Synthesis of environment friendly biosurfactants and characterization of interfacial properties for cosmetic and household products formulations

Accepted Manuscript Title: Synthesis of environment friendly biosurfactants and characterization of interfacial properties for cosmetic and household products formulations Authors: SuMin Lee, JuYeon Lee, HyonPil Yu, JongChoo Lim PII: DOI: Reference:

S0927-7757(17)30423-5 http://dx.doi.org/doi:10.1016/j.colsurfa.2017.05.001 COLSUA 21594

To appear in:

Colloids and Surfaces A: Physicochem. Eng. Aspects

Received date: Revised date: Accepted date:

21-9-2016 13-2-2017 2-5-2017

Please cite this article as: SuMin Lee, JuYeon Lee, HyonPil Yu, JongChoo Lim, Synthesis of environment friendly biosurfactants and characterization of interfacial properties for cosmetic and household products formulations, Colloids and Surfaces A: Physicochemical and Engineering Aspectshttp://dx.doi.org/10.1016/j.colsurfa.2017.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis of environment friendly biosurfactants and characterization of interfacial properties for cosmetic and household products formulations

SuMin Leea ·JuYeon Leea · HyonPil Yub ·JongChoo Lima*

a

Department of Chemical and Biochemical Engineering, Dongguk University-Seoul, Seoul 100-715, Korea b

AK ChemTech Central Research Lab., DaeJeon, Korea

1

Graphical Abstract

+

H 2N

O

+ Cl

N n

OH

+

Cl O

O P

OH Cl (a)

O-Na+

O (b)

(c)

Synthetic routes of LP(O) where plant oil corresponds to rapeseed oil; (a) intermediate 1, (b) intermediate 2, (c) LP(O).

2

Highlights ● Zwitterionic phospholipid biosurfactants LP(A) and LP(O) were prepared using 2 different raw materials such as rapeseed oil and rapeseed acid respectively and characterized by 1H NMR, 13C NMR, and FT-IR. ● Interfacial properties of newly synthesized surfactants were measured and environmental compatibility such as biodegradability and acute oral toxicity was also evaluated. ● Detergency, eye irritation and skin irritation tests indicated that LP(O) surfactant can be considered as a strong candidate for the potential applicability in cosmetic and household products formulations.

ABSTRACT In this study, 2 types of zwitterionic phospholipid biosurfactants LP(A) and LP(O) were prepared using 2 different raw materials such as rapeseed oil and rapeseed acid respectively. The structure of the resulting products was elucidated by FT-IR, 1H NMR, and 13C NMR spectroscopies and the interfacial properties of LP(A) and LP(O) have been examined such as CMC, static and dynamic surface tensions, interfacial tension, wetting property, emulsion stability, and foam property. Detergency test evaluated by using a Terg-o-tometer showed moderately good detergency compared with conventional nonionic and anionic surfactants used in detergent formulations. Biodegradability, acute oral toxicity, acute dermal irritation and acute eye irritation tests revealed that LP(O) surfactant possesses excellent mildness and superior environmental compatibility, indicating the potential applicability in cosmetic and household products formulations.

Keywords: Environment friendly, Biosurfactant, Environmental compatibility, Interfacial property, Cosmetic and household products

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1. Introduction

Surfactants have a wide variety of applications ranging from being the active ingredients in household cleaning formulations such as laundry detergents, soaps, shampoos, and cosmetic products to industrial use in petroleum production and textile processing. Applications in household cleaners and cosmetic products account for more than half of annual surfactant consumptions. The industrial applications of surfactants are quite diverse and there is virtually no industry that does not use them to some extent. For example, surfactants are used as additives in paints, coatings, adhesives, lubricants, and concrete mixtures and also have a great potential use in surfactant flooding for enhanced oil recovery [1]. Zwitterionic surfactants (amphoteric surfactants) are characterized by having 2 distinct and opposite charges on the same molecule at either adjacent or non-adjacent sites. In general, zwitterionic surfactants have been found to be compatible with other types of surfactants and polymers and impart interesting properties to solutions containing these molecules by formation of self-assembling complexes with polymers or anionic surfactants [2-6]. They can boost the foaming performance of anionic surfactant systems via a variety of mechanisms, by either increasing the speed at which foam is formed (flashing), improving the density and luxurious feel of the foam, or by increasing the foam stability (longevity) [7]. Recently, zwitterionic surfactants received great attention due to their synergistic effects with other surfactants or polymers in the solution, as well as an ability to alleviate undesirable properties of some surfactants, such as skin irritation and a tendency to strip the hair and skin of too much moisture [8]. The enormous market demand for surfactants is currently met by numerous synthetic, mainly petroleum-based, chemical surfactants [9]. These compounds are usually toxic, non-biodegradable and environmentally hazardous. Tightening environmental regulations and increasing awareness for the need to protect the ecosystem have effectively resulted in an increasing interest in biosurfactants as possible alternatives to chemical surfactants [9,10]. Biosurfactants can be defined as the surface-active biomolecules produced by microorganisms with wide-range of applications [11]. They have advantages over their chemical counterparts in specificity, relative ease of preparation, mildness, and effectiveness even at extreme temperature or pH. Biosurfactants also have the merit of diversity, environment friendly nature such as 4

nontoxicity and excellent biodegradability, possibility of large-scale production, selectivity, performance under extreme conditions, and potential applications in environmental protection [9]. Due to their unique functional properties, biosurfactants have been used in various industries including agriculture, fertilizers, petroleum, petrochemicals, cosmetics, pharmaceuticals, personal care products, food processing, beverages, textile manufacturing, metal treatment and processing, pulp and paper processing, paint industries and many others [10,11]. They can be used as emulsifiers as well as demulsifiers, wetting agents, foaming agents, spreading agents, functional food ingredients and detergents [11]. Biosurfactants are also considered as a potential candidate for the environmental cleanup of pollutants due to their environmental friendliness and diversity [9-12]. In this work, environmentally friendly zwitterionic phospholipid biosurfactants were synthesized using 2 different sources such as rapeseed oil and rapeseed acid respectively. The characterization of the resulting products was performed by 1H NMR,

13

C NMR, and FT-IR and environmental compatibility of the

surfactants such as biodegradability and acute oral toxicity was evaluated. The interfacial properties of newly synthesized surfactants were also measured such as CMC, surface tension, interfacial tension, contact angle, and foam stability. Detergency, eye irritation and skin irritation tests have been also performed with newly synthesized surfactants in order to test the potential applicability in cosmetic and household products formulations. Of particular interest was to compare interfacial properties and environmental compatibility of 2 types of phospholipid biosurfactants synthesized from rapeseed acid and rapeseed oil respectively.

2. Materials and methods

2.1. Materials Rapeseed acid and rapeseed oil, raw materials used in the synthesis 2 types of phospholipid biosurfactants as shown in Schemes 1 and 2, were purchased from LG Household & Health Care Ltd and used without any further purification. The rapeseed acid consists of 52.1% of cetoleic acid, 33.3% of stearic acid, 8.5 % of arachidic acid, 4.2 % of palmitic acid and 1.9% of cerebronic acid on a weight basis. On the 5

other hand, the alkyl chain R in the rapeseed oil as shown in Scheme 2 is made up of 49.1% of cetoleic acid, 16.8% of linolenic acid, 15.5% of linoleic acid, 13.5% of oleic acid, 4.0% of palmitic acid and 1.1% of stearic acid on a weight basis. Dimethylaminopropylamine (C5H14N2, 98% purity), hypophosphorous acid (H3O2P, 50% purity), p-toluenesulfonic acid (C7H8O3S, 99% purity), epichlorohydrin (C3H5ClO, 98% purity), phosphate monobasic (H2PO4, 95% purity), sodium carbonate (Na2CO3, 98% purity) and sodium hydroxide (NaOH, 98% purity) were purchased from Sigma-Aldrich Co and used as received. N-decane (C10H22) with a purity of greater than 99% was purchased from Sigma-Aldrich Co. and used as model nonpolar hydrocarbon oil for interfacial tension measurement. Water used for sample preparation was ultrapure having been double distilled and passed through a Nanopure (Sybron-Brinkman Inc.) ion exchange system. The pH of aqueous solution was adjusted by using 0.1N NaOH and 0.1N HCl respectively.

2.2. Methods

2.2.1. Synthesis Intermediate 1 for LP(A) The mixture containing 3.5 mol of dimethylaminopropylamine and 1.0 mol of rapeseed acid was added to a reactor equipped with a mechanical stirrer, thermometer, heating and cooling system. The reaction vessel was heated to 130°C and 0.01 wt% of the catalyst based on the total amount, made of equal weights of 50% hypophosphorous acid and p-toluenesulfonic acid, was added. The reaction proceeds for 10 hr. The final yield of intermediate 1 was higher than 96% and detailed 1H-NMR and

13

C-NMR spectrum signals of

intermediate 1 for LP(A) were as follows:

H-NMR (400MHz, CD3OD), δ: 0.91, 3H(-CH3, t): 1.24-1.41, 20H(-CH2-, m): 1.62-1.76, 2H(-CH2-, m):

1

2.06, 2H(-CH2-CH=, d): 2.12-2.38, 12H(-CH2-, m): 2.65-2.80, 2H(-CH2-, m): 3.18-3.20, 2H(-CH2-, m): 3.50-3.65, 2H(-O-CH2-, m): 5.23-5.32, 1H(-CH2=CH2-, m): 5.62, 1H(-CH2=CH2-, m): 5.93, 1H(-CH2=CH2-, m), 6.31, 1H(-CH2=CH2-, m) (Fig. 1(a) in supplementary material)

6

13

C-NMR (100MHz, CDCl3), δ: 176.2, 135.8, 131.5, 131.4, 131.1, 130.5, 130.2, 127.0, 58.5, 49.8, 49.6, 49.5,

46.4, 49.3, 49.2, 49.1, 45.6, 39.1, 37.8, 33.8, 33.7, 33.6, 33.4, 33.3, 33.0, 30.8, 30.6, 30.5, 30.4, 30.3, 29.2, 29.0, 27.1, 23.9, 23.5, 14.5 (Fig. 1(b) in supplementary material)

Intermediate 2 for LP(A) The mixture containing 1.95 mol of epichlorohydrin and 1 mol of sodium phosphate monobasic was added to a reactor equipped with a mechanical stirrer, thermometer, heating and cooling system. The reaction vessel was heated to 70°C and 0.01 wt% of the catalyst based on the total amount, made of equal weights of Na2CO3 and NaOH, was added. The reaction proceeds for 5 hr. The final yield of intermediate 2 was higher than 95% and detailed 1H-NMR and 13C-NMR spectrum signals of intermediate 2 for LP(A) were as follows:

H-NMR (400MHz, CD3OD), δ: 3.45-3.48 2H(-CH2-Cl, m): 3.49-3.53, 2H(-CH-OH, m): 3.55-3.62,

1

2H(-CH2-Cl, m): 3.78-3.82, 2H(-O-CH2-CH-, m): 3.91-3.98, 2H(-O-CH2-CH-, m) (Fig. 2(a) in supplementary material) 13

C-NMR (400MHz, CDCl3), δ: 73.5, 72.4, 68.8, 68.7, 48.4, 47.9 (Fig. 2(b) in supplementary material)

LP(A) LP(A) was obtained by reacting 1.2:1 weight ratio of intermediate 1 and intermediate 2 without catalyst at 85°C. The yield of the final product LP(A) was higher than 95% and detailed 1H-NMR and

13

C-NMR

spectrum signals of LP(A) were as follows:

H-NMR (400MHz, CD3OD), δ: 0.63, 6H(-CH3, t): 0.91, 42H(-CH2-, m): 1.08, 30H(-CH2-, m): 2.64,

1

4H(-CH2-CH=, d): 2.99, 6H(-CH2-NH-, m): 3.18-3.30, 18H(-N-CH2-CH3, m): 3.61-3.65, 8H(-CH2-OP, m) (Fig. 3(a) in supplementary material) 13

C-NMR (100MHz, CDCl3), δ : 176.5, 72.1, 72.0, 69.0, 67.7, 64.5, 63.9, 49.8, 49.6, 49.5, 49.4, 49.3, 49.2,

49.1, 47.0, 43.9, 37.8, 32.6, 31.0 23.8, 19.1, 14.9 (Fig. 3(b) in supplementary material) 7

Intermediate 1 for LP(O) The mixture containing 1 mol of dimethylaminopropylamine and 3.2 mol of rapeseed oil was added to a reactor equipped with a mechanical stirrer, thermometer, heating and cooling system. The reaction vessel was heated to 95°C and 0.01 wt% of the catalyst based on the total amount, made of equal weights of 50% hypophosphorous acid and p-toluenesulfonic acid, was added. The reaction proceeds for 8 hr. The final yield of intermediate 1 was higher than 98% and detailed 1H-NMR and 13C-NMR spectrum signals of intermediate 1 for LP(O) were as follows:

H-NMR (400MHz, CD3OD), δ: 0.91, 3H(-CH3, t): 1.29-1.40, 18H(-CH2-, m): 1.55-1.72, 2H(-CH2-, m):

1

2.05, 2H(-CH2-CH=, d): 2.12-2.18, 2H(-CH2-C=O, m): 2.21-2.23, 6H(CH3-N-, m) 2.32-2.39, 2H(-N-CH2-, m): 3.18-3.20, 2H(-CH2-NH-, m): 3.50-3.65, 2H(-O-CH2-, m): 5.31-5.41, 2H(-CH2=CH2-, m) (Fig. 4(a) in supplementary material) 13

C-NMR (100MHz, CDCl3), δ: 176.3, 131.1, 129.6, 74.0, 64.2, 49.8, 49.6, 49.5, 49.4, 49.3, 49.2, 49.1, 45.8,

40.4, 38.4, 37.8, 33.6, 33.0, 30.8, 30.6, 30.5, 30.3, 29.2, 27.0, 26.4, 23.9, 14.4 (Fig. 4(b) in supplementary material)

Intermediate 2 for LP(O) The mixture containing 1.95 mol of epichlorohydrin and 1 mol of sodium phosphate monobasic was added to a reactor equipped with a mechanical stirrer, thermometer, heating and cooling system. The reaction vessel was heated to 70°C and 0.01 wt% of the catalyst based on the total amount, made of equal weights of Na2CO3 and NaOH, was added. The reaction proceeds for 5 hr. The final yield of intermediate 2 was higher than 95% and detailed 1H-NMR and 13C-NMR spectrum signals of intermediate 2 for LP(O) were as follows:

H-NMR (400MHz, CD3OD), δ: 3.45-3.48 2H(-CH2-Cl, m): 3.49-3.53, 2H(-CH-OH, m): 3.55-3.62,

1

2H(-CH2-Cl, m): 3.78-3.82, 2H(-O-CH2-CH-, m): 3.91-3.98, 2H(-O-CH2-CH-, m) (Fig. 5(a) in 8

supplementary material) 13

C-NMR (400MHz, CDCl3), δ: 73.5, 72.4, 68.8, 68.7, 48.4, 47.9 (Fig. 5(b) in supplementary material)

LP(O) LP(O) was obtained by reacting 1.2:1 weight ratio of intermediate 1 and intermediate 2 at 85°C without catalyst. The yield of the final product LP(O) was higher than 95% and detailed 1H-NMR and

13

C-NMR

spectrum signals of LP(O) were as follows:

H-NMR (400MHz, CD3OD), δ: 0.64, 6H(-CH3, t): 0.91, 40H(-CH2-, m): 1.08, 24H(-CH2-, m): 1.79,

1

8H(-CH2-, m): 2.02, 2H(-P-OH, d): 2.50, 4H(-CH2-CH=, d): 2.67, 4H(-CH2-CH=, d): 2.98-3.05, 8H(-CH2-NH-, m): 3.18-3.43, 20H(-N-CH2-CH3, m): 3.60-3.68, 8H(-CH2-OP, m), 5.08, 2H(-CH=CH-, d) (Fig. 6(a) in supplementary material) 13

C-NMR (100MHz, CDCl3), δ: 176.3, 131.1, 129.6, 73.6, 72.1, 72.0 68.2, 67.8, 67.6 63.8, 63.6, 49.8, 49.6,

49.5, 49.4, 49.3, 49.2, 49.1, 46.8, 43.9, 38.4, 37.2, 32.9, 31.0, 30.9, 30.8 30.8, 30.6, 30.5, 29.2, 29.0, 27.1, 27.0, 23.9, 19.2, 15.1 (Fig. 6(b) in supplementary material)

2.2.2. Characterization All products were identified by 1H-NMR, 13C-NMR and FT-IR spectrophotometer. In order to determine the structure of the products, 1H-NMR and 13C-NMR spectra were recorded on a Bruker DPX 300 (300MHz) and expressed as δ units at room temperature in CDCl3. Digilab's FT-IR FTS-165 FT-IR spectrometer was used to obtain IR spectra of the products. The pH of synthesized surfactants was measured using a pH meter (S220-K, Mettler Toledo, USA) and the viscosity of 5 wt% surfactant solutions was measured using a DV-II+ digital viscometer. The zeta potential of 1.0 wt% of aqueous surfactant solution was measured at 25°C as a function of pH by using a zeta potential analyzer (Otsuka ELS-800, Japan) to determine the isoelectric point of synthesized zwitterionic surfactants.

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Surface tension measurements of the prepared aqueous surfactant solutions were made at 25℃ using a Du Nuoy ring tensiometer with a platinum ring (Kruss K100, Germany). The CMC was considered to reach when there was no further decrease in surface tension with an increase in surfactant concentration. The packing of surfactant molecules at the air–water interface was estimated on the basis of their surface excess concentration Γ and area occupied per molecule σ, determined from the Gibbs adsorption Eqn. [1]. Γ = - (1/RT) (dγ / d ln C)

(1)

Here, R is the gas constant, T is the absolute temperature, γ is the surface tension, and C is the surfactant concentration in the aqueous phase. The area occupied by one surfactant molecule at the air-water interface, σ in Å2, can be estimated from the following Eqn (2): σ = 1016 / (NA Γ)

(2)

Here NA is Avogadro's number. Dynamic surface tensions for shorter time periods were measured by a bubble pressure tensiometer (Kruss BP2, Germany), where the range of bubble life time used was from 5 to 60,000 msec. A spinning drop tensiometer (Kruss, Site 04, Germany) equipped with a video camera (Sony SSC-DC374, Japan) was utilized to measure the interfacial tension between 1 wt% surfactant solution and n-decane oil at 25℃. The elapsed time of the experiment was recorded from drop injection. Drop shape analysis system (Kruss DSA100, Germany) was used to measure contact angle at 25℃ by forming a drop of surfactant solution on a glass micro slide. A foam test apparatus (IFAC FoamScan, Germany) was used for foam stability measurement at 25℃. Average foam formation rate was obtained based on the time required for foam to reach the 20-cm mark above from an initial 0-cm marked position and foam stability was recorded by measuring foam decay with time. In this study, foam stability was determined by measuring percentage of foam volume decrease during initial 1500 sec. The stability of aqueous surfactant solutions was determined by conductometric measurement (DualConTM Ⅲ, ITEC) at 25℃ [12,13]. By measuring the voltage potential at the upper fill height and at the bottom of the sample 100 hr

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after preparation of the sample, the changes in structure of aqueous dispersions as well as coalescence, aggregation or creaming phenomena were determined. Biodegradability test has been performed at Korea Testing & Research Institute (KTR) in order to get qualified certification test report from international accredited testing laboratory. The primary biodegradability of a surfactant was evaluated using an activated sludge test based on Korean Standard Method (KSM) 2714 [14] and the detailed information on experimental procedure has been described in a previous study [15]. Acute toxicity test was performed at Biotoxtech Co. based on OECD/OCDE 423 in order to get qualified certification test report from international accredited testing laboratory. The lethal dose 50 (LD50) was used to measure the short-term poisoning potential (acute toxicity) of a newly synthesized surfactant where LD50 is defined as the amount of a material, given all at once, which causes the death of 50% (one half) of a group of test animals. In this study, LD50 testing has been done using rats by an oral administration method and the result was expressed as the amount of chemical administered (e.g., milligrams) per kilogram of the body weight of test animal rat. The detailed information on measurement procedure of LD50 has been described in a previous study [15]. Detergency of a test sample was evaluated by using an agitation/mixing type detergency tester (Terg-o-tometer, United States Testing Co., Inc.), generally used in a laboratory [16]. The test was carried out under the conditions of a temperature of 25℃, a rotation speed of 120 rpm, 1g of surfactant sample in 1L of water, washing time of 10 min and 2 rinsing cycles, each being 3 min long. Soiled fabrics used for the test were AS12 Japanese wet soiled fabrics. The artificial soil for a detergency test consisted of 0.5% of carbon black, 28.3% of oleic acid, 15.6% of triolein, 2.5% of paraffin, 2.5% of squalene, 1.6% of cholesterol, 12.2% of cholesterolate, 7.0% of ceratin, and 29.8% of mud on a mass basis. Soiled fabric was cut into a size of 4cm x 4cm and nine sheets of fabrics were used for each test. After washing, cleaning effect for each fabric was evaluated by measuring whiteness of each cloth with a colorimeter (Japan testing Co., Inc.). Then the detergency was calculated according to the following formula. Detergency (%) = [(C-B) / (A-B)] x 100

(3)

where A is the whiteness of a white cloth, B is the whiteness of a soiled cloth before washing, and C is the

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whiteness of a soiled cloth after washing [16,17]. Acute dermal irritation test was performed at Biotoxtech Co. based on OECD/OCDE404 method in order to get qualified certification test report from international accredited testing laboratory and the result was expressed in terms of primary irritation index (PII). Irritation scores for erythema, eschar and edema formation at 1, 24, 48 and 72 hr after patch removal were summed up and divided by the number of observations, to obtain the individual PII. For the calculation of PII, all individual PII’s were summed up and divided by the number of animals used during the test. The detailed information on experimental procedures of each test was described in the literature [18]. Acute eye irritation test was also performed at Biotoxtech Co. based on OECD/OCDE405 method in order to get qualified certification test report from international accredited testing laboratory and the result was expressed in terms of maximum mean total score (MMTS). Each eye response was scored at specified intervals and summed to calculate the individual total score (ITS). The ITS of each animal was summed and divided by the number of animals to produce the mean total score (MTS). The highest value among the MTS produced at specified intervals within 96 hr after test substance application was taken as MMTS. The detailed information on experimental procedures of each test was described in the literature [19].

3. Results and discussion

3.1. Synthesis All products including intermediate 1 and intermediate 2 were identified by 1H-NMR, 13C-NMR, and FT-IR spectrophotometer. The results of 1H-NMR,

13

C-NMR and FT-IR spectra for intermediates, LP(A)

and LP(O) are shown in Figs. 1-6 in supplementary material respectively. As shown in FT-IR spectra (Figs. 1(c)-6(c)), the functional groups of intermediates of LP(A) and LP(O) and final products could be determined with the bands at the specific absorption peak (cm-1). The specific bands likes N-H stretching band (3400~3300 cm-1), C-H stretching band (2928 cm-1), N-H bend amides

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(1628 cm-1), C-N amides (1464 cm-1), C-H bending band (1078cm-1) could be determined through IR spectra of the intermediates. Additionally the presence of C=H olefin (1047cm-1) as shown in Fig. 6(c) could qualitatively identify the difference in the final products of LP(A) and LP(O).

3.2. Interfacial properties The pH of LP(A) and LP(O) surfactants was measured using a pH meter and found to be 6.8 and 5.9 respectively. The viscosity of 5 wt% surfactant solutions was measured and the result is summarized in Table 1. As seen in Table 1, the viscosity values of LP(A) and LP(O) surfactant systems in cP are 27.2 and 15.5 respectively. The isoelectric point of 1.0 wt% of aqueous surfactant solutions was determined by measuring zeta potential as a function of pH. As summarized in Table 1, the isoelectric points of LP(A) and LP(O) surfactant systems were found to be 5.3 and 6.4 respectively. In this study, the CMC was determined by measuring the surface tension as a function of surfactant concentration. The CMC of a surfactant was considered as the concentration beyond which the surface tension of the aqueous solution does not change any more. In addition to CMC, other interfacial properties such as surface tension, interfacial tension, contact angle, foam stability, and emulsion stability were measured at 25℃ and the results are summarized in Table 1. As shown in Fig. 1 and Table 1, the CMC in mol/L of LP(A) and LP(O) surfactants are 4.79x10-4 and 3.43x10-4 respectively. Also seen from Fig. 1 and Table 1, the surface tensions of LP(A) and LP(O) surfactant systems at CMC condition are 35.60 mN/m and 34.61 mN/m respectively. This result indicates that LP(O) surfactant is more hydrophobic and surface active than LP(A) surfactant. Surface excess concentration Γ of each surfactant was calculated using Eqn. (1) from the slope of CMC curve vs concentration as shown in Fig. 1. The surface excess concentrations of LP(A) and LP(O) surfactant systems in mol/m2 are found to be 7.48x10-7 and 8.22x10-7 respectively. The area occupied per molecule of LP(O) and LP(A) was calculated using Eqn. (2) and found to be 221.96 Å2 and 201.98 Å2 respectively. These results indicate that the newly synthesized LP surfactants are surface active and effective in reducing surface free energy. The capacity to lower surface tension under dynamic conditions is also of great importance for practical 13

applications such as foaming or coating processes, where bubbles are rapidly generated and need to be stabilized [17,20]. Due to the molecular structure, surfactant molecules migrate to the liquid or solid surface bordering air until the surface is fully occupied and thus cannot accommodate extra molecules [14,21]. During this process, the adsorption of surfactant monomers at the air-water interface occurs through a sequence of following steps [17]: 1) The dissociation of a micellar aggregate into monomers; slow relaxation process with a lifetime of 10-3 to 1 sec. 2) The molecular diffusion of surfactant monomers from a bulk solution to the air-water interface. 3) The adsorption/desorption of surfactant molecules at the air-water interface. Since the diffusion coefficient of a surfactant monomer is in the order of 10 -6 cm2/sec in general, the molecular diffusion of surfactant monomers from a bulk solution to the air-water interface might be considered as a rate controlling step [17]. Maximum bubble pressure method has been commonly used to measure the dynamic surface tension for systems containing surfactants or other impurities since it takes more time to obtain a completely formed surface and this means that it is difficult to achieve the static equilibrium as a pure liquid does [17,21]. Dynamic surface tensions for shorter time periods were measured by a bubble pressure tensiometer and the result was shown in Fig. 2. As seen in Fig. 2, the surface tension of the aqueous surfactant solution decreases with an increase in surfactant concentration. In addition, relatively short time was required to reach an equilibrium value presumably due to the high mobility of surfactant molecules. This result indicates that any depletion of LP surfactant molecules from the air/water interface will be replenished by an instantaneous diffusion of molecules from the bulk aqueous solution presumably due to high mobility of surfactant molecules [17,20-24]. Dynamic interfacial tension plays an important role during an emulsification process which is the process of preparing emulsions. It has been well-known that fast-adsorbing emulsifier molecules at newly formed interfaces diminish the influence of coalescence and allow formation of smaller droplets of the emulsion produced [17,25,26]. Therefore, dynamic interfacial tension measurement is useful to determine the time needed to stabilize the droplets against aggregation and coalescence and to understand adsorption kinetics of the emulsifier molecules [17]. Interfacial tensions were measured as a function of time for 14

n-decane drops brought into contact with 1 wt% surfactant solutions at 25℃. For LP(A) system, Fig. 3(a) shows that the tension decreased slowly to an equilibrium value of 0.480 mN/m, which was somewhat higher than LP(O) system and. As Fig. 1(b) indicates, the tension for LP(O) system dropped to an equilibrium value of 0.317 mN/m over a period of about 5 min. As also seen in Table 1, the equilibrium interfacial tensions measured between surfactant solution and n-decane oil are in the same order of magnitude as those exhibited between micellar solutions and nonpolar hydrocarbon oils [17,20-24]. Contact angle for 3 wt % surfactant solution was measured at 25℃ by forming one drop of aqueous surfactant solution on a glass slide and determined by the angle formed between planes tangential to the surfaces of the solid and liquid at the wetting perimeter. As summarized in Table 1, the contact angles of LP(A) and LP(O) surfactant systems are 34.36° and 38.59° respectively. Stable foams are very important in a variety of applications such as in the case of hand dish washing detergents, soaps, shampoos, shaving creams, beer, bubble bath, and fire-fighting agents [27-29]. In this study, the foam stability of 1 wt% surfactant solution was studied at 25℃ by measuring the percentage of foam volume decrease during 1500 sec. As seen in Table 1, the percentages of foam volume decrease in LP(A) and LP(O) surfactant systems correspond to 12.5 and 11.9 respectively, indicating both LP surfactants are excellent foaming agents. Even though the difference is not that large, more stable foams were observed with LP(O) surfactant system. It is noteworthy that this result is consistent with that of surface tension measurement where LP(O) system exhibited a lower surface tension value than LP(A) system. Obviously, LP(O) surfactant molecules present in the aqueous solution are preferentially adsorbed at the gas/liquid interface and lower the surface tension at the gas/liquid interface, facilitate the dispersion of gas and reduce the size of bubbles, and change the velocity and regime of bubble rise [17,20-24,28-31]. The stability of 5 wt% aqueous surfactant solution was evaluated at 25℃ by measuring the electrical conductivities of top and bottom portions of a sample bottle of a surfactant solution. The difference between 2 conductivity values was used to estimate stability of surfactant solutions. As shown in the Table 1, aqueous solutions of LP(O) is considered to be more stable than LP(A) since the difference between 2 conductivity values of top and bottom portions is less with LP(O).

15

3.3. Performance test Environmental compatibility of LP surfactants such as acute oral toxicity and biodegradability has been evaluated. In this study, LD50 was used to measure the short-term poisoning potential (acute toxicity) of newly synthesized LP surfactants and the result is summarized in Table 2. As seen in Table 2, both LD50 values determined for LP(A) and LP(O) were found to be greater than 2000 mg/kg. This result indicates that both LP surfactants are very mild compared with polyoxyethylene (9) lauryl ether (PLA) and dodecylbenzene sulfonic acid (LAS) since LD50 values measured with PLA and LAS are known to be 1.19 mg/kg and 650 mg/kg respectively [17]. The primary biodegradability tests for LP(A) and LP(O) surfactants were performed using an activated sludge test based on KSM 2714 and the result is summarized in Table 2. As shown in Table 2, the primary biodegradability of LP(A) and LP(O) has been found to be 96.2% and 99.3% respectively. Even though no data is available in the literature for biodegradability measurement of conventional surfactants based on KSM 2714, biodegradability of phospholipid biosurfactants acceptable for cosmetic applications has been found to be in the range of 96% to 99% based on KSM 2714 [17,32]. It is also noteworthy that biodegradability of higher than 90% measured by KSM 2714 is acceptable for use in detergent formulation [17], indicating that synthesized LP surfactants have excellent biodegradability. Detergency test has been performed with newly synthesized surfactants by using an agitation/mixing type detergency tester at 25℃. As seen in Table 2, the newly synthesized LP surfactants show moderately good detergency. In particular, LP(A) surfactant shows better detergency result than LP(O) surfactant. For example, JIS detergency tests using LP(A) and LP(O) surfactants show soil removal of 94.0% and 81.8% respectively. Acute dermal irritation test was performed and the result was expressed in terms of primary irritation index (PII). The PII was classified to determine the degree of irritation according to the Draize’s method [33] such as PII = 0; non-irritant, 0 < PII ≦ 2; slightly irritant, 2 < PII ≦ 5; moderately irritant, and 5 < PII ≦ 8; severely irritant. As shown in Table 2, LP(O) surfactant is found to be almost non-irritant. Also, glycerol, by-product of LP(O) as shown in Scheme 2, has been found to be beneficial to skin due to its physical effects

16

on the status of water in the outer layers of the stratum corneum by interacting with stratum corneum lipid structure or proteins, altering their water-binding and/or hydrophilic properties [34]. Beyond hydrating properties, glycerol has other helpful effects on skin such as skin barrier repair effect, anti-irritant effect, penetration enhancing effect, skin mechanical properties improvement effect and so on [35]. For example, glycerol prevents the phase transition of the stratum corneum (SC) lipids from liquid to solid crystalline structure, thus preventing water loss and improving skin barrier properties [35]. It is also reported that glycerol (50% in water) is not irritating to subjects with dermatitis (n = 420) when administered for 20~24 hr under occlusion [36] On the other hand, LP(A) surfactant is found to be more irritating than LP(O), which results from remaining fatty acids during the synthesis. It is well-known that fatty acids increase skin permeability and might cause irritation problem by disrupting the packed structure of the lipids in the extracellular spaces of the stratum corneum of the normal skin [37-41]. Acute eye irritation test was carried out at Biotoxtech Co. and the result was expressed in terms of maximum mean total score (MMTS). The MMTS was classified to determine the degree of eye irritation according to the method of Kay and Calandra [37] such as 0.0-0.5; nonirritating, 0.5-2.5; practically nonirritating, 2.5-15; minimally irritating, 15-25; mildly irritating, 25-50; moderately irritating, 50-80; severely irritating, 80-100; extremely irritating, and 100-110; maximally irritating. As seen in Table 2, the MMTS’s for LP(A) and LP(O) are found to be 37.0 and 19.0 respectively, indicating that both surfactants are mild. LP(A) surfactant is more irritating than LP(A) surfactant mainly due to the remaining fatty acids during the synthesis.

4. Conclusions

In this study, 2 types of zwitterionic phospholipid biosurfactants LP(A) and LP(O) were prepared using 2 different raw materials such as rapeseed oil and rapeseed acid respectively and the structure of the resulting products was elucidated by FT-IR, 1H NMR, and

13

C NMR spectroscopies. The interfacial properties of

17

LP(A) and LP(O) surfactant systems have been measured such as CMC, static and dynamic surface tensions, interfacial tension, wetting property, emulsion stability, and foam property. The results indicated that both LP(A) and LP(O) surfactant systems have excellent interfacial properties. In particular, LP(O) has better interfacial properties than LP(A) such as low CMC, surface and interfacial tensions, excellent foam stability, and superior stability of aqueous surfactant solution. Acute oral toxicity (LD50) measurement showed that both LP surfactants are very mild compared with conventional nonionic and anionic surfactants used in detergent formulations such as PLA and LAS. The primary biodegradability of LP(A) and LP(O) has been found to be 96.2% and 99.3% respectively, suggesting that both surfactants are acceptable for cosmetic and detergent applications. Both acute dermal irritation and acute eye irritation tests revealed that both LP(A) and LP(O) surfactants are mild. However, LP(A) surfactant is more irritating than LP(O) surfactant mainly due to the remaining fatty acids during the synthesis. Since newly synthesized LP(O) surfactant is surface active, mild, and readily biodegradable, LP(O) surfactant can be considered as a strong candidate for the potential applicability in cosmetic and household products formulations.

Acknowledgements This work was supported by “the Technology Innovation Program” (10050496, Development of environment friendly biocompatible surfactants and their related products derives from natural resources) funded by the Ministry of Trade, Industry & Energy, Korea.

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List of Table

Table 1. Summary of Physical Properties of Phospholipid Surfactants Measured at 25℃ Table 2. Summary of Surfactant Performance of Phospholipid Surfactants Measured at 25℃

MW

pH

Viscositya (cP)

Isoelectri c Point

LP(A)

951

6.8

27.2

5.3

LP(O)

962

5.9

15.5

6.4

CMC (mol/L)

Surface Tensionb (mN/m)

4.79x104

3.43x104

Γc (mol/m2) 7.48x10-

35.60

7

8.22x10-

34.61

7

σd (Å2)

IFTe (mN/m )

Contact Anglef ( °)

Foam Stabilityg (%)

Emulsion Stabilityh (1/V) Top

Bottom

221.96

0.480

34.36

12.5

1.432

1.564

201.98

0.317

38.59

11.9

1.005

1.003

a

Measured with 5 wt% surfactant concentration

b

Measured at CMC

c

Surface excess concentration calculated using Gibbs adsorption equation.

d

Area occupied per surfactant molecule at the air–water interface.

e

Interfacial tension measured at 25℃ as a function of time between 1 wt% surfactant solution and n-decane using a spinning drop tensiometer

f

Measured with 3 wt% surfactant concentration at 25℃ by forming a drop of surfactant solution on a glass micro slide using a drop shape analysis

system 23

g

Foam stability measurement by determining percentage of foam volume decrease during 1500 sec, initially generated with1 wt% surfactant

concentration h

Stability of 3 wt% of surfactant solutions at 25℃ determined by measuring the voltage potential at the upper fill height and at the bottom of the sample 100 hr after preparation of the sample

24

Table 2. Summary of Surfactant Performance of Phospholipid Surfactants Measured at 25℃

Biodegradabilitya (%)

Toxicityb (LD50) (mg/kg)

Detergencyc (%)

Skin Irritation (PIId)

Eye Irritation (MMTSe)

LP(A)

96.2

> 2000

94.0

1.0

37.0

LP(O)

99.3

> 2000

81.8

0.7

19.0

a

Evaluated using an activated sludge test based on KSM 2714

b

The amount of substance required to kill 50% of the test rats within 24 hours

c

The artificial soil for a detergency test consisted of 0.5% of carbon black, 28.3% of oleic acid, 15.6% of triolein, 2.5% of paraffin, 2.5% of squalene,

1.6% of cholesterol, 12.2% of cholesterolate, 7.0% of ceratin, and 29.8% of mud on a mass basis. d

Measured the primary irritation index (PII) at 72 hours after patch removal

e

Measured the maximum mean total score (MMTS) within 96 hours after test substance application

25