STRUCTURE AND MOLECULAR WEIGHT OF KONJAC GLUCOMANNAN

3.2 STRUCTURE AND MOLECULAR WEIGHT OF KONJAC GLUCOMANNAN

The constitution of konjac glucomannan was the object of many previous investi- gations, and the results show that konjac glucomannan consists of glucose and

mannose, and the ratio is 1.0 to 1.6. 3 It has been believed that there are some branching points at the C-3 of the mannose unit. 4 However, a recent study shows that the branching point is C-6 of glucosyl units and the degree of branching is about 8%. The ratio of terminal glucosyl units to mannosyl units is ca. 2. 5

KGM contains some acetyl groups (about 1 acetyl group per 19 sugar residues) 6 that confer the water solubility to the konjac polymer. It forms a gel upon heating in the presence of alkali, and the role of alkali is believed to remove the acetyl groups. The peak at 1730 cm –1 in the Fourier-transformed infrared (FTIR) absorbance spectra of KGM film, which was attributed to the absorption by acetyl groups, disappeared by alkali treatment, 7,8,20 as shown in Figure 3.1.

3.2.1 T HE M AN /G LC R ATIO

The ratio of mannose (Man) to glucose (Glc) for KGM samples with different molecular weights, prepared by enzymatic degradation, was determined by high-

performance liquid chromatography (HPLC). 8 The Man/Glc ratio was calculated from the peak areas of mannose and glucose detected with a refractive index (RI) detector, and was approximately 2.0 for all the fractions, higher than the previously

reported 3 1.6. It was close to the ratio 2.1 reported for glucomannan extracted from Scotch pine. 9 The experimental fact that the Man/Glc ratio was not much different for a nondegraded specimen and for enzymatically degraded specimens suggests that there is no block structure in konjac glucomannan because glucose and mannose residues do not exhibit a difference in enzyme reactivity.

3.2.2 F RACTIONATION

To obtain fractions with different molecular weights, konjac glucomannan powder was dissolved in water. Methanol as a precipitant was added, and the solution was

Konjac Glucomannan

anc sorb

A b 3367

Wavenumber/cm − 1

FIGURE 3.1 Absorbance FTIR spectra of LM4 with the assignment of the main bands. A: Untreated. B: Treated with Na 2 CO 3 . (From Zhang, H. et al., Biopolymers, 59, 38, 2001. With permission.)

kept at 30˚C. Although a clear phase separation could not be achieved as has been done for many solutions of synthetic polymers in organic solvents, cloud-like flakes appeared in the solution after 1 day. The flakes were removed and methanol was added again. By repeating this procedure, four fractions (F1 to F4) were obtained. 10

Fractions with different molecular weights were also prepared by Shimizu Chemical Co. (Hiroshima, Japan) using an enzymatic degradation method. The native and nondegraded konjac glucomannan (ND) were treated with an enzyme (SP-249, Novo Nordisk A/S, Copenhagen, Denmark) for different reaction times at ambient temperature, and four fractions with low molecular weights, LM1 to LM4, were obtained. 11

3.2.3 M OLECULAR W EIGHT

The molecular weight of each KGM fraction was determined by gel permeation chromatography (GPC) at room temperature. KGM was dissolved in cadoxen to obtain a dilute solution because of its very low solubility in aqueous solutions. 1 Cadoxen is known to dissolve cellulose and to give a clear, colorless, and stable

solution. 12 A colorless solution allows the use of an RI detector, 13 and the much lower viscosity of the cadoxen solution compared to that of aqueous solution is a great advantage for GPC analysis. The peak corresponding to the highest molecular

weight fraction (8.53 × 10 5 ) of pullulan appeared at 11.73 min, and that corre- sponding to the lowest one (5.80 × 10 3 ) appeared at 15.07 min. The standard pullulan samples 14 gave a good linear calibration curve (data not shown). The weight-average molecular weight (Mw) for five KGM samples decreased with increasing the reaction time with the enzyme. The molecular weight distribution

Functional Food Carbohydrates

was not narrow judging from the chromatograms. The elution curve for ND showed the second peak in the lower molecular weight region. The sample did not flow

homogeneously through the columns, probably due to the high viscosity. This behavior was typically observed in a highly viscous solution, and the true average molecular weight could be higher. Even in cadoxen, which forms a KGM solution with far lower viscosity than in water, it was difficult to prepare a suitable solution for GPC analysis, especially in the case of the highest molecular weight fraction. Native (nondegraded) KGM is known as a gum with very high viscosity, and it is difficult to determine the molecular weight of native KGM by GPC. The ND

sample contains molecules with molecular weights higher than 1.0 × 10 6 , but no lower than 1.0 × 10 4 molecular weight fraction. A similar wide distribution of molecular weights was also reported for aqueous konjac mannan. 15 It ranged from

4.0 × 10 4 to more than 1.0 × 10 6 and the polymer eluted over almost the entire range of elution volumes for the GPC gels (4.0 × 10 4 to 2.0 × 10 7 ). The enzyme treatment increased the ratio of lower molecular weight polymers; however, the fraction of higher than 1.0 × 10 6 molecular weight still remained, even in the lower molecular weight fraction LM1. The exclusion limit for the GPC column was assumed to be 5.0 × 10 7 because there are no standard polymers with higher than

1.0 × 10 6 molecular weight. It is difficult to assign a molecular weight for the earlier eluted fraction. Therefore, high accuracy for the molecular weight obtained from GPC should not be expected. It is certain, however, that the KGM fractions have different molecular weights.