Utilization of rice bran to prepare oligosaccharides by enzymatic method

Trƣơng Thị Phƣơng Khanh1, Rumpagaporn Pinthip2  
1Vin Khoa hc ng dng, trường Đại hc Công nghTP. HChí Minh (HUTECH)  
2Khoa Agro-Industry, trường Đi học Kasetsart, Băng Cốc, Thái Lan  
Oligosaccharides (OS), các prebiotics tiềm năng, được chiết xut tarabinoxylan (AX) có trong cám go  
bng vic sdụng enzyme xylanase thương mại. Tuy nhiên, vic sdng các loi cám go khác nhau có  
thgây ảnh hưởng ti hiêu sut thu hi OS. Bài nghiên cứu này đã sử dng ba loi cám go khác nhau để  
chiết xut OS bằng enzyme Ultraflo Max và sau đó xác định các cu trúc OS có thcó. Cám gạo AX được  
chiết xut tcám gạo thương mại đã tách béo (COM) mang lại lượng OS ln nht (83,39 mg/g RBAX),  
tiếp theo là ging lúa Sanpahtawng (27,05 mg/g RBAX), và cui cùng là ging lúc Chainat1 (21,53 mg/g  
g RBAX), được chng minh qua quá trình thy phân enzyme Ultraflo Max. Kết quả đáng chú ý là A3X là  
sn phm chính trong tt ccác sn phm thy phân tAX khi sdng Ultraflo Max. Vy nên Ultraflo  
Max là một enzyme thương mại phù hợp để chiết xut OS chui ngn tcám go AX.  
Oligosaccharides (OS), potential prebiotics, can be produced from rice bran arabinoxylan (AX) using  
commercial xylanase enzyme. However, differences in rice bran cultivars may affect extracted  
oligosaccharides (OS) yields. This study investigated extracted OS structures derived from three different  
rice bran AX using Ultraflo Max. Rice bran AX extracted from commercially defatted rice bran (COM)  
yielded the greatest OS amount (83.39 mg/g RBAX), followed by that of the Sanpahtawng cultivar (27.05  
mg/g RBAX), and lastly, the Chainat1 cultivar (21.53 mg/g RBAX), as evidenced via Ultraflo Max  
enzyme hydrolysis. Interestingly, A3X was the primary OS product in all rice bran AX hydrolysates  
prepared by Ultraflo Max. Ultraflo Max was therefore a suitable commercial enzyme for short-chain OS  
conversion from rice bran AX.  
Keywords: Xylanase, prebiotic, oligosaccharides, rice bran, Ultraflo Max.  
Oligosaccharides (OS), consisting of xylooligosaccharides and arabinoxylooligosaccharides, are a partial  
hydrolysis product of arabinoxylan (AX) with beneficial prebiotic properties [3]. Enzymatic hydrolysis is  
potentially superior for OS preparation, being more environmentally-friendly with fewer undesirable by-  
products compared to other chemical and physical methods [10]. Xylanases were preferable due to their  
endo-action on xylan backbone [7]. Previous studies reveal influence and efficiency of xylanases on wheat  
bran [2] and its alkali-extractable AX [7]. However, xylanase impact research on rice bran arabinoxylan  
(RBAX) is limited. This study, therefore, focused on the influence and efficiency of commercial xylanases  
on alkali-extractable RBAX for OS preparation. The composition of OS obtained from different varieties  
of RBAX was investigated by high-performance anion-exchange chromatography coupled with pulsed  
amperomatic detector (HPAEC-PAD) and yields of OS were calculated. This information will be useful  
for rice bran OS production.  
2.1. Materials  
Two cultivars of Thai rice (Oryza sativa L.) paddy, Sanpahtawng (SPT) and Chainat1 (CN), were bought  
from the Rice Research Center, while commercially defatted rice bran (COM) was kindly provided by  
Thai Edible Oil Co., Ltd. All enzymes were kindly given by Novozymes (Bagsvaerd, Denmark)  
2.2. Methods  
2.2.1. Preparation of alkali-soluble arabinoxylans from different rice bran cultivars  
Alkali-soluble AX preparation from rice bran followed that of the previous publication [6]: n-hexane  
discarded the fat, both Termamyl 120L and amyloglucosidase (AMG 300L) hydrolyzed the starch, and  
Alcalase 2.4L removed the protein from rice bran. After that, delignification was applied by 72% H2SO4  
with NaClO2 prior to use 0.5M NaOH (at 40 °C for 6 h) for crude AX extraction [1]. Lastly, a dialysis bag  
(Spectra Por, cut-off 3.5 kDa) was used to enhance the purity of isolated AX, following the method  
described in [4]. The collected supernatant was dried in a hot-air oven, namely alkali-soluble AX, for  
further enzyme hydrolysis.  
2.2.2. Enzymatic treatment for alkali-soluble AX  
Alkali-soluble AX (3% w/v) was re-suspended in a sodium acetate buffer (25 mM) in an Erlenmeyer  
flask. The designated pH value was obtained with 0.1 N HCl and/or NaOH. Under continuous stirring at  
50 °C for 24 h, suspension with a suitable amount of Ultraflo Max was incubated. Later, the hydrolysate  
was boiled for 15 min for enzyme inactivation and centrifuged at 4,000 x g for 15 min to separate  
supernatant from residue.  
2.2.3. Determination of oligosaccharides  
Supernatants were characterized by HPAEC-PAD following Rivière‘s method [8]. CarboPac PA-200 with  
guard column identified and quantified extracted AX-OS and Chromelon 6.7 (Thermo Scientific) provided  
system control and data analysis. Gradients of 120 mM NaOAc in 100 mM NaOH, and 100 mM NaOH  
were used for AX-OS analysis with a total analysis time of 37.5 min. The external standard for  
oligosaccharides analysis was a mixture of arabinose, xylose, and AXOS (A2XX, A3X, XA3XX, A2+3XX)  
and an XOS (DP 2-6) mixture.  
2.2.4. Statistical analysis  
All statistical analysis was performed using SPSS software (Version 21, IBM Corp., USA). Analysis of  
variance with Turkey HSD was used to compare means. A level of 0.05 was set to determine statistical  
significance of differences.  
From our previous work, the monosaccharide compositions of all alkali-soluble AX were analyzed and  
arabinose/xylose (A/X) ratios were calculated to approximate their degree of branching. The A/X ratio of  
RBAX extracted from CN (1.09) was the highest, followed by that of COM (1.04), and SPT (0.76). OS  
structures derived from all AX by Ultraflo Max were characterized by HPAEC-PAD after 24 h of  
incubation (Figure 1). The high A/X ratio indicates a greater degree of branching and therefore a relatively  
higher solubility [9]. The A/X ratio of AX from SPT was lower than those from COM and CN, however,  
the COM hydrolysate contained the highest content of OS. The side chains of AX may hinder active  
xylanase sites, thereby affecting OS production. The different structures of the arabinose-substituted/-  
unsubstituted xylan backbone also affected OS yield.  
Figure 1. HPAEC-PAD chromatograms of OS extracted from COM AX 50 time dilution (a), Sanpahtawng  
(SPT) cultivar 10 time dilution (b) and Chainat1 (CN) cultivar 10 time dilution (c) by Ultraflo Max for 24  
h. Arabinose (1), Xylose (2), X2 (3), X3 (4), X4 (5), X5 (6), X6 (7), A2XX (8), A3X (9), XA3XX (10), A2+3XX  
(11) components were indicated  
Peaks corresponding to Ara, Xyl, X2, X3, X4, X5, X6, A2XX, A3X, XA3XX and A2+3XX were clearly  
observed in all RBAX hydrolysates. COM hydrolysate also yielded the most OS content (83.39 mg/g  
RBAX), followed by SPT (27.05 mg/g RBAX) and CN (21.53 mg/g RBAX) (Figure 2). Interestingly, the  
main OS product found in Ultraflo Max was A3X (61.22 mg/g RBAX for COM), followed by SPT (19.54  
mg/g RBAX), and CN (14.92 mg/g RBAX). The Ultraflo Max commercial enzyme is a mixture of  
xylanase (GHF10) and β-glucanases. The different accessory enzymes containing in Ultraflo Max and  
hydrolysis mechanism of GHF 10 xylanase may affect the structure of extracted OS.  
Figure 2. The amount of OS extracted from different rice varieties of rice bran arabinoxylan (RBAX) by  
Ultraflo Max for 24 h. Commercially defatted rice bran (CDRB), Chainat1 (CN1) and Sanpahtawng (SPT1)  
were used to extracted with Ultraflo Max. Arabinose (Ara), xylose (X1), xylobiose (X2), xylotriose (X3),  
xylotetraose (X4), xylopentaose (X5), xylohexaose (X6) and 2,3-α-L-Ara-(1-4)-β-D-xylotriose (A2XX), 3,2-α-  
L-Ara-(1-4)-β-D-xylobiose (A3X), 3,3-α-L-Ara-(1-4)-β-D-xylotetraose (XA3XX), and 2,3-di-α-L-Ara-(1-4)-  
β-D-xylotriose (A2+3XX) were analyzed.  
The presence of A3X in all rice bran hydrolysates clearly revealed the GHF10 xylanase mechanism.  
Similar AXOS structures were found in a study by Mathew [7]. including A3X, A2XX and A2+3XX when  
wheat bran AX was hydrolyzed with GHF10 xylanase. This also indicated that differences in substrate  
structure might also affect the final products. Furthermore, the high amount of arabinose and xylose  
produced from RBAX hydrolysis might be due to the side activities commercial enzyme. This could  
release the branched chain of AX and enhance the conversion efficiency of RBAX into OS by lowering  
the A/X ratio to favor the enzyme breakdown on the xylan main chain per the previous study [5].  
In conclusion, oligosaccharides (OS) from rice bran AX could be solubilized by Ultraflo Max. The  
amount and type of OS produced from rice bran AX could depend on the rice cultivar. Rice bran AX  
extracted from commercially defatted rice bran is the best source for OS production, compared to those  
from Sanpahtawng and Chainat1 cultivars.  
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