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Keywords:

  • Lactobacillus plantarum;
  • Oenococcus oeni;
  • Wine;
  • Stress

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

A total of 76 Lactobacillus plantarum and Oenococcus oeni wild strains were recovered from traditionally elaborated Spanish red wines and were investigated with respect to their response to acid pH, lyophilisation, temperature and ethanol concentrations which are normally lethal to lactic acid bacteria. Both L. plantarum and O. oeni strains were able to grow at pH 3.2, were highly resistant to lyophilisation treatment and proliferated in the presence of up to 13% ethanol at 18°C. Therefore, it is shown that both species are highly tolerant to stress conditions and that similarly to O. oeni strains, L. plantarum strains are of interest in beverage biotechnology.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Lactic acid bacteria (LAB) are widely used in food biotechnology and efficient control of these microbiological processes requires an increase in our knowledge of bacterial behaviour under stress conditions. Various physical-chemical factors, such as ethanol, pH and temperature, are known to affect the growth of the LAB responsible for the malolactic fermentation (MLF) of wine [1]. Ethanol is generally regarded as one of the principal inhibitors of bacterial growth. Ethanol resistance varies with a number of conditions in the medium (i.e. pH and temperature) and Oenococcus oeni has long been reported as the LAB species most resistant to the presence of ethanol in wine (for a review, see [2]). O. oeni is used as a starter culture for MLF in wine and cider. The conversion of l-malic acid to l-lactic acid and CO2 deacidifies wine, which leads to a significant influence on its quality and stability. MLF gives wines additional flavours and stability for ageing and is, therefore, sought for producing old red wines and some old white wines [1]. Many factors appear to affect MLF, which at the present cannot be fully controlled. This may lead to a number of processing problems, time consumption and risk of alteration of wine. When MLF is desired, the addition of bacteria is a general practice [3], but cells undergo rapid death due to the harsh environment (pH between 3.0 and 3.6, presence of ethanol and SO2). The resistance to ethanol varies from strain to strain and it is generally accepted that all O. oeni strains grow in a medium containing 10% ethanol at pH 4.7 [4] and that small quantities of ethanol (3–4%) can stimulate their growth [3]. The optimum pH reported for the growth of O. oeni is between 4.3 and 4.8 and a pH of 3 or lower prevents almost all growth [4]. The optimum growth temperature in wine seems to be between 20 and 25°C [4] and at 15 °C or lower temperatures the possibility of bacterial growth in wine is slight [5]. When MLF is to be induced, it is important to know which organism is best suited, as the strain or strains must have the ability to grow under rather adverse conditions.

It has long been reported that exposure to stress conditions such as heat, ethanol or acid pH can provide protection against further hostile environmental conditions [6,7]. This adaptive response requires the expression of some defence mechanisms, so that bacteria become more tolerant to adverse conditions following exposure to mild stress conditions. Thus, acclimatisation to cold temperatures can render LAB cryotolerant [8–11] and accumulation of certain osmoprotectant organic compounds is a response to osmotic stress in certain LAB [12,13] and a method of survival of LAB subjected to drying [14]. O. oeni resistance mechanisms to wine stress conditions have been studied [15–20]. Lactobacillus plantarum responses to osmotic stress, which includes salt and non-electrolyte stress, as well as to cold shock [6] and oxidoreduction potential [21], have also been studied. This species is widely used in food biotechnology of fermented products of animal origin and its behaviour after freezing and drying has been the object of great interest [22–24]. Nevertheless, to date no study has been reported on the ability of L. plantarum strains to grow and proliferate under stress conditions of ethanol presence in the medium and acid pH.

The purpose of this study was to determine which O. oeni strains were best adapted to the adverse growth conditions of wine, and our results demonstrated that L. plantarum strains were as well adapted to those conditions of acid pH and ethanol presence, revealing a high tolerance to lyophilisation, to ethanol concentration up to 13% (v/v) in the medium, and to low pHs in the range 3.2–4. A comparison is established between the behaviour of O. oeni and L. plantarum. Results indicate that L. plantarum strains could constitute starter cultures for induction of MLF in wines, and that strains possess resistance mechanisms to survive and proliferate in hostile wine media.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Bacterial strains and growth conditions

A total of 259 L. plantarum and O. oeni isolates were recovered from Spanish Rioja red wines produced during the period 1994–2000. Alcoholic fermentations were conducted in the presence of grape skins, seeds and stalks, after the addition of SO2, and until the residual reducing sugar content was below 2.20 g l−1. At this end point of alcoholic fermentation, wines (alcohol 12% by volume; pH≥3.4) were drawn off from the yeast lees and allowed to undergo spontaneous MLF with the endogenous microflora (no starter inoculum was used). Samples were collected during this spontaneous MLF. Wine samples of 10 ml were spun at 100×g for 3 min at 4°C (Sorwall RC-5 B refrigerated superspeed centrifuge). Pellets containing fermentation debris were discarded and supernatants were spun at 1000×g for 10 min under the same conditions. Pellets were collected and after appropriate dilutions in sterile saline solution (0.9% NaCl) they were seeded onto MRS agar (Scharlau Chemie, Barcelona, Spain) plates with 200 μg of nystatin per ml (Acofarma, Terrassa, Spain). Samples were incubated at 30°C under strict anaerobic conditions (GasPak, Oxoid, Basingstoke, UK). These anaerobic conditions were used to prevent the growth of acetic acid bacteria and to accelerate LAB growth. Colony reisolation was carried out and a total of 259 isolates were used for this study. It should be pointed out that all the isolates were recovered from wines that after MLF followed the normal elaboration processes at their corresponding wineries, were bottled and underwent quality and sensorial analysis before commercialisation.

2.2Bacterial species identification

Strain species were identified according to previously recommended methods, which included bacteria morphology, Gram staining, and carbohydrate fermentation patterns [25]. The API 50 CHL system (BioMérieux, La Balme, France) was also used. O. oeni and L. plantarum species were confirmed in all the studied isolates by the species-specific polymerase chain reaction (PCR) analyses described by Zapparoli et al. [26] and Quere et al. [27], respectively. L. plantarum strains were grown onto MRS agar (Scharlau) plates, at 30°C either under 98% humidity and 5% CO2 atmosphere, or under strict anaerobic conditions (GasPak, Oxoid). MRS medium pH was 6.2. O. oeni strains were grown onto MLO (Scharlau) agar and under the same conditions. MLO medium pH was 4.7. Strains were stored in 20% w/v sterile skim milk (Difco, Madrid, Spain) at −20°C.

2.3Clonal characterisation of strains by PFGE

Genomic DNAs were immobilised into agarose blocks, subjected to restriction enzyme digestion, and separated by pulsed field gel electrophoresis (PFGE) according to the following method. Bacterial cells from fresh cultures were recovered by centrifugation and immobilised in 1% agarose (pulsed field certified agarose, Bio-Rad, Hercules, CA, USA) in 0.5×TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0). Agarose blocks were incubated in bacterial lysis buffer [28] containing 2 mg ml−1 of lysozyme (Sigma-Aldrich, Madrid, Spain), and then incubated with proteinase K (1 mg ml−1) (Sigma) in digestion buffer [28]. After these enzyme treatments, agarose blocks were cut (slices 1–2 mm) and digested with SfiI restriction enzyme (Biolabs, Beverly, MA, USA) following the manufacturer's instructions. Gel blocks were loaded onto 1% (w/v) agarose D-5 (Pronadisa, Madrid, Spain) gels. DNA fragments were separated in 0.5×TBE buffer in a CHEF DR II system (Bio-Rad Laboratories, Hercules, CA, USA). PFGE was performed at 14°C at a constant voltage of 4.5 V cm−1 with a switch time ramped from 5 to 45 s over a 24-h period. The CHEF DNA size standard lambda ladder (Bio-Rad) was used as the molecular size standard. The GelCompar 2.5 software (Applied Maths, Kortrijk, Belgium) was used for conversion, normalisation, and further processing of images. Comparison of the obtained PFGE patterns was performed with Pearson's product–moment correlation coefficient and the unweighted pair group method using arithmetic averages (UPGMA)

2.4Lyophilisation

Twenty-five L. plantarum strains and 43 O. oeni strains were subjected to lyophilisation. Inocula with turbidity equivalents of 1 and 3 McFarland were prepared from fresh cultures of L. plantarum and O. oeni strains respectively (3×108 cfu ml−1 and 9×108 cfu ml−1 respectively). 25 μl of L. plantarum inoculum was added to 3 ml of MLO broth, and 100 μl of O. oeni inoculum was added to 6 ml of MLO broth. Cells were grown at 30°C with continuous shaking (Innova 4000 incubator shaker) and incubations were maintained until cells reached stationary phase. At this stationary phase colony enumeration was in the 108 cfu ml−1 range. Bacteria were collected by centrifugation at 2000×g (Megafuge 1.0, Heraeus Instruments) for 15 min. Pellets were resuspended in 0.5 ml sterile skimmed milk and stored at −80°C. Lyophilisation was performed at −55°C for 16 h under vacuum (Freeze mobile 3.3 lyophiliser, Virtis Company).

2.5Measurement of acid pH tolerance

Twenty L. plantarum strains and 22 O. oeni strains were included in the study of acid pH tolerance. HCl was added to MLO broth to final pHs of 4.7, 3.6, 3.3 and 3.2. Inocula with turbidity equivalents of 1 McFarland were prepared from fresh cultures of L. plantarum strains (3×108 cfu ml−1), and with turbidity equivalents of 3 McFarland for O. oeni strains (9×108 cfu ml−1). 100 μl and 200 μl of these inocula were added to 6 ml of growth medium in the case of L. plantarum and O. oeni, respectively. Cells were incubated at 30°C without shaking. Bacterial growth was followed by OD at 600 nm until a plateau was reached, this took an average of 3–4 days in the case of L. plantarum, and 4–5 days for O. oeni.

2.6Measurement of ethanol tolerance

Twenty-five L. plantarum strains and 51 O. oeni strains were included in the study of ethanol tolerance, for which MLO broth supplemented with ethanol to final concentrations of 7%, 12% and 13% (v/v) was used. Inocula with turbidity equivalents of 1 McFarland were prepared from fresh cultures of L. plantarum strains (3×108 cfu ml−1), and with turbidity equivalents of 3 McFarland for O. oeni strains (9×108 cfu ml−1). 25 μl of L. plantarum inoculum was added to 3 ml of growth medium, and 200 μl of O. oeni inoculum was added to 6 ml of growth medium. Samples were grown at 18°C and 10°C without shaking. Control samples were incubated without ethanol at 30°C, 18°C and 10°C. Bacterial growth was followed by OD at 600 nm until a plateau was reached. This took an average of 6–9 days.

2.7Growth parameters

OD at 600 nm was followed for determination of bacterial growth. Absorbance data were plotted versus time for each strain and for each tested condition. Absorbance slopes and absorbance values reached at the plateau at stationary phase were used respectively for growth rate and maximal bacterial population determinations. Relative maximal bacterial populations and growth rates were calculated as percentages relative to respectively maximal bacterial population and growth rate reached by each strain under defined standard conditions for each type of experiment. In lyophilisation experiments, growth parameters were calculated relative to the corresponding values before subjecting cells to the process of lyophilisation. In the case of acid pH tolerance, growth parameters were calculated relative to strain growth parameters in standard MLO broth pH 4.7. In the case of ethanol tolerance, growth parameters were calculated relative to the growth parameters reached by each strain in standard MLO broth without ethanol.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

3.1Strain identification

From the total 259 initial LAB isolates of this study, 211 isolates were identified as O. oeni and 48 as L. plantarum, as described in Section 2. The species-specific PCR analyses described by Zapparoli et al. [26] and Quere et al. [27] have been shown to be of high reliability and specificity for species identification, and the specific O. oeni 1025-bp and L. plantarum 280-bp amplicons were generated from all the investigated isolates (results not shown). After strain identification by PFGE, as described in Section 2, the total number of O. oeni strains was 56, and that of L. plantarum was 35. Fig. 1A shows the PFGE patterns obtained after SfiI digestion of total bacterial DNAs of 11 L. plantarum isolates belonging to nine unrelated and one closely related patterns. Fig. 1B shows the PFGE patterns of total bacterial DNA of 10 O. oeni strains with 10 unrelated patterns.

image

Figure 1. A: PFGE patterns of SfiI digests of genomic DNA from L. plantarum strains. Lanes: 1: Lambda DNA marker; 2: V-6; 3: V-8; 4: E-8; 5: E-14; 6: J-36; 7: J-36; 8: J-51; 9: J-66; 10: J-70; 11: J-34; 12: J-58. B: PFGE patterns of SfiI digests of genomic DNA from O. oeni strains. Lanes: 1: IS 33; 2: IS 138; 3: Lambda DNA marker; 4: IS 141; 5: IS 142; 6: IS143; 7: IS144; 8: IS 146; 9: IS 147; 10: IS 148; 11: IS 149.

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3.2Lyophilisation

A total of 43 O. oeni strains and 25 L. plantarum strains were subjected to lyophilisation and successfully survived after the process. This process is regarded as a gentle drying procedure when compared with other methods, such as spray drying, and in fact it is the preferred method for wine starter preparation. L. plantarum strains maintained the same growth rate and the same maximal bacterial population as before being subjected to lyophilisation, as shown in Table 1, which indicates that 90.0% (±9.0%) of maximal population values and 97.2% (±24.4%) of growth rates were recovered after the process in a total of 25 studied strains. These results are in agreement with those reported by Linders et al. [29,30] who observed that one L. plantarum strain was able to survive successfully (61% residual activity) after a process of drying, when a high initial cell concentration was used for drying and without addition of solutes, such as betaine or carnitine, which were supposed to be beneficial for LAB during drying [31]. In our experiments, bacterial concentrations were kept high before subjecting them to lyophilisation (around 6×109 cfu ml−1) and this could account for the high rate of recovery obtained.

Table 1.  Effect of lyophilisation on the growth of 68 L. plantarum and O. oeni wild strains
  1. aMaximal populations and growth rates were calculated for each strain as percentages relative to the corresponding values before lyophilisation. Bacterial growth was followed by OD at 600 nm as described in Section 2. Absorbance values reached at the plateau at stationary phase were used for maximal bacterial population determinations.

L. plantarum strainMaximal populationa after lyophilisation (%)Growth ratea after lyophilisation (%)O. oeni strainMaximal populationa after lyophilisation (%)Growth ratea after lyophilisation (%)
J-2199.9105.5IS 13220.0100.0
J-23103.6106.0IS 1776.478.7
J-30106.5109.1IS 2691.597.3
J-36107.2108.7IS 3168.671.4
J-39109.752.6IS 36106.5112.2
J-51107.0111.2IS 42149.963.2
J-53107.351.1IS 4585.0122.3
J-5597.7114.7IS 4768.973.8
J-58102.5105.4IS 4865.2134.2
J-61102.3106.3IS 53295.0148.6
J-6298.299.7IS 7591.5101.3
J-66100.7106.2IS 94131.473.7
J-6793.344.0IS 9799.688.6
J-6981.086.2IS 122112.0152.5
J-70101.0105.1IS 129304.2125.4
J-71107.9106.5IS 13581.3189.5
J-73103.8118.6IS 142107.593.1
T-1994.0147.6IS 143181.8175.6
T-2089.1118.1IS 14594.6117.4
T-4390.193.3IS 151110.7121.8
T-5388.6101.0IS 158100.8121.2
E-886.785.7IS 159125.450.7
E-14114.748.5IS 163114.1116.8
I-394.299.0IS 16485.6161.1
V-887.799.5IS 166121.874.4
Mean±S.D.90.0±9.097.2±24.4IS 16749.464.9
   IS 174166.3101.2
   IS 183174.891.6
   IS 186152.878.6
   IS 189151.3137.0
   IS 202173.575.7
   IS 209207.190.9
   IS 210195.4274.0
   J-10195.760.6
   J-10284.8109.5
   J-110125.1168.5
   J-11381.4107.8
   T-1462.862.0
   T-27135.184.7
   T-31133.8124.7
   T-38102.275.4
   T-4086.479.5
   T-5672.064.4
   Mean±S.D.124.2±57107.3±43

In the case of our O. oeni strains, they behaved similarly and maintained the same average growth rate and maximal population as before lyophilisation, 107.3% and 124.2% respectively in a total of 43 unrelated strains (Table 1). Nevertheless, in some cases O. oeni cells acquired an anomalous morphology reaching a bacterial width of 1 μm, as shown in Fig. 2, and they proliferated at a higher rate after lyophilisation. Forty-eight per cent of the studied O. oeni strains showed to some extent this larger morphology and the increased growth rate after lyophilisation. Probably this process, involving low temperatures (below −45°C) and high vacuum, would provoke cell death and most resistant bacteria, presumably those presenting larger morphology, survived successfully and rendered higher growth rates. To our knowledge, this is the first time that such an increase in cell growth rate and cell size is reported for LAB after lyophilisation. Maicas et al. [32] also reported high survival rates for O. oeni cells, up to 62.5%, after freeze-drying and 1 year of storage at 4°C.

image

Figure 2. Digitalised electron micrograph of O. oeni cells in growth phase after undergoing lyophilisation. Magnification: ×18000.

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Regarding lyophilisation, our results reveal that all the O. oeni and L. plantarum isolates from wines of this study did not decrease their growth rate after lyophilisation, which means a high degree of resistance to adverse conditions and, moreover, saves supplements of additional cryoprotectants to bacterial starters before lyophilisation.

3.3Acid pH resistance

Twenty different L. plantarum strains were grown to study the effect of pH on bacterial growth. In these experiments bacterial populations at growth plateau were in the order of magnitude of 108–109 cfu ml−1. Table 2 shows that when pH was decreased from 4.7, which is the standard pH of MLO broth, to 3.3, the average maximal population of L. plantarum strains only decreased to 80.8% and the average growth rate decreased to 56.9% with respect to optimal conditions at pH 4.7 in MLO broth. When pH was 3.2, the average maximal bacterial population dropped to 67.0% and the average growth rate decreased to 37.9% of the corresponding values under optimal conditions. These results all together showed that in the pH range from 3.2 to 3.6, which is the normal pH range for Rioja red wines, our L. plantarum strains proliferated successfully. This resistance may be a variable feature within the species and may be dependent on the strain and its source. Nevertheless, our whole collection of strains isolated from wines showed this resistance.

Table 2.  Effect of acid pH on the growth of 44 wild L. plantarum and O. oeni strains
  1. aRelative maximal bacterial populations and growth rates were calculated as percentages relative to the corresponding parameters for each strain growing in standard MLO broth (pH 4.7). Bacterial growth was followed by OD at 600 nm, as described in Section 2. Absorbance values at stationary phase and absorbance slopes were used for maximal bacterial population and growth rate determinations respectively.

L. plantarum strainRelative maximal populationa at pHRelative growth ratea at pH
 3.63.33.23.63.33.2
J-2679.778.250.990.198.226.5
J-3476.279.064.940.646.464.0
J-3961.662.454.462.746.737.3
J-4074.376.59.527.823.48.3
J-5181.774.063.358.144.731.7
J-6188.189.881.372.471.952.6
J-6270.572.076.556.644.635.7
J-6687.485.974.149.549.954.4
J-6780.674.063.341.666.758.7
J-6986.583.375.568.368.843.6
J-7070.273.867.655.347.335.2
J-7186.189.776.458.359.942.9
J-7270.075.328.765.871.514.1
J-7389.792.283.265.067.744.1
T-1980.881.679.361.644.237.3
T-2094.790.393.568.268.841.8
T-4377.079.865.140.047.722.9
T-5389.184.080.158.958.634.4
E-887.088.876.553.649.432.8
E-1484.284.875.950.762.039.1
Mean±S.D.80.8±8.480.8±7.767.0±19.557.3±13.756.9±15.837.9±13.8
O. oeni strain
IS 18100.294.9102.993.282.367.1
IS 4079.881.868.231.434.023.9
IS 59156.479.559.4173.039.462.5
IS 9556.058.552.749.644.038.0
IS 11168.460.166.243.940.931.0
IS 12777.898.178.050.476.947.1
IS 15555.558.354.350.658.759.9
IS 15965.958.557.843.931.331.3
IS 16798.3107.784.1115.8108.347.4
IS 17780.796.395.757.659.255.6
IS 18072.378.370.162.160.160.3
IS 18963.475.165.853.847.456.2
IS 20933.532.939.029.623.627.1
IS 21084.95.693.672.1105.797.1
J-10152.1131.163.743.146.160.0
J-10279.686.066.866.474.548.9
J-11391.687.979.858.255.256.6
J-11697.687.089.879.863.576.0
T-1478.278.887.664.057.6100.3
T-27109.976.268.558.960.958.5
T-3886.1133.3111.048.4111.553.8
T-5645.743.336.136.537.719.6
Mean±S.D.78.8±25.781.8±24.772.3±19.462.8±31.660.0±24.753.6±20.8

The 22 O. oeni strains, studied under the same pH conditions as for L. plantarum strains, behaved in a similar fashion, in that the average bacterial population decreased to 81.8% of the maximal value under standard conditions, and the average growth rate decreased to 60.0% when pH was lowered from 4.7 to 3.3, and average decreases to 72.3% and 53.6% in bacterial population and growth rates respectively were obtained at pH 3.2 (Table 2). Bacterial populations at stationary phase in the case of O. oeni strains were in the order of magnitude of 107–108 cfu ml−1. These results indicate that our strains were able to grow in the pH range 4–3.2 with high growth rates (relative values above 50%), and reveal a high adaptation of bacterial cells to wine acid pH. O. oeni resistance to wine low pH has been widely reported [4], in addition, our results show that also our collection of L. plantarum strains were able to growth successfully at wine pH 3.2, and that bacterial populations reached similar values as in the case of O. oeni strains, which traditionally had been considered as the most tolerant to acid pH among wine LAB species [1,3].

3.4Resistance to ethanol

All the studied strains (25 L. plantarum strains and 51 O. oeni strains) showed maximal growth rates and maximal populations when grown in a medium free of ethanol (MLO broth) and at 30°C, as expected. The combined effect of the presence of 12% ethanol in the medium and low incubation temperature (10°C) prevented bacterial growth of all strains of both species L. plantarum and O. oeni of this study, as shown in Fig. 3A,B, which represent growth curves of L. plantarum and O. oeni strains respectively. Sampling points were the average of three independent experiments corresponding to the growth of three different strains representative of the behaviour of all the studied L. plantarum and O. oeni strains. Similar results were obtained by Britz and Tracey [4] with a collection of 54 O. oeni strains. They showed that the combination of 15°C and 13% ethanol reduced to 15% the number of O. oeni strains able to grow under these conditions.

image

Figure 3. Effect of ethanol and temperature on (A) L. plantarum growth and (B) O. oeni growth. Cells were incubated in MLO broth in the following conditions: ♦ 30°C without ethanol; ? 18°C without ethanol; ▴ 18°C and 7% ethanol; ? 18°C and 12% ethanol; □ 18°C and 13% ethanol; ▵ 10°C without ethanol; ◯ 10°C and 12% ethanol. Sampling point is the average of three independent experiments corresponding to the growth of three different strains representative of the behaviour of all the studied L. plantarum and O. oeni strains.

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When we grew L. plantarum strains at 18°C in the absence of ethanol, their growth rate decreased 70%, a short lag phase reaching up to 24 h appeared in some cases, and bacterial population decreased 18% when compared with optimal conditions at 30°C (Fig. 3A). The presence of 12% ethanol in the medium affected L. plantarum growth rate, which decreased when compared with control samples incubated at 18°C without ethanol (Fig. 3A). Table 3 shows the results of relative maximal populations of the 25 L. plantarum strains that were studied. Bacterial population decreased in presence of 7%, 12% and 13% ethanol, to respectively 78.6%, 45.6% and 39.5% of the maximal population without ethanol at 18°C incubation temperature. Nevertheless, bacterial populations reached high values, around 108 cfu ml−1, under conditions of 13% ethanol and 18°C, which are values in the same range as those reached by O. oeni strains under the same growth conditions. Some L. plantarum strains have been described as able to grow in wine [1] and, therefore, they should develop some mechanisms of ethanol resistance, such as changes in membrane lipid composition. Nevertheless, this is the first time, to our knowledge, that similar bacterial populations are reported for L. plantarum and O. oeni strains grown in 13% ethanol and 18°C.

Table 3.  Effect of ethanol on the growth of 76 L. plantarum and O. oeni wild strains: relative maximal bacterial populationsa
  1. aRelative maximal bacterial populations were calculated as percentages relative to the corresponding value for each strain growing in MLO broth without ethanol. Strains were grown at 18°C in MLO broth in the presence on different concentrations of ethanol, as described in Section 2. Bacterial growth was followed by OD at 600 nm. Absorbance values reached at the plateau at stationary phase were used for maximal bacterial population determinations.

L. plantarum strain7% ethanol12% ethanol13% ethanolO. oeni strain7% ethanol12% ethanol13% ethanolO. oeni strain7% ethanol12% ethanol13% ethanol
J-2161.940.843.5J-10163.6150.2111.9IS 53116.0130.458.0
J-2373.244.053.7J-102353.9262.3172.5IS 63132.154.323.6
J-3072.343.636.0J-110103.992.584.9IS 7581.955.557.1
J-3686.037.838.8J-113100.680.776.1IS 9498.168.268.4
J-3980.847.629.3T-3139.585.667.2IS 9797.2107.615.8
J-5168.838.838.2T-14247.4134.4121.5IS 122122.538.940.1
J-5374.338.842.4T-2737.435.017.6IS 135162.977.030.7
J-5578.951.451.8T-31113.966.345.3IS 142215.4226.753.5
J-5879.251.546.5T-40102.058.755.5IS 143157.2137.9211.8
J-6182.145.836.6T-5646.619.916.8IS 14485.924.976.7
J-6289.138.735.7IS 1188.297.897.8IS 145108.575.737.9
J-6674.643.437.4IS 13129.068.2IS 14793.377.136.0
J-6781.240.938.0IS 17125.2105.1IS 151122.4109.726.7
J-6984.043.736.4IS 24178.347.9IS 159126.188.020.6
J-7082.834.625.2IS 26103.173.177.0IS 16197.274.5114.4
J-7183.845.726.7IS 3172.467.423.4IS 163100.872.079.4
J-7272.551.247.5IS 33221.1173.4IS 16498.053.681.0
J-7398.233.623.3IS 3688.278.4IS 16664.088.248.7
T-1986.160.750.9IS 4184.842.821.3IS 16777.282.284.8
T-2086.052.040.3IS 42152.791.697.4IS 174125.369.373.3
T-4385.064.150.1IS 4386.739.234.4IS 183112.183.187.1
T-5374.045.841.9IS 44115.782.463.9IS 189115.566.358.1
E-881.966.350.9IS 4588.526.225.8IS 20293.845.546.6
E-1462.640.543.6IS 4780.675.062.9IS 20456.783.4100.4
V-866.337.422.5IS 48145.6122.334.4IS 20983.058.074.8
        IS 21064.779.792.0
Mean±S.D.78.6±8.645.6±8.639.5±9.1    Mean±S.D.115.2±53.584.4±45.565.3±39.8

When O. oeni strains were incubated at 18°C, growth rates decreased 38% when compared with growth rates under optimal conditions of 30°C and absence of ethanol (Fig. 3B), and a lag phase of 48–72 h appeared. After lag phase and cell proliferation, bacterial populations at 18°C reached values very similar to those obtained under optimal conditions (data not shown). Table 3 shows the relative maximal populations of the 51 O. oeni strains that were studied in these experiments and reveals that O. oeni strain growth was activated when 7% ethanol was present in the medium, giving higher bacterial populations than in control experiments at 18°C without ethanol. Small percentages of ethanol had been previously reported to activate bacterial growth, and thus 3–4%[3] or 5–6%[1] had been reported to activate O. oeni growth. In our O. oeni strains, 7% ethanol gave higher bacterial populations than in the absence of ethanol in the medium. An increase in ethanol content up to 12–13% (which are normal alcohol content values in the original red wines from which strains were isolated) produced no major decrease in bacterial populations with respect to populations reached at 18°C without ethanol, which revealed the high degree of adaptation of our O. oeni strains to growth in wine and, therefore, to relatively high amounts of ethanol in the medium. Bacterial populations under these conditions reached values around 108 cfu ml−1, which was in the same range as the average bacterial population of L. plantarum strains grown under the same conditions.

We conclude that our collection of L. plantarum strains can tolerate the combination of acid pH and ethanol concentration in the medium, and proliferate under conditions that are normally lethal to LAB. They survive and proliferate at pH 3.2, they grow in the presence of 13% ethanol at 18°C reaching bacterial populations of 108 cfu ml−1, in the same range as O. oeni populations adapted to growing in wine. And, therefore, it is concluded that L. plantarum strains could constitute starters for inducing MLF and are of interest in wine production.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

This work was supported by FEDER-CYCIT Grant 2FD97-1475 of the European Community and the Spanish Ministry of Science and Technology, by I.N.I.A. Grant VIN00-043-C3 of the Spanish Ministry of Science and Technology, by University of La Rioja Grants API00/B30 and API01/B34, and by Autonomous Community of La Rioja Grant ACPI-ANGI/2000.

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  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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