363

TECHNICAL NOTES MAINTENANCE OF ACTIVE LACTIC CULTURES BY FREEZING AS AN ALTERNATIVE TO DAILY TRANSFER Daily p r o p a g a t i o n of lactic cultures involves considerable expenditure of time, media, and materials, and requires careful technique to avoid contamination during repeated transfers. F u r t h e r m o r e , the desirable balance of strains generally present in a freshly combined culture f r e q u e n t l y is lost a f t e r several propagations. The preservation of an active culture in its optimum condition f o r flavor and acid production has been attempted by Johns (3), Olson (5), and H e i n e m a n (1). This report deals with the technique studied by the writers in a similar attempt to maintain lactic cultures at optimmn activity without daily transfers. Cultures were p r e p a r e d by inoculating a sterile milk medium at the rate of 1 % with freshly ripened active starter. The inoculated milk was placed at once in an ice c r e a m - h a r d ening room at --20 ° F. A f t e r storage f o r up to 6 too., the previously inoculated milk was thawed in about 2 hr. in a water bath at 70 ° F., then incubated f o r 16 hr. at 70 ° F. Table 1 shows that Culture B, frozen in this lnanner and tested at monthly intervals, maintained practically the same activity during storage for 6 too. Cultures f r o m f o u r commercial sources were tested similarly and responded equally well a f t e r storage at --20 ° F. Table 2 emphasizes the need for freezing the culture medium soon a f t e r inoculation, as incubation for more than 6 hr. before freezing resulted in a less active culture a f t e r storage. V a r y i n g the level of inoeulum fronl 1 to 10% did not influence activity. Most modifications of milk media normally used for culture maintenanee were suitable for the frozen cultures. F r e s h fluid skimmilk plus 2 % dry skimmilk appeared slightly s u p e r i o r in these trials. The

cultures were frozen in 16-oz., wide-mouthed, Nalgene H H plastic containers. 1 This container was used repeatedly f o r sterilizing milk at 15 lb. steam pressure for 15 miu., without severely d a m a g i n g the c o n t a i n e r . Freezing, thawing, and subsequent incubation were performed in the same plastic container. Cultures p r e p a r e d as described above were supplied to a local commercial dairy, where they were used to inoculate bulk starter. B u t termilk was made successfully five times weekly over a period of 3 too., using a frozen culture to inoculate each bulk starter. F r o z e n cultures also were used in this m a n n e r f o r processing buttermilk and cottage cheese in the Clemson College Dairy. The activity of the frozen cultures compared f a v o r a b l y with fresh starters t r a n s f e r r e d six days per week. P r e l i m i n a r y trials i n d i c a t e d that several types of p a p e r milk cartons were suitable f o r the freezing, thawing, and incubation steps. I n these trials, commercially pasteurized and eartoned milk was used. Since serial t r a n s f e r s are eliminated in this system, it is felt that pasteurized r a t h e r t h a n sterilized milk can be used. B y inocuIating carefully pasteurized milk before filling the cartons, it should be practicable to p r e p a r e at one time enough cultures for 3-6 too. of operation. J. C. SIMMONS 2 D. M. GRAHAM s South Carolina A g r i c u l t u r a l E x p e r i m e n t Station, Clemson Made by the Nalge Company, Inc., Rochester, New York. '~Dairy Industry Supply Association Fellow. * Present address: Pet Milk Company, Research Laboratory, Greenville, Illinois.

TABLE 1 Culture B tested at monthly intervals during storage at --20 ° F. Storage time

Developed acidity

(qno.) 0 1 2 3 4 5 6

(%) 0.90 0.60 0.75 0.77 0.82 0.85 0.78

pH

Leber's test (4)

Elliker's Test (2)

Creatine Test ~

4.50 4.45 4.60 4.50 4.50 4.50 4.55

(~nin.) 30 30 30 30 30 30 30

( % acidity) 0.47 0.34 0.41 0.36 0.41 0.36 0.38

++ +++ +++ + + + +

. . . . indicates intense, "+ moderate, + slight, and - absence of red color. 363

Aroma Good Good Good Good Good Good Good

JOURNAL

364

OF D A I R Y

SCIENCE

TABLE 2 (h~lture A incubated for different periods of time before and after storage at --20 ° F. Incubation time (hr.ff Before freezing 0 2 4 ~ 8 1 (l 12 14 1 (i

After freezing

Total time

Developed acidity

16 16 1 (; l6 l (; 16 16 16 16

(%) 0.88 0.89 1.17 1.1.6 0.fi4 0.5!) 0,89 0.91 0.90

16 14 ]2 l0 8 6 4 2 0

pH

Leber's Test

Elliker's Test

4.60 4.65 4.65 4.50 4.95 4.90 4.65 4.60 4.70

Onin.) 30 30 30 30 45 60 45 60 45

( % acidity) 0.44 0.39 0.45 0.40 0.30 0.25 0.29 0.29 0.30

Creatine Test ~ ~+ ~~ + *+ + ~+ ++ ~"

Aroma Excellent Excellent Good Good iOair FMr Fair Fair Excellent

" A f t e r thawing the cultures which were incubated less than ] 6 hr. before freezing, incubation was resumed for the interval necessary to give a total incubation time of 16 hr. " See footnote ", Table 1. R E'F]'TR E N C E S (1) IIEINEMAN, P . ]?reserving the A c t i v i t y of l,actic (!u|turcs. d. l)air:q Sci., 41: 705. 195S. (2) lIORItAI,h, B. E., AND i']LI,IKEIt, P. IL A c t i v i t y T e s t for (~ht,ihlar a n d C o t t a g e Cheese Starters. J. Dairy Sci., 30: 523. 1!147. (3) ,braNS, C. K. P r e s e r v i n g the A c t i v i t y of

ABSORPTION

OF

COLOSTRUM

D1RECTLY

INTO

It has h m g been recognized t h a t t h e r e is intestimfl a b s o r p t i o n or' i m m u n e globulins f r o m the c . l o s t r u n , by the n e w b o r n calf f o r a limited time a f t e r birth. M a s o n et al. (4) suggested t h a t the i n t e s t i n a l a b s o r p t i o n of globulins b y the n e w b o r n m a y be the i n c a p a c i t y f o r digestion, or t h a t the g u t of the n e w b o r n is more p e r m e a b l e at b i r t h t h a n later, or both. Hill (2) suggested t h a t the cessation of int e s t i n ' d a b s o r p t i o n of i m m u n e p r o t e i n s f r o m colostrum by the n e w b o r n of several species coincides with the d e v e l o p m e n t of g a s t r i c p r o t e i n digestion. One e x p l a n a t i o n of this r e l a t i o n s h i p is t h a t a f t e r g a s t r i c digestion is i n i t i a t e d eolost r m n p r o t e i n s are digested. Colostrum contains an antitryptie factor that normally may p r o t e c t the p r o t e i n s f r o m p a n c r e a t i c proteases (3). Dcutseh a n d S m i t h (2) fed a n a l u m i n u m h y d r o x i d e gel a n d p r o b a n t h i n e to a c a l f f o r 32 hr. p o s t p a r t u m , to i n h i b i t g a s t r i c activity. A test g l o b u l i n was f e d a t 40 hr., b u t t h e r e was no a b s o r p t i o n of the globulin. H o w e v e r , no test was m a d e to d e t e r m i n e w h e t h e r t h e r e was dim i n i s h e d g a s t r i c activity. This s t u d y was u n d e r t a k e n to a s c e r t a i n the intestinal permeability to i m m u n e p r o t e i n s f r o m eolostrmn i n t r o d u c e d d i r e c t l y into the d u o d e n u m of ealves v a r y i n g i n age. H o l s t e i n calves were d e p r i v e d of eolostrum

Frozen Chccse Starter Cultures. Camulian Dairy a'ml lee Cream J., 35: 32. 1956. (4) IA,;tu,;u, H. A Rea:v/an'in Starter Activity Test. Milk Planl Monlhly, 39: 4(). 3950. (5) ()I,SON, lI. C. Influence of Frequency of Transfer and of lncub:ltion Period on the Activity of Imctic Cultures. ,I. Dairy Sei., 41: 346. 1958.

(ILOt{I;LINS TIlE

INTR()I)UCED

I)I~OI)ENUM

a n d m a i n t a i n e d on m a t u r e milk p r i o r to t r e a t ment. Age difference, served as the e x p e r i m e n tal variable. The calw~s were anesthetized with sodium p e n t a b a r b i t a l a n d the intestines exposed. The d u o d e n u m was ligated a p p r o x i m a t e l y 3 in. p o s t e r i o r to the pyloric sphincter. A b o u t 1.5 liters of colostrum were i n t r o d u c e d into the i n t e s t i n e p o s t e r i o r to the ligature via a p o l y e t h y l e n e tube. T h e n the d u o d e n u m was ]igated p o s t e r i o r to the p o i n t where colostrum was introduced, a n d the incision was closed b y s u t u r i n g the p e r i t o n e u m , muscles, a n d skin. Blood was w i t h d r a w n f r o m all calves p r i o r to a n d a t 4, 8, a n d 12 hr. f o l l o w i n g the introduction of colostrum. All s e r u m samples (0.01 ml.) were s u b j e c t e d to d u p l i c a t e electrophoretie f r a c t i o n a t i o n s in Spineo Model R B electrophoresis a p p a r a t u s on p a p e r s t r i p s in sodium veronal troffer ( p H 8.6, ~/ = 0.075) f o r 16 hr. The ages of the v a r i o u s calves ~t the time of i n f u s i o n w e r e : ( n ) 6 hr., ( B ) 18 hr., (C) 48-60 hr., (D) 48--60 hr., a n d ( E ) 48-60 hr. The operation a n d colostrum i n f u s i o n of Calf E was perf o r m e d w i t h o u t anesthesia. E l e e t r o p h o r e t i e p a t t e r n s of the blood serum p r o t e i n s before a n d a f t e r the i n t r o d u c t i o n of eolostrum into the d u o d e n u m of Calves A a n d E are s h o w n on the l e f t a n d right, respectively, of F i g u r e 1. S e r u m p r o t e i n p a t t e r n s of Calves