An Improved Method for Determination of the Activity of [3-Galactosidase in Yoghurt by High-Performance Liquid Chromatography

J. I. Gonz~i lez-Andrada / C. R o m e r o / E J. Mora les / S. J im6nez-P6rez*

Department of Dairy Products, lnstituto del Frio (C.S.I.C.), Ciudad Universitaria s/n, 28040 Madrid, Spain

Key Words Column liquid chromatography Yoghurt I~-Galactosidase activity Glucose Galactose

Summary A new method has been developed for the HPLC deter- mination of the activity of lactase from the microor- ganisms in yoghurt. The method is based upon the ability of t3-galactosidase to hydrolyze lactose bonds in glucose and galactose. To determine such activity in yoghurt, 1 mL of yoghurt was diluted 1:10 with 0.1 M phosphate buffer (pH 7) containing 2 % (w/v) lactose and 5 mM dithioerythritol as reducing agent; the mix- ture was the incubated and injected into the HPLC. Lac- tase activity remained high as long as the number of vi- able microorganisms did not fall below the minimum CFU �9 mL -1 (107). This method is more repeatable than conventional colorimetric determination, and may also be automated for routine analysis.

Introduction [~-D-galactosidase ([3-D-galactoside galactohydrolase, EC 3.2.1.23) [1] is an endoenzyme occurring in yoghurt microorganisms which is able to catalyze rupture of lac- tose glycoside bonds in glucose and galactose [2]. When lactic acid bacteria grow in milk, energy is derived from Conversion of lactose to lactate [3]. The catabolic path- Ways of lactose and galactose in lactic acid bacteria is thoroughly understood. Such degradation of lactose to lactate may be either homo- or heterofermentative,

most lactate being produced by homofermentative degradation [3]. Lactic acid bacteria possessing two dis- tinct systems for lactose uptake have been found:

- A phosphoenolpyruvate-dependent lactose phos- photransferase system (PTS tae) is utilized by strains of L, lactis, L. casei and other mesophilic lactobacilli. In these, PEP-PTS lac and phospho-~-galactosidase enzymes are induced during growth in lactose and galactose [4, 5].

- The second system, lactose permease, is used by ther- mophilic strains of lactobacillus, streptococcus, and leuconostoc. Lactose is taken up by lactose permease as a free sugar. Galactose is converted to galactose-6- phosphate in the Leloir pathway by the enzymes: galactokinase, galactose-l-phosphate-uridy!trans- ferase, diphosphogalactose-4-epimerase uridine, uridine diphosphoglucose synthetase, and phos- phoglucomutase [6]. The Leloir catabolic pathway is blocked in yoghurt microorganisms (Lactobacillus bulgaricus and Streptococcus thermophilus by low levels of galactokinase, and the result is stoichio- metric excretion of galactose to the medium via per- incase lactose, which acts as a lactose/galactose an- tiporter system [7]. Unstable galactose positive (Gal § mutants have, however, been described in which this effect does not occur [8].

Strains of S. thermophilus appear to be fairly homo- geneous as regards utilization of [3-galactosidase in the hydrolysis of lactose [9], because only phospho-13-D- galactosidase occurs in non-thermophilic microor- ganisms [10]. I]-Galactosidase from S. thermophilus is less active than that from L. bulgaricus but is less thermostable and is rendered inactive by heating at 70 ~ [11]. Maximum activity of the enzyme isolated from both microorganisms occurs within a temperature range of 55--60 ~ at pH 6.5-7 [11, 12] under opt imum experimental conditions, including the presence of monovalent and divalent cations and intense reducing activity (5 mM dithioerythritol) [2]. 13-Galactosidase is

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highly sensitive to inactivation byp-chloromercurioben- zoate, which envelops the SH groups in the active parts of the enzyme [13, 14].

Conventional methods for determining lactase activity are based upon the ability to break down the o-nitro- phenyl-13-D-galactopyranoside (ONPG) bond into o- nitrophenol (ONP), a colored compound which absorbs at 420 nm, and galactose [11]. As there was no estab- lished procedure for measuring I]-galactosidase activity in yoghurt by HPLC, our laboratory undertook the design of an alternative method to the (spectro- photometric) ONPG-breakdown method that would be as simple and easily automated as possible. The aim of the work described in this paper was to develop a new method with greater sensitivity for measurement of lac- tase activity while improving repeatability and repro- ducibility, because literature data are lacking on this topic.

Experimental

Reagents

Chromatography: Equipment and Conditions

The HPLC equipment (all from Kontron Instruments, Milan, Italy) consisted of a programmable pump (model 325) with ternary-gradient low-pressure system and helium purge, an automatic injector (model 465) and an oven (model 480). The system was controlled, via an RS o 232 interface, by DS450-MT2 (v. 3.00) application software, resident in a personal computer. Detection was performed with a refraction index detector (ERMA, model ERC-7522, Tokyo, Japan).

The chromatographic conditions were based, with minor modifications, on those used in the method of Richmond et al. [16] for HPLC analysis of sugars. A 300 mm x 7.6 mm analytical ion-exchange column con- taining Aminex HPX-87C resin in the Ca z+ form (Bio- Rad, CA, USA) with 30 mm x 4.6 mm precolumn (Bio- Rad 125--0131, with cartridges, Bio-Rad 125--0128) were used. Sample or standard (20 ~tL) were injected, and eluted with bidistilled water (Milli-Q, Millipore, USA) at a constant flow rate of 0.6 mL min -~ for a 20-rain run- ning time. The oven temperature was isothermal at 85 ~

All the chemicals used were of analytical grade. Dithioerythritol (ref. D8255) and galactose (ref. G6404) were purchased from Sigma (St. Louis, MO, USA). 13-D- galactosidase [EC.3.2.1.23] was purchased from Boeh- ringer-Mannheim (Mannheim, Germany; ref. 176303). Potassium dihydrogen phosphate was purchased from Probus (Badalona, Spain; ref. 145010). 12-Hydrate dibasic sodium phosphate (ref. 141678) and lactose (ref. 141375) were purchased from Panreac (Barcelona, Spain).

Sample Preparation

Yoghurt samples (1 mL) diluted t:10 with bidistilled water, were incubated overnight, in a bath at 55 ~ with potassium phosphate buffer solution (0.1 M, pH 7; 5 mL) containing lactose (2 %, w/v) and dithioerythritol (5 mM) as reducing agent [10]. Finally, samples were fil- tered through Whatman no. 42 filter paper and then through a 0.2 ~tm nylon matrix membrane (LIDA, Kenosha, WI, USA; ref. NY201300) before injection into the HPLC. Injected samples were checked against a blank yoghurt solution (1 mL yoghurt and 5 mL bidis- tilled water) and a blank buffer solution (1 mL bidis- tilled water and 5 mL of the buffer used). Both blanks were treated in the same way as the sample and injected into the HPLC. The average amount of galactose sub- tracted as blank was 0.039 + 0.007 g �9 100 mL -1 (n = 13). Commercial yoghurts were purchased at local markets.

ONPG Method

The yoghurt was diluted ten times with distilled water. The solution (1 mL) was analyzed as described by Citti et al. [15].

Results and Discussion

The basis of the method is that although thermophilic microorganisms will metabolize lactose, and failing that galactose, as a source of energy, they lack the enzyme galactokinase and are therefore incapable of utilizing galactose released by hydrolysis of lactose. Galactose is thus secreted as permease lactose and accumulated in the medium [17]. Galactose can thus be quantified by HPLC to measure the lactase activity of microor- ganisms in yoghurt. Such galactose determination could be an indirect means of measuring microbe count and hence the optimum moment for yoghurt consumption, that is the point at which its properties are most benefi- cial to human health and nutrition.

Several trials were performed to optimize sample preparation. Firstly, centrifugation of the yoghurt sample and freeze-drying of the supernatant were tested for sample preparation. The supernatant was then placed in contact with a 5 % (w/v) lactose solution and the sample was injected after varying incubation times up to 24 h, but residual hydrolysis was observed. There being no detectable 13-galactosidase activity, in sub- sequent assays the sample was concentrated by other means. In another test, the diluted yoghurt sample was separated and concentrated by vacuum rotary evapora- tion, then filtered and placed in contact with a 5 % (w/v) lactose solution as before, but again no residual 13-galac- tosidase activity was measured. To force the enzyme from inside the microorganism and thus secure a solu- tion with 13-galactosidase activity, yoghurt microor- ganisms were subjected to 30 min sonication (Labsonic U, B. Braun, 20 kHz, ref. 853811/5, USA). The sample was then centrifuged and the supernatant divided into

86 Chromatographia Vol.43,No. 1/2,July t996 Short Communication

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Figure 1 Effect of sonication during sample preparation on the release of lactase activity (mmol ONP. rain -1 �9 g-l) at 37 ~ and 55 ~ (dotted lines) from a commercial yoghurt (e) and a laboratory-made YOghurt (ll).

two equal parts, one of which was concentrated by Vacuum rotary evaporation and the other by freeze- drying. At the same time, a blank was prepared and not Sonicated. After filtering, the concentrates were placed in contact with a 2 % or 5 % (w/v) lactose solution then injected into the HPLC system. There were no appreci- able differences between the two procedures and hydrolysis was not significant. Further sonication trials Were subsequently run to compare the yoghurt microor- ganisms obtained in the laboratory with those occurring in a commercial yoghurt. Yoghurt (50 mL) diluted 1:10 With bidistilled water was sonicated for 30 min. Aliquots (2 mL) were collected at different times and incubated at 37 or 55 ~ The results are shown in Figure 1. The microbial strains from the laboratory-made yoghurts Were more resistant to sonication; as a result there was Very little [3-galactosidase activity. These findings established that intact cells of yoghurt microorganisms were necessary for measurement of J]-galactosidase activity. This is in accordance with legis- lation on the determination of yoghurt quality [18], be- cause no positive results were forthcoming either from Sonication trials, which released no enzyme owing to high resistance of strains, or from sample centrifugation or from filtration prior to incubation of the enzyme. The optimum preparation of the selected sample was finally Performed using 1 mL of yoghurt diluted 1:10 with bidistilled water. Incubation times were 0.25 h, 1 h, 6 h, 12 h, 17 h and 48 h at 55 ~ Samples were incubated

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with a solution of 2 % (w/v) lactose in a phosphate buff- er (pH 7) with 5 mM dithioerythritol, following the op- timum conditions indicated in the literature on purified 13-galactosidase [10,13]. Initially the reaction was halted with sodium carbonate, but the use of this compound was abandoned owing to incompatibility with the analytical column. A number of trials was therefore run, filtering and cooling at 4 ~ after incubation then check- ing to see that there was no variation in the lactose and galactose contents of samples. This procedure gave satis- factory results. Figure 2 shows the concentrations (g �9 100 mL -1) of lactose and galactose in samples of the same yoghurt incubated for different times for the pur- pose of optimization.

Finally, the incubation time selected was 17 h, as reac- tion lasted to the end and there was no alteration of the sugars analyzed. To check whether the reaction was caused by spontaneous hydrolysis of lactose rather than the enzyme itself, a yoghurt blank (1 mL yoghurt diluted 1:10 with 5 mL bidistiUed water) and a buffer blank (1 mL bidistilled water with 5 mL of the phosphate buff- er with 2 % lactose) were incubated at the same time as the sample. These were taken from the 55 ~ bath at the same time as sample and subjected to the same treat- ment. Figure 3 shows a characteristic chromatogram of the separation of the sugars for the lactase activity method�9

To quantify the enzymatic activity, a calibration curve was drawn using several dilutions of [3-D-galactosidase to simulate microbial activity. To quantify galactose release, a standard curve was constructed with a solution of 1 % (w/v) galactose in the buffer solution, varying amounts of which (40, 30, 20, 10, 5 and 2 ~tL) were in- jected into the chromatograph. The resulting regression equation was:

Galactose (g. 100 mL -1) = 0.1529 x Area (mV. min -1) + 0.0209 (r 2 = 0.9998, n = 10).

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Coefficients of variation (C.V. %, n = 10) for day-to-day repeatability were 3.9 % and 2.4 %, respectively, for measurement of fl-galactosidase activity by the tradi- tional ONPG method and the H P L C method here described. In the same way, lower C.V. values (n = 10) for run-to-run repeatability were calculated for the H P L C method ( 3 . 1 % ) compared with the spectro- photometric method (3.8 %). In conclusion, lactase activity can be measured in fer- mented milk with a simple and rapid isocratic H P L C method. This approach would enable reliable and automated control of the microbiological quality and culture activity of yoghurt. At present, the procedure is being tested on several commercial yoghurts.

Acknowledgments The authors gratefully acknowledge the valuable tech- nical assistance of Dolores Gomez. This work was finan- cially supported by the F E O G A , project No. 1116/ Esp4-I.

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(1979). [10] W. M. De Vos, G. Simons, Biochimie 70, 461 (1988). [11] N. Shah, P, Jelen, J. Food Sci. 55, 506 (1990). [12] P. Jelen, Bull. IDF 289, 54 (1993). [13] N.A. Greenberg, R. R. Mahoney, J. Food Sci. 47, 1824 (1982). [14] M. V. R. Rao, S. M. Dutta, J. Food Sci. 46, 1419 (1981). [15] J. E. Citti, W. E. Sandine, P. R. Elliker, J. Bacteriol. 89, 937

(1965). [16] M.L. Richmond, B. R. Harte, J. 1. Gray, C M. Stine, J. Dairy

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Received: Jan 19, 1996 Revised manuscripts received: Mar 25, and Apr 29,1996 Accepted: May 22, 1996

88 Chromatographia Vol. 43, No. 1/2,July 1996 Short Communication