Soil fungal communities affect the chemical quality of flue-cured tobacco leaves in Bijie, Southwest China.

Soil microorganisms could affect the quality of tobacco leaves, however, little is known about the association of tobacco chemical components and soil fungal communities. In the present study, the relationship between soil fungi and tobacco quality based on chemical components in Bijie was investigated. The results showed that the total harmony scores (THS) of the analyzed tobacco leaves ranged from 46.55 ± 3.5 to 91.55 ± 2.25. Analyses of chemical components revealed that high contents of nicotine (≥ 1.06%) and sugar (total sugar: ≥ 22.96%, reducing sugar: ≥ 19.62%), as well as low potassium level (≤ 2.68%) were the main factors limiting the quality of flue-cured tobacco leaves. Pearson correlation analysis indicated that soil nitrate, available potassium/phosphorous, and organic matter significantly correlated with tobacco nicotine, potassium, and chloride levels (p < 0.05). Besides, the analysis of alpha- and beta-diversity of soil fungal communities implied that fungal structure rather than the richness affected the chemical quality of tobacco. In detail, the relative abundance of Humicola olivacea species in soils was positively correlated with the THS of tobaccos (r = 0.52, p < 0.05). Moreover, the species including Mortierella alpina, Mortierella hyalina, Tausonia pullulan, and Humicola olivacea were negatively correlated with tobacco sugar (r ≤ - 0.45, p < 0.05) while, Codinaea acaciae and Saitozyma podzolica species were negatively correlated with tobacco nicotine (r ≤ - 0.51, p < 0.05). The present study provides a preliminary basis for utilizing fungal species in soils to improve the chemical quality of tobacco in the studied area.

Process optimization and characterization of arachidonic acid oil degumming using ultrasound-assisted enzymatic method.

Ultrasound assisted enzymatic method was applied to the degumming of arachidonic acid (ARA) oil produced by Mortierella alpina. The conditions of degumming process were optimized by response surface methodology with Box- Behnken design. A dephosphorization rate of 98.82% was achieved under optimum conditions of a 500 U/kg of Phospholipase A1 (PLA1) dosage, 2.8 mL/100 g of water volume, 120 min of ultrasonic time, and 135 W of ultrasonic power. The phosphorus content of ultrasonic assisted enzymatic degumming oil (UAEDO) was 4.79 mg/kg, which was significantly lower than that of enzymatic degumming oil (EDO, 17.98 mg/kg). Crude Oil (CO), EDO and UAEDO revealed the similar fatty acid compositions, and ARA was dominated (50.97 ~ 52.40%). The oxidation stability of UAEDO was equivalent to EDO and weaker than CO, while UAEDO presented the strongest thermal stability, followed by EDO and CO. Furthermore, aldehydes, acids and alcohols were identified the main volatile flavor components for the three oils. The proportions of major contributing components such as hexanal, nonanal, (E)-2-nonanal, (E, E)-2,4-decadienal, (E)-2-nonenal and aldehydes in UAEDO and EDO were all lower than CO. Overall, Ultrasound assisted enzymatic degumming proved to be an efficient and superior method for degumming of ARA oil.

In vitro cytotoxic effect of a chitin-like polysaccharide produced by Mortierella alpina on adrenocortical carcinoma cells H295R, and its use as mitotane adjuvant.

This study presents an in vitro evaluation of the antitumor potential of a chitin-like exopolysaccharide (EPS, produced by Mortierella alpina) on Adrenocortical carcinoma cells (ACC) compared to mitotane, a commercial drug commonly used in ACC treatment, and known for its side effects. Techniques of cellular viability determination such as MTT and fluorescence were used to measure the cytotoxic effects of the EPS and mitotane in tumoral cells (H295R) and non-tumoral cells (VERO), observing high cytotoxicity of mitotane and a 10% superior pro-apoptotic effect of the EPS compared to mitotane (p < 0.05). The cytotoxic effect of the EPS was similar to the effect of 50 µM mitotane on tumoral cells (p < 0.05). A decrement of the lysosomal volume was also noted in tumoral cells treated with the EPS. To enhance the antitumor effect, a combination of mitotane at a lower dosage and the EPS (as adjuvant) was also tested, showing a slight improvement of the cytotoxicity effect on tumoral cells. Therefore, the results indicate a cytotoxic effect of the EPS produced by Mortierella alpina on adrenocortical carcinoma, and a possible application in biomedical formulations or additional treatments.

Mortiamides A-D, Cyclic Heptapeptides from a Novel Mortierella sp. Obtained from Frobisher Bay.

Four new cyclic heptapeptides, mortiamides A-D (1-4), were obtained from a novel Mortierella sp. isolate obtained from marine sediment collected from the intertidal zone of Frobisher Bay, Nunavut, Canada. The structures of the compounds were elucidated by NMR spectroscopy and tandem mass spectrometry. The absolute configurations of the amino acids were determined using Marfey's method. Localization of l and d amino acids within each compound was ascertained by retention time comparison of the partial hydrosylate products of each compound to synthesized dipeptide standards using LC-HRMS. Compounds 1-4 did not exhibit any significant antimicrobial or cytotoxic activity.

Influence of Supplementation of Vegetable Oil Blends on Omega-3 Fatty Acid Production in Mortierella alpina CFR-GV15.

Objectives of this study were designed for improved production of mycelial omega-3 fatty acids with particular reference to EPA and DHA from the oleaginous fungus Mortierella alpina CFR-GV15 under submerged low temperatures fermentation supplemented with linseed oil and garden cress oil as an additional energy source. The fungus was grown at 20°C temperature for four days initially followed by 12°C temperature for next five days. The basal medium contained starch, yeast extract, and a blend of linseed oil (LSO) and garden cress oil (GCO) in the ratio 1 : 1. Results of the study revealed that, after nine days of total incubation period, the enhancement of biomass was up to 16.7 g/L dry weight with a total lipid content of 55.4% (v/w). Enrichment of omega-3 fatty acids indicated a significant increase in fatty acid bioconversion (ALA 32.2 ± 0.42%, EPA 7.9 ± 0.1%, and DHA 4.09 ± 0.2%) by 2.5-fold. The two-stage temperature cultivation alters the fatty acid profile due to activation of the desaturase enzyme in the cellular levels due to which arachidonic acid (AA) content reduced significantly. It can be concluded that Mortierella alpina CFR-GV15 is a fungal culture suitable for commercial production of PUFAs with enriched EPA and DHA.

Developmental and reproductive toxicological evaluation of arachidonic acid (ARA)-Rich oil and docosahexaenoic acid (DHA)-Rich oil.

The purpose of this study was to investigate the reproductive and developmental toxicity of dietary exposure to DHA-rich oil from Schizochytrium sp. and ARA-rich oil from Mortierella alpina. In a developmental toxicity study, pregnant Wistar rats were untreated (control) or administered corn oil (vehicle control), 1000, 2500, or 5000 mg/kg bw/day of DHA-rich oil or ARA-rich oil via gavage from gestation days 6 through 20. In the reproductive toxicity study, male and female Wistar rats were administered vehicle control (corn oil), or 1000, 2500, or 5000 mg/kg bw/day of DHA- or ARA-rich oil via gavage throughout the mating period, pregnancy, and the nursing and lactation period. Differences in the number of fetuses, fetal skeletal malformations, and external and visceral anomalies in the developmental study and mortality, clinical signs, fertility indices, physical observations, gross necropsy findings, and gestation period length in the reproductive toxicity study were not dose-related or significantly different from control groups, and were not considered to be treatment related. The no observed adverse effect level (NOAEL) for maternal toxicity and embryo/fetal development and for paternal or maternal treatment-related reproductive toxicity for the DHA-rich oil and ARA-rich oil administered by gavage, was 5000 mg/kg bw/day.

FTIR Spectroscopy for Evaluation and Monitoring of Lipid Extraction Efficiency for Oleaginous Fungi.

To assess whether Fourier Transform Infrared (FTIR) spectroscopy could be used to evaluate and monitor lipid extraction processes, the extraction methods of Folch, Bligh and Lewis were used. Biomass of the oleaginous fungi Mucor circinelloides and Mortierella alpina were employed as lipid-rich material for the lipid extraction. The presence of lipids was determined by recording infrared spectra of all components in the lipid extraction procedure, such as the biomass before and after extraction, the water and extract phases. Infrared spectra revealed the incomplete extraction after all three extraction methods applied to M.circinelloides and it was shown that mechanical disruption using bead beating and HCl treatment were necessary to complete the extraction in this species. FTIR spectroscopy was used to identify components, such as polyphosphates, that may have negatively affected the extraction process and resulted in differences in extraction efficiency between M.circinelloides and M.alpina. Residual lipids could not be detected in the infrared spectra of M.alpina biomass after extraction using the Folch and Lewis methods, indicating their complete lipid extraction in this species. Bligh extraction underestimated the fatty acid content of both M.circinelloides and M.alpina biomass and an increase in the initial solvent-to-sample ratio (from 3:1 to 20:1) was needed to achieve complete extraction and a lipid-free IR spectrum. In accordance with previous studies, the gravimetric lipid yield was shown to overestimate the potential of the SCO producers and FAME quantification in GC-FID was found to be the best-suited method for lipid quantification. We conclude that FTIR spectroscopy can serve as a tool for evaluating the lipid extraction efficiency, in addition to identifying components that may affect lipid extraction processes.

Single cell oil production by Mortierella isabellina from steam exploded corn stover degraded by three-stage enzymatic hydrolysis in the context of on-site enzyme production.

Single cell oil (SCO), promising as alternative oil source, was produced from steam exploded corn stover (SECS) by Mortierella isabellina. Different bioprocesses from SECS to SCO were compared and the bioprocess C using the three-stage enzymatic hydrolysis was found to be the most efficient one. The bioprocess C used the lowest enzyme input 20FPIU cellulase/g glucan and the shortest time 222h, but produced 44.94g dry cell biomass and 25.77g lipid from 327.63g dry SECS. It had the highest lipid content 57.34%, and its productivities and yields were much higher than those of the bioprocess B and comparable to the bioprocess A, indicating that the three-stage enzymatic hydrolysis could greatly improve the efficiency of the bioprocess from high solid loading SECS to SCO by Mortierella isabellina. This work testified the application value of three-stage enzymatic hydrolysis in lignocellulose-based bioprocesses.

Lipid Fraction and Intracellular Metabolite Analysis Reveal the Mechanism of Arachidonic Acid-Rich Oil Accumulation in the Aging Process of Mortierella alpina.

The mechanism of arachidonic acid (ARA) content increase during aging of Mortierella alpina was elucidated. Lipid fraction analysis showed that ARA content increased from 46.9% to 66.4% in the triacylglycerol (TAG) molecule, and ARA residue occupation increased in the majority of TAG molecules during the aging process. For the first time, intracellular metabolite analysis was conducted to reveal the pathways closely associated with ARA biosynthesis during aging. The main reason for the increased ARA content was not only at the expense of other fatty acids degradation but also at the expense of further ARA biosynthesis during aging. Furthermore, translocation played a vital role in ARA redistribution among the glycerol moiety, and mycelium did not die immediately with key pathways activated to maintain a relatively stable intracellular environment. This study lays a foundation for further improvement of ARA content in the oil product obtained from M. alpina.

Structural determination and antitumor activities of a water-soluble polysaccharide from Mortierella hepiali.

A water-soluble polysaccharide (MHP-1) isolated from cultured Mortierella hepiali was obtained by hot-water extraction, DEAE-cellulose 52 anion-exchange, and Sephadex G-25 gel permeation chromatography. Its structural characteristics were investigated by PMP pre-column derivation, partial hydrolysis with acid, methylation analysis, as well as FTIR and NMR spectroscopy. Data revealed that MHP-1 possessed a backbone consisting of the repeating unit→2)-ß-d-Manp-(1, 6)-ß-d-Glcp-(1→and 3)-ß-d-Manp-(1→, branching at O-4 of→2)-ß-d-Manp-(1, and terminated with ß-d-Manp. The antitumor effects of MHP-1 on H22 tumor-bearing mice were studied. Results showed that MHP-1 significantly inhibited the growth of implanted H22 tumors in mice in vivo, but not on H22, A549, and Hep-G2 cells in vitro. Serum and spleen cells were separated to evaluate the concentrations of TNF-α by ELISA kit. The release of TNF-α was markedly induced by MHP-1 in a dose-dependent manner.

Subchronic (13-week) oral toxicity study of dihomo-gamma-linolenic acid (DGLA) oil in rats.

Dihomo-gamma-linolenic acid (DGLA) is one of the essential fatty acids, and has anti-inflammatory and anti-allergic effects. To assess the toxicity of a novel DGLA oil produced by the fungus Mortierella alpina, we examined it in the Ames test and in acute and subchronic oral toxicity tests in rats. In the Ames test, no mutagenicity was found up to 5000 microg/plate. The acute toxicity test revealed no toxicity related to DGLA oil at 10 g/kg. In the subchronic toxicity test, DGLA oil (500, 1000, and 2000 mg/kg) was orally administered. Water and soybean oil (2000 mg/kg) were used for the no-oil control and soybean oil control groups, respectively. There was no death in either sex. Because of administration of large amounts of oil, food consumption was low in the soybean oil control and the three test groups, which appeared to mildly decrease urinary excretion of Na, K, and Cl, as well as total serum protein, albumin, and blood urea nitrogen levels. There were no toxicological changes in body weight, food consumption, ophthalmological examination, urinalysis, hematological examination, blood biochemical examination, necropsy, organ weight, or histopathological examination. These findings show that the no-observed-adverse-effect level of the DGLA oil was 2000 mg/kg.

Production of arachidonic acid and dihomo-gamma-linolenic acid from glycerol by oil-producing filamentous fungi, Mortierella in the ARS culture collection.

The filamentous fungi of the genus Mortierella are known to produce arachidonic acid from glucose, and the species alpina is currently used in industrial production of arachidonic acid in Japan. In anticipation of a large excess of the co-product glycerol from the national biodiesel program, we are trying to find new uses for bioglycerin. We screened 12 Mortierella species: M. alpina NRRL 6302, M. claussenii NRRL 2760, M. elongata NRRL 5246, M. epigama NRRL 5512, M. humilis NRRL 6369, M. hygrophila NRRL 2591, M. minutissima NRRL 6462, M. multidivaricata NRRL 6456, M. nantahalensis NRRL 5216, M. parvispora NRRL 2941, M. sepedonioides NRRL 6425, and M. zychae NRRL 2592 for their production of arachidonic acid (AA) and dihomo-gamma-linolenic acid (DGLA) from glycerol. With glucose as substrate all of the strains tested produced AA and DGLA. The total fatty acid content of 125 mg/g cell dry weight (CDW) and fatty acid composition for AA (19.63%) and DGLA (5.95%) in the mycelia of M. alpina grown on glucose were comparable with those reported by Takeno et al. (Appl Environ Microbiol 71:5124-5128, 2005). With glycerol as substrate all species tested grew on glycerol and produced AA and DGLA except M. nantahalensis NRRL 5216, which could not grow on glycerol. The amount of AA and DGLA produced were comparable with those obtained with glucose-grown mycelia. The top five AA producers (mg AA/CDW) from glycerol were in the following order: M. parvispora>M. claussenii>M. alpina>M. zychae>M. minutissima. The top five dry mycelia weights were: M. zychae>M. epigama>M. hygrophila>M. humilis>M. minutissima. The top five species for total fatty acids production (mg/g CDW) were: M. claussenii>M. parvispora>M. minutissima>M. hygrophila>M. maltidivaricata. We selected two species, M. alpina and M. zychae for further studies with glycerol substrate. Their optimum production conditions were determined. Time course studies showed that the maximum cell growth and AA production for both species were at 6 days of incubation. Therefore, glycerol can be considered for industrial use in the production of AA and DGLA.

Improvement of the fatty acid composition of an oil-producing filamentous fungus, Mortierella alpina 1S-4, through RNA interference with delta12-desaturase gene expression.

An oleaginous fungus, Mortierella alpina 1S-4, is used commercially for arachidonic acid production. Delta12-Desaturase, which desaturates oleic acid (18:1n-9) to linoleic acid (18:2n-6), is a key enzyme in the arachidonic acid biosynthetic pathway. To determine if RNA interference (RNAi) by double-stranded RNA occurs in M. alpina 1S-4, we silenced the Delta12-desaturase gene. The silenced strains accumulate 18:2n-9, 20:2n-9, and Mead acid (20:3n-9), which are not detected in either the control strain or wild type strain 1S-4. The fatty acid composition of stable transformants was similar to that of Delta12-desaturation-defective mutants previously identified. Thus, RNAi occurs in M. alpina and could be used to alter the types and relative amounts of fatty acids produced by commercial strains of this fungus without mutagenesis or other permanent changes in the genetic background of the producing strains.

Evaluation of a subchronic (13-week) oral toxicity study, preceded by an in utero exposure phase, with arachidonic acid oil derived from Mortierella alpina in rats.

Arachidonic acid oil (ARA-oil) derived from the fungus Mortierella alpina for use in infant nutrition was tested in a subchronic (13-week) oral toxicity study in rats, preceded by an in utero exposure phase. The ARA-oil was administered as admixture to the rodent diet at dose levels of 3000 ppm, 15,000 ppm and 75,000 ppm. An additional high-dose group received 75,000 ppm ARA-oil in combination with 55,000 ppm fish oil containing docosahexaenoic acid (DHA), at a ratio of ARA to DHA, comparable to the ratio in mother's milk of 2:1. The total levels of fat in each diet were kept constant by adding the appropriate amounts of corn oil. A concurrent control group received 130,000 ppm corn oil in the diet. An additional carrier control group was fed unsupplemented rodent diet. Administration of the test substances from 4 weeks prior to mating, throughout mating, gestation, lactation of parental (F(0)) animals and weaning of the F(1) pups did not affect fertility or reproductive performance, nor the general condition of pups, viability, sex ratio or number of pups. Pup weight gain in the ARA/DHA-oil group was lower than the controls administered equal amounts of corn oil. In the subsequent subchronic study survival, clinical signs, body weight gain and food consumption were not adversely affected by the test substances. Ophthalmoscopic examination did not reveal any treatment-related changes. There were no treatment-related effects observed up to dietary test substance concentrations of 15,000 ppm. The following statistically significant differences were found in the ARA high-dose group and /or in the ARA/DHA group compared to the corn oil control group: decreased alkaline phosphatase activity, decreases in cholesterol, triglycerides and phospholipids concentrations, increased creatinine and urea concentrations. Furthermore, these groups showed increased adrenal, spleen and liver weights. The incidence of hepatocellular vacuolation was increased in females of the ARA high-dose group and the ARA/DHA group. Oil droplets were observed in the mesenteric lymph nodes and in the intestinal villi in the ARA high-dose group and the ARA/DHA group. In addition, lipogranulomas were observed in the mesenteric lymph nodes in these groups. The observed changes in the high-dose groups may be effects of the high intake of high-fat levels, rather than specific effects of the ARA-oil. The no-observed-effect level in this study was placed at 15,000 ppm ARA-oil. This level is equivalent to approximately 970mg ARA-oil/kg body weight/day.

Dielectric analysis for estimation of oil content in the mycelia of Mortierella alpina.

The dielectric behavior of the filamentous fungi Mortierella alpina SAM2104 and 1S-4, which produce polyunsaturated fatty acid enriched oil in the mycelia, was investigated. During the cultivation carried out in a 10-kL fermentor for 12-15 days, the relative permittivity and conductivity of the broth were measured in the frequency range of 100 kHz to 30 MHz. The dielectric parameters, i.e., the amplitude of dielectric relaxation (Deltaepsilon) and the characteristic frequency (f(c)), were obtained by fitting the Cole-Cole equation to the observed dielectric relaxation, and the conductivity of the medium (kappa(a)) was also measured. The value of Deltaepsilon gradually increased from the second day through the end of cultivation, suggesting that volume fraction of the cell increased with oil accumulation. The conductivity of the cytoplasm (kappa(i)) was calculated from the experimental values of f(c) and kappa(a), using a theoretical equation based on an ellipsoidal cell model. As a result, good correlation between the calculated kappa(i) and the oil content was obtained. These findings indicate that dielectric analysis enables us to estimate the oil content in the mycelia of oleaginous fungi and also provides a useful tool for monitoring cell growth and for controlling the cultivation process.