Otimização da fermentação acética para a produção de vinagre de farelo de arroz e avaliação do potencial antioxidante

Abstract

INTRODUCTION AND AIMS ─ Rice is one of the most consumed cereal in the world. The processing results in rice bran, a byproduct derived from the stripping step. Currently, the bran is only used for oil extraction; the defatted bran is generally used in animal feed production. The manufacturing of vinegar provides a means of using raw materials underutilized in industrial establishments, therefore, the development of process that add value to rice bran, as the possibility of use as substrate in fermentation process, like the vinegar production, is an alternative to increase the applications of this agro-industrial residue, which has a high nutritional and antioxidant potential due to the presence of proteins (15 %), carbohydrates (47 %) and phytic acid (6 %) in considerable concentrations. Phytic acid is a current research topic of studies in different fields such as in food preservation, antioxidant metals and human health, with anticancer activity, in the treatment of diabetes, renal calculus and Parkinson's disease. The defatted rice bran (DRB) can be applied as substrate for fermentation processes, but a preliminary step of hydrolysis is necessary to convert complex carbohydrates into fermentable sugars. After the hydrolysis, a double fermentation is performed: the substrate is consumed by yeasts in the alcoholic fermentation step, producing ethanol for the subsequent acetification (acetic fermentation). Among the steps of the vinegar processing, clarification aims to eliminate all substances suspended in solution, to make it clear. Bentonite is a widely used clarifier, being efficiently used in the clarification of white wine and vinegar industry, mainly to eliminate the protein turbidity. In view of these considerations, the aim of this study was to obtain DRB vinegar by submerged fermentation, the evaluation of its antioxidant activity, the optimization of clarification step and sensory analysis of obtained vinegar. METHODS ─The DRB was ground and hydrolyzed enzymatically, using three commercial enzymes: protease (ALCALASE 2.4L), α-amylase (TERMAMYL 2X) and amyloglucosidase (AMG 300L). After centrifugation of the hydrolysed medium, the supernatant was submitted to alcoholic fermentation with a culture of Saccharomyces cerevisiae (Saf-instant®), in a 3L erlenmeyer flasks containing 1.5 L of hydrolysed medium, pH adjusted 5.0, and 5.0 % (w/v) inoculum. The incubation was carried out under stationary conditions at 30 °C for 48 h. Subsequently, the wine (product of alcoholic fermentation) with 3.62±0.23 % of ethanol was centrifuged and frozen for use in the acetic fermentation, in a bench fermentor. The conditions used for activation of acetic bacteria were: 30 °C, aeration flow of 0.25 VVM (volume of air / volume of medium x minute) and agitation of 300 rpm. The inoculum used was the strong alcohol vinegar, assigned by the company Chemim Foods at the ratio of 1.5:1.0 of the base wine, plus 1.0 g·L-1 of Acetozyn (mixture of inorganic salts, sugars, plant extracts, amino acids and vitamins, food grade) for the supply of nutrients necessary for acetic bacteria. During acetic fermentation sample collection was carried out every eight hours for monitoring the acidity (acetic acid content) and alcohol content. The end of acetification process occurred when the alcohol content reached close to 0.5 %. Upon completion of the acetification, the vinegar was clarified by vacuum filtered and pasteurized at 65 °C for 30 minutes. The acetic fermentation was evaluated according to the performance and process productivity. In the optimization stage of acetic fermentation, a Full Factorial design (FFD) (22 with 3 central points, 7 runs) was applied to evaluate the influence of agitation (100-500 rpm) and aeration (0.25 to 1.0 VVM) over the stoichiometric yield and TC yield (TC - sum of the concentration of ethanol and acetic acid). The characterization of the vinegar was performed by determinations of total acidity, alcohol content, total solids and ash); the antioxidant activity was made by DPPH• and ABTS+ methods. To optimize the clarification step of the DRB vinegar, two Central Composite Rotatable Designs (CCRD) were applied to evaluate the influence of the concentration of clarifying agent (bentonite) and time of process under the vinegar color parameters (L*, a*, b*). The DRB vinegar and two other commercial rice vinegars was also evaluated by sensorial analysis using the Hedonic Scale method (nine points) and Preference Ranking Test with 112 judges not trained. MAIN RESULTS─The yield obtained by the total concentration (TC) for DRB vinegar ranged from 74.21±1.96 % to 97.60±0.67 %. In the optimization of acetic fermentation step, both variables had a positive effect on the answers within the range studied. To the antioxidant activity, the DPPH• method resulted in EC50 = 10.62±1.42 µg·ml-1, which corresponding to the sample required to reduce by 50 % the initial concentration of DPPH• radical. The ABTS+ method resulted in 0.010±0.001 mM Trolox/ml. The step of DRB vinegar clarification was optimized in the follow conditions: 30-48 hours to time of process, and 2 to 3.5 % for the concentration of bentonite, where was obtained higher values for L* (lightness). In relation to the physical-chemical analyzes with the non-clarified and clarified vinegar was observed significant differences between the trials (p<0.05). In sensory evaluation the evaluated attributes was color (6.34), consistency (6.81), aroma (5.41), flavor (5.30) and overall evaluation (5.74); the acceptability rate of the three samples were close: 77.8 %, 70.6 % and 63.8 % (two commercial vinegar and DRB vinegar, respectively). Under Brazilian law, the vinegars were produced within the required parameters: real alcohol content <1 %; volatile acidity>4.0 g of acetic acid·100 ml-1; reduced dry extract>7.0 g∙L-1; with the exception of ash content, which should be between 1-5 g∙L-1, which can be explained by the raw material used(defatted rice bran) that has a high ash content. DISCUSSION AND CONCLUSION─According to the results obtained it was concluded that the defatted rice bran can be used as a substrate in submerged fermentation processes, in this case, to obtain vinegar. The submerged fermentation was efficient regarding the conversion of ethanol to acetic acid (74.21±1.96 % to 97.60±0.67 %) during the eleven process repetitions (30 ºC, 300 rpm, 0.25 VVM), indicating that losses by evaporation or super oxidation during the process acetification were low. By the results of CCRD applied to optimize the step of acetic fermentation it was observed positive effects of the two variables (aeration and agitation) on the answers (stoichiometric yield and TC yield), within the range studied. The stoichiometric yield (79.97±7.97 %) and CT yield (100.50±1.27 %) showed superior results where the aeration and agitation were 1.0 VVM and 500 rpm, respectively. About the antioxidant activity the result (EC50 = 10.62±1.42 µg·ml-1) was satisfactory for the DPPH• method, because it lower than the values found in the literature, ie, showed higher antioxidant activity. On the other hand, for the ABTS+ method the result found (0.010 mM Trolox/mL) was not satisfactory, because has less antioxidant activity compared with other vinegars analysed by this method. From the defatted rice bran it was possible to produce vinegar with important antioxidant activity, as confirmed by DPPH• method and by the presence of phytic acid, which makes a product with functional properties. Added to this it is necessary to evaluate their sensory attributes in order to improve them and ensure the highest consumer acceptability, since the acceptability of DRB vinegar was below the recommended, however similar to the assessed commercial vinegars. In the step of clarification was possible to determine an optimum range for the variables studied (bentonite concentration and time of process), to increase the brightness (L*).