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Fig. 2. Dendrogram of 16S rDNA sequences of Zymomonas mobilis pomaceae AN 0101, Zymomonas mobilis
mobilis ATCC 10988, Zymomonas mobilis PROIMI A1, Zymomonas mobilis pomaceae ATCC 29192, Zymo-
monas mobilis mobilis ATCC 29191, Zymomonas mobilis ZM4 and Sphingomonas sp. JRL-5 obtained by us-
ing the Pearson correlation on Bionumerics (Applied Maths, Belgium).
clear correlation was observed between the “framboisé”
and total and volatile acidity or polyphenol content.
The evolution of the acetaldehyde concentration, as a
marker of “framboisé”, and the populations of acetic acid
bacteria and other bacteria which grew on ZPP, were
plotted to look for correlations (Fig. 1). In cider A, acet-
aldehyde was stable, and acetic acid bacteria were present
at 103 CFU/mL on day 0 and stabilized at 104 CFU/mL at
day 6. No bacteria grew on ZPP medium. In cider B, the
acetaldehyde concentration was 350 mg/L on day 13.
Acetic acid bacteria increased from 103 CFU/mL on day
0 to 105 CFU/mL on day 6, and remained stable. Bacteria
which grew on ZPP medium were detected at around 103
CFU/mL on day 1 and reached 2.107 CFU/mL on day 9.
Then the population decreased rapidly and was no longer
detectable after day 16. In cider C, the evolution was simi-
lar to cider A.
These results showed a strong correlation between clas-
sical symptoms of “framboisé” (acetaldehyde formation,
decrease in sugar content, increase of ethanol content, high
pressure and persistent foam) and the presence of bacteria
growing on ZPP media. Sensory evaluation of the ciders
during the experiment confirmed the “framboisé” charac-
ter of cider B, while no alteration in flavor was detected in
ciders A and C. The one log difference of acetic acid bac-
teria populations observed between ciders B and C was
probably due to the addition of SO2 in the latter. The re-
sults of this experiment suggest that the bacteria which
grew under anaerobic conditions on ZPP medium were
able to form ethanol, CO2 and acetaldehyde from sugar,
and thus could be responsible for the “framboisé” spoil-
age in French ciders.
weak assimilation of 5-ceto-gluconate were observed. To
better identify the bacteria, the gene corresponding to the
16S rDNA was obtained after a PCR experiment and then
sequenced. The 1436 bp long sequence (Genbank acces-
sion number: AY350737) was compared to international
sequences in databases and showed 98% identities with
the sequences of 5 strains of Zymomonas mobilis, namely
ZM4 (AF117351), PROIMI A1 (AF088897), ATCC 10988
(AF281033), ATCC 29191 (AF281034) and ATCC 29192
(AF28281032), and only 92% with Sphingomonas sp.
JRL-5 (AF1851572) (Fig. 2). Taxonomically, Zymomonas
mobilis has been divided into two subspecies Zymomonas
mobilis mobilis and Zymomonas mobilis pomaceae17. Z.
mobilis mobilis has been isolated from bees, from ripening
honey in Spain, from fermenting sap of Agave americana
in Mexico, from fermenting palm juice (Arenga pinnata)
in Java and Indonesia, and from Elaeis guineensis and
Raphia vinifera in Zaire and Nigeria17. It has also been
isolated from spoiled beers in England5. Z. mobilis poma-
ceae was isolated in England from sick cider3,14 and apple
pulp4. No differences in the 16S rDNA sequences were
observed between Zymomonas mobilis mobilis phenotypic
centrotype strain ATCC 29191 and Zymomonas mobilis
pomaceae type strain ATCC 29192, while 20 differences
were observed between the Z. mobilis AN 0101 sequence
and the sequences of the previously mentioned strains. To
identify the strain at the subspecies level, two tests
(growth in presence of 0.5% NaCl and growth at 36°C), as
described by Swing and De Ley17, were used. According
to the authors, strains of Z. mobilis mobilis can grow in
the presence of salt and at 36°C while Z. mobilis poma-
ceae cannot. Strain AN 0101 showed no growth after 48 h
in liquid medium in the presence of 0.5% NaCl and at
36°C, while the type strain of Z. mobilis pomaceae ATCC
29192 and the reference strain of Z. mobilis mobilis ATCC
29191 grew in the presence of 0.5% NaCl and at 36°C.
These results combined with the DNA sequence of the
reference strain Z. mobilis pomaceae ATCC 29192 brought
into question how to differentiate the two subspecies and
the validity of the identification of this strain as a Z. mo-
bilis pomaceae. The strain isolated during this study was
designated as Zymomonas mobilis pomaceae AN 0101.
Identification of bacteria
The bacteria isolated on ZPP, coded AN 0101, appeared
as typical colonies on this media; round, white, glossy, 0.5
to 2 mm diameter, with a small peak in the middle. The
colonies resembled an anaerobic Gram negative large ba-
cillus. The catalase test was positive and the oxidase test
was negative. An API gallery 50CHE was employed to
investigate the metabolism of AN 0101, and after 48 h,
only a strong assimilation of glucose and fructose and a
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JOURNAL OF THE INSTITUTE OF BREWING