Production of high-content galacto-oligosaccharide by enzyme catalysis and fermentation with Kluyveromyces marxianus / Chao. Chun Cheng

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Abstract Of three b-galactosidases from Asper-gillus oryzae, Kluyveromyces lactis and Bacillus sp., used for the production of low-content galacto-oligosaccharides (GOS) from lactose, the latter produced the highest yield of trisaccharides and tetrasaccharides. GOS production was enhanced by mixing b-galactosidase glucose oxidase. The low-content GOS syrups, produced either by b-galacto-sidase alone or by the mixed enzyme system, were subjected to the fermentation by Kluyveromyces marxianus, whereby glucose, galactose, lactose and other disaccharides were depleted, resulting in up to 97% and 98% on a dry weight basis of high-content GOS with the yields of 31% and 32%, respectively. Keywords Batch fermentation Ỉ Galacto-oligosaccharide Æ b-galactosidase Æ Glucose oxidase Æ Kluyveromyces marxianus

Ingested galacto-oligosaccharides (GOS), as a prebiotic, can stimulate the proliferation of lactic acid bacteria and bifidobacteria in the intestines to promote human health (Sako et al. 1999). GOS has been produced through the transgalactosyla-tion of various b-galactosidases (lactase, EC.3.2.1.23) on lactose (Za´rate and Lo´pez-Leiva 1990). b-Galactosidase can catalyze the transga-lactosyl reaction as well as the hydrolysis of lac-tose. The proportion of transgalactosylation to hydrolysis reaction varies, depending on different sources of the enzyme. b-Galactosidase from E. coli or Aspergillus niger exerts strong hydro-lytic activity, whereas b-galactosidase from Aspergillus oryzae or Bacillus circulans exerts strong transgalactosylation (Mahoney 1998). GOS comprises transgalactosyl disaccharides (GOS-2), trisaccharides (GOS-3), tetrasaccha-rides (GOS-4) and pentasacchatetrasaccha-rides. Currently, commercial GOS products contain large amounts of glucose and unreacted lactose and conse-quently, are not appropriate for the people suf-fering from diabetes mellitus or lactose intolerance. This study has produced high-content GOS by fermentation with Kluyveromyces marxianus of the low-content GOS syrups, pro-duced either by b-galactosidase alone or when mixed with glucose oxidase, to remove digestible sugars including glucose, galactose and lactose. C.-C. Cheng Ỉ D.-C. Sheu (&) Ỉ K.-J. Duan Ỉ

Department of Chemical Engineering, TatungUniversity, Taipei 104, Taiwan

T.-C. Cheng

Department of Chemical Engineering, NorthernTaiwan Institute of Science and Technology, Taipei112, Taiwan

DOI 10.1007/s10529-006-9002-1 O R I G I N A L P A P E R

Production of high-content galacto-oligosaccharide by enzyme catalysis and fermentation

with Kluyveromyces marxianus

Chao-Chun Cheng Æ Mei-Ching Yu Æ Tzu-Chien Cheng Æ Dey-Chyi Sheu Æ Kow-Jen Duan Ỉ Wei-Lun Tai

Received: 19 October 2005 / Accepted: 8 February 2006 / Published online: 10 June 2006 Springer Science+Business Media B.V. 2006

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Materials and methods Microorganism and materials

Kluyveromyces marxianus ATCC 56497, a milk yeast, was used in this work. The b-galactosidases from A. oryzae and K. lactis were purchased from Sigma. The b-galactosidase from Bacillus sp. (Cheng et al. 2006) was a kind gift from Taiwan Fructose Co. (Taoyuan, Taiwan). Gluzyme, a mixture of glucose oxidase and catalase, was purchased from Novo Nordisk. Other chemicals were obtained from commercial sources.

Analysis of carbohydrates

Carbohydrate analysis was performed by HPLC with a refractive detector and a Lichrospher 100 NH2 (250 · 4 mm, particle size 5 lm) column (Merck). The mobile phase was acetonitrile/water (75:25, v/v) at 1 ml min)1. Because glucose and galactose ran at the similar retention time on HPLC, glucose was to be assayed by YSI model 2700 biochemical analyzer (Yellow Springs Instrument Co. USA) and galactose was deter-mined by the subtraction method.

Enzyme assay

For the enzyme assay and the GOS production, a 0.01 M potassium phosphate buffer (pH 7) was used for the b-galactosidase from K. lactis. A 0.01 M sodium acetate buffer (pH 5) was used for the b-galactosidases from A. oryzae, Bacillus sp., and the mixed enzyme system with gluzyme and Bacillus b-galactosidase. The b-galactosidase from K. lactis was assayed at 40C, using 1 M lactose as substrate, whereas b-galactosidases from A. oryzae and Bacillus sp. were assayed at 50C. After 60-min, the reaction mixture was held at 100C to inactivate b-galactosidase and termi-nate the reaction. One unit of b-galactosidase activity was defined as 1 lmol glucose liberated per min under the above-described conditions.

To assay glucose oxidase activity, gluzyme, 1 g, was dissolved in 100 ml 0.01 M sodium acetate buffer (pH 5) and 1 ml added to 50 ml of prein-cubated 5% (w/v) glucose in the same buffer. After 30 min at 37C with shaking, the reaction

mixture was held at 100C to terminate the reac-tion. The residual glucose was determined by YSI model 2700 analyzer. One unit of glucose oxidase activity was defined as 1 lmol glucose consumed per min under the above-described conditions. Production of low-content GOS

To produce GOS using b-galactosidase alone, 6.2 U b-galactosidase from A. oryzae, 10 and 13 U b-galactosidase from K. lactis, 4.5 and 5.6 U of b-galactosidase from Bacillus sp., respectively, were used for 1 g lactose. The reactions were carried out at 30, 40, or 50C.

In the mixed enzyme system, 4.5 U Bacillus b-galactosidase was used for 1 g of initial lactose and 15 U gluzyme per gram of initial lactose was added at 0, 6, 12 and 18 h. The reaction was carried out for 24 h in a stirred tank reactor with a working volume of 2 l, at an aeration rate of 2 vvm, an agitation rate of 300 rpm, 50C, with the pH was controlled at 5.0 by adding 40% (w/w) CaCO3.

Unless otherwise specified, a 330 g lactose l–1 was used for the GOS production catalyzed either by the b-galactosidase alone or by the mixed en-zyme system.

Yeast fermentation

To carry out the fermentation of low-content GOS, K. marxianus was cultured in a shake-flask containing 1% (w/v) yeast extract and 1% (w/v) malt extract at 200 rpm, 30C for 24 h. The cul-ture was then inoculated into a jar fermenter with a working volume of 2 l. The medium was com-posed of 20% (w/w) low-content GOS and 0.5% (w/v) yeast extract. The fermentation was carried out at an aeration rate of 2 vvm, an agitation rate of 300 rpm, 30C, and the pH was controlled at 5.0–5.5 by 5MNaOH.

Results and discussion

Comparison in GOS composition by the catalysis of b-galactosidases from various sources

As shown in Table 1, three b-galactosidases upon 3 or 5 h of reaction resulted in GOS with

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content always lower than 36% on a dry weight basis. The b-galactosidase from A. oryzae pro-duced only GOS-3. The enzyme from K. lactis produced GOS-2 and GOS-3, mainly GOS-2. However, the enzyme from Bacillus sp. pro-duced GOS-2, GOS-3 and GOS-4, mainly GOS-3. That the b-galactosidase from K. lactis produced comparably large amounts of glucose and galactose indicated its strong hydrolytic activity. Upon various enzyme doses and reac-tion temperatures, the b-galactosidase from K. lactis resulted in the highest yield of GOS. However, this GOS is largely disaccharides and most of them will be depleted during the fer-mentation by K. marxianus. Among various re-sults listed in Table 1, a relatively higher yield of GOS on average was obtained by the b-galac-tosidase from Bacillus. The low-content GOS production upon 4.5 U (per gram lactose) of Bacillus b-galactosidase at 50C for 5 h with the highest concentration of GOS-3 was subjected to yeast fermentation for the production of high-content GOS.

Production of GOS either by b-galactosidase alone or by the mixed enzyme system

Fig. 1a and b shows the batch kinetics of GOS production catalyzed by Bacillus b-galactosidase alone (4.5 U enzyme per gram of lactose) and the mixed enzyme system with Bacillus b-galactosidase and gluzyme, respectively. For

both reactions, maximal amount of GOS-3 was achieved after 5 h with Bacillus b-galactosidase alone, the slight increase in total GOS from 5 Table 1 Comparison of low-content GOS produced by three b-galactosidases under various conditions. The results wereobtained from duplicated experiments

Fig. 1 Batch kinetics of GOS formation catalyzed by (a)Bacillus b-galactosidase alone; (b) the mixed enzymesystem with Bacillus b-galactosidase and gluzyme. Theresults were obtained from duplicated experiments

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to 20 h was attributed primarily to the increase in GOS-2, whereas GOS-3 remained nearly constant and GOS-4 peaked at 9 h and then gradually declined. With the mixed enzyme system, the total GOS peaked at 5 h and then decreased. This might be because the byprod-uct, glucose, had been oxidized and in its absence the reaction equilibrium of b-galactosi-dase brought about more hydrolytic activity. Previously (Sheu et al. 2001), a mixed enzyme system with b-fructofuranosidase and glucose oxidase was applied to produce high-content fructo-oligosaccharides (FOS), with only 3% of initial sucrose remaining and up to 93% on a dry weight basis of FOS was achieved. How-ever, in this study, as shown in Fig. 1a and b, up to 28% and 12% of initial lactose remained unreacted during the GOS production catalyzed either by Bacillus b-galactosidase alone or by the mixed enzyme system, respectively. The difference in the yields between GOS and FOS might result from the nature of the enzymes, and galactose, a product of b-galactosidase, might be a competitive inhibitor for the b-galactosidase (Mahoney 1985). During the catalysis of various b-galactosidases, a large fraction of substrate lactose was always left in the GOS products (Za´rate and Lo´pez-Leiva 1990). In the present study, even by the mixed enzyme system, a large amount of lactose was left in the product, resulting in a low-content GOS at, less than 53% on a dry weight basis. Conversion of low-content GOS into

high-content GOS by yeast fermentation

The low-content GOS produced upon 4.5 U (per g lactose) of Bacillus b-galactosidase at 50C for 9 h with the highest concentration of GOS-3 and GOS-4 (Fig. 1a) was subjected to yeast fermen-tation for the production of high-content GOS. Fig. 2 shows the time-course of K. marxianus fermentation of the low-content GOS produced by Bacillus b-galactosidase. The consumption of lactose began after 12 h of fermentation when glucose and galactose had been nearly depleted. Thus the metabolism of lactose by K. marxianus is an inducible process and repressed by glucose and galactose. It took 30 h to remove all digestible

sugars in the GOS syrup, accompanied with the formation of ethanol. However, in Fig. 2b, most GOS-2 also disappeared, indicating that GOS-2 is consumed by K. marxianus as well as other digestible sugars.

In Table 2, the GOS syrups produced by the enzymatic processes were compared with that by combinations of enzymatic catalysis and yeast fermentation. After yeast fermentation, higher than 97% on a dry weight basis of high-content GOS was always obtained. Fig. 3a and b shows the HPLC analysis of the GOS ob-tained before and after the yeast fermentation, respectively. Compared to the GOS produced by b-galactosidase alone, the mixed enzyme system led to an increase in final mass produc-tion of GOS by 3%. This is because more GOS-4 and less GOS-2 are obtained by the mixed enzyme system. During the fermentation, GOS-2 is consumed by K. marxianus, whereas GOS-4

Fig. 2 Time-course of K. marxianus fermentation of low-content GOS produced by the catalysis of Bacillus b-galactosidase. (a) changes of digestible sugars, ethanol andDCW (dry cell weight); (b) the change of GOS. The resultswere obtained from duplicated experiments

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High-content GOS has been successfully pro-duced through enzymatic process and yeast fer-mentation. Either a b-galactosidase alone or a mixed enzyme system with b-galactosidase and glucose oxidase can be applied for the production of the low-content GOS from lactose. After the fermentation of low-content GOS by K. marxi-anus, most GOS-2 as well as all digestible sugars, including glucose, galactose and lactose were re-moved, forming high-content GOS. Compared to the pretreatment by b-galactosidase alone, the mixed enzyme system resulted in an increase in mass production of high-content GOS. The

low-content GOS produced by the b-galactosidase from K. lactis was not ideal for conversion to high-content GOS by K. marxianus fermentation since it produced predominantly GOS-2.

AcknowledgementThe authors are thankful for thefinancial support provided by the National Science Councilof Republic of China under Contract NSC 93-2214-E-036-005.

Cheng TC, Duan KJ, Sheu DC (2006) Application of tris(hydroxymethyl) phosphine as a coupling agent for b-galactosidase immobilized on chitosan to producegalactooligosaccharides. J Chem Technol Biotechnol81:233–236

Mahoney RR (1985) Modification of lactose and lactose-containing dairy products with b-galactosidase. In:Fox PF (ed) Developments in Dairy Chemistry 3,Amsterdam. Elsevier Applied Science, The Nether-lands, pp 69–108

Mahoney RR (1998) Galactosyl-oligosaccharide formationduring lactose hydrolysis: a review. Food Chem63:147–154

Sako T, Matsumoto K, Tanaka R (1999) Recent progresson research and applications of non-digestible galac-to-oligosaccharides. Int Dairy J 9:69–80

Sheu DC, Lio PJ, Chen ST, Lin CT, Duan KJ (2001)Production of fructo-oligosaccharides in high yieldusing a mixed enzyme system of b-fructofuranosidaseand glucose oxidase. Biotechnol Lett 23:1499–1503Za´rate S, Lo´pez-Leiva MH (1990) Oligosaccharide

for-mation during enzymatic lactose hydrolysis: a litera-ture review. J Food Prot 53:262–268

Table 2 Comparison of GOS produced by enzyme catalysis and succeeded yeast fermentationGOS produced from

100 g of initial lactose

Before fermentationAfter fermentationBefore fermentationAfter fermentation

The results were obtained from duplicated experiments. Bacillus b-galactosidase and gluzyme were used in the experiments

amass ratio of GOS to the initial lactose

GOS content on a dry weight basis

Retention time (min)

Fig. 3 HPLC chromatograms of (a) the low-content GOSproduced by the catalysis of Bacillus b-galactosidase; (b)the high-content GOS obtained after yeast fermentation

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Production of high-content galacto-oligosaccharide by enzyme catalysis and fermentation with Kluyveromyces marxianus / Chao. Chun Cheng

Production of high-content galacto-oligosaccharide by enzyme catalysis and fermentation

with Kluyveromyces marxianus

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