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研究概要

高オリゴ糖を含有したオカラ繊維の生産技術及び機能性評価

研究機関名

中国天津科技大学・食品工程及び生物技術学院

代表者

陳野、高辰、閻暁光

本研究の主旨

1. Introduction
Okara, the main by-product in the processing of soy milk, tofu, and other soybean products, is a good source of dietary fiber. The content of dietary fiber in okara is about 60%; however, the content of SDF is very low. In China, only a small amount of the okara produced is used as feed; most is discarded as waste, resulting in a tremendous waste of resources. In order to increase the SDF content of okara, different approaches were developed, such as chemical or enzymatic treatments. Of these methods, extrusion is regarded as the most effective and fastest, in addition to its already widespread usage in food processing applications. In this research, fresh okara was taken as raw to obtain the Okara dietary fiber with high oligosaccharide content by the combination of extrusion and β- mannanase enzymatic. This provides a good way to take high-value advantage of okara and other agricultural by-pruducts.

2. Materials and Experiments
2.1. Optimization of extrusion parameters
Fresh okara with a moisture content of 82% was extruded to produce specimens with a moisture content of 55-65% by a laboratory scale twin-screw extruder (SYSLG32-Ⅱ, Jinan Saibainuo Science and Technology Development Co. Ltd., Jinan, China). The die diameter was 4mm. The extruder was equipped with three barrels. The barrel diameter (D) and its length (L) to diameter ratio (L/D) were 32 mm and 18:1, respectively. The extrusion temperature was set to 170℃ and the screw speed was set to 150 r/min. The extruded okara specimens were collected, dried at 60°C, and then ground and sieved (60 mesh) to obtain extruded okara powder. The same method was used to obtain fresh okara powder as the control.
2.2. Extraction of SDF
10 g of okara samples was mixed with 400 mL of water and refluxed at 121°C for 60 min. The mixture was centrifuged at 3000×g for 20 min. All supernatants were combined and hydrolyzed with α-amylase and amyloglucosidase for 30 min. The pH of solution was adjusted to 4.5. Then, the solution was hydrolyzed by adding protease for 1 hour. After high-temperature enzyme inactivation, the solution was centrifuged at 5000×g for 30 min. The supernatants were concentrated to 10% of the original volume under a vacuum. Then, it was precipitated by adding four times the volume of 95% (v/v) ethanol at 4°C for 24 h. The sediment was centrifuged (3000×g, 15 min) and vacuum freeze dried. The SDF samples from the extruded okara and untreated okara were thus obtained.
2.3. Preparation of crude oligosaccharides
3 g Okara SDF was dissolved in 150 mL distilled water. Enzymatic the SDF with β-mannanase to improved the content of oligosaccharides. The enzymatic parameters were: enzymatic temperature: 60℃; hydrolysis time: 4 h; enzyme and substrate mass ratio: 1:50. The hollow fiber ultra-filtration method was used to separate the crude oligosaccharides in the hydrolysates. The cut off molecular weight was 10 kDa; ultra-filtration pressure was under 0.1 MPa; Ultra-filtration 2 times. The filtered fluid were collected then concentrated and lyophilized to obtain the crude oligosaccharides solid.

3. Results and discussion
3.1 Effect of extrusion parameters on the SDF content of Okara
After extrusion, the okara SDF content increased from 6.7% to 30.1%. This may be explained by the accelerated depolymerization of the polysaccharide’s glucosidal bonds induced by high temperature and high pressure, resulting in improved solubility of the dietary fiber
3.2 Molecular weight of okara SDF
The SDF molecular weight extruded okara was determined and the results are shown in Table 1.

As shown in the above, content of the mainly component in extraction SDF was 91.87% which molecular weight was 117 kDa. The oligosaccharides content was only 0.63%.
3.3 Crude oligosaccharide
The crude oligosaccharide was obtained after the SDF was hydrolyzed and ultra-filtrated. The yield of it was about 37.67g per 100g SDF. Molecular weight of the crude oligosaccharide was shown in Table 2.

Compared with the SDF, the molecular weight of the crude oligosaccharide has changed greatly. There are four components of oligosaccharide in the crude oligosaccharide, the molecular weight were 1349, 847, 682 and 349 Da respectively. The total content was 40.31%. The highest content component was 1349 Da, the content of it was 27.65%, it may be speculated was a kind of oligosaccharide with the polymer of 8 according to its molecular weight. In addition, small-molecule polysaccharides (molecular weight 8357 and 4711 Da) also accounted for about 12.99% of the total crude oligosaccharide product. This indicated that β-mannanase really degraded part of the SDF, converted it to oligosaccharides and small-molecule polysaccharides. This result suggested that the combination of extrusion and enzymatic to obtain high oligosaccharides-containing okara dietary fiber is feasible. After extrusion and enzymatic, the oligosaccharide content in okara was about 3.70% (30.10%×37.67%×40.31%). It was far beyond the original oligosaccharide content in okara.
3.4 Separation of the main oligosaccharide
The molecular weight of the main component in crude oligosaccharide was 1349 Da, therefore, Sephadex G-25 gel was chosen to separate this component of oligosaccharide. In dextran gel column chromatography, the larger molecular weight components were early eluted by mobile phase, so the tubes of it were at front. In this research, the molecular weight of the target component was 1349 Da, the content of it was 27.65%. So the first big-eluting peak after No.52 tube was the target component. The liquid in No.59 - No.62 were collected as the main oligosaccharide in okara SDF. The elution curve of the collection was a single and symmetrical peak. This indicated that the collection was a single component oligosaccharide.

3.5 Monosaccharide composition of the main component oligosaccharide

Fig. 3 GC results, (a): complex standard monosaccharide derivative, (b): The main oligosaccharide component in okara SDF
Fig. 3(a) is the GC chromatogram of a standard monosaccharide. As the results show, the capillary column OV-1701 could separate the nine types of monosaccharide well. The elution order was: fucose (20.044 min), ribose (20.335 min), rhamnose (20.943 min), arabinose (21.446 min), xylose (22.842 min), sorbitol (32.090 min), mannose (33.048 min), galactose (33.591 min), and glucose (34.749 min).
Fig. 2(b) is the GC chromatogram of the oligosaccharide. As the results shows, the main component oligosaccharide in SDF was mainly composed of four kinds of monosaccharide. Based on the relative content, the concentrations of galactose and mannose were very high, 39.81% and 31.78%, respectively.

Conclusion
Extrusion significantly increased the content of SDF in Okara. Okara dietary fiber with high oligosaccharide content was obtained by β-mannanase enzymatic. The oligosaccharide content increased from 0.63% to 3.70%, increased nearly five-fold. The molecular weight of the major oligosaccharide component was 1349 Da, the content of it was about 27.65%. It was mainly composed of rhamnose, sorbitol, mannose and galactose. The concentrations of galactose and mannose were very high, 39.81% and 31.78%, respectively.

Acknowledgement
This research was kindly supported by Key Projects of Tianjin Science and Technology Support program. (10ZCKNC01900)

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