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Human milk technology
HUMAN MILK TECHNOLOGY CLEMENT A.
SMITI-I,
M.D.
BOSTON, MASS.
E standards for directories for mothers' milk prepared by MacT HPherson and Talbot 1 and published in 1939 presented the methods then in use at the Boston Directory for the selection of mothers, the collection of their milk, its pasteurization, preservation, and distribution. I t had been f o u n d b y considerable experience that b y the use of those s t a n d a r d methods a hygienic p r o d u c t was obtained with efficiency and dispensed without possibility of deterioration or contamination. The Standards, or others applicable to all communities, such as are now being developed by the American Academy of Pediatrics and shortly to be published, may well be made the basis of any legal regulations governing the commercial sale of human milk, should the application of such legislation ever be deemed necessary. Besides the more orless administrative aspects of human milk supply to which they refer, there are the more technologic procedures which have been investigated and in certain instances adopted as part of the operation of the Boston Directory. With each new consideration of this sort, difficulty has been encountered in searching out and surveying a r a t h e r scattered literature. This fact, and the o p p o r t u n i t y for discussing a few procedures on the basis of experience with them, has p r o m p t e d the present article. I t is concerned p r i m a r i l y with (1) methods of identification and tests for adulteration of human milk ; (2) pasteurization and ~tests of pasteurization; (3) methods of p r e s e r v a t i o n ; and (4) the effect of some of these procedures on the food and vitamin values of the milk. IDENTIFICATION
It is obvious that in organizations to which lactating" women come for periodic pumping" or expression of milk, questions of adulteration will hardly arise. If,. on the other hand, the milk is expressed or pumped by the women at home and kept there for collection at daily intervMs, these questions will arise often enough so that some fairly simple means of ruling' out adulteration is necessary. The first need is to determine adulteration with water, the second, to ascertain the presence of milk from another species. Of these, the f o r m e r is much the more difficult, for the addition of water to milk is not easily detected. Methods which depend upon alterations of the freezing point and refractometrie analysis 2 are sometimes used in the dairy i n d u s t r y but present obvious difficulties. Variations in total solids or specific gravity will not, by themselves, be v e r y helpful in a physical system whose w a t e r content is n o r m a l l y from 85 to 90 per cent, since much more w a t e r m a y be added F r o m the Department of Pediatrics, H a r v a r d Medical School, and the Directory for Mother's Milk, Ine,, Boston, Mass. 616
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with a r e l a t i v e l y slight change in p r o p o r t i o n s resulting. F o r example, the adultera~ion of a q u a r t of milk h a v i n g an 85 p e r cent w a t e r content b y the addition of an additional pint of w a t e r will raise the p e r c e n t a g e o~ w a t e r in the t o t a l only to 90 per cent. The specific g r a v i t y would be r e c o g n i z a b l y a l t e r e d b y such a iorocedure, b u t this is also affected b y v a r i a t i o n s in the f a t content of the milk. Because m i l k f a t is lighter t h a n water, increases in the f a t content will lower the specific g r a v i t y ; thus a w o m a n m a y send in milk which has a low specific g r a v i t y not because i~ contains too m u c h w a t e r b u t because it contains an exces,s of one of its n o r m a l solid components. StrSm '~ has f o u n d t h a t a n o r m a l w o m a n ' s m i l k m a y v a r y in f a t content f r o m 2.2 to 8.8 p e r cent in samples taken on the same day, indeed only a :few hours apart, so t h a t any d e t e r m i n a t i o n s of specific g r a v i t y are of little value as evidence of milk dilution unless the f a t content of the sample is known. This can best be learned b y the s t a n d a r d B a b c o c k test, s a p r o c e d u r e in r e g u l a r use in d a i r y ] a b o r a t o r i e s P SP. G. i.034 3Z :50 Q i : : : : ; : " : : : - : ' " .%
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F i g . 1 . - - C h a r t d e v i s e d b y S t r D m ~ to i n d i c a t e a d u l t e r a t i o n of h u m a n m i l k w i t h water. The c e n t e r iine r e p r e s e n t s the m e a n of specific g r a v i t y a n d f a t content m e a s u r e m e n t s i n s e v e n t y - f i v e s a m p l e s of n o r m a l h u m a n m i l k , m o s t o f w h i c h r e s u l t e d in p o i n t s f a l l i n g w i t h i n t h e s h a d e d a r e a . P o i n t s outside this a r e a , if still w i t h i n the solid lines, a r e also c o m p a t i b l e w i t h n o r m a l v a r i a t i o n s , a l t h o u g h t h e y r a i s e s u s p i c i o n of d i l u t i o n . A n y m i l k w h o s e specific g r a v i t y a n d f a t c o n t e n t m e a s u r e m e n t s g i v e a p o i n t o u t s i d e t h e s o l i d l i n e s is c e r t a i n l y d i l u t e d w i t h w a t e r .
W i t h the f a t content thus d e t e r m i n e d and the specific g r a v i t y of the milk m e a s u r e d b y a m i l k h y g r o m e t e r ( l a c t o m e t e r ) , one can plot a point on the c h a r t devised by StrSm ~ (Fig. 1) a n d determine w h e t h e r it fails w i t h i n the area of normal, v a r i a t i o n or outside this in the region *So r o u t i n e a p r o c e d u r e is t h i s i n s u c h l a b o r a t o r i e s a n d so s t a n d a r d i z e d is t h e e q u i p m e n t r e q u i r e d t h a t m u c h t i m e a n d .effort m a y b e s p a r e d i f d u b i o u s s a m p l e s of b r e a s t m i l k c a n b e a n a l y z e d b y o n e of t h e s e i n s t i t u t i o n s .
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which signifies a diluted milk. It becomes clear from the chart that in m i l k which' originally had a high specific g r a v i t y and a m o d e r a t e f a t content (i.e., S.G. 1.032, fat 4 per cent), a considerable dilution with w a t e r could be p r a c t i c e d without detection; in fact, w a t e r m i g h t be a d d e d until the specific g r a v i t y is m a r k e d l y lowbred and still the c h a r t would show a point within the bounds of ~mrmal variation. Actually StrSm has calculated t h a t in certain milks a 32 p e r cent addition of w a t e r would still result in fat and specific g r a v i t y within the r a n g e of normal. On the other hand, one can u n d e r s t a n d f r o m the c h a r t how in milk with a 4 p e r cent f a t content a n d an original specific g r a v i t y of 1.028 a v e r y slight dilution would be discovered. In the d a i r y industry, the test f o r a d u l t e r a t i o n of c o w ' s milk by the addition of w a t e r is usually p e r f o r m e d b y d e t e r m i n i n g the f a t percentage and then a p p l y i n g the f o r m u l a :~ Lactometer o" 6
+ 1.2 f a t = p e r cent of total solids
P e r cent of total solids - per cent of f a t ~ per cent of solids not fat I f the figure for solids not fat be less t h a n 8 per cent, water.dilution is suspected ; if less than 7.7 per cent, w a t e r dilution is almost certain. The same formuJa could be applied to h u m a n milk, and under some circumstances might give a more aceurate check on water dilution t h a n the use of S t r a i n ' s diagram. Both methods of course require the determination of f a t content. More numerous procedures have been advocated for determining adulteration of h u m a n milk by the addition of cow's milk. Those with which we have had experience are discussed below, while several others mentioned are reviewed in the p a p e r b y Gnosspe!ius. ~ Since the average hydrogen-ion concentration of h u m a n milk gives a pI-I of about 7.0 or slightly above, and t h a t of cow's milk about 6.7, it is n a t u r a l that several reactions based u p o n this difference should have been advocated. The range of p H n o r m a l l y encountered in h m n a n milk extends, however, f r o m 6.9 to 7.4, and t h a t of cow's milk f r o m 6.5 to 7.2, Gso t h a t a wide degree of overlapping is possible. The nile blue sulfate test, modified from B a u e r ' s method, ~ is one of those dependent upon differences of acidity; it is p e r f o r m e d very simply by mixing equal p a r t s of the milk to be tested and a 0.25 per .cent solution of nile blue sulfate dye in water. I n the presence of h u m a n milk, the mixture in the test tube assumes a rosy pink or magenta color, but if cow's milk is present, the resultant color is a dull purplish blue. I n the past this reaction was a standa r d procedure at the Directory, even though samples known to be h u m a n milk sometimes gave a color practically indistinguishable f r o m the blue produced b y cow's milk. Studies carried out with the help of Dr. Eric Peters showed t h a t when such confusing specimens were boiled the color changed toward the pink one characteristic of h u m a n milk, while this *In this sense, lactometer
ing,
Usually
about
meal, s the last
32 (i.e.S.G.
~
1.032).
two figures of the specific gravity
r,ead-
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did not occur on heating those blue mixtures of known cow's milk and dye. The test p e r f o r m e d with this modification, and done by mixing one drop of milk and one of dye upon a white glass plate, seemed fairly rapid, economical of milk, and reasonably accurate. However, the reaction a p p a r e n t l y depends at least in p a r t on some unusual characteristic of certain stocks of nile blue sulfate, since a f t e r the international situation made it no longer possible to get the dye f r o m Germany, it was discovered t h a t the domestic material did not give reliable color changes. Another dye test advocated by Sol~ ~ consists of adding to a small sample of milk an equal portion of saturated solution of hematoxylin, such as is used in histologic work. A purple color, deepening upon standing, is to be expected in the case of h u m a n milk, while a bright yellow-red color should a p p e a r with cow's r_.~ilk. I n a series of determinations carried out with the assistance of Dr. Peters, the end-point of this test' was f o u n d to be less clear and the n u m b e r of confusing reactions somewhat more frequent t h a n had been encountered with the nile blue sulfate dye. Difference in hydrogen-ion concentration is also the basis of the neut r a l red reaction, advocated by Moro ~, and mentioned b y Gnosspelius '~ but not tried in our laboratory. I t is said to fail in detecting cow's milk unless it is present in ~more t h a n 10 p e r cent concentration. ,Jacobi 1~ advocated a color reaction supposedly due to differences in the t y p e of casein, because of which a purple color a p p e a r s when concentrated sulfuric acid is added to cow's milk but a brown color when the milk is human. A n o t h e r test based on casein differences has been described by Z i m m e r m a n n ~ a n d modified by Urbaeh, ~2 but since it requires fortyeight hours for the complete precipitation of clotted casein f r o m cow's milk ~upon the addition of dilute acid), it has not aroused our interest enough to stimulate its experimental use. The light refraction method of Kapeller and Gottfried, aa as described by Gnosspelius, requires expensive and complicated apparatus. Our interest was for some time a t t r a c t e d by the papers of K a y s e r ~ who a d v o c a t e d a differ'ent sort of color reaction, a p p e a r i n g when the m i l k is seen ~u n d e r ultra-violet illumination. Most samples of h u m a n milk assume a blue or violet flu0reseenee u n d e r such light, while cow's milk as a rule takes on a flaxen yellow shade. The method was found to be quick and easy of application in our laboratory, but there were usually a few specimens in every m o r n i n g ' s collection of breast milk which gave a more or less yellow color. Therefore, a large n u m b e r of specimens f r o m nursing mothers in the Boston L y i n g - I n Hospital were examined, but even irt these proved samples of h u m a n milk the yellow color occasionally appeared. We were never able to learn what interfering substance caused the peculiar reactions. K a y s e r ~5 later suggested t h a t they followed the addition of liver to the m a t e r n a l diet and m i g h t indieate the presence of lactoflavine, b u t t h a t d~d n.ot always p r o v e to be the ease in o u r experience.
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Such biologic tests that are based on anaphylactic shock and complement fixation are c o m p h c a t e d and time-consuming. The injection of milk f r o m the cow into the blood s t r e a m of a n o t h e r species (rabbit) produces precipitins, which arc, according to Gnosspelius, prinlarily dne to the casein portion of the milk protein though also to the less specific laetalbumin and laetoglobulin fractions. Since the caseins of human and cow's milk have certain f u n d a m e n t a l differences, rabbit serum containing the precipitins so induced will react specifically with cow's milk to form a definite precipitate not appearing when such serum is mixed with milk from any other, such as the h u m a n species. The titer of r a b b i t serum, and thus the sensitivity of the test, can be raised in direct p r o p o r t i o n to the n u m b e r of times the rabbit is injected with milk. On the basis of some experimentation., Gnosspelins advised the use of five inoculations of 5 e.c. of cow's milk each, on alternate days, a f t e r which the serum is tested for its titer ; if satisfactory, the rabbit is bled and the serum mixed with a preservative and stored for use. Serum so prepared by Gnosspelius and mixed on a glass slide with an equal a m o u n t of milk gave a definite and grossly visible precipitate f o r m a t i o n in the presence of as little as a 5 per cent dilution with cow's milk. Since casein is thermostable, the reaction is also given with milk t h a t has been boiled. The caseins are so consistently and definitely specific t h a t the mother, even b y consuming large quantities of cow's milk, cannot secrete breast milk w h i c h gives a i a l s e positive reaction. This test has been found v e r y satisfactory in recent use at the D~rect o r y in Boston. Serum from rabbits given only three intravenous inoculations on alternate days has been of such strength that w h e n one drop of it is mixed with a drop of breast milk adulterated with 20 per cent cow's milk, a precipitate forms, and with more sensitizing" doses a higher titer is produced. One rabbit will yield up to 50 e.c. of serum ; for such an amount the addition of 0.5 e.e. of a 1 per cent aqueous solution of neutral acriflavine is a satisfactory preservative, after which the serum m a y be stored in sealed capillary tubes of about 15 cm. in length. One such tube, containing 0.2 e.c. of serum or less, can be used for testing some t w e n t y samples of milk if the drops d~elivered from it are not too large. Preciloitation, when it occurs, can be observed easily if the m i x t u r e with milk is made with some stirring and the glass plate on which this is done is obliquely illuminated from slightly below but held with a d a r k b a c k g r o u n d directly behind it. It is well to v e r i f y this b y testing one drop of known cow's milk each time human milk is so examined. I t is obvious t h a t this test will not d'etect the presence of milk from some other species, such as that from the goat, although of course a serum for that purpose could be made. I t m a y also be said f r o m a practical s t a n d p o i n t that the adulteration of breast milk with cow's milk, if practiced for economic gain by a w o m a n selling her milk, might well be detected or at least suspected from the bacteria count in the a d u l t e r a t e d portions,. It would be diffi-
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cult to dilute with cow's milk without raising the bacteria count at the same time unless such an expensive grade of milk was used for the a d u l t e r a n t that the f r a u d would not be economically worth attempting. PASTEURIZATION
Milk collected under satisfactory home conditions and even that p u m p e d or expressed u n d e r the most ideal conditions at dispensaries m a y contain a certain n u m b e r of bacteria, the count being from 500 to 3000 colonies per cubic centimeter in that kept oil ice in the home f o r daily colleetion2 So rapidly may organisms increase t h e r e a f t e r t h a t some f o r m of sterilization is required even though the m~lk be dispensed at once. F o r this, o r d i n a r y boiling may be used as an expedienL In the 1939 Standards, MaePherson and Talbot ~ advise pasteurization at between 140 ~ and 145 ~ F. for t h i r t y minutes, a process whieh has p r o v e d s a t i s f a c t o r y during" m a n y years of operation, since immediately t h e r e a f t e r the milk is sterile and if k e p t p r o p e r l y ehilled will remain so for at least forty-eight hours. I n Massachusetts the legal s t a n d a r d . f o r the pasteurization of cow's milk has been decreed as a t e m p e r a t u r e of 142 ~ F. or above, maintained for at least t h i r t y minu t e s ; b u t the so-called -" flash " pasteurization of 161 ~ P. for sixteen seconds is atso and might be applied to h u m a n milk. The ba, eteria count constitutes a fair eheek on the efflcaey of the pasteurization process. A n o t h e r cheek is the so-called phosphatase test as devised b y K a y and Graham 1~ and later improved and simplified b y Scharer. ~ The reaction depends upon t h e phosphomono-esterase n a t u r a l l y present in raw milk and' its almost complete destruction at the t e m p e r a t u r e of pasteurization. At the D i r e c t o r y in Boston the efficacy of pasteurization is checked b y this method by testing a few random samples from the pasteurizer every two weeks.*
legal
PRESERVATION
[Phe difficulties of meeting a sudden demand for breast milk and the advantages of saving a temporarily excessive supply have called forth various methods of preservation. The dairy i n d u s t r y has met the same problem by condensation with sugar, b y partial evaporation, and by complete d r y i n g to a powder. There is no reason why these same processes cannot be applied to the handling of human milk, but a search for practical and simple procedures using m o r e easily available apparatus has led to experimentation with and sometimes adoption of other methods. Methods such as t h e so-called "lyophilization ! ' s or d r y i n g from the frozen state, which are increasingly used for the preservation of sera and other biologic materials, are being studied as possible means of milk preservation. In m a n y cities some form of apparatus for this purpose *A c o n v e n i e n t set of a p p a r a t u s f o r t h e t e s t c a n b e o b t a i n e d f r o m t h e Applied R e s e a r c h I n s t i t u t e , 15 W e s t 34th ,Street, N e w Y o r k , N . Y.
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is available at one or more medical centers; by its use, milk in units of S ounces or more can he reduced to a d r y or somewhat oily powder or cake in a process requiring f r o m twelve to twenty-four hours. The milk can then either be stored in the original containers f r o m which evaporation took place or the powder f r o m several of these containers can be scraped out under sterile precautions and combined in a single larger one. The volume of the original milk must be known so that p r o p e r dilution or reeonstitution for use can be p e r f o r m e d b y the addition of the p r o p e r a m o u n t of sterile distilled water. I n our experience with a few samples so p r e p a r e d f o r us b y Dr. S. H. A r m s t r o n g , ~ the reconstituted milk f o r m e d a r a t h e r poor emulsion, with collection of the f a t into larger, coarser globules t h a n in the unprocessed milk. This effect, although quite obvious, was less noticeable in samples of h u m a n milk t h a n in those of cow's milk and did not seem to disturb the digestion of an infant to whom 16 ounces of such human milk was fed. The taste of cow's milk thus dried and then reconstituted was abnormal. Possibly if h u m a n milk were homogenized by forcing it t h r o u g h a very fine a p e r t u r e before freezing' and d r y i n g processes were applied, this f a t separation might be prevented. Folsom, ~9 working at the Memorial Hospital in New York, has noted a tendency for cow's milk (and p a r t i c u l a r l y cream) desiccated from 'the frozen state to become rancid during storage unless Mr is kept f r o m it. F o r this reason and others it might well be preferable to keep the dried powder in v a c u u m in the original container f r o m which its water portion was d r a w n off. This of course requires t h e expense of larger receptacle s and ones which m a y be sealed off, as. by the melting of a narrow neck, to preserve pressure conditions within. On the whole, it appears t h a t any such desiccating process, whether it be of the "lyophile, ''~s " c r y o chem,"~~ or " d e s i v a c " ~ l type, will p r o b a b l y prove too expensive for the regular processing of breast milk. All such methods of course do offer the distinct advantage of dispensing with specially chilled space for the product during storage or transportation. Breast milk m a y be processed in several ways if it is to be held therea f t e r n e a r or at subfreezing temperatures. One simple method was that described in 1933 by B r o a d h u r s t and Duncan. 22 The milk, t r i p l y pasteurized by heating to 175 ~ F. for t h i r t y minutes on three successive days, could then be kept in a r e f r i g e r a t e d (but not frozen) state two years or longer without bacteriologic or chemical change b e i n g noted, and, as Scheuer and Duncan ~3 showed, could t h e r e a f t e r be successfully fed to svaall infants. D u r i n g a 10rig series of studies carried on at the Directory for Mothe r ' s Milk between 1922 and 1933, E m e r s o n and others, 24 a f t e r first reporting" t h a t c o n t e m p o r a r y processes for d r y i n g breast milk h a d not proved satisfactory, then described a method for the solidification of milk b y quick freezingY 4t' I n that f o r m it could be held at subfreezing * D e p a r t m e n t of P h y s i c a l Chemistry, H a r v a r d Medical School.
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temperatures with suspension of bacterial or chemical activity more or less indefinitely and thawed to a state entirely similar to its prefrozen condition. This freezing procedure *.~ attained its r a p i d i t y of action by virtue of the small size of units frozen and by the use of cakes of solidified carbon dioxide (dry ice) to transmit very low temperatures to the milk as poured into the shallow depressions of an aluminum mold. The small cakes o r wafers o]~ frozen milk (each one about 10 c.e. in volume) a f t e r being taken f r o m t h e mold e0uld be collected in sterile glass jars~ 8 ounces o f milk occupying a 16-ounce jar, because of the loose packing of the wafers. 'This process proved entirely successful and was in regular use at this and Other mother's m i l k stations for eight or~ years, d u r i n g which time the only modifications of the process involved have been changes in the temperature of the storage space. It was thought at first that an important amount of slow bacterial multiplication might be avoided if the frozen milk were held at an extremely low temperature, and for t h i s r e a s o n a storage at -26 ~ C. (-15 ~ F.) was f o r m e r l y advoeated. T h r o u g h the kindness and i n t e r e s t of Prof. B. E. Proctor, of t h e Massachusetts I n s t i t u t e of Tecl~nology, studies were made in ]937 by B e r t r a n d Bennison of that institution which indicated that a holding temperature of -12 ~ C. (10 ~ F./ could be cgnsidered safe and satisfactory. The recent development of frozen food lockers in which f a m i l y provisions may he kept has made excellent holding s p a c e available in certain cities. The Variety Club Mother's Milk B a n k in Memphis, Tenn., keeps some 2,400 ounce s of frozen milk in this way a t a temperature of between L18 ~ and -23 ~ C. {0 ~ to -10 ~ F.). Since the q u i c k - f r e e z i n g method has the drawback of requiring considerable time and handling a s well as excessive storage space because the frozen units store so loosely, a number of experiments have. been carried out at the Directory to determine whether mil,k might be frozen in larger units, perhaps with somewhat less r a p i d i t y but nevertheless quickly enough so. that its digestibility on thawing might be unimpaiored. So general is the impression that milk frozen slowly is indigestible ~ that every, attemp t v~as made to keep the process as rapid as possible. Thus it was found that fourteen minutes would suffice for the freezing of milk in an 8-ounce unit if the process were carried out in a d r y ice-acetone bath at a t e m p e r a t u r e of -70 ~ C. (-94 ~ F.), the milk being first sealed in a tin can or a p y r e x bottle and then immersed in such a bath. 2~ At the suggestion of Professor Proctor and with the cooperation of Mr. David Oreenlie, it was also found possible to freeze breast milk in conveniently rectangular 8-ounce bricks by p o u r i n g it into latex or "cryoVae" bagst and setting these in a metal mold s u r r o u n d e d b y t h e dry ice-acetone mixture. All of these preparations, even though their freezing required perhaps five times as long as did the original process, re* T h e ~ o r d e n C o m p a n y of N e w Y o r k C i t y c o n t r o l s t h e p a t e n t on t h i s f r e e z i n g prdcesS and the molds used. t F u r n i S h e d b y t h e ]Dewey a n d A l m y C h e m i c a l Co.
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suited in milk which was of satisfactory appearance and bacteria content upon thawing, and which was fed to a number of premature infants with complete success. In 1934 a brief and not v e r y detailed article by Stern and Lieberman "6 announced that those authors had fed a number of premature infants with breast milk which had a p p a r e n t l y been frozen in the ice compartment of an ordinary electric refrigerator, and in 1940 we learned that at the University Hospital at Iowa City it was the practice to preserve breast milk by freezing it comparatively slowly at about -7 ~ C. (18 ~ F.) in the hardening compartment of an ice cream machine. On the assurance ~of Dr. E. D. Plass that the product had been fed with success to p r e m a t u r e and mature newborn infants, the same process was instituted at the Boston Directory. A t the temperature used at the University of Iowa Hospital, it was found that 8- or 16-ounce bottles of breast milk became solidly frozen in from t h i r t y to sixty minutes. The milk upon thawing presented an emulsion of fat not quite so fine as that of the original fluid, the ~at globules being larger when examined microscopically. There was also a slight tendency for the protein to separate in a few fine threads of curd, but both of these manifestations became less noticeable when the milk was warmed for feeding. The preservation procedure is particularly simple if the laboratory maintains a cold cabinet or locker for holding frozen milk, since any surplus amount of pasteurized milk may simply be left in a sterile (Mason) j a r at this holding t e m p e r a t u r e until frozen. To prevent breakage of glass, it is wise to cover the jar with sterile gauze d u r i n g the process or to set the glass lid on loosely. A f t e r the milk is solidified, the jar can be sealed with its glass lid and rubber ring, wrapped, labeled, and replaced in the storage. Thus far, more than a thousand ounces have been preserved in this way at the Boston Directory to be thawed and fed to small prem a t u r e infants with entirely satisfactory results, even though the freezing time is ten to twenty times that which the earlier workers strove to achieve. Besides the experience at Iowa City an article 27 has recently been published describing a similar r e f r i g e r a t o r technique for preservation of breast milk that is in satisfactory operation at the Presbyterian Hospital in Chicago. THE EFFECTS OF PROCESSING ON ]?OODVALUE AS shown by the practical tests of successful ntilization by small infants, there is no evidence that the protein, carbohydrate, or fat content of milk is altered importantly by pas,teurization or by quick freezing. 2~g Although slowly frozen cow's milk is traditionally indigestible by the human infant, there is strikingly little in the way of controlled observation upon that point in the literature of pediatrics. Two studies of the effect of feeding frozen milk to animals have been found in the dairy journals. In one, 2s evaporated milk allowed to congeal at -5 ~ F. and held at that temperature for several months was melted and fed to young
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rats. During a twelve-week period on this diet, the rats gained as well as did a control group. In another series of experiments by Munkwitz, Berry, and Boyer, ~9 pasteurized cow's milk, frozen slowly, thawed, and fed to rats was shown to be well tolerated. Thus there seems to be no positive evidence available to prove that freezing disturbs the digestibility or nutritional value of milk. " A l l the known vitamins are present in breast milk, the quantity being greatly influenced by the intake of the mother.' '~ That at least one of these substances, vitamin C, is more or less destroyed by the ternperatures of'pasteurization is well known. Since breast milk is usually pasteurized before being preserved by freezing, the question of the effect of cold preserving processes on this vitamin in breast milk is somewhat academic. In a few studies made at the Boston Directory by Dr. Harold Teel, little effect could~be shown to have resulted by rapid freezing on the small amount of ascorbie acid in pasteurized milk. MunselP ~ states that vitamin A is not altered by the temperatures of boiling and thus of pasteurization, and also that the content of this vitamin is not decreased by freezing and thawing. Some feeding experiments with human milk preserved by various methods led E ddy and Morris ~1 to the conclusion that frozen human milk retained its vitamin A content better than that which had been preserved by evaporation to dryness. A few measurements made for us directly from preserved and freshly pasteurized specimens of human milk by Dr. J. F. McCreary in 1940 showed a loss of about bne-third' of the vitamin A to occur whether the milk was frozen v e r y rapidly or comparatively slowly. The various components of the B complex which are present in human milk have not been studied with respect to the effects of temperature, although Rose ~2 gives some evidence to suggest that whereas pasteurization might lower the thiamine and riboflavin values, freezing would probably bring about no further reduction. Although human milk does possess a certain antirachitic value, its vitamin D content is so low that no investigations of the effect of physical processes upon that substance seem warranted. In general, there is reason to conclude that the necessary process of pasteurization lowers the amount of vitamin C in human milk just as in cow's milk but does not otherwise alter its nutritional value, while the useful preservation process of slow freezing may also diminish the content of vitamin A. Whether this is sufficiently depressed to require a vitamin A supplement (as well as one for vitamins C and D) might well be investigated. REFERENCES 1. M a e P h e r s o n , Cornelia, and Talbot, F . B . : J. PEDIAa'. 15: 461, 1939. 2. S t a n d a r d Methods for the E x a m i n a t i o n of D a i r y Products, ed. 7, Hew York, 1939, A m e r i c a n Public H e a l t h Association. 3. StrSm, Y.: Nord. reed. 2: 1938, 1939. 4. Woodman, A. G.: Food Analysis, ed. 3, New York, 1931, McGraw-Hill Book Co., Inc.
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5. Gnosspelius, A.: Arch. Dis. Childhood 15: 171, 1940. 6. Holt, L. E., Jr, and Macintosh, R.: Holt's Diseases of Infancy "tnd Childhood~ ed. 11, New York~ 1940, D. Appleton-Century Company, Inc. 7. Bauer: Monatschr. f. I(inderh. II: 474~ 1912. 8. So]~, A.: ~onatschr. f. I~inderh. 63: 383, 1935. 9. Z/lorD, E.: iKiinchen, reed. Wchnschr. 59: 2553~ 1912. i0. Jacobi~ W.: IYionatsehr, f. i~inderh. 23: 44, 1922. 11. Zimmermann~ ~/[.: Ztsehr. f. ~inderh. 45: 310~ 1928. 12. Urbach, If.: Klin. Wchnschr. 17. 633, 19,38. 13. ](appeller~ G., and Gottfried~ A.: ;LYliinchen. Ined. Wchnsehr. 67: 82[3, 1920. 14. I~ayser~ M . E . : ]V[iinehen. reed. w c h n s e h r . 74: 2142, 1927. 15. I(ayser, IVL E.: Deutsche med. Wchnschr. 63: 712, 1937. 16. I~ay, H. D., and Graham, W. 1~.~ Jr.: J. Dairy Research 6: 191, 1935. 17. Scharer, It.: J. Dairy Research 21: 21~ 1938. 18. Folsom, T. 1~.: W a r Medicine 1: 342, 1941. 19. Folsom, T . R . : Personal communication. 20. Flosdorf, E. W., and ]V[udd, S.: J. Immuno]. 34: 469, 1938. 21. Flosdorf, E. W , Stokes~ F. J., and Z[udd, S.: J . A . ~I. A. 115: 1095, 1940. 22. Broadhurst, J , a n d Duncan~ J . E . : Am. J. N u r s i n g 33: 453, 1933. 23. Schener~ L. A., and Duncan, ~. E.: Am. J. Dis. Child. 51: 249, 1936. 242 a. Emerson, P . W . : J . A . 1Vi. A. 78: 641, 1922. b. Smith~ L. W.~ and Emerson~ P. W.: Boston IYl. and S. J. 191: 938, 1924. c. Smith~ L.W.: J. Biol. Chem. 61: 625, 1924. d. Emerson, P.W.: Am. J. Dis. Child. 30: 769, 1925. e. Smith, L. W., and EmerSon~ P.W.: Am. *J. Dis. Child. 31: 1, 1926. f. Emerson, P. W., and Platt ~ W.: J. PEDIAT. 2: 472, 1933. g. Emerson~ P.W.: ~ew England J. )/led. 209: 893, 1933. 25. Boland, F. E.: The Technology of Frozen Milk. Thesis submitted to the IV[assachusetts Institute of Technology, 1938. 26, Stern, A, and Lieberman~ D.P.: Arch. Pediat. 51: 799~ 1934. 27. Watson~ lY[ary L.: Am. J. Nursing 41: 672, 1941. 28. Louder, E. A., and Smith~ L.F.: J. Dairy Science 15: 113, 1932. 29. ]Yiunkwitz~ R. C., Berry, IV[. II.~ and B'oyer, W. C. : IV[d. Agrie. Exper. Station Bulletin 344~ 1933. 30. Munsell, If. E.: In The Vitamins, a Symposium, Chicago, 1939, American 1V[edical Association. 31. Eddy~ W. I-I, and ~orris~ S . G . : J. PE])IAT. 4: 208, 1934. 32. l~ose, lYL S.: J . A . ~ . A. 114: 1356~ 1940.
SMITI-I,
M.D.
BOSTON, MASS.
E standards for directories for mothers' milk prepared by MacT HPherson and Talbot 1 and published in 1939 presented the methods then in use at the Boston Directory for the selection of mothers, the collection of their milk, its pasteurization, preservation, and distribution. I t had been f o u n d b y considerable experience that b y the use of those s t a n d a r d methods a hygienic p r o d u c t was obtained with efficiency and dispensed without possibility of deterioration or contamination. The Standards, or others applicable to all communities, such as are now being developed by the American Academy of Pediatrics and shortly to be published, may well be made the basis of any legal regulations governing the commercial sale of human milk, should the application of such legislation ever be deemed necessary. Besides the more orless administrative aspects of human milk supply to which they refer, there are the more technologic procedures which have been investigated and in certain instances adopted as part of the operation of the Boston Directory. With each new consideration of this sort, difficulty has been encountered in searching out and surveying a r a t h e r scattered literature. This fact, and the o p p o r t u n i t y for discussing a few procedures on the basis of experience with them, has p r o m p t e d the present article. I t is concerned p r i m a r i l y with (1) methods of identification and tests for adulteration of human milk ; (2) pasteurization and ~tests of pasteurization; (3) methods of p r e s e r v a t i o n ; and (4) the effect of some of these procedures on the food and vitamin values of the milk. IDENTIFICATION
It is obvious that in organizations to which lactating" women come for periodic pumping" or expression of milk, questions of adulteration will hardly arise. If,. on the other hand, the milk is expressed or pumped by the women at home and kept there for collection at daily intervMs, these questions will arise often enough so that some fairly simple means of ruling' out adulteration is necessary. The first need is to determine adulteration with water, the second, to ascertain the presence of milk from another species. Of these, the f o r m e r is much the more difficult, for the addition of water to milk is not easily detected. Methods which depend upon alterations of the freezing point and refractometrie analysis 2 are sometimes used in the dairy i n d u s t r y but present obvious difficulties. Variations in total solids or specific gravity will not, by themselves, be v e r y helpful in a physical system whose w a t e r content is n o r m a l l y from 85 to 90 per cent, since much more w a t e r m a y be added F r o m the Department of Pediatrics, H a r v a r d Medical School, and the Directory for Mother's Milk, Ine,, Boston, Mass. 616
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with a r e l a t i v e l y slight change in p r o p o r t i o n s resulting. F o r example, the adultera~ion of a q u a r t of milk h a v i n g an 85 p e r cent w a t e r content b y the addition of an additional pint of w a t e r will raise the p e r c e n t a g e o~ w a t e r in the t o t a l only to 90 per cent. The specific g r a v i t y would be r e c o g n i z a b l y a l t e r e d b y such a iorocedure, b u t this is also affected b y v a r i a t i o n s in the f a t content of the milk. Because m i l k f a t is lighter t h a n water, increases in the f a t content will lower the specific g r a v i t y ; thus a w o m a n m a y send in milk which has a low specific g r a v i t y not because i~ contains too m u c h w a t e r b u t because it contains an exces,s of one of its n o r m a l solid components. StrSm '~ has f o u n d t h a t a n o r m a l w o m a n ' s m i l k m a y v a r y in f a t content f r o m 2.2 to 8.8 p e r cent in samples taken on the same day, indeed only a :few hours apart, so t h a t any d e t e r m i n a t i o n s of specific g r a v i t y are of little value as evidence of milk dilution unless the f a t content of the sample is known. This can best be learned b y the s t a n d a r d B a b c o c k test, s a p r o c e d u r e in r e g u l a r use in d a i r y ] a b o r a t o r i e s P SP. G. i.034 3Z :50 Q i : : : : ; : " : : : - : ' " .%
,28 26
!.'x \
Z4 22 J.02O
0
I
2
3
4
5
6
7
8
9
I 0 ~i~ F A T .
F i g . 1 . - - C h a r t d e v i s e d b y S t r D m ~ to i n d i c a t e a d u l t e r a t i o n of h u m a n m i l k w i t h water. The c e n t e r iine r e p r e s e n t s the m e a n of specific g r a v i t y a n d f a t content m e a s u r e m e n t s i n s e v e n t y - f i v e s a m p l e s of n o r m a l h u m a n m i l k , m o s t o f w h i c h r e s u l t e d in p o i n t s f a l l i n g w i t h i n t h e s h a d e d a r e a . P o i n t s outside this a r e a , if still w i t h i n the solid lines, a r e also c o m p a t i b l e w i t h n o r m a l v a r i a t i o n s , a l t h o u g h t h e y r a i s e s u s p i c i o n of d i l u t i o n . A n y m i l k w h o s e specific g r a v i t y a n d f a t c o n t e n t m e a s u r e m e n t s g i v e a p o i n t o u t s i d e t h e s o l i d l i n e s is c e r t a i n l y d i l u t e d w i t h w a t e r .
W i t h the f a t content thus d e t e r m i n e d and the specific g r a v i t y of the milk m e a s u r e d b y a m i l k h y g r o m e t e r ( l a c t o m e t e r ) , one can plot a point on the c h a r t devised by StrSm ~ (Fig. 1) a n d determine w h e t h e r it fails w i t h i n the area of normal, v a r i a t i o n or outside this in the region *So r o u t i n e a p r o c e d u r e is t h i s i n s u c h l a b o r a t o r i e s a n d so s t a n d a r d i z e d is t h e e q u i p m e n t r e q u i r e d t h a t m u c h t i m e a n d .effort m a y b e s p a r e d i f d u b i o u s s a m p l e s of b r e a s t m i l k c a n b e a n a l y z e d b y o n e of t h e s e i n s t i t u t i o n s .
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which signifies a diluted milk. It becomes clear from the chart that in m i l k which' originally had a high specific g r a v i t y and a m o d e r a t e f a t content (i.e., S.G. 1.032, fat 4 per cent), a considerable dilution with w a t e r could be p r a c t i c e d without detection; in fact, w a t e r m i g h t be a d d e d until the specific g r a v i t y is m a r k e d l y lowbred and still the c h a r t would show a point within the bounds of ~mrmal variation. Actually StrSm has calculated t h a t in certain milks a 32 p e r cent addition of w a t e r would still result in fat and specific g r a v i t y within the r a n g e of normal. On the other hand, one can u n d e r s t a n d f r o m the c h a r t how in milk with a 4 p e r cent f a t content a n d an original specific g r a v i t y of 1.028 a v e r y slight dilution would be discovered. In the d a i r y industry, the test f o r a d u l t e r a t i o n of c o w ' s milk by the addition of w a t e r is usually p e r f o r m e d b y d e t e r m i n i n g the f a t percentage and then a p p l y i n g the f o r m u l a :~ Lactometer o" 6
+ 1.2 f a t = p e r cent of total solids
P e r cent of total solids - per cent of f a t ~ per cent of solids not fat I f the figure for solids not fat be less t h a n 8 per cent, water.dilution is suspected ; if less than 7.7 per cent, w a t e r dilution is almost certain. The same formuJa could be applied to h u m a n milk, and under some circumstances might give a more aceurate check on water dilution t h a n the use of S t r a i n ' s diagram. Both methods of course require the determination of f a t content. More numerous procedures have been advocated for determining adulteration of h u m a n milk by the addition of cow's milk. Those with which we have had experience are discussed below, while several others mentioned are reviewed in the p a p e r b y Gnosspe!ius. ~ Since the average hydrogen-ion concentration of h u m a n milk gives a pI-I of about 7.0 or slightly above, and t h a t of cow's milk about 6.7, it is n a t u r a l that several reactions based u p o n this difference should have been advocated. The range of p H n o r m a l l y encountered in h m n a n milk extends, however, f r o m 6.9 to 7.4, and t h a t of cow's milk f r o m 6.5 to 7.2, Gso t h a t a wide degree of overlapping is possible. The nile blue sulfate test, modified from B a u e r ' s method, ~ is one of those dependent upon differences of acidity; it is p e r f o r m e d very simply by mixing equal p a r t s of the milk to be tested and a 0.25 per .cent solution of nile blue sulfate dye in water. I n the presence of h u m a n milk, the mixture in the test tube assumes a rosy pink or magenta color, but if cow's milk is present, the resultant color is a dull purplish blue. I n the past this reaction was a standa r d procedure at the Directory, even though samples known to be h u m a n milk sometimes gave a color practically indistinguishable f r o m the blue produced b y cow's milk. Studies carried out with the help of Dr. Eric Peters showed t h a t when such confusing specimens were boiled the color changed toward the pink one characteristic of h u m a n milk, while this *In this sense, lactometer
ing,
Usually
about
meal, s the last
32 (i.e.S.G.
~
1.032).
two figures of the specific gravity
r,ead-
SMITH:
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619
did not occur on heating those blue mixtures of known cow's milk and dye. The test p e r f o r m e d with this modification, and done by mixing one drop of milk and one of dye upon a white glass plate, seemed fairly rapid, economical of milk, and reasonably accurate. However, the reaction a p p a r e n t l y depends at least in p a r t on some unusual characteristic of certain stocks of nile blue sulfate, since a f t e r the international situation made it no longer possible to get the dye f r o m Germany, it was discovered t h a t the domestic material did not give reliable color changes. Another dye test advocated by Sol~ ~ consists of adding to a small sample of milk an equal portion of saturated solution of hematoxylin, such as is used in histologic work. A purple color, deepening upon standing, is to be expected in the case of h u m a n milk, while a bright yellow-red color should a p p e a r with cow's r_.~ilk. I n a series of determinations carried out with the assistance of Dr. Peters, the end-point of this test' was f o u n d to be less clear and the n u m b e r of confusing reactions somewhat more frequent t h a n had been encountered with the nile blue sulfate dye. Difference in hydrogen-ion concentration is also the basis of the neut r a l red reaction, advocated by Moro ~, and mentioned b y Gnosspelius '~ but not tried in our laboratory. I t is said to fail in detecting cow's milk unless it is present in ~more t h a n 10 p e r cent concentration. ,Jacobi 1~ advocated a color reaction supposedly due to differences in the t y p e of casein, because of which a purple color a p p e a r s when concentrated sulfuric acid is added to cow's milk but a brown color when the milk is human. A n o t h e r test based on casein differences has been described by Z i m m e r m a n n ~ a n d modified by Urbaeh, ~2 but since it requires fortyeight hours for the complete precipitation of clotted casein f r o m cow's milk ~upon the addition of dilute acid), it has not aroused our interest enough to stimulate its experimental use. The light refraction method of Kapeller and Gottfried, aa as described by Gnosspelius, requires expensive and complicated apparatus. Our interest was for some time a t t r a c t e d by the papers of K a y s e r ~ who a d v o c a t e d a differ'ent sort of color reaction, a p p e a r i n g when the m i l k is seen ~u n d e r ultra-violet illumination. Most samples of h u m a n milk assume a blue or violet flu0reseenee u n d e r such light, while cow's milk as a rule takes on a flaxen yellow shade. The method was found to be quick and easy of application in our laboratory, but there were usually a few specimens in every m o r n i n g ' s collection of breast milk which gave a more or less yellow color. Therefore, a large n u m b e r of specimens f r o m nursing mothers in the Boston L y i n g - I n Hospital were examined, but even irt these proved samples of h u m a n milk the yellow color occasionally appeared. We were never able to learn what interfering substance caused the peculiar reactions. K a y s e r ~5 later suggested t h a t they followed the addition of liver to the m a t e r n a l diet and m i g h t indieate the presence of lactoflavine, b u t t h a t d~d n.ot always p r o v e to be the ease in o u r experience.
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Such biologic tests that are based on anaphylactic shock and complement fixation are c o m p h c a t e d and time-consuming. The injection of milk f r o m the cow into the blood s t r e a m of a n o t h e r species (rabbit) produces precipitins, which arc, according to Gnosspelius, prinlarily dne to the casein portion of the milk protein though also to the less specific laetalbumin and laetoglobulin fractions. Since the caseins of human and cow's milk have certain f u n d a m e n t a l differences, rabbit serum containing the precipitins so induced will react specifically with cow's milk to form a definite precipitate not appearing when such serum is mixed with milk from any other, such as the h u m a n species. The titer of r a b b i t serum, and thus the sensitivity of the test, can be raised in direct p r o p o r t i o n to the n u m b e r of times the rabbit is injected with milk. On the basis of some experimentation., Gnosspelins advised the use of five inoculations of 5 e.c. of cow's milk each, on alternate days, a f t e r which the serum is tested for its titer ; if satisfactory, the rabbit is bled and the serum mixed with a preservative and stored for use. Serum so prepared by Gnosspelius and mixed on a glass slide with an equal a m o u n t of milk gave a definite and grossly visible precipitate f o r m a t i o n in the presence of as little as a 5 per cent dilution with cow's milk. Since casein is thermostable, the reaction is also given with milk t h a t has been boiled. The caseins are so consistently and definitely specific t h a t the mother, even b y consuming large quantities of cow's milk, cannot secrete breast milk w h i c h gives a i a l s e positive reaction. This test has been found v e r y satisfactory in recent use at the D~rect o r y in Boston. Serum from rabbits given only three intravenous inoculations on alternate days has been of such strength that w h e n one drop of it is mixed with a drop of breast milk adulterated with 20 per cent cow's milk, a precipitate forms, and with more sensitizing" doses a higher titer is produced. One rabbit will yield up to 50 e.c. of serum ; for such an amount the addition of 0.5 e.e. of a 1 per cent aqueous solution of neutral acriflavine is a satisfactory preservative, after which the serum m a y be stored in sealed capillary tubes of about 15 cm. in length. One such tube, containing 0.2 e.c. of serum or less, can be used for testing some t w e n t y samples of milk if the drops d~elivered from it are not too large. Preciloitation, when it occurs, can be observed easily if the m i x t u r e with milk is made with some stirring and the glass plate on which this is done is obliquely illuminated from slightly below but held with a d a r k b a c k g r o u n d directly behind it. It is well to v e r i f y this b y testing one drop of known cow's milk each time human milk is so examined. I t is obvious t h a t this test will not d'etect the presence of milk from some other species, such as that from the goat, although of course a serum for that purpose could be made. I t m a y also be said f r o m a practical s t a n d p o i n t that the adulteration of breast milk with cow's milk, if practiced for economic gain by a w o m a n selling her milk, might well be detected or at least suspected from the bacteria count in the a d u l t e r a t e d portions,. It would be diffi-
SMITH:
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cult to dilute with cow's milk without raising the bacteria count at the same time unless such an expensive grade of milk was used for the a d u l t e r a n t that the f r a u d would not be economically worth attempting. PASTEURIZATION
Milk collected under satisfactory home conditions and even that p u m p e d or expressed u n d e r the most ideal conditions at dispensaries m a y contain a certain n u m b e r of bacteria, the count being from 500 to 3000 colonies per cubic centimeter in that kept oil ice in the home f o r daily colleetion2 So rapidly may organisms increase t h e r e a f t e r t h a t some f o r m of sterilization is required even though the m~lk be dispensed at once. F o r this, o r d i n a r y boiling may be used as an expedienL In the 1939 Standards, MaePherson and Talbot ~ advise pasteurization at between 140 ~ and 145 ~ F. for t h i r t y minutes, a process whieh has p r o v e d s a t i s f a c t o r y during" m a n y years of operation, since immediately t h e r e a f t e r the milk is sterile and if k e p t p r o p e r l y ehilled will remain so for at least forty-eight hours. I n Massachusetts the legal s t a n d a r d . f o r the pasteurization of cow's milk has been decreed as a t e m p e r a t u r e of 142 ~ F. or above, maintained for at least t h i r t y minu t e s ; b u t the so-called -" flash " pasteurization of 161 ~ P. for sixteen seconds is atso and might be applied to h u m a n milk. The ba, eteria count constitutes a fair eheek on the efflcaey of the pasteurization process. A n o t h e r cheek is the so-called phosphatase test as devised b y K a y and Graham 1~ and later improved and simplified b y Scharer. ~ The reaction depends upon t h e phosphomono-esterase n a t u r a l l y present in raw milk and' its almost complete destruction at the t e m p e r a t u r e of pasteurization. At the D i r e c t o r y in Boston the efficacy of pasteurization is checked b y this method by testing a few random samples from the pasteurizer every two weeks.*
legal
PRESERVATION
[Phe difficulties of meeting a sudden demand for breast milk and the advantages of saving a temporarily excessive supply have called forth various methods of preservation. The dairy i n d u s t r y has met the same problem by condensation with sugar, b y partial evaporation, and by complete d r y i n g to a powder. There is no reason why these same processes cannot be applied to the handling of human milk, but a search for practical and simple procedures using m o r e easily available apparatus has led to experimentation with and sometimes adoption of other methods. Methods such as t h e so-called "lyophilization ! ' s or d r y i n g from the frozen state, which are increasingly used for the preservation of sera and other biologic materials, are being studied as possible means of milk preservation. In m a n y cities some form of apparatus for this purpose *A c o n v e n i e n t set of a p p a r a t u s f o r t h e t e s t c a n b e o b t a i n e d f r o m t h e Applied R e s e a r c h I n s t i t u t e , 15 W e s t 34th ,Street, N e w Y o r k , N . Y.
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is available at one or more medical centers; by its use, milk in units of S ounces or more can he reduced to a d r y or somewhat oily powder or cake in a process requiring f r o m twelve to twenty-four hours. The milk can then either be stored in the original containers f r o m which evaporation took place or the powder f r o m several of these containers can be scraped out under sterile precautions and combined in a single larger one. The volume of the original milk must be known so that p r o p e r dilution or reeonstitution for use can be p e r f o r m e d b y the addition of the p r o p e r a m o u n t of sterile distilled water. I n our experience with a few samples so p r e p a r e d f o r us b y Dr. S. H. A r m s t r o n g , ~ the reconstituted milk f o r m e d a r a t h e r poor emulsion, with collection of the f a t into larger, coarser globules t h a n in the unprocessed milk. This effect, although quite obvious, was less noticeable in samples of h u m a n milk t h a n in those of cow's milk and did not seem to disturb the digestion of an infant to whom 16 ounces of such human milk was fed. The taste of cow's milk thus dried and then reconstituted was abnormal. Possibly if h u m a n milk were homogenized by forcing it t h r o u g h a very fine a p e r t u r e before freezing' and d r y i n g processes were applied, this f a t separation might be prevented. Folsom, ~9 working at the Memorial Hospital in New York, has noted a tendency for cow's milk (and p a r t i c u l a r l y cream) desiccated from 'the frozen state to become rancid during storage unless Mr is kept f r o m it. F o r this reason and others it might well be preferable to keep the dried powder in v a c u u m in the original container f r o m which its water portion was d r a w n off. This of course requires t h e expense of larger receptacle s and ones which m a y be sealed off, as. by the melting of a narrow neck, to preserve pressure conditions within. On the whole, it appears t h a t any such desiccating process, whether it be of the "lyophile, ''~s " c r y o chem,"~~ or " d e s i v a c " ~ l type, will p r o b a b l y prove too expensive for the regular processing of breast milk. All such methods of course do offer the distinct advantage of dispensing with specially chilled space for the product during storage or transportation. Breast milk m a y be processed in several ways if it is to be held therea f t e r n e a r or at subfreezing temperatures. One simple method was that described in 1933 by B r o a d h u r s t and Duncan. 22 The milk, t r i p l y pasteurized by heating to 175 ~ F. for t h i r t y minutes on three successive days, could then be kept in a r e f r i g e r a t e d (but not frozen) state two years or longer without bacteriologic or chemical change b e i n g noted, and, as Scheuer and Duncan ~3 showed, could t h e r e a f t e r be successfully fed to svaall infants. D u r i n g a 10rig series of studies carried on at the Directory for Mothe r ' s Milk between 1922 and 1933, E m e r s o n and others, 24 a f t e r first reporting" t h a t c o n t e m p o r a r y processes for d r y i n g breast milk h a d not proved satisfactory, then described a method for the solidification of milk b y quick freezingY 4t' I n that f o r m it could be held at subfreezing * D e p a r t m e n t of P h y s i c a l Chemistry, H a r v a r d Medical School.
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temperatures with suspension of bacterial or chemical activity more or less indefinitely and thawed to a state entirely similar to its prefrozen condition. This freezing procedure *.~ attained its r a p i d i t y of action by virtue of the small size of units frozen and by the use of cakes of solidified carbon dioxide (dry ice) to transmit very low temperatures to the milk as poured into the shallow depressions of an aluminum mold. The small cakes o r wafers o]~ frozen milk (each one about 10 c.e. in volume) a f t e r being taken f r o m t h e mold e0uld be collected in sterile glass jars~ 8 ounces o f milk occupying a 16-ounce jar, because of the loose packing of the wafers. 'This process proved entirely successful and was in regular use at this and Other mother's m i l k stations for eight or~ years, d u r i n g which time the only modifications of the process involved have been changes in the temperature of the storage space. It was thought at first that an important amount of slow bacterial multiplication might be avoided if the frozen milk were held at an extremely low temperature, and for t h i s r e a s o n a storage at -26 ~ C. (-15 ~ F.) was f o r m e r l y advoeated. T h r o u g h the kindness and i n t e r e s t of Prof. B. E. Proctor, of t h e Massachusetts I n s t i t u t e of Tecl~nology, studies were made in ]937 by B e r t r a n d Bennison of that institution which indicated that a holding temperature of -12 ~ C. (10 ~ F./ could be cgnsidered safe and satisfactory. The recent development of frozen food lockers in which f a m i l y provisions may he kept has made excellent holding s p a c e available in certain cities. The Variety Club Mother's Milk B a n k in Memphis, Tenn., keeps some 2,400 ounce s of frozen milk in this way a t a temperature of between L18 ~ and -23 ~ C. {0 ~ to -10 ~ F.). Since the q u i c k - f r e e z i n g method has the drawback of requiring considerable time and handling a s well as excessive storage space because the frozen units store so loosely, a number of experiments have. been carried out at the Directory to determine whether mil,k might be frozen in larger units, perhaps with somewhat less r a p i d i t y but nevertheless quickly enough so. that its digestibility on thawing might be unimpaiored. So general is the impression that milk frozen slowly is indigestible ~ that every, attemp t v~as made to keep the process as rapid as possible. Thus it was found that fourteen minutes would suffice for the freezing of milk in an 8-ounce unit if the process were carried out in a d r y ice-acetone bath at a t e m p e r a t u r e of -70 ~ C. (-94 ~ F.), the milk being first sealed in a tin can or a p y r e x bottle and then immersed in such a bath. 2~ At the suggestion of Professor Proctor and with the cooperation of Mr. David Oreenlie, it was also found possible to freeze breast milk in conveniently rectangular 8-ounce bricks by p o u r i n g it into latex or "cryoVae" bagst and setting these in a metal mold s u r r o u n d e d b y t h e dry ice-acetone mixture. All of these preparations, even though their freezing required perhaps five times as long as did the original process, re* T h e ~ o r d e n C o m p a n y of N e w Y o r k C i t y c o n t r o l s t h e p a t e n t on t h i s f r e e z i n g prdcesS and the molds used. t F u r n i S h e d b y t h e ]Dewey a n d A l m y C h e m i c a l Co.
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suited in milk which was of satisfactory appearance and bacteria content upon thawing, and which was fed to a number of premature infants with complete success. In 1934 a brief and not v e r y detailed article by Stern and Lieberman "6 announced that those authors had fed a number of premature infants with breast milk which had a p p a r e n t l y been frozen in the ice compartment of an ordinary electric refrigerator, and in 1940 we learned that at the University Hospital at Iowa City it was the practice to preserve breast milk by freezing it comparatively slowly at about -7 ~ C. (18 ~ F.) in the hardening compartment of an ice cream machine. On the assurance ~of Dr. E. D. Plass that the product had been fed with success to p r e m a t u r e and mature newborn infants, the same process was instituted at the Boston Directory. A t the temperature used at the University of Iowa Hospital, it was found that 8- or 16-ounce bottles of breast milk became solidly frozen in from t h i r t y to sixty minutes. The milk upon thawing presented an emulsion of fat not quite so fine as that of the original fluid, the ~at globules being larger when examined microscopically. There was also a slight tendency for the protein to separate in a few fine threads of curd, but both of these manifestations became less noticeable when the milk was warmed for feeding. The preservation procedure is particularly simple if the laboratory maintains a cold cabinet or locker for holding frozen milk, since any surplus amount of pasteurized milk may simply be left in a sterile (Mason) j a r at this holding t e m p e r a t u r e until frozen. To prevent breakage of glass, it is wise to cover the jar with sterile gauze d u r i n g the process or to set the glass lid on loosely. A f t e r the milk is solidified, the jar can be sealed with its glass lid and rubber ring, wrapped, labeled, and replaced in the storage. Thus far, more than a thousand ounces have been preserved in this way at the Boston Directory to be thawed and fed to small prem a t u r e infants with entirely satisfactory results, even though the freezing time is ten to twenty times that which the earlier workers strove to achieve. Besides the experience at Iowa City an article 27 has recently been published describing a similar r e f r i g e r a t o r technique for preservation of breast milk that is in satisfactory operation at the Presbyterian Hospital in Chicago. THE EFFECTS OF PROCESSING ON ]?OODVALUE AS shown by the practical tests of successful ntilization by small infants, there is no evidence that the protein, carbohydrate, or fat content of milk is altered importantly by pas,teurization or by quick freezing. 2~g Although slowly frozen cow's milk is traditionally indigestible by the human infant, there is strikingly little in the way of controlled observation upon that point in the literature of pediatrics. Two studies of the effect of feeding frozen milk to animals have been found in the dairy journals. In one, 2s evaporated milk allowed to congeal at -5 ~ F. and held at that temperature for several months was melted and fed to young
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rats. During a twelve-week period on this diet, the rats gained as well as did a control group. In another series of experiments by Munkwitz, Berry, and Boyer, ~9 pasteurized cow's milk, frozen slowly, thawed, and fed to rats was shown to be well tolerated. Thus there seems to be no positive evidence available to prove that freezing disturbs the digestibility or nutritional value of milk. " A l l the known vitamins are present in breast milk, the quantity being greatly influenced by the intake of the mother.' '~ That at least one of these substances, vitamin C, is more or less destroyed by the ternperatures of'pasteurization is well known. Since breast milk is usually pasteurized before being preserved by freezing, the question of the effect of cold preserving processes on this vitamin in breast milk is somewhat academic. In a few studies made at the Boston Directory by Dr. Harold Teel, little effect could~be shown to have resulted by rapid freezing on the small amount of ascorbie acid in pasteurized milk. MunselP ~ states that vitamin A is not altered by the temperatures of boiling and thus of pasteurization, and also that the content of this vitamin is not decreased by freezing and thawing. Some feeding experiments with human milk preserved by various methods led E ddy and Morris ~1 to the conclusion that frozen human milk retained its vitamin A content better than that which had been preserved by evaporation to dryness. A few measurements made for us directly from preserved and freshly pasteurized specimens of human milk by Dr. J. F. McCreary in 1940 showed a loss of about bne-third' of the vitamin A to occur whether the milk was frozen v e r y rapidly or comparatively slowly. The various components of the B complex which are present in human milk have not been studied with respect to the effects of temperature, although Rose ~2 gives some evidence to suggest that whereas pasteurization might lower the thiamine and riboflavin values, freezing would probably bring about no further reduction. Although human milk does possess a certain antirachitic value, its vitamin D content is so low that no investigations of the effect of physical processes upon that substance seem warranted. In general, there is reason to conclude that the necessary process of pasteurization lowers the amount of vitamin C in human milk just as in cow's milk but does not otherwise alter its nutritional value, while the useful preservation process of slow freezing may also diminish the content of vitamin A. Whether this is sufficiently depressed to require a vitamin A supplement (as well as one for vitamins C and D) might well be investigated. REFERENCES 1. M a e P h e r s o n , Cornelia, and Talbot, F . B . : J. PEDIAa'. 15: 461, 1939. 2. S t a n d a r d Methods for the E x a m i n a t i o n of D a i r y Products, ed. 7, Hew York, 1939, A m e r i c a n Public H e a l t h Association. 3. StrSm, Y.: Nord. reed. 2: 1938, 1939. 4. Woodman, A. G.: Food Analysis, ed. 3, New York, 1931, McGraw-Hill Book Co., Inc.
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5. Gnosspelius, A.: Arch. Dis. Childhood 15: 171, 1940. 6. Holt, L. E., Jr, and Macintosh, R.: Holt's Diseases of Infancy "tnd Childhood~ ed. 11, New York~ 1940, D. Appleton-Century Company, Inc. 7. Bauer: Monatschr. f. I(inderh. II: 474~ 1912. 8. So]~, A.: ~onatschr. f. I~inderh. 63: 383, 1935. 9. Z/lorD, E.: iKiinchen, reed. Wchnschr. 59: 2553~ 1912. i0. Jacobi~ W.: IYionatsehr, f. i~inderh. 23: 44, 1922. 11. Zimmermann~ ~/[.: Ztsehr. f. ~inderh. 45: 310~ 1928. 12. Urbach, If.: Klin. Wchnschr. 17. 633, 19,38. 13. ](appeller~ G., and Gottfried~ A.: ;LYliinchen. Ined. Wchnsehr. 67: 82[3, 1920. 14. I~ayser~ M . E . : ]V[iinehen. reed. w c h n s e h r . 74: 2142, 1927. 15. I(ayser, IVL E.: Deutsche med. Wchnschr. 63: 712, 1937. 16. I~ay, H. D., and Graham, W. 1~.~ Jr.: J. Dairy Research 6: 191, 1935. 17. Scharer, It.: J. Dairy Research 21: 21~ 1938. 18. Folsom, T. 1~.: W a r Medicine 1: 342, 1941. 19. Folsom, T . R . : Personal communication. 20. Flosdorf, E. W., and ]V[udd, S.: J. Immuno]. 34: 469, 1938. 21. Flosdorf, E. W , Stokes~ F. J., and Z[udd, S.: J . A . ~I. A. 115: 1095, 1940. 22. Broadhurst, J , a n d Duncan~ J . E . : Am. J. N u r s i n g 33: 453, 1933. 23. Schener~ L. A., and Duncan, ~. E.: Am. J. Dis. Child. 51: 249, 1936. 242 a. Emerson, P . W . : J . A . 1Vi. A. 78: 641, 1922. b. Smith~ L. W.~ and Emerson~ P. W.: Boston IYl. and S. J. 191: 938, 1924. c. Smith~ L.W.: J. Biol. Chem. 61: 625, 1924. d. Emerson, P.W.: Am. J. Dis. Child. 30: 769, 1925. e. Smith, L. W., and EmerSon~ P.W.: Am. *J. Dis. Child. 31: 1, 1926. f. Emerson, P. W., and Platt ~ W.: J. PEDIAT. 2: 472, 1933. g. Emerson~ P.W.: ~ew England J. )/led. 209: 893, 1933. 25. Boland, F. E.: The Technology of Frozen Milk. Thesis submitted to the IV[assachusetts Institute of Technology, 1938. 26, Stern, A, and Lieberman~ D.P.: Arch. Pediat. 51: 799~ 1934. 27. Watson~ lY[ary L.: Am. J. Nursing 41: 672, 1941. 28. Louder, E. A., and Smith~ L.F.: J. Dairy Science 15: 113, 1932. 29. ]Yiunkwitz~ R. C., Berry, IV[. II.~ and B'oyer, W. C. : IV[d. Agrie. Exper. Station Bulletin 344~ 1933. 30. Munsell, If. E.: In The Vitamins, a Symposium, Chicago, 1939, American 1V[edical Association. 31. Eddy~ W. I-I, and ~orris~ S . G . : J. PE])IAT. 4: 208, 1934. 32. l~ose, lYL S.: J . A . ~ . A. 114: 1356~ 1940.