Application Of Atomic Force Microscopy In Structural Analysis Of Food-Borne Protein Nanofibers

Food proteins are nontoxic,nutritionally adequate,digestible,readily available,and agriculturally sustainable proteins for human nutritional intake.Food proteins are important diets for human health and are intaked by human body every day.Common food proteins are mainly from four types of food sources.?1?Animal sources?2?Botanical sources?3?Macro-algae sources?4?Micro-organisms sources.The structures of food proteins may be changed during food processing process or a storage process,which may affect their in vivo bioavailability,metabolism,and nutrition abilities.Therefore,it is a great need to study the structure characteristics of food proteins and their behaviors during different ingredient preparation,processing or preservation processes.In order to make better use of food proteins.AFM as a powerful nanotech imaging tool and has been successfully and broadly applied in the field of food protein researches.Among the application methods of AFM,height image is the most popular method for studying food proteins.Therefore,this research topic focuses on collagen and zein in food proteins.AFM was used to study food-borne proteins from nanometer scale to micrometer scale.The following work was carried out:?1?We extract collagens from tilapia skin by three types of extraction methods:acetic acid method,hot water method,and sodium hydroxide method.The production yields?based on the wet weight of fish skin?were:acetic acid method?11.7%?>hot water method?10.7%?>sodium hydroxide method?8.5%?.Tilapia fish skin contained the collagen at the level of 21.89g/100g.Therefore,the yield based on the initial collagen presented in the skin or recovery?%?for acetic acid method,hot water method,and sodium hydroxide method were 53.4%,48.9%,and 38.8%,respectively.SDS-PAGE electrophoresis was used to determine the molecular weight of collagen obtained by three extraction methods.The protein pattern showed that these extracted collagens had different molecular weights and the collagen molecules were mostly hydrolyzed during the sodium hydroxide method.ATR-FTIR spectra showed that they had similar molecular structures.?2?The polymorphism and stability of collagen extracted from acetic acid extracted Tilapia Skin Collagen and I type bovine tendon collagen and I type mouse tail collagen were studied.The sodium dodecyl sulfate-polyacrylamide gel electrophoresis?SDS-PAGE?was used to study three kinds of collagen from the same concentration,different sample size and different concentration.The results show different loading volumes had no obvious effects on the protein bands of three types of collagens except the intensity.At the same sample sizes,different concentration had obvious effects on the protein bands of three types of collagens.The attenuated total reflection Fourier transform infrared?ATR-FTIR?spectra were used to analyze the secondary structure of three collagen proteins in the 1600-1700 cm^-1 band after different heat treatments.The results showed that the secondary structure of BFTC did not change significantly with time,while that of RTC and TSC changed significantly with time.AFM was used to observe the polymorphism of three collagen proteins at different concentrations.It was concluded that the formation of nanostructures was related to the concentration.The stocked collagen solutions were diluted to 0.4mg/mL and incubated at 37?.At different time points?0,7,14,21,and 28d?,the collagen solution samples were deposited onto mica until dry,and examined by ex situ AFM.The results showed that during the incubation process,BFTC nanofibers were assembled into left-handed helix BFTC nanofibers,and then nanoparticles.RTC nanostructures?protofibrils,nanoparticles,and disordered aggregates?were assembled into nanoparticles and protofibrils.TSC nanostructures?nanofilaments,nanoparticles,and disordered aggregates?were assembled into nanoparticles and protofilaments.?3?Zein was used to prepare the protein fiber membrane,and ordinary optical microscope was used to determine the film concentration.A large number of long nanobelts and almost no microparticles were fabricated using 35%?w/v?zein solution,Therefore,35%zein solution was chosen for the preparation of electrospun curcumin-loaded zein membranes in further studies.Scanning electron microscopy?SEM?was used to determine the successful preparation of nanofibrous/nanobelts membranes.AFM results showed the widths were larger than the heights according to the sectional analyses,which further confirmed that these membranes were consisted of nanobelts.The prepared fiber membrane was used for Fe³⁺detection experiment,The prepared zein-loaded curcumin nanobelts membranes can be used for the detection of Fe³⁺more quickly and efficiently,and can also be used for the detection of Fe³⁺in drinking water.The detection temperature has a significant impact on the rapid detection of Fe³⁺and the prepared nanobelts membranes have good storage stability.It provides guidance for the preparation of protein band membrane and its application in practical production,which provided guidance for better application of protein membrane in actual production.We believe it will attract increasing attentions in scientific and engineering fields such as materials,environmental,and food science.