Several Weeks Of Storage Research Articles

Various cold storage-backslopping cycles show the robustness of Limosilactobacillus fermentum IMDO 130101 as starter culture for Type 3 sourdough production

Type 3 sourdoughs, which are starter culture-initiated and subsequently backslopped, are less studied than other sourdough types. Yet, they can serve as a model to assess how competitive starter culture strains for sourdough production are and how the microbial composition of such sourdoughs may evolve over time. In the present study, Limosilactobacillus fermentum IMDO 130101 was used to produce Type 3 sourdoughs, prepared from wheat and wholemeal wheat flours. Therefore, an initial fermentation of the flour-water mixture was performed at 30 °C for 48 h. This was followed by cold storage-backslopping cycles, consisting of refreshments (50 %, v/v), fermentation steps of 16 h, and storage at 4 °C each week, every three weeks, and every six weeks. The microbial dynamics (culture-dependent and -independent approaches) and metabolite dynamics were measured. In all sourdoughs produced, starter culture strain monitoring, following an amplicon sequence variant approach, showed that Liml. fermentum IMDO 130101 prevailed during one month when the sourdoughs were refreshed each week, during 24 weeks when the sourdoughs were refreshed every three weeks, and during 12 weeks when the sourdoughs were refreshed every six weeks. This suggested the competitiveness and robustness of Liml. fermentum IMDO 130101 for a considerable duration but also showed that the strain is prone to microbial interference. For instance, Levilactobacillus brevis and Pediococcus spp. prevailed upon further cold storage and backslopping. Also, although no yeasts were inoculated into the flour-water mixtures, Kazachstania unispora, Torulaspora delbrueckii, and Wickerhamomyces anomalus were the main yeast species found. They appeared after several weeks of storage and backslopping, which however indicated the importance of an interplay between LAB and yeast species in sourdoughs. The main differences among the mature sourdoughs obtained could be explained by the different flours used, the refreshment conditions applied, and the sampling time (before and after backslopping). Finally, the metabolite quantifications revealed continued metabolite production during the cold storage periods, which may impact the sourdough properties and those of the breads made thereof.

Effect of moisture on the reactivity of alpha-tricalcium phosphate

The ability of the high-temperature polymorph of tricalcium phosphate, α-TCP, to hydrolyse to calcium-deficient hydroxyapatite underlies many developments in the field of synthetic bone grafts, including calcium phosphate cements, foams, and self-setting inks. The objective of the present work was to investigate the effect of humidity on α-TCP powder reactivity. The results showed that a 3-week incubation at high relative humidity (80%) had no impact on reactivity, but, as the incubation was prolonged, the powder started to hydrolyse. This reactivity was associated to the presence of defects and to an amorphous phase induced during powder milling. Moisture studies performed under static/dynamic conditions gave comparable water adsorption percentages. The dynamic studies further proved irreversible water sorption, indicating that some water molecules reacted with the powder after short incubation times. Taken together, these results demonstrate that, although α-TCP powder adsorbs water from moisture immediately, the impact on reactivity appears only after several weeks of storage.

Evaluation of Storage and Freezing, Baking, and Boiling Treatments on Total Carotenoids Content in the Fruits of Selected Cucurbita moschata Duch. Varieties

Cucurbita moschata Duch. is a seasonal crop, appreciated for its nutrimental and bioactive components especially carotenoids. From the whole edible plant, fruit is most often consumed which can be stored and also processed. Six varieties—Liscia, Matilda, Orange, Serpentine, UG 205 F1, and Waltham—of Cucurbita moschata Duch. were investigated, in order to analyze and evaluate the changes in total carotenoids content in the pulp during storage and after the effect of heat treatments—freezing, baking, and cooking (water boiling). The average total carotenoids content ranged from 3.32 mg/100 g (Serpentine) to 9.35 mg/100 g fresh matter (FM) (Orange). After 60 days of storage, a slight increase in the total carotenoids content of all analyzed varieties was observed. The values of the monitored varieties ranged from 4.18 mg/100 g FM (Serpentine), which represents a 26% increase to 10.96 mg/100 g FM (Orange), where a 17% increase was observed. After 120 days of storage, the results were mixed. In some varieties (Liscia and Matilda), the total carotenoid content decreased, while in the varieties Serpentine, UG 205 F1, and Waltham, its content increased slightly (11%, 3%, and 11%), but the content of the Orange variety remained unchanged. The total carotenoids increment after 60 and 120 days of storage in the dry matter (DM) was statistically significant, as well as the effect of the variety. The total carotenoid content of cooked samples of all varieties increased, from 119.78 mg/100 g DM (Waltham) to 255.19 mg/100 g DM (Orange). After baking, an average of 12% increase in the total content of carotenoids was recorded, after freezing, a decrease in its level of 5% was indicated. The findings of this work show that Cucurbita moschata Duch. is a good source of carotenoids, even after several weeks of storage and after exposure to heat treatments.

The effect of storage and processing on vitamin C content in Japanese quince fruit

Abstract Stability of vitamin C in stored fruit and sugar syrup of Japanese quince was tested. Vitamin C was measured in the fruit at harvest and after a few weeks of storage under normal atmospheric conditions. Sugar syrup obtained from the fruit was subjected to analysis before and after pasteurisation, and after several weeks of storage. The Chaenomeles genotypes tested were valuable in terms of vitamin C content. Storage significantly reduced the vitamin C content in the fruit, by 20% on average after 2 weeks of storage. The mechanical treatment and preparation of sugar syrup decreased the vitamin content by nearly two-thirds when compared with the fruit at harvest. Pasteurisation and storage of the sugar syrup contributed to further losses of vitamin C, reducing it to a very low level.

Effects of ergothioneine-rich mushroom extracts on lipid oxidation and discoloration in salmon muscle stored at low temperatures

The effects of supplementing ergothioneine-rich mushroom extracts (ME) on discoloration and lipid oxidation in astaxanthin-pigmented salmon muscles were evaluated. ME (Flammulina velutipes) were added (in vitro) to minced Oncorhynchus mykiss muscles and stored at −10°C. During several weeks of storage, the ME-treated group showed lower lipid hydroperoxide (HPO) accumulation and higher retained astaxanthin levels than in the control group. The effects of adding concentrated ME (Pleurotus cornucopiae) to Oncorhynchus kisutch diets were also tested over a 2-month feeding trial. No adverse effects on fish growth or pigmentation were observed. Muscle samples were collected, stored (−2 or −18°C), and evaluated. At the end of the storage period, the ME-treated group had lower HPO and higher retained astaxanthin levels in the muscle samples than the levels in the control group. Thus, the addition of ergothioneine from MEs successfully controlled lipid oxidation and stabilized astaxanthin during post-harvest storage at low temperatures.

Use of FTA® card methodology for sampling and molecular characterization of Echinococcus granulosus sensu lato in Africa

Cystic Echinococcosis is a parasitic disease caused by the cestode Echinococcus granulosus widely distributed in Africa. Monitoring of this parasite requires access to cyst samples on intermediate hosts observed at the slaughterhouse. In order to facilitate sampling in the field and analysis, the French National Reference Laboratory for Echinococcus spp. has developed a tissue derived from DNA sampling with FTA® card technology. The DNA samples were taken by applying the FTA® paper on the germinal layer after opening the cysts. The sampling technique was validated using frozen cysts (n = 76) stored in the laboratory and from field samples (n = 134) taken at the slaughterhouse by veterinarian technicians during meat inspection in Morocco, Mali and Mauritania. DNA was extracted after several weeks of storage at room temperature. PCR assays were performed using primers for generic cestode (cox1) and amplified fragments were sequenced. All samples taken in the lab and 80% of field samples were capable of molecular characterization. Cyst-derived DNA from FTA® samples can be useful for easy sampling, storage and rapid, safe and cheap shipment. The use of the FTA methodology will facilitate studies in the field to investigate the presence and genetic characterization of E. granulosus sensu lato in African countries.

Low Temperature Irreversible Poly(DiMethyl) Siloxane Packaging of Silanized SU8 Microchannels: Characterization and Lab-on-Chip Application

In this paper, we describe and characterize a novel method, based on silanization, for strong bonding of SU8 microchannels to poly(dimethyl)siloxane (PDMS) flexible covers. First, the SU8 surface treatment process (silanization) is characterized through atomic force microscopy and contact angle measurements. The aging study proves grafting stability during more than two days. Silanized SU8 patterns and PDMS cover are finally bonded to seal the microchannel network. Such assembled microdevices can be used without leakage at flow rates above 2.4 mL/min, corresponding to 1.2 MPa if the PDMS deformation is neglected. The bonding tensile pressure exceeds 1.5 MPa, proving the packaging strength. Furthermore, SU8-PDMS composite devices display stable bonding after several weeks of storage. This rapid low cost and low temperature bonding technique is finally successfully employed to fabricate a fully packaged biochip for electric and fluidic handling of biological cells.

Chilling-related cell damage of apple (Malus × domestica Borkh.) fruit cortical tissue impacts antioxidant, lipid and phenolic metabolism.

'Soggy breakdown' (SB) is an internal flesh disorder of 'Honeycrisp' apple (Malus × domestica Borkh.) fruit that occurs during low temperature storage. The disorder is a chilling injury (CI) in which visible symptoms typically appear after several weeks of storage, but information about the underlying metabolism associated with its induction and development is lacking. The metabolic profile of flesh tissue from wholly healthy fruit and brown and healthy tissues from fruit with SB was characterized using gas chromatography-mass spectrometry (GC-MS) and liquid chromatograph-mass spectrometry (LC-MS). Partial least squares discriminant analysis (PLS-DA) and correlation networks revealed correlation among ester volatile compounds by composition and differences in phytosterol, phenolic and putative triacylglycerides (TAGs) metabolism among the tissues. anova-simultaneous component analysis (ASCA) was used to test the significance of metabolic changes linked with tissue health status. ASCA-significant components included antioxidant compounds, TAGs, and phytosterol conjugates. Relative to entirely healthy tissues, elevated metabolite levels in symptomatic tissue included γ-amino butyric acid, glycerol, sitosteryl (6'-O-palmitoyl) β-d-glucoside and sitosteryl (6'-O-stearate) β-d-glucoside, and TAGs containing combinations of 16:0, 18:3, 18:2 and 18:1 fatty acids. Reduced metabolite levels in SB tissue included 5-caffeoyl quinate, β-carotene, catechin, epicatechin, α-tocopherol, violaxanthin and sitosteryl β-d glucoside. Pathway analysis indicated aspects of primary metabolism differed according to tissue condition, although differences in metabolites involved were more subtle than those of some secondary metabolites. The results implicate oxidative stress and membrane disruption processes in SB development and constitute a diagnostic metabolic profile for the disorder.

Short-Term Storage of Human Spermatozoa in Electrolyte-Free Medium Without Freezing Maintains Sperm Chromatin Integrity Better Than Cryopreservation

Previous attempts to maintain human spermatozoa without freezing were based on short-term storage in component-rich medium and led to fast decline in motility and increased incidence of chromosome breaks. Here we report a new method in which sperm are maintained without freezing in an electrolyte-free medium (EFM) composed of glucose and bovine serum albumin. Human sperm were stored in EFM or human tubal fluid medium (HTFM) or were cryopreserved, and their motility, viability, and DNA integrity were examined at different intervals. Cryopreservation led to significant decline in sperm motility and viability and induced DNA fragmentation. Sperm stored in EFM maintained motility and viability for up to 4 and 7 wk, respectively, much longer than sperm stored in HTFM (<2 and <4 wk, respectively). DNA integrity, assessed with comet assay, was also maintained significantly better in EFM than in HTFM. One-week storage in EFM yielded motility and viability similar to that of cryopreserved sperm, but DNA integrity was significantly higher, resembling that of fresh sperm. After several weeks of storage in EFM, sperm were able to activate oocytes, undergo chromatin remodeling, and form normal zygotic chromosomes after intracytoplasmic sperm injection. This study demonstrated that human spermatozoa can be stored in EFM without freezing for several weeks while maintaining motility, viability, and chromatin integrity and that 1-wk storage in EFM offers better protection of sperm DNA integrity than cryopreservation. Sperm storage in EFM may become a viable option for the physicians working in assisted reproduction technology clinics, which would avoid cryodamage.

Stable recombinant alpaca antibodies for detection of Tulip virus X

For detection of the plant pathogenic Tulip virus X (TuVX), a panel of six recombinant antibodies was identified. To this end, a repertoire of variable domains from heavy-chain immunoglobulins (VHH) was cloned from an alpaca, which had been immunized with TuVX. Binding domains were selected by phage display and panning on immobilized TuVX particles. Recombinant VHH antibodies were tested for sensitivity in a sandwich ELISA, and were demonstrated to be readily able to distinguish TuVX-infected tulip leaf material from uninfected leafs. No cross-reactivity of the VHH antibodies to related flexiviridae was observed. Recombinant VHHs maintained their reactivity upon storage at −20°C for over a year. The effect of incubation at higher temperatures for prolonged time was studied. Two out of three VHH proteins retained activity after several weeks of storage at 37°C.

Modification of the Nanoscale Structure of the J-Aggregate of a Sulfonate-Substituted Amphiphilic Carbocyanine Dye through Incorporation of Surface-Active Additives

The amphiphilic dye 3,3'-bis(2-sulfopropyl)-5,5',6,6'-tetrachloro-1,1'-dioctylbenzimidacarbocyanine (C8S3) self-aggregates in aqueous solution to form tubular J-aggregates with a diameter of 17.0 +/- 0.5 nm, a wall thickness of approximately 4 nm, and a length exceeding several hundred nanometers. The absorption spectrum shows the typical features expected for tubular J-aggregates with several sharp and red-shifted absorption bands. Morphological investigations using cryo-transmission electron microscopy (cryo-TEM) and spectroscopic investigations reveal a high stability of the tubular morphology but a tendency of the aggregates to assemble into ropelike bundles after several weeks of storage. It is found that aggregation in solutions containing additives such as alcohols or surfactants results in the formation of new types of aggregates. A second type of tubular aggregate with a diameter of 13.0 +/- 0.5 nm is observed when the solutions contain more than 10 wt % MeOH. On the time scale of days these tubular aggregates transform into ribbonlike structures characterized by a new absorption spectrum, and they convert after several weeks into giant tubes with diameters of up to 500 nm.

A novel metal-protected plasma treatment for the robust bonding of polydimethylsiloxane

We describe a method for the irreversible bonding of PDMS-based microfluidic components by exploiting the first reported "shelfable" plasma treatment of PDMS. Simultaneous plasma activation and protection of PDMS surfaces are achieved via RF magnetron sputtering of thin aluminium films in the presence of an argon plasma. In this process, Ar plasma exposure generates a hydrophilic, silanol-enriched polymer surface amenable to irreversible bonding to glass, PDMS or silicon substrates, while the aluminium film functions as a capping layer to preserve the surface functionality over several weeks of storage in ambient conditions. Prior to bonding, this protective aluminium layer is removed by immersion in an aqueous etchant, exposing the adhesive surface. Employing this technology, PDMS-glass and PDMS-PDMS microfluidic devices were fabricated and the adhesive strength was quantified by tensile and leakage testing. Bonding success rates in excess of 80% were demonstrated for both PDMS-glass and PDMS-PDMS assemblies sealed 24 h and 7 days following initial polymer surface activation. PDMS-glass microdevices performed optimally, displaying maximum adhesive strengths on the order of 5 MPa and burst flow rates of approximately 1 mL min(-1) (channel dimensions: l = 25 mm; w = 300 microm; h = 20 microm). These data demonstrate a significant improvement in performance over previously reported bonding technologies, resulting in the production of more robust, longer-lasting microfluidic systems that can withstand higher pressures and flow rates.

Regulatory Aspects of Ethanol Fermentation in Immature and Mature Citrus Fruit

Molecular aspects of ethanol fermentation in citrus fruit were investigated in immature and mature ‘Star Ruby’ grapefruit (Citrus paradisi Macf.) and ‘Murcott’ mandarin (Citrus reticulata Blanco). Transcript levels of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH), which play a central role in ethanol fermentation, were detectable in all stages of fruit development, but accumulation of acetaldehyde (AA) and ethanol was evident only as fruit approached maturation or after several weeks of storage. Treatment of mature fruit with ethylene enhanced ethanol fermentation in grapefruit but not in mandarin. Immature fruit of both cultivars, on the other hand, responded to ethylene by prominent, although transient, enhancement of ethanol fermentation. Exposure of mature or immature fruit to anaerobic conditions (N2 atmosphere) upregulated the expression of PDC and ADH, and increased the levels of AA and ethanol. Exposure of mature fruit to anaerobic conditions also increased the enzymatic activities of PDC and ADH. The data indicate that the potential for ethanol fermentation exists in citrus fruit throughout development, even under aerobic conditions, but AA and ethanol are detected mainly toward maturation or under prolonged storage. However, prominent, long-term molecular induction of ethanol fermentation occurs only under anaerobic conditions imposed by N2 atmosphere.

Survival and Possible Spread of Erwinia amylovora and Related Plant-Pathogenic Bacteria Exposed to Environmental Stress Conditions

The fire blight pathogen Erwinia amylovora was assayed for survival under unfavourable conditions such as on nitrocellulose filters, in non-host plants as well as in inoculated mature apples and in infested apple stem sections. In a sterile dry environment, an E. amylovora EPS (exopolysaccharide) mutant, and to a lesser extent its parental wild-type strain decreased within 3 weeks to a low titre. However, under moist conditions the decrease of viable cells occurred only partially for both strains. Very low cell titres were recovered after application of E. amylovora onto the surface of tobacco leaves, whereas infiltration into the leaves produced lesions (hypersensitive response, HR), in which the bacteria survived in significant amounts. A similar effect was found for the necrotic zones of HR in tobacco leaves caused by E. pyrifoliae, by Pseudomonas syringae pathovars and HR-deficient E. amylovora mutants or mutants deficient in EPS synthesis and disease-specific genes. During 7 years of storage, the viability of E. amylovora in wood sections from fire blight-infested apple trees declined to a low titre. In tissue of mature apples, E. amylovora cells slowly dispersed and could still be recovered after several weeks of storage at room temperature. A minimal risk of accidental dissemination of E. amylovora apart from infested host plants can experimentally not be excluded, but other data confirm a very low incidence of any long distance distribution.

Skin adhesion in sweetpotato and its lack of relationship to polygalacturonase and pectinmethylesterase during storage

Sweetpotatoes have a tendency to experience epidermal loss during harvest and postharvest handling. Epidermal detachment from underlying periderm (skin loss) causes weight loss, shriveling of the root surface, increased susceptibility to pathogen attack and inferior appearance. ‘Beauregard’ is very prone to skin loss, while ‘Jewel’ is thought to have stronger skin. Our objective was to determine the variation in skin strength in relation to cell wall enzyme activity and to determine possible correlations among these variables that could explain skin loss on the basis of enzyme activity during storage of the roots. Skin adhesion, polygalacturonase (PG) and pectinmethylesterase (PME) activity were measured during storage of ‘Beauregard’ and ‘Jewel’ roots in 1999 and again in 2000. Skin adhesion varied among years and cultivars. Overall, roots of ‘Beauregard’ were more susceptible than ‘Jewel’ to skin loss after several weeks of storage. Roots of ‘Jewel’ had lower skin adhesion at harvest, which increased after several weeks of storage and decreased again at the end of the storage period. Enzyme activity exhibited a random pattern during the storage period, and skin adhesion did not correlate with PG or PME activity.

Formation of Luminescent CdTe–Silica Nanoparticles through an Inverse Microemulsion Technique

Abstract CdTe quantum dots (QDs, 3–6 nmφ) were incorporated into silica nanoparticles (20–30 nmφ) by an inverse microemulsion technique. The transmission electron microscope image shows that the QDs are situated on the surface of silica particles. These QD-silica particles in solution exhibited a stable luminescence with at least the similar quantum efficiency (ca. 5%) as that of the initial colloidal solution over several weeks of storage. The particles taken out from the solution as a powder form showed the same quantum efficiency for a long time as well. This is because silica acts as an effective protective environment for the QDs.

Synthesis and Characterization of Active Ester-Functionalized Polypyrrole−Silica Nanoparticles: Application to the Covalent Attachment of Proteins

Novel ester-functionalized polypyrrole-silica nanocomposite particles were prepared by oxidative copolymerization of pyrrole and N-succinimidyl ester pyrrole (50/50% initial concentrations), using FeCl3 in the presence of ultrafine silica nanoparticles (20 nm diameter). The N-succinimidyl ester pyrrole monomer was prepared in aqueous solution using 1-(2-carboxyethylpyrrole) and N-hydroxysuccinimide in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The resulting nanocomposites (N-succinimidyl ester polypyrrole-silica) are raspberry-shaped agglomerates of silica sol particles "glued" together by the insoluble poly(pyrrole-co-N-succinimidyl pyrrole). The N-succinimidyl ester polypyrrole-silica particles were characterized in terms of their size, density, copolymer content, and polydispersity. Scanning electron microscopy and disk centrifuge sedimentometry confirmed that the nanocomposite particles had narrow size distributions. X-ray photoelectron spectroscopy analysis indicated a silica-rich surface and a high surface concentration of N-succinimidyl ester groups. These nanoparticles exhibited good long-term dispersion stability. The chemical stability of the ester functions in aqueous media after several weeks of storage was monitored by FTIR spectroscopy. The functionalized nanocomposites were tested as bioadsorbents of human serum albumin (HSA). The very high amount of immobilized HSA determined by UV-visible spectroscopy is believed to be due to covalent binding. Incubation of the HSA-grafted nanocomposite with anti-HSA resulted in immediate flocculation, an indication that they are alternative candidates for visual diagnostic assays.

Comparison of techniques for enzyme immobilization on silicon supports

Enzyme immobilization onto silicon substrates has been investigated by five different coupling procedures. The methods included covalent coupling either through a metal link reagent or silane reagents containing pendant amino or epoxide linkers, an entrapment technique using a thin layer of gelatin, or an adsorption technique using poly- l-lysine. These immobilization procedures were evaluated using glucose oxidase and a simple spectrophotometric method employing Fenton’s reagent. Retention of enzyme activity and surface loading were assessed. The immobilization techniques were also evaluated by electron microscopy to characterize the evenness of the surface coatings. All of the covalent coupling procedures led to surface loadings, approaching 1 pmol mm −2; however, the surfaces appeared irregular on a microscopic scale. The poly- l-lysine adsorption technique provided the smoothest surface. With the exception of the entrapment technique, all immobilization procedures provided immobilized enzyme that retained >75% activity after several weeks of storage.

Cationic Liposomes Coated with Polyethylene Glycol As Carriers for Oligonucleotides

Modification of liposome surface with polyethylene glycol was used to improve oligodeoxyribonucleotide (ODN) loading, stability of the resulting complexes, and specificity of cellular delivery of ODN by cationic liposomes. Liposomes composed of a cationic lipid (DOTAP, DOGS, DDAB), a neutral lipid (DOPE), and a phospholipid derivative of polyethylene glycol (PEG-PE) formed a complex with 18-mer phosphorothioate up to ODN/lipid molar ratio of 0.25. The complexes showed intact vesicular structures similar to original liposomes and their size (100-130 nm) was unchanged after several weeks of storage, whereas complexes lacking PEG-PE showed progressive aggregation and/or precipitation. After exposure to human plasma, PEG-modified cationic liposomes retained over 60% of the originally bound ODN. PEG-coated complexes resulted in 4-13-fold enhancement of the ODN uptake by human breast cancer cells in serum-supplemented growth medium, relative to free ODN. Complexes containing conjugated anti-HER2 F(ab') fragments at the distal termini of PEG chains efficiently delivered ODN primarily into the cytoplasm and nuclei of HER2 overexpressing cancer cells and greatly enhanced the biological activity of antisense ODN. The development of PEG-modified cationic liposomes may lead to improved ODN potency in vivo.

Physiological and biochemical aspects of the acid survival of Listeria monocytogenes.

The physiological aspects of the response to acidic conditions and the correlated protein synthesis were studied by using Listeria monocytogenes grown in a chemically defined synthetic medium. This growth was greatly affected by pH of the medium. It decreased when pH declined and was arrested at pH 4. When pH went under 4, the bacteria began to die. If the bacteria had been adapted to an intermediary sublethal pH before imposition of lethal pH stress, they would have resisted better lethal pH. A prolonged treatment at intermediary pH, however, rendered the bacteria more sensitive to subsequent lethal pH. Organic volatile acids exerted a more deleterious effect on L. monocytogenes than inorganic acids at the same stressing pH. The acquired acid tolerance was conserved after several weeks of storage of the adapted bacteria at 4 degrees C. Acid stress and acid adaptation (tolerance) affected the synthesis patterns of bacterial proteins: Many proteins were repressed and several others increased in expression level. These acid-induced proteins were separated by two-dimensional (2D-) electrophoresis and analyzed by a computer-aided 2D-gel analysis system. The results obtained suggested that acid tolerance and acid stress responses require the synthesis of a certain number of shared proteins and that additional acid-induced proteins are needed when the bacteria must face more severe acidic pH.