Short communication

Enhancement of the yield of c-aminobutyric acid by Aspergillus

oryzae and antioxidant activities of rice bran through explosion

puffing processing

Jiaofei Wang,1 Gao Chen,1 Yong Han,1 Ye Chen1* & Ran Ye2*

1 National Key Laboratory of Food Nutrition and Safety of Ministry of Education, Tianjin University of Science and Technology, Tianjin

300222, China

2 Department of Biosystems Engineering and Soil Science, University of Tennessee, 2506 E.J. Chapman Drive, Knoxville, TN 37996-4531,

USA

(Received 18 October 2013; Accepted in revised form 29 November 2013)

Keywords Antioxidant activity in vitro, explosion puffing processing, rice bran, c-aminobutyric acid.

Introduction

As a common cereal grain, rice is considered as one ofthe most principal foods around the world. In China,the annual yield of rice reaches up to approximately 2billion tons. Rice bran is a main by-product obtainedfrom rice processing, accounting for approximately12% of the yield of rice (Hoke et al., 2007; Huanget al., 2013). It contains lipopolysaccharide, oryzanol,squalene, ceramide, tocopherols (Laokuldilok et al.,2011) and numerous other bioactive substances, all ofwhich have great positive significance on human healthand disease prevention. Hence, rice bran can be poten-tially used as an inexpensive starting material for theproduction of multiple bioactive substances, which areviewed as high-value-added products with variousapplications in food and agricultural industries (Jang& Yang, 2008). c-aminobutyric acid (GABA), a bioac-tive substance in rice bran, is an efficient inhibitionneurotransmitter with beneficial health effects onhypertension, cancer and diabetes (Jun et al., 2012).

Microbial fermentation has been employed toimprove GABA content in rice bran by decarboxyl-ation of microbial cells. In general, microorganismssuch as lactic acid bacteria, yeast and Escherichia colicontain the glutamic acid decarboxylase (GAD) (Rata-naburee et al., 2011, 2013) that can transform thedecarboxylation of glutamate into GABA and CO2.

Explosion Puffing Process (EPP), a new processingtechnology, has been utilised in the processing ofapple, potatoes (Nath et al., 2007) and winter jujube(He et al., 2013). Briefly, the samples with different

water contents are heated at an elevated temperatureand pressure in a sealed chamber by superheatedsteam or electric heater. As the temperature andpressure inside the chamber reach a certain point, thepressure was suddenly released, leading to the occur-rence of explosion and evaporation of the moisturerapidly from food. Compared to the conventional dry-ing technology, EPP can efficiently offer the high qual-ity of the dehydrated food products, such asmaintaining original colour and flavour of the product(Hoke et al., 2007; Bi & Wei, 2008; He et al., 2013).In addition, EPP caused the production of food mate-rials with porous textures, resulting in the release ofinternal bioactive compounds (Lee et al., 2010). In thisstudy, EPP equipment used (Fig. 1) was designed con-sisting of three components: a steam generator, a vac-uum tank and a puffing tank. Initially, the rawmaterials were loaded into the puffing tank to beheated by steam, followed by the high pressure. Whenreaching upon the desired temperature and pressuresettings, the tank pressure was suddenly releasedresulting in explosion puffing of the raw material,which changes its microscopic structure and nutrientcontent (He et al., 2013).Most of studies pertaining to EPP centred on drying

of fruits and vegetables (Bi & Wei, 2008; He et al.,2013). No attempt in literature has been made to pre-treat rice bran using EPP for fermentation. Herein, thepurpose of this research was to study the effect of anEPP system on the pretreatment of rice bran and fur-ther assessment of the yield of GABA obtained fromthe fermentation by Aspergillus oryzae under the opti-mal conditions. In addition, the antioxidant activity ofthe fermented rice bran was also evaluated.

*Correspondent: Fax: +86 22 60601425;

e-mails: chenye@tust.edu.cn and ran.ye.email@gmail.com

International Journal of Food Science and Technology 2014, 49, 1420–1424

doi:10.1111/ijfs.12478

© 2013 Institute of Food Science and Technology

1420

Materials and methods

Materials

Rice bran was provided by Jinyuanhai Rice IndustryCo., Ltd. (Tianjin, China); A. oryzae was obtained fromChina’s Microbiological Center (Tianjin, China). All theother reagents in the experiment were of analyticalgrade. The EPP device (TQDPH10-1) in Fig. 1 was man-ufactured by Tianjin Qinde Co., Ltd. (Tianjin, China).

Explosion puffing processing treatment of rice bran

Initially, a batch of rice bran and the required amountof DI water were poured into the puffing tank(U1500 9 U1000 9 1850 mm) for achieving differentmoisture contents (14–60%). Then, the puffing tankwas heated to the required temperature (50–80 °C) for30–120 min. Subsequently, the pressure in the tankwas increased gradually. When reaching a certain pres-sure (0.4 MPa in this study), the pressure was rapidlyreleased. The vacuum pressure in the puffing tank was�0.1 MPa. The volume of the vacuum tank was 4 m3,and the steam production capacity and the maxpressure of the steam generator were 28 kg h�1 and0.4 MPa, respectively. The specifications of the vac-uum system: the power and pumping speed of rootsvacuum pump were 1.1 Kw and 70 L s�1, respectively.The compression of air compressor was 0.6 m3 min�1,and the pressure was 0.8 MPa. The instant reductionin pressure leaded to a quick evaporation of waterfrom rice bran, leading to a porous structure.

Culture medium and liquid fermentation

The A. oryzae (As 3.951) was grown in a PDA slantmedium at 30 °C and 80% relative humidity for 7day. The liquid activation medium was composed of:200 g L�1 of potato juice, 2.0 g L�1 of yeast extract,1.0 g L�1 of KH2PO4 and 0.6 g L�1 of MgSO4. Slantcolonies were cultured at 30 °C for 16 h, at 130 rpm.Ten gram (10 g) of puffed rice bran and of 100 mL

water were packed into a flask and autoclaved at121 °C for 20 min. Seven point five per cent (7.5%) ofthe liquid medium was inoculated at 28 °C for 2 dayat 130 rpm.

Determination of c-aminobutyric acid

Two thousand microlitre of borate buffer solution(0.4 M, pH 10.4); 400 lL of the sample, and 400 lL ofthe OPA derivative reagent were gently mixed for5 min. The solution was then filtered through a 0.45-lmfilter (EMD Millipore Corp., Billerica, MA, USA) andanalysed by high-performance liquid chromatography(HPLC). HPLC separations were performed in aD-2000 Elite Series instrument (Hitachi, Schaumburg,IL, USA) with an Agilent TC-C18 column (4.6 9 250 mm,5 lm). The column temperature was at 40 °C and theultraviolet detector was set at 338 nm. The mobile phaseconsisted of a mixture of 1.6 g of CH3COONa • 3H2O,600 mL of water, 200 mL of methanol and 200 mL ofacetonitrile at a pH of 7.2 (Yang et al., 2008).

Study of antioxidant activity in vitro

One hundred microlitre (100 lL) of fermented prod-uct was added to 900 lL of 100% ethanol contain-ing 100 lmol of DPPH. After being mixed andstored at room temperature in darkness for 30 min,the absorbance of DPPH was determined by a spec-trophotometer at 517 nm. The DPPH radical-scav-enging activity was calculated according to thefollowing equation:

DPPH ð%Þ ¼ ½ðA� BÞ=A� � 100%;

where A is the absorbance at 517 nm with blank andB is the absorbance at 517 nm with products.

Statistical analysis

Averages and standard deviations (SD) from duplicatemeasurements of each sample were reported. ANOVA

12

3Figure 1 The explosion puffing processing

system: (1) Steam generator; (2) Vacuum

tank; (3) Puffing tank.

© 2013 Institute of Food Science and Technology International Journal of Food Science and Technology 2014

Explosion puffing processing J. Wang et al. 1421

and Student’s t-test were applied to analyse theresults, at the significance level of P-value <0.05 usingthe SAS software (SAS Institute Inc, Cary, NC,USA).

Results and discussion

Effects of the puffing parameters on c-aminobutyric acidyield

Figure 2 showed that GABA content increased ini-tially until the water content reached 50%. After-wards, a slight decrease in GABA occurred. Thepuffing impact was closely related to the moisture con-tents of the raw materials because the release ofexpanded water vapour pressure in the system resultedin structural changes in the raw material (Lee et al.,2010). The presence of the appropriate moisture con-tent in the system enabled sufficient water vapour forpuffing. Puffed rice bran showed a honeycomb or sheetstructure, with a starch chain and peptide chainexposed, allowing the formation of a large area withsmaller steric hindrance for enzymatic reaction duringfermentation (Lee & Lee, 2009). When the moisturecontent was >60%, rice bran failed to absorb extrawater in the system, leading to the exudation of thesoluble nutrients with water, and thereby resulting inthe loss of GABA content.

With the increase in the puffing temperature, GABAcontent in fermented rice bran increased until 70 °C(Fig. 2b). The moderate high temperature can promotethe breakage of internal macromolecular, leading to therelease of soluble polysaccharides, lipids, crude proteinsand other chemicals simultaneously (Spears et al., 2004;Hoke et al., 2007; Chen et al., 2014). The moderate tem-peratures (50–70 °C) during EPP facilitated saccharifi-cation and degradation of insoluble ingredients instarch, depending upon the amount of water in thesystem (Sagol et al., 2006; Kashaninejad et al., 2007;Briffaz et al., 2014). When the temperature was too high(>70 °C), it caused the presence of protein denaturationand sugars–protein coagulation due to the Maillardreaction and further the reduction of reducing sugarcontent in fermentation media (Wang et al., 2013).

For the heating time, GABA content reached themaximum value at 90 min and showed a steadydecreasing trend (Fig. 2c). Heating provides sufficientenergy for the removal of extra water and the degra-dation of internal macromolecular for the release ofsoluble sugars and proteins (Singkhornart et al.,2013), benefiting microbial growth and GABA accu-mulation. Insufficient heating time allowed the pres-ence of redundant water. Conversely, excessive heatingtime caused the formation of by-products from theMaillard reaction and hindered the production ofGABA.

(a)

(b)

(c)

Figure 2 Effects of (a) moisture content: The puffing temperature

was 700 °C for 90 min. (b) puffing temperature: The sample contain-

ing 60% of moisture was heated for 90 min and (c) heating time:

The sample containing 60% of moisture was heated at 700 °C on

GABA content in fermented media using rice bran. All experiments

were performed in duplicate.

© 2013 Institute of Food Science and TechnologyInternational Journal of Food Science and Technology 2014

Explosion puffing processing J. Wang et al.1422

Under the optimal conditions (water content of 47%,puffing temperature of 74 °C, and heating time of83 min, which were optimised by response surface meth-odology and preliminary data is not shown), the highestyield of 144.2 mg (c-aminobutyric acid)/100 g (the reac-tion media) was achieved. It is a more than twofoldincrease compared to the control (67.9 mg per 100 g).The chromatograms of GABA obtained from the pre-treated sample and control were displayed in Fig. 3a, b.

DPPH radical-scavenging rate

The positive relationships between mass concentrationand the radical-scavenging rate are shown in Fig. 4.The antioxidant capacity of the fermented rice brandisplayed the highest scavenging activity (~90%) whenthe concentration of fermented rice bran reached8 mg mL�1, which is significantly higher (P < 0.05)than the control (~65%). Indeed, some natural antioxi-dants in rice bran, such as tocopherols, tocotrienolsand oryzanols, favoured the high scavenging activity(Laokuldilok et al., 2011; Pitija et al., 2013). Further-more, the different types and concentrations ofphenolic compounds in rice bran gave rise to theantioxidant activity (Jun et al., 2012; Sukhonthara &Theerakulkait, 2012). It was illustrated that EPP essen-tially contributes to the release of natural antioxidantsfrom rice brans, thereby accounting for the improve-ment of the antioxidant activity of the treated sample.

Conclusions

In summary, our results suggest that EPP can signifi-cantly promote the final concentration of GABA bypretreating rice bran. Under the optimal conditions, ayield of 144.2 mg per 100 g was obtained from thepretreated rice bran, more than twofold higher thanthat of the control. Moreover, the fermented rice branshowed high scavenging activity, indicating that thefinal products could be used as a high-value-addedfood and feed ingredients. This study advances aninnovative food processing technology for the produc-tion of useful chemicals from cereal grains.

Conflict of interest

Authors have no conflict of interest in this study.

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(a)mV300

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0

mV300

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0

0 2 4 6 8 10 12 14 16 min

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