Whev as a Source of Vitamins J
and Vitamin Products G. C. SWPLEE The Borden Company, Bainbridge, N. Y.
Whey or milk serum following removal of the fat and casein may serve as the starting material for the further isolation of lactalbumin, milk sugar, milk phosphates, mixed sterols, cholesterol, fat-soluble vitamin K (prothrombin), and water-soluble vitamins, particularly commercial quantities of lactoflavin (riboflavin). The presence of an oestrogenic substance has been demonstrated in fractions derived from whey. The multiplicity of vitamins found in the whey fraction after removal of protein and milk sugar is adequate in water-soluble factors to support growth, reproduction, and lactation. Eight successive generations of white rats have been maintained with a normal life cycle on a restricted experimental diet in which the whey vitamin fraction supplemented with rice polish served as the sole source of all vitamins except the fat-soluble factors carried by a small percentage of cod liver oil.
T
HE status of milk as a food and especially as the sole diet of the very young necessarily implies that it contains all of the entities essential to sustain life and promote growth. Nutritional and biochemical research has been the means by which specific knowledge of the properties and physiological merits of the components of milk has become available. The integration of milk into its gross constituents may be accomplished by a sequence of steps which yield products of dietary or industrial value in themselves or which serve as the basic raw materials for further processing, concentration, or isolation of specific entities. These steps for which practical procedures have been worked out or are in the course of perfection may be briefly enumerated as follows: The fat in the form of cream is removed by centrifugal means, followed by removal of the casein from the skimmed milk by any one of numerous precipitation procedures. The resulting whey or milk serum may be considered as a secondary starting material which can be desiccated and conserved for utilization as such, or as the basic material for the recovery of lactalbumin, milk sugar, a substantial proportion of the original milk calcium, and phosphorus and vitamin concentrates. The milk serum remaining after removal of the fat and casein contains substantially 94 per cent water, 0.8 per cent protein, 0.2 per cent lipide matter, 4.85 per cent lactose, 0.5 per cent mineral ash, and a variety of trace substances, including known vitamin entities. Further integrated, this mixture yields derivatives which are rapidly becoming primary objects of physiological investigations and extended use in dietary and pharmaceutical products. The lactalbumin may be removed by proper pH adjustment and heat coagulation or, depending upon the physical condition in which it is desired to obtain this protein fraction, precipitation by pH adjustment alone may be employed. If appropriate methods are used, the lactalbumin fraction is obtained with a minimum of occluded material. Following the removal of the albumin, the serum may be subjected to a further pH adjustment, preferably without heat; it then yields other proteins, a
large portion of the soluble calcium, and substantially all of the organic and inorganic phosphorus. This fraction will also contain as high as 18 to 20 per cent (dry basis) of prosthetically bound lipide matter other than butterfat. After the removal of this protein fraction, the serum may be concentrated under vacuum for the removal of milk sugar according to well established methods. Following the removal of the milk sugar, a readily workable mixture of water-soluble substances remains, including the water-soluble milk minerals, highly dispersed protein in small amounts, some lactose, a trace of sterols prosthetically bound with the protein, and a mixture of concentrated entities, including the water-soluble vitamins inherent in the original milk. It is this final concentrated mixture, as well as the albumin fraction, which will be discussed from the standpoint of vitamin content and dietary value.
Vitamin Activity The first investigations relating to the vitamin activity of this material were carried out in the early twenties when knowledge of these dietary essentials was substantially limited to the fabsoluble and water-soluble classification; also, methods of study lacked the precision inherent in many of the biological methods perfected since that time. Nevertheless the results obtained from the inclusion of small amounts of this concentrated water-soluble milk fraction, in basal rations purified by the best means then available, were so conclusive in yielding evidence of a high concentration of vitamins that systematic investigations with improved methods have proceeded continually since that time. One of the striking early experiments was somewhat elementary. A basal ration was compounded with the gross components of mdk, butterfat, casein, and lactose, further fortified with minerals and cod liver oil. Animals receiving this mixture failed to grow, became emaciated, and died within a few weeks. Other animals receiving the same basal mixture to which was added a small percentage of the concentrated whey fraction grew a t a phenomenal rate and re238
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mained in good condition during observation periods of several weeks or months. This evidence was adequate to prove that growth-promoting and life-sustaining substances inherent in milk could be segregated from the gross components and retain a high degree of activity. These results invited comparison with other products known to contain the water-solulile vitamins. It was determined that basal rations containing from 7 to 12 per cent of the crude milk vitamin fraction promoted growth as satisfactorily as an equivalent amount of dry yeast (4),and also permitted reproduction, lactation, and normal rearing of the young. As the nature of the vitamin B complex became better understood, more detailed studies of the milk fraction were necessary. It was revealed that, even though it contained all water-soluble factors, some were present in greater amounts than others. Comparative experiments involving the use of rice polish, known to contain a relatively high amount of the antineuritic vitamin, showed that this material contained more vitamin B1than the milk fraction, but that this latter material contained substantially large amounts of other essential substances (6). The complementing effect from the combined use of the two products gave more satisfactory results than were obtained by any other single material. Varying combinations of these two products were used in an extended series of growth and life cycle experiments with white rats of both sexes. The gross components of the basal diet consisted of purified protein, dextrin as the carbohydrate, mineral salt mixture, Crisco, and cod liver oil to supply vitamins A and D. The two test products served as the sole source of all other vitamins. Typical results are summarized in Table I. The ration containing 7.5 per cent of the milk fraction and 7.0 per cent of rice polishings maintained normal development and a high degree of fecundity in both sexes through eight successive generations, when the observations mere discontinued. All animals vere maintained under controlled experimental conditions which extended over a period somei~liatin excess of 3 years. TABLE I. FECUNDITY OF WHITE RATSTHROUGH EIGHTSucCESSIVE GENERATIONS RECEIVING A CONTROLLED BASALDIET, RICE POLISH, A N D THE CONCENTRATED VITAMINFRACTION OF WHEYAS THE SOLESOURCE OF WATER-SOLUBLE VITAMISS Milk vitamin fraction. yo Crude rice polishings, 70 No. of matings Successful matings. % No. young born Av. No. per litter Young reared, % Av. at.of young a t 18 days, grams
Ration 175 7.5 2.0 43 65 147 5.3 17 24
Ration 210 7.5 7.0 120 74 568 6.4 76 30.8
In developing the evidence concerning the vitamin characteristics of the concentrated whey fraction, it has been necessary to rechart or establish new objectives from time to time as improved techniques and further knowledge of the mode of action and chemistry of the vitamin entities became knonm. Increasing familiarity with the properties and limitations of the material has permitted the demonstration of some phenomenal results in the matter of growth proniotion and control. In 1931 (5) this laboratory published data from experiments with animals where a basal ration was employed which would permit maintenance of the animal a t constant weight for periods as long as 7 to 8 months. Animals maintained on this restricted dietary showed a phenomenal growth response upon the addition of a small percentage of the milk vitamin fraction. In some instances gains of 55 to 59 grams were shown during the first week following the addition of the milk fraction. It was not unusual for animals t o sho~van average gain of 43 to 48 grams per week for a 3-week
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period following the addition of 5 to 7 per cent of the milk vitamin fraction to the restricted basal diet.
Fecundity The degree to which this material is effective in maintaining satisfactory physical condition and even improving the fecundity of experimental animals is illustrated by the performance of our rat colony prior to and for 6 years following incorporation of this concentrate in a stock diet previously assumed to be adequate in all regards. The colony was established in 1923 from animals obtained from the Wistar Institute; there has been no introduction of animals other than those obtained from this source. The stock diet from 1923 to 1931 consisted of the following: ground whole yellow corn, 76 parts; linseed oil meal, 16; crude casein, 5 ; ground alfalfa leaf meal, 2; sodium chloride, 0.5; calcium carbonate, 0.5; and fluid whole milk, ad lib. During the first few years no unusual degree of infertility or difficulties in the rearing of the young were encountered, During TABLE 11. STATISTICAL ANALYSES OF OVERFOUR THOWSAXD LITTERSOF WHITERATSSHOWING IMPROVEMENT IN FECUNDITY RESULTIXGFROM IMPROVED DIET CONTAINIXG INCREASED AMOUNT OF MILKCOMPONENTS A v . KO. Standard Year per Litter Deviation 1923-30 1931 1932 1933 1934 1935 1936
7.058 7.407 7,543 8.123 8.186 8.369 8.377
2.780 2.851 2.570 2.769 2.607 2.568 2.524
First Quartile Probable % of Error No. 7-yr. BY. 1 0 . 1 4 3 4 86 87.0 *0.079 88.6 4.95 1 0 . 0 7 4 5.33 95.4 *0.067 5.97 106.9 -0.069 5.96 106.7 *0.064 6.19 110.8 10.067 5.84 104.5
Third Quartile % of No. 7-yr. av. 8.70 93.6 9.00 96.8 6.77 94.3 9.48 101.9 9.59 103.1 9.68 104.1 9.87 106.1
subsequent years, however, difficulties of this character became increasingly prevalent, particularly from the standpoint of inability of the mother to raise the young after birth. Various expedients were resorted t o by supplementing the stock diet mixture with green vegetables, butterfat, meat, cooked cereals, ultraTiolet irradiation, and modification of the mating routine. No significant and consistent improvements resulted from these expedients. In March, 1931, the stock diet was changed to the following: ground whole yellow corn, 51 parts; linseed oil meal, 16; whole milk powder, 25; ground alfalfa leaf meal, 2; water-soluble milk vitamin conccntrate, 6; and calcium carbonate, 0.5 part. The principal modification of the ration involved the introduction of whole milk solids in lieu of fluid milk and the introduction of the milk vitamin fraction. The feeding of further supplements was discontinued. During the 6-year period from 1931 to 1936, inclusive, there was consistent improvement in breeding performance and fecundity of the colony as a whole. The average size of the litters has shown a constant increase, and little or no difficulty has been encountered in the rearing of the young. In March, 1935, a litter of twenty-three wellnourished normal pups was cast by a young female a t the first mating. This is believed to be the largest litter on record. Table I1 contains a summary of statistical records involving over four thousand litters and over thirty thousand animals.
Vitamin Entities Present The evidence presented in regard t o the vitamin value of whey obviously demands a more detailed consideration of the particular entities involved. Direct experimental data have shown the following factors to be present in whey or in the concentrated fraction previously described : lactoflavin (riboflavin vitamin BPor G), thiamine (vitamin Bl), the antiacrodynia factor (vitamin &), prothrombin (vitamin K), pro-
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vitamin D (ergosterol or other antirachitically activable substances), the oestrogenic hormone, and obviously by permissible deduction, vitamin E and factor W. In brief, probably all known vitamins or other unidentified dietary essentials are present in whey to a greater or less degree; otherwise maintenance, growth promotion, and reproduction directly attributable to the concentrated fraction would be impossible. The lactalbumin and protein fractions of whey are carriers of prosthetically bound provitamin D and vitamin K. The albumin precipitated from whey by appropriate pH adjustment yields a protein which carries with marked constancy substantially 7 per cent of lipide matter which is different from butterfat (2) ; it is characterized particularly by about 6 per cent of nonsaponifiable matter of which 40 to 50 per cent is recoverable as cholesterol with melting point of 145-147’ C. This cholesterol fraction has been activated with ultraviolet rays to a vitamin D potency of as high as 1000 U. s. P. units per gram. The provitamin D may also be activated by irradiation of the protein itself as well as by irradiation of the whey, either in fluid or dry form. The unsaponifiable portion of the lipides associated with the lactalbumin show substantial vitamin K activity, This factor may be further concentrated by the removal of the cholesterol. The properties of the active material are readily demonstrated by oral feeding of a few milligrams to properly prepared chicks, the blood clotting time being decreased markedly within 3 t o 6 hours. The vitamin K component of whey is not confined solely to the lipide matter associated with the albumin. The water-soluble fraction from which the albumin has been removed likewise contains the prothrombin factor. Fractions have been obtained from this product with even greater activity than has been observed in the lipide matter associated with the protein. Notwithstanding the presence of readily demonstrable amounts of the fat-soluble vitamin factors in whey, the evidence is not to be construed as supporting any contention that whey is a rich or relatively potent source of these factors. Nevertheless, attention may properly be directed to these matters in view of the popular belief that, since whey is deficient in milk fat, it contains none of the fat-soluble vitamins. The fat-soluble vitamins found in whey are not of primary importance, quantitatively considered in comparison with the water-soluble factors, a t least from a competitive or industrial utilization point of view. The fractionation of whey as previously outlined permits a concentration of the watersoluble factors in amounts which endow the crude concentrate itself with significant dietary merit because of the multiplicity of factors which it contains; and also because such a concentrate serves as a convenient starting material for the further concentration, isolation, and purification of the individual entities. It contains vitamin B1 in significant amounts, as demonstrated by its specificity for the prevention and cure of polyneuritis in rats and chickens, and for the cure of this condition in pigeons by the administration of small doses, as well as by the abundance of data showing growth response directly attributable to the presence of this factor. Vitamin Be, the substance known as the antiacrodynia factor which prevents the characteristic florid or wet type of dermatitis readily produced in experimental animals, is also contained in whey and the whey concentrate under consideration. The relative amount of this factor in whey is difficult to state because fats, in the dietary or as stored in the body, influence its efficacy. However, under comparable test conditions the whey concentrate in 200-300 mg. quantities per day has been shown to prevent this condition in white rats; 50100 mg. of dry yeast, 35-50 mg. of a water-soluble concentrate from rice polish, and 4-5 cc. of fluid whole milk are also
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protective; wheat germ oil in excess of 200 mg. per day afforded little or no protection. Among the various entities comprising the vitamin B complex, the status of lactoflavin (riboflavin) has been clarified to a marked degree during the past few years. Whey is recognized as one of the richest sources of this dietary substance. Its inherent lactoflavin content may be conserved without destruction in the final water-soluble fraction. This fraction as well as the original whey itself has served as the starting material for both laboratory and commercial methods of isolation and preparation of the purified crystalline material and commercial concentrates (1, 3). Such lactoflavin preparations are obtainable from whey or whey derivatives by appropriate absorption and elution procedures, followed by simple or elaborate purification methods, depending upon the comparative degree of purity desired. The preparation of lactoflavin products from whey derivatives has made possible a rapid increase in the progress of biological research relating to the water-soluble vitamins and has also permitted a constantly expanding utilization of this factor in pharmaceutical and medicinal preparations. The utilization of whey, whey concentrates, or whey derivatives as sources of vitamins has reached a certain degree of established status as an industrial enterprise. This development has reached its present state from comparatively meager knowledge, in view of the potentialities which such a product holds as a basic material for further research and development. What may be revealed by further investigations directed specifically to whey cannot be fully predicted. Nevertheless, evidence is already a t hand that vitamin entities other than those specifically mentioned here are present. An oestrogenic substance in whey associated with the lactalbumin and present in the water-soluble vitamin fraction has been demonstrated. However, methods for the concentration of such material in highly or competitively potent form have not yet been perfected. The active material in the crude concentrates obtained thus far has apparently been in an unstable form and consistently reproducible results have not been obtained. This development, as yet in an elementary stage, serves to illustrate the potential field for technical investigations presented by the complex mixture of the constituents of milk. Milk and its derivatives are, in fact, basic raw materials which yield a series of products for direct consumption as well as starting materials for the preparation of still other products with proved or potential therapeutic, dietary, and industrial use.
Literature Cited (1) Ansbacher, S., “Lactoflavin”, Borden Co., 1936. (2) Ansbacher, S., and Supplee, G. C., J. Bid. Chem., 105,391 (1934). (3) Supplee, G. C.,Ansbacher, S., Flanigan, G. E., and Hanford, 8. M., J. D a i w Sci., 19, 215 (1936). (4) Supplee, G. C., Dow, 0. D., and Flanigan, G. E., Ibid., 11, 420 (1928). (5) Supplee, G. C., Kahlenberg, 0. J., and Flanigan, G. E., J . Bid. Chem., 93, 705 (1931). PREUENTED before the Division of Agricultural and Food Chemistry at the 97th Meeting of the American Chemical Society, Baltimore, Md.
Gases in the Commercial Handling of Citrus Juices-Correction It has been brought t o our attention that there is an error in one of the references cited in our paper which appeared in October, 1939, pages 1275-8. Reference (6) on page 1278, “Toulouse, IND.ENG.CHEM.,28, 480 (1936)” should read “Toulouse, IND. ENG.CHEM.,26, 769 (1934)”. GEORGE N. PULLEY AND HARRY W. VON LOESECKB
