ESTIMATION 



hours or by titration after twenty-four to thirty hours. Other bacteria 

 that have been used for the assay of pantothenic acid are Strepto- 

 bacterium plantarum ^^' ^* and Lactobacillus bulgaricus}^ 



Yeast was used by Atkin et al.}^ with a medium containing am- 

 monium sulphate as the source of nitrogen, together with sufficient 

 asparagine to prevent interference by /3-alanine, which also stimulates 

 the growth of yeast. Saccharomyces cerevisiae var. ellipsoideus was 

 found to be suitable for the assay of pantothenic acid by Emery et al.}"^ 

 who also obtained promising results with another yeast, Kloeckera 

 brevis. 



A possible complication in the microbiological assay of panto- 

 thenic acid is that the two products formed by the hydrolysis of 

 pantothenic acid, namely /3-alanine and pantoic acid, may stimulate 

 the growth of the micro-organism used. For this reason, only organisms 

 that respond specifically to pantothenic acid should be used, or else 

 some method must be introduced of suppressing the effect of the 

 degradation products. An example of this is the use of asparagine 

 to suppress the response of yeast to ^-alanine. 



IViicrobiological Response to ^-Alanine and Pantoic Acid 



j3- Alanine can be estimated by measuring first the growth response 

 of yeast, which is stimulated by pantothenic acid and jS-alanine, and 

 then that of Streptobacterium plantarum, which responds to panto- 

 thenic acid only and subtracting the second result from the first. ^* 

 Cory neb acterium diphtheriae, like yeast, also responds to both j8-alanine 

 and pantothenic acid.^^ Acetobacter suboxydans, on the other hand, 

 responds to pantoic acid but not to ^-alanine,^^ and can therefore be 

 used for the assay of pantolactone. As already noted, Proteus morganii 

 responds to both pantothenic acid and pantolactone. 



Preparation of Solutions for Assay 



It is particularly important to prevent hydrolysis of pantothenic 

 acid during its liberation from foodstuffs, in which it generally occurs 

 in the bound state. In their original method, Pennington et al} 

 autoclaved the material with or without previous autolysis under 

 benzene, whilst other authors have used enzymes, such as clarase or 

 " mylase P ", to liberate pantothenic acid.^» i^» 20, 21, 22 ^ mixture of 

 chicken liver enzyme and intestinal phosphatase is said to be even 

 more effective. ^^ With some sources of pantothenic acid, such as 

 malt products, treatment with cold dilute alkali solution prior to 

 digestion with papain and takadiastase increased the extraction 2- to 

 4-fold.24 



A further difficulty encountered in the assay of foodstuffs with 



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