Mannosylerythritol Lipids: Production and Applications

Transcription

1 Journal of Oleo Science Copyright 2015 by Japan Oil Chemists Society doi : /jos.ess14185 REVIEW Mannosylerythritol Lipids: Production and Applications Tomotake Morita *, Tokuma Fukuoka, Tomohiro Imura and Dai Kitamoto Research Institute for Innovation in Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5-2, Higashi 1-1-1, Tsukuba, Ibaraki , JAPAN Abstract: Mannosylerythritol lipids (MELs) are a glycolipid class of biosurfactants produced by a variety yeast and fungal strains that exhibit excellent interfacial and biochemical properties. MEL-producing fungi were identified using an efficient screening method for the glycolipid production and taxonomical classification on the basis of ribosomal RNA sequences. MEL production is limited primarily to the genus Pseudozyma, with significant variability among the MEL structures produced by each species. Outside of Pseudozyma, one recently isolated strain, Ustilago scitaminea, has been shown to exhibit abundant MEL-B production from sugarcane juice. Structural analyses of these compounds suggest a role for MELs in numerous cosmetic applications. MELs act as effective topical moisturizers and can repair damaged hair. Furthermore, these compounds have been shown to exhibit both protective and healing activities, to activate fibroblasts and papilla cells, and to act as natural antioxidants. In this review, we provide a brief summary of MEL research over the past few decades, focusing on the identification of MEL-producing fungi, the structural characterization of MELs, the use of alternative compounds as a primary carbon source, and the use of these compounds in cosmetic applications. Key words: glycolipid, biosurfactant, mannosylerythritol lipid, yeast, sugarcane, cosmetics 1 MANNOSYLERYTHRITOL LIPIDS MELs MELs are long chain fatty acids that contain either 4-Oβ - D - m a n n o p y r a n o s y l - e r y t h r i t o l o r 1 - O - β - D - mannopyranosylerythritol as the hydrophilic head group, which is attached to a variety of fatty acids as the hydrophobic chain Fig. 1. These compounds are known to act as functional glycolipids, and are produced almost exclusively by yeast strains of the genus Pseudozyma 1 3. MELs exhibit excellent interfacial properties 4, as well as a variety of other biochemical functions, including the induction of differentiation in mammalian cells and the binding of various classes of immunoglobulins 5. As with many lipids, differences in chemical structure directly affect their biological activity; for example, MEL-A, a diacetylated MEL, has been shown to strongly increase the efficiency of gene transfection using cationic liposomes 6, 7. Furthermore, certain MELs have been shown to possess anti-inflammatory properties, such as inhibiting the secretion of inflammatory mediators from mast cells When combined with their high biodegradability and relative ease of production, these compounds hold tremendous promise for use in a wide range of applications, including food production, cosmetics, and pharmaceuticals 5, 13. Here, we provide a brief summary highlighting recent advances in MEL research, including the identification of MEL-producing fungi, structural characterizations of MELs, the use of alternative compounds as a primary carbon source, and the use of MELs in cosmetic applications. 2 IDENTIFICATION OF MEL-PRODUCING FUNGI To identify a wide variety of MEL structures, we first sought to develop a rapid and efficient method for detecting MEL production on the basis of decreasing surface tension in cultures containing biosurfactants. The surface tension of each culture was assessed by evaluating the diameter of droplets on the surface of a hydrophobic film Fig MELs were then detected by thin-layer chroma- * Correspondence to: Tomotake Morita, Research Institute for Innovation in Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5-2, Higashi 1-1-1, Tsukuba, Ibaraki , JAPAN morita-tomotake@aist.go.jp Accepted September 3, 2014 (received for review August 22, 2014) Journal of Oleo Science ISSN print / ISSN online This is the review by the winner of 48th Award for Prominent Studies, The Japan Oil Chemists Society (JOCS). 133

2 T. Morita, T. Fukuoka, T. Imura et al. Fig. 1 Chemical structure of glycolipids produced by yeast strains of the genus Pseudozyma. (a) Tri-acylated MEL. (b) di-acylated MEL. (c) mono-acylated MEL. (d) diastereomer type of MEL. (e) mono-acylated and tri-acetylated MEL. (f) mannosyl-mannitol lipid. (g) mannosyl-arabitol lipid. (h) mannosyl-ribitol lipid. MEL-A: R 1 =Ac, R 2 =Ac; MEL-B: R 1 =Ac, R 2 =H; MEL-C: R 1 =H, R 2 =Ac; MEL-D: R 1 =H, R 2 =H; n = 4-16; m = Fig. 2 The shape of the culture supernatant containing MELs (modified from Morita et al., 2006) 9). The surface tension of the culture medium of P. antarctica grown with glucose, glycerol, and soybean oil were visualized on the hydrophobic surface of Parafilm M, reported previously. 134

3 Mannosylerythritol lipids tography TLC. This novel screening method resulted in the identification of numerous MEL-producing fungi, as well as many previously undescribed MEL structures. For example, an MEL-producing species of the genus Pseudozyma, Pseudozyma churashimaensis, isolated from sugarcane, was found to produce not only MEL-A as the major product, but also two novel compounds, monoactylated and triacetylated MELs, as the minor fraction Fig. 1e 15. Alternatively, the smut fungus Ustilago scitaminea, also isolated from sugarcane, was found to produce MEL-B 16. Other fungi shown to produce diastereomers of MEL-B include Pseudozyma tsukubaensis strains 1D9, 1D10, 1D11, and 1E5, all of which were isolated from Perilla frutescens leaves 17. Fifteen additional strains of MEL-producing fungi, representing a variety of Pseudozyma yeasts, were isolated from various vegetables and fruits using this new screening method TAXONOMY OF MEL-PRODUCING FUNGI The vast majority of MEL-producing fungi belong to the genus Pseudozyma. The first-known MEL producer, Pseudozyma antarctica T-34, was first identified over two decades ago; it produces mainly MEL-A, along with smaller amounts of MEL-B and MEL-C 19. More recently identified MEL-producing fungi include Pseudozyma rugulosa, Pseudozyma aphidis, Pseudozyma parantarctica, P. tsukubaensis, Pseudozyma fusiformata, Pseudozyma graminicola, Pseudozyma shanxiensis, Pseudozyma siamensis, and Pseudozyma crassa 2, 20 24, in addition to the smut fungus Ustilago cynodontis, which produces primarily MEL-C when grown in the presence of vegetable oil 25. Not surprisingly, the taxonomic distribution of these fungi is strongly associated with MEL production Fig. 3. Fungi that produce mainly MEL-A, including P. antarctica, P. aphidis, P. rugulosa, and P. parantarctica, are closely linked within a discrete branch of the phylogenetic tree. A producer of MEL-A diastereomers, P. crassa, also clustered closely with the MEL-A producers, albeit in a separate taxonomic branch. The producer of a diastereomeric MEL-B, Fig. 3 Molecular phylogenic tree constructed using ITS1, 5.8S rrna gene, and ITS2 sequences of the genus Pseudozyma and Ustilago (from Morita et al., 2009) 3). The DDBJ/GenBank/EMBL accession numbers are indicated in parentheses. 135

4 T. Morita, T. Fukuoka, T. Imura et al. P. tsukubaensis, was independently positioned in the tree, while MEL-C-producing fungi were scattered throughout the tree, indicative of less conservation between these species. Additional genetic studies will be necessary to better characterize the structures of less common MEL variants, and to determine the relationship between the fungi that produce them. 4 STRUCTURAL DIVERSITY OF MELs As described previously, P. antarctica, P. aphidis, P. rugulosa, and P. parantarctica are the largest producers of MELs, mainly MEL-A a diacetylated MEL along with smaller percentages of MEL-B and MEL-C Fig. 1b 3, 13. These fungi can produce 100 g/l of MELs using vegetable oil as a substrate 19, 21, Pseudozyma tsukubaensis selectively produces a diastereomer of MEL-B. The sugar moiety found within this alternative MEL-B consists of 1-Oβ-D-mannopyranosyl-erythritol Fig. 1d, an isomer of the traditional 4-O-β-D-mannopyranosyl-erythritol found in other MELs 30. Pseudozyma crassa produces diastereomers of MEL-A, MEL-B, and MEL-C, all of which are stereochemically distinct from conventional MELs 20. Pseudozyma hubeiensis, P. graminicola, P. shanxiensis, and P. siamensis produce MEL-C exclusively 23, 24, 31, 32. A synthetic MEL, MEL-D, has been also synthesized via the lipasecatalyzed hydrolysis of MEL-B 33. The use of alternative substrates as the primary carbon source can be used to produce MELs with hydrophobic chains of different lengths, including triacylated MELs Fig. 1a 34, 35, which are produced in the presence of excess soybean oil, or monoacylated MELs Fig. 1c, which are produced using glucose as the sole carbon source 36. The diastereomeric form of MEL-B containing a hydroxy fatty acid was produced by P. tsukubaensis using castor oil as the carbon source 37, while P. parantarctica has been shown to produce mannosyl-mannitol lipids containing a C6 sugar alcohol as the hydrophilic part of the molecule when grown in the presence of olive oil containing excess amounts of D-mannitol Fig. 1f 38. Other homologs containing C5 sugar alcohols, including mannosyl-arabitol and mannosyl-ribitol lipids, are produced by P. parantarctica Fig. 1g, h 39. A detailed list of MEL-producing fungi, the production yield, and the interfacial properties of each MEL homolog are listed in Table 1. 5 PRODUCTION OF MEL-B USING SUGARCANE JUICE AS THE PRIMARY CARBON SOURCE Vegetable oils are often the best carbon source for MEL production; however, the use of these oils in place of more hydrophilic compounds presents significant challenges for many downstream applications due to the presence of chemical by-products such as free fatty acids, monoglycerols, and diacylglycerols. Significant attention has therefore been placed on finding a suitable water-soluble carbon source e.g., glucose or glycerol for use in downstream applications. Among the MEL-producing fungi, we identified a smut fungus, U. scitaminea NBRC32730, which was able to produce large amounts of MEL-B using only sucrose, glucose, fructose, or mannose as the sole carbon source 40, 41. This strain was also able to produce MEL-B using sugarcane juice supplemented with urea as the main nutrient source, reaching a maximum yield of 25 g/l Fig. 4. Furthermore, TLC revealed a single spot corresponding to MEL-B, indicative of high baseline purity without the need for additional purification steps COSMETIC APPLICATIONS 6.1 Moisturizing activity In the course of our research, we assumed that MELs are able to express the moisturizing activity similar to ceramide-3 Fig. 5a, an essential component of the stratum corneum 42, 43, because both of the lipids consist of two fatty acids and sugar moieties, and are capable of forming various lyotropic liquid crystals, including the lamellar phase 5. As previously reported, the moisturizing activity was evaluated using a three-dimensional cultured human skin model, TESTSKIN TM Toyobo, Japan based on the cell viability 44, 45. The sodium dodecyl sulfate SDS -damaged human skin cells were prepared, and then the effects of different lipids on viability of the SDS-damaged cells. Consequently, MELs were found to exhibit moisturizing properties similar to those of ceramide-3 Fig. 5b. Furthermore, the diastereomeric form of MEL-B exhibited excellent water retention properties, increasing the water content within the stratum corneum, while visibly suppressing perspiration on the skin surface 46. These results indicate that MELs may hold promise as an alternative, cost-effective skincare ingredient, capable of replacing higher priced ingredients such as natural ceramides. 6.2 Repair of damaged hair The ceramide-like properties of MELs were also found to extend into the realm of hair care. Ceramides are present not only in the stratum corneum, but also the cuticle of hair, protecting and repairing hair fibers in the face of a variety of environmental stresses. Electron microscopy showed that both MEL-A and MEL-B were able to repair fine cracks on the surface of artificially damaged hair in a manner equivalent to that of ceramides 47. Furthermore, MEL treatment increased the overall strength and sustained the average friction coefficient of damaged hair. These results suggest that MELs are a useful ingredient in 136

5 Mannosylerythritol lipids Table 1 MEL producers and their products (modified from Morita et al., 2013) 13). Microbial producers Carbon sources Glycolipids Yield (g/l) cmc (M) gcmc (mn/m) References Pseudozyma aphidis Soybean oil and glucose MEL-A (main), MEL-B, MEL-C 165 a, b, c ND , 28, 29 Pseudozyma atarctica Soybean oil MEL-A (main), MEL-B, MEL-C 40 a , 4, 19 Soybean oil MEL-B (purified) , 19 n-alkane MEL-A (main), MEL-B, MEL-C 140 a, b ND ND 27 Glucose Mono-acylated MEL (purified) Soybean oil Try-acylated MEL (purified) ND ND ND 34, 35 Pseudozyma churashimaensis Soybean oil Mono-acylated/tri-acetylated MEL (purified) 3.8 a Pseudozyma crassa Oliec acid and glucose Diastereomer MEL-A (main), MEL-B, MEL-C 4.6 a Pseudozyma fujiformata Soybean oil MEL-A (main), MEL-B, MEL-C 4 a ND ND 2 Pseudozyma graminicola Soybean oil MEL-A, MEL-B, MEL-C (main) 9.6 a Pseudozyma hubeiensis Soybean oil MEL-A, MEL-B, MEL-C (main) 76.3 a, b, c Pseudozyma parantarctica Soybean oil MEL-A (main), MEL-B, MEL-C a, b ND ND 2, 34 Glucose Mono-acylated MEL (purified) 1.2 ND ND 36 Soybean oil Try-acylated MEL (purified) 22.7 ND ND 34 Olive oil and mannitol Mannosyl-mannitol lipids (purified) Olive oil and arabitol Mannosyl-arabitol lipids (purified) ND Olive oil and ribitol Mannosyl-ribitol lipids (purified) ND Pseudozyma rugulosa Soybean oil and erythritol MEL-A (main), MEL-B, MEL-C 142 a, b ND ND 2, 21 Soybean oil Try-acylated MEL (purified) ND ND ND 35 Pseudozyma shanxiensis Soybean oil MEL-C 2.72 ND ND 23 Pseudozyma siamensis Safflower oil MEL-B, MEL-C (main) 18.5 a Pseudozyma tsukubaensis Soybean oil Diastereomer MEL-B 73.1 b, c , 17, 30 Castor oil Diastereomer MEL-B containing a hydroxy fatty acid Ustilago cynodontis Soybean oil MEL-C 1.4 ND ND 25 Ustilago maydis Sunflower oil MELs and cellobiose lipids 30 d ND ND 50 Ustilago scitaminea Sugarcane juice MEL-B a As a mixture of MELs b Feeding using resting cells c Large scale production with jer-fermenter d As a mixture of MELs and cellobiose lipids ND: no data hair care products due to their ability to both strengthen and repair damaged hair. 6.3 Activation of fibroblasts and papilla cells In addition to their reparative functions, MELs have been shown to activate fibroblasts and papilla cells, leading to follicle formation and hair growth via transdifferentiation of the adult epidermis 48. Treatment with MEL-A markedly increased the viability of both fibroblasts and papilla cells by more than 150 relative to controls. Consequently, MEL-A may hold promise as a hair growth treatment on the basis of its cell activation properties. 6.4 Antioxidative effects Protection against oxidative stress is essential for longterm skin health, and is therefore an important topic in the cosmetics industry. MELs harboring unsaturated fatty acids in the hydrophobic portion of the molecule exhibit both 1,1-diphenyl-2-picryl hydrazine radical- and superoxide anion-scavenging activities, albeit at levels below that of arbutin, a strong scavenger of reactive oxygen species 49. Further investigation into the antioxidant properties of MELs using cultured human skin fibroblasts under H 2 O 2 -induced oxidative stress revealed significant cytoprotective activity similar to that of arbutin. MELs may therefore be useful as an anti-aging ingredient in skin care products due to their combination of both antioxidant and cytoprotective effects. 7 FURTHER PERSPECTIVES Naturally occurring extracellular glycolipids, including MELs, have been investigated for their use as environmentally friendly biosurfactants with excellent interfacial and biochemical properties. To facilitate their use in a wide 137

6 T. Morita, T. Fukuoka, T. Imura et al. Fig. 4 Production of MEL-B from sugarcane juice supplemented with urea by Ustilago scitaminea NBRC (modified from Morita et al., 2011) 11). (a) The cells of strain NBRC were cultivated in 700 ml of the sugarcane juice supplemented with urea (1 g/l) in a jar-fermenter (1.5 L) at 25 for 7 d. MEL-B was extracted from the cultured medium using an equal amount of ethyl acetate, and quantified by HPLC (closed circle). The amounts of sugars were quantified by HPLC after filtration: sucrose (closed square), glucose (closed diamond), fructose (open square). (b) After 7 d cultivation, the ethyl acetate extract from the culture medium showed a single peak corresponding to MEL-B on HPLC analysis. The purified MELs (MEL-A, MEL-B, and MEL-C) were used as the standard. The retention time (min) of each peak is given in parentheses. (c)the extract also showed a single spot corresponding to MEL-B on TLC. The purified MELs (MEL-A, MEL-B, and MEL-C) were used as the standard. Fig. 5 Moisturizing property of MELs (modified from Morita et al., 2009) 37). (a) Chemical structure of ceramide 3. (b) Relative viability of the cultured skin cells treated with SDS. The cultured skin cells were treated with 1% SDS, washed out the SDS solution, and then re-treated with MELs-A dissolved in olive oil. The viability of cells was determined with a colorimetric method (MTT assay) at 570 nm. Ceramide was used as the positive control. -SDS: non-treated with SDS, +SDS: treated with SDS. 138

7 Mannosylerythritol lipids range of industrial applications, we developed an efficient method for the detection and isolation of MEL-producing fungi, from which we were able to expanded both the structural and functional varieties of MELs. Since then, our investigations have focused primarily on the interfacial properties and practical characteristics of MELs, with the goal of using MELs in cosmetic applications. From this work, we have been able to dramatically reduce the production cost of MELs through the use of an improved fermentation process, and the identification of fungal strains capable of using raw sugarcane juice as the main carbon source. Further decreases in the overall production cost will be necessary for the expanded use of MELs outside of the cosmetics industry, as described previously 5, 13. Additional investigations regarding the production, biosynthesis, and physicochemical properties of these functional glycolipids will be necessary to fully exploit MELs in commercial applications. References 1 Kitamoto, D.; Isoda, H.; Nakahara, T. Functional and potential application of glycolipid biosurfactants. J. Biosci. Bioeng. 94, Morita, T.; Konishi, M.; Fukuoka, T.; Imura, T.; Kitamoto, H.; Kitamoto, D. Characterization of the genus Pseudozyma by the formation of glycolipid biosurfactants, mannosylerythritol lipids. FEMS Yeast Res. 7, Morita, T.; Fukuoka, T.; Imura, T.; Kitamoto, D. Production of glycolipid biosurfactants by basidiomycetous yeasts. Biotechnol. Appl. Biochem. 53, Kitamoto, D.; Yanagishita, H.; Shinbo, T.; Nakane, T.; Kamisawa, C.; Nakahara, T. Surface active properties and antimicrobial activities of mannosylerythritol lipids as biosurfactants produced by Candida antarctica. J. Biotechnol. 29, Kitamoto, D.; Morita, T.; Fukuoka, T.; Konishi, M.; Imura, T. Self-assembling properties of glycolipid biosurfactants and their potential applications. Curr. Opin. Colloid Interface Sci. 14, Inoh, Y.; Furuno, T.; Hirashima, N.; Kitamoto, D.; Nakanishi, M. The ratio of unsaturated fatty acids in biosurfactants affects the efficiency of gene transfection. Int. J. Pharm. 398, Ueno, Y.; Inoh, Y.; Furuno, T.; Hirashima, N.; Kitamoto, D.; Nakanishi, M. NBD-conjugated biosurfactant MEL- A shows a new pathway for transfection. J. Control Release 123, Isoda, H.; Shinomoto, H.; Kitamoto, D.; Matsumura, M.; Nakahara, T. Differentiation of human promyelocytic leukemia cell line HL60 by microbial extracellular glycolipids. Lipids 32, Isoda, H.; Kitamoto, D.; Shinomoto, H.; Matsumura, M.; Nakahara, T. Microbial extracellular glycolipid induction of differentiation and inhibition of the protein kinase C activity of human promyelocytic leukemia cell line activity of human promyelocytic leukemia cell line HL60. Biosci. Biotechnol. Biochem. 61, Wakamatsu, Y.; Zhao, X.; Jin, C.; Day, N.; Shibahara, M.; Nomura, N.; Nakahara, T.; Murata, T.; Yokoyama, K. K. Mannosylerythritol lipid induces characteristics of neuronal differentiation in PC12 cells through an ERKrelated signal cascade. Eur. J. Biochem. 268, Zhao, X.; Wakamatsu, Y.; Shibahara, M.; Nomura, N.; Geltinger, C.; Nakahara, T.; Murata, T.; Yokoyama, K. K. Mannosylerythritol lipid is a potent inducer of apoptosis and differentiation of mouse melanoma cells in culture. Cancer Res. 59, Zhao, X.; Murata, T.; Ohno, S.; Day, N.; Song, J.; Nomura, N.; Nakahara, T.; Yokoyama, K. K. Protein kinase C alpha plays a critical role in mannosylerythritol lipidinduced differentiation of melanoma B16 cells. J. Biol. Chem. 276, Morita, Y.; Tadokoro, S.; Sasai, M.; Kitamoto, D.; Hirashima, N. Biosurfactant mannosylerythritol lipid inhibits secretion of inflammatory mediators from RBL- 2H3 cells. Biochim. Biophys. Acta. 1810, Morita, T.; Fukuoka, T.; Imura, T.; Kitamoto, D. Production of mannosylerythritol lipids and their application in cosmetics. Appl. Microbiol. Biotechnol. 97, Morita, T.; Konishi, M.; Fukuoka, T.; Imura, T.; Kitamoto, D. Physiological differences in the formation of the glycolipid biosurfactants, mannosylerythritol lipids, between Pseudozyma antarctica and Pseudozyma aphidis. Appl. Microbiol. Biotechnol. 74, Morita, T.; Ogura, Y.; Takashima, M.; Hirose, N.; Fukuoka, T.; Imura, T.; Kondo, Y.; Kitamoto, D. Isolation of Pseudozyma churashimaensis sp. nov., a novel ustilaginomycetous yeast species as a producer of glycolipid biosurfactants, mannosylerythritol lipids. J. Biosci. Bioeng. 112, Morita, T.; Ishibashi, Y.; Hirose, N.; Wada, K.; Takahashi, M.; Fukuoka, T.; Imura, T.; Sakai, H.; Abe, M.; Kitamoto, D. Production and characterization of a glycolipid biosurfactant, mannosylerythritol lipid B, from sugarcane juice by Ustilago scitaminea NBRC Biosci. Biotechnol. Biochem. 75, Morita, T.; Takashima, M.; Fukuoka, T.; Konishi, M.; Imura, T.; Kitamoto, D. Isolation of basidiomycetous yeast Pseudozyma tsukubaensis and production of glycolipid biosurfactant, a diastereomer type of mannosy- 139

8 T. Morita, T. Fukuoka, T. Imura et al. lerythritol lipid-b. Appl. Microbiol. Biotechnol. 88, Konishi, M.; Maruoka, N.; Furuta, Y.; Morita, T.; Fukuoka, T.; Imura, T.; Kitamoto, D. Biosurfactant-producing yeasts widely inhabit various vegetables and fruits. Biosci. Biotechnol. Biochem. 78, Kitamoto, D.; Haneishi, K.; Nakahara, T.; Tabuchi, T. Production of mannosylerythritol lipids by Candida antarctica from vegetable oils. Agric. Biol. Chem. 54, Fukuoka, T.; Kawamura, M.; Morita, T.; Imura, T.; Sakai, H.; Abe, M.; Kitamoto, D. A basidiomycetous yeast, Pseudozyma crassa, produces novel diastereomers of conventional mannosylerythritol lipids as glycolipid biosurfactants. Carbohydr. Res. 343, Morita, T.; Konishi, M.; Fukuoka, T.; Imura, T.; Kitamoto, D. Discovery of Pseudozyma rugulosa NBRC as a novel producer of glycolipid biosurfactants, mannosylerythritol lipids, based on rdna sequence. Appl. Microbiol. Biotechnol. 73, Morita, T.; Konishi, M.; Fukuoka, T.; Imura, T.; Yamamoto, S.; Kitagawa, M.; Sogabe, A.; Kitamoto, D. Identification of Pseudozyma graminicola CBS as a producer of glycolipid biosurfactants, mannosylerythritol lipids. J. Oleo Sci. 57, Fukuoka, T.; Morita, T.; Konishi, M.; Imura, T.; Kitamoto, D. Characterization of new types of mannosylerythritol lipids as biosurfactants produced from soybean oil by a basidiomycetous yeast, Pseudozyma shanxiensis. J. Oleo Sci. 56, Morita, T.; Konishi, M.; Fukuoka, T.; Imura, T.; Kitamoto, D. Production of glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma siamensis CBS 9960 and their interfacial properties. J. Biosci. Bioeng. 105, Morita, T.; Konishi, M.; Fukuoka, T.; Imura, T.; Kitamoto, D. Identification of Ustilago cynodontis as a new producer of glycolipid biosurfactants, mannosylerythritol lipids, based on ribosomal DNA sequences. J. Oleo Sci. 57, Kitamoto, D.; Fuzishiro, T.; Yanagishita, H.; Nakane, T.; Nakahara, T. Production of mannosylerythritol lipids as biosurfactants by resting cells of Candida antarctica. Biotechnol. Lett. 14, Kitamoto, D.; Ikegami, T.; Suzuki, T.; Sasaki, A.; Takeyama, Y.; Idemoto, Y.; Koura, N.; Yanagishita, H. Microbial conversion of n-alkanes into glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma antarctica. Biotechnol. Lett. 23, Rau, U.; Nguyen, L. A.; Schulz, S.; Wary, V.; Nimtz, M.; Roeper, H.; Koch, H.; Lang, S. Formation and analysis of mannosylerythritol lipids secreted by Pseudozyma aphidis. Appl. Microbiol. Biotechnol. 66, Rau, U.; Nguyen, L. A.; Roeper, H.; Koch, H.; Lang, S. Fed-batch bioreactor production of mannosylerythritol lipids secreted by Pseudozyma aphidis. Appl. Microbiol. Biotechnol. 68, Fukuoka, T.; Morita, T.; Konishi, M.; Imura, T.; Kitamoto, D. A basidiomycetous yeast, Pseudozyma tsukubaensis, efficiently produces a novel glycolipid biosurfactant. The identification of a new diastereomer of mannosylerythritol lipid-b. Carbohydr. Res. 343, Fukuoka, T.; Morita, T.; Konishi, M.; Imura, T.; Kitamoto, D. Characterization of new types of mannosylerythritol lipids as biosurfactants produced from soybean oil by a basidiomycetous yeast, Pseudozyma shanxiensis. J. Oleo Sci. 56, Konishi, M.; Morita, T.; Fukuoka, T.; Imura, T.; Kakugawa, K.; Kitamoto, D. Efficient production of mannosylerythritol lipids with high hydrophilicity by Pseudozyma hubeiensis KM-59. Appl. Microbiol. Biotechnol. 78, Fukuoka, T.; Yanagihara, T.; Imura, T.; Morita, T.; Sakai, H.; Abe, M.; Kitamoto, D. Enzymatic synthesis of a novel glycolipid biosurfactant, mannosylerythritol lipid-d and its aqueous phase behavior. Carbohydr. Res. 346, Morita, T.; Konishi, M.; Fukuoka, T.; Imura, T.; Sakai, H.; Kitamoto, D. Efficient production of di- and tri-acylated mannosylerythritol lipids as glycolipid biosurfactants by Pseudozyma parantarctica JCM T. J. Oleo Sci. 57, Fukuoka, T.; Morita, T.; Konishi, M.; Imura, T.; Kitamoto, D. Characterization of new glycolipid biosurfactants, tri-acylated mannosylerythritol lipids, produced by Pseudozyma yeasts. Biotechnol. Lett. 29, Fukuoka, T.; Morita, T.; Konishi, M.; Imura, T.; Sakai, H.; Kitamoto, D. Structural characterization and surfaceactive properties of a new glycolipid biosurfactant, mono-acylated mannosylerythritol lipid, produced from glucose by Pseudozyma antarctica. Appl. Microbiol. Biotechnol. 76, Yamamoto, S.; Fukuoka, T.; Imura, T.; Morita, T.; Yanagidani, S.; Kitamoto, D.; Kitagawa, M. Production of a novel mannosylerythritol lipid containing a hydroxy fatty acid from castor oil by Pseudozyma tsukubaensis. J. Oleo Sci. 62, Morita, T.; Fukuoka, T.; Konishi, M.; Imura, T.; Yamamoto, S.; Kitagawa, M.; Sogabe, A.; Kitamoto, D. Production of a novel glycolipid biosurfactant, mannosylmannitol lipid, by Pseudozyma parantarctica and its interfacial properties. Appl. Microbiol. Biotechnol. 83,

9 Mannosylerythritol lipids 39 Morita, T.; Fukuoka, T.; Imura, T.; Kitamoto, D. Formation of the two novel glycolipid biosurfactants, mannosylribitol lipid and mannosylarabitol lipid, by Pseudozyma parantarctica JCM 11752T. Appl. Microbiol. Biotechnol. 96, Morita, T.; Ishibashi, Y.; Fukuoka, T.; Imura, T.; Sakai, H.; Abe, M.; Kitamoto, D. Production of glycolipid biosurfactants, mannosylerythritol lipids, by a smut fungus, Ustilago scitaminea NBRC Biosci. Biotechnol. Biochem. 73, Morita, T.; Ishibashi, Y.; Fukuoka, T.; Imura, T.; Sakai, H.; Abe, M.; Kitamoto, D. Production of glycolipid biosurfactants, mannosylerythritol lipids, using sucrose by fungal and yeast strains, and their interfacial properties. Biosci. Biotechnol. Biochem. 73, Morita, T.; Kitagawa, M.; Suzuki, M.; Yamamoto, S.; Sogabe, A.; Yanagidani, S.; Imura, T.; Fukuoka, T.; Kitamoto, D. A yeast glycolipid biosurfactant, mannosylerythritol lipid, shows potential moisturizing activity toward cultured human skin cells: the recovery effect of MEL-A on the SDS-damaged human skin cells. J. Oleo Sci. 58, Lévêque, J. L.; Rigal, J.; Saint-Léger, D.; Billy, D. How does sodium lauryl sulfate alter the skin barrier function in man? A multiparametric approach. Skin pharmacol. 6, Imokawa, G.; Akasaki, S.; Minematsu, Y.; Kawai, M. Importance of intracellular lipids in water-retention properties of the stratum corneum: induction and recovery study of surfactant dry skin. Arch. Dermatol. Res. 281, Yamamoto, S.; Morita, T.; Fukuoka, T.; Imura, T.; Yanagidani, S.; Sogabe, A.; Kitamoto, D.; Kitagawa, M. The moisturizing effects of glycolipid biosurfactants, mannosylerythritol lipids, on human skin. J. Oleo Sci. 61, Sugibayashi, K.; Hayashi, T.; Matsumoto, K.; Hasegawa, T. Utility of a three-dimensional cultured human skin model as a tool to evaluate the simultaneous diffusion and metabolism of ethyl nicotinate in skin. Drug Metab. Pharmacokinet. 19, Morita, T.; Kitagawa, M.; Yamamoto, S.; Sogabe, A.; Imura, T.; Fukuoka, T.; Kitamoto, D. Glycolipid biosurfactants, mannosylerythritol lipids, repair the damaged hair. J. Oleo Sci. 59, Morita, T.; Kitagawa, M.; Yamamoto, S.; Suzuki, M.; Sogabe, A.; Imura, T.; Fukuoka, T.; Kitamoto, D. Activation of fibroblast and papilla cells by glycolipid biosurfactants, mannosylerythritol lipids. J. Oleo Sci. 59, Takahashi, M.; Morita, T.; Fukuoka, T.; Imura, T.; Kitamoto, D. Glycolipid biosurfactants, mannosylerythritol lipids, show antioxidant and protective effects against H 2 O 2 -induced oxidative stress in cultured human skin fibroblasts. J. Oleo Sci. 61, Spoeckner, S.; Wray, V.; Nimtz, M.; Lang, S. Glycolipids of the smut fungus Ustilago maydis from cultivation on renewable resources. Appl. Microbiol. Biotechnol. 51,