International Journal of Food Microbiology 116 (2007) 186 189 www.elsevier.

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Cultural condition affecting the growth and production of -galactosidase by Bifidobacterium longum CCRC 15708 in a jar fermenter
C.A. Hsu a , R.C. Yu a , S.L. Lee b , C.C. Chou a,
a

Graduate Institute of Food Science and Technology, National Taiwan University 59, lane 144, Keelung Rd., Sec. 4, Taipei, Taiwan b Department of Bioengineering, Tatung University, Taipei, Taiwan Received 15 December 2005; received in revised form 7 September 2006; accepted 10 December 2006

Abstract In the present study, the growth and production of -galactosidase by Bifidobacterium longum CCRC 15708 in a 5-L jar fermenter as influenced by cultivation temperature (2742 C), medium pH (4.57.5) and agitation speed (5200 rpm) were evaluated. In general, it was found that a cultivation temperature of 37 C proved optimal for both growth and -galactosidase production by the test organism. Although the growth of the test organism was the highest in the culture with pH controlled at 4.56.5, the culture with pH controlled at 6.5 resulted in the highest production of -galactosidase. Further, agitation at 100 rpm or more was found to enhance both the growth and production of -galactosidase. Fermentation conducted in a jar fermenter having the pH of the culture medium, the cultivation temperature, and the agitation speed controlled at 6.5, 37 C, and 100 rpm, respectively, a maximum -galactosidase activity of 36.7 U/ml and a maximum transgalactosylation activity of 0.49 U/ ml was achieved in 10 h of fermentation. There are ca 2.0 and 12.3 fold greater than the reported maximum -galactosidase and transgalactosylation activity, respectively, produced by B. longum CCRC 15708 in a flask culture system. 2007 Elsevier B.V. All rights reserved.
Keywords: Bifidobacterium longum; -galactosidase; Fermentation variables; Jar fermenter

1. Introduction -Galactosidase has long been used to hydrolyze lactose to glucose and galactose in the manufacture of dairy products (Mahoney, 1998). Further, this enzyme can also catalyze a transgalactosylation reaction, which leads to the formation of di-, tri-, or higher galacto-oligosaccharides (GOS) (Prenosil et al., 1987; Yang and Tang, 1988; Zarate and Lpez-Leiva, 1990). GOS were found to stimulate the growth and establishment of bifidobacteria in the human intestine (Mitsuoka, 1990; Sako et al., 1999) and suppress potentially harmful bacteria such as clostridia and Bacteriodes species in the gut and as such are now regarded as a prebiotic food ingredient. (Oku, 1996; Fooks et al., 1999).

Corresponding author. Tel.: +886 2 3366 4111; fax: +886 2 2362 0849. E-mail address: fstcchou@ntu.edu.tw (C.C. Chou). 0168-1605/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.12.034

Previously, we noted that B. longum CCRC 15708, owing to its GRAS nature, coupled with its high yields of -galactosidase with high levels of transgalactosylation activity, may potentially be a industrial strain for the production of -galactosidase (Hsu et al., 2005). In addition, properties of -galactosidase produced by B. longum CCRC 15708 was further characterized (Hsu et al., 2006). In this study, we extend this line of research using a fermenter system for enzyme production instead of a flask culture system. Since a fermenter system allows for the strict control of environmental conditions, it follows that a careful control of the variables that determine these conditions may improve the production of enzyme by microorganism. Besides, compared to other organisms, to this point reports on the cultivation of bifidobacteria at fermenter scale, and on the development of optimal cultural conditions for its growth and production, are still rather limited. For these reasons, this study evaluated the growth behavior and production of -galactosidase by B. longum CCRC

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15708 in a 5-L jar fermenter system. Specifically, the fermentation parameters including cultivation temperature, pH and agitation speed were examined. 2. Materials and methods

These data were then compared by the Duncan's multiple range method (SAS, 2001). 3. Results and discussion 3.1. Effect of cultivation temperature

2.1. Microorganism B. longum CCRC 15708 obtained from the Food Industry Research and Development Institute, Hsinchu, Taiwan was used in this study as the test organism. 2.2. Culture condition Fermentation for the growth and production of -galactosidase was performed in a 5-L jar fermenter (BR 5, Exon Science Inc., Taipei, Taiwan) consisting of a cylindrical culture vessel with a working volume of 3 L. This fermenter was equipped with Exon BR 5 software (BR 5, Exon Science Inc.) to control temperature, pH, and agitation speed. The culture medium consisted of 4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO47H2O and 0.03% L-cysteine. The amount of lactose and yeast extract present in the fermentation medium has been found to be optimal for growth and -galactosidase production by test organism (Hsu, 2006). The organism was inoculated into MRS broth (Difco) supplemented with 0.05% cysteine (MRSC broth) at 37 C for 12 h. Cells were harvested by centrifugation (10,000 g for 10 min at 4 C), then diluted with the same medium to a population of ca 109 CFU/ml and used as the inoculum. When fermentation was conducted, fermentation medium was inoculated with B. longum CCRC 15708 at an inoculum size of 20%. To examine the effect of pH, the pH of the medium was maintained at various values (4.5, 5.5, 6.5 and 7.5) by the addition of sterile 6N NaOH or HCl. Additionally, fermentation conducted without controlling pH during the fermentation process was examined by using a medium with an initial pH of 6.5. To examine the effects of culture temperature and agitation, the culture was maintained at 2742 C and at an agitation speed of 5200 rpm, respectively. 2.3. Determination of - galactosidase and transgalactosylation activity and cell growth To determine -galactosidase activity and transgalactosylation activity, the detailed procedure described in our previous paper (Hsu et al., 2005) was followed. One unit of -galactosidase activity and transgalactosylation activity was defined as the amount of enzyme producing 1 mol of o-nitrophenol and trisaccharide, respectively, per min under the assay condition. Cell growth was determined by measuring the absorbance at 540 nm using a spectrophotometer. 2.4. Statistical analysis In this study, the mean values and the standard deviation were calculated from the data obtained with triplicate trials. B. longum CCRC 15708 showed the highest growth at 37 C followed by 32 C with significantly less, but similar growth at 42 C and 27 C after 12 h of fermentation. At all the cultivation temperatures examined, except 42 C, the activity of -galactosidase in the culture increased as the fermentation proceeded with the most marked increase of galactosidase activity at 37 C. After 12 h of fermentation, this culture showed the highest level of -galactosidase activity at ca 35.9 U/ml followed by those cultivated at 32 C, 27 C and 42 C. At the last temperature, although a -galactosidase activity of ca 2.2 U/ml was detected after 2 h of fermentation, no further marked increase of enzyme activity was noted thereafter (Fig. 1B), despite the observed continuous growth (Fig. 1A). Variation in the optimal temperature for growth and enzyme production by microorganisms in different culture systems has been previously reported (Wu, 1994; Chou et al., 1999), while the optimal temperature of 37 C for growth and -galactosidase production by B. longum CCRC 15708 in the 5-L jar fermenter observed in the present study is consistent with that noted in the system of flask culture (Hsu et al., 2005). 3.2. Effect of pH Controlling the pH of the culture during fermentation has been reported to enhance microorganism growth and enzyme production (Buckland et al., 1976; Chou et al., 1999). Relatively rapid growth and greatest cell yields were noted in cultures with pH controlled at 4.5, 5.5, or 6.5, in comparison with cultures at pH 7.5 or without pH control (Fig. 2A). -Galactosidase activity increased in all the pH-controlled cultures as the fermentation proceeded. Among the various pHcontrolled cultures examined, the culture with pH controlled at 6.5 exhibited the highest -galactosidase activity, while the activity was the least in culture with pH controlled at 7.5 (Fig. 2B). In the culture without pH control, the pH dropped from 6.5 at the beginning to ca 3.9 after 6 h of fermentation which may be the cause of the lowest observed growth and galactosidase production after 12 h fermentation. 3.3. Effect of agitation speed During the initial 4 h, growth of test organism showed no significant difference in media at the various agitation speeds examined (Fig. 3A), but after 810 h, an agitation speed of 100 rpm or more was optimal for growth of B. longum CCRC 15708. Maximum growth (Fig. 3A) and maximum -galactosidase production (Fig. 3B) were observed after 10 h of cultivation in the media at an agitation of 100 rpm or more. On

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Fig. 1. Effect of temperature on the growth (A) and -galactosidase activity (B) of B. longum CCRC 15708. Fermentation was conducted at 20% (v/v) inoculum size, controlled pH 6.5 and 50 rpm in a medium containing: 4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO47H2O and 0.03% Lcysteine. Bars indicate standard deviations. , 27 C; , 32 C; , 37 C; , 42 C.

Fig. 3. Effect of agitation speed on the growth (A) and -galactosidase activity (B) of B. longum CCRC 15708. Fermentation was conducted at 20% (v/v) inoculum size, 37 C and controlled pH 6.5 in a medium containing: 4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO47H2O and 0.03% Lcysteine. Bars indicate standard deviations. , 5 rpm; , 50 rpm; , 100 rpm; ,150 rpm; , 200 rpm.

the other hand, the test organism showed the maximum growth and -galactosidase production after a longer cultivation period of 12 h in medium at an agitation speed of 5 or 50 rpm. Higher agitation speeds have been reported to stimulate the dispersion of macromolecules in the medium (Buckland et al.,

1976), to improve the utilization of poorly dispersed substrate by the organism (Confer and Logan, 1991) and decrease the viscosity of high-protein components that might hinder the growth of a microorganism (Umasankar et al., 1996). These effects may all lead to the highest growth and -galactosidase in

Fig. 2. Effect of pH on the growth (A) and -galactosidase activity (B) of B. longum CCRC 15708. Fermentation was conducted at 20% (v/v) inoculum size, 37 C and 50 rpm in a medium containing: 4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO47H2O and 0.03% L-cysteine. Bars indicate standard deviations. , uncontrolled pH; , pH 4.5; , pH 5.5; , pH 6.5; , pH 7.5.

Fig. 4. Time course of the growth and residual lactose concentration (A) and galactosidase and transgalactosylation activity (B) of B. longum CCRC 15708. Fermentation was conducted at 20% (v/v) inoculum size, 37 C, controlled pH 6.5 and 100 rpm in a medium containing 4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO47H2O, and 0.03% L-cysteine. Bars indicate standard deviations.

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a shorter cultivation period as observed in cultures with a higher speed of agitation in the present study. 3.4. Culture under optimal culture conditions The various periods of growth, lactose content -galactosidase activity and transgalactosylation activity in the culture under optimal culture conditions are shown in Fig. 4. Growth of test organism began as the fermentation was started and reached its maximum after ca 10 h of cultivation in the jar fermenter (Fig. 4A). In general, a similar tendency was observed with galactosidase activity and transgalactosylation activity. In addition, there was a rapid reduction of lactose in the culture after ca 6 h of cultivation that further reduced to a nondetectable level after 10 h of cultivation. Lactose is considered the best carbon source to induce the maximum production of galactosidase by Rhizomucor sp. (Shaikh et al., 1997), K. fragilis (Fiedurek and Szczodrak, 1994) and B. longum CCRC 15708 (Hsu et al., 2005). Therefore, the lack of lactose in the culture medium may be one of the important factors contributing to the no further increase in the activity of galactosidase and transgalactosylation after 10 h of fermentation (Fig. 4B). Maximum -galactosidase activity and transgalactosylation activity of 36.7 and 0.49 U/ml, respectively, were obtained through fermentation conducted in a fermenter under optimal conditions (20% inoculum size; medium containing 4.0% lactose and 3.5% yeast extract as carbon and nitrogen source, respectively; temperature, pH and agitation speed controlled at 37 C, 6.5 and 100 rpm, respectively). This is respectively ca 2.0 and 12.3 fold greater than that noted in a flask culture system by Hsu et al. (2005) in 10 h compared with 1416 h. The optimized culture conditions discussed in this study provide a basis for industrial fermentation of B. longum CCRC 15708 to produce -galactosidase. Acknowledgment This work was supported by the National Science Council (NSC 93-2313-B-002-002), Taiwan, ROC. References
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