Cork-borne bacteria and yeasts as potential producers of off-flavours in wine

Bacteria and yeasts were found to be present within cork lenticels, covered by mucous or fibrous substances. They survived heating, peroxide treatment and contact with the alcohol and sulfur dioxide of wine. 187 bacteria and 36 yeast strains were isolated from cork stoppers of wine bottles and, during various stages of production, from corkwood and new cork stoppers. After culturing, a number of isolates showed the ability to modify the aroma of model systems consisting of dilute or full strength wine and pulverised cork. The aromas produced by isolates of varying cork origin are tabled. A small number of isolates methylated 2,4,6-trichlorophenol, yielding 2,4,6-trichloroanisole, responsible for the typical cork taint. During the boiling of cork slabs, the internal temperature on the inside of a box made from cork slices did not exceed 87°C.     

Antagonism of Trichoderma or Gliocladium Species on Two Phytopathogenic Species of Fusarium

SUMMARY

The antagonistic actions of Trichoderma and Gliocladium species against two phytopathogenic species of Fusarium (a serious disease in cotton production) were studied in vitro. The antagonist induced a substantial lysis of mycelium of the target fungi which became malformed and thickened. Bulging or swollen hyphal strands occurred and sometimes with clamydospores production. Antagonists produced chitinase and β-1,3-glucanase in liquid culture contained cell wall of Fusarium spp. as a sole carbon source. Crude enzyme preparations of the antagonists with lytic enzyme activities greatly reduced the germinated conidia of Fusarium spp. Moreover, aberrant morphology of conidia was observed with germ tube lysis. On the other hand, the resulted mycelia showed wilt mycelium, coagulated protoplasm, heavy vacuolization and swelling followed by the complete destruction of the hyphae.     

Acidic pH as a determinant of TRI gene expression and trichothecene B biosynthesis in Fusarium graminearum

Reducing production of type B trichothecenes by Fusarium graminearum on cereals is necessary to control contamination, prevent yield reduction and protect human and animal health. Thus, an understanding of how trichothecene biosynthesis is induced is essential. The effect of ambient pH on fungal growth, toxin biosynthesis and expression of TRI genes was studied during in vitro liquid culture of F. graminearum on minimal medium. Fungal development stopped at day 3 after a sharp pH drop in the medium. At the same time, induction of TRI gene expression was observed and toxin began accumulating 1 day later. Acidification seems a determinant of induction, as neither the toxin nor the TRI genes were detected when the pH was maintained neutral. Shifting from neutral to acidic pH by mycelium transfer induced TRI gene expression and toxin accumulation. The regulation of toxin production by ambient pH appears to be specific to some TRI genes since TRI5, located in the core FgTRI5 cluster, showed an immediate induction while TRI101, located elsewhere in the genome, showed a more progressive response. The regulation of trichothecene biosynthesis by the ambient pH appears to be a general mechanism, independent of strain or chemotype, as all tested strains, including F. graminearum and F. culmorum species, showed a regulation of toxin production in response to the ambient pH. We conclude that, in vitro, external acidification is required for induction of TRI gene expression.     

Fungal isolates and metabolites in locally processed rice from five agro-ecological zones of Nigeria

This study reports the distribution of fungal isolates and fungal metabolites that naturally contaminate locally processed rice from five agro-ecological zones of Nigeria. The fungal species were isolated by the dilution plate technique and identified by appropriate diagnostics, while metabolites were determined by a liquid chromatographic tandem mass spectrometric method. Aspergillus and Penicillium species were the predominant isolates found in the rice samples while Fusarium spp. were not isolated. The mean fungal count differed significantly (p < 0.05) across the zones and ranged from 9.98 × 10² cfu g^?1 in the Southern Guinea Savannah to 96.97 × 10² cfu g^?1 in the Derived Savannah. For 16 fungal metabolites, selected from 63 positively identified fungal metabolites based on their concentration and spread across the zones, an occurrence map was constructed. The Northern Guinea Savannah recorded the highest contamination of fungal metabolites while the Sudan Savannah zone recorded the least.     

Fumonisin detection and analysis of potential fumonisin‐producing Fusarium spp. in asparagus (Asparagus officinalis L.) in Zhejiang Province of China

BACKGROUND: Fumonisins are mycotoxins produced by a number of Fusarium species, including several pathogens of asparagus plants. China is one of the largest asparagus producers in the world. In this study, we analysed the contamination of fumonisins and fumonisin‐producing fungi in asparagus spear samples from Zhejiang Province, the major asparagus production province in China. RESULTS: The asparagus did not contain a detectable level of fumonisins. However, the recovery of Fusarium in asparagus was 72.7%, including F. proliferatum (40.9%), F. oxysporum (22.7%), F. acuminatum (4.55%) and F. equesti (4.55%). A multiplex PCR targeting the internal transcribed spacer sequence (ITS), translation elongation factor 1‐α (TEF), and key biosynthetic genes FUM1 and FUM8, was used to simultaneously determine the identity and the biosynthetic ability of the fungal isolates. Fungal isolates containing the FUM genes also produced fumonisins in cultures, ranging from 28 to 4204 µg g^?1. F. proliferatum was the only fumonisin‐producing Fusarium species in asparagus. CONCLUSION: Although no fumonisin contamination was detected in asparagus in the current survey, we found that the majority of samples contained Fusarium spp. Because F. proliferatum is a high fumonisin‐producing species, potential health risks for human consumption of asparagus exist, if the appropriate environmental conditions are present for this fungus. Copyright © 2010 Society of Chemical Industry     

Transformation ability of fungi isolated from cork and grape to produce 2,4,6-trichloroanisole from 2,4,6-trichlorophenol

The ability of eight fungal strains to transform 2,4,6-trichlorophenol (TCP) to 2,4,6-trichloroanisole (TCA) was studied. These fungi were isolated from cork, belonging to the genera Penicillium, Aspergillus, Trichoderma and Chrysonilia, and from grapes Botrytis cinerea. All, except Chrysonilia, produced TCA when grown directly on cork in the presence of TCP, Aspergillus and Botrytis cinerea being the ones with the highest level of production. It is the first time that Botrytis cinerea, a microorganism often present on grapes and in winery environments, has been shown to transform TCP into TCA. This result can partially explain the wine cork taint before being bottled.     

In vitro experimental environments lacking or containing soil disparately affect competition experiments of Aspergillus flavus and co-occurring fungi in maize grains

In vitro experimental environments are used to study interactions between microorganisms, and to predict dynamics in natural ecosystems. This study highlights that experimental in vitro environments should be selected to match closely the natural environment of interest during in vitro studies to strengthen extrapolations about aflatoxin production by Aspergillus and competing organisms. Fungal competition and aflatoxin accumulation were studied in soil, cotton wool or tube (water-only) environments, for Aspergillus flavus competition with Penicillium purpurogenum, Fusarium oxysporum or Sarocladium zeae within maize grains. Inoculated grains were incubated in each environment at two temperature regimes (25 and 30°C). Competition experiments showed interaction between the main effects of aflatoxin accumulation and the environment at 25°C, but not so at 30°C. However, competition experiments showed fungal populations were always interacting with their environments. Fungal survival differed after the 72-h incubation in different experimental environments. Whereas all fungi incubated within the soil environment survived, in the cotton wool environment none of the competitors of A. flavus survived at 30°C. With aflatoxin accumulation, F. oxysporum was the only fungus able to interdict aflatoxin production at both temperatures. This occurred only in the soil environment and fumonisins accumulated instead. Smallholder farmers in developing countries face serious mycotoxin contamination of their grains, and soil is a natural reservoir for the associated fungal propagules, and a drying and storage surface for grains on these farms. Studying fungal dynamics in the soil environment and other environments in vitro can provide insights into aflatoxin accumulation post-harvest.     

Microbiological Quality of Raw Dried Pasta from the German Market,with Special Emphasis on Cronobacter Species

The microbiological quality of 132 dried pasta products available on the German market, originating from 11 different countries, was studied. Sample materials included soft or durum wheat products, some of which produced with other ingredients such as eggs, spices, or vegetables. Parameters included hygiene indicators (aerobic plate count, mold count, the presence of Enterobacteriaceae) and pathogenic/toxinogenic bacterial species (Salmonella spp., Staphylococcus aureus, presumptive Bacillus cereus, and Cronobacter spp.). The overall results of hygiene parameters indicated a satisfactory quality. Salmonella was not found in any sample. Three samples were positive for S. aureus (10² to 10⁴ colony forming unit (CFU)/g). Presumptive B. cereus at levels of 10³ to 10⁴ CFU/g were detected in 3 samples. Cronobacter spp. were isolated from 14 (10.6%) products. Of these, 9 isolates were identified as C. sakazakii, 2 each as C. turicensis and C. malonaticus, and 1 as C. muytjensii. The isolates were assigned to 9 multilocus sequence typing (MLST) sequence types and to 14 different PFGE profiles. Although pasta products are typically cooked before consumption, some consumers, and children in particular, may also eat raw pasta as nibbles. Raw pasta seems to be a relevant source of exposure to dietary Cronobacter spp., although health risks are probably restricted to vulnerable consumers. High numbers of presumptive B. cereus as found in some samples may be a risk after improper storage of cooked pasta products because toxinogenic strains are frequently found within this species.     

Internal transcribed spacer (ITS) sequence-based characterization of fungal isolates from multiple yogurt facilities—A case study

Fungal spoilage remains a significant issue in dairy product quality, especially for cultured dairy products such as yogurt formulated without preservatives such as potassium sorbate. Fungal contamination can occur throughout the processing continuum, from the dairy farm environment to the finished product processing environment. As molecular characterization of fungal isolates is used more frequently, we obtained fungal isolates obtained in 2 yogurt processing facilities as part of routine fungal testing of raw materials (e.g., fruit preparations, added ingredients), in-process product samples, environmental samples (e.g., air plates, equipment surfaces such as valves, face plates, air nozzles), and finished product samples, to determine whether internal transcribed spacer (ITS) barcoding data would be helpful to support source tracking of fungal contamination issues. Internal transcribed spacer PCR amplification and sequencing allowed us to classify the 852 isolates from these 2 facilities into 200 unique ITS allelic types (AT), representing the phyla Ascomycota (743 isolates), Basidiomycota (97 isolates), and Mucoromycota (12 isolates). Thirty ITS AT were isolated from both facilities; 62 and 108 ITS AT were isolated from only facility A or only facility B, respectively. Nine ITS AT were each represented by more than 20 isolates; these AT comprised 53% of the 852 isolates. The considerable diversity of fungal isolates even within a single facility illustrates the challenge associated with controlling fungal contamination of dairy products. The ITS barcoding technique, however, did show promise for facilitating the source tracking of fungal contamination, particularly for ITS AT over-represented in a given facility. For example, we found evidence for equipment-specific reservoirs for 2 AT (14 and 219) in facility B. Our data suggest that despite its limited discriminatory power, ITS sequencing can provide initial information that can help trace fungal contamination along the processing continuum. However, development and implementation of discriminatory subtyping methods will be needed to further improve the ability to identify sources of fungal contamination in dairy facilities. Developing and implementing sampling plans that comprehensively capture yeast and mold diversity in a given processing facility remain a considerable challenge.     

A field survey of pistachio (Pistacia vera) nut production and storage in Turkey with particular reference to aflatoxin contamination.

A field study of soils and pistachio nuts in three major pistachio-growing areas of Turkey has been undertaken. The predominant soil fungus isolated from all three areas was Aspergillus niger; isolates of Fusarium, Trichoderma, Mucor and Rhizopus species were also common. A flavus and A. ochraceus and related organisms were isolated only from the plateau of Gaziantep. The dominant external mycoflora of the immature nuts from all sites consisted of A. niger, A. flavus and Penicillium spp. There was no evidence for fungal contamination of immature endosperms nor of freshly dehulled nuts, but stored dehulled nuts and dust samples taken from ware-houses were extensively contaminated with A. flavus, A. niger and A. ochraceus. Of 43 A. flavus isolates tested, 20 produced aflatoxins on laboratory media or on rice; all 11 isolates of A. ochraceus tested produced ochratoxin A on laboratory media. The results obtained are discussed in relation to agricultural and marketing practices for Turkish pistachio nuts.     

A further study of the use of date extract "dibis" for mycological fat production in still and shaken cultures

Fungal growth and fat formation on date extract “dibis” medium were poor and slow. Addition of corn-steep liquor (c.s.l.) as a source of nitrogen and other nutritive substances led to a remarkable increase in growth and in fat yield. The fat content in fungal mycelium increased about 4 times. Shaken cultures were superior to still cultures with regard to growth and fat yield only when a high concentration of c.s.l. was added to dibis medium. Under other experimental conditions, still cultures led to heavier mycelium and higher fat contents than shaken cultures.     

Some Pectinolytic and Cellulolytic Enzyme Activities of Fungi Causing Rots of Cocoyams

Representative isolates of Aspergillus niger, Botryodiplodia theobromae, Corticium rolfsii, Geotrichum candidum, Fusarium oxysporum and F solani recovered from rotten cocoyams were studied for the production of pectinolytic and cellulolytic enzymes. All the isolates elaborated high levels of hydrolase, lyase and pectinesterase in cocoyam tissue medium and lyase and pectinesterase in pectin medium. There were no significant differences in the overall levels of lyase and pectinesterase activities produced by all the isolates in both media. The level of hydrolase, lyase and pectinesterase activities individually produced by A niger, B theobromae and C rolfsii in both media was significantly higher than that of any other isolate. The highest hydrolase activity was produced by C rolfsii in cocoyam medium while A niger produced the highest lyase activity in pectin medium. Maximum pectinesterase activity was obtained from B theobromae and C rolfsii in pectin medium. All the test isolates produced cellulase in a medium containing carboxymethyl cellulose with C rolfsii showing significantly high activity followed by F oxysporum and A niger. © 1997 SCI.     

Occurrence of ochratoxin A-producing fungi in grapes grown in Italy

A study was carried out to investigate fungi present on grapes grown in Italy. Aspergillus and Penicillium spp. isolates were identified and studied in vitro, and their ability to produce ochratoxin A (OA) was investigated. The survey involved nine vineyards, three located in northern Italy and six located in southern Italy. In 1999 and 2000, bunches of grapes at different growth stages were collected from all nine vineyards, and berry samples were placed in moist chambers and incubated. The resultant fungal colonies were then transferred to petri dishes containing Czapek yeast agar and incubated at 25 degrees C for 7 days; the fungal isolates were identified and then cultivated in liquid Czapek yeast medium and evaluated for their ability to produce OA. During the survey, 508 isolates were collected, with 477 belonging to Aspergillus spp. and 31 belonging to Penicillium spp. Among the aspergilli, species of the Fumigati, Circumdati, and Nigri sections were identified, with species of the Nigri section (464 isolates) largely predominating; for species of the Nigri section, 108 isolates were uniseriate, 270 were biseriate, and 86 were identified as Aspergillus carbonarius. Black aspergilli isolated over the 2 years of the study showed a very similar pattern. On average, the biseriates represented about 60% of the isolates collected in both years and were followed by uniseriates (21%) and A. carbonarius (19%). The most toxigenic strains proved to be those of A. carbonarius; about 60% of these isolates were OA producers and produced the highest levels of OA. A. carbonarius was more frequent in the south, but in both areas the percentages of OA-producing isolates remained the same.     

Characterization of molds isolated from smoked paprika by PCR-RFLP and micellar electrokinetic capillary electrophoresis

Molds are common contaminants of paprika meat products. The drying and storage stages of paprika processing are critical because they can provide molds with the conditions particularly appropriate for their growth and proliferation. Thus, an efficient and accurate characterization of the toxigenic molds of paprika is necessary. An RFLP analysis of the rRNA genes was performed by using a TaqI restriction enzyme. In addition, a micellar electrokinetic capillary chromatography (MECC) method was tested to analyze secondary metabolites produced by mold strains commonly found in paprika. This study was confirmed with a 5.8S-ITS region sequence analysis. A total of 31 isolates were identified by RFLP and MECC analysis. These showed stable RFLP profiles that were clearly different for the different genera and species, and were grouped into clusters together with the profiles of the 16 reference strains. MECC analysis provided additional characteristic peak patterns for the characterization of the mold species present. The characterized isolates were species of the genera Fusarium spp., Aspergillus spp., Penicillium spp., Cladosporium spp., Mucor spp. and Phlebia spp. The identifications were confirmed by the 5.8S-ITS region sequence analysis and by a BLAST search of the GenBank database. RFLP patterns with TaqI restriction enzyme and MECC profiles, either singly or combined, could be of great interest to distinguish molds in paprika.     

Growth and mycotoxin production by fungi in atmospheres containing 80% carbon dioxide and 20% oxygen

The effect of atmosphere containing 80% CO₂ and 20% O₂ on growth of Mucor plumbeus, Fusarium oxysporum, Byssochlamys fulva, Byssochlamys nivea, Penicillium commune, Penicillium roqueforti, Aspergillus flavus, Eurotium chevalieri and Xeromyces bisporus was investigated. Production of aflatoxin by A. flavus, patulin by B. nivea, roquefortine C by P. roqueforti, and cyclopiazonic acid by P. commune was also studied. Fungal growth was evaluated by three methods: colony diameter, hyphal length or mycelium dry weight and ergosterol content. Among the nine fungal species examined, two E. chevalieri and X. bisporus, did not grow under these conditions. In this study, fungi differed in their response to modified atmospheres in biomass, ergosterol content, mycotoxin production and morphology. Reductions of 57.8-96.9%, 73.7-99.6% and 91.5-99.9% were obtained in colony diameter, hyphal length and ergosterol content, respectively, under this atmosphere compared to air. Ergosterol content was more affected in most species than other measurements. Patulin, cyclopiazonic acid and roquefortine C were produced in this atmosphere, although levels were very low and aflatoxin was not produced at all. Growth was quite extensive as measured by colony diameters, but hyphal lengths were low and ergosterol production was also affected in all species of this study.     

Pulsed light for the inactivation of fungal biofilms of clinically important pathogenic Candida species

Microorganisms are naturally found as biofilm communities more than planktonic free‐floating cells; however, planktonic culture remains the current model for microbiological studies, such as disinfection techniques. The presence of fungal biofilms in the clinical setting has a negative impact on patient mortality, as Candida biofilms have proved to be resistant to biocides in numerous in vitro studies; however, there is limited information on the effect of pulsed light on sessile communities. Here we report on the use of pulsed UV light for the effective inactivation of clinically relevant Candida species. Fungal biofilms were grown by use of a CDC reactor on clinically relevant surfaces. Following a maximal 72 h formation period, the densely populated biofilms were exposed to pulsed light at varying fluences to determine biofilm sensitivity to pulsed‐light inactivation. The results were then compared to planktonic cell inactivation. High levels of inactivation of C. albicans and C. parapsilosis biofilms were achieved with pulsed light for both 48 and 72 h biofilm structures. The findings suggest that pulsed light has the potential to provide a means of surface decontamination, subsequently reducing the risk of infection to patients. The research described herein deals with an important aspect of disease prevention and public health.     

A study on Iraqi date extract: "Dibis" as substrate for mycological fat production

A date extract known locally in Iraq as “dibis” was used as substrate for mycological fat formation by Penicillium lilacinum, P. soppii and Aspergillus nidulans. The three fungi were grown on dibis medium at three concentrations. Carbohydrates readily disappeared but this was accompanied by poor fungal growth and low yields of fat. Fungal growth on dibis media enriched with asparagine led to heavy mycelial yields together with higher fat contents. Both economic and fat coefficients rose considerably. It is concluded that nitrogen deficiency in dibis media is at least one of the major factors against good fungal growth and high fat formation.     

Development of Molecular Typing Methods for Bacillus spp. and Paenibacillus spp. Isolated from Fluid Milk Products

Bacillus spp. and related sporeformers are important food spoilage organisms. While use of molecular subtyping methods has provided important information on the ecology and transmission of foodborne pathogens, the lack of rapid, reliable, and affordable subtyping methods for Bacillus spp. has limited our ability to understand and control their transmission throughout the food chain. We used a previously described collection of Bacillus spp. and Paenibacillus spp. isolated from dairy products to develop a DNA sequencing‐based subtyping approach for these spoilage microorganisms. After optimization of polymerase chain reaction (PCR) parameters, primers targeting the rpoB housekeeping gene allowed for successful amplification in all isolates. rpoB sequencing allowed differentiation of 29 subtypes (that is, sequence types) among the 57 isolates characterized. Phylogenetic analyses of rpoB sequences revealed distinct monophyletic lineages that correlated with bacterial genera (Bacillus and Paenibacillus) as well as with species or species‐like assemblages within each genus. rpoB sequencing provided improved subtype discrimination over 16S rDNA sequencing; therefore, rpoB sequencing allows for both sensitive subtype discrimination as well as for species and genus identification. Analysis of subtypes isolated over time in dairy products revealed the presence of both persistent and transient bacterial subtypes, indicating that application of these methods can improve our understanding of the ecology of these spoilage organisms and can help in identification of bacterial niches that may contribute to the persistence of these spoilage organisms in food systems.     

Bioproduction of mushroom mycelium of Agaricus bisporus by commercial submerged fermentation for the production of meat analogue

BACKGROUND As worldwide interest in healthy and delicious meat analogues increases, the texture of these products has become an important indicator of quality. Mycoprotein as fungal mycelium could provide a distinctive chewing sensation; however, the unfavorable consumer perception of fungal mycelium demands the production of meat analogues with true mushroom mycelium. RESULTS: The industrial and economical bioprocess was developed using an inexpensive medium (30 g L^?1 sugar cane extract (SCE), 10 g L^?1 NaNO₃ and 5 g L^?1 yeast extract) and A. bisporus Suksung. The SCE was maintained at around 10 g L^?1 to minimize osmotic shock. The maximum mycelium production of 15.0 g L^?1 (dry weight) was reached within 4 days. Scanning electron microscopic analysis showed fibrous and directional structure rather than a more typical pellet structure. Meat analogues with mushroom mycelium had better textural properties, being higher in hardness, springiness, and chewiness and with preferable umami characteristics compared to meat analogues utilizing soy protein. The overall acceptance of meat analogues prepared with mycelium and soy protein, and a ground beef patty, were 5, 2 and 10, respectively. CONCLUSION: The development of an industrial bioprocess for A. bisporus mycelium allowed the production of a highly acceptable meat analogue having not only superior textural properties but also umami characteristics when compared to that of soy protein. Copyright © 2011 Society of Chemical Industry