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Applied and Environmental Microbiology, July 2006, p. 4503-4514, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.01829-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Response of Lactobacillus helveticus PR4 to Heat Stress during Propagation in Cheese Whey with a Gradient of Decreasing Temperatures

Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi di Bari, Bari, Italy,¹ Department of Food and Nutritional Sciences, University College Cork, Cork, Ireland²

Received 3 August 2005/ Accepted 16 March 2006

	ABSTRACT

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The heat stress response was studied in Lactobacillus helveticus PR4 during propagation in cheese whey with a gradient of naturally decreasing temperature (55 to 20°C). Growth under a gradient of decreasing temperature was compared to growth at a constant temperature of 42°C. Proteinase, peptidase, and acidification activities of L. helveticus PR4 were found to be higher in cells harvested when 40°C was reached by a gradient of decreasing temperature than in cells grown at constant temperature of 42°C. When cells grown under a temperature gradient were harvested after an initial exposure of 35 min to 55°C followed by decreases in temperature to 40 (3 h), 30 (5 h 30 min), or 20°C (13 h 30 min) and were then compared with cells grown for the same time at a constant temperature of 42°C, a frequently transient induction of the levels of expression of 48 proteins was found by two-dimensional electrophoresis analysis. Expression of most of these proteins increased following cooling from 55 to 40°C (3 h). Sixteen of these proteins were subjected to N-terminal and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses. They were identified as stress proteins (e.g., DnaK and GroEL), glycolysis-related machinery (e.g., enolase and glyceraldehyde-3-phosphate dehydrogenase), and other regulatory proteins or factors (e.g., DNA-binding protein II and ATP-dependent protease). Most of these proteins have been found to play a role in the mechanisms of heat stress adaptation in other bacteria.

	INTRODUCTION

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Lactobacillus helveticus is a homofermentative thermophilic lactic acid bacterium widely used in the dairy industry. In particular, it is used as primary natural whey starter for the manufacture of Swiss and Italian hard and extrahard (e.g., Provolone, Parmigiano Reggiano, and Grana Padano) cheese varieties (16, 18, 31). Natural whey starters are prepared daily at the cheese plant by incubating overnight some of the whey drained from the cheese vat under more or less selective conditions. For Parmigiano Reggiano and Grana Padano cheeses, whey is traditionally fermented at a naturally decreasing temperature from ca. 55°C (cooking temperature of the curds) to 30°C or below (especially in the winter) in approximately 20 h until a pH of ca. 3.3 is reached. The resulting whey starter is dominated by aciduric and/or thermophilic strains: mainly L. helveticus (>85%), Lactobacillus delbrueckii subsp. lactis, Lactobacillus fermentum, and Streptococcus thermophilus (31). Since this technique of propagation at cheese plants usually leads to marked starter heterogeneity, several studies have used molecular tools to define the biodiversity of L. helveticus strains (8, 16, 17) and others have focused on the relevant contribution of L. helveticus to proteolysis during cheese ripening (25, 37, 41). No studies have considered the effect of the heat stress to which cells of L. helveticus are subjected during propagation of the natural whey starter culture.

Overall, the use of lactic acid bacteria in foods implies that they are exposed to various environmental stresses such as extremes of temperature, pH, osmotic pressure, oxygen, high pressure, and starvation. It is essential to know not only which conditions are favorable or detrimental for the life of lactic acid bacteria but also which mechanisms permit their survival and metabolic activities (10). Heat stress has been studied in several lactobacilli, including L. helveticus (6). Induction of heat tolerance to pasteurization (e.g., 63°C for 20 min) was related to the transient induction of heat shock proteins (HSPs), the major groups of which consist of the DnaK and GroEL families. The groE operon has been sequenced in L. helveticus (5). The gene htrA, which codes for a putative membrane serine-proteinase from the HtrA/DegP family, was also shown to be involved in the heat stress response of L. helveticus (36). High hydrostatic pressure stress activates the aminopeptidase and X-prolyl dipeptidyl aminopeptidase activities of L. helveticus after a treatment of 400 MPa at 30°C for 10 min (27), and the increase of the oxygen-consuming desaturase system, with a consequent increase in fatty acid desaturation, was found to be the cellular response of L. helveticus when it is subjected to toxic oxygen species and high temperatures (21). Nevertheless, the heat shock during propagation of L. helveticus in the natural whey starter culture is very different from the above studies and may be relevant for the performance of the whey starter in cheese making. The population of L. helveticus recovered after propagation in whey may consist of a mixture of cells which show different mechanisms of adaptation and differences in technological properties depending on the exposure to heat during the transient decrease of temperature from ca. 55 to 30°C or below. Knowledge about these proteomic aspects may permit the optimization of growth, acidification, and proteolysis during cheese making (10). A recent proteomic study on the bacterial proteins released in Emmental cheese showed that during manufacture thermophilic lactic acid bacteria and Propionibacterium strains liberated proteins involved in proteolysis, glycolysis, stress response, DNA and RNA repair, and oxidoreduction (15).

This study was aimed at describing some aspects of the molecular heat response and cheese-related phenotypic properties of L. helveticus PR4 grown in whey cheese with a gradient of decreasing temperature from 55 to 20°C, which mimics the manufacture of natural whey starter. Two-dimensional electrophoresis and identification of stress proteins by N-terminal sequencing and matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry were used to highlight the mechanism of stress response.

	MATERIALS AND METHODS

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Strains and culture condition.
L. helveticus strains PR4, PR5, and PR6, which were identified by 16S rRNA gene sequence analysis from natural whey starters for Parmigiano Reggiano (Culture Collection of the Dipartimento di Protezione delle Piante e Microbiologia Applicata, University of Bari, Bari, Italy), were used. Strains were propagated routinely in MRS broth (Oxoid Ltd., Basingstoke, United Kingdom) at 42°C for 24 h.

Fresh whey from cheese making was centrifuged at 9,000 x g for 30 min at 4°C, filtered onto a Whatman apparatus (Polycarp 75 SPF; Maidstone, England), sterilized by filtration on 0.45-µm-pore-size membrane filters (Millipore Corp., Bedford, MA), and stored at –20°C before use. The pH of the whey was ca. 5.5. Twenty-four-hour-old cells of L. helveticus strains grown in MRS broth were inoculated (4%, vol/vol) into whey and cultured twice before use.

Growth under a temperature gradient.
The naturally decreasing temperature which occurs during propagation of the natural whey starter used for Parmigiano Reggiano cheese making was mimicked. Twenty-four-hour-old cells of L. helveticus grown in whey were inoculated into fresh whey at 55°C (initial cell density of ca. 6.8 log CFU ml^–1) and incubated under a gradient of decreasing temperature. The gradient was as follows: 55°C for 35 min, 55 to 40°C in 2 h 25 min, 40 to 30°C in 2 h 30, 30 to 20°C (room temperature) in 8 h, and further incubation at 20°C up to a total of 20 h. Aliquots of cell biomasses were harvested by centrifugation (9,000 x g for 10 min at 4°C) after 35 min of exposure to 55°C; after a temperature of 40 (3 h), 30 (5 h 30 min), or 20 (13 h 30 min) was reached; or after incubation for 20 h under a temperature gradient. Harvested cells were used for the assays (see below).

Two other conditions of cultivation in whey were chosen and used as the controls. One corresponded to cells of L. helveticus strains grown at a constant temperature of 42°C (optimum temperature for growth of the strains used), which were harvested at times corresponding to those used for growth with the temperature gradient: after 35 min, 3 h, 5 h 30 min, 13 h 30 min, or 20 h of incubation. The other condition corresponded to cells exposed to temperature shifts but without a naturally decreasing gradient. The shift in temperature was as follows: 55°C for 35 min, rapid cooling (1 min) at 40°C and incubation for 6 h, rapid cooling to 30°C and incubation for 6 h, and rapid cooling at 20°C and incubation for a total of 20 h. A time of 6 h at 40°C and at 30°C was used to have stationary phase cells (12 h) grown at two subsequent temperatures. Aliquots of cell biomass were harvested before each shift in temperature and after 20 h of incubation.

Kinetics of cell growth.
Growth data were modeled according to the Gompertz equation as modified by Zwietering et al. (48): y = k + A exp{– exp[(µ_max e/A)( – t) + 1]}, where y is the extent of growth as log CFU ml^–1 at the time t, k is the initial cell density as log CFU ml^–1, A represents the difference in cell density between inoculation and the stationary phase, µ_max is the maximum growth rate as log CFU ml^–1 h^–1, is the length of the latency phase expressed in hours, and t is the time. The experimental data were modeled through the nonlinear regression procedure of the statistics package Statistica for Windows (Statsoft, Tulsa, Okla.).

Protein extraction and two-dimensional electrophoresis.
For two-dimensional electrophoresis analyses, harvested cells of L. helveticus were washed in 0.05 M Tris HCl, pH 7.5, centrifuged (15,000 x g for 15 min at 4°C), and frozen or directly resuspended in denaturing buffer composed of 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), 40 mM Tris base, and 65 mM dithiothreitol. To extract total proteins, cells were disrupted with a Branson model B15 sonifier by three cycles of sonication (1 min each). After unbroken cells were pelleted (15,000 x g for 15 min at 4°C), the protein content of the supernatant was measured by the method of Bradford (4).

Two-dimensional electrophoresis was performed using the immobiline-polyacrylamide system, essentially as described by Görg et al. (20) and Hochstrasser et al. (24) using a Pharmacia 2D-EF system (Uppsala, Sweden). The same amount (30 µg) of total protein was used for each electrophoretic run. Isoelectric focusing was carried out on Immobiline strips providing a nonlinear gradient from pH 3 to 10 (IPG strips; Amersham Pharmacia Biotech) by IPG-phore at 15°C. Voltage was increased from 300 to 5,000 V during the first 5 h and then stabilized at 8,000 V for 8 h. After electrophoresis, IPG strips were equilibrated for 12 min against 6 M urea, 30% (vol/vol) glycerol, 2% (wt/vol) sodium dodecyl sulfate (SDS), 0.05 M Tris-HCl (pH 6.8), and 2% (wt/vol) dithiothreitol, and for 5 min, against 6 M urea, 30% (vol/vol) glycerol, 2% (wt/vol) SDS, 0.05 M Tris-HCl (pH 6.8), 2.5% (vol/vol) iodioacetamide, and 0.5% (wt/vol) blue bromophenol. For the second dimension, 12.5% homogeneous SDS-polyacrylamide gel electrophoresis gels were used. Gels were calibrated with three molecular mass markers: comigration of the cell extracts with markers for two-dimensional electrophoresis (pI range, 7.6 to 3.8; molecular mass range, 17 to 89 kDa) from Sigma Chemical Co.; human serum proteins for the molecular mass range 200 to 10 kDa, and markers from Pharmacia Biotech for the low-molecular-mass range (16.9, 14.4, 10.7, 8.2, 6.2, and 2.5 kDa). The electrophoretic coordinates used for serum proteins were according to the method of Bjellqvist et al. (3). The gels were silver stained, as described by Hochstrasser et al. (24) and Oakley et al. (29). The protein maps were scanned with a laser densitometer (Molecular Dynamics 300s) and analyzed with Image Master 2D elite computer software (Pharmacia). Three gels from three independent experiments were analyzed, and spot intensities were normalized as reported by Bini et al. (2). In particular, the spot quantification for each gel was calculated as relative volume, i.e., the volume of each spot divided by the total volume over the whole image. In this way, differences in color intensities among the gels were eliminated (1). The induction factor for individual proteins was expressed as the ratio between spot intensity of the same protein in the adapted cells and in the nonadapted cells. All the induction factors were calculated based on the average of the spot intensities of each of the three gels, and standard deviation was calculated.

N-terminal amino acid sequencing.
Spots from two-dimensional electrophoresis gels were transferred onto polyvinylidene difluoride membranes by passive absorption as described by Messer et al. (26), with some modifications. Protein bands were excised and dried in a Speed Vac for 30 min, and then the gel pieces were allowed to swell again in 30 µl of 2% (wt/vol) SDS in 0.2 M Tris-HCl, pH 8.4, for 30 min. After swelling, 150 µl of high-pressure liquid chromatography water was added, and then a piece of prewet (methanol) polyvinylidene difluoride membrane (4 by 4 mm; Problott Applied Biosystems, Foster City, Calif.) was added to the solution. The procedure required 2 days at room temperature (23°C) with gentle vortexing. At the end of this transfer time, the gel piece and the solution were clear, and the membrane was blue. The membrane was washed five times with 1 ml of 10% methanol with vortexing. N-terminal Edman sequencing was performed on an Applied Biosystems Procise 494HT by using reagents and methods as recommended by the manufacturer. Sequence comparison was performed by using SWALL and BLAST at NCBI nonredundant databases.

MALDI-time of flight mass spectrometry.
Slices of gel were destained by several washings with 5 mM NH₄HCO₃-acetonitrile (ACN) (50/50) and subsequently dried with pure ACN. The gel slices were rehydrated for 45 min at 4°C in 20 µl of digestion buffer containing 10 ng/µl of trypsin and 5 mM NH₄HCO₃ (45). The excess of protease solution was removed, and the volume was adjusted with 5 mM NH₄HCO₃ to cover the gel slices. Digestion was allowed to proceed overnight at 37°C. The mass spectrometer used was a Tofspec SE (Micromass, Manchester, United Kingdom) equipped with a delayed extraction unit. The laser wavelength was 337 nm, and the accelerating voltage was 20 kV (23). Peptide spectra were obtained in reflectron mode in the range of 800 to 4,000 Da. The peptide solution was loaded onto the MALDI target plate by mixing 1.5 pl of each solution with the same volume of a matrix solution, prepared by dissolving 10 mg/ml alpha-cyano-4-hydroxycinnamic acid solution in 40% ACN-0.1% trifluoroacetic acid (vol/vol), and allowed to dry. External calibration was done by using the fragment ions from the standard peptides, adrenocorticotropic hormone 18 to 39, and angiotensin I. Each mass spectrum was generated by accumulating data from 100 to 120 laser shots (14). Database searches were done with the peptide masses against the nonredundant NCBI database using the search program ProFound (http://www.prowl.rockefeller.edu/cgibin/ProFound) from Rockefeller University and ProteoMetrics.

Proteinase and peptidase activities.
Harvested cells of L. helveticus were washed in 50 mM phosphate buffer, pH 7.0, centrifuged at 9,000 x g for 15 min at 4°C, and resuspended in the same buffer at a cell density of ca. 8.0 log CFU ml^–1. All the enzyme activities were determined at 42°C. Proteinase activity was measured by the method of Twinning (40) using fluorescent casein as a substrate. One unit (arbitrary unit [AU]) of proteinase activity was expressed as an increase of 0.1 unit of fluorescence 10 min^–1 (19). Leu-p-nitroanilide (p-NA), Pro-p-NA, Gly-Pro-p-Na, and Leu-Leu were used as synthetic substrates relatively specific for general aminopeptidase type N (PepN), iminopeptidase (PepI), X-prolyl dipeptidyl aminopeptidase (PepX), and dipeptidase (PepV) activities, respectively. The enzyme activities toward p-NA derivatives and Leu-Leu were determined as reported by Gobbetti et al. (19). One unit (AU) of PepN, PepI, and PepX activity corresponded to an increase in absorbance at 410 nm of 0.01 min^–1 (PepN) and 5 min^–1 (PepI and PepX). PepV activity was measured by the Cd-ninhydrin method (19). One unit (AU) of PepV activity was defined as the amount of enzyme that produced an increase in absorbance at 505 nm of 0.1 min^–1.

Kinetics of acidification.
Harvested cells of L. helveticus were washed in 50 mM phosphate buffer (pH 7.0), centrifuged at 9,000 x g for 15 min at 4°C, and resuspended in sterile skim milk at a cell density of ca. 6.8 log CFU ml^–1.

Samples were incubated at 42°C for 18 h, and the pH was recorded on line. Acidification data were modeled according to the Gompertz equation as modified by Zwietering et al. (48), where y is dpH/dt (units of pH h^–1), k is the initial level of the dependent variable to be modeled, A (pH) is the difference in pH (units) between the initial value (pH₀) and the value reached after 18 h, µ_max is the maximum acidification rate (pH h^–1), is the length of the latency phase of acidification expressed in hours, and t is time.

Fermentative characterization by the Biolog system.
Harvested cells of L.helveticus were washed in 50 mM phosphate buffer (pH 7.0), centrifuged at 9,000 x g for 15 min at 4°C, and resuspended in AN Inoculating Fluid (Biolog Inc., Hayward CA) at a value of transmittance of 65 ± 2%. An aliquot (100 µl) of the cell suspension was pipetted into Biolog 96-well AN MicroPlates, which were assessed by the Biolog MicroStation Reader after anaerobic incubation at 42°C for 24 h. For this characterization, cells grown at a constant temperature of 20 or 30°C for 20 h were included also.

	RESULTS

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Kinetics of growth.
Cells of L. helveticus PR4 were grown in cheese whey for 20 h under different temperatures: a naturally decreasing gradient of temperature from 55 to 20°C, shifted temperatures without a naturally decreasing gradient (55 to 20°C), or a constant temperature of 42°C (Fig. 1). Parameters of growth were calculated by using the Gompertz equation. The increase in cell density was similar under all the conditions of cultivation; it ranged from 1.91 ± 0.18 to 2.12 ± 0.15 log CFU ml^–1. The same was found for the final value of pH, which ranged from 3.3 to 3.5. Cells grown at a constant temperature of 42°C had the lowest values of µ_max (0.32 ± 0.02 log CFU ml^–1 h^–1) and (0.93 ± 0.04 h). Growth under a temperature gradient gave values of µ_max and higher than those found in growth under shifted temperatures (0.62 ± 0.02 versus 0.43 ± 0.01 log CFU ml^–1 h^–1 and 2.80 ± 0.05 versus 2.14 ± 0.05 h, respectively). The stationary phase of growth was reached earlier with the temperature gradient (ca. 7.5 h) than at a constant temperature of 42°C (ca. 16 h). Growth under shifted temperature reached the stationary phase after ca. 12 h. For this and further experiments, the behavior of strains PR5 and PR6 was similar to PR4; therefore, results for strain PR4 only are shown.

View larger version (9K): [in this window] [in a new window]   FIG. 1. Kinetics of growth of L. helveticus PR4 grown in cheese whey under a gradient of naturally decreasing temperature (55 to 20°C) (), shifted temperature (55 to 20°C) without a naturally decreasing gradient (•), or constant temperature of 42°C (). Data are the means of three independent experiments.

View larger version (130K): [in this window] [in a new window]   FIG. 2. Two-dimensional electrophoresis analysis of protein expression in L. helveticus PR4 grown in cheese whey for 20 h at a constant temperature of 42°C. Numbered ovals refer to proteins that were present at an increased level under a gradient of naturally decreasing temperature (55 to 20°C). The position of the proteins identified by N-terminal sequencing and peptide mass fingerprinting are indicated: DnaK, GroEL, heat shock low-molecular-weight protein (HSLMWp), putative DNA topoisomerase III (PDNA III), Dps family protein (Dps fp), DNA-binding protein II HB (DNA II HB), N-acetyltransferase (NAT), pyruvate kinase (PK), enolase, glyceraldehyde-3-phosphate dehydrogenase type I (GAPDH I), thioredoxin reductase (TrxR), putative teichuronic acid biosynthesis glycosyl transferase TuaH (tuaH), 50S ribosomal protein L13 (L13), ATP-dependent protease with one transmembrane helix (ATPdp), and ATP synthase (ATPase).

View larger version (88K): [in this window] [in a new window]   FIG. 3. Portions of two-dimensional electrophoresis analysis of the protein expression in L. helveticus PR4 grown in cheese whey under a gradient of naturally decreasing temperature (55 to 20°C). Cells were harvested after 35 min of exposure to 55°C (A) and after the temperature reached 40°C (3 h) (B), 30°C (5 h 30 min) (C), or 20°C (13 h 30 min) (D) (see Materials and Methods). Spot designations correspond to those of the gel shown in Fig. 1.

Proteinase and peptidase activities.
Cells of L. helveticus PR4 grown under a gradient of naturally decreasing temperature were used for enzyme assays at a cell density of ca. 8.0 log CFU ml^–1. The same assay was carried out for cells grown with shifted temperatures. The proteinase activity of cells harvested at 40°C was markedly higher than that found for cells exposed to the other temperatures (Fig. 4A). Cells of L. helveticus PR4 subjected to shifted temperature also showed the highest proteinase activity after exposure to 40°C for 6 h (Fig. 4B). The PepN activity towards Leu-p-NA of cells harvested at 40°C was at least twice that of cells from the other conditions of the temperature gradient(Fig. 5A). The same was found for cells grown under shifted temperatures (Fig. 5B). The same results were found by using substrates relatively specific for PepI, PepX, and PepV enzymes (Table 3). Cells harvested at 40°C either from the naturally decreasing temperature gradient or from shifted temperature showed markedly higher proteinase and peptidase activities than cells grown at a constant temperature of 42°C (data not shown).

View larger version (8K): [in this window] [in a new window]   FIG. 4. Proteinase activity (•) of L. helveticus PR4 cells grown in cheese whey under a gradient of naturally decreasing temperature (55 to 20°C) (A) or shifted temperature (55 to 20°C) (B). The dashed line shows the gradient or shifted temperature during cultivation. One unit of proteinase activity was expressed as an increase of 0.1 unit of fluorescence 10 min–1. Data are the means of three independent experiments.

View larger version (7K): [in this window] [in a new window]   FIG. 5. Aminopeptidase type N (PepN) activity (•) of L. helveticus PR4 cells grown in cheese whey under a gradient of naturally decreasing temperature (55 to 20°C) (A) or shifted temperature (55 to 20°C) (B). The dashed line shows the gradient or shifted temperature during cultivation. One unit of PepN activity corresponded to an increase of absorbance at 410 nm of 0.01 min–1. Data are the means of three independent experiments.

View larger version (10K): [in this window] [in a new window]   FIG. 6. Kinetics of acidification (pH) in skim milk of L. helveticus PR4 cells grown in whey under a gradient of naturally decreasing temperature (55 to 20°C) or at constant temperature of 42°C for 3 h (). Cells were harvested and inoculated in skim milk after 35 min of exposure to 55°C () and after the temperatures of 40°C (3 h) (•), 30°C (5 h 30 min) (), or 20°C (13 h 30 min) () were reached (see Materials and Methods). Data are the means of three independent experiments.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

As expected, no major differences were found in terms of cell yield by comparing growth of L. helveticus PR4 under a temperature gradient, shifted temperature (55 to 20°C) without a naturally decreasing gradient, or a constant temperature of 42°C. Nevertheless, cells grown under a temperature gradient had the highest growth rate and reached the stationary phase of growth rapidly (ca. 7.5 h), probably due to the rapid decrease of the pH in cheese whey. To propagate whey starter L. helveticus PR4 under a temperature gradient or under constant temperature is not a problem for cell biomass recovery.

When cells grown under a temperature gradient were harvested after 35 min of exposure to 55°C or after the temperature reached 40 (3 h), 30 (5 h 30 min), or 20 (13 h 30 min) and then were compared with cells grown for the same time at a constant temperature of 42°C, a transient induction of the expression of several proteins was found by two-dimensional electrophoresis analysis. The level of expression of the major part of these proteins increased during 3 h of incubation from 55 to 40°C. Differences were attributed to heat stress response mainly. HSPs such as DnaK, GroEL, and low-molecular-weight HSPs (small HSPs [sHPSs]) were transiently induced in L. helveticus PR4 until the temperature of 40°C was reached (Table 1). In situ reconstitution experiments showed that DnaK/DnaJ and GroES/GroEL chaperones can interact successively to bind substrate proteins in a transient noncovalent manner, prevent premature folding, and promote the attainment of the correct state (7). The sHSPs form a family with a conserved -crystallin homology domain and molecular masses between 10 and 30 kDa. Their main function appears to be the prevention of the accumulation of unfolded protein intermediates during stress periods (43). Classical response to heat stress includes ATP-dependent protease, ATP synthase, and 50 ribosomal proteins such as L7-L12 and L13, which were overexpressed in L. helveticus PR4. ATP synthase beta chain was found to be induced after heat exposure of GroESL-overproducing Lactococcus lactis and probiotic Lactobacillus paracasei NFBC 338 (12) and, with ATP-dependent protease, is involved in the cell energy metabolism. Biochemical and structure studies have revealed that the GroEL chaperone acts with its partner, cochaperone GroES, as a two-stroke ATP-regulated folding machine, thus involving ATP-related enzymes (44). Protein L7-L12 was also identified in the proteome analysis of HSP expression in Bradyrhizobium japonicum (28). This protein was supposed to be the binding site for several of the factors involved in protein synthesis and appeared to be essential for accurate translation, acting as one of the primary sensors of conditions that evoke the heat shock response (42). Studies on Bacillus subtilis showed that 50S ribosomal protein L13 could act as a possible mediator of Obg, an essential GTP binding protein for this microorganism (35). Members of the Obg subfamily of GTP binding proteins are thought to monitor the state of GTP level in bacteria and serve as a switch to promote growth when bound to GTP but not when associated with GDP (30).

Several other proteins somewhat related to heat shock were identified in L. helveticus PR4 grown under a temperature gradient. Glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase, enolase, and pyruvate kinase were induced in L. helveticus PR4 also. While enolase overexpressed only in the range 55 to 40°C, the level of expression of the glyceraldehydes-3-phosphate dehydrogenase decreased transiently at 30°C and increased again in correspondence with the temperature gradient range from 30 to 20°C. Pyruvate kinase was induced at 40°C, and the level of expression was maintained rather constant at the lower temperatures of the gradient. The same glycolytic enzymes were induced in Lactobacillus rhamnosus HN001 (34) and Streptococcus thermophilus (22) under stress conditions or different culture conditions. Overall, an elevated number of proteins involved in glycolysis-related machinery is an important factor for survival under conditions of stress for either increasing the level of ATP synthesis (46) or compensating the low glycolytic capacity (47). DNA-binding protein II HB was specifically induced in L. helveticus PR4 as the temperature decreased from 55 to 40°C. DNA-binding protein II HB, also called HU-type protein, was overexpressed in Lactobacillus plantarum subjected to heat shock (11). It is a small basic protein which binds DNA without sequence of specificity but recognizes special DNA structures, such as cruciforms and bulges (32). It plays a role in cell division and modulates the interaction of regulatory proteins with their specific sites on DNA.

To our knowledge, the other proteins (putative teichuronic acid, Dps, thioredoxin reductase, putative DNA topoisomerase, and N-acetyltransferase) induced during the growth of L. helveticus PR4 under a temperature gradient were not attributed previously to heat shock response. Overall, resistance by defense systems may be divided into two classes: specific systems for response to specific stress and more general system for response to a large variety of stress conditions. The borderline between the two classes is sometimes difficult to define. Putative teichuronic acid biosynthesis glycosyl transferase TuaH is involved in substitution of phosphate-rich teichoic acids by phosphate-free teichuronic acid under starvation and/or phosphate-limited growth to maintain the cell's shape, irrespective of the chemical nature of the cell wall anionic polymer (33). Dps family protein plays a major role in the protection of bacterial DNA against damage by reactive oxygen species (38). Two major functions have been suggested: binding of Dps to DNA to decrease the number of single-stranded breaks induced by a hydroxyl radical and sequestering iron to protect DNA from damage by acting as a physical shield. The gene trxB encoding the thioredoxin reductase has been identified in Lactobacillus johnsonii (13). Overall, thioredoxin reductase has a role in maintaining the reduced state of cytoplasmic proteins and protects Lactobacillus cells against the accumulation of detrimental cytoplasmic disulfide bonds. Putative DNA topoisomerase III is efficient in the decatenation of gapped, multiply interlinked DNA dimers and DNA replication intermediates. Based on this observation, it has been proposed that DNA topoisomerase III has a role in chromosomal segregation (9). N-Acetyltransferase has been shown to confer freeze tolerance by reducing the intracellular levels of reactive oxygen species, and, more generally, to be involved in the adjustment mechanisms linking fatty acid composition to various stress factors (21). As a first part of this work, it was concluded that L. helveticus PR4 during growth under a gradient of naturally decreasing temperature responded through the synthesis, in most cases transiently (from 55 to 40°C), of stress proteins, glycolysis-related machinery, and other regulatory proteins of factors which confer a competitive advantage of cell resistance.

Therefore, the resulting population of L. helveticus at the end of propagation in whey under a temperature gradient is a mixture of cells that had been subjected to transient heat adaptation and may show differences in technological properties. Proteinase and peptidase (PepN, PepI, PepX, and PepV) activities were found to be higher for cells harvested when the temperature reached 40°C. The same was found for the acidification rate in skim milk. These activities were also higher than those found in cells grown at a constant temperature of 42°C for the same time. Studies (39) on the effect of temperature (controlled or not) on lactic acid production from cheese whey using L. helveticus showed that a temperature above 40°C induced maximum specific growth, rate of lactose utilization, and lactic acid production. For L. helveticus PR4, it seemed that growth at a temperature higher than 40°C is important, but, especially, the exposure to a transient decrease in temperature from 55 to 40°C for a certain time (ca. 3 h) is necessary to express the highest proteolytic and acidification activities. Overall, starters provide the most significant contribution to the microbial biomass in young curd, typically attaining a density of 10⁸ CFU g^–1 within 1 day (31). Therefore, the contribution of primary starters to cheese ripening is fundamental, especially for cheeses such as Parmigiano Reggiano and Grana Padano, which do not use secondary starters. To use strains of L. helveticus which had been propagated in cheese whey under a modified temperature gradient, compared to the traditional gradient (e.g., by maintaining the temperature constant at 40°C after the initial decrease from 55°C), could be a competitive advantage in terms of acidification rate and proteolysis during cheese manufacture and proteolysis. As shown by the Biolog system characterization, cells harvested at 40°C under a temperature gradient achieved the complete phenotypic traits of the species.

This is the first study of the heat stress response of L. helveticus during propagation in cheese whey under a temperature gradient and shows the transient induction of several proteins and technological properties that are related to heat adaptation.

	FOOTNOTES

 
* Corresponding author. Mailing address: Dipartimento di Protezione delle Piante e Microbiologia Applicata, Facoltà di Scienze Biotecnologiche di Bari, Via G. Amendola 165/a, 70126 Bari, Italy. Phone: 39 080 5442948. Fax: 39 080 5442911. E-mail: m.deangelis{at}agr.uniba.it.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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