| This study was supported by the National Key Research and Development Program of China(2017YFD0400603).α-Lactalbumin is a functional protein in milk,which can promote the growth and development of infants and children and regulate the quality of sleep.It is a functional protein in milk,which has many activities such as promoting growth and development of infants and children and regulating sleep quality.However,its separation and preparation process is monopolized by foreign technology,so the establishment of a process for α-lactalbumin separation and preparation is also an urgent technical problem to be solved.In addition,α-lactalbumin,as a natural amphiphilic substance,can be combined with hydrophobic small molecule compounds to improve the functional activity of small molecule compounds.The use of carvacrol and thymol as natural bacterial antagonists is bioenvironmentally friendly and safe,but their highly water-insoluble and volatile properties limit their application in the food industry.their application in the food industry.Based on this,a process for the isolation and preparation of α-lactalbumin was established in this study,using raw milk as raw material,to provide a research basis for the production and preparation of industrial α-lactalbumin.The mechanism of the interaction between α-lactalbumin and carvacrol and thymol was also investigated using techniques such as multispectroscopy.Combined with molecular docking technique and kinetic simulation technique,the binding sites of α-lactalbumin with carvacrol and thymol were explained at the molecular level,and the driving force of their binding was clarified to reveal the mechanism of action of α-lactalbumin-carvacrol/thymol complexes.On this basis,the inhibitory effects of α-lactalbumin-carvacrol complex and α-lactalbumin-thymol complex on Staphylococcus aureus and Escherichia coli were investigated,and the antibacterial activities of α-lactalbumin formed with carvacrol and thymol respectively were clarified,which laid the foundation for the further development and application ofα-lactalbumin-carvacrol/ thymol complex in the food field.The experimental contents and conclusions of this study are summarized as follows:(1)The separation and preparation process of α-lactalbumin from raw milk was established: the experiments firstly investigated the best method for the separation and extraction of whey protein,and the study innovatively proposed the enzyme followed by acid method to obtain whey protein from raw milk.In contrast to the traditional acid precipitation and enzyme coagulation methods,the yield of whey protein recovered by the enzyme followed by acid method could be increased by 17.11%.On this basis,α-lactalbumin was purified by membrane technology combined with chromatographic techniques.The protein content of the filtrate was 6.53 ± 0.22 mg/m L.Polysulfone membrane was selected as the best membrane material for the ultrafiltration of the microfiltration filtrate,and 35℃ and 0.3 MPa were selected as the best ultrafiltration parameters to enrich α-lactalbumin.The purity of α-lactalbumin after ultrafiltration was24.28 ± 0.62%.The purity of α-lactalbumin after ultrafiltration was 24.28 ± 0.62%.The crude protein was further purified by combined DEAE-Sepharose Fast Flow ion exchange chromatography to obtain chromatographic grade α-lactalbumin with a purity of 90.89 ± 0.31% and a recovery of 79.02 ± 0.13%,and the obtained production parameters could be realized for industrial scale up production.The high purity α-lactalbumin was examined physicochemically,and the α-lactalbumin standard was used as a control,and the obtained high purity α-lactalbumin was of good quality by SDSPAGE band comparison,UV spectroscopy,circular dichroism spectroscopy and NIR spectroscopy,and can be applied in the development and production of fortified functional foods.(2)Preparation of α-lactalbumin-carvacrol/thymol non-covalent complexes and evaluation of their interaction mechanism: The high-purity α-lactalbumin was prepared as a carrier and combined with carvacrol and thymol,respectively,and the complexes were evaluated by UV spectroscopy,fluorescence spectroscopy,simultaneous fluorescence spectroscopy,three-dimensional fluorescence spectroscopy and circular dichroism spectroscopy,etc.to determine the structural changes of α-lactalbumin with different concentrations of carvacrol/thymol(0-24 μg/ m L).To assess the mechanism of complex formation.The results of the study showed that the amino acid microenvironment of the chromogenic group of α-lactalbumin was altered and the internal hydrophobicity of α-lactalbumin was enhanced after the interaction with carvacrol and thymol,respectively.This change could prove the formation of α-lactalbumin-carvacrol/thymol complex and,at the same time,indicate that in this binding,carvacrol changes the structure of α-lactalbumin.Simultaneous fluorescence assay analysis demonstrated that Tyr is more involved in the interaction between α-lactalbumin and carvacrol compared to Trp.The results of three-dimensional fluorescence and circular dichroism spectroscopy showed that the complexes formed and increased the diameter of α-lactalbumin,while thymol successfully induced conformational changes in α-lactalbumin,while the polar effects near Tyr and Trp residues were greater than those of the parsley phenol experimental group.And the comparison of the morphology of α-lactalbumin before and after the action of carvacrol and thymol by scanning electron microscopy technique showed that the complexes were larger in diameter compared to the natural α-lactalbumin and attached to the protein surface,making the spherical α-lactalbumin surface rougher,again proving the formation of the complexes.(3)Assessment of the mechanism of action of α-lactalbumin-carvacrol/thymol non-covalent complexes using molecular docking techniques and kinetic simulations:Based on multispectral analysis,molecular docking was used to predict the binding mode of carvacrol to α-lactalbumin.The relevant residues of α-lactalbumin involved in hydrophobic contacts by carvacrol were Phe53,Tyr103,Trp104,Leu105 and Ala106.Meanwhile,carvacrol formed hydrogen bonds with two residues of α-lactalbumin(His32 and Thr33),and the binding energy of the former was-4.82 kcal/mol;while the protein involved in hydrophobic interactions by thymol residues were Tyr36,Phe31,Trp118,and Gln117 with a binding energy of-5 kcal/mol;and formed a hydrogen bond with one residue of α-lactalbumin(Lys5).After docking,the hydroxyl positions of carvacrol and thymol were rotated to an orientation more favorable for electrostatic interaction.To investigate the binding stability of the α-lactalbumin-carvacrol/thymol complex,we selected the most stable mode of α-lactalbumin with carvacrol and thymol for molecular dynamics simulations,and the results showed that after the dynamics simulations,the binding positions of carvacrol and thymol to α-lactalbumin were shifted and the binding position shifted to a more stable binding state.To obtain more information about the α-lactalbumin-vanillin complex,the total binding free energy of the α-lactalbumin-vanillin complex and their detailed energetic contributions were calculated using Amber software.The experimental results indicate that the main source of contribution in the α-lactalbumin-vanillin/ thymol complex is van der Waals forces,followed by electrostatic interaction forces.(4)The antibacterial activity of α-lactalbumin-carvacrol/ thymol complexes was evaluated: based on the α-lactalbumin-carvacrol complex and α-lactalbumin-thymol complex prepared in Chapter 3,the inhibitory activity of both complexes against Staphylococcus aureus and Escherichia coli was further investigated.The MICs of carvacrol and thymol alone and with α-lactalbumin against Staphylococcus aureus were determined by measuring the minimum inhibitory concentrations and plotting the growth curves,and the changes in the inhibitory activities before and after the effects were quantified by plate counting.The results showed that the complexes of carvacrol and thymol non-covalently bound to α-lactalbumin showed higher inhibitory activity against Staphylococcus aureus and Escherichia coli than when acting alone.This may be due to the fact that α-lactalbumin can increase the water solubility of carvacrol and thymol.In addition,the inhibitory activity of thymol experimental group against S.aureus was slightly higher than that of carvacrol experimental group.The disruptive effects of α-lactalbumin-carvacrol complex and α-lactalbumin-thymol complex on the cell membranes of Staphylococcus aureus and Escherichia coli were further investigated by bacterial cell membrane potential assay,bacterial cell membrane integrity assay,morphological and structural analysis of the bacteria,and crystalline violet assay analysis.From the perspective of biofilm formation,α-lactalbumincarvacrol/ thymol complexes were more effective on the cell membranes of Staphylococcus aureus and Escherichia coli.Staphylococcus aureus and Escherichia coli may be inhibited by disrupting biofilm formation to cause bacterial death.The cytotoxicity assay of the complexes on He La cells demonstrated the safety of the prepared complexes. |
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