Effect of CaCl 2 on 2 heat-induced whey protein concentrate fibrillation pathways: Spontaneous and nuclear induction

Amyloid fibrils have many excellent functional properties that facilitate their applications in the food industry. There are 2 pathways for whey protein concentrate (WPC) to form amyloid fibril aggregates: spontaneous pathway and nuclear induction pathway. Low ionic strength is a necessary condition for the spontaneous pathway to proceed successfully. In this paper, the effect of salt ions on 2 WPC fibrillation pathways was investigated by adding CaCl 2 . The re-sults demonstrated WPC fibrils were unable to form normally through spontaneous pathway as adding CaCl 2 ; but still could form through nuclear induction pathway with 20 to 30 m M CaCl 2 , the nuclei accelerated the fibrillation process led to the resistance to the disordered aggregation brought by CaCl 2 . Moreover, divalent cations (Ca 2+ , Mg 2+ ) had much stronger effects than monovalent cations (Na + ) on fibril formation, and the results of X-ray photoelectron spectrum together with Fourier-transform infrared spectroscopy suggested that Ca 2+ had a greater effect on the fibril formation than Cl − .


INTRODUCTION
In recent years, Gosal et al. (2004) proposed that whey protein concentrate (WPC) is capable of selfassembling into amyloid fibril aggregates with a diameter around 1 to 10 nm and a length 1 to 20 μm under certain denaturing conditions, such as high temperature with low pH and low ionic strength (Dong et al., 2016;Farjami et al., 2016).Compared with conventional protein aggregates, the fibril aggregates have many excellent properties, such as gelation, emulsifica-tion, foam stability, antioxidant activity, moreover, the highly resistant to acids and heat as well as greatly reducing the allergenicity are also the outstanding properties (Adamcik et al., 2010;Loveday et al., 2012b;Mohammadian and Madadlou, 2016;Peng et al., 2016).The functional properties of whey proteins can be significantly improved after fibrillation treatment, which opens up new areas of application for whey proteins in the food industry, thus better serving mankind and society.
Protein fibrillation is the process of native soluble proteins misfolding into insoluble fibrils comprising a cross-β-sheet in which individual β-strands run perpendicular to the fibril axis (Lee et al., 2007;Espargaró et al., 2020), which can be divided into the lag, elongation and saturation phases (Michaels and Knowles, 2015); in particular, the lag phase is the main rate-limiting phase lasting for hours or even days (Šarić et al., 2016).In general, the self-assembly aggregation of fibrillation proceeds via a rate-limiting nucleation and an elongation of these formed nuclei, which is a nucleation-dependent aggregation process (Harper and Lansbury, 1997;Knowles et al., 2009;Kumar et al., 2017).In other words, the nucleation is a key step for the formation of amyloid fibrils.Foderà et al. (2008) found that the possible mechanisms for a nucleation-elongation process include homogeneous nucleation and secondary nucleation.As the initial step of fibril formation, the homogeneous nucleation process occurs several activated protein monomers span energy barrier in lag phase then self-assemble into the nuclei called homogeneous nuclei (HN; Arosio et al., 2015).The secondary nucleation process involves the formed nuclei acting as an activator to interact with protein monomers and elongate into protofilaments, subsequently some protofilaments twist into multistranded helical mature amyloid fibrils also called secondary nuclei (SN; Foderà et al., 2008;Tan et al., 2018).
The lag phase can be eliminated by adding preformed nuclei as "seeds" (Kalapothakis et al., 2015), which can accelerate the WPC aggregated rate and increase

Effect of CaCl 2 on 2 heat-induced whey protein concentrate fibrillation pathways: Spontaneous and nuclear induction
the number of fibrils.The same results were found in our previous study (Tan et al., 2018).The additional nuclei have the induction effect, which make protein monomers aggregate into amyloid fibrils immediately (Krebs et al., 2004;Knowles et al., 2009).Therefore, in addition to the conventional spontaneous pathway, the nuclear induction pathway provides another effective method for the preparation of fibrils, which can significantly accelerate the fibrillation process and lead to the formation of more fibrils.
Both pH and ionic strength can regulate the strength of electrostatic interactions between fibril-building blocks (Cao and Mezzenga, 2019).Multiple literature reported that low ionic strength is a necessary condition for proteins to form long and characteristic fibrils.Adding salt ions can accelerate (WPC) aggregated rate to some extent, resulting in the formation of short and soft fibrils (Loveday et al., 2012a); for instance, long semiflexible β-lactoglobulin fibrils are formed at low ionic strength, whereas short worm-like fibrils prevail at higher ionic strengths and with a faster assembly rate (Loveday et al., 2010b(Loveday et al., , 2017)).Arnaudov and de Vries (2006) also found that fibrils obtained at a higher ion strength are shorter and more curved as opposed to the longer and straighter fibrils obtained at a lower ionic strength.Taken together, these results reflect that ionic strength increases with strengthening electrostatic repulsion and disrupts fibrils formation.
Some studies investigating the effect of adding CaCl 2 on whey proteins show that CaCl 2 can influence the formation of network structures between proteins by shielding negatively charged molecules.In addition, divalent cations can combine with free carboxyl groups of Asp acids and Glu acids to form salt bridges (Hongsprabhas and Barbut, 1997a;Kastuta, 1998).Meanwhile, there are related reports showing that Asp, Asn, and Glu participate in the formation of a structural motif consisting of 2-residue segments, which appears to be important in the local folding patterns of proteins (Dhar and Chakrabarti, 2018).In recent years, it has been reported that the addition of CaCl 2 to the aggregation process of WPC spontaneous pathway has a certain effect on the formation of fibrils.Great progress has been made in this field; however, there is still a lack of related reports describing whether the nuclear induction pathway is affected by ionic strength or which pathway demonstrates better resistance to additional ions.Based on the existing literature, we carried out studies in an effort to obtain insight into the effects of different concentrations of CaCl 2 on nuclear formation, compared the resistance to CaCl 2 between the nuclear induction pathway and spontaneous pathway, and explored the mechanism of action between ions and fibrils.

Materials
The WPC was purchased from Hilmar Cheese Co.The protein content of the WPC was 79.15% (measured by Kjeldahl determination: N × 6.38), the fat content was 5.60%, the lactose content was 5.65%, and the ash content was 4.91%.In our previous study, the β-LG and α-La in WPC were extracted by a method adapted from Alomirah and Alli (2004), the content of β-LG and α-LA, respectively, was 53 and 27% (Gao et al., 2021).Thioflavin T (ThT) powder was purchased from Sigma-Aldrich.All other reagents and chemicals were of analytical grade and purchased from a local market.A flowchart that summarized the preparation of fibrils and nuclei was given in Figure 1.No animals were used in this study, and ethical approval for the use of animals was thus deemed unnecessary.

Preparation of WPC and Nuclei
The WPC (5 g) was dissolved in Millipore water (Wa Ha Ha; 100 mL), and the pH was set to 2.0 by adding 6 M and 1 M HCl solutions.To remove undissolved protein, the solution was centrifuged at 16,000 × g for 20 min at 4°C, and the protein content was determined by Kjeldahl determination.The protein content of the supernatant was diluted to 3.0 wt% by Millipore water (pH 2.0), and the samples were stored at 4°C in a refrigerator.
According to the results pertaining to induction ability from our previous study, HN and SN were screened out and formed by 3.0 wt% WPC (pH 2.0) heated at 90°C for 2 h (HN) and 10 h (SN; Tan et al., 2018;Bolder et al., 2007).

Preparation of Fibrils
In this experiment, the spontaneous pathway and nuclear induction pathway were used to prepare fibrils.
Nuclear Induction Pathway.The HN+WPC fibrils (HNF) or SN+WPC fibrils (SNF) referred to the addition of 7 mL of 3 wt% HN or SN to 21 mL of 3 wt% WPC, which was mixed evenly, and the mixture was added to 2 mL of Millipore water (pH 2.0), the final protein concentration was 2.8 wt%; subsequent preparation methods were the same as WF.The blank sample, HN+H 2 O (HNH) or SN+H 2 O (SNH), refers to the addition of 7 mL of 3 wt% HN or SN to 23 mL of Millipore water (pH 2.0), which was mixed evenly, the final protein concentration was 0.7 wt%, and the subsequent preparation methods were the same as HNF and SNF.
The Ca-HN+WPC fibrils (Ca-HNF) or Ca-SN+WPC fibrils (Ca-SNF) referred to the application of Ca-HN or Ca-SN to induce WPC to form fibrils. First, 7.5 mL of 2.8 wt% Ca-HN or Ca-SN was mixed with 22.5 mL of 2.8 wt% WPC, and then the mixtures were incubated in 90°C for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 h.The blank sample, Ca-HN+H 2 O (Ca-HNH) or Ca-SN+H 2 O (Ca-SNH), refers that 7.5 mL of 2.8 wt% Ca-HN or Ca-SN was mixed with 22.5 mL of Millipore water (pH 2.0), the final protein concentration was 0.7 wt%, and the subsequent preparation methods were the same as Ca-HNF and Ca-SNF.
Control 1 and control 2 were used to exclude the interference of CaCl 2 on the experiment.Control 1 referred the addition of 28 mL of 3.0 wt% WPC (pH 2.0) sample to 2 mL of 0.15, 0.30, 0.45, 0.60, and 0.75 M CaCl 2 , the final protein concentration was 2.8 wt%, then 7.5 mL mixture was added 22.5 mL 2.8 wt% WPC, and the subsequent preparation methods were the same as Ca-HNF and Ca-SNF.Control 2 referred the addition of 28 mL of 3.0 wt% HN or SN (pH 2.0) sample to 2 mL of 0.15, 0.30, 0.45, 0.60, and 0.75 M CaCl 2 , the final protein concentration was 2.8 wt%, then 7.5 mL mixture was added 22.5 mL 2.8 wt% WPC, and the subsequent preparation methods were the same as Ca-HNF and Ca-SNF.Blank sample: the addition of 28 mL of 3.0 wt% WPC or HN or SN (pH 2.0) sample to 2 mL of 0.15, 0.30, 0.45, 0.60, and 0.75 M CaCl 2 , the final protein concentration was 2.8 wt%, then 7.5 mL mixture was added 22.5 mL H 2 O (pH 2.0), and the subsequent preparation methods were the same as Ca-HNF and Ca-SNF.
The HN+CaCl 2 +WPC fibrils (HNCaF) or SN+CaCl 2 +WPC fibrils (SNCaF) referred to CaCl 2 was added after nuclei had formed.First, 7 mL of 3.0 wt% HN or SN was mixed with 21 mL of 3.0 wt% WPC, and 28 mL of the mixtures were added to 2 mL of 0.15, 0.30, 0.45, 0.60, and 0.75 M CaCl 2 , the final protein concentration was 2.8 wt%, and the final concentrations of CaCl 2 were 10, 20, 30, 40, and 50 mM, respectively; subsequent preparation methods were the same as those for WCaF.The blank sample, HN+CaCl 2 +H 2 O (HNCaH) or SN+CaCl 2 +H 2 O (SNCaH), refers to 7 mL of 3.0 wt% HN or SN was mixed with 21 mL of Millipore water (pH 2.0) and 28 mL of mixtures were added to 2 mL of 0.15, 0.30, 0.45, 0.60, and 0.75 M CaCl 2 , the final protein concentration was 0.7 wt%; the subsequent preparation methods were the same as HNCaF and SNCaF.

ThT Fluorescence Assay
According to the method of Akkermans et al. (2008) with some modifications, 0.80 g of ThT was dissolved in 1 L of NaCl-phosphate buffer (20 mM NaCl, 10 mM phosphate buffer solution, pH 7.0).This stock solution was filtered through a 0.22-μm syringe filter (Millex-GS; Millipore Corp.) and stored in brown glass bottles covered with tin foil in a 4°C refrigerator.The working solution was obtained by diluting the stock solution 50-fold in NaCl-phosphate buffer before use.
In the assay, 0.8 μL of sample was mixed with 10 mL of working solution and maintained for 1 min of whirlpool oscillation before the fluorescence measurement (Hitachi F4500 fluorescence spectrometer; Hitachi High, Technologies Corp.).The excitation wavelength for ThT was 460 nm, and the emission wavelength was 490 nm (Loveday et al., 2012b).All measurements were performed in triplicate.
where the ThT is the ThT fluorescence intensity of the sample (ThT sample ) subtracted from that of the corresponding blank sample (ThT blank sample ).

Polymerization Kinetics
Referencing previous studies (Loveday et al., 2012b) with some modifications, ThT fluorescence data were fitted with Equation 2, given initially by Morris et al. (2008).Note that we used this equation as an empirical curve-fitting function, wherein f t was fluorescence intensity at time t, and α, β, and γ were arbitrary constants.The t lag (lag time), the t 1/2 max (time for fluorescence to increase to half of its maximal value), and the (df/dt) max (the maximum rate of increase in fluorescence) were calculated with the analytical expressions in Equations 3 to 5, which were derived from Equation 2 from a previous study (Loveday et al., 2010a): [5]

Transmission Electron Microscopy
The microstructure of the protein dispersions incubated for 10 h at 90°C in the absence of CaCl 2 (WF; HNF; SNF) and in the presence of 50 mM CaCl 2 (WCaF; HNCaF; SNCaF) was observed using transmission electron microscopy (H-7650; Hitachi High Technologies Corp.).The samples were diluted 20-fold, and a droplet of the diluted sample was placed on a carbon support film on a copper grid.After 20 min, the excess droplet was removed with filter paper.After the grid was dried at room temperature for 30 min, the samples were observed using a transmission electron microscope operated at 100 kV (Akkermans et al., 2008).

Thermogravimetry Analysis
According to the method of Mantovani et al. ( 2018), with some modifications, analysis was performed in triplicate on freeze-dried samples (WF, WCaF, and SN-CaF with 30 mM CaCl 2 heated 10 h) set in aluminum pans heated from 200 and 600°C at 8°C/min under a N 2 atmosphere using a TGA SDT650 (TA Instruments).

Fourier-Transform Infrared Spectroscopy
The WF, HNF, SNF and WCaF, HNCaF, SNCaF (adding 50 mM CaCl 2 ) were freeze-dried for 12 h.An appropriate amount of KBr was added to the samples at a mass ratio of 1:100, ground to powder, and then pressed into thin sheets.Then, we set the resolution to 4 cm −1 , scan time to 32, and full-wave band to 4,000 to 400 cm −1 (FTIR-8400S; Hitachi High, Technologies Corp.; Qi et al., 2015).

X-Ray Photoelectron Spectrum
According to the method of Ikeuba et al. (2019), with some modifications, the contents of Ca 2+ and Cl − on the surface of WCaF, HNCaF, and SNCaF with 30 mM CaCl 2 were measured in the assay, the peak area of a certain element (Ca 2+ , Cl − ) in the figure was obtained by Origin, which represents the amount of the element combining with fibrils.The combined amount of the same element could be directly compared through the peak area.

Statistical Analysis
The results are presented as the means ± standard deviations.One-way ANOVA was used to determine the significant differences.Significant differences between means were identified using Duncan's multiple range test (P < 0.05).Origin 2021 was used to plot the data.

Effects of CaCl 2 on Nuclei Formation
The effects of CaCl 2 on nuclei formation were judged according to whether the Ca-nuclei had induction ability which was reflected by comparing the number of Ca-HNF or Ca-SNF with WCaF.The ThT is a dye that shows enhanced fluorescence when bound to intermolecular β-sheets present in aggregates, which represents a useful and convenient tool for the fast and reliable diagnosis of the presence of amyloid fibrils in disease-affected tissues and organs (Goers et al., 2002;Turoverov et al., 2007).From the ThT intensity of fibrils formed by Ca-nuclei induction, it could be seen the nuclei formation was interfered greatly by the addition of CaCl 2 to the nucleation process (Figures 2 and 3).The results indicated that upon 10 mM CaCl 2 , the HN were unable to form normally and lost induction ability (Figure 2).More serious situations were observed for SN, which could not form properly once CaCl 2 was added (Figure 3).This indicated the formation of nuclei would be influenced if the CaCl 2 was added before nuclei formed, but in comparison to SN, HN still could be formed at low concentrations of CaCl 2 (10 mM).In addition, it was speculated that the addition of CaCl 2 influenced the formation of HN, thereby causing SN cannot form normally at any CaCl 2 concentrations.

Effects of CaCl 2 on Fibrils Formation with the Nuclear Induction Pathway
Both HN and SN were destroyed to different degrees by adding CaCl 2 before nuclear formation, the HN formed under 10 mM CaCl 2 still had weak induction ability, whereas SN could not form normally under any concentrations of CaCl 2 and accompanied by the loss of inductive effect.If nuclei had formed then mixed with WPC, what effect did the increase in ionic strength during aggregation process have on the nuclear induction pathway?As described in Figure 4A-C, in the absence of CaCl 2 , the ThT fluorescence intensity of HNF or SNF was greater than that of WF, indicating the nuclei possessed induction ability and whose addition could increase the number of fibrils.Inversely, in the presence of CaCl 2 , the nuclear induction ability changed with the increasing concentrations of CaCl 2 , moreover, there was a difference in the resistance to CaCl 2 between the HN and SN.For example, in the presence of 10 to 20 mM CaCl 2 for HN and 10 to 30 mM CaCl 2 for SN, they still could induce WPC to form more fibrils than spontaneous pathway (WF), it was obvious that the SN could resist more CaCl 2 ; however, the nuclear induction ability was lost gradually as adding more CaCl 2 to the aggregation process.Low ionic strength is necessary for the formation of amyloid fibrils, the increase of ionic strength may lead to changes of fibril morphology and the formation of amorphous aggregates (Loveday et al., 2011(Loveday et al., , 2017;;Serfert et al., 2014).Accordingly, most of the observed aggregates were presumed to be amorphous aggregates as adding 50 mM CaCl 2 (Figure 4D-E).These results indicated that for the spontaneous pathway, once CaCl 2 was added during aggregation process, WPC was unable to assemble into fibrils properly; on the contrary, WPC could still form fibrils through the nuclear induction pathway despite adding a certain concentration of CaCl 2 .

Polymerization Kinetics
The nuclear induction pathway could resist higher ionic strength in contrast to spontaneous pathway, especially SN still equipped with induction ability even at the condition of adding 30 mM CaCl 2 , we next investigated the effect of increasing ionic strength on the aggregated rate (df/dt) of 2 fibrillation pathways (Figure 5).In the absence of CaCl 2 , a comparative view of the 2 fibrillation pathways showed that the maximum of df/dt for WF, HNF and SNF was at the third, second, and first hours, respectively, suggesting that the self-assembly process was accelerated by the addition of nuclei (HN and SN).Simultaneously, the addition of CaCl 2 could accelerate the early stage of aggregation process regardless of spontaneous pathway and nuclear induction pathway, both maximums of df/ dt were advanced to first, this was consistent with previous reports (Loveday et al., 2010b(Loveday et al., , 2017)).
The increase of ionic strength had an accelerate effect on aggregation process, a comparison of the kinetic parameters between the 2 fibrillation pathways under the different concentrations of CaCl 2 was shown in Table 1.Similarly, the kinetic parameters also showed the nuclei were able to increase the number of fibrils and eliminate the lag phase thereby accelerating aggregated rate.Nevertheless, the kinetic parameters changed significantly with increasing CaCl 2 concentrations, where t lag , t 0.5max , and f max were all gradually shortened, and still the nuclear induction pathway (HNCaF and SN-CaF) t lag and t 0.5max were shorter and f max was higher than those of the WPC spontaneous pathway (WCaF), similar to the regulation trends between HNF or SNF and WF.In general, both the polymerization kinetic and the final fibril morphology were largely influenced by the ionic strength.Especially, the formation of amorphous aggregates was observed under 50 mM CaCl 2 , it was speculated that rapid aggregate brought by CaCl 2 interfered the sequential self-assembly of proteins.However, the nuclear induction pathway could resist more CaCl 2 , which might be caused by the addition of preformed nuclei could shorten the rate-limiting lag phase of the conventional spontaneous pathway so that the WPC self-assembly rate was increased and could better resist the disordered aggregation reaction produced by CaCl 2 .The above results revealed that HN and SN could not form normally and lost their induction ability when CaCl 2 was added before nuclei formation.In contrast, once nuclei had formed, they had better resistance to disordered aggregation produced by CaCl 2 , which meant the nuclear induction pathway was not as demanding on ionic strength as the spontaneous pathway.Then we explored the reasons why the increase of ionic strength during aggregation process influenced the orderly self-assembly of proteins.It should be mentioned that in this experiment, the self-assembly of WPC into amyloid fibrils occurred at pH 2.0, consequently the interaction between CaCl 2 and proteins may be different from that reported under normal pH conditions.

Thermostability
The induction ability of SN was about to disappear with the addition of 30 mM CaCl 2 , and the number of fibrils formed by SN induction pathway (SNCaF) was similar to that of spontaneous pathway (WF).Based on this result, the 30 mM CaCl 2 was added to the aggregation process to compare thermal stability of fibrils formed by the 2 pathways, thermogravimetric (TG) plot of WF, WCaF, and SNCaF was shown in Figure  6.The mass decrement (%) during the heating process was determined from TG curves and the temperature of the maximum speed of the process (T max ) was determined from the derivation of TG curves (Dandurand et al., 2014).As shown in Table 2, the WF as the control sample, the T max of WCaF and SNCaF were lower than that of WF.Moreover, the SNCaF and WF with close ThT intensity also had similar mass decrement (%), and both were lower than that of WCaF, indicating the WCaF was not as stable as WF and SNCaF.Among the multiple aggregation states of proteins, such as the unfolded state, partially folded states, native state, amorphous aggregates, and amyloid fibrils, it has been reported that the ordered stacking of β-sheets makes amyloid fibrils possess the best stability, especially extremely resistant to acid and heat (Balchin et al., 2016).Accordingly, it could be seen from the results of ThT fluorescence intensity that most of monomeric WPC in the spontaneous pathway probably aggregated into nonfibrous aggregates without an ordered stacking structure under the addition of 30 mM CaCl 2 , thus showing the poorer thermal stability of formed Different letters indicate significant differences within a group (P < 0.05; ANOVA-Duncan). A-C Different letters indicate significant differences between groups (P < 0.05; ANOVA-Duncan).aggregates, in comparison, the SN had better resistance to CaCl 2 and could still induce WPC to form fibrous aggregates similar to those formed by the normal spontaneous pathway (WF), which resulted in the similar thermal stability between SNCaF and WF.

Action of Anions and Cations
The TG results indicated that the presence of CaCl 2 during the aggregation process led to changes in the structure of the formed aggregates.In addition, whether different ionic valence states would have different degrees of influence on protein aggregation?It was evident that there were no marked differences in the effect of the same concentration divalent cations (Ca 2+ and Mg 2+ ) on aggregation process, but all had a greater influence than monovalent cation (Na + ) at the same Cl − concentration (Figure 7 A-C).The same phenomenon was observed in Figure 7D-F, in which the influence of SO 4 2-on the aggregate process was more pro-nounced than that of Cl − .In summary, it was speculated that the higher the ionic valence state, whether cation or anion, the greater the effect on the protein fibrillation process.The presence of salt ions in the aggregation environment interfered with the protein fibrillation process, in this case, did the anion or the cation play a major role?With the addition of CaCl 2 , all peaks showed a reduced intensity (Figure 8A-C), indicating that the added salt might restrict the motion of the organic group (Yang et al., 2019;Guan et al., 2022), and then interfered the fibrillation process.The amide I band (1,600-1,700 cm −1 ) of FTIR spectra is highly sensitive to the formation of fibrils, the strong peak at 1,611 to 1,630 cm −1 ascribed to β-sheets structure represented the existence of amyloid fibrils (Farrokhi et al., 2020;Cao and Mezzenga, 2019).Both the samples with and without CaCl 2 contained a major component at 1,626 cm −1 (Figure 8D-I), it was speculated that the addition of 50 mM CaCl 2 was adverse for the fibril formation and accompanied by the decrease of the number of fibrils, but there were still some proteins that could aggregate into fibrils.Previous study has reported that the peak of amide II at 1,550 cm −1 is associated with N-H and C-N (Andrade et al., 2019).The intensity of band at 1,536 cm −1 (N-H bending) decreased and shifted to lower wavenumbers after adding CaCl 2 (Figure 8A-C).Farjami et al. (2016) proposed that Cl − is a good proton receptor that can combine with NH− and inhibit the formation of additional intermolecular N-H•••C=O hydrogen bonds from amide groups.Thus, perhaps the shift of peak was brought by the combination between Cl − and N-H.
Additionally, we questioned the connection between the degree of binding of fibrils to Ca 2+ or Cl − and the number of fibrils, and whether the protein monomers in spontaneous pathway or nuclear induction pathway had different action modes with CaCl 2 .Based on the above conjecture, X-ray photoelectron spectrum (XPS) was measured to quantitatively compare the amount of Ca 2+ and Cl − bound to WF formed by the 2 fibrillation pathways.The peak area in the XPS was used to rep- resent the element content bound to the sample surface (Ikeuba et al., 2019).The percentage in Figure 9 was calculated by dividing the peak area of one sample by total that of 3 samples, respectively.The XPS results showed that fibrils formed by the nuclear induction pathway bound less Ca 2+ (Figure 9A).However, unlike Ca 2+ , there was no correlation between the binding amount of Cl − and the number of fibrils, where HNCaF had the highest binding amount with Cl − (Figure 9B).Consequently, the disordered aggregation caused by the addition of excess salts may be related to the binding of cations rather than anions.It was proposed previously that Ca 2+ will combine with the free carboxyl groups of Asp acid and Glu acid to form a salt bridge (Hongsprabhas and Barbut, 1997b;Loveday et al., 2012a).Moreover, Dhar and Chakrabarti (2018) reported that a structural motif of 2-residue segments (m and m+1) could form hydrogen-bonded 5-membered fused-ring motifs, which appear to be important in the local folding patterns of proteins and occur near protein active sites.Remarkably, this motif may be an important component of fibrillation, and Gly, Asn, and Asp have high propensities to occur at the first position (m).Therefore, perhaps the rate-limiting lag phase of the spontaneous pathway occurred the free carboxyl groups of protein monomers first bound with Ca 2+ , thus cannot participate in the assembling process, on the contrary, the self-assembly rate of protein monomers in nuclear induction pathway was sufficiently fast to bind with less CaCl 2 , so that the nuclear induction pathway could resist a certain concentration of salt ions.

CONCLUSIONS
There was a significant effect on the spontaneous pathway with the addition of CaCl 2 , evidenced by the notable decrease in the number of fibrils.Interestingly, WPC still could form more fibrils through the nuclear induction pathway despite adding CaCl 2 .The SN induction pathway formed fibrils at higher levels than the normal WPC spontaneous pathway even up to 30 mM CaCl 2 , whereas the nuclear induction pathway substantially accelerated the rate of fibril formation, eliminated the lag phase, and induced fibrils to form immediately, resulting in better resistance to rapid and disorderly aggregation brought by CaCl 2 .For the ions selected in this experiment, the divalent cation had a stronger effect on fibrils formation that the monovalent cation; moreover, the effect of CaCl 2 on the number of fibrils decreasing was mainly connected with Ca 2+ .

1t
lag = lag time; (df/dt) max = the maximum rate of fluorescence increase; t 0.5max = time for fluorescence to increase to half its maximal value; f max = maximum fluorescence intensity.WCaF = whey protein concentrate (WPC) formed fibrils under different CaCl 2 concentrations.HNCaF = homogeneous nuclei (HN) induced WPC to form fibrils under different CaCl 2 concentrations.SNCaF = secondary nuclei (SN) induced WPC to form fibrils under different CaCl 2 concentrations.AU = arbitrary units.Data are mean ± SD.
10 h.The

Table 1 .
Yang et al.:WHEY PROTEIN CONCENTRATE FIBRILLATION PATHWAYS The kinetic parameters derived from fitting Equation 2 to thioflavin T (ThT) fluorescence data of samples formed in different CaCl 2 concentrations and 90°C heat incubation for 10 h 1