Chun-Chi Chen

The effects of transglutaminase (TGase), reductant, and thermal treatment on the cross-linking of white proteins in soft-shell turtle eggs were investigated. Egg white proteins were denatured by reductant (0.83% 2-mercaptoethanol, 2-ME) pretreatment and thermal pretreatment (95 °C and 5 min), and the denatured proteins were then catalyzed by TGase (1.0 unit/mL). SDS–PAGE showed that without any pretreatments, three major egg white proteins (210 kDa, 115 kDa, and 76 kDa proteins) were inferior substrates for TGase. Only portions of the 210 kDa protein (7.9%), 115 kDa protein (11.4%), and 76 kDa protein (42.9%) were polymerized by TGase into high-molecular-weight (MW) protein polymers (>180 kDa) after incubation for 3 h at 40 °C. However, the combined use of TGase with 0.83% 2-ME and thermal pretreatment led to a significant increase (p < 0.05) in the rate of white protein polymerization after 3 h: 210 kDa protein (90.8%), 115 kDa protein (69.5%), and 76 kDa protein (72.2%). Particle size analysis indicated that these cross-linked high-MW protein polymers were 2000–10,000 nm in size. Based on the experimental results, egg white proteins denatured by 2-ME and heat pretreatment are more prone to TGase-induced cross-linking.

Soybeans are a good source of protein and functional nutrition for humans and are used in the production of many foods, such as soymilk and tofu, which are popular in Asia. Soymilk is a beverage with a milky appearance and is produced by grinding soybeans in water; this raw soymilk is then filtered and heated. Soymilk can be used to produce tofu, a gelatinous food, in a process that generally involves coagulating, forming, and pressing soymilk. Tofu is also a source of functional ingredients, such as isoflavones, that have received much attention for their antioxidant properties. These isoflavones bind to soy protein to form soy protein–isoflavone complexes in tofu. In this review, we examined the processing, gelation and functional components of tofu. First, we introduced novel processing technologies (such as thermal and nonthermal processing) and hydrocolloids that affect the rheological property and texture of tofu. Then, because the coagulation and gelation of soymilk are important steps in the tofu manufacturing process, we also described detailed coagulation and gelation mechanisms of soybean proteins in tofu. Finally, we described the functional compounds and ingredients in tofu. This review provides basic knowledge for the further application of novel processing technology in tofu processing.

The effects of chymosin on the physicochemical and hydrolysis characteristics of casein micelles and individual caseins were investigated. Adding 0.03 units of chymosin/mL led to the casein micelles in skim milk coagulating after a 3 h incubation period at 30 °C. SDS–PAGE investigation showed that β-CN, κ-CN, α_s-CN, and a portion of β-lactoglobulin (β-LG) in the milk supernatant fraction (MSF) were precipitated into the milk pellet fraction (MPF). The mean particle size of the MSF with chymosin decreased from 254.4 nm to 179.2 nm after a 3 h incubation period. Mass spectrometry and SDS–PAGE analysis suggested that chymosin hydrolyzed individual β-CN, κ-CN, and α_s-CN, but not β-LG. Chymosin hydrolysis led to a decrease in the molecular weights of the hydrolyzed β-CN, κ-CN, and α_s-CN. Particle size analysis indicated that there was no difference in the particle size distribution of hydrolyzed β-CN and α_s-CN. Moreover, our outcomes demonstrated that the hydrolysis of κ-CN by chymosin occurs before that of β-CN and α_s-CN.

The effects of ultrasonic treatment (UT)-assisted transglutaminase (TGase) catalysis on the physicochemical properties of individual α_s-casein (α_s-CN), β-casein (β-CN), and κ-casein (κ-CN) were investigated. After 60 min of incubation at 30 °C, α_s-CN, β-CN, and κ-CN were cross-linked with TGase (6.0 units/mL), and high molecular weight polymers (>200 kDa) were formed. The use of TGase in conjunction with UT (20 kHz, power of 400 W, and amplitude 20%) led to an increase in the rate of α_s-CN, β-CN, and κ-CN polymerization compared to the individual casein that contained TGase but did not undergo UT. SDS-PAGE scrutiny showed that the intensities of α_s-CN, β-CN, and κ-CN incubation with regard to TGase and UT at 30 °C for 60 min noticeably decreased to 5.66 ± 0.39, 3.97 ± 0.43, and 26.07 ± 1.18%, respectively (p < 0.05). Particle size analysis results indicated that the molecule size appropriation for the cross-linking of α_s-CN, β-CN, and κ-CN ranged from 6000 to 10,000 nm after 60 min incubation with TGase and UT. Transmission electron microscopy investigation showed network structures of cross-linking α_s-CN, β-CN, and κ-CN were formed from α_s-CN, β-CN, and κ-CN, respectively. As our results show, the comprehensive utilization of TGase and UT will be a superior method for the polymerization of α_s-CN, β-CN, and κ-CN.

The effects of microbial transglutaminase (MTGase) cross-linking on the physicochemical characteristics of individual caseins were investigated. MTGase was used to modify three major individual caseins, namely, κ-casein (κ-CN), α_S-casein (α_S-CN) and β-casein (β-CN). The SDS-PAGE analysis revealed that MTGase-induced cross-linking occurred during the reaction and that some components with high molecular weights (>130 kDa) were formed from the individual proteins κ-CN, α_S-CN and β-CN. Scanning electron microscopy (SEM) and particle size analysis respectively demonstrated that the κ-CN, α_S-CN and β-CN particle diameters and protein microstructures were larger and polymerized after MTGase cross-linking. The polymerized κ-CN (~749.9 nm) was smaller than that of β-CN (~7909.3 nm) and α_S-CN (~7909.3 nm). The enzyme kinetics results showed K_M values of 3.04 × 10⁻⁶, 2.37 × 10⁻⁴ and 8.90 × 10⁻³ M for κ-CN, α_S-CN and β-CN, respectively, and, furthermore, k_cat values of 5.17 × 10⁻⁴, 1.92 × 10⁻³ and 4.76 × 10⁻² 1/s, for κ-CN, α_S-CN and β-CN, respectively. Our results revealed that the cross-linking of β-CN catalyzed by MTGase was faster than that of α_S-CN or κ-CN. Overall, the polymers that formed in the individual caseins in the presence of MTGase presented a higher molecular weight and larger particles.

The chitosan-induced coacervation of milk proteins was investigated using a proteomic approach. The addition of 0.8% chitosan to milk caused the milk proteins to coacervate after a 1 h incubation period. Approximately 86% of the milk proteins were present in the milk pellet fraction (MPF), and the protein concentration of the milk supernatant fraction (MSF) decreased from 29.4 ± 0.2 to 4.2 ± 0.6 mg/mL. SDS-PAGE analysis showed that the total intensities of serum albumin (BSA), α_S-casein (α_S-CN), β-casein (β-CN), κ-casein (κ-CN) and β-lactoglobulin (β-LG) in the MSF decreased to 8.5% ± 0.2%, 0.9% ± 0.3%, 0.7% ± 0.3%, 0.5% ± 0.2% and 15.0% ± 0.5%, respectively. Two-dimensional electrophoresis analysis indicated that α_S1-, α_S2-, β- and κ-CN and a fraction of the β-LG and BSA were found in the MSF following incubation with 0.8% chitosan. Isothermal titration calorimetry analysis indicated that binding of chitosan to milk proteins is an exothermic reaction based on binding titration curves of milk proteins dispersions with chitosan, and the enthalpy of binding (ΔH) and binding constant (Ka) were −7.85 × 10⁴ cal/mol and 1.06 × 10⁵/mol, respectively. These results suggested that the addition of 0.8% chitosan causes milk proteins to coacervate due to polysaccharide-protein interactions.