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Journal of Biotechnology 150 (2010) 140–148

Contents lists available at ScienceDirect

Journal of Biotechnology

journa l homepage: www.e lsev ier .com/ locate / jb io tec

comprehensive investigation of the synthesis of prebioticalactooligosaccharides by whole cells of Bifidobacterium bifidum NCIMB 41171

li Osmana, George Tzortzisb, Robert A. Rastall a, Dimitris Charalampopoulosa,∗

Department of Food and Nutritional Sciences, The University of Reading, PO Box 226, Whiteknights, Reading, BerkshireRG6 6AP, UKClasado Ltd., 5 Canon Harnett Court, Wolverton Mill, Milton Keynes, MK12 5NF, UK

r t i c l e i n f o

rticle history:eceived 7 April 2010eceived in revised form 20 July 2010ccepted 3 August 2010

eywords:rebiotic

a b s t r a c t

The synthesis of galactooligosaccharides (GOS) by whole cells of Bifidobacterium bifidum NCIMB 41171was investigated by developing a set of mathematical models. These were second order polynomialequations, which described responses related to the production of GOS constituents, the selectivity oflactose conversion into GOS, and the relative composition of the produced GOS mixture, as a function ofthe amount of biocatalyst, temperature, initial lactose concentration, and time. The synthesis reactionswere followed for up to 36 h. Samples were withdrawn every 4 h, tested for �-galactosidase activity,and analysed for their carbohydrate content. GOS synthesis was well explained by the models, whichwere all significant (P < 0.001). The GOS yield increased as temperature increased from 40 ◦C to 60 ◦C,

alactooligosaccharides

ifidobacterium-Galactosidaseransgalactosylation

as transgalactosylation became more pronounced compared to hydrolysis. The relative composition ofGOS produced changed significantly with the initial lactose concentration (P < 0.001); higher ratios of tri-,tetra-, and penta-galactooligosaccharides to transgalactosylated disaccharides were obtained as lactoseconcentration increased. Time was a critical factor, as a balanced state between GOS synthesis and hydrol-ysis was roughly attained in most cases between 12 and 20 h, and was followed by more pronounced

hesis

GOS hydrolysis than synt

. Introduction

Prebiotics are defined as selectively fermented ingredients thatllow specific changes, both in the composition and/or the activ-ty in the gastrointestinal microbiota, that confer benefits upon the

ost’s well-being and health (Gibson et al., 2004). To date, only

nulin, fructooligosaccharides (FOS), lactulose, and galactooligosac-harides (GOS) are considered as established prebiotics (Bouhnik etl., 1999, 2004; Gibson et al., 1995, 2004; Kruse et al., 1999). GOS are

Abbreviations: GOS, galactooligosaccharides; HPLC, high performance liquidhromatography; HPAEC-PAD, high performance anion exchange chromatogra-hy coupled with pulsed amperometric detector; DP, degree of polymerisation;NOV, Aanalysis of variance; o-NPG, ortho-nitrophenyl-�-galactoside; YAL, pro-uction yield of allolactose; YO.T.DP2, production yield of other transgalactosylatedisaccharides; YT.DP2, production yield of all transgalactosylated disaccharides; YDP3,roduction yield of tri-galactooligosaccharides; YDP4, production yield of tetra-alactooligosaccharides; YDP5, production yield of penta-galactooligosaccharides;GOS ≥ 3, production yield of galactooligosaccharides with DP ≥ 3; YDP ≥ 4, productionield of galactooligosaccharides with DP ≥ 4; YGOS, production yield of total galac-ooligosaccharides; YP, purity yield of total galactooligosaccharides; SGOS, selectivityf lactose conversion into galactooligosaccharides; PIGOS, profile index of galac-ooligosaccharides; PE, process efficiency; C, consumption yield of lactose; U, lossf enzymatic activity in the synthesis reaction (calculated as units of activity).∗ Corresponding author. Tel.: +44 0 118 378 8216; fax: +44 0 118 931 0080.

E-mail address: d.charalampopoulos@reading.ac.uk (D. Charalampopoulos).

168-1656/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.jbiotec.2010.08.008

.© 2010 Elsevier B.V. All rights reserved.

oligomers of galactose linked to a terminal end of glucose or galac-tose. A number of positive health effects are associated with theirconsumption. These vary, and can include enhancing calcium andmagnesium absorption (Chonan et al., 1996; Van den Heuvel et al.,2000), assisting in the inhibition of the attachment of pathogenicbacteria to the colonic epithelium (Sinclair et al., 2009; Tzortziset al., 2005b), having potential as a therapeutic agent in irritablebowel syndrome (IBS) (Silk et al., 2009), preventing the incidenceand symptoms of travelers’ diarrhea (Drakoularakou et al., 2010),and stimulating the immune system (Vulevic et al., 2008).

GOS are produced by transgalactosylation reactions catalysed by�-galactosidases (EC 3.2.1.23) using lactose as a substrate. Duringlactose hydrolysis, �-galactosidase cleaves the �-(1 → 4) glycosidiclinkage between galactose and glucose; this leads to the releaseof glucose into the reaction medium. Subsequently, the enzymetransfers the galactosyl moiety into acceptor molecules that containhydroxyl groups. When the acceptor is water, galactose is formedand the pathway is referred to as hydrolysis. The transfer of thegalactosyl moiety to an acceptor molecule containing a hydroxylgroup other than water is known as transgalactosylation and is

observed under conditions of high lactose concentration. A draw-back of such transgalactosylation reactions is the competition withhydrolysis, which depends on the ratio of the transferase activity tothe hydrolytic activity of the enzyme. This is affected by the origin ofthe enzyme, the relative concentration of the galactosyl acceptors

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A. Osman et al. / Journal of B

n the reaction medium, and the conditions (e.g., temperature, time,nd pH) (Boon et al., 2000; Mahoney, 1998; Martinez-Villaluengat al., 2008). This type of transgalactosylation is known as indirectransgalactosylation. The other type, direct transgalactosylation,eads to the formation of isomers of lactose, such as allolactose,-d-galactopyranosyl (1 → 6)-d-glucose. This isomer is formed by

he internal transfer of the galactose moiety from the position 4 tohe position 6 of the glucose, with no release of glucose from thective site, which results in a change in the glycosidic linkage from-(1 → 4) found in lactose into �-(1 → 6) (Huber et al., 1976).

�-Galactosidases of microbial origin, especially those frompecies belonging to Kluyveromyces species (Kim et al., 2001;artinez-Villaluenga et al., 2008), Aspergillus species (Leiva anduzman, 1995; Iwasaki et al., 1996), and Bacillus species (Boon etl., 1999; Mozaffar et al., 1984), have been extensively used for GOSynthesis. Of great interest is the use of �-galactosidases found inifidobacteriium species for GOS production. The rationale for these of bifidobacterial enzymes for GOS synthesis is that GOS mix-ures produced using bifidobacterial �-galactosidases might haveetter selectivity for bifidobacteria in the gut, and thus the growthnd/or the activity of these beneficial bacteria in the gut is moreikely to be supported (Depeint et al., 2008; Rabiu et al., 2001). Inhis context, a novel mixture of GOS produced by the enzymaticctivity of Bifidobacterium bifidum NCIMB 41171 resulted in a sig-ificant increase in the bifidobacterial population ratio in the gut ofealthy humans (Depeint et al., 2008). The GOS mixture was pro-uced in a preliminary study with a yield of up to 20% (w/w) GOS perotal carbohydrates and consisted of (in (w/w)): 25% disaccharides,5% trisaccharides, 25% tetrasaccharides, and 15% pentasaccharidesTzortzis et al., 2005a). Subsequently, Goulas et al. (2007a), aftermproving the process in terms of the buffer used and the initialactose concentration, reported a GOS yield of 43% (w/w) using theame biocatalyst. The GOS mixture consisted mainly of transgalac-osylated disaccharides, trisaccharides, and to a lesser extent higheralactooligosaccharides, with mainly �-d-(1 → 3), but �-d-(1 → 4)nd �-d-(1 → 6) glycosidic linkages as well (Depeint et al., 2008;zortzis et al., 2005b). However, studies on the effect of the operat-ng conditions on the production of GOS constituents, the relativeomposition of the GOS mixture, and the selectivity of lactose con-ersion into GOS have not been reported yet. These characteristicsre of vital importance for large scale GOS production, as they affecthe process efficiency and the relative product composition, which

ight affect the GOS efficacy.This work investigates the synthesis of GOS by whole cells of B.

ifidum NCIMB 41171 through the development of mathematicalodels that describe a variety of responses related to the process

fficiency and the relative product composition as a function of themount of biocatalyst, temperature, the initial lactose concentra-ion, and time.

. Materials and methods

.1. Materials

All chemicals were purchased from Sigma (Dorset, UK) andere of the highest purity unless otherwise stated. Freeze dried B.

ifidum NCIMB 41171 cells were provided by Clasado Inc. (Jersey,K). Each gram of freeze dried cells had a �-galactosidase activityf 1877.7 ± 12.9 units, calculated following the assay described inection 2.4.

.2. Experimental design and modeling

GOS were synthesized batch-wise in 0.05 M sodium phosphateuffer solution with a pH of 6.8 ± 0.1 and operated at 150 rpm in

nology 150 (2010) 140–148 141

Duran bottles (500 ml). A full factorial design was conducted study-ing the effect of the amount of biocatalyst, temperature, initiallactose concentration, and time on GOS synthesis. The levels foreach factor that were included in the design were: 0.5, 0.75, and1.0 g per 100 g of synthesis solution for the amount of biocatalyst,40, 45, 50, 55, and 60 ◦C for temperature, 35, 39, 43, 47, and 51%(w/w) for the initial lactose concentration, and 4, 8, 12, 16, 20, 24,28, 32, and 36 h for time. Fifty-one test runs were conducted in total,which amounted to 459 experimental points. Multiple regressionanalysis was performed in order to fit a second order polynomialequation described below to the data:

Y = ˇ0 + ˇ0X1 + ˇ2X2 + ˇ3X3 + ˇ4X4 + ˇ11X21 + ˇ22X2

2 + ˇ33X23

+ ˇ44X24 + ˇ12X1X2 + ˇ13X1X3 + ˇ14X1X4 + ˇ23X2X3

+ ˇ24X2X4 + ˇ34X3X4

where Y is the dependent response (variable), ˇ0, ˇ1, ˇ2, ˇ3,. . ., ˇ34are the regression coefficients, and X1, X2, X3, X4 are the amountof biocatalyst, temperature, initial lactose concentration, and time,respectively.

The following responses were measured: production yield ofallolactose, YAL; production yield of other transgalactosylated dis-accharides, YO.T.DP2; production yield of all transgalactosylated dis-accharides, YT.DP2; production yield of tri-galactooligosaccharides,YDP3; production yield of tetra-galactooligosaccharides, YDP4; pro-duction yield of penta-galactooligosaccharides, YDP5; productionyield of galactooligosaccharides with DP ≥ 3 (DP: degree of poly-merisation), YGOS ≥ 3; production yield of galactooligosaccharideswith DP ≥ 4, YDP ≥ 4; production yield of total galactooligosac-charides, YGOS; purity yield of total galactooligosaccharides, YP;selectivity of lactose conversion into galactooligosaccharides, SGOS;profile index of galactooligosaccharides, PIGOS; process efficiency,PE; and consumption yield of lactose, C. The above responses weremathematically expressed using the following equations:

YAL = [AL][Lac]

× 100 (1)

YO.T.DP2 = [O.T.DP2][Lac]

× 100 (2)

YT.DP2 = [T.DP2][Lac]

× 100 (3)

YDP3 = 2[DP3][Lac]

× 100 (4)

YDP4 = 3[DP4][Lac]

× 100 (5)

YDP5 = 4[DP5][Lac]

× 100 (6)

YGOS≥3 = YDP3 + YGOS≥4 (7)

YGOS≥4 = YDP4 + YDP5 (8)

YGOS = YT.DP2 + YDP3 + YDP≥4 (9)

Yp = GOS produced (g)total carbohydrates (g)

× 100 (10)

SGOS = ([T.DP2] + 2[DP3] + 3[DP4] + 4[DP5])([T.DP2] + 2[DP3] + 3[DP4] + 4[DP5] + [GAL])

× 100 (11)

PIGOS = (YDP3 + YGOS≥4)YT.DP2 + YDP3 + YGOS≥4

× 100 (12)

PE = YGOSSGOSC

U(13)

C = [Lac]c

[Lac]× 100 (14)

1 iotechnology 150 (2010) 140–148

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42 A. Osman et al. / Journal of B

here [Lac] is the molar concentration of initial lactose, [T.DP2]s the molar concentration of all transgalactosylated disaccharides,AL] is the molar concentration of allolactose, [O.T.DP2] is the molaroncentration of other transgalactosylated disaccharides, [DP3] ishe molar concentration of tri-galactooligosaccharides, [DP4] is the

olar concentration of tetra-galactooligosaccharides, [DP5] is theolar concentration of penta-galactooligosaccharides, [Gal] is theolar concentration of galactose, [Lac]c is the molar concentration

f lactose catalysed in the reaction, and U is the enzymatic activitysed during synthesis. Total transgalactosylated disaccharides con-isted of allolactose and other transgalactosylated disaccharides. Ofhese responses, six models were principally developed, describingGOS, YGOS ≥ 3, YP, SGOS, PIGOS, and PE as a function of the four con-rolling factors. The rest of the responses were used to support the

ain findings.The Minitab statistical software (Release 15, State College, PA,

SA) was used to analyse the data and to calculate the effects ofach factor on the responses. The data were statistically treatedy analysis of variance (ANOVA). Three- and higher-order interac-ions were neglected. The mathematical models were validated byonducting a set of 20 independent experimental runs.

.3. Carbohydrate analysis

The carbohydrate composition of the synthesis reaction solu-ion was determined by high performance liquid chromatographyHPLC) using a system consisting of a G1322A degasser (Agilentechnologies, Cheshire, UK), a G1310A isocratic pump (Agi-ent Technologies, Cheshire, UK), and a Shodex RI-71 refractivendex detector (Kawasaki, Japan). Separation of carbohydrates waserformed using a Rezex RCM-Monosaccharide Ca2+ (8%) col-mn (300 mm × 7.8 mm) supplied by Phenomenex (Macclesfield,heshire, UK). The column was maintained at 84 ◦C; HPLC-gradeater was used as the mobile phase at a flow rate of 0.5 ml/min.uantitative determination of each peak was performed using

tandard calibration curves of maltopentaose, maltotetraose, iso-altotriose (Supelco, Bellefonte, PA, USA), lactose, glucose and

alactose, respectively.The carbohydrate composition of the reaction mixture was also

etermined by high performance anion exchange chromatogra-hy coupled with pulsed amperometric detector (HPAEC-PAD) inrder to quantify lactose, which was co-eluted with the trans-alactosylated disaccharides in the same peak using the HPLCethod. A Dionex system (Dionex corporation, Surrey, UK) con-

isting of a GS50 gradient pump, an ED50 electrochemical detectorith a gold working electrode, an LC25 chromatography oven,

nd an AS50 autosampler was used. Separation was performedsing a pellicular anion-exchange resin based column, CarboPacA-1 analytical (4 mm × 250 mm), connected to a CarboPac PA1uard (4 mm × 50 mm) (Dionex corporation, Surrey, UK). The col-mn was maintained at 25 ◦C; elution was performed at a flow ratef 1 ml/min using gradient concentrations of sodium hydroxide andodium acetate solutions. Under these conditions, allolactose wasetected and quantified using a standard calibration curve of allo-

actose (Carbosynth, Berkshire, UK). In addition, lactose was eluteds a separate peak from the other disaccharides, which allowedts quantification using a standard calibration curve. All chromato-raphic analyses were performed in duplicates.

.4. ˇ-Galactosidase activity

�-Galactosidase activity was measured during the synthesiseaction by withdrawing 0.5 ml from the synthesis solution andixing it with 0.5 ml of sodium phosphate buffer (0.05 M, pH 6.8).

he mixture was centrifuged at 8000 × g for 5 min. The supernatantrom the mixture was directly used to measure �-galactosidase

Fig. 1. Typical curve of GOS synthesis. This synthesis reaction was carried out at55 ◦C and 43% (w/w) initial lactose concentration using 1 g of biocatalyst per 100 gof synthesis solution.

activity after appropriate dilution with sodium phosphate buffer(0.05 M, pH 6.8). The cell pellet was re-suspended in 1 ml of thesame buffer and centrifuged under the above mentioned con-ditions. This step was repeated twice; the cell pellet was thenre-suspended in 15 ml of the phosphate buffer. The cell suspen-sion was then sonicated at 4 ◦C (twice for 30 s each time at 26amplitude microns) using a Soniprep 150 (SANYO Gallenkamp PLS,UK). The resultant sonicated cell solution was used to measure �-galactosidase activity. The total enzymatic activity was the sum ofthe activity found in the cells and in the supernatant.

The reaction mixture consisted of 250 �l of o-nitrophenol-ˇ-galactoside (o-NPG) (20 mM), 200 �l of sodium phosphate buffer(0.05 M, pH 6.8), and 10 �l of magnesium chloride (0.05 M). Thereaction was initiated by the addition of 40 �l of the sonicatedcell solution or the diluted supernatant solution, and incubatedat 40 ◦C for 15 min. The reaction was terminated by the additionof 500 �l of sodium carbonate (1 M). The absorbance was imme-diately measured at 420 nm against a suitable blank. One unit ofactivity was defined as the amount of enzyme that liberates 1 �molof o-nitrophenol (o-NP) per minute, under the above mentionedconditions. The molar extinction coefficient under these conditionswas 4616.6 M−1 cm−1. The protein concentration of the sampleswas measured using the Bradford method (Bradford, 1976) withbovine serum albumin (BSA) as a standard. All enzymatic assaymeasurements were carried out in triplicates.

3. Results

3.1. Characteristics of GOS synthesis curves

All synthesis reactions followed the typical curve depicted inFig. 1. This curve illustrates the hydrolysis of lactose with theconcomitant formation of a mixture of mono-, di, tri-, tetra-, andpenta-galactooligosaccharides. In the beginning of the reaction, theamount of lactose decreased considerably and was accompaniedby a significant increase in the formation of the products. As thereaction progressed, the lactose continued to decrease, while tri-,

tetra-, and penta-galactooligosaccharides showed a slow increaseuntil they leveled off, as a result of the balance between theirhydrolysis and synthesis driven by the thermodynamics of the reac-tion. Subsequently (in the case of Fig. 1 after approximately 16 h),they showed a slight decrease. The total transgalactosylated dis-

A. Osman et al. / Journal of Biotechnology 150 (2010) 140–148 143

Table 1Analysis of variance (ANOVA) for the responses: YGOS, YGOS ≥ 3, YP, and SGOS. DF and Adj MS stand for degrees of freedom and adjusted mean squares, respectively.

Source DF YGOS YGOS ≥ 3 YP SGOS

Adj MS F-value P Adj MS F-value P Adj MS F-value P Adj MS F-value P

Regression 14 6324.8 632.75 <0.001 2962.13 429.14 <0.001 3749.53 681.65 <0.001 7627.7 1571.15 <0.001Linear 4 13271.4 1327.71 <0.001 6703.48 971.18 <0.001 7665.22 1393.5 <0.001 19746.3 4067.31 <0.001Square 4 2253.3 225.42 <0.001 766.26 111.01 <0.001 1353.45 246.05 <0.001 316.9 65.28 <0.001Interaction 6 1015.3 101.57 <0.001 611.01 88.52 <0.001 560.48 101.89 <0.001 278 57.27 <0.001Residual error 458 10 6.9 5.5 4.9Lack of Fit 444 10.3 58.29 <0.001 7.12 99 <0.001 5.67 93.62 <0.001 5 6.3 <0.001Pure error 14 0.2 0.07 0.06 0.8Total 472

Table 2Analysis of variance (ANOVA) for the responses: PIGOS and PE. DF and Adj MS stand for degrees of freedom and adjusted mean squares, respectively.

Source DF PIGOS PE

Adj MS F-value P Adj MS F-value P

Regression 14 2369.52 222.89 <0.001 302915 248.58 <0.001Linear 4 5255.99 494.41 <0.001 497584 408.32 <0.001Square 4 211.77 19.92 <0.001 104305 85.59 <0.001Interaction 6 489.55 46.05 <0.001 82749 67.90 <0.001

anctmdrcpd

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Residual error 458 10.63Lack of Fit 444 10.94 13.83Pure error 14 0.79Total 472

ccharides followed a similar pattern as well; however, in a fewumber of runs, they did not reach the maximum during the timeourse of the reaction (36 h). The amounts of glucose and galac-ose increased continuously throughout the reaction since these

onosaccharides were the final hydrolytic products of all of thei-, tri-, tetra-, and penta-galactooligosaccharides present in theeaction medium. Overall, although the patterns of all synthesisurves were similar to that in Fig. 1, differences in the levels of theroduced galactooligosaccharides were observed, as a result of theifferent reaction conditions.

.2. Models development and statistical analysis

ANOVA analysis (Tables 1 and 2) of the quadratic regression

odels demonstrated that all models were significant (P < 0.001).ore specifically, the overall effect of the independent variables,

he squared terms of the variables, and the interaction terms wasignificant for all models. Moreover, the R2 values of all modelsere high, i.e., 0.950, 0.929, 0.954, 0.979, 0.882, and 0.884 for

able 3egression coefficients and the corresponding P values for the responses: YGOS, YGOS ≥ 3

emperature, initial lactose concentration, and time, respectively.

Term YGOS YGOS ≥ 3 YP

Coefficient P Coefficient P Coefficient P

Constant 50.222 <0.001* 30.507 <0.001* 40.682 <0.00X1 4.374 <0.001* 2.059 <0.001* 3.647 <0.00X2 14.432 <0.001* 9.192 <0.001* 11.165 <0.00X3 2.781 <0.001* 5.253 <0.001* 1.005 0.01X4 8.496 <0.001* 2.808 <0.001* 7.387 <0.00X2

1 −4.199 <0.001* −3.139 <0.001* −2.689 <0.00X2

2 0.298 0.461 0.043 0.898 0.04 0.89X2

3 −4.251 <0.001* −1.376 0.002* −3.763 <0.00X2

4 −10.06 <0.001* −5.494 <0.001* −8.052 <0.00X1X2 0.0185 0.949 0.488 0.041* −0.083 0.69X1X3 4.293 <0.001* 3.157 <0.001* 3.215 <0.00X1X4 −3.886 <0.001* −2.699 <0.001* −2.963 <0.00X2X3 −1.989 0.002* −2.162 <0.001* −1.459 0.00X2X4 1.1 0.003* 0.448 0.141 0.957 <0.00X3X4 4.878 <0.001* 4.235 <0.001* 3.44 <0.00

* Significant at the 95% level.

1219<0.001 1257 321.41 <0.001

4

YGOS, YGOS ≥ 3, YP, SGOS, PIGOS, and PE, respectively. Furthermore,the residual plots did not show any trend in the distribution ofthe residuals around the zero line, further confirming the goodnessof fit. Based on the regression coefficient estimates and the cor-responding Prob > F values shown in Table 3, it was deduced thatall the independent variables, i.e., the amount of biocatalyst, tem-perature, the initial lactose concentration, and time, significantlyaffected all models. Nearly all the squared terms of the variableshad also significant effects on all models, as shown in Table 3. Themajority of the interaction terms were also significant.

According to the estimated regression coefficients (Table 3),YGOS and YP were affected mainly by temperature, followed by time,the amount of biocatalyst, and lactose, respectively. This order waschanged for YGOS ≥ 3 and SGOS, where lactose was the second most

influential factor, followed by time and the amount of biocatalyst.In the case of PE, temperature was the main influential factor, fol-lowed by time, lactose, and the amount of biocatalyst. In contrast tothe above responses, PIGOS was primarily influenced by lactose, fol-lowed by time, the amount of biocatalyst and temperature (Table 3).

, YP, SGOS, PIGOS, and PE. X1, X2, X3, and X4 stand for amount of biocatalyst used,

SGOS PIGOS PE

Coefficient P Coefficient P Coefficient P

1* 62.858 <0.001* 61.077 <0.001* 258.296 <0.001*

1* −0.335 0.035* −2.241 <0.001* −17.072 <0.001*

1* 15.433 <0.001* 1.394 0.003* 92.968 <0.001*

8* 10.49 <0.001* 10.361 <0.001* 24.275 <0.001*

1* −2.704 <0.001* −7.595 <0.001* 33.075 <0.001*

1* −3.056 <0.001* −0.699 0.028* −29.796 <0.001*

4 −1.316 <0.001* −1.8052 <0.001* 5.139 0.2511* −2.103 <0.001* 2.091 <0.001* −50.599 <0.001*

1* −0.474 0.089 2.626 <0.001* −59.822 <0.001*

6 −0.868 <0.001* 1.438 <0.001* −20.879 <0.001*

1* 0.901 <0.001* 1.051 0.002* 33.584 <0.001*

1* −1.997 <0.001* −0.547 0.058 −42.987 <0.001*

2* −3.411 <0.001* −6.113 <0.001* −14.445 0.038*

1* 0.83 0.001* 2.133 <0.001* 20.577 <0.001*

1* 2.825 <0.001* 2.877 <0.001* 30.196 <0.001*

144 A. Osman et al. / Journal of Biotechnology 150 (2010) 140–148

Table 4Final equations of YGOS, YGOS ≥ 3, YP, SGOS, PIGOS and PE, based on the uncoded values of the four controlling factors. X1, X2, X3, and X4 stand for the amount of biocatalyst (g/100 gof the synthesis solution), temperature, initial lactose concentration, and time, respectively.

Model name Equation

YGOS YGOS = −188.394 + 45.038X1 + 2.07113X2 + 493207X3 + 0.848662X4 − 67.1863X21 + 0.00298425X2

2 − 0.0664278X23 − 0.039298X2

4 +0.00773849X1X2 + 2.14639X1X3 − 0.971364X1X4 − 0.0248738X2X3 + 0.00688159X2X4 + 0.0381096X3X4

YGOS≥3 YGOS≥3 = 3 = −107.656 + 19.452 X1 + 1.836 X2 + 2.011 X3 − 0.023 X4 − 50.233 X21 + 0.0004 X2

2 − 0.0215 X23 − 0.022 X2

4 + 0.195 X1X2 +1.578 X1X3 − 0.675 X1X4 − 0.027 X2X3 + 0.00279 X2X4 + 0.0331 X3X4

YP YP = −155.813 + 26.4966 X1 + 1.76567 X2 + 4.34986 X3 + 0.819514 X4 − 43.0279 X21 + 0.000401734 X2

2 − 0.0587929 X23 −

0.0314520 X24 − 0.0332784 X1X2 + 1.60735 X1X3 − 0.740856 X1X4 − 0.0182334 X2X3 + 0.00598091 X2X4 + 0.0269029 X3X4

SGOS SGOS = −261.691 + 79.976 X1 + 4.8496 X2 + 5.48961 X3 − 0.929063 X4 − 48.8926 X21 − 0.0131634 X2

2 − 0.0328595 X23 − 0.0018505 X2

4 −0.347196 X1X2 + 0.450424 X1X3 − 0.499284 X1X4 − 0.0426343 X2X3 + 0.00518741 X2X4 + 0.0220735 X3X4

PIGOS PIGOS = −67.4584 − 40.7894 X1 + 4.53245 X2 + 1.46295 X3 − 2.41532 X4 − 11.1989 X21 − 0.0180522 X2

2 + 0.0326694 X23 +

0.1333 X3

80569

TP

3

cPwtrswttTttca

3

Ymeo

TRt

0.01025579 X24 + 0.575316 X1X2 + 0.525363 X1X3 −

PE PE = −2011.74 + 557.293 X1 + 15.6137 X2 + 62.748.35175 X1X2 + 16.7920 X1X3 − 10.7467 X1X4 − 0.1

able 4 shows the final equations of YGOS, YGOS≥3, YP, SGOS, PIGOS andE based on the non coded values of the four controlling factors.

.3. The effect of the amount of biocatalyst

As indicated by the regression coefficients, the amount of bio-atalyst positively affected YGOS, YGOS ≥ 3, and YP, whereas SGOS,IGOS, and PE were negatively affected (Table 3). All these effectsere initially linear but became less obvious as the amount of

he biocatalyst increased. Increasing the amount of the biocatalystesulted in an increase in YT.DP2 compared to YGOS ≥ 3, as demon-trated by the fact that the regression coefficient of the biocatalystas higher in the case of YT.DP2 than YGOS ≥ 3 (Tables 3 and 5); this led

o lower PIGOS values. Transgalactosylated disaccharides were par-ially produced from the hydrolysis of tri-galactooligosaccharides.his was accompanied with the release of galactose into the reac-ion medium, which resulted in lower SGOS values as the amount ofhe biocatalyst increased. PE also decreased as the amount of bio-atalyst increased, due to the fact that PE is proportional to SGOS,nd inversely proportional to U (Eq. (13)).

.4. The effect of temperature

Temperature was the most important factor influencing YGOS,P, YGOS ≥ 3, SGOS, and PE, with the exception of PIGOS, which wasainly affected by the initial lactose concentration (Table 3). The

ffects of temperature were primarily linear and positive. Thenly significant quadratic effects of temperature were noticed for

able 5egression coefficients and the corresponding P values for the responses: YDP ≥ 4, YDP3,emperature, initial lactose concentration, and time, respectively.

Term YDP ≥ 4 YDP3 YAL

Coefficient P Coefficient P Coeffici

Constant 7.783 <0.001* 22.725 <0.001* 6.884X1 1.224 <0.001* 0.833 <0.001* 0.636X2 3.291 <0.001* 5.901 <0.001* 2.797X3 1.424 <0.001* 3.829 <0.001* −1.885X4 1.328 <0.001* 1.479 <0.001* 2.86X2

1 −1.269 <0.001* −1.869 <0.001* 0.166X2

2 0.472 0.001* −0.429 0.043* 2.487X2

3 −0.468 0.013* −0.909 0.001 −1.147X2

4 −1.968 <0.001* −3.528 <0.001* −2.629X1X2 0.506 <0.001* −0.0164 0.913 0.198X1X3 1.434 <0.001* 1.722 <0.001* 0.12X1X4 −0.738 <0.001* −1.961 <0.001* −0.671X2X3 −0.598 0.007* −1.563 <0.001* −0.719X2X4 0.352 0.006* 0.0964 0.613 0.823X3X4 1.475 <0.001* 2.761 <0.001* 0.09

* Significant at the 95% level.

6635 X1X4 − 0.0764176 X2X3 + 0.0133293 X2X4 + 0.0224723 X3X4

+ 2.90015 X4 − 476.743 X21 + 0.0513929 X2

2 − 0.790614 X23 − 0.233681 X2

4 −X2X3 + 0.128604 X2X4 + 0.235910 X3X4

SGOS and PIGOS. In both cases, after certain temperatures (generally≥55 ◦C), the increases in the responses leveled off.

3.5. The effect of the initial lactose concentration

All the models were initially affected by lactose concentrationlinearly and positively. Increasing the initial lactose concentrationfavored the formation of GOS with DP ≥ 3 over transgalactosylateddisaccharides (Table 3). To further support this finding, YAL, YO.T.DP2,YDP3, and YDP ≥ 4 were studied as a function of the four controllingfactors. The regression analysis showed that both YAL and YO.T.DP2were negatively influenced by increasing the initial lactose concen-tration, while YDP3 and YDP ≥ 4 were positively affected (Table 5).For this reason, PIGOS increased as the initial lactose concentrationincreased. Transgalactosylation was also favored over hydrolysiswhen the initial lactose concentration increased, as demonstratedby the SGOS model (Table 3). Since YGOS and SGOS were positivelyaffected by the initial lactose concentration, PE was also positivelyinfluenced.

The initial lactose concentration had also significant quadraticeffects on all models. These were mostly negative, indicating thatthe positive effects of this factor decreased, in most cases, whenlactose increased above certain levels (usually >43–47% (w/w)).

3.6. The effect of time

Time influenced YGOS, YP, YGOS ≥ 3, and PE positively as it isrequired for the synthesis of considerable amounts of GOS, whereas

YAL, YO.T.DP2, and YT.DP2. X1, X2, X3, and X4 stand for amount of biocatalyst used,

YO.T.DP2 YT.DP2

ent P Coefficient P Coefficient P

<0.001* 12.663 <0.001* 19.707 <0.001*

<0.001* 1.588 <0.001* 2.324 <0.001*

<0.001* 2.653 <0.001* 5.24 <0.001*

<0.001* −0.797 0.003* −2.469 <0.001*

<0.001* 2.962 <0.001* 5.691 <0.001*

0.025* −1.376 <0.001* −1.066 <0.001*

<0.001* −2.136 <0.001* 0.259 0.192<0.001* −1.732 <0.001* −2.868 <0.001*

<0.001* −2.214 <0.001* −4.556 <0.001*

0.004* −0.568 <0.001* −0.479 0.001*

0.134 0.891 <0.001* 1.137 <0.001*

0.316 −1.031 <0.001* −1.189 <0.001*

<0.001* 1.027 <0.001* 0.169 0.583<0.001* −0.154 0.361 0.649 <0.001*

0.376 0.644 0.001* 0.643 0.002*

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GOS and PIGOS were negatively influenced (Table 3). In most cases,s the synthesis progressed, SGOS and PIGOS decreased (P < 0.05). Theecrease in SGOS was higher at low temperatures and low initial

actose concentrations, indicating a preference towards hydrolysiss the synthesis progressed under such conditions. In the case ofynthesis reactions at high temperatures and high lactose concen-rations (usually ≥43% (w/w) lactose and >50 ◦C), SGOS was foundo be fairly stable until a certain time point (between 12 and 20 h),fter which it decreased slightly. The decrease indicated that theormed galactooligosaccharides started to hydrolyse at a fasterate than they were formed. The decrease in PIGOS as the reac-ion progressed was probably due to the fact that the formationf tri-, tetra-, and penta-galactooligosaccharides was accompaniedy their hydrolysis, which took over their synthesis at later stagesf the reaction.

Time had mainly linear effects on all models at the early stages ofhe reactions, usually up to 4–8 h. Then, apparent quadratic effectsere observed as the reactions progressed towards the balance

tate between GOS synthesis and hydrolysis. A decrease in mostesponses was observed at later stages of the synthesis reactions.

.7. The effect of factors interactions

In most cases, the interactions between the four factors affectedhe models significantly (Table 3). The interaction term of themount of biocatalyst and time negatively influenced all models.ikewise, the interaction term of temperature and the initial lac-ose concentration negatively affected all models. The rest of thenteraction terms showed mainly positive effects on all models.

.8. Model validation

In order to validate the models, 20 synthesis reactions wereonducted under various conditions within the range of the val-es that were used to generate the models. Table 6 presents theifferent conditions used along with the observed and predictedalues for the responses. Linear regression between the observednd the predicted values indicated that the models had a good pre-ictive ability, as the regression coefficients, (R2), were in all casesigh (>0.90). In addition, t-test analysis showed that both sets of theredicted and the observed values were not significantly differentP > 0.05) (Table 6).

. Discussion

In the presence of lactose, �-galactosidases are involved in sev-ral types of reactions that occur simultaneously. These include theydrolysis of lactose, the direct and the indirect transgalactosyla-ion, which leads to the formation of galactooligosaccharides, andhe hydrolysis of the formed galactooligosaccharides (Mahoney,998). The complexity of such a system was even more pronounced

n this study, as a whole cell biocatalyst was used, i.e., B. bifidumCIMB 41171. The aim of this study was to investigate the effectsf production conditions on several types of responses relatedo the production of GOS, through the development of a set of

ultivariate mathematical models. The reason for selecting fourndependent variables, namely lactose concentration, temperature,ell biomass and time, was to develop a comprehensive model thatakes into account the independent and the interactions effects ofhese variables on the GOS yields, and equally importantly, on the

OS profiles.

It has been previously shown that lactose concentration is theain factor influencing transgalactosylation, and that the higher

he lactose concentration the higher the production of GOS (Boont al., 2000; Cho et al., 2003; Monsan and Paul, 1995; Rabiu et al.,

nology 150 (2010) 140–148 145

2001). This work confirmed the importance of having high ini-tial lactose concentration for achieving high yields of GOS usingB. bifidum NCIMB 41171 as the biocatalyst. However, the initiallactose concentration was less important in its influence on YGOS,YP, YGOS ≥ 3, SGOS, and PE compared to temperature. One likely rea-son for this is the very high lactose concentrations included in theexperimental design, ranging from 35 to 51% (w/w). It is likelythat the effect of lactose concentration would have been more pro-nounced if the design considered a wider range of concentrations,starting well below 35% (w/w), taking into account the fact that theresponses increased linearly between 35 and 43% (w/w) lactose,and then leveled off. The effect of lactose was also more pronouncedon YGOS ≥ 3 compared to YGOS and YP, as both YGOS and YP modelscontain YAL and YO.T.DP2, which were negatively influenced by theinitial lactose concentration. The positive effect of lactose on SGOSwas most likely attributed to the fact that high initial lactose con-centration ensured that high concentrations of donor and acceptormolecules of the galactosyl moieties were available in the reac-tion medium. This decreased the availability of water molecules asacceptors of the galactosyl moieties, and hence less hydrolysis tookplace.

Temperature is a very important factor from an industrial pointof view as it influences the solubility of lactose in the reactionmedium, besides its effect on the thermodynamics of the enzy-matic reactions taking place. In this study, temperature was themain factor influencing YGOS, YP, YGOS ≥ 3, SGOS, and PE, instead oflactose. This was expected to a degree, as temperature controls thekinetics of enzyme catalysed reactions. However, the continuousincrease in these responses as temperature increased was mostlikely due to the type of the biocatalyst used in this study. The B.bifidum NCIMB 41171 cells consist of four �-galactosidases, namely,BbgI, BbgII, BbgIII and BbgIV (Goulas et al., 2007b). Previous stud-ies showed that BbgIV has the highest ability to convert lactoseinto GOS, followed by BbgI and BbgIII. On the other hand, BbgII hadmainly hydrolytic rather than transgalactosylation activity (Goulaset al., 2009). The optimum temperature of the activity of BbgIV wasfound to be 50 ◦C, while the other �-galactosidases had an optimumtemperature of 40 ◦C. Moreover, BbgIII and BbgII were more suscep-tible to high temperatures (>50 ◦C for BbgIII and >45 ◦C for BbgII)compared to BbgI and BbgIV (Goulas et al., 2008). The above suggestthat increasing the temperature most likely favored the activity ofBbgIV mainly and to a lesser extent BbgI, at the expense of the othertwo enzymes. Thus, transgalactosylation was essentially enhanced,and for this reason the aforementioned responses increased as thetemperature increased. The effect of temperature on GOS synthesishas also been investigated by other researchers. Cruz et al. (1999),using a �-galactosidase from Penicillium simplicissimum, found thatthe best production of GOS was at 50 ◦C, whereas reactions carriedout at 55 ◦C resulted in a rapid inactivation of the enzyme and alower GOS production. Boon et al. (2000) reported slightly highertrisaccharide yields at higher temperatures, within the 20–50 ◦Crange; however, no effect on the yield of GOS ≥ 4 was found.Martinez-Villaluenga et al. (2008) found that 6′-galactosyllactose,a trisaccharide, was produced with higher yields at 40 ◦C comparedto 50 ◦C by a Kluyveromyces lactis �-galactosidase, whereas disac-charides like galactobiose and allolactose were best produced at50 ◦C. In the present study however, both transgalactosylated dis-accharides and tri-galactooligosaccharides were better producedas temperature increased. The difference between the above men-tioned studies and the present study is most likely due to thedifferent microbial origins of the enzymes and the fact that a whole

cell biocatalyst was used in this study rather than isolated enzymes.

The reason for using the amount of cells as an independentvariable rather than the total �-galactosidase activity is that, asmentioned previously, the cells contained four �-galactosidaseswith different properties, and therefore the biocatalytic process

146A

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al./JournalofBiotechnology150 (2010) 140–148

Table 6Conditions for validation experiments and experimental/predicted values of the models. YGOS, YGOS ≥ 3, YP, SGOS, PIGOS, and PE. X1, X2, X3, and X4 stand for the amount of biocatalyst (g/100 g of the synthesis solution), temperature,initial lactose concentration, and time, respectively. P and E stand for predicted and experimental values.

Run number Reaction parameters YP YGOS YGOS ≥ 3 SGOS PIGOS PE

X1 X2 X3 X4 P E P E P E P E P A P E

1 0.65 53 45 22 42.80 43.87 53.29 52.04 33.18 34.54 69.14 71.05 63.39 66.36 296.74 319.642 0.6 43 37 27 28.53 29.19 33.42 33.26 16.86 16.88 37.78 40.78 47.31 50.75 145.43 167.423 0.6 58 47.5 23 46.07 48.35 58.04 59.21 36.92 39.46 77.4 79.19 64.18 66.64 340.51 359.054 0.65 53 45 27 44.43 45.15 55.16 53.31 33.81 34.14 68.76 70.55 61.97 64.04 306.22 291.635 0.85 58 48.5 9.5 40.01 40.83 51.36 49.81 35.36 36.34 78.44 78.37 69.37 72.96 251.13 237.966 0.95 53 46 22 46.53 47.81 57.68 57.37 35.72 37.23 68.08 72.12 62.49 64.89 260.43 279.87 0.6 58 47.5 7 29.17 31.71 37.30 39.73 26.32 28.14 76.36 77.99 69.76 70.83 210.81 225.658 0.75 47 38 9.5 28.23 29.18 34.46 33.49 21.14 20.81 52.99 52.09 61.41 62.12 162.19 155.149 0.85 58 48.5 7 36.71 38.96 47.31 47.59 33.32 34.82 78.47 79.96 70.62 73.17 229.10 239.6410 0.6 58 47.5 9.5 32.88 34.62 41.87 41.87 28.69 30.40 76.58 76.45 68.54 72.62 238.96 216.7911 0.6 43 37 7 17.36 19.36 20.71 22.11 13.12 13.38 42.82 45.69 62.19 60.52 90.12 95.8712 0.85 58 48.5 23 51.07 51.99 64.78 62.79 41.75 42.50 77.87 80.11 64.84 67.69 319.59 337.2313 0.95 53 46 7 35.16 32.91 44.13 41.56 29.95 27.85 70.58 71.11 70.11 67.01 206.71 224.2614 0.7 47 41.5 27 37.73 37.17 45.84 42.78 26.22 23.49 54.36 53.51 55.54 54.90 227.55 210.0715 0.9 53 44 27 46.69 46.85 57.41 54.87 34.01 32.62 65.59 68.71 58.65 59.45 262.23 246.4516 0.9 53 44 7 35.01 33.09 43.81 40.26 29.12 27.47 69.49 69.43 68.53 68.23 212.64 229.417 0.7 47 41.5 9.5 28.09 27.95 34.65 32.47 22.25 20.92 57.62 57.19 65.65 64.43 180.6 160.418 0.6 43 37 22 28.09 28.36 33.19 31.21 17.54 16.62 39.18 41.22 50.26 53.24 149.13 169.3419 0.9 53 44 22 46.13 45.55 56.95 54.67 34.39 33.07 66.71 68.78 60.35 60.49 267.36 252.8620 0.95 53 46 27 47.17 47.39 58.28 55.77 35.49 34.95 67.06 70.63 60.98 62.67 254.97 239.01

R2 values 0.9792 0.9811 0.9697 0.9869 0.9065 0.9295t-Test (P values) 0.827 0.745 0.992 0.721 0.507 0.912

A. Osman et al. / Journal of Biotech

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ig. 2. PE and YGOS as a function of time during GOS synthesis. This synthesis reactionas carried out at 55 ◦C and 39% (w/w) initial lactose concentration using 1 g of

iocatalyst per 100 g of synthesis solution.

annot be compared to a process where a pure enzyme is used ashe biocatalyst. As expected, both time and the amount of biocata-yst showed mainly positive effects on YGOS, YGOS ≥ 3, and YP. On thether hand, responses that measure quality characteristics of theeactions, such as SGOS and PIGOS, were negatively affected by timend the amount of biocatalyst. Time, in particular, was a criticalactor, as a balanced state between GOS synthesis and hydrolysisas roughly attained in most cases between 12 and 20 h, followed

y more pronounced GOS hydrolysis than synthesis. Therefore, arocess efficiency term (PE), able to identify the best time point totop the reactions, was devised, taking into account that such a timeoint guaranteed a high GOS yield, a high lactose consumption, andt the same time a high selectivity of its conversion into GOS, withinimal losses in enzymatic activity. Fig. 2 shows an example of

he best time point of a particular GOS synthesis reaction based onhe PE value rather than the GOS yield. As observed, based on PE,he synthesis should be stopped approximately at 12 h rather thant 16 h, the time point corresponding to the maximum YGOS.

Of special interest is the PIGOS model, which measured the vari-tions in the composition of the produced GOS mixture as theeaction conditions varied, as changes in the relative composition ofhe produced GOS mixture might affect its efficacy. From the model,t was shown that high ratios of GOS ≥ 3 to total GOS were producedt high initial lactose concentration, as well as in early stages of theynthesis reactions. This feature is important, taking into accounthat in vitro fermentation studies indicated that �-linked di- andri-galactooligosaccharides were the main bifidogenic componentsf this GOS mixture (Goulas et al., 2009). The presence of four �-alactosidases in the biocatalyst used in the present study was mostikely responsible for the differences observed in PIGOS when theeaction conditions were changed, as these �-galactosidases hadifferent properties. Different �-galactosidases, in this regard, haveeen shown to produce different profiles of galactooligosaccharidesBoon et al., 2000; Chockchaisawasdee et al., 2005; Iwasaki et al.,996; Mozaffar et al., 1984).

. Conclusions

The set of mathematical models that was developed describ-ng the synthesis of GOS by whole cells of B. bifidum NCIMB 41171ndicated that all factors studied, i.e., temperature, lactose con-entration, amount of biocatalyst and time were significant, withhe former being the most important one. Among the various

esponses, the relative composition of the produced GOS mixtureas of critical importance, since it changed significantly as the syn-

hesis conditions changed, suggesting that the efficacy of GOS couldlso be affected. As a balanced state between GOS synthesis andydrolysis was always attained at some points in the reaction, time

nology 150 (2010) 140–148 147

was critically looked at, and for this reason a process efficiency termwas devised to be able to select the most appropriate time point tostop the reaction.

Acknowledgments

The authors would like to thank EPSRC and Clasado Ltd. forfunding this project.

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