L-lysine moderates thermal aggregation of coconut proteins induced by thermal treatment

This work attempts to investigate the inhibitory effect of L-lysine (Lys) on the thermal aggregation of coconut protein (CP). The results showed that under neutral conditions (pH = 7), temperature reduced the solubility and enhanced the thermally induced gel formation of CP. In addition, Lys reduced the fluorescence properties, particle size and increased the turbidity of CP, which had an inhibitory effect on heat induced gels. The results indicate that Lys plays an important role in inhibiting protein thermal aggregation by interacting with CP to create steric hindrance and increase protein electrostatic repulsion.

Lys-CP mixture properties.Changes in turbidity reflect the degree of protein aggregation, while solubility reflect the number of insoluble aggregates in the protein solution 33 .Turbidity and solubility were measured for Lys-CP mixtures with different levels of heat treatment (25 °C, 100 °C, and 121 °C).After heat treatment, the turbidity of the CP solution increased from 0.57 to 0.72; the solubility decreased from 63.09 to 46.39% (Fig. 2).It is possible that the heat treatment can denature and aggregate the proteins, producing aggregates 34 .The presence of aggregates reduced the solubility and increased the turbidity of the CP solution 35 .When Lys was added and heat treated, turbidity decreases from 0.57 to 0.44 at 25 °C; from 0.63 to 0.47 at 100 °C; and from 0.72 to 0.50 at 121 °C (Fig. 2A).The solubility increased from 63.1 to 95.4% at 25 °C; from 55.4 to 93.8% at 100 °C; and from 46.4 to 84.4% at 121 °C (Fig. 2B).The results showed that the addition of Lys could improve the solubility and reduce the turbidity of the CP solution after heat treatment.Similarly, Lys has been reported to increase the solubility of proteins.A few authors have hypothesized that Lys can increase the electrostatic repulsion of proteins through interactions with them (e.g., electrostatic interactions) 36 .Due to spatial site resistance, protein interactions are reduced, and solubility is increased, which can slow down protein thermal aggregation 19,37 .Therefore, Lys may increase CP solubility and alleviate CP thermal aggregation.
Subsequently the effect of heat treatment temperature and Lys content on low concentration CP solutions was further investigated by changes in particle size distribution and protein tertiary structure.

Effect of Lys on the fluorescence characteristics of CP solutions. Tryptophan (Try) is typically used
to measure changes in the tertiary structure of proteins due to its sensitivity to the hydrophilic environment 38 .It is widely accepted that folded proteins exhibit high fluorescence intensity (FI) 39 .When proteins undergo denaturation unfolding, try was exposed to a polar microenvironment (λ max > 330 nm), resulted in a decrease in FI 22 .As shown in Fig. 3A, the FI of CP decreased after heat treatment.Attributed to the heat treatment caused the protein denaturation to unfold, and the fluorescence quenching occurred by exposing the internal tryptophan residues 40 .Heat treatment after the addition of Lys to the CP solution revealed a decrease in FI and red shift (Fig. 3B-D).It is possible that the addition of Lys caused the unfolding of the CP tertiary structure 41,42 .Similarly, Li et al. 42 induced unfolding of myosin by adding Lys, leading to a decrease in FI and the appearance of red shift.www.nature.com/scientificreports/Probably, this is due to the cation-π interaction of Lys with tryptophan residues, which changes the microenvironment around the tryptophan residues 37 .After heat treatment, the decrease in FI was more pronounced.Perhaps the interaction of Lys with the tryptophan residues unfolded by protein denaturation was caused.Our results suggest that the presence of Lys can induce changes in the tertiary structure of CP and a more polar environment for tryptophan residues.www.nature.com/scientificreports/Effect of Lys on the particle size of CP solutions.The particle size distribution maps can reflect the degree of protein aggregation and characterize the spatial structural changes of protein 43 .Figure 4A shows the particle size distribution of the CP solution after heat treatment.We can find that the particle size distribution of the protein solution was significantly changed (P < 0.05) and the number of aggregates of macromolecules increased with the increase of heat treatment temperature.Temperature is proportional to the size of the thermal aggregates.Thus, high temperature treatment can cause a series of covalent and non-covalent interactions between adjacent proteins, which leads to the aggregation of proteins and the formation of aggregates 44 .Figure 4B-D shows the effect of Lys content on the particle size of CP solutions at different temperature conditions.Under neutral conditions (pH = 7), the average particle size of the protein solution decreased with increasing Lys content, indicating that the aggregation of the Lys-CP mixture was inhibited after heat treatment.On the one hand, Lys undergoes cation-π interactions with tryptophan residues, changes the protein surroundings to be more polar 37 .On the other hand, weak interaction of Lys with proteins leads to a reduction in protein-protein interactions and a spatial site blocking effect 45 .This spatial site blocking effect provides favorable conditions for inhibiting the formation of thermal aggregates 36,46,47 .When the formation of aggregates is improved, the particle size of CP thermal aggregates decreases, which is consistent with previous results on the effect of Lys on the turbidity and solubility of CP thermal aggregates.
Further to demonstrate that Lys can increase the solubility of CP and alleviate the formation of thermal aggregates.We added Lys to 5% (w/v) and 9% (w/v) CP solutions for different temperature heat treatments.It was verified whether the thermally induced gel could also be formed.

Rheological properties.
The energy storage and loss modulus of Lys-CP thermally induced gels versus angular frequency (Fig. 5).When the angular frequency increased, the G′ and G″ of the gel increased, showing a strong frequency dependence.Throughout the measurement range, the higher the concentration of Lys added, the smaller the G′ and G″ values, which was attributed to the interaction of Lys with the proteins (caption-π interactions), reducing the interactions between the proteins 37 .With fewer protein interactions, the size and number of thermal protein aggregates decreased, and ultimately the chance of thermally induced gel generation is suppressed 37 .Guo et al. 16 showed that the protein stabilizer Lys significantly increased the solubility of  www.nature.com/scientificreports/proteins, thereby altering the texture and water retention of meat products.In addition, Lys raises the pH of the protein solution 48 .Lys is an alkaline amino acid that regulates the pH to keep proteins away from pI 19 .As the pH moved away from pI, the negative charge in protein solutions increases, repulsion between proteins increases, sites for binding water are enhanced, and the ability to form protein gels is diminished 49 .Thus, the strongly hydrophilic Lys enhances protein-water interactions.However, Li et al. 24 reported that the addition of Lys and Arg, with pH at myosin pI also increased their solubility.As a result, the increase in protein solubility may be the result of a combined effect.

SEM.
At different temperature conditions (100 °C, 121 °C), the formation of CP thermally induced gels diminished with increasing amounts of Lys added.The CP solution was unable to form thermally induced gels at Lys content of 0.2% (w/v).The SEM images also confirmed this view (Fig. 6).CP thermally induced gels show rough surfaces, large cavities and uneven porosity.The three-dimensional surface of the thermally induced protein gels formed a flatter and more uniform structure with smaller pores after the addition of Lys 50 .It was attributed to the fact that the addition of Lys increased the solubility of the proteins (Fig. 2), altered the tertiary structure of the protein (Fig. 3), reduced the particle size of the protein (Fig. 4), decreased the rheological properties of the protein (Fig. 5), and induced the cross-sectional area of the protein to exhibit a smaller cavity distribution and smoother planes.These results further elucidate that Lys increases CP solubility, alleviates the generation of thermal aggregates and inhibits the formation of thermally induced gels.We followed up by exploring the mechanism of action of Lys inhibition of thermal aggregation by the formation of heat-induced gels from the main proteins of the different CP fractions.Subsequently, we explored the mechanism of action of Lys inhibition of thermal aggregation by the formation of thermally induced gels from different fractions of CP proteins.
Exploring mechanisms.Natural proteins of plant and animal origin are thermally processed to produce insoluble thermal aggregates with reduced solubility during thermal processing.It is known as protein denaturation in food processing.When proteins undergo heat treatment conditions, the higher structure undergoes denaturation and unfolding, and the tight and ordered structure becomes a disordered structure peptide chain, which causes a loss of biological activity 51 .The physicochemical properties of denatured proteins are then changed, such as loose the peptide chain, reduced solubility, and precipitation 52 .When the advanced structure  www.nature.com/scientificreports/ of a protein changes, the surface structure of the molecule changes, the hydrophilic groups are relatively reduced, and the hydrophobic groups hidden inside the molecule are exposed on the surface, making the protein particles insoluble with water.It is easy to cause molecules to collide and entangle with each other, and the phenomenon of dissociation and aggregation occurs in the thermal aggregation of proteins 53 .
Several studies have shown that Globulin (Glo) are closely associated with the process of thermal denaturation of proteins, the primary mechanism of which is the formation of disulfide bond (S-S) from free sulfhydryl (-SH) groups in globulins through the oxidation of sulfhydryl groups, resulting in the formation of stable aggregates of proteins 27,46,54 .We have further graded the CP and determined the content of different fractions, as shown in Fig. 7A.As shown in Fig. 7B-C, Glo has the highest total sulfhydryl content, followed by CP, Glutelin (Glu) and Albumin (Alb), indicating that the Glo contain more sulfhydryl groups within the molecule compared to other coconut fraction proteins, which is a prerequisite for thermal aggregation of CP to occur.
Glo contain not only more disulfide bond (S-S) but also more acidic amino acids (Asp and Glu, both containing γ-COOH) [55][56][57] .Some studies have reported that alkaline amino acids (lysine, arginine, histidine) to improve protein solubility, slow heat induced aggregation, and improve gel structure 16,17,25,42,58 .Ma et al. 59 added L-Arg, L-Lys to soybean isolate to increase the solubility, emulsifying activity, and emulsion stability of soy protein.The mechanism of this action may be that after high temperature heat treatment, the acidic amino acid (Asp and Glu) residues within the Glo are exposed to the protein surface and surroundings, providing conditions for the interaction of Lys with Glo.
We hypothesize that the mechanism of action of Lys are shown in Fig. 7D.The possible mechanisms of action that exist are as follows: Firstly, Lys is an alkaline amino acid which raises the pH of the protein environment.The increase in pH raises the negative charge on the protein surface, resulting in greater electrostatic repulsion between proteins.As the electrostatic repulsion increases, the monomeric structure of the protein also increases, lead to a reduction in protein interactions and a reduction in the binding sites for protein interactions, result in a reduction in the conversion of the (-SH) group into a disulfide bond (S-S) 60,61 .
Secondly, Lys contains charged groups such as ε-NH 2 , -COOH, -NH 2 , which readily interact with water and are highly hydrophilic.The positively charged Lys interacts with negatively charged amino acid residues on the protein surface to form complexes.The formation of complex leads to an increase in the hydrophilicity of the protein, which results in a weakening of the conversion of the hydrophilic group (e.g., -SH)) into a disulfide bond (S-S) 17 .
The experimental results of this study validate that Lys can alleviate thermal aggregation of CP induced by heat treatment.Extraction of CP.CP: CP was extracted from coconut endosperm by isoelectric point precipitation.Solid coconut endosperm was removed from the coconut, cut into small pieces, mixed with phosphate buffer (PBS, pH = 8), and stirred.The mixture obtained was stirred continuously at room temperature for 6 h and filtered through 120 mesh gauze.CP mixture was centrifuged (6000 × g, 10 min) (CR22N, Hitachi, Japan), and the supernatant extracted.The supernatant was adjusted to pH = 4.5 with 1 mol/L hydrochloric acid solution, then left for 2 h to precipitate the protein.Proteins were precipitated by centrifugation at 10,000 × g for 10 min to extract the protein precipitate.Protein precipitates were dialyzed in dialysis bags (3800 Da retention capacity) for 48 h and then freeze-dried.The dry protein powder was defatted in hexane for 48 h to obtain CP.The total protein content of CP was determined by Kjeldahl nitrogen determination and using the protein conversion factor (N × 6.25) for all samples 62 .Further graded extraction of coconut protein (CP) was carried out by the method of Deng et al. 63 to obtain albumin (Alb), globulin (Glo), gliadin(Gli) and glutelin (Glu).Protein content measured in solid endosperm was approximately 3.1 ± 0.21%, similar to the results of Kwon et al. 64 .

Methods
Preparation of protein samples.A quantity of lyophilized CP powder was dissolved in deionized water and stirred magnetically at room temperature until all the protein had dissolved to make a protein solution with a mass concentration of 20% (w/v) and adjusted to pH = 7.A portion of the protein solution was diluted into different concentrations (3-13%) of CP solution, packed into sample bottles and subjected to different temperatures (100 °C, 121 °C) heat treatment for 20 min.The heat treatment CP dispersion was removed and placed in ice water, then stored in a refrigerator at 4 °C for 12 h before being removed.The fluidity of the protein samples was observed by inverting the vials.CP diluted into a low concentration (1%) and a high concentration protein suspension (5% and 9%).Various levels of Lys (0-0.3%) are added to the protein suspension to form a Lys-CP mixture and adjusted to pH = 7.The mixture was loaded into sample bottles and heat treatment for 20 min.Heat treatment samples were placed in a water bath in ice water and refrigerated at 4 °C for 12 h before removal.Completed samples were prepared and used for various studies.All samples in triplicate were processed.All samples were processed in triplicate.

DSC thermal analysis.
The protein samples were dissolved in deionized water to form a mixture, sodium azide (0.02%) was added as a preservative and mixed and transferred to a crucible.Samples evolved at 4 °C for 12 h and then the CP samples were measured by DSC (SETARAM, DSC131EVO, France) to obtain a thermal analysis profile.Sample conditions were as follows: A standard heating thermostat procedure consisting of heating the crucible containing the sample with the sample crucible (control) from 20 to 120 °C at a rate of 10 °C/min followed by a constant temperature at 120 °C for 20 min was set.Obtained images were analyzed using Calisto thermal analysis software.All samples were processed in triplicate.

Effect of Lys on low concentration CP solutions. Determination of turbidity and solubility. Prepared
Lys-CP mixture at 2.5 mg/L diluted with distilled water, and the turbidity of the solution at 350 nm was determined to use an enzyme marker (Biotek Instruments, Biotek, USA) and expressed as A350 nm.All samples were treated in triplicate.
Solubility (%) of the Lys-CP mixture as modified from Taşkın et al. 65 .Allow 10 mL of the prepared Lys-CP mixture to be centrifuged at 5000 × g for 10 min in a centrifuge (CR22N, Hitachi, Japan) and extract the supernatant.To 1 mL of supernatant, add 5 mL of Bradford's dye solution.After 10 min the absorbance of the protein was measured at 595 nm using an enzyme marker (Biotek Instruments, Biotek, USA).The soluble protein content (mg/mL) calculated using the standard curve for bovine serum albumin (BSA).Solubility (%) calculated as the percentage of protein concentration after centrifugation to the initial total protein concentration before centrifugation.All samples treated in triplicate.
Fluorescence spectroscopy scanning.The fluorescence spectroscopy scanning of the Lys-CP mixture was measured using an F-7000 fluorescence spectrophotometer (Hitachi Limited, Japan) at room temperature (25 °C).The measurement parameters were excitation wavelength 280 nm, emission wavelength 300-500 nm, excitation and emission wavelength slits 5 nm; PMT voltage 400 V; scanning speed 240 nm/min.All protein samples were measured three times in parallel.
Particle size distribution of the Lys-CP mixture.The particle size distribution of the Lys-CP mixture was measured using a laser particle size analyzer (Ineas Physical Optics Instrument, Co. Ltd., WJ-60, Shanghai, China).Lys-CP solution was homogeneously mixed, poured into a stirred tank containing deionized water, and measured at a shading rate 1.50.All samples treated in triplicate.

Effect of Lys on high concentration CP solutions. Determination of the dynamic rheology of
Lys-CP.Frequency scan tests were carried out using a rheometer (HAAKE MARS40, Thermo Fisher Scientific, USA).Plates with a diameter of 35 mm and a rotor model P35/Ti-02180953 for use.The sample with a thickness of approximately 1 mm was placed on the plate.Set the temperature to 25 °C, the frequency range to 0.1-10 Hz, and the strain setting to 1%.The G′ and G″ values of the gel samples were measured and all samples tested were within the linear viscoelastic region.All samples were processed in triplicate.
SEM.Heat treatment Lys-CP samples were freeze-dried (LGJ-10, Songwon Huaxing Technology Development Co., Ltd., China) for 48 h.The dried samples were gold sprayed and observed under a field emission scanning electron microscope (ZEISS sigma 300, Germany) operating at 15 kV and a magnification of 5 kV.

Figure 1 .
Figure 1.Thermal analysis of CP. (A) DSC profiles of CP; (B) Effect of 20 min heat treatment at different temperatures (100 °C, 121 °C) on CP solutions.

Figure 3 .
Figure 3. Change in fluorescence spectroscopy scanning of Lys-CP solution.(A) FI of heat-treated CP solutions at different temperatures; (B-D) FI of heat-treated Lys-CP mixture at different temperatures (25 °C, 100 °C, 121 °C).

Figure 4 .
Figure 4. Effect of Lys on the particle size distribution of CP particles.(A) Particle size distribution of CP solutions under heat treatment at different temperatures; (B-D) Particle size distribution of Lys-CP solutions under heat treatment at different temperatures (25 °C, 100 °C, 121 °C).

Materials.
Coconut was purchased from the aquatic wharf market in Haikou, Hainan, China.The collection of plant material and the experimental study of these plants comply with the national guidelines of China; Lys was purchased from Shanghai Yuanye Biotechnology Co., Ltd.(Shanghai, China).All solutions were prepared to use deionized water.All other chemicals are of analytical grade.

Figure 7 .
Figure 7. Exploration of the mechanism of Lys on CP heat aggregation.(A) Protein content of different fractions of CP. (B) Free sulfhydryl and total sulfhydryl content of different fractions of CP. (C) Apparent diagram of thermally induced gel formation in different fractions of CP at 121 °C for 20 min.(D) Diagram of the mechanism of CP thermal aggregation inhibition by Lys.