The Acid Hydrolysis of Lactose and the Preparation of Hydrolyzed Lactose Sirup

T H E A C I D H Y D R O L Y S I S OF L A C T O S E AND T H E P R E P A R A T I O N OF HYDROLYZED LACTOSE SIRUP G. A. l ~ A M S D E L L AND B. H . W E B B

Division of Dairy l~eseareh Laboratories, Bureau of Dairy Industry, A gr,~cultural t~esearch Administration, U. S. Department of Agriculture

This paper reports an investigation of factors concerned in the hydrolysis of lactose by acids, including a study of the solubility of mixtures of glucose and galactose a n d of methods for the production of hydrolyzed lactose sirups. B y hydro!yzing lactose into its more soluble hexose constituents, higher sugar concentrations could be obtained in some dairy products which otherwise might develop a"sandy" texture. However, the destructive effect of acid hydrolysis on proteins precludes this treatment on dairy products such as milk or whey. The milder method of enzyme hydrolysis is impractical because there are no known methods of econ(;mically preparing and concentrating large quantities of lactose. Browne and Ramsdell in a recent study (2) found the enzyme p r e p a r e d f r o m lactosefermenting yeasts to be unstable and weak in hydrolytic power. EXPERIMENTAL

U.S.P. grade lactose was used unless otherwise stated. Most of the samples were hydrolyzed by batch heating but a few were hydrolyzed in a high-temperature circulating heater. F o r one method of batch heating i50-ml, portions of the lactose test solutions were sealed in small, well-tinned cans coated with an acid-resisting lacquer. The cans were heated in a pilot evaporated-milk sterilizer with the reel revolving at 4 r.p.m. Fifteen minutes was used to bring the cans to the required temperature and this time was not included in the reported holding times. The other method of batch heating involved the use of a 2-gallon, glass-lined, steam-jacketed, reaction kettle equipped with an agitator and with an outlet valve for removing samples. The highest hydrolysis temperatures used in this work were attained in a Mallory (5, 10) heating unit. When holding periods longer than the time required for passage of the liquid through the heater were needed, the sirup was recirculated until the desired heating time was reached. The flow time through the different parts of the heater were : heating u n i t - - 3 seconds, holding p i p e - - 1 minute, cooling unit---3 seconds. The polariscopic method for determining the degree of hydrolysis was tried, but the results indicated that there was some interference dne to the by-products of hydrolysis; so the Munson and Walker gravimetric method (7) was used for most of the work. The concentrations of the different sugars in the finished hydrolyzed sirups were determined b y a combination l~eeelved f o r p u b t i c a t i m l ?¢Iarch 26, 1945. 677

678

G.A.

R A M S D E L L A N D B. I t .

WEBB

of methods described in another p a p e r (9). The effect of the by-products of hydrolysis on the reduction of copper, was not studied but none of the values obtained showed more than the theoretical yield of glucose and galactose. Mixtures of lactose and water containing as much as 800 grams of lactose per 1000 g r a m s of m i x t u r e were used. Since the lactose only p a r t l y dissolved until the t e m p e r a t u r e of hydrolysis was approached, the concentrations of lactose and acid were expressed as the n u m b e r of grams per 1000 grams r a t h e r than per liter. Solubility determination of galactose and glucose in each other's presence were made by placing an excess of galactose in glucose solutions of various concentrations. The mixtures were placed in 125-ml. ground-glassstopped bottles which were fastened to a motor-driven metal f r a m e submerged in a water b a t h held at 25 ° C. Five days were allowed for the mixtures to attain equilibrium. Solubility measurements were made by carefully t r a n s f e r r i n g filtered samples f r o m the bottles to a weighing dish containing d r y asbestos and d r y i n g to a constant weight in a vacuum oven maintained at 100 ° C. RESULTS

F o r certain food purposes the use of an organic acid as catalyst d u r i n g lactose hydrolysis might be advantageous. The effect of citric acid at different concentrations upon the reaction and the amount of inversion is shown in table 1. W h e n the p H values Obtained with citric acid ~tre comp a r e d with the values for hydrochloric acid given in table 2 and with hydrolysis d a t a presented in other p a r t s of this paper, the superiority of the mineral acid as catalyst becomes a p p a r e n t . The p H measurements given in these tables were made on the sirups a f t e r hydrolysis and, in consequence, there was a slight buffei~ing action by. the decomposition products produced during the heating. The effects of different concentrations of lactose and of hydrochloric acid and of the t e m p e r a t u r e of heating for 60 minutes in small cans by the batch method, upon the hydrolysis of lactose are presented in figures 1 to 3. The acid concen{ration was plotted on a logarithmic scale. The effects of highly concentrated lactose solutions were first studied because the p r o d u c t was to be p r e p a r e d for food m a n u f a c t u r e and the use of high concentrations would eliminate the need of subsequent condensing. D a t a on the hydrolysis of lactose solutions of different concentrations have been plotted in figure 1. The copper-reducing values of the 80 per cent and 55 per cent solutions indicated a r a p i d destruction of the sugars which increased with increased acid concentration. The data plotted in figure 2 show the effect of the concentration of acid upon the hydrolysis of 10 per cent lactose solutions at t e m p e r a t u r e s f r o m 100 ° C. to 145 ° C. At each temperature, over 90 per cent of the lactose was

679

ACID HYDROLYSIS OF LACTOSE TABLE 1 The effect of time of heating and the concentration of citric acid upon the hydrolysis of 50 per cent lactose sohttions

Heating time at 121 ° C.

Citric acid per 1000 grs.

Reaction a f t e r heating

Inversion

Velocity constant

~nin. 15

mole None O.0476 0.0951 0.1904 None 0.0476 0.0951 0.1904 None 0.0476 0.0951 0,1904 None 0.0476 0.0951 0.1904 None 0.0476 0.0951 0.1904

pH 3.56 1.85 1.67 1.45 3.44 1.80 1.67 1.41 2.87 1.78 1.65 1.41 2.80 1.69 1.51 1.32 2.41 1.78 1.62 1.37

% None 14.5 14.5 28.0 None 24,0 23.0 41.0 None 26.0 29.5 47.0 7,5 35.5 47.0 61.0 7.5 47.0 70.0 89.0

K x 10'

30

45

60

120

104 104 219 91 87 176 67 78 138 13 73 106 157 6 53 100 184

hydrolyzed when the maximum reducing value was reached and a destruction of reducing sugars became noticeable. The relationship (Fig. 3) bet w e e n t e m p e r a t u r e o f h y d r o l y s i s a n d t h e c o n c e n t r a t i o n of a c i d r e q u i r e d t o a t t a i n o v e r 90 p e r c e n t h y d r o l y s i s w a s l o g a r i t h m i c f o r t h e d a t a o f f i g u r e 2. A s t u d y w a s m a d e to d e t e r m i n e t h e a m o u n t o f h y d r o l y s i s w h i c h c o u l d b e o b t a i n e d i n a p r e s s u r e k e t t l e u s i n g a 7 0 0 0 - g r a m c h a r g e c o n t a i n i n g 30 p e r c e n t l a c t o s e b y w e i g h t a t a n a c i d c o n c e n t r a t i o n o f 0,007 m o l e s p e r 1 0 0 0 grams of solution. R e s u l t s o f a t y p i c a l e x p e r i m e n t a r e g i v e n i n f i g u r e 4. TABLE 2 The hydrogen-ion concentration of hydrolyzed lactose solutions

Lactose concentration HC1 per liter mole 0.002 0.006 0.01 0.02 0.04 0.08 0.2 0.5

10 per cent

30 per cent

pH 2.65 2.26 2,02 1.65 1.37 1.08 0.74 0.35

pH 2.46 2.04 1.75 1.42 1.11 0.89 0:54 0.20

10 per cent solution heated for 60 minutes at 130 ° C. 30 per cent solution heated for 60 minutes at 134 ° C.

680

G.A.

RAMSDELL AND B. H. WEBB ~eo

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FIG. 1. The effect of the c o n c e n t r a t i o n of HC1 u p o n the h y d r o l y s i s of lactose in solutions of different lactose concentrations. E a c h m i x t u r e w a s h e a t e d 60 m i n u t e s in cans at I 0 0 ° C., except the 80% lactose m i x t u r e w h i c h w a s heated to 108 ° C.

Thirty minutes were required to attain a temperature of 140 ° C., but at this point almost 50 per cent of the lactose was already hydrolyzed. The results shown in table 3 were obtained by hydrolysis with the circu-

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Fze. 2. The effect of the c o n c e n t r a t i o n of I-IC1 u p o n the h y d r o l y s i s of lactose in 10% lactose solutions. S a m p l e s were h e a t e d f o r 60 m i n u t e s in cans at different temperatures.

lating method. It was possible to use temperatures up to 165 ° C. with this method of heating. 3_ residue of milkstone was found coating the heating coil after the test in the]ast experiment of table 3 was started. This probably raised the pit slightly and in consequence lowered the reaction velocity.

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F~(~. 3. l~elationshIpbetween temperatnre and the concentration of l:f01 necessary to attain m a x i m u m hydrolysis (91% t o 9 3 % ) of the lactose in a 1 0 % solution durin~ h e a t i n g 60 m i n u t e s in cans.

ACID HYDROLYSIS OF LACTOSE

681

TABLE 3 ttesults f r o m e x p e r i m e n t s on the circulating m e t h o d of h y d r o l y z i n g lactose in pure solutions ~

Lactose in solution % 33.6 29.0 28.4 23.2 9.8t

HCI per 1000 grams lactose solution mole

0.034 0.023 0.023 0.019 0.020

Heating conditions

Reaction

Temperature

Time

heating

Lactose hydrolyzed

o C. 130 130 140 165 165

m~n. 36.0 58.8 30.0 8.2 10.5

pH 1.23 1.46 1.47 1.60 1.70

% 82.0 79.7 84.5 79.0 79.0

after

K × 104

Calculated time to invert 99.5%

476 271 622 1904 1487

111.3 195.4 85.3 27.8 35.6

min.

The authors are indebted to R. W. Bell of these laboratories for assistance in the operation of the Mallory heater with which the samples in this table were hydrolyzed. t A slight formation of milksto~e formed from a previous batch of milk was found to have been present on the heating coil. T h e effect o f t h e t e m p e r a t u r e of h y d r o l y s i s u p o n t h e v e l o c i t y of t h e r e a c t i o n f o r b o t h t h e r e t o r t a n d c i r c u l a t i n g m e t h o d s is p l o t t e d i n f i g u r e 5. The velocity constants K were calculated from the equagion for a unimolecular reaction : 1 a~ K = ~ × 2.303 × l o g a - x w h e r e x r e p r e s e n t s t h e a m o u n t o f h y d r o l y s i s a t t a i n e d i n t i m e t, a n d a t h e i n i t i a l c o n c e n t r a t i o n of l a c t o s e . W h e n K is c a l c u l a t e d f o r a p e r i o d b e g i n n i n g w i t h z e r o t i m e a n d a is e x p r e s s e d in p e r c e n t h y d r o l y s i s , a = 100. F o r c o n v e n i e n c e of e x p r e s s i o n K is m u l t i p l i e d b y 104. T h e v e l o c i t y c o n s t a n t s of h y d r o l y s i s of 10 p e r c e n t l a c t o s e s o l u t i o n s b y the batch method have been calculated for three acid levels and plotted in

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:Fz~.. 4. Heating curve for hydrolysis of 30% lactose solutions in pressure kettle, Values indicating the degree of hydrolysis and the reaction velocity constant are given at 4 points on the curve. The concentration of tIC1 was 0.007 mole per 1000 grams of solution.

682

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:FIG. 5. The effect of the temperature of hydrolysis upon the rate of inversiou of iactose during h e a t i n g by batch and circulating methods. The values obtained with the circulating heater were taken from table 3 ; a curve has been drawn through three of these points representing samples containing approximately the same lactose and acid concentrations.

figure 5. All the K values given in table 3 f o r the circulating method have also been plotted on figure 5. The solubility of galaetose in glucose solutions at 25 ° C. was determined and the results are plotted in figure 6. 6O,

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Solubility of galaetose in aqueous glucose solutions at 25 ° C.

ACID HYDROLYSIS OF LACTOSE

683

The solubility of glucose is 50.8 per cent (4), of galactose 32.09 p e r cent (3), while the solution containing the m a x i m u m q u a n t i t y of sugar consisted of 49.8 per cent glucose and 8.5 per cent galactose. The data indicate t h a t an equimolecular m i x t u r e of the two sugars would be s a t u r a t e d with respect to each other when the concentration of each reached 21 per cent. The combined total of 42 p e r cent glucose plus galactose represents an i m p o r t a n t increase in solubility over the value of 17.7 per cent for lactose. A procedure was developed for the p r e p a r a t i o n of a hydrolyzed lactose sirup suitable for use in foods. A charge consisting of 2100 g r a m s of a good grade of recrystallized lactose, 49 g r a m s of n o r m a l ttC1 and 4851 g r a m s of water was introduced into a 3-gallon, glass-lined, double-jacket, steamheated pressure kettle. The HC1 concentration was 0.007 moles per 1000 g r a m s of solution. The amount of acid used was held at a m i n i m u m to avoid the high salt concentration which would have resulted f r o m neutralization of large quantities of acid. The heating was carried out in accordance with the t i m e - t e m p e r a t u r e curve shown in figure 4, the m i x t u r e being stirred until the lactose dissolved. Seventy pounds of steam pressure was maintained t h r o u g h o u t the heating period of 65 minutes, but 60 minutes of this time were required for the m i x t u r e to attain the desired t e m p e r a t u r e of 147 ° C. Upon completion of the heat t r e a t m e n t the m i x t u r e was treated with decolorizing carbon, filtered, a n d condensed u n d e r v a c u u m to 60 per cent solids content. The sirup was neutralized with sodium bicarbonate to give a reaction of p H 4.9-5.0. Sirups p r e p a r e d in this m a n n e r contained practically no lactose, a p p r o x i m a t e l y 26 per cent glucose, 29 per cent galactose and 4 to 5 per cent of unidentified b y - p r o d u c t s (9). The p r o d u c t k e p t well at room t e m p e r a t u r e but showed a tendency to darken with age. The development of color in storage was more r a p i d when the reaction was adjusted to a higher pl=[,, especially above p H 6. The concentration of hexoses in water was 58 per cent, but, according to figure 6, the sirup should be s a t u r a t e d with these sugars at about 42 per cent. Occasionally in sirups made in this way some galactose crystallized d u r i n g storage, b u t most of the samples remained free of crystal f o r m a t i o n even a f t e r months of storage. l=Iydrolyzed lactose sirup was tried in food products where sweet sirups were needed. Good results were obtained when it was~used as a sweetening agent in such products as ice cream mixes, candy, sweet baked goods and f r u i t whips. I t had a pleasing flavor when used on hot cakes or as a t o p p i n g for frozen desserts. A t t e m p t s were made to hydrolyze the lactose in whey and in various p r e p a r a t i o n s of crude lactose. These experiments produced dark caramelized products which contained much caramel and c h a r r e d material. The protein and salts present in whey a n d crude lactose buffered the acid added to cataiyze the reaction with the result t h a t excessive quantities of acid were necessary. The results indicated that a hydrolyzed lactose p r e p a r a t i o n

684

G. A. RA]YISDELL AND B. I~. WEBB

acceptable for food uses should be p r e p a r e d f r o m a high grade technical or U.S.P. sugar. DISCUSSION

comparison of the effectiveness of hydrochloric acid and of various organic acids as catalysts in hydrolyzing lactose indicated t h a t hydrochloric acid was best suited for this purpose. I n view of the considerably lower degree of ionization of citric acid as compared with hydrochloric acid, it was found necessary to use 0.19 g r a m moles of citric acid to approach the reaction obtained with only 0.01 g r a m moles of hydrochloric acid. I n the p r e p a r a t i o n of a hydrolyzed product for food purposes a low salt content was desirable but this did not result when a large q u a n t i t y of organic acid was used since a correspondingly large q u a n t i t y of salt was produced by neutralization. Other investigators (1, 6, 8) have determined velocity constants for lactose hydrolysis in the presence of various quantities of acid and at temperatures below 100 ° C. A r m s t r o n g a n d Caldwell (1) hydrolyzed 18 per cent lactose with 0.5 molar HC1 at 99 ° C. and found K × 104 = 334.2. Polariscopic methods of analysis were used. D a t a f r o m figure 2 show that a 10 per cent lactose solution heated at 100 ° C. with 0.5 molar tIC1 gave a value for K × 104 = 399.7. The reaction velocity for lactose hydrolysis at 145 ° C., the highest t e m p e r a t u r e used in the batch method of heating, gave a value of K × 104 = 518 (Fig. 4). I n one experiment with the circulating heater at a t e m p e r a t u r e of 165 ° C., K x 104 was 1904 (Table 3). F r o m the data it was clear that a g r a d u a l destruction of the newly formed hexoses accompanied the hydrolysis reaction. A n analysis (9) of the sirups of 30 per cent sugar concentration p r e p a r e d b y heating in accordance with the heating curve in figure 4 showed that as the heating time progressed through 35, 50, 65 and 80 minutes the glucose concentrations were 8.6, 11.9, 13.1 and 13.1 per cent and the galactose concentrations were respectively 10.2, 13.1, 14.6 and 14.7 per cent. These figures indicate that the galactose was quite stable and most of the loss in hexose sugar was f r o m destruction of the glucose. Another indication of sugar destruction was the formation of acid during hydrolysis as indicated by the differences in the p H of the 10 per cent and 30 per cent solutions shown in table 2. Complete hydrolysis was not obtained as long as the curves of figures 1, 2, and 4 continued to rise toward the 100 per cent level. B u t none rose above 94 per cent of the theoretical yield which indicated, some destruction of sugars had taken place. Loss in reducing sugar a p p e a r e d to be greatest when a t t e m p t s were made to hydrolyze highly concentrated solutions such as the 80 p e r cent. sample, figure 1. The calculated values for K were subject to a certain uncorrected error which was dependent u p o n the extent of destruction of the sugars during hydrolysis. The per cent hydrolysis was calculated f r o m the theoretical

ACID HYDROLYSIS OF LACTOSE

685

yield of hexoses f o r m e d and the actual values obtained by analysis (9). The effect of sugar destruction on the calculated K values is illustrated in the reduction of K at different heating periods, other conditions r e m a i n i n g constant (Table 1). A substantial degree of hydrolysis of lactose in products such as whey or solutions of crude lactose was not attained without producing charred and caramelized by-products to an excessive degree. The occasional claim in d a i r y m a n u f a c t u r i n g literature and in some p a t e n t specifications t h a t the lactose in whey or skim milk undergoes a significant degree of hydrolysis a f t e r a relatively mild processing at reactions of p i t 4.0 or above would seem to be in error. The acid hydrolysis of the lactose in such products, containing, as they do, large quantities of milk protein, would be accompanied by a drastic breakdown in this protein material. M a n y samples of hydrolyzed lactose sirup were p r e p a r e d f r o m good grades of lactose. I t was f o u n d t h a t a hydrolyzed m i x t u r e with a total solids content of 60 per cent or greater would generally keep well bacteriologically but would darken in color and in some cases show crystal f o r m a tion. Although it was shown that an equimoleeular m i x t u r e of glucose and galactose was s a t u r a t e d at 42 per cent, it was possible to raise the hexose concentration of the sirups as high as 55-60 per cent before crystallization of galactose occurred spontaneously. E v i d e n t l y the presence of considerable quantities of hydrolytic by-products r e t a r d e d crystal growth. Sirups which contained a total solids content in excess of 70 p e r cent often developed crystals a f t e r a few weeks storage. SUMMARY

1. A study has been made of the factors affecting the hydrolysis of p u r e solntion~ of lactose b y heat. The velocity constant ( K × 104) for the reaction varied with the time and t e m p e r a t u r e of hydrolysis and the acid and lactose concentration of the solutions. Values for K × 10 ~" r a n g e d f r o m 11 at 100 ° C. to 1904 at 165 ° C. Hydrochloric acid was used in concentrations of 0.001 to 1.0 mole per 100'0 g r a m s of solution and it was f o u n d to be a more effective catalyst t h a n citric acid. 2. There was a progressive destruction of lactose a n d of the newly formed hexoses during hydrolysis. This destruction was accelerated by conditions of excessive or prolonged heating and when high sugar and acid concentrations were used. 3. tiexose sugars to the extent of 93 per cent of the theoretical yield were obtained b y hydrolyzing a 30 p e r cent lactose solution using 0.007 mole of HC1 per 1000 g r a m s of m i x t u r e and heating to 147 ° C:'" The process, conducted in a glass-lined pressure kettle required a tot£i time of 65 minutes, of which 60 minutes were used to heat the b a t c h ' t o 147 ° C. A circulating heater which would quickly raise the sirup to the hydrolyzing t e m p e r a t u r e would shorten the process.

686

G. A. RAMSDELL AND B. H. WEBB

4. A s o l u b i l i t y c u r v e f o r galactose i n a q u e o u s glucose solutions a t 25 ° C. has been p r e s e n t e d . The s o l u b i l i t y of glucose a t 25 ° C. is 50.8 p e r cent, galactose 32.09 p e r cent a n d a m i x t u r e of e q u a l p a r t s of both hexoses 42 p e r cent. T h e m a x i m u m c o n c e n t r a t i o n of the two hexoses which is soluble a t 25 ° C. is 58.3 p e r cent, c o n s i s t i n g of 49.8 p e r c e n t glucose a n d 8.5 p e r c e n t galactose. 5. The lactose i n skim milk, whey or c r u d e lactose, c a n n o t be h y d r o l y z e d w i t h o u t the use of excessive q u a n t i t i e s of acid a n d the p r o d u c t i o n of u n d e s i r a b l e p r o t e i n d e c o m p o s i t i o n products. REFERENCES

(1) ARMSTRONG, E. F., AND CALDWELL, R. J'.

Studies on Enzyme Action. IV. The Sacroclastic Action of Acids as Contrasted with that of Enzymes. Proc. Roy. Soc. London, Eng., 73: 526-537. 1904.

(2) BROWNE, H. ~-~., AND RAiM:SDELL, G . A .

Dairy Res. Labs., B.D.L, A.R.A., U.S.D.A.

Unpublished Results. (3) GOULD,STEPHEN P. The Final Solubility of D-Galactose in Water. SCI., 23: 227. 1940.

JOUR. DAIRY

(4) JACKSON, RICHARD F., AND SILSBEE, CLARA GILLIS. Saturation Relations in Mixtures of Sucrose, Dextrose and Levulose. Bur. Standards Tech. Paper, No. 259.

1924. (5) MALLORY~W . E .

U. S. P a t e n t 2,270,540.

1942.

(6) MOELWYN-HUGHES,E . A . The Kinetics of the Hydrolysis of Certain Glucosides. Trans. Faraday Soc., 24: 309-321. 1928. (7) Official and Tentative Methods of Analysis of the Association of Official Agricultural Chemists~ Washington, 5th Edition, p. 501. 1940. (8) PHELPS, F. P., AND HUDSON, C.S.

Relation between Rotary Power and Structure

in the Sugar Group. Jour. Am. Chem. Soc., 48: 503-507. 1926. (9) RAMSDELL~ G.A. Determination of Glucose, Galactose, and Lactose in Their Mixtures. Jolm. DAIRY ScI., 28: 671-676. 1945. (10) WEB•, B. H., BELL, R. W., DEYSHEE, E. F., AND HOLM, G . E . The Effeqt of Various Degrees of Forewarming upon the Heat Stability of Milks of Different Solids Concentrations. JOUR.DAIRY SCI., 26: 571-578. 1943.