Protective Mechanism Of β-lactoglobulin On The Stability Of Dandelion Flavonoids And Preparation Of Fermented Beverage

Dandelion is a medicinal and food homologous plant with physiological functions such as anti-oxidation,hypoglycemic,antibacterial,and anti-cardiovascular and cerebrovascular diseases.It is used in food and medicine fields.However,flavonoids are easily degraded by their own structure and external physical and chemical factors,which reduces their biological activity and affects their application in food.Therefore,it is of great significance to protect the stability of flavonoids.Dandelion has many kinds of flavonoids,among which quercetin（QUE）,luteolin（LUT）and rutin（RUT）have high content and similar structure.Therefore,this experiment took QUE,LUT and RUT as the research objects.The protective effect ofβ-lactoglobulin（β-LG）on the stability and antioxidant activity of three flavonoids was investigated under different temperatures（25℃,65℃,75℃,90℃）and natural light conditions on sunny days in summer.The protective mechanism ofβ-LG on three flavonoids was studied by thermodynamic analysis,multispectral analysis and molecular docking.Finally,dandelion fermented beverage was developed on the basis of the above research.The main research contents and results are as follows:（1）The protective effect ofβ-LG on the stability of QUE,LUT and RUTThe compounds of QUE,LUT and RUT with different concentrations（0μM,20μM,40μM,60μM and 80μM）ofβ-LG were used as the research objects,and the flavonoid retention rate was used as the index.The protective effects ofβ-LG on the stability of three flavonoids were studied under different temperatures（25℃,65℃,75℃,90℃）and sunny days in summer.The results showed that different temperatures（25℃,65℃,75℃,90℃）reduced the retention rates of the three flavonoids.The protective effect ofβ-LG on the three flavonoids increased first and then leveled off with the increase ofβ-LG concentration,and the optimal concentration ofβ-LG was 60μM.Within the studied temperature range,the protective effect ofβ-LG on the stability of QUE first increased and then decreased with the increase of temperature,and reached the maximum at 65℃,because the structure ofβ-LG began to unfold at 65℃,exposing more binding sites of small molecules and able to bind more QUE,but this binding force was relatively weak and partially destroyed when the temperature was too high.The protective effect ofβ-LG on LUT and RUT reached the maximum at 75°C,because LUT and RUT were more stable than QUE,degraded more at temperatures≥75°C,and the protective effect ofβ-LG was more obvious,and the structure ofβ-LG was fully unfolded at 75°C,and it was easy to bind more flavonoids.The protective effect ofβ-LG on the stability of the three flavonoids at 75°C was LUT>RUT>QUE,which may be related to the binding strength ofβ-LG to the three flavonoids.That is,the protective effect ofβ-LG on the thermal stability of three flavonoids was LUT>RUT>QUE.The stability of the three flavonoids was higher than that of LUT.The retention rate of the three flavonoids was increased by 5.00%（QUE）,11.30%（LUT）and 6.61%（RUT）with 60μMβ-LG under natural light conditions for 8 hours.The protective effect ofβ-LG on the stability of the three flavonoids LUT>RUT>QUE.（2）The protective effect ofβ-LG on the antioxidant activity of QUE,LUT and RUTThe concentrations of QUE,LUT,RUT and different concentrations（0μM,20μM,40μM,60μM,80μM）were measuredThe complex ofβ-LG was used as the research subject,and ABTS⁺free radical scavenging rate and reducing power were used as indexes to study the effects ofβ-LG on the antioxidant activities of three flavonoids under different temperatures（25℃,65℃,75℃,90℃）and sunny days in summer.It was concluded that the antioxidant activity of QUE alone decreased significantly at 65°C,75°C and 90°C,andβ-LG slowed down the decrease of antioxidant activity of QUE.At 65℃,the antioxidant activity of QUE combined withβ-LG is significantly lower than that of QUE alone.This is because the structure ofβ-LG begins to unfold at 65℃and binds more hydroxyl groups on the QUE structure,resulting in its antioxidant activity being temporarily masked.However,the binding ability ofβ-LG to QUE is weak,and it will be destroyed at high temperature.Therefore,β-LG had the strongest protective effect on QUE at 65°C.At 75℃,the antioxidant activity of LUT and RUT combined withβ-LG was significantly lower than that of LUT and RUT alone,because the structure ofβ-LG was fully unfolded at 75℃and bound more LUT and RUT,and this binding force was strong,which could stably exist at 75℃,which was consistent with the thermal stability experiment.The antioxidant activities of QUE and LUT decreased by 16.22%and 5.20%,and the antioxidant activities ofβ-LG-QUE andβ-LG-LUT decreased by 7.02%and1.85%,respectively,which were less than the control.The free radical scavenging rate of RUT increased by 8.78%,and that ofβ-LG-Rut increased by 2.28%,which indicated thatβ-LG could protect the photostability of the three flavonoids.（3）The protective mechanism ofβ-LG against QUE,LUT and RUTDifferential scanning calorimetry,UV absorption spectroscopy,fluorescence quenching spectroscopy,fluorescence resonance energy transfer,synchronous fluorescence spectroscopy,three-dimensional fluorescence spectroscopy,circular dichroism spectroscopy,infrared spectroscopy,and molecular docking were used to explore the protective mechanism ofβ-LG on QUE,LUT,and RUT,respectively.The results showed thatβ-LG could enhance the thermal stability of the three flavonoids by increasing the denaturation temperature of the three flavonoids,and the enhancement effect was LUT>RUT>QUE.β-LG protects the stability of three flavonoids by forming non-covalent complexes with them through hydrogen bonding,hydrophobic interaction,and van der Waals force.After binding to the three flavonoids,the hydrophilicity of tryptophan residue（Trp）ofβ-LG increased,indicating that the binding site was near Trp and the conformation ofβ-LG unfolded.Flavonoids with different structures had different affinity forβ-LG.The planar structure of flavonoids was more likely to enter the hydrophobic pocket of protein.The C₃ hydroxyl substitution and sugar group substitution could weaken the binding of flavonoids toβ-LG.The binding constants of three flavonoids toβ-LG were LUT（6.0×10⁴ L/mol）>RUT（5.09×10⁴ L/mol）>QUE（4.92×10⁴ L/mol）.The binding ofβ-LG to QUE,LUT and RUT was spontaneous.After binding with QUE and LUT,theα-helix and random coil contents ofβ-LG increased,theβ-sheet andβ-turn contents decreased,and the peptide chain unfolded,probably due to the interaction between the two flavonoids andβ-sheet,respectively.After binding with RUT,theα-helix content decreased and theβ-sheet content increased,which may be due to the fact that the binding site of RUT was close to theα-helix and the molecular structure contained a large sugar group,which destroyed the hydrogen bond network of the protein,loosened the conformation of the protein,and partially expanded the structure,which could bind more flavonoids.The three flavonoids could bind toβ-LG by conformation induction.（4）Preparation of dandelion fermented beverageOn the basis of the research in the previous three chapters,with dandelion fermented stock solution andβ-LG as the main raw materials,white granulated sugar as sweetener,citric acid as acidity regulator,the optimal formula of dandelion fermented beverage was obtained by orthogonal experiment on the basis of single factor:Dandelion fermentation stock solution added 5%,sugar added 5%,citric acid added 0.05%,β-LG added 0.15%,sodium carboxymethylcellulose added 0.03%,xanthan gum added 0.05%,sodium alginate added 0.03%,d-sodium erythorbate added 0.15%,According to this formula,the dandelion fermented beverage is light yellow in color,clear and transparent,with dandelion scent,sweet and sour,delicate and fresh taste,and uniform texture without impurities.