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The ripening of cheddar cheese Ingledew, N.H. 1934

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•CAT: m. L^2zJM^M^MAi THE RIPENING OF CHEDDAR CHEESE by N. H. Ingle dew and W. J . C. T a i t A J o i n t Thesis Submitted f o r the Degree of MASTER'OF SCIENCE IN AGRICULTURE . i n the Department of D a i r y i n g 1 T H E R I P E N I N G . D E G H E D D A R GHSESE • INTRODUCTION. A r i s i n g from the general t r e n d to improve and standardize d a i r y products, recent years have seen a movement towards the p a s t e u r i s a t i o n of m i l k f o r dieddar cheese-making. From the observations of Atkinson ( l ) and Stevenson (2) the p a s t e u r i z a t i o n of m i l k f o r cheese-making r e s u l t s i n an i n c r e a s -ed y i e l d of cheese, a more uniform product, and an improved q u a l i t y more s u i t e d to longer periods of storage. Sammis and Bruhn (3) showed that p a s t e u r i z a t i o n (JTlash at i66°E.) reduced the v a r i a t i o n s i n the q u a l i t y of the cheese from day to day by n e a r l y one h a l f and i n n i n e t y - s i x percent of the cases under t h e i r observations the cheese from p a s t e u r i z e d m i l k scored higher than those made from raw m i l k . These r e s u l t s are sub-s t a n t i a t e d by P r i c e (4) who pasteurized at 14^°E. f o r t h i r t y minutes. Other workers c l a i m d e f i n i t e disadvantages f o r p a s t e u r i z a t i o n . In the r e p o r t s of the Department of S c i e n t i f i c and I n d u s t r i a l Research of New Zealand (5) the op i n i o n i s ex-pressed that i n usi n g the "Holder Method" of p a s t e u r i z a t i o n at a temperature of 1^0°E• a d i s t i n c t i n f l u e n c e was evident i n the f l a v o u r of the cheese; the cheese d i d not mature i n the ordinary way and at f o u r months had a b i t t e r t a s t e . Moir (6) u s i n g the F l a s h Method at temperatures ranging from l 6 5 ° E . to l8j>°E. 2 concluded that p a s t e u r i z a t i o n produced a h i t t e r f l a v o u r i n the r e s u l t i n g cheese and the u l t i m a t e p r o t e i n decomposition pro-ducts were reduced. At k i n s o n ( l ) expressed the o p i n i o n that p a s t e u r i z i n g m i l k f o r eheese-making r e s u l t s i n a slower matur-i n g cheese than that made from raw m i l k . These i n v e s t i g a t i o n s have shown that o n l y low tem-peratures of p a s t e u r i z a t i o n were s a t i s f a c t o r y f o r cheese-making; h i g h temperatures of p a s t e u r i z a t i o n were apparently detrimental to the r e s u l t i n g cheese. Considering the disadvantages of high temperature p a s t e u r i z a t i o n , i t has seemed advisable to endeavour to obtain seme data i n regard to the i n f l u e n c e of p a s t e u r i z a t i o n on the m i c r o f l o r a of the r e s u l t i n g cheese; and to determine whether or not the disadvantages p r e v i o u s l y mentioned can be a t t r i b u t e d to d i f f e r e n c e s i n fermentation or to other changes, p h y s i c a l or chemical, r e s u l t i n g d i r e c t l y from the high temperatures of p a s t e u r i z a t i o n , or from a combination of both. HISTORICAL. ^vans, Hastings and Hart (7), 1924, found that the predominating organisms at the d i f f e r e n t stages of the r i p e n i n g of raw m i l k cheddar cheese were included i n f o u r groups, Bacterium l a o t i s a c i d i , Bacterium o a s e i ^ Streptococcus, and Micrococuss, and that the f l o r a of p a s t e u r i z e d m i l k cheese, w i t h the exception of B. c a s e i was s i m i l a r to the types of or-ganisms i n the s t a r t e r used i n making the cheese. They s t a t e : "The B. oasei group i s apparently responsible f o r the pungent 5 t a s t e which, develops..late i n the r i p e n i n g p e r i o d of both raw and p a s t e u r i z e d m i l k cheeses." F u r t h e r the yri showed th a t when, only B. l a c t i s a c i d i was used as s t a r t e r i n making cheese from p a s t e u r i z e d m i l k , no Cheddar f l a v o u r was obtained. However, when both B. l a c t i s a c i d i and B. oasei were added to pastuer-i z e d m i l k f o r cheese-making a sour f l a v o u r was produced.during the e a r l y p a r t of the r i p e n i n g process. I n c o n c l u s i o n they s t a t e : " I t does not seem unreasonable to hope that s t a r t e r s may be obtained, which w i l l give the c h a r a c t e r i s t i c f l a v o u r to cheese made from p a s t e u r i z e d m i l k . " To overcome l a c k of f l a v o u r i n cheddar cheese made from p a s t e u r i z e d m i l k , Hastings and Sammis (8) 1920, used a s p e c i a l s t a r t e r , which, i n a d d i t i o n to the l a c t i c a c i d b a c t e r i a c h a r a c t e r i s t i c of s t a r t e r s used i n cheese-making, contained other organisms which they d i d not d e f i n e , i s o l a t e d from s o i l and faeces. The cheese developed f l a v o u r more r a p i d l y than d i d the c o n t r o l cheese made from p a s t e u r i z e d m i l k w i t h s t a r t e r which d i d not c o n t a i n these organisms i s o l a t -ed from s o i l and f a e c e s . P a n f i l o v (9) 1928, i n a study of the e f f e c t of pa s t e u r i z e d m i l k on the m i c r o f l o r a of cheese 7 subjected batches of m i l k to temperatures of 70°C f o r twenty minutes, 65°0 f o r t h i r t y minutes, and 65°C f o r f i f t y minutes. At the lower tem-perature a g r e a t e r percentage of the l a c t i c a c i d organisms, e s p e c i a l l y B. e a s e l . were to be found i n the r e s u l t i n g cheese. Moreover, the cheese made from m i l k p a s t e u r i z e d at 6S°C f o r f i f t y minutes rip e n e d more q u i c k l y than those made from m i l k p a s t e u r i z e d at the higher temperatures. A l l m i l k used was obtained from the herd of the u n i v e r s i t y of B r i t i s h Columbia, ( c h i e f l y A y r s h i r e ) . The m i l k of the previous night and of the morning of each day of cheese-making, was thoroughly mixed and d i v i d e d between two v a t s . A s e r i e s of s i x l o t s of cheese was made from m i l k t r e a t e d as i n d i c a t e d i n Table I . TABLE I Nov. 15/30 Lot 1 Raw m i l k . Nov. " Lot 2 P a s t e u r i z e d 140 0! 1. - 30 min. Nov. 22/30 Lot 3 P a s t e u r i z e d 150°E. - 30 min. Nov. .'• L o t 4 P a s t e u r i z e d 160°I. - 30 min. Nov. 29/30 Lot 5 P a s t e u r i z e d 14Q°P. - 30 min. Nov. ": L o t 6 P a s t e u r i z e d 150°P. - 30 min. The p a s t e u r i z e r used was a g l a s s - l i n e d " Pfaudler" of the holder type with a capacity of s i x t y g a l l o n s , PROCESS Off MAITOffACTllKE. The cheese were manufactured a f t e r the manner of t ; the cheddar process f o l l o w i n g i n d e t a i l the u s u a l procedure of the D a i r y Department L a b o r a t o r i e s - a process very s i m i l a r to the Western Ontario method. Complete records of the process of manufacture are given i n Table 11. The s t a r t e r , cheese c o l o r and rennet were the standard commercial products of the Hansen L a b o r a t o r i e s . No attempt was made to determine the.types of organisms present i n these products. However, from the r e s u l t s of work done by students i n the advanced course i n Da i r y B a c t e r i o l o g y under the s u p e r v i s i o n of P r o f e s s o r W. Sadl e r , on the types of organ-isms present i n the s t a r t e r , showed that only coccus types c h a r a c t e r i s t i c of cheese s t a r t e r were to be found. I t i s to be seen from Table 11. that the e f f e c t of the p a s t e u r i z i n g temperature on the cheese-making process o d i d not become evident u n t i l the m i l k was heated to 160 IT. When t h i s temperature was used, however, a very s o f t coagulum was obtained r e q u i r i n g two hours to f i r m s u f f i c i e n t l y f o r c u t t i n g . The curd on the rack was spongy, h e l d moisture and d i f f i c u l t to mat. PROCESS.Off RIPENING. During the f i r s t f o u r t e e n days of r i p e n i n g the cheese were h e l d at approximately 62°ff.; sub se quent -ripening -was'-carried on at approximately 49°ff. The constancy i n tem-perature of the r i p e n i n g rooms i n which the cheese were h e l d , EXPERIMENT CHEDDAR CHEESE RECORD OF PROCESS o c. ^ t b b OF MANUFACTURE TABLE JJ i *Cut threa limes, alowing 10-ntInuta IntsrvaU-CHEDDAR CHEESE RECORD OF PROCESS OF MANUFACTURE T A B L E S — may be seen when the f i g u r e s g i v i n g the maximum and minimum v a r i a t i o n s are considered. ' Table 1 1 1 . HIGH: ROOM LOW ROOM Monthly Mean Temp. Monthly . Max.. °E. Min. °F. .Mean Temp. Monthly °E. Max. ^ . Min. Of f . Nov. T30 6 I . 5 64 58 5 0 . 8 5 9 43 Dec. f30 61.1 63 5 8 48.8 5 4 43 Jan.* 31 6 0 . 4 63 58 4 8 . 6 5 9 44 Feb. ? 31 63.0 66 6 0 47.1 5 1 43 Mar. f 31 63.4 66 6 0 48.6 5 9 42 B A O T E R I O L O G I O A L E X A M I N A T I O N . The cheese were sampled f o r b a c t e r i o l o g i c a l exam-i n a t i o n at the age of t h i r t y days and again at seventy-three days. Sampling: In sampling the cheese the surface was seared w i t h a,hot i r o n and a bore was taken with a s t e r i l e t r i e r and trans-f e r r e d to a s t e r i l e p e t r i d i s h . D i l u t i o n s : Representative p o r t i o n s of the bore amounting to one gram, were taken and t r a n s f e r r e d to a s t e r i l e g r i n d e r con-t a i n i n g 9c.c. of water and thoroughly e m u l s i f i e d . From t h i s g r i n d e r the r e q u i r e d d i l u t i o n s were made f o r the p l a t e method of examination. The d i l u t i o n s found most s a t i s f a c t o r y i n p r a c t i c e , u s i n g p l a t e s f i v e and one h a l f inches i n diameter, were 1:100,000 f o r the f i r s t examination and 1:10,000 f o r the second. Media and Incubation Temperatures: The medium used i n p l a t i n g throughout the experiment was Peptonized M i l k G e l a t i n , at a P.H. 6.0. A l l d i l u t i o n s were p l a t e d i n d u p l i c a t e and incubated at room temperature (21°-23°c) f o r ten days. Depending on the number of c o l o n i e s on a p l a t e , e i t h e r a complete p l a t e or a r e p r e s e n t a t i v e p o r t i o n of a p l a t e was picked. The c o l o n i e s were t r a n s f e r r e d d i r e c t l y i n t o l i t m u s m i l k c o n t a i n i n g 0.15% yeast e x t r a c t , * and were incubated at 3Q°G. A l l c u l t u r e s , as soon as clotted,were removed from the incubator and h e l d at room temperature. S l i d e s were made of a l l c u l t u r e s at twenty-four hours and were st a i n e d by Grams' method to deter-mine the Morphological c h a r a c t e r i s t i c s of the organisms. These c u l t u r e s were a l s o i n o c u l a t e d i n t o 10c.c. q u a n t i t i e s of m i l k and yeast-milk and incubated w i t h c o n t r o l s , f o r fourteen days, eocous forms at 2 3 o G , rod forms at 30°G. When in c u b a t i o n was completed, the c u l t u r e s were t i t r a t e d w i t h N/4 sodium hydroxide us i n g phenolphthalein as i n d i c a t o r ; the t i t r a t i o n of the con-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 . I t can be seen, that i n the manner of i n c u b a t i n g , t i t r a t i n g , and recording of r e s u l t s we have f o l l o w -ed procedure employed by Orla-Jensen. * Standard Products of the is-igestive Ferments Co. D e t r o i t , U. S.• A. ' ' 8 . SCORING OF CHEESE''; The cheese at the age of three months were scored by a judge,* who has s e v e r a l times taken part i n Domin-i o n competitions. These scores are submitted i n Table 1¥. TABLE lvY, SCORE OF CHEDDAR CHEESE. Feb. 2 3, 1931. No. of cheese 1 2 4 • •• 5 6 M i l k past. F • raw 140°F. 160°F. 140°F. 150°F F l a v o u r . ; 45 - . 43 .5 43 .25 43 .25 44 .25 43 .0 Texture. 25 24.0 23 .75 22.75 24 .5 24 .25 Closeness. 15.0 15.0 14 .75 13 .0 14 .75 14 .5 C o l o r . 10 9.75 9.5 9.5 9«8 9.7 F i n i s h . '• 5 ? : / 5 r— 7 5 T o t a l . 100.0 97.25 96.25 93.0 98.4 96.65 Remarks• Pasty. Crumbly. B i t t e r . EXPRESSION OF RESULTS-. The t i t r a t i o n s of the c u l t u r e s i n lOc.o • q u a n t i t i e s of m i l k and yeast-milk r e s p e c t i v e l y , are given i n grams, per m i l l e i n Charts 1.—X. There i s a l s o to he seen a b r i e f resume of the procedure f o l l o w e d throughout the deter-minations* Tables V. and 71. give a summary of the r e -s u l t s of the b a c t e r i a l examination of the cheese at t h i r t y and seventy-three days r e s p e c t i v e l y , and f u r t h e r give the num-ber and percentage of coccus forms whose v i t a l a c t i v i t y i n A.P. Slade of A.P. Slade and. Co., Vancouver, B.C. Canada. 9 to pq w Q O to EH CQ CO H 53 «1 cU «•• o O CO PH |H EH ti is! f-H H > EH >H CQ a o 4* PI CO P< O • •rl S O 'ri H « i tO. o» -e «D 0> 70.4 rH , tO * 00 o Orgai Hot I Addii H • o to O H 03 03 rH c-m E> rH C~ S If a ri -rt Pi • rt I 4 3 1 1 O (5 SI to H ... to to 03 H ' • CO to rH ft H 03 G 525 03 © 03 o> H t 1 1 1 1 P 1 1 1 I ( slogy o CO o 1 1 ts • o ; tO 03 .rt^ p. FH o ts . to to 03 03 CO . o 03 ts CO -• 03-O CO • rH . to o 9 03 . ts .', ID O in H • rH 03 O e in 03 Colon-ies Picked lO 03 rH .j: £S 03 r-i in 00 OS ' to o> Total Count per Gram o o o . o o « r-i co o o o o 03 to_ o o o o o co . «o rH. o. o o o o 03 o rH o • o o o o ro rH 03 oo rt © -p a +» 03 cd a) o O f t w cd fa o o r-i •; fa o o , «o rH fa o O rH Fq o o in rH <!-) 1 o rt • H rt ©. 0 O •p «d - H <d M. +» p r*j cd o to • • : OJ H in O to 03 rH \ m H O to 03 H *-s. 03 03 O to -\ 03 rH \ OJ 03 o to \ 03 r-i o> 03 Date of Make o to \ r-i r-i \ m rH o to r-i rH lO rH O to \ rH ' rH V, 03 03 O to rH r-i 03 o to r-i rH o> 03 © CQ © • © O 1 g * • -rH . 01 •• • LO CQ rt •H CO o h o ^ i CQ CO rt rt cd cd xi ft o (0 JH u bO • H • H rt R) cd o P< Pi H rt rt rt CQ • • H • H • H S fH a CO CO CQ O rt 0 a a «H o in fH (4 <r< O O o <rl <M <t-l O fH o co © « o «> f-l tl0 o o o o rt o o o ,rt o o o o to 1-^  Ii it II II II <j m o p 10 Organisms Not Respond-ing to the Addition of 75.0 CO O OJ 1 CO o CO 13.4 Organisms Not Respond-ing to the Addition of c CO H in H 1 I H O H in H to 60 O ® * * s •H rj-J © O • Pi PS * g H & „ g ^ H <D o ti O P5 -P «ai o O • in 02 E> ... a> O O o H •, 03 .« o H tQ • tO CO $ tO •in H ol £> cn >> -60 6 • o P. (4 O a to >• • CO 03 l i ! »• 1 fi i to ' » to ' in I i ! • rH 1 O • o 03 .« CO ! C-•« tD OJ o o pq cn i. o H o • in m • to . to H • to to tO ... t> O « O rH CO c-: to in « to in • in • r-i 03 ; Colon-ies i Picked! CO H H O CO . «o m t> 03 . r-i r-i to rH Total i Count per Gram j O O O o co r-l r-T o o o" o co o o o„ o o tO O O Q . O o of O O o_ cT m 03" OO P) • - H © -P 0 •P 01 ctf cd o Q PM CO cd w • N o o H O o tD r-i • pq o o r-i • pq ; 0 O in H Date of Examin-ation H to H V . C-03 CO 03 r-i CO CM CO r-i to 03 \ H rH rH CO 03 H rH o © M -P cd O •s H "in H § H H o 03 o OS-03 0 < 02 Cheese: No. • H * 03 • in • • to to a •H o •p H o ^ » ra to P) rH -ri Pi cd td o 01 to 14 fH 60 • H - H P| •'• cd £d O Pi PiH © -Cl p| p| p| m . p-*H - H »H a ra I—1 0 to to d O f-| Pi g fi fi^ 0 • H r l r l r l <rH O O O t3 +» ?H l(H O *d O U O Pi 01 to to m 3 pi 3 01 O O O rH 60 n O O O O Pi O O O O 0 0 0 0 co 1^ <H II II It ,11 II rH O «aj pq O Pt P£) « 11 m i l k was increased by the a d d i t i o n of yeast e x t r a c t . IMERPRETATIQH' OF TABLES.. . From an examination of Table IV. i t i s to be seen that: ( l ) Cheese 5 , p a s t e u r i z e d to 140°F. f o r t h i r t y minutes, scored h i g h e s t . (2) P a s t e u r i z i n g temperatures had no d i r e c t c o r r e l a t i o n w i t h the f l a v o u r scores of the cheese, due p o s s i b l y to the f a c t that i t was impossible to make a l l the cheese on the same day from the same m i l k . (3) Cheese 4, p a s t e u r i z e d to l60°F. f o r t h i r t y minutes, had a f a u l t y t e x t u r e , t h i s f a u l t y texture may very probably be a t t r i b u t e d to the temperature of p a s t e u r i z a t i o n s i n c e , as i s to be seen i n the making of the cheese, Table 11., the a c t i o n of the rennet was ap p r e c i a b l y delayed, thus a f f e c t i n g the e n t i r e process of manufacture. This i s i n accord w i t h the i n v e s t i g a t i o n s of numerous other workers, who have shown that h i g h temperatures of p a s t e u r i z a t i o n a f f e c t the p h y s i c a l and chemical nature of the m i l k to the detriment of the q u a l -i t y of the r e s u l t i n g cheese. Table V. shows that at t h i r t y days, the organ-isms i s o l a t e d from a l l the cheese were e n t i r e l y coccus types c h i e f l y i n p a i r s ; comparatively high counts were obtained. However, cheese 5 was an exception to t h i s and had, at t h i s age, a lower count per gram, c o n s i s t i n g c h i e f l y of coccus forms i n p a i r s and short chains. Moreover, when the t i t r a t i o n s of the organisms i s o l a t e d from t h i s cheese are considered, i t 12 i s to be seen that a l a r g e r p r o p o r t i o n of organisms % showing the yeast incidence were found. When the work of Sadler and Eagles (10) on the r i p e n i n g of the Kingston Cheese i s consider-ed, the p o s s i b i l i t y suggests I t s e l f that the f i n d i n g of a l a r g e p r o p o r t i o n of organisms, at t h i s age, showing the incidence of yeast e x t r a c t might, i n a measure, account f o r the hig h score. I t i s to he seen from f a b l e Yl» t h a t : (1) She t o t a l count per gram of a l l the cheese, was much l e s s at seventy-three days than i t was at t h i r t y days* (2) At the second examination of the raw m i l k cheese the pre-v a i l i n g type found were rod forms; i n a l l the other cheese, the p r e v a i l i n g type found we re coccus forms i n p a i r s and short chains. (5) Cheese 5, the highest s c o r i n g cheese, i s d i s t i n c t again, both i n the number and types of b a c t e r i a found. Between the t i : time of the f i r s t and second examinations the count per gram had decreased r e l a t i v e l y l e s s than that of any of the other oheese. D i s t i n c t from the p i c t u r e presented i n the case of cheeses Ko Ts 1 , 2,4 , 6 , i t i s to he seen that among the organisms i s o l a t e d from cheese 5 there were found an appreciable number which m o r p h o l o g i c a l l y were lar g e c e l l e d organisms i n long chains, resembling those of commercial s t a r t e r . Moreover, the number of organisms showing the incidence of yeast e x t r a c t has r e l a t i v e l y decreased r a t h e r than Increased as i n the other cheese» We a l s o f i n d i n d i c a t i o n s of the appearance of rod organisms i n t h i s cheese. This data would seem to suggest, that there may he three p o s s i b l e f a c t o r s to one of which may be a t t r i b u t e d the s u p e r i o r i t y of cheese 5 . (1) The d i f f e r e n c e i n the types of coccus forms found at the two examinations. (2) Rod organisms may have been present i n apprec i a b l e numbers, s u f f i c i e n t to have i n f l u e n c e d the r i p e n i n g of the cheese, at the time the cheese were judged, (3) Or a combination of both these f a c t o r s * But i n order to determine to which of these f a c t -ors the s u p e r i o r i t y of cheese 5 i s to be a t t r i b u t e d , i t would be necessary to examine the cheese more f r e q u e n t l y ever a l o n g er p e r i o d of time, to c l a s s i f y the organisms i s o l a t e d , to determine at the var i o u s examinations the amount of p r o t e i n , breakdown I n the cheese and to c o r r e l a t e t h i s w i t h the easein s p l i t t i n g powers of the organisms i s o l a t e d . CONCLUSIONS. The p a s t e u r i z i n g temperature of l60°P. f o r t h i r t y minutes had a decided de t r i m e n t a l e f f e c t on the rennet o l o t of the m i l k and on the texture of the r e s u l t i n g cheese. R o d forms, were the p r e v a i l i n g type at seventy-three days i n cheddar cheese made from raw m i l k . Rod forms were not found to be present at seventy-three days i n cheddar cheese made from m i l k p a s t e u r i z e d to. 150°P. and l60°F. f o r t h i r t y minutes. 14 At k i n s o n , T.H. 1924 The p a s t e u r i z a t i o n of m i l k f o r cheese manufacture. Agr. Gazette of New South Wales, pp. l?8-202. Stevenson, G. 1920 P a s t e u r i z a t i o n i n Cheese Manufacture, pp. 5-9. J r . of A g r i c u l t u r e , New Zealand. Sammis, J.L. and Bruhn, A.T. 1912 The manufacture of cheddar cheese from past-e u r i z e d m i l k . Wisconsin Agr. Exp. S t a . Res. B u i . No. 27. P r i c e , W.V. I927 The manufacture of Cheddar Cheese from m i l k p a s t e u r i z e d by the holder method. C o r n e l l u n i v e r s i t y Agr. Exp. S t a . Mem. 10 5 I927. Report of the Department of S c i e n t i f i c and I n d u s t r i a l Research. D a i r y Re search New Zealand, 1930. M o i r , G..M. 1930 . P a s t e u r i z e d m i l k f o r cheddar cheese-making. Reprint from J r . of D a i r y Research Vol.1,No.2. Evans, A.C., Hastings, E.Y. and Hart, E.B. 1914 B a c t e r i a concerned i n the production of the c h a r a c t e r i s t i c f l a v o u r In cheese of the cheddar type. • J r . Agr. Res. 2, pp. 167-192. 15 (8) Hastings, E.Y. and Sammis, J.L. 1920. Wisconsin Agr. Exp. S t a . B u i . 319. (9) F a n f i l o v , S.B. 1928. (10) E a g l e s , B.A. and S a d l e r , W i l f r i d . Cheese Ripening S t u d i e s . Canadian Journal o f Research, 1933. 16 AQmOWLETJOMEETS . We wish to express our a p p r e c i a t i o n f o r the guidance and a s s i s t a n c e of the l a t e P r o f e s s o r W i l f r i d S a d l e r , of the Department of D a i r y i n g , U n i v e r s i t y of B r i t i s h Columbia. We are indebted a l s o , to Dr. ff.S* Golding, f o r advise and a s s i s t a n c e i n the experimental work and to Dr<> B.A. Eagles f o r h i s c o n t r i b u t i o n s i n the arrangement of the m a t e r i a l and the p r e p a r a t i o n of the paper. _Oho.eBiL.i_ . Made 15/. "-Examined . ( R a u J l l .l/S •Age SO d iys -Ripened- r: .4~a£yB i t-61 and zema.naeJ at]<50°$ - H e a i a - P - I L G . Inoxibate 1 21 Oourits: flll. a t ten t p la te koMa. Jjfto _ m i l k . Init iba f - ransferted __tubes of I m i l l m i l k f o r Inc-ubatei a t I / f >o,o_d Cul tu ro Ho £7 it- sd 0 1') oe . a n d yeAst Tat^a-tTirTr ay_7 " X T .itmils " 0 . ?n M i l k 6& ?0 0:7 |aifc a-7 £ _£6_ £ / '.L^ L ££ 706 O-i.[\77 0'7 At •ex. ill 5 ^ .fea-ture :JTo / / : HUM I-ilJ <?7 l / - r £_5-l ?± i l •72 777 «7s 1177 7 7 r t \//<i *7o IL 7Z if. if f . - S 'c-i i i i ii_L. ! i t . i f l i £_>_. ______ < £ l / £ j£ £_ __95_ ?? 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