Microbiological and chemical profiles during the production of Japanese domestic (Kyoto prefecture) and traditional salted pickles (Suguki) were investigated. As the producing process proceeded, numbers of bacteria belonging to gram negative bacteria (coliforms)decreased and those of lactic acid bacteria increased. Raw materials were found to be soiled with various microorganisms such as Pseudomonad. Whereas in Araduke(1st Pickling), Microbacterium bacteria were dominant. In Oiduke process (3rd Pickling), bacteria of Lactobacillus genus such as L. sakei and L. brevis were dominant, and in Muro process (Fermentation at 40°C), L. plantarum became dominant. Salt concentration showed almost 3% expect for 6% in Araduke process, and pH decreases to 4.2 as the process proceeded. Dominant state of L. plantarum in the Muro process was thought to be due to its ability to grow under high temperature up to 42°C. L. plantarum is one of the most popular lactic acid bacteria that concern and contribute to various fermentations and brewing. In Suguki production, L. plantarum also would also play a significant role for generating its good and characteristic flavor and taste.
In Japan, foodborne illness caused by Salmonella have decreased year by year recently, but Salmonella is an important causative agent of potential risks associated with death. However, the standard method applying food hygiene is not fully established at present. Then, we evaluate the advanced method for detecting Salmonella from foods which was improved the official method for pasteurized liquid egg established on Nov. 1998. Sensitivity of the method was tested by two kinds of Salmonella strain (H2S producing and non-H2S producing strain). Both kinds of Salmonella strains inoculated in foods (3 or 7 cfu/25 g sample) were clearly detected by the advanced method. Moreover, practical use for detection of Salmonella in ground chicken meat and unpasteurized whole liquid egg was tested. Retailed ground chicken meat was naturally contaminated by Salmonella at a ratio of 40/70 at Tokyo and Osaka. However, concentration of Salmonella was not so high. Mean of MPN (most probable number)/g was under 0.33/g. On the other hand, unpasteurized whole liquid egg was naturally contaminated by Salmonella. It was shown higher MPN number than that of ground chicken meat. Ground chicken meat was mainly contaminated by S. Infantis and non-sterilized liquid whole egg was mainly contaminated by S. Enteritidis. Food causative agent of Salmonella is mainly S. Enteritidis in Japan. Egg contamination of Salmonella needs to be monitored now and in the future. PCR and LAMP detection after BPW enrichment was fully effective for Salmonella in non-sterilized liquid whole egg but that was less effective for Salmonella in chicken ground meat. The new advanced detection methods for Salmonella in food are useful and sufficient for detecting few Salmonella in food.
The official method for screening Escherichia coli O157 and O26 in food involves the detection of the VT genes by PCR. In this study, we evaluated the efficacy of the application of an internal control (EC3 DNA is coamplified with the VT gene) in preventing false-negative results of VT gene screening. A modular approach was applied to consider the effect of each parameter: (1) 4 food samples were tested by using 3 types of cultures artificially inoculated with E. coli O157 before or after enrichment; (2) 2 commercial kits were used; and (3) 2 DNA extraction methods in the official method were employed. We found that the PCR performance was greatly affected by each experimental parameter; in particular, the DNA extraction method that influenced the concentration of PCR inhibitors in each food sample. Hence, the use of internal control was found to be critical for avoiding false-negative results. Isolation of bacteria is the final assessment in the official method. If it is true-negative, however, any steps for isolating the E. coli will not be required. In conclusion, the results of our study may be useful in establishing an overall rapid and efficient test.
The Light Upon eXtension fluorogenic (LUX) primers were designed to detect the hip gene specific for Campylobacter jejuni, the glyA gene specific for C. coli, and the glyA gene specific for both C. jejuni and C. coli. The real-time quantitative PCR assay using LUX primer (LUX-qPCR) was performed with the primer sets of J-hip-FU/RL for detection of C. jejuni, C-glyA-FU/RL for detection of C. coli, and JC-glyA-FL/RU for detection of C. jejuni/coli. Those LUX primers were specific for C. jejuni, C. coli, and C. jejuni/coli, respectively; the detection limit of the LUX-qPCR was 2,200 to 3,800 CFU/ml (11 to 19 CFU/reaction mixture) and coefficient (r2) for the correlation between amount of DNA and Ct value was calculated to be more than 0.99. The multiplex LUX-qPCR with J-hip-FU/RL and C-glyA-FU/RL allowed simultaneous detection and differentiation of C. jejuni and C. coli. The LUX-qPCR and the multiplex LUX-qPCR were carried out in food after enrichment culture and compared to the cultural method. The results between these LUX-qPCR assays and the cultural method were mostly corresponding: C. jejuni/coli strains were isolated from most of the enrichment cultures which were PCR-positive and not isolated from all of the cultures which were PCR-negative, however the bacteria were not isolated from part of cultures which were PCR-positive. These rapid and sensitive LUX-qPCR assays provide useful tools for specific screening test of C. jejuni/coli in food after enrichment culture and rapid identification of Campylobacter isolate.