Oct., 1916
T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
capillary tubes closed a t t h e t o p a n d projecting above t h e liquid surface are placed in B . I n order t o give a n idea of t h e efficiency of this apparatus, t h e following may be s t a t e d : Redistilled alcohol was p u t in B a n d a mixture of I O cc. alcohol a n d 240 cc. water was boiled i n t h e flask; when I O cc. of distillate h a d been collected, t h e operation was stopped. T h e distillate proved t o be 95.6per cent alcohol a n d there was no alcohol in t h e residual liquid i n t h e flask. A mixture of 180 cc. aromatic sulfuric acid a n d 580 cc. water was placed in t h e flask a n d boiled. Ordinary alcohol was placed in B . At t h e e n d of each halfhour t h e thermometer readings a n d t h e volume of t h e distillate were noted, after which 5 cc. of t h e mixt u r e were withdrawn from t h e flask, cooled, diluted a n d titrated, while t h e distillation was resumed. I n Table X are given t h e results. T h e respective weights TABLEX Time Hrs. 0.0 0.5 1 .o 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5
Distil- Thermom- Reading late eter in in boiling Cc. vapor liquid 789 0.0 89.10’ 78 43.50 91.40 78 80.00 93.50 78 109.20 95.90 78 129.60 97.90 78 141.80 99.30 78 148.70 99.80 78 152.20 100.10 74 154.30 100.30 69 155.80 100.50 60 in0.70 156.30 60 156.90 100.85 50 157.20 101 . O O 48 157.50 101.15 48 101.30 157.70 i57.8n 48 101.45 158.40 48 101.70 158.80 48 101.85 101.90 159.00 48 102,05 159.00 48 41 102.10 159.00 40 102.10 159.00
Ratio of Weights of HzS04 Free to Total in 2 cc. of Liquid HnSO, Free Combined Per cent 0.0488 0.0343 79.35 0.0532 0,0328 79.51 0.0593 0.0314 82.70 0.0652 0.0304 84.10 0.0721 0.0255 86.93 0,0758 0.0231 ’ 88.34 0.0821 n.0181 91.44 0.0870 0.0152 92.56 0.0888 0.0147 92.89 0.0118 0.0917 94.31 n.. 0093 95 0.0939 ~ . . . .. 49 .. 0.0956 0,0088 95.77 0.0964 0.0073 96.46 0.0991 0.0050 97.64 0.1003 0.0034 98.34 0.1005 0.0029 98.58 0,1010 0.0019 99.05 0.1020 0.0015 99.52 0.1033 0.0003 99.86 0.1040 0,0000 100.00 0.0000 0.1040 100.00 0.0000 100.00 0.1040
of “free H2S04”a n d “combined H2S04”were obtained in this way: t h e j cc. of liquid withdrawn from t h e flask were made u p t o 2 5 cc. with water; of this dilute solution I O cc. were titrated a t once a n d I O cc. were completely hydrolyzed in a n open beaker on t h e water b a t h a n d t h e n titrated. F r o m t h e d a t a t h u s secured t h e amount of free sulfuric acid a n d t h a t of t h e ethylsulfuric acid present in each 2 cc. of t h e liquid i n t h e flask are calculated. It will be noted t h a t throughout t h e distillation t h e temperature of t h e vapor in t h e side-tube C never rose above 7 8 ” ; after t h e bulk of t h e alcohol h a d been removed, t h e reading of this thermometer slowly fell, since t h e q u a n t i t y of vapor passing through C became less a n d less a n d t h e loss of heat through radiation from t h e thermometer bulb was not compensated for. At t h e same time t h e temperature of t h e boiling liquid slowly rose as alcohol was distilled off. T h e s u m of t h e “free” a n d t h e “combined” acid also slowly increases, for t h e percentage of total acid in t h e boiling liquid rises as t h e alcohol is removed. Finally, we may note t h a t under t h e conditions of t h e experiment over 9 hrs.’ boiling was needed for t h e complete hydrolysis of t h e ethyl-sulfuric acid present. LABORATORYOFTHB MARYLANDSTATE DEPARTMENT O F HEALTH, BALTIMORE
RENNIN-A
909
NOTE AND A CORRECTION By HOWARDT. GRABER Received May 27, 1916
Rennin is not official in t h e sense t h a t i t is recognized b y either t h e United States Pharmacopoeia or t h e National Formulary, hence there is a wide variation in t h e article as found upon t h e market, as well as i n t h e methods used for its standardization. I t is universally recogniFed as a milk coagulant a n d all methods use milk as a medium for standardization. I n 1911 I published an article’ entitled “Some Observations upon t h e Assay of Digestive Ferments.” I n this article I included t h e assay used b y me in t h e standardization of rennin. There was a n error in this script which has since been corrected. This error t o which I want t o call especial attention was i n t h e amount of milk specified t o be used for t h e test. I t was just twice t h e volume intended, so t h a t t h e results b y this error were just half what t h e y should be, or in other words t h e rennin seemed t o be just half its real strength. I have tried other methods proposed, b u t I recommend t h e method as published b y me both for its simplicity of operation a n d t h e reliability of t h e results obtained when one uses a standard of known value as control. T h e enzyme rennin, like all other enzymes, is distinctly specific in its reaction a n d it shows its selective characteristics as t o t h e nature of t h e proteid, t e m perature a n d reaction of t h e media as well as t h e presence or absence of organic a n d inorganic salts, especially those of calcium a n d t h e phosphates. Results often vary when samples of milk are used from different cows, as well as different lots from t h e s a m e cow, a n d t h e best results are obtained when using a mixt u r e from t h e milking of many cows. There also seems t o be a seasonal variation in t h e ability of t h e milk t o coagulate. T h e following table gives t h e results of many of t h e tests: DATE 4/ 1 to 4/ 15/ 14 8/ 7/14 8
10 15 9/ 1/14 1/24/15 21 2/16/15 22 3/ 8/15 31 5 / 5/15 13 17 27 29 30 6/ !/I5 1
12 17 22 22 29 7 / 2/15 7 9
IO 21 22
MONTHLY ASSAY AVERAGE 1 : 30,000 in Min. Sec. Min. 7.5 10
io
7
7 9 16 11 8
30 30 30 30 30 30
10 10 11
8 11 11 10 10 10 9 11 8 10 12 12 11 8 8 8 8 12 10
9 9 13.75 9.25 11
30 10
7 10.3
45 45 10 5 30 15
10.6
30 45 45
DATE 11/ 5/15 12/ 3/15 22 23 24 27 28 29 30 31 1/ 3/16 4 5 6
9.3
8 2/14/16 3/ 6/16 7 8 9 10 11 12 14 23 24 28 29 30 31
MONTHLY ASSAY AVERAGE 1 : 30,000 in Min. Sec. Min. 10 30 10 30 11 30 8
16 13 7 11 11 11 10 11 10 11 11 10 11 10 9
7
7 5 9 9 8 1;
30 30
10.5
30 30
10.5
11 45 30 30 30 30 30
7.5
15
4
6 4 7
Several years ago I selected a rennin t o be used as a standard in t h e assay of this enzyme, a n d I have 1
THISJOURNAL,3 (1911), 919.
910
T H E J O l r R N A L O F I N D L ‘ S T R I A L A N D E~TGINEERIiVG C H E M I S T R Y
followed it quite consistently ever since. I n t h e spring of t h e year 1914i t was tested for a period of 14 days and was found t o assay I : 30,000 in 7 1 / 2 min. and was accepted as such. I n every assay made since then it has been run as a check upon t h e unknown sample. There are 74 assays here enumerated extending over a period of about z years. ,4 series of 14 assays min.; a second series of in April, 1914,averaging 14 in December, 1915,and January, 1916,averaging 101/2 min.. and a third series of 14 assays in March, 1916,averaging 71,’2 min. This is certainly a very interesting state of affairs a n d t h e natural question t o be asked is, “ W h a t is t h e cause of this variation?” There are many causes: some are knom-n and some we are still studying. M y experience seems t o show t h a t the rennin coagulates more quickly in t h e fall and spring t h a n in t h e winter and summer, and this fact seems t o prove my contention t h a t there is a seasonable variation in the milk. There may be several explanations of this phenomenon. T h e cattle may get more exercise and more organic and inorganic salts in t h e spring, summer and early fall, t h a n in t h e winter, hence their milk supply may vary, especially in relation t o its coagulability. If this be so, the question rises, why does not the summer supply show this same increment in coagulability? M y answer is t h a t i t would if we could regulate t h e supply, b u t during t h e hot summer months t h e milk is so prone t o temperature changes t h a t this offsets the other advantages. Then, too, the period of lactation is a n important consideration and milk will show great variations in composition during this time. It is an established fact t h a t t h e rennet coagulation proceeds in three’ stages: ( I ) T h e calcium casein is changed into t h e para casein; ( 2 ) t h e rennet enzyme seems t o act upon t h e insoluble calcium salts of t h e milk, converting t h e m into a form sufficiently soluble t o enable t h e m t o coagulate t h e calcium para casein; (3) the true coagulation. I t is t h e second change or conversion of insoluble calcium salts t o soluble salts which seems t o retard t h e progress of coagulation, and I have noticed t h a t in those cases where t h e time of coagulation was prolonged. t h e viscosity of t h e milk gradually changes; it seems t o thicken and acts as if i t almost wants t o coagulate, b u t something seems t o hold it backeither calcium salts are lacking or t h e insoluble calcium salts are with difficulty made soluble a n d available. I have also noticed t h a t if a milk, acting in this manner, is divided into two portions, t o one of which a soluble calcium salt is added, t h e coagulation period is much advanced in this portion when compared with t h e portion t o which no soluble calcium salt has been added. Hence my statement t h a t t h e seasonal variation in milk is a n important factor. There are other factors which, without a doubt, influence this coagulation period. Various samples of milk will show a variation in t h e f a t content and 1
Allen’s “Commercial Organic Analysis,” 8, 126.
T‘ol. 8, NO. IO
although t h e large creameries standardize t h e per cent of f a t in their milk, the smaller creameries do not, and t h e inclusion o€ a superabundance of fat globules in one sample of milk undoubtedly will have some bearing upon t h e ease of coagulation. T h e effect of bacterial content is a n important factor. T h e milk examined b y t h e Detroit Board of Health ranges in bacterial count from 3,000t o ~ , j o o , o o o bacteria per cc., which variation must certainly exert some influence upon rennet action. T h e range of reaction of t h e milk within t h e perceptible limits of litmus test is a n important item for consideration. In some cases I have found t h a t a milk which has started t o sour and thus had dereloped acidity above normal, seems t o accelerate t h e coagulation period of rennin; in other cases it has inhibited it. The “hydrogen-ion” concentration, a fact overlooked in t h e past by most of us b u t receiving more or less consideradon a t present, may help us clear u p some of these peculiarities in enzyme activity. I look forEard t o t h e time when we can eventually adopt a standard milk of normal hydrogen-ion concentration and adjust our enzyme solution t o a hydrogen-ion concentration found t o give its optimum activity, and thus have our method under absolute and scientific control. Until such time as this arrives, t h e results here enumerated lead me t o say, “Know your standard.” Do not adopt it on a single test or even a single series of tests. b u t follow it through a series of months as I have mine, then assign a strength which seems a fair average. T h e method I use for testing follows, with t h e correction before cited. (I) Only fresh, whole, unpasteurized milk is t o be used, preferably from a herd of many cows. (2) Milk-Heat 2 qts. gradually t o 43’ C.; mix well and take I q t . for t h e unknown sample and t h e other for t h e standard. Do not overheat t h e milk and if b y chance i t has been heated high enough t o produce a thin skin, discard and use a new lot. (3) Rennin Solution-Dissolve 0.4843 g. rennin in I jo cc. distilled water, mix thoroughly by inversion gently, and take I O cc. for a test ( I O cc. upon I qt. of milk gires a proportion of I : 30,000). D O not shake t h e rennin solution violently or t h e agitation will injure t h e strength of rennin. (4) LIeasure I O cc. rennin solution into a widemouth precipitating jar of about I//z gal. capacity; a d d I q t . of milk a t 42’ C., mix thoroughly by pouring rennin solution and milk 3 times from one jar t o another, taking time with stopwatch after t h e first pouring. T h e milk should now be just 40’ C. Keep t h e temperature at this point a n d test t h e milk for curd formation a t half-minute intervals b y tipping t h e precipitating jar forward gently; note t h e time required t o separate t h e casein from t h e liquid whey; report t h e sample as I : 30,000in this time. R E S E A R C H LABORATORY,
DIGESTIVEF E R M E N T S
DETROIT,
MICHIGAN
COMPANY
