TOFU OBTAINED WITH LEMON COAGULANT AND SOYBEAN CONCENTRATE TOFU OBTIDO COM COAGULANTE DE LIMÃO E CONCENTRADO DE SOJA

The objective of this work was to develop and characterize tofu, using vegetable coagulant (5-15%) and soy protein concentrate SPC (0-10%) applying an experimental design to maximize the yield of tofu. Also were evaluated the characteristics of acidity, moisture, total solids, proteins, syneresis and ash in the first and seventh days of storage. In the first day of storage, high percentage of coagulant resulted in an increase in the acidity of the tofu. High moisture contents were observed in high concentrations of coagulant without soy protein concentrate. In the total solids the high values were verified in low concentration of coagulant and SPC. Protein, syneresis and ash were high when added low concentrations of coagulant and SPC. The high yield was observed in the experiment with high SPC and low amount of coagulant. On the seventh day of storage, the high concentration of lemon resulted in a significant effect (p <0.05) on syneresis and protein. Therefore, tofu made with vegetable coagulant (5%) and addition of 10% SPC showed high amounts of protein, ash and better yield, as well as low acidity and could be an alternative for consumers looking for products with high protein content.

Revista Tecnológica -Universidade Estadual de Maringá -ISSN 1517-8048 DOI: 10.4025/revtecnol.v28i1.48693 ___________________________________________________________________________ The soybean used in this study were provided by Granja Três, from conventional cultivars, category S1, from the Jacutinga -RS region, 2017/2018 harvest. SWE was obtained by processing the soybean grains, according to the methodology of Benassi et al. (2011), with adaptations. Initially 150g of soybean grains were selected, sorted, weighed and washed, and then was left macerating (immersed) in 500 mL of distilled water at ±25°C for 16 h. After, the grains were drained and weighed and was evaluate the amount of water absorbed, by the difference in mass of grains macerated by the initial mass of grains. In the sequence, it was added distilled water at 90 °C, considering water absorbed by the grain so that they complete a volume of 1200 mL, with a final ratio of 1: 8 (beans: water). Then the mixture was triturated (M. Vitrory, 1/2 HP model) for 3 min, and after the SWE was separated by vacuum filtration (Tecnal, TE-058 model) and the okara (residue) was removed. Then it separates the SSE of the okara (residue) by filtration vacuum (Tecnal, TE-058 model). Were performed analyzes of moisture, protein, pH, acididy and total solids.

Vegetable coagulant
The coagulant used for production of tofu was the lemon Taiti, which was purchased in local shop (Erechim-RS-Brazil) and the extract was obtained according to the methodology of Adetunji et al. (2008). The lemons were washed, cut and extracted the juice, to obtain 1L of juice and an equal portion of 20 mM sodium phosphate buffer (pH 7.2) (1:1 v/v) was added. After centrifugation (Centrifuge MPW®, model 351R) at 5000 g for 30 min at 4 °C and vacuum filtration (Tecnal, model TE-058) was performed. The samples were stored at 4 °C until the time of tofu preparation, approximately 2 h.

Elaboration of tofu
Tofu was obtained from methodology of Benassi et al. (2011), with adaptations, made de coagulation with lemon extract. 1.5L of SWE was heated to 90 °C in a water bath (Marconi®, MA126 model).
After heating, the SWE was transferred to a glass vessel and cooled to the coagulation temperature (80ºC), according to Schmidt et al. (2017). After coagulant (lemon) was added, adjusting the pH of the lemon extract to 2.0 with citric acid. Also was added soy protein concentrate (purchased from Cerelus local food with 40% protein), homogenized with mixer and allowed to coagulate for 40 min. The concentrations of coagulant extract (lemon) and soy protein concentrate (SPC) were evaluated by experimental design. The levels of variables were defined according to preliminary tests, and Table 1 presents the levels and variables of experimental design 2². The amount of SWE was kept fixed (1.5 L). According to the matrix of the experimental design (2²) 5 different formulations of tofu were prepared, with runs 5, 6 and 7 being the triplicates of the central point. After coagulation the mass was cut by lire, and inserted into cheese molds (500 mL), which remained for 30 min to syneresis. After the tofu was removed of the mold, and packed in plastic packages without adding water and without vacuum, and stored under refrigeration (4 °C) for 7 days. During the coagulation of tofus were measured the pH and acidity. Revista Tecnológica -Universidade Estadual de Maringá -ISSN 1517-8048 DOI: 10.4025/revtecnol.v28i1.48693 ___________________________________________________________________________ In the tofu was carried out the analysis of moisture, protein, ash, acidity, total solids and syneresis according to the methodology of AOAC (2000), in the 1 st and 7 th days of storage. The yield was obtained according to Benassi et al. (2011) in the 1 st day of storage.

Characterization of WSE and tofu
Moisture and total solids were determined by the gravimetric method, in a drying chamber (Fanem®, a Model 320) at 105°C for about 4 h to constant weight. Protein was determined by Kjeldahl method. Ashes were obtained by gravimetric method, after calcination in a muffle oven (Quimis), at 550°C for 6 h. The pH was determined by potentiometry (Digimed, Model DM-22), while the acidity was analyzed by titrimetric method. The syneresis of tofu was determined by the method of drainage.

Statistical analysis
The results obtained from the experimental design where used to evaluated the differences between means variance analysis (ANOVA) follow by Tukey´s test using Statistic software, version 5.0, at a significance level of 95% of confidence.
The value of protein found in the WSE is in accordance with the Resolution RDC N. 268 of 2005(Brasil, 2005, which provides a minimum protein of 3%. Ciabotti et al. (2009) found 3.56% of protein in common soybeans, also near to obtained in this study.
According to Lambrech et al. (1996) the pH of the WSE for production of tofu, should be between 6.4 and 6.6, value close to that obtained in this work. Martins et al. (2013) found acidity between 0.1 and 0.2% of lactic acid in of common soybeans, and the acidity of the present study was between the range studied by the authors (10.5°D-0.105% of lactic acid).
The physicochemical parameters of SWE can vary according to soybean type, grain storage time and conditions, initial moisture content, extraction procedures, water-soy ratio and extraction temperature, as well as the climate where is produced, among others. Table 2 shows the combinations between the independent variables (lemon concentration and SPC) for each run, as well as the results obtained from the response variables (acidity, moisture, total solids, yield, protein, syneresis and ashes) for the 1 st and 7 th days of storage, respectively.
The acidity of tofu ranged from 1.67 to 4.21% on the 1 st day and from 1.24 to 3.52% on the 7 th day of storage. In the formulations developed on the 1 st day, among the variables studied, it was observed that the lemon concentration had a significant positive effect (p <0.05), and the interaction between the variables had a significant negative effect at a level of significance of 95% in the acid content of tofu ( Figure 1a). Thus, the high concentration of lemon added to the formulations resulted in high acidity. The ions and the hydrogen ionic strength influence the water-holding capacity of the tofu (TAY et al. 2006 However, on the 7 th days of storage only a significant positive effect (p <0.05) was observed between the interaction of the variables (Figure 1b). This effect can be resulted from hydrolysis of neutral lipids to fatty acids and also the oxidation of fatty acids during storage (LIU and CHANG, 2008). Schmidt et al. (2017), studied tofu with lemon coagulant, not found significant differences (p <0.05) between the 1 st and the 7 th day of storage on the acidity. According to Fasoyiro (2014), the acidity values of tofu are dependent on the coagulant used. In Table 2 is observed that in run 2 (tofu with 15% of lemon extract and 0% of SPC), the highest content of moisture at 1 st and 7 th days of storage. The results were statistically treated by analysis of variance (ANOVA). For tofu on the 1 st first and 7 th days of storage is observed by the Pareto chart (Figure 2a and 2b) that the variables studied did not present a significant effect (p >0.05) on the moisture content.
The use of acidic coagulant (lemon) provided a soft tofu, presenting a fragile texture due to the high moisture. This behavior was also observed by Ciabotti et al. (2009) that studied glucone-δ-lactone and lactic acid to obtain tofu and obtained 79-80% of moisture. In relation to the total solids contents of the tofu, it can be observed that run 4 presented higher values on the 1 st and 7 th days of storage ( Table 2). The results were statistically treated by analysis of variance (ANOVA). For tofu on the 1 st first and 7 th days of storage is observed by the Revista Tecnológica -Universidade Estadual de Maringá -ISSN 1517-8048 DOI: 10.4025/revtecnol.v28i1.48693 ___________________________________________________________________________ Pareto chart (Figure 3a and 3b) that the variables studied did not present a significant effect (p >0.05) on total solids contents. The similar values found can be due to the use of same coagulation process (temperature and time) (HOU and CHANG, 2004). This result is very interesting for practical operation and manufacturer that can achieve high quality tofu without economic loss. Benassi et al. (2011) studied soft tofu purchased commercially and obtained total solids content of 15.3%. It is observed that this value is lower than that obtained in the present study, which may be associated with the amount soy protein concentrate used in the tests.
The high protein content was obtained in run 3 (with 5% of lemon extract and 10% of SPC) ( Table 2). This high content is very interesting, that resulted in a product with high yield due the coagulation process, when occurs the cross linking of protein molecules with the divalent cations of the coagulant. Also, the high value of protein is very interesting for the consumer Revista Tecnológica -Universidade Estadual de Maringá -ISSN 1517-8048 DOI: 10.4025/revtecnol.v28i1.48693 ___________________________________________________________________________ health once is the main functional component. The major proteins are 7S and 11S that have different isoelectric points, molecular weights and properties to form gel in soybean milk with the coagulant resulting in a electrostatic interactions between the cations and the proteins (YUAN et al., 2002).
Equations 1 and 2 show the first order coded models for protein in the 1 st and 7 th days of storage, respectively. The models were validated by variance analysis (ANOVA), with correlation coefficient of 0.92 and 0.94 and calculated F were 2 and 3 times greater that of the F tabulated, respectively. The results presented significant difference (p<0.05), allowing the construction of the contour curves (Figure 4) Ciabotti et al. (2009), obtained a 9.8% of protein in tofu made with bleached common soybeans and coagulated with glucagon-δ-lactone. Schmidt et al. (2017) obtained 10.5% of protein tofu coagulated with lemon. The present study presented higher levels of protein in relation to these authors, which is associated with the addition of soy protein concentrate.
The run 2 presented the highest values for syneresis on the 1 st and 7 th days of storage (Table 2). Schmidt et al. (2017) also found high syneresis on the 1 st day of storage in relation to the 7 th of tofu coagulated with lemon. This behavior is due to the precipitation of proteins during tofu processing, causing greater serum release soon after coagulation, consequently, less serum will be released during storage. Run 2, with a higher concentration of lemon and without addition of soybean protein concentrate, presented higher syneresis at both storage times. This can be due to the fact that in the other experiments water absorption occurs due to the addition of the protein concentrate soybean, causing less serum release. In addition, the concentration of lemon provided a softer tofu mass. According to Pereira et al. (2003), the phenomenon of syneresis occurs due to the continuous rearrangements of protein molecules, leading to stress in the network and subsequent breakage of protein bonds. Several factors influence syneresis, such as incubation temperature and low pH.
In relation to syneresis of the tofu, the results were statistically treated by analysis of variance (ANOVA), and is observed on the 1 st first and 7 th days of storage by the Pareto chart (Figure 5a and 5b) that the variables studied did not present a significant effect (p >0.05). In relation to ash content of tofu, it can be observed that run 3 presented higher content on the 1 st and 7 th day of storage ( Table 2). The values found in the present study are higher than those obtained by Schmidt et al. (2017) in tofu coagulated with lemon, which may be due to the addition of soy protein concentrate.
Equation 3 present the first order coded model for ash of tofu on the 1 st day of storage where both lemon concentration and soy protein concentrate were significant. However, on the 7 th day of storage, according to Equation 4, only the soy protein concentrate was significant. The models were validated by analysis of variance (ANOVA), presenting a correlation coefficient of 0.99 and 0.98 and calculated F of 6.02 and 2.93 times greater that the F tabulated on the 1 st and 7 th days of storage of tofu, respectively. Thus, it was possible to construct the contour curves for the ashes shown in Figure 6. Ash (%) 1 st day = 1.67 + 0.04X1 + 0.13 X2 (3) Ash (%) 7 th day = 1.65 + 0.14X2 Where X1 is the lemon concentration and X2 is SPC concentration. According to Figure 6, it is possible to verify that the highest ash content was obtained with the highest concentrations of lemon and soy protein concentrate, on the 1 st and the 7 th day of storage.  The ash values of tofu found in the present study were similar to those obtained by Li et al. (2015) that elaborated tofu with organic soybean, coagulated with MgCl2, which found 1.85%.
For the yield it was observed that run 3 presented the highest value ( Table 2). The results were statistically treated by analysis of variance (ANOVA), where it was possible to obtain a first order coded model (Equation 5), which describes the yield as a function of the analyzed variables and it is observed that the concentrated soybean protein had a significant positive effect (p<0.05). Was found a correlation coefficient of 0.92 and calculated F of 1.96 times greater than the tabulated F, allowing the construction of the contour curve (Figure 7). Yield (%) = 28.02 + 21.03X2  The yields obtained in this study are high than those obtained by Schmidt et al. (2017), due to the addition of soybean protein concentrate, because in the tests with great addition of soybean protein concentrate resulted in high yield. In general, comparing tofus on the 1 st and 7 th days of storage was observed a decrease in acidity, moisture and syneresis and increase in solids and ash, and the protein content was not altered. Figure 8 shows the visual appearance of the tofus of the 5 runs of the experimental design, on the 1 st day of storage. All samples of experiments had similar white colour. According to Hou and Chang (2004) the white color is very acceptable for tofu.
It can be observed in Figure 8 (b), run 2, that is the tofu that presented a soft aspect, which is related to the higher moisture content, this occurred due to the low cross-linking between protein molecules resulting in a product softer. The low linking loose the network encompassing, resulting in many air gaps within the network (Jayasena et al., 2014). This aspect can affect the consumers acceptability of tofu.
The tofu of run 3 (Figure 8 c) was the one with the highest yield, protein and ash content, and lower acidity on the first day of storage. The tofu from runs 4 and 5 presented a hardness visual aspect (Figure 8 d and e), that can resulted from the number of protein particles proportion and structure of 11S in the soybeans. The high amount of 11S subunits causes leads to the complete coagulation of soy-curd (SYAH et al., 2015). Regarding the visual aspect, the tofu of run 1 presented the best consistency. Revista Tecnológica -Universidade Estadual de Maringá -ISSN 1517-

CONCLUSIONS
The tofu of run 3, made with vegetable coagulant (5%) and SPS (10%) presented better results in relation to protein, ash and yield, where this formulation is recommended for