Lipid Stability of Soybeans in Grains and Soybeans Processed as Tofu

Soybeans (Glycine max (L.) Merrill) contain bioactive substances. They are a functional, important food associated with reduced risks of chronic and degenerative diseases. This study assesses lipid stability and antioxidant in soy grains and processed soy tofu. The two soybean brands differed in antioxidant activity and total phenolic compounds, which ranged from 188.4 mg EAG.100g and 3.17 to 1.56 μmol of ferrous sulfate g. Both tofu samples only showed differences in total phenolic compounds, which ranged from 9.6 to 18.3 mg EAG.100g. Analysis of DPPH free radicals has not shown significant differences (P < 0.05) amongst analyzed soybean in grain and tofu brands; yet, we could identify antioxidant activities with an inhibition level above 50%. There was no significant difference among total lipid contents of the tested brands. Polyunsaturated and monounsaturated fatty acids found to be denser in soy and tofu samples were: linoleic, linolenic and oleic acid. The n-6/n-3 ratio values were satisfactory for soy and tofu. Thus, both soybean and tofu display significant antioxidant effects and are sources of polyunsaturated fatty acids.


Introduction
Soybeans ([Glycine max (L.) Merrill] are widely produced in Brazil (Neves et al., 2013;Loss et al., 2012).The last years have seen a rise in their consumption due to their chemical and nutritional characteristics.They are a source of two essential fatty acids-linoleic and α-linolenic-and plentiful protein.Health benefits are associated with the presence of phenolic compounds such as isoflavonoids.Such substances are important antioxidants that prevent noncommunicable chronic-degenerative diseases such as cancer (Wang et al., 2006).
The processing of food may alter the nutrients it contains (Tsai et al., 1981).Soybean grains often must soak in water to soften.With a proper heat treatment, this makes proteins more digestible while safeguarding the beans' nutritional value (Rockland & Radke, 1981;Escueta et al., 1986;Wang & Murphy, 1996;Bayran et al., 2004).
Despite its nutritional qualities, tofu is still little demanded in the Brazilian market.Likewise, only a few publications address its physicochemical characteristics, especially the quality and quantity of fatty acids that compose its lipid fraction (Axerold et al., 1981;Davies & Nielsen, 1986;Rackis et al., 1979).
Many have investigated the relationship between soy consumption and human health on the basis of soy's nutritional characteristics.Breast, prostate, and colon cancer cases have decreased in Asian populations, wherein the estimated per capita intake of soybean is 20-50 times higher than that of Western populations (Crouse et al., 1999).
This study aimed to explore the chemical transformations that occur as soybean becomes tofu, including the evaluation of phenolic content, antioxidant activity (AA), and lipid composition of soybean before and after processing. www.ccsen

Obtain
Were anal marketed i analysis time was 41.50 min.The gas flow rates were 30 mL min -1 for hydrogen, 30 mL min -1 for nitrogen and 250 mL min -1 for synthetic air.

Obtain
Injections (1.2 µL) were performed and the peak areas of methyl esters of fatty acids were determined by the software ChromQuest 4.1 (Thermo Electron, Italy).
The identification of fatty acids (FA) was carried out after verification of equivalent chain length of peaks and comparison of the retention times of samples with a pattern containing a mixture of fatty acid methyl esters (Sigma 189-19, USA), as described By Simionato et al. (2008).The quantification of fatty acids in g per 100 g of total lipids was performed by normalization of the peak areas of fatty acid methyl esters.

Extraction
Were obtained extracts of soybean and tofu as proposed by Hung et al. (2009), in triplicate by homogenizing about 1 g of soybean and tofu samples in 20 mL of methanol/water in a 4:1 ratio.We stirred for 20 minutes at room temperature, then filtered the extracts, centrifuged the suspension, and collected the supernatant.We stored the extracts in amber glass bottles at -12 °C until the time of probable use.

Determination of Total Phenolic Contents
The spectrophotometric method determined the total phenolic contents of the methanol extracts, using the Folin-Ciocalteu reagent mix (Swain & Hillis, 1959;Wettasingle & Shahidi, 1999) and the standard curve of gallic acid (GAE).We expressed the results as mg of total phenolic contents in GAE per 100 g of sample.We mixed a 0.350 mL aliquot of the extract with 0.250 mL of Folin-Ciocalteu reagent and 0.5 mL of 20% sodium carbonate in amber tubes, thus adjusting the volume to 5 mL with distilled water.After the mix settled for 25 minutes, we read the absorbance at 773 nm to measure the total phenolic contents.

Determination of Antioxidant Activity -DPPH
We measured antioxidant activity by reducing free radical levels of DPPH, as described by Brand-Williams et al. (1995) with some modifications (Sánchez-Moreno et al., 1998).Methanol extract aliquots of soybean and tofu at different concentrations (4.5, 7.5 and 10.0 mg mL -1 ) were added to 200 µL of methanol solution of DPPH 60 µM.After stirring, the tubes stood in the dark for 30 minutes.After the reaction time elapsed, we obtained the absorbance of samples at 517 nm, using methanol as the blank.We measured AA in two ways: via inhibitory concentration (IC 50 ), the amount of antioxidant necessary to halve the initial concentration of DPPH , and via the oxidation inhibition percentage of the radical calculated from Equation 1:

Determination of Iron Reducing Power (FRAP)
Were assessed the extracts' reducing power (FRAP) according to Benzie & Strain (1996), with some modifications.We obtained FRAP reagent from the combination of 2.5 mL of a 10 mM TPTZ solution in HCl 40 mM, 2.5 mL of ferric chloride and 20 mM and 25 mL of 0.3 mM acetate buffer (pH 3.6), used immediately after preparation.To evaluate antioxidant capacity, we transferred 350 µL of a methanol extract aliquot to amber test tubes containing 270 µL of distilled water and 2.7 mL of previously prepared FRAP reagent.After a shaker tube homogenized the samples, they incubated at 37 °C for 30 minutes.Then we made a reading at 595 nm.Using solutions with different concentrations of ferrous sulfate, we constructed a calibration curve.Accordingly, we expressed the results as μmol of ferrous sulfate/g of the sample.Analyses were done in triplicate.

Statistical Analyses
The results were expressed as mean ± standard deviation (SD).We compared means with analysis of variance (ANOVA) and Tukey's test (p < 0.05) using Statistical Software 7.0 (2005).

Fatty Acids Composition
Percentage of lipids in the soybean samples was 3.6%, in line with the nutritional table labeled on the packaging.Tofu samples showed an average fat content of 5.5%, below the values found by Benassi et al. (2011), which ranged between 23% and 30%.There were no significant differences between the total lipid contents of both soybean and tofu brands evaluated.
Were identified a total of 15 fatty acids in the tofu and soybean samples have been identified (Table 1).Both soybean and tofu have higher levels of polyunsaturated fatty acids (PUFA), ranging from 56.57% to 60.37% of total fatty acids, followed by monounsaturated (AGM) (23.62-25.04%)and saturated fatty acids (AGP) (15.77-17.95%).Means followed by different letters differ statistically by the Tukey's test at 5% significance.SFA = Total Saturated Fatty Acids; PUFA = Total Polyunsaturated Fatty Acids; MUFA = Total monounsaturated fatty acids nsThere is no significant difference at 5% probability by F test.
The high degree of unsaturation previously addressed was due to the predominance of linoleic (51.34-51.69%)and oleic acids (21.38-22.65%), the former being an essential fatty acid.
The amounts of fatty acids in tofu resembled those in the raw material, indicating that processing does not promote important qualitative or quantitative changes (p<0.05) in this class of compounds.Nevertheless, soybean and tofu samples of brand A have more omega-3 and total polyunsaturated fatty acids than those of brand B.
For A and B respectively, n-6:n-3 ratios were 5.75 and 7.62 for soybean samples, whereas this ratios for tofu were 5.95 and 8.72.The linoleic and α-linolenic fatty acid values in both tofu and brand A soybeans were near the optimum values for a human diet, as recommended by the Department of Agriculture of the United States (FAO, 2007), suggesting that this should lie between 5 and 10.Also known as omega-9, oleic acid had the second highest concentration with no significant differences between soybean samples or derivatives.This monounsaturated acid is hyperlipidemic: it reduces cholesterol and low density lipoprotein (LDL) responsible for the formation of atheromas (Mcnamara, 1990).Saturated fatty acids were in the samples, but in small quantities.The fatty acid profile suggests elevated palmitic (10.73-12.52%)and stearic saturated fatty acid values (3.96-3.97%).The United Kingdom Ministry of Health recommends the PUFA/SFA ratio of the lipid profile be above 0.4 to prevent diseases associated with saturated fat intake (Wood et al., 2003).The studied ratio is in line with this and can be used in low-cholesterol diets.All fatty acid values are in line with the reference table RDC No. 482 of 23/09/1999, the National Health Surveillance Agency (ANVISA).
For A and B respectively, n-6:n-3 ratios were 5.75 and 7.62 for soybean samples, whereas this ratios for tofu were 5.95 and 8.72.The linoleic and α-linolenic fatty acid values in both tofu and brand A soybeans were near the optimum values for a human diet, as recommended by the Department of Agriculture of the United States (FAO, 2007), suggesting that this should lie between 5 and 10.Also known as omega-9, oleic acid had the second highest concentration with no significant differences between soybean samples or derivatives.This monounsaturated acid is hyperlipidemic: it reduces cholesterol and low density lipoprotein (LDL) responsible for the formation of atheromas (Mcnamara, 1990).Saturated fatty acids were in the samples, but in small quantities.The fatty acid profile suggests elevated palmitic (10.73-12.52%)and stearic saturated fatty acid values (3.96-3.97%).The United Kingdom Ministry of Health recommends the PUFA/SFA ratio of the lipid profile be above 0.4 to prevent diseases associated with saturated fat intake (Wood et al., 2003).The studied ratio is in line with this and can be used in low-cholesterol diets.
All fatty acid values are in line with the reference table RDC No. 482 of 23/09/1999, the National Health Surveillance Agency (ANVISA).

Determination of Total Phenolic Content and Antioxidant Capacity
Table 2 shows the results of FRAP analysis of total phenolics and antioxidant activity.
The two brands of soybeans differed in FRAP antioxidant activity and total phenolic content.Tofu samples differed in total phenolic content, with 9.6 and 18.3 mg GAE 100g -1 .These differences may spring from plant genotype, environmental variations, sampling periods, or other factors (Shan et al., 2005).In this study, soybean grains showed more antioxidant activity (AA) and phenolic compounds than soybeans processed as tofu.According to Wang and Murphy (1996), the presence and concentration of isoflavonoids in soy-based products depend upon the processing conditions.
The FRAP method allowed us to verify that the AA values of soya were 1.56 and 3.17 μmol of ferrous sulfate/g of sample.Thus, antioxidant activity of brand A soya appeared above that of brand B, with a 5% greater probability by Tukey's test.In processed products such as tofu, AA tends to be 50% lower than in raw soybeans, indicating some soybean processing methods affect antioxidant capacity (LEE et al., 2004).The results in table 2 represent decreases much higher than 50% for AA.

Table 1 .
Fatty acid composition (%) of soybean in grains and tofu.

Table 2 .
Antioxidant activity and total phenolic content in soybeans and tofu