[Effect of Cooking and Processing on Quantitation of Soybean Proteins].

We examined the effects of cooking and processing on the quantitation of soy protein in various soy-based foods. For the phosphate-buffered saline (PBS) extraction, the total protein content was measured by bicinchoninic acid assay, and the buffer extraction containing sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (ME) was measured by the 2-D Quant Kit, and SDS-polyacrylamide gel electrophoresis analysis (SDS-PAGE) of each extraction was performed. Furthermore, measurements were performed by various ELISAs. During the tofu cooking process, the protein concentrations of soaked soybeans and Seigo (soybean homogenized with water) fluctuated- the change in protein solubility due to the amount of water during sample homogenization was considered to be a factor. It was thought that the decrease in protein concentration due to heating of Seigo during soymilk production was considered to indicate thermal denaturation of the protein, and that SDS and ME extraction in tofu may affect the measurement system. In cooking excluding roasted beans, proteins with a mass of 50 kDa or above and around 20 kDa were denatured, and in twice-fried tofu, proteins around 40 kDa were denatured, but the protein concentration excluding boiled soybeans did not decreased. Furthermore, the protein concentration from roasted beans, yuba, roasted okara and fried tofu increased with the cooking time, suggesting that the denaturation temperature of the protein shifted to a high temperature as the water content decreased. Both of the two types of ELISAs that comply with the official labeling system of foods containing allergenic substances were useful for detecting soybean protein by detecting proteins and peptides in processed soybean products, fermented foods excluding natto, and health foods.

Identification of Datura Species Involved in a Food-Poisoning Case Using LC-MS/MS and DNA Barcording.

A food-poisoning case due to eating the roots of Datura occurred in Kawasaki City, Japan in 2014. The Datura plant was mistakenly collected instead of burdock in a domestic garden. The roots of these plants are quite similar to each other. We presumed that the specimen was the root of Datura, but it was difficult to classify it only from the morphology. Using LC-MS/MS, we detected atropine and scopolamine from the remaining plant specimen. Therefore, we applied the DNA barcoding method. The results showed that the specimen was classified into Solanaceae family, but not Asteraceae family. Thus, the specimen was confirmed to be Datura species based on both chemical and genetic analyses.

Mouse models for assessing the cross-protective efficacy of oral non-typhoidal Salmonella vaccine candidates harbouring in-frame deletions of the ATP-dependent protease lon and other genes.

In BALB/c mouse models of Salmonella enterica serovar Typhimurium infection, a single oral immunization with a mutant strain with an insertion of the chloramphenicol resistance gene into the ATP-dependent protease clpP or lon gene decreased the number of salmonellae in each tissue sample 5 days after oral challenge with virulent S. Typhimurium at weeks 26 and 54 post-immunization. These data suggested that an oral immunization with the ClpP- or Lon-disrupted S. Typhimurium strain could provide long-term protection against oral challenge with virulent S. Typhimurium. Accordingly, recombinant oral non-typhoidal Salmonella (NTS) vaccines were constructed by incorporating mutants of both S. Typhimurium and S. enterica serovar Enteritidis harbouring stable in-frame markerless deletions of the clpP-lon-sulA (suppressor of lon), lon-sulA or lon-msbB (acyltransferase) genes. Amongst these orally administered vaccine candidates, those with the lon-sulA gene deletion mutants of S. Typhimurium and S. Enteritidis protected BALB/c and C57BL/6J mice against oral challenge with both virulent S. Typhimurium and virulent S. Enteritidis. Therefore, the in-frame markerless lon-sulA gene deletion mutant of S. Typhimurium or S. Enteritidis could be a promising cross-protective NTS live vaccine candidate for practical use in humans.

[Detection of allergenic substances (shrimp, crab) in processed seafood].

We have carried out a study (2009-2012) on processed seafood products in order to determine the level of contamination with shrimp and crab. In 2010-2012, after the Allergy Labeling Regulation went into effect, the detection rate of crustacean protein in processed seafood products including small fish, such as niboshi, tukudani and so on (both boiled and dried), was 63%. Detection rates for processed seafood products in which crustacean protein levels were below 1 µg/g were 36% with and 58% without advisory labels, allowing us to conclude that 60% of labels were adequate. On the other hand, the detection rate for processed seafood products with crustacean protein levels higher than the baseline of 10 µg/g was 9%, of which 60% carried no advisory labels. The rate of shrimp DNA detection using the Akiami primer in processed foods containing shrimp and crab was high (73%). This suggests that it is necessary to test these products using the Akiami primer for supplemental analyses of shrimp DNA. The PCR analysis for crab DNA detection failed due to combined detection of mantis shrimp DNA, which accounted for 8% of the total detected.

[Denaturation of egg antigens by cooking].

Changes in egg protein contents by cooking were measured with an ELISA kit using Tris-HCl buffer in model foods including cake, meatballs, pasta and pudding made with whole egg, egg-white and egg-yolk. The egg protein contents were lowest in the deep-fried model foods of cakes and meatballs. Ovalbumin (OVA) was undetectable (<1 µg/g) and ovomucoid (OVM) was lowest in pouched meatballs, suggesting that processing temperature and uniform heat-treatment affect the detection of egg protein. Furthermore, egg protein contents were below 6 µg/g in the pouched meatballs and pasta made with egg-yolk, and OVA and OVM were not detected by Western blotting analysis with human IgE from patients' serum. On the other hand, processed egg proteins were detected with an ELISA kit using a surfactant and reductant in the extract buffer.

[Comparison between old and new methods for detection of allergenic substances (egg and milk)].

The old ELISA method for detection of allergenic substances (egg and milk) in Kanagawa prefecture from 2003 to 2007, employed before improvement of the food allergen labeling system, yielded detection rates of 20% for egg and 30% for milk. In 2005, after improvement of the labeling system, the detection rate using the new ELISA in solutions containing 1% SDS and 7% 2-mercaptoethanol increased by about 10% for egg, but decreased by half for milk. There were 4 positive samples (over 10 µg/g) for both egg and milk proteins, on account of contamination by ingredients at the manufacturing line and the lack of proper food labeling. In 2009, the contamination levels of egg and milk proteins in labeled commercial foods were low. In a comparison between the new and old methods with the same samples, both the new ELISA and Western-blot analyses showed an increase in the detection rate of egg protein. In relation to milk protein, the detection rates were decreased with the new ELISA, although the ELISA detection rate and consistency rates with Western-blot analysis were increased. On the other hand, in the case of a protein content below 5 µg/g, it was impossible to determine ovomucoid and casein by Western-blot analysis.