A laboratory process for separating glycinin and β-conglycinin from soybean flakes was successfully scaled up to the pilot-plant scale (15 kg soy flakes). Average yields of the glycinin and β-conglycinin fractions were both 9.4% on a dry basis (db). The protein contents of glycinin and β-conglycinin fractions were 92.8 and 97.7% db, respectively. The glycinin and β-conglycinin purities were 90.4 and 72.7% of the protein content, respectively, which were very comparable to those of the laboratory-scale process. The total sulfhydryl plus half cystine content of the glycinin fraction was 37.8 mol/mol protein and 14.8 mol/mol protein for the β-conglycinin fraction. The native glycinin structure loss in the glycinin fraction was negligible. The native β-conglycinin loss in the β-conglycinin fraction was 10%, as estimated by rocket immunoelectrophoresis analysis. Hydrophobicity index value showed that hydrophobic properties of the pilot-plant protein fraction were ordered, from high to low: β-conglycinin fraction > glycinin fraction > intermediate mixture fraction.Paper no. J8950 in JAOCS 76, 285-293 (March 1999).KEY WORDS: β-Conglycinin, glycinin, pilot-plant process, protein, protein physicochemical properties, protein yield and purity, soybean proteins, soybeans, soy protein separation.Glycinin and β-conglycinin, the two major soybean storage proteins, apparently play different roles in food and nonfood soy protein products due to their different functional properties such as hydrophobicity, solubility, sulfhydryl cross-linking, gelation, and film formation. Wolf et al. in 1962 (1) first obtained 91-93% ultracentrifugally pure glycinin (11S) by cryoprecipitation followed by fractionation with ammonium sulfate. They reported 25% yield for the purified glycinin. Factors influencing yield and purity of the glycinin fraction, such as extraction ratio, temperature, pH, salt and sugar content, and reducing reagent (2-mercaptoethanol, 2-ME), have been investigated (2). Koshiyama (3) reported a procedure for glycinin and β-conglycinin (7S) fractionation. The glycinin fraction was first cryoprecipitated, and then 0.025 M CaCl 2 was added to remove the residual cold-insoluble proteins. The 7S fraction, which contained mostly β-conglycinin, was precipitated by adjusting the pH of the supernatant to 4.5. After gel filtration, a homogeneous β-conglycinin fraction was obtained as evaluated by ultracentrifugal analysis, which does not differentiate among different proteins of comparable mass. Thanh et al. in 1975 and 1976 (4,5) developed a straightforward process for glycinin and β-conglycinin separation based on differential solubilities of glycinin and β-conglycinin at pH 6.1-6.6. Tris (THAM) buffer (pH 8.0) containing 10 mM 2-mercaptoethanol (ME) was used to extract soy proteins. Glycinin was separated by adjusting the pH to 6.4 and collecting the precipitate after centrifuging at 2-5°C. β-Conglycinin was precipitated at pH 4.8 and purified by redissolving the precipitate in the 0.03 M Tris buffer and adjusting the pH to 6.2. ...