594 BIOTIN 



ill the Ijiotiii-free basal medium used inci-eases with the hiotin roncentration 

 in the range from to about 2 m7 per 10 ml. of medium. Pure biotin and 

 samples to supply biotin at several levels within this range are added to 

 individual tubes containing 5 ml. of the double-strength basal medium; 

 each tube is then diluted to 10 ml., capped, autoclaved, cooled, and inocu- 

 lated. Response of the test organism is customarily detei'mined by acid 

 titration after 72 hours of incubation at 30 to 37°, or turbidimetric estima- 

 tions of growth can be made as early as 24 hours. 



The specificity of the test organism to various derivatives of biotin which 

 might occur naturally has been summarized elsewhere." None of these, 

 including soluble bound forms of biotin, such as biocytin,'^ or the more 

 complex insoluble combinations of this vitamin that occur naturally, can 

 support growth in place of biotin. It is necessary, therefore, to liberate 

 biotin fi'om these combinations by hydrolysis if figures from the total biotin 

 content of natural materials are desired. In most products, hydrolysis 

 with 6 A'^ H2SO4 at 120° for 1 hour liberates a maximum amount of biotin; 

 in some products, however, this treatment destroys some biotin.^' " Some 

 investigation of extraction procedures should therefore be made prior to 

 extensive study of any given product. Hydrochloric acid destroys biotin 

 under some conditions and should not be used in place of sulfuric acid. 

 Unsaturated fatty acids, such as oleic and linoleic acids, replace biotin for 

 L. arahinosus when present in high amounts'^ and interfere with biotin 

 assay in considerably lowei' concentration. They are readily removed by 

 filtration of the acid-hydrolyzed sample through paper, or by ether extrac- 

 tion.^ 



Yeast assay methods for biotin are somewhat simpler and recjuire less 

 time than that using L. arahinosus, but require more complex apparatus 

 and turbidimetric estimation of growth. An unpublished procedure of Atkin 

 and coworkers^ gives excellent results, as does that of Hertz. ^' '^ The same 

 extraction procedures used for L. arahinosus are required for liberation of 

 biotin. Yeasts are less specific than lactic acid bacteria in their response 

 to biotin; biotin sulfoxide, for example, which may (X'cur naturally in 

 rancid foods, is active in place of biotin.-' Fatty acids, on the other hand, 

 do not replace biotin for yeast and thus show less interfering action when 

 this procedure is used. 



By slight modifications in techni([ue, including aseptic addition d the 

 iniheated samples, any of the methods recommended abo\'e can be adapted 

 to determination of a\'idin, the iMotin-hindiug i)rotein of egg white. In 



13 L. D. Wright, K. L. C^rcsson, 11. R. Skeggs. U. i>. Peck, 1). K. Wolf, T. R. Wood, 



J. Valiant, and K. Folkers, Scnmce 114, (535 (li)51 ). 

 1' H. V. Brofiuist and 1']. K. Siiell, ./. Biol. ('hem. 188. lol il!»51i. 

 15 n. llciiz, /'/or. Sor. Kxptl. Hlol. Mai. 52, 15 iHHI^i. 



J 



