A Study of Certain Ferments with a View to Determining a Method for

T H E J O U R N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y

360

(3) IMMERSION TEST-The joints are completely immersed in water a t 20’ C. for 1 2 hrs., and are then pulled apart, and the breaking strain recorded. Tests ( 2 ) and ( 3 ) are intended t o throw light on t h e behavior of a n airscrew under t h e extreme conditions of a tropical climate a n d high humidity. Tests were also made with moist heat, and on immersion followed by dry heat, b u t were discontinued. The last named test was found t o be too drastic, all glues giving very low results. The whole procedure outlined above is an arbitrary one, and for this reason it is necessary rigidly t o standardize and adhere t o t h e technique of the method, in order t o obtain comparable results. When t h e break occurs in t h e wood, as frequently happens i n t h e regular test, one can, of course, only say t h a t t h e glue is stronger t h a n t h e wood, and record t h e figure a t which t h e wood breaks. TABLEI-INFLUENCE OF ADDITIONOF PHENOLOR CONCENTRATED AMMONIA TO GLUE ON STRENGTH OF GLUEDJOINT GLUE TESTS: REGULAR HEAT IMMERSION Gelatin A . . 644 627 459 . . . ... Gelatin A 5 % Phenol.. 532* 616 476* 627 369 660 10% Phenol.. . . . . . . . 677* 845 ... 487 Gelatin A ... “Propeller” Glue. . . . . . . . . . . . . . . . . 464 ... 504 470 464 “Propeller” Glue 5 7 Phenol.. 526 593 395* 506 560 565 “Propeller” Glue 108J,Phenol.. 610 632 315 429 548 571 “Scotch” Glue.. 470 448 470 548 “Scotch” Glue 5 % Phenol.. 688* 723 425 425 10% Phenol.. 694 694 “Scotch” Glue 476 414 453 , 627 644 Gelatin A . . ..................... 549 459 565 875 2% Ammonia 610 655 Gelatin A 616 688 504 “Propeller” Glue. . . . . . . . . . . . . . . . . 464 464 470 580 609 783 “Propeller” Glue 2% Ammonia.. 520 5j2 The figures represent breaking strain in Ibs. per sq. in. of glued surface of the standardized joint. Breaks in the wood are indicated thus *,

++..................... ........ .. ++ .. ................. + .... + ... + ........ +

...

Addition of phenol t o t h e glue improves t h e regular test. T h e influence on heat tests and on immersion tests is not marked, but t h e tendency is t o raise t h e m slightly. The addition of 5 per cent phenol t o a I : 2 gelatine solution, depressed t h e setting point of t h a t solution from about 26 t o 18’ C., while I O per cent phenol caused t h e solution t o be still viscous a t 5’ C. The addition of 5 per cent phenol t o glue solutions t o be used on aeroplane work is, therefore, t o be recommended, both because of its tendency t o increase t h e strength of t h e joint, and also on account of its action in depressing t h e setting point. Ammonia causes t h e glue t o set more rapidly. It was found t o have t h e unexpected effect of raising t h e figures for heat and immersion tests, while leaving regular tests little affected. The Germans appear t o have used a casein glue on some of their aircraft. An analysis of a Swiss glue of this t y p e showed its composition t o be about 66 per cent casein and 23 per cent mineral matter. The latter was composed of soda, silica, lime and alumina. About I per cent of petroleum was present in t h e powder. This casein glue is prepared for use b y rubbing up with cold water. It requires about 3 days for t h e joint t o set, but has the advantage t h a t all t h e laminae of a n airscrew can be glued together a t once. T h e following test figures were obtained on joints made with t h e Swiss casein glue: Regular 551 655

Dry Heat 526 661

Moist Heat 448 465

The immersion test is particularly high.

Immersion 672 862

Vol. 9, No. 4

Casein glues are very generally used for cementing together the “veneers” on ply-wood which finds extended use on t h e fuselage and other parts of t h e aeroplane. One English firm uses a mixture of casein, lime, and blood, which yields a cement very resistant t o water. A three-ply board made with such a cement will withstand an immersion test in water a t 50’ C. for 1 2 hours without any separation of t h e plies, though t h e strength of this cement on a “regular” test is inferior t o t h a t of a hide glue. Another firm of ply-wood manufacturers uses lime and casein only, in t h e proportion approximately of 4 parts by weight of lime t o 7 parts by weight of casein. Casein and borax form a good mixture, b u t are, of course, more expensive t h a n casein and lime. Casein glues cannot be kept more t h a n a few hours after mixing with water, so t h a t a batch when mixed must be completely used, or t h e residue wasted. When a new glue is intended for use on airscrews, t h e tests described above should be supplemented b y a practical test of spinning a trial airscrew made with t h e new glue. HIOHLAND PARK,LLANERCK, PA.

A STUDY OF CERTAIN FERMENTS WITH A VIEW TO DETERMINING A METHOD FOR THE DIFFERENTIATION OF PASTEURIZED MILK FROM RAW MILK I. REDUCTASES B y RICHARD EDWINLEE AND MELVINGUY MELLON Received May 1, 1916 INTRODUCTORY

The enthusiasm and skill with which many of t h e problems relating t o t h e distribution, composition a n d action of a class of substances known as e n z y m e s or f e r m e n t s have been attacked in recent years have contributed much data of importance t o the biological chemist a n d sanitarian. Some of these ferments are so widely distributed in living tissues of members of both the animal a n d vegetable kingdoms t h a t it has been suggested by one well-known investigator “ t h a t t h e properties of these substaiices might almost be t u r n e d to account as a general chemical test f o r vital activity.” While this may seem like an exaggeration of their significance and although the precise r61e of these substances in the life of the cell has not yet been determined, it is undoubtedly true t h a t they are concerned in a great many of the most important biochemical processes with which we are familiar. And furthermore, regardless of the fact t h a t very few, if any, of these substances have been isolated in a pure condition,’ they have, without doubt, as have other compounds, a definite chemical composition; and through t h e exercise of definite chemical affinities, they are able t o produce alterations in other compounds. Holding t o this view, Traube2 formulated his theory of fermentation, for example, upon two distinct chemical propositions: first, t h a t the ferments are defi1 It would probably be more accurate to say that we do not know whether a specific enzyme has, or has not been prepared in the pure state. 2 Theorie der Fermentwirkungen, Berlin, 1868; Ueber Akfivrrung des Saurrstoffs., 16, 659-675.

Apr.9 I 9 1 7


nite chemical compounds elaborated from protein as a result of t h e combined action of heat, water, a n d oxygen, a n d are present not only in t h e lower organisms b u t also in t h e tissues of t h e higher forms, where t h e y are responsible for biochemical processes; second, t h a t t h e ferments are powerful reducing agents a n d oxygen carriers, capable, in t h e capacity of chemical go-betweens ( V e r m i t t l e r ) , of effecting t h e transfer not only of free oxygen t o easily oxidizable substances, b u t also t h e transfer of combined oxygen from one compound t o another. I n accordance with this theory, he was led t o divide ferments into three classes:’ (a) T/’erwesungsfermeizte,-those combined loosely with oxygen, forming unstable compounds which give u p their oxygen t o other substances less readily oxidized. ( b ) Reductionsfermente-those which combine with t h e oxygen of water, t h e hydrogen going t o effect t h e reduction of some passive body. ( c ) Hochste Fiiulnissjermente-those which cause putrefactions in which hydrogen is set free. Although there is not complete agreement among investigators as t o t h e exact mechanism b y which t h e alterations are effected, there is very general belief t h a t t h e y act catalytically, and, therefore, partake of t h e nature of ferments; t h a t is, enzymes may be regarded as organic catalysts. As is well known, they are very unstable, being generally destroyed b y a n exposure t o a temperature outside of relatively narrow limits; a n d inasmuch as they have optimum, maximum, a n d minimum temperatures, a n d thermal death points, t h e y resemble microorganisms. Usually action ceases at o o C.; t h e optimum for most types lies between 30 a n d j o o C., t h e y are soon destroyed at temperatures above 70’ C., a n d almost instantaneously b y boiling water. Likewise t h e y are remarkably sensitive t o t h e action of mineral acids such as HC1 a n d various poisons3 such as H C N , SO,, HgC12, CzHsNHz a n d CHC1,. It is in virtue, however, of these facts which have been pointed out, viz., t h e wide distribution of these enzymes in t h e animal a n d vegetable kingdoms a n d their sensitiveness t o various physical a n d chemical agents, t h a t t h e y have become of great importance t o t h e biological chemist a n d sanitarian, as enabling t h e m by means of specific tests for these enzymes t o form correct conclusions regarding t h e character a n d condition of certain foodstuffs; i. e., these tests have enabled t h e chemist t o say whether t h e food is r a w or has been heated. Among t h e first of these enzymes t o be discovered a n d t o have its properties studied is t h e group now known as t h e oxidases. They are among t h e most widely distributed of all t h e ferments. These, together with another group known as t h e reductases, Kastle, Bull. S9, Hygienic Laboratory. Traube in his later writings used the term Osydalionsfermenle a s preferable to the term Verwesungsfermente. This term is employed t o signify those ferments which possess the power of taking up free oxygen and carrying i t t o other passive substances, thereby accomplishing the oxidation of the latter. 3 For a more complete list see article by Harris and Creighton, J . B i d . Chem., 22 (1915), ,535. 1

2

361

are of particular interest t o us in t h e consideration of t h e problem presented i n this paper, owing t o t h e presence of t h e former (oxidases) in milk, and t h e probable bacterial origin of t h e reductases. For a number of years i t has been a most interesting question as t o whether these enzyme reactions could be made t h e basis of methods for differentiating old milk from new milk, a n d pasteurized milk from raw milk. Regardless of t h e controversy which has arisen, a numbet of investigators have proposed tests in which t h e reactive properties of these enzymes have been utilized with a view t o formulating methods for ascertaining t h e “sanitary condition” of milk. These investigations seem t o be entirely justified when we recall t h e following well-known facts: ( I ) Normal fresh milk has t h e property of decomposing hydrogen peroxide into free oxygen gas a n d water; ( 2 ) ordinary milk possesses t h e power of decoloring, within certain limits of time, various coloring substances by reduction or removal of oxygen. Although t h e exact nature of these reactions and t h e source of t h e substances in milk influencing t h e m have not been clearly established, there is much evidence which indicates t h a t t h e y are of a n enzymic nature. It is scarcely necessary t o point out t h a t there is much uncertainty a t t h e present time as t o t h e accuracy a n d significance of these proposed tests. There is no question, however, as t o t h e need of them. Inasmuch as i t has often become necessary t o pasteurize milk t o be sold t o t h e public, because i t has not been produced under t h e required sanitary conditions, t h e need of a test which will quickly a n d easily show whether a given sample of milk has been heated or not, is at once obvious. I n t h e effort t o meet this want a considerable number of methods have been formulated in t h e last few years. Unfortunately, most of those proposed are not characterized b y exactness or simplicity. However, there seem t o be a few methods which have received neither t h e use nor t h e study t o which they are apparently entitled. It was this view of t h e situation which led t h e authors of this paper t o t h e examination of various methods which have been proposed for t h e differentiating of raw milk from pasteurized milk with regard t o determining their relative accuracy. T h e methods which have been proposed may be conveniently divided into t w o main groups:‘ ( I ) M e t h o d s based upon changes which the protein i n m i l k undergoes w h e n the latter i s heated. ( 2 ) M e t h o d s based upon reactions i n j u e n c e d b y the presence of certain chemical f e r m e n t s i n m i l k . An examination of t h e literature dealing with this subject reveals t h e fact t h a t only those methods which are included in t h e second group have proven a t all satisfactory. And i t will be noted t h a t these are t h e methods which are based upon t h e action exerted by t h e presence of certain enzymes. I n this investigation we have been concerned with b u t three groups of these chemical ferments: ( I ) a peroxidase, which causes hydrogen peroxide t o react 1

Barthel, “Milk and Dairy Products,” p. 9 i .


Vol. 9 , No. 4

with certain (oxidase) reagents, thereby producing change of colors; ( 2 ) a catalase, which decomposes hydrogen peroxide (with t h e liberation of oxygen) b u t is incapable of effecting t h e oxidation of oxidase reagents by means of t h e peroxide; ( 3 ) t h e reductases, which combine with t h e oxygen of t h e water, the liberated hydrogen effecting t h e reduction of a passive body like methylene blue with attendant decoloration. Owing t o the fact t h a t the peroxidases and catalases are t o be taken up in another paper, they will not be considered further a t this time as this paper is concerned only with t h e nature, source, and action of t h e enzyme which influences t h e reactions upon which are based t h e reductase methods. REDUCTASES I N RELATION TO PROBLEM

7

\

N

In

18 I

d

d

The reductases (Reductionsjermente) are usually defined as those ferments which combine with the oxygen of water, the hydrogen going t o effect t h e reduction of a passive body like methylene blue. The reductase test is based upon t h e fact, first noticed by Duclaux,’ t h a t ordinary normal cow’s milk has t h e power of converting certain coloring matters, as indigo-carmine, into the corresponding leuco-compound by reduction. He also showed t h a t this property of milk depends upon t h e microorganisms which it contains. Neisser and Wechsberg2 proposed the use of methylene blue as a reagent for testing t h e quality of milk. H. Smidt,3 P. Th. Muller,4 and BartheP also worked on this method and came t o the conclusion t h a t there existed a distinct parallel between t h e number of organisms in t h e milk and t h e time required, under certain conditions, for a solution of methylene blue t o be decolorized. According t o BartheP the reductase test gives approximately t h e relative number of bacteria in t h e milk. Van Slyke’ seems t o agree with this as he maintains t h a t this reducing property appears t o depend upon t h e presence of microorganisms in milk since the larger t h e number of bacteria, t h e shorter t h e time required t o produce decoloration. There seems t o be some dispute, however, among other investigators as t o t h e exact origin of the reductase. Konning,* Seligman,S and GrimmerlO are of the opinion t h a t t h e reductases are produced by bacteria. However, Seligman states t h a t possibly some reductases may exist as enzymes in milk. Romer and Samesll are opposed t o the enzyme nature of reductase, and state t h a t i t is produced by t h e destruction of t h e cells of the mammary glands during milking, as t h e first milkings have but slight reducing properties and t h e last milkings are highly reducing. Sames12 is not only opposed t o t h e enzyme nature of 1

Le Lait, “8tudes chimiques et microbiologiques,” Paris, 1887.

:Analyst, 26 (1901). 148.

Milnch. Med. Wochschr., 1900, No. 37. Hyg. Rundschau, 14 (1904), 1-137. * Arch. Hyg., 66 (1906). 108. * Z . Nahr. Ccnussrn.. 16 (1908). 385; “Milk and Dairy Products,“ p. 128. 7,“Modern Methods of Testing Milk and Milk Products,” p. 158. 8 Milchwirtschaft Zcntr.. 4, 156. 8 4

Z Hyg.. 68, 1. Milchwirtschaff Zentr., 6 , 243. 11 Z . Nahr. Genussm., PO, 1 . 1: Milchwirtschaft Zcntr., 6 , 462.

1)

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

363

TABLEI Sample

No. 1

2

3

4

5

6

Age Hrs. 4 24 48 72 96 120 4 24 48 72 96 120 8 24 48 72 96 120 3 24 48 72 96 120 4 24 48 72 96 120 8 24 48 72 96 120

Degree of Acidity 16.5 27 82 93 100 105 16 26 43 65

... 85

Time to Decolorize BACTERIAL COUNT(a) Methylene Blue Plain Agar L. I,. Agar 90,000 30,000 No Decol. 10,000,000 7,000,000 10 Min. 450,000,000 300 000 000 7 M/n. 10,000,000 8 '000'000 12 M!n. 4,000,000 2 000: 000 25 Min. 50 Min. 500,000 400,000 No Decol. 5,000,000 8,000,000 30 Min. 10,000,000 7,000,000 25 Min. 1 ,000,000 600,000 15 Min.

19 21 25

...

95 103 15 21

... 78

87 95 18 22 76 87 92 97 19 20 72 88 99

...

4 19 24 22 74 48 72 79 96 97 120 16 4 8 17 24 48 75 72 96 90 120 96 15 4 9 18 24 48 76 72 96 88 90 120 19 8 10 24 59 48 95 72 96 102 107 120 (0) "Standard Methods" all bacterial counts. 7

...

... ...

...

.......

........

.......

:

.......

50 Min. No Decol. Several Hrs. Several Hrs.

1,000,000 150,000 160,000 6,000,000

40,000 4,000,000

35 Min. 45 Min. No Decol. 28 Min.

3,000,000 1 ,000,000 20,000 80,000

2,000,000 800,000 10,000 50,000

35 Min. 55 Min. 75 Min. 4 Hrs. 1 Hr. 25 =in. 30 Min. 30 Min. 35 Min. 5 Hrs. 4 Hrs. 10 M/n. 18 M!n. 60 Mm.

2,000,000 100.000 200,000 400,000 1,000,000 800,000 220,000 180,000 20,000 160,000 4,000,000 800,000 600,000

1,000,000 60.000 100,000 150,000 700,000 300,000 75,000 50.000 15,000 40,000 750,000 100,000

........

........

........

Slightly 6 Hrs. 2 Hrs. 6 Min. 40 Min.

........

30 Min. No Decol. 35 Min. 10 Min.

........

15 Min. 12 Min. 150 Min. 90 Min.

........ 10 Min.

30 Min. 35 Min. No Decol.

........ 4 Min.

.......

....... 2,000,000

1 ,000,000

.......

.......

....... 1,000,000

....... .......

20,000 100,000,000 1,000,000 750,000

10,000 75,000,000 50,000 40,000

500,000 10,000 600,000 10,000,000

60,000 4,000 150,000 500,000

.......

....... 1,000,000 1,000,000 10,000 100,000

....... 1 ,000,000

200,000 120,000 10,000

.......

....... .......

400,000 500,000

....... 40,000

....... 80,000 60,000 50,000

....... .......

1,350,000 100.000 10 Min. 1,300,000 80,000 14 Min. 500,000 80,000 18 Min. 300.000 10.000 as formulated by the American Public Health

reductase b u t insists t h a t Seligman's assumption t h a t t h e reduction is due t o bacteria is too far-reaching. Salusl believes t h a t reductase is a product of cell transformation. Oppenheimer2 is of t h e opinion t h a t t h e reductase is in no way related t o t h e bacteria a n d t h a t i t is merely difficult t o distinguish from bacterial reductases. However, on t h e basis of t h e probable bacterial origin of reductase, there have been two tests proposed for its detection: ( I ) Schardinger's M . Reductase which is carried o u t b y adding I cc. of Schardinger's reagent M. (viz., j cc. of a saturated alcoholic solution of methylene blue a n d 195 cc. of water) t o 20 cc. of milk in a test t u b e a n d placing i t in a water b a t h a t 4 j t o 50" C. With this reagent a number of hours is required t o produce decoloration. (2) Schardinger's F . M . Reductase Test; which is carried out b y adding I cc. of Schardinger's F. M. 1

Arch. Hyg., 10, 371. Kel. Inst. Expcrim. Thcrap. eu Frankfort a / M , 1908, 75. 2. Nohr. Gcnussm..4 (1906), 377. THISJOURNAL, 6 (1913), 922-927.

' Arb. 8

Sample Age

No. 11

Hrs.

Degree of Acidity 16 16.5 43 95 97 100 17 18 44 90 97

Time t o Decolorize Methylene Blue No Decol. Several Hrs. 8 Min. 15 Min. 20 Min. 30 Min. No Decol. 65 Min. 4 Min. 15 Min. 14 Min.

4 24 48 72 96 120 12 8 24 48 72 96 120 17 13 4 No Decol. 24 40 Min. 20 48 10 Min. 64 72 18 Min. 84 96 120 17 14 8 No Decol. 24 25 Min. 18 4 Min. 61 48 72 90 30 Min. 96 93 60 Min. 96 60 Min. 120 15 8 15 Slightly, 3 Hrs. Partly, 3 Hrs. 19 24 10 Min. 72 48 70 Min. 83 72 93 96 75 Min. 120 19 Partly, 3 Hrs. 16 4 22 2 5 Min. 24 86 20 Min. 48 96 72 30 Min. 96 35 Min. 120 102 19.5 Partly, 3 Hrs. 17 4 23 20 M/n. 24 18 Min. 86 48 96 15 Min. 72 96 45 Min. 120 100 16 Slightly, 150 Min. 18 8 60 Min. 19 24 15 Min. 64 48 72 40 Min. 89 96 50 Min. 120 98 150 Min. 16.5 19 8 120 Min. 17 24 40 Min. 82 48 ........ 72 ... 60 Min. 95 96 60 Min. 103 120 Slightly, 3 Hrs. 17 20 4 15 Min. 33 24 ... 48 15 Min. 100 72 30 Min. 107 96 60 Min. 98 120 Association for the bacterial examination of milk

...

.... ..

...

...

...

...

........

........ ........

........

........ ........

........

........

BACTERIAL COWNT(U) Plain Agar I,. L. Agar 15,000 4,000 20,000 10.000 5,000,000 2,000,000 370,000 350.000 300,000 250.000 200,000 9,000 3,000 400,000 150,000 1,500,000 1,000,000 500,000 350,000 400,000 150,000

.......

.......

.......

15,000 90,000 8,000,000 2,000,000

6,000 10,000 3 ,000,000

....... .......

1,000,000

13,000 1,500,000 5,000,000 1,500,000 50,000 30,000 8,000 20,000 900,000 350,000 150,000

1,700 30,000 60,000

....... ....... ....... 5.000 .......

.......

3,000 50,000 70,000 10,000

.......

.......

8,000 20,000 100,000 20,000

1,000 3,000 20,000 16,000

....... 10,000

10,000 100,000 130,000 100,000

....... 10,000

5.000 1,200,000 2,000,000

.......

.......

3,000 1,000 3,500

iO.000

55,000 ....... 2,000 ....... 100,000 120,000

.......

400,000 350,000 5.000 75,000 100,000

100,000 80,000

80,000 30,000 30,000 400,000

50,000 10,000 8,000 40,000

....... 20,000 25,000

.......

.......

.......

.......

140,000 20,000 100,000 10,000 20,000 were employed in making

.......

reagent (viz., j cc. of a saturated alcoholic solution of methylene blue, j cc. of 40 per cent formaldehyde, a n d 190 cc. of water) t o 20 cc. of milk in a test tube, placing it in a water b a t h a t 45 t o 50" C.,and covering t h e contents of t h e t u b e with a layer of liquid petroleum t o prevent t h e access of air. T h e formaldehyde serves t o produce a n acceleration in t h e time of reduction. Lythgoel states t h a t raw milk will decolor this reagent in less t h a n 20 min. a n d pasteurized milk will t a k e a longer time. T h e reaction depends upon t h e supposed presence of a specific enzyme in milk called aldehyde-reductase, which is more or less quickly destroyed at a temperat u r e above 70' C.; when in milk heated above 80" C., its destruction is complete.2 Barthe13 states t h a t t h e enzyme is destroyed more or less quickly at temperatures above 70' C.; and t h a t milk heated t o 80' C. does not discharge t h e color at all; or milk heated t o 7 5 ' C. for a few minutes or pasteurized for 15 minutes a t 70" C., discharges t h e color in 30 minutes. 1

THISJOURNAL, 6 (1913). 922-927.

Van Slyke, "Modern Methods of Testing Milk and Milk Products," p. 163. 8 "Milk and Dairy Products," p. 128. 2

3 64

Vol. 9, No. 4


Age of milk, Hrs.

Age of milk, Mrs. FIG.6

FIG.7

By means of these reagents i t is possible, according t o Lythgoe,' t o get a good idea of t h e temperature t o which t h e milk has been heated, and also how long it has been heated, since heating t h e milk destroys t h e enzyme. Thus, he maintains, this method affords a very good means of distinguishing between raw and pasteurized milk. As stated previously, there seems t o be a difference of opinion as t o the origin of t h e reductase; likewise investigators are far from a n y agreement regarding any relationship which may exist between the time of methylene blue reduction and t h e number of bacteria present. Angelici2 states t h a t there is absolutely no parallelism between t h e two. Fred,3 however, holds t o t h e view t h a t most, b u t not all, milk bacteria reduce methylene blue; and t h a t milk reducing methylene blue in from 15 minutes t o an hour contains from 15 t o 50 millions of bacteria per cc., and milk requiring 7 hours or more contains less t h a n one million bacteria per Cc. Barthe14 states t h a t fresh milk containing 10,000bacteria per cc. decolorized t h e methylene blue solution in 11 hrs.; and t h e same milk four days old containing 17 millions of bacteria per cc. decolored t h e solution in 14 min. He further6 maintains t h a t when t h e reduction takes place in less t h a n one hour, t h e milk has more t h a n I O million bacteria per cc., and when t h e time required is from I t o 3 hrs., t h e number of bacteria present is from 4 t o I O millions per cc. As a conclusion he asserts t h a t when t h e reduction requires less

t h a n I hour, the milk is bacterially too impure for food; and t h a t good commercial milk should require not less t h a n 3 hours for decoloration. As a result of the foregoing discussion, therefore, we may conclude t h a t there exists a considerable difference of opinion: ( I ) as t o whether t h e reductase is of bacterial origin or of animal metabolism; a n d (2) as t o t h e relationship existing between t h e time of methylene blue reduction and t h e number of bacteria present.

1

THISJOURNAL, 1 (1913), 922-927.

2

Clin. Val., 84, 388. Centr. Bakl. Parasitcnk.. I I Ab1 , 81, 491. Z . Nahr. Genussm.. 11, 385. Ibid.. 21, 513.

J

4 6

EXPERIMEKTAL

The following experimental work was carried on with a view t o determining by means of the Schardinger F. M . test: I-The relationship existing between the number of bacteria and t h e time required t o decolorize t h e methylene blue solution. a-The influence of time and temperature of pasteurization on milk in its relation t o Schardinger's reagent, F. M. RELATIONSHIP

BETWEEN

NUMBER

OF

BACTERIA

AND

TIME REQUIRED TO DECOLORIZE METHYLENE B L U E

Twenty samples of milk were tested as follows: ( I ) t h e acidity was determined by titration with N / I O NaOH, 0.1 cc. of t h e solution being equivalent t o I * of acidity; (2) t h e time required t o decolor methylene blue solution was observed; (3) bacterial counts were made a t t h e time of each test both on plain agar and on lactose litmus agar. Each sample was tested daily for a period of five days, during which time t h e milk was allowed t o remain a t the temperature of t h e laboratory. The results of this work appear i n Table I. The full significance of this d a t a becomes more easily

Apr.9 1917


apparent when i t is presented graphically. I n Figs. I t o 5, graphs have been drawn of 5 of t h e representative samples of t h e above series, using separate curves t o show t h e increase in degrees of acidity, t h e time of decoloration .of methylene blue, t h e plain agar bacterial count, a n d t h e lactose litmus agar bacterial count. T h e dotted portion of t h e curves indicates t h e probable course covering t h e period during which no tests were made, as, for example, on Sundays. The age of t h e milk i n hours is plotted as abscissas. There are three sets of ordinates: first, t h e column on t h e left b y which both t h e plain agar a n d t h e lactose litmus agar counts are plotted; second, t h e middle column expressed in minutes, b y which t h e time of decoloration is plotted; a n d third, t h e column on t h e right expressed in degrees, b y which t h e degree of acidity is plotted. This gives four curves for each sample. From t h e d a t a presented in t h e preceding table a n d b y means of graphs t h e following conclusions have been formulated: ( I ) An increase of acidity occurs with increase of age. I n t h e periods examined this increase was greatest in t h e majority of cases in t h e period between 24 a n d 1 8 hrs. ( 2 ) Normal fresh milk of good quality does not reduce Schardinger’s F. M. reagent in less t h a n 2 0 min.-the shortest time observed being much longer. When t h e decoloration was effected in I O min. or less time, t h e milk was found t o contain a t least I,OOO,OOO microorganisms per cc. ( 3 ) Up t o a certain point a n increase in t h e number of bacteria in a given sample is accompanied b y a corresponding decrease in t h e t i m e required b y it t o decolor t h e reagent. At this point t h e maximum bacterial count a n d t h e minimum time required for decoloration coincide. This relationship seems t o point t o t h e conclusion t h a t t h e reductase is of bacterial origin. The tables will show, however, if d a t a for diJerent samples are compared, t h a t no absolute parallelism exists between t h e time required for decoloring t h e reagent a n d t h e number of bacteria present. This is in agreement with results obtained b y Ange1ici.l (4) As t h e acidity increases beyond this point of coincidence t h e number of bacteria decreases a n d t h e time required for decoloration increases. (5) The final decrease in t h e number of bacteria and t h e increase in t h e time of decoloration are probably d u e t o t h e production of acid b y acid-forming bacteria, t h e acid t h u s produced probably tending t o remove t h e effect produced b y t h e aldehyde-reductase a n d t o make t h e medium unfit for t h e further growth of certain bacteria. The foregoing conclusions are in accord with t h e d a t a obtained in this laboratory over two years ago b y F. W. Fabian2 working on a similar problem. I n this connection it is noted t h a t i t is a matter of record t h a t Lythgoe3 found t h a t normal fresh milk reduced Schardinger’s F. AI. reagent in less t h a n 2 0 min. The authors are unable a t this time t o account Clin. Vaf., 84, 388. Working in this laboratory in 1912 in collaboration with the senior author. THISJOURNAL, 6 (1913). 922-927. 1

2

J

365

for this discrepancy in t h e time element, unless it can be attributed t o a difference in t h e standard of milk which is t o be regarded as “normal.” Table I1 is quoted from t h e report of Mr. Fabian’s work t o which reference has just been made. TABLE I1 (Results by M r . Fabian) Sample Minutes Required to Bacterial Count Decolorize M.-Blue on Plain Agar No. 3 17,000,000 lo... 35,000,000 3 7... 5... 6 12,000,000 14.. . . . . . . . . . . . . . . 7.5 44,000,000 1,900,000 11 . . . . . . . . . . . . . . . . . 9 810,000 2.. . . . . . . . . . . . . . . . 13 14 720,000 1 . .. 15 370,000 12. .. 4. . . . . . . . . . . . . . . . . 15 600,000 17 560,000 3... 19 300,000 6... 20 300,000 13. .. 220,000 S . . . . . . . . . . . . . . . . . 27 34 490,000 9... 32 360,000 15... 37 300,000 19.. 42 240,000 20... 300,000 1 6 . . . . . . . . . . . . . . . . 45 59 120,000 18.. 95 55,000’ 17 ... 120 80,000 25. . . . . . . . . . . . . . . . . 150 110,000 22 90,000 24.. . . . . . . . . . . . . . .155 40,000 2 3 . . . . . . . . . . . . . . . . . 180 184 20,000 21...

..............

. ..

...

.

Twenty-five different samples of raw milk were tested with Schardinger’s reagent, F. M. A bacterial count was also made of each sample. E F F E C T O F T I M E AND T E M P E R A T U R E O F PASTEURIZAT I O N O N M I L K I N I T S R E L A T I O N TO S C H A R DINGER’S

REAGEKT

Four separate portions of each of three different samples of milk were heated gradually-a rise of z o C. per min.-in a water b a t h t o 6 0 , 6 j, j o and j j o C., a n d then maintained a t these respective temperatures for 30 min. Portions were removed every I O min. a n d tested with t h e reagent. Bacterial counts were made of each sample tested a t t h e various intervals of time. Milk I O hrs. old was used, as fresh milk 4 hrs. old produced no decoloration of t h e reagent. T h e results obtained are given in Table 111. TABLE I11 PASTEURIZATION Time Required Temp, Time to Decolorize C. Min. Min. 0 30 . . . . . 60 7. 0_ IO 95 20 150 30 60 0 65 100 10 120 20 160 30 65 0 io 10 20 30 0 65 75 10 No decolor. 20 No decolor. No decolor. 30 28 0 Z...... . . 60 65 10 87 20 135 30 55 0 65 qn 10 _. 20 120 155 30 56 0 i0 No decolor. 10 No decolor. 20 No decolor. 30 0 55 i5 10 No decolor. No decolor. 20 Pjo decolor. 30 0 48 3 . . . . . . . . i5 No decolor. 10

Sample No. I...

BACTERIAL COUNT ON AGAR Plain 40,000 12,000 6,000 2,000 40,000 10,000 400 200 35,000 200 None None 36,000 300 None

65 ;000 15,000 1,000 300 44,000 7 00 None None 30,000 200 None None 35,000 150

L. L.

12,000 6,500 3,500 1,200 11,000 4,000 None None 11,000 None None None 10,000 None None None 25,000 9,000 5,000 3,000 31 .OOO

s;ooo

200 -..

None 23,000 35 None None 18,000

None None None 21.000 Xone

These d a t a indicate t h a t both t h e temperature a n d t h e duration of t h e pasteurization process affect milk

T H E JOURNAL OF INDUSTRIAL A N D ENGINEERING CHEMISTRY

366

with reference t o t h e time required by t h e latter t o decolorize methylene blue. I n general, as either factor is increased t h e time required for decoloration is increased. Furthermore, t h e authors found as t h e result of a larger number of tests t h a n are recorded here t h a t milk heated t o a temperature of 70’ C. for I O min. failed t o decolor t h e reagent in several hours. I n this latter respect t h e behavior of samples of milk pasteurized under t h e stated conditions was identical with t h a t of freshly drawn milk of good grade. Mr. Fabian found t h a t milk pasteurized a t 80’ C. for I min. behaved towards t h e reagent in t h e same manner as milk pasteurized a t 7 5 ’ C. for 3 min. or a t 70’ C. for I O min., and t h a t in no case was t h e reagent decolored in several hours. Table IV is quoted from Mr. Fabian’s report. Samples of raw milk, eight hours old, were used in making the tests indicated.

t h a t reductase is not only destroyed by t h e pasteurization process but t h a t it is of bacterial origin, t h e presumption being t h a t no decoloration of t h e reagent takes place until t h e bacteria have again multiplied sufficiently. Mr. Fabian in his work for a similar purpose collected a number of samples of raw milk and after making the bacterial count of each subjected t h e m t o a similar set of tests, with the results given in Table VI.

NO.

...

l......

Duration of Pasteurization 20 15 10 5 0 20 10 5 40 42 0 3 4 75 10 5 0 80 5 3 2 1 0 Signifies t h a t no decolorization occurred in several hours.

Temp. (” C.)of Pasteurization 60 60 60 60 60 70

+

++ + t ++ + +3-

F U R T H E R EVIDENCE

AS T O THE ORIGIN

++

+ t ++ + +7

OF REDUCTASE

With a view t o obtaining more evidence in regard t o t h e origin of reductase in milk, 5 samples of t h e latter were pasteurized a t 70’ C. for I O min. and then set aside. The samples, in t h e usual paper-diskcovered bottles, were allowed t o “age” a t t h e usual laboratory temperatures. They were tested a t intervals of 2 4 hrs. with t h e Schardinger reagent and bacterial counts made. The d a t a obtained by testing t h e first two samples are presented graphically in Fig. 6. TABLEV

No. 1

Age Hrs. 8 24 48 72 96 120 4 24 48 72 4 24 48 72 4 24 48 72 4 24 48 72

Acidity Degrees 20 20.5 72 95 100 106 17 17 78 95 16 18 76 93 19 20 74 98 20 23 76 96

Time to Decolorize No decol. No decol. 8 min. 15 min. 18 min. 60 min. No decol. N o decol. 10 min. 12 min. No decol. No decol. 25 min. 30 min. No decol. No decol. 14 min. 16 min. No decol. N o decol. 8 min. 12 min.

BACTERIAL COUNT ON AGAR Plain L...I 500 300 5,000 2,000,000 1,iiIo: 000 1,000,000 400,000 40,000 20,000 30,000 10,000 2,500 500 100,000 5,000 150,000 100,000 200,000 125,000 1.000 300 10,000 4,000 100,000 30,000 60,000 25,000 800 450 11,400 4.500 700,000 250,000 500,000 250,000 400 300 8,000 5,000 1 ,000,000 800,000 800,000 300,000

The experiments reported in Table V yielded results which indicate t h a t milk pasteurized a t 70’ C. for I O min. will not decolor methylene blue solution a t t h e expiration of 24 hrs. but will after being allowed t o stand for 48 hrs. This points t o t h e conclusion

TABLEVI (Resulls by M I . Fabian) BACTERIAL ON AGAR COUNT Age Time Required Hrs. to Decolorize Plain L. L. 110,000 3 (raw) Several hrs. 300,000 24 Several hrs. 92 min. 48 17 min. 72 2 min. 96 4 min. 120 220,000 110,000 3 (raw) Several hrs. Several hra. 24 ....... 70 min. 48 6 min. 72 3 min. 96 120 6 min. 3 (raw) Several hrs. 3 10,000 100.000 24 Several hrs. 48 35 min. 7,ObO:OOO 304,000,000 72 6 min. 92 7 min. 120 8 min. 200,000 150,000 3 (raw) Several hrs. . Several hrs. 24 48 10 min. 72 6 min. 96 9 min. ~

2. .....

...

TABLE IV (ResuEfs b y Mr. Fabian) Minutes Required t o Decolorize Duplicates 20 19 16 18 13 17 7 10 3 7

Vol. 9, No. 4

3

........

4..

.......

..... ..... ..... ..... .....

~~

....... ....... ....... .......

..... ..... ....... ....... ....... ....... ....... ....... ....... ....... .......

.....

..... ..... ..... ..... ..... ..... .....

The conclusions t o be made here are obviously t h e same as those formulated in t h e preceding paragraph. EFFECT

OF

THE

PRESENCE

OF

FORMALDEHYDE

ON

MILK W I T H R E F E R E N C E T O T H E T I M E R E Q U I R E D T O DECOLORIZE

METHYLENE BLUE

The following work was undertaken in order t o ascertain if a so-called “preservative” like formaldehyde influences t h e action of milk in its relation t o t h e Schardinger reagent. Five different samples of milk, each 8 hours old, were examined as follows: Each sample was tested as regards its acidity, time required t o decolor t h e reagent, and bacterial count; then each sample was treated with formaldehyde (0.5 cc. per pint of milk) and t h e enumerated tests repeated a t intervals of 2 4 hours. The results are given in Table V I I . TABLEVI1 No. 1

2

3

4

5

Age Hrs. 8 24 48 72 96 120 8 24 72 96 120 8 24 72 96 120 8 24 48 72 96 8 24 48 72 96

Acidity Degrees 20 20.5 21 22 23 23 19 19 20 21 21 17 19 20 22 22 20 23 23.5 23.5 24 16.5 18 19 21 21

Time t o Decolorize N o decolorization

BACTZRIAL COUNT ON AGAR Plain L. L.

Slight 5 hrs. No dicolorization No decolorization No decolorization Slight, 3 hrs. Slight 7 hrs. No dicolorization No decolorization N o decolorization Slight, 4 hrs. Slight, 4 hrs. N o decolorization N o decolorization No decolorization

CONCLUSIONS

The relationships indicated, if not proved, by this rather limited series of experiments are extremely interesting. For example:

Apr., 1917


367

colored by normal milk allowed t o “age” under ( I ) T h e germicidal properties of formaldehyde i n relation t o microorganisms found in milk are shown ordinary conditions of temperature for 24 t o 48 hrs. in a general way. (3) Pasteurization increases t h e time required for t h e decoloration of t h e reagent. ( 2 ) Unless i t be proven t h a t reductase is formed within t h e milk by purely chemical changes, this (4) I n general, no proportionality exists between series, considered in relation t o t h e foregoing series of t h e time required for t h e decoloration of t h e reagent a n d tests, points t o t h e conclusion t h a t reductase is of t h e number of bacteria in milk. I n a given sample, bacterial origin, as t h e time required for decoloring however, a general relation seems t o exist between t h e t h e reagent was not reduced b y allowing t h e milk two up t o a given point of acidity. t o “age,” owing presumably t o t h e fact t h a t bacterial ( 5 ) Inasmuch as there is no absolute parallelism growth in t h e samples of milk was inhibited b y t h e between number of bacteria present in milk and t h e formaldehyde. time required t o decolor t h e reagent b u t t h a t t h e re(3) T h e partial decoloration which occurred at first , lationship seems t o exist in a given sample of milk, was probably effected b y t h e reductase present in t h e i t would indicate t h a t reductase is of bacterial origin milk before t h e formaldehyde was added. T h e final b u t t h a t not all bacteria found in milk produce this loss of power of these same samples t o effect t h e same enzyme. (This latter conclusion is in accord with reaction suggests t h a t t h e formaldehyde may interact t h e views of Fred.’) with t h e reductase or counteract its influence in some (6) It seems probable t h a t formaldehyde either way. T h e fact t h a t both of these substances are gradually retards t h e action of t h e reductase or destrong reducing agents does not t e n d t o render t h e stroys it. This is a matter, however, for more careful problem more easy of solution. investigation in t h e future. Since t h e completion of this work t h e report of It will be noted t h a t although t h e conclusions formut h e investigation of Harris a n d Creightonl on t h e lated in this paper are not in accord with t h e entire influence of certain poisons on reductase has appeared. body of conclusions of a n y previous worker in this Although t h e list of poisons reported b y t h e m as either field, yet many of t h e m are in close agreement with destroying t h e reductase or retarding its action does certain conclusions of a number of investigators. n o t include formaldehyde, t h e latter may act in a HYGIENIC LABORATORY similar manner. This, however, will be a matter for CARNEGIE HALL OF CHEMISTRY future investigation. ALLEGHENY COLLEGE. MEADVILLE, PA.

____~__

GEXERAL SUMMARY

I-A brief outline has been made of t h e classification, distribution a n d reactions of certain enzymes ; t h e possibility of making their sensitiveness t o various physical a n d chemical agents t h e basis of methods for determining t h e sanitary condition of certain foodstuffs has been considered. 11-A survey has been made of t h e work done concerning t h e source, nature a n d action of reductase in its relation t o certain methods which have been proposed for t h e differentiation of pasteurized\ milk from raw milk. 111-The experimental investigation undertaken by t h e authors of this paper has been described. As t h e result of this work certain conclusions have been formulated. T h e y are as follows: ( I ) Methylene blue as i t occurs in Schardinger’s reagent, F. M., is not decolored b y : ( a ) Normal fresh milk in less t h a n 20 min. When decoloration was effected in I O minutes or less time t h e milk was found t o contain I,OOO,OOO or more, microorganisms per cc. ( b ) Milk pasteurized at 70’ C. for I O min. unless approximately 48 hrs. have elapsed since t h e milk was pasteurized; or until t h e bacteria have had time t o multiply sufficiently. (c) Old milk i n which t h e “preservative,” formaldehyde, has inhibited t h e growth of bacteria. ( 2 ) Schardinger’s reagent, F. M., is as a rule de-

’ Harris and Creighton, J. Bid. Chem., 22 (1915), 535.

A STUDY OF THE VOLUMETRIC OR PEMBERTON METHOD FOR DETERMINING PHOSPHORIC ACID, WITH SOME EXPERIMENTS SHOWlNG THE INFLUENCE OF TEMPERATUFCE AND THE SULFURIC ACID RADICAL ON RESULTS* By PHILIPMcG. SHUSY Received October 20, 1916

There has been a great deal written a n d said of t h e volumetric method for determining phosphoric acid, b u t still many chemists have trouble in its use a n d manipulation. It has been found by most workers who employ this method t h a t a number of years of careful a n d patient experience is necessary t o master it, a n d owing t o t h e length of time necessary t o acquire this, many chemists have discarded i t altogether. The writer has h a d more t h a n I O years of practical a n d constant experience in determining phosphoric acid b y this method, both with a large fertilizer concern, a n d in t h e phosphate fields of Florida, and possibly some points already mentioned can be emphasized in this paper. On account of t h e extreme delicacy of t h e method, a n d in order t o show how i t may be rendered accurate a n d reliable, i t might be of interest t o include some experiments showing some of t h e principal causes t h a t bring about disturbances in results. T h e problem is most interesting, a n d while no pretense is made t h a t this article will cover t h e entire field, i t is hoped t h a t it will a t least serve t h e purpose of aiding 1 Ccntr. Bok;. Parasilenke. I I Abt., 35, 491. 3 Presented at 53rd Meeting of American Chemical Society, New York City, September 25 to 30, 1916.

A Study of Certain Ferments with a View to Determining a Method for

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