Transglutaminase includes enzymes that catalyze the acyl transfer reaction in which the Y-carboxaminde group of glutamine residues in proteins and peptides acts as an acyl donor, and the primary amine group in amine compounds or the ξ-amino group of lysine residues in proteins or peptides acts as an acyl receptor. It is derived from the liver of animals or the culture of actinomycetes (limited to Streptoverticillium mobaraense and species of the genus Streptomyces) or bacteria (limited to species of the genus Bacillus). It may contain foods used exclusively for bulking, powdering, diluting, stabilizing, or preserving it or for adjusting its activity. It may also contain food additives used for bulking, powdering, diluting, stabilizing, or preserving it or for adjusting its pH or activity.
Transglutaminase occurs as white to dark brown granules, powder, or paste, or as a colorless to dark brown liquid. It is odorless or has a characteristic odor.
Transglutaminase complies with the Transglutaminase Activity Test.
(1) Lead Not more than 5 μg/g as Pb (0.80g, Method 1, Control Solution: Lead Standard Solution 4.0mL, Flame Method).
If the residue does not dissolve in 5 mL of diluted nitric acid (1 in 100) in the preparation of the test solution, conduct the test using Method 3.
(2) Arsenic Not more than 3 μg/g as As (0.50g, Method 5, Standard color: Arsenic Standard Solution 3.0mL, Apparatus B).
Proceed as directed under the Microbial Limit Tests.
Total plate count: Not more than 50,000 per gram.
Escherichia coli: Negative per test.
Salmonella: Negative per test.
Sample Fluid Prepare as directed in Method 3 for total plate count.
Pre-enrichment Culture Prepare as directed in Method 3 for the Escherichia coli test and Method 2 for the Salmonella test.
Transglutaminase Activity Test
Perform the test using the method given below. If the identification test cannot be performed by the given method, appropriate replacement of the sample dilution factor, buffer solution, and temperature for the reaction is acceptable where this is scientifically justifiable.
Sample solution Weigh 0.10g of Transglutaminase, add Tris buffer (0.2mol/L) at pH6.0 to dissolve it or disperse it uniformly, and make 10 mL. As necessary, prepare a 10-fold or 100-fold dilution by adding the same buffer (0.2mol/L, pH6.0) to the resulting solution.
Substrate solution Weigh 4.048g of benzyloxycarbonyl-L-glutaminylglycine, 2.780g of hydroxylammonium chloride, 1.229g of glutathione (reduced form), 0.295g of calcium chloride dehydrate, and 9.688g of 2-amino-2-hydroxy methyl-1,3-propanedionl, and dissolve them in water, then adjust the pH of this solution to 6.0, and make 400mL.
Test solution Equilibrate 0.2mL of the sample solution at 37℃ for 1 minute, add 2 ml of the substrate solution, equilibrated at 37℃ for 10 minutes, and shake well immediately. Incubate the mixture at 37℃ for 10 minutes. Add 2 mL of iron (III) chloride (for the transglutaminase activity test), and shake well immediately. Centrifuge this solution at 3000 rpm, and use the supernatant as the test solution.
Control Solution Equilibrate 2 mL of the substrate solution at 37℃ for 10 minutes, add 2mL of iron (III) chloride (for the transglutaminase activity test) and shake well immediately. Then add 0.2mL of the sample solution, shake well, and centrifuge this solution. Use the supernatant as the control solution.
Procedure Measure the absorbance of the test solution and the control solution at a wavelength of 525 nm. The absorbance value of the test solution is higher than that of the control solution.