Comparison of gamma-glutamyl hydrolase (conjugase; EC 3.4.22.12) and amylase treatment procedures in the microbiological assay for food folates.
1. It has been suggested that deconjugation of food folates with pig kidney, compared with chicken pancreas, may increase measureable folate by approximately 50% (Phillips & Wright, 1983). Therefore deconjugation with conjugases from these two enzyme sources was optimized and compared. Folate was measured microbiologically with Lactobacillus casei (ATCC 7469) as the test organism at pH 5.6. 2. Treatment for 6 h with 200 mg pig kidney/200 mg sample was compared with the conventional assay employing overnight incubation with 20 mg chicken pancreas/5 g sample. Comparison of the deconjugation systems showed chicken pancreas to be superior for peas (Pisum sativum) and beans (Phaseolus vulgaris), while there was no difference for potatoes. 3. gamma-Glutamyl hydrolase (conjugase; EC 3.4.22.12) treatment for 2 and 20 h with pig kidney and chicken pancreas was optimized for raw potatoes and green frozen peas. Treatments with pig kidney were conducted at pH 4.6, which is optimal, and at pH 5.2. There was no significant difference between 2 and 20 h treatments at pH 5.2. Treatments with chicken pancreas were conducted at pH 6.1. Treatment for 2 h was preferred as it resulted in significantly higher measureable folate activity in peas and potatoes, and because overnight treatment can be influenced by microbial production of folate. 4. With optimal treatment conditions the source of enzymes did not significantly influence measureable folate activity. Chicken pancreas is the traditional source of conjugase in Scandinavia and was preferred for deconjugation. 5. Chicken pancreas, 20 and 60 mg, was used for deconjugation of sixteen different food samples, each containing approximately 200 ng folate. Chicken pancreas at 60 mg/sample gave significantly higher results. Further addition of enzyme did not increase the folate values. 6. A combined amylase treatment using heat-resistant alpha-amylase (EC 3.2.1.1) during extraction to ensure the gelatinization of the sample, and glucan 1,4-alpha-glucosidase (amyloglucosidase; EC 3.2.1.3), along with the incubation with chicken pancreas to complete the digestion, was shown to give a small but significant increase in folate values of starch-containing samples. 7. Folate results using the recommended procedure are given for raw potatoes, wheat bran, rolled oats, wheat flour and dark rye bread. 8. Chicken pancreas was shown to contain equally high amounts of amylases as did the alpha-amylase and amyloglucosidase sources. This finding may explain the relatively small benefit of a specific amylase addition.
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002645:Chickens; D003546:Cysteine Endopeptidases / Q000494:pharmacology*; D005492:Folic Acid / Q000032:analysis*; D005504:Food Analysis / Q000379:methods*; D007780:Lacticaseibacillus casei / Q000378:metabolism; D013552:Swine; D014020:Tissue Extracts / Q000494:pharmacology; D000516:alpha-Amylases / Q000494:pharmacology*; D011623:gamma-Glutamyl Hydrolase / Q000494:pharmacology*
D014020:Tissue Extracts; D005492:Folic Acid; D000516:alpha-Amylases; D011623:gamma-Glutamyl Hydrolase; D003546:Cysteine Endopeptidases