International Journal of Food Microbiology 112 (2006) 26 34 www.elsevier.

com/locate/ijfoodmicro

Importance of bacterial surface properties to control the stability of emulsions

Mai Huong Ly a,d , Murielle Natali-Bouchez b , Thierry Meylheuc c , Marie-Nolle Bellon-Fontaine b , Thanh Mai Le d , Jean-Marc Belin a , Yves Wach a,
c a Laboratoire de Microbiologie UMR UB/INRA 1232, ENSBANA, 1, Esplanade Erasme 21000 Dijon, France Unit d'Enseignement et de Recherche en Qualit et Scurit Microbiologiques des Aliments et Procds, ENSIA, France Unit de Recherche en Bioadhsion et Hygine des Matriaux, INRA, 25, Avenue de la Rpublique, 91744 Massy Cedex, France d Laboratoire de Technologie et Biologie Alimentaire de l'Institut Polytechnique de Hano, 1, Rue Dai Co Viet, Hano, Vietnam b

Received 24 October 2005; received in revised form 24 February 2006; accepted 24 May 2006

Abstract In colloidal media such as emulsions or food matrixes, the stability results from physicochemical interactions. The same type of interaction is involved in the attachment processes of microorganisms, through their surface properties, to interfaces. When bacteria are present in a food matrix, it is probable that their surface interacts with the other constituents. In this paper, the involvement of bacterial surface properties of Lactococcus lactis subsp lactis biovar diacetylactis (LLD) on the stability of model emulsions has been studied. The hydrophobic and electrostatic cell-surface properties were characterized by the MATH method and by microelectrophoresis, respectively. The oil-in-water emulsions were stabilized by various surface-active compounds, CTAB, SDS or Tween 20, giving differently charged droplets. Two strains with different surface characteristics were added to the emulsion. Contrasting with emulsions made with the non-ionic surfactant, for which the stability was not modified by the addition of bacteria, the emulsions made with ionic surface-active compounds were unstable in the presence of bacteria when the bacterial surface charge was opposite to the one of the emulsion droplets. Moreover, aggregation and flocculation phenomena were observed for emulsions stabilized with the cationic surfactant, particularly for more negatively charged bacteria. The effect of bacteria on the emulsion stability depended on the strain which shows the importance of the choice of the microorganism according to of the characteristics of the colloidal media to obtain a stable system. In addition, these results suggest that the interactions between bacteria and other food components can influence the position of bacteria in food matrixes. 2006 Elsevier B.V. All rights reserved.
Keywords: Lactic acid bacteria; Surface charge (zeta potential); Surface properties; Emulsion stability

1. Introduction Food matrixes, such as creams, sauces, mayonnaises, cheeses, and yogurts, are usually complex heterogeneous colloidal systems (Gupta and Muralidhara, 2001). The structure and stability of these emulsions determine the physicochemical and organoleptic characteristics of food products, such as texture, viscosity, and aroma perception. The dispersion of the components in food matrixes results from intermolecular interactions (e.g. van der Waals, electrostatic, structural) (Chenevier et al., 2000; Huang et al., 2001). Colloidal systems, such as oil-in-water emulsions, can be stabilized by surfactants adsorbed at the surface of the droplets. However, the association
Corresponding author. Tel.: +33 380396680; fax: +33 380396641. E-mail address: ywache@u-bourgogne.fr (Y. Wach). 0168-1605/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.05.022

of surfactants with other compounds present in the medium may change emulsion stability (Dickinson, 2003). In the case of electrostatic interactions, emulsion stability depends on the nature of the surfactant and on the conditions of the medium (pH and ionic strength) (Dickinson, 2003). For instance, emulsions stabilized by lecithin have negatively charged droplets. The addition of a cationic polymer (chitosan) to these emulsions results, under certain conditions, in a more stable structure by the formation of a lecithin-chitosan membrane (Ogawa et al., 2003). Conversely, the addition of Ca2+ in an emulsion stabilized by whey proteins, that also confer a negative charge to the droplets at pH 7, triggers the aggregation and coalescence of the globules due to the bridging of calcium between the proteins adsorbed at the surface of the droplets (Ye and Singh, 2000; van Aken, 2003). The flocculation of an emulsion may also be promoted by non-ionic interactions (Dickinson et al., 1999).

M.H. Ly et al. / International Journal of Food Microbiology 112 (2006) 2634

27

Many food products result from fermentation. Microbial processes have been widely studied particularly with respect to acidification and flavor production. The investigations have dealt mainly with the enzymatic potential of strains. However, bacteria possess physicochemical surface properties, such as hydrophobicity or charge (Boonaert and Rouxhet, 2000; van der Mei et al., 2001). These properties have been studied for their role in interactions between bacteria and interfaces in the formation of biofilms (Bellon-Fontaine et al., 1996) or in some steps in bioremediation (Briandet et al., 2001). It is also likely that the surfaces of bacteria interact physicochemically with other components of food matrixes such as fatty globules, proteins or emulsifiers. Up to now, a few investigations have been carried out in this field and these have concerned mainly on the hydrophobic characteristics. They reported for instance that the interactions between yeast wall extracts and aroma compounds modify the volatility of hydrophobic aroma due to the presence of lipids or mannoproteins (Lubbers et al., 1994). Another work dealt with the relationship between Lactobacillus casei surface properties and adhesion to lipids to enhance lipid metabolism and facilitate the production and retention of flavor compounds in fat (Kiely and Olson, 2000). The impact of electrostatic interactions is usually considered as dominant in the phenomena of microbial attachment to interfaces (BellonFontaine et al., 1996). Poortinga et al. (2002) showed that the bacterial cell surface is highly dynamic, responding strongly to environmental changes (pH, ionic strength or hydrophobicity) through proton dissociation or association. Bacteria may thus be regarded as charged colloidal particles. However, electrostatic interactions have scarcely been studied in food matrixes. With the objective of studying the direct physical impact of technological bacteria on food matrixes, we have investigated the physicochemical effect of bacteria on the stability of model emulsions stabilized by different surfactants. Lactococcus lactis subsp lactis biovar diacetylactis (LLD), which is found in the dairy industry for the production of cheese and other fermented milk products was chosen for this study. Three strains were selected after the preliminary evaluation of the properties of several strains from the bacterial collection of the laboratory. We observed a significant effect of bacteria on emulsion stability, an effect that was based on the opposition of charge between cells and emulsion droplets and which thus depended on the diversity of bacterial surface properties. These results made an interesting contribution to the understanding of bacterial adhesion to food components and thus food quality and maturation. 2. Materials and methods All chemicals were of the highest purity and were purchased from Sigma Aldrich (France). 2.1. Characterization of cell surfaces 2.1.1. Bacterial strains and growth conditions Three L. lactis subsp lactis biovar diacetylactis strains were studied: LLD16, LLD18 (formerly SD16 and SD18 from

Arilait, Paris, France) and LLD125 (CNRZ/INRA, Jouy-enJosas, France). The bacteria conserved at 70 C in MRS media (De Man et al., 1960) (without Tween 80 and for which glucose was replaced by lactose) containing 25% (vol/vol) glycerol were thawed, subcultured overnight, and grown in liquid MRS media. Cultures were grown at 27 C in static conditions until the early stationary phase. 2.1.2. Hydrophobicity of cell surfaces The hydrophobicity of the cell surface was evaluated by Microbial Adhesion To Hydrocarbons (MATH) according to the method proposed by Rosenberg (1991). Bacterial cells were harvested by centrifugation at 7000 g for 5 min and resuspended to Abs600 nm = 0.4 (A0) in 0.01 M potassium phosphate buffer (pH 6.5). 0.4 ml of hexadecane was added to 2.4 ml of cell suspension. The two-phase system was mixed by vortexing for 30 s and allowed to separate for 20 min. The aqueous phase was removed with a Pasteur pipette and its absorbance at 600 nm (A1) was measured. The percentage of microbial adhesion to solvent was calculated as follows: (1 A1 / A0) 100. 2.1.3. Zeta potential () of cell surfaces The electrical properties of the cell surfaces were assessed by microelectrophoresis. The electrophoretic mobility (EM) was determined at pH from 2 to 8. Cells in the early stationary phase were harvested twice by centrifugation at 7000 g for 5 min, and resuspended in citrate buffer, phosphate buffer or in physiological water (NaCl 9 g/l) at a concentration of about 107 cells/ml. In this last case, pHs were adjusted by the addition of KOH and HNO3 (100 mM). EMs were evaluated at room temperature on a laser zetaphoremeter II (CAD Instrumentation, France). The EM, expressed in 10 8 m2 V 1 s 1, was derived from the velocity of the bacteria in suspension under an applied electric field of 100 mV. 2.2. Characterization of emulsion stability 2.2.1. Preparation of the model emulsions Stock emulsions were prepared by mixing three times for 3 min 80% distilled water and 0.5% w/v surfactant (hexadecyltrimethylammonium bromide (CTAB), sodium dodecyl sulphate (SDS) or polyoxyethylensorbitan monolaurate 20 (Tween 20) and 20% hexadecane at a power level of + 200 V by an ultrasonic generator (Fisher scientific Transsonic 460H, Illkirch, France). The stock emulsions were diluted 10 fold in citrate (0.01 M, pH 3, 3.5 and 4.5) or phosphate (0.01 M, pH 7) buffer before use. Bacteria were added to the emulsions at a final concentration of approximately 109 cells/ml by vortexing for 5 s. 2.2.2. Preparation of the food model emulsion Stock emulsions were prepared by mixing 80% distilled water containing 6% w/v whey protein and 20% of sunflower oil with an ultra turrax (Kika T25 basic, Germany) at 16,000 rpm. The stock emulsions were diluted 10 fold in citrate buffer (0.01 M, pH 3, 4.5, 6) before caring. A bacterial concentration of approximately 109 cells/ml was added to the emulsions. In all cases, the pH was verified after the addition of bacteria.

28

M.H. Ly et al. / International Journal of Food Microbiology 112 (2006) 2634 Table 1 Stability of emulsions prepared with various surfactants 24 h after the addition of bacteria as evaluated by observation of glass tubes Surfactant pH LLD18 LLD16 Tween 20 3 O O 3.5 O O 4.5 O O 7 O O SDS 3 X O 3.5 O O 4.5 O O 7 O O CTAB 3 O X 3.5 O X 4.5 X X 7 X X

2.2.3. Determination of droplet size distribution A Malvern Mastersizer, model S2-01, laser diffractometer (Malvern instruments, Worcs, England) was used to evaluate droplet size distributions using 1.4345 as the refractive index of hexadecane. 2.2.4. Determination of droplet zeta potential () Previously described emulsions at various pH were diluted 20 times prior to analysis. 1 ml was injected into the cell of a Zetasizer Nano ZS (Malvern, Worcs) in which the droplets moved in the measurement chamber according to the electric field. was determined by measuring the direction and velocity of the droplet in the applied electric field. 2.2.5. Determination of the creaming height 4 ml of emulsion were put into a tube of 10 cm height and of 0.8 cm radius. The creaming stability in tubes was determined by the measurement of the cream layer height percentage which was calculated as follows: (total cream layer height / total emulsion height in the tube) 100. 2.2.6. Determination of emulsion turbidity 1 ml of the emulsion was placed into a 1-cm path length plastic spectrophotometer cuvette. The change in the emulsion turbidity at 600 nm was measured over 24 h of storage at 25 C. The oil-droplets of the emulsion moved upwards due to gravity. This movement led to the formation of a relatively clear serum layer at the bottom of the cuvette. The light beam passed through the emulsions at a height of about 10 mm from the cuvette bottom and always 30% lower than the height of the emulsion layer. The turbidity of the emulsion in the bottom part of the cuvette indicates the stability of the emulsion: reduced turbidities account for unstable emulsions. The diminution of absorbance was calculated as follows: (At / A0) 100. A0 and At are absorbance of the emulsion before and at time = t after the addition of bacteria, respectively.

X: creaming. O: no creaming.

2.2.7. Staining and microscopic observation The emulsions with or without bacteria were observed in fluorescence microscopy (Zeiss Axioplan 2 imaging, Zeiss, Iena, FRG). The emulsions were stained with Nile Red (8 g/ml, stock solution: 4 mg/ml in acetone) and the bacteria were stained with DAPI (10 g/ml, stock solution: 5 mg/ml in distilled water) for 15 min at 27 C under agitation at 140 rpm. The emulsion and the cells were stained separately and then cells were added to the emulsion under agitation at room temperature. The images from the Axiocam MRm camera were treated with Axio Vision 4 (Zeiss). All data represent the averages of three independent experiments with separate bacterial cultures unless stated otherwise. 3. Results In the first part of our study, we investigated the hydrophobicity and zeta potential () components of the cell surface properties of three strains of L. lactis subsp lactis biovar diacetylactis. Then, these bacteria were used to study the effect of bacterial surface on emulsion stability. 3.1. Cell surface characteristics 3.1.1. Hydrophobicity of cell surfaces Cell surface hydrophobicity was investigated through the partitioning of cells between water and hexadecane. The percentages of adhesion to hexadecane of the three strains of LLD showed the diversity of their surface hydrophobicity. LLD18 was the most hydrophobic strain with 45% adhesion to nhexadecane and LLD16 was the most hydrophilic with 0% adhesion. LLD125 exhibited intermediate adhesion percentages (30%). 3.1.2. Zeta potential of cell surfaces The of the three strains of LLD at different pH was first examined in physiological water (Fig. 1). depended upon the strains and pH. of all strains were negative at high pH values and became more positive with decreasing pH. The LLD18 strain had a net negative charge at pH 7.5 ( 32 mV) and a positive one (7 mV) at pH 2. The isoelectric point (IEP) of LLD18 was around pH 3.5. Contrasting with these results, the LLD16 strain was negatively charged at all pH values of the test and more negatively charged than LLD18 ( 30 mV at pH 2 and 40 mV from pH 4.5 to 7.5). The profiles of according to pH were similar for LLD125 and LLD18 at pHs above 5. At lower pH, of LLD125 was less positive than

Fig. 1. Zeta potential of LLD strains as a function of pH: () LLD18; () LLD125; () LLD16.

M.H. Ly et al. / International Journal of Food Microbiology 112 (2006) 2634

29

Fig. 2. Phase separation of emulsion made with CTAB at pH 4.5: 15 s (A); 30 min (B); 1 h (C) and 24 h (D) after cell addition. W Control without bacteria, 18 with strain LLD18 and 16 with strain LLD16.

that of LLD18 but more positive than that of LLD16. LLD125 strain possessed an isoelectric point at a pH lower than LLD18 (around pH 2.5). The EM was also examined in citrate or phosphate buffer (10 mM) (data not shown). The charge profiles were very similar to those obtained in water. In all cases, LLD16 was more negatively charged than LLD18. From these results, the more hydrophobic and weakly negatively charged LLD18 and the hydrophilic and more negatively charged LLD16 were selected as model bacteria to study the effect of microorganisms on emulsions. 3.2. Emulsion stability characteristics The evolution of emulsion stability for the three differently charged emulsions with the two strains was evaluated firstly through the observation of the apparition of a thick cream layer (phase separation). Then, the emulsions were studied in more detail by measures of the cream layer height, the turbidity of the serum phase, and the microscopic observation of the aggregation and the size distribution of oil-droplets. 3.2.1. Phase separation in tubes Phase separation was visually observed for the emulsions made with CTAB at all pHs in the presence of bacteria, except for LLD18 at pH 3 and 3.5. The emulsion with SDS was unstable only at pH 3 in the presence of LLD16. No phase separation was observed for the emulsions made with Tween 20. The results are summarized in Table 1. The phase separation phenomenon was most visible for emulsions made with CTAB at pH 4.5 (Fig. 2). The emulsion was immediately destabilized after the addition of bacteria (Fig. 2-A) with a recombination of droplets followed by coalescence. With time, this phenomenon was more and more visible (Fig. 2-B and C). After 24 h, the complete creaming of the emulsions with bacteria was almost attained (Fig. 2-D), most droplets had risen to the top and the aqueous phase was limpid. Whatever the time after the addition of bacteria, the creaming with LLD16 greater than with LLD18 while emulsions without bacteria were stable and no creaming was observed. Similar phenomena of phase separation were observed at pH 7, but the difference between LLD16 and LLD18 was slighter than the one observed at pH 4.5 (data not shown). In order to evaluate the phase separation due to bacteria more precisely, the turbidity of the serum phase and the height percentage of the cream layer were measured.

3.2.2. Turbidity of emulsions The instability of the emulsions inversely correlated with their turbidity (Fig. 3). In the presence of bacteria the turbidity of the emulsions was reduced while emulsions without bacteria remained stable. The difference in the turbidity of the emulsions was also observed between the two strains. In the presence of LLD16, the turbidity decreased immediately just after the addition of the bacteria. After 24 h, the turbidity of the serum phase was similar to that of water. With LLD18 in emulsion, the turbidity was reduced to a lesser extent than with LLD16. 3.2.3. Microscopic observation From the observation of the emulsions made with CTAB at pH 4.5, the oil-droplets of the emulsions without bacteria appeared to be homogeneous and stable (Fig. 4). Similar observations were made after 2h, 4h, 6h and 24h (results not shown). In the presence of bacteria, aggregation and coalescence were observed. The droplets were bigger and coated with the cells (Fig. 4 center and right). The bacteria adsorbed to the droplet surface formed bridges between them. These results were observed above all with the LLD16 strain although similar but less dramatic results were obtained with the LLD18 strain. Some non-adsorbed LLD18 cells were also encountered in the aqueous phase while all the cells of LLD16 were adsorbed to the emulsion droplets. With the non-ionic surfactant Tween 20, the emulsion droplets were similar with or without bacteria and no adhesion of bacteria to the droplets was observed (Fig. 4).

Fig. 3. Diminution of absorbance (At / A0) 100 of emulsions made with CTAB at pH 4.5 after the addition of bacteria: control without bacteria ( ); in presence of strain LLD18 () and of strain LLD16 ( ).

30

M.H. Ly et al. / International Journal of Food Microbiology 112 (2006) 2634

Fig. 4. Microscopic observation of emulsions: control without bacteria (left), with strain LLD18 (center) and with strain LLD16 (right) 1 h after cell addition: emulsions made with CTAB (up) and with Tween 20 (down) at pH 4.5. Alkanes stained with Nile Red and bacteria with Dapi.

3.2.4. Size droplet distribution In order to investigate the effect of bacteria on emulsions more precisely, the size of the emulsion droplets was monitored. Whatever the pH, the stability of the emulsions made with Tween 20 was not affected by the addition of bacteria as shown through the size distributions of oil-droplets at pH 3 (Fig. 5). This distribution was stable and the pH exhibited no detectable effect on this parameter (results not shown). For the emulsions made with SDS, the mean droplet diameter in the presence of LLD18 was higher than the one without bacteria or in the presence of LLD16 at pH 3 (Fig. 5). At the other pHs, the size distribution was similar with or without bacteria (results not shown).

Contrasting with the results obtained for emulsions made with SDS, those made with CTAB had the larger mean droplet diameter in the presence of LLD16 while these values were similar for emulsion without or with LLD18 at pH 3 and 3.5.

Fig. 5. Particle size distribution (1 h after the cell addition) of emulsions at pH 3 ( Control without bacteria); (.. Emulsion + strain LLD16); (.. Emulsion +strain LLD18).

Fig. 6. Effect of the concentration of bacteria added to the emulsion made with CTAB at pH 4.5 on: A the thickness of the cream layer, B the Zeta potential of the emulsion droplets: control without bacteria (), with strain LLD18 (), and strain LLD16 () (data obtained 24 h after cell addition).

M.H. Ly et al. / International Journal of Food Microbiology 112 (2006) 2634

31

Fig. 7. Effect of NaCl on: A the diminution of absorbance: control without bacteria (), in the presence of strain LLD18 ( ) and in the presence of strain LLD16 ( ); B on phase separation: W without bacteria, 18 with LLD18 and 16 with LLD16 of emulsions made with CTAB at pH 4.5 after 24 h.

3.2.5. Influence of the bacterial concentration on the emulsion characteristics Visible instability of the emulsions made with CTAB was observed when 109 bacteria/ml were added at the highest pHs (4.5 and 7). To examine the influence of the cell concentration on emulsion stability, different bacterial concentrations (107 to 5.109 cells/ml) were added to the emulsion at pH 4.5 (Fig. 6). The instability of the emulsion made with CTAB increased gradually with the increase in the bacterial concentration. At the lowest cellular concentration tested (107 cells/ml), no phase separation was observable. At 108 cells/ml concentration the separation was observed only with the more negatively charged LLD16 and the difference in the creaming of emulsions with or without bacteria was only slightly detectable for LLD18 strains. At 109 cells/ml the separation and creaming was observed for all strains but differently for LLD16 and LLD18. Over the 2.109 cells/ml concentration, the separation was promoted very rapidly and in a similar way for the two strains (Fig. 6A). These results indicate that the strong droplet aggregation in emulsions made with CTAB depended on the concentration of the charged bacteria. The effect of bacterial concentration on the of CTABemulsion droplets was measured (Fig. 6B). In the absence of bacteria, the of the emulsion was around +35 mV. When the bacteria were added at low concentrations (107 cells/ml and 108 cells/ml), the of droplets were similar to those emulsions without bacteria. The became progressively less positive with increasing cell concentrations. And at the highest cell concentration, the of the droplets became negative reaching 27 and 35 mVat 5.109 cells/ml for LLD18 and LLD16, respectively. In all the cases, the of emulsion droplets in the presence of LLD16 were more negative than in the presence of LLD18. 3.2.6. Effect of NaCl on emulsion stability The purpose of these experiments was to examine the influence of NaCl on the emulsion stability with the bacteria. The stock emulsions made with CTAB were diluted 10 fold in citrate buffer (10 mM, pH 4.5) containing 100 mM or 200 mM of NaCl. 109 cells/ml of LLD16 or LLD18 were added to the emulsion. With 100 mM NaCl, no phase separation was observed in the presence of LLD18 and, in the presence of LLD16, the creaming was reduced compared to the one without

NaCl (Fig. 7). When the concentration of NaCl was higher (200 mM), no phase separation or creaming was observed. 3.2.7. Aggregation of bacteria in the presence of surfactant To investigate interactions in the absence of alkane, both strains were suspended in citrate buffer (pH 4.5; 10 mM) in the

Fig. 8. Aggregation of bacteria in citrate buffer at pH 4.5 in presence of 0.5% of surfactant: A control without surfactants; B CTAB; C SDS; D Tween 20. Left column with strain LLD18, right one with strain LLD16.

32

M.H. Ly et al. / International Journal of Food Microbiology 112 (2006) 2634

Fig. 9. Phase separation of emulsions stabilized by whey proteins at different pH. W control without bacteria, 18 with strain LLD18 and 16 with LLD16.

presence of 0.5% CTAB, Tween 20 or SDS (Fig. 8). Bacteria aggregated only in the presence of CTAB (Fig. 8B). The aggregation of LLD18 was observed to a lesser extent than with LLD16. No aggregation of bacteria was observed in the presence of Tween 20 or SDS (Fig. 8C and D) at pH 4.5. The effect of NaCl on the surfactant-caused aggregation of bacteria was also examined. The bacteria were suspended in citrate buffer with CTAB and 0.15 M of NaCl. In that case, no aggregation of bacteria was observed in either case. 3.3. Stability of food emulsions In order to simulate the conditions of food matrixes, LLD16 and LLD18 bacterial strains were added to emulsions stabilized by whey proteins at pH 3; 4.5 and 6. At pH 3, the emulsions possessed positively charged droplets, they were stable in the presence of LLD18 but not in the presence LLD16 (Fig. 9). At pH 4.5, close to the isoelectric point of whey proteins, emulsions were unstable with and even without bacteria. At pH 6, the emulsion possessed negatively charged droplets and no effect of the bacteria was observed. At this pH, all of the emulsions, with or without bacteria were stable. 4. Discussion According to the MATH technique results, the three LLD strains possessed rather hydrophilic to moderately hydrophobic surfaces with the maximum adhesion percentage to hexadecane at around 45% for LLD18. Similar results concerning the cell surface of technological bacteria were reported in previous studies (Pelletier et al., 1997; Kiely and Olson, 2000). The profiles of the bacterial cells in relation to the pH were examined by microelectrophoresis. They showed a variety of electrostatic cell surface properties for the three LLD strains tested. In contrast, previous works (Pelletier et al., 1997; Boonaert and Rouxhet, 2000) observed a similarity of isoelectric points for the different strains of the same species. Surprisingly, the IEP of LLD16 was inferior to 2. To our knowledge, no similarly low IEP has been reported for lactic acid bacteria in the literature. The surface characteristics of bacteria are related to the chemical composition of the cellular surface with a special account to proteins, polypeptides and polysaccharides (PLS). The hydrophobicity of the strains is related to PLS and hydrocarbon concentration (Pelletier et al., 1997; Boonaert and Rouxhet, 2000). The bacterial surface charge results from the dissociation or protonation of three main ionizable groups, the phosphate-group of (lipo)teichoic acids and

the carboxyl- and amino-groups of proteins, which depend on pH. According to Millsap et al. (1996), the IEP may vary from strain to strain and it is directly correlated with the surface nitrogen concentration and inversely correlated with the surface oxygen concentration. At physiological pH between 5 and 7, the number of ionized carboxyl- and phosphate-groups exceeds the number of amino-groups and most bacterial strains are negatively charged (Boonaert and Rouxhet, 2000). The properties of the strains of this study confirm the general rule that hydrophobic strains have IEPs around pH 45 whereas more hydrophilic strains have lower IEPs or remain negatively charged at lower pH (van der Mei et al., 2003). Bacterial cell surface properties have been studied for the impact on bacterial adhesion to surface, which may be described by the DLVO (Derjaguin, Landau, Verwey, Overbeek) theory (Hermansson, 1999). According to this theory, particle adhesion is governed by long-range interactions between the adhering particle and macroscopic substratum surface and it includes hydrophobic, Lewis acid-base and electrostatic interactions. However, this theory is also used to describe the force governing colloid system stability (Yeung et al., 2003). So far, few paper have mentioned the impact of microbial adhesion in colloidal systems such as the adhesion of bacterial particles to droplets in emulsion systems through these interactions. The present study examined the impact of bacterial surface properties on emulsions with different characteristics by using different surface-active compounds to stabilize the emulsions: a cationic (CTAB), anionic (SDS) and non-ionic (Tween 20) one. The effect of bacteria on emulsion stability was observed for the emulsions made with the ionic surfactants, SDS and CTAB, but not for those made with the non-ionic one, Tween 20. This suggests that the interaction between bacteria and surfactantcoated droplets are mainly due to electrostatic forces. The instability of the emulsions made with ionic surfactants was restricted to the range for which the charge of bacteria was opposite to the droplets' one (Table 1). Thus, the emulsions made with the anionic surfactant SDS were destabilized by the addition of bacteria only if the medium pH was inferior to the strain's IEP. Inversely, with the cationic CTAB, the emulsion was unstable in the presence of bacteria for pHs superior to the strain's IEP. The instability of the emulsion made with CTAB at pH 4.5 is shown in Fig. 4. Bacteria formed bridge-like structures between droplets resulting first in globule aggregation and then in their coalescence in a way which could be similar to the effect of calcium on emulsions stabilized by whey proteins ( Ye and Singh, 2000; van Aken, 2003). According to these authors, Ca2+

M.H. Ly et al. / International Journal of Food Microbiology 112 (2006) 2634

33

adsorbed on the droplet-surface-localized whey proteins and reduced the surface charge of the external layer of the droplets. This decrease in the surface charge facilitated the connections between the droplets. The destabilization and flocculation of emulsions in the presence of bacteria were reduced by the presence of NaCl (Fig. 7). This may be explained by the competition between the Na+ ions and the cationic surfactant to bind to cell negative surfaces via electrostatic interactions (Goldberg et al., 1990). Indeed, adsorption of Na+ to the cell surface will reduce the CTABmediated interactions between cells and droplets. It may also be due to the decrease in the negative charge of the bacterial surface resulting from the specific ion adsorption to cell surfaces as shown with Ba2+, Ca2+ and Sr2+ by Poortinga et al. (2001). The droplet charge of emulsions made with CTAB was changed from positive without bacteria to negative with high concentrations of cells (Fig. 4B). This may be explained by the adsorption of bacteria onto droplets (Fig. 5) due to electrostatic interactions. With high concentrations of cells, a bacteriasurfactant structure coated the emulsion droplets. The phenomenon of formation of a surfactant-biopolymer envelope coating the droplets was observed when a cationic surfactant (chitosan) was added to a lecithin-stabilized emulsion (Ogawa et al., 2003). This surrounding membrane could provide better stability to the emulsion. In our study, bacteria could also form an envelope around the droplet surface but the emulsion was unstable. Unlike chitosan, bacteria appear to be able to adhere simultaneously to two droplets, forming bridges between them. The interactions between the cationic surfactant CTAB and bacteria were confirmed in solutions without alkane. Similar bacterial aggregations have been observed with ammonium sulfate (AMS) (Lin et al., 1995). According to these authors, AMS may neutralize electrostatic charges on the cells, thus allowing the hydrophobic forces to dominate the cell-to-cell interaction. These authors observed a high correlation between the rate of AMS-caused aggregation and the rate of adhesion to hexadecane by the bacteria. In the present study, the CTAB-caused aggregation of bacteria depended on the charge of the cells and on the ionic strength. At pH 4.5, aggregation was observed for the hydrophilic and negatively charged LLD16 strain and not for the more hydrophobic and less negatively charged LLD18 (Fig. 8). Theses results show that the aggregation of bacteria due to cationic surfactants depends essentially on the negative charge of bacteria suggesting that the binding of cationic surfactants to cells depends on this cell surface charge. The effect of bacteria on emulsions depends not only on the interfacial surface and the bacterial concentration but also on the bacterial strain especially with regard to and IEP. When the combination of cell concentration and surface charge is sufficient to neutralize the droplets charge, the addition of cells triggers complete phase separation. In conclusion, these results show the importance of the cell surface charge of bacteria on the stability of emulsions. The impact of bacteria on colloidal systems varies with the system composition, especially with the presence of charged components. Diversity between strains of the same species is particularly pointed out. Although strains are basically negatively charged at

neutral pH and positively charged at acidic ones, differences in and IEP between the strains enable to find for the same colloidal system, strains that can, by their presence, destabilize the system, inducing coagulation and flocculation, and other strains that cannot. As the utilization of new polymers and surfactants grows, possibilities to design new food emulsions increase as well. For example, Ogawa et al. (2003) have formulated a pH 3-emulsion containing cationic droplets stabilized by lecithin-chitosan membranes in order to avoid the oxidation of lipids. The addition of bacteria to these emulsions could result in different effects: negatively charged cells at this pH (e.g. LLD16) could destabilize the emulsion whereas positively charged ones (like LLD18) would not. Similar results could be expected for emulsions made with whey proteins with IEP around pH 5: at pH 3, the presence of negatively charged cells (like LLD16), but not of positively charged ones (LLD18), could provoke the creaming of the emulsion. The important amount of work on bacterial surface properties should thus concern more the utilization of bacteria in the food industry as, this study confirms that beside their biotechnological characteristics, bacteria can be considered as physical particles and their surface properties cannot only influence the food physicochemical stability but also the position of bacteria inside the colloidal system. It is indeed likely that, in the food matrix, bacteria can adhere to one or the other component of the matrix. This adhesion, especially if the compound of attachment of bacteria is an aroma precursor or a toxic surfactant, can have a high impact in the maturation of food and on bacterial survival. Acknowledgements This work was partly supported by the Conseil Rgional de Bourgogne. Ly possesses a grant from the Agence Universitaire de la Francophonie (AUF). References
Bellon-Fontaine, M.-N., Rault, J., Van Oss, C.J., 1996. Microbial adhesion to solvents: a novel method to determine the electron-donor/electro-acceptor or Lewis acid-base properties of microbial cell. Colloids and Surfaces. B, Biointerfaces 7, 4753. Boonaert, C.J., Rouxhet, P.G., 2000. Surface of lactic acid bacteria: relationships between chemical composition and physicochemical properties. Applied and Environmental Microbiology 66, 25482554. Briandet, R., Herry, J., Bellon-Fontaine, M.-N., 2001. Determination of the van der Waals, electron donor and electron acceptor surface tension components of static Gram-positive microbial biofilms. Colloids and Surfaces. B, Biointerfaces 21, 299310. Chenevier, P., Veyret, B., Roux, D., Henry-Toulme, N., 2000. Interaction of cationic colloids at the surface of J774 cells: a kinetic analysis. Biophysical Journal 79, 12981309. De Man, J.C., Rogosa, M., Sharpe, M.E., 1960. A medium for cultivation of Lactobacilli. Journal of Applied Bacteriology 23, 130135. Dickinson, E., 2003. Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocolloids 17, 2539. Dickinson, E., Ritzoulis, C., Povey, M.J., 1999. Stability of emulsions containing both sodium caseinate and Tween 20. Journal of Colloid and Interface Science 212, 466473. Goldberg, S., Doyle, R.J., Rosenberg, M., 1990. Mechanism of enhancement of microbial cell hydrophobicity by cationic polymers. Journal of Bacteriology 172, 56505654.

34

M.H. Ly et al. / International Journal of Food Microbiology 112 (2006) 2634 Poortinga, A.T., Bos, R., Busscher, H.J., 2001. Electrostatic interactions in the adhesion of an ion-penetrable and ion-impenetrable bacterial strain to glass. Colloids and Surfaces. B, Biointerfaces 20, 105117. Poortinga, A.T., Bos, R., Norde, W., Busscher, H.J., 2002. Electric double layer interactions in bacterial adhesion to surfaces. Surface Science Reports 47, 132. Rosenberg, M., 1991. Basic and applied aspects of microbial adhesion at the hydrocarbon:water interface. Critical Reviews in Microbiology, vol. 18, pp. 159173. van Aken, G.A., 2003. Competitive adsorption of protein and surfactants in highly concentrated emulsions: effect on coalescence mechanisms. Colloids and Surfaces. A, Physicochemical and Engineering Aspects 213, 209219. van der Mei, H.C., van de, B.B., Doyle, R.J., Busscher, H.J., 2001. Cell surface analysis and adhesion of chemically modified streptococci. Journal of Colloid and Interface Science 241, 327332. van der Mei, H.C., van de Belt-Gritter, B., Pouwels, P.H., Martinez, B., Busscher, H.J., 2003. Cell surface hydrophobicity is conveyed by S-layer proteinsa study in recombinant lactobacilli. Colloids and Surfaces. B, Biointerfaces 28, 127134. Ye, A., Singh, H., 2000. Influence of calcium chloride addition on the properties of emulsions stabilized by whey protein concentrate. Food Hydrocolloids 14, 337346. Yeung, A., Moran, K., Masliyah, J., Czarnecki, J., 2003. Shear-induced coalescence of emulsified oil drops. Journal of Colloid and Interface Science 265, 439443.

Gupta, R., Muralidhara, H.S., 2001. Interfacial challenges in food industry. Trends in Food Science and Technology 12, 382391. Hermansson, M., 1999. The DLVO theory in microbial adhesion. Colloids and Surfaces. B, Biointerfaces 14, 105119. Huang, X., Kakuda, Y., Cui, W., 2001. Hydrocolloids in emulsions: particle size distribution and interfacial activity. Food Hydrocolloids 15, 533542. Kiely, L.J., Olson, N.F., 2000. The physicochemical surface characteristics of Lactobacillus casei. Food Microbiology 17, 277291. Lin, L., Rosenberg, M., Taylor, K.G., Doyle, R.J., 1995. Kinetic analysis of ammonium sulfate dependent aggregation of bacteria. Colloids and Surfaces. B, Biointerfaces 5, 127134. Lubbers, S., Charpentier, C., Feuillat, M., Voillet, A., 1994. Influence of yeast walls on the behavior of aroma compounds in a model wine. American Journal of Enology and Viticulture 45, 2933. Millsap, K.W., van der Mei, H.C., Reid, G., Busscher, H.J., 1996. Physicochemical and adhesive cell surface properties of Lactobacillus strains grown in old formula and new, standardized MRS medium. Journal of Microbiological Methods 27, 239242. Ogawa, S., E.A., D., McClements, D., 2003. Production and characterization of O/W emulsions containing cationic droplets stabilized by lecithin-chitosan membranes. Journal of Agricultural and Food Chemistry 51, 28062812. Pelletier, C., Bouley, C., Cayuela, C., Bouttier, S., Bourlioux, P., BellonFontaine, M.-N., 1997. Cell surface characteristics of Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus strains. Applied and Environmental Microbiology 63, 17251731.