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The effect of cold, acid and ethanol shocks on synthesis of membrane fatty acid, freeze-drying survival and malolactic activity of Oenococcus oeni

The effect of cold, acid and ethanol shocks on synthesis of membrane fatty acid, freeze-drying... Ann Microbiol (2013) 63:477–485 DOI 10.1007/s13213-012-0492-x ORIGINAL ARTICLE The effect of cold, acid and ethanol shocks on synthesis of membrane fatty acid, freeze-drying survival and malolactic activity of Oenococcus oeni Guoqiang Zhang & Mingtao Fan & Qian Lv & Yahui Li & Yanlin Liu & Shuangfeng Zhang & Hua Zhang Received: 14 February 2012 /Accepted: 25 May 2012 /Published online: 16 June 2012 Springer-Verlag and the University of Milan 2012 Abstract The effects of stress shocks on the freeze-drying membrane fatty acid composition are not enough to explain viability, malolactic activity and membrane fatty acid com- the greater freeze-drying viability of cells shocked at 8% position of the Oenococcus oeni SD-2a cells were studied. ethanol. Thus, other mechanisms could be responsible for O. oeni SD-2a cells after 2 h of stress exposure exhibited this increase in the bacterial resistance to lyophilization. better freeze-drying viability and malolactic fermentation . . ability. A decrease in unsaturated fatty acids/saturated fatty Keywords Oenococcus oeni Stress shock Freeze-drying . . acids (UFA/SFA) ratio and in the C18:1 relative concentra- viability Fatty acid composition Malolactic activity tion, and an increase in cyclopropane fatty acids (CFA) content mainly due to the increase in C19cyc11 relative concentration were observed in all stress shocked cells. Introduction There was a significant negative correlation between C19cyc11 and C18:lcis11, C16:0 in all stress shocks. The Malolactic fermentation (MLF) following alcoholic fermentation freeze-drying viability exhibited a significant positive cor- in winemaking could reduce acidity, improve taste and relation with the levels of C19cyc11 in cold and acid shocks. biological stability. Different bacteria genera have been The only significant positive correlation between the ability reported to carry out MLF in wine. Among them, Oenococcus of O. oeni SD-2a to conduct malic acid degradation and oeni is recognized as the most advantageous and tolerant membrane composition existed with C14:0 in ethanol bacterium (Lonvaud-Funel 1999; Versari et al. 1999). MLF shocks. In general, freeze-drying viabilities were maximum deacidifies wine and results in a softer feeling in mouth, for cells with low UFA/SFA ratio and high CFA levels, and, also improves microbiological stability and organoleptic consequently, with low membrane fluidity. Moreover, CFA characteristics (Versari et al. 1999; Maicas 2001). For formation played a major role in protecting stress shocked many years, MLF has been recognized as an indispensable step cells from lyophilization. However, changes observed in in the elaboration of most wines (Nielsen et al. 1996; Maicas et al. 2000). However, due to very harsh environmental conditions in wine for bacterial survival and growth, induction of MLF has : : : : : : G. Zhang M. Fan (*) Q. Lv Y. Li Y. Liu S. Zhang failured in some processes (Garbay and Lonvaud-Funel 1996; H. Zhang Maicas et al. 2000; Zhao and Zhang 2009; Beltramo et al. College of Food Science and Engineering, 2006). There are two reasons for such failures. On one hand, Northwest A&F University, Yangling 712100, China strains lose their natural adaptation to survive and grow in wine, e-mail: mingtaofan@163.com which results in inoculation low viability. On the other hand, lyophilization offers stress conditions such as freezing, drying G. Zhang and concentration stress, which diminish cell viability. Tibet Agriculture and Animal Husbandary College, Therefore, expanding interest in freeze-dried ready-to-use Linzhi 860000, China 478 Ann Microbiol (2013) 63:477–485 malolactic starter cultures has placed more emphasis on The bacteria were statically cultured in ATB media at 25 °C. developing starter production and preservation methods Cell growth was monitored by absorbance readings at 600 that promote high cell viability and activity (Carvalho et nm using a spectrophotometer. When the cells had grown up al. 2004). to early stationary phase (A 01.9), the bacterial cells were Research in recent years has demonstrated that Oenococcus harvested by centrifugation (6,000×g for 10 min) at room oeni possesses remarkable resilience and adaptability in temperature. unfavorable environments (Maicas et al. 2000; Chu-Ky Cells were collected, inoculated in the same fresh medium et al. 2005;Beltramoetal. 2006;Sicoetal. 2009). and exposed for 2 h to different sublethal stresses. The Moreover, stress response, induced under one set of conditions and the amplitudes of stresses tested (various conditions, often provide the cell with cross-protection temperatures, pH and ethanol content) were chosen against other hostile and seemingly unrelated environments depending on real conditions in winemaking processes (van de Guchte et al. 2002;Spano andMassa 2006). Then a (Lonvaud-Funel 1999; Versari et al. 1999). For cold question was put forward as to whether stress response could shocks, the cell suspensions were incubated at 4, 15 take effect on the freeze-drying viability of O. oeni cells and and 25 °C, respectively. For ethanol shocks, ethanol be used in preparing freeze-dried ready-to-use malolactic was added to the same fresh medium under continuous starter cultures. The effect of stress response has been less stirring to final concentrations of 5, 8 and 12% (v/v). studied, although from data in literature for Lactobacillus ssp. During cold and ethanol stresses, the pH of the cell and Oenococcus oeni it can be deduced that their adaption in suspension was unchanged (pH 4.8). For acid shocks, low pH mediums is accompanied by an increase in freeze- different pH values (4.0, 3.5 and 3.2) were adjusted by drying viability (Schoug et al. 2008;Lietal. 2009a, b). addition of 1 M HCl solution. During acid shocks, the However, as far as we know little attention has been paid to temperature of the cell suspension was kept at 25 °C. the effects of stress shocks on the bacterial resistance to Two independent experiments with three repetitions each lyophilization and their MLF ability in wine-like medium. were performed. After 2 h, cell suspensions were centrifuged The mechanisms involved in the bacterial resistance to to harvest for lyophilization and FAs extraction. lyophilization are not fully understood. However, several reports have found the correlations between membrane fatty Freeze-drying assays acid compositions of different Lactobacillus spp. and their lyophilization tolerance (Schoug et al. 2008; Lietal. The stress-shocked cells were harvested by centrifugation at 2009a). In general, these authors have shown that cells with 6,000×g for 10 min and washed once in aseptic distilled a decreased concentration of unsaturated fatty acids or with water. Cell pellet was resuspended in sterile protective agents an increased content of saturated fatty acids have a decreased containing 2.5% sodium glutamate to make a cell suspension membrane fluidity, which is linked to a higher lyophilization allowed to equilibrate for 20 min at room temperature. tolerance. Furthermore, in a previous study performed in our Aliquots (10 mL) of each resuspension were transferred laboratory we found that the formation of cyoclopropane fatty into serum bottles (50 mL) and frozen at -20 °C overnight. acids plays an important role in protecting acid-adapted O. oeni Then, the samples were immediately freeze-dried in an cells from lyophilization inactivation (Li et al. 2009b). Edwards freeze dryer at a condenser temperature -56 °C and The objective of this study is to evaluate the effect of at a chamber pressure <0.06 mbar for 28 h. After freeze- stress shocks on the malolactic activity of Oenococcus oeni drying, samples were immediately brought to their original SD-2a in wine-like medium and its resistance to freeze- volume (10 mL) with ATB medium at 25 °C. Then, samples drying, meanwhile, try to clarify the stress response mech- were homogenized for 1 min with a Vortex mixer and incu- anism of O. oeni SD-2a related to membrane fatty acid bated at room temperature for 20 min. Serial dilutions were composition. spread-plated onto the surface of Petri plates containing ATB medium agar (ATB medium and 2.0% agar). These plates were incubated at 25 °C for at least 7 days, and the viability Materials and methods was then determined. Survival levels were expressed as the quotient of colony-forming units per millilitre (CFU/mL) on Bacterial strain and Stress treatments ATB medium before (N )and after(N ) freeze-drying. 0 f Viability0(N /N )×100. f 0 Oenococcus oeni strain SD-2a (Li et al. 2006) isolated from Chinesewines was used. Theculture mediawereATB MLF assays containing (g/L): glucose 10, yeast extract 5, peptone 10, MgSO ·7H O0.2,MnSO ·4H O 0.05, cysteine/HCl 0.5, The effect of stress shocks on the growth and MLF ability of 4 2 4 2 tomato juice 250 mL, pH of the medium was about 4.8. lyophilized Oenococcus oeni SD-2a were investigated. Ann Microbiol (2013) 63:477–485 479 Immediately after rehydration, a concentration of about cells were cells in the stationary phase incubated in fresh 1.0×10 CFU/mL of stress-shocked cells were inoculated into medium for 2 h while the control cells corresponded to the bottles containing 100 mL of the sterilized wine-like medium. same cells directly lyophilized. The wine-like medium (g/L) is a modified chemically defined Growth curves from rehydrated Oenococcus oeni SD-2a medium (Terrade et al. 2009) supplemented with L-malic acid suggested that the increased culturability of stress-shocked 2.5, tartaric acid 3.5, glucose 1.0, fructose 1.0, and the ethanol cells allowed these bacteria to reach mid-exponential phase content 10.0% (v/v). The medium was then adjusted to 3.5 more quickly than control cells. Specially rehydrated cells with HCl/KOH followed by sterile membrane filtration (0.22 shocked at pH 3.5 or 8% ethanol reached the mid- μm nylon, Millipore Corp., USA). Cell growth was monitored exponential phase significantly faster than rehydrated spectrophotometrically at A . Malic acid in samples was de- control cells. Stress-shocked cells started to grow almost termined by high performance liquid chromatography (HPLC). without lag phase except cold-shocked cells and 5% ethanol shocked cells. It was indicated that the appropriate Analysis of fatty acid composition stress shock was important for the application of lyophilized malolactic starters because stress shock affects the lag phase of The membrane fatty acid composition of the bacteria was the inoculation bacteria. Once the surviving bacteria start to determined using gas chromatography as described by multiply in the wine, the growth rates are similar. Rozes et al. (1993) and Li et al. (2009b). Concentrated cells Similar experiments were performed to investigate the were washed twice in sterile distilled water. Methylation and MLF ability of rehydrated stress-shocked cells. Rehydrated extraction were performed by adding 2.0 mL of sodium stress-shocked Oenococcus oeni SD-2a degradated malic methoxide (1 M in methanol) and shaking for 1 min. Fatty acid significantly faster than rehydrated control cells, acid methyl esters were extracted with 1 mL of hexane. specially cells shocked at pH 3.5 and 8% ethanol. After decanting for 2 min, the upper phase was taken out MLF was completed within 10 days with all treated and stored at 4 °C in an airtight glass bottle until analysis. cells, and malic acid degradation was as high as 50% The analyses were performed on a gas chromatographer within 2 days. It may also be worthwhile to note that (HP 6890, Hewlett Packard, Avondale, PA) equipped with malic acid degradation was higher for rehydrated control mass selective detector (Agilent 5973, Hewlett Packard). A cells than for fresh cells, but the A value was lower capillary column (BPX 70, 60 m×0.25 mm, SGE, Victoria, for rehydrated control cells than fresh cells. It may Australia) was used. Helium was used as carrier gas (1.2 reflect that lyophilization is beneficial for O. oeni SD-2a to mL/min), and the injection volume was 1 μL. Injection was achieve MLF. Moreover, it was indicated that appropriate done in splitless mode for 2 min. The oven temperature was stress shock plays an important role in increasing the MLF increased from 65 to 230 °C at 5 °C/min and maintained for ability of O. oeni SD-2a cells in wine. 10 min at 230 °C. Injection and detection temperatures were 230 °C. Effects of stress shocks on freeze-drying viability and fatty Results were expressed as relative percentages for each fatty acid composition acid based on the ratio of the surface area of the considered peak to the total area of all peaks. Analyses were made in triplicate. Three kinds of stress shocks (various temperatures, pH and ethanol content) depending on real conditions in winemaking Statistical analysis processes were carried out (Lonvaud-Funel 1999; Versari et al. 1999). The freeze-drying viability and the relative percentage The data presented are the mean of three replicates within of the membrane FAs of Oenococcus oeni SD-2a in the each experiment. The variations were <5%. Pearson corre- stationary phase (Sp), in the control (Co) corresponded lation analysis (SPSS software) was performed for all the to the same cells incubated in fresh medium for 2 h and stress shocks to relate the fatty acid composition to freeze- in cells exposed for 2 h to different stress conditions drying viability and malolactic activity of O. oeni SD-2a. were showninTable 1. Freeze-drying survival of cells harvested in stationary phase was 69.5% and control was 67.8% respectively Results (Table 1). This survival was almost in agreement with other reports on freeze-drying survival of Oenococcus oeni (Zhao MLF in wine-like medium and Zhang 2009; Li, et al. 2009b). It can also be seen from Table 1 that pretreatments at different stress treatments The results from direct inoculation of fresh cells, rehydrated before freeze-drying had different effect on the cell viability. control cells and rehydrated stress-shocked cells were In cold shocks, pretreatment at 4 °C and 15 °C improved the shown in Fig. 1. It is necessary to mention that the fresh cell viability lightly compared to the control. However, pH 480 Ann Microbiol (2013) 63:477–485 Fig. 1 Growth (solid curve) and malic acid degradation 0.15 2.5 (dotted curve) during culturing rehydrated Cold-shocked cells 0.12 2 (a), Acid-shocked cells (b) and fres h Ethanol-shocked cells (c)in control 0.09 1.5 wine-like medium 4 shocked 0.06 1 15 shocked 0.03 0.5 0 0 02 468 10 12 Time (d) 0.15 2.5 0.12 2 fres h control 0.09 1.5 pH3.2 s hocked pH3.5 s hocked 0.06 1 pH4.0 s hocked 0.03 0.5 0 0 0246 8 10 12 Time (d) 2.5 0.15 0.12 fres h control 1.5 0.09 5% ethanol s hocked 8% ethanol s hocked 0.06 12% ethanol s hocked 0.5 0.03 0 0 02 468 10 12 Time (d) of suspension medium had significant influence on the cell acid (C19cyc11). Their relative contents were between 3 and survival, which increased from 67.8 to 80.5% when pH of 45%, totaling to more than 90% of all fatty acids identified. suspension medium decreased from 4.8 to 3.5, but when pH These fatty acids are commonly found in other lactic acid of suspension medium was 3.2 the cell viability had a little bacteria strains (Beal et al. 2001; Gómez et al. 2000; Machado decrease. For ethanol shocks, the cell viability after freeze- et al. 2004; Taranto et al. 2003; Tymczyszyn et al. 2005;Wang drying increased by 14.2% when the ethanol concentration et al. 2005; Montanari et al. 2010). These results was in good increased from 0 to 8%. However, when the ethanol agreement with those of Garbay et al. (1995); Drici-Cachon et concentration further increased from 8 to 12%, the cell al. (1996)and Li et al.(2009b). Methylenhexadecanoic acid survival decreased, indicating higher ethanol concentration (C17cyc9), hexadecadienoic acid (C16:2cis9,12), stearic acid did not improve the cell viability. (C18:0) and octadecadienoic acid (C18:2cis9,12) were also The fatty acid compositions of Oenococcus oeni SD-2a after detected but in very low quantities. different stress shocks were determined (Table 1). In all cases, So as to determine the differences among membrane fatty nine fatty acids were found, the main fatty acids were: myristic acid composition of Oenococcus oeni SD-2a cells, total acid (C14:0), hexadecanoic acid (C16:0), hexadecenoic acid saturated fatty acids (SFA), total unsaturated fatty acids (C16:1cis9), cis-vaccenic acid (C18:1cis11) and lactobacillic (UFA) and total cyoclopropane fatty acids (CFA) were A600 A600 A600 Malic acid (g/L) Malic acid (g/L) Malic acid (g/L) Ann Microbiol (2013) 63:477–485 481 Table 1 Fatty acid composition and freeze-drying viability of O. oeni SD-2a in the stationary phase (Sp), in the control without stress shock (Co) and in cells exposed for 2 h to different stress shocks. The data are means of three independent experiments. The coefficients of variability were lower than 5% Fatty acids Sp Co Cold shocks Acid shocks Ethanol shocks 4°C 15°C pH 4.0 pH 3.5 pH 3.2 5% 8% 12% C14:0 2.96 2.90 2.91 3.11 3.33 3.57 3.84 3.67 4.28 4.01 C16:0 27.62 27.86 26.43 26.28 26.18 25.23 25.05 25.47 24.87 25.21 C16:1cis9 7.53 7.37 7.65 7.43 7.41 7.15 6.97 7.98 7.83 7.54 C16:2cis9,12 0.64 0.7 0.76 0.77 0.71 0.66 0.68 0.72 0.64 0.78 C17:0cyc9 0.36 0.35 0.41 0.48 0.56 0.68 0.72 0.39 0.55 0.52 C18:0 2.08 2.12 2.21 2.29 2.28 2.36 2.43 2.38 2.19 2.25 C18:1cis11 16.57 17.49 15.58 15.47 14.63 14.01 14.23 14.98 14.46 14.77 C18:2cis9,12 0.87 0.83 0.88 0.86 0.92 0.89 0.81 0.76 0.82 0.89 C19:0cyc11 41.37 40.28 42.97 43.31 43.98 45.45 45.27 43.65 44.36 44.03 SFA 32.66 32.88 31.75 31.68 31.79 31.16 31.32 31.52 31.34 31.47 UFA 25.97 26.74 25.28 25.01 24.23 23.39 23.41 24.83 24.3 24.5 U/S 0.8 0.81 0.80 0.79 0.76 0.75 0.75 0.79 0.77 0.78 Viability 69.5 67.8 71.1 72.1 74 80.5 77.2 72.3 82 79.1 estimated. The UFA/SFA ratio was used as an indicator and Hahn Hagerdal 2000). Under ethanol shocks, similar to assess membrane fluidity. It has been previously changes were observed in membrane fatty acid profile as those reported that membranes with high UFA/SFA ratio show observed in acid shocked cells but of smaller amplitude. There a high fluidity (Casadei et al. 2002;Wanget al. 2005; was an increase in the content of the saturated palmitic (C14: 0) Álvarez-Ordóňez et al. 2009). As expected, O. oeni SD-2a and a decrease in the content of CFA compared to that of acid exposed to different stress shocks resulted in differences in shocked cells. However, the UFA/SFA ratio of cells subjected membrane fatty acid composition, major changes as a to 8% and 12% ethanol shock was higher than that of cells response to different stress shocks were observed in the subjected to acid shocks. These results agreed with Teixeira et relative content of lactobacillic acids (C19cyc11) and al. (2002). UFA/SFA ratio. For control cells that corresponding to the cells in stational phase (Sp) incubated in fresh Relationship between fatty acid composition medium for 2 h, the lowest CFA content and the highest and freeze-drying viability, malolactic activity UFA/SFA ratio were observed, indicating there was no stress in control cells. The UFA/SFA ratio observed for cold shocked In order to seek a possible relationship among fatty acid cells was similar with the cells in stational phase (Sp), but the composition, freeze-drying viability and malolactic activity cold shock induced a slight increase in the proportion of CFA. (MA), all these results were subjected to Pearson correlation These results agreed with Li et al. (2009a), Wang et al. (2005), analysis with nine variables (C14:0; C16:0; C16:1cis9; C18: and Casadei et al. (2002). However, the opposite effect was lcis11; C19cyc11; U/S; freeze-drying viability; malolactic described by other authors (Fernández et al. 2000; Guillot et activity and stress shocks). MA was malic acid degraded per al. 2000). Under different acid shocks, the main changes day (expressed as g/L) in the first 8 days after wine-like occurred in the relative content of C16:0, C17cyc9, medium inoculation. C18:1cis11, and C19cyc11. It was observed that as the pH The correlation matrix (Table 2, 3 and 4) calculated by dropped, the levels of C19cyc11 distinctly increased at the SPSS software showed the significant negative correlations expense of C18:lcis11 due to the conversion of UFA (C18:1) between C19cyc11 and C18:lcis11,C16:0inall stress to CFA (C19cyc11). The amount of C16:0 decreased from shocks. It was indicated that the levels of C19cyc11 27.86 to 25.05%, and the levels of C17cyc9 slightly increased. increased at expense of C18:lcis11, which was a precursor In addition, the UFA/SFA ratio distinctly decreased as the acid fatty acid for C19cyc11, and the role of C16:0 could be stress intensified, those results were similar with some reports replaced by C19cyc1. This conversion of the unsaturated fatty (Álvarez-Ordóñez, et al. 2008; 2009). Contrary result in UFA/ acids to their cyclopropane derivatives is assumed to serve as a SFA ratio in Lactobacillus spp. cells adapted to low pH was protective measure against stress conditions (Brown et also found (Schoug et al. 2008;Wangetal. 2005;Palmfeldt al. 1997; Álvarez-Ordóñez et al. 2008;Li etal. 2009b). 482 Ann Microbiol (2013) 63:477–485 Table 2 Correlation values between fatty acids composition, freeze-drying viability and malic acid degradation (MA) of O. oeni SD-2a cells exposed for 2 h to cold shocks C14:0 C16:0 C16:1cis9 C18:1cis11 C19:0cyc11 U/S Viability MA Cold shocks C14:0 1 C16:0 -0.514 1 C16:1cis9 -0.189 0.639 1 C18:1cis11 -0.486 0.868 0.450 1 a a C19:0cyc11 0.548 -0.798 -0.725 -0.827 1 U/S -0.339 0.681 0.718 0.687 -0.773 1 Viability 0.686 -0.734 -0.656 -0.722 0.898 -0.755 1 MA 0.763 -0.625 -0.445 -0.702 0.652 -0.579 0.723 1 Cold shocks -0.128 0.717 0.892 0.729 -0.200 0.776 -0.714 -0.390 1 U/S cyc: Unsaturated/saturated fatty acid ratio. MA, g of malic acid degraded per day, calculated in the first 8 days after inoculation of wine-like medium. Correlation is significant at the 0.05 level. Correlation is significant at the 0.01 level. The freeze-drying viability exhibited a significant positive Discussion correlation with the levels of C19cyc11 in cold and acid shocks (Tables 2 and 3), which implied C19cyc11 could offer Stress response mechanism of Oenococcus oeni has gained stronger tolerance ability of O. oeni SD-2a cells to freeze- increasing research interest (Beltramo et al. 2006), but there drying and play an important role in cold and acid stress are nearly no reports aiming at applying their stress shocks adaptive response. Furthermore, the only significant positive in preparation of O. oeni freeze-dried ready-to-use malolactic correlations between the ability of O. oeni SD-2a to conduct starter cultures. From an industrial point of view, the malic acid degradation and membrane composition existed development of protocols for the preparation of starter cultures with C14:0 in ethanol shocks (Table 4), showing O. with highly active, viable cells tolerant to adverse conditions oeni SD-2a would be more efficient in MA when the would be advantageous. Moreover, stress responses may be carbon chain was relatively short and saturated. This result used to enhance the survival of lactic acid bacteria in stress was in agreement with the previous report (Garbay et al. conditions and to improve their technological properties (van 1995). de Guchte et al. 2002; Carvalho et al. 2004). In our studies, we Table 3 Correlation values between fatty acids composition, freeze-drying viability and malic acid degradation (MA) of O. oeni SD-2a cells exposed for 2 h to acid shocks C14:0 C16:0 C16:1cis9 C18:1cis11 C19:0cyc11 U/S Viability MA Acid shocks C14:0 1 C16:0 -0.632 1 C16:1cis9 -0.783 0.526 1 C18:1cis11 -0.233 0.884 0.555 1 a b C19:0cyc11 0.402 -0.858 -0.540 -0.894 1 a b a U/S -0.703 0.887 0.622 0.825 -0.861 1 Viability 0.682 -0.676 -0.662 -0.746 0.869 -0.538 1 MA 0.812 -0.733 -0.790 -0.770 0.765 -0.803 0.721 1 a a Acid shocks -0.718 0.709 0.732 0.519 -0.798 0.851 -0.610 -0.883 1 U/S cyc: Unsaturated/saturated fatty acid ratio. MA, g of malic acid degraded per day, calculated in the first 8 days after inoculation of wine-like medium. Correlation is significant at the 0.05 level. Correlation is significant at the 0.01 level. Ann Microbiol (2013) 63:477–485 483 Table 4 Correlation values between fatty acids composition, freeze-drying viability and malic acid degradation (MA) of O. oeni SD-2a cells exposed for 2 h to ethanol shocks C14:0 C16:0 C16:1cis9 C18:1cis11 C19:0cyc11 U/S Viability MA Ethanol shocks C14:0 1 C16:0 -0.477 1 C16:1cis9 0.743 0.028 1 C18:1cis11 -0.610 0.791 0.093 1 a a C19:0cyc11 0.750 -0.888 -0.183 -0.891 1 U/S -0.798 0.528 -0.760 0.773 -0.700 1 Viability 0.628 -0.730 0.340 -0.701 0.762 -0.770 1 MA 0.869 -0.727 0.206 -0.669 0.775 -0.659 0.739 1 Ethanol shocks 0.828 -0.790 0.098 -0.624 0.754 -0.650 0.561 0.782 1 U/S cyc: Unsaturated/saturated fatty acid ratio. MA, g of malic acid degraded per day, calculated in the first 8 days after inoculation of wine-like medium. Correlation is significant at the 0.05 level. Correlation is significant at the 0.01 level. observed that all stress-shocked cells promoted lyophilization conversion of the unsaturated fatty acids to their cyclopropane resistance and MLF ability, specially cells shocked at pH 3.5 derivatives is believed to serve as a protective measure against and 8% ethanol notably increased freeze-drying viability and cold shock, acid shock, ethanol shock and lyophilization MLF ability. The results showed that the pretreatments play an inactivation. However, CFA contribution to membrane important role in improving bacterial resistance to adverse properties is not yet understood, especially concerning conditions, which could result in enhancing the reliability of the modifications of membrane fluidity in response to freeze-dried ready-to-use starter cultures in terms of quality. environmental stress. Muňoz-Rojas et al. (2006)have Biomembranes are the primary contact site of cells with indicated that the presence of a cyclopropane ring within their environment, cell survival ability to a large extent membrane fatty acids increases the stability of the structural relies on the flexibility and the adaptation capability (Cosette and function as a resistance barrier against environmental et al. 2008). Membrane lipid and protein compositions are stresses. Other studies have suggested that an increase in recognized as some of the main factors involved in stress CFA content could cause a decrease in membrane fluidity tolerance (da Silveira et al. 2003, 2004). The UFA/SFA ratio (Annous et al. 1999), which could explain the increase in was used as an indicator to assess membrane fluidity. Low lyophilization resistance of O. oeni SD-2a with high CFA UFA/SFA ratio has previously been linked to less membrane levels. In general, freeze-drying viabilities were maximum fluidity (Casadei et al. 2002; Wang et al. 2005; Álvarez- for cells with low UFA/SFA ratio and high CFA levels, and, Ordóňez et al. 2009). It is generally admitted that cells regulate consequently, with low membrane fluidity. Moreover, CFA their lipid composition in order to achieve a degree of fluidity (C19cyc11) formation played a major role in protecting stress compatible with life (Teixeira et al. 2002). Our results showed shocked cells from lyophilization (Li et al. 2009b). However, that freeze-drying viabilities obtained were higher for cells changes observed in membrane fatty acid composition are not with low UFA/SFA ratio. Similar results have been previously enough to explain the greater freeze-drying viability of cells found for Lactobacillus ssp. (Schoug et al. 2008;Lietal. shocked at 8% ethanol. Thus, other mechanisms could be 2009a). Previous researchers concluded that the synthesis of responsible for this increase in the bacterial resistance to CFA in cell membrane phospholipids during acid adaptation is lyophilization. an important factor in the protection from several stress Our study showed the freeze-drying survival of Oenococcus conditions (Brown et al. 1997; Chang and Cronan 1999). The oeni SD-2a was correlated with the increase of C19cyc11 and formation of CFA is considered to be a postsynthetic the decrease of UFA/SFA ratio in the membrane lipids of those modification of the phospholipid bilayer, which predominantly cells. However, it is difficult to speculate the role of stress occurs as cultures enter into the stationary phase. The CFA are protein and compatible solutes in protecting the cells against formed by CFA synthase through the addition of a methylene damage during lyophilization. Nevertheless, we suggest that group from S-adenosyl-L-methionine to the cis double bond of the shift to a high percentage of cyclopropane fatty acids and the UFA moiety of the phospholipid (Brown et al. 1997). In low UFA/SFA ratio in membranes protect O. oeni SD-2a from our case, a significant proportion of the UFA (C18:1) were freeze-drying. To gain further insight into the potential role of C19cyc11 in freeze-drying, detailed transcriptional studies of converted to CFA (C19cyc11) during all stress shocks. This 484 Ann Microbiol (2013) 63:477–485 Garbay S, Rozes N, Lonvaud-Funel A (1995) Fatty acid composition cyclopropane fatty acids synthesis in O. oeni SD-2a cells is of Leuconostoc oenos, incidence of growth conditions and being performed, furthermore, the explanation of stress relationship with malolactic efficiency. 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The effect of cold, acid and ethanol shocks on synthesis of membrane fatty acid, freeze-drying survival and malolactic activity of Oenococcus oeni

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Springer Journals
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Copyright © 2012 by Springer-Verlag and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
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10.1007/s13213-012-0492-x
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Ann Microbiol (2013) 63:477–485 DOI 10.1007/s13213-012-0492-x ORIGINAL ARTICLE The effect of cold, acid and ethanol shocks on synthesis of membrane fatty acid, freeze-drying survival and malolactic activity of Oenococcus oeni Guoqiang Zhang & Mingtao Fan & Qian Lv & Yahui Li & Yanlin Liu & Shuangfeng Zhang & Hua Zhang Received: 14 February 2012 /Accepted: 25 May 2012 /Published online: 16 June 2012 Springer-Verlag and the University of Milan 2012 Abstract The effects of stress shocks on the freeze-drying membrane fatty acid composition are not enough to explain viability, malolactic activity and membrane fatty acid com- the greater freeze-drying viability of cells shocked at 8% position of the Oenococcus oeni SD-2a cells were studied. ethanol. Thus, other mechanisms could be responsible for O. oeni SD-2a cells after 2 h of stress exposure exhibited this increase in the bacterial resistance to lyophilization. better freeze-drying viability and malolactic fermentation . . ability. A decrease in unsaturated fatty acids/saturated fatty Keywords Oenococcus oeni Stress shock Freeze-drying . . acids (UFA/SFA) ratio and in the C18:1 relative concentra- viability Fatty acid composition Malolactic activity tion, and an increase in cyclopropane fatty acids (CFA) content mainly due to the increase in C19cyc11 relative concentration were observed in all stress shocked cells. Introduction There was a significant negative correlation between C19cyc11 and C18:lcis11, C16:0 in all stress shocks. The Malolactic fermentation (MLF) following alcoholic fermentation freeze-drying viability exhibited a significant positive cor- in winemaking could reduce acidity, improve taste and relation with the levels of C19cyc11 in cold and acid shocks. biological stability. Different bacteria genera have been The only significant positive correlation between the ability reported to carry out MLF in wine. Among them, Oenococcus of O. oeni SD-2a to conduct malic acid degradation and oeni is recognized as the most advantageous and tolerant membrane composition existed with C14:0 in ethanol bacterium (Lonvaud-Funel 1999; Versari et al. 1999). MLF shocks. In general, freeze-drying viabilities were maximum deacidifies wine and results in a softer feeling in mouth, for cells with low UFA/SFA ratio and high CFA levels, and, also improves microbiological stability and organoleptic consequently, with low membrane fluidity. Moreover, CFA characteristics (Versari et al. 1999; Maicas 2001). For formation played a major role in protecting stress shocked many years, MLF has been recognized as an indispensable step cells from lyophilization. However, changes observed in in the elaboration of most wines (Nielsen et al. 1996; Maicas et al. 2000). However, due to very harsh environmental conditions in wine for bacterial survival and growth, induction of MLF has : : : : : : G. Zhang M. Fan (*) Q. Lv Y. Li Y. Liu S. Zhang failured in some processes (Garbay and Lonvaud-Funel 1996; H. Zhang Maicas et al. 2000; Zhao and Zhang 2009; Beltramo et al. College of Food Science and Engineering, 2006). There are two reasons for such failures. On one hand, Northwest A&F University, Yangling 712100, China strains lose their natural adaptation to survive and grow in wine, e-mail: mingtaofan@163.com which results in inoculation low viability. On the other hand, lyophilization offers stress conditions such as freezing, drying G. Zhang and concentration stress, which diminish cell viability. Tibet Agriculture and Animal Husbandary College, Therefore, expanding interest in freeze-dried ready-to-use Linzhi 860000, China 478 Ann Microbiol (2013) 63:477–485 malolactic starter cultures has placed more emphasis on The bacteria were statically cultured in ATB media at 25 °C. developing starter production and preservation methods Cell growth was monitored by absorbance readings at 600 that promote high cell viability and activity (Carvalho et nm using a spectrophotometer. When the cells had grown up al. 2004). to early stationary phase (A 01.9), the bacterial cells were Research in recent years has demonstrated that Oenococcus harvested by centrifugation (6,000×g for 10 min) at room oeni possesses remarkable resilience and adaptability in temperature. unfavorable environments (Maicas et al. 2000; Chu-Ky Cells were collected, inoculated in the same fresh medium et al. 2005;Beltramoetal. 2006;Sicoetal. 2009). and exposed for 2 h to different sublethal stresses. The Moreover, stress response, induced under one set of conditions and the amplitudes of stresses tested (various conditions, often provide the cell with cross-protection temperatures, pH and ethanol content) were chosen against other hostile and seemingly unrelated environments depending on real conditions in winemaking processes (van de Guchte et al. 2002;Spano andMassa 2006). Then a (Lonvaud-Funel 1999; Versari et al. 1999). For cold question was put forward as to whether stress response could shocks, the cell suspensions were incubated at 4, 15 take effect on the freeze-drying viability of O. oeni cells and and 25 °C, respectively. For ethanol shocks, ethanol be used in preparing freeze-dried ready-to-use malolactic was added to the same fresh medium under continuous starter cultures. The effect of stress response has been less stirring to final concentrations of 5, 8 and 12% (v/v). studied, although from data in literature for Lactobacillus ssp. During cold and ethanol stresses, the pH of the cell and Oenococcus oeni it can be deduced that their adaption in suspension was unchanged (pH 4.8). For acid shocks, low pH mediums is accompanied by an increase in freeze- different pH values (4.0, 3.5 and 3.2) were adjusted by drying viability (Schoug et al. 2008;Lietal. 2009a, b). addition of 1 M HCl solution. During acid shocks, the However, as far as we know little attention has been paid to temperature of the cell suspension was kept at 25 °C. the effects of stress shocks on the bacterial resistance to Two independent experiments with three repetitions each lyophilization and their MLF ability in wine-like medium. were performed. After 2 h, cell suspensions were centrifuged The mechanisms involved in the bacterial resistance to to harvest for lyophilization and FAs extraction. lyophilization are not fully understood. However, several reports have found the correlations between membrane fatty Freeze-drying assays acid compositions of different Lactobacillus spp. and their lyophilization tolerance (Schoug et al. 2008; Lietal. The stress-shocked cells were harvested by centrifugation at 2009a). In general, these authors have shown that cells with 6,000×g for 10 min and washed once in aseptic distilled a decreased concentration of unsaturated fatty acids or with water. Cell pellet was resuspended in sterile protective agents an increased content of saturated fatty acids have a decreased containing 2.5% sodium glutamate to make a cell suspension membrane fluidity, which is linked to a higher lyophilization allowed to equilibrate for 20 min at room temperature. tolerance. Furthermore, in a previous study performed in our Aliquots (10 mL) of each resuspension were transferred laboratory we found that the formation of cyoclopropane fatty into serum bottles (50 mL) and frozen at -20 °C overnight. acids plays an important role in protecting acid-adapted O. oeni Then, the samples were immediately freeze-dried in an cells from lyophilization inactivation (Li et al. 2009b). Edwards freeze dryer at a condenser temperature -56 °C and The objective of this study is to evaluate the effect of at a chamber pressure <0.06 mbar for 28 h. After freeze- stress shocks on the malolactic activity of Oenococcus oeni drying, samples were immediately brought to their original SD-2a in wine-like medium and its resistance to freeze- volume (10 mL) with ATB medium at 25 °C. Then, samples drying, meanwhile, try to clarify the stress response mech- were homogenized for 1 min with a Vortex mixer and incu- anism of O. oeni SD-2a related to membrane fatty acid bated at room temperature for 20 min. Serial dilutions were composition. spread-plated onto the surface of Petri plates containing ATB medium agar (ATB medium and 2.0% agar). These plates were incubated at 25 °C for at least 7 days, and the viability Materials and methods was then determined. Survival levels were expressed as the quotient of colony-forming units per millilitre (CFU/mL) on Bacterial strain and Stress treatments ATB medium before (N )and after(N ) freeze-drying. 0 f Viability0(N /N )×100. f 0 Oenococcus oeni strain SD-2a (Li et al. 2006) isolated from Chinesewines was used. Theculture mediawereATB MLF assays containing (g/L): glucose 10, yeast extract 5, peptone 10, MgSO ·7H O0.2,MnSO ·4H O 0.05, cysteine/HCl 0.5, The effect of stress shocks on the growth and MLF ability of 4 2 4 2 tomato juice 250 mL, pH of the medium was about 4.8. lyophilized Oenococcus oeni SD-2a were investigated. Ann Microbiol (2013) 63:477–485 479 Immediately after rehydration, a concentration of about cells were cells in the stationary phase incubated in fresh 1.0×10 CFU/mL of stress-shocked cells were inoculated into medium for 2 h while the control cells corresponded to the bottles containing 100 mL of the sterilized wine-like medium. same cells directly lyophilized. The wine-like medium (g/L) is a modified chemically defined Growth curves from rehydrated Oenococcus oeni SD-2a medium (Terrade et al. 2009) supplemented with L-malic acid suggested that the increased culturability of stress-shocked 2.5, tartaric acid 3.5, glucose 1.0, fructose 1.0, and the ethanol cells allowed these bacteria to reach mid-exponential phase content 10.0% (v/v). The medium was then adjusted to 3.5 more quickly than control cells. Specially rehydrated cells with HCl/KOH followed by sterile membrane filtration (0.22 shocked at pH 3.5 or 8% ethanol reached the mid- μm nylon, Millipore Corp., USA). Cell growth was monitored exponential phase significantly faster than rehydrated spectrophotometrically at A . Malic acid in samples was de- control cells. Stress-shocked cells started to grow almost termined by high performance liquid chromatography (HPLC). without lag phase except cold-shocked cells and 5% ethanol shocked cells. It was indicated that the appropriate Analysis of fatty acid composition stress shock was important for the application of lyophilized malolactic starters because stress shock affects the lag phase of The membrane fatty acid composition of the bacteria was the inoculation bacteria. Once the surviving bacteria start to determined using gas chromatography as described by multiply in the wine, the growth rates are similar. Rozes et al. (1993) and Li et al. (2009b). Concentrated cells Similar experiments were performed to investigate the were washed twice in sterile distilled water. Methylation and MLF ability of rehydrated stress-shocked cells. Rehydrated extraction were performed by adding 2.0 mL of sodium stress-shocked Oenococcus oeni SD-2a degradated malic methoxide (1 M in methanol) and shaking for 1 min. Fatty acid significantly faster than rehydrated control cells, acid methyl esters were extracted with 1 mL of hexane. specially cells shocked at pH 3.5 and 8% ethanol. After decanting for 2 min, the upper phase was taken out MLF was completed within 10 days with all treated and stored at 4 °C in an airtight glass bottle until analysis. cells, and malic acid degradation was as high as 50% The analyses were performed on a gas chromatographer within 2 days. It may also be worthwhile to note that (HP 6890, Hewlett Packard, Avondale, PA) equipped with malic acid degradation was higher for rehydrated control mass selective detector (Agilent 5973, Hewlett Packard). A cells than for fresh cells, but the A value was lower capillary column (BPX 70, 60 m×0.25 mm, SGE, Victoria, for rehydrated control cells than fresh cells. It may Australia) was used. Helium was used as carrier gas (1.2 reflect that lyophilization is beneficial for O. oeni SD-2a to mL/min), and the injection volume was 1 μL. Injection was achieve MLF. Moreover, it was indicated that appropriate done in splitless mode for 2 min. The oven temperature was stress shock plays an important role in increasing the MLF increased from 65 to 230 °C at 5 °C/min and maintained for ability of O. oeni SD-2a cells in wine. 10 min at 230 °C. Injection and detection temperatures were 230 °C. Effects of stress shocks on freeze-drying viability and fatty Results were expressed as relative percentages for each fatty acid composition acid based on the ratio of the surface area of the considered peak to the total area of all peaks. Analyses were made in triplicate. Three kinds of stress shocks (various temperatures, pH and ethanol content) depending on real conditions in winemaking Statistical analysis processes were carried out (Lonvaud-Funel 1999; Versari et al. 1999). The freeze-drying viability and the relative percentage The data presented are the mean of three replicates within of the membrane FAs of Oenococcus oeni SD-2a in the each experiment. The variations were <5%. Pearson corre- stationary phase (Sp), in the control (Co) corresponded lation analysis (SPSS software) was performed for all the to the same cells incubated in fresh medium for 2 h and stress shocks to relate the fatty acid composition to freeze- in cells exposed for 2 h to different stress conditions drying viability and malolactic activity of O. oeni SD-2a. were showninTable 1. Freeze-drying survival of cells harvested in stationary phase was 69.5% and control was 67.8% respectively Results (Table 1). This survival was almost in agreement with other reports on freeze-drying survival of Oenococcus oeni (Zhao MLF in wine-like medium and Zhang 2009; Li, et al. 2009b). It can also be seen from Table 1 that pretreatments at different stress treatments The results from direct inoculation of fresh cells, rehydrated before freeze-drying had different effect on the cell viability. control cells and rehydrated stress-shocked cells were In cold shocks, pretreatment at 4 °C and 15 °C improved the shown in Fig. 1. It is necessary to mention that the fresh cell viability lightly compared to the control. However, pH 480 Ann Microbiol (2013) 63:477–485 Fig. 1 Growth (solid curve) and malic acid degradation 0.15 2.5 (dotted curve) during culturing rehydrated Cold-shocked cells 0.12 2 (a), Acid-shocked cells (b) and fres h Ethanol-shocked cells (c)in control 0.09 1.5 wine-like medium 4 shocked 0.06 1 15 shocked 0.03 0.5 0 0 02 468 10 12 Time (d) 0.15 2.5 0.12 2 fres h control 0.09 1.5 pH3.2 s hocked pH3.5 s hocked 0.06 1 pH4.0 s hocked 0.03 0.5 0 0 0246 8 10 12 Time (d) 2.5 0.15 0.12 fres h control 1.5 0.09 5% ethanol s hocked 8% ethanol s hocked 0.06 12% ethanol s hocked 0.5 0.03 0 0 02 468 10 12 Time (d) of suspension medium had significant influence on the cell acid (C19cyc11). Their relative contents were between 3 and survival, which increased from 67.8 to 80.5% when pH of 45%, totaling to more than 90% of all fatty acids identified. suspension medium decreased from 4.8 to 3.5, but when pH These fatty acids are commonly found in other lactic acid of suspension medium was 3.2 the cell viability had a little bacteria strains (Beal et al. 2001; Gómez et al. 2000; Machado decrease. For ethanol shocks, the cell viability after freeze- et al. 2004; Taranto et al. 2003; Tymczyszyn et al. 2005;Wang drying increased by 14.2% when the ethanol concentration et al. 2005; Montanari et al. 2010). These results was in good increased from 0 to 8%. However, when the ethanol agreement with those of Garbay et al. (1995); Drici-Cachon et concentration further increased from 8 to 12%, the cell al. (1996)and Li et al.(2009b). Methylenhexadecanoic acid survival decreased, indicating higher ethanol concentration (C17cyc9), hexadecadienoic acid (C16:2cis9,12), stearic acid did not improve the cell viability. (C18:0) and octadecadienoic acid (C18:2cis9,12) were also The fatty acid compositions of Oenococcus oeni SD-2a after detected but in very low quantities. different stress shocks were determined (Table 1). In all cases, So as to determine the differences among membrane fatty nine fatty acids were found, the main fatty acids were: myristic acid composition of Oenococcus oeni SD-2a cells, total acid (C14:0), hexadecanoic acid (C16:0), hexadecenoic acid saturated fatty acids (SFA), total unsaturated fatty acids (C16:1cis9), cis-vaccenic acid (C18:1cis11) and lactobacillic (UFA) and total cyoclopropane fatty acids (CFA) were A600 A600 A600 Malic acid (g/L) Malic acid (g/L) Malic acid (g/L) Ann Microbiol (2013) 63:477–485 481 Table 1 Fatty acid composition and freeze-drying viability of O. oeni SD-2a in the stationary phase (Sp), in the control without stress shock (Co) and in cells exposed for 2 h to different stress shocks. The data are means of three independent experiments. The coefficients of variability were lower than 5% Fatty acids Sp Co Cold shocks Acid shocks Ethanol shocks 4°C 15°C pH 4.0 pH 3.5 pH 3.2 5% 8% 12% C14:0 2.96 2.90 2.91 3.11 3.33 3.57 3.84 3.67 4.28 4.01 C16:0 27.62 27.86 26.43 26.28 26.18 25.23 25.05 25.47 24.87 25.21 C16:1cis9 7.53 7.37 7.65 7.43 7.41 7.15 6.97 7.98 7.83 7.54 C16:2cis9,12 0.64 0.7 0.76 0.77 0.71 0.66 0.68 0.72 0.64 0.78 C17:0cyc9 0.36 0.35 0.41 0.48 0.56 0.68 0.72 0.39 0.55 0.52 C18:0 2.08 2.12 2.21 2.29 2.28 2.36 2.43 2.38 2.19 2.25 C18:1cis11 16.57 17.49 15.58 15.47 14.63 14.01 14.23 14.98 14.46 14.77 C18:2cis9,12 0.87 0.83 0.88 0.86 0.92 0.89 0.81 0.76 0.82 0.89 C19:0cyc11 41.37 40.28 42.97 43.31 43.98 45.45 45.27 43.65 44.36 44.03 SFA 32.66 32.88 31.75 31.68 31.79 31.16 31.32 31.52 31.34 31.47 UFA 25.97 26.74 25.28 25.01 24.23 23.39 23.41 24.83 24.3 24.5 U/S 0.8 0.81 0.80 0.79 0.76 0.75 0.75 0.79 0.77 0.78 Viability 69.5 67.8 71.1 72.1 74 80.5 77.2 72.3 82 79.1 estimated. The UFA/SFA ratio was used as an indicator and Hahn Hagerdal 2000). Under ethanol shocks, similar to assess membrane fluidity. It has been previously changes were observed in membrane fatty acid profile as those reported that membranes with high UFA/SFA ratio show observed in acid shocked cells but of smaller amplitude. There a high fluidity (Casadei et al. 2002;Wanget al. 2005; was an increase in the content of the saturated palmitic (C14: 0) Álvarez-Ordóňez et al. 2009). As expected, O. oeni SD-2a and a decrease in the content of CFA compared to that of acid exposed to different stress shocks resulted in differences in shocked cells. However, the UFA/SFA ratio of cells subjected membrane fatty acid composition, major changes as a to 8% and 12% ethanol shock was higher than that of cells response to different stress shocks were observed in the subjected to acid shocks. These results agreed with Teixeira et relative content of lactobacillic acids (C19cyc11) and al. (2002). UFA/SFA ratio. For control cells that corresponding to the cells in stational phase (Sp) incubated in fresh Relationship between fatty acid composition medium for 2 h, the lowest CFA content and the highest and freeze-drying viability, malolactic activity UFA/SFA ratio were observed, indicating there was no stress in control cells. The UFA/SFA ratio observed for cold shocked In order to seek a possible relationship among fatty acid cells was similar with the cells in stational phase (Sp), but the composition, freeze-drying viability and malolactic activity cold shock induced a slight increase in the proportion of CFA. (MA), all these results were subjected to Pearson correlation These results agreed with Li et al. (2009a), Wang et al. (2005), analysis with nine variables (C14:0; C16:0; C16:1cis9; C18: and Casadei et al. (2002). However, the opposite effect was lcis11; C19cyc11; U/S; freeze-drying viability; malolactic described by other authors (Fernández et al. 2000; Guillot et activity and stress shocks). MA was malic acid degraded per al. 2000). Under different acid shocks, the main changes day (expressed as g/L) in the first 8 days after wine-like occurred in the relative content of C16:0, C17cyc9, medium inoculation. C18:1cis11, and C19cyc11. It was observed that as the pH The correlation matrix (Table 2, 3 and 4) calculated by dropped, the levels of C19cyc11 distinctly increased at the SPSS software showed the significant negative correlations expense of C18:lcis11 due to the conversion of UFA (C18:1) between C19cyc11 and C18:lcis11,C16:0inall stress to CFA (C19cyc11). The amount of C16:0 decreased from shocks. It was indicated that the levels of C19cyc11 27.86 to 25.05%, and the levels of C17cyc9 slightly increased. increased at expense of C18:lcis11, which was a precursor In addition, the UFA/SFA ratio distinctly decreased as the acid fatty acid for C19cyc11, and the role of C16:0 could be stress intensified, those results were similar with some reports replaced by C19cyc1. This conversion of the unsaturated fatty (Álvarez-Ordóñez, et al. 2008; 2009). Contrary result in UFA/ acids to their cyclopropane derivatives is assumed to serve as a SFA ratio in Lactobacillus spp. cells adapted to low pH was protective measure against stress conditions (Brown et also found (Schoug et al. 2008;Wangetal. 2005;Palmfeldt al. 1997; Álvarez-Ordóñez et al. 2008;Li etal. 2009b). 482 Ann Microbiol (2013) 63:477–485 Table 2 Correlation values between fatty acids composition, freeze-drying viability and malic acid degradation (MA) of O. oeni SD-2a cells exposed for 2 h to cold shocks C14:0 C16:0 C16:1cis9 C18:1cis11 C19:0cyc11 U/S Viability MA Cold shocks C14:0 1 C16:0 -0.514 1 C16:1cis9 -0.189 0.639 1 C18:1cis11 -0.486 0.868 0.450 1 a a C19:0cyc11 0.548 -0.798 -0.725 -0.827 1 U/S -0.339 0.681 0.718 0.687 -0.773 1 Viability 0.686 -0.734 -0.656 -0.722 0.898 -0.755 1 MA 0.763 -0.625 -0.445 -0.702 0.652 -0.579 0.723 1 Cold shocks -0.128 0.717 0.892 0.729 -0.200 0.776 -0.714 -0.390 1 U/S cyc: Unsaturated/saturated fatty acid ratio. MA, g of malic acid degraded per day, calculated in the first 8 days after inoculation of wine-like medium. Correlation is significant at the 0.05 level. Correlation is significant at the 0.01 level. The freeze-drying viability exhibited a significant positive Discussion correlation with the levels of C19cyc11 in cold and acid shocks (Tables 2 and 3), which implied C19cyc11 could offer Stress response mechanism of Oenococcus oeni has gained stronger tolerance ability of O. oeni SD-2a cells to freeze- increasing research interest (Beltramo et al. 2006), but there drying and play an important role in cold and acid stress are nearly no reports aiming at applying their stress shocks adaptive response. Furthermore, the only significant positive in preparation of O. oeni freeze-dried ready-to-use malolactic correlations between the ability of O. oeni SD-2a to conduct starter cultures. From an industrial point of view, the malic acid degradation and membrane composition existed development of protocols for the preparation of starter cultures with C14:0 in ethanol shocks (Table 4), showing O. with highly active, viable cells tolerant to adverse conditions oeni SD-2a would be more efficient in MA when the would be advantageous. Moreover, stress responses may be carbon chain was relatively short and saturated. This result used to enhance the survival of lactic acid bacteria in stress was in agreement with the previous report (Garbay et al. conditions and to improve their technological properties (van 1995). de Guchte et al. 2002; Carvalho et al. 2004). In our studies, we Table 3 Correlation values between fatty acids composition, freeze-drying viability and malic acid degradation (MA) of O. oeni SD-2a cells exposed for 2 h to acid shocks C14:0 C16:0 C16:1cis9 C18:1cis11 C19:0cyc11 U/S Viability MA Acid shocks C14:0 1 C16:0 -0.632 1 C16:1cis9 -0.783 0.526 1 C18:1cis11 -0.233 0.884 0.555 1 a b C19:0cyc11 0.402 -0.858 -0.540 -0.894 1 a b a U/S -0.703 0.887 0.622 0.825 -0.861 1 Viability 0.682 -0.676 -0.662 -0.746 0.869 -0.538 1 MA 0.812 -0.733 -0.790 -0.770 0.765 -0.803 0.721 1 a a Acid shocks -0.718 0.709 0.732 0.519 -0.798 0.851 -0.610 -0.883 1 U/S cyc: Unsaturated/saturated fatty acid ratio. MA, g of malic acid degraded per day, calculated in the first 8 days after inoculation of wine-like medium. Correlation is significant at the 0.05 level. Correlation is significant at the 0.01 level. Ann Microbiol (2013) 63:477–485 483 Table 4 Correlation values between fatty acids composition, freeze-drying viability and malic acid degradation (MA) of O. oeni SD-2a cells exposed for 2 h to ethanol shocks C14:0 C16:0 C16:1cis9 C18:1cis11 C19:0cyc11 U/S Viability MA Ethanol shocks C14:0 1 C16:0 -0.477 1 C16:1cis9 0.743 0.028 1 C18:1cis11 -0.610 0.791 0.093 1 a a C19:0cyc11 0.750 -0.888 -0.183 -0.891 1 U/S -0.798 0.528 -0.760 0.773 -0.700 1 Viability 0.628 -0.730 0.340 -0.701 0.762 -0.770 1 MA 0.869 -0.727 0.206 -0.669 0.775 -0.659 0.739 1 Ethanol shocks 0.828 -0.790 0.098 -0.624 0.754 -0.650 0.561 0.782 1 U/S cyc: Unsaturated/saturated fatty acid ratio. MA, g of malic acid degraded per day, calculated in the first 8 days after inoculation of wine-like medium. Correlation is significant at the 0.05 level. Correlation is significant at the 0.01 level. observed that all stress-shocked cells promoted lyophilization conversion of the unsaturated fatty acids to their cyclopropane resistance and MLF ability, specially cells shocked at pH 3.5 derivatives is believed to serve as a protective measure against and 8% ethanol notably increased freeze-drying viability and cold shock, acid shock, ethanol shock and lyophilization MLF ability. The results showed that the pretreatments play an inactivation. However, CFA contribution to membrane important role in improving bacterial resistance to adverse properties is not yet understood, especially concerning conditions, which could result in enhancing the reliability of the modifications of membrane fluidity in response to freeze-dried ready-to-use starter cultures in terms of quality. environmental stress. Muňoz-Rojas et al. (2006)have Biomembranes are the primary contact site of cells with indicated that the presence of a cyclopropane ring within their environment, cell survival ability to a large extent membrane fatty acids increases the stability of the structural relies on the flexibility and the adaptation capability (Cosette and function as a resistance barrier against environmental et al. 2008). Membrane lipid and protein compositions are stresses. Other studies have suggested that an increase in recognized as some of the main factors involved in stress CFA content could cause a decrease in membrane fluidity tolerance (da Silveira et al. 2003, 2004). The UFA/SFA ratio (Annous et al. 1999), which could explain the increase in was used as an indicator to assess membrane fluidity. Low lyophilization resistance of O. oeni SD-2a with high CFA UFA/SFA ratio has previously been linked to less membrane levels. In general, freeze-drying viabilities were maximum fluidity (Casadei et al. 2002; Wang et al. 2005; Álvarez- for cells with low UFA/SFA ratio and high CFA levels, and, Ordóňez et al. 2009). It is generally admitted that cells regulate consequently, with low membrane fluidity. Moreover, CFA their lipid composition in order to achieve a degree of fluidity (C19cyc11) formation played a major role in protecting stress compatible with life (Teixeira et al. 2002). Our results showed shocked cells from lyophilization (Li et al. 2009b). However, that freeze-drying viabilities obtained were higher for cells changes observed in membrane fatty acid composition are not with low UFA/SFA ratio. Similar results have been previously enough to explain the greater freeze-drying viability of cells found for Lactobacillus ssp. (Schoug et al. 2008;Lietal. shocked at 8% ethanol. Thus, other mechanisms could be 2009a). Previous researchers concluded that the synthesis of responsible for this increase in the bacterial resistance to CFA in cell membrane phospholipids during acid adaptation is lyophilization. an important factor in the protection from several stress Our study showed the freeze-drying survival of Oenococcus conditions (Brown et al. 1997; Chang and Cronan 1999). The oeni SD-2a was correlated with the increase of C19cyc11 and formation of CFA is considered to be a postsynthetic the decrease of UFA/SFA ratio in the membrane lipids of those modification of the phospholipid bilayer, which predominantly cells. However, it is difficult to speculate the role of stress occurs as cultures enter into the stationary phase. The CFA are protein and compatible solutes in protecting the cells against formed by CFA synthase through the addition of a methylene damage during lyophilization. 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Journal

Annals of Microbiology – Springer Journals

Published: Jun 16, 2012

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