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Production ofh-galactosidase by Bifidobacteria as influenced by

various culture conditions

C.A. Hsu, R.C. Yu, C.C. Chou*

Graduate Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan, ROC

Received 25 February 2004; received in revised form 16 August 2004; accepted 12 February 2005

Abstract

h-Galactosidase production by Bifidobacterium longum CCRC 15708, Bifidobacterium longum B6 and Bifidobacterium

infantis CCRC 14633 was first examined with B. longum CCRC 15708 showing the highest production ofh-galactosidase and

the highest specific activity. Further study with B. longum CCRC 15708 revealed that the highest level ofh-galactosidase was

produced with lactose and yeast extract as carbon and nitrogen sources, respectively. Optimal enzyme production occurred at an

initial pH of 6.5 and at 37 8C. Under these optimum culture conditions, a maximumh-galactosidase activity of 18.6 U/ml could

be obtained after 16 h of fermentation in a medium contain 4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1% KH2PO4,

0.05% MgSO4d

7H2O and 0.03%l

-cysteine. The highest transgalactosylation activity was also detected in this culture after 1416 h of fermentation.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Bifidobacteria; h-Galactosidase; Nitrogen and carbon sources; Culture conditions

1. Introduction

The enzyme, h-galactosidase (EC 3.2.2.23) is a

commercially important enzyme. It catalyzes the hy-

drolysis of h-d-galactopyranosides such as lactose.By hydrolyzing lactose with h-galactosidase, the pro-

blems associated with whey disposal, lactose crystal-

lization in frozen concentrated deserts and milk

consumption by lactose-intolerant individuals can be

alleviated (Kim and Rajagopal, 2000).

In addition to catalyzing the conversion of lactose

to glucose and galactose, h-galactosidase also cata-lyzes transgalactosylation reaction; lactose serves as

galactosyl donor and an acceptor to form di-, tri-, or

higher galactooligosaccharides (GOS) (Wallenfels and

Weil, 1972; Prenosil et al., 1987). GOS are now

considered as a probiotic food ingredient and have

been demonstrated to promote the growth and the

establishment of bifidobacteria in the intestine

(Tanaka et al., 1983; Mitsuoka, 1990) and thus exert

0168-1605/$ - see front matterD 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.ijfoodmicro.2005.02.010

* Corresponding author. Postal address: Graduate Institute of

Food Science and Technology, National Taiwan University 59,

lane 144, Keelung Rd., Sec. 4, Taipei, Taiwan, ROC. Tel.: +886 2

2363 0231x2717; fax: +886 2 2362 0849.

E-mail address: [email protected] (C.C. Chou).

International Journal of Food Microbiology 104 (2005) 197206

www.elsevier.com/locate/ijfoodmicro

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a beneficial effect on the human host (Ishibashi and

Shimamura, 1993).

So far, various studies concerning the production

of GOS have been reported (Iwasaki et al., 1996;Onishi and Kira, 1996; Mozaffar et al., 1987; Shin

et al., 1998; Yang and Bedmarcik, 2001). It was

indicated that depending on the source of h-galacto-

sidase, the resulting GOS production from lactose is

quite different in the final products and yields (Yang

and Bednarcik, 2001). Furthermore, most of the h-

galactosidase used in these studies are not approved

for food use, are expensive and many are not available

or not availa ble in sufficient quantities for industrial

application (Kim and Rajagopal, 2000; Albayrak and

Yang, 2002). Therefore, selection of microorganismswhich are safe for human use and are capable of

producing high levels ofh-galactosidase becomes an

urgent and attractive task.

Bifidobacterium, a probiotic organism, and its h-

galactosidase preparations are generally recognized as

safe (GRAS) for use in foods and food systems. In the

present study, various bifidobacteria were first

screened for the production of h-galactosidase. Cul-

ture conditions that affect enzyme production by the

selected strain were further investigated.

2. Materials and methods

2.1. Microorganisms

Microorganisms tested in the present study in-

cluded Bifidobacterium infantis CCRC 14633, Bifi-

dobacterium longum CCRC 15708 (obtained from

the Food Industry Research & Development Insti-

tute, Hsinchu, Taiwan) and Bifidobacterium longum

B6 (obtained from Prof. H. Y. Lin, Dept. of Food

Science, National Chung-Hsing University, Tai-chung, Taiwan).

2.2. Culture condition

Before the experiment, the test organisms were

activated by two successive transfers in Lactobacilli

MRS agar (Difco, Detroit, MI, USA) supplemented

with 0.05% cysteine (Sigma, St. Louis, MO, USA)

(MRSC agar) at 37 8C for 12 h. The activated

culture was again inoculated into MRS broth

(Difco) supplemented with 0.05% cysteine (MRSC

broth) at 37 8C for 12 h. Cells in the culture were

harvested by centrifugation (10,000g for 10 min at

4 8C, washed twice with saline solution, diluted to a population of ca 109 CFU/ml and used as the

inoculum.

Fermentation for the production ofh-galactosidase

was carried out by transferring an aliquot (1.0 ml) of

the inoculum to a 250-ml Erlenmeyer flask containing

100 ml culture medium which consisted of 10 g

glucose, 20 g lactose, 10 g peptone, 10 g yeast extract,

5.0 g (NH4)2SO4, 3.0 g K2HPO4, 1.0 g KH2PO4, 0.5 g

MgSO4d7H2O and 0.3 g l-cysteine per liter (Onishi

and Tanaka, 1997). After 12-h incubation at 37 8C, the

population of test organisms and h-galactosidase ac-tivity were determined.

To examine the effect of various carbon sources

on h-galactosidase production, lactose, galactose or

glucose (all are products of Sigma) was used as the

carbon source in the medium. To investigate the

effect of nitrogen sources on the h-galactosidase

production, peptone, yeast extract and (NH4)2SO4in the above mentioned medium formula were

replaced with various amount nitrogen sources, so

the medium contained 0.3% N. The nitrogen sources

tested included casein, peptone, tryptone, gelatin,

beef extract and yeast extract (all were Difco pro-

ducts) and (NH4)2SO4 (Baker J. T., Phillipsburg, NJ,

USA). Nitrogen contents of these nitrogen sources

listed in Difco Manual (Anonymous, 1998) were

used to calculate the amount of nitrogen source

required. To examine the effect of pH, the initial

pH of the medium was adjusted to various values

(5.07.5) with sterile 1.0 N NaOH or 1.0 N HCl

solution to investigate the effect of culture tempera-

ture. The culture was maintained at temperatures

from 22 to 47 8C and all fermentation experiments

were carried out for 12 h. Detailed culture conditionsare specified in Results and discussion.

2.3. Determination of b-galactosidase activity

For the determination of h-galactosidase activity,

cells of bifidobacteria in the culture were first har-

vested by centrifugation (10,000g for 10 min at 4

8C). After washing twice with 0.03 M sodium phos-

phate buffer (pH 6.8), they were suspended in phos-

phate buffer. The suspension, maintained in an ice

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bath, was treated by sonication with a sonicator

(Model 3000, Misonix, Farmingdale, NY, USA) and

was centrifuged (15,000g, 10 min). The supernatant

served as the enzyme source.Activity of h-galactosidase was then assayed es-

sentially according to the method described by Nagy

et al. (2001b). The reaction mixture was composed of

0.5 ml of enzyme source and 0.5 ml of 15 mM o-

nitrophenyl h-d-galactopyranoside (OPNG) in 0.03

M sodium phosphate buffer (pH 6.8). After 10 min

at 37 8C, 2.0 ml of 0.1 M sodium carbonate was added

to the reaction mixture to stop reaction. Absorbance

was measured at 420 nm with a spectrophotometer

(Model Helios a, Unicam Co., Cambridge, UK). A

unit ofh-galactosidase was defined as the amount ofenzyme catalyzing the formation of 1 Amol of o-

nitrophenyl per min under the assay condition.

2.4. Determination of transgalactosylation

Transgalactosylation activity was assayed accord-

ing to the method described by Dumortier et al.

(1994). The reaction mixture was composed of 100

Al of enzyme, 300 Al of 0.05 M sodium phosphate

buffer (pH 4.3), and 100 Al of 0.5 M lactose. After 4

h a t 4 5 8C, the enzyme reaction was stopped by

heating for 2 min on a boiling-water bath, then the

incubation mixture was centrifuged for 30 min at

15,000g. The supernatant was diluted with water

before injection (20 Al) onto an HPLC column. An

HPLC system consisting of a degassing system

(Model DG-2410, Sanwa Tsusho Co., Tokyo,

Japan), a pump (880-LC, Jasco Co., Tokyo, Japan),

a carbohydrate analysis column (Rezex RNM carbo-

hydrate column, 7.8300 mm, Phenomenex Co.,

CA, USA), a guard column (Rezex RNM carbohy-

drate column, 7.850 mm, Phenomenex Co., CA,

USA), a column heater (800-LC, Jasco Co., Tokyo,Japan), a refractive index detector (830-RI, Jasco

Co., Tokyo, Japan), and a chromatography data sys-

tem (SISC Co., CA, USA). The eluent was pre-

degassed distilled water at a flow rate of 0.4 ml/

min. The column temperature was maintained at 85

8C and the detector temperature was set at 45 8C. A

unit of transgalactosylation activity was defined as

the amount of enzyme catalyzing the formation of 1

Amol of trisaccharide per min at 45 8C and at pH

4.3.

2.5. Determination of protein

Protein was assayed according to the method de-

scribed by Smith et al. (1985) using the bicinchoninicacid protein assay kit (Sigma). Bovine serum albumin

(Sigma) was used as the standard for calibration.

2.6. Enumeration of bifidobacteria

To enumerate bifidobacteria, samples were serially

diluted with saline solution and pour plated on MRSC

agar. Colonies were counted after incubation at 37 8C

for 48 h.

2.7. Statistical analysis

The mean values and the standard deviation were

calculated from the data obtained with triplicate trials.

These data were then compared by the Duncans

multiple range method (SAS, 2001).

3. Results and discussion

3.1. b-galactosidase production by Bifidobacteria

In a preliminary study, no h-galactosidase activity

was detected in the cultures of Bifidobacterium

longum CCRC 14634, Bifidobacterium breve CCRC

11846, Bifidobacterium bifidum CCRC 14615 and

Bifidobacterium adolescentis CCRC 14608. Howev-

er, B. infantis CCRC 14633, B. longum CCRC 15708

and B6 were found to be capable of producing h-

galactosidase with transgalactosylation activity. h-Ga-

lactosidase production by these bifidobacteria after 12

h of cultivation at 37 8C is shown in Table 1. The h-

galactosidase activities detected in the cultures of B.

lognum B6 and CCRC 15708 were not significantlydifferent (p N0.05), but were significantly higher than

that of only 0.56 U/ml found in the otherh-galacto-

sidase-producing culture of B. infantis CCRC 14633.

On the other hand, h-galactosidase detected in the

culture of B. longum CCRC 15708 showed a specific

activity of 7.15 U/mg protein which was significantly

higher than that (6.48 U/mg protein) detected in the

culture of B. longum B6.

Previously, h-galactosidase biosynthesis by bacte-

ria, yeasts, and moulds has been reported by various

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investigators (Ramana Rao and Dutta, 1977; Park et

al., 1979; Itoh et al., 1982; Dumortier et al., 1994;

Kim and Rajagopal, 2000; Nagy et al., 2001b). h-

Galactosidase activity varied up to a maximum of 2.5

U/ml forLactobacillus crispatus (Kim and Rajagopal,2000). Therefore, h-galactosidase production by B.

longum B6 and CCRC 15708 reported here is mark-

edly higher than previously recorded across a wide

range of microorganisms.

3.2. Effect of carbon source on the growth and b-

galactosidase production

Several investigators have described the carbon

source regulation of h-galactosidase biosynthesis in

various microorganisms (Fantes and Roberts, 1973;

Montero et al., 1989; Fiedurek and Szczodrak, 1994;

Nikolaev and Vinetski, 1998; De Vries et al., 1999;

Nagy et al., 2001a; Fekete et al., 2002). All indicated

that the role of carbon source in the biosynthesis ofh-

galactosidase may vary and depend on the microor-ganisms tested.

To examine the effect of carbon source on the

production of h-galactosidase, B. longum CCRC

15708, which produced a high amount ofh-galacto-

sidase with the highest specific activity (Table 1) was

used as the test organism. Growth and h-galactosidase

detected in cultures containing lactose, glucose or

galactose as the sole carbon source is shown in Fig.

1. It was found that the final viable population of B.

longum CCRC 15708 was higher in cultures contain-

ing either lactose or glucose as the sole carbon source

Carbon source

lactose glucose galactos

Final

population(logCFU/ml)

0

2

4

6

8

10

12

-galac

tosidaseactivity(U/ml)

0

3

6a a

b

A

C

B

e

Fig. 1. Effect of carbon source on the growth and h-galactosidase production by B. longum CCRC 15708. Medium contained 1% yeast extract,

1% peptone, 0.5% (NH4)2SO4, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO4d 7H2O, 0.03% l-cysteine and 1% of various carbon sources.

Determinations were made after a 12-h cultivation at 37 8C. Bars indicate standard deviations. Different letters, within each type of

determination, indicate significant difference (p b0.05).

Table 1

Final pH, population size and h-galactosidase production by bifidobacteria*

Strain Final pH Final population

(log CFU/ml)

Activity (U/ml) Protein

(mg/ml)

Specific activity

(U/mg)B. longum CCRC 15708 4.0F0.0b** 8.2F0.0ab 4.96F0.42a 0.69F0.06b 7.15F0.16a

B. longum B6 3.9F0.0ab 8.2F0.0ab 4.81F0.66a 0.74F0.04a 6.48F0.70b

B. infantis CCRC 14633 4.2F0.0a 8.4F0.0a 0.56F0.04b 0.35F0.02c 1.63F0.17c

* Fermentation was conducted in a medium containing 2% lactose, 1% glucose, 1% yeast extract, 1% peptone, 0.5% (NH 4)2SO4, 0.3%

K2HPO4, 0.1% KH2PO4, 0.05% MgSO4d 7H2O and 0.03% l-cysteine at 37 8C for 12 h.

** Values in the same column with different letters were significantly different by Duncans multiple range test ( p b0.05).


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with the highesth-galactosidase activity (5.44 U/ml)

detected with lactose followed by galactose and the

lowest activity with glucose as the carbon source.

These results were different from the report of Kimand Rajagopal (2000) which indicated thatL. cripatus

grown in MRS broth containing galactose as the

carbon source showed the highest h-galactosidase

activity followed by moderate levels of enzyme pro-

duction on lactose and insignificant activity with glu-

cose or maltose. However, our results agree with those

observed for Kluyveromyces fragilis and Rhizomucor

sp. by Fiedurek and Szczodrak (1994) and Shaikh et

al. (1997), respectively, who demonstrated that lactose

is the best carbon source which induces the maximum

synthesis ofh-galactosidase by Rhizomucorsp., whileglucose is a poor inducer.

Amounts of carbon source in the medium may

affect the expression of h-galactosidase by microor-

ganisms (Fiedurek and Szczodrak, 1994; Inchaur-

rondo et al., 1998). As shown in Fig. 2, a final

viable population of the test organism ranging be-

tween 8.1 and 8.4 log CFU/ml was observed in the

culture originally containing 1.010.0% lactose. h-

galactosidase activity increased as the concentration

of lactose in the medium was increased up to 4.0%.

Further increasing the lactose content resulted in the

reduction of h-galactosidase activity. A similar phe-

nomenon was observed by Fiedurek and Szczodrak

(1994) who investigated the biosynthesis ofh-galac-

tosidase by K. fragilis. The decreased h-galactosi-

dase activity in the medium containing 5% or morelactose may be attributed to the increased concentra-

tion of internally released glucose which represses

biosynthesis of h-galactosidase by test organism

(Inchaurrondo et al., 1998). Furthermore, we also

demonstrated that 4% lactose was sufficient to induce

the highest expression of h-galactosidase under the

condition tested.

Kim and Rajagopal (2000) described that galactose

was the best carbon source for the biosynthesis of h-

galactosidase by L. crispatus, while addition of glu-

cose or lactose to the growth medium containinggalactose inhibited the synthesis of h-galactosidase.

We then further examined the effect of glucose and

galactose supplementation (0.55.0%) on the produc-

tion ofh-galactosidase in a medium containing 4.0%

lactose by B. longum CCRC 15708. We noted a

significantly (p b0.05) reduced enzyme activity in

the lactose-containing broth supplemented with

2.0% or more glucose or 0.5% galactose. Further-

more, as the amount of glucose or galactose was

increased in the growth medium, h-galactosidase ac-

tivity was repressed with galactose having a much

greater effect than glucose (data not shown).

Lactose concentration (%)

0.5 2 3 4 51 10

Finalpopulation(logCFU/ml)

0

2

4

6

8

10

12

-galac

tosidaseactivity(U/ml)

0

2

4

6

8

10

a cd D abab b d

C

D

BB

A A

D

Fig. 2. Effect of lactose concentration on the growth and h-galactosidase production by B. longum CCRC 15708. Medium contained 1% yeast

extract, 1% peptone, 0.5% (NH4)2SO4, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO4d 7H2O, 0.03% l-cysteine and different concentrations of

lactose. Determinations were made after a 12-h cultivation at 37 8C. Bars indicate standard deviations. Different letters, within each type of



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3.3. Effect of nitrogen source on the production of

b-galactosidase

Nitrogen sources may affect microbial biosynthesisof h-galactosidase (Ramana Rao and Dutta, 1977;

Shaikh et al., 1997). Viable populations of B. longum

CCRC 15708 after 12 h of fermentation in the m edia

containing different nitrogen sources are shown in Fig.

3. It was noted that the final viable population of the test

organism ranged from 5.9 to 8.7 log CFU/ml depend-

ing on the nitrogen source. A large variation in the

activity of h-galactosidase, ranging from 0.00 to

11.13 U/ml, was also detected in the cultures with

different nitrogen sources. Yeast extract was found to

support the highest production ofh-galactosidase byB.longum CCRC 15708. This may be attributed to the

growth factors in addition to the nitrogen compounds

present in yeast extract (Bridson and Brecker, 1970).

As shown Table 2, the concentration of yeast ex-

tract in the medium was also found to affecth-galac-

tosidase production by B. longum CCRC 15708. The

activity ofh-galactosidase increased upon increasing

the yeast extract concentration up to 10.0% in the

medium. Further increasing yeast extract content in

the medium resulted in a sharp reduction in the activ-

ity ofh-galactosidase and a reduced final population

of the test organism. As show in Table 2, a consider-

able amount ofh-galactosidase could be obtained in

the medium containing 3.5% yeast extract, and this

level was used in subsequent studies.

3.4. Effect of initial pH and temperature on the

production ofb-galactosidase

In media with an initial pH between 5.0 and 6.5, the

test organism showed a final population of 8.58.7 log

CFU/ml with a smaller yield in media having an initial

pH of 7.0 or 7.5 as shown in Fig. 4. At an initial pH 5.0,

h-galactosidase activity was found to be ca 10.95 U/ml

increasing as the initial pH of the culture medium

increased, reaching a maximum of ca 15.87 U/ml at pH 6.5. Higher initial pH values resulted in a sharp

reduction in the production ofh-galactosidase by the

test organism (Fig. 4).

Although the final population detected in the culture

grown at 2742 8C is similar, h-galactosidase activity

at 37 8C was significantly higher than that detected at

other cultivation temperatures (Fig. 5). The activity of

h-galactosidase was increased as the cultivation tem-

perature was increased from 22 8C, the lowest cultiva-

tion temperature tested in the present study, to 37 8C.

Further increases in the cultivation temperature led to a

Contr

ol

Casei

n

Pepto

ne

(NH4)2S

O4

Trypto

neGelati

n

Beefe

xtract

Yeast

extra

ct

Fina

lpopulation(logCFU/ml)

0

2

4

6

8

10

12

-galactosidaseactivity(U/ml)

0

3

6

9

12

15

ba

eg

d

h

f

c

B

G

E

H

C

F

D

A

Fig. 3. Effect of nitrogen source on the growth and h-galactosidase production by B. longum CCRC 15708. Medium contained 4% lactose,

0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO4d 7H2O, 0.03% l-cysteine and various nitrogen sources. Determinations were made after a 12-

h cultivation at 37 8C. Bars indicate standard deviations. Different letters, within each type of determination, indicate significant difference

(p b0.05).


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reduction of enzyme production accompanied by a

reduction in the final viable population. These observa-

tions agree with Fiedurek and Szczodrak (1994) and

Smith et al. (1985) and demonstrated that the highesth-

galactosidase production by B. longum CCRC 15708

was obtained with an initial medium pH of 6.5 at 37 8C.

The time course of h-galactosidase production,

transgalactosylation activity and growth of B. longum

CCRC 15708 for a period of 24 h is shown in Fig. 6.

Viable cells of the test organism increased gradually as

the fermentation started and reached a maximum after

ca 10 h of cultivation. Thereafter, the population of

viable cells showed no marked change. During the

fermentation, the pH of the medium declined rapidly

from 6.5 at the beginning to ca 3.8 after 16 h of fer-

mentation. On the other hand, h-galactosidase activity

increased sharply from the beginning of fermentation,

reached its maximum of ca 18.60 U/ml after 16 h of

fermentation, after which the level ofh-galactosidase

activity decreased. In general, changes of transgalacto-

Initial pH value5 65.5 6.5 7 7.5

Finalp

opulation(logCFU/ml)

0

3

6

9

12

-galactosidaseactivity(U/ml)

0

4

8

12

16

20

c

C

bc Ba

A

ab

A

d

C

e

D

Fig. 4. Effect of initial pH on the growth and h-galactosidase production by B. longum CCRC 15708. Medium with various initial pH contained

4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO4d 7H2O, and 0.03% l-cysteine. Determinations were made after a

12-h cultivation at 37 8C. Bars indicate standard deviations. Different letters, within each type of determination, indicate significant difference

(p b0.05).

Table 2

Effect of yeast extract concentration on the growth and h-galactosidase production by B. longum CCRC 15708*

Yeast extract (%) Final pH Final population

(log CFU/ml)

Activity (U/ml) Protein (mg/ml) Specific activity

(U/mg)0 6.7F0.0ab** 7.3F0.4e 0.00F0.00i 0.00F0.00f 0.00F0.00e

1.5 4.5F0.3d 8.0F0.3d 6.03F0.61e 0.59F0.08d 10.27F0.60b

3.5 4.4F0.2d 8.5F0.1ab 13.40F0.55d 1.26F0.17c 10.73F1.20b

4.5 4.4F0.1d 8.6F0.0a 15.71F0.56c 1.50F0.19b 10.61F1.04b

5.5 4.4F0.4d 8.5F0.1ab 17.32F0.44b 1.62F0.04a 10.70F0.37b

10 5.3F0.1c 8.2F0.1c 18.80F0.78a 1.53F0.12ab 12.32F0.45a

12 6.5F0.1b 7.2F0.1b 2.78F0.18f 0.55F0.01e 10.20F0.97bc

14 6.5F0.1b 7.0F0.1f 2.21F0.02g 0.47F0.01e 9.36F0.39c

16 6.8F0.0a 6.9F0.2f 0.62F0.05h 0.18F0.01f 6.93F0.69d

* Fermentation was conducted in a medium containing 4% lactose, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO4d 7H2O, 0.03% l-cysteine

and different concentrations of yeast extract at 37 8C for 12 h.

** Values in the same column with different letters were significantly different by Duncans multiple range test ( p b0.05).


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p

H

4

5

6

7

Finalpopulation(logCFU/ml)

6.5

7.0

7.5

8.0

8.5

9.0

Time (h)

0 4 8 12 16 20 24

-galactosidase

activity(U/ml)

0

5

10

15

20

Transgalactosylationactivity(U/ml)

0.00

0.01

0.02

0.03

0.04

Fig. 6. Time course of growth, final pH value, h-galactosidase activity and transgalactosylation activity of B. longum CCRC 15708.

Fermentation was conducted at 37 8C in a medium with an initial pH 6.5 containing 4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1%

KH2PO4, 0.05% MgSO4d 7H2O, an 0.03% l-cysteine. Bars indicate standard deviations.

Temperature (C)

22 27 30 37 42 47

Finalpopulation(logC

FU/ml)

0

3

6

9

12

-galactosidaseactivity

(U/ml)

0

5

10

15

20

b

E

a

C

a

B

a

A

a

D

b

E

Fig. 5. Effect of temperature on the growth and h-galactosidase production by B. longum CCRC 15708. Medium with an initial pH 6.5

contained 4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO4d 7H2O, and 0.03% l-cysteine. Determinations were

made after a 12-h cultivation at various incubation temperature. Bars indicate standard deviations. Different letters, within each type of



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sylation activity detected in the culture showed a sim-

ilar trend as that observed withh-galactosidase activity.

The transgalactosylation activity increased as the fer-

mentation started, reached a maximum after ca 1216h of cultivation and declined as the fermentation was

further extended.

4. Conclusion

The results of this study demonstrated that produc-

tion ofh-galactosidase by bifidobacteria varied with

the strains selected. B. longum CCRC 15708 is capa-

ble of producing a high level ofh-galactosidase with

high specific activity. After optimizing the mediacomposition and culture growth conditions for the

production ofh-galactosidase by B. longum CCRC

15708, we achieved a maximum of 18.6 U/ml, which

was about 3.8-fold over the initial values obtained

with the non-optimized medium. Considering the

high yield of h-galactosidase with transgalactosyla-

tion activity and its GRAS nature, B. longum CCRC

15708 may be a potentially useful industrial strain for

the production ofh-galactosidase. Thus characteriza-

tion ofh-galactosidase produced by B. longum CCRC

15708 and application of this enzyme for GOS pro-

duction with lactose are being investigated.

Acknowledgment

This work was supported by the National Science

Council (NSC 91-2313-B-002-305), Taiwan, ROC.

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