The effect of production parameters on the synthesis of the prebiotic trisaccharide, neokestose, by Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

Enzyme and Microbial Technology 32 (2003) 728–737

The effect of production parameters on the synthesis of the prebiotic trisaccharide, neokestose, by Xanthophyllomyces dendrorhous (Phaffia rhodozyma) S.M. Kritzinger a , S.G. Kilian a,∗ , M.A. Potgieter b , J.C. du Preez a a

Department of Microbiology and Biochemistry, University of the Free State, P.O. Box 339, Bloemfontein 9300, South Africa b FARMOVS-PAREXEL, Private Bag X09, Brandhof 9324, Bloemfontein, South Africa Received 5 July 2002; received in revised form 30 January 2003; accepted 6 February 2003

Abstract The effects of important process parameters on the production of the novel prebiotic trisaccharide neokestose from sucrose by Xanthophyllomyces dendrorhous cells suspended in buffer were investigated. Recycling of cells reduced the level and rate of neokestose production while cells harvested in the exponential growth phase produced neokestose more efficiently than stationary phase cells. A further investigation into neokestose production was carried out using a fractional factorial statistical design. The neokestose produced was hydrolyzed following cessation of sucrose conversion at 31 g l−1 initial substrate concentration but not at 110 g l−1 initial sucrose. Statistical analysis of the data showed that sucrose concentration had a large positive effect on the maximum concentration of neokestose produced, the maximum specific rate of neokestose production and the maximum yield coefficient of neokestose. Surprisingly, increased cell concentration decreased the maximum specific rate of product formation. Strong interactions involving pH and temperature were identified. Curvature plots indicated that the optima for the maximum specific rate of neokestose production and for maximum yield coefficient were within the experimental range, whereas the optimum conditions for the maximum concentration of neokestose were outside this range. This is the first report on the production of a prebiotic oligosaccharide of the 6 G series in high yields. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Phaffia; Xanthophyllomyces; Neokestose; Optimization; Bifidogenic; Prebiotic; Oligosaccharide; Trisaccharide; Transfructosylation

1. Introduction Simple oligosaccharides are not obvious candidates as health promoting agents but evidence indicating that some of these compounds can indeed improve general health and provide resistance to infection in humans and animals is accumulating [1–3]. The basis of many of these observations is the differential stimulation of the growth of beneficial bacteria, especially Bifidobacterium and Lactobacillus species, by prebiotic oligosaccharides [4]. Members of the beneficial group produce enzymes that break down and ferment the oligosaccharides to short chain fatty acids and lactic acid in the colon, whereas potential harmful bacteria do not [5]. Commercial production of food grade oligosaccharides is growing and 85,000 t was produced in 1995 [6]. Oligosaccharides obtained by transfructosylation from sucrose feature prominently as prebiotic compounds and are produced commercially for inclusion in some food∗

Corresponding author. Tel.: +27-51-4012780; fax: +27-51-4443219. E-mail address: [email protected] (S.G. Kilian).

stuffs and animal feeds [2,4]. These oligosaccharides can be produced from sucrose using either free or immobilized microbial cells or enzymes [1,2,6–11]. Fructooligosaccharides (FOS) of the 1 F (inulin) type have been most extensively investigated as prebiotic compounds. The production of 6 F type oligosaccharides from sucrose has also received some attention [12,13] but so far production of potential prebiotic oligosaccharides of the 6 G series, such as the trisaccharide neokestose, has not been investigated. The probable reason is that neokestose occurs only as a minor transfructosylation product of whole cells or enzymes from various plants, Saccharomyces cerevisiae [7,15] and some filamentous fungi [14,16–18]. Previous studies showed that cultures of the astaxanthin-producing yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) accumulated neokestose as a major transfructosylation product when growing on sucrose [19]. Subsequent investigations with both pure cultures and batch cultures using human feces as inoculum have demonstrated that neokestose has prebiotic effects that surpass those of a commercial fructooligosaccharide [20]. The yeast also produces neokestose

0141-0229/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0141-0229(03)00035-8

S.M. Kritzinger et al. / Enzyme and Microbial Technology 32 (2003) 728–737

when suspended in buffer, eliminating the need for purified enzymes. It seemed, therefore, worthwhile to investigate the effect of process variables on the parameters of production of neokestose by X. dendrorhous to establish its potential for large-scale production. The effect of the physiological condition of the culture at harvesting was investigated by comparing the efficiency of exponentially growing and stationary cells to convert sucrose to neokestose. The effect of cell recycling to improve the economy of sucrose conversion into neokestose in a batch conversion process was also investigated. A statistical experimental design was used to determine the effects of pH, temperature, as well as sucrose and cell concentrations, on production parameters. Depending on the relative contribution of different components towards the production cost, different production parameters become important. Three important responses were, therefore, measured: the maximum concentration of neokestose produced, the maximum yield coefficient and the maximum specific rate of production.

729

aseptically harvested and resuspended in a medium of the same composition as in the first cycle. This procedure was repeated twice for a total of three cycles. For the comparison of exponential and stationary phase cells, 0.6 g l−1 cells harvested from either the exponential or stationary phase were resuspended in shake flasks containing citrate-phosphate buffer (pH 6.55) and incubated at 30 ◦ C for 24 h. Samples were periodically withdrawn to monitor substrate conversion and product formation. For the investigation of neokestose production using the experimental design, cells were harvested aseptically in the exponential phase. These cells were resuspended in 500 ml shake flasks containing 100 ml citrate-phosphate buffer with sucrose as substrate and incubated according to the experimental design (Table 1). 2.2. Experimental design Experiments were set up according to a two-level Resolution IV fractional factorial design [21] for the variables sucrose concentration, cell concentration, pH and temperature. Intermediate values for these variables were used as center points in triplicate experiments to provide a better estimate of the experimental error (Table 1). The responses: maximum concentration of neokestose produced, maximum yield coefficient of neokestose and maximum specific rate of neokestose production were measured. This design allowed independent estimates of all the main effects without confounding by each other or by two-factor interactions, but two-factor interactions were partially confounded. Interaction plots were used to determine which interactions were most likely involved in an observed response. Statistical analysis was done using the SAS® software package (V 6.12, Cary, NC).

2. Materials and methods 2.1. Microorganism and cultivation X. dendrorhous UOFS Y-0175 was maintained on Yeast Malt (YM) agar containing (per l): 10 g sucrose, 5 g peptone, 3 g yeast extract, 3 g malt extract and 17 g agar. An inoculum was prepared by inoculating 500 ml shake flasks containing 100 ml YM medium containing 50 g l−1 sucrose as substrate from a fresh slant followed by incubation for 36–40 h at 21 ◦ C. Cultivation was stopped in either the exponential or stationary growth phase and the cells were harvested aseptically. For determination of the effect of recycling, cells were resuspended in 500 ml shake flasks containing 100 ml citrate-phosphate buffer (pH 6.55) and 50 g l−1 sucrose to a final cell concentration of 0.9 g l−1 . The flasks were incubated on an orbital shaker for 12 h at 30 ◦ C and the cells were

2.3. Analytical methods Culture turbidity was monitored with a Klett-Summerson colorimeter at 640 nm. Dry biomass was determined gravimetrically in triplicate. Sugars were analyzed by HPLC

Table 1 Factorial design for the determination of the effects of the variables sucrose concentration, cell concentration, pH and temperature on the production of neokestose from sucrose by X. dendrorhous Run

Sucrose concentration (g l−1 )

Cell concentration (g l−1 )

pH

Temperature (◦ C)

Maximum specific rate of production (g g−1 h−1 )

Maximum concentration of neokestose produced (g l−1 )

Yield coefficient for neokestose production (g g−1 )

1 2 3 4 5 6 7 8 9 10 11

31 31 31 31 110 110 110 110 70 70 70

5.1 5.1 20.5 20.5 5.1 5.1 20.5 20.5 11.3 11.3 11.3

4 7 4 7 4 7 4 7 5.5 5.5 5.5

25 40 40 25 40 25 25 40 33 33 33

1.59 1.12 0.32 0.42 2.93 2.60 0.44 0.70 1.66 1.71 1.81

16.2 11.4 12.9 17.2 48.7 75.1 68.0 64.3 39.5 43.3 41.4

0.60 0.43 0.48 0.61 0.85 0.82 0.77 0.68 0.65 0.71 0.68

730

S.M. Kritzinger et al. / Enzyme and Microbial Technology 32 (2003) 728–737

using a Biorad Aminex Carbohydrate HPX-42C column. Degassed deionized water containing 0.05 M CaEDTA at 0.5 ml min−1 served as eluent, the column temperature was 85 ◦ C, a refractive index detector was used and 20 ␮l sample was injected. The approximate concentrations of neokestose and an unidentified oligosaccharide were determined using sucrose, which elutes close to neokestose, as standard. 3. Results 3.1. The effect of growth phase and cell recycling on neokestose production The maximum concentration of neokestose produced by cells suspended in citrate-phosphate buffer (pH 6.55) de-

creased after every 12 h cycle, as did the maximum specific rate of neokestose production (Table 2). Three consecutive cycles resulted in decreases of 56 and 63% in the maximum specific rate of neokestose production and the maximum concentration of neokestose produced, respectively (Table 2). The rate of sucrose conversion also declined with each recycling and the amount of sucrose converted decreased from 63% in the first run to about 5% in the final run after 12 h incubation (results not shown). Cells harvested in the exponential phase achieved a higher maximum specific rate of neokestose production, a higher concentration of neokestose and a higher yield coefficient than stationary phase (Table 3). Exponentially grown cells were, therefore, used for the further investigation of neokestose production.

Fig. 1. The production of neokestose from sucrose by cells suspended in citrate-phosphate buffer in shake flasks. (䊊) Neokestose; (䉲) sucrose; () glucose; (䊉) unidentified oligosaccharide. (A) 31 g l−1 sucrose, 5.1 g l−1 cells, pH 4, 25 ◦ C. (B) 31 g l−1 sucrose, 5.1 g l−1 cells, pH 7, 40 ◦ C. (C) 31 g l−1 sucrose, 20.5 g l−1 cells, pH 4, 40 ◦ C. (D) 31 g l−1 sucrose, 20.5 g l−1 cells, pH 7, 25 ◦ C. (E) 110 g l−1 sucrose, 5.1 g l−1 cells, pH 4, 40 ◦ C. (F) 110 g l−1 sucrose, 5.1 g l−1 cells, pH 7, 25 ◦ C. (G) 110 g l−1 sucrose, 20.5 g l−1 cells, pH 4, 25 ◦ C. (H) 110 g l−1 sucrose, 20.5 g l−1 cells, pH 7, 40 ◦ C.

S.M. Kritzinger et al. / Enzyme and Microbial Technology 32 (2003) 728–737 Table 2 The effect of cell recycling on the production of neokestose from sucrose by X. dendrorhous during 12 h incubation in citrate-phosphate buffer (pH 6.55) containing 50 g l−1 sucrose in shake flasks Production parameter

Runa 1

Maximum specific rate of neokestose production (g g−1 h−1 ) Maximum concentration of neokestose produced (g l−1 )

2 1.8 (0.02)

1.1 (0.08)

21.2 (0.04)

13.0 (0.04)

3 0.80 (0.004)

731

to the statistical model (results not shown). The standard deviations for maximum specific rate of neokestose production, maximum concentration of neokestose produced and maximum yield coefficient for neokestose production at the center points were less than 5%. This is well within acceptable regions of experimental error. Plots of observed versus predicted values were linear for the maximum specific

7.9 (0.01)

Values given are the means of triplicate experiments with standard deviations in brackets. a Runs 2 and 3 are first and second recycling, respectively.

3.2. The effect of process variables on the time course of neokestose production Substrate concentration had a major influence on the pattern of neokestose production. At 31 g l−1 initial sucrose concentration, neokestose peaked within 4 h and then decreased rapidly (Fig. 1A–D). In all these cases the glucose concentration remained below 5 g l−1 throughout the conversion process. By contrast, little of the neokestose produced was utilized and high levels of residual sucrose and glucose were present at 110 g l−1 initial sucrose (Fig. 1E–H). At the intermediary initial sucrose concentration of 70 g l−1 neokestose conversion was slower than at 31 g l−1 initial substrate, whereas glucose accumulated to levels similar to those observed at 110 g l−1 (not shown). Temperature seemed to affect glucose accumulation at a starting sucrose concentration of 31 g l−1 , since no significant accumulation was observed at 25 ◦ C (Fig. 1A and D) whereas glucose accumulated at 40 ◦ C (Fig. 1B and C). 3.3. Analysis of the factorial design Residual plots did not show any trend in the distribution of the residuals around the zero line, indicating a good fit

Table 3 The effect of growth phase on the production of neokestose from 50 g l−1 sucrose by X. dendrorhous in citrate-phosphate buffer (pH 6.55) containing 50 g l−1 sucrose in shake flasks Response Maximum specific rate of neokestose production (g g−1 h−1 ) Maximum concentration of neokestose produced (g l−1 ) Maximum yield coefficient of neokestose production (g g−1 )

Exponential phase cells 2.3 (0.003)

21.5 (0.05) 0.63 (0.008)

Stationary phase cells 1.7 (0.007)

18.0 (0.02) 0.59 (0.004)

Values are the means of triplicate experiments with standard deviations in brackets.

Fig. 2. Observed vs. predicted values of responses. (A) Maximum concentration of neokestose produced (g l−1 ); (B) specific rate of neokestose production (g g−1 h−1 ); (C) maximum yield coefficient of neokestose production (g g−1 ).

732

S.M. Kritzinger et al. / Enzyme and Microbial Technology 32 (2003) 728–737

Fig. 3. Pareto charts depicting the influence of cell concentration (g l−1 ), sucrose concentration (g l−1 ), pH, temperature (◦ C) and possible interactions on the maximum concentration of neokestose produced (g l−1 ) (A), the maximum yield coefficient for neokestose production (B) and the maximum specific rate of neokestose production (g g−1 h−1 ) (C) from sucrose by Xanthophyllomyces dendrorhous cell suspensions incubated in shake flasks in citrate-phosphate buffer containing sucrose. Temp: temperature; cell: cell concentration; sucrose: sucrose concentration; (∗): interaction between variables. Interaction pairs separated by forward slashes indicate aliases. Shaded columns represent negative effects, unshaded columns represent positive effects.

S.M. Kritzinger et al. / Enzyme and Microbial Technology 32 (2003) 728–737

rate of neokestose produced, maximum concentration of neokestose produced and maximum yield coefficient while r2 values of 0.999, 0.999 and 0.988, respectively, were obtained (Fig. 2). The lines of unity indicate great similarity between the observed and predicted values. It was, therefore, concluded that the statistical model was sufficient. The highest maximum specific rate of production (2.93 g g−1 h−1 ) and yield coefficient (0.85 g g−1 ) were both obtained at 110 g l−1 sucrose, 5.1 g l−1 cell concentration, pH 4 and 40 ◦ C (Table 1, run 5). The highest maximum concentration of neokestose produced (75.11 g l−1 ), however, was obtained at 110 g l−1 sucrose, 5.1 g l−1 cell concentration, pH 7 and 25 ◦ C (Table 1, run 6). Pareto charts indicated that higher sucrose concentrations increased the levels of all three responses (Fig. 3). This was confirmed by ANOVA analysis, which indicated that the effect of sucrose concentration had a P-value of <0.05 for all responses (Table 4). Sucrose concentration was the only positive main effect affecting the responses significantly. The effect of cell concentration varied from a large, statistically significant negative effect on the specific rate of neokestose production (Fig. 3C; Table 4) to a small, statistically insignificant positive effect on the maximum neokestose concentration obtained (Fig. 3A; Table 4). Increased temperature negatively affected both the maximum neokestose concentration reached (Fig. 3A) and the yield coefficient (Fig. 3B), but ANOVA analysis indicated that only the former was significant at the 5% level (Table 4). pH did not significantly affect production parameters (Table 4).

733

Some significant interaction terms were also identified. Either sucrose concentration × pH or its alias, cell concentration×temp, had a positive effect on the neokestose concentration (Fig. 3A), the significance of which was confirmed by ANOVA analysis (Table 4). The lines on the interaction plot for sucrose concentration and pH crossed (Fig. 4A) whereas the cell concentration/temperature plot did not (Fig. 4B), indicating that the sucrose concentration× pH interaction is probably the most significant. Setting both factors at their higher levels, therefore, should enhance the maximum concentration of neokestose. Two statistically significant interaction terms affected the maximum specific rate of neokestose production. These were sucrose concentration × temperature or its alias, cell concentration × pH and either pH × temperature or sucrose concentration × cell concentration. Interaction plots did not allow clear distinction between sucrose concentration ×temperature/cell concentration ×pH (results not shown) but those for the second interaction showed that the pH×temperature interaction is probably more important than the sucrose concentration × cell concentration interaction (Fig. 4C and D). The latter is a particularly strong interaction and similar responses are obtained from the low pH/high temperature and the high pH/low temperature combination of settings (Fig. 4C). The middle level included in the experimental design enabled us to investigate the possibility of curvature in the responses. Polynomial relationships could be fitted to the responses maximum specific rate of neokestose production

Fig. 4. The effects of two-factor interactions between sucrose concentration and pH on the maximum concentration of neokestose (A), cell concentration and temperature on the maximum concentration of neokestose (B), pH and temperature on the maximum specific rate of neokestose production (C) and sucrose concentration and cell concentration on the maximum specific rate of neokestose production (D).

734

Source

DFa

Mean square Rate

Model Error Uncorrected total Intercept Sucrose concentration Cell concentration pH Temperature Sucrose concentration × cell concentrationb Sucrose concentration × pHc Cell concentration × pHd

F-value Concentration

Yield coefficient

Rate

P>F Concentration

Yield coefficient

Rate

Concentration

Yield coefficient

9 2 11 1 2 1 1 1 1

3.22382759 0.00550533

2529.67 3.60

0.5548478 0.0010293

585.58

702.85

539.04

0.0017

0.0014

0.0019

21.2447608 0.88592488 5.05779013 0.02409013 0.00001012 0.73629113

17440.84 2464.81 14.99 61.63 193.02 4.58

4.8246568 0.0638758 0.0034031 0.0037411 0.0161101 0.0104401

3858.94 160.92 918.71 4.38 0.00 133.74

4845.83 684.83 4.17 17.12 53.63 1.27

4687.17 62.06 3.31 3.63 15.65 10.14

0.0003 0.0062∗ 0.0011∗ 0.1716 0.9697 0.0074∗

0.0002 0.0015∗ 0.1780 0.0537 0.0181∗ 0.3764

0.0002 0.0159∗ 0.2106 0.1969 0.0584 0.0861

1 1

0.01058512 0.16907112

67.15 55.18

0.0007411 0.0067861

1.92 30.71

18.66 15.33

0.72 6.59

0.2999 0.0311∗

0.0496∗ 0.0595

0.4855 0.1241

Aliases: b pH × temperature; c cell concentration × temperature; d sucrose × temperature. a DF: degrees of freedom. ∗ Significant at the 5% level.

S.M. Kritzinger et al. / Enzyme and Microbial Technology 32 (2003) 728–737

Table 4 Analysis of variance for the responses specific rate of neokestose production, concentration of neokestose produced and yield coefficient of neokestose on sucrose from data presented in Table 1

S.M. Kritzinger et al. / Enzyme and Microbial Technology 32 (2003) 728–737

735

4. Discussion

Fig. 5. The effect of the low, mean and high levels of sucrose and cell concentrations, pH and temperature on responses. The models y = −0.58550x 2 − 0.4455x + 1.727 (R 2 = 1.00), y = −0.04250x 2 + 0.03850x + 0.6810 (R 2 = 1.00) and y = 24.03x + 40.64 (R 2 = 9.99) were fitted to A, B and C, respectively. Each point represents the average response at the low, medium or high level of all the experimental factors.

and maximum yield coefficient (Fig. 5A and B). This suggested that the optima for the maximum specific rate of neokestose production and the maximum yield coefficient lay between the low and the mean values and between the mean and the high values of the experimental factors, respectively. A linear response was detected for the neokestose concentration, indicating that the maximum response lay outside of the range tested (Fig. 5C).

Rapid conversion of sucrose, resulting in a high yield of neokestose, was obtained using non-growing cells suspended in a citrate-phosphate buffer as biocatalysts. At 30 g l−1 initial sucrose, neokestose production was followed by a sharp decrease, which indicated neokestose hydrolysis (Fig. 1A–D). Similar patterns of oligosaccharide production followed by hydrolysis were reported using cell suspensions of S. cerevisiae or Aspergillus sp. [15] and Fusarium oxysporum growing on sucrose [22]. By contrast, purified fungal as well as bacterial enzymes frequently lack the ability to hydrolyze oligosaccharides formed from sucrose [23,25–27]. At 110 g l−1 sucrose, however, the neokestose formed was not assimilated to a significant extend (Fig. 1E–H). This may have resulted from enzyme inhibition or repression by the higher level of glucose accumulated at 110 g l−1 sucrose (Fig. 1E–H). Using higher initial concentrations of sucrose will eliminate the necessity for very accurate timing of production termination to prevent product assimilation. Cell recycling could potentially reduce the overall cost of production, but it reduced production parameters to prohibitively low levels in our study. Although exponentially growing cells of X. dendrorhous cells produced neokestose significantly more efficiently than stationary cells, the difference was small and will probably not prohibit the use of stationary cells. Results obtained from the experimental design showed that increased sucrose concentration was the main consideration for the improvement of process parameters. This is in agreement with most reports for transferase activities from filamentous fungi [14,28–31], yeasts [32] and bacteria [25,33]. Surprisingly, sucrose was also the main factor enhancing the yield coefficient. This is in contrast with a report by Hang et al. [34], who found that increased initial sucrose levels decreased the yield coefficient of kestose from sucrose. The strong effect of substrate concentration observed in our experiments suggested that neokestose production could be substantially increased by increasing the sucrose concentration. The higher cell concentration had an unexpected large negative effect on the specific rate of neokestose production, indicating that efficient neokestose production can be achieved using a low cell concentration. The ability to use lower cell concentrations would reduce the cost of neokestose production. A similar observation was made by Chien et al. [11] for FOS production from sucrose using immobilized Aspergillus japonicus biomass. They speculated that mass transfer limitations at high cell concentrations may be involved. In addition, Bonnin and Thibault [24] found that the transglycosylation activity of purified exogalactanase from Aspergillus niger declined when the enzyme concentration was increased from 5 to 20 nkat ml−1 . Some authors reported that transglycosylating activities were strictly pH dependent [18,24], whereas no significant effect was observed in other studies [29]. Our results did

736

S.M. Kritzinger et al. / Enzyme and Microbial Technology 32 (2003) 728–737

not yield any statistically significant effect of pH as main effect. However, pH was involved in two significant interactions. Setting both pH and temperature at high levels was detrimental to the specific rate of production, the highest rate being observed at mixed settings of these two factors. This is especially important for process design, since the detrimental effect of high temperature can be compensated for by a low pH and vice versa. In addition, the positive effect of sucrose concentration on the neokestose concentration could be further enhanced by increased pH due to the interaction between sucrose concentration and pH. The higher temperature setting had a detrimental effect on the yield coefficient and the concentration of neokestose produced, with the latter being statistically significant. This may reflect temperature inactivation of the transfructosylating enzyme, in contrast to previous results showing that ␤-fructofuranosidases from several filamentous fungi were stable at 40 ◦ C [30,35,36] and had optima of between 50 and 60 ◦ C [10,28,30,37–40]. X. dendrorhous has a maximum growth temperature of 21 ◦ C, which may explain the apparent temperature sensitivity of transfructosylation. Analysis of curvature showed that, whereas the maximum responses of the yield coefficient and the specific rate of production lay within the experimental region, that of the maximum level of neokestose produced lay outside the range of the experimental design. The generalized curvature term of a fractional factorial design with center points cannot be ascribed to any single experimental factor. In view of its large effect on production parameters, however, sucrose concentration is a likely candidate for the enhancement of the neokestose concentration; in commercial applications sucrose concentrations of up to 800 g l−1 are used [10]. As reported for other oligosaccharide production processes, such as that of the commercially available neosugar [41], glucose, unreacted fructose and sucrose remained at the end of the conversion process. Neokestose comprised 95% of the total oligosaccharides formed, which is higher than values of between 27 and 82% reported before or calculated from results reported for 1-kestose, a prominent prebiotic trisaccharide [11,23,25,28,42–44]. Likewise, the highest yield coefficient of 0.85 g neokestose g sucrose−1 obtained compares very favorably to values of around 0.5 previously reported for 1-kestose [11,23,28]. The results reported here demonstrate, for the first time, the potential of a member of the 6 G fructose oligosaccharides for large-scale production as prebiotic substance. It is also the first time that efficient conversion of sucrose into prebiotic oligosaccharides by yeasts or yeast enzymes has been described. This opens up the field for further investigation into these prebiotic sources. In addition, important effects of process parameters have been identified that will be used in planning further optimization studies.

Acknowledgments Financial support was provided by the National Research Foundation of South Africa. Piet Botes assisted with sugar analysis by high performance liquid chromatography. References [1] Gibson GR, Willis CL, van Loo J. Non-digestible oligosaccharides and bifidobacteria: implications for health. Int Sugar J 1994;96: 381–7. [2] Hidaka H, Eida T, Takizawa T, Tokunaga T, Tashiro Y. Effects of fructooligosaccharides on intestinal flora and human health. Bifidobacteria Microflora 1986;5:37–50. [3] Tamime AY, Marshall VME, Robinson RK. Microbiological and technological aspects of milk fermented by bifidobacteria. J Dairy Res 1995;62:151–87. [4] Hidaka H, Hirayama M. Useful characteristics and commercial applications of fructooligosaccharides. Biochem Soc Trans 1991;19: 561–5. [5] Oku T, Tokunaga T, Hosoya N. Non-digestibility of a new sweetener, “neosugar” in the rat. J Nutr 1984;114:1281–574. [6] Crittenden RG, Playne MJ. Production, properties and applications of food-grade oligosaccharides. Trends Food Sci Technol 1996;7:353– 61. [7] Bacon JSD. The oligosaccharides produced by the action of yeast invertase preparations on sucrose. Biochem J 1954;57:320–8. [8] Crittenden RG, Doelle HW. Structural identification of oligosaccharides produced by Zymomonas mobilis levansucrase. Biotechnol Lett 1993;15:1055–60. [9] Hayashi S, Sasao S, Takasaki Y, Imada K. Immobilization of ␤-fructofuranosidase from Aureobasidium on DEAE-cellulose. J Ind Microbiol 1994;13:103–5. [10] Yun JW. Fructooligosaccharides: occurrence, preparation, and application. Enzyme Microb Technol 1996;19:107–17. [11] Chien C-S, Lee W-C, Lin T-J. Immobilization of Aspergillus japonicus by entrapping cells in gluten for production of fructooligosaccharides. Enzyme Microb Technol 2001;29:257–525. [12] Han YW, Watson MA. Production of microbial levan from sucrose, sugarcane juice and beet molasses. J Ind Microbiol 1992;9:257–60. [13] Marx SP, Winkler S, Hartmeier W. Metabolization of ␤-(2,6)-linked fructose oligosaccharides by different bifidobacteria. FEMS Microbiol Lett 2000;182:163–9. [14] Rehm J, Willmitzer L, Heyer AG. Production of 1-kestose in transgenic yeast expressing a fructosyltransferase from Aspergillus foetidus. J Bacteriol 1998;180:1305–10. [15] Hidaka H, Hirayama M, Sumi N. A fructooligosaccharide-producing enzyme from Aspergillus niger ATCC 20611. Agric Biol Chem 1988;52:1181–7. [16] Yasuda H, Shitoh T, Yamano T, Itoh Y, Shimura S. Identification of neokestose produced from sucrose by an enzyme of Penicillium oxalicum. Agric Biol Chem 1986;50:777–8. [17] Hayashi S, Yoshiyama T, Fujii N, Shinohara S. Production of a novel syrup containing neofructooligosaccharides by the cells of Penicillium citrinum. Biotechnol Lett 2000;22:1465–9. [18] Dickerson AG. A ␤-d-fructofuranosidase from Claviceps purpurea. Biochem J 1972;129:263–72. [19] Kilian SG, Sutherland FCW, Meyer PS, du Preez JC. Transportlimited sucrose utilisation and neokestose production by Phaffia rhodozyma. Biotechnol Lett 1996;18:975–80. [20] Kilian SG, Kritzinger SM, Rycroft C, Gibson GR, du Preez JC. The effects of the novel bifidogenic trisaccharide, neokestose, on the human colonic microbiota. World J Microbiol Biotechnol 2000;18:637–44.

S.M. Kritzinger et al. / Enzyme and Microbial Technology 32 (2003) 728–737 [21] Haaland PD. Experimental design in biotechnology. New York: Marcel Dekker; 1989. [22] Patel V, Saunders G, Bucke C. Production of fructooligosaccharides by Fusarium oxysporum. Biotechnol Lett 1994;16:1139–44. [23] Támbara Y, Hormaza JV, Pérez C, León A, Arrieta J, Hernández L. Structural analysis and optimised production of fructooligosaccharides by levansucrase from Acetobacter diazotrophicus SRT4. Biotechnol Lett 1999;21:117–21. [24] Bonnin E, Thibault J-F. Galactooligosaccharide production by transfer reaction of an exogalactanase. Enzyme Microb Technol 1996;19:99–106. [25] Fujita K, Hara K, Hashimoto H, Kitahata S. Transfructosylation catalyzed by beta-fructofuranosidase I from Arthrobacter sp. K-1. Agric Biol Chem 1990;54:2655–61. [26] Hayashi S, Nonoguchi M, Takasaki Y. Purification and properties of beta-fructofuranosidase from Aureobasidium sp. ATTCC 20524. J Ind Microbiol 1991;7:251–6. [27] van Laere KMJ, Hartemink R, Beldman G, Pitson S, Dijkema C, Schols HA, et al. Transglycosidase activity of Bifidiobacterium adolescentis DSM 20083 ␣-galactosidase. Appl Microbiol Biotechnol 1999;52:681–8. [28] Chiang CJ, Lee WC, Sheu DC, Duan KJ. Immobilization of ␤-fructofuranosidases from Aspergillus on methacrylamide-based polymeric beads for production of fructooligosaccharides. Biotechnol Prog 1997;13:577–82. [29] Smith NK, Gilmour SG, Rastall RA. Statistical optimization of enzymatic synthesis of derivatives of trehalose and sucrose. Enzyme Microb Technol 1997;21:349–54. [30] Hirayama M, Sumi N, Hidaka H. Purification and properties of a fructooligosaccharide-producing ␤-fructofuranosidase from Aspergillus niger ATCC 20611. Agric Biol Chem 1989;53:667– 73. [31] Jung KH, Lim JY, Yoo SJ, Lee JH, Yoo MY. Production of fructosyltransferase from Aureobasidium pullulans. Biotechnol Lett 1987;9:703–8. [32] Boon MA, Janssen AEM, Van’t Riet K. Effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides. Enzyme Microb Technol 2000;26:271–81.

737

[33] Rabiu BA, Jay AJ, Gibson GR, Rastall RA. Synthesis and fermentation properties of novel galactooligosaccharides by beta-galactosidases from Bifidobacterium species. Appl Environ Microbiol 2001;67:2526–30. [34] Hang YD, Woodams EE, Jang KY. Enzymatic conversion of sucrose to kestose by fungal extracellular fructosyltransferase. Biotechnol Lett 1995;17:295–8. [35] Hayashi S, Matsuzaki K, Takasaki Y, Ueno H, Imada K. Production of ␤-fructofuranosidase by Aspergillus japonicus. World J Microbiol Biotechnol 1992;8:155–9. [36] Wang X-D, Rakshit SK. Iso-oligosaccharide production by multiple forms of transferase enzymes from Aspergillus foetidus. Process Biochem 2000;35:771–5. [37] Hayashi S, Nonoguchi M, Imada K. Production of a fructosyltransferring enzyme by Aureobasidium sp. ATCC 20524. J Ind Microbiol 1990;5:395–400. [38] Barthomeuf C, Pourrat H. Production of high-content fructooligosaccharide by an enzymatic system from Penicillium rugulosum. Biotechnol Lett 1995;17:911–6. [39] van Balken JAM, van Dooren JGM, van den Tweel WJJ, Kamphuis J, Meijer EM. Production of 1-kestose with intact mycelium of Aspergillus phoenicis containing sucrose-1 F-fructosyltransferase. Appl Microbiol Biotechnol 1991;35:216–21. [40] Cairns CJ, Lee WC, Sheu DC, Duan KJ. Characterization of acid invertase from the snow mould Monographella nivalis: a mesophylic enzyme from a psychrophilic fungus. New Phytol 1997;130:391–400. [41] Mitsuoka T, Hidaka H, Eida T. Effect of fructooligosaccharides on intestinal microflora. Die Nahrung 1987;31:427–36. [42] Trujillo LE, Arrieta JG, Dafhnis F, Garcia J, Valdes J, Tambara Y, et al. Fructooligosaccharides production by the Gluconacetobacter diazotrophicus levansucrase expressed in the methylotrophic yeast Pichia pastoris. Enzyme Microb Technol 2001;28:139–44. [43] Yun JW, Song SK. The production of high-content fructooligosaccharides from sucrose by the mixed-enzyme system of fructosyltransferase and glucose oxidase. Biotechnol Lett 1993;15:573–6. [44] Barthomeuf C, Grizard D, Teulade JC. Assay and structural determination of fructooligosaccharides synthesized by an enzymatic system from Penicillium rogulosum. Biotechnol Tech 1997;11:845–8.