Degradation Of Antinutritional Factor β-Conglycinin In Soybean Meal Based On Aptamer Sensor

Soybean meal is one of the most commonly used protein sources in the animal feed industry.However,soybean meal contains a variety of antinutritional factors such as antigenic proteins,which limit its nutritional effectiveness in young animals.β-conglycinin is one of the major antigenic proteins causing soybean meal allergy in weaned piglets.Among them,theβsubunit ofβ-conglycinin is the most thermally stable and enzymatically tolerant,which is not easily removed during processing,but can be used to evaluate the degradation level of antigenic proteins.The bacterial enzyme synergistic fermentation process of soybean meal is gradually becoming a hot research topic nowadays.However,the existing process is difficult to solve the problems of antigenic protein content evaluation and fermentation endpoint control,which hinders the industrial production of fermented soybean meal.To address the above challenges,this study constructed a gold nanoparticles（Au NPs）-aptamer biosensor for the rapid detection ofβ-conglycininβ-subunit based on the previous research.The Au NPs-aptamer sensor was also used to evaluate the degradation ofβ-conglycinin during the fermentation of soybean meal,and to establish and optimize a bacterial enzyme synergistic processing method for the effective degradation of soybean meal antigenic protein.The study covers four main aspects as follows:（1）The feasibility of the application of an evaluation method for the degradation of antigenic proteins based on Au NPs-aptamer sensors was verified.This study first established the Au NPs-aptamer sensor for the detection ofβ-conglycininβsubunit concentration.The detection limit was 3.94 nmol·L^-1（3S/N）,and the detection range was 7～56 nmol·L^-1.The Au NPs-aptamer sensor can accurately detectβ-conglycinin in soybean meal simulating simple liquid fermentation as well as in actual soybean meal products,which demonstrated its good specificity,stability and applicability in evaluating the degradation of soybean meal antigenβ-conglycinin.Meanwhile,the conditions of soybean meal sample detection were optimized to achieve a more accurate and realistic response.The comparative validation by SDS-PAGE and Native-PAGE also demonstrated that the degradation trend of soybean meal antigenic protein was consistent with the results detected by Au NPs-aptamer sensor,which provides a basis for its process evaluation in the bacterial enzyme synergistic fermentation process.（2）The Au NPs-aptamer sensor guided the optimization of the bacterial enzyme synergistic fermentation process of Lactobacillus plantarum DY6/alkaline protease.In this study,the degree ofβ-conglycinin degradation was measured to guide the optimization of the bacterial enzyme synergistic fermentation process of Lactobacillus plantarum DY6/alkaline protease from the perspective of antigenic protein degradation.The optimum fermentation conditions were determined as follows:solid-liquid ratio 2∶1(g·m L^-1),Lactobacillus plantarum DY6 5%,alkaline protease 500 U·g^-1,molasses 3%,37°C for 72 h.Finally,the fermented soybean meal product that effectively degraded 80.75%ofβ-conglycinin was obtained.（3）The Au NPs-aptamer sensor guided the optimization of the bacterial enzyme synergistic fermentation process of Bacillus subtilis 20522/alkaline protease.In this study,Au NPs-aptamer sensor was used to determine the degradation ofβ-conglycinin,and guided the optimization of the bacterial enzyme synergistic fermentation process of Bacillus subtilis 20522/alkaline protease from the perspective of antigen protein degradation level.The optimum fermentation conditions were determined as follows:solid-liquid ratio 2∶1(g·m L^-1),Bacillus subtilis 20522 7%,alkaline protease 200 U·g^-1,molasses 3%,30°C for 72 h.The fermented soybean meal from this process was effective in degrading allergenic proteins,withβ-conglycinin degradation up to 75.27%.（4）The product quality indexes of fermented soybean meal with Lactobacillus plantarum DY6/alkaline protease and Bacillus subtilis 20522/alkaline protease synergistic fermentation processes were determined.The crude protein,protein solubility and acid soluble protein contents of the two fermentation processes of soybean meal were increased.The amino acid content tended to be nutritionally balanced.Organic acid and total acid content increased,and anti-nutritional factors such as trypsin inhibitors,urease,and oligosaccharides were blunted or removed.The nutritional value and flavor of soybean meal were improved.Meanwhile,Lactobacillus plantarum DY6/alkaline protease synergistic fermentation process had better performance in acid production and improvement of acid soluble protein,while Bacillus subtilis 20522/alkaline protease synergistic fermentation process was more advantageous in degrading protein and reducing the anti-nutritional effect of trypsin inhibitors.