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 Journal of Food Engineering 79 (2007) 1374–1382
www.elsevier.com/locate/jfoodeng

Survival and preservation after freeze-drying process

py
of thermoresistant acetic acid bacteria isolated from
tropical products of Subsaharan Africa

co
Bassirou Ndoye a,b,*, Frédéric Weekers c, Bréhima Diawara d,
Amadou Tidiane Guiro b, Philippe Thonart a,c
a
University of Gembloux, Faculty of Agronomic Sciences, Bioindustry Unit, 2 Passage des Déportés, B-5030 Gembloux, Belgium
b
Dakar Institute of Food Technology, Route des Pères Maristes, BP 2765 Dakar, Senegal
c
University of Liege, Wallon Centre of Industrial Biology, Sart-Tilman, B-40, B-4000 Liege, Belgium
d

al
Institute of Research in Applied Sciences and Technologies, CNRST, Department of Food Technology, 03, BP 7182 Ouagadougou, Burkina Faso

Received 1 December 2005; accepted 12 April 2006

Available online 6 May 2006
on
Abstract
rs

Two thermoresistant acetic acid bacteria (TAAB) were previously isolated and selected for a sustainable development of vinegar fer-
mentation in Subsaharan Africa. Their use as a starter culture in vinegar manufactures in such regions could reduce considerably water
cooling expenses. For optimising biomass preservation, the effect of 20% w/w mannitol as cryoprotectant on the cells viability after
freeze-drying process and during storage was evaluated. Results showed that freeze-dried cells could be conserved at 4 C for at least
pe

6 months without loss of viability. The main reasons were that cryoprotectant tends to lower the water activity (aw) and to maintain
a temperature of product weaker than that of the glass transition temperature Tg. Furthermore, the heat resistance of freeze-dried cells
during storage was all the more increased that strains were cryoprotected. In addition, intrinsically, an increase of saturated fatty acids
with the temperature is the essential modification in the lipidome level of membrane cells when the fermentation occured at a temperature
of 30 C. Tolerance to heat during storage was significantly enhanced under such mechanisms.
 2006 Elsevier Ltd. All rights reserved.
r's

Keywords: Thermoresistant acetic acid bacteria; Freeze-drying; Starter culture; Cryoprotectant; Survival; Heat stress; Fatty acids composition; Vinegar
fermentation
o

1. Introduction Particularly, depending on the strains, they produce a final

acetic acid concentration of up 17% to be achieved in mod-
th

Acetic acid bacteria (AAB) are Gram-negative strictly ern submerged fermentation processes as so called acetators
aerobic bacteria and commonly found in nature on various (Sokollek & Hammes, 1997; Sokollek, Hertel, & Hammes,
plants (fruits, cereals, herbs, etc.) (De Ley, Gillis, & Swings, 1998). Vinegar is an aqueous solution frequently used in
Au

1984). They have an ability to oxidize different kinds of
alcohols and sugars into commercially important foods
and chemical products like vinegar, cellulose, sorbose, glu-
conic acid, etc. (Deppenmeir, Hoffmeister, & Prust, 2002).
*
Corresponding author. Present address: University of Gembloux,
Faculty of Agronomic Sciences, Bioindustry Unit, 2 Passage des Déportés,
B-5030 Gembloux, Belgium. Tel.: +32 81 62 23 05; fax: +32 81 61 42 22.
E-mail address: ndoye.b@fsagx.ac.be (B. Ndoye).
African regions as food condiment or conservator. Conse-
quently, AAB cause an important industrial interest as well
as lactic acid bacteria and yeast. However, problems related
to environmental conditions such as temperature variations
and process technology limit the industrial applications in
Subsaharan regions. Recently, we have isolated and suited
two thermoresistant AAB from over-producing crops such
as mangos (Mangifera indica L.) and cereals, respectively in
Senegal and Burkina Faso (Subsaharan Africa), highly

0260-8774/$ - see front matter  2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2006.04.036
 B. Ndoye et al. / Journal of Food Engineering 79 (2007) 1374–1382
appropriate for a sustainable development of vinegar fer-
mentation (Ndoye et al., in press In: Enzyme and Microbiol
Technology). However, transport and conservation of the
liquid inoculum do not fit with the artisanal production of
vinegar in these regions. To overcome these problems,
Sokollek and Hammes (1997) and Sokollek et al. (1998) per-
formed for the first and only time the cultivation of AAB as
starters for an optimum acetification process as well as their
preservation via a dehydration process. Such studies have
widely described the microbiological conditions to obtain
a high level of biomass cells as much as possible and to revi-
talize these starters for a new acetification process as quickly
as possible. However, the YPM medium (Yeast, Polypep-
tone, Mannitol) reinforced with acetic acid and ethanol
(YPM-RAE) and used by these authors for the production
of biomass was not suitable for TAAB strains. Coloured
vinegar was reproducibly obtained, in a reproducible way,
a coloured vinegar at the end of the acetification process
probably due to the presence of the polypeptone. Further-
more, the determination of the microbiologically survival
duration of foods and ferments became a new tool to assess
their best stability after desiccation (Cerf, 2005; Uyttendaele
on
et al., 2004).
These terms were the principal objectives of this commu-
nication, to investigate:
rs
(i) freeze-drying process of thermoresistant strains as a
potential method for preserving them with regard to
the wild type strain;
pe
(ii) survival of strains by performing the stability and via-
bility of freeze-dried starters;
(iii) thermal inactivation kinetics of the dehydrated prod-
ucts and the impact of water activity and gel phase
transition temperature in the stability conditions of
products.
(iv) The influence of membrane fatty acid composition on
r's
cells viability after freeze-drying process.
2. Materials and methods
o
2.1. Cell cultivation
th
A mesophilic AAB Acetobacter cerevisae LMG 1625T
(Cleenwerck, Vandemeulebroecke, Janssens, & Swings,
Au
2002) was provided by the Laboratory of Microbiology
at the University of Ghent (Belgium). CWBI-B418T and
CWBI-B419T strains are thermoresistant strains isolated
from mango and cereal, respectively in Senegal and Burk-
ina Faso (Subsaharan regions). They were inoculated with
500 ll of seed culture into a 5 L embossed flasks containing
1 L of autoclaved (121 C for 20 min) YGM/Mg2+ medium
(yeast extract 10 g/l, glucose 20 g/l, mannitol 20 g/l,
MgSO4 1 g/l, citrate trisodique 1 g/l, NH4HPO4 1 g/l,
KH2PO4 1 g/l, ethanol 2.5% and glacial acetic acid 0.5%
after sterilization). Flasks (double for each strain) were
incubated on a rotary shaker with agitation of 130 rpm
1375
at 28 C for 24 h. When cells have reached the exponential
phase (until 1.2 and 1.5 of OD unit), they were then culti-
vated by batch process on a sterile pilot-fermentor of 20 L
(stainless steel Biolafitte, France) with YGM/Mg2+ med-
ium without ethanol nor glacial acetic acid at 30 C for
the mesophilic strain and 35 C for the thermoresistant
strains within 4 days corresponding of the total consump-
tion of glucose. Cells were then harvested by centrifugation
py
for 10 min at 9600g at 4 C, washed with a potassium phos-
phate buffer (KPB) 50 mM (pH 5.5–6) followed by centri-
fugation. The cell paste was stored at 4 C. Fresh cell
paste from each batch was resuspended into 20% w/w man-
co
nitol as cryoprotectant and frozen overnight at 80 C.
2.2. Freeze-drying process
To determine the effect of dehydration on viability, 20%
w/w mannitol as cryoprotectant was added to the cell paste
before freezing and freeze-drying. After a freezing process
al
for 3 h at 20 C, cells have been submitted to dehydration
at 15–20 C for 23 h.
Freeze-dried cells, with and without cryoprotectant
(control), were preserved under vacuum packaging at
4 C and safe from moisture, oxygen and light.
2.3. Dry cell weight (DCW) determination
The dry cell weight of the samples was determined based
on weight loss after drying at 105 C. Either 1 g of cell
paste or 1 g of powder was placed in an aluminium weigh-
ing boat and dried in a convection oven at 105 C for 24 h.
2.4. Water activity (aw) and glass transition temperature
(Tg)
aw were measured in a standard hygrometer Aqua Lab.
The principle of the measurement technique is based on the
measure of the dewpoint, detected on a mirror subjected to
a series of cycle of heating and cooling. The scale of mea-
surement lies between 0.100 and 1.000. The precision of
measurements is of ±0.003. The apparatus is gauged with
a distilled water solution (aw = 1.000 ± 0.003).
The glass transition temperatures Tg were determined
using Differential Scanning Calorimeter (DSC) at a heating
rate of 10 C/min. The estimated error was ±5 C. The Tg
was obtained using the slope intercept method.
2.5. Cell viability
Viable cells or countable colonies were enumerated onto
a modified YGM agar containing yeast extract 5 g/l, pep-
tone of soja 5 g/l, Glucose 20 g/l, agar 17 g/l and ethanol
2.5%, glacial acetic acid 0.5%, KOH 14 ml after steriliza-
tion. Freeze-dried powders (1 g) were rehydrated directly
in peptonized water by mixing thoroughly with a vortex
mixer for 10 min and spreaded onto the surface of YGM
agar after appropriate decimal dilutions. Colonies were
1376 B. Ndoye et al. / Journal of Food Engineering 79 (2007) 1374–1382
visible within 3 or 4 days at 28 C. Cell viability of the sam-
ples N0 was expressed as colonies forming unit per gramme
or ml (CFU/g ou ml) and comparing them with the viabil-
ity of the controls, which were samples that had been
freeze-dried without cryoprotectant.
2.6. Stability of freeze-dried cells
The Residual Viability (%) of the samples was measured
the same day as each experiment from 1 to 180 days (6
months). The freeze-dried samples were stored at 4 C
and the cell viability N was determined as above. Residual
Viability (%) was expressed as follow: RV (%) = 100 ·
N/N0 where N0 is the initial cell viability of the sample,
N is the cell viability at time t.
2.7. Thermal inactivation rate constant
Freeze-dried products (1 g) were filled into a vial (10 ml)
and incubated the same time in convection oven at 30, 35
and 40 C for 8 days. After each 24 h, samples were taken
out, placed in ice water and spreaded onto a modified
on
YGM agar. They are incubated at 30 C within 4 days to
favorise visible colonies. Survival (%) was obtained using
the following equation: Ln (% survival) = Ln
(100)  k · t, where k is the thermal inactivation rate con-
rs
stant and t is the heating time (Lievense, Verbeek, Meerd-
ink, & Van’t Riet, 1990). Percent survival was calculated as
100 · N/N0, where N is the CFU/ml of the heated sample
pe
and N0 is the CFU/ml of the unheated sample. A survivor
curve (log (N) vs. t) has a slope equal to k that gives rela-
tionship between temperature and the residual viable cell.
An exact mathematical model relationship between k and
D value was given by To and Etzel (1997).
2.8. Fatty acids methyl esters (FAME) analysis
r's
Total lipids extraction was performed according to a
standard protocol (Zelles, 1996). Briefly, the lipid fraction
o
th
Au
Fig. 1. Viability of cells freeze-dried and conserved at 4 C. Cells have been safe from oxygen and moisture. (A) Cells freeze-dried without cryprotectant.
(B) Cells freeze-dried with cryoprotectant.
was extracted from powdered freeze-dried cells (500 mg)
according to the adaptated method of White, Davis,
Nickels, King, and Bobbie (1979). Fatty acids methyl esters
were prepared by incubating the lipids extracts at 80 C for
4 h in 2 ml of methanol, containing 15% (v/v) of KOH. The
fatty acids were extracted with chloroform and analysed by
gas chromatography on a HP 6890 (Hewlett Pachard,
Waldbronn, Germany) equipped with a flame-fused silica
py
capillary column. The conditions were as follows: injector
temperature, 260 C; detector temperature, 260 C; carrier
gas (helium) flow rate, 3 ml/min. The oven temperature
was programmed from 140 C during 5 min to 240 C at
co
4 C/min. For peak identification, standard solution
(sigma) was used.
The results were relative percentages of fatty acids,
determined from peak areas of methyl esters. They are
means three independent experiments. The ratio between
the standard deviations and the means values was between
2% and 5%.
al
3. Results
3.1. Effects of the freeze-drying process
Freeze-drying process was used to investigate the effects
of dehydration on cell viability and stability. The use of
20% mannitol as cryoprotectant, added to the fresh paste
before freezing and freeze-drying, has been evaluated to
perform the efficiency of the desiccation process and com-
pared to cells submitted to thermal stress without cryopro-
tectant. The following figure (Fig. 1) showed the
dehydration effects of the cells for 6 months of conserva-
tion at 4 C under vacuum packaging and safe from mois-
ture, oxygen and light.
Thermoresistant strains freeze-dried with 20% mannitol
as cryoprotectant and conserved at 4 C showed a biomass
cell always higher than 1 · 1010 CFU/g of powder for 6
months of conservation whereas that the wild strain bio-
mass did not. However, this strain maintains its growth
 B. Ndoye et al. / Journal of Food Engineering 79 (2007) 1374–1382 1377

Table 1
Determination of the water activity aw and the glass transition temperature Tg of powder after freeze-drying
Strains Water activity aw (±0.001) Tg (±5)
WC Control WC Control
LMG 1625T 0.174 ± 0.001 0.200 ± 0.0011 87.68 ± 2.71 85.69 ± 2.32
CWBI-B418T 0.135 ± 0.0017 0.176 ± 0.0019 85.36 ± 4.02 81.10 ± 5.33
CWBI-B419T 0.168 ± 0.0013 0.201 ± 0.0009 86.06 ± 3.11 85.18 ± 2.71
The glass transition temperature was measured by DSC (Differential Scanning Calorimetry). WC, with cryoprotectant; C, control (without cryopotectant).

py
Data are presented as the average of two independent trials with SD.

potential to at least 1 · 109 CFU/g of powder necessary for Then, associated to the operating conditions, aw and Tg
any starter preparation (Sokollek & Hammes, 1997). The could help to formulate, choose and adapt the drying

co
absence of cryoprotectant before freeze-drying decreased process.
the cellular viability of the strains. But thermoresistant
strains maintain nevertheless a viability around of 3.2. Stability at 4 C and dry cell weight of freeze dried cells
1 · 109 CFU/g of powder. This faculty of resistance to
the desiccation could be allotted to a weaker value of water Survival of freeze-dried LMG 1625T (control strain),
activity after freeze-drying as well as a maintenance of tem- CWBI-B418T and CWBI-B419T strains was higher after
perature lower than that of the glass transition temperature freeze-drying process (Fig. 2). Without cryoprotectant, sta-

al
Tg (Table 1). Indeed, the cryoprotectant tends to lower the bility of freeze-dried powder decreased quickly with a sur-
water activity (aw) and to maintain a temperature of vival value around of 10% (<1 · 105 powder CFU/g in the
product weaker than that of the glass transition tempera- case of the control strain) in 6 months of conservation. The
on
ture Tg. cryoprotectant maintains a residual viability of the cells
The aw indeed expresses the availability of water in the from almost 50 ± 1% after 6 months of conservation at
products and then its aptitude to take part in the reactions +4 C with the absence of oxygen and moisture by stabiliz-
as solvent or reagent. It is then necessary to quantify this ing the dry cell weight equal to or higher than 95%
rs

water activity of which the degradation speed of the prod- (Table 2).
ucts strongly depends (Schuck et al., 2004). The products The viability is all the more significant as the strain is
were preserved better for aw egal to 0.2 (Le Meste, Lorient, thermoresistant. The strain was indeed stable at 4 C for
pe

3
& Simatos, 2002). In the same time, a variation in the tem- 4
of the year if 20% mannitol is used as cryoprotectant.
perature of the product around of the glass transition tem-
perature Tg is accompanied by a strong modification of the 3.3. Thermal inactivation rate constants
mechanical properties of the molecules of the product.
Roos (1993) suggested that certain physicochemical and The thermal inactivation rate k of the freeze-dried
structural processes are better correlated to the glass tran- strains were given by incubating them at the increasing
sition temperature through plasticization by water or tem- temperatures of 30, 35 and 40 C during 8 days of incuba-
r's

perature. The overriding mechanism of deteriorative tion (Fig. 3).

processes including stickiness, crispness, collapse, amor- This parameter makes it possible to show the resistance
phous-to-crystalline transformations is the molecular of the strains submitted to the thermal stress conditions
mobility that relates directly to Tg (Le Meste & Simatos, during conservation or storage. For all the freeze-dried
o

1990; Sablani, Kasapis, & Rahman, in press). strains, thermal inactivation followed first order kinetics.
th

100 100 Cp 100

W/o Cp Cp
Cp
W/o Cp
W/o Cp
Residual viability %

Au

10
10 10

1
1 1
1 15 30 60 90 120 150 180 1 15 30 60 90 120 150 180
1 15 30 60 90 120 150 180
A Time (day) B Time (day) C Time (day)

Fig. 2. Stability of freeze-dried powder, conserved at 4 C and safe from oxygen and moisture. (A) LMG 1625T. (B) CWBI-B418T. (C) CWBI-B419T. Cp,
with cryoprotectant; W/o for control.
1378 B. Ndoye et al. / Journal of Food Engineering 79 (2007) 1374–1382

Table 2
Dry cell weight of freeze-dried cells, conserved at 4 C and safe from oxygen and moisture
Periods of conservation Strains
LMG 1625T CWBI-B418T CWBI-B419T
% Dry cell weight (±1%)
WC C WC C WC C
1 day 98 ± 0.25 97 ± 0.65 98 ± 0.75 97 ± 0.85 98 ± 0.95 98 ± 0.85
15 days 98 ± 0.15 97 ± 0.54 98 ± 0.6 97 ± 0.65 98 ± 0.9 98 ± 0.75

py
1 month 97 ± 0.85 97 ± 0.35 98 ± 0.45 97 ± 0.54 98 ± 0.85 98 ± 0.12
2 months 97 ± 0.76 97 ± 0.12 98 ± 0.05 97 ± 0.17 98 ± 0.8 97 ± 0.95
3 months 97 ± 0.36 96 ± 0.58 97 ± 0.29 96 ± 0.12 98 ± 0.7 97 ± 0.6
4 months 97 ± 0.06 96 ± 0.03 97 ± 0.10 95 ± 0.98 98 ± 0.25 97 ± 0.15
5 months 96 ± 0.87 95 ± 0.78 96 ± 0.98 95 ± 0.75 97 ± 0.85 96 ± 0.88

co
6 months 96 ± 0.23 95 ± 0.08 96 ± 0.75 95 ± 0.21 97 ± 0.05 96 ± 0.01
WC, with cryoprotectant; C, control (without cryoprotectant). Data were presented as the average of two independent trials with SD.

Increasing the temperature increased k (Table 2). However, increase needed to reduce D value by 90%) from the slope
the addition of cryoprotectant as 20% mannitol decreased of the curve. Z values were higher for the cryoprotected
the thermal inactivation constant and, then, increased the products, and more thermoresistant the strain was, more

al
heat resistance of the strain (Table 3). important Z value was. The activation energy (EA) values
Fig. 4A inset shows the decrease of D values with the (Table 4) were found from an Arrhenius plot (Fig. 4B).
temperature. Fig. 3A plots the logarithm of D values The activation energy (EA) was determined from Arrhenius
on
against temperature to calculate Z values (temperature equation: k ¼ AeEA =RT where k is the rate constant (day1),

100 100
30 ºC 30 ºC
rs

35 ºC 35 ºC
Residual viability %

40 ºC 40 ºC

10 10
pe

1 1
1 2 4 6 8 1 2 4 6 8

A Time (day) B Time (day)

100 100
30 ºC 30 ºC
r's

35 ºC
Residual viability %

35 ºC
40 ºC 40 ºC

10 10
o

1 1
th

1 2 4 6 1 2 4 6 8

C Time (day) D Time (day)

100 100
Au

30 ºC 30 ºC
35 ºC 35 ºC
Residual viability %

40 ºC
40 ºC

10 10

1 1
1 2 4 6 1 2 4 6 8

E Time (day) F Time (day)

Fig. 3. Survival in function of time at different temperatures for freeze-dried strains stored under vacuum and protected from moisture. Survival of A.
cerevisae LMG 1625T freeze-dried without cryoprotectant (A) or with cryoprotectant (B). Survival of CWBI-B418T freeze-dried without cryoprotectant
(C) or without cryoprotectant (D). Survival of CWBI-B419T freeze-dried without cryoprotectant (E) or with cryoprotectant (F).
 B. Ndoye et al. / Journal of Food Engineering 79 (2007) 1374–1382 1379

Table 3 3 CWBI-B418 (W/o) CWBI-B418/Cp

CWBI-B419 (W/o) CWBI-B419/Cp
Temperature and cryoprotectant effects on thermal inactivation constant k LMG 1625 (W/o)
LMG 1625 /Cp
of freeze-dried cells 2.5

Strains Control With cryoprotectant

Log D value (s)

2
Temp. (C) k (day1) Temp. (C) k (day1) Z = 7.02 ºC
Z = 7.47 ºC
T Z = 5.4 ºC
LMG 1625 30 0.0495 30 0.0279 1.5
Z = 6.75 ºC
35 0.0813 35 0.0578
500 Z = 5.94 ºC
40 0.2340 40 0.1235 A

D value(s)
1 400
300

py
Z = 5.67 ºC
CWBI-B418T 30 0.0165 30 0.00603 200
100
35 0.0283 35 0.01054 0.5 0
25 30 35 40 45
40 0.0654 40 0.0364 Temperature ºC
0
CWBI-B419T 30 0.0109 30 0.0049 25 30 35 40 45

co
35 0.0276 35 0.0152 Temperature ºC
40 0.0627 40 0.0325
Note that experiment was not a quantitative method. 1 LMG 1625 (W/o) LMG 1625/Cp CWBI-B419 (W/o) B
CWBI-B419/Cp CWBI-B418 (W/o) CWBI-B418/Cp

Thermal inactivation constant (day-1)

A is the pre-exponential time (day1), R is the ideal gas
constant and T is the absolute temperature (K). EA repre- 0.1
sents the sensitivity of the phenomenon temperature

al
variations.
Activation energy values were higher at the wild-type
strain LMG 1625T, characterizing a thermal property of
on
0.01
sensitivity whereas the very low values of the EA of the
strains CWBI-B418T and CWBI-B419T specify their rela-
tive resistance in the case of thermal stress.
rs

3.4. Effects of heat stress on fatty acid composition 0.001

3.18 3.2 3.22 3.24 3.26 3.28 3.3

1/T (K-1 x 103)

Many authors reported that fatty acids composition was
pe

suggested to be a good tool for measuring the stress state of Fig. 4. Heat inactivation of freeze-dried strains with cryoprotectant (Cp)
and without cryoprotectant (W/o Cp). A. Determination of Z values
bacterial cells. Thermal stress could improve perturbations
(temperature increase needed to reduce D values by 90%) from Fig. A inset
in the lipidome level of membrane cells. These changes in which represents D values as function of heating temperature. B.
lipid composition enable the microorganisms to maintain Arrhenius plot for the heat inactivation of freeze-dried strains.
membrane function in the face of environmental fluctua-
tions (Joyeux, Fouchard, Llopiz, & Neunlist, 2004; Sow,
Dubois-Dauphin, Roblain, Guiro, & Thonart, 2005). The
r's

following table (Table 5) showed results recently obtained Table 4

for elucidating thermoresistant properties of CWBI- Activation energy (EA) values of freeze-dried strains found from the
Arrhenius plot
B418T and CWBI-B419T strains (Ndoye et al., in press
In: Enzyme and Microbiol Technology). Strains Activation energy (EA) (kcal mole1)
o

The major fatty acids found from AAB strains was Control With cryoprotectant
methyl oleate (C18:1) (Table 6). All strains exhibited also LMG 1625T 64.19 33.23
th

methyl meristate (C14:0), methyl palmitate (C16:0), methyl CWBI-B418T 16.91 10.55
stearate (C18:0), methyl linoleate (C18:2), methyl linole- CWBI-B419T 18.00 9.58
nate (C18:3). An increased temperature of growth Note that experiment was not a quantitative method.
Au

Table 5
Biochemical characteristics (growth temperature, bioconversion including acidification and over-oxydation) of the strains A. cerevisae LMG 1625T,
CWBI-B418T and CWBI-B419T (5)
Strains Growth temp. (C) Acidification Over-oxydation
25 30 40 25 C 30 C 40 C 25 C 30 C 40 C
A.LMG 1625T ++ ++  + + + +
CWBI-B418T ++ ++ ++ + + + + + +
CWBI-B419T ++ ++ ++ + + + + + +
(+ +) very good growth; (+) good growth; () no growth; (A) acidification: (+) positive test; () negative test; (O) over-oxidation: (+) positive test; ()
negative test.
1380 B. Ndoye et al. / Journal of Food Engineering 79 (2007) 1374–1382

Table 6
Total fatty acids composition and modification induced by growth temperature
Fatty acids Strains
A. cerevisae LMG 1625T CWBI-B418T CWBI-B419T
30 C 40 C 30 C 40 C 30 C 40 C
C12:0 1.97 ± 0.03 1.78 ± 0.13
C14:0 1.95 ± 0.028 5.99 ± 0.25 4.65 ± 0.22 11.46 ± 0.86 2.64 ± 0.15 5.55 ± 0.21
C15:0 1.36 ± 0.06 0.77 ± 0.03

py
C16:0 9.5 ± 0.18 17.71 ± 0.34 5.02 13.75 ± 0.23 2.69 ± 0.37 13.22 ± 0.71
C16:1 2.91 ± 0.07 5.15 ± 0.2 13.38 ± 0.14 2.45 ± 0.16 5.25 ± 0.19 3.05 ± 0.17
C17:0 4.37 ± 0.31 1.12 ± 0.03
C18:0 3.39 ± 0.04 5.44 ± 0.31 9.67 ± 0.14 6.33 ± 0.09 5.65 ± 0.35
C18:1 70.49 ± 0.33 60.1 ± 0.75 62.43 ± 0.61 46.13 ± 0.84 69.66 ± 0.69 60.64 ± 0.98

co
C18:2 0.85 ± 0.09 2.91 ± 0.19
C18:3 1.03 ± 0.02 7.24 ± 0.41 9.03 ± 0.01 3.32 ± 0.32 11.87 ± 0.27 9.99 ± 0.65
C20:0 10.72 ± 0.6 1.64 ± 0.05
C20:1 0.99 ± 0.04
*
SFA% 25.56 25.67 15.11 44.04 11.66 26.31
*
MUFA% 73.40 65.25 75.38 48.58 74.91 63.69
*
PUFA% 1.03 8.09 9.03 6.23 11.87 9.99
*
M + P% 74.43 73.34 84.41 54.81 86.78 73.69

al
(M + P/SFA)% 2.91 2.85 5.58 1.24 7.44 2.80
Cells were cultivated by batch fermentation at increased temperatures from 25 to 40 C and harvested by centrifugation. After freezing for 24 h, cells were
freeze-dried without cryoprotectant and fatty acids were extracted and analysed by CPG as described in Section 2.
*
on
SFA, Saturated fatty acids; MUFA, Mono-unsaturated fatty acids; PUFA, Poly-unsaturated fatty acids; M + P, Mono-unsaturated fatty acid-
s + Poly-unsaturated fatty acids. Data were presented as the average of two independent trials with SD. Note that experiment was not a quantitative
method.
rs

synthesis more sensitive to heat and acidic stress (Joyeux

et al., 2004).
pe

4. Discussion and conclusions

A fermentation process was designed for high AAB

biomass production. The cells could be freeze-dried and
preserved with good viability and stability to be used as star-
ter cultures for new acetification process. In the course of
r's

research, no more studies related to preservation of AAB

were done. The use of YGM medium (without polypeptone)
Fig. 5. Total cellular fatty acids evaluated by the % of pics areas in allowed us to obtain a very high viable biomass cells and a
relation with growth temperature.
clear vinegar without coloration. The cultivated and washed
o

cells could be frozen and freeze-dried for preservation.

Before freezing, 20% mannitol as cryoprotectant was added
th

improved saturated fatty acids (C12:0, C14:0, C16:0, to the paste to prevent any damage during dehydration as to
C17:0, C18:0, C20:0) synthesis (Fig. 5) and a decrease of maintain viability and stability of the cells. Results have
unsaturated fatty acids (mono and poly) mainly C18:1. shown that the cryoprotectant was determinant to enhance
Au

The unsaturated/saturated ratio decreased from low to
high temperature mainly with thermoresistant strains
CWBI-B418T and CWBI-B419T. These results were
recently found from Frateuria laurantia (DSMZ 6220), an
acetic acid bacteria isolated from acidic soils (Joyeux
et al., 2004). In contrast, the SFA synthesis decreased in
the wild strain A. cerevisae LMG 1625T at higher tempera-
ture and PUFA synthesis was observed. This fact could
explain why null growth and a loss of acetification ability
were observed at 40 C in this strain (Table 5) due to the
absence of thermal and acidic tolerance. The loss of
thermal and acidic tolerance was attributed to the PUFA
the viability of the cells during storage. Le Meste, Roudaut,
Simatos, and Colas (2001) have shown that, due to its similar
structure with water, cryoprotectant take place of the water
molecules in its associations with proteins and limited fold-
ings up and torsions which would have involved to the dam-
age of the cells and the lost of their stability.
Many authors (To & Etzel, 1997) reported that freeze-
dried cells were very unstable when stored in the presence
of oxygen and moisture at 4 C. However, some dehy-
drated bacteria stored in an environment with low oxygen
and moisture content improved storage stability (Heckly &
Quay, 1983; Samelis, Kakouri, & Rementzis, 2000).
 B. Ndoye et al. / Journal of Food Engineering 79 (2007) 1374–1382
Stability of the cells during storage was enhanced when
water activity aw was around 0.2. This parameter was very
necessary to be quantified due to its relation to the speed of
degradation of foods or ferments (Schuck et al., 2004).
Furthermore, the water activity (aw) strongly influenced
the physical properties extrinsic of the powders (glass tran-
sition temperature Tg). The glass transition temperature
was characterized to predict the stability of frozen or des-
hydrated products (Roos, 1993; Sablani et al., in press).
Cells were highly resistant to freezing, thawing and dehy-
dration during freeze-drying process and survival was
greater by taking into account of all physical intrinsic and
extrinsic parameters (Carvalho, Silva, Teixeira, Malcata,
& Gibbs, 2002; Miyamoto-Shinohara et al., 2000). How-
ever, thermal inactivation after down stream process could
be the main reason for reduced cell viability (Carcoba &
Rodriguez, 2000; De Giulo et al., 2005). There is mathemat-
ical models in which thermal inactivation and temperature
is a key component. Thermal inactivation rate constant k
was measured vs. temperature. Increasing the temperature
always increased k. But, k was all the more lower that the
strain is thermoresistant and cryoprotected. The present
on
of cryoprotectant decreased water activity value. Conse-
quently, water may not be sufficiently available for the inac-
tivation reactions at low activity (To & Etzel, 1997). Thus,
dehydration may protect cells from thermal inactivation
rs
through such mechanisms. The values of activation energy
EA found from an Arrhenius plot showed that the wild type
strain was more heat sensitive than the thermoresistant one
pe
and consequently less stable during preservation.
The beneficial effect of heat stress during storage of Ace-
tobacter strains can be attributed to the saturated fatty
acids synthesis (Suutari & Laasko, 1994; Ziadi, Touhami,
Achour, Thonart, & Hamdi, 2005). Basically, for the
higher temperature, unsaturated fatty acids concentrations
were significantly lower than for lower temperatures. Such
r's
effects were interpreted in several cases as the consequence
of the regulation of the membrane fluidity by a modifica-
tion of the fatty acids structures. It is noteworthy that only
a temperature increase leads to an appreciable effect on the
o
lipid composition whereas a temperature decrease leads to
less obvious effects (Joyeux et al., 2004; Sow et al., 2005).
th
In addition, the physiological and biochemical responses
of the microorganisms to all occasional thermal elevation
are universal and well known. A relationship between the
Au
induction of stress proteins, such as ‘‘heat shock proteins’’
(HSP) to various stressors has been described (Parsell &
Lindquist, 1993). These proteins have been believed to pro-
tect cells from various stresses that result from changes in
the surrounding environment. GroEL and GroES operons,
which are members of the HSP60 and HSP10 protein fam-
ilies found from Acetobacter genus, respectively, are among
the most extensively investigated stress proteins for acetic
acid bacteria (Akiko et al., 2002). These proteins are
members of the molecular chaperone family, commonly
known as chaperonins and are known to be induced not
only by temperature shifts, but also by other stresses such
1381
as exposure to ethanol. These HSP can act as biological
cryoprotectants.
Elucidating the mechanisms underlaying the resistance
of acetic acid bacteria to stressors is, therefore, important
for understanding the mechanisms of vinegar production,
and will be helpful in developing strains with more sophis-
ticated acetic acid fermentation abilities.
By taking into account of all the parameters described
py
above, it will be possible to preserve AAB by over a year
without loss of viability.
For a commercial processing view point, a phylogenetic
identification of these thermoresistant strains is necessary
co
to determine their taxonomic position. A polyphasic
approach including phenotypic and genotypic characteris-
tics will be our future investigation.
Acknowledgements
This research was supported by the International Foun-
al
dation for Science (IFS), Sweden, via a grant programme
awarded to B. Ndoye (grant No. E/3595-1). It was sup-
ported from the beginning by the partnership between the
Walloon region (Belgium) and Senegal (DRI contract
No. 27/11/2003-134-S). The authors gratefully acknowl-
edge Ir. Aldric Jean Marc for technical assistance on Fatty
acids analysis, Prof. Jerome. R at the Laboratory of Mac-
romolecular Chemistry and Organics Materials of the Uni-
versity of Liege for assistance in DSC analysis, Jourdan E.
for his help during preparation of manuscript and Ir. Delv-
igne Frank for critical reading of final manuscript.
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