Separation of Lactose and Soluble Proteins of Whey by Alcohol

hol precipitation results in no serious impairment, and below which alcohol precipi- tation causes serious im- pairment in the solubility in water of ...
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Separation of Lactose and Soluble Proteins of W h e y by Alcohol Extraction XPERIMENTAL stud-

From a commercial standpoint it would be advantaies of the solvent effect geous to develop a process so o n w h e y p o w d e r of that (a) a greater yield of alcoholic solutions of varilactose could be obtained, (b) ous strengths indicate that a lactose of high purity could in the region of 70.7 per cent be obtained as a result of by weight of alcohol, a narrow but one crystallization, and critical range of concentra(c) the protein could be tion exists above which alcoseparated from all or part hol precipitation results in of the lactose and salts withno serious impairment, and out any serious impairment below which alcohol precipiof its desirable properties] tation causes serious imABRAHAM LEVITON AND ALAN LEIGHTON p a r t i c u l a r l y i t s solubility pairment in the solubility in Bureau of Dairy Industry, and its ability to yield, water of w h e y p r o t e i n s . U. S. Department of Agriculture, w h e n d i s so 1ved , solutions These studies indicate also Washington, D. C. of marked frothing Dower. that the lactose in whcv The purpose oftilis paper powder exists in a glassy is to describe experiments leading to the development of a amorphous state; consequently the first effect on lactose process in which these ends have been realized. The procof the a,ddition of alcohol to whey powder, apart from the ess involves the use of alcohol in the extraction of lactose effect of change of solvent, is a diluting action whereby a from spray-dried whey powder. Although the method is solution supersaturated with respect to lactose is formed. capable of application not only to whey powder, but also to The stability of the supersaturated solution is such that comskim milk and whey protein powders, and not only to powders, plete extraction of the lactose and separation from insoluble material by filtration is easily realized. The lactose subbut also to concentrated whey, concentrated skim milk, and concentrated solutions of whey protein powder, this paper is sequently crystallizes from the filtrate and is recovered. limited to the discussion of the process only as it concerns These observations, primarily] and others form the basis sweet whey1 powder. for the development of an extraction process (7) for the fractionation of the essential ingredients of whey into the followMaterials and Analytical Methods ing fractions: (a) high-grade lactose as the result of one crystallization in yields as high as 80 per cent; (b) a tasteless, A series of preliminary experiments disclosed that in the lactose-free, soluble protein powder containing approximately neighborhood of 70.7 per cent by weight, a critical range of con50 per cent protein, 15 per cent ash, and 35 per cent solids, centration of alcohol in water existed, above which alcohol precipitation of whey powder constituents resulted in no serious volatile a t the ashing temperature; and (c) a food product impairment of the solubility in water of the whey proteins, and rich in its lactoflavin content. below which alcohol precipitation resulted in serious impairment. I n the manufacture of lactose from whey, the usual proInasmuch as one of the objectives of the experiments which cedure is to adjust the reaction and apply heat until the follow is the recovery of a soluble whey protein powder, the solvent employed in these experiments was made to contain at greater part of the proteins coagulate and some of the salts least 70.7 per cent by weight of alcohol and, in most instances, precipitate, separate the precipitate by decantation and filtraexactly 70.7 per cent by weight or 4 parts by volume of 95 tion, and concentrate the filtrate under vacuum until the per cent alcohol to 1 part of water. lactose crystallizes. The crystals are then removed and puriThe spray-dried whey powder used throughout was a KraftPhenix product derivcd from Cheddar cheese whey. It confied by a second crystallization. By this method about 50 tained 69.1 per cent lactose, 12.6 per cent protein, 7.0 per cent per cent of the sugar is recovered. The proteins are deash, and 11.3 per cent solids, volatile at the ashing temperature. natured by the heating and are no longer soluble. A 5 per cent aqueous solution of powder had a pH of 5.88. It is possible to adjust the reaction and temperature so Lactose was determined polarimetrically by means of a Schmidt-Haensch double-wedge, half-shadow saccharimeter that the sugar can be crystallized without removing the proaccording to a modified procedure based on the A. 0. A. C . method tein and without rendering it insoluble (3). If this is done, (1) for the determination of lactose in milk. Alcoholic solutions the mother liquor, after removal of the sugar, may be dried to of lactose when analyzed were first heated to remove the alcohol. a powder, usually designated as whey protein powder; it Acid mercuric nitrate t o the extent of 2 cc. per 50 cc. of solution contains 37 to 52 per cent lactose, 32 to 45 per cent protein, 1 Sweet whey is a term commonly used by cheese makers to describe the and 12 to 18 per cent ash. Some of the salts may be removed taste of whey relatively low in acid. In this paper “sweet whcy” meane from this powder by dialysis (11)to increase the proportion of whey derived from milk as a result of rennet rather than acid coagulation of the casein. protein and lactose. 1305

E

]Extraction from Spray-

Dried Whey Powder Derived from Sweet Whey

INDUSTRIAL AND ENGINEERING CHEMISTRY

1306

VOL. 30, NO. 11

TABLEI. EFFECTOF SUPERSATURATION ON RATEOF EXTRACTION OF WHEYPOWDER CONSTITUENTS FROM 10 GRAMS OF POWDER

Time

Min.

a Calculated as

Dissolved Solids c

Dissolved Dissolyed Lactose Protein G./i00 8 . solvent

Dissolved Material Other Than Lactose and Protein

Extd. Lactose Remaining in Soh.

Lactose Crystallizing after 4 Min.

Nitrogenous Materiala Separating after 4 Min.

Ratio between Nitrogenous Material and Separated Lactose

%

N.

was used as the protein coagulant. Equilibrium between the several forms of lactose was established at 70" C., and the protein coagulant was added to the cooled sample. The solution was then diluted to contain exactly 50 cc. and filtered. The filtrate was polarized at 25' C., and the percentage of lactose was determined from the measured rotation, weight of sample, and final volume of solution corrected for the volume of precipitate. Nitrogen was determined according to the Arnold-Gunning modification of the Kjeldahl method. Determinations of pH were made by means of the quinhydrone electrode referred to a saturated potassium chloridecalomel half-cell. The pH scale employed is based upon considerations of aqueous solutions. It is necessary, therefore, to bear this in mind in the interpretation of the data obtained with alcoholic solutions. For example, if the solute (extracted from whey powder) remains unchanged, the substitution of water for the alcohol contained in a 70.7 per cent alcoholic solution results in an increase in measured pH of approximately one-half unit. Turbidity measurements were made by means of a directreading photoelectric turbidimeter. As a source of illumination, a 6-volt flashlight bulb was used. The intensity of illumination was controlled by an electrical circuit identical with the one described by Evelyn (4). To concentrate and to center the illumination with respect to the cubical absorption cell and the Weston photronic cell, the light source was mounted in juxtaposition with the eyepiece of a microscope, the absorption cell was mounted on the stage, and the photronic cell was mounted below the diaphragm. Whenever filters were used, they were mounted on the stage between the absorption and photronic cells. The current generated by the light transmitted through the filled absorption cell and filter was measured by means of a sensitive galvanometer critically damped. In the measurement of lactose solutions, a standard solution was prepared from c. P . lactose, and the intensity of illumination was adjusted until a deflection of 10 cm. on the galvanometer scale was obtained. The unknown solution was then substituted for the standard, and the deflection was again measured. The difference in deflection multiulied bv " ten., then, is a measure of the uercentage of light absorb6d. In the measurement of the turbidity of solutions of whey protein powder, water was employed as a standard. I n the measurement of a series of filtered solutions containing the coloring material of whey powder, a yellow Noviol Corning glass filter No. 352 was used, and one of the filtered solutions free of any apparent turbidity was used as a standard. Total solids were determined under vacuum at 100" C. in a steam-jacketed vacuum oven. Determinations of carbonated ash in lactose were made according to methods of the A. 0. A. C. (2) for the determination of ash in sugars. Ashing temperatures never exceeded 600" C . The preparation of suspensions of whey powder in alcoholic solutions is accomplished with difficulty, unless the whey powder is first wet with 95 per cent alcohol. Caking results when the solution is added directly to the powder, and the efficiency of the extraction process is lowered. Consequently in the preparation of the suspensions, whey powder was first stirred into all or part of the 95 per cent alcohol required; and immediately thereafter the appropriate quantity of water or water and 95 er cent alcohol was added to give the desired concentration. fn this way uniform suspensions could be easily realized. I

Solvent Effect of 70.7 Per Cent Alcohol on Whey Powder Samples in glass-capped, glass-stoppered bottles containing 10 grams of whey powder suspended in 200 cc. of a 70.7 per cent alcoholic solution were rotated in a thermostat at 24.5"C.

Periodically samples were filtered under slight pressure through a sintered glass filter reinforced with a layer of asbestos, and the filtrate was analyzed for nitrogen, lactose, and total solids. The results are given in Table I. SurprXngly, with increasing time the quantity of dissolved lactose diminishes. The only plausible explanation of this phenomenon lies in the fact that the lactose in normal spray-dried whey powder is present in the amorphous state, as the experiments of Troy and Sharpe (9) and Tuckey, Ruehe, and Clark (IO) demonstrate qualitatively; consequently the first effect on lactose of the addition of alcohol and water to whey powder, apart from the effect of change of solvent, is a diluting action whereby a supersaturated solution of lactose in the solvent is formed. The lactose subsequently crystallizes from its supersaturated solution until the solution is just saturated with lactose. The stability of the supersaturated solution is such that separation of the filtrate from insoluble material is readily accomplished before any of the lactose crystallizes. Thus the results in Table I1 indicate that a t 30' C. all of the lactose in 10 grams of whey powder is extracted by 200 cc. of a 70.7 per cent solution of alcohol within 3 minutes after the mixing of the ingredients, and that the resulting supersaturated solution is stable for 7 and 15 minutes, respectively, depending upon whether or not continuous agitation is employed. Because the ingredients of whey which are insoluble in the solvent behave as a granular precipitate upon filtration, separation, within the interval during which the filtrate supersaturated with respect to the lactose is stable, is quite feasible. TABLE11. VARIATIONS I N LACTOSEEXTRACTED WITH 70.7 PERCENTALCOHOL AT 30" C. FROM WHEYPOWDER WITH TIME OF EXTRACTION Lactose E x t d . 7 Stirred Stirred continuously 2.5 min. 90.5 90.5 97.8 97.8 100,o 100.0 100.0 99.0 100.0 100.0

7 %

Time Min.' 0 1 2.5 3.5

5

Lactose Extd.Stirred Stirred continuously 2.5 min. 99.5 100.0 98.2 99.5 90.7 99.0 81.3 95.0

7-%,

Time, Min. 7 10 15 25

The solubility of lactose a t 24.5' C. in 70.7 per cent alcohol, according to determinations made in this laboratory, is 0.28 per cent, and if this value is taken into consideration, it may be calculated that approximately 90 per cent of the lactose contained in whey powder may be recovered from the filtrate. Inasmuch as the concentration of nitrogenous material calculated as proteins, and the concentration of ingredients other than lactose and protein in the filtrate is quite small (0.15 and 0.38 per cent, respectively, Table I), contamination with large amounts of occluded impurities such as occurs in the usual preparation of lactose is avoided. However, a source of contamination exists in that the filtrate supersaturated with respect to lactose is also slightly supersaturated with respect to the nitrogenous and other

INDUSTRIAL AND ENGINEERING CHEMISTRY

NOVEMBER, 1938

1307

TABLE111. EFFECTS OF SOLVENT-POWDER RATIOON EFFICIENCY OF EXTRACTION W , Weight

of Powder per 100 Cc. Solvent Grams 2.15 5.0 8.1 10.1 13.0

Weight of Lactose per 100 Go. Solvent Grams 1 49 3.47 5.62 7.00 8.98

PH 5.45 5.22 5.20 5.18 5.07

L , Lactose Extd. per 100 c c . Solvent Grams 1.48 3.43 4.50 4.68 4.33

Lactose Extd.

% 100

99.5 80.4 67.0 48.3

P , Protein per 100 c c . Solvent Grams 0.052 0.127 0.199 0.236 0.268

ingredients of whey. Table I amply illustrates this point. For example, corresponding to a separation of 3.21 grams of lactose in 1130 minutes, there is a separation of 0.0018 gram of nitrogenous material calculated as nitrogen. The calculation of the quantity of nitrogenous material is based on the assumption that the change in concentration after 4 minutes is due solely to the separation of the dissolved ingredients without any further dissolution of amorphous material. The data also show that minute quantities of ingredients other than lactose and protein have separated. The extent to which this source of contamination contributes to the contamination of the lactose is illustrated by the following experiment: A mixture containing 100 grams of whey powder suspended in 1750 cc. of 70.7 per cent alcohol was agitated; and samples were withdrawn a t intervals and filtered. The lactose samples crystallizing from the various filtrates were analyzed for nitrogenous material. The results show that the samples of lactose obtained a t later stages of the crystallization process are lower in nitrogenous material than those obtained a t earlier stages. Thus the lactose samples recovered after 2, 4, 7, 12, 18, 24, and 30 minutes contain, respectively, 0.049, 0.049, 0.047, 0.037, 0.030, 0.023, and 0.018 per cent nitrogen. The data also indicate that the precipitation of nitrogenous material along with the crystallization of the lactose is due to the supersaturation of mother liquor with respect to both, rather than to adsorption of the protein by the lactose. I n the event of adsorption, the concentration of nitrogenous material associated with the sugar would probably be independent of the stage a t which crystallization takes place.

Effect of Variations in Quantity of Solvent The following experiment was conducted at 21 C. to determine the optimum ratio of whey powder to solvent required t o obtain complete or nearly complete extraction of the lactose: Three and two minutes, respectively, were allotted arbitrarily for the agitation and filtration of the samples. Immediately following the suspension of the whey powder in 200 cc. of 95 per cent alcohol, 50 cc. of water were added. The mixture was agitated and then filtered. The filtrates obtained in this manner were analyzed for nitrogen, lactose, and total solids. The results are given in Table 111. When the quantity of powder per 100 cc. of solvent exceeds 5 grams, corresponding to a quantity of lactose in excess of 3.45'grams, lactose extraction becomes less and less complete (column 5); finally when the concentration of lactose reaches 9.0 grams per 100 cc. of solvent, the quantity of lactose extracted begins to diminish. Consequently, for the complete extraction of lactose under the conditions arbitrarily adopted, the concentration of lactose should not exceed 3.45 grams per 100 cc. solvent. However, this ratio varies with variations in temperature, and its value would also depend on the degree and time of agitation and the time required for filtration. The relations which hold a t various temperatures under various conditions of agitation have not been worked out. I n general, it is true that the stability of the supersaturated lactose solution increases with increasing temperature, while the time required

100

2.4 2.5 2.5 2.3 2.1

S, Total Solids Extd. per 100 cc. Solvent Grams 1.62 3.86 5.28 5.56 5.58

100 75.4 77.2 65.3 55.0 43.0

"per

'l'dofc~' 8 -(L+P) 100

Solvent Grams 0.09 0.30 0.58 0.64 0.98

W

4.2 6.0 7.2 6.3 7.5

to dissolve the amorphous lactose decreases; consequently a t low temperatures the extraction becomes less efficient. Table I11 is also interesting in that the results throw light on the applicability of the filtrate-that is, the mother liquorfor use as a solvent. Theoretically, a t least, the applicability of any solvent to the process depends upon the small solubility of the principal ingredients of whey in the solvent, together with the stability of supersaturated lactose solutions formed. Consequently, a solvent already saturated with respect to the principal ingredients of whey should be highly effective and applicable. Unfortunately, as the results in Table I1 indicate, the quantity of protein extracted with the lactose is nearly proportional to the quantity of powder extracted, and the quantity of solids extracted other than protein and lactose also increases markedly with an increase in the quantity of powder treated; consequently, although the mother liquor remaining after the recovery of the lactose may be used as a solvent, its efficiency, if the ratio between the lactose and the other solids extracted is considered as one of the criteria, is less than that of the original solution from which it was derived.

*

Variation of Purity of Lactose with p H of Filtrate It has already been shown that the applicability of the process for the production of "pure" lactose is limited by the tendency of minute quantities of nitrogenous and other insoluble materials to precipitate out of supersaturated solution along with the lactose. I n order to overcome this difficulty in part, the filtrate containing the lactose in solution may be acidified prior to the crystallization of the lactose. The solubility of the nitrogenous material and calcium salts contained in the filtrate is increased by an increase in the acidity of the liquor from which the lactose crystallizes; a t the same time the solubility of lactose is not appreciably affected. TABLEIV. VARIATIONS IN NITROGEN AND ASH CONTENT OF CRYSTALLIZED LACTOSE AND IN TURBIDITY OF 5 PER CENT SOLUTIONS WITH PH OF MOTHER LIQUOR 1.7 N Alcoholic HC1 Added

0.92 N NaOH Added

pH Mother Liquor

..

18.3

.. ..

3.7 2.4 0.0 0.6 10.6 2.9

5.33 4.87 4.24 3.65 0.75 .. 2.75 1.00 2.50 1.13 5.43 0:25 6.46 0.75 a CY? (c. P. lactose), 55.2. 0.0 0.2 0.5

..

..

Turbidity Light Ab: sorbed by 5% Soln.

..

12.2

Nitrogen Content of Lactose 0.058 0.045 0.024 0.020 0.011 0.012 0.048 0.032

Carbonated Specific'" Ash Rotation 0.30 0.20 0.19 0.15 0.14 0.16 0.11 0.09

.. .. ..

.. .. .. ..

55.1

The results in Table IV indicate that, as the p H of the mother liquor is lowered by the addition of hydrochloric acid, there is a progressive decrease in the ash and nitrogen content of the lactose which crystallizes. The turbidity of solutions prepared from the various lactose samples also shows a progressive decrease corresponding to decreases in

INDUSTRIAL AND ENGINEERING CHEMISTRY

1308

the p H a t which the sugars were prepared. The addition, however, of more than one per cent by volume of 1.7 N hydrochloric acid to the mother liquor results in no further improvement in sugar quality. _-

-

90 -

a 80-

---

pH 2.75

YIELD= l.DP

- pli5.33

YIELD-I.ISP

FIGURE1. CRYSTALLIZATION OF LACTOSEFROM ACIDIFIED AND UNACIDIFIED FILTRATES AS A FUNCTION OF TIME!

If sodium hydroxide is added to the mother liquor, a precipitate fprms. This precipitation evidently reduces supersaturation of the mother liquor, particularly with respect to the insoluble salts, for the sugar obtained after the removal of the precipitate prior to crystallization shows a marked reduction in ash and a less significant reduction in nitrogen. Both an increase and a decrease in the pH of the mother liquor, then, lead to significant reductions in the degree of contamination of the sugar crystallizing from the 1 iquor; the first leads primarily to a reduction in ash content, the second, to reduction in nitrogen. The percentage of crystallization or the ratio between the quantity of lactose which has separated a t any time and the total quantity separable is plotted as a function of time in Figure 1. I n Figure 2 the rate of crystallization is plotted as a function of the percentage of uncrystallized l a c t o s e . Figure 1 shows that, in order to bring about 80 per cent crystallization or better, no practical advantage, as far as economy in time is concerned, results from the a c i d i f i c a t i o n of the mother liquor to a p H of 2.75. Only at the PER CENT INCOM~LETECRYSTALLIZAT'ON onset of crystallization, FIGURE2. RATEOF CRYSTALLJand until about 50 per ZATION OF LACTOSE FROM ACIDIcent of the crystalliz% FIED AND UNACIDIFIED FILTRATES tion is complete, does the added acid markedly increase the rate of crystallization. This point is well illustrated in Figure 2. After 50 per cent crystallization, the ratio of crystallization is slightly more rapid in the unacidified solution. The data obtained are presented primarily in order to establish the time necessary to realize a satisfactory yield. On this point it is apparent that approximately 16 hours would be required, regardless of acidification, to obtain a yield of SO per cent. A 50 per cent yield, if considered satisfactory, may be realized at a p H of 2.75 in 200 minutes; a t a p H of 5.33, 300 minutes are required. The values presented were obtained under conditions of mild agitation, With no agitation, approximately 4 days are required for SO per cent crystallization. It is not known whether stronger agitation than that

VOL. 30, NO. 11

employed in these experiments would result in an increased rate of crystallization. Another point of interest lies in the fact that the solubility of lactose (taken as the quantity of lactose which remains in solution after 3 days of stirring) is slightly greater a t the lower pH. This would indicate possibly that a t the lower pH, lactose combines with the soluble calcium salts in the mother liquor. Compound formation between lactose and salts in aqueous solutions was observed by Herrington (6). Such compound formation may account largely for the ash content of the sugar which crystallizes out of the acidified mother liquor.

Effect of Alcohol Concentration on Solubility of W h e y Proteins The following experiments were undertaken in order to place on a firmer experimental basis the observation reported earlier in the manuscript that a critical range of concentration of alcohol exists in the neighborhood of 70.7 per cent by weight; above this range the solubility in water of the protein material contained in whey powder is largely unaffected by alcohol precipitation, and below it the solubility is seriously impaired. I n the first series of experiments 20-gram samples of whey powder were suspended in 200-cc. portions of solutions of alcohol differingin strength. These suspensions were rotated in a thermostat a t 24.5' C. for 10 minutes, and then filtered through a Biichner funnel. When the ratio between the volume of 95 per cent alcohol and water fell below 4 to 1, the rate of filtration became exceedingly slow. The residues recovered on filtration were mashed with absolute alcohol, and dried a t room temperature. Nitrogen determinations were made on the various residues, and aqueous suspensions of these powders were standardized to contain 0.51 gram of nitrogenous material, calculated as albumin, per 100 cc. of water. These suspensions were preserved with formaldehyde and stirred for 5 hours at 24.5" C. Portions of the suspensions were filtered and refiltered through No. 44 Whatman's filter paper. This paper retains the finest precipitates. Total solids and protein determinations were made on the original suspensions and their filtrates, and turbidity measurements were made on the filtrates. The results are presented in Table V. It is apparent that when the concentration of alcohol falls below 70.7 per cent by weight, alcoholic precipitation of whey solids results in a sharp increase in the quantity of filterable solids and proteins in aqueous suspensions of the precipitate. However, it is difficult to ascertain whether the differences observed are due to differences in solubility or to differences in the degree of dispersion of suspended material. Thus, examination of the turbidity of the various filtrates discloses that the filtrate corresponding to the residue obtained from 70.7 per cent alcohol shows the maximum turbidity; consequently, the fact that the quantity of filterable material in suspension becomes significant only for those suspensions corresponding to residues obtained from weaker alcoholic solutions may be interpreted to mean that the degree of dispersion in these suspensions has become low enough t o permit filtration by ordinary means. I n order to clarify the point discussed above, more concentrated suspensions were prepared from the following residues: (a) that obtained from 41.5 per cent alcohol, ( b ) that from 10 grams of whey powder treated with 70.7 per cent alcohol, and (e) that from filtered concentrated whey treated with sufficient alcohol to yield a 70.7 per cent solution. Residues (b) and (e) were found upon analysis to contain no lactose and 9.4 per cent lactose, respectively. All three powders contained approximately the same percentage of protein. The results of analyses for total solids and protein on the

NOVEMBER, 1938 TABLE V.

INDUSTRIAL AND ENGINEERING CHEMISTRY

RELATION BETWEEN QUANTITY OF INSOLUBLE MATERIAL IN ALCOHOL-PRECIPITATED WIIEY SOLIDS AND CONCENTRATION OF ALCOEOLIN SOLVENT

Source of Whey Powder

Protein Content

Spray-dried, untreated Treated with abs. alcohol Treated with 95% alcohol by vol. Treated with 81 3 alrohol by weight TieaLrrl with 7 0 : 7 2 alcohol by weight Treated w i t h 60.6% alcohol by weiaht Treated with 50 7 alcohol by weight Treated with 41:5% alcohol by weight

12.5 12.6 12.6 12.3 16.4 22.5 52.4 49.3

Total Solids in Original -Soln.

%

TABLE VI.

Total Solids in Filtrate -Grams

4.11 4.16 4.16 4.19 3.13 2.33 0.99 1.04

Concn. Original of Insol. Protein Solids Conon. p s r 100 cc. HnO 0.02 0.02 0.01 0.01 0 03 0.15 0 20 0.33

4.09 4.14 4.15 4.18 3.10 2.18 0.79 0.71

Protein Concn. in Filtrate

%

%

0.0 2.0 0.0 2.0 2.0 13 5 23.1 42.3

31.3 30 5 36.7 40 1 44 2 34 6 27 4 12.7

F

0.51 0.51 0.52 0.51 0.51 0.45 0.40 0.30

0.51 0.52 0.52 0.52 0 52 0.52 0.52 0.52

Turhidity of Fillrates (Light Absorbed)

Insol. Protein in Proteins

EFFECT OF CLARIFICATION OF WHEYox QUANTITY OF INSOLUBLE MATERIALIN ALCOHOL-PRECIPITATED WHEY SOLIDS, AND EFFECTOF ALCOHOL CONCEKTRATION Protein in Powder

Source of Whey Powder

Conrn. of Solids in Filtrate G./100 cc. H i 0

Concn. of Powder i n

HzO

% 20 Erams, treated with 200

CC.

41.5%

alcohol 10 grams, treated with 200 CC. 70.7% alcohul Clarified concentrated; treated t o contain 70.7% alcohol

TABLE VII.

1309

EFFECT OF

Insol. Solids in Powder

%

Concn. of Concn. Protein in of Protein Filtrate G./iOO cc. IIzO

Insol. Protein in Proteins

%

49.3

5.00

2.48

50.4

2.41

0.867

60.1

51.0'

4.83

4.00

15.5

2.46

2.07

15.8

52.0

9.55

0.49

0.6

4.96

4.96

0.0

ALCOHOLON QUANTITY O F INSOLUBLE MATERIAL I N SOLUTIONS ALCOHOLPRECIPITATED POWDER AND ALCOHOL-FREE MOTHERLIQUOR

CONCENTRATION O F FROM

Condition of Filtrate

Clear Clear Slightly turbid

RECONSTITUTED

Total Solids in Unfiltered Soh

Total, Solids in Filtrate

Conon. of Inqml. Solids

Protein in Unfiltered Soh.

Protein in Filtrate

Insol. Protein in Proteins

(Light Abaorbed)

Unfiltered Solng.

%

%

%

%

%

%

%

%

Spray-dried untreated Treated wit'! 02% alcohol by vol. Treated with 81,3y alcohol by weiaht Treated w i t h 7 0 . 7 3 alcohol by weight Treated with GO.CiU/C alaohol by weight

6.27 6.24 6.09 5.98 5.86

6.24 6.20 6.05 5.93 5.62

0.04 0.04 0.05 0.24

0.03

0.77 0.76 0.73 0.67 0.65

0.76 0.75 0.69 0.59 0.39

1.3 1.3 5.5 11.9 40.0

37 24 2.0 2.0 0 0

53 50

Treated with 50.9% alcohol by weiaht Treated with 41.5% alcohol by weight

5.31 5.26

5.08 5.01

0.23 0.25

0.64 0.62

0.40 0.41

37.5 34.0

Sourco of Whey Powder

original suspensions and filtrates are given in Table VI. The filtrates corresponding to residues (a) and (b) show no apparent turbidity, and that corresponding to (c) shows only a slight turbidity. The suspended material in the concentrated suspensions impregnates the pores of the filter upon filtration; in a sense, therefore, ultrafilterable as well as filterable material is removed t o yield clear filtrates upon filtration. The results presented in Table VI indicate that the presence of insoluble and coarsely dispersed materials in residues obtained from alcoholic solutions containing more than 70.7 per cent alcohol is due largely to the presence of insoluble and coarsely dispersed material in the original whey. Thus, when the residue obtained from the action of 70.7 per cent alcohol on filtered whey is dispersed in water to form approximately a 10 per cent suspension, the suspension is only slightly turbid and contains a negligible quantity of insoluble material. On the other hand, the suspensions formed from the residues derived from unfiltered whey (whey powder) are turbid, and contain 15.5 per cent of its solids and approximately the same percentage of its protein in various degrees of dispersion. Corresponding to 15.5 per cent insoluble protein in the residue obtained from 70.7 per cent alcohol, 60.1 per cent of the protein in the residue obtained from 41.5 per cent alcohol is insoluble. Also, clear filtrates are obtained upon filtration of suspension of the residues in both cases. These facts lead to the conclusion that the abrupt decrease in the quantity of filterable material as the concentration of alcohol falls below 70.7 per cent is due largely to the impairment of the solubility of the soluble proteins in whey rather than to unstabilization of the suspended proteins.

Turbidity

of Filtrate Turbidity of

(standard) 3.0 3.0

.. ..

".

..

..

PH 5.9 5.8 5.9 5.8 5.7 5.8 5.6

This conclusion is supported further by the results presented in Table VII. I n experiments leading to these results, 10-gram samples of whey powder were suspended and agitated for 3 minutes in alcoholic solutions (200 cc.) of varying strength. The suspensions were filtered, and thc residues were washed with absolute alcohol and dried in air. T h e filtrates were preserved with formaldehyde during evaporation a t room temperature; after all the alcohol had been removed, they were filtered. The residues were then suspended in their corresponding filtrates, agitated for several hours, and filtered. The suspensions and filtrates obtaincd from them were then analyzed for total solids and protein,. and the filtrates were examined for turbid material. The suspensions prepared in this way, unlike the suspensions of the residues in water, were practically identical in composition and differed only in the solubility and degree of dispersion of the residues as they had been affected by the alcohol. The filtrates, with the exception of those derived from untreated whey powder and whey powder treated with 95 per cent alcohol, were apparently clear; turbidity measurements indicated that no significant differences in turbidity existed between the filtrates corresponding to the residues obtained from 81.3, 70.7, 60.6, 50.9, and 41.5 per cent alcohol. As in preceding experiments, the results presented in Table VI1 show that, unless the concentration of alcohol exceeds 70.7 per cent, extraction of whey powder with alcoholic solutions results in serious impairment of the solubility of the soluble whey proteins. Consequently, the use of alcohol as a solvent in the extraction of whey powder is limited to concentrations equal to or exceeding 70.0 per cent alcohol if the. preparation of a soluble protein powder is sought.

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INDUSTRIAL AND ENGINEERING CHEMISTRY

Preparation of Lactose and Soluble Protein Powder EXANPLE 1. The following example is illustrative of the extraction of whey powder with 70.7 per cent alcohol: 100 grams of spray-dried sweet whey powder containing 69.1 per cent lactose and 12.5 per cent nitrogenous material calculated as albumin were stirred rapidly a t room temperature (25' C.) in about 300 cc. of 95 per cent alcohol. This operation should take no longer than the time required for thorough mixing. A mixture of alcohol and water was then added in such proportions that the resulting 2 liters of solvent contained 96 per cent alcohol and water in the ratio by volume of 4 to 1. The mixture was stirred for several minutes and then filtered rapidly. The filtrate contained all of the lactose in solution. It was acidified with 1.0 per cent by volume of a 1.7 N hydrochloric acid solution in absolute alcohol; as a result the p H was lowered from 5.38 to 2.78. After 15 hours of agitation 80 per cent of the lactose originally present in the whey had crystallized. One part of these crystals, which had been washed with four parts of 70.7 per cent alcohol, contained 0.011 per cent nitrogen and 0.14 per cent carbonated ash; when dissolved it yielded a clear 5 per cent solution neutral t o neutral litmus paper. The residue insoluble in the solvent and recovered on filtration was washed prior to drying a t room temperature with sufficient alcohol to render the strength of the occluded liquor equal to, or greater than, the strength of an azeotropic mixture; 15.6 grams of residue were recovered. It contained no measurable quantity of lactose, 47.8 per cent protein, and 16 per cent ash. A portion of an 8.66 per cent suspension of the powder in water, which had been centrifuged a t approximately 2000 r. p. m. in an International centrifuge (type ISB) yielded a centrifugate containing 8.27 per cent solids. These solids contained 48.7 per cent protein and 13.6 per cent ash. Another portion of the 8.66 per cent suspension was filtered twice to yield a clear filtrate containing 7.41 per cent solids, of which 47.3 and 13.5 per cent were protein and ash, respectively. These results indicate that 15.8 per cent of the solids in the residue are insoluble or in colloidal suspension, and that the insoluble salts comprise the major portion of the more coarsely suspended material. A 10 per cent suspension of the residue in water exhibited excellent whipping properties and was free of any salty taste. EXAMPLE 2. This example pertains to the use of 95 per cent alcohol by volume as solvent. The lactose obtained possessed a number of desirable properties, as indicated. One-fourth pound of the whey powder described in example 1 was stirred into 20 pounds of 95 per cent alcohol a t 60" C. After 1 minute the suspension was filtered. The filtrate contained 0.465 gram lactose, 0.027 gram ash, 0.020 gram protein, and 0.568 gram solids per 100 cc. The quantity of lactose in the filtrate constituted 68 per cent of the lactose in the original powder. The filtrate was acidified with 0.28 volume per cent of 1.7 N hydrochloric acid and cooled rapidly to room temperature. After several hours the lactose which had crystallized was separated and washed with 95 per cent alcohol. A 60 per cent yield was obtained. The sugar was found upon analysis to contain 0.005 per cent nitrogen and 0.09 per cent ash. This unusually sweet sugar dissolved rapidly in water to yield clear solutions, and polarimetric analysis showed that it contained 1.3 parts &lactose per part of a-lactose. Fifty-seven grams of the residue recovered in the process contained 25.0 per cent protein, 14.2 per cent ash, and 52.5 per cent lactose. Water suspensions of the residue exhibited excellent whipping properties and possessed a pleasant sweet kaste.

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Discussion of Results Whey is a product of variable composition. Variations in the exact numerical values presented in the previous part are reasonably to be expected with variations in compoaition of normal sweet whey. However, extraction experiments on a variety of sweet whey powders confirm the conclusions derived from the data presented, and consequently they may be considered as typical. From the facts already established it is possible to conclude that, with some modification in detail, the process used with sweet whey powder should also be applicable to acid whey powder and to concentrated whey. Recovery of solvents in the extraction of powder would require merely straight distillation; but unless the concentration of whey was great enough, solvent recovery in the treatment of concentrated whey would necessitate the use of a fractionating column, I n the treatment of concentrated whey, it might be feasible to recover part of the lactose according to the method of Bell, Peter, and Johnson ( 3 ) . I n this method whey is concentrated to the point where the solution is highly supersaturated with respect to lactose. The lactose crystallizes and is recovered. However, sufficient dissolved lactose remains in the mother liquor to constitute between 37 and 52 per cent of the solids. The fact of the adaptability of the extraction process to the extraction of the remaining lactose and of some of the salts responsible for the salty taste of the mother liquor has already been demonstrated in this laboratory. The process also lends itself to the preparation of lactose from concentrated skim milk and skim milk powder. The casein in skim milk, however, is denatured even in the strongest alcoholic solution. Considerable work remains to be done in the extension of the results obtained with sweet whey powder to the other powders and solutions. It is noteworthy that the neutralized mother liquor remaining after the separation of the lactose and the removal of alcohol contains solids of which approximately 58 per cent is lactose, 10 per cent is nitrogenous material, and 21.5 per cent is ash. I n addition, the solids have been enriched considerably in their lactoflapin content (if complete extraction is assumed) in comparison to the solids in whey. The protein in the mother liquor is not heat coagulable to any great extent and is presumably composed largely of the alcoholsoluble protein reported by Osborne and Wakeman (8). Further study is needed to clarify the nature of the solids extracted by alcohol and to reveal uses for these solids, particularly in connection with their nutritive value. The use of 95 per cent alcohol as a solvent is of exceptional interest in that the lactose which is obtained is extraordinarily pure, sweet, and soluble. It consists of a finely divided mixture of a- and 6-lactose in nearly the same proportions contained in an equilibrated solution of lactose. The volume of alcohol used, 10 liters for thp extraction of 100 grams of whey powder, is proportionately very great; and this consideration would seem to render the process impracticable. It is true, however, that the yield of lactose obtained after 2 hours is satisfactory and that the time required for crystallization may be reduced considerably if agitation is employed during crystallization; consequently the turnover of solvent in the extraction with 95 per cent alcohol is much greater than in the extraction with 70.7 per cent alcohol where a crystallization period of 15 hours is required to obtain a satisfactory yield. The conditions specified in example 2 for the use of 95 per cent alcohol as solvent do not necessarily represent optimum conditions. The relation which exists between the quantity of solvent employed and the quantity of lactose extracted, as well as other relations, have not been determined. The conditions set forth in example 2 mere adopted arbitrarily t o

NOVEMBER, 1938

INDUSTRIAL ANI) ENGINEERING CHEMISTRY

demonstrate the applicability of the process a t higher temperatures and with the use of 95 per cent alcohol as solvent. The relations which exist between the temperature of extraction, the concentration of alcohol in the solvent, the time of extraction, the quantity of solvent, and the yields and properties of lactose and protein powder resulting from the process require further investigation. The work of Ferry, Cohn, and Newman (6) on the influence of salts on the solubility of egg albumen in 25 per cent alcohol a t - 5 " C. indicates that the critical concentration of 70.7 per cent alcohol a t room temperature reported in this paper may vary with temperature. Incomplete data obtained in this laboratory tend to indicate that a t lower temperatures the advantages which accrue to the use of 95 per cent alcohol a t higher temperatures may be obtained with 70.7 per cent alcohol. It is unnecessary for the purpose of this paper to account theoretically for the effect of alcohol concentration on the solubility of the alcohol-precipitated whey proteins. Alcohol precipitation of lactalbumin from a practically salt-free solution gives a product readily soluble in water, and precipitation from a solution containing salts in considerable quantity gives a relatively insoluble albumin. On this basis, then, the solubility in water of alcohol-precipitated whey lactalbumin may depend on the solubility in alcohol of the soluble whey salts which, in turn, may depend on the concentration of alcohol. The whey protein powder produced may be made to contain approximately 50 per cent protein. The pretreatment of whey to remove insoluble materials (casein and insoluble calcium salts) results in a considerable improvement in the final products- lactose and protein powder. As a result the quantity of insoluble or coarsely dispersed material associated with these products becomes insignificant. However, the process is limited a t present to the preparation of a powder

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containing not more than 50 per cent protein. This powder contains in the neighborhood of 15 per cent ash; consequently about 35 per cent of the powder is lost in the ashing process. Presumably, therefore, the salts contained in the powder consist in part of the insoluble and soluble citrates and lactates originally present in whey. T o obtain a further increase in the protein content of the powder, it would evidently be necessary to treat the original whey for the removal of calcium citrate and calcium lactate. However satisfactory the results have been on a laboratory scale, the process has not been projected on a plant or pilotplant scale extensively enough to warrant any statement as to the possibilities of its commercial exploitation. The simplicity of the steps involved leads to the opinion that no particular difficulty should be encountered in the application of the process to a plant scale. The qualities and useful properties of the products obtained indicate that it has great potential value.

Literature Cited (1) Assoc. Official Agr. Chem., Methods of Analysis, 3rd ed., p. 216 (1932). (2) Ibid., 3rd ed., p. 365 (1932). (3) Bell, R. W., Peter, P. N., and Johnson, W. T., Jr., J . Dairy Sci., 11, 163 (1928). (4) Evelyn, K.A., S.Biol. Chem., 115, 63 (1936). ( 5 ) Ferry, R. M., Cohn, E . J., and Newman, E. S., J. Am. Chem. SOC.,58, 2370 (1936). ( 6 ) Herrington, B. L., J. Dairy Sci., 17, 805 (1934). ( 7 ) Leviton, Abraham, U. S. patents pending. (8) Osborne, T. B., and Wakeman, A. J., J. Biol. Chem., 33, 243 (1918). (9) Troy, H. C., and Sharpe, P. F., J . Dairy Sci., 13, 140 (1930). (10) Tuckey, S. L., Ruehe, H. A., and Clark, G. L., Ibid., 17, 587 (1934). (11) W7atson, P.D.,IND.ENG.CHEM.,26, 640 (1934). RBCEIVED May 24, 1938.

SEPARATION PROCESSES Correlation between the y us. x and the Molal Heat Content us. Mole Fraction Diagrams MERLE RANDALL AND BRUCE LONGTIN University of California, Berkeley, Calif.

P

REVIOUS papers (3, 4) have indicated the general validity of the molal property us. mole fraction diagrams as a representation of the behavior of separation processes. While they are valid, they are not always convenient. They will become most useful when we can transfer their implications to the simpler 21 us. z diagrams. This can be readily accomplished when we recognize a fundamental correspondence between geometrical elements of the diagrams. Contact Transformations The y 08. 5 diagram may be obtained from the molal property vs. mole fraction diagram by what is known mathe-

I n the McCabe and Thiele diagram for the design of fractionating columns and in similar diagrams, various simplifying assumptions are usually made. This paper indicates methods of transferring various operations which can be represented with no simplifying assumptions in a mole property V S . mole fraction diagram to diagrams of the McCabe and Thiele type. Thus a pair of liquids each with a different heat of vaporization is shown to give a curved instead of straight operating line in the McCabe and Thiele diagram. matically as a contact transformation ( 2 ) . Through each point (z, y) in the X Y plane there is an infinite number of line elements; each has a different slope, p . By the equations