Occurrence and involvement of yeast biota in ripening of Italian Fossa cheese

Abstract

A microbiological investigation on a typical Italian Fossa cheese during ripening was reported here. Two yeast isolation campaigns were conducted to investigate the yeast diversity on cheese and pit environment, before and at the end of cheese ripening in pit, using classical and molecular tools. Before the ripening, eight different yeast species were identified from pit environment: Candida zeylanoides, Candida norvegica, Pichia occidentalis, Pichia guilliermondii, Pichia jadinii, Cryptococcus albidus, Cryptococcus skinneri, and Sporobolomyces roseus. Only C. zeylanoides was also found at the end of the cheese-ripening stage, together with the new isolated species Wickerhamomyces anomalus, Saccharomyces cerevisiae, Debaryomyces hansenii, and Candida homilentoma. To evaluate the contributions of these autochthonous species found during ripening, they were inoculated into fresh cheeses. Results show that D. hansenii, C. zeylanoides, and W. anomalus drastically reduced the colonization of molds on the cheese surface, with excellent results of sensory evaluation of the ripened cheese. The cheese inoculated with these indigenous selected yeasts did not show any defects, and volatile organic compounds analysis showed a high concentration of methyl ketones, and butanoic, hexanoic, and octanoic acids, which typically enhance the taste of the highly matured Fossa cheese. These results highlight the positive role of these indigenous yeasts during ripening process of the Fossa cheese.

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European Food Research and Technology (2018) 244:1921–1931
https://doi.org/10.1007/s00217-018-3104-6
ORIGINAL PAPER
Occurrence andinvolvement ofyeast biota inripening ofItalian Fossa
cheese
ClaudiaBiagiotti1· MaurizioCiani1· LauraCanonico1· FrancescaComitini1
Received: 27 March 2018 / Revised: 28 May 2018 / Accepted: 2 June 2018 / Published online: 13 June 2018
© Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
A microbiological investigation on a typical Italian Fossa cheese during ripening was reported here. Two yeast isolation
campaigns were conducted to investigate the yeast diversity on cheese and pit environment, before and at the end of cheese
ripening in pit, using classical and molecular tools. Before the ripening, eight different yeast species were identified from
pit environment: Candida zeylanoides, Candida norvegica, Pichia occidentalis, Pichia guilliermondii, Pichia jadinii, Cryp-
tococcus albidus, Cryptococcus skinneri, and Sporobolomyces roseus. Only C. zeylanoides was also found at the end of
the cheese-ripening stage, together with the new isolated species Wickerhamomyces anomalus, Saccharomyces cerevisiae,
Debaryomyces hansenii, and Candida homilentoma. To evaluate the contributions of these autochthonous species found
during ripening, they were inoculated into fresh cheeses. Results show that D. hansenii, C. zeylanoides, and W. anomalus
drastically reduced the colonization of molds on the cheese surface, with excellent results of sensory evaluation of the
ripened cheese. The cheese inoculated with these indigenous selected yeasts did not show any defects, and volatile organic
compounds analysis showed a high concentration of methyl ketones, and butanoic, hexanoic, and octanoic acids, which
typically enhance the taste of the highly matured Fossa cheese. These results highlight the positive role of these indigenous
yeasts during ripening process of the Fossa cheese.
Keywords Fossa cheese· Yeast biota· Candida zeylanoides· Wickerhamomyces anomalus
Introduction
Fossa cheese is a traditional Italian cheese, named ‘cheese
of the pit’, since the process of ripening occurs in special
underground pits placed in a delimited area in the Center
of Italy (North of Marche region). Fossa cheese undergoes
initial ripening in the dairy for a period of about 60days,
at temperature between 6 and 14°C. At the end of the first
dairy seasoning, the cheeses are enclosed in cloth bags and
ripened in a pit. The pits have a truncated cone shape and
are dug into the sandstone, with a circular opening of about
70cm and about 3m in depth. The walls of the pits are
covered by straw, which is kept in place by reeds, and the
bottoms of the pits have a wooden grating to collect the lipid
components that are released. This maturation in the pits
gives this product its organoleptic character, and there is a
strict relation between the technological processes and the
ecosystem of the pits [1].
During the pit ripening, for 85days the cheese undergoes
a process of anaerobic fermentation at 30–35°C and at 100%
relative humidity, which makes the final product softer and
lactose-free, and gives it a more complex flavor. Hydrolysis
of proteins and lipids play an important role on aromatic
compounds of final product. In particular, lipolysis results
directly in the formation of flavor compounds by liberating
free fatty acids that may be also metabolized to alkan-2-
ones and fatty acid lactones. Proteolysis of the caseins to
a range of small- and intermediate-sized peptides and free
amino acids contributes to the background flavor of most
long matured cheese varieties [2].
Lactic acid bacteria starter cultures and secondary micro-
biota that originate from the pits have important roles during
the cheese ripening in the pits. The secondary flora includes
a complex mixture of bacteria, yeast, and molds that coexist
in a complex equilibrium, and together with environmen-
tal factors, these confer the specific characteristic of this
* Francesca Comitini
f.comitini@univpm.it
1 Dipartimento Scienze della Vita e dell’Ambiente,
Università Politecnica delle Marche, Via Brecce Bianche,
60131Ancona, Italy

1922 European Food Research and Technology (2018) 244:1921–1931
1 3
particular type of cheese. A lot of studies have been car-
ried out to characterize the bacteria and molds, but there are
no data readily available on the yeast biota of Fossa cheese
[1, 3, 4]. The resident microbiota during dairy seasoning is
mainly composed by mesophilic lactic acid bacteria (LAB);
autochthonous strains originated from raw milk and dairy
environmental or from inoculated starter cultures [5]. In
Fossa cheese, the disciplinary dossier provides the use of
pasteurized milk and the use of selected LAB that contrib-
ute to acidification and ripening during the first step (dairy
seasoning). However, the extreme environmental conditions
during the secondary maturation in pit (low water content,
high salt concentration, and pH) compromise the survival
of LAB, while molds are dominant biota in this maturation
step [3, 6]. Indeed, the role of Penicillium and Aspergillus
for rheological and sensory characteristic of Fossa cheese is
well established [4, 7]. However, non-filamentous fungi were
found in a wide variety of cheeses, but in most cases, their
involvement in ripening is uncertain [8]. Most of studies on
cheese microbiota did not report on the presence of yeasts,
probably for a lack of specific examination procedures of
these microorganisms [9]. Only Fox and co-workers [10]
described the ubiquitary presence of Debaryomyces hanse-
nii in some cheese types such as Roquefort, Cabrales, and
Camembert, while Roostita and Fleet [11] studied the posi-
tive contribution of yeasts to flavor and texture development.
However, there are no reports on the occurrence of yeast
and on their contribution in Pecorino cheese type such as
Fossa cheese.
In the present study, we quantified, isolated, and then
identified the yeast flora from Fossa cheese and the pit envi-
ronment before and after cheese ripening, using both classi-
cal and molecular approaches. The isolated yeasts were then
used as indigenous starters to inoculate cheeses, evaluating
their influence on the analytical and sensorial characteristics
of the ripened cheese.
Materials andmethods
Technological procedure forFossa cheese
production
Fossa cheese is a typical Pecorino only produced in a spe-
cific area of central Italy as required by the disciplinary that
accurately describe the phases of production. The detailed
cheese making process is reported in the Gazzetta Ufficiale
della Repubblica Italiana [12] and here briefly reported with
the aim to clarify the procedures used during experimental
plan. Fossa cheese can be produced from pasteurized whole
sheep or cow milk or from a mixture of them, with the addi-
tion of selected starter of LAB (Sacco, Como Italy, code
MA11/11 YO), and the coagulation is made at 42–45°C by
the addition of artisan ovine rennet. In this study, the cheeses
were produced in the dairy from a mixture of whole cow’s
milk and whole sheep’s milk in a ratio of 80:20. The first
maturation occurs in dairy for at least 60days at 6–15°C
and 72–90% humidity, to achieve the necessary consistence.
Cheeses are then wrapped in apposite cloth bags in natural
cotton and transferred in pit for about 3months for the sec-
ond ripening.
Sampling fromFossa cheese andthepit
environment
Samples from Fossa cheese and the pit environment were
supplied by a dairy in the Marche region. Two different iso-
lation periods were included, using the same procedures, as
described below. The first isolation period was from both
the Fossa cheese and the pit environment during the sum-
mer period (June 2013), before the cheese ripening, and the
second isolation period was in late fall (October 2013), after
the Fossa cheese maturation.
Before and after pit-ripening isolation campaign, the
cheese sampling was carried out considering ten different
cheeses each contained in ten different cloth bags, consid-
ering the position and the different oxygen concentration
in the pit (bottom, middle, and top). Since the final ripened
cheese is characterized by a high degree of hardness difficult
to homogenize, the sampling was made in the crust surface
using sterile non-absorbent cotton swabs, by rubbing an area
of about 10cm2.
The ten cloth bags (containing the cheeses previously
sampled) were also assayed after the cheese ripening, using a
1cm2 piece cut out of each cloth. The pit sandstone rock sur-
face (i.e., sidewall), wooden floor-boards, and straw placed
around the walls were sampled in ten different sites consid-
ering the representative positions, by rubbing with the sterile
non-absorbent cotton swabs over 1cm2 surfaces. Each of the
cotton swabs coming from a specific site, was immediately
placed into sterile physiological solution (cheese 10ml;
cloth bag 2ml; pit 1ml), and the samples were maintained
aseptically on a rotary shaker at 150rpm overnight at 4°C,
to facilitate the microbial release.
In the laboratory, tenfold dilutions were made from the
buffered rinse solution from the cotton swabs, and these
were spread onto Wallerstein Laboratories (WL) agar with-
out and with 0.02% biphenyl, to count molds and yeast, and
de Man, Rogosa, and Sharpe (MRS) agar medium (Oxoid)
supplemented with 0.005% cycloheximide. The plates were
incubated for 5days at 25°C and the colonies were counted.
Representative yeast colonies were selected on the basis of
micro-morphological and macro-morphological evaluation
and proportionally to their frequencies. Isolates were then
sub-cultured on yeast extract peptone dextrose (YPD) agar
and identified using the molecular methods described below.

1923European Food Research and Technology (2018) 244:1921–1931
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Molecular identification oftheyeasts
One-hundred yeast colonies (75 before and 25 after pit rip-
ening) selected on the basis of micro-morphological evalu-
ation and proportionally to their frequencies, were subjected
to molecular characterization for their identification. DNA
was extracted and purified following the procedure proposed
by Stringini etal. [13]. The DNA from all yeast strains (from
cheese and fossa environmental samples) were identified
using the internal transcribed spacer (ITS)-PCR procedure,
as described by Esteve-Zarzoso etal. [14]. The fragment
sizes were estimated by comparisons with DNA standard
markers (GeneRuler 100-bp DNA Ladder; AB Fermentas),
and the restriction patterns were compared with those of
previously published studies [15]. Strains with uncertain
profiles were analyzed on D1/D2 domain of 16S rDNA that
identifies specific nucleotide sequences of the individual
species [16].
PCR‑DGGE yeast detection fromcloth bags
andcheese afterripening
PCR-denaturing gradient gel electrophoresis (DGGE) analy-
sis was performed using the method described by Cocolin
etal. [17]. Before the DGGE analysis, PCR was carried out
using the eukaryotic universal primers NL1GC 5′-CGC
CCG CCG CGC GCG GCG GGC GGG GCG GGG GCC
ATA TCA ATA AGC GGA GGA AAA G-3′ (the GC clamp
sequence is underlined) and LS2, 5′-ATT CCC AAA CAA
CTC GAC TC-3′.
The PCR products were obtained by direct DNA extrac-
tion from the cloth bags and the cheese inside after the pit
opening. DNA extraction was carried out using Innu-SPEED
Soil DNA kits (Biosense, Italy), according to the following
procedure. The liquid samples both from the cloth bags and
cheese surface (see above) were centrifuged, and 150µl of
pellet was used for the DNA extraction.
The extracted DNA was then analyzed using a DGGE-1
Elettrofor apparatus (Elettrofor, Rovigo, Italy). The DGGE
for sequence-specific identification was performed in 8%
polyacrylamide gels (acrylamide/bis-acrylamide mix; 37.5:1,
w/v) (Elettrofor, Italy) containing a 20–50% urea–forma-
mide gradient. The electrophoresis was performed in 1×
Tris-acetate-EDTA (TAE) buffer at a constant voltage of
150V, for 8h, with a constant temperature of 60°C. After
electrophoresis, the gels were stained in 1.25× TAE buffer
containing ethidium bromide, and then photographed under
UV transillumination, using a digital camera (Canon Power
Shot G5; Canon INC, Tokyo, Japan). Purified extract DNA
from the strains directly isolated before and after the cheese
ripening were used as markers.
After the DGGE analysis, all bands were excised from
the gels using a sterile pipette tip, and the DNA was eluted
in 50µl TE buffer, pH 8.0, overnight at 4°C. The eluted
DNA was reamplified with primers without the GC clamp,
using the same reaction mixture described above. The PCR
products were sequenced, and DNA sequence similarity
analysis was performed on-line using the basic local align-
ment search tool program (WU-BLAST2) on the European
Bioinformatics Institute webpage (http://www.ebi.ac.uk/
bast2 /index .htlm).
Inoculum ofyeast forcheese ripened inanartificial
pit
On the bases of DGGE results, five cultures, belonging
to five different species, were chosen as representative of
yeast biota. In particular, S. cerevisiae, C. homilentoma,
D. hansenii, W. anomalus, and C. zeylanoides were used,
separately (as pure culture) and in a complete mixture, to
inoculate artificial pits. Specifically, to create the artificial
pit, that attempted to reproduce the normal pit environ-
ment, propylene boxes were used (68 × 50 × 24cm), which
had been previously sterilized using UV light, and which
were equipped with vacuum closing caps. Each box was
filled with a layer of crumbled sandstone and a wooden
grate was place in the bottom, while the walls of each box
were covered with straw. These artificial pits were filled
with cheeses, hermetically closed, and left at 30°C for
48days. Each artificial pit contained four cheeses (400g
each one) placed inside a new cotton bag, cheeses were
previously produced in a dairy according to the Fossa
cheese disciplinary until the dairy seasoning step. Pre-
liminary, three different yeast inoculation procedures were
tested: (1) together with starter LAB in pasteurized milk
(inoculation I); (2) after curd rupture (inoculation II); and
(3) sprayed onto the cheese surface after dairy maturation
(inoculation III). However, based on results obtained (data
not shown), the inoculation III procedure was applied. For
each inoculation procedure, 20ml of cell suspension in
peptonate water (106cell/ml) was added.
The experimental plan included six theses repeated in
triplicate. Each thesis corresponded to an artificial pit: (a)
inoculation with S. cerevisiae; (b) inoculation with C. homi-
lentoma; (c) inoculation with D. hansenii; (d) inoculation
with W. anomalus; (e) inoculation with C. zeylanoides, and
(f) inoculation of the yeast mix. Control trials, without yeast
inoculation, were also carried out.
After maturation time in artificial pit, one cheese per pit
was randomly chosen and analyzed by experienced testing
panel. The best-evaluated cheese from sensory analysis and
a control (cheese from natural pit maturation) were analyzed
by solid-phase microextraction–gas chromatography-mass
spectrometry (SPME–GC-MS) to determine their final ana-
lytical profile.

1924 European Food Research and Technology (2018) 244:1921–1931
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Sensory evaluation
A panel of ten assessors, eight males and two females, took
part in the descriptive sensory analysis. All were members
of an established sensory panel at Consorzio del Forma-
ggio di Fossa di Sogliano. The panel was recruited and
screened according to the international standards ISO/DIS
13299:1998 [18]. This experienced–trained panel possessed
a high level of discrimination, sensitivity, and consistency
in measurement. All assessors were trained to carry out
descriptive analysis and were involved in developing the
descriptive vocabulary for cheese. The development of a
full descriptive vocabulary, to describe the sensory char-
acteristics of the cheeses, was carried out for each product
category, and the panel of assessors evaluated the products
for flavor, texture, mouth feel, appearance, and odor using a
11-point hedonic scale (0–10; poor to excellent) [19]. The
data from the evaluation panels are expressed as means of
triplicate sensory evaluations, with significantly differences
reported. A second level of sensory evaluation was carried
out according to the Etana method described by Bozzetti
etal. [20] where the evaluated attributes were flavor and
aroma: sweet, salty, bitter, and acidic. The tactile sensations
as astringent, spicy, heat, softness, compactness, refresh-
ing, aftertasting, and persistence were also evaluated. In this
case, the hedonic scale was 6-point (0–5; poor to excellent).
SPME‑GC/MS analysis
10g cheese from the artificial ripening was assayed for the
analytical profile, which was compared with two cheeses
used as positive and negative controls: the first was matured
in a natural pit environment and the second was an un-inoc-
ulated cheese matured in artificial pit. For this analytical
evaluation was followed the procedure proposed by Gioac-
chini etal. [21] using a gas chromatograph mass spectrom-
eter GCMS-QP2010 SE Shimadzu (Tokyo, Japan).
Statistical analysis
The data from the plate counts are means ± SD of at least
three different environment and cheese samples. Differ-
ences in the sensory evaluation of the cheese samples in the
panel tests and the SPME-GC analyses were evaluated using
analysis of variance. The data were elaborated using Dun-
can ANOVA-tests. Differences were considered significant
at P < 0.05 using the Statistica 7 software.
Results
Microbial count, andyeast isolation
andidentification
The microbiota of the Fossa cheese surface assessed by
culture-dependent methods are given in Table1. Quantita-
tive and qualitative results did not show differences between
samples collected by different positions of the pit (bottom,
middle, and top). Instead, there were slight differences
between the mold content on the cheese surface comparing
the output from the dairy (1.5 × 103CFU/cm2) and before the
ripening in the pit (9.0 × 103CFU/cm2). As expected, strong
Table 1 Quantitative evaluation of the microbiota from the Fossa cheese and the pit environment
n.d. Not detected
Sample Sampling Isolation source Colony area (× 103CFU/cm2)
Yeast Molds LAB
Cheese After dairy seasoning Cheese surface 0.02 ± 0 1.58 ± 0.1 0.13 ± 0.01
Before cheese ripening Cheese surface 0.03 ± 0.01 9 ± 0 0.10 ± 0.1
After cheese ripening Cheese surface 2.70 ± 0.01 1000 ± 0.8 0.14 ± 0.2
Pit environment Before cheese ripening Sandstone rock (top wall) 0.72 ± 0.02 0.11 ± 0.01 1.60 ± 0.04
Sandstone rock (center wall) 0.44 ± 0.01 0.15 ± 0.03 0.85 ± 0.04
Sandstone rock (bottom wall) 0.23 ± 0.02 0.16 ± 0.04 3.80 ± 0.02
Straw 0.08 ± 0 0.34 ± 0.04 0.14 ± 0.07
Wood grate 0.12 ± 0.07 0.35 ± 0 0.45 ± 0
Cloth bags 0 0 0
After cheese ripening Sandstone rock (top wall) 0.84 ± 0.01 5.90 ± 0.5 0.52 ± 0.2
Sandstone rock (center wall) 0.50 ± 0 6.40± 0.46 ± 0.06
Sandstone rock (bottom wall) 0.34 ± 0.03 7.20 ± 0.01 0.98 ± 0.04
Straw 0.33 ± 0.20 4.70 ± 0.04 0.06 ± 0.01
Wooden grate 0.06 ± 0.03 54 ± 0.9 n.d.
Cloth bag 2.30 ± 0.3 58 ± 0.3 0.08 ± 0.01

1925European Food Research and Technology (2018) 244:1921–1931
1 3
enhancement was seen after the pit maturation. Indeed,
the mold content increased by three orders of magnitude
(1.0 × 106CFU/cm2). The levels of bacteria on the cheese
surface remained stable before and after the pit ripening (ca.
1.0 × 102CFU/cm2). In contrast, the yeast biota increased by
two orders of magnitude after the pit ripening, to achieve a
final level of 2.7 × 103CFU/cm2. This relevant enhancement
of the yeast biota highlighted the changes in the structure of
the microbial community on the cheese surface during the
secondary maturation in the pit.
The counting of the microbiota isolated from the pit envi-
ronment is also given in Table1. As expected, before the
ripening step, all of the samples showed high levels of molds
(ca. 1.0 × 102CFU/cm2), which rose to 1.0 × 103CFU/cm2
after ripening. The molds were particularly abundant on the
wooden grate after the ripening (5.4 × 104CFU/cm2). The
yeast and bacteria communities did not change significantly
in terms of the levels before and after the aging in the pit.
Indeed, these were at 1.0 × 102CFU/cm2, with the exception
of the cloth bag at the end of maturation (2.3 × 104CFU/
cm2).
While no quantitative differences in the yeast colonization
in pit environment was seen, the results of the identification
revealed large changes in the yeast biota composition before
and after the pit maturation (Table2).
A total of 100 colonies were isolated from pit environ-
ment and cheese after ripening based on micro-morpholog-
ical and macro-morphological aspects and proportionally
to their frequencies Most of these were isolated before the
filling of the pit with the cheeses, and they came from the
walls of the pit, the straw, and the wooden grate. The yeast
isolated in this step belongs mainly to the Basidiomycetes
group, such as Cryptococcus (wide occurrence), Filoba-
sidium, and Sporobolomyces together with the yeast-like
fungus Aureobasidium. Two species that belonged to the
Candida genera were also identified. Therefore, 11 species
were found before ripening and only 5 species were identi-
fied after ripening. Among these, only Candida zeylanoides
was found both before and after the cheese ripening, together
with the newly found species Wickerhamomyces anomalus,
Debaryomyces hansenii, Candida homilentoma, and Sac-
charomyces cerevisiae. In terms of frequency, D. hansenii
Table 2 Occurrence of the yeast
biota and the ITS identification
*Used method
a Average data for three batches of samples analyzed, considering ten different cheeses, in duplicate
b Pit environment (Sandstone rock, straw, wood grating)
Sampling Species Frequencya
(CFU/cm2)
Identification
ITS-RFLP D1/D2
Before cheese ripening
Pit environmentbCryptococcus hungaricus 82 ± 0.3 *
Pichia occidentalis 3 ± 0.1 *
Cryptococcus laurentii 100 ± 0.6 *
Candida norvegica 10 ± 0.1 *
Cryptococcus skinneri 206 ± 0.5 *
Candida zeylanoides 34 ± 0.3 *
Sporobolomyces roseus 20 ± 0.8 *
Aureobasidium pullulans 69 ± 0.4 *
Filobasidium globisporium 55 ± 0.1 * *
Cryptococcus chernovii 110 ± 0.4 * *
Cryptococcus victoriae 215 ± 0.3 * *
After cheese ripening
Pit environment Saccharomyces cerevisiae 95 *
Wickerhamomyces anomalus 110
Debaryomyces hansenii 603 *
Candida zeylanoides 301
Candida homilentoma 27 *
Cheese *
*
Wickerhamomyces anomalus 413 * *
Debaryomyces hansenii 930 * *
Candida zeylanoides 243 * *
Candida homilentoma 82 * *

1926 European Food Research and Technology (2018) 244:1921–1931
1 3
was the most abundant species, followed by W. anomalus
and C. zeylanoides and then C. homilentoma and S. cerevi-
siae, the most poorly represented.
PCR‑DGGE analysis
DNA samples from the cheese surface and the cloth bag
matrix after the pit ripening were analyzed by PCR-DGGE,
including the ladder profiles of the 26S rRNA gene frag-
ment, to compare the sample profiles with all five species
found in the pit environment after the cheese maturation
period (Fig.1).
The data obtained by culture-independent methods con-
firmed the very low biodiversity shown with the culture-
dependent approaches. Indeed, the sequenced gel bands from
the cheese surface matched with D. hansenii, C. zeylanoides,
and W. anomalus (Table3). In combination with the culture-
dependent approach, these data highlighted the simplifica-
tion of this specific ecological niche during the 85days of
cheese ripening. The stabilized yeast flora dominated the
environment and colonized the cheese surface with only a
few well-adapted species.
Preliminary evaluation ofinoculated cheeses
ripened intheartificial pit
The mold colonization on the cheese surface in the control
trial (i.e., without yeast inoculation) was controlled after
15days of pit closure, concurrent with the increase in the
humidity. Comparing the un-inoculated cheese (negative
control) with the other inoculated cheeses, an evident vari-
ation in the surface colonization was seen (data not shown).
The preliminary evaluation carried out at the end of the arti-
ficial ripening showed that the cheeses in the control trials
had irregular shapes, with distended areas and depressions,
with the surface wet and fatty, and in some cases covered
by greenish molds; in contrast, the inoculated cheeses had a
dry surface and appeared less colonized by molds (Table4).
In particular, cheeses matured under artificial inocula-
tion conditions showed higher scores and often similar to
that obtained by the positive control (i.e., the natural Fossa
cheese), especially for the flavor quality and intensity and the
low mold colonization. In particular, low mold colonization
was evident with the inocula of C. zeylanoides, D. hanse-
nii, and W. anomalus. The only exception was the cheeses
inoculated with S. cerevisiae and C. homilentoma, which
showed relatively little flavor and crust compactness, and
irregular body texture. Moreover, cheeses inoculated with W.
anomalus were particularly appreciated by the panel judges.
Indeed, these cheeses were judged positively for flavor qual-
ity, intensity, and body texture. Also, the cheeses ripened in
the artificial pit after inoculation of the mix of these yeasts
obtained very positive scores (Fig.2).
Sensory evaluation andanalytical profiles
ofthecheeses fromtheartificial pit
On the basis of the results during the preliminary evalu-
ation of the cheeses ripened in the artificial pits, cheeses
inoculated with only W. anomalus and with the mix of
yeast were subjected to a secondary sensory evaluation.
Fig. 1 DGGE profile of analyzed samples. Lanes 1, 3, samples from
cloth bag; lanes 2, 4, samples from the cheese surface. Reference
strains: Cz, C. zeylanoides; Wa, W. anomalus; Dh, D. hansenii; and
Sc, S. cerevisiae. Bands from A to F were excised, reamplified, and
subjected to sequencing
Table 3 Sequencing data for the bands excised from the yeast DGGE
gels
Band Size (bp) Origin Closest relative % Identity
A 213 Sandstone D. hansenii 97
B 204 Sandstone D. hansenii 99
C 214 Cheese D. hansenii 98
D 204 Cheese D. hansenii 94
E 196 Cheese C. zeylanoides 95
F 180 Sandstone W. anomalus 98

1927European Food Research and Technology (2018) 244:1921–1931
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This further evaluation considered a more accurate panel
with 12 descriptors according to the ETANA model,
accompanied by SPME–GC-MS analysis, with the data
from the W. anomalus alone inoculation given here. The
olfactory characteristics were indeed typical of aged
Fossa cheese, such as the smell of fermented grass and
rennet, and a toasty smell were present for the cheeses
inoculated with both W. anomalus alone (Fig.2) and the
mixture of these yeast (data not shown), unlike the nega-
tive control, which did not show these notes. Similar to
the olfactory evaluation, the analysis of taste revealed the
scent of sweetness, salty, bitter, and pungent only in these
inoculated cheeses. In contrast to the negative control, the
perception of the structure of these inoculated cheeses
indicated that they also had adequate softness and easy
swallowing friability (Fig.2).
The data for the analysis of the volatile organic com-
pounds are given in Table5, where the cheeses inoculated
with W. anomalus alone were compared with an un-inocu-
lated cheese (negative control) and a natural Fossa cheese
(positive control). The fatty acids and ketones were the most
abundant component, comparable in relative terms between
un-inoculated control cheese and in the yeast-inoculated
samples. Actually, while hexanoic and butyric acids were
relatively higher in the cheese inoculated with W. anomalus
compared to the negative control, octanoic acid was slightly
lower. Interestingly, comparing the inoculated cheese from
artificial pit with the natural Fossa cheese, the relative per-
centage of butyric and hexanoic acids and nonanone did not
show significant differences. These volatile compounds are
responsible of dairy-like and cheesy taste, and are the most
important components of cheese flavor [22].
The relative amount of isovaleric acid, associated with
an acidic taste, and phenyl ethyl alcohol associated with
the floral smell, in the W. anomalus inoculated cheese and
natural Fossa cheese were significantly comparable, and the
presence were confirmed to be still perceptible by the panel
test (Fig.2).
Among ketones, 2-pentanone (16.96%), 2-heptanone
(13.24%), 2-nonanone (9.85%), and 2-undecanone (0.13%)
were detected in artificial pit cheese, as shown in Table5.
Discussion
Several studies have investigated the microbial ecology
of traditional Italian cheeses (i.e., smear ripened), such
as Fontina, Taleggio, and Gorgonzola cheeses [6, 23, 24].
However, the attention is generally focused on the role of
LAB, autochthonous or selected cultures, that produce
acids during fermentation process and contribute to cheese
ripening through proteolysis activity [9]. Fossa cheese is
a niche product where the pit ripening is the longest and
most important phase where the cheese is transformed from
Pecorino to very typical product. Based on this, during pit
ripening the specific environment and the complex interac-
tion between microorganisms confer the peculiar sensory
characters to final product. However, the composition and
the peculiar role of microbial communities that characterize
the secondary microflora of the Fossa cheese environment
have been poorly investigated, and the studies available have
focused particularly on characterization of the mold [3, 4,
25]. In pit environment, only the mold colonization has been
widely investigated and reported [3, 6]. Only recently, Car-
doso etal., [26] investigating on the frequency and seasonal
diversity of yeasts during maturation of a traditional Brazil-
ian cheese, found an evident diversity in yeast communities
with an effective aromatic role, resulting from protease and
β-galactosidase activities.
In this study, investigating on the occurrence of yeasts,
we found that the wide biodiversity in the pit environment
before the ripening was reduced after cheese seasoning. On
the other hand, the quantitative presence of yeasts on the
cheese surface increased, indicating an effective coloniza-
tion by a few specific yeast species, such as S. cerevisiae, C.
homilentoma, D. hansenii, W. anomalus, and C. zeylanoides.
Table 4 Preliminary evaluation of the Fossa cheese ripened in the artificial pit, according to an ordinary ten-point scale (1–10; poor to excellent)
Data with different superscript letters (a,b) are significantly different (p < 0.05) for each character using Duncan’ test
c The yeasts (Ch, C. homilentoma; Cz, C. zeylanoides; Dh, D. hansenii; Sc, S. cerevisiae; Wa, W. anomalus; Mix, mixture of all five yeast) are
sprayed onto cheese surface after dairy maturation. Data are mean ± standard deviation of cheeses analyzed in duplicate. Negative control, un-
inoculated cheese ripened in the same way; positive control, natural Fossa cheese
Character Control Inoculated cheesec
Positive control Negative control Ch Cz Dh Sc Wa Mix
Mold on surface 7.5a ± 1.03 2.5b ± 0.91 2.4b ± 0.12 5.5a ± 1.55 7.8a ± 2.03 3.0b ± 0.05 7.7a ± 0.45 9.0a ± 0.23
Flavor quality 8.6a ± 1.47 2.6b ± 1.02 6.2a ± 1.97 8.8a ± 2.04 8.0a ± 0.89 1.0b ± 0.74 7.2a ± 0.55 8.7a ± 0.01
Flavor intensity 9.8a ± 1.06 2.0b ± 0.98 5.5a ± 0.02 7.5a ± 0.09 8.5a ± 1.47 4.8ab ± 1.08 9.0a ± 2.03 8.0b ± 1.74
Crust compactness 8.02a ± 0.02 1.9b ± 0.57 4.3a ± 0.27 6.9a ± 0.98 8.3a ± 0.04 3.15b ± 0.23 8.5a ± 2.00 8.12a ± 1.04
Body and texture 7.99a ± 0.55 3.4b ± 1.05 3.8b ± 0.02 8.2a ± 0.01 7.7a ± 1.88 4b ± 2.04 7.9a ± 2.03 9.4a ± 2.77

1928 European Food Research and Technology (2018) 244:1921–1931
1 3
0
1
2
3
4
5
Softness
Compactness
Sweet
Salty
Bitter
Spicy
Acid
Astringent
Heater
Refreshing
Aftertaste
Persistence
(a)
0
1
2
3
4
5
Softness
Compactness
Sweet
Salty
Bitter
Spicy
Acid
Astringent
Heater
Refreshing
Aftertaste
Persistence
(b)
0
1
2
3
4
5
Softness
Compactness
Sweet
Salty
Bitter
Spicy
Acid
Astringent
Heater
Refreshing
Aftertaste
Persistence
(c)
0
1
2
3
4
5
Softness
Compactness
Sweet
Salty
Bitter
Spicy
Acid
Astringent
Heater
Refreshing
Aftertaste
Persistence
(d)
0
1
2
3
4
5
Softness
Compactness
Sweet
Salty
Bitter
Spicy
Acid
Astringent
Heater
Refreshing
Aftertaste
Persistence
(e)
0
1
2
3
4
5
Softness
Compactness
Sweet
Salty
Bitter
Spicy
Acid
Astringent
Heater
Refreshing
Aftertaste
Persistence
(f)
Fig. 2 Panel evaluation of the cheese inoculated with selected indig-
enous yeasts (dashed lines, marker ●) ripened in an artificial pit: a
inoculation with S. cerevisiae; b inoculation with C. homilentoma;
c inoculation with D. hansenii; d inoculation with W. anomalus; e
inoculation with C. zeylanoides, and f inoculation of the yeast mix.
Continuous line with marker (♦) indicates the negative control (un-
inoculated cheese); continuous line with marker (▲) indicates a natu-
ral Fossa cheese

1929European Food Research and Technology (2018) 244:1921–1931
1 3
This reduced yeast diversity could be explained consider-
ing the pit an environment highly selective in terms of the
pH, low temperature, and anaerobic conditions [27]. DGGE
analysis corroborates and supports this finding. Indeed, only
three yeast species in cheese after ripening was found. On
the other hand, the microbiological patterns obtained after
DGGE analysis are strictly connected to the numerically
dominant species with a low detection limit and highly
matrix dependent [13].
When the isolated C. zeylanoides and W. anomalus yeast
were used as inoculum, this drastically reduced the mold
colonization of the cheese surface during cheese ripening,
while also providing excellent results in the panel evalua-
tions. Indeed, the cheese that was ripened here in artificial
pits following inoculation with W. anomalus, showed com-
parable characteristics to those of the Fossa cheese seasoned
in the natural environment, with higher concentrations of
acids, esters, and ketones.
The flavor of Fossa cheese is unique, sweet at first,
then becoming sharper after pit ripening that is the cru-
cial step that characterize the organoleptically properties.
Indeed, the appearance of the Fossa cheese is remarkably
Table 5 Volatile organic compounds identified by SPME–GC-MS in the cheese before (control) and after (sample) seasoning in the artificial pit,
for the cheeses inoculated with W. anomalus alone (inoculation III experiment)
Sensory description were in according with Bozzetti etal. [20]
Data are mean ± standard error of three batches of cheeses, each analyzed in duplicate
Data with different superscript letters (a,b) are significantly different (p < 0.05) for each character using Duncan’ test
Negative control, un-inoculated cheese ripened in the same way; positive control, natural Fossa cheese
Compound Relative area (%) Sensory description
Negative control Positive control Sample
2-Nonanone 20.17 ± 1.05a5.99 ± 1.25a,b 9.01 ± 1.69bFruity, sweet, waxy, soapy, cheesy, green, herbaceous, coconut-like
Hexanoic acid 17.39 ± 0.96a25.31 ± 2.04b23.28 ± 3.36bSour, fatty, sweat, cheesy
Octanoic acid 15.92 ± 1.52a9.01 ± 0.14b15.08 ± 1.25aFatty, waxy, rancid, oily, vegetable-like, cheesy
Butyric acid 12.42 ± 1.25a5.47 ± 0.36a,b 8.51 ± 2.87bSharp, dairy-like, cheesy, buttery with a fruity nuance
2-Pentanone 9.28 ± 0.77a20.58 ± 5.57b16.96 ± 5.01cEthereal, diffusive, and sweet banana-like with fermented woody
nuance
2-Heptanone 5.84 ± 1.04a11.4 ± 2.02b12.24 ± 2.35bCheese, fruity, spicy, ketonic, green banana-like, with a creamy
nuance
Decanoic acid 4.91 ± 0.24a0.89 ± 0.01c2.24 ± 0.95bUnpleasant, rancid, sour, fatty, citrus-like
Acetyl methyl carbinol 4.57 ± 0.03a0.94 ± 0.01c2.72 ± 0.03bSweet, buttery, creamy, dairy, milky, fatty
2,3-Butanediol 1.95 ± 0.01a1.56 ± 0.04a0.53 ± 0.02bFruity, creamy, buttery
2-Undecanone 1.74 ± 0.02aTrace 0.13 ± 0.01bWaxy, fruity, creamy, floral, with fatty pineapple nuances
Nonanoic acid 1.06 ± 0.03aTrace 0.86 ± 0.01aWaxy, dirty, and cheesy with a cultured dairy nuance
8-Nonen-2-one 0.87 ± 0.01aTrace 0.79 ± 0.02aFruity, baked
2-Nonanol 0.44 ± 0.02aTrace 0.08 ± 0.01bWaxy, green, creamy, orange-like, cheesy, slight fruity
2-Octanone 0.23 ± 0.01a1.06 ± 0.02b0.28 ± 0.07aMusty, ketonic, blue, and parmesan cheese-like, earthy, dairy
Heptanoic acid 0.18 ± 0.01a2.47 ± 1.07b0.27 ± 0.09aCheesy, waxy, sweaty, fermented, pineapple-like, fruity
Ethyl hexanoate 0.18 ± 0.01aTrace 0.11 ± 0.01aSweet, fruity, pineapple-like, waxy, fatty, green banana-like
Ethyl decanoate 0.17 ± 0.02aTrace 0.10 ± 0.05aSweet, waxy, fruity, apple/grape-like, oily, brandy-like
2-Decanone 0.13 ± 0.03a1.62 ± 0.36b0.02 ± 0.01cOrange-like, floral, fatty, peach-like
Isovaleric acid 0.12 ± 0.04a0.22 ± 0.57b0.25 ± 0.03bCheese, dairy, acidic, sour, pungent, fruity, stinky, ripe, fatty
Nonanal 0.12 ± 0.01a0.02 ± 0.01b0.06 ± 0.02bWaxy, with a fresh slightly green lemon peel like nuance, and a
cucumber fattiness
Ethyl octanoate 0.10 ± 0.02a0.14 ± 0.01a0.12 ± 0.01aWaxy, sweet, musty, brandy-like, pineapple-like, and fruity with a
creamy, dairy nuance
Fenylethyl alcohol 0.06 ± 0.01a0.33 ± 0.01b0.19 ± 0.01bSweet, floral, fresh, and bready with a rosey honey nuance
2-Heptanol 0.29 ± 0.02a2.04 ± 0.02b0.60 ± 0.05aFresh, lemon, grassy, sweet, floral, fruity, green
2-Esanone 0.78 ± 0.06a5.25 ± 0.3b1.93 ± 0.03aEthereal
Isoamylic alcohol 0.09 ± 0.01a1.02 ± 0.01a0.27 ± 0.01bFuel, alcoholic, pungent, ethereal, cognac-like, fruity, banana, and
molasses-like
2-methylbutyric alcohol 0.01 ± 0.01a0.78 ± 0.01b0.06 ± 0.01aAcidic, fruity, dirty, cheesy with a fermented nuance
γ-Dodecalactone Trace 1.35 ± 0.04a0.25 ± 0.01bFatty, peach-like, sweet, metallic, fruity

1930 European Food Research and Technology (2018) 244:1921–1931
1 3
different from factory ripened cheeses in terms of its hard-
ness, moistness, and flavor. Even if the origin of the milk
(cow, sheep or mixture) influences the final aroma, the
cheese taste is defined essentially during the long period
of maturation in the pits.
Free fatty acids were the main components of the total
volatile compounds in these analyzed cheeses. In particu-
lar, butyric acid, which originates from the action of an
endogenous lipase, was abundant in different types of rip-
ened cheeses, in particular in typical Roman cheeses, and
together with other acids, such as glutamic acid and the ace-
tic acid, these give the particular flavor to the cheeses [28,
29]. However, the concentration of butyric acid in a cheese
must remain below a threshold value to avoid becoming a
defect for a cheese [30]. Otherwise, the concentrations of
hexanoic and octanoic acids are directly related to the qual-
ity of a cheese [21]. Indeed, the free fatty acids are important
components of flavor, although their role vary according to
the cheese variety [22]. In the present study, the un-inocu-
lated cheeses showed higher amounts of butyric acid, while
higher levels of hexanoic and octanoic acids were produced
by cheeses inoculated with W. anomalus (comparable to nat-
ural Fossa cheese). In Fossa cheese, the most representative
ketones are 2-heptanone, which provides a typical cheese
note (major constituent) and 2-nonanone, found in very lim-
ited amounts [29]. This trend was confirmed in our study,
where both natural Fossa cheese and inoculated cheese with
W. anomalus completely reflected this feature. On the other
hand, the un-inoculated cheeses contained high amounts
of 2-nonanone, which gave excessive fruit and herbaceous
notes, indicating an actual positive effect of yeast in the rip-
ening process. Another important aspect is the high concen-
trations of esters, which are generally linked to high lipase
activity in dairy products [31]. The microorganisms involved
in ester formation are mainly yeasts [32], and secondary
LAB. These compounds confer floral and fruity notes and
strongly contribute to the cheese aroma, minimizing the
sharpness of fatty acids and the bitterness of amines [33].
Overall, the data on the aromatic profiles highlight the
role of yeast and support the purpose of using selected and
autochthonous yeast from the pits to improve and control
the cheese ripening. Indeed, the amounts of volatile organic
compounds in these cheeses ripened in artificial pits with
selected yeast confer a good balance to the final products,
with the perception of the right notes of fatty, sweat, and
vegetable-like, as confirmed by the panel evaluations.
Moreover, inoculation of selected yeast into the seasoned
cheese in the dairy can be used as a biocontrol tool, to inhibit
the growth of several dairy molds. Indeed, all three species
that were widely found here (i.e., W. anomalus, D. hansenii,
and C. zeylanoides) significantly reduced the mold coloniza-
tion on the cheese surface during the artificial pit ripening,
without altering the typical Fossa cheese aroma.
In conclusion, this study represents a characterization
of the yeast biota in the pit environment of Fossa cheese,
and shows the positive contribution of these yeasts during
the cheese ripening. Despite the complex microbial com-
munity, this study has allowed us to identify W. anomalus,
D. hansenii, and C. zeylanodes as the most represent yeast
species in the pit environment after Fossa cheese ripening.
Extending this experimental design to other dairies to rein-
force the results obtained, these yeasts could be useful to
better control the Fossa cheese-ripening process reducing
the management costs.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
Compliance with ethics requirements All authors declare that this ar-
ticle does not contain any studies with human or animal subjects.
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