Physiological requirements for growth and competitiveness of Dekkera bruxellensis under oxygen-limited or anaerobic conditions

The effect of glucose and oxygen limitation on the growth and fermentation performances of Dekkera bruxellensis was investigated in order to understand which factors favour its propagation in ethanol or wine plants. Although D. bruxellensis has been described as a facultative anaerobe, no growth was observed in mineral medium under complete anaerobiosis while growth was retarded under severe oxygen limitation. In a continuous culture with no gas inflow, glucose was not completely consumed, most probably due to oxygen limitation. When an air/nitrogen mixture (O₂‐content ca. 5%) was sparged to the culture, growth became glucose‐limited. In co‐cultivations with Saccharomyces cerevisiae, ethanol yields/g consumed sugar were not affected by the co‐cultures as compared to the pure cultures. However, different population responses were observed in both systems. In oxygen‐limited cultivation, glucose was depleted within 24 h after challenging with S. cerevisiae and both yeast populations were maintained at a stable level. In contrast, the S. cerevisiae population constantly decreased to about 1% of its initial cell number in the sparged glucose‐limited fermentation, whereas the D. bruxellensis population remained constant. To identify the requirements of D. bruxellensis for anaerobic growth, the yeast was cultivated in several nitrogen sources and with the addition of amino acids. Yeast extract and most of the supplied amino acids supported anaerobic growth, which points towards a higher nutrient demand for D. bruxellensis compared to S. cerevisiae in anaerobic conditions. Copyright © 2012 John Wiley & Sons, Ltd.     

Production of sensory compounds by means of the yeast Dekkera bruxellensis in different nitrogen sources with the prospect of producing cachaça

The distilled spirit made from sugar cane juice, also known as cachaça, is a traditional Brazilian beverage that in recent years has increased its market share among international distilled beverages. Several volatile compounds produced by yeast cells during the fermentation process are responsible for the unique taste and aroma of this drink. The yeast Dekkera bruxellensis has acquired increasing importance in the fermented beverage production, as the different metabolites produced by this yeast may be either beneficial or harmful to the end‐product. Since D. bruxellensis is often found in the fermentation processes carried out in ethanol fuel distillation in Brazil, we employed this yeast to analyse the physiological profile and production of aromatic compounds and to examine whether it is feasible to regard it as a cachaça‐producing microorganism. The assays were performed on a small scale and simulated the conditions for the production of handmade cachaça. The results showed that the presence of aromatic and branched‐chain amino acids in the medium has a strong influence on the metabolism and production of flavours by D. bruxellensis. The assimilation of these alternative nitrogen sources led to different fermentation yields and the production of flavouring compounds. The influence of the nitrogen source on the metabolism of fusel alcohols and esters in D. bruxellensis highlights the need for further studies of the nitrogen requirements to obtain the desired level of sensory compounds in the fermentation. Our results suggest that D. bruxellensis has the potential to play a role in the production of cachaça. Copyright © 2014 John Wiley & Sons, Ltd.     

On the catabolism of amino acids in the yeast Dekkera bruxellensis and the implications for industrial fermentation processes

In the last years several reports have reported the capacity of the yeast Dekkera (Brettanomyces) bruxellensis to survive and adapt to the industrial process of alcoholic fermentation. Much of this feature seems to relate to the ability to assimilate limiting sources of nutrients, or somehow some that are inaccessible to Saccharomyces cerevisiae, in particular the sources of nitrogen. Among them, amino acids (AA) are relevant in terms of beverage musts, and could also be important for bioethanol. In view of the limited knowledge on the control of AA, the present work combines physiological and genetic studies to understand how it operates in D. bruxellensis in response to oxygen availibility. The results allowed separation of the AA in three groups of preferentiality and showed that glutamine is the preferred AA irrespective of the presence of oxygen. Glutamate and aspartate were also preferred AA in anaerobiosis, as indicated by the physiological data. Gene expression experiments showed that, apart from the conventional nitrogen catabolic repression mechanism that is operating in aerobiosis, there seems to be an oxygen‐independent mechanism acting to overexpress key genes like GAP1, GDH1, GDH2 and GLT1 to ensure adequate anaerobic growth even in the presence of non‐preferential nitrogen source. This could be of major importance for the industrial fitness of this yeast species.     

First aspects on acetate metabolism in the yeast Dekkera bruxellensis: a few keys for improving ethanol fermentation

Dekkera bruxellensis is continuously changing its status in fermentation processes, ranging from a contaminant or spoiling yeast to a microorganism with potential to produce metabolites of biotechnological interest. In spite of that, several major aspects of its physiology are still poorly understood. As an acetogenic yeast, minimal oxygen concentrations are able to drive glucose assimilation to oxidative metabolism, in order to produce biomass and acetate, with consequent low yield in ethanol. In the present study, we used disulfiram to inhibit acetaldehyde dehydrogenase activity to evaluate the influence of cytosolic acetate on cell metabolism. D. bruxellensis was more tolerant to disulfiram than Saccharomyces cerevisiae and the use of different carbon sources revealed that the former yeast might be able to export acetate (or acetyl‐CoA) from mitochondria to cytoplasm. Fermentation assays showed that acetaldehyde dehydrogenase inhibition re‐oriented yeast central metabolism to increase ethanol production and decrease biomass formation. However, glucose uptake was reduced, which ultimately represents economical loss to the fermentation process. This might be the major challenge for future metabolic engineering enterprises on this yeast.     

Fermentation profiles of the yeast Brettanomyces bruxellensis in d-xylose and l-arabinose aiming its application as a second-generation ethanol producer

The yeast Brettanomyces bruxellensis is able to ferment the main sugars used in first-generation ethanol production. However, its employment in this industry is prohibitive because the ethanol productivity reached is significantly lower than the observed for Saccharomyces cerevisiae. On the other hand, a possible application of B. bruxellensis in the second-generation ethanol production has been suggested because this yeast is also able to use d -xylose and l -arabinose, the major pentoses released from lignocellulosic material. Although the latter application seems to be reasonable, it has been poorly explored. Therefore, we aimed to evaluate whether or not different industrial strains of B. bruxellensis are able to ferment d -xylose and l -arabinose, both in aerobiosis and oxygen-limited conditions. Three out of nine tested strains were able to assimilate those sugars. When in aerobiosis, B. bruxellensis cells exclusively used them to support biomass formation, and no ethanol was produced. Moreover, whereas l -arabinose was not consumed under oxygen limitation, d -xylose was only slightly used, which resulted in low ethanol yield and productivity. In conclusion, our results showed that d -xylose and l -arabinose are not efficiently converted to ethanol by B. bruxellensis, most likely due to a redox imbalance in the assimilatory pathways of these sugars. Therefore, despite presenting other industrially relevant traits, the employment of B. bruxellensis in second-generation ethanol production depends on the development of genetic engineering strategies to overcome this metabolic bottleneck.     

Factors affecting the production of 4-ethylphenol by the yeast Dekkera bruxellensis in enological conditions

The conversion of p-coumaric acid into 4-ethylphenol was studied in Dekkera bruxellensis ISA 1791 under defined conditions in synthetic media. The production of 4-ethylphenol occurred roughly between mid-exponential growth phase and the beginning of the stationary phase. This behaviour was observed when glucose was the only energy and carbon source, the conversion rate being close to 90%. Ethanol, as the single energy source, yielded conversion rates close to 80% while in the presence of trehalose and acetic acid conversion rates lower than 10% were obtained. The production of 4-ethylphenol was not observed when the cells were maintained in buffer solution without carbon and energy sources. The precursor of 4-ethylphenol, p-coumaric acid, was not utilized as energy and carbon source. Furthermore, it was shown that 4-vinylphenol may be used as a precursor of 4-ethylphenol in the absence of p-coumaric acid.Growth and 4-ethylphenol production were inhibited by increasing concentrations of ethanol, being fully prevented at 13% (v/v) ethanol.The cultivation of strain ISA 1791 in mixed culture with Saccharomyces cerevisiae, in synthetic medium, showed that the cell numbers of D. bruxellensis increased from 10⁴ cfu/ml to 5×10⁹ cfu/ml. Laboratory microvinifications of white and red juices inoculated with as low as 10 cfu/ml of D. bruxellensis and 10⁷ cells/ml of S. cerevisiae showed growth of D. bruxellensis to levels of about 5×10⁸ cfu/ml. In addition, 4-ethylphenol production by D. bruxellensis was observed only after complete fermentation of the grape juices.     

Impact of Australian Dekkera bruxellensis strains grown under oxygen-limited conditions on model wine composition and aroma

Spoilage of red wine by the yeast species Dekkera bruxellensis is a common problem for the global wine industry. When conditions are conducive for growth of these yeasts in wine, they efficiently convert non-volatile hydroxycinnamic acids into aroma-active ethylphenols, thereby reducing the quality of the wine. It has been demonstrated previously that dissolved oxygen is a key factor which stimulates D. bruxellensis growth in wine. We demonstrate that whereas the presence of oxygen accelerates the growth of this species, oxygen-limited conditions favour 4-ethylphenol production. Consequently, we evaluated wine spoilage potential of three D. bruxellensis strains (AWRI1499, AWRI1608 and AWRI1613) under oxygen-limited conditions. Each strain was cultured in a chemically-defined wine medium and the fermentation products were analysed using HPLC and HS-SPME–GC/MS. The strains displayed different growth characteristics but were equally capable of producing ethylphenols. On the other hand, significant differences were observed for 18 of the remaining 33 metabolites analysed and duo-trio sensory analysis indicated significant aroma differences between wines inoculated with AWRI1499 and AWRI1613. When these wines were spiked with low concentrations of 4-ethylphenol and 4-ethylguaiacol, no sensorial differences could be perceived. Together these data suggest that the three predominant D. bruxellensis strains previously isolated during a large survey of Australian wineries do not differ substantively in their capacity to grow in, and spoil, a model wine medium.     

Physiological profiles relevant for novel alcoholic beverage design among Dekkera bruxellensis strains from different provenances

A selection of D. bruxellensis strains from different geographical and beverage sources were tested for their potential to develop novel alcoholic beverages. Selected strains were initially clustered based on genetic similarities determined by PCR fingerprinting. Physiological profiles were subsequently determined during the fermentation experiments that were carried out in a defined synthetic medium supplemented with glucose and 4‐vinylphenol for 22 days, as static cultures under microaerobic conditions. There were significant differences (p ≤ 0.05) in ethanol, glycerol and acetic acid yields and in the growth rates between the strains. During prolonged fermentation, a reduction in ethanol and acetic acid was observed, ranging from 43 to 54% for ethanol and from 4 to 45% for acetic acid, which was strain or genetic group specific. Consumption of ethanol and acetic acid occurred during the stationary phase, suggesting that ethanol and acetic acid were utilized for processes other than growth and must have had an impact on the formation of the aromatic profile. The conversion of 4‐vinylphenol to 4‐ethylphenol was much more efficient and was completed within 4 days of fermentation. Although further investigation is needed, the results indicate a potential of this previously undesired microorganism to be useful for a wide range of applications. Copyright © 2016 The Institute of Brewing & Distilling     

Diversity in Spoilage Yeast Dekkera/Brettanomyces bruxellensis Isolated from French Red Wine. Assessment During Fermentation of Synthetic Wine Medium

Yeast Brettanomyces bruxellensis is a contaminant found worldwide and is responsible for red wine spoilage due to the development of animal and phenolic off‐odours. During this study, 24 Brettanomyces bruxellenis isolates were obtained from red wine samples from two French wineries and these were discriminated as 23 strains. Nine strains coming from 2 wineries and 4 vintages were cultivated in synthetic wine medium for 1500 hours and they gave nine different behaviours. Four main growth patterns (with different growth steps and durations) and three main different sugar consumption profiles were obtained. Glucose and fructose were not limiting substrates for all strains. The production level of 4‐ethylphenol was found to vary from strain to strain (from 0.350 to 2.773 mg L^?1) and was independent of the biomass concentration. Some strains presented a coupled‐to‐growth production of volatile phenols, others did not. This study showed that different strains of Brettanomyces bruxellensis behaved differently, one from another, under the given conditions taking into consideration several aspects. The results thus demonstrate a large intraspecific diversity.     

Effect of nitrogen source on oxalate accumulation in Setaria sphacelata (cv. Kazungula)

Setaria sphacelata (cv. Kazungula) was grown under controlled environment conditions on sand provided with mineral nutrient solutions containing nitrate, ammonium, urea, or nitrate plus ammonium as sole nitrogen sources. Growth in urea or ammonium resulted in small but significant decreases in oxalate concentrations of foliage compared with growth in nitrate, but nitrogen source had little effect on plant growth rates. Similar levels of potassium and total nitrogen were found in foliage from all treatments. Nitrate concentration was highest in leaves of plants supplied nitrate plus ammonium and lowest in leaves of plants supplied only ammonium as the nitrogen source. Nitrate reductase activity was virtually absent from new fully expanded leaves from all treatments, but readily detectable in young regrowth when highest activities were associated with the nitrate plus ammonium treatment and lowest with ammonium alone. Leaves continued to accumulate oxalate after reaching maturity and it is considered that anions other than nitrate must be responsible for the cation excess which prompts the synthesis of this oxalate. No diurnal variation in oxalate content of controlled environment or field grown Setaria sphacelata was observed.     

Nitrate modifies urea root uptake and assimilation in wheat seedlings

BACKGROUND: Some authors suggest that nitrate improves the effects of ammonium and urea nutrition on plant growth via an increase in both the root uptake of ammonium and urea, and the further activation of its assimilation. In order to verify these hypotheses, wheat (Triticum aestivum L.) seedlings were grown with various nitrogen supplies containing the main nitrogen forms (ammonium, nitrate and urea). Root nitrogen uptake and the concentration of each of the nitrogen forms in roots and shoots were studied. RESULTS: Results indicated that nitrate increases root uptake of ammonium and urea, with this effect being highly significant when the three nitrogen forms are supplied simultaneously. Nitrate significantly increased both urea and ammonium assimilation in the shoot of plants fed urea‐and‐nitrate. However, nitrate did not significantly affect ammonium assimilation in either shoots or roots in plants fed ammonium‐and‐nitrate. CONCLUSION: The beneficial effect of nitrate seems to be related to an enhancement of root uptake of ammonium and urea, although their assimilation was significantly increased in urea‐fed plants, but not in ammonium‐fed plants. Copyright © 2008 Society of Chemical Industry     

Ustilago maydis killer toxin as a new tool for the biocontrol of the wine spoilage yeast Brettanomyces bruxellensis

Brettanomyces bruxellensis is one of the most damaging species for wine quality, and tools for controlling its growth are limited. In this study, thirty-nine strains belonging to Saccharomyces cerevisiae and B. bruxellensis have been isolated from wineries, identified and then tested against a panel of thirty-nine killer yeasts. Here, for the first time, the killer activity of Ustilago maydis is proven to be effective against B. bruxellensis. Mixed cultures in winemaking conditions show that U. maydis CYC 1410 has the ability to inhibit B. bruxellensis, while S. cerevisiae is fully resistant to its killer activity, indicating that it could be used in wine fermentation to avoid the development of B. bruxellensis without undesirable effects on the fermentative yeast. The characterization of the dsRNAs isolated and purified from U. maydis CYC 1410 indicated that this strain produces a KP6-related toxin. Killer toxin extracts were active against B. bruxellensis at pH values between 3.0 and 4.5 and temperatures comprised between 15 °C and 25 °C, confirming their biocontrol activity in winemaking and wine aging conditions. Furthermore, small amounts (100 AU/ml) of killer toxin extracts from U. maydis significantly reduced the amount of 4-ethylphenol produced by B. bruxellensis, indicating that in addition to the growth inhibition observed for high killer toxin concentrations (ranging from 400 to 2000 AU/ml), small amounts of the toxin are able to reduce the production of volatile phenols responsible for the aroma defects in wines caused by B. bruxellensis.     

Effect of nitrogen source in the fertilizing solution on nutritional quality of three members of the Portulaca oleracea aggregate

BACKGROUND: Portulaca oleracea (purslane) is nutritious but, in addition to the essential α‐linolenic acid, vitamin C and tocopherols, it contains undesirable oxalic acid. Knowing the effects of nitrate and ammonium on oxalate accumulation, we tested the agronomic potential of three members of the P. oleracea aggregate under various nitrogen fertilization conditions, by measuring biomass production and accumulation of fatty acids, organic acids and tocopherol in the commercial P. sativa (Pos) and two natural members: P. nitida (Pon) and P. papillato‐stellulata (Pop). RESULTS: With nitrate as the sole N source, we measured differences between Pon and Pos in concentrations of the essential ω‐3 fatty acid α‐linolenic acid. Pos also gained less dry biomass under these conditions, implying a higher agronomical and nutritional value for Pon. Increasing the fertilizer ammonium concentration and reducing that of nitrate significantly decreased oxalic acid by factors of up to 1.7, 2.6 and 3.4 in Pos, Pop and Pon, respectively, significantly increased concentrations of tocopherol and malic acid, had no effect on fatty acids or ascorbic acid, but reduced biomass. CONCLUSION: In spite of the recumbent growth habit of Pon, the present findings indicate its agronomic potential. Because early flowering and seed production may be the limiting factors in purslane agriculture, growing Pon in nitrate‐poor conditions might be agriculturally favorable. Copyright © 2010 Society of Chemical Industry     

Nitrogen fertiliser source effects on the growth and mineral nutrition of pepper (Capsicum annuum L.) and wheat (Triticum aestivum L.)

BACKGROUND: The presence of stable mixed nitrogen forms (such as nitrate/ammonium/urea or nitrate/urea) in the soil solution is due to the use of nitrification and/or urease inhibitors in urea‐based fertilisers. However, there is no specific information in the literature comparing the efficiency of these urea mixed nitrogen forms as a nitrogen source for plants with that of nitrate and ammonium/nitrate. The aim of this study was to compare the effects on plant growth and mineral nutrition of different nitrogen forms, including mixed nitrogen forms containing urea. RESULTS: The results indicated that for both wheat (Triticum aestivum L.) and pepper (Capsicum annuum L.) the growth of plants fed mixed nitrogen forms containing urea was generally similar to that of plants receiving nitrate and nitrate/ammonium. Only in the case of pepper did ammonium/urea nutrition cause a significant decrease in plant growth. The presence of nitrate corrected the negative effects of mixed nitrogen forms containing ammonium and/or urea on the growth of pepper plants. CONCLUSION: Mixed nitrogen forms containing urea did not cause any negative effect on plant growth or mineral nutrition. In fact, plants fed mixed nitrogen forms containing urea had higher shoot concentrations of potassium, phosphorus, iron and boron than plants receiving nitrate. Copyright © 2007 Society of Chemical Industry     

Regulation of alcoholic fermentation in batch and chemostat cultures of Kluyveromyces lactis CBS 2359

Kluyveromyces lactis is an important industrial yeast, as well as a popular laboratory model. There is currently no consensus in the literature on the physiology of this yeast, in particular with respect to aerobic alcoholic fermentation (‘Crabtree effect’). This study deals with regulation of alcoholic fermentation in K. lactis CBS 2359, a proposed reference strain for molecular studies. In aerobic, glucose-limited chemostat cultures (D=0·05–0·40 h⁻¹) growth was entirely respiratory, without significant accumulation of ethanol or other metabolites. Alcoholic fermentation occurred in glucose-grown shake-flask cultures, but was absent during batch cultivation on glucose in fermenters under strictly aerobic conditions. This indicated that ethanol formation in the shake-flask cultures resulted from oxygen limitation. Indeed, when the oxygen feed to steady-state chemostat cultures (D=0·10 h⁻¹) was lowered, a mixed respirofermentative metabolism only occurred at very low dissolved oxygen concentrations (less than 1% of air saturation). The onset of respirofermentative metabolism as a result of oxygen limitation was accompanied by an increase of the levels of pyruvate decarboxylase and alcohol dehydrogenase. When aerobic, glucose-limited chemostat cultures (D=0·10 h⁻¹) were pulsed with excess glucose, ethanol production did not occur during the first 40 min after the pulse. However, a slow aerobic ethanol formation was invariably observed after this period. Since alcoholic fermentation did not occur in aerobic batch cultures this is probably a transient response, caused by an imbalanced adjustment of enzyme levels during the transition from steady-state growth at μ=0·10 h⁻¹ to growth at μ_max. It is concluded that in K. lactis, as in other Crabtree-negative yeasts, the primary environmental trigger for occurrence of alcoholic fermentation is oxygen limitation. © 1998 John Wiley & Sons, Ltd.     

The temperature dependent functionality of Brettanomyces bruxellensis strains in wort fermentations

In recent years, Brettanomyces bruxellensis has found increasing application in brewery fermentations. Indeed, B. bruxellensis contributes to the flavour profile of many Belgian beers, typically during secondary or spontaneous fermentation. In North America, the yeast is used in primary fermentation to produce beers with ‘Brett’ characteristics with ‘fruity’ and/or ‘funky’ sensory profiles associated with the production of volatile esters and phenols. However, little is understood about the factors that influence flavour metabolite production or fermentation rate in this yeast. Here, the impact of temperature is reported on fermentation efficiency, flavour metabolite production and carbon utilisation of one commonly used and eight poorly characterised B. bruxellensis strains during wort fermentation. A high degree of strain and temperature‐dependent variability was found in fermentation efficiency and metabolite production amongst B. bruxellensis strains. Further, fermentation efficiency and carbon utilisation were temperature dependent, while ester production increased at higher temperature and phenol production was strain and temperature independent. These results indicate significant strain and temperature dependent variation, suggesting the potential application of strain variability as a tool to achieve product diversity in B. bruxellensis primary fermentations. © 2019 The Institute of Brewing & Distilling     

The yeasts of the genus Spathaspora: potential candidates for second‐generation biofuel production

Yeasts of the Spathaspora clade have the ability to convert d ‐xylose to ethanol and/or xylitol. This is an important trait, as these yeasts may be used to produce bioethanol from lignocellulosic biomass or as a source of new d ‐xylose metabolism genes for recombinant industrial strains of Saccharomyces cerevisiae. The core group of the genus Spathaspora has 22 species, both formally described and not yet described. Other species, such as Sp. allomyrinae, Candida alai, C. insectamans, C. lyxosophila, C. sake, Sp. boniae and C. subhashii are weakly associated with this clade, based on LSU rRNA gene D1/D2 sequence analyses. Spathaspora passalidarum, Sp. arborariae, Sp. gorwiae and Sp. hagerdaliae produce mostly ethanol from d ‐xylose, whereas the remaining species within the Spathaspora clade already tested for this property may be considered xylitol producers. Among the d ‐xylose‐fermenting Spathaspora species, Sp. passalidarum is the best ethanol producer, displaying high ethanol yields and productivities when cultured in media supplemented with this pentose under oxygen‐limited or anaerobic conditions. The species also exhibits rapid d ‐xylose consumption and the ability to ferment glucose, xylose and cellobiose simultaneously. These characteristics suggest that Sp. passalidarum is a potential candidate for domestication and use in the fermentation of lignocellulosic materials. Copyright © 2017 John Wiley & Sons, Ltd.     

Osmotic stress response in the wine yeast Dekkera bruxellensis

Dekkera bruxellensis is mainly associated with lambic beer fermentation and wine production and may contribute in a positive or negative manner to the flavor development. This yeast is able to produce phenolic compounds, such as 4-ethylguaiacol and 4-ethylphenol which could spoil the wine, depending on their concentration. In this work we have investigated how this yeast responds when exposed to conditions causing osmotic stress, as high sorbitol or salt concentrations. We observed that osmotic stress determined the production and accumulation of intracellular glycerol, and the expression of NADH-dependent glycerol-3-phosphate dehydrogenase (GPD) activity was elevated. The involvement of the HOG MAPK pathway in response to this stress condition was also investigated. We show that in D. bruxellensis Hog1 protein is activated by phosphorylation under hyperosmotic conditions, highlighting the conserved role of HOG MAP kinase signaling pathway in the osmotic stress response.     

Characterization and expression analysis of a gene cluster for nitrate assimilation from the yeast Arxula adeninivorans

In Arxula adeninivorans nitrate assimilation is mediated by the combined actions of a nitrate transporter, a nitrate reductase and a nitrite reductase. Single‐copy genes for these activities (AYNT1, AYNR1, AYNI1, respectively) form a 9103 bp gene cluster localized on chromosome 2. The 3210 bp AYNI1 ORF codes for a protein of 1070 amino acids, which exhibits a high degree of identity to nitrite reductases from the yeasts Pichia anomala (58%), Hansenula polymorpha (58%) and Dekkera bruxellensis (54%). The second ORF (AYNR1, 2535 bp) encodes a nitrate reductase of 845 residues that shows significant (51%) identity to nitrate reductases of P. anomala and H. polymorpha. The third ORF in the cluster (AYNT1, 1518 bp) specifies a nitrate transporter with 506 amino acids, which is 46% identical to that of H. polymorpha. The three genes are independently expressed upon induction with NaNO₃. We quantitatively analysed the promoter activities by qRT–PCR and after fusing individual promoter fragments to the phytase (phyK) gene from Klebsiella sp. ASR1. The AYNI1 promoter was found to exhibit the highest activity, followed by the AYNT1 and AYNR1 elements. Direct measurements of nitrate and nitrite reductase activities performed after induction with NaNO₃ are compatible with these results. Both enzymes show optimal activity at around 42°C and near‐neutral pH, and require FAD as a co‐factor and NADPH as electron donor. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of Initial Headspace O2 Level on the Growth and Volatile Metabolite Production of Leuconostoc Mesenteriodes and the Microbial and Sensorial Quality of Modified Atmosphere Packaged Par‐Fried French Fries

This study evaluated the effect of residual O₂ level (0% to 5%) on microbial growth and volatile metabolite production on par‐fried French fries packaged in a modified atmosphere with 60% CO₂ (rest N₂) at 4 °C. The results obtained showed that the initial headspace (IH) O₂ level had an effect on growth of Leuconostoc mesenteroides on French fry simulation agar, whereby growth was slightly faster under 5% O₂. In terms of quantity, ethanol, 2‐methyl‐1‐propanol, and dimethyl disulphide were the most significant volatile metabolites produced by L. mesenteroides. The production of ethanol by L. mesenteroides was highest on simulation agar packaged under low IH O₂ levels (0% to 1%), indicating that the fermentative metabolism was induced under these conditions. In agreement with the results observed on the simulation medium, growth of native lactic acid bacteria was faster under an IH O₂ level of 5%. In addition, ethanol, 2‐methyl‐1‐propanol, and dimethyl disulphide were also quantitatively the most important volatile metabolites. However, in contrast, greater quantities of ethanol and dimethyl disulphide were produced on par‐fried French fries packaged under 5% O₂. This was attributed to the limited growth of the native flora on the par‐fried French fries under residual O₂ levels of 0% and 1%. Although some significant differences (P < 0.05) occurred between the French fries packaged in 0%, 1%, and 5 % residual O₂ during storage, all products were considered to be acceptable for consumption. The results of this study can be used to optimize the shelf‐life of packaged chill stored potato products.