Preparation,Characterization,Digestive Properties And Antibacterial Activity Of Allicin-Whey Protein Isolate Conjugates

Allicin?Diallyl thiosulfinate?,namely diallyl thiosulfinate,has a broad-spectrum bacteriostatic,tumor suppressing and anti-oxidant physiological activity.Due to its huge health and medical value,it is widely used in food and medicine,agriculture,aquaculture and other fields.However,due to the presence of thiosulfinic acid groups and allyl groups in the structure of allicin,allicin is extremely unstable.Allicin is sensitive to air,temperature,pH and organic solvents,and is easily degraded into various organic sulfur compounds.This makes allicin's activity greatly reduced,which limits its application.The sulfinic acid group in allicin can be combined with the sulfhydryl group on polypeptide or protein through disulfide bond to form stable conjugates of allcin and polypetide/protein.Whey protein isolate is a high-quality natural protein that is easily to be obtained and can be used to prepare allicin-protein conjugates.However,in order to obtain the ideal combination effect with allicin,it is necessary to increase the sulfhydryl content in whey protein isolate.Studies have shown that proper ultrasonic treatment can increase the sulfhydryl content in proteins.In this study,the extracted fresh allicin was used as the raw material,the whey protein isolate was pretreated by ultrasound,and then reacted with allicin to form conjugates,and the reaction conditions were optimized;the differences in the molecular structure and functional characteristics of the conjugate with different binding rates were analyzed.And the in vitro simulated digestion test and antibacterial activity study of the conjugates were carried out to provide a theoretical basis for the development of new stabilization technology of allicin.The main research contents and results of the paper are as follows:?1?Study on preparation of conjugates of allicin?TS?and whey protein isolate?WPI?.There was no significant difference among the extraction rate of allicin extracted with pure water?1.21 mg/g?and the extraction rates of 40%and 80%ethanol solutions?1.22 and 1.26 mg/g,respectively?.Ultrasonic treatment?40?,20+40 kHz,20 min,and 50 W/L?increased the sulfhydryl content of whey protein isolate by 35.05%?p<0.05?compared with the control,thereby improved the binding capacity of the protein and allicin.The single factor and response surface optimization experiments showed that the best reaction conditions of allicin and WPI were:TS_mol:-SH_mol=2.2,pH 4.5,the reaction was performed at 25?for 34 min,and the highest binding rate was61.56%.Fitting the binding process of allicin and protein,the Elovich model?q_t=m+nln?t?,R²=0.9778?can better reflect the changing law of the binding process of allicin and protein.After storage at 70?for half an hour,the mass retention rate of allcin was 7.45%.The higher temperature,the more unstable of the unbound status allicin.After stored at different temperatures for 14 days,the mass retention rate of the conjugates are all kept more than 90%,and the conjugates of allicin and whey protein isolate is more stable and not easy to decompose.?2?The effect of binding rate on the structural and functional properties of allicin-whey protein isolate conjugates?TS-WPI?.The higher the binding rate of the protein-allicin conjugates,the greater the surface hydrophobicity and the more obvious the decrease in fluorescence intensity.Circular dichroism chromatography showed that the protein structure tended to expand after the protein was combined with allicin.Infrared spectroscopy showed that the absorption peak of the TS-WPI conjugates in the amide I region were significantly enhanced.As the binding rate increases,the absolute value of the zeta potential of the conjugate gradually decreases.This may be due to the influence of allicin on the electrostatic and hydrophobic interaction of the whey protein isolate dispersion system,which reduces the homosexual charge.After WPI binding to allicin,the small molecular weight components increase,and there is no significant difference in the molecular weight distribution of the conjugates with different binding rates.Scanning electron microscopy showed that after the combination of WPI and TS,the surface was smoother and changed from spherical to sheet-like distribution.The reason may be that the secondary structure in the protein was destroyed and the spherical structure was lost.The solubility of the TS-WPI conjugate is significantly improved compared to TS?solubility of 2.5%,10??.The conjugate of a 61%binding rate with ultrasonic pretreatment showed the highest emulsifying activity?49.56 m²/g?and stability?10.06 min?.?3?Analysis of in vitro digestion characteristics of allicin-whey protein isolate conjugates.Whey protein and conjugates are more easily hydrolyzed and digested during the trypsin digestion stage.Allicin does not affect the protein hydrolysis degree in the simulated gastrointestinal tract.There is no significant difference in the digestibilities of conjugates with different binding ratios.The average particle size of the gastrointestinal digestion product of the conjugates were larger than that before unconjugation?p<0.05?,and the particle size distribution range becomes wider.After the simulated gastrointestinal digestion,the molecular weight?MW?distribution range of TS-WPI conjugates are close to the small MW,and the content of the small MW components increased,indicating that the conjugates are easily absorbed and utilized compared to the protein.With the increase of digestion time,the content of sulfur compounds decreases in volatile components of allicin extract and conjugates digestion products,and the volatile components of in the intestine digested products are more complicated.The composition of sulfur compounds in gastrointestinal digestive products of allicin extract and the conjugates are similar.?4?Study on antibacterial activity and mechanism of allicin water extract and TS-WPI conjugates.There is no significant difference between the antibacterial activities of the conjugates and allicin water extract against Escherichia coli and Staphylococcus aureus.Allicin water extract can dissolve the cell membrane and cell wall of the bacterial cell,leading to the outflow of cell contents,and the conjugate can also damage the cell structure to a certain extent.The conjugate and allicin water extract will increase the conductivity of the bacterial suspension,the leakage of macromolecular substances,the increase of intracellular calcium ion concentration,and the increase of the permeability of the bacterial cell membrane.Allicin water extracts and conjugate have comparable inhibitory effects on bacterial protein synthesis.Compared with the allicin water extract,the?-galactosidase activity of the Staphylococcus aureus treated with the conjugate decreased faster and the respiratory inhibition rate of the bacteria was higher,thereby affecting the physiological metabolism and respiratory metabolism of the bacteria.