1. Folate was measured microbiologically with Lactobacillus casei in extracts from a range of different foods at two incubation pH values, 6.2 and 6.8. 2.The values for folate content obtained at pH 6.2 were, in several instances, considerably higher than at pH 6.8. The 'positive drift' seen in the values for dilutions incubated at pH 6.8 were absent from results at pH 6.2.3. A comparison was made of the ability of the two main sources (hog kidney and chicken pancreas) of deconjugase enzyme to produce measurable folate and the hog-kidney enzyme was shown to produce consistently higher values.4. The results presented here will have significance for studies of folate intake using calculations from food composition tables, in the light of the apparent discrepancy between calculated folate intake and the recommended daily allowances for the UK.

1. The response of Lactobacillus cmei was measured for a number of the monoglutamyl forms of folate derivatives.2. At the concentrations of folate commonly used in the assay of folate vitamin in foods the response of L. cuseito folk acid, (pteroyl glutamicacid) and 5-formyl-tetrahydrofolic acid was similar, but 5-methyl-tetrahydrofolic acid gave as little as half the response of folic acid.3. present.

1. The comparative and absolute growth response of Lactobacillus casei was measured nephelometrically for time periods of 17-23 h in the microbiological assay of folic acid and 5-methyl-tetrahydrofolic acid at concentrations of 0-8 ng/lO ml basal medium at pH 6.8.2. At concentrations of G I ng/lO ml the comparative growth response to 5-methyl-tetrahydrofolic acid was markedly depressed whereas growth was the same at 2 ng/lO ml and above. Comparative growth was unaffected by the length of assay incubation, depressed growth being due to differences in log-phase growth rates with the rate-plot for 5-methyl-tetrahydrofolic acid being sigmoidal and for folk acid being a rectangular hyperbola with linearity only in the 0-1 ng/lO ml range. The reciprocal rate-plot for folic acid was linear whereas that for 5-methyl-tetrahydrofolic acid was coincidental only in part, giving rise to the same estimate of maximum velocity and substrate concentration for half-maximum velocity, with the exhibition of a strong threshold at low concentration. & Wright, 1982) that the L. casei growth response to 5-methyl-tetrahydrofolic acid may be significantly less than that to folic acid is confirmed as is the long-established view that the response to both folates may be equal. In the light of current knowledge regarding folate-binding, transport and metabolism by L. casei, it is argued that the intracellular oxidation of 5-methyl-tetrahydrofolic acid to 5, 10-methylenetetrahydrofolic acid is a rate-limiting step at low substrate concentrations, subsequently giving rise to a threshold growth response peculiar to 5-methyl-tetrahydrofolic acid. Since the rate of L. cusei growth with folk acid is not linearabove 1 ng/lO ml, itisrecommended thatmicrobiologicalfolateassaysbeconductedonlyin t h e G l ng/lO ml range and at a pH that elicits the same growth response from L. casei to 5-methyl-tetrahydrofolic acid as to folic acid and other folate monoglutamates. A previous observation (PhillipsMicrobiological assay in a basal medium of approximately pH 6.5-6.8 using Lactobacillus casei ATCC 7469 (NCIB 6375) is the most widely used method for the measurement of folate concentrations in foods and biological fluids. In a recent study, Phillips & Wright (1 982) demonstrated that L. casei has a reduced growth response to 5-methyl-tetrahydrofolic acid at low concentrations (0-1 ng/lO ml assay), where the initial pH of the assay medium was 6.5-6.8, when compared with other folate monoglutamates such as folic acid which is commonly used as the calibration standard for the microbiological assay. Since 5-methyl-tetrahydrofolate (as polyglutamates) is one of the most important forms of folate in foods, it was argued that this reduced response of L. casei would result in an underestimation of total food folate. The present paper describes further studies of the microbiological assay of folic acid and 5-methyl-tetrahydrofolic acid with a view to establishing the cause and extent of the reduced L. casei growth response to 5-methyltetrahydrofolic acid. E X P E R I...

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