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.     

Comparison of bioactive phytochemical content and release of isothiocyanates in selected brassica sprouts

The consumption of brassica sprouts as raw vegetables provides a fair amount of glucosinolates (GLs) and active plant myrosinase, which enables the breakdown of GLs into health-promoting isothiocyanates (ITCs). This study reports the determination of the main constituents related to human health found in edible sprouts of two Brassica oleracea varieties, broccoli and Tuscan black kale, and two Raphanus sativus varieties, Daikon and Sango. Radish sprouts exhibited the highest ability to produce ITCs, with Daikon showing the greatest level of conversion of GLs into bioactive ITCs (96.5%), followed by Sango (90.0%). Tuscan black kale gave a value of 68.5%, whereas broccoli displayed the lowest with 18.7%. ITCs were not the exclusive GL breakdown products in the two B. oleracea varieties, since nitriles were also produced, thus accounting for the lower conversion observed. Measuring the release of plant ITCs is a valuable tool in predicting the potential level of exposure to these bioactive compounds after the consumption of raw brassica sprouts.     

Microdroplet-based multiplex PCR on chip to detect foodborne bacteria producing biogenic amines

The development of fast, reliable and culture-independent molecular tools to detect bacteria producing biogenic amines deserves the attention of research and ultimately of the food industry in order to protect consumers' health. Here we present the application of a simple, low-cost, fast and sensitive method to perform microdroplet-based multiplex PCR, directly on a food matrix, for the simultaneous detection of bacterial genes involved in biogenic amine biosynthesis. After inoculating wine with Lactobacillus brevis IOEB 9809, cell lysis and DNA amplification are performed in one single step, without preliminary nucleic acid extraction or purification treatments. The assay is performed in about 30 min, requiring 150 nL of starting sample and it enables the detection of down to 15 bacterial cells. With respect to traditional culture techniques, the speed, the simplicity and the cheapness of this procedure allow an effective monitoring of microbial cells during food-making and processing.     

Development of Real-time PCR Assays for the Detection of the <Emphasis Type="Italic">pin</Emphasis> II Terminator (t<Emphasis Type="Italic">pin</Emphasis>II) Used in GM Constructs and Its Donor Organism,Potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>)

Two singleplex TaqMan methods were developed for the detection of potato targets: one for the detection of the tpinII terminator, which is an emerging terminator used in GM constructs, and one for the detection of the endogenous StLS gene of potato. Performance criteria such as specificity and sensitivity were successfully tested for the two methods, taking into account the recommendations of international guidelines. The presence of the StLS target was checked in 16 potato cultivars. The StLS target is present at low copy number and can be used for quantitation purposes, as demonstrated on transgenic potatoes in this paper. The StLS target is an excellent candidate to replace the presently recommended endogenous target based on the UGP gene, which shows several disadvantages due to its high copy number and lack of specificity. The research also indicates that DNA can easily be extracted from different parts of potato tubers with a classical cetyltrimethylammonium bromide method.     

The potential of volatile organic compound analysis in cervicovaginal mucus to predict estrus and ovulation in estrus-synchronized heifers

Cervicovaginal mucus is a mixture of mucins, ions, salts, and water, the proportions of which change during the reproductive cycle. It is suspected that this mucus emits an important volatile signal indicative of the reproductive state of the female. The objective of this study was to identify volatile organic compounds (VOC) in bovine cervicovaginal mucus that are modulated during the estrous cycle and could potentially be used as biomarkers of estrus and ovulation. Cervicovaginal mucus was collected from crossbred beef heifers (n = 8), which were synchronized using an 8-d controlled internal drug release (CIDR) protocol and in which onset of estrus and time of ovulation were determined by visual observation and ultrasonography, respectively. Mucus samples were collected between 0 and 96 h after CIDR removal (estrus onset occurred at 49.1 ± 3.3 h after CIDR removal). A validation study was performed on an independent group of 15 heifers from which cervicovaginal mucus samples were collected every 8 h from 40 to 80 h after CIDR removal. The VOC in mucus were identified using gas chromatography-mass spectrometry and selected compounds were quantified using selected-ion flow-tube mass spectrometry. The presence of 47 VOC was detected in mucus samples by gas chromatography-mass spectrometry with those exhibiting highest abundance including 2-butanone, acetone, 2-pentanone, 4-methyl-2-pentanone, 1-(1-methylethoxy)-2-propanone, ethanol, 2-methyl-2-propanol, and 2-butanol. All VOC peaked between 24 to 47 h after the onset of estrus (ovulation occurred 26.6 ± 5.6 h after estrus onset). Two VOC, 2-pentanone and 4-methyl-2-pentanone, exhibited a significant increase at the onset of estrus, whereas concentration of 2-butanone increased significantly just after estrus onset, indicating that these VOC may be used as putative biomarkers of estrus. The results of our study may contribute to the development of a sensor device based on VOC to aid the detection of estrus and ovulation in cattle, with particular relevance for the dairy industry where the majority of females are bred by artificial insemination.     

Use of PCR-based methods and PFGE for typing and monitoring homofermentative lactobacilli during Comté cheese ripening

In order to obtain a better understanding of the biochemical events taking place in Saccharomyces cerevisiae during the lag phase, the proteins expressed during the first hours after inoculation were investigated by two-dimensional (2-D) gel electrophoresis and compared to those expressed in late respiratory growth phase. The studies were performed on a haploid strain (S288C) grown in defined minimal medium. Some of the abundant proteins, whose expression relative to total protein expression was induced during the lag phase, were identified by MALDI MS, and the expression of the corresponding genes was assessed by Northern blotting. The rate of protein synthesis was found to increase strongly during the lag phase and the number of spots detected on 2-D gels increased from 502 spots just after inoculation to 1533 spots at the end of the lag phase. During the first 20 min, the number of detectable spots was considerably reduced compared to the number of spots detected from the yeast in respiratory growth just prior to harvest and inoculation (747 spots), indicating an immediate pausing or shutdown in synthesis of many proteins just after inoculation. In this period, the cells got rid of most of their buds. The MALDI MS-identified, lag phase-induced proteins were adenosine kinase (Ado1p), whose cellular role is presently uncertain, cytosolic acetaldehyde dehydrogenase (Ald6p) and (DL)-glycerol-3-phosphatase 1, both involved in carbohydrate metabolism, a ribosomal protein (Asc1p), a fragment of the 70-kDa heat shock protein Ssb1, and translationally controlled tumour protein homologue (Yk1056cp), all involved in translation, and S-adenosylmethionine synthetase I involved in biosynthesis reactions. The level of mRNA of the corresponding genes was found to increase strongly after inoculation. By pattern matching using previously published 2-D maps of yeast proteins, several other lag phase-induced proteins were identified. These were also proteins involved in carbohydrate metabolism, translation, and biosynthesis reactions. The identified proteins together with other, yet unidentified, lag phase-induced proteins are expected to be important for yeast growth initiation and could be valuable biological markers for yeast performance. Such markers would be highly beneficial in the control and optimisation of industrial fermentations.     

Estrogenic activity and steroid hormones in swine wastewater through a lagoon constructed-wetland system

Anaerobic lagoons and treatment wetlands are used worldwide to treat wastewater from dense livestock production facilities; however, there is very limited data on the hormonal activity of the wastewater effluent produced by these treatment systems. The objectives of this experiment were to measure (1) the hormonal activity of the initial effluent and (2) the effectiveness of a lagoon-constructed wetland treatment system for producing an effluent with a low hormonal activity. Wastewater samples were taken in April, July, and November 2004 and July 2005 from a lagoon-constructed wetland system at a swine farrowing facility. Estrogenic activity (in vitro E-screen assay), 17 beta-estradiol (E2), and testosterone concentrations (LC/MS-MS) were measured. A high correlation was found between estradiol equivalents determined by E-screen and LC/MS-MS (R2 = 0.82). Nutrient removal was measured to ensure that the wetlands were functioning in a manner similar to literature reports. Nutrient removals were typical for treatment wetlands: TKN 59-75% and orthophosphate 0-18%. Wetlands decreased estrogenic activity by 83-93%. Estrone was the most persistent estrogenic compound. Constructed wetlands produced effluents with estrogenic activity below the lowest equivalent E2 concentration known to have an effect on fish (10 ng/L or approximately 37 x 10(-12) M).     

Yoghurt fermented by Lactobacillus delbrueckii subsp. bulgaricus H+ -ATPase-defective mutants exhibits enhanced viability of Bifidobacterium breve during storage

Persistent acid production by Lactobacillus delbrueckii subsp. bulgaricus during refrigerated storage is a major cause of reduced viability of probiotic strains such as Bifidobacterium breve in yoghurt. It was established that H+ -ATPase-defective mutants of lactic acid bacteria have reduced growth and metabolism in low pH environments. Therefore, the aim of this study was to evaluate inhibition of post-acidification and maintenance of B. breve viability in yoghurt fermented by L. delbrueckii subsp. bulgaricus mutants with reduced membrane-bound H+ -ATPase activity during refrigerated storage. Spontaneous neomycin mutants of L. delbrueckii subsp. bulgaricus that had a significantly (P < or = 0.05) reduced H+ -ATPase activity were successfully isolated. Yoghurt fermented using L. delbrueckii subsp. bulgaricus SBT0164 No. 55-1 (mutant) starter culture had markedly reduced post-acidification and maintained viability (> or = 10(8) CFU/ml) of both Bifidobacteruim breve JCM 1192(T) and Bifidobacteruim breve JCM 7017 during storage at 10 degrees C for 21 days. These results clearly showed that yoghurt fermented by mutants of L. delbrueckii subsp. bulgaricus with reduced membrane-bound H+ -ATPase activity has reduced post-acidification that prolongs viability of B. breve in yoghurt during refrigerated storage.     

Development of an epitope-specific analytical tool for the major peanut allergen Ara h 2 using a high-density multiple-antigenic peptide strategy

Using the major peanut allergen Ara h 2 as an example, an analytical tool enabling the determination of immunoglobulin E (IgE)-epitopes in processed food allergens was developed. We synthesized a multiple-antigenic peptide (MAP) of the IgE-reactive linear epitope 3 (amino acid positions 27-36) of Ara h 2 and raised a monospecific antiserum against this epitope to obtain a positive control for future epitope resolved diagnostics. First, a MAP of epitope 3, having a molecular mass of 7770 Da, was synthesized, purified, and its structure confirmed by liquid chromatography-mass spectrometry (electrospray ionization) (LC-MS(ESI)), matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), and Edman sequencing. The MAP was then used to raise high titer antibodies in rabbits using the adjuvant Titermax and to characterize the specificity of IgE from allergenic patients sensitized to Ara h 2. The antiserum exclusively detects Ara h 2 in crude peanut extract with a titer of 10(7) by Western blot and reacts specifically with epitope 3 shown by epitope mapping for a library of solid-phase-bound synthetic 15-mer peptides covering the entire sequence of Ara h 2. Such IgE-reactive epitopes are of high analytical relevance as they could constitute the basis for epitope-specific detection systems for use in quality control in the food industry or for forensic purposes in cases of fatal reactions to otherwise undetected peanut proteins.