Chemicals were purchased from Merck, soybean seeds were from local markets, edible soy flour was provided by Progressive Exim, refined rice bran oil was from Shethia Oil Mill, and refined sesame oil was from the local market.

Soy milk from whole soy seeds was heated to 97 °C, covered with aluminum foil, and cooled to 87 °C. It was coagulated with different coagulating agents, formed a gel, compressed, and left overnight at room temperature. The resulting four different types of tofu were used as control and designated:

De-oiled soy flour provided by M/S Progressive Exim, Raipur, M.P., India, was used in the preparation of Tofu. Soy milk from edible soy flours (meal) was prepared according to the methods described by Sengupta et al. (2016). The production of RBO and SO incorporated soy milk from edible de-oiled soy flour was prepared by Sengupta et al. (2016) with slight modifications. Soy milk from edible soy flour was homogenized and pasteurized at 80 °C for 15 minutes. RBO and SO were added, blended in different ratios (20:80, 50:50, and 80:20), and stored in amber bottles. The fat content in the tofu was maintained by adding 5% soy milk, and mixtures of soy milk and oils were homogenized. Citric acid, lactic acid, tartaric acid, and calcium lactate were used as coagulants, each dissolved separately in distilled water to create a coagulant solution.

The RBO and SO-fortified soy milk prepared from the de-oiled edible soy flour was subsequently used in the preparation of fortified tofu following the same procedure described in the preparation of the control tofu from soy milk obtained from whole soy seeds. A schematic diagram for the preparation of fortified tofu is given in Figure 1. All the control and experimental tofu were stored at 4 °C in the refrigerator for 9 days for further analysis. Twelve different types of experimentally fortified tofu were manufactured and designated:

The proximate compositions of all tofu samples prepared by using different coagulants were carried out in triplicate using the standard methods of AOAC (2005). The tofu sample’s initial weight was determined, dried in an oven, and then desiccated. The moisture content was determined by subtracting the dry weight from the original weight, while ash content was estimated by incinerating the sample in a muffle furnace for 24 hours and was then expressed as g/100g dry weight of tofu. The protein level in the defatted tofu sample was evaluated using the Bradford (1976) method at 595 nm after extraction, centrifugation, and protein level quantification. Fat was determined according to Bligh and Dyer’s (1959) method by some process modifications and the total lipid content was determined using the formula in Equation (1).

Carbohydrate content was calculated by difference [100 − (moisture + crude protein + lipid +ash)]. The Atwater formula was used to calculate energy values, with fat, protein, and carbohydrates contributing 9, 4, and 3.75 kcal g^-1, respectively (Merrill and Watt 1973).

The penetration properties of tofu were determined by a Penetrometer (Stanhope-Seta Surrey, England) using the cone-form penetration body with an apical angle of 45° and a weight of 72.5 g. The depth of penetration was measured at 5 s at a product temperature of 25 °C.

The color intensities in the tofu were measured by use of the colorimeter (Konica Minolta CR 10), which gave the Hunter parameter (L ^* , a ^* , b ^* ) and also c ^* and h ^* values directly. L ^* indicated lightness which describes the light reflecting or transmitting capacity of an object. Color analysis was also performed by the determination of a ^* (−green to +red component), b ^* (−blue to +yellow), c ^* (chroma) and h ^* (hue angle) values in triplicate.

According to APHA (2005), to assess the microbiological quality of tofu, ten grams of each sample were homogenized in a sterile saline solution until a 10⁶-fold dilution was achieved. The dilutions were then distributed onto Petri dishes, and the total plate count was calculated using Plate Count Agar. Potato Dextrose Agar was used for yeasts and molds.

Freshly prepared tofu samples were kept at 37 °C for sensory evaluations. 20 members were chosen from the School of Community Science and Technology, IIEST, Shibpur, Howrah, West Bengal. They developed a consensus evaluation for flavor attributes for tofu and the evaluation was carried out on a Nine-Point Hedonic Scale (ISO, 2014). The quality properties that were evaluated were color, taste, flavor and overall acceptance.

The oxidative stabilities of tofu were examined based on the determination of acid (FFA %) content, peroxide value (PV), thiobarbituric acid value (TBA) and p-anisidine value (p-AV) and Totox value (TV) of the oil extracted from tofu. The FFA content (%) of the tofu samples was determined using a method described by the AOCS Ca 5a-40 Official Method (1997) with some modification in the weight of sample used (5 g oil sample). Oil extracted from the samples was mixed with ethanol and phenolphthalein, titrated with NaOH, and analyzed to determine its color retention. The % FFA was expressed using the following Equation (4)

The PV (meq O₂·Kg⁻¹) of the oils was measured by the acetic acid-chloroform method AOCS Cd 8-53(1997) to determine primary lipid oxidation products. A 5 g oil sample was weighed, mixed with acetic acid, chloroform, starch, and potassium iodide, and titrated with thiosulfate until the blue color disappeared. The PV was determined according to Equation (5):

whereB,S,N, andW are ml of sodium thiosulfate titrated for the control, ml of sodium thiosulfate titrated for the sample, sodium thiosulfate normality, and weight of the sample, respectively.

The TBA analysis of tofu was conducted following the procedure described by Hekmat and McHamon (1997). The TBA reagent was prepared by dissolving 200 mg TBA in 1-butanol, filtered, and stored. The oil sample was dissolved, heated, and measured for absorbance. A reagent blank was also prepared and the TBA value was expressed using the following Equation (6)

Where, A, B, M are absorbance of the solution, absorbance of TBA reagent and mass of the mg of the oil sample, respectively.

The Official method AOCS Cd 18-90 (1997) was used to determine p-AV. The study involved dissolved p-anisidine in glacial acetic acid, diluted with isooctane, and measuring its absorbance using an isooctane-based spectrophotometer. The solution was then pipetted into separate test tubes, combined with 1 ml of p-AV solution, and the absorbance was measured after 10 minutes. Equation (7) was used to calculate p-AV

where As and Ab are absorbances of the solutions before and after the reaction with the p-AV solution, respectively.Wis the weight of sample.

TOTOX values (TV) were calculated using the following Equation (8) .

(PV: Peroxide Value; AV: Anisidine Value)

Data are presented as means ± SD. Statistical analysis was performed using multivariant analysis of variance (ANOVA) with the Origin Pro 8 software package for windows. The means were compared between groups by Tukey’s post-hoc test. All analyses were carried out in triplicate. Values of p < 0.05 were considered significant.

The study compared the composition of control tofu samples from soy milk and tofu samples from RBO and SO-fortified soy milk using the same coagulating agents. The proximate compositions of the control and the experimental tofu samples are shown in Table 1.

Table 1 revealed that there were significant differences in moisture content between the control and fortified tofu prepared by using the above-mentioned coagulants. The citric acid-coagulated tofu samples (both control and fortified) had the highest moisture content, and the tartaric acid-coagulated tofu had the lowest moisture content among the tofu samples. Among fortified tofu, CIT TII (4.59±0.04; p < 0.05) showed the highest moisture value, and TAR TIV (4.14±0.04; p < 0.001) showed the lowest moisture value. Joshi et al. (1991) also observed that the addition of tartaric acid as a coagulant increased moisture retention in chhana manufactured from animal sources, which is in contrast to our findings because we developed fortified tofu from plant sources. Additionally, the variation in moisture content of fortified tofu with different coagulants was probably due to the differences in gel networks within the fortified tofu particles which were influenced by different ratios of RBO and SO incorporation towards the water holding capacity of de-oiled soy protein gels. It may also be due to the unique coagulating properties of different coagulating agents (Yakubu et al., 2013). The protein content of different tofu samples is given in Table 1, showing that there were significant differences (p < 0.05) in protein content between the fortified tofu and the control tofu. Fortified tofu prepared by using citric acid, lactic acid, and tartaric acid had a much higher protein content than the control tofu obtained by using the same coagulants. The protein content of fortified tofu was higher than that of the control tofu, except for the tofu prepared using calcium lactate. These values for proteins were higher than the values (13-17.6%) obtained by Yakubu et al. (2013) and lower than the values (56.89-59.98%) obtained by Shokunbi et al. (2011). Sengupta et al. (2016) also noted that the protein content of non-dairy soy yogurt with sesame and rice bran oil had a greater percentage of protein in comparison with the control soy yogurt without the addition of vegetable oil. Our research supports these observations. Table 1 indicated that there were no marked differences in fat content between the control and fortified tofu prepared by using the same coagulating agents. Lactic acid-coagulated tofu samples (both control and fortified) had the highest fat value, and tartaric acid coagulated tofu had the lowest fat content among the tofu samples. The decrease in fat content was due to the decrease in fat recovery from lactic acid, followed by calcium lactate, citric acid, and tartaric acid tofu (Khan et al., 2014). It can be observed in Table 1 that the same trend in ash content was observed between the control and fortified tofu prepared by using different coagulating agents. Citric acid-coagulated tofu samples (both control and fortified) had the highest ash value, and lactic acid-coagulated tofu had the lowest ash content among all the tofu samples prepared by using different coagulating agents. Our findings are comparable to those of Khan et al. (2014), who found no significant differences in the ash level of paneer coagulated by the three different forms of coagulant, although their citric acid-coagulated paneer had a greater value of ash content. Table 1 indicates that the same trend in moisture contents was observed between the control and fortified tofu prepared by using citric acid, lactic acid, tartaric acid, and calcium lactate. Table 1 reveals that the carbohydrate content of fortified tofu was lower than that of control tofu, except for tofu prepared using calcium lactate. The presence of carbohydrates is responsible for these energy values. The projected energy values increased when the amount of carbohydrates increased, and vice versa. When compared to the data from Shokunbi et al. (2011), the computed range of carbohydrates in the current study was from 25.17 to 38.17%, which was somewhat higher.

The penetration properties of the different tofu samples prepared by different coagulating agents are shown in Table 2 , showing that the penetration properties of the control tofu were lower than those of the fortified tofu. Each coagulant used in the preparation of fortified tofu influenced the texture of the tofu, particularly the hardness of the fortified tofu. The penetration of tofu indicated its resistance to compressive forces. The results showed that the hardness of the control tofu made from whole soy beans was very high and the addition of citric acid, lactic acid, tartaric acid and calcium lactate was not able to alter the hardness values. The difference in hardness in fortified tofu had a close correlation with the ratio of RBO and SO in the gel of the protein network. Based on the penetration property, tofu made with tartaric acid was less rigid and stretchy than tofu cured with citric and lactic acids. Hanáková et al. (2013) made a similar study and discovered that gel hardness decreased with increasing vegetable oil concentrations.

The effects of different coagulants and the effects of vegetable oil fortification on the color (L*, a*, b *, c, and h) of the control and fortified tofu are shown in Table 3.

Table 3 indicates that brightness (L*), redness (a*), and yellowness (b*) showed highly significant (p < 0.05) differences between the control and fortified tofu. It was observed that there was a decreasing trend for L* values in the de-oiled seed flour-based fortified tofu compared to the control tofu. Citric acid-coagulated control and fortified tofu had the highest L*, followed by lactic and tartaric acid-coagulated tofu, respectively. The lowest L* values were obtained in the cases of calcium lactate-coagulated control and fortified tofu. With an increase in RBO content in blended oil-incorporated tofu, an increase in L* values were observed. This may be due to the bleaching properties of the fatty acids present in the oil. There were significant differences in a* values (p < 0.05) among the different fortified tofu (-0.35±0.01<a*<-0.15±0.05) and among different control tofu (-0.18±0.01<a*<-0.12±0.15). The a* values of fortified tofu were lower than those of the control tofu. Table 3 shows that citric acid-coagulated fortified tofu had higher b* values, followed by lactic acid, tartaric acid, and calcium lactate, respectively. On the other hand, tartaric acid-coagulated control tofu had the highest b* values compared to other control tofu. Fortified tofu showed a marked decrease in the yellow component (+11.90±0.01< b*<+14.80±0.04) in relation to tofu (control) (+12.62±0.29<b*<+15.20±0.03) and the b* values of fortified tofu were lower than tofu (control). The results indicate significant differences among the color indices measured with respect to the different types of coagulants used. CIT TIII becomes more yellow. Food deteriorative processes, including the oxidation of fat, browning, and color oxidation, are all facilitated by oxygen. These findings highlight the need for effective packaging in order to preserve enriched tofu. Our findings are consistent with those of Jain et al. (2015), who similarly noted a decline in the color value of the Kalakand during an atmospheric oxygen attack. De-oiled soy flour would have increased the amount of amine compounds that combine with aldehydes during the Maillard process to create dark pigments (melanoidins).

The microbiological qualities of control tofu and fortified tofu are presented in Table 5 . Control and fortified tofu’s microbiological quality may be influenced by the soy milk’s quality, heat treatment, unsanitary manufacturing practices, and conditions after production, such as handling, packaging, and storage. The results concerning TPC (log CFU. g^-1) of all treatments (tofu) were not significantly (p > 0.05) different. After 9 days of storage, there were no appreciable changes in the TPC values of any treatment during the course of storage, which saw counts gradually rise in all treatments. Yeast and mold count levels grew throughout storage in all treatments; however, they did not differ significantly from one another. Additionally, the results showed that neither the control nor the fortified tofu had any coliform, Salmonella, or E. coli during storage (data not shown). It has been found that whereas boiling milk at 80 °C for 15 minutes normally totally destroys coliforms, yeast, and mold, these organisms may reappear in a sample of tofu under post-manufacturing conditions. The increasing trend of TPC and yeast and mold count during the storage of tofu has also been reported in some studies (Das et al., 2018). The TPC, yeast, and mold results revealed that oryzanol and lignan, which are retained in the enriched tofu matrix, had an inhibitory effect on microorganisms. In contrast to the control tofu, microorganisms multiplied less after storage in fortified tofu that had RBO and SO. After 12 days of storage, it was noticed that fungus had begun to grow on the fortified tofu’s surface. Our findings were in line with those made by Das et al. (2018), who observed that paneer or tofu surfaces showed obvious sliminess and reddish-brown or yellowish-brown degradation after 12 days of storage due to microbial buildup, damaged paneer or the fact that the tofu was discarded.

The sensory evaluation of the control and fortified tofu produced using various coagulants is given in Table 6 , showing that fortified tofu showed significantly (p < 0.05) higher acceptability than control tofu as typified by the color, taste, and aroma. Overall acceptability was highest in TAR TIII (8.14±0.39; p < 0.05) and lowest in LAC TIV (6.37±0.29; p < 0.001). This result was in accordance with the results of the sensory evaluation of tofu by Mitra et al. (2013) and Bandyopadhyay et al. (2005), who showed that tofu coagulated with calcium salt and tartaric acid was rated superior in terms of color, taste, aroma, and overall acceptability to the tofu obtained from citrus juices. The organoleptic properties of tofu are significantly influenced by the combination of RBO and SO. Therefore, the quality of the milk and vegetable oil used to make tofu depend on each other. According to certain research, cheese made with palm oil was substantially softer than cheese made with whole milk fat when milk fat was replaced at 100% (Bandyopadhyay et al., 2005; Mitra et al., 2013). We noticed the same pattern with a fresh soft-fortified tofu called TAR TIII that contained a 100% blend of RBO and SO (80:20). It was explained that this was because the presence of fat causes the protein bonds to break down, reducing rigidity and bringing about smoothness and a softer texture, which led to a higher level of acceptability overall than with the control tofu.

The effect of citric acid, lactic acid, tartaric acid, and calcium lactate as coagulants on FFA, PV, TBA, PAV, and TV of the control and fortified tofu over 9 days of storage is given in Table 7 (A, B, C, and D). It was observed that FFA, PV, TBA, PAV, and TV of both the control and fortified tofu increased during the total storage period of 9 days. The results revealed that the above-mentioned values for fortified tofu were lower than those of the control tofu due to the presence of antioxidants. The results were in agreement with Kumar and Bector, (1991), who studied the effect of synthetic antioxidants on the shelf-life of tofu. The control and fortified tofu’s peroxide values both steadily rose throughout storage, whereas the control tofu’s value significantly dropped at the end of the storage period. Thiobarbituric acid (TBA) values indicated significant increasing trends after storage for 9 days in the generation of secondary lipid peroxidation products (Table 7: A, B, C, and D). A fast rate of increase in AV or TBA values was observed after up to 9 days of storage. This would have been feasible as a result of the breakdown or volatilization of secondary oxidation products. In comparison to the control tofu, RBO and SO-enriched tofu exhibited better oxidative stability, as seen by the decreased concentration of degradative compounds found in these products. Since the PAV monitors secondary oxidation products, which are more stable during the heating process, it is a more accurate and useful test than the PV. The significantly lower concentration of secondary reaction products as indicated by PAV in RBO and SO-fortified tofu compared to control tofu might be due to the increase in SFA or MUFA with the decrease in PUFA in control tofu, which are the primary targets of thermal oxidative reactions. The TV in the control and fortified tofu increased during storage. However, the levels of TV in the current investigation did not rise continuously with longer storage times. This improved oxidative stability may have been brought about by the nutritional value of minor components such as tocopherols, tocotrienols, and oryzanol in RBO and sesamin and sesamolin in SO (Gulla and Waghray, 2011).