Lactose hydrolysis by free and fibre-entrapped (3-galactosidase from Streptococcus thermophilus Zhennai Yang, Eero Pahkala and Tuomo Tupasela Yang, Z., Pahkala, E. & Tupasela, T. 1993.Lactose hydrolysis by freeand fibre-en- trapped -galactosidase from Streptococcus thermophilus. Agric. Sci. Finl. 2: 395-401, (Jilin Academy of Agric. Sci., Branch of Animal Husbandry, Lab of Animal Products Processing, Gong-Zhu-Ling, Jilin Province, 136100P.R. China and Agric. Res. Centre of Finland, Food Res. Inst., FIN-31600 Jokioinen, Finland.) To study lactose hydrolysis by (3-galactosidase, this enzyme was produced from Strep- tococcus thermophilus strain 11F and partially purified by acetone and ammonium sulphate fractionation, and ion exchange chromatography on a Q Sepharose FF column. Lactose hydrolysis by the enzyme was affected by lactose concentrations, sugars and milk proteins. The maximum extent of lactose hydrolysis in buffer was obtained with a 15% lactose concentration. Addition of 2% of lactose, glucose, galactose or sucrose in milk inhibited the enzymatic hydrolysis. The enzyme was activated by bovine serum albumin and a combination of as-casein and (3-casein. Of the casein fractions, the principal fraction, as-casein, was less effective than (3-casein and K-casein. The fibre- entrapped enzyme had a temperature optimum of 57°C, and a pH optimum from 7.5 to at least 9.0 with 0-nitrophenyl-(3-D-galactopyranoside as substrate. By recycling with whey and skim milk through a jacketed glass column (1.6 cm x 30 cm) loaded with fibre-entrapped enzyme at 55°C, a lactose hydrolysis of 49.5% and 47.9% was achieved in 11 h and 7 h respectively. Key words: lactose hydrolysis. Streptococcus thermophilus, (3-galactosidase, fibre-en- trapped enzyme Introduction Hydrolysis of lactose in milk and milk products by (3-galactosidase has been regarded as a potential solution to lactose maldigestion, which occurs in the majority of adults all over the world. Manufac- ture of dairy products with lactose-hydrolyzed milk or whey has many advantages, including enhanced sweetness, improved carbohydrate solubility and digestibility, more readily fermentable sugars, pre- vention of lactose crystallization etc. Enzymatic processes of lactose hydrolysis by both free and immobilised [3-galactosidase are already commer- cially available (Mahoney 1985, Harju 1987). The utilization of (3-galactosidase available from yeast and mould, however, is relatively limited, either due to the low temperature optimum or the low pH optimum of the enzyme. Attention has recently been shifted to finding a heat-stable, neut- ral-pH (3-galactosidase that would be suitable for the hydrolysis of lactose in milk and facilitate the enzyme reactors with high operating temperatures to prevent microbial contamination. A thermo- stable (3-galactosidase from Streptococcus thermo- philus (Str. thermophilus) is considered such an enzyme and appears promising in this respect. The 395 Agric. Sei. Finl. 2 (1993) https://www.c-info.fi/en/info/?token=cdZt7aIfiuf5DSV6.JWCrTCgFncmqNl6RQGUrYg.gvk14qhJ4YSLmZD2iRF-V0P69ZpwXc3WAWS9vq9X0dHlD4Vs_TY0vf6l8Go16uP7udaIRXLDL5Q0bV3JJ3VngOZIa24ocffxQtWRlbcgm924irGewLPF8ruW3Ydj_yk2xnTDW_qUYMCsy-_pY0aq-p3NvfKbwK764RB-bDu4LOqYHqzAb7UBna9sdibANqakr2SZkilPJ5c3BvyKIhUoRVesgEMJTAv_-i1OwwG1tHev-SO7tHqHu1R89g-JAyUi2VAWU6smmt6I2CLJ4Bu7lSfgCQ8qzyHpngPApRUxbHvJNDzF purification and characterization of the enzyme has been the subject of several recent studies (Ramana and Dutta 1981, Greenberg and Mahoney 1982, Greenberg et al. 1985, Smart et al. 1985, Chang and Mahoney 1989a, b). However, the research on the immobilization of the enzyme is still lacking and certain factors influencing the en- zymatic hydrolysis of lactose need to be determined in more detail. The present study was carried out to assay the hydrolysis of lactose in milk and buffer by the (3-galactosidase from Sir. thennophilus , and to evaluate the enzyme immobilized with cellulose triacetate. Material and methods Sir. thennophilus strain HE was donated by the Research and Development Centre of Valio Ltd., Helsinki. Cellulose triacetate was purchased from Fluka Chemie AG, Switzerland, O- nitrophenyl-f) D-galactopyranoside (ONPG) from Sigma Chem- ical Company, USA, and Q Sepharose FF and the glass column were from Pharmacia Fine Chem- icals, Sweden. The chemicals and reagents used were of analytical grade. Double-distilled water was used to make the buffers. Raw milk and whey from edam cheese manufacture were obtainedfrom the dairy of the Food Research Institute, Jokioinen, Finland. adding ammonium sulphate to 65% saturation, and the pellet was dissolved in about 10 ml buffer A and dialyzed at 4°C against buffer A with several changes of the buffer. The enzyme was stored at -25°C. The above procedures of enzyme prepara- tion were carried out and repeated five times. The final enzyme products were used for the following assays of lactose hydrolysis and enzyme immobil- ization. Assay of (J-galactosidase activity and protein concentration Enzymatic activity was spectrophotometrically measured at 420 nm as described by Greenberget al. (1985). One unit of (3-galactosidase activity is defined as the amount of enzyme which will liber- ate 1 pmol of o-nitrophenol (ONP) per minute under the assay conditions. Protein concentration was determined by the method described by Lowry et al. (1951). Bovine serum albumin was used as standard protein. Immobilization of the enzyme The partially purified enzyme preparations were entrapped in cellulose triacetate fibres (thickness 0.2-0.3 mm) according to the procedures reported by Morisi et al. (1973). Enzyme preparation The procedures ofbacterial cultivation and enzyme purification by acetone and ammonium sulphate precipitation basically followed an earlier method described by Chang and Mahoney (1989a). The enzyme extract obtained was further par- tially purified by ion exchange chromatography with Q Sepharose FF in a glass column (1.6 cm x 30 cm). The elution was carried out at 5 ml/min with a linear increasing gradient of 0.5 M NaCl in buffer A (Chang and Mahoney 1989a) from 30% to 80%. The fractions (10 ml) containing enzyme ac- tivity were pooled, the protein was precipitated by Determination ofthe optimum temperature and pH of the fibre-entrapped enzyme The activity of the fibre-entrapped enzyme during the assays was examined as follows: 0.15 g fibres were added to 2 ml 2.2 mM ONPG in buffer at 55°C in a water bath. The absorbance of the solu- tion at 420 nm was measured after 2 minutes. The pH optimum was determined with 2 ml 2.2 mM ONPG in 0.01 M potassium phosphate buffer ad- justed to the pH of the assay with phosphoric acid. The temperature optimum was determined with 2 ml 2.2 mM ONPG in buffer A at the assay tem- peratures. 396 Agric. Sei. Finl. 2 (1993) Assay of lactose hydrolysis Lactose hydrolysis was assayed with a rapid method based on the measurement of freezing point depressing of the solution as described by Ramet et al. (1979). 2 ml ofsample (or dilutedsample) was taken, and the freezing point determined with a cryoscope. Results and discussion Enzyme purification Elution of the enzyme extract by ion exchange chromatography on a Q Sepharose FF column is shown in Figure I. The dialysate from the ammo- nium sulphate step had to be centrifuged because of its thickness, and only the supernatant (about 58% ofthe total activity) was applied to the column. The precipitate, with a specific activity of 16 units/mg protein, could be directly freeze-dried as a crude enzyme. Elution of the enzyme started shortly after the specified gradient was introduced. One main peak ofprotein coincident with the enzyme activity was eluted. The pooled fractions from the main peak, however, accounted for 86% of the total ac- tivity recovered, with a four-fold increase in spe- cific activity compared with the applied enzyme sample. Thus, further purification of the enzyme was not carried out. The final partially purified enzyme had a specific activity of 117 units/mg protein. Lactose hydrolysis in milk and buffer containing carbohydrates or milk proteins Effect oflactose concentrations As shown in Figure 2, the process of lactose hydro- lysis by the enzyme was examined at different concentrations of lactose in 0.025 M potassium phosphate buffer, pH 6.8, containing 0.8 mM MgC12.6H20, 0.125 mM dithioerythritol, 0.005 % NaN3 and 10% glycerol (buffer B). Maximum hydrolysis was found in a 15% lactose solution, in which 100% of lactose was hydrolyzed in 4 hours. With lactose concentrations of5%, 10% and 25%, the degrees of hydrolysis after 4 hours were about 70%, 85% and 50%, respectively. Simi- lar results were observed by using Saccharomyces fragilis lactase to hydrolyze reconstituted whey and milk, wherethe highest degree of lactose hydrolysis was achieved at 10-15% lactose concentrations (WENDORFF et al. 1971). It seems that optimal in- Fig. 1.Purification of the enzyme by ion exchange chromatography on a Q Sepharose FF column. ( ) absor- bance, (—) NaCl gradient, (••••) en- zyme activity 397 Agric. Sei. Finl. 2 (1993) teraction between the enzyme and lactose occurres when lactose concentration was about 15%. Effect ofsugars Figure 3 shows that lactose hydrolysis was remark- ably reduced when 2% of lactose, glucose, galac - tose or sucrose was added to the milk. Lactose gave the slightest inhibition to the hydrolysis, whereas there was no evident difference between inhibition caused by the other sugars. The results in Figure 2 indicate that lactose hydrolysis increased along with increasing lactose concentration in buffer (except 25%). Adding lac - tose to milk, however, decreased the lactose hydro - lysis. Lactose, galactose and glucose have been found to stabilize P-galactosidase from Sir. thermo- philus (Chang and Mahoney 1989b), but the lac - tose hydrolysis in milk by the enzyme may be reversely affected by these sugars. Effect ofmilk proteins The effect of several casein fractions and bovine serum albumin on lactose hydrolysis in buffer B containing 15% lactose is shown in Figure 4. The concentrations of as-casein, k- casein and p-casein in buffer B were similar to their corresponding concentrations in milk. After 3 hours, the hydroly- sis degrees in buffer B containing as-casein and p- casein, or bovine serum albumin, reached 95%, almost the same as achieved in 15% lactose solu- tion within the same timebut using a higher enzyme concentration (shown in Figure 2). This suggests that the enzyme was activated by bovine serum albumin and by the combination of as-casein and P-casein. Previous work (Mahoney and Adam- chuk 1980) has also shown that P-galactosidase from S. fragilis is strongly activated by bovine serum albumin. Casein has been found to activate P-galactosidase from S. fragilis and stabilize P-ga- lactosidase from Str. thermophilus (CHANG and Mahoney 1989b). However, the results inFigure 4 indicate that the individual effects of casein frac- tions on the enzyme were different, as-casein was less effective than P-casein and K-casein, although it is the principal fraction of casein. K-casein, com- prising only about 13% of the total casein, pro- duced the greatest effect. Immobilization of the enzyme pH optimum The results (Figure 5) indicate a wide pH optimum range from 7.5 to at least 9.0 with a sharp decrease ofenzyme activity below pH 7.5. The optimum pH Fig. 2. Hydrolysis of lactose at 55°C in buffer B , 10 ONPG units of enzyme activity were added per ml of buffer B. Fig. 3. Hydrolysis of lactose at 55°C in milk with added sugars . 8 ONPG units of enzyme activity were added per ml of milk. 398 Agric. Sei. Fin!. 2(1993) for this free enzyme has been reported to be be- tween 7.0 and 7.5 (Smart et al. I985). The broader pH optimum of the fibre-entrapped enzyme was probably due to the rate limiting of the substrate diffusion. In addition, since the enzyme preparation was not entirely pure, some protein impurities en- trapped in the fibres could also have contributed to the broader pH optimum. Morisi et al. (1973) re- ported a similar wider range of pH optimum for (J-galactosidase from Escherichia coli. Immobil- ized crude pepsin has also been found to have a wider pH optimum range than another more pure immobilized pepsin preparation (Hustad et al. 1973). Temperature optimum The effect of different temperatures on the activity of the fibre-entrapped enzyme is shown in Figure 6. Of the temperatures studied, the optimum appeared to be at about 57°C. This was an upward shift of about 2°C compared with the corresponding free enzyme, for which the optimum temperature has been reported at 55°C (Smart et al. 1985). Al- though the enzyme was actually entrapped in sol- uble form within the microcavities of the fibres, it may be that the outer layer of the fibres provided heat protection for the entrapped enzyme, thus res- ulting in a higher temperature optimum. Lactose hydrolysis in milk products by the fibre-entrapped enzyme Treating 15 ml skim milk, whole milk or whey with 1.0gof fibre-entrapped enzyme for 3 hours at 50°C or 53°C resulted in lactose hydrolysis of about 70%, 65% and 27%, respectively (Figure 7). Skim milk and whole milk appeared to be more suitable than whey for lactose hydrolysis by the immobil- ized enzyme. The low extent of lactose hydrolysis in whey may be mainly due to the low pH of the Fig. 4. Hydrolysis of lactose at 55°C in buffer B with added milk proteins . 7 ONPG units of enzyme activity were added per ml of buffer B. Fig. 5. pH profile of fibre-entrapped (i-galactosidase with ONPG as substrate. Fig. 6. Temperature profile of fibre-entrapped (i-galactosi- dase with ONPG as substrate. 399 Agric. Sei. Fint. 2 (1993) whey (about pH 6.3). As indicated in Figure 5, the activity of the immobilized enzyme was strongly affected by pH values below 7.5. Column operation Trials were made to assay the performance of the enzyme entrapped in the fibres which were parallel- packed in the jacketed glass column (1.6 cm x 30 cm). The first assay indicated that lactose hydro- lysis ofonly 8.4% in whole milk was obtained with the column containing 9 g fibres at 45°C and at a flow rate of 38 ml/h. The substrate was then changed by feeding whey at a lower flow rate, but no improvement was observed. The column was packed again using more fibres (13 g). After run- ning with whey, skim milk and lactose solution in buffer B at above 50°C and at even lower flow rates, higher degrees of lactose hydrolysis were achieved, but they were still less than 30%. When whole milk or skim milk was passed through the column at flow rates less than 20 ml/h, column plugging by fat and protein became easier. The substrates were, therefore, recycled through the col- umn at 55°C and at much higher flow rates (about 70 ml/h), and lactose hydrolysis of 49.5%, 47.9% was reached with whey (90 ml) and skim milk (90 ml) after 11 and 7 hours respectively. Acknowledgements. This work was initiated by the Agricul- tural Research Centre of Finland and JiLin Academy of Agricultural Sciences of P.R. China. The authors would like to thank the Finnish Ministry of Trade and Industry for financial support to this study. References Chang, B. & Mahoney, R.R. 1989a, Purification and thermostability of (i-galactosida.se (lactase) from an auto- lytic strain ofStreptococcus salivarius subsp. thermophi- lus. J. Dairy Res. 56: 117-127. & Mahoney, R.R. 1989b. Factors affecting the thermo- stability of (i-galactosidase (Streptococcus salivarius subsp. thermophilus) in milk: a quantitative study. J. Dairy Res. 56: 785-792. Greenberg, N.A. & Mahoney, R.R, 1982. Production and characterization of [i-galactosidase from Streptococcus thermophilus. J. Food Sci. 47: 1824-1828, 1835. —, Wilder, T. & Mahoney, R.R. 1985. Studies on the thermostability of lactase (Streptococcus thermophilus) in milk and sweet whey. J. Dairy Res. 52: 439-449. Harju, M. 1987.Lactose Hydrolysis, IDF Bull. 212: 50-55. Hustad, G.0., Richardson, T. & Olson, N.F. 1973. Immo- bilization of (i-galactosidase on an insoluble carrier with a polyisocyanate polymer. 11. Kinetics and stability. J. Dairy Sci. 56: 1118-1122, Lowry, 0.H., Rosebrough, N.J., Farr, A.L. & Randall, RJ. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 251-275. Mahoney, R.R. 1985. Modification of lactose and lactose- containing dairy products with (5-galactosidase. In: Fox P. (ed.). Developments in Dairy Chemistry-3. Elsevier Applied Science Publishers, London and New York. p. 69-109. & Adamchuk, C. 1980. Effect of milk constituents on the hydrolysis of lactose by lactase from Kluyveromyces fragilis. J. Food Sci. 45: 962-964,968. Morisi, F., Pastore, M. & Viglia, A. 1973. Reduction of lactose content of milk by entrapped (i-galactosidase. I. Characteristics of (i-galactosidase from yeast and Es- cherichia coli. J. Dairy Sci. 56: 1123-1127. Ramana Rao, M.V. & Dutta, S.M. 1981. Purification and properties of (i-galactosidase from Streptococcus ther- mophilus. J. Food Sci. 46: 1419-1423. Ramet, J.P., Novak , G,, Evers, P.A. & Nupels, H.N. 1979. Application de la cryométrie ä la mesure de I’hydrolyse enzymatique du lactose. Le Lait 59: 46-55. Fig. 7. Lactose hydrolysis in milk products by fibre-entrapped (i-galactosidase (1.0 g) (skim milk 50°C, whole milk 50°C, whey 53°C). 400 Agric. Sei. Fint. 2(1993) Smart, J.8., Crow, V.L. & Thomas, T.D. 1985. Lactose hydrolysis in milk and whey using (i-galactosidase from Streptococcus thermophilus. New Zealand J. Dairy Sci. and Technol. 20: 43-56. Wendorfi-, W.L., Amundson, C.H. & Olson, N.F. 1971. Use of yeast P-galactosidase in milk and milk products. J. Milk Food Technol. 34: 294-299. Manuscript received June 1993 Zhennai Yang Jilin Academy of Agricultural Science Branch of Animal Husbandry Laboratory of Animal Products Processing Gong-Zhu-Ling, Jilin Province 136100P.R.China Eero Pahkala Tuomo Tupase la Agricultural Research Centre of Finland Food Research Institute FIN-31600 Jokioinen, Finland SELOSTUS Laktoosin hydrolyysi vapaalla jakuituun sidotulla Streptococcus thermophilus-ft-galaktosidaasi\\a Zhennai Yang, Eero Pahkala jaTuomo Tupasela Jilin Academy of Agricultural Sciences ja Maatalouden tutkimuskeskus Streptococcus thermophilus I IF kannalla tuotettiin (i-galak- tosidaasia, joka osittain puhdistettiin asetoni- ja ammonium- sulfaattifraktioinnilla sekä ioninvaihtokromatografialla Q Sepharose FF-pylväässä. Entsyymin aiheuttamaan laktoosin pilkkoutumiseen vaikuttivat laktoosin pitoisuus, eri sokerit ja maidon valkuaisaineet. Korkein hydrolyysiaste puskuriliuok- sessa saavutettiin 15 % laktoosipitoisuudessa. Lisättäessä maitoon 2 % joko laktoosia, glukoosia, galaktoosia tai sakka- roosia laktoosin hydrolyysi heikkeni. Seerumin albumiini sekä as- ja p-kaseiinien yhdistelmä aktivoi entsyymiä. Mai- don kaseiinien pääkomponentin, as-kaseiinin vaikutus hydro- lyysiin oli vähäisempi kuin P- tai K-kaseiinin. Kuituun sidotun entsyymin lämpötilaoptimi oli 57°C japH optimi välillä 7,5- 9,0 käytettäessä O-nitrofenyyli-P-D-galactopyranosiidia sub- traattina. Kierrätettäessä kuorittua maitoa tai heraa kuituun sidotulla entsyymillä täytetyssä lasikolonnissa saavutettiin 55°C lämpötilassa 7 tunnissa 47,9 %ja 11 tunnissa 49,5 % laktoosin hydrolyysiaste. 401 Agric. Sei. Fint. 2 (1993)