Guilliermondii Var Research Articles

ENZYMATIC CHARACTERIZATION OF YEAST STRAINS ORIGINATED FROM TRADITIONAL MIHALIC CHEESE

Yeasts, associated with secondary flora of many cheese types, are important microorganisms for cheese ripening process. The aim of this study was to identify the yeasts isolated from traditional Mihalic cheese and to determine their enzymatic activities as a tool for their technological characteristics. Phenotypic identification was performed by using API ID 32C test system and some complementary morphological, physiological and biochemical tests. Enzyme profiles of the isolates were determined by using API-ZYM strips. In this study, 72 yeast isolates were obtained from 29 Mihalic cheese samples. Fifty-six (78%) of the isolates could be identified at species level, and one isolate at genus level. The identified yeast species belonged to three genera; Candida, Geotrichum and Trichosporon. It was determined that Candida famata var. famata was the dominant species in Mihalic cheese. The yeast isolates had variable enzyme activities including acid phosphatase, esterase, esterase lipase, lipase, β-galactosidase, leucine arylamidase, valine arylamidase and cysteine arylamidase, which could have important attributes during cheese ripening. C. famata var. famata M22, Candida guilliermondii var. membranefaciens M54 and Candida tropicalis M2 were selected to be superior strains on the basis of their enzyme profiles. Identification and enzymatic characterization of the yeasts originated from Mihalic cheese was performed for the first time in this study.

Two new species in the clade: sp. nov., the ascosporic state of , and 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.

Taxonomic relationships among the taxa in the Candida guilliermondii complex, as revealed by comparative electrophoretic karyotyping.

Electrophoretic karyotypes of 15 type strains of the taxa in the Candida guilliermondii complex including Candida fukuyamaensis Nakase et al. and Candida xestobii Yarrow et S. A. Meyer were comparatively analysed by using the CHEF (contour-clamped homogeneous electric field) method of PFGE. Eighteen strains (isolated from various natural sources in China) which were originally identified as C. guilliermondii by conventional methods were also included. Six electrophoretic karyotype groups were recognized among the strains compared. The following type strains were grouped together with the type strains of C. guilliermondii (Castellani) Langeron et Guerra and Pichia guilliermondii Wickerham: Blastodendrion arztii Ota, Blastodendrion krausi Ota, Candida amidovorans Balloni et al., C. guilliermondii var. japonica Sugiyama et Goto, Candida mamillae S. Goto, Candida parapsilosis (Ashford) Langeron et Talice var. tokyoensis Suzuki et al., C. parapsilosis var. tuxtlensis Herrera et al. and six Chinese strains. The type strain of Torulopsis kestonii Scarr et Rose was classified into the group together with the type strain of Candida fermentati (Saito) Bai and seven Chinese strains. The group represented by the type strain of C. fukuyamaensis included five other strains isolated in China. The type strains of Candida xestobii, C. guilliermondii var. carpophila Phaff et M. W. Miller and Trichosporon appendiculare Batista et al. were separated into three different groups, respectively. Taxonomic relationships among the taxa studied are discussed.

Demonstration of antibodies against Candida guilliermondii var. guilliermondii in asymptomatic infertile men.

Candida guilliermondii (C.g.) occurs mostly in animals, but it can affect humans too. Using serological screening methods studies have been carried out in 57 (30 normozoospermic and 27 oligozoospermic) asymptomatic infertile men, of whom 15 (26.3%; 9 oligozoospermic and 6 normozoospermic) were found to have responded to Candida guilliermondii var. guilliermondii. Sperm parameters (cell count, motility, morphology) were determined and various tests (hypo-osmotic swelling test, bovine mucus penetration, swim-up and testosterone examinations) were also performed. In the C.g.-infected and non-infected groups no significant difference was observed in any of the parameters. Precipitation was performed with a special antigen and in the positive group agglutination titres ranged from 0 to 1/320. Comparing C.g. titres with sperm parameters, only the swim-up technique showed noticeable alteration (P = 0.003). One month following ketoconazole treatment, the patients showed no precipitation reactions and their agglutination titres were < 1/80. Our results suggest that for the diagnosis of male infertility a serologic screening for C.g. is essential. As C.g. can also be sexually transmitted, both the husband and wife must be treated, if infection occurs.

Candida guilliermondii var. guilliermondii infection in infertile women.

The sera of 263 women--217 infertile and 46 pregnant--were examined by various serological methods (precipitation test, agglutination, indirect immunofluorescence) to detect Candida guilliermondii var. guilliermondii (C.g.) infection. The precipitation reaction was performed with extracellular C. guilliermondii antigen, the agglutination reaction was employed parallel with C. albicans. In the infertile group 122 (56.2%) proved to be C.g. positive, while in the fertile 11 women (23.9%) proved to be so, the level of significance being p less than 0.0001 between the two groups. A one-month ketoconazole treatment (one tablet, 200 mg/day) was adequate for eliminating the C.g. infection. In a few cases hystological examinations were also performed according to Gomori-Grocott and yeast cells could be detected in the stroma of the ovary. IgA, IgG, IgM, Gc-globulin, transferrin and ferritin determinations were carried out before and after the ketoconazole treatment, and there were significant differences in the IgM and transferrin levels between the infected and non-infected groups. The authors achieved 5 pregnancies of 56 treated women in 6 months.

Lysine degradation in Candida. Characterization and probable role of l-norleucine-leucine, 4-aminobutyrate and σ-aminovalerate: 2-oxoglutarate aminotransferases

Three enzymes partially purified that catalyze respectively the transamination of l-norleucine, 4-aminobutyrate and σ-aminovalerate with α-ketoglutarate as aminoacceptor were characterized and isolated from l-lysine adapted cell of Candida guilliermondii var. membranaefaciens. The transaminases have a maximum activity in the pH range of 7.8–8.5 and at 55°C, 45°C and 40°C respectively. α-Ketoglutarate and to a lesser extent pyridoxal-5′-phosphate were effective protecting agents against rise in temperature. The enzymes exhibit absorption maximum at 280 nm, 330 nm and 410 nm. The fact that l-norleucine-leucine aminotransferase, 4-aminobutyrate aminotransferase and σ-aminovalerate aminotransferase are strongly induced by growing the yeast Candida on l-lysin suggests new hypothetic pathways for the catabolism of l-lysine where the main substrate of each aminotransferase could be an intermediary metabolite.

Candida L-norleucine,leucine:2-oxoglutarate aminotransferase. Purification and properties.

A new enzyme which catalyzes the transamination of L-norleucine (2-aminohexanoic acid) and L-leucine with 2-oxoglutarate was purified to homogeneity from cells of Candida guilliermondii var. membranaefaciens. The relative molecular mass determined by gel filtration was estimated to be close to 100,000. The transaminase behaved as a dimer which consists of two subunits identical in molecular mass (Mr 51,000). The enzyme has a maximum activity in the pH range of 8.0-8.5 and at 55 degrees C. 2-Oxoglutarate, and to a lesser extent pyridoxal 5'-phosphate, were effective protecting agents against increasing temperature. The enzyme exhibits absorption maximum at 330 nm and 410 nm. L-Norleucine, and L-leucine to a lesser extent, are the best amino donors with 2-oxoglutarate as amino acceptor. The Km values for L-norleucine, L-leucine and 2-oxoglutarate determined from the Lineweaver-Burk plot were 1.8 mM, 6.6 mM and 2.0 mM respectively. A ping-pong bi-bi mechanism of inhibition with alternative substrates is found when the enzyme is in the presence of both L-norleucine and L-leucine. The inhibitory effect of various amino acid analogs on the transamination reaction between L-norleucine and 2-oxoglutarate was studied and Ki values were determined.

Candida delta-aminovalerate: alpha-ketoglutarate aminotransferase: purification and enzymologic properties.

A new enzyme that catalyzes the transamination of delta-aminovalerate with alpha-ketoglutarate was purified to homogeneity from adapted cells of Candida guilliermondii var. membranaefaciens. The relative molecular mass determined by gel filtration was estimated to be close to 118,000. The transaminase behaved as a dimer with two similar subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a maximum activity in the pH range of 7.8-8.5 and at 40 degrees C. alpha-Ketoglutarate and to a lesser extent pyridoxal 5'-phosphate were effective protecting agents toward temperature raising. The enzyme exhibits absorption maximum at 330 and 410 nm. The enzyme catalyzes the transamination between omega-amino acids and alpha-ketoglutarate. delta-Aminovaleric acid is the best amino donor. The Km values for delta-aminovalerate, alpha-ketoglutarate, and pyridoxal 5'-phosphate determined from the Lineweaver-Burk plot were 4.9 mM, 3.6 mM, and 22.7 microM, respectively. The inhibitory effect of various amino acids analogues on the transamination reaction between delta-aminovalerate and alpha-ketoglutarate was studied, and Ki values were determined.

4-Aminobutyrate:2-oxoglutarate aminotransferase from Candida. Purification and properties.

An enzyme which catalyzes the transamination of 4-aminobutyrate with 2-oxoglutarate was purified 588-fold to homogeneity from Candida guilliermondii var. membranaefaciens, grown with 4-aminobutyrate as sole source of nitrogen. An apparent relative molecular mass of 107,000 was estimated by gel filtration. The enzyme was found to be a dimer made up of two subunits identical in molecular mass (Mr 55,000). The enzyme has a maximum activity in the pH range 7.8-8.0 and a temperature optimum of 45 degrees C. 2-Oxoglutarate protects the enzyme from heat inactivation better than pyridoxal 5'-phosphate. The absorption spectrum of the enzyme exhibits two maxima at 412 nm and 330 nm. The purified enzyme catalyzes the transamination of omega-amino acids; 4-aminobutyrate is the best amino donor and low activity is observed with beta-alanine. The Michaelis constants are 1.5 mM for 2-oxoglutarate and 2.3 mM for 4-aminobutyrate. Several amino acids, such as alpha,beta-alanine and 2-aminobutyrate, are inhibitors (Ki = 38.7 mM, Ki = 35.5 mM and Ki = 33.2 mM respectively). Propionic and butyric acids are also inhibitors (Ki = 3 mM and Ki = 2 mM).

Water relations and metabolism of propionate in two yeasts from hay

Many yeasts and bacteria were isolated from moist hays (&gt;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.

In vitro determination of phagocytic indices of Candida berkhout species by rat peritoneal macrophages.

The aims of this investigation were to study and describe the behaviour of 13 different species of Candida, as compared with C. albicans, by means of phagocytosis assays in vitro. Tests were carried out with rat peritoneal macrophages in contact with quantified suspensions of live yeasts. Phagocytic indices, candidacidal activity and filamentation rat were tested microscopically after 3 h incubation at 37 degrees C. The phagocytic indices obtained allowed us to separate the fungi into four groups. Candida albicans and tropicalis belong to Group I; diddensii and shehatae, among others, belong to Group II; sake, krusei, viswanathii, etc., Group III; and C. glaebosa and haploid strains of Pichia ohmeri (C. guilliermondii var. membranaefaciens), Group IV. These data would suggest a possible correlation between pathogenesis and phagocytic indices. There were no evidences of any phagocytes ability to kill yeasts. Candidacidal activity was absent in the species assayed. Yeast lysis may have been observed if our assays would have taken longer than 3 h.

Association of Candida guilliermondii with amylolytic filamentous fungi on preharvest corn

Candida guilliermondii var. guilliermondii was the dominant yeast isolated from preharvest Georgia corn and comprised 3.6–47.0% of the total fungi as measured by dilution plating. The yeast was confined mainly to kernels visibly molded by filamentous fungi, primarily Fusarium moniliforme and Aspergillus flavus. When grown in liquid shake culture, Cand. guilliermondii was unable to utilize starch. Growth of the yeast increased considerably when associated with amylolytic F. moniliforme or Asp. flavus. Utilization of starch hydrolyzates by Cand. guilliermondii in mixed cultures was reflected by a decrease in reducing sugar concentrations and a reduction in growth of F. moniliforme and Asp. flavus. When glucose replaced starch as a carbon source, F. moniliforme and Asp. flavus did not affect the maximum growth of Cand. guilliermondii. The association between Cand. guilliermondii and amylolytic filamentous fungi on preharvest corn is probably commensalistic.

発酵母液の再資源化研究 ＩＩＩ アルコール発酵母液を用いた海洋酵母の培養およびその菌学的性質

Marine yeats isolated from microbial flocks produced from alcohol fermentation slop (AFS), were an effective food for the rotifer Brachionus plicatilis. Large scale cultures of threes strains of the isolated yeasts, which are very effective as food, were carried out with a medium containing AFS as the sole source of carbon and energy using a 10-l jar fermentor. The yields of the isolated yeast cells from one liter of culture broth were in the range of 15-24g (dry cell weight). The dried yeast cells contained crude protein amounting to 33-38%. General compositions, amino acid and fatty acid compositions, and vitamin contents of the isolated yeasts cultured with AFS were compared with those of bakers's yeast. The results showed that except for crude ash there was no distinct difference to be found between the isolated yeasts and baker's yeast. Four representative strains of the isolated yeasts were identified as Torulopsis inconspicua YU-1, Candida guilliermondii var. guillermondii YA-3, Candida krusei ZA-3 and Candida valida KU-1, respectively.

Candida guilliermondii, an Actidione‐resistant Yeast from Wine*

Candida guilliermondii var. guilliermondii, a yeast which showed high resistance to actidione, was isolated from a red wine sample. Growth in 5000 parts/106of actidione was equal to that of the control. Sorbic acid and sodium benzoate were most effective in checking the growth at 250 parts/106but growth occurred at 750 parts/106of potassium metabisulphite. Fermentation of glucose, galactose, sucrose and raffinose was not inhibited in the presence of 1000 parts/106actidione. This is the first report on the occurrence of actidione‐resistant yeasts in the genus Candida and on the isolation of C. guilliermondii var. guilliermondii from wine.

The genus Candida Berkhout. XI. Electron microscopy and roentgen-phase analysis of cell walls of three strains of Candida guilliermondii (Cast.) Langeron et Guerra.

The aim of the study was to verify the seem'soy of the taxonomic classification of rough variants of the species Candida guilliermondii on the basis of comprehensive study of phonetic manifestation and to determine differences in cell wall structure with special reference to polysaccharides (1) According to their phenotypo, the test strains Candida guilliermg~dii (Cant.) Laageroa et Guarra and O:~niida guillier- mondii var. membranaefaciens Lodder et Kregor-Van Rij belong to the species C,zndida guillier,nondii, while Candida guilliermondii var. nitratophila Diddons et Lodder is phenotypically closer to Candida peUieutosa. (2) Observation of native and hydrolysed cell walls in the electron microscope showed no differences between the test strains. (3) The results of X-ray phase analysis of cell walls differentiated Candida guilliermondii var. nitratophila from the other two, however. (4) Electron microscopy photo- micrographs and diffractograms of cell walls indicated that, after 2% I-IC1 extraction at 100 C, the cell walls contain chitin, which is isolated by further extraction with 30~0 HC1. After 3% NaOH hydrolysis the chitin diffractogram is not clear.

Microbial Production of Xylitol from Glucose1

A microbiological method is described for the production of xylitol, which is used as a sugar substitute for diabetics. A sequential fermentation process yielded 9.0 g of xylitol from 77.5 g of glucose via D-arabitol and D-xylulose. Candida guilliermondii var. soya (ATCC 20216) consumed 5.1 g of D-xylulose and produced 2.8 g of xylitol per 100 ml. Pentitol production from D-xylulose by yeasts was divided into three types: I, yeast-produced xylitol; II, yeast-produced D-arabitol; and III, yeast-produced xylitol and D-arabitol. D-Xylulose, but not glucose, was dissimilated to xylitol by yeasts under aerobic conditions.

Successive Microbial Populations in Calimyrna Figs

Smyrna-type (Calimyrna) figs have essentially sterile internal tissue until visited by the pollinating fig wasp, Blastophaga psenes, which introduces a specific microflora consisting of Candida guilliermondii var. carpophila and Serratia plymuthica. This flora persists and develops in numbers throughout the ripening period until maturity of the fruit. These organisms do not cause spoilage. The presence of C. guilliermondii var. carpophila appears to increase the attractiveness of the fruit to drosophilae. Drosophila (mainly D. melanogaster) carry spoilage yeasts and bacteria on their exterior body parts, and introduce these organisms during ovipositing in the fruit cavity. The spoilage yeasts consist almost entirely of apiculate yeasts (Hanseniaspora valbyensis, H. uvarum, and Kloeckera apiculata) and of Torulopsis stellata, which cause active fermentative spoilage. Spoilage bacteria (primarily Acetobacter melanogenus) are also introduced with the yeasts. Organic acids are produced by these yeasts as well as by the Acetobacter. A number of minor spoilage yeasts were also identified.