Industrial Biotechnology

In recent years, outbreaks of infections associated with raw and minimally processed fruits and vegetables have been reported. The objective of this study was to analyse the growth/survival of Salmonella Enteritidis at spot-inoculated or stem-injected cherry tomatoes during passive modified atmosphere packaging (MAP), controlled atmosphere (CA) and to compare the results with those of air storage at 7 and 22 1C. During MAP, the gas composition equilibrated to 6% O 2 /4% CO 2 . CO 2 level was maintained as 5% through the term of CA storage at 7 and 22 1C. The results demonstrate that S. Enteritidis can survive and/or grow during the storage of tomatoes depending on the location site of the pathogen on fruit, suspension cell density and storage temperature. During MAP, CA and air storage, S. Enteritidis with initial population of 7.0 log 10 cfu/tomato survived on tomato surfaces with an approximate decrease of 4.0-5.0 log 10 cfu/ tomato in population within the storage period; however, in the case of initial population of 3.0 log 10 cfu/tomato, cells died completely on day 4 during MAP storage and on day 6 during both CA and air storage. The death rate of S. Enteritidis on the surfaces of tomatoes that were stored in MAP was faster than that of stored in air and in CA. Storage temperature was effective on the survival of S. Enteritidis for the samples stored at ambient atmosphere; cells died completely on day 6 at 7 1C and on day 8 at 22 1C. Stem scars provided protective environments for Salmonella; an approximate increase of 1.0 log 10 cfu/tomato in stem-scar population was observed during MAP, CA and air storage at 22 1C within the period of 20 days. Cells survived with no significant change in number at 7 1C. During the research, the effect of ozone treatment (5-30 mg/l ozone gas for 0-20 min) was also considered for surface sanitation before storage. Gaseous ozone treatment has bactericidal effect on S. Enteritidis, inoculated on the surface of the tomatoes and can be used for surface sanitation of S. Enteritidis on tomatoes before storage at different conditions. Ten mg/l ozone gas treatment with different time intervals of 5 and 15 min was found to be effective respectively on low and high dose inoculum levels of S. Enteritidis attached for 1 h. Another variable considered during ozone treatment was the 4 h attachment time.

Waikato Environment for Knowledge Analysis (WEKA) is a comprehensive suite of Java class libraries that implement many state-of-the-art machine learning/data mining algorithms. Non-programmers interact with the software via a user interface component called the Knowledge Explorer.

Consumers are becoming increasingly health conscious and therefore more discerning in their food choices. The production of fermented food products with elevated levels of B-vitamins increase both their commercial and nutritional value, and eliminate the need for subsequent fortification with these essential vitamins. Such novel products could reduce the incidence of inadequate vitamin intake which is common in many parts of the world, not only in developing countries, but also in many industrialised countries. Moreover, the concept of in situ fortification by bacterial fermentation opens the way for development of food products targeted at specific groups in society such as the elderly and adolescents. This review looks at how vitamin overproduction strategies have been developed, some of which have successfully been tested in animal models. Such innovative strategies could be relatively easily adapted by the food industry to develop novel vitamin-enhanced functional foods with enhanced consumer appeal.

As the structure of biofilms plays a key role in their resistance and persistence, this work presents for the first time the numerical characterization of the temporal evolution of biofilm structures formed by three Listeria monocytogenes strains on two types of stainless-steel supports, AISI 304 SS No. 2B and AISI 316 SS No. 2R. Counting methods, motility tests, fluorescence microscopy and image analysis were combined to study the dynamic evolution of biofilm formation and structure. Image analysis was performed with several well-known parameters as well as a newly defined parameter to quantify spatio-temporal distribution. The results confirm the interstrain variability of L. monocytogenes species regarding to biofilm structure and structural evolution. Two types of biofilm were observed: homogeneous or flat and heterogeneous or clustered. Differences in clusters and in attachment and detachment processes were due mainly to the topography and composition of the two surfaces although an effect due to motility was also found.

Optimisation of nutrient feeding was developed to overcome the limitation in batch fermentation and to increase the CGTase production from Bacillus sp. TS1-1 in fed batch fermentation. Optimisation of the C/N ratio in the feed stream was conducted in a 5 l fermenter, where feeding was initiated at constant rate of 0.02 h −1. In our initial screening process, the addition of nitrogen source boosted the growth of the microbes, but on the other hand reduced the CGTase production. The amount of tapioca starch and yeast extract was optimised in order to obtain a sufficient growth and thus, increased the CGTase production. Results were analysed using three-dimensional response surface plot, and the optimised values of carbon and nitrogen concentration of 3.30% (w/v) and 0.13% (w/v) were obtained, respectively. CGTase activity increased up to 80.12 U/ml, which is 13.94% higher as compared to batch fermentation (70.32 U/ml). This also led to 14.54% increment of CGTase production in fed batch culture as compared to the production before the optimisation. The CGTase activity obtained was close to the predicted value, which is 78.05 U/ml.

Hybridization probes produced from DNA sequences have proven to be a powerful tool in the rapid and sensitive analysis of natural microbial communities. By using function-specific probes, such as those identifying genes coding for photosynthesis, the potential a microbial community has for performing a given function may be rapidly determined. Gene probes have also been used in the idelatification and isolation of a specific catabolic genotype in less than one-fourth the time required for the conventional culture enrichment technique. Species-specific probes constructed from portions of genes coding for ribosomal RNA have been used for the rapid identification and enumeration of bacterial species in environmental samples. The use of reassociation kinetics as a measure of community diversity and complexity is also discussed. The successful application of this technique to community analysis may reduce the time required from 1 year, for conventional analysis, to 2 weeks.

We investigated the heat resistance of a four-strain mixture of Escherichia coli O157:H7 in raw ground beef in both the absence and presence of white and green tea powders and an apple skin extract. Inoculated meat was cooked using the sousvide technique, i.e., the meat was packaged in sterile bags and completely immersed in a circulating water bath at low temperature for a period of time. The bags were cooked for 1 h to an internal temperature of 55, 58, 60, or 62.5ЊC, and then held from 240 min at 55ЊC to 10 min at 62.5ЊC. The surviving bacteria were enumerated by spiral plating onto tryptic soy agar overlaid with sorbitol-MacConkey agar. Inactivation kinetics of the pathogens deviated from first-order kinetics. D-values (time, in minutes, required for the bacteria to decrease by 90%) in the control beef ranged from 67.79 min at 55ЊC to 2.01 min at 62.5ЊC. D-values determined by a logistic model ranged from 36.22 (D 1 , the D-value of a major population of surviving cells) and 112.79 (D 2 , the D-value of a minor subpopulation) at 55ЊC to 1.39 (D 1) and 3.00 (D 2) at 62.5ЊC. A significant increase (P Ͻ 0.05) in the sensitivity of the bacteria to heat was observed with the addition of 3% added antimicrobials. D-value reductions of 62 to 74% were observed with apple powder and 18 to 58% with tea powders. Thermal death times from this study will assist the retail food industry to design cooking regimes that ensure the safety of beef contaminated with E. coli O157:H7.

N-acetyl-β-D-hexosaminidase was purified from wheat bran and characterized. The purified enzyme showed two protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular mass of 75 and 78 kDa. The enzyme exhibited optimum pH and temperature at 5.0 and 50 o C, respectively. The enzyme was active on the substrates of p-nitrophenyl-Nacetyl-β-D-glucosaminide (pNP-GlcNAc) and p-nitrophenyl-Nacetyl-β-D-galactosaminide (pNP-GalNAc), whereas inactive on pNP-β-D-glucopyranoside, pNP-β-D-galactopyranoside, swollen chitin, and colloidal chitin, suggesting high substrate specificity. The enzyme activity for pNP-GlcNAc was stable at pH 3-6 and under 50 o C. The K m , V max and K cat for pNP-GlcNAc were 0.014 mM, 0.05 µmol/min, and 3.01×10 6 min −1 , respectively. The enzyme could be completely inhibited at 1-10 mM HgCl 2 and AgNO 3, suggesting that the intact thiol group is essential for activity. β-N-Acetylhexosaminidase from wheat bran could inhibit the conidial germination and digest the hyphae of Fusarium solani.

Background: Gold nanoparticles (AuNPs) have found wide range of applications in electronics, biomedical engineering, and chemistry owing to their exceptional opto-electrical properties. Biological synthesis of gold nanoparticles by using plant extracts and microbes have received profound interest in recent times owing to their potential to produce nanoparticles with varied shape, size and morphology. Marine microorganisms are unique to tolerate high salt concentration and can evade toxicity of different metal ions. However, these marine microbes are not sufficiently explored for their capability of metal nanoparticle synthesis. Although, marine water is one of the richest sources of gold in the nature, however, there is no significant publication regarding utilization of marine micro-organisms to produce gold nanoparticles. Therefore, there might be a possibility of exploring marine bacteria as nanofactories for AuNP biosynthesis.

Solid-state fermentation (SSF) has built up credibility in recent years in biotech industries due to its potential applications in the production of biologically active secondary metabolites, apart from feed, fuel, food, industrial chemicals and pharmaceutical products and has emerged as an attractive alternative to submerged fermentation. Bioremediation, bioleaching, biopulping, biobeneficiation, etc. are the major applications of SSF in bioprocesses which have set another milestone. Utilization of agro-industrial residues as substrates in SSF processes provides an alternative avenue and value-addition to these otherwise under-or non-utilized residues. Innovation is the key to success and it is imperative to be up-to-date with the changing demands of the industries and meet their needs for better product and services. Better understanding of biochemical engineering aspects, particularly on mathematical modeling and design of bioreactors (fermenters) has made it possible to scale-up SSF processes and some designs have been developed for commercialization, making the technology economically feasible. In future, SSF technology would be well developed at par with SmF if rationalization and standardization continues in current trend. This review describes the state-of-art scenario in totality on SSF although the focus is on the most recent developments of last 5 years or so on SSF processes and products developments.

Salmonella enteritidis (SE) is a common foodborne pathogen, the transmission of which is primarily associated with the consumption of contaminated Grade A shell eggs. In order to estimate the level of SE present in raw shell eggs, it is necessary to consider the protective effects of the egg albumin, which effectively inhibits SE growth in a time-and temperature-dependent manner. In this study, a SE growth model was produced by combining two mathematical equations that described both the extended lag phase of SE growth (food component) and a SE growth model (pathogen component). This biphasic growth model was then applied to various egg handling scenarios based on the farm-to-table continuum, including in-line and off-line processing facilities with consideration of key events in production, processing, transportation, and storage. Seasonal effects were also studied. Monte Carlo simulation was used to characterize variability in temperature and time parameter values influencing the level of SE to which individuals are exposed. The total level of SE consumed was estimated under best, most likely, and time -temperature abusive handling scenarios. The model estimated that, in most cases, there was no SE growth in contaminated eggs handled under most likely practices, because 10 -70% of the yolk membrane remained intact. Under abusive handling scenarios, complete loss of yolk membrane integrity frequently occurred by the time eggs reach the distribution phase, followed by subsequent SE growth, which was often quite rapid. In general, the effect of season and processing method (in-line vs. off-line) was minimal. Further sensitivity analysis demonstrated that the initial SE contamination level significantly influenced the final exposure levels only under no-abuse or mildly abusive conditions. The results of our study suggest that, for maximum reduction of SE exposure level, cooling strategies should not only focus on the on-farm or processing phases, but should emphasize the importance of cooling strategies at the distribution and consumer phases of the farm-to-fork continuum. D

On the study of the scientific basis for the use of products of processing of oak wood in the wine industry of the Republic of Azerbaijan Tariyel Panahov The Ministry of Agriculture of the Republic of Azerbaijan; the Centre of Agrarian Science and Innovation

Increasing its concentration from 1 8 to 5 mmol l significantly enhanced the heat inactivation rate of B. cereus F4165⁄75 spores. The thermodynamic data were used to speculate on the mechanism of heat inactivation of the spores. These thermodynamic values were not significantly different in the presence or absence of monolaurin. This suggested that the enhancing effect due to increasing concentrations of monolaurin on the thermal inactivation of spores is essentially identical at any sterilization temperature, although it allows the heating temperature for sterilization to be reduced.

The composition of flavonoids in plants is thought to be genetically determined. Thus it is characteristic for plant species. Here, proanthocyanidins are used to identify barley (Hordeum vulgare L.) varieties: six barley varieties and the corresponding malts in 61 samples of the growing years 1998-2001 from three locations in Germany are examined. Their absolute polyphenolic composition is quite similar, but the ratios of proanthocyanidins among each othertermed here "relative quantitative fingerprint" (RQF)-are characteristic for the variety independent from growing year, provenience and status (barley/malt). The analytical method presented is highly automated and robust, thus it allows analyzing a high number of samples and to detect small differences between samples unambiguously. Hence the polyphenols can be used as a tool for food tracing, to prove authenticity and to discover frauds of food from plant origin.

Background The objective of this work was to engineer Deinococcus radiodurans R1 as a microbial cell factory for the production of pinene, a monoterpene molecule prominently used for the production of fragrances, pharmaceutical products, and jet engine biofuels. Our objective was to produce pinene from glycerol, an abundant by-product of various industries. Results To enable pinene production in D. radiodurans, we expressed the pinene synthase from Abies grandis, the geranyl pyrophosphate (GPP) synthase from Escherichia coli, and overexpressed the native 1-deoxy-d-xylulose 5-phosphate synthase. Further, we disrupted the deinoxanthin pathway competing for the substrate GPP by either inactivating the gene dr0862, encoding phytoene synthase, or substituting the native GPP synthase with that of E. coli. These manipulations resulted in a D. radiodurans strain capable of producing 3.2 ± 0.2 mg/L pinene in a minimal medium supplemented with glycerol, with a yield of 0.13 ± 0.04 mg/g glycer...

The microbiological standard for detection of Salmonella relies on several cultural steps and requires more than 5 days for final confirmation, and as consequence there is a need for an alternative rapid methodology for its detection. The aim of this study was to compare different detection strategies based on real-time PCR for a rapid and sensitive detection in an ample range of food products: raw pork and poultry meat, ready to eat lettuce salad and raw sheep milk cured cheese. Three main parameters were evaluated to reduce the time and cost for final results: the initial sample size (25 and 50 g), the incubation times (6, 10 and 18 h) and the bacterial DNA extraction (simple boiling of the culture after washing the bacterial pellet, the use of the Chelex resin, and a commercial silica column). The results obtained demonstrate that a combination of an incubation in buffered peptone water for 18 h of a 25 g-sample coupled to a DNA extraction by boiling and a real-time PCR assay detected down to 2-4 Salmonella spp. CFU per sample in less than 21 h in different types of food products. This RTi-PCR-based method is fully compatible with the ISO standard, providing results more rapidly and cost-effectively. The results were confirmed in a large number of naturally contaminated food samples with at least the same analytical performance as the reference method.

In the present investigation, a previously isolated Enterococcus faecium KH 24 strain was evaluated for the presence of virulence determinants (agg, esp, efaAfm, gelE, cylA, cylB, clyM, cpd, cob, ccf, ace and hyl), sensitivity to various antibiotics and production of biogenic amines. No virulence determinants were detected, except efaAfm. KH 24 was found to be sensitive to most of the tested antibiotics and none of the biogenic amines were produced by it. Moreover, KH 24 showed good in vitro tolerance to biological barriers and furthermore, its survival in gut of mice was also evaluated. Mice group fed with E. faecium KH 24 strain showed better weight gain and nearly 1 log cfu/g decrease in Salmonella enteritidis counts in the intestines as compared to control (p b 0.05). Enhanced growth of lactobacilli (p b 0.05) and decrease in coliform counts (p b 0.05) were also observed in test group. E. faecium KH 24 is, therefore, found to be a safe strain and it may be used as protective culture or as a probiotic in food preparations.

A study of 25 paper mill slime deposits and one additive revealed nine pink-pigmented bacterial isolates, eight of which were different from pink-pigmented bacteria identified in the paper industry in the middle 1900s. The pinkpigmented bacteria described previously in pulp and paper included Micrococcus agilis, Bacillus subtilis, Serratia sp. and Alcaligenes viscosus. With the exception of one isolate, Micrococcus sp., these isolates possessed many cultural, biochemical and chemical properties which were different from the ones previously reported for paper mills. Eight of these bacteria were Gram-negative rods or filamentous, aerobic and positive for catalase production. Two isolates were methylotrophic, oxidizing methanol and identified as Methylobacterium zatmanii. Cellular fatty acid analysis and other characteristics showed one isolate to be Roseomonas sp. Using 16S rRNA gene sequencing, one isolate which was a Gram-negative rod was identified as Deionococcus grandis. Four bacteria had cells that were long or filamentous and these were isolated from mills with pink slime problems. The identity of one of the filamentous bacteria was determined by 16S rRNA gene sequencing to be close to Flectobacillus sp. strain MWH38. Most of the isolates were susceptible to 11 industrial biocides.

Minimum Inhibitory Concentrations (MICs) were determined for nisin and an aqueous garlic extract (AGE) for six strains of Listeria monocytogenes, including a nisin-resistant mutant.When used in combination in broth a synergistic e¡ect was observed between nisin and AGE in all strains. Increasing the pH reduced the activity of both nisin and garlic. During storage of the aqueous garlic extract at 48C and at 208C for14 days, the MIC increased over the ¢rst 8 days but remained stable thereafter, indicating at least two antimicrobial components, one of which was relatively stable. Aqueous garlic extract was not appreciably bactericidal although there was a positive interaction with nisin in terms of its bactericidal e¡ect in broth at 48C. The e¡ect of combining nisin and garlic in a food system, hummus, was also studied. Sub-MIC combinations of AGE and nisin were e¡ective at preventing listerial growth and did enhance the slight bactericidal e¡ect of nisin.The results indicate that combined use of nisin with garlic could help overcome problems of nisin resistance in Gram-positive organisms. Such interactions could also be a signi¢cant factor in traditional lactic fermented foods where garlic is an ingredient.

The technological effect of wheat fibre, as a healthy ingredient in which consumers are interested, was studied in surimi gel products. Three and six per cent wheat fibre with different particle sizes was added. Functional properties of wheat fibre, such as water retention capacity (WRC), swelling (SW) and fat adsorption capacity (FAC), were determined. Elastic modulus (G′) of surimi with 6% wheat fibre was lower throughout thermal gelling. Under scanning electronic microscopy (SEM), gel samples with added fibre presented uneven distribution of fibre. This induced the formation of a non-homogeneous protein net associated with lower gel strength, cohesiveness and water binding capacity (WBC). The sensory panel found no differences in appearance but differences in texture among samples containing different proportions and types of fibre. Fibre with large particle sizes protected surimi from loss of gel strength and hardness during freezing. There were very few variations during frozen storage of different samples.

The effects of normal and controlled atmosphere combinations on tuber components responsible for acrylamide formation were studied during prolonged storage at 9±1 °C using the tubers of “Agria” and “Russet Burbank.” The effects of low-dose irradiation (50, 200 Gy) prior to normal atmosphere storage were also studied. There was only a limited increase in the concentrations of sugars during 6 month of storage under normal atmosphere conditions. Low-dose irradiation slightly decreased the rate of sweetening in the tubers. The potential of acrylamide formation remained almost the same; however, the loss of firmness became clearer during 6 month of storage under normal atmosphere conditions. Controlled atmosphere storage in which O2 was deficient to a sufficient respiration increased the concentrations of sugars, and thus, the potential of acrylamide formation in potatoes upon frying at 170 °C for 10 min. The sum of glucose and fructose concentrations showed a good correlation (r 2∼0.90) with the potential of acrylamide formation for both cultivars.

In this study we identified the culturable population of mesophilic lactic acid bacteria (LAB) from a French cheese Maroilles made either with raw or pasteurized milk using MALDI-TOF mass spectrometry (MS). Samples from rind and heart of Maroilles cheese were used, the LAB were selected on MRS agar at 30°C and 197 Grampositive and catalase-negative strains were subjected to identification by MALDI-TOF MS profiling. All strains were unambiguously identified: 105 strains from Maroilles made with raw milk (38 on the rind and 67 in the heart) and 92 strains from Maroilles made with pasteurized milk (39 on the rind and 53 in the heart). MALDI-TOF MS identification allowed identification of three genera belonging to LAB including Lactobacillus, Enterococcus and Leuconostoc. Lactobacillus was the most represented genus with seven species: Lactobacillus plantarum (L. plantarum), L. paracasei, L. curvatus, L. rhamnosus, L. fructivorans, L. parabuchneri, L. brevis found in Maroilles made with both kind of milk. The correlation between the 16S rDNA-based identification performed on selected strains and those obtained by MALDI-TOF-MS demonstrates that this fast, economically affordable, robust and reliable method for bacteria characterisation stands as an attractive alternative to the commonly-used methods and its application in food industry is discussed.

Six different blends of lactic acid bacterial cultures (Lactobacillus plantarum or Pediococcus cerevisiae) combined with Saccharomyces cerevisiae were used as co-culture for preparing sourdoughs. These prefermented sourdoughs were applied for producing pan breads. Acidification power, leavening ability, and microbial counts were conducted at different sour fermentation intervals. The highest acidification power in sourdoughs were recorded when 2% wd/wd (dry weight

mThe amount of resistant starch (RS) in raw and processed legumes, lentils, chickpeas and beans was analysed. In addition, the effect of cooking on the availability of legume starches to enzymes, which may influence the glycaemic response, was studied. Raw legumes were found to have higher RS values (16±21%) than retrograded or cooked ones (4±8%). Cooling increased RS yields, whereas reheating maintained or decreased the value depending on the sample. On the other hand, the kinetics of starch hydrolysis revealed that the rate of digestion was higher for cooked seeds and retrograded flours (15±21% of total starch digested) than for raw flours (5±8% of total starch digested). The estimated glycaemic indices (GIs), i.e. GI1 from the Hydrolysis Index (HI) and GI2 from the percentage of hydrolysis within 90 min (H90), were well correlated with the reported GI values (r = 0.96 and r = 0.95 respectively, P # 0.05).

A microbial survival curve is constructed by plotting the number of recoverable cells or their logarithm vs. the exposure time to the hostile agent, be it high or low temperature, a chemical preservative or disinfectant, etc. Since the recovery is usually done in a medium and under conditions that favor growth, the result is insensitive to whether the counted survivors are intact or injured. If or when both the total number of survivors and those remaining intact follow a Weibullian decay pattern (with different parameters), then the momentary number of injured cells will be the momentary difference between the two. Such a scenario can be easily modeled mathematically and the resulting model enables to simulate a variety of survived-injury patterns in thermal and non-thermal food preservation processes. Under certain conditions according to this model, almost all the survivors would be injured to at least some extent and hence may perish during the food's storage and transportation. Isothermal survival-injury curves generated with the Weibullian model based on the above considerations were in general agreement with published experimental data. In principle, the methodology can be extended to simulate mortality-injury patterns under dynamic conditions, i.e., when the temperature or chemical agent's concentration vary with time. Whether a cell is considered injured depends on the recovery method, e.g., on whether it can or cannot grow in a saline medium. Thus recovery in different media may yield somewhat different quantitative results but very unlikely a qualitatively different pattern. Although the model used was based on that microbial mortality and injury both follow the Weibullian model, very similar results would have been obtained had other survival modes been assumed.

Growth curves were generated for Escherichia coli O157:H7 in brain±heart infusion broth incubated at 37 or158C in the presence of individual and combinations of competing micro£ora. Broths were inoculated with E. coli O157:H7 (log 10 3Á00 cfu ml 71) and competitors (log 10 4Á00 cfu ml 71) and the initial pH of the broth was either neutral (7Á0) or adjusted to 5Á8 and then sequentially reduced to 4Á8 over 10 h to simulate fermentation conditions. Growth curves were also generated for the competitors in these cultures, including Pseudomonas fragi, Hafnia alvei, Pediococcus acidilactici (pepperoni starter culture) and Brochothrix thermosphacta. Gompertz equations were ¢tted to the data and growth kinetics including lag phase duration, exponential growth rates and maximum population densities (MPD) calculated. In pure culture, the growth parameters for E. coli O157:H7 in neutral pH broths were signi¢cantly di¡erent from those recorded in simulated fermentation broths (P50Á05).The presence of competitors in the broth also had a signi¢cant e¡ect on the growth kinetics of the pathogen. H. alvei signi¢cantly inhibited the growth (lag phase, growth rate and MPD) of E. coli O157:H7 at 378C, neutral pH and outgrew the pathogen under these conditions. In neutral pH cultures, two other competitors, B. thermosphacta and P. acidilactici also inhibited the lag phase of the pathogen but had no e¡ect on the other growth parameters. In simulated fermentation broths, the growth rate of E. coli O157:H7 was consistently slower and the MPD lower in the presence of a competitive micro£ora than when grown individually. At158C, only one competitor, P. fragi signi¢cantly inhibited the lag phase of the pathogen. The implications of these ¢ndings for food safety are discussed.

In this work, we report results on the functional analysis of Saccharomyces cerevisiae ORF YGR224w, predicted to code for an integral membrane protein, with 14 potential transmembrane segments, belonging to the major facilitator superfamily (MFS) of transporters which are required for multiple-drug resistance (MDR). This MFS±MDR homologue is required for yeast adaptation to high stress imposed by low-chain organic acids, in particular by acetic acid, and for resistance to azoles, especially to ketoconazole and¯uconazole; the encoding gene was thus named the AZR1 gene. These conclusions were based on the higher susceptibility to these compounds of an azr1D deletion mutant strain compared with the wild-type and on the increased resistance of both azr1D and wildtype strains upon increased expression of the AZR1 gene from a centromeric plasmid clone. AZR1 gene expression reduces the duration of acetic acid-induced latency, although the growth kinetics of adapted cells under acetic acid stress is apparently independent of AZR1 expression level. Fluorescence microscopy observation of the distribution of the Azr1±GFP fusion protein in yeast living cells indicated that Azr1 is a plasma membrane protein. Studies carried out to gain some understanding of how this plasma membrane putative transporter facilitates yeast adaptation to acetic acid did not implicate Azr1p in the alteration of acetic acid accumulation into the cell through the active ef¯ux of acetate.

The use of moulds as a seasoning for sausage can have both desirable and undesirable consequences. The desirable consequences are the creation of a successful product that appeals to consumers. The undesirable consequences are due to the growth of undesirable moulds that produce highly toxic secondary metabolites referred to as mycotoxins. The aim of the paper was to investigate the presence of moulds producing ochratoxin A (OTA) on the surface of sausages from northern Italy. A total of 757 mould strains were isolated from sausage casings. The most frequently identified species were Penicillium nalgiovense, Penicillium oxalicum, Eurotium amstelodami, Penicillium olsonii, Penicillium chrysogenum, Penicillium verrucosum, Penicillium viridicatum, and Eupenicillium crustaceum. Aspergillus ochraceus was detected in only one production lot. Approximately 45% of these samples were positive for the presence of OTA. On the casings of the investigated sausages, the lowest and highest OTA values were 3 and 18 mg/kg, respectively.

A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray, has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target elements first amplified by PCR, followed by direct hybridisation of the amplicons on a predefined microarray (DualChip Ò GMO, Eppendorf, Germany). The validation was performed within the framework of a European project (Co-Extra, contract no 007158) and in collaboration with 12 laboratories specialised in GMO detection. The present study reports the strategy and the results of an ISO complying validation of the method carried out through an inter-laboratory study. Sets of blind samples were provided consisting of DNA reference materials covering all the elements detectable by specific probes present on the array. The GMO concentrations varied from 1% down to 0.045%. In addition, a mixture of two GMO events (0.1% study to test the robustness of the assay in extreme conditions. Data were processed according to ISO 5725 standard. The method was evaluated with predefined performance criteria with respect to the EC CRL method acceptance criteria. The overall method performance met the acceptance criteria; in particular, the results showed that the method is suitable for the detection of the different target elements at 0.1% concentration of GMO with a 95% accuracy rate. This collaborative trial showed that the method can be considered as fit for the purpose of screening with respect to its intra-and inter-laboratory accuracy. The results demonstrated the validity of combining multiplex PCR with array detection as provided by the DualChip Ò GMO (Eppendorf, Germany) for the screening of GMO. The results showed that the technology is robust, practical and suitable as a screening tool.

Biofilms have been of considerable interest in the context of food hygiene. Of special significance is the ability of microorganisms to attach and grow on food and food-contact surfaces under favourable conditions. Biofilm formation is a dynamic process and different mechanisms are involved in their attachment and growth. Extracellular polymeric substances play an important role in the attachment and colonization of microorganisms to food-contact surfaces. Various techniques have been adopted for the proper study and understanding of biofilm attachment and control. If the microorganisms from food-contact surfaces are not completely removed, they may lead to biofilm formation and also increase the biotransfer potential. Therefore, various preventive and control strategies like hygienic plant lay-out and design of equipment, choice of materials, correct use and selection of detergents and disinfectants coupled with physical methods can be suitably applied for controlling biofilm formation on food-contact surfaces. In addition, bacteriocins and enzymes are gaining importance and have an unique potential in the food industry for the effective biocontrol and removal of biofilms. These newer biocontrol strategies are considered important for the maintenance of biofilm-free systems, for quality and safety of foods.

We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4 + gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C-or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C-or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was d50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe.

In previous studies it was shown that deletion of the HXK2 gene in Saccharomyces cerevisiae yields a strain that hardly produces ethanol and grows almost exclusively oxidatively in the presence of abundant glucose. This paper reports on physiological studies on the hxk2 deletion strain on mixtures of glucose/sucrose, glucose/galactose, glucose/ maltose and glucose/ethanol in aerobic batch cultures. The hxk2 deletion strain co-consumed galactose and sucrose, together with glucose. In addition, co-consumption of glucose and ethanol was observed during the early exponential growth phase. In S. cerevisiae, co-consumption of ethanol and glucose (in the presence of abundant glucose) has never been reported before. The specific respiration rate of the hxk2 deletion strain growing on the glucose/ethanol mixture was 900 mmolemin x1 e(g protein) x1 , which is four to five times higher than that of the hxk2 deletion strain growing oxidatively on glucose, three times higher than its parent growing on ethanol (when respiration is fully derepressed) and is almost 10 times higher than its parent growing on glucose (when respiration is repressed). This indicates that the hxk2 deletion strain has a strongly enhanced oxidative capacity when grown on a mixture of glucose and ethanol.

The antioxidant and antimicrobial activity of Myrtus communis extract was determined before and after its encapsulation in liposomes of diVerent composition. Evaluation of the sunXower oil oxidation by the Rancimat stability test and malondialdehyde formation by HPLC were used to measure the antioxidant action in comparison with common commercial antioxidants, such as butylated hydroxytoluene andtocopherol. The thermal-oxidative decomposition of the samples, the modiWcation of the main transition temperature for the lipid mixture and the splitting of the calorimetric peak in the presence of the antioxidants were studied by diVerential scanning calorimetry. The size and the surface charge of liposomes were also studied. The extract showed antioxidant and antimicrobial activity. t concentrations up to 160 ppm, the extract showed superior activity than -tocopherol. When the extract was encapsulated in liposomes, its antioxidant as well as its antimicrobial activity proved to be superior from that of itself in pure form.

The occurrence of d-amino acids in native and processed plant products is brought into context with the harshness of their treatment condition. It was found that already a small increase in processing harshness such as the milling efficiency of wheat straw or the increased pressure and duration of pumpkin seed oil extraction leads to traceable changes in the overall amino acid content as well as the racemization rate of free and protein bound amino acids.

The effects of ozone in aqueous solution on the shelf life of whole, vacuum-packaged rainbow trout, stored under refrigeration (4±0.5 • C) were studied by monitoring the microbiological, chemical and sensory changes for a period of 15 days. Vacuum-packaged non-ozonated trout served as the control sample. Ozonation affected populations of bacteria namely, mesophilic aerobic bacteria, Pseudomonas spp. and H 2 S-producing bacteria until day 11 of storage, Brochothrix thermosphacta, lactic acid bacteria and Enterobacteriaceae until day 8 of storage. Trimethylamine (TMA) values of all rainbow trout samples remained low (<3 mg N/100 g) until day 11 of storage, and then increased to 12.2, 8.9 and 4.7 mg N/100 g for the control and the samples ozonated for 60 and 90 min, respectively on day 15 of storage. Total volatile basic nitrogen (TVB-N) values remained relatively constant (20-25 mg N/100 g) until day 11 of storage, but increased to 61.1, 37.6 and 39.4 mg N/100 g flesh for the control and ozonated specimen for 60 and 90 min, respectively on day 15 of storage. Thiobarbituric acid (TBA) values remained relatively constant (1-3 mg MA/kg flesh) until day 12 of storage but increased to 8.4, 6.4 and 3.8 mg MA/kg flesh on day 15 of storage for the control and the ozonated trout for 60 and 90 min, respectively. Sensory evaluation (odor, taste and texture) of cooked rainbow trout showed a very good correlation with bacterial populations. On the basis of both sensory and microbiological data, a shelf life of 10 and 12 days was obtained for ozonated, vacuum-packaged and refrigerated rainbow trout at 60 and 90 min, respectively.

Ethnicity can confound results in pharmacogenomic studies. Allele frequencies of loci that influence drug metabolism can vary substantially between different ethnicities and underlying ancestral genetic differences can lead to spurious findings in pharmacogenomic association studies. We evaluated the application of principal component analysis (PCA) in a pharmacogenomic study in Canada to detect and correct for genetic ancestry differences using genotype data from 2094 loci in 220 key drug biotransformation genes. Using 89 Coriell worldwide reference samples, we observed a strong correlation between principal component values and geographic origin. We further applied PCA to accurately infer the genetic ancestry in our ethnically diverse Canadian cohort of 524 patients from the GATC study of severe adverse drug reactions. We show that PCA can be successfully applied in pharmacogenomic studies using a limited set of markers to detect underlying differences in genetic ancestry thereby maximizing power and minimizing false-positive findings.

Milk samples from 248 Pag ewes, belonging to 14 different flocks and located through the Pag Island (Croatia), were analyzed by isoelectrofocusing and PCR-RFLP. Two genetic variants (A and B) and three genotypes (AA, AB and BB) of b -lactoglobulin have been identified. According to the allele frequency (A=0.48, B=0.52) and occurrence of genetic variants, the Pag breed is similar to other Mediterranean dairy sheep breeds. The observed genotype frequencies at the b -lactoglobulin locus (AA=0.185, AB=0.589 and BB=0.226) were significantly different from those expected from Hardy-Weinberg equilibrium. In comparison with 12 different studies related to Mediterranean dairy sheep populations, the significant departure from Hardy-Weinberg was also obtained in Valle del Belice sheep. However, the sample size required for detection of the same amount of departure from the Hardy-Weinberg as it was observed in Pag sheep was only sufficient in four studies (including Pag sheep). The superiority of the AB b -lactoglobulin genotype for milk production could be one of possible reasons for the observed excess of heterozygotes and allele frequencies at intermediate level.

The effect of different sulphur dioxide concentrations on culturability and viability of seven strains of Brettanomyces bruxellensis was tested in a synthetic wine medium (SWM) and a different response to molecular SO 2 among strains was detected. Sulphur dioxide induced a viable but non culturable (VBNC) state in all the strains. The greater percentage of VBNC cells were identified for five strains at molecular SO 2 concentrations of 0.2 mg/L and for two strains at the concentration of 0.4 mg/L. Vinyl phenols were detected in media containing VBNC or not viable B. bruxellensis, suggesting that its spoilage metabolism could be maintained during wine storage. Overall, this study indicates that SO 2 is a chemical stressor inducing VBNC state in B. bruxellensis grown in synthetic wine medium. Further studies are needed to evaluate the effects of SO 2 on the metabolism of this yeast in wine spoilage.

Xylanase was produced by Aspergillus niger LPB 326 cultivated on lignocellulosic substrate composed by sugarcane bagasse and soybean meal in solid-state fermentation. The effects of various variables were observed and optimized by applying statistical experimental designs. The best xylanase activity was obtained in a medium containing 10 g of sugarcane bagasse and soybean meal in the ratio of 65 and 35 %, respectively, moistened to 85 % of initial water content with a nutrient salt solution composed of (in g/L): CuSO 4 0.4, KH 2 PO 4 1.5 and CoSO 4 0.0012, and incubated for 4 days at 30°C. Under these optimized conditions, a xylanase activity of 3099 IU/g of dry matter was obtained.

Sodium hypochlorite and sodium chlorite are commonly used as disinfectants, and understanding the mechanisms of microbial resistance to these compounds is of considerable importance. In this study, the role of oxidative stress and antioxidant enzymes in the sensitivity of the yeast Saccharomyces cerevisiae to hypochlorite and chlorite was studied. Yeast mutants lacking Cu-Zn superoxide dismutase, but not mutants deficient in cytoplasmic and peroxisomal catalase, were hypersensitive to the action of both hypochlorite and chlorite. Both compounds depleted cellular glutathione, induced the production of reactive oxygen species and decreased the viability of the cells. The toxicity of hypochlorite and chlorite was abolished by hypoxic and anoxic conditions and ameliorated by thiol antioxidants and ascorbate. The results demonstrated that the action of hypochlorite and chlorite involves the formation of superoxide and peroxide and that SOD1 is protective, probably by limiting the formation of hydroxyl radicals and damage to proteins.

Bifidobacteria represent important human commensals because of their perceived contribution to the maintenance of a balanced gastro intestinal tract (GIT). In recent years bifidobacteria have drawn much scientific attention because of their use as live bacteria in numerous food preparations with various health-related claims. For such reasons these bacteria constitute a growing area of interest with respect to genomics, molecular biology and genetics. This review will discuss the current knowledge on the molecular players that allow bifidobacteria to contend with heat-, osmotic-, bile-and acidic stress. Here, we describe the principal molecular chaperones involved in such stresses, as well as their use as phylogenetic markers for gaining insight into the evolutionary history of high G+C Gram positive bacteria.