Formation of Oligosaccharides During #-Galactosidase Action on Lactose L. E. WIERZBICKI and F. V. KOSIKOWSKI Department of Food Science Cornell University, Ithaca, New York 14850 Abstract The formation of oligosaccharides or polysaccharides by transgalactosidation reactions during hydrolysis of lactose in acid whey by fi-galactosidase from Asper- gillus niger was studied. Five benzidine- positive carbohydrates, other than lactose, galactose, or glucose, were formed after fl-galactosidase action on 4% lactose at pH 4.5. These compounds, tentatively identified as oligosaccharides, comprised 1 to 2% of the total lactose in acid whey. Oligosaccharides were influenced by sub- strate concentration and reaction time. At the initial stage, oligosaccharides with Rf values less than lactose attained maxi- mum production in 18 to 25g lactose, whereas those with Rf values higher than lactose attained maximum production in 7 to 10% lactose in acid whey concen- trates. Introduction fi-galactosidase splits glycosidic linkages of lactose to produce glucose and galactose and may transfer some monosaccharide units to ac- tive acceptors, such as monosaccharides, poly- saccharides, or alcohol. This side reaction is called transgalactosidation and can occur in enzymatically or chemically catalyzed proc- esses. New glycosidic bonds are formed at dif- ferent positions of individual carbohydrates leading to oligosaeeharides of varying molecu- lar weights. The mechanism of transgalactosidation dur- ing lactose hydrolysis is not well understood. Early studies showed that the number and type of oligosaccharides formed are affected by enzyme source, substrate concentration, pH, temperature, and inorganic ions (9). The significance of oligosaccharides in foods at high concentration may be important nutritionally because of man's inability to digest them. Oligosaccharides formed from enzymatically hydrolyzed lactose in milk or whey have been Received November 30, 1979.. reported by several investigators (1, 5, 7, 8, 9). The number of oligosaccharides in milk varies from 3 to 11 (5, 8). While fi-galactosidase from A. niger (12) shows promise for the development of food in- gredients from acid whey and fermented milks, no information exists on the extent of oligo- saccharide formation by/~-galactosidase under acid conditions. The present study shows the extent of oligosaccharide formation during lac- tose hydrolysis by fl-galactosidase from A. niger. Methods and Materials Enzyme and substrate. A fl-galactosidase (lactase) preparation from A. niger with op- timum activity at pH 3.5 to 4.5 and 37 to 55 C was obtained from the Wallerstein Company (Morton Grove, Illinois). The substrate was acid whey (pH 4.5) pre- pared from rennet cottage cheese as described by Kosikowski (2). This whey was stored at --20 C and as required concentrated to 50% total solids under vacuum at 45 C in a rotary flash evaporator (Buchler Model FE-2 C, Fisher Scientific Co.). Enzyme assay was composed of 10 ml of acid whey (6 to 50% total solids) containing 4 to 35% lactose. The whey, containing spe- cific amounts of lactase preparations, was incu- bated at pH 4.5 and 55 C for 5 h. One milli- liter was analyzed for carbohydrates by a mod- ified Nelson-Somogyi method (4, 11) and by thin-layer chromatography (6). Oligosaccharide and monosaccharide separa- tion. Carbohydrate separation was carried out on earbon/celite columns (30 mmx 420 mm) (3, 10). Desorption of the carbohydrate from the column was obtained by stepwise elution at a flow rate 2 ml/min with water and ethanol/water mixtures to give 0, 2.5, 5, 7, 15, 25, 50, 75, and 95% ethanol. Fractions of 10 to 15 ml were obtained by an ISCO fraction collector. The 1-ml fractions were confirmed for carbohydrate by color after reacting with 1 ml of 1.0% orcinol in H2SO4. Concentrated fractions were then analyzed. Thin-layer chromatography. Five 20 x 201 1400
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Formation of Oligosaccharides During -Galactosidase Action on LactoseFormation of Oligosaccharides During #-Galactosidase Action on Lactose
L. E. WIERZBICKI and F. V. KOSIKOWSKI Department of Food Science
Cornell University, Ithaca, New York 14850
Abstract
The formation of oligosaccharides or polysaccharides by transgalactosidation reactions during hydrolysis of lactose in acid whey by fi-galactosidase from Asper- gillus niger was studied. Five benzidine- positive carbohydrates, other than lactose, galactose, or glucose, were formed after fl-galactosidase action on 4% lactose at pH 4.5. These compounds, tentatively identified as oligosaccharides, comprised 1 to 2% of the total lactose in acid whey. Oligosaccharides were influenced by sub- strate concentration and reaction time. At the initial stage, oligosaccharides with Rf values less than lactose attained maxi- mum production in 18 to 25g lactose, whereas those with Rf values higher than lactose attained maximum production in 7 to 10% lactose in acid whey concen- trates.
Introduction
fi-galactosidase splits glycosidic linkages of lactose to produce glucose and galactose and may transfer some monosaccharide units to ac- tive acceptors, such as monosaccharides, poly- saccharides, or alcohol. This side reaction is called transgalactosidation and can occur in enzymatically or chemically catalyzed proc- esses. New glycosidic bonds are formed at dif- ferent positions of individual carbohydrates leading to oligosaeeharides of varying molecu- lar weights.
The mechanism of transgalactosidation dur- ing lactose hydrolysis is not well understood. Early studies showed that the number and type of oligosaccharides formed are affected by enzyme source, substrate concentration, pH, temperature, and inorganic ions (9). The significance of oligosaccharides in foods at high concentration may be important nutritionally because of man's inability to digest them.
Oligosaccharides formed from enzymatically hydrolyzed lactose in milk or whey have been
Received November 30, 1979..
reported by several investigators (1, 5, 7, 8, 9). The number of oligosaccharides in milk varies from 3 to 11 (5, 8).
While fi-galactosidase from A. niger (12) shows promise for the development of food in- gredients from acid whey and fermented milks, no information exists on the extent of oligo- saccharide formation by/~-galactosidase under acid conditions. The present study shows the extent of oligosaccharide formation during lac- tose hydrolysis by fl-galactosidase from A. niger.
Methods and Materials
Enzyme and substrate. A fl-galactosidase (lactase) preparation from A. niger with op- timum activity at pH 3.5 to 4.5 and 37 to 55 C was obtained from the Wallerstein Company (Morton Grove, Illinois).
The substrate was acid whey (pH 4.5) pre- pared from rennet cottage cheese as described by Kosikowski (2). This whey was stored a t --20 C and as required concentrated to 50% total solids under vacuum at 45 C in a rotary flash evaporator (Buchler Model FE-2 C, Fisher Scientific Co.).
Enzyme assay was composed of 10 ml of acid whey (6 to 50% total solids) containing 4 to 35% lactose. The whey, containing spe- cific amounts of lactase preparations, was incu- bated at pH 4.5 and 55 C for 5 h. One milli- liter was analyzed for carbohydrates by a mod- ified Nelson-Somogyi method (4, 11) and by thin-layer chromatography (6).
Oligosaccharide and monosaccharide separa- tion. Carbohydrate separation was carried out on earbon/celite columns (30 m m x 420 mm) (3, 10). Desorption of the carbohydrate from the column was obtained by stepwise elution at a flow rate 2 ml/min with water and ethanol/water mixtures to give 0, 2.5, 5, 7, 15, 25, 50, 75, and 95% ethanol. Fractions of 10 to 15 ml were obtained by an ISCO fraction collector. The 1-ml fractions were confirmed for carbohydrate by color after reacting with 1 ml of 1.0% orcinol in H2SO4. Concentrated fractions were then analyzed.
Thin-layer chromatography. Five 20 x 201 1400
OLIGOSACCHAB.IDES 1401
cm plates were prepared by mixing 40 g of sil- ica gel G with 80 ml of water and applying to the plates. A .25-mm layer was most suitable for general analysis. Plates, after drying for a few hours at room temperature, were activated at 100 C for 30 rain and held in a desiccator containing silica gel according to Randorath (6). The developing solvent was n-butanol; acetic acid; diethylether; water (9:6:3:1).
To .5 g of benzidine dissolved in 10 ml of acetic acid were added 10 ml of 40% w/v tri- ehloroaeetic acid in water and 80 ml of ethanol used as a color developing reagent.
A volume of sugar solution (1 to 5 /~g sug- ar) was applied to the plate whichl was devel- oped in a closed jar with so vent. The plate was then dried in an oven at 100 C for 10 to 15 min, sprayed with color reagent, and heated at 100 C for 25 rain. With monosaccharides and oligosaccharides, benzidine gave yellow- brown spots under daylight and green under short wavelength UV.
Estimation of saccharides. The individual benzidine-positive spots corresponding to sug- ars were extracted directly from thin-layer chromatography (TLC) plates by a mixtttre of 40~ trichloroacetic acid in water, acetic acid, and ethanol ( 1:1:8). Optical density extracted material was measured at 400 nm in a Beck- man Model DU spectropho~ometer and sugar calculated from a standard curve prepared for various concentrations of lactose solution. Di- rect optical density readings of the spots were obtained in a Photovolt Model 520A automati- cally recording photodensitometer (Photovolt Corp., New York). Reducing (glucose) equiv- alents of the separated fractions were deter- mined by a modified Nelson-Somogyi proce- dure (4, 11).
Results
Lactose in various acid whey e~ncen- trates was hydrolyzed by fl-galactosidase (.4 mg/ml) from A. niger at 55 C for 5 h. Samples were analyzed at zero time and after 2 and 5 h incubation. The rate of lactose hydrolysis and formation of new benzidine positive com- pounds in treated samples is in Fig. 1. Zero time samples showed traces of monosaccba- rides. Eight different carbohydrates were de- tected in an acid whey sample (6% total sol- ids) containing about 4% lactose treated with fl-galactosidase for 5 h at pH 4.5. These in- cluded glucose, galactose, and lactose. The other five, presumably oligosaccharides, were separated by their Itf values and reducing sugar reactions (Tables I and 2).
TABLE 1. The Bf values of carbohydrate com- pounds" separated on thin-layer chromatograms b.
Rf Compound value
Oligosaecharide- 1 .48 Glucose .46 Galaetose .41 Oligosaccharide - 2 .32 Lactose .20 Oligosaccharide - 3 .09 Oligosaccharide - 4 .05 Oligosaccharide - 5 .03
Fractionated on carbon/celite column. b Using thin-layer chromatography with silica
gel G and n-butanol; acetic acid; diethylether; water (9:6:3:1).
Sugar fractions separated on the column af- ter TLC analysis showed two oligosaccharides in the fraction eluted with 5% ethanol (Areas B and E, Fig. 2). Fractions eluted with water, 2.5, 7.0, and 15% ethanol contained no oligo- saceharides (Fig. 2). A third and fourth oligo- saccharide was separated on the second col- umn with 15% ethanol (No. 7, Areas A and C, Fig. 3). A fifth oligosaccharide was ob- served (No. 2, Area B, Fig. 3).
Concentrations of oligosaccharides produced in acid whey by fi-galactosidase are shown (Fig. 4). Total oligosaccharides increased with increasing substrate concentration between 4 to 21% lactose. Maximum oligosaccharides in acid whey represent 1 to 2% of the original lactose. The highest rate of production oc-
Fro. 1. Thin-laver ehromatogram showing the progressive hydrolysis and formation of oligosac- charities in acid whey containing different total whey solids by a O-galactosidase from A. niger.
Legend: A, B, C, D, E correspond respectively to oligosaccharides, lactose, oligosaccharides, ga- lactose, glucose; A', B' represent 1 mg/ml of lac- tose and glucose/galactose mixture; 1-4 correspond to 5, 10, 15, and 25g total solids, respectively.
JOURNAL OF DAIRY SCIENCE VoL. 56, NO. i i
1402 W l E R Z B I C K I A N D K O S I K O W S K I
TABLE 2. Recovery of reducing compounds ~ from hydrolyzed acid whey.
Fraction eluted Fractions on TLC Orcinol from earbon/eelite Benzidine test
column color UV reaction h
Reducing glucose equivalents
Ethanol 3 1 Red 1,379 2.5~ + + +
Ethanol 5 1 Brown-yellow 4,000 5.0~ + +
Ethanol 5 2 Red-yellow 275 7.0% + +
Ethanol 5 3 Brown-red 212 15.0% + + +
Ethanol 3 2 Brown-green 30 25% + +
Ethanol 2 2 Brown-greenish 45 50~ + +
Ethanol 1 1 Yellow 35 75% 4-
Ethanol 1 1 Yellow 30 95~g 4-
Eluted from carbon/celite column. b Intensity increases with number of 4-'s. TLC = thin-layer chromatography.
curred in the first 15 min of hydrolysis at all substrate levels, and the rate increased with increases in substrate concentration (Table 3). One group of oligosaccharides (Area A, Fig. 4) was produced in largest concentration at 18 to 25% while a second group (Area C, Fig.
4) achieved this state at 7 to 10% lactose in acid whey concentrates.
Discussion
Acid whey has not been used as a ]3-galac- tosidase substrate because of a lack of suitable
Ftc. 2. Thin-layer chromatogram showing sepa- ration of sugars on carbon/celite column obtained by ethanol elution and the products of lactose hy- drolysis using/3-galactosidase from A. niger.
Legend: A, B, C, D, E correspond respectively to lactose, oligosaccharide, galaetose, glucose, oligo- saccharide; 1-4 control spots of galactose, glucose, lactose, and mixture of glucose and galactose; 5 and 6 fractions eluted with water; 7-10 corre- spond to 2.5~ ethanol fractionation; 11-14 corre- spond to 5~; ethanol fractionation; 15 and 16 eluted with 7% ethanol; and 17 corresponds to 15-50% ethanol pool fraction. JOUR~AL OF DArRY SCIENCE VOL. 56, NO. 11
Fie. 3. Thin-layer chromatogram showing sepa- ration of sugars on earbon/eelite column obtained by ethanol elution and the products of lactose hydrolysis by fl-galactosidase from A. niger.
Legend: A to G spots correspond respectively to oligosaeeharide, oligosaccharide, oligosaccha- ride, lactose, oligosaccharide, galactose, glucose; a-d controls of glucose, galactose, mixture of glu- cose and galactose, and lactose; 1 corresponds to fraction eluted with water; 2 to fraction eluted with 5% ethanol; 3 fraction elnted with water; 4-10 correspond to 2.5, 5, 7, 15, 25-50, 75, and 95~ ethanol eluted fractions.
OLIGOSAGCHARIDES 1 4 0 3
TABLE 3. Relative optical density of spots corre- sponding to oligosaccharides occurring in acid whey following lactose hydrolysis by fl-galactosi- dase from A. niger.
Whey total solids
peak area (ram 2) ~ 5 91.5 80.0 108.0
10 165.0 180.0 200.0 20 187.5 198.0 217.0 30 201.0 210.0 270.0
Spots.
lactase preparation functioning maximally at pH 4.5 or lower. Applying a new food grade, experimental, fl-galactosidase preparation to acid whey provided a realistic means of hydro- lyziug lactose under acid conditions (11, 12, 13). However, it simultaneously caused prob- lems concerning oligosaccharide production.
Oligosaceharides may be important physio- logically in metabolic processes as noted by Roberts and McFarren (7) in testing fecal contents of rats fed a high lactose diet. There- fore, when microbial fi-galactosidases hydro- lyze the lactose of milk or milk products, the extent of oligosaceharide formation is impor- tant. Much of the early work was confined to milk, sweet whey, or lactose at neutral pH, us- ing fl-galaetosidase from Saccharomyces fragil- /s. The maximum number of oligosaccharides reported for skim milk using an S. fragiIis en-
Fro. 4. Oligosaecharide formation in acid whey at different whey solids concentration during lac- tose hydrolysis by fl-galactosidase from A. niger.
Legend: A, B, C, D, E correspond respectively to oligosaeeharides, lactose, oligosaeeharides, galae- tose, glucose; 1 corresponds to 6% of acid whey solids without enzyme treatment; 2-5 correspond to 10, 15, 25, and 35% acid whey total solids with enzyme treatment.
zyme preparation was 10 and 11 (7, 8). These oligosaccharides were as large or larger than lactose and usually contained a galactose moiety.
The present study tentatively showed the presence of only five oligosaccharides result- ing from the activity of fi-galactosidase, and these appeared at very low levels estimated at 1 to 2% of the total lactose. However, at sub- st_rate concentrations higher than 4% lactose the total oligosaccharides appeared in larger amounts. It is conceivable that at the. higher level of substrate, the small amount of unhy- drolyzed lactose which appeared in our studies may include newly formed reducing carbohy- drates. An example of such compounds might be mellibiose possessing a molecule similar to lactose except for a difference in type of gly- cosidic linkage between galactose and glucose and an almost identical Rf value on silica gel TLC (6).
References
(1) Aronson, M. 1952. Transgalactosidation dur- ing lactose hydrolysis. Arch. Biochem. Bio- phys. 39:370.
(2) Kosikowski, F. V. 1970. Page 429 in Cheese and fermented milk foods. Edwards Broth- ers, Inc., Ann Arbor, Michigan.
(3) Miller, C. L. 1960. Micro-column chromato- graphic method for analysis of oligosac- eharides. Anal. Bioehem. 2:133,
(4) Nelson, N. 1944. Nelson-Somogyi modified eolorimetrie method for determination of reducing sugar. J. Biol. Chem. 153:875.
(5) Pazur, J. H., T. M. Tipton, J. Budovieh, and J. M. Marsh. 1958. Structural characteriza- tion of products o~ enzymatic dispropor- tionation of lactose. J. Amer. Chem. See. 80:119.
(6) Randorath, K. 1964. Thin layer chromatog- raphy. Academic Press, New York.
(7) Roberts, H. R., and E. F. McFarren. 1953. The chromatographic observation of oligo- saecharides formed during the lactase hy- drolysis of lactose. J. Dairy Sci. 36:620.
(8) Roberts, H. R., and J. D. Pettinati. 1957. Conversion of lactose to oligosaccharides. J. Agr. Food Chem. 5:130.
(9) Webb, B. H., and A. H. Johnson. 1965. Fundamentals of dairy chemistry. The AVI Publishing Co., Inc., Westport, Connecticut.
(10) Whistler, K L., and D. F. Durso. 1950. Chromatographic separation of sugars on charcoal, j. Amer. Chem. See. 72:677.
(11) Wierzbicki, L. E. 1970. Biological behavior of microbial lactases and the development of new foods from their application. Ph.D. Thesis, Cornell University, Ithaca, New York.
JOURNAL OF DAIRY SCIENCE VOL. 56, NO. 11
1404 W I E R Z B I C K I A N D K O S I K O W S K I
(12) Wierzbicki, L. E., and F. V. Kosikowski. 1973. Kinetics of lactose hydrolysis in acid whey by 3-galactosidase from Aspergilhts niger. J. Dairy Sci. 56:1396.
(13) Wierzbieki, L. E., and F. V. Kosikowski. 1973. Lactase potential of various micro- organisms grown in whey. J. Dairy Sci. 56:26.
JOURNAL OF DAIRY SCIENCE VOL. 56, NO. 11
L. E. WIERZBICKI and F. V. KOSIKOWSKI Department of Food Science
Cornell University, Ithaca, New York 14850
Abstract
The formation of oligosaccharides or polysaccharides by transgalactosidation reactions during hydrolysis of lactose in acid whey by fi-galactosidase from Asper- gillus niger was studied. Five benzidine- positive carbohydrates, other than lactose, galactose, or glucose, were formed after fl-galactosidase action on 4% lactose at pH 4.5. These compounds, tentatively identified as oligosaccharides, comprised 1 to 2% of the total lactose in acid whey. Oligosaccharides were influenced by sub- strate concentration and reaction time. At the initial stage, oligosaccharides with Rf values less than lactose attained maxi- mum production in 18 to 25g lactose, whereas those with Rf values higher than lactose attained maximum production in 7 to 10% lactose in acid whey concen- trates.
Introduction
fi-galactosidase splits glycosidic linkages of lactose to produce glucose and galactose and may transfer some monosaccharide units to ac- tive acceptors, such as monosaccharides, poly- saccharides, or alcohol. This side reaction is called transgalactosidation and can occur in enzymatically or chemically catalyzed proc- esses. New glycosidic bonds are formed at dif- ferent positions of individual carbohydrates leading to oligosaeeharides of varying molecu- lar weights.
The mechanism of transgalactosidation dur- ing lactose hydrolysis is not well understood. Early studies showed that the number and type of oligosaccharides formed are affected by enzyme source, substrate concentration, pH, temperature, and inorganic ions (9). The significance of oligosaccharides in foods at high concentration may be important nutritionally because of man's inability to digest them.
Oligosaccharides formed from enzymatically hydrolyzed lactose in milk or whey have been
Received November 30, 1979..
reported by several investigators (1, 5, 7, 8, 9). The number of oligosaccharides in milk varies from 3 to 11 (5, 8).
While fi-galactosidase from A. niger (12) shows promise for the development of food in- gredients from acid whey and fermented milks, no information exists on the extent of oligo- saccharide formation by/~-galactosidase under acid conditions. The present study shows the extent of oligosaccharide formation during lac- tose hydrolysis by fl-galactosidase from A. niger.
Methods and Materials
Enzyme and substrate. A fl-galactosidase (lactase) preparation from A. niger with op- timum activity at pH 3.5 to 4.5 and 37 to 55 C was obtained from the Wallerstein Company (Morton Grove, Illinois).
The substrate was acid whey (pH 4.5) pre- pared from rennet cottage cheese as described by Kosikowski (2). This whey was stored a t --20 C and as required concentrated to 50% total solids under vacuum at 45 C in a rotary flash evaporator (Buchler Model FE-2 C, Fisher Scientific Co.).
Enzyme assay was composed of 10 ml of acid whey (6 to 50% total solids) containing 4 to 35% lactose. The whey, containing spe- cific amounts of lactase preparations, was incu- bated at pH 4.5 and 55 C for 5 h. One milli- liter was analyzed for carbohydrates by a mod- ified Nelson-Somogyi method (4, 11) and by thin-layer chromatography (6).
Oligosaccharide and monosaccharide separa- tion. Carbohydrate separation was carried out on earbon/celite columns (30 m m x 420 mm) (3, 10). Desorption of the carbohydrate from the column was obtained by stepwise elution at a flow rate 2 ml/min with water and ethanol/water mixtures to give 0, 2.5, 5, 7, 15, 25, 50, 75, and 95% ethanol. Fractions of 10 to 15 ml were obtained by an ISCO fraction collector. The 1-ml fractions were confirmed for carbohydrate by color after reacting with 1 ml of 1.0% orcinol in H2SO4. Concentrated fractions were then analyzed.
Thin-layer chromatography. Five 20 x 201 1400
OLIGOSACCHAB.IDES 1401
cm plates were prepared by mixing 40 g of sil- ica gel G with 80 ml of water and applying to the plates. A .25-mm layer was most suitable for general analysis. Plates, after drying for a few hours at room temperature, were activated at 100 C for 30 rain and held in a desiccator containing silica gel according to Randorath (6). The developing solvent was n-butanol; acetic acid; diethylether; water (9:6:3:1).
To .5 g of benzidine dissolved in 10 ml of acetic acid were added 10 ml of 40% w/v tri- ehloroaeetic acid in water and 80 ml of ethanol used as a color developing reagent.
A volume of sugar solution (1 to 5 /~g sug- ar) was applied to the plate whichl was devel- oped in a closed jar with so vent. The plate was then dried in an oven at 100 C for 10 to 15 min, sprayed with color reagent, and heated at 100 C for 25 rain. With monosaccharides and oligosaccharides, benzidine gave yellow- brown spots under daylight and green under short wavelength UV.
Estimation of saccharides. The individual benzidine-positive spots corresponding to sug- ars were extracted directly from thin-layer chromatography (TLC) plates by a mixtttre of 40~ trichloroacetic acid in water, acetic acid, and ethanol ( 1:1:8). Optical density extracted material was measured at 400 nm in a Beck- man Model DU spectropho~ometer and sugar calculated from a standard curve prepared for various concentrations of lactose solution. Di- rect optical density readings of the spots were obtained in a Photovolt Model 520A automati- cally recording photodensitometer (Photovolt Corp., New York). Reducing (glucose) equiv- alents of the separated fractions were deter- mined by a modified Nelson-Somogyi proce- dure (4, 11).
Results
Lactose in various acid whey e~ncen- trates was hydrolyzed by fl-galactosidase (.4 mg/ml) from A. niger at 55 C for 5 h. Samples were analyzed at zero time and after 2 and 5 h incubation. The rate of lactose hydrolysis and formation of new benzidine positive com- pounds in treated samples is in Fig. 1. Zero time samples showed traces of monosaccba- rides. Eight different carbohydrates were de- tected in an acid whey sample (6% total sol- ids) containing about 4% lactose treated with fl-galactosidase for 5 h at pH 4.5. These in- cluded glucose, galactose, and lactose. The other five, presumably oligosaccharides, were separated by their Itf values and reducing sugar reactions (Tables I and 2).
TABLE 1. The Bf values of carbohydrate com- pounds" separated on thin-layer chromatograms b.
Rf Compound value
Oligosaecharide- 1 .48 Glucose .46 Galaetose .41 Oligosaccharide - 2 .32 Lactose .20 Oligosaccharide - 3 .09 Oligosaccharide - 4 .05 Oligosaccharide - 5 .03
Fractionated on carbon/celite column. b Using thin-layer chromatography with silica
gel G and n-butanol; acetic acid; diethylether; water (9:6:3:1).
Sugar fractions separated on the column af- ter TLC analysis showed two oligosaccharides in the fraction eluted with 5% ethanol (Areas B and E, Fig. 2). Fractions eluted with water, 2.5, 7.0, and 15% ethanol contained no oligo- saceharides (Fig. 2). A third and fourth oligo- saccharide was separated on the second col- umn with 15% ethanol (No. 7, Areas A and C, Fig. 3). A fifth oligosaccharide was ob- served (No. 2, Area B, Fig. 3).
Concentrations of oligosaccharides produced in acid whey by fi-galactosidase are shown (Fig. 4). Total oligosaccharides increased with increasing substrate concentration between 4 to 21% lactose. Maximum oligosaccharides in acid whey represent 1 to 2% of the original lactose. The highest rate of production oc-
Fro. 1. Thin-laver ehromatogram showing the progressive hydrolysis and formation of oligosac- charities in acid whey containing different total whey solids by a O-galactosidase from A. niger.
Legend: A, B, C, D, E correspond respectively to oligosaccharides, lactose, oligosaccharides, ga- lactose, glucose; A', B' represent 1 mg/ml of lac- tose and glucose/galactose mixture; 1-4 correspond to 5, 10, 15, and 25g total solids, respectively.
JOURNAL OF DAIRY SCIENCE VoL. 56, NO. i i
1402 W l E R Z B I C K I A N D K O S I K O W S K I
TABLE 2. Recovery of reducing compounds ~ from hydrolyzed acid whey.
Fraction eluted Fractions on TLC Orcinol from earbon/eelite Benzidine test
column color UV reaction h
Reducing glucose equivalents
Ethanol 3 1 Red 1,379 2.5~ + + +
Ethanol 5 1 Brown-yellow 4,000 5.0~ + +
Ethanol 5 2 Red-yellow 275 7.0% + +
Ethanol 5 3 Brown-red 212 15.0% + + +
Ethanol 3 2 Brown-green 30 25% + +
Ethanol 2 2 Brown-greenish 45 50~ + +
Ethanol 1 1 Yellow 35 75% 4-
Ethanol 1 1 Yellow 30 95~g 4-
Eluted from carbon/celite column. b Intensity increases with number of 4-'s. TLC = thin-layer chromatography.
curred in the first 15 min of hydrolysis at all substrate levels, and the rate increased with increases in substrate concentration (Table 3). One group of oligosaccharides (Area A, Fig. 4) was produced in largest concentration at 18 to 25% while a second group (Area C, Fig.
4) achieved this state at 7 to 10% lactose in acid whey concentrates.
Discussion
Acid whey has not been used as a ]3-galac- tosidase substrate because of a lack of suitable
Ftc. 2. Thin-layer chromatogram showing sepa- ration of sugars on carbon/celite column obtained by ethanol elution and the products of lactose hy- drolysis using/3-galactosidase from A. niger.
Legend: A, B, C, D, E correspond respectively to lactose, oligosaccharide, galaetose, glucose, oligo- saccharide; 1-4 control spots of galactose, glucose, lactose, and mixture of glucose and galactose; 5 and 6 fractions eluted with water; 7-10 corre- spond to 2.5~ ethanol fractionation; 11-14 corre- spond to 5~; ethanol fractionation; 15 and 16 eluted with 7% ethanol; and 17 corresponds to 15-50% ethanol pool fraction. JOUR~AL OF DArRY SCIENCE VOL. 56, NO. 11
Fie. 3. Thin-layer chromatogram showing sepa- ration of sugars on earbon/eelite column obtained by ethanol elution and the products of lactose hydrolysis by fl-galactosidase from A. niger.
Legend: A to G spots correspond respectively to oligosaeeharide, oligosaccharide, oligosaccha- ride, lactose, oligosaccharide, galactose, glucose; a-d controls of glucose, galactose, mixture of glu- cose and galactose, and lactose; 1 corresponds to fraction eluted with water; 2 to fraction eluted with 5% ethanol; 3 fraction elnted with water; 4-10 correspond to 2.5, 5, 7, 15, 25-50, 75, and 95~ ethanol eluted fractions.
OLIGOSAGCHARIDES 1 4 0 3
TABLE 3. Relative optical density of spots corre- sponding to oligosaccharides occurring in acid whey following lactose hydrolysis by fl-galactosi- dase from A. niger.
Whey total solids
peak area (ram 2) ~ 5 91.5 80.0 108.0
10 165.0 180.0 200.0 20 187.5 198.0 217.0 30 201.0 210.0 270.0
Spots.
lactase preparation functioning maximally at pH 4.5 or lower. Applying a new food grade, experimental, fl-galactosidase preparation to acid whey provided a realistic means of hydro- lyziug lactose under acid conditions (11, 12, 13). However, it simultaneously caused prob- lems concerning oligosaccharide production.
Oligosaceharides may be important physio- logically in metabolic processes as noted by Roberts and McFarren (7) in testing fecal contents of rats fed a high lactose diet. There- fore, when microbial fi-galactosidases hydro- lyze the lactose of milk or milk products, the extent of oligosaceharide formation is impor- tant. Much of the early work was confined to milk, sweet whey, or lactose at neutral pH, us- ing fl-galaetosidase from Saccharomyces fragil- /s. The maximum number of oligosaccharides reported for skim milk using an S. fragiIis en-
Fro. 4. Oligosaecharide formation in acid whey at different whey solids concentration during lac- tose hydrolysis by fl-galactosidase from A. niger.
Legend: A, B, C, D, E correspond respectively to oligosaeeharides, lactose, oligosaeeharides, galae- tose, glucose; 1 corresponds to 6% of acid whey solids without enzyme treatment; 2-5 correspond to 10, 15, 25, and 35% acid whey total solids with enzyme treatment.
zyme preparation was 10 and 11 (7, 8). These oligosaccharides were as large or larger than lactose and usually contained a galactose moiety.
The present study tentatively showed the presence of only five oligosaccharides result- ing from the activity of fi-galactosidase, and these appeared at very low levels estimated at 1 to 2% of the total lactose. However, at sub- st_rate concentrations higher than 4% lactose the total oligosaccharides appeared in larger amounts. It is conceivable that at the. higher level of substrate, the small amount of unhy- drolyzed lactose which appeared in our studies may include newly formed reducing carbohy- drates. An example of such compounds might be mellibiose possessing a molecule similar to lactose except for a difference in type of gly- cosidic linkage between galactose and glucose and an almost identical Rf value on silica gel TLC (6).
References
(1) Aronson, M. 1952. Transgalactosidation dur- ing lactose hydrolysis. Arch. Biochem. Bio- phys. 39:370.
(2) Kosikowski, F. V. 1970. Page 429 in Cheese and fermented milk foods. Edwards Broth- ers, Inc., Ann Arbor, Michigan.
(3) Miller, C. L. 1960. Micro-column chromato- graphic method for analysis of oligosac- eharides. Anal. Bioehem. 2:133,
(4) Nelson, N. 1944. Nelson-Somogyi modified eolorimetrie method for determination of reducing sugar. J. Biol. Chem. 153:875.
(5) Pazur, J. H., T. M. Tipton, J. Budovieh, and J. M. Marsh. 1958. Structural characteriza- tion of products o~ enzymatic dispropor- tionation of lactose. J. Amer. Chem. See. 80:119.
(6) Randorath, K. 1964. Thin layer chromatog- raphy. Academic Press, New York.
(7) Roberts, H. R., and E. F. McFarren. 1953. The chromatographic observation of oligo- saecharides formed during the lactase hy- drolysis of lactose. J. Dairy Sci. 36:620.
(8) Roberts, H. R., and J. D. Pettinati. 1957. Conversion of lactose to oligosaccharides. J. Agr. Food Chem. 5:130.
(9) Webb, B. H., and A. H. Johnson. 1965. Fundamentals of dairy chemistry. The AVI Publishing Co., Inc., Westport, Connecticut.
(10) Whistler, K L., and D. F. Durso. 1950. Chromatographic separation of sugars on charcoal, j. Amer. Chem. See. 72:677.
(11) Wierzbicki, L. E. 1970. Biological behavior of microbial lactases and the development of new foods from their application. Ph.D. Thesis, Cornell University, Ithaca, New York.
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(12) Wierzbicki, L. E., and F. V. Kosikowski. 1973. Kinetics of lactose hydrolysis in acid whey by 3-galactosidase from Aspergilhts niger. J. Dairy Sci. 56:1396.
(13) Wierzbieki, L. E., and F. V. Kosikowski. 1973. Lactase potential of various micro- organisms grown in whey. J. Dairy Sci. 56:26.
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