Diversity of culturable microorganisms and occurrence of Listeria monocytogenes and Salmonella spp. in Tuber aestivum and Tuber melanosporum ascocarps

The aim of this study was to investigate the total mesophilic microorganisms, Pseudomonas genus, Enterobacteriaceae family, mold and yeast counts and the presence of Listeria monocytogenes and Salmonella spp on Tuber aestivum and Tuber melanosporum ascocarps. The results confirmed that the major percentage of the microorganisms, approximately 9.0 log ufc/g, were present in the peridium, the glebas of healthy truffles being practically free of microorganisms. The predominant microbial group was the Pseudomonas averaging 8.3 and 8.4 log cfu/g on T. aestivum and T. melanosporum whole ascocarps, respectively. The Enterobacteriaceae also achieved high populations, especially in T. aestivum truffles, with 6.3 log cfu/g. Molds and yeasts never exceeded 5.0 log cfu/g. The characterization of the isolates revealed that the fluorescens pseudomonads were the most prevalent. Raoultella terrigena and Enterobacter intermedius were the dominant Enterobacteriaceae. The identification of the yeast isolates revealed five species: Debaryomyces hansenii, Issatchenkia scutulata, Rhodotorula aurantiaca, Saccharomyces dairensis and Trichosporon beigelii subspecies A and B. The mold genera detected in both species of truffles were Aspergillus, Cladosporium, Penicillium and Fusarium, Trichoderma being present only in T. aestivum. L. monocytogenes was found in 10% of the samples of T. aestivum analysed but Salmonella spp. was not detected. Knowledge of the microbial population in terms of possible food borne and pathogen microorganisms is very useful for establishing successful disinfection and storage methods to prolong the shelf-life of ascocarps of T. aestivum and T. melanosporum.     

Evaluation of lactic acid bacteria and yeast diversity in traditional white pickled and fresh soft cheeses from the mountain regions of Serbia and lowland regions of Croatia

The goal of this study was the characterisation of indigenous lactic acid bacteria (LAB) and yeasts isolated from nine white pickled (BG) and nine fresh soft (ZG) artisanal cheeses collected in Serbia and Croatia. While LAB were present in all of the cheeses collected, yeasts were found in all BG cheeses but only in three ZG cheese samples. High LAB and yeast species diversity was determined (average H′_L = 0.4 and H′_Y = 0.8, respectively). The predominant LAB species in white pickled (BG) cheeses were Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides, while in fresh soft (ZG) cheeses the most dominant LAB species were L. lactis, Enterococcus faecalis, and Leuconostoc pseudomesenteroides. Among the 20 yeast species found, Debaryomyces hansenii, Candida zeylanoides, and Torulaspora delbrueckii were found to be predominant in BG cheeses, while Yarrowia lipolytica was predominant in ZG cheeses. The characterisation of metabolic and technological potentials revealed that 53.4% of LAB isolates produced antimicrobial compounds, 44.3% of LAB strains showed proteolytic activity, while most of the yeast species possessed either lipolytic or proteolytic activity. In conclusion, the results obtained in this study showed that the composition of LAB and yeast populations in white pickled and fresh soft cheeses is region specific. The knowledge gained in this study could eventually be used to select region specific LAB and yeast strains for the production of white pickled and fresh soft artisanal cheeses with geographically specific origins under controlled conditions.     

Stable and non-competitive association of Saccharomyces cerevisiae, Candida milleri and Lactobacillus sanfranciscensis during manufacture of two traditional sourdough baked goods

The microbiota occurring in all the manufacturing phases of two Italian sourdough sweet-leavened baked goods (a typical Genoese dry biscuit, Lagaccio, and a soft stuffed North Italian typical cake, Panettone) were investigated over a period of three years. The two sourdough mother sponges were characterized by the stable presence of three dominant microbial species in potential competition for carbohydrates: Lactobacillus sanfranciscensis, Candida milleri, and Saccharomyces cerevisiae. Genotypic and phenotypic characterizations of microbial isolates pointed out that each mother sponge harbored its own strains, well distinguishable by molecular methods of analysis but not differing in their main metabolic properties from those known for the corresponding species. The microbial and biochemical evolution during the whole production protocol of both manufactures demonstrated that the three microbial species grew at almost the same growth rates, without exhausting any of the main carbon substrates (maltose, glucose and fructose). The quite similar growth dynamics under practical conditions and the constant presence of all fermentable carbohydrates were recognized as responsible for the stable non competitive association of maltose-positive and maltose-negative species in both sourdoughs. However, the two sourdoughs were characterized by quite different LAB to yeast ratio, with values significantly higher in Panettone than in Lagaccio. The cause of this difference could mainly be ascribed to the temperature of the mother sponge regeneration phase, that, in the case of Panettone manufacture, occurred under conditions of moderate refrigeration.     

Isolation and quantitative determination of ergosterol peroxide in various edible mushroom species

Ergosterol peroxide, the steroidal derivative with cytotoxic activity, has been isolated for the first time from the mycelium of edible and medicinal mushroom Hericiumerinaceum (lion’s mane mushroom) together with erinacine A. The new densitometric method was applied for the quantitative determination of ergosterol peroxide in n-hexane extracts of H. erinaceum, Laetiporus sulfureus (chicken mushroom), and Morchella esculenta (common morel) mycelia, as well as in Boletus edulis (king bolete), Suillus bovinus (Jersey cow mushroom), and B. badius (bay bolete) fruiting bodies. The ergosterol peroxide content reached 15.98 ± 0.78, 10.07 ± 0.75, 13.37 ± 0.56, 29.32 ± 1.43, 17.27 ± 0.84, and 12.60 ± 0.59 mg per 100 g, respectively. What is significant was that ergosterol peroxide was identified for the first time, to the best of our knowledge, in edible mushrooms mentioned above.     

Prevalence and comparison of Streptococcus infantarius subsp. infantarius and Streptococcus gallolyticus subsp. macedonicus in raw and fermented dairy products from East and West Africa

Streptococcus infantarius subsp. infantarius (Sii) and Streptococcus gallolyticus subsp. macedonicus are members of the Streptococcus bovis/Streptococcus equinus complex (SBSEC) associated with human infections. SBSEC-related endocarditis was furthermore associated with rural residency in Southern Europe. SBSEC members are increasingly isolated as predominant species from fermented dairy products in Europe, Asia and Africa. African variants of Sii displayed dairy adaptations to lactose metabolism paralleling those of Streptococcus thermophilus including genome decay. In this study, the aim was to assess the prevalence of Sii and possibly other SBSEC members in dairy products of East and West Africa in order to identify their habitat, estimate their importance in dairy fermentation processes and determine geographic areas affected by this potential health risk. Presumptive SBSEC members were isolated on semi-selective M17 and SM agar media. Subsequent genotypic identification of isolates was based on rep-PCR fingerprinting and SBSEC-specific16S rRNA gene PCR assay. Detailed identification was achieved through application of novel primers enhancing the binding stringency in partial groES/groEL gene amplification and subsequent DNA sequencing. The presence of S. thermophilus-like lacS and lacZ genes in the SBSEC isolates was determined to elucidate the prevalence of this dairy adaptation. Isolates (n = 754) were obtained from 72 raw and 95 fermented milk samples from Côte d'Ivoire and Kenya on semi-selective agar media. Colonies of Sii were not detected from raw milk despite high microbial titers of approximately 10⁶ CFU/mL on M17 agar medium. However, after spontaneous milk fermentation Sii was genotypically identified in 94.1% of Kenyan samples and 60.8% of Kenyan isolates. Sii prevalence in Côte d'Ivoire displayed seasonal variations in samples from 32.3% (June) to 40.0% (Dec/Jan) and isolates from 20.5% (June) to 27.7% (Dec/Jan) present at titers of 10⁶–10⁸ CFU/mL. lacS and lacZ genes were detected in all Kenyan and 25.8% (June) to 65.4% (Dec/Jan) of Ivorian Sii isolates. Regional differences in prevalence of Sii and dairy adaptations were observed, but no clear effect of dairy animal, fermentation procedure and climate was revealed. Conclusively, the high prevalence of Sii in Kenya, Côte d'Ivoire in addition to Somalia, Sudan and Mali strongly indicates a pivotal role of Sii in traditional African dairy fermentations potentially paralleling that of typical western dairy species S. thermophilus. Putative health risks associated with the consumption of high amounts of live Sii and potential different degrees of evolutionary adaptation or ecological colonization require further epidemiologic and genomic investigations, particularly in Africa.     

Chemical composition,antimicrobial potential against cariogenic bacteria and cytotoxic activity of Tunisian Nigella sativa essential oil and thymoquinone

We investigate in this work the chemical composition by GC–EIMS, the antibacterial and the cytotoxic activities of Tunisian Nigella sativa essential oil and its bioactive compound, thymoquinone, were tested against various clinical cariogenic bacteria (n = 30). Eighty-four compounds were identified in the essential oil. The major one was p-cymene (49.48%) whereas thymoquinone represented only 0.79%. The essential oil (2.43 mg/disc) containing only 3.35 μg of thymoquinone showed pronounced dose dependant antibacterial activity against Streptococcus mitis, Streptococcus mutans, Streptococcus constellatus and Gemella haemolysans (15.5 ± 0.707 mm). However, pure thymoquinone compound (150 μg/disk) was active against all the studied strains especially S. mutans and S. mitis (24.5 ± 0.71 and 22 ± 1.41 mm inhibition zones, respectively).     

Chlorogenic acids and related compounds in medicinal plants and infusions

The consumption of plant infusions for prevention and treatment of health disorders is a worldwide practise. Various pharmacological activities inherent to medicinal plants have been attributed to their phenolic composition, including chlorogenic acids (CGA). Studies have shown potential beneficial properties of CGA to humans such as antioxidant, hepatoprotective, hypoglycaemic. In the present study, the CGA composition of 14 dried medicinal plants was determined by HPLC-UV and LC-DAD–ESI-MS. The plants with the highest CGA contents were Ilex paraguariensis, Bacharis genistelloides, Pimpinella anisum, Achyrochine satureioides, Camellia sinensis, Melissa officinalis and Cymbopogon citratus, with 84.7 mg/100 g–9.7 g/100 g, dry weight. Plant infusions were prepared (at 0.5%) in order to evaluate the actual consumption of CGA through these beverages. Total CGA contents in the infusions were similar to those in the methanolic extracts and indicated that a satisfactory extraction occurs during the preparation of infusions. These CGA-rich plants deserve attention regarding the pharmacological properties attributed to CGA.     

Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: A strategy to enhance acidity and improve the overall quality of wine

In the last few years there is an increasing interest on the use of mixed fermentation of Saccharomyces and non-Saccharomyces wine yeasts for inoculation of wine fermentations to enhance the quality and improve complexity of wines. In the present work Lachancea (Kluyveromyces) thermotolerans and Saccharomyces cerevisiae were evaluated in simultaneous and sequential fermentation with the aim to enhance acidity and improve the quality of wine.     

Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt--a review

Yoghurt is increasingly being used as a carrier of probiotic bacteria for their potential health benefits. To meet with a recommended level of ≥ 10⁶ viable cells/g of a product, assessment of viability of probiotic bacteria in market preparations is crucial. This requires a working method for selective enumeration of these probiotic bacteria and lactic acid bacteria in yoghurt such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lb. acidophilus, Lb. casei and Bifidobacterium. This chapter presents an overview of media that could be used for differential and selective enumerations of yoghurt bacteria. De Man Rogosa Sharpe agar containing fructose (MRSF), MRS agar pH 5.2 (MRS 5.2), reinforced clostridial prussian blue agar at pH 5.0 (RCPB 5.0) or reinforced clostridial agar at pH 5.3 (RCA 5.3) are suitable for enumeration of Lb. delbrueckii subsp. bulgaricus when the incubation is carried out at 45 °C for 72 h. S. thermophilus (ST) agar and M17 are recommended for selective enumeration of S. thermophilus. Selective enumeration of Lb. acidophilus in mixed culture could be made in Rogosa agar added with 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside (X-Glu) or MRS containing maltose (MRSM) and incubation in a 20% CO₂ atmosphere. Lb. casei could be selectively enumerated on specially formulated Lb. casei (LC) agar from products containing yoghurt starter bacteria (S. thermophilus and Lb. delbrueckii subsp. bulgaricus), Lb. acidophilus, Bifidobacterium spp. and Lb. casei. Bifidobacterium could be enumerated on MRS agar supplemented with nalidixic acid, paromomycin, neomycin sulphate and lithium chloride (MRS-NPNL) under anaerobic incubation at 37 °C for 72 h.     

Determination of viable wine yeast using DNA binding dyes and quantitative PCR

The detection and quantification of wine yeast can be misleading due to under or overestimation of these microorganisms. Underestimation may be caused by variable growing rates of different microorganisms in culture media or the presence of viable but non-cultivable microorganisms. Overestimation may be caused by the lack of discrimination between live and dead microorganisms if quantitative PCR is used to quantify with DNA as the template. However, culture-independent methods that use dyes have been described to remove the DNA from dead cells and then quantify the live microorganisms. Two dyes have been studied in this paper: ethidium monoazide bromide (EMA) and propidium monoazide bromide (PMA). The technique was applied to grape must fermentation and ageing wines. Both dyes presented similar results on yeast monitoring. Membrane cell recovery was necessary when yeasts were originated from ethanol-containing media. When applied to grape must fermentation, differences of up to 1 log unit were seen between the QPCR estimation with or without the dye during the stationary phase. In ageing wines, good agreement was found between plating techniques and QPCR. Most of the viable cells were also culturable and no differences were observed with the methods, except for Zygosaccharomyces bailii and Dekkera bruxellensis where much higher counts were occasionally detected by QPCR. The presence of excess dead cells did not interfere with the quantification of live cells with either of the dyes.     

Characterization of Longissimus thoracis,Semitendinosus and Masseter muscles and relationships with technological quality in pigs. 1. Microscopic analysis of muscles

Three porcine muscles (Longissimus thoracis, Semitendinosus, Masseter), known to have large differences in biochemical and histological traits, were fully characterized and the link between muscle structure and quality evaluated. The oxidative Masseter had more pigment, higher content of metmyoglobin, haem iron, protein and collagen, and was redder with higher fibre numbers, fibre circularity, pH and water holding capacity than the glycolytic Longissimus. Fibre type distribution showed predominance of type IIB in Longissimus and Semitendinosus white, type I in Semitendinosus red and IIA in Masseter. Type I fibres were larger than type IIB and IIA in Semitendinosus and Masseter, respectively, but not in the Longissimus, indicating that fibre size is muscle dependent. Muscle redness was positively correlated with type I fibre traits, haem iron and metmyoglobin, and negatively associated with type II fibre characteristics, non-haem iron and oxymyoglobin. Expressible juice had positive correlation with fibre size and negative with fibre number and connective tissue.     

Microbiological conditions of meats from large game animals and birds

Large game animals and birds used for the commercial production of meat include deer of various species, wild boar and feral pigs, ostriches, emus and rheas, crocodiles and alligators, bison, and kangaroos. Meat from feral pigs and kangaroos is obtained from wild animals only, but much or most meat from the other game animals or birds is obtained from farmed animals. The microbiological conditions of meats from hunted animals can be compromised by poor placement of shots, the usual evisceration and sometimes further dressing of carcass in the field, and ageing of carcasses at ambient temperatures. However, the general microbiological conditions of carcasses from farmed game animals or birds slaughtered and dressed at suitable abattoirs can be comparable with or better than the microbiological conditions of carcasses from domestic animals or birds. The incidences of enteric pathogens on meat from wild or farmed game animals or birds can be less than those for meat from intensively reared domestic animals, but infection of some game meats with Trichinella or other foodborne parasites may occur.     

Antioxidant capacity and phenolic compounds in commercially grown native Australian herbs and spices

The antioxidant capacities and phenolic composition in six native, commercially grown, Australian herbs and spices were investigated. Tasmannia pepper leaf, followed by anise myrtle and lemon myrtle contained the highest levels of total phenolics (TP; 102.1; 55.9 and 31.4 mg gallic acid equivalents (GAE)/g dry weight (DW), respectively). Tasmannia pepper leaf exhibited the highest oxygen radical absorbance capacity (ORAC assay) followed by lemon myrtle and anise myrtle. Anise myrtle exhibited the highest total reducing capacity [TRC; Ferric Reducing Antioxidant Power (FRAP) assay], followed by Tasmannia pepper leaf and lemon myrtle. Australian bush tomato, with TP content of 12.4 ± 0.9 mg GAE/gDW and TRC of 206.2 μMol Fe⁺²/gDW, resembled the Chinese Barbary Wolfberry fruit. The TP content of Tasmannia pepper berry (16.86 mg GAE/gDW) was similar to that of black pepper, but it’s TRC was 25% lower. Cinnamic acids and flavonoids, tentatively identified by mass spectrometry, were identified as the main sources of antioxidant activities.     

Alternative microbial methods: An overview and selection criteria

This study provides an overview and criteria for the selection of a method, other than the reference method, for microbial analysis of foods. In a first part an overview of the general characteristics of rapid methods available, both for enumeration and detection, is given with reference to relevant bibliography. Perspectives on future development and the potential of the rapid method for routine application in food diagnostics are discussed. As various alternative “rapid” methods in different formats are available on the market, it can be very difficult for a food business operator or for a control authority to select the most appropriate method which fits its purpose. Validation of a method by a third party, according to international accepted protocol based upon ISO 16140, may increase the confidence in the performance of a method. A list of at the moment validated methods for enumeration of both utility indicators (aerobic plate count) and hygiene indicators (Enterobacteriaceae, Escherichia coli, coagulase positive Staphylococcus) as well as for detection of the four major pathogens (Salmonella spp., Listeria monocytogenes, E. coli O157 and Campylobacter spp.) is included with reference to relevant websites to check for updates. In a second part of this study, selection criteria are introduced to underpin the choice of the appropriate method(s) for a defined application. The selection criteria link the definition of the context in which the user of the method functions – and thus the prospective use of the microbial test results – with the technical information on the method and its operational requirements and sustainability. The selection criteria can help the end user of the method to obtain a systematic insight into all relevant factors to be taken into account for selection of a method for microbial analysis.     

Phylogenetic analyses and toxigenic profiles of Fusarium equiseti and Fusarium acuminatum isolated from cereals from Southern Europe

Fusarium equiseti and Fusarium acuminatum are toxigenic species that contaminate cereal crops from diverse climatic regions. They are common in Spanish cereals. The information available on their phylogenetics and toxigenic profiles is, however, insufficient to assist risk evaluation. In this work, phylogenetic analyses were performed using partial sequences of the translation elongation factor gene (EF-1α) of F. equiseti and F. acuminatum strains isolated from barley and wheat from Spain and other countries. The Northern and Southern European F. equiseti strains largely separated into two phylogenetically distinct clusters. This suggests the existence of two distinct populations within this species, explaining its presence in these regions of markedly different climate. Production of type A and B trichothecenes by the Spanish strains, examined in wheat cultures using a multitoxin analytical method, indicated that F. equiseti could produce deoxynivalenol and nivalenol and other trichothecenes, at concentrations that might represent a significant risk of toxin contamination for Southern European cereals. F. acuminatum showed low intraspecific genetic variability and 58% of the strains could produce deoxynivalenol at low level. Neither species was found to produce T-2 or HT-2 toxins. The present results provide important phylogenetic and toxigenic information essential for the accurate prediction of toxigenic risk.     

Multiplex conventional and real-time PCR for fish species identification of Bianchetto (juvenile form of Sardina pilchardus), Rossetto (Aphia minuta), and Icefish in fresh,marinated and cooked products

A conventional and a realtime multiplex PCR were developed to detect fraudulent substitutions of Bianchetto (juvenile form of Sardina philcardus) and Rossetto (Aphia minuta) with Icefish (Neosalanx spp.), which show similar morphological characteristics. Since it is the major by-catch species, Engraulis encrasicolus was also included in the analytical procedure. A common reverse primer and forward species-specific primer were designed on the mitochondrial cytochrome b gene to amplify sequences of different lengths by conventional PCR. Specific peaks were therefore also provided after melting temperature analysis in real-time PCR, thus enabling each species to be clearly differentiated. The two PCR methods were validated on fresh and processed products after preparing four typical dishes: two marinades (from raw or lightly boiled fish), a pasta sauce, and batter-fried fish cakes. All samples were correctly identified, although there was some DNA degradation after processing.     

Effect of antioxidants and competing mycoflora on Fusarium verticillioides and F. proliferatum populations and fumonisin production on maize grain

This study was carried out to determine the temporal effect of the antioxidants butylated hydroxyanisol (BHA) and propyl paraben (PP) at doses of 500 and 1000 μg/g on the growth of Fusarium verticillioides and F. proliferatum inoculated on natural maize grain in the presence of the competing mycoflora and fumonisin production at 0.98 and 0.95 water activity (a_w) over a 28-day storage period. The reduction in the log colony forming units (CFU) of Penicillium, Aspergillus and Fusarium populations was 10-100 fold depending on dose of BHA or PP, a_w and time. However, the populations of all three groups were higher at 0.98 a_w than 0.95 a_w. BHA at 500 μg/g and 0.95 a_w reduced the fumonisin content by 82% after 7-14 days incubation, but at the end of the experimental period the reduction was only 32%. A higher reduction in the level of fumonisin produced (77%) was achieved with BHA at 1000 μg/g after 28 days. PP at 500 and 1000 μg/g decreased fumonisin production throughout the incubation period in the drier treatment, but at 0.98 a_w control of toxin production was only achieved after 7-14 days. The reduction in the fumonisin levels could be due to the combined effect of antioxidants, and the competing mycoflora, mainly Aspergillus and Penicillium species.     

Disruption of ptsG gene and manXYZ operon of ethanol-producing Escherichia coli KO11: Effects on glucose and xylose utilization and ethanol production

Ethanol-producing Escherichia coli strain KO11 consumed 99% of the glucose and only 13% of the xylose in a mixture of glucose (60g/L) and xylose (40g/L) during the 72-h fermentation at 30°C. The deletion mutants ΔptsG, ΔmanXYZ, and ΔptsG/manXYZ utilized 42%, 78%, and 35% of the glucose and 50%, 32%, and 32% of the xylose, respectively.     

Chemical composition and insecticidal properties of some aromatic herbs essential oils from Algeria

The biological effects on Sitophilus granarius were evaluated for three aromatic herbs essential oils: Foeniculum vulgare, Rosmarius officinalis and Lippia citriodora. Stored grain pests were currently controlled by chemical pesticides. This control method leads to pollution of the environment and intoxication of consumers. Essential oils of aromatic plants are more considered as good control alternative tools. Filter papers treated with 5, 50 and 500 μl of test oil were placed in the bottom cover of 1 l plastic bottle. The insects, 50 adults per bottle, were exposed for 1–5 days. Cumulative mortalities were determined 24 and 120 h after treatment. All treatments were replicated five times. The components of the essential oils were identified through GC and GC–MS. The identity of the constituents was confirmed and their relative proportions determined. The results indicate that these natural products may find potential application as useful, environmentally safe insect control and crop protectant agents.     

Assessment of the microbiological safety of dried spices and herbs from production and retail premises in the United Kingdom

A study of dried spices and herbs from retail and production premises to determine the microbiological status of such products was undertaken in the UK during 2004. According to EC Recommendation 2004/24/EC and European Spice Association specifications, 96% of 2833 retail samples and 92% of 132 production batches were of satisfactory/acceptable quality. Salmonella spp. were detected in 1.5% and 1.1% of dried spices and herbs sampled at production and retail, respectively. Overall, 3.0% of herbs and spices contained high counts of Bacillus cereus (1%, ≥10⁵ cfu g⁻¹), Clostridium perfringens (0.4%, ≥10³ cfu g⁻¹) and/or Escherichia coli (2.1%, ≥10² cfu g⁻¹). Ninety percent of samples examined were recorded as being ‘ready-to-use’, 96% of which were of satisfactory/acceptable quality. The potential public health risk of using spices and herbs as an addition to ready-to-eat foods that potentially undergo no further processing is therefore highlighted in this study. Prevention of microbial contamination in dried herbs and spices lies in the application of good hygiene practices during growing, harvesting and processing from farm to fork, and effective decontamination. In addition, the importance of correct food handling practices and usage of herbs and spices by end users cannot be overemphasised.