Journal of Oleo Science Copyright ©2015 by Japan Oil Chemists’ Society doi : 10.5650/jos.ess14185 J. Oleo Sci. 64, (2) 133-141 (2015)
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 305-8565, 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-mannopyranosyl-erythritol or 1-O-β-Dmannopyranosylerythritol 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 Pseudozyma1−3）. MELs exhibit excellent interfacial properties4）, as well as a variety of other biochemical functions, including the induction of differentiation in mammalian cells and the binding of various classes of immunoglobulins5）. 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 liposomes6, 7）. Furthermore, certain MELs have been shown to possess anti-inflammatory properties, such as inhibiting the secretion of inflammatory mediators from mast cells8−12）. 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 pharmaceuticals5, 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. 2）14）. MELs were then detected by thin-layer chroma-
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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 305-8565, JAPAN E-mail: [email protected] Accepted September 3, 2014 (received for review August 22, 2014)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
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This is the review by the winner of 48th Award for Prominent Studies, The Japan Oil Chemists’ Society (JOCS).
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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: R1=Ac, R2=Ac; MEL-B: R1=Ac, R2=H; MEL-C: R1=H, R2=Ac; MEL-D: R1=H, R2=H; n = 4-16; m = 6-16.
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
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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-B16）. 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 leaves17）. Fifteen additional strains of MEL-producing fungi, representing a variety of Pseudozyma yeasts, were isolated from various vegetables and fruits using this new screening method18）.
3 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-C19）. 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 crassa2, 20−24）, in addition to the smut fungus Ustilago cynodontis, which produces primarily MEL-C when grown in the presence of vegetable oil25）. 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
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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 substrate19, 21, 26−29）. 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 MELs20）. Pseudozyma hubeiensis, P. graminicola, P. shanxiensis, and P. siamensis produce MEL-C exclusively23, 24, 31, 32）. A synthetic MEL, MEL-D, has been also synthesized via the lipasecatalyzed hydrolysis of MEL-B33）. 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 source36）. The diastereomeric form of MEL-B containing a hydroxy fatty acid was produced by P. tsukubaensis using castor oil as the carbon source37）, 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.
OF MEL-B USING SUGARCANE 5 PRODUCTION 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 source40, 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 steps16）.
6 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 phase5）. As previously reported, the moisturizing activity was evaluated using a three-dimensional cultured human （Toyobo, Japan）based on the cell skin model, TESTSKINTM viability44, 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 surface46）. 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
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Table 1 MEL producers and their products (modified from Morita et al., 2013)13). Glycolipids
Yield (g/L)
Soybean oil and glucose
MEL-A (main), MEL-B, MEL-C
165a, b, c
ND
26.2
2, 28, 29
Soybean oil
MEL-A (main), MEL-B, MEL-C
40a
2.7×10–6
28.4
2, 4, 19
Soybean oil
MEL-B (purified)
10
4.5×10–6
28.2
4, 19
n-Alkane
MEL-A (main), MEL-B, MEL-C
140a, b
ND
ND
27
Glucose
Mono-acylated MEL (purified)
1.3
3.6×10–4
33.8
36
Soybean oil
Try-acylated MEL (purified)
ND
ND
ND
34, 35
Soybean oil
Mono-acylated/tri-acetylated MEL (purified)
3.8a
1.7×10–6
29.2
15
Oliec acid and glucose
Diastereomer MEL-A (main), MEL-B, MEL-C
4.6a
5.2×10–6
26.5
20
ND
2
24.2
22
Microbial producers
Carbon sources
Pseudozyma aphidis Pseudozyma atarctica
Pseudozyma churashimaensis Pseudozyma crassa
a
cmc (M)
gcmc (mN/m)
Pseudozyma fujiformata
Soybean oil
MEL-A (main), MEL-B, MEL-C
4
ND
Pseudozyma graminicola
Soybean oil
MEL-A, MEL-B, MEL-C (main)
9.6a
4.0×10–6 a, b, c
–6
References
Pseudozyma hubeiensis
Soybean oil
MEL-A, MEL-B, MEL-C (main)
76.3
6.0×10
25.1
32
Pseudozyma parantarctica
Soybean oil
MEL-A (main), MEL-B, MEL-C
106.7a, b
ND
ND
2, 34 36
Glucose
Mono-acylated MEL (purified)
1.2
ND
ND
Soybean oil
Try-acylated MEL (purified)
22.7
ND
ND
34
Olive oil and mannitol
Mannosyl-mannitol lipids (purified)
18.2
2.6×10–6
24.2
35
Olive oil and arabitol
Mannosyl-arabitol lipids (purified)
ND
1.5×10–6
24.2
39
ND
–6
1.2×10
23.7
39
ND
ND
2, 21
Olive oil and ribitol Pseudozyma rugulosa
Mannosyl-ribitol lipids (purified)
Soybean oil and erythritol
MEL-A (main), MEL-B, MEL-C
142
a, b
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)
18.5a
4.5×10–6
Pseudozyma tsukubaensis Ustilago cynodontis Ustilago maydis Ustilago scitaminea
30.7
24
Soybean oil
Diastereomer MEL-B
73.1
3.1×10–6
26.1
2, 17, 30
Castor oil
Diastereomer MEL-B containing a hydroxy fatty acid
22
2.2×10–5
28.5
37
Soybean oil
MEL-C
1.4
ND
ND
25
Sunflower oil
MELs and cellobiose lipids
30d
ND
ND
50
Sugarcane juice
MEL-B
25.1
3.7×10–6
25.2
16
b, c
a
As a mixture of MELs Feeding using resting cells Large scale production with jer-fermenter d As a mixture of MELs and cellobiose lipids ND: no data b c
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 epidermis48）. 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 species49）. Further investigation into the antioxidant properties of MELs using cultured human skin fibroblasts under H2O2-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
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Fig. 4 Production of MEL-B from sugarcane juice supplemented with urea by Ustilago scitaminea NBRC 32730 (modified from Morita et al., 2011)11). (a) The cells of strain NBRC 32730 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
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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 previously5, 13）. Additional investigations regarding the production, biosynthesis, and physicochemical properties of these functional glycolipids will be necessary to fully exploit MELs in commercial applications.
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lerythritol lipid-B. Appl. Microbiol. Biotechnol. 88, 679-688 （2010） . 18）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, 516-523 （2014）. 19）Kitamoto, D.; Haneishi, K.; Nakahara, T.; Tabuchi, T. Production of mannosylerythritol lipids by Candida antarctica from vegetable oils. Agric. Biol. Chem. 54, 37-40 （1990） . 20）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, 29472955 （2008） . 21）Morita, T.; Konishi, M.; Fukuoka, T.; Imura, T.; Kitamoto, D. Discovery of Pseudozyma rugulosa NBRC 10877 as a novel producer of glycolipid biosurfactants, mannosylerythritol lipids, based on rDNA sequence. Appl. Microbiol. Biotechnol. 73, 305-315 （2006） . 22）Morita, T.; Konishi, M.; Fukuoka, T.; Imura, T.; Yamamoto, S.; Kitagawa, M.; Sogabe, A.; Kitamoto, D. Identification of Pseudozyma graminicola CBS 10092 as a producer of glycolipid biosurfactants, mannosyl（2008） . erythritol lipids. J. Oleo Sci. 57, 123-131 23）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, 435-442 （2007） . 24）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, 493-502 （2008） . 25）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, 549-556 （2008） . 26）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, 305-310 （1992） . 27）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, 1709-1714 . （2001） 28）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, 551-559 （2005）. 29）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, 607-613（2005）. 30）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, 555560（2008）. 31）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, 435-442（2007）. 32）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, 37-46 （2008）. 33）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, 266-271（2011）. 34）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 11752（T）. J. Oleo Sci. 57, 557-565（2008）. 35）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, 11111118（2007）. 36）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, 801-810（2007）. 37）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, 381-389（2013）. 38）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, 1017-1025（2009）.
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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, 931-938 （2012） . 40）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 32730. Biosci. Bio（2009） . technol. Biochem. 73, 788-792 41）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, 2352-2355 （2009）. 42）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, 639-642 （2009） . 43）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, 111-115 （1993） . 44）Imokawa, G.; Akasaki, S.; Minematsu, Y.; Kawai, M. Importance of intracellular lipids in water-retention properties of the stratum corneum: induction and re-
covery study of surfactant dry skin. Arch. Dermatol. Res. 281, 45-51（1989）. 45）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, 407-412（2012）. 46）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, 352-362（2004）. 47）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, 267-272（2010）. 48）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, 451-455（2010）. 49）Takahashi, M.; Morita, T.; Fukuoka, T.; Imura, T.; Kitamoto, D. Glycolipid biosurfactants, mannosylerythritol lipids, show antioxidant and protective effects against H2O2-induced oxidative stress in cultured human skin fibroblasts. J. Oleo Sci. 61, 457-464（2012）. 50）Spoeckner, S.; Wray, V.; Nimtz, M.; Lang, S. Glycolipids of the smut fungus Ustilago maydis from cultivation on renewable resources. Appl. Microbiol. Biotechnol. 51, 33-39（1999）.
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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 305-8565, 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-mannopyranosyl-erythritol or 1-O-β-Dmannopyranosylerythritol 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 Pseudozyma1−3）. MELs exhibit excellent interfacial properties4）, as well as a variety of other biochemical functions, including the induction of differentiation in mammalian cells and the binding of various classes of immunoglobulins5）. 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 liposomes6, 7）. Furthermore, certain MELs have been shown to possess anti-inflammatory properties, such as inhibiting the secretion of inflammatory mediators from mast cells8−12）. 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 pharmaceuticals5, 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. 2）14）. 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 305-8565, JAPAN E-mail: [email protected] Accepted September 3, 2014 (received for review August 22, 2014)
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
This is the review by the winner of 48th Award for Prominent Studies, The Japan Oil Chemists’ Society (JOCS).
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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: R1=Ac, R2=Ac; MEL-B: R1=Ac, R2=H; MEL-C: R1=H, R2=Ac; MEL-D: R1=H, R2=H; n = 4-16; m = 6-16.
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
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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-B16）. 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 leaves17）. Fifteen additional strains of MEL-producing fungi, representing a variety of Pseudozyma yeasts, were isolated from various vegetables and fruits using this new screening method18）.
3 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-C19）. 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 crassa2, 20−24）, in addition to the smut fungus Ustilago cynodontis, which produces primarily MEL-C when grown in the presence of vegetable oil25）. 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
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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 substrate19, 21, 26−29）. 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 MELs20）. Pseudozyma hubeiensis, P. graminicola, P. shanxiensis, and P. siamensis produce MEL-C exclusively23, 24, 31, 32）. A synthetic MEL, MEL-D, has been also synthesized via the lipasecatalyzed hydrolysis of MEL-B33）. 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 source36）. The diastereomeric form of MEL-B containing a hydroxy fatty acid was produced by P. tsukubaensis using castor oil as the carbon source37）, 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.
OF MEL-B USING SUGARCANE 5 PRODUCTION 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 source40, 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 steps16）.
6 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 phase5）. As previously reported, the moisturizing activity was evaluated using a three-dimensional cultured human （Toyobo, Japan）based on the cell skin model, TESTSKINTM viability44, 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 surface46）. 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
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Table 1 MEL producers and their products (modified from Morita et al., 2013)13). Glycolipids
Yield (g/L)
Soybean oil and glucose
MEL-A (main), MEL-B, MEL-C
165a, b, c
ND
26.2
2, 28, 29
Soybean oil
MEL-A (main), MEL-B, MEL-C
40a
2.7×10–6
28.4
2, 4, 19
Soybean oil
MEL-B (purified)
10
4.5×10–6
28.2
4, 19
n-Alkane
MEL-A (main), MEL-B, MEL-C
140a, b
ND
ND
27
Glucose
Mono-acylated MEL (purified)
1.3
3.6×10–4
33.8
36
Soybean oil
Try-acylated MEL (purified)
ND
ND
ND
34, 35
Soybean oil
Mono-acylated/tri-acetylated MEL (purified)
3.8a
1.7×10–6
29.2
15
Oliec acid and glucose
Diastereomer MEL-A (main), MEL-B, MEL-C
4.6a
5.2×10–6
26.5
20
ND
2
24.2
22
Microbial producers
Carbon sources
Pseudozyma aphidis Pseudozyma atarctica
Pseudozyma churashimaensis Pseudozyma crassa
a
cmc (M)
gcmc (mN/m)
Pseudozyma fujiformata
Soybean oil
MEL-A (main), MEL-B, MEL-C
4
ND
Pseudozyma graminicola
Soybean oil
MEL-A, MEL-B, MEL-C (main)
9.6a
4.0×10–6 a, b, c
–6
References
Pseudozyma hubeiensis
Soybean oil
MEL-A, MEL-B, MEL-C (main)
76.3
6.0×10
25.1
32
Pseudozyma parantarctica
Soybean oil
MEL-A (main), MEL-B, MEL-C
106.7a, b
ND
ND
2, 34 36
Glucose
Mono-acylated MEL (purified)
1.2
ND
ND
Soybean oil
Try-acylated MEL (purified)
22.7
ND
ND
34
Olive oil and mannitol
Mannosyl-mannitol lipids (purified)
18.2
2.6×10–6
24.2
35
Olive oil and arabitol
Mannosyl-arabitol lipids (purified)
ND
1.5×10–6
24.2
39
ND
–6
1.2×10
23.7
39
ND
ND
2, 21
Olive oil and ribitol Pseudozyma rugulosa
Mannosyl-ribitol lipids (purified)
Soybean oil and erythritol
MEL-A (main), MEL-B, MEL-C
142
a, b
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)
18.5a
4.5×10–6
Pseudozyma tsukubaensis Ustilago cynodontis Ustilago maydis Ustilago scitaminea
30.7
24
Soybean oil
Diastereomer MEL-B
73.1
3.1×10–6
26.1
2, 17, 30
Castor oil
Diastereomer MEL-B containing a hydroxy fatty acid
22
2.2×10–5
28.5
37
Soybean oil
MEL-C
1.4
ND
ND
25
Sunflower oil
MELs and cellobiose lipids
30d
ND
ND
50
Sugarcane juice
MEL-B
25.1
3.7×10–6
25.2
16
b, c
a
As a mixture of MELs Feeding using resting cells Large scale production with jer-fermenter d As a mixture of MELs and cellobiose lipids ND: no data b c
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 epidermis48）. 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 species49）. Further investigation into the antioxidant properties of MELs using cultured human skin fibroblasts under H2O2-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
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Fig. 4 Production of MEL-B from sugarcane juice supplemented with urea by Ustilago scitaminea NBRC 32730 (modified from Morita et al., 2011)11). (a) The cells of strain NBRC 32730 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
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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 previously5, 13）. Additional investigations regarding the production, biosynthesis, and physicochemical properties of these functional glycolipids will be necessary to fully exploit MELs in commercial applications.
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