Enzymatic production and characterization of manno-oligosaccharides from Gleditsia sinensis galactomannan gum

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Abstract

Enzymatic hydrolysis of Gleditsia sinensis gum was performed to produce manno-oligosaccharides having functional applications as dietary fiber and prebiotics. The optimum hydrolysis conditions, including enzyme loading, temperature and time, from response surface methodology were 8.1 U/g, 57.4 °C and 34.1 h, respectively. The yield of DP 1–5 oligosaccharides was 75.9% (29.1 g/L). The Michaelis–Menten kinetics and molecular weight distribution were determined. The obtained oligosaccharides were further separated by HPLC and SEC, and the galactose distribution of G. sinensis gum was elucidated. Results indicated that G. sinensis gum has potential to produce value-added oligosaccharides in food industries.

Highlights

Gleditsia sinensis gum was hydrolyzed by β-mannanase to produce oligosaccharides. ► The hydrolysis conditions were optimized by response surface methodology. ► 75.9% (29.1 g/L) oligosaccharides were yielded from the galactomannan gum. ► Enzyme kinetics and changes of MWD and apparent viscosity were determined. ► Structural characterization was conducted for the resulting oligosaccharides.

Introduction

Galactomannan is a kind of natural and water-soluble polysaccharide consisting of a β-1,4-linked d-mannose backbone randomly substituted with single-unit α-1,6-linked d-galactose residues [1], [2]. The functional properties of polysaccharide hydrocolloids are directly related with their structure. Polysaccharides could be depolymerized through various methods, such as γ-irradiation [3], ionizing radiation [4], microwave irradiation [5], chemical [6], [7] and enzymatic hydrolysis [8], [9], [10]. In general, enzymatic hydrolysis can be kinetically controlled to produce desired end products. Degraded galactomannan with reduced molecular weight and viscosity could be applied as soluble dietary fiber in functional foods [11].

The susceptible enzymatic hydrolysis of galactomannan involves three types of enzymes: β-mannanase, β-mannosidase and α-galactosidase. Endo-1,4-β-d-mannanase (EC 3.2.1.78) randomly cleaves the β-1,4-d-mannosidic linkages within the main chain of mannan and mannan-type polysaccharides such as galactomannans and glucomannans to produce manno-oligosaccharides [12]. Only a few β-mannanases can release mannose in the hydrolysis of mannan [13]. The hydrolysis of galactomannan by β-mannanase is greatly affected by the extent and pattern of galactose substitution on the mannose backbone [14], [15]. The β-mannanase is preferred to act on the region with low galactose substitution, and the liberated fractions mainly consist of non-substituted mannose residues [6], [16].

Manno-oligosaccharides are non-digestible and could be potentially applied as dietary fiber and prebiotics [17]. Previous studies have confirmed the prebiotic activities of manno-oligosaccharides to human intestinal beneficial microflora (mainly bifidobacteria and lactobacilli) [18], [19], [20]. On the other hand, manno-oligosaccharides could prevent the fat storage through inhibiting the intestinal absorption of dietary fat in a high fat diet [21]. The researches with healthy adults as subjects revealed that the intake of 3.0 g manno-oligosaccharides per day could increase the amount of excreted fat and decrease the fat utilization [22], [23], [24]. Moreover, it was found that manno-oligosaccharides could suppress the elevation in blood pressure [21]. Manno-oligosaccharides have potential to be used as non-nutritional and functional food additives for selective growth of intestinal beneficial microflora, reduction of dietary fat absorption, and inhibition of the elevation of blood pressure.

Gleditsia sinensis Lam., a woody species (genus Gleditsia) in Leguminosae family, is widely distributed in China. Galactomannan as a storage polysaccharide exists in the endosperm of the G. sinensis seeds [25]. G. sinensis gum, similar to guar and locust bean gums both structurally and functionally, is a non-traditional source of industrial gums. At present, it usually served as thickener, stabilizer and flocculant in various industries of food, pharmaceuticals, petroleum drilling, paper and printing [26].

This work aimed at the production of manno-oligosaccharides from G. sinensis gum by enzymatic hydrolysis. The optimum hydrolysis conditions were obtained using response surface methodology (RSM). The G. sinensis gum-derived oligosaccharides were further separated by size exclusion chromatography (SEC) and identified by high performance liquid chromatography (HPLC). The galactose substitution degree of G. sinensis galactomannan and the fractions with different degree of polymerization (DP) were determined to obtain galactose distribution information of G. sinensis galactomannan. Furthermore, the Michaelis–Menten kinetics and changes in apparent viscosity and molecular weight distribution (MWD) were investigated. The results of this study contribute to new applications of G. sinensis seed gum based on prebiotic activities, which are helpful to transform plant seed gum into high value-added oligosaccharides in food industry. The characterization of hydrolysis process and products can provide a better understanding of the G. sinensis galactomannan gum.

Section snippets

Materials

Locust bean gum, guar gum and standard monosaccharides, including arabinose, mannose, galactose and glucose, were purchased from Sigma–Aldrich, Saint Quentin Fallavier, France. Standard manno-oligosaccharides of β-(1→4)-linked mannobiose (M2), mannotriose (M3), mannotetraose (M4), and mannopentaose (M5) were purchased from Megazyme International, Bray, Ireland. The endo-β-mannanase (EC 3.2.1.78), a commercial available enzyme purified from Aspergillus niger, was offered by Beijing Bosar

Enzyme kinetics

The β-mannanase had a specific activity of 2.5 × 103 U/g toward locust bean gum. The Michaelis–Menten parameters calculated from the double reciprocal plot are summarized in Table 1. The β-mannanase showed a higher affinity toward locust bean gum, because of a lower Km value than that of G. sinensis gum. However, the catalytic efficiency (kcat/Km) of β-mannanase to locust bean gum was a little lower than that to G. sinensis gum. Locust bean gum has a higher mannose/galactose (M/G) ratio compared

Conclusions

In the present study, G. sinensis seed gum was subjected to enzymatic hydrolysis for the production of manno-oligosaccharides to develop its functional applications as dietary fiber and prebiotics. The hydrolysis conditions including enzyme loading, temperature and time were optimized by RSM. Characterization and separation of the hydrolysis products of G. sinensis gum afforded a series of oligosaccharides with DP 1–5. It was found that the distribution patterns of galactose in G. sinensis

Contributions

Hong-Lei Jian mainly made contributions to design and conduct the research, analysis and processing of data, and drafting the article.

Li-Wei Zhu mainly contributed to the conception and participation of the research, and acquisition of data.

Wei-Ming Zhang and Da-Feng Sun revised the draft critically and proposed lots of good suggestions.

Jian-Xin Jiang made great contributions to conception and design the research and revising the paper seriously for improvement.

All authors have approved the

Acknowledgements

This research was supported by the Specific Programs in Graduate Science and Technology Innovation of Beijing Forestry University (BLYJ201111), the National Natural Science Foundation of China (31270624) and the Beijing Natural Science Foundation (6122023).

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