Rice bran (RB) is a by-product of the rice milling process, it amounts to nearly 7-8% of the rice grain ^[1], and is a source of protein, fiber and lipids, basically unsaturated fatty acids ^[2]. After its production in the mill it undergoes rapid enzymatic hydrolysis caused by lipases activity on the bran lipids released from the cells in the milling process, which results in the decomposition of lipids into free fatty acids (FFA) ^[3]. Therefore, because of the biochemical instability of rice bran, occurring immediately after the polishing of rice grain, it has a short shelf life due to the decomposition of its lipids which renders it unsuitable for human consumption. However, if the bran is subject to a thermal treatment of high temperature and short time immediately after polishing, the lipase activity is reduced, thus producing a bran with longer shelf life which is suitable for human consumption. Thermal treatments to bran rice for stabilization has been applied throw microwaving, extruding, steaming and parboiling ^[4-11]. Chemical treatments have been also used to prevent rancidity of rice bran ^[12,13]. This study was conducted to determine the effect on the content of FFA resulting from any residual enzyme activity in fresh rice bran after being thermally treated by a double screw extruder and stored for a period of time under certain specific conditions. The treated rice bran was stored for a period of two months, packed following two different packaging methods (with and without vacuum) and two ambient conditions (temperature and relative humidity). Rice bran samples preserved by the procedure described above were used in other project, oriented to the development of a ready-to-eat cereal processed by the extrusion and enriched with fibers from rice and maize brans.

The effects of extrusion on bran stability as measured by the FFA content of stored samples are shown in Figs. 2 and 3.

Figure 2 Source: The authors.

Figure 3 Source: The authors.

It was found that there was a difference between the two levels of each factor during storage of the samples of bran in function to the contents of FFA. The statistical analysis indicated that the packaging method showed a significant difference (p <0.05) in the content of FFA in the stored bran samples, in both raw and extruded ones, and at both ambient storing conditions. The untreated rice bran showed a significantly higher increase in FFA content compared to the heat-treated rice bran extrusion, regardless of packing methods at both ambient storage conditions. Throughout the experimental period, the lower values of FFA content were observed for the extruded samples stored at 18° C - 70% RH and packaged without vacuum.

Figs. 2 and 3 it is show a constant increase in FFA levels for both packaging methods during the four-week storage period; week 6 and week 8, an accelerating rate of increase of the FFA content was observed in the bran extruded and packed under vacuum and a significantly higher levels of FFA were presented in the extruded sample, packaged without vacuum when stored at 30° C - 78% RH (Fig. 2). The FFA content in bran extruded samples kept in a vacuum packaging was higher than those of the samples stored in laminated bags metalized without vacuum and kept at 18° C - 70 % RH for 8 weeks (Fig. 3). Changes in FFA contents in samples were significant between the packing methods at both ambient conditions. Additionally, the FFA content of samples kept in the vacuum packaging was consistently higher than that in the samples without vacuum packaging over an 8-week storage period at both ambient conditions 18° C - 70 % RH and 30° C - 78 % HR (Fig. 2 and 3).

The increased content of FFA of rice bran without heat treatment by extrusion during the storage test in this study is similar to the results reported in other studies ^[3,5,17-19]. It is a typical trend the rapid development of FFA caused by hydrolytic rancidity in raw rice bran makes this material unsuitable in food formulations for human consumption ^[18,20] also observed that in rice bran without being processed by the extruder, there was a decrease in the enzymatic activity of lipases as the storage temperatures were lower.

The FFA content in samples stored in both packaging methods was lower when the samples were kept at 18° C - 70 % (Fig. 3). While in the sample stored at 30° C - 78% the level of FFA increased in a non-vacuum packaging, the increase of FFA was higher in samples kept under the vacuum-packing (Fig. 2). Sharp and Timme ^[21] noted the same pattern in brown rice stored in bags without vacuum versus those in vacuum packaging. Ramezanzadeh et al. ^[5] reported a similar behavior in the raw rice bran treated by microwave heating and stored in bags with hermetic sealing and in bags with vacuum sealing and temperatures of 4-5° C and 25° C for 16 weeks. This could be due to the removal of air and oxygen in vacuum packaging, thus observing that the bran samples untreated by extrusion had a possible activation of anaerobic microorganisms that caused increased lipase enzyme activity in the samples of rice bran.

Chahinian et al. ^[22] in studies on lipase production by microbial metabolism noticed that in the type I lipase, which specifically acts on triacylglycerols, the microbial culture grew without aeration. Lipase type II, which is only activated partially on acylglycerols, had an optimum growth in microbial culture with aeration, thus suggesting that in the storage process of rice bran, lipases with greater proportion of type I are activated. Additionally, Randall et al. ^[20] performed a count of anaerobic microorganisms in raw rice bran and extruded one, with results of 82 x 10³ and 9 x 10² bacteria/g, respectively, which certifies high levels of microorganisms growing in a culture medium without the presence of air in the raw bran.

After 8 weeks of storage, in extruded rice bran, a FFA content level of 13.9 and 9.7% in samples packaged with and without vacuum was obtained, respectively, when stored at 30° C - 78 % (Fig. 2) and a FFA level of 9.3 and 7.8% for samples packaged with and without vacuum, respectively, and stored at lower temperature and relative humidity storage (18° C - 70%) (Fig. 3). Bran oil with an excess of 10% of FFA is considered unfit for human consumption ^[18]. The rate of formation of FFA in the bran or brown rice flour is high. Approximately 30% of lipids may be hydrolyzed to FFA within a week under conditions of high humidity and temperature ^[3]. From the above it can be said that at the end of a period of two months, the bran extruded and stored at the conditions of 30 ° C - 78% does not meet the requirements for human consumption. On the other hand, it comes from storage with acceptable levels of FFA at an environment of lower temperature and relative humidity. In this case, rice bran is suitable for later use in the manufacture of products for human consumption, for example mixtures of ready-to- eat extruded cereals.

FFA values obtained in this study showed that a heat treatment by a twin screw extrusion process may be used as a method for inactivation of lipases to extend the shelf life of the rice bran. Storage at 18° C - 70 % RH resulted in lower values of FFA for an 8-week storage as compared with values of FFA at 30° C - 78 % RH. Vacuum packaging of rice bran showed significant disadvantage as compared with packaging it without vacuum. Therefore, based on the tests used in this study, it can be concluded that the recommended storage conditions to prevent hydrolytic rancidity in the stabilized rice bran by extrusion is the use of laminated bags metalized, packaged without vacuum and at temperature - humidity storage of 18° C - 70 %, for up to 8 weeks of storage.