Effect Mechanism Of β-lactoglobulin And Polyphenol Interaction On The Bioavailability Of Polyphenol Under Thermal Treatment

As public health awareness continues to rise and the functional food market expands,the demand for polyphenols has experienced rapid growth in recent years.Polyphenol-enriched dairy drinks and protein powders with functional properties have gained popularity among consumers.For the body to derive the health advantages of polyphenols,they must be absorbed and utilized effectively.The poor bioavailability of polyphenols to humans can be attributed to the structure of polyphenols,the food matrix,and food processing.By investigating factors such as food processing and interactions between different ingredients,this research aims to explore effective approaches for improving the bioavailability of polyphenols,which is essential for designing and developing health products with high utilization of polyphenols.Recently,many studies have been conducted on the impact of interactions between polyphenols and proteins on the bioavailability of polyphenols.However,there is limited research on the effects of interactions under food processing,especially on the patterns and changes in protein-polyphenol interactions after thermal processing and their impact on polyphenol bioavailability mechanisms,which has yet to be explored.Therefore,this study chose β-Lactoglobulin(β-Lg)as the research subject due to its distinctive structure resulting from thermal treatment,as well as its compatibility with polyphenols and four commonly consumed dietary polyphenols,namely chlorogenic acid(CGA),cyanidin-3-O-glucoside(C3G),epigallocatechin gallate(EGCG),and Naringin(Nari).This study extensively investigated the effect of β-Lg and dietary polyphenol interactions under thermal processing on the bioavailability of polyphenols.Indepth research will be conducted on the interaction mechanisms and changes between β-Lg and polyphenols under variable thermal processing condition.The impact of these interactions on the antioxidant activity and stability and the in vitro bioavailability,cellular transport rate,and in vivo utilization of polyphenols in the hybrid system will be explored.This study aims to provide a theoretical basis and guidance for improving the bioavailability of food polyphenols and developing healthy foods containing polyphenols.This study investigated the impact of thermal induced interaction between β-Lg and foodderived polyphenols using various techniques,such as fluorescence spectroscopy,circular dichroism,mass spectrometry,molecular docking,and physicochemical analysis.The results indicate that polyphenols can cause protein structural unfolding.EGCG has the most significant impact on β-Lg’s secondary structure,followed by C3G and CGA,while Nari has no significant effect.CGA,C3G,EGCG,and Nari mainly formed non-covalent complexes with β-Lg through spontaneous hydrophobic or hydrogen bonding interactions,with an average binding site number of around 1.2～1.5.With thermal treatment at 60℃～121℃,the non-covalent binding strength between β-Lg and C3G,CGA(at 60℃ and 121℃)was reduced,while the interaction strength between β-Lg and EGCG,Nari,CGA(at 85℃ and 100℃)was increased to varying degrees.Thermal processing can promote the formation of covalent compounds between β-Lg and EGCG or CGA.The binding strength of interaction between the four polyphenols and β-Lg at room temperature was ranked as Nari > C3G > CGA > EGCG,while after heat treatment,it was ranked as Nari > EGCG > C3G > CGA.This study investigates the impact of thermal processing on the interactions between β-Lg and food-grade polyphenols from multiple tests,including fluorescence spectroscopy,circular dichroism,mass spectrometry,molecular docking,and physicochemical analysis.The results showed that thermal processing decreased the antioxidant capacity of CGA,C3G,EGCG,and Nari.After heat treatment at 60℃～121℃,the interaction between β-Lg and CGA showed a synergistic effect on both ABTS and FRAP assays,resulting in an increase in antioxidant capacity from 9.13% to 56.18%.However,the interaction between β-Lg and C3G,EGCG,and Nari showed antagonistic effects on ABTS scavenging capacity,decreasing it by13.90%～23.38%,11.17%～19.18%,and 12.00%～18.45%,respectively.On the other hand,theβ-Lg-C3G and β-Lg-EGCG systems exhibited a synergistic effect and an antagonistic effect on FRAP,respectively.There is a significant correlation between the strength of interactions and the degree of synergistic or antagonistic antioxidant activity in the four complex.The interaction between β-Lg and polyphenols generally reduces the stability of the complex.However,there is no correlation between the strength of interactions and the stability of the complex.Both interactions and changes in protein structure determine the stability of the complex.The impact of β-Lg-polyphenol interactions with varied thermal processing on β-Lg digestibility,polyphenol in vitro digestibility stability,and bioaccessibility was investigated through in vitro digestion experiments.The results showed that thermal processing significantly increased the solubility of β-Lg and β-Lg-polyphenol complexes.At the same time,the interaction between the four polyphenols and β-Lg hindered its hydrolysis and digestion.For the β-Lg-CGA,β-Lg-C3G,β-Lg-EGCG,and β-Lg-Nari,the interaction increased in vitro bioaccessibility by 21.24%～58.76%,26.09%～354.62%,66.74%～220.99%,and3.95%～13.07%,respectively.The addition of β-Lg has the most substantial effect on improving the bioaccessibility of the thermally sensitive EGCG and C3G,followed by CGA,and the weakest effect on Nari.When the interaction strength is too high,or the interaction at high temperatures causes the complex aggregates,the bioaccessibility of polyphenols is reduced due to incomplete release from the complex.The impact of β-Lg-polyphenol interactions under heat treatment on polyphenol cellular transport rate was explored using the Caco-2 cell absorption model.This was done by examining cellular transport rate,internalization pathway,and drug transport protein gene expression in the tested results.The factors that affect the transport of β-Lg-polyphenol complexes by Caco-2 cells were also studied.The results indicate that adding β-Lg after heat treatment can,to some extent,suppress the expression of efflux genes and enhance the expression of uptake genes,as well as promote the transport of the complex by clathrinmediated endocytosis and increase the transepithelial transport of CGA by 62.38%(85℃)and55.76%(100℃).For Nari,the corrected cell transport rate increased by 25.88% to 72.86%under heat treatment at 60℃ to 121℃,and an increase in interaction strength can somewhat improve the cell transport rate.For EGCG and C3G,the interaction at 25℃,60℃,and 100℃led to a significant decrease in the corrected transport rate of EGCG by 86.85%,72.06%,and60.61%,respectively.At the same time,the remaining complexes had no significant effect.For C3G and EGCG,as well as the four β-Lg-polyphenol complexes at high temperatures,the strong interaction induced by the complexes caused high aggregation,which led to a decrease in cell uptake or an increase in efflux,resulting in no significant improvement in cell transport rate of polyphenol.Finally,this study analyzed the pharmacokinetic parameters of single polyphenols and β-Lg-polyphenol complexes in the circulatory system of rats,aiming to explore and compare the relationship between β-Lg-polyphenol interactions under thermal processing and changes in polyphenol bioavailability in vivo.The results showed that heat treatment generally did not affect the absorption rate of the four polyphenols and their complexes but decreased their bioavailability.During heat treatment at 60℃～121℃,the interaction between β-Lg and CGA led to an increase in corrected bioavailability of 69.68%～123.95%,and Nari by 41.64%(at121℃).In comparison,no significant effect was observed on the corrected bioavailability of C3G and EGCG.Increasing the strength of the interaction between β-Lg and polyphenols can somewhat improve the bioavailability of polyphenols.However,when the interaction causes the complex to aggregate,factors such as low-release rate in the gastrointestinal tract,ineffective cellular uptake,and high efflux rate can limit the enhancement of polyphenol bioavailability.Although there was no significant interaction effect on the corrected bioavailability of C3G and EGCG,the addition of β-Lg can increase their bioavailability by reducing heat degradation and gastrointestinal losses.In addition to slowing down thermal degradation and gastrointestinal loss,the interaction between β-Lg and CGA/Nari can enhance their bioavailability by increasing endocytosis and uptake via relevant transport proteins and reducing efflux.