Isolation of a bioemulsifier from Candida lipolytica

The yeast Candida lipolytica produced an inducible extracellular emulsification activity when it was grown with a number of water-immiscible carbon substrates. Negligible emulsification activity was produced by this yeast when it was grown with glucose as the carbon substrate. In hexadecane-supplemented cultures, emulsification activity was first detected after 36 h of growth, with maximum production after 130 h. A water-soluble emulsification activity was partially purified by repeated solvent extractions of the culture filtrate. This emulsifier, which we named liposan, was primarily composed of carbohydrate. Maximum emulsification activity was obtained when the ratio of hexadecane to liposan was 50:1. Maximum emulsification activity was obtained from pH 2 to 5. Liposan was heat stable to temperatures up to 70 degrees C, with a 60% loss in activity after heating for 1 h at 100 degrees C. Liposan effected stable oil-in-water emulsions with a variety of hydrocarbons.     

Isolation of a bioemulsifier from Candida lipolytica.

The yeast Candida lipolytica produced an inducible extracellular emulsification activity when it was grown with a number of water-immiscible carbon substrates. Negligible emulsification activity was produced by this yeast when it was grown with glucose as the carbon substrate. In hexadecane-supplemented cultures, emulsification activity was first detected after 36 h of growth, with maximum production after 130 h. A water-soluble emulsification activity was partially purified by repeated solvent extractions of the culture filtrate. This emulsifier, which we named liposan, was primarily composed of carbohydrate. Maximum emulsification activity was obtained when the ratio of hexadecane to liposan was 50:1. Maximum emulsification activity was obtained from pH 2 to 5. Liposan was heat stable to temperatures up to 70 degrees C, with a 60% loss in activity after heating for 1 h at 100 degrees C. Liposan effected stable oil-in-water emulsions with a variety of hydrocarbons.     

Formation of Fine Oil-in-Water Emulsions by the Gel Emulsification Method with Saccharide and Protein

An emulsification method using a gel-like phase of a saccharide and protein mixture has been developed. In the method, which is called a gel emulsification method, an oil is added to the highly concentrated saccharide solution containing protein to form a clear gel-like phase, which followed by dilution with water to form a fine oil-in-water emulsion. This emulsion was investigated as to its emulsifying activity and emulsion stability as compared with that obtained by high-shear equipment, which was called a homomixer method. The emulsifying activity of the emulsions prepared by the gel emulsification method was much higher than that of the emulsions prepared by the homomixer method.

The emulsions prepared by both methods were highly stable in terms of the stability against coalescence. On the other hand, the stability against creaming of the emulsions prepared by the gel emulsification method was much higher than that of the emulsions prepared by the homomixer method.

The surface hydrophobicity of the protein and the unfreezable water content in the highly concentrated saccharide solution containing protein were not correlated to the emulsifying properties of the emulsions prepared by the gel emulsification method, which appeared to be dependent on the viscosity of the highly concentrated saccharide solution containing protein.     

Characterization of hydrocarbon emulsification and solubilization occurring during the growth of Endomycopsis lipolytica on hydrocarbons

A comparative characterization of the phenomena of hydrocarbon emulsification and solubilization taking place during the growth of Endomycopsis lipolytica on n-alkanes and alkenes was made. Evidence was obtained for the cellular production of different factors involved in emulsification and solubilization of hydrocarbons. It was shown that the production of these factors closely followed cell growth. The inducible nature of the alkane solubilizing factor was demonstrated using actidione as inhibitor. Whereas emulsifying factor was demonstrated using actidione as inhibitor. Whereas emulsifying factors showed a broad affinity to some particular hydrocarbons, solubilizing factor was found to be highly specific for the particular hydrocarbon on which the cells were grown. The emulsifying factor was heat stable whereas the solubilizing factor was highly unstable even at ?4°C. Metal-ion chelating agents strongly inhibited the activity of both of the factors. A crude isolate of the alkane emulsifying factor was obtained and its peptide characteristics were demonstrated. Using EDTA as an inhibitor for the emulsification–solubilization activity, evidence was obtained for the predominent role played by the emulsification–solubilization mechanism in the uptake of alkane by yeast cells.     

Production of bioemulsifier by a SCP-producing strain ofCandida tropicalis during hydrocarbon fermentation

Summary SCP producingCandida tropicalis, when grown in fed batch culture using n-hexadecane as carbon substrate, exhibited extracellular emulsifier production. The emulsifier showed activity against various hydrocarbons, maximum with aromatics and least with normal paraffins. Higher emulsification activity was noted in nitrogen-limiting growth conditions than in substrate- limiting conditions. The hot water extract of the cells also showed significant emulsification activity.     

A practical approach to biosurfactant production using nonaseptic fermentation of mixed cultures

Non-aseptic production of biosurfactant from molasses by a mixed culture was investigated in stirred batch reactors. Biosurfactant production was quantified by surface tension reduction, critical micelle dilution (CMD), and emulsification capacity (EC). Biosurfactant production was directly correlated with biomass production, and was improved by pH control and addition of yeast extract. Centrifugation of the whole broth increased emulsifying capacity and reduced surface tension. Acidification of the whole broth increased the emulsification capacity but reduced the apparent biosurfactant concentration (CMD), without affecting the surface tension. The emulsification capacity of the cell-free broth was equivalent to that of a 100 mg/L solution of sodium dodecyl sulfate. The emulsification capacity of the whole broth and cell-free broth were reduced by about 50% at and above NaCl concentrations of 100mM. Preliminary characterization suggests that the biosurfactant activity is primarily associated with one or more protease-sensitive species, released from cells in larger quantities after more vigorous centrifugation.     

1株耐高温生物表面活性剂产生菌的特性研究

研究了耐高温生物表面活性剂产生菌ZY-3的生理生化特性,并通过测定发酵液的菌体密度、表面张力和乳化活性等指标,研究不同碳源和初始pH对菌株ZY-3生长和产生物表面活性剂的影响,同时对其所产生物表面活性剂进行了初步分离和性质分析。菌株ZY-3被初步鉴定为芽胞杆菌属(Bacillus),具有产酸、不产H_2S、还原硝酸盐等特性。在以淀粉为碳源、初始pH 6.0的培养基中发酵,产生物表面活性剂多且稳定;在种子培养基和发酵培养基中都有淀粉的条件下,菌体生长较多,降低表面张力和乳化的作用均较强,所产生物表面活性剂可以使发酵液的表面张力从72.1 mN/m降到53.1 mN/m,乳化活性从0升高到24%。初步判断产物为糖脂类阴离子表面活性剂。     

Studies on bioemulsifier production by Acinetobacter strains isolated from healthy human skin

AIMS: In recent years, interest has been growing in the search for novel bioemulsifiers. Many bacterial genera including Acinetobacter have been reported to produce bioemulsifiers. The present study aims to screen Acinetobacter isolates from healthy human skin for bioemulsifier production. Methods and Results: Acinetobacter junii SC14 produced maximum bioemulsifier in the presence of almond oil during stationary growth phase at 37 degrees C and pH 7.2. Partially purified, nondialysable bioemulsifier from SC14 was a proteoglycan. The protein and polysaccharide fractions resulted in 95.2% reconstitution of the emulsification activity. The role of esterase in the release of cell-bound emulsifier and the contribution of capsular polysaccharide to the emulsification activity were observed. CONCLUSION: Acinetobacter strains from human skin exhibited better emulsification activity than that by burn wound or soil isolates, owing to the inherent differences in chemical microenvironment of their habitats. SIGNIFICANCE AND IMPACT OF THE STUDY: Investigation of skin commensals, especially acinetobacters, would lead to the discovery of novel bioemulsifiers with interesting properties. Attempts of screening and strain improvement directed towards skin commensals will open up new avenues for strains producing bioemulsifier on a commercial scale.     

Anionic lipopeptides from Bacillus mojavensis I4 as effective antihypertensive agents: Production,characterization, and identification

A new isolated Bacillus mojavensis strain I4 was found as producer of biosurfactants by different screening methods, such as parafilm M test, hemolytic activity, oil displacement test, emulsification index, surface tension, and lipase production assay. Enhanced biosurfactants production was obtained using glucose and glutamic acid as carbon and nitrogen sources, respectively. The optimal production of the biosurfactants was obtained by using a C/N ratio of 17, pH of 7.0, and temperature of 37°C. The surface tension was reduced to 29 mN/m and the emulsification index E24 of 62% was achieved after 72 h of culture. The purified biosurfactants showed stability with regard to surface tension reduction and emulsification in a wide range of temperatures (4–120°C), pH (4–10), and salinity (2–12% of NaCl). The thin‐layer chromatography showed that the produced biosurfactants were lipopeptides. The biosurfactants were characterized as a group of anionic lipopeptides with zeta potential measurement. Chromatographic characterization using HPLC revealed that I4 lipopeptides contained numerous isoforms and surfactin was the major component. Moreover, the I4 lipopeptides showed interesting angiotensin‐converting enzyme‐inhibitory activity.     

银耳粗多糖对桉叶油的乳化性研究

对不同浓度、温度、pH、NaCl浓度条件下,银耳粗多糖对桉叶油的乳化能力,以及乳化体系的稳定性进行了研究。结果表明,银耳粗多糖对桉叶油的乳化性随浓度增大上升,在浓度1%时达到最大,随后呈下降趋势;在pH 4.0~8.0之间;有较好的乳化性及乳化稳定性,在此范围以外,随pH的升高、降低,银耳粗多糖的乳化和乳化性均呈下降趋势;随着温度和NaCl浓度升高,银耳粗多糖的乳化活性和乳化稳定性有所降低。     

In vitro assessment of antimicrobial activity of carvacrol, thymol and cinnamaldehyde towards Salmonella serotype Typhimurium DT104: effects of pig diets and emulsification in hydrocolloids

AIMS: To determine the effect of pig diets in vitro on the antimicrobial activity of carvacrol, thymol and cinnamaldehyde, and to identify an emulsifier/stabilizer that can stabilize the essential oil (EO) components in aqueous solution and retain their antimicrobial activity in the presence of the diets. METHODS AND RESULTS: Emulsification of essential oil components with hydrocolloid solution was achieved by blending with a Polytron. Antimicrobial activity was measured through in vitro assays to determine the inhibition of bacterial growth by measuring the optical density at 600 nm or plating on nutrition agar after incubation of the mixtures of an EO component with the culture of Salmonella serotype Typhimurium DT104 in the presence or absence of pig diets. The results generated through the in vitro assays indicated that pig diets were able to abolish the antimicrobial activity of EOs. Xanthan, fenugreek and yellow mustard gums were the best in forming stable emulsions of five different EO components among ten different plant polysaccharides and surfactants examined. Emulsification of all the EO components in the fenugreek gum solution did not alter their antimicrobial activity. However, the antimicrobial activity of geraniol was significantly reduced when emulsified with other polysaccharides and surfactants. Both fenugreek and xanthan gum solutions were unable to protect the antimicrobial activity of carvacrol and thymol when mixed with the diets. Although cinnamaldehyde required no emulsification, but a high concentration (equivalent to at least three times of minimum bactericidal concentration for cinnamon oil) to inhibit Salmonella growth significantly in the presence of the diets, emulsification in fenugreek gum appeared to be essential for cinnamaldehyde solution to retain its antimicrobial activity during storage. CONCLUSIONS: The diets for newly weaned pigs were a significant factor limiting the antimicrobial activity of EOs and their components. Cinnamaldehyde required a high concentration to retain its antimicrobial activity in the diets, in addition to its requirement for emulsification to stabilize its activity during the storage. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay with the diets used in this study for measuring the antimicrobial activity can be used in vitro for rapid and effective screening of potential antimicrobials for swine production. This study has identified polysaccharides that are able to stabilize EO component solutions. It has also identified cinnamaldehyde for further in vivo studies that may have potential in future application in controlling Salmonella and possibly other enteric pathogens in swine production.     

Relationship between the Molecular Structures and Emulsification Properties of Edible Oils

Emulsification properties are very important to control the texture of foods. However, the relationship between the molecular structure and emulsification properties of edible oils is not understood. To analyze this relationship, the emulsification susceptibilities of various kinds of single triacylglycerol molecular species and edible oils were systematically measured. The emulsification susceptibility increased as the carbon number and double bond number of triacylglycerol molecular species consisting oils increased. In addition, the effect of the double bond number was predominant. These results demonstrate that the emulsification property is affected by the molecular structure of oils. Furthermore, the emulsification susceptibilities of edible oils modified by enzymatic interesterification were changed as compared with those of native oils. This shows that emulsification property can be changed by the modification of the molecular structure of edible oils.     

The Acinetobacter outer membrane protein A (OmpA) is a secreted emulsifier

Acinetobacter strains use hydrophobic carbon sources and most of them are efficient oil degraders. They secrete a variety of emulsifiers which are efficient in producing and stabilizing oil-in-water emulsions. The bioemulsifier of Acinetobacter radioresistens KA53 (Alasan) is a high-mass complex of proteins and polysaccharides. The major emulsification activity of this complex is associated with a 45 kDa protein (AlnA), which is homologous to the outer membrane protein OmpA. The emulsification ability of AlnA depends on the presence of hydrophobic residues in the four loops spanning the transmembrane domains. The finding of a secreted OmpA was unexpected, in view of the fact that this protein is essential in all Gram-negative bacteria, has four trans-membrane domains and is considered to be an integral structural component of the outer membrane. However, secretion of an OmpA with emulsifying ability could be of physiological importance in the utilization of hydrophobic substrates as carbon sources. Here we examined the possibility that secretion of OmpA with emulsifying activity is a general property of the oil-degrading Acinetobacter strains. The results indicate that OmpA is secreted in five strains of Acinetobacter, including strain Acinetobacter sp. ADP1 whose genome has been sequenced. The ompA genes of ADP1 and an additional strain, Acinetobacter sp. V-26 were cloned and sequenced. Structure analysis of the sequence of the two proteins indicated the existence of the hydrophobic regions, previously shown to be responsible for the emulsification activity of AlnA. Further examination of the recombinant OmpA proteins indicated that they are, indeed, strong emulsifiers, even when produced in Escherichia coli. The finding that Acinetobacter OmpA has emulsifying activity and that it is secreted in five strains of Acinetobacter may be physiologically significant and suggests the involvement of this protein in biodegradation of hydrophobic substrates, including hydrocarbons.     

Development of a microchannel emulsification process for pancreatic beta cell encapsulation

In this study, we developed a high-throughput microchannel emulsification process to encapsulate pancreatic beta cells in monodisperse alginate beads. The process builds on a stirred emulsification and internal gelation method previously adapted to pancreatic cell encapsulation. Alginate bead production was achieved by flowing a 0.5–2.5% alginate solution with cells and CaCO₃ across a 1-mm thick polytetrafluoroethylene plate with 700 × 200 μm rectangular straight-through channels. Alginate beads ranging from 1.5–3 mm in diameter were obtained at production rates exceeding 140 mL/hr per microchannel. Compared to the stirred emulsification process, the microchannel emulsification beads had a narrower size distribution and demonstrated enhanced compressive burst strength. Both microchannel and stirred emulsification beads exhibited homogeneous profiles of 0.7% alginate concentration using an initial alginate solution concentration of 1.5%. Encapsulated beta cell viability of 89 ± 2% based on live/dead staining was achieved by minimizing the bead residence time in the acidified organic phase fluid. Microchannel emulsification is a promising method for clinical-scale pancreatic beta cell encapsulation as well as other applications in the pharmaceutical, food, and cosmetic industries.     

A unique polypeptide from the C-terminus of the exocellular esterase of Acinetobacter venetianus RAG-1 modulates the emulsifying activity of the polymeric bioemulsifier apoemulsan

An exocellular esterase from the oil-degrading Acinetobacter venetianus RAG-1 was previously shown to enhance the emulsification and emulsion stabilization properties of the amphipathic, aminopolysaccharide bioemulsifier, emulsan [Bach H, Berdichevsky Y, Gutnick D (2003) An exocellular protein from the oil-degrading microbe Acinetobacter venetianus RAG-1 enhances the emulsifying activity of the polymeric bioemulsifier emulsan. Appl Environ Microbiol 69:2608–15]. This enhancement was specific for the RAG-1 esterase and was independent of catalytic activity. In this report, fragments from both the N′- and C′-termini were cloned as fusions to the C-terminus of the maltose-binding protein (MBP) and were tested for enhancement activity in the presence of the deproteinated form of emulsan, apoemulsan. The activity could be localized to the C-terminal third of the protein which exhibited the same activity as the intact enzyme. MBP itself was completely inactive and could be cleaved from the fusion without affecting the subsequent emulsification. However, the enhancement completely depended on the presence of a unique C-terminal 20 amino acid peptide not found in any other protein in the databases. In addition, progressive removal of amino acids from the N-terminus of the active MBP polypeptide resulted in a concomitant loss of activity, indicating that enhancement is also proportional to the size of the peptide fragment. The middle third and the C-terminal third of the enzyme each contained a copy of the conserved Cardin–Weintraub consensus sequence for protein binding to heparin. These sequences were not detected in homologous esterases from a closely related strain, Acinetobacter calcoaceticus BD413.     

Optimizing the medium components in bioemulsifiers production by Candida lipolytica with response surface method

A response surface methodology was used to study bioemulsifier production by Candida lipolytica. A 2(4) full experimental design was previously carried out to investigate the effects and interactions of the concentrations of corn oil, urea, ammonium sulfate, and potassium dihydrogen orthophosphate on the emulsification activity (EA) of the bioemulsifier produced by C. lipolytica. The best EA value (3.727 units of emulsification activity (UEA)) was obtained with a medium composed of 0.4 g of urea, 1.1 g of ammonium sulfate, 2.04 g of potassium dihydrogen orthophosphate, 5 mL of corn oil, 50 mL of distilled water, and 50 mL of seawater. A curvature check was performed and revealed a lack of fit of the linear approximation. The proximity of the optimum point was evident, as was the need for quadratic model and second-order designs that incorporate the effect of the curvature. Medium constituents were then optimized for the EA using a three-factor central composite design and response surface methodology. The second-order model showed statistical significance and predictive ability. It was found that the maximum EA produced was 4.415 UEA, and the optimum levels of urea, ammonium sulfate, and potassium dihydrogen orthophosphate were, respectively, 0.544% (m/v), 2.131% (m/v), and 2.628% (m/v).     

Membrane formation by interfacial cross-linking of chitosan for microencapsulation of Lactococcus lactis

Lactic acid bacteria were microencapsulated within cross-linked chitosan membranes formed by emulsification/interfacial polymerization. The technique was modified and optimized to provide biocompatible conditions during encapsulation involving the use of mineral oils as the continuous phase and chitosan as the membrane material. Chitosan cross-linked with hexamethylene diisocyanate or glutaraldehyde resulted in strong membranes, with a narrow size distribution about a mean diameter of 150 mum. Cell viability and activity was demonstrated by the acidification of milk. Loss of acidification activity during microencapsulation was recovered in subsequent fermentations to levels similar to that of free cell fermentations. (c) 1993 John Wiley & Sons, Inc.     

Physicochemical and structural characterization of biosurfactant produced by <Emphasis Type="Italic">Pleurotus djamor</Emphasis> in solid-state fermentation

Biosurfactants are amphiphilic compounds produced by several microorganisms that reduce the surface tension. Low toxicity, optimal activity in extreme conditions, biodegradability and production from several wastes are main advantages of biosurfactants as compared to synthetic surfactants. Production of biosurfactant by a white rot fungus Pleurotus djamor on sunflower seed shell in solid-state fermentation was determined by emulsification indexes, oil spreading activity and surface tension (28.82 ± 0.3mN/m) measurement. The critical micelle concentration was detected as 0.964 ± 0.09 mg/mL. Also, the chemical and physicochemical properties of the biosurfactant produced were investigated. Considering the results of the chemical contents analysis, HPLC, FT-IR and ¹H-NMR, it can be concluded that the produced biosurfactant has a complex structure. Besides, resistance of its activity to environmental factors such as temperature, pH and salt concentration, as well as its thermal stability, were investigated. Additionally, the produced biosurfactant formed stabile emulsions with different hydrocarbons. Lastly, the performance of removing waste frying oil from contaminated sand of produced biosurfactant was detected as 76.57 ± 6%. Owing to its high emulsification capacity, low surface tension and critical micelle concentration, the biosurfactant, shows great potential for use in hydrocarbon removal applications.     

The Potential of Bacterial Isolates for Emulsification with a Range of Hydrocarbons

A study was undertaken to investigate the distribution of biosurfactant producing and crude oil degrading bacteria in the oil contaminated environment. This research revealed that hydrocarbon contaminated sites are the potent sources for oil degraders. Among 32 oil degrading bacteria isolated from ten different oil contaminated sites of gasoline and diesel fuel stations, 80% exhibited biosurfactant production. The quantity and emulsification activity of the biosurfactants varied. Pseudomonas sp. DS10‐129 produced a maximum of 7.5 ± 0.4 g/l of biosurfactant with a corresponding reduction in surface tension from 68 mN/m to 29.4 ± 0.7 mN/m at 84 h incubation. The isolates Micrococcus sp. GS2‐22, Bacillus sp. DS6‐86, Corynebacterium sp. GS5‐66, Flavobacterium sp. DS5‐73, Pseudomonas sp. DS10‐129, Pseudomonas sp. DS9‐119 and Acinetobacter sp. DS5‐74 emulsified xylene, benzene, n‐hexane, Bombay High crude oil, kerosene, gasoline, diesel fuel and olive oil. The first five of the above isolates had the highest emulsification activity and crude oil degradation ability and were selected for the preparation of a mixed bacterial consortium, which was also an efficient biosurfactant producing oil emulsifying and degrading culture. During this study, biosurfactant production and emulsification activity were detected in Moraxella sp., Flavobacterium sp. and in a mixed bacterial consortium, which have not been reported before.     

Characterization of new biosurfactant produced by Klebsiella sp. Y6-1 isolated from waste soybean oil

To obtain predominant bacteria degrading crude oil, we isolated some bacteria from waste soybean oil. Isolated bacterial strain had a marked tributyrin (C4:0) degrading activity as developed clear zone around the colony after incubation for 24h at 37 degrees C. It was identified as Klebsiella sp. Y6-1 by analysis of 16S rRNA gene. Crude biosurfactant was extracted from the culture supernatant of Klebsiella sp. Y6-1 by organic solvent (methanol:chloroform:1-butanol) after vacuum freeze drying and the extracted biosurfactant was purified by silica gel column chromatography. When the purified biosurfactant dropped, it formed degrading zone on crude oil plate. When a constituent element of the purified biosurfactant was analyzed by TLC and SDS-PAGE, it was composed of peptides and lipid. The emulsification activity and stability of biosurfactant was measured by using hydrocarbons and crude oil. The emulsification activity and stability of the biosurfactant showed better than the chemically synthesized surfactant. It reduced the surface tension of water from 72 to 32 mN/m at a concentration of 40 mg/l.