Eur Food Res Technol (2017) 243:1943–1955
DOI 10.1007/s00217-017-2899-x
ORIGINAL PAPER
Lactobacillus delbrueckii subsp. lactis as a starter culture
significantly affects the dynamics of volatile compound profiles
of hard cooked cheeses
Solange Buchin1 · Gabriel Duboz1 · Jean‑Christophe Salmon1 
Received: 24 December 2016 / Revised: 6 April 2017 / Accepted: 22 April 2017 / Published online: 2 May 2017
© Springer-Verlag Berlin Heidelberg 2017
Abstract  Lactobacillus delbrueckii (LD) is a species usually
found in raw milk hard cooked cheeses. Present in raw milk,
it grows in cheeses during ripening, but it can also be added in
large concentrations with acidifying starter cultures. Its role in
cheese proteolysis has been largely demonstrated, but its role
in the formation of volatile compounds in cheeses remains
to be documented. For this purpose, 16 experimental hard
cooked cheeses were made from raw milk using adjunct cul-
tures made up of Streptococcus thermophilus and Lactobacil-
lus helveticus with the addition or not of LD, and ripened for
184 days. Bacterial populations and volatile compounds were
monitored throughout ripening. The LD addition affected
the early growth of the starters but not the dynamics of the
native microbiota. LD had a major effect on proteolysis and
on the composition of volatile compounds. Of the 85 volatile
compounds identified in the cheeses, 6 of the 7 acids, 10 of
the 16 aldehydes, 11 of the 17 alcohols, 10 of the 17 ketones,
10 of the 23 esters, and 4 of the 5 sulphur compounds were
affected by the LD addition at one or more stages of ripening.
At the end of ripening, levels of the 31 compounds affected
by LD were higher in the cheeses with LD added, except one
secondary alcohol and 2 methyl-ketones. The question of the
effective involvement of LD in these observations, whether
due to the metabolic activity of LD or interactions with other
microbial populations, is raised.
Keywords  Hard cooked cheese · Lactobacillus
delbrueckii · Volatile compound · Thermophilic acidifying
starter
* Solange Buchin
solange.buchin@inra.fr
1
INRA, UR342 Technologie et Analyses Laitières,
39800 Poligny, France
Introduction
Cheese flavour develops during ripening as a result of fla-
voured compounds that form largely due to the growth
and metabolic activity of microbial populations. These
compounds are part of volatile fraction made of numer-
ous acidic and neutral compounds whose balance is deter-
minant for cheese flavour. Their occurrence in cheese and
their impact on flavour have been extensively described
[1–5].
In hard cooked cheeses, the secondary microbiota that
develops in the cheese core during ripening plays a decisive
role in the production of these compounds, whether it is the
raw milk microbiota [6, 7] or a secondary starter [8]. Fur-
thermore, large amounts of acidifying starters are added to
the vat milk; counts then decrease as ripening progresses,
so their role during ripening is less obvious. In hard cooked
cheese varieties that use thermophilic starters, Lactobacil-
lus delbrueckii (LD) - in general, the subspecies lactis - is
commonly used, in addition to Lactobacillus helveticus
(LH) and Streptococcus thermophilus (ST). Among lacto-
bacilli, LD counts persist much longer than LH counts dur-
ing cheese ripening [7, 9]. LD also develops when it is not
added as a starter, as it may still be present as a subdomi-
nant population in cheeses made from raw milk [10].
The role of LD in shaping sensory characteristics and
the extent of proteolysis was previously investigated in
experimental Swiss cheeses [9, 11–13]. The use of LD
as an acidifying starter for cheese manufacture was thus
associated with higher secondary proteolysis, a less sweet
taste and a better, more cohesive and elastic texture. These
results held regardless of whether LH was added as another
acidifying starter. The presence of LD in starters was also
related in Swiss cheeses to different volatile fatty acid
13

1944
profiles, including acetic and propionic acids [11, 14–16].
However, apart Biede et al. [11], the starter combinations
that were compared differed by more than one strain, which
may explain that the results about LD effect were not con-
sistent. Regarding the impact of LD on the neutral volatile
fraction of hard cooked cheeses, it has not yet been studied
with a dedicated experimental design. Controlling cheese
flavour using LD as a starter requires understanding how
this species may affect the development of acidic as well as
neutral volatile fractions.
The aim of this work was to determine the effect of the
presence of LD, specifically Lb delbrueckii subsp. lactis, as
a starter in raw milk hard cooked cheeses on the microbial
populations and on the composition of volatile compounds.
This effect was studied in model cheeses made in con-
trolled conditions, by comparing the inoculation of thermo-
philic starters with or without LD.
Materials and methods
Cheese manufacture
Four batches of Swiss-type cheeses were manufactured
from raw milk over four days. Four cheeses were made
each day from 40 L per vat of the same partly skimmed
raw milk (fat/protein = 1.12), with the addition of CuSO4
(140 mg per vat) to mimic the amount of copper measured
in milk when traditional copper vats are used. In addi-
tion, a mix of commercial terpenes was added to half the
vats to study the effect of milk terpenes on the dynamics
of bacteria and volatile compounds, and the interactions
with the LD addition. The results are not presented here
because of the negligible effect and absence of interac-
tions. These vats were therefore considered as biological
replicates. Two mixes of thermophilic starters made using
strains from the URTAL collection were added by cross-
ing with terpene addition. Two Streptococcus thermophilus
strains (S3 and S79) (5 × 105 cells ­mL−1 each) (ST), one
Lactobacillus helveticus strain (L122) ­ (105 cells ­
mL−1)
(LH), and one Lactobacillus delbrueckii subsp. lactis strain
(L139) ­(105 cells ­mL−1) (LD) were added to two vats (the
so-called LD+). Only ST and LH were added to the two
others (the so-called LD0). Rennet (Marshall 700 mg/L,
Danisco, Dangé Saint Romain, France) was added (6.9 mL
per vat), and the milk was left to curdle. After cutting the
curd, the vats were heated to 53 °C. After moulding, the
cheeses were pressed for 6 h at 150 g c­ m−2. The pH was
determined at 20 h after moulding with a penetration elec-
trode (Mettler). The cheeses were then salted by rubbing
with salt after removing from the mould and 13 times dur-
ing the first 30 days. The cheeses were ripened for 184 days
at 11 °C, 92–94% RH.
13
Eur Food Res Technol (2017) 243:1943–1955
Gross composition
Analyses were performed as described by Demarigny
et al. [17]. For cheeses aged 1 and 184 days, the pH of
grated cheese (Radiometer combined electrode, Neuilly-
Plaisance, France), dry matter (DM) and fat (butyromet-
ric method) contents were measured. The fat/dry matter
(FDM) and moisture in the non-fat substance (MNFS)
ratios were calculated. For cheeses aged 184 days, the chlo-
ride content was determined using a Corning 926 Chloride
Coulter (Bagneux/Loing, France) and expressed as salt in
water (NaCl/W); total nitrogen (TN), water-soluble nitro-
gen (WSN) and nitrogen soluble in phosphotungstic acid
(PTASN) were determined using the Kjeldahl method after
adequate extractions. The WSN/TN and PTASN/TN ratios
were calculated.
Microbiological analyses
The total aerotolerant flora was enumerated in milk before
processing on PCA medium. Thermophilic lactobacilli
(LT), thermophilic streptococci (ST), mesophilic faculta-
tively heterofermentative lactobacilli (FHL) and propionic
acid bacteria (PAB) were enumerated in milk after addition
of the starter, as well as in cheeses aged 1 and 7 days. All
the strains were enumerated in cheeses after 48, 92, 139
and 184 days of ripening. MRS, M17, FH and SLAC media
were used, respectively, according to the culture conditions
described by Charlet et al. [10]. In accordance with these
authors, in milk and cheeses until 7 days, colonies of LH
and LD were distinguished by their morphology on plates.
In addition, 511 colonies (from 2 batches of cheeses at
48 days and from 3 batches at 92 and 139 days of ripen-
ing) were collected from the MRS agar plates used for the
enumeration of thermophilic lactobacilli. These colonies
were Rep-typed [10] to determine if they were i/LD or ii/
LD strain L139 added to the milk as a starter.
Volatile compounds
Volatile fatty acids were analysed in 15 g of cheese by sol-
vent extraction/saponification, followed by gas chroma-
tography GC/FID (CE8160, ThermoScientific, Dardilly,
France) performed with a Stabilwax DA column (Restek,
Lisses, France) 30 m in length, 0.53 mm in diameter, and
1 µm in thickness, as described by Bugaud et al. [18]. In
addition, to discriminate 2-methyl-butyric and 3-methyl-
butyric acids, a second GC separation was conducted in
the same conditions on a RTX5 column (Restek, Lisses,
France) 30 m in length, 0.53 mm in diameter, and 1 µm in
thickness. Neutral volatile compounds were analysed in
12 g of cheese by Purge and Trap extraction (Tekmar 3000,
Agilent instruments, Les Ulis, France), followed by GC/MS
Eur Food Res Technol (2017) 243:1943–1955 1945
(GC6890/MSD5973, Agilent instruments, Les Ulis, France)
as described by Bugaud et al. [18]. Compounds were identi-
fied according to Kovat’s retention index and the NIST/EPA/
MSDC mass spectral database (Royal Society of Chemis-
try, Cambridge, UK). The quantification of compounds was
expressed in arbitrary area units of a specific ion.
Statistical analyses
An analysis of variance was performed on all the data using
GLM analysis in S ­ YSTAT® to assess the effects of LD,
batch and interactions. Microbiological and volatile com-
pound data were log transformed before statistical treat-
ment. Means were compared using Bonferroni’s test at
P < 0.05. A principal component analysis (PCA) was per-
formed using ­XLSTAT® software. The volatile compounds
measured in cheeses at day 184 were used as active varia-
bles and physico-chemical data and microbial numerations
measured at different stages of ripening were introduced as
additional variables.
Results and discussion
Physico‑chemical composition
Regarding the gross composition of the cheeses, the val-
ues were as expected at 1 and 184 days of ripening, i.e.
an increase in pH from 5.17 ± 0.04 to 5.89 ± 0.09, and
DM from 61.9 ± 0.5% to 64.2 ± 0.6%; a decrease in
MNFS from 56.2 ± 0.4% to 53.9 ± 0.9%, and FDM from
52.1 ± 0.6% to 52.4 ± 0.8%. This composition did not dif-
fer with the LD addition. On day 184, the NaCl/W content
was slightly higher in LD0 cheeses than in LD+ cheeses
(3.68  ± 0.25% vs. 3.51 ± 0.24%, P < 0.05), whereas the
extent of proteolysis was dramatically affected by the LD
addition. The fraction that contained small peptides and
free amino acids, represented by the ratio PTASN/TN, was
enhanced by 40% in LD+ cheeses (9.21 ± 0.51%) com-
pared with LD0 cheeses (6.14 ± 0.39%) (P < 0.001). The
fraction WSN/TN that contained both large and small pep-
tides was enhanced by 4%, with values of 24.6 ± 0.8%
in LD+ cheeses and of 23.7 ± 0.8% in LD0 cheeses
(P < 0.01). This dramatic increase in proteolysis conforms
with the presence of numerous proteinases and peptidases
in this species [19], and the observations of Bouton et al.
[12] and Bouton and Grappin [20] in Comté and model
Swiss-type cheeses.
Microbiology
The milk samples contained low levels of indigenous
microbiota, with a mean total aerobic microbiota count of
3.27 log cfu mL−1. The quantities of Lactobacillus helve-
ticus (LH) and Lactobacillus delbrueckii (LD) measured in
the vat milk reached the expected values (around 5.0 log
cfu mL−1) (Table 1). The lower average count of Strep-
tococcus thermophilus (ST) in the LD0 milk was due to
low values found in batch B4 (data not shown). This is in
accordance with the observations of Fornasari et al. [21] on
ST cultivability, altered in unfavourable conditions, milk
of batch B4 having the lowest protein content (data not
shown). This interaction was no longer observed.
Maximal counts of all starters were observed in cheeses
on day 1, with dramatic differences whether LD was
added as a starter or not (P < 0.001): on average 8.34
vs. 6.73 log cfu g−1 for ST, 5.76 vs. 7.96 log cfu g−1 for
LH, and 8.61 vs. 6.61 log cfu g−1 for LD. As no LD was
added in LD0 milk, we could consider that those found in
cheese originated from raw milk. After day 1, ST and LH
counts dropped, and on day 7, the effect of the LD addi-
tion remained for ST (P < 0.01), and in a lesser extent for
LH (P  = 0.06). In contrast, LD counts remained high in
LD+ cheeses, with numbers between 7.76 and 8.34 log
cfu g−1 on day 7.
From day 48, counts on MRS medium and M17 medium
at 45 °C could not be definitely attributed to thermo-
philic lactobacilli and Streptococcus thermophilus popu-
lations. PCR analysis revealed that on day 48 no LH was
observed on MRS medium at 45 °C, whereas LD repre-
sented around 45% of total colonies in LD0 cheeses and
60% in LD+ cheeses. Counts on MRS were significantly
higher on day 48 with the LD addition (P < 0.001). This
effect was due to the presence of cells of strain L139, found
only in cheeses LD+ where it represented 70–80% of LD
(around 6.86 log cfu ­g−1) (data not shown). Besides these
cells, the counts of LD were the same in cheeses LD0 and
LD+ (around 6.43 log cfu ­g−1), showing that non-starter
LD developed in both cheeses at the same levels. Another
population that was not identified as LD was also present
on MRS-45 °C agar plates at around 6.70 log cfu ­g−1.
This unidentified microbiota was probably non-starter
lactic acid bacteria (NSLAB) [22, 23]. Afterwards, LD
represented only around 20% (on day 92) to 30% (on day
139) of counts on MRS-45 °C agar plates in both LD0 and
LD+ cheeses, the inoculated strain L139 being no longer
found (data not shown). In a similar way, as starter cells
dramatically dropped on M17 medium at day 7, cells found
on this medium afterwards were probably ST cells present
in raw milk.
FHL were present at lower levels in LD+ cheeses com-
pared with LD0 on days 1 and 7, but with a slight differ-
ence. No effect of the LD addition on the native populations
was observed during ripening, except on day 184 when
FHL and PAB levels were slightly higher in LD+ and LD0
cheeses, respectively.
13

1946 Eur Food Res Technol (2017) 243:1943–1955

Table 1  Counts of microbial groups: means without (LD0, n  = 8) Table 1  continued

and with (LD+, n = 8) LD addition in vat milk after starter inocula-
tion and in cheeses at six ripening steps and results of GLM analysis Microorganisms LD0 LD+ Pc
 Presumptive thermophilic lacto- 6.43 6.48 NS
Microorganisms LD0 LD+ Pc
bacilli
Milk after starter ­inoculationa  Facultatively heterofermentative 7.35 7.46 **
 Streptococcus thermophilus 5.41 5.84 *** lactobacilli
 Lactobacillus helveticus 5.11 4.97 NS  Propionic acid bacteria 5.78 5.42 *
 Lactobacillus delbrueckii <4.00 5.15 NS Mean comparison: different letters indicate different means by Bon-
 Facultatively heterofermentative 2.11 2.34 *** ferroni’s test, P < 0.05
lactobacilli a
 Counts expressed in log (cfu mL−1); others counts expressed in
 Propionic acid bacteria 2.45 2.43 NS log(cfu g−1)
b
Cheese day 1   In brackets LD numerated by PCR expressed in  % of the counts of
 Streptococcus thermophilus 6.73 8.34 *** presumptive thermophilic lactobacilli
c
 Lactobacillus helveticus 7.96 5.76 ***   P value of GLM analysis: NS not significant; *P < 0.05;
** P < 0.01; *** P < 0.001
 Lactobacillus delbrueckii 6.61 8.61 ***
 Facultatively heterofermentative 3.28 2.94 *
lactobacilli
 Propionic acid bacteria 2.90 2.99 NS The microbial dynamics observed conform to previ-
Cheese day 7
ous descriptions for raw milk hard cooked cheeses [22,
 Streptococcus thermophilus 4.64 5.16 **
23], with particularly low counts of PAB due to the low
 Lactobacillus helveticus 5.82 5.12 NS
ripening temperature and the relatively high salt content
observed in this study [24]. In addition, a break in the
 Lactobacillus delbrueckii 6.27 8.04 ***
PAB dynamics was observed with levels of about 1 log
 Facultatively heterofermentative 4.43 4.09 ***
lactobacilli less observed at day 139, compared to days 92 and 184.
 Propionic acid bacteria 2.88 2.87 NS This probably means that PAB cells present during the
Cheese day 48 first part of ripening lost cultivability at this time due to
 Presumptive Streptococcus thermo- 7.15 7.18 NS stressful conditions. Hence, these cells could be replaced
philus by strains that were able to develop or a switch occured in
 Presumptive thermophilic lactoba- 6.81 (45%) 7.21 (60%) *** their metabolism that made them able to survive. Unsur-
cilli ­(LDb) prisingly, LD was the only starter species that remained
 Facultatively heterofermentative 7.46 7.42 NS in a cultivable state after the early stages of ripening [9]
lactobacilli but at 92 days of ripening, cells of the added strain L139
 Propionic acid bacteria 3.48 3.53 NS were no more cultivable. Hence, they could be present in
Cheese day 92 cheese matrix in different physiological states: cells were
 Presumptive Streptococcus thermo- 7.16 7.10 NS dead and some of them lysed, or cells remained metaboli-
philus
cally active but not cultivable (ABNC) [25].
 Presumptive thermophilic lactoba- 7.17 (23%) 7.19 (19%) NS
Counts in milk and early growth of microbiota revealed
cilli (LD)
interactions between LD and other species, many of them
 Facultatively heterofermentative 7.77 7.65 NS
lactobacilli already described. According to Charlet et al. [10], LD
 Propionic acid bacteria 5.60 5.77 NS providing ST with peptides or amino acids may explain
Cheese day 139 this positive interaction. Hence, the major impact of the
 Presumptive Streptococcus thermo- 6.71 6.74 NS LD addition as a starter we observed on cheese gross
philus composition was the dramatic increase in proteolysis. As
 Presumptive thermophilic lactoba- 7.02 (32%) 7.01 (27%) NS hypothesized by Charlet et al. [10], the negative interac-
cilli (LD) tion between LD and LH may come from competition for
 Facultatively heterofermentative 7.51 7.48 NS carbohydrate availability. The antagonism between LD
lactobacilli and native FHL species in early ripening is consistent with
 Propionic acid bacteria 4.12 4.12 NS the explanation of Bouton et al. [7] regarding the nega-
Cheese day 184 tive impact of FHL on LD due to competition for limiting
 Presumptive Streptococcus thermo- 7.06 7.07 NS metabolites. However, after early stages, the impact of LD
philus
addition as a starter on microbial counts was limited.

13
 Table 2  Cheese volatile compounds: mean without (LD0, n = 8) and with (LD+ , n = 8) LD addition at four ripening steps and results of GLM analysis
Compounds Iona Kovat’s ­indexb Day 1 Day 92 Day 139 Day 184
c
LD0 LD+ P LD0 LD+ P LD0 LD+ P LD0 LD+ P

VFAs
 Acetic acid (C2) – – – 90 104 NS 95 108 NS 126 145 *
 Propionic acid (C3) – – – 69 97 *** 156 192 * 180 232 ***
 Butyric acid (C4) – – – 15.1 14.8 NS 16.6 17.5 * 17 19 **
 Caproic acid (C6) – – – 5.1 4.4 NS 5.6 5.7 NS 5.6 6.0 NS
 Isobutyric acid (iC4) – – – 0.63 1.22 *** 0.61 1.37 ** 0.57 0.81 *
 2-Methyl butyric acid (2MeC4) – – – 0.95 1.28 ** 0.85 1.77 * 0.82 1.14 **
 3-Methyl butyric acid (3MeC4) – – – 1.5 2.2 *** 2.3 3.4 * 2.5 3.0 NS
Eur Food Res Technol (2017) 243:1943–1955

Aldehydes
 Acetaldehyde 44 205 252 NS 241 269 NS 270 422 ** 279 399 ***
 Propanal 29 523 – – – 124 89 * 84 76 NS 103 76 NS
 Butanal 72 594 11 15 NS 23 31 * 22 32 * 23 36 ***
 Pentanal 58 699 168 222 NS 206 135 * 125 140 NS 130 107 NS
 Hexanal 72 801 19 30 NS 52 61 NS 41 44 NS 48 56 **
 Heptanal 96 902 7 7 NS 9 11 NS 6 8 NS 5 4 NS
 Octanal 84 1004 14 16 NS 33 37 NS 34 40 NS 26 21 NS
 Nonanal 57 1106 22 20 NS 66 73 NS 68 73 NS 73 53 **
 Decanal 57 1208 nd nd – 10 9 NS 5 7 NS 11 8 NS
 Propenal 56 486 34 41 NS 63 57 NS 73 68 NS 61 74 **
 2-Methyl-propanal 72 554 98 111 NS 120 201 ** 258 388 ** 231 376 **
 2-Methyl-butanal 58 662 66 42 ** 268 333 * 562 798 ** 405 701 ***
 3-Methyl-butanal 58 653 149 98 ** 370 444 * 578 746 ** 409 661 ***
 Benzaldehyde 106 969 20 42 NS 130 226 NS 126 177 NS 117 97 NS
 Benzeneacetaldehyde 91 1053 nd nd – 19 21 NS 20 27 NS 18 19 NS
 2-Hydroxy-benzaldehyde 122 1055 nd nd – 12 19 NS 3 6 NS TR ND NS
Alcohols
 Ethanol 45 448 4202 5757 NS 22,979 29,336 ** 30,326 30,245 * 41,461 38,360 NS
 1-Propanol 59 557 14 7 NS 7212 12,156 ** 23,518 35,060 ** 34,248 52,068 **
 1-Butanol 56 663 13 18 * 1625 2889 * 4087 6247 * 7,675 10,393 *
 1-Pentanol 55 769 111 100 NS 171 98 ** 104 100 NS 82 80 NS
 1-Hexanol 56 870 17 27 NS 268 293 NS 255 382 * 415 536 *
 1-Penten-3-ol 57 683 ND ND – 50 43 NS 49 46 NS 19 19 NS
 2-Propanol 45 504 756 958 NS 17,439 18,258 NS 22,119 24,223 NS 28,253 30,256 NS
 2-Butanol 59 605 5 6 NS 24,102 13,392 NS 48,638 26,446 ** 61,643 33,801 **

13
1947


Table 2  continued
1948

Compounds Iona Kovat’s ­indexb Day 1 Day 92 Day 139 Day 184
c
LD0 LD+ P LD0 LD+ P LD0 LD+ P LD0 LD+ P

13
 2-Pentanol 45 703 ND ND – 2180 2493 NS 2657 3108 * 2459 2838 *
 2-Heptanol 45 900 ND ND – 371 341 NS 1005 1124 NS 768 743 NS
 2-Methyl-1-propanol 74 628 ND ND – 25 35 ** 108 102 NS 96 118 *
 2-Methyl-1-butanol 70 739 ND ND – 219 306 ** 726 902 NS 630 902 **
 3-Methyl-1-butanol 70 736 25 11 * 773 1907 NS 1788 1847 NS 1565 2132 *
 3-Methyl-2-butanol 45 683 ND ND – 156 185 NS 288 584 NS 189 440 NS
 4-Methyl-2-pentanol 45 792 ND ND – 248 304 NS 323 397 * 197 220 NS
 3-Methyl-3-buten-1-ol 68 735 58 68 ** 114 123 NS 149 180 * 141 169 ***
 Phenol 94 979 Nd Nd – 66 80 NS 26 37 NS 21 19 NS
Esters
 Methyl acetate 43 526 4 4 NS 44 61 NS 56 86 * 70 86 NS
 Methyl propanoate 88 630 ND ND – ND TR – 2 9 *** 5 15 **
 Methyl butanoate 74 723 ND ND – 5 4 NS 1 6 NS 4 8 *
 Ethyl acetate 61 614 3 4 NS 49 53 NS 82 124 NS 137 210 *
 Ethyl propanoate 57 712 ND ND – 84 107 NS 439 693 ** 728 1304 ***
 Ethyl butanoate 88 801 ND ND – 93 126 NS 179 262 ** 309 413 *
 Ethyl hexanoate 88 997 ND ND – 98 38 NS 66 96 ** 111 146 **
 Propyl acetate 61 715 ND ND – 28 62 ** 293 448 NS 593 1102 *
 Propyl propanoate 75 810 ND ND – 2 5 NS 68 130 ** 128 345 ***
 Propyl butanoate 89 897 ND ND – 3 7 NS 44 69 * 81 167 ***
 Butyl acetate 43 815 ND ND – 69 99 NS 162 260 NS 356 559 NS
 1-Methyl-propyl propanoate 57 850 ND ND – ND TR – 17 10 NS 31 24 NS
 1-Methyl-propyl butanoate 71 937 ND ND – ND ND – ND ND – 66 1 NS
 3-Methyl-butyl acetate 70 877 ND ND – 10 6 NS 52 59 NS 81 105 NS
Ketones
 2-Propanone 58 495 29,871 28,554 NS 1688 1078 NS 2281 1720 NS 1499 2089 ***
 2-Butanone 72 600 1296 1396 NS 9988 3837 ** 11,758 7127 ** 9396 5626 ***
 2-Pentanone 43 687 1205 918 *** 862 650 NS 1112 789 NS 609 580 NS
 2-Hexanone 58 791 1 2 NS 72 13 *** 18 15 NS 7 7 NS
 2-Heptanone 58 891 488 329 *** 1035 672 *** 1424 1105 NS 619 579 NS
 3-Heptanone 57 887 ND ND – 99 9 NS 16 6 NS 15 8 NS
 2-Octanone 58 992 ND ND – 89 9 NS 22 14 NS 5 4 NS
 2-Nonanone 58 1097 17 10 ** 124 80 *** 271 135 NS 105 80 NS
 3-Methyl-2-butanone 86 659 ND ND – 9 10 NS 16 58 NS 5 43 NS
 4-Methyl-2-pentanone 100 740 ND ND – 5 6 NS 15 21 NS 1 4 NS
Eur Food Res Technol (2017) 243:1943–1955
 Table 2  continued
Compounds Iona Kovat’s ­indexb Day 1 Day 92 Day 139 Day 184
c
LD0 LD+ P LD0 LD+ P LD0 LD+ P LD0 LD+ P
 3-Methyl-2-pentanone 72 754 ND ND – 64 54 NS 44 38 NS 11 10 NS
 Acetoin 88 712 984 1054 NS 80 39 * 62 27 NS 18 2 NS
 Diacetyl 86 589 1761 1338 ** 773 470 *** 940 592 * 438 415 NS
 2,3-Pentanedione 100 695 188 215 NS 13 6 * 39 44 NS 29 39 NS
 2,3-Hexanedione 71 795 ND ND – 2 2 NS 6 6 NS 3 3 NS
 Cyclopentanone 84 744 ND ND – 27 6 NS 12 28 ** 4 26 ***
 1-Phenyl ethanone 105 1077 ND ND – 31 44 NS 3 18 NS 15 13 NS
Sulphur compounds
Eur Food Res Technol (2017) 243:1943–1955

 Carbon disulphide 76 535 174 192 NS 280 263 NS 309 382 NS 250 290 *
 Dimethyl sulphide 62 518 346 497 ** 1134 1223 NS 1748 2015 NS 1626 2490 *
 Dimethyl disulphide 94 747 52 62 NS 1111 1424 NS 2307 2911 NS 1966 1582 NS
 S-methyl thioacetate 90 702 ND ND – 8 17 *** 20 90 ** 15 34 NS
 2,4-Dithiapentane 61 896 ND ND – ND ND – 20 23 ** 24 28 NS

VFAs are expressed as mg/100 g cheese; neutral compounds are expressed as area value × 10 −3
NS not significant, – not analysed, ND not detected, TR traces
* P < 0.05; ** P < 0.01; *** P < 0.001. Mean comparison: different letters indicate different means by Bonferroni’s test, P < 0.05
a
  Specific ion used for area value record
b
  Retention index
c
  P value of GLM analysis

13
1949

1950
Variations in volatile compounds
Except terpenes (not shown), a total of 85 volatile com-
pounds was detected in the cheeses: 7 volatile fatty acids
(VFAs), 16 aldehydes, 17 alcohols, 23 esters, 17 ketones
and 5 sulphur compounds (Table 2), including 9 esters
found only in trace amounts and not listed in the table.
Only 68 of them were detected in cheeses on day 1 (75%
of aldehydes, 60% of sulphur compounds, 53% of alco-
hols and ketones, 2 esters). Among the aldehydes, propanal
could not be quantified at this stage due to coelution with
2-propanone in high amounts. Globally, most compounds
increased as ripening progressed, particularly esters and
alcohols, except 5 ketones that clearly decreased from day
1 (2-propanone, 2-pentanone, diacetyl, acetoin, 2,3-pentan-
edione) and 2 aldehydes (propanal, pentanal) that remained
at similar levels. One VFA (isobutyric acid), 5 aldehydes
(heptanal, octanal, 2- and 3-methyl-butanal, benzaldehyde),
4 alcohols (1-pentanol, 1-penten-3-ol, 4-methyl-2-pen-
tanol, phenol), 7 ketones (2-hexanone, 2- and 3-heptanone,
2-octanone, 2-nonanone, 3-methyl-2-pentanone, 1-phenyl-
ethanone) and 3 sulphur compounds (carbon disulphide,
dimethyl disulphide, S-methylthioacetate) reached maxi-
mum levels during ripening, and then decreased. These
variations were consistent with the results of Bosset et al.
[26, 27] in Gruyere or Thierry et al. [28] in Emmental, even
if the range of variation differs between studies. Propionic
acid concentrations were globally higher than expected
owing to the low temperature of ripening [18] and the low
levels of PAB [23], whereas the levels of other volatile fatty
acids were conform with values previously observed in
such cheeses by Bugaud et al. [18] (Abondance) and Berd-
agué et al. [29] (Comté ripened at 10 °C). It is interesting
that branched compounds (acids, aldehydes, alcohols) were
at their maximum at 139 days and then decreased. This
was the time PAB and also presumptive ST were at a mini-
mum level, before they increased again. Such compounds
may arise from branched amino acid catabolism. Following
Ganesan et al. [30], such compounds may serve as inter-
mediates in energy production, transcription and translation
in nonculturable cells of lactococci. Following Dherbecourt
et al. [31], branched fatty acids from amino acids may be
used in membrane-branched fatty acids synthesis in PAB.
This observation may support the hypothesis of a metabol-
ical switch at 139 days that would favour the survival of
PAB cells.
Variability of volatile compounds: effect of LD as a
starter
LD added as starter, through its own activity or through
interactions with other microorganisms, seems to be a
determining contributor to cheese ripening, as seen by
13
Eur Food Res Technol (2017) 243:1943–1955
Fig. 1  PCA representation of cheeses at the end of ripening (184 days) ▸
according to their composition in volatile compounds at 184 days, phys-
ico-chemical and microbial data at different ripening times. a List of
volatile compounds added as active variables (in bold, compounds sig-
nificantly affected by LD addition): C2, acetic acid; C3, propionic acid;
iC4, isobutyric acid; C4, butyric acid; 2MeC4, 2-methyl-butyric acid;
acetald, acetaldehyde; butanal; hexanal; nonanal; propenal; 2Mpro-
panal, 2-methyl-propanal; 3Mbutanal, 3-methyl-butanal; 2Mbutanal,
2-methyl-butanal; propanol, 1- propanol; butanol, 1-butanol; hexanol,
1-hexanol; 2butanol, 2-butanol; 2pentanol, 2-pentanol; 2Mpropanol,
2-methyl-1-propanol; 3Mbutanol, 3-methyl-1-butanol; 2Mbutanol,
2-methyl-1-butanol; 3M1buten3ol, 3-methyl-1-buten-3-ol; propanone,
2-propanone; butanone, 2-butanone; M-C4, methyl butyrate; E-C2,
ethyl acetate; E-C3, ethyl propionate; E-C4, ethyl butyrate; E-C6,
ethyl hexanoate; P-C2, propyl acetate; P-C3, propyl propionate; P-C4,
propyl butyrate; CDS, carbon disulphide; DMS, dimethyl sulphide; (in
italics, compounds not significantly affected by LD addition): 3MeC4,
3-methyl-butyric acid; C6, hexanoic acid; propanal; pentanal; heptanal;
octanal; benzald, benzaldehyde; ethanol; pentanol, 1-pentanol; 2pro-
panol, 2-propanol; 2heptanol, 2-heptanol; phenol; pentanone, 2-pen-
tanone; heptanone, 2-heptanone; octanone, 2-octanone; nonanone,
2-nonanone; acetoin; diacetyl; ptdione, 2,3-pentanedione; phethanone,
phenylethanone; M-C2, methyl acetate; B-C2, butyl acetate; 3 MB-C2,
3-methyl-butyl acetate; DMDS, dimethyl disulphide; MT-C2, methyl-
thio acetate. List of supplementary variables (underlined): microbial
counts: FHL, facultatively heterofermentative lactobacilli, PAB, propi-
onic acid bacteria; LT, presumptive thermophilic lactobacilli; LD, Lac-
tobacillus delbrueckii; LH, Lactobacillus helveticus; ST, Streptococ-
cus thermophilus; physico-chemical data at 184 days: DM, dry matter;
MNFS, moisture in non-fat substance; FDM, fat in dry matter; NFDM,
non-fat dry matter; Cl/W, NaCl/Water; WSN, water-soluble nitrogen in
total nitrogen; PTASN, phosphotungstic acid soluble nitrogen in total
nitrogen. b Observations: different symbols were given for different
cheese-making batches, black symbols for cheeses LD+, open symbols
for cheeses LD0
its globally enhancing effect on over half the volatile
compounds, at one stage of ripening or more. All VFAs
were enhanced with the LD addition, except caproic acid
(C6), which was never affected. Propionic (C3), isobu-
tyric (iC4) and 2-methyl-butyric (2MeC4) acids were
affected throughout ripening, whereas butyric acid (C4)
was affected after day 92, acetic acid (C2) only on day
184 and 3-methyl-butyric acid (3MeC4) before day 184
(Table 2). Among the aldehydes, the addition of LD was
associated with enhanced levels of butanal throughout
ripening, acetaldehyde on days 139 and 184, and hexa-
nal on day 184, as well as with decreased levels of pro-
panal, pentanal and nonanal. On day 1, branched alde-
hydes 3- and 2-methyl-butanal were more concentrated in
LD0 cheeses, but during ripening concentrations of the
three branched aldehydes (including 2-methyl-propanal)
were higher in LD+ cheeses (from 33 to 84%). As far
as alcohols were concerned, concentrations of 1-butanol
were higher in LD+ cheeses throughout the process,
ethanol and 1-propanol from day 92, 1-hexanol from day
139, and 2-pentanol on days 139 and 184. Higher levels
of 1-pentanol were observed in LD0 cheeses on day 92
and 2-butanol on days 139 and 184. On day 1, higher
Eur Food Res Technol (2017) 243:1943–1955 1951

Variables (axes F1 & F2 : 57,68 %)

a
P-C4
propanol
E-C4
PTASN
butanol acetald
E-C6 E-C2
P-C2
hexanol C4 LD1
B-C2
P-C3
C6 butanal E-C3
ST184 PAB48 C2
ST1 M-C4
DMS iC4 LT1
NFDM
propenal
2propanol 2MeC4
PAB184 M-C2 hexanal CDS C3
MT-C2 2Mpropanal
LT48
2butanol DM LT184 propanone
F2 (24,14 %)

2pentanol
3MeC4 2Mbutanal
ethanol WSN 3Mbutanal
acetoin PAB1 pH FHL184 3MB-C2
ST139 2Mbutanol
2heptanol 3M1buten3ol
LT92 2Mpropanol
PAB92 FHL139
FHL48 ST48
3Mbutanol
MNFS LT139
Cl/W
butanone FHL1 ptdione FDM
PAB139 pentanal benzald pentanone
FHL92
DMDS nonanal heptanal
pentanol
phenol octanal
propanal phethanone ST92 diacetyl
heptanone
octanone
LH1
nonanone

F1 (33,55 %)

Observaons (axes F1 & F2 : 57,68 %)

b 8

2
F2 (24,14 %)

-2

-4

-6

-8
-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12
F1 (33,55 %)

amounts of 3-methyl-1-butanol, the only branched alco-
hol present at this stage, were found in LD0 cheeses. On
the contrary, during ripening, the effect of the LD addi-
tion observed on the three branched alcohols resulted in
globally higher levels in LD+ cheeses (from 37 to 60%).
The LD addition increasingly affected esters as ripen-
ing progressed (1/11 on day 92, 8/13 on day 139), with
a major effect on day 184 (13/14). When significant,
the effect of the LD addition always resulted in higher
concentrations in LD+ cheeses. Ketones, when affected

13

1952
by the LD addition, were found in lower amounts in
the LD+ cheeses, except for 2-propanone on day 184,
whereas 4/5 sulphur compounds increased with the LD
addition at one or more ripening stages. There is no evi-
dence of a direct role of the LD strain L139 in the for-
mation of the compounds that were enhanced because of
decreasing plate counts as ripening progressed and loss
of cultivability after day 48. However, even if acidify-
ing starter cells lose their cultivability, they may remain
metabolically active during ripening [32, 34], so a direct
role of LD added as a starter cannot be excluded. Oth-
erwise, LD may favour the activity of other microbial
groups by providing them with nutrients. Some cells may
have died and lysed, and then have released molecules in
the matrix. Sgarbi et al. [34] have shown that mesophilic
lactobacilli can use the lysis products of LH cells as a
unique energy source. Irrespective of cell lysis, the nitro-
gen compounds produced by LD in our cheeses may have
affected the activity of the secondary microbiota. Fur-
thermore, compounds released by LD may serve as pre-
cursors for some enzymatic activities. Lastly, LD added
as a starter may have modified the balance between other
microbial populations. The counts of microbial groups on
M17, MRS or FH agar plates are not informative about
the species present during ripening.
Variability of volatile compounds: effect of other
bacterial groups
A principal component analysis (PCA) (Fig. 1) was used to
illustrate the correlations between volatile compounds of
cheeses at the end of ripening and other compositional data,
in particular the dynamics of the different bacterial groups.
We can distinguish three main groups of compounds. The
positive end of the first axis was characterized by the pres-
ence of branched compounds (aldehydes, alcohols, acids),
C3, carbon disulphide, 2-propanone, and 2-pentanol that
were enhanced by LD addition and positively correlated to
presumptive thermophilic lactobacilli (LT) at day 1, FHL at
days 139 and 184, ST at day 139, PAB at day 92 and con-
versely to PAB at day 184. pH at day 184 was positively
correlated to aldehydes and alcohols of this group, whereas
NaCl/W was negatively correlated to acids and some other
compounds (C3, iC4, 2MeC4, 2-methyl-propanal, 2-pen-
tanol, carbon disulphide). A second group was correlated
with the negative end of the second axis, not affected by
LD addition nor by LD at day 1 and LT at day 1, and was
mostly made of methyl- and di- ketones, which were simi-
larly correlated to the above-cited bacterial groups and fat
content. Moreover, LH at day 1 was negatively correlated to
these ketones, and also to some aldehydes (propanal, pen-
tanal, heptanal, octanal, benzaldehyde). The compounds
of the third group were enhanced by LD addition and
13
Eur Food Res Technol (2017) 243:1943–1955
positively correlated to LD at day 1 and LT at day 1. They
were mainly esters (ethyl, propyl), aldehydes (acetaldehyde,
butanal, hexanal, propenal), 1-propanol and dimethyl sul-
phide. This last group was positively correlated to PTASN/
TN (esters, acetaldehyde) and negatively to NaCl/W content
(hexanal, propenal, propyl acetate and propyl propionate).
This figure highlights the difficulty to interpret correlations
between bacterial counts and volatile compounds. Whereas
the relationships with FHL at the end of ripening seem to
match, observations made with PAB at the same time are
not relevant and may result from the above-mentioned break
in their dynamics. Hence, the production of expected com-
pounds from PAB metabolism, i.e. propionic fermentation
and isoleucine catabolism could be related to PAB counts
at the end of logarithmic phase, at day 92, at the maximum
of counts. Physico-chemical variables that were involved
in correlations may be linked also to the formation of com-
pounds, because pH, salt or free amino acid contents may
be important for metabolic activities. In the case of salt, the
compounds that were involved reflected PAB activity, i.e.
propionic acid fermentation (C3) and isoleucine catabolism
(2MeC4). This is consistent with the particular sensitivity of
PAB to NaCl level. We can then suppose that besides these
compounds, iC4, 2-methyl-propanal, 2-pentanol, carbon
disulphide may also originate from PAB metabolism. As an
example, Richoux et al. [35] described an effect on propi-
onic acid fermentation with 4–5 times less VFA production
when NaCl/W increased from 1 to 3%. In our cheeses, vari-
ations of NaCl/W from 3.25 to 3.96% did not modify the
growth of PAB, but were sufficient to influence PAB activity
and decrease C3 and 2MeC4 production (respectively, from
304 to 138 and from 1.9 to 0.4 mg/100 g). Hence, correla-
tions identified with microbial and physico-chemical vari-
ables may be of interest.
Effect of LD addition on the formation of volatile
compounds
Propionic acid fermentation
In hard cooked cheeses, propionic fermentation by PAB
provides the major part of C3 together with C2. C3 and
related esters are important flavour compounds because
of their characteristic sweet aroma typical of Swiss-type
cheeses. An indirect impact of LD, via cooperation with
PAB, may explain the strong positive relation between
the LD addition and C3. In our cheeses, in the presence
of LD, levels of C3 increased throughout ripening from
38 to 57%, while the growth of PAB was not affected, and
even decreased at the end of ripening. Bouton et al. [14]
in Comté and Kerjean et al. [15] and Thierry et al. [16]
in Emmental have already observed higher levels of C3
and/or C2 when LD was added to starters. However, PAB
Eur Food Res Technol (2017) 243:1943–1955 1953
growth and metabolism are not necessarily commensurate,
as already mentioned by Kerjean et al. [15], which would
explain the effect of adding LD on C3 but not on PAB
counts. In this study, LD possibly favoured PAB activity by
providing peptides, as suggested by Thierry et al. [16, 36].
Amino acid catabolism
The catabolism of amino acids in cheese is of major inter-
est because after sugar exhaustion, amino acids remain the
main source of energy and nutrients for bacterial survival
[37]. Branched VFAs (iC4, 2MeC4, 3MeC4), aldehydes
(2-methyl-propanal, 2-methyl-butanal, 3-methyl-butanal),
and alcohols (2-methyl-1-propanol, 2-methyl-1-butanol,
3-methyl-1-butanol) are compounds from the catabolism
of branched amino acids valine, isoleucine and leucine.
Many of these volatile compounds may be involved in
cheese flavour, in particular VFAs with cheesy notes, and
aldehydes with a malty flavour [38]. In our cheeses, all the
bacterial groups studied may contribute to such catabo-
lism, as revealed by the enhancing effect of LD on many
of these compounds from day 92 to 184, and the correla-
tions observed between FHL and these same compounds
at the end of ripening (not shown). According to Helinck
et al. [39] and Williams et al. [40], cells of thermophilic
starters and of NSLAB were able to produce in vitro
branched volatile compounds from branched amino acids
via transamination. This metabolic route requires the suc-
cession of different enzymes, aminotransferase, decarboxy-
lase, dehydrogenase, with α-ketoacid availability being a
limiting substrate for transamination, the first reaction. The
occurrence and activity of amino acid converting enzymes
in lactobacilli are species and strain dependent [41, 42]
and possibly the realisation of such a metabolic route
requires complementation between strains, as suggested
by Helinck et al. [39] for LD and ST. Indeed, aminotrans-
ferase activities have been measured in vitro in our L139
strain (unpublished results), which hence may be able to
perform transamination, but not glutamate dehydrogenase
activity that may produce α-ketoglutarate as a substrate for
transamination.
The sulphur compounds resulting from methionine
catabolism are key flavour compounds in all cheeses
because of their potent sulphur odour. Carbon disulphide
and S-methyl-thioacetate in our cheeses on day 184 were
correlated with branched volatile compounds and glob-
ally showed the same patterns of variation, so the mecha-
nisms of formation may be the same. Indeed, methionine in
cheese may be catabolised by lactic acid bacteria through
transamination [43]. However, lyases can also cleave the
lateral chain and cystathionine lyase activity that poten-
tially degrades methionine has been shown in vitro in LD,
principally subspecies bulgaricus [44]. Besides, the effect
of the LD addition on dimethyl sulphide in our cheeses
seems to confirm the ability of LD to catabolise methio-
nine, as shown in fermented milk by Imhof et al. [45], with
the production of (di)methyl-sulphide and (di)methyl-disul-
phide by Lb. delbrueckii subsp. bulgaricus. However, a
contribution of LD by providing free methionine as precur-
sor cannot be excluded.
Esters and related compounds
Ethanol, 1-propanol, 1-butanol and 1-hexanol are of lit-
tle interest in themselves as flavourful compounds, but
are important as precursors of many esters in Swiss-type
cheeses. They come from the reduction of the correspond-
ing aldehydes by alcohol dehydrogenase. Such an activity
has been measured in vitro in some LD strains by Keenan
and Lindsay [46], and Helinck et al. [39] as well as in our
starter LD strain L139 (unpublished results). The enhancing
effect of the LD addition on these alcohols in our cheeses
suggests that this enzyme may operate in cheese conditions
provided starter strain remained metabolically active. As
acetaldehyde, butanal and hexanal were similarly affected,
LD may also contribute to the formation of these precursor
compounds, as already observed for acetaldehyde [43–48].
Propanal was inversely affected, so another population may
produce this compound [49], and then LD reduces it. LH at
day 1 was positively correlated with this compound.
With their characteristic fruity flavours, esters are key
compounds for the desirable sweet and fruity notes of
Swiss-type cheeses [50]. Their formation in cheese would
be due to enzymatic activity, as lactic acid bacteria and
PAB possess esterases. In most cheese varieties, the major-
ity of esters are ethyl esters, whereas in our cheeses we
recovered a noteworthy higher diversity, in accordance
with Liu et al. [50]. Such diversity may require long rip-
ening periods because of the late formation, as observed
in our cheeses. This delay may be due to the need for suf-
ficient amounts of precursors, acids and alcohols. A glob-
ally enhancing effect of the LD addition was observed for
esters, which increased over time, so on day 184, all the
major esters had been significantly affected, except for the
esters derived from the three branched alcohols, which
were in low amounts. On the one hand, LD may have
favoured the formation of esters because they enhanced the
production of alcohols and/or VFAs as precursors. On the
other hand, these bacteria may also possess esterase activi-
ties, although weaker than ST and PAB [50], as evidenced
by El Soda et al. [51], Oliszewski et al. [52] or Zourari
et al. [48] in strains of both Lb. delbrueckii subsp. bulga-
ricus and Lb. delbrueckii subsp. lactis. Of all populations
of our cheeses, only LD has been identified as positively
correlated with esters, which supports LD involvement in
this synthesis.
13

1954
Ketones and related compounds
From the concentration dynamics in our cheeses, we can
observe that diacetyl, present in high concentrations in fresh
cheeses and characteristic of the butter flavour in young
cheeses was progressively reduced to acetoin, 2-butanone and
2-butanol [53] as ripening progressed. A significant decreas-
ing effect of the LD addition was observed on these four com-
pounds on day 1, day 92, from days 92 to 184, and on days
139 and 184, respectively. 2-propanone also appeared to be
reduced to 2-propanol, but the effect of LD was the opposite.
All these compounds are from pyruvate metabolism, which
may be modulated by LD. With respect to lipid metabolism,
LD addition had a limited effect on fatty acids from lipolysis
(C4 and C6), but was linked to decreased levels of methyl-
ketones formed from fatty acid oxidation in the first half of
ripening. For 2-pentanone, this may be explained by a higher
reduction into secondary alcohol 2-pentanol.
Conclusion
The addition of Lactobacillus delbrueckii subsp. lactis
clearly influenced proteolysis and the levels of volatile com-
pounds during the ripening of raw milk hard cooked cheeses.
Many metabolic pathways were involved such as amino acid
catabolism, esterification, and reduction. However, in most
cases it was not possible to determine how LD was involved,
whether it was through its own enzymatic activities or by
providing precursors or nitrogen compounds that favoured
other species. Hence, further investigations are needed to
understand the exact role of this species in the production
of volatile compounds, in particular its interactions with the
secondary microbiota. However, these results suggest that
LD may affect flavour formation in hard cooked cheeses.
Acknowledgements We are grateful to Dominique Lefier, Francis
Faurie, Romain Palme, and Marie-Odile Coquillard for their technical
assistance, Eric Beuvier for his read-through of the manuscript and
Helen Lamprell for her review of English language.
Compliance with ethical standards  Propionibacterium freudenreichii strains quantitatively affect
Conflicts of interest  The authors declare no conflicts of interest.
Compliance with ethics requirements  This article does not contain
any studies with human or animal subjects.
References
1. Curioni PMG, Bosset JO (2002) Key odorants in various
cheese types as determined by gas chromatography-olfactome-
try. Int Dairy J 12:959–984
13
Eur Food Res Technol (2017) 243:1943–1955
2. McSweeney PLH, Sousa MJ (2000) Biochemical pathways for
the production of flavour compounds in cheeses during ripen-
ing: a review. Lait 80:293–324
3. Preininger M, Warmke R, Grosch W (1996) Identification of
the character impact flavour compounds of Swiss cheese by
sensory studies of models. Z Lebensm Unters For 202:30–34
4. Rychlik M, Bosset JO (2001) Flavour and off-flavour com-
pounds of Swiss Gruyère cheese. Identification of key odorants
by quantitative instrumental and sensory studies. Int Dairy J
11:903–910
5. Urbach G (1997) The flavour of milk and dairy products. II.
Cheese: contribution of volatile compounds. Int J Dairy Technol
50:79–89
6. Beuvier E, Buchin S (2004) Raw Milk Cheeses. In: Fox P, Mc
Sweeney PLH, Cogan TM, Guinee TP (eds) Cheese chemistry,
physics and microbiology. Vol 1 General aspects, 3rd ed. Else-
vier Ltd, London, pp 319–345
7. Bouton Y, Buchin S, Duboz G, Pochet S, Beuvier E (2009) Effect
of mesophilic lactobacilli and enterococci adjunct cultures on the
final characteristics of a microfiltered milk Swiss-type cheese.
Food Microbiol 26:183–191
8. Thierry A, Deutsch S, Falentin H, Dalmasso M, Cousin F, Jan G
(2011) New insights into physiology and metabolism of Propi-
onibacterium freudenreichii. Int J Food Microbiol 149:19–27
9. Chamba JF (2000) Emmental cheese: a complex microbial eco-
system. Consequences on selection and use of starters. Sci Ali-
ments 20:37–54
10. Charlet M, Duboz G, Faurie F, Le Quéré JL, Berthier F (2009)
Multiple interactions between Streptococcus thermophilus,
Lactobacillus helveticus and Lactobacillus delbrueckii strongly
affect their growth kinetics during the making of hard cooked
cheeses. Int J Food Microbiol 131:10–19
11. Biede S, Reinbold G, Hammond E (1977) Influence of Lacto-
bacillus bulgaricus on commercial Swiss cheese. J Dairy Sci
60:123–125
12. Bouton Y, Guyot P, Dasen A, Grappin R (1994) Proteolytic
activity of thermophilic lactobacilli strains isolated from start-
ers and Comte II. Applications in cheese plants. Lait 74:33–46
13. Richoux R, Aubert L, Roset G, Kerjean JR (2009) Impact
of the proteolysis due to lactobacilli on the stretchability of
Swiss-type cheese. Dairy Sci Technol 89:31–41
14. Bouton Y, Guyot P, Dasen A (1996) Effects of interactions
between milk and lactic starters on the ripening and quality of
Comte cheese. Int Dairy J 6:997–1013
15. Kerjean JR, Condon S, Lodi R, Kalantzopoulos G, Chamba
JF, Suomalainen T, Cogan T, Moreau D (2000) Improving the
quality of European hard-cheeses by controlling of interac-
tions between lactic acid bacteria and propionibacteria. Food
Res Int 33:281–287
16. Thierry A, Maillard MB, Richoux R, Kerjean JR, Lortal S (2005)
production of volatile compounds in Swiss cheese. Lait 85:57–74
17. Demarigny Y, Beuvier E, Buchin S, Pochet S, Grappin R
(1997) Influence of raw milk microflora on the characteristics
of Swiss-type cheeses. 2. Biochemical and sensory characteris-
tics. Lait 77:151–167
18. Bugaud C, Buchin S, Hauwuy A, Coulon JB (2001) Relation-
ships between flavour and chemical composition of Abon-
dance cheese derived from different types of pastures. Lait
81:757–773
19. Christensen JE, Dudley EG, Pederson JA, Steele JL (1999)
Peptidases and amino acid catabolism in lactic acid bacteria.
Anton Leeuw 76:217–246
20. Bouton Y, Grappin R (1995) Comparison of the final quality of
a Swiss-type cheese made from raw or microfiltered milk. Lait
75:31–44
Eur Food Res Technol (2017) 243:1943–1955 1955
21. Fornasari ME, Rossetti L, Carminati D, Giraffa G (2006) Culti-
vability of Streptococcus thermophilus in Grana Padano cheese
whey starters. FEMS Microbiol Lett 257:139–144
22. Depouilly A, Dufrene F, Beuvier E, Berthier F (2004) Genotypic
characterisation of the dynamics of the lactic acid bacterial pop-
ulation of Comte cheese. Lait. 84:155–167
23. Grappin R, Beuvier E, Bouton Y, Pochet S (1999) Advances in
the biochemistry and microbiology of Swiss-type cheeses. Lait
79:3–22
24. Carcano M, Todesco R, Lodi R, Brasca M (1995) Propionibacte-
ria in Italian hard cheeses. Lait 75:415–426
25. Nocker A, Camper AK (2009) Novel approaches toward prefer-
ential detection of viable cells using nucleic acid amplification
techniques. FEMS Microbiol Lett 291:137–142
26. Bosset JO, Collomb M, Sieber R (1993) The aroma composition
of Swiss Gruyère Cheese IV. The acidic volatile components and
their changes in content during ripening. Lebensm Wiss Technol
26:581–592
27. Bosset JO, Liardon R (1984) The aroma composition of Swiss
Gruyère Cheese. II. The neutral volatile components. Lebensm
Wiss Technol 17:359–362
28. Thierry A, Maillard MB, Le Quéré JL (1999) Dynamic head-
space analysis of Emmental aqueous phase as a method to quan-
tify changes in volatile flavour compounds during ripening. Int
Dairy J 9:453–463
29. Berdague JL, Jeunet R, Grappin R, Duboz G (1987) Ripening
and quality of Gruyère of Comté cheese III. Influence of the
ripening conditions on lactic acid and fermentation and on the
amount of volatile fatty acids (Affinage et qualité du Gruyère de
Comté III. Fermentation lactique et teneur en acides gras volatils
des fromages de Comté). Lait 67:249–263
30. Ganesan B, Dobrowolski P, Weimer BC (2006) Identification of
the Leucine-to-2-Methylbutyric Acid Catabolic Pathway of Lac-
tococcus lactis. Appl Environ Microb 72:4264–4273
31. Dherbécourt J, Maillard MB, Catheline D, Thierry A (2008)
Production of branched-chain aroma compounds by: links with
the biosynthesis of membrane fatty acids. J Appl Microbiol
105:977–985
32. Falentin H, Henaff N, Bivic P, Deutsch S, Parayre S, Richoux R,
Sohier D, Thierry A, Lortal S, Postollec F (2012) Reverse tran-
scription quantitative PCR revealed persistency of thermophilic
lactic acid bacteria metabolic activity until the end of the ripen-
ing of Emmental cheese. Food Microbiol 29:132–140
33. Ganesan B, Stuart MR, Weimer BC (2007) Carbohydrate starva-
tion causes a metabolically active but nonculturable state in Lac-
tococcus lactis. Appl Environ Microb 73:2498–2512
34. Sgarbi E, Bottari B, Gatti M, Neviani E (2014) Investigation of
the ability of dairy nonstarter lactic acid bacteria to grow using
cell lysates of other lactic acid bacteria as the exclusive source of
nutrients. Int J Dairy Technol 67:342–347
35. Richoux R, Faivre E, Kerjean JR (1998) Effect of NaCl content
on lactate fermentation by Propionibacterium freudenreichii in
small scale Swiss-type cheeses. Lait 78:319–331
36. Thierry A, Salvat-Brunaud D, Maubois J (1999) Influence
of thermophilic lactic acid bacteria strains on propionibacte-
ria growth and lactate consumption in an Emmental juice-like
medium. J Dairy Res 66:105–113
37. Ganesan B, Weimer B (2007) Amino acid metabolism in rela-
tionship to cheese flavor development. In: Weimer BC (ed)
Improving the flavour of cheese. CRC Press, Woodhead Publish-
ing Limited, Cambridge, pp 70–101
38. Smit G, Smit BA, Engels WJM (2005) Flavour formation by
lactic acid bacteria and biochemical flavour profiling of cheese
products. FEMS Microbiol Rev 29:591–610
39. Helinck S, Le Bars D, Moreau D, Yvon M (2004) Ability of ther-
mophilic lactic acid bacteria to produce aroma compounds from
amino acids. Appl Environ Microb 70:3855–3861
40. Williams AG, Noble J, Banks JM (2001) Catabolism of amino
acids by lactic acid bacteria isolated from Cheddar cheese. Int
Dairy J 11:203–215
41. Gobbetti M, De Angelis M, Di Cagno R, Mancini L, Fox PF
(2015) Pros and cons for using non-starter lactic acid bacte-
ria (NSLAB) as secondary/adjunct starters for cheese ripening.
Trends Food Sci Tech 45:167–178
42. Williams AG, Withers SE, Brechany EY, Banks JM (2006)
Glutamate dehydrogenase activity in lactobacilli and the use of
glutamate dehydrogenase-producing adjunct Lactobacillus spp.
cultures in the manufacture of Cheddar cheese. J Appl Microbiol
101:1062–1075
43. Amarita F, Requena T, Taborda G, Amigo L, Pelaez C (2001)
Lactobacillus casei and Lactobacillus plantarum initiate
catabolism of methionine by transamination. J Appl Microbiol
90:971–978
44. Aubel D, Germond JE, Gilbert C, Atlan D (2002) Beta-Cysta-
thionase activity within the Lactobacillus delbrueckii specium.
Sci Aliments 22:161–166
45. Imhof R, Glaettli H, Bosset JO (1995) Volatile organic com-
pounds produced by thermophilic and mesophilic single strain
dairy starter cultures. Lebensm Wiss Technol 28:78–86
46. Keenan TW, Lindsay RC (1967) Dehydrogenase activity of Lac-
tobacillus species. J Dairy Sci 50:1585–1588
47. Ott A, Germond J, Chaintreau A (2000) Origin of acetaldehyde
during milk fermentation using 13C-labeled precursors. J Agric
Food Chem 48:1512–1517
48. Zourari A, Accolas J, Desmazeaud M (1992) Metabolism and
biochemical characteristics of yogurt bacteria. A review. Lait
72:1–34
49. Keenan TW, Bills DD (1968) Volatile compounds produced by
Propionibacterium shermanii. J Dairy Sci 51:797–799
50. Liu SQ, Holland R, Crow VL (2004) Esters and their biosynthe-
sis in fermented dairy products: a review. Int Dairy J 14:923–945
51. El Soda M, Abd El Wahab H, Ezzat N, Desmazeaud MJ, Ismail
A (1986) The esterolytic and lipolytic activities of lactobacilli.
II. Detection of the esterase system of Lactobacillus helveticus,
Lactobacillus bulgaricus, Lactobacillus lactis and Lactobacillus
acidophilus. Lait 66:431–443
52. Oliszewski R, Medina RB, Gonzalez SN, Perez Chaia AB (2007)
Esterase activities of indigenous lactic acid bacteria from Argen-
tinean goats’ milk and cheeses. Food Chem 101:1446–1450
53. Urbach G (1993) Relations between cheese flavour and chemical
composition. Int Dairy J 3:389–422
13