β-Glucanases as a Tool for the Control of Wine Spoilage Yeasts

ABSTRACT:  In this study, the antimicrobial activity of a commercial β-glucanase preparation against wine spoilage yeasts such as  Cryptococcus albidus ,  Dekkera bruxellensis ,  Pichia membranifaciens ,  Saccharomyces cerevisiae ,  Zygosaccharomyces bailii , and  Zygosaccharomyces bisporus  has been evaluated. Yeast species tested showed different sensitivities to the enzyme preparation.  In vitro  assays in laboratory medium (GPY) showed inhibition by the β-glucanase preparation of  D. bruxellensis  and  Z. bailii  growth with IC₅₀ and MIC values approximately 3 to 4-fold greater than the recommended dose for improving wine filtration. Wine spoilage experiments showed antimicrobial action against  D. bruxellensis  and  Z. bailii  although with reduced effectiveness to that showed in laboratory medium. Under the conditions tested, the addition of β-glucanase did not affect wine enological parameters. Our data suggest the potential use of β-glucanases as antimicrobial agents in wine and indicate that the application of antimicrobial enzymes for yeast spoilage control deserves further investigation.     

Inhibition of the wine spoilage yeast Dekkera bruxellensis by bovine lactoferrin-derived peptides

The antimicrobial action of lactoferrin (LF)-derived peptides against Dekkera bruxellensis strains isolated from spoiled wines has been examined. The study included a fifteen-residue peptide (LfcinB(17-31)) derived from bovine lactoferricin B and a bovine LF pepsin hydrolysate (LFH). In vitro assays showed the inhibitory properties of LfcinB(17-31) on D. bruxellensis growth with IC(50) and MIC values in the micromolar range. Strains tested showed different sensitivity to the peptide. LfcinB(17-31) showed fungicidal properties towards all strains tested in laboratory growth medium. However, the extent of fungicidal activity was strain-dependent in must and wine, confirming the different antimicrobial action of peptides depending on both the food matrix and the target micro-organism. The binding of LfcinB(17-31) to D. bruxellensis cells was visualized by fluorescence microscopy and correlated with the fungicidal activity in the different matrixes. LfcinB(17-31) and LFH showed growth inhibitory properties in wine suggesting their potential use for spoilage control.     

Molecular cloning and sequence analysis of the Zygosaccharomyces bailii HIS3 gene encoding the imidazole glycerolphosphate dehydratase

Zygosaccharomyces bailii is a spoilage yeast belonging to the Zygosaccharomyces genus. In recent years these yeasts, due to their exceptional resistance to several stresses, have become more and more interesting as model organisms to study the molecular basis of the said resistance. A Z. bailii cDNA library has been built and the 672 bp nucleotide sequence coding for the HIS3 gene was cloned by complementation of a Saccharomyces cerevisiae his3 mutant strain. The deduced 223 amino acid sequence shares a high degree of homology with His3p homologues in other non-conventional yeast species. The GeneBank Accession No. is AY050224.     

Physiological characterization of spoilage strains of Zygosaccharomyces bailii and Zygosaccharomyces rouxii isolated from high sugar environments

Two isolates of spoilage yeasts Zygosaccharomyces bailii and Zygosaccharomyces rouxii were obtained from a high sugar environment, in a factory producing candied fruit and nougat. Other strains, Z. bailii from other environments and other isolates from high sugar/salt environments were obtained for comparison (Zygosaccharomyces lentus, Candida magnoliae, Candida halophila and Pichia guilliermondii). A full physiological assessment of these isolates was carried out, of preservative and biocide resistance, osmotolerance, ethanol-tolerance, low pH resistance, degree of fermentation, and growth temperature and survival to pasteurisation. Results showed that the strains isolated from high sugar environments did not show extreme physiology. These were robust strains but within the normal parameters expected for the species. One exception to this was that the Z. bailii strains were abnormally able to grow at 37 degrees C. In all strains other than C. magnolia and C. halophila, cells were able to adapt to high levels of sugar. Cultures grown in high glucose concentrations were subsequently able to tolerate higher concentrations of glucose than previously. Similarly, high sugar was found to confer a degree of protection against pasteurisation, enabling survival in what would have otherwise been a lethal treatment. Isolates of Z. bailii showed a high level of resistance to preservatives such as sorbic acid, benzoic acid, acetic acid, cinnamic acid, and ethanol, and also to heat. Unexpectedly Z. bailii isolates were not exceptionally resistant to biocides such as peracetic acid, or hypochlorite. These results indicate that spoilage by yeasts such as Z. bailii may be better prevented by use of biocidal cleaning agents in the factory, rather than treating the food with preservatives.     

Antifungal activity of octyl gallate

Antifungal activities of propyl (C3), octyl (C8) and dodecyl (C12) gallates (3,4,5-trihydroxybenzoate) were tested against Saccharomyces cerevisiae ATCC7754 and Zygosaccharomyces bailii ATCC 60483. Octyl gallate was found to be the only active compound with the minimum fungicidal concentration of 25 microg/ml (89 microM) against S. cerevisiae and of 50 microg/ml (177 microM) against Z. bailii, respectively. The inactivation study showed that octyl gallate was fungicidal against both S. cerevisiae and Z. bailii at any stage of growth. These fungicidal activities were not influenced by pH values. Octyl gallate at 100 microg /ml reduced plasma membrane fluidity to 48% of control. On the other hand, dodecyl gallate at the same concentration reduced it to 76% of control. Only octyl gallate inhibited glucose-induced medium acidification, indicating direct or indirect inhibition of plasma membrane H +-ATPase. The primary fungicidal activity of octyl gallate comes from its ability to act as a nonionic surface-active agent (surfactant), though it can not be inferred that membrane damage, such as a decrease in the membrane fluidity, is the only cause of the lethal effect.     

Analysis of the inhibition of food spoilage yeasts by vanillin

The antimicrobial potential of vanillin, the major component of vanilla flavour, was examined against the growth of three yeasts associated with food spoilage, Saccharomyces cerevisiae, Zygosaccharomyces bailii and Zygosaccharomyces rouxii. Minimum inhibitory concentration (MIC) values of 21, 20 and 13 mM vanillin were determined for the three yeast strains, respectively. The observed inhibition was found to be biostatic. During fermentation, the bioconversion of sub-MIC levels of vanillin in the culture medium was demonstrated. The major bioconversion product was identified as vanillyl alcohol, however low levels of vanillic acid were also detected. Neither the vanillyl alcohol nor the vanillic acid was found to be antagonistic to yeast cell growth. The results indicate the importance of the aldehyde moiety in the vanillin structure regarding its antimicrobial activity and that the bioconversion of vanillin could be advantageous for the yeasts, but only at levels below MIC. These bioconversion activities, presumably catalysed by non-specific dehydrogenases, were shown to be expressed constitutively. It was observed that increased vanillin concentrations inhibited its own bioconversion suggesting that the activity required intact cells with metabolic capacity.     

Molecular monitoring of spoilage yeasts during the production of candied fruit nougats to determine food contamination sources

In the present work, we have analysed the yeast microbiota present in a manufacturing plant of candied fruits and nougats. Four yeasts species (Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Sporobolomyces roseus, and Debaryomyces hansenii) and a filamentous fungi (Nectria mauriiticola) were identified according to restriction analysis of 5.8S-ITS rDNA. These identifications were subsequently confirmed by sequencing the D1/D2 domain of the 26S rRNA gene. Z. rouxii and Z. bailii were isolated at high frequency along the whole manufacturing process. Since food alteration by Z. bailii and Z. rouxii is the cause of important economic losses for the food industry, there is a need for differentiating yeasts at the strain level as an essential part of quality control programs in this industry. For this purpose, we have tested the performance of three molecular techniques (RFLP mtDNA, RAPD-PCR, and microsatellite with (GAC)5 and (GTG)5 primers) to differentiate strains belonging to these two Zygosaccharomyces species. Those techniques with the best discriminatory power were applied to differentiate Zygosaccharomyces species isolates. The results of this analysis indicate that one strain of Z. bailii and two strains of Z. rouxii were involved in the spoilage of candied fruits. Moreover, the Z. bailii strain was also present in the spoiled nougat, hence being responsible of this alteration.     

Potential of using real-time PCR-based detection of spoilage yeast in fruit juice--a preliminary study

A real-time PCR system was used to differentiate between the common spoilage yeasts, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Candida krusei, Rhodotorula glutinis and Saccharomyces cerevisiae, based on melting peak Tm analysis of the 5.8S rDNA subunit and the adjacent ITS2 region of these yeasts. By using the real-time PCR system and by targeting the citrate synthase (cs 1) gene of C. krusei, it was possible to develop a sensitive detection system to both identify and quantitate the level of C. krusei growth in an artificially contaminated apple juice sample.     

Activity of essential oils from Mediterranean Lamiaceae species against food spoilage yeasts

The essential oils from aerial parts of Melissa officinalis, Lavandula angustifolia, Salvia officinalis, and Mentha piperita were analyzed by gas chromatography and gas chromatography-mass spectrometry. Their antimicrobial activities were evaluated against five food spoilage yeasts, Torulaspora delbrueckii, Zygosaccharomyces bailii, Pichia membranifaciens, Dekkera anomala, and Yarrowia lipolytica. Saccharomyces cerevisiae was also used as a reference. The oils were preliminarily screened by a disc diffusion technique, with the most active being the oil from M. officinalis. MICs were determined by the broth dilution method, and the main components of the oils were also tested by this method. The essential oil of M. officinalis at 500 microg/ml completely inhibited the growth of all yeast species. The main component of the oil of M. officinalis is citral (neral plus geranial) (58.3%), which showed a marked fungitoxic effect, contributing to its high activity.     

Sorbic acid resistance: the inoculum effect

Zygosaccharomyces is a genus associated with the more extreme spoilage yeasts. Zygosaccharomyces spoilage yeasts are osmotolerant, fructophiles (preferring fructose), highly-fermentative and extremely preservative-resistant. Zygosaccharomyces bailii can grow in the presence of commonly-used food preservatives, benzoic, acetic or sorbic acids, at concentrations far higher than are legally permitted or organolepically acceptable in foods. An inoculum effect has been described for many micro-organisms and antimicrobial agents. The minimum inhibitory concentration (MIC) increases with the size of the inoculum; large inocula at high cell density therefore require considerably higher concentrations of inhibitors to prevent growth than do dilute cell suspensions. A substantial inoculum effect was found using sorbic acid against the spoilage yeast Zygosaccharomyces bailii NCYC 1766. The inoculum effect was not caused by yeasts metabolizing or adsorbing sorbic acid, thereby lowering the effective concentration; was not due to absence of cell-cell signals in dilute cell suspensions; and was not an artefact, generated by insufficient time for small inocula to grow. The inoculum effect appeared to be caused by diversity in the populations of yeast cells, with higher probability of sorbic acid-resistant cells being present in large inocula. It was found that individual cells of Zygosaccharomyces bailii populations, grown as single cells in microtitre plate wells, were very diverse, varying enormously in resistance to sorbic acid. 26S ribosomal DNA sequencing did not detect differences between the small fraction of resistant 'super cells' and the average population. Re-inoculation of the 'super cells' after overnight growth on YEPD showed a normal distribution of resistance to sorbic acid, similar to that of the original population. The resistance phenotype was therefore not heritable and not caused by a genetically distinct subpopulation. It was concluded that resistance of the spoilage yeast Zygosaccharomyces bailii to sorbic acid was due to the presence of small numbers of phenotypically resistant cells in the population.     

The effect of citric acid and pH on growth and metabolism of anaerobic Saccharomyces cerevisiae and Zygosaccharomyces bailii cultures

The effects of citric acid at pH values of 3.0, 4.0, and 4.5 on growth and metabolism of anaerobic Saccharomyces cerevisiae and Zygosaccharomyces bailii cultures were investigated. S. cerevisiae and Z. bailii exhibited similar tolerances to citric acid, as determined by growth measurements, at all three pH values investigated. The citric-acid-induced growth inhibition of both yeast species increased with increasing pH values, indicating that the antimicrobial mechanism of citric acid differs from that of classical weak-acid preservatives. In S. cerevisiae, citric acid shifted the primary energy metabolism towards lower ethanol production and higher glycerol production, thus resulting in lower ATP production. These metabolic changes in S. cerevisiae were pH-dependent; i.e. the higher the pH, the lower the ATP production, and they may explain why growth of S. cerevisiae is more inhibited by citric acid at higher pH values. In Z. bailii, citric acid also caused an increased glycerol production, although to a lesser extent than in S. cerevisiae, but it caused virtually no changes in ethanol and ATP production.     

Application of proanthocyanidins from peanut skins as a natural yeast inhibitory agent

Proanthocyanidins were extracted from peanut skins and investigated for their antimicrobial activity against Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Zygosaccharomyces bisporus in traditional growth media (Sabouraud Dextrose and Maltose broth) and a simulated apple juice beverage. Peanut skins extracts (PSE) were prepared through a multisolvent extraction procedure. The PSE extended the lag phase growth of the 3 yeasts studied at a concentration of 1 mg/mL and at 10 mg/mL yeast growth was totally inhibited for 120 h. PSE was fractionated by normal phase high performance liquid chromatography and the active components/fractions were determined. Compounds present in the fractions were identified by liquid chromatography-mass spectrometry to determine the compounds responsible for inhibition. Fractions consisting mostly of A-type proanthocyanidin dimers, trimers, and tetramers showed the highest percent inhibition toward the yeasts tested in this study. Both optical density (OD) and standard enumeration plating methods were performed in this study. The OD method led to an overestimation of the inhibitory effects of PSE, the 2 methods agreed in respect to treatment effects but not the severity of the inhibition. PRACTICAL APPLICATION: There is a growing consumer demand for "fresh like" products containing reduced amounts of chemical preservatives without compromising food safety and quality. Therefore, the goal of this study was to determine if an extract of peanut skins containing flavonoid rich compounds could function as a natural antimicrobial in a model beverage system. Proteins were removed through the process of producing the peanut skin extract, thus it is unlikely to contain peanut allergens. The antimicrobial compounds mentioned in this study were successfully integrated into a model beverage system, and were found to have antimicrobial effect. However, the incorporation of these compounds would likely lead to negative sensory attributes at the concentration needed to achieve an appreciable antimicrobial effect alone.     

Yeasts associated to Traditional Balsamic Vinegar: ecological and technological features

Traditional Balsamic Vinegar (TBV) is an Italian homemade vinegar made with cooked grape must through a three-step process: conversion of sugars to ethanol by naturally occurring yeasts; oxidation of ethanol to acetic acid by acetic acid bacteria (AAB); and, finally, at least 12-years ageing. The cooked must is a selective and stressful medium for yeasts growth, due to its high sugar content and low pH values. Recent studies have shown that a large number of yeast species are involved in the fermentation, among them there are Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Zygosaccharomyces pseudorouxii, Zygosaccharomyces mellis, Zygosaccharomyces bisporus, Zygosaccharomyces lentus, Hanseniaspora valbyensis, Hanseniaspora osmophila, Candida lactis-condensi, Candida stellata, Saccharomycodes ludwigii and Saccharomyces cerevisiae. Nevertheless, the TBV-associated yeast population could be even more complex and many other slow-growing or poorly cultivable species might contribute to cooked must fermentation. In this review the main TBV yeast species are described, pointing out their role in TBV production and their influence on final product quality. Finally, both future developments in TBV yeast community studies (culture-independent and metagenomic techniques) and technological advances in TBV making (use of starter culture) are discussed.     

Potassium transport at the plasma membrane of the food spoilage yeast Zygosaccharomyces bailii

Zygosaccharomyces bailii is a commercially important spoilage yeast capable of growth at low pH in the presence of weak organic acid preservatives, such as benzoic acid. A patch-clamp electrophysiological analysis of plasma membrane K+ transport revealed a high conductance pathway for low-affinity K+ uptake. In contrast to the equivalent K+ transporter in Saccharomyces cerevisiae, this system remained operative at low extracellular pH and may therefore facilitate K+ uptake in K(+)-rich and acidic beverages. Benzoate inhibited growth, increased intracellular K+ content, yet decreased the magnitude of the K+ uptake conductance; specifically, the hyperpolarization-activated inwardly-rectifying component was reduced. It is proposed that this adaptation helps maintain a hyperpolarized membrane voltage to effect continued ATPase-mediated H+ extrusion and so combat preservative-induced cytosolic acidosis. Again in contrast to S. cerevisiae, the K+ conductance was relatively insensitive to increased extracellular Ca2+. Paradoxically (and unlike S. cerevisiae) increasing extracellular Ca2+ inhibited growth, suggesting a simple expedient to limit spoilage by Z. bailii.     

Targeted gene deletion in Zygosaccharomyces bailii

Yeasts of the genus Zygosaccharomyces are notable agents of large-scale food spoilage. Despite the economic importance of these organisms, little is known about the stress adaptations whereby they adapt to many of the more severe conditions of food preservation. In this study it was shown that genes of Z. bailii, a yeast notable for its high resistances to food preservatives and ethanol, can be isolated by complementation of the corresponding mutant strains of Saccharomyces cerevisiae. It was also discovered that the acquisition by S. cerevisiae of a single small Z. bailii gene (ZbYME2) was sufficient for the former yeast to acquire the ability to degrade two major food preservatives, benzoic acid and sorbic acid. Using DNA cassettes containing dominant selectable markers and methods originally developed for performing gene deletions in S. cerevisiae, the two copies of ZbYME2 in the Z. bailii genome were sequentially deleted. The resulting Zbyme2/Zbyme2 homozygous deletant strain had lost any ability to utilize benzoate as sole carbon source and was more sensitive to weak acid preservatives during growth on glucose. Thus, ZbYME2, probably the nuclear gene for a mitochondrial mono-oxygenase function, is essential for Z. bailii to degrade food preservatives. This ability to catabolize weak acid preservatives is a significant factor contributing to the preservative resistance of Z. bailii under aerobic conditions. This study is the first to demonstrate that it is possible to delete in Z. bailii genes that are suspected as being important for growth of this organism in preserved foods and beverages. With the construction of further mutant of Z. bailii strains, a clearer picture should emerge of how this yeast adapts to the conditions of food preservation. This information will, in turn, allow the design of new preservation strategies. GenBank Accession Nos: ZbURA3 (AF279259), ZbTIM9 (AF279260), ZbYME2 (AF279261), ZbTRP1 (AF279262), ZbHHT1(AF296170).     

Comparison of the activity of antifungal hexapeptides and the fungicides thiabendazole and imazalil against postharvest fungal pathogens

In this study, we evaluated the activity of short antimicrobial peptides against different fungal isolates that cause postharvest decay of fresh fruits. The previously identified hexapeptides PAF19, PAF26 and LfcinB4-9 inhibited the in vitro growth of isolates from Penicillium digitatum and P. italicum, and from Alternaria and Geotrichum genera, being no active against Rhizopus, Mucor and Aspergillus. The results extend our previous observations on the specific and distinct activity profiles of this class of antifungal peptides. In addition, peptide activities were compared with that of two fungicides used for citrus fruit preservation, thiabendazole (TBZ) and imazalil (IMZ). We observed a lack of correlation between peptide and fungicide sensitivity among different species. Importantly, P. digitatum and P. italicum isolates resistant to fungicides were susceptible to peptides and our data suggest that common multiple drug resistance mechanisms are not active against this class of peptides. The in vitro peptide inhibition was correlated with a retard of the decay caused by Penicillium on citrus fruits, and this effect was comparable for both fungicide-resistant and -sensitive isolates. Comparison of PAF26 and TBZ in vitro minimum inhibitory concentration (MIC) values and their in vivo effect on citrus decay indicated that PAF26 performed in vivo better than TBZ.     

昌黎产区产酶酵母多样性及其应用潜力分析

利用优选酵母混菌发酵,是改善葡萄酒品质及增香的有效途径。以赖氨酸培养基、WL培养基和产酶筛选培养基为基础,分析了昌黎4个葡萄园土样中产酶酵母的多样性,结果表明,该地区产酶酵母丰富,50%的产酶酵母能同时产2种或2种以上的酶。对产酶酵母酿造因子耐性分析表明,菌株GY1、M9和J24具有高糖耐性,适合于高糖低醇果酒酿造;菌株GY13和M9具有高酒精耐性,酒精含量为15.000%时活性较强;菌株J24和GY1具有高酸耐性,pH 2.5时具有较高活性;7株菌15℃时具有较高活性。综合分析发现,菌株M9(Torulaspora delbrueckii)同时产β-葡萄糖苷酶、果胶酶和纤维素酶,除酸耐性较差外,具有高糖、高酒精度和低温耐性,在高糖低醇果酒酿造中具有很大应用潜力。     

Rapid identification of Saccharomyces cerevisiae, Zygosaccharomyces bailii and Zygosaccharomyces rouxii.

Most strains of Saccharomyces cerevisiae, Zygosaccharomyces bailii and Zygosaccharomyces rouxii have been found to contain plasmid DNA. The sequences of the plasmids from these three yeasts are known to be different. We have used two primers within the plasmid from each yeast species in a multiplex polymerase chain reaction (PCR) to discriminate between these three yeasts. The primers were designed to give easily distinguishable fragment sizes when run on a simple agarose gel. Due to the sensitivity of PCR, crude cells can be used with no need to isolate DNA. The method is rapid when compared with current methods.     

A differential medium for the enumeration of the spoilage yeast Zygosaccharomyces bailii in wine

A collection of yeasts, isolated mostly from spoiled wines, was used in order to develop a differential medium for Zygosaccharomyces bailii. The 118 selected strains of 21 species differed in their origin and resistance to preservatives and belonged to the genera Pichia, Torulaspora, Dekkera, Debaryomyces, Saccharomycodes, Issatchenkia, Kluyveromyces, Kloeckera, Lodderomyces, Schizosaccharomyces, Rhodotorula, Saccharomyces, and Zygosaccharomyces. The design of the culture medium was based on the different ability of the various yeast species to grow in a mineral medium with glucose and formic acid (mixed-substrate medium) as the only carbon and energy sources and supplemented with an acid-base indicator. By manipulating the concentration of the acid and the sugar it was possible to select conditions where only Z. bailii strains gave rise to alkalinization, associated with a color change of the medium (positive response). The final composition of the mixed medium was adjusted as a compromise between the percentage of recovery and selectivity for Z. bailii. This was accomplished by the use of pure or mixed cultures of the yeast strains and applying the membrane filtration methodology. The microbiological analysis of two samples of contaminated Vinho Verde showed that the new medium can be considered as a differential medium to distinguish Z. bailii from other contaminating yeasts, having potential application in the microbiological control of wines and probably other beverages and foods.     

The antifungal properties of chitosan in laboratory media and apple juice.

The antimicrobial properties of chitosan glutamate, a derivative of chitin, were investigated in laboratory media and apple juice against 15 yeasts and moulds associated with food spoilage in order to assess the potential for using chitosan as a natural food preservative. Of the seven strains of filamentous fungi studied, chitosan reduced the growth rate of Mucor racemosus at 1 g/l whilst concentrations of 5 g/l were required to completely prevent growth of three strains of Byssochlamys spp. on agar plates incubated at 25 degrees C for 3 weeks. Three strains of filamentous fungi were resistant to the antifungal effects of chitosan at 10 g/l. The presence of chitosan in apple juice (pH 3.4) at levels ranging from 0.1 to 5 g/l inhibited growth at 25 degrees C of all eight spoilage yeasts examined in this study. The initial effect of chitosan in apple juice was biocidal with viable numbers reduced by up to 3 log cycles. Following an extended lag phase, some strains recovered and resumed growth to levels similar to those observed in unsupplemented apple juice. The most sensitive strain was an isolate of Zygosaccharomyces bailii obtained from a spoiled carbonated beverage; this yeast was completely inactivated by chitosan at 0.1 and 0.4 g/l for 32 days of storage at 25 degrees C. The most resistant strain was Saccharomycodes ludwigii, an isolate from spoiled cider: a level of addition of 5 g/l of chitosan was required to inactivate this strain and to maintain yeast-free conditions in apple juice for 14 days at 25 degrees C. Growth inhibition and inactivation of filamentous moulds and yeasts, respectively, was concentration-, pH- and temperature-dependent. It was concluded that chitosan was worthy of further study as a natural preservative for foods prone to fungal spoilage.