January, 1930
IA’DCSTRIAL AKD ENGINEERING CHEMISTRY
3, 4, and 5, while zeolite-softened water was used in runs 6, 7 , and 8. This feed water averaged about 7 p. p. m. dissolved
oxygen.
1. 2.
Table V - D e t e r m i n a t i o n of Concentration“ of Feed Water [At 10.54 kg. per sq. cm. (150 lbs. per sq. in.) pressure] ALKATOTAL C1 SO& LINITY NaCl WATER SOLIDS P.p.m. P.p.m. P.p.m. P.p.m. P . p . m . P , p . m . Feed water 145.2 Concentrate Distilled water 17.1 Concentration 8,5 Feed water 30.0 275.0 115.0 382.6 126.0 569.0 Concentrate 245.0 2232.0 140.0 3137.0 15.2 4115.0 Concentration 8.16 8.12 ... , 8.19 8.3 7.2 Feed water 29.0 275.0 110.0 382.3 163.9 607.0 Concentrate 262.4 2474.0 180.0 3461.0 1 8 . 3 4437.0 Concentration 9,05 9.0 , ,. , 9.06 9.0 7.3 Feed water 29.6 274,8 86,5 400.3 183.5 571.0 Concentrate 360.0 3359.0 274.0 4884.0 15.1 6132.0 Concentration 12.16 12.23 .,, ,. 12.2 12.2 10.7 Feed water 29.3 276.0 69.0 4 0 1 , 3 161.9 554.0 Concentrate 385.0 3616.0 350.0 5237.0 1 2 . 4 6654.0 Concentration 13.1 13.1 .,, ., 13.0 13.0 12.0 732.5 1581 784.0 287.0 Feed water 38.0 286.0 1 7 . 2 6716.0 2582.0 2543.0 6450.0 Concentrate 346.0 9.0 9.2 8.6 Concentration 9.1 8.9 S.95 144.5 1035.0 336.0 285.0 Feed water 147.0 984.6 17.2 8274.0 Concentrate 1 2 4 0 . 0 2 8 4 8 . 0 2 3 2 3 . 0 8287.0 8.49 8.16 8.4 8.0 Concentration 8.45 8.42 145.2 1331.0 Feed water 371.0 293.0 285.0 1326.0 Concentrate 3180.0 2549.0 2406.0 11256.0 1 7 . 0 10860.0 Concentration 8.58 8.56 8.45 8.50 8.5 8.1 a Coccentration equals ratio concentrate to feed water.
.
3.
.
4. 5. 6.
7. 8.
Distilled water gave the least corrosion. The amount of residual alkalinity in runs 2, 3, 4, and 5 increased and the
39
corresponding values of corrosion decreased accordingly. Zeolite-softened water always gave less corrosion than the lime-soda-softened water. This is probably due t o the higher alkalinity. Concentration of Feed Water
Various methods have been used in determining the coilcentration of boiler water. The determination of the ratio of chloride in the feed water to that in the boiler water seems to be in the best favor. A partial analysis of the feed mater and boiler water is given in Table V for the purpose of calculation of the concentrations. Neither the alkalinity nor the total solids could be considered with the lime-soda-softened water. However, the ratios of the values of chlorides, sulfate, sodium chloride, and water agree quite well. The values for sulfate could not be used on account of deposition of calcium sulfate within the boiler. Good agreement is obtained between all the values, with exception of total solids, in the case of the zeolite-softened water. This discrepancy is caused by the hydrolysis of sodium carbonate t o sodium hydroxide. Literature Cited Bosshard and Pfenniger, Chem - Z l g , 40, 5, 46, 63, 91 (1916) Fager and Reynolds, IND ENG CHEM, 21,357 (1929). Gunderson, Razlway R e s , 79, 335 (1926). Jacob and Kaesbohrer, Chem - Z t g , 35, 877 (1911). Lloyd, Trans A m lnst Mzn E n s , 39, 814 (1908) ( 6 ) Whitney, J. A m Chem Soc , 25, 394 (1903) (1) (2) (3) (4) (5)
DAIRY CHEMISTRY SYMPOSIUM Papers presented before the Division of Agricultural and Food Chemistry at the 78th Annual Meeting of the American Chemical Society, Minneapolis, Minn., September 9 to 13, 1929
Some Recent Advances in the Chemistry of Milk’ L. S. Palmer DIVISIONO F AGRICULTURAL BIOCHEMISTRY, UNIVERSITY
M
ILK and its various products continue to be of interest to chemists all over the world. During 1928 Chemical Abstracts covered 336 articles under the general headings of hlilk, Milk Analysis, Butter, Cheese, Ice Cream, Cream, and Buttermilk. These are exclusive of books and patents. Although I have arbitrarily selected the work reported in 1928 and 1929, it is obviously necessary to limit myself to just a few of the recent papers. For convenience, I have grouped the papers to be mentioned under several topical headings. Milk Analysis The determination of the fat content of milk and its products would appear to be so well established that no new problems would arise in connection Kith it. However, Thurston and Peterson (15) have shown that very large errors may be involved in applying to buttermilk any of the well-known gravimetric methods for fat, because buttermilk has a relatively high concentration of compound and derived lipides of the lecithin and sterol types, especially the former. Agricultural chemists, of course, have recognized for years that the ether extract of many plant and animal products is not true fat, but dairymen seemingly have assumed that the gravimet1 Received October 9, 1929. Published with the approval of the Director, as Paper No. 890, Journal Series, Minnesota Agricultural Experiment Station.
OF
MINNESOTA, ST. PAUL, MINN
ric procedures have given the true fat content of their products. Apparently dairymen will have to decide whether true fat or “ether extract” is what is desired and apply only such methods as give the correct value for the particular dairy product under examination. The Babcock test, which has long been held to be a practical rather than a strictly accurate method, apparently, under proper conditions will show the true fat content with greater accuracy than the more highly esteemed gravimetric methods. The problems presented here find special application in the estimation of fat losses in churning, as buttermilk seems to be especially rich in ether-extract substances that are not true fat. Factors Influencing the Composition of Milk
Variations that may be produced experimentally or that occur naturally in the composition of cow’s milk continue to attract workers both here and abroad. New light is being thrown on some very old problems. For instance, Davies and Provan ( 2 ) show that the changes which are supposed to occur almost invariably when cows go to pasture depend to a considerable extent on the system of winter feeding and are especially pronounced when the winter diet has been low in protein. The increased concentrations of casein, total and inorganic phosphorus, and total calcium following the change from low-protein winter feeding to pasture, were
INDUSTRIAL A N D ENGINEERING CHEMISTRY
40
accompanied by an increased flow of milk. These studies were made in Wales. Another old problem recently studied anew is the seasonal variation in the fat content of milk. Weaver and Matthews (17) show, by applying statistical methods to a large number of data collected by them at Iowa State College, that the fat test is affected chiefly by changes in the environmental temperature. This seems to explain the well-known fact that milk has a higher fat content in winter than in summer. The composition and properties of milk as a rule are not changed by slight variations in the food. Oil feeding is frequently reflected in the constitution of the milk fat, but not in the milk itself. However, fairly large additions of codliver oil (8 ounces daily) to a cow's diet appear to depress the fat content, a fact first observed by a group of British workers, including Golding and Drummond. This problem has recently been studied again by Mattick (6), who finds that the effect of the cod-liver oil is rather specific, other oils2 not showing this effect. Besides the fat depression, Mattick finds a lower titratable acidity, greater total calcium and total ash, lower diffusible calcium, and greater time required for coagulation with rennet. Some very interesting problems are suggested by these findings. Chemical and Physical Properties of Milk
Schneck (14) suggests a new method of studying the now well-known fact that changes in the degree of dispersion of milk constituents are dominantly characteristic of many processes which milk undergoes, such as creaming, churning, and cheese making. Schneck employs light transmittance for a study of changes in dispersion of both fat and casein. He finds that milk must be diluted 250 times before there is any definite relation between depth of solution and transmittance. The buffer action of milk has been another physico-chemical property studied. Come1 (1) employed goat's milk for a study which is of interest primarily because of the method employed. He adds increasing quantities of milk to a mixture of NaH2P04 and Na2HP04 and also to Na3P04 and plots the pH of the phosphates alone against the pH of the phosphates plus milk. Whittier (18) titrates milk and whey, the whey being prepared by acids or rennet, to determine the buffer action of casein. He plots (dB/dpH) against pH. He finds that casein exerts its maximum buffering a t pH 5.2 and concludes from his studies that casein is one of the chief buffers in milk. Of interest is his finding that paracasein does not show a point of maximum buffer intensity of any such magnitude as that exhibited by casein. Furthermore, in his study, it differs from casein in showing several dissociation constants, whereas casein shows only one in the region that he studied. Pyne (11) has published an important contribution to the subject of milk viscosity in a study of the action of viscogen (calcium sucrate) on milk and cream. He uses an ingenious technic to show that the higher viscosity resulting from viscogen additions is due primarily to precipitated Ca3(P0& and a mechanical carrying down of some of the suspended casein. Pyne discusses the view that greater cream layers rising in viscogen-treated milk are due to clumping of the fat globules and concludes that it is more probable that the effect is the result of the difficulty with which the phosphatecasein precipitate is "floated" by the entangled fat globules. This study was made in Cork. Inorganic Constituents of Milk
The recent discovery that under certain conditions copper is of importance in nutrition has raised again the question a Aractus oil was used.
Vol. 22, No. 1
of the copper content of milk. Quam and Hellwig (12) report that normal cow's milk may contain 0.37 to 0.50 mg. copper per liter. Commercially pasteurized milk appears to be considerably richer in copper. Buttermilk contains from 2.4 to 2.5 mg. per liter. Values similar to the latter were obtained for condensed or evaporated milk, probably owing to the copper surface of the vessels used in the processing. Citric acid has long been an interesting as well as important constituent of milk. Jerlor (4), however, has found that it does not occur in milk until the second day after parturition. This fact may be of some value in future studies on milk synthesis. One of the unsettled problems in connection with the salts of milk is the true effect of heat on the calcium and phosphorus compounds. Mattick and Hallett (7) held samples of milk for 30 minutes a t various temperatures between 105" and 209" F. (41" and 98" C.) and compared the total and diffusible phosphorus and calcium with that in unheated milk. No significant effects on phosphorus were noted up to 178" F. (81" C.) Above this about 3.5 per cent of the diffusible phosphorus was made nondiffusible. Higher temperatures produced no greater effects. More marked effects were noted on the calcium. At 135-140" F. (57-60" C.) about 0.6 per cent of the diffusible calcium was made nondiffusible. This rose to 2.0 per cent at 145-150" F. (63-66" C.), and above this temperature the results varied between 2.5 and 3.6 per cent, indicating that the effect of heat reaches a maximum, as in the case of phosphorus, but at a lower temperature. If diffusible calcium also represents ionized calcium, it is difficult to reconcile these relatively small changes with the general effects of heat on milk that have been frequently alleged to be caused by effects of heat on the calcium salts of milk. Chemical Constitution of Butter Fat
The most significant recent contribution in this field is the extensive study by Hilditch and Jones (3) of the constitution of Kew Zealand butter. These investigators have developed new methods for determining the ratios of the fatty acids present and also a semi-quantitative method for determining the manner in which the fatty acids are combined to form glycerides. Their method of study showed that the approximate composition of the fatty acids is butyric 3, caproic 2, caprylic 1, capric 2, lauric 4, myristic 11, palmitic 28, stearic 9, oleic 33.5, and linoleic 4.5 per cent. Acids less saturated than oleic were consistently present. These investigators found that butter fat contains about 30 per cent of fully saturated glycerides containing the same fatty acids as are found in the whole fat and in about the same proportions. Their study indicates the presence of about 36 per cent of mixed glycerides of the mono-oleo-disaturated type and the remainder of the di-oleo-monosaturated type. No appreciable quantities of simple glycerides were found. Chemical Composition of Butter
Although the skilled butter maker is now able to manufacture a product with remarkable uniformity of composition, there are still some very interesting problems dealing with the influences of the concentration of the various cream constituents on the churnability. Van Dam and Holwerda (16) in Holland have studied especially the effects of varying the concentration of casein and albumin and also of varying the concentration of acid (natural souring) on the completeness of churning. It is somewhat surprising to learn that a high-casein cream churns more exhaustively with less fat in the buttermilk than low-casein cream, whereas the
January, 1930
INDUXTRIAL A N D ENGINEERIhrG CHEMISTRY
reverse is true of the high- versus low-albumin cream. The differences in fat losses were much greater, however, for the casein experiment than for the albumin experiment, amounting to about 0.25 per cent fat for the former and to 0.020.05 per cent for the latter. The results of the casein experiment do not support Rahn's foam theory of churning because the high-casein cream churned more quickly than the low-casein cream. According to the foam theory, the longer churning should be more exhaustive because it allows more time for the fat to be drawn into the foam, where the agglutination of the fat is supposed to occur. I n the study of varying acidity the decrease in pH from 4.68 to 4.38 cut down the churning time and also the fat loss, the lower acid cream giving buttermilk averaging 0.54 per cent fat, against 0.37 per cent fat in the buttermilk from the higher acid cream.
41
the paracasein compounds present in milk a t the time of rennin action. It also remains to be determined how rennin effects these changes. It seems doubtful that the mere loss of 4 per cent of its nitrogen is sufficient to account for the known differences between casein and paracasein. One of the problems of great interest in connection with the clotting of milk by rennin is raised by the fact that the heating of milk changes its rapidity of clotting and also the character of the clot. Some new facts in connection with these phenomena have been discovered by Mattick and Hallett ( 7 ) . They heated different samples of milk for 30 minutes at various temperatures between 105" and 209' F. (41' and 98" C.) and observed the time required for thickening with rennin under standard conditions after cooling to 84" F. (29" C.) They observed that the lower heating temperatures employed 105-140" F. (41-60" C.) accelerate the time reChemistry of Casein quired for clotting the milk which has just been cooled to the standard coagulating temperature, but after the milk has Biological chemists are interested in the work of Linder- stood for a time the coagulating time becomes normal. The strom-Lang (5) on the individuality of casein, as this pro- time required for the recovery of the normal clotting time tein has long been held to be a specific substance. I n his becomes less as the heat-treating temperatures are increased. most recent paper on this subject Linderstrom-Lang gives Above 145' F. (63" C.) a retarding effect was noted at further evidence for his belief that casein is not an individual once. Moreover, there was a slowing-down effect at or above substance. This evidence is based on the fact that pure this temperature, the clotting time reaching a definite level casein permits a fractionation with slightly acidified 60 per only after some time. For instance, when the 30-minute cent alcohol (0.001-0.002 N HCl), the fractions showing a heat treatment was a t 165-170" F. ( 7 4 7 7 " C.), the full effect phosphorus content varying between 0.15 and 1.0 per cent, on the clotting time was not noted until after the milk had a tryptophane content varying between 1.4 and 2.3 per cent, been cooled for about 21/2 hours. When the heat treatment and a tyrosine content between 3.8 and 6.1 per cent. Smaller was a t 198" F. (92" C.) the milk would no longer clot after but significant variations were noted also in the arginine and it had stood cooled for 45 minutes. It seems obvious that lysine content. When the various fractions obtained were these remarkable effects are not explained by the old hypothecombined again with the undissolved portion and the casein sis that the effect of heat on the coagulability of milk by recovered, its physical and chemical properties were said to rennin is caused by the removal of soluble calcium salts. be identical with the original casein. These results, if verified, The recent studies of Mattick and Hallett show that the appear to have an important bearing on some of the differences amount of diffusible calcium removed from milk by heat obtained by investigators in studies on certain physicoreaches a maximum a t a temperature of 145-150' F. (63chemical properties of casein. It remains to be determined 66" C.). On the other hand, these results appear to show whether this plurality of casein is characteristic of the casein a very striking example of hysteresis of a colloidal system as it exists in milk or whether it is solely a characteristic and furnish further proof for the much more obvious fact of the substance after isolation. that the important heat effects in milk are on its chief colloidal system, its casein. Chemistry of Rennin Action These thoughts raise the question as to the extent to which The substance that results from the action of rennin on the action of rennin can be explained as a colloidal phenomecasein is known as paracasein. The reaction is not reversible. non. Richardson and Palmer (IS) approached this question Among the most important questions presented by this fact from the standpoint of electrokinetics, with very suggestive is the nature of this reaction. Is it purely biochemical or results. They found that calcium caseinate solutions a t or is it largely colloidal or are both types of reactions involved? below a pH of 6.8 exhibit a marked retardation in the cataWhich is the more important? Recent studies have thrown phoretic velocity of their protein after having been in contact with active rennin under milk clotting conditions, in new light on these questions. From the biochemical viewpoint, Porcher (IO) has offered comparison with control tests using boiled or inactivated evidence that Hammarstein's old view is, in part a t least, rennin. Above this pH no effects on migration velocity correct-namely, that rennin effects a partial hydrolysis were noted. Moreover, these investigators obtained evidence of the casein molecule during the conversion of casein to para- that below a pH of 6.9 rennin solutions alone show a catacasein. Porcher finds that a relatively constant, although phoresis of their colloidal material in the opposite direcsmall (about 4 per cent), fraction of the nitrogen always tion to that of casein, while above this pH the rennin colremains uncoagulated and may be recovered as a definite loids migrate in the same direction as casein. These facts substance with proteose-like properties. It remains to be indicate that active rennin carries the opposite electric charge determined, however, whether this splitting is essential for to that on casein micellae. I n addition, it appears that one the production of paracasein or whether it is merely inci- of the major effects of the rennin, perhaps the most impordental and reflects the close analogy between rennin and tant one in explaining milk clotting, is its power to reduce pepsin. the electric charge on the casein. As the casein charge is The outstanding chemical differences between casein and negative, the casein micellae would be rendered more sensiparacasein are in solubility, maximum acid- and base-bind- tive to other positively-charged cations, especially di-valent ing capacity, and titration curve. These differences, most ones, thus accounting for the coagulation. of which were pointed out by Palmer and Richardson (8), These results also led Richardson and Palmer to seek an have recently been confirmed by Pertzoff (9). It is ad- explanation of the effects of heat on milk in relation to rennin mittedly a matter of hypothesis rather than a demonstrated action. They found that heat increases the cataphoretic fact that these differences explain the great instability of velocity of casein solutions, which means that it increases
I N D U S T R I A L A N D ENGINEERING CHEMISTRY
42
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 T H E BORDENCOMPANY, 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
T
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
