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Retention of aroma compounds by lactic acid bacteria in model food media
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HYDROCOLLOIDS Food Hydrocolloids 22 (2008) 211–217 www.elsevier.com/locate/foodhyd
Retention of aroma compounds by lactic acid bacteria in model food media M.H. Lya,c,, M. Covarrubias-Cervantesb, C. Dury-Brunb, S. Bordeta, A. Voilleyb, T.M. Lec, J.-M. Belina, Y Wache´a, a Laboratoire de Microbiologie UMR UB/INRA 1232, ENSBANA, 1, Esplanade Erasme 21000 Dijon, France Laboratoire IMSAPS, Universite´ de Bourgogne, IFR 92, ENSBANA, 1, Esplanade Erasme 21000 Dijon, France c Laboratoire de Technologie et Biologie Alimentaire de l’Institut Polytechnique de Hanoı¨, 1, Rue Dai Co Viet, Hanoı¨, Vietnam b
Received 1 June 2006; accepted 6 November 2006
Abstract The interactions between aroma compounds and other particles in foods, particularly with macromolecules, have been greatly studied in order to better understand the binding of flavors in food matrices. Bacteria possess many macromolecules on their cellular surface that provide them surface properties which are involved in the physicochemical interactions between cells and interfaces. However, the interactions between bacteria and aroma compounds have not received so much attention despite the presence of bacteria in many fermented products. In order to study the retention of aroma compounds by bacteria, we have investigated the retention of esters by lactic acid bacteria with static headspace techniques. Two strains of Lactococcus lactis subsp. lactis biov. diacetylactis reflecting the natural diversity of the bacterial surface properties and two ethyl esters generally involved in the cheese flavor (ethyl acetate and ethyl hexanoate) were chosen for the experiments. The results have shown that bacteria, through their surface physicochemical properties, can interact directly with aroma compounds or in an indirect way, by changing the emulsion characteristics. However, these effects depend on the physicochemical properties of both aroma compounds and bacterial surfaces. r 2006 Elsevier Ltd. All rights reserved. Keywords: Aroma compound; Retention; Surface properties; Lactic acid bacteria; Interactions
1. Introduction The retention of aroma compounds in food matrices is important for the perception of food products (de Roos, 2003; Druaux and Voilley, 1997). In aqueous systems, the retention of aroma compounds can be modified by physicochemical interactions between macromolecules such as proteins and polysaccharides, lipids and aroma compounds (Guichard, 2006; Seuvre et al., 2000; Seuvre et al., 2006; Terta et al., 2006). Specific interactions resulting in the binding of aroma compounds to food components have been demonstrated such as covalent and Corresponding authors. Laboratoire de Microbiologie UMR UB/ INRA 1232, ENSBANA, 1, Esplanade Erasme 21000 Dijon, France. E-mail addresses: [email protected] (M.H. Ly), [email protected] (Y. Wache´).
0268-005X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2006.11.001
hydrogen bonding and hydrophobic interactions (Le Thanh et al., 1992; Meynier et al., 2004). However these interactions depend on the physicochemical properties and formulation of both aromas and macromolecules (Reiners et al., 2000). Food products have generally heterogeneous structures with water-insoluble compounds dispersed in the matrix which is often emulsified or gelified. The retention of aroma compounds can also be affected by the food microstructure and rheology (Terta et al., 2006). For instance, the size of the droplets of emulsions can have an impact on the release of aromas, particularly on hydrophobic compounds (Meynier et al., 2005). Moreover, food matrices such as fermented products can also contain an important number of microbial cells. Bacterial concentrations can exceed 109 cultivable cells/ml at the end of fermentations, amount that is usually
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maintained over 108 cells=ml for probiotic products (Yoon et al., 2006). Bacterial cells are micrometer-scale organisms bearing at their surface many macromolecules, such as proteins, mannoproteins, peptidoglycan, teichoic acid and polysaccharides (Boonaert and Rouxhet, 2000; van der Mei et al., 2000). These compounds confer to cells physicochemical properties that enable bacteria to interact with other compounds through Lewis acid/base, electrostatic and hydrophobic interactions (Hermansson, 1999; Scha¨r-Zammaretti and Ubbink, 2003). The microbial surface properties have been widely studied in order to understand the interactions between bacteria and interfaces and, more particularly, their implication in the formation of biofilms, a phenomenon important in many fields such as biomedical and food safety, corrosion and environment (Bellon-Fontaine et al., 1996; Briandet et al., 2001; Strevett and Chen, 2003; van der Mei et al., 2000). Hydrophobic interactions are important to explain the affinity of bacteria for apolar compounds (Bouchez-Naı¨ tali et al., 2001; Bruinsma et al., 2001; Ly et al., 2006; Pascual et al., 2000). In a recent study (Ly, Vo et al., 2006), we have studied the interactions between bacteria and food components observing that cells with more hydrophobic surfaces had a higher affinity for milk fat. On the other hand, electrostatic interactions between bacteria and droplets of emulsions can modify the emulsion stability (Li et al., 2001; Ly, Naı¨ tali et al., 2006). Up to now, few studies investigated the retention of aroma compounds by microorganism. Some authors (Chalier et al., 2007; Lubbers et al., 1994) have shown that the retention of aroma compounds by mannoproteins of yeast extracts depends on the hydrophobicity of aromas and on mannoprotein fractions. The present study aims at investigating the impact of bacteria on the retention of aroma compounds. In the first part, the study deals with the direct effect of bacteria on the retention of esters with different hydrophobicity in aqueous solutions. In the second part, the effect of bacteria on the retention of esters is investigated in a model emulsion that can be destabilized by bacteria. A species of lactic acid bacteria largely employed in fermentation processes, Lactococcus lactis subsp. lactis biov. diacetylactis, was used (McKay and Baldwin, 1990; Ross et al., 2002) with two strains exhibiting different surface properties selected from preceding studies (Ly, Vo et al., 2006). The retention of aroma compounds was evaluated by a static headspace technique. 2. Material and methods 2.1. Material 2.1.1. Aroma compounds The aroma compounds used in this study are esters, ethyl acetate and ethyl hexanoate (both purchased from Sigma with purities 498%), which are present in typical flavors of cheese, (Liu et al., 2004) and have already been character-
ized in several studies concerning retention (Landy et al., 1995; Seuvre et al., 2006). The hydrophobic constants of volatile compounds are expressed by the logarithm of the partition coefficient between water and n-octanol: log P of ethyl acetate is 0.7 and log P of ethyl hexanoate is 2.8 (Seuvre et al., 2006). 2.1.2. Bacterial strains Two strains of L. lactis ssp. lactis subv. diacetylactis (noted LLD16 and LLD18 and previously SD (S. diacetylactis)) from the CNRZ/INRA (Jouy-en-Josas) collection were used. Their surface properties had been previously characterized, hydrophobicity by the microbial adhesion to hydrocarbon (MATH) test which consists in evaluating the percentage of cells extracted by a solvent phase after vortex, and the z potential and isoelectric point, after the electrophoretic mobility of the cells (Ly, Naı¨ tali et al., 2006). The surface of the strain LLD18 was hydrophobic with 50% of the cells adhering to the apolar solvent hexadecane and the isoelectric point was about 4.5. The strain LLD16 adhered weakly to alkanes and its isoelectric point was inferior to 2. 2.2. Methods 2.2.1. Cell culture conditions Bacteria were cultured in MRS media (De Man et al., 1960) (In g/l: proteose peptone, 10, yeast extract, 5, meat extract, 10, trisodium citrate, 3.1, magnesium sulfate, 0.1, manganese sulfate, 0.05, dipotassium phosphate, 2 without Tween 80 and for which glucose was replaced with lactose, 15). Bacteria conserved at 70 C in MRS media containing 25% (vol/vol) glycerol were thawed, subcultured overnight, and grown in liquid MRS media. Cultures were performed at 27 C in static conditions until the early stationary phase. 2.2.2. Static headspace analysis Quantification of esters in the gas phase was carried out for aqueous solutions and emulsions containing one or the other strain. 2.2.2.1. Preparation. 50 ppm (v/v) of ethyl acetate and ethyl hexanoate were added in solutions and emulsions. Solutions consisted in citrate buffer (0.01 M, pH 4.5) or 9 g/l NaCl containing 109 cells=ml of bacteria LLD18 or LLD16, corresponding to an optical density at 600 nm of OD600 nm ¼ 1. The emulsion was prepared by mixing three times for 3 min, 80% distilled water, 20% hexadecane and 0.5 % w/v of hexadecyltrimethylammonium bromide (CTAB) by an ultrasonic generator at a power level of þ200 V (Fisher scientific Transsonic 460H, Illkirch, France). This emulsion was stocked at 4 1C and diluted ten folds in citrate buffer (0.01 M, pH 4.5) before use.
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2.2.2.2. Headspace analysis. 35 ml of the medium to analyze were put into a 44 ml glass flask (Supelco Bellefonte USA) equipped with a mininert valve (Supelco Bellefonte USA) and were stocked at 25 1C for 45 min before injecting in the chromatograph. A 1 ml sample of the vapor phase was harvested and injected into the gas chromatograph at regular intervals. The vapor–liquid equilibrium was considered to be attained when the concentration of aroma compounds in the gas phase remained constant. The vapor–liquid partition coefficient K partition is the ratio of the mass fraction of the component i in the vapor and the liquid phases, respectively, at equilibrium. y K partition ¼ , x where x is the Mass fraction of the aroma compound in the solution at equilibrium, and y the Mass fraction of the aroma compound in the gas phase at equilibrium. Gas chromatographic conditions: A Chrompack CP 9000 with a flame ionization detector (FID) and a Hewlett Packard 3380A integrator were used. The stainless steel column (3 m 2:2 mm internal diameter) was packed with 100/120 mesh Carbowax 20M-10%. The injector and detector temperatures were maintained at 190 and 200 1C, respectively. The column isotherm was at 130 1C. The nitrogen flow rate was 34 ml/min, the hydrogen flow rate was 32 ml/min and the air flow rate was 263 ml/min. The results of headspace analysis were expressed by the ratio R, which is the ratio of the partition coefficient of volatile compounds in solution or emulsion with bacteria versus the partition coefficient of this compound in sample without bacteria. R¼1
K sample in presence of strain K sample without bacteria
X
100%
Ro0 means that the aroma compound is more released in presence of bacteria than in preparations without bacteria and inversely if R40, aroma compound is more retained with bacteria than without (Landy et al., 1995). All experiments were at least carried out 10 independent times and the results presented are mean values of all the results obtained for which the standard deviation was calculated and is shown in the figures.
2.2.3. Microscopic observation of emulsions and staining procedures The emulsions were observed in fluorescence microscopy (Zeiss Axioplan 2 imaging, Zeiss, Iena, FRG). The emulsions were stained with Nile Red (8 mg=ml, stock solution: 4 mg/ml in acetone) and the bacteria were stained with DAPI (10 mg=ml, stock solution: 5 mg/ml in distilled water) for 15 min at 27 1C under a 140 rpm agitation. The emulsion and the cells were stained separately and then cells were added to the emulsion under agitation at room temperature. Esters were added before or after cells as stated above. The images from the Axiocam MRm camera were treated with the software AxioVision 4 (Zeiss).
3. Results and discussion 3.1. Retention of aroma compounds by cells in solution In the presence of strain LLD18 in both 9 g/l NaCl and citrate buffer solutions, the ratios R were markedly positive
ethyl acetate 30
ethyl hexanoate
a
20
10
Ratio R (in %)
Two different orders of addition of bacteria and aroma compounds were used: bacteria LLD18 or LLD16 were first added to each emulsion at a final concentration of approximately 109 cells=ml by vortexing for 2 s and the aroma compounds were then added and dispersed by manual agitation. aroma compounds were first added to each emulsion and bacteria were added at a final concentration of approximately 109 cells=ml afterwards and vortexed for 2 s.
213
0 30
b
20
10
0
18
16 Bacterial strain
Fig. 1. Retention of ethyl acetate ð Þ and ethyl hexanoate ð Þ expressed by the percentage of retention R in 9 g/l NaCl (A) and citrate buffer 0.01 M, pH 4.5 (B) solutions.
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for ethyl hexanoate (12–15%), the most hydrophobic aroma compound tested, and only slightly positive for ethyl acetate (1–8%) (Fig. 1). This means that ethyl hexanoate was more retained in presence of LLD18 than in presence of LLD16 in aqueous solution. For ethyl acetate the retention by the two strains was similar at about 5%. The hydrophobic volatile compounds are little retained in solution except in the presence of compounds that can establish hydrophobic bonding such as proteins or lipids (Druaux and Voilley, 1997). In this study, the most hydrophobic compound (ethyl hexanoate) was more retained by the strain which was found to be the most hydrophobic (LLD18) suggesting the existence of hydrophobic interactions between them. The surface properties of microorganisms depend on the chemical composition of the cellular surface involving proteins, teichoic acid, polysaccharides (Boonaert and Rouxhet, 2000; van der Mei, Busscher et al., 2000; van der Mei, de Vries et al., 2000). According to Latrache et al. (2002), van der Mei, Busscher, et al. (2000) and van der Mei, de Vries et al. (2000), there is a good correlation between the hydrophobicity and isoelectric point of cells and the concentration of proteins and peptidoglycans as deduced from X-ray photoelectron spectroscopy. These observations suggest that the surface of LLD 18 (more hydrophobic and with a higher isoelectric point than LLD16) is richer in proteins and peptidoglycans than the one of LLD16. As peptidoglycans are rather polar compounds, the hydrophobic interactions between cell surfaces and aroma compounds could thus be mainly due to the presence of proteins on the cellular surface. Lubbers et al. (1994) and Chalier et al. (2007) have found that the binding of aroma compounds to mannoproteins extracted from the cell wall of Saccharomyces cerevisiae strains increases with the log P of the aroma compound. A similar correlation between the log P of esters and their binding to b-lactoglobulin was observed (Reiners et al., 2000). However, the interactions between proteins and aroma compounds depend on the nature of proteins and on the structure of aroma compounds (Reiners et al., 2000). Apolar carbon chain can interact with hydrophobic parts of proteins whereas alcohols as well as carboxylates with hydroxyl groups can develop hydrogen bonding with proteins. On the other hand, the presence of salts increases the volatility of aroma compounds (Pionnier et al., 2002; Voilley et al., 1977). In model media consisting of water containing amino acids, mineral salts and lactic acid, ethyl hexanoate is released by the effect of salts (Pionnier et al., 2002). According to our own results there is no significant difference of percentage of retention R between NaCl 9 g/l and citrate buffer 0.01 M pH 4.5 (Fig. 1). Moreover, the adhesion to alkanes of LLD18 and LLD16 was not different in NaCl 9 g/l and the citrate buffer at pH 4.5 (results not shown). According to previous results (Ly, Naı¨ tali et al., 2006), the ionic strength rather affects the
electrostatic interactions between Lactococci and other compounds. Our present results indicated that, in this range of pH and ionic strength, NaCl does not affect the interactions between the cell surface of the strains tested and ethyl esters. In solution, our results confirmed the adhesion of L. lactis strains to aroma compounds and lipids (Ly, Vo et al., 2006), the more hydrophobic ethyl hexanoate was retained by the bacteria with the most hydrophobic surface whereas the retention of ethyl acetate was low and was not affected by the differences in the surface properties of the two strains.
3.2. Retention of aroma compounds by cells in emulsion The interactions of aroma compounds with other components are usually studied in aqueous systems. However, food matrices are complex media composed of a lot of different compounds and generally consisting in heterogeneous phases. Most food products are emulsified or gelified and their structure can also affect the release and perception of flavor compounds (de Roos, 2003; Druaux and Voilley, 1997). In a recent study (Ly, Naı¨ tali et al., 2006), we have shown that the presence of bacteria in an emulsion can change its stability. The addition of negatively charged bacteria to the emulsion droplets stabilized by the cationic surfactant CTAB led to the adsorption of bacteria on the surface of droplets by electrostatic interactions. For this reason, we have investigated in the present study the effect of bacteria on the retention of aroma compounds in emulsions. As shown in Fig. 2, in emulsions ethyl acetate was little retained whereas ethyl hexanoate was markedly released particularly in the presence of LLD16. To better understand the reasons for behavior of esters in emulsions with bacteria, the retention of aroma compounds were evaluated with two different orders of addition of bacteria and esters. In the first one bacteria were added to the emulsion before the esters (Fig. 2A) and in the second one, bacteria were added after the esters (Fig. 2B). Interestingly, the ratios R of ethyl hexanoate were more negative when bacteria were added before the ester (52% for LLD16 and 23% for LLD18) compared to when bacteria were added after (19% for LLD16 and 8% for LLD18) (Fig. 2). This means that the most hydrophobic compound was less retained in the media if bacteria were added before. To explain these results, the structure of the emulsions was observed carefully (Figs. 3 and 4). In microscopy (Fig. 3), we observed that the addition of cells to lipids provoked the aggregation and coalescence of droplets. The size of the droplets was thus increased. Moreover, bacteria were localized at the surface of the droplets forming an interfacial layer particularly observable for LLD16 (Fig. 3). From experiments carried out with ethyl esters in emulsions in the presence of sodium caseinate, Charles
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ethyl acetate 30 20
215
ethyl hexanoate
a 16
10
18
0 -10 -20 -30 -40
Ratio R (in %)
-50 -60 -70 30 20
b 16
10 0 -10 -20
Fig. 3. Microscopic observations of the localization of bacteria on the alkane phase surface for emulsions made with CTAB in citrate buffer pH 4.5: strain LLD18 (up) and strain LLD16 (down). Hexadecane (L) stained with Nile Red and bacteria (B) with Dapi. bar: 20 mm.
18
-30 -40 -50 -60 -70 Bacterial strain Fig. 2. Percentage of retention of ethyl acetate ð Þ and ethyl hexanoate ð Þ in emulsions of hexadecane in water with CTAB in the presence of strain LLD18 or LLD16. Results are presented for two different modes of preparation: A-(ðemulsion þ bacteriaÞ þ aroma compounds) and B(ðemulsion + aroma compoundsÞ þ bacteria) (see the method part for details).
et al. (2000) and Druaux and Voilley (1997) observed that the size of the droplets had little effect on the retention of aroma compounds whereas sodium caseinate adsorbed at the oil–water interface could act as a barrier able to decrease the aroma transferred through the oil–water interface. In that case, aromas interact with the surfactant on the interface before going into the lipid phase. As for surfactants, bacteria located at the lipid interface could make a barrier which could prevent the contact between aroma compounds and droplets when the bacteria were added before the aroma compounds. On the contrary, when bacteria were added after the hydrophobic aroma compound, the aroma compound could get inside the droplets first, before the formation of the bacterial barrier which could prevent their release from lipids. In our study, when bacteria were added before aroma compounds, ethyl
hexanoate was more released in the presence of LLD16 than in the presence of LLD18. These results could be explained by the level of adsorption of the two strains which was different, due to the charge of their surface. As shown in Fig. 3, LLD16, with a more negative charge, adsorbed more strongly to droplets breaking the emulsions and resulting in a barrier surrounding the oil phase while LLD18 adhered to the oil phase without forming an integral barrier (Fig. 3). In emulsions containing bacteria, a creaming layer was formed at the top of the tube (Fig. 4). This layer could have an impact on the retention of aroma compounds. The creaming layer was above all observable for emulsions with LLD16. With this strain, ethyl acetate was more retained particularly when bacteria were added before the aroma compound (Fig. 2A). This can be explained by the fact that, when the cream layer was constituted before the addition of aroma compounds, ethyl acetate had to cross this layer to reach the gas phase. However, bacteria were very concentrated in this layer due to their adsorption to lipids (Fig. 3). Ethyl acetate molecules were likely to encounter cells before being released in the gas phase. As shown with solutions, ethyl acetate was slightly retained in the presence of 109 bacteria=ml. In the cream layer, the concentration of bacteria is likely to be significantly higher favoring thus the interactions between bacteria and ethyl acetate. Our results suggest thus that the binding of ethyl acetate to bacteria is promoted by the presence of a top layer rich in bacteria.
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Acknowledgements This work was partly financed by Re´gion Bourgogne, Ly´ Mai Huong possesses a grant from the Agence Universitaire de la Francophonie in the frame of a collaboration between Hanoi University of Technology (Viet Nam) and the Ensbana/University of Burgundy. Fig. 4. Creaming of emulsions made with CTAB in citrate buffer pH 4.5 15 min after the addition of bacteria: control without bacteria (left), with strain LLD18 (center) and with strain LLD16 (right).
For ethyl hexanoate, the release with LLD16 (with the more separated cream layer) was always higher than for LLD18. This hydrophobic aroma compound had an affinity for the alkane phase. If the alkane phase formed a layer at the top of the sample, ethyl hexanoate could go directly from the alkane to the gas phase without crossing any aqueous phase. Several studies have discussed the release of flavor in biphasic systems (Charles et al., 2000; Druaux and Voilley, 1997; Everett and Olson, 2003; Meynier et al., 2005). In oilin-water emulsions, the volatile compounds distribute in three phases: aqueous, lipid and interface (Charles et al., 2000). The affinity of volatile compounds for the lipid phase depends on their hydrophobicity but also on the structure of emulsions which is dependent on the nature and amount of the surfactant adsorbed at the interface. Our results show that bacteria can play a role in the distribution of volatile compounds in emulsions. Particularly the adsorption of cells to interfaces seem to have an impact, promoting interactions between bacteria and aroma compounds during the transfer of aroma compounds from one phase to the other. In conclusion, bacteria, through their surface physicochemical properties, can interact directly with aroma compounds or in an indirect way, by modifying the emulsions characteristics leading to changes in the transfers of aroma. With the great diversity of bacterial surface properties (Ly, Vo et al., 2006), it is possible to select strains possessing adequate surface properties in addition to the right metabolic characteristics to reach the optimal behavior in food matrix. This study constitutes a first step in the field of interactions between cells and aroma compounds. After this demonstration of the impact of bacteria on flavor retention, it will be interesting to characterize the nature of the interactions particularly by working with different bacterial strains and aroma compounds from different chemical classes and different physicochemical properties. The evaluation of the physiological consequences of interactions between living bacteria and aroma compounds, i.e. how bacteria modulate their surface properties depending on the presence of aroma compounds, would also be of interest to enable to predict the retention of volatile compounds by microorganisms.
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FOOD
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Retention of aroma compounds by lactic acid bacteria in model food media M.H. Lya,c,, M. Covarrubias-Cervantesb, C. Dury-Brunb, S. Bordeta, A. Voilleyb, T.M. Lec, J.-M. Belina, Y Wache´a, a Laboratoire de Microbiologie UMR UB/INRA 1232, ENSBANA, 1, Esplanade Erasme 21000 Dijon, France Laboratoire IMSAPS, Universite´ de Bourgogne, IFR 92, ENSBANA, 1, Esplanade Erasme 21000 Dijon, France c Laboratoire de Technologie et Biologie Alimentaire de l’Institut Polytechnique de Hanoı¨, 1, Rue Dai Co Viet, Hanoı¨, Vietnam b
Received 1 June 2006; accepted 6 November 2006
Abstract The interactions between aroma compounds and other particles in foods, particularly with macromolecules, have been greatly studied in order to better understand the binding of flavors in food matrices. Bacteria possess many macromolecules on their cellular surface that provide them surface properties which are involved in the physicochemical interactions between cells and interfaces. However, the interactions between bacteria and aroma compounds have not received so much attention despite the presence of bacteria in many fermented products. In order to study the retention of aroma compounds by bacteria, we have investigated the retention of esters by lactic acid bacteria with static headspace techniques. Two strains of Lactococcus lactis subsp. lactis biov. diacetylactis reflecting the natural diversity of the bacterial surface properties and two ethyl esters generally involved in the cheese flavor (ethyl acetate and ethyl hexanoate) were chosen for the experiments. The results have shown that bacteria, through their surface physicochemical properties, can interact directly with aroma compounds or in an indirect way, by changing the emulsion characteristics. However, these effects depend on the physicochemical properties of both aroma compounds and bacterial surfaces. r 2006 Elsevier Ltd. All rights reserved. Keywords: Aroma compound; Retention; Surface properties; Lactic acid bacteria; Interactions
1. Introduction The retention of aroma compounds in food matrices is important for the perception of food products (de Roos, 2003; Druaux and Voilley, 1997). In aqueous systems, the retention of aroma compounds can be modified by physicochemical interactions between macromolecules such as proteins and polysaccharides, lipids and aroma compounds (Guichard, 2006; Seuvre et al., 2000; Seuvre et al., 2006; Terta et al., 2006). Specific interactions resulting in the binding of aroma compounds to food components have been demonstrated such as covalent and Corresponding authors. Laboratoire de Microbiologie UMR UB/ INRA 1232, ENSBANA, 1, Esplanade Erasme 21000 Dijon, France. E-mail addresses: [email protected] (M.H. Ly), [email protected] (Y. Wache´).
0268-005X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2006.11.001
hydrogen bonding and hydrophobic interactions (Le Thanh et al., 1992; Meynier et al., 2004). However these interactions depend on the physicochemical properties and formulation of both aromas and macromolecules (Reiners et al., 2000). Food products have generally heterogeneous structures with water-insoluble compounds dispersed in the matrix which is often emulsified or gelified. The retention of aroma compounds can also be affected by the food microstructure and rheology (Terta et al., 2006). For instance, the size of the droplets of emulsions can have an impact on the release of aromas, particularly on hydrophobic compounds (Meynier et al., 2005). Moreover, food matrices such as fermented products can also contain an important number of microbial cells. Bacterial concentrations can exceed 109 cultivable cells/ml at the end of fermentations, amount that is usually
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maintained over 108 cells=ml for probiotic products (Yoon et al., 2006). Bacterial cells are micrometer-scale organisms bearing at their surface many macromolecules, such as proteins, mannoproteins, peptidoglycan, teichoic acid and polysaccharides (Boonaert and Rouxhet, 2000; van der Mei et al., 2000). These compounds confer to cells physicochemical properties that enable bacteria to interact with other compounds through Lewis acid/base, electrostatic and hydrophobic interactions (Hermansson, 1999; Scha¨r-Zammaretti and Ubbink, 2003). The microbial surface properties have been widely studied in order to understand the interactions between bacteria and interfaces and, more particularly, their implication in the formation of biofilms, a phenomenon important in many fields such as biomedical and food safety, corrosion and environment (Bellon-Fontaine et al., 1996; Briandet et al., 2001; Strevett and Chen, 2003; van der Mei et al., 2000). Hydrophobic interactions are important to explain the affinity of bacteria for apolar compounds (Bouchez-Naı¨ tali et al., 2001; Bruinsma et al., 2001; Ly et al., 2006; Pascual et al., 2000). In a recent study (Ly, Vo et al., 2006), we have studied the interactions between bacteria and food components observing that cells with more hydrophobic surfaces had a higher affinity for milk fat. On the other hand, electrostatic interactions between bacteria and droplets of emulsions can modify the emulsion stability (Li et al., 2001; Ly, Naı¨ tali et al., 2006). Up to now, few studies investigated the retention of aroma compounds by microorganism. Some authors (Chalier et al., 2007; Lubbers et al., 1994) have shown that the retention of aroma compounds by mannoproteins of yeast extracts depends on the hydrophobicity of aromas and on mannoprotein fractions. The present study aims at investigating the impact of bacteria on the retention of aroma compounds. In the first part, the study deals with the direct effect of bacteria on the retention of esters with different hydrophobicity in aqueous solutions. In the second part, the effect of bacteria on the retention of esters is investigated in a model emulsion that can be destabilized by bacteria. A species of lactic acid bacteria largely employed in fermentation processes, Lactococcus lactis subsp. lactis biov. diacetylactis, was used (McKay and Baldwin, 1990; Ross et al., 2002) with two strains exhibiting different surface properties selected from preceding studies (Ly, Vo et al., 2006). The retention of aroma compounds was evaluated by a static headspace technique. 2. Material and methods 2.1. Material 2.1.1. Aroma compounds The aroma compounds used in this study are esters, ethyl acetate and ethyl hexanoate (both purchased from Sigma with purities 498%), which are present in typical flavors of cheese, (Liu et al., 2004) and have already been character-
ized in several studies concerning retention (Landy et al., 1995; Seuvre et al., 2006). The hydrophobic constants of volatile compounds are expressed by the logarithm of the partition coefficient between water and n-octanol: log P of ethyl acetate is 0.7 and log P of ethyl hexanoate is 2.8 (Seuvre et al., 2006). 2.1.2. Bacterial strains Two strains of L. lactis ssp. lactis subv. diacetylactis (noted LLD16 and LLD18 and previously SD (S. diacetylactis)) from the CNRZ/INRA (Jouy-en-Josas) collection were used. Their surface properties had been previously characterized, hydrophobicity by the microbial adhesion to hydrocarbon (MATH) test which consists in evaluating the percentage of cells extracted by a solvent phase after vortex, and the z potential and isoelectric point, after the electrophoretic mobility of the cells (Ly, Naı¨ tali et al., 2006). The surface of the strain LLD18 was hydrophobic with 50% of the cells adhering to the apolar solvent hexadecane and the isoelectric point was about 4.5. The strain LLD16 adhered weakly to alkanes and its isoelectric point was inferior to 2. 2.2. Methods 2.2.1. Cell culture conditions Bacteria were cultured in MRS media (De Man et al., 1960) (In g/l: proteose peptone, 10, yeast extract, 5, meat extract, 10, trisodium citrate, 3.1, magnesium sulfate, 0.1, manganese sulfate, 0.05, dipotassium phosphate, 2 without Tween 80 and for which glucose was replaced with lactose, 15). Bacteria conserved at 70 C in MRS media containing 25% (vol/vol) glycerol were thawed, subcultured overnight, and grown in liquid MRS media. Cultures were performed at 27 C in static conditions until the early stationary phase. 2.2.2. Static headspace analysis Quantification of esters in the gas phase was carried out for aqueous solutions and emulsions containing one or the other strain. 2.2.2.1. Preparation. 50 ppm (v/v) of ethyl acetate and ethyl hexanoate were added in solutions and emulsions. Solutions consisted in citrate buffer (0.01 M, pH 4.5) or 9 g/l NaCl containing 109 cells=ml of bacteria LLD18 or LLD16, corresponding to an optical density at 600 nm of OD600 nm ¼ 1. The emulsion was prepared by mixing three times for 3 min, 80% distilled water, 20% hexadecane and 0.5 % w/v of hexadecyltrimethylammonium bromide (CTAB) by an ultrasonic generator at a power level of þ200 V (Fisher scientific Transsonic 460H, Illkirch, France). This emulsion was stocked at 4 1C and diluted ten folds in citrate buffer (0.01 M, pH 4.5) before use.
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2.2.2.2. Headspace analysis. 35 ml of the medium to analyze were put into a 44 ml glass flask (Supelco Bellefonte USA) equipped with a mininert valve (Supelco Bellefonte USA) and were stocked at 25 1C for 45 min before injecting in the chromatograph. A 1 ml sample of the vapor phase was harvested and injected into the gas chromatograph at regular intervals. The vapor–liquid equilibrium was considered to be attained when the concentration of aroma compounds in the gas phase remained constant. The vapor–liquid partition coefficient K partition is the ratio of the mass fraction of the component i in the vapor and the liquid phases, respectively, at equilibrium. y K partition ¼ , x where x is the Mass fraction of the aroma compound in the solution at equilibrium, and y the Mass fraction of the aroma compound in the gas phase at equilibrium. Gas chromatographic conditions: A Chrompack CP 9000 with a flame ionization detector (FID) and a Hewlett Packard 3380A integrator were used. The stainless steel column (3 m 2:2 mm internal diameter) was packed with 100/120 mesh Carbowax 20M-10%. The injector and detector temperatures were maintained at 190 and 200 1C, respectively. The column isotherm was at 130 1C. The nitrogen flow rate was 34 ml/min, the hydrogen flow rate was 32 ml/min and the air flow rate was 263 ml/min. The results of headspace analysis were expressed by the ratio R, which is the ratio of the partition coefficient of volatile compounds in solution or emulsion with bacteria versus the partition coefficient of this compound in sample without bacteria. R¼1
K sample in presence of strain K sample without bacteria
X
100%
Ro0 means that the aroma compound is more released in presence of bacteria than in preparations without bacteria and inversely if R40, aroma compound is more retained with bacteria than without (Landy et al., 1995). All experiments were at least carried out 10 independent times and the results presented are mean values of all the results obtained for which the standard deviation was calculated and is shown in the figures.
2.2.3. Microscopic observation of emulsions and staining procedures The emulsions were observed in fluorescence microscopy (Zeiss Axioplan 2 imaging, Zeiss, Iena, FRG). The emulsions were stained with Nile Red (8 mg=ml, stock solution: 4 mg/ml in acetone) and the bacteria were stained with DAPI (10 mg=ml, stock solution: 5 mg/ml in distilled water) for 15 min at 27 1C under a 140 rpm agitation. The emulsion and the cells were stained separately and then cells were added to the emulsion under agitation at room temperature. Esters were added before or after cells as stated above. The images from the Axiocam MRm camera were treated with the software AxioVision 4 (Zeiss).
3. Results and discussion 3.1. Retention of aroma compounds by cells in solution In the presence of strain LLD18 in both 9 g/l NaCl and citrate buffer solutions, the ratios R were markedly positive
ethyl acetate 30
ethyl hexanoate
a
20
10
Ratio R (in %)
Two different orders of addition of bacteria and aroma compounds were used: bacteria LLD18 or LLD16 were first added to each emulsion at a final concentration of approximately 109 cells=ml by vortexing for 2 s and the aroma compounds were then added and dispersed by manual agitation. aroma compounds were first added to each emulsion and bacteria were added at a final concentration of approximately 109 cells=ml afterwards and vortexed for 2 s.
213
0 30
b
20
10
0
18
16 Bacterial strain
Fig. 1. Retention of ethyl acetate ð Þ and ethyl hexanoate ð Þ expressed by the percentage of retention R in 9 g/l NaCl (A) and citrate buffer 0.01 M, pH 4.5 (B) solutions.
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for ethyl hexanoate (12–15%), the most hydrophobic aroma compound tested, and only slightly positive for ethyl acetate (1–8%) (Fig. 1). This means that ethyl hexanoate was more retained in presence of LLD18 than in presence of LLD16 in aqueous solution. For ethyl acetate the retention by the two strains was similar at about 5%. The hydrophobic volatile compounds are little retained in solution except in the presence of compounds that can establish hydrophobic bonding such as proteins or lipids (Druaux and Voilley, 1997). In this study, the most hydrophobic compound (ethyl hexanoate) was more retained by the strain which was found to be the most hydrophobic (LLD18) suggesting the existence of hydrophobic interactions between them. The surface properties of microorganisms depend on the chemical composition of the cellular surface involving proteins, teichoic acid, polysaccharides (Boonaert and Rouxhet, 2000; van der Mei, Busscher et al., 2000; van der Mei, de Vries et al., 2000). According to Latrache et al. (2002), van der Mei, Busscher, et al. (2000) and van der Mei, de Vries et al. (2000), there is a good correlation between the hydrophobicity and isoelectric point of cells and the concentration of proteins and peptidoglycans as deduced from X-ray photoelectron spectroscopy. These observations suggest that the surface of LLD 18 (more hydrophobic and with a higher isoelectric point than LLD16) is richer in proteins and peptidoglycans than the one of LLD16. As peptidoglycans are rather polar compounds, the hydrophobic interactions between cell surfaces and aroma compounds could thus be mainly due to the presence of proteins on the cellular surface. Lubbers et al. (1994) and Chalier et al. (2007) have found that the binding of aroma compounds to mannoproteins extracted from the cell wall of Saccharomyces cerevisiae strains increases with the log P of the aroma compound. A similar correlation between the log P of esters and their binding to b-lactoglobulin was observed (Reiners et al., 2000). However, the interactions between proteins and aroma compounds depend on the nature of proteins and on the structure of aroma compounds (Reiners et al., 2000). Apolar carbon chain can interact with hydrophobic parts of proteins whereas alcohols as well as carboxylates with hydroxyl groups can develop hydrogen bonding with proteins. On the other hand, the presence of salts increases the volatility of aroma compounds (Pionnier et al., 2002; Voilley et al., 1977). In model media consisting of water containing amino acids, mineral salts and lactic acid, ethyl hexanoate is released by the effect of salts (Pionnier et al., 2002). According to our own results there is no significant difference of percentage of retention R between NaCl 9 g/l and citrate buffer 0.01 M pH 4.5 (Fig. 1). Moreover, the adhesion to alkanes of LLD18 and LLD16 was not different in NaCl 9 g/l and the citrate buffer at pH 4.5 (results not shown). According to previous results (Ly, Naı¨ tali et al., 2006), the ionic strength rather affects the
electrostatic interactions between Lactococci and other compounds. Our present results indicated that, in this range of pH and ionic strength, NaCl does not affect the interactions between the cell surface of the strains tested and ethyl esters. In solution, our results confirmed the adhesion of L. lactis strains to aroma compounds and lipids (Ly, Vo et al., 2006), the more hydrophobic ethyl hexanoate was retained by the bacteria with the most hydrophobic surface whereas the retention of ethyl acetate was low and was not affected by the differences in the surface properties of the two strains.
3.2. Retention of aroma compounds by cells in emulsion The interactions of aroma compounds with other components are usually studied in aqueous systems. However, food matrices are complex media composed of a lot of different compounds and generally consisting in heterogeneous phases. Most food products are emulsified or gelified and their structure can also affect the release and perception of flavor compounds (de Roos, 2003; Druaux and Voilley, 1997). In a recent study (Ly, Naı¨ tali et al., 2006), we have shown that the presence of bacteria in an emulsion can change its stability. The addition of negatively charged bacteria to the emulsion droplets stabilized by the cationic surfactant CTAB led to the adsorption of bacteria on the surface of droplets by electrostatic interactions. For this reason, we have investigated in the present study the effect of bacteria on the retention of aroma compounds in emulsions. As shown in Fig. 2, in emulsions ethyl acetate was little retained whereas ethyl hexanoate was markedly released particularly in the presence of LLD16. To better understand the reasons for behavior of esters in emulsions with bacteria, the retention of aroma compounds were evaluated with two different orders of addition of bacteria and esters. In the first one bacteria were added to the emulsion before the esters (Fig. 2A) and in the second one, bacteria were added after the esters (Fig. 2B). Interestingly, the ratios R of ethyl hexanoate were more negative when bacteria were added before the ester (52% for LLD16 and 23% for LLD18) compared to when bacteria were added after (19% for LLD16 and 8% for LLD18) (Fig. 2). This means that the most hydrophobic compound was less retained in the media if bacteria were added before. To explain these results, the structure of the emulsions was observed carefully (Figs. 3 and 4). In microscopy (Fig. 3), we observed that the addition of cells to lipids provoked the aggregation and coalescence of droplets. The size of the droplets was thus increased. Moreover, bacteria were localized at the surface of the droplets forming an interfacial layer particularly observable for LLD16 (Fig. 3). From experiments carried out with ethyl esters in emulsions in the presence of sodium caseinate, Charles
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ethyl acetate 30 20
215
ethyl hexanoate
a 16
10
18
0 -10 -20 -30 -40
Ratio R (in %)
-50 -60 -70 30 20
b 16
10 0 -10 -20
Fig. 3. Microscopic observations of the localization of bacteria on the alkane phase surface for emulsions made with CTAB in citrate buffer pH 4.5: strain LLD18 (up) and strain LLD16 (down). Hexadecane (L) stained with Nile Red and bacteria (B) with Dapi. bar: 20 mm.
18
-30 -40 -50 -60 -70 Bacterial strain Fig. 2. Percentage of retention of ethyl acetate ð Þ and ethyl hexanoate ð Þ in emulsions of hexadecane in water with CTAB in the presence of strain LLD18 or LLD16. Results are presented for two different modes of preparation: A-(ðemulsion þ bacteriaÞ þ aroma compounds) and B(ðemulsion + aroma compoundsÞ þ bacteria) (see the method part for details).
et al. (2000) and Druaux and Voilley (1997) observed that the size of the droplets had little effect on the retention of aroma compounds whereas sodium caseinate adsorbed at the oil–water interface could act as a barrier able to decrease the aroma transferred through the oil–water interface. In that case, aromas interact with the surfactant on the interface before going into the lipid phase. As for surfactants, bacteria located at the lipid interface could make a barrier which could prevent the contact between aroma compounds and droplets when the bacteria were added before the aroma compounds. On the contrary, when bacteria were added after the hydrophobic aroma compound, the aroma compound could get inside the droplets first, before the formation of the bacterial barrier which could prevent their release from lipids. In our study, when bacteria were added before aroma compounds, ethyl
hexanoate was more released in the presence of LLD16 than in the presence of LLD18. These results could be explained by the level of adsorption of the two strains which was different, due to the charge of their surface. As shown in Fig. 3, LLD16, with a more negative charge, adsorbed more strongly to droplets breaking the emulsions and resulting in a barrier surrounding the oil phase while LLD18 adhered to the oil phase without forming an integral barrier (Fig. 3). In emulsions containing bacteria, a creaming layer was formed at the top of the tube (Fig. 4). This layer could have an impact on the retention of aroma compounds. The creaming layer was above all observable for emulsions with LLD16. With this strain, ethyl acetate was more retained particularly when bacteria were added before the aroma compound (Fig. 2A). This can be explained by the fact that, when the cream layer was constituted before the addition of aroma compounds, ethyl acetate had to cross this layer to reach the gas phase. However, bacteria were very concentrated in this layer due to their adsorption to lipids (Fig. 3). Ethyl acetate molecules were likely to encounter cells before being released in the gas phase. As shown with solutions, ethyl acetate was slightly retained in the presence of 109 bacteria=ml. In the cream layer, the concentration of bacteria is likely to be significantly higher favoring thus the interactions between bacteria and ethyl acetate. Our results suggest thus that the binding of ethyl acetate to bacteria is promoted by the presence of a top layer rich in bacteria.
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Acknowledgements This work was partly financed by Re´gion Bourgogne, Ly´ Mai Huong possesses a grant from the Agence Universitaire de la Francophonie in the frame of a collaboration between Hanoi University of Technology (Viet Nam) and the Ensbana/University of Burgundy. Fig. 4. Creaming of emulsions made with CTAB in citrate buffer pH 4.5 15 min after the addition of bacteria: control without bacteria (left), with strain LLD18 (center) and with strain LLD16 (right).
For ethyl hexanoate, the release with LLD16 (with the more separated cream layer) was always higher than for LLD18. This hydrophobic aroma compound had an affinity for the alkane phase. If the alkane phase formed a layer at the top of the sample, ethyl hexanoate could go directly from the alkane to the gas phase without crossing any aqueous phase. Several studies have discussed the release of flavor in biphasic systems (Charles et al., 2000; Druaux and Voilley, 1997; Everett and Olson, 2003; Meynier et al., 2005). In oilin-water emulsions, the volatile compounds distribute in three phases: aqueous, lipid and interface (Charles et al., 2000). The affinity of volatile compounds for the lipid phase depends on their hydrophobicity but also on the structure of emulsions which is dependent on the nature and amount of the surfactant adsorbed at the interface. Our results show that bacteria can play a role in the distribution of volatile compounds in emulsions. Particularly the adsorption of cells to interfaces seem to have an impact, promoting interactions between bacteria and aroma compounds during the transfer of aroma compounds from one phase to the other. In conclusion, bacteria, through their surface physicochemical properties, can interact directly with aroma compounds or in an indirect way, by modifying the emulsions characteristics leading to changes in the transfers of aroma. With the great diversity of bacterial surface properties (Ly, Vo et al., 2006), it is possible to select strains possessing adequate surface properties in addition to the right metabolic characteristics to reach the optimal behavior in food matrix. This study constitutes a first step in the field of interactions between cells and aroma compounds. After this demonstration of the impact of bacteria on flavor retention, it will be interesting to characterize the nature of the interactions particularly by working with different bacterial strains and aroma compounds from different chemical classes and different physicochemical properties. The evaluation of the physiological consequences of interactions between living bacteria and aroma compounds, i.e. how bacteria modulate their surface properties depending on the presence of aroma compounds, would also be of interest to enable to predict the retention of volatile compounds by microorganisms.
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