UBC Theses and Dissertations

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UBC Theses and Dissertations

The nature of the activators required by the lactic acid bacteria Kadzielawa, Arthur Stephen 1939

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L .ft THE NATURE OP THE ACTIVATORS HEQUIBED BY THE LACTIC ACID BACTERIA by A r t h u r Stephen Kgtas ie lawa T h e s i s Submitted i n P a r t i a l Fulfilment of the l e q u i r e m e n t s for the Degree of MASTER OF SCIENCE-IN AGRICULTURE • in the Department of Dairying The U n i v e r s i t y of A p r i l , B r i t i s h Co lumb ia 1939. TABLE Off CONTENTS I n t r o d u c t i o n Page 1 Part... I '• , The S e l a t i o a s h i p o f B i o s "to the Y i t a m i a s , , Pag© 5 f he na t u r e o f B i o s Page 35 ?®rt H i _ '. fhe ' E f f e c t o f M e t a l l i c l o a i on the "Ac id -P roduc t i on of .tbe" l a - o t i c A e i d B a o t e r i a v , . . P a g e 44 gf*-t IY . ,,• ' S t u d i e s oa S y n t h e t i c Med ia f o r the Growth .. of the I»act ie Ac id . , ,B@ote r i a . . , . . . . . » . , > . . . , . . Page 4? BIBLIOGRAPHY Page 70 ms mrtJBB OF THE ACTIVATORS EECPIHSD BY * ' THE LAOTia.AQID BACTERIA IMTRODUGTION It i s now only a l i t t l e over a quarter of a century since the concept prevailed that an adequate supply of proteins, f a t s , .carbohydrates, Inorganic s a l t s and water constitxited a complete diet f o r the n u t r i t i o n of animals. In the e a r l i e r l i t e r a t u r e however evidence i s to be found strongly suggest-ing the necessity of providing other food factors essential for the normal growth and development of the animal organism. These unknown factors were characterized by a disparity be-.tween the.amounts required and t h e i r metabolic e f f e c t s . Many investigators believed that the animal body i s adjusted to l i v e either upon plant tissue or the tissues of other animals and that: these "tissues contain countless substances oth§r than proteins, fats and carbohydrates., A great stimulus to the. research which led to the vitamin concept and established the modern science of n u t r i t i o n was furnished by the investigations into the chemistry of the pro-teins which not only contributed to our knowledge of the com-position of the proteins but also to the relationship that exists between the individual constituents of the dietary. It was shown that the various proteins -exhibited, q u a l i t a t i v e l y as well as quantitatively a varied composition and that t h i s Variation was reflected i n t h e i r b i o l o g i c a l value. Even though i t has been realized for some time that cer-tain diseases were of n u t r i t i o n a l origin, i t was not suspected that they were deficiency diseases. The general opinion p r i o r to the development of the vitamin concept was that these d i -seases were due to some positive agency and that cure could be obtained only by supplying some substance which would neutralize the toxic effects of the materials used i n the diet causing the condition.. It was not u n t i l Gasimere Punk pub-lished his volume on llThe Etiology of Deficiency Diseases" i n 1912 was i t cl e a r l y established that beri^beri was due to the lack of an essential dietary factor. Funk gave the name "Vitacrine" to t h i s component, . He also advanced the hypothesis that certain other n u t r i t i o n a l diseases. were to be c l a s s i f i e d as deficiency diseases and postulated that other factors whose absence from the diet was responsible for. the occurence of these conditions would be found. Long before the vitamins were discovered a considerable amount of work had been done on the n u t r i t i o n a l requirements of micro-organisms required for t h e i r most intensive meta-bolism substances to be found i n the extracts of animal and vegetable tissues. It was shown i n some of the older much neglected work, which has since been repeated and confirmed that;the capacity of the yeast-cell f o r growth depended very much upon the size of the inoculum. Pasteur had clearly shown that In addition to the size of the inoculum that cer-t a i n organic substances ascelerated the fermentation of eer-t a i n types of yeasts. .Wildiers' Interpreted correctly Pas-teur's observations and developed further the o r i g i n a l hy-pothesis advanced by him, Wildiers studies demonstrated that upon the addition of a small quantity of s t e r i l e yeast ex-tract to the n u t r i t i v e solution a small inoculum was,suffic-ient to provide good growth. He recognized that t h i s growth stimulus could not be attributed to the presence of hitherto knovra factors and named the yeast growth promoting factor "Bios", , ' Following Wildiers. c l a s s i c a l work the study of the nu-t r i t i o n a l requirements of micro-organisms was again neglected u n t i l - i t was revived by the vitamin workers when they rea-l i z e d that the activators essential f o r the yeast growth were found i n the same materials that had been used f o r the pre-paration of the Vitamin B concentrates, This led to the hy-^  pothesis that -Bios and the Vitamin B were identical.. This hypothesis provided a great stimulus to the work on the n u t r i t i o n a l requirements of yeasts because, the vitamin workers welcomed any method of testing their, vitamin concen-trates which would provide a simpler method for the testing of their fractions than that afforded by the use of animals. As a result of work involving the comparison of the two procedures f o r the testing of the.vitamin and Bios fractions i t became clear that the factor required by yeasts was not iden t i c a l with the antiberi-beri vitamin. .Nevertheless as hew discoveries have been made regarding the multiple nature of Bios and the complexity of the Vitamin B f r a c t i o n the hope of establishing a relationship "between certain of the-Bios and 'Vitamin B -components has. "been revived. ' The work on the n u t r i t i o n a l requirements of yeasts set a precedent to those working with other organisms. It i s now a d e f i n i t e l y established fact that most micro-organisms require f o r . t h e i r optimum development certain substances which might be related to Bios. ; The l a c t i c acid bacteria are no exception and i t has been shown that they are as fastidious i n their n u t r i t i o n a l requirements as are the higher animals. In the following work an attempt has been mads to determine the nature of the activator requirements of this group of mioro-organIsms. THE NATURE OF THE ACTIVATORS REQUIRED B Y THE L A C T I C AOID BACTERIA PART .1... The, R e l a t i o n s h i p o f B i o s to the. V i t a m i n s ' 1'HE RELATIONSHIP OF BIOS TO THE- VITAMINS At the .time vitamins were disoovered i t was an estab-lis h e d faot that micro-organisms required for their most i n -tensive metabolism small quantities of certain organic sub-stances the nature of which has been very d i f f i c u l t to de-termine, as the raw materials used i n their preparation con-tain only traces of them. Outside of some scattered refer -ences work on this phase of the n u t r i t i o n a l requirements of micro-organisms was much neglected u n t i l i t was shown that the higher animals also required s i m i l a r substances i n t h e i r d i e t . It did not take long for the early workers to r e a l i z e that the crude extracts used i n the vitamin research had many points i n common with the,extracts used i n Bios studies and a relationship between the two substances was hypothe-sized. This question of the relationship of Bios to the v i t a -mins has been brought up from time to time as new develop-ments i n the p u r i f i c a t i o n and fractionation of either Bios or the vitamins have been made. Many of the d i f f i c u l t i e s that have arisen i n attempts to e s t a b l i s h a relationship be-tween these fractions have been due, to varying interpreta-tions that have been placed on the essential nature of the respective fractions and their metabolic functions. Wildiers introduced the term "Bios" i n his paper which bears the t i t l e "Nouvelle Substance indispensable au de-veloppement de l a levure.f Although the t i t l e indicates that the substance i s indispensable, the author, does not claim in. the' text of. the paper that i n the absence of Bios repro-duction f a i l s to ocour, but merely that budding i s very ..slow and the resulting c e l l s are not healthy. If the word "nor-mal" had been prefixed to development the t i t l e of his paper would have expressed i t s contents more accurately. As w i l l be seen l a t e r a great deal of work has;been done, to; determine whether or not yeasts w i l l grow in; a purely synthetic medium. Many authors have shown that continuous growth of yeast can be maintained i n media which they considered to contain only pure chemicals and that any attempts at further p u r i f i c a t i o n of the culture f l u i d s do not affeot the yeast growth. The reproduction i s very slow but i s greatly increased by the addition of only small quantities of "Bios" as o r i g i n a l l y obtained and described by Wildiers, In the l i g h t of the ex-perimental evidence provided by Wildiers. and of sub sequent workers i t would appear that reference to Bios as a growth stimulant o r a c t i v a t o r rather than as a substance absolutely essential to the growth of yeast more cl e a r l y portrays i t s true function, -A c c o r d i n g t o some o f the o b s e r v a t i o n s of P a s t e u r (1871) y e a s t s d i f f e r e d f r om many organ i sms i n t h e i r n u t r i t i o n a l r e -qu i r emen t s . He found t h a t s o l u t i o n s o f i n o r g a n i c s a l t s p l u s sugar .would be f e rmen ted w i t h an i n c r e a s e i n the number of f e r m e n t i n g o r gan i sms . However, W i l d i e r s (1901) m a i n t a i n e d t h a t some o t h e r e lement must he added t o P a s t e u r ' s s o l u t i o n f o r the c u l t i v a t i o n of y e a s t i f t he amount of inoculum,;, was s m a l l . I f t he amount o f i n o c u l a t i n g m a t e r i a l was l a r g e f e r -men t a t i o n soon f o l l o w e d , W i l d i e r s e x p l a i n e d t h i s by s ugge s t -i n g t h a t t h e r e was i n t r o d u c e d w i t h t h e l a r g e i no cu l um some subs tance wh ioh was r e a d i l y s o l u b l e and e s s e n t i a l f o r t h e r a p i d ; f e r m e n t a t i o n by y e a s t . W i l d i e r s c a l l e d t h i s g rowth p r omo t i ng subs tance B i o s and s t a t e d t h a t i t was an o r g a n i c subs tance s o l u b l e i n w a t e r and 80$ a l c o h o l , s t a b l e t o hea t and a c i d , no t p r e c i p i t a t e d by n e u t r o l o r b a s i c l e a d a c e t a t e , p ho spho t ung s t i c a c i d , , phosphomo lybd i c a c i d o r by s i l v e r n i t r a t e i n a c i d s o l u t i o n o r i n t he p r e sence o f ammonia. I n an a t tempt t o de te rm ine t he n a t u r e o f B i o s he t e s t e d a s e r i e s o f p u r i n e s and p y r i m i d i n e s but f a i l e d t o f i n d any of them t h a t would r e p l a c e B i o s . Amand (1902) showed t h a t f a i l u r e t o o b t a i n g r ow th w i t h t h e s m a l l i n o c u l a t i o n s was no t due : to any t o x i c subs tance i n t he medium. Most of these s t a t emen t s were s uppo r t e d by Dev loo (1906) who sugges ted t h a t the subs tance i s r e l a t e d t o c h o l i n e and p o s s i b l y a s s o d a t e d w i t h c h o l i n e i n n a t u r e . Funk (1912) w h i l e e x pe r imen t i n g w i t h t he a n t i b e r i - b e r i v i t a m i n obse rved t h a t the m a t e r i a l s r i o h i n the v i t a m i n were a l s o r i c h i n t h e y ea s t g rowth s t i m u l a t i n g f a c t o r . Th i s l e d t o the i d e a t h a t the subs t ances may be s i m i l a r . However i n h i s e xpe r imen t s t o see i f t he a n t i b e r i - b e r i v i t a m i n c o u l d a c t as a ooferment i n a l c o h o l i c f e r m e n t a t i o n he was u n s u c c e s s f u l . I t was no t u n t i l the work of W i l l i a m s ( 1 9 1 9 B a o h m a n ( 1 9 1 9 ) ? a n d Eddy and S tevenson (1919-20) t h a t . the s u b j e c t had a g a i n a t t a i n e d new i n t e r e s t . T h e i r r e s u l t s s t r o n g l y sugges t ed t h a t the v i t a m i n a c t i v i t y o f e x t r a c t s c o u l d be measured by the growth o f y e a s t c e l l s and wou ld p e rm i t the d i s p e n s i n g o f a n ima l e x p e r i m e n t a t i o n i n t he e a r l y s t a ge s o f t he v i t a m i n f r a c t i o n a t i o n . W i l l i a m s R, J . (1919) showed t h a t t he g rowth p romo t i ng subs tance o f y e a s t o c c u r s i n t h e same m a t e r i a l s as t ho se i n wh i ch v i t a m i n B has been f ound , namely p r o t e i n f r e e m i l k , wheat germ, l a c t o s e , y e a s t , egg y o l k and p a n c r e a t i n . The subs tance i s none of the commoner am i no - ao i d s c o n t a i n e d i n a c i d d i g e s t of c a s e i n w i t h t r y p t phane added . I t has the : same p r o p e r t i e s o f s o l u b i l i t y , p r e c i p i t a t i o n by phospho-t u n g s t i c a c i d a b s o r p t i o n on f u l l e r ' s e a r t h , hea t s t a b i l i t y and b e h a v i o r toward a c i d and a l k a l i , as the wa t e r s o l u b l e v i t a m i n B and f u r t h e rmo r e t he t?>/o subs t ance s had no d i v e r - •• gent p r o p e r t i e s . M i s s Baohman (1919) a r r i v e d a t a s i m i l a r c o n c l u s i o n bu t p roposed the use o f the amount o f ca rbon d i o x i d e l i b e r a t e d d u r i n g the g rowth of y e a s t a s an i ndex o f t he v i t a m i n e f f e c t - 1 0 -of the p r e p a r a t i o n s unde r s t u d y . Funk and Dub in (1920) de s -c r i b e d a s i m i l a r t e s t f o r the a n t i b e r i - b e r i v i t a m i n . H i s p rocedu re i s a l i t t l e s i m p l e r t han t h a t d e s c r i b e d by W i l l i a m s . A l t h o u g h a c o n s i d e r a b l e amount o f work t ended to i n -d i c a t e t h a t B i o s and t he w a t e r s o l u b l e v i t a m i n were i d e n t i c a l t h e r e were s t i l l some a u t h o r s who d i s a g r e e d w i t h t h i s v i e w . Consequen t l y f o r the nex t two o r t h r e e y e a r s t h e r e was con?-s i d e r a b l e c o n t r o v e r s y as t o whethe r or no t the two subs t ance s were i d e n t i c a l . Souza and McOol lum (1920) were among the f i r s t t o show t h a t e x t r a c t s o f c e r t a i n f oods - - wheat germ, r o l l e d o a t s , musc le t i s s u e e t c . even when t r e a t e d so as t o c o n t a i n so l i t t l e o f t h e a n t i n e u r i t i c subs t ance t h a t i t s p re sence cannot be demons t ra ted e x p e r i m e n t a l l y w i t h r a t s , s t i l l e x e r t a p r o f ound i n f l u e n c e on the p r o l i f e r a t i o n o f y ea s t c e l l s . The same i s t r u e o f the a d d i t i o n o f m i x t u r e s o f am i no - a c i d s and of g l u c o s e . From t h i s d a t a they conc l uded t h a t the use o f y e a s t a s a t e s t o rgan i sm f o r d e t e r m i n i n g the p re sence o r absence of t h e a n t i n e u r i t i c d i e t a r y f a c t o r i s c o m p l i c a t e d by so many d i s t u r b i n g f a c t o r s as t o make i t o f l i t t l e i f any v a l u e . Eddy, H e f t , S t e venson and Johnson (1921) showed t h a t when the y e a s t t e s t i s a p p l i e d to m a t e r i a l s a l r e a d y e v a l u a t -ed as to v i t a m i n con ten t by r a t f e e d i n g e xpe r imen t s t h e r e i s o n l y app rox ima te agreement . The agreement i s more marked when the e x t r a c t s a r e d i l u t e . - 1 1 -I n 1923 W i l l aman and O l s en came to the o o n o l u s i o n t h a t B i o s and wa te r s o l u b l e B a r e not i d e n t i c a l because o f the f o l l o w i n g c o n s i d e r a t i o n s : V i t a m i n B i s r e a d i l y removed by 95$ a l c o h o l w h i l e B i o s i s n o t , B i o s i s l e s s r e a d i l y abso rbed by f u l l e r ; ? 1 e a r t h . I t i s r e c o g n i z e d t h a t wa t e r s o l u b l e B i s no t s t a b l e i n d i l u t e b o i l i n g a l k a l i e s , but s e v e r a l i n v e s t i g a t o r s W i l l i a m s (• 1919.) ,• Fu lmar , N e l s o n , and Sherwood ( 1921 ) , and Wh ipp l e (1920) have f ound t h a t e x t r a c t s thus t r e a t e d s t i l l r e -t a i n t h e i r po tency f o r y e a s t g r ow th . T h i s l e a d s to t he c on -c l u s i o n t h a t t h e r e a r e two subs t ance s i n v o l v e d . Fu lme r , N e l s o n , and Sherwood (1920) a t t a c k e d the p rob l em f rom ano the r p o i n t o f v iew and p r e s e n t e d d a t a wh i c h showed t h a t wa t e r s o l u b l e B i s no t a n e c e s s a r y c o n s t i t u e n t of a med-ium f o r the g rowth of y e a s t , t h a t wa t e r s o l u b l e B i s no t the y ea s t g rowth s t i m u l a n t i n t he e x t r a c t s s t u d i e d , t h a t a l f a l f a and wheat embryo c o n t a i n n i t r o g e n o u s and i n o r g a n i c m a t e r i a l s wh i ch w i l l m a i n t a i n the g rowth o f y e a s t and t h a t t he r e l a t i v e , p o t e n c i e s of t hese m a t e r i a l s f o r the optimum growth o f y ea s t cannot be a r r i v e d a t on an e qua l we igh t b a s i s . They had d e v e l -oped a medium of known c o n s t i t u e n t s wh i c h p romotes the g rowth of y e a s t w i t h o u t the a d d i t i o n o f v i t a m i n s . The a d d i t i o n o f wa t e r s o l u b l e B does no t improve the medium. Th i s work was f u r t h e r s u b s t a n t i a t e d by Eddy, K e r r and W i l l i a m s (1924) when they compared S e i d e l l ' s a n t i n e u r i t i c f a c t o r w i t h a " B i o s " t hey had p r e p a r e d , on b o t h y e a s t g rowth and r a t g r ow th . These authors and MaoDonald and MoQollum (1920-23 ) con-tend that yeast.not only does not require vitamin B "but that i t also does not require Bios f o r growth. She proof depends upon .obtaining a medium completely free from: unknown sub-stances especially those of b i o l o g i c a l o r i g i n which might oome under the term "Bios". . . < The necessity of Bios for the growth of yeast was f i r s t seriously questioned by Fulmer and co-workers (1921, 1923, 1 & 2} and by MacDonald and MoGcllum (1920), Of this work Fulmer (1923) says these investigators came to the conclu-sion that Bios i s not a necessary component of media for the growth of yeast, but place i t i n a class of growth stimu-lants. However much of the evidence that Bios i s non-essen-t i a l to yeast growth has been v i t i a t e d by the discovery by Eddy, Kerr and Williams £1924) that cane sugar carries an appreciable amount of Bios, Fulmer and Nelson's (1923) work in which they state that extraction of the sugar f o r seven days by continuous extraction with 95% alcohol did not re-move any active material from the sugar may be explained by the fact that Bios i s not readily extracted by 95fo alcohol. They also indicated that the presence of foreign organisms may lead to an. overturning of the opimum conditions i n the medium resulting i n l o s s of growth. They also found that even sligh t contaminations of certain metals such as lead decreased the growth, ioew (1889) prepared a synthetic sugar and showed that - 1 3 -i t was f e rmen t ab l e by y e a s t . T h i s p r o v i d e d Fu lme r , N e l s o n and Whi te (19 83) w i t h an e x c e l l e n t o p p o r t u n i t y t o p r ove t h e i r p o i n t t h a t y e a s t c o u l d be s u b e u l t i v a t e d on a medium w h o l l y s y n t h e t i c i n o r i g i n , : The work on the r e l a t i o n s h i p o f B i o s t o t he v i t a m i n s g r a d u a l l y came to a s t a n d s t i l l as e v i d en ce was b rought f o r -ward t ha t bo th B i o s and the wa t e r s o l u b l e v i t a m i n were no t s i n g l e subs t ances b u t were complexes o f a t l e a s t two compon-e n t s . N e v e r t h e l e s s , Eddy, K e r r , and W i l l i a m s (1924) p u b l i s h e d a paper d e s c r i b i n g the i s o l a t i o n o f B i o s i n c r y s t a l l i n e fo rm and i n d i c a t i n g t h a t t h e y had o b t a i n e d no e v i d en ce i n s up -p o r t o f the c o n t e n t i o n t h a t B i o s was o f a m u l t i p l e n a t u r e , . I n c o n t r a d i c t i o n of the r e s u l t s o b t a i n e d by Eddy et a l Fu lmer and co -wo rke r s (1924) c i t e d ev i dence sugges l i ng the h y p o t h e s i s t h a t B i o s was of a m u l t i p l e n a t u r e . They f r a c -t i o n a t e d a wa t e r e x t r a c t of a l f a l f a w i t h v a r y i n g c on cen -t r a t i o n of e t h y l a l c o h o l and o b t a i n e d f o u r f r a c t i o n s wh ich when t e s t e d on yea s t e x h i b i t e d a d i f f e r e n c e i n a c t i v i t y . - 1 4 -Expe r imen t s c a r r i e d out on a more e x t e n s i v e b a s i s and s t r o n g l y s u g g e s t i n g t h a t t h e r e a r e at l e a s t two B i o s e s have been made the s ub j e c t o f a p r e l i m i n a r y r e p o r t by M i l l e r (1924) Lucas (1924) w h i l e wo r k i n g on t h e t o x i c i t y , of v a r i o u s compounds found t h a t of the d i f f e r e n t media d e s c r i b e d i n the l i t e r a t u r e none p roved comparab le t o wor t as a c u l t u r e medium f o r y e a s t . T h i s was e x p l a i n e d by t he p r e s en ce o f B i o s i n the w o r t , The s tudy was t a k en up by O l a r k (1922) who d e v i s e d a b i o l o g i c a l / m e t h o d f o r t he d e t e r m i n a t i o n 0 f B i o s i n s o l u t i o n s . Then came a de te rm ined a t tempt to i s o l a t e the a c t i v e p r i n c i p l e n e ce s s a r y f o r t he r a p i d g r ow th o f y e a s t , O l a r k (•19ly) had obse rved t h a t when a t t e m p t i n g to d e c o l o r i z e wor t w i t h c h a r c o a l t he e f f i c i e n c y of t h e medium f o r t he growth o f y e a s t s was g r e a t l y r e d u c e d . Many e xpe r imen t s were c a r r i e d out i n an a t tempt t o r e c o v e r t he a c t i v e p r i n c i p l e f rom t h e . c h a r c o a l but a l l p r o v e d u n s u c c e s s f u l , Lucas (1924) showed t h a t B i o s c o n s i s t e d o f two f r a c -t i o n s wh i ch he c a l l e d B i o s I and B i o s , I I , Each o f t hese f r a o t i o n s a l one wou ld not s t i m u l a t e yea s t g rowth b u t the two t o g e t h e r were a c t i v e . He a l s o showed, t h a t n e i t h e r B i o s I no r I I a l one no r b o t h t o g e t h e r c ou l d r e p l a c e the v i t a m i n s i n p r e v e n t i n g p o l y n e u r i t i s i n p i g e o n s , o r s c u r v y i n gu i nea p i g s , o r I n m a i n t a i n i n g the g rowth o f r a t s . - 1 5 -.. F i n a l l y i n . 192-8. l a s t cot t i s o l a t e d , and i d e n t i f i e d B i o s I as i n a c t i v e i n o s i t o l , •:' :.--Williams^ W i l s o n and Ah© (1927) a f t e r a t t e m p t i n g f r a c -t i o n a t i o n s o f . B i o s as d e s c r i b e d i n the ' l i t e r a t u r e up t o t h a t t ime found most o f the p r o c edu r e s u n s a t i s f a c t o r y , They found t h a t by t r e a t i n g the e x t r a c t s w i t h p ho spho t ung s t l o a c i d and t h en w i t h f u l l e r ' s e a r t h t hey c o u l d o b t a i n two f r a c t i o n s . The f r a c t i o n absorbed on f u l l e r ' s e a r t h was conoen t r a t ed to . such an e x t e n t t h a t ,008 mg p e r cc . wou ld g i v e marked, s t i m u -l a t i o n of g r o w t h , W i l l i a m s (1931) p roposed the t e r m ' n u t r i -l i t e s — w h i c h means a subs tance t h a t p l a y s an impo r t an t p a r t i n the n u t r i t i o n o f b a c t e r i a , f u n g i and y e a s t s . The t e rm does no t imp l y the i n d l s p e r i s a b l l l t y as do t he te rms v i t a m i n and B i o s , C o n t i n u i n g t h e i r work on the f r a c t i o n a t i o n o f B i o s , W i l l i a m s , Warner and Roehm (1929) s t u d i e d the a c t i v a t o r r e -qu i rements of a number o f y e a s t s and compared t h e i r f r a c t i o n s w i t h t ho se o f o t h e r w o r k e r s . The e f f e c t s o f t h e l a t t e r f r a c -t i o n s on the s i x y e a s t s t e s t e d a re of a l e s s e r o r d e r t han those o f Hl l ] fer and i n v o l v e t he p r e s en ce o f r e l a t i v e l y l a r g e amounts o f t he s u b s t a n c e s . I t appeared t o them w i t h i n the r ea lm of p o s s i b i l i t y t h a t such a c t i v i t i e s as t he se p r e p a r a -t i o n s po s s e s s ed may have been due t o accumu la ted o r abso rbed i m p u r i t i e s , Narayanan {1929) a f t e r g i v i n g a comprehens ive r e -v iew o f t h e work oh B i o s and a f t e r d e s c r i b i n g a method comes t o the c o n c l u s i o n t h a t t h e r e i s on l y one B i o s , An e x t e n s i v e e x am i n a t i o n of E a s t c o t t ' s s t a t emen t s t ha t i n o s i t o l i s an e s -s e n t i a l c o n s t i t u e n t o f B i o s was made but no e v i d en ce had been ob t a i n ed t o suppo r t h e r c l a i m . E&mamrara and Gh ikamatsu a l s o oame t o t he C o n c l u s i o n t h a t o n l y B i o s I I had a g rowth p r o -mo t i ng a c t i o n on y e a s t . > . F rom a s t udy of the d i f f i c u l t i e s a r i s i n g i n the i n t e r -p r e t a t i o n o f t h e r e s u l t s W i l l i a m s and Bradway d e s c r i b e dif^-f e r e noe s i n t h e y e a s t s Used by. W i l d i e r s , M i l l e r and o t h e r w o r k e r s . F u r t h e r s t u d i e s ,on t he n a t u r e of B i o s , c a r r i e d out by the Toron to wo r k e r s [ M i l l e r 1932 and 1933) show t h a t B i o s I I c o n s i s t s o f two f r a c t i o n s I I A and I I B . The s e p a r a t i o n i s e f f e c t e d by c h a r c o a l a b s o r p t i o n . The B i o s I I B i s abso rbed on t h e c h a r c o a l and e l u t e d w i t h S p a r l i n g ' s a ce t one ammonia r e a g e n t . A t t h i s t ime they a l s o showed tha t the B i o s I I ob -t a i n e d f r om tomato, j u i c e i s much more r e a d i l y f r a c t i o n a t e d t h a n t h a t f rom ma l t combings a s . i t does not r e q u i r e the p r e -l i m i n a r y p u r i f i c a t i o n w i t h a o e t o n e . M i l l e r [1936) d e s c r i b e d a method f o r the f u r t h e r p u r i -f i c a t i o n of t h e B i o s I I f r a c t i o n s and came to the c o n c l u s i o n t h a t the p r o p e r t i e s o f " B i o s I I A " a re due : t o i t s con ten t of ^5-a lan ine and 1 - l e u e i n e . A l t h o u g h most of the work conce rned w i t h a s t udy o f t he : r e l a t i o n s h i p of B i o s t o t h e v i t a m i n s has been c a r r i e d out emp loy ing y ea s t as the t e s t o rgan i sm, c o n s i d e r a b l e a t t e n t i o n has been p a i d t o the q u e s t i o n of t h e n u t r i t i v e r equ i r emen t s of o t he r m i c r o o r g a n i s m s . I t i s a w e l l known f a c t t h a t c e r t a i n o f t h e p a t h ogen i c -17-bacteria produce either a very scanty growth or no growth at a l l when grown on some" of the commonly used culture media. If certain of the body f l u i d s are present i n these media the organisms produce an abundant growth. Davis 1917 has shown that/on add!tion of hemoglobin to the Culture media for the growth of hemophylio bacteria a substance i s incorporated the action of rwhich i s s t r i k i n g l y similar to that of the vitamins required in. animal n u t r i t i o n , Lloyd (1917) has shown that such substances are necessary for the successful c u l t i v a t i o n of the menigococous. Ha eke. r -idge (1915-^17) found that the presence of • soluble humus, es-p e c i a l l y that produced by bacterial decomposition, i n the soil,, increases the rate of nitrogen f i x a t i o n and n i t r i f i c a -t i o n and that the effect of these soluble decomposition pro--ducts upon the nitrogen f i x i n g and n i t r i f y i n g , bacteria i s due to the presence i n the humus of growth promoting substances or vitamins, Hughes (19132) described a growth factor, -obtained from hydrolysed casein, which was required for the growth, of Staphylococci, Knight (1935) isol a t e d another similar substance es-se n t i a l f o r the growth of Staphylococcus Aureus. This f r a c -tion, isolated from mormite was obtained by employing the usual p r e c i p i t a t i n g reagents for preparing similar extracts, and f i n a l l y p u rifying by d i s t i l a t i o u i n a high vacuum. In l a t e r papers Knight (1936, 1937) shows that i n addition to -18-. this f r a c t i o n , which he called the Staphylococcus Aureus fac-tor 1, t h i s organism requires Vitamin B-j_ and n i c o t i n i c acid or i t s .amidei Nielsen and Hartelius (1937) describe two groups of fac-tors dif f e r e n t i a t e d by their s u c c e p t i b i l i t y to oxidation. The f i r s t e a s i l y oxidized, affected the growth of yeasts while the other, very resistant to oxidation,. affected the growth of" moulds (Aspergillus Niger). The l a t t e r group re-quired the presence of metals as co-factors for complete ac-tivation,, They concluded that the requirements of yeasts and moulds for different factors depended on the a b i l i t y of the organisms to synthesize these substances. Leofbourow, 5;err is and Morgan (1937) i n studying the s p e c i f i c i t y of Bios preparations f o r bacteria obtained a, f r a c t i o n from yeast and three fractions from malt oombings and tested them on various bacteria, •.The results varied from marked stimulation to i n h i b i t i o n of growth, and i n general, the effects of the different preparations were similar for any one organism. Buston and Pramanik (1931) and Buston,and Kasinathan (1933) made a study of the factors necessary for the growth , of Nematospora G-ossypii. In the f i r s t paper'they showed that the f r a c t i o n was active only i n the presence.of i n o s i t o l * In the second paper they describe more of i t s properties. The substance appears to be associated with proteins,.chiefly seed proteins. In studies on the quantitative determination of the sugar fermenting a b i l i t i e s of the l a o t i o acid bacteria. Orla-Jensen (1919), Orla-Jensen, Orla-Jensen and Spur (1925) and Enudsen and S^rensen (1929 ) have shown that for certain strains, included i n the genus Betacoceus or Betabacterium Orla-Jensen, the enrichment of the nitrogen source with auta-lysed yeast extract exerts a marked Increase innthe amount of acid produced from many of the carbohydrates submitted to fer -mentation. Sadler, Eagles, Bowen and Wood (1936) and Eagles, Wood and Bowen (1936) while studying the sugar fermentations of the l a c t i c acid bacteria observed that extracts of materials other than yeast also produced a stimulation for. the growth and acid production of these organisms. Eohn and Hegarty ,,(1938) showed that the formation of l a c -t i c acid by washed c e l l s of Streptococcus Lactis i n a phos-phate buffer solution containing 2$ glucose was increased by the addition of peptone to the. medium, The presence of as-carbic acid increased the fermentation of damaged c e l l s and; n i c o t i n i c acid stimulated both damaged and normal c e l l s , while the. two substances together showed an additive effect on l a c -t i c fermentation, Their effect however was less than that of peptone. 1 mixture of n i c o t i n i c acid, ascarbic acid, cystein and adenine also increased fermentation, but cystein and ade-nine alone had no e f f e c t . - 2 0 -The r e v i ew of l i t e r a t u r e on the growth s t i m u l a n t s s ug -ge s t ed t h a t t h e r e m igh t e x i s t a r e l a t i o n s h i p "between t h e a c -t i v a t o r s f o r y e a s t and f o r t he l a o t i o a c i d b a c t e r i a . Con-s equen t l y of f r a c t i o n a t i o n by S add l e r E a g l e s , Bowen and Wood o f - W i l d i e r s B i o s a f t e r the manner o f M i l l e r (1936) showed t h a t the f r a c t i o n s o b t a i n ed produoed a s t i m u l a t i o n o f growth Q r l a - J emsen (1936) made a t ho rough s tudy o f t he n u t r i -t i o n a l r equ i r emen t s o f t he l a c t i c a c i d b a c t e r i a . These o r -ganisms a re as f a s t i d i o u s i n t h e i r r equ i r emen t s as a re the . h i g h e r a n i m a l s . They were found t o ' g i v e a good response when s m a l l amounts o f a c t i v a t i n g subs t ances were added t o t h e i r c u l t u r e med i a . I n some of the e a r l i e r work o f O r l a -Jensen (1931) i t was shown t h a t a l a r g e number o f l a c t i c a c i d b a c t e r i a w i l l no t f e rment c a rbohyd ra t e s w i t h o u t . t h e p r e sence of c e r t a i n a l d e h y d e - l i k e a c t i v a t o r s . These a r e n o r m a l l y fo rmed when t h e c a r bohyd r a t e I s s t e r i l i z e d t oge t he r w i t h t h e n i t r o g e n o u s compounds. C o n s i d e r i n g the v i t a m i n s wh i ch may have an e f f e c t on t h e s e organ isms O r l a - J e n s e n s t a t e s t h a t t h e f a t s o l u b l e v i t a m i n s may be e l i m i n a t e d as the l a c t i c a c i d b a c t e r i a grow j u s t as w e l l i n s k im m i l k a s i n who l e m i l k . Of the wa te r s o l u b l e v i t a m i n s V i t a m i n e may be d i s r e g a r d e d because the l a c t i c a c i d b a c t e r i a .as a whole t h r i v e j u s t as w e l l , i n m i l k wh i c h has3 been hea ted c o n s i d e r -a b l y as they do i n raw m i l k . O r l a - J e n s e n t h e r e f o r e comes t o t h e c o n c l u s i o n t h a t among the v i t a m i n s o c c u r i n g i n m i l k on l y t he v i t a m i n s o f the B group may be o f s i g n i f i c a n c e i n t he g r ow th -o f t he l a c t i c a c i d b a c t e r i a , E. Kuhn (1934) has d e -scribed Bg as an orange f l a v i n g with a greenish fluorescence giving to both whey, and raw white of egg their characteristi color, * Other,/workers have characterized vitamin Bg as the antipellagra vitamin. However, the f l a v i n s alone do not seem to counteract pellagra; thus Vitamin Bgmust contain s t i l l another component called Vitamin Bg which i s a l k a l i fast and which Orla-Jensen. claims may be identical with the . main part of Bios, the pantothenic acid described by Williams and Saunders. (1934) From his studies Orla-Jensen draws the following con-clusions, that the l a c t i c acid bacteria require i n addition ,>to l a c t o f l a v i n e a thermostable, a l k a l i - f a s t substance that i s adsorbed by activated charcoal i n the acid range and i s easily eluted by pyridine methyl alcohol; that this substance stimulates the growth of yeast and thus may be considered identical with pantathenlc acid; that i t may also be i d e n t i -c a l with Yitamin Bg, In a study of the n u t r i t i v e factors required by the l a c -t i c acid bacteria, Snell, Strong and Peterson (1937) have recently reported the presence i n an alcohol extract of l i v e r of an acid ether-extraotable substance essential for the normal growth of 14 species of the l a c t i c acid bacteria. This substance i s rather l a b i l e to heat, acid and especially to a l k a l i , i s adsorbed by charcoal but not by Lloyd's re- ••• agent, and i s not precipitated,by.phosphotungst 10 acid. • In t h i s lab. the. work on the activators gradually evol-pn ved out of work started by Saddler and co-workers(1932/the c - 2 2 -; n u t r i t i o n a l requirements of certain of the l a c t i c acid bac-t e r i a . Eo'r some time i t had been known that the nitrogen source has a marked influence on the sugar-fermenting a b i l i -t i e s of the l a c t i c acid bacteria. It was shown that the kind and amount of nitrogen are both c r i t i c a l and that when yeast extract i s used as an enriching entity,, s p e c i f i c l a c t i c acid .strains demand a certain part of the nitrogen f r a c t i o n for the fermentat ion of a particular carbohydrate, ,Sadler, Eagles, Bowen, and Wood (1936) have shown that the enriching [ entity has no effect on acid production of Streptococci i n sugar broth but the enrichment with yeast or a l f a l f a ex-tracts causes not only a marked increase In the acid produc-t i o n "by Betacoccl but also a definite stimulating effect on the rate of acid production. Ihey also showed that extracts •of other forage" c r o p s exerted to a certain extent a stimulat- • ing effect on the n i t a l a c t i v i t y of the organisms studied * In another paper Eagles, Wood and Bowen (1936) showed that there was a d e f i n i t e relationship between the active p r i n c i p l e s i n yeast and a l f a l f a extracts with the Bios-of Wildiers, and that the Betaeocci demand, for their most i n -tensive metabolism, activators corresponding to .those re-quired by yeasts, e.g. Bios I , I I I and IIB as described by M i l l e r and co-workers. As t h i s section of the work deals with a relationship of Bios .to. the vitamins, p a r t i c u l a r l y those of the water soluble Vitamin B complex i t may be.well at t h i s point to make some reference to the progress made i n th e i r i s o l a t i o n and p u r i f i c a t i o n . However as' spaoe i s limited the reader i s referred to some of the reviews of the l i t e r a t u r e on the vitamins suoh as: (a) The Vitamins by Funk, translated by Dubin; (b) Recent Advances i n the knowledge of the Vitamins; (o) A detailed summary of the properties and constituent of the Vitamin B complex found i n the paper by Idgar and Macrae (Biochem J". 31, 1937, 8 8 6 - 9 0 2 ) . From a review of the l i t e r a t u r e i t i s seen that i t was only a short time aft e r the work on the vitamins and a c t i -vators f o r micro-organisms.was d e f i n i t e l y under way that attempts ?/ere made to establish a relationship between these substances. The early Work was not successful partly due to the c o n f l i c t i n g results obtained by the numerous authors and to the fact that the fractions studied were very impure and more than l i k e l y consisting of varying amounts of the d i s t i n c t fractions which are now known to constitute Bios and the Vitamin B complex. , ' \ -. As the work progressed on the study of the Bios of W i l d i e r T s evidence of i t s multiple nature gradually accumu-lated. M i l l e r , East cott and Maconachie (1933) showed that Bios consisted of at least three fractions which they called Bios I, Bios IIA and Bios IIB. At the same time the work on " -24-the V i t a m i n B complex has.made tremendous p r o g r e s s . V i t a m i n B^ and V i t a m i n B g have been o b t a i n e d i n c r y s t a l i n e form. Edgar and Macrae have d e s c r i b e d the s e p a r a t i o n from an a n t o c l a v e d y e a s t e x t r a c t of two heat s t a b l e f a c t o r s necessary f o r the optimum growth of r a t s m a i n t a i n e d on a V i t a m i n B f r e e d i e t and r e c e i v i n g V i t a m i n B, and l a c t o f l a v i n e . One f a c t o r i s p r e s e n t I n the f i l t r a t e a f t e r e x t r a c t i o n w i t h f u l l e r ' s " e a r t h , w h i l e the second i s c o n t a i n e d i n the e l u a t e from the f u l l e r ' s e a r t h adsorbate a f t e r t h e removal of l a o t o f l a v i n e . The f u l l e r ' s e a r t h f i l t r a t e f a c t o r i s ab-sorbed by N O T i t e c h a r c o a l a t pH 1.2 and i s not preciiJltatrea?, oy phospho-tuTig-stio a c i d , l l v e h j e m and Koehn (1935) d e s c r i b e d a f u l l e r ' s e a r t h - f i l t r a t e f a c t o r p r e s e n t i n an aqueous l i v e r e x t r a c t and n e c e s s a r y f o r the growth' of the c h i c k . T h i s f a c t o r was l a t e r i n v e s t i g a t e d by Lepkovsky and Jukes (1936). U n l i k e the y e a s t f i l t r a t e f a c t o r of Edgar and Macrae, the l i v e r f i l t r a t e f a c t o r i s not adsorbed by c h a r c o a l . The y e a s t f u l l e r ' s e a r t h adsorbate f a c t o r may be s i m i -l a r t o , or i d e n t i c a l w i t h the f a c t o r o b t a i n e d by Lepkovsky and oo-workers (1936)'from the f u l l e r ' s e a r t h adsorbate from r i c e b r a n e x t r a c t s . - I n c e r t a i n r e s p e c t s Edgar, and Macrae's adsorbate f a c t o r - resembles the V i t a m i n B^ i n v e s t i g a t e d by B i r c h and Gyorgy (193 6 ) . W i t h r e g a r d s t o the r e l a t i o n s h i p of B i o s t o the V i t a m i n B complex the r e v i e w o f the l i t e r a t u r e shows t h a t the e v i -dence i s overwhelmingly a g a i n s t the suggested i d e n t i t y of os and Vitamin B l a In t h e i r early work on the f r a c t i o n a t i of the Bios, M i l l e r and co-workers .showed that neither Bios I nor Bios II cured a v i a n " p o l y n e u r i t i s or promoted the growth of rats. However, i n the l i g h t of the more recent work on a l heat-stahle dietary factors essential for the n u t r i t i o n of the rat, i t appeared not improbably, to assume that a re-l a t i o n might exist between the Bios factors and certain of -the B-vitamins. A study of the factors constituting the Bios of Wildiers and the Vitamin-B complex strongly suggested to us that the Bios activators which were shown to be re-quired by the l a c t i c acid bacteria are identical with cer-r t a i n of the heat stable components ;of the vitamin-B complex. In Order to establish the v a l i d i t y of t h i s hypothesis fractionation of tomato juice, by means of a combination of the procedures used for the d i f f e r e n t i a t i o n of the Bios con-stituents and for the characterization of the heat stable components of the Vitamin B complex, was undertaken. The be-havior of the respective e n t i t i e s towards adsorption by char-coal and f u l l e r ' s earth, and towards pre c i p i t a t i o n by phos-photungstic was used as a basis for separation. . - 2 6 ~ EXPERIMENTAL The . f i l t r a t e resulting from the treatment of the contents of two tins of tomato juice with tannic acid and lead acetate, with subsequent removal of hydrogen sulphide was used as the starting material for the preparations of the various frac-tions ( M i l l e r 1936), This f i l t r a t e was divided Into/two equal fractions, 1 and 2, • . , F i l t r a t e 1 was treated after the manner of M i l l e r for the -separation of Bios IIA and Bios IlB, These Bios preparations were concentrated to 100 cc, and 50 cc»quantities respectively, A f u l l e r ' s earth f i l t r a t e factor and a f u l l e r ' s earth ad-sorbate factor were prepared from F i l t r a t e 2 after the manner of Edgar and Macrae, These vitamin factors were also con-centratedto 100 co, and 50 c c quantities respectively. An aliquot equivalent to 1/5 of the volume of each of the Bios and vitamin concentrates was retained for the purpose of de-termining i t s influence on the acid producing a b i l i t i e s of the l a c t i c acid bacteria. F u l l e r ' s earth f i l t r a t e and adsOrbate fractions were prepared by the procedure,of Edgar and Macrae from the re-mainder of each of the Bios concentrates after they had been made up to a volume of 600 cc. Each Of the resulting f r a c -tions was concentrated to the volume of the solution from which i t was prepared. The four fractions thus obtained were: . F u l l e r ' s earth f i l t r a t e f of Bios IIA Fraction 1 F u l l e r ' s earth adsorbate of Bios IIA Fraction 2 F u l l e r ' s earth f i l t r a t e of Bios IIB Fraction 3 F u l l e r ' s earth adsorbate of Bios lIB Fraction 4 - 2 7 -. Gharooal adsorbate and f i l t r a t e fractions were prepared by the procedure of M i l l e r from the remainder of the f u l l e r ' s earth f i l t r a t e and f u l l e r ' s earth adsorbate factors. Each solution was made up to a volume o f ' 6 0 0 oo»prior to treatment with charcoal, and each of the resulting fractions was con-centrated to the volume of the solution from which i t was pre-pared, -The four fractions thus obtained were:--Gharooal f i l t r a t e of f u l l e r ' s earth f i l t r a t e Fraction 5 Oharooal adsorbate of f u l l e r ' s earth f i l t r a t e Fraction 6 Charcoal f i l t r a t e of f u l l e r ' s dearth adsorbate Fraction 7 Charcoal adsorbate of f u l l e r ' s earth adsorbate F r a c t i o n 8 An aliquot equivalent to j of the volume of eaoh of the eight fractions was retained for the purpose of determining i t s influence on the acid producing a b i l i t i e s of the l a c t i c acid bacteria, A phosphotungstic acid p r e c i p i t a t i o n was carried out on the remainder of each of the eight fractions. Each fracti o n was diluted to a volume of 200 cc, adjusted to contain 5$> sulphuric acid by volume, and pre c i p i t a t i o n was effected with 20^ phosphotungstic a c i d i n 5fo sulphuric acid, The phospho- . tungstio acid precipitates and f i l t r a t e s were freed from Phosphotungstic aoid i n the, usual manner and ,each of the re-sulting solutions was concentrated to the volume of the solu-tion from which It was prepared.- Fraction 3 f a i l e d to y i e l d a phosphotungstic acid precipitate, The influence of each of the preparations, when added to milk as an enriching entity, on the aoid-producing a b i l i t i e s of cultures E. M. B t 173 and -23-E. S.-Bg 173 was determined. The enrichment was added at the rate of Vfo i n a l l cases. Milk and milk enriohed with 0.15$ amtolyse'd'yeast extract served as controls, • Culture E. M. 173 i s an atypical s t r a i n of Streptococcus Oremoris (Orla-. Jensen) responding markedly to the enrichment of milk with yeast extract. Culture E. M. B 173 i s to be c l a s s i f i e d as a s t r a i n of Betaooocus Oremoris (Knudsen and S^rensen). Pro-cedures followed throughout the fermentation have been de-scribed by Sadler, Eagles and Pendray (1932). The results of the determinations of the t o t a l t i t r a t a b l e a c i d i t y pro-duced by each of the cultures are given i n table I, When the data for the acid production of cultures E. M. B^ 173 and S, I, Bg 173 are considered, i t i s obvious that enrichment of milk with either Bios IIA or Bios IIB has a. marked stimulating effect on the v i t a l a c t i v i t y of the or-ganisms, and that, the influence of Bios IIA, p a r t i c u l a r l y i n the case, of culture E. M. B 173, is more marked than that of Bios IIB, The addition of the f u l l e r ' s earth f i l t r a t e factor or, of the f u l l e r ' s earth adsorbate factor has a marked stimu-l a t i n g effect on the v i t a l a c t i v i t y of both micro-organisms« The t o t a l titratable' acidity produced by culture E. M. B i 173 in,milk enriched with the f u l l e r ' s earth f i l t r a t e factor i s equal to that produced when Bios IIA i s employed as the en-r i c h i n g entity. It would appear that culture E. M, B g 173 iss stimulated to a greater extent by Bios IIA than by the f u l l e r ' s earth f i l t r a t e factor. It Is d i f f i c u l t at this time -28-1 a) TAB-LB" I T.ITR&TABL1S AGIDTTY' I N G3MMS LAO TIG AG IB PER LITff l C u l t u r e number Emails EMBgl73 3.6 1,6 6.5 3.4 7,4 7.4 7.2 6,3 3 .6 1.8 4 . 5 4.7. 3 .6 1,8 3 .6 1.6 3 .6 1,6 5,4 3 .6 4 .7 3 .6 3,4 1.6 6.3 3«S 5.6 2.7 3.6 1.6 6,b 3.6 •5.6 4 . 1 3 .6 1.6 5.9 4 .7 5.9 2.7 4 , 3 •1.6 6 ,1 1.8 5.2 3.2 i . i 3 .6 1.6 4 ,3 .1.6 5,4 • 3.4 5.9 2,9 3,6 2.0 Medium employed as s u b s t r a t e M i l k M i l k f 0,1.5$ y e a s t • e x t r a at M i l k -f B i o s ITA M i l k 4 F r a c t i o n 1 M i l k f P T A * p r e c i p i t a t e of F r a c t i o n 1 M i l k 4 PTA f i l t r a t e o f F r a c t i o n 1 M i l k 4 F r a c t i o n 2 M i l k 4 PTA p r e c i p i t a t e of F r a c t i o n 2. M i l k . 4 PTA f i l t r a t e of F r a c t i o n 2. M i l k 4 B i o s TIB M i l k 4 F r a c t i o n 3 M i l k 4 PTA. p r e c i p i t a t e of F r a c t i o n 8 M i l k f PTA f i l t r a t e of F r a c t i o n 3 M i l k 4 F r a c t i o n 4 . M i l k 4 PTA p r e c i p i t a t e , o f F r a c t i o n 4 M i l k * PTA f i l t r a t e o f F r a c t i o n 4 M i l k 4 F u l l e r : 1 s e a r t h f i l t r a t e f a c t o r M i l k 4 F r a c t i o n 5 M i l k 4 PTA p r e c i p i t a t e of F r a c t i o n 5 M i l k ,4 PTA f i l t r a t e o f F r a c t i o n 5 M i l k 4 F r a c t i o n 6 M i l k . 4 PTA p r e c i p i t a t e o f F r a c t i o n 6 M i l k 4 PTA f i l t r a t e o f F r a c t i o n 6 M i l k . 4 F u l l e r ' s e a r t h adso rba te f a c t o r M i l k 4 F r a c t i o n . : 7 M i l k 4 PTA p r e c i p i t a t e of. F r a c t i o n 7 M i l k . 4 PTA f i l t r a t e o f F r a c t i o n 7 M i l k 4 F r a c t i o n 8 ' M i l k 4 PTA p r e c i p i t a t e o f F r a c t i o n 8 M i l k 4 PTA f i l t r a t e o f F r a c t i o n 8 -*PTA s tands f o r phospho t u n g s t i o a c i d . I ' -29-to p r o v i d e an adequate e x p l a n a t i o n f o r t h i s phenomenon; I t I s p o s s i b l e , however, t h a t the s e n s i t i v i t y of the organism t o v a r y i n g ' c o n c e n t r a t i o n of t h e a c t i v e p r i n c i p l e s accounts f o r the apparent d i s c r e p a n c y observed i n t h e s t i m u l a t i n g a b i l i -t i e s of a c c e s s o r y f a c t o r s , w h i c h resemble each other i n many r e s p e c t s . The a c t i v a t i o n produced by enrichment of m i l k w i t h the f u l l e r ' s e a r t h a dsorbate f a c t o r i s p r a c t i c a l l y i d e n t i c a l w i t h t h a t o b t a i n e d by the a d d i t i o n of B i o s I I B i n the case of b o t h micro-organisms, A study of t h e i n f l u e n c e of the e i g h t f r a c t i o n s prepared from B i o s I I A and B i o s I I B , the f u l l e r ' s e a r t h f i l t r a t e f a c -t o r , and the f u l l e r ' s e a r t h a dsorbate f a c t o r on the a c t i v i t y of the micro-organisms shows c l e a r l y t h a t a r e l a t i o n e x i s t s between the B i o s c o n s t i t u e n t s and the heat s t a b l e components of the Vitamin-B complex. On the b a s i s of t h e a d s o r p t i o n r e a c t i o n s employed f o r the p r e p a r a t i o n of these f r a c t i o n s a c l o s e resemblance i n the a o i d - s t i m u l a t i n g a b i l i t i e s of F r a c t i o n s 1 and 5, 2 and 7, 3 and 6, 4 and 8, r e s p e c t i v e l y i s to be e x p e c t e d . The r e -s u l t s d e t a i l e d i n t a b l e I bear out t h i s e x p e c t a t i o n . I t i s e v i d e n t t h a t we are d e a l i n g w i t h only f o u r d i s t i n c t f r a c -t i o n s . The f r a c t i o n s that are adsorbed by f u l l e r ' s e a r t h but not by c h a r c o a l , F r a c t i o n s 2 and 7, are i n a o t i v e . Marked s t i m u l a t i n g a b i l i t y i s t o be found, however, i n a l l o t h e r f r a c t i o n s . I t i s apparent t h a t we a r e concerned w i t h t h r e e d i s t i n c t a c t i v a t i n g e n t i t i e s : a f a c t o r adsorbed by c h a r c o a l and by f u l l e r ' s e a r t h , F r a c t i o n s 4 and 8; a f a c t o r -so-adsorbed by charcoal but not by f u l l e r ' s earth, Fractions 3 and '6; and a factor that i s not adsorbed by either charcoal or f u l l e r ' s earth, Fractions 1 and 5. Confirmation of these r e s u l t s was obtained when the solutions r e s u l t i n g from the treatment of the respective a'Ct ive f r a c t i o n s with phosphotungst i c acid were employed as enriching e n t i t i e s . Of the active factors, only the one ad-sorbed by both f u l l e r ' s earth and charcoal i s precipitated by phosphotungstic acid. Regardless of the order i n which the adsorbents are employed f o r the separation of the re-spective fractions, corresponding pairs of factors react si m i l a r l y i n so far as their p r e c i p i t a b i l i t y by: phospho-tungstic acid i s concerned. Although neither of the factors that are adsorbed by charcoal but not by f u l l e r ' s earth i s precipitated by phosphotungstic acid, a difference i n the behavior of-.the respective solutions containing t h i s factor to?(/ards phosphotungst i c a c i d was observed, F r a c t i o n s , which was.prepared by adsorption on charcoal prior to treatment with f u l l e r ' s earth, yielded no precipitate, and the factor became inactivated by the addition of phosphotungstio aoid, . Fraction 7 , on the other hand, yielded a phosphotungstic acid precipitate, the f i l t r a t e retaining the stimulating a c t i v i t y of the fraction,, Fractionation of Bios IIA yielded two' fractions,, only one of.which was aetive, Fraction, 1-, Fraction 5, obtained from the f u l l e r ' s earth f i l t r a t e factor, exhibited similar chemical, physical, and b i o l o g i c a l characteristics. On the - 3 L -basis of i t s chemical and physical properties, the Bios III of M i l l e r would appear to he i d e n t i c a l with the l i v e r f i l -trate factor, described by Elvehjem and Koehn, and Lepkovsky and Jukes. Treatment of Bios IIB with f u l l e r ' s earth yields two . active components Fractions 3 and 4 , a f i l t r a t e factor not •pre-oi p i table by phosphotungstio acid, and an adsorbate fac-t o r precipitable by t h i s reagent. Fraction 6, obtained from the f u l l e r ' s earth f i l t r a t e factor was, similar to Fraction 3 . Fraction 8, the f r a c t i o n adsorbed by charcoal from the f u l l e r ' s earth adsorbate factor, resembled Fraction 4 , The activating constituent of Bios IIB not adsorbed by f u l l e r ' s earth Is similar to the f u l l e r ' s earth f i l t r a t e factor described by Edgar and Macrae„ ' . From a study of the properties of the other active com-ponent, of Bios IIB, adsorbed by f u l l e r ' s earth and p r e c i p i -tated by phosphotungstic acid, i t would appear that this activator i s d i s t i n c t from any of the dietary factors of the Vitamln-B complex described by various workers. It resembles in certain respects the vitamin B of Birch and Gybrgy and the factor obtained from rice bran extracts by Lepkovsky and co-workers. Unlike- Vitamin B , t h i s component of Bios IIB 6 i s adsorbed by No r i t e charcoal even at pH. 6 . To e s t a b l i s h more clearly the relation between the Bios factors and the constituents of the Vitamin-B complexj f r a c -tionation of l i v e r and yeast extracts, the p r i n c i p l e materials used as sources of the B-vitamin, was undertaken. When an aqueous extract of either of these materials, antoclaved for f i v e hours at 120° G., was fractionated, the stimulating a b i l i t i e s of the resulting activators were found to be similar to those Of corresponding e n t i t i e s prepared from canned tomato jui c e . Thus, canned tomato juice, anto-elaved yeast.extract or antoclaved l i v e r extract may serve as sources of the three d i s t i n c t activators. In an attempt to oh t a i n further evidence supporting the hypothesis that two of the three activators required by the l a c t i c acid bacteria are similar to the factors described by Edgar and Macrae, fra c t i o n a t i o n of an aTatoclaved extract of brewer's yeast was carried out., the. method described by these workers being followed i n s t r i c t d e t a i l . In their procedure, adsorption with f u l l e r ' s earth i s carried out d i r e c t l y on an acid extract of the material, without an i n -tervening tannic acid orlle&cd acetate p r e c i p i t a t i o n , Fractions corresponding to their, f u l l e r ' s earth adsorbate and f i l t r a t e factors were prepared* 'The f i l t r a t e remaining a f t e r successive treatment with f u l l e r ' s earth and charcoal should contain the Bios IIA en-. t i t y . This f i l t r a t e did,exhibit s l i g h t stimulating a b i l i t y , which however did not approach that exhibited by Fractions 1 and 5, "obtained when the procedure of M i l l e r , using a tannic acid and lead, acetate precipitation, was employed. It would appear that in the procedure of Edgar and Macrae, th i s factor may be p a r t i a l l y removed by treatment with -fuller's earth. Although we, have not carried out tests on the a c t i v i t y of the washings of the f u l l e r ' s earth prior to elution, i t Is possible that the IIA may he.found therein. Of the two factors described by Edgar and Macrae, one i s ad-sorbed by charcoal and the other by both charcoal and f u l l e r ' earth. It Is to be noted that Edgar and Macrae do not de-scribe a factor possessing the properties of Fractions 1 and •5. As shown above, eaoh of these fractions i s identical with Bios IIA and the l i v e r f i l t r a t e factor of Elvehjem and Koehn, and i s not to be found i n appreciable quantities i n the f i l t r a t e after treatment of an activated yeast extract ' with f u l l e r ' s earth and charcoal a f t e r the manner of Edgar and Macrae, If the Bios IIB fr a c t i o n , which contains the two fac-tors described by Edgar and Maorae, was prepared from an unautoclaved extract of brewer's yeasty i t was found to exert only a sligh t influence on the acid producing a b i l i -t i e s of the micro-organisms. Subsequent antoclaving of this f r a c t i o n , however, resulted i n an increased a c t i v i t y ap-proximating the a c t i v i t y possessed by Bios IIB prepared, from an extradt of yeast a&toclaved prior to fractionation. The three activators were also separated from an axLto-claved extract of fresh hog's l i v e r , by use of the'proced-ures employed in the fractionation of brewer's yeast. Fo Bios IIA a c t i v i t y was obtained when direct adsorption with f u l l e r ' s earth and charcoal was employed. Unless the l i v e r extract was autoclaved p r i o r to fractionation, the Bios IIB possessed only s l i g h t stimulating a c t i v i t y * - 3 4 -DISQUSSIOff On, consideration of the results as a whole, i t i s ev i -dent that three di s t i n c t growth stimulants other than Bios I ( i n o s i t o l ) are present i n tomato juice, autoclaved yeast extract, and i n autoclaved l i v e r extract, and that these activators are essential for the most intensive metabolism of certain l a c t i c acid bacteria. One of these activators i s Bios IIA, and the other two have been shown to be components constituting Bios IIB, Although i t has been impossible to carry out confirma-tory b i o l o g i c a l testing of the factors on laboratory animals, the nature of the physical and chemical characteristics of the activators, combined with the results obtained when l a c -t i c acid micro-organisms have been used as an index of the activating power of the e n t i t i e s , strongly suggests that these factors required by the l a c t i c acid bacteria are iden-t i c a l with certain of the heat-stable accessory food factors of the Vitamin-B complex. THE NATURE OF THE ACTIVATORS REQUIRED BY THE LACTIC ACID BACTERIA PART I I . . The Nature of B i o s -35-THE NATURE OF BIOS Having defined the activator requirements of the l a c t i c a d d 'bacteria and shown the relationship that existed be-tween these factors and those reported to be essential i n the n u t r i t i o n of animals, i t was considered advisable to attempt a fractionation and i s o l a t i o n of the respective p r i n c i p l e s employing micro-organisms as indicators of -the a c t i v i t y of the- prepared fractions. The work of Ylokery (1924, 1927} on the determination of the nature of the basic nitrogen i n the a l f a l f a plant i n which he successfully em-ployed the Neuberg Reagent used by M i l l e r i n h i s work on the fractionation of Bios strongly suggested that the ap-p l i c a t i o n of the technique of ¥ickery i n our work might lead to the elucidation of the nature of certain of the Bios en-t i t i e s . By means of a series of fractionations Viokery divided the basic nitrogen into three fractions (a) purines and d i am ine acids; (b) amides and amino acids and (c) -Deta ins among which was stachydrini, EXPERIMENTAL One t i n of tomato juice was centrifuged and the residue washed with 400 oe. water. The total extract of 2335 cc, was then made to contain 53$ by weight of alcohol (3230 co. of 90$ alcohol were necessary). After standing for one hour the precipitate was f i l t e r e d off and the f i l t r a t e concen-trated' to about 500 cc. The concentrate was then made to contain 30$ alcohol by volume and 20$ normal lead acetate - 3 6 -solution was added u n t i l no further precipitate was formed. The precipitate was Oentrifuged off and the f i l t r a t e was treated With 20$ Na s 00 3 u n t i l alkaline to litmus but s t i l l acid to phenolphthalin. The precipitate was f i l t e r e d off and concentrated to 500 cc. The last traces of lead were removed with HgS and then HgS removed in vacuo, 20$ Na 2 GOg "Gvg and 20$ mercuric acetate were added alternately u n t i l an orange precipitate was obtained^ and an equal volume a l -cohol was then added. The precipitate was oentrifuged off, . the f i l t r a t e a c i d i f i e d and freed of mercury with HgS, The f i l t r a t e from the Neuberg pre c i p i t a t i o n was treated with a hot saturated solution of Hg Gig i n alcohol u n t i l no further precipitate was formed. The f i l t r a t e was l e t stand over night during which time more precipitate was formed. The combined precipitates were decomposed with HgS yielding the Betain f r a c t i o n . On concentration of the f i l t r a t e there was .a syrupy reddish brown solution, not unlike that obtained f o r Bios IIA, which was retained for further study.. The precipitate from the Neuberg reagent was decomposed with HgS and the excess HgS removed i n vacuo,, The solution was: made to contain .5$. sulphuric acid and ,then was treated with 20$ phosphotungstic acid i n 5$ sulphuric,acid. The pre-cipi t a t e and f i l t r a t e were freed from phosphotungstic acid and sulphuric acid i n the usual manner, ..The phosphotungstic acid precipitate yielded the purine and diamine ac i d fraction while the phosphotungstic acid f i l t r a t e yielded the amide and amino acid f r a c t i o n . In aliquot of each of these fractions was retained f o r testing,, The remaining portion of-each f r a c t i o n was made acid to pH 1 . 4 and treated with charcoal after the manner of M i l l e r et a l , and corresponding charcoal adsorbate and f i l -trate fractions were obtained, . Each of'these twelve fractions separately and in com-bination were then added to milk as'an enrichment, and the acid-producing a b i l i t i e s of cultures 1 , M, Bj_ 173 and E« M, Bg 1 73 determined i n the respective media. !heheartchmsnt" was added at the rate of 2 $ i n a l l cases. Milk and milk enriched with 0 , 1 5 $ gutolysed yeast extract served as con-t r o l s . . The results of the total t i t r a t a b l e acidity recorded in grams of l a c t i c acid per l i t r e produced by each of the cultures are given i n table II« When the data f o r the acid, production of the cultures E. M. B^ 173 and E. M, B g 175' are considered, i t i s seen .that with the exception of the ferine f i l t r a t e factor which i s inactive a l l the fractions exhibit a stimulating influence on one of the organisms and i n most instances on both or-ganisms, Considering these results as a whole i t does not appear that a satisfactory separation of the active constituents has been made as i t i s hardly l i k e l y that there would "be at least seven d i s t i n c t active fract i o n s . For this reason this procedure was not investigated further. However, the results - 3 8 -TABLE II TIT11ATABLE AGILITY IN GRAMS. LAO TIP ACID PER LITRE Medium employed as substrate Milk Milk 4 yeast O . J 5 % Milk. 4 Purine Milk ,4 Amines Milk 4 Betain Milk 1 F i l t r a t e f a c t o r (5$) Milk= I Purine;. 4 Amides Milk. 4 Betain 4 f i l t r a t e faotor Milk 4 a l l four Milk 4 Purine f i l t r a t e Milk- 4 Purine adsorbate Milk i Amide f i l t r a t e Milk 4 Amide adsorbate Milk 4 Betain f i l t r a t e , Milk 4 Betain adsorbate Milk 1 f i l t r a t e faotor f i l t r a t e Milk 4 f i l t r a t e faotor adsorbate Culture number EMBT173 3,8' 1.8 5>Q 5,2 7,0 2,7 7.2 3,4 . 5.4 2.7 7.2 4.5 7,9 5.9 5.4 3.4 • 6*8 5.6 3.8 1.8 2^5 3.8 6,8 4.1 2.3 3.8 3,2 4,7 1.8 3.6 • 4 . 3 5,0 1.8 Elffi2173 -39-seemed to indicate that certain of the Betains, amino acids and purines might have a stimulating action on the acid pro-duction of the l a c t i c acid "bacteria. With this thought i n mind experiments were carried out employing a number of these compounds and related substances i n an attempt to determine i f they had any influence on the acid production by the l a c t i c acid bacteria. Numerous other compounds were also used. The solutions of these substances were based on work reported i n the l i t e r a t u r e dealing with the nature of Bios and certain of the constituents contain-ing the Vitamin-B complex, Wildiers who f i r s t described Bios also used this method of approach to the problem and tested a series of purines and pyrimidines but f a i l e d to find any of them to replace Bios, Devioo (1906) suggested that the substance is related to choline and i s possibly associated with choline i n nature but neither choline nor, any of i t s derivatives have yet been shown to possess Bios a c t i v i t y . About 1920 many workers indicated that Vitamin-B was identical with Bios. This, however, was also disproved since the extracts containing the Vitamin could be so treated that they contained no Vita-min-B a c t i v i t y for animals yet could stimulate yeast growth, Then as Vitamin B-^  was obtained i n pure form i t was shown by Orla-Jensen to contain no Bios a c t i v i t y and was not es-s e n t i a l for the most intensive metabolism of the l a c t i c acid "bacteria. Then Vitamin Bg (lactoflavin) although i t stimu-lated the acid production of certain strains of the l a c t i c -40-acid bacteria (Orla-Jensen 1936), was not the most important factor although lactoflavine was shown to exert a marked stimulating effect on the metabolism of the l a c t i c aoid bac-t e r i a , i t was without influence i n the absence of an unknown factor designated by Orla-Jensen as "milk Bios" and suggested by him.to be i d e n t i c a l with pantothenic aoid described by Williams. Williams (1919) showed that the substance promoting the growth, of yeast i s none of the commoner amino acids contained i n an acid.digest of casein with tryptophane added, Souza and MoGollum (1920) showed that the addition of mixtures of amino acid and glucose exerted a profound i n -fluence on the p r o l i f e r a t i o n of yeast c e l l s . MaoBonald and. MoGollum's be l i e f "that many other sub-stances have a favourable influence on the growth of yeast" was shown to be erroneous by Ide (1921), who showed that urea, amino acids, nuolein bases, oholine and i t s derivatives, the four ethyl and methyl amines, many alkaloids and gluoosides did not exert any Bios a c t i v i t y . Bevloo found Biosine i n a fat soluble form i n l e c i t h i n but i t i s neither choline, nor gly c o l amine and i s quite insoluble in'ethyl alcohol. Miss Reader (19 27) while.-studying the organisms st.rep-tothrix coralline's'" and Saroina aurantiaca showed that the following substances could replace glucose as a source of carbon without a loss i n the efficiency of the medium, gly-cerol, mannltol> arabinose, lactate, c i t r a t e * pyruvate and glycogen. Amino acids do not supply an improved source of - 4 1 -n i t r o g e n or oarbon t o t h e medium. G l y c i n e , a s pa r ag i n e , a l a -n i n e , and p o s s i b l y c y s t i n e may supp l y ca rbon but ammonium s a l t s a re a more e f f i c i e n t , source o f N**. Tne work of E r a n k e l and Sohwarz (1920) and Su zu k i e t a l (19 23-25) tended to s t r e n g t h e n the o p i n i o n t ha t B i o s was a n i t r o g e n o u s base and t ha t i t i s r e l a t e d to c h o l i n e t o I n h i b i t t h e growth of y e a s t . W i l l i a m s et a l (1927) i s o l a t e d a s i n g l e e r y s t a l i n e substance wh i c h they c l a imed to pos se s s B i o s a c t i v i t y and wh i ch was b e l i e v e to be r e l a t e d t o p r o l i n e . I n t he s t u d i e s c a r r i e d out by Narayanan (1930) no a c t i v i t y was shown by c h o l i n e ph reno -s i n e , s p h i n g o m y e l i n , the base sph ingos i r te and he drew the con -c l u s i o n t h a t the a c t i v i t y i n D e v l o o ' s m a t e r i a l was due to an i m p u r i t y and t ha t Dev loo was i n c o r r e c t i n r e l a t i n g B i o s t o c h o l i n e and- l e c i t h i n . H e . a l s o found s t a c h y d r i n , l y s i n e j spe rm ine , g u a n i d i n e , me thy l g u a n i d i n e , p u t r e s c i n e , c ada -v e r i h e , hexose d i phospha t e and po t a s s i um pyrophosphate t o be i n a c t i v e , E a s t c o t t (1928) showed t ha t B i o s I and i n o s i t o l were i d e n t i c a l , and t ha t f o r maximum s t i m u l a t i o n i n o s i t o l p l u s the unknown f a c t o r ,B ios I I was e s s e n t i a l . -M i l l e r (1936) a f t e r a s tudy of the n a t u r e of B i o s I I A and a f t e r t r y i n g a l a r g e number of amino a c i d s came to the c o n c l u s i o n t h a t the p r o p e r t i e s of B i o s I I A are due to i t s c on t en t o f ^ - a l a n i n e and 1-leuciiB . . Kn i gh t (1936, 1937) has r e p o r t e d t ha t the b a s i c f a c t o r f o r the g rowth of S t a phy l o c o c cu s aureus i n a c i d - h y d r o l y s e d media can be replaced by mixtures of vitamin B l and nicotinic acid or i t s : amide. Bichardson 11936) added that for anaero-bic growth this organism requires u r a c i l and pyruvic aoid i n addition to the above basic faotor, Lwoff and Lwoff (1937) have discovered that factor V, which i s necessary for certain haemophilio organisms, can be completely replaced by very small quantities of Warburg.' coenzyme or cozymase, Mueller (1937) has i d e n t i f i e d pimelic acid as an accessory substance needed for growth of a strain of the diptheria b a c i l l u s , Confirming the results of Orla-Jensen (1936) Wood, Anderson and Workman (1937) have shown that the propionic: acid bacteria required lactoflavine i n addition to the ether soluble factor previously reported by Wood, latum and Peter-son, : . EXP3RIMSIM1 The following series of compounds, Titamin B^, lacto-flavine ?Kadiostol (made with pure crystal i n vitamin D)yh'toi»»ic3«A Stachydrin, choline, i n o s i t o l , 1-leucine, 1-histidine, l y -sine , cystein, urocanic acid, iminazol aerylic acid, er-1 gothionehei dl-methionine, 1-aspartic acid, e g l u t o r i a acid, pimelic aoid, adipic aoid, asparagine, serine, u r a c i l * car-nosine, ergostero.l, cholesterol, leoithiniand vitamin G: were tested for Bios a c t i v i t y . Each Jof the compounds was added singly and i n various, combinations as an enrichment to milk, charcoaled milk, and to synthetic media. - 4 3 -The c h a r c o a l e d m i l k was p repa red by t r e a t i n g f r e i h ski® mills, w i t h charcoal a f t e r the manner of Orla-Jensen (1936)* The s y n t h e t i c medium used was the same as tha t d e s c r i b ed below* Fo r the t e s t i n g of these compounds th® f o l l o w i n g o r g -anisms were u s ed , E .M .B j 173 an a t i p i c a l s t r a i n of S t r e p t o -coccus oremoris i s o l a t e d f r om K i n g s t o n Cheese; E.M.Bi 195 ano t he r s t r a i n of S t r e p t o c o c c u s o remor i s i s o l a t e d f rom K i n g s t o n Cheese; E.2fl.Bg 173 a Betaoooovs i s o l a t e d f rom K i n g s t o n Cheese; SA30 a t y p i c a l S t r e p t o c o c cu s l a c t i s ( L i s t e r ) except that i t produces a ca rame l aroma and f l a v o u r i n milk, I t was i s o l a t e d and described by Sadler (1926) ; and 0088 a Streptobacterium c a s e i i s o l a t e d f rom Chesh i r e cheese by S a i l e r (1929). The f i n d i n g s were u n i f o r m l y nega t i v e and consequently no t a b l e s a re d e t a i l e d h e r e i n . The r e s u l t s show ths/t none of the above compounds e x e r t e d any s t i m u l a t i n g e f f e c t on the a c i d p r o d u c t i o n of the organisms s t u d i e d . A l s o various comb i na t i on s o f these compounds had no e f f e c t on the a c i d p r o d u c t i o n . The r e f o r e I t may be conc luded t ha t none of the known compounds t e s t e d c o n s t i t u t e the a c t i v e p r i n c i p l e of B i o s . THE NATURE OF THE ACTIVATORS REQUIRED BY THE LACTIC ACID BACTERIA PART H I . The Effect of Metallic Ions on the Acid. Proa-action of the Lactic Acid Bacteria,, ^ 4 4 -THE EFFECT OF METALLIC IONS ON THE AGID  PRODUCTION BY THE LACTIC. ACID BACTERIA It i s a well known faot that minerals play a very im-portant part i n the n u t r i t i o n of the higher animals and i t seemed reasonable to suspeot that the minerals might have 'some effect on the acid producing a b i l i t i e s of the laotio acid bacteria. From a careful examination of the methods used i n preparing the active fractions i t is noted that there i s nothing to suggest an absence of traces of minerals. Some unpublished data, f i l e d by the late Prof. Sadler, i n which a study of the effect of the addition of various Ca-j_ Mgc and Fe salts Casein Digest Broth (described by Eagles and Sadler (1932) was made, on a number of l a c t i c acid bac-t e r i a , added stimulus to t h i s work, • EXPERIMENTAL The elements most often mentioned as causing certain of the deficiency diseases i n both plants and animals, fer -r i c and ferrous iron, caloium, magnesium, cobalt, manganous added and manganic manganese, copper, zinc and iodine were/alone and i n a l l possible combinations to synthetic medium and to milk as an enrichment. The t i t r a t a b l e acidity was determined in grams of l a c t i c acid per l i t r e for the organisms E. M. B-j_ 173 and E. M. Bg 173, The results are detailed i n table III (a) and III (b) . Milk and milk enriched with .15$ auto-lysed yeast extract served as controls. The following metals iron, copper, manganese, cobalt, calcium, zinc, iodine and copper and a l l possible combina--45-TABLS I11(a) 0.1 ABATABLE API PI TY IK GRAMS IA.0TIQ AOID PER BITOT? Medium employed as substrate ' -Culture 3 number EIffi1173 EMB£173 Milk 3,6 1,4 Milk 4 0.15$ yeast extract 6.8 5.9 Milk 4 .03 Mg/oo Fe CFe01„) Milk - 4 ,1 Mg/ccFe CEeSo|) 5.9 6.8 2.7 3.4 •> TABES; i i i C b ) ., : ../.EITRAIABIiE: ACIDITY IH GRAMS LAQTIQ AOID PER LITRE 1 ~ Culture number Medium emulo'ved as sub str'atfi -EMB-,173 EMBgl73 Milk .  5.9 2.0. Milk 4:Q,15$ yeast extract 7 . 7 3.8 Milk 4 0.1 .mg/cc Fe Cferrous lactate)* Milk 4 0.01 mg/cc Go (Co(N0 ) ) Milk 4 0.01.mg/cc .Mn (;HhGlgJ A. Milk,.4 0.1 mg/oc MnfMnGla) Milk 4,0.01 mg/cc Mn {MnSO^) Milk 4 0.1 mg/cc MnfMnSO^) . Milk _* .0.001 mg/cc On. ( G U S O 4 ) Milk * 0.01 mg/cc Fe * 0.01 mg/oo Gu M l k 4 0.01 mg/cc Fe -f 0.001 mg/cc Gu 7.9 6,5 7,7 7,0 7.4 7,7 5,9 5,9 6.8 3 .8 2.0 3.8 5,2 4.1 4,7 2,0 ' 2.2 2.2 The fomulae i n parenthesis indicate the salt used. -46-t i o n s of them were added as enrichment to s y n t h e t i c medium but a l l were w i t h o u t e f f e c t on the growth o r a c i d p r o d u c t i o n i n t h i s medium, DISCUSSION An e x a m i n a t i o n of t a b l e s I I I (a) and I I I (b) i n d i c a t e s 'that metal i o i o n s when added to m i l k have a s t i m u l a t i n g e f -f e c t on the a c i d p r o d u c t i o n of the organisms s t u d i e d . How-ever when the same s a l t s a r e added to a s y n t h e t i c medium no s t i m u l a t i o n i s shown, T h i s may mean th a t the l a c t i c a c i d b a c t e r i a r e q u i r e these elements but they must be present e i t h e r as a component of an o r g a n i c compound or t h a t i t s a c t i o n must be a s s o c i a t e d w i t h some unknown o r g a n i c sub-s t a n c e . THE NATURE OF THE ACTIVATORS REQUIRED BY THE LACTIC ACID BACTERIA FART IV. S t u d i e s on S y n t h e t i c Media, f o r the Growth of the L a o t i o A o i d B a c t e r i a , , - 4 7 -SffXIDIBS ON SYNTHETIC MEDIA FOB THE GEO WTH ..OF'' THE LAOTIO ACID B A P . T O T a In order to obtain a clearer insight into the nature of the activator requirements of the l a c t i c , acid bacteria i t was realized a medium composed entirely of known constituents must be employed as the basic substrate. With this object in view a" search through the l i t e r a t u r e f a i l e d to reveal any synthetic medium suitable for the purpose, The medium used by ..Orla-Jen sen (1936) although a near approach to a synthetic medium s t i l l contained unknown factors, . EXPERIMENTAL The f i r s t synthetic medium decided upon was made to con-t a i n a l l of the inorganic s a l t s thought to be essential f o r the metabolism of the l a c t i c acid bacteria. Those substances which'are found only i n very small quantities were added i n the same proportion as they are found in milk, some of the other salts were added i n the same proportion as Orla-Jensen used i n his medium, ammonium chlorideammonium sulphate and ammonium c i t r a t e were added to give together ,2$ nitrogen and lactose was added at the same rate as to the usual casein digest broth e . g . 2$. " ' '• • For f i v e l i t r e s of medium the following quantities of the respective salts were weighed out, 5.0 gms. NaGl P.048gms, ZnGlg 0.083gms, F e C l g ..6H20 : -48-9.55 gms. NH4OI 0.01 gms. aaS04.5H20 11.785 gms. (KH4)-2S04 _ 28.95 gms, ammonium oitrate 10.20 gms. MgS04.7H20 25,00 gms. .K3HPO4 * The ammonium oitrate was made up by dissolving 25,03 gms. c i t r i c aoid i n water, adding 357.5 oe, one normal ammonium hydroxide and then making up the volume to 500 cov Each of the above sal t s was dissolved i n water and the volumes made up to 500 GO, .Starting with the sodium, chloride the salts were added together i n the,above order to prevent p r e c i p i t a t i o n of some of the constituents. When a l l the s a l t s solutions were mixed together the to t a l volume was 4500 cc* This was l e f t as such thus allowing 500 co, for dilutiom on adding ,the various materials to be tested, lactose was added at the rate of 2$ and the medium adjusted to pH7,0 be-fore autoclaving. , This synthetic medium would not support the growth of l a c t i c acid bacteria thus the activating power of the Bios fractions, l a c t o f l a v i n e and autolyzed yeast extract eould be determined i n a medium which would not normally support their growth. In Table IY results of the acid produced i n grams per l i t r e by f i v e strains of l a c t i c a c i d bacteria employing various combinations of the activating e n t i t i e s are detailed. In a l l cases each of the Bios fractions was added at the rate TABLE I F TITRA TABLE ACIDITY IH .GRAMS LAGTIC. AOID PER LITRE Medium employed as substrate SM SM SM yeast 2A i SM t EA2B I SM •» EF2B ! SM 2A t EA2B 1 SM t aa...--+-BP2B' • S M f EE2B .* EA2B , S M !' ' • ' 2A * E A 2 B * EF2B SM •» * L» SM * • L, yeast • SM L« * 2A S M EA2B S M ! f 2B . SM * L, -.4 2A * EA2B SM 2A f EE2B SM L. •* 1E2B ;* .BA2B SM + I i . + •2A.: 1* EA2B + EF2B Culture number EMBx EMB 2 •EMBJL" SA30 ( GG88 173 173 195 0 .0 0 ,0 0 .0 0 ,0 0 ,0 2.3 0.0 1,8 0 ,0 o.b 1.4 0 ,0 0 .5 1.4 0 .0 0 .0 0 .0 0 .0 0.0 0 ,0 0 .0 0 .0 0 .0 0 .0 0.7 0 .2 1.1 1,8 0.7 0.5 0,0 0 .5 1.6 .0.8 0 .0 0 .0 0 ,0 0.0 1.6 .0.0 0 .0 0 .0 0.0 0,0 0 ,0 0 .0 0 ,0 ..0.0 1,4 : 0 .0 1.4 0.9 0 .5 0 ,7 1 .1 2 ,0 0 .7 0 .0 0 ,0 0.2 . 0..7 0 .7 0 .2 0 ,0 0 .0 0. .7 1.1 0 . 2 0.2. 1 ,4 1 , 8 0,9 0 ,3 0.7 3,4 2,9 0,9 0 .2 0 .0 0 .2 1.1 0,7 2 «S 0 .0 . 2 . 9 .1.8 L. stands f o r laotoflavlne -50-of 3$ and lactoflavine at the rate of 0.5 mg per l i t r e , DISGUSSIOH It i s evident from the results obtained that the medium, even when enriohed with those substances which are found to increase the acid production of these organisms i n milk, does not support growth or acid production to the same extent as do othe'icasein digest broth (Eagles and Sadler 1932) or skim milk o r d i n a r i l y used f o r t h e i r c u l t i v a t i o n . Therefore one may safely draw the conclusion that a synthetic medium pre-pared i n the above manner Is not suitable for the optimum growth of the l a c t i c acid bacteria. The results also confirm Orla-Jensen's statements that the l a c t i c acid bacteria re-quire lactoflavine as well as the unknown constituent he called "milk Bios". In Orla-Jensen 1s (1936) monograph on the n u t r i t i o n a l re-quirements of the l a c t i c acid bacteria he has shown that i n his synthetic medium which contained charcoaled whey as i t s basis that certain strains of the l a c t i c acid bacteria re-quired certain amino acids f o r t h e i r optimum development. It was shown that leucine, h i s t i d i n e , lysine and cysteine were required by the large number of the l a c t i c acid bacteria. From th i s work i t was assumed that i f these four amino acids were added i n the optimum concentrations suggested by Orla-Jensen that the medium would be considerably improved. Table V shows the results obtained on t h e i r addition together with lactoflavine and the Bios f r a c t i o n s . TABLE V TgRATABLE AO I PITY IN ffMs LACTIC APIS PER. LITRE ••.Medium' employed as s u b s t r a t e • C u l t u r e number EMB-j^  172 EMBg 173 19 5 SA30 CO 88 SM : 0 ,0 0 .0 0 .0 0 ,0 0 .0 SM •+ L* 0,0 0 .0 0 .0 o.o 0.0 SM + L + Amino a o l d s 0 .0 •0.0 0.0 0.9 0.9 SM + L . +. AA* + 2A 0 ,5 0.7 2 .0 3 . 4 3,8 SM + L, + AA EA2B Q.Q 0.0 0 ,0 1.4 0.9 SM 1- L . i- AA EE2B 0 .5 0*0 1.3 0 ,5 1,8 . SM + L . +: AA EA2B , •.1.8 0 .2 3 ,8 5,0 1,8 SM + L . + :. AA 2A -+ EE2B 2.7 0.7 . 3 ,6 .5 .0 1,4 SM + L, +. AA + EA2B+EE2B 2.7 0.0 0,7 1,1 1,6 SM + L . + , AA •K 2A + EA2B t EF2B 4 . 1 0 ,2 4 . 3 5 e S 4 . 1 * i , s t ands f o r l a o t o f l a v i n e f A A s tands f o r t h e m i x t u r e o f amino a o i d s u s e d . - 5 2 -DISOUSSIOW From an examination of Table V i t i s seen that although the synthetic medium containing amino acids and lactoflavine does not provide a suitable substrate for the optimum growth of the l a c t i c acid bacteria the addition of the three Bios fractions provides a medium more nearly approaching a casein digest broth or milk i t s e l f f o r the growth of the organisms studied. This medium however i s s t i l l inadequate for the optimum growth of Betacoocus culture E. M. Bgl73, Since the combination of the four amino acids had such a marked effect on the acid production of the organisms stu-died i t ?ras decided to determine i f any one of them or com-binations of two or three of them would also have a similar' effect, Using the same concentrations as employed previously each of the amino acids was tested as detailed i n Table VI, DISCUSSION From a study of the results i t i s evident that none of the amino acids with the possible exception of h i s t i d i n e when added alone has a marked effect on the medium but as the num-ber of amino acids i n any,comb inat ion i s increased there i s an improvement i n the medium. However, any combination i n which h i s t i d i n e makes up one of the constituents i s d e f i n i t e l y superior to one. from which i t i s withheld, A mixture of h i s -t i d i n e and lysine seems to satisfy the requirements f o r E. M. B i l 7 3 j _ E s M, B]_195 and SA30 but not for B:.M. Bgl73 and cc, 88. For cc. 88 the complete mixture seems to be preferable. 53 a fcri o CO © ra © S~« 01 rf-K* P> © e> IB W> (0 •03' if*-» at o e it* CJt if* « CJ) o a C J ri-03 W rf- pa a ts P-» Hj CJ O . ^ O H> ^ S3 o S> ri-O N ID *H p © a S-1J fcd H » O 03 W B fe! >=3 o © © © pt, ri-O rf-cr © © CQ CO 03 to CO C IS) S l S M g @ HI- + + + + + + ^ + + + ir* tr* L~* ir* tf« f !r< i r ' t * f tr' l r ' jg sg to co m N ^  tN "i- -r -J-fc-« fc-' Er< tr* t-» H « H " S-« H > W » H » I J . J J . [ j . J J . J J . G o + + fcr* o o fe" <^ H » ts to P» ri-fe CD p i + +4- + -^ + + + + .^ w ri-© a ri-© CD O <^ + CO ri- !r> © <=! (J» CO ^ h"* © © a B a © ri-© i r 5 ©, ^  S « © H* © + + Q ca ri-1 cl- «• {j) K*: H» H< p !r* ca d rf-H» ® W W t< f 1* w- cn o ra co ji tj |M?4.'"}**». P> S3 ES CD CD 4- -}--f + O 5r> <y *4 ri- w. <B CO © ,fa> efr H» W» © © P f3 f ci tr 4 ©. ^  P ca ra H * © pi S hi* o> © p -?• o tr o' td (-• to ^ o H» (5i H * p. ©' p> H * © © W t d tx) bd |.<J« ^£i. 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C^> <LD CTJ G> 03 ~a -^ 2 O O I n r e c en t pape r s by S n e l l * Tatum and P e t e r s o n (193?) and S n e l l , S t r o ng and P e t e r s o n (1937) a s l i g h t l y d i f f e r e n t m i n e r a l m i x t u r e t h an t ha t employed i n t he work o u t l i n e d above was used and I t was c on s i d e r ed a d v i s a b l e t o de te rm ine the s u i t a b i l i t y o f t h i s medium as a b a s i c s u b s t r a t e f o r t h e work upon wh i ch we wer-e engaged, The m i x t u r e as o u t l i n e d by S n e l l and oo-workers c o n s i s t e d o f 0 .05$ K 2 HPQ4; 0 .05$ ' EE2PO4; 0 .02$ MgS04«7H 20; 0 . ; 001$'-NaGl; 0 ,001$. MnSQ4.3H 2 0r and 0 .001$ I , eS04 . 7H 2 0 . . To t h i s m i x t u r e was added 0,9 5$ :(NH4) 2S04( c o r r e s pond i ng t o 0 ,2$ ET) and 0 . 6$ NaAof L a c t o f l a v i n e and B i o s .were added i n the u s u a l manner t h i s c o n s t i t u t e d medium I , From the r e s u l t s based on the f i r s t s y n t h e t i c medium i t was though t h a t I f ammonium c i t r a t e was s u b s t i t u t e d f o r the ammonium s u l p h a t e a b e t t e r medium would r e s u l t as the c i t r a t e wou ld tend t o i n c r e a s e the b u f f e r c a p a c i t y o f the medium. The ammonium c i t r a t e was added to g i v e a 0,2$ET s o l u t i o n and B i o s and l a c t o f l a v i n e were added as b e f o r e . T h i s c o n s t i t u t e d medium B, 'As a c o n t r o l the s y n t h e t i c medium f i r s t d e s c r i b e d w i t h B i o s and l a c t o f l a v i n e added c o n s t i t u t e d medium 4 , These media were t e s t e d i n t h e u s u a l manner emp loy ing the c u l t u r e s - E . M. B ] 173 , E. M. B 2 1 7 3 , E . Mi' :'-B;jl9.5, SA30 and 0.0, .88.. The r e s u l t s a r e d e t a i l e d in. Tab l e Y I I . ? MaNo /s tands f o r .Sodium A c e t a t e . -54-TABLE T i l ;j?IT.BA!DABLl! ACIDITY IH, GRAINS. LACTIC ACID PER LITRE .. Culture number Medium : EMBi.173 ,EMB.p,17.5 EMBi 19,5,..:SA3Q . CC 88 1 3,2 0.0 2.5 - S « & 2.5 2 2,9 0.0 2.5 2.0 2.3 3 4,7 0.2 2.9 2,9 4 2,2 0,0 2 a S 2,9 1.8 Biscussiog From the results given i n Table VII i t i s seen that the medium f i r s t described i s superior. However, when ammonium ' oitrate was substituted for the ammonium sulphate i t was found that f o r E. M.. 3^173 the aoid production was increased from 3.2 to 4.7 grams l a c t i c acid per l i t r e there was some increase i n the acid production In the case of SA30 E. M. B^ 195 and CO 88 but E-, M, B 2173 s t i l l would not produce acid although a slight amount of, growth could be detected i n the bottom of the tube. The medium f i n a l l y decided on and used throughout the subsequent work and i n the work referred to on page 43 was made up as follows: N a d .001$ '•" % H P % .05 fa KH2P04. • ,05 fa Sodium acetate „6 fo EeS0 4 .001$ Mg S0 4 .02 $ H U S O 4 ,001$ Ammonium c i t r a t e 1,5 $ Lactose 2.0 $ - 5 5 -The ammonium cit r a t e was added to give ,2$N i n the f i n a l medium. Using t h i s medium as a basis salts of the following metals were added iron, ( f e r r i c and ferrous) manganese (man-ganic and manganous), calcium, magnesium, cobalt, zinc, cop-per and iodine. Each \¥as added alone and i n a l l possible combinations but none had any effect on the acid production, of E. M, Bxl72, E. M, B2173 and SA30, Yeast ash was also tested but found to.be without effect thus emphasizing the point suggested e a r l i e r i n the work that the effect of the metals may best be shown when they are i n combination with unknown organic compound. In order to determine the effect of various other com-pounds which have, from time to time, been reported to have Bios a c t i v i t y a number of these compounds alone, together with Bios and i n combinations were tested for their influence on the acid production of E. M, Bil73 and E. M, 3 2173, De-t a i l s of concentrations and combinations are given i n Tables VIII and IX, ' . DISCUSSION From the tables i t i s readily seen that the compounds tested did not increase the acid productions of the two strains of l a c t i c acid bacteria i n synthetic media. Even i n the presence of Bios these compounds showed l i t t l e or no effect and often tended to i n h i b i t the stimulation due to Bios, The presence, of amino acids tends to improve the medium to such an extent that even E, M. Bgl73 which seems to be very p a r t i -cular as to i t s n u t r i t i o n a l requirements i s stimulated to pro-HABIE/: VIII .TORAIABEE AGILITY IH .GRAMS LACTIC AGID PER LITRE Medium employed as substrate Culture number EMB-,173 BMB g173. Stt 1 SM. SM SM SM •SM + yeast extract (0.15$) - SA S SM SM SM SM SM Bios EF2B EA2B; 2A + 2A + EF2B 2A EF2B EA2B + EA2B 1P2B EA2B SM SM SM SM SM SM SM + + + B i o s 3 -+• ,1$L¥ as Lysine dihydrochloride .03^1 as lysine dihydrochloride Bios + ,03$N as Histidine monhydroch-l i d e ,03$ His t i d i n e monochloride Bios + ,03$N as cystein hydrochloride ,03$N as Cystein hydrochloride Bios +• .03'$KF as 1-aspartic acid ,G3$N as 1-aspartic acid Bios + 0.1$!? as 1-leueina .. 03$ET as 1-leuci as SM+Bios + «03:$1? as asparagine SM. •+- ,Q3$N as asparaginei, SM+Bios +• ,03$H as alanine SM + »03$N"as alanine. SM+Bios + ,03$N as Serine SM + ,03$H" as cerine SM+Bios + 0e03$H as choline as eholiae + ,001$F as n i c o t i n i c acid 0.1$ n i c o t i n i c acid «001$H as' n i c o t i n i c a c i d 0,1$BF as n i c o t i n i c acid Bios + ,01$N as methionine ,01$H as methionine <•  Bios +• ,1$N as u r a c i l ,1$H" u r a c i l Si>0,03$N s SM SM SM SM SIC SM SM + + + + Bios Bios 1 2 3 Bios 0.,0 5.. 6 0.2 0.2 0,0 0,9 0,9 0,0 1.6 2,0 0,7 2,3 0.0 2 .,5 0.0 1.6 0,5 2,3 0,0 1.4 0.0 1,6 0.5 1,4 •0.0 1.4 0*0 1.8 0.7 0,2 0.0 1.1 0,0 2,0 Q,2 0.0 2,0 0,0 0.0 Q'Q 0,0 0,0 0,0. 0,0 0*5 0.5 0,9 0.0 0,2 •0.0 0.2 0,2 0,7 0,0 0,0 0,0 0.5 0,0 0.0 0,0 0*0 0.0 0.0 0,5 0,0 0,0 0,0 0,0 0,5 0.0 SM Synthetic Medium + lactoflavine A l l Bios added at rate of 20$ complete mixture of .2$ each of Bios 2A,; EA2B and EF2B, - 5 6 - ( a ) TABES I X £EITRATABLE. AQ1D1TY I N GRAMS .LAQTIO AOID .Pin? LITRE Medium employed as s u b s t r a t e C u l t u r e number EMBn 173 SM SM + ^ SM > -sk -SM + SM + SM SM -t-y e a s t , e x t r a c t 0 .15$ g l u t a r i o a o i d 0 . 1 to 0 ,001 mg pe r c c a d i p i c a o i d 0 . 1 to 0 ,001 mg. pe r c o , p i m e l i c a c i d 0 , 1 t o 0 ,00001 mg, p e r c c i n o s i t o l 0 , 1 to 0 ,0001 mg/cc , o a ruo s i ne 0 , 1 t o 0 .001 mg/oc. p i m e l i c a c i d 0.000S5 m g . / c c . -t- n i o o ^ i t i n i c a c i d 0 ,001 mg./cc* SM + B- ra lan ine ,001 m g . / o c . + n i c o t i n i c a o i d 0 .001 mg . /o c . . SM + oa rnos i ne 0.005 m g . / c c . + n i c o t i n i c a o i d 0 .001 m g . / c c . SM + p i m e l i o a o i d 0 .00025 mg . / o c . + B~ala< n i n e 0 . 001 mg . / o c , •+- n i o o t i n i c a c i d 0 , 001 mg . / o c . SM •*• p i m e l i c a c i d 0.00025 mg . / o c . + carno-s i n e 0,005 mg . / o c . -t- n i c o t i n i c a c i d 0 .001 m g . / c c . 0 .0 2.9 0 ,0 0 .0 . 0 ,0 0.0 0,0 0 . 0 0*0 . 0 ,0 0 ,0 0 ,0 EMB 2173 0.0 1.1 0.0 0 .0 0 .0 0.0 0 .0 0 ,0 0 ,0 0.0 0 ,0 0 . 0 duce j u s t s l i g h t amounts of a c i d t The improved c o n d i t i o n of the medium due t o the a d d i t i o n of amino a c i d s suggested t h a t the' n i t r o g e n source may be i n -adequate. S i n c e pure amino a c i d s are very expensive i t was d e c i d e d t o p r e pare a mixture of amino a c i d s by making a com-p l e t e a c i d h y d r o l y s i s of c a s e i n l a c t a l b u m i n and u s i n g t h i s m i x t u r e i n the medium to r e p l a c e the ammonium c i t r a t e as the n i t r o g e n source. EXPERIMENTAL 85,71 gms, of B r i t i s h Drug Houses' L i g h t White S o l u b l e C a s e i n and 14,29 gms, of Borden's l a c t a l b u m i n w e r e.digested i n one l i t r e o f 20$ (by weight) s u l p h u r i c a c i d . The d i g e s -t i o n was c a r r i e d on u n t i l t h e f i l t r a t e gave a n e g a t i v e B i -u r e t t e s t . T h i s r e q u i r e d 17 hours of d i g e s t i o n under a r e -f l u x condenser, • To the r e s u l t i n g m i x t u r e 10 grams of c h a r c o a l were added and the m i x t u r e shaken, f i l t e r e d , and t r e a t e d w i t h barium h y d r o x i d e to remove a l l of the s u l p h u r i c a c i d . The f i l t r a t e was d i s t i l l e d i n vacuo t o two l i t r e s and then the voliime made to 2500 C C r - c o n t a i n i n g 0.4$ t o t a l n i t r o g e n . U s i n g the c a s e i n - l a c t a l b u m i n h j i d r o l y s a t e as a b a s i s the f o l l o w i n g s a l t s were added: KH2PO4 0,05$, KgHPO^ o.05$ sodium a c e t a t e 0.6$, EeS0 40.001$ Mg S 0 4 0,02$ and MnS0 4 0.001$, T h i s medium (SMg) was t e s t e d i n t h e u s u a l way u s i n g the c u l -t u r e s E. M. B]_173 s E. M. .B2173, and SA30. The r e s u l t s are d e t a i l e d i n Table X, - 5 8 -IABLE 2 T ITMTABLE AOIB I IY I H GRAMS LAGTIQ AGTD PER. LITHE. Medium -employed as s u b s t r a t e SMg SMg + y e a s t ( 0 . 1 5 $ ! SMg •+- 2A SMg + EA2B SMg +• BE2B SMg * 2A + -EA2B + EF2B SMg + l a o t o f l a v i n e Slvlg -t- l a o t o f l a v i n e y e a s t ( 0 , 15$ SMg + l a o t o f l a v i n e + 2A SMg l a o t o f l a v i n e + EA2B SMg -»- l a o t o f l a v i n e + EF2B SMg t- l a o t o f l a v i n e + 2A 0*7 4 , 1 0 .7 i a 0.7 0 ,7 1,6 4 , 1 1 . 4 ; 2.3 2. 7 SA2B EE2B 1.6-G u l t u r e number EMB-,173 EMB g l 7 3 0 ,0 7 .4 0 , 2 0,9 0 ,2 0 .2 0 .0 7 ,4 0,5 0 ,2 0 ,2 0 .2 SA30 0,0 1.6 0,5 1.1 J L 6 !L 0.7 0.0 2,3 0,5 1.4 0,9 0,5 The B i o s f r a c t i o n s were added a t the r a t e of 2$ and l ao to -f l a v i n e a t 0 ,5 mg, p e r l i t r e , . ' : 1 1 - 5 9 -PISOIXSSION From Table X i t i s seen that even with the complete mix-ture of amino acids and salts there i s no acid production a l -though slight traces of growth might have been present i n some of the cultures. However when the activators, Bios and laetoflavine are added a good growth and a small acid pro-duction i s obtained, The addition of yeast extract produces a very good growth and a high acid production. ,. .QffNGLUSIQHS From an examination of Tables T i l and X i t i s seen that the medium containing amino acids i s more suitable for the growth of the l a c t i c acid bacteria studied than i s a medium in whioh they are absent. The tremendous difference i n the acid production i n the medium containing the autolysed yeast extract and a mixture of the three Bioses indicates that there i s s t i l l another important factor present i n autolysed yeast extract and which i s not Bios IIA nor either of the constitu-ents of Bios IIB, e.g. EASB and EF2B, THE NATURE OF THE AOTIYATORS REQUIRED BY THE LACTIC ACID BACTERIA BART- 7 . The P u r l f l o a t i o n of B i o s -60-THE PURIFICATION OF BIOS It oan readily be understood why Bios has not been iso -lated i n the pure form and i d e n t i f i e d when one considers the d i f f i c u l t i e s which confront those working with i t . F i r s t l y , since i t exists i n very small quantities in the starting ma-t e r i a l s large amounts of these substances are required i n order to obtain enough of the concentrate to enable one to study i t . Then the removal of the impurities requires very careful treatments to prevent large losses of the active con-stituents. Since the only methods of testing the concen-trates obtained are b i o l o g i c a l i n nature i t i s readily seen that countless d i f f i c u l t i e s might arise not only for the i n -dividual-worker but p a r t i c u l a r l y so when an attempt i s made to correlate, the findings detailed with results reported up-on by other investigators. Recent: work has tended to substantiate the hypothesis advanced i n Bart I (pages 26-34) that the EA2B fraction shown to be essential for tlie most intensive metabolism of the l a c -t i c acid bacteria i s iden t i c a l with the Vitamin Bg. Lepkovsky '(1938), ayorgy (1938), Kuhn and Wendt (1938), Keresztesy and Stevens (1938) and Ichiba and Michi (1938) i s o -lated Vitamin B & i n c r y s t a l l i n e form and showed: that i t s em-p i r i c a l formula was O^H^^Og.HGl, The properties of the cry-s t a l l i n e Bg.are iden t i c a l with those given f o r the concen-trated EA2B fractions. The formula given f o r Vitamin B 6 also suggests that i t -61-might be related to the factor isolated by Eddy, Kerr, and Y/illiams ••(1924). Hoy/ever their f r a c t i o n was not adsorbed by f u l l e r ' s earth and not precipitated by phosphotungstic acid while 'Vitamin: Bgis adsorbed by f u l l e r ' s earth at pH 1.4 and precipitated by phosphotungstio acid. • . The work reported upon below deals with an account of attempts made to purify the Bios IIA f r a c t i o n of M i l l e r . EXPEBIMEFIAL Three tins of tomato juice were treatedwith 67 grams tannic acid dissolved i n 300 ml, hot water and l e t stand 24 hours, The. precipitate was oentrifuged off and the f i l t r a t e treated with 320 gms, lead acetate dissolved i n 400 cc. of hot. water, The precipitate was oentrifuged off, , the excess lead removed with 27 cc. concentrated HgS0 4 (and the remainder with BaS, The HgS was.removed by evaporation in,vacuo to about 1500 cc. To the f i l t r a t e 3 cc, of concentrated sulphuric acid were added and then treated with.two portions of charcoal, -the f i r s t 50 gms. and the second 37 gms. The charcoal fil«* trate was evaporated to about 300 cc,, then 16.6 c.c. of con-centrated sulphuric and the mixture digested for,5 hours at 150°0:, This treatment "chars a large portion of the inactive material without affecting the a c t i v i t y . The char was thor-oughly washed with 1 l i t r e of d i s t i l l e d water, I t jac.? the f i i - r trate and v/ashing 0a(0H)g was added u n t i l the solution was only s l i g h t l y acid, i n order to remove most of the sulphuric • -62-acid. The precipitate was f i l t e r e d off, the f i l t r a t e de-colorized with charcoal and made alkaline to P o i r r i e r ' s Blue with Ga(OH) The solution was evaporated on a steam hath i n vacuo to remove ammonia and v o l a t i l e "bases. The excess calcium was precipitated as the carbonate by saturating the solution with GOg, b o i l i n g and f i l t e r i n g . The f i l t r a t e from the calcium" precipitation was diluted to €66 cc. and 166 gms, copper acetate i n 866 cc. boiling water were added, A small precipitate was formed which was f i l t e r e d off and the copper removed from the solution by pre-c i p i t a t i o n with HgS. The f i l t r a t e was evaporate din vacuo, to dryness, the residue was dissolved i n water and neutralized with Na(OH) and made to a volume of 500 cc. The f i l t r a t e was then treated with Neu berg's Reagent. The solutions consists of 10 gms. anhydrous NagGOg and 25 grams mercuric acetate per 100 cc. solution. They were added a l -ternately beginning with 16.6 cc. of the sodium carbonate followed by 20,6 cc. of mercuric acetate u n t i l 50 oc, of the carbonate and 62 cc. of the acetate had been added. Then 3666 oc, of 95% alcohol were added and the mixture l e t stand two hours, the precipitate was f i l t e r e d off, the excess mer-cury removed with HgS and the f i l t r a t e evaporated to dryness, i n vacuo on a steam bath, The residue was taken up i n water, the volume made to 460 cc, and the p r e c i p i t a t i o n was carried out as above but using 220 cc. of sodium carbonate solution, 274 oc, mercuric -63-aoetate solution and 4333 oo, of 25fo alcohol. The precipitate was suspended i n 666 cc« ?;ater and decomposed with H _ S — f i l -2 tered and washed the precipitate, evaporated the f i l t r a t e and washings to dryness and made up to 50 cc« with water. This solution constituted the p u r i f i e d Bios IIA. An aliquot of the f i l t r a t e s , at the different steps in the p u r i f i c a t i o n , was retained for testing of the a c t i v i t y of the f r a c t i o n . The amount added as enrichment to milk was calculated to contain a proportionate amount of the active p r i n c i p l e i n every case e.g. 3% of the Bios IIA after the charcoal treatment. The acid production i n grams l a c t i c acid per l i t r e was determined for the cultures E. M. 173, E. M. Bgl73 and SA30. The results are detailed i n Table X I . PIS GIT SSI ON From the data presented i n Table XI i t i s seen that the active p r i n c i p l e i s destroyed even under such drastic treat-ment as autoclaving at 150°0 i n a strongly acid solution. Furthermore on p u r i f i c a t i o n and removal of other materials the f r a c t i o n appears to become increasingly more active. 25 c c of the p u r i f i e d Bios IIA solution was evaporated to dryness and tested for s o l u b i l i t y . The white residue was insoluble i n absolute alcohol, ether and acetone. Since i t was insoluble i n these solvents i t was decided to extract i t with claloroform. After 8 hours of -continuous extraction and evaporation of the chloroform there remained a brown oi l y l i q u i d which was p r a c t i c a l l y insoluble i n water. - 6 4 -TABLS S I .TITRATABLE AOILI.TT m/GRAMS MCTIG APIS PER LITRE Medium employed as s u b s t r a t e C u l t u r e number SMB 117 3 E¥R g 173 SA30 M i l k . 1.1 , 0.9 2.5 M i l k + y e a s t e x t r a c t (0 .15$) 2,9 3,6 M i l k + 2A a f t e r c h a r c o a l t r ea tment 1.6 2 . 3 3*8 M i l k -+ 2A a f t e r a u t o c l a v i n g and Ca (OH)g t r ea tmen t 2.3 1,4 3 .8 M i l k + 2A a f t e r - Cu t r ea tmen t 2,9 1.8 4 ,3 M i l k + 2A a f t e r - 1 s t Heuherg p r e e i p i t a t i o n | 5.0 2,7 ' 7,7 M i l k + 2A a f t e r 2nd Neuberg p r e o i p i t a t i o n | 4 , 3 3 ,2 9 , 2 -65-The residue insoluble i n water was extracted with 95% alcohol forming a yellow solution, The alcohol was evaporated • of f-and an o i l y l i q u i d remained, The residue- insoluble i n alco-hol was dissolved i n chloroform and the•chloroform evaporated slowly, From the chloroform solution brownish yellow needle shaped orystals separated out. The fractions r e s u l t i n g from the chloroform extraction were added as enrichment to milk and the acid production i n grams l a c t i c acid' per l i t r e was determined for cultures E, M. B.jJ.73, E . I . B g173 and SA30. The results are detailed i n Table XII, Sodium acetate which might have been a contami-. nant i n the fractions was also added to milk, DISCUSSION A study of the results detailed i n Table XII sho?/s that extraction of the dried Bios IIA results i n a complete loss of a c t i v i t y . Neither the extract,, residue or combination of the fractions exhibited any stimulating power. Furthermore an examination of the fractions obtained shows that the chloroform extract Is of an o i l y nature and i s insoluble i n water. The crystals obtained from the water and of alcohol insoluble f r a c t i o n are not unlike those^cholesterol. A They also give a positive Salkowski's reaction for cholesterol, However i t s melting point i s 114°G, while that of cholesterol i s 148.5°G. : In this connection a recent paper by Devloo (1938) i s of paramount importance. He shows that certain of the steroles TITRATABLE AGILITY IH" GRAMS LACTIC A0I3) PER LITRE Medium employed as s u b s t r a t e M i l k M i l k . -»-. y e a s t M i l k +• c h l o r o f o r m s o l . c r y s t a l s M i l k + a l c o h o l s o l u b l e o i l M i l k + • wa te r s o l u b l e e x t r a c t M i l k + c h l o r o f o rm i n s o l . r e s i d u e M i l k +• sodium a c e t a t e M i l k + a l l f r a c t i o n s t oge t he r C u l t u r e number EMB.^73 2 ,5 6 , 1 2«5 2 ,5 2,5 2 ,5 2«5 2,5 EMB 2173 1.1 4 .5 1,1 1.1 1.1 1,1 1.1 10 SA30 6,1 7,3 6 ,1 6,1 6,1 6,1 6 ,1 6 .1 and. V i t a m i n Dg possess B i o s a c t i v i t y . However, attempts t o s t i m u l a t e the a c i d p r o d u c t i o n of t h e l a c t i c a c i d "bacteria s t u d i e d "by e n r i c h i n g the medium w i t h the s t e r o l e s e r g o s t e r o l and c h o l e s t e r o l , l e c i t h i n and a commercial p r e p a r a t i o n of V i t a m i n D ( H a d i o s t o l ) have "been u n s u c c e s s f u l . I t would ap-p e a r t h a t the a c t i v e component of B i o s I I A i n so f a r as the l a c t i c a c i d b a c t e r i a are concerned i s not a s t e r o l , or t h a t t h e r e might "be two f r a c t i o n s i n v o l v e d or t h a t when the f r a c -t i o n i s changed to the i n s o l u b l e form i t l o s e s i t s a c t i v i t y o r t h a t the method used i n adding t h e f r a c t i o n s t o the media d i d not produce a s u f f i c i e n t d i s p e r s i o n of t h e m a t e r i a l t o i n f l u e n c e the metabolism o f . t h e organisms s t u d i e d , N e v e r t h e l e s s the chemical and p h y s i c a l p r o p e r t i e s of the f r a c t i o n s e s p e c i a l l y the m e l t i n g p o i n t s of the c r y s t a l i n e f r a c t i o n , w h i c h i s 114°C and of c a l c i f e r o l ( V i t a m i n Dg) which i s 114-11?°C". s t r o n g l y suggests t h a t t h e r e e x i s t s a r e l a t i o n -ship, between B i o s I I A and V i t a m i n D, -68-STJMMARY 1 . A c r i t i c a l review of the l i t e r a t u r e on the relationship of Bios to the Vitamins is presented,, 2, A method for the fractionation of Bios IIA and IIB of M i l l e r i s described, 3 . It has been shown that Bios IIB is composed of two dis-t i n c t e n t i t i e s which have been designated as Fractions EA2B and EF2B, 4 . On the basis of th e i r physical and chemical properties, i t i s suggested that the growth stimulants required by the l a c t i c acid bacteria are identical with certain of the heat-stable accessory food factors of the Vitamin-B complex es-se n t i a l f o r the growth of animals, 5, In an attempt to define more e x p l i c i t l y the nature of the respective Bios fractions the procedure of Viokery has been applied to-the separation of the nitrogenous constituents of tomato juice, 6, The following compounds Vitamin B 1 ? laotoflavine, Radios-t o l , stachydrin, choline, i n o s i t o l , n i c o t i n i c acid, 1-leuoine, 1 - h i s t i d i n e , lysine, cystein, urocanic acid, imlnazol a c r y l i c acid, ergothioneine, dl-methionine, 1-aspartic acid, g l u t a r i c acid, pimelio acid, adipic .acid, aspargine, serine, u r a c i l , carnosine, ergosterol, l e c i t h i n and Vitamin 0 singly and in combinations were tested for Bios a c t i v i t y but were found to be without influence on the v i t a l a c t i v i t y of the l a c t i c acid bacteria. 7» The influence of the ions f e r r i c , ferrous, oaloium, mag-nesium, manganic, manganous, cobalt, oopper, sine and iodine on the acid producing a b i l i t y of the l a c t i c acid bacteria was determined. Iron and manganese have been shown to exart a stimulating effect when added to milk but to be without influence when incorporated in synthetic media. 8 . 1 basic synthetic medium for th© study of the influence of activators on the l a c t i c acid bacteria has been desoribed. 9* A method Is described for the p u r i f i c a t i o n of Bios IIA with the resultant i s o l a t i o n of a sterol tike substance. On the basis of i t s properties i t i s probable that this material i s i d e n t i c a l with Vitamin D g. This substance is without apparent influence on the laot i o acid bacteria. The possib-i l i t y exists however that this substance may play th© role of an activator for the se organisms* BIBLIOGRAPHY 1. Adler, E,, arid Euler, H. Y. (1934) Ztschr. f, physiol. chem. 22.5, 41-45, 2. Amand, A, (1902) La Cellule 20, 225, 3. Baohman, F. M, (1919) jr. B i o l , ohem, 39, 235-258, 4. Biroh, I, ¥., and Gyorgy, P. (1936) Biochem J. 30, 304-315. • 5. Buston, H, W., and Rasinathan, S. 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(1919) J, B i o l . Chem. 38, 465^-486. 83. Williams, R. J . (1920) J . B i o l . Chem. 42, 259-265. '84. Walllaeis, R. 3. (1928) Science 67, 607-608. 85. Williams (.1931) 86. Williams, R. j . and Bradway, E. M. (1931) J . Amer, Ghem. Soc. 53, 783-789. 87, Williams, R. J,, Lyman, G. M. Goodyear, C, H», Trues-" d a i l , J . H . and'Haladay, B. (1933) J", Amer. Chem. Soc. 55, 2912-2927, 88. Williams B J and Saunders (19 34 ) . B ioohem. J . 28. 1887. 89, Williams, R. J,, 'Warner, M. E., and Roehm, R. S. (1929) J. Am. Ghem. Soc. 51, 2764-2774, : 90, Williams, R. J,, Wilson, J. L., and Ahe, F. H. v, der. (1927) J . Am. Chem. Soc. 49 , 2 27-235, 91, Wood Anderson and Workman, 92. . Wood, H. G., Taturn, E. L., and Peterson, W, H., (1937) J. Bact. 33, 227. I w i s h t o extend my sincere thanks to B r , B.A. E a g l e s for h i s gu idance in.the l a b o r a t o r y work and h i s h e l p f u l suggest ion® in the p r epa r a t i o n , o f this thesis, So M i s s 0. O k u l i t c h f o r her v a l u a b l e a s s i s t a n c e in xh. the l a b o r a t o r y work • and to the U n i v e r s i t y o f B r i t i s h Co lumbia f o r p r o v i d i n g a g ran t wh i c h made p o s s i b l e some of the work© 

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