Film-forming Yeasts Research Articles

Effects of film-forming Pichia and Candida yeasts on cider and wine as post-fermentation contaminants.

Film-forming yeasts are potential sources of defects in alcoholic beverages. The aim of this study is to assess the growth capacity of Pichia and Candida film-forming yeasts in cider and wine and the effects on their chemical composition. Cider, partially and fully fermented wine were inoculated with strains of C. californica, P. fermentans, P. kluyveri, P. kudriavzevii, P. manshurica, and P. membranifaciens to simulate a post-fermentative contamination. The former three species grew only in cider. Pichia manshurica and P. kudriavzevii displayed high viability in wine up to 13.18% (v v-1) ethanol. Significant changes in odour-active molecules from different chemical groups were observed in cider and wine in the inoculated samples, compared to the non-inoculated ones. Cider is more susceptible to contamination by all of the species tested, due to its low alcohol content, while P. membranifaciens, P. manshurica, and P. kudriavzevii are additionally potential spoilage agents of wine. This study highlights the risk of cider and wine contamination by film-forming yeasts. Their impact on aroma profiles depends on their ability to grow and their metabolism. This study contributes to an understanding of the possible physiological and metabolic mechanisms responsible for film formation and chemical changes in alcoholic beverages.

Fabrication of Gold Nanoparticles and Cinnamaldehyde-Functionalized Paper-Based Films and Their Antimicrobial Activities against White Film-Forming Yeasts.

During storage and fermentation of kimchi, white film-forming yeasts (WFY) are generated on the surface of kimchi under various conditions. These yeasts include Candida sake, Pichia kudriavzevii, Kazachstania servazzii, Debaryomyces hansenii, and Hanseniaspora uvarum. Because of the off-odor and texture-softening properties of WFY that degrade the quality of kimchi, a method to prevent WFY is required. In this study, cinnamaldehyde (CIN) and gold nanoparticles (AuNPs) with a large surface area were grafted on a paper surface, which was termed the "Paper_AuNPs_CIN" film. CIN is an antimicrobial agent that is approved for use in food applications. In the as-fabricated Paper_AuNPs_CIN film, antimicrobial CIN molecules were physically adsorbed to the surface of AuNPs and simultaneously chemically synthesized on the paper surface via the imine reaction. The Paper_AuNPs_CIN film exhibited greater antimicrobial activity against the three WFY strains than a Paper_CIN film (which contains only CIN molecules). Since more CIN molecules were adsorbed to the large surface area of the paper-reduced AuNPs, the Paper_AuNPs_CIN film exhibited a higher antimicrobial activity. Using AuNPs and CIN simultaneously to inhibit the growth of WFY is a novel approach that has not yet been reported. The morphology and elemental mapping of the functionalized films were examined via scanning electron microscopy and energy-dispersive spectroscopy, elemental composition was analyzed via inductively coupled plasma optical emission spectroscopy, and chemical bonding and optical properties were investigated via Fourier transform infrared spectroscopy and diffuse reflectance spectroscopy. Additionally, agar-well diffusion assays were used to determine the antimicrobial activity against three representative WFY strains: C. sake, P. kudriavzevii, and K. servazzii.

Prenylated flavonoids from the stems and leaves of Desmodium caudatum and evaluation of their inhibitory activity against the film-forming growth of Zygosaccharomyces rouxii F51.

In order to provide scientific evidence for the relationship between the traditional usage, stems and leaves of Desmodium caudatum being used for protecting miso from spoilage, and its Japanese name (miso-naoshi), phytochemical study on the stems and leaves of this plant was carried out. Seven new prenylated flavonoids (1-3, 15-18), together with 19 known compounds (4-14, 19-26), were isolated, and the structures of new compounds were elucidated by extensive spectroscopic analyses. The minimum inhibitory concentrations (MICs) of 28 flavonoids, including 17 compounds (1, 2, 4, 5, 7-14, 20-22, 24, 25) isolated in this study and 11 flavonoids (27-37) previously isolated from the roots of this plant, against the film-forming yeast of Zygosaccharomyces rouxii F51 were determined. Fifteen compounds (2, 4, 5, 11, 12, 14, 21, 22, 25, 27, 28, 32-35) inhibited the film-forming growth of Z. rouxii F51 (MIC values, 7.8-62.5 μg/mL), among which 2",2"-dimethylpyran-(5",6":7,8)-5,2'-dihydroxy-4'-methoxy-(2R,3R)-dihydroflavonol (11) demonstrated potent inhibitory activity with an MIC value of 7.8 μg/mL.

Purification and characterization of antifungal compounds from Lactobacillus plantarum HD1 isolated from kimchi

Strain HD1 with antifungal activity was isolated from kimchi and identified as Lactobacillus plantarum. Antifungal compounds from Lb. plantarum HD1 were active against food- and feed-borne filamentous fungi and yeasts in a spot-on-the-lawn assay. Antifungal activity of Lb. plantarum HD1 was stronger against filamentous fungi than yeast. Antifungal compounds were purified using solid phase extraction (SPE) and recycling preparative-HPLC. Structures of the antifungal compounds were elucidated by electrospray ionization-mass spectrometry and nuclear magnetic resonance. Active compounds from Lb. plantarum HD1 were identified as 5-oxododecanoic acid (MW 214), 3-hydroxy decanoic acid (MW 188), and 3-hydroxy-5-dodecenoic acid (MW 214). To investigate the potential application of these antifungal compounds for reduction of fungal spoilage in foods, Korean draft rice wine was used as a food model. White film-forming yeasts were observed in control draft rice wine after 11 days of incubation. However, film-forming yeasts were not observed in draft rice wine treated with SPE-prepared culture supernatant of Lb. plantarum HD1 (equivalent to 2.5% addition of culture supernatant) until 27 days of incubation. The addition of antifungal compounds to Korean draft rice wine extended shelf-life up to 27 days at 10 °C without any sterilization process. Therefore, the antifungal activity of Lb. plantarum HD1 may lead to the development of powerful biopreservative systems capable of preventing food- and feed-borne fungal spoilage.

Enological Behaviour of Biofilms Formed by Genetically-Characterized Strains of Sherry Yeast

The flor yeasts (Saccharomyces cerevisiae) form a biofilm, known as flor velum, on the surface of fino-type sherry wine at the end of the alcoholic fermentation. These film-forming yeasts are responsible for the oxidative transformation of alcohol to acetaldehyde, together with other reactions, which produce the characteristic flavours and aromas of these wines. In this study, we examine the enological behaviour of eight flor yeast strains biofilms in biological aging experiments carried out in the laboratory. Strains with identical chromosomal and mitochondrial DNA patterns and the same origin showed a more closely-related enological behaviour. But the kinetics of growth and acetaldehyde accumulation in the wine were found to be strain-dependent. Moreover, some strains were marked by high acetaldehyde accumulation in their pure cultures during the various phases of the biofilm development. These results provide valuable knowledge for planning technical strategies to improve the biological aging process in the sherry wine industry.

Effect of a Leuconostoc mesenteroides strain as a starter culture isolated from the kimchi

To investigate the effect of starter isolated from kimchi, we selected the most effective strain among the 121 strains examined and identified the strain as Leuconostoc mesenteroides LK93 through 16S rDNA sequencing. This strain exhibited resistance to acid and bile salts with good survival capacity and showed antimicrobial and antifungal activity. We investigated the effects of starter on kimchi fermentation. The kimchi was fermented at 25°C for 25 d. Furthermore, the LK93 strain was examined for its ability to inhibit the growth of film-forming yeast. It was determined that the LK93 strain could be a potential starter in kimchi.

저염 장류에서 증식하는 산막 효모에 길항 작용을 갖는 Bacillus 균주의 분리

한국인의 저염 섭취 필요성에 부응하기 위해 14% (w/w) 염도의 된장 대신 8% (w/w) 저염 된장을 제조하였다. 숙성 후 8% 저염 된장 시료들은 14% 염 된장과 달리 모두 산막을 형성하였고, 이 산막 형성균들을 동정한 결과 8종 모두 효모인 Pichia kudriavzevii에 속하였다. 장류 고유한 풍미를 유지하기 위해 발효 특성이 우수한 11종의 Bacillus 균들을 전통 장류에서 분리했고, 이들의 생화학적 특성 및 16S rRNA 염기 서열 분석 결과 B. subtilis, B. licheniformis, B. methylotrophicus로 동정되었다. 이 균주들은 8종의 산막 효모에 대해 증식 억제 능력을 보였으며, 항균 계면활성제인 lichenysin 또는 surfactin 유전자들을 함유하고 있었다. Soybean pastes with 8% (w/w) salinity were prepared instead of soybean paste with 14% (w/w) salinity to meet the growing demands of Korean's low sodium diet. After aging, white films had appeared on the surface of all low-salted soybean pastes [8% (w/w) salinity] unlike high-salted soybean pastes [14% (w/w) salinity]. All of eight microbes isolated from the surface film were identified as Pichia kudriavzevii. Eleven Bacillus strains with good characteristics of fermentation were isolated from traditionally fermented soybean pastes in order to preserve their unique flavors and aromas after aging, and as a result of analyzing the biochemical characteristics and 16S rRNA sequences, those were identified as B. subtilis, B. licheniformis, and B. methylotrophicus. All of the Bacillus isolates had antagonistic activities against 8 isolates of the film-forming yeasts and harbored the genes for synthesis of antimicrobial surfactants including lichenysin and/or surfactin.

Fermentation of non-sterilized fish biomass with a mixed culture of film-forming yeasts and lactobacilli and its effect on innate and adaptive immunity in mice

Non-sterilized fish waste containing fish bones was fermented using combined starter cultures of film-forming yeast (Candida ethanolica) and lactic acid bacteria (LAB; Lactobacillus casei and Lactobacillus rhamnosus) in order to obtain a liquefied fermented broth without spoiling. During the entire fermentation, the number of LAB cells was maintained at a high level (6 × 10(8)-5 × 10(7) cells/ml). Although the number of general bacteria was 10(6)cell/ml after adding non-sterilized fish biomass, its growth was suppressed to be 1-3 × 10(4) cells/ml. The entire biomass had completely liquefied and the fermented broth contained all 20 α-amino acids composed of protein and also various kinds of minerals in abundance. The weight of mice group fed the fermented broth content feed (sample feed) for 31 days significantly increased compared with that fed no broth feed (control feed) (21.37 g vs 20.76 g (p < 0.05). No abnormal behavior and appearance were observed. All internal organs (the heart, the liver, the lung, the intestines, and the spleen) of both groups were confirmed to be normal by visual observation. In peripheral blood, the percentages of NK cells and CD8+ T cells of the mice in the sample feed group increased significantly relative to those in the control feed group (NK cells: 19% vs 11%, CD8+ T cells: 9% vs 5%, p < 0.05). In the spleen, the percentage of NK cells in the sample feed group also increased significantly compared to that in the control feed group (p < 0.05). The fermented fish biomass is expected to be effective for innate and adaptive immunity and thus fit for animal feed.

Yeasts and wine off-flavours: a technological perspective

In wine production, yeasts have both beneficial and detrimental activities. Saccharomyces cerevisiae is the yeast mainly responsible for turning grape juice into wine but this species and several others may also show undesirable effects in wines. Among such effects, technologists are particularly concerned with the production of off-flavours that may occur during all stages of winemaking. Typical spoiling activities include the production of ethyl acetate by apiculate yeasts before fermentation, hydrogen sulphide by S. cerevisiae during fermentation phases, acetaldehyde by film-forming yeasts during bulk storage, and volatile phenols by Dekkera bruxellensis during storage or after bottling. The occurrence of these hazards depends on the technological operations designed to obtain a given type of wine and most can be avoided by current preventive or curative measures. On the contrary, good manufacturing practices must be strengthened to deal with the problem of volatile phenol production in red wines. Appropriate monitoring of D. bruxellensis populations and quantification of 4-ethylphenol is advised during storage, particularly when oak barrels are used, and absence of viable cells must be guaranteed in bottled wines. This work, which is based on our experience at winery level, aims to provide information on appropriate technological strategies to deal with the problem of off-flavours produced by yeasts.

Antiyeast steroidal saponins from Yucca schidigera (Mohave yucca), a new anti-food-deteriorating agent.

A saponin fraction from the stems of Yucca schidigera (Mohave yucca) exhibited potent growth-inhibitory activities against certain food-deteriorating yeasts, film-forming yeasts, and dermatophytic yeasts and fungi. From this fraction, a number of new anti-yeast monodesmosidic spirostanol saponins, named schidigera-saponins A1 (1), A2 (2), A3 (3), B1 (4), C1 (5),C2 (6); 25(R and S) schidigera-saponins D1 (7), D2 (8), E1 (12), F1 (13); and 25(S) schidigera-saponins D3 (9), D4 (10), D5 (11), and F2 (14) were isolated, together with several related known saponins, and the structures were elucidated by spectroscopic methods (see Chart 1). The relationship between the antiyeast activities and the structures of these saponins is described.

Structure of Antibacterial Substance in Extract of Cocklebur Leaves Used for Preparation of Wheat-Koji(qu) Described in the Chinese Classic "Chi-min-yao-shu" and Its Effect on Growth of Bacteria and Yeasts.

In our previous publications, we have reported that extract of cocklebur containsan antibacterial substance. This substance was purified using silica gel column chromatography and TLC; it was dissolved in acetone and successively crystallized by adding hexane. The crystal appeared needle-like and white, with a melting point of 60°C and molecular weight of 246. IR, UV and 1H-NMR spectra revealed that the compound was xanthatin. Minimum inhibitory concentrations (MICs) of xanthatin obtained were 12.5-100 μg/ml against Bacillus sp. which usually contaminates koji (qu) used in the preparation of alcoholic beverages. MICs of xanthatin were 25-100 μg/ml against Candida sp., Pichia sp., Saccharomycopsis sp. and Torulaspora sp.. Therefore, the inhibitory effect exhibited by xanthatin from cocklebur leaf extract against Bacillus sp. and some film-forming yeasts would be very useful to prevent contamination in koji and during production of alcoholic beverages.

Growth Inhibition of Film-Forming Yeasts During Production of Ume-Zuke (Pickled Japanese Apricots) by an Antimicrobial Fraction Extracted from Paprika Seeds.

Paprika extract (50 to 100 μg/ml) obtained by suspending ground paprika seeds in water, adding ethanol (50%, v/v), and then filtering the suspension, inhibited the growth of film-forming yeasts (Kloeckera, Pichia, Debaryomyces, and Candida species) isolated during the production of ume-zuke, a processed food product made by salting Japanese apricots (Prunus mume). The antimicrobial activity of the extract towards Pichia anomala was influenced by the initial number of yeast cells, as well as the temperature, pH, and sodium chloride concentration of the culture medium (ume vinegar). The inhibitory effect was cumulatively enhanced, although not synergistically, when the paprika seed extract was used in combination with either SO2, sorbic acid, thiamine dilauryl sulfate, or acetic acid. The antimicrobial activity of the extract was not influenced by the vinegar components. The extract proved to be very effective as a preservative to prevent the contamination and spoilage of ume-zuke by film-forming yeasts.

清酒生翫仕込み過程におけるキラー酵母の変動およびキラー菌株の性質

Killer yeasts which showed killer activity toward Candida glabrata IFO 0622, Saccharomyces cerevisiae Y 15, and S. cerevisiae Y-222, were isolated in the process of Ki-moto making. The isolates showd no killer activity toward S. cerevisiae Kyokai No.7. Each isolate didn't show killer activity to all other isolates.The killer yeasts accounted for about 50% of the total wild yeasts on the 4th day of the Ki mob but were not detected at the mid to the late stage of the Ki-moto making. All of the isolated killer strains were found to be film-forming yeasts. Most of them showed high killer activity and were identified as Pichia anomala. And these killer strains showed high killer activity to Zygosaccharomyces rouxii IFO 0505 in the presence of NaCl. The results indicated that the killer yeasts seemed not only to be harmless during the sake brewing process but also to have much the same spectra of killer activity against sensitive yeasts in the eary stage of Ki-moto making.

Inhibitory Effect of Yucca Extract on the Growth of Film-Forming Yeasts Isolated from Ume-Zuke, Salted Japanese Apricot Fruit

Inhibitory Effect of Yucca Extract on the Growth of Film-Forming Yeasts Isolated from Ume-Zuke, Salted Japanese Apricot Fruit

ワインから分離した産膜酵母に対するパプリカ種子抽出物の抗菌性

ワインから分離した産膜酵母に対するパプリカ種子抽出物の抗菌性

Interaction between film-forming yeasts and phenolic acids

Ninety strains of film-forming yeasts of the Saccharomyces genus were screened for their ability to use phenolic acids. None were able to use phenolic acids as a sole carbon source. When ethanol at 2% was added to the medium, however, the yeasts were able to grow, exhibiting florization accompanied by decolouration.

Dynamics of Flor Yeast Populations During the Biological Aging of Sherry Wines

Molecular and physiological analyses were applied for the study of film-forming yeasts (flor yeasts) over the period of two years in sherry wines. According to the molecular criteria used for distinguishing flor yeasts, six different strains were identified. Some of these strains, which differed in their molecular karyotype, yielded identical mitochondrial DNA restriction patterns, and some others showing distinguishable mitochondrial genomes exhibited identical karyotype, suggesting that the different flor yeast strains identified are closely related. By means of these molecular techniques, a single and stable strain was usually identified in the yeast film of individual barrels; this strain may, however, vary in different barrels. According to physiological properties two main types of flor yeasts were identified, one overproducing acetaldehyde and a second type (dominant strain) showing a high rate of film formation. These two properties might influence the yeast ecology in sherry wines, being the rate of film formation the main factor driving the dynamic of flor yeast populations.

Fatty acid patterns of film-forming yeasts and new evidence for the heterogeneity of Pichia membranaefaciens

Total fatty acids (C14:0 to C18:3) of 50 strains assigned by classical identification methods to Pichia membranaefaciens (28), P. anomala (15), Dekkera anomala (2), D. bruxellensis (2) and Candida vini (3) were determined and data analysed by multivariate statistical procedures. Principal components cluster analysis defined six groups of strains. Thirteen strains of P. anomala formed a well-defined cluster, whereas P. membranaefaciens was split into three groups. The taxonomic status of the P. membranaefaciens strains was evaluated by determination of the G + C content and DNA-DNA hybridization. The results provided evidence in support of P. membranaefaciens encompassing distinct species. Only eight strains were certified as P. membranaefaciens and six were included in the same cluster, indicating that numerical analysis of fatty acid profiles points to genetic differences which remain undetectable by conventional phenotypic tests.

Characterization of yeasts isolated from red wine surface film.

We have isolated 6 morphologically different axenic yeast cultures from the film surface of red wine. Based on morphological, physiological and biochemical characteristics we have identified the strains as follows: isolates 1-4 are morphologically different strains of the anamorph basidiomycetous film-forming yeast Candida humicola (DASZEWSKA) DIDDENS et LODDER, syn. Apiotrichum humicola (DASZEWSKA) VON ARX WEISMAN. Isolates 5 and 6 belong to the genus Saccharomyces of the associated species S. cerevisiae (isolate 5 originally S. bayanus, isolate 6 S. capensis). These do not participate in the surface film formation.

Improved Production of a Spanish-Type Sherry by Using Selected Indigenous Film-Forming Yeasts as Starters

In a series of experiments aimed at improving production of an unfortified Spanish-type sherry (Vernaccia di Oristano), the yeast flora were identified in various samples of must and flor. Thirty yeast strains isolated from the flor were subjected to functional selection, and the most suitable were used as starters in winery aging trials. The functional selection showed that <i>Saccharomyces prostoserdovii</i> and <i>Sacch. bayanus</i> (the species most frequently found in the flor) were almost equally strong fermenters, but <i>Sacch. prostoserdovii</i> was superior in the maturation phase. The aging trials showed that <i>Sacch. prostoserdovii</i> used alone produced a wine of the highest quality with more reliable and more uniform aging in one third of the time normally taken.