Detection Of Soybean Proteins In Surimi Products

In recent years, with the production of surimi products increasing, the low-value proteinsespecially soybean proteins are frequently added to surimi proteins or raw materials, whichinjurious to the interests of consumers. The aim of this study was to establish sensitive andreliable methods for determination of soybean proteins in surimi products. Soybean trypsininhibitor (STI) and β subunit of β-conglycinin were chosen as the target proteins which havehigh thermal stability. This study would provid the technical support for preparing a kit fordetection soybean proteins in surimi products.STI was purified from soybean meal by a series of steps, including acetone extraction,H2SO4treatments, ammonium sulfate fractionation DEAE-Sephacel, and SP-Sepharose ionexchange column chromatography. Purified STI was heated at110℃for30min as theimmunogen. Then, an anti-STI pAb were prepared by immunized the New Zealand white rabbitsfed with soybean-free diet after weaning. Moreover, anti-STI mAb was prepared by immunizedthe female BALB/c mice fed with soybean-free diet. The pAb and mAb were purified by ProteinA Sepharose and Protein G Sepharose affinity column, respectively. Western blot, c-ELISA ands-ELISA using these two antibodies were then established to determine the soybean proteins insurimi products. The limit of detection (LOD) of c-ELISA established with anti-STI pAb was24mg SPI/kg food, and the recoveries of SPI in surimi (contain5,10,20mg/g SPI) ranged from76.0%to109.0%. The LOD of Western blot and immunofluorescence established by anti-STImAb were50mg SPI/kg food and187.5mg SPI/kg food, respectively. The recoveries rangedfrom81.9%to98.7%analysis by Western blot. These two methods could be used tosemi-quantitative of soybean proteins in surimi products. Western blot is sensitive thanimmunofluorescence, however, immunofluorescence displayed the characteristics of shorttime-consuming and safety. In the s-ELISA, anti-STI mAb (A11-6) was used as coating antibody,and anti-STIⅡ pAb was used as secondary antibody. The LOD and the limit of quantitation(LOQ) were13.6mg SPI/kg food and61.7mg SPI/kg food, respectively. The recoveries ofsoybean proteins in surimi products were ranged from100.1%to122.2%. The results indicatedthat s-ELISA showed high accuracy. According to the established the s-ELISA，the kit for thequantitative detection of soybean proteins in surimi products was prepared.On the other hand, the protein with molecular weight of48kDa was also purified fromsoybean meat by isoelectric precipitation and Q-Sepharose column chromatography.9peptidefragments with a total of175amino acid residues was obtained by Peptide mass figerprinting (PMF) analysis, which was100%identical to β subunit of β-conglycinin from Glycine max,suggesting that it is β subunit of β-conglycinin. Chosen β subunit as the target protein, pAb andmAb were prepared by immunized New Zealand white rabbit and BALB/c mice fed withsoybean-free diet after weaning, respectively. The pAb was obtained with high specificity butlow affinity to β subunit, so it was unsuited to establish the c-ELISA. The β subunit subcloneshowed low specific to β subunit of β-conglycinin. Therfore, the preparation of monoclonalantibody was not continued.