Enhanced lactic acid bacteria viability with yeast coincubation under acidic conditions

ABSTRACT

The enhancing effects of yeasts on the viability of lactic acid bacteria (LAB) under acidic conditions were investigated. Meyerozyma guilliermondii, coaggregative with both LAB strains under acidic conditions, significantly enhanced the viability of Lactobacillus pentosus and L. paracasei in pH 3.0 lactic acid (LA) buffer at 10°C (p < 0.05). Non-coaggregative yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Cyberlindnera saturnus) also significantly enhanced the LAB viability (p < 0.05), and physical contact between LAB and yeasts was not essential for the viability-enhancing effect, indicating that the coaggregation had no relation to the enhancing mechanism. Although yeast metabolites and LA assimilation had no enhancing effect, hydrogen peroxide (H₂O₂) decreased after yeast coincubation, and H₂O₂ elimination improved L. pentosus viability. H₂O₂ elimination alone did not sufficiently improve L. paracasei viability, but the addition of antioxidants was effective. These results suggest that the antioxidant activity of yeast increased the LAB viability under acidic conditions.     

Modification of physiological properties in Saccharomyces uvarum and Kluyveromyces bulgaricus grown in presence of polyoxyalkylene glycoĺ-oleic acid condensates

Summary Condensates of polyoxyalkylene glycol of diverse molecular weight esterified by oleic acid, used as antifoam agents in fermentors, were tested on Saccharomyces uvarum and Kluyveromyces bulgaricus. These compounds, used at a concentration of 0.1% (V/V) in the culture medium, stimulated the aerobic growth of the yeasts, and adding oleic acid (up to a concentration of 0.005% V/V in the medium) to the antifoam compounds further increased the final biomass.the presence of the antifoam agents during the development of yeasts increased their viability at the end of the culture and reinforced this viability for a further conservation by freezing. Antifoam agents also stimulated respiration in K. bulgaricus and to a lesser degree in S. uvarum. Flocculation of both yeasts was decreased.Over and above their physico-chemical foam — inhibiting action, polyoxyalkylene glycol compounds had a beneficial effect on the metabolism of yeasts. These compounds have a more positive action on yeasts than colza oil, another industrial antifoam agents.     

Enhancing viability of two biocontrol yeasts in liquid formulation by applying sugar protectant combined with antioxidant

Survivals of Cryptococcus laurentii and Pichia membranaefaciens in liquid formulations with sugar protectants (trehalose and galactose) and L-ascorbic acid (Vc) were investigated during storage at 4°C and 25°C. When galactose or trehalose was used alone as protectant, C. laurentii maintained relatively high viability in potassium phosphate buffer. Addition of Vc to trehalose improved its protective effect. P. membranaefaciens maintained viability >60% after 90 days at 4°C when 5% galactose served as a protectant, and its combination with Vc was the most effective at maintaining viability. Moreover, liquid formulation kept higher viability of the two yeasts at 4°C than at 25°C. Biocontrol efficiency of the two yeasts was maintained after formulation and storage. The results indicate that trehalose is considered as a suitable protectant for liquid formulation of C. laurentii, while galactose is better for P. membranaefaciens. Combining Vc with the sugars improves the protective efficiency.     

The diversity of Pityrosporum (Malassezia) yeasts in vivo and in vitro

Conclusions In considering the diversity of the lipophilic yeasts it has been shown that in vivo both spherical and oval yeasts may be found in normal conditions on the skin and also associated with hyphae in scales from pityriasis versicolor. There is however generally a different distribution pattern on the body for two forms. This may indicate a different ecology for two distinct varieties or the varying conditions at each site may influence changes in the cell shape of a single species. It is striking that the spherical yeasts (P. orbiculare) have not been found in animals. In vitro, several morphological variants can be maintained, but the change from one form to another which is the subject of a number of reports will be one deciding factor leading to the opinion that P. orbiculare and P. ovale are synonymous. However, physiological differences such as growth rate, their viability in subculture and the analysis of proteins, are all characteristics which alone would be insufficient to support a taxonomic division but when added together confirm the morphological separation of isolates. It remains to be seen if DNA studies, which have so far unified the anthropophilic, lipid dependant Pityrosporum yeasts, will in fact continue to show that they should be confined to a single species.This paper was presented at the X^th congress of the International Society for Human and Animal Mycology at Barcelona, Spain from June 27 to July 1, 1988.     

Kinetics and metabolic behavior of a composite culture of <Emphasis Type="Italic">Kloeckera apiculata</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine related strains

The kinetics and metabolic behavior of Kloeckera apiculata mc1 and Saccharomyces cerevisiae mc2 in composite culture was investigated. K. apiculata showed a higher viability through the fermentation; however the maximum cell density of both yeasts decreased. This behavior was not due to ethanol concentration, killer toxins production or competition for assimilable nitrogenous compounds between both yeasts. Despite the consistent production of secondary products by single culture of K. apiculata, an increase of these compounds was not observed in mixed culture. These results contribute to a better understanding of the behavior of non-Saccharomyces yeasts and their potential application in the wine industry.     

Combined effects of endo- and exogenous trehalose on stress tolerance and biocontrol efficacy of two antagonistic yeasts

Effects of endo- and exogenous trehalose on viability of two antagonistic yeasts, Cryptococcus laurentii (Kuffer.) Skinner and Rhodotorula glutinis (Fresen.) Harrison, were investigated after being treated with rapid-freezing, slow-freezing and freeze-drying, respectively. The accumulation of intracellular trehalose in the two yeasts was induced by culturing the yeast cells in trehalose-containing medium, which significantly enhanced viabilities of both yeasts in the slow-freezing test. Trehalose, as an exogenous protectant, at the concentration of 5% or 10% could markedly increase survivals of the two yeasts when subjected to freeze-drying. When combined with exogenous trehalose as a protective substance, the yeasts containing high intracellular trehalose level showed higher viabilities as compared to those containing low levels under both freezing and freeze-drying stresses. The highest survival of C. laurentii and R. glutinis were 90% and 97% after freeze-drying, respectively, compared to 63% and 28% for the yeasts with lower intracellular trehalose levels. These results may be due to the fact that a combined effect occurred between endo- and exogenous trehalose of yeast cells. The combined effect on C. laurentii and R. glutinis also resulted in the highest level of biocontrol efficacy against blue mold in apple fruit caused by Penicillium expansum Link, and reduced the disease indexes to 45 and 56, respectively, compared to 94 and 81 in the untreated control. Meanwhile, the combination of endo- and exogenous trehalose significantly increased population of both yeasts in apple wounds, especially at the first 48 h after inoculation, which might explain the reason of the improvement in biocontrol effects of the two yeasts.     

Yeast viability related to water potential variation: influence of the transient phase

The decrease rate of the water potential was found to have a great effect on yeasts submitted to hypertonic shifts. The application of slow and linear decreases of the water potential of the medium to cells of Saccharomyces cerevisiae demonstrated that the cells could survive (90 to 100% of viability) very low levels of water potential (=–101 MPa). Resistance of the cells was examined through viability measurements and cell volume changes. The kinetics of cell volume variation were measured continuously using an on-line image analysis system coupled to a microscope. No biological response of the cells occurred, due to the lack of a usable carbon-energy source in the medium. The viability level was found to be a function of the water exit flow rate from the cells. The denaturation of the membrane structure was assumed to be involved in such phenomena.     

Recent Developments in the Zymomonas Process for Ethanol Production

Abstract

The bacterium Zymomonas mobilis, which is used in the tropics to make pulque and alcoholic palm wines, appears to have considerable potential for industrial alcohol fermentations. Some of the advantages of the Zymomonas process reported in studies from our laboratory^1-24 are

1. There are significantly higher specific rates of sugar uptake and ethanol production compared to those found for yeasts.

2. Considerably higher volumetric ethanol productivities found in continuous cell recycle systems (up to 120 to 200 g/hr).

3. There are higher ethanol yields and lower biomass production than for yeasts. The lower biomass concentrations would seem to be a consequence of the lower metabolic energy available for growth. Zymomonas metabolize glucose via the Entner-Doudoroff pathway while yeasts convert glucose to ethanol via glycolysis.

4. Zymomonas cultures grow anaerobically and, unlike yeasts, do not require the controlled addition of oxygen to maintain viability at high cell concentrations.

5. The ethanol tolerance of some selected strains of Zymomonas is comparable if not higher than strains of Saccharomyces cerevisiae. Ethanol concentrations of 85 g/(up to 11% v/v) have been achieved in continuous culture and up to 130 g/(16% v/v) in batch culture.     

Effects of live Saccharomyces cerevisiae cells on zoospore germination,growth, and cellulolytic activity of the rumen anaerobic fungus,Neocallimastix frontalis MCH3

The effects of a live yeast strain of Saccharomyces cerevisiae have been investigated on zoospore germination, metabolism, and cellulolytic activity of the anaerobic rumen fungus Neocallimastix frontalis MCH3. The addition of yeast cells to a vitamin-deficient medium stimulated the germination of fungal zoospores, increased cellulose degradation and hydrogen, formate, lactate, and acetate production. Responses depended on the concentration of yeast cells added and on their viability. Yeast supplementation provided vitamins such as thiamine, which is essential for fungal growth and activity. These results demonstrate that yeasts could enhance plant cell wall colonization by N. frontalis. With certain diets, yeasts could therefore be a good tool to optimize the microbial degradation of lignocellulosic materials, but more research is needed to understand their mechanisms of action, so that they can be used with maximum efficiency as feed supplements.     

Electrophoretic karyotyping ofCandida albicans strains isolated from premature infants and hospital personnel in a neonatal intensive care unit

Electrophoretic karyotyping was used to compare DNA probes of yeasts isolated from blood of preterm neonates (n=66) in a neonatal intensive care unit (NICU) and from the hands of healthy hospital personnel (n=10). The yeasts were identified asCandida albicans using standard laboratory methods. DNA was extracted from yeasts and isolation of identical DNA strains from the pairs nurse-neonate suggested that one nurse transmitted one yeast strain by her hands to three neonates. Four neonates harbored two identical strains originating from two nurses,i.e. each nurse transmitted the same strain to two neonates. In the additional 7 cases transmission of 1 yeast strain by 1 nurse, to 1 neonate was observed. Our data suggest that nonperinatal nosocomial transmission ofC. albicans occurs in neonates. possiblyvia cross-contamination being transferred on hands of health care workers The importance of careful hand washing of staff (health care workers) and other infectioncontrol procedures (to prevent the nosocomial transmission of pathogens in the NICU environment) is emphasizeded.     

Yeasts and filamentous fungi carried by the gynes of leaf-cutting ants

Insect-associated microbes exhibit a wide range of interactions with their hosts. One example of such interactions is the insect-driven dispersal of microorganisms, which plays an essential role in the ecology of several microbes. To study dispersal of microorganisms by leaf-cutting ants (Formicidae: Attini), we applied culture-dependent methods to identify the filamentous fungi and yeasts found in two different body parts of leaf-cutting ant gynes: the exoskeleton and the infrabuccal pocket. The gynes use the latter structure to store a pellet of the ants’ symbiotic fungus during nest founding. Many filamentous fungi (n = 142) and yeasts (n = 19) were isolated from the gynes’ exoskeleton. In contrast, only seven filamentous fungi and three yeasts isolates were recovered from the infrabuccal pellets, suggesting an efficient mechanism utilized by the gynes to prevent contamination of the symbiotic fungus inoculum. The genus Cladosporium prevailed (78%) among filamentous fungi whereas Aureobasidium, Candida and Cryptococcus prevailed among yeasts associated with gynes. Interestingly, Escovopsis, a specialized fungal pathogen of the leaf-cutting ant-fungus symbiosis, was not isolated from the body parts or from infrabuccal pellets of any gynes sampled. Our results suggest that gynes of the leaf-cutter ants Atta laevigata and A. capiguara do not vertically transmit any particular species of yeasts or filamentous fungi during the foundation of a new nest. Instead, fungi found in association with gynes have a cosmopolitan distribution, suggesting they are probably acquired from the environment and passively dispersed during nest foundation. The possible role of these fungi for the attine ant–microbial symbiosis is discussed.     

The shelf life and effectiveness of granular formulations of Metschnikowia pulcherrima and Pichia guilliermondii yeast isolates that control postharvest decay of citrus fruit

Our overall objectives were to prepare commercially acceptable formulations of the postharvest biological control yeasts, Metschnikowia pulcherrima and Pichia guilliermondii, which have a long storage life and to determine the effectiveness of these formulations to control postharvest green and blue moulds on citrus fruit. Yeasts, grown on a cane molasses-based medium, were combined with talc or kaolin carriers and various adjuvants and the viability of yeast in 12 formulations was determined over a 6 month period. Formulation no. 11, containing talc, sodium alginate, sucrose, and yeast extract, for both yeasts had a significantly higher viable yeast cell content over a 6 month storage period. Among the formulations, three formulations (formulations no. 5, 6, and 11) were selected for additional in vivo testing because they had higher levels of viability amongst yeast cell populations during storage and were easier to resuspend remained in suspension more easily. These formulations were tested on Satsuma mandarin and grapefruit to control green and blue moulds. Formulations no. 5, 6, and 11 for both yeasts effectively controlled green mould, while only formulation no. 11 with either yeast isolate M. pulcherrima (isolate M1/1) or P. guilliermondii (isolate P1/3) effectively controlled both blue and green moulds.     

Two new species of the genus Candida in the Zygoascus clade, Candida lundiana sp. nov. and Candida suthepensis sp. nov., isolated from raw honey in Thailand

During a survey of yeasts associated with raw honey collected in Thailand, two strains of the Zygoascus clade were isolated from the Asian cavity-nesting honeybee Apis cerana and the stingless bee Homotrigona fimbriata. Phylogeny based on 26S rDNA D1/D2 sequences placed these yeasts as members of a clade including Candida bituminiphila, Candida patagonica and Candida polysorbophila. The strains of the two novel species, CBS 12271^T and CBS 12270^T, respectively, could be unquestionably distinguished from their relatives by rDNA sequences and other taxonomic characteristics. Therefore, the novel anamorphic species, Candida lundiana sp. nov. (type strain CBS 12271^T = JCM 16823^T) and Candida suthepensis sp. nov. (type strain CBS 12270^T = JCM 16822^T) are described.     

Differential response to UV stress and DNA damage during the yeast replicative life span

The yeast Saccharomyces cerevisiae is mortal. Before they die, individual yeasts bud repeatedly producing a finite number of progeny, which have the capacity for a full life span. A feature of aging in many species is the waning of resistance to stress. To determine whether this is the case in yeast, we have examined the survival (viability) of age-synchronized populations of yeasts of various ages, spanning youth, midlife, and old age, after irradiation with ultraviolet light (UV). Resistance to UV was biphasic. There was an increase through midlife, followed by a precipitous decline. For comparison, another mutagenic agent, ethyl methanesulfonate (EMS), was tested in the same way. The response was very different. A uniphase decrease in resistance to this DNA-alkylating agent was found with a plateau later in life. The results argue that the increase in resistance to UV with age is an active process and not simply a monotonic age change. RAS2 is among the genes that determine yeast longevity. This gene is preferentially expressed in young cells and has a life span-extending effect on yeasts. One known function of RAS2 is to mount a protective response to irradiation by UV, which occurs independently of DNA damage. The distinction between UV and EMS found here is consistent with the notion that resistance to UV plays a role in yeast longevity in a manner not related to DNA damage. Furthermore, it suggests that RAS2 may participate in this response. We have found that RAS2 expression and UV resistance coincide in middle-aged yeasts bolstering this possibility. These data and the eclipse in activity of several longevity determining genes at midlife in yeasts also raise the possibility that active life maintenance processes function through this period, after which the organism operates on any remaining reserves until death. © 1996 Wiley-Liss, Inc.     

Yeast communities in Sphagnum phyllosphere along the temperature-moisture ecocline in the boreal forest-swamp ecosystem and description of Candida sphagnicola sp. nov.

The effects of the temperature-moisture factors on the phylloplane yeast communities inhabiting Sphagnum mosses were studied along the transition from a boreal forest to a swamp biotope at the Central Forest State Biosphere Reserve (Tver region, Russia). We tested the hypothesis that microclimatic parameters affect yeast community composition and structure even on a rather small spatial scale. Using a conventional plating technique we isolated and identified by molecular methods a total of 15 species of yeasts. Total yeast counts and species richness values did not depend on environmental factors, although yeast community composition and structure did. On average, Sphagnum in the swamp biotope supported a more evenly structured yeast community. Relative abundance of ascomycetous yeasts was significantly higher on swamp moss. Rhodotorula mucilaginosa dominated in the spruce forest and Cryptococcus magnus was more abundant in the swamp. Our study confirmed the low occurrence of tremellaceous yeasts in the Sphagnum phyllosphere. Of the few isolated ascomycetous yeast and yeast-like species, some were differentiated from hitherto known species in physiological tests and phylogenetic analyses. We describe one of them as Candida sphagnicola and designate KBP Y-3887^T (=CBS 11774^T = VKPM Y-3566^T = MUCL 53590^T) as the type strain. The new species was registered in MycoBank under MB 563443.     

Selenium Tolerance of Yeasts

Selenium tolerance of yeasts widely varies: the growth of some yeasts can be inhibited by a selenium concentration as low as 10^–4 M, whereas others can grow in the presence of 10^–1 M selenium. Homogeneous yeast taxa are characterized by a certain level of selenium tolerance, and heterogeneous taxa show a variable level of tolerance to selenium. In general, ascomycetous yeasts are more tolerant to selenium than basidiomycetous yeasts. Among the ascomycetous yeasts, the genera Dekkera and Schizosaccharomyces exhibited the lowest and the species Candida maltosa, Hanseniaspora valbyensis, Kluyveromyces marxianus, and Yarrowia lipolytica the highest tolerance to selenium. Among the basidiomycetous yeasts, the genera Bullera, Cryptococcusand Holtermannia showed the lowest and the species Cryptococcus curvatus, Cr. humicola, and Trichosporon spp. the highest tolerance to selenium. The selenium tolerance of yeasts depends on the composition of the growth medium, in particular, on the presence of sulfate, sulfur-containing amino acids, and glutamine in the medium.     

Effect of rate of inbreeding on inbreeding depression in Drosophila melanogaster

Summary This experiment was designed to study the relationship between rate of inbreeding and observed inbreeding depression of larval viability, adult fecundity and cold shock mortality in Drosophila melanogaster. Rates of inbreeding used were full-sib mating and closed lines of N=4 and N=20. Eight generations of mating in the N=20 lines, three generations in the N=4 lines and one generation of full-sib mating were synchronised to simultaneously produce individuals with an expected level of inbreeding coefficient (F) of approximately 0.25. Inbreeding depression for the three traits was significant at F=0.25. N=20 lines showed significantly less inbreeding depression than full-sib mated lines for larval viability at approximately the same level of F. A similar trend was observed for fecundity. No effect of rate of inbreeding depression was found for cold shock mortality, but this trait was measured with less precision than the other two. Natural selection acting on loci influencing larval viability and fecundity during the process of inbreeding could explain these results. Selection is expected to be more effective with slow rates of inbreeding because there are more generations and greater opportunity for selection to act before F=0.25 is reached. Selection intensities seem to have been different in the three traits measured. Selection was most intense for larval viability, less intense for fecundity and, perhaps, negligible at loci influencing cold shock mortality.     

Occurrence and Diversity of Yeasts in the Mid-Atlantic Ridge Hydrothermal Fields Near the Azores Archipelago

The yeast community associated with deep-sea hydrothermal systems of the Mid-Atlantic Rift was surveyed for the first time. This study relied on a culture-based approach using two different growth media: a conventional culture medium for yeasts supplemented with sea salts (MYPss) and the same medium additionally supplemented with sulfur (MYPssS). For the evaluation of species diversity, a molecular approach involving minisatellite-primed polymerase chain reaction (MSP-PCR) strain typing and sequence analysis of the D1/D2 domains of the 26S rDNA was followed. In the seven water samples that were studied, the number of colony-forming units per liter (cfu/L) ranged from 0 to 5940. The nonpigmented yeasts were much more abundant than the pink-pigmented ones. This disproportion was not observed in studies of other marine systems and may be due to the unique conditions of hydrothermal vents, characterized by a rich animal and microbial diversity and therefore by the availability of organic compounds utilizable by yeasts. Higher counts of nonpigmented yeast were obtained using MYPss, whereas for pink yeasts, higher counts were obtained using MYPssS. Moreover, among pink yeasts, some of the MSP-PCR classes obtained were composed of isolates obtained only on MYPssS, which might be an indication that these isolates are adapted to the ecosystems of the hydrothermal vents. Twelve phylotypes belonged to the Ascomycota and seven phylotypes belonged to the Basidiomycota. The nonpigmented yeasts were identified as Candida atlantica, C. atmosphaerica, C. lodderae, C. parapsilosis, Exophiala dermatitidis, Pichia guilliermondii, and Trichosporon dermatis, whereas the pigmented yeasts were identified as Rhodosporidium diobovatum, R. sphaerocarpum, R. toruloides, and Rhodotorula mucilaginosa. Some of the yeasts that were found belong to phylogenetic groups that include species reported from other marine environments, and eight phylotypes represent undescribed species. The new phylotypes found at Mid-Atlantic Ridge hydrothermal fields represent 33% of the total number of yeast taxa that were found.     

Inhibition of Virulence Factors of <Emphasis Type="Italic">Candida</Emphasis> spp. by Different Surfactants

Candida yeasts are opportunistic pathogens responsible for infections in immunocompromised individuals. Among the virulence factors present in these yeasts we can mention the ability to adhere to host cells, exoenzyme production and germ tube formation. Several compounds, such as antifungal agents, plants extracts, protein inhibitors and surfactants, have been tested regarding their capacity in inhibit Candida spp. virulence factors. Among these compounds, a significant lower number of works are focused on the inhibition action caused by different types of surfactant. The present work aimed to evaluate the effect generated by the surfactants cetyltrimethylammonium chloride (CTAC), sodium dodecyl sulfate (SDS), N-hexadecyl-N–N′-dimethyl-3-ammonio-1-propane-sulfonate (HPS) and octylphenoxypolyethoxyethanol (Triton X-100) on the viability, adhesion ability and exoenzyme production by Candida species. CTAC and HPS were capable to inhibit Candida spp. growth at very low concentrations. All surfactants demonstrated to be capable to inhibit the adhesion of Candida species to buccal epithelial cells (BEC) and the proteinase production. On the other hand, the phospholipase production remained unaltered after the treatment with these compounds. The present data denote that cationic and zwitterionic surfactants are interesting prototypes of inhibitory agents against Candida spp., which is probably associated with the cationic punctual charge of both surfactants. The results are discussed in details in agreement with recent reports from literature.     

Isolation and characterization of rat primary lung cells

Summary Lung cell culture may be useful as anin vitro alternative to study the susceptibility of the lung to various toxic agents. Lungs from female Wistar rats were enzymatically digested by recirculating perfusion through the pulmonary artery with a sequence of solutions containing deoxyribonuclease, chymopapain, pronase, collagenase, and elastase. Lung tissue was microdissected and resuspended and the cells obtained were washed by centrifugation. By this isolation method, 2×10⁸ cells per rat lung were obtained with an average viability of 97%. Lung cells cultured in medium containing antibiotics and serum maintained a viability of >70% for 5 d. Rat primary lung cells were exposed to various toxic agents and their viability was assessed by formazan production capacity after 18 h of incubation. Compared to rat and mouse hepatocyte cultures (EC⁵⁰=5.8 mM), rat primary lung cells were much more susceptible to hydrogen peroxide (EC₅₀=0.6 mM). All cell types were equally sensitive to the more potent toxicanttert-butylhydroperoxide (EC₅₀=0.1 mM). Paraquat was more toxic to lung cells (EC₅₀=0.03 mM) than to rat (EC₅₀=2.8 mM) and mouse (EC₅₀=0.2 mM) hepatocytes. In contrast, rat lung cells were less sensitive to sodium nitroprusside (EC₅₀=2.6 mM) compared to rat (EC₅₀=0.2 mM) and mouse (EC₅₀=0.03 mM) hepatocytes. Nitrofurantoin and menadione (at EC₅₀=0.04 mM and 0.006 mM, respectively) were more toxic to rat lung and liver cells than to murine hepatocytes (EC₅₀=0.2 mM and 0.04 mM, respectively). Our findings demonstrate the applicability of this rat primary lung cell culture for studying the effects of lung toxicants. Parts of the study had been presented orally at the meeting of the German Society of Toxicology and Pharmacology in Mainz (FRG), March 15–17, 1994.