Accepted Article
Article Type : Original Article
EFFECT OF THE FATTY ACID COMPOSITION OF ACCLIMATED OENOLOGICAL LACTOBACILLUS PLANTARUM ON THE RESISTANCE TO ETHANOL
Bárbara Mercedes Bravo-Ferrada1, Andrea Gómez-Zavaglia2, Liliana Semorile1 and E. Elizabeth Tymczyszyn1*
1
Laboratorio de Microbiología Molecular, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal, Argentina
2
Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA) (CONICET La Plata, UNLP), La Plata, Argentina
Running head: Fatty acid composition of Lb plantarum This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/lam.12350 This article is protected by copyright. All rights reserved.
Accepted Article
*
Corresponding author: E. Elizabeth Tymczyszyn
Address: Roque Saenz Peña 352, Buenos Aires. Argentina (B1876BXD) Telephone: Tel. (+54 11) 4365 7100 Fax: (+54 11) 4365 7101 E-mail: [email protected]
Significance and Impact of the Study Understanding the adaptation of oenological strains to ethanol has strong impact on winemaking as it allows defining the most appropriate conditions to maintain bacteria viability and improve the efficiency of malolactic fermentation. The acclimation of oenological Lb. plantarum in media containing ethanol produces a drastic decrease of C18:1 with an increase in the content of saturated short-chain length membrane fatty acids that are correlated with a higher resistance to the adverse environmental conditions of wine.
Abstract The aim of this work was to evaluate the changes due to acclimation to ethanol on the fatty acid composition of three oenological Lactobacillus plantarum strains and their effect on the resistance to ethanol and malic acid consumption (MAC). Lb plantarum UNQLp 133, UNQLp 65.3 and UNQLp 155 were acclimated in the presence of 6 or 10% v/v ethanol, for 48 h at 28 °C. Lipids were extracted to obtain fatty acid methyl esters, and analyzed by gas chromatography interfaced with mass spectroscopy. The influence of change in fatty acid
This article is protected by copyright. All rights reserved.
Accepted Article
composition on the viability and MAC in synthetic wine was analyzed by determining the Pearson correlation coefficient. Acclimated strains showed a significant change in the fatty composition with regard to the non-acclimated strains. Adaptation to ethanol led to a decrease in the unsaturated/saturated ratio, mainly resulting from an increase in the contribution of short length fatty acid C12:0 and a decrease of C18:1. The content of C12:0 was related to a higher viability after inoculation of synthetic wine. The MAC increased at higher contents in saturated fatty acid, but its efficiency was strain dependent.
Keywords: Lactobacillus plantarum, fatty acid, ethanol stress, acclimation, wine.
INTRODUCTION Malolactic bacteria are lactic acid bacteria that are able to carry out malolactic fermentation (MLF). The control of their activity is an important technological aspect in the commercial production of wine (Bastianini et al. 2000). The viability of malolactic starters must be high enough to ensure that the harsh wine conditions are not detrimental for MLF (Henick-Kling 1995).
The acclimation of starter cultures with sublethal concentrations of ethanol and low pH (from 3.5 to 4.6) is necessary to increase their ethanol resistance during winemaking (Cecconi et al. 2009; Solieri et al. 2009; Lerm et al. 2010).
This article is protected by copyright. All rights reserved.
Accepted Article
Even when Oenococcus oeni is the main species used as commercial starter for MLF, some Lactobacillus plantarum strains can also survive in the harsh conditions of wine (Bravo-Ferrada et al 2013, Lerm et al. 2011). In addition, different studies have demonstrated that Lb plantarum strains are able to grow in wine conditions (Du Plessis et al. 2004; G-Alegría et al. 2004, López et al. 2008; Miller et al. 2011) and have resistance mechanisms to tolerate high concentrations of ethanol and low pHs (G-Alegría et al. 2004; Lee et al. 2012; van Bokhorst-van de Veen et al., 2011). The viability of bacterial cells depends on their adaptation capacity to the environment, which is mainly regulated by the membrane fluidity (da Silveira et al 2003, Chu-ky et al 2005). Several events are related with the ability of cells to survive in wine conditions and metabolize malic acid, one of them being the synthesis of small heat shock proteins and membrane proteins (Guzzo et al 1997, Silveira et al 2004). Another strategy is the adjustment of the membrane lipid composition. Different authors investigated the effect of stress factors on the lipid composition of Oenococus oeni. In this regard, it has been reported that the increase in the unsaturated/saturated fatty acids ratio counteract the toxic effect of ethanol (Garbay et al 1995, Bastianini et al 2000, da Silveira et al 2003). Nevertheless, Grandvalet et al. 2008 found a decrease in the unsaturated/ saturated fatty acids ratio when O. Oeni was grown in the presence of ethanol.
The increase in the resistance to ethanol has been associated with high concentrations of cycC19:0 fatty acid (Garbay et al 1995, Teixeira et al. 2002, Bastianini et al 2000, Grandvalet et al 2008). CycC19:0 is produced by enzymatic conversion of C18:1. However, the role of cycC19:0 is controversial. Some authors consider this fatty acid as unsaturated
This article is protected by copyright. All rights reserved.
Accepted Article
and responsible for the increase of membrane fluidity (Garbay et al 1995, Bastianini et al 2000). On the contrary, other authors ascribe its protective effect against ethanol to its capacity to rigidize lipid membranes, thus reducing bacterial membrane permeability (Grandvalet et al 2008).
In our previous paper we reported that acclimation of oenological strains of Lb plantarum with 6 and 10% v/v ethanol prevents membrane damage after inoculation in wine conditions (Bravo-Ferrada et al. 2014). However, to our knowledge the effect of acclimation on the fatty acid composition of Lb plantarum has not been studied. For this reason, the aim of this work was to evaluate the changes due to acclimation to ethanol on the membrane fatty acid composition of three oenological strains of Lb plantarum, and correlate these changes with the malic acid consumption (MAC) and the bacterial resistance to harsh wine environment.
RESULTS AND DISCUSSION Different authors reported alterations in membrane properties of O. oeni grown in the presence of ethanol (da Silveira et al 2002, 2003 and 2009, Chu ky et al 2005). It was also reported that acclimation of Lb plantarum leads to decrease in membrane damage when grown in the presence of ethanol (Bravo-Ferrada et al., 2014). Lipid membranes are the main target of damage by ethanol and microorganisms have different strategies to prevent this damage (da Silveria et al 2003 and 2004). One of them is the modification of the fatty acid composition to regulate the membrane fluidity (Garbay et al 1995, Teixeira et al 2002,
This article is protected by copyright. All rights reserved.
Accepted Article
da Silveira et al 2003, Gandvalet et al 2008). However, to our knowledge, a correlation between the fatty acid composition of Lb plantarum strains and acclimation has not been reported hereto.
The fatty acid composition of Lb plantarum UNQLp 133, 65.3 and 155 non-acclimated and acclimated in the presence of 6 and 10% v/v ethanol is depicted in Figures 1A, B and C (controls: non-acclimated strains). The fatty acid composition of the non-acclimated strains was similar, 16:0 and 18:1 being the most abundant fatty acids. CycC19:0, a typical lactobacilli fatty acid, was ~10-20%.
Upon acclimation, important changes were observed in the fatty acid composition. Acclimation of Lb plantarum UNQLp 133 with 6 and 10% v/v ethanol led to an increase of 12:0 and a decrease of 16:0, 18:1 and cycC19:0 (Figure 1A). A significant increase of C14:0 was observed after acclimation with ethanol 6%, and C10:0 significantly increased when UNQLp 133 was acclimated with ethanol 10% (P
Article Type : Original Article
EFFECT OF THE FATTY ACID COMPOSITION OF ACCLIMATED OENOLOGICAL LACTOBACILLUS PLANTARUM ON THE RESISTANCE TO ETHANOL
Bárbara Mercedes Bravo-Ferrada1, Andrea Gómez-Zavaglia2, Liliana Semorile1 and E. Elizabeth Tymczyszyn1*
1
Laboratorio de Microbiología Molecular, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal, Argentina
2
Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA) (CONICET La Plata, UNLP), La Plata, Argentina
Running head: Fatty acid composition of Lb plantarum This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/lam.12350 This article is protected by copyright. All rights reserved.
Accepted Article
*
Corresponding author: E. Elizabeth Tymczyszyn
Address: Roque Saenz Peña 352, Buenos Aires. Argentina (B1876BXD) Telephone: Tel. (+54 11) 4365 7100 Fax: (+54 11) 4365 7101 E-mail: [email protected]
Significance and Impact of the Study Understanding the adaptation of oenological strains to ethanol has strong impact on winemaking as it allows defining the most appropriate conditions to maintain bacteria viability and improve the efficiency of malolactic fermentation. The acclimation of oenological Lb. plantarum in media containing ethanol produces a drastic decrease of C18:1 with an increase in the content of saturated short-chain length membrane fatty acids that are correlated with a higher resistance to the adverse environmental conditions of wine.
Abstract The aim of this work was to evaluate the changes due to acclimation to ethanol on the fatty acid composition of three oenological Lactobacillus plantarum strains and their effect on the resistance to ethanol and malic acid consumption (MAC). Lb plantarum UNQLp 133, UNQLp 65.3 and UNQLp 155 were acclimated in the presence of 6 or 10% v/v ethanol, for 48 h at 28 °C. Lipids were extracted to obtain fatty acid methyl esters, and analyzed by gas chromatography interfaced with mass spectroscopy. The influence of change in fatty acid
This article is protected by copyright. All rights reserved.
Accepted Article
composition on the viability and MAC in synthetic wine was analyzed by determining the Pearson correlation coefficient. Acclimated strains showed a significant change in the fatty composition with regard to the non-acclimated strains. Adaptation to ethanol led to a decrease in the unsaturated/saturated ratio, mainly resulting from an increase in the contribution of short length fatty acid C12:0 and a decrease of C18:1. The content of C12:0 was related to a higher viability after inoculation of synthetic wine. The MAC increased at higher contents in saturated fatty acid, but its efficiency was strain dependent.
Keywords: Lactobacillus plantarum, fatty acid, ethanol stress, acclimation, wine.
INTRODUCTION Malolactic bacteria are lactic acid bacteria that are able to carry out malolactic fermentation (MLF). The control of their activity is an important technological aspect in the commercial production of wine (Bastianini et al. 2000). The viability of malolactic starters must be high enough to ensure that the harsh wine conditions are not detrimental for MLF (Henick-Kling 1995).
The acclimation of starter cultures with sublethal concentrations of ethanol and low pH (from 3.5 to 4.6) is necessary to increase their ethanol resistance during winemaking (Cecconi et al. 2009; Solieri et al. 2009; Lerm et al. 2010).
This article is protected by copyright. All rights reserved.
Accepted Article
Even when Oenococcus oeni is the main species used as commercial starter for MLF, some Lactobacillus plantarum strains can also survive in the harsh conditions of wine (Bravo-Ferrada et al 2013, Lerm et al. 2011). In addition, different studies have demonstrated that Lb plantarum strains are able to grow in wine conditions (Du Plessis et al. 2004; G-Alegría et al. 2004, López et al. 2008; Miller et al. 2011) and have resistance mechanisms to tolerate high concentrations of ethanol and low pHs (G-Alegría et al. 2004; Lee et al. 2012; van Bokhorst-van de Veen et al., 2011). The viability of bacterial cells depends on their adaptation capacity to the environment, which is mainly regulated by the membrane fluidity (da Silveira et al 2003, Chu-ky et al 2005). Several events are related with the ability of cells to survive in wine conditions and metabolize malic acid, one of them being the synthesis of small heat shock proteins and membrane proteins (Guzzo et al 1997, Silveira et al 2004). Another strategy is the adjustment of the membrane lipid composition. Different authors investigated the effect of stress factors on the lipid composition of Oenococus oeni. In this regard, it has been reported that the increase in the unsaturated/saturated fatty acids ratio counteract the toxic effect of ethanol (Garbay et al 1995, Bastianini et al 2000, da Silveira et al 2003). Nevertheless, Grandvalet et al. 2008 found a decrease in the unsaturated/ saturated fatty acids ratio when O. Oeni was grown in the presence of ethanol.
The increase in the resistance to ethanol has been associated with high concentrations of cycC19:0 fatty acid (Garbay et al 1995, Teixeira et al. 2002, Bastianini et al 2000, Grandvalet et al 2008). CycC19:0 is produced by enzymatic conversion of C18:1. However, the role of cycC19:0 is controversial. Some authors consider this fatty acid as unsaturated
This article is protected by copyright. All rights reserved.
Accepted Article
and responsible for the increase of membrane fluidity (Garbay et al 1995, Bastianini et al 2000). On the contrary, other authors ascribe its protective effect against ethanol to its capacity to rigidize lipid membranes, thus reducing bacterial membrane permeability (Grandvalet et al 2008).
In our previous paper we reported that acclimation of oenological strains of Lb plantarum with 6 and 10% v/v ethanol prevents membrane damage after inoculation in wine conditions (Bravo-Ferrada et al. 2014). However, to our knowledge the effect of acclimation on the fatty acid composition of Lb plantarum has not been studied. For this reason, the aim of this work was to evaluate the changes due to acclimation to ethanol on the membrane fatty acid composition of three oenological strains of Lb plantarum, and correlate these changes with the malic acid consumption (MAC) and the bacterial resistance to harsh wine environment.
RESULTS AND DISCUSSION Different authors reported alterations in membrane properties of O. oeni grown in the presence of ethanol (da Silveira et al 2002, 2003 and 2009, Chu ky et al 2005). It was also reported that acclimation of Lb plantarum leads to decrease in membrane damage when grown in the presence of ethanol (Bravo-Ferrada et al., 2014). Lipid membranes are the main target of damage by ethanol and microorganisms have different strategies to prevent this damage (da Silveria et al 2003 and 2004). One of them is the modification of the fatty acid composition to regulate the membrane fluidity (Garbay et al 1995, Teixeira et al 2002,
This article is protected by copyright. All rights reserved.
Accepted Article
da Silveira et al 2003, Gandvalet et al 2008). However, to our knowledge, a correlation between the fatty acid composition of Lb plantarum strains and acclimation has not been reported hereto.
The fatty acid composition of Lb plantarum UNQLp 133, 65.3 and 155 non-acclimated and acclimated in the presence of 6 and 10% v/v ethanol is depicted in Figures 1A, B and C (controls: non-acclimated strains). The fatty acid composition of the non-acclimated strains was similar, 16:0 and 18:1 being the most abundant fatty acids. CycC19:0, a typical lactobacilli fatty acid, was ~10-20%.
Upon acclimation, important changes were observed in the fatty acid composition. Acclimation of Lb plantarum UNQLp 133 with 6 and 10% v/v ethanol led to an increase of 12:0 and a decrease of 16:0, 18:1 and cycC19:0 (Figure 1A). A significant increase of C14:0 was observed after acclimation with ethanol 6%, and C10:0 significantly increased when UNQLp 133 was acclimated with ethanol 10% (P
