Antimicrobial Activity of Aroma Compounds against Saccharomyces cerevisiae and Improvement of Microbiological Stability of Soft Drinks as Assessed by Logistic Regression

The combined effects of a mild heat treatment (55°C) and the presence of three aroma compounds [citron essential oil, citral, and (E)-2-hexenal] on the spoilage of noncarbonated beverages inoculated with different amounts of a Saccharomyces cerevisiae strain were evaluated. The results, expressed as growth/no growth, were elaborated using a logistic regression in order to assess the probability of beverage spoilage as a function of thermal treatment length, concentration of flavoring agents, and yeast inoculum. The logit models obtained for the three substances were extremely precise. The thermal treatment alone, even if prolonged for 20 min, was not able to prevent yeast growth. However, the presence of increasing concentrations of aroma compounds improved the stability of the products. The inhibiting effect of the compounds was enhanced by a prolonged thermal treatment. In fact, it influenced the vapor pressure of the molecules, which can easily interact within microbial membranes when they are in gaseous form. (E)-2-Hexenal showed a threshold level, related to initial inoculum and thermal treatment length, over which yeast growth was rapidly inhibited. Concentrations over 100 ppm of citral and thermal treatment longer than 16 min allowed a 90% probability of stability for bottles inoculated with 10⁵ CFU/bottle. Citron gave the most interesting responses: beverages with 500 ppm of essential oil needed only 3 min of treatment to prevent yeast growth. In this framework, the logistic regression proved to be an important tool to study alternative hurdle strategies for the stabilization of noncarbonated beverages.     

Modeling yeast spoilage in cold-filled ready-to-drink beverages with Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Candida lipolytica

Mathematical models were developed to predict the probability of yeast spoilage of cold-filled ready-to-drink beverages as a function of beverage formulation. A Box-Behnken experimental design included five variables, each at three levels: pH (2.8, 3.3, and 3.8), titratable acidity (0.20, 0.40, and 0.60%), sugar content (8.0, 12.0, and 16.0 degrees Brix), sodium benzoate concentration (100, 225, and 350 ppm), and potassium sorbate concentration (100, 225, and 350 ppm). Duplicate samples were inoculated with a yeast cocktail (100 microl/50 ml) consisting of equal proportions of Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Candida lipolytica (approximately 5.0 x 10(4) CFU/ml each). The inoculated samples were plated on malt extract agar after 0, 1, 2, 4, 6, and 8 weeks. Logistic regression was used to create the predictive models. The pH and sodium benzoate and potassium sorbate concentrations were found to be significant factors controlling the probability of yeast growth. Interaction terms for pH and each preservative were also significant in the predictive model. Neither the titratable acidity nor the sugar content of the model beverages was a significant predictor of yeast growth in the ranges tested.     

Modeling Yeast Spoilage in Cold-Filled Ready-To-Drink Beverages with Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Candida lipolytica

Mathematical models were developed to predict the probability of yeast spoilage of cold-filled ready-to-drink beverages as a function of beverage formulation. A Box-Behnken experimental design included five variables, each at three levels: pH (2.8, 3.3, and 3.8), titratable acidity (0.20, 0.40, and 0.60%), sugar content (8.0, 12.0, and 16.0 °Brix), sodium benzoate concentration (100, 225, and 350 ppm), and potassium sorbate concentration (100, 225, and 350 ppm). Duplicate samples were inoculated with a yeast cocktail (100 μl/50 ml) consisting of equal proportions of Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Candida lipolytica (~5.0 × 10⁴ CFU/ml each). The inoculated samples were plated on malt extract agar after 0, 1, 2, 4, 6, and 8 weeks. Logistic regression was used to create the predictive models. The pH and sodium benzoate and potassium sorbate concentrations were found to be significant factors controlling the probability of yeast growth. Interaction terms for pH and each preservative were also significant in the predictive model. Neither the titratable acidity nor the sugar content of the model beverages was a significant predictor of yeast growth in the ranges tested.     

Characterization of spore germination of a thermoacidophilic spore-forming bacterium, Alicyclobacillus acidoterrestris

The germination behaviors of spores of Alicyclobacillus acidoterrestris, which has been considered to be a causative microorganism of flat sour type spoilage in acidic beverages, were investigated. The spores of A. acidoterrestris showed efficient germination and outgrowth after heat activation (80 degrees C, 20 min) in Potato dextrose medium (pH 4.0). Further, the spores treated with heat activation germinated in McIlvaine buffer (pH 4.0) in the presence of a germinative substance (L-alanine) and commercial fruit juices, although not in phosphate buffer (pH 7.0). Heat activation was necessary for germination. The spores of A. acidoterrestris, which easily survived the heat treatment in acidic conditions, lost their resistance to heat during germination. Our results suggest that the models obtained from spore germination of A. acidoterrestris might be beneficial to determine adequate thermal process in preventing the growth of potential spoilage bacteria in acidic beverages.     

Prevention of Aerobic Spoilage of Maize Silage by a Genetically Modified Killer Yeast, Kluyveromyces lactis, Defective in the Ability To Grow on Lactic Acid

In this study, we propose a new process of adding a genetically modified killer yeast to improve the aerobic stability of silage. Previously constructed Kluyveromyces lactis killer strain PCK27, defective in growth on lactic acid due to disruption of the gene coding for phosphoenolpyruvate carboxykinase, a key enzyme for gluconeogenesis, inhibited the growth of Pichia anomala inoculated as an aerobic spoilage yeast and prevented a rise in pH in a model of silage fermentation. This suppressive effect of PCK27 was not only due to growth competition but also due to the killer protein produced. From these results, we concluded that strain PCK27 can be used as an additive to prolong the aerobic stability of maize silage. In the laboratory-scale experiment of maize silage, the addition of a killer yeast changed the yeast flora and significantly reduced aerobic spoilage.     

Prevention of aerobic spoilage of maize silage by a genetically modified killer yeast, Kluyveromyces lactis, defective in the ability to grow on lactic acid.

In this study, we propose a new process of adding a genetically modified killer yeast to improve the aerobic stability of silage. Previously constructed Kluyveromyces lactis killer strain PCK27, defective in growth on lactic acid due to disruption of the gene coding for phosphoenolpyruvate carboxykinase, a key enzyme for gluconeogenesis, inhibited the growth of Pichia anomala inoculated as an aerobic spoilage yeast and prevented a rise in pH in a model of silage fermentation. This suppressive effect of PCK27 was not only due to growth competition but also due to the killer protein produced. From these results, we concluded that strain PCK27 can be used as an additive to prolong the aerobic stability of maize silage. In the laboratory-scale experiment of maize silage, the addition of a killer yeast changed the yeast flora and significantly reduced aerobic spoilage.     

A new strategy for inhibition of the spoilage yeasts Saccharomyces cerevisiae and Zygosaccharomyces bailii based on combination of a membrane-active peptide with an oligosaccharide that leads to an impaired glycosylphosphatidylinositol (GPI)-dependent yeast wall protein layer

Glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in yeast are connected to the beta-1,3-glucan network via a beta-1,6-glucan moiety. Addition of gentiobiose or beta-1,6-glucan oligomers to growing cells affected the construction of a normal layer of GPI-dependent cell wall proteins at the outer rim of the Saccharomyces cerevisiae cell wall. Treated S. cerevisiae cells secreted significant amounts of cell wall protein 2, were much more sensitive to the lytic action of zymolyase 20T and displayed a marked increase in sensitivity to the small amphipathic antimicrobial peptide MB-21. Similar results in terms of sensitization of yeast cells to the antimicrobial peptide were obtained with the notorious food spoilage yeast Zygosaccharomyces bailii. Our results indicate that treating cells with a membrane-perturbing compound together with compounds that lead to an impaired construction of a normal GPI-dependent yeast wall protein layer represents an effective strategy to prevent the growth of major food spoilage yeasts.     

Lactic acid bacteria with potential to eliminate fungal spoilage in foods

Aims: To investigate antifungal activity produced by lactic acid bacteria (LAB) isolated from malted cereals and to determine if such LAB have the capacity to prevent fungal growth in a particular food model system. Methods and Results: The effect of pH, temperature and carbon source on production of antifungal activity by four LAB was determined. Pediococcus pentosaceus was used to conduct a trial to determine if it is feasible to eliminate Penicillium expansum, the mould responsible for apple rot, using an apple model. Penicillium expansum was incapable of growth during the trial on apple‐based agar plates inoculated with the antifungal‐producing culture, whereas the mould did grow on apple plates inoculated with an LAB possessing no antifungal activity. Conclusion: Partial characterization of the antifungal compounds indicates that their activity is likely to be because of production of antifungal peptides. The trial conducted showed that the antifungal culture has the ability to prevent growth of the mould involved in apple spoilage, using apples as a model. Significance and Impact of the study: The ability of an LAB to prevent growth of Pen. expansum using the apple model suggests that these antifungal LAB have potential applications in the food industry to prevent fungal spoilage of food.     

Survival of Candida parapsilosis yeast in olive oil

Candida parapsilosis is a human opportunistic pathogen yeast isolated from different habitats like animals, man, pickled cucumber, fruit juices, and water. Recent studies have demonstrated that C. parapsilosis can survive in olive oil for very long periods even exceeding 24 months. The survival of two strains of C. parapsilosis named DAPES 1890 and 1892, previously isolated from extra virgin olive oil, was influenced by the state of hydration of the cells and the polyphenols concentration of olive oil. When the cells of the two strains of C. parapsilosis were inoculated under a liophilized form into olive oil containing 45–312 mg/kg of total polyphenols, their survival in some olive oil samples reached approximately 18 months. However, if the above-mentioned inoculum was rehydrated with 1 % of distilled water, then the survival of both yeast strains in some samples of oil exceeded 24 months. The two yeast strains, recovered from the olive oil samples after 24 months of storage, showed, under SEM, spherical shapes with and without buds according to whether the inoculum was made up of rehydrated or lyophilized cells. The survival of all the C. parapsilosis strains was also negatively influenced by the polyphenols concentration of the olive oil samples inoculated both with lyophilized and rehydrated yeast cells. In the oily habitat, the polyphenols sorption to the C. parapsilosis yeast surface was observed, and during storage the polyphenols reacted with the yeast cell walls according to their concentration in the inoculated olive oil.     

小果蔷薇净油化学成分的研究

从贵州的小果蔷薇(Rosa cymosa Tratt.)花制备净油。用毛细管色谱/质谱/计算机联用方法进行了化学成分分析,鉴定了34个化合物。占净油的93.75％。主要芳香成分为苯甲醇(46.26％).丁香酚(10.02％),苯乙醇(9.17％),肉桂醛(6.87％),苯丙酸乙酯(4.11％)。芳樟醇(3.76％),苯甲酸乙酯(3.19％),柠檬醛(2.07％)等。该油具有特殊芳香。适用于调配化妆品、食品、饮料等香精,具有增香、定香的作用。     

Histochemical Localization of Citral Accumulation in Lemongrass Leaves (Cymbopogon citratus(DC.) Stapf., Poaceae)

Lemongrasses (Cymbopogonspp., Poaceae) are a group of commerciallyimportant C₄tropical grasses. Their leaves contain up to 1.5%(d.wt) essential oils with a typical lemon-like aroma, consistingmainly of citral (a mixture of the isomeric acyclic monoterpenealdehydes geranial and neral). To specifically locate the sitesof citral accumulation in lemongrass we employed Schiff's reagent,which reacts with aldehydes and gives a purple-red colorationwith citral. Using this technique, single oil-accumulating cellswere detected in the adaxial side of leaf mesophyll, commonlyadjacent to non-photosynthetic tissue, and between vascularbundles. Cell walls of these oil cells are lignified. Our resultssuggest that citral accumulation takes place in individual oilcells within the leaf tissues.Copyright 1998 Annals of BotanyCompany Lemongrass;Cymbopogon citratus; Poaceae; oil cells; histochemistry; citral; aldehydes; Schiff's-reagent.     

Transcriptional profiling of wine yeast in fermenting grape juice: regulatory effect of diammonium phosphate

The nitrogen composition of grape musts affects fermentation kinetics and production of aroma and spoilage compounds in wine. It is common practice in wineries to supplement grape musts with diammonium phosphate (DAP) to prevent nitrogen-related fermentation problems. Laboratory strains of Saccharomyces cerevisiae preferentially use rich nitrogen sources, such as ammonia, over poor nitrogen sources. We used global gene expression analysis to monitor the effect of DAP addition on gene expression patterns in wine yeast in fermenting Riesling grape must. The expression of 350 genes in the commercial wine yeast strain VIN13 was affected; 185 genes were down-regulated and 165 genes were up-regulated in response to DAP. Genes that were down-regulated encode small molecule transporters and nitrogen catabolic enzymes, including those linked to the production of urea, a precursor of ethyl carbamate in wine. Genes involved in amino acid metabolism, assimilation of sulfate, de novo purine biosynthesis, tetrahydrofolate one-carbon metabolism, and protein synthesis were up-regulated. The expression level of 86 orphan genes was also affected by DAP.     

Mechanisms of Antiviral Action of Plant Antimicrobials against Murine Norovirus

Numerous plant compounds have antibacterial or antiviral properties; however, limited research has been conducted with nonenveloped viruses. The efficacies of allspice oil, lemongrass oil, and citral were evaluated against the nonenveloped murine norovirus (MNV), a human norovirus surrogate. The antiviral mechanisms of action were also examined using an RNase I protection assay, a host cell binding assay, and transmission electron microscopy. All three antimicrobials produced significant reductions (P ≤ 0.05) in viral infectivity within 6 h of exposure (0.90 log₁₀ to 1.88 log₁₀). After 24 h, the reductions were 2.74, 3.00, and 3.41 log₁₀ for lemongrass oil, citral, and allspice oil, respectively. The antiviral effect of allspice oil was both time and concentration dependent; the effects of lemongrass oil and citral were time dependent. Based on the RNase I assay, allspice oil appeared to act directly upon the viral capsid and RNA. The capsids enlarged from ≤35 nm to up to 75 nm following treatment. MNV adsorption to host cells was not significantly affected. Alternatively, the capsid remained intact following exposure to lemongrass oil and citral, which appeared to coat the capsid, causing nonspecific and nonproductive binding to host cells that did not lead to successful infection. Such contrasting effects between allspice oil and both lemongrass oil and citral suggest that though different plant compounds may yield similar reductions in virus infectivity, the mechanisms of inactivation may be highly varied and specific to the antimicrobial. This study demonstrates the antiviral properties of allspice oil, lemongrass oil, and citral against MNV and thus indicates their potential as natural food and surface sanitizers to control noroviruses.     

Prevention of yeast spoilage in feed and food by the yeast mycocin HMK

The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts.     

Prevention of Yeast Spoilage in Feed and Food by the Yeast Mycocin HMK

The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts.     

Monoterpenic aldehydes as potential anti-Leishmania agents: activity of Cymbopogon citratus and citral on L. infantum, L. tropica and L. major

In order to contribute for the search of new drugs for leishmaniasis, we study the susceptibility of Leishmania infantum, Leishmania tropica and Leishmania major to Cymbopogon citratus essential oil and major compounds, mrycene and citral. C. citratus and citral were the most active inhibiting L. infantum, L. tropica and L. major growth at IC(50) concentrations ranging from 25 to 52 μg/ml and from 34 to 42 μg/ml, respectively. L. infantum promastigotes exposed to essential oil and citral underwent considerable ultrastructural alterations, namely mitochondrial and kinetoplast swelling, autophagosomal structures, disruption of nuclear membrane and nuclear chromatin condensation. C. citratus essential oil and citral promoted the leishmanicidal effect by triggering a programmed cell death. In fact, the leishmanicidal activity was mediated via apoptosis as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, and cell-cycle arrest at the G(0)/G(1) phase. Taken together, ours findings lead us to propose that citral was responsible for anti-Leishmania activity of the C. citratus and both may represent a valuable source for therapeutic control of leishmaniasis.     

Oxygen requirements of the food spoilage yeast Zygosaccharomyces bailii in synthetic and complex media

Most yeast species can ferment sugars to ethanol, but only a few can grow in the complete absence of oxygen. Oxygen availability might, therefore, be a key parameter in spoilage of food caused by fermentative yeasts. In this study, the oxygen requirement and regulation of alcoholic fermentation were studied in batch cultures of the spoilage yeast Zygosaccharomyces bailii at a constant pH, pH 3.0. In aerobic, glucose-grown cultures, Z. bailii exhibited aerobic alcoholic fermentation similar to that of Saccharomyces cerevisiae and other Crabtree-positive yeasts. In anaerobic fermentor cultures grown on a synthetic medium supplemented with glucose, Tween 80, and ergosterol, S. cerevisiae exhibited rapid exponential growth. Growth of Z. bailii under these conditions was extremely slow and linear. These linear growth kinetics indicate that cell proliferation of Z. bailii in the anaerobic fermentors was limited by a constant, low rate of oxygen leakage into the system. Similar results were obtained with the facultatively fermentative yeast Candida utilis. When the same experimental setup was used for anaerobic cultivation, in complex YPD medium, Z. bailii exhibited exponential growth and vigorous fermentation, indicating that a nutritional requirement for anaerobic growth was met by complex-medium components. Our results demonstrate that restriction of oxygen entry into foods and beverages, which are rich in nutrients, is not a promising strategy for preventing growth and gas formation by Z. bailii. In contrast to the growth of Z. bailii, anaerobic growth of S. cerevisiae on complex YPD medium was much slower than growth in synthetic medium, which probably reflected the superior tolerance of the former yeast to organic acids at low pH.     

Inactivation of Escherichia coli by citral

Aims: The aim was to evaluate (i) the resistance of Escherichia coli BJ4 to citral in a buffer system as a function of citral concentration, treatment medium pH, storage time and initial inoculum size, (ii) the role of the sigma factor RpoS on citral resistance of E. coli, (iii) the role of the cell envelope damage in the mechanism of microbial inactivation by citral and (iiii) possible synergistic effects of mild heat treatment and pulsed electric fields (PEF) treatment combined with citral. Methods and Results: The initial inoculum size greatly affected the efficacy of citral against E. coli cells. Exposure to 200 μl l^?1 of citral at pH 4·0 for 24 h at 20°C caused the inactivation of more than 5 log₁₀ cycles of cells starting at an inoculum size of 10⁶ or 10⁷ CFU ml^?1, whereas increasing the cell concentration to 10⁹ CFU ml^?1 caused <1 log₁₀ cycle of inactivation. Escherichia coli showed higher resistance to citral at pH 4·0 than pH 7·0. The rpoS null mutant strain E. coli BJ4L1 was less resistant to citral than the wild‐type strain. Occurrence of sublethal injury to both the cytoplasmic and outer membranes was demonstrated by adding sodium chloride or bile salts to the recovery media. The majority of sublethally injured cells by citral required energy and lipid synthesis for repair. A strongly synergistic lethal effect was shown by mild heat treatment combined with citral but the presence of citral during the application of a PEF treatment did not show any advantage. Conclusions: This work confirms that cell envelope damage is an important event in citral inactivation of bacteria, and it describes the key factors on the inactivation of E. coli cells by citral. Significance and Impact of the Study: Knowledge about the mechanism of microbial inactivation by citral helps establish successful combined preservation treatments.     

Spoilage yeasts.

Yeasts are best known for their beneficial contributions to society, and the literature abounds with discussions of their role in the fermentation of alcoholic beverages, bread, and other products. Yeasts also cause spoilage, but, with a few exceptions, this unwanted activity often goes unrecognized and underestimated as a major problem in the food and beverage industries. In some cases, there is only a fine line between what is perceived as either a spoilage or beneficial activity. This review examines the occurrence and growth of yeasts in foods and beverages with respect to their spoilage activities, the biochemistry of this spoilage, and technologies for the enumeration and identification of spoilage yeasts.     

Antifungal activity of essential oils evaluated by two different application techniques against rye bread spoilage fungi

AIMS: To study how antifungal activity of natural essential oils depends on the assay method used. METHODS AND RESULTS: Oils of bay, cinnamon leaf, clove, lemongrass, mustard, orange, sage, thyme and two rosemary oils were tested by two methods: (1) a rye bread-based agar medium was supplemented with 100 and 250 microl l-1 essential oil and (2) real rye bread was exposed to 136 and 272 microl l-1 volatile oil in air. Rye bread spoilage fungi were used for testing. Method 1 proved thyme oil to be the overall best growth inhibitor, followed by clove and cinnamon. On the contrary, orange, sage and rosemary oils had very limited effects. Mustard and lemongrass were the most effective oils by the volatile method, and orange, sage and one rosemary showed some effects. Oil compositions were analysed by gas chromatography-mass spectrography. CONCLUSIONS: Antifungal effects of the essential oils depended on the application method. Larger phenolic compounds such as thymol and eugenol (thyme, cinnamon and clove) had best effect applied directly to medium, whereas smaller compounds such as allyl isothiocyanate and citral (mustard and lemongrass) were most efficient when added as volatiles. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proves that the method used for screening essential oils as potential antimicrobials should correspond with the application sought.