The Effects Of High Hydrostatic Pressure On The Stability And Bioavailability Of Natural Folate In Soybean

Folate is one of the B-group water-soluble vitamins which is necessary for human growth and development.However,as a common folate supplement and fortifier,chemically synthesized folic acid has the potential risk to mask vitamin B₁₂deficiency and induce cancer.Countries such as the European Union recommend to intake folate in its natural form.Soybean is rich in natural folate（144.36±0.39μg/100g）,which can provide at least 10%of the daily recommended intake of folate and can be regarded as a good source of folate.However,compared to chemically synthesized folic acid,natural folate has poor stability（e.g.,the biological half-life of tetrahydrofolate<5 h）and low bioavailability（10%-98%）.Therefore,how to improve the stability and bioavailability of natural folate has always been an important direction of scientific research.High hydrostatic pressure（HHP）,a new type of nonthermal food processing technology,may change the stability and bioavailability of natural folate by changing the structure of different substrates in food and the form of natural folate,but without related research reports.Therefore,this study will explore the effect of HHP on the stability and bioavailability of natural folate in soybeans,the research content are as follows:1.An ultra-high performance liquid chromatography-tandem mass spectrometry（UHPLC-MS/MS）method was established and validated for the efficient determination of one-carbon metabolites,and the extraction method was also optimized.The advantages of the established method include:high efficiency（7.5min/sample）,good linearity（0.992≤R²≤0.999）,low limit of detection（0.33-1.35nmol/L）,good accuracy（intraday 92.17%-112.53%,interday 92.10%-115.29%）,high recovery（84.39%-121.47%）,little matrix effect（89.98%-115.03%）,simultaneous quantification of multiple metabolites（11 species）,as well as,wide range of applications（serum,plasma,whole blood lysate,embryos,soybeans and feed）.The optimization experiment showed that the optimal enzymatic hydrolysis time for extracting one-carbon folate metabolites and amino acids from embryos was 30 min.The optimal method for extraction from whole blood lysate is:protein isoelectric point precipitation（33.33%-300%increase）.2.Based on the UHPLC-MS/MS method,the effects of HHP on the stability and in vitro bioaccessibility of natural folate in soybean were explored,and the bioaccessibility of natural folate in raw beans and germinated beans at different periods were compared.The results showed that HHP treatment at 100 MPa/10 min could improve the stability of natural folate,then significantly increased the natural folate concentration in soybean（15.36%increase）,and folate bioaccessibility（7.28%increase）.The bioaccessibility of natural folate in germinated beans on the 4^thday increased significantly by 20.66%（p<0.05）.Thus,low pressure HHP has the effect of stabilizing natural folate and increasing its bioaccessibility.3.The composition of stabilizing folate in soybean was confirmed,and the stabilizing effect of soybean protein isolate（SPI）on 5-methyltetrahydrofolic acid（5-CH₃THF）was investigated,which can provide a theoretical basis for supplying SPI that has the effect of stabilizing folate.The results showed that HHP treatment at 100MPa/10 min,200 MPa/10 min and 300 MPa/10 min could increase the apparent binding constant（4.83-26.78 fold）and the number of binding sites（9.22%-15.60%）of 5-CH₃THF to SPI.The reaction between 5-CH₃THF and SPI was an exothermic,spontaneous and enthalpy-driven reaction.Van der Waals force and hydrogen bonding played a major role in the combination process of 5-CH₃THF and SPI.SPI was the key substance to stabilize folate in soybean,and the low pressure HHP treatment on SPI resulted in the exposure of hydrophobic groups and polar groups by changing the secondary,tertiary and other advanced structures of SPI,which improved the binding effect between SPI and 5-CH₃THF,thus improving the stability of natural folate.4.Animal experiments were used to investigate the effect of HHP treatment of SPI on the bioavailability of natural folate in vivo.The results showed that the plasma folate concentration of rats after 28 days of folate-deficiency was 15.68±2.47 nmol/L,and the plasma folate concentration of rats with normal diet was 64.95±12.75nmol/L,indicating that the model of folate-deficient was successful.Unfortunately,the combination of SPI and 5-CH₃THF in a mass ratio of 10:1 did not significantly affect the bioavailability of folate in deficient rats,and the combination of HHP-treated SPI and 5-CH₃THF in the same mass ratio did not significantly affect the bioavailability of folate in deficient rats（p>0.05）.