| Soybean is one of the rich resources in China, and many various bioactive components are present in the soybean whey wastewater byproduct of soy protein concentrate. However, there are more 3 million tons whey wastewater abandoned every year, which have caused the waste of resources and environment pollution, such as the functional trypsin inhibitors and flavonoids. Trypsin inhibitor (TI) mainly distributing in cultivated and wild-type soybean or other plant seeds, possesses various specialized functions, which has been applied in pharmacological and medical fields owning to the ability to prevent or suppress carcinogen-induced transformation, as detected in various in vitro and in vivo model systems. However, very few studies on the purification of TI are available and often the extraction procedures are long and tedious, such as extracted with water or acid, salting out, gel filtration chromatography and affinity purification with sepharose gel.Aspergillus flavus is one of the major spoilage moulds of intermediate moisture foods that can infect several agricultural crops such as peanuts, cotton, tree nuts, maize, rice, peppers, spices, and figs, resulting in the production of one of the most toxic and potent carcinogenic metabolites known to mammals, aflatoxin. Therefore, many research approaches are being used to reduce and eliminate A. flavus contamination with some physical or chemical methods. However, there are rarely questioned about cultivated and wild soybean TI anti-A. flavus activity.Therefore, the objectives of this study is to purify TI from the cultivated and wild soybean seeds with a quick, effective and scalable affinity chromatography, and further seek to determine if the trypsin inhibitor resistant to A. flavus spore germination and mycelial growth.1) Chitosan resin was prepared by reverse suspension cross-linking method with glutaraldehyde as the cross-linking agent. The influences of several parameters (stirring rate, cross-linking and chemical modifying agent) on the chitosan resin were investigated. The results showed that chitosan resin demonstrated a spherical shape and showed the appropriate pile-up density and porosity degree at levels of 300 r/min stirring rate,3.6% glutaraldehyde (v/v) and 0.06 M NaBH4. The results of FTIR, XRD and DSC showed that the cross linking reaction was took place between the amino groups of chitosan and aldehyde groups of glutaraldehyde to generate the Shicff structure, further to form the chitosan beads. The concentrations of glutaraldehyde and NaBH4 played an important role in the porosity values, cross-linking degree and microstructure of the prepared chitosan resins.2) Chitosan resin-trypsin was prepared with the chitosan resin as the carrier, epichlorohydrin as the activator and trypsin as the coupling agent. When the concentration of epichlorohydrin arrived at 15%, activating temperature at 60℃with NaBH4 and 1,4-Dioxane added, and coupling temperature at 30℃, the prepared resin-trypsin obtained the higher trypsin activity. Furthermore, the trypsin activity assay indicated that chitosan resin-trypsin could tolerate relatively high temperature (65℃) and wide pH range (5.0-9.0), compared with free trypsin (45℃, pH 6.0-8.0), caused by the immobilization of trypsin inside the porous resins increased the enzyme stability by reducing the overlapping, preserving tertiary structure and protecting the enzyme from conformational changes.3) With prepared chitosan resin-trypsin as matrix on affinity chromatography column, a Bowman-Birk TI from cultivated and wild soybean was purified (adsorption capacity of chitosan resin-trypsin for TI was about 1.33 mg/g wet matrix), with homogeneous MW of 8.2 kDa estimated by SDS-PAGE. The affinity purification factor and recovery rate of TI were much improved over established procedures. And it was further demonstrated that the purified TI belonged to the family of Bowman-Birk TI, based on the evidences of its amino acid composition, two independent binding sites for trypsin and chymotrypsin, and lysine residue as the active site for trypsin-inhibitory.4) Studying on the inhibition of A. flavus showed that the effect of TI from the cultivated and wild soybean on A. flavusα-amylase and proteases activity and aflatoxin B1 production depended on TI's concentrations, and with inhibition IC50 at 1.6μM and 1.0μM, respectively. The resistance to A. flavus infection was partially due to the inhibition ability of TI for the endogenous a-amylase and proteases activity, thereby limiting the availability of the hydrolyzed reducing sugar for fungal growth and suppressing aflatoxin B1 biosynthesis.5) chitosan-based blend films were prepared from chitosan, TI extract and glycerol solutions, the properties of which were also investigated, including thickness, mechanical property, water vapor transmission, optical transmittance, and solubility. In addition, the resulting blend films were characterized by SEM, XRD and FTIR. The result of SEM images showed the surface and cross section of the blend films had more smooth and dense morphology than pure chitosan film. XRD and FTIR spectra indicated that the possible interaction force among the components might be the hydrogen bonds of N-H…O=C and O-H…O=C. Furthermore, the facts that the germination and growth of A. flavus were strongly inhibited by the blend films indicated that it might be useful as potential bio-control packaging against A. flavus during the peanuts and other cereals storage.Nowadays, China has become one of the major peanut production, consumption and export countries in the world. However, the peanut export volume has been hovering around 200 million dollars, due to the pollution of aflatoxins in recent years. According to determination of aflatoxins for 1685 copies of peanuts and 1172 copies of peanut oil samples from the 22 provinces by Ministry of Health Food Supervision and Inspection Institute, there were 26.3 percent and 47.3 percent of peanuts and peanut oil, which showed much more aflatoxins than the standard limit. Therefore, the objectives of this study is to purify TI from the cultivated and wild soybean seeds with a quick, effective and scalable chitosan resin-trypsin affinity chromatography, and further seek to determine if TI inhibit A. flavus spore germination and mycelial growth by inhibiting a-amylase and proteases, to provide a theoretical basis for the biological control of A. flavus technology during the peanut, corn or other grains storage.
|
PDF Full Text Request