Exploring the characteristics of Burkholderia gladioli pathovar cocovenenans: Growth, bongkrekic acid production, and potential risks of food contamination in wet rice noodles and vermicelli.

This research investigated the presence of Burkholderia gladioli pathovar cocovenenans (BGC) in wet rice and starch products, Tremella, and Auricularia auricula in Guangzhou, China. It examined BGC growth and bongkrekic acid (BA) production in wet rice noodles and vermicelli with varying rice flour, edible starch ratios, and oil concentrations. A qualitative analysis of 482 samples revealed a detection rate of 0.62%, with three positive for BGC. Rice flour-based wet rice noodles had BA concentrations of 13.67 ± 0.64 mg/kg, 2.92 times higher than 100% corn starch samples (4.68 ± 0.54 mg/kg). Wet rice noodles with 4% soybean oil had a BA concentration of 31.72 ± 9.41 mg/kg, 5.74 times higher than those without soybean oil (5.53 ± 1.23 mg/kg). The BA concentration correlated positively (r = 0.707, P < 0.05) with BGC contamination levels. Low temperatures (4 °C and -18 °C) inhibited BGC growth and BA production, while higher storage temperatures (26 °C and 32 °C) promoted BGC proliferation and increased BA production. Reducing edible oil use and increasing edible starch can mitigate the risk of BGC-related food poisoning in wet rice noodles and vermicelli production. Further research is needed to find alternative oils that do not enhance BA production. Strengthening prevention and control measures is crucial across the entire production chain to address BGC contamination and BA production.

Pan-genome analysis of the Burkholderia gladioli PV. Cocovenenans reveal the extent of variation in the toxigenic gene cluster.

Burkholderia gladioli has been reported as the pathogen responsible for cases of foodborne illness in many countries. The poisonous bongkrekic acid (BA) produced by B. gladioli was linked to a gene cluster absent in non-pathogenic strains. The whole genome sequence of eight bacteria strains, which were screened from the collected 175 raw food and environmental samples, were assembled and analyzed to detect a significant association of 19 protein-coding genes with the pathogenic status. Except for the common BA synthesis-related gene, several other genes, including the toxin-antitoxin genes, were also absent in the non-pathogenic strains. The bacteria strains with the BA gene cluster were found to form a single cluster in the analysis of all B. gladioli genome assemblies for the variants in the gene cluster. Divergence of this cluster was detected in the analysis for both the flanking sequences and those of the whole genome level, which indicates its complex origin. Genome recombination was found to cause a precise sequence deletion in the gene cluster region, which was found to be predominant in the non-pathogenic strains indicating the possible effect of horizontal gene transfer. Our study provided new information and resources for understanding the evolution and divergence of the B. gladioli species.

Development and validation of a multianalyte method for quantification of aflatoxins and bongkrekic acid in rice and noodle products using PRiME-UHPLC-MS/MS method.

An analytical method based on PRiME (process, robustness, improvements, matrix effects, ease of use) HLB purification followed by the ultra-high liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection has been developed for the determination aflatoxin B1, B2, G1 and G2 and bongkrekic acid in rice and noodle products. Five toxins were separated on a Waters BEH C18 column by gradient elution, scanned by ESI+ and ESI- dynamic switching and detected with MRM mode. LOD, LOQ, matrix effects, accuracy and precision of the developed method were investigated. Under the optimal sample pretreatment conditions, high sensitivity (LOQs: 0.20-0.40 µg/kg), good recoveries (80.5%-106.6%) and acceptable precision (2.4%-7.2%) were obtained for the analysis of the four aflatoxins and bongkrekic acid. This method was successfully applied to the analysis of rice and noodle products, demonstrating its applicability and suitability for the routine analysis of aflatoxins and bongkrekic acid in rice and noodle products.

Determination of bongkrekic acid and isobongkrekic acid in rice noodles by HPLC-Orbitrap HRMS technology using magnetic halloysite nanotubes.

The existing extraction and detection methods of bongkrekic acid (BKA) and isobongkrekic acid (IBKA) are complex, time-consuming and solvent-consuming. In this work, a simple and fast pre-concentration procedure based on Fe3O4/HNTs was developed for the determination of BKA and IBKA in rice noodles using HPLC-Orbitrap HRMS. The structure and morphology of Fe3O4/HNTs was characterized by means of XRD, SEM, FT-IR and VSM. Parameters affecting the extraction efficiency including adsorbent amount, pH, extraction time, type and volume of eluent were investigated by employing the response surface method. Results indicated that the proposed method had favorable linearity in the concentration range of 2-200 µg/L with a correlation coefficient >0.998. Method LOD and LOQ were 0.3 µg/kg and 1.0 µg/kg, respectively. Finally, the method was successfully applied to determine BKA and IBKA in rice noodle samples from southern China with recoveries ranging from 79.8% to 102.6% and relative standard deviation (RSD) of 4.2%-7.1%.

[Rapid determination of bongkrekic acid in Liushenqu by ultra performance liquid chromatography-tandem mass spectrometry].

A method for the rapid determination of biotoxin (bongkrekic acid) in the Liushenqu was established using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The samples were extracted with methanol by ultrasonication, then the pH was adjusted to 8 using ammonia. After filtration, the extract was purified using an Oasis Max strong anion exchange resin column. The Waters HSS T3 column (100 mm×2.1 mm, 1.8 µm) was used for the UPLC-MS/MS analysis. The mobile phase was acetonitrile-10 mmol/L ammonium formate solution (containing 0.1% (v/v) formic acid). MS analysis was performed using an electrospray ionization (ESI) source in the negative and multiple reaction monitoring (MRM) modes. Under the optimum conditions, the linear range of bongkrekic acid was 0.5-100 µg/L (correlations coefficient (R2)>0.99). The recoveries of the bongkrekic acid were 80.6%-85.3%. The intra-day and inter-day relative standard deviations (RSDs) were in the range of 4.2%-6.8% and 8.2%-13.2%, respectively. The limit of detection (LOD) and limit of quantification (LOQ) were 0.4 µg/kg and 1.2 µg/kg, respectively. The results showed that bongkrekic acid residues were detected in Liushenqu, thereby confirming the supporting role of our method for the risk monitoring of biotoxins in health foods and Chinese herbal medicines. This method is simple, easy, sensitive, and suitable for the determination of the bongkrekic acid residues in Liushenqu.

An Investigation of Bongkrekic Acid Poisoning Caused by Consumption of a Nonfermented Rice Noodle Product without Noticeable Signs of Spoilage.

Bongkrekic acid (BKA) is a tricarboxylic fatty acid that inhibits adenine nucleotide translocase as a kind of mitochondrial toxins. BKA is produced by the bacterium Burkholderia gladioli pathovar cocovenenans. An investigation was performed to determine the source of possible BKA poisoning of a family in H City, Guangdong Province, People's Republic of China, who consumed a commercially produced rice noodle product that was not fermented or noticeably spoiled. Clinical and food samples were tested. BKA concentration was detected by liquid chromatography-tandem mass spectrometry. We isolated and identified the suspicious strains from the rice noodles and performed toxicity determination through an animal experiment. BKA detected in the cases and the dead dog was 2.15 to about 343 µg/kg. The cases and dead dog shared a unique history of food exposure. The BKA in the factory's food samples was 150 and 160 µg/kg. All mice given the BKA extract by gavage died within 24 h. In conclusion, the food poisoning was caused by the high BKA concentration of expired (4 days over the 24-h shelf life) wet rice noodle products, with corn and wheat starch contaminated by B. gladioli cocovenenans. Different from traditional BKA poisoning caused by fermented and spoiled corn or coconut products, there was no noticeable spoilage because of the nonfermentation process and overused sodium dehydroacetate. The risk of BKA in wet rice noodle products and application of antiseptics, such as sodium dehydroacetate, in such food should be quantitatively evaluated to prevent the recurrence of similar events.

Identification of the potent toxin bongkrekic acid in a traditional African beverage linked to a fatal outbreak.

In January 2015, 75 people died and 177 were hospitalized in the Mozambique village of Chitima after attending a funeral. The deaths were linked to the consumption of a traditional African beverage called pombe. Samples of the suspect pombe were subjected to myriad analyses and compared to a control sample. Ultimately, non-targeted liquid chromatography-mass spectrometry screening revealed the presence of the potent toxin bongkrekic acid, and its structural isomer, isobongkrekic acid. Quantitative analysis found potentially fatal levels of these toxins in the suspect pombe samples. Bongkrekic acid is known to be produced by the bacterium Burkholderia gladioli pv. cocovenenans. This bacterium could not be isolated from the suspect pombe, but bacteria identified as B. gladioli were isolated from corn flour, a starting ingredient in the production of pombe, obtained from the brewer's home. When the bacteria were co-plated with the fungus Rhizopus oryzae, which was also isolated from the corn flour, synergistic production of bongkrekic acid was observed. The results suggest a mechanism for bongkrekic acid intoxication, a phenomenon previously thought to be restricted to specific regions of Indonesia and China.

[Determination and analysis of cellular fatty acids composition of Burkholderia gladioli].

OBJECTIVE: To determine and analyse the composition of cellular fatty acids of Burkholderia gladioli. METHODS: The cellular fatty acids composition of different pathovar strains of Burkholderia gladioli were determined by GC method and analyzed by MIDI-FAME. RESULTS: The results showed that the composition of cellular fatty acids of these strains, including original food poison strains of, were similar and were identified as, and this confirmed the conclusion that Pseudomonas cocovenenans subsp farinofermentans and Burkholderia cocovenenan were the junior synonym of Burkholdria gladioli. It was interesting to find that fatty acids of C16: 0.20H, C18: 1.20H and C16: 1.20H were related to the Bongkrekic acid (BA) producing of Pseudomonas cocovenenans subsp farinofermentans. CONCLUSION: It provide more data for the study of the mechanism of BA production and pathogenesis.

Simultaneous determination of toxic metabolites by linear combination derivative spectrophotometry.

A linear combination derivative spectrophotometric method is described. The method overcomes the problem of overlapping in derivative spectrophotometry and allows the maximum use of quantitative information. In addition, the method can be used to increase the selectivity, sensitivity and accuracy of the simultaneous analysis of multicomponent mixtures. The application of the method to the simultaneous determination of bongkrekic acid and toxoflavin, the toxic metabolites produced by Pseudomonas farinofermentans, is described.