Molecular identification and relative abundance of microorganisms in douchi koji and salted egg white sufu during processing

A novel animal protein-based douchi koji-inoculated steamed salted egg white sufu (SEWS) has been developed. This study determined the relative abundance of microorganisms in the douchi koji and semi-finished (5-day fermentation) and finished (5-day fermentation and 14-day ripening) SEWS by using 16S and 18S ribosomal DNA and gene-cloning methods. The results revealed that Bacillus spp. and Aspergillus oryzae were dominant in the douchi koji. In the semi-finished SEWS, the percentages of Bacillus amyloliquefaciens and Bacillus subtilis were considerably lower, whereas those of Enterococcus and Staphylococcus were substantially higher. In the finished SEWS, Bacillus spp. became dominant again and A. oryzae was the only fungus detected. In conclusion, by using molecular techniques, microbial population dynamics in SEWS can be evaluated. During processing, the relative abundance of microorganisms in SEWS changed and Bacillus spp. and A. oryzae remained dominant. This study provides crucial information for designing starter cultures for producing SEWS.     

Efficacy of avermectin B1 given orally against equine intestinal strongyles and Onchocera microfilaria

Three groups of horses and ponies (N = 13, 13 and 12) were treated with ivermectin paste (0.2 mg/kg p.o.), avermectin B1 solution (0.2 mg/kg p.o.), or fenbendazole suspension (10 mg/kg via nasogastric tube). The avermectin B1 was a 1% solution in a propylene glycolglycerol formal base. Faecal strongyle egg counts were performed before, and 14, 28, 42, 56 and 70 d, after treatment. Full-thickness skin biopsies from the neck, pectoral and umbilical regions were examined for Onchocera microfilaria before treatment, and again 14 and 70 d later. Ivermectin therapy produced a significant (P less than 0.01) decrease in mean strongyle egg counts 14, 28, 42 and 56 d after treatment. Avermectin B1 therapy resulted in significant (P less than 0.01) decreases in mean strongyle egg counts 14, 28 and 42 d after treatment. All horses given ivermectin or avermectin B1 had zero strongyle egg counts 14 and 28 d after treatment. Fenbendazole failed to significantly decrease strongyle egg counts. Both ivermectin and avermectin B1 resulted in zero microfilaria counts in all horses 14 d after treatment. On day 70 the percentage decrease in microfilaria counts were 100% and 99.6% respectively. Fenbendazole failed to significantly decrease microfilaria counts. The oral administration of this formulation of avermectin B1 appeared to be highly efficacious against intestinal strongyles and Onchocera microfilaria. The duration of anti-strongyle activity was, however, significantly (P less than 0.01) shorter than that of ivermectin paste.     

Monitoring distances travelled by horses using GPS tracking collars

Objective The aims of this work were to (1) develop a low-cost equine movement tracking collar based on readily available components, (2) conduct preliminary studies assessing the effects of both paddock size and internal fence design on the movements of domestic horses, with and without foals at foot, and (3) describe distances moved by mares and their foals. Additional monitoring of free-ranging feral horses was conducted to allow preliminary comparisons with the movement of confined domestic horses. Procedures A lightweight global positioning system (GPS) data logger modified from a personal/vehicle tracker and mounted on a collar was used to monitor the movement of domestic horses in a range of paddock sizes and internal fence designs for 6.5-day periods. Results In the paddocks used (0.8–16 ha), groups of domestic horses exhibited a logarithmic response in mean daily distance travelled as a function of increasing paddock size, tending asymptotically towards approximately 7.5 km/day. The distance moved by newborn foals was similar to their dams, with total distance travelled also dependent on paddock size. Without altering available paddock area, paddock design, with the exception of a spiral design, did not significantly affect mean daily distance travelled. Feral horses (17.9 km/day) travelled substantially greater mean daily distances than domestic horses (7.2 km/day in 16-ha paddock), even when allowing for larger paddock size. Conclusions Horses kept in stables or small yards and paddocks are quite sedentary in comparison with their feral relatives. For a given paddock area, most designs did not significantly affect mean daily distance travelled.     

Cloning and characterization of monacolin K biosynthetic gene cluster from Monascus pilosus

Monacolin K is a secondary metabolite synthesized by polyketide synthases (PKS) from Monascus, and it has the same structure as lovastatin, which is mainly produced by Aspergillus terreus. In the present study, a bacterial artificial chromosome (BAC) clone, mps01, was screened from the BAC library constructed from Monascus pilosus BCRC38072 genomic DNA. The putative monacolin K biosynthetic gene cluster was found within a 42 kb region in the mps01 clone. The deduced amino acid sequences encoded by the nine genes designated as mokA- mokI, which share over 54% similarity with the lovastatin biosynthetic gene cluster in A. terreus, were assumed to be involved in monacolin K biosynthesis. A gene disruption construct designed to replace the central part of mokA, a polyketide synthase gene, in wild-type M. pilosus BCRC38072 with a hygromycin B resistance gene through homologous recombination, resulted in a mokA-disrupted strain. The disruptant did not produce monacolin K, indicating that mokA encoded the PKS responsible for monacolin K biosynthesis in M. pilosus BCRC38072.     

Isotope-edited nuclear magnetic resonance: novel methodologies for investigating metabolism

While the use of NMR and stable isotopes in metabolism studies is hardly new, it is only recently that isotope-edited NMR spectroscopy has been applied in kinetic studies of glyphosate metabolism of soil microbes. NMR can detect multiple species simultaneously and non-destructively, yielding valuable information on structural identification of metabolites. T riple R esonance I sotope ED ited spectroscopy (TRIED), [²H]NMR, and [²H–¹³C] INEPT (I nsensitive N ucleus E nhancement through P olarization T ransfer) are three isotope-edited techniques which have been used in combination to examine the microbial degradation of glyphosate (N-phosphonomethylglycine). Using ¹³C- and ¹⁵N-labeled glyphosate, TRIED can detect multiple metabolites in crude matrices at submicrogram levels, an improvement over earlier techniques where milligrams were needed. It can detect 500 nanograms of ¹³C–¹⁵N-labeled compound in a crude sample (1 : 1400 mass ratio), only a few hours work being required. [²H]NMR and [²H–¹³C]INEPT were also used as complementary techniques to further examine metabolites whose ¹³C–¹⁵N bond has been cleaved. The three-isotope-edited methods produced results consistent with both radioactivity and HPLC analyses. Accordingly, we are able to detect minute levels of metabolites in the presence of complex mixtures, minimizing the costs and time of sample purification. ©1999 Society of Chemical Industry