a study of the factors that influence the

8/7/2019 A STUDY OF THE FACTORS THAT INFLUENCE THE

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A S T U D Y O F T H E F A C T O R S T H A T I N F L U E N C E T H EC O A G U L A T I O N O F M I L K I N T H E

A L C O H O L T E S T I

H. H. SOMMER Ann T. H. BIN NEY ~Department of Dairy Husban dry, College of Ag riculture, University of WisconsinMadison, Wisconsin

Received for publ icati on July 1, 1922T h e a l c o ho l te s t i s b e i n g u s e d t o s o m e e x t e n t a s a t e s t f o r

g r a d i n g m i l k . T h e t e s t is u s u a l l y m a d e b y a d d i n g 2 c c. o f m i l kt o 2 c c. o f a l c o h o l i n a t e s t t u b e . T h e t e s t t u b e is t h e n c l o s e dw i t h t h e t h u m b , in v e r t e d s e v e ra l t i m e s a n d e x a m i n e d . I f n op r e c i p i t a t e is fo r m e d t h e t e s t i s sa i d t o b e n e g a t i v e , a n d i f ap r e c i p i t a t e i s f o r m e d t h e t e s t i s s a i d t o b e p o s i t iv e . T h e s t r e n g t ho f t h e a l c oh o l u s e d i s u s u a l ly 6 8 o r 7 0 p e r c e n t b y v o l u m e .

W h e n t h e t e s t f i r s t c a m e i n t o u s e i n 1 8 9 0 i t w a s r e g a r d e dl a rg e l y a s a m e a s u r e o f a c i d i t y . H o w e v e r i t w a s s o o n f o u n dt h a t f r e s h m i l k f r o m i n d i v i d u a l c o w s f r e q u e n t l y c o a g u l a t e s a n dt h a t t h e t e s t w a s d e p e n d e n t u p o n o t h e r f a c to r s a s w e ll a s u p o na c i d i t y . N e v e r t h e l e s s t h e t e s t w a s s ti ll u s e d fo r i t w a s t h o u g h tt h a t b e s i d e s a c i d i t y i t d e t e c t e d o t h e r u n d e s i r a b l e c h a r a c t e r i s t i c s .T h u s E r n s t (1 ) b e l i e v e d t h a t a p o s i t iv e t e s t o f f r e sh m i l k w a sd u e t o i r r i t a t i o n o f t h e m i l k gl a n d s. R u h m (2 ), R u l l m a n a n dT r o m m s d o r f (3 ), a n d C a m p b e l l ( 4) c l a i m e d t h a t a p o s i ti v e te s tw a s d u e t o i n f e c t e d o r d i s e a s e d u d d e r s . M e t z g e r ( 5) f o u n dt h a t i n f e c t i o n o f t h e u t e r u s a n d a n i m a l s l e a n f r o m d i se a s e c a u se da p o s i t i v e t e s t . A u z i n g e r (6 ) s t a t e d t h a t m i l k f r o m c o w s w i t hi n f e c te d v a g i n a s a n d c o w s t h a t h a d a b o r t e d w a s p o s it i v e t o t h et e s t . H e n k e l (7 ), M e t z g e r (5 ) a n d A u z i n g e r (6 ) f o u n d t h a tc o l o s t r u m m i l k i n f l u e n c e d t h e t e s t .

t Published with permission of the Director of Wisconsin Agricultural Experi-ment Station.2 The work presented in this paper c onsti tutes the thesis of T. H. Binney,submitt ed in parti al fulfillment of the requir ements for the degree of Master ofSc i enc e in the graduate school of the Unive rsit y of Wisconsin, 1922.176


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C O A G U L A T I O N O F M I L K IN A L C O H O L T E S T 1 7 7

T h e s e v a r i o u s c a u s e s l i s te d i n t h e l i te r a t u r e a r e o n l y r e m o t ec a u s e s , t h e y a r e o p e r a t i v e o n l y in s o f a r a s t h e y a f f e ct t h e c h e m i -c a l c o m p o s i t i o n a n d p r o p e r t i e s o f t h e m i l k . A s f a r a s w e a r ea w a r e t h e o n l y o t h e r d i r e c t c a u s e s m e n t i o n e d b e s i de s a c i d i t y a r et h e e ff e ct o f r e n n e t f o r m i n g o r g a n is m s s t u d i e d b y A y e r s a n dJ o h n s o n ( 8) , a n d t h e e f f e c t of t h e m i l k s a lt s, e s p e c i a l ly c a l c iu m ,o b s e r v e d b y A u z i n g e r ( 6 ).

T h u s i t is a p p a r e n t t h a t o u r k n o w l e d g e of t h e d i r e c t c a u se s fo rt h e a l c o h o l c o a g u l a t i o n i s i n c o m p l e t e . I t i s t h e p u r p o s e o f t h ew o r k r e p o r t e d i n t h i s p a p e r t o d e t e r m i n e t h e f a c to r s i n fl u en c i ngt h e a l c o h o l t e s t a n d t o s t u d y t h e i r r e la t iv e i m p o r t a n c e .

METHOD USED

I n t h is w o r k b o t h 7 0 a n d 7 5 p e r c e n t a l c o h o l b y v o l u m e w e r eu s e d . T h e a l c o h o l u s e d w a s d is t il l e d i n th e p r e s e n c e o f s o d i u mh y d r o x i d e t o r e m o v e a n y a c id w h i c h m i g h t b e p r e s e n t. T h i sd i s ti ll e d al c o h o l w a s t h e n a d j u s t e d t o t h e d e s i r e d s t r e n g t h b yt h e a d d i t io n o f d i st il le d w a t e r . T h e s t r e n g t h w a s d e t e r m i n e db y m e a n s o f t h e W e s t p h a l b a l a n c e . T h e 7 0 p e r c e n t a l c o h o l b yv o l u m e w a s a d j u s t e d t o a s p e c if ic g r a v i t y o f 0. 89 0 0 a t 1 5 C .a n d t h e 7 5 p e r c e n t a l c o h o l b y v o l u m e t o a sp e c if ic g r a v i t y o f0 .8 7 7 3 .

I n e a c h c a s e , e x ce p t w h e r e o t h e rw i s e i n d i c a t e d t h r o u g h o u tt h i s w o r k , 2 c c . o f m i l k w e r e a d d e d t o 2 c c . o f a l c o h o l i n a t e s tt u b e . T h e te s t tu b e w a s t h e n c lo s e d w i t h t h e t h u m b , i n v e r t e ds e v e r a l t im e s a n d e x a m i n e d f o r t h e a p p e a r a n c e o f t h e p r e c i p i ta t e .T h e m i x t u re s w e r e th e n v e r y c a r e f u ll y e x a m i n e d a n d o n t h ea p p e a r a n c e o f t h e s l i g h t e s t p r e c i p i t a t e t h e t e s t s w e r e c a ll e dp o s i ti v e . A n y a d d i t i o n a l m e t h o d s u s e d i n th i s w o r k w il l b ee x p l a i n e d w h e n t h e e x p e r i m e n t s i n w h i c h t h e y w e r e u s e d a r ed i s c u s s e d .

THE E FFEC T OF MILK SALTS

I t i s a w e l l k n o w n f a c t t h a t e l e c t r o ly t e s h a v e a v e r y m a r k e de f f e c t o n t h e s t a b i l i t y o f c o l l o i d s , e s p e c i a l l y e l e c t r o l y t e s t h a ty i e l d d o u b l y a n d t r i p l y c h a r g e d i o n s o n d i s so c i a ti o n . I n t h ec a s e o f m i l k w e a r e d e a l i n g w i t h a c o l l o id a l s o l u t i o n o f a l b u m i n


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178 H. H. SOMMER AND T. H. BIN NEYand casein in the presence of the milk salts containing the biva-lent calcium and magnesium and the trivalent phosphate andcitrate radicles. We would expect, then, that the stability ofthe milk proteins would be influenced by the amounts of thesesalts present. Van Slyke and Hart (9) and Van Slyke andBosworth (10) actually found in working with calcium com-pounds of casein that the presence of small amounts of solublecalcium salts have a very marked effect upon the solubility ofthe casein compounds.More recently it has been shown by Sommer and Hart (11)tha t the calcium and magnesium and the citrates and phosphatespresent in the milk have a very marked effect upon the heatcoagulation of milk, as in the sterilization of evaporated milk.It was shown that there is a balancing effect between calciumand magnesium on the one hand, and the citrates and the phos-phates on the other, the proper balance of these producing themost stable condition, an excess of either of these causing themilk to coagulate more readily on heating.That the milk salts are also of importance in the alcohol testwas indicated by the work of Auzinger (6). Working with themilk from three cows he was able to change the alcohol test fromnegative to positive by feeding each cow 120 grams of calciumphosphate per day. However, other investigators have con-tinued to give other reasons for the test and as a result haveeither contradicted or obscured Auzinger's conclusions.In order to determine experimental ly what effect the variousmilk salts have on the alcohol test they were added to milk invarying amounts and the alcohol test applied. Fourth-molarsolutions of calcium acetate, magnesium chloride, potassiumchloride, di-potassium phosphate and sodium citrate were pre-pared and added to 25 cc. of fresh mill~ in a series, increasing theamounts of the salt solution by 0.1 cc. In each series the dilu-tion was equalized by the addition of the proper amount ofdistilled water. The results are given in table 1. Table 1shows that 0.2 cc. of ~/4 calcium acetate and 0.1 cc. of ~/4magnesium chloride added to 25 cc. of fresh milk was sufficientto cause a positive reaction with the alcohol test. These addi-


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C O A G U L A T I O N O F M I L K I N A L C O H O L T E S T 179t i o n s a r e e q u i v a l e n t t o a n i n c r e a s e o f 0 .0 1 1 2 a n d 0 .0 0 4 0 p e r c e n tC a O a n d M g O , r e s p e c t i v e l y . A s h i g h a s 0 .9 c c . o f M / 4 p o t a s s i u mc h lo r id e , d i -p o t a s s i u m p h o s p h a t e , a n d s o d i u m c i t ra t e w e r ea d d e d t o 2 5 c c . of m i l k a n d t h e m i l k s ti ll g a v e a n e g a t i v e t e s tw i t h a l c o h o l .

T h e r e s u l ts s h o w t h a t a s l i g h t i n c re a s e in t h e c a l c i u m o r m a g -n e s i u m c o n t e n t o f t h e m i l k w il l c a u s e a p o s i t iv e r e a c t io n w i t h t h ea l c o h o l t e s t , w h i l e a n i n c r e a s e o f p o t a s s i u m , s o d i u m c h l o r i d e ,c i t ra t e a n d p h o s p h a t e w i l l n o t c a u s e a p o s i t i v e t e s t .

T A B L E 1Effect of salts on alcohol t e s t

I W A T E R , (X ~. . . .. )5 CC. MILK PLU S ~SOLU'PION, CC.

~ / 4 c a l c i u m a c e t a t e . . . {~ / 4 m a g n e s i u m { - -c h l o r i d e . . .

z / 4 p o t a s s i u m c h lo r id e { - / 4 d i - p o t a s s i u m I -p h o s p h a t e .~ / 4 s o d i u m c i t r a t e . . . . {

0.90.0

0.8 0.70.I 0.2- +

+ +- +

0.6 0.50.3 0.4+ ++ ++ ++ +

0.4 0.30.5 0.6+ ++ ++ ++ +

0.2 0.I0.7 0.8+ ++ ++ ++ +

0 . 0 S T R E N G T HO F A L C O H O lP E R C E N T0 . 9 B Y Y O L U M ]

+ 7 5+ 7 0

+ 7 5+ 7 0

7 57 0

7 57 0

7 57 0

* P l u s a n d m i n u s m g n s i n d i c a t e p o s i t iv e a n d n e g a t i v e a l c o h o l t e s t s r e s p e c -t i v e l y , u n d e r t h e c o n d i t i o n s i n d i c a t e d i n t h e t a b l e .

S A L T B A L A N C EI n o r d e r t o d e t e r m i n e w h e t h e r t h e e f f ec ts of th e m i l k s a l ts

o n t h e a l c o h o l t e s t c o u n t e r a c t e a c h o t h e r i n a m a n n e r s im i l a r t ot h e s a l t b a l a n c e d e m o n s t r a t e d i n t h e h e a t c o a g u l a t io n , t h e e ff ec to f v a r i o u s c o m b i n a t i o n s o f sa l ts w a s s t u d i e d .

T a b l e 2 s h o w s t h e r e s u l t s o f t h e a l c o h o l t e s t o n t h r e e d i f f e r e n tc o m b i n a t i o n s of s a lt s i n v a r y i n g p ro p o r t io n s . U s i n g th e s a m ef r e s h m i l k t h r o u g h o u t t h e e x p e r i m e n t t h r e e s er ie s of s a m p l e s


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180 H . H . S O M M E R A N D T . H . B I N N E Yw e r e p r e p a r e d . E a c h s e r ie s c o n s i s t e d o f t e n p o r t i o n s o f 2 5c u b i c c e n t i m e t e r s e a c h . T o e a c h o f t h e s a m p l e s i n a l l t h r e es e ri es 0 . 5 e e . o f ~ / 4 c a l c iu m a c e t a t e w a s a d d e d . T h i s w a ss u f fi c ie n t t o m a k e th e a l c o h o l t e s t d i s t i n c t l y p o s i ti v e . T o t h e s et h r e e s er ie s M / 4 d i- p o t a s s iu m p h o s p h a t e , p o t a s s i u m chloride,

T A B L E 2T h e e f fec t of s (~l ts on the a lcohol t e s t

t M / c ~ A ~ E T A T E ,T I O N , ( ~ . . . . .

~ / 4 p o t a s s i u m {p h o s p h a t e .

M / 4 p o t a s s i u m c h l o r i d e (

M / 4 s o d i u m c i t r a t e . . . {

0.5 0 .5 0 .50 .1 0 .2 0 .3+* + ++ + +

++ + ++ + +

0 .50 .4

0 .50 .5+

0 .50 .6

0 .5 0 .50 .9 1 .0

0 .5 0 .50 .7 0 .8

+ ++ +

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

S T R E N G T HO F A L C O H O lB Y Y O L U M I

757075707570

* S e e t a b l e 1 .TABLE 3

T h e e f f e c t o f sa l t s on t he a l c oho l t e s tM / 4 M g C H L O -

.)~ C O . M I ~ K P I OU S R I D E , C C . . . .Iz~14 SOLU-TIO1V,C C . . . .~/4 potassium chloride(~/4 potassiumphosphate .t~ / 4 s od ium c it ra t e . . . . {

0.5 0 .5

0. 1 0.2+* ++ ++ ++ ++ ++ +

0 .5

0. 3++++++

0.5 0 .5

0 .4 0 .5+ ++ ++ ++ ++

0 .6 0 .5 0 .5 0 .5 0 .5

0 .6 0 .7 0 .8 0 .9 1 .0+ + + + ++ + + + ++ ++

S T R E N G T ] ~O F A L C O H O '

P E R C E N TB Y V O L U M |

757075707570

* S e e t a b l e .and sodium citra te, r e s p e c t i v e l y , w e r e added in amounts rangingfro m 0.1 to 1.0 cc. On applyin g the alcohol test it was foun dth at the coagul ation caused by t he 0.5 cc. of M/4 calcium acetat ewas prevented by 0.5 cc. of 5/4 sodium citrate and by 0.6 cc.


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COAGULATION OF MILK IN ALCOHOL TEST 181of di-potassium phosphate. The potassium chloride did notcounteract the effect of the calcium salt.Table 3 shows the results of the alcohol test on three seriessimilar to those in table 2. Instead of using calcium aceta te tocause the milk to become distinctly positive, M/4 magnesiumchloride was used in table 3. It was found that the coagulationcaused by the 0.5 cc. of M/4 magnesium chloride was preventedby 0.8 cc. of M/4 di-potassium phosphate, and 0.5 cc. of M/4sodium citrate, respectively. The potassium chloride did notprevent the coagulation caused by the magnesium chloride.

These experiments demonstrate that the salt composition ofthe milk has a marked effect upon the alcohol coagulation. Smallincreases in the amount of CaO (0.0112 per cent) and MgO(0.0040 per cent) cause a positive alcohol test. The effect ofthe calcium and magnesium is counteracted by the citrates andphosphates in the milk, so that a positive test with fresh milkdepends upon the relative amounts of these four salts presentin it.The importance of the salt composition of the milk as a factorinfluencing the alcohol coagulation becomes more apparentwhen we compare the amounts of the salts that will produce aneffect on the coagulation with the amounts of these salts normal lypresent in milk. In the above experiments it was demonstratedthat an increase of 0.0112 per cent in the CaO content changeda negative sample to a positive one. Compare this with thenormal differences that are found between samples of milk,Sommer and Hart (11) in an analysis of 30 different samplesfound the CaO content to vary from 0.132 to 0.210 per cent.

FEEDING EXPERIMENT

The effect of the salts on the alcohol coagulation has thus farbeen demonstrated entirely by their addition to various samplesof milk. The additions of salts which were found effective werevery small, well within the limits of normal variation of thesecompounds in milk. In spite of these facts there might be doubtin the minds of some people because these salts were added andwere not secreted by the cow as a part of the milk. In order to


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182 H. H. SOMMER AND T. H. BIN NEYovercome this objection a feeding experiment was conducted todetermine what effect the feeding of calcium would have on thealcohol test.Three heavy producing cows in the middle of the lactationperiod were selected for the experiment. The time of the experi-ment was divided into four periods. The first or pre-periodwas an observation period to determine how far from the coagu-lating point the milk of the animals was, and to note fluctuationsand irregularities in the test. During the second period whichwas from the eighth to the fourteenth day, 50 grams of calciumcarbonate was fed to each animal per day. The third period,from the fourteenth to the twenty-second day, the animals werefed 100 grams of calcium carbonate per day. During the fourthperiod, from the twenty-second to the twenty-eighth day, eachanimal was fed 200 grams of calcium carbonate per day. Thefifth period immediately followed the feeding of the last of thecalcium carbonate, up to the time the milk reached the samecondition as in the pre-period. The basal ration during theentire experiment consisted of 14 to 16 pounds of grain mixture(500 pounds corn; 200 pounds oats; 200 pounds bran; 100 poundsoil meal) ; 10 pounds of alfalfa ha y and 30 pounds of corn silage.On the evening of the twenty-fourth day the cows refused toeat all of the calcium carbonate . The next day only 100 gramswere fed. On the twenty-sixth day 200 grams were again fedand the feeding continued without any changes in amounts.On the basis of the preceding demonstration th at the milk saltsare an important factor in the alcohol coagulation, an attemptwas made to measure the extent to which a sample was positiveor negative. If a sample was negat ive 5 cc. of milk was takenand ~/20 calcium acetate was added in amounts just sufficientto cause a positive test. This amount is indicated in column 3of table 4. Similarly, if the sample was positive 5 cc. of milk wastaken and M/20 potassium phosphate was added in amountsjust sufficient to cause a negat ive test. This amount is re-corded in column 4 of table 4. The results are more strikinglypictured in graphs 1, 2 and 3.


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TABLE 4Ef f e c t o f f e e d i ng c a l c i um c arbonate on t he al cohol t e s t

D A T E

"8

C O W

Alice Bud Doll

r . ) o

,,

a study of the factors that influence the

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