Elsevier

Process Biochemistry

Volume 46, Issue 1, January 2011, Pages 298-303
Process Biochemistry

Continuous production of oligofructose syrup from Jerusalem artichoke juice by immobilized endo-inulinase

https://doi.org/10.1016/j.procbio.2010.08.028Get rights and content

Abstract

A commercially available endo-inulinase from Aspergillus niger was successfully immobilized onto a chitin carrier with 66% yield. The immobilized endo-inulinase showed maximal activity at 65 °C that was 5 °C higher than the optimum temperature of the free enzyme. Also, the optimum pH shifted from 4.5 to 5.0 for free form and 5.5 to 6.0 for the immobilized form. The immobilized endo-inulinase was stable at 4 °C for at least 1 year. The residual activity of 90% and 95% of free and immobilized enzymes were recovered after 5 days of incubation without substrate at 60 °C and pH between 4.5 and 6.5. Kinetic parameters (Km and Vmax) for free and immobilized endo-inulinase were 2.04% (w/v) and 80.88 U/mg protein, and 2.19% (w/v) and 291.58 U/g support, respectively. The half-life time of immobilized endo-inulinase in a packed-bed column reactor was estimated to be 48 days. A continuous production system of inulo-oligosaccharides (IOS) from inulin and Jerusalem artichoke (JA) juice was set up and operated. Using this system, syrups with high IOS content (from 35% to 65%) were successfully prepared.

Introduction

Oligo- and polysaccharides that contain specific β(2  1) glycosidic linkages have an important role in nutrition, because the upper part of the human intestinal tract lacks enzymes that cleave these linkages [1]. Inulin and fructo-oligosaccharides (FOS) are known as non-digestible food ingredients that beneficially affect the health status of a human body by selectively stimulating the growth and/or activity of bifidobacteria already resident in the colon, considered to be prebiotic [2]. Many health-beneficial effects of fermentable fructans, especially the pentamers (DP5) and the hexamers (DP6) have been reported including the prevention of diabetes, cancer and the control of lipid metabolism [3], [4], [5], [6]. Prebiotic effectiveness of inulin-type fructans depend not only on the dietary dosage, but also on the degree of polymerization (DP). Coudray et al. [7] reported that only the combination of oligofructose (DPav 4) and HP-inulin (DPav 25) at 1:1 ratio resulted in synergistic effects on internal calcium absorption and balance in rats. Relating to the growth and utilization of inulin-type fructans, Biedrzycka and Bielecka [8] found that the effects of highly polymerized inulins were more diverse and depended on the presence of other bacteria to initiate degradation, followed by the possible subsequent stimulation of growth of Bifidobacteria. The effects of fructo-oligosaccharides (DP2–4), oligofructose (DP2–8), and mildly polymerized inulin (DP9–22) were evident than the effects of HP-inulin [9]. The β(2  1) fructans with a chain length of up to 10 residues are soluble and particularly ‘bifidogenic’ [1]. Moreover, there is no doubt that carbohydrates with small molecular size are able to pass through a cell wall easily and serve as a substrate for probiotic bacteria. Long chain inulins (10–65 monomers) are poorly soluble in water and have less pronounced bifidogenic properties [1]. The utilization of these long chain inulins are expected to more time in the colon. Nevertheless, the relationship between the DP of fructans and prebiotic effects has not been clearly defined.

Endo-inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) cleaves the internal β(2  1) fructosyl linkages of inulin to produce short-chain oligofructose/inulo-oligosaccharides (IOS). In recent years, some researchers have concentrated their efforts on separating endo-inulinase from invertase and exo-inulinase in order to hydrolyze inulin and produce oligosaccharides [10], [11]. However, others have focused on the process yields [12] and percentage of total amount of inulo-oligosaccharides, and gave less attention to individual IOS. For example, the production of high content IOS (DP2–7) was carried out by Cho et al. [13] using dual endo-inulinase system (from Xanthomonas sp. and from Pseudomonas sp.). Mutanda et al. [6], however, have purified endo-inulinase from a Novozym 960 preparation and optimized process parameters for the production of IOS (F3, F4 and F5). In both studies, free enzyme technology was used that limited the adaptability to industrial scale production because of high cost, stability of operation and the lack of reusability of the enzyme. In contrast, the advent of immobilized enzyme technology has led to increase in efforts to replace conventional enzymatic process with immobilized enzyme preparations. The advantages of immobilization technology include (a) allowing the enzymes to process large amounts of substrate since it can be separated easily from the mixture of substrate and product(s) thus enabling the enzyme to be reused [14]; (b) imparting greater stability to the enzyme [15], [16], so that it can be used for the development of continuous process; (c) affording greater control of the catalytic process; and (d) permitting the economical utilization of an otherwise cost-prohibitive [17]. Powerful and economical effects of immobilized enzyme preparation are demonstrated by immobilized glucose isomerase in food processing [18] or glucose oxidase/peroxidase in clinical diagnostic [19]. Taking into consideration that chitin is a biopolymer from natural sources (shellfish) and its promising characteristics including biocompatibility, hydrophilic feature, biodegradability, and anti-bacterial properties [20], [21], this material has the potential as a low cost, bifunctional support for enzyme immobilization in both food and pharmaceutical applications. According to Chang and Juang [21], more than 10 enzymes could be effectively immobilized by chitosan. However, the major advantages of this method are industrial applicability and environmental friendly. The immobilization yield depends largely on the method applied as well as on the nature of the enzyme. The present study reports the production of short-chain inulo-oligosaccharides from Jerusalem artichoke juice using immobilized endo-inulinase from Aspergillus niger.

Section snippets

Materials

Endo-inulinase preparation from A. niger was purchased from Megazyme International Ireland Ltd. (Co. Wicklow, Ireland). According to the information provided by the supplier, this enzyme has higher activity on dahlia fructan (65 U/mg) than on kestose (3.7 U/mg). Therefore, it could be classified as an endo-acting enzyme. Chitin (Product No. C7170) was obtained from Sigma–Aldrich Inc. (Budapest, Hungary). The inulin with high DP (DPav 25) from dahlia tubers was a Fluka product (F 57614). All other

Results and discussion

After the immobilization process, neither inulinase activity nor protein content was detected in the supernatant fraction, confirming that the endo-inulinase covalently bonded to the carrier. The activity on chitosan (immobilized enzyme fraction) was assayed and 66% of inulinase activity was recovered. Higher yield (82.60%) of immobilization was reported by de Paula et al. in 2008 [27] when immobilized the crude inulinase from Kluyveromyces marxianus var. bulgaricus onto gelatin. Gill et al.

Conclusion

A commercially available endo-inulinase from A. niger was successfully immobilized onto a chitin carrier. The optimum pH of endo-inulinase from A. niger was shifted by 1 unit higher after immobilization. The immobilized preparation was more stable at higher temperature than free enzyme, opening possibilities to operate continuously at 60 °C for a long period. Also, with immobilized endo-inulinase the bioconversion rate was high, which would be an advantage to prevent microbial contamination. A

Acknowledgments

Authors would like to thank Dr. M.K. Bhat for carefully read the manuscript. This work was supported by the National Office for Research and Technology (Project No. NKFP4/002/2004), Hungarian Scientific Research Fund (Project No. OTKA F 67717) and National Development Agency (Project No. TÁMOP-4.2.1./B-09/1-KMR-2010-0005). Dr. Quang D. Nguyen received the Bolyai János Research Grant from the Hungarian Academy of Sciences.

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