Food Research International 116 (2019) 1344–1356

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Use of autochthonous mesophilic lactic acid bacteria as starter cultures for T

making Pecorino Crotonese cheese: Effect on compositional, microbiological
and biochemical attributes
⁎
Ilaria De Pasqualea, Raffaella Di Cagnob, , Solange Buchinc, Maria De Angelisa, Marco Gobbettib
a
Department of Soil, Plant and Food Science, University of Bari Aldo Moro, Bari 70126, Italy
b
Faculty of Science and Technology, Libera Università di Bolzano, Bolzano, Italy
c
Technologie et Analyses Laitières, INRA, Poligny UR 342, France

A R T I C LE I N FO A B S T R A C T

Keywords: The use of selected autochthonous mesophilic lactic acid bacteria as starter cultures was investigated according
Non-starter lactic acid bacteria to the traditional protocol for making Pecorino Crotonose (PC). Leuconostoc mesenteroides subsp. mesenteroides
Starter cultures 2A, Lactobacillus casei 23C and Lactobacillus plantarum 18C (Autochthonous Starter, AS1) and Leuc. mesenteroides
Ripening subsp. mesenteroides 2A, and L. casei 25D and 16A (AS2) were isolated and identified from aged ewes' milk PC
Volatile components
cheeses, selected based on several enzymatic activities, and used as starter cultures. As shown by the in vitro
Pecorino Crotonese
kinetic of acidification, selected starter cultures had suitable capabilities to acidify. The manufacture of PC
cheeses was carried out at an industrial plant scale. A control cheese (CC) was also made, using commercial
starters consisting of mesophilic and thermophilic species. Ripening lasted 105 days at 10 °C. A poly-phasic
approach was used to compare cheeses during manufacture and ripening, mainly based on pyrosequencing of the
16S rRNA targeting DNA, proteolysis and volatile component analyses. Compared to CC, both autochthonous
starter cultures slightly affected the gross chemical composition of PC cheese. The cell density of thermophilic
starters of CC progressively decreased throughout ripening. Plate count and RAPD-PCR showed that the cell
number of autochthonous lactobacilli cultures of PC cheeses, made with AS1 and AS2, was almost constant
throughout ripening and abundantly higher than that observed in CC. As shown by culture-independent analysis,
the OTUs found during ripening varied depending on the manufacture with or without autochthonous starter
cultures. The major chemical differences among cheeses were the concentration of free amino acids and the
synthesis of some key volatile components (e.g., 2-methyl-1-propanol, 2-methyl-1-butanol, isobutyric, isovaleric,
and isocaproic acids). Compared to CC, the use of AS1 positively affected the overall cheese quality.

1. Introduction
Pecorino Crotonese (PC) is an Italian ewes' milk cheese, which
gained the status of Protected Designation of Origin (PDO) (CEE
Regulation n. 1262/2014). PC cheese is only manufactured by artisanal
cheesemakers who associated in a Consortium and follow traditional
protocols of cheesemaking in a well-defined area of southern Calabria
(Italy). In 2017, the PC production has been estimated to be ca. 15 t
(http://www.bioagricert.org/it/). Renneting by kid rennet paste and
the use of raw ewes' milk from two to four daily milking are the primary
typical features of PC cheesemaking. As reported in the Regulatory
Board of the Consortium, pasteurization or thermization of milk is also
permitted. The use of natural whey cultures and that of commercial
starters (mixture of mesophilic and thermophilic species/strains) has
been a technology option for long-time. Recently, the Consortium made
the decision to prohibit the use of starter cultures. Consequently, the
mild acidification of cheese milk is expected to be mainly by auto-
chthonous lactic acid bacteria. Nevertheless, the Regulatory Board of
the Consortium is encouraging the exploitation of the potential of se-
lected autochthonous mesophilic lactic acid bacteria to be used as
starter cultures. This would help to avoid flavour flattening and to fa-
vour the standardization of the overall cheese quality.
Primary commercial or natural whey/milk starters are mostly lim-
ited to a few species of lactic acid bacteria (LAB), mainly Lactococcus
lactis subsp. lactis, Streptococcus thermophilus, Lactobacillus delbrueckii
spp. and Lactobacillus helveticus, which do not fully explain the diversity
of a high number of cheese varieties. The well-established observation
that raw milk cheeses develop a more intense flavour than pasteurized

⁎
Corresponding author.
E-mail address: raffaella.dicagno@uniba.it (R. Di Cagno).

https://doi.org/10.1016/j.foodres.2018.10.024
Received 19 June 2018; Received in revised form 5 October 2018; Accepted 7 October 2018
Available online 09 October 2018
0963-9969/ © 2018 Published by Elsevier Ltd.
I. De Pasquale et al.
cheeses (Lynch, McSweeney, Fox, Cogan, & Drinan, 1996) and the
growing interest to preserve the features of traditional raw milk cheeses
has stimulated the use of selected autochthonous starter cultures for
making pasteurized milk cheeses (Gobbetti, De Angelis, Di Cagno,
Mancini, & Fox, 2015). Depending on the environmental and techno-
logical factors, all ripened cheese varieties harbour an autochthonous
microbiota, which mainly diversifies cheeses throughout processing
(Montel et al., 2014). The abundant literature data of the last two
decades showed that the autochthonous cheese microbiota commonly
consists of so-called non-starter lactic acid bacteria (NSLAB), mainly
lactobacilli, enterococci, and to a lesser extent Leuconostoc spp. and
thermophilic lactic acid bacteria (Bautista-Gallego et al., 2014;
Gobbetti et al., 2015; Lynch et al., 1996). Although milk and the house
microbiota ensure an almost constant supply of NSLAB (Calasso et al.,
2016), their cell density and diversity markedly varies between cheese
batches. Besides, the use of NSLAB as starters may had given rise to off-
flavours or quality defects (Herreros et al., 2007). Both the above
considerations impose the careful screening among potential starter
cultures before their introduction into large-scale model fermentations.
Starter cultures may originate from raw milk and, especially, from high
quality ripened cheeses (Gobbetti et al., 2015). To be suitable as starter
cultures, mesophilic NSLAB have to meet several criteria. They have to
acidify, to reach and maintain high cell densities in cheese milk, to
prevent defects in cheese, and to affect positively the overall cheese
quality (Crow, Curry, & Hayes, 2001; Di Cagno et al., 2011; Gobbetti
et al., 2015). The screening based on various and complementary en-
zyme activities often preceded the use of mesophilic lactobacilli as
starters (Di Cagno et al., 2011; Di Cagno, De Pasquale, De Angelis, &
Gobbetti, 2012; Gobbetti et al., 2015). To the best of our knowledge,
the diversity and dynamics of NSLAB harboured by artisanal PC cheeses
underwent to characterization and genotyping during the manufacture
and ripening, showing a composition that mainly consisted of meso-
philic lactobacilli (e.g., Lactobacillus rhamnosus, Lactobacillus plantarum
and Lactobacillus buchneri) and Leuconostoc mesenteroides (Randazzo,
Pitino, Ribbera, & Caggia, 2010). No published reports have concerned
the selection of autochthonous NSLAB isolated from PC cheeses in order
to establish mesophilic starter cultures suitable for the manufacture of
PC cheese.
This study aimed at isolating and identifying autochthonous NSLAB
from aged PC cheese, and at selecting the best performing strains based
on enzyme activities. Selected strains were used in the formulation of
two starter cultures for the industrial manufacture of PC cheese. PC
cheesemaking was carried out according to the traditional protocol
using commercial primary starters or selected autochthonous starters,
comprising mesophilic NSLAB. A poly-phasic approach compared the
cheeses through manufacture and ripening, mainly including pyr-
osequencing of the 16S rRNA targeting DNA, proteolysis and volatile
component analyses.
2. Materials and methods
2.1. Enumeration and isolation of non-starter LAB from Pecorino Crotonese
cheeses
Ewes' pasteurized milk PC cheese samples, made with natural whey
starter cultures, were manufactured by two representative dairies as-
sociated to the Consortium. Three cheeses, each one from a different
batch, were collected from each dairy, and subjected to analysis at 75,
90, 105 and 120 days of ripening. Analyses were in duplicate for each
batch of cheese (twelve analyses for each time of ripening). Twenty
grams of cheese were diluted into 180 mL of a sodium citrate (2%, wt/
vol) solution, and homogenization was carried out with a Stomacher
Lab-Blender 400 (PBI International, Milan, Italy). Serial dilutions were
performed in quarter's strength Ringer's solution, and plating was on de
Man, Rogosa and Sharpe (MRS) agar media (Oxoid Ltd., Basingstoke,
Hampshire, UK). The enumeration of mesophilic lactic acid bacteria
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Food Research International 116 (2019) 1344–1356
was carried out incubating at 30 °C for 48 to 72 h, under aerobic con-
ditions. At least 10 colonies, possibly with different morphologies, were
isolated from the last plate dilution. Gram-positive, catalase-negative,
non-motile rod and cocci isolates were cultivated in MRS broth (Oxoid
Ltd.) at 30 °C for 24 h, and re-streaked onto MRS agar. All the isolates
considered for further analyses showed the capacity of acidifying the
culture medium and grew at 15 °C but not at 45 °C.
2.2. Genotyping of LAB isolates by randomly amplified polymorphic DNA-
polymerase chain reaction (RAPD-PCR)
The extraction of genomic DNA was from 2 mL of MRS culture broth
of each isolates, as described by de Los Reyes-Gavilán, Limsowtin,
Tailliez, Séchaud, and Accolas (1992). Two primer pairs (Sigma Che-
mical Co. Milan, Italy), LacbF/LacbR and LpCoF/LpCoR, were used to
amplify 16S rRNA gene fragment of lactic acid bacteria (De Angelis
et al., 2006). The expected amplicons of ca. 1400 and 1000 bp eluted
from the gel and the purification was carried out by the GFXTM PCR
DNA Gel Band Purification Kit (GE Healthcare, Buckinghmshire, UK).
RAPD-PCR analysis was carried out using primers M13, P7 and P4
(Invitrogen Life Technologies, Milan, Italy), as described by Di Cagno
et al. (2007).
2.3. Identification of LAB isolates by 16S rRNA gene sequencing
PCR products separated through electrophoresis were subjected to
purification (as described above), and sequenced. Taxonomic strain
identification was performed by comparing sequences of each isolate
with those reported in the BLAST database (Altschul et al., 1997).
Strains showing homology of at least 97% belonged to the same species
(Goebel & Stackebrandt, 1994). Cultures were maintained as stocks in
15% (vol/vol) glycerol at −80 °C and routinely propagated at 30 °C for
24 h in MRS broth (Oxoid).
2.4. Selection of autochthonous starters based on enzymatic activities
Seventy-seven strains were selected based on aminopeptidase, pro-
line iminopeptidase, endopeptidase type O, glutamate dehydrogenase
and cystathionine lyase activities. Aminopeptidase (EC 3.4.11.11) type
N activity on Leu-p-nitroanilide (p-NA), proline iminopeptidase (EC
3.4.11.9) activity on Pro-p-NA, and endopeptidase type O (EC 3.4.23)
activity on Z-Gly-Pro-NH-trifluoromethyl-coumarin were determined as
described by Gobbetti et al. (1999). An arbitrary unit of enzymatic
activity was defined as the amount of enzyme that caused an increase in
absorbance at 410 nm of 1 (aminopeptidase type N) and 0.1 (proline
iminopeptidase and endopeptidase type O) per minute at 37 °C and
pH 7.0. Glutamate dehydrogenase (EC 1.4.1.2) activity was determined
on glutamate by measuring the glutamate-dependent reduction of
NADP or NAD at 340 nm, as described by De Angelis, Calasso, Di Cagno,
Minervini, and Gobbetti (2010). An arbitrary unit of enzymatic activity
was defined as the amount of enzyme that gave an increase of absor-
bance of 0.1 per minute at 37 °C and pH 7.0. Cystathionine lyase (EC
4.4.1.1) activity was determined by measuring the amount of ketoacids,
ammonia and free thiols released from cystathionine, as described by
De Angelis, Curtin, McSweeney, Faccia, and Gobbetti (2002). An arbi-
trary unit of enzymatic activity was defined as the amount of enzyme
that caused an increase of absorbance at 412 nm of 1 per minute at
37 °C and pH 7.0.
2.5. Kinetic of acidification of autochthonous starter cultures
After overnight cultivation in MRS broth (Oxoid), selected meso-
philic autochthonous starter cultures were harvested by centrifugation
(10,000 ×g, 10 min at 4 °C), washed twice with sterile 50 mM po-
tassium phosphate buffer, pH 7.0, and re-suspended in sterile distilled
water at the cell count of ~9.0 log cfu mL−1. This cell suspension was
I. De Pasquale et al.
used to inoculate sterile skim milk (Oxoid), which was incubated at
37 °C for 24 h and, the kinetic of acidification was determined. The pH
was measured by a Foodtrode electrode (Hamilton, Bonaduz,
Switzerland). Kinetics of acidification were determined and modeled
according to the Gompertz equation as modified by Zwietering,
Jongeberger, Roumbouts, and Van't Riet (1990): y = k + A exp.{−exp
[(Vmax e/A)(λ-t) + 1]}, where k is the initial level of the dependent
variable to be modeled (pH units), A is the difference in pH units (ΔpH)
between inoculation and the stationary phase, Vmax is the maximum
acidification rate (expressed as pH/h), λ is the length of the lag phase
(expressed in hours), and t is the time.
2.6. Preparation of autochthonous starter cultures and manufacturing of
Pecorino Crotonese cheese
After overnight cultivation in MRS broth (Oxoid), cells of selected
mesophilic LAB were harvested by centrifugation (10,000 x g, 10 min at
4 °C), washed twice with sterile 50 mM potassium phosphate buffer,
pH 7.0, and re-suspended into sterile milk at a cell density of approxi-
mately log 9.5 log cfu mL−1. Because of the complementary enzyme
activities, two mixtures of Autochthonous Starter (AS1 and AS2) were
formulated, each one consisting of three strains. AS1 comprised
Leuconostoc mesenteroides subsp. mesenteroides 2A, Lactobacillus casei
23C and Lactobacillus plantarum 18C, while AS2 consisted of Leuc. me-
senteroides subsp. mesenteroides 2A, and L. casei 25D and 16A. As shown
by RAPD-PCR typing, selected L. casei strains did not correspond to that
used in the commercial preparation (data not shown). These mixtures
were used separately to inoculate 500 L of cheese milk. The cell count of
each species/strain was ca. log 7 cfu mL−1. The commercial starter
culture, customarily employed by dairies associated to the Consortium,
was used as a control. According to the manufacturer (Mediterranea
Biotecnologie, Termoli, Italy), starter culture consisted of Streptococcus
thermophilus, Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris,
Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus and L.
casei (initial cell density of ca. log 9.2 ± 0.2 cfu mL−1). Daily, the same
milk was used for making three types of cheese (Experimental Cheese 1,
EC1, inoculated with AS1; EC2, inoculated with AS2; and Control
Cheese, CC, inoculated with commercial primary starters) at the dairy
plant (Caseificio APOCC) located in Cutro (Crotone), Calabria region,
Italy. Cheese making was in triplicate for three consecutive days (total
three batches for each type of cheese). Ewes' milk from two daily
milking was used. After pasteurization (71 °C for 15 s), the milk was
cooled to 37 °C and subjected to inoculation (see above). After 20 min at
37 °C, kid rennet paste was added and coagulation took place within
30–40 min. After cutting the coagulum (size of ca. 0.5 to 1.0 cm), the
curd-whey mixture was held at 42 °C for ca. 5 min. After whey drainage
and moulding, the curd was kept at 40 °C for 120 min, and cooked in
hot whey at 55 °C for 2–3 min. After storage for approximately 24 h at
room temperature, the curd was dry salted (cheese post-dry salting).
Ripening was at ca. 15 °C, with a relative humidity of 70%, for
105 days. The weight of the cheese was approximately 1.0 kg.
Raw ewes' milk and cheese samples after 1 day (post-dry salting)
(C1), 7 days (C7), 15 days (C15), 30 days (C30), 45 days (C45), 60 days
(C60), 75 days (C75), 90 days (C90) and 105 days (C105) of ripening
were collected from each batch. All samples were transported to the
laboratory into thermal plastic bag under refrigerated conditions (ca.
4 °C), 250 g of the inner part of each sample were collected and ana-
lysed immediately (microbiological analysis) or kept frozen (−80 °C)
(biochemical analysis and extraction of total DNA).
2.7. Compositional and microbiological analyses of PC cheese
manufactured with commercial starters and autochthonous starter cultures
Cheese samples were analysed for protein (IDF, 1964), fat (IIRS,
1955), moisture (oven drying at 102 °C) (IDF, 1982) and salt (Fox,
1963). The determination of the pH was by Foodtrode electrode
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(Hamilton, Bonaduz, Switzerland). Microbiological analyses were per-
formed as described previously (Di Cagno et al., 2007). The enumera-
tion of presumptive mesophilic lactobacilli and lactococci was onto
MRS and M17 agar media (Oxoid, Basingstoke, Hampshire, UK) at 30 °C
for 48 h under anaerobiosis, respectively. The enumeration of pre-
sumptive thermophilic streptococci was onto M17 agar (Oxoid) at 42 °C
for 48 h under anaerobiosis. Counting of enterococci was onto Slanetz &
Bartley Agar (Oxoid) at 37 °C for 48 h. Except for enterococci, the media
for plating bacteria were supplemented with cycloheximide at
0.1 g L−1. Typing and monitoring of selected autochthonous starter
cultures was carried out by RAPD-PCR analysis as before described.
2.8. Assessment of proteolysis
The pH 4.6-insoluble and -soluble nitrogen fractions of the cheeses
were analysed by urea-polyacrylamide gel electrophoresis (Urea-PAGE)
and reverse phase high-pressure liquid chromatography (RP-HPLC), as
described by Andrews (1983), and Gobbetti et al. (2002), respectively.
Total and individual free amino acids (FAA) from the pH 4.6-soluble
fraction were determined by a Biochrom 30 series Amino Acid Analyser
(Biochrom Ltd., Cambridge Science Park, UK), as described by Di Cagno
et al. (2007).
2.9. Extraction of total dead and viable bacterial genomic DNA,
pyrosequencing and data analyses
Ninety millilitres of potassium phosphate (50 mM [pH 7.0]) buffer
was added to 10 g of cheese sample and homogenized for 5 min, and
DNA extraction was carried out as previously described (De Pasquale
et al., 2014). Fifty millilitres of this suspension were centrifuged at
1000 x g for 5 min at 4 °C. To harvest the cells, the supernatant was
centrifuged for a second time (5000 x g for 15 min at 4 °C) and the cell
pellet was stored at −20 °C. An aliquot of the pellet of ca. 500 mg was
used for DNA extraction (Bornay-Llinares et al., 1999) using the Fas-
tDNA Pro Soil-Direct Kit (MP Biomedicals, CA., United States) and
FastPrep instrument (BIO 101), according to the manufacturer's in-
structions. Three DNA samples, corresponding to the three batches for
each cheese, were pooled and used for 16S based bacterial diversity
analysis. The assessment of bacterial diversity was done by pyr-
osequencing on an Illumina MiSeq (Illumina, Inc. San Diego, California)
and performed by Research and Testing Laboratories (Lubbock, TX),
according to standard laboratory procedures. Primers targeting the
V3–V4 region (Escherichia coli position 357–785, forward 357F: CCT-
ACGGGNGGCWGCAG and reverse 785R: GACTACHVGGGTATCTAA-
TCC) of the 16S rRNA gene (Herlemann et al., 2011) were used. Pyr-
osequencing procedures were carried out based upon RTL protocols
http://www.researchandtesting.com (Research and Testing Labora-
tories, Lubbock, TX).
2.10. Bioinformatics
Sequence data for each cheese were processed using Research and
Testing Laboratory's in-house pipeline, described at http://www.
rtlgenomics.com/docs/Data_Analysis_Methodology.pdf (version2.2.3).
Briefly, sequences were grouped using their barcodes and any sequence
that contained a low quality barcode or that failed to be at least half the
expected amplicon length (or 250 bp, whichever was shortest) was re-
moved from the data pool. Sequences that passed the quality filter were
denoised using an algorithm based on USEARCH pipeline (Edgar,
2010), and then checked for chimeras using UCHIME (Edgar, Haas,
Clemente, Quince, & Knight, 2011). Finally, sequence data were sepa-
rated into operational taxonomic units (OTUs) at 97% similarity using a
USEARCH and all OTUs were used for classification by using UBLAST
global alignment against a custom16S database comprised of well-
characterized sequences from nr/nt. The output was then analysed
using a internally developed Python pipeline that parses the assigned
I. De Pasquale et al.
taxonomic information to create the final analysis files. The evaluation
of alpha- and beta-diversities was by QIIME, as described (Lozupone &
Knight, 2005).
2.11. Determinations of neutral volatile components and volatile free fatty
acids
The determination of neutral volatile components (VOC) was by
Purge and Trap (PT), coupled with Gas Chromatography-Mass
Spectrometry (PT-GC/MS). Prior to PT, 3 g of cheese were mixed with
27 g of ultra-high quality (UHQ) deodorized water, with an ultra-turrax
during 4 × 40 s separated by 10 s rest, in a glass flask with a narrow
neck plunged into ice. Ten mL of this suspension were placed in a glass
extractor connected to the PT apparatus (Tekmar 3000, Agilent
Instruments, NY, USA). Extraction was with helium at a flow rate of
40 mL min−1 on a tenax trap at 37 °C. The desorption of the trap was at
225 °C and injection used a cryo-concentrator at −150 °C. The chro-
matograph (6890 Agilent Instruments) was equipped with a DB5-like
capillary column (RTX5 Restek, Lisses, France), 60 m length, 0.32 μm
internal diameter, and 1 μm thickness. The helium flow rate was 2 mL
min−1, the oven temperature was 40 °C during the first 6 min and then
was increased at 3 °C min−1 to 230 °C. The mass detector (MSD5973,
Agilent Instruments) has used in electronic impact at 70 eV and in scan
mode, from 29 to 206 atomic mass. Quantification of compounds has
expressed in arbitrary units of area.
For solid-phase microextraction (SPME) extraction of volatile free
fatty acids (VFFA), each sample (15 g) was mixed with valeric acid
(Sigma-Aldrich, St Quentin-Fallavier, France) as internal standard and
50 mL of sulphuric acid 10% (v/v). The suspension was poured into
a10-mL flask and poured into a Jalade apparatus to the bottom of which
was attached a balloon containing 60 mL of diethyl ether (Carlo Erba,
Val de Reuil, France) and 60 mL of petroleum spirit (40 to 60 °C)
(Normapur Prolabo, Fontenay S/Bois, France), and at the top was a
refrigerator. Extraction was performed for 6 h by ebullition of the sol-
vent. The solvent phase was separated from water phase, then mixed
with 50 mL of a solution ethanol/water (4:1) (v/v) and two drops of 1%
phenolphthalein. VFFA were saponified by addition of 1% NaOH (w/v)
until neutralization. The water phase was kept and, desiccated by
heating at 103 °C. Twenty milligrams of volatile fatty acids soaps were
dissolved in 0.5 mL of 12% TCA in ethanol. Injection was by a splitless
chromatograph injector at 240 °C (10 mL min−1 split, split ratio of
20:1). The chromatograph (8160CE, Thermo-Finnigan, Villebon/
Yvette, France) was equipped with a FFAP column with a 30-m length,
0.53-μm diameter, and 1-μm thickness (Stabilwax DA Restek) and a
flame ionization detector (FID) detector. The helium flow rate was
6 mL min−1; the oven temperature was 120 °C for 1 min, was increased
to 162 °C at 1.8 °C min−1 and to 240 °C at 10 °C min−1, and lastly held
at 240 °C for 5 min.
Concentrations of VFFAs were calculated from calibration curves
established with external standards (Sigma, L'Isle d'Abeau, France).
2.12. Sensory analysis
Sensory analysis of cheeses after 105 days of ripening was carried
out by ten trained panellists, with an equal distribution of males and
females and age ranging between 21 and 40 years. Cheese samples were
randomly coded and served at 18–20 °C in aliquots of 20 g together with
non-salted table biscuits and still water to panellists placed separately
in rooms for unbiased evaluation of sensory attributes. Cheeses were
scored (Madkor, Tong, & El Soda, 2000) from 0 to 10 for quality
parameters that included flavour intensity (0 = none, 10 = aged),
aroma intensity (0 = none, 10 = aged), texture (0 = hardest,
10 = softest) and overall acceptability (also including typical features
of the PC cheese) (0 = dislike very much, 10 = like very much).
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Food Research International 116 (2019) 1344–1356
2.13. Statistical analysis
All the results from compositional, microbiological and biochemical
analyses were the average of 3 batches for each cheese; each batch was
analysed in triplicate (total 9 samples analysed for each cheese).
According to previous studies (De Filippis, La Storia, Stellato, Gatti, &
Ercolini, 2013; De Pasquale, Calasso, et al., 2014; De Pasquale, Di
Cagno, Buchin, De Angelis, & Gobbetti, 2014) data from pyrosequen-
cing result from the pooling of three DNA samples, corresponding to the
three batches for each cheese. Data were subjected to one-way ANOVA,
and pairwise of treatment means was achieved by Tukey's procedure at
P < 0.05, using the statistical software Statistica for Windows (Sta-
tistica 7.0, per Windows). Data sets related to VOC (arbitrary units of
area) were analysed through Principal Component Analysis (PCA),
using the software XLSTAT® 2016. Only compounds mostly correlated
with principal components 1 and 2 (r ≥ 0.70) were considered.
2.14. Nucleotide sequence accession number
Sequence data have submitted to the sequence read archive data-
base of the National Center for Biotechnology Information under ac-
cession no. PRJNA379167.
3. Results
3.1. Isolation, genotyping and identification of non-starter lactic acid
bacteria (NSLAB) from Pecorino Crotonese cheeses
As estimated by plating on MRS agar, the cell number of pre-
sumptive mesophilic non-starter lactic acid bacteria (NSLAB) in
Pecorino Crotonese (PC) cheeses did not significantly (P > 0.05) vary
between 75, 90, 105 and 120 days of ripening (range of 7.55 to 7.94 log
cfu g−1). One-hundred-twenty Gram-positive, catalase-negative, non-
motile cocci and rods, able to grow at 15 °C and to acidify MRS broth
were recovered from the last plate dilutions and subjected to pre-
liminary RAPD-PCR analysis by using single primers (Fig. 1). The as-
sessment of the reproducibility of RAPD fingerprints was made by
comparing PCR products from three separate cultures of the same
strain. At the similarity level of 85%, isolates grouped into 23 clusters
(A – W).
The identification of all isolates was done by partial sequencing of
the 16S rRNA. The following species were identified: Lactobacillus casei,
Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp.
lactis, Lactobacillus brevis, Pediococcus pentosaceus, Lactobacillus rham-
nosus, and Lactobacillus plantarum. Lc. lactis subsp. lactis and, especially,
L. casei were the species found most frequently, which accounted for 12
and 97 of the 120 isolates, respectively. Apart from the time of ripening,
strains of the same species did not frequently group in a unique cluster.
Forty-three isolates showed identical fingerprints and, therefore, were
excluded from the further characterization.
3.2. Selection of autochthonous starter cultures based on enzymatic
activities
The selection among the remaining 77 strains concerned enzyme
activities responsible for secondary proteolysis and catabolism of FAA,
which mainly affect the acceleration of cheese ripening and improve
cheese flavour. Most of the strains showed an intense aminopeptidase
type N activity. L. casei 25D had the highest activity (5.1 ± 0.3 U),
followed by L. plantarum 18C and L. casei 3C (4.9 ± 0.4 U) (Fig. S1).
Proline iminopeptidase and endopeptidase type O activities did not
significantly (P > 0.05) vary among the strains. Leuc. mesenteroides
subsp. mesenteroides 2A, and L. casei 23C and 22C had very high values
of glutamate dehydrogenase (4.6 ± 1.2, 4.3 ± 1.0 and 4.0 ± 1.2 U,
respectively) and cystationine lyase (4.3 ± 1.4, 4.6 ± 1.2 and
3.9 ± 1.1 U, respectively) activities. L. casei 16A showed the highest
I. De Pasquale et al.
Fig. 1. Dendrogram obtained by combined random amplification of polymorphic DNA patterns for the isolates from Pecorino Crotonose cheeses. Dendrogram
obtained by combined RAPD-DNA patterns for the isolates from PC cheeses using primer P4, M13 and P7. Cluster analysis was based on the simple matching
coefficient and unweighted pair grouped method with arithmetic average. Cheese samples were collected at 75, 90, 105 and 120 days of ripening.
cystationine lyase activity (5.3 ± 1.7 U).
Based on the above results and aiming at guarantying full enzyme
patterns, the decision was to formulate two mixtures of autochthonous
starters (AS). AS1 consisted of Leuc. mesenteroides subsp. mesenteroides
2A, L. plantarum 18C and L. casei 23C. AS2 comprised Leuc. mesenter-
oides subsp. mesenteroides 2A, and L. casei 25D and 16A. Using these two
formulations, we had almost similar enzyme activities in presence of
different species/strains, which, on the other hand, could have diverse
and non-predictable effects on other cheese quality attributes (e.g.,
synthesis of volatile components).
3.3. Kinetic of acidification of autochthonous starter cultures
The acidification kinetics of the single strains that were present in
the two selected mixtures were assessed on sterile skim milk at 37 °C for
24 h. The lowest latency phase of acidification (λ) was found for L.
plantarum 18C and L. casei 22C (0.54 ± 0.02 and 0.79 ± 0.01 h, re-
spectively), followed by L. casei 23C and 3C (1.06 ± 0.01 and
1.05 ± 0.01 h, respectively), and L. casei 25D and Leuc. mesenteroides
subsp. mesenteroides 2A (1.15 ± 0.02 and 1.22 ± 0.02 h, respec-
tively). The values of Vmax did not significantly (P > 0.05) vary
among the strains (range of 0.24 to 0.27 ΔpH h−1). At the end of in-
cubation, the values of ΔpH were almost similar for most of the strains
(range of 2.23 to 2.29 units). Leuc. mesenteroides subsp. mesenteroides 2A
and L. casei 22C showed the lowest (2.04 ± 0.02 units) and highest
(2.40 ± 0.03 units) values, respectively.
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3.4. Compositional and microbiological characteristics of PC cheese
manufactured with commercial and autochthonous starter cultures
The raw ewes' milk used had the following characteristics: pH 6.61,
protein 6%, fat 6.8% and 1.1% salt. No significant (P < 0.05) differ-
ences were found between the three batches. Table 1 shows the main
chemical attributes of PC cheeses at the beginning and after 105 days of
ripening. The use of autochthonous starter cultures interfered only
slightly with pH and moisture. The pH decreased moderately during
ripening. At 105 days, the pH of the control cheese (CC) was 5.7 ± 0.4.
AS1 and AS2 caused a slight decrease of pH after manufacture, which
reached significantly (P < 0.05) lower values (5.2 ± 0.2 and
5.4 ± 0.3, respectively) at 105 days of ripening. Therefore, both AS1
and AS2 showed a suitable capability to acidify. Differences in pH in-
fluenced the levels of moisture. The percentages of moisture were
29.0 ± 1.4, 31.0 ± 1.5 and 33.3 ± 1.7 for PC cheeses manufactured
with AS1 and AS2, and CC, respectively.
Although the commercial starter preparation also included L. casei,
CC had the lowest initial value of presumptive mesophilic lactobacilli
(5.8 ± 0.2 log cfu g−1), which increased during time (Table 2).
Cheeses manufactured with AS1 and AS2 had the highest (P > 0.05)
initial cell number of presumptive mesophilic lactobacilli (9.5 ± 0.3
and 9.3 ± 0.1 log cfu g−1, respectively). This number remained almost
constant up to 45 days, and then decreased by ca. 1.0–0.7 log cycles at
105 days, without significant (P < 0.05) differences between the two
cheeses manufactured with autochthonous starter cultures. Pre-
sumptive mesophilic LAB were randomly isolated from MRS agar plates
(ca. 30 isolates from the highest plate dilutions) at 105 days of ripening
and subjected to RAPD-PCR. The selected NSLAB persisted in both the
cheeses manufactured with autochthonous starter cultures (data not
I. De Pasquale et al. Food Research International 116 (2019) 1344–1356

Table 1
Main chemical compositiona at beginning (1 days) and the end (105 days) of ripening of Pecorino Crotonese cheese made with commercial primary starters (control
cheese, CC) or with Autochthonous Starter (AS1 and AS2) mixturesb.
CC AS1 AS2

Days of ripening

1 105 1 105 1 105

A C B E B
pH ± SD 6.1 ± 0.3 5.7 ± 0.4 5.9 ± 0.3 5.2 ± 0.2 5.9 ± 0.3 5.4 ± 0.3D
Moisture content (%) ± SD 45.4 ± 1.9A 33.3 ± 1.7D 42.5 ± 1.5C 29.0 ± 1.4F 43.2 ± 1.7B 31.0 ± 1.5E
Fat content (%) ± SD 23.1 ± 1.1F 29.1 ± 1.4C 25.5 ± 1.3D 33.3 ± 1.6A 25.1 ± 1.2E 31.5 ± 1.3B
Protein content (%) ± SD 22.4 ± 1.4E 25.3 ± 1.1C 23.5 ± 1.3D 29.7 ± 1.4A 23.1 ± 1.1D 28.5 ± 1.5B
NaCl content (%) ± SD 0.12 ± 0.06E 2.5 ± 1.6B 0.17 ± 0.05C 3.3 ± 0.6A 0.15 ± 0.06D 3.1 ± 0.4A

Data in the same row with different superscript letters (A-F) are significantly different (P < .05).
a
Mean values ± standard deviations for three batches of each type of cheese, analysed in duplicate (n = 6).
b
AS1 consisted of Leuconostoc mesenteroides subsp. mesenteroides 2A, Lactobacillus casei 23C and Lactobacillus plantarum 18C; AS2 consisted of Leuc. mesenteroides
subsp. mesenteroides 2A, L. casei 25D and 16A.

shown). As expected, CC made with commercial starters had the highest
initial cell densities of thermophilic lactobacilli and streptococci, and
mesophilic lactococci. These values progressively decreased during ri-
pening. Cheeses manufactured with autochthonous starter cultures had
the lowest (P < 0.05) initial cell number of presumptive thermophilic
lactobacilli (5.3 ± 0.1 and 5.2 ± 0.2 log cfu g−1, respectively) and
streptococci (5.6 ± 0.2 and 5.4 ± 0.2 log cfu g−1, respectively),
which decreased throughout ripening (Table 2).
3.5. Proteolysis
Urea-PAGE electrophoresis of the pH 4.6-insoluble nitrogen fraction
showed a few differences among primary proteolysis of the three
cheeses (Fig. 2, panel A, B, C). The hydrolysis of αS1-casein (CN) was
limited, with a presumptive residual activity of chymosin. The main
degradation of αS1-CN started earlier in the cheese made with AS1. At
the end of ripening, a large amount of non-hydrolysed β-CN persisted in
all cheeses. The formation of protein bands with low electrophoretic
mobility, which presumably corresponded to γ-CN, was evident. This
indicated plasmin activity. The urea-PAGE electrophoretogram of the
pH 4.6-soluble fraction showed differences over time for all three
cheeses (Fig. 2, panel D, E, F). Characteristic polypeptide bands ap-
peared at 7 days of ripening and persisted in cheeses made with auto-
chthonous starter cultures during late ripening. CC showed a more
complex profile only after 45 days. Complementary information
emerged by RP-HPLC analysis (data not shown). The number of peaks,
which were recognized and matched visually with the Unicorn program
(Amersham Biosciences), varied (quantitatively and qualitatively)
during ripening. CC differed from cheeses made with autochthonous
starter cultures. From day 60 onward, the differences became most
evident. CC showed 12 peaks, which remained almost constant. Within
the same interval of time, cheeses made with autochthonous starter
cultures had the highest number of peaks, which ranged from 17 to 23.
Principal component analysis (PCA) was applied to RP-HPLC data
(number and the area of hydrophobic and hydrophilic peptide peaks
from 60 to 105 days) (Fig. 3). The two PCs explained 73.4% of the total
variance. The cheese manufactured with AS1 showed the highest con-
centration of peptides, whereas that made with AS2 was located in the
centre of the plane. CC separated due to the lowest concentration of
peptides and the highest area of hydrophilic peaks. At the beginning of
ripening, the concentration of total free amino acids (FAA) of the three
PC cheeses was almost the same (663–732 mg kg−1) (Table 3). During
ripening, CC showed the highest increase (8902 ± 210 mg kg−1). The
concentration was approximately 2.5 and 2.2 times higher than those
found in cheeses made with AS1 (3538 ± 177 mg kg−1) and AS2
(4040 ± 202 mg kg−1). At 105 days, the FAA found at the highest
concentrations (> 100 mg kg−1) were Leu, Phe, Val, Lys, Ala and Pro.
Both cheeses manufactured with autochthonous starter cultures showed

Table 2
Cell numbers (log CFU g−1)a of various microbial groups during ripening of Pecorino Crotonese cheese made with commercial primary starters (control cheese, CC)
or Autochthonous Starter (AS1 and AS2) mixturesb.
Days of ripening 1 15 45 75 105

Microbial group CC
Thermophilic lactobacilli 7.3 ± 0.2E 8.7 ± 0.2A 8.3 ± 0.1B 7.7 ± 0.3D 7.9 ± 0.2C
Mesophilic lactobacilli 5.8 ± 0.2E 7.4 ± 0.1D 7.9 ± 0.2A 7.8 ± 0.1B 7.7 ± 0.3C
Mesophilic lactococci 7.7 ± 0.2D 8.5 ± 0.1A 8.2 ± 0.1B 8.3 ± 0.1C 7.7 ± 0.2D
Thermophilic streptococci 7.9 ± 0.3C 8.5 ± 0.3A 7.9 ± 0.2C 8.3 ± 0.2B 7.1 ± 0.1D

AS1
Thermophilic lactobacilli 5.3 ± 0.1E 6.7 ± 0.1A 6.1 ± 0.3B 5.8 ± 0.1C 5.5 ± 0.4D
Mesophilic lactobacilli 9.5 ± 0.3A 9.2 ± 0.2B 9.2 ± 0.2B 8.2 ± 0.1D 8.5 ± 0.1C
Mesophilic lactococci 7.3 ± 0.1A 7.1 ± 0.2B 6.4 ± 0.1C 6.5 ± 0.2C 6.0 ± 0.2D
Thermophilic streptococci 5.6 ± 0.2C 6.0 ± 0.1A 6.1 ± 0.3A 5.8 ± 0.4B 5.5 ± 0.3C

AS2
Thermophilic lactobacilli 5.2 ± 0.2D 6.7 ± 0.1B 6.9 ± 0.3A 5.5 ± 0.2C 5.2 ± 0.1D
Mesophilic lactobacilli 9.3 ± 0.1B 9.3 ± 0.3B 9.8 ± 0.1A 8.3 ± 0.3D 8.5 ± 0.3C
Mesophilic lactococci 7.0 ± 0.2B 7.2 ± 0.4A 6.5 ± 0.1C 6.4 ± 0.1C 6.3 ± 0.4D
Thermophilic streptococci 5.4 ± 0.2C 6.9 ± 0.1A 6.6 ± 0.3B 5.6 ± 0.4C 5.2 ± 0.3D

Data in the same row with different superscript letters (A-E) are significantly different (P < 0.05).
a
Mean values ± standard deviations for two batches of each type of cheese, analysed in triplicate (n = 6).
b
AS1 consisted of Leuconostoc mesenteroides subsp. mesenteroides 2A, Lactobacillus casei 23C and Lactobacillus plantarum 18C; AS2 consisted of Leuc. mesenteroides
subsp. mesenteroides 2A, L. casei 25D and 16A.

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Fig. 2. Urea-polyacrylamide gel electrophoresis (PAGE) of pH 4.6-insoluble (panels A, B and C), and –soluble (panels D, E and F) nitrogen fractions during ripening of
Pecorino Crotonese cheese made with commercial primary starters (control cheese, CC) (panels A and D), or with Autochthonous Starter (AS) 1 (Leuconostoc
mesenteroides subsp. mesenteroides 2A, Lactobacillus casei 23C and Lactobacillus plantarum 18C) (panels B and E), or with AS2 (Leuc. mesenteroides subsp. mesenteroides
2A, L. casei 25D and 16A) (panels C and F). Lanes: M, ewe casein (CN) standard; 1, raw ewes' milk; 2-10, cheese after 1, 7, 15, 30, 45, 60, 75, 90 and 105 days of
ripening, respectively.

the highest concentration of Cys. of microbial diversity at day 1 (post-dry salting), and spread in the right
part of the plot. Cheeses made with AS1 and AS2 formed two separate
subgroups throughout ripening. As the time of ripening proceeded (day
3.6. Structure and changes of the microbiota
15 onward), cheeses made with the same starter grouped together. PC
cheese made with AS2 showed the lowest variability of microbial di-
Overall, the structure of the microbiota did not significantly
versity. Raw ewes' milk separated from cheeses.
(P > 0.05) vary among the three batches of cheeses analysed. After
The bacterial sequences from DNA assigned to bacterial phyla and
pyrosequencing, there were 148,232 (CC), 107,945 (cheese made with
their relative abundance varied depending on cheese and time of ri-
AS1), and 164,132 (cheese made with AS2) raw sequence reads of 16S
pening (data not shown). DNA from raw ewes' milk included mainly
rRNA gene amplicons (average length, 473 bp). Table S1 shows the
Proteobacteria (89–90%) and Firmicutes (10–11%). At day 1 of ripening,
parameters of calculated alpha diversity. At the beginning and after
CC still had a high abundance of Proteobacteria (93%), which later (day
15 days of ripening, CC had the highest values of Chao1 richness and
15 onward) was replaced by Firmicutes (ca. 99%). At day 15,
Shannon diversity indices (54.25 and 27.86, and 1.9 and 0.84, re-
Actinobacteria (0.6%) and Bacteroidetes (1%) occurred. The phylum
spectively). These indices decreased from day 45 onward. Chao1 rich-
Firmicutes dominated (ca. 95 to 99%) the microbiota of the cheeses
ness and Shannon diversity decreased throughout ripening of cheese
made with autochthonous starter cultures throughout ripening. The
made with AS1, while, after 15 days, these indices remained almost
phylum Proteobacteria was present at a very low frequency.
constant in the cheese made with AS2. Raw ewes' milk had values of
Fig. 4 reports the distribution of the OTUs that were classified at
Chao1 richness and Shannon diversity of 41.0 and 1.51, respectively.
genus level. Although 20 OTUs were identified in raw ewes' milk, only 7
Good's estimated sample coverage (ESC) was above 99% for all the
had a relative abundance higher than 0.3% in at least one sample.
samples, indicating a satisfactory description of the microbial diversity.
Pseudomonas (85%), Lactococcus (8.8%), Citrobacter (3.6%), Strepto-
The community structure was analysed also using three phylogeny-
coccus (0.6%), Acinetobacter (0.6%), Chryseobacterium (0.4%) and
based beta-diversity measures (Fig. S2). The two principal coordinate
Trueperella (0.3%) were the main genera. Subdominant genera be-
analyses (PCoA), based on the unweighted and weighted UniFrac dis-
longed to the phyla Proteobacteria (Janthinobacterium, Enterobacter,
tance matrix, clearly differentiated CC. This had the highest variability

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Fig. 3. Score plot and loading plot of the first and secondary principal components after PC analysis based on area and number peaks obtained from reversed-phase
high-protein liquid chromatograms of the pH 4.6-soluble fractions fraction of Pecorino Crotonese cheese. Score plot (A) and loading plot (B) of the first and secondary
principal components after PC analysis based on area and number peaks obtained from reversed-phase high-pressure liquid (RP-HPLC) chromatography of the
pH 4.6-soluble fractions fraction of PC cheese with commercial primary starters (control cheese, CC) or with Autochthonous Starter (AS) 1 (Leuconostoc mesenteroides
subsp. mesenteroides 2A, Lactobacillus casei 23C and Lactobacillus plantarum 18C) and AS2 (Leuc. mesenteroides subsp. mesenteroides 2A, L. casei 25D and 16A) mixtures
during ripening (60, 75, 90 and 105 days).

Escherichia, Moraxella, Serratia and Stenotrophomonas) (0.02 to 0.26%),
Firmicutes (Macrococcus, Staphylococcus, Enterococcus and Leuconostoc)
(0.02 to 0.04%) and Actinobacteria (Corynebacterium and Kocuria) (0.02
to 0.04%). At day 1 of ripening, the bacterial profile of CC had a high
abundance of contaminant Pseudomonas (73%), Acinetobacter (15%)
and Citrobacter (3.3%), followed by Lactococcus (2.3%), Streptococcus
(1.7%) and Lactobacillus (0.2%). Several contaminants flanked these
genera: Chrysobacterium at 1% and Staphylococcus, Serratia, Kokuria and
Enterobacter at values < 1%. From day 15 onward, Streptococcus
(85–92%), Lactobacillus (2.9–11.4%) and Lactococcus (3.3–4.5%)
dominated the microbiota. Most of the other genera were not anymore
detectable. In agreement with the numbers of presumptive mesophilic
lactic acid bacteria (Table 2), Lactobacillus was the dominant genus
throughout ripening (43.2 to 56.8%) of both the cheeses manufactured
with autochthonous starter cultures. The house contaminant Strepto-
coccus ranged from 2.2 to 12.7%. Other sub-dominant genera (< 0.4%)
belonged to Actinobacteria (Kocuria), Firmicutes (Enterococcus, Lacto-
coccus, Leuconosctoc, Macroccus and Pediococcus) and Proteobacteria
(Citrobacter and Enterobacter).
Table 4 reports the abundance of OTUs, with taxonomic details up
to species level when such assignment was possible. Pseudomonas sp.
(63%) was the most abundant species in raw ewes' milk, followed by
Streptococcus thermophilus (22%) and Lactococcus lactis (7.7%). After the
inoculation of commercial starters, St. thermophilus (31.4%), Lc. lactis
(3.7%) and cluster L. casei/L. plantarum (3%) dominated the microbiota
of CC (day 1), still flanked by a high abundance of Pseudomonas sp.
(60%). After 15 days, the microbiota changed. Contaminant Pseudo-
monas sp. completely disappeared, whereas the relative abundance of
St. thermophilus increased (ca. 2.5 times) and remained almost constant
throughout ripening. At the same time, Lc. lactis remained almost
constant, whereas the cluster L. casei/L. plantarum became dominant
within mesophilic lactobacilli and persisted until the end of ripening
(6.41%). The cheeses manufactured with AS1 and AS2 harboured the
cluster L. casei/L. plantarum (ca. 60 to 70%) throughout ripening. Al-
though at a less abundance (ca. 6 to 0.02%), the cluster L. delbruecki/
Leuc. mesenteroides flanked the above one and stably persisted. St.
thermophilus (ca. 7.8 to 26%) and Streptococcus sp. (ca. 2.2 to 12.7%)
were also found as contaminants. Based on 16S sequences and using the
standard of 97% OTU similarity, members of each cluster were indis-
tinguishable.
3.7. Volatile components
Usually, the literature (Gobbetti et al., 2015; Montel et al., 2014)
reports the highest effect of mesophilic lactobacilli on volatile compo-
nents (VOC) at the late ripening. In agreement, VOC were analysed
from 60 to 105 days. The identification of VOC (154 in total) was
through PT-GC/MS. VOC belonged to the following chemical classes:
esters (34 compounds), ketones (31), alcohols (18), aldehydes (21),
sulphur compounds (7), terpenes (20), furans (11), hydrocarbons (10)
and miscellaneous (2).
The levels of 62 VOC and 3 volatile free fatty acids (VFFA) sig-
nificantly (P < 0.05; r ≥ 0.7) differentiated CC and the cheeses made
with autochthonous starter cultures (Table S2 and Fig. 5). Cheeses
manufactured with AS1 and AS2 had concentrations of acetic acid
(143–116 and 169–131 ppm, respectively) significantly higher
(P < 0.05) than that found in CC (31–40 ppm). On the contrary, the
levels of butanoic and hexanoic acids and of most unsaturated or
branched aldehydes, alcohols, branched ketones, methyl esters, di-
methyl-sulfide, furfural and 2-propyl-furan were the highest in CC. The
same was found for the levels of butyl esters, and the hexyl ester and
ethyl 3-methyl-butanoate at 45 days ripening. CC showed the lowest
levels of some unsaturated or methyl-ketones, sulphur compounds such
as dimethyl-disulfide, dimethyl-triulfide, 2,4-dithiapentane and S-me-
thyl thioacetate, and some furans. During ripening, the cheese manu-
factured with AS2 had the lowest contents of several aldehydes and
ketones. After 75 days ripening, branched acids and branched alcohols
were significantly (P < 0.05) the highest in the cheese made with AS1.
At the end of ripening, the same cheese was the richest in many alde-
hydes (13 of 21), ketones (21 of 31), furans (7 of 12), hydrocarbons (4

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Table 3
Operational taxonomic unit (OTUs) assigned to the species level during ripening of Pecorino Crotonese cheese with commercial primary starters (control cheese, CC)
or with Autochthonous Starter (AS1 and AS2) mixturesa.
Taxon Incidence of OTU (%)

Days of ripening

CC AS1 AS2

Milk 1 15 45 75 105 1 15 45 75 105 1 15 45 75 105

Trueperella pyogenes 0.32 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Kocuria kristinae 0.04 0 0.01 0.37 0 0 0.02 0 0 0 0 0.07 0.03 0 0 0
Chryseobacterium sp. 0.20 0 0 0.21 0 0 0 0 0 0 0 0 0 0 0 0
Macrococcus caseolyticus 0.03 0 0 0.05 0 0.02 0.06 0.05 0.09 0.17 0.06 0.02 0.04 0.01 0.03 0.02
Staphylococcus chromogenes 0.02 0 0 0.40 0 0 0 0 0.01 0 0 0 0 0 0 0
Staphylococcus saprophyticus 0.01 0.88 0.02 0.35 0.10 0.54 0.03 0.04 0.07 0.09 0.04 0.01 0.10 0 0 0.03
Enterococcus faecalis 0 0 0.01 0 0 0.01 0.03 0.01 0.05 0.03 0.02 0.04 0.04 0.03 0.03 0.02
Lactobacillus brevis 0 0 0.21 0 0 0 0.01 0 0 0 0
Lactobacillus casei clusterb 0.02 2.91 5.51 0 3.08 6.41 69.24 69.82 81.64 79.30 79.22 66.08 72.56 65.17 75.28 71.44
Lactobacillus coryniformis 0 0 5.62 0.01 0 0 0 0 0 0 0 0 0 0 0 0
Lactobacillus curvatus 0 0 0 0 0.61 0 0 0 0 0 0 0 0 0 0 0
Lactobacillus delbrueckii cluster 0 0.03 0.08 0 0 0.04 6.36 1.63 1.51 1.30 1.22 0.07 0.16 0.07 0.02 0.21
Lactobacillus fermentum 0 0 0 0 0 0 0.24 0.02 0.01 0 0 0 0.01 0 0 0
Pediococcus pentosaceus 0 0.01 0.54 0.01 0.02 0.01 0 0.01 0.02 0.01 0 0.04 0.03 0.02 0.01 0.03
Lactococcus garvieae 0.05 0 0 0.16 0 0 0.01 0 0 0 0 0 0 0 0 0
Lactococcus lactis 7.67 3.68 3.32 1.79 4.20 4.44 0.06 0.05 0.08 0.18 0.14 0.03 0 0 0 0
Lactococcus raffinolactis 1.08 0.04 0.03 0.37 0.07 0.06 0 0 0.01 0 0 0.03 0 0.05 0.02 0.01
Streptococcus dysgalactiae 0.14 0 0 0.43 0 0 0 0 0 0 0 0 0 0 0 0
Streptococcus parauberis 0.28 0 0 0.95 0 0.01 0 0 0 0 0 0.01 0 0 0 0
Streptococcus sp. 0.17 0.67 5.45 0.27 0.79 1.74 3.84 6.06 5.03 2.22 4.63 6.11 7.37 11.69 12.76 6.90
Streptococcus suis 0 0 0 0.06 0 0 0.08 0.07 0.11 0.05 0.04 0.03 0.03 0.04 0.08 0.05
Streptococcus thermophilus 22.6 31.38 79.13 89.56 91.10 82.41 15.54 19.28 7.82 7.98 11.05 26.28 19.23 22.76 11.11 21.01
Janthinobacterium sp. 0.02 0 0 0.10 0 0 0 0 0 0 0 0 0 0 0 0
Citrobacter freundii 3.51 0 0 2.93 0 0.01 0.84 0.58 1.13 2.79 0.73 0.15 0.01 0.01 0.01 0.03
Citrobacter sp. 0.05 0 0 0.37 0 0.01 0.53 0.48 0.62 1.42 0.32 0.15 0.01 0 0.01 0
Enterobacter hormaechei 0.11 0 0 0.36 0.01 0.02 0.10 0.12 0.05 0.28 0.05 0.01 0 0.01 0 0
Klebsiella sp. 0 0.01 0.02 0.08 0 0 1.84 1.20 1.05 2.77 1.51 0.45 0.22 0.04 0.45 0.18
Serratia sp. 0.26 0 0 0.39 0 0 0 0 0 0 0 0 0 0 0 0
Acinetobacter baumannii 0.10 0.39 0.02 0.01 0.02 4.27 0.17 0.02 0.01 0.02 0.03 0 0 0 0 0
Acinetobacter guillouiae 0.05 0 0 0.34 0 0 0 0 0 0 0 0 0 0 0 0
Acinetobacter sp. 0.48 0 0 14.47 0 0 0 0 0 0 0 0 0 0 0 0
Acinetobacter ursingii 0 0 0 0.11 0 0 0 0 0 0 0 0 0 0 0 0
Moraxella sp. 0.11 0 0 0.32 0 0 0 0 0 0 0 0 0 0 0 0
Pseudomonas sp. 62.89 60 0.01 0 0 0 0 0 0 0 0 0 0 0 0 0
Stenotrophomonas maltophilia 0.07 0 0 0.37 0 0 0 0 0 0 0 0 0 0 0 0

Incidences of OTUs assigned to the Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria species level during ripening of Pecorino Crotonese cheese. Only OTUs
occurring at 0.1% abundance in at least one sample were included. Based on 16S rRNA gene pyrosequencing analysis of all DNA samples directly from Pecorino
Crotonese cheese.
a
AS1, consisted of Leuconostoc mesenteroides subsp. mesenteroides 2A, Lactobacillus casei 23C and Lactobacillus plantarum 18C; AS2, consisted of Leuc. mesenteroides
subsp. mesenteroides 2A, L. casei 25D and 16A.
b
Cluster, grouped members indistinguishable based on their 16S sequences in the region amplified using the standard 97% OTU similarity.

of 10), terpenes (19 of 20) and all esters, apart from methyl esters and
2-methyl-butyl acetate. PCA elaborated the VOC data (Fig. 5A and B).
The two PCs explained ca. 82% of the total variance. CC and cheeses
manufactured with AS1 and AS2 clearly differed. According to PC1
(47.8%), CC distributed in the opposite zone with respect to cheeses
made with autochthonous starter cultures. According to PC2 (33.8%),
the cheese made with AS1 separated according to the increase of the
time of ripening. The cheese made with the AS2 did not differ from day
60 onward, and CC was conversely distributed.
3.8. Sensory analysis
The overall acceptability (6.9 ± 0.5), also referring to typical at-
tributes of PDO PC cheese, was significantly the highest (P < 0.05) for
the cheese manufactured with AS1, followed by CC (5.8 ± 0.3) and the
cheese made with AS2 (5.2 ± 0.2). This result mirrored the scores for
aroma (7.8 ± 0.4, 7.1 ± 0.1 and 6.5 ± 0.3) and flavour (7.0 ± 0.4,
6.6 ± 0.1 and 6.2 ± 0.1) intensities. The texture (5.3 ± 0.2 and
5.1 ± 0.3) did not significantly vary (P > 0.05) between the cheese
manufactured with autochthonous starter cultures, which agreed with
levels of acidification and moisture. CC was the softest (6.0 ± 0.2).
4. Discussion
It is well documented that the high quality aged raw milk cheeses
are undoubtedly the best sources for isolation of LAB strains suitable to
be used as starter cultures for cheesemaking (Gobbetti et al., 2015). In
agreement, the autochthonous NSLAB of this study were isolated from
aged (75 to 120 days) PC cheeses. Although NSLAB caused very rarely
cheese defects, adventitious NSLAB may introduce variability into ri-
pening processes, either within the same dairy plant or for consecutive
cheese batches (Antonsson, Molin, & Ardö, 2003). The most obvious
and suitable option to control adventitious NSLAB is to pre-empt their
cheese colonization via deliberate introduction of selected NSLAB as
starters or adjunct cultures. In this study, the selection for starter can-
didates was within a large number of mesophilic NSLAB. Lactococcus
lactis and, especially, Lactobacillus casei dominate the aged PC cheeses
but also Leuconostoc mesenteroides subsp. mesenteroides, Lactobacillus
plantarum, Lactobacillus rhamnosus, Lactobacillus brevis and Pediococcus
pentosaceus were present. This bacterial composition almost agreed

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Fig. 4. Pseudo-heatmap depicting the distribution

(%) of bacterial genera during ripening of Pecorino
Crotonese cheese. Pseudo-heatmap depicting the
distribution (%) of bacterial genera during ripening
of PC cheese. Only OTUs occurring at 0.3% abun-
dance in at least one samples were included.
Clustering of samples and taxa was obtained using
hierarchical clustering of weighted UniFrac distances
between samples or Euclidean distance measure be-
tween taxa. Raw ewes' milk (M), Pecorino Crotonese
cheese made with commercial primary starters
(control cheese, CC) or with Autochthonous Starter
(AS) 1 (Leuconostoc mesenteroides subsp. mesenter-
oides 2A, Lactobacillus casei 23C and Lactobacillus
plantarum 18C) and AS2 (Leuc. mesenteroides subsp.
mesenteroides 2A, L. casei 25D and 16A) mixtures
during ripening (1, 15, 45, 75 and 105 days).

Table 4 mesenteroides 2A, and L. casei 16A, 23C, and 22C. These results con-
Mean valuesa for the level of free amino acids (mg kg−1) found in Pecorino firmed that NSLAB and, especially, L. casei were suitable reservoirs of a
Crotonese cheese made with commercial primary starters (control cheese, CC) wide enzymatic portfolio (Nongonierma, Abrlova, & Kilcawley, 2013).
or with Autochthonous Starter (AS1 and AS2) mixturesb. The selection allowed the formulation of two mixed Autochthonous
Days of ripening CC AS1 AS2 Starter (AS1 and AS2), which guaranteed an almost complete enzyme
pattern as well a suitable acidification rate.
B A
1 663 ± 19 715 ± 21 732 ± 17A PC cheeses manufactured at industrial level had a gross chemical
7 917 ± 22B 1140 ± 19A 1124 ± 23A
15 1394 ± 38B 1516 ± 40A 1301 ± 43B
composition, which approached those of other Italian ewes' milk
30 1561 ± 39A 1526 ± 41A 1436 ± 43B cheeses (Coda et al., 2006). Compared to control cheese (CC) made with
45 2801 ± 72A 1581 ± 64B 1583 ± 65B commercial starters, the use of both autochthonous starter cultures had
60 3738 ± 84A 1788 ± 75C 2391 ± 81B only a slight influence. This is one of the main pre-requisite for their
75 3829 ± 88A 1860 ± 78C 2051 ± 89B
successful use (Upadhyay & McSweeney, 2003). The microbial cell
90 3746 ± 120A 2758 ± 111C 3048 ± 104B
105 8902 ± 210A 3538 ± 177C 4040 ± 202B density, composition and ratio between mesophilic and thermophilic
lactobacilli of CC reflected the most common succession, which char-
Data in the same row with different superscript letters (A-C) are significantly acterizes the cheeses during ripening. As expected, mesophilic lacto-
different (P < 0.05). bacilli were at the highest cell density in the cheeses manufactured with
a
Mean values ± standard deviations for three batches of each type of autochthonous starter cultures. The OTUs found during ripening by
cheese, analysed in duplicate (n = 6). culture-independent analysis provided a picture on how the auto-
b
AS1, consisted of Leuconostoc mesenteroides subsp. mesenteroides 2A,
chthonous starter cultures established in the cheese matrix throughout
Lactobacillus casei 23C and Lactobacillus plantarum 18C; AS2, consisted of Leuc.
ripening. The presence of bacterial phyla in raw ewes' milk reflected the
mesenteroides subsp. mesenteroides 2A, L. casei 25D and 16A.
outcome of environmental contamination. Firmicutes and, especially,
Proteobacteria dominate different farm surfaces, including teat, milking
with a previous microbiological characterization (Randazzo et al.,
parlours, hay, air and dust (Quigley et al., 2013). Psychrotrophic
2010) of PC cheese. In general, the bacterial biota identified in this
Pseudomonas spp., which dominated the microbiota of CC at the be-
study was consistent with other reports (Coda et al., 2006; De Angelis
ginning of ripening (day 1), are the most common cause of milk spoi-
et al., 2001; Macedo, Tavaresa, & Malcata, 2004; Mannu, Comunian, &
lage (Ercolini, Russo, Ferrocino, & Villani, 2009) and may constitute 70
Scintu, 2000) that described the microbial composition of ewes' milk
to 90% of the microbial population during milk storage at low tem-
cheeses.
peratures and, in limited cases, may persist during cheese ripening
The selection of autochthonous starter cultures concerned enzyme
(Raats, Offek, Minz, & Halpern, 2011). When autochthonous starter
activities responsible for secondary proteolysis and catabolism of FAA
cultures were used, most of these contaminants decreased drastically
(Gobbetti et al., 1999). Except for aminopeptidase type N, which was
(day 1) with a rate higher than in the presence of the commercial starter
the highest for L. casei 25D and L. plantarum 18C, all the other enzyme
(day 15). The synthesis of inhibitory compounds (e.g., bacteriocins) by
activities were present at the highest levels in Leuc. mesenteroides subsp.

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I. De Pasquale et al. Food Research International 116 (2019) 1344–1356

Fig. 5. Score plot and loading plot of first and second principal components of volatile components and volatile free fatty acids. Score plot (A) and loading plot (B) of
first and second principal components after PC analysis based on volatile components (log of units of area) and volatile free fatty acids (ppm) that mainly differ-
entiated (P < 0.05; r ≥ 0.7) the three Pecorino Crotonese cheeses made with commercial primary starters (control cheese, CC) or with Autochthonous Starter (AS) 1
(Leuconostoc mesenteroides subsp. mesenteroides 2A, Lactobacillus casei 23C and Lactobacillus plantarum 18C) and AS2 (Leuc. mesenteroides subsp. mesenteroides 2A, L.
casei 25D and 16A) mixtures during ripening (60, 75 and 105 days). C2, acetic acid; C4, butanoic acid; C6, caproic acid; butenal, 2-butenal E; pentenal, 2-pentenal;
hexenal, 2-hexenal; hexadienal, 2,4-hexadienal; heptenal, 2-heptenal; heptadienal, 2,4-heptanedienal; octenal, 2-octenal; 1pentanol, 1-pentanol; 1heptanol, 1-
heptanol; 1pentan3ol, 1-penten-3-ol; 1octen3ol, 1-octen-3ol; 2butanol, 2-butanol; 2pentanol, 2-pentanol; 2M1propanol, 2-methyl-1-propanol; 2M1butanol, 2-me-
thyl-1-butanol; 2butanone, 2-butanone; 3hexanone, 3-hexanone; 4heptanone, 4-heptanone; 3heptanone, 3-heptanone; 2heptanone, 2-heptanone; 3octanone, 3-
octanone; 2octanone, 2-octanone; 2nonanone, 2-nonanone; 1octen3one, 1-octen-3-one; hexanedione, 2,3-hexanedione; octanedione, 2,3-octanedione; 3M2butanone,
3-methyl-2-butanone; 5M2hexanone, 5-methyl-2-hexanone; 6M2heptanone, 6-mehyl-2-heptanone; 6M3heptanone, 6-methyl-3-heptanone; M-C2, methyl acetate; M-
C3, methyl propanoate; M-C4, methyl butanoate; M-3MC4, methyl 3-methyl-butanoate; M-2MC4, methyl 2-methyl-butanoate; M-C5, methyl pentanoate; M-C6,
methyl hexanoate; M-C7, methyl heptanoate; M-C8, methyl ocatnoate; M-benzoate, methyl benzoate; E-C2, ethyl acetate; E-2MC3, ethyl 2-methyl-propanoate; E-
2MC4, ethyl 2-methyl-butanoate; P—C6, propyl hexanoate; B—C2, butyl acetate; H—C2, hexyl acetate; 2MP-C2, 2-methyl-propyl acetate; 3 MB-C2, 3-methyl-butyl
acetate; 2 MB-C2, 2-methyl-butyl acetate; 2MP-C4, 2-methyl-propyl butanoate; DMS, dimethyl-sulfide; dithiapentane, 2,4-dithiapentane; thiophenone, 2-methyl-
tetrahydrothiophen-3-one; Efuran, 2-ethyl-furan; Bfuran, 2-butyl-furan; Ptfuran, 2-pentyl-furan; diMfuran, 2,5-dimethyl-furan.

autochthonous starter cultures might had explained this difference
during ripening. The bacterial community of PC cheeses manufactured
with AS1 and AS2 was almost constant throughout ripening. It con-
sisted of L. casei/L. plantarum and L. delbruecki/Leuc. mesenteroides
clusters, and St. thermophilus, as the only house contaminant, thus
confirming the robustness of the selected autochthonous starter cul-
tures. These findings were in agreement with the literature (Heunis,
Deane, Smit, & Dicks, 2014), showing that NSLAB grow at low tem-
peratures and adapt to lack of fermentable carbohydrates, low pH,
water activity, and the presence of bacteriocins.
Proteolysis is the most complex and important biochemical event,
which occurs during ripening of almost all cheese varieties (Gobbetti,
Angelis, Cagno, & Rizzello, 2007). Primary proteolysis of PC cheese
showed a slight and late hydrolysis of αs1-CN, probably due to the re-
sidual activity of chymosin. Unlike other Italian ewes' milk cheeses
(e.g., Pecorino Romano, Canestrato Pugliese), the PC manufacture did
not include cooking of the curd, which allowed residual chymosin ac-
tivity. A considerable amount of β-CN persisted at the end of ripening.
The presence of γ-CN indicated plasmin activity. Chymosin activity
toward β–CN is usually limited, mainly because of hydrophobic inter-
actions between salt and proteins (Fox, 1963). The urea-PAGE elec-
trophoretographs and chromatograms of the pH 4.6-soluble fraction
and the concentration of FAA showed an increased extent of secondary
proteolysis in the presence of autochthonous starter cultures. In parti-
cular, the use of AS1 favoured an increase of the number and area of
hydrophilic and hydrophobic peptide peaks from day 60 onward. Dif-
ferently from cheesemaking with the combination of primary starters
and adjunct cultures (De Pasquale, Calasso, et al., 2014; Di Cagno et al.,
2011), PC cheeses made with autochthonous starter cultures alone
showed lower levels of total FAA than those found in CC. This finding
may reflect an accelerated liberation of FAA because of the high pro-
teolytic activity of thermophilic lactobacilli present in the commercial
starter, and their further metabolic fate during ripening (Ardo, Larsson,
Lindmark-Manson, & Hedenberg, 1989; Katalari, Voutainas, & Kondyli,
2002; Michaelidou, Katsiari, Voutsinas, & Alichanidis, 2003). Leu, Phe,
Val, Lys, Ala and Pro were found at the highest concentrations, which is
typical for several Italian semi-hard and extra-hard cheese varieties
(Albenzio et al., 2001; Coda et al., 2006; Gobbetti et al., 1999). An
accelerated liberation of FAA promotes an increased synthesis of vo-
latile compounds (VOC), which has repercussions on cheese flavour
(Fox & Wallace, 1997). The levels of 62 VOC differentiated CC from the
cheeses manufactured with autochthonous starter cultures, which
highlighted their complementary enzymatic activity. The levels of most
unsaturated or branched aldehydes, branched ketones, many alcohols,
all methyl esters, butanoic and hexanoic acids were the highest in CC,
whereas sulphur compounds were globally the lowest. The higher levels

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I. De Pasquale et al.
of branched compounds, which occur from branched amino-acid cata-
bolism, aldehydes (Yvon & Rijnen, 2001) and ketones (Thierry,
Maillard, Richoux, Kerjean, & Lortal, 2005), may be the consequence of
an accelerated liberation of precursors FAA in CC cheese. As reported
for Gouda and Cheddar cheeses, such compounds generate fruity and
nutty notes, but when present at high levels they are responsible for
defects (Engels, Dekker, de Jong, Neeter, & Visser, 1997). Other com-
pounds, such as butyric and hexanoic acids, may had been liberated
through lipolysis by kid rennet paste. Aliphatic alcohols such as 2-bu-
tanol and 1-pentanol, which generate green and alcoholic notes
(Curioni & Bosset, 2002), were present in CC cheese at the highest le-
vels probably because of the metabolism by primary starters such as
Lactococcus lactis (Randazzo et al., 2010). Overall, the cheeses manu-
factured with AS1 and AS2 were quite similar at 60 days of ripening,
showing the lowest amounts of VOC. Only acetic acid was at the highest
levels, probably because of the presence of Leuconostoc species (Keenan,
1968). After 75 days of ripening, the cheese manufactured with AS1
had the highest levels of many alcohols, including primary (ethanol, 1-
propanol and 1- hexanol) and branched (2-methyl-1-propanol, 2-me-
thyl-1-butanol, 3-methyl-1-butanol) alcohols, and branched acids (iso-
butyric, isovaleric, isocaproic). These branched compounds, which de-
rive from the catabolism of branched FAA (Yvon & Rijnen, 2001), show
that the degradation of FAA in the presence of mesophilic species of
AS1 favours the synthesis of compounds that differed from those ob-
tained using commercial starters. At the end of ripening, many differ-
ences emerged between AS1 and AS2. The cheese made with AS2
harboured mainly neutral VOC, whereas that made with AS1 had the
highest levels of many VOC such as esters (e.g., ethyl, propyl, hexyl, 2-
methyl-propyl and 3-methyl-butyl esters), ketones (e.g., 4-heptanone,
2-pentanone, 2-nonanone), aldehydes (e.g., hexanal, octanal) and
furans (e.g., 2-butyl-furan, 2,5-dimethyl-furan). Overall, esters were
among the most abundant VOC in PC cheese, which confer floral and
fruity notes (Randazzo et al., 2010). In particular, ethyl-hexanoate has
an important role in the aroma of many aged cheeses (Engels et al.,
1997). Ketones are intermediate compounds, further reduced to sec-
ondary alcohols. Usually, the synthesis of methyl ketones occur from
fatty acids via oxidative degradation (McSweeney and Sousa, 2000).
Various aroma notes associated with methyl ketones, such as in the
cases of 4-heptanone (blue cheese flavour), 2-heptanone (musty, sweet,
mouldy, varnish) and 2-nonanone (floral, fruity, peach). Usually, these
methyl ketones derive from the metabolism of mesophilic NSLAB
(Rothe, Engst, & Erhardt, 2006). Besides, the simultaneous higher levels
of aldehydes and furans indicated a globally higher oxidation of fatty
acids occurring with AS1. During ripening, the microbial catabolism of
methionine and cysteine leads to the formation of hydrogen sulphide
and methanethiol, which reflects the metabolic interaction of the se-
lected autochthonous mesophilic strains. After oxidation, these latter
compounds generate methional and dimethyl-disulphide, which oc-
curred at the highest levels when AS1 and AS2 were used for cheese-
making. These sulphur compounds are indispensable for the char-
acteristic aroma of several cheese varieties. The preliminary screening
of mesophilic NSLAB also considered cystathionine lyase activity,
which is consistent with the highest levels of sulphur compounds found
in the cheeses manufactured with autochthonous starter cultures
(Smacchi & Gobbetti, 1998). According to the most diversified VOC
profiles of the cheeses, attributes for sensory analysis and overall
quality confirmed that PC cheese manufactured with AS1 was preferred
with respect to CC.
The Regulatory Board of the Consortium is discouraging the use of
commercial starters for PDO PC cheesemaking, which usually consists
of a mixture of thermophilic and mesophilic species/strains. This study
allowed to understand and explain the population dynamics with re-
spect to interactions between autochthonous starter cultures and other
bacteria dominating the ecosystem of Pecorino Crotonese cheese.
Cause-effect relationships between autochthonous starter cultures and
secondary proteolysis and catabolism of free amino acids during
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Food Research International 116 (2019) 1344–1356
ripening were established. According to the main expectations of the
Pecorino Crotonese Consortium, a promising pool of autochthonous
starter cultures consisting of NSLAB was selected.
Acknowledgements
The authors thank the industrial plant Caseificio APOCC located in
Cutro (Crotone), Calabria region, Italy and the Consortium of Pecorino
Crotonese cheese. The authors are grateful to Dr. Isabella Filannino, Dr.
Vincenzo Verdoliva and Dr. Pasquale De Francesco of the Azienda
Officina gbs of Roma for technical support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodres.2018.10.024.
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