Impact of hot alkali modification conditions on secondary structure of peanut protein and embedding rate of curcumin

Wei Li, Shugng Li, Yong Hu, Mengzhou Zhou, Cho Wng, Dongsheng Li,?,Deyun Li

a Hubei University of Technology, Wuhan, 430068, China

b Hubei Cooperative Innovation Center for Industrial Fermentation, Wuhan, 430068, China

c Function Food Engineering Center of Hubei province, Hubei University of Chinese Medicine, Wuhan, 430068, China

Keywords:

A B S T R A C T

1. Introduction

Peanuts are rich in unsaturated fatty acids and linolenic acid, and they contain 8 essential amino acids and up to 25%–30% crude protein. Peanut protein isolate (PPI) produced by alkaline extraction and subsequent isoelectric precipitation technology using slightly denatured and defatted peanut protein powder is a proteinaceous material with relatively uniform components and good physicochemical properties [1]. PPI is composed mainly of arachin (14S)and conarachin (7.8S and 2S) [2]. General properties of arachin and conarachinare shown in Table 1 [3]. They occupy an important economic position in world agricultural production and trade.In recent years, peanut protein has interested researchers because it is the best source of wall material for preparing nano-particles and microcapsule particles due to its good biocompatibility and biodegradability, excellent nanometer granulation, and protective slow-release effects [2,3]. However, research has found that certain proteins in peanuts have compact four-stage structure, with rarely exposed active sites.It is necessary to improve the functionsof concentrated peanut protein to extend its application Protein molecules must be modified to expose more active groups and increase contact surfaces with core material to improve embedding activity [4].

Table 1 General properties of arachin and conarachin.

Fig.1. Chemical structure of curcumin.

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)1,6-heptadiene-3,5-dione) is one of the most popular natural polyphenols derived from Curcuma longa L. Its compound demonstrates a wide spectrum of properties, especially appreciated in the pharmaceutical and food industries [5–7]. Chemical structure of curcumin shown in Fig.1 [8].

However, bioavailability is low due to low solubility, poor stability and other factors; thus, the application of curcumin in food and medicine is limited [8,9]. In order to improve curcumin bioavailability and take advantage of it in functional foods and supplements,several carriers including emulsions [6], protein nanoparticles[7,16], protein nanotubes [8], complex coacervates [9], solid lipid nanoparticles [10], filled hydrogel beads [11], liposomes [12], and casein micelles [13–15] have been investigated. This experiment modified isolated and extracted peanut protein with hot alkali to study the impact of pH, heating temperature, processing time and other alkali liquor conditions on the molecular structure of the peanut protein and the embedding rate of curcumin. Finally,orthogonal tests were used to explore optimal technological conditions for the embedding of curcumin with modified peanut protein.The breakthrough and innovation in this study focus on the research method by embedding of curcumin. By modifying peanut protein, improving its properties and extending its application range, we can open up a new method adapted to produce, it is important in practice.

2. Materials and methods

2.1. Preparation of peanut protein isolate

We referred to the methods of Zhang et al. [2]. The peanut(purchased from the vegetable market at the South Gate of Hubei University of Technology, moisture content ≤ 5%) was removed after drying the peanut kernel; then the pent kernel was crushed and screened with 60 mesh screen. Five times mass volume of petroleum ether was added for degreasing. The mixture underwent extraction for 2 h at 4°C (DF-101S heat collecting thermostatic heating magnetic stirrer, Gongyi Yuhua Instrument Co., Ltd.Henan)and centrifugation for 5 min at 6000 r/min (TGL-20 M desktop high speed refrigerated centrifuge from Hunan Xiangyi Laboratory Instrument Development Co., Ltd). Then, 10% NaOH was added to the solid residue from three times of repetitive extraction, degreasing, and centrifugation, according to a mass ratio of 1:5. The pH was adjusted to 9. The mixture was stirred and centrifuged at 6000 r/min for 5 min to collect the filtrate. Then, 8% HCl was used to adjust the pH of the filtrate to 4, after which the mixture was allowed to stand for 1 h to collect the sediment. The sediment was washed with deionized water until the pH was neutral, and then it was frozen. The sample was obtained by drying with the vacuum freeze drier (LGJ-12 vacuum freeze drier, Zhengzhou Honglang Instrument Equipment Co., Ltd.).

2.2. Modification of peanut protein

A 10 g sample of peanut protein was weighed and dissolved in 100 ml 0.5 M NaOH solution. The test was conducted as follows: (1)10% NaOH and 8% HCl were used to adjust the pH of the protein solution to 8, 9, 10, 11 or 12, after which the tubes containing protein sample were immersed in a water bath at 85°C for 15 min; (2)10% NaOH and 8% HCl were used to adjust the pH of the protein solution to 10, after which the tubes containing aprotein sample were immersed in a water bath at 75, 80, 85, 90 or 95°C for 15 min;and (3) 10% NaOH and 8% HCl were used to adjust the pH of the protein solution to 9, after which the tubes containing aprotein sample were immersed in a water bath at 85°C for 5, 10, 15, 20 or 25 min.

Upon the completion of the above operations, the solution was allowed to cool to the ambient temperature, after which it was centrifuged at 6000 r/min for 5 min to collect the filtrate. The pH of the filtrate was adjusted to 4 with 8% HCl, after which the solution was allowed to stand for 1 h to collect the sediment. The sediment was washed with deionized water until the pH was neutral, and then it was frozen. The sample was obtained by drying with the vacuum freeze drier for 72 h. Modified peanut proteins prepared under different conditions was obtained. Then, 6 g modified peanut protein was weighed and dissolved in 100 ml double distilled water to prepare modified peanut protein solution.

2.3. Determination of secondary structure

Using the methods of Ye et al. [17] Fourier infrared transform spectrum (FTIR)(Nicolet5700, Thermo Fisher Scientific Inc., USA)was adopted to analyze the structural information of peanut protein before and after modification. Then, the 2 mg sample was accurately weighed and added to 100 mg KBr. The mixture was evenly ground in a mortar, after which it was tableted and analyzed 128 times with full-band scanning (400 –4000cm?1) with an infrared spectrometer.The data (indoleamine I,1600 –1700 cm?1)from the infrared spectrum were processed with the software Peak-Fit 4.12 (SPSS Inc., Chicago, IL, USA).

2.4. Preparation of curcumin granule embedded with modified protein

A curcumin sample was weighed to be 0.5 g and dissolved in 1 L absolute ethyl alcohol to prepare curcumin ethanol solution with a mass concentration of 0.05%. Then, 30 ml curcumin ethanol solution was collected to mix with the peanut protein solution prepared under different modification conditions in 1.3.2. The mixture was stirred and maintained in a dark place for reaction for 2 h. Then,80 ml 5 mmol/L CaCl2was added to the mixed solution, and the mixture was allowed to stand at ambient temperature for 16 h, after which it underwent desalting treatment with 14,000 KD dialysis bag for 72 h. Solid peanut protein granules with curcumin embedded therein were obtained by freeze drying.

Fig.2. Deconvolved and curve fit bands of amide I of peanut protein isolate.

2.5. Determination of embedding rate

Using the methods of Xun et al. [18] a certain amount of protein grains with curcumin embedded therein was collected and placed in 5 ml centrifuge tube. Then, ethanol was added, and the mixture was violently vibrated for 5 min and centrifuged at a speed of 10,000 r/min for 5 min; the supernatant was collected to determine its absorbency with ultraviolet-visible spectrophotometer(UV2450/2550 ultraviolet spectrophotometer from Shimazu International Trade (Shanghai) Co., Ltd.). For the ultraviolet analysis of the retained curcumin, the absorbance at 424 nm was measured.The sample in the centrifuge tube was further repeatedly washed by the same method until minimal changes in the curcumin content were observed. The sum of the curcumin measured in the supernatant, each time, was the amount of curcumin absorbed on the surface of the sample. The amount of residual curcumin in the sample was obtained by dissolving the sample in ethyl acetate.The embedding rate of curcumin was calculated with Formula (1):where m1is the total amount of curcumin in the sample, and m2is the amount of curcumin absorbed on the surface of the sample.

2.6. Data processing

The data were processed with OriginPro 2007 (OriginLab Corporation, USA) and SAS 8.1(North Carolina State University,US). The measurement results were expressed with average value ± deviation(x ± std). The significance level was 0.05, and the highly significant level was 0.01.

3. Results

3.1. Identification of structure of infrared Spectrum of unmodified peanut protein

Table 2 Fitted results of amide I band of peanut protein.

Fig.2 shows the atlas of Amide Band I in the infrared spectrum of unmodified peanut protein after curvefitting. From Fig.2, there were 10 structure-relevant peaks after deconvolution, indicating that the secondary structure of the peanut protein was very complicated. Table 2.1 shows the results of the fitting analysis of the figure.According to the structural features of peanut protein and the pertinent literature [17], the structural information and percentage corresponding to the above 10 peaks are shown in Table 2.

3.2. Impact of modification conditions on secondary structure of peanut protein

3.2.1. Impact of pH of alkali liquor on secondary structure of peanut protein

Table 3 demonstrates the fitting results of Acylamide Band I of the sample from the alkali liquor with different pH values, heated by the water bath at 85°C for 15 min. When the pH was less than 11, as the pH of the alkali liquor increased, the percentage of AAR (1605–1610cm-1) gradually increased from 4.32 ± 0.43% to 5.21 ± 0.33% (p < 0.01). The percentage of random coil(1645–1648 cm-1) gradually increased from 10.21 ± 1.96%to 11.24 ± 0.87% (p < 0.05), while the percentage of β-sheets at 1637–1639 cm-1 gradually decreased from 12.27 ± 0.87% to11.85 ± 0.97% (p < 0.01) and that of the α-helices gradually decreased from 11.76 ± 2.04% to 10.85 ± 2.67% (p < 0.05)

Table 3 Fitted results of amide I band of peanut protein treated with alkaline liquor.

Fig.3. Changes in secondary structure of peanut protein with increasingly alkaline pH.

Fig.3 demonstrates the superposition results of the same secondary structure classification identified at different peak positions in Table 3. When pH was within the range of 8–12, the percentage of random coils and β-turns gradually increased with increasing pH, while the percentages of α-helices and β-sheets gradually decreased with increasing pH. When pH was below 11, the percentage of AAR gradually increased with increasing pH.

3.2.2. Impact of heating time on secondary structure of peanut protein

Table 4 demonstrates the fitting results of Acylamide Band I of the sample from the treated alkali liquor with pH = 10, heated by a water bath at 85°C for different times. Within 20 min,with increased heating time, the percentage of AAR (1605–1610 cm?1) gradually increased from 4.56 ± 1.01% to 5.22 ± 1.54% (p <0.05). The percentage of random coils (1645–1648 cm?1) gradually increased from 10.49 ± 3.12% to 11.43 ± 2.16% (p < 0.05),and that of β-sheets at 1617–1618 cm?1gradually increased from 9.31 ± 0.09% to 10.7 ± 0.97% (p<0.01). The percentage of βsheets at 1691–1692 cm?1gradually decreased from 5.57 ± 0.61%to 4.82 ± 0.57% (p<0.05). Within 25 min, the percentage of α-helices decreases from 11.83 ± 1.04% to 10.75 ± 2.34% (p < 0.05).

Fig.4 demonstrates the superposition results of the same secondary structure classification identified at different peak positions in Table 4. Within the range of 5–25 min, the percentage of random coils and AAR increased with increased heating time, and the percentage of α-helices gradually decreased from 11.77 ± 0.37% to 10.41 ± 1.18% (p<0.01).

3.2.3. Impacts of heating temperature on secondary structure of peanut protein

Table 5 demonstrates the fitting results of Acylamide Band I of the sample from the treated alkali liquor with a pH = 10, heated by a water bath at different temperatures for 15 min. When the heating temperature was within the range of 75–95°C, with increasing the heating temperature, the percentage of AAR (1605–1610 cm?1)gradually decreased from 4.94 ± 1.11% to 3.92 ± 1.54% (p<0.01),and the percentage of random coils(1645–1648 cm-1) gradually increased from 10.01 ± 2.37% to 11.93 ± 2.16% (p<0.05). While the percentage of β-sheets at 1627–1628 cm-1 gradually increasedfrom 11.29 ± 0.06% to 12.14 ± 1.46% (p<0.01), the percentage of βsheets at 1682–1684 cm-1 gradually increased from 9.43 ± 0.61%to 10.03 ± 0.38% (p<0.05). The percentage of α-helices gradually decreased from 11.76 ± 2.04% to 10.85 ± 2.67% (p<0.05).

Table 4 Fitted results of amide I band of peanut protein treated with heating time.

Fig.4. Changes in secondary structure of peanut protein with increased heating time.

Table 5 Fitted results of amide I band of peanut protein treated with heating temperature.

Fig.5 demonstrates the superposition results of the same secondary structure classification identified at different peak positions in Table 4. When the heating temperature was within the range of 75–95°C, the percentage of random coils and β-sheets gradually increased with increasing of heating temperatures, while the percentage of α-helices and AAR gradually decreased with increasing heating temperature (Table 6).

Fig.5. Changes in secondary structure of peanut protein with increasing of heating temperature.

Table 6 Factors and levels.

Fig.6. Changes in embedding rate of curcumin with increased alkaline pH.

3.3. Embedding rate of curcumin

3.3.1. Single factor experiment

Fig.6 demonstrates the impact of the peanut protein sample treatment(from alkali liquor at different pH values, heated by a water bath at 85°C for 15 min) on the embedding rate of curcumin. When the pH of alkali liquor was within the range of 8–12,increased pH first increased the embedding rate of curcumin and then decreased it; when the pH was 10, the embedding rate was the highest, 82.09 ± 1.64%. Therefore, pH values of 9, 10 and 11 were selected for the orthogonal test.

Fig.7. Changes in embedding rate of curcumin with increased heating time.

Fig.7 demonstrates the impacts of the peanut protein sample(obtained from alkali liquor at pH = 10 heated with a water bath at 85°C for different times) on the embedding rate of curcumin.When the heating time was within the range of 5–25 min, with increasing time, the embedding rate of curcumin increased first and then remained the same. When the heating time was 20 min, the embedding rate was highest, 84.73 ± 1.65%. Therefore, the heating times of 15, 20 and 25 min were selected for the orthogonal test.

Fig.8 demonstrates the impacts of the peanut protein sample(obtained from alkali liquor at pH = 10 and heated by a water bath at different temperatures for 20 min) on the embedding rate of curcumin. When the heating temperature was within the range of 85–95°C, increased heating temperature first increased and then decreased the embedding rate of curcumin. When the heating tem-perature was 90°C, the embedding rate was highest, 85.23 ± 1.08%.Therefore, the heating temperatures 85°C, 90°C and 95°C were selected for the orthogonal test.

Table 7 Arrangement and results of orthogonal test of L9 (34).

Fig.8. Changes in embedding rate of curcumin with increased heating temperature.

Table 8 Result of orthogonal test variance analysis.

3.3.2. Design of orthogonal test

Using the embedding rate of curcumin as the optimizing index for the single factor experiments, the three levels of water bath temperatures (A), heating times (B) and the pH of alkali liquor (C)were selected for the orthogonal design according to orthogonal table L9(34): see Table 5 for the factor level table; see Table 7 for experimental results; and see Table 8 for variance analysis results.

From the range analysis in Table 7, the order of influence level of three factors on the embedding rate of curcumin was as follows: the pH of alkali liquor (C) > heating time (B) > heating temperature (A).Within the range of the experimental design, the optimum conditions for hot alkali modification of peanut protein were B2A2C3.That is to say, the conditions of heating temperature of 90°C,heating time of 20 min and pH of alkali liquor of 11, enabled a maximum embedding rate of curcumin. From Table 8, the pH of alkali liquor had extremely significant impacts on the embedding rate of curcumin, and the heating time and temperature had significant impacts of the embedding rate of curcumin.

3.3.3. Confirmation experiment

Three parallel experiments were conducted according to the composition conditions A2B2C3for the orthogonal test. The embedding rate of curcumin reached 88.32 ± 1.29%, which was higher than all test results in Table 7. Therefore, A2B2C3was the optimum modification condition for the embedding of curcumin with peanut protein.

4. Discussion

Peanuts are usually consumed by millions of people in the world mainly as toasted or salted snacks and as peanut butter or sweets.Peanuts are also an important source of cooking oil considered premium [2]. This material is used mainly in the Asian continent for the preparation of meals and the residue of the process is a flour with high protein content (47%–55%) commonly used as ingredient in animal feedstocks. Despite its mild taste, good color and digestibility, the defatted peanut flour is rarely used in human foods [3]. The feasibility of using peanut protein is often limited because of its poor solubility,gel property and this feature is associated with the high molecular weight of the protein structure and the low number of ionizable groups. Such functionalities can be modified to improve solubility by Mechanical or chemical action.

Secondary structure elements of protein include α-helices, βsheets, β-turns, random coils and other conformations, α-helices and β-sheets are ordered arrangements in proteins that are maintained by hydrogen bonds [20], the secondary structure of a protein directly impacts its functional properties. The secondary structure and functional properties of protein may be affected by factors that affect the stability of the hydrogen bonds between protein molecules, facilitating the gel [21], emulsifying [17,22] and foaming properties [23] of a protein.

Some studies showed that the electrostatic field [24,25] and pH[26] affect the distribution of amino acid side chain charges and the stability of hydrogen bonds in salt-soluble myofibrin molecules.Ying et al. [27] found that increased pH promoted the conformation of tilapia myosin; Liu et al. [28,29] found that, under acidic conditions, changes in electrostatic interactions of myofibrillar protein molecules affect the stability of hydrogen bonds in pork and cause the loss of α-helices in these proteins. When studying the impacts of heating temperature and the pH of alkali liquor on the secondary structure of peanut protein, the α-helix was easily lost under acidic conditions and gradually decreased with increasing pH, that is, alkaline conditions. Based on previous studies [28–30],it is likely that intermolecular electrostatic interactions and the stability of hydrogen bonds in peanut protein are weaker in alkaline or acidic condition than neutral condition. Therefore, when modifying peanut proteins, determining appropriate pH conditions will be beneficial for opening the compact four-stage structure of peanut protein. The increased content of AAR in Table 3 and Fig.3 further verified that the compact and stable long-chain structure of peanut protein began to depolymerize, and the modification was in progress.

Ling et al. [31] found, by studying the impacts of heating on the structure of myofibrin in chicken breast, that changes in heating temperature significantly changed the secondary structure of protein; Jian et al. [32] studied the impact of heating conditions on the molecular structure of soybean protein isolate. With increased heating temperature, they found that hydrogen bonds in the αhelix structure gradually fractured, and the structure uncoiled,decreasing the proportion of α-helix structures; Stathopulos et al.[33] studied temperature increases during heating and observed the polymerization of group R, and that the stability of α-structure was damaged when group R was too large. The results in Table 4 and Fig.4 demonstrated that, within the range of 75°C–95°C,the percentage of α-helices decreased with increased temperature,which may indicate that the degree of molecular expansion of the peanut protein increased. The increased percentage of β-sheets and decreased percentage of AAR may indicate increased aggregation between protein molecules.

Peanut protein isolates were successfully modifed using hot alkali. Within the levels evaluated, reaction parameters such as alkali pH, temperature and time affected the conditions of secondary structure. All these are the efforts to make innovations.The optimum conditions for hot alkali modification of peanut protein as followed: heating temperature of 90°C, heating time of 20 min and a pH of alkali liquor of 11. Under these optimum conditions,the embedding rate of curcumin by the modified protein can reach 88.32 ± 1.29%. HPLC and peanut protein isolate.will be used separating and purifying in the next step, It is expected that results will be useful to assess the feasibility of these ingredients as novel food components for human consumption,

Conflict of interest statement

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, “Impact of Hot Alkali Modification Conditions on Secondary Structure of Peanut Protein and Embedding Rate of Curcumin.

Acknowledgements

This work was financially supported by The foundation for young scientists of hubei province (grant number 610112246), the foundation for Doctoral startup project of Hubei University of Technology (grant number 337/338). We would like to thank LetPub(www.letpub.com) for providing linguistic assistance during the preparation of this manuscript.