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A detailed study of the lactic acid bacteria constituting a commercial cheese starter Okulitch, Olga 1935

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A DETAILED STUDY OF THE LACTIC ACID^RACTERIA CONSTITUTING A COMMERCIAL CHEESE STARTER  Olga O k u l i t e h , B.A.  A T h e s i s submitted f o r the Degree o f MASTER OF ARTS i n t h e Department of • BACTERIOLOGY  The U n i v e r s i t y of B r i t i s h Columbia A p r i l , 1935  f  CONTENTS Introduction....... Methods. Media Employed Throughout the Work........ Experimental. Discussion.  •  Sugar-Fermenting A b i l i t i e s . Rotary Power o f the l a c t i c A c i d . C a s e i n - S p l i t t ing A b i l i t y . Rate o f A c i d P r o d u c t i o n . . . .... ............. 0 Is, ss ific&"fc.Lon* •••»«»•  «l« • • • • • • • • • • • • • «  • • • o • • •« «  Summary and ConelusIon *....................... Acknowledgments............................... References Tables and F i g u r e s .  A DETAILED STUDY OF THE  LACTIC ACID BACTTCRTA  CONSTITUTING A COMMERCIAL CHEESE STARTER  INTRODUCTION As the  our [knowledge o f the processes concerned w i t h  r i p e n i n g of cheese has progressed, the r o l e p l a y e d  the l a o t i c  a c i d s t r e p t o c o c c i present i n the s t a r t e r employed  i n the making o f cheese has The  by  importance of the  r i p e n i n g of cheese was  assumed a g r e a t e r  significance.  l a c t i c a c i d s t r e p t o c o c c i i n the first  only d i d he demonstrate the  shown by B a r t h e l  (1).  Not  importance of these organisms  during the cheese-making process but a l s o showed t h a t throughout the r i p e n i n g process the breakdown o f c a s e i n  and  the development of f l a v o u r were i n l a r g e measure dependent upon enzymes elaborated  by these l a c t i c a c i d  a c t i n g i n a s s o c i a t i o n w i t h the Further,  the great  streptococci  rennet.  importance that may  t o t h e p a r t played by d i f f e r e n t species  be  of l a c t i c  attached  acid  b a c t e r i a i n the cheese r i p e n i n g process i s to be seen when we  contemplate the statement made by Orla-Jensen when he  began h i s work on the l a c t i c a c i d b a c t e r i a .  In h i s  intro-  d u c t i o n t o the Monograph on the L a c t i c A c i d B a c t e r i a  he  s t a t e s : " I t i s p a r t i c u l a r l y d i f f i c u l t to understand  how  various  s o r t s of hard cheese, apparently c o n t a i n i n g  the  same m i c r o f l o r a , should each have i t s own  characteristic  t a s t e and  doubt, t h a t  smell.  There can h a r d l y  be any  these s o r t s o f cheese i n r e a l i t y  contain d i f f e r e n t  species  o f b a c t e r i a , only we are unable t o d i s t i n g u i s h them by the methodshitherto employed.  The object  o f the present work i s  p r i m a r i l y to meet t h i s want by d e s c r i b i n g the u s e f u l b a c t e r i a o f the d a i r y i n d u s t r y ,  so thoroughly that i t may be p o s s i b l e  i n the future t o i d e n t i f y the s t r a i n s encountered." (2) The  succeeding years have shown how p r o p h e t i c a l l y  true  the statement o f Orla-Jensen has proved t o be. As f u r t h e r knowledge has emerged the s i g n i f i c a n c e t o be attached t o the l a c t i c a c i d b a c t e r i a concerned w i t h the r i p e n i n g o f t h e cheese has made i t more imperative than ever t h a t  i n order t h a t we  may be the b e t t e r able t o determine the r e l a t i o n s h i p o f organisms from the s t a r t e r t o organisms found i n the the r i p e n i n g ,  cheese  during  i t i s necessary t h a t we study i n d e t a i l the  organisms c o n s t i t u t i n g the s t a r t e r . E a r l y i n the h i s t o r y o f the study o f s t a r t e r s i t was  c l e a r l y shown that a s i n g l e s t r a i n s t a r t e r f o r both b u t t e r  and  cheese making was never as s a t i s f a c t o r y as a mixed  starter.  culture  I n the monumental work o f S t o r c h (3), and the papers  o f Boekhout and de V r i e s  (4), Hammer and B a i l e y  ;(>5, 6,7),  Enudsen and S i r e n s o n (8,9), t h i s fundamental conception o f t h e a s s o c i a t i v e a c t i o n o f organisms has been v e r i f i e d .  The work  of the above i n v e s t i g a t o r s has been c h i e f l y concerned w i t h t h e Betaoooci and t h e i r r e l a t i o n s h i p t o the S t r e p t o c c i present i n the s t a r t e r . Or la-Jens en - i n 1919 (2), c l e a r l y showed that two  -3s p e c i e s o f S t r e p t o c o c c i c Streptococcus cremoris and Streptococcus lactis*»eonstitute the m a j o r i t y o f the t o be found i n a commercial s t a r t e r .  organisms  Although he d i d not  demonstrate the presenoe i n s t a r t e r s o f d i s t i n c t species c l o s e l y a l l i e d t o these forms, he p o s t u l a t e d t h e i r p o s s i b l e e x i s t e n c e and h i s system o f c l a s s i f i c a t i o n i s f l e x i b l e enough t o a l l o w o f t h e i r i n c l u s i o n i  The presence of other  species o f S t r e p t o c o c c i i n s t a r t e r s was  shown d e f i n i t e l y f o r  the f i r s t time by Knudsen ( 8 ) , and by Orla-Jensen and Hansen (10).  That other i n t e r m e d i a t e s p e c i e s o r s t r a i n s might  be  found seemed probable; w i t h t h i s o b j e c t i n view, the present work was undertaken. study was  Of s e t purpose the s t a r t e r chosen f o r  one which had been propagated i n m i l k t o which  yeast e x t r a c t had been added as an enrichment.  I t was  that a s t a r t e r c a r r i e d under such c o n d i t i o n s might  thought  contain  species o f S t r e p t o c o c c i which had not been encountered previously. METHODS For the study o f the morphology o f the  organisms  a l l c u l t u r e s were i n o c u l a t e d i n t o y e a s t l i t m u s m i l k and casein digest broth, respectively.  into  A f t e r 24 hours i n c u b a t i o n  at 23°C m i c r o s c o p i c examination of the c u l t u r e s was made, u s i n g the Gram method o f s t a i n i n g . I n the study o f the sugar fermenting a b i l i t i e s o f the organisms, the procedure f o l l o w e d throughout was  that  employed by Orla-Jensen (2) and by S a d l e r , Eagles and Pendray  (11, 12).  For the p r e p a r a t i o n of each sugar b r o t h the r e q u i r e d  carbohydrate  i s added at the r a t e o f B^,  the b r o t h i s then  tubed and plugged—10 c c . i n each t e s t t u b e — a n d i s s t e r i l i z e d at 12 pounds pressure f o r 20 minutes.  After  sterilization  each tube i s i n o c u l a t e d w i t h the d e s i r e d c u l t u r e . a 3mm.  Uniformly,  loop i n o c u l a t i o n from a vigorous growth i n m i l k or  c a s e i n d i g e s t b r o t h i s made--the m i l k used f o r t h i s purpose b e i n g enriched w i t h a t r a c e o f yeast e x t r a c t .  After  inocula-  t i o n , the s e r i e s w i t h c o n t r o l s are incubated f o r 14 days at the a p p r o p r i a t e temperature--in t h i s case 23°0. t i o n i s completed  When incuba-  the c u l t u r e s are t i t r a t e d w i t h 3J/4 Sodium  Hydroxidej u s i n g p h e n o l p h t h a l e i n as an i n d i c a t o r ; the t i t r a t i o n of the c o n t r o l s deducted,  and the r e s u l t s worked out  and  recorded as grams l a c t i c a c i d per m i l l e . To determine  the r a t e o f a c i d p r o d u c t i o n i n m i l k and i n  mills enriclied w i t h y e a s t e x t r a c t at the r a t e o f 0.15;$ the methods used by S a d l e r and E a g l e s were f o l l o w e d (13).  For  t h i s phase of the work seven tubes o f each r e s p e c t i v e m i l k are u n i f o r m l y i n o c u l a t e d w i t h the d e s i r e d c u l t u r e .  After  i n o c u l a t i o n , the s e r i e s - w i t h c o n t r o l s are Incubated a t the a p p r o p r i a t e temperature--in t h i s case 23°C.  At d e f i n e d  i n t e r v a l s , one tube o f each c u l t u r e and a c o r r e s p o n d i n g c o n t r o l tube are removed from the incubator and the amount of t i t r a t a b l e a c i d i t y produced  determined.  The r e s u l t s of the determinations  o f the p t o t a l t i t r a t a b l e a c i d i t y produced throughout  at d e f i n e d times  the 14 days' p e r i o d of i n c u b a t i o n by each o f the  twenty c u l t u r e s i n m i l k and i n m i l k enriched w i t h yeast extract,  .'. -5- • are g i v e n i n F i g u r e s 1-20. For the determination o f the c a s e i n s p l i t t i n g a b i l i t i e s o f the organisms the method used "by Orla-Jensen (2) i n h i s study on the l a c t i c a c i d "bacteria was f o l l o w e d .  The  c u l t u r e s are i n o c u l a t e d a t the r a t e o f ifo i n t o 1200 c c . o f s t e r i l e m i l k t o which zfo o f c h a l k has "been added. m i l k c u l t u r e s a r e incubated f o r s i x weeks a t 23° C.  The c h a l k They a r e  then f i l t e r e d and t h e f i l t r a t e used f o r the d e t e r m i n a t i o n o f s o l u b l e n i t r o g e n and amino n i t r o g e n .  As i t i s necessary t o  a s c e r t a i n the amount o f s o l u b l e n i t r o g e n and amino n i t r o g e n formed by the organisms, determinations  of t o t a l n i t r o g e n ,  s o l u b l e n i t r o g e n and amino n i t r o g e n are a l s o made on the control f l a s k s of chalk-milk. The t o t a l n i t r o g e n i s determined 5 c c . p o r t i o n s o f the c o n t r o l m i l k . of s o l u b l e n i t r o g e n 50 c c .  i n t r i p l i c a t e on  F o r t h e determination  of the chalk-milk f i l t r a t e of  the c u l t u r e i s taken, and t o I t are added 15 c c o f 1$ a c e t i c a c i d and 100 c c . water. water bath f o r 30 min., and f i l t e r e d .  The s o l u t i o n i s heated  i n a boiling  cooled, made up to volume o f 250 c c  Two 50 c c p o r t ions o f t h i s f i l t r a t e ( a c e t i c  a c i d f i l t r a t e ) are taken f o r the determination o f s o l u b l e nitrogen. To 100 c c . o f the a c e t i c a c i d f i l t r a t e are added 30 c c . o f 25$ by volume s u l p h u r i c a c i d , 10 c c water, and 10 c c o f 19% phosphotungstic  acid.  A f t e r s t a n d i n g 24 hours  the p r e c i p i t a t e i s f i l t e r e d o f f , and two 50 c e . p o r t i o n s o f the f i l t r a t e (phosphotungstic  f i l t r a t e ) are taken f o r the  -6-  determinat i o n o f amino n i t r o g e n . In c a l c u l a t i n g the amount o f s o l u b l e n i t r o g e n formed and the a meant o f amino n i t r o g e n formed "by the organisms, the r e s u l t s obtained f o r t h e s o l u b l e n i t r o g e n and amino n i t r o g e n .'v *f • ;  o f the c o n t r o l s are s u b t r a c t e d from the corresponding d e t e r minations  f o r the cultures.  The r e s u l t a n t f i g u r e  represents  the amount o f s o l u b l e n i t r o g e n formed o r the amount o f amino n i t r o g e n formed, as t h e case may be, and i s expressed of the t o t a l n i t r o g e n .  i n terms  As c a l c u l a t e d i n t h i s manner the  r e s u l t s are shown i n Table 7. For the determination o f the amount o f v o l a t i l e  acid  produced and the type o f l a c t i c a c i d formed by t h e c u l t u r e s , 400 c.o. o f the chalk-milk f i l t r a t e are taken. (The u s u a l procedure i s t o employ 200 c c o f f i l t r a t e ) .  F o r the l i b e r -  a t i o n o f the organic a c i d s from t h e i r calcium s a l t s as they are present i n the c u l t u r e f i l t r a t e ,  s u l p h u r i c a c i d i s added,  thereby p r e c i p i t a t i n g the calcium as i n s o l u b l e calcium sulphate. From the p e r cent sugar,as determined by the S o x h l e t - A l l i h n copper method, the amount o f s u l p h u r i c a c i d r e q u i r e d f o r the l i b e r a t i o n o f the organic a c i d s i s c a l c u l a t e d from the f o l l o w i n g formula where X equals fo sugar fermented c.c, HgS0  4  r e q u i r e d f o r each 400 c c serum » X x O*6-1.0i  When the c a l c i u m sulphate p r e c i p i t a t e has s e t t l e d , the s o l u t i o n i s decanted and subjected t o steam d i s t i l l a t i o n ,  1800 c c . o f  d i s t i l l a t e are o o l l e c t e d and t h e v o l a t i l e a c i d content  deter-  mined by t i t r a t i n g w i t h N/10 NaOH u s i n g methyl r e d as i n d i c a t o r . The r e s u l t s are expressed  as c c o f K/lO UaOH r e q u i r e d t o  -7-  n e u t r a l i s e 900 c . o i of d i s t i l l a t e . The remaining l i q u i d i s used f o r the d e t e r m i n a t i o n o f the r o t a r y power of the l a c t i c a c i d produced.  With the  gradual a d d i t i o n of pure s i l i c a sand the s o l u t i o n i s evaporated to dryness on a water bath.  The r e s i d u e i s then e x t r a c t e d by  t r i t u r a t i o n w i t h e t h e r i n a mortar.  The ether i s then removed  by d i s t i l l a t i o n and a y e l l o w syrupy s o l u t i o n c o n t a i n i n g the l a c t i c a c i d remains.  The s o l u t i o n i s taken up w i t h a s m a l l  volume o f hot water and f i l t e r e d .  For the c o n v e r s i o n of the  l a c t i c a c i d i n t o the z i n o s a l t and f o r the removal o f c o l o u r i n g matter, the f i l t r a t e i s heated w i t h a n excess o f z i n c oxide and c h a r c o a l on a b o i l i n g water bath.  When the r e a c t i o n  i s complete, as evidenoed by the i n s oluble z i n c oxide remaini n g u n d i s s o l v e d , the hot s o l u t i o n i s f i l t e r e d and the  filtrate  evaporated u n t i l c r y s t a l s o f z i n c l a c t a t e b e g i n t o form.  The  s o l u t i o n i s t h e n cooled, the z i n c l a c t a t e i s f i l t e r e d o f f , pressed between f i l t e r paper, and t h e n d r i e d i n a d e s i c c a t o r over concentrated s u l p h u r i c a c i d .  A f t e r s e v e r a l days d r y i n g  i n the d e s i c c a t o r , the z i n c l a c t a t e i s d r i e d to constant weight at 90° 0.  As a p r e l i m i n a r y means o f d i f f e r e n t i a t i n g  between e i t h e r o f the a c t i v e z i n o s a l t s and the i n a c t i v e form, the percent water o f c r y s t a l l i z a t i o n o f the s a l t i s determined* I t has been shown t h a t the a c t i v e s a l t s — d e x t r o and l a e v o — * c o n t a i n 2 molecules o f water of c r y s t a l l i z a t i o n corresponding t o 12.9$  o f the molecular weight, and t h a t the i n a c t i v e  salt  contains molecules o f water of c r y s t a l l i z a t i o n corresponding  -8to 18.18$ o f the molecular weight.  F o r the d e t e r m i n a t i o n o f  the water o f c r y s t a l l i z a t i o n the s a l t s are heated a t 140° C, for2 hours and the l o s s i n weight determined. The  r o t a r y power o f the z i n c s a l t s i s determined on  the anhydrous s a l t . i s opposite  The o p t i c a l r o t a t i o n o f the z i n c  i n s i g n t o that o f the corresponding f r e e  salt lactic  . acid, MEDIA EMPLOYED THROFGrHOTJT THE WORK C a s e i n d i g e s t agar a f t e r Orla-^Jensen (2) w i t h a trace o f mannose ( 1 4 ) . Peptonized m i l k g e l a t i n e  (Dlfco) ( 1 5 ) .  Peptonized m i l k g e l a t i n e enriched w i t h yeast at t h e r a t e o f 0 . 1 5 $ . Nutrient  extract  gelatine;  Sucrose n u t r i e n t g e l a t i n e — n u t r i e n t g e l a t i n e ifo and 5?o sucrose, r e s p e c t i v e l y .  containing  P e p t i c c a s e i n d i g e s t b r o t h a f t e r Orla-Jensen (2) and E a g l e s and S a d l e r (16). M i l k , f r e s h l y separated. M i l k , f r e s h l y separated and enriched w i t h yeast e x t r a c t at the r a t e o f 6 * 1 5 $ . The yeast e x t r a c t i s d i s s o l v e d i n a minimum amount o f water^ f i l t e r e d , and the f i l t r a t e added t o the m i l k . Yeast litmus m i l k — f r e s h l y separated m i l k enriched w i t h yeast e x t r a c t at the r a t e o f 0.15$ and a r e q u i s i t e amount o f A z o l i t m i n added. The f o l l o w i n g n i t r o g e n  sources were employed: the  numbers a s c r i b e d t o the p a r t i c u l a r source are those used by Eagles and Sadler  (16)j-  ' -9.-  N i t r o g e n Source ! • — P e p t i c c a s e i n d i g e s t "broth, c o n t a i n i n g approximately 1$ t o t a l n i t r o g e n (Eagles and Sadler (16). T h i s broth i s prepared as f o l l o w s ; to 300 cc,* o f tap water are added £80 gms. o f commercial c a s e i n ( W i l l C o r p o r a t i o n ) , 16 gm. of p e p s i n and 56 cc, o f concentrated h y d r o c h l o r i c a c i d - - 0 r l a •• Jensen uses 8 gm, o f pepsin. With frequent shaking the whole i s allowed t o d i g e s t f o r 10 days at 37°C. The digest i s then f i l t e r e d , u s i n g Munktell's No. 8 f i l t e r paper, made up t o £500 cc. w i t h water, and t o i t a r e added 10 gm. o f dipotassium hydrogen phosphate and 5 gm. o f magnesium sulphate. S u f f i c i e n t I8N sodium hydroxide i s added t o adjust the broth t o a pH o f 6,8. Note: t h i s b r o t h approximates, that from which Orla-Jensen prepares, by d i l u t i o n w i t h an e q u a l volume o f water, the standard c a s e i n d i g e s t b r o t h used by him i n h i s fermentation s t u d i e s ( 2 ) . N i t r o g e n Source 2 — N i t r o g e n Source 1 d i l u t e d w i t h an equal volume o f water, c o n t a i n i n g approximately 0.5$ t o t a l n i t r o g e n (16). N i t r o g e n Source 2 - N i t r o g e n Source 2 enriched w i t h yeast e x t r a c t a t the r a t e o f 0.15$--0.01$ t o t a l n i t r o g e n (Eagles and S a d l e r ) ( 1 7 ) . a  N i t r o g e n Source 20-Tryptic c a s e i n d i g e s t broth prepared as f o l l o w s : t o 500 cc, o f 1$ sodium carbonate s o l u t i o n i s added 45 gm. o f commercial c a s e i n ( W i l l Corp • ) , The s o l u t i o n i s allowed t o r e m i n at 37QC. f o r 24 hr., and 50 c c . o f an 80$ g l y c e r o l e x t r a c t (10 p a r t s 87$ g l y c e r o l t o 1 p a r t dry gland) o f d r i e d p i g s pancreas prepared a c c o r d i n g t o the method o f W i l l s t a t t e r (18) i s t h e n added. D i g e s t i o n i s continued f o r 10 days, the pH o f the d i g e s t being adjusted t o pH 7.6 from time t o time. The d i g e s t i s then f i l t e r e d , made up t o 420 cc. w i t h water, and t o i t are added 1.67 gm. o f potassium hydrogen phosphate and 0.88 gm. o f magnesium s u l p h a t e . Sufficient 6N,hydrochloric a c i d i s then added to adjust the broth t o a pH o f 6v8. N i t r o g e n Source 21-No. £0 d i l u t e d w i t h an e q u a l volume o f water. N i t r o g e n Source 31-Hydrolyzed c a s e i n D i f c o broth, 0.5$ t o t a l n i t r o g e n content (16).  -10ErPERIMENTAL The  s t a r t e r , from which the organisms f o r t h i s study  were i s o l a t e d , was obtained from a cheese manufacturing laboratory.  I t was b u i l t up from Hansen's d e s i c c a t e d  commer-  c i a l s t a r t e r and propagated f o r 20 days i n mixed skim m i l k t o v/hioh 0.1$ Yeast E x t r a c t had been added.  That yeast  extract  may exert some i n f l u e n c e on the v i t a l i t y o f the organisms c o n s t i t u t i n g the s t a r t e r may be seen when the paper on the Butter Aroma B a c t e r i a by Orla-Jensen, Orla-Jensen and Spur (19)  i s considered. In p l a t i n g out the organisms, s u c c e s s i v e  d i l u t i o n s of  the c u l t u r e s were m a d e — d i l u t i o n s 1:1,000,000 and 1:10,000,000 were p l a t e d on peptonized m i l k g e l a t i n e (Difco) and on peptonized milk gelatine  (Difco) enriched  w i t h yeast  e x t r a c t at the  r a t e o f 0 . 1 5 $ — e q u i v a l e n t i n terms o f n i t r o g e n t o 0.01$ t o t a l nitrogen  (17). A f t e r incubation  at room temperature f o r seven  days, from an e n t i r e 1:1,000,000 d i l u t i o n peptonized m i l k g e l a t i n e p l a t e and from an e n t i r e 1:10,000,000 d i l u t i o n e x t r a c t enriched p l a t e , 230 c o l o n i e s and 53 c o l o n i e s , t i v e l y , were picked  i n t o yeast  litmus m i l k .  yeast  respec-  The c o l o n i e s  from the peptonized m i l k g e l a t i n e p l a t e were designated as EMBg and given numbers 1 t o 230; those from the peptonized m i l k g e l a t i n e enriched w i t h yeast  e x t r a c t p l a t e were l a b e l l e d  C u l t u r e S^ 1 to 53.  The yeast  incubated a t 30° G.  f o r 24 hours and then at 23° C. u n t i l the  cultures clotted.  litmus m i l k c u l t u r e s were  A f t e r 48 hours i n c u b a t i o n a l l c u l t u r e s  except EMBg 26 and 130 and  5,7,10,11,15,22,32,49 and 52  were c l o t t e d .  The t e n c u l t u r e s o f the S]_ p l a t e which had not  c l o t t e d i n yeast l i t m u s m i l k a f t e r 48 hours i n c u b a t i o n were r e l a b e l l e d EMBg 231-240, r e s p e c t i v e l y , the remaining 43 c u l t u r e s o f t h i s s e r i e s being d i s c a r d e d ; •9  The 240 c u l t u r e s were t r a n s f e r r e d from yeast  litmus  m i l k i n t o c a s e i n d i g e s t broth and then were stabbed i n t o  casein  d i g e s t agar, incubated f o r 48 hours at 23° C. and then h e l d as stock cultures. The morphology  o f a l l c u l t u r e s was examined i n yeast  l i t m u s m i l k a f t e r i n c u b a t i o n f o r 24 hours at 23° G.  The organ-  isms are a l l Gram p o s i t i v e coccus forms, some appearing as c h a i n s , and some as p a i r e d or s i n g l e c e l l s . of the organisms are elongated. morphology  In b r o t h a few  A d e t a i l e d d e s c r i p t i o n o f the  o f the 20 organisms which were chosen f o r thorough  study i s g i v e n i n Tables l a and l b .  Two o f the organisms  c u l t u r e s EMBg 26 and 130 l i q u e f y n u t r i e n t g e l a t i n e and sugar g e l a t i n e j t h e y a l s o form slime i n these media. A p r e l i m i n a r y study o f the t o t a l t i t r a t a b l e  acidity  produced by the organisms i n m i l k and i n m i l k enriched w i t h yeast e x t r a c t a t the r a t e of. •0*15$, showed t h a t l e s s than 5 $ of the organisms respond to any extent t o the yeast enrichment. S i n c e time d i d not permit t o make a d e t a i l e d study o f a l l c u l t u r e s i s o l a t e d , a study whioh would a l l o w a rough grouping o f the organisms was undertaken.  For t h i s purpose  determinations were made o f the a b i l i t y o f the organisms to ferment mannitol, g l u c o s e , sucrose, maltose, l a c t o s e , d e x t r i n and s t a r c h , i n N i t r o g e n Source 2.  -12- • On t h e sum o f the c h a r a c t e r i s t i c s thus the organisms were d i v i d e d 240 c u l t u r e s , were maltose  determined.,  i n t o twelve g e n e r a l groups.  Of the  4.6.2$ were Streptococcus cremoris types; 14.1$ fermenting s t r a i n s o f Streptoooocus  15$ were maltose  and s t a r c h  cremoris;  fermenting cremoris types; 0.4$  were mannitol fermenting and 2$ maltose, s t a r c h and sucrose fermenting s t r a i n s .  0.4$ were t y p i c a l Streptococcus  s t r a i n s ; 16$ Were s t a r c h  fermenting; 2 $ — m a n n i t o l  lactis  and s t a r c h  fermenting, 0;4$--sucrose and s t a r c h fermenting; and 0.4$ non-maltose s t a r c h fermenting s t r a i n s o f Streptococcus 0.4$ o f the s t r a i n s f a i l e d t o ferment glucose, sucrose, maltose lactose,  and s t a r c h .  l a c t o s e , hut fermented Except  i n t h e case o f  0.4$ o f the s t r a i n s were unable t o ferment  carbohydrates used. i s seen that  lactis.  any o f the  From the study o f the data presented i t  the percentage  o f s t a r c h fermenting s t r a i n s i s  s u r p r i s i n g l y high; 35.8$ o f the c u l t u r e s  are s t a r c h  fermenting  s t r a i n s o f e i t h e r Streptococcus cremoris or Streptococcus lactis.  This f i n d i n g i s e s p e c i a l l y remarkable,  of the f a c t that  and i n view  i n h i s monograph Orla-Jensen (2) does not  d e s c r i b e any s t a r o h fermenting s t r a i n s o f Streptoooocus of Streptoooocus  cremoris. or o f the B e t a c o c o i , i s o l a t e d  s t a r t e r s , the f i n d i n g of such a l a r g e percentage  lactis, from  of starch  fermenting s t r a i n s among the organisms i s o l a t e d from a commerc i a l s t a r t e r assumes a s i g n i f i c a n c e  o f considerable  F o r the purpose o f d e t a i l e d  importance.  study and c l a s s i f i c a t i o n ,  20 r e p r e s e n t a t i v e s t r a i n s were chosen, u s i n g the t e n t a t i v e grouping o f the organisms as a guide.  -13.  .  .  .  »  In the d e t a i l e d study o f the £0 organisms,  deter-  minations were made o f : (1)  the morphology, Tables l a and l b ;  (£)  the a b i l i t y o f the organisms t o ferment carbohydrates  18 d e f i n e d  i n s i x d i f f e r e n t n i t r o g e n sources,  Tables 2-6; (3)  the c a s e i n - s p l i t t i n g a b i l i t y o f the organisms a f t e r  (4)  Orla-Jensen ( S ) , Table 7; the r o t a r y power o f the l a c t i c a c i d produced, Table S;  (5)  the r a t e o f a p i d p r o d u c t i o n i n m i l k and i n m i l k enriched w i t h yeast e x t r a c t at the r a t e o f 0.15$, F i g u r e s 1-20. DISCUSSION  Sugar-Fermenting  Abilities:  On examining and comparing the amount o f t o t a l t i t r a t a b l e a c i d i t y produced when u s i n g the s i x d i f f e r e n t n i t r o g e n sources, i t i s seen t h a t none o f the s t r a i n s  ferments  any o f the sugars when t r y p t i o c a s e i n d i g e s t i s employed as the source o f n i t r o g e n (no t a b l e g i v e n ) •  When t r y p t i o c a s e i n  d i g e s t b r o t h i s d i l u t e d w i t h an equal volume o f water, however, two c u l t u r e s , EMB  2  26 and 130 show a s l i g h t i n d i c a t i o n of  fermenting s e v e r a l of the c a r b o h y d r a t e s — s u c r o s e ,  raffinose  and s t a r c h g i v i n g the h i g h e s t t i t r a t i o n f i g u r e s , Table 6, f o r a l l the s t r a i n s s t u d i e d ^  N i t r o g e n Source 1 i s , on the  whole, the most s u i t a b l e source o f n i t r o g e n f o r the fermentat i o n of a l l the sugars except d e x t r i n , d e x t r i n being to a g r e a t e r extent v/hen N i t r o g e n Source  fermented  31 i s used, Tables S  -14and 5.  N i t r o g e n Sources 2 and Ea are comparable i n t h e i r  suitability  f o r the fermentation o f a l l the carbohydrates,  the amount o f a c i d produced being i n every case lower than when N i t r o g e n Source 1 i s used.  For the fermentation o f  g l y c e r i n e , arabinose, sucrose and d e x t r i n , N i t r o g e n Source 31 i s more s u i t a b l e than e i t h e r N i t r o g e n Sources E or 2a, the amount o f a c i d produced to,  from these carbohydrates being equal  or i n some cases even h i g h e r than, the amount  produced  when N i t r o g e n Source 1 i s employed. Rotary Power o f the L a c t i c A c i d : From Table 2, i t i s seen that a l l the organisms s t u d i e d produce dextro l a c t i c  acid.  G a s e i n - S p l i t t jng A b i l i t y : From the study of the o a s e l n - s p l i t t ing a b i l i t y o f the organisms, i t i s shown that the m a j o r i t y o f the s t r a i n s are not able t o b r i n g about an a p p r e c i a b l e c a s e i n breakdown.  In  the case o f these organisms the extent of the p r o t e i n decomp o s i t i o n as r e v e a l e d by the percent of s o l u b l e n i t r o g e n and amino n i t r o g e n formed tively.  i s seen t o be under 5$ and ifo respec-  C u l t u r e EMBg 130 possesses the most marked c a s e i n -  s p l i t t i n g a b i l i t y , the decomposition i n the case o f t h i s organism b e i n g 83.6$ s o l u b l e n i t r o g e n formed and 44o7$ amino n i t r o g e n formed.  C u l t u r e s EMBg 26, 43, 45, 114, 142, and 240  e x h i b i t c o n s i d e r a b l e p r o t e i n breakdown and are quite comparable one w i t h another i n t h e i r o a s e i n - s p l i t t i n g  abilities—as  judged by t h e i r a b i l i t y to form s o l u b l e n i t r o g e n ; however, the a b i l i t y o f the r e s p e c t i v e organisms  When,  t o form amino  -15n i t r o g e n i s considered, i t i s evident from Table 7 t h a t only c u l t u r e s EMBg 114 and 142 are s t r i c t l y comparable, c u l t u r e s EMBg 45, 26 and 240 forming c o n s i d e r a b l y l e s s amino n i t r o g e n . C u l t u r e EMBg 43 forms approximately  one-half the amount o f  'r  s o l u b l e n i t r o g e n produced by the other c a s e i n - s p l i t t i n g s t r a i n s and the extent o f the p r o t e i n breakdown as shown by the amount of amino n i t r o g e n i s s m a l l .  I t i s i n t e r e s t i n g t o note t h a t i n  the ease o f c u l t u r e s EMBg 45, 114, and 142, there would appear to be a r e l a t i o n s h i p between the r a t e o f a c i d p r o d u c t i o n i n m i l k and the r a t e o f c a s e i n - s p l i t t i n g as evidenoed  by v i s i b l e  d i g e s t i o n o f the chalk-milk. Rate o f A c i d P r o d u c t i o n ; On examining t h e r a t e s o f a c i d p r o d u c t i o n , i t i s seen that f o r c u l t u r e s EMBg 45, 114 and 142 the curves  for acid  p r o d u c t i o n i n m i l k and i n m i l k e n r i c h e d w i t h yeast e x t r a o t are p r a c t i c a l l y i d e n t i c a l .  A t no stage o f t h e i n c u b a t i o n  p e r i o d does t h e e n r i c h i n g e n t i t y appear t o exert a s t i m u l a t i n g i n f l u e n c e on the a c i d producing a b i l i t y o f these  organisms.  W i t h i n 48 hours the maximum t i t r a t a b l e a c i d i t y produced by these c u l t u r e s i n both m i l k and m i l k enriched w i t h yeast e x t r a c t has been reached. i s t o be noted  F i g s . 7, 9, 13.  A yeast  inoidence  i n the case o f a l l other c u l t u r e s s t u d i e d .  The p r o d u c t i o n o f l a c t i c a c i d by c u l t u r e EMBg 26 i s low even i n m i l k enriched w i t h yeast, but i t ier to be observed s t i m u l a t i n g i n f l u e n c e o f yeast e x t r a c t continues the e n t i r e p e r i o d o f i n c u b a t i o n , F i g , 5.  t h a t the  throughout  C u l t u r e EMBg 130  f a i l s to produce a measurable amount o f a c i d i n m i l k and the  amount produced  i n yeast enriched mill?: i s very low, F i g . 11.  The s t i m u l a t i n g e f f e c t o f yeast e x t r a c t on the v i t a l  activity  of t h e other c u l t u r e s i s most marked d u r i n g the f i r s t of incubation.  few days  For c e r t a i n o f t h e c u l t u r e s the maximum  t a b l e a c i d i t y produced  titra-  i n both m i l k and i n m i l k enriched with  yeast e x t r a c t i s reached a f t e r 48 hours i n c u b a t i o n , F i g s , 2, 3j4;10;12, w h i l s t i n others the maximum i s n o t a t t a i n e d u n t i l a f t e r three t o f i v e days' growth, F i g s . 1, 6, 8, 14, 15, 16, 17,'18, 19, 20.  I n the case o f c u l t u r e s EMBg 210 and 219  i n both m i l k and milk enriched with yeast e x t r a c t and i n the case o f c u l t u r e EMBg 240 i n y e a s t e x t r a c t enriched m i l k only, a d e c l i n e i n t h e t o t a l t i t r a t a b l e a c i d i t y f i g u r e i s t o be observed  a f t e r the t e n t h day o f i n c u b a t i o n . CLASSIFICATION In the c l a s s i f i c a t i o n o f the organisms,  o f c l a s s i f i c a t i o n evolved by Orla-Jensen throughout.  the system  (2) has been f o l l o w e d  The b r i n g i n g t o g e t h e r o f t h e s t r a i n s on the sum  of t h e i r c h a r a c t e r i s t i c s has been the g u i d i n g p r i n c i p l e . t o t h e d i s t i n c t v a r i a t i o n encountered  Due  i n the c h a r a c t e r i s t i c s  o f organisms u s u a l l y to be found w i t h i n a s i n g l e s p e c i e s , f o r the purpose of t h i s work i t has been found expedient t o e s t a b l i s h a b a s i s f o r the naming of the organisms s t u d i e d . Although,  according to the monograph o f Orla-Jensen  (2), there  are c e r t a i n s t r a i n s o f Streptococcus cremoris r e p o r t e d upon which a r e capable o f fermenting maltose  and s a l i c i n , or e i t h e r  of these sugars alone, i n t h i s study a t y p i c a l Streptococcus  -17oremoris  i s considered as a s t r a i n which ferments n e i t h e r  maltose nor s a l i c i n "but i s capable o f fermenting l a e v u l o s e , dextrose, mannose, g a l a c t o s e and l a c t o s e ; and, although a c c o r d i n g to Orla-Jensen the a b i l i t y t o ferment ferment  or not t o  mannitol i s a v a r i a b l e c h a r a c t e r i s t i c o f the s t r a i n s  o f Streptococcus l a c t i s s t u d i e d by him, i n t h i s paper a t y p i c a l Streptococcus l a o t i s i s c o n s i d e r e d as a s t r a i n which i s incapable o f fermenting mannitol but i s capable o f fermenting l a e v u l o s e , dextrose, mannose, g a l a c t o s e , maltose, l a c t o s e , d e x t r i n and s a l i c i n .  And i n t h e c l a s s i f i c a t i o n o f  both s p e c i e s an i n a b i l i t y t o ferment  glycerine, xylose,  arabinose, rhamnose, s o r b i t o l , sucrose, r a f f i n o s e ^  inulin  and s t a r c h i s taken as c h a r a c t e r i s t i c o f the two s p e c i e s . EMBgl—The c h a r a c t e r i s t i c s as a whole o f t h i s p l a c e i t w i t h i n the genus S t r e p t o c o c c u s .  strain  The morphology—  Tables l a and l b — t h e p r o d u c t i o n o f dextro r o t a r y l a c t i c  acid,  the fermentation o f maltose, d e x t r i n and s a l i c i n , and the h i g h a c i d p r o d u c t i o n i n milk, determine  the p l a c i n g o f c u l t u r e  EMBg 1 w i t h i n t h e s p e c i e s Streptococcus l a c t i s .  But i t cannot  be considered a t y p i c a l s t r a i n o f Streptococcus l a o t i s i n t h a t i t e x h i b i t s the a b i l i t y t o ferment it  starch.  In t h i s r e s p e c t  i s more l i k e the s t r a i n s d e s c r i b e d by Orla-Jensen and  Hansen (10) as Streptococcus a m y l o l a c t i s and the s t a r c h fermenting s t r a i n s of i - B a c t e r i a described by Knudsen (9), but i t d i f f e r s from both o f the above-named s p e c i e s i n i t s i n a b i l i t y t o ferment  sucrose and mannitol.  On the sum o f t h e  c h a r a c t e r i s t i c s c u l t u r e EMBg 1 may be t h e r e f o r e c l a s s i f i e d as  -18a s t a r c h fermenting s t r a i n o f Streptococcus l a c t i s , or, f o l l o w i n g the nomenclature e s t a b l i s h e d by Orla-Jensen and Hansen (10), the c u l t u r e may be termed a non-sucrose; nonmannitol fermenting s t r a i n o f Streptococcus a m y l o l a c t i s . F o l l o w i n g e x p l i c i t l y the system o f c l a s s i f i c a t i o n  proposed  by Orla-Jensen w i t h the p r o v i s o s p r e v i o u s l y o u t l i n e d ,  this  species may be more c o r r e c t l y d e f i n e d as a t r u e Streptococcus amylolaotis. EMBg 4 - - S h e morphology and the c h a r a c t e r i s t i c s as a whole suggest t h a t t h e s t r a i n be p l a c e d among the S t r e p t o c o c c i and, i n p a r t i c u l a r , i t s v i g o u r i n c e r t a i n o f the sugars and i t s a b i l i t y t o produoe a c i d i n m i l k i n d i c a t e that i t i s a s t r a i n o f Streptococcus cremoris. O r l a - J e n s e n s Streptocoocus 1  I t aligns i t s e l f with  cremoris s t r a i n No. 1 ( 2 ) .  On  the sum o f the c h a r a c t e r i s t i c s , c u l t u r e EMBg 4 i s c l a s s i f i e d as a s a l i c i n and maltose fermenting s t r a i n o f Streptococcus cremoris. EMBg 9 — T h e main c h a r a c t e r i s t i c s o f t h e organism would appear to guide i t i n t o the genus Streptococcus. I t s s t r o n g a c t i v i t y i n milk, i t s morphology, and i t s sugar  fermenting  a b i l i t i e s p l a c e the c u l t u r e w i t h i n the s p e c i e s Streptococcus cremoris. resembles  I n i t s sugar fermentations the c u l t u r e o l o s e l y s t r a i n No. 9 o f Orla-Jensen ( 2 ) , d i f f e r i n g  i n the amount o f a c i d produced  i n the d i f f e r e n t  slightly  sugars.  C u l t u r e EMBg 9 i s thus to be p l a c e d s t r i c t l y w i t h i n the species Streptococcus cremoris.  -19EMBg 1 4 — T h e p r i n c i p a l c h a r a c t e r i s t i c s o f t h i s p l a c e i t w i t h i n the gemis S t r e p t o c c u s .  This culture  from c u l t u r e EMBg 4 i n i t s a b i l i t y t o ferment i n a b i l i t y to ferment  strain  differs  starch, i n i t s  s a l i c i n and i t s g r e a t e r a c i d i t y i n maltose  No s t a r c h f e r m e n t i n g s t r a i n s o f S t r e p t o c c u s cremoris have been as y e t d e s c r i b e d by Orla-Jensen ( 2 ) , Orla-Jensen and Hansen (10), o r by Knudsen ( 9 ) . o f nomenclature proposed  T h e r e f o r e , proceeding on the b a s i s and adopted by Orla-Jensen and Hansen  (10), i t i s suggested t h a t c u l t u r e EMBg 4 be c l a s s i f i e d as a maltose  fermenting s t r a i n o f Streptococcus  amylocremoris.  EMBg 2 6 — T h e morphology and the c h a r a c t e r i s t i c s as a whole suggest t h a t the s t r a i n be p l a c e d w i t h i n the genus Tetracoocus. Orla-Jensen  The c u l t u r e i s d i f f e r e n t from any d e s c r i b e d by ( 2 ) . I t does not ferment  extent, but does ferment  starch.  g l y c e r i n e to any great  I n i t s case i n - s p l i t t i n g  a b i l i t i e s i t i s i n t e r m e d i a t e between the s t r a i n s o f Tetracoocus l i q u e f a c i e n s and Tetracoocus c a s e i d e s c r i b e d "by Orla-Jensen ( 2 ) . The v a r i a t i o n s n o t w i t h s t a n d i n g , c u l t u r e EMBg 26 i s c l a s s i f i e d as a s t a r c h f e r m e n t i n g s t r a i n o f Tetracoocus EMBg 4 3 — T h e  liquefaciens.  c h a r a c t e r i s t i c s as a whole suggest t h a t  the s t r a i n be p l a c e d w i t h i n t h e genus S t r e p t o c o c c u s .  The  f e r m e n t a t i o n o f mannitol, d e x t r i n and s t a r c h would appear t o suggest t h e p l a c i n g o f the s t r a i n w i t h i n the s p e c i e s S t r e p t o c o c c u s a m y l o l a c t i s (Orla-Jensen and Hansen). organism  The  d i f f e r s , however, from the s t r a i n s d e s c r i b e d by  Orla-Jensen and Hansen (10) i n n o t being a b l e t o ferment sucrose and s a l i c i n .  Culture  EMB2  43 i s c l a s s i f i e d as a non-  -20s a l i c i n fermenting s t r a i n o f Streptococcus mannito-amylolactis. EMBg 45--Except f o r i t s marked c a s e i n - s p l i t t i n g a b i l i t y and i t s a b i l i t y t o form c o n s i d e r a b l e v o l a t i l e a c i d when grown i n c h a l k - m i l k c u l t u r e , c u l t u r e EMBg 45  resembles  c u l t u r e EMBg 9 i n a l l r e s p e c t s and i s t h e r e f o r e to be c l a s s i f i e d as a s t r o n g case i n - s p l i t t i n g , h i g h v o l a t i l e a c i d producing s t r a i n o f Streptocoecus cremoris... EMBg 5 1 — A s a r e c u l t u r e s EMBg 219 and EMBg 232 (see below),  c u l t u r e EMBg 51 may be regarded as a t r a n s i t i o n a l  s t r a i n b e a r i n g i n c e r t a i n o f i t s c h a r a c t e r i s t i c s a resemblance to Streptococcus cremoris and i n other r e s p e c t s a r e l a t i o n s h i p t o Streptococcus l a c t i s .  I t i s to be noted, Table 2  u n l i k e any o f t h e other s t r a i n s studied, exoept  9  that  culture  EMBg 43, c u l t u r e EMBg; 51 e x h i b i t s marked a b i l i t y t o ferment the a l c o h o l , mannitol.  I f the s t r a i n i s t o be grouped as a  type o f Streptococcus l a c t i s ,  i t may be c l a s s i f i e d a f t e r O r l a -  Jensen as a n o n - s a l i c i n fermenting s t r a i n o f Streptococcus mannito-amylolactis  (see c l a s s i f i c a t i o n o f EMBg 1 ) , I f , on the  other hand, t h e organism  i s to be c l a s s i f i e d as a s t r a i n o f  Streptococcus cremoris--and i t s i n a b i l i t y t o ferment  salicin  may be taken as a j u s t i f i c a t i o n f o r t h i s a c t i o n — t h e c u l t u r e may be c l a s s i f i e d as a d e x t r i n fermenting s t r a i n o f Streptoooocus  mannito-amylooremoris (see c l a s s i f i c a t i o n o f EMBg14,  EMBg 114--The c h a r a c t e r i s t i c s as a whole o f t h i s s t r a i n p l a c e i t w i t h i n t h e genus Streptococcus.  I t s fermenta-  t i o n o f carbohydrates and i t s a c t i v i t y i n m i l k i n d i c a t e that it  i s a s t r a i n o f Streptococcus cremoris.  I t aligns  itself  -Elvery c l o s e l y to Orla-Jensen s 1  characteristicsj  s t r a i n No. 9.  On the sum o f the  c u l t u r e EMBg 114 i s c l a s s i f i e d as a s t r a i n o f  ••.Stre.ptocoocus cremoris. EMBg 1 2 7 — A s i s seen from Tables S and 8, on the sum of i t s c h a r a c t e r i s t i c s , c u l t u r e EMBg 127 i s c l a s s i f i e d as a s a l i c i n - f e r m e n t i n g s t r a i n of Streptococcus cremoris. EMBg ISO--Culture EMBg 130 p r e s e n t s c o n s i d e r a b l e d i f f i c u l t y when a c l a s s i f i c a t i o n of the organism The morphology of the organism,  i s attempted.  i t s extremely low a c i d pro-  d u c t i o n i n m i l k , i t s weak fermentation o f most o f the sugars, i t s s t r o n g c a s e i n - s p l i t t i n g a b i l i t y , as w e l l as i t s l i q u e f a c t i o n o f g e l a t i n e w i t h f o r m a t i o n o f slime, s t r o n g l y suggest t h a t the c u l t u r e be p l a c e d w i t h i n the genus Tetracoocus.  The  d e f i n i n g o f the p a r t i c u l a r s p e c i e s t o which the organism belongs i s f o r the present i m p o s s i b l e . 'The i n a b i l i t y o f the organism  t o ferment  l a c t o s e and mannose i s t o be n o t e d — a n d  the a b i l i t y t o ferment  s t a r c h i s noteworthy i n t h a t none o f  the s t r a i n s o f T e t r a c o c c i r e p o r t e d upon by Orla-Jensen (2) e x h i b i t the a b i l i t y t o ferment  this  carbohydrate.  EMBg 1 3 8 — I n i t s sugar-fermenting a b i l i t i e s EMBg 138 would appear t o be a composite  culture  o f c u l t u r e s EMBo 4  and EMBg 14, f o r i t i s t o be seen, Table 8, t h a t c u l t u r e EMBg 138 i s capable o f fermenting maltose, s t a r c h and s a l i c i n ; On the sum o f i t s c h a r a c t e r i s t i c s and f o l l o w i n g the system o f c l a s s i f i c a t i o n used by Orla-Jensen and Hansen (10), i t i s suggested  t h a t t h i s organism  be c l a s s i f i e d as a maltose and  s a l i c i n fermenting s t r a i n o f Streptococcus  amylocremoris.  -22EMBg 142—-Except  f o r i t s a b i l i t y t o ferment  salicin,  c u l t u r e EMBg 142 i s very s i m i l a r t o c u l t u r e EMBg 114. sum  On the  o f i t s c h a r a c t e r i s t i c s i t i s c l a s s i f i e d as a s a l i c i n  fermenting s t r a i n o f Streptoooocus cremoris. EMBg 1 4 6 — I n a l l c h a r a c t e r i s t i c s determined,  culture  EMBg 146 resembles c u l t u r e EMBg 1, and thus i s t o be c l a s s i f i e d as a t r u e s t r a i n o f Streptococcus a m y l o l a o t i s a f t e r O r l a Jensen ( 2 ) . EMBg 2 1 0 — I n a l l c h a r a c t e r i s t i c s determined, EMBg 210 resembles  culture  c u l t u r e EMBg 1 and thus i s to be c l a s s i f i e d  as a t r u e s t r a i n of Streptoooocus a m y l o l a o t i s a f t e r O r l a Jensen ( 2 ) . EMBg 2 1 9 — C u l t u r e EMBg 219 resembles  c u l t u r e EMBg 138  i n a l l i t s c u l t u r a l c h a r a c t e r i s t i c s , except that evidence o f an a b i l i t y t o ferment d e x t r i n i s t o be observed. might  The o u l t u r e  be c l a s s i f i e d — a s was c u l t u r e EMBg 1 3 8 — a s a maltose and  s a l i c i n fermenting s t r a i n o f Streptocooous amyloc.remoris.or. i f s i g n i f i c a n c e i s to be attached t o i t s d e x t r i n fermenting a b i l i t y , i t may be c l a s s i f i e d as a weak d e x t r i n fermenting s t r a i n o f Streptococcus a m y l o l a o t i s (see c l a s s i f i c a t i o n o f c u l t u r e EMBg 1 ) . EMBg 2 3 2 — W i t h the added a b i l i t y t o ferment s u c r o s e , c u l t u r e EMBg 232 i s seen to resemble c u l t u r e EMBg 219.  As i n  the case o f o u l t u r e EMBg 219, t h e d e f i n i t e p l a c i n g o f t h e organism w i t h i n a system o f c l a s s i f i c a t i o n i s an extremely d i f f i c u l t question.  I f the c u l t u r e i s t o be considered as o f  the "cremoris" s p e c i e s , i t i s t o be c a l l e d a maltose,  salicin  -23and sucrose fermenting s t r a i n o f Streptococcus On the other hand, i f the organism s t r a i n o f Streptococcus l a c t i s .  amylocremorls.  i s t o he c l a s s e d as a  i t may he c l a s s i f i e d as a  s t a r c h fermenting s t r a i n o f S t r e p t o c o c c u s s a c o h a r o l a c t i s a f t e r Orla-Jensen and Hansen (10). EMBg 2 3 6 — I n a l l c h a r a c t e r i s t i c s determined, EMBg 236 resembles  culture  c u l t u r e EMBg 1, and thus i s t o he c l a s s i f i e d  as a t r u e s t r a i n o f S t r e p t o c o c c u s a m y l o l a o t i s .• EMBg 2 3 7 — T h i s teristics  c u l t u r e resembles  i n a l l i t s charac-  c u l t u r e EMBg 9, and i s thus c l a s s i f i e d as a s t r a i n  of S t r e p t o c o c c u s cremoris. EMBg 240-—Except f o r i t s marked  casein-splitting  a b i l i t i e s and i t s a b i l i t y to form c o n s i d e r a b l e v o l a t i l e  acid  when grown i n c h a l k - m i l k c u l t u r e , c u l t u r e EMBg 240 resembles c u l t u r e EMBg 232, and i s t h e r e f o r e t o be c l a s s i f i e d as a s t r o n g c a s e i n - s p l i t t i n g , h i g h v o l a t i l e a c i d p r o d u c i n g maltose, and sucrose fermenting s t r a i n o f Streptococcus  salicin  amylocremoris  (see c a l s s i f i c a t i o n o f EMBg 232). SUMMARY AND CONCLUSION The present work,which has i n v o l v e d a d e t a i l e d study o f the r e p r e s e n t a t i v e s t r a i n s s e l e c t e d from 240 s t r a i n s i s o l a t e d from a commercial  s t a r t e r which had been c a r r i e d i n  m i l k t o which y e a s t e x t r a c t had been added, has l e d t o the d i s c o v e r y of s e v e r a l new s t r a i n s o f l a c t i c a c i d That there are s t r a i n s o f l a c t i c a c i d  streptococci. streptococci  intermediate between the t y p i c a l Streptococcus cremoris and  "-24- '  '  Streptococcus l a c t i s . as d e f i n e d by Orla-Jensen ( 2 ) , has been shown a l r e a d y by Orla-Jensen and Hansen (10). study confirms the f i n d i n g s o f these workers.  The present C e r t a i n of the  new s p e c i e s d e f i n e d by them have been found among the organisms here s t u d i e d , and, i n a d d i t i o n , s e v e r a l new intermediate forms have been d e s c r i b e d , and a c l a s s i f i c a t i o n o f them has been attempted. I t i s t o be seen, Table 8, that o f the 18 r e p r e s e n t a t i v e Streptococcus s t r a i n s submitted t o d e t a i l e d  study,  only four were to be c l a s s i f i e d as t y p i c a l Streptococcus cremoris.  two o f t h e s t r a i n s , c u l t u r e s EMBg 9 and 237, being  weak c a s e i n - s p l i t t i n g and low v o l a t i l e a c i d producing v a r i e t i e s , and two o f the s t r a i n s , c u l t u r e s EMBg 45 and 142, s t r o n g c a s e i n - s p l i t t i n g and h i g h v o l a t i l e a c i d producing  forms.  The same r e l a t i o n s h i p i n r e g a r d t o c a s e i n - s p l i t t i n g a b i l i t y and v o l a t i l e a c i d p r o d u c t i o n i s t o be seen when c u l t u r e s EMBg 127 and EMBg 114 a r e compared the one w i t h the other.  I n the system o f c l a s s i f i c a t i o n adopted  the a b i l i t y t o ferment  i n this  study,  the g l u c o s i d e s a l i c i n i s taken as one  o f the c h a r a c t e r i s t i c s o f Streptococcus l a c t i s , s e r v i n g to d i s t i n g u i s h t h i s species from a t y p i c a l Streptococcus cremoris. For t h i s reason, c u l t u r e s EMBg 127 and 114 can not be c l a s s i f i e d as t y p i c a l s t r a i n s o f Streptococcus cremoris, and are to be regarded as s a l i c i n - f e r m e n t i n g s t r a i n s o f Streptococcus cremoris. For s i m i l a r reasons, c u l t u r e s EMBg 4 , 14, and 138  -25are to be regarded as maltose  and s a l i c i n , maltose  and s t a r c h ,  maltose, s t a r c h and s a l i c i n fermenting s t r a i n s o f Streptococcus cremoris. r e s p e c t i v e l y ,  i t i s t o be observed t h a t a l l o f these  s t r a i n s are weak c a s e i n - s p l i t t i n g and low v o l a t i l e a c i d producing  forms. When the c l a s s i f i c a t i o n o f c u l t u r e s  219,  232, and 240 i s undertaken,  EMB2  43. 51.  considerable d i f f i c u l t y i n  the p r e c i s e d e f i n i n g o f s p e c i e s p r e s e n t s i t s e l f .  ,For, i t i s  t o be seen, Table 8, t h a t w h i l s t c e r t a i n c h a r a c t e r i s t i c s o f these organisms would serve d e f i n i t e l y t o p l a c e them w i t h i n the s p e c i e s "cremoris", other e q u a l l y Important would put .them among the " l a c t i s " s t r a i n s . a b i l i t i e s t o ferment  or not t o ferment  characteristics  And when t h e i r  c e r t a i n sugars are  c o n s i d e r e d , almost every p o s s i b l e v a r i a t i o n i n r e g a r d to t h e f e r m e n t a t i o n o f mannitol, sucrose, maltose, and s a l i c i n i s t o be seen. among t y p i c a l  dextrin, starch  And, as was seen to be the case  c r e m o r i s " s t r a i n s and a l s o among s a l i c i n -  fermenting s t r a i n s o f cremoris, t h e r e i s t o be observed i n c e r t a i n o f the t r a n s i t i o n a l forms, which, i n a l l other c h a r a c t e r i s t i c s , r e s e m b l e one another c l o s e l y , a d i s t i n c t v a r i a t i o n i n c a s e i n - s p l i t t i n g a b i l i t y and v o l a t i l e  acid  production; I t i s to be e s p e c i a l l y noted t h a t no t y p i c a l Streptococcus l a c t i s s t r a i n s were encountered.  F o r , when  c u l t u r e s which can be d e f i n i t e l y c l a s s i f i e d as Streptococcus l a c t i s a r e considered, i t i s seen (Table 8) t h a t a l l the s t r a i n s were o f one type and were s t a r c h - f e r m e n t i n g s t r a i n s  -26of t y p i c a l Streptoooocus  lactis*  There i s no evidence i n the  l i t e r a t u r e o f the occurrence i n s t a r t e r s o f s t r o n g s t a r c h fermenting s t r a i n s o f Streptococcus l a c t i s .  I n every sense o f  the word c u l t u r e s EMBg 1, 146, 210, and 236 are t o he regarded as t y p i c a l r e p r e s e n t a t i v e s o f Streptococcus a m y l o l a o t i s and r e p r e s e n t a d i s t i n c t new s p e c i e s o f l a c t i c  streptococci.  The  use o f the term, Streptococcus a m y l o l a o t i s , i n t h i s study i s to-be d i s t i n g u i s h e d from the sense i n which the term i s used by Orla-Jensen and Hansen (10). In t h e i r work a Streptococcus. a m y l o l a o t i s i s a s t r a i n o f Streptoooocus  l a c t i s , which, i n  a d d i t i o n t o fermenting s t a r c h , i s capable o f fermenting mannitol and s u c r o s e . I t i s a l s o t o be observed t h a t w i t h i n the genus Streptococcus s t a r c h - f e r m e n t i n g s t r a i n s o f Streptococcus cremoris were encountered; ferment  and, whenever t h i s a b i l i t y t o  s t a r c h was evidenced by "oremoris" s t r a i n s , the  a b i l i t y t o ferment  the d i s a c c h a r i d e maltose was t o be seen.  From the p o i n t o f view o f the chemical c o n s t i t u t i o n o f the carbohydrates, maltose, d e x t r i n and s t a r c h i n r e l a t i o n t o the sugar-fermenting a b i l i t i e s  o f the L a c t i c A c i d B a c t e r i a , i t i s  i n t e r e s t i n g to observe that s t r a i n s were found which were capable o f fermenting maltose to ferment  dextrin;  and s t a r c h without the a b i l i t y  One s t r a i n fermented maltose  o n l y and the  t r a n s i t i o n a l " l a c t i s " or "cremoris" types showed s l i g h t to d e f i n i t e fermentation o f d e x t r i n i n a d d i t i o n to fermenting maltose  and s t a r c h .  -27When the c l a s s i f i c a t i o n o f the two Tetracoocus —i  s t r a i n s encountered i s considered,  i t i s t o "be e s p e c i a l l y  noted t h a t , as was the case i n many o f the S t r e p t o c o c c i s t u d i e d , an a b i l i t y t o ferment s t a r c h by the organisms c l a s s i f i e d w i t h i n the genus Tetracoocus was observed. When the considered  organisms I s o l a t e d from the s t a r t e r are  as a whole, the most noteworthy f i n d i n g s a r e that  no -member o f the genus Betacoccus was encountered, and that so many s t a r c h - f e r m e n t i n g  organisms were found t o c o n s t i t u t e  the s t a r t e r . It i s d i f f i c u l t of starch-fermenting  to see why such a l a r g e percentage  s t r a i n s should make t h e i r appearance.  I t i s p o s s i b l e t h a t the i n f l u e n c e o f the yeast  e x t r a c t used  as an e n r i c h i n g e n t i t y o f the m i l k employed f o r t h e c a r r y i n g o f the  s t a r t e r was such that the c h a r a c t e r i s t i c s o f t h e  organisms" u s u a l l y t o be found i n s t a r t e r s became a l t e r e d . The  other p o s s i b l e  explanation  i t s e l f i s that yeast  o f the phenomenon that p r e s e n t s  e x t r a c t stimulated,at  t h e expense o f t h e  types u s u a l l y to be found i n s t a r t e r s propagated i n m i l k , c e r t a i n species predominance.  o f organisms which o r d i n a r i l y f a i l t o g a i n I t i s suggested t h a t the  starch-fermenting  s t r a i n s do n o t f i n d o r d i n a r y m i l k as d e s i r a b l e a medium as m i l k enriched w i t h yeast  e x t r a c t , and, conversely,  yeast  e x t r a c t enr I ched m i l k i s not a s u i t a b l e medium f o r the propag a t i o n o f the t y p i c a l Streptococcus cremoris s t r a i n s which u s u a l l y c o n s t i t u t e the s t a r t e r f l o r a *  -28As M nas been p o i n t e d  out by Orla-Jensen  r e f e r r i n g to the species Streptococcus  cremoris,  (2),when that "the  best s t a r t e r s seem to have l e a s t power o f fermenting  these  sugars (sucrose, maltose, d e x t r i n ) " and f o r our purpose we may i n c l u d e " s t a r c h and s a l i c i n ' ! , i t i s i n t e r e s t i n g to note that the p a r t i c u l a r s t a r t e r s t u d i e d i n t h i s work has been used s u c c e s s f u l l y f o r the making o f cheese f o r a short p e r i o d but t h a t those cheese made about the time the s t a r t e r was taken f o r s t u d y — a n d f o r a short time t h e r e a f t e r were o f an inferior quality,  F o r t h i s r e a s a i the s t a r t e r was d i s c a r d e d .  I t was f e l t by those r e s p o n s i b l e f o r t h e making off the cheese that the presence o f yeast  i n the s t a r t e r m i l k was r e s p o n s i b l e  f o r the d e t e r i o r a t i o n o f the s t a r t e r i n s o f a r as i t s a b i l i t y to make cheese was concerned.  The whole question as t o the  i n f l u e n c e o f yeast e x t r a c t on the b a c t e r i a l f l o r a o f the s t a r t e r has now been r a i s e d . to the i n f l u e n c e o f yeast  Before  d e f i n i t e c o n c l u s i o n s as  e x t r a c t can be drawn, however,  much f u r t h e r work i s r e q u i r e d ,  -29-  ACKNOWLEDGMENTS 1 wish t o express my a p p r e c i a t i o n and. thanks t o Dr, B.A. E a g l e s f o r h i s guidance and help throughout the course o f t h i s work and f o r h i s advice i n the p r e p a r a t i o n o f t h i s manuscript.  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N c o c o o b - t x ) ^ c o L O M  M M  M  — (1  i—I  o « in  p H ^ c n ^ t D t n i o H ^ t - o c o w to-© cn « I D r> o *-• H W ^ < l i n H M K ) N < | < | H H B B l < J ^ M M M M M M C a c V W W W W  9, . iritroKm Source- 2. Mtrogen Source P.. Cultures dumber £[  1 2 3 4 5 6 7 6 •9 10 11 13 13 14 15  7.4 7.4 7.2 7.2 7.4 7.2 7.7 7.4 8.3 7.7 7.8 7.0 7.4 7.4 7.2 7.4 7.4 7.4 7.7 7.4 7.4 7.4 7.2 7.4 7.4 7.4 7.4 7.4 8.1 7.7 :is 7.2 7.7 17 7.4 7.4 18 7.7 7.4 19 7.2 7.2 20 7.4 7.2 21 7.4 7.4 22 7.4 7.4 23 7.2 7.2 24 6.5 6.8 25 7.7 6.5 26 0.7 2;0 27 7.0 7.2 28 7.0 7.2 29 7.4 7.7 30 7.7 7.4 31 7.7 7.2 32 7.7 7.4 . 33 7.4 7.7 34 7.2 7.2 35 5.9 7.0 36 7.4 7.2 37 7.2 7.0 38 7.2 7.7 39 7.7 77.7 40 7.4 7.7 41 7.9 7.7 42 7.2 7.4 43 8.1 8.1 3.8 44 8.1 7.9 3.8 45 7.9 7.7 3.4 46 7.2 7.4 0.2 47 7.9 7.7 48 7.9 7.7 00.2 49 7.4 7.7 0.2 50 7.2 7.2 51 7.4 7.4 3.4 52 7.4 7.7 0.2 53 B.4 7.2 54 6.5 6.5 55 7.2 7;4 56 7.2 7.4 57 7.7 7.2 0.2 58 7.4 7.2 59 7.9 6.8 60 6.5 6.8 0.2  -Oulture If umber  5.6 4.5 5.2 2.9 4.5 5.8 4.5 5.0 2.7 4.5 5.4 5.2 5.2 1.4 4.5 5.6 0.7 5.6 5.0 0.7 5.2 0.7 5.4 0.2 5.0 0.2 5.2 0.2 4.-7 5.9 0.2 5.2 5.4 0.2 5.4 5.4 0.5 5.2 5.2 0.7 4.7 5.2 0.7 4.5 0.7 5.2 3.8 4.7 2.0 5.2 3.6 5.0 2.3 5.2 2.9 5.2 0.5 5.4 0.7 5.0 0.5 5.2 0.5 4.5 0.2 5.4 0.7 3.4 4.7 2.0 6.2 4.3 5.0 2.5 4.5 5.4 .#r6- 5.8 4.7 2.9 5.2 3.6 4.7 2.0 5.2 0.2 4.7 0.2 5.2 5.0 0.2 5.2 4.5 4.7 2.9 4.5 2.7 2.9 2.7 2.0 0.2 0.9 5.2 0.2 4.7 5.2 0.5 5.0 5.6 0.5 5.4 5.4 0.5 5.0 5.4 1«4 4.5 5.4 2.3 4.5 0.2 0.7 5.2 0.2 4.1 4.5 5.4 4.5 4.7 1.1 4.5 5.2 0.2 0.2 4.5 5.4 0.2 0.'5 4.7 5.2 0.2 0.5 4.75.4 0".5 5.0 5.4 0.2 4.7 5.2 0.2 5.0 5.0 0.2 4.3 5.2 0.5 4.5 5.2 4.3 5.-0 -1.4 4.3 5.4 3.6 4.5 2.0 3.8 5.3 0.2 2.9 5.2 3.6 4.7 1.6 5.2 4.1 4.7 5.0 4-1 4.7 1.8 5.2 4.1 5.2 0.2 0.5 5.4 5.2 5.2 4.5 5.0 2.0 4.5 5.2 0.2 5.2 5.6 0.2 4.7 5.2 0.2 4.3 5.2 3-8 4.7 2.0 5.0 3.8 4.7 1.8 5.2 3.8 5.0 1.6 5.2 5.2 5.2 0.2 ,5.4 5.2 0.2 4.7  - 61 7.2 7.2 5.0 62 7.7 7.4 5.2 63 8.1 7.4 4.7 •64, 7.4 7.4 5.2 65 7.4 7.4 6.2 66 7.4 7.4 5.2 67 7.7 7.2 0.2 5.2 68 7.4 7.2 5.2 69 7.2 7.0 5.2 70 7.7 7.4 . 5.2 71 6.8 7.0 5.2 72 7.7 7;4 5.2 73 744 8.1 8.3 5.4 75 76 7.7 7.7 0.2 5.4 77 7.2 7.4 0.2 5;4 78 8.1 7.9 5.4 79 7.2 7.4 0.2 5;2 80 7.0 7.7 0.2 5.4 81 7.4 7;2 5.2 82 7.4 7.7- 0.2 5.2 83 7.2 7.0 5.0 84 7.4 7.0 5.0 85 8.1 8.3 5.4 86 7.4 7.4 0.2 5.0 87 7.9 7.7 "5.2 88 7.0 7.2 5.0 89 7.2 7.2 5.2 90 7.7 7.i 5.6 91 7.2 7.4 5.0 922 7.7 7.7 5.Z 93 6.5 7.2 5.0 94 .6.8 7.7 5.2 95 7.9 -7.7 -5.4 96 7.4 7.2 5.2 0 97 7.4 7.7 0.2 '5.6 98 7.0 7.7 0.2 5.4 99 7.4 7.7 0.2 5.4 100 7.7 7.7 0.2 5.4 101 7.4 7.7 0.2 5.2 102 103 7.7 7.7 5.4 104 7.4 7.7 5.6 105 7.9 8.1 5.2 106 7.2 7.7 0.2 4.5 107 108 7.2 7.7 0.2 5.2 109 7.4 7.7 5.2 110 7.2 7.7 5.2 111 7.4 7.4 6.4 112 6.8 6.8 4.7 113 7.9 7.9 5.4 114 8.1 8.8 5.2 115 7.7 7.9 5.6 116 7.4 7,9 5.2 117 7.7 7.7 5.6 118 7.4 7.4 5.4 119 7.7 7.7 5.4 120 7.4 7.9 0.2 5.6  Hote: Results reooraed as grams l a c t i c AeM per mille.  0.2 '4.7,.:.: •„• ' 4.7 3.8 1,6 4.3 ' '4.3 4.7 3.6 4.7 1.6 3.8 4.7 2.5 4.5 4.7 1.1 4. ' 4.3 4.7 3.4 3.8 3.8 4.7 0.5 •3.8 4.7 1.8 3-8 5.6 1.8 4.3 -4.5 3.8 4.13.8 4.7 2.5 3  0.2  5.6  0.2 4.5 ' • 0.5 5.0 4.3 5.2 3.4 KS ii3 4.7 3.4 4.5 4.3 5i0 8*4 '4.1 2; 3 4.7 0;2 4.7 2.3 4.7 0.7 0:9'- 4.5 '•" , 0i2 5.6 0.2 2.3 4.5' 1.4 4.5 . 3.8 4.5 ;... 4.1.' 4.7 3.6 4.3 0.2 5.2 0.2 4.7 0.2 4.7 0.2 0.2: 4.5 2.9 5.0 B.-4 0.2 4.5 0.5 4.7 0.2' 6.0 4.5 5.0 • 3.8 4.7 0.2 5.0 6.2 5.0 4.5. 4.7 - 2. •: :  2  0  3  0.2 5.2 0.5 0.2 5.4" 4.1 5.6 2.7 4.5'•4.7 . •/.' S-4 3.8 3.4 :'• 2.'3 1.8  5.0,  2.3  5.6 3.2 4 . 3  4.7 5/6 4.7 5.0 •S-.4 5.4 3.6 5.2 5.0 4 . 3 5.Q 4.6' 5.6 0.2 5.2  -  6.2  0.7  2.0  3.2  3.7 2.5  4.3 3.8  9; (continued)  Culture ITumo er  gitrogen Sourcs 2. Jfitroffeh Source 2.  5 "B'  Guipure Number  EMB  2 7.2 5.2 0.2 4.7 121 7.4 7.4 5.4 0.2 5.2 122 7.2 7.2 5.2 0.5 5.0 : 123 7.2 7.2 5.6 0.2 5.4 124 7.2 7.4 5.6 1.4 5.6 1.6 4.1 125 7.4 7.4 5.2 3.6 5.4 1.6; 4.1 126 6.5 6.6 5.2 5.0 127 7.7 7.2 5.2 3.4 4.7 128 1.1 8.1 7.4 2.9 5.2 4.1 5.2 1.8 . 3,6 129 0.2 0.2 1.4 ,1.6 0.2 130: 7.4 0.9 7.0 5.2 0.2 5.0 131 7.7 7.4 5.2 5.2 132 8.1 8.1 5.4 5.2 133 7.2 7.2 0.2 5.2 0.2 134 7.2 7.2 '5.2 5.2 135 7.7 7.4 5.2 5.2 136 7.7 7.4 5.2 5.0 .. 187 7.4 7.4 5.2 5.0 138 7.4 7.7 5.2 4.7 139' 7.2 7.2 5.2 5.0 140 7.2 7.4 5.2 5i0 ' 141 8.3 . 8.6 5.6 1.4 142 3.2 5.6 6.5 7.4 4.7" 143 4.5 7.0 7.0 5.0 144 5.2 6.8 7.0 5;0 145 5.2 7.2 7.4 4.7 146 5.2 7.4 7.7 2.7 4.3 4.7 147 5.2 •754. 7.2 148 5.2 3.8 4.7 0.9 2.7 7.0 7.4 149 5.2 4.7 4.7 2.5 4.1, 150 7.7 - '7.4 5.6 •151 5.4 8.1 8.1 5.2 152 0.2 5.67.7 7.4 5.4 153 0.2 5.0 8.1 8.1 5.6 0.2 5.2 154 7.0 7.4 5.2 3.4 4.7 155 1.1 7.2 7.4 5.2 156 3.4 4.7 0.9 7.2 7.4 5.2 '157 :5.0 8.1 7.9, 5.2 158 0.5 4.7 7.2 7.0 5.2 159 4.1. 4.7 2.9 4.1^ 7i4 -7.4 - 5.2 160 5.0 5.2 161 ' 8.1 7.4 : : 4.1 5.0 2.7 4.-77.4 -•7.4.' : 5.2 162 0.2 5.0. 0.2 5.4 163. 7.2': 7.7, •4.5 4.7 3.4 -4.5' 5.4 ••: 164"'7.-2 7.7 4; 3 .4.5 2.9' 4.7 5.2 :i65 ; 7.7 7.4 4.5 8.3 7.9 5.4 '166 0.5 5.2 7.4 :7.4 5.4 4.5 167:' 7.7 7.4 5.4 5.6 168 .7.-4.' '7.7' 5.4 6;0 169 7,4 7.'7., 5.4 5.0 170 •8.1 7.9 5.2: 4.7 171 8.6 '8.3 5.6 5.0 172 ' .7.4" 7.2.5.6' 5V0 173 8.3, •7,9 ..." 5.2 •". 4.7. 174 7.7 ' 7.7 5.4 4.7 4.5 4.5 175 8.1 7;9 ' 5.2 "5.6 176 •' .7.7 7.9 '5.6 • 5.4 7.7 17,7 7.4 "5.6 5.0 '178 ~. 7.4 7.4' ., 5.6 5.0 3.8: 4.5 ' 7.7' ; .179 714' " 5.6 5.2 •180 : :  :  181 6.5 7.4 5.6 182 7.7 7.4 5,6 •183 7.7 6.5 5.6 184 7.9 7.7 6.1 185 7.2 7.9 •6.6 186 7.2 7.4 5.2 187 7.2 7.7 , 5.0 188 7.4 7.7 5.2 ,189 6.8 7.0 5.6 190 ,7.9 7.9 .5.4' 191 6.8 6.8 5.2 192 7.2 7.4 '5,6 193 7.4 7.7 55 9 194 7.4 7.7 5.6 195 '7.4 7.9 5.4 196 7.4 7.9 , 5.2 ' 197' 7.4 7.7 5.6 188 7.* 7.7 6.6 199 6.8 7.2 : ' ,:5.4 " 200 7.2 7.4 : 5.6-.' 20i 7.4 7.7 6.6 202 7.4 ,'7.7 0.6 203 7.4 7.2 5.6 : 204 7.2 •7.7 5.6- , 205 7.4 7.9 4.7 206 6.8 7.7 5.6 , 207 7.4 7.7 . ,5.6 , 208 7.4 •7.7 ' " .6.1 '• 209 7.4 7.7 5.6 210 %.Z 8.1 . 5.4 - 211 7.9 .. 7~.>7.6.0 212 7.2 7.4 5.2 213 6.1 6.8 . 5.6 214 7.7 7.7 .5.6: 215 7.4 7.4 5.4 216 6.8 6.8 5.2 217 8.3 7.9 , 5.6 218 7.2 7.-4 ' 5.6 219 7.9 7.9 5.4- . ,220: '8.3 8.3 '-. ,5.6 . 221 •'7.4-' 7.7 •:5.4-'::.'. .222 '7.2 7.7 ,•'5.4 '•• ,223 . 7.4 7.4- •", 5.6'' "• '224 .7.7 •7.9 '.-• 5.4 : 225 7.7' 7.4 , •5.4 226 7.7 - 7.7 :, ' 5.4 227 7.7, 7.7 5.4 ,, 228 7.9 7.7 ' •: :: 5.4:. •229 7.7 7.7 ' 5.4, "230 7.7 7.7' "5i4 . 231- T.0 7i6 ' 5.2 . "" 232 :6.1 6."3'-.:.- '• 4 . 3 ' '2.< 233" 7.4 . 234 7.2 7}7 •.'.'. '4.1 235 6.1 7.4 .'•' 5.6 236' '7.2 7.4:, 6.4 237 7.2 6.8' • 238'" 6.3 6.5,.-" :.'• 239 : 6.5 4.3 3.2, 240 6.1 '6.3: .4.'5 \2."7  "„4.,7 4.5 5.2 5.6 0.5 5 .2 '3.6 4 ;7: '3.6 5 .0 •2.7' '4.7 2.0 4.-5 iiB•'4i7'" 4.7 ,0.9 4 .7 6.7 : . 4.5"' 0.5 4 ;7 ." 3.8 : 4 .7»S 4';5. 0.9 " 5 . 0: 0.5 ' 5.0 :0.5= • 5 . 0 : 4 . 5 4.7 ' •3.8 4 . 7 4.3 4 .7' 4 , 3 . 1.8 ' '•,4.7',4.1. •' •0.5 4 . 7 0.5 2.3- 4 . 7 ,3.8: ;-4.s 2.9... '3.8- 4 . 7 , 4..27 2.7 .. &'e•3.6 , 4.5 5 2.7 4 . 5 , 4 . 3 , ;5.o 2.9 4.1... 5. .0. 4.7 4 4.3 4 . 77 3.8 5.0 2.5  f  '3.8 6.7 4.1  ' 6.5 4.3  :  :  :  •4:i.  !  Ifote: Results'recorded as gram l a c t i c A c i d per mllle.  2.9  . ' 3.4  ni'  ' .4.1  ' 4.1 4.1'  4.1  3.8:  .' 5.0 . '5.0 5.0 4.7 2.5 5.2 4.7 • 4.7 2.3 5.2 .5.2 5.0: ",4."3 '4.5' 4.7 .-,2X9 4.7: 5.6 ••;2.-9' 3.2' - 4 . 3 : 4.7 • 4.7," 2.'9 ' 4 . 3 . 4 . 7 " 3.2: "4^3; •4*7., -3.8 -:.4. 7" 4 . 3 0.2' * - 3 5.0 . ;  ;  3.8  :  3.8:: 4.5 4^7 3.8, 4'. 7 3.8' . ? • ? ' O.S . 4 . 3 „ 4.1 4.7,' 0.7 ••' .2,3 : V  • ,4."3; " 0."2 :' ili 6.''  EMB  Z  /  (Fig. I)  10  <? e 7 6  /  I  i  /i  X4  I  1 —  /  -  /  /  I  / 1  Legend: MitK Milky?  Days  —  to  7"/7<s above /egend opp//es ?o all other graphs.  fag  BJ  — —  -4— /  /  1  / /  / ^  i  2  —  // /  /  -il— if § §  A 5 Days  /o  /3 Z4-  fag 3)  10 9  3  $> 6  *3  i £MB  IO  II  /O  t/  is.  13 /4-  f4  3  IO  8  2  3  4  £>ays  S  6  7  6  9  12  A3  14-  EMB  s  4  ae  EMB /u a  9  a r"  7 —  - •— * "  / /  1  X  8  1  if  i /O  — •  //  /£>  /g  —-" •  / / —  /  / /  // /  .u  I.  —  /  /  ••Soys  /c7  //  /.e  A3  £MB  I  a  /30  fag. //)  3  k  r .0  I, I 3 <4 JDai/s  /o  £MB  Z  a/o  £MB  Z  237  -30REFERENTS  B a r t h e l , Chr., 1914. Meddelande N:r 97 f r a n f o r forsoksvasendet pa jordbruksomrldet. l a b o r a t o r i e t . H:r 11. . 2.  3.  Centralanstalten Bakteriologiska  Orla-Jensen, S. 1919. The l a c t i c a c i d b a c t e r i a ( i n E n g l i s h ) . D. K g l . Daiiske Yidensk. S k r i f t e r , l a t u r og Mathematisk, 8, Raekke, v» 2, Copenhagen. S t o r c h , Y , 1919. F o r s t a t t e Unders^gelse over F r e r a s t i l l i n g e n a f Syrevaekkere 108 de Beretnung f r a F o r s ^ g s l a b o r a t o r i e t . K g l . Y e t e r i n a e r - og Landobohjzfj sko1e s Laboratorium f o r landj^konosiske Forspg. Kp'benhavn. S  4.  5;  Boekhout, F.W.G., and Ott de T r i e s , J . J . , 1919. Aromabildnen b e i den Rahmsaurung. C e n t r a l b l . f . Bakt. I I abk,, 49, 373-382. Hammer, B.W., and B a i l e y , D.E., 1919. The v o l a t i l e a c i d p r o d u c t i o n o f s t a r t e r s and o f organisms i s o l a t e d from them. Iowa Agr. Exp. S t a . Res. B u i . 55.  6.  Hammer, B.W., 1920. V o l a t i l e a c i d p r o d u c t i o n o f S t r e p t o coccus l a c t i c u s and t h e organisms a s s o c i a t e d with i t i n s t a r t e r s . Iowa Agr. Exp. S t a . Res. B u i . 63.  7.  Hammer, B.W., 1920. The t y p e o f l a c t i c a c i d produced by s t a r t e r s and by organisms i s o l a t e d from them. Iowa Agr. Exp. S t a . Res. B u i . 65.  8.  Knudsen, Sc/ncke, 1931. S t a r t e r s ( t r a n s l a t e d by E.R. H i s c o x ) . The J o u r n a l o f D a i r y Research, 2, 137-161.  9.  Knudsen, So'ncke, og S^renson,A., 1929. Den K g l . Yeterinaerog Landbohp^skole A a e r s s k i f t , p. 64. ( E n g l i s h summary).  10.  11. 12.  Orla-Jensen, A.D. and Hansen, A., 1932. The B a c t e r i o l o g i c a l f l o r a of spontaneously ..scured m i l k and o f commercial s t a r t e r s f o r butter making. Sonderabdruck aus dem Z e n t r a l b l a t t f u r B a c t e r i o l o g i e , Parasitunkunde und I n f e k t i o n k r a n k h e i t e n . I I A b t e i l u n g . 1932, Bd. 86. (Translated by G. F i s h e r ) . S a d l e r , W., E a g l e s , B.A. and Pendray, G., 1932. Canadian J . Research, 7:370-377. S a d l e r , W., E a g l e s B.A. and Pendray, G., 1932. Biochemical Journal, x x v i , 5, 1532-1535. t  -31-  13.  Eagles, B.A. and S a d l e r , W., for publication*  manuscript  14.  B a r t h e l , Ghr. und Sandberg, E., 1919. B a k t e r i o l . I I Afct. 49.  15.  D i f c o D i g e s t i v e Ferments Co., D e t r o i t , Michigan.  16.  E a g l e s , B.A. and S a d l e r , W., 7:364-369,  17.  S a d l e r , W. and E a g l e s , B.A., unpublished  18.  W i l l s t a t t e r , R., and Waldschmidt-Leitz, P h y s i o l . Chem. 125:132-198.  19.  Orla-Jensen, S., Orla-Jensen, A.D., and Spur, Bernhard, 1926. The "butter aroma "bacteria. Jour. Bact. 12, 333-342.  1932.  i n preparation Centrabl. f a r  Canadian J . Research resalts. E.Z. 1923.  

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