Oil Gelation Ability of a Rice Bran Wax and Botanical High-Melting-Point

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Oil Gelation Ability of a Rice Bran Wax and Botanical High-Melting-Point Alcohol Mixture Midori Endo and Masashi Shibata＊ School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura-machi, Hachioji-shi, Tokyo 192-0982, JAPAN

Abstract: Due to its availability and relatively high melting point, rice bran wax can be used in foods and cosmetics as an oil solidifying agent. The addition of high-melting-point alcohols to the wax to optimize its performance was investigated. Such alcohols were prepared by the hydrolyzation of rice bran wax or carnauba wax. These alcohols had melting points of ～80℃, higher than that of rice bran wax, and consisted of hydrocarbons with alkyl chain lengths ranging from C24 to C34, much longer than the alkyl chains found in commercially available higher alcohols. Oil gel made with only rice bran wax as a solidifying agent has lower hardness than the conventional hydrocarbon wax gel, too low for practical usage in stick cosmetics such as lipsticks and lip creams. Blending high-melting-point alcohols with rice bran wax at 10–20% led to marked increase in gel hardness, equivalent to the gel hardness of the hydrocarbon wax. This effect on rice bran wax was not observed in commercially available higher alcohols and esters with lower melting points. Based upon the change in microstructure observed with SEM, the improved gel hardness of the rice bran wax upon addition of high-melting-point alcohols was probably induced by the disappearance of spherical clusters, originally presented in the gel, resulting in close to uniform morphology.

Key words: rice bran wax, high-melting-point alcohol, carnauba wax, gel hardness, scanning electron microscope

1 Introduction the rheological properties or gel hardness of rice bran wax Wax gels obtained by solidifying liquid oils with small and other botanical waxes6－11（） Table 1）and it has been re- amounts of solid oils（waxes）are widely used for stick cos- ported that the ratio of free alcohol and wax ester in metics, such as lipsticks and lip creams. Hydrocarbon natural waxes influences oil gel physical properties12－14）. waxes, such as paraffin wax and polyethylene, are mainly However, it has been known empirically that the simple ad- used as solidifiers（gelling agents）for these oil gels1, 2）. dition of various commercial higher alcohols, wax esters, or Since these waxes are derived from petroleum, they may other botanical waxes to rice bran wax cannot improve its be viewed unfavorably by cosmetics users. Further depen- gel hardness to a level comparable to the hardness of hy- dence on fossil fuels leads to environmental burden. drocarbon wax gels. On the other hand, botanical waxes like candelilla wax, We have reported that when candelilla wax, which has a carnauba wax, and rice bran wax are used in cosmetics as lower melting point（54℃）than rice bran wax, was blended oil thickeners, emulsion stabilizers, and as gelling agents with behenyl behenate（m.p. 67℃）, a high-purity wax ester mixed with hydrocarbon waxes3）. However, when they are synthesized from C22 alcohol and C22 fatty acid, the ob- used as gelling agents, botanical waxes tend to form gels tained gel exhibited far higher hardness than that of can- that are much softer than those formed from hydrocarbon delilla wax15）. The significance of this technology is that the waxes4）. botanical wax（candelilla wax）and the additive（behenyl be- Rice bran wax, a botanical wax containing an ester as henate）crystallize simultaneously in the gel preparation the main component, is obtained as a by-product of edible process. For this reason, the melting point of the additive grain rice processing5）and is used both as a cosmetic raw must be equal to or higher than that of the botanical wax. material and a food additive. Among botanical waxes, rice Therefore, even if behenyl behenate was used as an addi- bran wax is attractive as an oil gelling agent for cosmetics tive, the hardness of the rice bran wax would not increase. due to its relatively high melting point（72℃）and rather Unfortunately, it is difficult to obtain wax esters with high gel hardness. Many studies have been conducted on higher melting points than that of behenyl behenate

＊Correspondence to: Masashi Shibata, School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura-machi, Hachioji-shi, Tokyo 192-0982, JAPAN E-mail: [email protected] Accepted May 8, 2019 (received for review Match 3, 2019) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs

739 M. Endo and M. Shibata

10 Table 1 Botanical waxes used in cosmetics ）. Component Rice bran wax Carnauba wax Candelilla wax Melting point 72℃ 81℃ 54℃ Long chain ester 93%－97% 80%－85% 24%－30% Free fatty acid 4%－7% 3%－4% 10%－20% Free higher alcohol － 10%－12% 10%－15% Hydrocarbon － 1%－3% 40%－50% Other phospholipid resin 3%－4% resin 15%－18% squalene lactone 3%－5%

because fatty acids with chains longer than C22 occur nat- at 80℃. The mixture was allowed to cool down and stand urally in small amounts only. On the other hand, it is possi- at room temperature. The resultant oil layer was removed ble to obtain higher alcohols, with longer chains than C22 using a dropper and deposited into a 200-mL beaker. from plants, whose melting points are higher than that of Pure water（50 g）was added to this oil solution and the behenyl behenate16）. Hence, these higher alcohols should mixture was stirred at 80℃ to remove the water-soluble function as additives to rice bran wax. component. This step was repeated three times. Finally, In this study, we sought to develop a new oil solidifying the oil solution was transferred to a 100-mL beaker and agent using a mixture of rice bran wax and a botanically crystallization was carried out at 10℃ for 12 h. The crystal- derived long-chain alcohol. Long-chain alcohols were ob- lized product was removed from the solution by suction fil- tained by the hydrolyzation of rice bran wax or carnauba tration and washed three times with ethanol. This product wax. We evaluated whether gel hardness could be im- was left standing at room temperature and then dried to proved by blending these long-chain alcohols with rice evaluate the alcohol content. The obtained alcohols were bran wax. either rice alcohol or carnauba alcohol.

2.3 Preparation of oil gels and gel hardness measurement 2 Experimental Procedures Different amounts of rice bran wax, higher alcohol, and 2.1 Materials macadamia nut oil（10 g in total）were placed in a 100-mL Rice bran wax（Rice bran wax S-100, Yokozeki Oil & Fat glass beaker and stirred on a hot plate at 100℃ for 30 min. Industries Co., Ltd.）, carnauba wax（TOWAX-1P2, Cerarica The total amount of rice bran wax and higher alcohol was Noda Co., Ltd.）, and candelilla wax（Candelilla wax MK-4, set to be 15％ of the gel. Subsequently, the liquid mixture Yokozeki Oil & Fat Industries Co., Ltd.）were the botanical was poured into a polycarbonate cylindrical container with waxes used. For comparison, paraffin wax（HNP-9, Nippon a diameter of 3 cm and a height of 1 cm and allowed to Seiro Co., Ltd.）was used. Commercially available higher al- stand at 25℃ for 24 h. cohols, cetanol（Hainol 16ss, Kokyu Alcohol Kogyo Co., The hardness of the gel in the container was measured Ltd.）, stearyl alcohol（Hainol 18SS）, arachidyl alcohol by using a gel hardness meter（Rheotex SD 700, Sun Scien- （Hainol 20SS）, and behenyl alcohol（Hainol 22SS）were also tific Co., Ltd.）equipped with a spherical plunger of 5 mm used. Macadamia nut oil（Fujifilm Wako Pure Chemical diameter. A spherical plunger was inserted into the gel to a Corporation）was used as the oil for gel preparation. depth of 2.5 mm at a speed of 30 mm/min, and the maximum stress was measured. Nine samples were pre- 2.2 Preparation of botanically derived long-chain alcohols pared for each composition, and the hardness was mea- （carnauba alcohol and rice alcohol） sured seven times at random locations in the container, Pure water（10 g）, ethanol（10 g）, and potassium hydrox- and the average value was taken. ide（1.4 g）were added to 5 g of rice bran wax or carnauba wax in a 300-mL 2-necked round-bottomed flask. Hydroly- 2.4 Characterization sis was carried out under reflux conditions for 1 h. The composition of the botanically derived long-chain al- Oil（caprylic/capric triglyceride, The Nisshin OilliO cohols was analyzed using a gas chromatograph with a Group, Ltd., 100 g）and pure water（50 g）were placed in a flame ionization detector（GC-17A, Shimadzu Corporation; 500-mL glass beaker. The contents of the 300-mL 2-necked column: DB-17, Agilent Technologies International Japan, round-bottomed flask（refer to the previous paragraph） Ltd.）after an acetylation treatment. The crystal structure were then added to 500-mL glass beaker and stirred for 2 h of the wax inside the gels was observed by scanning elec-

740 J. Oleo Sci. 68, (8) 739-745 (2019) Gel Hardness of Rice Bran Wax and High Melting Point Alcohol

tron microscopy（SEM）. Oil was removed from the gels Figure 1 shows the differential scanning calorimetry pat- before SEM observation. A piece of gel（1 g）was cut out terns recorded during the cooling and heating processes of and added to a 9-mL glass jar; 8 mL of ethanol was then rice bran wax, rice alcohol, candelilla alcohol and mixture added and the jar was mixed gently. The mixture was then of rice bran wax and rice alcohol（50:50）. The respective allowed to stand at room temperature for 24 h. Subse- melting points estimated based on the DSC profiles are quently, the supernatant was removed, and fresh ethanol shown in Table 2. Commercially available higher alcohols was added again and mixed gently. This procedure was such as cetanol（C16）, stearyl alcohol（C18）, arachidyl conducted once a day for 2 weeks. The oil-free sample was alcohol（C20）, and behenyl alcohol（C22）, which are ob- finally dried at room temperature. After drying, the sample tained by the hydrolysis and reduction of plant-derived tri- was deposited with gold and observed with SEM（JSM- glycerides, had melting points lower than 70℃. The rice 6060LV, JEOL Ltd.）at an accelerating voltage of 15 kV. alcohol and carnauba alcohol obtained in this study have The melting point and crystallization temperatures of much higher melting temperatures（77–78℃）than those of the samples were measured using a differential scanning the commercially available higher alcohols. Comparing the calorimeter（DSC-60, Shimadzu Corporation）. The temper- two higher alcohols（referred to as high-melting-point alco- ature was raised from room temperature to 100℃, lowered hols herein）, the carnauba alcohol exhibited a slightly to －10℃, and increased again to 100℃. The scanning higher melting point than rice alcohol. speed was set at 5℃/min. The melting points of various waxes are shown in Table 3. Among the listed waxes, the carnauba wax exhibited the highest melting point of 81℃, while the paraffin and rice bran waxes exhibited similar values. The candelilla wax ex- 3 Results and Discussion hibited the lowest melting temperature. 3.1 Physical properties of botanically derived higher alco- As shown in Fig. 1, the precipitation temperature of rice hols alcohol（81.0℃）was slightly higher than that of rice bran To obtain botanically derived long-chain alcohols having wax（77.4℃）, but when they were mixed the temperature high melting points, rice alcohol and carnauba alcohol were was dropped to 72.2℃, which suggested that both of them prepared by the hydrolysis of rice bran wax and carnauba were simultaneously precipitated. wax. The alcohol compositions of the two high-melting-point

Fig. 1 DSC patterns recorded during the cooling（a）and heating（b）processes of rice bran wax（RBW）, rice alcohol（RA）, candelilla alcohol（CA）and RBW – RA mixture（TBA/RA, 50/50）.

741 J. Oleo Sci. 68, (8) 739-745 (2019) M. Endo and M. Shibata

Table 2 Melting points of higher alcohols. Sample Melting point (℃) Rice alcohol 77 Carnauba alcohol 78 Cetanol 48 Stearyl alcohol 56 Arachidyl alcohol 59 Behenyl alcohol 68

Table 3 Melting temperatures of different waxes.

Sample Melting point (℃) Candelilla wax 54

Rice bran wax 72 Fig. 2 Hardness of gels constructed with rice bran wax or Carnauba wax 81 paraffin wax. Paraffin wax 75 ●: rice bran wax, ■: paraffin wax

Table 4 Alcohol composition of botanically derived to a level equivalent to that of paraffin wax. The compari- long-chain alcohols. son of the gel hardness of practical hydrocarbon waxes re- vealed that the gel hardness of rice bran wax, having a Carbon number Rice alcohol Carnauba alcohol concentration of 15％, must be improved to a hardness of C22 －－ more than 150 g, and close to 200 g. C24 2.4% 1.6% 3.3 Effect of blending high-melting-point alcohols with C26 5.7% － rice bran wax C28 16.3% 2.0% Although we have reported that the gel hardness in- C30 32.7% 11.3% creased upon blending behenyl behenate with candelilla C32 25.0% 69.9% wax, a highly crystalline long-chain ester wax, the addition of behenyl behenate to rice bran wax did not increase the C34 17.9% 15.2% gel hardness. We attribute this result to the much lower C36 －－ melting point of behenyl behenate（67℃）than that of rice bran wax. Therefore, in this study, the effect of blending alcohols are shown in Table 4. The main components of rice alcohol, which has a melting point close to that of rice rice alcohol were C28–C34 alcohols, while that of carnauba bran wax, was investigated. alcohol was C32 alcohol. These alcohol chains are longer Figure 3 shows the relationship between gel hardness than those of commercially available alcohols（maximum and the ratio of rice bran wax and rice alcohol. The total C22）. Since carnauba alcohol has longer alkyl chains in amount of rice bran wax and rice alcohol was set at 15％ of than rice alcohol, it has a higher melting point. the gel. The hardness of the gel containing only rice bran wax was approximately 20 g, i.e. it was very soft. Moreover, 3.2 Gelling ability of rice bran wax rice alcohol alone could not gel the oil. Gel hardness in- The relationships between the wax concentration and creased when a small amount of rice alcohol was blended the hardness of wax gels constructed using paraffin wax with rice bran wax. When the ratio of rice alcohol to rice （typical hydrocarbon wax）and rice bran wax are shown in bran wax was 20:80, the gel hardness reached a maximum Fig. 2. value; gel hardness at this ratio exceeded the target value In both cases, the gel hardness increased with increasing of 150 g. wax amount. The hardness of the rice bran wax gel was To confirm whether this blending effect was due to the less than that of the paraffin wax gel at the same wax con- high melting point of the alcohols, commercially available centration. The rice bran wax did not gel at 10％ wax and higher alcohols with hydrocarbon chains shorter than those the hardness of the 20％ rice wax gel was almost the same of rice alcohol（stearyl alcohol, arachidyl alcohol, and as that of the 15％ paraffin wax gel. The aim of this study behenyl alcohol）were blended with rice bran wax. Their is therefore to improve the gelling ability of rice bran wax gel hardnesses were measured in the same manner as for

742 J. Oleo Sci. 68, (8) 739-745 (2019) Gel Hardness of Rice Bran Wax and High Melting Point Alcohol

Fig. 5 Gel hardness of mixtures of carnauba wax and long-chain alcohol. Fig. 3 Gel hardness of mixtures of rice bran wax and rice ○: rice alcohol, □:carnauba alcohol alcohol.

respect to gel hardness. However, when compared to rice alcohol, carnauba alcohol achieved maximum gel hardness at a lower blend ratio（carnauba alcohol/rice alcohol＝ 10/90）. Its maximum gel hardness was higher than that of rice alcohol as well. In summary, esters or alcohols having low melting points （less than 70℃ and lower than that of rice bran wax）did not exhibit a blending effect; only alcohols having high melting points（～77℃）were useful in this respect. In order to raise the gel hardness of vegetable waxes, we propose that it is necessary for the additives（high-melting- point alcohols）and plant waxes to crystallize in the oil almost simultaneously when preparing the gels. To confirm such proposal, carnauba wax with composition that is similar to rice bran wax, differing only in its higher melting point（81℃）, was used instead of rice bran wax. The effect of blending the high melting point alcohols was investigated in the same manner. As shown in Fig. 5, the gel hardness of carnauba wax did not increase even when blended with Fig. 4 Gel hardness of mixtures of rice bran wax and rice alcohol or carnauba alcohol as expected. carnauba alcohol. 3.4 Microstructure of the gels and mechanism of gel rice alcohol. We confirmed that higher alcohols with hardness improvement melting points lower than 68℃ could not improve the gel The gel hardness of rice bran wax was remarkably in- hardness of the rice bran wax gels. creased by adding 10％ rice alcohol having poor gelation Next, the effect of blending carnauba alcohol with rice ability, which indicates that this increase in gel hardness bran wax was investigated（Fig. 4）. Rice alcohol and car- was not simply due to the increase in the proportion of nauba alcohol have similar melting points, although the components having high gelling ability. It is likely due to main alcohol chain of carnauba alcohol is slightly longer the interaction between rice bran wax and rice alcohol. than that of rice alcohol. When blended with rice wax, car- The microstructure, composed of wax crystals in the rice nauba alcohol and rice alcohol performed similarly with bran wax gel, was therefore observed using a scanning

743 J. Oleo Sci. 68, (8) 739-745 (2019) M. Endo and M. Shibata

Fig. 6 SEM images of the microstructures of gels with botanical wax. （a）rice bran wax only,（ b）blend of rice bran wax and rice alcohol（80/20）,（ c）blend of rice bran wax and carnauba alcohol（90/10）

Fig. 7 SEM images of the microstructures of gels with high-melting-point alcohols. （a）rice alcohol only,（ b）carnauba alcohol only electron microscope（SEM）in this study. cal clusters is not clear at this stage. The preferred structure of the wax gel is a uniform At this point, the spherical clusters still exist to some structure composed of plate wax crystals, known as a card extent and could not form a uniform structure like those house structure1）. Hydrocarbon waxes always form the formed by hydrocarbon wax gels. If the substance that pro- card house structure and their gel hardness is high. In gels duces the spherical clusters is eliminated, the gel hardness prepared with rice bran wax only, spherical clusters existed of the rice bran wax is expected to improve. The structure which had not been observed in the hydrocarbon wax gels. of an oil gel prepared with rice alcohol alone is shown in These spherical clusters were composed of plate micro- Fig. 7（a）. Even in the rice alcohol, spherical clusters exist crystallites（Fig. 6（a））. We assumed that this heteroge- like those in the rice bran wax, which is the raw material neous structure containing many spherical clusters pre- for the alcohol. Hence, it is thought that the causative sub- vented rice bran wax gel from becoming hard. stance of the spherical clusters was conveyed from the rice When rice alcohol was blended with rice bran wax as an bran wax to the rice alcohol. additive, the sizes of these spherical clusters became Based upon the X-ray diffraction measurement, we con- larger, ranging from 20–40 μm to 50–100 μm, and their firmed that the crystal system of the high-melting-point al- number decreased. The inner structure of the gel also cohols and rice bran wax were both orthorhombic. This changed, to a certain extent, from a clustered morphology crystal system tends to produce plate shape crystals. Fur- to a homogeneous morphology（Fig. 6（b））. The effect of thermore, as seen in Fig. 7（b）, the spherical cluster was eliminating spherical clusters was more pronounced with formed with plate crystallites. This observation implies that carnauba alcohol than with rice alcohol. The rice bran wax impurities, behaving as crystal nuclei, were present in both gel obtained after blending with carnauba alcohol was the high-melting-point alcohols and the rice bran wax, al- closer to the uniform card house structure（Fig. 6（c））. lowing many plate crystallites to grow from them and form It was considered that the blending of the high-melting- spherical clusters. point alcohols to rice bran wax made it difficult to form the Since this impurity may behave as a crystal nucleus for spherical clusters in the gels, whereby the hardness of the higher alcohols, it is difficult to remove it from the rice gels was increased. This mechanism of eliminating spheri- alcohol even by using the most efficient purification

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method, recrystallization. A technique for purifying high- and Fat Products（Shahidi, F. ed.）Part 2. Chapter 10. melting-point alcohol and removing nucleation material is John Wiley & Sons, Inc. p. 465（2005）. desired. 6） Dassanayake, L.S.K.; Kodali, D.R.; Ueno, S.; Sato, K. Physical properties of rice bran wax in bulk and organogels. J. Am. Oil Chem. Soc. 86, 1163-1173 （2009）. 4 Conclusion 7） Sabale, V.; Sabale, P.M.; Lakhotiya, C.L. Comparative To improve the gel hardness of rice bran wax, botanically evaluation of rice bran wax as an ointment base with derived high-melting-point alcohols, obtained by the hy- standard base. Indian J. Pharm. Sci. 71, 77-79 drolysis of rice bran wax or carnauba wax, were used as （2009）. additives to rice bran wax. The obtained alcohols were 8） Dassanayake, L.S.K.; Kodali, D.R.; Ueno, S.; Sato, K. named rice alcohol and carnauba alcohol. Their melting Crystallization kinetics of organogels prepared by rice points were approximately 80℃, which were higher than bran wax and vegetable oils. J. Oleo Sci. 61, 1-9 that of the rice bran wax. They consisted of alcohols with （2012）. hydrocarbon chain lengths ranging from C24 to C34, which 9） Hwang, H.-S.; Singh, M.; Bakota, E.; Winkler-Moser, J.; were much longer than those of the commercially available Kim, S.; Liu, S. Margarine from organogels of plant higher alcohols. Carnauba alcohol consisted of alcohols wax and soybean oil. J. Am. Oil Chem. Soc. 90, 1705- with longer chains and its melting point was higher than 1712（2013）. that of rice alcohol. 10） Doan, C.D.; Van de Walle, D. Dewettinck K.; Patel, A.R. When blended with rice bran wax at a concentration of Evaluating the oil-gelling properties of natural waxes 10–20％, both rice alcohol and carnauba alcohol showed in rice bran oil: Rheological, thermal, and microstruc- marked increases in gel hardness equivalent to that of the tural study. J. Am. Oil Chem. Soc. 92, 801-811（2015）. hydrocarbon wax. Such an effect was not observed with 11） Patel, A.R.; Babaahmadi M.; Lesaffer, A.; Dewettinck, commercially available higher alcohols and esters with K. Rheological profiling of organogels prepared at crit- lower melting points. ical gelling concentrations of Natural waxes in a triac- Based upon the microstructure change observed with ylglycerol solvent. J. Agric. Food Chem. 63, 4862- SEM, we believe that the improvement in the gel hardness 4869（2015）. of the rice bran wax by adding high-melting-point alcohols 12） Wang, M.F.; Lian, H.Z.; Mao, L.; Zhou, J.P.; Gong, H.J.; was induced by the disappearance of spherical clusters Qian, B.Y.; Fang, Y.; Li J. Comparison of various extrac- present in the gel, which led to the formation of a uniform tion methods for policosanol from rice bran wax and structure. establishment of chromatographic fingerprint of policosanol. J. Agric. Food Chem. 55, 5552-5558（2007）. 13） Doan, C.D.; To, C.M.; De Vrieze, M.; Lynen, F.; Dan- thine, S.; Brown, A.; Dewettinck, K.; Patel, A.R. Chem- References ical profiling of the major components in natural wax- 1） Shibata, M. Control of the physical properties of wax es to elucidate their role in liquid oil structuring. Food gels and their application to cosmetics. Oleoscience Chem. 214, 717-725（2017）. 17, 633-642（2017）. 14） Doan, C.D.; Tavernier, I.; Okuro, P.K.; Dewettinck, K. 2） Imai, T.; Nakamura, K.; Shibata, M. Relationship be- Internal and external factors affecting the crystalliza- tween the hardness of an oil-wax gel and the surface tion, gelation and applicability of wax-based oleogels structure of the wax crystals. Colloids Surf. A 194, in food industry. Innov. Food Sci. Emerg. Technol. 233-237（2001）. 45, 42-52（2018）. 3） Fukui, H. Cosmetics Made Absolutely Simple. Al- 15） Shimizu, T.; Tanabe, T.; Kachi, H.; Shibata, M. Petro- lured Pub Co., Carol Stream, Illinois（2013）. leum-ingredient-free lipsticks consisting of long-chain 4） Kugimiya, M.; Hibi, H. Structure in lipsticks and con- wax ester and botanical wax. Proceedings of IFSCC trol of its stability. Fragrance Journal 35（4）, 88-94 Congress 2018, Munich. P-S8-433（2018）. （2007）. 16） Fusegawa, K. Properties and applications of waxes. 5） Orthoefer, F.T. Rice Bran Oil. in Bailey's Industrial Oil Saiwai Shobo Co., Ltd., Tokyo（1993）.

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