Utilization of rice bran to prepare oligosaccharides by enzymatic method

UTILIZATION OF RICE BRAN TO PREPARE

OLIGOSACCHARIDES BY ENZYMATIC METHOD

Trƣơng Thị Phƣơng Khanh¹, Rumpagaporn Pinthip²

¹Viện Khoa học Ứng dụng, trường Đại học Công nghệ TP. Hồ Chí Minh (HUTECH)

²Khoa Agro-Industry, trường Đại học Kasetsart, Băng Cốc, Thái Lan

TÓM TẮT

Oligosaccharides (OS), các prebiotics tiềm năng, được chiết xuất từ arabinoxylan (AX) có trong cám gạo

bằng việc sử dụng enzyme xylanase thương mại. Tuy nhiên, việc sử dụng các loại cám gạo khác nhau có

thể gây ảnh hưởng tới hiêu suất thu hồi OS. Bài nghiên cứu này đã sử dụng ba loại cám gạo khác nhau để

chiết xuất OS bằng enzyme Ultraflo Max và sau đó xác định các cấu trúc OS có thể có. Cám gạo AX được

chiết xuất từ cám gạo thương mại đã tách béo (COM) mang lại lượng OS lớn nhất (83,39 mg/g RBAX),

tiếp theo là giống lúa Sanpahtawng (27,05 mg/g RBAX), và cuối cùng là giống lúc Chainat1 (21,53 mg/g

g RBAX), được chứng minh qua quá trình thủy phân enzyme Ultraflo Max. Kết quả đáng chú ý là A³X là

sản phẩm chính trong tất cả các sản phẩm thủy phân từ AX khi sử dụng Ultraflo Max. Vậy nên Ultraflo

Max là một enzyme thương mại phù hợp để chiết xuất OS chuỗi ngắn từ cám gạo AX.

ABSTRACT

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.

1. INTRODUCTION

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

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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. MATERIALS AND METHODS

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 NaClO₂prior 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.

3. RESULTS AND DISCUSSION

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

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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

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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.

LITERATURE CITED

[1] Bataillon M, Mathaly P, Cardinali A-PN, Duchiron F (1998) Extraction and purification of

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