Sylvain Caillol
email: sylvain.caillol@enscm.fr

Cardanol: a small giant of the future of biobased aromatic polymers and additives

Keywords: Cardanol, cashew nut shell liquid, biobased polymers, surfactant, plasticizer

Among the renewable resource materials, cashew nut shell liquid (CNSL) is considered as an important starting material due to its unique structural features, abundant availability and low cost. A large number of chemicals and products have been developed starting from CNSL by taking advantage of the three reactive sites, namely, phenolic hydroxyl, aromatic ring and unsaturation(s) in the alkenyl side chain. Increasing attention is paid to promising cardanol-based products that could be have potential interest in industry, such as epoxy and acrylic monomers, plasticizers and surfactants.

INTRODUCTION

The use of renewable resources for chemistry and the manufacture of materials represents a very important subject, and has been attracting the growing attention of academic and industrial communities for several years 1 . This development is due to the volatility of petrochemical prices associated with growing environmental concerns.

Renewable raw materials are already widely used such as natural rubber, cellulose but also vegetable oils and proteins as well as chitin or other polysaccharides. One of the challenges of chemistry derived from renewable resources consists in particular in identifying aromatic resources to replace phenol and provide mechanical and thermal resistance properties to biosourced polymers. The use and choice of renewable resources is closely linked to their availability and the impacts generated for their development. This is notably why access to aromatic resources is an issue 2 . Indeed many research studies have focused on the use of lignin or its derivatives such as vanillin, or eugenol or tannins, but in general the technological access routes are not mature, the processes are pollutants or availability is limited. There is an aromatic resource which has a certain future, it is cardanol and its derivatives. Cardanol comes from Cashew Nutshell Liquid CNSL, co-produced during of cashew nuts industry. CNSL being a non-edible oil, its use as a chemical raw material does not potentially put undue pressure on the food supply chain unlike other renewable chemical feed-stocks such as edible oils. Moreover, as a co-product, its environmental assessment is very favorable.

CNSL appears as a liquid greenish yellow viscous substance present in the thickness of the cashew nut shell. Cashew is a product of the cashew tree, Anacardium occidental [START_REF] Tyman | Synthetic and Natural Phenols[END_REF][START_REF] Harvey | [END_REF] , native to Brazil and abundantly available in many parts of the world, such as Brazil, India, Bangladesh, Benin, Tanzania, Kenya, Mozambique, tropical regions of Africa, Southeast Asia and the Far East. In some of these countries, cashew nuts are a popular agricultural product, while some others import cashew nuts for processing. CNSL belongs to the family of long-chain hydrocarbon phenols which are phenolic lipids found in plants of these families such as Anacardiaceae 5 . The plant can reach 12 m in height. Cashew nuts are the cash crop produced by the tree, but it also produces cashew apples (edible), a gum similar to gum arabic, and the wood is used for building boats or making charcoal. The cashew apple is used in drinks, jams and jellies. Cashew apple juice has anti-tumor and antimicrobial properties 6 . The cashew nut is greenish gray and is attached to the cashew apple, which when ripe turns bright yellow or red. The pericarp of the nut consists of a leathery epicarp, a spongy mesocarp and an endocarp. The core is inside the shell and is covered by a brown skin called the testa. The nut is kidney-shaped and its size varies from 2.5 to 4 cm. CNSL constitutes 18-27% of the total weight of raw nuts.

Traditionally, a number of methods have been used to extract CNSL from cashew nuts.

Two processes are mainly used in the extraction of CNSL: the hot oil process and the roasting process in which the CNSL oozes out of the shell [START_REF] Tyman | Synthetic and Natural Phenols[END_REF][START_REF] Manjula | [END_REF] . Oil extraction uses solvents such as benzene, toluene, petroleum hydrocarbons or alcohols 9 . Other techniques are also considered, such as the use of a mixture of supercritical CO 2 and isopropyl alcohol 10 .

The presence of sulphides, nitrogenous materials and minerals contained in the crude CNSL directly affects the quality of the oil. CNSL is therefore often subjected to a preliminary treatment with compounds such as hydrocarbon sulphates and sulfuric acid before industrial use. Aqueous solutions of acids such as hydrochloric acid, sulfuric acid, acetic acid, chloroacetic acid or phosphoric acid, or acid sulfates, such as sodium hydrogen sulfate, are used to this effect. This produces two main effects: the precipitation of the mineral salts of ammonium, calcium and potassium as well as the reduction of the content of cutaneous vesicant present in the CNSL such as urushiol. The chemical treatment is accompanied by the release of hydrogen sulphide. It has been found that the reduction in sulphide content roughly corresponds to the elimination of the causative agent of the skin irritation. Treatment with amines or Group IA or II metal hydroxides has been identified to reduce cardol content.

Refined CNSL can be easily distilled under reduced pressure or hydrogenated, both processes leading to products with good color stability. Steaming of CNSL treated with H2SO4, followed by distillation, has been found to deodorize the oil to a great extent 11 .

During extraction or refining processes, the anacardic acid present in CNSL is decarboxylated to cardanol 12 .

In nature, CNSL contains four main components: anacardic acid, cardanol, cardol and 2methyl cardol. They are all mono-phenols or di-phenols with an aliphatic chain in the meta position. Figure 1 shows the chemical structures of these compounds. Commercial-grade CNSL contains virtually no anacardic acid due to the decarboxylation reaction occurring during the roasting process, which converts anacardic acid to cardanol 13 . CNSL components are themselves mixtures of four constituents differing in the number of side chain unsaturations. Indeed this chain can be saturated (5-8%), or in the form of monoene (48-49%), diene (16-17%) or triene (29-30%) 14 . The composition of natural and technical CNSLs has been studied by several researchers 15,16,17,18,19,20 . Using primarily chromatographic techniques, Tyman et al. 21,22 reported the presence of oligomers and polymers in natural and engineered CNSLs. The composition of natural and technical CNSL 23 is given in Figure 1. Cardanol is very difficult to obtain according to conventional routes of chemical synthesis insofar as the aliphatic side chain is in the meta position with respect to the phenol group which generally directs all the substitutions in ortho and/or para positions. Cardanol lends itself to many functionalizations to lead to specific properties of interest for high additives or value-added polymers. Hence, there is more and more academic work on cardanol including to develop products for industrial applications. Additionally, several review papers 24,25,26,27,28 have already been reported. However, all these works do not have the same approach or the same maturity, which is why this perspective article proposes to select the most promising works from cardanol.

CARDANOL FOR CHEMISTRY

Cardanol has intrinsic antioxidant and antimicrobial properties. It is thus used for these properties but also as an additive in many formulations (surfactants, etc.). Currently, cardanol is mainly used in friction particles [START_REF] Dai | Patent cardolite WO[END_REF] . However, the structure of cardanol is unique for many reasons which are linked to the properties provided or to the possible polymers obtained thereof. Hence, cardanol-derived polymers have exceptional properties that are unique for many applications (Figure 2). Polymers derived from [START_REF] Blazdell | [END_REF]31 exhibit flexibility due to "internal plasticization" resulting from the presence of a long side chain which also exhibits hydrophobicity towards materials. Polymer derived from cardanol have also high thermal, chemical even fire resistance due to the presence of the aromatic ring. The phenol groups entails fast curing reaction and adhesion properties. The presence of polar phenol group and long alkyl chain leads to a "surfactant" effect that could also be interestingly used as compatibilizer in formulations 32,33 . Cardanol is also compatible when mixed with a wide variety of polymers such as alkyds, melamines, polyesters… allowing homogeneous formulations to be obtained. Structural modifications can be made at the hydroxyl group, on the aromatic ring and on the side chain (Figure 2). During extraction or refining processes, anacardic acid decarboxylates to cardanol by simple heating (Figure 3, d) 34 . Hydrogenation (Figure 4,e) is usually carried out using hydrogen and catalysts such as copper, nickel, palladium or platinum 35 . Hydrogenated cardanol can be separated by distillation, as for crude CNSL. Perdriau et al. 36 performed an interesting conversion of CNSL polyenes to monenes by RuCl3-catalyzed transfer hydrogenation. CNSL derivatives can also be sulfonated to give alkylarylsulphonic acids or their metal salts (Figure 3, f) 37 . Direct nitration of hydrogenated cardanol leads to nitrate derivatives, which are very useful products (Figure 3, g) 38 halogenation can be accomplished very easily. For example, chlorine gas can be passed through a solution of CNSL in kerosene to achieve 15% (mass) chlorination 39 . The reaction of cardanol with acid chlorides in the presence of a base can lead to the corresponding cardanyl esters (Figure 3, h) 40 . The epoxidation of cardanol (Figure 3, i) can be carried out using epichlorohydrin in the presence of caustic soda as a catalyst 41 . Finally etherification has also been reported with various alkyl halides (Figure 3, j) 42 . Various techniques can also be used to polymerize CNSL to obtain various resins. The presence of the aliphatic side chain gives these resins a pronounced hydrophobic character, which is a valuable property for many applications such as resistance to polar solvents.

CARDANOL IN ADDITIVES

Cardanol is widely used for the synthesis of many additives, such as antioxidants 43,44 , doping agents 45,46 or rubber additives 47,48 . The most dynamic fields remain that of plasticizers and surfactants.

Plasticizing agents

Poly (vinyl chloride) (PVC) is one of the most widely used polymers in the world due to its great versatility, adaptation to many additives, good mechanical properties and low cost production. Annual PVC production reached around 60 million tons in 2020. Applications range from very rigid PVC (window profiles, computer cases, outdoor products, containers, blisters) to soft PVC using certain additives, such as plasticizers, with a mass ratio between 30 and 50%. Soft PVC formulations can be used in the medical field, for packaging materials, artificial leather and toys... 49,50 . Over the past decade, global production of plasticizers has been around 6.4 million tonnes per year. More than 80% of these additives are phthalates with long alkyl chains such as diisononyl phthalate (DINP) or bis(2-ethylhexyl) phthalate (DEHP). Thanks to their low cost and good performance with PVC, these additives are produced in large quantities. However, the reprotoxicity of phthalates on animals and humans is reported and some of them have recently been banned by European regulations 2000/60/EC and European regulations 1907/2006/EC 51 . During the last ten years, many studies have been devoted to the synthesis of cardanol-based plasticizers.

Indeed, due to its aromatic ring providing thermal stability and its C15 alkyl chain which can confer a plasticizing effect, cardanol is a promising substituent for phthalates. The phenol esterification of cardanol is necessary to obtain good miscibility of cardanol in PVC. Cardanol esterified with short alkyl chains have already shown good results as secondary plasticizers in the presence of DEHP 52 . Epoxidation of the double bonds further improves the plasticizing properties and thermal stability of PVC 53 . Furthermore, Chen et al. studied cardanol-based epoxidized plasticizers using a mild synthetic route 54,55 . The same team studied the internal plasticization of PVC after the partial modification of PVC by the Mannich base of cardanol 56 . Recently, cardanol derivatives epoxidized and modified with various fatty acids have demonstrated very relevant PVC plasticization properties, similar to those obtained with DINP (Figure 4). In addition, these plasticizers derived from cardanol did not demonstrate toxicity 57 . Their biodegradability has not been studied, however, the easily hydrolysable aromatic ester bond makes it easy to return to the starting reagents which are natural compounds entering natural metabolic cycles. In addition, cardanol has made it possible to develop plasticizers for polymers other than PVC. Thus, the properties of natural rubber modified with phosphorylated cardanol were found to be superior to those obtained with DEHP 58 . Additionally, phosphorylated cardanol has demonstrated properties as an effective plasticizing agent for EPDM (ethylenepropylene-diene-monomers) rubber copolymers 59 , polychloroprene and polybutadiene rubber 60 , and LLDPE/EVA copolymer blends.

(linear low density polyethylene/ethylene-vinylacetate) 61 . The use of CNSL in polyaniline-based conductive composites has also been reported 62 . The CNSL has thus improved the processability of blends of SBS (styrene-butadiene-styrene), polyaniline and dodecylbenzenesulfonic acid copolymers.

Surfactants

A recent review reports the uses of cardanol for surfactants synthesis 63 . Hence, cardanol has been widely studied for the elaboration of various surfactants, such as cationic surfactants 64,65 , nonionic 66,67 and anionic surfactants 68,69 . There are two main structures of cardanol-based anionic surfactants, one with sulfonate directly on the aromatic ring, and one with aromatic grafted on phenol. Similarly, two main structures of cationic cardanol-based surfactants have been reported, either with alcoolate or carboxylate in the molecule, or with a halogenated counterion (Figure 5). Peungjitton et al. 70 synthesized sodium cardanol sulfonate and found similar properties than dodecylbenzene sulfonate. Thus, cardanol sodium sulfonate can be used as a raw material for the commercial production of detergents. All these works reported that cardanol is easily sulfonated with excess sulfuric acid in halogenated solvents at room temperature 71,72,73 . It has been demonstrated that cardanol easily reacts with ethylene sulphate, to yield sulphonated derivatives which have excellent surfactant properties 74 .

The use of ethylene sulfate with the phenoxide interestingly avoids both the reaction with sulfuric acid and initial hydroxyethylation step. Moreover, several cardanol-based amphiphilic molecules have successfully studied for many industrial applications 75,76,77,78 . Cardanol-derived amphiphilic azobenzenes generated stable emulsions 79,80,81 .

Cardanol-based sodium-5-n-pentadecylphenol 2,4-disulfonate, allowed micellization, and the authors reported that the presence of multiple CH 2 groups was important for micelle formation 82,83 . The presence of the phenoxy group in addition to the long alkyl hydrocarbon chain significantly influences the hydrophobic character of these new amphiphilic molecules. Tyman et al. 84,85 reported the synthesis of cardanol and cardol polyethoxylates. Cardanol polyethoxylate and its saturated analogue demonstrated similar surfactant properties. Carboxylate derivatives of cardanol and anacardic acid have also been prepared and tested as anionic surfactants 86 . They lower the surface tension, have a critical micellar concentration and produce microemulsions when mixed with dodecyl sulphate. Cationic surfactants containing imidazolium and pyridinium units have been synthesized from cardanol 87,88,89,90,91 . In 2017, Faye et al proposed a platform approach to allow the synthesis of cardanol-based anionic, cationic and zwitterionic surfactants 32 .

CARDANOL IN POLYMERS

Cardanol has intrinsic antioxidant and antimicrobial properties. It is thus used for these properties but also as an additive in many formulations. Polymers derived from CNSL have some exceptional properties that make them unique for many applications 92,93 . Hence these cardanol derived polymers exhibit flexibility due to "internal plasticization" resulting from the presence of a long alkyl chain which also imparts hydrophobicity to the materials. Cardanol is also compatible in mixtures with a wide variety of structures such as vegetable oils, natural phenols or polymers such as alkyds, polyesters, etc., allowing homogeneous formulations to be obtained 63,94 .

Polymers obtained by step polymerization (polyaddition and polycondensation) constitute one of the most important classes of thermoplastic materials used in industry. Most of these polymers are obtained from difunctional monomers derived from fossil fuels. Cardanol was used as a starting reagent for the synthesis of polycondensation monomers. Various difunctional monomers such as diamines, diacids, diols, dialdehydes, diacylhydrazide, etc. were synthesized from cardanol 95,96,97,98 . These difunctional monomers have been used for the synthesis of a wide variety of high performance polymers such as aromatic polyimides, polyamides, poly(azomethine), poly(amideimides), poly(esterimides), polyesters, polyhydrazides, polyoxadiazoles, etc.. The presence of the C15 alkyl chain along the polymer backbone disrupted the organization of the polymer chains, and provided additional sites for interaction with solvents, thus improving the solubility of these polymers compared to reference polymers. The cardanol-derived polymers also exhibited lower Tgs than the reference polymers, attributed to the presence of pentadecyl chains which decrease intermolecular interactions and increase the segmental movement of the polymer chains through an "internal plasticization" effect.

Moreover, cardanol was used in the synthesis of various monomers for the synthesis of various polymers. Indeed, cardanol allowed to synthesize aromatic monomers such as benzoxazines 99,100 or Mannich-based polyols 101 for highly crosslinked polyurethanes. Cardanol was also used for the synthesis of BPA substitutents in vinyl esters 102,103 , cyanate esters 104,105 . Additionally, recent papers reported its use in hybrid and flame retardant alkyd resins 106,107 . Recently, interesting biobased latex have been synthesized by homo-or co-polyerization of cardanol-based acrylates with petro-or bio-based comonomers in direct or mini-emulsions 108,109,110 .

The phenolic and the epoxy networks constitute the most dynamic domain of cardanol-based use in polymers, including for industrial products.

Phenolics

The phenol group in cardanol allows it to react with formaldehyde under various conditions to form resole or novolac-like resins 111,112,113 . Compared to conventional phenolic (phenol-formaldehyde) resins, cardanol-formaldehyde resins have greater flexibility (due to the "internal plasticizing" effect of the C15 side chain). The side chain allows a higher processability and gives also a more hydrophobic character to the network, more resistant to erosion. These polymers have also demonstrated a very low loss of efficiency during friction, which makes them very interesting polymers as additives for the formulation of organic materials for brakes 114 . In order to reinforce the cardanol-formaldehyde resins, natural fibers have been added such as ramie, flax, hemp or jute fibers 115 . Several teams have studied cardanol phenolic resins, and particularly with the addition of various polymers. Hence, Chuayjuljit et al. 116 reported the synthesis of cardanol-formaldehyde resins for the reinforcement of natural rubber (NR). The aliphatic side chain of cardanol reduced the polarity of phenolic resins and, therefore, increased their compatibility with NR. Shibata et al. 117 synthesized thermosetting resins composed of cardanol-novolac and bismaleimide. Shukla et al. 118 synthesized a phenolic resin using cardanol and maleic anhydride and studied its reaction kinetics. Nanocomposite foams of cardanol phenolic resin and organomodified montmorillonite were prepared and the results showed that cardanol could reduce the crosslinking density of the phenolic foams while improving its mechanical properties 119 . Cardanol-based phenolic resins are particularly studied since cardanol could advantageously replace phenol that is a toxic substance classified carcinogenic, mutagenic, reprotoxic, hence subject to ban due to Reach regulations.

Epoxy resins

Some cardanol-based epoxy monomers are already commercialized by

Companies such as Cardolite NC-514 supplied by Cardolite® Corporation which led to epoxy resins with interesting properties (Figure 6). Jaillet et al., studied networks obtained by reaction between NC-514 and various diamines 120 . The structural characterization led to the conclusion that NC-514 was an oligomer mainly composed of diepoxy monomers, with a small percentage of unsaturated compounds. The presence of C15 alkyl chain, endowed plastifying properties that led to a low glass transition temperatures of the epoxy network. However, the reactivity of NC-514 was comparable to the one of Bisphenol A diglycidyl ether (BADGE). GX-2551 was also described 121 . This is a molecular cardanol-based epoxy synthesized by epoxidation of both phenol (with epichlorohydrin) and double bonds of alkyl chain (with hydrogen peroxide). The reactivity of internal double bonds is poor with amines, but the curing with anhydrids or acids led to high Tg, since the dangling chain is part of the crosslinked network (Figure 6). It is also possible to obtain a cardanol-novolac prepolymer from the condensation reaction of cardanol with formaldehyde 122 . Subsequently, the epoxidation is carried out with ECH in a basic medium. This novolac epoxy resin is also marketed by Cardolite® Corporation as NC-547 (Figure 6). It has a higher viscosity than NC-514 (between 20,000 and 50,000 mPa s) and a slightly lower density. The materials obtained with this epoxy resin are very flexible hydrophobic elastomeric materials, even at low temperatures. The tensile strength is lower and the Tg higher than that obtained with NC-514 due to the higher aromatic density of NC-547 123 . The reactivity is similar since it also contains glycidyl ether functions, and could possibly be fully cured at room temperature.

CONCLUSION

Cardanol is a major constituent of CNSL which is a by-product of the cashew nut industry whose current production exceeds 900kt per year. Cardanol, is a nonfood, cheap and abundantly available versatile industrial raw material of great promise for the replacement of certain products based on fossil resources. The presence of phenolic hydroxyl group and olefinic linkages in the C15 alkyl chain, combined with the aromatic ring makes cardanol an interesting starting material for the synthesis of

Figure 1 :

 1 Figure 1: chemical structure of cardanol

Figure 2 :

 2 Figure 2: Reactive site and properties of cardanol

Figure 3 :

 3 Figure 3: functionalization routes of cardanol

Figure 4 :

 4 Figure 4: Cardanol-based PVC plastcizers

Figure 5 :

 5 Figure 5: Generic structures of main CNSL-based surfactant a) anionic, b) cationic, c) non-ionic

Figure 6 :

 6 Figure 6: Commercialized cardanol-based epoxy monomers

  various value added products by making use of reactivity of each of these functionalities. Hence, dynamic research is developed by several academic teams[98]. In industry Cardolite Corporation (Bristol, PA, USA) is the world's leading developer, manufacturer and marketer of CNSL products124,125 . Palmer International is one of CNSL's leading suppliers in North America. In India, several companies are involved in the production of CNSL and derivative products such as: Satya Cashew Chemicals Ltd., Adarsh Industrial Chemicals, Akshar Cashew Industries… Since the beginning of the 2010s, new players have appeared such as Senesel in Poland or Orpia Innovation in Montpellier, France.Currently, the most promising products from cardanol are epoxy monomers, phenolics, PVC-plasticizers and emulsion surfactants. But very interesting development recently gained attention, such as the work of Lemaire et al. who succeeded in the synthesis of 3-pentadecylcyclohexanone, which has structural similarity with the cyclohexanone, a very important building block of the petro-based chemical industry 126 . Thus, it is only a question of time and the demand for renewables until CNSL-based products would find widespread applications especially for the production of polymers with a unique combination of properties.

  . CNSL

	Esterification, allylation, epoxydation, alkylation, ethoxylation, propoxylation, phosphorylation…	Hydrogenation, hydrosylilation, metathesis, epoxidation…
	Surfactant effect
	Fast cure, adhesion
	Thermal and chemical resistance
	Condensation, bromination, nitration hydrogenation, amination, Mannich reaction…	Water resistance, flexibility, low viscosity, surface tension


M Sahihi

A K Bordbar

Y Ghayeb

N Fani

Structure-function relationship of β-lactoglobulin in the presence of sodium dodecylbenzenesulfonate

Keywords:

Bovine β-lactoglobulin (β-lg) present in milks has been found "in vivo" in complexes with lipids such as butyric and oleic acids. To elucidate the still unknown structure-function relationship in this protein, the structural changes of β-lactoglobulin variant A (β-lg A) were investigated in the presence of sodium dodecylbenzenesulfonate (SDBS) as an anionic surfactant using spectrofluorimetry. Subsequently, the retinol binding was investigated by β-lg in the presence of various amounts of this surfactant as its extrinsic functional binding fluorophore. The comparison of the results allowed for determining the binding of retinol by β-lg in the presence of SDBS.

The results of fluorescence studies showed a higher denaturating effect of SDBS at acidic pH that can be due to the positive charge density of β-lg at this pH which was calculated using the Henderson-Hasselbalch equation and pKa values of its ionizable groups. For each transition curve, the conventional method of analysis which assumed a linear concentration dependence of the preand post-transition base lines gave the most realistic values for Δ�Do(H2O). The value of about 21.6 kJ • mol -1 was obtained for Δ�Do(H2O) at various pH from transition curves. The results of retinol binding studies represented the substantial enhancement of retinol binding affinity of β-lg in the presence of this surfactant at various pH levels. Moreover, the obtained results confirmed that the β-lg/retinol binding was pH-dependent.

Highlights

► Stability parameters and retinol binding property of β-lg in the presence of SDBS have been determined at various pH. ► Higher denaturating effect of SDBS at acidic pH can be due to positive charge density of β-lg at this pH. ► SDBS enhances the retinol binding affinity of β-lg in all of its concentration range. ► The β-lg/retinol binding is pH-dependent.

Introduction

Surfactant is a group of amphipathic substance composed of both hydrophilic and hydrophobic groups. Surfactants can induce the unfolding of proteins and, in some special cases, stabilize proteins at very low concentration [START_REF] Ananthapadmanabhan | Interactions of Surfactants with Polymers and Proteins[END_REF]. They are also solubilizing agents for membrane proteins [START_REF] Ananthapadmanabhan | Interactions of Surfactants with Polymers and Proteins[END_REF]. Studies on protein-surfactant interactions have been carried out for many decades [START_REF] Jones | [END_REF].

The great number of recent studies, however, indicates that this issue continues to interest the researchers in academics [3], [4], [5], [6], [7], [8]. That is because, first, the heterogeneity of surfactant binding sites in protein imparts significant changes in the physicochemical properties of protein molecule, which reflects that solute, i.e. the protein-surfactant complex, and solvent structures are mutually modified. Second, more importantly, because (protein + surfactant) systems are qualitatively consistent with the biological membrane, the information with respect to the intermolecular protein-surfactant interactions may have important implications for the behavior of biological membrane. Recent studies on such systems have used NMR [9], SANS [10], fluorescence [4], [8], [11], [12], ESR [13], microcalorimetry [START_REF] Jones | [END_REF], [14], [15], light scattering [16], and other techniques for advancing the current understanding of protein-surfactant interactions.

However, the classical techniques of physical chemistry still remain relevant [14], [16], [17].

It is generally accepted that binding of ionic surfactant molecules to proteins disrupts the native structure of most globular proteins. This initially involves the ionic binding of surfactants to the ionic sites of the protein. Further binding occurs by hydrophobic cooperative interactions. Hence, ionic surfactants interact with proteins through a combination of electrostatic and hydrophobic forces [18]. It is well known that anionic surfactants can interact strongly with proteins, modify protein conformation and denature proteins [19], [20].

β-Lactoglobulin (β-lg) is a globular whey protein with the molar mass of 1.84 • 10 4 g • mol -1 and isoelectric point, pI, of around 5.2 [21], [22]. β-lg may play important functions in the binding and the transportation of hydrophobic ligands, such as retinoids, alkenes, and fatty acids, attracts more and more attention [6], [23], [24], [25], [26], [27], [28], [29]; also, its interactions with amphiphilic and hydrophobic ligands such as retinoids, long chain fatty acids and surfactants have been investigated by different methods. This small globular protein has a three-dimensional structure consisting of one α-helix and nine anti-parallel β-strands with eight β-sheets folded into a cone-shaped barrel forming a hydrophobic pocket [30]. β-lg is composed of 162 amino acid residues and has two disulfide bonds at the positions of Cys66-Cys160 and Cys106-Cys119 and one sulfhydryl residue at Cys121 in each molecule [START_REF] Hambling | Advanced Dairy Chemistry 1: Proteins[END_REF].

Interactions between amphiphiles (surfactants/emulsifiers) and β-lg are highly diverse and have received much attention because of their relevance to e.g. two-component food systems [START_REF] Dickinson | [END_REF], [33].

At the pH 7, the amphiphilic palmitic acid binds as a natural ligand in the central calyx according to heteronuclear NMR spectroscopy and fluorescence spectroscopy [34]. Affinity is mediated by hydrophobic interactions within the calyx with the head group protruding toward the solvent.

Therefore, ionic surfactants are thought to bind in a similar manner at low concentrations. At higher concentrations, ionic surfactants such as the cationic n-decyltrimethyl ammonium bromides (CnTABs) unfold the protein [4]. In our previous investigations, a series of systematic studies were done on the interaction of various surfactants on β-lg [3], [4], [5], [6]. The results represented the induction of cooperative unfolding in β-lg by some cationic surfactants. β-lg had conformational changes during its interaction with sodium n-dodecyl sulfate (SDS) and Triton X-100 while retinol binding properties of β-lg did not show significant changes in the presence of these surfactants.

However, the variation of structural index parameters of β-lg versus these surfactants concentration did not show sigmoidal feature so that no cooperative unfolding transitions were detected. The values of Δ�Do(H2O) as the quantitative measure of the thermodynamic stability of β-lg were extracted from the transition denaturation curves in the presence of cetylpyridinium chloride (CPC) and n-decyltrimethyl ammonium bromides (CnTABs). The results represented the increase in the denaturating power of surfactants with an increase in hydrocarbon chain length (n).

Moreover, the retinol binding affinity was measured in the presence of CnTABs. The relevant results represented the enhancement of retinol binding affinity in the presence of these surfactants.

To continue previous studies, the interaction of sodium dodecylbenzenesulfonate (SDBS) (scheme 1), as an anionic surfactant, with β-lg was investigated at various pH levels using the spectrofluorimetry technique. The results of this study not only determine the nature of forces in the interaction process but also provide the sufficient results for evaluating the accuracy and validity of the applied data analysis method. Moreover, the effect of electrostatic forces on the structure and stability of β-lg was investigated by running the measurement at various pH levels. SCHEME 1. Chemical structure of SDBS.

Material and methods

Materials

β-lg genetic variant A, retinol, and SDBS were purchased from Sigma Chemical Company and were used without further purification. The purities of these materials were ⩾90%, ⩾95%, and 98% for BLG, retinol, and surfactant, respectively. All salts used for buffer preparation were of analytical grade and were dissolved in double distilled water. All solutions were used freshly after preparation.

Methods

Fluorescence spectroscopy is used for studying the binding and conformational changes of proteins exploiting the intrinsic fluorescence of tryptophan (Trp) residues, which are particularly sensitive to the changes of their microenvironments [35]. During fluorescence measurements, the samples were put in the quartz cuvettes of 1 cm optical path length. In typical experiments, 2.0 mL of β-lg solution was placed into the cuvette. Emission spectra were recorded after each addition of SDBS stock solution (1 mM) at 298 K. The excitation was performed at 295 nm and the emitted light was recorded between 300 and 400 nm for SDBS binding studies. The observed fluorescence intensities were corrected for dilution. The band slits for excitation and emission were set at 5 nm.

To avoid the inner filtering effect, absorbance of the β-lg solutions in 279 nm should not exceed 0.1.

The binding of retinol was measured by following the differences of retinol fluorescence at λmax.

The following procedure was used during the titration of retinol solutions or various

[surfactant]/[retinol] solutions with β-lg: 1 mL of retinol solutions or various [surfactant]/[retinol] solutions was placed in a cuvette and small increments (0 μL to 100 μL) of the β-lg solution were injected in the cuvette. These spectra were recorded from 350 nm to 550 nm with an excitation wavelength of 325 nm. Spectral resolution was 5 nm for both excitation and emission wavelengths.

Results and discussion

Influence of various anionic surfactant concentrations on the structure of β-lg

When ionic surfactants are added to the β-lg solutions, the ionic surfactant monomers first bind electrostatically to the oppositely charged residues on the β-lg surface (site-specific binding) and this binding induces the expansion of the β-lg structure. This expansion allows for more hydrophobic interactions of the surfactant hydrophobic tails with the exposed β-lg non-polar interior (nonspecific, cooperative binding) sites. To investigate the influence of protein net charge on the denaturating power of SDBS, the net charge of β-lg was calculated using the net charge of its ionizable groups. Their net charge as a function of pH was calculated using the Henderson-Hasselbalch equation and their pKa values [36]. Figure 1 shows the β-lg net charge at different pH levels. The isoelectric point (the pH associated with zero net charge) was about 4.6 which was in good agreement with the experimental reported data [37]. The net charges of β-lg were about 17.8, -11.1, and -15.5 in the pH 2.0, 6.7, and 8.0, respectively. At the pH 2.0, below the pKa of aspartates and glutamates, β-lg was positively charged and could constitute the binding sites for anionic surfactant; so, it could form precipitates with oppositely charged surfactants because of the creation of neutral complexes between β-lg and anionic surfactant. During the further increase of concentrations of anionic surfactant, more surfactants were bound to the electrostatically neutral complexes, so the secondary complexes of β-lg/anionic surfactant gradually became negatively charged and the precipitate was dissolved. At the pH 6.7 and 8.0, β-lg was negatively charged and when anionic surfactant was bounded to β-lg, the β-lg/anionic surfactant complex was always negatively charged and the precipitation was not observed.

Denaturation of β-lg

As mentioned above at the pH 2.0, β-lg was positively charged, so it can form precipitates with oppositely charged surfactant SDBS because of the formation of neutral complex β-lg/SDBS. With the further increase of SDBS concentration, more anionic surfactant was bound to the already electrostatically neutral complexes, so the complex of β-lg/SDBS became negatively charged and the precipitate was dissolved. Therefore, upon the increase of SDBS concentration, the mixtures of β-lg/SDBS underwent a transition from homogeneous solution to precipitates, and then to another homogeneous solution. It should be noted that the transition curve was recorded after dissolving the precipitate at this pH. No precipitate was observed at the pH 6. The sigmoidal feature of the curves in figure 2 can be related to the characteristic of a cooperative transition. Similar sigmoidal transitions that have been also reported for thermal and chemical denaturation of β-lg [3], [38] can be taken as another reason for the cooperative denaturation of βlg by this anionic surfactant. It seems that this surfactant can induce an unfolding transition in the tertiary structure of β-lg. With respect to the position of the transition curves in figure 2, it can be judged that the denaturating power of surfactant in the pH 2.0 is greater than that of other pHs. As mentioned before, β-lg is positively charged at this pH and can constitute the binding sites for SDBS. It represents the significant role of electrostatic interaction in the process of surfactant binding.

The standard free energy of denaturation, Δ�Do, was calculated as a function of surfactant concentration by assuming a two-state mechanism [39], [40] and using equations ( 1), (2) [4], [38] where K is the equilibrium constant, FD is the fraction of denatured protein, R is the gas constant, yobs is the observed optical property and yN and yD are the optical properties of the native and denatured protein molecules, respectively. The two latter ones were extrapolated from the preand post-transition baselines to the considered denaturant concentration. These pre-and posttransition baselines were determined by a linear fit of the corresponding data before and after the transition according to the following equations:

Figure 3 shows the standard free energy of denaturation, Δ�Do (which was calculated from equation ( 2) based on the data in figure 2) varies linearly with surfactant concentration. Δ�Do was plotted against [surfactant] and a linear least-squares analysis was used to fit the Δ�Do and [surfactant] data to the following relation [41],

where Δ�Do(H2O) is the value of Δ�Do at 0 M denaturant and m is the slope of the curves which gives the linear dependence of Δ�Do on surfactants concentration.

Retinol binding of β-lg

The gradual increase in retinol fluorescence intensity was observed at various pH levels as β-lg concentration increased, which suggested that retinol transferred from the hydrophilic environment of the aqueous solution to a more hydrophobic environment.

Generally, complexes involving noncovalent bonds are reversible. For example, the binding of retinol to retinol binding protein is involved in the equilibrium between retinol and retinol-protein complexes [42]. Likewise, there may be equilibrium between free and β-lg-bound retinol. Song [23] who claimed that the binding of retinol to β-lg is pH-independent in the pH range of 2.0 to 7.5 but agrees with the fluorescence spectroscopy results reported by Dufour et al. [25]. In the case of this study, the obtained results show that the β-lg/retinol binding is pH-dependent. All the obtained data suggest the change of conformation and retinol binding properties of β-lg with pH.

The obtained values for Ka in the presence of SDBS at various pH levels are given in table 2. These values represent a large enhancement of retinol binding affinity of β-lg in the concentrations range of this surfactant located in the pre-transition state of denaturation curves (figure 2). Although, at all concentrations of surfactant, the tertiary structure of protein changes in such a manner that the retinol binding site of β-lg is more accessible and causes an increase in the retinol binding affinity of β-lg. Moreover, the Ka values in the presence of SDBS are lower than CnTABs [4]. Thus, it can judge conclude that, in comparison with SDBS, the increase of hydrophobic patches in the protein surface is more in the presence of CnTABs.

Conclusions

The studied surfactant denatured β-lg in a cooperative manner at various pH levels. The denaturation effect of this surfactant in the pH 2.0 was greater than that in other pHs because of the positive net charge of BLG at this pH and represented the predominate role of electrostatic interaction.

The values of about 21.92 kJ • mol -1 , 21.89 kJ • mol -1 , and 21.00 kJ • mol -1 were obtained at the pH 2.0, 6.7, and 8.0, respectively, for Δ�Do(H2O), as thermodynamic stability of β-lg in the absence of surfactant. These values were consistent with the previously reported data for the interaction of β-lg and CnTABs. This similarity approved the validity of the applied data analysis method. The values of Δ�Do(surfactant) and Δ�tro represented the less stability of β-lg in the presence of SDBS in comparison with CnTABs. The apparent β-lg/retinol binding constants decreased between the pH levels of 2.0 and 8.0. The obtained data suggested that β-lg conformation changed in the studied pH range and binding of retinol by β-lg was pH-dependent.

The used surfactant enhanced the retinol binding affinity of β-lg in all of their concentration range at various pH levels. In the presence of surfactant, the tertiary structure of protein was changed in such a manner that its retinol binding site became more accessible and obtained higher affinity.

However, this enhancement was much more in the region of pre-transition. It seems that binding of initial surfactant ions increases the hydrophobic patches in the protein surface and, subsequently, increases the hydrophobic interaction of retinol molecule with β-lg. This enhancement for the pH 6.7 was more than others. However, the authors' previous study showed more enhancement of Ka in the presence of CnTABs.

FIGURE 1 .

 1 FIGURE 1. Net charge versus pH curve of β-lg.

  7 and 8.0. The relative emission intensity of β-lg solution (F/F0) at 330 nm and various concentrations of surfactants ([surfactant]) is shown in figure 2, where F and F0 are the emission intensities in the presence and absence of surfactant, respectively.

FIGURE 2 .

 2 FIGURE 2. SDBS denaturation curves for β-lg in various pH. The excitation wavelength was 295 nm and band slits were 5 nm for both excitation and emission wavelengths. The relative fluorescence intensities were measured at 330 nm. F and F0 are the emission intensities in the presence and absence of surfactant, respectively.

FIGURE 3 .

 3 FIGURE 3. The variation of Δ�Do versus SDBS concentration. Δ�Do is the standard free energy of denaturation.

Figure 4

 4 Figure 4 shows the linearity of the plot of Δ�Do versus ln[surfactant]. The standard free energy in the surfactant solution, Δ�Do(surfactant), was obtained at ln[surfactant] = 0 from the leastsquares analysis of these plots. The concentration of surfactant of 1 M is in good agreement with the transition of the native state of protein (from water) to the denatured state of protein (to surfactant solution). The estimated value of Δ�Do(surfactant), Δ�Do(H2O), and Δ�tro are shown in table 1. Δ�Do(H2O) has positive values but Δ�Do(surfactant) and Δ�tro have negative values. The values of Δ�Do(H2O) (about 21.6 kJ • mol -1 ) are in good agreement withthe previously reported data for the denaturation of β-lg in the presence of n-decyltrimethyl ammonium bromides[4]. This similarity confirms the validity and accuracy of the applied data analysis method. The values of Δ�Do(surfactant) show that the minimum stability occurs at the pH 2.0 (the protein is completely surrounded by denaturant). Δ�tro is also less negative at this pH, which is the best state for the denatured protein. The comparison of Δ�Do(surfactant) and Δ�tro values with the results of the our previous study on the interaction of β-lg with CnTABs represents that β-lg has less stability in the presence of SDBS.

FIGURE 4 .TABLE 1 . 1

 411 FIGURE 4. The variation of Δ�Do versus ln CSDBS. Δ�Do is the standard free energy of denaturation.

  For 1:1 complexes, the retinol fluorescence data can be analyzed by the following equation [43]: ΔF is the change of retinol fluorescence intensity in the presence and absence of β-lg ΔFmax is the maximal change of fluorescence intensity, Ka is the binding constant and [β-lg] is the concentration of β-lg. The linear plots of 1/ΔF as a function of 1/[β-lg] in the presence of SDBS at the pH 2.0, 6.7, and 8.0 are given in FIGURE5, FIGURE6, FIGURE7, respectively. From the slope of the straight line, Ka can be calculated according to equation(7). The values of 12.8 • 10 4 M -1 , 9.8 • 10 4 M -1 , and 5.0 • 10 4 M -1 were obtained for Ka in the absence of any surfactant at the pH 2.0, 6.7, and 8.0, respectively. The apparent β-lg/retinol binding constants decrease between the pH levels 2.0 and 8.0. This observation does not support the suggestions of Fugate and

FIGURE 5 .

 5 FIGURE 5. The linear plots of 1/ΔF as a function of 1/Cβ-lg based on equation (7) and in the presence of various SDBS concentrations at pH 2.0; (a) 0 μM, (b) 50 μM and (c) 150 μM. ΔF is the change of retinol fluorescence intensity in the presence and absence of β-lg.

FIGURE 6 .

 6 FIGURE 6. The linear plots of 1/ΔF as a function of 1/Cβ-lg based on equation (7) and in the presence of various SDBS concentrations at pH 6.7; (a) 0 μM, (b) 200 μM, and (c) 330 μM. ΔF is the change of retinol fluorescence intensity in the presence and absence of β-lg.

FIGURE 7 .

 7 FIGURE 7. The linear plots of 1/ΔF as a function of 1/Cβ-lg based on equation (7) and in the presence of various SDBS concentrations at pH 8.0; (a) 0 μM, (b) 30 μM, and (c) 120 μM. ΔF is the change of retinol fluorescence intensity in the presence and absence of β-lg.

TABLE 2 .

 2 The obtained values for Ka in the presence of SDBS. Ka is the binding constant.

	pH [Surfactant]/(μM)	Ka • 10 -4 /(M -1 )	pH [Surfactant]/(μM)	Ka • 10 -4 /(M -1 )
	2.0 0	12.8	6.7 280	174.3
	10	32.5	330	130.0

Acknowledgment

The financial support of Research Council of Isfahan University of Technology is gratefully acknowledged.