Elucidation of molecular interactions of theaflavin monogallate with camel milk lactoferrin: detailed spectroscopic and dynamic simulation studies

Lactoferrin is a heme-binding multifunctional glycoprotein known for iron transportation in the blood and also contributes to innate immunity. In this study, the interaction of theaflavin monogallate, a polyphenolic component of black tea, with camel milk lactoferrin was studied using various biophysical and computational techniques. Fluorescence quenching at different temperatures suggests that theaflavin monogallate interacted with lactoferrin by forming a non-fluorescent complex, i.e., static quenching. Theaflavin monogallate shows a significant affinity towards lactoferrin with a binding constant of ∼104–105 M−1 at different temperatures. ANS binding shows that the binding of polyphenol resulted in the burial of hydrophobic domains of lactoferrin. Moreover, thermodynamic parameters (ΔH, ΔS and ΔG) suggested that the interaction between protein and polyphenol was entropically favored and spontaneous. Circular dichroism confirmed there was no alteration in the secondary structure of lactoferrin. The energy transfer efficiency (FRET) from lactoferrin to theaflavin was found to be approximately 50%, with a distance between protein and polyphenol of 2.44 nm. Molecular docking shows that the binding energy of lactoferrin–theaflavin monogallate interaction was −9.7 kcal mol−1. Theaflavin monogallate was bound at the central cavity of lactoferrin and formed hydrogen bonds with Gln89, Tyr192, Lys301, Ser303, Gln87, and Val250 of lactoferrin. Other residues, such as Tyr82, Tyr92, and Tyr192, were involved in hydrophobic interactions. The calculation of various molecular dynamics simulations parameters indicated the formation of a stable complex between protein and polyphenol. This study delineates the binding mechanism of polyphenol with milk protein and could be helpful in milk formulations and play a key role in the food industry.


Introduction
Lactoferrin (MW ¼ $80 kDa) is an iron-binding protein present in mammalian milk and other exocrine secretions such as tears, nasal and bronchial mucous and saliva. 1,2 It belongs to the transferrin family of non-heme protein. Lactoferrin plays various roles in the innate immunology of the host, 3 inhibition of neutrophil priming by bacterial lipopolysaccharide, 4 and modulating inammation by amplifying apoptotic signals. 5 Moreover, lactoferrin has also been documented to exhibit anti-tumor, anti-fungal, anti-viral, and anti-bacterial properties. 6 Lactoferrin has recently come under the spotlight, particularly with regards to the new coronavirus pandemic that started in 2019 . A recently published literature study suggested that lactoferrin can bind to at least some of the receptors used by coronaviruses thereby blocking their entry. 7 Structurally, a simple polypeptide chain of lactoferrin is folded to form two symmetrical lobes. These two lobes (N and C) are highly homologous of approximately 33-41%. 8 The N lobe of this polypeptide chain ranges from 1-332 amino acids, and C lobes range from 344-703 amino acids. Both these lobes are comprised of a-helices and b-pleated sheets, creating two domains for each lobe viz. domains I and II. 9 These lobes are connected by a hinge region which provides additional exibility to the protein. 10 Both lobes adopt open conformations indicating wide distances between the iron binding residues in the native iron-free form of lactoferrin. The structure of human apolactoferrin was more intriguing in which the N-lobe adopted an open conformation while the C-lobe stayed in the closed conformation similar to its holo-form. Bovine and human lactoferrins can bind to the HCV envelope proteins E1 and E2. This binding inhibits any possible interaction of the virus with its cellular receptors. Similar results have recently been reported for camel lactoferrin, demonstrating complete inhibition of virus entry when camel lactoferrin and HCV were preincubated together. In contrast, lactoferrin pre-incubation with human leukocytes, HepG2 cells and Huh7.5 cells before HCV infection did not affect viral entry. 11 Moreover, in comparison to cow milk, camel milk is rich in vitamin C, niacin, vitamin A and E. 12,13 Camel milk has a high content of a-lactoalbumin and lactoferrin but lacks b-lactoglobulin. 14 In the past, camel milk has been used as a nutritional supplement in chronic pulmonary tuberculosis. 15 It has no allergenic properties and can be taken by lactase-decient and immune-decient people. 16 Theaavins are a class of polyphenols and is a major component of black tea. They are regarded as 'golden molecules' owing to their therapeutic attributions. 17 They have been shown to have various physiological actions, including antioxidant, 18 anticancer, 19 anti-atherosclerotic, 20 anti-inammatory, 21 antiviral, 22,23 anti-periodontitis 24 and the inhibition of osteoporosis. 25 Furthermore, these compounds have been shown to possess human health benets including glucose-lowering 26 anti-obesity 27 as a prevention of lifestyle-related diseases.
Studies delineating the interaction of clinically signicant proteins with natural compounds are on a high in present times owing to the fact natural compounds possesses enormous structural and chemical diversity 28 coupled with their clinical potential thereby playing a key role in drug discovery. 29 Previous literature suggested the superiority of camel milk and the use of lactoferrin in various marketing products such as infant formulas, probiotics, supplemental tablets, cosmetics and as a natural solubilizers of iron in food 30 prompted us to explore its interactions with theaavin monogallate. Several biophysical and computational tools were performed to reveal the conformational changes and mechanism of binding between proteinpolyphenols complex.

Materials
Lactoferrin from camel milk was isolated and puried from a previous procedure. 31 Theaavin monogallate and ANS (8anilinonaphthalene-1-sulfonic acid) dye were obtained from Sigma-Aldrich (MO, USA). All other chemicals were of a high standard and analytical grade. A stock solution of ANS was prepared in double-distilled water. The stock solution of thea-avin monogallate was prepared in DMSO. The concentration of DMSO used in the experiment was less than 1%.

Steady-state uorescence study
The steady-state uorescence was performed using a spectro-uorometer (Jasco FP-750, Japan). Briey, lactoferrin (5 mM) was excited at 280 nm, and its uorescence emission spectrum was recorded from 290 to 450 nm. Theaavin monogallate (0-15 mM) was titrated to lactoferrin solution, and uorescence emission spectra were recorded at each titration. The experiment was performed at 298, 303, and 308 K for the analysis of thermodynamic parameters. The uorescence of the buffer solution was subtracted from the uorescence spectra of lactoferrin. Moreover, the inner lter effect on the uorescence was corrected using the following relation (eqn (1)).
where F c and F o are the corrected and measured uorescence, respectively; A ex and A em are the absorptions of protein and ligand complex at the excitation and emission wavelengths, respectively.

Circular dichroism measurements
The circular dichroism (CD) measurements were carried out using Applied Photophysics, ChirascanPlus, UK spectropolarimeter attached with Peltier. The CD spectra of native lactoferrin (5 mM) and their complex with theaavin monogallate (1 : 5 and 1 : 10 protein : ligand ratio) were recorded in the far-UV range between 200-250 nm. Sodium phosphate buffer (20 mM, pH 7.4) was used for the baseline correction. Each spectrum presented is the average of 3 scans.

Hydrophobicity analysis using ANS
The hydrophobicity of lactoferrin in the presence of varying concentrations of theaavin monogallate was determined by measuring ANS uorescence. To the xed concentration of lactoferrin (5 mM), a 50 times higher concentration of ANS dye was added. The uorescence emission spectrum was recorded for native lactoferrin by exiting at 380 nm. Further, theaavin monogallate (0-15 mM) was titrated to the solution (protein-ANS), and the emission signals were obtained from 400 to 650 nm. All measurements were carried out at 298 K.

Forster resonance energy transfer (FRET)
The UV-visible absorption spectrum of theaavin monogallate (10 mM) was recorded using UV-vis Spectrophotometer (Amersham Bioscience, Sweden). The uorescence emission of spectra of lactoferrin (5 mM) was recorded by exciting at 295 nm. All the uorescence and absorbance readings were normalized to 1. The overlap between the uorescence spectrum of lactoferrin and the absorption spectrum of the theaavin monogallate was determined, and the FRET parameters were determined as reported previously. 32

Molecular docking analysis
The interaction between lactoferrin and theaavin monogallate was elucidated by performing molecular docking using Auto-Dock4.2, as described previously. 31 The three-dimensional coordinates of camel lactoferrin (PDB Id: 1I6Q, resolution: 2.70Å) were obtained from the RSCB database, while the structure of theaavin monogallate was obtained from PubChem (CID: 169167). Before molecular docking, lactoferrin's structure was optimized by deleting crystallographic water molecules and any other heteroatoms, adding missing hydrogen atoms, and dening Kollman united atom type charge using AutoDock Tool (ADT). Theaavin monogallate was pre-processed by dening rotatable bonds, number of torsions, and adding Gasteiger partial charges. The energy of the theaavin monogallate was minimized using a universal force eld (UFF). A grid map of 103 Â 76 Â 71Å centered at 39.8 Â 1.9 Â 8.4Å with 0.375Å spacing was created using AutoGrid. The parameters in AutoDock were set to their default values, and distance-dependent dielectric parameters were employed to calculate van der Waals' and electrostatic energies. Lamarck Genetic Algorithm (LGA) along with Solis and Wets methods, were employed to perform docking calculations. For each run, a maximum of 2 500 000 energy terms was calculated, and a total of 10 runs were computed. The initial position, torsion, and orientation of theaavin monogallate were set randomly. The population size, translational step, quaternions, and torsions were set to 150, 0.2Å, and 5, respectively. The binding affinity (K d ) of theaavin monogallate towards lactoferrin was calculated from binding energy (DG) using the following relation. 32 where R and T were the Boltzmann gas constant and temperature, respectively.

Molecular dynamics (MD) simulations
The stability and dynamics of the lactoferrin-theaavin monogallate complex were evaluated by performing MD simulation using GROMACS version 2020.2, as described earlier. 33 The topology of lactoferrin was generated using the pdb2gmx command and selecting OPLS (Optimized Parameters for Liquid Simulation) forceeld. Conversely, the topology parameters of theaavin monogallate were generated in the LigParGen server and combined with the lactoferrin topology. The lactoferrin and lactoferrin-theaavin monogallate complex systems were placed in a cubic box and solvated with an spc216 water model to mimic the aqueous environment. The energies of both the systems were minimized for 1000 ps using 1500 steps of the steepest descent method. The temperature of both the systems was gradually increased under periodic boundary conditions from 0 to 300 K through an equilibration period of 100 ps at constant volume and pressure (1 bar). For both the systems, the nal production MD run was performed for 50 ns, and the resulting trajectories were analyzed as described previously. 33 3. Results and discussion

Steady-state uorescence
The steady-state uorescence spectroscopy is a valuable tool to study proteins' interaction with small molecules or drugs. In this technique, small molecules' effect on the native uorescence emission spectrum of protein is analyzed. The uorescence emission signal of camel milk lactoferrin in the absence and presence of varying concentrations (0.5-15 mM) of thea-avin monogallate is shown in Fig. 1A. The uorescence quenching of lactoferrin in the presence of theaavin monogallate at temperatures 303 and 308 K is also shown in ESI, Fig. 1. † Native lactoferrin exhibited uorescence maxima at 335 nm, a characteristic signal of lactoferrin protein. 34 The addition of theaavin monogallate resulted in a progressive quenching of lactoferrin uorescence signal, indicating an interaction between protein and polyphenol. 35 Moreover, thea-avin monogallate just caused the quenching without changing the emission maximum and shape of the peak. The similar trend was earlier observed in BSA protein aer interaction with theaavins. 36 Further, quenching uorescence of lactoferrin-theaavin monogallate binding was mathematically evaluated using Stern-Volmer eqn (3) to obtain K sv . 37 where F o is the uorescence maxima of free or native lactoferrin; F is the uorescence maxima of lactoferrin in the presence of theaavin monogallate; K SV is Stern-Volmer constant, and [Q] is the concentration of theaavin monogallate. The Stern-Volmer plot for the interaction of theaavin monogallate with lactoferrin is shown in Fig. 1B, and the values of K SV are illustrated in Table 1. The K SV values for the interaction of theaavin monogallate with lactoferrin were 7.16 Â 10 4 , 5.54 Â 10 4 , and 4.06 Â 10 4 M À1 at 298, 303, and 308 K, respectively. However, the values of K SV alone cannot ensure the mode of uorescence quenching. To analysis the mode of uorescence quenching, bimolecular quenching rate constant (k q ) using eqn (4). 38 where s 0 is the uorescence lifetime whose value is approximately 5.78 Â 10 À9 s. The values of k q as a function of temperature varied in the range of 0.71-1.25 Â 10 13 M À1 s À1 ( Table 1). The bimolecular quenching rate constant (k q ) for all tested system are higher than the maximum scatter collision constant (2 Â 10 10 M À1 s À1 for dynamic quenching). 38 Thus, our results illustrated the static mode of quenching in lactoferrin uorescence occurred due to the formation of a non-uorescent complex between theaavin monogallate and lactoferrin. The quencher physically interacts with the excited molecule through chemical bonds in static quenching. 40 The mode of quenching can further be conrmed by analyzing the dependence of K sv on temperature. The values of K q and K sv (Table 1) were inversely correlated with temperature for theaavin monogallate, which again indicated that the nature of quenching was static. Earlier reports also validated the static mode of quenching between polyphenols and proteins. 36,39 In static quenching, K sv decreases with increasing temperature due to the breakdown of weakly bound complexes. However, in dynamic quenching, K sv increases with an increase in temperature due to a higher diffusion rate. 41

Determination of binding parameters
The uorescence quenching data was further used to determine the binding constant (K) and number of binding sites. These binding parameters were calculated using the modied Stern-Volmer eqn (5). 42 where K a is the association, or binding constant, and n is the number of binding sites. The modied Stern-Volmer plot is shown in Fig. 1C, and the values obtained is enlisted in Table 1.
The values K a were found to be 4.28 Â 10 4 , 7.58 Â 10 4 , and 12.97 Â 10 4 M À1 at 298, 303, and 308 K, respectively. These values in the order of 10 4 M À1 suggested that theaavin monogallate binds with signicant affinity to lactoferrin. The number of binding sites was estimated to be close to 1, signifying that there was only one binding site of theaavin monogallate on lactoferrin. Previously, EGCG and theaavins were reported to possess single binding domain with albumin. 36 Moreover, there was an increase in binding constant values with increasing temperature, showing that the protein-polyphenol complex was relatively more stable at higher temperatures.

Binding thermodynamics: lactoferrin-theaavin interactions
The values of binding constant at different temperatures were used for the calculation of thermodynamic parameters. The nature of forces or type of bonds responsible for the complexation of theaavin monogallate to lactoferrin was determined using thermodynamic parameters. The active force between phenolic compounds and biomolecules may include electrostatic interactions, van der Waals interactions, and hydrophobic effect and so on. The model of interaction between quencher and a protein molecule can be concluded according to DH 0 and DS 0 data. 43 More specically, (1) DH 0 > 0 and DS 0 > 0, the main force would be hydrophobic (2) DH 0 < 0 and DS 0 > 0, it would be electrostatic force; (3) DH 0 < 0 and DS 0 < 0, it would be hydrogen bond and van der Waals interactions. 36,44,45 The values and the mathematical sign of thermodynamic parameters provide insight regarding the various interactions. The values of entropy change (DS) and enthalpy change (DH) were obtained using van't Hoff's eqn (6). 46,47 where T is temperature and R is the universal gas constant (1.987 cal mol À1 K À1 ). The van't Hoff's plot is shown in Fig. 1D, and values of DS and DH are presented in Table 2. The positive value of entropy change suggests that water molecules were arranged in an orderly fashion around the lactoferrin that got randomized aer the interaction of theaavin monogallate. 45 A positive value of enthalpy change conrms that the interaction of theaavin monogallate with lactoferrin was endothermic. Moreover, the positive values of both entropy and enthalpy change indicate the formation of hydrophobic interaction between lactoferrin and theaavin monogallate. 48 The value of Gibb's free energy change (DG) was calculated using eqn (7).
The values of DG at all tested temperatures were negative and ranged from À7.20 to À6.31 kcal mol À1 ( Table 2). The negative value of DG conrms the binding of lactoferrin with theaavin monogallate to be a spontaneous reaction. 46 Based on the sign and values of these binding parameters, various literature suggested the hydrophobic forces played a signicant role in the bindings of polyphenols-protein. 36,49

Circular dichroism spectroscopy
Circular dichroism is one of the most sensitive spectroscopic techniques used to determine the secondary structure of proteins. CD spectra of lactoferrin in the absence and presence of theaavin monogallate are shown in Fig. 2A. The free lactoferrin exhibited two negative peaks, one at 208 nm and the other at 222 nm. These two negative ellipticities are attributed to the a-helical content of proteins. 50 The data was rst converted into mean residue ellipticity (MRE) using eqn (8) where C p is the molar concentration of protein, n is the number of amino acids, and l is the path length of the cuvette. The percentage of a-helix was calculated from MRE using eqn (9). 51 a-helixð%Þ ¼ ÀMRE 208 À 4000 33 000 À 4000 Â 100 (9) where MRE 208 is MRE at 208 nm, 4000 is MRE of the random coil conformation, and b-form at 208 nm, and 33 000 is MRE of pure a-helix at 208 nm. The presence of theaavin monogallate slightly increased the ellipticity at both negative peaks. The increase in ellipticity indicates a slight increase in the a-helical content of lactoferrin. The amount of a-helix in free lactoferrin was 32.04% which increased to 33.04% and 33.72% aer the addition of theaavin monogallate in 1 : 5 and 1 : 10 molar ratios, respectively. Epigallocatechin gallate (EGCG) changed the conformation of albumin and increase the a-helical content has been earlier reported. 36 Moreover, Roy et al. (2013) also demonstrated that polyphenol (genistein) caused the similar increase in a-helical content of albumin. 52 Contrary to our results, theaavins has been shown to decrease the a-helical content of alphaglucosidase and albumin. 36,53 The dissimilarity in the results could be due to low concentration of theaavins, incubation time and different structure of proteins and ligand.

8-Anilino-1-naphthalene sulfonic acid (ANS)
uorescence assay ANS uorescence assay was performed to analyze the effect of theaavin monogallate on the hydrophobic domains or patches of lactoferrin. ANS is a uorescent probe that gives negligible uorescence when present in a polar solution. However, ANS gives strong uorescence when bound to the hydrophobic domains of protein. 54 The ANS uorescence emission spectra of lactoferrin in the absence and presence of theaavin monogallate are presented in Fig. 2B. Free ANS did not produce much uorescence in the solution. In contrast, the addition of lactoferrin to ANS solution resulted in remarkable enhancement of the ANS uorescence showing the attachment of ANS to the hydrophobic domains of lactoferrin. The addition of theaavin monogallate resulted in a gradual decrease of the ANS uorescence; saturated at 15 mM theaavin monogallate. The results indicate that binding of theaavin monogallate competed for the binding site of ANS. Moreover, this also shows the burial of the hydrophobic domain of lactoferrin on the addition of the-aavin monogallate.

Förster resonance energy transfer (FRET) analysis
FRET is a phenomenon in which the transfer of energy occurs when two molecules interact with each other. This phenomenon is used to determine the energy transfer efficiency and distance between the two interacting molecules. FRET only occurs when the donor molecule's emission spectrum overlaps with the absorption spectrum of the acceptor molecule. For a successful FRET, donor and acceptor molecules must remain in the proximity of less than 10 nm; the donor molecule must have a high quantum yield, and there should be a proper orientation of transition dipole moment of both molecules. 55 If there is considerable overlap between the acceptor molecule's absorption spectrum and the donor molecule's emission spectrum, then there is a probability of energy transfer between the interacting molecules. 56 The spectral overlap of uorescence emission of lactoferrin and absorption spectrum of theaavin monogallate is shown in Fig. 3. The amount of energy transfer is directly proportional to the degree of spectral overlap. Various parameters of FRET were calculated using eqn (10)-(12). 57,58 R o 6 þ r 6 (10) where F o and F are the uorescence intensities of lactoferrin in the absence and presence of theaavin monogallate; R o is the critical distance at 50% transfer efficiency, r is the distance between donor and acceptor molecules, K 2 is orientation factor (2/3), f is uorescence quantum yield of the donor (0.118), n is the refractive index of the medium (1.336), J is the degree of spectral overlap, F(l) is the uorescence intensity of donor in wavelength ranging from l to l + Dl, and 3(l) is molar absorptivity of acceptor at l. The parameters for the interaction of theaavin monogallate to lactoferrin obtained using FRET are enlisted in Table 3. J and R o values were found to be 1.20 Â 10 À14 cm 3 M À1 and 2.54 nm, respectively. The distance between theaavin monogallate and lactoferrin was found to be 2.44 nm, and the energy transfer efficiency was obtained found as 49.6%. These values indicate a high probability of energy transfer from lactoferrin to theaavin monogallate. As per the non-radiative energy transfer theory of Förster, the R o and r values lies below 10 nm. 59 Therefore, the values of R o and r conrms a nonradiative energy transfer mechanism occurring between lactoferrin and theaavin monogallate. Moreover, FRET parameters also validate that there was static quenching mechanism energy transfer that contributed to the decrease of uorescence intensity lactoferrin. 60

Molecular docking
Molecular docking was performed to obtain a closer insight into the binding of theaavin monogallate to lactoferrin. Such studies provide detailed information regarding the nature of forces involved in interaction and the amino acids interacting with the ligand. The lowest binding energy docked complex of theaavin monogallate with lactoferrin is shown in Fig. 4. The details of interacting amino acid residues and the nature of forces involved in the interaction are enlisted in Table 4. The binding energy for the interaction of theaavin monogallate with lactoferrin was À9.7 kcal mol À1 , which corresponds to the binding affinity of 2.03 Â 10 6 M À1 . Theaavin monogallate formed seven hydrogen bonds with Gln87, Gln89, Tyr192, Val250, Lys301, and Ser303 of lactoferrin, while Tyr82, Tyr92, and Tyr192 interacted with theaavin monogallate via three hydrophobic interactions (Pi-Pi T-shaped). Also, several residues such as Gly83, Thr84, Pro88, Thr90, His91, Arg121, Lys210, Pro251, Ser252, His253, and Arg280 formed van der Waals' interactions. Moreover, Ser212 was involved in an unfavorable interaction with theaavin monogallate.

Molecular dynamics (MD) simulation
The stability of the theaavin monogallate-lactoferrin complex was further studied by molecular dynamics (MD) simulation. The protein-ligand complex is dynamic in nature; therefore, its stability was assessed by simulating at the physiological  conditions (Fig. 5). The complex obtained in molecular docking was used as initial conformation, and MD simulation was performed for 50 ns. The RMSD (root mean square variation) of lactoferrin and its complex with theaavin monogallate with respect to the original frame is presented in Fig. 5A. There was a little deviation in RMSD of the complex from 0 to 20 ns, which at a later part (20-50 ns) of the simulation gets stabilized. The small deviation might be due to the entry of theaavin monogallate into the cavity of lactoferrin. The mean RMSD values of lactoferrin and lactoferrin-theaavin monogallate systems for 20-50 ns were 0.3693 and 0.3781 nm, respectively. These results conrmed the formation of a stable complex between lactoferrin and theaavin monogallate. Moreover, RMSF (root mean square uctuation) along the lactoferrin chain were calculated to analyze the local conformational alterations caused by the binding of theaavin monogallate (Fig. 5B). It was found that the uctuation in residues of lactoferrin-theaavin monogallate complex exhibited a similar uctuation pattern as exhibited by lactoferrin alone. The results conrm that no signicant structural changes occurred in lactoferrin due to the binding of theaavin monogallate. The radius of gyration (R g ) of lactoferrin and its complex was also calculated to ascertain the structural stability as a function of simulation time (Fig. 5C). The R g of lactoferrin and lactoferrin-theaavin monogallate complex remained constant approximately at 3.01 and 3.02 nm, indicating that lactoferrin  did not undergo any signicant conformational alterations. Finally, the stability of the complex between theaavin monogallate and lactoferrin was further validated by computing the solvent-accessible molecular surface area (SASA) as a function of simulation time (Fig. 5D). It was found that SASA of lactoferrin and complex remained constant within limits. The average values of SASA of lactoferrin alone and in complex with theaavin monogallate were estimated as 135.45 and 136.58 nm 2 , respectively. Thus, the results of molecular dynamics simulations indicate that theaavin monogallate formed a stable complex with lactoferrin.

Conclusion
The ndings of this study provide detailed insights into the interaction between theaavin monogallate and lactoferrin. The-aavin monogallate interacted with lactoferrin with a moderate binding affinity. The mode of uorescence quenching was found to be static. The values of various thermodynamic parameters showed that a change in entropy drove the binding. The interaction between lactoferrin and theaavin monogallate resulted in forming a stable complex in which a-helical content was not altered. The conformation of lactoferrin remained nearly uncaged, and energy transfer from lactoferrin to theaavin monogallate was efficient. Theaavin monogallate binds the central cavity of lactoferrin and interacts with key amino acid residues. Hydrogen bonding and hydrophobic interactions were predominant forces in the formation of a stable protein-polyphenol complex. The parameters of molecular dynamics simulations further validated the stability of theaavin monogallate-lactoferrin complex. These ndings are signicant in understanding the nature and mechanism of interaction for the binding of theaavin monogallate with lactoferrin at the molecular level.

Conflicts of interest
There are no conicts to declare.