 48 h later and measured on a cm scale. The
experiments were conducted in triplicate.

2.5. Identification of Isolate by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass

Spectrometry (MALDI-TOF MS) Fingerprinting
LAB was identified by extracting isolated cultures according to Maier et al. [25].
The MALDI-TOF MS was a Microflex L20 (Bruker Daltonics, Billerica, MA, USA) mass
spectrophotometer equipped with an N2 laser. The sample spectra were acquired in positive
linear ion mode, with a voltage acceleration of 20 kV, and the mass ranged from 2000 to
20,000 Da. Moreover, the spectra were obtained in triplicate as suggested by MALDI
Biotyper Realtime Classification (RTC). The identification of the samples was the spectra
of the largest log score organism. Finally, the results were compared with the MBT 7854 y
MBT 7311_RUO database (Bruker Daltonics).

2.6. Preparation of the Bacteria-Free Supernatant (BFS) and Development of a Postbiotic

Antifungal Ingredient
Meat broth (MB) was formulated by modifying the composition of the MRSb, as plotted
in Supplementary Material Table S1. Meat extract and yeast extract were substituted with
freeze-dried pork loin at different concentrations: 2.00 (MB2), 4.00 (MB4), 8.00 (MB8), and
10.00 (MB10) g/L. The ingredients were mixed and dissolved in distilled water for 10 min,
and the pH was adjusted to 6. Then, the media were autoclaved at 121 ◦ C for 21 min.
Afterward, the isolated LAB were defrosted and incubated in MRSb at 37 ◦ C for 12 h to
reach the exponential growth phase. Subsequently, the LAB were placed at 107 CFU/mL
in 50 mL of the MB and incubated at 37 ◦ C for 48 h. After fermentation, the LAB were
mechanically separated by centrifugation at 4000× g for 10 min to separate the BFS. Finally,
BFS were kept at −80 ◦ C for 24 h and freeze-dried to obtain the postbiotic antifungal
ingredient [26]. Freeze-dried BFS of fermented MRSb was carried out as a control group.

2.7. Determination of Sensitivity of Food-Borne Toxigenic Fungi to Bacteria Isolates

2.7.1. In Vitro Antifungal Activity of Samples by Agar Diffusion Method
The agar diffusion test was used initially to determine the sensitivity of food spoilage
and toxin-producing fungi to the BFS. The method was previously described by Fredua-
Agyeman et al. [27] with some modifications.
Petri dishes containing PDA were infected with collected fungal conidial grown on
PDA plates. The conidia of the fungal strains described in Section 2.3 were scraped off
the agar and distributed on another PDA plate using sterile cotton swabs. Then, using
sterile pipette tips, 10 mm diameter wells were created, and each well was filled with
100 µL of freeze-dried BFS resuspended in sterile water at a concentration of 250 g/L. A
sterile water solution containing freeze-dried MRSb was elaborated as a control. After that,
the PDA plates were incubated at 25 ◦ C for 72 h to enable either fungal growth or BFS
diffusion through the agar. Finally, the antifungal activity was determined by measuring
the inhibition zone around the well on a cm scale.
Foods 2023, 12, 1427 5 of 22

2.7.2. Microdilution Susceptibility Test to Freeze-Dried BFS

To establish the Minimum Inhibitory Concentration (MIC) and the Minimum Fungi-
cidal Concentration (MFC), the microdilution test was carried out in 96-well microplates
following the CLSI M38-A2 [28] criteria, with a few changes. Firstly, the microplates were
filled with 100 µL of PDB. Next, plates were filled with PDB containing freeze-dried BFS at
a concentration of 340 g/L and then serially diluted 2-fold to reach the final concentration
of 0.33 g/L. A volume of 100 µL of PDB containing 5 × 103 conidia/mL of the toxigenic
fungi described by Section 2.3 was added to each well. The positive control consisted of
contaminated PDB with fungal spores, whereas the negative control used PDB that had not
been infected. Then, the microplates were incubated for 72 h at 25 ◦ C. Each dose was tested
in four replicates, and the experiment was carried out two times (n = 8).
Three parameters were determined: MIC50, MIC100, and MFC. After the incubation
period, the MIC50 was established as the minimum concentration at which the BFS inhibited
50% of the fungal colonies, whereas the MIC100 was defined as the minimum concentration
of BFS at which no growth was seen in the microplate. The MFC was then determined by
reculturing 10 µL of the concentration matching the MIC100 and higher concentrations
assessed on PDA plates. After 72 h of incubation at 25 ◦ C, the MFC concentration that
prevented fungal growth was determined. The plates were rechecked after seven days
of incubation.

2.8. Extraction and Quantification of Organic Acids

The organic acids were extracted from the fermented MRSb and MB10, according
to Özcelik et al. [29]. First, 5 mL of the BFS was aliquoted into a separated tube and
treated with 1 mL of metaphosphoric acid. After 2 min of homogenization, the tubes were
centrifuged at 4000× g for 10 min. The BFS was filtered using a membrane filter (0.45 µm).
Finally, the BFS was diluted in water: formic acid (0.1%) and injected into an HPLC system.
The system applied for the chromatographic determination was an Agilent 1100 (Santa
Clara, CA, USA) HPLC equipped with an autosampler, binary pump, and vacuum degasser.
Results were expressed in g/L. The column used as the stationary phase was a Rezex™
ROA-Organic Acid H+ (8%) (150 × 7.8 mm, Ea Phenomenex® , Torrance, CA, USA). The
mobile phases consisted of water as solvent A and ACN as solvent B, both acidified with
0.1% formic acid and eluted using the gradient (5% B at 0 min, 95% B at 30 min, and 5%
at 35 min). Each analysis began with a 3-min equilibration of the column. The flow rate
was set at 0.3 mL/min, and the sample volume was set to 20 µL. The photo diode array
detector (DAD) (Agilent G1315B) was applied at 210 nm to monitor the chromatogram.
Individual stock solutions of each examined organic acid were produced by dissolving
the compounds in the mobile phase. At least six-point calibration curves were created for
acetic and lactic acid. The concentrations of organic acids were determined by comparing
the peak area to the calibration curve standard area. Results were expressed in g/L.

2.9. Extraction and Quantification of Phenolic Acids

The phenolic acids of the fermented MRSb and the MB10 were purified using the
QuEChERS extraction described by Brosnan et al. [30]. In 50-mL tubes, a solution of 4 g
MgSO4 , 1 g NaCl, and 10 mL ethyl acetate with 1% formic acid (v/v) were added, followed
by the addition of 10 mL of the BFS. For 1 min, the tubes were vortexed and cooled on
ice. Next, the solution was centrifuged at 4 ◦ C and 4000× g for 10 min. The supernatant
was combined with 900 mg of MgSO4 and 150 mg of C18 and vortexed for 1 min before
centrifuging as previously described. Finally, the samples were filtered with a 0.22 µm pore
filter and evaporated under nitrogen flow.
The phenolic acids analysis was performed in an Agilent 6450 Ultra High-Definition
Accurate Mass QTOF-MS equipment, coupled to an Agilent Dual Jet Stream Electrospray
Ionization (ESI) interface in negative ionization mode under the conditions described by
Denardi-Souza et al. [31]. MassHunter Qualitative Analysis software version B.08.00 was
used to handle data integration and elaboration. The results were expressed in µg/L.
Foods 2023, 12, 1427 6 of 22

2.10. Volatile Organic Compounds (VOCs) Determination

Volatile Organic Compounds (VOCs) emitted by the bacteria after fermentation in MRSb
and MB10 media were determined by head-space solid phase microextraction (HS-SPME)
technique and subsequent analysis in a gas chromatograph coupled to a mass spectrometer
(GC-MS) following the methodology of Luz et al. [32] with minor modifications.
For this purpose, 10 mL of the BFS, obtained as described in Section 2.6, was placed
in a 20 mL glass vial. The VOCs were extracted for 45 min at 50 ◦ C with a divinyl-
benzene/carbon wide range/polydimethylsiloxane (DVB/C-WR/PDMS) coated fiber
(80 µm × 10 mm) (Agilent Technologies, Santa Clara, CA, USA). Then, the fiber was des-
orbed for 10 min at 250 ◦ C in splitless mode on an Agilent 7890A gas chromatograph
coupled to an Agilent 7000A triple quadrupole mass spectrometer equipped with an elec-
tron impact (EI) source. The column used for chromatographic separation was an HP-5MS
(30 m × 0.25 mm, 0.25 µm) (Agilent Technologies). The temperature ramp was programmed
as follows: 40 ◦ C held for 2 min and raised to 160 ◦ C at 6 ◦ C/min; then, it was ramped
up to 260 ◦ C at 10 ◦ C/min and held for 4 min. The carrier gas was helium (99.99%) at a
flow rate of 2.5 mL/min. Compound detection was performed in Full Scan mode in an m/z
range of 40–450 Da.
The compounds were identified by comparison of their mass spectra with those
recorded in the NIST 09 library. In addition, linear retention indices (LRI) were calculated
based on the retention time of a solution of alkanes (C8–C20) tested under the same
conditions as the samples and compared with the existing literature.

2.11. Statistical Analysis

For statistical analysis, GraphPad Prism version 3.0 software was employed. The dif-
ferences (p ≤ 0.05) between the groups of the organic acids and phenolic acids composition
were analyzed by a one-way ANOVA followed by the Tukey post hoc test for multiple
comparisons.

3. Results and Discussion

3.1. Isolation, Screening, and Identification of Antifungal Bacteria Strains
A total of 102 bacteria were isolated from different handmade dry Spanish sausages.
Among these, 42 isolates were classified as LAB since they were gram-positive, could grow
in micro-aerobic conditions, and lacked catalase activity (Table 1). Further characterization
of bacteria strains highlighted that these isolates grew in the following conditions: temper-
ature ranging from 15 to 45 ◦ C; pH ranging from 3.5 to 6; and growth in MRS-NaCl at 3,
6.5, and 10%. Therefore, these bacteria were selected, and after the characterization, a first
screening was performed using the overlay approach to assess the antifungal properties
of the 42 bacteria isolated; they were assayed against six spoilage fungal strains in vitro
that commonly contaminate cured sausage products. As presented in Table 2, only 14
bacteria isolates (C11, C12, C13, C15, C20, C28, C56, C58, C60, C66, C69, C71, C72, and
C79) demonstrated inhibitory activity against all fungi examined, with varying degrees
of inhibition depending on the fungal strain tested. In particular, P. griseofulvum CECT
2605–T and P. commune CECT 20767 were the most susceptible strains, with inhibition
zones ranging from 1.5–3.0 cm and 1.0–3.0 cm, respectively. In contrast, Aspergillus strains
(A. parasiticus CECT 2681 and A. flavus ITEM 8111) were the most resistant to the LAB, with
inhibition zones ranging from 0.3–1.7 cm. An example of overlay methodology is given in
Figure 1. These findings agree with those obtained by previous authors [33], which noticed
that Aspergillus strains are more resistant to LAB than other fungal genera.
Foods 2023, 12, 1427 7 of 22

Table 1. Characterization of LAB isolated from dry-cured sausages.

Growth at Growth in Growth in MRS-NaCl

Preliminary Characterization
Temperature pH (%)
Bacteria Morphology Gram * Catalase 15 to 45 ◦ C 3.5 to 6 3 to 6.5 10
C1 Coccoid P - + + + -
C4 Coccoid P - + + + -
C5 Coccoid P - + + + -
C6 Coccoid rod P - + + + -
C11 Coccoid P - + + + -
C12 Coccoid P - + + + -
C13 Coccoid P - + + + -
C14 Coccoid rod P - + + + +
C15 Coccoid P - + + + +
C16 Rod-shaped P - + + + +
C17 Rod-shaped P - + + + +
C19 Coccoid P - + + + +
C20 Rod-shaped P - + + + +
C27 Rod-shaped P - + + + +
C28 Coccoid P - + + + +
C29 Rod-shaped P - + + + +
C30 Rod-shaped P - + + + +
C31 Coccoid P - + + + +
C35 Coccoid P - + + + -
C36 Coccoid P - + + + +
C37 Coccoid P - + + + +
C38 Coccoid P - + + + -
C39 Coccoid P - + + + +
C44 Coccoid P - + + + +
C45 Coccoid P - + + + +
C46 Coccoid rod P - + + + -
C47 Coccoid P - + + + +
C48 Coccoid P - + + + +
C50 Coccoid P - + + + +
C56 Coccoid P - + + + +
C57 Coccoid P - + + + +
C58 Coccoid P - + + + +
C59 Coccoid P - + + + -
C60 Rod-shaped P - + + + +
C66 Coccoid P - + + + +
C68 Coccoid P - + + + +
C69 Coccoid P - + + + +
C70 Coccoid P - + + + -
C71 Coccoid P - + + + +
C72 Coccoid P - + + + +
C79 Coccoid P - + + + +
C80 Coccoid P - + + + +
* P means positive; + signifies visual growth, and - means no visual growth.
Foods 2023, 12, 1427 8 of 22

Table 2. Antifungal activity of LAB by overlay technique.

Aspergillus Aspergillus Penicillium Penicillium Penicillium Penicillium

Selected
parasiticus flavus commune verrucosum griseofulvum nordicum
Bacteria
Inhibition Zone (cm)
C1 0.0 0.0 2.4 2.0 3.0 1.0
C4 0.0 0.1 1.5 1.7 3.0 0.9
C5 0.3 0.0 2.5 1.4 3.0 1.0
C6 0.3 0.0 2.7 1.7 3.0 1.1
C11 1.0 0.4 3.0 2.0 3.0 1.4
C12 0.5 0.3 3.0 1.7 2.3 1.3
C13 0.7 0.6 3.0 1.8 2.1 1.0
C14 0.5 0.0 2.0 1.4 3.0 1.1
C15 0.4 0.6 3.0 1.6 3.0 1.2
C16 0.5 0.0 3.0 1.5 0.6 0.7
C17 0.3 0.0 0.6 2.0 3.0 0.8
C19 0.3 0.0 1.6 1.7 3.0 1.0
C20 0.8 0.9 3.0 1.7 1.9 1.3
C27 0.5 0.0 0.9 1.7 1.3 0.5
C28 1.0 0.8 3.0 1.7 1.8 1.3
C29 0.4 0.0 2.9 1.7 1.2 0.9
C30 0.6 0.0 3.0 1.4 1.0 0.9
C31 0.5 0.0 3.0 1.7 1.8 0.9
C35 0.4 0.0 3.0 1.7 1.6 1.1
C36 0.0 0.0 2.7 1.6 3.0 1.0
C37 0.4 0.0 3.0 2.5 1.0 0.8
C38 0.2 0.0 2.4 1.5 3.0 1.1
C39 0.4 0.0 0.0 1.8 1.6 0.4
C44 2.0 0.0 1.4 1.7 3.0 1.0
C45 0.4 0.0 1.1 1.6 3.0 0.8
C46 0.2 0.0 0.5 2.0 3.0 0.7
C47 0.3 0.0 3.0 1.6 2.5 1.1
C48 0.5 0.0 3.0 1.7 1.5 1.1
C50 0.9 0.0 2.5 1.5 1.8 0.8
C56 1.5 1.2 3.0 2.1 1.6 1.1
C57 1.0 0.0 2.8 1.8 0.2 0.6
C58 1.5 1.4 3.0 1.5 3.0 1.5
C59 0.5 0.0 0.6 2.0 1.5 0.5
C60 1.2 1.7 1.6 1.5 2.5 1.2
C66 1.0 1.1 1.0 2.1 0.8 0.6
C68 1.0 0.0 3.0 2.2 2.5 1.1
C69 0.4 0.5 3.0 2.5 3.0 1.1
C70 0.0 0.0 3.0 1.3 3.0 1.2
C71 1.5 1.2 2.6 1.5 3.0 1.3
C72 1.0 1.1 3.0 1.5 1.5 0.9
C79 0.4 0.3 2.7 1.7 3.0 1.1
C80 0.0 0.0 2.7 1.7 3.0 1.0
Foods 2023, 12, 1427 9 of 22

Figure 1. Screening of lactic acid bacteria (LAB) through overlay method. Strong inhibitory action
(a); slightly inhibitory effect (b); no inhibitory action (c); control (d).

The 14 gram-positive bacteria with antifungal activity against all fungal strains were
identified by MALDI-TOF-MS and classified regarding the MTB 7854 and MBT 7311_RUO
databases (Bruker Daltonics). The classification to species level was the following: Pediococ-
cus pentosaceus C11, P. pentosaceus C12, P. pentosaceus C13, P. pentosaceus C15, Lactiplantibacil-
lus plantarum C20, P. pentosaceus C28, P. pentosaceus C56, P. pentosaceus C58, L. plantarum
C60, P. pentosaceus C66, P. pentosaceus C69, P. pentosaceus C71, P. pentosaceus C72, and P.
pentosaceus C79. Thus, the bacteria identified with potential inhibitory properties were
studied to develop an antifungal ingredient against dry-cured meat spoilage agents.
The LAB with antifungal activity are poorly documented, while more attention has
been exploited its antibacterial activity. The LAB that produces bacteriocins are isolated
from a severe category of food. For example, Delcarlo et al. [34] isolated 22 LAB with antimi-
crobial action from mussels on the Argentina coast. Parlindungan et al. [35] identified novel
probiotic candidates from fermented meats and characterized them by bacteriocin produc-
tion. Furthermore, Ivanovic et al. [36] isolated, identified, and characterized antibacterial
LAB from traditional cheese.
Only a few studies from scientific literature cite a broad spectrum of antifungal activity
for LAB. Most of them showed higher strain-specific antifungal activity against one or
two mold species [37]. For this reason, one of the goals of this study is to isolate a large-
spectrum strain. In this work, we observed that antifungal activity was strain-dependent,
as well as fungal species and methodologies analyzed. Moreover, it seems that dry-cured
sausages can be used as a potential source of antifungal LAB because they have significant
antagonistic properties against the microorganisms tested.
Foods 2023, 12, 1427 10 of 22

3.2. In Vitro Antifungal Study of the MB Formulated

Two methods were performed to determine the fungal sensitivity to bacterial isolates:
agar diffusion test and microdilution of BFS in 96-well plates.
The MB was formulated by changing the composition of the MRSb by subtracting
the yeast extract and meat extract and adding freeze-dried pork loin as a protein source
at 2.00, 4.00, 8.00, and 10.00 g/L (MB2, MB4, MB8, and MB10, respectively). These new
formulations were fermented with the 14 bacteria strains identified in Section 3.1, and
a first qualitative evaluation was performed using the agar-diffusion test to assess the
antifungal properties. Moreover, the fermented MRSb was also prepared with the same
bacteria strains to compare the effectiveness of the newly formulated ingredient.
After fermentation, only the MB10 exhibited antifungal activity similar to the fer-
mented MRSb used as a control reference. As presented in Table 3, the inhibition halos
obtained varied according to the fungal strain tested and the bacteria used as inoculum. In
particular, the most sensitive strains to the MB10 were P. commune CECT 20767, P. nordicum
CECT 2320, and P. verrucosum VTT–D01847. These strains were susceptible to all the for-
mulated MB, with inhibition halos observed on the PDA plates ranging from 0.2 cm (+)
to inhibition halos greater than 0.4 cm (+++). The fungal strains with the most significant
resistance were those of the Aspergillus genera, in which only Pediococcus pentosaceus C12,
Pediococcus pentosaceus C15, and Lactiplantibacillus plantarum C60 showed inhibitory activ-
ity. This finding agrees with the previous characterization of the LAB isolates by overlay
method since Aspergillus strains showed higher resistance to the LAB than Penicillium
strains. For this reason, the formulation MB10, P. pentosaceus C12, P. pentosaceus C15, and L.
plantarum C60 isolates were selected, and the preliminary results of the antifungal activity
were confirmed through the MIC50, MIC100, and MFC determination in vitro. The MB10
and MRSb that were fermented for 48 h obtained the highest inhibition halo zone. The
antifungal effect of MB10 fermented for 24, 48, and 72 h is plotted in Table S2.

Table 3. Antifungal activity of bacterial free supernatant (BFS) extract in agar diffusion method. LAB
fermented MRS broth (MRSb) and a meat broth (MB10) for 48 h at 37 ◦ C. Then, the supernatant was
freeze-dried and resuspended at a concentration of 250 g/L. These bacteria were previously selected
due to their antifungal effect in the overlay method.

Antifungal Activity of the BFS of the Fermented MRS Broth and the Meat Broth (MB10)
Selected Fungal Strain
Bacteria A. flavus A. parasiticus P. commune P. griseofulvum P. nordicum P. verrucosum
MRSb MB10 MRSb MB10 MRSb MB10 MRSb MB10 MRSb MB10 MRSb MB10
C11 + - - - + + + + + + + +
C12 + + ++ + ++ ++ ++ ++ + + ++ ++
C13 - - + - + + + + +++ +++ +++ +++
C15 ++ ++ ++ ++ +++ +++ +++ +++ +++ +++ +++ +++
C20 - - + - + + + + ++ ++ + +
C28 + - - - + + + + +++ +++ +++ +++
C56 - - + - + + + + + + + +
C58 - - - - + + ++ ++ +++ +++ +++ +++
C60 + + ++ ++ ++ ++ ++ ++ +++ +++ ++ ++
C66 + - - - ++ ++ +++ +++ ++ ++ +++ +++
C69 - - + - + + - - ++ ++ + +
C71 - - + - + + - - + + + +
C72 + - - - ++ + ++ ++ ++ ++ +++ +++
C79 + - - - ++ + ++ ++ ++ ++ +++ +++
(-) Represents no halo inhibition; (+) Represents a growth inhibition halo of 0.2 cm; (++) represents a growth
inhibition halo of between 0.2 to 0.4 cm; (+++) represents a growth inhibition halo greater than 0.4 cm.

Similar findings in the literature indicated that a range of theories might explain LAB’s
antifungal activities; Schnürer and Magnusson [38] found that the suppression of mold
Foods 2023, 12, 1427 11 of 22

growth on an agar plate is the consequence of a complex interaction of multiple chemicals

and metabolites, all of which contribute to the overall antifungal activity.
The microdilution assay demonstrated that the BFS of the fermented MRSb was the
most active extract against all fungi tested, regardless of the bacteria isolated (Table 4). The
MIC50 obtained ranged from 5–21 g/L; the MIC100 ranged from 21–85 g/L; and the MFC
ranged from 21–170 g/L. However, for MB10 formulation, the MIC50, MIC100, and MFC
parameters ranged from 11–43 g/L, 21–85 g/L, and 43–170 g/L, respectively. It is important
to underline that the BFS of the MRSb and the MB10 fermented by P. pentosaceus C15 showed
lower concentrations required to achieve fungal growth against all the toxigenic strains
tested compared to P. pentosaceus C12 and L. plantarum C60 fermented BFS. Although the
BFS of the MRSb evidenced a higher antifungal effect, a poor difference was evidenced
when compared to MB10. In some cases, there were no significant differences (p ≤ 0.05)
between the treatments, e.g., MB10 fermented by P. pentosaceus C15 had no statistical
difference to MRSb comparing the average values of MIC100 and MFC (Table 4). In other
words, P. pentosaceus C15 was able to ferment MB10 and produce antifungal compounds,
which inhibited fungal growth at similar conditions to MRSb. In addition, MRSb fermented
by P. pentosaceus C15 obtained MIC50, MIC100, and MFC values lower than L. plantarum
C60 and P. pentosaceus C12, regardless of the fungal strain essayed.

Table 4. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of
bacterial-free supernatant (BFS). MRS broth (MRSb) and meat broth (MB10) were fermented by LAB
for 48 h at 37 ◦ C and centrifuged to obtain the BFS.

BFS Concentration (g/L) of the Fermented MRSb

Fungi Lactiplantibacillus plantarum C60 Pediococcus pentosaceus C12 Pediococcus pentosaceus C15
MIC 1 MIC 2 MFC MIC 1 MIC 2 MFC MIC 1 MIC 2 MFC
A. flavus 11 85 170 21 85 170 11 21 85
A. parasiticus 11 43 170 11 43 170 11 21 85
P. commune 21 43 85 21 43 85 11 21 85
P. griseofulvum 21 43 170 21 43 170 5 21 170
P. nordicum 11 43 85 11 43 85 5 21 21
P. verrucosum 21 43 85 21 43 85 11 43 43
Mean 16 50 128 18 50 128 9 25 82
BFS concentration (g/L) of the MB10
Fungi 1 2
MIC MIC MFC MIC 1 MIC 2 MFC MIC 1 MIC 2 MFC
A. flavus 21 85 170 43 85 170 21 43 85
A. parasiticus 21 43 170 21 43 170 21 43 85
P. commune 43 85 85 43 85 85 21 21 85
P. griseofulvum 43 85 170 43 85 170 11 21 170
P. nordicum 21 43 85 43 85 85 11 21 43
P. verrucosum 43 85 85 43 85 85 11 43 43
Mean 32 * 71 128 39 * 78 * 128 16 * 35 85
1: 50% of visual inhibition growth; 2 : 100% of visual inhibition growth. *: results were statistically analyzed by the
t-test, comparing the Bacterial free supernatant (BFS) of MRSb with the BFS of MB10. Means are significantly
different if p ≤ 0.05.

The MRSb was applied in both experiments as a control group since previous studies
have demonstrated its antifungal activity while being fermented by LAB and producing
antimicrobial substances. For instance, Taroub et al. [39] identified P. pentosaceus and L.
plantarum strains that showed good antifungal activity against A. niger and A. carbonarius.
In addition, the strains showed a high capacity for the degradation of OTA, one of the main
toxins produced by these fungi. However, the authors could not identify the compounds
responsible for the antifungal and antimicotoxigenic activities of the strains. In other
studies, the application of MRS fermented by L. plantarum inhibited the A. flavus and F.
Foods 2023, 12, 1427 12 of 22

verticillioides growth and reduced the production of aflatoxin and fumonisin in cereals such
as maize [26].
Therefore, MRSb has proven to be an interesting medium for fermenting bacteria as
antimicrobial metabolites are produced. However, the use of MRSb has some disadvantages,
such as its high cost. In addition, the complexity of some of their ingredients that convert
MRSb might not be allowed as an ingredient in meat products. Therefore, one of the
objectives of the work was to develop a meat broth with similar antifungal characteristics
but, at the same time, could be incorporated as an antifungal ingredient.
Overall, the P. pentosaceus C15 showed higher antifungal potential in the agar diffu-
sion test and microdilution of BFS; hence, these results led us to select this strain for the
fermentation of MB10 and elaborate a postbiotic antifungal ingredient.

3.3. Chemical Characterization of the Postbiotic Antifungal Ingredient

3.3.1. Organic and Phenolic Acid Production
The metabolites generated after fermentation of the antifungal strains selected were
investigated in the BFS of the MRSb and MB10. As plotted in Figure 2, two organic
acids, lactic acid and acetic acid, were identified in both formulations. For lactic acid, the
mean values of this metabolite ranged from 2.49 to 3.00 g/L, whereas for acetic acid, the
concentration detected was lower, with mean values ranging from 0.24 to 0.38 g/L. The
highest amount of lactic acid was detected in the fermented MRSb (2.973 g/L) and MB10
(3.00 g/L) fermented by the P. pentosaceus C15 strain. There were no statistical differences
in lactic acid production between these two formulations (p ≤ 0.05). Regarding acetic
acid, the higher concentration (p ≤ 0.05) was also quantified in the fermented MB10 with
P. pentosaceus C15 (0.38 g/L), followed by the MRSb of P. pentosaceus C15 (0.32 g/L) and the
fermented MRSb with L. plantarum C60 (0.31 g/L).

Figure 2. The concentration of organic acids produced by LAB in meat broth (MB10) and MRS broth
(MRSb) after incubation for 48 h at 37 ◦ C. The meat broth was prepared with 10% freeze-dried loin
pork and fermented by P. pentosaceus C12, P. pentosaceus C15, and L. plantarum C60. Different letters
represent statistical differences in the same group of molecules between treatments (p ≤ 0.05).

Concerning the phenolic acids, a total of 12 different metabolites, described in the

literature as antifungals, were identified and quantified through the UHPLC Q-TOF MS
technique. Among them, it is noteworthy that the concentration of benzoic acid, DL–3–
phenyllactic acid, syringic acid, and vanillic acid stood out regardless of the fermented
broth and bacteria tested (Table 5). In general, it could be noted that MRSb produced higher
concentrations of phenolic acids compared to its analog MB10. However, in most cases
the difference was not significant (p ≤ 0.05). In view of these results, we suggest further
Foods 2023, 12, 1427 13 of 22

studies to evaluate the synergistic effect of these four compounds in order to evaluate their
antifungal activity and to increase the understanding about the mechanism of antimicrobial
action of LAB.

Table 5. The concentration of phenolic compounds in Man, Rogosa and Sharpe broth (MRSb) and
meat broth (MB10) fermented by antifungal LAB. The broths were fermented for 48 h at 37 ◦ C.

The Concentration of Phenolic CompoundsMean ± SD (µg/L)

Phenolic Compound Pediococcus pentosaceus C12 Pediococcus pentosaceus C15 Lactiplantibacillus plantarum C60
MRSb MB10 MRSb MB10 MRSb MB10
9.39 ± 2.02 a b cd cd d 4.64 ± 1.39 c
1,2–Dihydroxybenzene 2.51 ± 0.41 5.86 ± 2.74 5.64 ± 2.22 7.12 ± 1.3
Propionic acid 7.14 ± 1.79 a 7.08 ± 1.34 a 9.66 ± 2.64 a 9.36 ± 0.62 a 6.19 ± 2.85 a 13.44 ± 5.58 b
Benzoic acid 31.65 ± 4.42 a 18.09 ± 5.35 b 24.69 ± 0.72 c 22.12 ± 7.32 bc 19.38 ± 5.91 bc 23.18 ± 0.35 bc
Caffeic acid 4.07 ± 1.46 a 3.99 ± 2.46 a 8.37 ± 1.26 b 7.72 ± 1.87 bc 3.40 ± 0.97 a 6.27 ± 1.55 c
DL–3–Phenyllactic acid 18.17 ± 3.52 a 18.30 ± 1.54 ab 31.90 ± 7.49 c 22.68 ± 3.25 b 16.98 ± 3.66 a 15.85 ± 0.61 a
Ferulic acid n.d n.d 4.79 ± 1.81 ab 5.85 ± 4.02 ab 3.70 ± 0.72 a 6.18 ± 1.26 b
Gallic acid n.d n.d 8.04 ± 2.83 a 6.06 ± 1.88 a 6.58 ± 2.05 a 7.44 ± 1.25 a
p–Coumaric acid 22.59 ± 9.91 a 9.37 ± 1.72 b 6.28 ± 0.07 b 7.56 ± 0.44 b 5.29 ± 1.47 b 4.99 ± 2.03 b
Sinapic acid 4.87 ± 1.04 ab 6.43 ± 0.44 ac 5.85 ± 2.38 ad 7.86 ± 3.66 cd 3.54 ± 0.86 b 7.22 ± 1.32 cd
Syringic acid 26.78 ± 3.52 a 19.47 ± 5.28 b 17.73 ± 4.12 b 21.12 ± 0.39 b 17.42 ± 1.40 b 12.83 ± 3.40 c
Vanillic acid 20.84 ± 4.56 ac 18.81 ± 2.82 a 30.83 ± 6.19 b 25.18 ± 1.13 d 27.60 ± 2.94 bd 24.00 ± 2.12 cd
Vanillin 11.68 ± 1.26 a 13.76 ± 1.08 a 16.85 ± 5.47 a 13.73 ± 1.72 a 7.57 ± 1.65 b 8.46 ± 1.51 b
Different letters mean a significant statistical difference in the concentration of phenolic compounds among
fermented broths (p ≤ 0.05). n.d means not detected.

The selected microorganisms grew on the MB10 and produced a broad variety of
metabolites previously described in the literature as antimicrobial compounds. The pheno-
lic acid most produced by P. pentosaceus C12 in MB10 was syringic acid, showing values
of 19.47 µg/L: these values are significantly higher than bacteria C60. Aziz et al. [40]
demonstrated that syringic acid at 300 mg/L inhibited A. flavus and A. parasiticus growth
and aflatoxin production. In addition, Ren et al. [41] demonstrated that lower doses of
syringic acid (100 mg/L) avoided the growth of A. niger. Therefore, it seems that a higher
concentration of phenolic acids is required to achieve an antifungal effect when they are
applied in isolation. These findings reinforce the hypothesis that the LAB antifungal effect
is obtained via a synergistic effect.
Regarding P. pentosaceus C15 and L. plantarum C60, vanillic acid was the most pro-
duced phenolic acid, reaching values of 25.18 and 24.00 µg/L in MB10 and 30.83 and
27.60 µg/L in MRSb, respectively. An oxidized derivative of vanillin, vanillic acid, is a
monohydroxybenzoic acid composed of a 4–hydroxybenzoic acid with a methoxy group at
position 3 [41]. It has been noted for its antioxidant properties, but its antifungal activity
is poorly reported, and more research is required. In contrast, its precursor, vanillin, has
been the subject of extensive research, and its antifungal activity varies depending on the
microorganism tested [42,43].
These findings also suggest that the antifungal ingredient obtained from MB10 could
be incorporated into meat food products to provide a significant source of phenolic and
organic acids.
LAB can synthesize a wide variety of antifungal compounds such as organic acids,
phenolic acids, antimicrobial peptides, diacetyl, and reuterin [44]. During fermentation,
carbon metabolism produces organic acids such as lactic acid, acetic acid, and propionic
acid [45]. Among these metabolites, the most studied are organic acids. It is important to
mention that these metabolites can constitute synergistic activity. However, this synergistic
activity’s exact mechanism is unknown [46].
The results also suggest that the combination of organic acids and phenolic acids
produced by LAB could be responsible for the inhibitory activity of the spoilage fungi
in vitro. However, it is essential to underline that the antifungal properties of the MB10
are probably not exclusively produced by organic and phenolic acids; for instance, several
volatile substances can act synergistically and potentialize the antifungal properties [16].
Foods 2023, 12, 1427 14 of 22

Moreover, the decrease in pH collaborates to a more efficient antifungal activity [47]. In

this context, Peyer et al. [48] found that organic acids and phenolic acids generated by
some LAB strains as antifungal metabolites have synergistic actions against F. culmorum.
Likewise, in bakery products, organic acids and antifungal peptides produced by LAB
showed a significant synergistic effect which allowed biopreservation and enhanced the
shelf life of quinoa and rice bread [49].
In addition to the synergistic compound effect, the combination of different microor-
ganisms can also promote a positive effect. For instance, Ruggirello et al. [50] associated
yeast with LAB and obtained a great antifungal effect against strains of Aspergillus and
Penicillium genera, fermenting cocoa beans. The authors reported that the antifungal po-
tential could be a result of association between organic acids of LAB with proteinaceous
substances of yeasts. According to Christ-Ribeiro et al. [51], phenolic chemicals avoid
fungal development by inhibiting the production of cell wall components including glucan,
chitin, and mannoproteins, as well as cell membrane components like ergosterol. This
process happens by damaging the cell wall and cell membrane, affecting nutrient influx
regulation. As a result, phenolic chemicals impede fungal cell production of proteins, amino
acids, and sphingolipids. Moreover, they obstruct electron transit and the preservation of
cellular integrity.

3.3.2. VOC Analysis in MB10 Formulation

The fermented meat broth VOCs (MB10) were analyzed through HS-SPME coupled to
the GC-MS technique. A total of 24 compounds were identified through MS comparison
with the NIST library and calculation of the LRI and belonged to several chemical classes:
acid (2), alcohol (8), aldehyde (6), ketone (5), and pyrazine (2) (Table S3). Moreover, the
percentage peak area (%PA) of each identified compound was calculated, and the results
obtained are reported in Table 6.

Table 6. Percentage area (%) of the volatile organic compounds (VOCs) identified in the formu-
lated meat broth (MB10) fermented with different lactic acid bacteria strains (P. pentosaceus C12,
P. pentosaceus C15, and L. plantarum C60). Results are expressed as mean ± standard deviation.

N◦ Rt Compound Control C15 C60 C12

Acid
1 3.11 Acetic acid n.d 13.74 ± 1.12 a 11.11 ± 0.62 ab 8.95 ± 2.63 b
2 15.49 Nonanoic acid n.d 5.45 ± 1.69 a 6.94 ± 1.00 a 3.70 ± 1.13 a
Alcohol
3 8.97 2–Octanol 8.12 ± 2.16 a 1.77 ± 0.16 b 0.39 ± 0.05 b 0.87 ± 0.28 b
4 11.19 1–Octanol 2.16 ± 0.07 a 2.70 ± 0.10 ab 3.03 ± 0.25 ab 4.16 ± 1.11 b
5 11.62 2–Nonanol n.d 0.58 ± 0.08 a 0.53 ± 0.14 a 0.66 ± 0.06 a
6 13.43 Phenylethyl alcohol 1.24 ± 0.13 a 6.57 ± 0.45 b 1.60 ± 0.20 a 2.44 ± 0.88 a
7 13.6 1–Nonanol 1.26 ± 0.17 a 3.65 ± 0.05 b 1.95 ± 0.18 ab 3.80 ± 1.49 b
8 15.88 1–Decanol n.d 1.22 ± 0.12 a 2.18 ± 0.70 a 1.86 ± 0.35 a
9 16.27 2–Undecanol 0.86 ± 0.03 a 1.15 ± 0.16 a 1.09 ± 0.03 a 1.76 ± 0.42 b
Aldehyde
10 6.25 Heptanal 3.51 ± 0.43 ab 1.04 ± 0.21 b 2.13 ± 0.35 b 5.71 ± 2.66 a
11 8.89 Octanal 13.45 ± 2.80 a 3.81 ± 0.33 b 3.75 ± 0.63 b 4.98 ± 0.06 b
12 11.02 Benzeneacetaldehyde 20.31 ± 2.76 a 19.27 ± 2.39 a 19.82 ± 3.34 a 18.36 ± 3.74 a
13 11.42 Nonanal 1.24 ± 0.13 a 2.91 ± 0.53 a 2.43 ± 0.16 a 2.94 ± 1.44 a
14 15.82 2–Decenal 0.97 ± 0.18 a 0.53 ± 0.07 a 1.44 ± 0.13 a 1.47 ± 0.72 a
15 18.5 Dodecanal 1.28 ± 0.07 a 0.27 ± 0.05 b 0.81 ± 0.19 ab 0.94 ± 0.56 ab
Foods 2023, 12, 1427 15 of 22

Table 6. Cont.

N◦ Rt Compound Control C15 C60 C12

Alkane
16 10.77 Decane, 2–methyl n.d 0.85 ± 0.05 a 2.42 ± 0.04 b 1.81 ± 0.63 b
17 11.69 Undecane 1.66 ± 0.41 ab 1.47 ± 0.31 ab 0.78 ± 0.22 a 2.21 ± 0.66 b
Ketone
18 5.78 2–Heptanone n.d 1.73 ± 0.40 a 2.80 ± 0.53 a 2.82 ± 0.72 a
19 8.71 2–Octanone 2.52 ± 0.45 a 2.89 ± 0.19 a 1.92 ± 0.15 a 2.32 ± 0.95 a
20 11.28 2–Nonanone 1.59 ± 0.03 ab 2.82 ± 0.55 b 0.88 ± 0.05 a 2.13 ± 1.28 ab
21 16.11 2–Undecanone n.d 0.63 ± 0.10 a 1.29 ± 0.38 b 0.82 ± 0.12 ab
22 20.45 2–Tridecanone n.d 0.60 ± 0.08 a 2.13 ± 0.35 b 1.13 ± 0.20 a
Pyrazine
23 4.55 Pyrazine, methyl– 10.24 ± 1.00 a 5.30 ± 0.09 b 6.91 ± 1.73 b 4.34 ± 1.11 b
24 6.37 Pyrazine, 2,5–dimethyl 29.58 ± 2.79 a 19.05 ± 1.87 b 21.68 ± 0.80 b 19.79 ± 0.07 b
n.d = no-detected. Different letters represent statistical differences in the same molecules between treatments
(p ≤ 0.05).

Aldehydes were the most abundant compounds in the samples analyzed and repre-
sented a proportion between 27.8–40.8% of the total VOCs detected (Figure 3). In particular,
five linear aldehydes (heptanal, octanal, nonanal, 2–decenal, and dodecanal) were iden-
tified in all MB10; nonetheless, the greatest observed concentration was of an aromatic
aldehyde, benzeneacetaldehyde, which %PA ranged between 18.4 and 20.3% depending
on the formulation analyzed. Saturated aldehydes are lipid-derived volatiles produced
mainly by the oxidation of oleic acid, a characteristic fatty acid of raw pork meat [52–54].
Regarding benzeneacetaldehyde, this compound could be synthesized using phenylalanine
as a precursor through the Maillard reaction during the sterilization step of the MB10 since
it is mainly detected in cooked meat [55,56]. It was noted that aldehyde content in the
fermented MB10 was statistically lower (p ≤ 0.05) in comparison with the control group,
which corroborated the findings of Kwaw et al. [57], which described a decline of aldehydes
in mulberry juice when LAB fermentation was applied.

Figure 3. Total percentage area (%) of the chemical classes identified in the volatile fraction of
the Meat Broth formulated with 10% of lyophilized pork loin and fermented by P. pentosaceus C12
(MB12), P. pentosaceus C15 (MB15), and L. plantarum C60 (MB60). Different letters represent statistical
differences in the same group of molecules between treatments (p ≤ 0.05).
Foods 2023, 12, 1427 16 of 22

Pyrazines were the second abundant group detected in MB10 formulations (%PA rang-
ing from 24.1 to 39.8). It was observed that the %PA statistically decreased in the fermented
MB10 formulations (p ≤ 0.05) compared to control formulations. This phenomenon was
also described by Kurt et al. [58], which evidenced that the pyrazine content in spirulina
water solutions (4% w/v) was reduced after LAB fermentation.
The greatest variety of chemical compounds found in the MB10 were alcohols, rep-
resenting a mean %PA value ranging from 10.8 to 17.6%. Among the alcohols identified,
phenylethyl alcohol (PEA) was detected in a higher proportion (6.6 ± 0.5 %PA) in the MB10
fermented with C15 (MB10-C15) in comparison with other formulations (p ≤ 0.05). This
active compound has been studied for its antifungal potential and could explain the lower
MIC and MFC values detected in the MB10-C15, as previously reported in Section 3.2.
For instance, Gong et al. [59] determined that the antifungal properties of Enterobacter
absuriae Vt–7 were mainly due to the volatile antifungal PEA, and it effectively controlled
the development of the toxigenic fungi Aspergillus flavus in peanuts. Similar results were
obtained by Wonglom et al. [60], who associated the antifungal potential of Trichoderma sp.
T76–12/2 against Sclerotium fruit rot due to the synthesis of PEA and other VOCs.
Regarding ketones, LAB not only significantly increased (p ≤ 0.05) the ketone levels
in the formulated MB10, but also introduced three new ketones that were not present in
the control group, such as 2–heptanone, 2–undecanone, and 2–tridecanone (Table 6). Some
ketones may be synthesized through microbial oxidation of fatty acids, and this could
explain its higher proportion in the MB10 formulations when compared to the control
formulation (MB10 without fermentation) [61].
Only two acids, acetic acid and nonanoic acid, were identified in the fermented MB10.
Nonanoic acid is mainly produced from the degradation of unsaturated fatty acids such as
oleic acid, whereas acetic acid is produced because of the heterofermentative metabolism of
LAB [62]. Previous studies conducted observed an increase in these chemical compounds
when LAB are employed in different food matrices such as pumpkin and watermelon
juices [63,64]. Regarding its biological properties, it is important to emphasize that nonanoic
acid has evidenced antimicrobial properties against several pathogens such as Alternaria
alternata, Botrytis cinerea [65], Candida albicans [66], Salmonella enterica [67], and Escherichia
coli O157:H7 [68]. Thus, volatile acids combined with the other biological compounds
found in the MB10 (such as PEA, organic acids, and phenolic acids) could contribute to the
antifungal properties of the formulated ingredient since LAB antifungal potential is related
to the synergistic action of the different metabolites synthesized [38].

3.3.3. Principal Component Analysis (PCA) of the MB10 with LAB

In order to understand the differences between bioactive metabolite production (or-
ganic acids, phenolic acids, and VOCs) in the fermented MB formulations, a PCA was
realized, and the results obtained are outlined in Figure 4. The sum of the first two principal
components (PC) achieved 90.1% of the total explained variance, in which PC1 explained up
to 64.5% of the total variance, while PC2 explained 25.6% (Figure 4a). The PC1 distributed
on the positive axis both P. pentosaceus strains (C12 and C15), whereas L. plantarum C60
strain was positioned on the negative axis. The PC2 allowed the distinction of the C12 and
C15 strains according to their bioactive metabolite profile since C12 was positioned on the
negative axis and C15 was positioned on the positive axis.
Foods 2023, 12, 1427 17 of 22

Figure 4. Principal component analysis (PCA) scores plot of the bioactive compounds (organic acids,
phenolic acids, and volatile organic compounds) found in the meat broth fermented by P. pentosaceus
C12 (MB C12), P. pentosaceus C15 (MB C15), and L. plantarum C60 (MB C60) (a) and relative loadings
of the variables employed (b).
Foods 2023, 12, 1427 18 of 22

According to the loading plot that represents the relative importance of the variables
analyzed (Figure 4b), MB10-C12 was distinguished from MB10-C15 for its higher volatile
aldehyde production; specifically, this was found for specific compounds such as hep-
tanal (V24), 2–decenal (V28), and dodecanal (V29). The MB10 formulated with C15 was
mainly characteristic from the other formulations due to the higher production of different
antifungal compounds such as nonanoic acid (V16) and phenyl ethyl alcohol (V20).
Regarding the MB10 formulated with L. plantarum strain (MB10-C60), the main variable
that distinguishes this formulation from those prepared with P. pentosaceus strains was
the higher production of the phenolic compound 3–(4–hydrodoxy–3–methoxyphenyl)
propionic (V2). Furthermore, the ferulic acid (V6) and gallic acid content (V7) positioned
on the positive axis according to the second component the MB10-C60 formulation and
permitted the distinction between the MBC12 formulation. The variable that was positioned
on the negative axis according to the first component of the MB C60 was the lower content
in volatile alcohols in comparison with C12 and C15, such as 1–nonanol (V21) and 2–
undecanol (V23).

4. Conclusions
In the study, 42 antifungal LAB isolated from dry-cured sausages were evaluated
against 6 fungi of the Aspergillus and Penicillium genera. Firstly, a battery of bacteria was
isolated from dry-cured meat products and characterized according to their antifungal
properties. Among them, 14 presented antifungal capacity and 3 inhibited the growth of
all fungi assayed. The P. pentosaceus C15 showed higher antifungal activity in vitro; these
bacteria were selected for the preparation of several fermented-meat broth (MB2, MB4,
MB8 and MB10). MB were elaborated changing de MRSb formulation. The MB10 and MB
formulated with 10% of freeze-dried loin pork demonstrated higher antifungal activity
through agar diffusion and microdilution method.
In particular, the MB10 fermented with P. pentosaceus C15 was selected to elaborate
a postbiotic antifungal ingredient due to their higher antifungal activity. The chemical
characterization highlighted that the MB10 formulation was rich in phenolic acids and
organic acids such as lactic acid and acetic acid. Moreover, the postbiotic product changed
the VOCs composition and increased the concentration and number of antifungal com-
pounds detected.
Discovering the synergistic process amongst antifungal agents might provide under-
standing in increasing the effect while modifying the implicated bacterial or nutritional
compositions, subsequently leading to food applications. PCA analysis indicated that
some antifungal agents enabled us to differentiate strains by their production. For in-
stance, P. pentosaceus C15 and C12 were distinguished by the production of aldehydes
such as heptanal, decenal, and dodecanal. In contrast, L. plantarum C60 was differentiated
from P. pentosaceus strains due to the production of 3–(4–hydrodoxy–3–methoxyphenyl)
propionic.
Further research will focus on the application of the postbiotic antifungal ingredient
(MB10-C15) in the development of dry-cured meat products, as well as study c inhibition
of mycotoxins synthesis that can potentially harm the consumers health.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/foods12071427/s1, Table S1: Elaboration of meat broths for
fermentation for lactic acid bacteria; Table S2: Antifungal activity of formulated meat broths (MB)
and MRS broth fermented by Pediococcus pentosaceus C15 during 24, 48, and 72 h at 37 ◦ C. The
bacterial-free supernatant (BFS) was freeze-dried, resuspended at a concentration of 500 g/L, and
tested against six toxigenic fungi; Table S3: Identification of Volatile Organic Compounds (VOCs)
of the fermented Meat Broth 10, with retention time, chemical class, calculated LRI (LRI exp.), and
references [69–86].
Author Contributions: Conceptualization, J.M. and G.M.; methodology, T.d.M.N.; formal analysis,
T.d.M.N., J.C. and C.L.; investigation, T.d.M.N.; resources, J.M. and G.M.; data curation, T.d.M.N., J.C.
Foods 2023, 12, 1427 19 of 22

and C.L.; writing—original draft preparation, T.d.M.N., J.C., C.L., J.M. and G.M.; visualization, J.C.
and C.L.; writing—review and editing, T.d.M.N., J.C., C.L., J.M. and G.M.; supervision, G.M.; project
administration, J.M. and G.M.; funding acquisition, J.M. and G.M. All authors have read and agreed
to the published version of the manuscript.
Funding: This research was funded by the Spanish Ministry of Science and Innovation, grant number
PID2019-108070RB-100.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The authors would like to thank the program of the University of Valencia
(Atracció de Talent UV-INV-PREDOC19F1-1006684).
Conflicts of Interest: The authors declare no conflict of interest.

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