Formation of volatile sulfur compounds and metabolism of methionine and other sulfur compounds in fermented food

The formation of volatile sulfur compounds (VSC) in fermented food is a subject of interest. Such compounds are essential for the aroma of many food products like cheeses or fermented beverages, in which they can play an attractive or a repulsive role, depending on their identity and their concentration. VSC essentially arise from common sulfur-bearing precursors, methionine being the most commonly found. In the first section of this paper, the main VSC found in cheese, wine, and beer are reviewed. It is shown that a wide variety of VSC has been evidenced in these food products. Because of their low odor threshold and flavor notes, these compounds impart essential sensorial properties to the final product. In the second section of this review, the main (bio)chemical pathways leading to VSC synthesis are presented. Attention is focused on the microbial/enzymatic phenomena—which initiate sulfur bearing precursors degradation—leading to VSC production. Although chemical reactions could also play an important role in this process, this aspect is not fully developed in our review. The main catabolic pathways leading to VSC from the precursor methionine are presented.     

Influence of anxiety on the production of oral volatile sulfur compounds

Since many patients complain about halitosis without there being any clinical evidence of its cause, psychological symptoms have been pointed out as halitosis-inducing factors. The aim of this study was to evaluate the influence of anxiety on the production of volatile sulfur compounds (VSC). Seventeen undergraduate men in good oral and general health participated in this study, after approval by the ethics committee. The volunteers were requested to refrain from toothbrushing, using mouth rinse and eating on the experimental day. Before presenting the anxiogenic condition, the volunteer was asked to fill out the Beck Anxiety Inventory questionnaire, to check whether he had been exposed to stressors during the previous week. The Video-Recorded Stroop Color-Word Test (VRSCWT) was used to elicit anxiety. The VSC (halimeter), blood pressure, heart rate and salivary flow measurements were taken before and after the VRSCWT. The volunteers presented a minimal or slight level of anxiety before the test. There was an increase in the oral concentration of VSC, Systolic Blood Pressure and of heart rate (p < 0.05) after the VRSCWT, and no changes in the salivary flow. The results of the present study showed that the anxiogenic condition (VRSCWT) induced increases in VSC concentration, which might contribute to halitosis.     

葡萄酒中重要挥发性硫化物的代谢及基因调控

葡萄酒中的挥发性硫化物是由酿酒微生物在葡萄酒发酵过程中代谢所产生的,主要包括硫化氢、硫醇、硫醚、硫醇酯、含硫杂醇油及杂环化合物等,它们对葡萄酒的风味会产生重要影响。本综述介绍了葡萄酒中重要的挥发性硫化物的主要代谢途径及相关基因的调控机制,并提出酿酒微生物的相关研究是提高优良风味物质含量,同时抑制不良风味产生的有效途径。     

Selection of non-conventional yeasts and their use in immobilized form for the bioremediation of olive oil mill wastewaters

The yeast population dynamics in olive wastewaters (OMW), sampled in five mills from Salento (Apulia, Southern Italy), were investigated. Three hundred yeasts were isolated in five industrial mills and identified by molecular analysis. Strains belonging to Geotrichum, Saccharomyces, Pichia, Rhodotorula and Candida were detected. Five G. candidum strains were able to grow in OMW as the sole carbon source and to reduce phenolics, chemical oxygen demand (COD) and antimicrobial compounds. One G. candidum isolate was selected for whole-cell immobilization in calcium alginate gel. The COD and phenolic reduction obtained with immobilized cells showed a 2.2- and 2-fold increase compared to the removal obtained with free cells, respectively. The immobilization system enhanced yeast oxidative activity by avoiding the presence of microbial protease in treated OMW. To our knowledge, this is the first report on G. candidum whole-cell immobilization for OMW bioremediation.     

A nonradioactive 96-well plate assay for screening of trans-sialidase activity

Trans-sialidase (E.C. 3.2.1.18) catalyzes the transfer of preferably alpha2,3-linked sialic acid to another glycan or glycoconjugate, forming a new alpha2,3 linkage to galactose or N-acetylgalactosamine. Here, we describe a nonradioactive 96-well plate fluorescence test for monitoring trans-sialidase activity with high sensitivity, specificity, and reproducibility using sialyllactose and 4-methylumbelliferyl-beta-D-galactoside as donor and acceptor substrates, respectively. The assay conditions were optimized using the trans-sialidase from Trypanosoma congolense and its general applicability was confirmed with recombinant trans-sialidase from Trypanosoma cruzi. Using this procedure, a large number of samples can be tested quickly and reliably, for instance in monitoring trans-sialidase during enzyme purification and the production of monoclonal antibodies, for enzyme characterization, and for identifying potential substrates and inhibitors. The trans-sialidase assay reported here was capable of detecting trans-sialidase activity in the low-mU range and may be a valuable tool in the search for further trans-sialidases in various biological systems.     

Identification of Geotrichum candidum at the species and strain level: proposal for a standardized protocol

In this study, the M13 primer was used to distinguish Geotrichum candidum from the anamorphic and teleomorphic forms of other arthrospore-forming species (discriminatory power = 0.99). For intraspecific characterization, the GATA4 primer showed the highest level of discrimination for G. candidum among the 20 microsatellite primers tested. A molecular typing protocol (DNA concentration, hybridization temperature and type of PCR machine) was optimized through a series of intra- and interlaboratory trials. This protocol was validated using 75 strains of G. candidum, one strain of G. capitatum and one strain of G. fragrans, and exhibited a discrimination score of 0.87. This method could therefore be used in the agro-food industries to identify and to evaluate biodiversity and trace strains of G. candidum. The results show that the GATA4 primer might be used to differentiate strains according to their ecological niche.     

Improvement of the cryopreservation of the fungal starter Geotrichum candidum by artificial nucleation and temperature downshift control

Food industry tends towards the use of controlled microorganisms in order to improve its technologies including frozen starter production. The fungus Geotrichum candidum, which is currently found in various environments, is widely used as ripening agent in some specific cheese making process. In order to optimize the cryopreservation of this microorganism, freezing experiments were carried out using a Peltier cooler-heater incubator, which permits to control the temperature downshift from +20 to -10 degrees C in time period ranges from 20 to 40min depending on the experiments. Concomitantly, study of the effect of an industrial ice nucleator protein derived from Pseudomonas syringae (SNOMAX) on the dynamic of freezing of G. candidum was carried out. Our results showed that the addition of this protein in the microbiological suspension has different complementary effects: (i) the synchronization of the different samples nucleation, leading to an homogeneous and earlier freezing, (ii) the increase of the freezing point temperature from -8.6 to -2.6 degrees C, (iii) a significant decrease of the lethality of G. candidum cells subjected to a freezing-thawing cycles challenge.     

Diffusible and volatile compounds produced by an antagonistic Bacillus subtilis strain cause structural deformations in pathogenic fungi in vitro

An efficient antagonistic strain of Bacillus subtilis, originally isolated from the rhizosphere of established tea bushes, was found to cause structural deformities in six pathogenic fungi under in vitro culture conditions. This effect was attributed to the production of diffusible and volatile antifungal compounds. Out of the selected test fungi four were phytopathogenic, while the remaining two were of clinical importance. The bacterial strain successfully restricted the growth of all test fungi in dual cultures, and induced morphological abnormalities such as mycelial and conidial deviations. The inhibitory effect caused by volatiles was greater than that by diffusible compounds.     

Kinetics of an extracellular exo-inulinase production from a 5-flourocytosine resistant mutant of Geotrichum candidum using two-factorial design

In the present study, eight different strains of Geotrichum candidum were isolated and screened for an extracellular exo-inulinase production using chemically enriched sucrose–mineral media. The isolate (Zool-3i) with a better enzyme activity (1.38 IU/ml) was subjected to induced mutagenesis using methyl methane sulphonate (MMS) and a mutant with an enzyme activity of 32.06 IU/ml was obtained. Further exposure to ethyl methane sulphonate (EMS) and ultraviolet (UV) radiations yielded a mutant exhibiting an improved activity of 39.34 IU/ml. The potential mutant was cultured overnight and plated on 5fc–YPR agar medium and thus made resistant against 5-flourocytocine. Over 50-fold enhancement in enzyme production (71.85 IU/ml) was achieved when the process parameters including incubation period (48 h), sucrose concentration (5.0 g/L), pH (6.0), inoculum size (2.0%, 16 h old) and urea (0.2%) were identified using Plackett–Burman design. On the basis of kinetic variables, notably Q_p (0.723 U/g/h), Y_p/s (2.036 U/g) and q_p (0.091 U/g cells/h), the mutant MEU-5fc-6 was found to be a hyper producer of exo-inulinase (HS, LSD 0.045, p ? 0.05).     

Muscodor fengyangensis sp. nov. from southeast China: morphology, physiology and production of volatile compounds

The fungal genus Muscodor was erected on the basis of Muscodor albus, an endophytic fungus originally isolated from Cinnamomum zeylanicum. It produces a mixture of volatile organic compounds (VOCs) with antimicrobial activity that can be used as mycofumigants. The genus currently comprises five species. Here we describe the isolation and characterization of a new species of Muscodor on the basis of five endophytic fungal strains from leaves of Actinidia chinensis, Pseudotaxus chienii and an unidentified broad leaf tree in the Fengyangshan Nature Reserve, Zhejiang Province, Southeast of China. They exhibit white colonies on potato dextrose agar (PDA) media, rope-like mycelial strands, but did not sporulate. The optimum growth temperature is 25°C. The results of a phylogenetic analysis based on four loci (ITS1-5.8S-ITS2, 28S rRNA, rpb2 and tub1) are consistent with the hypothesis that these five strains belong to a single taxon. All five strains also produce volatile chemical components with antimicrobial activity in vitro, which were different from those previously described for other Muscodor species.     

Development of a high-throughput screening system for the compounds that inhibit collagen–protein interactions

Collagen-binding proteins (CBPs) play important roles in various physiological events. Some CBPs are regarded as targets for drug development; for example, platelet glycoprotein VI (GPVI) and heat shock protein 47 (HSP47) are promising targets for the development of novel antiplatelet and antifibrotic drugs, respectively. However, no systematic screening method to search compounds that inhibit collagen–CBP interactions have been developed, and only a few CBP inhibitors have been reported to date. In this study, a facile turbidimetric multiwell plate assay was developed to evaluate inhibitors of CBPs. The assay is based on the finding that CBPs retard spontaneous collagen fibril formation in vitro and that fibril formation is restored in the presence of compounds that interfere with the collagen–CBP interactions. Using the same platform, the assay was performed in various combinations of fibril-forming collagen types and CBPs. This homogeneous assay is simple, convenient, and suitable as an automated high-throughput screening system.     

Spatial and temporal variability of dissolved sulfur compounds in European estuaries

The spatial and temporal distribution of Dimethylsulfide (DMS),Dimethylsulfoniopropionate (DMSP) – its precursor –,Dimethylsulfoxyde (DMSO) – one of its oxidation products –Carbonyl sulfide (COS) and Carbonyl disulfide (CS₂) wereinvestigated during nine oceanographic cruises carried out in six majortidal European estuaries between July 1996 and May 1998. Low levels ofDMS were recorded (mean of 0.6 nM, standard deviation () 0.3 nM)during these 9 cruises. Positive correlations between DMS and salinitywere frequently observed, with the highest DMS concentrations in theplume of estuaries, which could be explained by change in phytoplanktonspeciation from estuarine to shelf waters. Strong correlation betweenDMSP and DMS was reported for most of the estuaries denoting a simpleconservative mixing of riverine and marine waters controlled by tide. Incontrast to DMS, significant levels of COS and CS₂ with meanconcentrations of 220 ± 150 (pM; pM =10^–12 M) and 25 ± 6 pM respectively wererecorded in four estuaries, indicating that estuaries could be asignificant source of these compounds.     

Between plant and diurnal variation in quantities and ratios of volatile compounds emitted by Vicia faba plants

Ratios of volatile phytochemicals potentially offer a means for insects to recognise their host-plant species. However, for this to occur ratios of volatiles would need to be sufficiently consistent between plants and over time to constitute a host-characteristic cue. In this context we collected headspace samples from Vicia faba plants to determine how consistent ratios of key volatile phytochemicals used in host location by one of its insect pests, the black bean aphid, Aphis fabae, were. These were (E)-2-hexenal, (Z)-3-hexen-1-ol, 1-hexanol, benzaldehyde, 6-methyl-5-hepten-2-one, octanal, (Z)-3-hexen-1-yl acetate, (R)-linalool, methyl salicylate, decanal, undecanal, (E)-caryophyllene, (E)-β-farnesene, (S)-germacrene D, and (E, E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, which had previously been found to be electrophysiologically and behaviourally active to A. fabae. Although the quantities of volatiles produced by V. faba showed large between plant and diurnal variation, correlations between quantities of compounds indicated that the ratios of certain pairs of volatiles were very consistent. This suggests that there is a host-characteristic cue available to A. fabae in the form of ratios of volatiles.     

Tandem screening of toxic compounds on GFP-labeled bacteria and cancer cells in microtiter plates

A 96-well fluorescence-based assay has been developed for the rapid screening of potential cytotoxic and bacteriocidal compounds. The assay is based on detection of green fluorescent protein (GFP) in HeLa human carcinoma cells as well as gram negative (Escherichia coli) and gram positive bacteria (Mycobacterium avium). Addition of a toxic compound to the GFP marked cells resulted in the loss of the GFP fluorescence which was readily detected by fluorometry. Thirty-nine distinct naphthoquinone derivatives were screened and several of these compounds were found to be toxic to all cell types. Apart from differences in overall toxicity, two general types of toxic compounds were detected, those that exhibited toxicity to two or all three of the cell types and those that were primarily toxic to the HeLa cells. Our results demonstrate that the parallel screening of both eukaryotic and prokaryotic cells is not only feasible and reproducible but also cost effective.     

Seasonal changes in fluxes of methane and volatile sulfur compounds from rice paddies and their concentrations in soil water

Rice paddies emit not only methane but also several volatile sulfur compounds such as dimethyl sulfide (DMS: CH₃SCH₃). However, little is known about DMS emission from rice paddies. Fluxes of methane and DMS, and the concentrations of methane and several volatile sulfur compounds including hydrogen sulfide (H₂S), carbonyl disulfide (CS₂), methyl mercaptan (CH₃SH) and DMS in soil water and flood water were measured in four lysimeter rice paddies (2.5 × 4 m, depth 2.0 m) once per week throughout the entire cultivation period in 1995 in Tsukuba, Japan. The addition of exogenous organic matter (rice straw) was also examined for its influence on methane or DMS emissions. Methane fluxes greatly differed between treatments in which rice straw had been incorporated into the paddy soil (rice straw plot) and plots without rice straw (mineral fertilizer plot). The annual methane emission from the rice straw plots (37.7 g m^-2) was approximately 8 times higher than that from the mineral fertilizer plots (4.8 g m^-2). Application of rice straw had little influence on DMS fluxes. Significant diurnal and seasonal changes in DMS fluxes were observed. Peak DMS fluxes were found around noon. DMS was emitted from the flood water in the early growth stage of rice and began to be emitted from rice plants during the middle stage. DMS fluxes increased with the growth of rice plants and the highest flux, 15.1 µg m^-2 h^-1, was recorded before heading. DMS in the soil water was negligible during the entire cultivation period. These facts indicate that the DMS emitted from rice paddies is produced by metabolic processes in rice plants. The total amount of DMS emitted from rice paddies over the cultivated period was estimated to be approximately 5–6 mg m^-2. CH₃SH was emitted only from flood water during the first month after flooding.     

Development of a plate technique for screening of polysaccharide-degrading microorganisms by using a mixture of insoluble chromogenic substrates

A plate assay based on the visible solubilization of small substrate particles and the formation of haloes on Petri dishes, containing a mixture of different dye-labelled polysaccharides as substrates, provides a specific, reliable and rapid simultaneous detection of corresponding polysaccharide-degrading microorganisms. It has potential for increasing the efficacy of screening of microorganisms, utilizing different polysaccharides, in large numbers of natural samples. Diversely colored insoluble forms of amylose, xylan and hydroxyethyl-cellulose (HE-cellulose) were prepared as chromogenic substrates by using the cross-linking reagent 1,4-butanediol diglycidyl ether and the dyes Brilliant Red 3B-A, Cibacron Blue 3GA and Reactive Orange 14. Using the method, the bacteria with amylase or xylanase or cellulase or a combination of these activities were screened from soil and sludge samples, selected and identified according to 16S rDNA sequencing.     

一种鉴定多糖水解酶类及其产生菌的新方法

利用曲里苯蓝能与多糖形成复合物的原理,建立起一种简便、快捷、灵敏的筛选多糖水解酶类及其产生菌的平板鉴定方法。曲里苯蓝对供试微生物无毒害作用,不影响酶的活性,并可高温灭菌。其最适浓度在0.005%~0.01%(W/V)之间。可用于纤维素水解酶类、淀粉水解酶类、甘露聚糖酶、普鲁兰酶等多糖水解酶,与传统方法相比,该方法不仅有着同样高的灵敏度,而且能够提高筛选效率,避免污染问题,同时适合于多种多糖底物。但不能用于木聚糖酶和菊粉酶的检测。     

Reduction of ammonia and volatile organic compounds from food waste-composting facilities using a novel anti-clogging biofilter system

The performance of a pilot-scale anti-clogging biofilter system (ABS) was evaluated over a period of 125 days for treating ammonia and volatile organic compounds emitted from a full-scale food waste-composting facility. The pilot-scale ABS was designed to intermittently and automatically remove excess biomass using an agitator. When the pressure drop in the polyurethane filter bed was increased to a set point (50 mm H₂O m⁻¹), due to excess biomass acclimation, the agitator automatically worked by the differential pressure switch, without biofilter shutdown. A high removal efficiency (97-99%) was stably maintained for the 125 days after an acclimation period of 1 week, even thought the inlet gas concentrations fluctuated from 0.16 to 0.55 g m⁻³. Due the intermittent automatic agitation of the filter bed, the biomass concentration and pressure drop in the biofilter were maintained within the ranges of 1.1-2.0 g-DCW g PU⁻¹ and below 50 mm H₂O m⁻¹, respectively.     

Identification of volatile markers for indoor fungal growth and chemotaxonomic classification of Aspergillus species

Microbial volatile organic compounds (MVOCs) were collected in water-damaged buildings to evaluate their use as possible indicators of indoor fungal growth. Fungal species isolated from contaminated buildings were screened for MVOC production on malt extract agar by means of headspace solid-phase microextraction followed by gas chromatography-mass spectrometry (GC-MS) analysis. Some sesquiterpenes, specifically derived from fungal growth, were detected in the sampled environments and the corresponding fungal producers were identified. Statistical analysis of the detected MVOC profiles allowed the identification of species-specific MVOCs or MVOC patterns for Aspergillus versicolor group, Aspergillus ustus, and Eurotium amstelodami. In addition, Chaetomium spp. and Epicoccum spp. were clearly differentiated by their volatile production from a group of 76 fungal strains belonging to different genera. These results are useful in the chemotaxonomic discrimination of fungal species, in aid to the classical morphological and molecular identification techniques.     

Isolation and characterization of microorganisms able to produce 1,3-propanediol under aerobic conditions

Microbial fermentation under strictly anaerobic conditions has been conventionally used for the production of 1,3-propanediol, a key raw material required for the synthesis of polytrimethylene terephthalate (PTT) and other polyester fibers. In the current study, we have identified eight strains of microorganism which are able to produce 1,3-propanediol under aerobic condition. Those strains were isolated from garden soil, which were enriched by culturing in LB medium with glycerol added under aerobic condition. The identities of those strains were established based on their 16S rRNA sequences and physiological characteristics. Results indicated 6 strains are Citrobacter freundii and 2 strains are Klebsiella pneumoniae subsp Penumoniae. One of Klebsiella pneumoniae subsp Penumoniae strains, designated as TUAC01, demonstrated comparable levels of 1,3-propanediol oxidoreductase, glycerol dehydratase and glycerol dehydrogenase activity to the anaerobic microorganisms described in the literature. Accordingly, in larger scales (5 l) fed-batch culture the TUAC01 strain showed a remarkable 1,3-propanediol producing potency under aerobic conditions. 60.1 g/l of 1,3-propanediol was yield after 42 h incubation in an agitating bioreactor; and in air-lift bioreactor 66.3 g/l of 1,3-propanediol was yield after 58.5 h incubation. The aerobic ferment process, reduced the product cost and made the biological method of 1,3-propanediol production more attractive.