International Journal of Food Microbiology 191 (2014) 135–143

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Cold stress improves the ability of Lactobacillus plantarum L67 to survive freezing Sooyeon Song a, Dong-Won Bae b, Kwangsei Lim c, Mansel W. Griffiths d, Sejong Oh a,⁎ a

Division of Animal Science, Chonnam National University, 77 Yongbong-ro, Gwangju 500-757, Republic of Korea Central Instrument Facility, Gyeongsang National University, 900 Gajwa-dong, Jinju, Gyeongnam 660-701, Republic of Korea Dairy Food R&D Center, Maeil Dairies Co., Ltd., 480, Gagok-ri, Jinwi-myun Pyungtaek-si, Republic of Korea d Department of Food Science, University of Guelph, Canadian Research Institute for Food Safety, Guelph, ON, Canada b c

a r t i c l e

i n f o

Article history: Received 27 March 2014 Received in revised form 11 August 2014 Accepted 14 September 2014 Available online 18 September 2014 Chemical compounds studied in this article: Sodium chloride (PubChem CID: 5234) Phenol (PubChem CID: 996) Acetone (PubChem CID: 180) Urea (PubChem CID: 1176) CHAPS (PubChem CID: 107670) Dithiothreitol (PubChem CID: 19001) Isopropanol (PubChem CID: 3776) Glycerol (PubChem CID: 753) Bromophenol blue (PubChem CID: 8272) Ammonium bicarbonate (PubChem CID: 14013)

a b s t r a c t The stress resistance of bacteria is affected by the physiological status of the bacterial cell and environmental factors such as pH, salts and temperature. In this study, we report on the stress response of Lactobacillus plantarum L67 after four consecutive freeze–thaw cycles. The cold stress response of the cold-shock protein genes (cspC, cspL and cspP) and ATPase activities were then evaluated. The cold stress was adjusted to 5 °C when the bacteria were growing at the mid-exponential phase. A comparative proteomic analysis was performed with twodimensional gel electrophoresis (2D SDS-PAGE) and a matrix assisted laser desorption/ionization-mass spectrometer. Only 56% of the L. plantarum L67 cells without prior exposure to cold stress survived after four consecutive freeze–thaw cycles. However, 78% of the L. plantarum L67 cells that were treated with cold stress at 5 °C for 6 h survived after freeze–thaw conditions. After applying cold stress to the culture for 6 h, the cells were then stored for 60 days at 5 °C, 25 °C and 35 °C separately. The cold-stressed culture of L. plantarum L67 showed an 8% higher viability than the control culture. After applying cold stress for 6 h, the transcript levels of two genes (cspP and cspL) were up-regulated 1.4 (cspP) and 1.2 (cspL) times compared to the control. However, cspC was not upregulated. A proteomic analysis showed that the proteins increased after a reduction of the incubation temperature to 5 °C. The importance of the expression of 13 other relevant proteins was also determined through the study. The exposure of L. plantarum cells to low temperatures aids their ability to survive through subsequent freeze–thaw processes and lyophilization. © 2014 Published by Elsevier B.V.

Keywords: Lactobacillus plantarum L67 Cold stress response cspC cspP cspL

1. Introduction Lactobacilli and bifidobacteria are well known as the most frequently and safely used starter probiotic bacteria, even for children and immunocompromised individuals (Borriello et al., 2003). In particular, Lactobacillus plantarum belongs to the facultatively heterofermentative group of lactobacilli and is a heterogeneous and versatile species that is encountered in a variety of environmental niches, including dairy, meat, fish, and many vegetable or plant fermentations. It has a proven ability to survive gastric transit and colonize the intestinal track of humans and other mammals (Sartor, 2004; de Vries et al., 2006). ⁎ Corresponding author at: Division of Animal Science Chonnam National University, 77 Yongbong-ro Puk-gu, Gwangju 500-757, Republic of Korea. Tel.: +82 62 530 0822; fax: +82 62 5302129. E-mail address: [email protected] (S. Oh).

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.09.017 0168-1605/© 2014 Published by Elsevier B.V.

During the manufacture of lactic acid fermented products, bacterial cells are exposed to various environmental stresses such as extreme temperatures, pH, osmotic pressure, oxygen, high pressure and starvation, which may affect the physiological status and the properties of the cells. However, bacterial cells are naturally equipped with a plethora of defense mechanisms, such as chaperone proteins (Gro ES/GroEL and DnaK/ DnaJ/Grp E), proteases, transport systems and proton pumps to enhance survival in stressful environments (Carcoran et al., 2008). Moreover, many research studies have reported the positive effects of the stress response. For example, to improve the viability of the probiotic strain Lactobacillus paracasei NFBC 338 during spray-drying, Desmond et al. (2004) demonstrated that pre-stressing the culture by exposure to 52 °C for 15 min improved the survival of the strain 700-fold (in reconstituted skim milk) during heat stress and 18-fold during spraydrying compared to unadapted cells. Exposure to salt stress also afforded a level of thermo-tolerance. Indeed, following exposure to

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0.3 M NaCl, the survival of the strain improved by 16-fold during spraydrying (Desmond et al., 2001). Freezing is generally used to preserve a starter culture for an extensive amount of time while still maintaining its viability and acidification activity. After freezing and thawing, many cells are often metabolically damaged or even killed. The freezing resistance of Lactobacillus is enhanced by applying cold stress conditions before freezing. In contrast with Escherichia coli, Bacillus subtilis, and Lactococcus lactis, the function of the cold response in L. plantarum has not been extensively studied (Barria et al., 2013). Cold-shock response is classically exhibited when an exponentially growing culture is shifted from its optimum growth temperature to a lower temperature. In most bacteria, such as B. subtilis, a temperature downshift causes a transient cell growth arrest, during which the general protein synthesis is severely inhibited. However, under these conditions, the synthesis of CIPs is triggered. Eventually, the synthesis of these proteins decreases, the cells become acclimated to the low temperature, and growth resumes (Jones et al., 1987). Recently, it has been established that CSPs might regulate the expression of cold-induced genes such as anti-terminators (Bae et al., 2000). The regulation of csp genes takes place at several levels. Additionally, the regulation of CspAE expression seemed to occur transcriptionally and post-transcriptionally through protein stability effectors (Goldenberg et al., 1997). The chromosome of L. lactis contains two sets of cold-inducible csp genes (cspA/cspB and cspC/cspD) (Wouters et al., 2000a, 2000b). Small heat shock (shs) genes are also induced by cold in L. plantarum, and a role for the sHsps in preventing damage by low temperature has been suggested and L. plantarum strains overproducing Hsp 18.5, Hsp 18.55 and Hsp 19.3 have improved growth at low temperature (Spano et al., 2005; Fiocco et al., 2007). L. plantarum is usually exposed to harsh, stressful environments during handling and storage. These harsh environments include being either freeze-dried or just subjected to a regular freeze during the food process through the GI tract. In the case of L. plantarum, cold stress genes such as cspC, cspL, and cspP have been identified (Mayo et al., 1997; Derzelle et al., 2000). The purpose of this study was to determine the survival rate of csp mRNA levels and the inductive proteins associated with L. plantarum L67 under cold stress. 2. Materials and methods 2.1. Bacteria L. plantarum L67 from infant feces were selected according to their colony characteristics, and underwent a gram stain to be tested for catalase activity. These isolates were further screened for their acid and bile salt tolerance capacities according to Park et al. (2002). For identification of L. plantarum L67, the biochemical properties were first examined using an API 50CHL kit (BioMerieux, France) and, finally, 16s rDNA sequencing data, as described by Kim et al. (2004). L. plantarum L67 was grown in MRS broth at 37 °C for 18 h (de Man et al., 1960). The bacterial cells were separated by centrifugation at 3000 ×g for 15 min at room temperature. Subsequently, the precipitated cells were washed twice with sterile saline (0.85% sodium chloride). One milliliter of 10% skim milk was added to the sediments, and the suspension was stored at −80 °C until use. 2.2. Growth conditions of L. plantarum L67 and the cold stress treatments The cells were inoculated in 1% of the overnight culture of the strain in 10 ml of fresh MRS broth, incubated at 37 °C and harvested when they reached the mid-exponential phase of growth (OD 600 nm of 0.6). Growth kinetic experiments were performed with MRS broth at 37 °C. For cold shock experiments, the exponentially growing cells were incubated at 5 °C for 1, 4 or 6 h, and then growth kinetic experiments were performed at 37 °C. All experiments were carried out in triplicate.

2.3. Freeze–thaw challenge Two hundred milliliters of MRS broth was inoculated (1%) and incubated at 37 °C. The cells grown to the mid-exponential phase were cold shocked at 5 °C for 1, 4 and 6 h. Then, the cells were quickly frozen and stored at − 70 °C. The control group consisted of cells frozen directly without pre-adaptation for 24 h of freezing. After, the cells were thawed by placing the tubes in a 37 °C water bath for 5 min. Aliquots of each sample were taken out and analyzed for viability before they were frozen again at −70 °C. A total of four freeze–thaw cycles were performed. Each freeze–thaw cycle was performed after 24 h of the previous one. The thawed cells were serially diluted with 1% peptone water, and the viable cells were counted.

2.4. Storage stability of freeze-dried L. plantarum L67 L. plantarum L67 was grown in 1 l of MRS broth at 37 °C. When the growth reached the mid-exponential phase (OD600 of 0.6), the cells were subjected to 6 h of cold shock treatment at 5 °C in fresh MRS broth. The cell pellets were collected by centrifugation (5000 ×g at 4 °C) and then re-suspended in skim milk before being frozen at −70 °C. The frozen cells were freeze-dried for 48 h at 0.2 mbar with a collecting temperature of −50 °C (IlShin Lab, Yangju, Korea). The cell suspensions were freeze-dried in duplicates. Duplicate samples of each treatment were placed in glass vials (40 ml) and purged with nitrogen gas (10 ml/min) for 10 min. The glass vial lots were stored at 5 °C, 25 °C or 35 °C in the dark for 60 days.

2.5. RNA isolation and RT-qPCR Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA) with the manufacturer's instructions. The cDNA was prepared from 1 μl of total RNA (1 μg/20 μl) using Maxime RT Premix Oligo (dT) for the RT-qPCR kit (Intron, Seongnam, Korea). The primers used in this study were designed by Primer3 software program (www.bioinformatics.nl/primer3plus) based on the genome sequence of L. plantarum WCFS1 (GenBank accession number NC_ 004567). The primers used are shown in Table 1. The expression levels of the respective genes were measured by real-time PCR using 2× Prime QMaster Mix (Kapa Biosystems, Boston, USA) and were analyzed using a CFX96TM Real-Time system (Bio-Rad, California, USA). The reaction parameters for the real-time PCR analysis were 94 °C for 5 min, followed by 35 cycles of 94 °C for 40 s, 58 °C for 40 s, and 72 °C for 60 s. The final elongation step was at 72 °C for 5 min. The sample ΔCt (SΔCt) value was calculated as the difference between the Ct values of the csp gene before and after applying the cold shock. The ΔCt value of the undifferentiated L. plantarum WFSC1 rpoD (Kleerebezem et al., 2003; Duary et al., 2012) gene was used as the control ΔCt (CΔCt) value. The relative gene expression levels between the sample and the control were determined using the formula: 2−(SΔCt − CΔCt) (Livak and Schmittgen, 2001).

Table 1 Oligonucleotides used for real-time PCR in this study. Gene

Sequence of PCR primers (5′ to 3′)

cspP

Forward: 5′GTGAAGACGGTACCGATGTCTT-3′ Reverse: 5′GTGGTTGAACGTTCGTTGCT-3′ Forward: 5′ATCACTCGCGAAAACGGTAG-3′ Reverse: 5′CCACGATCGCTTTCTTCAAC-3′ Forward: 5′TACTGGTGAAGATGGCAACG-3′ Reverse: 5′GAACAACGTTAGCAGCTTGTGG-3′ Forward: 5′GTGAAGAGGACGATTCACACCT-3′ Reverse: 5′GGTATCAAGGACACCTTCCAG-3′

cspC cspL rpoD

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2.6. Protein extraction of L. plantarum L67 For the proteomic study, 200 ml of the mid-exponential phase (OD600 of 0.6) cultures was obtained as described above at 37 °C. For the cold shock experiments, the cells were cultivated at 37 °C at the mid-exponential phase (OD600 of 0.6) and subjected to 1, 4, or 6 h (respectively) of cold shock treatment at 5 °C. The cell pellets were collected by centrifugation (12,000 ×g, 4 °C). The collected cell pellets were washed 3 times using 0.85% NaCl. The soluble protein fractions were extracted twice with phenol (Sigma Chemical Co, St. Louis, USA) (Kim et al., 2008). Then, 2 ml aliquots of each individual extract were transferred to 15 ml polypropylene tubes. One milliliter of phenol (Sigma Chemical Co.) was then added. The sample was vortexed after and was heated at 70 °C for 10 min. The sample was cooled on ice for 5 min, and the phases were separated by centrifugation at 12,000 ×g for 20 min. The top aqueous phase was discarded and replaced by 1 ml of distilled H2O. The sample was then vortexed and heated at 70 °C for 10 min. The sample was cooled on ice for 5 min, and the phases were separated again by centrifugation, as before. Then, the top aqueous phase was discarded. The proteins were precipitated by the addition of cold acetone at 4 °C for 2 h. After centrifugation, the supernatant was discarded, and cold acetone was added. The pellet was disrupted by vigorous vortexing, and the precipitated proteins were pelleted by a final centrifugation of 12,000 ×g for 20 min. The resulting pellet was airdried for 2 h under a hood. The protein concentrations were measured by a Bradford assay (Bradford, 1976) using the Bio-Rad Protein Assay (Bio-Rad, California, USA).

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Mini Bead beater (BioSpec Products, Seoul, Korea). After the addition of DNase and RNase (10 μg/ml), the solutions were maintained at room temperature for 45 min. The unbroken cells and debris were subsequently removed by centrifugation at 15,000 ×g for 20 min. The supernatant was further centrifuged at 20,000 ×g for 20 min, and the ATPase activity in the supernatant was determined. 2.10. ATPase activity assay The ATPase activity was estimated from the release of inorganic phosphate, as measured according to the method of Fiske and Subbarow (1925). The membrane protein was incubated at 37 °C for 10 min in Tris maleate buffer (pH 7.5, 50 mM) containing 10 mM MgCl2 and 5 mM ATP (Sigma Chemical Co, St. Louis, USA). The reaction was stopped by addition of 1 ml of sterile water containing 3.5 N sulfuric acid, 1 ml of 3.5% ammonium molybdate and 1 ml of 2.1% sodium bisulfite with 0.7% Developer (Kodak Korea, Seoul, Korea) subsequently added. After 20 min, the absorbance at 660 nm was measured. One unit of ATPase activity was defined as the amount of enzyme that 1 μmol of inorganic phosphate released in 1 min. The calibration was performed with a series of Pi standards. 2.11. Statistical analysis All the experiments were performed in triplicate. The data were analyzed using Duncan's multiple range test (Duncan, 1955). A P value of b0.05 in all replicate experiments represented statistical significance.

2.7. Two-dimensional gel electrophoresis 3. Results Protein loads of 1 mg/ml in 315 μl of rehydration buffer (8 M urea, 2% CHAPS, 50 mM dithiothreitol, 10% isopropanol, 5% glycerol, 0.5% ampholytes at pH of 3 to 10, and a trace of bromophenol blue) were used to load the Ready Strip IPG Strips (17 cm, pH 3 to 10, Genomine, Pohang, Korea) under mineral oil at 20 °C for 15 h. Isoelectric focusing (IEF) was performed using a PROTEAN IEF cell (Bio-Rad, California, USA) according to the manufacturer's instructions. The two dimensional gel electrophoresis (2D SDS-PAGE) was performed at 150 constant volts using 12.5% SDS-PAGE gels. The separation was conducted using a PROTEAN II xi system (Bio-Rad, California, USA) with 10 mA per gel for 1 h and thereafter with 20 mA per gel at 4 °C. The protein spots were visualized by Coomassie blue staining (Candiano et al., 2004). The stained gels were scanned with a densitometric scanner (800 by 1600 dpi; UTA 2100XL, UMAX, Techville Inc., Dallas, TX), and the spots were analyzed with PD Quest software (Bio-Rad, California, USA).

3.1. Effect of temperature on L. plantarum L67 growth As a first step to understanding the cold stress response in L. plantarum L67, we constructed a growth curve for L. plantarum L67 at 5 °C, 37 °C and under cold stress at 5 °C following growth to the mid-exponential phase at 37 °C. The cell viability at 37 °C after cold shock treatment for 1, 4 or 6 h was also determined. Fig. 1 shows the growth curve of L. plantarum L67 at 37 °C and 5 °C. The L. plantarum on the MRS broth at 37 °C reached an OD of 1.2 (OD600) after 6 h, whereas the L. plantarum cells did not grow at 5 °C. The effect of prior cold stress on the growth kinetics of L. plantarum L67 at 37 °C is shown in Fig. 2. The cell density reached 1.2 (OD600) at 2.0

2.8. Analysis of matrix assisted laser desorption/ionization-mass spectrometer (MALDI-MS)

2.9. Crude enzyme extract of L. plantarum L67 for the ATPase activity assay The L. plantarum L67 cultures at the mid-exponential phase of growth (OD600 of 0.6) at 37 °C were harvested by centrifugation at 7000 ×g for 10 min. Then, they were washed three times at room temperature in sterile water. The cells were suspended in Tris HCl buffer (pH 7.5, 75 mM) containing 0.4 mM sucrose and 2 mM MgCl2 and were disrupted by grinding with 0.1 mm glass beads using the Micro

OD at 600 nm

The protein spots of interest were manually excised. The proteolytic digestion was performed overnight at 37 °C using trypsin (20 μg/ml; Promega, WI, USA) in the presence of 25 mM ammonium bicarbonate. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) was employed using the method described by Kim et al. (2008) for protein identification. The obtained spectra from the Ettan MALDI-TOF (Amersham Pharmacia Biotech, Uppsala, Sweden) were subjected to analysis by Mascot software.

1.5

1.0

0.5

0.0

0

3

5

6

9

12

24

Incubation time (h) Fig. 1. Growth curve of L. plantarum L67 at 37 °C (□), 5 °C (*) and cold stress at 5 °C (△) after growth at 37 °C until reaching an OD600 of 0.6. The arrow (→) indicates a temperature change shift to 5 °C. The cells were inoculated in 1% of 10 ml of overnight culture in 10 ml of fresh MRS broth and incubated at the appropriate temperature (37 °C and 5 °C). For the cold stress experiments, when the cultures reached 0.6 (OD600) at 37 °C, they were transferred to incubation at 5 °C. The optical density (OD) was measured with a Synergy HT multimode plate reader (BioTek, Winooski, VT). The results are the means of triplicated trials, and the error bars indicate the standard errors.

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2.0 1.8 1.6 OD at 600 nm

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0

3

6

9 12 Incubation time (h)

15

18

24

Fig. 2. Effect of cold stress on the growth kinetics of L. plantarum L67. The cells were cultured in MRS broth at 37 °C prior to cold stress; control ( ); 5 °C (□); cold stress at 5 °C for 1 h (▲), 4 h (×), and 6 h (●). The cells were inoculated in 1% of 10 ml of overnight culture in 10 ml of fresh MRS broth and were incubated at 37 °C. For the cold stress, the inoculated culture was incubated at 5 °C for 1, 4 or 6 h, and then growth kinetic experiments were performed at 37 °C. For the control, the growth kinetic experiments were performed without cold stress. The OD was measured with a Synergy HT multimode plate reader (BioTek, Winooski, VT). The results are the means of triplicated trials, and the error bars indicate the standard errors.

37 °C in 6 h, whereas it reached only 0.6 after prior exposure to 5 °C for 1 h. However, after 9 h of pre-exposure at the lowest temperature, the cells grew at a similar rate to those of the control. The fastest resumption of growth occurred for the cells that had been subjected to cold stress for 6 h.

3.2. Freeze–thaw challenge of L. plantarum L67 To measure the freezing tolerance, the freeze and thaw cycle was repeated for 4 cycles. The survival ratio of the cells after the freeze–thaw was then determined and is shown in Fig. 3. Only 56% of the control cells without cold shock survived after 4 consecutive freeze–thaw cycles. However, the cells adapted for 4 and 6 h at 5 °C exhibited increased survival (up to 69 and 78%, respectively). The cells exposed to cold stress for 6 h exhibited an almost constant level of survival after freeze–

thaw following an initial reduction in count. This result indicates that cryotolerance is improved by subjecting the cells to prior cold stress.

3.3. Storage stability of dried L. plantarum L67 The viabilities of the L. plantarum L67 cultures were initially 3.8 × 1011 CFU/g and 2.21 × 1011 CFU/g for the cold-stressed cultures and the controls, respectively. At 5 °C, the cell population of the coldstressed L. plantarum L67 remained stable at 1.05 × 1010 CFU/g, while the cell numbers in the control samples declined (1.82 × 109 CFU/g). L. plantarum L67 was stored for 60 days. At 25 and 35 °C storage, dramatic losses in cell count were observed in the freeze dried cultures of the probiotic, with levels of L. plantarum L67 cells that were subjected to prior cold-stress decreasing to 4.6 × 108 CFU/g (at 25 °C) and 5.4 × 108 CFU/g (at 35 °C), whereas the

100

Survival (%)

90

80

70

60

50

40 0

1

2

3

4

Freeze-Thaw Cycle (Times) Fig. 3. Survival ratio of the L. plantarum L67 cells after freeze–thaw cycles prior to cold stress. Control (□); cold stress at 5 °C for 1 h ( ), 4 h ( ), and 6 h (■). Two hundred milliliters of MRS broth was inoculated (1%) and incubated at 37 °C. The cells were grown to the mid-exponential phase and were treated with cold stress at 5 °C for 1, 4 or 6 h; then the cells were frozen and stored at −70 °C. The control cells were frozen directly without the cold stress. After 24 h of freezing, the cells were thawed by placing the tubes in a 37 °C water bath for 5 min. A total of four freeze–thaw cycles were carried out. The 4 cycles of the freeze–thaw cycle was performed every 24 h. The thawed cells were serially diluted with 1% peptone water, and the viable cells were counted. The results are the means of triplicated trials, and the error bars indicate the standard errors.

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cspP

Transcription fold level (normalized by rpo D)

1.5

*

*

*

1.0

0.5

0.0 1h

4h

cspL

Transcription fold level (normalized by rpo D)

1.5

6h

*

1.0

0.5

0.0 1h

Transcription fold level (normalized by rpo D)

cell counts in the control sample fell to 8.7 × 107 CFU/g (at 25 °C) and 9.8 × 107 CFU/g (at 35 °C) following 60 days of storage (Fig. 4).

6h

cspC

1.5 Fig. 4. Survival of the freeze-dried L. plantarum L67 during storage for 60 days. L. plantarum L67 was grown in 1 l of MRS broth at 37 °C. The white bar indicates the powders prepared with the control cultures, while the black bar indicates the powders prepared with the cold-stress cultures. When the growth reached the mid-exponential phase (OD600 of 0.6), the cells were subjected to 6 h of cold shock treatment at 5 °C. The cell pellets were collected by centrifugation (5000 ×g at 4 °C) and were then re-suspended in skim milk before being frozen at −70 °C. The frozen cells were freeze-dried. Duplicate samples of each treatment were stored at 5 °C, 25 °C or 35 °C in the dark for 60 days. The cells were diluted with 1% peptone water, and the viable cells were counted after 15 days. The results are the means of triplicate drying trials, and the error bars indicate the standard errors.

4h

1.0

0.5

0.0 3.4. mRNA expression and ATPase activity under cold stress RT-qPCR was performed to determine the expression of the coldshock genes in L. plantarum L67 (Fig. 5). After applying the cold stress, two genes (cspP and cspL) were significantly expressed, but cspC was not expressed at 6 h compared to the control.

1h

4h

6h

Fig. 5. Relative mRNA expression of the cold shock protein genes of L. plantarum L67. The mRNA levels (transcription fold level (normalized by rpoD)) were calculated relative to the transcription level detected in the control. Total RNA from mid-exponential phase cultures (OD600 of 0.6) prior to cold stress. * indicates a significant difference between the control and the treatment of cold stress, P b 0.05.

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APTase activity (pi μmol/min/mg protein)

10.0

*

8.0 *

6.0

4.0 Standard

1h

4h

6h

Fig. 6. ATPase activity of L. plantarum L67 under cold stress. ATPase activity was estimated from the release of inorganic phosphate as measured according to the method of Fiske and Subbarow (1925). * indicates a significant difference between the control and the treatment of cold stress, P b 0.05.

The ATPase activity of L. plantarum L67 subjected to cold stress is shown in Fig. 6. The ATPase activity of L. plantarum L67 grown at 37 °C was 8.5 unit enzymes (UE). The ATPase activity in the cells that were cold stressed for 1 h at 5 °C was 7.2 UE, while following cold stress for 4 h, the enzyme activity reached the maximum observed level of 9.2 UE. The degree of ATPase activity (8.7 UE) after cold stress for 6 h was slightly lower compared to the levels in cells that underwent cold stress for 4 h. In the early stages of cold stress, ATPase activity was decreased, but after 4 h, the ATPase activity of the cells returned to normal levels. 3.5. Inductive protein by cold stress in L. plantarum L67 Fig. 7 displays the 2D SDS-PAGE gels of the CBB stained proteins extracted from L. plantarum L67 following growth at 37 °C (OD600 of 0.6) and subjected to cold stress for 6 h, which resulted in the highest survival rate of cells after the freeze–thaw cycles. Under cold stress, 24 spots were significantly overexpressed compared with the control. The proteins identified by MALDI-TOF/MS were classified according to their functions (Table 2). The thirteen identified proteins were classified into six groups: cell growth (31%), unknown function (23%), energy (15%), stress response (15%), signal transduction (8%) and transcription (8%) (Fig. 8). 4. Discussion L. plantarum is important within the food industry as a starter strain for the fermentation of food. When foods are processed, bacteria are exposed to low temperature stresses. Hence, it is important to understand the cold shock response and cold adaptation phenomenon of bacteria such as L. plantarum L67. As a first step to understanding the cold stress

response in L. plantarum L67, we determined the L. plantarum L67 viability after cold shock treatment for 1, 4 and 6 h before incubation again at the optimal temperature by measuring the growth kinetics. The results of the study assumed that the cells might have a tolerance against freezing upon preadaptation to cold stress due to the expression of stress proteins such as CSPP and CSPL (Fig. 3). The relevance of cold shock and cryotolerance was first reported in B. subtilis, where it was determined that the removal of the cold shock protein B resulted in increased sensitivity to freezing (Willimsky et al., 1992). In many bacteria, survival to cold shock prior to freezing increases survival because cold adaptation likely results in a greater proportion of unsaturated fatty acids in the plasma membrane of the affected cells compared to untreated cultures. However, the exact function of cold shock proteins in relation to cryotolerance is still unknown (El kest and Marth, 1992). In a recent report (Derzelle et al., 2000), it was shown that the overproduction of each CSP causes distinct phenotypic effects in L. plantarum. It is speculated that the different CSP proteins may antiterminate the transcription of the three csp genes of L. plantarum, thus allowing the cell to adequately respond to stressful environmental challenges. In this study, RT-qPCR was performed to measure the expression of cold-shock genes. After cold stress, two genes (cspP and cspL) were significantly expressed, but cspC was not significantly expressed at 6 h compared to the control. Recent reports have shown that cold inducible CSP proteins may also be expressed at specific stages of the normal growth cycle (Weber et al., 2001; Graumann and Marahiel, 1999). The highest mRNA level of cspC is expressed during early exponentially growing cells at the optimal temperature, while the cspC mRNA level declines in the stationary phase (Derzelle et al., 2000). Derzelle et al. (2003) have suggested that cspC overproduction improves growth resumption at the optimal temperature. Although cspC is not primarily implicated in cold shock adaptation, it may play a positive role during the acclimation phase of growth at cold temperatures. To better understand the mechanisms involved in this cold response, the role of the cspC gene of L. plantarum will be further studied in the future. This result is similar to that described by Derzelle et al. (2003). Derzelle et al. (2002) also demonstrate that cold shock has induced the cspL gene. This is a key result of the present study because it demonstrates that csp transcripts may be more stable and more efficiently expressed than other cellular messengers even if their absolute level does not significantly increase during cold exposure. It has been reported that the overproduction of cspL after cold shock transiently augments growth, while the overproduction of cspP and cspC has no effect on growth compared to the control (Derzelle et al., 2003). Additionally in this study, as the mRNA levels of cspP increased, the survival ratio of the cells after freeze–thaw also increased. However, we could not observe the CSP protein in the proteomic analysis. While the csp gene was overexpressed, the CSP protein was not expressed. It may be because

Fig. 7. Comparative two-dimensional gel electrophoresis patterns of the intracellular proteins of L. plantarum L67 growth at 37 °C (A) and under cold stress at 5 °C for 6 h (B). The proteins were subjected to isoelectric focusing (pH 3–10) and resolved in the second dimension by 12.5% SDS-PAGE; the proteins were visualized with CBB staining.

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Table 2 Differentially expressed proteins in L. plantarum L67 with cold shock for 6 h at 5 °C. The proteins were identified by MALTI-TOF/MS. Spot no. Accession number

Homology

% coverage Matched Mascot score Mr value Species peptides

2 3 4 5 6 7 11 15 18 19 21

F4XMM6 A8S6V7 B3X7G0 C3KZR4 gi|167622455 F7MRE9 C1FJ41 UPI0001E29AA2 D3HC60 Q334H4 A8TV77

20 17 12 19 10 13 21 21 54 99 46

11 10 13 12 7 4 8 9 9 5 10

59 59 61 59 60 61 59 59 54 52 55

23509 68556 38641 41066 34112 5412 15350 10913 31481 4285 28634

Lyngbya majuscula Faecalibacterium prausnitzii Shigella dysenteriae Clostridium botulinum Shewanella halifaxensis HAW-EB4 Clostridium botulinum C str. Stockholm Micromonas sp. RCC299 Pseudomonas syringae pv. syringae Streptococcus gallolyticus subsp. gallolyticus Triticum monococcum Alpha proteobacterium

22 24

F0S3W5 F3KX52

Transposase Putative uncharacterized protein Pantothenate kinase Putative YqaJ hypothetical protein Shal_0515 Arsenate reductase-like protein Predicted protein phage integrase family protein Pantothenate synthetase Heat shock protein 101 Universal stress protein UspA and related nucleotide-binding protein Multi-sensor signal transduction histidine kinase Regulatory protein, LysR:LysR, substrate-binding

29 38

11 10

57 56

74423 36076

Desulfurobacterium thermolithotrophum DSM Hylemonella gracilis ATCC 19624

each CSP protein has a different induction time; for example, the csp gene induction time was different. Although the protein was not expressed under cold stress for 6 h, we observed other stress response proteins such as Hsp and the universal stress protein UspA. The activity of ATPase in the cells exposed to cold stress for 1 h showed a significantly reduced activity compared to the control cells. However, the cells exposed for 4 h showed significantly higher activities compared to the control cells. The results indicated that when the cells were exposed to low temperatures, ATPase activity decreased, whereas the ATPase activity of the adapted cells increased. We suggest that ATPase activity is related to the maintenance of membrane fluidity. When the growth temperature decreases, there is an increase of

Unknown funtion 23%

unsaturated fatty acids and methyl branching. Thus, the decrease of average chain length improves the membrane fluidity (Casanueva et al., 2010). For the proteomic analysis, we performed 2D SDS-PAGE (Fig. 7). Under cold stress, twenty four spots were overexpressed and thirteen spots were identified. The proteins were classified into six groups: energy metabolism, cell growth, unknown function, signal transduction, stress response and transcription (Fig. 8). Thirty-one percent of the overexpressed proteins were related to cell growth. We reported that spot 4 and spot 18 were related to pantothenate kinase and synthetase. Pantothenic acid is used in the synthesis of coenzyme A (CoA). CoA is important to energy metabolism for

Energy metabolism 15% Signal transduction 8%

Stress response 15% Cell growth 31% Transcription 8%

Fig. 8. Functional classification of the identified proteins from L. plantarum L67 analyzed by MALDI-TOF/MS using Map Man ontology as described by Beven et al. (1998).

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pyruvate to enter the tricarboxylic acid cycle (TCA cycle) as acetyl-CoA and for α-ketoglutarate to be transformed to succinyl-CoA in the TCA cycle (Gropper et al., 2008). CoA is also important for the biosynthesis of many important compounds such as fatty acids, cholesterol, and acetylcholine (Gropper et al., 2008). Spot 2 had homology with a transposase protein, which is an enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another part of the genome by a cut and paste mechanism or through a replicative transposition mechanism (Jones et al., 2011). Spot 5 had homology with a putative protein, YqaJ. The function of this protein domain is to digest DNA for recombination. This recombination is crucial to viral replication (Vellani and Myers, 2003). DNA exonucleases play roles in DNA metabolism, e.g., in replication, repair, and recombination. Spot 15 was associated with the homology phage integrase family protein. Spots 2, 5 and 15 were related to chromosome structuring. The proteins related to energy metabolism were identified as spots 3 and 7. Spot 3 was recognized as a protein involved in electron transport (Sudarsanam et al., 2007). Spot 7 was related to arsenate reductases such as oxidoreductases. We observed an up-regulation of the proteins involved in the stress response, which included spots 19 and 21. Spot 19 was identified as a heat shock protein (Hsp), which is a stress response protein. Wu et al. (2009) have reported that heat shock proteins respond to low pH stress; the proteins have the function of preventing the accumulation of unfolded protein intermediates under stress. Additionally, Fiocco et al. (2007) have reported that sHsp may respond to specific environmental stresses. L. plantarum transformed strains were able to tolerate high and low temperature stresses. Taken together, these observations suggest that signal transduction machinery and transcription factors that mediate the genetic response to stressful conditions appear to be shared by different stress-regulative networks (Capozzi et al., 2011). The universal stress protein UspA-related nucleotide-binding protein was induced (spot 21). The levels of UspA have been evaluated in the response to the cell status of E. coli under stressful conditions (Gustavsson et al., 2002; Kvint et al., 2003). The UspA protein of E. coli has a role in resisting damage to DNA. Based on our conclusions, the induced expression of the nucleotide-binding protein UspA may be associated with protecting the DNA of cells during cold stress. However, the exact physiological role of UspA in L. plantarum L67 requires further study. Spot 22 had homology with multi-sensor signal transduction histidine kinase. Histidine kinases (HK) are multifunctional and typically transmembrane; they are proteins of the transferase class that plays a role in signal transduction across the cellular membrane (Wolanin et al., 2002). Spot 24 was a regulatory protein involved in regulated transcription (Chen et al., 2011). Among the identified proteins, spots 6 and 11 have yet to be identified according to their function (Worden et al., 2009). Many L. plantarum L67 proteins were overexpressed in the cells exposed to low temperatures, and these proteins may interact with each other, resulting in cryotolerance. Cold stress induced proteins play roles in a variety of cellular processes such as fatty acid metabolism, chromosome structuring, transcription, translation, general metabolism, energy metabolism, and stress response (Graumann et al., 1997; Wouters et al., 2000a, 2000b; Yamanaka et al., 1998). Additionally, the induced proteins in this study were related to energy metabolism, cell growth, signal transduction, stress response and transcription. In conclusion, we suggest that L. plantarum L67 adapts to low temperatures by a constitutive expression of the potentially cryoprotective cspP gene and proteins related to metabolism and stress response. In a bio-preservation strategy, the protective strains are generally cultivated at their optimum temperatures, eventually freeze-dried, and then directly inoculated into products that are stored at chilled temperatures. Cold acclimation thus constitutes a demonstrable advantage in bacterial competition against spoilage and pathogenic psychrotrophic bacteria in terms of food preservation. This property would enable

cells to multiply at optimal temperatures and to be successfully inoculated into chilled foodstuffs without delaying the food preservative effect.

Acknowledgments This research was supported by iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (112121-2).

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Cold stress improves the ability of Lactobacillus plantarum L67 to survive freezing.

The stress resistance of bacteria is affected by the physiological status of the bacterial cell and environmental factors such as pH, salts and temper...
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