Codon Optimization of the "Bos Taurus Chymosin" Gene for the Production of Recombinant Chymosin in Pichia pastoris.

Codon optimization of the Bos taurus Chymosin gene (CYM) for its expression in Pichia pastoris was performed in this study. A synthetic CYM gene was designed in silico by replacing codons rarely used by P. pastoris with equivalent nucleotide combinations that codify for the same amino acid but that are more frequently encountered in the genome of P. pastoris. A total of 332 nucleotides were modified to optimize 289 codons. The synthetic CYM gene was cloned into the expression vector pPICZαA and transformed into P. pastoris. The transformed strains were grown in artificial media supplemented with glycerol as a carbon source to increase biomass and then cultured in a similar medium replacing glycerol with methanol as a carbon source to initiate gene induction. Raw extracts of the growth media exhibited milk-clotting activity of 146.11 SU/mL. Produced recombinant chymosin showed coagulant activity from 25 to 50 °C, and within a pH range of 5-6.9, having optimum activity at 35-40 °C, and pH 5.0. These results show that codon optimization is a viable strategy to improve CYM gene expression levels in P. pastoris for the production of recombinant chymosin.

Assessing the yield, microstructure, and texture properties of miniature Chihuahua-type cheese manufactured with a phospholipase A1 and exopolysaccharide-producing bacteria.

Chihuahua cheese or Mennonite cheese is one of the most popular and consumed cheeses in Mexico and by the Hispanic community in the United States. According to local producers the yield of Chihuahua cheese ranges from 9 to 9.5 kg of cheese from 100 kg of milk. Cheese yield is a crucial determinant of profitability in cheese-manufacturing plants; therefore, different methods have been developed to increase it. In this work, a miniature Chihuahua-type cheese model was used to assess the effect of a phospholipase A1 (PL-A1) and exopolysaccharide (EPS)-producing bacteria (separately and in combination) on the yield, microstructure, and texture of cheese. Four different cheeses were manufactured: cheese made with PL-A1, cheese made with EPS-producing bacteria, cheese with both PL-A1 and EPS-producing bacteria, and a cheese control without PL-A1 or EPS-producing bacteria. The compositional analysis of cheese was carried out using methods of AOAC International (Washington, DC). The actual yield and moisture-adjusted yield were calculated for all cheese treatments. Texture profile analyses of cheeses were performed using a texture analyzer. Micrographs were obtained by electron scanning microscopy. Fifty panelists carried out sensorial analysis using ranking tests. Incorporation of EPS-producing bacteria in the manufacture of cheese increased the moisture content and water activity. In contrast, the addition of PL-A1 did not increase fat retention or cheese yield. The use of EPS alone improved the cheese yield by increasing water and fat retention, but also caused a negative effect on the texture and flavor of Chihuahua cheese. The use of EPS-producing bacteria in combination with PL-A1 improved the cheese yield and increased the moisture and fat content. The cheeses with the best flavor and texture were those manufactured with PL-A1 and the cheeses manufactured with the combination of PL-A1 and EPS-producing culture.

Microsomal oxidative stress induced by NADPH is inhibited by nitrofurantoin redox biotranformation.

Nitrofurantoin is used in the antibacterial therapy of the urinary tract. This therapy is associated with various adverse effects whose mechanisms remain unclear. Diverse studies show that the nitro reductive metabolism of nitrofurantoin leads to ROS generation. This reaction can be catalyzed by several reductases, including the cytochrome P450 (CYP450) reductase. Oxidative stress arising from this nitro reductive metabolism has been proposed as the mechanism underlying the adverse effects associated with nitrofurantoin. There is, however, an apparent paradox between these findings and the ability of nitrofurantoin to inhibit lipid peroxidation provoked by NADPH in rat liver microsomes. This work was aimed to show the potential contribution of different enzymatic systems to the metabolism of this drug in rat liver microsomes. Our results show that microsomal lipid peroxidation promoted by NADPH is inhibited by nitrofurantoin in a concentration-dependent manner. This suggests that the consumption of NADPH in microsomes can be competitively promoted by lipid peroxidation and nitrofurantoin metabolism. The incubation of microsomes with NADPH and nitrofurantoin generated 1-aminohidantoin. In addition, the biotransformation of a classical substrate of CYP450 oxidative system was competitively inhibited by nitrofurantoin. These results suggest that nitrofurantoin is metabolized through CYP450 system. Data are discussed in terms of the in vitro redox metabolism of nitrofurantoin.

Determination of absolute threshold and just noticeable difference in the sensory perception of pungency.

Absolute threshold and just noticeable difference (JND) were determined for the perception of pungency using chili pepper in aqueous solutions. Absolute threshold and JND were determined using 2 alternative forced-choice sensory tests tests. High-performance liquid chromatography technique was used to determine capsaicinoids concentration in samples used for sensory analysis. Sensory absolute threshold was 0.050 mg capsaicinoids/kg sample. Five JND values were determined using 5 reference solutions with different capsaicinoids concentration. JND values changed proportionally as capsaicinoids concentration of the reference sample solutions changed. Weber fraction remained stable for the first 4 reference capsaicinoid solutions (0.05, 0.11, 0.13, and 0.17 mg/kg) but changed when the most concentrated reference capsaicinoids solution was used (0.23 mg/kg). Quantification limit for instrumental analysis was 1.512 mg/kg capsaicinoids. Sensory methods employed in this study proved to be more sensitive than instrumental methods. Practical Application: A better understanding of the process involved in the sensory perception of pungency is currently required because "hot" foods are becoming more popular in western cuisine. Absolute thresholds and differential thresholds are useful tools in the formulation and development of new food products. These parameters may help in defining how much chili pepper is required in a formulated product to ensure a perceptible level of pungency, as well as in deciding how much more chili pepper is required in a product to produce a perceptible increase in its pungency.