Antimicrobial action of synthetic peptides towards wine spoilage yeasts

The antimicrobial action of selected short synthetic peptides against wine spoilage yeasts such as Cryptococcus albidus, Dekkera bruxellensis, Pichia membranifaciens, Saccharomyces cerevisiae, Zygosaccharomyces bailii and Zygosaccharomyces bisporus has been examined. Peptides analyzed include nine sequence-related antifungal hexapeptides (PAFs) previously developed by a combinatorial approach, and two representative lactoferricin B (LfcinB)-derived peptides. Different peptides had distinct activity profiles. In vitro assays identified the peptides PAF26, PAF36, and LfcinB(17-31), as having growth inhibitory properties towards several of the yeasts at low micromolar concentrations. Z. bailii and Z. bisporus were the most sensitive yeasts. In addition to their fungistatic activity, the three peptides showed fungicidal properties towards Z. bailii, Z. bisporus, and S. cerevisiae in laboratory growth medium. Remarkably, only LfcinB(17-31) against Z. bisporus had inhibitory and fungicidal properties in wine at the concentrations assayed, showing that the antimicrobial action of each peptide is dependent on both the food matrix and the target micro-organism. Lack of fungicidal activity of peptides against Z. bailii in wine is related to the presence of salt ions other than divalent cations. On the contrary, fungicidal activity of LfcinB(17-31) towards Z. bisporus was not significantly affected by wine salts. Our data identify a bioactive peptide from natural origin with potential use against the food spoilage yeast Z. bisporus, and indicate that the application of antimicrobial peptides in wine preservation 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 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.     

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.     

我国葡萄酒中布鲁塞尔酒香酵母的检测和鉴定

为快速且准确地检测葡萄酒中布鲁塞尔酒香酵母的存在情况,本研究比较了酒香酵母鉴别培养基（Dekkera bruxellensis differential medium,DBDM）和WLN（wallersteins laboratory nurtrient）培养基分离检测布鲁塞尔酒香酵母的效果,并利用特异聚合酶链式反应（polymerase chain reaction,PCR）扩增法对上述两种培养基的分离检测效果作了进一步验证。结果表明,上述两种培养基均能对葡萄酒中的布鲁塞尔酒香酵母进行分离鉴别,而DBDM培养基较少出现假阳性菌落,具有更好的选择性,结合特异PCR扩增可准确鉴定布鲁塞尔酒香酵母。此外,利用DBDM培养基结合特异PCR扩增法对国内5 个主要葡萄酒产区的酒样进行布鲁塞尔酒香酵母感染情况的调查发现,河北、山东和吉林的葡萄酒中存在布鲁塞尔酒香酵母感染的情况,尤其是在橡木桶中陈酿的葡萄酒。因此,国内葡萄酒行业应对酒香酵母感染问题足够重视。     

Survival patterns of Dekkera bruxellensis in wines and inhibitory effect of sulphur dioxide

The wine spoilage yeast species Dekkera bruxellensis, after inoculation in red wines, displayed three survival patterns characterized by: i) initial lag phase followed by growth and sequential death; ii) initial death phase leading to reduced viable counts followed by growth and sequential death; and iii) death phase leading to complete loss of viability. These survival patterns were observed for the same strain in different dry red wine blends with 12% (v/v) ethanol and pH 3.50, in the absence of free sulphur dioxide. For the same wine blend, these patterns also varied with the tested strain. Under laboratory conditions the addition of 150 mg/l of potassium metabisulphite (PMB) to dry red wine with 12% (v/v) ethanol and pH 3.50 reduced initial cell counts by more than 6 logarithmic cycles, inducing full death within less than 24 h. Winery trials showed that D. bruxellensis blooms were only prevented in the presence of about 40 mg/l of free sulphur dioxide in dry red wine, with 13.8% (v/v) ethanol and pH 3.42, matured in oak barrels. These different amounts of PMB and sulphur dioxide corresponded to about 1 mg/l of molecular sulphur dioxide. Our results therefore demonstrate that the control of populations of D. bruxellensis growing in red wine can only be achieved under the presence of relatively high doses of molecular sulphur dioxide.     

Mousy off-flavour production in grape juice and wine by Dekkera and Brettanomyces yeasts

The ability of four known species of Dekkera and Brettanomyces yeasts to produce mousy off-flavour in grape juice and wine was investigated for the first time. Using a sensory assessment technique the twelve type strains of Dekkera and Brettanomyces , representing four species, were found to grow and to be capable of producing mousy off-flavour in a grape juice medium; however, differences between strains were apparent. Four strains representing the two species of Dekkera (D. bruxellensis and D. anomala ) known to be associated with the spoilage of wine and other fermented beverages were further investigated for mousy off-flavour production in a red and white wine supplemented with nutrients. D. anomala and only one of the three D. bruxellensis strains tested grew in both wines and generally produced a moderate level of off-flavour, whereas the remaining two strains of D. bruxellensis , despite slowly losing viability (10- to 100-fold) over the 52–55 day period, produced detectable off-flavour. This work demonstrates the general ability of Dekkera and Brettanomyces yeasts to produce mousy off-flavour and confirms the importance of these yeasts for off-flavour production in grape juice and wine.     

INHIBITION OF MICROORGANISMS IN SALAD DRESSING BY SUCROSE AND METHYLGLUCOSE FATTY ACID MONOESTERS

The antimicrobial activity of sucrose and methylglucose esters of medium to long chain fatty acids was studied with two microorganisms involved in the spoilage of salad dressings, Zygosaccharomyces bailii and Lactobacillus fructivorans. The microorganisms were inhibited to various degrees by 0.1, 0.5, and 1.0% synthesized sucrose or methylglucose monoesters using a modified broth dilution method. Sucrose monoesters were most inhibitory when the esterified fatty acid was myristic (C₁₄) or palmitic acid (C₁₆). Methylglucose monoesters with lauric (C₁₂) or myristic acid (C₁₄) exhibited greater inhibition than those with longer chain fatty acids. The least inhibition was generally observed with sucrose and methylglucose oleate (C_18:1). Sucrose monoesters were usually more inhibitory than methylglucose monoesters of the same fatty acid, especially for palmitic and stearic (C₁₈) acids. In salad dressing, 1% sucrose monoesters of lauric, myristic, or palmitic acid significantly (P < 0.05) inhibited the growth of Z. bailii and L. fructivorans, and were comparable with or more effective than 0.1% sodium benzoate. Z. bailii growth was nearly completely inhibited by sucrose laurate, myristate and palmitate by 9 days of salad dressing storage. Sucrose monoesters did not delay the lag phase of L.     

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.     

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.     

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.     

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.     

The boundary for growth of Zygosaccharomyces bailii in acidified products described by models for time to growth and probability of growth

Models to predict days to growth and probability of growth of Zygosaccharomyces bailii in high-acid foods were developed, and the equations are presented here. The models were constructed from measurements of growth of Z. bailii using automated turbidimetry over a 29-day period at various pH, NaCl, fructose, and acetic acid levels. Statistical analyses were carried out using Statistical Analysis Systems LIFEREG procedures, and the data were fitted to log-logistic models. Model 1 predicts days to growth based on two factors, combined molar concentration of salt plus sugar and undissociated acetic acid. This model allows a growth/no-growth boundary to be visualized. The boundary is comparable with that established by G. Tuynenburg Muys (Process Biochem. 6:25-28, 1971), which still forms the basis of industry assumptions about the stability of acidic foods. Model 2 predicts days to growth based on the four independent factors of salt, sugar, acetic acid, and pH levels and is, therefore, much more useful for product development. Validation data derived from challenge studies in retail products from the U.S. market are presented for Model 2, showing that the model gives reliable, fail-safe predictions and is suitable for use in predicting growth responses of Z. bailii in high-acid foods. Model 3 predicts probability of growth of Z. bailii in 29 days. This model is most useful for spoilage risk assessment. All three models showed good agreement between predictions and observed values for the underlying data.     

Genetic interrelationship among species of the genus Zygosaccharomyces as revealed by small-subunit rRNA gene sequences

The phylogenetic interrelationship of species of the genus Zygosaccharomyces was examined by 18S rRNA gene sequencing. Comparative analysis of the sequence data revealed the genus to consist of a number of distinct subdivisions. The most prevalent species associated with food spoilage, Z. bailii, Z. bisporus and Z. rouxii, along with Z. mellis were found to form one subdivision. Zygosaccharomyces cidri and Z. fermentati formed a distinct species pair, as did Z. microellipsoides and Z. mrakii. Zygosaccharomyces florentinus formed a separate line displaying no specific relationship with any of the other Zygosaccharomyces species examined. Comparison with nine published ascosporogenous yeast 18S rRNA gene sequences showed that Z. microellipsoides and Z. mrakii were genealogically very close to Torulaspora delbrueckii (both displaying 99·8% 18S rRNA sequence similarity), raising the possibility that these two Zygosaccharomyces species should be moved to the genus Torulaspora. The topologies of trees derived from complete 18S rRNA gene sequences and from individual domains within the gene were compared and the implications of using partial sequence data for inferring phylogenetic relationships discussed.     

High Pdr12 levels in spoilage yeast (Saccharomyces cerevisiae) correlate directly with sorbic acid levels in the culture medium but are not sufficient to provide cells with acquired resistance to the food preservative

Sorbic acid is a commonly used food preservative against yeast and fungal food spoilage. Understanding its effect on the molecular physiology of yeast cells will allow the food industry to develop knowledge-based strategies to make more optimal use of its preservative action. Here we show that the yeast membrane protein Pdr12, previously shown to be prominently involved in sorbic acid resistance development in laboratory strains, was strongly induced by the presence of sorbic acid in the culture medium in Saccharomyces strains isolated from spoiled foods. Induction of Pdr12 expression was seen both under laboratory conditions and upon growth in a commercial soft drink. Induction was rapid and maintained for the duration of the stress. No Pdr12-like protein induction was seen in Zygosaccharomyces bailii or Zygosaccharomyces lentus, two well-known beverages spoilage organisms. Finally, unexpectedly, our studies showed for the first time that pre-inducing Pdr12p to maximal levels by subjecting cells to a mild sorbic acid stress did not lead to cells with an acquired resistance. Neither more rapid growth in the presence of the acid nor growth at higher sorbic acid concentrations at a given environmental pH was observed. Thus we have shown that while important in resistance development against sorbic acid, by itself induction of the pump is not sufficient to acquire resistance to the preservative.     

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).     

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.     

红茶菌中醋酸菌和酵母菌的分离鉴定及其相互作用

采用涂布平板法分离红茶菌中的醋酸菌和酵母菌，然后将分离得到的醋酸菌和酵母菌按照一定比例分别进行共培养，比较细菌纤维素的产量差别。结果表明，从红茶菌中分离出1株醋酸菌，经鉴定为居间驹形杆菌（Komagataeibacter intermedius）；3株酵母菌，经鉴定分别为布鲁塞尔酒香酵母（Brettanomyces bruxellensis）、二孢接合酵母（Zygosaccharomyces bisporus）和核果梅奇酵母（Metschnikowia fructicola），其中布鲁塞尔酒香酵母为优势酵母。当居间驹形杆菌与布鲁塞尔酒香酵母以1∶100的比例共培养时，产生的细菌纤维素最多达4.70 g/L；当二孢接合酵母和核果梅奇酵母以相同比例分别与居间驹形杆菌共培养时产生的细菌纤维素分别为3.59 g/L（高于对照组）和1.14 g/L（低于对照组）。由此可知，布鲁塞尔酒香酵母和Z. bisporus均能够促进居间驹形杆菌产细菌纤维素，而核果梅奇酵母对居间驹形杆菌没有明显的促进作用。     

Effect of Sweet Humectants on Stability and Antimicrobial Action of Sorbates

ABSTRACT: The effect of glucose or polyols on sorbate stability and on its antimicrobial action were studied in aqueous systems (pH 3.0). All humectants promoted preservative degradation, but polyols decreased browning development. Depression of water activity did not inhibit the growth of Zygosaccharomyces bailii , but xylitol increased the lag phase and decreased the growth rate and the stationary phase level. Sorbate minimum inhibitory concentration (MIC) concerning Z. bailii growth acquired slightly lower values (0.020% w/w) when sorbitol or xylitol were present for systems with water activity of 0.971. The residual level of the preservative after 3 wk of storage at 35 °C was smaller than the MIC for all studied systems with the exception of the humectant-free system.     

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.