Efficient chemical hydrophobization of lactic acid bacteria – One-step formation of double emulsion
Graphical abstract
Introduction
Emulsions are one of the most important food structures, because the final products possess favorable sensory properties such as texture, flavor and appearance. Due to the kinetic and thermodynamic instability of emulsion, surfactants or emulsifiers are required to prevent destabilization during long time storage (Dickinson, 2010b, Nushtaeva, 2016, Tavernier et al., 2016). Single W/O or O/W emulsions can be created using predominantly hydrophobic or hydrophilic stabilizers, while the combination of stabilizers with complementary hydrophilic-lipophilic balance values can produce double emulsions, where the compartmentalized primary dispersions contain even smaller droplets of a different phase (Bhattacharjee et al., 2018, Dickinson, 2011, Dickinson, 2015).
Besides conventionally low molecular emulsifiers, emulsions can also be stabilized by adsorbed fine solid particles, which is referred to as Pickering stabilization (Dickinson, 2010a, Murray et al., 2011). Pickering stabilization is able to produce foams and emulsions with high stability as the desorption is considered to be impossible due to much higher desorption energy than thermal energy (Dickinson, 2015, Hua et al., 2016, Jin et al., 2012). Microorganisms such as bacteria (Dorobantu, Yeung, Foght, & Gray, 2004), yeasts (Firoozmand & Rousseau, 2015) and viruses (Kaur et al., 2009, Russell et al., 2005) have exhibited their ability of stabilizing foams and emulsions. Although applications were mainly reported in non-food area (Heard, Harvey, Johnson, Wells, & Angove, 2008), recent efforts have been made to utilize food grade microorganisms for developing food foams and emulsions (Firoozmand and Rousseau, 2015, Rayner et al., 2014).
Lactic acid bacteria, as important constitutional and nutritional components in dairy products, demonstrate the potential to serve as also structural building blocks based on Pickering principles. While certain strains showed their inherent surface activity as Pickering particles for emulsions and gels (Dorobantu et al., 2004), most lactic acid bacteria as Gram positive bacteria, still exhibit a dominantly hydrophilic nature attributed to large presence of peptidoglycan with a high ratio of polysaccharides to hydrocarbons (Boonaert and Rouxhet, 2000, Chapot-Chartier and Kulakauskas, 2014, Schär-Zammaretti and Ubbink, 2003). Hence, modification is necessary to alter their physiochemical properties towards suitable interfacial materials. Biologically, fermentation conditions like media composition (Schär-zammaretti et al., 2005), growth time (Rosenberg & Rosenberg, 1985) and temperature (Deepika, Karunakaran, Hurley, Biggs, & Charalampopoulos, 2012) can change the chemical composition and cell surface properties. Physical coating of bacteria with oppositely-charged chitosan (Wongkongkatep et al., 2012) and milk proteins (Falco, Geng, Cárdenas, & Risbo, 2017) was also capable of modifying the surface charge and cell hydrophobicity.
Chemical hydrophobization has been previously reported for polysaccharide nanoparticles like starches (Balic et al., 2017, Neelam et al., 2012, Yusoff and Murray, 2011), celluloses (Jin et al., 2012) and chitosans (Fink, Höhne, Spange, & Simon, 2009) using carboxylic acid derivatives. The mechanism is that the hydrophobic chains of these chemicals covalently condense with hydroxyl or amine groups on the surface of polysaccharide particles through esterification or amidation (Ačkar et al., 2015, Cunha and Gandini, 2010). Considering the large presence of amino groups and hydroxyl groups in bacterial cell wall peptidoglycan, their surface can be potentially modified with the similar principle. Yet, studies on the chemical modification of bacterial cell surface are very limited. Recently, the surface hydrophobicity of lactic acid bacteria Lactobacillus acidophilus (La5) was increased using octenyl succinic anhydride, and the modified bacteria were able to stabilize foams and emulsions (Jiang, Falco, Dalby, Siegumfeldt, Arneborg, & Risbo, 2019).
The present study aims to develop an efficient approach to chemically modifying the surface of lactic acid bacteria Lactobacillus rhamnosus (LGG) in a non-aqueous environment. The idea is that by using lauroyl chloride (LC), the hydrophilic bacteria can be hydrophobically modified through connecting the alkyl chains of LC to bacterial surface functional groups. Surface properties of the bacteria were evaluated using microbial adhesion to hexadecane (MATH), water contact angle (WCA) measurement, zeta potential measurement and the observation of bacterial aggregating behavior. Finally, unmodified and modified bacteria were used for emulsion preparation and droplet size measurement, optical microscopy and confocal microscopy were applied to characterize the produced emulsion.
Section snippets
Materials and chemicals
Lauroyl chloride (LC), glycerol, dimethyl sulfoxide (DMSO), hexadecane, sodium chloride (NaCl), potassium chloride (KCl), disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), TWEEN® 80, sodium carbonate (Na2CO3) and 40,6-Diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich, Steinheim, Germany. BODIPY™ 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) was bought from ThermoFisher Scientific, Molecular Probes, Eugene, OR, USA.
Culturability of modified bacteria
The effect of lyophilization and LC modification on bacterial culturability was investigated using plate counting method (Fig. 1). An instantaneously negative effect of lyophilization on culturability was not observed for unmodified bacteria based on the unchanged culturability before and after lyophilization (Fig. 1A). Likewise, no obvious decrease in culturability was observed for bacteria immediately after LC modification regardless of LC concentration (Fig. 1B). A similar observation was
Discussion
In the present work, an efficient scheme for modifying surface properties of lactic acid bacteria was presented. The method was based on reaction with acid chlorides in non-aqueous media to ensure solubility of the long chain acid chloride and avoid unwanted side reaction into free carboxylic acid. The formed HCl was neutralized by suspended insoluble particles of NaCO3. When remediated back into aqueous solvent, the bacteria could be characterized and used for stabilization of emulsions. The
Conclusion
Chemical modification of lactic acid bacteria LGG with LC was achieved efficiently in a non-aqueous bacteria-friendly environment. Significant improvement in terms of cell hydrophobicity was demonstrated by larger WCAs and higher bacterial adhesion to hexadecane, with the improved surface properties stable over one month. Lyophilization posed a time-hidden effect on the bacterial culturability over two-week storage while no instantaneous effect was observed after modification. Consequently, all
CRediT authorship contribution statement
Xiaoyi Jiang: Conceptualization, Investigation, Methodology, Formal analysis, Writing - original draft. Elhamalsadat Shekarforoush: Conceptualization, Methodology, Writing - review & editing. Musemma Kedir Muhammed: Methodology, Writing - review & editing. Kathryn Whitehead: Writing - review & editing. Adam Cohen Simonsen: Resources. Nils Arneborg: Writing - review & editing, Supervision. Jens Risbo: Conceptualization, Writing - review & editing, Supervision, Project administration, Funding
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The research leading to these results has received funding from The Danish Independent Research Foundation under framework grant n° 8022-00139B and financial support of Chinese Scholarship Council (CSC) grant n° 201807940009.
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2022, Food Research InternationalCitation Excerpt :There are different routes to alter the physicochemical properties of the cell wall of LAB such as changes in the fermentation conditions (Schär-Zammaretti et al., 2005, Deepika et al., 2012, Rosenberg and Rosenberg, 1985), adsorption of macromolecules such as polysaccharides, polyphenols, and proteins (Schär-Zammaretti and Ubbink, 2003, Morata et al., 2003, Falco et al., 2017) to the surfaces of LAB, which is based on the electrostatic attraction between the oppositely charged macromolecules and bacterial cell surfaces. The hydrophobicity of LAB has also been increased by chemical modification of the bacterial cell wall using carboxylic acid anhydrates (Jiang et al., 2019) and acid chloride (Jiang et al., 2021). Cell lysis enzymes, such as lysozyme, lysostaphin and proteinase K degrade the cell wall components and are used to release the inter-cellular materials such as DNA, RNA, protein, or organelles (Shehadul Islam et al., 2017, Lavigne et al., 2004, Salazar and Asenjo, 2007, Giovannoni et al., 2020).