In vitro Evaluation of the Digestive Tolerance of Water-Soluble Sodium β-(1→4)-Polyglucuronate

Abstract

A water-soluble sodium β-(1→4)-polyglucuronate (or sodium cellouronate, Na-CUA) was prepared from regenerated cellulose by catalytic oxidation in water at pH ~10. Digestive behavior of Na-CUA was evaluated in vitro using three enzymes, α-amylase, pepsin, and pancreatin in water at 80 or 40 °C for 30 or 60 min. The results showed that 98.95 % Na-CUA remained undigested, indicating that Na-CUA is categorized as water-soluble dietary fibers.

Abbreviations

CMC, carboxymethyl cellulose; CUA, sodium cellouronate or sodium β-(1→4)-polyglucuronate; DT, digestive tolerance

Water-soluble sodium β-(1→4)-polyglucuronates (or sodium cellouronates, Na-CUAs) were prepared from regenerated cellulose by catalytic oxidation with TEMPO/NaBr/NaOCl in water at pH ~10 (TEMPO is an abbreviation of 2,2,6,6-tetramethyl piperidine N-oxyl radical, which is water soluble and stable nitroxyl radical) (Fig. 1) [1, 2, 3]. In the oxidation system, TEMPO and NaBr behave as catalysts, while NaOCl is consumed as a primary oxidant. The C6-OH groups in regenerated cellulose are converted to sodium C6-carboxylate groups, resulting in the preparation of water-soluble Na-CUAs in high yields [4].

Fig. 1. Preparation of sodium cellouronate (Na-CUA) from regenerated cellulose by TEMPO/NaBr/NaOCl system in water at pH ~10 and room temperature.

　n, m: degree of polymerization.

Although Na-CUAs are artificially prepared from regenerated cellulose by catalytic oxidation, they are completely biodegradable and metabolizable to CO₂ and water by microorganisms in soil [5, 6], differing from synthetic sodium acrylates or sodium carboxymethyl cellulose (Na-CMC). Na-CMC is biodegradable at the continuously glucose unit-linked positions in each CMC molecule but not completely metabolizable because of the presence of artificially introduced carboxymethyl ether groups [5]. Therefore, Na-CUAs are expected as new water-soluble and biodegradable/metabolizable sodium polycarboxylates prepared from abundant and atmospheric CO₂-accumulated cellulose resources, partly in place of sodium acrylates or CMCs used as polymeric builders (or calcium catchers in tap water) in laundry detergents for improving washing efficiency.

It was reported that Na-CMC is not degraded by human digestive enzymes [7]. The Japanese National Institute of Health Sciences reported that no toxicity of Na-CMC was observed for human bodies, and consequently Na-CMC is approved to be used as a food additive. Furthermore, oral administration tests of Na-CMC to rats and guinea pigs proved to be harmless to these animals [7]. Na-CMC and Na-CUA are prepared from cellulose by etherification and oxidation, respectively. Water-soluble Na-CMC and Na-CUA have different but similar chemical structures containing sodium carboxylate groups. Therefore, it is anticipated that Na-CUA is also non-degradable by human digestive enzymes as in the case of Na-CMC.

Na-CUAs are enzymatically depolymerized by exo-type Na-CUA lyases, and one of the enzymes has been isolated and identified from filamentous fungus Trichoderma reesei [8, 9, 10, 11]. Na-CUAs have homogeneous chemical structures consisting of sodium glucuronate units, similar to heterogeneous natural polyuronates, such as pectin, alginates, and hyaluronic acid. In this study, we evaluated the digestibility of Na-CUA in vitro using three representative enzymes by a gravimetric method. The three enzymes used in this study were commercial products.

The Na-CUA sample with an average molar mass of 10 kDa (or degree of polymerization of ~52) was prepared from viscose rayon by the TEMPO/NaBr/NaOCl system in water at pH ~10 [2]. Its molar mass was determined by gel-permeation chromatography using pullulan standards [12]. The dietary fiber evaluation of Na-CUA was performed in vitro with reference to the literature [13], in which pullulan was treated with different enzymes secreted in the human digestive system in the same order as food passes through in humans. The detailed experimental conditions were, however, set independently used in this study, as described below, considering the optimal treatment conditions such as pH, temperature, and time for each enzyme.

First, α-amylase was applied to Na-CUA to simulate decomposition by saliva. Next, pepsin was used to simulate degradation of Na-CUA in the stomach. Finally, pancreatin, i.e., a mixture of α-amylase, trypsin, and lipase secreted by the pancreas, was applied to Na-CUA.

70 mL of 0.1 M Na₂HPO₄ and 40 mL of 0.1 M KH₂PO₄ were mixed to prepare a 0.1 M phosphate buffer at pH 7.0. This buffer (5 mL) was added to an accurate mass of dried Na-CUA (0.2010 g), and a transparent Na-CUA/buffer solution was prepared. A Termamyl enzyme solution (20 μL) (thermally stable α-amylase, International Registration Number 1210685, Novozymes A/S, Bagsværd, Denmark) was added to the Na-CUA/buffer solution and incubated at 80 °C for 30 min. 4 N HCl (0.48 mL) was added to the Termamyl enzyme-treated Na-CUA solution to adjust the pH to 1.5, where the transparent solution turned to translucent because protonated CUA-COOH fine particles were formed. Deionized water (3.52 mL) was added to the mixture to adjust the total volume of the solution to be 9 mL. Pepsin (20 mg) was added to the mixture and incubated at 40 °C for 60 min.

4 M sodium hydroxide (0.46 mL) was added to the above mixture to adjust the pH to 7.0, where the mixture turned into a clear solution because CUA molecules were dissolved in the mixture at pH 7.0, forming CUA-COONa. Deionized water (3.54 mL) was added to the solution to adjust the total volume to be 13 mL. Pancreatin (20 mg) was added to the solution, and incubated at 40 °C for 60 min. Ethanol (52 mL) was added to the solution, and incubated at 60 °C for 60 min to precipitate Na-CUA from the ethanol-rich solution (~80 % ethanol/water containing enzymes and salts). The Na-CUA precipitate-containing mixture was filtered on a filter paper (0.2904 g on dry mass measured beforehand), and the precipitate on the filter paper was washed twice (2 mL each) with 78 % ethanol, 95 % ethanol, and acetone in this order. After removal of acetone in the filter paper by natural evaporation, the sample with the filter paper was dried at 105-110 °C until a constant mass was reached. After cooling, the dry mass of the Na-CUA and enzymes insoluble in ethanol-rich solutions collected on the filter paper was measured. A blank test using enzymes without Na-CUA was also conducted at the same time for measuring the mass of enzymes insoluble in the ethanol-rich solutions and collected on another filter paper (0.2919 g on dry mass measured beforehand), assuming that the used enzymes partly precipitated in the ethanol-rich solutions to cause an increase in mass of the enzyme-treated Na-CUA insoluble in the ethanol-rich solutions.

The results are as shown in Table 1. The digestive tolerance (DT) or dietary fiber mass ratio is calculated according to the following equation,

$$\mathrm{DT}(\text{\%})=[(\mathrm{C}\mathrm{-}\mathrm{B})]/(\mathrm{A})\times 100=98.95\text{\%}$$

where C is the dry mass of enzyme-treated Na-CUA and enzymes insoluble in the ethanol-rich solutions, B is the dry mass of the blank test, and A is the dry mass of the initial Na-CUA tested (Table 1). As a result, 98.95 % of the initial Na-CUA was collected as the fraction insoluble in the ethanol-rich solutions without almost mass loss by enzymatic degradation, showing that Na-CUA is categorized as water-soluble dietary fibers. In this study, the non-digestibility of Na-CUA was evaluated from its mass loss after treatments with three enzymes. However, changes in the amount of reducing ends of enzyme-treated Na-CUA and/or those in its molar mass/molar mass distribution should be measured for more accurate evaluation of digestibility of Na-CUA. Furthermore, safety issue of Na-CUAs as food additives for human beings or other animals should be evaluated by some in vivo tests separately from this digestive tolerance experiment.

Table 1. Dry masses of enzyme-treated Na-CUA and blank test, and Na-CUA digestibility.

Dry mass of initial Na-CUA (A)	Dry mass of enzymes insoluble in ethanol-rich solutions (blank test) (B)	Dry mass of enzyme-treated Na-CUA and enzymes insoluble in ethanol-rich solutions (C)	Digestive tolerance (DT)
0.2010 g	0.0356 g	0.2345 g	98.95 %

CONFLICTS OF INTERESTS

IS and EK have already received permission from their company, JNC Corporation Yokohama Reseach Center, to submit this manuscript.