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Journal of Food Engineering 79 (2007) 1374–1382

Survival and preservation after freeze-drying processof thermoresistant acetic acid bacteria isolated from

tropical products of Subsaharan Africa

Bassirou Ndoye a,b,*, Frederic Weekers c, Brehima Diawara d,Amadou Tidiane Guiro b, Philippe Thonart a,c

a University of Gembloux, Faculty of Agronomic Sciences, Bioindustry Unit, 2 Passage des Deportes, B-5030 Gembloux, Belgiumb Dakar Institute of Food Technology, Route des Peres Maristes, BP 2765 Dakar, Senegal

c University of Liege, Wallon Centre of Industrial Biology, Sart-Tilman, B-40, B-4000 Liege, Belgiumd 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 2006Available online 6 May 2006

Abstract

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 watercooling expenses. For optimising biomass preservation, the effect of 20% w/w mannitol as cryoprotectant on the cells viability afterfreeze-drying process and during storage was evaluated. Results showed that freeze-dried cells could be conserved at 4 �C for at least6 months without loss of viability. The main reasons were that cryoprotectant tends to lower the water activity (aw) and to maintaina temperature of product weaker than that of the glass transition temperature Tg. Furthermore, the heat resistance of freeze-dried cellsduring storage was all the more increased that strains were cryoprotected. In addition, intrinsically, an increase of saturated fatty acidswith the temperature is the essential modification in the lipidome level of membrane cells when the fermentation occured at a temperatureof 30 �C. Tolerance to heat during storage was significantly enhanced under such mechanisms.� 2006 Elsevier Ltd. All rights reserved.

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

1. Introduction

Acetic acid bacteria (AAB) are Gram-negative strictlyaerobic bacteria and commonly found in nature on variousplants (fruits, cereals, herbs, etc.) (De Ley, Gillis, & Swings,1984). They have an ability to oxidize different kinds ofalcohols and sugars into commercially important foodsand chemical products like vinegar, cellulose, sorbose, glu-conic acid, etc. (Deppenmeir, Hoffmeister, & Prust, 2002).

0260-8774/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jfoodeng.2006.04.036

* Corresponding author. Present address: University of Gembloux,Faculty of Agronomic Sciences, Bioindustry Unit, 2 Passage des Deportes,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).

Particularly, depending on the strains, they produce a finalacetic acid concentration of up 17% to be achieved in mod-ern submerged fermentation processes as so called acetators(Sokollek & Hammes, 1997; Sokollek, Hertel, & Hammes,1998). Vinegar is an aqueous solution frequently used inAfrican regions as food condiment or conservator. Conse-quently, AAB cause an important industrial interest as wellas lactic acid bacteria and yeast. However, problems relatedto environmental conditions such as temperature variationsand process technology limit the industrial applications inSubsaharan regions. Recently, we have isolated and suitedtwo thermoresistant AAB from over-producing crops suchas mangos (Mangifera indica L.) and cereals, respectively inSenegal and Burkina Faso (Subsaharan Africa), highly

B. Ndoye et al. / Journal of Food Engineering 79 (2007) 1374–1382 1375

appropriate for a sustainable development of vinegar fer-mentation (Ndoye et al., in press In: Enzyme and MicrobiolTechnology). However, transport and conservation of theliquid inoculum do not fit with the artisanal production ofvinegar 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 asstarters for an optimum acetification process as well as theirpreservation via a dehydration process. Such studies havewidely described the microbiological conditions to obtaina high level of biomass cells as much as possible and to revi-talize these starters for a new acetification process as quicklyas possible. However, the YPM medium (Yeast, Polypep-tone, Mannitol) reinforced with acetic acid and ethanol(YPM-RAE) and used by these authors for the productionof biomass was not suitable for TAAB strains. Colouredvinegar was reproducibly obtained, in a reproducible way,a coloured vinegar at the end of the acetification processprobably due to the presence of the polypeptone. Further-more, the determination of the microbiologically survivalduration of foods and ferments became a new tool to assesstheir best stability after desiccation (Cerf, 2005; Uyttendaeleet al., 2004).

These terms were the principal objectives of this commu-nication, to investigate:

(i) freeze-drying process of thermoresistant strains as apotential method for preserving them with regard tothe wild type strain;

(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 phasetransition temperature in the stability conditions ofproducts.

(iv) The influence of membrane fatty acid composition oncells viability after freeze-drying process.

2. Materials and methods

2.1. Cell cultivation

A mesophilic AAB Acetobacter cerevisae LMG 1625T

(Cleenwerck, Vandemeulebroecke, Janssens, & Swings,2002) was provided by the Laboratory of Microbiologyat the University of Ghent (Belgium). CWBI-B418T andCWBI-B419T strains are thermoresistant strains isolatedfrom mango and cereal, respectively in Senegal and Burk-ina Faso (Subsaharan regions). They were inoculated with500 ll of seed culture into a 5 L embossed flasks containing1 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) wereincubated on a rotary shaker with agitation of 130 rpm

at 28 �C for 24 h. When cells have reached the exponentialphase (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 forthe mesophilic strain and 35 �C for the thermoresistantstrains within 4 days corresponding of the total consump-tion of glucose. Cells were then harvested by centrifugationfor 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 cellpaste from each batch was resuspended into 20% w/w man-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 pastebefore freezing and freeze-drying. After a freezing processfor 3 h at �20 �C, cells have been submitted to dehydrationat 15–20 �C for 23 h.

Freeze-dried cells, with and without cryoprotectant(control), were preserved under vacuum packaging at4 �C and safe from moisture, oxygen and light.

2.3. Dry cell weight (DCW) determination

The dry cell weight of the samples was determined basedon weight loss after drying at 105 �C. Either 1 g of cellpaste 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 themeasure of the dewpoint, detected on a mirror subjected toa series of cycle of heating and cooling. The scale of mea-surement lies between 0.100 and 1.000. The precision ofmeasurements is of ±0.003. The apparatus is gauged witha distilled water solution (aw = 1.000 ± 0.003).

The glass transition temperatures Tg were determinedusing Differential Scanning Calorimeter (DSC) at a heatingrate 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 ontoa 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 ethanol2.5%, glacial acetic acid 0.5%, KOH 14 ml after steriliza-tion. Freeze-dried powders (1 g) were rehydrated directlyin peptonized water by mixing thoroughly with a vortexmixer for 10 min and spreaded onto the surface of YGMagar 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 grammeor ml (CFU/g ou ml) and comparing them with the viabil-ity of the controls, which were samples that had beenfreeze-dried without cryoprotectant.

2.6. Stability of freeze-dried cells

The Residual Viability (%) of the samples was measuredthe same day as each experiment from 1 to 180 days (6months). The freeze-dried samples were stored at 4 �Cand the cell viability N was determined as above. ResidualViability (%) 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, 35and 40 �C for 8 days. After each 24 h, samples were takenout, placed in ice water and spreaded onto a modifiedYGM agar. They are incubated at 30 �C within 4 days tofavorise visible colonies. Survival (%) was obtained usingthe following equation: Ln (% survival) = Ln(100) � k · t, where k is the thermal inactivation rate con-stant and t is the heating time (Lievense, Verbeek, Meerd-ink, & Van’t Riet, 1990). Percent survival was calculated as100 · N/N0, where N is the CFU/ml of the heated sampleand N0 is the CFU/ml of the unheated sample. A survivorcurve (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 andD value was given by To and Etzel (1997).

2.8. Fatty acids methyl esters (FAME) analysis

Total lipids extraction was performed according to astandard protocol (Zelles, 1996). Briefly, the lipid fraction

Fig. 1. Viability of cells freeze-dried and conserved at 4 �C. Cells have been sa(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 esterswere prepared by incubating the lipids extracts at 80 �C for4 h in 2 ml of methanol, containing 15% (v/v) of KOH. Thefatty acids were extracted with chloroform and analysed bygas chromatography on a HP 6890 (Hewlett Pachard,Waldbronn, Germany) equipped with a flame-fused silicacapillary column. The conditions were as follows: injectortemperature, 260 �C; detector temperature, 260 �C; carriergas (helium) flow rate, 3 ml/min. The oven temperaturewas programmed from 140 �C during 5 min to 240 �C at4 �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 aremeans three independent experiments. The ratio betweenthe standard deviations and the means values was between2% and 5%.

3. Results

3.1. Effects of the freeze-drying process

Freeze-drying process was used to investigate the effectsof dehydration on cell viability and stability. The use of20% mannitol as cryoprotectant, added to the fresh pastebefore freezing and freeze-drying, has been evaluated toperform the efficiency of the desiccation process and com-pared to cells submitted to thermal stress without cryopro-tectant. The following figure (Fig. 1) showed thedehydration 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% mannitolas cryoprotectant and conserved at 4 �C showed a biomasscell always higher than 1 · 1010 CFU/g of powder for 6months of conservation whereas that the wild strain bio-mass did not. However, this strain maintains its growth

fe from oxygen and moisture. (A) Cells freeze-dried without cryprotectant.

Table 1Determination 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 ± 2.32CWBI-B418T 0.135 ± 0.0017 0.176 ± 0.0019 85.36 ± 4.02 81.10 ± 5.33CWBI-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).Data are presented as the average of two independent trials with SD.

B. Ndoye et al. / Journal of Food Engineering 79 (2007) 1374–1382 1377

potential to at least 1 · 109 CFU/g of powder necessary forany starter preparation (Sokollek & Hammes, 1997). Theabsence of cryoprotectant before freeze-drying decreasedthe cellular viability of the strains. But thermoresistantstrains maintain nevertheless a viability around of1 · 109 CFU/g of powder. This faculty of resistance tothe desiccation could be allotted to a weaker value of wateractivity after freeze-drying as well as a maintenance of tem-perature lower than that of the glass transition temperatureTg (Table 1). Indeed, the cryoprotectant tends to lower thewater activity (aw) and to maintain a temperature ofproduct weaker than that of the glass transition tempera-ture Tg.

The aw indeed expresses the availability of water in theproducts and then its aptitude to take part in the reactionsas solvent or reagent. It is then necessary to quantify thiswater activity of which the degradation speed of the prod-ucts strongly depends (Schuck et al., 2004). The productswere preserved better for aw egal to 0.2 (Le Meste, Lorient,& Simatos, 2002). In the same time, a variation in the tem-perature of the product around of the glass transition tem-perature Tg is accompanied by a strong modification of themechanical properties of the molecules of the product.Roos (1993) suggested that certain physicochemical andstructural processes are better correlated to the glass tran-sition temperature through plasticization by water or tem-perature. The overriding mechanism of deteriorativeprocesses including stickiness, crispness, collapse, amor-phous-to-crystalline transformations is the molecularmobility that relates directly to Tg (Le Meste & Simatos,1990; Sablani, Kasapis, & Rahman, in press).

1

10

100

1 15 30 60 90 120 150 180

Time (day)1 15 30 60

Time

Res

idua

l via

bilit

y %

Cp

W/o Cp

A

1

10

100

B

Fig. 2. Stability of freeze-dried powder, conserved at 4 �C and safe from oxygewith cryoprotectant; W/o for control.

Then, associated to the operating conditions, aw and Tg

could help to formulate, choose and adapt the dryingprocess.

3.2. Stability at 4 �C and dry cell weight of freeze dried cells

Survival of freeze-dried LMG 1625T (control strain),CWBI-B418T and CWBI-B419T strains was higher afterfreeze-drying process (Fig. 2). Without cryoprotectant, sta-bility of freeze-dried powder decreased quickly with a sur-vival value around of 10% (<1 · 105 powder CFU/g in thecase of the control strain) in 6 months of conservation. Thecryoprotectant maintains a residual viability of the cellsfrom almost 50 ± 1% after 6 months of conservation at+4 �C with the absence of oxygen and moisture by stabiliz-ing the dry cell weight equal to or higher than 95%(Table 2).

The viability is all the more significant as the strain isthermoresistant. The strain was indeed stable at 4 �C for34

of the year if 20% mannitol is used as cryoprotectant.

3.3. Thermal inactivation rate constants

The thermal inactivation rate k of the freeze-driedstrains were given by incubating them at the increasingtemperatures of 30, 35 and 40 �C during 8 days of incuba-tion (Fig. 3).

This parameter makes it possible to show the resistanceof the strains submitted to the thermal stress conditionsduring conservation or storage. For all the freeze-driedstrains, thermal inactivation followed first order kinetics.

90 120 150 180

(day)1 15 30 60 90 120 150 180

Time (day)

1

10

100

C

Cp

W/o Cp Cp

W/o Cp

n and moisture. (A) LMG 1625T. (B) CWBI-B418T. (C) CWBI-B419T. Cp,

Table 2Dry 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 ± 0.8515 days 98 ± 0.15 97 ± 0.54 98 ± 0.6 97 ± 0.65 98 ± 0.9 98 ± 0.751 month 97 ± 0.85 97 ± 0.35 98 ± 0.45 97 ± 0.54 98 ± 0.85 98 ± 0.122 months 97 ± 0.76 97 ± 0.12 98 ± 0.05 97 ± 0.17 98 ± 0.8 97 ± 0.953 months 97 ± 0.36 96 ± 0.58 97 ± 0.29 96 ± 0.12 98 ± 0.7 97 ± 0.64 months 97 ± 0.06 96 ± 0.03 97 ± 0.10 95 ± 0.98 98 ± 0.25 97 ± 0.155 months 96 ± 0.87 95 ± 0.78 96 ± 0.98 95 ± 0.75 97 ± 0.85 96 ± 0.886 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.

1378 B. Ndoye et al. / Journal of Food Engineering 79 (2007) 1374–1382

Increasing the temperature increased k (Table 2). However,the addition of cryoprotectant as 20% mannitol decreasedthe thermal inactivation constant and, then, increased theheat resistance of the strain (Table 3).

Fig. 4A inset shows the decrease of D values with thetemperature. Fig. 3A plots the logarithm of D valuesagainst temperature to calculate Z values (temperature

1 2 4 6 81

10

100

Time (day)

Res

idua

l via

bilit

y %

30 ºC35 ºC 40 ºC

A

1

10

100

1 2 4 6

Time (day)

Res

idua

l via

bilit

y %

30 ºC35 ºC40 ºC

C

1

10

100

1 2 4 6

Time (day)

Res

idua

l via

bilit

y %

30 ºC35 ºC40 ºC

E

Fig. 3. Survival in function of time at different temperatures for freeze-driedcerevisae LMG 1625T freeze-dried without cryoprotectant (A) or with cryopro(C) or without cryoprotectant (D). Survival of CWBI-B419T freeze-dried with

increase needed to reduce D value by 90%) from the slopeof the curve. Z values were higher for the cryoprotectedproducts, and more thermoresistant the strain was, moreimportant Z value was. The activation energy (EA) values(Table 4) were found from an Arrhenius plot (Fig. 4B).The activation energy (EA) was determined from Arrheniusequation: k ¼ Ae�EA=RT where k is the rate constant (day�1),

1

10

100

1 2 4 6 8

Time (day)

30 ºC35 ºC40 ºC

D

1

10

100

1 2 4 6 8

Time (day)

30 ºC35 ºC40 ºC

B

1

10

100

1 2 4 6 8

Time (day)

30 ºC35 ºC40 ºC

F

strains stored under vacuum and protected from moisture. Survival of A.

tectant (B). Survival of CWBI-B418T freeze-dried without cryoprotectantout cryoprotectant (E) or with cryoprotectant (F).

Table 3Temperature and cryoprotectant effects on thermal inactivation constant k

of freeze-dried cells

Strains Control With cryoprotectant

Temp. (�C) k (day�1) Temp. (�C) k (day�1)

LMG 1625T 30 0.0495 30 0.027935 0.0813 35 0.057840 0.2340 40 0.1235

CWBI-B418T 30 0.0165 30 0.0060335 0.0283 35 0.0105440 0.0654 40 0.0364

CWBI-B419T 30 0.0109 30 0.004935 0.0276 35 0.015240 0.0627 40 0.0325

Note that experiment was not a quantitative method.

0.001

0.01

0.1

1

3.18 3.2 3.22 3.24 3.26 3.28 3.3

1/T (K-1 x 103)

The

rmal

inac

tiva

tion

con

stan

t (d

ay-1

)

LMG 1625 (W/o) LMG 1625/Cp CWBI-B419 (W/o)

CWBI-B419/Cp CWBI-B418 (W/o) CWBI-B418/Cp

0

0.5

1

1.5

2

2.5

3

25 30 35 40 45

Temperature ºC

Log

D v

alue

(s)

CWBI-B418 (W/o) CWBI-B418/CpCWBI-B419 (W/o) CWBI-B419/CpLMG 1625 (W/o) LMG 1625 /Cp

Z = 7.02 ºC

Z = 5.94 ºC

Z = 7.47 ºCZ = 5.4 ºC

Z = 6.75 ºC

Z = 5.67 ºC

0

100200

300400

500

25 30 35 40 45Temperature ºC

D v

alue

(s) A

B

Fig. 4. Heat inactivation of freeze-dried strains with cryoprotectant (Cp)and without cryoprotectant (W/o Cp). A. Determination of Z values(temperature increase needed to reduce D values by 90%) from Fig. A insetwhich represents D values as function of heating temperature. B.Arrhenius plot for the heat inactivation of freeze-dried strains.

Table 4Activation energy (EA) values of freeze-dried strains found from theArrhenius plot

Strains Activation energy (EA) (kcal mole�1)

Control With cryoprotectant

LMG 1625T 64.19 33.23CWBI-B418T 16.91 10.55CWBI-B419T 18.00 9.58

Note that experiment was not a quantitative method.

B. Ndoye et al. / Journal of Food Engineering 79 (2007) 1374–1382 1379

A is the pre-exponential time (day�1), R is the ideal gasconstant and T is the absolute temperature (K). EA repre-sents the sensitivity of the phenomenon temperaturevariations.

Activation energy values were higher at the wild-typestrain LMG 1625T, characterizing a thermal property ofsensitivity whereas the very low values of the EA of thestrains CWBI-B418T and CWBI-B419T specify their rela-tive resistance in the case of thermal stress.

3.4. Effects of heat stress on fatty acid composition

Many authors reported that fatty acids composition wassuggested to be a good tool for measuring the stress state ofbacterial cells. Thermal stress could improve perturbationsin the lipidome level of membrane cells. These changes inlipid composition enable the microorganisms to maintainmembrane function in the face of environmental fluctua-tions (Joyeux, Fouchard, Llopiz, & Neunlist, 2004; Sow,Dubois-Dauphin, Roblain, Guiro, & Thonart, 2005). Thefollowing table (Table 5) showed results recently obtainedfor elucidating thermoresistant properties of CWBI-B418T and CWBI-B419T strains (Ndoye et al., in pressIn: Enzyme and Microbiol Technology).

The major fatty acids found from AAB strains wasmethyl oleate (C18:1) (Table 6). All strains exhibited alsomethyl meristate (C14:0), methyl palmitate (C16:0), methylstearate (C18:0), methyl linoleate (C18:2), methyl linole-nate (C18:3). An increased temperature of growth

Table 5Biochemical 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.

Table 6Total 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 ± 0.13C14:0 1.95 ± 0.028 5.99 ± 0.25 4.65 ± 0.22 11.46 ± 0.86 2.64 ± 0.15 5.55 ± 0.21C15:0 1.36 ± 0.06 0.77 ± 0.03C16:0 9.5 ± 0.18 17.71 ± 0.34 5.02 13.75 ± 0.23 2.69 ± 0.37 13.22 ± 0.71C16:1 2.91 ± 0.07 5.15 ± 0.2 13.38 ± 0.14 2.45 ± 0.16 5.25 ± 0.19 3.05 ± 0.17C17:0 4.37 ± 0.31 1.12 ± 0.03C18:0 3.39 ± 0.04 5.44 ± 0.31 9.67 ± 0.14 6.33 ± 0.09 5.65 ± 0.35C18:1 70.49 ± 0.33 60.1 ± 0.75 62.43 ± 0.61 46.13 ± 0.84 69.66 ± 0.69 60.64 ± 0.98C18:2 0.85 ± 0.09 2.91 ± 0.19C18:3 1.03 ± 0.02 7.24 ± 0.41 9.03 ± 0.01 3.32 ± 0.32 11.87 ± 0.27 9.99 ± 0.65C20:0 10.72 ± 0.6 1.64 ± 0.05C20:1 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(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 werefreeze-dried without cryoprotectant and fatty acids were extracted and analysed by CPG as described in Section 2.

* 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 quantitativemethod.

Fig. 5. Total cellular fatty acids evaluated by the % of pics areas inrelation with growth temperature.

1380 B. Ndoye et al. / Journal of Food Engineering 79 (2007) 1374–1382

improved saturated fatty acids (C12:0, C14:0, C16:0,C17:0, C18:0, C20:0) synthesis (Fig. 5) and a decrease ofunsaturated fatty acids (mono and poly) mainly C18:1.The unsaturated/saturated ratio decreased from low tohigh temperature mainly with thermoresistant strainsCWBI-B418T and CWBI-B419T. These results wererecently found from Frateuria laurantia (DSMZ 6220), anacetic acid bacteria isolated from acidic soils (Joyeuxet al., 2004). In contrast, the SFA synthesis decreased inthe wild strain A. cerevisae LMG 1625T at higher tempera-ture and PUFA synthesis was observed. This fact couldexplain why null growth and a loss of acetification abilitywere observed at 40 �C in this strain (Table 5) due to theabsence of thermal and acidic tolerance. The loss ofthermal and acidic tolerance was attributed to the PUFA

synthesis more sensitive to heat and acidic stress (Joyeuxet al., 2004).

4. Discussion and conclusions

A fermentation process was designed for high AABbiomass production. The cells could be freeze-dried andpreserved with good viability and stability to be used as star-ter cultures for new acetification process. In the course ofresearch, no more studies related to preservation of AABwere done. The use of YGM medium (without polypeptone)allowed us to obtain a very high viable biomass cells and aclear vinegar without coloration. The cultivated and washedcells could be frozen and freeze-dried for preservation.Before freezing, 20% mannitol as cryoprotectant was addedto the paste to prevent any damage during dehydration as tomaintain viability and stability of the cells. Results haveshown that the cryoprotectant was determinant to enhancethe viability of the cells during storage. Le Meste, Roudaut,Simatos, and Colas (2001) have shown that, due to its similarstructure with water, cryoprotectant take place of the watermolecules 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 presenceof oxygen and moisture at 4 �C. However, some dehy-drated bacteria stored in an environment with low oxygenand moisture content improved storage stability (Heckly &Quay, 1983; Samelis, Kakouri, & Rementzis, 2000).

B. Ndoye et al. / Journal of Food Engineering 79 (2007) 1374–1382 1381

Stability of the cells during storage was enhanced whenwater activity aw was around 0.2. This parameter was verynecessary to be quantified due to its relation to the speed ofdegradation of foods or ferments (Schuck et al., 2004).Furthermore, the water activity (aw) strongly influencedthe physical properties extrinsic of the powders (glass tran-sition temperature Tg). The glass transition temperaturewas 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 wasgreater by taking into account of all physical intrinsic andextrinsic parameters (Carvalho, Silva, Teixeira, Malcata,& Gibbs, 2002; Miyamoto-Shinohara et al., 2000). How-ever, thermal inactivation after down stream process couldbe 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 temperatureis a key component. Thermal inactivation rate constant k

was measured vs. temperature. Increasing the temperaturealways increased k. But, k was all the more lower that thestrain is thermoresistant and cryoprotected. The presentof 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 inactivationthrough such mechanisms. The values of activation energyEA found from an Arrhenius plot showed that the wild typestrain was more heat sensitive than the thermoresistant oneand consequently less stable during preservation.

The beneficial effect of heat stress during storage of Ace-

tobacter strains can be attributed to the saturated fattyacids synthesis (Suutari & Laasko, 1994; Ziadi, Touhami,Achour, Thonart, & Hamdi, 2005). Basically, for thehigher temperature, unsaturated fatty acids concentrationswere significantly lower than for lower temperatures. Sucheffects were interpreted in several cases as the consequenceof the regulation of the membrane fluidity by a modifica-tion of the fatty acids structures. It is noteworthy that onlya temperature increase leads to an appreciable effect on thelipid composition whereas a temperature decrease leads toless obvious effects (Joyeux et al., 2004; Sow et al., 2005).

In addition, the physiological and biochemical responsesof the microorganisms to all occasional thermal elevationare universal and well known. A relationship between theinduction 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 inthe surrounding environment. GroEL and GroES operons,which are members of the HSP60 and HSP10 protein fam-ilies found from Acetobacter genus, respectively, are amongthe most extensively investigated stress proteins for aceticacid bacteria (Akiko et al., 2002). These proteins aremembers of the molecular chaperone family, commonlyknown as chaperonins and are known to be induced notonly by temperature shifts, but also by other stresses such

as exposure to ethanol. These HSP can act as biologicalcryoprotectants.

Elucidating the mechanisms underlaying the resistanceof acetic acid bacteria to stressors is, therefore, importantfor 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 describedabove, it will be possible to preserve AAB by over a yearwithout loss of viability.

For a commercial processing view point, a phylogeneticidentification of these thermoresistant strains is necessaryto determine their taxonomic position. A polyphasicapproach including phenotypic and genotypic characteris-tics will be our future investigation.

Acknowledgements

This research was supported by the International Foun-dation for Science (IFS), Sweden, via a grant programmeawarded to B. Ndoye (grant No. E/3595-1). It was sup-ported from the beginning by the partnership between theWalloon region (Belgium) and Senegal (DRI contractNo. 27/11/2003-134-S). The authors gratefully acknowl-edge Ir. Aldric Jean Marc for technical assistance on Fattyacids 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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