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CyTA - Journal of Food
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Physico-chemical characterization of fermented rice bran biomassCaracterización fisico-química de la biomasa del salvado de arroz fermentado
M. dos S. Oliveiraa; V. Fedderna; L. Kupskia; E. P. Cipolattia; E. Badiale-Furlonga; L. A. de Souza-Soaresa a Laboratório de Ciência de Alimentos, Escola de Química e Alimentos, Universidade Federal do Rio Grande (FURG), Rua Eng, Brasil Online publication date: 23 September 2010
To cite this Article Oliveira, M. dos S. , Feddern, V. , Kupski, L. , Cipolatti, E. P. , Badiale-Furlong, E. and de Souza-Soares,
L. A.(2010) 'Physico-chemical characterization of fermented rice bran biomassCaracterización fisico-química de la biomasa del salvado de arroz fermentado', CyTA - Journal of Food, 8: 3, 229 — 236 To link to this Article: DOI: 10.1080/19476330903450274 URL: http://dx.doi.org/10.1080/19476330903450274
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CyTA – Journal of Food Vol. 8, No. 3, November 2010, 229–236
Physico-chemical characterization of fermented rice bran biomass Caracterizacio´n fisico-quı´ mica de la biomasa del salvado de arroz fermentado M. dos S. Oliveira*, V. Feddern, L. Kupski, E.P. Cipolatti, E. Badiale-Furlong and L.A. de Souza-Soares Laborato´rio de Cieˆncia de Alimentos, Escola de Quı´mica e Alimentos, Universidade Federal do Rio Grande (FURG), Rua Eng. Alfredo Huch, 475, Pav. 06, Centro, CEP 96201–900, Rio Grande – RS, Brasil
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(Received 10 June 2009; final version received 26 October 2009) The experiment was conducted to evaluate the fermentation effect with Rhizopus oryzae in the composition of whole rice bran, which was used as substratum for the fermentative procedure in tray bioreactors at 30 8C for 120 h. During the fermentation, samples were withdrawn in different times (0, 24, 48, 72, 96, and 120 h) for physico-chemical determination using standard procedures. Reductions in moisture, fat, phytic acid, and reducing sugars content of rice bran were respectively 24.6%, 40%, 50%, and 60%. The fermented bran presented an increase of 30% in ash content, 50% in fibers, and 40% in proteins. The digestible amino acid determination indicated 27.6% increase in the digestibility of produced proteins. Keywords: Rhizopus oryzae; solid-state fermentation; rice bran; protein; phytic acid; digestible amino acid El experimento fue llevado a cabo para evaluar el efecto de la fermentacio´n con Rhizopus oryzae en la composicio´n de salvado de arroz, el cual fue usado como sustrato para el proceso de fermentacio´n en biorreactores de bandeja a 30 8C durante 120 horas. Durante la fermentacio´n, se tomaron muestras en diferentes momentos (0, 24, 48, 72, 96 y 120 h) para su determinacio´n fı´ sico-quı´ mica usando me´todos esta´ndar. La reduccio´n en contenido de humedad, grasa, a´cido fı´ tico y azu´cares reductores del salvado de arroz fueron respectivamente 24,6%, 40%, 50% y 60%. El salvado fermentado presento´ un incremento en 30% de contenido de cenizas, 50% de fibras y 40% de proteı´ nas. La determinacio´n de aminoa´cidos digeribles indico´ un aumento de 27,6% en la digestibilidad de proteı´ nas producidas. Palabras clave: Rhizopus oryzae; fermentacio´n en estado so´lido; salvado de arroz; proteı´ na; a´cido fı´ tico; aminoa´cidos digeribles
Introduction The agroindustrial sectors produce a great amount of wastes, residues, and by-products which can be recovered and often upgraded to higher value and useful products. Wastes increase pollution problems and represent a loss of valuable biomass and nutrients. In opposition of what happened in the past, currently, concepts of minimization, by-product recovery, and bioconversion of residues are more and more spread out and necessary for the agroindustrial chains (Laufenberg, Kunz, & Nystroem, 2003). The use of fermentative processes, which implies employing microorganisms to get resultant transformations of their metabolic activity, is among the forms to increase the availability of nutrients in raw materials (Pelizer, Pontieri, & Moraes, 2007). Amongst these processes, the solid-state fermentation has been adopted in recent years, for the biotechnological industry, due to its potential application in the production of the active secondary metabolites, for the industries of animal food, fuel, foods, chemical *Corresponding author. Email:
[email protected] ISSN 1947-6337 print/ISSN 1947-6345 online Ó 2010 Taylor & Francis DOI: 10.1080/19476330903450274 http://www.informaworld.com
and pharmaceutical, conferring aggregate value to residues not or underutilized (Singhania, Patel, Soccol, & Pandey, 2009). The fungi are among the more appropriate microorganisms utilized in solid fermentation, as they can produce a variety of biochemical products, many of which are required for their proper growth and metabolism. They also produce secondary metabolites frequently in the stationary phase, thanks to their ability of reproducing in low water activity environments (Esposito & Azevedo, 2004). The fungi of the Rhizopus genera are classified as zygomycetes, of the Mucorales order and they are considered the most primitive fungi (Esposito & Azevedo, 2004; Pitt & Hocking, 1997). Some species, such as R. oryzae, R. oligosporus, and R. stolonifer, can produce enzymes, such as phytases, lipases and glucoamylases and others, mainly using agroindustrial residues as solid medium for their development (Ramachandran, Roopesh, Nampoothiri, Szakacs, & Pandey, 2005; Sabu et al., 2002). Characteristically,
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this genera of fungi does not include toxic species, being Generally Recognized As Safe (GRAS) by the Food and Drug Administration (FDA). Rice is amongst the original sources of residues that can be called by-products. Rice milling results in 5 to 8% of rice bran. The 2007/2008 rice harvest in Rio Grande do Sul – Brazil was of 7.5 million tons, with an estimated record harvest in 2009 (www.irga.rs.gov.br, 2008). This production generates consequently, 0.4–0.6 million tons of rice bran. This bran contains between 11 and 13% of basic protein, *11.5% of crude fiber, being able to reach 20% of its weight in oil, and antioxidant and functional components (Silva, Sanches, & Amante, 2006). Rice bran is the subject of different researches in the area of feed, oil components extraction, protein extraction, and due to its low cost and abundance, it presents great potential to be used in biotechnological processes in obtaining metabolites of interest (Adebiyi, Adebiyi, Jin, Ogawa, & Muramoto, 2007; Chandi & Sogi, 2007; Pandey, Szakacs, Soccol, Rodriguez-Leon, & Soccol, 2001; Vali, Ju, Kaimal, & Chern, 2005; Yun & Hong, 2007). The objective of this work was to evaluate the effect of R. oryzae in physico-chemical characteristics of rice bran applied as substratum in a solid-state fermentation process, aiming to identify the potential of chemical components from fermented biomass. Materials and methods Rice bran The rice bran used was supplied by a rice institute named IRGA (Instituto Rio Grandense de Arroz), located in Rio Grande do Sul, Brazil. The bran was packed in polypropylene bags of 5 kg, being kept at the temperature of 710 8C, until the moment of its use in the fermentative process. The rice bran preparation, as a substrate of the solid-state fermentative process, consisted of standardizing its granulometry between 0.35 and 0.60 mm, using an electromagnetic agitator (Bertel) of 60 Hz, equipped with sieves of different meshes. Solid-state fermentation Inoculum preparation The strain of R. oryzae, used as fermentative agent, was isolated from rice bran and identified in the Laboratory of Microbiology at the Food Processing Center of the Passo Fundo University, RS, Brazil. The cultures were kept at 4 8C in potato-dextrose agar medium and the spores were scraped from the slopes into aqueous emulsion of Tween 80 (0.2%). The same medium was used for spores incubation during 7 d at 30 8C until new and complete sporulation of the fungi in the culture. Spores were enumerated in Neubauer chamber.
Fermentation The fermentation was carried out in tray bioreactors, with dimensions of 2961765.5 cm3. The rice bran substratum (100 g) was placed in bioreactors, in the form of a fine layer of *2 cm, after the substratum homogenization with 45 mL of the saline solution (KH2PO4 2 g L71, MgSO4 1 g L71, NH2CONH2 1.8 g L71 in HCl 0.4 M). Bran initial spores concentration was 4.06106 spores g71 (Badiale-Furlong, Cacciamani & Garda-Buffon, 2007). According to Hasan, Costa, and Sanzo (1998), the moisture was adjusted to 50% with sterilized water addition and trays were covered with sterilized gauze to allow aeration, before being incubated at 30 8C for 120 h. To carry out physico-chemical characterization, samples were withdrawn at the beginning of the process and each 24 h, being stored at 718 8C. Physico-chemical characterization of fermented biomass Proximate composition The moisture, ash, and fat determination were gravimetrically determined, according to conditions recommended by AOAC (2000). The total protein content was determined as total nitrogen, by Kjeldahl method, described in AOAC 2000 (955.04C), using the protein conversion factor of 6.25. The crude fibers were determined through gravimetrical measurement of the residue obtained by the acid and basic digestion of the samples. The residue ashes were estimated, following the recommendations of the coordinated collaborative study by the Ministry of Agriculture, Livestock and Provisioning (1991). Reducing sugars content The sample (10 g) added of 40 mL of distilled water was taken to a 50 8C water-bath for 30 min with agitation. The solution was filtered, and 10 mL of Carrez I solution mixed with 10 mL of Carrez II solution were added making up the volume to 100 mL with distilled water. The liquid and flocculated phases were separated by centrifugation at 2800g. An aliquot (1 mL) of the supernatant, added with 1 mL of 2 M HCl, was kept in a 50 8C waterbath for 40 min. The mixture was neutralized with 2 M NaOH, being the colorimetrical reaction carried out with addition of 1 mL dinitrosalicylic acid (DNS), completing to a final volume of 10 mL. The absorbances were measured in a UV–Visible spectrophotometer (model Cary 100, Varian, Palo Alto, CA, USA) at 546 nm. A glucose standard curve of concentration between 0.01 and 0.1 mg mL71 was used for quantification (Miller, 1959). Digestible amino acid content The digestible amino acid content was estimated after enzymatic hydrolysis of the biomass protein with
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CyTA – Journal of Food pepsin (specific activity of 56.7 mgtyrosine min71 mgprot71) and pancreatin (specific activity of 320 mgtyrosine min71 mgprot71). The sample (2.5 g) was dissolved in 10 mL pepsin solution and agitated (90 rpm) at 37 8C for 3 h. Finally, the pH of the sample was elevated to 7.0 and 10 mL of pancreatin solution was added (1.5 mg mL71 phosphate buffer pH 8.0). The samples were kept under orbital agitation (130 rpm) at 37 8C for 24 h. After hydrolysis, the undigested fraction was separated by centrifugation (2800g) and filtration (Silveira & Badiale-Furlong, 2007). The filtrated fraction (10 mL) was mixed with 10 mL of 40% TCA solution and remained at rest for 1 h in refrigeration temperature (4 8C). After that period, the mixture was centrifuged and filtrated. The second filtrate was diluted for released amino acids determination using the Lowry, Rosenbough, Fair, and Randall (1951) method. The absorbance was measured in a UV–Visible spectrophotometer (model Cary 100, Varian, Palo Alto, CA, USA) at 660 nm and the amino acid content estimated by a standard tyrosine curve varying from 1.6 to 15 mg mL71. Phytic acid content The phytic acid extraction was carried out with 5 g of sample and 50 mL of 0.8 M HCl solution under orbital agitation (130 rpm) for 1 h. The mixture was separated by centrifugation at 2800g for 10 min. The supernatant was eluted in ion-exchange chromatography column (the stationary phase was prepared with 0.50 g of DowexAGX-4 resin dissolved in 5 mL of deionized water, conditioned in the column and eluted with 10 mL of deionized water, 10 mL of NaCl 0.7 M solution and again with 10 mL of deionized water). Sample of 1 mL of the supernatant was diluted to 25 mL with deionized water, of which 2 mL were eluted in the column with 10 mL of NaCl 0.1 M solution, followed by 10 mL of NaCl 0.7 M solution. The last aliquot was collected and 3 mL of the eluate reacted with 1 mL of the Wade reagent (ferric chloride 0.03% and sulfosalicylic acid 0.3%). The absorbance was measured at 500 nm in a UV–Visible spectrophotometer (model Cary 100, Varian, Palo Alto, CA, USA). The concentration of phytic acid standard curve varied between 6.6 and 39.8 mg mL71 (Latta & Eskin, 1980). Statistical analysis of data The data generated from this study were subjected to a one-way analysis of variance (ANOVA) at 5% level of significance. Means were compared by Tukey test. All the determinations were carried out in duplicates. Results and discussion The granulometry is an important factor in the physicochemical determinations; therefore it can influence in
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the efficiency of the extraction methods of studied components, and also allow the standardization of the rice bran as medium for the solid-state fermentation. The granulometry effect, heat transference, and other parameters of process were studied by Hasan et al. (1998) and the results showed that this affects fermentative process development. Table 1 shows the rice bran granulometry distribution used as fermentation substratum. For the experiment, the fractions from Tyler 24 to 42 were chosen. The removal of bran fractions with granulometry less than 0.355 mm avoids medium compaction during fermentation. The bran fraction removed in Tyler 14 consisted of endosperm fragments and rice hulls. They could cause analytical errors and not uniform chemical composition. Several authors point out that the growth of microorganism on a substrate can change its chemical composition due to the production of exocellular enzymes to obtain nutrients, in addition, production of other metabolites. This metabolism can enrich the substrate, depending on the intrinsic components of the fermentation agent or by nutrients availability presented in the substrate. The microbial action turns the substrate components available to chemical or enzymatic extractive processes (Blandino, Al-Aseeri, Pandiella, Cantero, & Webb, 2003; Olanipekun, Otunola, Adelakun, & Oyelade, 2009; Othman, Roblain, Chammen, Thonart, & Hamdi, 2009; ParedesLo´pes, Gonza´lez-Castan˜eda, & Ca´rabez-Trejo, 1991; Prinyawiwatkul, Beuchat, Mcwatters, & Phillips, 1996). Table 2 shows the rice bran physico-chemical composition before and during the fermentative process by R. oryzae. The results were expressed in dry basis (db). Among the authors who mentioned the R. oryzae action, Amadioha (1998) found that R. oryzae growth caused a decrease in starch, maltose, sucrose, protein and lipid content from stored potatoes. BadialeFurlong, Cacciamani, and Garda-Buffon (2007) found for rice bran that the action of Rhizopus spp. reduced
Table 1. Rice bran granulometry used in solid-state fermentation. Tabla 1. Taman˜o de salvado de arroz utilizado en la fermentacio´n en estado so´lido. Granulometry Ty 14 (1.18 mm) Ty 24 (0.71 mm) Ty 28 (0.6 mm) Ty 32 (0.5 mm) Ty 35 (0.425 mm) Ty 42 (0.355 mm) Bottom Total
Mass (g)* 8.7 23.7 9.3 3.3 6.3 5.7 41.6 98.6
+ + + + + + + +
0.63 0.63 0.43 0.49 0.49 0.5 1.0 2.0
Note: *The results are expressed as Means + SD. Nota: *Los resultados se expresan como Medios + SD.
Yield (%) 8.7 23.7 9.3 3.3 6.3 5.7 41.6
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Table 2.
Physico-chemical composition of fermented and unfermented rice bran.
Tabla 2.
Composicio´n fı´ sico-quı´ mica del salvado de arroz fermentado y sin fermentar.
Rice bran NF 0h 24 h 48 h 72 h 96 h 120 h
Moisture (g kg71) 118 479 463 421 425 404 361
+ + + + + + +
0.05a 0.2b 0.3b 2.1b 7.3b 2.2b 3.5b
Ash (g kg71) 104 93 114 135 133 141 136
+ + + + + + +
1.1ab 0.05a 0.20b 0.44c 0.11c 0.10c 0.14c
Fiber (g kg71) 85 81 110 128 126 145 131
+ + + + + + +
0.3a 0.2a 0.1b 0.03c 0.3bc 0.5d 0.8cd
Fat (g kg71) 189 204 195 159 131 110 112
+ + + + + + +
0.2a 1.2a 0.5a 0.2b 0.4c 0.1c 0.5c
Reducing sugars (mgglucose g71db) 21.0 22.0 47.6 13.2 8.5 7.7 7.9
+ + + + + + +
2.7a 0.2a 0.5b 0.3c 0.3d 0.4d 0.1d
Phytic acid (g kg71) 10 9.0 3.0 4.0 4.0 4.0 4.0
+ + + + + + +
0.03a 0.03b 0.02c 0.02d 0.03d 0.02d 0,03d
Notes: The values in each column with the same overwritten letter are not significantly different (p 5 0.05). Values are expressed as Means + SD. NF, bran not fermented; bran fermented for 0 h, 24 h, 48 h, 72 h, 96 h and 120 h.
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Notas: Los valores en cada columna con el mismo superı´ ndice no son significativamente diferente (p 5 0,05). Los valores se expresan como Medios + SD. NF, salvado no fermentado; salvado fermentado durante 0 h, 24 h, 48 h, 72 h, 96h y 120 h.
mycotoxins contamination levels in 80% and increased protein content in 17%. Silveira and Badiale-Furlong (2007) verified that the action of Rhizopus spp. increased protein, digestible amino acids and available methionine content in rice bran. There was a temperature increase during the fermentation process, probably because of moisture loss observed in Table 2, despite the moisture excess in the fermentation chamber. Statistical analysis indicated that the fermented bran did not differ in free water content along the time. The ash content of rice bran was similar to the results found by other authors which were 11.7%, 10.5%, and 10% (Feddern, Badiale-Furlong, & SouzaSoares, 2007; Oduguwa, Edema, & Ayeni, 2008; Silveira & Badiale-Furlong, 2007). There was a significant increase in fermented rice bran ash content from 48 h on; this result is probably due to fungus inherent ash content. The increase in ash content reached 30% in 120 h. Similar phenomenon was observed by Oduguwa et al. (2008) in relation to Rhizopus oligosporus and Saccharomyces cerevisiae, using rice bran as substrate, they found an increase of 24.5% after 48 h of fermentation in ash content. The fungi ash content can vary between 1% and 29%, depending on species and growth conditions (Griffin, 1994). According to Chaud and Sgarbieri (2006), the cell wall of Saccharomyces cerevisiae presents 1.4% ash, 2% of total lipids and 3.8% insoluble fiber. The bran fiber content fermented for 120 h showed a 60% significant increase comparing to time zero. This fiber increase comes from the intrinsic chitin production of R. oryzae. The fungi produce several polysaccharides; some are similar to other sources structures. These compounds may be exocellular associated with the membrane and cell wall or they can be intracellular. The fungi polysaccharides are commonly cellulose and chitin (Griffin, 1994). Yoon, Nam, and Kang (2008) obtained more than 100%
increase in dietary fiber content in fermented rice bran with ethanolic extracts by four different fungi. The mycelia of various fungi including Absidia coerulea, Absidia glauca, Aspergillus niger, Colletotrichum lindemuthianum, Gangronella butleri, M. rouxii, Phycomyces blakesleeanus, Pleurotus sajo-caju, R. oryzae, Lentinus edodes, and Trichoderma reesei has been suggested as alternative sources of chitosan (Suntornsuk, Pochanavanich, & Suntornsuk, 2002; Wu, Zivanovic, Draughon, & Sams, 2004). The rice bran lipid content was 18.9% in agreement with values obtained by other authors (Amissah, Ellis, Oduro, & Manful, 2003; Silva, Sanches, & Amante, 2006). The results determined that the unfermented and fermented brans until 24 h were not statistically different at a 5% level. The reduction of lipid content from 48 h on was significantly different from the first ones; these results are due to lipid use by the fungus for chitin synthesis. Oduguwa et al. (2008) reported a 40% reduction in fat content in rice bran fermented by R. oligosporus and Saccharomyces cerevisiae. The phytic acid is the primary storage compound of phosphorus (80% of total) in seeds, accounting for 1.5% of its dry weight. The undesirable change of phosphate to phytic acid, attached to metal cations of Ca, Fe, K, Mg, Mn and Zn make them insoluble and unavailable as nutritional factors. The phytates accumulate mainly in vacuoles of storage proteins, predominantly located in the aleurone layer (wheat, rice, and barley) or germ (embryo-corn). During germination, phytate is hydrolyzed by the endogenous phytases and other phosphatases to release phosphate, inositol, and micronutrients for plant growth support (Bohn, Meyer, & Rasmussen, 2008). The phytates have several important physiological functions in the plant during its life cycle, including storage of cations and phosphorus, providing material for the formation of cell walls, after the germination of seeds. Furthermore, the
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CyTA – Journal of Food phytic acid would protect the seed against oxidative damage during storage. Phytases are phosphohydrolases which begin the phosphate removal stage from phytate. These enzymes are used in feed to improve the nutrition of phosphorus to reduce its pollution from animal waste. The potential of phytases in improving human nutrition in trace mineral elements coming from foods of vegetable sources is being explored (Lei & Porres, 2003). The phytic acid content in rice bran was 10 g kg71, expected to occur in rice, which has a range from 1 to 5% of the total grain weight. The phytic acid extraction method used in this study showed recovery of 84% and variation coefficient of 9%. After 24 h of fermentation the phytic acid content decreased significantly, 66–55%, in relation to the beginning of the fermentation, resulting in fungi phytase production. Some species of Rhizopus spp. as R. oryzae, R. oligosporus, and R. ostolonifer and some species of Aspergillus are listed as phytase producers (Ramachandran et al., 2005). Kadan and Phillippy (2007) determined the content of phytates in rice bread containing defatted rice bran and different amounts of yeast. The researchers observed a degradation of phytates by 42% when the product contained 3.7% of rice bran after bread fermentation, regardless of the amount of yeast. Figure 1 shows the performance of reducing sugars content during fermentation. In 24 h there was an increase of these compounds, but as the time passed the reducing sugar content decreased significantly from 48 h to 120 h. The results showed the fungus ability in hydrolyzing the substrate carbohydrates to glucose, which is for its own use. Similar behavior was found by other authors (Alam et al., 2009; Ge´linas & Barrete, 2007). The effect of fermentation can be exploited even on vitamins, minerals, amino acid profile and other compounds, complementing the studies to enable the recovery and use of these formed compounds.
Knowledge of the fermentation agent composition helps elucidate the changes in raw material. The bioconversion of agroindustrial raw materials is a potential for protein production, especially for feed nutritional supplementation. These products are also sources of high added value compounds such as enzymes, vitamins, amino acids, antioxidants, and minerals (Ghorai et al., 2009). Some fungi are referenced in various studies by helping to increase the protein content of different agroindustrial raw materials (Anupama & Ravindra, 2000; Ge´linas & Barrete, 2007; Oduguwa et al., 2008). Table 3 presents fermented and unfermented rice bran results of protein levels and digestible amino acids. The values for unfermented bran are in agreement with the literature (Feddern et al., 2007; Pestana, Mendonc¸a, & Zambiazi, 2008; Silva, Sanches, & Amante,
Figure 1. Reducing sugars content in rice bran fermented by Rhizopus oryzae. Figura 1. Contenido de azu´cares reductores en el salvado de arroz fermentado con Rhizopus oryzae.
Table 3.
Biomass protein content, percentage of protein increase and digestible amino acids.
Tabla 3.
Contenido de proteı´ nas, porcentaje de aumento de proteı´ na y aminoa´cidos digeribles de la biomasa.
Fermentation time NF 0h 24 h 48 h 72 h 96 h 120 h
Protein (g kg71) 147 164 210 220 230 234 230
+ + + + + + +
0.7a 0.17b 0.15c 0.1cd 0.5de 0.5e 0.4de
Increase in protein content (%)
28.2 34.6 40.6 42.8 38.6
Digestible AA (mg gmedium71) 11.0 11.9 20.1 19.9 18.7 19.8 20.4
+ + + + + + +
0.29a 0.38a 0.38b 0.44bc 0.11cd 0.31bd 0.43b
Notes: The values in each column with the same overwritten letter are not significantly different (p 5 0.05). Values are expressed Means + SD (n ¼ 3). NF, bran not fermented; bran fermented for 0 h, 24 h, 48 h, 72 h, 96 h and 120 h; AA, amino acid. Notas: Los valores en cada columna con el mismo superı´ ndice no son significativamente diferentes (p 5 0.05). Los valores se expresan como Medios + SD (n ¼ 3). NF, salvado no fermentado; salvado fermentado durante 0 h, 24 h, 48 h, 72 h, 96 h y 120 h; AA, aminoa´cido.
Digestible AA (mg g protein71) 74.9 72.7 95.7 90.4 81.2 84.7 88.9
+ + + + + + +
2.0a 2.3a 1.8b 1.9bc 0.5a 1.3ac 1.8bc
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2001). The development of fungal biomass resulted in a fermented bran protein content increase which was significantly different, comparing the protein values from 24 h on in relation to the beginning of the process. This increase reached 42.8% after 96 h of fermentation compared to the beginning. However, it can be noted that within 24 h there was an increase of 4.6 percentage points in relation to the beginning of the fermentation time and in 48 h the increase was one percentage point over the same interval, keeping this behavior for 72 h and decreasing at 96 h and 120 h. These results indicate that in the first 24 h of the fermentation process the fungi are in full metabolic activity. Rudravaram, Chandel, Linga, and Pogaku (2006) studied protein enrichment of deoiled rice bran by solid-state fermentation using Aspergillus oryzae. They achieved the maximum protein enrichment (100%) in 3 days, while the yield achieved in the present work in the same period was 34.6% using whole rice bran. However, fermented rice bran application on animal feed or food may contribute to increase the nutritional quality of the product, once its lipid content is rich in antioxidant compounds and unsaturated fatty acids. Lee, Hong, and Cho (2008), mentioned that fermented rice bran can be source of compounds with antibacterial and antioxidant activity, besides presenting tyrosinase inhibition. Kim et al. (2002) reported that mice administered with hot water extract of fermented rice bran showed anti-stress and anti-fatigue effects. Due to the significant increase in protein content of the biomass, the quality of this produced compound
was the target of interest. So the content of amino acids was estimated after a process of enzymatic digestion. The determined digest amino acids, when expressed on mass of fermented bran, had increased *50% from the 24 h and were maintained throughout the experiment. This result in relation to protein bran showed that the digested amino acids increased from 18.5% to 27.6%, indicating an increase in digestibility of the biomass on the non-fermented rice bran. Figure 2 shows the original protein content of the bran and the contents of digested protein and solubilized after the process of digestion, measured and quantified by an albumin standard curve. The results showed an increased content of soluble proteins during fermentation. This component can be the subject to further studies to verify its quality. Conclusion The results of this study showed that the fermentation process caused changes in the physico-chemical composition of fermented rice bran in solid medium by R. oryzae. The fermentation caused a reduction of 24.6%, 40%, and 50% in moisture, lipids and phytic acid in bran, respectively. The reducing sugars were 60% consumed during fermentation. The fermented bran had higher content of ash, fiber and protein, with an increase of 30%, 50%, and 40%, respectively. The levels of digestible amino acids showed an increase of 27.6% in the digestibility of the produced proteins. References
Figure 2. Comparison among protein content, soluble protein and digested protein after enzymatic digestion process. Figura 2. Comparacio´n del contenido de proteı´ nas del salvado de arroz y de la biomasa, proteı´ na digerible e soluble despue´s del proceso de digestio´n enzima´tica.
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