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

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

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 Æ

Galacto-oligosaccharide Æ b-galactosidase Æ Glucose

oxidase Æ Kluyveromyces marxianus

Introduction 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 Æ D.-C Sheu ( &) Æ K.-J Duan Æ

W.-L Tai

Department of Bioengineering, Tatung University,

Taipei 104, Taiwan

e-mail: dcsheu@ttu.edu.tw

M.-C Yu

Department of Chemical Engineering, Tatung

University, Taipei 104, Taiwan

T.-C Cheng

Department of Chemical Engineering, Northern

Taiwan Institute of Science and Technology, Taipei

112, 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 Æ Wei-Lun Tai

Received: 19 October 2005 / Accepted: 8 February 2006 / Published online: 10 June 2006

Springer Science+Business Media B.V 2006

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

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 were obtained from duplicated experiments

Reaction temperature

and time

30C 5 h 50C 5 h 30C 3 h 40C 3 h 50C 3 h 50C 5 h 50C 3 h 50C 5 h

a mass ratio of GOS to the consumed lactose

b

mass ratio of GOS to the initial lactose

0 100 200

300 Glucose Galactose

Lactose GOS-2 GOS-3 GOS-4 Total GOS

Reaction Time (h)

0 100 200 300

(a)

(b)

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

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

is not

0 10 20 30

0 10 20 30

0 20 40 60 80 100

0 10 20 30 40

Glucose Galactose Lactose Ethanol DCW

Fermentation (h)

GOS-2 GOS-3 GOS-4

(a)

(b)

0 20 40 60 80 100

−1)

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 and DCW (dry cell weight); (b) the change of GOS The results were obtained from duplicated experiments

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

Acknowledgement The authors are thankful for the financial support provided by the National Science Council

of Republic of China under Contract NSC 93-2214-E-036-005.

References Cheng TC, Duan KJ, Sheu DC (2006) Application of tris (hydroxymethyl) phosphine as a coupling agent for b-galactosidase immobilized on chitosan to produce galactooligosaccharides J Chem Technol Biotechnol 81:233–236

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 formation during lactose hydrolysis: a review Food Chem 63:147–154

Sako T, Matsumoto K, Tanaka R (1999) Recent progress

on 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 yield using a mixed enzyme system of b-fructofuranosidase and glucose oxidase Biotechnol Lett 23:1499–1503 Za´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 fermentation

GOS produced from

100 g of initial lactose

Before fermentation After fermentation Before fermentation After fermentation

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

a mass ratio of GOS to the initial lactose

b

GOS content on a dry weight basis

Retention time (min)

glucose

galactose lactose

GOS-2

GOS-3

GOS-4

(a)

(b)

GOS-2

GOS-3

GOS-4

Fig 3 HPLC chromatograms of (a) the low-content GOS

produced by the catalysis of Bacillus b-galactosidase; (b)

the high-content GOS obtained after yeast fermentation