Fabrication Of Kaempferol-loaded Deamidated Zein-Pectin Nanoparticles And Its Biological Activity Studies

Kaempferol is a natural flavonoid with antioxidant,anti-inflammatory,anticancer,antibacterial and antiviral biological activities,which are beneficial to human health.The deamidation of zein promotes the change of the spatial configuration of the protein,thereby increasing the solubility of the protein and improving its functional properties.In this paper,the molecular structure changes of zein after alkali deamidation treatment and the characteristics of deamidated zein nanoparticles were analyzed,and kaempferol-loaded deamidated zein-anionic polysaccharide nanoparticles were prepared by anti-solvent method.Explore its stability in different environments（such as p H,ionic strength,temperature）to determine the optimal preparation conditions;the release behavior of kaempferol was explored by simulating in vitro digestion,and the changes of biological activity of kaempferol were analyzed by free radical scavenging experiment and intracellular anti-inflammatory activity evaluation method.The main conclusions are as follows:1.The degree of deamidation increased with the increase of the alkali heat treatment time of the samples（p<0.05）.The degree of deamidation was about 25.94%after 10 hours of alkali heat treatment.2.Circular dichroism spectroscopy indicated that deamidated zein had a reducedα-helix content and more protein segments were opened compared to native zein.In Fourier transform infrared spectroscopy,the stretching vibrations of zein in the fingerprint region,amide I and amide II were enhanced after deamidation,and red-shifted to different degrees.The fluorescence spectrum showed that with the increase of the degree of deamidation of zein,the degree of protein unfolding changed,and the fluorescence intensity also increased.The wavelength corresponding to the strongest fluorescence intensity of natural zein is 303 nm.The maximum peak of zein was shifted to long wavelength at 304 nm,and the above results confirmed that the secondary structure of zein was changed during alkali treatment.3.The results of particle size,PDI and scanning electron microscopy showed that the particle size distribution of deamidated zein nanoparticles was unimodal,and the size distribution of nanoparticles was uniform.Compared with the untreated samples,the deamidated nanoparticles were spherical or nearly spherical under SEM,with smooth surface and uniform size.The hydrophobicity results also showed that the hydrophobicity of the nanoparticles decreased after deamidation（p<0.05）.4.Pectin and xanthan gum were used as stabilizers to study their effects on the stability of deamidated zein nanoparticles loaded with kaempferol.The results show that pectin has better stability to nanoparticles,the average particle size of nanoparticles increased from about 164.03 nm to 332.43 nm in the range of p H2～7.5,and the polydispersity index（PDI<0.167）was still relatively low,indicating that the nanoparticles have good anti-settling properties.In addition,with the increase of sodium chloride concentration to 50 m M,the particle size of nanoparticles gradually increased（153.00 nm to 377.80 nm）,but the PDI was still less than 0.263.When the nanoparticles were heated at 80°C for 60 minutes,the average particle size was in 131.00-140.57 nm,indicating that the distribution of nanoparticles is still relatively uniform under high salt and heating environments.The particle size of the nanoparticles is about 158.30 nm,and the particle size distribution shows a narrow unimodal distribution,and the particle yield is 99.55%,and the loading rate of kaempferol reaches 89.54%,indicating that the anti-solvent precipitation method can prepare small and uniform particles.Nanoparticles with higher loading rate.5.The formation of nanoparticles is mainly due to hydrogen bonding and electrostatic interaction between zein,kaempferol and pectin.XRD results showed that the crystallization peaks of kaempferol in the nanoparticles disappeared and existed in an amorphous state.6.The simulated digestion results of the gastrointestinal tract showed that the nanoparticle was gradually released during the gastric digestion stage,and the release rate reached 6.66%,which was higher than that of kaempferol or physical mixture.After the intestinal digestion stage,the bioavailability of kaempferol-loaded nanoparticles was 39.78%,which was significantly higher than that of kaempferol（20.34%）and physical mixture（23.73%）（p<0.05）.7.With the increase in the content of kaempferol in the simulated digestion in vitro,the DPPH·free radical scavenging ability of kaempferol,kaempferol physical mixture and kaempferol nanoparticles all showed an upward trend.Nanoparticles had the strongest scavenging ability,ranging from 27.00±0.06 to 38.55±0.17μmol TE/g DW.Similarly,with the increase of kaempferol content,the oxygen radical absorbance capacity of kaempferol changed little,while kaempferol mixture and kaempferol nanoparticles basically showed an upward trend.Among them,the ORAC values of kaempferol nanoparticles ranged from 1180.13±170.18 to1607.64±27.73μmol TE/g DW.8.The anti-inflammatory results of the cells are as follows:Compared with the blank control group,the expressions of the inflammatory mediator PEG₂ and the inflammatory factor TNF-αin the lipopolysaccharide（LPS）treatment group were significantly increased（p<0.05）.Kaempferol,kaempferol physical mixture and kaempferol nanoparticles could significantly inhibit the expression of PEG₂ and TNF-αinduced by LPS（p<0.05）.However,there are differences in their inhibitory effects.Kaempferol nanoparticles showed stronger activity in inhibiting the production of inflammatory mediators PEG₂ and inflammatory factor TNF-α,and its inhibitory effects were significantly higher than those of kaempferol（p<0.05）.At the same time,kaempferol nanoparticles also promoted the release of IL-10 anti-inflammatory factors,while the release of IL-10 anti-inflammatory factors in the lipopolysaccharide group was relatively weak.