Two new species in the Pichia guilliermondii clade: Pichia caribbica sp. nov., the ascosporic state of Candida fermentati, and Candida carpophila comb. nov

Pichia caribbica sp. nov. (type strain DBVPG 4519, NRRL Y-27274, CBS 9966) is described as the ascosporic state of Candida fermentati, and Candida guilliermondii var. carpophila (type strain DBVPG 7739, NRRL Y-17905, CBS 5256) is elevated to species status as Candida carpophila comb. nov. These new taxa, which are indistinguishable on the basis of conventional taxonomic criteria, differ from one another and from Pichia guilliermondii by low DNA base sequence relatedness, different electrophoretic karyotypes, and nucleotide divergence in domains D1/D2 of 26S rDNA. Pichia caribbica produces one, rarely two, saturn-shaped ascospores in persistent asci. On the basis of molecular criteria, C. carpophila comb. nov., C. fukuyamaensis, and C. xestobii are conspecific, with the name C. carpophila having taxonomic priority.     

Yeast succession in the Amazon fruit Parahancornia amapa as resource partitioning among Drosophila spp.

The succession of yeasts colonizing the fallen ripe amapa fruit, from Parahancornia amapa, was examined. The occupation of the substrate depended on both the competitive interactions of yeast species, such as the production of killer toxins, and the selective dispersion by the drosophilid guild of the amapa fruit. The yeast community associated with this Amazon fruit differed from those isolated from other fruits in the same forest. The physiological profile of these yeasts was mostly restricted to the assimilation of a few simple carbon sources, mainly L-sorbose, D-glycerol, DL-lactate, cellobiose, and salicin. Common fruit-associated yeasts of the genera Kloeckera and Hanseniaspora, Candida guilliermondii, and Candida krusei colonized fruits during the first three days after the fruit fell. These yeasts were dispersed and served as food for the invader Drosophila malerkotliana. The resident flies of the Drosophila willistoni group fed selectively on patches of yeasts colonizing fruits 3 to 10 days after the fruit fell. The killer toxin-producing yeasts Pichia kluyveri var. kluyveri and Candida fructus were probably involved in the exclusion of some species during the intermediate stages of fruit deterioration. An increase in pH, inhibiting toxin activity and the depletion of simple sugars, may have promoted an increase in yeast diversity in the later stages of decomposition. The yeast succession provided a patchy environment for the drosophilids sharing this ephemeral substrate.     

Yeasts in foods and beverages: impact on product quality and safety

The role of yeasts in food and beverage production extends beyond the well-known bread, beer and wine fermentations. Molecular analytical technologies have led to a major revision of yeast taxonomy, and have facilitated the ecological study of yeasts in many other products. The mechanisms by which yeasts grow in these ecosystems and impact on product quality can now be studied at the level of gene expression. Their growth and metabolic activities are moderated by a network of strain and species interactions, including interactions with bacteria and other fungi. Some yeasts have been developed as agents for the biocontrol of food spoilage fungi, and others are being considered as novel probiotic organisms. The association of yeasts with opportunistic infections and other adverse responses in humans raises new issues in the field of food safety.     

The effect of sulphur dioxide and oxygen on the viability and culturability of a strain of Acetobacter pasteurianus and a strain of Brettanomyces bruxellensis isolated from wine

AIMS: The objective of this study was to investigate the effects of free molecular and bound forms of sulphur dioxide and oxygen on the viability and culturability of a selected strain of Acetobacter pasteurianus and a selected strain of Brettanomyces bruxellensis in wine. METHODS AND RESULTS: Acetic acid bacteria and Brettanomyces/Dekkera yeasts associated with wine spoilage were isolated from bottled commercial red wines. One bacterium, A. pasteurianus strain A8, and one yeast, B. bruxellensis strain B3a, were selected for further study. The resistance to sulphur dioxide and the effect of oxygen addition on these two selected strains were determined by using plating and epifluorescence techniques for monitoring cell viability in wine. Acetobacter pasteurianus A8 was more resistant to sulphur dioxide than B. bruxellensis B3a, with the latter being rapidly affected by a short exposure time to free molecular form of sulphur dioxide. As expected, neither of these microbial strains was affected by the bound form of sulphur dioxide. The addition of oxygen negated the difference observed between plate and epifluorescence counts for A. pasteurianus A8 during storage, while it stimulated growth of B. bruxellensis B3a. CONCLUSIONS: Acetobacter pasteurianus A8 can survive under anaerobic conditions in wine in the presence of sulphur dioxide. Brettanomyces bruxellensis B3a is more sensitive to sulphur dioxide than A. pasteurianus A8, but can grow in the presence of oxygen. Care should be taken to exclude oxygen from contact with wine when it is being transferred or moved. SIGNIFICANCE AND IMPACT OF THE STUDY: Wine spoilage can be avoided by preventing growth of undesirable acetic acid bacteria and Brettanomyces/Dekkera yeasts through the effective use of sulphur dioxide and the management of oxygen throughout the winemaking process.     

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, a_w) in the medium. Growth rates were slightly greater when glycerol rather than NaCl was used to alter a_w in the range 0.995 to 0.93. At both 0.995 and 0.95 a_w optimum growth was at pH 6. The significance of these findings with regard to the preservation of moist hay is discussed.     

Symbiotic interactions of culturable microbes with the nickel hyperaccumulator Berkheya coddii and the herbivorous insect Chrysolina clathrata

Chrysolina clathrata is a specialized phytophagous beetle feeding exclusively on the herbaceous nickel hyperaccumulating plant Berkheya coddii. These organisms appear impervious to the toxic levels of nickel in their environment. In the current study we aimed to identify microorganisms that may have symbiotic relationships with these organisms. Culture techniques were used to isolate bacteria and fungi from plants and the faeces of beetles reared under laboratory conditions. The identity of isolates was determined using morphology and molecular techniques. Several genera of filamentous fungi (Alternaria, Aspergillus, Bipolaris, Cladosporium, Epicoccum, Fusarium, and Penicillium), yeasts (Cryptococcus, Meyerozyma, and Rhodotorula), as well as endophytic bacteria (Bacillus and Lysinibacillus) were isolated from the leaves of the plant. We also selectively isolated yeasts (Candida, Cryptococcus, Debaryomyces, Meyerozyma and Wickerhamomyces) from the beetle’s faeces. Subsequently we determined the minimum inhibitory Ni-concentration (MIC) of all isolates. The endophytic bacteria, filamentous fungi and the yeasts Candida intermedia, Cryptococcus flavescens and Meyerozyma guilliermondii, showed notable Ni resistance, while the beetle’s gut seems to select for Ni resistant yeasts.     

Monitoring Seasonal Changes in Winery-Resident Microbiota

During the transformation of grapes to wine, wine fermentations are exposed to a large area of specialized equipment surfaces within wineries, which may serve as important reservoirs for two-way transfer of microbes between fermentations. However, the role of winery environments in shaping the microbiota of wine fermentations and vectoring wine spoilage organisms is poorly understood at the systems level. Microbial communities inhabiting all major equipment and surfaces in a pilot-scale winery were surveyed over the course of a single harvest to track the appearance of equipment microbiota before, during, and after grape harvest. Results demonstrate that under normal cleaning conditions winery surfaces harbor seasonally fluctuating populations of bacteria and fungi. Surface microbial communities were dependent on the production context at each site, shaped by technological practices, processing stage, and season. During harvest, grape- and fermentation-associated organisms populated most winery surfaces, acting as potential reservoirs for microbial transfer between fermentations. These surfaces harbored large populations of Saccharomyces cerevisiae and other yeasts prior to harvest, potentially serving as an important vector of these yeasts in wine fermentations. However, the majority of the surface communities before and after harvest comprised organisms with no known link to wine fermentations and a near-absence of spoilage-related organisms, suggesting that winery surfaces do not overtly vector wine spoilage microbes under normal operating conditions.     

A note on shelf-life extension of British fresh sausage by vacuum packing

Vacuum packing of British fresh sausage in a low oxygen permeability film (Diolon) extended the product shelf-life at 6 degrees C to more than 20 d compared with 9-14 d in conventional packs. After 10 d storage, counts of key spoilage organisms such as yeasts and Brochothrix thermosphacta were generally 2 log cycles lower in vacuum packs. Vacuum-packed sausages also displayed a slower rate of loss of free sulphite. Variations in pack permeability to SO2 were not responsible for this. Losses of free SO2 in stored sausages are largely due to the production of sulphite-binding agents by yeasts. Selective enumeration of these yeasts showed them to be inhibited by conditions of vacuum packing. The extension of shelf-life observed is ascribed to the reduction in growth rate of the spoilage flora in vacuum packs coupled with the consequent maintenance of inhibitory levels of sulphite for a longer period.     

The effect of acetic acid bacteria upon the growth and metabolism of yeasts during the fermentation of grape juice

The influence of species of Acetobacter and Gluconobacter upon growth of the wine yeasts Saccharomyces cerevisiae, Kloeckera apiculata and Candida stellata was examined during mixed culture in grape juice. Acetobacter pasteurianus, A. aceti and Gluconobacter oxydans grew in conjunction with yeasts during juice fermentation. As determined by viable counts, yeast growth was only slightly impaired by the presence of bacteria. However, as judged by the concentrations of glucose, fructose, ethanol, glycerol, acetaldehyde, ethyl acetate, iso -amyl alcohol and organic acids in the fermented juice, acetic acid bacteria significantly influenced the alcoholic fermentation by yeasts.     

Do yeasts and Drosophila interact just by chance?

The fruit fly Drosophila melanogaster and the baker's yeast Saccharomyces cerevisiae are classic research model organisms that are also associated in nature, at least around vineyards. Sharing the same ephemeral fruit niche, winged Drosophila feed on immotile yeasts. That a yeast diet is essential for larval development, and that saprophagous fruit flies are attracted to a suite of yeast volatiles, has been well established over the last century. Recently, research has focussed on the potential mutual benefit of this interaction hypothesising yeasts also benefit via dispersal from ephemeral fruits. It now appears that the concept of a co-evolved mutualism between yeasts and Drosophila has permeated the literature. However, until robust evidence regarding the evolution and maintenance of this yeast-fly association has been provided, we suggest there is no compelling evidence to reject the more simplistic null hypothesis that these interactions are due to exaptation, and not a mutualism driven by natural selection.     

Biological preservation of plant derived animal feed with antifungal microorganisms: safety and formulation aspects

During storage of moist animal feed, growth of detrimental fungi causing spoilage, or being mycotoxigenic or pathogenic, is a severe problem. Addition of biopreservative yeasts or lactic acid bacteria can significantly reduce this problem. However, their use requires several careful considerations. One is the safety to the animal, humans and the environment, tightly connected to legal aspects and the need for pre-market authorisation when supplementing feed with microorganisms. Although both yeasts and lactic acid bacteria are considered comparatively safe organisms due to low production of toxic metabolites, it is of great importance to understand the mechanisms behind the biopreservative abilities. Another important issue concerns practical aspects, such as the economic production of large amounts of the organisms and the development of a suitable formulation giving the organisms a long shelf life. These aspects are discussed and a recommendation of this review is that both safety and formulation aspects of a specific microbe should be considered at an early stage in the selection of new organisms with biopreservation potential.     

Physical factors influencing microbial interactions and biochemical changes during the Baceman stage of Indonesian Kecap (soy sauce) production

The composition of samples taken from the baceman stage of traditional Indonesian kecap (soy sauce) production can vary greatly from batch to batch and from manufacturer to manufacturer. This variability could be caused by physical factors such as salt concentration, temperature, dissolved oxygen tension and pH. The effect of these factors on the changes in microflora and biochemical composition during the baceman stage are described in this report. Salt concentration was found to have a large influence on the spoilage of the baceman. At low salt concentrations pellicle-forming yeasts were able to grow. The pellicle on the liquid surface formed by these yeasts provided an acrobic environment in which coryneform bacteria could grow. These bacteria consumed amino acids and fermentation products like acetate and lactate, which resulted in a rise in pH and subsequent spoilage of the baceman. An aerobic baceman showed the same development as those with a low salt concentration. Salt concentration as well as temperature had a large influence on the rate of growth of bacteria. Growth of bacteria and associated lactate and acetate production were also stimulated by a high pH, while growth of yeasts and ethanol and glycerol formation was better at low pH. Also, the production of formol nitrogen by enzymes resulting from the previous bungkil stage was greatly influenced by temperature and pH, with higher temperature and pH giving rise to higher formol nitrogen contents. The consequences of these physical factors on the production of traditional Indonesian kecap are discussed.     

Fermentation and aerobic metabolism of cellodextrins by yeasts.

The fermentation and aerobic metabolism of cellodextrins by 14 yeast species or strains was monitored. When grown aerobically, Candida wickerhamii, C. guilliermondii, and C. molischiana metabolized cellodextrins of degree of polymerization 3 to 6. C. wickerhamii and C. molischiana also fermented these substrates, while C. guilliermondii fermented only cellodextrins of degree of polymerization less than or equal to 3. Debaryomyces polymorphus, Pichia guilliermondii, Clavispora lusitaniae, and one of two strains of Kluyveromyces lactis metabolized glucose, cellobiose, and cellotriose when grown aerobically. These yeasts also fermented these substrates, except for K. lactis, which fermented only glucose and cellobiose. The remaining species/strains tested, K. lactis, Brettano-myces claussenii, B. anomalus, K. dobzhanskii, Rhodotorula minuta, and Dekkera intermedia, both fermented and aerobically metabolized glucose and cellobiose. Crude enzyme preparations from all 14 yeast species or strains were tested for ability to hydrolyze cellotriose and cellotretose. Most of the yeasts produced an enzyme(s) capable of hydrolyzing cellotriose. However, with two exceptions, R. minuta and P. guilliermondii, only the yeasts that metabolized cellodextrins of degree of polymerization greater than 3 produced an enzyme(s) that hydrolyzed cellotretose.     

Fermentation and aerobic metabolism of cellodextrins by yeasts

The fermentation and aerobic metabolism of cellodextrins by 14 yeast species or strains was monitored. When grown aerobically, Candida wickerhamii, C. guilliermondii, and C. molischiana metabolized cellodextrins of degree of polymerization 3 to 6. C. wickerhamii and C. molischiana also fermented these substrates, while C. guilliermondii fermented only cellodextrins of degree of polymerization less than or equal to 3. Debaryomyces polymorphus, Pichia guilliermondii, Clavispora lusitaniae, and one of two strains of Kluyveromyces lactis metabolized glucose, cellobiose, and cellotriose when grown aerobically. These yeasts also fermented these substrates, except for K. lactis, which fermented only glucose and cellobiose. The remaining species/strains tested, K. lactis, Brettano-myces claussenii, B. anomalus, K. dobzhanskii, Rhodotorula minuta, and Dekkera intermedia, both fermented and aerobically metabolized glucose and cellobiose. Crude enzyme preparations from all 14 yeast species or strains were tested for ability to hydrolyze cellotriose and cellotretose. Most of the yeasts produced an enzyme(s) capable of hydrolyzing cellotriose. However, with two exceptions, R. minuta and P. guilliermondii, only the yeasts that metabolized cellodextrins of degree of polymerization greater than 3 produced an enzyme(s) that hydrolyzed cellotretose.     

Flocculation and coflocculation of bacteria by yeasts

Biotransformations in natural environments frequently involve interactions between microorganisms. Although there are many reports on the interactions between bacteria, interactions between yeasts and bacteria have not been extensively studied. Previously we reported on the flocculation and coflocculation of Pediococcus damnosus by Saccharomyces cerevisiae. Now we report that several other yeasts, such as Candida utilis, Dekkera bruxellensis, Hanseniaspora guilliermondii, Kloeckera apiculata, and Schizosaccharomyces pombe, induce flocculation with several industrially or medically relevant bacteria, including Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus. Candida utilis was one of the best flocculation inducers. The results are discussed with respect to interactions between yeasts and bacteria and their applications in industry and medicine.     

Spoilage yeasts.

Yeasts are best known for their beneficial contributions to society, and the literature abounds with discussions of their role in the fermentation of alcoholic beverages, bread, and other products. Yeasts also cause spoilage, but, with a few exceptions, this unwanted activity often goes unrecognized and underestimated as a major problem in the food and beverage industries. In some cases, there is only a fine line between what is perceived as either a spoilage or beneficial activity. This review examines the occurrence and growth of yeasts in foods and beverages with respect to their spoilage activities, the biochemistry of this spoilage, and technologies for the enumeration and identification of spoilage yeasts.     

Identification and characterization of non-saccharomyces spoilage yeasts isolated from Brazilian wines

The industry of fine wines and also locally consumed table wines is emerging in Brazil with an increasing volume and economic impact. Enologists in this region currently lack information about the prevalence and characteristics of spoilage yeasts, which may contaminate and potentially undervalue Brazilian wines. Herein, we analyzed 50 local red wines including 27 fine wines (V. vinifera) and 23 table wines (V. labrusca). Presumptive spoilage yeasts were isolated on differential medium, and classified by RFLP-PCR and sequencing of the ITS1-5.8S-ITS2 and D1/D2 26S rDNA loci. The prevalence of spoilage yeasts in fine wines (11 %) was comparable to that reported in European and US wines, and significantly lower than that observed for local table wines (70 %). The majority of isolates belonged to Brettanomyces bruxelliensis, followed by Pichia guillermondii, and more rarely Candida wickerhamii and Trigonopsis cantarelli. The Brettanomyces isolates varied greatly in off-flavor production, displayed ethanol tolerance (>10 % by volume), tolerated sulfite (≥0.68 mg/l mSO₂), and 39 % of them grew on ethanol as sole carbon source. We discuss the causes and consequences of spoilage yeasts in relation to the Brazilian wine industry.     

A note on shelf-life extension of British fresh sausage by vacuum packing

Vacuum packing of British fresh sausage in a low oxygen permeability film (Diolon) extended the product shelf-life at 6°C to more than 20 d compared with 9–14 d in conventional packs. After 10 d storage, counts of key spoilage organisms such as yeasts and Brochothrix thermosphacta were generally 2 log cycles lower in vacuum packs. Vacuum-packed sausages also displayed a slower rate of loss of free sulphite. Variations in pack permeability to SO₂ were not responsible for this. Losses of free SO₂ in stored sausages are largely due to the production of sulphite-binding agents by yeasts. Selective enumeration of these yeasts showed them to be inhibited by conditions of vacuum packing. The extension of shelf-life observed is ascribed to the reduction in growth rate of the spoilage flora in vacuum packs coupled with the consequent maintenance of inhibitory levels of sulphite for a longer period.     

红茶菌抗菌蛋白产生方式的初步研究*

从红茶菌液中分离得到膜醋酸菌与裂殖酵母。采用单菌培养与混合菌培养,探讨了各菌对红茶菌抗菌蛋白的产生所起的作用。初步推断,裂殖酵母先合成相关蛋白,该蛋白经膜醋酸菌产生的酶修饰后,抗菌活力显著提高。不同属酵母与膜醋酸菌共培养,结果表明裂殖酵母优于其它酵母。