CRM197 is the diphtheria toxin mutant used in many conjugate vaccines. A fusion CRM197 (fCRM197) containing all the tags conferred by the pET32a vector was produced as a soluble protein in Escherichia coli co-expressing several chaperone proteins in conjunction with low temperature cultivation. Trigger factor (Tf) enhanced formation of soluble fCRM197 (150.69 ± 8.95 μg/mL) to a greater degree than other chaperones when fCRM197 expression was induced at 25 °C for 12 h. However, prolonged cultivation resulted in a progressive reduction of fCRM197 accumulation. In contrast, at 15 °C cells, with or without Tf, fCRM197 accumulated to the highest level at 48 h (153.70 ± 13.14 μg/mL and 150.07 ± 8.13 μg/mL, respectively). Transmission electron microscopy (TEM) demonstrated that the formation of inclusion protein as well as cell lysis was reduced in cultures grown at 15 °C. Cell viability was substantially reduced in cells expressing Tf, compared to cultures without Tf, when fCRM197 was induced at 25 °C. The viability of Tf-expressing cells was enhanced when cultured at 15 °C. Both purified fCRM197 and CRM197 efficiently digested lambda DNA (λDNA) at 37 °C (92.78 and 97.45 %, respectively). Digestion efficiency of fCRM197 and CRM197 was reduced at 25 °C (80.80 and 62.73 %, respectively) and at 15 °C (7.34 and 24.79 %, respectively). These results demonstrating nuclease activity, enhanced cell lysis, and reduced cell viability are consistent with the finding of lower fCRM197 yield when cultivation and induction times were prolonged at 25 °C. The present work provides a procedure for the high-level production of soluble fCRM197 using E. coli as a heterologous host.

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CRM197 is a diphtheria toxin (DT) mutant (G52E) which has been used as a carrier protein for conjugate vaccines. However, it still possesses cytotoxicity toward mammalian cells. The goal of this project was to produce a non-toxic and soluble CRM197EK through introduction of triple amino acid substitutions (K51E/G52E/E148K) in Escherichia coli. The expression of CRM197EKTrxHis was optimized and co-expressed with different molecular chaperones. The soluble CRM197EKTrxHis was produced at a high concentration (97.33 ± 17.47 μg/ml) under the optimal condition (induction with 0.1 mM IPTG at 20 °C for 24 h). Cells containing pG-Tf2, expressing trigger factor and GroEL-GroES, accumulated the highest amount of soluble CRM197EKTrxHis at 111.24 ± 10.40 μg/ml after induction for 24 h at 20 °C. The soluble CRM197EKTrxHis still possesses nuclease activity and completely digest λDNA at 25 and 37 °C with 8- and 4-h incubation, respectively. Molecular modeling of diphtheria toxin, CRM197 and CRM197EK indicated that substitutions of two amino acids (K51E/E148K) may cause poor NAD binding, consistent with the lack of toxicity. Therefore, CRM197EK might be used as a new potential carrier protein. However, further in vivo study is required to confirm its roles as functional carrier protein in conjugate vaccines.

Budu is one of the most popular fermented fish products in Thailand's southern area due to its distinctive flavor. It is being manufactured in large quantities for usage in cuisine as seasonings and sauces. The objective of this study was to determine the effect of amino acids on the distinctive taste components of Budu in southern Thailand. The amino acids in Budu were determined using GC-MS after fish was fermented for 6–12 months as recommended by the manufacturer. Lysine, glutamic acid, and aspartic acid are the three most abundant amino acids, with 1600, 1,540, and 1,260 mg/100g, respectively. Additionally, it was revealed that the umami taste was formed by a group of amino acids (glutamic acid and aspartic acid) followed by sweetness and bitterness. The sensory analysis discovered salty tastes, followed by umami, sour, sweet, and bitter. Four Budu samples generate a salty and umami flavor. Salt is mixed with cleaned fresh fish and fermented to enable native enzymes to auto-digest the protein and produce amino acid-rich products. Fish enzymatic fermentation produces short-chain peptides and amino acids that contribute to the umami flavor and taste. Additionally, the fermentation process creates a high glutamic acid concentration, as well as other amino acids and nucleotides that add to the umami flavor of the products. The study findings will be information that is particularly beneficial to consumers and manufacturers to promote Budu products in the country's region.

Meat adulteration has become a serious problem in global which directly affects to food consumers and producers. Therefore, it requires a tool to authenticate meat species to ensure safety of food products. Next generation sequencing (NGS) coupled with ribosomal RNA mitochondrial DNA gene can be used to analyze mixture of meat species in multiple meat samples. Therefore, this study aims to utilize NGS coupled with rRNA gene to identify 4 meat species (cattle, chicken, fish, and pig). Three primer sets (12S-Ki, 16S-KH, and 16S-Ki) were used to amplify DNA from the four meat species. All primer sets could be successfully amplified DNA fragments which corresponded to their size expectation. 16S-KH showed better detection effect in all species comparing with others. While the 12S-Ki and 16S-Ki could not be used to amplify in fish and chicken species. This may occur due to mismatches between sequences of primers and annealed regions of these species. Library construction of all PCR amplicons were prepared and sequenced by NGS. Amplicons amplified by 12S-Ki (fish) and 16SKi (chicken and fish) could not be mapped to the database because no PCR amplicons could not be amplified. NGS coupled with 16S-KH was then evaluated for precision test. The experimental precision was directly investigated comparing the results obtained from libraries that derives from DNA of four meat species which separately amplified for 3 different runs. As expected, the number and proportion of mapped reads between three different runs were also concordant. The percentage of mapped reads ranged from 14.05% to 31.04%, 15.14% to 31.98%, and 14.21% to 33.05% (1st, 2nd, and 3rd run, respectively). This demonstrated that NGS coupled with rRNA mtDNA gene could be reliably implemented as a routine testing. This developed technique can be applied to control and monitor meat adulterations in halal meat production and industry.