- Open Access
The role of protein modifications in senescence of freeze-dried Acetobacter senegalensis during storage
© Shafiei et al.; licensee BioMed Central Ltd. 2014
- Received: 7 October 2013
- Accepted: 7 February 2014
- Published: 19 February 2014
Loss of viability is one of the most important problems during starter culture production. Previous research has mostly focused on the production process of bacterial starters, but there are few studies about cellular protein deterioration causing cell defectiveness during storage. In the present study, we investigated the influence of storage temperature (−21, 4, 35°C) on the cellular protein modifications which may contribute to the senescence of freeze-dried Acetobacter senegalensis.
Heterogeneous populations composed of culturable cells, viable but non-culturable cells (VBNC) and dead cells were generated when freeze-dried cells were kept at −21 and 4°C for 12 months whereas higher storage temperature (35°C) mainly caused death of the cells. The analysis of stored cell proteome by 2D-DiGE demonstrated a modified pattern of protein profile for cell kept at 4 and 35°C due to the formation of protein spot trains and shift of Isoelectric point (pI). Quantification of carbonylated protein by ELISA showed that the cells stored at 4 and 35°C had higher carbonylated protein contents than fresh cells. 2D-DiGE followed by Western blotting also confirmed the carbonylation of cellular proteins involved in translation process and energy generation. The auto-fluorescent feature of cells kept at 35°C increased significantly which may be an indication of protein glycation during storage. In addition, the percentage of cellular unsaturated fatty acid and the solubility of cellular proteins decreased upon storage of cells at higher temperature suggesting that peroxidation of fatty acids and possibly protein lipidation and oxidation occurred.
High storage temperature induces some deteriorative reactions such as protein oxidation, lipidation and glycation which may cause further protein modifications like pI-shift, and protein insolubility. These modifications can partly account for the changes in cell viability. It can also be deduced that even moderate carbonylation of some critical cellular proteins (like ribosomal proteins) may lead to VBNC formation or death of freeze-dried bacteria. Moreover, it seems that other mechanisms of biomolecule deterioration preceding protein carbonylation lead to VBNC formation under very low storage temperature.
- Acetic acid fermentation
- Oxidative stress
A starter culture may be defined as a preparation containing large numbers of viable and culturable microorganisms, which may accelerate a favorable fermentation process . Vinegar technology is one of those fermentation technologies which still suffer from the lack of cost-effective starter cultures  due to the genetic instability of acetic acid bacteria during preservation  and the susceptibility of cells to downstream processes [4, 5].
The viable cells which are used as vinegar starter must be able to tolerate acetic acid and metabolize ethanol efficiently under aggressive conditions of acetic acid fermentation . The availability of appropriate vinegar starter cultures is desirable when a new fermentation run has to be started or when it needs to be restarted due to a sudden interruption of fermentation . In recent years, Acetobacter senegalensis, a thermo-tolerant bacterium, has been used for starter production and vinegar production in a pilot plant scale acetifier [4, 7]. Use of this strain in vinegar industry can decrease the cooling cost of bioreactors especially in tropical regions. In addition, since this strain can remain viable and active in a wide range of temperature during acetous fermentation , any fluctuation of fermentation temperature can be tolerated readily.
There are different industrial techniques to preserve microbial starters; however despite being an expensive and long process, freeze-drying is one of the most convenient and applicable methods compared to other drying methods . However, as with other drying methods, loss of viability is one of the main problems during freezing and drying processes as well as storage period .
Intrinsic and environmental factors have considerable influence on the viability of microbial starters . Generally, the survival rate of gram-positive bacteria immediately after freeze-drying tends to be higher than that of gram-negative bacteria . In addition, pre-adaptation of cells in different culture media, usage of protectants and storage conditions affect bacterial viability significantly [5, 11]. In this context, there are evidences that damage to DNA, cell wall and cell membrane of bacterial cells under different storage conditions occur [12, 13], and it is now well accepted that membrane lipid peroxidation and change in the degree of un-saturated lipids strongly affect the survival of bacteria [7, 9, 13]. Furthermore, damage to ribosomes and their functions are speculated as the primary reasons of cell viability loss .
Cellular proteins are also subjected to deteriorating reactions during downstream processes. They are very susceptible to various modifications due to the range of functional groups displayed by amino acids . Damage to proteins such as oxidation during preservation process and storage period has been studied in animal cells, plant cells, seeds as well as in therapeutic products [15–19]. Oxidative changes of cellular proteins can lead to diverse detrimental consequences in structure and function of proteins such as inhibition of enzymatic activities, polymerization, loss of solubility, increased susceptibility to aggregation and proteolysis [14, 20].
Although several protein oxidative modifications exist, most oxidized proteins exhibit carbonyl groups (aldehydes and ketones) . However, compared to other oxidative modifications, carbonylation is relatively difficult to be induced and in contrast to, for example, methionine sulfoxide and cysteine disulfide bond formation, carbonylation is an irreversible oxidative process [21, 22]. Carbonyl groups are introduced into the proteins through a variety of oxidative pathways: (I) a decline in the antioxidant defense system, (II) an increased production of reactive oxygen species (ROS), (III) a diminished capacity for removal of oxidized proteins, or (IV) an increased susceptibility of proteins to oxidative attack . In addition, carbonyl derivatives are formed by a direct metal catalyzed oxidative (MCO) attack on the amino-acid side chains of proline, arginine, lysine, and threonine. Furthermore, carbonyl derivatives on lysine, cysteine, and histidine can be formed by secondary reactions with reactive carbonyl compounds on carbohydrates, lipids, and Advanced Glycation/lipoxidation End Products (AGEs) .
Advanced Glycation End products (AGEs) and AGE pigments (known also as lipofuscin) are created through non-enzymatic reactions (the Maillard reaction) between reducing sugars and free amino groups of proteins, lipids, or nucleic acids. These biomolecules contain transition metals such as iron, zinc, manganese and copper. These metals can cause a redox-active surface that can catalyze reactive releasing process . AGEs alter the structure of proteins and compromise their functions. The rate of accumulation of AGEs in proteins may be viewed as an index of the rate of damage to other biomolecules, including lipids, glycol-conjugates and DNA . A decreased enzymatic activity through oxidative reaction of proteins and Maillard reaction products has been demonstrated in plant seeds during storage [26, 27]. However, there are few studies which address the protein modifications in bacterial cells during desiccation .
Despite the progress made so far in the context of bacterial starter production, to our knowledge there are only few studies on the cellular protein modifications occurring during storage of bacterial starters. The aim of this study was to investigate the influences of storage temperature on cellular viability and senescence of freeze-dried A. senegalensis. Specifically, the occurrence of modifications in protein content and properties affecting the survival of stored bacteria was studied. In this regard, three storage temperatures (−21, 4, and 35°C) were chosen and freeze-dried cells were stored for 12 months in sealed vials at the three mentioned temperatures. Then, the oxidative damage and its subsequent effects on some cellular protein properties were analyzed. In addition, the consequence of protein oxidation on some other cellular features such as AGEs formation or cell respiration was studied.
Heterogeneous populations are formed during storage of freeze-dried cells
Prolonged stability of freeze-dried starters is crucial for fermentation industry. Studies from our laboratory and others have shown that culturability and the viability of freeze-dried starters are dependent upon freeze-drying process as well as storage conditions [4, 9, 11, 13, 28–30]. We have also previously shown that storage temperature affect the viability and culturability of freeze-dried starter, and that even at low storage temperature (−21, 4°C), cells enter into VBNC state .
Culturable cells and moisture content of freeze-dried cells kept at various temperatures for 12 months
After freeze-drying process
Direct count (cfu/g)
5.96E + 11
6.01E + 11
6.21E + 11
5.89E + 11
cfu/g on GY agar
1.90E + 11
1.11E + 11
1.03E + 10
<E + 2
Dry matter (%) 1
91.60 ± 1.64
91.7 ± 2.14
92.53 ± 1.18
93.29 ± 2.34
Storing of freeze-dried cells at 4°C for 12 months resulted in a heterogeneous population of bacteria according to CTC reduction ability (Figure 1B). This may indicate that some cellular enzymes involved in respiration system such as dehydrogenases were subjected to detrimental conditions which disabled the respiration system. However, about 23% of cells were able to carry out that reaction completely, resulting in high absorbance on FL3. It has been already shown that dehydrogenases are especially heat sensitive . In our previous study, it was shown that storage of freeze-dried A. senegalensis at 4°C for nine months did not change the cell envelope integrity considerably , therefore the changes in dehydrogenase activity during storage at 4°C may not be due to the leakage of cellular components or entrance of liquids into the cells. In contrast, it could be due to direct inactivation of enzymes.
Formation of fluorescent compounds is enhanced during storage at high temperature
Recently, the evaluation of fluorescent compounds generated by the Amadori rearrangement product has become a routine practice. Besides its use in food quality control, fluorescence measurement is also employed to evaluate Maillard reactions causing AGEs generation under physiological conditions, and also to assess AGEs development under pathological conditions . The results obtained in this study are in agreements with those of Kurtmann and coworkers. They showed that browning of freeze-dried Lactobacillus acidophilus during storage under relatively mild conditions resulted in various types of non-enzymatic browning reactions including carbonyl-protein (or carbonyl-DNA) interactions and carbohydrate condensation/polymerization . Furthermore, in our study, since the cells were not washed prior to freeze-drying (to avoid possible damage to the cells by low osmotic buffers) [4, 5], residual reducing sugar (glucose) and proteins or even DNA or RNA released by cell lysis during fermentation or freeze-drying can enhance the non-enzymatic browning reactions .
It is believed that formulation strategies during freeze-drying process have great influence on the stability and viability of bacteria. Addition of compounds containing carbonyl groups has been suggested to be a cause of mortality occurring during storage of dried microorganisms .
Form these results, it can be deduced that some temperature-dependent reactions induce the formation of fluorescent compounds in the stored cells. These fluorescent compounds can be a sign of protein glycation.
The proteome is markedly affected in freeze-dried cells stored at high temperature
To study the molecular mechanisms associated with the loss of viability and culturability during storage, a differential proteomic analysis approach was used. First, we determined the distribution pattern of the cellular proteome by two-dimensional difference gel electrophoresis (2D-DiGE) for cell kept at different temperatures. In the second part, the extent of protein carbonylation was studied using ELISA test and 2D-DiGE followed by immunoblotting (Western blotting).
Some cellular proteins modifications of freeze-dried A. senegalensis stored at 35°C for 12 months
Modified amino acid and site of carbonylation
Score/number of peptides/Percentage of coverage
Shift of pI
LSU ribosomal protein L17P
Shift of pI to about 4.5-5
LSU ribosomal protein L21P
Shift of’ pI to about 4.5-5
LSU ribosomal protein L22P
Shift of’ pI to about 4.5-5
Translation elongation factor Tu
Translation elongation factor G
Pro: 651, 681, 683
Translation elongation factor G
Pro: 681 and 683
SSU ribosomal protein S1
Pyruvate phosphate dikinase
Proline carbonylation Threonine carbonylation Arginine carbonylation
Pro: 758, 623, 670
Thr: 290 Arg:579
Threonine carbonylation Lysine carbonylation
Lys: 86, 236
ATP synthase F1 beta subunit
Proline carbonylation Arginine carbonylation
Pro: 238, 359, 364
Arg: 68, 289
ATP synthase F1 alpha subunit
Pro: 460, 464
Carbonylation of proteins increases massively during storage
By comparing the results of ELISA and western blotting, it was found that the results of Western blotting for samples kept at −21 and 35°C are in accordance with the results of ELISA test. In contrast, a contradiction was observed for cells stored at 4°C. As already explained, a moderate increase in the amount of carbonylated proteins was observed by ELISA method for these cells, however no carbonylated protein were detected using 2D-DiGE followed by western blotting. It is believed that the latter method has significantly more sensitivity and specificity than all other total carbonyl assays such as spectrophotometric DNPH assay and ELISA method . It should however be noted that there is a detection limit for oxidized proteins by immune-blotting methods, and oxidized proteins in low quantity cannot be detected easily . Therefore, it can be assumed that some proteins may undergo very slight carbonylation which is not detectable by these methods. In addition, as already discussed, some other protein oxidation reactions may occur prior to carbonylation which may cause VBNC formation in cells. In order to identify the proteins that were oxidized when cells were kept at 35°C, 88 protein spots were selected and picked from preparative gel maps that had been matched to the 2-4-DNP-derivatized protein spots maps and subjected to identification by MSMS analysis. Proteins were identified in only 13 of the 88 selected spots from Figure 10 of which 10 contained carbonylated amino acids (Table 2). This low number technically results from the limited coverage of sequence (generally between 15 and 50%) that was achieved allowing only a small number of peptides to be analyzed. Thus, the lack of detection of carbonylated residues by MSMS analysis does not necessarily means that the identified proteins were not carbonylated. Lysine, Arginine, Threonine and Proline were identified as target amino acids where carbonylation occurred. Both structural proteins and enzymes were found among the carbonylated proteins.
Our results are considerably consistent with the studies showing the susceptibility of cell envelope protein and translational proteins to oxidative stress [47–50]. It has already been shown that exposure of E. coli to different stress conditions such as H2O2, iron overloading or super oxide generating compounds causes carbonylation of Elongation factor G (EF-G), outer membrane protein A (ompA), β-subunit of F0F1- ATPase, heat shock proteins and enolase. In addition, following such oxidative stress, the viability was affected to different extents depending on the type of exerted stress . One group of carbonylated proteins identified in this study indeed belongs to the cell translation machine (Figure 10 and Table 2). SSU ribosomal protein S1 presumed function is to participate in the initiation of translation, possibly by binding mRNA and directing it to the ribosome . There are also evidences that heat stress causes important irreversible reactions in the cell ribosomes; however more experimental evidence is still needed to determine whether or not ribosome is the critical component responsible for the thermal death of microorganisms [11, 52, 53]. Thus, the obtained results can be used as an indication and also confirmation that during cell death at high storage temperature, ribosomal proteins are oxidized.
Other proteins involved in translation process which were also carbonylated during storage of cells at 35°C are Elongation factor G (EF-G) and Elongation factor TU (EF-TU). In the protein synthesis process by ribosome, loading of tRNA into the A site of 50S subunit is assisted by the elongation factor EF-Tu. Elongation factor G (EF-G) is involved in translocation of the tRNA holding the polypeptide . Recently, it has been shown that the latter protein is very susceptible to oxidation during oxidative stress in E. coli. Under in vitro conditions, treatment of EF-G with H2O2 resulted in a complete loss of activity due to oxidation of cysteine residues . In our previous study on storage of freeze-dried A. senegalensis, we have shown that cell envelope integrity was subjected to changes during freeze-drying process and storage at 35°C . Those findings are consistent with the observations of the present study and some other studies stating that essential targets on which survival depends during ROS stress include membrane lipid integrity and ROS-susceptible proteins, including proteins required for faithful translation of mRNA .
Another group of oxidized proteins identified in this study is involved in cell energy generation. The α and β subunits of F1 ATP synthase were detected as heavily carbonylated proteins (Table 2). ATPases consist of two components, a multiprotein cytoplasmic complex called F1 that carries out the chemical function (ATP synthesis), connected to a membrane-integrated component called F0 that carries out the ion-translocating function . As already mentioned, the respiration system of cells kept at 4 and 35°C was affected during storage (Figure 1). In addition, in our previous study on stored freeze-dried A. senegalensis, we observed that cellular respiration was impaired during storage at high temperature , thus the results of the present study suggest that respiration disability of these cells may be due to defective ATP synthase involved in respiration chain.
In our previous study on storage of freeze-dried A. senegalensis, we have also shown that the cell envelope integrity was subjected to changes during freeze-drying process and storage at 35°C . Those findings are consistent with the observations of the present study and some other studies stating essential targets on which survival depends during ROS stress include membrane lipid integrity and ROS-susceptible proteins, including proteins required for faithful translation of mRNA . According to the obtained results from the ELISA test and immunoblotting, with regard to the results of viability and culturability (Figure 2), it can be inferred that carbonylation of proteins is not the main reason for the entrance of bacteria to VBNC at low storage temperature. Thus, it can be assumed that other deleterious reactions preceding carbonylation can lead to the death or VBNC formation at low temperatures.
Solubility of cellular proteins is reduced during storage
Soluble protein contents (mg/g dried cells) of cells kept at different storage temperatures for 12 months
9.54 ± 1.41
11.05 ± 3.33
11.14 ± 2.98
74.1 ± 6.15
66.67 ± 4.05
58.75 ± 6.38
0.74 ± 0.43
1.26 ± 0.21
0.44 ± 0.18
0.39 ± 0.13
0.24 ± 0.12
42.49 ± 0.71
41.99 ± 3.29
34.99 ± 5.65
One direct consequence of oxidative damage (such as carbonylation) to proteins is a change in protein solubility. High molecular-weight aggregates are formed when proteins are heavily carbonylated whereas the proteasomal system only moderately degrades carbonylated proteins . Since carbonylation of proteins is an irreversible and irreparable oxidative damage [57, 58], the generated aggregates accumulate as damaged or unfolded proteins . An important question is whether protein insolubility which is observed in many aging processes (as well as in the present study) is also responsible for the death of bacterial cells into VBNC state. Whatever the causal relationship between protein oxidation and temperature, oxidative stress has been recognized to play a major role in cell viability by direct or indirect protein modifications . Maisonneuve and coworkers have shown the presence of aggregated proteins in aerobically growing healthy E. coli. As the level of aggregation was correlated with the amount of ROS produced during growth, they finally concluded that aggregates may function as temporary trash organelles for detoxifications . In addition, it has been shown that protein aggregates increase during population senescence of E. coli, and reach a maximum in stationary phase. The amount of protein aggregates is proportional to the ratio of dead cells . Cao and et al. showed that viability of invading bacteria to plant cells was reduced through oxidation of special membrane transporters . It was also shown that E. coli VBNC state during stationary phase is preceded by damage to proteins such as carbonylation of proteins which affects various bacterial compartments and proteins .
According to these results and in general agreements with earlier studies, it can be assumed that insolubility of proteins which can be one of the consequence of protein carbonylation increases during senescence of bacterial population in storage period, and thus can be considered as a cause of death.
Changes in fatty acid profile may enhance protein modification
The same trend of fatty acid change was observed for cells kept at lower storage temperature; but, the extent of change was temperature dependent. The percentage of oleic acid moderately decreased during storage at 4°C (p < 0.05) and was even less reduced at −21°C (p < 0.05, range 0.004). Change of fatty acid ratio (unsaturated/saturated) of different lipid products during storage conditions have been reported by many authors [11, 30, 63–65]. It is well-known that the loss of bacterial viability during storage is increased following lipid oxidation [11, 49]. α, β-unsaturated alkenals such as 4-hydroxynonenal produced during the peroxidation of polyunsaturated fatty acids have been shown to react with proteins  and to form stable covalent thiolether adducts carrying a carbonyl function .
Considering the storage condition of our samples (normal atmosphere, low and high temperature) and the intrinsic factors in the formulation (presence of glucose, and spent growth culture media), several sources of ROS formation might exist. Formation of ROS during freeze-drying process and storage of freeze-dried products has indeed been reported [34, 35]. At low moisture content, non-enzymatic reactions are known to occur, such as Amadori and Maillard reactions , as well as lipid peroxidation [34, 66]. Enzymatic oxidation of lipids through lipoxygenase is also possible in low water activity (about 0.4) . Indeed, it is widely accepted that presence of lipid moiety, specially unsaturated lipids may enhance free radical-mediated oxidative damage [34, 67] it has also been shown that presence of lipids in freeze-drying formulation can enhance the ROS formation during storage .
This work demonstrated that the protein content of freeze-dried A. senegalensis is subjected to different changes during storage period. In addition, storage temperature affects the total cellular proteome significantly. Although many studies attempted to improve viability in bacterial starter, modifications of bacterial cellular proteins during storage of freeze-dried starter have not been considered in details. An association between storage temperature and protein carbonylation was established in our study. Moreover, we showed that samples mainly composed of dead cells contained higher amount of carbonylated proteins including some critical proteins such as ribosomal proteins or proteins involved in energy generation. Moreover, since protein carbonylation is a reaction which normally occur after other oxidative reactions, it seems that some other reactions (such as lipid peroxidation or protein lipidation and glycation) happen prior or in parallel to carbonylation. These reactions may be involved in VBNC formation even at very low storage temperature.
Formation of fluorescent substances in the stored cells, the changed pattern of 2D-DiGE and the modifications in composition of cellular fatty acids can be considered as indications of Maillard reaction progression, AGEs formation and protein lipidation. Therefore, from a practical point of view, it is first suggested to decrease the residues of spent growth medium and the metals content in the freeze-drying formula (e.g. by washing cells with appropriate buffers before freezing process to eliminate the fermentation supernatant). Secondly, because a part of the cells already has been entered into VBNC state during drying process, it seems that the deterioration reactions were first initiated during freeze-drying process and enhanced during storage. Accordingly, to diminish the deterioration reactions during storage period, any process that may causes bacterial injuries during fermentation and freeze-drying process must be avoided. Finally, since the deterioration reactions are considerably temperature dependent it is recommended to keep the freeze-dried bacteria at low temperature.
Microorganisms and production of biomass
A. senegalensis CWBI-B418T (=LMG 23690 T = DSM 18889 T), a thermo-tolerant acetic acid bacterium, isolated from mango fruit in Walloon Center of Industrial biology (CWBI), was used through this study .
A. senegalensis was grown in a 15 L laboratory-scale bioreactor (Bio Biolaffite, France) containing glucose (20 g/l), yeast extract (7.5 g/l), MgSO4.7 H2O (1 g/l), and (NH4)2HPO4 (1 g/l), K2HPO4 (1 g/l). The fermentation was performed under conditions previously described .
Production and storage of freeze-dried cells
Production of freeze-dried starter culture was performed as previously described  except that the cells were not mixed with mannitol. Instead, the cells were mixed with culture supernatant to reach the right dry weight. Freeze-dried cells were crushed and then dispensed into glass vials. The vials were sealed with septum and caps. They were then kept at three different temperature (−21, 4 and 35°C) up to 12 months in the dark.
Determination of total cell number and culturable cells after freeze-drying process
Total cell number of fresh cells and rehydrated cells was determined by using Brucker slides (Lo-Laboroptic Ltd, Lancing, UK). Briefly, 100 mg of freeze-dried cells were resuspended in 10 ml of GY medium. After 5 min of incubation at 30°C, it was mixed vigorously. The number of cells was then counted by using phase contrast microscope (Olympus, Tokyo, Japan).
Culturable cells in freeze-dried samples were determined by using spread plate technique. GY medium was used as the medium for enumeration of culturable cells. GY agar medium contained: glucose 20 g/l, yeast extract 7.5 g/l, MgSO4.7 H2O 1 g/l, and (NH4)2HPO4 1 g/l, KH2PO4 1 g/l, agar 15 g/l. pH of medium was set at 6.0 ± 0.05.
GY broth (the same components as GY agar) were used as rehydration medium and diluting medium as well.
Determination of total dehydrogenase activity of stored cells
5-cyano-2,3-ditolyl tetrazolium chloride (CTC) (λex 450 nm; λem 630 nm) and thiazole orange (TO) (λ ex 509 nm; λ em 530 nm) were used to determine the total dehydrogenase activity. CTC is a redox dye which is converted to a solid formazan dye by cellular respiratory chain. Solid formazan emits red fluorescence.
A stock solution of 50 mM CTC in filtrated deionized water, and a stock solution of 42 μmol/L TO in DMSO were prepared. The TO solution was diluted 10 times in DMSO just before staining of cells [4, 5]. All experiments were performed using a BD FACSCalibur™ flow cytometer (Becton, Dickinson, USA) equipped with an air-cooled argon laser and a red diode laser. Data were analyzed using BD CellQuest™ Pro software (Becton, Dickinson, USA). Instrument settings and staining procedure were performed as already described . Briefly, freeze-dried A. senegalensis were rehydrated and harvested by centrifugation, and the pellets were washed twice with saline phosphate buffer solution containing 18 mM glucose (PBSG). 45 μl of CTC solution was mixed with 450 μl of cell suspension and incubated at 30°C for 90 min on shaker (130 rpm) in the dark, then 5 μl of diluted TO was added to each sample and incubation was continued for 5 min at 30°C in the dark before passing the samples to FACS Cytometry.
Assessment of fluorescent substance formation in stored cells
Auto-fluorescence of cells was detected according to the procedure explained by Sheehy . Briefly, freeze-dried cells were suspended in PBS (100 mM, pH 7.4) for 10 min and washed three times to remove extracellular components. Then, the cell suspensions were delivered to BD FACSCalibur™ flow cytometer (Becton, Dickinson, USA) at low flow rate, corresponding to 500–700 events/s (in the Forward Scatter vs. Side Scatter plot). The autofluorescence signal was collected on FL3. For each cell population, three independent samples were prepared and introduced to the FACS. The total counted events for each sample were set on 55000.
Sequential extraction of cellular proteins
30 mg of freeze-dried cells were suspended in 10 ml of 50 mM phosphate buffer (pH 6.8), mixed vigorously and incubated at 25°C for 10 min, and then the cells were washed with the phosphate buffer solution twice. Washed cells were re-suspended in 1 ml of 50 mM Tris–HCl, 200 mM NaCl buffer (pH 8.3) and sonicated (50% power, 5 cycles) for 135 seconds on ice. Afterwards, the sequential extraction was performed in protein free Eppendorf® tubes as follows: 50 mM Tris–HCl, 200 mM NaCl buffer (pH 8.3) (Low Salt, LS) for 20 min on ice (twice); 50 mM Tris– HCl, 1 M NaCl buffer (pH 8.3) (High Salt, HS) for 20 min on ice; 70% (v/v) ethanol for 30 min at 65°C (E); and 0.1 M NaOH solution for 30 min on ice. At each extraction stage, 0.6 μl of protease inhibitor cocktail for general use (Sigma-Aldrich) was added to the solutions. Supernatants were obtained after 10 min centrifugation (14,000 g and 4°C), and then the amount of soluble protein in each fraction was determined by Bradford method.
2D-DiGE analysis of freeze-dried cells stored at different temperature
2D-DIGE proteomic analysis has been performed on freeze-dried cells kept at different temperatures for 12 months according to the procedures already used and published by Wislet-Gendebien et al. . Proteins were extracted in a lysis buffer containing 7 M urea (GE Healthcare, Diegem, Belgium), 2 M thio-urea (GE Healthcare), 30 mM Tris (pH 8.5) (GE Healthcare) and 2% ASB14 (Sigma-Aldrich). The supernatant containing the extracted solubilized proteins was precipitated (2-D Clean- Up Kit; GE Healthcare) and proteins resolubilized in lysis buffer were quantified using RC-DC Protein Assay (Bio-Rad). Each 25 μg of sample proteins was labeled with 200 pmol CyDye (GE Healthcare), either Cy3 or Cy5, and left for 30 min in the dark. The labeling reaction was stopped by adding 10 mM lysine for 10 min at 4°C. An internal standard was prepared by mixing equal quantities of all the experimental samples and was labeled with Cy2. Then, all the samples within the experiment were mixed in pairs together with 25 μg of the labeled internal standard and were separated by isoelectric focusing using pH 3–11 (24 cm) IPG strips in an Ettan IPGphor focusing system (GE Healthcare) in the first dimension.
Before initiating the second dimension step, proteins in IPG strips were reduced for 15 min in an equilibration buffer (50 mM Tris–HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS) containing 1% DTT and then they were alkylated in the same equilibration buffer containing 5% iodoacetamide. IPG strips were placed on top of classical 12.5% SDS-PAGE gels and the electrophoretic migration was completed in an Ettan Dalt apparatus (GE Healthcare) at 2 W/gel for 30 min, and then 25 W for 18 h. After scanning the gels with a Typhoon 9400 Laser Scanner (GE Healthcare) at three different wavelengths corresponding to the different CyDyes, 2-D gel analysis software (DeCyder version 7.0; GE Healthcare) was used for spot detection, spot quantification relative to the corresponding spot in the internal standard and gel matching. Protein spots that showed a significant variation in their abundance of at least twofold (Student’s t test, p < 0.05) between storage conditions were selected for further identification and were automatically picked from preparative gels run in parallel with the Ettan Spot Picker (GE Healthcare). Proteins in spots were identified by MALDI-TOF-MS-MS at the GIGA-Research Proteomic platform. Gels plugs were subjected to automatic tryptic digestion (PROTEINEER dp automated digester; Bruker Daltonics, Bremen, Germany). Gel pieces were washed three times in 50 mM NH4HCO3 followed by 50% ACN/50 mM NH4HCO3. Two other washes were carried out with 100% ACN to dehydrate the gels. In-gel digestion was performed with freshly activated trypsin (Roche, Basel, Switzerland) at a concentration of 10 ng l-1 in 50 mM NH4HCO3/5% ACN. After rehydration of the gel pieces at 8°C for 60 min, tryptic digestion was carried out at 30°C for 4 h. The resulting digested peptides were extracted with 1% trifluoroacetic acid (TFA) for 30 min at 20°C with occasional shaking. A volume of 3 μl of protein digests was adsorbed for 3 min on Prespotted AnchorChip plates with a-Cyano- 4-hydroxycinnamic acid (CHCA) as a matrix, using the PROTEINEER dp automat. Then the spots were briefly desalted with 10 mM dihydrogen ammonium phosphate in 0.1% TFA. MS fingerprints of the samples were acquired using the Ultraflex II MALDI-TOF–TOF mass spectrometer (Bruker Daltonics) in the mass range 700–3,500 Da. The Peptide Mass Fingerprinting (PMFs) was searched against the NCBI database. The variable and fixed modifications were methionine oxidation and cysteine carbamylation, respectively, with a maximum number of missed cleavages of 1. Mass precision tolerance error was set to 100 ppm. Peaks with the highest intensities, obtained in TOF/MS mode, were next analyzed by LIFT MS/MS. Proteins were identified with the Biotools 3.0 software (Bruker Daltonics) using the Mascot search engine (Matrix Science, Boston, MA, USA).
Quantification of carbonylated proteins
Carbonyl assay was performed according to the procedure explained in the manual of protein carbonyl ELISA kit (Enzo® Life science catalog # ALX-850-312-KI01). Briefly, after extraction of total cellular proteins in extraction buffer (Tris 100 mM pH 7.4, 100 mM NaCl, 1 mM EDTA, 1% Triton X100, 0.5% Sodium Deoxycholate) by sonicator (50% power, 6 cycles, 5 min, on ice) and removal of DNA by streptomycin solution, the extracted proteins were reacted with Dinitrophenyl hydrazine (DNP); then the proteins were nonspecifically adsorbed to an ELISA plate. Unconjugated DNP and non-protein constituents were washed away. The adsorbed proteins were probed with biotinylated anti-DNP antibody followed by streptavidin-linked horseradish peroxidase. Adsorbances were related to a standard curve prepared with serum albumin containing increasing proportions of hypochlorous acid-oxidized proteins that has been calibrated colorimetrically.
Determination of carbonylated proteins by western blotting
For detection and identification of carbonylated proteins in freeze-dried cells, a modified 2D-DiGE analysis (200 μg of one individual sample per gel together with 25 μg internal standard) was run, followed by western blotting. 2D-DiGE procedure was performed as mentioned in the previous section except that before running the second dimension, the IPG strips were incubated in a 10 mM 2,4-dinitrophenyl hydrazine (DNPH) solution for 20 min at 25°C. The IPG strips were then rinsed with Tris base/glycerol (2 M/30% (v/v)) solution and equilibration buffer. Each strip was then treated for reduction/alkylation and the second dimension of electrophoresis was run as mentioned above.
Three gels prepared from each cell samples (nine gels for the three storage temperature) were transferred to activated PVDF membranes. Carbonyl groups in the transferred derivatized proteins were immunodetected with anti-2,4-DNP primary antibody (1/500) overnight at 4°C in skimmed milk-TTBs buffer. Then, the membranes were washed three times in TTBS buffer and incubated with the second antibody (1/2500) at room temperature for 1 h. Finally, after three washings with TBS buffer, the membranes were scanned with a Typhoon 9400 Laser Scanner (GE Healthcare) at three different wavelengths corresponding to the different CyDyes and Anti-DNP antibody. 2-D gel analysis software (DeCyder version 7.0; GE Healthcare) was used for spot detection, gel matching and spot quantification relative to the corresponding spot in the internal standard.
Total fatty acid analysis
Analysis of the fatty acid content was performed after trans-esterification of extracted fatty acid. Briefly, 100 mg of freeze-dried cells was suspended in 10 ml of 50 mM phosphate buffer (pH 6.8), mixed vigorously and incubated at 25°C for 10 min, and then the cells were washed twice with the same phosphate buffer solution. The washed cells were then mixed with 300 μl of a solution containing Chloroform/Methanol (2:1). After addition of glass beads to the cell suspension, the cells underwent three cycle of heat shock (Liquid nitrogen-37°C). They were then mixed vigorously for 10 min. The supernatant was mixed with 1 ml of methylation mixture (33% methanolic 3 N HCl, 67% methanol, 10 μg/ml BTH), and methylation was performed at 85°C for 35 min. After cooling of the samples, 900 μl of 0.9% NaCl solution was added to each sample, and finally the lipid phase was dissolved in Heptan.
The gas chromatography analysis of the fatty acids contents was performed with a GC-2010 Shimadzu Gas chromatograph equipped with a SGE-capillary BPX70 column (30 m length). Supelco-FAME mix-37 (Sigma–Aldrich) was used as standard for fatty acids identifications and quantifications.
All the experiments were performed at least in three independent replicates. Kolmogorov-Smirnov normality test was used for checking the normality of data. The test showed that in all the experiments, the data are approximately normally distributed. Two-Factors Repeated Measures ANOVA was performed on the data by R.3.0.10 Software. After doing ANOVA, we performed Duncan’s multiple range test (MRT) to investigate significant levels for the difference between any pair of means, regardless of whether a significant F resulted from ANOVA.
The authors thank Dr. Sandra Ormenese and Mr. Raafat Stephan for their help, scientific and technical advice during flow-cytometric analyses. PL, is a Senior Research Associate at the National Fund for Scientific Research (FNRS), Belgium. This work was supported by Iranian Ministry of Science, Research and Technology (MSRT) and also by the Belgian Fund for Medical Scientific Research (FRSM).
- Holzapfel WH: Appropriate starter culture technologies for small-scale fermentation in developing countries. Int J Food Microbiol. 2002, 75: 197-212. 10.1016/S0168-1605(01)00707-3.View ArticleGoogle Scholar
- Sokollek SJ, Hammes WP: Description of a starter culture preparation for vinegar fermentation. Syst Appl Microbiol. 1997, 20: 481-491. 10.1016/S0723-2020(97)80017-3.View ArticleGoogle Scholar
- Azuma Y, Hosoyama A, Matsutani M, Furuya N, Horikawa H, Harada T, Hirakawa H, Kuhara S, Matsushita K, Fujita N, Shirai M: Whole-genome analyses reveal genetic instability of Acetobacter pasteurianus. Nucleic Acids Res. 2009, 37: 5768-5783. 10.1093/nar/gkp612.View ArticleGoogle Scholar
- Shafiei R, Delvigne F, Thonart P: Flow-cytometric assessment of damages to Acetobacter senegalensis during freeze-drying process and storage. Acetic Acid Bacteria. 2013, 2 (s1): e10-View ArticleGoogle Scholar
- Shafiei R, Delvigne F, Babanezhad M, Thonart P: Evaluation of viability and growth of Acetobacter senegalensis under different stress conditions. Int J Food Microbiol. 2013, 163: 204-213. 10.1016/j.ijfoodmicro.2013.03.011.View ArticleGoogle Scholar
- Gullo M, Mamlouk D, De Vero L, Giudici P: Acetobacter pasteurianus strain AB0220: cultivability and phenotypic stability over 9 years of preservation. Curr Microbiol. 2012, 64: 576-580. 10.1007/s00284-012-0112-9.View ArticleGoogle Scholar
- Ndoye B, Weekers F, Diawara B, Guiro AT, Thonart P: Survival and preservation after freeze-drying process of thermoresistant acetic acid bacteria isolated from tropical products of Subsaharan Africa. J Food Eng. 2007, 79: 1374-1382. 10.1016/j.jfoodeng.2006.04.036.View ArticleGoogle Scholar
- Ndoye B, Lebecque S, Dubois-Dauphin R, Tounkara L, Guiro AT, Kere C, Diawara B, Thonart P: Thermoresistant properties of acetic acids bacteria isolated from tropical products of Sub-Saharan Africa and destined to industrial vinegar. Enzyme Microb Technol. 2006, 39: 916-923. 10.1016/j.enzmictec.2006.01.020.View ArticleGoogle Scholar
- Santivarangkna C, Kulozik U, Foerst P: Alternative drying processes for the industrial preservation of lactic acid starter cultures. Biotechnol Prog. 2007, 23: 302-315. 10.1021/bp060268f.View ArticleGoogle Scholar
- Miyamoto-Shinohara Y, Imaizumi T, Sukenobe J, Murakami Y, Kawamura S, Komatsu Y: Survival rate of microbes after freeze-drying and long-term storage. Cryobiology. 2000, 41: 251-255. 10.1006/cryo.2000.2282.View ArticleGoogle Scholar
- Santivarangkna C, Kulozik U, Foerst P: Inactivation mechanisms of lactic acid starter cultures preserved by drying processes. J Appl Microbiol. 2008, 105: 1-13. 10.1111/j.1365-2672.2008.03744.x.View ArticleGoogle Scholar
- Morgan CA, Herman N, White PA, Vesey G: Preservation of micro-organisms by drying: a review. J Microbiol Methods. 2006, 66: 183-193. 10.1016/j.mimet.2006.02.017.View ArticleGoogle Scholar
- Lievense LC, Van’t Riet K: Convective drying of bacteria. Adv Biochem Eng Biotechnol. 1994, 51: 72-86.Google Scholar
- Shacter E: Quantification and significance of protein oxidation in biological samples. Drug Metab Rev. 2000, 32: 307-326. 10.1081/DMR-100102336.View ArticleGoogle Scholar
- Lund MN, Heinonen M, Baron CP, Estévez M: Protein oxidation in muscle foods: a review. Mol Nutr Food Res. 2011, 55: 83-95. 10.1002/mnfr.201000453.View ArticleGoogle Scholar
- Ragoonanan V, Aksan A: Protein Stabilization. Transfus Med Hemother. 2007, 34: 246-252. 10.1159/000104678.View ArticleGoogle Scholar
- Fredrickson JK, Li SMW, Gaidamakova EK, Matrosova VY, Zhai M, Sulloway HM, Scholten JC, Brown MG, Balkwill DL, Daly MJ: Protein oxidation: key to bacterial desiccation resistance?. ISME J. 2008, 2: 393-403. 10.1038/ismej.2007.116.View ArticleGoogle Scholar
- Castellión M, Matiacevich S, Buera P, Maldonado S: Protein deterioration and longevity of quinoa seeds during long-term storage. Food Chem. 2010, 121: 952-958. 10.1016/j.foodchem.2010.01.025.View ArticleGoogle Scholar
- Claudette Job LR, Lovigny Y, Belghazi M, Job D: Patterns of protein oxidation in Arabidopsis seeds and during germination. Plant Physiol. 2005, 138: 790-802. 10.1104/pp.105.062778.View ArticleGoogle Scholar
- Linares MA, Marín-García P, Méndez D, Puyet A, Diez A, Bautista JM: Proteomic approaches to identifying carbonylated proteins in brain tissue. J Proteome Res. 2011, 10: 1719-1727. 10.1021/pr101014e.View ArticleGoogle Scholar
- Dalle-Donne I, Giustarini D, Colombo R, Rossi R, Milzani A: Protein carbonylation in human diseases. Trends Mol Med. 2003, 9: 169-176. 10.1016/S1471-4914(03)00031-5.View ArticleGoogle Scholar
- Møller IM, Jensen PE, Hansson A: Oxidative modifications to cellular components in plants. Annu Rev Plant Biol. 2007, 58: 459-481. 10.1146/annurev.arplant.58.032806.103946.View ArticleGoogle Scholar
- Nystrom T: Role of oxidative carbonylation in protein quality control and senescence. EMBO J. 2005, 24: 1311-1317. 10.1038/sj.emboj.7600599.View ArticleGoogle Scholar
- Jung T, Höhn A, Grune T: Lipofuscin: Detection and quantification by microscopic techniques. Advanced Protocols in Oxidative Stress II. Edited by: Armstrong D. 2010, Humana Press, Springer New York Dordrecht Heidelberg London, 173-193. Methods in Molecular Biology. ISBN 978-1-60761-410-4, 594View ArticleGoogle Scholar
- Baynes JW: The role of AGEs in aging: causation or correlation. Exp Gerontol. 2001, 36: 1527-1537. 10.1016/S0531-5565(01)00138-3.View ArticleGoogle Scholar
- Murthy UMN, Liang Y, Kumar PP, Sun WQ: Non-enzymatic protein modification by the Maillard reaction reduces the activities of scavenging enzymes in Vigna radiata. Physiol Plant. 2002, 115: 213-220. 10.1034/j.1399-3054.2002.1150206.x.View ArticleGoogle Scholar
- Murthy UMN, Kumar PP, Sun WQ: Mechanisms of seed ageing under different storage conditions for Vigna radiata (L.) Wilczek: lipid peroxidation, sugar hydrolysis, Maillard reactions and their relationship to glass state transition. J Exp Bot. 2003, 54: 1057-1067. 10.1093/jxb/erg092.View ArticleGoogle Scholar
- Kurtmann L, Skibsted LH, Carlsen CU: Browning of freeze-dried probiotic bacteria cultures in relation to loss of viability during storage. J Agric Food Chem. 2009, 57: 6736-6741. 10.1021/jf901044u.View ArticleGoogle Scholar
- Nocker A, Fernández PS, Montijn R, Schuren F: Effect of air drying on bacterial viability: a multiparameter viability assessment. J Microbiol Methods. 2012, 90: 86-95. 10.1016/j.mimet.2012.04.015.View ArticleGoogle Scholar
- Coulibaly I, Amenan AY, Lognay G, Fauconnier ML, Thonart P: Survival of freeze-dried Leuconostoc mesenteroides and Lactobacillus plantarum related to their cellular fatty acids composition during storage. Appl Biochem Biotechnol. 2009, 157: 70-84. 10.1007/s12010-008-8240-1.View ArticleGoogle Scholar
- Wesche AM, Gurtler JB, Marks BP, Ryser ET: Stress, sublethal injury, resuscitation, and virulence of bacterial foodborne pathogens. J Food Prot. 2009, 72: 1121-1138.Google Scholar
- Hoefman S, Van Hoorde K, Boon N, Vandamme P, De Vos P, Heylen K: Survival or revival: long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria. PloS One. 2012, 7: e34196-10.1371/journal.pone.0034196.View ArticleGoogle Scholar
- Vriezen JA, de Bruijn FJ, Nusslein KR: Desiccation induces viable but non-culturable cells in Sinorhizobium meliloti 1021. AMB Express. 2012, 2: 6-10.1186/2191-0855-2-6.View ArticleGoogle Scholar
- Molina MD, Anchordoquy TJ: Degradation of lyophilized lipid/DNA complexes during storage: the role of lipid and reactive oxygen species. Biochim Biophys Acta. 2008, 1778: 2119-2126. 10.1016/j.bbamem.2008.04.003.View ArticleGoogle Scholar
- Molina MC, Anchordoquy TJ: Metal contaminants promote degradation of lipid/DNA complexes during lyophilization. Biochim Biophys Acta Biomembr. 2007, 1768: 669-677. 10.1016/j.bbamem.2006.12.004.View ArticleGoogle Scholar
- Narayana Murthy UM, Sun WQ: Protein modification by Amadori and Maillard reactions during seed storage: roles of sugar hydrolysis and lipid peroxidation. J Exp Bot. 2000, 51: 1221-1228. 10.1093/jexbot/51.348.1221.View ArticleGoogle Scholar
- Cohen-Or I, Katz C, Ron EZ: AGEs secreted by bacteria are involved in the inflammatory response. PloS One. 2011, 6: e17974-10.1371/journal.pone.0017974.View ArticleGoogle Scholar
- Markowicz Bastos D, Monaro E, Siguemoto E, Séfora M: Maillard reaction products in processed food: Pros and Cons. Food Industrial Processes - Methods and Equipment. Edited by: Valdez B. 2012Google Scholar
- Kurtmann L, Carlsen CU, Skibsted LH, Risbo J: Water activity-temperature state diagrams of freeze-dried Lactobacillus acidophilus (La-5): influence of physical state on bacterial survival during storage. Biotechnol Prog. 2009, 25: 265-270. 10.1002/btpr.96.View ArticleGoogle Scholar
- Marshall BJ, Coote GG, Scott WJ: Some factors affecting the viability of dried bacteria during storage in vacuo. Appl Microbiol. 1974, 27: 648-652.Google Scholar
- Löster K, Kannicht C: 2-Dimensional electrophoresis: detection of Glycosylation and influence on spot pattern. Post-translational Modifications of Proteins. Edited by: Kannicht C. 2008, Totowa Nj: Humana Press, 199-214. Methods in Molecular Biology™, 446View ArticleGoogle Scholar
- Dalle-Donne I, Rossi R, Giustarini D, Milzani A, Colombo R: Protein carbonyl groups as biomarkers of oxidative stress. Clin Chim Acta. 2003, 329: 23-38. 10.1016/S0009-8981(03)00003-2.View ArticleGoogle Scholar
- Oliver JD: The viable but nonculturable state in bacteria. J Microbiol. 2005, 43 Spec No: 93-100.Google Scholar
- Cabiscol E, Tamarit J, Ros J: Oxidative stress in bacteria and protein damage by reactive oxygen species. Int Microbiol. 2000, 3: 3-8.Google Scholar
- Møller IM, Rogowska-Wrzesinska A, Rao RSP: Protein carbonylation and metal-catalyzed protein oxidation in a cellular perspective. J Proteomics. 2011, 74: 2228-2242. 10.1016/j.jprot.2011.05.004.View ArticleGoogle Scholar
- Grune T, Shringarpure R, Sitte N, Davies K: Age-related changes in protein oxidation and proteolysis in mammalian cells. J Gerontol A: Biol Med Sci. 2001, 56: B459-B467. 10.1093/gerona/56.11.B459.View ArticleGoogle Scholar
- Tamarit J, Cabiscol E, Ros J: Identification of the major oxidatively damaged proteins in Escherichia coli cells exposed to oxidative stress. J Biol Chem. 1998, 273: 3027-3032. 10.1074/jbc.273.5.3027.View ArticleGoogle Scholar
- Cabiscol E, Piulats E, Echave P, Herrero E, Ros J: Oxidative stress promotes specific protein damage in Saccharomyces cerevisiae. J Biol Chem. 2000, 275: 27393-27398.Google Scholar
- Teixeira P, Castro H, Kirby R: Evidence of membrane lipid oxidation of spray-dried Lactobacillus bulgaricus during storage. Lett Appl Microbiol. 1996, 22: 34-38. 10.1111/j.1472-765X.1996.tb01103.x.View ArticleGoogle Scholar
- Williams P, Winzer K, Chan WC, Cámara M: Look who’s talking: communication and quorum sensing in the bacterial world. Philos Trans Royal Soc B: Biol Sci. 2007, 362: 1119-1134. 10.1098/rstb.2007.2039.View ArticleGoogle Scholar
- Wilson DN, Gupta R, Mikolajka A, Nierhaus KH: Ribosomal proteins: Role in ribosomal functions. eLS. 2001, Chichester, Chichester: John Wiley & Sons, LtdGoogle Scholar
- Teixeira P, Castro H, Mohacsi-Farkas C, Kirby R: Identification of sites of injury in Lactobacillus bulgaricus during heat stress. J Appl Microbiol. 1997, 83: 219-226. 10.1046/j.1365-2672.1997.00221.x.View ArticleGoogle Scholar
- Fu N, Chen XD: Towards a maximal cell survival in convective thermal drying processes. Food Res Int. 2011, 44: 1127-1149. 10.1016/j.foodres.2011.03.053.View ArticleGoogle Scholar
- Madigan Michael T, Martinko John M, Stahl David A, Clark David P: Molecular biology of bacteria. Brock biology of microorganisms. 2012, 150-235. 13Google Scholar
- Nagano T, Kojima K, Hisabori T, Hayashi H, Morita EH, Kanamori T, Miyagi T, Ueda T, Nishiyama Y: Elongation factor G is a critical target during oxidative damage to the translation system of Escherichia coli. J Biol Chem. 2012, 287: 28697-28704. 10.1074/jbc.M112.378067.View ArticleGoogle Scholar
- Avery SV: Molecular targets of oxidative stress. Biochem J. 2011, 434: 201-210. 10.1042/BJ20101695.View ArticleGoogle Scholar
- Dalle-Donne I, Aldini G, Carini M, Colombo R, Rossi R, Milzani A: Protein carbonylation, cellular dysfunction, and disease progression. J Cell Mol Med. 2006, 10: 389-406. 10.1111/j.1582-4934.2006.tb00407.x.View ArticleGoogle Scholar
- Maisonneuve E, Fraysse L, Lignon S, Capron L, Dukan S: Carbonylated proteins are detectable only in a degradation-resistant aggregate state in Escherichia coli. J Bacteriol. 2008, 190: 6609-6614. 10.1128/JB.00588-08.View ArticleGoogle Scholar
- Maisonneuve E, Fraysse L, Moinier D, Dukan S: Existence of abnormal protein aggregates in healthy Escherichia coli cells. J Bacteriol. 2008, 190: 887-893. 10.1128/JB.01603-07.View ArticleGoogle Scholar
- Maisonneuve E, Ezraty B, Dukan S: Protein aggregates: an aging factor involved in cell death. J Bacteriol. 2008, 190: 6070-6075. 10.1128/JB.00736-08.View ArticleGoogle Scholar
- Cao B, Liu J, Qin G, Tian S: Oxidative stress acts on special membrane proteins to reduce the viability of Pseudomonas syringae pv tomato. J Proteome Res. 2012, 11: 4927-4938. 10.1021/pr300446g.View ArticleGoogle Scholar
- Desnues B, Cuny C, Gregori G, Dukan S, Aguilaniu H, Nystrom T: Differential oxidative damage and expression of stress defence regulons in culturable and non-culturable Escherichia coli cells. EMBO Rep. 2003, 4: 400-404. 10.1038/sj.embor.embor799.View ArticleGoogle Scholar
- Castro HP, Teixeira PM, Kirby R: Evidence of membrane damage in Lactobacillus bulgaricus following freeze drying. J Appl Microbiol. 1997, 82: 87-94. 10.1111/j.1365-2672.1997.tb03301.x.View ArticleGoogle Scholar
- Pimpo MT, Seri S: Study of lipid changes in freeze-dried fish during storage: I: the interaction of relative humidity and tissue lipids. Boll Soc Ital Biol Sper. 1992, 68: 735-739.Google Scholar
- Yao AA, Coulibaly I, Lognay G, Fauconnier ML, Thonart P: Impact of polyunsaturated fatty acid degradation on survival and acidification activity of freeze-dried Weissella paramesenteroides LC11 during storage. Appl Microbiol Biotechnol. 2008, 79: 1045-1052. 10.1007/s00253-008-1497-z.View ArticleGoogle Scholar
- Oracz K, El-Maarouf Bouteau H, Farrant JM, Cooper K, Belghazi M, Job C, Job D, Corbineau F, Bailly C: ROS production and protein oxidation as a novel mechanism for seed dormancy alleviation. Plant J. 2007, 50: 452-465. 10.1111/j.1365-313X.2007.03063.x.View ArticleGoogle Scholar
- Araseki M, Yamamoto K, Miyashita K: Oxidative stability of polyunsaturated fatty acid in phosphatidylcholine liposomes. Biosci Biotechnol Biochem. 2002, 66: 2573-2577. 10.1271/bbb.66.2573.View ArticleGoogle Scholar
- Sheehy MRJ: A flow-cytometric method for quantification of neurolipofuscin and comparison with existing histological and biochemical approaches. Arch Gerontol Geriatr. 2002, 34: 233-248. 10.1016/S0167-4943(01)00217-5.View ArticleGoogle Scholar
- Wislet-Gendebien S, Laudet E, Neirinckx V, Alix P, Leprince P, Glejzer A, Poulet C, Hennuy B, Sommer L, Shakhova O, Rogister B: Mesenchymal stem cells and neural crest stem cells from adult bone marrow: characterization of their surprising similarities and differences. Cell Mol Life Sci. 2012, 69: 2593-2608. 10.1007/s00018-012-0937-1.View ArticleGoogle Scholar
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