D O C T O R A L D IS S E R T A T IO N SERIES A Synthetic M e d iu m F o r Microbiological A ssay O f R ib o fla v in t PanlothenU Acid,___ TITLE R join t, N icotinic A cid, Ijrrrdoxin e _______A n d F olic R eid ___________ AUTHOR £ I M U $ jC 1! ¥ V I * U NIVE RS\T V _ _ JM L DEGREE . DATE icingan Stale College n.x>. J . /# - «LA PUBLICATION NO. 1I 1| 1I I I 1I 1I 1| 1 i I I I I I f | I[ I I I | I i l ' 1 I 11 ' 1 12 iM! 1 1 13 ^ U N IV ERSITY M I C R O F I L M S M ANN ARBOR - MICHIGAN A SYNTHETIC ".1EIIUY FOB. MICROBIOLOGICAL AS : ay OF RIBOFLAVIN, PANTOTHENIC ACID, BIOTIN NICOTINIC ACID, PYRIDOXINE AND FOLIC ACID t)v Andrew Hy;na -X . The v a r i a b l e s o f c a s e i n h y d r o l y s a t e i n m i c r o b i o l o g i c a l a s s a y m e dia " 'e r e removed by t h e u s e o f p u r e araino a c i d s f o r t h e g r o w t h r e q u i r e m e n t o f L. a r a b i n o s u s and h . c a . s e i . D e t a i l e d s t u d i e s were made on t h e minimum number and q u a n t i t i e s o f amino a c i d s e s s e n t i a l f o r g r o w t h o f t h e s e o r g a n i s m s . I n u s i n g L . a r a b i n o s u s i t was f ound t h a t t h e f o l l o w i n g n i n e amino a c i d s ; try o to p h a n e , c y s ti n e , glutam ic s c id , a r g in in e , t h r e o n i n e and v a l i n e p r o d u c e d a good g r o w t h . tyro sine, leu cin e, iso leucine, The p r a c t i c a l u s e o f t h i s medium f o r t h e a s s a y o f b i o t i n , n i c o t i n i c a c i d and p a n t o t h e n i c acid, was d e m o n s t r a t e d w i t h L. a r a b i n o s u s a s t e s t o r g a n i s m . L. e a s e l was more f a s t i d i o u s , and r e c t i i r e d i n a d d i t i o n t o t h e a bo ve amino a c i d s f a c t o r s h i o t i r . and f o l i c a c i d . p resen t in y e a s t as On a d d i t i o n o f t h e s e f a c t o r s L» c a s e i g a v e c o n s i s t e n t r e s u l t s on t h e e s s a y o f s i x f a c t o r s o f B complex v i t a m i n s a s r i b o f l a v i n , p a n t o ­ then ic acid, r y r i d o x i n e , n i c o t i n i c a c i d , b i o t i n and f o l i c a c i d . a l l t h e sm ino a c i d s , A m ixture o f a s o c c u r i n g i n c a s e i n h y d r o l y s a t e , i s recommended i n o r d e r t o b a l a n c e o x i d a t i o n and r e d u c t i o n r e a c t i o n s b e t w e e n p a i r s o f amino a c i d s b e c a u s e a n a b s o l u t e d i s t i n c t i o n c a n n o t be m a i n t a i n e d b e t w e e n H d o n a t i n g and H a c c e n t i n g amino a c i d s . T he se amino a c i d s i n t h e q u a n t i t i e s shown p l u s t h e p u r i n e and o y r i m i d i n e b a s e s a r e - t h o r o u g h l y mixed i n a. d e s i r e d am oun t and :-i may be s t o r e d i n amber b o t t l e s w i t h o u t a p p a r e n t d e p r e c i a t i o n . The amount n e c e s s a r y f o r m e dia o r e o a r a t Lon may be w ei ghe d from t h i s m i x t u r e when and a s required. I n t h e u s e o f t' rii s m i x t u r e t h e v a r i a b l e s p r e s e n t i n c a s e i n h y d r o ­ l y s a t e and y e a s t s u m l e m e n t a r e removed a nd c o n s i s t e n t r e s u l t s o r e o b t a i n e d . S t u d i e s w e re rna.de on t h e e f f e c t o f a d d e d c a r b o h y d r a t e s , s u c h a s manr.ose, g a l a c t o s e , and i n o s i t o l , a s w e l l a s n u c l e i c a c i d a nd p a r a amino b e n z o i c a c i d . The a n t i v i t a m i n a c t i v i t y o f m e t h i o n e a l s o was i n v t s L i g a t e d . The medium, i s o f known c h e m i c a l c o m o o s i t i o n e nd r e s u l t s o r e more r e p r o d u c i a b l e and t h e procedure g r e a t l y s im p lif ie d . A SYNTHETIC MEDIUM FOR MICROBIOLOGICAL ASSAY OF RIBOFLAVIN, PANTOTHENIC ACID, BIOTIN, NICOTINIC ACID, PYRIDOXINE AND FOLIC ACID.* By Andrew M. Hyraa A THESIS Submitted t o th e Graduate School o f Michigan S t a t e College o f A g r i c u l t u r e and Applied Scie nce i n p a r t i a l f u l f i l m e n t o f the r e q u ir e m e n ts f o r th e degree of DOCTOR OF PHILOSOPHY Department o f B a c t e r i o l o g y and P u b l i c Health Ea s t Lans ing , Michigan 1945 T/'BLE OF CONTENTS INTRODUCTION. ...................................................................... r NPEFI '.’"NTAL. . ............................... . ..................... .... riFOBEFIOE, ....................................... . . . . CONCLUSION................................................................................... T.JT-HRtTnFE CTT'FD. ................................................ Introduction M i c r o b i o l o g i c a l ass ay ? f o r th e v a r i o u s f a c t o r s o f the B complex v i t a m i n s have become well e s t a b l i s h e d . These methods a r e base d on th e e s s e n t i a l n a t u r e o f t h e B complex f a c t o r s as r i b o f l a v i n , p a n t o t h e n i c s a i d , b i o t i n , n i c o t i n i c a c i d , py rid ox in e and f o l i c a c i d f o r the i n i t i ­ a t i o n =-nd maintenance of gravrth of c e r t a i n ty p e s of b a c t e r i a . While 1 £ 3 many micro -or gan is ms - - b a c t e r i a ; molds ; fu n g i j a r e a b le t o s y n t h e s i s e one or more o f t h e s e f a c t o r s , t h e r e a r e a few organisms such as th e l a c t i c a c i d b a c t e r i a , s t r e p t o c o c c i and o t h e r s t h a t a r e unable t o grow u n l e s s an o u t s i d e su eoly o f ore or more o f t h e B comolex f a c t o r s a re added. This h«? become th e b a s i s f o r the m i c r o b i o l o g i c a l a ss a y o f t h e B c enrol ex vi ten-ins. Lee t i c a cid b a c t e r i a 4 J 5 end s t r e p t o c o c c i a r e m c u r r e n t use f o r r i b o f l a v i n a s s a y and P r o te u s mor g a n i C, 7 l a c t i c acid • organisms P, 9 , 10, 11 and hemolytic s t r e p t o c o c c i. 12 ff o r p a n4t o+t'n e m•c a c i*J d. , . T 4. J. J. 15 K Pacch-3'romyces c e r e v l e i s e , f ta phy 3,ococ cue a u r e u s , C l o s t r i c i u m but y l l ]5 10 cum " and Lact'qhac i l l u s _ a r a b l n o s u s ‘ have been suggested, and used f o r b io tin assay. L. n r a b i n osus *' i s a l s o used f o r n i c o t i n i c a c i d d e te r m in - . . IB , La c to be.c 111 u s c a.ae i to r nyridoxme 19 _ 20 St r e p t o c o c c u s I s c t i s and L. c a s e i are used in a s s a y f o r a t ion s and f a c e t , c e r e v i s i a e assay, 16 17 f o l i c ncid.From the above i t becomes obvious t h a t th e s e m i c r o b i o l o g i c a l s s m y s e r e numerous with & wide veri.ety of c u l t u r e media and p r o c e d u r e s . Although many of t h e '-bove methodt have become s ta n d a rd p ro c e du re s a method which enrol oys a s i m p l i f i e d b a s a l medium -and a s i n g l e t e s t organism would be of g r e a t a d v a n ta g e . With t h i s in mind t h e l a c t i c a c i d b a c t e r i a have been ir-ves t i t r a t e d and L, c a s e i and a r a b i n o s u s have been employed. Very s a t i s f a c t o r y r e s u l t s have been a t t a i n e d w it h I,, a r a b i n o s u s f o r tie a ss a y of p a n t o t h e n i c a c i d , b i o t i n and n i c o t i n i c a c i d , L. c a s e i , however, g i v e s t h e g r e a t e s t promise as t h e d e s i r e d t e s t organism be cause i t s 4 9 n u t r i t i o n a l requirements include r i b o f l a v i n , pantothenic acid , . +. . .9 . , . £1 £0 , „ .. £0 n i c o t i n i c a c i d , pyricioyme , biotir.i ana f o l i c a c i d A s tu d y o f media m s made t o d e v i s e a s i m p l i f i e d medium t h a t could be used f o r t h e e ss a y o f t h e v a r i o u s B com pi ex v i t a mi jus. In p r a c tic a lly a l l t h e p r o c e d u re s p r o t e i n m a t e r i a l i n t h e form of c a s e i n , pe ptone and v e r s t i s used i n c o n j u n c t i o n w it h c e r t a i n amino a c i d s . Hydrolyzed c a s e i n i s a v e r y prominent c o n s t i t u e n t o f many of t h e media. Various h y d r o l y s a t e s o f casei?'- and g e l a t i n have been used t o g e t h e r with a r a b i n o s us p-:d L. c a s e i as t e s t orgy-riisms. i b r e a l down o f t h e c a s e i n h y d r o i y s a t e mediurrg of S n e l l and " r i g h t i r t h e i r a s s a y o f n i c o t i n i c a c i d u s i n g L. a r a b in oe us v-.r.s .node. This was done i n o r d e r to i n v e s t i g a t e th e c o n s t i t u e n t s e s s e n t i a l or r e s p o n ­ s i b l e f o r g ro w th . T h e i r medium i s o f t h e f o l l o w i n g com pos it ion : i c i d h y d r o ly z e d c a s e i n ------------- 0,5* C y s ti n e 0.01 $ —■ -------- Tryptoohane 0.0 1$ Glucose ------------- 1.0% Sodium -^ c e tr te ------------ 0.6% Adenine ------------- 10 p .p . m . Guanine---------------------------------------- 10 p . p . m . Uracil — 10 p.p .m . R ib o f la v in --------------------- ----------- 0 .2 p.p.m. B i o t i n c o n c e n t r a t e -------- ----------- 0.4 p . p . b i l l i o n of pure c o n c e n t r a t e In o r g a n ic s a l t s ----------- ------------ trace Tryptophane was added bythem because of h y d r o l y s i s of c a s e i n . Cystine was i t s d e s t r u c t i o n on the a cid added because the amount n a t u r a l l y ore r e n t in -■ c a s e i n hydrolyro.te i s i n s u f f i c i e n t t o meet the sulphur re q u ir e m e n ts o f t h e t e s t organism used. One c o n s t i t u e n t of the medium v.&s o fl i tt ed a t each t r i a l with th e r e s u l t s as shown i n th e graph. Thus i t becomes e v i d e n t t h a t i n a d d i t i o n to t h e B complex f a c t o r s as p a n to th e n ic ? c i d , b i o t i n ana n i c o t i n i c a c i d , t h i s t e s t organism r e q u i r e s the amino a c i d s p r e s e n t in c a s e i n h y d r o l y s a t e f o r growth. i s no growth on the omission o f t r y p t o p h a n e , A small amount of growth re s po nse i s noted on th e omission of c y s t i n e . of some c y s t i n e in the c a s e i n h y d r o l y $ f t e . There This i n d i c a t e s the presence The omission of casein h y d r o l y s a t e r e s u l t s in no growth ana shov.s the importance o f the amino a c id s f o r growth r e s p o n s e . Under proper c o n d i t i o n s , c a s e i n hydroly a t e could be r e p l a c e d by a mixture of n u r i f i e d amino a c i d s , compounded to y i e l d l i k e r e s u l t s . Gladstone (?5) in 1957 i n a study of th e n u t r i t i o n a l re qu ire m e nts of St a o h . a ure us form ulat ed a medium c o n ta i n in g o u r i i i e d amino a c i d s , with satisfactory re su lts. The s i m u l s r i t y o f G la d s to n e 1s median to the compo­ s i t i o n of c a s e in h y d r o l y s a t e i s shown i n Table 1. The amount of c a s e i n h y d r o l y s a t e and sodium hydroxide t r e a t e d 16, EG, 4, 8 peptone g e n e r a l l y recommended i s 0 . 5 per c ent TABLE I Comparison o f a one h a l f o f one p e r cent- c a s e i n h y d r o l y s a t e s o l u t i o n w i t h Glad :t o n e ' s medium p e r 10 c c. Amino a c i d s c a s e i n hydrolysate _soluticn, .Amino a c i d s G l a d s t o n e ' s medium Mgs. pe r 10 c c . p e r 10 Glycine (0.4*) * 0.2 Glycine 0.1 A la ni ne ( 1 . 8 $ ) 0.9 Alanine d 1 0.4 Serine (0.5$) 0. 2 5 Serine d 1 3.2 Valine (7.9$) 5.9 5 Valine d 1 1.6 Leucine d 1 1.0 4 .8 5 Iso leucine d 1 1.0 1.95 Phenylalanine d 1 1.0 Leucine ) ) ) Iso leucine Phenylalanine 9.7$ (5.9$) Tyrosine (6.5$) 3.25 Tyrosine 1 0.7 C"stine (9.5$) 0.1 5 Cystine 0.1 T r yptop ha ne (2 . 2%.) 1.1 T r y p to p h a n e 1 0.5 Proline 4.0 Proline 1 1.0 Hydroxy P r o l i n e (0.2%) 0.1 Hydroxy P r o l i n e 1 0.1 A spartic acid (4.1$) ofv.• nc vj ^ A spartic acid 0.5 G l u t a m i c nc i d (£1.8$) G lu ta m ic a c i d 2.5., (8.0$) 10.9 1 Hydroxy g l u t a m i c ( 1 0 . 5 $ ) 5.25 Hydroxy glutamic: H istidine ( 2 .6 $ ) 1.5 H istidine 1 0.4 'A r g i n i n e (5.2$) 2.6 Arginine 0.8 Lysine (7.6$) 3.8 L y s i n e d.i HL d 1 2.0 Methionine (5.1$) 1.5 5 M e th i o n in e cl 1 0.8 Ammonia 0.8 Threonine d 1 0.6 (1.6$) d ■* P e r c e n t a g e of amino s c i d c o n t e n t of c a s e i n ^ ^ . BXPIfliLWKTAL The f i r s t i n v e s t i g a t i o n s were concerned with the use o f v a r i o u s hydrolysates. The proce dure f o r the removal of v ita m in f a c t o r s was 4, 8, 9, 10, i x c a r r i e d out a cc o rd in g t o st u n o ar d p r a c t i c e . Casein a cid h y d r o l y s n t e prervored from n v it a m in f r e e c a s e i n * , an enzymatic c a s e in h y d ro ly s a te * * and a c id h y dr ol ys e d g e l a t i n were i n v e s t i g a t e s i n t h e p r e p a r ­ a t i o n o f s t a n d a r d b a s a l media. The enzymatic c a s e in b y d r o l y s a t e gave a maximum a c id pro d u c ti o n e q u i v a l e n t to I f . 6 c c . of 0 . 1 N. sodium hydroxide. The a c i d hy d ro ly ze d c a s e i n e q u i v a l e n t t o 9 .2 c c . and g e l a t i n acid hydro­ l y s a t e e q u i v a l e n t to 7 . 0 cc. V a li ne, tr y p t o p h a n e and hydroxy g lu ta m ic pcid were added t o the g e l a t i n hydroly rate medium because the y a r e no t n a tu ra lly oresent in g e la tin . The amount o f g lu ta m ic was i n c r e a s e d t o t h e amount p r e s e n t in c a s e i n h y d r o l y s a t e as t h e amount p r e s e n t i n g e l a t i n i s much l e s s th a n i n c a s e i n . Varying r e s u l t s , however, were o b t a i n e d depending upon th e amount o f N o r i t e treo.trr.ent f o r the removal of t r a c e s of vita mi n c o n t e n t and th e completeness o f h y d r o l y s i s . Complete h y d r o l y s i s of c a s e i n i s d i f f i c u l t . In t h e N o r it e t r e a t m e n t T i s i l u i s has r e p o r t e d t h e a b s o r p t i o n of amino •7 do a c i d s end p e o t i d e s by a c t i v a t e d c h a r c o a l and carbon . Because o f the inc om ple te ne s s of h y d r o l y s i s , the v a r i a b i l i t y i n the composition o f th e o r i g i n a l c a s e i n , and the l o s s of amino a c i d s on vitamin removal, t h e r e i s much u n c e r t a i n t y of the f i n a l amounts of each amino i n the h y d ro ly s a te . * From: R esearch L a b o r a t o r i e s , Corporation, Chagrin f a l l s , Ohio. -** From; L a b o r a t o r i e s , ulead & Johnson, Z v a n s v i l l e , I n d i a n a , I t would. 3 opesr, f i n a l medium. t h e r e f o r e , t h a t t h e r e e r e too many v a r i a b l e s i n th e Any change i n q u a n t i t y i n each amino a c id p a r t i c u l a r l y i f t h e amount i s lov.er th a n t h e r e q u i r e d minimun o f the t e s t organism would r e s u l t i n marked v a r i a t i o n s i n a s s a y r e s u l t s . An a s s a y t e s t showed t h a t t h e c a s e i n h y d r o l y s a t e gave s a t i s ­ factory r e s u lts . However as s t a t e d , i t i s d i f f i c u l t to o b t a i n hy d ro - l y s r t e s o f unifo rm c o m p o s i t i o n . The n e x t s t u d i e s were l o g i c a l l y examin­ a t i o n s f o r the v s l u e of t h e amino a c i d s p r e s e n t i n c a s e in h y d r o l y s a t e , l o t h as t o t h e s p e c i f i c amino a c id and q u a n t i t i e s of each p r e s e n t . Accordingly media, mere prepare;! o f pure amino a c i o s based on the c a s e i n hydrolysate amino a c i d c o n t e n t . Ad e t e r m i n a t i o n mas made o f the e s s e n - t i a l amino a c i d s n e c e s s a r y t o produce and m a i n t a i n growth by w i t h h o l d i n g , i n t u r n , one of t h e amino a c i d s p r e s e n t i n c a s e i n h y d r o l y s a t e . Is fa r es p o s s i b l e t h e n a t u r a l l y oecur fng o p t i c a l l y a c t i v e forms of sraino a c i d s a s found i n c a s e i n were employed. Tfhere i t was n e c e s s a r y t o use s y n t h e t i c . racomic forms th e amounts were i n c r e a s e d , A b a s a l medium was f i r s t p r e p a r e d a c c o rd in g to Table I I . In p r e v i o u s exper imen ts th e need f o r t r y p t o p h a n e , c y s t i n e and c a s e i n h y d r o l y s a t e have been d e m o n s t r a t e d , as no growth oc cu rs on tile om is si on o f any one of t h e s e c o n s t i t u e n t s from th e medium. The b a s a l medium (T abl e I I ) with a l l the amino a c i o s (19) p r e s e n t i n th e r e q u i r e d amounts, ? s i n c a s e i n h y d r o l y s a t e , gave a maximum a c i d p r o d u c t i o n e q u i v a l e n t to 9.8 c c. 0 . 1 b NaQH. R e s u l t s on th e w it h h o ld in g of one o f t h e amino a c i d s i n t u r n a r e shown on t h e accompanying g r a p h . There i s p r a c t i c a l l y no growth w it h o u t t r y p t o p h a n e ( 2 ) , c y s t i n e ( 5 ) , g l u ta m ic a c i d (4) .and v a l i n e (16) and v e ry l i t t l e growth w it h o u t - 7- TABLE I I L a c t i c acid b a c t e r i a , L. c a s e i and L. a r a b i n o sus were s e l e c t e d as t e s t or ga ni s m s. These organisms were s e l e c t e d because of t h e i r f a s t i d i o u s n u t r i t i o n a l requirements. l(-) Tryptophane 0.100 gms. l(-) C y st i n e 0.100 gms. 10.0 gms. Sodium Acetate 6.0 gms. So la . A* (inorganic s a l t s 5 .0 ml. S oln . B* ( 5.0 ml. Glucose " " Adenine 0.005 gms. Quanine 0.005 gms. Uracil 0.005 gms. N i c o t i n i c a c id 0.0001 gms. Thiamine h y d r o c h lo r id e 0.0001 gms. Calcium p a n t o t h e n a t e 0.0001 gms. Pyridoxine 0.0001 gms. Fiboflavin o . ooq;1 B i o t i n (F re e a c id ) 0.0002 gms. l e a s t supplement** Water gms. 10.000 cc. 1000 cc to . (pH a d j u s t e d to 6.8) 1 *So ln . B -*Soln. A KgHPO rngs, %so4 .7H20 0 .5 gms, KHgPO^ 5 mgs. NaCl Water 50 cc. FeS0..7H<-0 4 £ 0 .5 °gms. MnS04 .4HgO 0.557 Water 250 c c . Tlie y e a s t supplement e q u i v a l e n t t o 2 .0 gms. o f whole a u t o l y z e d y e a s t . Yeast was added f o r s t u d i e s with L. c a s e i . t a c < 0 :),] Sodium Hyaro>ide - 8- E x p l a n a t i o n of Graph 1. BaS6l Tiediun Table I I complete w it h a l l amino a c i d s s s i n c a s e i n hydrolysate. O f• ■z 4. Jfi s e l medium Table I I minus 1 - tr y p t o p h a n e i tt tr tr It tt 1 - cystine ft tt tt tt Tt d - g l u ta m ic ac id tt 11 tt TT TT d - arginine 6. tr It t! I! T? 1 - tyrosine 7. tt Tt 11 It tr 1 8. tr tt tt tt ft 1 - leucine. 9. tr tt Tt ti Tt 1 - isoleucine 10. tr Tt It Tt " d 1 - phenyl a l a n i n e 11. it tt IT tt " d 1 - alanine IE. ti ti tt tt TT d - lysine 13. tt IT tt tt IT 1 - serine 14. tr Tt tt Tt tt i X c, Tt - a s p a rtic acid - histidine " d 1* - t h r e o n i n e ... 16. n tt tt ft " d 1 - va l i n e 17. tt tt tt Tt tt 1 - methonine 18. tt TT ft TT ti 1 - proline 19. tt Tt T1 tt IT 1 - hydroxy p r o ] i n e 20. ft tt Tt Tt tt glycine arginine (5), tyrosine (6), a s p a rtic acid (7), leucine (8), iso l e u c i n e (9) and t h r e o n i n e ( 1 5 ) . The omission of phenyl a l a n i n e ( 1 0 ) , alc-.nine ( l l ) , l y s i n e ( i f ) , methionine ( 1 7 ) , c u t down a cid p ro d u c ti o n to a g r e a t e x t e n t . Acid p r o d u c t i o n was only lowered to th e e q u i v a l e n t o f l e s s than 1 c c . of 9.1 N sodium hydroxide on th e omission of s e r i n e ( 1 3 ) , h i s t i d i n e ( 1 4 ) , p r o l ine ( 1 8 ) , hydroxy p r o l i n e (19) and glycine (20). This would i n d i c a t e t h a t t r y p t o p h a n e , c y s t i n e , g l u ta m ic a c id , isoleucir.e, le u cin e, v a lin e , a rg in in e , ty ro s in e , threonine are e s s e n t i a l f o r growth of th e l a c t i c a c id b a c t e r i a . The a d d i t i o n of a s p a r t i c a c i d , phenyl a l a n i n e and methonine i n c r e a s e a c i d p r o d u c t i o n . Maximum p r o d u c t i o n of a c i d i s o b ta in e d when a l l th e amino a c i d s i n the q u a n t i t i e s p r e s e n t i n c a s e i n h y d r o l y s a t e a r e employed. only f o r L. a r a b i n o s u s . y e a s t supplement 52 Thi s hold s t r u e P r a c t i c a l l y e q u i v a l e n t r e s u l t s oc curea when was added to t h e medium m th e case o f L. c a s e i . F u r t h e r i n v e s t i g a t i o n of t h e n u t r i e n t re q u ir e m e n ts of L. c a s e i and L. a r a b i n o s u s f o r t h e v a r i o u s amino a c i d s were c a r r i e d o u t using i n d i v i d u a l amino a c i d s i n c o n ju n c t io n with the b a s a l medium. combinations were t r i e d i n t h i s e x p e r i m e n t a t i o n . employed ' i s t h a t shown i n Table I I above. Many The b a s a l medium To th e b a s a l medium was «dded one amino a c i d at. a time t o a s c e r t a i n which amino a c i d would s t i m u l a t e growth, and in such a manner t h a t th e r e s u l t i n g medium was augmented by each s u c c e s s iv e a d d i t i o n . To the b a s a l medium was added (1) g l u t a m i c a c i d , the next, t e s t medium was ( l ) g l u t a m i c p l u s (H) l y s i n e . Each a d d i t i o n was made i n th e o r d e r as shown on g r a p h . While t h r e o n i n e CO ^ •x * i s not re p o rte d as a n a tu ra l c o n stitu e n t of casein hydrolysate i t is an aid i n promoting g r e a t e r grow th. amino a c i d combination. Gladstone 23 . . . in c l u d e d i t i n h i s F u r t h e r i t has been found t h a t on a th oro ugh Series T tr y p t o p h a n e tryptophane cystine Sodiua. a c e t a t e glucose : _ :: a d e n in e p u a n in e u racil S o l* n. A. S o l 1n . 5• S o l ’ n. Bt Bo C-tt. P a n t o . S o l 1n . . 5 2 . . : ° . . .................................................. S o l rn , F i o t i n S o l ' n . . N ic o tin ic acid A «.C-lutP tdc a c id M :q O ► *) it) O 13. l y s i n e , leucine r tyrnsine Arginine .• g l y c i n e .! p r o l i n e . tethienine ; . aianire •T.‘ v a lin e K.: s e r i n e ■ : I.; i s o l e u c i n e *«?. phenyl e l ? n i n e 1. 7 V \ 2. : ?. SfeBC 4 . Ip B C P 5. i'li'.crF’ 6- fc y D K F 7 , 3&BSDEFG 3. IlBODKFGH 9- -jtiBGDUFGHI 10. # i BCM:FGHIJ 11. MBCDFFGHIJK 12- J:\ BCDEFGHIJKL I F . 1/iBCDEFGUIJK1M ; 11. } aBCDE'FGHI JKLMN 15. y AF-CBEFGRIJKLMNO 16. 1 FFCDEfGHIKJ1MN0? 17. 1 /.BGDRFGHIJKLMNOPG IS - 7 ABCPF:FGHIJKLMNOpQF TI, a s p a r tic ecid 0. h i s t i d i n e hydroxy; p r o l i n e 0. t h r e o n i n e P. a s p a r a g i n e . ' 1 < r; | 5 4 5 6 _ 7 8 9 10 11 12 12 14 15 10 17 18 - 10- i n v e s t i g a t i o n o f t h e h y d r o l y t i c p r o d u c ts o f c a s e i n , a mixture o f t h e 19 amino a c i d s commonly oc cu ri ng in p r o t e i n s does not o e rm it growth, o r su p p o rt uniform body weight in r a t s . On t h e a d d i t i o n of t h r e o n i n e t o th e above 19 amino a c i d s , r a t s were a b l e to su p p o rt e x c e l l e n t growth. Omission o f t h r e o n i n e r e s u l t e d in a l o s s of body weight 36 . For t h e s e r e a s o n s and th e f a c t t h a t i n p re v io u s e x p e r i m e n t a t i o n s , i t had been found to be i n d i s p e n s a b l e f o r growth of L. n r r b i n o s u s , we in c lu d e d i t in the i n v e s t i g a t i o n . A.sovrgine v/as a l s o added because many i n v e s t i ­ g a t o r s hove used i t i n c o n j u n c t i o n with a c i d hydrolyzed c a s e i n media. No a p p a r e n t growth was noted u n t i l th e a d d i t i o n o f phenyl­ a l a n i n e (13) to t h e twelve pre c ed in g a c i d s . Of the twelve a ci ds f i r s t used no one amino acid or combination of t h e twelve produced growth. P h e n y la la n in e in combination with t h e f i r s t twelve a c i d s produced some growth which was brought up t o a h i g h e r l e v e l bv the a d d i t i o n of (14) a s p a r t i c a c i d , and s t i l l h i g h e r by the a d d i t i o n o f (17) t h r e o n i n e . The a d d i t i o n o f (15) h i s t i d i n e and (16) h y d ro x y - p r o l i n e caused l i t t l e i n c r e a s e i n growth, maximum growth was a t t a i n e d when a combination of a l l t h e amino a c i d s through 17' were added. This would i n d i c a t e t h a t p h e n y l a l a n i n e , a s p a r t i c a c i d , t h r e o n i n e and a s p a r a g in e were growth promoting f a c t o r s i n c o n ju n c t io n w it h one or more of the fo ll ow in g amino a c i d s ; g lu ta m ic a c i d , l y s i n e , l e u c i n e , t y r o s i n e , a r g i n i n e , g l y c i n e , p r o l i n e , m e th io n in e , a l a n i n e , v a l i n e , s e r i n e and i s o l e u c i n e . Another grouping o f amino a c i d s shows t h a t p r o l i n e , bydroxyp r o l i n e and a l a n i n e aid somewhat i n growth p ro d u c ti o n . Four hundred and. e i g h t combinations of t h e v a r i o u s amino a c i d s were run t h e r e a f t e r i n t r i p l i c a t e with i n t e r e s t i n g r e s u l t s . I t was - 11 - found t h a t good growth could be a t t a i n e d by using a combination o f t he fo ll ow in g seven amino a c i d s in c o n j u n c t i o n with the b a s a l medium (which c o n t a i n s th e amino a c i d s , try p t o p h an e and c y s t i n e ) . The seven amino a c i d s a.re glu ta m ic a c i d , a r g i n i n e , t y r o s i n e , l e u c i n e , i s o l e u c i n e , t h r e o n i n e and v a l i n e . Maximum growth and a c i d p r o d u c t i o n were a t t a i n e d on the a d d i t i o n o f m e th io nin e , a s p a r t i c a c i d , l y s i n e , end p h e n y l a l a n i n e . This conbin- a t i o n o f e lev e n imino a c i d s olu s the b a s a l medium, ( t a i r t e e n with t r y p t o p h a n e and c y s t i n e in the b a s a l medium) produced e x c e l l e n t growth. The omission of any one o f t h e 13 a c i d s produced from p r a c t i c a l l y no growth on the omission of t r y p t o p h a n e , c y s t i n e , v a l i n e , i s o l e u c i n e , t h r e o n i n e , g l u t a m i c a c i d t o from a 15 t o a 60 per c e n t d e cr ea se on the omission of any of t h e o t h e r s , f e a s t e x t r a c t had to be added t o the medium i n t h e case of L. c a s e i t o produce s a t i s f a c t o r y r e s u l t s . L. arnb in o s us d i d n o t r e q u i r e t h e y e a s t supplement. Vcolley *md Hutchings amino a c i d 24 have found t n a t t h e s i m p l e s t e f f e c t i v e c o n bln at io n c o n s i s t e d of t r y p t o p h a n e , g lu ta m ic a c i d , i s o - l e u c i n e , l y s i n e , a r g i n i n e , t y r o s i n e , c y s t i n e and methionine i n the growth and a cid p r o d u c ti o n o f b t r e p t o c o c c u s zymogenes. Clifton 25 work- °6 ing with C l o s t r i d i u m hotulinum*' '' used a combination of a l a n i n e , p r o l i n e , l e u c i n e , s e r i n e , g l u ta m ic a c i d , l y s i n e , c y s t i n e and: t r y p t o p h a n e . PR ' Tolley'-"' found t h a t seven amino " c i d s , namely: g l u ta m ic acxd, t r y p t o ­ phane, i s o l e u c i n e , l y s i n e , a r g i n i n e , c y s t i n e and t y r o s i n e ere e s s e n t i a l f o r growth o f he moly tic s t r e p t o c o c c i . This would i n d i c a t e t h a t the amino a c i d r e q u ir e m e n ts of the l a c t i c a c i d b a c t e r i a a r e s i m i l a r t o th e re q u i r e m e n t s o f t h e s e m ic ro -o r ga ni s m s . The s i m p l e s t combination of - 12 - amino a c i d s >vr>fc found t o c o n s i s t of g l u t a m i c a c i d , a r g i n i n e , t y r o s i n e , l e u c i n e , i s o l e u c i n e , t h r e o n i n e , v a l i n e , -tryptophane and c y s t i n e . An 57 i n t e r e s t i n g comparison with t h e 1 i n c i n g o f Rose on e s s e n t i a l amino c e i d s f o r growth produ ct ior . may be ras.de a t t h i s p o i n t . I t was found t h a t t h e f o l l o w i n g 10 amino a c i d s ; l y s i n e , t r y p t o p h a n e , h i s t i d i n e , p h e n y l a l a n i n e , l e u c i n e , i s o l e u c i n e : , t h r e o n i n e , m e th io n in e , v a l i n e and a r g i n i n e a r e i r d l e "*p e r s ' ; t i e r a g u ir e ia e n ts f o r -Uthe grev.irg an imal. rn Although t h i s combination i s s u f f i c i e n t f o r gro wth o f t h e l a c t i c a c i d b a c t e r i a , t h e re ma in in g amino - c i d s i n c a s e i n h y d r o l y p a t e c m be added w i t h o u t a g r e a t d e a l of d i f f i c u l t y and er penso • nd t h e growth l e v e l r a i s e d . Furt her mor e t h e a d d i t i o n of e l l o f th e amino a c i d s p r e s e n t in c a s e i n hydrolysate may a id t o b a l a n c e o x i d a t i o n and r e d u c t i o n r e a c t i o n s between £7 p a i r s o f d i f f e r e n t amino a c i d s as in t h e 1t i c k l a n d r e a c t i o n r>7 S t i c k l a n d * - showed I n s t -ashed suspensions- o f C l o s t ri di um s por og e r e s a c t i v a t e c e r t a i n amino a c i d r as h y d ro g e n -d o n » to r s and o t h e r s a s hybro m! ->• or e n t e r s , te0 t h a t r o u n > d r . a c t i o m - L-ke ulace between, p a i r s o f them r e s u l t i n g in t h e i r deemir at,.! o n . th is function. amino ?cid~ 50 'Woods1 c o n ti n u e d t h e stu dy o f The r e s u l t s o f t h e s e two workers snow, t h a t t n e f o ll o w i n g a c t as H -d o n " to rs : a la n in e , valine, le u c in e , phenylalunine, c y s t e i n e , s e r i n e , h i s t i d i n e , k s p a r t i c a c i d , and g l u t - m i c a c i d ; and th e f o l l o w i n g ■?£ H - n c c e n t e r s : arginine. g l y c i n e , p r o l i n e , h y r i r o r y - p r o l i n e and 27 S t i c k l e n d ” a l s o shoved t h a t r e a c t i o n between g l y c i n e and a l a n i n e g i v e s r i s e to two noleculeL of a c e t i c a c i d , CO,, and NK*, and t h a t L - p r o l i n e i s re duc ed by 1 - a l a n i n e i n t h e p r e s e n c e o f C. so oro genes t o oroduce d e l t a amino v a l e r i c n c i t p l u s p y r u v i c a c i d ana Met hionine 28 g r o w t h due tn i t s has been r e p o r t e d t o be an a n t i - v i t a m i n v ni e h i n h i b i t s ■•nti-biotin a c t i v i t y . A s e r i e s of t e s t s were run o m i t t i n g m e t h i o n i n e front t h e m i x t u r e o f g l u t a m i c e c i d , a r g i n i n e , ty ro sin e , a s p a r t i c a c id , le u c in e , is o le u c in e , pbenyl a la n in e , ly s in e , th re o n in e , tryptophane nd c y s t i n e . The maximum g r o w t h a t t a i n a b l e * a s I =s t v' an 50 n o r c e n t on t h e o m i s s i o n o f m e t h i o n i n e . It. h a s b e e n f o u n d , h o w e v e r , t h e t w h i l e m e t h i o n i n e may r e t a r d g r o w t h a t l e a s t f o r t h e f i r s t t v e n ty f o u r h o u rs , in the case o f l a c t i c i n p r o m o t i n g maximum g r o w t h . acid b e c te n ia , i t aid s g re a tly I t did n o t ap p ear to g r e a t l y h in d e r the nrovrth o f ju. o.ra b i n o g u 3 v.hich nas be en shown t o r ^ ^ i i r e b i o t i n f o r growth. D»rf' -uni no be V'-oic .acid ha^ b e e n r e p o r t e d at, a n e t n b c l i t e e s s e n 19 tla-'l f o r b a c t e r i a l g r o w t h . No a p p r e c i a b l e e f f e c t on gr ov.tn was n o t e d o r t h e a d d i t i o n o f p a r a amino, b e n c o i c a c i d t o t h e aodiura o f amino a c i d co't’Mnnti'-,r>t added c ar bo hy dr at es ar e n o t e s s e n t i a l f o r t h e st im u li ti c -n of grov.tb and .acid pro duct ion of t h e l a c t i c a c id bnct*-. r i e . These carbohydra t e s were added t o a s c e r t a i n t h e i r e f f e c t or. g r o v i h r*xto «cir> pr oduc t!o n i n the media. Car bohydra tes 0 ' have been ronor-teri to hove -j spar ing a c t i o n on the ae.valuation o f p r o t e i n . In some c o e s t h e presence- oi g lu c os e hen l i t t l e or no e f f e c t upon a c t u a l deam in atio n bv' • ashed suspensions grov tvrre Glucose 3 0.000 gms » Bod i urn n.cet ■3 t e 10.003 gms . B«lt Sol. S a l t So l. A* 5.000 cc. P-:k - 5.000 cc. Water 1.000 cc. to pH a d j u s t e d to 6.8 *The i n o r g a n i c s a l t s o l u t i o n s ore the same as p re v io u s ly reported. To complete the medium the v it a m in c o n s t i t u e n t s were .added i n the f o l i o w i r y amounts per 103 c c . ; Stock S o lu t io n Riboflavin gamma per 100 c c . (130 ganma/cc. i n 3.2N a c e t i c a ci d) 0 .2 c c . or 20 gamma Cn. p a n to th e n a te ( " i n wafer ) 3.1 c c . or 10 " - i c o t i r i c a c id ( " ,f ” ) 0.1 c c - o r 10 " Thiamine HC1 ( " " " ) 0. 1 cc. or 10 " Py r id ox in e ( " " ) 0.}. c c . or 10 " Biotin (0. 2 gamma/cc. Tl " •) 0.2 cc. or 0.04 " The b a s a l medium gave e x c e l l e n t r e s u l t s on a cid pro d u c ti o n with L. a.rabinosus b u t not w it h h . c a s e i . Using t h i s medium and o m it ti n g - 17 - one o f t h e v ita m in c o n s t i t u e n t s in t u r n , i t was found t h a t i n using L. a r a t i n o s u s a s the t e s t organism no growth oc cur s on t h e omission of n i c o t i n i c a c i d o r b i o t i n . of calcium p a n t o t h e n a t e . Some growth was noted on t h e omission I t fchov s c l e a r l y t h a t L. a r a t i n o s u s does n o t r e o u i r e r i b o f l a v i n , p y r i d o x i n e and t h i a m i n e . Thi s medium mas used mith good r e s u l t s f o r t h e a ss a y of n i c o t i n i c a c i d and b i o t i n . The f a i l u r e t o e s t a b l i s h growtn vat-h L. c a s e i c l e a r l y shows t h a t some c o n s t i t u e n t o r c o n s t i t u e n t s f o r growth, i s l a c k i n g . For th e purpose o f i n v e s t i g a t i o n v a r i o u s s u b s t a n c e s were used i n c o n j u n c t i o n w it h t h e b a s a l medium.. Nuc leic a c id was auued to th e medium w it h the t h o u g h t i n mind t h a t i t might supply t h e r e q u i r e d f a c t o r as a c ar bo­ h y d r a t e o r s. la c k i n g p u r in e o r p yr im id in e ba se. ”-ere added to t h e medium a s l e a s t supplements rt’epsu'ed by s t r o n g , Feeny and Z a r l 33 . In t h e a d d i t i o n o f the Strong and C a r p e n t e r ' s y e a s t supplement ( e q u i v a l ­ e n t t o 20 mgs. o f whole e u t o l y r e d y e a s t per 10 c c . ) t h e above medium ~ T his w i l l giv e p r a c t i c a l l y a s y n t h e t i c neaium o f known chemical co mp os it io n f o r m i c r o b i o l o g i c a l a s s a y . supplement by The re p la c e m e n t o f th e y e a s t f a c t o r of ■mown co mp osi tio n would g i v e an e n t i r e l y s y n t h e t i c medium o f c h e m i c a ll y d e f i n e d co m posi tio n. Ot S n e l l and Peterson*"1" found t h a t t . c a s e i r e q u i r e d two s e p a r a t e f a c t o r s f o r growth o b t a i n e d from, y e a s t e x t r a c t by a b s o r p t i o n on N o n t A. They termed th e se f a c t o r s t h e " f i l t r a t e ” end " e l u a t e " f r a c t i o n s , Landy and Dicker. f o r L. c a s e i . r e p o r t b i o t i n as a growth f a c t o r e s s e n t i a l They employed the basal medium o f S n e l l and P e t e r s o n m od if ied by t h e re p la c e m e n t c f t h e t f o r i t e l u a t e f r a c t i o n with f o l i c 31 - - a.cid i n e x c e s s . 18 - They found t h a t b i o t i n would r e p l a c e th e f i l t r a t e f r a c t i o n and produce e q u i v a l e n t gr ow th . They a l s o found t h a t t h e 34 amount o f b i o t i n used by S n e l l and P e t e r s o n was i n s u f f i c i e n t to 36 s u p p o r t maximum gr ow th . M i t c h e l l , S n e l l and Viillinms also rep o rt f o l i c a c i d as a f a c t o r s t i m u l a t i n g growth f o r L. c s s e i . These f i n d i n g s su g g es te d the use of f o l i c a c i d ?nd b i o t i n i n p la c e of th e y e a s t e x t r a c t a s a growth f a c t o r f o r L. ense i . medium i s g iv e n in Table IV. medium when f r e s h l y p r e p a r e d . The formula o f the complete Best r e s u l t s a re o b ta in e d w it h t h i s Heating on p. steam b a t h f o r a s h o r t p e r i o d a i d s in th e s o l u t i o n o f t h e amino a c i d s . b e f o r e t h e a d d i t i o n o f th e v i t a m i n s , This should be done A s l i g h t s e d i m e n t a t i o n and t u r b i d i t y may remain which c m e a s i l y be removed by f i l t r a t i o n . The amount of g l u c o s e and sodium, a c e t a t e {,%%) i s double t h a t used i n th e S n e l l and S tr on g medium. These -'mounts g i v e bettei* r e s u l t s . A ll v i t a m i n s o l u t i o n s should be f r e s h l y p r e p a r e d . (U .S .P .R ef e re n c e) were used, in «nd amino a c i d s were o b t a i n e d from Merck and C o m p a n y , F o l i c a c i d was k i n d l y s u p p l i e d by R. J . W il li a m s , Department o f C hem istry, U n i v e r s i t y of Texas. The a s s a y pro c e d u re s a r e p r a c t i c a l l y t h e same as in o t h e r micro­ b io lo g ical assays. For any one e s s e n t i a l vi ta m in a Quantitative re s p o n se t o growth and acid p r o d u c t i o n was found t o be c o n s i s t e n t l y p r o p o r t i o n a l t o t h e amount o f v i t a m i n s u p p l i e d . I n making t h e a s s a y f o r any one v i t ­ amin, t h e v i t a m i n under t e s t is: o m itt e d from t h e b a s a l medium. The b a s a l medium p r e v i o u s l y mentioned i s o f double s t r e n g t h and onl y 5 c c . ip r e q u i r e d f o r e r c h c u l t u r e t u b e , .Assay? '••re c a r r i e d o u t it! 5/8 x 6 -1 9 - TABLE IV Complete ?£edium 7 ,4 8 0 gms. Amino a c i d m i x t u r e ( T a b l e I I I ) O lu c o s e 10.000 gms. Sodium > 10.0 00 gms. S a l t Soln. A 5.0 S a l t Soln. B 5.0 cc. R iboflavin B iotin (100 gamma/cc. i n 0.2N a c e t i c a c i d ) (Free acid ) 0 . 5 ge-ima /c c. i n 7’h t e r cc. £.0 cc. 10 .0 c c . S a l. Pantotnenate (100 gamma/cc.) 2 . 0 c c. N icotinic acid (100 gai&ma/cc.) 2.0 cc. Soln. Pyridoxine (100 gamma/cc.) 4.0 cc. S o l n . Thiamine (100 g a m n a / c e . ) 1.0 cc. Soln. F o lic acid (10 1.0 cc. D i s t i l l e d wa t e r gjimma./cc.) to pH a d j u s t e d t o 6. 8 500 cc. -gOinch c u ltu re tubes. D u p l i c a t e t u b e s a r e s e t up i n t h e range o f concen­ tr e tio n re q u ire d as standard. C o n c e n t r a t i o n s of t h e v i t a m i n i n q u e s t i o n i n amounts to meet t h e r e q u i r e d r a nge ? r e added t o t h e c u l t u r e t u b e s and b r o u g h t t o a volume o f 5 c c . w i t h d i s t i l l e d w a t e r . (The s o l u t i o n must be o f such a c o n c e n t r a t i o n t r o t t h e h i g h e s t r e q u i r e d amount w i l l be c o n t a i n e d i n no more t a a n 5 c c . ) F iv e c c . of b a s a l medium i s t h e n added t o each t u b e t o b r i n g t h e t o t a l volume to 10 c c . ssmole un de r t e s t a r e o r e o a r e d . I n l i k e manner s o l u t i o n s o f th e The t u b e s a r e plugged w it h c o t t o n and s t e r i l i z e d a t f i f t e e n pounds o r e s s u r e f o r 15 m i n u t e s . A f t e r c o o l i n g tn e y a r e p l a n t e d w it h a s u s p e n s i o n o f L. c a s e i and i n c u b a t e d a t 57°C. f o r 7Sc hours. A f t e r i n c u b a t i o n t h e s c i d p r o d u c t i o n i s d e te r m in e d by t i t r a t i o n w it h 0 . 1 N. W&OH. Values o b t a i n e d w it h t h e s t a n d a r d s o l u t i o n s a r e s e t up i n a s t a n d a r d curve from which t h e v i t a m i n c o n t e n t o f any d i l u t i o n o f sample may be c a l c u l a t e d . The s t o c k c u l t u r e i s c a r r i e d i n y e a s t d e x t r o s e a g a r s t a b s . The o r i g i n a l c u l t u r e s ware o b t a i n e d from t h e ^ m e r i c a n Type C u l t u r e C o l l e c t i o n o f t h e iTeorgetown t i n ' § c h o o T , v ffasffihg' E' oh^: used were L. a r a b i n o s u s 3014 and L. c a s e i 7469. ore por ed tH’*" 'Tlie'"’c u l t u r e The y e a s t a g a r s t a b s a r e 9 k e n t ac cording t o the method of S n e l l h S tr o n g . In place o f o l a n t i n g i n t o y e a s t d e x t r o s e b r o t h o r the b a s a l medium o f ^ n e l l & S t r o n g , p l a n t i n g s a r e made i n th e b a s a l s y n t h e t i c medium (T ab le I V ). In t h i s way t h e r e can be no c a r r y o v e r of any t r a c e s of v i t a m i n c o n t e n t from y e a s t nr c a s e i n h y d r o l y s a t e t o i n t e r f e r e w i t h t h e a s s a y . A f t e r 74 hour in cubation t h i s c u lt u r e i s c e n tr ifu g e d , the su p ern ata n t l i q u i d i s decanted and t h e n r e s u s p e n d e d i n 10 c c . o f 0 . 8 5 per c e n t sodium c h l o r i d e s o l u t i o n . One t e n t h c c . o f t h i s s u s p e n s i o n i s u s e d f o r s ee di ng o f th e a s s a y t u b e s and c u l t u r e t u b e s under t e s t . The b a s a l medium used f o r s e e d i n g shou ld be f r e s h l y p r e p a r e d i n o r d e r t o o b t a i n 4»ximu.n r e s u l t s . In order to i n s u r e t h e p o t e n c y and t o p r e v e n t s p o i l a g e t h e b i o t i n and t h e f o l i c a c i d s r e d i s s o l v e d i n s t e r i l e d i s t i l l e d w a t e r i n t h e c o n c e n t r a t i o n s named, rod a s e p t i c n l l y t r a n s f e r e d t o 10 c c . sm o u le s. s e a l e d and used a s o c c a s i o n a r i s e s . These amoules n r e t h e n Care s h o u ld be e x e r c i s e d i n t h e s t o r a g e and t r . a t. a e n t o f t h e v i t a m i n s o l u t i o n s , e s p e c i a l l y r i b o f l a v i n . There s h ould be no undue e x p o s u r e t o l i v h t f o r any l e n g t h o f t i m e . A ll t h e v i t a m i n t e s t s o l u t i o n s s h o u ld be f r e e u e n t l y r e o l a c e d i n o r d e r t o m a i n t a i n t h e i r po tency and t o p r e v e n t d e c o m o o s i t i o n . S t a n d a r d c u r v e s f u r n i c o t i n i c a c i d , b i o t i n and c a lc iu m p a n t o t h e n a t e r e s u l t u s i n g L. n r n b i n o s u s a c c o r d i n g t o t h e f o l l o w i n g g r a p h s . The f o l l o v d n g s t a n d a r d g r a p h s r e s u l t e d f o r r i b o f l a v i n , Calcium p a n t o t h e n a t e , b i o t i n , n i c o t i n i c a c i d , p y r i d o x i n e and f o l i c a c i d , when 1. c a s e i was u s e d a s t h e t e s t org a n is m . The v i t a m i n c o n t e n t of s e v e r a l p r o d u c t s o f t h e ! rhenta.uin D i v i s i o n , De P r e e Company, H o l l a n d , liic liguu vu-, dett-rmined by t h e s u g g e s t e d a s s a y procedure. Be s u i t ? were w e l l i n a gre em en t with o t h e r e s t a b l i s h e d m ic ro ­ b i o l o g i c a l and sp ec t r o oho t o m e t r i c a s s a y methods. A more l i n e a r c urv e a ooe nr ed t o r e s u l t from t i e u s e o f t h i s method. I n soue c a s e s p e r f e c t b l a n k s were n o t o b t a i n e d ; how ever, b l a n k s e q u i v a l e n t t o o n l y 1 . 0 c c . o f 0.1 K a c id w i l l n o t g r e a t l y i n t e r f e r e with a c c u ra te assay d e te rm in a tio n s The p r o d u c t s t e s t e d were a l l w a t e r s o l u b l e and i n f r e e form. Products i n Which t h e v i t a m i n s a r e i n combined form would r e q u i r e s p e c i a l h yd ro ­ l y s i s previous to m icro b io lo g ical assay. The f o l l o w i n g g r a p h s , i n d i c a t e t h a t t h e p r o c e d u r e i s a d a p t a b l e and n m c t i c n l f o r a s s a y p r o d u c t s o f t h i s n a t u r e . p o i n i ! H .lffffl u -u i-fm ijjr^ M X T rn rtrrrr.rT T T . B io tin cc, 0.1 B. NaOH i.om o.oo? |o . ) fR T F m I u m i n l i -HI V - m ^ h ^ M J r n : |j x : : n - u ,, I • u u , l . i 11 LU30x r 33X i.'|fn rn j( i f n r . n P an to th en ate L. atah tn o sn s n . f' Gamma Ca. Pantothenate } t I N ic o tin ic acid o Ga.iKa N ic o tin ic Acid R iboflav in L. c a se i cc. 0.1 N. NaOH (kiiama R ib o flav in jix£njxrrm.i: Pyridoxine L. c s s e i " c c t 0.1 N. ISaOH o p i 1. 1. p ej Gamma P y r i d o x i n e 1 " V f-( (! 1111ITIifrmil IjTlTM'f'fl IHI11ITTTIH44+)-! 111l:f-H1111l-lfll!IH11 1ri' rr' ' ’!: Ga, P an to th en ate L« e a s e l 1~ O P.02M o 7 r > 1 r ). 1 Gamma Ca. Pen Let senate VI l1Mxl 'i t - o x m i n ru"i-!Jii'H ! ; i.n.i.'n m HTt n i n V n i.v r r r n r f i HH44iTOi+H-H111II! I'MI mil IIUn11 B io tin VII L .c a s e i H 1*0 a 7 3. B io tin 1 c mm i n -' r n ' i i f c r |.^-flAM4..|-|-l-lW4W ^U ffn xps • M c o t i n l c Acid L. cc. 0.1 o N. JfaOH r / 7 Gamma N ic o tin ic Acid Li >r n m mt Liijsm Inim Lro iiXli i.mii V II I f i m-Fi-H11h 11111.ii- f o l i c Acid L. c a se i 10 cc. 0.1 N. NaOH 9 8 7 6 5 U 3 1 0 005 .015 Gamma F o lic Acid 025 1111II111111t:l i-h-i i t hu s m m f m w fflMIWiFFBR 'HIT' nfri' [|:I'HI R ib o flav in Assay L. e a se l 10 C ontrol cc. 0.1 N. NaOH Monogards - Whefitomin - OePree 0..1 0.2 0.3 Gamma R iboflavin 0 .4 t^TTPTf [1111111 tWWffff f t[W l tl 1!-W HH-W-i 111i fh m11i Mh h-H-) , 111 Ca. P an to th en ate Assay- 10 cc. 0.1 N. NaOH C ontrol 9 II S 7 6 5 3 2 1 0 0.1 0 . 2! .....0.3 ' 0 Gamma Ca. Pantothenate Pantocaps - De P ree H T r r r 'r p i m I fT.-r r n t i'| n m r n t h 11 ) i >>I >I' P 'n i" r m ‘i'H ' | 1UTI 171 i.t H T H iJ 'j:!lh i:t± t:n ^ llllL L C 'n ^ f± U U lJ lilU J S J X L J jJ ..fX T l4J^-J-Q-^SJ-U^iTlT44 N ic o tin ic Acid Assay k^SM Sl ; C ontrol II Gamma N ic o tin ic Acid 3-Complex Capsules : (Wheatamin Brand, : De 11iTj-frmmjm^i 1111i11111n n 1111ri i.rom f f m t n t T Pyridoxine Assay L. ease l 10 ; cc. 0.1 N. NaOII 9 : -© -€> oT6~~ ...... ' Gamma Pyridoxine I C ontrol III Wheatamin 1'ablets - De Pree r-i.... ............ !§iif 1 a i m Iwnn ml!;:1!m »m ■11/ -© II 0 0.005 0.01 0.015 Gamma F o lic Acid 0.02 0.025 Wheatamin. F l i x i r pe Pree !*""! t r i i H"f""ft t t t tt 'i11; i i j i i i n T r n r r r f TTTT H * i j i m m V i < p t r n T T T H u i Ll . l i IT, t.U .L t L f X i X I T r H T T l L l N ic o tin ic Acid a ssay L. arabinosus I Control cc. 0.1 N. NaQH ' ■■■- II ?• j ■; • 0.3 Gamma N ic o tin ic Acid 1.0 ■■ : " ' ' T ' ! N ic o tin ic Acid Tabs n rt-n h hhi mmi i f M ^ w f w B io tin Assay L. arabinosus 10 cc. 0.1 N. NaQH C ontrol Rice Bran E x tra c t; (Wheatamin - De Free 0 0.001 0.002 0.003 Gamma B io tin 0.004 0.005 *1ii w w r Bio t i n . Assay L.«. a r a p i r o s u s ....... "O o I ’ Control Xo I I ...... F l e e Bran E x t r a c t Yjheut&niin - De Dree T7 1i . 3.301 3 . OOS? 3.30? -JO.OO/ ). :>r -2 2 - DISCUSSION The amino a c i d s e s s e n t i a l f o r grovrth find a c i d p ro d u c ti o n o f th e l a c t i c a cid b a c t e r i a have been demonstrated thr ough c a r e f u l s t u d / . It h a s been shorn t h a t t h e amino a c i d s , i n the combination and q u a n t i t i e s shown, are more e f f i c i e n t t h a n the r e g u l a r h y d r o l y s a t e s o f c a s e i n i n m icrobiological assays. D et er m in at io n s were .made by a d e t a i l e d o ro c e ss o f a d d i t i o n and e l i m i n a t i o n . More th a n f i v e hundred combinations of the amino a c i d s have been i n v e s t i g a t e d . The s i m p l e s t combination t h a t w i l l oroduce ° good growth c o n s i s t s o f t h e follo wing s 9 amino a c i d s ; t r y p t o p h a n e , c y s t i n e , g lu U m ie a c i d , v a l i n e , t y r o s i n e , t h e o n i n e , l e u c i n e , i s o l e u c i n e and a r g i n i n e . l r e « t e r a c i d -nroduction and h i g h e r growth l e v e l s a r e a t t a i n e d by t h e use of a l l t h e smino a c i d s shown in Table I I I . No r e s u l t a n t growth oc cu rs on t h e omission o f any one o f the f o ll o w i n g amino a c i d s ; t r y p t o p h a n e , c y s t i n e , g lu ta m ic a c id o r v a l i n e , and s u ' ,re s t s the p o s s i b i l i t y of m i c r o b i o l o g i c a l d e t e c t ion and d e t e r m i n a t i o n of these a cid s. ^nd f o l i c a c i d had v e r y l i t t l e e f f e c t upon t n e growth and a c i d p r o d u c ti o n o f L. a r a h i n o g u s , w hile the omission of n i c o t i n i c a c i d , p a n t o t h e n i c a c i d and b i o t i n r e s u l t e d i n no grow th. The omission o f any one o f th e s e s i x f a c t o r s above r e s u l t e d i n no growth resp ons e f o r L. e a s e l . This would I n d i c a t e t h a t L. s.rabinosns does n o t r e q u i r e r i b o f l a v i n , pyrid oxin e or f o l i c a c i d b u t t h e t L. e a s e l does r e q u i r e a l l s i x of the vit a m in f a c t o r s a s r i b o f l a v i n , o&ntothenic a c i d , b i o t i n , n i c o t i n i c a c i d , p y ri do xi ne and fo lic acid. No r e s u l t s were o b t a . i n a b l e i n t h e c a s e o f t h i a m i n e . assay 01 A m icrobiological t h i s f a c t o r i s d i f f i c u l t due t o t h e f a c t t h a t i t i s e a s i l y d e s t r o y e d and w i l l n o t s t a n d a u t o c l a v i n g a t IB pounds p r e s s u r e f o r 15 minutes... Assay o f t h i s f a c t o r may De r e a d i l y a cco mp lis he d by s p e c t r o p h o t o m e t r i c measure­ ments u s i n g t h e t.hiochrome method v.lth t h e a d a p t a t i o n o f t h e Hennessey and Cerecedo o r o c e ■i u r e . •‘• r e c e d i n g g r - p h s show t h a t growth and a c i d p r o d u c t i o n a r e p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n o f the v i t a m i n unde r t e s t . n e c e s s a r y f o r grcv.th v a r i e s with each v i t a m i n . The amount o f v i t a m i n Only ve ry minute amounts a r e r e q u i r e d i n ea c h c a s e and t h e t e s t s a r e v e r y s e n s i t i v e . Parallel ' eo monr Ison vh.tn r e s u l t s o f o t h e r methods o f t h e v i t a m i n a s s a y s f o r t h e v a r i o u s f a c t o r s now in use a r e i n c l o s e a g r e e m e n t . A s lig h t v ariatio n in t h e extreme uo o e r and l o n e r c a r t s o f th e c u r v e may o c c u r ; however, t h a t o o r t i o n o f t h e c ur ve which i s l i n e a r i s r e l i a b l e and o nl y .can be used f o r t h e c a l c u l a t i o n o f as s a y v«1uhs. In the exoerim entation throughout t h i s o°r»er a l l t e s t s were run i n t r i o l i c a t e i n o r d e r t o o b t a i n r e l i a b l e c he c k s . " The use o f Leucon o s t o c m e s e n t e c l o i a e ' b as fa i'h-bvorganism: was ' a i s e ' ... tried. I n some i n s t a n c e s i t -mve promise t h a t i t could be used b u t r e s u l t s were n o t e n t i r e l y s a t i s f a c t o r y when used i n c om bin at ion w i t h t h e orooosed medium. L. e a s e l gave by f a r the most s a t i s f a c t o r y r e s u l t s . L. a r a b i n o s u s can o n ly be used f o r t h r e e of t h e s e f a c t o r s . The p r o c e d u r e ’" as been g r e a t l y s i m p l i f i e d by t h e us e o f t h e amino acid combination. The time and e f f o r t r e q u i r e d f o r t h e h y d r o l y u a t i o n o f c a s e i n has been removed cud t h e r e s u l t s a r e more c o n s i s t e n t . t h e y e o s t nunolfiTient a l s o h s ” been done away w i t h , The use o f ’f h i l e x-:e nave no f i g u r e s on the c o s t d i f f e r e n t i a l , th e expense i n c u r r e d s hould n o t g r e a t l y -? 4 exceed th e c o s t of R medium using hyd rol yz ed c s s e l n end y e n s t e x t r a c t . The sore o f p r e o n r o t i o n would f o r o f f s e t «ny s l i g h t i n c r e a s e i n c o s t . - 25- COHCLUSION A medium hn ■ been pro d u c e d c o n s i s t i n g of e n t i r e l y known c he m i c a l ooooosition. The o r o c e d u r e hos been g r e a t l y s i m p l i f i e d by t h e u s e o f the o r e m i x l u r e o f ,-amino ac'ido -aid o t h e r r e q u i r e d compounds. may be used f o r t h e s i x v i t a m i n a s s a y s . h. casei R e s u l t s a r e .uore a c c u r a t e and r e l i a b l e ' s. t i e i n t e r f e r e n c e s n a t u r a l l y o r e s e n t i n h y d r o l y s a t e s ■v‘ c a s e i n r o * s t , end oe nto ne a re n o t o r e s e n t . Gimole procedure u s in g The medium p r e s e n t s a noun c h e m i c a l co.-apounas and a s i n g l e t e s t org a n is m f o r t h e a s s a y o f t h e s i x most i m p o r t a n t v i t a m i n s o f t h e B com lex. -2 6 - Rlt'FERENCES 1. " r r b u r g , 0 . end C h r i s t i a n , Vs. Biochem. Z. 26R, 577 (19?,5). 2. La v o l l e y , 7. , and F r i a ' a t , L.D. P r o c . Amer. Soc. B i o l . Chem., CXIX, (1941) 12. Subbarow, Y. and Cane, L. J r . Amer. Chem. Soc. 61:1615 (1959). 13. S n e l l , F . F . ; Fa k in , P . F . , and l i l L i s n s , R..J. J r . A u e r . Che;n. Soc. 62:175, ( 1 9 4 0 ) . 14, P o s t e r , T.R. '-rod f e l c z a r , Sc ie n c e , 91:576, (194 0) . I F . L a m e n , O . J . ; B a h l e r , G . ? . and P e t e r s e n , r . r l . J o u r n . N u t r i t i o n , 25:11 , (.1942). .07 _ 16. S n e l l , F . F . and ''r’l g h t , L.D. J r . P i o l . Che,a. 139 : 67 5, (1941). 17. F a l l La/as, R . J . j F s k i n , P . E . , and HcJahnn, J . R . P u b l i c a t i o n Eo. 4137, 24, (1941). IS. Bohonos, N e s t o r , Hu tchings , B.L. and P e t e r s o n , J r . P e c t . 44:479, ( l 9 4 2 ) . 19. “':i t c h e l l , H.R.j S n e l l , F . E . , nnd r i l l i * r ’.s, F . J . J r . *ner. Chea. no c . 33:2234, (1941) . •''S. Landy, B.L. end Dickens, D.vl. J r . Lab. and C l i n , l e d . , 2 7 : 1 0 8 6 , • ( 1 3 4 2 ) . ne Li , F.F . "nd ne t e r s o n , ”'.H. TV 22. ? t . , 39:573, (1940). n-’V.’k a ad P e rg e in P r a c t i c a l P h y s io lo g - i c e l Cher.. E l e v e n t h E d i t i o n , "age 104:105, (1957). G l a d s t o n e , G.P. ^ r i t . J r . i x p t l . P a t h . , 16:522, (1957) " 'o l le y , P.V. and H u t c h l i g s , B.L. J r . F a c t . 59:264, (1940). i l i i t o n , C.E. J r . - n e t . , 39:465, J r . "Fret.., 4 2:155, 71941) . " 27. S t i e k l n n a , L. H. J r . P.iol. ^he/n., 29:283, 839, 893, (1955). 29. F i s h e r , V. and m a r t i n , G.J., J r . r o c t . 45:35, (1943). 29. Pa rk , C.R. and Y'ood, 7.B. Joan Ho old is Hosp. J r . B u l l . 70:19, (19<3?) . ?0. Roods, P.O. J-r. B i o l . Chern. 29:349, (1935). 31. H i l l e r , V.; Lash, ana n i o s . J r . Chem. I d . 7;?B7, (1950). 32. S+.rong, F.H. and Carrier.ter, L.E. I n d . and E g . Chem., Anal. F d . , 14:909, (1942) pt r o n g , F . v ' . ; Fe en y, R . F . , and I n r l e . A na l. R d ., 1 5 : 5 6 0 , ( 1 9 4 1 ) . 5-1 , F e e n y , TR. E . , a n d s t r o n g , F...I. •T, F . i o l . Chen., 1 * 0 ; 3 H , ( 1 9 4 1 ) . 5 5. P n e l l , P . P . , ->nd - i t c h e l l , ii.R . ’’r o c . N a t . A c - d . S c . , 27 ; 1 , ( 1 9 4 1 ) . v vicrniLdt C h e m is tr y o f Amino and P r o t e i n s , 2nd Fd. "7. Pone, ”V7. n h y - i o l . r' ^ . , —, 1:109, (192R), 0 •-1 e , 17. F. P i n l . F e v . , Vol. 4, No. 5, 135,