Lipase in Raw, Heated, and Desiccated Milk'

in control unheated solutions. This result offers the first logical colloidal explanation of why it is necessary to add highly active cations to heate...
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I N D U S T R I A L A N D ENGINEERING CHEMISTRY

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the electric charge on the micellae. Although rennin was able to reduce this charge, as shown in these studies, the reduction was not sufficient to lower the velocity to that found in control unheated solutions. This result offers the first logical colloidal explanation of why it is necessary to add highly active cations to heated milk (CaCL) in order to cause heated milk to clot normally with rennin. These results throw no light on the hysteresis-like effects of heat noted by Mattick and Hallett, because the casein solutions employed were heated only a t one temperature (that of boiling water) for 1 hour, and sufficient time elapsed before the heated sols were studied in the cataphoretic tube to have secured the maximum effects. The writer ventures to predict, however, that these hysteresis effects will be found to be chiefly on the zeta potentials of the casein micellae.

Vol. 22, No. 1

Literature Cited (1) (2) (3) (4) (5) (6) (7) (8)

(9) (10) (11) (12) (13) (14) (15) (16) (17) (18)

Comel, Bull. soc. ital. b i d . sper., 3, 908 (1928). Davies and Provan, Welsh J . Agr., 4, 114 (1928). Hilditch and Jones, Analyst, 64, 75 (1929). Jerlor, Svensk Lakarelidning, May, 1929. LinderstrZm-Lang, Z . physiol. Chem., 176, 78 (1928). Mattick, Biochem. J., 22, 144 (1928). Mattick and Hallett, J . Agr. Sci., 19, 452 (1929). Palmer and Richardson, Colloid Symposium Monograph, Vol. 111, p. 112 (1925). Pertzoff, J . Gen. Physiol., 11, 239 (1928). Porcher, Chimie & industrie, 19, 589, 809 (1928). Pyne, J . Agr. Sci., 19, 463 (1929). Quam and Hellwig, J . B i d . Chem., 78, 681 (1928). Richardson and Palmer, J . Phys. Chem., 33, 567 (1929). Schneck, ~lililchwirtschaft.Forsch., 7, 1 (1928). Thurston and Peterson, J . Dairy Science, 11, 270 (1928). Van Dam and Holwerda, Lait, 8, 698, 768, 897 (1928). Weaver and Matthews, Iowa Agr. Expt. Sta., Res. Bull. 107 (1928). Whittier, J . B i d . Chem., 83, 79 (1929).

Lipase in Raw, Heated, and Desiccated Milk‘ John H. Nair THEBORDENCOMPANY, SYRACUSE, N. Y.

HE development of undesirable odors and flavors in butter, sweetened condensed milk, desiccated milk, and other manufactured milk products has long been a matter of concern to manufacturer and consumer alike. Various descriptive terms, such as rancidity, tallowiness, fishiness, and cheesiness have been applied somewhat indiscriminately to these undesirable changes. An understanding of the mechanism of the reactions giving rise to these flavors and odors is essential to modifications in processes of manufacture, packaging, and storage aimed a t their repression or elimination. Powdered whole milk has proved exceptionally difficult to maintain in a palatable condition over any extended period of time. Numerous studies of the oxidative, enzymic, and bacterial changes which occur in desiccated milk have been carried on in this laboratory. Some results of an investigation on the hydrolysis of milk fat through enzyme action are presented in this paper. There are several recognized lipolytic enzymes capable of inducing hydrolysis of fats and the simpler esters. The term lipase used in this paper is limited to those enzymes which hydrolyze the natural glycerol fats (17). The general method for the detection and determination of lipase consists in adding the substance to be tested to a suitable substrate and measuring the increase in acidity in the mixture over some suitable period of time. I n the absence of bacterial growth, it is assumed that the increase is due to a splitting off of the acids by the enzyme. I n applying this method to the study of lipase in milk, it is essential to employ a natural fat, well emulsified in aqueous solution, as a substrate and to inhibit bacterial action. Previous Work Reported

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Reference to the literature on the occurrence of true lipase in milk and milk products fails to establish conclusive evidence as to its presence. Much of the earlier work was confined to detection of an esterase capable of splitting monobutyrin. Marfan ( 7 ) , Marfan‘ and Gillett (@, Spolverini (I?’), Friedjung and Hecht (4), and Gillett (5) reported positive results, although Marfan and Gillett stated that this was not an index of the presence of a true lipase. Mor0 (9) and Hippius (6) used olive or almond oil as a substrate and obtained positive results with human milk. Thatcher and Dahlberg (19) mixed the curd of butter with 1

Received October 9,1829.

butterfat or olive oil, adding 2.5 per cent of chloroform as a preservative, and obtained no increases of acidity that could be attributed to lipase activity. Vandevelde (20) worked with cow’s milk to which an acetone solution of iodoform had been added. He found no increase in acidity on either a steam distillate or an alcohol-ether solution of the milk, and concluded that the lipolytic decomposition reported by other investigators resulted from poorly conducted studies or bacterial contamination. As Palmer ( I d ) has shown that chloroform, acetone, and iodoform have a marked retarding effect on lipase activity, however, the results of these workers are open to criticism. Davidsohn’s work (S), which led him to the conclusion that lipase is present in human but not in cow’s milk, is unconvincing. Rogers (15) worked with butterfat, using formaldehyde as a preservative. I n one series of experiments he added fresh milk and obtained a marked increase in acidity over a check sample in which boiled milk was used. I n another experiment butter was churned from fresh and from heated cream. The butter from the unheated cream showed an increase in acidity, as compared with the heated sample, after storage a t room temperature. Later Rogers, Berg, and Davis ( I 6 ) , using ethyl butyrate as a substrate, chloroform as a preservative, and milk, pasteurized a t different temperatures, as a source of enzyme, established that cow’s milk has the ability to hydrolyze ethyl butyrate. Palmer (IO), using an artificial milk prepared by emulsifying butterfat with gum arabic in water, formaldehyde being employed as a preservative, was unable to demonstrate the presence of lipase. Later Palmer ( I I ) , using the technic of Rogers, Berg, and Davis (16), but without the addition of ethyl butyrate, failed to find an active lipolytic ferment in cow’s milk. Beumer ( I ) reported that lipase is not present in the milk of cows, goats, rabbits, or dogs, but is found in considerable amounts in human milk. Rice and Markley (IS), using cream of high fat content sat,urated with sucrose, and employing titration as a measure of lipolysis, concluded that milk normally contains a lipase which splits butterfat. Experimental Procedure

The technic of the foregoing investigators was followed in the experiments conducted in this laboratory and their results with raw milk were confirmed experimentally. A substrate

INDUSTRIAL A N D ENGINEERING CHEMISTRY

January, 1930

was prepared by adding 1500 grams of sucrose to 1000 grams of raw cream containing 44 per cent of butterfat. The mixture was boiled for 5 minutes to destroy all enzymes and to facilitate solution of the sugar. After cooling to 37" C.. 100-gram portions were transferred to 250-cc. Erlenmeyer flasks, the flasks being stoppered with cotton plugs. Several series of flasks containing 1, 3, 5, 10, 15, and 20 per cent additions of the milk to be tested were prepared. The milk was thoroughly mixed with the substrate and the initial titratable acidity of each mix was determined. The flasks were then incubated a t 37" C. for 30 days or longer, with occasional titration of sample portions from each flask. -4s the object of the work was to establish the presence of a fat-splitting enzyme, it was felt that any increases in water-soluble acid would serve as an index of such activity. To measure this, 10 grams of the digestion mixture was weighed off into a beaker and 50 cc. of warm distilled water was added. After thorough stirring, the mixture was titrated to neutrality with 0.1 X sodium hydroxide using 4 drops of 1.25 per cent phenolphthalein. A preliminary experiment was made with a substrate composed of equal parts by weight of cream and sucrose, using 1, 5, and 20 per cent of added raw and pasteurized milks. Although there was a marked increase in acidity in the flasks containing raw milk, as compared with those containing pasteurized milk, results are not reported here because it was found that the proportion of sugar present was not sufficient to keep down the growth of microorganisms with the higher percentages of added milk. This was corrected by the use of 1500 grams of sucrose in succeeding experiments. As a check on bacterial growth, all samples containing 20 per cent of added milk in each series were plated in dilutions of 1 to 100 with standard lactose beef agar. Counts were made at the beginning and at the end of the incubation period. In one series certified raw whole milk less than 24 hours old was used. Portions of the same milk were pasteurized as shown in Table I. Table I

TEMPERATURE

c.

PERIOD Msnutes

61 64.5 67.5 74 74 85

30 30 1 5 1

Series of flasks were inoculated from each of these milks in the proportion of 1, 3, 5, 10, 15, and 20 per cent. -------TITRATION MILK 5 ADDED Start days Per cent Control 4 . 0 4.0 4.0 4.0 4.0 Raw whole milk: 1 4.0 6.5 8.0 9.0 13.0 15.0 3 4.0 8.5 16.0 19.5 5 4.2 11.0 .. 10 4.5 16.0 , . 23.0 28.0 15 5.0 20.0 , 30.0 35.5 20 5.2 22.0 .. 34.0 40.0 Raw skim milk: 1 4.0 .. 7.0 9.0 3 4.0 10.5 15.0 5 4.5 .. 13.5 19.5 10 4.8 . 21.0 ,. 28.5 35.3 15 5.0 , 26.0 .. 30.0 41.5 20 6.0 a Expressed as cc. 0.1 A' NaOH per 100 grams of

..

.. .

.. .. ..

...... ..

33 days 4.2

the titration tests, physical inspection was used to determine whether detectable rancidity had developed in any given sample. I n the absence of fat splitting all samples gave a distinctly tallowy odor, as was to be expected, owing to oxidation of the fat which would proceed rapidly a t the temperatures used for incubation. Experimental Results

The data on the titration values of the various series, together with those for the control flasks, which contained only the cream-sugar substrate with no added milk, are given in Tables 11, 111, IV, and V. MILK PAS-

-----TITRATION

Table 111 VALUES~----

Start 5 days 1 2 days 19 days 33 days Per cent Control 3.5 ... 3.5 3.5 At 61' C. for 30 minutes: 1 3.5 3.7 3.5 4.0 4.3 3 4.0 4.5 5.0 4.0 4.0 5 4.5 4.3 5.0 4.3 6.0 10 4.7 5.5 ;,? 6.0 4.8 15 5.0 6.5 5.0 J.J 7.5 5.2 7.0 5.5 9.5 20 6.2 At 64.5"C. for 30 minutes: 1 3.5 3.5 3.5 ... 3.7 4.0 4.0 4.0 3 ... 4.0 4.3 4.2 4.5 4.2 5 ... 5.2 10 ;.? 4.7 4.5 18 5.0 5.0 5.0 ... 3.3 20 5.5 5.5 7.0 9.0 14.0 0 Expressed as cc. 0.1 N NaOH per 100 grams of mix. TEURIZED

...

...

MILK PAS-

REMARKS Tallowy

12.0 17.0 24.0 33.0 41.0 47.0

Rancid Rancid Rancid Rancid Rancid Very rancid

11.5 18.0 22.0 32.0 40.0 47.0 mix.

Rancid Rancid Rancid Rancid Rancid Very rancid

Another series was carried out with raw skim milk taken direct from the separator in a local milk distributing plant. The course of the reaction was followed by a titration of 10gram samples as indicated above, at intervals of 5, 9, 12, 19, and 33 days. The results of titration are expressed on the basis of 100 grams of digestion mixture. I n conjunction with

Tallowy Tallowy Tallowy Tallowy Tallowy Tallowy Slightly rancid Tallowy Tallowy Tallowy Tallowy Tallowy Rancid

Table IV TITRATION VALUES4----

Start 5 days 12 days 19 days 33 days Per cent Control 4.0 4.0 4.0 4.0 4.2 At 67.5' C. for 30 minutes: 1 4.0 4.0 ... ... 3.8 3 4.2 4.0 ,. . ... 4.3 5 4.3 4.3 ... ... 4.2 10 4.5 4.5 ... 5.5 6.0 15 5.0 5.2 ... 6.0 7.0 20 5.5 6.0 6.5 7.5 8.5 At 74' C. for 1 minute: 1 3.5 3.5 ... ... 3.5 4.0 4.0 4.0 3 ... 4.2 4.5 5 ... ... 4.3 4.8 4.8 10 ... 5.0 6.0 5.0 5.2 15 ... 7.0 9.5 5.7 5.7 20 10.0 7.5 15.0 0 Expressed as cc. 0.1 N iYaOH per 100 grams of mix. TEURIZED

MILK PAS-

REMARKS

7---

...

30

Table I1 VALUES~-----9 12 19 days days days

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-----TITRATION

REMARKS Tallowy Tallowy Tallowy Tallowy Tallowy Tallowy Sliehtlvrancid - . Tallowy Tallowy Tallowy Tallowy Slightly rancid Rancid

Table V VALUESO----

Start 5 days 12 days 19 days 33 days Per cenl Control 3.5 3.5 3.5 At 74' C. for 5 minutes: 1 3.7 3.5 ... 4.0 3 4.0 4.0 ... , . 4.0 5 4.0 4.0 ... . , 4.5 10 4.5 4.5 ... 5.0 5.0 15 5.0 4.8 ... 5,0 7.5 20 5.5 5.5 6.5 7.5 10.0 At 8 5 O C. for 1 minute: 1 3.5 3.5 ,. 3.5 3 4.3 4.0 ... 4.0 ... 5 4.0 4.0 4.0 10 4.5 4.5 4.5 5.0 15 5.0 6.0 5.5 6.0 20 5.5 5.5 7.0 7.5 10.5 a Expressed a s cc. 0.1 N NaOH per 100 grams of mix. TEURIZED

...

...

... . .

... ... ...

. ... ...

REMARKS Tallowy Tallowy Tallowy Tallowy Tallowy Tallowy Slightly rancid Tallowy Tallowy Tallowy Tallowy Tallowy Slightly rancid

The bacterial counts per gram of the control flasks and of the various experimental flasks are given in Table VI. Table VI SAMPLE Control Raw whole milk Raw skim milk Milk pasteurized: At 61' C. At 64.5' C. At 67.5' C. At 74' C. for 1 minute At 74' C. for 5 minutes At 85' C.

TOTALBACTERIAL COUNT Start After 33 days 2400 300 500 300 200,000 400

...

100 200 100 100 200 200

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Table I1 shows a very marked increase in titratable acidity of water-soluble acids, the increase being roughly proportional to the percentage of added raw milk, the rate of increase falling off with the increasing time of incubation. These results confirm the findings of Rice and Markley (IS) and indicate that the samples of raw milk examined contained an enzyme capable of producing fat splitting. This is further borne out by the appearance of a rancid odor, in the production of which it is recognized that the lower fatty acids, most of which are water-soluble to some extent, are involved. Inspection of Tables 111, IV, and V indicates that the fatsplitting enzyme is very susceptible to heat destruction and that holding for 1 minute at 85" C. produces as much inactivation as holding for 30 minutes at 61 O C. It is possible that the slight increases observed in some flasks, particularly those in which the milk was subjected to severe heat treatment, were not the result of fat splitting, but were due to slight alterations in the minor constituents of the digestion medium, reactions which would be accelerated at the incubation temperature used. On the other hand, slight lipolysis may have occurred in some samples without being detected, as the titration samples in most mixes containing heated milk required less than 1 cc. of alkali for neutralization. There was also the further difficulty in obtaining the same shade of color in the opaque titration mixture when titrating a t intervals.

Vol. 22, No. 1

of this type would show an entirely different type of curve. Furthermore, Rice and Markley (14) showed that raw skim milk which produced lipolysis on their cream-sugar substrate failed to cause an increase in the acidity of a similar skim milk-sugar substrate during a 6-week incubation. This'indicates that the butterfat, not the lactose, is the source of the acid developed. Experiments with Powdered Milk

Experiments were carried out to ascertain whether or not lipase was present in dried milk, although it was anticipated from the earlier results with pasteurized milk that no enzyme activity would be detected. The milk used in this experiment was mixed herd milk which was pasteurized a t 63-64.5" C. for 30 minutes, followed by desiccation in a commercial drying unit by the Merrell-Merrell Gere spray process. I n the case of skim milk, the milk was separated prior to pasteurization. The powder was reliquefied with tap water to give fluid milk of normal solids content. One ounce by weight of powdered milk was used to 7.5 fluid ounces of water, and 0.75 ounce of powdered skim milk was used with the same quantity of water. A substrate was prepared as in the earlier experiments. Bacterial counts were also made on this series at the beginning and end of the incubation period. Three series of flasks were prepared as follows: 1, 3, 5, 10, 15, and 20 per cent of raw whole milk; 1,3,5,10, 15, and 20 per cent of reliquefied whole milk powder; 3, 10, and 20 per cent of reliquefied skim milk powder; cream-sugar controls. The results obtained during a 35-day incubation period are given in Table VII. Table VI1 MILK

ADDED

r---~ITRATION

VALUESn-----

Start

8days

4days

35 days

Per cent Control Raw milk: 1 3 5

10

15 20

6.5

6.5

6.5

6.5

6.8 7.0 7.0 7.5 8.0 8.9

8.0 9.0 13.0 17.5 21.5 24.0

12.0 19.5 21.5 28.5 36.5 36.5

16.0 26.0 23.0 32.0 34.0 30.0

Reliquefied skim milk: 3 7.3 10 7.2 20 8.5 a Expressed as cc. 0.1 N NaOH

Days of lncubafion

1

I

The decrease in the number of microorganisms shown in Table V I indicates that the acidity increase observed is not the result of bacterial action on the lactose. I n addition, no trace of yeast or mold growth was observable. The rate of acid production in the raw milk series is proportional to the quantities of raw milk added, a different condition than would result if it were a matter of inoculation with bacteria in varying quantities. In Figure 1 the amount of alkali required for the neutralization of the acid in each series is plotted against the time of incubation. The rate is rapid at first, but decreases markedly after a few days, owing probably to a decrease in enzyme activity. This is further indication that the increase in acidity is not the result of bacterial growth, as a reaction

6.0 7.5 7.5 7.5 9.0 11.0 per 100 grams of mis.

6.0 8.5 9.5

Bacterial counts were less than 100 per gram on all samples except that containing added 20 per cent reliquefied skim milk powder, which had an initial count of 400, decreasing to less than 100 at the end of the incubation period. Inspection of Table VI1 indicates that the sample of raw milk used here was not as high in lipolytic acitivity as those in the previous series. There was, nevertheless, a large increase in acidity. The reliquefied whole milk gave no increase, and the slight variations in titratable acidity of digestion mixes containing reliquefied skim milk are within the experimental error. It is evident that powdered milk made by this system of desiccation contains no lipase detectable by the methods of measurement employed. Hence lipolysis of the fat cannot be an important factor in the development of objectionable odors and flavors in this product. These experiments indicate that raw cow's milk normally contains an enzyme capable of splitting the natural glycerol fats, resulting in rancid odors and flavors. Heat treatment of the milk by either the "flash" or holding method of pasteurization results in destruction of this enzyme, the extent depending on the temperature and time of holding. It is more

January, 1930

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INDUSTRIAL A N D ENGINEERING CHEMISTRY

than likely that the natural lipase of milk is carried over into butter, cheese, and sweetened condensed milk, causing rancidity to develop during long periods of storage. Blanchet (2) found that lipase from castor seeds was sufficiently active to saponify the fat of castor oil even at -5” C. Hence cold storage will not serve to prevent rancidity development if heat insufficient to inactivate the lipase is employed during the process of manufacturing milk products. Summary

A true lipase is n normal constituent of raw cow’s milk, as indicated by increase in titratable acidity of high butterfat cream preserved with sucrose and by the development of rancid odors. Pasteurization inactivates lipolytic enzymes, the completeness of destruction depending on the time and temperature of holding. No lipase could be detected in powdered whole or skim milk desiccated by a drying system in which the fluid milk had undergone a preliminary pasteurization at 63-64.5” C. for 30 minutes. Rancid odors and flavors may be expected to develop in manufactured milk

products if the fluid milk is not subjected to sufficient heat treatment for inactivation of lipase. Literature Cited Beumer, Z . Kinderheilk., 38, 593 (1924). Blanchet, Bull. Agr. Intelligence, 9,767 (1917). Davidsohn, Z Kinderheilk., 8, 14 (1913). Friedjung and Hecht, Arch. Kinderheilk., 37, 233 (1903). Gillett, J . physiol. path. gln., 5, 503 (1903). Hippius, Jahrb. Kinderheilk., 61, 365 (1905). Marfan, Presse mbd., January 9, 1901, p. 13. Marfan and Gillett, Monafsschr. Kinderheilk., 1, 57 (1902). Moro, Jahrb. Kinderheilk., 56, 391 (1902). Palmer, Mo. Agr. Expt. Sta., Bull. 171, 20 (1920). Palmer. J . Dairy Sci., 5 , 51 (1922). Palmer, J . A m . Chem. Soc., 44, 1527 (1922). Rice and Markley, J . Dairy Sci., 6 , 64 (1922). Rice and Markley, Ibid., 5, 74 (1922). Rogers, U. S. Dept. Agr., Bur. Animal Ind., Bull. 57 (1904). Rogers, Berg, and Davis, U. S. Dept. Agr., Circ. 89, 319 (1902). Sammely, Oppenheimer’s “Handbuch der Biochemie,” Vol. I, p. 533 (1909). Spolverini, Reo. hyg. m l d . infant., 1, 262 (1902). Thatcher and Dahlberg, J . Agr. Research, 11, 437 (1917). Vandevelde, Rev. gbn. l a i t , 6, 414 (1907).

Evaporated and Condensed Milk from the Chemical and Nutritional Point of View‘ Frank E. Rice* EVAPORATED MILKASSOCIATION, CHICAGO, ILL.

Evaporated Milk milks at her grocer’s; the quantity buyer uses three or Evaporated milk is used to a greater extent than any of four more. With the many varieties of concentrated the other concentrated milks. It is important because, being milk on the market it is unfortunate that better methods of sterilized, it can be shipped to consumers thousands of miles naming them have not been adopted. In referring to any away from the production source. For evaporated milk the one it is usually necessary to employ one or more descriptive Government requires 7.8 per cent fat and 25.5 per cent total terms. milk solids, and the consumer expects a smooth, creamy con“Evaporated milk,” also called “unsweetened evaporated sistency without cream separation, and without sediment or milk,” is that form of concentrated milk which has been curd. I n fulfilling these specifications the manufacturer has doubly concentrated by evaporation of water, hermetically -succeeded, after years of experience, in processing what is sealed in cans, and sterilized by heat; no sugar or any other probably the most complex raw material there is. substance has been added. In order to consider more intelligently the chemistry of ‘(Sweetened condensed milk” is the name given to the evaporated milk, it is necessary to have clearly in mind the sweetened variety of concentrated milk. It is sometimes process of manufacture. A good quality of raw milk must be called just “condensed milk.” Cane or beet sugar acts as used. It must be clean and must have been kept cold from the preservative, from 40 to 46 per cent being present. Sweet- the time of production until it reaches the plant, for the prodened condensed milk is sold in 14- or 15-ounce tins and ucts of bacterial growth render the milk unstable to heat (36). also in barrels. That found in tins is practically always Government Standards of Concentrated whole milk while the barreled milk is usually skimmed. I n Table I-Consumption a n d Liquid Mllks some markets, however, there are demands for canned sweetCoNsUMPTIoN GOVERNMENT STANDARD ened condensed skim milk and condensed whole milk in KINDOF MILK 1928 Fat Milk solids Sucrose bulk. “Plain condensed milk” is one of the terms used to describe Pounds Per cent Per cent liquid concentrated milk sold in ordinary 10-gallon milk cans. Evaporated 1,248,492,000 7.8 25.5 None The container is not sealed and the milk is not sterile. It Sweetened condensed: 8.0 28.0 112,007,000 In tins Added is usually skim milk concentrated 3 or 4 to 1 and contains no I n barrels: 8.0 28.0 38,660,000 Whole Added sugar. This milk is sometimes called “evaporated milk154,723,000 . . . 2 4 . 0 Added Skim bulk goods;” government statistical reports use this term. N o standard None Plain condensed 236,961,000 These three are the most important of the concentrated Dry 151,262,000 liquid milks. They alone will be considered in this paper. The figures given in Table I show the extent to which these PREHEATING-The first step in the transformation of raw forms of milk are used. The government standards are also milk into evaporated milk consists in heating it either by inoutlined in this table. Table I1 gives also comparative data jecting live steam into a vat of the milk or by passing the which will be useful in obtaining a clear picture of the differ- milk through a heated coil. Careful control is exercised over ences between the concentrated milks. the preheating operation since it so largely influences the behavior of the milk later. When the incoming milk is pre1 Received October 9. 1929. heated at about 95’ C., the finished evaporated milk with2 Executive Secretary, Evaporated Milk Association.

T

HE housewife finds two kinds of liquid concentrated

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1