| ?-conglycinin is an important soybean antigen protein composed of three subunits of?'(76k Da),?(72k Da)and?(54k Da),which can cause severe allergic reactions in piglets and other animals such as diarrhea,decreased growth function and even death.Among them,?subunit is the most stable and difficult to remove,which is one of the main nutritional inhibitors and can be used to evaluate the content of?-conglycinin.However,there is no effective and accurate method for its rapid detection.Therefore,this study intends to design an efficient,stable,and convenient?subunit detection method for quality control of soybean and its by-products.First,according to the difference of isoelectric point,the 7S globulin?-conglycinin was obtained by isoelectric point precipitation method.Among the subunit trimers,?'and?subunits are hydrophilic peptide,?subunit is hydrophobic peptide with different molecular weight,so?subunit was purified sequentially by phenyl hydrophobic column and sephadex column(sephadex 200),the purity(97.8%)and the mass concentration(0.6 g·L-1)were calculated by Quantity One software and Bradford method,which both met the requirements for aptamer screening.Then,the high-affinity modified aptamer screening kit X-Aptamer was used to obtain aptamer that specifically bind to the?subunit after preliminary screening and re-screening.The bound sequences were dissociated and eluted for next-generation sequencing,and the 10 most abundant sequences were obtained,with an average length of 40bp.The most abundant sequence was selected(?A-1)with dissociation constant 6.9 nmol·L-1determined by q PCR,indicating that?A-1 has a higher affinity that can be used for the establishment of aptamer sensors.With the best aptamer?A-1,the determination of?-conglycinin?subunit combined with gold nanoparticles(AuNPs)-based colorimetric method was established.The best detection conditions of AuNPs proportion,Na Cl concentration,aptamer concentration were 62.5%,0.3mol·L-1,0.5?mol·L-1,respectively.The sensor was used to detect purified?subunit.When the?subunit concentration was in the range of 0-87 nmol·L-1,A600/A520 gradually increased with the increase of the concentration of?subunit,which showed a good correlation in the range of 7-57 nmol·L-1(R2=0.98)and the detection limit was 2.58 n M(3S/N).The specificity of the method was verified by detecting the interference rate of the interfering proteins.The?subunit with final concentrations of 20,50,and 70 nmol·L-1 were added to the diluted soymilk solution to verify the practicability of the method.The recovery rate of?subunit was95.55%-104.88%,which showed that the method can be applied to the detection of actual samples.Finally,the alkaline protease was used to simulate the fermentation of soybean meal to verify the feasibility of the sensor for the detection of actual samples.Then,the key influence factors were analyzed,and finally used for the detection of soybean meal samples that treated by synergistic fermentation method of bacteria and enzyme.The optimal dilution ratio for detection of?-conglycinin?subunit in fermented soybean meal was 10-50 times.The degradation rate of?-conglycinin?subunit of soybean meal was about 87.81%,treated by synergistic fermentation of bacteria and enzyme for 24 h,which was 6.91%higher than that treated with alkaline protease alone.Indicating the aptamer sensor can be applied to the detection of?-conglycinin?subunit in soybean meal fermentation. |
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