Soybean Oil Processing Research Articles

Model for prediction of pesticide residues in soybean oil using partial least squares regression with molecular descriptors

We developed a partial least squares regression (PLSR) model based on competitive adaptive reweighted sampling (CARS) to predict the processing factors of 54 pesticides during soybean oil processing. Characteristic variables were selected to improve the performance of the model. Four calculators were used to compute the molecular descriptors used in the model, and the model based on values computed with ChemoPy produced the best results: Rc ​= ​0.94, RMSEc ​= ​0.67, Rp ​= ​0.91, and RMSEp ​= ​0.54 for hot-pressed oil and Rc ​= ​0.93, RMSEc ​= ​0.73, Rp ​= ​0.93, and RMSEp ​= ​0.59 for cold-pressed oil. A rapid and quantitative model of processing factors was established to predict the behaviour and distribution of pesticide residues during food processing. The model was further validated using data from field-grown soybeans; it demonstrated a high correlation coefficient between predicted and measured residue concentrations (Rp ​> ​0.93, RMSEp ​< ​0.72) and successfully predicted the distribution and behaviour of pesticide residues. Our model provides a reference for assessing safety risk and determining the maximum residue limits for pesticides in processed products.

Development of soybean meal protein isolate-based active edible film incorporating bioactive compounds rich kinnow (Citrus reticulata) peel

Soybean meal, a reservoir of high-quality protein has been produced in large quantities from the soybean oil processing industry and is mostly used as animal feed. The present work was aimed at the development and characterization of soybean meal protein-based antimicrobial edible film incorporating debittered kinnow peel powder (KPP) and valorizing the underutilized soybean meal protein as well as kinnow peel bioactive compounds. The incorporation of kinnow peel powder reduced the solubility, moisture content and swelling ability of the film than the film prepared without KPP. The addition of KPP up to 20 % (w/w) increased the mechanical and barrier properties of the films. The antioxidant and antimicrobial activity of the film increased by 19 to 20 %, and 75 to 79 %, respectively after the incorporation of KPP. The results of structural characteristics (Fourier transform infrared spectroscopy and X-ray diffraction), and morphology (Scanning electron microscopy) showed the good compatibility of KPP and soybean protein isolate. Thermal properties studied through differential scanning colorimetry showed higher melting temperature of KKP added films as compared to control film. The present work indicated that KPP can be successfully used to develop an active edible film and can be used for storing a variety of food products like cheese, paneer, chocolate bars, cut fruits, etc.

Application of Material Flow Cost Accounting to Soybean Oil Production Process: A Systematic Review

Purpose –This study aims to provide an in-depth review of the application of the material flow cost accounting (MFCA) technique. It also provides a detailed account of how the technique can be applied in soybean oil processing, emphasizing its significance, key components, and potential benefits. Methodology - This paper adopts the literature review approach to explain the impact of applying the MFCA technique to the soybean oil production process. It provides insights into how MFCA can be effectively applied and implemented in this context. It aims to identify and quantify the environmental and economic costs associated with material usage, waste generation and management, and energy consumption. Findings - When applied to soybean oil production, MFCA provides insights into process efficiency, waste reduction opportunities and potential cost savings. It is concluded that applying the MFCA technique to the soybean oil production process will bring about cost reduction, waste reduction and environmental sustainability. Implications – The study’s scope and focus did not include a practical application of the MFCA technique to the soybean oil production process. However, if all the steps discussed in this study are diligently followed, it is believed that the technique can be successfully applied in soybean oil production. Originality – The study provides an original insight into the application of the MFCA technique, which is yet to be done to the best of the researchers’ knowledge.

Effect of pH‐shifting treatment on the structural characteristics and nutritional quality of the ultrasonically extracted protein isolate from soybean meal

AbstractSoybean meal is an underutilized byproduct of the soybean oil processing industry, which is rich in high quality protein but mostly used as an animal feed. Valorization of soybean meal on a large scale is possible by extracting protein isolate and modifying its structural characteristics through pH‐shifting or ultrasonic treatment, which has a direct association with the better functional properties of protein isolate. The present study aimed to evaluate the impact of pH‐shifting treatment on the structural characteristics and nutritional quality of ultrasonically extracted soybean meal protein isolate (SMPI). The results showed that the pH shifted SMPI had higher hydrophobicity (60.5–104.3) and free sulfhydryl group (22.67–24.45 μmol/g) than without pH shifted SMPI (53.5 and 19.67 μmol/g, respectively). The fluorescence intensity of the SMPI treated at extreme acidic and alkaline pH was higher than that of other pH‐treated samples. The ꞵ‐sheet (39.68%) components of SMPI treated at pH 10 showed the highest value when compared to other pH‐treated (38.18–39.26%). The alkaline pH‐treated sample had higher concentration of essential amino acids (36.16 g/100 g) than other acidic or near neutral pH‐treated sample (30.06–32.30 g/100 g). The quality parameters such as biological value (67.62), and amino acid index (72.77) were higher for the SMPI treated at pH 10 as compared to the SMPI treated at other pH conditions. Hence, the pH‐treatment method can be utilized as an effective technique to improve the characteristics of SMPI, which will increases its applications in various food sectors.Practical ApplicationsThe current investigation demonstrated that pH‐shifting treatment had a substantial impact on the structural and nutritional quality of the soybean meal protein isolate extracted by ultrasonication. Improvements in soybean meal protein's surface hydrophobicity, free sulfhydryl groups, and secondary structural components will expand its uses in the dairy, bread, meat, and infant food development industries, among other food sectors as these structural changes improve the functionality of protein. Furthermore, the treatment of soybean meal protein isolate with pH shifting led to a considerable increase in nutritional quality, as measured by the essential amino acid composition, essential amino acid index, and protein efficiency ratio, which is expected to increase its utilization in novel food product development.

Ultrasonic extraction of soy protein isolate: Characterization and comparison with microwave and enzymatic extraction methods.

Soy meal as an agro-industrial by-product produced by the soybean oil processing industry is rich in protein. To valorize soy meal, the present study was aimed at the optimization of soy protein isolate (SPI) extraction by ultrasound treatment, its characterization, and comparison with microwave, enzymatic, and conventionally extracted SPI. Maximum yield (24.17%±0.79%) and protein purity (91.6%±1.08%) of SPI were obtained at the optimized ultrasound extraction conditions of 15.38:1 (liquid-solid ratio), 51.85% (amplitude), 21.70°C (temperature), 3.49s (pulse), and 11.01min (time). The SPI extracted with ultrasound treatment showed a smaller particle size (27.24±0.33µm) as compared to that extracted with microwave, enzymatically, or conventional treatment. Functional characteristics, namely, water and oil binding capacity, emulsion properties, and foaming properties of ultrasonically extracted SPI were increased by 40%-50% as compared to SPI extracted with microwave treatment, enzymatically, or conventionally. Structural and thermal properties studied by Fourier-transform infrared spectroscopy, X-ray diffraction, and differential scanning colorimeter showed amorphous, secondary structural change, and high thermal resistance of ultrasonically extracted SPI. Increased functionality of ultrasonically obtained SPI can enhance its application in the development of various new food products. PRACTICAL APPLICATION: Soybean meal is one of the richest sources of protein and has huge potential to lessen protein-based malnutrition. Most of the studies on soy protein extraction were found to be based on the conventional methods that yield less quantity of protein. Hence, ultrasound treatment which is one of the novel nonthermal techniques has been selected for the present work and optimized for soy protein extraction. The ultrasound treatment showed significant improvement in extraction yield, proximate composition, amino acids profile, and improvement of functional properties of SPI as compared to the conventional, microwave, and enzymatic methods which proved the novelty of the work. Hence, the ultrasound technique could be used to increase the applications of SPI for developing a wide range of food products.

Non-GMO-high oleic soybean meal value addition and studying the functional and reconstitution behavior

ABSTRACT Soy meal, a substantial byproduct of soy-oil processing, is rich in crude protein (44–49%), crude ash (5.2–9.1%), minerals, and vitamins. Only 2% of the total soy meal is consumed by humans, while 97% is utilized for animal feed and other industrial purposes. In this study, we use soy meal from non-genetically modified high oleic (Non-GMO-HO) soybean oil processing to develop value-added food products. In this study, soy meal was spray-dried using 5–15% maltodextrin and gum Arabic as a drying aid because trial investigations revealed that the sticky behavior of soy meal powder, which impacts product yield. The physical properties, such as colour, flowability (m3/h), moisture content (%), water activity (Aw), hygroscopicity (%), reconstitution properties, viscosity (mPa-s), thermal degradation behavior, and particle size distribution, were investigated to analyze the impact of drying aid on the quality of soy meal powder. JMP 14.0.0 Pro software was used to conduct the statistical analysis. Spray-dried soy meal powder can be used in reconstituted beverages, infant formulas, soy-based cookies, meat analogs, carrier agents, and more.

Effects of total dissolved solids and organic loadings on the chemical oxygen demand removal by yeast-activated sludge system.

Typically, the soybean oil processing industry generates complex wastewater containing high total dissolved solids (TDS), chemical oxygen demand (COD), and fat oil and grease (FOG) contents with low pH. Therefore, treating this industrial wastewater with the conventional activated sludge (AS) wastewater treatment system is challenging. Yeast can be co-cultured with the activated sludge (AS) called yeast-activated sludge (YAS) to degrade high COD and salinity in the wastewater. However, little is known about the effects of TDS and organic loading (OLRs) on the performance of YAS system with the presence of both Rhodotorula mucilaginosa and Candida xylanilytica. In this study, we evaluated the effects of TDS and organic loading rates (OLRs) on the performances of the YAS system for soybean oil wastewater treatment. Four sequencing batch reactors (SBRs) were operated under aerobic conditions at different TDS concentrations 25%, 50%, 75%, and 100% increasing rate. The results indicated that the TDS at the loading rate of 50% resulted in maximum biomass production. The overloading of the clarifier was observed and possibly caused by the accumulation of solids contained in the influent wastewater. At the OLR of 0.67 kg BOD5/m3-day and the TDS loading rate of 15.5 kg TDS/m3-day, the YAS system achieved the average COD removal efficiencies of 71±1%

A comparative life cycle assessment of phytosterol and meadowsweet (Filipendula ulmaria) oncoprotective functional food ingredients

The urgent and relevant goals of providing a healthier diet and sustainable food systems taken together require a systematic approach that considers a broader environment for food choice. The paper compares three technologies to produce functional ingredients for preventing oncological diseases - each of them involving different sources of raw materials - a by-product of soybean processing, wild meadowsweet extract and planted meadowsweet extract.Life cycle assessment (LCA) has been selected to justify the choice of raw ingredients and the best technology for the stakeholders. Life cycle inventory analysis (LCIA) of technologies was carried out based on the SimaPro 9.1.1.1. To confirm the results of the LCIA of technologies to produce biologically active substances (BAS) from by-products of soybean oil processing, the following models of the SimaPro 9.1.1.1 software were used: EPD (2018) V1.01, ReCiPe 2016 (Midpoint (H) V1.04 / World (2010) H) and IPCC 2013 (GWP 500a V1.01). The validation of the results and the assessment of the impact of uncertainties in the initial data on the LCA calculations of the technologies were carried out using Monte Carlo method. The results showed that production of BAS from a by-product of soybean oil processing is the most environmentally friendly technology, and the data obtained could add to the life cycle database for the category of “Functional food ingredients” or serve as an example for further research and comparison with other targeted functional ingredients.

QTL Mapping of Palmitic Acid Content Using Specific-Locus Amplified Fragment Sequencing (SLAF-Seq) Genotyping in Soybeans (Glycine max L.).

Soybeans are essential crops that supply protein and oil. The composition and contents of soybean fatty acids are relevant to human health and have a significant relationship with soybean oil processing and applications. Identifying quantitative trait locus (QTL) genes related to palmitic acid could facilitate the development of a range of nutritive soybean cultivars using molecular marker-assisted selection. In this study, we used a cultivar with higher palmitic acid content, ‘Dongnong42’, and a lower palmitic acid content cultivar, ‘Hobbit’, to establish F2:6 recombinant inbred lines. A high-density genetic map containing 9980 SLAF markers was constructed and distributed across 20 soybean chromosomes. The genetic map contained a total genetic distance of 2602.58 cM and an average genetic distance of 0.39 cM between adjacent markers. Two QTLs related to palmitic acid content were mapped using inclusive composite interval mapping, explaining 4.2–10.1% of the phenotypic variance in three different years and environments, including the QTL included in seed palmitic 7-3, which was validated by developing SSR markers. Based on the SNP/Indel and significant differential expression analyses of Dongnong42 and Hobbit, two genes, Glyma.15g119700 and Glyma.15g119800, were selected as candidate genes. The high-density genetic map, QTLs, and molecular markers will be helpful for the map-based cloning of palmitic acid content genes. These could be used to accelerate breeding for high nutritive value cultivars via molecular marker-assisted breeding.

Establishment of a predictive model to identify suitable soybean genotypes for oil processing

Various characteristics of soybean play important roles in soybean oil quality. However, the relationship between soybean chemical traits and soybean oil processing quality characteristics is unclear. In this study, the chemical traits of soybean and ten characteristic parameters of soybean oil from 58 genotypes were determined. An evaluation model for the overall soybean oil quality was established according to ten parameters, based on which soybean oil of 58 genotypes was classified into three quality grades by cluster analysis. A prediction model for the quality of soybean oil based on the chemical traits of soybeans was constructed by stepwise regression. The relative errors between the actual values and the predicted values of the validation genotypes were less than 10 %. The predicted quality for 11 of the 14 tested soybean varieties corresponded to the actual oil quality, suggesting that the model is reliable. Our study provides a method for selecting ideal soybean genotypes for producing high-quality soybean oil.

Distribution and degradation of DNA during industrial soybean oil processing

In recent years, soybean has become the most widely grown genetically modified (GM) crop worldwide, and the most extensive manner of transgenic soybean processing is oil production. Determining the distribution and degradation of DNA during oil processing can facilitate the detection, monitoring, and traceability of GM soybean. In this study, we systematically sampled industrial soybean oil extraction and refining processes and performed ultraviolet spectrophotometry, real-time polymerase chain reaction (PCR), atomic force microscopy, and qualitative PCR to evaluate the DNA status. The results indicated that the DNA concentrations in soybean seeds, kernels, conditioned soybean, laminated soybean, extracted soybean, and meal (2.035 × 106–2.682 × 106 ng/g) were significantly higher than those in crude oil (194.800 ng/g) and in subsequently refined oils (0.026–0.664 ng/g) (P < 0.05). Organic solvent extraction and degumming were the two main steps in removing DNA from refined soybean oil. Combined treatment with organic solvent and heating (110 °C) and heating at 240–250 °C were key steps in degrading lectin and cp4epsps during industrial soybean oil processing. Our analyses of the DNA status during soybean oil processing will contribute to the detection, traceability, and labelling of GM soybean.

Enzymatic synthesis of biolubricants from by-product of soybean oil processing catalyzed by different biocatalysts of Candida rugosa lipase

The present work studied the synthesis of biolubricants from by-products of soybean oil processing using the lipase from Candida rugosa (CRL) both in free and immobilized forms, as biocatalysts. Soybean fatty acid distillate (SFAD) was used for first time as source of free fatty acids (FFA) to produce biolubricants via their enzymatic esterification with neopentyl glycol (NPG) and trimethylolpropane (TMP) alcohols in a solvent-free medium. The immobilization of CRL was performed via interfacial activation using a commercial hydrophobic support (Accurel) and a home-made core-shell matrix of poly(methyl methacrylate) (PMMA). Conversions of about 80 % were obtained using lyophilized CRL after 30 or 180 min using NPG and TMP, respectively. When CRL-PMMA/PMMA was used to catalyze the synthesis of NPG esters, 90 % conversion was achieved after only 6 h, whilst CRL-Accurel were more active using TMP. The biocatalysts maintained the reaction conversion during eight batches of 24 h. The lubricant properties of the products were also characterized, showing that it can be an environmentally friendly alternative for petrol lubricants

Effect of chemical refining on the levels of bioactive components and hazardous substances in soybean oil

Tocopherols and phytosterols are bioactive components naturally present in vegetable oils, while trans fatty acids (TFAs), polycyclic aromatic hydrocarbons (PAHs) and 3-monochloropropane-1,2-diol esters (3-MCPD esters) are harmful components that frequently occurring in edible oils. The study is aiming to evaluate changes of these components in soybean oil at each step during chemical refining. After refining, the concentrations of tocopherols, phytosterols and PAHs decreased from 1411.1 mg/kg, 2578.0 mg/kg, and 45.3 µg/kg to 1063.6 mg/kg, 1354.6 mg/kg, and 3.7 µg/kg, respectively. However, the concentrations of TFAs and 3-MCPD esters increased from 0.11% and 25.5 µg/kg to 1.08% and 320.2 µg/kg, respectively. The most significant reductions in phytosterols (56.5% of total reduction) and PAHs (67.0% of total reduction) were attributed to neutralization, while the most significant reduction in tocopherols (47.8% of total reduction) was attributed to deodorization. Meanwhile, deodorization contributed most to the increases of TFAs (96.9% of total increment) and 3-MCPD esters (64.9% of total increment). This work could provide guidance for soybean oil processing with respect to minimize the amounts of hazardous substances while maintain the contents of bioactive components.

Intensification of degumming of crude soybean oil using ultrasound in combination with oxidizing agents

Intensified degumming of crude soybean oil was investigated using ultrasound assisted combination approaches based on longitudinal ultrasonic horn at a capacity of 5 L with comparison with the conventional processing based on only ultrasound. The combination of ultrasound and oxidizing agents such as hydrogen peroxide (H2O2), potassium persulphate (KPS), Peroxymono sulphate (PMS) and Fenton reagent have been investigated for establishing the efficacy for intensified degumming. Combination of ultrasound and H2O2 loading at 100 ppm resulted in 94.15% phospholipid reduction at 150 W power and ambient temperature. Enhanced extent of degumming was observed for combination of ultrasound with KPS and PMS as 95.10 and 95.55% respectively. Ultrasound combined with Fenton reagent resulted in significant increase in extent of degumming (98.28%). Best degumming protocol was established as the combination of ultrasound with UV and hydrogen peroxide giving 99.18% phospholipid removal in only 120 min of treatment. The degumming was also supported by positive changes in the oil characteristics in terms of reduction in acid value (0.74 as the final value) and better oxidative stability (TOTOX value of ˂ 4). The present work demonstrated intensified degumming protocol for crude soybean oil processing for obtaining more than 99% degumming with better characteristics of processed oil.

Ultrasound assisted enzymatic degumming of crude soybean oil

The present work deals with ultrasound assisted enzymatic degumming (UAED) of crude soybean oil quantifying the extent of degumming (EOD), cavitational yield and synergistic index (f) for the combination approaches. The effect of different operating parameters such as enzyme loading, pH, presence of water, temperature and ultrasonic power on the EOD has been investigated. Ultrasound combined with enzyme at loading of 2.0 ml/L resulted in EOD as 92.2% under ambient conditions. Addition of water (5%) in combination with ultrasound and enzyme at 2.0 ml/L loading and pH of 5 resulted in maximum EOD (98.4%) in 120 min of treatment. The extent of phospholipid separation was also observed to be dependent on the power dissipation and maximum phospholipids separation was obtained at 100 W. Scale-up studies were performed at 500 ml and 1 L operating volume under optimized conditions of 2.0 ml/L as the enzyme loading, pH of 5, 5% water addition and ultrasonic power of 100 W where 93.63% and 91.15% phospholipid separation respectively was obtained. The effects of ultrasonic treatment were also quantified in terms of the acid value reduction and oxidative stability for the processed oil. It was demonstrated that suitable reduction in acid value (final value less than 1) and oxidative stability (TOTOX less than 4) is effectively obtained using UAED. Overall the approach of UAED was established to show much higher efficacy for soybean oil processing as compared to only ultrasound or only enzymatic treatment.

Intensified degumming of crude soybean oil using cavitational reactors

A novel cavitation assisted degumming approach for the purification of crude soybean oil in terms of the removal of the total phospholipid content has been reported in the work with a detailed study into the effect of operating parameters such as temperature, hydrogen peroxide loading and introduction of ozone gas at varying flow rate. Ultrasonic horn, ultrasonic bath and hydrodynamic cavitation reactors have been used for comparing the efficacy of different types of cavitational reactors. Initially, extent of degumming was quantified at different temperatures and 60 °C was established as optimum to give significant extent of degumming in terms of phospholipids removal. Degumming in the presence of ozone gas gave unsatisfactory results because of the specific physical and chemical properties of crude soybean oil. Combination of ultrasound and hydrogen peroxide resulted in complete phospholipid degradation at capacity of 250 ml at optimized temperature in 100 min of treatment. Ultrasonic horn and bath were also used at 1 L operating volume and the extent of degumming was found to be 90.81% and 93.50% respectively. For large scale studies, hydrodynamic cavitation was operated at 4 bar pressure using slit venturi as the cavitating device which gave 93.65% as the extent of degumming. Overall, intensified degumming approach for processing of crude soybean oil was demonstrated to yield high quality oil in terms of the reduced phospholipids content. Cavitation based degumming process can be an excellent alternative approach to the conventional method with major benefits like reduction in time and energy requirements as demonstrated in the present work.

Thermal processing of soybean oil to obtain bio-based polymers and bio-oil

In this study we evaluated the use of triacylglycerides to produce bio-based resins and bio-oils suitable to be used, respectively, as a binder in printing inks (offset) and as diesel-like fuel. Soybean oil was kept under nitrogen atmosphere at temperatures ranging from 260°C to 370°C up to 12h in the presence or absence of a nickel complex as a catalyst precursor. It was observed that the reaction occurs in two steps. In the first one, occurs the consumption of the double bonds via Diels–Alder to form a polymer increasing the viscosity of the material. In a second step, the pyrolysis of ester groups and the alkyd chains takes place, reducing a viscosity of the polymers. Besides, using Nickel complex as a catalyst precursor it was observed a high activity to produce polymers with higher viscosity in a shorter time than when comparing with reactions without catalyst. It were also analyzed the bio-oil formed during the reaction. It was observed that without catalyst the pyrolysis leads to the formation of high amounts of carboxylic acids with short chain. However, the presence of Nickel complex increased the formation of hydrocarbons and reduced the amount of formed carboxylic acids, strongly indicating its activity in the deoxygenation.

Industrial Applications of Selected JFS Articles

Industrial Applications of Selected <i>JFS</i> Articles

Soybean cake as a source of stable bioactive compounds (647.43)

Soybean cake is a byproduct of soybean oil processing rich in bioactive compounds. In this study we evaluated the effect of soybean cake extracts on cell viability, cycle and apoptosis in human colon adenocarcinoma cell line (HT‐29) and their stability under different storage conditions. Soybean cake from soybean oil industry (SCI) and produced under laboratory conditions (SCL) were extracted with a ternary mixture (water:ethanol:ethyl acetate), lyophilized and incubated with HT‐29 cells at concentrations (10 to 400 mg/g) from 6 to 96 h. The dry extracts were stored in freezer or room temperature (light or dark) and the following analysis were conducted at regular intervals during 120 days: spectrophotometric assays for phenolic compounds, flavonoids and saponins; antioxidant capacity (AC) by FRAP and TEAC assays; isoflavones and soyasaponins by LC‐DAD‐MS. SCL and SCI extracts showed high levels of phenolic compounds (12.36 and 10.45 mg gallic acid/g), flavonoids (7.24 and 9.98 mg genistein/g), saponins (224.86 and 253.99 mg/g) and AC (FRAP: 71.35 and 91.57 µmol Fe+2/g; TEAC: 105.02 and 103.90 µmol Trolox/g). LC‐DAD‐MS analysis revealed that SCI extract presented higher levels of β‐glycosylated isoflavones (3.67 mg/g) than SCL (0.72 mg/g). Aglycone isoflavones were present in higher levels in SCL extract (3.16 mg/g) than in SCI (0.82 mg/g). Both extracts reduced in a time‐dose dependent fashion cell viability when compared to control at concentrations starting from 100 mg/g. These reductions ranged from 4% to 84% and from 24% to 85% when cells were incubated with SCL and SCI, respectively. After incubation with SCL a decrease in the percentage of cells at G0/G1 phase and an increase in G2/M phase was observed, as well as an increase in the apoptosis rate. After 120 days of storage, bioactive compounds’ levels did not change, with the exception of FRAP values. Extracts showed anticancer effects in vitro and soybean cake is a potential source of stable bioactive compounds for in vivo studies.Grant Funding Source: FAPERJ, CNPq, CAPES, UFRJ

The Integral and Extrinsic Bioactive Proteins in the Aqueous Extracted Soybean Oil Bodies

Soybean oil bodies (OBs), naturally pre-emulsified soybean oil, have been examined by many researchers owing to their great potential utilizations in food, cosmetics, pharmaceutical, and other applications requiring stable oil-in-water emulsions. This study was the first time to confirm that lectin, Gly m Bd 28K (Bd 28K, one soybean allergenic protein), Kunitz trypsin inhibitor (KTI), and Bowman-Birk inhibitor (BBI) were not contained in the extracted soybean OBs even by neutral pH aqueous extraction. It was clarified that the well-known Gly m Bd 30K (Bd 30K), another soybean allergenic protein, was strongly bound to soybean OBs through a disulfide bond with 24 kDa oleosin. One steroleosin isoform (41 kDa) and two caleosin isoforms (27 kDa, 29 kDa), the integral bioactive proteins, were confirmed for the first time in soybean OBs, and a considerable amount of calcium, necessary for the biological activities of caleosin, was strongly bound to OBs. Unexpectedly, it was found that 24 kDa and 18 kDa oleosins could be hydrolyzed by an unknown soybean endoprotease in the extracted soybean OBs, which might give some hints for improving the enzyme-assisted aqueous extraction processing of soybean free oil.