Production of methylanthranilate by the basidiomycete Pycnoporus cinnabarinus (Karst.)

Summary Pyncnoporus cinnabarinus (Polyporaceae) is able to produce methylanthranilate in liquid cultures. Study of the culture conditions of P. cinnabarinus IP I-937 has permitted increase in the aroma productivity by a factor of 16. A low nitrogen concentration, with maltose as carbon source, was required; the culture pH was uncontrolled. The inoculum nature and concentration greatly influence on production: best results were obtained with conidia from a late harvest, used at a rate of 2 × 10⁵ spores/ml. Under these conditions, 18.7 mg methylanthranilate/l was produced after 5 days of culture. Aroma production is probably connected with the biosynthesis of phenoxazinones, which are characteristic pigments of the genus Pycnoporus. Offprint requests to: B. Gross     

Characterization of volatile fraction of typical Irpinian wines fermented with a new starter yeast

Non-Saccharomyces yeasts are microorganisms that play an important role in the fermentation dynamics, compositions and flavour of wine. The aromatic compounds responsible for varietal aroma in wine are mainly terpenes, of which the most important group are the monoterpenes because of their volatility and odour if present in a free form. In fact, some terpenyl-glycosides do not contribute to the aroma unless they are hydrolysed. The glycosylated form of terpenes can be converted by hydrolysis with β-glycosidases produced by yeasts during the winemaking process, into aromatic compounds. In this study we utilized a non-Saccharomyces yeast, with a high extra-cellular glycosidase activity, isolated from grapes of cultivars typical of Irpinia region. This strain, identified as a Rhodotorula mucillaginosa (strain WLR12), was used to carry out an experimental winemaking process and the results were compared with those obtained with a commercial yeast starter. Chemical and sensorial analysis demonstrated that the wines produced with WLR12 strain had a more floral aroma and some sweet and ripened fruit notes compared to those obtained with commercial yeast. The data also showed an increasing of the free terpenes fraction that, however, did not significatively modify the bouquet of the wines.     

Application of bioreactors for large-scale micropropagation systems of plants

Summary The application of bioreactor culture techniques for plant micropropagation is regarded as one of the ways to reduce production cost by scaling-up and automation. Recent experiments are restricted to a small number of species that, however, demonstrate the feasibility of this technology. Periodic immersion liquid culture using ebb and flood system and column-type bubble bioreactors equipped with a raft support system to maintain plant tissues at the air and liquid interface were found to be suitable for micropropagation of plants via the organogenic pathway. Balloon-type bubble bioreactors proved to be fit for micropropagation via somatic embryogenesis with less shear stress on cultured cells. Several cultivars of Lilium were successfully propagated using a two-stage culture method in one bioreactor. A large number of small-scale segments were cultured for 4 wk with periodic immersion liquid culture to induce multiple bulblets from each segment, then the bulblet induction medium was changed into bulblet growth medium by employing a submerged liquid bioreactor system. This culture method resulted in a nearly 10-fold increase in bulblet growth compared to conventional culture with solid medium. About 20 000 cuttings of virus-free potato could be obtained from 120 singlenode explants in a 20-liter balloon-type bubble bioreactor after 8 wk of culture. The percentage of ex vitro survival and root induction of the cuttings was more than 95%. Other successful results were obtained from the micropropagation and transplant production of chrysanthemum, sweetpotato, Chinese foxglove. Propagation systems via somatic embryogenesis in Acanthopanax koreanum and thornless Aralia elata were established using a liquid suspension of embryogenic determined cells. More than 500 000 somatic embryos in different stages were harvested from a 10-liter balloon-type bubble bioreactor after a 6-wk culture. Further development of these embryos in solid medium and eventually in the field was successful. The bioreactor system could reduce initial and operational cost for micropropagation, but further development of sophisticated technology might be needed to apply this system to plant micropropagation industries.     

Extracellular proteolytic activity ofCryptococcus neoformans

Eight strains ofCryptococcus neoformans var.neoformans isolated from AIDS patients in the Infectious Disease Institute, University of Turin, Italy, were examined for growth and extracellular proteolytic activity in culture with solid and liquid media. All of the strains grew well on Yeast Carbon Base (YCB) agar medium supplemented with both 0.1% (w/v) bovine serum albumin (BSA) and 0.01% (w/v) polypeptone (Pp), and produced a clear proteolytic zone around their colonies, whereas they exhibited less growth and proteolytic activity on YCB medium supplemented with BSA alone. Strain #8 with a strong proteolytic activity was cultured in three different liquid media. Its growth was limited in YCB medium supplemented with 0.1% BSA, but was moderate in that with 0.01% Pp. Enhanced growth was supported by the addition of both BSA and Pp to the YCB medium. The relative value of the final cellular yields obtained with the above YCB-0.1% BSA, YCB-0.01% Pp and YCB-0.1% BSA-0.01% Pp media was approximately 1:10:20. In the culture with YCB medium containing both BSA and Pp, a rapid decrease in the amount of BSA was demonstrated by a spectrophotometric assay and gel electrophoresis of the culture supernatant after the log-to-stationary phase. The proteolytic activity in the culture supernatant became detectable after the log phase when tested with skim milk agarose plates. These results allowed us to conclude thatCr. neoformans var.neoformans is able to secrete protease and to utilize protein as a source of nitrogen.     

A Thin‐Layer Liquid Culture Technique for the Growth of Helicobacter pylori

Background and Aims: Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin‐layer liquid culture technique for the growth of H. pylori. Methods: A thin‐layer liquid culture system was established by adding liquid media to a 90‐mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined. Results: Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI‐1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum‐free RPMI‐1640 supported the growth of H. pylori when supplemented with dimethyl‐β‐cyclodextrin (200 μg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD₆₀₀ with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD₆₀₀ contained 1.3 ± 0.1 × 10⁹ CFU/mL. γ‐Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin‐layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours. Conclusions: Thin‐layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.     

Temporal Aroma Release Profile of Solid and Liquid Droplet Emulsions

The release kinetics of four model aroma compounds from coarse (d ₃₂ = 1.0 μm) and fine (d ₃₂ = 0.25 μm) eicosane and hydrogenated palm stearin (HPS) emulsions prepared with either solid or liquid lipid droplets were measured using a model mouth instrument. For both lipids, the release of aroma compounds from emulsions with solid droplets was higher than from emulsions with liquid droplets. This difference was greater for less polar aroma compounds. The release from solid eicosane droplets increased with particle size but no such effect was observed for HPS emulsions, however, the release from solid eicosane was higher than solid HPS. The initial aroma release profile of the solid droplet emulsion matches that of a similar liquid oil emulsion but requires much less added aroma. Meeting presentation: Presented at 98th AOCS Annual Meeting and Expo in Quebec City, Canada.     

Growth and gender in the gametophyte of Blechnum spicant L

The growth and gender in the gametophyte of Blechnum spicant L. were strongly affected by its origin, spore or mature homogenized gametophytes, and also by the addition of plant growth regulators to the culture medium. Spore-derived gametophytes cultured in Murashige and Skoog liquid medium were females and heart-shaped, whereas those derived from mature homogenized gametophytes were shorter and male or asexual (1:3) because of the release of antheridiogen to the culture medium. In the latter, maleness was especially increased by the addition of BA. This cytokinin is a strong inductor of maleness in homogenized cultures; however, even though BA influenced sexual organ formation in spore-derived gametophytes, it does not change the␣female sexual pattern that these gametophytes have. Separate male and female populations of gametophytes were obtained in this work.     

CARBONYL COMPOUNDS PRODUCED BY CERATOCYSTIS FAGACEARUM

Ceratocystis fagacearum produces both in nature and in laboratory culture a characteristic fruity aroma. It is assumed that the odor may serve as an attractant for insects which are vectors for the disease as well as important agents in the fertilization mechanism. A number of carbonyl compounds were identified from liquid cultures of C. fagacearum and included: acetaldehyde, furfuraldehyde, acetone, 2-hexenal and 2-methylbutanal. The 2,4-dinitrophenylhydrazones of the compounds were identified by means of thin-layer chromatography, melting-point determination, gas chromatography and infrared spectroscopy.     

Influence of age of mycelium and water activity of the medium on aroma production by Trichoderma viride grown on solid substrate

The production of 2-heptanone (cheese aroma) by Trichoderma viride TS cultivated on agar media was evaluated using headspace gas chromatographic analysis. The radial growth rate of the filamentous fungi increased with high water activity values, but the mycelial density was higher for lower water activity. Maximum aroma production of a culture was obtained at a_w = 0.96. An apparatus intended to measure the aroma production of different areas of a mycelial colony was set up. The study of the aroma production of these areas showed that the production values were greatly different and were evolving with time. It was shown that the mycelium aroma production was maximum when mycelia were about 3.5 to 6.5 d old.     

Formation of Nonculturable Cells of Mycobacterium tuberculosis and Their Resuscitation

Nonculturable cells were found to occur in populations of Mycobacterium tuberculosis cells during the long poststationary phase. These cells were small (0.6–0.8 m) ovoid and coccoid forms with intact cell walls and negligible respiratory activity, which allows them to be regarded as dormant cells. Nonculturable cells were characterized by low viability after plating onto solid medium; a minor part of the population of these cells could be cultivated in liquid medium. Cell-free culture liquid of an exponential-phase Mycobacterium tuberculosisculture or the bacterial growth factor Rpf exerted a resuscitating effect, increasing substantially the growth capacity of the nonculturable cells in liquid medium. During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell shape occurred. At early stages of resuscitation, ovoid cells formed small aggregates. The recovery of culturability was associated with the formation of rod-shaped cells in the culture. The data obtained demonstrate the in vitro formation of dormant cells of Mycobacterium tuberculosis, which do not grow on solid media but can be resuscitated in liquid medium under the effect of substance(s) secreted by actively growing cells.     

Clonal Growth of Leukaemic Cells In Vitro

Human leukaemic cell specimens were obtained from patients and directly plated into soft agar (t= 0) or cultured for 1 week in liquid phase and then plated in soft agar. Growth for 1 week in liquid phase allowed the clonal growth in agar of leukaemic specimens which were unable to clone at t= 0. Clonal growth after liquid culture consisted of the usual leukaemic type of cluster-colonies, growth of a new type of ‘syncytial’ cell colony or a mixture of colony types. In addition, marrow from a patient with acute lymphocytic leukaemia produced normal-appearing colonies after 1 week of growth in liquid phase. These studies suggest a similarity in the growth requirements of some leukaemic cells and normal CFUd cells.     

Magnetite formation by a magnetic bacterium capable of growing aerobically

Summary A helically shaped magnetic bacterium was isolated from freshwater sediment and a pure culture was obtained. The growth medium contained succinate, nitrate and ferric malate as the carbon, nitrogen and iron sources, respectively. The magnetic bacterium, designated AMB-1, was able to grow in free gaseous exchange with an air atmosphere. When cells were grown aerobically on agar, oxidase activity was present, and white non-magnetic colonies, which did not show catalase activity, were formed. The stationary phase of growth was reached 4–5 days later at a cell concentration of 1.4×10⁹ cells/ml in liquid culture when an initial cell concentration of 10⁵ cells/ml was employed. After ultrasonic disruption of harvested cells, 2.6 mg bacterial magnetite was obtained from a 11 culture. Offprint requests to: T. Matsunaga     

<Emphasis Type="Italic">Brettanomyces</Emphasis> as a Starter Culture in Rice-Steamed Sponge Cake: A Traditional Fermented Food in China

The potential use of Brettanomyces anomalus PSY-001 as an additional starter culture for the production of Rice-steamed sponge cake (RSSC), a traditional fermented food in China, was investigated. Two productions of RSSC, each containing batches of experimental cakes with Brettanomyces added and reference cakes with the leavened liquid added were carried out. For both experimental and reference cakes, chemical analysis and sensory evaluation were carried out during the fermentation period. The results showed that experimental cakes had desirable aroma and taste. The observed differences indicate a positive contribution to the overall quality of RSSC by B. anomalus PSY-001.     

Effect of leucine on aroma volatiles production from Ceratocystis fimbriata grown in liquid culture

Aroma volatiles produced by Ceratocystis fimbriata on a defined liquid synthetic medium with and without the addition of leucine were identified by gas chromatography–mass spectrometry and quantified by gas chromatography-flame ionisation detection in the liquid medium as well as in the headspace. Volatiles were extracted from the liquid by simultaneous steam distillation–solvent extraction. Ceratocystis fimbriata produced a complex set of volatile intermediary metabolites, of which ethanol was the dominant compound (92–95% of total volatiles). Low molecular weight esters, alcohols, aldehydes, ketones, alkanes, and carboxylic acids were identified in the liquid broth. Alcohols and esters were the most abundant aroma volatiles. Leucine addition effected further growth and higher volatiles production. In the headspace, ethanol and ethyl acetate accounted for 92% of total volatiles over the synthetic medium and 89% when leucine was added. Aroma perception (fruity and banana) correlated closely with liquid and headspace total volatiles.     

黑微5-3已酸菌种的分离和培养条件的探讨

黑微5-3己酸菌是从我省富裕老窖酒厂的廿多年窖泥中,分离到的一株高产菌株:最高产酸量为540毫克/100ml,活菌数5亿/ml以上,经20代连续培养没有发现菌种性能下降和退化现象,本文较详细的叙述了黑微5-3己酸菌的分离方法以及形态和培养特性,已酸菌在液态法白酒和优质酒(浓香型)的生产上,被广泛地应用,它对增加成品酒中的己酸乙酯的含量起着主导作用。     

Establishment of embryogenic suspension cultures of a wild relative of cotton (Gossypium klotzschianum Anderss.)

Summary Maintainable, highly embryogenic suspension cultures of a wild relative of cotton (Gossypium klotzschianum Anderss.) have been obtained. Callus with no apparent organization was used to establish the liquid culture. Callus growth conditions as well as suspension medium composition were optimized. A visual selection scheme was beneficial for the maintenance of the embryogenic suspension. These liquid cultures have been maintained for over 10 mo. with no loss in embryogenic capacity. The somatic embryos developed after transfer of the embryogenic tissues to a hormone-free liquid medium. Salaries and research support were provided by State and Federal funds appropriated to OSU-OARDC. This is journal article No. 71-87.     

Direct shoot and cormlet regeneration from leaf explants of ‘Silk’ banana (AAB)

Summary Direct shoot and cormlet regeneration from leaf explants were obtained in triploid dessert banana cultivar Nanjanagud Rasabale (NR) that is classified under the group ‘Silk’ and has the genotype AAB. The response for both cormlet and direct shool formation was observed only in leaf explants obtained from shoots cultured in liquid medium but not in similar explants obtained from shoots grown on gelled medium. Shoot initiation occurred after a sequential culture of leaf (sheath) explants on modified Murashige and Skoog (MS) medium supplemented with different growth regulators. In the sequence, the leaf explants were cultured first on medium with a high level (22.4 μM) of benzyladenine (BA), second on indolc-3-butyric acid (IBA) supplemented medium, and third on reduced BA medium under incubation in the dark. The highest adventitious shoot regeneration in 24% of the explants, with the number of shoots ranging from 2 to 3 per explant, occurred in the explants incubated at the first step in medium with 22.4 and 0.198 μM IBA. Further growth and complete shoot formation occurred under incubation in a 16-h photoperiod. While keeping the culture conditions constant and replacing BA with picloram (0.83–20.71 μM) in the initial step, adventious origin of cormlets occurred in 12% of the explants. However, when rhizome explants (also obtained from shoots grown in liquid medium) were cultured with various growth regulators in the first step, medium containing 2,4,5-trichlorophenoxyacctic acid (7.82 μM) produced friable callus that re-differentiated into roots only. Physical forms of the medium, ie.e. agar-gelled or liquid, imparted specific effects on the extent of multiplication of leaf-regenerated shoots with no differences in morphology and growth patterns when compared to those of meristem-derived plants.     

The composition of volatile components of dry cepe and oyster mushroom

The composition of aroma compounds in dry cepe mushroom (Boletis edulis Fr.) and oyster mushroom (Pleurotus ostreatus Fr.) was studied using capillary gas chromatography and chromatography—mass spectrometry. In dry cepe, 53 volatile compounds were identified, and in dry oyster mushroom 41 compounds were identified. Volatile organic substances with various functional groups formed the flavor of dry mushrooms. Unsaturated alcohols and ketones with eight carbon atoms were responsible for the mushroom notes of products. Their content in dry cepe was much higher than in dry oyster mushroom. The specific aroma of dry cepe was formed by the complex mixture of methional, substituted furans, pyrazines, and pyrroles. The content of these compounds was higher in dry cepe than in dry oyster mushroom. The content of aromatic and aliphatic aldehydes with six, nine, and ten carbon atoms was higher in dry oyster mushroom. The differences in the qualitative and quantitative composition of volatile compounds are responsible for more intensive and pleasant aroma of dry cepe in comparison to that of dry oyster mushroom.     

Biodegradation of unvulcanized natural rubber by microorganisms isolated from soil and rubber surface: A preliminary study

The Natural rubber (NR) biodegradation by three microorganisms has been evaluated: a yeast (Rhodotorula mucilaginosa) and a bacterium (Pseudomonas sp.), isolated in a liquid culture from soil, and a filamentous fungus (Alternaria alternata), isolated on a solid culture from an NR surface, were tested. The biodegradation was conducted for four months in liquid culture, at 30°C, in agitated and stationary conditions, using a Mineral Salt Medium with NR as the only carbon source. The growth behaviour of the yeast and the bacterium was evaluated by means of optical density measurements (OD₆₅₀). At the end of the incubation, the dry weight biomass of the microorganisms was measured. R. mucilaginosa showed a higher biomass production in the agitated culture, while a more efficient production was observed in static conditions for the Pseudomonas and A. alternata strains. The highest enzymatic activity of Lignin peroxidase (LiP) and Manganese-dependent peroxidase (MnP) was obtained in static conditions for A. alternata. The laccase production was probed by guaiacol oxidative polymerization on agar plates. The microorganism biodegradation capability was assessed through a combination of SEM analysis, FTIR-ATR spectroscopy, and Size Exclusion Chromatography techniques. An extended mycelium-substrate interphase and a decrease in the NR molecular weight were observed.