Adsorption of the bacteriocins produced by Lactobacillus curvatus CRL705 on a multilayer-LLDPE film for food-packaging applications

Mariana Blanco Massania, , , Graciela M. Vignolob, c, Patricia Eisenberga, d, Pedro J. Morandoc, e, f a INTI-Plásticos, Gral Paz 5445, Buenos Aires, Argentina b Centro de Referencia para Lactobacilos (CERELA), CONICET, Chacabuco 145, Argentina c CONICET, Argentina Tucumán, Argentina d 3iA-UNSAM, Argentina e Gerencia Química Centro Atómico Constituyentes, Comisión Nacional de Energia Atómica (CNEA), Av. Gral Paz y Constituyentes, Buenos Aires, Argentina f Instituto Sábato, Argentina

LWT - Food Science and Technology

Volume 53, Issue 1, September 2013, Pages 128–138

Received 24 August 2012, Revised 28 December 2012, Accepted 21 January 2013, Available online 30 January 2013

https://doi.org/10.1016/j.lwt.2013.01.018

© <2017>. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/



*Manuscript Click here to view linked References



1 Adsorption of the bacteriocins produced by Lactobacillus curvatus



2 CRL705 on a multilayer-LLDPE film for food-packaging applications



3



4



Mariana Blanco Massani a*, Graciela M. Vignolo b,c Patricia Eisenberg a,d and



5



Pedro J. Morando c,e, f



6



7 aINTI-Plásticos. Gral Paz 5445. Buenos Aires, Argentina; bCentro de Referencia para



8 Lactobacilos (CERELA), CONICET. Chacabuco 145, cCONICET, Argentina Tucumán,



9



Argentina. d3iA-UNSAM, Argentina, eGerencia Química Centro Atómico



10



Constituyentes, Comisión Nacional de Energia Atómica (CNEA). Av. Gral Paz y



11



Constituyentes, Buenos Aires, Argentina; fInstituto Sábato, Argentina.



12



13



14



15



16



17 *Corresponding author: Mariana Blanco Massani, E-mail: blanco@inti.gob.ar. Tel/Fax:



18 +54 11 4753 5773



19



20



1



*Manuscript Click here to view linked References



1 ABSTRACT



2



Adsorption of bacteriocins produced by Lactobacillus curvatus CRL705, lactocin



3 705 (whose activity depends upon complementation of two peptides, lac705 and



4 lac705β) and bacteriocin/s with strong anti-Listeria activity, on a multilayer film was



5 investigated. Lactocin 705 adsorption equilibrium at 30ºC was reached from 1 hour of



6 film contact. This bacteriocin exhibited a Langmuir-type adsorption, showing a mass



7 adsorption maximum of



cm-2 and a minimum inhibition concentration of



8 1 µg ml-1. The influence of impurities generated from the growth of bacteriocinogenic



9 strains on bacteriocins adsorption to the film was investigated by inhibition area



10 evaluation in semisolid agar. Impurities from LAB growth strongly influenced



11 adsorption and lactocin 705 antimicrobial activity on the film, while antilisterial



12 bacteriocin/s adsorption remained unaffected. To explain these results, a lack of lac705



13 and lac705 peptides complementation necessary for antimicrobial activity, while no



14 interactions among impurities and antilisterial bacteriocin/s during adsorption was



15 suggested. Antilisterial bacteriocin/s activity on the film was not influenced by lactocin



16 705 adsorption; conformational reorganization of adsorbed antilisterial bacteriocin/s in



17 the presence of lactocin 705 could allow the adsorption of both bacteriocins while



18 maintaining antilisterial antimicrobial activity. This study highlights the technological



19 importance of adsorption optimization to obtain effective antimicrobial food packaging



20 systems.



21



22



Keywords: Bacteriocins adsorption; Food packaging; Antimicrobial multilayer-



23 LLDPE film; Anti-Listeria activity; Lactobacillus curvatus CRL705.



24



25



1



26 Abbreviations:



27 705



Lactocin 705



28 AB



Antilisterial bacteriocin/s



29 ATR-IR



Attenuated total internal reflectance infrared spectroscopy



30 AU



Arbitrary units



31 BU



Bacteriocin units



32 C-AB



Concentrated antilisterial bacteriocin/s



33 CD



Circular dichroism



34 CE



Crude extract



35 CFU



Colony forming units



36 C-Sac7



Concentrated impurities from Bac- variant growth



37 FTIR



Fourier Transform Infrared



38 Imp



Impurities generated from the growth of LAB in MRS broth



39 LAB



Lactic acid bacteria



40 LLDPE



Linear low density polyethylene



41 MIC



Minimum inhibitory concentration



42 P-AB



Purified antilisterial bacteriocin/s



43 RSA



Random Sequence Adsorption



44 S-705



Synthetic lactocin 705



45



2



46 1. Introduction



47



The use of proper packaging materials to minimize food losses and provide safe and



48 wholesome food products has always been the focus of food packaging. Consumer



49 trends for better quality, fresh-like, and convenient foods have been intensified in recent



50 decades. As a consequence, a variety of active food packaging technologies have been



51 developed, among which antimicrobial containing as well as inherently antimicrobial



52 films offer new opportunities for the food industry (Cho, Lee, & Han, 2009; Aider,



53 2010). Antimicrobial packaging systems constitute an emerging technology designed to



54 control the microbial population and target specific microorganisms, thus providing



55 higher safety and quality products. A range of chemical preservatives have been used in



56 active-antimicrobial releasing systems among which bacteriocins and particularly nisin



57 was the most commonly incorporated into films (Joerger, 2007).



58



Bacteriocins are ribosomally synthesized antimicrobial peptides produced by



59 bacteria, from which three main classes have been recently proposed for Gram-positive



60 microorganisms (Rea, Ross, Cotter, & Hill, 2011). Among them, Class II bacteriocins



61 encompass pos-transductionally unmodified peptides and include IIa pediocin like



62 (antilisterial bacteriocins) and IIb two-peptide bacteriocins.



63



The genome sequence of Lactobacillus curvatus CRL705, isolated from dry-



64 fermented sausages, revealed the presence of the genes encoding for five bacteriocins



65 production (Hebert, Saavedra, Taranto, Mozzi, Magni, Nader, Font de Valdez, Sesma,



66 Vignolo, & Raya, 2012). Among them, the bacteriocin lactocin 705, characterized as



67 belonging to class IIb bacteriocins, and whose activity depends upon the



68 complementation of two peptides lac705α and lac705β with 33 amino acid residues



69 each (Cuozzo, Sesma, Palacios, Pesce de Ruiz Holgado, & Raya, 2000), exerted



70 antimicrobial activity against some Lactic acid bacteria (LAB) and Brochothirx



3



71 thermosphacta (Castellano & Vignolo, 2006). Although neither lac705α nor lac705β



72 displayed bacteriocin activity by itself when the growth of sensitive cells was



73 monitored, both peptides showed the ability to interact with a zwitterionic membrane at



74 different bilayer levels (Castellano, Vignolo, Farías, Arrondo, & Cheín, 2007) and



75 bactericidal effect on the indicator strain Lactobacillus plantarum CRL691 was



76 exhibited with a 1 to 4 optimal lac705α/lac705β peptides ratio (Cuozzo, Castellano,



77 Sesma, Vignolo, & Raya, 2003). ¨Lactocin AL705¨, even when it was not yet



78 sequenced, broth and meat slurry assays demonstrated high specific activity against



79 Listeria species (Castellano, Holzapfel, & Vignolo, 2004; Castellano & Vignolo, 2006),



80 thus it may be ascribed to antilisterial (AB) class IIa bacteriocins. Moreover, since L.



81 curvatus CRL705 genome encodes for the production of sakacin P and sakacin X, class



82 IIa bacteriocins, antilisterial activity could be ascribable to those antilisterial



83 bacteriocins (Drider, Fimland, Héchard, McMullen, & Prévost, 2006; Hebert et al.,



84 2012).



85



The strong anphiphatic nature of proteins and peptides gives them great stability



86 in the adsorbed state. Thus, protein adsorption is a common event that takes place in



87 areas such as medicine, pharmaceutical sciences, analytical sciences, biotechnology,



88 cell biology, or biophysics (Hlady & Buijs, 1996; Rabe, Verdes, & Seeger, 2011).



89 Conformational rearrangements involved in adsorption could cause bacteriocin structure



90 alteration and negatively affect its antimicrobial activity (Roach, Farrar, & Perry, 2005;



91 Drider et al., 2006; Nissen-Meyer, Oppegård, Rogne, Hauguen, & Kristiansen, 2010).



92 Vast investigation on understanding protein adsorption can be found in the literature



93 and techniques to detect adsorbates presence on the surface include ellipsometry, quartz



94 crystal microbalance measurements, and analytical methods such as Lowry method or



95 absorbance determination, among others (Sarkar & Chattoraj, 1993; Nakanishi,



4



96 Sakiyama, & Imamura, 2001; Roach et al., 2005; Wei, Huang, Hou, Yuan, & Fang,



97 2007). Techniques that specifically focus on the secondary structure of adsorbed



98 proteins such as attenuated total internal reflectance infrared spectroscopy (ATR-IR)



99 and Circular dichroism (CD) spectroscopy are valuable tools to study conformational



100 changes (Rabe et al., 2011). However, for bacteriocins less information on solid surface



101 interactions is available and techniques used for adsorption determinations include



102 ellipsometry (Bower, McGuire, & Daeschel, 1995; Tai, McGuire, & Neff, 2008) and



103 those taking into account biological activity of the proteinaceous substances, such as



104 turbidimetry or inhibition on semisolid medium (Bower et al., 1995; Guerra, Macías,



105 Agrasar, & Castro, 2005a; Guerra, Araujo, Barrera, Agrasar, Macías, Carballo, &



106 Pastrana, 2005b; Ibarguren, Audisio, Farfán Torres, & Apella, 2010).



107



Langmuir model is the most basic adsorption model that accounts for the



108 adsorption and desorption of particles at distinct surface sites. Although there is general



109 accordance in the community that this formalism is inadequate to accurately describe



110 protein adsorption, it is a kind of starting point for the development of theoretical



111 descriptions of protein adsorption events, since is it has a simple mathematical format



112 (Rabe et al., 2011). Langmuir model application to descript proteins and bacteriocins



113 adsorption behavior has been earlier reported (Daeschel, McGuire, & Al-Makhlafi,



114 1992; Wei et. al, 2007).



115



Allowing bacteriocins to adsorb to food contact surfaces may have the potential to



116 prevent spoilage and pathogenic colonization of foods. Several works have addressed



117 surfaces activation using bacteriocins accompanied by impurities from the culture



118 medium of production (Bower et al. 1995; Scannell, Hill, Ross, Marx, Hartmeier, &



119 Arendt, 2000; Guerra et al. 2005a, b; Ibarguren et al. 2010). However, no studies on the



120 interaction of impurities and bacteriocins during adsorption were performed in the



5



121 above mentioned works. In our previous studies, a CE obtained from L. curvatus



122 CRL705 and containing lactocin 705, antilisterial bacteriocin/s and impurities from the



123 producer bacterium was used to adsorb on a multilayer-Linear low density polyethylene



124 (LLDPE) film, to render antimicrobial activity (Blanco Massani, Fernandez, Ariosti,



125 Eisenberg, & Vignolo, 2008) and film surface properties before and after activation



126 treatment were determined (Blanco Massani, Morando, Vignolo, & Eisenberg, 2012). In



127 order to understand and control bacteriocins adsorption on the multilayer-LLDPE film



128 as well as to predict antimicrobial film effectiveness, synthetic lactocin 705 was



129 adsorbed on the multilayer-LLDPE film and the interaction with antilisterial



130 bacteriocin/s and impurities from L. curvatus CRL705 growth during the adsorption



131 process was investigated.



132



133 2. Materials and Methods



134



135 2.1. Bacterial strains and growth conditions



136



Lactobacillus curvatus CRL705, lactocin 705 (705) and antilisterial bacteriocin/s



137 (AB) producer, and Lactobacillus plantarum CRL691, used as an indicator of 705



138 activity, were isolated from dry-fermented sausages (Vignolo, Suriani, Ruiz Holgado, &



139 Oliver, 1993). Sac7 strain is a derivative from L. curvatus CRL705 unable to produce



140 either 705 or AB (Bac- variant) (Cuozzo unpublished results). L. curvatus CRL1579, a



141 derivative of CRL705 which only produces AB, was obtained as reported by Castellano



142 & Vignolo (2006). All lactobacilli strains were grown in MRS broth (Britania,



143 Argentina) at 30 ºC. Listeria innocua 7, used as indicator of AB activity, was obtained



144 from the Unité de Recherches Laitières et Génétique Appliquée, INRA (France) and 145 grown in trypticase soy broth (TSB, Britania) with 5mg ml-1of added yeast extract (YE,



6



146 Britania) at 30 ºC. All strains were maintained and stored at -20ºC in 0.15 g ml-1 of



147 glycerol.



148



149 2.2. Sorbent and adsorbates



150



A 100 µm multilayer-LLDPE film composed of an external polypropylene layer, an



151 internal polyamide-polyethylene structure, a barrier layer of ethylene vinyl alcohol



152 copolymer, and a linear low density polyethylene food contact layer (Cryovac; Sealed



153 Air Co, Argentina), was used as sorbent in this study. The bacteriocins lactocin 705, the



154 AB, and impurities from L. curvatus CRL705 growth obtained from different sources



155 were used as adsorbates (Table 1).



156



157 2.3. Adsorbates characterization by Fourier Transform Infrared (FTIR) spectroscopy



158



Each adsorbate was characterized by FTIR spectroscopy using a Thermo Nicolet



159 6700 spectrometer equipped with a DTGS KBr detector and a Smart iTR ATR sampling 160 accessory. Sixty four scans were taken for each sample from 4000 to 650 cm-1 at a 161 resolution of 4 cm-1.



162



163 2.4. Bacteriocins quantification in solution



164



For lactocin 705 kinetic and equilibrium of adsorption studies (see below),



165 bacteriocin activity was quantified against L. plantarum CRL691 with a turbidimetric



166 bioassay (Cabo, Murado, González, & Pastoriza, 1999) based on growth inhibition of



167 the target bacterium caused by serial dilution of bacteriocin samples. Briefly, one



168 volume of S-705 and its two-fold dilutions were mixed in different tubes with one 169 volume of the target bacterium (L. plantarum CRL691, 105 CFU ml-1) suspended in



170 MRS broth (Britania, Argentina). The tubes were then incubated at 30 ºC during 16 h,



7



171 growth inhibition was measured spectrophotometrically at 600 nm (Cuozzo et al., 2003) 172 and dose-response curves were obtained. Controls consisted of tubes in which S-705 173 was replaced by sterile distilled water. Bacteriocin activity was calculated as bacteriocin 174 units (1 BU: bacteriocin needed to obtain 50% growth inhibition compared with control 175 tubes) (Guerra et al 2005b). Active lactocin 705 concentration was determined from a 176 standard curve constructed with BU versus different S-705 concentration (µg ml-1). 177 For bacteriocins adsorption study under different conditions (see below), bacteriocins 178 activity in solution (titer) was determined by a modification of the agar well diffusion 179 method (Pongtharangkul & Demirci, 2004). Fifteen μl of serial two-fold dilutions of the 180 bacteriocins solutions were added to 5 mm diameter wells cut in semisolid MRS agar 181 plates seeded with L. plantarum CRL691 for lactocin 705, and TSB + YE agar seeded 182 with L. innocua 7, for AB titration. The agar plates were stored at 4 ºC for 24 h to allow 183 pre-diffusion, then incubated for 16-18 h at 30 ºC and examined for inhibition zones. 184 Bacteriocins titer, expressed in arbitrary units (AU ml-1), was defined as the reciprocal 185 of the highest dilution yielding a visible zone of inhibition on the sensitive strain. All 186 determinations were performed in triplicate. 187 188 2.5. Bacteriocins quantification on the multilayer LLDPE film 189 Antimicrobial activity on the multilayer-LLDPE film (see below) was determined as 190 described earlier (Blanco Massani et al., 2008). Film circles (0.95 cm2) with and without 191 bacteriocins were placed face down on semisolid agar plates seeded with the sensitive 192 organisms (L. plantarum CRL691 for 705 and L. innocua 7 for AB). Film activity was 193 evidenced as an inhibition zone of the indicator organisms beneath and around the 194 packaging material and was expressed as relative inhibition area (inhibition zone around 195 packaging film/film area). Four replicates for each sample were run.

8



196



197 2.6. Lactocin 705 adsorption on the multilayer-LLDPE film



198 2.6.1 Minimum inhibitory concentration (MIC).



199 Lactocin 705 MIC necessary to assure uniform inhibition area on the multilayer film



200 LLDPE surface was determined by contacting 0.260 ml of S-705 (0.5, 1, 2, 3, 4, 5, 6 201 and 8 µg ml-1) with 0.95 cm2 of the film. After contact, films were rinsed with sterile



202 distilled water and antimicrobial activity on their LLDPE surface was determined in



203 semisolid agar as earlier described.



204 2.6.2. Adsorption kinetic and equilibrium. 205 To optimize lactocin 705 adsorption temperature, a S-705 bacteriocin solution (1 µg ml206 1) was contacted with the multilayer-LLDPE film food contact face during pre-



207 established times ranging from 10 to 120 min, at 20, 30 and 40 ºC. Lactocin 705



208 adsorption isotherm was obtained by contacting the film with different concentrations of



209 S-705 solution at 30 ºC during 1 h. In all cases, in order to investigate whether a loss of



210 lactocin 705 activity occurred during the active film preparation, control solutions of S-



211 705 were subjected to the adsorption conditions in the absence of the multilayer-LLDPE



212 film (Scannell et al., 2000). Bacteriocin active concentration, of control and film-



213 contacted solutions was examined by BU determination as described above. The



214 amount of active lactocin 705 adsorbed on the multilayer film LLDPE contact face, Γ 215 (µg cm-2) at each time/concentration/temperature set, was determined from the



216 difference between bacteriocin concentration in the controls and in the film-contacted



217 solutions, as expressed by equation [1],



218



  (Cc  C f )v



[1]



A



219 where Cc: lactocin 705 concentration in the control solution, Cf: bacteriocin



220 concentration after the adsorption process, v: volume of bacteriocin solution to which A



9



221 (cm2) of multilayer-LLDPE film food contact face were contacted (Sarkar & Chattoraj,



222 1993). For all experiments lactocin 705 activity on the multilayer film food contact



223 surface was confirmed on semisolid agar. Experiments were run in triplicates.



224



225 2.7. Bacteriocins adsorption under different conditions



226



Adsorbates were combined in order to study bacteriocins and impurities interaction



227 during the adsorption process (Table 2). Bacteriocins adsorption curves were



228 constructed from the relative inhibition areas exerted by bacteriocins adsorbed on



229 multilayer-LLDPE film versus bacteriocin titer (AU ml-1) after adsorption.



230



S-705 (8 µg ml-1, 6400 AU ml-1) was also contacted (1 h, 30 ºC) with the multilayer-



231 LLDPE film surface in the presence of C-Sac7 (0.1, 1, 20 and 40 mg ml-1) and P-AB



232 (AB, 12800 AU ml-1) to simulate the conditions presented by 40 mg ml-1 of the CE



233 (lactocin 705, 6400 AU ml-1; AB, 12800 AU ml-1). After contact, S-705 adsorption



234 performance was checked by relative inhibition area evaluation in semisolid agar as



235 earlier described. The same experiment was conducted for antilisterial bacteriocin/s 236 adsorption from P-AB (AB, 12800 AU ml-1), C-AB (AB, 12800 AU ml-1), the 237 combination of P-AB and S-705 (AB, 12800 AU ml-1; 705, 6400 AU ml-1), and the CE 238 (40 mg ml-1). A sequential adsorption study was also performed; the multilayer-LLDPE 239 film was treated with S-705 (6400 AU ml-1, 1 h, 30 ºC), rinsed with sterile water, 240 contacted with Sac7 (40 mg ml-1, 1 h, 30 ºC), rinsed again and assayed for antimicrobial



241 activity. The same experiment was performed inverting the adsorbates order.



242



243 2.8. Statistical analysis



10



244 In all experiments data were subjected to analysis of variance (ANOVA), and the Tukey



245 test was applied at the 0.05 level of significance. All statistical analyses were performed



246 using Minitab Statistic Program, release 12 (Pennsylvania, USA).



247



248



249 3. Results and Discussion



250



251 3.1. FTIR adsorbates characterization



252



Bacteriocins and impurities used in this study were obtained from different sources



253 (CE, C-Sac7, C-AB, P-AB and S-705), FTIR spectroscopy was used as a tool to



254 characterize them, looking for molecular groups associated with proteins, fatty acids



255 and polysaccharides (Fig. 1). FTIR bands assignment were carried out according to



256 those reported by Quinteiro Rodríguez (2000), Barth (2000), Maquelin, Kirschner,



257 Choo-Smith, van den Braak, Endtz, Naumann, & Puppels (2002) and Motta, Flores,



258 Souto, & Brandelli (2008) for microorganisms, peptides and amino-acids



259 characterization (Table 3). From these results, the bands exhibited at around 3500 and 260 3200 cm-1 as well as those in the amide I (1700-1610 cm-1) and II (1520-1500 cm-1)



261 regions were present in all analyzed adsorbates (Table 3) and could be associated with



262 hydroxyl groups, proteins or protein compounds which is in agreement with the peptide



263 nature of bacteriocins. On the other hand, the C-H stretching vibration of lipid acyl 264 chains in the spectral region between 2900 and 2800 cm-1, C-H deformation of 265 aliphatics at 1450 cm-1, stretching vibration from esters at 1740 cm-1 as well as bands 266 associated with carbohydrates deformation between 900 and 1200 cm-1 were present in



267 adsorbates from Lactobacilus cultures (CE, C-Sac7, C-AB, and P-AB), but were absent



268 in synthetic lactocin 705 (Fig. 1, Table 3). This result suggests the presence of various



11



269 LAB impurities (metabolites from the LAB growth and MRS medium components) as 270 well as cellular debris, nucleic acids and aliphatic molecules among others. These 271 results are in agreement with those reported by Vodnar, Paucean, Duluf, & Socaciu 272 (2010) who were able to fingerprint probiotic LAB using FTIR by the specific bands 273 located around 2845 and 2929 cm-1, characteristic to the bacterial wall fatty acids, and a 274 specific absorption peak at 1127 cm-1, for lactic acid. The spectrum corresponding to P275 AB that was obtained after purification of C-AB (obtained from L. curvatus CRL1579) 276 showed to lack a band at 1400 cm-1 (Fig. 1), assigned to C=O stretching symmetric of 277 COO- groups (Table 3); in addition, the bands between 900 and 1200 cm-1 experienced 278 a marked decrease in P-AB spectrum when compared with that of C-AB. These results 279 suggest that part of acids compounds and polysaccharides from the culture media have 280 been removed after adsorbate purification. On the other hand, the bands at 1438, 1200 281 and 1122 cm-1 were present in the synthetic lactocin 705, as well as in the CE spectrum 282 (Table 3). According to Barth (2000), these bands may be assigned to C-N stretching 283 from Histidine (1439 cm-1), Tyrosine Tyr-OH bending (1169-1260 cm-1) and C-O 284 stretching from Aspartate (1120-1253 cm-1), this being in coincidence with the 285 determined lactocin 705 amino acid sequence in which these amino acids are involved 286 (Cuozzo et al., 2000). The infrared adsorption of amino acids side chain in a protein 287 may deviate significantly from their absorption in solution or in a crystal (Barth, 2000). 288 Although from amino acid side chains infrared bands of Barth’s compilation (2000) 289 absorption values for His, Tyr and Asp are in the spectral zone of those found for S-705 290 differential bands, these values were regarded only as guidelines for spectra 291 interpretation, since experimental conditions used for IR determinations by Barth (2000) 292 were different from that used in our work (amino acids in water in contrast to S-705 as 293 solid powder).

12



294



295 3.2. Lactocin 705 adsorption on the multilayer-LLDPE film



296



3.2.1. MIC determination



297



Lactocin 705 antimicrobial activity of the multilayer-LLDPE film assayed in



298 semisolid agar after contact with different concentration of S-705 is shown in Figure 2. 299 Results showed an uneven inhibition area when S-705 at a concentration of 0.5 µg ml-1



300 was applied, while uniform areas were observed when the multilayer film LLDPE 301 surface was treated with S-705 concentrations from 1 to 8 µg ml-1. Thus, 1 µg/ml was



302 chosen as the lactocin 705 MIC.



303



3.2.2. Effect of temperature on bacteriocin adsorption



304



The variation of lactocin 705 active concentration in the activation solution, at three



305 different temperatures, in absence (control) and in presence of the multilayer-LLDPE



306 film was evaluated to determine the influence of temperature on lactocin 705 adsorption



307 (Fig. 3a,b). A decrease of lactocin 705 active concentration as temperature increased



308 from 20 to 40 ºC during 120 min was recorded, both in the control solution and when



309 contacted with multilayer-LLDPE film. For the different assayed temperatures (20, 30



310 and 40 ºC), the higher the temperature, the sharper the active lactocin 705 concentration



311 decrease in the absence (control) and in the presence of multilayer-LLDPE film (Fig. 3a



312 and Fig. 3b, respectively). When the adsorbed mass of active lactocin 705 at 20, 30 and



313 40 ºC was evaluated on the multilayer-LLDPE film, it was observed to be maximal at



314 30 ºC (Fig 3c). Even when thermal resistance of class II peptides is widely accepted,



315 structural changes in the helical region observed at elevated temperatures may account



316 for the loss of activity of these small peptides (Kaur, Andrew, Wishart, & Vederas,



317 2004; Soliman, Wang, Bhattacharjee, & Kaur, 2010). Besides bacteriocin degradation



318 with temperature, the more pronounced decrease in concentration, when the bacteriocin



13



319 solution was contacted with the multilayer-LLDPE film, indicated that there was a



320 remaining concentration adsorbed on the film.



321



Protein adsorption is controlled by many types of interactions, the main constituents



322 being dehydration of the sorbent surface and parts of the protein molecule, electrostatic



323 interactions between the protein and the sorbent, and changes in the conformational



324 entropy of the protein (Norde, 1996). In the adsorbed state, hydrophobic amino acid



325 residues may rearrange its structure in order to optimize interaction with the sorbent,



326 preventing contact with water. Such structural rearrangements involve an entropy gain



327 related to an increased rotational mobility along the polypeptide chain. This entropy



328 increase may be sufficiently large to compensate for the positive adsorption enthalpy



329 (Norde, Macritchie, Nowicka, & Lyklema, 1986). The amount of surface adsorbed



330 proteins generally increases at elevated temperatures (Nakanishi et al., 2001).



331 Temperature has an effect on both, the equilibrium state and the kinetics of protein



332 adsorption. At higher temperatures structural arrangements increase significantly, and



333 adsorption rates increases can be expected due to an accelerated diffusivity of proteins



334 towards the sorbent surface (Kondo, & Fukuda, 1998; Rabe et al., 2011). In our study,



335 an increase in the active adsorbed mass of lactocin 705 when temperature changed from



336 20 to 30 ºC was observed. However, the active adsorbed mass at 40ºC was lower than



337 that obtained at 30ºC (Fig. 3c). Bacteriocin degradation in the solution as temperature



338 increases from 30 to 40ºC (Fig 3a), could led to a decrease in bacteriocin active



339 concentration, available to be adsorbed on the film. This antagonist effect allowed



340 defining 30ºC as an optimal adsorption temperature.



341 3.2.3. Effect of contact time in bacteriocin adsorption



342



The antimicrobial activity of lactocin 705 when adsorption plateau was attained on



343 the film at 20, 60 and 120 min for 40, 30 and 20 ºC, respectively determined on



14



344 semisolid agar is shown in Figure 3d. A lack of antimicrobial activity on the multilayer-



345 LLDPE film surface up to 120 min of contact at 20 ºC and after 20 min at 40 ºC were



346 observed, while an activated multilayer-LLDPE film was obtained after contacting 60



347 min with S-705 at 30 ºC. These results are in agreement with the higher bacteriocin 348 adsorbed mass after 60 min of film contact at 30 ºC (0.07 ± 0.02 µg cm-2) compared



349 with the values obtained at 120 min and 20 min (20 and 40 ºC, respectively) (Figure 3c).



350 Consequently, 60 min was defined as the minimal contact time for lactocin 705



351 equilibrium attainment in the multilayer-LLDPE film at 30ºC; this result is in



352 coincidence with previously reported exposure time for lactocin 705 and AB adsorption



353 from L. curvatus CRL705 CE (Blanco Massani et al., 2008). Greater contact times were



354 necessary to homogenously adsorb other antimicrobials such as the antilisterial



355 bacteriocin from Enterococcus faecium CRL1385 and nisin to silicates and other



356 hydrophilic and hydrophobic surfaces (Guerra et al., 2005a, b; Ibarguren et al., 2010).



357 This could suggest a higher lactocin 705 bacteriocin affinity for the multilayer-LLDPE



358 film.



359 3.2.4. Isotherm construction



360



Figure 4 shows the adsorption isotherm of active lactocin 705 on the multilayer-



361 LLDPE film at 30 ºC. Modeling of experimental results from protein adsorption studies



362 often requires the adaptation of different adsorption isotherms models (Hlady & Buijs,



363 1996). Several reports on nisin adsorption to surfaces with different hydrophobicity



364 degrees showed monolayer (Daeschel et al., 1992), multilayer (Bower et al., 1995; Tai



365 et al., 2008) adsorption isotherms or a combination of both (Guerra et al., 2005a).



366 Different adsorption models have been proposed during the past decades for protein



367 adsorption (Rabe et al., 2011); among them the Random Sequence Adsorption (RSA)



368 model (Talbot, Tarjus, Van Tassel, & Viot, 2000) has been applied for protein



15



369 adsorption modeling at solid surfaces (Ramsden, 1993; Guemouri, Ogier, Zekhnini, &



370 Ramsden, 2000). Nevertheless, to our knowledge RSA model has not been yet applied



371 for bacteriocins adsorption modeling. Here, for comparative purposes with other studies



372 on bacteriocin adsorption, Langmuir adsorption model was applied. This model



373 assumes a monolayer adsorption, a homogeneous surface and no lateral interaction



374 among adsorbed peptides molecules. Although this theory is too simplistic to explain



375 the complex behavior of bacteriocins adsorption and data are not necessarily well



376 described by the model, lactocin 705 adsorption may be empirically interpreted by



377 Langmuir-type equation [2].



378



  c K Ceq



[2]



1  K Ceq



379 in which, Γ, is the equilibrium concentration in the solid phase, Ceq,, concentration in the 380 liquid phase, K, apparent association constant representing interaction between



381 adsorbate (lactocin 705) and the sorbent film surface (LLDPE multilayer film face).



382



Adsorption capacity of lactocin 705 on the multilayer-LLDPE film, as calculated



383 from the curve plateau (Fig. 4, eq 2, R=0.9319) showed a value of 0.72±0.05 µg cm-2,



384 this being similar to those found by Guerra et al., (2005b) who, using biological 385 methods, determined similar nisin adsorption abilities (0.665 and 0.697 µg cm-2) to 386 polyethylene-terephthalate and rubber, respectively, while a lower value (0.396 µg cm-2)



387 was found for stainless steel. However, using ellipsometry, an adsorption capacity of 388 0.4 µg cm-2 was reported for nisin on hydrophobic silicon surface (Daeschel et al.,



389 1992). Lactocin 705 adsorption plateau was reached from an S-705 contact solution 390 with a concentration above 4 µg ml-1 (: 0.61±0.05 µg cm-2; Ceq: 0.91 µg ml-1);



391 multilayer-LLDPE film constant inhibition area on semisolid agar was also obtained



392 from this concentration (Fig. 2).



16



393 394 3.3. Bacteriocins adsorption under different conditions 395 3.3.1 Impurities and AB influence on lactocin 705 adsorption



396



Qualitatively, it can be seen from Figure 2 that the amount of lactocin 705



397 antimicrobial activity associated with bacteriocin adsorbed (as indicated by diameters of



398 respective inhibition areas) corresponds to the mass of lactocin 705 actually adsorbed to



399 the respective multilayer films (Fig. 4); i.e., the smallest inhibition zone corresponded



400 with the smallest adsorbed mass of bacteriocin. Base on this trend, adsorption of



401 lactocin 705, checked by relative inhibition area determinations was subjected to an



402 empirical treatment according to Langmuir equation (Fig. 5). Since bacteriocins are



403 produced during bacterial growth, various types and amounts impurities, encompassing



404 metabolites produced during growth of the bacteriocinogenic LAB strains (L. curvatus



405 CRL705 and CRL1579) as well as growth medium components, are present in bacteria



406 extracts. Consequently the influence of impurities on lactocin 705 adsorption was



407 investigated. Lactocin 705 relative inhibition area of multilayer-LLDPE film decreased 408 in the presence of impurities from Bac- variant (C-Sac7, 1 mg ml-1), from 3.7±0.1 (S-



409 705 alone) to 2.6±0.3 at adsorption equilibrium (Fig. 5). However, a higher relative



410 inhibition area (3.0±0.3) was exerted by lactocin 705 when adsorbed in the presence of 411 P-AB (AB, 2000 AU ml-1). In addition, to simulate the conditions of bacteriocin crude 412 extract (CE); different amounts of impurities (C-Sac7, 0.1, 1, 20 and 40 mg ml-1) and P413 AB (AB, 12800 AU ml-1) were added to S-705 (705, 6400 AU ml-1), and contacted with



414 the multilayer-LLDPE film. Even when lactocin 705 titer in solution did not change



415 upon C-Sac7 addition; as the impurities concentration increased a significant decreasing



416 effect (P<0.05) on lactocin 705 activity on the multilayer-LLDPE film was observed



417 (Table 4). When the film was treated with S-705 in the presence of P-AB, the exerted



17



418 relative inhibition area (3.0±0.3) was not significantly different (P≥0.05) from that 419 produced in the presence of Sac7 0.1 mg ml-1 (3.2±0.3). Multilayer-LLDPE film treated 420 with S-705 added with C-Sac7 (20 and 40 mg ml-1), showed the same inhibition area 421 (1.6±0.3 and 1.1±0.5, respectively) than that treated with CE (P≥0.05) (Table 4). The 422 reduction in the adsorption maximum of lactocin 705 added with P-AB and C-Sac7 423 (containing impurities from LAB growth) may be explained by the presence of proteins, 424 fatty acids and polysaccharides as showed by FTIR analysis of the bacterially produced 425 adsorbates. Lactocin 705 inactivation on the multilayer-LLDPE film surface was 426 previously reported when contacted with sunflower oil (Blanco Massani et al., 2012); 427 lipid acyl chains present in C-Sac7, CE and P-AB may also interfere with lactocin 705 428 adsorption, contributing to the observed antimicrobial activity decrease. Since the 429 derivative Sac7 strain differs from the parental L. curvatus CRL705 on its ability to 430 ferment sucrose and to produce lactocin 705 and AB, and since the growth MRS 431 medium used do not contain sucrose in its formulation, metabolites produced by both 432 bacteria were assumed to be the same.



433



When considering protein and peptides mixtures, the adsorption behavior is often a



434 result of an overlap of transport, adsorption and repulsion processes (Rabe et al., 2011).



435 Small proteins diffuse faster than large ones and are the dominating species in the early



436 adsorption stage. However, larger proteins typically bind stronger to the surface because



437 of a larger contact area, and can even repel other pre-adsorbed proteins during spreading



438 on the surface (Lutanie, Voegel, Shaaf, Freund, Cazenave, & Schmitt, 1992; Nasir &



439 McGuire, 1998). Consequently, the total mass of adsorbed proteins passes through a



440 maximum during the course of adsorption (Andrade & Hlady, 1986). In our study, the



441 pronounced relative inhibition areas reduction for lactocin 705 with increasing C-Sac7



442 (impurities from LAB growth) concentration might suggest a competitive adsorption



18



443 between lactocin 705 (synthetic two-peptide bacteriocin) and the molecules present in



444 C-Sac7 adsorbate (fatty acids, peptides or proteins). Similarly, the decreased



445 antimicrobial activity of lactocin 705 on the multilayer-LLDPE film surface in the



446 presence of PAB and from the CE may be ascribed to the presence of such impurities.



447 In order to check a competitive adsorption, a sequential adsorption study was performed



448 and inhibition areas were compared to that of the obtained by CE (Table 4). No



449 significant differences (P≥0.05) in multilayer-LLDPE film relative inhibition areas were 450 obtained after the sequential adsorption of S-705 and C-Sac7 (40 mg ml-1) (1.5±0.1),



451 and from treatment with CE (1.4±0.4). Conversely, relative inhibition area obtained 452 from sequential treatment of multilayer-LLDPE film with C-Sac7 (40 mg ml-1) and S-



453 705 (3.3±0.4) showed no significant difference (P≥0.05) from that of the film treated



454 with S-705 alone (3.7±0.1). These results could suggest that impurities from C-Sac 7 are



455 not being adsorbed directly on multilayer-LLDPE film.



456 3.3.2 Impurities content and 705 influence on AB adsorption



457



AB adsorption on the multilayer-LLDPE film was studied on semisolid agar (Fig. 6,



458 Table 4). No differences in AB relative inhibition areas on the film were observed



459 (P≥0.05) when the bacteriocin was adsorbed from C-AB, P-AB alone or combined with 460 S-705 (8 µg ml-1), (Fig 6). The effect of the impurities content on lactocin AB



461 adsorption was analyzed by comparing relative inhibition areas of the multilayer-



462 LLDPE film contacted with P-AB and C-AB. No significant differences (P≥0.05) were



463 found when AB adsorbed from these adsorbate sources (P-AB, 2.1±0.2 and C-AB,



464 2.2±0.1), (Table 4). In addition, no influence of S-705 on AB adsorption was observed,



465 since the obtained relative inhibition area (2.1±0.2) was similar (P≥0.05) to that of the



466 adsorption from P-AB alone (2.1 ±0.2).



467 3.3.3. Results rationalization



19



468



From the obtained results (Figs. 5, 6 and Table 4) a rationalized scheme was carried



469 out (Fig. 7) representing the different bacteriocins adsorption processes, on the basis of



470 previously reported interactions between Class II a and b bacteriocins and biological



471 membranes (Castellano et al., 2007; Drider et al. 2006; Nissen-Meyer et al., 2010). A



472 conformational change of lac705 peptide during the hydrophobic interaction with the



473 multilayer-LLDPE film (Fig 7a) and a further interaction between this adsorbed peptide



474 and the impurities from C-Sac7, P-AB and CE might have been occurred (Fig. 7b).



475 Thus, a lack of lac705 and lac705 peptides complementation necessary for



476 antimicrobial activity may be suggested, this leading to a decrease of lactocin 705



477 activity on the multilayer-LLDPE film. Conformational changes upon protein



478 adsorption on hydrophobic surfaces have been earlier reported (Norde et al., 1986;



479 Roach et al., 2005). Moreover, from a previous study reporting the interaction between



480 S-705 and a lipid bi-layer, important conformational reorganization was observed for



481 lac705β; while lac705α interacted with the interfacial region inducing dehydration,



482 lac705β peptide interacted with the hydrophobic core of the bi-layer (Castellano et al.,



483 2007).



484



For AB adsorption on the multilayer-LLDPE, similar relative inhibition areas were



485 obtained regardless impurities and lactocin 705 presence. Due to its strong antilisterial



486 activity, AB produced by L. curvatus CRL705 are believed to belong to class IIa



487 bacteriocins, which are small single-molecule peptides in contrast to class IIb (two-



488 component bacteriocins). From the results, it may be suggested that AB adsorb on



489 multilayer-LLDPE film surface and no further interactions with impurities occur during



490 adsorption (Fig. 7c). In addition, since no differences in relative inhibition areas were



491 observed when P-AB was adsorbed in combination with S-705, no changes in the



492 number of AB molecules neither adsorbed alone nor in the presence of lactocin 705,



20



493 may be suggested. Therefore, AB adsorption with its long axis parallel to the surface



494 might have been occurred, thus covering the surface with a number of molecules (Fig.



495 7c). In the presence of S-705, no changes in the number of AB adsorbed molecules



496 would be expected if a rearrangement to a perpendicular orientation were produced,



497 hence lactocin 705 would adsorb on the uncovered surface, maintaining multilayer-



498 LLDPE film AB antimicrobial activity (Fig. 7d). Similar results were reported for



499 fibrinogen adsorption on a hydrophobic surface which in an initial stage adsorbed with



500 its long axis parallel to the surface and then, due to high protein concentration on the



501 surrounding medium, rearrangement of the protein to a perpendicular orientation



502 occurred allowing further protein molecules to adsorb on the uncovered surface (Roach



503 et al., 2005).



504



505 4. Conclusions



506



507



From the antagonist effect of temperature on lactocin 705 activity and adsorption,



508 60 min and 30ºC were established as optimal conditions for bacteriocin adsorption on



509 the multilayer-LLDPE film. A Langmuir-type treatment allowed determining lactocin



510 705 active adsorbed mass. Different bacteriocin sources were characterized and



511 compared regarding their ability to adsorb on the film producing inhibitory activity



512 against food pathogen involving anti-Listeria activity. Impurities generated during



513 growth of L. curvatus CRL705 and CRL1579, used as bacteriocin-producers, strongly



514 influenced the adsorption and antimicrobial activity of lactocin 705 on the multilayer-



515 LLDPE film, while no evidence of their effect was found for AB adsorption. These



516 results were rationalized and an adsorption mechanism was proposed for the



517 bacteriocins, from which lack of lac705 and lac705 peptides complementation



21



518 necessary for lactocin 705 antimicrobial activity, while no interactions among 519 impurities and AB during adsorption was proposed. AB activity on the film was not 520 influenced by lactocin 705 adsorption; conformational reorganization of adsorbed AB in 521 the presence of 705 could allow the adsorption of both bacteriocins while maintaining 522 antilisterial antimicrobial activity.



523



The study developed in our work contributes to the understanding of bacteriocins



524 adsorption and interactions with metabolites that could negatively affect the adsorption



525 process, decreasing antimicrobial activity on the film. The awareness of these



526 interactions could help to understand the film performance in contaminated food, which



527 is part of current work. Further studies (e.g. circular dichroism) should give detailed



528 information on conformational changes upon adsorption to LLDPE surface of the



529 multilayer film.



530 The bacteriocins obtained after L. curvatus CRL705 growth in MRS medium may be



531 used to be adsorbed to multilayer-LLDPE films offering a promising and simple



532 alternative for anti-Listeria packaging development.



533



534 FUNDING SOURCE



535 This study was supported by funds from 3iA-UNSAM-2006 from Argentina.



536



537 ACKNOWLEDGEMENTS



538



The authors would like to acknowledge the technical assistance of INTI-Carnes,



539 Beatriz De Rito, Mariela Giberti, Vanesa Molina and Gabriel Ybarra.



540



22



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683 684



685



29



686 Figure legends 687 688 Figure 1. FTIR spectra for the different adsorbates. For better comprehension the 689 Figure has been divided in spectral zones. 690 691 Figure 2. Inhibition areas exerted by multilayer-LLDPE film treated with 0.5; 1; 2; 3; 4; 692 5; 6; 8 µg ml-1 of S-705. 693 694 Figure 3. Active lactocin 705 concentration changes during 120 min at (●) 20, (▼) 30 695 and (■) 40 ºC. (a) S-705 solution; (b) S-705 solution contacted with multilayer-LLDPE 696 film; (c) active lactocin 705 mass adsorbed on the multilayer-LLDPE film and (d) 697 multilayer-LLDPE film 705 antimicrobial activity on semisolid agar. Continuous lines 698 mark tendencies. Error bars indicate standard deviations 699 700 Figure 4. Lactocin 705 adsorption isotherm on the multilayer-LLDPE film food contact 701 face at 30 ºC. The curve drawn through the data follows Langmuir equation [2] 702 (R=0.9399). Error bars indicate standard deviations. 703 704 Figure 5. Lactocin 705 adsorption on the multilayer-LLDPE film at 30 ºC from (■) S705 705 alone; in the presence of (▼) P-AB (2000 AU ml-1) and (●) C-Sac 7 (1 mg ml-1). 706 The curves drawn through the data follow Langmuir equation [2]. Error 707 bars indicate standard deviations 708 709 Figure 6. Antilisterial bacteriocin/s (AB) adsorption on the multilayer-LLDPE film at 710 30 ºC from (●) P-AB alone; (▼) in the presence of S-705 (6400 AU ml-1) and (■) from

30



711 C-AB. The curve drawn through the data follows Langmuir equation [2]. Error bars 712 indicate standard deviations 713 714 Figure 7. Scheme to allow visualization of bacteriocins adsorption from different 715 sources. (a) lactocin 705 from S-705; (b) 705 in the presence of impurities (Imp); (c) 716 AB in the presence of Imp and (d) 705 in the presence of AB and Imp. X and white 717 parts of the graphs denote respectively hydrophilic and hydrophobic parts of the 718 peptides. For a detailed explanation see text. 719

31



Table



Table 1. Used adsorbates and their source



Adsorbates



Source a



Producer microorganisms



Preparation method



Lactocin 705,

antilisterial CE

bacteriocin/s and

impuritiesb



antilisterial bacteriocin/s and C-AB impuritiesb



Metabolites



C-Sac7



L. curvatus CRL705

L. curvatus CRL1579

Sac7 strain



An overnight culture of the



producer microorganism was



centrifuged (2500 g, 15 min);



the



supernatant



was



precipitated using 0.44 g cm-3



ammonium



sulphate,



centrifuged (20000 g, 20 min)



and freeze-dried (Blanco



Massani et al., 2008).



Lactocin 705



S-705



Lac705α and lac705β peptides were synthetized according to Palacios, Vignolo, Farías, Ruiz Holgado, Oliver, & Sesma (1999) and Cuozzo et al. (2000).



antilisterial bacteriocin/s and impuritiesb



P-AB c



L. curvatus CRL1579



C-AB was applied to a solid phase extraction cartridge (C18) as earlier described (Blanco Massani et al., 2008) and freeze-dried.



a CE, crude extract; C-AB, concentrated antilisterial bacteriocin/s; C-Sac7, concentrated impurities from

Bac- variant; S-705, synthetic lactocin 705; P-AB, purified antilisterial bacteriocin/s. b Impurities include MRS medium components and bacterial metabolites c P-AB has lower impurities content than C-AB



Table



Table 2. Combination of adsorbates used in adsorption tests



Adsorbate I (source)



Adsorbate II (source)



Study developed



Sensitive strain



Lactocin 705, 27 to 4815 AU ml-1 (S-705)



-



Lactocin 705 adsorption



Impurities (C-Sac7, 1 mg ml-1)



Impurities influence on 705 adsorption



AB influence on 705 2000 AU ml-1 AB (P-AB a)

adsorption



L. plantarum CRL691



AB, 27 to 8717 AU ml-1 (P-AB a)



6400 AU ml-1 lactocin 705 Lactocin 705 influence on



(S-705)



AB adsorption



AB, 27 to 8717 AU ml-1 (P-AB a) compared to AB, 27 to 8717 AU ml-1 (C-AB)



Impurities influence on AB adsorption



L. innocua 7



a P-AB has lower impurities content than C-AB, but both adsorbates had the same AB titer.



Table



Table 3. Tentative assignment of FTIR bands obtained for the different adsorbates, following (Quinteiro Rodriguez (2000), Barth (2000), Maquelin et al. (2002) and Motta et al. (2008).



Frequency (cm-1) CE C-Sac7 C-AB P-AB S-705

3447 3428 3443 3466 3470



Bibliografy frequency



Possible assignmenta



~ 3500



O-H stretch of OH- groups



3215 3203 3207 3237 3280



3200



2925 2926 2926 2927 -



2920



2871 2872 2872 2871 -



2870



N-H stretch (amide A from proteins)

CH2 asymmetric stretch (fatty acids)

CH3 symmetric stretch (fatty acids)



1740 1740 1743 1743 -



1740



C=O stretch of esters (fatty acids)



1640 1636 1635 1633 1654



1700-1610 amide I (proteins)



1520 1539 1538 1524 1530



1550-1520 amide II (proteins)



1450 1450 1450 1446 -



1450



C-H def in aliphatics (fatty acids)



1400 1400 1400 -



-



1400



C=O symmetric stretch of COO-



1200b -



-



-



1438, 1200, 1122



1438, 1200, C-N, C-O vibrations from amino



and 1122



acids side chains



√



√



√



√



-



900-1200



C-O, C-C stretch and C-O-H, C-O-C def (glycopeptides, phosphodiester, polysaccharides)



a Stretch, stretching; def, deformation. b band at 1439, included in band observed arround 1450, band at 1198 and 1122 cm-1 are included in bands between 900-1200 cm-1 (See Fig. 1).



Table



Table 4. Antimicrobial activity of the multilayer-LLDPE film contacted (1 h, 30 ºC) with different adsorbates combination. Values in a column followed by different uppercase letters are statistically different (P<0.05)a.



Adsorbate I



Adsorbate II \_



Relative inhibition area exerted by



lactocin 705 a AB a



3.7A ± 0.1



\_



C-Sac7 (0.1 mg ml-1)



3.2B ± 0.3



\_



S-705



C-Sac7 (1 mg ml-1)



(6400 AU ml-1 lactocin



705)



C-Sac7 (20 mg ml-1)



2.6C ± 0.3



\_



1.6D ± 0.3



\_



C-Sac7 (40 mg ml-1)



1.1D ± 0.5



\_



P-AB (12800 AU ml-1 AB) 3.0B ± 0.3



CE (6400 and 12800 AU ml-1, respectively 705 and AL705)



1.4D ± 0.4



2.1A ± 0.2 2.3A ± 0.2



P-AB (12800 AU ml-1 AL705)



\_



C-AB (12800 AU ml-1 AL705)



\_



S-705 (6400 AU ml-1 705) b



C-Sac7 (40 mg ml-1) b



1.5 D ± 0.1



C-Sac 7 (40 mg ml-1) b S-705 (6400 AU ml-1 705) b 3.3 A ± 0.4



a Mean of four replications ± standard deviation b sequential adsorption (See materials and methods)



2.1A ± 0.2 2.2A ± 0.1 \_ \_



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