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Lactose Crystallization in Dry Products of Milk. I. A Method for Estimating the Degree of Crystallization
JOURNAL OF DAIRY SCIENCE VOLUME X X X I V
SEPTEMBER, 1 9 5 1
NUMBEI~ 9
L A C T O S E C R Y S T A L L I Z A T I O N I N DRY P R O D U C T S OF MILK. I. A METHOD FOR ESTIMATING THE DEGREE OF CRYSTALLIZATION R. P. CHOI, C. W. TATTER1 AND C. ~¢I. O ' M A L L E Y 2 Thv A~nerican Dry Mille Institute, Inc., Chicago, Illinois
I n continuing the study (2) on the state of lactose in d r y products of milk, a different approach to determine the degree of crystallization of lactose has been investigated. I n a saturated solution of lactose the alpha and beta forms of lactose are in equilibrium with one another and with the crystalline a-lactose h y d r a t e solid phase. Crystalline a-lactose h y d r a t e when added to such a solution will not dissolve. On the other hand, amorphous or "glass" lactose, which is a supersaturated solution of lactose, will dissolve because this essentially amounts to diluting the lactose " g l a s s . " Likewise, crystalline fl-lactose will dissolve because of its greater solubility in water than lactose h y d r a t e (5). Therefore, when a d r y product of milk, which may contain a- and B-lactose in either the amorphous or crystalline state or both, is added to a saturated solution of lactose, only crystalline m-lactose h y d r a t e will remain undissolved. I f the suspension is centrifuged quickly to prevent any appreciable seeding or crystallization from occurring, then, by analyzing the s u p e r n a t a n t liquid for the amount of lactose which has gone into solution and also for the total lactose in the d r y sample, the degree of crystallization can be calculated. METHOD
A solution of lactose, saturated at 25 ° C., was p r e p a r e d by dissolving exactly 22.8 g. of lactose h y d r a t e (c.p.) in 100.0 g. of distilled water. The solntion was covered with a thin layer of toluene and allowed to stand at least 24 hr. before use. E x a c t l y 25.00 g. of the saturated solution were weighed into a 50-ml. P y r e x centrifuge tube and the content brought to the temperature of 25 ° C. in a constant-temperature water bath. An accurately weighed quantity of d r y product of milk (5.00 g. for nonfat d r y milk solids or 3.5 g. for dry whey solids) was quickly added to the saturated solution. The tube was tightly stoppered with a r u b b e r stopper, shaken in the water bath for I rain. at the rate of about two short excursions per second and then immediately centrifuged for 2 rain. at about 1,500 r.p.m. The supernatant liquid was carefully decanted into a dry test tube. Total solids were determined on the s u p e r n a t a n t liquid using the Received for publication March 2, 1951. 1 Present address: Beatrice Foods Co., Chicago, Ill. 2 Present address: Dry Milks Inc., 308 W. Washington St., Chicago, Ill. 845 Copyright 1951, by the A~EEICAN DAIRY SCIENCE ASSOCIATION
846
R. t,. cHoI ET XL
A.O.A.C. method for fluid milk (1). Lactose in the s u p e r n a t t m t liquid was determined by the H i n t o n - M a c a r a method (3) by accurately weighing an aliquot of 3.5 to 4.0 g. into a 100-ml. volumetric flask and diluting to volume a f t e r precipitating the proteins with 5.00 ml. of p r e c i p i t a t i n g reagent. Twenty-five ml. of the clear filtrate were oxidized with the ehloramine-T reage`nt. F r o m the per cent of water in the s u p e r n a t a n t liquid, as calculated f r o m the total solids content, the lactose concentration can be expressed as g r a m s of lactose m o n o h y d r a t e per 100 g. of water. The per cent of the total lactose t h a t is in the crystalline a-hydrate f o r m m a y be calculated as follows: Crystalline lactose hydrat: ~ (as per cent of total lactose) = 100
20.4 (C - 22.8) 100 WL
C is the concentration of lactose in the s u p e r n a t a n t liquid in g r a m s per 100 g. of water, W is the weight of d r y sample used, and L is the per cent of lactose in the d r y milk. The factor 20.4 represents the m u n b e r of grams of w a t e r in 25 g. of the s a t u r a t e d lactose solution. RESULTS AND DISCUSSION The accuracy of the present method for the determi.nation of crystalline a-laetese h y d r a t e is dopendent, to a large ~,xteut, upon the amount of crystallization that m a y take place when the dr'y milk product is shaken with the s a t u r a t e d lactose solution. E x c e p t in the case where all of the lactose is present as the crystalline a-hydrate, the resultant solution is s u p e r s a t u r a t e d to various degrees with respect to lactose. I`n the absence of nuchd of a-lactose hydrate, spontaneous crystallization p r o b a b l y will not occur, since the degree of supers a t u r a t i o n is well below the super-solubility of lactose (7). Ilowever, with products containing both a-lactose h y d r a t e and amorphous lactose, crystallization m a y take place in the l-rain, shaking period and during p a r t of the c e n t r i f u g i n g period. Therefore, a series of experiments was conducted to investigate this factor. Crystalline a-lactose h y d r a t e powder (c.p.), crystalline fl-anhydride (c.p.) and a spray-process nonfat d r y milk solids in which the lactose is amorphous were used to p r e p a r e nlixtures of v a r y i n g co,ncentrations of crystalline a-hydrate to correspond to different degrees of crystallization of the lactose. E a c h m i x t u r e contained a total of about 2.5 g. of lactose, a p p r o x i m a t e l y the same q u a n t i t y as present in the amount of d r y milk sample used for analysis. The concentration of fl-lactose was m a i n t a i n e d at the equilibrium value and assumed to be unaltered until all of the a-lactose has been crystallized. This assumption is reasonable, since the rate of crystallization of a-lactose in d r y products of milk p r o b a b l y is much faster t h a n the rate of conversion of beta to alpha form. The m i x t u r e s were analyzed by the method described above. The a m o u n t of lactose which dissolved in the s a t u r a t e d solution is compared with the theoretical soluble lactose, which is the sum of the ~-lactose and the lactose contributed by the n o n f a t d r y milk solids. D a t a are presented in table 1. I t m a y be noted t h a t lactose h y d r a t e (mixture 1) does not dissolve in the s a t u r a t e d solution. F o r mixture 7 containing only 5.00 g. of ,nonfat d r y milk
LACTOSE CRYSTALLIZATION.
847
I
solids 97 to 99 per cent of the total lactose dissolved, indicating little or no crystallization. Mixture 6 consisting of 1.0 g. of crystalline a-hydrate and 1.5 g. of B-lactose shows slight crystallization, since o.nly 94 to 96 per cent of the B-lactose actually dissolved. On the other hand, for m i x t u r e s containing crystalline ~-laetose hydrate, B-anhydride and a m o r p h o u s lactose, which is an equilibr i m n mixture of a- and B-lactose, recoveries are not as good, a fact which definitely indicates crystallization of the a-lactose f r o m solution. The lower degree of crystallization in the ease of m i x t u r e 6 m a y be a t t r i b u t e d to the fact t h a t the B-lactose must undergo m u t a r o t a t i o n to the alpha f o r m before crystallization can take place. TABLE
I
Results o f seedi~tq experiments .~t,sing c'rystalli'ne ~-laetose hydrate, spray process nonfat dry mille solids (ndms) and ~-lactose Composition Mixture
1 2 3 4 5 6 7
Lacrose - t t , O n d m s
Beta
(g.)
(g.)
(.q.)
2.5 0.2 0.4 0.6 0.8 1.0 0
0 4.0 3.0 2.0 1.0 0 5.0
0 0.3 0.6 (1.9 1.2 :1.5 0
L a c t o s e soluble ( a s g. lactose h y d r a t e / 1 0 0 g, H ,O )
Recovery in t r i a l :
Theoretieal
1
1
2
3
(%)
(%)
(%)
0 11.6 10.6 9.60 8.72 7.59 ] 2.6
0 10.8 9.8 9.6 8.0 7.3 12.4
93 93 ] 00 92 96 99
93 86 90 93 94 97
91
Found in t r i a l : 2 3
0 10.8 9.1 8.6 8.1 7. l ] '2.'2
0 1.0.6 8.8 7.8 7.2 12.'2
92 89 95 97
I n an effort to minimize this seeding effect, various experin,ental conditions were studied. A worki.ng t e m p e r a t u r e of 39 ° C. as well as doubling the amount of each con~ponent was investigated, but no i m p r o v e m e n t was found. Also studied was the effect of addition of a dye, crystal violet. Leighton and t ' c t e r s (7) fom~d t h a t ('.rystal violet was one of the six dyes out of 3!) studied t h a t have a r e t a r d i n g effect on laetose crystallization. The concentration of the (lye used was not reported by the authors. Accordingly, a fairly strong solution (0.5 per cent) was p r e p a r e d in a s a t u r a t e d lactose solution and different aliquots of this diluted with s a t u r a t e d lactose solution to give a total of 25.0 g. The results obtained indicate no r e t a r d i n g action on crystallization. The method has beeti applied to a study of the degree of lactose crystallization in various d r y products of milk. T r o y and S h a r p (8) have previously used the seeding test of t t u d s o n and Brown (6) and examination b y means of a polarizing microscope to s t u d y the state of lactose in d r y milks. Th(,se tests show only the prese.nce or absence of lactose h y d r a t e crystals but give no indication of the q u a n t i t y present. D a t a in table 2 show the degrees of crystallization of lactose in n o n f a t d r y milk solids and d r y whey solids as determined by the present method. Nonfat d r y milk solids samples 1 to 6 inclusive, were relatively fresh products containing less t h a n 4 per cent moisture. Samples 7 to 11, inclusive, which had been stored in p a p e r bags u n d e r high relative humidity, had
848
R . P . CIIOI ET AL
m o i s t u r e c o n t e n t s of 8 to 9 p e r cent. F o r t h e s a m p l e s of d r y w h e y s o l i d s t h e a- a n d fl-lactose c o n t e n t s w e r e d e t e r m i n e d b y t h e s o l u b i l i t y m e t h o d ( 3 ) . I n all s a m p l e s of s p r a y - a n d r o l l e r - p r o c e s s n o n f a t d r y m i l k solids of low m o i s t u r e c o n t e n t a n d in d r y w h e y solids s a m p l e s I to 3, inclusive, t h e lactose is a l m o s t e n t i r e l y in t h e a m o r p h o u s s t a t e . I n these p r o d u c t s t h e r a t i o of fl- to a-lactose is a b o u t 1,6 to 1, as d e t e r m i n e d b y t h e s o l u b i l i t y m e t h o d ( 3 ) . T h e s e r e s u l t s c o n f i r m the o b s e r v a t i o n s of T r o y a n d S h a r p ( 8 ) . i n t h e s a m p l e s of TABLE 2 Degree of crystallization of lactose iJt nonfat dry milk solids and dry whey solids
S,~mple
(spray) (spray) (spray) (roller) 5 ( roller ) 6 (roller) 7 8 9 10 11 1 2
3 4 5 6 7 8 9 l0 ]1
% o f totallactose cryst 'd as alpha hydrate
Alpha-beta lactose (as % total lactose) Alpha
Beta
38.0 37.4 40.4 71.5 69.9 89.0 86.8 82.7 77.2 79.8 84.l
62.0 62.6 59.6 28.5 30.1 11.0 13.2 17.3 22.8 20.2 15.9
Nonfat dry milk solids 5.2 3.3 4.2 2.6, 1.1 1.4 0.5 39.6 49.4 51.6 63.2 50.(; Dry whey solids 6.7 4.1, 4.1 5.8, 5.8 70.0, 69.5 70.0, 70.0 91.1 87.2 82.6 76.0 78.3 83.6
n o n f a t d r y nfilk solids of h i g h m o i s t u r e c o n t e n t the lactose is c r y s t a l l i z e d to t h e e x t e n t of f r o m 39.6 to 63.2 p e r cent. I n t h e remaini.ng s a m p l e s of d r y w h e y solids i n w h i c h c ~ y s t a l l i z a t i o n of t h e l a c t o s e was i n d u c e d d u r i n g p r o c e s s i n g to r e d u c e t h e h y g r o s c o p i c i t y of t h e final p r o d u c t , the a m o u n t of c r y s t a l l i n e a-lactose h y d r a t e v a r i e s f r o m 69.7 to 91.1 p e r c e n t of the t o t a l lactose. These v a l u e s a g r e e closely w i t h the t o t a l a-lactose as d e t e r m i n e d b y t h e s o l u b i l i t y m e t h o d ( 3 ) . T h i s f a c t i n d i c a t e s t h a t t h e a-lactose in these s a m p l e s h a s b e e n c o m p l e t e l y c r y s t a l l i z e d a n d t h a t t h e r a t e of c r y s t a l l i z a t i o n of a-lactose is g r e a t e r t h a n t h e r a t e of i s o m e r i z a t i o n of t h e beta to t h e a l p h a in t h e l a t e r s t a g e of c r y s t a l l i z a t i o n . F o r t h i s t y p e of d r y m i l k p r o d u c t a d e t e r m i n a t i o n of t h e a m o u n t of c r y s t a l l i n e a-lactose h y d r a t e also w o u l d i n d i r e c t l y give t h e fl-laetose c o n t e n t . A p p l i c a t i o n of this m e t h o d to t h e s t u d y of the effects of m o i s t u r e a n d 95 p e r c e n t e t h y l alcohol on l a c t o s e c r y s t a l l i z a t i o n w i l l be d e s c r i b e d in a n o t h e r paper.
LACTOSE CRYSTALL[Z&TION.
1
849
SUMMARY
A method is described for estimating the amount of crystalline a-lactose h y d r a t e in d r y products of milk. I t is based upon the fact that crystalline a-lactose hydrate is insoluble in a saturated lactose solution, while amorphous lactose and crystalline fl-anhydride are soluble. The simultaneous presence of crystalline lactose h y d r a t e and amorphous lactose decreases somewhat the accuracy of the method due to crystallization. I t has been shown by this method that the lactose is non-crystalline in fresh nonfat d r y milk solids of low moisture content and also in some d r y whey solids. F o r n o n f a t d r y milk solids which had been allowed to absorb considerable amounts of moisture at high relative humidities and for d r y whey solids in which (~rystallization had been induced during m a n u f a c t u r i n g the method shows a large percentage of crystalline a-lactose hydrate. REFERENCES ( t ) ASSOCIATION 0F OF~'ICIAL AGRICULTURAL CHEMISTS. O~eial and Tentative Methods o/ Analysis. 6th ed. p. 207. Washington, D . C . ]945. (2) CHoI, R. P., O'MALLEY, C. M., ~-ND F~IRBA.~KS, B . W . Moisture Studies on Dry Products of Milk. II. Estimating Water of Crystallization of Alpha Lactose in Dry Whey Solids. J. Dairy Sci., 31: 619-625. 1948. (3) Cl-IOI,R. P., TATTEI¢, C. W.~ O ~MALLEY, C. M., AND FAIRBANKS, ]~. W. A Solubility Method for the Determination of Alpha and :Beta Lactose in Dry Products of Milk. J. Dairy Sci., 32: 39]-397. ]949. (4) ttI~To~, C. L., AND MACAaA, T. The Determination of Aldose Sugar by Means of Chloramine-T with Special Reference to the Analysis of Milk Products. Analyst, 52: 668688. 1927. (5) ttUDSON, C . S . Further Studies on the Forms of Milk Sugar. J. Am. Chem. Soe., 30: 1767-1783. 1908. (6) HVDSON, C. S., AND BROWN, F . C . The IIeat of Solution of the Three Forms of Milk Sugar. J. Am. Chem. Soc., 30: 960-97]. ]908. (7) |~EIGHTON,A., AND PETER, p . N . Factors I~flue~wblg the Cryst~llizatiou of L:wtose. Pro('. World's Dairy Congress, 1: 477~i85. 1923. (8) TROY, It. C., ANI) SI.IARP, P. F. Alpha and ]%ta Lactose in Some Milk Products. J. Dairy Sci., 13: 140-157. 1930.
SEPTEMBER, 1 9 5 1
NUMBEI~ 9
L A C T O S E C R Y S T A L L I Z A T I O N I N DRY P R O D U C T S OF MILK. I. A METHOD FOR ESTIMATING THE DEGREE OF CRYSTALLIZATION R. P. CHOI, C. W. TATTER1 AND C. ~¢I. O ' M A L L E Y 2 Thv A~nerican Dry Mille Institute, Inc., Chicago, Illinois
I n continuing the study (2) on the state of lactose in d r y products of milk, a different approach to determine the degree of crystallization of lactose has been investigated. I n a saturated solution of lactose the alpha and beta forms of lactose are in equilibrium with one another and with the crystalline a-lactose h y d r a t e solid phase. Crystalline a-lactose h y d r a t e when added to such a solution will not dissolve. On the other hand, amorphous or "glass" lactose, which is a supersaturated solution of lactose, will dissolve because this essentially amounts to diluting the lactose " g l a s s . " Likewise, crystalline fl-lactose will dissolve because of its greater solubility in water than lactose h y d r a t e (5). Therefore, when a d r y product of milk, which may contain a- and B-lactose in either the amorphous or crystalline state or both, is added to a saturated solution of lactose, only crystalline m-lactose h y d r a t e will remain undissolved. I f the suspension is centrifuged quickly to prevent any appreciable seeding or crystallization from occurring, then, by analyzing the s u p e r n a t a n t liquid for the amount of lactose which has gone into solution and also for the total lactose in the d r y sample, the degree of crystallization can be calculated. METHOD
A solution of lactose, saturated at 25 ° C., was p r e p a r e d by dissolving exactly 22.8 g. of lactose h y d r a t e (c.p.) in 100.0 g. of distilled water. The solntion was covered with a thin layer of toluene and allowed to stand at least 24 hr. before use. E x a c t l y 25.00 g. of the saturated solution were weighed into a 50-ml. P y r e x centrifuge tube and the content brought to the temperature of 25 ° C. in a constant-temperature water bath. An accurately weighed quantity of d r y product of milk (5.00 g. for nonfat d r y milk solids or 3.5 g. for dry whey solids) was quickly added to the saturated solution. The tube was tightly stoppered with a r u b b e r stopper, shaken in the water bath for I rain. at the rate of about two short excursions per second and then immediately centrifuged for 2 rain. at about 1,500 r.p.m. The supernatant liquid was carefully decanted into a dry test tube. Total solids were determined on the s u p e r n a t a n t liquid using the Received for publication March 2, 1951. 1 Present address: Beatrice Foods Co., Chicago, Ill. 2 Present address: Dry Milks Inc., 308 W. Washington St., Chicago, Ill. 845 Copyright 1951, by the A~EEICAN DAIRY SCIENCE ASSOCIATION
846
R. t,. cHoI ET XL
A.O.A.C. method for fluid milk (1). Lactose in the s u p e r n a t t m t liquid was determined by the H i n t o n - M a c a r a method (3) by accurately weighing an aliquot of 3.5 to 4.0 g. into a 100-ml. volumetric flask and diluting to volume a f t e r precipitating the proteins with 5.00 ml. of p r e c i p i t a t i n g reagent. Twenty-five ml. of the clear filtrate were oxidized with the ehloramine-T reage`nt. F r o m the per cent of water in the s u p e r n a t a n t liquid, as calculated f r o m the total solids content, the lactose concentration can be expressed as g r a m s of lactose m o n o h y d r a t e per 100 g. of water. The per cent of the total lactose t h a t is in the crystalline a-hydrate f o r m m a y be calculated as follows: Crystalline lactose hydrat: ~ (as per cent of total lactose) = 100
20.4 (C - 22.8) 100 WL
C is the concentration of lactose in the s u p e r n a t a n t liquid in g r a m s per 100 g. of water, W is the weight of d r y sample used, and L is the per cent of lactose in the d r y milk. The factor 20.4 represents the m u n b e r of grams of w a t e r in 25 g. of the s a t u r a t e d lactose solution. RESULTS AND DISCUSSION The accuracy of the present method for the determi.nation of crystalline a-laetese h y d r a t e is dopendent, to a large ~,xteut, upon the amount of crystallization that m a y take place when the dr'y milk product is shaken with the s a t u r a t e d lactose solution. E x c e p t in the case where all of the lactose is present as the crystalline a-hydrate, the resultant solution is s u p e r s a t u r a t e d to various degrees with respect to lactose. I`n the absence of nuchd of a-lactose hydrate, spontaneous crystallization p r o b a b l y will not occur, since the degree of supers a t u r a t i o n is well below the super-solubility of lactose (7). Ilowever, with products containing both a-lactose h y d r a t e and amorphous lactose, crystallization m a y take place in the l-rain, shaking period and during p a r t of the c e n t r i f u g i n g period. Therefore, a series of experiments was conducted to investigate this factor. Crystalline a-lactose h y d r a t e powder (c.p.), crystalline fl-anhydride (c.p.) and a spray-process nonfat d r y milk solids in which the lactose is amorphous were used to p r e p a r e nlixtures of v a r y i n g co,ncentrations of crystalline a-hydrate to correspond to different degrees of crystallization of the lactose. E a c h m i x t u r e contained a total of about 2.5 g. of lactose, a p p r o x i m a t e l y the same q u a n t i t y as present in the amount of d r y milk sample used for analysis. The concentration of fl-lactose was m a i n t a i n e d at the equilibrium value and assumed to be unaltered until all of the a-lactose has been crystallized. This assumption is reasonable, since the rate of crystallization of a-lactose in d r y products of milk p r o b a b l y is much faster t h a n the rate of conversion of beta to alpha form. The m i x t u r e s were analyzed by the method described above. The a m o u n t of lactose which dissolved in the s a t u r a t e d solution is compared with the theoretical soluble lactose, which is the sum of the ~-lactose and the lactose contributed by the n o n f a t d r y milk solids. D a t a are presented in table 1. I t m a y be noted t h a t lactose h y d r a t e (mixture 1) does not dissolve in the s a t u r a t e d solution. F o r mixture 7 containing only 5.00 g. of ,nonfat d r y milk
LACTOSE CRYSTALLIZATION.
847
I
solids 97 to 99 per cent of the total lactose dissolved, indicating little or no crystallization. Mixture 6 consisting of 1.0 g. of crystalline a-hydrate and 1.5 g. of B-lactose shows slight crystallization, since o.nly 94 to 96 per cent of the B-lactose actually dissolved. On the other hand, for m i x t u r e s containing crystalline ~-laetose hydrate, B-anhydride and a m o r p h o u s lactose, which is an equilibr i m n mixture of a- and B-lactose, recoveries are not as good, a fact which definitely indicates crystallization of the a-lactose f r o m solution. The lower degree of crystallization in the ease of m i x t u r e 6 m a y be a t t r i b u t e d to the fact t h a t the B-lactose must undergo m u t a r o t a t i o n to the alpha f o r m before crystallization can take place. TABLE
I
Results o f seedi~tq experiments .~t,sing c'rystalli'ne ~-laetose hydrate, spray process nonfat dry mille solids (ndms) and ~-lactose Composition Mixture
1 2 3 4 5 6 7
Lacrose - t t , O n d m s
Beta
(g.)
(g.)
(.q.)
2.5 0.2 0.4 0.6 0.8 1.0 0
0 4.0 3.0 2.0 1.0 0 5.0
0 0.3 0.6 (1.9 1.2 :1.5 0
L a c t o s e soluble ( a s g. lactose h y d r a t e / 1 0 0 g, H ,O )
Recovery in t r i a l :
Theoretieal
1
1
2
3
(%)
(%)
(%)
0 11.6 10.6 9.60 8.72 7.59 ] 2.6
0 10.8 9.8 9.6 8.0 7.3 12.4
93 93 ] 00 92 96 99
93 86 90 93 94 97
91
Found in t r i a l : 2 3
0 10.8 9.1 8.6 8.1 7. l ] '2.'2
0 1.0.6 8.8 7.8 7.2 12.'2
92 89 95 97
I n an effort to minimize this seeding effect, various experin,ental conditions were studied. A worki.ng t e m p e r a t u r e of 39 ° C. as well as doubling the amount of each con~ponent was investigated, but no i m p r o v e m e n t was found. Also studied was the effect of addition of a dye, crystal violet. Leighton and t ' c t e r s (7) fom~d t h a t ('.rystal violet was one of the six dyes out of 3!) studied t h a t have a r e t a r d i n g effect on laetose crystallization. The concentration of the (lye used was not reported by the authors. Accordingly, a fairly strong solution (0.5 per cent) was p r e p a r e d in a s a t u r a t e d lactose solution and different aliquots of this diluted with s a t u r a t e d lactose solution to give a total of 25.0 g. The results obtained indicate no r e t a r d i n g action on crystallization. The method has beeti applied to a study of the degree of lactose crystallization in various d r y products of milk. T r o y and S h a r p (8) have previously used the seeding test of t t u d s o n and Brown (6) and examination b y means of a polarizing microscope to s t u d y the state of lactose in d r y milks. Th(,se tests show only the prese.nce or absence of lactose h y d r a t e crystals but give no indication of the q u a n t i t y present. D a t a in table 2 show the degrees of crystallization of lactose in n o n f a t d r y milk solids and d r y whey solids as determined by the present method. Nonfat d r y milk solids samples 1 to 6 inclusive, were relatively fresh products containing less t h a n 4 per cent moisture. Samples 7 to 11, inclusive, which had been stored in p a p e r bags u n d e r high relative humidity, had
848
R . P . CIIOI ET AL
m o i s t u r e c o n t e n t s of 8 to 9 p e r cent. F o r t h e s a m p l e s of d r y w h e y s o l i d s t h e a- a n d fl-lactose c o n t e n t s w e r e d e t e r m i n e d b y t h e s o l u b i l i t y m e t h o d ( 3 ) . I n all s a m p l e s of s p r a y - a n d r o l l e r - p r o c e s s n o n f a t d r y m i l k solids of low m o i s t u r e c o n t e n t a n d in d r y w h e y solids s a m p l e s I to 3, inclusive, t h e lactose is a l m o s t e n t i r e l y in t h e a m o r p h o u s s t a t e . I n these p r o d u c t s t h e r a t i o of fl- to a-lactose is a b o u t 1,6 to 1, as d e t e r m i n e d b y t h e s o l u b i l i t y m e t h o d ( 3 ) . T h e s e r e s u l t s c o n f i r m the o b s e r v a t i o n s of T r o y a n d S h a r p ( 8 ) . i n t h e s a m p l e s of TABLE 2 Degree of crystallization of lactose iJt nonfat dry milk solids and dry whey solids
S,~mple
(spray) (spray) (spray) (roller) 5 ( roller ) 6 (roller) 7 8 9 10 11 1 2
3 4 5 6 7 8 9 l0 ]1
% o f totallactose cryst 'd as alpha hydrate
Alpha-beta lactose (as % total lactose) Alpha
Beta
38.0 37.4 40.4 71.5 69.9 89.0 86.8 82.7 77.2 79.8 84.l
62.0 62.6 59.6 28.5 30.1 11.0 13.2 17.3 22.8 20.2 15.9
Nonfat dry milk solids 5.2 3.3 4.2 2.6, 1.1 1.4 0.5 39.6 49.4 51.6 63.2 50.(; Dry whey solids 6.7 4.1, 4.1 5.8, 5.8 70.0, 69.5 70.0, 70.0 91.1 87.2 82.6 76.0 78.3 83.6
n o n f a t d r y nfilk solids of h i g h m o i s t u r e c o n t e n t the lactose is c r y s t a l l i z e d to t h e e x t e n t of f r o m 39.6 to 63.2 p e r cent. I n t h e remaini.ng s a m p l e s of d r y w h e y solids i n w h i c h c ~ y s t a l l i z a t i o n of t h e l a c t o s e was i n d u c e d d u r i n g p r o c e s s i n g to r e d u c e t h e h y g r o s c o p i c i t y of t h e final p r o d u c t , the a m o u n t of c r y s t a l l i n e a-lactose h y d r a t e v a r i e s f r o m 69.7 to 91.1 p e r c e n t of the t o t a l lactose. These v a l u e s a g r e e closely w i t h the t o t a l a-lactose as d e t e r m i n e d b y t h e s o l u b i l i t y m e t h o d ( 3 ) . T h i s f a c t i n d i c a t e s t h a t t h e a-lactose in these s a m p l e s h a s b e e n c o m p l e t e l y c r y s t a l l i z e d a n d t h a t t h e r a t e of c r y s t a l l i z a t i o n of a-lactose is g r e a t e r t h a n t h e r a t e of i s o m e r i z a t i o n of t h e beta to t h e a l p h a in t h e l a t e r s t a g e of c r y s t a l l i z a t i o n . F o r t h i s t y p e of d r y m i l k p r o d u c t a d e t e r m i n a t i o n of t h e a m o u n t of c r y s t a l l i n e a-lactose h y d r a t e also w o u l d i n d i r e c t l y give t h e fl-laetose c o n t e n t . A p p l i c a t i o n of this m e t h o d to t h e s t u d y of the effects of m o i s t u r e a n d 95 p e r c e n t e t h y l alcohol on l a c t o s e c r y s t a l l i z a t i o n w i l l be d e s c r i b e d in a n o t h e r paper.
LACTOSE CRYSTALL[Z&TION.
1
849
SUMMARY
A method is described for estimating the amount of crystalline a-lactose h y d r a t e in d r y products of milk. I t is based upon the fact that crystalline a-lactose hydrate is insoluble in a saturated lactose solution, while amorphous lactose and crystalline fl-anhydride are soluble. The simultaneous presence of crystalline lactose h y d r a t e and amorphous lactose decreases somewhat the accuracy of the method due to crystallization. I t has been shown by this method that the lactose is non-crystalline in fresh nonfat d r y milk solids of low moisture content and also in some d r y whey solids. F o r n o n f a t d r y milk solids which had been allowed to absorb considerable amounts of moisture at high relative humidities and for d r y whey solids in which (~rystallization had been induced during m a n u f a c t u r i n g the method shows a large percentage of crystalline a-lactose hydrate. REFERENCES ( t ) ASSOCIATION 0F OF~'ICIAL AGRICULTURAL CHEMISTS. O~eial and Tentative Methods o/ Analysis. 6th ed. p. 207. Washington, D . C . ]945. (2) CHoI, R. P., O'MALLEY, C. M., ~-ND F~IRBA.~KS, B . W . Moisture Studies on Dry Products of Milk. II. Estimating Water of Crystallization of Alpha Lactose in Dry Whey Solids. J. Dairy Sci., 31: 619-625. 1948. (3) Cl-IOI,R. P., TATTEI¢, C. W.~ O ~MALLEY, C. M., AND FAIRBANKS, ]~. W. A Solubility Method for the Determination of Alpha and :Beta Lactose in Dry Products of Milk. J. Dairy Sci., 32: 39]-397. ]949. (4) ttI~To~, C. L., AND MACAaA, T. The Determination of Aldose Sugar by Means of Chloramine-T with Special Reference to the Analysis of Milk Products. Analyst, 52: 668688. 1927. (5) ttUDSON, C . S . Further Studies on the Forms of Milk Sugar. J. Am. Chem. Soe., 30: 1767-1783. 1908. (6) HVDSON, C. S., AND BROWN, F . C . The IIeat of Solution of the Three Forms of Milk Sugar. J. Am. Chem. Soc., 30: 960-97]. ]908. (7) |~EIGHTON,A., AND PETER, p . N . Factors I~flue~wblg the Cryst~llizatiou of L:wtose. Pro('. World's Dairy Congress, 1: 477~i85. 1923. (8) TROY, It. C., ANI) SI.IARP, P. F. Alpha and ]%ta Lactose in Some Milk Products. J. Dairy Sci., 13: 140-157. 1930.