7,10-Dihydroxy-8(E)-octadecenoic acid (DOD) is a dihydroxy fatty acid produced by Pseudomonas aeruginosa PR3 from oleic acid. Present study investigated preventive effects of dietary DOD on obesity, hyperlipidemia and hyperglycemia in diabetic/obese KK-A(y) mice. After one week of acclimation, mice (5-week-old) were divided three groups fed with AIN-93G-based high fat diet (20% fat, w/w) as follows; Control group: 3% soybean oil + 17% lard, DOD 1% group: 3% soybean oil + 16% lard +1% DOD, DOD 2% group: 3% soybean oil + 15% lard + 2% DOD. Four weeks feeding with 1% and 2% DOD diets suppressed body weight gain and visceral fat accumulation, and improved blood glucose and serum lipids levels as well as hepatic lipid accumulation compared to control group. RT-PCR analysis revealed no changes were observed in mRNA of adiponectin (Adipoq) and proinflammatory TNF-alpha (Tnfa) and MCP-1 (Ccl2), but a decreasing trend in leptin mRNA expression in mesenteric white adipose tissue (WAT), suggesting that suppressed fat accumulation by DOD contribute to prevention of over production of leptin. We also observed a reduction of mRNA expression of hepatic insulin-target gluconeogenic enzymes such as PEPCK and G6Pase and the transcription factor sterol regulatory element-binding protein-1. These results showed that dietary DOD is effective to improve glucose and lipid metabolism in KK-A(y )mice. The present study provides new insights showing potential use of dihydroxy fatty acids as functional lipids.

Cell-free synthesis has been adopted in the bioconversion process due to its known advantages, such as fast production rate, high product content, and no substrate/product inhibition effect. In this study, the cell-free supernatant of Pseudomonas aeruginosa was used to improve the production of 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) from oleic acid. DOD production using cell-free supernatant demonstrated reduction in bioconversion duration and higher product concentration than conventional method using whole cell culture. The maximum DOD concentration (6.41 g/L) was obtained after 36 h of biotransformation using 1 % v/v oleic acid as a substrate with a productivity of 0.178 g/L/h and a yield of 74.8 %. DOD concentration, productivity, and yield using cell-free supernatant were 2.12, 7.12, and 2.22 times higher, respectively, than using the conventional whole cell culture method. Of the carbon and nitrogen sources used in pre-culture, galactose and sodium glutamate along with diammonium phosphate were found to be the most effective for DOD production. An incubation temperature of 27 degrees C and pH 8.0 were found to be most favorable for DOD production. In addition, sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis demonstrated the presence of enzymes related to DOD production in the cell-free supernatant, which was substantiated by performing DOD production experiment using the supernatant enzymes extracted from protein gel bands with oleic acid as a substrate. To the best of our knowledge, this is the first report on DOD production using a cell-free supernatant and verifying the existence of the relevant enzymes in the cell-free supernatant. Compared to whole cell process, cell-free DOD production holds several advantages, including higher DOD productivity which could be beneficial for largescale production.

Laminaria japonica biomass was investigated as an untapped carbon source for poly hydrox yalkanoates (PHA) accumulation via batch and fed-batch cultivation of three different bacterial strains including Cupriavidus necator NCIMB 11599, Para coccus sp. LL1, and Bacillus megaterium ALA2. Algal bio mass (10% (w/v)) was treated with different sulfuric and hydrochlo ric acid concentrations (0.1, 0.15, 0.2, and 0.3 N). Increas ing acid concentration led to a significant in crease in reducing sugar production, giving rise to a maximum production of 6.1 and 5.9 g/L for H2SO4 and HCl pre treatment, respectively. Shake flask culture revealed that C. necator, Para coccus sp. LL1, and B. megaterium ALA2 were able to pro duce PHA up to 19-32% of their dry cell weight (DCW) under 2% of reducing sugar supple mentation. Overall, C. necator showed the high est level of PHA accumulation (1.58 g/L, 32% of DCW). By batch and fed-batch culture, PHA con tent was enhanced to 44 and 49%, respectively. Along with PHA, Para coccus sp. LL1 was able to pro duce 0.9 mg/L of carotenoids after 96 h of cultivation in flask culture. Carotenoid production was enhanced by batch and fed-batch culture to 1.45 and 2.3 mg/L, respectively. Based on Fourier trans form infrared spectroscopy, gas chromatography, and H-1 nu clear magnetic resonance spectroscopy, the produced PHA polymers were identified as poly(3-hydroxybutyrate) or poly (3-hydroxybutyrate-co-3-hydroxyvalerate).

Furan fatty acids (F-acid) are known to have strong antioxidant properties, including radical scavenging activity, which play important roles in biological systems including humans. F-acids are widely distributed in most biological systems as trace components and their biosyntheses are complicated. Although several studies were reported on the chemical synthesis of various types of F-acids, it required complicated and multistep chemical reactions. Recently, a simple one-step heat treatment method was developed for the production of a novel furan fatty acid, 7,10-epoxy-octadeca-7,9-dienoic acid (7,10-EODA) from 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). (Journal of Agricultural and Food Chemistry, 2011, 59, 8175-8179). In this report, we optimized reaction conditions for the efficient production of 7,10-EODA from DOD by heat treatment. Under optimal conditions tested, the maximum production yield of 7,10-EODA was 80% with incubation in hexane for at least 48 h at 85 degrees C or above.

The effects of liquid culture media, pH, temperature, and illumination on the basidiospore germination of Lentinus swartzii and Lentinus strigosus as well as their morphogenesis were studied and documented. Coconut water and potato broth with pH of 5.0 – 7.0, incubated at 30°C and total dark, were the optimum nutritional and physical factors in liquid culture condition as activators for successful basidiospore germination of the two Lentinus species. A peculiar germination process was observed in which the basidiospores germinated through swelling and elongated to become hyphae. The vegetative phases of both mushrooms occurred in four significant stages: swelling and clumping (a mechanism for plasmogamy), elongation, septation, and branching. On the other hand, their reproductive phases had six distinct stages: mycelial coat formation, browning and hardening stage (only in L. swartzii), popcorn-like formation (only in L. strigosus), primordia initiation, pinhead stage, pileus expansion stage, and maturation stage. This study established that the two Lentinus spp. have the same vegetative phase (i.e., basidiospore germination and development of hypha and mycelia), but have unique cultivation characteristics in their reproductive phase (i.e., formation of basidiocarp). The obtained information on the developmental biology of the two Lentinus spp. is very useful not only in the generation of effective mushroom biomass production technologies but also in many fungal biotechnological applications.

Soy-polyol oils (oxygenated acylglycerols) are important starting materials for the manufacture of polymers such as polyurethane. We reported methods for microbial screening and production of polyol oils from soybean oil through bioprocessing (Hou and Lin, 2013). Using this screening method, we screened 650 cultures isolated and found 50 cultures were positive for converting soybean oil to polyol oil. The two most active of these positive cultures are Acinetobacter haemolyticus A01-35 and Pseudomonas aeruginosa E03-12 (Hou et al., 2015). The polyol oil produced by strain A01-35 was a mixture of hydroxy fatty acids-containing DAG. Strain E03-12 produced more polyol oil than strain A01-35 and its product contains both hydroxy fatty acids-containing DAG and TAG. We employed one-factor-at-a-time method to develop an optimized culture medium composition and reaction conditions for polyol oils production from soybean oil by strain E03-12. We found that among the different carbon sources studied, glucose at 12.5 g/L is the best. A combination of both tryptone at 12.5 g/L and yeast extract at 10 g/L serves as the best nitrogen source. Both iron and magnesium were studied for the effect of metal on the production of polyol oils. MgSO4 7H2O at 0.75 g/L and FeSO4 7H2O at 0.30 g/L produced the best result. Using this optimum medium composition, the best pH for the polyol oils production was found at 6.8. The optimum temperature for polyol oil production is 28 C. The optimum substrate concentration is 183 mg/30 mL. In the time course studies with these optimum reaction conditions, the best yield is 18.5 mg/30 mL at 48 h. Longer incubation time leads to the decrease in product polyol oils and increase in DAGs. The information obtained from this study is important for developing scale up production of polyol oils directly from soybean oil.

Oxylipins are known to have several biological functions. The oxylipin 7,10-dihydroxy-8(E)-octadecenoic acid (DOD), produced from oleic acid by Pseudomonas aeruginosa, was reported to have strong antibacterial activities against broad range of food-borne and plant pathogenic bacteria. Among DOD producers, P. aeruginosa PR3 shows efficient production of DOD from oleic acid, triolein, and olive oil. In this study, we report the serendipitous isolation and characterization of a variant strain of PR3, which upon examination showed higher efficiency for DOD production from olive oil than the original PR3 strain. Compared with the PR3 strain, the variant strain displayed enhanced DOD production by 47.6% and different carbon requirements for maximum DOD production. Considering the advantageous differences seen in this study, we propose the further characterization of this new strain and optimization of culture conditions for efficient DOD production.

Conversion of free fatty acids into monoacylglycerol gives rise to new structural properties, particularly amphipathic property. Therefore, monoacylglycerols are widely used in pharmaceutical and food industries and are also reported to facilitate better absorption into the human body. A functional fatty acid when transformed into a monoacylglycerol will possibly conserve both the original functionality and amphipathic property. The compound 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was generated from oleic acid by Pseudomonas aeruginosa PR3 and was known to contain antimicrobial activities against a broad range of food-borne and plant pathogenic bacteria. Here, we attempted to convert DOD into its monoacylglycerol form using lipase for producing an amphipathic antibacterial agent. Consequently, the monoacylglycerol of DOD (DOD-MAG) was successfully produced by coincubating DOD, glycerol, and lipase at 30 °C. The maximum conversion yield reached 70% after 12 h of incubation. Antibacterial activity of DOD-MAG was enhanced by 8 times from the original activity of DOD against food-borne bacteria.

Bio-based new polyurethanes (PU) were synthesized from microbially converted castor oil. Castor oil was used as a raw material to synthesize 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD) by the strain Pseudomonas aeruginosa PR3. Subsequently, hexamethylene diisocyanate (HMDI) was used in different ratios and reacted with TOD to produce new PU. Fourier transform-infrared spectroscopy, nuclear magnetic resonance spectroscopy, and gas chromatography/mass spectrometry were used to confirm the identity of TOD. Differential scanning calorimetry, thermogravimetric analysis, and tensile property testing were used to investigate the thermal and mechanical properties of PU. PU synthesized based on TOD had a tensile strength of 45.4 MPa with low elongation at break of 8.16% at an isocyanate/hydroxyl ratio of 2.0. To modify the properties of PU, TOD was blended separately with polyethylene glycol or polycaprolactone diol at different weight ratios before reacting with HMDI. The modified polymer showed improved thermal stability and comparatively higher elongation at break. This is the first study demonstrating the conversion of castor oil into TOD that can be used stand alone or as a blend for PU synthesis.

New polyurethanes were synthesized based on dihydroxy fatty acid obtained by the microbial conversion of olive oil. Monounsaturated 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was produced from olive oil by Pseudomonas aeruginosa PR3 and reacted with hexamethylene diisocyanate (HMDI) at different ratios to form polyurethanes. Fourier transform infrared spectroscopy and gas chromatography/mass spectrometry confirmed the synthesis of DOD. The thermal and tensile properties of the polyurethanes were investigated by differential scanning calorimetry, thermogravimetric analysis, and a universal testing machine. At an isocyanate/hydroxyl ratio of 1.4, the polyurethane exhibited an elongation at break of 59.2% and a high tensile strength of 37.9 MPa. DOD was also mixed with polycaprolactone diol or polyethylene glycol at different weight ratios and then reacted with HMDI to produce new polyurethanes of various properties. These polyurethanes displayed higher elongation at break and good thermal stability. This is the first report on the synthesis of polyurethanes based on DOD produced by the microbial conversion of vegetable oil.

The progress in the understanding of the microbiology and biochemistry of methylotrophs can be attributed largely to Whittenbury and co-workers who isolated and characterized many methane-oxidizing bacteria, and to Quayle and co-workers who unveiled the metabolic pathways of C1 compounds in methylotrophs. Methylotrophic bacteria are recognized by their ability to use, as sole carbon and energy sources for growth, compounds that contain no carbon-carbon bonds and to assimilate carbon as formaldehyde or a mixture of formaldehyde and carbon dioxide. They are different from methanogenic bacteria which produce methane from organic compounds. Methylotrophic bacteria contain two different types of formaldehyde (aldehyde) dehydro genase activity in crude soluble extracts. There are two routes for the oxidation of methylamine in methylotrophic bacteria. The direct route is catalyzed by methylamine dehydrogenase. The second route involves N-methylglutamate as an intermediate. Methylotrophs play an important role in carbon recycling in the biosphere.

Lipase is one of the widely used biocatalysts, which is well studied for its application in industrial production. Recently, lipases with special characteristics such as thermo-stability, alkaline-, acidic nature, and cold-activity, have gained attention for effective applications in specific purposes. Previously, Pichia lynferdii NRRL Y-7723 was reported to produce high amounts of extracellular cold-active lipase (Kim et al., 2010). In this study, we report the identification of lip1 gene that encodes an extracellular cold-active lipase from P. lynferdii NRRL Y-7723. The open reading frame of the gene consisted of 1122bp of nucleotides that encoded a protein with 373 amino acids. The deduced molecular weight and isoelectric point were 41.8kDa and pH 5.82, respectively. The lip1 gene was cloned into a bacterial expression vector, and the recombinant lipase was successfully expressed and purified. Several parameters of the recombinant lipase were analyzed, and Lip1 showed high activity at low temperature.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) infections with multi-drug resistance needs effective and alternative control strategies. In this study we investigated the adjuvant effect of a novel furan fatty acid, 7,10-epoxyoctadeca-7,9-dienoic acid (7,10-EODA) against multidrug-resistant S. aureus (MDRSA) strain 01ST001 by disc diffusion, checker board and time kill assays. Further the membrane targeting action of 7,10-EODA was investigated by spectroscopic and confocal microscopic studies. 7,10-EODA exerted synergistic activity along with β-lactam antibiotics against all clinical MRSA strains, with a mean fractional inhibitory concentration index below 0.5. In time-kill kinetic study, combination of 7,10-EODA with oxacillin, ampicillin, and penicillin resulted in 3.8–4.2 log10 reduction in the viable counts of MDRSA 01ST001. Further, 7,10-EODA dose dependently altered the membrane integrity (p < 0.001) and increased the binding of fluorescent analog of penicillin, Bocillin-FL to the MDRSA cells. The membrane action of 7,10-EODA further facilitated the uptake of several other antibiotics in MDRSA. The results of the present study suggested that 7,10-EODA could be a novel antibiotic adjuvant, especially useful in repurposing β-lactam antibiotics against multidrug-resistant MRSA.

Edible Philippine mushrooms including Ganoderma lucidum have many health benefits. We have recently reported the identities and the contents of 77 molecular species of acylglycerols containing hydroxy fatty acids (HFA) in this mushroom. The structures of these HFA were proposed using the electrospray ionization mass spectrometry of the lithium adducts of acylglycerols in the HPLC fractions of the lipid extract. The proposed structures of the HFA were OH1319:110, OH1319:114, 2OH11,1218:0, 2OH11,1218:19, 2OH11,1218:29,13, 3OH11,12,1318:19 and 3OH11,12,1318:114. The locations of the hydroxyl groups and double bonds were near the middle of the HFA chains. The structures were the same or similar to that of HFA in castor oil except that the HFA of odd numbered carbon atoms were not detected in castor oil. OH18:1 was identified in the mushroom and its structure was not OH1218:19 (ricinoleate) as that in castor oil. Ricinoleate and/or HFA (OH18:1) structurally similar to ricinoleate was likely the biological precursors of these HFA in mushroom.

Edible Philippine mushrooms including Ganoderma lucidum have many health benefits. Seventy-two molecular species of triacylglycerols and five molecular species of diacylglycerols containing hydroxy fatty acids (FA) in the lipid extract of this mushroom were identified by HPLC and MS. The mono-, di-and tri-hydroxy FA constituents of the molecular species of acylglycerols were identified by various degrees of dehydration of [FA + Li](+). Many odd carbon numbered hydroxy FA constituents of acylglycerols were also identified. The contents of the 77 molecular species of acylglyerols and their constituent hydroxy FA in the lipid extract were estimated by the relative ion signal intensities of the molecular species of acylglycerols and the HPLC peak area % (ELSD). The contents (%) of the molecular species of acylglycerols containing hydroxy FA in the lipid extract were as: P-19: 02OH18: 2, 0.69%; 2OH18: 2-2OH18: 2-2OH18: 2, 0.60%; P-OH19: 1-OH19: 1, 0.48%; S-OH19: 1-OH19: 1, 0.43%; 19: 0-19: 0-2OH18: 2, 0.27% and the total of about 5.8%. "2OH18: 2" stands for dihydroxy FA with 18 carbon atoms and two double bonds. The mushroom lipid extract was 1.67% of freeze-dried mycelium. The contents (%) of individual hydroxy FA in the mushroom lipid extract in decreasing order were: 2OH18: 2, 1.4%; OH19: 1, 1.1%; OH18: 1, 0.38%; 2OH19: 0, 0.19%; 2OH18: 0, 0.13% and the total of about 3.5%. The odd carbon numbered hydroxy FA was about 1.44% of the mushroom lipid extract. The high contents of acylglycerols containing hydroxy FA, odd carbon numbered FA and odd carbon numbered hydroxy FA in edible mushroom might be related to one or more of its health benefits. As far as we are aware of, this mushroom contains the highest amounts of hydroxy FA and odd carbon numbered FA among the human foods.

This paper highlighted the antioxidant and antibacterial activities of Lentinus tigrinus and Pleurotus djamour. Extracts of mushroom fruiting bodies were obtained using hexane and acetonitrile solvents. Acetonitrile extracts of both mushrooms exhibited higher biological activities than hexane extracts. L. tigrinus acetonitrile extract had 39.2% (EC50 value 637.75mg/L) radical scavenging activity, contained 451mg AAE (ascorbic acid equivalent)/g sample total phenolics, and showed 9.48mm diameter zone of inhibition against Staphylococcus aureus. On the other hand, P. djamour acetonitrile extract had 32.8% (EC50 value 762.19mg/L) scavenging activity, contained 982mg AAE/g sample total phenolics, and inhibited S. aureus growth with 9.37mm diameter zone of inhibition. Both hexane extracts did not show any inhibitory effect against the two bacteria used. Collectively, the acetonitrile extracts of L. tigrinus and P. djamour could be valuable resource of antioxidant and anti-S. aureus compounds which we need to isolate, characterize and identify.

With remarkable bioactivities and delightful taste, mushrooms have been a commercial nutraceutical around the world. Mushrooms are cultivated on solid materials. Here we report the successful cultivation of four Philippine edible mushrooms in liquid medium. This work highlights the optimal liquid culture conditions with reference to the nutritional and physical growth factors, and preliminarily elucidated their wide variety of lipids by thin-layer chromatography. Sabouraud dextrose broth (SDB) was the most suitable culture medium for all studied mushrooms. Maximum mycelial biomasses of Ganoderma lucidum, Pleurotus cystidiosus, Volvariella volvacea and Schizophyllum commune favorably produced in SDB at pH 7, 7, 6 and 8, respectively, when incubated at 28°C and 30°C as optimum temperatures. The mycelia biomass yields obtained in the present study 0.8g/30ml are superior to the reported biomass yields of other basidiomycetes such as Russula sp. and Pycnoporus cinnabarinus at 0.3g/50ml (Shittu et al., 2005). Agitation did not improved mycelial growth of mushrooms except G. lucidum, which the biomass yield was peaked at 100rpm (500ml medium in 2.8L) shake-flask culture. Cholesterol, triglycerides, free fatty acids, and polar lipids were detected in all the four mushrooms. The potential biological activities as well as molecular species of these lipid materials from the four basidiomycetes are currently under investigation.

The objective of this study is to identify the chemical species of the polyol oil products produced from soybean oil by Pseudomonas aeruginosa E03-12 NRRL B-59991. We reported earlier the polyol products produced from soybean oil by Acinetobacter haemolyticus A01-35 (NRRL B-59985) (Hou and Lin, 2013). The polyol oil produced by strain A01-35 were a mixture of 57 molecular species of DAG containing tri-, di-, mono-hydroxy FA and normal FA. In this study, tricaprylin was selected as internal standard for HPLC quantitative estimation of products. A CombiFlash chromatographic method was established for separation of the polyol oil products. The molecular species of the polyol oils produced from soybean oil by strain E03-12 were identified with HPLC/MS. We identified 41 derivatives of DAG, among them 32 molecular species containing one hydroxy FA and one normal FA, 8 molecular species containing two hydroxy FA without normal FA, and one molecular specie containing two normal FA without hydroxylated FA. The hydroxy FA included mono-, di- and tri-hydroxy FA. Eight molecular species of DAG containing one trihydroxy FA and 14 molecular species of DAG containing one dihydroxy FA. We have also identified 64 molecular species of TAG, among them 13 molecular species containing two hydroxy FA, 42 molecular species containing one hydroxy FA and 9 molecular species containing no hydroxylated FA. This is different from our previous findings with A. haemolyticus A01-35 which produced only DAG polyol oils. E03-12 is a better strain for developing into an industrial bioprocess.