Hyphopichia Burtonii Research Articles

Autochthonous yeasts as potential biocontrol agents in dry-cured meat products

This work aimed to evaluate the ability of selected yeasts, previously isolated from dry-cured hams, to compete with Penicillium nordicum and to inhibit ochratoxin A (OTA) accumulation in a dry-cured pork meat model system in the perspective of their use as surface starter cultures. Two strains of Debaryomyces hansenii and one strain of Hyphopichia burtonii were used. A dry-cured pork meat model system was prepared using meat portions at 0.88 and 0.92 aw; meat surface disinfection (disinfected or non disinfected) was considered to take into account the possible role of naturally occurring microbial population. P. nordicum (105 spores/ml) was co-inoculated with each yeast strain (around 106 cells/cm2 on meat surface) one at a time. Meat portions were incubated in the dark at 18 °C for 30 days. The co-inoculation of P. nordicum and yeasts allowed a decrease in penicillia counts between 1 and 3 Log compared to the control, irrespective of the investigated conditions. Ochratoxin A content in meat portions was significantly reduced when yeasts were co-inoculated with P. nordicum. D. hansenii 147 showed the greatest antagonistic activity and proved to be effective in the investigated conditions. This strain is supposed to be appropriate for typical flavour development, being native to the production plants. Therefore, it is eligible to be tested as biocontrol agent in the dry-cured meat production chain.

Effect of ethanol on growth of Chrysonilia sitophila (‘the red bread mould’) and Hyphopichia burtonii (‘the chalky mould’) in sliced bread

Contamination of food industrial environments and recontamination of finished products by Chrysonilia sitophila and Hyphopichia burtonii have long represented serious problems for the bakery industries. As one of the most common ways to slow down or avoid fungal spoilage on bakery products is the use of ethanol, in the present work the effect of this substance has been assessed on growth of two of the most frequently occurring associated moulds, C.sitophila and H.burtonii, by means of tests on both synthetic media and sliced bread. Test on synthetic media: H.burtonii was less markedly affected in lag-phase duration and radial growth rates by the addition of ethanol to DG18 and the reduction in incubation temperature than C.sitophila that failed to grow at the highest concentrations of ethanol tested (2·0 and 4·0% at 15°C; 4·0% at 25°C). Test on sliced bread: ethanol proved to be effective to prevent spoilage by C.sitophila even at the lowest concentration tested (0·8%, w/w), while higher concentrations (2·0%, w/w) were needed to prevent spoilage by H.burtonii. This study shows that ethanol could represent an effective barrier to prevent spoilage of bakery products by associated moulds such as Chrysonilia sitophila and Hyphopichia burtonii, whose growth on packed and sliced bread was inhibited at very low (0·8%) or medium (2·0%) ethanol concentrations, respectively. The results obtained represent a fundamental point of reference for the bakery industries, as they can apply them in the productive practice to avoid spoilage by C.sitophila and H.burtonii on their products.

Cutaneous mycosis in a Barbastelle bat (Barbastella barbastellus) caused by Hyphopichia burtonii

A rare barbastelle bat (Barbastella barbastellus) died shortly after being found in emaciated condition in Devon, England. The skin over the muzzle and face was grossly thickened, crusty, and in places was sloughing and ulcerated. There were numerous nodules up to 3 mm in diameter on both wings and ear pinnae. Histologically, multiple foci of epidermal hyperplasia, hyperkeratosis, and crateriform erosions containing masses of fungal spores and septate hyphae were found in the wing. Epidermal hyperplasia and follicular hyperkeratosis, with fungal masses within keratinized follicles and also in fissured stratum corneum, were found in the pinna. Hyphae did not invade the dermis, and there was no inflammation, but there was multifocal serous exudation and crusting. No parasites or other significant organisms were identified. Microscopic and multiple cultural analyses of face and wing lesions demonstrated (10/10) a fine, septate fungus bearing laterally oval to clavate conidia; morphologically and culturally this was entirely consistent with Hyphopichia burtonii, and polymerase chain reaction analysis and sequencing gave 100% identity with the type strain. The organism isolated was morphologically consistent with that repeatedly seen in histology sections and demonstrates that although H. burtonii has not previously been recognized as a dermatophyte, it clearly has the ability to invade the skin of live bats. Although not identical, the lesions in this case show similarity with those of white nose syndrome and therefore H. burtonii should be considered as a potential pathogen of bats.

Assessment of the yeast species composition of cocoa bean fermentations in different cocoa-producing regions using denaturing gradient gel electrophoresis

The yeast species composition of 12 cocoa bean fermentations carried out in Brazil, Ecuador, Ivory Coast and Malaysia was investigated culture-independently. Denaturing gradient gel electrophoresis of 26S rRNA gene fragments, obtained through polymerase chain reaction with universal eukaryotic primers, was carried out with two different commercial apparatus (the DCode and CBS systems). In general, this molecular method allowed a rapid monitoring of the yeast species prevailing during fermentation. Under similar and optimal denaturing gradient gel electrophoresis conditions, the CBS system allowed a better separated band pattern than the DCode system and an unambiguous detection of the prevailing species present in the fermentation samples. The most frequent yeast species were Hanseniaspora sp., followed by Pichia kudriavzevii and Saccharomyces cerevisiae, independent of the origin of the cocoa. This indicates a restricted yeast species composition of the cocoa bean fermentation process. Exceptionally, the Ivorian cocoa bean box fermentation samples showed a wider yeast species composition, with Hyphopichia burtonii and Meyerozyma caribbica among the main representatives. Yeasts were not detected in the samples when the temperature inside the fermenting cocoa pulp-bean mass reached values higher than 45 °C or under early acetic acid production conditions.

Identification and characterization of yeasts causing chalk mould defects on par-baked bread

Pichia anomala, Hyphopichia burtonii and Saccharomycopsis fibuligera are spoilage yeasts causing chalk mould defects on par-baked breads packaged under modified atmosphere. The first objective of this study was to identify yeasts isolated from spoiled par-baked breads by means of a RAPD protocol and to determine the dominant spoilers amongst identified strains. The second objective was to determine the effects of water activity ( a w) and pH value on the growth rates and lag phase durations of P. anomala, H. burtonii and S. fibuligera. 95% of the yeasts tested were identified as P. anomala and 5% as S. fibuligera, H. burtonii was not detected. In order to investigate the effect of a w and pH the growth of the three yeasts was tested within an a w range of 0.88–0.98 and a pH range of 2.8–8.0. P. anomala was able to grow from pH 2.8 to 8 without a clear optimum. S. fibuligera and H. burtonii showed a pH optimum for growth of 5. The optimum water activity for growth was different for the three strains and varied between 0.96 and 0.98. These growth data were further used to develop secondary models that describe the relationship between a w and the radial or colony growth rate (g, mm/d) or the lag phase duration ( λ, d). The identification of the spoilage organisms and a good understanding of the effects of a w and pH on the growth behavior is essential for future development of adequate conservation strategies against chalk mould defects.

Re-examination of strains formerly assigned to Hyphopichia burtonii, the phylogeny of the genus Hyphopichia, and the description of Hyphopichia pseudoburtonii sp. nov.

On the basis of the nucleotide divergence in the internal transcribed spacer (ITS) domain of the rRNA gene and the D1/D2 domain of the 26S rRNA gene, species of the genus Hyphopichia can be divided into different phylogenetic groups. In this study, three distinct phylogenetic clusters as well as a novel species, Hyphopichia pseudoburtonii sp. nov. with type strain CBS 2455(T) (=JCM 16346(T)), are introduced. Mating studies indicate that strains belonging to Hyphopichia pseudoburtonii are heterothallic.

Dynamics and characterization of yeasts during ripening of typical Italian dry-cured ham

The evolution of the yeast population during manufacturing and ripening of dry-cured Parma ham was investigated. Contamination levels ranged from 10 5 to 10 7 cfu/g on muscle surface, 10 4 to 10 6 cfu/g on covering fat and exceeded 10 7 cfu/g on spreadable fat mince (“sugna”). Two hundred and sixty one yeast isolates underwent identification test, showing that the predominant species of yeast population during the whole maturing process were Debaryomyces hansenii, Candida zeylanoides, Debaryomyces maramus, and to a lesser extent, Candida famata and Hyphopichia burtonii. The species Candida catenulata, Candida guilliermondii, Candida edax and other genera like Cryptococcus and Wingea were occasionally found. The yeast counts and species distribution changed according to the stage of processing and to the ham sampling location. At the end of the cold phase, the washing procedure was effective in lowering the yeast count in muscle and fat surface layers, but during the next ageing stages, yeast colonization of unskinned ham muscle increased again, though species distribution changed if compared to previous manufacturing phases. The ripening steps taken into account from the end of the cold phase to the final outcome, were always characterized by more than one yeast species, suggesting that yeasts other than Debaryomyces spp. could play a remarkable role on the sensory and safety properties of typical Italian dry-cured ham.

Microflora of banh men, a fermentation starter from Vietnam

In this study Banh mensamples obtained from Vietnam were analysed in terms of pH, moisture content and fungal composition. The banh men pH proved to be acidic with a mean pH of 5.76. Moisture content was 13.6%. Total mould and yeast counts yielded 1.3 × 106 and 4.3 × 106 c.f.u./g-fresh sample, respectively. A total of 53 fungal isolates were obtained from 20 moulds and 33 yeasts. The mould isolates were identified as Rhizopus oryzae, Mucor indicus, Mucor circinilloides, and Amylomyces rouxii. The yeast isolates were identified as Saccharomyces cerevisiae, Hyphopichia burtonii, Saccharomycopsis fibuligera, Pichia anomala, and Candida sp. Based on the parameters used in this study, it can be deduced that banh men is similar to ragi and other Asian fermentation starters.

Colonization of Barley Grain by Penicillium verrucosum and Ochratoxin A Formation in the Presence of Competing Fungi

Colonization of barley grain by Penicillium verrucosum and the formation of ochratoxin A were studied, both in pure culture and when paired with Aspergillus flavus, Fusarium sporotrichioides, and Hyphopichia burtonii, at 20° and 30°C and at 0.97, 0.95 and 0.90 aw over a 3-week period. Grain colonization was assessed on the basis of visible molding, seed infection, and numbers of CFU and by observing hyphal extension on the grain surface by scanning electron microscopy. Ochratoxin A concentrations were assayed by enzyme-linked immunosorbent assay using a monoclonal antibody. Germination of P. verrucosum spores was unaffected by the presence of other species. However, seed infection under most conditions was markedly decreased, relative to pure culture, by the presence of A. flavus and H. burtonii, but only slightly by F. sporotrichioides. The number of CFU of P. verrucosum was only slightly decreased in the presence of other species under most conditions. Generally, production of ochratoxin A by P. verrucosum was inhibited, sometimes significantly, in the presence of A. flavus and H. burtonii, but was changed only slightly by the presence of F. sporotrichioides. There was occasionally temporary enhancement in ochratoxin A production with all species during the 3-week incubation period.

Aspergillus flavus colonization and aflatoxin B1 formation in barley grain during interaction with other fungi

Colonization of barley grain by Aspergillus flavus and formation of aflatoxin B(1) in the presence of Penicillium verrucosum, Fusarium sporotrichioides, and Hyphopichia burtonii were studied over a three-week period in all combinations of 20 or 30 °C and 0.97, 0.95 or 0.90 a(w). Grain colonization was assessed initially by observing hyphal extension on the grain surface, using scanning electron microscopy, and then from the proportion of seeds infected and numbers of colony forming units (cfu) formed. Aflatoxin b(1) concentrations were determined by enzyme linked immunosorbent assay using a monoclonal antibody. These studies showed that interaction between A. flavus and other fungi in paired culture had different effects on both colonization and aflatoxin formation depending on the species involved and environmental conditions. Germination of A. flavus spores was unaffected by the presence of other species on the grain surface. Subsequently, three principal patterns of A. flavus colonization of barley grain were observed through the incubation period in the presence of other fungal species: (a) colonization unaffected by the presence of other species; (b) colonization initially slower in the presence of other species but later differing little from pure cultures; and (c) colonization adversely affected by the presence of other species. Five main patterns of aflatoxin B(1) production were observed relative to pure culture but with no consistent relationship with species, a(w), temperature or incubation period; (a) little changed; (b) increased slowly; (c) decreased; (d) enhanced; and (e, f) increased initially but later decreased to (e) the same level as in pure culture or (f) to less than in pure culture. Generally, production of aflatoxin B(1) by A. flavus was less than in pure culture but sometimes was changed only slightly by the presence of P. verrucosum, F. sporotrichioides or H. burtonii or was temporarily enhanced.

Killer activity of yeasts isolated from the water environment.

The killer activity of 46 strains belonging to 12 yeast and yeast-like species isolated from water or sediment samples was studied. Only two strains of the genus Cryptococcus did not show killer activity. Killer activity of yeast-like species Aureobasidium pullulans, Hyphopichia burtonii and Geotrichum candidum, and yeast species Candida krusei and Candida lambica was low. Sporobolomyces salmonicolor, Cryptococcus laurentii and Cryptococcus albidus had better activity against basidiomycetous than ascomycetous species. Hansenula anomala strains showed good activity against Geotrichum candidum strains, Cryptococcus albidus, and Sporobolomyces salmonicolor. Rhodotorula species showed activity against the majority of both ascomycetous and basidiomycetous species.

Effects of water activity and temperature on the growth of fungi interacting on barley grain

Germination, growth and interactions of Aspergillus flavus, Penicillium verrucosum, Fusarium poae and Hyphopichia burtonii, either alone or in the presence of another species, were observed at water activities of 0·97, 0·95 or 0·90 aw and at 20 or 30 °C, both on the surface of individual barley grains by scanning electron microscopy and on thin layers of grain with a stereomicroscope. By both methods, each species exhibited characteristic linear growth rates which changed with aw and temperature. Spore germination was unaffected by the presence of another species, but distinct interaction patterns between competing species on the grain surface were identified, which were determined by rate of hyphal extension and branching, namely (a) faster growth of one species causing progressive inhibition of the slower-growing species; (b) faster growth initially of one species which is then inhibited by the slower-growing species; (c) one species grew faster than the other but with no adverse effects; (d) one species grew faster than the other initially, but growth rates of both declined later during interaction; (e) both species grew at similar rates initially but growth rate of one declined during competition; and (f) both species grew at similar rates initially but later reduced the growth of each other. On thin layers of barley grain, competing species generally showed mutual inhibition on contact, forming discrete colonies whose size was determined by the relative radial growth rates of the two species and positions of inocula. However, at 30°, A. flavus overgrew colonies of all other species.

Stimulation by Hyphopichia burtonii and Bacillus amyloliquefaciens of aflatoxin production by Aspergillus flavus in irradiated maize and rice grains.

Aspergillus flavus was grown on maize and rice extract agars and on irradiated viable cracked maize and rice grains, either in pure culture or in dual culture with wild strains of either Hyphopichia burtonii or Bacillus amyloliquefaciens. Aflatoxin production by A. flavus and its growth and interactions with the other microorganisms were studied at three water activities (aw) (0.98, 0.95, and 0.90) and two temperatures (25 and 16 degrees C). Both H. burtonii and B. amyloliquefaciens markedly stimulated growth and aflatoxin production by A. flavus on cracked maize, especially at 25 degrees C and 0.95 and 0.98 aw. No aflatoxin was detected in pure cultures of A. flavus on cracked rice after 12 days of incubation at 25 degrees C, but some was produced by mixed cultures at 16 degrees C and 0.98 aw. The morphological interactions among A. flavus, H. burtonii, and B. amyloliquefaciens were also examined on maize and rice extract agars under similar controlled conditions.

The biochemistry and immunology of yeasts that produce true mycelia: Ambrosiozyma, Hormoascus, and Hyphopichia.

Ambrosiozyma, yeasts that produce true mycelia with transverse septa that communicate by a dolipore type septum, are compared with Hyphopichia and Hormoascus. This study includes intracellular oxidases, nitrite and nitrate reductases, guanine plus cytosine content in DNA (GC-content), cytochrome spectra (a+a3, b, c), coenzyme Q, and serological comparison by immunodiffusion and immunoelectrophoresis, with A. monospora and Hormoascus platypodis as references. The results support the division of the genus Ambrosiozyma into 2 groups based upon GC content, vitamin requirements, and serological homologies. Affinities between Hormoascus platypodis and A. philentoma (vitamin requirements, intracellular nitrite and nitrate reductases, β-galactosidase, serological homology, cytochrome spectra, and GC content) leads to the placing of Hormoascus platypodis in synonymy with Ambrosiozyma philentoma. Ambrosiozyma ambrosiae is compared with the other Ambrosiozyma and with the non-fermentative Pichia, and Hansenula silvicola. These comparisons show that A. ambrosiae has more affinity with the other Ambrosiozyma than with Pichia or Hansenula. Hyphopichia burtonii differs from the other species in regard to vitamin requirements, coenzyme Q8, α2 band of cytochrome c and serological homologies.

Water relations and metabolism of propionate in two yeasts from hay

Many yeasts and bacteria were isolated from moist hays (>30% water content) treated with up to 3% propionic acid‐based preservatives. Predominant yeasts were Candida guilliermondii var. guilliermondii and Hyphopichia burtonii. Growth of both species was decreased more than 50% in liquid medium containing 54 mmol/l ammonium propionate but some still occurred in 135 mmol/l propionate. Both metabolized between 80 and 85% of 27 mmol/l ammonium propionate in 1% malt broth within four weeks at 25°C. Growth on solid malt extract agar containing ammonium propionate was decreased by decreasing the water availability (water activity, aw) in the medium. Growth rates were slightly greater when glycerol rather than NaCl was used to alter aw in the range 0.995 to 0.93. At both 0.995 and 0.95 aw optimum growth was at pH 6. The significance of these findings with regard to the preservation of moist hay is discussed.

Classification of ascoideaceous yeasts based on the electrophoretic comparison of enzymes and coenzymes Q systems.

Sixty one strains of ascoideaceous yeasts belonging to twenty species of nine genera were examined from a taxonomical viewpoint based on a study of the electrophoretic comparison of the enzymes and the coenzymes Q (Co-Q) systems. The fifty strains, excluding those yeasts in which the detected number of enzymes was 3 or less, were divided into 4 clusters and 2 subclusters by numerical analysis of the relative electrophoretic mobilities (Rm) of 8 enzymes. On the other hand, the Co-Q systems of the ascoideaceous yeasts were Q-7, Q-8, or Q-9. These Co-Q systems and the septal ultrastructures coincided with each other. It is possible to divide these yeasts into four groups on the basis of similarity value according to the Rm of 8 enzymes: Saccharomycopsis lipolytica, other Saccharomycopsis spp. and related genera, Hyphopichia burtonii and/or Stephanoascus ciferrii, and Ambrosiozyma and Hormoascus. Saccharomycopsis lipolytica should be excluded from the genus Saccharomycopsis and is assigned to the genus Yarrowia. Ambrosiozyma and Hormoascus are considered to be cogeneric. Our data revealed close taxonomic relationship in the numerical classification based on the Rm of enzymes, Co-Q systems, and the septal ultrastructure.

Survival of micro-organisms in deep-frozen barley and figfeed

Deep freezing at −23°C was not found to have any effect on colony counts of field and storage fungi and bacteria in barley under several combinations of conditions. The factors studied were time in deep-freeze, moisture content, size of sample and thawing rate. Deep-freezing also had no effect on the count of a storage yeast Hyphopichia burtonii in a pig feed.

A note on the yeast flora associated with fermentation of mango

Fifteen morphologically different groups of yeasts consisting of Metschnikowia pulcherrima, Trichosporon cutaneum, Kloeckera apiculata, Torulopsis Candida, Tor. glabrata. Tor. apicola, Candida tropicalis, Cand. krusei, Cand. sorbosa, Cand. diversa, Pichia terricola, Pic. membranaefaciens, Hyphopichia burtonii, Rhodotorula graminis and Aureobasidium pullulans were isolated from fresh, fermenting and fermented juice of two varieties of mango. This is the first report on the study of yeast flora of mango and also on the occurrence of Hyp. burtonii on fruits.