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Synthesis of three types of purpose-designed mannosylerythritol lipid (MEL)-D analogues with decanoyl groups, β-GlcEL-D, α-GlcEL-D, and α-MEL-D, was accomplished utilizing our boron-mediated aglycon delivery (BMAD) methods. Their self-assembling properties, recovery effects on damaged skin cells, and antibacterial activity were evaluated. It was revealed, for the first time, that α-GlcEL-D and α-MEL-D only generated giant vesicles, indicating that slight differences in the steric configuration of an erythritol moiety and fatty acyl chains affect the ability to form vesicles. Analogue α-MEL-D exhibited significant recovery effects on damaged skin cells. Furthermore, α-MEL-D exhibited antibacterial activity as high as that for MEL-D, indicating that α-MEL-D is a promising artificial sugar-based material candidate for enhancing the barrier function of the stratum corneum, superior to a known cosmetic ingredient, and possesses antibacterial activity., (© 2022 Wiley-VCH GmbH.)

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a promising tool for the screening of glycolipid-type biosurfactants (BSs) from a crude extract of microbial products. However, it is unsuitable for the detection of lower molecular weight products because the observed ions are overlapped with matrix-derived ions at lower mass range. In this study, we applied a "matrix-free" surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) analysis using a through-hole alumina membrane as an ionization-assisting substrate. Using this method, we could detect a variety of lower molecular weight products in an extract of a glycolipid BS producer with good sensitivity. In addition, the culture solution could be analyzed directly by this method.

Biosurfactants produced by a variety of microorganisms show attractive properties (e.g., higher surface activity and biodegradability, lower toxicity, and environmental compatibility) compared to chemically synthesized counterparts. The numerous advantages of biosurfactants have prompted their application to not only the food, cosmetic, and pharmaceutical industries, but agriculture and environmental protection disciplines as well. Among different types of biosurfactants, glycolipids are the most practically useful, due to their high product titers from renewable resources and versatile interfacial and biochemical properties. Mannosylerythritol lipids (MELs) are characteristic glycolipid biosurfactants that are produced by different yeast strains of the genus Pseudozyma. MELs exhibit different lyotropic liquid crystalline phases, such as sponge (L 3 ), reverse bicontinuous cubic (V 2 ), or lamellar (L α ) phases; and they have high levels of surface activity at very low concentrations. MELs also show excellent moisturizing effects on human skin and hair, with comparable performance to natural ceramides. Today, MELs are commercially produced by a Japanese company and their use is rapidly expanding around the world. In this review, we will briefly describe the current R&D status of glycolipid biosurfactants, with a focus on the interfacial properties of MELs and their applications in cosmetic and personal care products.

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by various yeasts. Mmf1, a putative transporter of MELs, is conserved in the MEL biosynthesis gene clusters of diverse MEL producers, including the genera Ustilago, Pseudozyma, Moesziomyces, and Sporisorium. To clarify the function of Mmf1, we generated the gene-deleted strain of P. tsukubaensis ΔPtMMF1 and evaluated its MEL production. Using thin-layer chromatography analyses, we detected most MELs produced by ΔPtMMF1 in the culture supernatant. The spot size of diacylated MEL-B (the only product of the parental strain) was significantly smaller for strain ΔPtMMF1 than for the parental strain, and a mono-acylated MEL-D spot was detected. In addition, an unknown glycolipid was detected in the sample extracted from strain ΔPtMMF1. Liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses revealed that the unknown glycolipid was a novel MEL homologue, mono-acylated MEL-B. KEY POINTS: • P. tsukubaensis is able to secrete MELs without PtMMF1p. • Strain ΔPtMMF1 mainly produced mono-acylated MELs.

We applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) to screen for glycolipid-type biosurfactant (BS) producers. A crude extract of Pseudozyma antarctica, a well-known mannosylerythritol lipid (MEL) producer, was initially subjected to MALDI-TOF/MS. The spectrum of the extract showed the accumulation of diacylated MELs in culture. We then screened 80 environmental samples for BS-producing yeasts, and extracts from broth cultures of the selected five strains were examined using MALDI-TOF/MS. The results showed that all five strains produced MELs, whereas four strains also produced cellobiose lipids (CLs). By D1/D2 region sequence analysis, the MEL-producing strain was assigned to P. antarctica while the four MEL- and CL-producing strains were assigned to P. hubeiensis. These results demonstrate that MALDI-TOF/MS is a rapid and reliable tool to detect BS molecules in crude extracts of broth cultures to screen for glycolipid-type BS producers.

Surface properties of cast films of poly(lactic acid) (PLA) containing 1 wt% of several glycolipid-type biosurfactants (BSs) were investigated. The wettability of PLA films containing a homologue of mannosylerythritol lipids (MEL-B), lactone-form sophorolipid (LSL), or cellobiose lipid (CL) was drastically higher than that of untreated PLA and several synthetic surfactants-containing PLA. Surface wettability was also dependent on the hydrophilicity of the substrate used during solvent casting of the PLA films. The wetting behavior of the opposing sides of MEL-B-containing films prepared on glass substrates differed significantly; the contact angle on the side of the film that had been in contact with the glass surface was significantly lower than that obtained on the side of the film that had been in contact with air. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis results showed that the MEL in MEL-B-containing thin PLA cast films was localized to a thin surface layer. These results suggest self-assembly of MEL-B and micro-phase separation between the PLA matrix and MEL-B domains. This resulted in the localization and orientation of MEL-B at the surface of the cast PLA film, which determined its specific wetting behavior.

We discovered that Candida floricola ZM1502 is capable of selectively producing the promising hydrophilic biosurfactants, acid-form sophorolipids (SLs), from glycerol. However, productivity was very low (approximately 3.5 g L -1 ) under the initial culture conditions. Here, we describe the design of culture medium for abundant production of acid-form SLs by C. floricola ZM1502 using waste glycerol and hydrophobic substrates in order to develop a method for SL production and disposal of waste glycerol produced by oleo-chemical industries. Urea provided the best nitrogen source for acid-form SL production from glycerol among four nitrogen sources tested [urea, NaNO 3 , NH 4 NO 3 , and (NH 4 ) 2 SO 4 ]. Among carbon sources we compared, hydrophobic substrates (soybean oil and oleic acid) led to productivities of approximately 20 g L -1 , indicating that hydrophobic substrates provided fatty acid moieties for SL production. Addition of olive oil and oleic acid to waste glycerol enhanced acid-form SL production to 42.1 ± 0.9 and 37.5 ± 3.4 g L -1 , respectively. To develop a potential industrial process, we explored other suitable hydrophobic substrates for SL production, which were obtained on site from oleo-chemical industries. Alkyl C 18 esters (Pastell M-182), along with waste glycerol, increased acid-form SL production to 48.0 ± 3.4 g L -1 over a 7-d period. Furthermore, we demonstrated abundant production of acidic SLs at the mini-jar fermenter scale, obtaining 169 g L -1 over 180 h using a fed-batch cultivation technique. Efficient acid-form SL production by C. floricola could have a great impact on the development of bio-industrial processes using waste glycerol as a substrate.

The basidiomycetous yeast genus Pseudozyma produce large amounts of mannosylerythritol lipids (MELs), which are biosurfactants. A few Pseudozyma strains produce mono-acylated MEL as a minor compound using excess glucose as the sole carbon source. Mono-acylated MEL shows higher hydrophilicity than di-acylated MEL and has great potential for aqueous applications. Recently, the gene cluster involved in the MEL biosynthesis pathway was identified in yeast. Here, we generated an acyltransferase (PtMAC2) deletion strain of P. tsukubaensis 1E5 with uracil auxotrophy as a selectable marker. A PtURA5-mutant with a frameshift mutation in PtURA5 was generated as a uracil auxotroph of strain 1E5 by ultraviolet irradiation on plate medium containing 5-fluoro-orotic acid (5-FOA). In the mutant, PtMAC2 was replaced with a PtURA5 cassette containing the 5' untranslated region (UTR) (2000 bp) and 3' UTR (2000 bp) of PtMAC2 by homologous recombination, yielding strain ΔPtMAC2. Based on TLC and NMR analysis, we found that ΔPtMAC2 accumulates MEL acylated at the C-2' position of the mannose moiety. These results indicate that PtMAC2p catalyzes acylation at the C-3' position of the mannose of MEL.

Biosurfactants (BSs) are produced in abundance from various feedstocks by diverse microorganisms, and are used in various applications. In this paper, we describe a new yeast isolate that produces glycolipid-BSs from glycerol, with the aim of enhancing the utilization of the surplus glycerol produced by the oleo-chemical industry. As a result of the screening, strain ZM1502 was obtained as a potential producer of BS from glycerol. Based on TLC analysis, the strain produced glycolipid BSs. According to structural analyses (NMR, MALDI-TOF MS, and GC-MS), the main component of the glycolipids was 6',6"-di-O-acetylated acid-form sophorolipid (SL). Interestingly, the strain produced only acid-form SL, without lactone-form SLs, although the conventional SL-producing yeast, Starmerella bombicola, produces lactone-form SLs with small amounts of the acid-form. Based on taxonomy, the strain was identified as Candida floricola. It produced 3.5 g L -1 of acid-form SLs in 20% (w/v) glycerol. In addition, C. floricola CBS7290 and NBRC10700 T also produced only acid-form SLs from glycerol. These results suggest that C. floricola would enhance the utilization of waste glycerol as a fermentation feedstock and facilitate a broad range of applications for SLs.

Cutinase-like esterase from the yeasts Pseudozyma antarctica (PaE) shows strong degradation activity in an agricultural biodegradable plastic (BP) model of mulch films composed of poly(butylene succinate-co-adipate) (PBSA). P. antarctica is known to abundantly produce a glycolipid biosurfactant, mannosylerythritol lipid (MEL). Here, the effects of MEL on PaE-catalyzed degradation of BPs were investigated. Based on PBSA dispersion solution, the degradation of PBSA particles by PaE was inhibited in the presence of MEL. MEL behavior on BP substrates was monitored by surface plasmon resonance (SPR) using a sensor chip coated with polymer films. The positive SPR signal shift indicated that MEL readily adsorbed and spread onto the surface of a BP film. The amount of BP degradation by PaE was monitored based on the negative SPR signal shift and was decreased 1.7-fold by MEL pretreatment. Furthermore, the shape of PBSA mulch films in PaE-containing solution was maintained with MEL pretreatment, whereas untreated films were almost completely degraded and dissolved. These results suggest that MEL covering the surface of BP film inhibits adsorption of PaE and PaE-catalyzed degradation of BPs. We applied the above results to control the microbial degradation of BP mulch films. MEL pretreatment significantly inhibited BP mulch film degradation by both PaE solution and BP-degradable microorganism. Moreover, the degradation of these films was recovered after removal of the coated MEL by ethanol treatment. These results demonstrate that the biodegradation of BP films can be readily and reversibly controlled by a physical approach using MEL.

Aims: To investigate the effects of mannosylerythritol lipids (MELs) on the hydrophobicity of solid surfaces, their suppressive activity against the early infection behaviours of several phytopathogenic fungal conidia, and their suppressive activity against disease occurrences on fungal host plant leaves., Methods and Results: The changes in the hydrophobicity of plastic film surfaces resulting from treatments with MEL solutions (MEL-A, MEL-B, MEL-C and isoMEL-B) and synthetic surfactant solutions were evaluated based on the changes in contact angles of water droplets placed on the surfaces. The droplet angles on surfaces treated with MELs were verified to decrease within 100 s after placement, with contact angles similar to those observed on Tween 20-treated surfaces, indicating decreases in surface hydrophobicity after MEL treatments. Next, conidial germination, germ tube elongation and the formation of appressorium of Blumeria graminis f. sp. tritici, Colletotrichum dematium, Glomerella cingulata and Magnaporthe grisea were evaluated on plastic surfaces that were pretreated with surfactant solutions. On the surfaces of MEL-treated plastic film, inhibition of conidial germination, germ tube elongation, and suppression of appressoria formation tended to be observed, although the level of effect was dependent on the combination of fungal species and type of MEL. Inoculation tests revealed that the powdery mildew symptom caused by B. graminis f. sp. tritici was significantly suppressed on wheat leaf segments treated with MELs., Conclusions: MELs exhibited superior abilities in reducing the hydrophobicity of solid surfaces, and have the potential to suppress powdery mildew in wheat plants, presumably due to the inhibition of conidial germination., Significance and Impact of the Study: This study provides significant evidence of the potential for MELs to be used as novel agricultural chemical pesticides., (© 2015 The Society for Applied Microbiology.)

Glycolipid biosurfactant-producing bacteria were isolated from deep-sea sediment collected from the Okinawa Trough. Isolate BS15 produced the largest amount of the glycolipid, generating up to 6.31 ± 1.15 g l(-1) after 4 days at 20 °C. Glucose was identified in the hydrolysate of the purified major component of the biosurfactant glycolipid. According to gas chromatography/mass spectrometry analysis, the hydrophobic moieties in the major component were hexadecanoate, octadecanoate, 3-hydroxyhexadecanoate, 2-hydroxyoctanoate, and succinate. The molecular weight of the purified major glycolipid was calculated to be 1,211, while (1)H and (13)C nuclear magnetic resonance spectra confirmed that the major component consisted of 2 mol of α-glucoside and 1 mol of β-glucoside. The molecular structure was assigned as novel trisaccharide-type glycolipid biosurfactant, glucotriose lipids. The critical micelle concentration of the purified major glycolipid was 2.3 × 10(-6) M, with a surface tension of 29.5 mN m(-1). Phylogenetic analysis showed isolate BS15 was closely related to a Rhodococcus strains isolated from Antarctica, and to Rhodococcus fascians, a phytopathogen. PCR analysis showed that the fasA, fasB, fasC, fasD, fasE, and fasF genes, which are involved in phytohormone-like cytokinin production, were not present in the genome of BS15; however, analysis of a draft genome sequence of BS15 (5.5 Mb) identified regions with 31 %, 53 %, 46 %, 30 %, and 31 % DNA sequence identity to the fasA, fasB, fasC, and fasD genes, respectively.

The synergic effect and miscibility of the lactonic and acidic forms of sophorolipids (SLs) produced by Starmerella bombicola NBRC 10243 were first evaluated through atomic force microscopy (AFM), together with the Langmuir monolayer technique. The π-A isotherm of a pure lactonic sophorolipid (LS) monolayer mostly exhibited a liquid expanded monolayer, while that of an acidic sophorolipid (AS) monolayer showed a liquid condensed monolayer, suggesting that the lactonization of SLs makes the molecules more bulky and prevents them from adopting a close-packed arrangement. Plots of the mean area per molecule of mixed LS/AS monolayers gave positive deviations from the ideal curves, implying that the LS and AS molecules are miscible. Interestingly, the positive deviation of excess area (Aex) from ideality was most significant at a mole fraction (XAS) of 0.3, which closely resembles the composition of the LS/AS mixture secreted by S. bombicola in culture. The AFM images of mixed LS/AS monolayers transferred at 20 mN/m revealed no phase-separated microdomain structures, but rather showed small protruding objects for all compositions, indicating that LS and AS are partially miscible, as predicted by the positive deviations from the ideal curves. Cross-section analysis of the AFM images indicated that the observed protruding objects are AS-rich monolayers formed on the LS/AS monolayer. Our results clearly demonstrate that AFM combined with the Langmuir technique is useful for the exploration of the miscibility and synergic effects of microbial products.

The isolation of biosurfactant-producing yeasts from food materials was accomplished. By a combination of a new drop collapse method and thin-layer chromatography, 48 strains were selected as glycolipid biosurfactant producers from 347 strains, which were randomly isolated from various vegetables and fruits. Of the producers, 69% were obtained from vegetables of the Brassica family. Of the 48 producers, 15 strains gave relatively high yields of mannosylerythritol lipids (MELs), and were identified as Pseudozyma yeasts. These strains produced MELs from olive oil at yields ranging from 8.5 to 24.3 g/L. The best yield coefficient reached 0.49 g/g as to the carbon sources added. Accordingly, MEL producers were isolated at high efficiency from various vegetables and fruits, indicating that biosurfactant producers are widely present in foods. The present results should facilitate their application in the food and related industries.

In this study, spontaneous vesicle formation from the sodium salt of acidic sophorolipid (SLNa) was observed, and its potential application as a skin penetration enhancer for triterpene glycosides extracted from the fruits of Siraitia grosvenorii Swingle was then investigated. Dynamic light scattering (DLS) measurements of the SLNa assemblies prepared by the gentle mixing of SLNa with water (1%) showed their hydrodynamic radius (Rh) to be 96.2 nm, and their structure was assigned to be vesicles by freeze-fracture electron microscopy (FF-TEM). DLS and FF-TEM also revealed that the size of the vesicles increased with an increase in the concentration of the triterpene glycosides, indicating that the triterpene glycosides were incorporated into the SLNa vesicles. The results of an in vitro skin permeation assay, after loading the SLNa vesicles on a 3D cultured skin model, showed that the amount of SLNa that penetrated though the skin model increased with time. It was also found that the amount of permeated mogroside, which is the main active component of triterpene glycosides, was significantly enhanced by the SLNa vesicle formulation. These results clearly demonstrated that spontaneously formed vesicles composed of the bolaamphiphile SLNa are useful for application as penetration enhancers for active ingredients such as mogroside V.

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants abundantly produced by different basidiomycetous yeasts such as Pseudozyma, and show not only excellent interfacial properties but also versatile biochemical actions. These features of MELs make their application in new technology areas possible. Recently, the structural and functional variety of MELs was considerably expanded by advanced microbial screening methods. Different types of MELs bearing different hydrophilic and hydrophobic parts have been reported. The genes responsible for MEL biosynthesis were identified, and their genetic study is now in progress, aiming to control the chemical structure. The excellent properties leading to practical cosmetic ingredients, i.e., moisturization of dry skin, repair of damaged hair, activation of fibroblast and papilla cells and antioxidant and protective effects in skin cells, have been demonstrated on the yeast glycolipid biosurfactants. In this review, the current status of research and development on MELs, particularly the commercial application in cosmetics, is described.

To facilitate the development of bio-based chemicals from renewable and inexpensive natural resources, we sought to produce biosurfactants using non-edible jatropha oil. Twenty yeasts known to produce biosurfactants were tested in this study, and Stamerella bombicola NBRC 10243 was found to use jatropha oil efficiently to produce sophorolipids (SLs) as a mixture of lactone-form SL (L-SL) and acid-form SL (A-SL). Under culture conditions using rice bran as the source of organic nutrients, the yield of SLs reached 122.6 g/L in 5-L jar fermentors after 9 d in culture. HPLC analysis of the culture medium showed that the levels of phorbol esters (PEs), major toxic components of the oil, decreased markedly with an increase in culture time, suggesting that the yeast degrades PEs. Although the SLs obtained by solvent extraction of the culture medium contained a small amount of PEs, the sodium salt of A-SL (A-SL-Na) obtained by alkaline treatment (5N NaOH, 80°C) showed no PE peaks upon HPLC analysis. A-SL-Na had excellent surface activity with low CMC (9.0×10⁻⁴ M) and γ(CMC) (29.6 mN/m), which are lower than that of sodium dodecyl sulfate (SDS). The solubilizing ability of A-SL-Na toward for octanoic acid ([octanoic acid]/[A-SL-Na]) was found to be 2.0, which is half that of SDS. Our findings should help improve SL production from non-edible feedstock and broaden the use of promising bio-based surfactants.

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by various yeasts belonging to the genus Pseudozyma, which exhibit excellent surface activities as well as versatile biochemical activities. A study on P. tsukubaensis NBRC1940 as a mono-acetylated MEL (MEL-B) producer revealed that the yeast accumulated a novel glycolipid from castor oil at a yield of 22 g/L. Its main chemical structure was identified as 1-O-β-(2'-O-alka(e)noyl-3'-O-hydroxyalka(e)noyl-6'-O-acetyl-D-mannopyranosyl)-D-erythritol designated as "new MEL-B." The new MEL-B, comprising a hydroxy fatty acid had a reduced surface tension of 28.5 mN/m at a critical micelle concentration (CMC) of 2.2×10⁻⁵ M in water. The observed CMC was 5-fold higher than that of conventional MEL-B. When conventional MEL-B was dispersed in water, it self-assembled to form the lamellar (L(α)) phase at a wide range of concentrations. In contrast, new MEL-B formed spherical oily droplets similar to the sponge (L₃) phase, which is observed in aqueous solutions of di-acetylated MEL (MEL-A). The data suggest that the newly identified MEL-B is likely to have a different structure and interfacial properties compared to the conventional MELs, and could facilitate an increase in the application of glycolipid biosurfactants.

In order to develop novel glycolipid biosurfactants, Pseudozyma parantarctica JCM 11752(T), which is known as a producer of mannosylerythritol lipids (MEL), was cultivated using different sugar alcohols with the presence of vegetable oil. When cultivated in a medium containing 4 % (w/v) olive oil and 4 % D-ribitol or D-arabitol, the yeast strain provided different glycolipids, compared to the case of no sugar alcohol. On TLC, both of the extracted glycolipid fractions gave two major spots corresponding to MEL-A (di-acetylated MEL) and MEL-B (mono-acetylated MEL). Based on (1)H NMR analysis, one glycolipid was identified as MEL-A, but the other was not MEL-B. On high-performance liquid chromatography after acid hydrolysis, the unknown glycolipid from the D-ribitol culture provided mainly two peaks identical to D-mannose and D-ribitol, and the other unknown glycolipid from the D-arabitol culture did two peaks identical to D-mannose and D-arabitol. Accordingly, the two unknown glycolipids were identified as mannosylribitol lipid (MRL) and mannosylarabitol lipid (MAL), respectively. The observed critical micelle concentration (CMC) and surface tension at CMC of MRL were 1.6 × 10(-6) M and 23.7 mN/m, and those of MAL were 1.5 × 10(-6) M and 24.2 mN/m, respectively. These surface-tension-lowering activities were significantly higher compared to conventional MEL. Furthermore, on a water-penetration scan, MRL and MAL efficiently formed not only the lamella phase (L(α)) but also the myelins at a wide range of concentrations, indicating their excellent self-assembling properties and high hydrophilicity. The present two glycolipids should thus facilitate the application of biosurfactants as new functional materials.

Glycolipid biosurfactants, such as mannosylerythritol lipids (MELs), are produced by different yeasts belonging to the genus Pseudozyma and have been attracting much attention as new cosmetic ingredients owing to their unique liquid-crystal-forming and moisturizing properties. In this study, the effects of different MEL derivatives on the skin were evaluated in detail using a three-dimensional cultured human skin model and an in vivo human study. The skin cells were cultured and treated with sodium dodecyl sulfate (SDS), and the effects of different lipids on the SDS-damaged cells were evaluated on the basis of cell viability. Most MEL derivatives efficiently recovered the viability of the cells and showed high recovery rates (over 80%) comparable with that of natural ceramide. It is interesting that the recovery rate with MEL-A prepared from olive oil was significantly higher than that of MEL-A prepared from soybean oil. The water retention properties of MEL-B were further investigated on human forearm skin in a preliminary study. Compared with the control, the aqueous solution of MEL-B (5 wt%) was estimated to considerably increase the stratum corneum water content in the skin. Moreover, perspiration on the skin surface was clearly suppressed by treatment with the MEL-B solution. These results suggest that MELs are likely to exhibit a high moisturizing action, by assisting the barrier function of the skin. Accordingly, the yeast glycolipids have a strong potential as a new ingredient for skin care products.

Mannosylerythritol lipids (MELs) are biosurfactants known for their versatile interfacial and biochemical properties. To broaden their application in cosmetics, we investigated the antioxidant properties of different MEL derivatives (MEL-A, -B, and -C) by using a 1,1-diphenyl-2-picryl hydrazine (DPPH) free-radical- and superoxide anion-scavenging assay. All MEL derivatives tested showed antioxidant activity in vitro, but at lower levels than those of arbutin. Of the MELs, MEL-C, which is produced from soybean oil by Pseudozyma hubeiensis, showed the highest rates of DPPH radical scavenging (50.3% at 10 mg/mL) and superoxide anion scavenging (>50% at 1 mg/mL). The antioxidant property of MEL-C was further examined using cultured human skin fibroblasts (NB1RGB cells) under H(2)O(2) induced oxidative stress. Surprisingly, MEL-C had a higher protective activity against oxidative stress than arbutin did: 10 µg/mL of MEL-C and arbutin had protective activities of 30.3% and 13%, respectively. Expression of an oxidative stress marker, cyclooxygenase-2, in these cells was repressed by treatment with MEL-C as well as by arbutin. MEL-C was thus confirmed to have antioxidant and protective effects in cells, and we suggest that MELs have potential as anti-aging skin care ingredients.

Forty-three fungal producers for glycolipid biosurfactants, mannosylerythritol lipids (MELs), were isolated from leaves and smuts of sugarcane plants. These isolates produced MELs with sugarcane juice as nutrient source. The strains were taxonomically categorized into the genera Pseudozyma and Ustilago on the basis of partial sequences of the ribosomal RNA gene.

Mannosylerythritol lipids (MELs) are secreted by yeasts and are promising glycolipid biosurfactants. In our study on the non-aqueous phase behaviors of MEL homologues, we found that MEL-D (4-O-[2',3'-di-O-alka(e)noyl-β-D-mannopyranosyl]-(2R,3S)-erythritol) forms aggregates in decane. The microscopic observation and the X-ray scattering measurement of these aggregates revealed that they are reverse vesicles that consist of bilayers whose hydrophilic domains are located in the interior of the bilayers. In addition, MEL-D formed reverse vesicles without co-surfactants and co-solvents in various oily solutions, such as n-alkanes, cyclohexane, squalane, squalene, and silicone oils at a concentration below 10 mM. This is the first report on the reverse vesicle formation from biosurfactants.

Cellobiose lipids (CLs) are bolaform glycolipid biosurfactants, which are produced from natural resources by a yeast strain and show fungicidal activity. In this study, the gelation properties of CL in solvents were investigated by several techniques including rheology and atomic force microscopy (AFM). The yeast CL was found to gelate 6 out of 26 solvents. Although it did not provide gels in ethanol or 1, 3-butanediol which are widely used for cosmetic industries, we succeeded in producing gels by mixing ethanol or 1, 3-butanediol with water. AFM observation of the gels on a silicon substrate provided 3D supramolecular structures with an entangled fibrous network. Moreover, it was also found that some of fibrous structures were twisted helical ribbons. This should be due to the cellobiose backbone having several chiral functional groups. The sol-gel phase transition temperatures for gels in mixed ethanol/water and 1, 3-butanediol/water systems were below 100°C, indicating that the gels can be obtained with rather mild preparation conditions. The present CL gels would be useful for novel multifunctional materials applicable to various industries.

Glyceric acid (GA) is one of the most promising functional hydroxyl acids, and it is abundantly obtained from glycerol by a bioprocess using acetic acid bacteria. In this study, several monoacyl GAs were synthesized by esterification of GA and saturated fatty acyl chlorides (C12, C14, C16, and C18), forming a new class of bio-based surfactants. By the present method, a mixture of two isomers, namely 2-O-acyl and 3-O-acyl GAs, was produced, in which the 2-O-acyl derivatives were obtained as a major product. These isomers were isolated, and their surface-active properties were investigated for the first time. The surface tensions of 2-O-acyl GAs with different chain lengths were determined by the Wilhelmy method. At concentrations below 10(-4) M, the 2-O-acyl GAs exhibited higher surface-active properties compared to commercially available synthetic surfactants. For example, 2-O-lauroyl GA reduced the surface tension of water to around 25 mN/m above the critical micelle concentration (3.0×10(-4) M). In addition, 2-O-acyl derivatives showed higher surface-tension-lowering activity than 3-O-acyl GAs. The monoacyl GAs synthesized herein can potentially be used as "green surfactants."

An ustilaginomycetous anamorphic yeast species isolated from the leaves of Saccharum officinarum (sugarcane) in Okinawa, Japan, was identified as a novel Pseudozyma species based on morphological and physiological aspects and molecular taxonomic analysis using the D1/D2 domains of the large subunit (26S) rRNA gene and the internal transcribed spacer 1 (ITS1)-5.8S-ITS2 regions. The name Pseudozyma churashimaensis sp. nov. was proposed for the novel species, with JCM 16988(T) as the type strain. Interestingly, P. churashimaensis was found to produce glycolipid biosurfactants, a mixture of mannosylerythritol lipids (MELs), including a novel tri-acetylated derivative (MEL-A2), from glucose. The observed critical micelle concentration (CMC) and the surface tension at CMC of MEL-A2 were 1.7 × 10⁻⁶ M and 29.2 mN/m, respectively. Moreover, on a water-penetration scan, MEL-A2 efficiently formed different lyotropic liquid crystalline phases, including the lamella phase at a wide range of concentrations, indicating its excellent surface-active and self-assembling properties. The novel strain of the genus Pseudozyma should thus facilitate the application of glycolipid biosurfactants in combination with other MEL producers., (Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

We improved the culture conditions for a biosurfactant producing yeast, Pseudozyma hubeiensis SY62. We found that yeast extract greatly stimulates MEL production. Furthermore, we demonstrated a highly efficient production of MELs in the improved medium by fed-batch cultivation. The final concentration of MELs reached 129 ± 8.2g/l for one week., (Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Mannosylerythritol lipids (MELs) produced by yeasts are one of the most promising glycolipid biosurfactants. In this study, we succeeded in the preparation of a novel MEL homolog having no acetyl groups, namely MEL-D. MEL-D was synthesized by lipase-catalyzed hydrolysis of acetyl groups from a known MEL, and identified as 4-O-[2',3'-di-O-alka(e)noyl-β-d-mannopyranosyl]-(2R,3S)-erythritol. The obtained MEL-D showed a higher critical aggregation concentration (CAC=1.2 × 10(-5)M) and hydrophilicity compared to known MELs, retaining an excellent surface tension lowering activity (the surface tension at the CAC was 24.5mN/m). In addition, we estimated the binary phase diagram of the MEL-D-water system based on a combination of visual inspection, polarized optical microscopy, and SAXS measurement. From these results, MEL-D was found to self-assemble into a lamellar (L(α)) structure over all ranges of concentration. Meanwhile, the one-phase L(α) region of MEL-D was extended wider than those of known MELs. MEL-D might keep more water between the polar layers in accordance with the extension of the interlayer spacing (d). These results suggest that the newly obtained MEL-D would facilitate the application of MELs in various fields as a lamellar-forming glycolipid with higher hydrate ability., (2010 Elsevier Ltd. All rights reserved.)

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants excreted by fungal strains. They show not only excellent surface-active properties but also versatile biochemical actions. Ustilago scitaminea NBRC 32730 has been reported mainly to produce a mono-acetylated and di-acylated MEL, MEL-B, from sucrose as sole carbon source. In order to make biosurfactant production more efficient, we focused our attention on the use of sugarcane juice, one of the most economical resources. The fungal strain produced MEL-B at the yield of 12.7 g/L from only sugarcane juice containing 22.4% w/w sugars. Supplementation with organic (yeast extract, peptone, and urea) and inorganic (sodium nitrate and ammonium nitrate) nitrogen sources markedly enhanced the production yield. Of the nitrogen sources, urea gave the best yield. Under optimum conditions, the strain produced 25.1 g/L of MEL-B from the juice (19.3% sugars) supplemented with 1 g/L of urea in a jar fermenter at 25 °C over 7 d. The critical micelle concentration (CMC) and the surface-tension at the CMC for the present MEL-B were 3.7×10(-6) M and 25.2 mN/m respectively. On water-penetration scan, the biosurfactant efficiently formed the lamella phase (L(α)) and myelins over a wide range of concentrations, indicating excellent surface-active and self-assembling properties. More significantly, the biosurfactant showed a ceramide-like skin-care property in a three-dimensional cultured human skin model. Thus, sugarcane juice is likely to be effective in glycolipid production by U. scitaminea NBRC 32730, and should facilitate the application of MELs.

Cryptococcus humicola JCM 1461 efficiently produced cellobiose lipids (CLs), bolaform biosurfactants. The main product was identified as 16-O-(2″,3″,4″,6'-tetra-O-acetyl-β-cellobiosyl)-2-hydroxyhexadecanoic acid. The production yield of CLs reached 13.1 g/L under the intermittent feeding of glucose. The critical micelle concentrations (CMC) of the main product at pH 4.0 and 7.0 were 3.3×10(-5) M and 4.1×10(-4) M respectively.

Biosurfactants (BS) are produced by a variety of microorganisms from renewable resources, and have unique properties compared to chemical surfactants. In order to attain efficient production of BS from low-cost materials, we focused our attention on the use of sugarcane molasses. Fifteen yeast strains that are known as BS producers were examined for BS productivity from a culture medium consisting of only molasses and water. Among the strains tested, only Starmerella bombicola NBRC 10243 produced sophorolipids (SL), which are glycolipid BS. The culture conditions for the yeast were then investigated in a shake-flask culture. SL production was significantly affected by the pH of the medium and was highly accelerated at pH 6. Under the optimum conditions, the amount of SL reached 14.4 g/L after 120 h from a medium containing 150 g/L of total sugars. We tried to improve the production of SL further by feeding the molasses using a jar fermentor. Interestingly, the amount of SL increased up to 22.8 g/L after 120 h; the production rate was 1.6-fold higher than that in the shake-flask culture. These results suggest that the present yeast should have great potential for the low-cost production of SL, and facilitate the application of BS in various fields.

The producers of glycolipid biosurfactant, mannosylerythritol lipid-B (MEL-B), were isolated from leaves of Perilla frutescens on Ibaraki in Japan. Four isolates, 1D9, 1D10, 1D11, and 1E5, were identified as basidiomycetous yeast Pseudozyma tsukubaensis by rDNA sequence and biochemical properties. The structure of MEL-B produced by these strains was analyzed by (1)H nuclear magnetic resonance and gas chromatography-mass spectrometry methods, and was determined to be the same as the diastereomer MEL-B produced by P. tsukubaensis NBRC 1940. Of these isolates, P. tsukubaensis 1E5 (JCM 16987) is capable of producing the largest amount of the diastereomer MEL-B from vegetable oils. In order to progress the diastereomer MEL-B production by strain 1E5, factors affecting the production, such as carbon and organic nutrient sources, were further examined. Olive oil and yeast extract were the best carbon and nutrient sources, respectively. Under the optimal conditions, a maximum yield, productivity, and yield coefficient of 73.1 g/L, 10.4 g L(-1) day(-1), and 43.5 g/g were achieved by feeding of olive oil in a 5-L jar-fermenter culture using strain 1E5.

We describe a detailed structure determination of biosurfactant produced by Pseudozyma hubeiensis SY62, which was newly isolated from Calyptogena soyoae (deep-sea cold-seep clam, Shirouri-gai) at 1156 m in Sagami bay. P. hubeiensis SY62 was taxonomically slightly different from the P. hubeiensis type strain, which produces biosurfactants. Glycolipid production by the strain was also slightly different from those of previously reported strains. BS productivity was estimated to be around 30 g/l from the weight of the crude extract. At least five different spots of glycolipid biosurfactants (BSs) were detected by TLC. Results of nuclear magnetic resonance spectroscopies indicated the major product, namely MEL-C (4-O-[4'-O-acetyl-2',3'-di-O-alka(e)noil-beta-d-mannopyranosyl]-d-erythritol), as a promising BS. By further structural determination, the major fatty acids of MEL-C were estimated to be saturated C(6), C(10), and C(12) acids, which were shorter than those of previously reported MEL-C. Furthermore, (1)H-NMR spectra implied the presence of C(2) acids as acyl groups. According to surface tension determination, the novel MEL-C showed larger critical micelle concentration (1.1x10(-5) M) than conventional MEL-C which bound C(10) and C(12) acids (9.1x10(-6) M). From these results, shorter fatty acids would confer hydrophilicity onto the novel MEL-C., (Copyright 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Sophoroselipids (SL) are bolaform biosurafactants which are abundantly produced by microorganisms from renewable resources. In this study, four kinds of bolaform biosurfactants were produced, and these derivatives were chemoenzymatically synthesized from "acid form" diacetylated SL (SLdiAc), which are preferentially produced by Candida floricola TM 1502 which was newly found in our group. The effects of the structure of sugar moiety on their surface-active properties were investigated by surface tension measurement. After microbial production of SLdiAc from oleic acid/glucose, SLdiAc was converted into acetylated glucoselipid (GLAc). Among twelve species of glucosidases, pectinase and pectolyase including polygalacturonase were found to cleave the beta-1,2-glycosidic linked disaccharide, especially pectolyase produced GLAc effectively at 40 degrees C and pH 4.0. The structure of the major component of purified GLAc was assigned as 17- [(beta-D-glucopyranosyl)oxy]-cis-9-octadecenoate 6'-acetate by using NMR analyses, MALDI-TOF/MS and GC-MS. Glucoselipid (GL) without acetyl group was also enzymatically converted from SL obtained from alkaline hydrolysis of SLdiAc. Interestingly, the estimated CMC values of SLdiAc, SL, GLAc, GL indicated almost the same values despite their difference in hydrophilic structure. Although the difference in monosaccaride and disaccharide also did not affect gammaCMC, the presence of acetyl group on sugar moiety was found to lower the gammaCMC value slightly, suggesting that the acetyl group on produced bolaform biosurfactant is likely to play more important role to reduce the free energy of air/water interface.

Mannosylerythritol lipids (MELs), are produced from feedstock by the genus Pseudozyma, and are the most promising biosurfactants known due to its versatile interfacial and biochemical actions. In order to broaden the application in cosmetics, the hair care properties of MELs were investigated using damaged hair. On electron microscopic observation, the damaged hair was dramatically recovered with applying MEL-A and MEL-B. The tensile strength of the damaged hair increased by treatment with MEL-A (122.0 +/- 13.5 gf/p), MEL-B (119.4 +/- 7.6 gf/p) and ceramide (100.7 +/- 15.9 gf/p) compared with only lauryl glucoside (96.7 +/- 12.7 gf/p), indicating the advantage of MELs on hair care treatment. In addition, the average friction coefficient of the damaged hair was maintained after treatment with MEL-A (0.108 +/- 0.002), MEL-B (0.107 +/- 0.003) and the ceramide (0.111 +/- 0.003), although lauryl glucoside treatment increased the average friction coefficient (0.126 +/- 0.003). The increase of bending rigidity by treatment with lauryl glucoside (0.204 +/- 0.002) was prevented by treatment with MEL-A (0.129 +/- 0.002), MEL-B (0.176 +/- 0.003) and the ceramide (0.164 +/- 0.002). Consequently, MELs are proposed to be the new hair care ingredient, which are the highly useful agent for not only for the recovery of damaged hair but also for providing the smooth and flexible hair.

Mannosylerythritol lipids (MELs), the extracellular glycolipids produced from feedstock by yeasts belonging to the genus Pseudozyma, are the most promising biosurfactants known due to its versatile interfacial and biochemical actions. In order to broaden the application in cosmetics, the cell activating property of MELs was investigated using cultured fibroblast and papilla cells, and a three-dimensional cultured human skin model. The di-acetylated MEL (MEL-A) produced from soybean oil significantly increased the viability of the fibroblast cells over 150% compared with that of control cells. On the other hand, no cell activation was observed by the treatment with MEL-A produced from olive oil. The mono-acetylated MEL (MEL-B) hardly increased the cell viability. The viability of the fibroblast cells decreased with the addition of more than 1 microg/L of MELs, whereas the cultured human skin cells showed high viability with 5 microg/L of MELs. Interestingly, the papilla cells were dramatically activated with 0.001 microg/L of MEL-A produced from soybean oil: the cell viability reached at 150% compared with that of control cells. Consequently, the present MEL-A produced from soybean oil should have a potential as a new hair growth agent stimulating the papilla cells.

Glycolipid biosurfactants, mannosylerythritol lipids (MELs), were produced from glucose and sucrose without vegetable oils. Pseudozyma antarctica JCM 10317, Ustilago maydis NBRC 5346, U. scitaminea NBRC 32730, and P. siamensis CBS 9960 produced mainly MEL-A, MEL-A, MEL-B, and MEL-C respectively. The sucrose-derived MELs showed excellent interfacial properties: low critical micelle concentration as well as that of oil-derived MELs.

The development of a novel glycolipid biosurfactant was undertaken using the high-level producers of mannosylerythritol lipids (MELs) such as Pseudozyma parantarctica, Pseudozyma antarctica, and Pseudozyma rugulosa. Besides the conventional MELs (MEL-A, MEL-B, and MEL-C), these yeasts produced an unknown glycolipid when they were cultivated in a medium containing 4% (w/v) olive oil and 4% (w/w) mannitol as the carbon source. The unknown glycolipid extracted from the culture medium of P. parantarctica JCM 11752(T) displayed the spot with lower mobility than that of known MELs on TLC and provided mainly two peaks identical to mannose and mannitol on high-performance liquid chromatography after acid hydrolysis. Based on structural analysis by (1)H and (13)C nuclear magnetic resonance, the novel glycolipid was composed of mannose and mannitol as the hydrophilic sugar moiety and was identified as mannosylmannitol lipid (MML). Of the strains tested, P. parantarctica JCM 11752(T) gave the best yield of MML (18.2 g/L), which comprised approximately 35% of all glycolipids produced. We further investigated the interfacial properties of the MML, considering the unique hydrophilic structure. The observed critical micelle concentration (CMC) and the surface tension at CMC of the MML were 2.6 x 10(-6) M and 24.2 mN/m, respectively. In addition, on a water-penetration scan, the MML efficiently formed not only the lamella phase (Lalpha) but also the myelins at a wide range of concentrations, indicating its excellent self-assembling properties and high hydrophilicity. The present glycolipid should thus facilitate the application of biosurfactants as new functional materials.

BSs (biosurfactants) produced by various micro-organisms show unique properties (e.g. mild production conditions, lower toxicity, higher biodegradability and environmental compatibility) compared with chemically synthesized surfactants. The numerous advantages of BSs have prompted applications not only in the food, cosmetic and pharmaceutical industries but also in environmental protection and energy-saving technology. Among BSs, glycolipid types are the most promising, owing to their high productivity from renewable resources and versatile biochemical properties. MELs (mannosylerythritol lipids), which are glycolipid BSs abundantly produced by basidiomycetous yeasts such as strains of Pseudozyma, exhibit not only excellent interfacial properties, but also remarkable differentiation-inducing activities against human leukaemia cells. MELs also show high binding affinity towards different immunoglobulins and lectins. Recently, a cationic liposome bearing MEL has been demonstrated to increase dramatically the efficiency of gene transfection into mammalian cells. These features of BSs should broaden their application in new advanced technologies. In the present review the current status of research and development on glycolipid BSs, especially their production by Pseudozyma yeasts, is described.

A smut fungus Ustilago scitaminea NBRC 32730 on sugar cane (Saccharum) was found to accumulate a large amount of glycolipids in the culture medium. As a result of structural characterization, the main glycolipid was identified as MEL-B, 4-O-beta-(2',3'-di-O-alka(e)noyl-6'-O-acetyl-D-mannopyranosyl)-erythritol. The MEL-B was sufficiently produced from a variety of sugars such as sucrose, glucose, fructose, and mannose. Olive oil and methyl oleate were also available as carbon sources to produce MEL-B. However, these residual oils made product recovery very complicated. Under optimal conditions, a maximum MEL yield of 12.8 g/l was achieved by feeding of sucrose.

Mannosylerythritol lipids (MELs) are produced in large amounts from renewable vegetable oils by Pseudozyma antarctica, and are the most promising biosurfactants known due to its versatile interfacial and biochemical actions. In order to broaden the application in cosmetics and pharmaceuticals, the skin care property of MEL-A, the major component of MELs, was investigated using a three-dimensional cultured human skin model. The skin cells were cultured and treated with sodium dodecyl sulfate (SDS) solution of 1 wt%, and the effects of different lipids on the SDS-damaged cells were then evaluated on the basis of the cell viability. The viability of the damaged cells was markedly recovered by the addition of MEL-A in a dose-dependent manner. Compared to the control, MEL-A solutions of 5 wt% and 10 wt% gave the recovery rate of 73% and 91%, respectively, while ceramide solution of 1 wt% gave the rate of over 100%. This revealed that MEL-A shows a ceramide-like moisturizing activity toward the skin cells. Considering the drawbacks of natural ceramides, namely limited amount and high production cost, the yeast biosurfactants should have a great potential as a novel moisturizer for treating the damaged skin.

Mannosylerythritol lipids (MELs) are one of the most promising glycolipid biosurfactants produced by yeast strains of the genus Pseudozyma. In this study, the aqueous-phase behavior of a new monoacetyl MEL derivative, 1-O-beta-(2',3'-di-O-alka(e)noyl-6'-O-acetyl-d-mannopyranosyl)-d-erythritol (MEL-B), was investigated using polarized optical microscopy, small-angle X-ray scattering (SAXS), confocal laser scanning microscopy (CLSM), and differential scanning calorimetry (DSC). The present MEL-B was found to self-assemble into a lamellar (L(alpha)) phase over remarkably wide concentration and temperature ranges. According to SAXS measurement, the interlayer spacing (d) was estimated to be almost constant (about 4.7 nm) at the low MEL-B concentration (60 wt.%) region, the d-spacing gradually decreased to 3.1 nm with an increase in the MEL-B concentration. The thermal stability of the liquid crystalline phase was investigated by DSC measurement. The obtained L(alpha) phase was found to be stable up to 95 degrees C below a MEL-B concentration of 85 wt.%; then, the melting temperature of the liquid crystalline phase dramatically decreased with an increase in MEL-B concentration (above 85 wt.%). Furthermore, we found relatively large vesicles (1-5 microm) at the low MEL-B concentration using CLSM observation. The trapped volume of the obtained MEL-B vesicle was estimated to be about 0.42 microL/mumol by glucose dialysis method. These results suggest that the natural glycolipid biosurfactant, the newly found MEL-B, would be useful in various fields of applications as an L(alpha) phase- and/or vesicle-forming lipid.

The search for a novel producer of glycolipid biosurfactants, mannosylerythritol lipids (MELs), was undertaken on the basis of the analysis of ribosomal DNA sequences of yeast strains of the genus Pseudozyma. In the course of the investigation, Pseudozyma siamensis CBS 9960, which is closely related to Pseudozyma shanxiensis, a known MEL-C producer but with a different morphology, was found to accumulate a large amount of glycolipids. On thin layer chromatography, the extracellular glycolipids showed nearly the same spots as those of the MELs produced by P. shanxiensis. However, the result of high-performance liquid chromatography analysis revealed that the present strain has a much higher glycolipid production yield than P. shanxiensis. From the structural characterization by (1)H and (13)C NMR, the major glycolipid (more than 84% of the total) was identified as a mixture of 4-O-[(2',4'-di-O-acetyl-3'-O-alka(e)noyl)-beta-D-mannopyranosyl]-D-erythritol and 4-O-[(4'-O-acetyl-3'-O-alka(e)noyl-2'-O-butanoyl)-beta-D-mannopyranosyl]-D-erythritol, both of which are types of MEL-C. The present MEL-C possessed a short-chain acid (C(2) or C(4)) at the C-2' position and a long-chain acid (C(16)) at the C-3' position of the mannose moiety, and thus, the hydrophobic part was considerably different from that of conventional MELs, which mainly possess two medium-chain acids (C(10)) at the C-2' and C-3' positions. Under optimal growth conditions with safflower oil in a shake culture, the total amount of MELs reached approximately 19 g/l after 9 d at 25 degrees C. We further investigated the interfacial properties of the present MEL-C, considering its unique hydrophobic structure. The observed critical micelle concentration (CMC) and the surface tension at the CMC of the MEL were 4.5 x 10(-6) M and 30.7 mN/m, respectively. In addition, on a water penetration scan, the MEL efficiently formed the liquid crystal phases such as hexagonal (H) and lamella (L(a)) at a wide range of concentrations. These results demonstrated that the newly identified MEL-C produced by P. siamensis exhibits not only high surface activity but also excellent self-assembling properties, and should facilitate the development of promising yeast biosurfactants.

Mannosylerythritol lipid-A (MEL-A) is one of the most promising glycolipid biosurfactants, and abundantly produced by Pseudozyma yeasts. MEL-A gives not only excellent self-assembling properties but also a high binding affinity toward human immunoglobulin G (HIgG). In this study, three kinds of MEL-A were prepared from methyl myristate [MEL-A (m)], olive oil [MEL-A (o)], and soybean oil [MEL-A (s)], and the effect of interfacial properties of each MEL-A monolayer on the binding affinity toward HIgG was investigated using surface plasmon resonance (SPR) and the measurement of surface pressure (pi)-area (A) isotherms. Based on GC-MS analysis, the main fatty acids were C(8) and C(10) acids in all MEL-A, and the content of unsaturated fatty acids was 0% for MEL-A (m), 9.1% for MEL-A (o), 46.3% for MEL-A (s), respectively. Interestingly, the acid content significantly influenced on their binding affinity, and the monolayer of MEL-A (o) gave a higher binding affinity than that of MEL-A (m) and MEL-A (s). Moreover, the mixed MEL-A (o)/ MEL-A (s) monolayer prepared from 1/1 molar ratio, which comprised of 27.8% of unsaturated fatty acids, indicated the highest binding affinity. At the air/water interface, MEL-A (o) monolayer exhibited a phase transition at 13 degrees C from a liquid condensed monolayer to a liquid expanded monolayer, and the area per molecule significantly expanded above 13 degrees C, while the amount of HIgG bound to the liquid expanded monolayer was much higher than that bound to liquid condensed monolayer. The binding affinity of MEL-A toward HIgG is thus likely to closely relate to the monolayer packing density, and may be partly controlled by temperature.

A basidiomycetous yeast, Pseudozyma graminicola CBS 10092, was found to accumulate a large amount of glycolipids in the cultured medium when grown on soybean oil as the sole carbon source. Based on thin layer chromatography, the extracellular glycolipids gave spots corresponding to those of mannosylerythritol lipids (MELs), which are highly functional and promising biosurfactants. From the structural characterization by 1H and 13C NMR, the main product was identified as 4-O-[(4'-mono-O-acetyl-2', 3'-di-O-alka(e)noyl)-beta-D-mannopyranosyl]-D-erythritol, which is a highly hydrophilic derivative of MELs known as MEL-C. According to high-performance liquid chromatography analysis, the main product, MEL-C, comprised approximately 85% of all the MELs, and the total amount reached approximately 10 g/L for 7 days. The fatty acids of the present MEL-C consisted of mainly C6, C8 and C14 acids, considerably different from those of MEL-C produced by other Pseudozyma strains such as P. antarctica and P. shanxiensis. The observed critical micelle concentration (CMC) and the surface-tension at CMC of the MEL-C were 4.0 x 10(-6) M and 24.2 mN/m, respectively, while those of MEL-A, the most intensively studied MEL, were 2.7 x 10(-6) M and 28.4 mN/m, respectively. This implied that the MEL-C has higher hydrophilicity than conventional MELs hitherto reported. In addition, on a water-penetration scan, the MEL-C efficiently formed the lamella phase (Lalpha) at a wide range of concentrations, indicating its excellent self-assembling properties. From these results, the newly identified MELs produced by P. graminicola are likely to have great potential for use in oil-in-water type emulsifiers and/or washing detergents, and would thus facilitate a broad range of applications for the promising yeast biosurfactants.

Mannosylerythritol lipids (MELs) are one of the most promising biosurfactants known, because of their multifunctionality and biocompatibility. In order to attain an efficient production of MELs, Pseudozyma parantarctica JCM 11752(T), which is a newly identified strain of the genus, was examined for the productivity of MELs at different culture conditions. The yeast strain showed significant cell growth and production of di-acylated MELs even at 36 degrees C. In contrast, on conventional high-level MEL producers including P. rugulosa, the MEL yield considerably decreased with an increase of the cultivation temperature at over 30 degrees C. On P. parantarctica, soybean oil and sodium nitrate were the best carbon and nitrogen sources, respectively. Under the optimal conditions on a shake-flask culture at 34 degrees C, the amount of di-acylated MELs reached over 100 g/L by intermittent feeding of only soybean oil. Interestingly, the yeast strain produced tri-acylated MELs as well as di-acylated ones when grown on the medium containing higher soybean oil concentrations than 8% (vol/vol). The production of tri-acylated MELs was significantly accelerated at between 34 and 36 degrees C. With 20 % (vol/vol) of soybean oil at 34 degrees C, the yield of tri-acylated MELs reached 22.7 g/L. The extracellular lipase activity considerably depended on the culture temperature, and became the maximum at 34 degrees C; this would bring the accelerated production of tri-acylated MELs. Accordingly, the present strain of P. parantarctica provided high efficiency in MEL production at elevated temperatures compared to conventional MEL producers, and would thus be highly advantageous for the commercial production of the promising biosurfactants.

Mannosylerythritol lipid-A (MEL-A) is a glycolipid biosurfactant abundantly produced from soybean oil by microorganisms at a yield of up to 100 g/L. In this study, the formation of water-in-oil (W/O) microemulsion based on the single component of MEL-A was confirmed using dynamic light scattering (DLS) and freeze fracture electron microscopy (FF-EM). DLS and FF-EM measurements revealed that the diameter of the microemulsion increases with an increase in water-to-surfactant mole ratio (W(0)) ranging from 20 to 60 nm, and the maximum W(0) value was found to be 20, which is as high as that of soybean lecithin. Glycolipid biosurfactant has a great potential for the formation of W/O microemulsion without using any cosurfactants.

Mannosylerythritol lipids (MELs) are one of the most promising glycolipid biosurfactants known because of their multifunctionality and biocompatibility. The search for novel producers of MELs was undertaken based on the analysis of ribosomal DNA sequences on basidiomycetous yeasts. The bermuda grass smut fungus Ustilago cynodontis NBRC 7530, which taxonomically relates to Pseudozyma shanxiensis known as a MEL-C producer, was found to accumulate glycolipids in the cultured medium. Under a shake flask culture with soybean oil, the amount of the glycolipids was 1.4 g/L for 7 days at 25 degrees C. As a result of the structural characterization, the main glycolipids was identified as 4-O-[(4'-O-acetyl-3'-O-alka(e)noyl-2'-O-butanoyl)-beta-D-mannopyranosyl]-D-erythritol, and the major fatty acids were C(14) and C(16) ones. The glycolipid was highly hydrophilic MEL-C, and very similar to those produced by P. shanxiensis. The fungi of the genus Ustilago are thus likely to be potential producers of MELs as well as the yeasts of the genus Pseudozyma.

Mannosylerythritol lipids (MELs), which are glycolipid biosurfactants produced by Pseudozyma yeasts, show not only excellent interfacial properties but also versatile biochemical actions. In the course of MEL production from glucose as the sole carbon source, P. antarctica was found to produce unknown glycolipids more hydrophilic than conventional "di-acylated MELs," which have two fatty acyl esters on the mannose moiety. Based on a detailed characterization, the most hydrophilic one was identified as 4-O-(3'-O-alka(e)noyl-beta-D: -mannopyranosyl)-D: -erythritol namely, "mono-acylated MEL." The mono-acylated MEL reduced the surface tension of water to 33.8 mN/m at a critical micelle concentration (CMC) of 3.6 x 10(-4) M, and its hydrophilic-lipophilic balance was tentatively calculated to be 12.15. The observed CMC was 100-fold higher than that of the MELs hitherto reported. Interestingly, of the yeast strains of the genus Pseudozyma, only P. antarctica and P. parantarctica gave the mono-acylated MEL from glucose, despite a great diversity of di-acylated MEL producers in the genus. These strains produced MELs including the mono-acylated one at a rate of 20-25%. From these results, the new MEL is likely to have great potential for use in oil-in-water-type emulsifiers and washing detergents because of its higher water solubility compared to conventional MELs and will thus contribute to facilitating a broad range of applications for the environmentally advanced surfactants.

Kinetic studies on the interactions between self-assembled monolayers of mannosylerythritol lipids (MELs), which are glycolipid biosurfactants abundantly produced by microorganisms, and various classes of immunoglobulins including human IgG, IgA, and IgM were performed using surface plasmon resonance (SPR). The effect of the MEL structure on the binding behavior of HIgG was examined. Assembled monolayers of MEL-A having two acetyl groups on the headgroup gave a high affinity (K(d)=1.7x10(-6)M) toward HIgG, while those of MEL-B or MEL-C having only one acetyl group at C-6' or C-4' position gave little affinity. Our kinetic analysis revealed that the binding manner of HIgG, HIgA (K(d)=2.4x10(-7)M), and HIgM (K(d)=2.2x10(-7)M) to the assembled monolayers of MEL-A is not the monovalent mode but the bivalent mode, and both the first and second rate association constants (k(a1), k(a2)) increase with an increase in the number of antibody binding sites, while those for dissociation (k(d1), k(d2)) changed little. Moreover, we succeeded in directly observing great amounts of HIgG, HIgA, and HIgM bound to MEL-A monolayers using atomic force microscopy (AFM). Finally, we found that MEL-A assembled monolayer binds toward various IgG derived from mouse, pig, rabbit, horse, goat, rat, and bovine as well as human IgG (HIgG), and the only exception was sheep IgG. These results clearly demonstrate that MEL-A assembled monolayers would be useful as noble affinity ligand system for various immunoglobulins.